BuffaloBulletinVol 32 SP2

See discussions, stats, and author profiles for this publication at: https://www.researchgate.
net/publication/261065145
Quality evaluation of olive oil coated Labneh cheese mixed with culinary herbs
Article in Buffalo Bulletin · January 2013
CITATIONS READS
5 1,612
4 authors, including:
Sarfraz Ahmad Faqir Muhammad Anjum

Engro Foods Limited University of Gambia
35 PUBLICATIONS 970 CITATIONS 329 PUBLICATIONS 9,731 CITATIONS
SEE PROFILE SEE PROFILE
Mian Anjum Murtaza

University of Sargodha
131 PUBLICATIONS 1,613 CITATIONS
SEE PROFILE
Some of the authors of this publication are also working on these related projects:
Research work of my Msc(hons.) thesis View project
Pesticide residues and Heavy metal in vegetables Punjab Pakistan View project
All content following this page was uploaded by Mian Anjum Murtaza on 13 October 2015.
The user has requested enhancement of the downloaded file.

Editors
Wisitiporn Suksombat Rangsun Parnpai
Mariena Ketudat-Cairns Kanokwan Srirattana
Kanchana Punyawai Arthip Limcharoen
Congresses Secretariat
Embryo Technology and Stem Cell Research Center
School of Biotechnology
Institute of Agricultural Technology
Suranaree University of Technology
E-mail: info@wbc2013, www.wbc2013.com
Editorial Board
Buffalo Reproduction Symposium
1. Prof. Yindee Kitiyanant
2. Prof. Dr Kehuan Lu
3. Prof. Dr. Dheer Singh
4. Dr. Sunpetch Sophon
5. Assoc. Prof. Petai Pongpiachan
6. Assoc. Prof. Dr. Yupaporn Chaiseha
7. Assoc. Prof. Dr. Bunlue Kornmatitsuk
8. Assoc. Prof. Dr. Suneerat Aiumlamai
9. Assoc. Prof. Dr. Siriwat Suadsong
10. Asst. Prof. Pongthorn Suwannatada
11. Asst. Prof. Dr. Theerawat Tharasanit
12. Dr. Takashi Nagai
13. Dr. Kei Imai
14. Dr. Tamas Somfai
15. Dr. Giorgio Antonio Presicce
16. Dr. Vibuntita Chankitisakul
17. Dr. Nutthee Amin
18. Dr. Anucha Sathanawongs
19. Dr. Krittiya Lertchunhakiat
20. Dr. Thuchadaporn Chaikhun
21. Mr. Anawat Sangmalee
22. Ms. Jakkhaphan Chasombat
23. Mr. Ashit Pual

Buffalo Genetics and Breeding Symposium
1. Prof. Dr. Kanchana Markvichitr
2. Prof. Masroor Ellahi Babar
3. Prof. Leopoldo Iannuzzi
4. Assoc. Prof. Dr. Monchai Duangjinda
5. Assoc. Prof. Dr. Marina Ketudat-Cairns
6. Assoc. Prof. Dr. Neramit Sukmanee
7. Asst. Prof. Dr. Sirinporn Sindhuvanich
8. Asst. Prof. Dr. Skorn Koonawootrittririon
9. Asst. Prof. Dr. China Supakorn
10. Asst. Prof. Dr. Amornrat Molee
11. Asst. Prof. Dr. Wuttigrai Boonkum
12. Dr. Nalinee Imboonta
13. Dr. Supawadee Manatrinon
14. Dr. Warangkana Kitpipit
15. Dr. Yuanyuan Liang
16. MS. Kanokwan Srirattana
17. Mr. Phakphume Saowaphak
Buffalo Nutrition and Feeding Symposium
1. Assoc. Prof. Dr. Wisitiporn Suksombat
Buffalo Health Symposium
1. Prof. Dr. Chaleow Salakij
2. Assoc. Prof. Dr. Anudep Rungsipipat
3. Assoc. Prof. Dr. Wijit Banlunara
4. Asst. Prof. Dr. Somporn Techangamsuwan
5. Asst. Prof. Dr. Burin Nimsuphan
6. Asst. Prof. Khamphee Pattanatanang

7. Asst. Prof. Dr. Sonthaya Tiawsirisup
8. Asst. Prof. Dr. Nareerat Viseshakul
9. Asst. Prof. Dr. Worakij Cherdchutham
10. Asst. Prof. Dr. Sirikachorn Tangkawattana
11. Asst. Prof. Dr. Piyanan Taweethavonsawat
12. Dr. Kidsadagon Pringproa
Buffalo Physiology Symposium
1. Assoc. Prof. Dr. Apassara Choothesa
2. Assoc. Prof. Dr. Jatuporn Kajaysri
3. Assoc. Prof. Dr. Witaya Suriyasathaporn
4. Asst. Prof. Dr. Siriwan Prapong
5. Dr. Chaiwat Boonkaewwan
6. Dr. Narudee Kashemsant
Buffalo Production and Management Symposium
1. Prof. Dr. Ibrahim Soliman
Buffalo Socio-Economic and Sustainable Production Symposium
1. Assoc. Prof. Dr. Kwunkamol Donkwa
2. Asst. Prof. Dr.Kanchana Sukanthasirikul
3. Asst. Prof. Chanisa Maneeratrungrord
4. Mrs. Ancharlie Na Chiangmai
5. Dr. Kampanat Vijitsrikamol
6. Dr. Worapote Suthisai
7. Dr. Boonchauy Boonme
8. Dr. Kulapa Kuldilok

Buffalo Meat and Meat Products Symposium
1. Professor Dr. Sanchai Jaturasitha
2. Assoc. Prof. Dr. Suthipong Uriyapongson
3. Asst. Prof. Dr. Sujate Chaunchom
4. Asst. Prof. Dr. Chaiyawan Wattanachant
Buffalo Milk and Milk Products Symposium
1. Assoc. Prof. Dr. Somkiert Prasanpanich
2. Asst. Prof. Dr. Manote Sutheerawattananonda
3. Asst. Prof. Dr. Piyawan Gasaluck
4. Asst. Prof. Dr. Surasak Kochapakdee
5. Asst. Prof. Dr. Pipat Lounglawan
6. Dr. Pisut Niumsup
7. Dr. Dumrong Leenanuruksa

Contents
Page
Buffalo Reproduction
Effect of Different Doses of hCG at AI on Pregnancy Rates of Muhammad Saleem 362

Repeat Breeder Nili-Ravi Buffalo AKHTAR
Pregnancy Rate in Lactating Buffaloes Treated with or Muhammad Saleem 366

without Estradiol after Estrus Synchronization Protocols at AKHTAR
Timed AI
Cholesterol Enhances Post-Thaw Semen Quality in Buffaloes Abdul SATTAR 370

(Bubalus bubalis)
Assessment of the Rate of Pregnancy in Buffaloes Crossbred Nestor Simon Montiel 375
Lactation using Two Protocols CIDR-SYNCH® in 6 and 8 URDANETA
Days
Increasing Efficiency of Artificial Insemination (AI) in Felomino V. MUMUAD 376

Buffalo Upgrading Program in Nueva Ecija, Philippines
Thyroid Hormone Levels during Oestrous Cycle in Santosh Hirba DALVI 380
Pandharpuri Buffalo
Efficiency of OPU-IVEP-ET of Fresh and Vitrified Embryos Wilson SALIBA 385

in Buffaloes
Pregnancy Monitoring of In Vitro Produced Embryos in Wilson SALIBA 389

Buffaloes
Development of Frozen Buffalo Semen Production in the Felomino MAMUAD 392

Phillippines
Morphology and Biometrics of Spermatozoa of Two Breeds Sofronio P. KALAW 393

of Water Buffalo (Bubalus bubalis L.)
Development of Assessing Motility in Computer Assisted Emma VENTURINA 397

Sperm Analyzer and Phase Contrast Microscopy
A Two-step Culture System Sustains Individual Buffalo Wenxin ZHANG 401

Embryo Development
Comparison of Motility, Morphology, Acrosome Integrity, Nutthee AM-IN 405

Membrane Integrity and Fertilizing Ability of Frozen-Thawed
Buffalo Sperm Separated by a Percoll® Gradient or
PureSperm®
Effect of Percoll® Density, Duration and Force of Vibuntita 409

Centrifugation on Sperm Motility, Morphology, Acrosome CHANKITISAKUL
Integrity, Membrane Integrity and Sperm Recovery Rate of
Frozen-Thawed Buffalo Semen
Determination of Pregnancy Associated Glycoproteins Levels Rafael PAIVA 413

in Water Buffalo Cows after Calving
Controlled Breeding and Reproductive Management in Shivayogayya HIREMATH 418
Buffaloes using EAZI Breed CIDR
A Comparison of In Vitro Development of Buffalo (Bubalus Jianghua SHANG 423

bubalis) Embryos Produced either by Somatic Cell Nuclear
Transfer or In Vitro Fertilization using Oocytes Obtained by
Different Methods
Optimization of Culture and Cryopreservation of Hand-Made Jianghua SHANG 427

Clone (HMC) Buffalo (Bubalus bubalis) Embryos
The Impact of Linolenic Acid on In Vitro Development of Jianghua SHANG 432

Buffalo Embryos
A Preliminary Study on Stable Transfection of EGFP in Jianghua SHANG 436

Buffalo Cumulus Cells
Scrotal Circumference Growth Curves of Buffalo Bulls of Marc HENRY 439

Different Breeds Raised in Brazil
Validation of an ELISA for Detection of Pregnancy Rafael PAIVA 443

Associated Glycoprotein in Water Buffalo Cows
Profiles of Pregnancy-Associated Glycoprotein (PAG) Van Hanh NGUYEN 448

Concentrations during Gestation in Swamp Buffalo
Effect of Povidone-Iodine Uterine Flushing on Insemination Caro B. SALCES 452

Success Rate for Grazing Dairy Buffaloes
Effects of Curcumin on Buffalo Embryonic Development Jiang-Hua SHANG 456

In Vitro
Supplementation of Epidermal Growth Factor into In Vitro Nguyen VIET LINH 460
Maturation Medium Improves Fertilization Efficiency of
Swamp Buffalo Oocytes
Effect of Improving Reproductive Management on Dairy Nasim AHMAD 464

Farm Economics
Localization of GnRH Receptors in Buffalo Cow Pituitary Thuchadaporn CHAIKHUN 468

Gland in Follicular and Luteal Phases
Progestin-Based Protocol for Synchronization of Follicular Lindsay Unno GIMENES 473

Wave Emergence in Buffaloes during Summer and Winter
Influence of Parity and Season of the Year on Oocyte Quality Lindsay Unno GIMENES 476
and Number in Buffaloes
Comparison of Two Synchronization Protocols for Timed Domenico VECCHIO 479

Artificial Insemination in Acyclic Italian Mediterranean
Buffalo Cows out of the Breeding Season
Morphometric and Functional Characteristics of Buffaloes and Eunice OBA 480

Cows Corpus Luteum at Different Stages of the Estrous Cycle
Response to the First GNRH and Pregnancy Outcome in Gianluca NEGLIA 483
Buffaloes Underwent Ovsynch and Fixed Timed Artificial
Insemination
Comparison of Botu-Bov and Tris as Freezing Extenders of Eunice OBA 484
Buffalo Sperm Recovered from Epididymal Cauda
Adding Motility Stimulants to Improve Freezing of Buffalo Eunice OBA 487

Sperm Recovered from Epididymal Cauda
Effect of Testicular Thermoregulation on the Quality of Carlos Ramires NETO 489

Buffalo Sperm
Attenuation of LPS Induced Proinplammatory Gene Vengala Rao YENUGANTI 493

Expression by Conjugated Linoleic Acid (CLA) in Buffalo
Granulosa Cells
Frozen-Thawed Epididymal Sperm Quality and the Success Yusnizar YULNAWATI 494
Rate of Artificial Insemination in Spotted Buffaloes (Bubalus
bubalis carabanensis)
Supplementation of Buffalo Follicular Fluid: Beware of Other Kitiya SRISAKWATTANA 498

Sources of Steroid Hormones during Culture
Use of Commercially Available Bovine Semen Sexing Agent Vittoria Lucia BARILE 502
in Buffalo: Preliminary Report of the Effect on the Conception
Rate
Leptin and Pregnancy: Preliminary Results in Buffalo Cows Vittoria Lucia BARILE 505
(Bubalus bubalis)
Preliminary Evidence on Effect Induced by HeifersPlusTM Vittoria Lucia BARILE 509

after In Vitro Exposure on Functional Parameters of Buffalo's
Spermatozoa
Pre-ovulatory Follicle Size, LH Peak Values and Pregnancy Giuseppina 513

Induced by Three Synchronization Treatments in Buffalo MariaTERZANO
Cows, during Non Breeding Season
Progesterone Treatment during the Periovulatory Period Júlia Gleyci SOARES 517
Decreases Embryo Production in Superovulated Buffaloes
PGF2α Treatment during the Periovulatory Period Increases Júlia Gleyci SOARES 522
the Number of Embryos Recovered from Superovulated
Buffaloes
Use of Different Progestagens for Ovulation Synchronization Nelcio Antonio Tonizza de 527
and TAI in Buffaloes during the Non Breeding Season CARVALHO
Use of Intravaginal Progesterone Devices during Eight or Nelcio Antonio Tonizza de 532
Nine Days in the Ovulation Synchronization Protocol for TAI CARVALHO
in Buffaloes during the Non Breeding Season
Plasma Progesterone Radioimmunoassay as a Tool to Confirm Wanvipa SUTHIKRAI 537

Ovarian Cyclicity of Recipients Following Nonsurgical
Transfer of Cloned Swamp Buffalo (Bubalus bubalis Lin.)
Embryo
Seasonal Effect on Oocytes Recovery Rate and Maturation Kriengsak TASRIPOO 541
Rate of Swamp Buffalo Ovaries Collected from
Slaughterhouse in Thailand
In Vitro Embryo Production and Transfer of Bubaline Flocerfida AQUINO 538
Embryos Using Oocytes Derived from Transvaginal
Ultrasound-Guide Follicular Aspiration (TUFA)
L-Carnitine Influences the Developmental Competence of Marlon B. OCAMPO 542

Buffalo Oocytes from Aged Donors
ART’s for In Vitro Production of Buffalo Embryos (Philippine Marlon B. OCAMPO 543
Experience)
Developmental Competence of Water Buffalo Oocytes In Marlon B. OCAMPO 544

Vitro in Single Base Medium-Synthetic Oviductal Fluid
Blastocyst Formation after Intracytoplasmic Sperm Injection Prudencio B. PEDRO 545

in Bovine and Buffalo Oocytes Derived from Slaughterhouse
Somatic Cell Nuclear Transfer as a Tool for the Multiplication Edwin C. ATABAY 549
of Genetically Superior Water Buffaloes: The Philippine
Initiatives
Improving Ovulation and Conception Rates in Oestrus Bahareldin-Ali ABDALLA 553

Synchronized and Artificially Inseminated Water Buffalo
Cows by Immunization against Inhibin
Uterine Microbial Flora of Nili-Ravi Buffalo during Estrus Masood RABBANI 557
and Its Relationship with Pregnancy Rate in Pakistan
Reproductive Performance of Murrah Buffaloes under Jamlong MITCHAOTHAI 561

Intensive Farming System in Thailand
Fetal Loss in Dairy Buffaloes in Eastern of Thailand Jatuporn KAJAYSRI 565
Study of Frequency Distribution of Calving of River Nestor Simon Montiel 568

Buffaloes (Bubalus bubalis) in Twelve Farms in Different URDANETA
Ecological Areas in Venezuela
Generation of Genetically Modified Buffalo Embryos using Chunying PANG 569

Multiple-Locus Gene Targeting
Effect of Frozen Semen from Italian Mediterranean Buffalo Guangsheng QIN 570
on Some Reproductive Parameters and Conception Rate
Performed to Different Species Water Buffalo in Southern
China
Early Embryo Development in Buffalo Adriana Caroprezo MORINI 575

Glucose, Cholesterol, Total Protein and Growth Factor Carlos GALLEGO 579
Insulin-like Type I in Follicular Fluid of River Buffaloes
(Bubalus bubalis)
Uterine Involution, Fluid Accumulation and Ovarian Activity Eunice OBA 583
after Injection of Prostaglandin at Different Periods of Buffalo
Puerperium
Values of 13,14-dihydro-15-keto-PGF2α, Progesterone and Eunice OBA 586

Oestradiol after Injection of Prostaglandin at Different Periods
of Buffalo Puerperium
Molecular Evaluation of Developmental Competence of Laís Mendes VIEIRA 596
Oocytes Collected In Vivo from Buffalo and Bovine Heifers
during Winter and Summer
The Carabao Development Program as the Cornerstone of the Eufrocina P. ATABAY 601
Livestock Biotechnology Program in the Philippines
Factors Affecting the Performance of an Artificial Jesus BERDUGO 604

Insemination Program in North Coast Colombia
First Report of an Artificial Insemination Program with Sexed Juan ANGEL 607
Semen in North Coast Colombia
Assessment Buffalo Spermatozoa Parameters in using Flow Jesus BERDUGO 609

Cytometry
Effects of L-carnitine Supplemented in Maturation Medium Teewara PHONGNIMITR 613

on the Maturation Rate of Swamp Buffalo Oocytes
Influence of Growth Factors on Survival and Development of Kwanrudee 617

Swamp Buffalo Early Antral Follicle Cultured In Vitro KAEWMUNGKUN
Abstracts Buffalo Genetics and Breeding
Genetic Parameters for Reproductive Traits of Crossbred Carlos Henrique Mendes 623
Buffaloes from Brazil, Estimated by Bayesian Inference MALHADO
Genetic Parameters for Growth Traits of Mediterranean Vanius FALLEIRO 627

Buffaloes from Brazil, Estimated by Bayesian Inference
Growth Traits of Anatolian and Anatolian x Italian Crossbred Özel ŞEKERDEN 632
Buffalo Calves under the Village Conditions
Population Parameters Based on Known Pedigree Records Carlos Henrique Mendes 637
from Brazilian Jaffarabadi Buffaloes MALHADO
Inbreeding, Average Relatedness Coefficient and Effective Carlos Henrique Mendes

Population Size in Jaffarabadi Buffaloes Raised in Brazil MALHADO 641
Genetic Parameters for Milk Yield and Lactation Length of Carlos Henrique Mendes 646
Crossbred Buffaloes from Brazil by Bayesian Inference MALHADO
Genetic Correlation for Pre-Weaning and Post-Weaning Traits Vanius FALLEIRO 650
of Mediterranean Buffaloes from Brazil, Estimated by
Bayesian Inference
Productive and Reproductive Traits in Murrah Breed from Alcides Amorim RAMOS 654
Brazil
Genomic Exploration of Pakistani Buffalo: Our Black Gold Masroor Ellahi BABAR 658
Application of Reactions Norms in Study of Genotype Humberto TONHATI 662
Environmental Interaction for Milk Yield of Buffaloes
High Conservation of SRY Gene in Buffalo Compared to Tanveer HUSSAIN 666

Other Bovids
The Swamp Buffalo: Domestication, Dispersal, and Genetic Yi ZHANG 671
Differentiation
Single Nucleotide Variations in the Buffalo Kappa-Casein Nedenia Bonvino 675

Gene (CSN3) STAFUZZA
Allelic Variant Analysis of the DRB3 Gene by PCR-RFLP in Nedenia Bonvino 680
Mediterranean Buffaloes Breed STAFUZZA
Cattle-Derived Microsatellite Markers to Study the Water Nedenia Bonvino 681

Buffalo Genome STAFUZZA
Genetic Estimates for Productive Parameters in Buffaloes in Nestor Simon Montiel 686
Different Ecological Areas in Venezuela URDANETA
Index of Heritage's Weight at Birth and Effects of Some Nestor Simon Montiel 687
Environmental Factors on Buffalo Calf using the Animal URDANETA
Model
Genetic Parameters for Productive and Reproductive Traits for Camila da Costa BARROS 688
Milk Buffalo in Brazil
Genetic Variants of POU1F1 Gene in Azakheli Buffalo Breed Asif NADEEMA 692
of Pakistan
Single Nucleotide Polymorphisms in Bovine CYP11B1 Gene Maryam JAVED 697

in Nili Ravi Buffalo Breed of Pakistan
Identification of Single Nucleotide Polymorphisms in OLR1 Maryam JAVED 701

Gene in Nili Ravi Buffalo
POU1 Transcription Factor 1 DNA Polymorphism in Nili Rashid MAJEED 706

Ravi Buffalo
Bovine Stearoyl-CoA Desaturase Gene Polymorphism in Nili Maryam JAVED 710

Ravi Buffalo
Genetic Parameter Estimates for Milk Yield and Lactation Yenny GARCÍA 714
Length in Buffalo
Genetic Parameters and Trends for Weaning Weight and Nikorn SANGHUAYPHRAI 717
Calving Interval of Department of Livestock Development
Swamp Buffalo
Genetic Polymorphism in Caspase Activating Recruitment Archana VERMA 723

Domain 15 and Its Association with Incidence of Mastitis in
Murrah Buffalo
Genetic Variability in Production and Immune Function Archana VERMA 729

Genes Associated with Production Traits and Incidence of
Mastitis in Indian Murrah Buffalo
Genotyping and Molecular Characterization of NRAMP1/-2 Lawrence P. BELOTINDOS 730

Genes as Location of Markers for Resistance and/or
Susceptibility to Mycobacterium bovis in Swamp and Riverine
Type Water Buffaloes
Buffalo Genetic Improvement Programme in Nepal-Current Bhola Shankar SHRESTHA 734
Status and Future Prospects
Optimization of the Transfection Efficiency of Buffalo Fetal Tingxian DENG 744

Fibroblasts Cells using Liposome
The Construct of Fat-1 Gene Targeted Buffalo Kidney Yingyin ZHANG 745
Fibroblast Cell
Multiple-trait Genomic Evaluation for Milk Yield and Milk Humberto TONHATI 746
Quality Traits using Genomic and Phenotypic Data in Buffalo
in Brazila
Preliminary Results on the Growth Performance of F1 Guangsheng QIN 750

Mediterranean Buffalo Offspring Crossed in China
Karyotype Analysis of Mediterranean Buffalo and Its Hybrids Fenxiang HUANG 755
Estimation of Genetic Parameters for Growth Traits of Three Agapita J. SALCES 760
Genotypes of Water Buffalo Bulls Raised on a Ranching
Operation
Some Environmental Factors Affecting Performance Traits in Khalid JAVED 764

Registered Nili Ravi Buffalo Population at Field Area of
Pakistan
Genetic Parameters for Milk Yield and Milk Component Ester B. FLORES 768
Traits Estimated from Test Day and 305D Lactation Records
of Philippine Dairy Buffaloes
Improvement, Utilization and Conservation of Buffaloes Muhammad Nawaz SAEED 773

under New Legislation of Pakistan
Genetic Parameters for Test-Day Fat Yield Estimated by Humberto TONHATI 774
Random Regression Models in Dairy Buffaloes using
Bayesian Inference
Development of Multiplex PCR for Sexing Buffalo Embryos Wisut NUALCHUEN 775
Studies on Linear Type Traits and Morphometric Khalid JAVED 780

Measurements in Nili Ravi Buffaloes of Pakistan
Identification of Amino Acid Substitutions and Structural Asif NADEEM 784

Model Prediction of POU1F1 Gene in Azakheli Buffalo
Breed of Pakistan
Polymorphisms in Osteopontin Gene in Amazon Buffaloes Sebastião Tavares ROLIM 788

FILHO
Relationship between Body Measurements and Milk Nisar AHMAD 792

Production in Nili-Ravi Buffaloes Maintained at Commercial
Farms in Peri-urban Vicinity of Lahore
Buffalo Milk Transcriptomics Christophe LEFEVRE 796

Compare of Phenotypic Variation with the Polymorphism of Qingkun ZENG 805
One Coagulation Related Gene Locus in Buffalo Kappa-
Casein
Comparative Proteomic Analysis of the Changes of Milk Qingkun ZENG 811
Protein Associated with Different Breeds of Buffalo
Productive and Reproductive Parameters in Buffaloes and N. MONTIEL-URDANETA 812

Cows in a Farm Located in Tropical Dry Forest in Venezuela
Productive and Reproductive Parameters in Buffaloes and N. MONTIEL-URDANETA 813

Cows in a Farm Located in Tropical Humid Forest in
Venezuela
Microsatellite Analysis of Thai Swamp Buffalo Cloned Calf Supitchaya 814

Derived from Ear Fibroblasts TREEBONMUANG
The Comparison of Some Reproductive Traits of Anatolian Özel ŞEKERDEN 819

and F1 Crossbred (Anatolian X Italian) Buffalo under Village
Conditions in Turkey
Abstracts Buffalo Nutrition and Feeding
Minerals Status of Soil, Fodder and in Lactating Nili-Ravi Muhammad Saleem 824
Buffaloes in Irrigated Agro-ecological Zone of Punjab, AKHTAR
Pakistan
Digestibility and Performance of Buffalo Fed Total Mixed Suthipong 829

Ration with Different Levels of Citric Waste URIYAPONGSON
Milk Yield Response of Bypass Protein Feeding (Soybean Netra Prasad OSTI 834
Meals) in Dairy Animals
Body Composition and Net Energy Requirements for André Mendes JORGE 840
Maintenance of Non-Castrated Water Buffaloes (Bubalus
bubalis)
Allometry of Organs and the Gastrointestinal Tract from André Mendes JORGE 844
Water Buffaloes (Bubalus bubalis) Finished in Feedlot
Distribution of Beta-Catenin in Colon Precarcinomatous Marcial SÁNCHEZ 848

Lesions of Rats Fed with Functional Buffalo Milk NEGRETTE
Anticancer Effects of Bubaline Functional Milk with Higher Gabriela Verónica 853
Concentration of Conjugated Linoleic Acid and Omega-3 RAMIREZ
Fatty Acids
Virtues of the Milk from Water Buffalo Muhammad YOUNAS 857
Improving Oat Grass Silage Quality through using Exogenous Mahr-un-NISA 866
Enzyme in Cannulated Buffalo Bulls
Effects of Roughage Types on Feed Intake, and Nutrient Walailuck KAEWWONGSA 871
Digestibilities in Swamp Buffaloes (Bubalus bubalis)
Comparative Performance of Calves Fed Milk and/or Milk Muhammad ABDULLAH 874
Replacer Supplemented with Calf Starter up to Weaning Age
in Nili-Ravi Buffaloes
Changes in Molecular Diversity of Rumen Methanogens in Gondelina A. RADOVAN 878

Buffalo and Cattle in Response to Dietary Tannin
Comparative Utilization of Different Types of Roughage in Thongsuk JETANA 883
Thai Swamp Buffalo and Thai Brahman Cattle Based on In
Vivo Nutrient Utilization, Nitrogen Balance and Purine
Derivatives Excretion in the Urine
454 GS FLX Pyrosequencing Reveals Rumen Bacterial Chengjian YANG 888

Diversity of Chinese Water Buffalo
Rumen Bacterial Diversity of Murrah and Nili-Rivi Buffalo Chengjian YANG 889
(Bubalus bubalis) Assessed by 454 GS FLX Pyrosequencing
Novel Methods to Improve the Nutritive Value of Low Faisal SHAHZAD 890
Quality Roughages for Nili Ravi Buffalo Calves
Effect of Cysteamine Hydrochloride on In Vitro Methane Caixia ZOU 894

Emission in Water Buffalo
Evaluation of Nutritive Value of Agricultural By-Products and Junhua ZHOU 895

Industrial By-Products for Buffalo by Cornell Net
Carbohydrate and Protein System
Effects of Beer Lees and Cassava Residues Respectively Caixia ZOU 900
Substituting for Soybean Meal and Grassiness on Milk
Performance in Lactating Water Buffalo
Graded Replacement of Corn Grain by Wheat Grain in Muhammad Aasif 904

Sahiwal Calves: Influence on Nutrients Intake, Digestibility, SHAHZAD
Blood Metabolites and Growth Performance
Response and Ruminal Characteristics of Buffalo Bulls Fed Muhammad SARWAR 908
Urea-Molasses Treated Wheat Straw Inoculated with Rumen
Digest
Response of Sahiwal Heifers Receiving Maize Fodder with Nasir Ali TAUQIR 913
Supplementation of Urea Molasses Block
Performance of Nili Ravi Buffalo Calves Fed Urea-Corn Muhammad Aasif 918
Steep Liquor Treated Corn Cobs SHAHZAD
Estimation of In vitro Methane Production in Buffalo and Federico INFASCELLI 924

Cow
Chemical Composition, Rumen Fermentation Kinetics, Patricia. M. Guimaraes 928

Digestibility and Energy Value of Cassava Leaves Hay at BEELEN
Different Storage Times
Effects of substituting Beer Lees and Cassava Residues Zhongsheng XIA 929
Respectively for Buffalo Dietary Soybean Meal and
Grassiness on Rumen Fermentation In Vitro
Nutritive Value and In Situ Digestion Kinetics of Some Nawaz SAEED 938
Leguminous and Non-leguminous Fodder Baled Silages in
Buffalo Bulls
Potential Benefits from the Utilization of Some Natural Feed Thongsuk JETANA 942
Resources in Thai Swamp Buffaloes
Effects of Lasia spinosa Thw. and Season on Plasma Leptin Ratree JINTANA 947
and Glucose of Weaned Female Murrah X Swamp Buffalo
Calves
Rumen Bacterial Diversity of Water Buffalo (Bubalus Chengjian YANG 951

bubalis) as Influenced by Concentrate Levels
Effect of Rumen-Protected Methionine and Reduced Crude Antonella CHIARIOTTI 952

Protein in Lactating Mediterranean Buffaloes Diet
Effect of Varying Levels of NDF on Voluntary Intake, Saeed AHMED 957

Nutrients Digestibility for Nili Ravi Buffalo Heifers
Effects of Augmented Feeding with By-Passed Amino Acid Daniel Lopez AQUINO 961
and Slow-Released Non-Protein Nitrogen Supplements on
Milk Peak, Lactation Persistency and Post-partum
Reproductive Performance of Brazilian Buffaloes
Effect of Protein on Microbial Protein Synthesis and Pramote PAENGKOUM 966

Productive Performances of Thai Swamp Buffalo (Bubalus
bubalis)
Effect of Protein Level and Urea Source in Concentrate on Sungchhang KANG 970
Feed Intake and Rumen Ecology in Swamp Buffalo Fed Rice
Straw
Effect of Dried Leucaena Leaf Supplementation on Rumen Kampanat PHESATCHA 975

Ecology, Nutrient Digestibility and Urinary Excretion of 2,3-
Dihydroxy Pyridone (2,3-DHP) and 3,4-Dihydroxy Pyridone
(3,4-DHP) in Swamp Buffaloes
Effects of Eucalyptus Crude Oils Supplementation on Nguyen The THAO 980

Nutrients Digestibility of Swamp Buffaloes
Influence of Urea-Calcium Mixture in High-Quality Feed Anusorn CHERDTHONG 984

Block on Ruminal Fermentation in Swamp Buffalo
Nutritional Status of Some Trace Minerals of Water Buffaloes Maha Mohamed HADY 988
in Egypt
Effect of Roughage Sources and Fibrolytic Enzyme Chalermpon 993

Supplementation on Nutrient Digestion and Rumen YUANGKLANG
Fermentation in Buffaloes
Buffalo Health
Antigen Based Detection of Cystic Echinococcosis in Arumugam SANGARAN 999

Buffaloes using ELISA and Dot – EIA
Comparative Efficacy of Enrofloxacin and Oxytetracycline as Muhammad KASHIFA 1002

Systemic Dry Period Therapy for the Control of Bubaline
Mastitis
Incidence and Organ Wise Involvement of Hydatidosis in Arumugam SANGARAN 1009

Buffaloes
Emphysemated Necrotic Skin Disease (Patakha): a Newly Abdul SHAKOOR 1011
Emerging Disease in Buffaloes
Comparative Efficacy of Three Indigenous Plants (Fumaria Sayyed AUN 1016

parviflora, Artemesia maritima & Swertia chirata) Alone or in MUHAMMAD
Combination for the Treatment of Toxaemia
In Vivo Comparison of Specific Activity of Egg Yolk Tanveer AHMAD 1017

Immunoglobulins (IgY) and Antibiotic Against
Staphylococcus aureus Causing Mastitis in Buffaloes
(Bubalus bubalis)
Antibiogram Analysis of Staphylococcus aureus isolated from Abid HUSSAIN 1021

Mastitic Milk Samples of Buffaloes in District Bhimber Azad
Kashmir
Molecular Identification of Brucella abortus Bv5 and Strain Diana MARTINEZ 1029
19 in Water Buffaloes (Bubalus bubalis) in Northeast
Argentina
Efficacy Analysis of Parasitic Integrated Control in Buffaloes Eduardo BASTIANETTO 1033
Detection of Toxin Genes by PCR in Clostridium perfringens Esterina DE CARLO 1035

Isolates Collected from Water Buffaloes (Bubalus bubalis)
Affected by Lethal Enterotoxemia
BUN and Total Protein Levels of Buffalo Population in Chanachai BOONPERM 1038
Udonthani Province of Thailand
Prevalence and Molecular Diagnosis of Staphylococcus Waseem SHAHZAD 1041

aureus Subclinical Mastitis in Lactating Nili-Ravi Buffaloes
(Bubalus Bubalis) at Livestock Experiment Station,
Bahadurnagar, Okara, Pakistan
Prevalence, Molecular Diagnosis and Treatment of Field Waseem SHAHZAD 1046

Isolates of Toxogenic Pasteurella multocida in a Hemorrhagic
Septicemia Outbreak in Nili-Ravi Buffalo Calves at Livestock
Experiment Station, Bahadurnagar, Okara, Pakistan
Management of Khari Disease Syndrome with Pentasulfates Doj RAJ KHANAL 1051
Supplementation in Lactating Buffaloes
Isolation of Mycoplasma capricolum subspecies capricolum Esterina DE CARLO 1056

from Dairy Buffalo (Bubalus bubalis)
Bubaline Herpesvirus 1 Associated with Abortion in a Esterina DE CARLO 1059

Mediterranean Water Buffalo
Doramectin Resistance in Helminths from Buffaloes Euardo BASTIANETTO 1063
Breeding Techniques, Welfare and Mammary Gland Esterina DE CARLO 1066

Pathologies in Buffalo
Comparison between Two Gamma-Ifn Assays and Esterina DE CARLO 1071

Intradermal Tuberculin Test for the Diagnosis of Tuberculosis
in Water Buffalo (Bubalus bubalis)
Carrier Status of Foot and Mouth Disease in Ruminants Muhammad Hassan 1075
through Reverse Transcription Polymerase Chain Reaction MUSHTAQ
The Metaphylactic Efficacy of Toltrazuril (Baycox® Bovis - Giovanna CAPPELLI 1076

Bayer) and Diclazuril (Vecoxan® - Esteve Veterinaria) in
Natural Infections of Eimeria spp. in Buffalo Calves: a
Multicenter Trial in Southern Italy
Gastrointestinal Parasitic Diseases of Buffaloes and Bhoj Raj JOSHIP 1082

Implications of Climate Change for these Diseases in Nepal
Detection of Bovine Viral Diarrhea Virus Prevalent in Dairy Masood RABBANI 1088
Herds of Punjab, Pakistan
Clinical Evaluation of Hypertonic Saline Solution for Frederico Augusto Mazzocca 1091
Treatment of Lactic Acidosis in Water Buffaloes Lopes RODRIGUES
Influence of Storage Temperature on Blood Gas Analyses on Antonio Humberto Hamad 1092
Buffalo Venous Blood MINERVINO
Experimental Induction of Lactic Acidosis in Buffaloes with Raimundo Alves 1093

Sucrose BARRÊTO-JÚNIOR
Mineral Status of Buffaloes Raised in the Wetlands of the Enrico Lippi ORTOLANI 1094
Lower Amazon Basin Ecosystem
Serological Profile of Buffalo (Bubalus bubalis) Female Geraldo de NARDI JÚNIOR 1095
Calves Vaccinated with Standard Brucella abortus Strain 19
Vaccine using Rose Bengal, 2-Mercaptoethanol and
Complement Fixation Tests
Interference of Vaccinal Antibodies on Serological Diagnostic André Mendes JORGE 1099

of Leptospirosis in Vaccinated Buffalo using Two Types of
Commercial Vaccines
Vaccine Trial of Recombinant Schistosoma japonicum Mario Antonio L. JIZ II 1103

Paramyosin in Water Buffaloes
Seroepidemiological Investigation, Risk Factors Analysis of Mansur-ud-Din AHMAD 1104

Brucellosis in Ruminants and Their Owners in District Buner
of Pakistan
Disease Susceptibility of Buffalo in Pakistan Sidra MANZOOR 1110
Epidemiological Studies on Mastitis in Pakistani Buffalo Sidra MANZOOR 1114
Excretion of Aflatoxin M1 in Milk of Mediterranean Italian Carlo BOSELLI 1119

Buffalo Cow Fed Diet Naturally Contaminated of Aflatoxin
B1
Preliminary Results on the Utilization of Phytotherapy in Domenico VECCHIO 1123

Organic Italian Mediterranean Buffaloes
Buffalo Physiology
Bone Marrow´s Harvest in the Coxal Tuberosity for Isolation Eunice OBA 1125
and Culture of Mesenchymal Stem Cells of Buffaloes
(Bubalus Bubalis)
Isolation, Culture and Differentiation of Buffaloes Bone Eunice OBA 1128

Marrow Mesenchymal Stem Cells Obtained from the Coxal
Tuberosity
Haematochemical and Hormonal Parameters Related to Cristina RONCORONI 1131

Buffalo Calves Welfare
A Preliminary Study about Lymphocyte Subset of Water Cristina RONCORONI 1136

Buffalo Calves
Impact of Thermal Stress on Rectal, Skin Surface Vijay KUMAR 1141

Temperatures, Respiration Rate, Heat Load Index and Heat
Storage in Lactating Murrah Buffaloes (Bubalus bubalis)
Scrotal Thermography and Doppler Ultrasonography of the Carlos Ramires NETO 1145
Testicular Artery of Buffaloes Subjected to Environmental
Heat Stress
Blood Biochemical Profiles of Mehsana Riverine and Thai Suvit BOONPRONG 1146
Swamp Buffaloes under Tropical Conditions in the Northeast
Thailand
Changes in Insulin-Like Growth Factor-Binding Proteins in Sirima THONGRUAY 1151

Swamp Buffalo Uterus during Estrous Cycle
Use of Oxytocin and Milking Management of Buffaloes in Muhammad TARIQ 1155

(Urban) Peri-Urban Area of Faisalabad
Environmental Factors Affecting Live Weight and Khalid JAVED 1161

Morphological Traits in Nili Ravi Buffaloes of Pakistan
BUN and Total Protein levels of Buffalo Population in Chanachai BOONPERM 1165
Udonthani Province of Thailand
Preliminary Study on Energy Metabolism and Energy Caixia ZOU 1168

Requirement of Early Lactation Water Buffalo
Buffalo Production and Management
Inter-relationship of Milk Constituents with Body and Udder Khalid JAVED 1170
Measurements in Nili-Ravi Buffaloes Raised at Commercial
Farms of Pakistan
An Integrated Study on Milk and Beef Production Conducted Luis Mateo FRAGA 1174
at Macún Buffalo Enterprise in Cuba, Some Results and BENITEZ
Recommendations
Productivity of Mehsana Riverine Buffalo under Tropical Prapawan SAWASDEE 1175
Conditions of Thailand
Status and Perspectives of Buffalo in Bangladesh Quazi M EMDADUL 1179

HUQUE
Buffalo Farming in Bhutan: Challenges & Opportunities Nar Bahadur TAMANG 1184
Transition of Milk Production and Reproduction of Dairy Yoshiaki HAYASHI 1188

Buffaloes in Nepal
Buffalo Socio-economic and Sustainable Production
Community Management for Buffalo Eco-Tourism in Udon Krisdakorn WONGWAI 1193

Thani
Economical and Social Importance of Buffalo in a Small Milk Eduardo BASTIANETTO 1197
Production System
Coordination of the Chain of Buffalo Milk in São Paulo State Fabrício Pini ROSALES 1200
(Brazil)
Swamp Buffalo Production System and Needs for Extension Sornnarong 1204
on Local Scale Farmers in the Lower Northeast of Thailand SUPHACHAVALIT
The Problems and Obstacles on Raising Buffaloes of Local Sirisuk SAPAPANAN 1208
Farmers in Central Thailand: A Case Study of Saraburi
Province
Assessment of Village-Based Artificial Insemination Sonia D. POL 1212

Technician (VBAIT) Scheme as a Strategy Towards
Privatization of Artificial Insemination (AI) Services in Nueva
Ecija
Condition and Specifics of Buffalo Breeding in Republic of Bone PALASHEVSKI 1218

Macedonia
Assessing the Performance of Village-Based Artificial Eric PALACPAC 1222

Insemination Technicians for Water Buffaloes in Nueva Ecija
Province, Philippines
Sustainability of Philippine Carabao Center and Primary Wilma T. Del ROSARIO 1226
Cooperative Partnership in Carabao-Based Enterprise
A Model of Sustainable Herd Buffalo Farming in Songkhram Tanapat SURANARAKUL 1230

Wet Land, Nakhon Phanom
Contextualizing the Gatasng Kalabaw Festival in Support to Marlowe U. AQUINO 1233

the Carabao-based Enterprise in Nueva Ecija, Philippines
Economics of Raising Calves on Milk and/or Milk Replacer in Muhammad SAADULLAH 1237
Nili-Ravi Buffaloes
Biohydrogen Production from Buffalo Manure Codigested Antonella CHIARIOTTI 1241

with Agroindustrial By-products in an Anaerobic Reactor
The Herd Size and Production Performances of Buffalo in Livia VIDU 1245
Romania
Buffalo Meat and Meat Products
Fatty Acids in the Muscle and Fat Layer of Buffaloes Exequiel Maria PATIÑO 1250
Supplemented with Fish Oil
Using Slaughter Weight to Predict Weight and Yield of Primal André Mendes JORGE 1254
Cuts of Carcass from Buffaloes
Carcass Composition and Meat Quality of Buffalo by Raised Tanom TATHONG 1258
Alongside Mekong River: Nakhon Phanom Province
Effect of Gender on Carcass Composition and Meat Quality of Tanom TATHONG 1262
Buffalo in Wet-Land in Nakhorn Phanom
Tenderization Effect on the Physical Characteristics of Laura Marcela MENDOZA 1266

Commercial Cuts of Second and Third Buffalo´s Meat ARAGÓN
(Bubalus bubalis) during Ripening Process
Conjugated Linoleic Acid and Fatty Acids Profile in Buffalo Federico INFASCELLI 1270
Meat
Determination of Muscularity and Correlation with Body Gladis REBAK 1274

Weight in Buffalo in the Northeast of Argentina
Relation of the Approximate Age and Ultrasound Data in Gladis REBAK 1277
buffalo in the Northeast of Argentina
Research regarding the dynamics of body development in Livia VIDU 1280

young buffaloes, depending on various factors
Estimation of Fine Cuts of Meat Production Performance in Roberta VITTORIA 1284

Mediterranean Italian Buffalo Young Bulls
Body Condition Score (BCS) System in Murrah Buffaloes Kappa Sarjan RAO 1290
Buffalo Milk and Milk Products
Flavored Probiotic (Acidophilus) Buffalo Milk: Development Muhammad JUNAID 1300

and Quality Assessment
Effective Environmental Factors on Milk Composition, Özel ŞEKERDEN 1305

Rennet Coagulation Time and Urea Content of in Anatolian
Buffaloes Milk of Ilikpinar Village Hatay Province
A Study on the Composition and Microbiology of Raw Milk Jitkamol THANASAK 1311
from Three Breeds of Buffalo in Thailand
Quality Evaluation of Olive Oil Coated Labneh Cheese Mixed Sarfraz AHMAD 1316
with Culinary Herbs
Seasonal Variations in Chemical Composition of Buffalo Milk Sarfraz AHMAD 1324
Milk CLA Content and ∆9 Desaturase Activity in Buffalo Federico INFASCELLI 1330
Cows along the Lactation
Milk Yield and Milking Characteristics in Murrah Buffaloes Alberto de GUSMÃO 1334
Submitted to Machine Milked with or without Calf COUTO
Relationship of Udder and Teat Morphology with Milk Muhammad ABDULLAH 1335
Production in Nili-Ravi Buffaloes of Pakistan
Differences in Lactation Curves and Peak Production in Angelo COLETTA 1339

Mediterranean Italian Buffaloes Bred According to Three
Levels of Production
Microstructure, Rheological and Textural Characteristics of Mian Anjum MURTAZA 1346

Low Fat Buffalo Milk Cheddar Cheese
Buffalo Bulletin 2013 Vol.32 (Special Issue 2): 362-365
Effect of Different Doses of hCG at AI on Pregnancy Rates of Repeat Breeder

Nili-Ravi Buffalo
Muhammad Saleem AKHTAR, *Laeeq Akbar LODHI, a Abdul Asim FAROOQ, Muhammd
Mazhar AYAZ, Saeed MURTAZA, Muhammad ARSHAD, Irtaza HUSSAIN, Ijaz AHMADa
and Mushtaq HUSSAINb
Faculty of Veterinary Sciences, Bahauddin Zakariya University, Multan, Pakistan, aUniversity of

Agriculture, Faisalabad, Pakistan, bInstitute of Pure and Applied Biology, Bahauddin Zakariya
University, Multan, Pakistan.
*Corresponding e-mail: drsaleem46@hotmail.com
ABSTRACT
The efficacy of different doses of hCG administered at the time of artificial insemination (AI) for
the treatment of repeat breeder buffaloes was evaluated by using a total of 140 Nili-Ravi buffaloes.
The animals were assigned at random to four treatments (n=35) administered i.m at the time of
artificial insemination (AI) following estrus synchronization with a single dose of PGF2α. The
treatments were, 3000 IU hCG (group A), 2250 IU hCG (group B), 1500 IU hCG (group C) and 2ml
saline (control group D). AI was performed about 12 hours following the onset of observed estrus.
Blood samples were obtained from all buffaloes 5 and 12 days after AI. The conception rate at first
service in group A, B, C and D was 48, 28, 25, 17 percent respectively. Conception rate increased as
the dose of hCG increased at first service. The differences between treatment A and other treatments
were significant (P< 0.05). The treatment effects followed the same pattern at the second and third AI
but the differences were not significant. Mean progesterone concentrations were significantly higher
(P< 0.05) in animals that become pregnant to the first AI for between group A on day 5 (1.90 ng/ml)
and day 12 (2.10 ng/ml) than treatments group B, C and D on day 5 (1.01-1.02 ng/ml) and day 12
(1.02-1.03 ng/ml). It was concluded that administration of hCG at the time of insemination increased
progesterone concentrations and improved the pregnancy rate of repeat breeder buffaloes.
Keywords: Buffalo, Repeat breeder, hCG, Pregnancy rate, Progesterone
INTRODUCTION
Repeat breeding is one of the most important reproductive problem in buffalo which distresses
fertility and results huge economic loss to buffalo farmers. Several etiological factors have been
explored and documented, which mainly focused around infection of the uterus (Shukla and Wani,
2002) and delayed ovulation (Dolelel et al., 1998) as major determinants of repeat breeding in bovines.
Since several factors affect the incidence of repeat breeding in dairy cows, it is difficult to make
generalizations regarding predominant causes. Accordingly, many treatment regimens involving
antibiotics (Shukla and Wani, 2002), chemotherapeutic agents and hormones (Stevenson et al., 1990)
have been reported with varying results.
Gonadotropin-releasing hormone (GnRH) has been used over the past 20 years on the day of
insemination or between days 11 and 14 after insemination to improve pregnancy rates, particularly in
repeat-breeder dairy cows. GnRH and hCG have similar effects on the ovary. An injection of hCG
given at the time of mating in cow may improve embryo viability and therefore fertility (Khan et al.,
2003). The effectiveness of hCG in inducing ovulation and the formation of a functional CL has been
described by several authors (Schmitt et al., 1996; Sianangama and Rajamahendran, 1996). There is
scanty information regarding efficacy of hCG by using different doses for treatment of repeat breeder
Accepted April 10, 2013; Online February 24, 2014.

362
buffaloes. Therefore, the present study was intended to evaluate the efficacy of different doses of hCG
at the time of artificial insemination for treatment of repeat breeder buffaloes.
MATERIALS AND METHODS

The present study was under taken at Buffalo Research Institute (BRI), Pattoki, District Kasur,
Pakistan, from October 2010 to March 2011. All animals were free from any reproductive abnormality
when examined per rectally before the start of experiment. These animals had normal estrous cycle
with clear vaginal mucous discharge at estrus but failed to conceived after 3 or more inseminations.
To synchronize estrus all buffaloes were given a single intramuscular injection of prostaglandin
F2α (0.150 mg, Cloprostenol, Fatro, Pharmaceutical Veterinary Industry, Italy, Marketed by Prix
Pharmaceutica, Lahore, Pakistan) on day 12 of the oestrous cycle (oestrus = day 0). Estrus detection of
experimental animals was performed twice daily (morning and evening) by visual observation with the
help of a vasectomized teaser buffalo bull. The artificial insemination (AI) was done approximately
12 h after the onset of estrus by using frozen thawed semen.
One hundred and forty Nili-Ravi buffaloes with a history of repeat breeding were randomly
divided into four groups. Group A (n = 35) buffaloes were treated intramuscularly with each 3000 IU
of hCG (2 ml Pregnyl, N. V. Organon Oss Holland) at the time of artificial insemination (AI). Group B
(n = 35) buffaloes were given intramuscularly with each 2250 IU of hCG whereas group C (n = 35)
buffaloes were treated intramuscularly with each 1500 IU of hCG at the time of artificial insemination.
Group D (n = 35) buffaloes were administered intramuscularly with saline as control at the time of
artificial insemination. Animals returning to estrus were re-inseminated at the subsequent estrus. Those
which did not return to estrus were examined per-rectum on day 60 post-insemination for pregnancy.
The first, second, third service and overall pregnancy rates were calculated.
Blood samples were obtained from all buffaloes 5 and 12 days after treatment. Serum was
pipetted and progesterone was determined through ELISA by using a commercially available kit.
Statistical analysis
Pregnancy rates among different treatment groups and control were compared using χ2-test
(Steel et al., 2006). The mean values of progesterone in various treatment groups were compared by Z-
test. The level of significance was observed at 5%.
RESULTS AND DISCUSSIONS

There were significant variations in first service after hCG treatment and overall pregnancy rate
in different groups of Nili-Ravi buffaloes. Higher dose of hCG significantly increased the pregnancy
rate. In group A, first service pregnancy rate was significantly higher (P < 0.05) in comparison with
groups B, C and D. The overall pregnancy rate was also significantly higher (P < 0.05) in buffaloes of
group A as compared to buffaloes of other groups (B, C and D) (Table-1).
The first service pregnancy rate and overall pregnancy rate differed non-significantly (P > 0.05)
among group B, C and D. There was non-significant (P > 0.05) difference in second and third service
pregnancy rate among buffaloes of all four groups. Similarly the overall pregnancy rate also varied
non-significantly (P > 0.05) between group B, C and D. In the present study, there were enhancement
in first service pregnancy rates from 8 to 31% in different treatment groups (A, B & C) when
compared with group D. Increased pregnancy rates by use of hCG has also been reported by
Rajamahendran and Sianangama (1992). Higher difference in buffaloes of group A may be attributed
to higher doses of hCG which might had enhanced development of the small antral follicle destined to
be ovulated in the second and third phase of estrous cycle. Overall pregnancy rate was also
significantly higher in group A buffaloes in comparison with other groups. The higher dose of hCG
may have boosted LH secretion which in sequence augmented ovulation rate as Chenault et al. (1990)
also reported similar findings by using GnRH analogue. Usefulness of hCG in inducing ovulation and
363
the formation of a functional CL has been reported by many other workers (Schmitt et al., 1996;
Sianangama and Rajamahendran, 1996).
Mean serum progesterone concentrations on day 5 and 12 after AI were significantly higher
(P<0.05) in group A animals in comparison with group B, C and D whereas non-significant difference
in progesterone concentrations were observed among buffaloes of group B, C and D (Table-2).
Buffaloes those were pregnant in their 2nd or 3rd service, the progesterone concentrations at day 5 and
12 after hCG treatment were below 1ng/ml.
Higher progesterone concentrations in blood during pregnancy have also been reported by other
workers (Roy and Prakash, 2009). Human chorionic gonadotrophin (hCG) treatment in cow
(Rajamahendran and Sianangama, 1992) has been linked to elevated numbers of large luteal cells and a
concomitant reduction in the number of small luteal cells, accompanied by increased serum
progesterone the early luteal stage induces the formation of accessory corpora lutea and increases the
surface area and the volume of the CL (Santos et al., 2001). Luteal cells also become larger (Schmitt et
al., 1996) and serum progesterone concentrations rise in response to hCG, mainly due to secretion by
accessory CL but also through stimulation of the spontaneous CL (Rajamahendran and Sianangama,
1992). The high overall pregnancy rate observed following hCG treatment in group A may also be due
to all these above mentioned factors as all these effects may increase serum progesterone
concentrations at the time of maternal recognition of pregnancy and enhance embryo survival
(Rajamahendran and Sianangama, 1992; Santos et al., 2001).
In conclusion, this study demonstrated that the use of 3000 IU hCG at the time of AI increased
serum progesterone levels and improved the pregnancy per artificial insemination in repeat breeder
buffaloes.
REFERENCES
Chenault, J.R., D.D., Kratzer, R.A., Rzepkowski and M.C., Goodwin. 1990. LH and FSH response of
Holstein heifers to fertirelin acetate, gonadorelin and buserlin. Theriogenology 34: 81–84.
Dolelel, R. and M. Aech Sand Lopataova. 1998. Delayed ovulation and embryonic mortality in
subfertile cows. Reproduction in Domestic Animals Suppl. 5:127.
Khan, T.H., P.M. Hastie, N.F.G. Beck and M. Khalid. 2003. hCG treatment on day of mating improves
embryo viability and fertility in ewe lambs. Anim. Reprod. Sci. 76: 81–89.
Rajamahendran, R. and P.C. Sianangama. 1992. Effect of human chorionic gonadotrophin (hCG) on
dominant follicles in cows: accessory corpus luteum formation, progesterone production and
pregnancy rate. J. Reprod. Fertil. 95: 577–584.
Roy, K.S. and B.S. Prakash. 2009. Plasma progesterone, oestradiol-17β and total oestrogen profiles in
relation to oestrous behaviour during induced ovulation in Murrah buffalo heifers. J. Anim.
Physiol. Anim. Nutr. 93: 486–495.
Santos, J.E.P., W.W. Thatcher, L. Pool and M.W. Ovrton. 2001. Effect of human chorionic
gonadotropin on luteal function and reproductive performance of high producing lactating
Holstein dairy cow. J. Anim. Sci. 79: 2881–94.
Schmitt, E.J.P., C.M. Barros, P.A. Fields, M.J. Fields, T. Diaz and J.M. Kluge. 1996. A cellular and
endocrine characterization of the original and induced corpus luteum after administration of a
gonadotropin-releasing hormone agonist or human chorionic gonadotropin on Day 5 of the
estrous cycle. J. Anim. Sci. 74: 1915–1929.
Shukla, M.K. and N.A. Wani. 2002. Treatment of repeat breeding condition using oxytetracycline and
gentamicin therapy in cattle. Anim. Reprod. Sci. 26: 269-270.
Sianangama, P.C. and R. Rajamahendran. 1996. Effect of hCG administration on Day 7 of the estrous
cycle on follicular dynamics and cycle length in cows. Theriogenology 45: 583–592.
364
Sianangama, P.C. and R. Rajamahendran. 1992. Effect of human chorionic gonadotrophin

administered at specific times following breeding on milk progesterone and pregnancy in cows.
Theriogenology 38: 85–96.
Steel, R. G. D., J. H. Torrie and D. A. Dickey. 2006. Principles and Procedures of Statistics. A
biometrical approach. 3rd Ed., McGraw Hill Co. New York, USA.
Stevenson, J.S., E.P. Call, R.K. Scoby and A.P. Phatak. 1990. Double insemination and gonadotropin
releasing hormone treatment of repeat breeding dairy cattle. J. Dairy Sci. 73: 1766-1772.
Table 1. Pregnancy rates of repeat breeder Nili-Ravi buffaloes treated with different doses of hCG at
artificial insemination (AI).
Parameter Group A Group B Group C Group D
(n=35) (n=35) (n=35) (n=35)
Service Rate (%) 100 100 100 100
1st Service Conception Rate (%) 48 a 28b 25b 17b
2nd Service Conception Rate 22 20 17 14
(%)
3rd Service Conception Rate (%) 20 17 14 17
Pregnancy Rate (%) 91 a 65b 57b 48b
Within a row, values with different superscripts are significantly different (P < 0.05).
Table 2. Mean (±SE) serum progesterone concentrations (ng/ml) at day 5 and 12 after AI in buffaloes
that becomes pregnant at first AI.
Groups Day 5 after Day 12 after AI
AI
A (n=17) 1.90±0.08a 2.10±0.10a
b
B (n=10) 1.01±0.09 1.03±0.09b
b
C (n=09) 1.02±0.07 1.03±0.06b
D (n=06) 1.01±0.06b 1.02±0.06b
Within a column, values with different superscripts are significantly different (P < 0.05).
365
Pregnancy Rate in Lactating Buffaloes Treated with or without Estradiol after

Estrus Synchronization Protocols at Timed AI
Muhammad Saleem AKHTAR, * Saleem ULLAH, aAbdul Asim FAROOQ, Muhammd Mazhar
AYAZ, Saeed MURTAZA, Muhammad ARSHAD, Irtaza HUSSAIN and Mushtaq HUSSAINb
Faculty of Veterinary Sciences, Bahauddin Zakariya University, Multan, Pakistan, a Department of

Animal Sciences, Allama Iqbal Open University, Islamabad, Pakistan. bInstitute of Pure and Applied
Biology, Bahauddin Zakariya University, Multan, Pakistan.
*Corresponding e-mail: drsaleem46@hotmail.com
ABSTRACT
The present study was conducted to compare the reproductive performance of forty lactating
Nili-Ravi buffalos treated with GnRH and PGF2α for synchronization of estrus and ovulation with or
without supplemental Estradiol. At Buffalo Research Institute, Pattoki, District Kasur, forty buffaloes
received 25 mg PGF2α at 51 day post-partum for pre-synchronization of estrus cycles. After 13 days
of PGF2α injection, buffaloes were given one of 4 treatments; Group A were given an intramuscular
injection of 100 μg of GnRH followed by an injection of PGF2α 7 days later and a final injection of
GnRH at timed artificial insemination (AI) 48 hour after PGF2α (OvSynch48) whereas Group B were
given same treatment as given to group A buffaloes but with an injection of 1 mg of Estradiol 24 hour
after PGF2α injection. Group C (n=10) were given an intramuscular injection of 100 μg of GnRH
followed by an injection of PGF2α 7 days later and a final injection of GnRH at timed artificial
insemination (AI) 72 hour after PGF2α (OvSynch72) whereas Group D were given same treatment as
given to group C buffaloes but with an injection of 1 mg of Estradiol 24 hour after PGF2α injection.
The estrus expression was 30 %, 60 %, 50 %, 80 % in group A, B, C and D. There was significant
difference (P < 0.05) in estrus expression between all groups. At day 40 and 68, 40 %, 50 %, 50 %, 40
% animals were detected pregnant in group A, B, C, D, respectively. There was significant difference
(P < 0.05) between group A and B, C, whereas non-significant difference (P > 0.05) between group A,
D and group B, C, respectively. There was non-significant difference (P > 0.05) in serum progesterone
concentrations between group A, B, C, D, buffaloes at 7 days before, at the first GnRH, at final GnRH
and 7 days after GnRH of the OvSynch protocols, respectively. It was concluded, that in buffaloes,
extending the period of proestrus and supplementing with estradiol increased display of estrus
primarily at 72 hours after PGF2α but did not improve fertility.
Keywords: Buffalo, Estrus synchronization, GnRH, Estradiol
INTRODUCTION
Various estrus synchronization protocols have been tried among many other reproductive
technologies for improving the fertility of buffaloes. A novel synchronization protocol named Ovsynch
was developed in cows, which requires a three injection schedule (GnRH-PGF2α-GnRH) for
synchronization of ovulation. The technique was successfully carried out in cycling buffaloes (Paul
and Prakash, 2005) for synchronization of ovulation and fixed timed artificial insemination (AI).
Ovsynch is a timed artificial insemination (AI) protocol that reduces days open and increases
pregnancy rates by allowing for control of first and subsequent inseminations. It has now been a trend
to administer GnRH and or PGF2alpha in early postpartum cows and buffaloes in order to hasten early
resumption of cyclic ovarian activity and thereby to increase the reproductive efficiency (Barkawi et
al., 1995; Shah et al., 2002). Studies by Pursley et al. (1995) verified that, administration of GnRH
after PGF2α injection increases the rate of synchronized ovulation in cattle. It has been observed that

366
when PGF2α is administered on palpation of functional carpus leutum about 60-70% of treated
animals, were detected in estrus within 4 days post PGF2α injection.
Synchronization of ovulation with 1 mg of estradiol cypionate increased conception rate in
high producing dairy cows compared with artificial insemination (AI) after a synchronized estrus
(Cerri et al., 2004). No such studies have been undertaken in buffalo. It was hypothesized that
extending the period of proestrus and delaying the time of artificial insemination (AI) from 48 to 72
hour after induced luteolysis would increase expression of estrus, and fertility of buffalo. Therefore,
the study was conducted to compare the reproductive performance of lactating Nili-Ravi buffalos
treated with GnRH and PGF2α for synchronization of estrus, ovulation and conception rate with or
without supplemental Estradiol.

The study was carried out on forty lactating Nili-Ravi buffaloes at Buffalo Research Institute,
Pattoki, District Kasur, Pakistan during December 2010 to March 2011. Animals were divided into 4
groups (A, B, C, D). Each buffalo received 25 mg of PGF2α (Dinoprost tromethamin; 5 ml of Lutalyse
Sterile Solution, Pfizer Animal Health, Ghazi brothers, Pakistan) at 51 days in milk (DIM) for pre-
synchronization of estrus cycles. After 13 days of PGF2α injection, buffaloes were given one of 4
treatments.
OvSynch48
Group A (n=10) were given an intramuscular injection of 100 μg of GnRH (lecirelin acetate; 4 ml of
DalmarelinTM, Fatro Co., Italy) followed by an injection of PGF2α 7 days later and a final injection of
GnRH at timed artificial insemination (AI) 48 hour after PGF2α injection (Without estradiol).
Group B (n=10) were given same treatment as given to group A buffaloes but with an injection of 1
mg of Estradiol 24 hour after PGF2α injection (With estradiol).
OvSynch72
Group C (n=10) were given an intramuscular injection of 100 μg of GnRH followed by an injection of
PGF2α 7 days later and a final injection of GnRH at timed artificial insemination (AI) 72 hour after
PGF2α injection (Without estradiol).
Group D (n=10) were given same treatment as given to group C buffaloes but with an injection of 1
mg of Estradiol 24 hour after PGF2α injection (With estradiol).
The artificial insemination (AI) was done once daily in the morning. After the PGF2α injection
buffaloes were observed for estrus, after every 6 hours a day using teaser bull and animals which stood
to be mounted were considered to be in estrus.
Blood Sampling for Progesterone Analysis
Blood samples (10 ml) were collected from all buffaloes 7 days before and again at the first
GnRH of the OvSynch protocols, at 48 and 72 hour after the PGF2α injection of the OvSynch and at 7
days after the final GnRH of synchronization protocols, respectively. Progesterone was determined
through ELISA by using a commercially available kit (BioCheck, Inc. Foster City, USA, Lot. RN-
34859).
Pregnancy Diagnosis
All buffaloes were examined for pregnancy by palpation per rectum of the uterus and its
contents for detection of an embryonic vesicle at 40 ± 1 day after artificial insemination (AI), and
pregnant buffaloes were reexamined 4 week later at 68 ± 1 day after artificial insemination (AI).
The data regarding estrus response and pregnancy per artificial insemination was analyzed by
Chi square test whereas progesterone concentrations during different days were analyzed by ANOVA
(Steel et al., 2006).
367

At 51 days post-partum, when PGF2α injection was given for estrus synchronization, 80 %, 70
%, 70 %, 70 % buffaloes become cyclic in group A, B, C, D, respectively. There was significant
difference (P < 0.05) between group A and other groups i.e. B, C, D, whereas non-significant
difference (P > 0.05) was observed between group B, C, D (Table-1). Cerri et al. (2004) in his studies
reported that when cows received estradiol 24 hours after final PGF2α, estrus distribution for the 310
cows observed in estrus was 7.1, 26.5, and 66.5% for day 1, 2, and 3 after PGF2α, respectively.
Therefore, 3 days after a presynchronized GnRH-PGF2α program is when most cows are observed in
estrus. Similarly, the current study also demonstrated that the majority of buffaloes in estrus were
observed at 72 hours after the injection of PGF2α. It is important to indicate that in our studies,
detection of estrus was limited to the first 3 days after induced luteolysis, which might have
underestimated display of estrus, because some buffaloes might have exhibited estrus after 72 hours.
After OvSynch48 and OvSynch72, the estrus expression was 30 %, 60 %, 50 %, 80 % in group
A, B, C and D. There was significant difference (P < 0.05) in estrus expression between all groups.
The estrus expression was highest in group D indicating that estrus expression can be increased by
postponing the time of artificial insemination from 48 to 72 hours (Table-1).
At day 40 and 68, 40 %, 50 %, 50 %, 40 % animals were detected pregnant in group A, B, C, D,
respectively. There was significant difference (P < 0.05) between group A and B, C, whereas non-
significant difference was observed (P > 0.05) between group A, D and group B, C, respectively.
These findings are inagreement with Hillegass et al. (2008) and Rensis et al. (2005). These authors
reported similar pregnancy per artificial insemination in cows given GnRH injections and timed
artificial insemination at either 48 or 72 hours after luteolysis. However, Warriach et al. (2008)
reported 36.3 % and 30.4 % pregnancy rates in Nili-Ravi buffaloes in OvSnch protocols during peak
and low breeding season. The higher pregnancy rate in our study may be due to increased duration
from last GnRH treatment to fixed time artificial insemination, because Warriach et al. (2008)
inseminated buffaloes after 24 hours of final GnRH treatment. In the present study, between 40 and 68
day of gestation, none of the buffalo lost pregnancy.
There was non-significant difference (P > 0.05) in serum progesterone concentrations between
group A, B, C, D, buffaloes at 7 days before, at the first GnRH, at final GnRH and 7 days after GnRH
of the OvSynch protocols, respectively (Table-2). The data varied non-significantly (P > 0.05) when
comparisons were made within each group at 7 days before, at the first GnRH, at final GnRH and 7
days after GnRH of the OvSynch protocols, respectively. However, the progesterone concentrations in
cycling buffaloes of all groups remained above 1 ng/ml whereas in acyclic buffaloes the progesterone
concentrations were less than 1 ng/ml. Ullah et al. (2006) also reported > 1.0 ng/ml serum
progesterone concentrations in cyclic Nili-Ravi buffaloes.
In our study, supplementation with estradiol did not influence pregnancy per artificial insemination.
Therefore, extending the period of proestrus and supplementing buffaloes with estradiol increased
display of estrus primarily at 72 hours after PGF2α but did not improve fertility. It is concluded, from
the results of the present study, that in buffaloes, extending the period of proestrus and supplementing
with estradiol increased display of estrus primarily at 72 hours after PGF2α but did not improve
fertility. It is therefore recommended that that timed artificial insemination programs in buffaloes
should be designed to increase expression of estrus either at the day of timed artificial insemination or
after as it is related to increased fertility.
REFERENCES
Barkawi, A.H., H.M. Farghaly and A.M. El-Borady. 1995. Effect of treatment with GnRH analogue on
postpartum reproductive performance of suckling Egyptian buffalo cows. Buffalo J. 11: 117-
123.
368
Cerri, R.L.A., J.E.P. Santos, S.O. Juchem, K. N. Galvao and R. C. Chebel. 2004. Timed artificial
insemination with estradiol cypionate or insemination at estrus in high-producing dairy cows. J.
Dairy Sci. 873704–3715.
Hillegass, J., F.S. Lima, M.F. Filho and J.E.P. Santos. 2008. Effect of time of artificial insemination
and supplemental estradiol on reproduction of lactating dairy cows. J. Dairy Sci. 91:4226-4237.
Paul, V. and B. S. Prakash. 2005. Efficacy of the Ovsynch protocol for synchronization of ovulation
and fixed-time artificial insemination in Murrah buffaloes (Bubalus bubalis). Theriogenology
64: 1049-1060.
Pursley, J. R., M. O. Mee and M. C. Wiltbank. 1995. Synchronization of ovulation in dairy cows using
PGF2α and GnRH. Theriogenology 44:915-923.
Rensis, F.D., G. Ronci, P. Guarneri, B.X. Nguyen, G.A. Presicce, G. Huszenicza and R.J. Scaramuzzi.
2005. Conception rate after fixed time insemination following ovsynch protocol with and
without progesterone supplementation in cyclic and non-cyclic Mediterranean Italian buffaloes
(Bubalus bubalis). Theriogenology 63: 1824-1831.
Shah, R.G., V. B. Kharadi, A. J. Dhami, P. M. Desai and F. S. Kavani. 2002. Effect of gonadotrophin
releasing hormone on reproductive performance and steroid profile of postpartum suckled Surti
buffaloes. Indian J. Anim. Sci. 72: 1076–1082.
Steel, R.G.D., J.H. Torrie and D.A. Dickey, 2006. Principles and Procedures of Statistics. A
biometrical approach. 3rd Ed., Mcgraw Hill Co., New York, USA.
Ullah, N., M. Anwar, S. Rizwan and S. Murtaza. 2006. Blood plasma progesterone concentrations in
two different veins and comparison of progesterone concentrations and rectal palpation
findings to different ovarian cyclicity in the Nili-Ravi buffalo. Pak. Vet. J. 26: 118-120.
Warriach, H. M., A.A. Channa and N. Ahmad. 2008. Effect of oestrus synchronization methods on
oestrus behaviour, timing of ovulation and pregnancy rate during the breeding and low
breeding seasons in Nili-Ravi buffaloes. Anim. Reprod. Sci. 107: 62-67.
Table 1. Effect of OvSynch treatments on estrus responses and pregnancy per artificial insemination
(AI) of Nili-Ravi buffaloes.
Items Group A Group B Group C Group D
Cyclic 80.0a 70.0b 70.0b 70.0b
c a b c
Detected estrus 30.0 60.0 50.0 80.0d
Pregnancy per AI (day40) 40.0a 50.0b 50.0b 40.0a
a b b
Pregnancy per AI (day 68) 40.0 50.0 50.0 40.0a
Values sharing different superscripts in a row differed significantly (P < 0.05)
Table 2. Serum progesterone concentrations (ng/ml) before, during and after OvSynch treatments in
Nili-Ravi buffaloes.
Items Group A Group B Group C Group D
ax ax ax
7 days before OvSynch protocols 1.28±0.04 1.26±0.04 1.27±0.03 1.26±0.03a
x
at the first GnRH of the OvSynch protocols 1.29±0.03ax 1.25±0.03ax 1.26±0.03ax 1.24±0.03a
x
ax ax ax
at the final GnRH of the OvSynch protocols 1.27±0.03 1.26±0.03 1.26±0.02 1.26±0.03a
x
ax ax ax
7 days after the final GnRH of the OvSynch 1.20±0.02 1.22±0.03 1.23±0.02 1.21±0.03a
x
Values sharing similar superscript “a” in a row differed non-significantly (P > 0.05)
Values sharing similar superscript “x” in a row differed non-significantly (P > 0.05)
369
Cholesterol Enhances Post-Thaw Semen Quality in Buffaloes (Bubalus bubalis)
Ahmad Yar QAMAR, a Abdul SATTARa* and Nasim AHMADa

a
Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore 54000,
Pakistan
*Corresponding email: masattar@uvas.edu.pk
ABSTRACT
The objective of the present study was to determine if addition of cholesterol in semen
extender has a beneficial effect on its post-thaw semen quality in buffalo bulls. Cholesterol was
added to Tris-citric acid semen extender in the form of cholesterol-loaded cyclodextrin (CLC). Split
pooled ejaculates (n=7) from four buffalo bulls were diluted in extender containing CLC either 3
mg (LOW), 4 mg (MED), 5 mg (HIGH) /ml or without CLC, i.e., control (CON). Motility before
freezing (MBF), post-thaw motility (PTM), live percentage (LP), plasma membrane integrity
(PMI), normal acrosomes (NA) and morphological abnormalities (MA) were assessed by using
Phase contrast microscope and hypo osmotic swelling assays. Analysis of variance revealed that
PTM & PMI from semen samples containing MED concentration of CLC were the highest (P<0.05)
followed by LOW, HIGH and CON semen samples, respectively. Live percentage (LP) was the
highest (P<0.05) in MED followed by LOW, HIGH and CON semen samples, respectively. Percent
normal acrosomes (NA) values from semen samples containing MED concentration of CLC was the
highest followed by LOW, HIGH and CON, respectively. Mean NA values in all the groups
differed significantly (P<0.05) except LOW and MED which differed non-significantly with each
other. Mean MA values were the highest in CON followed by HIGH, LOW and MED semen
samples. Mean values for MA amongst all the groups differed significantly (P<0.05) except HIGH
and CON which differed non-significantly. It is concluded that addition of cholesterol in MED
concentration to buffalo bull semen can improve post-thaw semen quality.
Keywords: cholesterol, sperm, buffalo bulls
INTRODUCTION
Nili-Ravi buffalo (Bubalus bubalis) has a pivotal role in the agricultural economy of
Pakistan. Presently, buffalo population is 32.7 million with contribution to the milk production is
61.65% of total milk production (Anonymous, 2012). Buffalo has relatively poor reproductive
efficiency and lower conception rates especially with artificial insemination due to non-perfection
of semen processing techniques (Gordon, 1996). Similarly, buffalo bulls have lower daily sperm
production rate and lower extra gonadal reserve of spermatozoa. There is greater activity of
phosphatase in buffalo bull semen as compared to that of cow bull resulting into excessive
liberation of phosphate ions and inhibition of respiration affecting viability of spermatozoa.
Concentration of calcium ions is also higher in buffalo bull semen as compared to that of cow bull
adversely affecting the viability of stored diluted semen (Usmani and Shah, 1985). Frozen semen
utilization has some disadvantages because it decreases fertility potential after cryopreservation.
Incubation of cholesterol loaded cyclodextrin (CLC) with sperm cells of bull prior to
cryoprocessing led to higher post-thaw percentage of live and motile spermatozoa as compared with
control group (Purdy and Graham, 2004). Keeping in view these advantages of the cholesterol, this
project was designed with the following objective: 1) to attain better post-thaw sperm survival
parameters with CLC addition in buffalo bull semen extender during cryopreservation 2) to
optimize the CLC concentrations for buffalo bull semen cryopreservation.

370

Selection of Animals
The study was carried out at Al-Haiwan Sires Semen Production Unit, Pakpattan Road,
Sahiwal (30°45'N latitude and 73°8'E longitude), Pakistan during August to October. Randomly
selected four buffalo bulls (Bubalus bubalis) of Nili-Ravi breed routinely used as semen donors
were included in this study. The age of these bulls ranged from 5-7 years.
Extender Preparation
Tris-citric acid extender was prepared by mixing 3.93% (w/v) N-Tris-(hydroxymethyl)-
aminomethane (Fluka) with 6.25% (w/v) citric acid (Fluka, Buchs, Switzerland) to pH 6.8. The
buffer was used in combination with 20% (v/v) fresh egg yolk, 8% (v/v) glycerol (Merck),0.2%
(w/v) fructose (Merck, Darmstadt, Germany), 100 µg/mL streptomycin sulfate (Sigma) and1000
IU/ml benzyl penicillin (Sigma, St Louis, Mo). Cholesterol-loaded Cyclodextrin (CLC) was
prepared as explained by Purdy and Graham (2004). Preparation of working solution of
Cholesterol-loaded Cyclodextrin was done by mixing 50 mg of CLC crystals to 1 ml TALP at 37°C.
Semen Collection and Processing
Semen collection was done from four mature Nili Ravi Buffalo bulls. Semen was collected
(two ejaculates from each bull) by using an artificial vagina having temperature 42ºC. Each
ejaculate was transported to the laboratory within 5 minutes. The semen was kept in a water bath at
37ºC before assessment and then evaluated for sperm motility. The ejaculates from different bulls
having at least 60% visual motility were used in experiments.At each collection day, 1st and 2nd
ejaculates of all the four buffalo bulls were pooled to minimize the bull effect. Pooled semen
samples was diluted at a rate of 50 × 106/ml with the extender (TCA) containing CLC in four
groups; Control with no CLC in the extender. LOW, MED and HIGH have 3, 4 and 5 mg of CLC
per ml of extender, respectively. Afterwards cooling, equilibration and freezing was done with
standard cryopreservation procedures. Semen samples thus processed were stored in liquid nitrogen
container. Post-thaw semen evaluation assays were performed at Theriogenology Laboratory at
UVAS, Lahore.
Preparation of Reagents used for semen evaluation assays
Hypo-osmotic, Formal citrate and Formal saline solutions were prepared as described by
Rasul et al. (2000).
Post-thaw semen Evaluation Assays
Different post-thaw evaluations and assays such as motility before freezing, post-thaw
motility, live percentage, plasma membrane integrity, normal acrosomes and morphological
abnormalities were performed following the procedures previously described by Nasrullah (2011).
Post-thaw semen evaluation parameters of CLC treated and untreated buffalo bull
semensamples were determined and compared by analysis ofvariance (ANOVA) and the correlation
between different parameters was also determined (Steel et al. 1997).

Conception rate of AI in buffaloes is generally lowered than in cows. It might be due to
greater activity of phosphatase in buffalo bull semen resulting into excessive liberation of phosphate
ions and inhibition of respiration affecting viability of spermatozoa. Moreover concentration of
calcium ions is also higher in buffalo bull semen, adversely affecting the viability of stored diluted
semen (Usmani and Shah, 1985). During cryopreservation of semen, spermatozoa undergo both
intracellular and extracellular stresses. These stresses can cause injury to plasma membrane of
spermatozoa leading to cell death. Membrane damage takes place when sperm plasma membrane
passes through a transition from liquid phase to gel state. Survival of spermatozoa during cold
shock is compromised in species like bull and rams having lower cholesterol to phospholipid
concentrations in their semen (Darin-Bennett and White, 1977). As cholesterol is an important
component in the regulation of membrane fluidity. This role becomes more important in the
cryopreservation of sperm cells where destabilization of plasma membrane leads to intracellular ice
371
formation causing death of the cell. Keeping in view the beneficial effects of cholesterol addition to
the semen extender in bulls (Purdy and Graham, 2004), Bucks (Farshad et al. 2011), rams (Moce et
al. 2010) and stallion (Zahn et al. 2002), this study was planned to observe the effect of cholesterol
addition in semen extenders on post thaw quality of cryopreserved Buffalo bull semen.
Motility Before Freezing (%)
In this study, motility before freezing was significantly(P<0.05) higher in semen samples of
MED and LOW as compared to other groups (Table 1).
Post-thaw Motility (%)
Semen samples of MED group (4 mg of CLC per ml of extender) showed significantly
highest (P<0.05) post-thaw motility as compared to all other groups (Table 1) in this study. Purdy
and Graham (2004) reported higher post-thaw motility in Holstein bull using 1.5 mg CLC per ml of
extender. But Zahn et al. (2002) reported addition of CLC did not significantly increase post-thaw
motility of equine spermatozoa.
Live Percentage (%)
Buffalo bull semen in MED group had significantly (P<0.05) higher live sperm percentage
as compared with other groups but the values of HIGH &CON did not differ significantly with each
other (Table 1) in this study. Purdy and Graham (2004) reported in Holstein bull that the
concentrations of 1.5, 3.0 or 4.5 mg CLC per ml of extender had live spermatozoa percentage after
freezing and thawing as 55, 60 and 57%, respectively. Similarly, Alvarez et al. (2006) also reported
that CLC treatments produced an increase in viable spermatozoa percentage than Control group in
donkey semen.
Plasma Membrane Integrity (%)
Plasma membrane integrity was significantly (P<0.05) highest in MED group as compared
with all other groups in this study (Table 1). Zahn et al. (2002) reported in equine that addition of
cholesterol (0.125 mM) significantly increased membrane integrity (48.7%) as compared with
control (43.9%). Moce et al. (2010) reported in rams that sperm cells treated with CLC showed
increased percentage of plasma membrane integrity (+16%) than the control group. The CLC
concentrations 1 or 2 mg/ml showed greater percentages of plasma membrane integrity after
processing as those of 0.5 or 4 mg/ml.
Normal Acrosomal Integrity (%)
Buffalo bull semen in MED and LOW group had significantly (P<0.05) higher normal
acrosomal integrity than other groups (Table 1) in this study. Zahn et al. (2002) reported in equines
that addition of cholesterol significantly increased acrosome integrity as compared with control.
Amidi et al. (2010) reported in goats that supplementation of 1.5 mg/ml CLC increased greatly (60
%) the percentages for post-thaw spermatozoa with intact acrosome. Farshad et al. (2011) reported
in Markoz buck that acrosomal integrity and vitality were significantly better for CLC
concentrations 1.5 and 2.25 mg/ml as compared with other levels of CLC (0, 0.75 and 3 mg/ml).
Morphological Abnormalities (%)
In this study, MED group had significantly (P<0.05) lower morphological abnormalities of
spermatozoa as compared with other groups but HIGH and CON groups did not significantly with
each other (Table 1).
In this study, all the post-thaw semen evaluation parameters were highly (P<0.01) and
positively correlated (Table 2) with each other except morphological abnormalities. In conclusion,
the addition of cholesterol, using CLC technology, to buffalo bull spermatozoa before freezing
increased the viability, motility and integrity of acrosome of spermatozoa after freezing and
thawing. However, additional probing is needed to find out the mechanism by which added
cholesterol affects the sperm during freezing and thawing, in particular the interaction between
cholesterol and sperm membrane. There is also a need of in vivo fertility trials of semen treated with
CLC.
372
REFERENCES
Alvarez, A.L., C. Serres, P. Torres,F. Crespo, E. Mateos, C.G. Mez-Cuétara. 2006. Effect of
cholesterol-loaded cyclodextrin on the cryopreservation of donkey spermatozoa. Anim.
Reprod. Sci. 94: 89-91.
Amidi, F., A. Farshad, A.K. Khor. 2010. Effects of Cholesterol-loaded cyclodextrin during freezing
step of cryopreservation with TCGY extender containing bovine serum albumin on quality
of goat spermatozoa. Cryobiology 61: 94-99.
Anonymous. 2012. Pakistan Economic Survey 2011-12, Ministry of Finance Pakistan.
Darin-Bennett, A., I.G. White. 1977. Influence of the cholesterol content of mammalian
spermatozoa on susceptibility to cold shock. Cryobiology 14 (4): 466-470.
Farshad, A., F. Amidi, A.K. Khor, A. Rashidi. 2011. Effect of Cholesterol-loaded-cyclodextrin in
Presence and Absence of Egg Yolk during Freezing Step on Quality of Markhoz Buck’s
Spermatozoa. Asian-Aust. J. Anim. Sci. 24(2): 181-189.
Gordon, I. 1996. Controlled reproduction in cattle and buffaloes. CAB International. Wallingford,
Uk.pp:440-445.
Moce,E., P.H. Purdy, J.K. Graham. 2010. Treating ram sperm with cholesterol-loaded cyclodextrins
improves cryosurvival. Anim. Reprod. Sci. 118: 236-247.
Nasrullah, R. 2011. Effects of cryopreservation and equilibration time on characteristics of Buck
semen. MPhil Thesis Department of Theriogenology, University of Veterinary and Animal
Sciences Lahore.
Purdy, P.H., J.K. Graham. 2004. Effect of cholesterol-loaded cyclodextrin on the cryosurvival of
bull sperm. Cryobiology 48: 36-45
Rasul, Z., M. Anzar, N. Ahmad. 2000.Effect of buffering systems on post-thaw motion
characteristics, plasma membrane integrity, and acrosome morphology of buffalo
spermatozoa. Anim. Reprod. Sci. 59: 31-41.
Steel, R.G.D., J.H. Torrie, D.A. Dickey. 1997. Principle and procedures of statistics. A biochemical
approach 3rd edition McGraw Hill Book Co.Inc, New York, USA.
Usmani, R.H., S.K. Shah. 1985. Processing and use of buffalo semen: Proceedings of a national
workshop held at the National Agriculture Research Centre, 27-28 May, 1984.
Zahn, F.S., F.O. Papa, J.A. Dell Aqua Jr. 2002. Cholesterol incorporation on equine sperm
membrane: effects on post thaw sperm parameters and fertility. Theriogenology. 58: 237-
240.
Table 1. Effect of different CLC concentrations adding in semen extender (0, 3, 4 and 5 mg per ml of semen
extender) on motility before freezing and post-thaw semen quality in Nilli Ravi Buffalo bulls (Mean ± SE).
Different Concentrations of CLC
0 mg 3 mg 4 mg 5 mg
Variables
(CON) (LOW) (MED) (HIGH)
(n = 7) (n = 7) (n = 7) (n = 7)
Motility before freezing (%) 61.79 ± 1.41a 68.21 ± 0.90 b 68.93 ± 0.51b 55.71 ± 2.36 c
Post-thaw motility (%) 32.86 ± 2.07a 50.36 ± 1.15b 61.43 ± 0.74c 38.57 ± 2.66 d
a b c
Live percentage (%) 50.14 ± 2.23 61.43 ± 1.62 69.64 ± 1.84 51.07 ± 2.56a
Plasma membrane integrity (%) 41.14 ± 1.71 a 51.93 ± 1.32b 59.36 ± 1.32c 44.00 ± 1.65 d
a b b
Normal acrosomes (%) 34.93 ± 2.89 49.36 ± 0.78 52.93 ± 1.78 40.57 ± 2.01 c
a b c
Morphological abnormalities (%) 9.86 ± 0.14 5.29 ± 0.38 4.29 ± 0.47 9.50 ± 0.24 a
Values with different superscripts within each row differ significantly (P < 0.05).
373
Table 2. Correlation coefficient between different post-thaw variables of buffalo bull semen treated with
CLC.
No. Variables Correlation between values
1 2 3 4 5 6
1 Motility before freezing (%) - 0.67** 0.59** 0.69** 0.60** -0.73**
2 Post-thaw motility (%) - - 0.75** 0.87** 0.85** -0.83**
3 Live percentage (%) - - - 0.80** 0.57** -0.76**
4 Plasma membrane integrity (%) - - - - 0.73** -0.85**
5 Normal acrosomes (%) - - - - - -0.84**
6 Morphological abnormalities (%) - - - - -
**Correlation coefficient was significant when P < 0.01.
374
Buffalo Bulletin 2013 Vol.32 (Special Issue 2): 375
Assessment of the Rate of Pregnancy in Buffaloes Crossbred Lactation using Two

Protocols CIDR-SYNCH® in 6 and 8 Days
Nestor Simon Montiel URDANETA, *C. C.Ch. MONTIEL M., N. BERRIOS, N. MORILLO S.,
J. BELANDRIA, M. ANDARA and J. ARIAS
Department of Production and Industry Animal. School of Veterinary Sciences. The University of the
Zulia. Maracaibo, Zulia State. Venezuela.
*Corresponding e-mail: nsmontiel@gmail.com; nmontiel@cantv.net
ABSTRACT
To assess the rate of pregnancy in crossbred buffaloes were synchronized with two protocols:
T1: 6 days CIDR-Synch and T2: CIDR-SYNCH® for 8 days with a second dose of prostaglandin
inseminated Fixed timedartificial insemination (FTAI). 72 Buffaloes with 4 lactations and a body
condition (BC) of 3.9 to 4.1 in grazing with 40 days of lactation when the initial were used protocol.
The buffaloes received a pre-synchronization with prostaglandin (PGF2α, 25 mg of Lutalyse®) all those
buffaloes which had a body luteum functional being incorporated randomly to each protocols; 11 days
later (day 0), received 10 µg of busereline (GnRH) and an intravaginal device with 1.9 g. progesterone
(CIDR). The buffaloes examined by tranrectal ultrasonography (SCANER 100, PIE MEDICAL) the
day of the first dose of PGF in the pre-synchronization, in the first GnRH, the withdrawal of the device
(to determine the presence and number of CL) and 32 days after the IATF determine pregnancy. The
data were analyzed by analysis of variance to determine the effect of the protocols, the number of CL at
the time of the CIDR removal and pregnancy rate. There were no significant differences in the rate of
pregnancy of the two protocols: T1: 38.9% (14/36); T2: 42.3% (15/36). It is concluded that the two
protocols were similar for pregnancy rate in crossbred buffaloes in production and that the application
of a second dose of PGF in the protocol of 8 days had no significant effect.
Keywords: synchronization, buffaloes, CIDR-SYNCH
This paper has only abstract

375
Increasing Efficiency of Artificial Insemination (AI) in Buffalo Upgrading

ProgramIn Nueva Ecija, Philippines
Felomino V. MUMUAD
Philippine Carabao Center, Science City of Muñoz, Nueva Ecija, Philippines

*Corresponding Email: fvmamuad@yahoo.com
ABSTRACT
This work demonstrated an effective system of increasing efficiency of Artificial
Insemination as a tool in Buffalo Upgrading Program in Nueva Ecija. The effectiveness of
village-based AI technicians (VBAIT) and Local Government Unit AI technicians (LGUAIT)
was analyzed in terms of number of buffalo and cattle examined inseminated, % CR and %
calves produced. There are 66 technicians that are trained to serve the province. Out of this,
58 are trained as VBAIT; 8 are LGUAIT. Only 38 of the VBAIT are successfully performing
AI in the villages, while 14 LGUAIT are performing. Reports showed a mean of 20 cows per
month are examined by individual VBAIT in 2004 and 14 cows per year in 2004 to 2008.
This was due to new performing VBAIT during that year. The number of buffaloes that are
examined in 2004 is 20 and decreased to 16 in 2008 or a mean of 15.2. It decreases because
some are still learning and this will slowly increase and reach up to 30 cows/mos to 60
cows/mos. However, more animals are examined and inseminated. The LGU technicians
examined an average of 7 cows in 2004 to 2008. Results show that VBAIT have a mean %
non-return estrus for the year 2004 to 2008 of 90% and 95.44% for buffalo and cattle
respectively and % CR of 77.49% for buffalo and 71% for cattle. Calf production shows
71.21% for buffaloes and 83.97% for cattle. On the other hand LGUAIT gave a mean percent
non-estrus of 93.34% for buffalo and 95.91% for cattle. Mean CR shows a mean of 54.54%
and 55.44% for buffalo and cattle respectively, Calf production shows a mean of 71% for
buffalo and 83% for cattle. In conclusion, performance of both VBAIT and LGUAIT shows
that they could serve quality AI services in the villages. However, VBAIT are more efficient
because they are always available and have more time thus more animals are served, and more
animals are produced with high genetics. A combination of LGU and VBAI technicians could
be promising. It is recommended that all possible support from the government should sustain
the privatization of AI services to the farmers. The improvement in the income and nutrition
of farmer’s animals will be reported as soon as their animals have reached their mature age or
have been producing milk.
Keywords: (AI) =Artificial Insemination, (VBAIT)=Village Based Artificial Insemination

Technician, (LGU AIT)=Local Government Unit Artificial Insemination Technician,(CUP)=
Carabao Upgrading Program
INTRODUCTION
Artificial insemination (AI) offers an economically feasible means of increasing the
productive performance of dairy buffaloes and cattle. Semen is deposited to the female
genitalia through the use of instruments. AI is the most effective way of magnifying and
prolonging the usefulness of a genetically superior bull. Its actual conduct is very simple but
necessitates a sound management program. Factors must be considered to effectively
implement AI Program whether the animals are in a farm or separately owned by individual
farmers. The AI technician must be knowledgeable, well-trained and committed to served as
AI technician.The use of artificial insemination (AI) as a tool for the buffaloes upgrading
program of the Philippine Carabao Center (PCC). The initiative and cooperation of farmers in

376
the villages and the community itself are also important in the buffalo development program.
It was realized the values of their participation in the AI program. By localizing the AI
Program down to the village level, the PCC might be able to increase the efficiency of
artificial insemination in the province. The program objectives will compare the effectiveness
of implementing AI through a village-based approach and with LGU-led approach or a
combination thereof; increase the calving rate by at least 10%.
Program Implementation
Identification and selection of program areas and VBAIT; Training of selected
VBAIT, conduct of AI by the VBAIT thru 1). Distribution of AI equipment, supplies and
materials, 2). Provision of technical support, 3) Establishment of the reproductive cycle of
buffaloes, 4) Monitoring.
RESULTS AND DISCUSSION

Nineteen (19) batches of AI trainings were conducted and produce a total of 84 AI
technicians for N.E. Only 4 trainings was conducted in 2006 to 2008.We tapped other
previously trained and interested AI technicians both from the private and government sectors.
There are 57 trained technicians who are performing, (38 VBAIT, 14 LGU, and 5 PCC
technicians) and conduct AI services. The program covers 181 villages in the province. AI
guns were given to technician’s right after they completed the training course on AI. Other
than this, the straw cutters, forceps and thermometers and other logistics are also provided.
Table1. Summary of animals examined and artificially inseminated per month by LGUAIT
and VBAI technicians on CY 2004 to 2008.
Examined Artificially Inseminated

No. of No. of No. of No. of No. of
System Year AI animals animals animals animals
technician per yr/ per Buffalo Cattle per yr per Buffalo Cattle
tech months/ Tech months/
tech tech
VBAI 5 236 20 925 257 210 18 859 190
2004
LGU 7 98 8 557 133 79 7 432 119
VBAI 9 231 19 1,479 597 196 16 1,273 491
2005
LGU 6 68 6 240 168 62 5 209 161
VBAI 29 115 10 2,187 140 114 10 2,157 1,135
2006
LGU 7 74 6 300 217 73 6 295 213
VBAI 36 127 11 2,995 1,591 131 11 2,979 1,686
2007
LGU 7 72 6 349 153 49 4 203 142
VBAI 2008 48 189 16 4,568 4,527 154 13 4,563 2,837
LGU 7 92 8 282 364 93 8 282 369
VBAIT Mean 25.4 179.6 15.2 2,430.8 1,422.4 161 13.6 2,366.2 1,267.8
LGU Mean 6.8 80.8 6.8 345.6 207 71.2 6 284.2 200.8
The AI technicians will submit their AI accomplishment reports and calves born. They
have to provide this information every time AI supplies are distributed once a week. The
VBAIT appear to have more time in examining animals compared to LGUAIT. When more
animals were examined by them, more animals are diagnose to be in estrus, thus, more
animals are inseminated per year by VBAI technicians as was shown in Table 1.
In both the LGUAIT and VBAIT, the buffaloes and cattle presented by farmers were
most likely to be in estrus. About 80 to 99% of them were inseminated. In 2004 to 2008, 14
head per month is AI’d by VBAIT. On the other hand LGU AI technicians appear to have
inseminated an average of 6 heads per month. Their performance appears to be affected by
377
time to spend in the performance of their functions as AIT. Their availability at all times when
buffaloes of farmers are in estrus is another factor that is good for their function.
Table 2. Percent non-return to estrus, % CR and % Calf Production of buffalo and cattle after
first service base on monitored animals and AI’ed by LGU and VBAI technicians on
CY 2004-2008.
AI’ed AI’ed % Non- % % Calf

Buffalo Cattle return to Conception
Production
Technician Year estrus Rate of
Buffalo& Buffalo and (C/b)* 100
Cattle Cattle Buffalo Cattle
LGU 2004 1,139 635 94.05/96.26 54.54/55.44
to 71.74 83.60
2007
VBAI 2004 11,831 6,339 90.00/95.44 77.49/71.18 71.21 83.97
to
2008
Animals that were inseminated were also monitored for estrus after 21 and 42 days
post AI to determine if the animals were considered as suspect pregnant. The performances of
LGUAIT have an average of 94% and 96% non-return to estrus for buffaloes and cattle
respectively for 2004 to 2008. The number of reported animals returned to estrus is low and
the perceived pregnancy is higher. The VBAIT have a mean % non-return to estrus of 90% for
buffaloes and 95% for cattle for the period of 2004 to 2008. The % non-return to estrus for
cattle and buffaloes for the period of five years is higher. The impregnated animals through AI
were monitored for conception rate 90 days after. The LGU AI technicians obtain a mean of
11, 78, 75 and 54% CR for the year 2004 to 2007 respectively, giving a grand mean % CR of
54% for buffaloes. Cattle had a mean of 18, 85 and 63% for year 2005 to 2007, giving an
average % CR of 55%.The VBAIT had a mean CR of 93, 82. 64% and 70 % respectively for
year 2004 to 2007 giving an average of 77% CR for four years, for buffaloes and an average
grand mean of 71% for cattle. The LGUAIT obtains a mean percent calf production of 72%
and 84% for buffaloes and cattle. The VBAIT had a mean calf production of 71% for
buffaloes and 84% for cattle. The % calf production performance of LGU and VBAIT were
almost the same. The apparently low % calf production could be attributed to the fact that
both LGU and VBAIT are not so interested to monitor the number of calves born out of their
inseminations. They see to it that the animals had been impregnated to get full payment of
their services. Farmers pay their services a minimum of 500 pesos when confirmed pregnant.
AI technicians keep on looking for animals to be inseminated to get more pay for the services.
When the UNAIP program and PCC will be implemented especially on incentives for every
calf born reported, the % calf production report will be expected to surely improve.
CONCLUSION AND RECOMMENDATIONS

Base on the foregoing studies, it is concluded that the availability of VBAIT in the
villages assured buffaloes farmers readily available breeder of their estrus cows, 24 hours
throughout the year, thus become more effective. The farmers could get more offspring to
replace their former animals. The government saved significantly because, the VBAIT are not
government employee thus, are not given salary, TEV, fuel, repair and maintenance. Their
requirement to have mobility for their services has been provided by them and they have
formed an association to have better performance at the higher level. The farmer becomes part
of the program because they invest by paying the VBAIT per successful service. The
378
investment of the government in providing training and logistics in the implementation

becomes more efficient in terms of output than when it is done by government technician. For
the LGUAIT, they are given so many responsibilities by the government for they are hired as
their technicians. The VBAIT became a private technician that provided service to the
carabaos/cows of farmers for a fee. The number of buffaloes in the villages dictates the
income a technician could get each day or month.The incentive per calf drop promised by the
government under UNAIP and PCC must be implemented. The incentive per calf produced
will trigger higher monitoring rate among serviced cows till calving. Other forms of incentive
must be in place to sustain the program. Better reporting system must also be included in the
program to get the real picture of the program.
REFERENCES
Hafez, E.S.E. 1953. Conception rate and periodicity in buffalo. Emp. Journal. Exp. Agri. 21,
15.
Tomar, S.P.S. 1984. Artificial Insemination and Reproduction of Cattle and Buffaloes.
College of Veterinary Science and Animal Husbandry, Chandra Shekhar Azar
University of Agriculture and Technology, Mathura, U.P. India. pp. 1-82.
379
Thyroid Hormone Levels during Oestrous Cycle In Pandharpuri Buffalo
Santosh Hirba DALVI, a Bhanudas Tukaramji DESHMUKHb and J. KUMARASAMY c
a Associate Professor, Department of Veterinary Biochemistry, Bombay Veterinary College, Parel,

Mumbai-400 012. Maharashtra Animal & Fishery Sciences University, Nagpur, India
b Ex-Professor, Department of Veterinary Physiology & Biochemistry, Bombay Veterinary College,
Parel, Mumbai-400 012. Maharashtra Animal & Fishery Sciences University, Nagpur, India
c Scientific Officer- C , Radiation Medicine Centre, Bhabha Atomic Reasearch Centre, Parel,
Mumbai-400 012
Corresponding Email: santdalvi@yahoo.co.in, drdalvi5@gmail.com
ABSTRACT
The study was conducted on 15 multiparous Pandharpuri buffaloes in their 4th to 6th lactation
exhibiting regular oestrous cycle and five regular cycling heifers aged 42 to 48 months to monitor the
serum levels of Triiodothyronine (T3) and Thyroxine (T4) through entire oestrous cycle. The average
oestrous cycle length in heifers and multiparous Pandharpuri buffaloes in the present study was 19.2 ±
concentration was higher (1.51  0.13 ng/ml) on the day of estrus. The average serum triiodothyronine
0.73 days and 20.8 ± 0.34 days respectively with range of 18 -22 days. The triiodothyronine
concentrations in heifers and multiparous buffaloes were 1.45  0.21 and 1.30  0.07ng/ml,
profile. The maximum concentration of 41.20  3.27 ng/ml was recorded on the day of estrus. The
respectively. The thyroxine concentration exhibited almost identical trend to that of triiodothyronine
average serum thyroxine concentrations in heifers and multiparous buffaloes were 36.32  4.53 and
36.38 2.86 ng/ml. On the day of estrus, the T4: T3 ratio was 29.32  2.50. The T4: T3 ratio from the
day 2 to 12 of the oestrous cycle remained within the narrow range of 28.97  1.35 to 30.96  2.06,
without exhibiting any particular trend.
Keywords: Triiodothyronine, Thyroxine, Oestrous cycle, Pandharpuri buffalo
INTRODUCTION
Thyroid hormones affects many diverse tissues and influence major processes such as
metabolism, growth, differentiation, reproduction and lactation. Thyroid hormones are thought to be
necessary for normal secretion and utilization of gonadotropic and gonadal hormones (Dalvi et.al.,
1995). Abnormalities in reproduction are common when breeding animal develops hypothyroidism.
Lack of libido and reduction in sperm count may occur in males, where as abnormal oestrous cycles
and reduced conception rates may results in females. Obesity and changes in behaviour resulting from
hypothyroidism often have detrimental effects on reproduction. Studies on thyroid hormones in blood
indicate that their concentrations as affected by age (Sharma and Agarwal, 1985), season (Kumar and
Ratan,1992), oestrous cycle ( Sarvaiya et. al., 1992 ) and stage of lactation ( Jindal and Ludri, 1991)
have been reported. The hormonal profile of thyroid gland varies during different reproductive stages
(Baruah et.al. 1990).Many workers have reported the hormonal profile of thyroid gland during
different reproductive stages but in Pandharpuri buffalo, little information is available on the
circulating levels of thyroid hormones during various reproductive processes.

The study was conducted on 15 multiparous Pandharpuri buffaloes in their 4th to 6th lactation
exhibiting regular oestrous cycle and five regular cycling heifers aged 42 to 48 months from Livestock
Farm, KNP College of Veterinary Science, Shirval, Dist. Satara, Maharashtra, India. The estrus in the
380
buffalo was detected by signs of estrus . Blood Samples from these buffaloes were collected by jugular
venipuncture on the day of estrus and on every alternate day in the morning during 10–11 A.M. till
they exhibited subsequent estrus. Clear serum was separated by centrifugation and stored at -20°C until
used for hormone assay. The serum levels of Thyroid hormone (T3 & T4) estimated by
radioimmunoassay using and kits procured from Board of Research in Isotopes Technology (BRIT),
BARC, Mumbai. Analysis of variance of the data was done according to Snedecor and Cochran (1994)
using complete randomised design and Randomized block design. Differences in means were tested
using critical difference (CD) test. (T4/T3) ratio was calculated.
As depicted in Figure 1, the triiodothyronine concentration was higher (1.51  0.13 ng/ml) on
Triiodothyronine
the day of estrus. It showed slight declining trend and reached to 1.28  0.10 ng/ml on day 2 of the
fluctuated within narrow range of 1.25  0.09 to 1.39  ng/ml, without exhibiting any definite trend.
oestrous cycle. The triiodothyronine concentration from day 2 onward to day 18 of the oestrous cycle
to a value of 1.49  0.16 and 1.60  0.14ng/ml on day 20 and 22 of oestrous cycle. The average serum
The serum triiodothyronine concentration thereafter, showed increasing trend and reached respectively
triiodothyronine concentrations in heifers and multiparous buffaloes were 1.45  0.21 and 1.30  0.07
ng/ml, respectively. The concentration was significantly higher (P<0.01) in heifers than in multiparous
buffaloes as depicted in Figure 4.
Thyroxine
triiodothyronine profile. The maximum concentration of 41.20  3.27 ng/ml was recorded on the day of
As depicted in Figure 2, the thyroxine concentration exhibited almost identical trend to that of
estrus. The concentration declined and reached to 36.13  2.54 ng/ml, on day 2 of oestrous cycle. The
33.31 2.43 and 38.78  2.47ng/ml without exhibiting any specific trend, except on day 18, 20 and 22
thyroxine concentration from day 2 onwards through the entire oestrous cycle fluctuated between
which respectively rose to 36.01  2.13, 37.38  2.32, and 38.78 2.47 ng/ml . The average serum
thyroxine concentrations in heifers and multiparous buffaloes were 36.32  4.53 and 36.38  2.86
ng/ml. The thyroxine concentration did not significantly differ and remained almost similar in heifers
and multiparous buffaloes as depicted in Figure 4.
The observation of the present study that the T3 and T4 concentration was higher on the day of
estrus corroborates the findings in Black Bengal goats (Reddy et al.,1999). Baruha et al. (1990)
indicated that the higher value of T3 and T4 during estrus and day 20 onwards, just before the next
estrus may possibly be associated with the increase of estrogenic activity during those periods of the
cycle. Ingbar and Woeber (1974) opined that, T3 and T4 concentrations appear to be closely associated
with increase in the concentration of circulating estrogen. Vadodaria et al. (1980) observed increased
thyroid activity of the thyroid follicles during follicular phase than during luteal phase and higher level
of thyroxine during estrus is considered to cause a change in the sensitivity of the gonads to
gonadotropic hormones because in a variety of species like mice, rabbit, swine, sheep and cattle higher
level of thyroid hormones during estrus has improved the ovarian function. Soliman and Reineke
(1954) suggested that increased thyroid activity at estrus in ewes may either be due to action of an
increase in estrogen level at estrus or due to direct release of TSH from pituitary coincident with
release of FSH and LH.
As depicted in Figure 3, on the day of estrus, the T4:T3 ratio was 29.32  2.50. The T4:T3 ratio
T4: T3 ratio
from the day 2 to 12 of the oestrous cycle remained within the narrow range of 28.97  1.35 to 30.96 
identical ratio of 34.40  3.23 and 34.34  3.64 on day 14 and 16 of the oestrous cycle. The T4:T3 ratio
2.06, without exhibiting any particular trend. The T4:T3 ratio thereafter, increased and recorded almost
381
declined from day 18 (32.52  3.26) through day 22 of the oestrous cycle. The ratio remained almost
similar (27.47  2.31 and 27.56  2.91) on day 20 and 22 of the oestrous cycle. The small variations in
the T4:T3 ratio recorded during the oestrous cycle period were statistically non-significant. The
average serum T4:T3 ratio was significantly lower (P<0.05) in heifers than in multiparous buffaloes.
The literature regarding the pattern of T4:T3 ratio observed in the present study could not be traced
for comparison. The values of serum T4 : T3 ratio recorded in the present study were higher than those
reported on the day of estrus in Murrah buffaloes (Nagvekar, 2012) and lower than reported in buffalo
calves and heifers (Ingole , 2009).
REFERENCES
Baruah, K.K., A. Baruah and R.N. Baruah. 1993. Circulating levels of thyroid hormones during oestrous
cycle in dairy cows. IJAR 14(2): 72 -73.
Dalvi, S. H., B.T. Deshmukh, A. M. Mantri and B. A. Talvelkar .1995. Concentration of blood serum
thyroid hormones during late pregnancy, parturition and early lactation in crossbred cows. Indian
J. Anim.Sci.65 (1): 15-19.
Ingbar, H.S. and A.K .Woeber. 1974. “The thyroid gland”. In: Text book of endocrinology, edited by
Williams H. Roberts, 5th ed., Saunders Company, Philadelphia:195.
Ingole, S.D. (2009) Endocrine profile from birth to puberty in buffalo calves and heifers. PhD thesis submitted to
Maharashtra Animal and Fishery Sciences University, Nagpur,India.
Jindal, S.K. and R.S. Ludri.1991. Circulating thyroxine and triiodothyronine levels in lactating
crossbred cows and buffaloes as affected by stage of lactation and time of sampling. Inter. J.
Anim. Sci. 6: 122-127.
Kumar, R. and P.J.S. Rattan.1992. Plasma thyroidal and adrenocortical hormones during different
developmental stages in buffalo heifers. Indian J. Anim. Sci. 62: 747 – 748.
Lepinot, J.B., R.F. Nachreiner, and E. M. Convey.1980. Effect on genetic, age and sex on thyroid
function in dairy calves. J. Anim. Sci. 51: 297.
Nagvekar, A. S., B. T. Deshmukh, D. B. Jagtap and S. D. Ingole .2012. Hormonal profile during
pregnancy stages in Murrah buffalo. Indian Vet. J. 89 (4): 63 – 66.
Reddy I. J., V P. Varshney and P.C. Sanwal.1999.. Plasma T3 and T4 profile during estrous cycle in Black
Bengal Female goats. Indian Vet. J. 76 : 565.
Refsal, K.R., Nachreiner, R.F. and Anderson, C.R.1984. Relationship of season herd, lactation, age and
pregnancy with serum thyroxine and triiodothyronine in Holstein cows. Domestic animal
Endocrinology. 1: 225 - 234. Dairy Sci. Abstr. 47: 4132 (1985)
.Sarvaiya, N.P., M.M. Pathak and A.V. Patel.1992.. Study on circulating thyroid hormones in pubertal
Surti buffalo heifers. Int. J. Anim. Sci. 7: 211- 213.
Sharma, I. J. and S. P. Agarwal,1985. Serum thyroid hormones levels in male buffalo calves as related to
age and sexual development. Theriogenology, 24: 509 – 517.
Shoda, Y. and T. Ishii.1976. Effect of season, pregnancy and lactation on serum thyroxine level in dairy
cattle. Japanese J. Zootechnical Sci., 47 659 – 664. Dairy Sci. Abstr., 39: 3802.
Snedecor,G.W. and W.G.Cochran. 1994. Statistical methods, 8th ed. Oxford and IBH Publishing
Company, New Delhi.
Soliman ,F.A. and E.P. Reineke.1954. Amer. J. Physol. 178:89. Cited by Baruah et al.1990. IJAR:11:1:44-
46.
Sutherland, R. L. and C. H. G. Irvine, 1974. Efffect of season and pregnancy on total plasma thyroxine in
sheep. Am. J. Vet. Res. 35: 311- 312.
Vadodaria,V.J., K.C.Jankiraman and N.C.Buch .1980.Thyroid activity in relation to reproduction in Surti
buffaloes heifers. Cited by Baruah et. al . 1993. IJAR.14 (2) 72-73.
.
382
1.90
Triiodothyronine (ng/ml)
1.70
1.50
1.30
1.10
0.90
0.70
0.50
0 2 4 6 8 10 12 14 16 18 20 22
Days of oestrous cycle
0= Day of estrus
Figure 1. Profile of serum triiodothyronine during oestrous cycle in Pandharpuri buffaloes.
60
55
50
Thyroxine (ng/ml)
45
40
35
30
25
20
0 2 4 6 8 10 12 14 16 18 20
0= Day of estrus
Figure 2. Profile of serum thyroxine during oestrous cycle in Pandharpuri buffaloes.
383
50
45
40
35
30
T4:T3 ratio
25
20
15
10
0
0 2 4 6 8 10 12 14 16 18 20 22
0= Day of estrus
Figure 3 Profile of serum T4:T3 ratio during oestrous cycle in Pandharpuri buffaloes.
1.8 42
Triiodothyronine(ng/ml)
1.6
40
1.4
Thyroxine(ng/ml)
1.2 38
1
36
0.8
0.6 34
0.4
32
0.2
0 30
Heifers Buffaloes Heifers Buffaloes
Figure 4 Triiodothyronine and thyroxine profile during oestrous cycle in heifers and multiparous
Pandharpuri buffaloes.
384
Efficiency of OPU-IVEP-ET of Fresh and Vitrified Embryos in Buffaloes
Wilson SALIBA a*, Lindsay GIMENES b, Roberti DRUMONDa, Henrique BAYÃOa, Múcio
ALVIM a, Pietro BARUSELLIc, Eduardo BASTIANETTOd, Rômulo LEITEd and Bianca
GASPARRINIe
a
Cenatte Embriões LTDA, Pedro Leopoldo - M G, Brazil
b
Departamento de Medicina Veterinária Preventiva e Reprodução Animal, FCAV- UNESP,
Jaboticabal – SP, Brazil
c
Departamento de Reprodução Animal, FMVZ-USP, São Paulo - SP, Brazil
d
Departamento de Medicina Veterinária Preventiva, UFMG, Belo Horizonte - MG, Brazil
e
Dipartimento di Scienze Zootechniche e Ispezione degli Alimenti, Università degli Studi di Napoli
Federico II (UNINA), Napoli, Italy
*Corresponding e-mail: wilson@cenatte.com.br
ABSTRACT
The present study aims to report ovum pickup (OPU), in vitro embryo production (IVEP)
buffalo donors were submitted to 11 OPU sessions (n = 201). A total of 998 oocytes (5.0  0.5/
and embryo transfer (ET) outcomes of fresh and vitrified buffalo embryos. For this purpose, 36
donor/ session) and 584 viable oocytes (2.9  0.3/ donor/ session) were recovered. Viable oocytes
(grades 1, 2 and 3) were subjected to IVM, IVF (D0) and IVC. On D2, 54.5% of cleavage rate was
obtained. Embryo yield on D7 was 44.9% (grade 1: 229 embryos, grade 2: 5 embryos and grade 3:
28 embryos). From this total, 115 fresh (grades 1 to 3) and 70 vitrified embryos (only grade 1) were
transferred into recipients previously synchronized with fixed time embryo transfer (FTET)
protocol. Vitrification was performed using the cryotop method. Pregnancy diagnosis in fresh and
in vitrified groups were, respectively: 43.5% (50/115) and 37.1% (26/70) on 30 days after embryo
transfer, and 41.7% (48/115) and 31.4% (22/70) on 60 days after embryo transfer. In conclusion,
our results demonstrate the possibilities for commercial use of the techniques of OPU, IVEP and ET
of fresh and vitrified embryos in buffaloes.
Keywords: Buffalo, Vitrification, Pregnancy
INTRODUCTION
OPU and IVEP are promising techniques in buffalo, inversely to recovery of embryos in
vivo which is still unsatisfactory. Scientific studies show that buffaloes are able to respond to
superovulatory protocols, however, embryo recovery is low (Baruselli et al., 2007). Moreover,
success in the transfer of fresh or vitrified embryos has reached low conception rates varying from
10.5 to 22.7% (Sá Filho et al., 2005; BonDurant et al., 2007; Hufana-Duran et al., 2007). The
present study aims to report OPU-IVEP-ET outcomes of fresh and vitrified embryos in buffaloes in
a commercial laboratory in Brazil.

For this purpose, 36 buffalo donors were submitted to 11 OPU sessions (n = 201) between
April and December 2010. Oocytes were classified according to number of cumulus cells layers and
ooplasm homogeneity in viable oocytes (grades 1, 2 and 3) and non-viable oocytes (denuded, atretic
and expanded cumulus oocytes). Only viable oocytes (i.e. with at least one cumulus cell layer and
ooplasm homogenous or heterogeneous) were subjected to IVM, IVF (D0) and IVC. The cleavage
rate was obtained on day 2 (D2) after IVF. Embryo yield was evaluated on day 7 (D7) after IVF and
embryos were classified according to stage of development, and according to blastomers extrusion
and color in grades 1 to 3. Part of the embryos were transferred fresh (n=115; embryos grades 1 to
3) and part of them (n= 70; only grade 1 embryos) were vitrified by the cryotop method (De Rosa et
al, 2007) and then transferred to synchronized recipients (Baruselli et al., 2007) on days 6 and 7
385
transfer. Results were expressed as means  S.E.M or as percentage. Qui-square test was done to
after ovulation. Ultrasound pregnancy diagnosis was performed 30 and 60 days after embryo
compare pregnancy rates obtained when embryos were transferred on day 6 or 7 after ovulation.
P<0.05 was considered as significant.

In buffaloes, superovulation and embryo transfer (SOV-ET) efficiency is lower compared to
bovines, due to few numbers of follicles and oocytes, and also to the low embryo quality (Baruselli
et al., 2007). Because of this, OPU-IVP appears as more suitable biotechnologies for buffalo
reproduction.
finding was similar to that obtained by Sá Filho et al. (2009; 4.1  0.5 in control group), however
Our results of OPU-IVEP outcomes are shown in Table 1. Concerning total oocytes, our
was greater than those of Gupta et al. (2006; 2.0  0.3) and Manjunatha et al. (2008; 1.2  0,07),
and lower than that described by Gimenes et al. (2010; 14.8  1.0). These divergences maybe can
be attributed to individual variation in follicular pool or differences in nutrition or management of
Gimenes et al. (2010; 7.9  0.7). This could be attributed to criteria of oocyte selection, which is a
animals. Viable oocytes were also greater than these previous reports, except in comparison to
subjective analysis, or to differences attributed to parity among studies (i.e. heifers and cows).
Cleavage rates ranging from 40 and 66% were already described (Nandi et al., 2002; Neglia
et al., 2003; Suresh et al., 2009), corroborating with our results.
Satisfactory blastocyst rates varies from 15 to 30% in buffaloes (Nandi et al., 2002;
reviewed in Suresh et al., 2009), therefore our embryo yield was greater than this range. Multiple
factors can be related to these differences, as oocyte quality, culture medium, use of antioxidants,
gas atmosphere, and others (reviewed in Suresh et al., 2009).
No difference between day of embryo transfer (day 6 or 7 after ovulation) was found
(P>0.05), therefore results concerning pregnancy rates of fresh and vitrified embryos were
combined (Table 2). Although several OPU-IVP studies in buffaloes had been performed, there are
few works describing pregnancy outcomes in the literature. Overall, our results were higher than
those reported on day 30 of pregnancy for fresh (25%: Madan et al., 1994; 26.4%: Misra et al.,
1999; 26.9%: Liang et al., 2008) and vitrified embryos (16.4%: Hufana-Duran et al., 2004; 37.5%:
Neglia et al., 2004; 10.5%: Sá Filho et al., 2005; 22.7%: BonDurant et al., 2007; 12.5%: Hufana-
Duran et al., 2007). It is important to note that all previous experiments were conducted with a
reduced number of embryos, inversely to the present study. Additionally, monitoring of pregnancy
beyond 50 days were only described for vitrified ET, resulting in total pregnancy loss in studies of
Neglia et al. (2004) and BonDurant et al. (2007), and low calving rates observed by Hufana-Duran
et al. (2004; 10.9%: 6 calves from 55 recipients), Sá Filho et al. (2005; 10.5%: 2 calves from 19
recipients), and Hufana-Duran et al. (2007; 10%: 4 calves form 40 recipients).
In conclusion, our results demonstrate possibilities for commercial use of the techniques of
OPU, IVEP and ET of fresh and vitrified embryos in buffaloes.
ACKNOWLEDGEMENT
FAPEMIG (APQ - 0591-5.04/08), FINEP, FIEMG.
REFERENCES
Baruselli, P.S., L.U. Gimenes, N.A.T. Carvalho, M.F. Sá Filho, M.L. Ferraz and R.C. Barnabe.
2007. O estado atual da biotecnologia reprodutiva em bubalinos: perspectiva de aplicação
comercial. Revista Brasileira de Reprodução Animal 31: 285-292.
BonDurant, R.H., M. Drost, J. Zambrano-Varon, G. Campanile, B. Gasparrini and L. Zicarelli.
2007. Importation of in vitro-produced Bubalus bubalis embryos from Italy into the
United States: a case report. Theriogenology 68: 454-460.
386
De Rosa A., L. Attanasio, L. Boccia, D. Vecchio, G. Campanile and B. Gasparrini. 2007. Cryotop
vitrification for in vitro produced bovine and buffalo (Bubalus bubalis) embryos at
different stage of development. Italian Journal Animal Science 6(Suppl. 2):747-750.
Gimenes, L.U., M.L. Ferraz, A. Araújo, P. Fantinato Neto, M.R. Chiaratti, L.G. Mesquita, J.S.P.
Arango, M. Raposo, D.C. Souza, G.D. Calomeni, R. Gardinal, C.L.V. Rodriguez, L.A.
Trinca, F.V. Meirelles and P.S. Baruselli. 2010. OPU at different times of a synchronized
follicular wave did not affect IVP in Bos indicus, Bos taurus and Bubalus bubalis. Reprod.
Fertil. Dev. 22: 293.
Gupta, V., R.S. Manik, M.S. Chauhan, S.K. Singla, Y.S. Akshey and P. Palta. 2006. Repeated
ultrasound-guided transvaginal oocyte retrieval from cyclic Murrah buffaloes (Bubalus
bubalis): Oocyte recovery and quality. Animal Reproduction Science 91: 89–96.
Hufana-Duran D., P.B. Pedro, H.V. Venturina, R.D. Hufana, A.L. Salazar, P.G. Duran and L.C.
Cruz. 2004. Post-warming hatching and birth of live calves following transfer of in vitro-
derived vitrified water buffalo (Bubalus bubalis) embryos. Theriogenology. 61: 1429-1439.
Hufana-Duran D., P.B. Pedro, H.V. Venturina, P.G. Duran and L.C. Cruz. 2007. Full-term delivery
of river buffalo calves (2n = 50) from in vitro-derived vitrified embryos by swamp
buffalo recipients (2n = 48). Livestock Science. 107: 213–219.
Liang X.W., Y.Q. Lu, M.T. Chen, X.F. Zhang, S.S. Lu, M. Zhang, C.Y. Pang, F.X. Huang and K.H.
Lu. 2008. In vitro embryo production in buffalo (Bubalus bubalis) using sexed sperm and
oocytes from ovum pick up. Theriogenology 69: 822-826.
Madan M.L., M.S. Chauhan, S.K. Singla and R.S. Manik. 1994. Pregnancies established from water
buffalo (bubalus bubalis) blastocysts derived from in vitro matured, in vitro fertilized
oocytes and co-cultured with cumulus and oviductal cells. Theriogenology 42: 591-600.
Manjunatha, B.M., J.P. Ravindra, P.S.P. Gupta, M. Devaraj and S. Nandi. 2008. Oocyte recovery
by ovum pick up and embryo production in river buffaloes (Bubalus bubalis). Reprod
Domest Anim. 43: 477-480.
Misra, A.K., M. Mutha, R. Kasiraj, N.S. Ranga Reddy and H. C. Pant. 1999. Factors affecting
pregnancy rate following nonsurgical embryo transfer in buffalo (Bubalus bubalis): a
retrospective study. Theriogenology 52: 1-10.
Nandi, S., H.M. Raghu, B.M. Ravindranatha and M.S. Chauhan. 2002. Production of buffalo
(Bubalus bubalis) embryos in vitro: premises and promises. Reprod Domest Animal.
37: 65-74.
Neglia, G., B. Gasparrini, V. Caracciolo di Brienza, R. Di Palo, G. Campanile, G.A. Presicce and L.
Zicarelli. 2003. Bovine and buffalo in vitro embryo production using oocytes derived from
abattoir ovaries or collected by transvaginal follicle aspiration. Theriogenology 59: 1123-
1130.
Neglia, G., B. Gasparrini, V. Caracciolo di Brienza, R. Di Palo and L. Zicarelli. 2004. First
pregnancies carried to term after transfer of vitrified buffalo embryos entirely produced in
vitro. Veterinary Research Communications. 28: 233–236.
Sá Filho, M.F., N.A. T. Carvalho, L.U. Gimenes, J.R.S. Torres-Junior, L.F. Nasser, H. Tonhati, J.
M. Garcia, B. Gasparrini, L. Zicarelli and P.S. Baruselli. 2009. Effect of recombinant bovine
somatotropin (bST) on follicular population and on in vitro buffalo embryo production.
Anim. Reprod. Sci. 113:51-59.
Sá Filho, M.., N.A.T. Carvalho and L.U. Gimenes, J. R. S. Torres-Junior, C. R. Ferreira, F. Perecin,
A. P. Perini, T. A. D. Tetzner, R. Vantini, G. F. Soria, J. M. Garcia, H. Tonhati, B.
Gasparrini, P. S. Baruselli. 2005. Prenhezes de embriões bubalinos frescos e vitrificados
produzidos in vitro. Acta Scientae Veterinariae. 33: 431.
Suresh, K.P., S. Nandi and S. Mondal. 2009. Factors affecting laboratory production of buffalo
embryos: a meta-analysis. Theriogenology 72: 978-985.
387
Table 1. Outcomes of OPU-IVP in buffalo.
Donors 36
Number of OPU sessions 11
Total number of OPU sessions 201
TOTAL OOCYTES
5.0  0.5
N 998
Mean ( S.E.M) per donor/ session
VIABLE OOCYTES
2.9  0.3
N 584
CLEAVED STRUCTURES
1.6  0.2
N 318
Cleavage rate (%) 54.5
BLASTOCYSTS
N 262
Grade 1 229
Grade 2 5
1.3  0.1
Grade 3 28
Blastocyst rate (%) 44.9
Table 2. Pregnancy rates 30 and 60 days after transfer of fresh or vitrified embryos.
30 days 60 days
Fresh Vitrified Fresh Vitrified
Pregnancies/ total of embryos transferred 50/115 26/70 48/115 22/70
Pregnancy rate (%) 43.5% 37.1% 41.7% 31.4%
388
Pregnancy Monitoring of In Vitro Produced Embryos in Buffaloes
Wilson Saliba1*, Lindsay Gimenes2 , Roberti Drumond1, Henrique Bayão1, Múcio Alvim1,
Pietro Baruselli3, Eduardo Bastianetto4, Rômulo Leite4, Bianca Gasparrini5
1
Cenatte Embriões LTDA, Pedro Leopoldo - MG, Brazil
2
3
4
Departamento de Medicina Veterinária Preventiva, UFMG, Belo Horizonte - MG, Brazil
5
Dipartimento di Scienze Zootechniche e Ispezione degli Alimenti, Università degli Studi di Napoli
Federico II (UNINA), Napoli, Italy
*Corresponding e-mail: wilson@cenatte.com.br
ABSTRACT
In the present study, pregnancies obtained from 115 in vitro produced embryos were monitored by
ultrasonography on days 30 and 60 after embryo transfer (ET), and at calving. Additionally, the
health of newborns and recipients were also evaluated. On day 30 after ET, positive pregnancy was
diagnosed in 50 animals (43.5%). A total of 8 fetal mortalities (16.0%) were verified from 30 days
until calving, in which 2 occurred from 30 to 60 days after ET (4.0%), and 6 occurred from 60 days
until calving (12.0%). In this last period of pregnancy, 3 pregnancy losses were due to abortion, and
the other 3 were stillbirth. One additional animal was eliminated from the study, remaining 41
pregnancies. From these 41 pregnancies, a total of 20 female calves (48.8%) and 21 male calves
(51.2%) were born. Pregnancies from female and male calves had a mean length of 309.8 and 310.9
days, respectively (range 300 to 328 days, and 297 to 320 days, respectively). Weight at calving
was a mean of 31.4 and 33.8 kg for female and male calves, respectively. All calving occurred
without intervention and dystocia was not observed in any case. No large offspring syndrome,
hydramnios, hydroallantois, or umbilical cord anomalies were observed in calves. Delivery was
normal in all recipients, and no puerperal infections, or retained placenta occurred. Suckling
assistance was not necessary in any newborn. All genetic pedigree was confirmed later by DNA
tests.
Keywords: Buffalos, IVP, ET, Calving
INTRODUCTION
In vitro embryo production (IVP) associated to ultrasound-guided ovum pickup (OPU) has
been proved to be an important tool for reproduction in buffaloes. However, even with several
studies being conducted in the species, there are few trials evaluating pregnancy and calving rates
after embryo transfer (Presicce, 2007). For this reason, the aims of the present study were to
monitor pregnancy after transfer of fresh embryos and to collect data of calves and recipients after
delivery.

The present study was conducted at Cenatte farm (Pedro Leopoldo - MG - Brazil) from
May, 2010 until November, 2011. A total of 80 crossbred Murrah heifers and cows were selected to
be embryo recipients, and were kept under pasture (Brachiaria decumbens and B. brizantha) with
free access to water and mineral salt. During autumn and winter all animals were supplemented
with sorghum silage.
A total of 115 in vitro produced embryos were transferred to synchronized recipients
(association of progestagen plus estradiol benzoate protocol; Baruselli et al., 2007) 6 and 7 days
after ovulation. Pregnancies were monitored by ultrasonography (Aloka 500, Tokio, Japan) on days

389
30 and 60 after ET, and at calving. Additionally, the health of newborns and recipients were also
evaluated. Data were expressed as mean or as percentage.

On day 30 after ET, positive pregnancy was diagnosed in 50 animals (43.5%). A total of 8
fetal mortalities (16.0%) were verified from 30 days until calving, in which 2 occurred from 30 to
60 days after ET (4.0%), and 6 occurred from 60 days until calving (12.0%; Table 1). In this last
period, 3 pregnancy losses were due to abortion, and the other 3 were stillbirth. In previous works,
pregnancy loss varying from 26.3 to 50% was reported for transfer of fresh embryos (Madan et al.,
1994; Liang et al., 2008). Lower mortality found in the present study could be attributed to
differences in breed of embryos and recipients compared to other studies, to management, or maybe
due to synchronization of recipients, which was previously indicated as an important factor
affecting pregnancy rate after ET (Misra et al., 1999).
One additional animal was eliminated from the study, remaining 41 pregnancies. From these
41 pregnancies, a total of 20 female calves (48.8%) and 21 male calves (51.2%) were born, which is
in accordance with Sá Filho et al. (2005) whose obtained two pregnancies from vitrified embryos
(one of each gender). Nevertheless, et al. (2008) described a higher rate of males (66.6%) than
females (33.3%), inversely to our findings.
Pregnancies from female and male calves had a mean length of 309.8 and 310.9 days,
respectively, with average of 310.4 days (Table 2), which is similar with other studies [312.8 days
for riverine vitrified embryos transferred to riverine recipients (Hufana-Duran et al., 2004); 302.3
days for riverine vitrified embryos transferred to swamp recipients (Hufana-Duran et al., 2007);
309.7 days for fresh riverine embryos transferred to swamp recipients (Liang et al., 2008)].
Weight at calving was a mean of 31.4 and 33.8 kg for female and male calves, respectively,
with average of 32.6 kg (Table 2), corroborating with other works (38.8 kg - Hufana-Duran et al.,
2004; 27 kg - Sá Filho et al., 2005; 30.5 kg - Liang et al., 2008).
All calving occurred without intervention, and dystocia was not observed in any case,
inversely to Liang et al. (2008), who’s observed 14.2% of parturition problems. Other reports of
dystocia were described for pregnancies of vitrified embryos, resulting in stillbirth (25%: Hufana-
Duran et al., 2007). No large offspring syndrome, hydramnios, hydroallantois, or umbilical cord
anomalies were observed in calves. Delivery was normal in all recipients, and no puerperal
infections, or retained placenta occurred. Suckling assistance was not necessary in any newborn. All
genetic pedigree will be confirmed later by DNA tests.
ACKNOWLEDGEMENTS
FAPEMIG (APQ - 0591-5.04/08), FINEP, FIEMG.
REFERENCES
Baruselli, P. S., L. U. Gimenes, N. A. T. Carvalho, M. F. Sá Filho, M. L. Ferraz and R. C. Barnabe.
2007. O estado atual da biotecnologia reprodutiva em bubalinos: perspectiva de aplicação
comercial. Revista Brasileira de Reprodução Animal. 31:285-292.
Hufana-Duran D., P. B. Pedro, H. V. Venturina, R. D. Hufana, A. L. Salazar, P. G. Duran and L. C.
Cruz. 2004. Post-warming hatching and birth of live calves following transfer of in vitro-
derived vitrified water buffalo (Bubalus bubalis) embryos. Theriogenology. 61: 1429-1439.
Hufana-Duran D., P. B. Pedro, H. V. Venturina, P. G. Duran and L. C. Cruz. 2007. Full-term
delivery of river buffalo calves (2n = 50) from in vitro-derived vitrified embryos by swamp
buffalo recipients (2n = 48). Livestock Science. 107: 213–219.
Liang X. W., Y. Q. Lu, M.T. Chen, X. F. Zhang, S. S. Lu, M. Zhang, C. Y. Pang, F. X. Huang and
K.H. Lu. 2008. In vitro embryo production in buffalo (Bubalus bubalis) using sexed
sperm and oocytes from ovum pick up. Theriogenology 69: 822-826.
Madan M. L., M. S. Chauhan, S. K. Singl and R. S. Manik. 1994. Pregnancies established from
water buffalo (Bubalus bubalis) blastocysts derived from in vitro matured, in vitro
390
fertilized oocytes and co-cultured with cumulus and oviductal cells. Theriogenology. 42:
591-600.
Misra A. K., M. Mutha, R. Kasiraj, N. S. Ranga Reddy and H. C. Pant. 1999. Factors affecting
pregnancy rate following nonsurgical embryo transfer in buffalo (Bubalus bubalis): a
retrospective study. Theriogenology. 52: 1-10.
Presicce G. A. 2007. Reproduction in the water buffalo. Reproduction of Domestic Animals. 42
(Suppl.2): 24–32.
Sá Filho, M. F., N. A. T. Carvalho, L. U. Gimenes, J. R. S. Torres-Junior, C. R. Ferreira, F. Perecin,
A. P. Perini, T. A. D. Tetzner, R. Vantini, G. F. Soria, J. M. Garcia, H. Tonhati, B.
Gasparrini and P. S. Baruselli. 2005. Prenhezes de embriões bubalinos frescos e vitrificados
produzidos in vitro. Acta Scientae Veterinariae. 33:431.
Table 1. Total number of pregnancies confirmed on days 30 and 60 after embryo transfer, and at
calving.
30 days 60 days At calving

Pregnancies/ total of embryos transferred 50/115 48/115 42/115
Pregnancy rate (%) 43.5% 41.7% 36.5%
Table 2. Gestational lenght (days) and weight (kg) at calving in male and female newborns.
Male Female
n 21 20
Gestational lenght (range in days) 310.9 (297 to 320) 309.8 (300 to 328)
Birth weight (range in kg) 33.8 (29 to 40) 31.4 (29 to 35)
391
Development of Frozen Buffalo Semen Production in the Phillippines
Felomino MAMUAD,a Emma VENTURINA,b

a
Philippine Carabao Center, Science City of Muñoz, Nueva Ecija 3120, Philippines
b
Philippine Carabao Center at Central Luzon State University, Science City of Muñoz,Nueva Ecija
3120, Philippines
* Corresponding e-mail: fvmamuad@yahoo.com
ABSTRACT
Artificial Insemination (AI) in mammals has been tried in almost all types of animals during
1780 through Mr. Lazzaro Spallanzani, an Italian Physiologist then to Russia, India, U.S, France, a
Germany and throughout the world. There are several techniques developed for cattle and Buffalo. AI
could be done using fresh or extended semen, but frozen buffalo semen is preferred in countries like
India, Pakistan and China. The equipment used for semen processing were mostly on Europe (France),
Germany and Japan. Frozen semen for AI in the Philippines started using imported frozen semen from
Pakistan, India and Thailand. Through the FAO-UNDP project and JOCV-JICA which procured
equipment for semen processing, we started producing extended and frozen semen from buffalo.
Researchers go along with the introduction of different extenders, cooling, equilibration time and
freezing of semen. Different method has been used in freezing of semen, i.e. ice chest freezing, wide
mouth liquid nitrogen tank freezing and freezing method introduced by Japanese volunteer using
freezing tank FHK 1652 which give us good results especially progressive motility by subjective
evaluation. Frozen buffalo semen continuously produced from 1984 to present using new equipment
given by JICA in 2001 and KOICA in 2012. Our production increased meeting our target every year.
This year, we had already produced 87.5% of the 200,000 doses for requirements. We are analyzing the
quality of semen using computer assisted sperm analyzer (CASA, Sperm VisionTM). The CASA
system combines a high quality microscope with digital image technology that offers accurate sperm
analysis. The average motility of buffalo semen that we analyzed through CASA is 85% - 90% initial
progressive motility and 70-80% progressive motile sperm in post thaw evaluation, Because of
machine this new method of evaluation and with other new equipment (Filling and sealing machine
MPP QUATTRO), our production increased by 30-40% and reduced the risk of improper handling of
semen. Now, the laboratory will be able to produce the requirements of the agency, PCC could
accommodate more buffalo bulls and cattle bulls that could inseminate here in the Philippines and also
abroad thru the ASEAN
Keywords: Artificial Insemination, Frozen buffalo semen, Semen production in the Philippines

392
Morphology and Biometrics of Spermatozoa of Two Breeds of Water Buffalo

(Bubalus Bubalis L.)
*
Sofronio P. KALAW a, F. G. CLAVERIA b AND F. V. MAMUADc
a
Central Luzon State University, Science City of Muñoz, Nueva Ecija 3120 Philippines, b De La
Salle University, Taft Ave., Metro Manila, cPhilippine Carabao Center, Science City of Muñoz,
Nueva Ecija 3120 Philippines
*Corresponding email: fvmamuad@yahoo.com
ABSTRACT
Bulls of Murrah buffalo, 4 of Indian (IMB) and 4 of Bulgarian (BMB) were used to
evaluate the semen quality of the two breedtype, specifically, the color, pH, volume, sperm
concentration, sperm motility, total concentration and sperm abnormality was evaluated. The
semen were collected as follows: a) two successive ejaculation per day at an interval of 4 days;
b) four successive ejaculation per day at an interval of 4 days; and c) daily collection of two
successive ejaculates for a period of 7 days. Also the biometrics of spermatozoa was compared
and the ultra structure of normal spermatozoa of the two breeds was described. In both IMB and
BMB, the semen color ranged from light creamy to creamy. The IMB showed higher semen
volume, sperm density, sperm motility, total concentration of motile spermatozoa and sperm
abnormality but lower pH value compared to the BMB. Although significant differences in the
semen parameters evaluated were noted among individual bulls these were insignificant between
the two breeds in the three different semen collection procedures used. In both IMB and BMB,
semen pH values, sperm concentration and sperm motility were significantly influenced by the
four successive ejaculation and daily collection procedures. However, semen color, volume and
sperm abnormality were not significantly affected. Increased frequency of ejaculation and semen
collection resulted in a significant reduction in sperm count with no appreciable decrease in
semen volume but with significant increase in pH toward the alkaline value, and increased
sperm motility. The IMB sperm was found to be longer (60.57 ± 3.50 um), with shorter (7.84±
1.09 um) and narrower head ( 5.0 ± 0.75 um), shorter midpiece (12.0 ± 1.02 um) and longer
tailpiece (44.70 ± 3. 53 um) than those of the BMB (58.4 ± 4.27 um, 8.10 ± 0.60um, 6.0 ±
0.50um. 12.26 ± 1.06um, and 38.0 ± 3.43, respectively). The sperm of both breeds consists of
the head, neck and flagellum. The head has a cone-shaped blunt ended acrosome. The flagellum
is characterized as a typical cartwheel pattern of 9 doublets and 2 central pairs of microtubules.
Results of the study show that both IMB and BMB can be used as a source of quality semen for
artificial insemination. When there is an urgent need for semen, three successive ejaculations in
BMB and two successive ejaculations in IMB or daily collection for three consecutive days for
both IMB and BMB are highly recommended.
Keywords: Sperm Concentration, Sperm motility, Spermatozoa
INTRODUCTION
The water buffalo is considered as one of the most important domestic animal in the
Philippines and other Asian countries. It is raised as a source of farm power, meat and milk.
Although researches on the quality and other semen parameters of bubaline semen have been
studied by several researchers on riverine buffalo, the present study focused on two breeds of

393
riverine buffalo, the Indian Murrah and Bulgarian Murrah that are presently maintained at
Philippine Carabao Center. The information obtained in this research could be valuable in
assessing the need to modify the present method of semen collection.

Four IMB and four BMB bulls with an age range of 4-6 years old were used as the source
of semen. Semen samples were collected from each bull at 6:00 to 7:00 a.m. using artificial
vagina. The semen quality was evaluated in the semen processing laboratory using standard
procedures.
The study was divided into four studies. Study 1, evaluated the semen quality of the two breed
using normal collection procedure. Study 2, determined the effect of successive ejaculation on
semen quality. Four successive ejaculates were collected from each bull. The procedure was
repeated after four days. Study 3, assessed the effect of daily collection on semen quality.
Semen samples were collected daily for one week from each bull. Study 4, described the
spermatozoa using scanning electron microscope and transmission electron microscope.

Semen Quality
The color of the semen ranged from light creamy to creamy. However, marked difference
in color was observed between the two breeds and between ejaculates.
In BMB, the semen volume mean values were 2.14 (first ejaculate) and 2.0 ml (second
ejaculate); and in IMB, mean values were 2.29 (first ejaculate) and 3.45 + 1.98 ml (second
ejaculate). The IMB produced higher semen volume than BMB and in both breeds, higher sperm
volume was observed in the first ejaculate. Insignificant differences in sperm volume were noted
between the first and second ejaculates and between the two breed.
The mean sperm count in BMB were 112.0 x 107 per ml in the first ejaculate and 105 x 10 7 per
ml in the second ejaculate. In IMB, the mean sperm concentrations were 118.0 x 10 7 (first
ejaculate) and 107.75 x 107 (second ejaculate) per ml. In both breeds more spermatozoa were
recorded in the first ejaculate with IMB producing higher sperm count than BMB. Differences in
sperm count, however, were significant between the first and second ejaculate but not between
the two breeds. Interestingly, individual bulls within each breed registered significant differences
in sperm concentration.
In both ejaculates, IMB registered higher sperm motility with mean values of 55 and 56.88%,
than in BMB with mean of 51.25 and 54.38% in the first and second ejaculates, respectively.
Higher sperm motility was noted in second ejaculate of the two breeds. Comparison of sperm
motility between the ejaculates and bulls are insignificant.
The BMB has lower % abnormal sperm with mean values of 9.27 (first) and 12.90 (second)
compared to IMB which registered mean values of 12.73 (first) and 14.56 (second) ejaculate. In
IMB, the more common primary sperm abnormalities consisted of the microhead, pyriform head,
screw midpiece, abaxial midpiece and knobbed sperm. In both breedtype, secondary sperm
abnormalities were those of coiled midpiece and tail, free head, midpiece and tail, bent midpiece,
bent tail and coiled tail. Percent abnormal sperm and the types did not differe significantly
between the ejaculates, among bulls, and breeds.
394
Effect of Four Successive Ejaculations on Semen Quality

In both IMB and BMB, the color ranged from creamy to light creamy. In both breeds, an
increase in pH was noted with subsequent ejaculation, among individual bulls and between
ejaculates.
In general, a decrease in semen volume with subsequent ejaculation was noted in both IMB and
BMB. Differences were insignificant. The amount of semen, however, produced by individual
bulls in both breeds differed significantly.
A decreasing trend in the sperm concentration with increasing frequency of ejaculation was
observed in both breeds. In IMB, the sperm count decreased from 124.75 x 10 7 to 65.58 x 10 7 in
the fourth ejaculate. In IMB the reduction was from 110.25 x 10 7 to 61.62 x 10 7 in the fourth
ejaculate. Sperm concentration differed significantly among individual bulls and between
ejaculates but not between the breed.
In both breeds an increasing trend in sperm motility with subsequent ejaculation was observed.
In IMB, the increase was from 49.38% in the first ejaculate to 61.25% in the fourth ejaculate. In
BMB, the increase ranged from 51.88% in the first ejaculate to 61.88% in the fourth ejaculate.
Significant difference in sperm motility between ejaculates and among individual bulls but not
between the breed was noted.
The highest sperm abnormality in IMB was recorded in the fourth ejaculate with a mean of
9.74%, while the lowest percent abnormality was observed in the second ejaculate with a mean
of 8.35%, Likewise, in BMB, the fourth ejaculate had the highest percent sperm abnormality
with a mean of 11.94% while the second ejaculate registered the lowest percent sperm
abnormality with a mean of 9.35%. While an increase in percent sperm abnormality was
observed in subsequent ejaculations, and were insignificant.
Effect of Daily Collection for Seven Days on Semen Quality
The color of semen in both breeds ranged from creamy to light creamy to colorless or
watery. A progressive increase in semen pH with daily collection of semen was observed. The
increase for IMB was from 6.60 on the first day of collection to 7.11 on the last day of collection.
In BMB pH ranged from 6.69 in the first day of collection to 7.218 in the last day of collection.
In both breeds, the pH ranged from slightly acidic to slightly neutral. Mean pH values differed
significantly between days of collection.
In IMB, highest semen volume was obtained on the fourth collection day with a mean of 2.845
ml. In BMB, the highest volume was noted on the 5th day with a mean of 1.96ml. Semen volume
differed significantly among individual bulls but not between breeds and between collection
days.
There was an appreciable decrease in the sperm concentration as a result of daily
collection. The sperm concentration decreased from 155 x 10 7 per ml on the first day to 31.75 x
10 7 on the last collection day for IMB. In BMB, the reduction was from 111 x 10 7 per ml on the
first day to 51.50 x 10 7 per ml on the last day of collection. In IMB, the lowest mean motility
rating was recorded on the first day with a mean of 53.75% while the highest mean of 65.62%
was observed on the 7th day with a mean of 65.62. In BMB, the lowest mean motility was noted
in the 2nd day with a mean of 55% while the highest motility rating of 61.25% was noted in the
7th day of collection.
In both breeds, the lowest percent sperm abnormality was noted in the first day of
collection while the highest percent was recorded on the 7th day of collection. The values
obtained, however, were insignificantly different between the breeds and among collection
periods but not among individual bulls.
395
Structure and Measurement of Spermatozoa

The normal sperm consisted of an ovate head and tail, which is divided into midpiece,
principal tailpiece and tail endpice. The tail end piece was better observed in SEM processed
sample. The neck or connecting piece appeared inconspicuous entity at the base of the head and
joined with the midpiece. The IMB sperm is longer, with shorter and narrower head, shorter
midpiece and longer tail piece while BMB sperm is shorter and with longer and wider head,
longer midpiece and shorter principal tailpiece.
The sperm head has a cone shaped blunt ended acrosome. This houses the compact nucleus with
electron dense chromatin materials enclosed in a double layered nuclear membrane. At the base
of the head is a depression which houses the centriole and exhibits the base of of the intranuclear
axoneme that joins with axial filament of the midpiece. The flagellum is composed of an
axoneme characterized as a typical cartwheel pattern of 9 doublets and 2 central microtubule.
Surrounding the midpiece axial filament is an outer ring of nine electron dense coarse fibers.
Prominent mitochondrion arranged into a sheath encloses only the axoneme of the midpice but
not the axoneme of the principal tail endpice.
CONCLUSIONS
1. The semen quality of IMB and BMB are comparable indicating that either breeds can be
used as the source of semen for artificial insemination.
2. Semen quality such as pH, volume, sperm concentration, sperm motility and percent
sperm abnormality differed significantly among bulls within breeds.
3. In both breeds, four successive ejaculation and daily semen collection reduced sperm
count, increased semen pH and sperm motility without influencing semen volume and
percent sperm abnormality.
REFFERENCES
Cuaresma, R.O. 1990. Morphological Examination of Spermatozoa. In Handbook for Semen
Processing Lab. PCRDC. Central Luzon State University, Muñoz, N.E
Hafez, E. and Y. darwish. 1956. Effect of successive ejaculation on semen characteristics in
buffalo. Journal of Agricultural Science.
Kodagali, S.B., B. K. Bhavsar and A. A. Deshpande. 1973. Biometrics of Surti buffalo
Spermatozoa. Indian Veterinary Journal. 50 (1): 50-54.
Tomar, N. S. 1984. Reproduction of cattle and buffaloes. Seroy Prakashan. India.
396
Development of Assessing Motility in Computer Assisted Sperm Analyzer and

Phase Contrast Microscopy
Emma VENTURINAa, Matt Daniel PERALTAa, C. RINGORa, R. J. BALANTUCAN ANDa

Felomino V. MAMUADa
a
PCC at CLSU, National Bull Farm, Nueva Ecija, Philippines bPCC National Headquarters and Gene
Pool, Science City of Munoz, Nueva Ecija, Philippines
*Corresponding email: fvmamuad@yahoo.com
ABSTRACT
This study was conducted to determine the efficiency of subjective method of evaluation of
motility and computer assisted sperm analyser (CASA) in the evaluation of motility of fresh and frozen
buffalo semen. Semen production of the Philippine Carabao Center, Department of Agriculture started
last 1983. The system was base on the knowledge obtain from Thailand, India, Pakistan, Japan, Korea
and Germany. Some are trained to do subjective method of evaluation via phase contrast microscopy
and train other staff of the center and officers of other agencies. In 1983 to 1986, subjective method of
evaluation and horizontal freezing was made. In 1987, the evaluation was also subjective method but
used vertical freezing as introduced by volunteers under JOCV/JICA, Japan. A better microscope was
used, phase contrast microscopy with TV monitor. This will enable us to see sperm cells that are
floating, die and go with water. Progressive motility evaluation was done by a well trained staff.
However, this staff is supported by other methods, like the percent live and dead spermatozoa and
percentage of sperm passages by sephadex filtration. The progressive motility of 63.7-72.75 % has
91.48-92.78 % live sperm and per cent sperm passage of 75-80 %. Freezing of semen was continuously
done thru vertical method by FHK 1652. The frozen semen production was 70-85 per cent of the total
semen doses subjected to freezing. The change in equipment was base on availability in CLSU, FAO-
UNDP, JOCV/JICA up to CY 2000. In 2001, JICA experts almost change all the equipment and this
was used up to 2011. In 2012, KOICA added some of the equipment and buy from Germany. Training
was made to our staff and started the CASA system from March of 2012. The mean PM in per cent in
CASA was 87.14-89.65 %, LM was 67.83-78.35 percent. In SM the PM was 63.7-72.75 percent. The
morphology was 91.48-92.78 % live sperm, 86.46-87.61% normal sperm for both. The PTM in CASA
system was 81.92-83.36% progressive motility and 68-70.26 % linear motility. In subjective method,
the PTM was 34.47-36.35 percent. Frozen semen production increases significantly during the study.
Keywords: Progressive motility (PM); Linear motility (LM); CASA; Subjective method(SM).
INTRODUCTION
For the last thirty years, we learn frozen semen processing in Thailand, India and Pakistan
utilizing ordinary electron microscope and Phase contrast microscopy. The evaluations have at least
two months or more in looking after the microscope and assess self of real evaluation. Other means like
eosine- nigrosin stain or the sephadex filtration test to correct any disparities in evaluation.
After several years, the CASA systems arrive and show its development over the Phase contrast
microscopy. The improvement is reviewed. To determine any improvement of different methods use in
the production of frozen buffalo semen. To compare the efficiency of phase contrast microscopy and
computer assisted sperm analyzer and to demonstrate the ability of subjective method and CASA in the
production of quality spermatozoa preserved and use for breeding.

397

The records of the semen production laboratory of Philippine Carabao Center from 1983-1986,
1987-2000, 2001- 2011 and 2012 has been reviewed. The equipment use has been identified and the
method used has been known. Evaluation was all by Phase contrast microscopy. Freezing was change
from horizontal to vertical freezing. The evaluator however improved due to experience and able to
determine whether good, better, best spermatozoa and pass the freezing to 30% PTM.
When the CASA arrived, the evaluator used the Phase contrast microscopy and CASA in the
evaluation. The evaluation was at the same time in one sample. There was a big lift on the evaluation
process that pass. All available animals at the National Bull Farm, Joson (Digdig), Carranglan, Nueva
Ecija was used in the study. The results base on number of ejaculates collected, number of ejaculates
with a passing progressive motility of 60 percent under subjective method will be processed into frozen
semen processing. In similar manner, all ejaculate with 60% motility under Phase contrast microscopy
and have 70% progressive motility under the CASA will be process into frozen semen.
The frozen semen production in the subjective method of evaluating semen and the CASA
system under the semen processing method will be characterize. The improvement in the production of
frozen semen will be determine as better in the procedure of frozen semen processing. Environmental
changes will be recorded.
All data will be analyzed in completely randomized design. Any differences will be analyzed by
DMRT.
The following main equipment used:
1. AV have water and place in a warmer to heat up to 42ºC - 46ºC. Determine sperm concentration
after collection. Samples for CASA and PCM were made for motility.
2. The amount of diluter is calculated or seen in the CASA System. The amount of diluter was based
on the sperm concentration needed. It is diluted at one time.
3. The Cold Handling Cabinet is used when it is working. The temperature is 2 to 7ºC or about 5ºC.
After about two hours, the straws that will be needed is printed and subjected to MPPQuatro for
filling and Sealing.
4. It is place in straw basket for freezing in FHK 1625 for 8 minutes and transfer to the temporary
storage tank.
5. On the following day, it is evaluated for Post Thaw motility. The passing mark under Phase contrast
microscopy is 30% or higher and evaluated to CASA. Usually, the PTM of PCM of 30% is 70%
under CASA system. The frozen semen that passed are stored in their own liquid nitrogen tank.
parameter
The Animals are: 3 Phil. Carabao, 5 Indian Murrah Buffalo, and 25 Bulgarian Murrah Buffalo.
The Semen extender was TRIS (Hydroxy) methyl amino methane(3.028g),Raffinose(1.25g), Citric
Acid(1.675g), fructose(2.7g), glycerol(7ml), distilled water(73ml), egg yolk(20ml),
Penecillin(500mg/ml) and Streptomycin (1mg/ml).

Semen were collected twice a week and two ejaculations per collection day. The average
volume, initial motility and post thaw motility of Philippine Carabao bulls is significantly lower than
the Indian and Bulgarian Murrah Buffalo (Table 1).
398
Table 1. Semen Production of three breed types utilizing phase contrast microscopy and casa system
(3 Months).
BREEDTYPE BMB IMB PC
No. of bulls 25 5 3
Ave. Vol. of ejaculate* 3.2 4.6 2.5
Semen concentration * 133 131 140
INITIAL MOTILITY
PCM 72.75 а 72.71 a 63.70 b
CASA/Linear*
76.33 a 78.35 a 67.83 b
CASA/Progressive*
89.65 89.24 87.14
PT Motility
PCM* 34.47 36.35 35.83
CASA/PROGRESSIVE* 81.92 83.36 83.07
CASA/Linear* 68 70.26 69.60
FS Produced (estimated) at 100M/ml
PCM 74,309 21,031 6,420
CASA 91,572 25,812 8,783
FS Produced (estimated) at 70M/ml
PCM 106.156 30.044 9.172
CASA/Progressive 130.817 36.874 11.396
Morphological
% Live* 92.52 92.78 91.48
% Normal* 86.46 87.61 86.54
*Averages
When the semen ejaculates are collected to evaluate base on Phase Contrast microscopy and
CASA, (Table 1) the initial motility in PCM is almost the same including the linear motility under
CASA system. However, the linear motility shows a significant difference of BMB and IMB to PC
bulls with 76.33, 78.35 and 67.83 percent respectively. The number under PCM is 72.75, 72.71 and
63.70% for BMB, IMB and PC bulls and the progressive motility under CASA was almost same as
89.65, 89.24 and 87. 14% respectively, However progressive motility is higher than the linear
motility and phase contrast microscopy.
In the post thaw motility, under PCM and CASA, the BMB, IMB and PC bull have 34.47, 36.35
and 35.83% while for the CASA, the linear motility was 68.00, 70.26 and 69.60% and the progressive
motility was 81.92, 83.36 and 83.07% respectively. It could be noted that the PCM is significantly
lower than the CASA system of linear motility and progressive motility.
The estimated frozen semen production in 0.5ml semen straws under phase contrast microscopy
or subjective method was 74,309, 21,031 and 6,420 doses for BMB, IMB and PC respectively. It is
significantly lower than CASA system with 91,572, 25,812 and 8,783 doses for BMB, IMB and PC
bulls respectively. The difference is 24,407 doses respectively. This is a semen dose with 50millions
sperm per doses already. However will be higher by 33.715 doses at 35 million motile sperm per dose
by using the computer assisted sperm analyzer system. This lower sperm concentration is being
followed now.
399
CONCLUSIONS
The computer assisted sperm analyzer is very useful to the evaluator who used to be using
his/her eyes in analysis of semen. This was base on several persons who knows the subjective method
of evaluation. However, we have to still continue in doing research activity of real sperm responses to
temperature and should have better glass slide for analysis in flat or concave microscope slides. Other
equipment, like the MPPQuatro has to be tested also for easiness in evaluation. The present study
clearly shows how CASA was able increase the number of frozen semen produce and will be use of
good conception and calving in the farm or field.
REFERENCES
F.V. Mamuad, H.V. Venturina, E.V. Venturina, R.T. Morcoso, E.C. Atabay, K. Kudo. 2005.
Artificial Insemination Manual for Water Buffaloes. Water Buffaloes and Beef Cattle
Improvement Project. JICA, BAI and PCC. October 2, 2000 – October 1, 2005.
Minitube Sperm Vision. Computerize Digital Sperm Analyzer for Semen Production and Research.
84184 Tiefenback, Germany.
HAFEZ, E.S.E. 1987. Reproduction in Farm Animals, 5th Ed. Lea and Febiger Pub., Philadelphia.
400
A Two-step Culture System Sustains Individual Buffalo Embryo Development
Di LI, Juan LOU, Wenxin ZHANG, Xiaogan YANG, Yangqing LU, Kehuan LU*
Animal Reproduction Institute; State Key Laboratory for Conservation and Utilization of
Subtropical Agro-bioresources, Guangxi University, Nanning, Guangxi 530004, China
*Corresponding email: khlu@gxu.edu.cn
ABSTRACT
Non-invasive methods of individual embryo research have a great potential in the study of
embryo nutritional requirement and embryo developmental potentiality. The use of metabolomics
methods is a significant approach to analyze the preimplantation embryo development during
cultured in chemically defined media. The aim of the present study was to establish a study model
of individual buffalo embryo development from the morula stage to the blastocyst stage. The result
showed that even though the feeder and serum free culture system had a significantly lower
blastocyst formation rate from morula-stage to blastocyst compared to the group culture system
with feeder cells and serum (P<0.05, 51.2%, 63/123 VS. 40.4%, 59/146) it could support individual
buffalo embryo development from morula-stage to blastocyst when SOF medium was used.
Keywords: Buffalo, individual embryo culture, oxygen tension, serum-free medium
INTRODUCTION
Previous studies in many species have demonstrated that embryos cultured in groups have a
higher blastocyst formation rate than that cultured individually. This is thought to be due to the
accumulation of secreted beneficial autocrine and/or paracrine factors from the grouped embryos
themselves, whereas the paracrine factors do not exist in the individual embryo production (iIVP)
system (Lane et al., 1992; O’Doherty et al., 1997). On the other hand, the use of iIVP systems is
expedient, since they make individual embryo tracking possible. Several strategies of iIVP have
been employed for mammalian embryos such as micro-droplets (Nagao et al., 2008), micro-wells
(Vajta et al., 2000), microfluidic technology (Beebe et al., 2002; Wheeler et al., 2007). The
improvement of iIVP systems would be of interest to several fields of research such as embryo
metabolism, nuclear cloning, ovum pick-up or clinical in vitro fertilization.
The embryo culture system has led to the evolution of two major types, one in which
chemically-defined media are supplemented with protein additives (e.g., BSA), the other in which
the embryosare cultured in the presence of feeder layers of somatic cells (Nancarrow et al., 1994).
In buffalo species, although many researchers still prefer the co-culture system for embryo
production, the utilization of defined media for embryo culture has become necessary to
comprehend the requirements of buffalo embryos in vitro which, in turn, would allow the
formulation of an optimal species-specific culture system.
Culturing buffalo embryos to the blastocyst stage has gained popularity for the past few years.
In many laboratories, blastocyst production in vitro is 15-30% of inseminated oocytes (Nandi et al.,
2002). Culture of bovine embryos individually in vitro is generally associated with poorer
developmental rates (Donnay et al, 1997; Nagao et al., 2008).
The aim of the present study was to establish a routine culture protocol without feeder layer and
serum for individual buffalo embryo development so as to further study in the buffalo embryo

401
metabolomics.

In vitro oocyte maturation (IVM) and In vitro fertilization (IVF)
Buffalo ovaries were collected at a local slaughterhouse and transported to the laboratory in
saline within 2 hours. Cumulus Oocyte Complexes (COCs) were aspirated from 2-8 mm diameter
follicles with10 ml disposable syringes fitted with 12-gauge needles. Only homogeneous cytoplasm
COCs with at least 3 compact layers of cumulus cells were selected and cultured for IVM in
modified TCM199 medium under 5% CO2 in humidified air at 39℃ for 22-24h. Matured COCs
were stripped of cumulus cells by gentle pipetting and washed twice in TALP medium and
transferred into 50μl droplets for in vitro fertilization (IVF). Sperm was treated by swimming-up
procedure for a final sperm fertilizing concentration of 2x106/ml. Fertilizing droplets were
incubated under the same gas atmosphere as for IVM.
In vitro culture (IVC)
The IVC procedure was composed of two steps and carried out in a 5% CO2 incubator at 39 ℃
with maximum humidity. Eight to ten hours after insemination, fifteen presumptive zygotes were
grouped in a 20μL-droplet and co-cultured with feeder cells (buffalo cumulus cell monolayer) in
TCM-199 medium (10% FBS) for initial 96 hours. Half volume of medium was replaced every 48
hours. After 96 hours, only morula-stage embryos were selected for the second culture step. The
morulae were individually cultured in 20 μl droplets which were adopted by either of two
chemically defined mediua, SOF or KSOM. The SOF we modified was based on the recipe reported
by Tervit (Tervit et al., 1972) and the KSOM was the same recipe as the report by Lawitts (Lawitts
et al., 1991).
Experimental design
In the second culture step, the morulae were divided into three groups and cultured in 20 μl-
droplet for another 72 hours: (1) Morulae (15 morulae/20 μl droplet) were co-cultured with feeder
cells in TCM-199 medium supplemented with 10% FBS as control group. (2) Morulae were
individually cultured in SOF medium or (3) in KSOM medium. The data were statistically analysed
by T-test.

For the morula production, a total of 769 presumptive zygotes were cultured, of which 383
were developed to morula-stage with a morula formation rate of 49.8%. The development rate from
the morula stage to the blastocyst stage in modified SOF was significantly lower than that in control
group (40.4%, 59/146vs. 51.2%, 63/123). None of the morulae cultured in KSOM developed to the
blastocyst stage (Table 1).
Bovine embryos cultured in vitro often undergo a ‘‘8- to 16-cell developmental block’’ which
result in a decrease of blastocyst formation rate. Feeder cells, conditioned medium or the presence
of serum in a complex medium, are often used to stimulate embryo development in vitro (Eyestone
et al., 1989; Myers et al., 1994; Rorie et al., 1994). However, complex culture media containing
serum and/or co-culture with feeder cells may contain unknown components, thereby troubling
metabolomic studies. We applieda two-step culture system to overcomethe developmental block
and obtain an acceptable blastocyst formation rate. In the first step, the feeder cell layer and group
culture were used which are beneficial for the embryos to reach the morula stage. After the morula
402
stage, we applieda serum-free culture media, which are expected to enable future studies on
metabolomics in individual embryos and greatly facilitate non-invasive studies on in vitro embryo
quality.
A previous report showed that KSOM was feasible for the culture of buffalo embryos (Zicarelli
et al., 2003). Although both of SOF and KSOM areconsidered as simple culture media, there was a
great difference in embryo development in these two culture media. As a general rule, simple
culture mediaare suggested to be used under low oxygen concentration (5% O2) because the simple
media lack serum or other macromolecules that serve as scavengers of reactive oxygen species. The
results of the present study revealed, that individual culture of buffalo embryos from the morula
stage to the blastocyst stage was less effective than group culture in terms of blastocyst yield.
However, a remarkable amount of blastocysts could be produced in the iIVP system using SOF
medium. On the other hand, KSOM did not support blastocyst formation in the iIVP system. The
reason why none of blastocyst developed in KSOM medium in our study might be that the KSOM
medium contained glucose (0.2mmol/L) and under high O2 tension glucose metabolism causes ROS
formation which is toxic to the embryos. However, glucose was eliminated from our modified SOF
medium.
In conclusion, the two-step culture system using SOF medium sustained the individual
development of buffalo embryos from the morula to the blastocyst stage and therefore is
recommended for further studies on embryo metabolomics.
REFERENCES
Beebe, D., M. Wheeler, H. Zeringue, E. Walters and S. Raty. 2002. Microfluidic technology for
assisted reproduction.Theriogenology 57: 125–135.
Donnay, I., A. Van Langendonckt, P. Auquier, B.Grisart, A. Vansteenbrugge, A. Massip, and F.
Dessy. 1997. Effects of co-culture and embryo number on the in vitro development of bovine
embryos. Theriogenology 47: 1549–1561
Eyestone, W.H. and N.L. First. 1989. Coculture of early cattle embryos to the blastocyst stage with
oviductal tissue or in conditioned medium. J. Reprod. Fertil. 85: 715-720.
Lane, M., and D.K Gardner. 1992. Effect of incubation volume and embryo density on the
development and viability of mouse embryos in vitro. Hum. Reprod. 7: 558–562.
Lawitts, J.A. and J.D. Biggers. 1991. Optimization of mouse embryo culture media using simplex
methods. Journal of reproduction and fertility 91: 543–556.
Myers, M.W., J.R. Broussard, Y. Menezo, S.G. Prough, J. Blackwell, R.A. Godke. and J.K.
Thibodeaux. 1994. Established cell lines and their conditioned media support bovine embryo
development during in-vitro culture. Hum. Reprod. 9: 1927-1931.
Nagao,Y., R. Iijima and K. Saeki. 2008. Interaction between embryos and culture conditions during
in vitro development of bovine early embryos. Zygote 16: 127–133.
Nancarrow, C.D. and J.L. Hill. 1994. Co-culture, oviduct secretion and the function of
oviduct-specific glycoproteins. Cell Biol Inter 18: 1105-1114.
O’Doherty, E,M,, M.G. Wade, J.L. Hill andM.P. Boland. 1997. .Effects of culturing bovine oocytes
either singly or in groups on development to blastocysts. Theriogenology 48: 161–169.
Rorie, R.W., T.D. Lester, G.F. Miller, D.W. Gliedt and R.W. Mcnew. 1994. Effect of protein source
and co-culture on bovine embryo development in synthetic oviductal fluid medium.
Theriogenology 42: 385-395.
Nandi S., H.M. Raghu, B.M. Ravindranatha and M.S. Chauhan. 2002. Production of Buffalo
(Bubalus bubalis) Embryos in vitro: Premises and Promises. Reprod. Dom. Anim. 37: 65–74.
Tervit, H. 2002 . Successful culture in vitro of sheep and cattle ova. Journal of Reproduction
493–497.
Vajta, G., T.T. Peura, P. Holm, A. Paldi, T. Greve, A.O. Trounson and H. Callesen. 2000, New
403
method for culture of zona-included or zonafree embryos: the Well-of-the-Well (WOW)

system. Mol. Reprod. Dev. 55: 256–264.
Zicarelli, L. and G. Neglia. 2003. Buffalo (Bubalus bubalis) in vitro embryo production in two
different defined culture media. Italian Journal of Animal Science 2: 136–138.
Funding sources:
This research was jointly supported by the Opening Project of Guangxi Key Laboratory of
Buffalo Genetics, Reproduction and Breeding (SNKF-2011-01); Guangxi Science and Technology
R&D Program (No. 1123005-1) and Guangxi Science Foundation Key Program (No.
2010GXNSFD013023).
Table1. Blastocyst development in two different culture media

Culture Culture Feeder cell No. embryo developed to Morula to blastocyst
Groups type* co-culture* (%)
Morula Blastocyst
1) Control Group + 123 63 51.2a
2) SOF Individual - 146 59 40.4b
3) KSOM Individual - 114 0 0
Three replicates were performed.
a, b; P<0.05
*Embryo culture from the morula stage
404
Comparison of Motility, Morphology, Acrosome Integrity, Membrane Integrity

and Fertilizing Ability of Frozen-Thawed Buffalo Sperm Separated
by a Percoll® Gradient or Puresperm®
Nutthee AM-IN,a* Vibuntita CHANKITISAKULb and Mongkol TECHAKUMPHUa
a
Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science,
Chulalongkorn University, Bangkok 10330, Thailand
b
Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002,
Thailand
*Corresponding e-mail: nutthee.a@chula.ac.th
ABSTRACT
The aim of this study was to evaluate sperm quality following separation and selection using a
silane-coated silica colloid method (PureSperm®) or Percoll®. Frozen/thawed sperm from buffalo bulls
(n=5) were assigned to two groups. The first group was separated using a Percoll® discontinuous
gradient (45 and 90%) and the second group by a single layer of PureSperm®. Sperm motility,
concentration, morphology, acrosome integrity and membrane integrity were evaluated and compared
before and after sperm separation. Ability of sperm to fertilize was tested by hyaluronan binding assay
(HBA) score. Sperm motility and morphology improved (P < 0.001) after the separation process with
no differences between methods. The Percoll® gradient increased the mean (± SEM) percentage of
sperm recovered (8.27±0.79) compared to PureSperm® (5.25 ± 1.41; P = 0.007). However, membrane
integrity (61.4 ± 1.3 vs. 56.8 ± 1.3) and acrosome integrity (59.8 ± 1.4 vs. 55.3 ± 0.6) were higher
following selection using PureSperm® than Percoll® (P = 0.03). The HBA score was higher when
semen was selected by PureSperm ®than by Percoll® (49.5 ± 1.6 vs. 40.0 ± 1.1; P = 0.02). We conclude
that PureSperm® separation resulted in lower sperm recovery than by Percoll® but was more effective
for selecting high quality sperm for in vitro fertilization for embryo production in buffalo.
Keywords: buffalo, sperm quality, Percoll®, PureSperm®
INTRODUCTION
Sperm selection using colloidal substance centrifugation is an efficient method to select good
quality sperm for in vitro fertilization (IVF). In buffaloes, Percoll® has been used as the colloidal
substance in a few studies in preparation for IVF (Mehmood et al., 2009) and intracytoplasmic sperm
injection (ICSI) (Chankitisakul et al., 2012). Percoll® has been reported to have variable degrees of
toxicity for human sperm (Carrell et al., 1998; De Vos et al., 1997). In October 1996, Percoll® was
withdrawn from the market for clinical use in assisted reproduction due to the risk of contaminations
with endotoxins (Andersen and Grinsted, 1997; Scott and Smith, 1997), possible membrane alterations
(Strehler et al., 1998) and inflammatory responses that could be induced by the insemination of sperm
populations contaminated with Percoll®. In consequence, some studies have chosen PureSperm® as an
alternative choice for sperm selection (Mousset-Simeon et al., 2004; Srisombut et al., 1998).
PureSperm® is a silane-coated colloidal substance yielding similar sperm parameters to those of
Percoll® and can be considered a suitable substitute for Percoll® (Simeon et al., 2004). PureSperm® is
able to reduce significantly the percentage of sperm with nuclear abnormalities and poor sperm plasma
membrane integrity (Sakkas et al., 2000). The sperm-hyaluronan–binding assay (HBA) was developed
and used as a commercial diagnostic kit to evaluate sperm maturity and function (Cayli et al., 2003;
Cayli et al., 2004). This test is based on previous reports of hyaluronic acid (HA) selectively binding to
mature sperm with intact acrosome and better morphology (Cayli et al., 2004). Some studies found that
405
the HBA can predict the ability of sperm to fertilize oocytes in vitro and is helpful in distinguishing
semen samples suitable for IVF or ICSI (Pregl Breznik et al.). Since, to our knowledge, there is no
published report of PureSperm® used with centrifugation of buffalo sperm, the present study was
performed to compare Percoll® or PureSperm® for sperm selection.

Semen source
Frozen sperm from 5 buffalo bulls was procured from a commercial facility in Thailand and
kept at -196C until required for use. The sperm was thawed at 37ºC in a water bath for 30 sec and
assigned to separation by one of 2 colloidal substances. The comparisons were carried out using a total
of 25 sperm samples (five per animal).
Sperm selection
Each thawed sperm sample was assigned to separation by 1) layering on top of two layers of
Percoll density gradient in a 15 ml plastic conical centrifuge tube (45 and 90%). The tube was
centrifuged at 800 × g for 15 min after which the supernatant was removed, leaving the sperm pellet.
The sperm pellet was washed using 1 ml Tyrode’s albumin lactate pyruvate (TALP) and centrifuged
again at 800 × g for 5 min. The supernatant was removed leaving 100 µl of sperm suspension in the
tube, which was then used for semen evaluation. 2) the sperm was layered on top of single layer of
PureSperm® and thereafter treated as for Percoll.
Sperm Evaluation
Sperm motility was investigated under light microscope at × 100 magnification. Eosin-nigrosin
staining was used to assess sperm viability. The sperm morphology was checked by Williams’ solution
(carbol–fuchsin–eosin) and buffered formalin solution (Koonjaenak et al., 2007). The hypo-osmotic
sperm swelling test (HOST) was used to evaluate the sperm membrane integrity (Imam et al., 2010).
The integrity of sperm acrosomes was evaluated using FITC-PNA staining (Chankitisakul et al., 2012;
Cheng et al., 1996).
Hyaluronan binding assay (HBA)
The percentage of sperm with the ability to bind to hyaluronan (HBA; Biocoat) was determined
following the manufacturer's instructions. Ten microliters of thawed semen was added to the HBA
chamber, and the Cell-Vu grid cover slip was attached. The cover slip provided a grid of 100 squares
within a viewing circle. After incubating the slide for 15 minutes, unbound motile sperm were counted
and the bound motile spermatozoa, and at least 200 spermatozoa in the same square or the entire 100
squares were counted. The percentage of spermatozoa binding to the hyaluronan was calculated by
dividing the number of motile bound spermatozoa by the sum of motile bound and unbound
spermatozoa (Pregl Breznik et al.).
All comparisons were analysed using SAS 9.2 (SAS®, NC., USA) and are presented as
mean±SEM. The sperm parameters were tested for normal distribution of residuals from the statistical
models using the UNIVARIATE procedure with the NORMAL option and compared between groups
of treatment by t-test. P≤ 0.05 was defined as statistically significant.

Sperm motility, vitality and morphology improved (P < 0.001) after the selection process with
no differences between methods. Percoll® gradient increased the mean (± SEM) percentage of sperm
recovered (8.27±0.79) compared to PureSperm® (5.25 ± 1.41; P = 0.007). Sperm separation using
PureSperm® compared with Percoll® resulted in a considerable variation among many studies for
recovery rate, motility, viability and normal sperm morphology. Some previous studies did not find
differences in the recovery rate between Percoll ® and PureSperm® (Claassens et al., 1998; Söderlund
and Lundin, 2000). However, in the present study membrane integrity (61.4 ± 1.3 vs. 56.8 ± 1.3) and
406
acrosome integrity (59.8 ± 1.4 vs. 55.3 ± 0.6) were higher after separation on PureSperm® compared to
Percoll® (P = 0.03; Table 1). Percoll® adheres to the sperm plasma membranes (Pickering et al., 1989)
and may change them by removing coating envelopes (Tanphaichitr et al., 1988). Therefore, intensive
washing of the spermatozoa after sperm separation with Percoll® was recommended. This requires
additional centrifugation which, in turn, can be detrimental to sperm because of the action of reactive
oxygen species (Aitken and Clarkson, 1988). PureSperm® was used instead of Percoll® because it has
less deleterious effects on sperm membranes (Strehler et al., 1998). PureSperm® was reported to give
higher motility than Percoll® (Chiamchanya et al., 2010). However, PureSperm® did not result in better
sperm motility in the present study. PureSperm® for sperm preparation has been reported to result in
higher motility and morphologically normal sperm than Percoll®. The percentage of HBA score was
higher when semen was selected by PureSperm® than by Percoll® (49.5 ± 1.6 vs. 40.0 ± 1.1; Table 1; P
= 0.02). In conclusion, separation using PureSperm® resulted in a lower sperm recovery compared to
Percoll® separation. In contrast, PureSperm® was more effective for selecting high quality sperm for in
vitro fertilization embryo production in the buffalo species.
REFERENCES
Aitken, R.J. and J.S. Clarkson. 1988. Significance of reactive oxygen species and antioxidants in
defining the efficacy of sperm preparation techniques. J. Androl. 9: 367-376.
Andersen, C.Y. and J. Grinsted. 1997. A new method for the purification of human motile spermatozoa
applying density-gradient centrifugation: polysucrose media compared to Percoll media. J.
Assist. Reprod. Genet. 14: 624-628.
Carrell, D.T., P.H. Kuneck, C.M. Peterson, H.H. Hatasaka, K.P. Jones and B.F. Campbell. 1998. A
randomized, prospective analysis of five sperm preparation techniques before intrauterine
insemination of husband sperm. Fertil. Steril. 69: 122-126.
Cayli, S., A. Jakab, L. Ovari, E. Delpiano, C. Celik-Ozenci, D. Sakkas, D. Ward and G. Huszar. 2003.
Biochemical markers of sperm function: male fertility and sperm selection for ICSI. Reprod.
BioMedicine. Online. 7: 462-468.
Cayli, S., D. Sakkas, L. Vigue, R. Demir and G. Huszar. 2004. Cellular maturity and apoptosis in
human sperm: creatine kinase, caspase‐3 and Bcl‐XL levels in mature and diminished maturity
sperm. Mol. Hum. Reprod. 10: 365-372.
Chankitisakul, V., N. Am-In, T.Tharasanit, T. Somfai, T. Nagai and Techakumphu, M. 2012. Sperm
Pretreatment with Dithiothreitol Increases Male Pronucleus Formation Rates After
Intracytoplasmic Sperm Injection (ICSI) in Swamp Buffalo Oocytes. J. Reprod. Dev. Inpress.
Cheng, F.P., A. Fazeli, W.F. Voorhout, A. Marks, M.M. Bevers and B. Colenbrander. 1996. Use of
peanut agglutinin to assess the acrosomal status and the zona pellucida-induced acrosome
reaction in stallion spermatozoa. J. Androl. 17: 674-682.
Chiamchanya, C., N. Kaewnoonual, N. Visutakul, S. Manochantr and J. Chaiya. 2010. Comparative
study of the effects of three semen preparation media on semen analysis, DNA damage and
protamine deficiency, and the correlation between DNA integrity and sperm parameters. Asian
J Androl. 12: 271-277.
Claassens, O.E., R. Menkveld and K.L. Harrison. 1998. Evaluation of three substitutes for Percoll in
sperm isolation by density gradient centrifugation. Hum. Reprod. 13: 3139-3143.
De Vos, A., Z.P. Nagy, H. Van de Velde, H. Joris, G. Bocken and A. Van Steirteghem. 1997. Percoll
gradient centrifugation can be omitted in sperm preparation for intracytoplasmic sperm
injection. Hum. Reprod. 12: 1980-1984.
Imam, S., M.R. Ansari, A. Kumar, C. Singh, V.K. Bharti and A. Kumaresan. 2010. Effect of Oviductal
Proteins on Structural and Functional Characteristics of Cryopreserved Sperm in Murrah
Buffaloes*. Reprod. Domest. Anim. 45: 302-306.
407
Koonjaenak, S., V. Chanatinart, H. Ekwall and H. Rodriguez-Martinez. 2007. Morphological Features

of Spermatozoa of Swamp Buffalo AI Bulls in Thailand. J. Vet. Med. Series. A. 54: 169-178.
Mehmood, A., M. Anwar and S.M.S. Naqvi. 2009. Motility, acrosome integrity, membrane integrity
and oocyte cleavage rate of sperm separated by swim-up or Percoll gradient method from
frozen–thawed buffalo semen. Anim. Reprod. Sci. 111: 141-148.
Mousset-Simeon, N., N. Rives, L. Masse, F. Chevallier and B. Mace. 2004. Comparison of Six Density
Gradient Media for Selection of Cryopreserved Donor Spermatozoa. J. Androl. 25: 881-884.
Pickering, S.J., T.P. Fleming, P.R. Braude, V.N. Bolton and G.A. Gresham. 1989. Are human
spermatozoa separated on a Percoll density gradient safe for therapeutic use? Fertil. Steril. 51:
1024-1029.
Pregl Breznik, B., B. Kovačič and V. Vlaisavljević, Are sperm DNA fragmentation, hyperactivation,
and hyaluronan-binding ability predictive for fertilization and embryo development in in vitro
fertilization and intracytoplasmic sperm injection? Fertil. Steril. Inpress.
Sakkas, D., G.C. Manicardi, M. Tomlinson, M. Mandrioli, D. Bizzaro, P.G. Bianchi and U. Bianchi,
2000. The use of two density gradient centrifugation techniques and the swim-up method to
separate spermatozoa with chromatin and nuclear DNA anomalies. Hum Reprod. 15:1112-1116.
Scott, L. and S. Smith. 1997. Mouse in vitro fertilization, embryo development and viability, and
human sperm motility in substances used for human sperm preparation for assisted
reproduction. Fertil. Steril. 67: 372-381.
Simeon, N.M., N. Rives, L. Masse, F. Cherallier and B. Mace. 2004. Comparison of six density
gradient media for selection of cryopreserved donor spermatozoa. J Androl. 25: 881–884.
Söderlund, B. and K. Lundin, 2000. The use of silane-coated silica particles for density gradient
centrifugation in in-vitro fertilization. Hum. Reprod. 15: 857-860.
Srisombut, C., M. Morshedi, M.H. Lin, A. Nassar and S. Oehninger. 1998. Comparison of various
methods of processing human cryopreserved-thawed semen samples. Hum. Reprod. 13: 2151-
2157.
Strehler, E., B. Baccetti, K. Sterzik, S. Capitani, G. Collodel, M. De Santo, L. Gambera and P.
Piomboni. 1998. Detrimental effects of polyvinylpyrrolidone on the ultrastructure of
spermatozoa (Notulae seminologicae 13). Hum. Reprod. 13: 120-123.
Tanphaichitr, N., C.F. Millette, A. Agulnick and L.M. Fitzgerald. 1988. Egg-penetration ability and
structural properties of human sperm prepared by Percoll-gradient centrifugation. Gamete. Res.
20: 67-81.
Table 1. Thawed sperm parameters following separation by Percoll® or PureSperm® (mean±SEM)
After centrifugation by
Semen parameters (%) Thawed semen
Percoll® PureSperm®
Motility 38.0±3.4 a 86.0±2.1 b 84.0±3.6 b
Viability 45.5±2.4 a 88.4±2.9 b 85.4±1.5 b
Normal morphology 75.5±4.2a 93.6±1.7 b
95.7±1.0 b
Normal acrosome 40.4±2.2 a 55.3±0.6 b 59.8±1.4 c
Normal plasma membrane 34.8±1.3 a 56.8±1.3 b
61.4±1.3 c
Recovery of motile sperm - 8.3±0.8a 5.3 ± 1.4b
HBA scores 20.0 ± 2.6 a 40.0 ± 1.1 b 49.5 ± 1.6 c
a,b,c
Rows with different superscripts differ P ≤ 0.05.
408
Effect of Percoll® Density, Duration and Force of Centrifugation on Sperm

Motility, Morphology, Acrosome Integrity, Membrane Integrity and Sperm
Recovery Rate of Frozen-Thawed Buffalo Semen
Vibuntita CHANKITISAKULa, Nutthee AM-INb* and Mongkol TECHAKUMPHUb
a
Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002,
Thailand
b
Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science,
Chulalongkorn University, Bangkok 10330, Thailand
*Corresponding email: nutthee.a@chula.ac.th
ABSTRACT
The aim of this study was to evaluate the effect of Percoll® density, duration and force of
centrifugation on sperm quality characteristics and sperm recovery rate in frozen-thawed buffalo
semen. Frozen/thawed semen from five buffalo bulls (n=5) were used in this present study. In
experiment 1, the semen was selected according to one of two protocol: (1) Percoll® discontinuous
gradient (45 and 90%) and Percoll® discontinuous gradient (40 and 80%). Both groups were
centrifuged at 800×g for 15 min. In experiment 2, the semen was selected following the best protocol
of Exp1 except changing the force of centrifugation to 700×g to compare between 800 and 700×g. In
experiment 3, the semen was select following the best protocol of Ex1 and 2 except changing the
duration of centrifugation to 10, 15 and 20 min. The results of Ex1 showed the higher sperm recovery
rate of 45 and 90% Percoll® than 40 and 80% Percoll® (p≤0.05). The centrifugation at 800×g has a
higher sperm recovery rate than 700×g (p≤0.05).There is no effect on sperm motility, morphology,
acrosome integrity membrane integrity between groups both in Ex1 and 2. In experiment 3, there is no
effect on sperm recovery rate, sperm motility, morphology, acrosome integrity and membrane integrity
between 15 and 20 min of duration. However, the 10 min of duration has a lower sperm recovery rate
than 15 and 20 min of duration (p≤0.05). In conclusion, the Percoll® density, duration and force of
centrifugation can affect on sperm recovery rate but have no effect on sperm motility, morphology,
acrosome integrity and membrane integrity in this present study.
Keywords: buffalo semen, Percoll®, sperm recovery rate, centrifugation
INTRODUCTION
Swamp buffaloes in Thailand are becoming the extinct species. Many people used many
methods to conserve them. In vitro embryo production (IVEP) technique is a choice which is applied to
preserve genetics (Chankitisakul et al., 2012a,b; Chankitisakul et al., 2010; Liang et al., 2012; Pandey et
al., 2010). In vitro fertilization (IVF) needs the highly motile sperm to produce the high quality of
embryos (Mehmood et al., 2009; Silva and Gadella, 2006). One technique using to select the motile
sperm from thawing semen is Percoll® gradient technique (Machado et al., 2009; Mehmood et al.,
2009). This technique was used in many experiments to prepare sperm for IVF in buffaloes. However,
vary forces and durations of centrifugation were used and no comparison of them. The success of IVF
requires a sperm which have a good motility and the intact acrosome up to time it fertilize with oocyte.
Therefore, sperm which can fertilize oocyte have to be a good motility, intact acrosome, membrane and
penetration. Selecting viable population by sperm separation methods could also be informative to
predict in vitro or in vivo fertilizing ability of sperm. This present study was conducted to compare the
efficacy of Percoll® density, duration and force of centrifugation on sperm motility, morphology,
acrosome integrity membrane integrity and sperm recovery rate of frozen-thawed buffalo semen.
409
MATERIAL AND METHODS

Semen source
kept in -196 C until use in this experiment. The semen was thawed at 37 ºC in a water bath for 30 sec and
Frozen semen of five buffalo bulls was procured from semen commercial unit in Thailand and
subjected into each experiment.

Sperm Evaluation
The sperm motility was investigated under light microscope × 100 magnification. Eosin-
nigrosin staining was used to assess buffalo spermatozoa viability. The sperm morphology was checked
by Williams solution (carbol–fuchsin–eosin) and buffered formalin solution (Koonjaenak et al., 2007).
The hypo-osmotic sperm swelling test (HOST) was used to evaluate the sperm membrane integrity
(Imam et al., 2010). The integrity of sperm acrosomes was evaluated using FITC-PNA staining
(Chankitisakul et al., 2012a; Cheng et al., 1996). Percent recovery of motile sperm was calculated as
(Mehmood et al., 2009):
(Concentrationfinal×volumefinal×motilityfinal)
Recovery of motile sperm (%) = × 100
(Concentrationinitial×volumeinitial×motilityinitial)
Experimental design
Experiment 1:
The thawed semen was then layered on top of two layers of Percoll® density gradient in
a 15 ml plastic conical centrifuge tube according to one of two protocol: (1) Percoll® discontinuous
gradient (45 and 90%) and Percoll® discontinuous gradient (40 and 80%). The tube was then
centrifuged at 800 × g for 15 min, after which the supernatant was removed, leaving only the sperm
pellet. The sperm pellet was washed using 1 ml Tyrode’s albumin lactate pyruvate (TALP) and
centrifuging at 800 × g for 5 min. The supernatant was removed leaving 100 µl containing the sperm
suspension in the tube, which was then used for semen evaluation
Experiment 2:
The semen was selected following the best protocol of Exp1 except changing the force
of centrifugation to 700×g to compare between 800 and 700×g.
Experiment 3:
The semen was select following the best protocol of Ex1 and 2 except changing the
duration of centrifugation to 10, 15 and 20 min.
All parameter were present as mean±S.D. The sperm parameter were tested for normal
distribution of residuals from the statistical models using the UNIVARIATE procedure with the
NORMAL option and compared between groups of treatment by t-test in Ex 1 and 2 and among groups
of treatment by ANOVA in Ex 3. P≤ 0.05 was defined as statistically significant.

The results of Ex1 (Table 1) showed the higher sperm recovery rate of 45 and 90% Percoll®
than 40 and 80% Percoll® (8.27±0.79 vs. 5.63±0.66; P=0.04). In human, the 40 and 80% Percoll®
density was used to select the sperm in IVF and show the lower motile sperm recovery rate than this
present study show (Hossain et al., 1996). The centrifugation at 800×g has a higher sperm recovery rate
than 700×g (8.27±0.79 vs. 5.87±0.59; P=0.009). The increasing force of centrifugation to 800×g can
improve the recovery motile sperm agreed with previous study presented lower percentage of recovery
motile sperm than this present study (Mehmood et al., 2009).However, the different Percoll® density
gradient has no effect on sperm motility, viability, morphology, acrosome integrity membrane integrity.
The duration and force of centrifugation have no effect on sperm motility, viability, morphology,
410
acrosome integrity and membrane integrity. However, the 10 min of duration has a lower sperm
recovery rate than 15 and 20 min of duration (P=0.02). The shorter duration of centrifugation used in
bovine sperm could have reduced the percentage of cells recovered after Percoll® centrifugation but it
can be compensated by the higher centrifugal force (Machado et al., 2009). In this present study, there
were no significant differences among treatments for all sperm end points evaluated except the
percentage of sperm recovered. It has also been demonstrated that pass semen through a Percoll®
gradient can damage the plasma membrane and cause a premature acrosome reaction (Cesari et al.,
2006), due to membrane destabilization. Percoll® volumes, force and duration of centrifugation did not
significantly affect sperm quality. That bovine and equine sperm were somewhat less sensitive to
centrifugation than sperm from other mammals (Silva and Gadella, 2006) may have accounted for the
apparent lack of damage. In conclusion, the Percoll® density, duration and force of centrifugation can
affect on sperm recovery rate but have no effect on sperm motility, morphology, acrosome integrity
and membrane integrity in this present study.
REFERENCES
Cesari, A., G.G. Kaiser, N. Mucci, A. Mutto, A. Vincenti, M.W. Fornés and R.H. Alberio. 2006.
Integrated morphophysiological assessment of two methods for sperm selection in bovine
embryo production in vitro. Theriogenology 66: 1185-1193.
Chankitisakul, V., N. Am-In, T. Tharasanit, T. Somfai, T. Nagai and M. Techakumphu. 2012a. Sperm
pretreatment with dithiothreitol increases male pronucleus formation rates after
intracytoplasmic sperm injection (ICSI) in swamp buffalo oocytes. J. Reprod. Dev. (In press).
Chankitisakul, V., T. Tharasanit, N. Phutikanit, K. Tasripoo, T. Nagai and M. Techakumphu. 2012b.
Lacking expression of paternally-expressed gene confirms the failure of syngamy after
intracytoplasmic sperm injection in swamp buffalo (Bubalus bubalis) Theriogenology 77: 1415-
1424.
Chankitisakul, V., T. Tharasanit, K. Tasripoo and M. Techakumphu. 2010. Chronological
reorganization of microtubules, actin microfilaments, and chromatin during the first cell cycle
in swamp buffalo (Bubalus bubalis) embryos. Vet. Med. Int. 2010: doi: 10.4061/2010/382989.
Cheng, F.P., A. Fazeli, W.F. Voorhout, A. Marks, M.M. Bevers and B. Colenbrander. 1996. Use of
peanut agglutinin to assess the acrosomal status and the zona pellucida-induced acrosome
reaction in stallion spermatozoa. J. Androl. 17: 674-682.
Hossain, A.M., C. Huff and B.Rizk. 1996. Fractions of Percoll eliminated sperm: increased sperm yield
in percoll wash. Arch. Androl. 37: 189-195.
Imam, S., M.R. Ansari, A. Kumar, C. Singh, V.K. Bharti and A. Kumaresan. 2010. Effect of oviductal
proteins on structural and functional characteristics of cryopreserved sperm in Murrah
buffaloes. Reprod. Domest. Anim. 45: 302-306.
Koonjaenak, S., V. Chanatinart, H. Ekwall and H. Rodriguez-Martinez. 2007. Morphological features
of spermatozoa of swamp buffalo AI bulls in Thailand. J. Vet. Med. Series A. 54: 169-178.
Liang, Y.Y., K. Srirattana, T. Phermthai, T. Somfai, T. Nagai and R. Parnpai. 2012. Effects of
vitrification cryoprotectant treatment and cooling method on the viability and development of
buffalo oocytes after intracytoplasmic sperm injection. Cryobiology. 65: 151-156.
Machado, G.M., J.O. Carvalho, E.S. Filho, E.S. Caixeta, M.M. Franco and R. Rumpf, M.A.N. Dode.
2009. Effect of Percoll volume, duration and force of centrifugation, on in vitro production and
sex ratio of bovine embryos. Theriogenology 71: 1289-1297.
Mehmood, A., M. Anwar and S.M.S. Naqvi, 2009. Motility, acrosome integrity, membrane integrity
and oocyte cleavage rate of sperm separated by swim-up or Percoll gradient method from
frozen–thawed buffalo semen. Anim. Reprod. Sci. 111: 141-148.
411
Pandey, A., S.C. Gupta, N. Singh, J.S. Rana and N. Gupta. 2010. Efficiency of SCNT buffalo (Bubalus
bubalis) embryos in different culture medium and analysis of mRNA expression of insulin-like
growth factors during embryogenesis. Reprod. Domest. Anim. 45: 786-795.
Silva, P.F.N. and B.M. Gadella, 2006. Detection of damage in mammalian sperm cells. Theriogenology
65: 958-978.
Table 1. Semen parameters compared between two types of Percoll® density of sperm separated by
centrifugation method (mean±S.D.).
Percoll density
Semen parameters (%)
45 and 90% 40 and 80%
Motility 86.00±5.48 a 88.00±4.47 a
Viability 86.40±2.88 a 84.40±1.82 a
Normal acrosome 78.20±4.44 a 81.00±3.94 a
Normal plasma membrane 63.80±3.35 a 64.20±3.96 a
Normal mophology 93.63±4.35a 92.51±3.95a
Recovery motile sperm 8.27±0.79 a 5.63±0.66 b
a,b
Rows with different superscripts differ p ≤ 0.05.
Table 2. Semen parameters compared between two forces of centrifugation of sperm separated method
(mean±S.D.).
forces of centrifugation
800×g 700×g
Motility 86.00±5.48 a 84.00±8.94 a
Viability 86.40±2.88 a 84.80±1.92 a
Normal acrosome 78.20±4.44 a 82.80±5.22 a
Normal plasma membrane 63.80±3.35 a 63.20±1.92 a
Normal mophology 93.63±4.35a 92.57±2.31a
Recovery motile sperm 8.27±0.79 a 5.87±0.59 b
a,b
Rows with different superscripts differ p ≤ 0.05.
Table 3. Semen parameters compared among three durations of centrifugation of sperm separated
method (mean±S.D.).
durations of centrifugation (min)

10 15 20
a a
Motility 82.00±8.37 86.00±5.48 84.00±8.94 a
Viability 86.20±3.42 a 86.40±2.88 a 83.80±2.17 a
a a
Normal acrosome 78.40±4.16 78.20±4.44 81.00±4.24 a
Normal plasma membrane 62.40±3.65 a 63.80±3.35 a 64.20±2.39 a
a a
Normal mophology 93.71±3.87 93.63±4.35 91.09±3.36a
a b
Recovery motile sperm 5.65±0.62 8.27±0.79 8.63±0.47 b
a,b
Rows with different superscripts differ P ≤ 0.05.
412
Determination of Pregnancy Associated Glycoproteins Levels in

Water Buffalo Cows after Calving
Rafael PAIVAa, Rosaura PEREZb, Simon ZAMBRANOb, Silvia ZIMMERMANa and Christoph
EGLI a
a
IDEXX Laboratories Inc., Westbrook, Maine, USA
b
Centro Diagnostico Veterinario, Mérida-Estado Mérida and Acarigua-Estado Portuguesa, Venezuela
*Correspondind e-mail: Rafael-Paiva@idexx.com; rperezgil@gmail.com;
simonerbe_12@hotmail.com; Silvia-Oliveirazimmerman@idexx.com; christoph-egli@idexx.com
ABSTRACT
Pregnancy Associated Glycoproteins (PAG) can be detected in serum or plasma by ELISA
technique in cows after day 35 to 40 of calving. PAG have been isolated from water buffalo (WB)
placentas (Barbato O., et al 2008); no study have been done in water buffalo to determine when PAG
levels disappear in blood afetr calving. The aim of this study is to determine the day after calving when
PAG’s are detectable in blood using a commercial ELISA (IDEXX Bovine Pregnancy Test). Forty nine
WB cows with less than 60 days after calving and diagnosed open by transrectal ultrasonography were
sampled. The ELISA test was run as described by the manufacturer. Sample-Negative ratios (S-N)
average for the 49 WB cows were calculated for each day after calving. PAG levels were found in
blood until day 23 after calving. PAG levels in blood until 23 days after calving will not interfere with
PAG from a next pregnancy detection using the commercial ELISA. These findings suggest that
synchronization and embryo transfer programs can be started early in WB cows.
Keywords: ELISA, Pregnancy Associated Glycoprotein, Transrectal Ultrasonography
INTRODUCTION
PAG can be detected in serum, plasma, and milk until day 60 post calving when they return to
baseline this allows the kit to detect a new pregnancy as soon as 60 days after the birth of a calf (3).
The measure of the PAG curve after calving will help in the determination of the earliest day
after calving that the ELISA can be used with no interference with the residual PAG’s after calving. No
studies had being performed in WB cows to determine when PAG’s are detectable in serum, plasma or
milk after calving.
PAG are powerful pregnancy markers in domestic cattle. These proteins are expressed in mono-
and bi-nucleate trophoblast cells from the first days of gestation until calving. In maternal blood and
milk, PAG’s rise to detectable levels from days 24 to 28 after fertilization. (2)
PAG extracted from mid and late placentas in water buffalo cows where isolated and characterized.
Three different types of water buffalo PAG were identified: P85048, P85049, and P85050 and deposit
in the SwissProt data base. (4)
A commercial ELISA was developed to detect PAG in bovine serum or plasma as a marker for
determination of pregnancy in cows. The assay uses an anti-PAG antibody coated onto the solid phase
to bind PAGs that may be present in the sample. A second anti-PAG antibody, coupled with biotin is
used as the detection reagent along with streptavidin-horseradish peroxidase (SA-HRP). TMB substrate
is used as a colorimetric indicator for PAG containing samples, and the enzymatic reaction is stopped
with stop solution. After reading the plate at 450nm, wells with color development above the assay
threshold are considered positive, indicating a pregnant animal, while wells with little or no color
development indicate open animals. (Figure 1). (3)

413
In the water buffalo industry the early pregnancy detection with the use of these techniques is
new, there are no commercial ELISA tests validated to be used in this species.

Forty nine water buffalo cows (Bubalus bubalis), with less than 60 days post calving and
diagnosed open by ultrasound palpation where sampled, blood sample from each animal was collected
just after the ultrasonography.
In the laboratory, the samples were centrifuged to obtain serum, this serum was separated from
the cloth frozen and used to perform the commercial ELISA test (IDEXX Bovine Pregnancy test),
calculations and interpretation as described by the manufacturer in its correspondent insert:
a) Test Protocol
All reagents must be allowed to come to 18–26°C before use. Reagents should be mixed by gentle
swirling. Use a separate pipette tip for each sample.
1. Obtain coated plate(s) and record the sample position.
2. Use only as many micro-well strips as required for the assay; place the unused strips in a sealed bag
with desiccant and return it to 2–8°C.
3. Dispense 25 μL of sample diluent into wells which will be used for samples and controls.
4. Dispense 100 μL of negative control into two wells of the assay plate.
5. Dispense 100 μL of positive control into two wells of the assay plate.
6. Dispense 100 μL of samples into appropriate wells.
7. Gently tap plate to mix.
8. Cover the wells and incubate 60 minutes (± 5 minutes) at 37°C (±2°C). The plates should be tightly
sealed to avoid evaporation.
9. Aspirate liquid contents of all wells into an appropriate waste reservoir.
10. Wash each well 3 to 5 times with approximately 300 μL of washing solution. Aspirate liquid
contents of all wells after each wash. Following the final aspiration, firmly tap residual wash fluid
from each plate onto absorbent material. Avoid plate drying between plate washings and prior to the
addition of the next reagent.
11. Dispense 100 μL of detector solution into each well.
12. Cover the wells and incubate for 30 minutes (± 2 minutes) at 18–26°C.
13. Repeat steps 9 and 10.
14. Dispense 100 μL of conjugate into each well.
17. Dispense 100 μL of TMB substrate N.12 into each well.
18. Incubate for 15 minutes (± 1 minute) at 18–26°C.
19. Dispense 100 μL of stop solution N.3 into each well to stop the reaction. Add the stop solution in
the same order as the Substrate solution was added in step 17.
20. Measure and record the A(450 nm)–A(REF) for samples and controls. The reference wavelength is
A (620 nm-650 nm).
21. Calculate results.
b) Test results
For the assay to be valid, the following specifications must be met. The positive control mean minus
the negative control mean must be greater than or equal to 0.300. In addition, the negative control mean
must be less than or equal to 0.150
c) Interpretation of test results
The pregnancy status of the animal is determined by calculating the S–N (Sample value for each
sample.
1. If the S–N value is less than 0.300, the animal is considered not pregnant (open).
414
2. If the S–N value is equal to or greater than 0.300, the animal is considered pregnant.

The average PAG S/P ratios for the 49 animals obtained in the ELISA were calculated. Table 1.
The PAG’s S/P ratios over 0.300 are considered as positives or pregnant, these levels of PAG’s are
present until day 23 after calving in Figure 2.
CONCLUSIONS
In WB cows, PAG are in blood until day 23 after calving in levels detected by the ELISA.
With this new tool for pregnancy detection breeding programs can be more efficient and open days will
be lowered because pregnancy detection can be done earlier.
The PAG-ELISA can be used by veterinarians to determine early pregnancies as soon as 24 days after
calving when it is too soon after breeding for an accurately palpation.
More studies need to be performed to obtain more data about interference of the PAG in the estrus
cycle after calving.
IMPLICATIONS
The findings in this study could help the reproduction specialists and farmers by the detection
of early pregnancies in Water Buffalo Cows, with a non invasive technique, less stress for the animals,
were Pregnancy Associated Glycoprotein of the previous pregnancy will interfere with the diagnostic.
REFERENCES
Silva E., R. A. Sterry, D. Kolb, N. Mathialagan, M. F. McGrath, J. M. Ballam and P. M. Fricke.
Accuracy of a Pregnancy-Associated Glycoprotein ELISA to Determine Pregnancy Status of
Lactating Dairy Cows Twenty-Seven Days After Timed Artificial Insemination. J. Dairy Sci.
90:4612–4622. doi:10.3168/jds.2007-0276. American Dairy Science Association, 2007.
Gajewski Z., M. Pertajitis, N. Sousa, J. Beckers, B. Pawliński, F. Janett. Pregnancy - associated
glycoproteins as a new diagnostic tool in cattle reproduction. Department of Clinical Sciences,
Faculty of Veterinary Medicine, WULS, Warsaw, Poland. zgajewski@supermidia.pl
Velek1, K., S. Michaud1, K. Boucher1, A. Rice1, L. Plourde1, N. Djuranovic1, C. Egli2, P. Welles1
and V. Leathers1. IDEXX Laboratories Inc., Westbrook, Maine, USA IDEXX Switzerland AG,
Liebefeld-Bern Switzerland. Development of an Early and Accurate ELISA for Detection of
Bovine Pregnancy.
Barbato1, O., N.M. Sousa2, K. Klisch3, E. Clerget2, A. Debenedetti1, V. Barile4, A. Malfatti5 and J.F.
Beckers2. 1 Dpt of Biopathological Veterinary Science, Fac. of Veterinary Medicine,
University of Perugia, Italy 2 Physiology of Animal Reproduction, Fac. of Veterinary Medicine,
University of Liege, Belgium 3 Microscopical Anatomy, Medical School Hannover, Germany.
4 Zootechnic Experimental Institute, Monterotondo, Italy. 5 Dpt of Veterinary Science, Faculty
of Veterinary Medicine, University of Camerino, Italy. Corresponding author: O. Barbato. Dpt
of Biopathological Veterinary Science, Fac. of Veterinary Medicine, University of Perugia. Via
S. Costanzo 4, Perugia, 06126, Italy - Tel. +39 075 585 7640- Fax: +39 075 585 7654 - Email:
barbato@unipg.it. Isolation of pregnancy-associated glycoproteins (PAG) from water buffalo
(Bubalus bubalis) placenta by use of Vicia villosa bound agarose affinity chromatography.
IDEXX Bovine Pregnancy Test Kit inserts. Reference number 06-41169-05. Manufacturer IDEXX
Switzerland AG, Stationsstrasse 12, 3097 liebefeld-Bern, Switzerland. EU-Representative:
IDEXX Europe B.V. NL-2130 EK Hoofddorp.
415
Acid Stop
Read at 450nm-ref
Substrate (TMB)
Incubate 15’ RT
WA SH 3 -4X
WA SH 3 -4X
Detector
(Biotinylated Ab)
Incubate 30’ RT
WA SH 3 -4X
Serum
or EDTA Plasma
(Early PAG)
+ Sample diluent
Incubate 1hr 37°C
Solid Phase (plate)
Anti-PAG Capture Ab
Figure 1. the procedure for the ELISA starts with the addition of the serum or plasma to the plate
coated with anti-PAG antibody, after incubation of the test sample in the coated well, captured PAG is
detected with a PAG-specific antibody (detector solution) and horseradish peroxidase conjugate
(HRPO conjugate). Unbound conjugate is washed away and TMB substrate is added to the wells. Color
development is proportional to the amount of PAG in the sample (5).
Figure 2. Pregnancy Associated Glycoprotein levels 60 day after calving in Water Buffalo cows.
416
Table 1. Average S/P ratio for Pregnancy Associated Glycoprotein after calving in Water buffalo
cows.
DAY S/P Result DAY S/P Result

0 2.418 Positive 27 0.290 Negative
24 0.119 Negative 57 0.027 Negative
26 0.221 Negative 60 0.034 Negative
417
Controlled Breeding and Reproductive Management in Buffaloes – Using EAZI

Breed CIDR
DR. S. S. HIREMATH. DEPUTY DIRECTOR (HRD & TRG)
Karnataka Milk Federation, Training Cetnre, Rayapur, Dharwad580009Karnataka, India.

*Corresponding email: hshivayogi@yahoo.com
ABSTRACT
The profitability of buffalo farming mainly depends upon their regular and efficient
breeding. Buffalo reproduction is considerably affected on account of late maturity, poor estrus,
symptoms and long post partum intervals. The present study was undertaken with the objective of
buffaloes to evaluate the efficiency of EAZI Breed CIDR – an intra vaginal progesterone release
device at a controlled rate into the blood stream of buffaloes in relation to estrus and fertility. For
the study purpose 500 buffalo cows of age group 4-6 years in 10 villages of Dharwad in Karnataka
state in India were randomly selected. True anestus buffaloes were inserted the intra vaginal device
EAZI Breed CIDR and remained in the vaginal cavity for 9 days. On 10th day CIDR was removed
and the 2 ml of cidirol(1 mg estradiol benzoate) intramuscular injection was given to all animals.
Ninety percent of the animals showed the pronounced estrus symptoms and they were inseminated
at mid heat period. Those buffaloes which failed to show estrus were again inserted the device and
inseminated after the expression of estrus. After 45-60 days animals are inseminated by AI and NS,
later on buffaloes were examined per rectally for pregnancy and observed that 80-85 percent were
pregnant and again after 90 days buffaloes were re-examined for reconfirmation of pregnancy.
Present study revealed that the use of EAZI Breed CIDR device in buffaloes could be one of the
possible ways to improve anoestrus reproductive performance, thus helping in profitable dairy
farming with buffaloes.
Keywords: Buffalo cows, Anoestrus, Progesterone, EAZI Breed CIDR, Intra vaginal device,
Cidirol injection
INTRODUCTION
In India, buffaloes contribute to food security through 60 million tones of milk and more
than 1 million tones of meat, besides work energy for agricultural purposes. They are found in
widely differing geographical conditions, which suggests that this species is adaptable to wide range
of environmental conditions. Buffalo reproduction is considerably affected on account of late
maturity, poor estrus, symptoms, and long post partum intervals. Besides genetic makeup, several
factors such as nutrition, management, environment, physiology, pathology and psychology affects
the conception rate in buffaloes both in farm and field conditions and pose serious threat to
profitable dairy farming. Ideally, buffalo cow should produce a calf once in 14-16 months. The
profitability of buffalo farming mainly depends upon their regular and efficient breeding.
Anoestrus due to ovarian inactivity is considered to be most important cause for lowered
fertility in buffaloes and is responsible for tremendous economic losses to the farmers by decreasing
milk yield besides the number calves produced in her lifetime. In true anestrus condition both
ovaries are small, smooth, and inactive with the absence of corpus luteum and characterized by
cessation of sexual cycle and psychic manifestation of estrus, this condition ismore in buffalo as
compared to cows (Tanwar et al. 2003). Chede (1990) reported the pattern of estrus, estrus behavior
and estrus synchronization in buffaloes with varying success. Agrawal, etal. (1999) reported
progesterone profile in anoestrus cattle using a systemic progesterone regimen-Crestar. Kathiresan
et al. (1995) observed the influence of ovarian status and lactational stress on induction of estrus
through norgestomet treatment in buffaloes. Totewad et al. (2009) reported induction of oestrus
418
using Cloprostenol by Intra vulvo-sub mucosal route in sub-oestrus buffaloes. Patel et al. (2003)
reported induction of estrus in buffaloes using norgestromet ear implant. Hormonal treatment like,
estrogen, progesterone 75 and GnRH alone or in combination has been tried with variable success
(Rao and Rao, 1984, Rao, et al. 1985, Hafez, 2000, Singh et al. 2004). Under this background, the
present study was undertaken on true anoestrus buffaloes to see the efficiency of eazi breed cidr
intra-vaginal progesterone release device at a controlled rate into the blood stream of animals in
relation to estrus and fertility.

The present study was conducted at Dharwad milk shed area. Dharwad district is situated in
the Western sector of the northern half of Karnataka State. The District encompasses an area of
4263 sq. kms lying between the latitudinal parallels of 15002’ and 15084 51’ North and longitudes
of 73043’ and 75085 35’ East. A baseline survey was conducted and nutritional and reproductive
conditions of each buffalo cow was examined and recorded. The buffaloes which did not show
estrus symptoms for 8-10 months after calving were examined per rectally and inactive ovaries with
no cyclic activity were considered as true anoestrus. 500 anoestrus buffaloes aged between 4 to 6
years belongings to 10 different villages with 45-55 buffaloes from each village were selected as
experimental animals for the study. These animals were inserted eazi breed cidr (1.9 gms of natural
hormone progesterone) device were easily administered using the specially designed applicator. The
wings of the device fold inward and were held in the folded position by the applicator. Squeezing of
the plunger handle then expelled the device when in the correct position in the anterior of the
vagina. When in place, the tail of the device protrudes from the valve lip.This device remained in
the vaginal cavity for 9 days. In some animals second insertion was required due to incorrect
positioning in the vagina or other animals in the group / cow boys pulling on the tail of the device
and thereby removing them. The intra-vaginal progesterone release devices deliver 98 progesterone
at a controlled rate into the blood stream of the animals. This progesterone is released by diffusion
from a silicon rubber easterner molded over a nylon spine which is shaped to retain the device in the
vaginal cavity. Removal at the end of the insertion period was easily achieved by gently but firmly
pulling on the tail and the estrus symptoms were observed. Twenty-four hrs after the removal of
device, all the animals were injected with 2 ml cidirol (1mg estradiol benzoate) and the pronounced
external estrus symptoms were more pronounced. Estrus symptoms were detected both by
external symptoms and per rectal examination. Buffaloes showing estrus symptoms were
inseminated during mid heat using fresh semen brought from KMF Nandini sperm station at
Hessaraghatta, Bangalore. Estrus was detected by observing behavioral symptoms and confirmed
by rectal examination of gentile. Estrus intensity was recorded as intense (standing discharge and
mucus flow), intermediate and weak. All the inseminated buffaloes were examined per rectally on
12th and 15th 110 day post artificial insemination, for the presence of corpus luteum. Pregnancy was
confirmed by per-rectal examination on 50th to 60th day after artificial Insemination while in
doubtful cases reexamined on 90th 112 day. Those buffaloes which failed to show estrus symptoms
were repeated again with the same treatment.

On the removal of implants, different external estrus symptoms were observed (Table 1).
Majority of the animals showed hyperemia of vagina and frequent urination. Per cent of animals
which showed different estrus symptoms are depicted in Table 1. Normally buffaloes does not
exhibit pronounced external estrus symptoms, however, after the injection of cidirol (2ml), the
external symptoms of bellowing, free flowing mucus, frequent 120 urination and hyperemia of
vagina got pronounced (Figure 1).
After the removal of the device and 2 ml of injection of cidirol, all the animals exhibited
estrus with duration of 36 to 48 hrs in intense estrus 67.8 %, intermediate estrus 25.8 % and weak
6.4 % as shown in table 2, with highest conception rate 85.16 %, 60.46 % and 40.75 % (table 3)
respectively in intense, intermediate & weak estrus.
419
The better estrus symptoms and fertility could be attributed to the eazi breed cidr cattle
device and cidirol injection which stimulated the follicular development and ovulation. The present
study findings are in agreement with the earlier reports of Chede (1990) and Nayak et al. (2009)
who observed 38.70 % and 32.25 % buffaloes in intense and intermediate estrus.
The present study showed that buffaloes which are having problem of true anoestrus could
be easily treated by the use of eazi breed cidr device which will improve the reproductive efficiency
in buffaloes and economical conditions of the rural dairy farmers. Controlled breeding under
scientific managemental conditions could be one of the possible way to improve reproductive
performance of buffaloes.
ACKNOWLEDGEMENTS
The authors are thankful to Dr. Peter Jellinek, International Livestock Breeding Consultant,
Australia, Dr. A. S. Premanath, Managing Director KMF Bangalore, Dr. Dayanand, Director (AH),
KMF, Bangalore and Dr Bernad Earnest (Additional Director) KMF, Bangalore, Dr. D.N.Hegde,
(Additional Director) KMF, Bangalore, Sri. Suresh Kulkarni (Additional Director) KMF,
Bangalore, for all the help extended for the study.
REFERENCES
Agrawal, S.K., U. Shankar, U. Kumar and G. Mohan. 1999. Studies on induction of ovarian
cyclicity and progesterone profile in anoestrus cattle using a systemic progesterone regimen-
Crestar. XVth 146 ISSAR Convention Compendium, 21-22. Chede, S.A. 1990.
Pattern of estrus, estrus behavior and synchronization in buffaloes. Ph. D. Thesis, Punjabrao Krishi
Vidyapeeth, Akola, India. Hafez, E.S.E. 2000.
Reproduction in Farm Animals, 7th 149 ed., Lippincott Williams and Wilking Co., Philadelphia.
Kathiresan, D. D. Ezekial Napolean, J. Antonie, L. Dowson and S.R. Pattabiraman. 1995.
Influence of ovarian status and lactational stress on the effect of norgestomet, treatment of
buffaloes. The Blue Cross Book, 4: 25.
Patel, D.M., N.P. Sarvaiya, A.V. Patel and A.P. Parmar. 2003. Induction of estrus and hormonal
profile in buffalo treated with norgestromet ear implant. Indian J. Anim. Reprod. 24(1): 67-
68.
Rao, A.R. and V.S. Rao. 1984. Improved conception rate in buffaloes after administration of
receptal. Indian Vet. J. 61: 813.
Rao, A.V.N., O. Srimanarayana and K.P. Rao. 1985. Estrus response and fertility in post partum
anoestrus buffaloes treated with progesterone, pregnant mare serum gonadatrophin and
prostaglandin during the low breeding season. Anim. Reprod. Sci. 8: 129-135.
Singh, A., M.S. Saxena and S. Prasad. 2004. Efficacy of Crestar and its combination with folligon
on postpartum anestrus buffaloes. Indian Journal of Animal Research 25(1): 43-49.
Tanwar, P.S., N.K. Rakha and J.B. Phogat. 2003. Challenges in buffalo infertility. Intas Polivet.
4(11): 121-127.
Totewad, G.D., Dhoble, R.L., Sawale, A.G., Naik, P.M. 165 and Ambore, M. N. 2009. Induction of
Oestrus using Cloprostenol by Intra vulvo-sub mucosal route in Sub-Oestrus Buffalo.
Veterinary 7 World 2(10): 381-382.
420
Hyperemia of Bellowed , 68
vagina , 86
Free flowing
mucus , 76
Frequent
urination , 83
Figure 1. Percent of animals showing 1 the particular estrus symptom after the injection of cidriol
2.
Table 1. Classification of buffaloes based 14 on the estrus symptoms in different villages.
Sl.No Name of the village No of animals Animals showed estrus symptoms
Intense Intermediate weak

1 Byhatti 52 37 13 02
2 Hebbasur 46 28 16 02
3 Kusgal 48 30 15 03
4 Lokur 45 32 12 01
5 Mugali 53 34 15 04
6 Madhanbhavi 56 36 16 04
7 Padesur 54 38 12 04
8 Shirol 54 38 10 06
9 Yadawad 45 32 11 02
10 Yamanoor 47 32 09 06
Total 500 337 129 32
Percentage 67.80 25.80 6.40
Table 2. Per cent of animals showing 1 the particular estrus symptom after the injection of cidriol.
SL. Estrus symptoms Percentage
No
1 Hyperemia of vagina 86
2 Frequent urination 83
3 Free flowing mucus 76
4 Bellowing 68
5 Raised tail 63
6 Excitement/Restlessness 62
7 Reduced feed & water intake 50
8 Milk yield low 40
9 Swollen valve 38
10 Allowing mounting 20
11 Licking other animals 12
12 Mounting on other animals 10
421
Table 3. Conception percentage in buffaloes showing intense, intermediate and weak estrus signs.
Conception percentage
Intense Intermediate Weak
28 07 01
23 08 01
25 08 01
27 07 -
30 08 20
30 12 01
32 10 02
34 06 03
28 07 01
30 05 03
287 78 14
85.16% 60.46% 40.55%
422
A Comparison of in vitro Development of Buffalo (Bubalus bubalis) Embryos
Produced either by Somatic Cell Nuclear Transfer or in vitro Fertilization using
Oocytes Obtained by Different Methods
Chunyan YANG, Chunying PANG, Haiying ZHENG, Fenxiang HUANG, Jianghua SHANG,
Jian WANG , Bingzhuang YANG, Xianwei LIANG*
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of

Agriculture and Guangxi, Buffalo Research Institute, Chinese Academy of Agricultural Sciences,
24-1Yongwu Road, Nanning 530001, P.R. China.
*Corresponding e-mail：liangbri@126.com
ABSTRACT
This study was conducted to investigate the developmental competence and blastocyst formation speed
of three kinds of in vitro produced buffalo embryos: in vitro fertilized (IVF) embryos derived from oocytes
collected either by aspirating of abattoir ovaries (abattoir-IVF) or by ovum pick up (OPU-IVF) matured in
vitro, and somatic cell nuclear transferred (SCNT) embryos. The cleavage rate, blastocyst rate and blastocyst
formation time of these embryos were recorded. The results showed that: (1) the cleavage rates of
abattoir-IVF, OPU-IVF and SCNT embryos were not different form each other (52.5%, 63.4%, 56.4%,
respectively), whereas the blastocyst rate of OPU-IVF embryos was higher than that of SCNT embryos
(36.4% vs 19.8%，p<0.05）; (2) as for abattoir ovaries-IVF embryos, the percentage of blastocysts harvested
on Day 7 (46.8%) was higher than those harvested on Days 6, 8 and 9 (17.7%, 23.0%, 12.5%, respectively ;
p<0.01); as for OPU-IVF embryos, the percentage of blastocysts harvested on Day 7 (44.5%) was higher
than those harvested on Days 6, 8 and 9 (17.6%, 27.8%, 9.7%, respectively ; p<0.01); as for SCNT embryos,
the percentages of blastocysts harvested on Day 6 and Day 7 (49.6% and 31.3%) was higher than those
harvested on Days 5, 8 and 9 (7.4%, 8.5%, 1.7%, respectively ; p<0.01). In conclusion, the developmental
competence of SCNT embryos was poorer than that of OPU-IVF embryos, whereas the abattoir-IVF and
OPU-IVF groups showed similar developmental competence. Furthermore, the blastocyst formation of
SCNT embryos occurs earlier than the IVF embryos.
Keywords: Buffalo, IVF, SCNT, Embryo, Developmental competence
INTRODUCTION
Reproductive efficiency of female buffalos is hampered by the delayed puberty, silent estrus, long
postpartum anoestrus period and calving interval, and low conception rate. Therefore, assisted reproductive
technologies such as IVF and SCNT have been employed to improve the reproductivity of buffalos. The
combination of OPU with IVF allows repeated production of embryos from live donors of particular value.
SCNT holds the promise of bypassing conventional breeding procedures to allow thousands of identical
duplicates of transgenic animals to be created in a single generation. Buffalo calves have also been
successfully cloned by SCNT in recent years. This study was conducted to investigate the cleavage rate,
blastocyst rate, and blastocyst formation time of abattoir-IVF embryos, OPU-IVF embryos and SCNT

423
embryos, in order to facilitate the improvement of method for cytopreservation of these embryos.

Production of IVF embryos
Pluriparous Nili-Ravi and Murrah buffalos were adopted as ovum pick-up (OPU) donors, with OPU
performed as described by Liang et al. (2008). Buffalo ovaries were collected at a local abattoir and
transported to the laboratory (within 3 h) in sterile 0.9% NaCl solution at 30 to 35 ºC. Follicles 2 to 10 mm in
diameter were aspirated using a 21-gauge needle attached to a vacuum pump. The cumulus oocyte
complexes (COCs) recovered by OPU or aspiration of abattoir-derived ovaries were identified and recovered
with the aid of a stereomicroscope. Only COCs with a homogeneous cytoplasm and at least one layer of
compact cumulus cells were selected, and then in vitro maturated in medium (TCM199 + 10% FBS + 5
µg/mL FSH + 10 µg /mL LH + 0.2 mM sodium pyruvate + 1 µg/mL E2 + 50 µM cysteine + 25 ng/mL EGF)
under a humidified atmosphere of 5% CO2 at 39 ºC for 22 to 24 h.
Frozen straws containing semen from Nili-Ravi or Murrah bulls were used for IVF. Following thawing,
swim-up was performed by layering semen at the bottom of a vial containing 3 mL sperm-washed medium
(modified Tyrode’s with 0.3% BSA). After 30 min, the semen suspension was washed by centrifugation at
260 × g for 5 min at room temperature. Thereafter, the sperm pellet was washed again with 3 mL IVF
medium (modified Tyrode’s supplemented with 60 µg/mL heparin and 0.6% BSA) by centrifugation. Sperm
concentration was assessed with a hemocytometer and a final concentration of 2×106 sperm/mL was
prepared.
Matured COCs were washed twice with IVF medium, and then transferred into 30-µL droplets of IVF
medium (10 to 15 COCs per droplet) where final sperm suspension was added with a volume of 10 µL. The
COCs and sperm were then co-cultured for 22 to 24 h.
Production of SCNT embryos
In this experiment, river buffalo ear fibroblast nucleus was fused into swamp buffalo oocyte cytoplasm
derived from abattoir ovaries. SCNT was performed according to the methods reported by Yang et al. (2010).
In vitro culture (IVC) of IVF and SCNT embryos
The IVF-presumptive zygotes and SCNT-reconstructed embryos (in groups of 10 to 20) were
co-cultured in 40-μL droplets of culture medium (CM) with a single layer of granulosa cells under a
humidified atmosphere of 5% CO2 in air at 39 ºC. Approximately half the media was replaced every 2 d.

The developmental competence of IVF and SCNT embryos
The cleavage rates of the two kind of IVF and the SCNT embryos were not significantly different from
each other, while the blastocyst rate of OPU-IVF was significantly higher than that of SCNT embryos
(36.4% vs19.8, p<0.05). (Table 1).
Percentage of IVF and SCNT blastocysts of various days
The percentage of Day 7 (46.8%) abattoir-IVF were significantly higher than those of Day 6 (17.7%),
Day 8 (23.0%) and Day 9 blastocysts (12.5%) (p<0.01). As for OPU-IVF embryos, the percentage of Day 7
(44.5%) blastocysts were higher than those of Day 6 (17.6%), Day 8 (27.8%) and Day 9 blastocysts (9.7%)
(p<0.01). As for SCNT embryos, the percentage of Day 6 (49.6%) and Day 7 (31.3%) blastocysts were
significantly higher than those of days 5, 8 and 9 blastocysts (7.4%, 8.5% and 1.7%, respectively) (p<0.01),
and the percentage of Day 6 blastocysts were also significantly higher than that of Day 7 blastocysts
(p<0.01). (Table 2).
The IVF embryos derived from abattoir ovaries and OPU may differ in developmental competence.
Manjunatha et al. (2008) reported that in vitro embryo development and blastocyst hatching rates following
424
vitrification of buffalo embryos produced from oocytes collected by OPU was significantly higher than those
oocytes collected from slaughterhouse ovaries. On the contrary, previous reports in cattle have not shown a
different developmental competence when the two sources of oocytes were compared (Galli et al., 2001). In
this study, the cleavage and blastocyst rate of IVF embryos derived from OPU appeared to be higher than
those of IVF embryos derived from abattoir ovaries, but the difference was not significant. The lower
blastocyst yield from oocytes derived from slaughterhouse ovaries might be due to heterogeneous quality of
oocytes recovered from the ovaries of slaughtered animals coming from follicles at different stages of growth
and atresia. The ovarian follicles in buffaloes develop in waves, the stages of the estrous cycle, phases of
folliculogenesis, follicular atresia or the presence or absence of a dominant follicle affect the oocyte
developmental competence in vitro (Manjunatha et al., 2008; Neglia et al., 2003). However, in buffaloes,
OPU repeated twice a week resets follicular population with a diameter >2mm to 0, resulting in a new
follicular wave characterized by homogenously sized follicles (Gasparrini et al., 2002). The occurrence of
follicular atresia is highly reduced. The in vitro developmental competence of SCNT embryos was also
lower than that of IVF embryos. This may be attributed to either (1) inappropriate cell cycle of donor cells
(Poehland et al., 2007), (2) suppressed micronucleus formation caused by activation of reconstructed
embryos with 6-DMAP (a protein kinase inhibitor) (Well et al., 1999), (3) or insufficient DNA synthesis in
the unqualified recipient cytoplasm (Alberio et al., 2001).
In this study, the SCNT embryos developed earlier than the IVF embryos; SCNT blastocysts could be
harvested from Day 5. This may be attributed to the breakdown of membrane boundary between the donor
cell and the cytoplast during electrical fusion, which leads to a direct contact of the nucleus of donor cell
with the recipient cytoplasm causing fast development of SCNT reconstructed embryos. However, during
IVF, spermatozoa need a certain time to enter the cytoplasm of the oocyte and to form male pronucleus, and
development of the IVF embryo can only be directed the after fusion with the female pronucleus.
In conclusion, the in vitro developmental competence of OPU-IVF embryos is higher than SCNT
embryos, while SCNT embryos develop earlier than the IVF embryos.
ACKNOWLEDGEMENTS
This research was supported by grants from the National Natural Science Foundation of China
(31160456) and the Natural Science Foundations of China (GuiKeZi0991011 and No. 2011GXSFB018045).
REFERENCES
Alberio, R., V. Zakhartchenko, J. Motlik and E. Wolf. 2001. Mammalian oocyte activation: lessons from
the sperm and implications for nuclear transfer. Int. J. Dev. Biol. 45: 797-809.
Galli, C., G. Crotti, C. Notari, P. Turini, R. Duchi and G. Lazzari. 2001. Embryo production by ovum pick
up from live donors.. Embryo production by ovum pick up from live donors. Theriogenology. 55:
1341–1357.
Gasparrini, A. 2002. In vitro embryo production in buffalo species: state of the art. Theriogenology 57(1):
237-256.
Liang, X.W., Y.Q. Lu, M.T. Chen, X.F. Zhang, S.S. Lu, M. Zhang, C.Y. Pang, F.X. Huang and K.H. Lu.
2008. In vitro embryo production in buffalo using sexed sperm and oocytes from ovum pick up.
Manjunatha, B.M., P.S. Gupta, J.P. Ravindra, M. Devaraj and S. Nandi. 2008. In vitro embryo development
and blastocyst hatching rates following vitrification of river buffalo embryos produced from oocytes
recovered from slaughterhouse ovaries or live animals by ovum pick-up. Anim. Reprod. Sci. 104(2-4):
419-426.
Neglia, G., B. Gasparrini, V. Caracciolo di Brienza, R. Di Palo, G. Campanile and G. Antonio Presicce and L
425
Zicarelli. 2003. Bovine and buffalo in vitro embryo production using oocytes derived from abattoir
ovaries or collected by transvaginal follicle aspiration. Theriogenology 59: 1123-1130.
Poehland, R., F. Al-Rostum, F. Becker, T. Viergutz, R.M. Brunner, W. Kanitz and S. Bhojwani. 2007. Donor
cell lines considerably affect the outcome of somatic nuclear transfer in the case of bovines. J. Reprod.
Dev. 53: 737-748.
Wang, Z.G., W.Wang, S.D.Yu and Z.R.Xu. Effects of different activation protocols on preimplantation
development, apoptosis and ploidy of bovine parthenogenetic embryos. Anim. Reprod. Sci. 105: 292-301.
Wells, D.N., P.M. Misica and H.R. Tervit. 1999. Production of cloned calves following nuclear transfer with
cultured adult mural granulosa cells. Biol. Reprod. 60: 996-1005.
Yang, C.Y., R.C. Li, C.Y. Pang, B.Z. Yang, G.S. Qin, M.T. Chen, X.F. Zhang, F.X. Huang, H.Y. Zheng,
Y.J. Huang and X.W. Liang. 2010. Study on the inter-subspecies nuclear transfer of river buffalo somatic
cell nuclei into swamp buffalo oocyte cytoplasm. Anim. Reprod. Sci. 121:78-83.
Table 1. The developmental competence of IVF and SCNT embryos (n=6).
Embryo Cleavage rate (%) Blastocyst rate (%)

Abattoir -IVF embryos 52.513.9 (134/254) 21.610.1 (50/254)ab
OPU-IVF embryos 63.417.6 (119/186) 36.417.6 (64/186)a
SCNT embryos 56.413.3 (146/269) 19.83.3 (53/269)b
a,b
Within a column, values without common letters differed (P<0.05).
Table 2. Percentage of IVF and SCNT blastocysts of various days.
No. of embryos (%)

Embryo age Abattoir -IVF
OPU-IVF embryos SCNT embryos
embryos
5d 19 (7.411.6)A
6d 107 (17.715.1)AB 39 (17.615.4)abA 113 (49.622.8)B
7d 245 (46.813.5)C 109 (44.515.4) cB 81 (31.320.5)C
8d 119 (23.010.9)A 61 ( 27.815.6) aA 20 (8.515.9)A
9d 61 (12.510.9)B 23 (9.710.9) bA 5 (1.74.3)A
Total 532 (100) 232 (100) 238 (100)
a,b
A,B
426
Optimization of Culture and Cryopreservation of Hand-Made Clone (HMC)
Buffalo (Bubalus bubalis) Embryos
Chunyan YANG, Chunying PANG, Haiying ZHENG, Fenxiang HUANG, Jian WANG ,
Jianghua SHANG, Bingzhuang YANG and Xianwei LIANG*
Key Laboratory of Buffalo Genetics, Breeding and Reproduction technology, Ministry of

ABSTRACT
This study was conducted to optimize the culture and cryopreservation conditions for buffalo
hand-made clone (HMC) embryos. Microdrop (MD), well of the well (WOW), flat surface (FS)
systems were adopted to culture HMC reconstructed embryos in vitro. The derived HMC
blastocysts were vitrified either with 40% EG, 25% EG + 25% DMSO or 20% EG + 20% DMSO +
0.5 M sucrose. Furthermore, the efficiencies of HMC and traditional somatic cell nuclear transfer
(SCNT) were also compared. The results showed that: (1) the cleavage rate of HMC embryos
cultured in WOW was higher than those cultured on FS (70.5% vs 55.8%, p<0.05) and in MD
珙70.5% vs 50.0%, p<0.01); also, the blastocyst rate of HMC embryos derived from WOW system
(40.0%) was higher than those derived from FS (19.8%) and MD (8.3%) systems (p<0.01) ; (2) the
cryosurvival rate of blastocysts vitrified with 20% EG + 20% DMSO + 0.5 M sucrose was higher
than those vitrified with 40% EG (90.9% vs 62.0%, p<0.01); (3) both the fusion and blastocyst rates
of HMC reconstructed embryos were higher than those of SCNT reconstructed embryos (89.1% vs
79.8%, p<0.05; 40.0% vs 19.8%, p<0.01), whereas the cryotolerances of HMC and SCNT
blastocysts were not different from each other (90.9% vs 92.3%). In conclusion, WOW is the most
suitable method for culture of HMC embryos, and vitrification of HMC blastocysts with 20% EG +
20% DMSO + 0.5M sucrose results in a high cryosurvival rate. HMC can be an alternative to
traditional SCNT in buffalo.
Keywords: buffalo, hand-made clone, in vitro culture, cryopreservation
INTRODUCTION
Cloning through traditional somatic cell nuclear transfer (SCNT) requires expensive
micromanipulator and skilled operators. An alternative technology called handmade cloning (HMC)
has been developed in recent years, which enable a simple and efficient production of blastocysts.
HMC has also resulted in cloned offspring in cattle, sheep, horse, mouse, pig and buffalo (Yang et
al., 2011). The culture system is particularly important for the in vitro culture (IVC) of HMC
reconstructed embryos; as for zona-free cloned embryos, the blastomeres of cleaved embryos
should closely contact with each other to form intact individual blastocysts. Several different culture
systems have been adopted for the isolated/individual culture of HMC embryos such as, well of the
well (WOW), agarose well, glass oviduct, microdrops (MD), and flat surface (FS) culture systems.
WOW has been proven to be most effective for IVC of HMC embryos in bovine (Vajta et al., 2000).

427
However, the most suitable culture system for buffalo HMC embryos remained unknown.
Furthermore, the cryopreservation of zona-free HMC blastocysts is crucial for the application of
this technology in practice. Nevertheless, the most efficient cryoprotectant agents (CPA) and
cryopreservation methods for the preservation of buffalo HMC blastocysts also remained unknown.
This study was conducted to optimize the IVC and cryopreservation systems for HMC
embryos. We compared the efficacies of the MD, FS and WOW culture systems for HMC embryos
in terms of blastocyst production. Resultant blastocysts were vitrified in different CPA combinations
to optimize cryopreservation of HMC blastocysts. The efficacies of HMC and traditional SCNT
were also compared.

Preparation of donor cells
Cumulus cells were used as donor cells. Their preparation was performed according to our
previous report (Yang et al., 2010).
Production of HMC embryos
The oocytes were derived from abattoir ovaries. IVM was performed as reported previously [3].
After IVM, denuded oocytes were incubated in 0.2% pronase for 10 min. Zona-free oocytes were
cultured in TCM199 + 20% FBS+ 7.5µg/mL cytochalasin B (CB) for 30 min. The oocytes with
protrusions along the edge of membrane were then transferred into enucleation medium (TCM199+
20% FBS+ 5µg/ml CB) and the partial cytoplasm with the protrusion was cut of.
The fusion of cytoplasts with donor cells was performed according to the report of Shah et al.
(2008) One hour later, the fused reconstructed embryos were activated by exposing to 5µM
ionomycin in CM medium for 5 min and followed by a subsequent incubation in CM supplemented
with 2 mM 6-dimethylaminopurine (6-DMAP) for 3-4 h.
After activation, reconstructed embryos were cultured in the following systems:
(1) WOW: 20 microwells (300µm-400µm wide and 300µm-400µm deep) were made by
unheated darning needle, and filled with 40µL CM and then covered with mineral oil. Each
microwell contained one reconstructed embryo.
(2) MD: 5 µL droplets of CM were made on a plastic dish and covered with mineral oil. A
single reconstructed embryo was cultured in each droplet.
(3) FS: 1.5 mL CM were added to a plastic dish (30 mm) and covered with mineral oil
Production of SCNT embryos
In this experiment, river buffalo cumulus cells and swamp buffalo oocytes were used for SCNT,
SCNT was performed according to the methods reported by Yang et al. (2010).
Vitrification of embryos
The vitrification was performed according to our previous report (Yang et al., 2012).

Effects of different culture systems on in vitro developmental competence of HMC embryos
As shown in Table 1, the cleavage rate of HMC embryos cultured in WOW was significantly
higher than those cultured in MD 70.5% vs. 50.0%, respectively; p<0.01）and on FS (70.5% vs.
55.8%, respectively; p<0.05). Furthermore, the blastocyst rate of HMC embryos cultured in WOW
(40.0%) was significantly higher than those cultured on FS (19.8%) and in MD (8.3%) (P<0.01).
Effects of CPA composition and embryo type on survival of buffalo HMC blastocysts cryopreserved
by vitrification
428
As shown in Table 2, the cryosurvival rate of HMC blastocysts vitrified with 20% EG + 20%
DMSO + 0.5 M sucrose was significantly higher than those vitrified with 40% EG (90.9% vs.
62.0%, respectively; p<0.01), and did not differ statistically from those vitrified with 25% EG + 25%
DMSO (76.4%). When 20% EG + 20% DMSO + 0.5 M sucrose was adopted to vitrify HMC and
SCNT blastocysts, the cryosurvival rate of SCNT blastocysts was not different form that of HMC
blastocysts (92.3% vs. 90.9%, respectively).
Embryo production efficiencies of HMC and SCNT
As shown in Table 3, the fusion rate of HMC reconstructed couplets was significantly higher
than that of SCNT reconstructed couplets (89.1% vs. 79.8%, respectively; p<0.05), the cleavage
rate of HMC embryos was higher than that of SCNT embryos (70.5% vs. 56.4%, respectively).
Furthermore, the blastocyst rate of HMC embryos was significantly higher than that of SCNT
embryos (40.0% vs. 19.8%, respectively; p<0.01).
DISCUSSIONS
Zona enclosed cloned embryos derived from traditional SCNT are generally cultured in 30-100
µL droplets, in which HMC embryos show a compromised developmental competence (Shah et al.,
2008; Vajta et al., 2003; 2005). The FS system is effective to keep trophoblast of blastocysts from
adhesion to the plastic surface; however, zona-free HMC embryos must be cultured individually,
avoiding any shaking to prevent aggregation of embryos. The in vitro development of HMC
embryos to the blastocyst stage cultured in WOW was higher than that of those cultured on FS.
Previously, Shah et al. (2008) found FS to be more efficient than WOW for in vitro development of
HMC buffalo embryos. This discrepancy may be due to the different culture media and conditions
between the two studies.
WOW is a generally preferred and very efficient system for the culture of zona-free embryos
with an approximate blastocyst yield of 50% in cattle. Also, WOW showed the highest blastocyst
yield in this study. The WOW system provides a constant in vitro microenvironment for zona-free
embryos, and it avoids the formation of giant chimeras when direct contact between individual
zona-free embryos occurs. Furthermore, the advantages due to group effect of embryos (such as the
secretion of autocrine and paracrine factors) are maintained in WOW system. Therefore WOW is
suitable for buffalo HMC embryo culture.
The present study examined the effect of different vitrification medium compositions (40% EG,
25% EG + 25% DMSO, 20% EG + 20% DMSO + 0.5 M sucrose) on the post-warming
cryosurvival of buffalo blastocysts. The blastocysts vitrified with 20% EG + 20% DMSO + 0.5 M
sucrose resulted in the highest cryosurvival rate, and the cryosurvival rate of blastocysts vitrified
with 25% EG + 25% DMSO was higher than those vitrified with 40% EG. Over the last decade, EG
had been effectively employed as a CPA for cattle embryo cryopreservation.
The ability of embryos to hatch 48 h post-thawing is improved when two (EG, DMSO) or
three (EG, DMSO and 1,3-butanediol) CPAs were included in the vitrification medium rather than a
single one. EG has minimal toxicity, and has been effectively used as a cryoprotectant for
cryopreservation of buffalo embryos. The combination of DMSO and EG has at least two
advantages: firstly, the permeability of each CPA is enhanced in the presence of the other (Ali et al.,
1993); Secondly, it is assumed to reduce not only the toxicity of each cryoprotectant, but also
osmotic damage at warming, since EG is more likely to diffuse out of the cell rapidly, whereas
DMSO is less permeable (Taniguchi et al., 2007),. Furthermore, previous reports indicated that
addition of sucrose to the vitrification medium could allow the reduction of the optimal
concentration of the permeable CPA and increase the potential of embryos to tolerate it. The results
429
of this study demonstrate that a combination of premeating and non-premeating CPAs in the
vitrification medium will achieve reasonable cryopreservation efficiency. Cryopreservation of
SCNT embryos is more difficult than IVF embryos, which may be due to the broken zona pellucida.
In this study, the cryosurvival rate of HMC blastocysts was similar to SCNT blastocysts, which
might indicate that the zona pellucida is not necessary for cryopreservation (Lagutina et al., 2007).
In this study, the fusion rate of HMC reconstructed couplets was higher than SCNT ones, and
the blastocyst rate of HMC embryos was also higher than SCNT embryos. This result was
consistent with those of Saikhun et al. (2004) and Vajta et al. (2003). Since two demi-cytoplasts
were fused together, the content of one HMC reconstructed couplet could reach approximately the
150 % of a single SCNT reconstructed couplet. Increased cytoplasm volume might contribute to the
high in vitro development ability of HMC embryos. In conclusion, HMC can be an alternative to
traditional SCNT, WOW system was suitable for culture of HMC embryos, and vitrification of
HMC blastocysts with 20% EG + 20% DMSO + 0.5 M sucrose could result in a high cryosurvival
rate.
ACKNOWLEDGEMENTS
(31160456) and the Natural Science Foundations of China under Grant No. 0991011 and No.
2011GXSFB018045.
REFERENCES
Ali, J. and J.N. Shelton. 1993. Design of vitrification solutions for the cryopreservation of embryos.
J. Reprod. Fertil. 99: 471-477.
Lagutina, I., Lazzari, G., Duchi, R., Turini, P., Tessaro, I., Brunetti, D., Colleoni, S., Crotti, G. and
C. Galli. 2007. Comparative aspects of somatic cell nuclear transfer with conventional and
zona-free method in cattle, horse, pig and sheep. Theriogenology 67: 90-98.
Saikhun, J., N. Kitiyanant, C. Songtaveesin, K. Pavasuthipaisit and Y. Kitiyanant. 2004.
Development of swamp buffalo (Bubalus bubalis) embryos after parthenogenetic activation
and nuclear transfer using serum fed or starved fetal fibroblasts. Repord. Nutr. Dev. 44: 65-78.
Shah, R.A., A. George, M.K. Singh, D. Kumar, M.S. Chauhan, R. Manik, P. Palta and S.K. Singla.
2008. Hand-made cloned buffalo (Bubalus bubalis) embryos: comparison of different media
and culture systems.Cloning. Stem. Cells. 10:435-442.
Taniguchi, M., A. Ikeda, E. Arikawa, P. Wongsrikeao, B. Agung, H. Naoi, T. Nagai and T. Otoi.
2007. Effect of cryoprotectant composition on in vitro viability of in vitro fertilized and
cloned bovine embryos following vitrification and instraw dilution. J.Reprod.Dev. 53:963–
969.
Vajta, G., Peura T.T., Holm P., Páldi, A., Greve, T., Trounson, A.O. and H. Callesen. 2000. New
method for culture of zona-included or zona-free embryos: the well of the well (WOW)
system. Mol. Reprod. 55: 256-264.
Vajta, G., I.M. Lewis, A.O. Trounson, S. Purup, P. Maddox-Hyttel, M. Schmidt, H.G. Pedersen, T.
Greve and H. Callesen. 2003. Handmade somatic cell cloning in cattle: analysis of factors
contributing to high efficiency in vitro. Biol. Reprod. 68: 571-578.
Vajta, G., P.M. Kragh and N.R. Mtango. Handmade cloning approach: potentials and limitations.
2005. Reprod. Fertil. Dev. 17: 97-112.
Yang, C.Y., R.C. Li, C.Y. Pang, B.Z. Yang, G.S. Qin, M.T. Chen, X.F. Zhang, F.X. Huang, H.Y.
430
Zheng, Y.J. Huang and X.W. Liang. 2010. Study on the inter-subspecies nuclear transfer of
river buffalo somatic cell nuclei into swamp buffalo oocyte cytoplasm. Anim. Reprod. Sci.
121: 78-83.
Yang, C.Y., C.Y. Pang, F.X. Huang, H.Y. Zheng, B.Z. Yang and X.W. Liang. 2011. Research on
hand-made clone in buffalo. China Animal Husbandry & Veterinary Medicine. 4: 147-150.
Yang, C.Y., C.Y. Pang, B.Z. Yang, R.C. Li, Y.Q. Lu and X.W. Liang. 2012. Optimization of
cryopreservation of buffalo (Bubalus bubalis) blastocysts produced by in vitro fertilization
and somatic cell nuclear transfer. Theriogenology. 15;78: 1437-1445.
Table 1. Effects of different culture systems on in vitro developmental competence of HMC

embryos珙n=6珩
Culture system
Development
MD FS WOW
56/112 68/124 99/140
Cleavage %）
50.09.6）aA (55.85.6)aAB (70.56.1)bB
9/112 23/124 57/140
Blastocyst %）
(8.32.7) A (19.82.1) B (40.06.3)C
a,b
Within a raw, values without common letters differed (P<0.05).
A,B
Table 2. Effects of various CPA combinations and embryo types on survival of buffalo HMC
blastocysts cryopreserved by vitrification n=6）
Item Cryosurvival rate %）

25%EG+25%DMSO 20% EG+20% DMSO+0.5M
Vitrification medium 40%EG %）
%） sucrose %）
32/53 31/41 38/42
HMC blastocyst
62.011.1）A 76.410.2）AB 90.96.58）B
55/60
SCNT blastocyst
92.38.5）
A,B
Table 3. Embryo production efficiencies of HMC and SCNT
Method Replicate Fusion rate %） Cleavage rate %） Blastocyst rate %）

269/336 146/269 53/269
SCNT 7 a
79.86.3） 56.413.3） 19.83.3）A
140/156 99/140 57/140
HMC 4
89.12.8）b 70.56.1） 40.06.3）B
a,b
A,B
431
The Impact of Linolenic Acid on in vitro Development of Buffalo Embryos
Haiying ZHENG, Chunyan YANG, Jianghua SHANG, Chunying PANG, Fenxiang HUANG,
Jian WANG, Bingzhuang YANG, Xianwei LIANG *
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo
Research Institute, Chinese Academy of Agricultural Sciences, Nanning 530001, P.R. China
*Corresponding email：liangbri@126.com
ABSTRACT
The present study investigated the effect of linolenic acid supplementation on the early
development of buffalo embryos in vitro. The presumptive zygotes were transferred to the vitro
culture medium (TCM199, 10%FBS) supplemented with 0 (control), 10, 50, 100 and 200 µM
linolenic acid. Cleavage and blastocyst development rates were recorded on Day 2 and Day 6 to 9
after insemination, respectively. A total of 1482 COCs were used in ten independent replicates. The
results showed that the cleavage rates in the groups of 0 (control), 10, 50, 100 and 200 µM linolenic
acid were 59.0±7.2%, 62.7±6.3%, 63.5±6.1%, 58.1±7.9% and 60.7±6.7%, respectively. No
statistical difference was observed regarding cleavage rates among treatments (p 0.05). The
treatment of COCs with 50 µM linolenic acid resulted in a significantly higher percentage of
blastocyst development rate compared to the control group and those supplemental with 10, 100 or
200 µM linolenic acid (34.5±4.1% vs 25.4±8.2%, 28.85±7.59%, 26.41±9.51%, 29.45±10.60%,
respectively, p﹤0.05). In contrast, no significant differences were found between the control group
and the other treatments. In conclusion, the results of this study indicated that supplementation of
linolenic acid in the vitro culture medium could enhance the blastocyst development in buffalo
species and the optimal concentration of linolenic acid in the present procedures was 50 µM.
Keywords: buffalo, linolenic acid, embryo development
INTRODUCTION
Buffalo is an important livestock resource in many Asian and Mediterranean countries. In
vitro embryo production (IVEP) and transfer of the embryos to produce calves with high genetic
merit would be of great interest in buffalo species. The efficiency of the IVEP in buffalo is low
compared to that in bovine. It may be due to the reproductive physiology of buffalo or the technical
factors in IVEP procedures. Recent studies have indicated that dietary polyunsaturated fatty acids
(PUFAs) supplementation can influence reproductive performance in cattle (Bilby et al., 2006).
Some high fat diets can result in higher blastocyst rates and improve embryo quality
(Fouladi-Nashta et al., 2007). PUFAs constitute the major portion of the fatty acid content of the
follicular fluid in small and large follicles (Homa et al.,1992). Changing the fatty acid content of the
diet was found to reflect on the fatty acid distribution in reproductive tissues (Bilby et al., 2006).
These effects may be partially mediated by improvements in oocyte maturation, which is essential
for successful fertilization and further embryo development (Marei et al., 2009). Linolenic acid
(ALA; 18:3 n-3) is the main dietary source of n-3 PUFAs. In bovine, supplementation of linolenic

432
acid during oocyte maturation can affect the molecular mechanisms controlling oocyte nuclear
maturation leading to an increased number of MII stage oocytes and improve subsequent early
embryo development (Marei et al., 2009). This effect may is mediated both directly through MAPK
pathway and PGE2 synthesis (Marei et al., 2009). Neverheless, little is known on the effects of
linoleic acid during embryo culture. Also, the use of linoleic acid to improve the productivity of
IVEP in buffaloes has not been reported. Hence, the aim of this study was to assess the direct effect
of linolenic acid addition to in vitro culture on embryo development after IVF in buffalo.

Unless otherwise stated, all chemicals were purchased from Sigma Chemical Company (St.
Louis, MO, USA).
Collection of oocytes and In Vitro Maturation
Cumulus-oocyte complexes(COCs) were retrieved from 2–8 mm antral follicles from
slaughterhouse ovaries, and washed two times in TCM199. The COCs were incubated in TCM199
supplemented with 10% FBS, 10µg/ml FSH, 10 IU/ml LH, 1µg 17β-estradiol and 25 ng/ml EGF for
20-22 h at 39℃, 5% CO2 in humidified air.

In Vitro Fertilization and Embryo Culture
In vitro-matured oocytes were fertilized for 24 h with motile buffalo sperm prepared from
semen frozen in straws, separated after thawing by 30 min swim-up in Tyrode’s medium with 10
mg/ml Heparin, 0.6%(w/v) BSA and PHE (20 µM penicillamine,10 µM hypotaurine and 0.5µM
epinephrine). The presumptive zygotes were then transferred to 30 µl droplets of the vitro culture
medium (TCM199, 10% FBS) supplemented with 0 (control), 10, 50, 100 and 200 µM linolenic
acid. Cleavage and blastocyst development rates were recorded on Day 2 and Day 6 to 9 after
insemination, respectively.
Experimental design
In this experiment, presumptive zygotes obtained after in vitro maturation and in vitro
fertilization in the absence of linolenic acid (see above) were pooled and randomly allocated to one
of the following treatment groups: in vitro culture with 0 (control), 10, 50, 100 and 200 µM
linolenic acid. Cleavage and blastocyst development rate was recorded on Day 2 and Day 6 to 8
after insemination.
Statistical Analysis
Data were analyzed using the SPSS program for Windows (SPSS16.0). Differences in embryo
development (cleavage and blastocyst rate) between experimental groups were analyzed using
one-way repeated measures ANOVA with arcsine transformation. All the results are expressed as
the mean±SEM. and statistical significance was accepted for P≤0.05.

Effect of linolenic acid supplementation on the early development of buffalo embryos in vitro.
The cleavage rates in the groups of 0 (control), 10, 50, 100 and 200 µM linolenic acid were
59.0±7.2%, 62.7±6.3%, 63.5±6.1%, 58.1±7.9% and 60.7±6.7%, respectively. No statistical
difference was observed regarding cleavage rates among treatments (p 0.05). The treatment of
COCs with 50 µM linolenic acid resulted in a significantly higher percentage of blastocyst
development rate compared to the control group and those supplemental with 10, 100 or 200 µM
433
linolenic acid (34.5±4.1% vs. 25.4±8.2%, 28.85±7.59%, 26.41±9.51%, 29.45±10.60%, respectively,
p﹤0.05). In contrast, no significant differences were found between the control group and the other
treatments. (Table1)
It has been reported that ALA supplementation during oocyte maturation enhances the quality
of resultant blastocyst and reduces apoptosis in them. However, in the bovine model, the effect of
ALA supplementation during IVC had not been addressed. In cattle, the concentration of ALA was
reported to be 53.9 - 143.66 μM in plasma and 35.9 -71.8 μM in follicular fluid (Childs et al., 2008).
Under our experimental conditions, the concentration of ALA used was in the range from 0 to 200
μM and so reflected the normal physiological situation. Our results have revealed that addition of
50 μM ALA during IVC enhances the developmental competence of buffalo embryos in vitro,
which results in significantly increased blastocyst yield.
In conclusion, the results of this study indicated that supplementation of linolenic acid in the
vitro culture medium could enhance the blastocyst development in buffalo species and the optimal
concentration of linolenic acid in the present procedures was 50 µM.
ACKNOWLEDGEMENTS
(31160456), the Natural Science Foundation of Guangxi, China (GuiKeZi 0991011,
2011GXSFB018045) and the Fundamental Research Funds for Guangxi Buffalo Research Institute
(ShuiNiuJi 1101008).
REFERENCES
Bilby, T.R., J. Block, B.C. doAmaral, O. Sa Filho, F.T. Silvestre, P.J. Hansen, C.R. Staples and W.W.
Thatcher. 2006. Effects of dietary unsaturated fatty acids on oocyte quality and follicular
development in lactating dairy cows in summer. Dairy Sci.89: 3891-3903.
Fouladi-Nashta, A.A., C.G. Gutierrez, J.G. Gong, P.C. Garnsworthy and R. Webb. 2007. Impact of
dietary fatty acids on oocyte quality and development in lactating dairy cows. Biol. Reprod.
77: 9-17.
Homa, S.T. and C.A. Brown. 1992. Changes in linoleic acid during follicular development and
inhibition of spontaneous breakdown of germinal vesicles in cumulus-free bovine oocytes.
Reprod. Fertil. 94: 153-160.
Bilby, T.R., T. Jenkins, C.R. Staples and W.W. Thatcher. 2006. Pregnancy, bovine somatotropin, and
dietary n-3 fatty acids in lactating dairy cows: III. Fatty acid distribution. J. Dairy Sci. 89:
3386-3399.
Marei, W.F., D.C. Wathes and A.A. Fouladi-Nashta. 2009. The effect of linolenic acid on bovine
oocyte maturation and development. Biol.Reprod. 81(6):1064–1072
Childs, S., A.A. Hennessy, J.M. Sreenan, D.C. Wathes, Z. Cheng, C. Stanton, M.G. Diskin and D.A.
Kenny. 2008 Effect of level of dietary an-3 polyunsaturated fatty acid supplementation on
systemic and tissue fatty acid concentrations and on selected reproductive variables in cattle.
434
Table 1. Effect of linolenic acid supplementation on embryo development in vitro.
ALA (M) replicate Oocyte Cleavage rate Blastocyst rate

IVC N N n (%) n (%)
0 10 325 193(59.08±7.26) 80(25.48±8.24)a
10 10 331 206(62.77±6.30) 94(28.85±7.59)a
50 10 339 215(63.54±6.19) 118(34.52±4.19)b

100 10 298 178(58.16±7.94) 73(26.41±9.51)ac
200 10 189 110(60.70±6.71) 53(29.45±10.60)a
a,b,c
Within a column, values without common letters differed significantly (P<0.05)
435
A Preliminary Study on Stable Transfection of EGFP in Buffalo Cumulus Cells
Jiang-Hua SHANG, a Chun-Yan YANG, a Jing QIN, a Hai-Ying ZHENG, a Fen-Xiang HUANG,
a
Jian WANG, a and Hua-Zhong LIU a b*
a
Nanning 530001, b Modern Biochemistry Centre, Guangdong Ocean University, Zhanjiang 524088,
P.R. China.
*Corresponding email：zj902030@163.com
ABSTRACT
Transgenic somatic cell nuclear transfer (SCNT) is currently one of effective ways for
producing transgenic animals, but the efficiency of the exogenous gene integrating into somatic
cells is still low. To obtain stably transfected buffalo somatic cells for transgenic SCNT for further
experiments, in this study, buffalo cumulus cells were transfected with pEGFP-N1 by using
Lipofectamine 2000 and selected by exposing to G418 (600 μg/ml) for 3 weeks. Selected
transfected cells were imaged under a fluorescence microscope. After 24 hours following
transfection, approximate 5% of cells were transiently transfected. Following G418 treatment for 3
weeks, 15% of the survived cells expressed EGFP with fluorescence intensity several folds larger
than that in the cells without G418 selection (control). EGFP expressing cells, even part of
non-EGFP expressing cells with G418 selection (treatment) were detected to have complex shapes
in their morphology which is different from the typical fusiform shape of cumulus cells (control).
This study demonstrates that EGFP can successfully be expressed in buffalo cumulus cells and
stably transfected cells can be enriched by G418 selection for multiple purposes. Future study
should be done to improve the integration rate of exogenous gene and the percentage of stably
transfected cells.
Keywords: buffalo, cumulus cells, EGFP, transfection, G418 selection
INTRODUCTION
The possibility of transfecting somatic cells used as donor karyoplasts for somatic cell
nuclear transfer (SCNT) provides a powerful tool for the production of transgenic livestock (Hyun
et al., 2003). Several methods, such as electroporation, lipofection, adenovirus, adeno-associated
virus, and lentivirus are available to achieve introduction of foreign DNA that carries a gene of
interest for ectopic expression in cells (Lakshmipathy et al., 2004). The lipofection
(liposome-mediated) is one of the most commonly used methods for generation of transiently and
stably transfected somatic cells in animals. However, their transient transfection efficiency is still
low.
Genetically modified cells can be selected in vitro, and only cells with stable, integrated
transgenes are used as donor cells. Compared to the comparatively fast transient transfection, stable
transfection takes a longer time and is possible to obtain a homogeneous and durable gene
expression at about the same level by simply cell passage since the inserted gene is inherited to the
daughter cells (Sitton et al., 2006).

436
Fluorescently labeled mammalian cell cultures are very frequently used in vitro experimental
models. Enhanced green fluorescent protein (EGFP) gene was successfully used as an expression
marker without any adverse biological effects on in vitro development of transfected embryos
(Stauber et al., 1998). Stable transfection of EGFP might significantly enhance the efficiency of
transgenic animal production, because expression of the reporter gene allows the selection of
transfected cells as donor karyoplasts for SCNT and positive embryos for transfer into the surrogate
mother (Arat et al., 2002). In this study, buffalo cumulus cells were cultured and transfected with
pEGFP-N1 by using Lipofectamine 2000 and than selected by exposing to G418 for 3 weeks to
evaluate the possibility and efficiency of stable transfection of EGFP in buffalo somatic cells for
transgenic SCNT for further experiments.

Chemicals
Lipofectamine™ 2000 (abbreviated here as LF2000) was purchased from Invitrogen
(Carlsbad, CA, USA) and pEGFP-N1 (containing the enhanced green fluorescence protein and
neomycin-resistant genes) from Clontech (Palo Alto, CA, USA), and other chemicals and media
were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Life Technologies (GIBCO) (Grand
Island, NY, USA), respectively.
Cell culture
The cumulus cells of water buffalo were isolated by stripping off the cumulus cells from in
vitro matured cumulus-oocytes complexes (COCs) and grown to 75-80% confluence in DMEM
supplemented with 10% fetal bovine serum in T25 flasks at 39℃, 5% CO2 in air atmosphere.
Transfection
One day prior to transfection, cells were harvested by trypsinization and plated into 12-well
tissue-cultured plates at a 0.2-0.5 million cells/ml. Plasmid pEGFP-N1 isolated with GeneJET™
PCR Purification Kit (Fermentas) from E. coli DH5α was used in this trial. Cells were transiently
transfected with DNA using LF2000 according to the manufacturer's instruction. 2 μg of purified
DNA (pEGFP-N1) was used for transfection, and G418 was added to the cell media 24 hours post-
transfection at a final concentration of 600 μg/ml for 3 weeks to select for cells with stably
integrated plasmid according to Arat et al. (2002). Media was changed once in every two days.
The plates were imaged under by using an epifluorescence microscope with a standard FITC
filter (Eclipse TE, Nikon) to assess the efficiency of transfection.

EGFP selection of donor cells has been used to produce transgenic offspring by SCNT in
mice, pigs, goats and cattle. Furthermore, EGFP gene can be used to confirm the presence of a
functional transgene in embryos derived from transgenic animals, which would greatly enhance the
efficiency of propagating transgenic livestock (reviewed by Sharma and Chopra, 2007). In the
present study, water buffalo cumulus cells were cultured and transfected with pEGFP-N1 plasmid
DNA by using LF2000, and then selected in G418 at the final concentration of 600 μg/ml for 3
weeks following a routine G418 selection procedure (Arat et al., 2002). After 24 hours following
transfection, cells were imaged under a fluorescence microscope. The percentage of transiently
transfected cumulus cells were approximate 5% and decreased gradually within 4 days. Following
G418 treatment for 3 weeks, 15% of the survived cells expressed EGFP with fluorescence intensity
several folds larger than that in the cells without G418 selection (control). EGFP expressing cells,
437
even part of non-EGFP expressing cells with G418 selection (treatment) were detected to have
complex shapes in their morphology which is different from the typical fusiform shape of cumulus
cells (control).
The efficiency of EGFP positive in cumulus cells after transient transfection is approximate
5%, which is similar to that (5%) in Chinese Hamster Ovary (CHO) cells obtained by using
activated dendrimers (Celtikci et al., 2010), but is lower than 11% in bone marrow stromal cells
(BMSC) by the same LF2000 (Clements et al., 2007).
This study demonstrates that EGFP can successfully be expressed in buffalo cumulus cells
and stably transfected cells can be enriched by G418 selection for multiple purposes. Future study
should be done to improve the integration rate of exogenous gene and the percentage of stably
transfected cells. Futhermore, single colonies should be isolated in the presence of G418 and
expanded for further transgenic SCNT experiments.
ACKNOWLEDGEMENTS
This research was supported by grants from the National Natural Science Foundation of
China (31160456), the Natural Science Foundation of Guangxi, China (GuiKeZi 0991011,
2011GXSFB018045) and the Scientific Project of Guangxi Bureau of Fisheries, Animal Husbandry
and Veterinary (GuiMuYuKe 1204910).
REFERENCES
Arat, S., J. Gibbons, S. Jacek Rzucidlo, D.S. Respess, M. Tumlin and S.L. Stice. 2002. In vitro
development of green fluorescent protein (GFP) transgenic bovine embryos after nuclear
transfer using different cell cycles and passages of fetal fibroblasts. Biol. Reprod. 66(6):
1768-1774.
Celtikci, B., N. Puralı and H. Asuman Özkara. 2010. Establishment of Green Fluorescent Protein
Expressing CHO Cells by Stable Transfection Using Activated Dendrimers and G418
Selection. Turkish Journal of Biochemistry 35 (4): 340-343.
Clements, B.A., V. Incani, C. Kucharski, A. Lavasanifar, B. Ritchie and H. Uludağ. 2007. A
comparative evaluation of poly-l-lysine-palmitic acid and Lipofectamine ™ 2000 for
plasmid delivery to bone marrow stromal cells. Biomaterials 28(31): 4693-4704.
Hyun, S., G. Lee, D. Kim, H. Kim, S. Lee, D. Nam, et al. 2003. Production of nuclear
transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green
fluorescent protein. Biol. Reprod. 69: 1060-1068.
Lakshmipathy, U., B. Pelacho, K. Sudo, J.L. Linehan, E. Coucouvanis, D.S. Kaufman and C.M.
Verfaillie. 2004. Efficient Transfection of Embryonic and Adult Stem Cells. Stem Cells 22:
531–543.
Sharma, A. and A. Chopra. 2007. Role of green florescent protein (GFP) gene in somatic cell
cultures: A Review. Agric. Rev. 28(2): 79-92.
Sitton, G., A. Hansgate, F. Srienc. 2006. Transient gene expression in CHO cells monitored with
automated flow cytometry. Cytotechnology 52: 13–24.
Stauber, R.H., K. Horie, P. Carney, E.A. Hudson, N.I. Tarasova, G.A. Gaitanaris and G.N. Pavlakis.
1998. Development and applications of enhanced green fluorescent protein mutants.
BioTechniques 24(3): 462-471.
438
Scrotal Circumference Growth Curves of Buffalo Bulls of Different Breeds Raised

in Brazil
Marc HENRYa, Mayara Ferreira BRITOa, Ana Maria LOAIZA-ECHEVERRIa, Cairo Henrique
Sousa DE OLIVEIRAb , André Felipe Bagarrão GIBSONc, Beatriz Parzewski NEVESa,
Guilherme De Oliveira ANDRADEa, Isabela Oliveira MELOa, Eduardo BASTIANETTOb
a
Departamento de Clínica e Cirurgia Veterinária, Escola de Veterinária, Universidade Federal de
Minas Gerais, Belo Horizonte, Minas Gerais, Brasil, b Departamento de Medicina Veterinária
Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, Minas
Gerais, Brasil, cPrograma de Pós-graduação em Saúde Animal na Amazônia, Curso de Medicina
Veterinária, Universidade Federal do Pará, Belém, Pará.
*
Corresponding email:henrym2601@gmail.com
Abstract
The aim of the present study was to compare the scrotal circumference (SC) growth curve of
three buffalo breeds. The nonlinear model used was the Logistic where parameter A is the estimated
testis size at maturity, B is the integration constant and k is a maturating index. A total of 65, 189 and
197 SC records of Jaffarabadi, Mediterranean and Murrah buffalo bulls were used. The SC size at
maturity was 32.94, 32.46 and 31.51 cm for Jaffarabadi, Mediterranean and Murrah buffalo bulls. The
SC and the age at inflection point were: 16.47 cm at 427 days for Jaffarabadi, 16.23 cm at 199 days for
Mediterranean and 15.75 at 330 days for Murrah buffalo bulls. The absolute growth rate increased from
0.016 cm/d and reached a maximum value of 0.023 cm/d between 358 and 497 days of age in
Jaffarabadi; increased from 0.020 cm/d and reached a maximum value of 0.022 cm/d between 156 and
243 days of age in Mediterranean; and increased from 0.017 cm/d and reached a maximum value of
0.020 cm/d between 167 and 493 days of age in Murrah buffalo bulls. The SC at inflection point was
similar among the breeds; however, the age at the inflection point was early in the life in Mediterranean
buffalo bulls. Scrotal circumference development was characterized by a phase of accelerated growth
followed by a decreasing growth rate. The SC and the age at the inflection point could be used to
buffalo bull’s selection for early sexual development.
Keywords: Buffalo bulls, Growth curve, Scrotal circumference
Introduction
Scrotal circumference (SC) is frequently used in breeding programs because of its easy
measurement, high repeatability and moderate to high heritability. It is also favorably associated with
physical semen characteristics, age at puberty, sexual precocity and weight gain (Brinks, 1994). One
way to describe testicular growth is using nonlinear regression models. The advantage of nonlinear
models is that they can accommodate a large number of measurements in some parameters and, thus,
permit appropriate biological interpretation (Loaiza-Echeverri et al., 2013). The information available
on age-related changes in testicular size, in water buffalo is limited and almost exclusively based on
cross-sectional studies (Ahmad et al., 2010). This information could assist in estimating the age at
puberty and maturity at buffalo bulls and to establish some norms for breeding soundness evaluation of
different breeds of buffalo raised in Brazil. The aim of the present study was to compare the scrotal
circumference growth curve of three buffalo breeds using a nonlinear model.

439
Material and Methods

This study was conducted in 5 farms located in the state of Minas Gerais, 4 farms located in the
state of Pará and one farm located in the state of Bahia, Brazil. A total of 65, 189 and 197 SC records
of Jaffarabadi, Mediterranean and Murrah buffalo bulls were used. Scrotal circumference was
measured in the region of the greatest diameter of the testes and included both testes positioned
symmetrically side by side, leaving the skin of the scrotum distended. In the Logistic model (SCt=
A/1+B-k*t)SCt is the scrotal circumference (SC) to t days of age, A is the estimated SC at maturity, B
indicates the proportion of the asymptotic mature testis size to be obtained after birth (established by
the initial value of SC and t); and k is a maturing index, establishing the earliness with which SC
approaches A. The inflection point for SC and age at the inflection point were calculated using the
formulas: and . The absolute growth rate (AGR) for SC was calculated by the
following formula: where . In this formula, is the SC estimated by
the model in t age, and and are the parameters estimated by the Logistic model. The absolute
growth rate represents the size gained per time unit.
Results and discussion

According to the parameter A, the SC size at maturity was high for the Jaffarabadi, followed by
the Mediterranean and Murrah (Table 1). We are not aware of any earlier reports of nonlinear models
used for describe the SC growth in buffaloes. The sexual maturity shows variations in buffalo breeds
from different continents. In Brazil, crossbreeds derived from Mediterranean x Jaffarabadi reached the
sexual maturity to 27 months with 30 cm of SC (Ohashi et al., 2001), that is similar to the results report
for SC at maturity in the present study. In Australia, the Swamp buffaloes reach the sexual maturity at
30 to 33 months of age, when SC is in the 17 to 20 cm range (McCool & Entwistle, 1989), and in India,
Murrah bulls reach the sexual maturity from 42 to 48 months (Rana & Bilaspuri, 2004).
Parameter k is related to growth rate and determines the slope of the curve. The value of k was
greater for Jaffarabadi bulls, and these animals had a more sigmoid curve (Figure 1). The size of SC at
the inflection point was similar between the three breeds; however, the age at the inflection point was
lower for Mediterranean buffaloes, indicating that Mediterranean buffaloes have early sexual
development. This value was similar to the age at inflection point reported for European and
crossbreeds bulls (Delgado et al., 2000; Nieto et al., 2006). On the other hand, Jaffarabadi buffaloes
had the greater age at the inflection point, and the value found was even high than the age at inflection
point reported for Zebu bulls (Neves, 2007; Loaiza-Echeverri et al., 2013) which reaches sexual
maturity later than European breeds bulls.
In Brazil, the age at puberty in crossbreeds derived from Mediterranean x Jaffarabadi bulls was
from 11 to 14 months with 21.7 ± 1.9 cm of SC (Ohashi et al., 2001) and in Pakistan, the Nili-Ravi
buffaloes reach the puberty with 22 months with 22.8 cm of SC (Ahmad et al., 1989). The parameters
used to define puberty in those studies were that ejaculates should contain at least 50x106 sperm with
more than 10% motility.
The absolute growth rate increased from 0.016 cm/day and reached a maximum value of 0.023
cm/day between 358 and 497 days of age in Jaffarabadi; increased from 0.020 cm/day and reached a
maximum value of 0.022 cm/day between 156 and 243 days of age in Mediterranean; and increased
from 0.017 cm/day and reached a maximum value of 0.020 cm/day between 167 and 493 days of age in
Murrah buffalo bulls (Figure 2). This results could indicate that the high age at inflection point in
Jaffarabadi buffalo bulls was due to the delay in the onset of the phase of fast SC growth.
In the same phases of the sexual development, the size of the SC at inflection point and at
maturity (parameter A) in the three buffaloes breeds analyzed in the present study were lower than the
values reported for European, crossbreeds and Zebu bulls (Delgado et al., 2000; Nieto et al., 2006;
440
Neves, 2007; Loaiza-Echeverri et al., 2013), and this could be indicate that the testis development in
this two species is different.
In conclusion, the scrotal circumference development in buffaloes breeds evaluated in this study
was characterized by a phase of accelerated growth followed by a decreasing growth rate, and the onset
in the accelerated SC growth could be associated with the early or late sexual development. The SC and
the age at the inflection point could be used to buffalo bull’s selection for early sexual development.
References
Ahmad N., M. Sahab, S. Khurshid and M. Arslan. Pubertal Development in the Male Buffalo:
Longitudinal Analysis of Body Growth, Testicular Size and Serum Profiles of Testosterone and
Oestradiol. 1989. Anim. Reprod. Sci. 19:161-170.
Ahmad N., S. Umair, M. Sahab and M. Arslan. 2010. Testicular development and establishment of
spermatogenesis in Nili-Ravi buffalo bulls. Theriogenology. 73:20-25.
Brinks J.S. Relationships of scrotal circumference to puberty and subsequent reproductive performance
in male and female offspring. 1994. In: Fields M. J., Sand R. S. (editor). Factors affecting calf
crop. Boca Raton: CRC, 363–370.
Delgado C., M. Valera, A. Molina, J.M. Jiménez and A. Rodero. 2000. Scrotal circumference as
indicator of reproductive performance in authoctonous beef cattle: growth curve analysis in
Retinto bull. Archivos de Zootecnia. 49:229-240.
McCooll C.J. and K.W. Entwistlez. 1989. The development of puberty and sexual maturity in the
australian swamp buffalo bull. Theriogenology. 32:171-184.
Neves, A.L.A., A.J. Del Rei, M.P. and M. P. Santos. 2011. Crescimento testicular de touros da raça
Nelore. Livestock Research for Rural Development. 23:93-100. ISSN/ISBN: 01213784.
Nieto L.M., L.O.C. Silva and A. Gondo. 2006. Análise da curva de crescimento do perímetro escrotal
de touros Canchim em diferentes sistemas de criação [Canchim bulls’ scrotal circumference
growth curve analysis in different breeding systems]. Arq. de Ciências Vet. e Zool. da Unipar.
9:99-103.
Loaiza-Echeverri A.M., Bergmann J.A.G., Toral F.L.B., Osorio J.P., Carmo A.S., Mendonça L.F.,
V.S. Moustacas and M. Henry. 2013. Use of nonlinear models for describing scrotal
circumference growth in Guzerat bulls raised under grazing conditions. Theriogenology. 1-9.
(In Press).
Ohashi O.M., E. Oba, J.C. Nogueira, J.S. Sousa and A.O.A. Silva. 2001. Characteristics of the
reproductive development of male buffalo: testicular development, puberty and sexual maturity.
Rev. Bras. de Med. Vet. 23:103-107 [in portugues].
Rana, B.K. and G.S. Bilaspuri. 2004. A quantitative study of seminiferous tubular cells in the
developing Murrah buffalo testis. The Vet. J. 167:95–103.
441
Table 1. Parameter estimation by the Logistic model and scrotal circumference and age at the
inflection point
Parameters Inflection point

A b k SC Age
Jaffarabadi 32.9445 3.3392 0.00282 16.47 427.5
Mediterranean 32.4696 1.7204 0.00272 16.23 199.5
Murrah 31.5173 2.4074 0.00266 15.75 330.2
Figure 1. Scrotal circumference growth curves estimated by the Logistic model for buffalo bulls of
three breeds.
Figure 2.Absolute growth rate of the scrotal circumference in buffaloes bulls based on the Logistic
model.
442
Validation of an ELISA for detection of Pregnancy Associated Glycoprotein in

Water Buffalo Cows
Rafael PAIVAa, Rosaura PEREZb, Simon ZAMBRANO b, Silvia ZIMMERMANa and Christoph
EGLIa
a
IDEXX Laboratories Inc., Westbrook, Maine, USA
b
Centro Diagnostico Veterinario, Mérida-Estado Mérida and Acarigua-Estado Portuguesa, Venezuela
*Corresponding e-mail:Rafael-Paiva@idexx.com; rperezgil@gmail.com;
simonerbe_12@hotmail.com; Silvia-Oliveirazimmerman@idexx.com; christoph-egli@idexx.com
ABSTRACT
Pregnancy Associated Glycoprotein (PAG) is produced by cotyledonary placenta after day 21
of pregnancy. The aim of this research was to determine the validation of pregnancy detection by an
ELISA test kit for PAG. 81 open and 40 pregnant water buffalo cows were diagnosed by transrectal
ultrasonography. The WB cows where between 25 and 300 days of pregnancy with an average of 193
days. At the same time of ultrasound examination a serum sample was taken and analyzed in the
laboratory performing the ELISA Pregnancy test kit as described by the manufacturer. The results were
analyzed for sensitivity and specificity. The values obtained for sensitivity and specificity was 100 %
for both parameters. Based on the results, the commercial ELISA Pregnancy test kit is valid for
detection of PAG’s in WB cows. In conclusion the ELISA for PAG’s detection can be used as an
effective tool that will help producers and veterinarians in the improvement of early pregnancy
detection and thus could help improve reproductive performance in WB herds.
Keywords: Pregnancy Associated Glycoprotein, target antigen, sensitivity, specificity
INTRODUCTION
Pregnancy diagnosis has being done routinely by trans-rectal palpation and ultrasound,
performed by veterinarians in dairy and beef production system with water buffalo. Several laboratory
diagnostic tests where developed in the past with the goal of early pregnancy detection.
Pregnancy Associated Gglycoproteins are powerful pregnancy markers in domestic cattle.
These proteins are expressed in mono- and binucleate trophoblast cells from the first days of gestation
until calving. In maternal blood and milk, PAGs rise to detectable levels from days 24 to 28 after
fertilization. (2)
Pregnancy Associated Glycoproteins extracted from mid and late placentas in water buffalo
cows where isolated and characterized. Three different water buffalo Pregnancy Associated
Glycoproteins where identified: P85048, P85049, and P85050 and deposit in the SwissProt data base.
(3)
A commercial ELISA was developed to detect Pregnancy Associated Glycoproteins in bovine
serum or plasma as a marker for determination of pregnancy in cows. The assay uses an anti-PAG
antibody coated onto the solid phase to bind PAGs that may be present in the sample. A second anti-
PAG antibody, coupled with biotin is used as the detection reagent along with streptavidin-horseradish
peroxidase (SA-HRP). TMB substrate is used as a colorimetric indicator for PAG containing samples,
and the enzymatic reaction is stopped with stop solution. After reading the plate at 450nm, wells with
color development above the assay threshold are considered positive, indicating a pregnant animal,
while wells with little or no color development indicate open animals. (Figure 1). (4)

443
In the water buffalo industry the early pregnancy detection with the use of these techniques is new,
there are no commercial ELISA tests validated to be used in this species.

One hundred twenty one (121) water buffalo cows (Bubalus bubalis), where diagnosed by
ultrasound palpation, blood sample from each animal was collected just after the ultrasound.
In the laboratory the samples where centrifuged to obtain serum, this serum was separated from the
cloth frozen and used to perform the commercial ELISA test (IDEXX Bovine Pregnancy test),
calculations and interpretation as described by the manufacturer in its correspondent insert:
a) Test Protocol
All reagents must be allowed to come to 18–26°C before use. Reagents should be mixed by gentle
swirling. Use a separate pipette tip for each sample.
1. Obtain coated plate(s) and record the sample position.
2. Use only as many microwell strips as required for the assay; place the unused strips in a sealed bag
with desiccant and return it to 2–8°C.
3. Dispense 25 μL of Sample Diluent into wells which will be used for samples and Controls.
4. Dispense 100 μL of Negative Control into two wells of the assay plate.
5. Dispense 100 μL of Positive Control into two wells of the assay plate.
6. Dispense 100 μL of samples into appropriate wells.
7. Gently tap plate to mix.
8. Cover the wells and incubate 60 minutes (± 5 minutes) at 37°C (±2°C). The plates should be tightly
sealed to avoid evaporation.
9. Aspirate liquid contents of all wells into an appropriate waste reservoir.
10. Wash each well 3 to 5 times with approximately 300 μL of Wash Solution. Aspirate liquid contents
of all wells after each wash. Following the final aspiration, firmly tap residual wash fluid from each
plate onto absorbent material. Avoid plate drying between plate washings and prior to the addition of
the next reagent.
11. Dispense 100 μL of Detector Solution into each well.
14. Dispense 100 μL of Conjugate into each well.
17. Dispense 100 μL of TMB Substrate N.12 into each well.
18. Incubate for 15 minutes (± 1 minute) at 18–26°C.
19. Dispense 100 μL of Stop Solution N.3 into each well to stop the reaction. Add the Stop Solution in
the same order as the Substrate solution was added in step 17.
20. Measure and record the A(450 nm)–A(REF) for samples and Controls. The reference wavelength is
A(620 nm-650 nm).
21. Calculate results.
b) Test Results
For the assay to be valid, the following specifications must be met. The Positive Control mean minus
the Negative Control mean must be greater than or equal to 0.300. In addition, the Negative Control
mean must be less than or equal to 0.150
c) Interpretation of test results

The pregnancy status of the animal is determined by calculating the S–N (Sample value for each
sample.
1. If the S–N value is less than 0.300, the animal is considered not pregnant (open).
444
2. If the S–N value is equal to or greater than 0.300, the animal is considered pregnant.

In the ultrasound palpation 81(66.9%) water buffalo cows where pregnant and 40 (33.05%)
where open. The same results for pregnant and open WB cows were obtained after the ELISA test.
Table 1.
The range of pregnant days was between 25 and 300 days with an average of 193 days.
Sensitivity and specificity with these 121 samples demonstrate that for pregnant animals the sensitivity
is 100% for 81 samples from pregnant water buffalo cows and specificity is 100% for 40 samples from
open water buffalo cows. Table 2.
Sensitivity 100%
Specificity 100%
The curve made with the average S-N value for each pregnancy day, showed that PAG levels are above
the cut off value after day 25 and during all gestation period. Graphic 1.
CONCLUSIONS
This study demonstrates that the commercial ELISA (IDEXX Bovine Pregnancy test) is valid
and can be used in water buffalo cows.
The ELISA test kit detects PAG’s at the beginning and throughout the period of gestation with
levels over the cut off recommended by the manufacturer. Farmers and veterinarians can save days
open and optimize calving interval by the early diagnose of pregnant water buffalo cows. It can also
improve fixed time artificial insemination and embryo transfer programs. The test can be used to detect
pregnant animals in early days when trans-rectal ultrasound and palpation are less accurate, in between
veterinarian’s visits to the farms or when there is no access to veterinarians or experienced palpators. It
is a useful tool that can improve reproduction, production levels and profit in the water buffalo
industry.
REFERENCES
Silva, E., R. A. Sterry, D. Kolb, N. Mathialagan, M. F. McGrath, J. M. Ballam, and P. M. Fricke
Accuracy of a Pregnancy-Associated Glycoprotein ELISA to Determine Pregnancy Status of
Lactating Dairy Cows Twenty-Seven Days After Timed Artificial Insemination. J. Dairy Sci.
90:4612–4622. doi:10.3168/jds.2007-0276.
Gajewski Z, M. Pertajitis, N. Sousa, J. Beckers, B. Pawliński and B. Janett. Pregnancy - associated
glycoproteins as a new diagnostic tool in cattle reproduction. Department of Clinical Sciences,
Faculty of Veterinary Medicine, WULS, Warsaw, Poland. zgajewski@supermidia.pl
Barbato1, O., N.M. Sousa2, K. Klisch3, E. Clerget2, A. Debenedetti1, V. Barile4, A. Malfatti5 and J.F.
Beckers2.. 1 Dpt of Biopathological Veterinary Science, Fac. of Veterinary Medicine,
University of Perugia, Italy 2 Physiology of Animal Reproduction, Fac. of Veterinary Medicine,
University of Liege, Belgium 3 Microscopical Anatomy, Medical School Hannover, Germany.
4 Zootechnic Experimental Institute, Monterotondo, Italy. 5 Dpt of Veterinary Science, Faculty
of Veterinary Medicine, University of Camerino, Italy. Corresponding author: O. Barbato. Dpt
of Biopathological Veterinary Science, Fac. of Veterinary Medicine, University of Perugia. Via
S. Costanzo 4, Perugia, 06126, Italy - Tel. +39 075 585 7640- Fax: +39 075 585 7654 - Email:
barbato@unipg.it. Isolation of pregnancy-associated glycoproteins (PAG) from water buffalo
(Bubalus bubalis) placenta by use of Vicia villosa bound agarose affinity chromatography.
Velek1, K., S. Michaud1, K. Boucher1, A. Rice1, L. Plourde1, N. Djuranovic1, C. Egli2, P. Welles1
and V. Leathers1. 1. IDEXX Laboratories Inc., Westbrook, Maine, USA 2. IDEXX Switzerland
AG, Liebefeld-Bern Switzerland. Development of an Early and Accurate ELISA for Detection
of Bovine Pregnancy.
445
Figure 1.
Acid Stop
Read at 450nm-ref
Substrate (TMB)
Incubate 15’ RT
WA SH 3 -4X
WA SH 3 -4X
Detector
(Biotinylated Ab)
Incubate 30’ RT
WA SH 3 -4X
Serum
or EDTA Plasma
(Early PAG)
+ Sample diluent
Incubate 1hr 37°C
Solid Phase (plate)
Anti-PAG Capture Ab
Graphic 1.
446
Table 1. US Palpation and ELISA results

US PALPATION ELISA
No % No %
PREGNANT 81 67 81 67
OPEN 40 33 40 33
TOTAL 121 100 121 100
Table 2. Sensitivity and specificity of the ELISA Bovine

Pregnancy Test in Water Buffalo cows
US PALPATION
PREGNANT OPEN
PREGNANT 81 0
ELISA
OPEN 0 40
447
Profiles of Pregnancy-Associated Glycoprotein (Pag) Concentrations during

Gestation in Swamp Buffalo
Van Hanh NGUYEN a*, N. Viet Linh a ,N. M. Sousab, J. F. Beckers b, X. N. Buia
a
Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi, Vietnam;
b
Faculty of Veterinary Medicine, University of Liege, Belgium,
*Corresponding email: nvhanh@ibt.ac.vn
ABSTRACT
The pregnancy-associated glycoproteins (PAG) are synthesized in the mono- and binucleate cells
of the ruminant’s trophectoderm. It was used as marker not only for early diagnosis pregnancy but also
for determination stage of pregnancy. In this report, we present the results of establishing the profiles
PAG concentration during time of gestation in swamp buffalo. A total 236 samples including maternal
plasma, fetal plasma, allantoid and amniotic fluids were collected from buffalo slaughtered. The age of
fetus were estimated from 6 - 48 weeks of pregnancy by base on crown-rum length measurement
method. The PAG concentration was detected by RIA using antibody anti-buffalo PAG. The results
shown that the mean of PAG concentration were variances in differences samples kind at the same
animal. The PAG concentration there is a linear correlation between PAG concentration in plasma
samples and time of pregnancy (Y = Ax2 + Bx + C) whereas the A, B and C are depend on the maternal
or fetus. In conclusion: the PAG concentrations in swamp buffalo are strictly correlated with the time
of gestation.
Keywords: gestation, pregnancy-associated glycoprotein, profiles, swamp buffalo
INTRODUCTION
Buffalo in Vietnam are almost swamp buffalo, in which population is 2.95 million and it has
decreased in the years recently. Reproductive technologies such as synchronization of oestrus, artificial
insemination, superovulation, in vitro maturation and fertilization and embryo transfer have been
investigated on swamp buffaloes (Uoc et al, 1997; Nguyen, 2006). The early embryonic mortality in
buffalo after synchronization and AI was observed from 22.9 to 49% (Campanile et al., 2005).
Although most pregnancy failures are due to early embryo mortality, fetal mortality and subsequent
abortion may occur due to a lesser extent until the end of pregnancy. The foregoing are major sources
of low reproduction rate and long interval between calving in buffaloes.
Since the PAGs have been discovered (Butler et al., 1982), it was reported in many species.
The measurement of the PAG concentration in plasma samples withdrawn from pregnant cows and
heifers throughout different periods following artificial insemination (AI) was estimated (Perényi et
al., 2002). The profiles of PAG concentration was established in many species. The purpose of PAG
profiles research throughout pregnancy is not only to utilize them for the purpose of pregnancy
detection but also to determinate the state of fetal health. In the cow, the PAG decrease is due to an
increased traffic of PAG to the mammary gland or to an increased clearance due to a higher metabolic
activity (Lopez-Gatius et al., 2007). In the goat, the analysis of the profiles clearly shows marked drops
in concentration that could indicate placental distress at different times (Zarrouk et al., 1999). PAG
profiles in pregnant ewes (Ledezma-Torres et al., 2006) are quite different than those obtained in cattle
(Zoli et al., 1992). However, till now, there was no reported to elucidate the PAG concentration
profiles and hypothetical role of these proteins during pregnancy in swamp buffalo. In this present the
buffalo PAG concentrations were detected during gestation and the regression analysis of them with
monthly gestation.
448

Samples collection
This study was conducted in 67 swamp buffaloes native from Vietnam. All samples including
fetal plasma (n = 67), maternal plasma (n = 51), allantoic fluid (n = 62) and amniotic fluid (n = 56)
were collected at slaughterhouse. After slaughter, the uterus was opened for collection of amniotic and
allantoic fluids. The umbilical cord was tied off, the fetus was cleaned and measured (crown rump
length or CRL) in order to estimate fetal age (Singh et al., 1963). Next, a large-bore needle was inserted
through the skin and between the ribs, directly into fetal heart in order to fetal blood collected.
Maternal blood was collected from the heart immediately after slaughter. The blood was usually
withdrawn using a sterile blood collection syringe (Monovette®, 9 ml, Sarstedt, Germany), and placed
in a cool box until centrifugation. The plasma was collected after centrifugation at 1,500 x g for 20 min
(at 4°C), and then separated and stored at –20°C until assay. Amniotic and allantoic fluids were
collected by aspiration prior to fetal blood collection. All samples were collected by sterile syringes (5
ml) and kept at 4°C during their transfer to the laboratory. Samples were centrifuged at 1,500 x g for 20
min (at 4°C) and the supernatant was separated and stored at –20°C until assay.
Radioiodination of PAG tracers
Iodination (NaI125, PerkinElmer, Boston, USA) was carried out following the chloramine T
pH 7.5) to obtain 1 g/l concentration. The radioiodination mixture was prepared by adding 10 µl of
method (Greenwood et al., 1963). The antigen (boPAG67kDa) was dissolved in phosphate buffer (0.2 M,
chloramine T solution (5 mg/ml dissolved in water) and 10 µl of Na-I125 (1 mCi, approximately 3.7 x
107 disintegrations per second) to 10 l of the antigen solution. After one minute of stirring, 10 µl of
metabisulphite solution (30 mg/ml dissolved in water) was added in order to terminate the reaction.
This mixture was loaded onto a Sephadex G-75 column (Amersham Biosciences, Uppsala, Sweden),
which was previously equilibrated with Tris-BSA buffer (0.025 M, pH 7.5), in order to separate the
free I125 and the labeled antigen. Eluted fractions of 1 ml were collected. The fractions were tested
before binding using the Geiger mini counter. Only fractions of more than 1,000 counts per second
were selected for use.
Procedure radioimmunoassay
PAG radioimmunoassays were performed by using the methods previously described by
Perenyi et al. (2002) and Nguyen et al. (2012), slightly adapted to buffalo samples. Briefly, 0.1 ml of
PAG standard (8 dilutions ranging from 100 to 0.8 ng/mL) and 0.05 ml of different fluid samples were
diluted in 0.3 ml of Tris buffer containing 1% BSA. After adding 0.1 ml of radiolabelled 125I-PAG, an
appropriate dilution of one of aforementioned antisera (0.1 ml) was poured in. Antiserum AS#859 was
obtained by immunizing a rabbit against buffalo PAG (molecular mass ranging from 61 to 73 kDa)
isolated from late pregnancy placentas (Barbato et al., 2008). Both the serum samples and standard
tubes were incubated overnight at room temperature. The next day, 1.0 ml of second antibody-
polyethylene glycol (PEG) solution was added to each tube (0.17 % (vol/vol) normal rabbit serum, 0.83
% (vol/vol) sheep anti-rabbit IgG, 0.3 % (wt/vol) BSA, 4 % (wt/vol) PEG 6000 diluted in Tris-buffer).
Incubation was carried out for 30 min at room temperature. Finally, 2 ml of Tris-BSA buffer was added
to the tubes before they were centrifuged (20 min at 1,500 x g). After centrifugation, the tubes were
aspirated and the radioactivity of the pellet was counted using a gamma counter.
Data analysis
The correlation between the concentrations was calculated versus estimation of time gestation
using the ANOVA single factor in Excel office program. Regression and the variance analysis were
done by Excel.

The correlation between PAG concentration of maternal plasma, fetal plasma, allantoic fluid,
amniotic fluid and estimate age of fetus was shown in Fig. 1 and Table 1. The mean PAG
449
concentrations in allantoic fluid, amniotic fluid were significantly lower than that in maternal plasma,
fetal plasma (Nguyen et al, 2012).
.
REFERENCES
Barbato, O., N. M. Sousa, K. Klisch, E. Clerget, A. Debenedetti, V. L. Barile, A. Malfatti and J. F.
Beckers. 2008. Isolation of new pregnancy- associated glycoproteins from water buffalo (Bubalus
bubalis) placenta by Vicia villosa affinity chromatography. Res. Vet. Sci. 85 (3):457-466.
Bui, X. N. 2006. Current status and trends of animal reproductive biotechnology in Vietnam. Embryo
Transfer Newsletter, Reprod. Fert. Develop. 24 (2):5-10.
Butler, J. E., W. C. Hamilton, R. G. Sasse, C. A. Ruder, G. M. Hass and R. J. Williams. 1982.
Detection and partial characterization of two bovine pregnancy-specific proteins. Biol. Reprod.
26:925-933.
Campanile, G., G. Neglia, B. Gasparrini, G. Galiero, A. Prandi, R. Di Palo, M. J. D’Occhio and L.
Zicarelli. 2005. Embryonic mortality in buffaloes synchronized and mated by AI during the
seasonal decline in reproductive function. Theriogenology 63:2334-2340.
Dosogne, H., C. Burvenich, A. E. Freeman, M. E. Kehrli, J. C. Detilleux, J. Sulon, J. F. Beckers and D.
Hoeben. 1999. Pregnancy-associated glycoprotein and decreased polymorphonuclear leukocyte
function in early post-partum dairy cows. Vet. Immun. Immunop. 67:47-54.
Greenwood, F. C., W. M. Hunter, J. S. Glover. 1963. The preparation of I131 labelled human growth
hormone of high specific radioactivity. J. Biochem. 89:114–123.
Ledezma-Torres, R. A., J. B. Beckers and W. Holtz. 2006. Assessment of plasma profile of pregnancy
associated glycoprotein (PAG) in sheep with a heterologous (anti-caPAG(55+59)) RIA and its
potential for diagnosing pregnancy. Theriogenology 66:906-912.
Lopez-Gatius, F., J. M. Garbayo, P. Santolaria, J. Yániz, A. Ayad, N. M. de Sousa and J. F. Beckers.
2007. Milk production correlates negatively with plasma levels of pregnancy-associated
glycoprotein (PAG) during the early fetal period in high producing dairy cows with live fetuses.
Domest Anim Endocrin. 32:29-42.
Nguyen, V. H., O. Barbato, X. N. Bui, J. F. Beckers and N. M. Sousa. 2012. Assessment of pregnancy-
associated glycoprotein (PAG) concentrations in swamp buffalo samples from fetal and maternal
origins by using interspecies antisera. J. Anim. Sci. 83:683–689.
Patel, O. V., J. Sulon, J. F. Beckers, T. Takahashi, M. Hirako, N. Sasaki and I. Domeki. 1997. Plasma
bovine pregnancy-associated glycoprotein concentrations throughout gestation in relationship to
fetal number in the cow. Eur. J. Endocrin. 137:423–428.
Perenyi, Z., O. Szenci, J. Sulon, P. V. Drion and J. F. Beckers. 2002. Comparison of the Ability of
three Radioimmunoassay to Detect Pregnancyassociated Glycoproteins in Bovine Plasma.
Reprod. Dom. Anim. 37:100–104.
Singh, S., O. P. S. Sengar and S. N. Singh. 1963. Prenatal development of buffalo (Bos bubalis L).
Agra. Univ. J. Res. 12:197–245.
Uoc, N.T., D. D. Long, L. V. Ty, D. Chupin, J. R. Renard and X. N. Bui. 1997. Effects of estradiol-17
and hCG supplementation on superovulatory responses and embryo quality in swamp buffalo
(Bubalus bubalisi) implanted with norgestomet. Animal. Reprod. Sci. 47:181- 187.
Zarrouk, A., I. Engeland, J. Sulon and J. F. Beckers. 1999. Determination of pregnancy-associated
glycoprotein concentrations in goats (Capra hircus) with unsuccessful pregnancies: a
retrospective study. Theriogenology 51:1221-1231.
450
Table 1. Buffalo PAG concentration during gestation.

Kind of Mean of PAG concentration (ng/ml) in estimate of gestation (month) (Mean±SEM)
samples 3 4 5 6 7 8 9 10 11
Maternal 24.63± 19.44± 8.22±3. 12.55 12.06± 15.21± 14.98 13.91± 25.06±
plasma 2.35 6.67 18 ±3.97 2.74 2.38 ±3.02 4.30 4.93
Fetal 16.21± 38.6±2 20.23± 19.15 28.66± 23.3±3. 13.6± 17.98± 11.48±
plasma 3.14 7.82 13.21 ±3.87 12.45 89 3.26 6.44 2.35
Allantoic 5.29±1. 4.95±1. 2.92±0. 3.64± 3.92±1. 3.56±0. 5.99± 8.19±3. 9.63±3.
fluid 86 76 52 0.75 35 62 1.90 94 79
Amniotic 5.71±0. 6.10±0. 6.96±6. 11.17 18.17± 7.71±2. 5.77± 4.98±1. 4.09±1.
fluid 96 92 09 ±4.22 14.41 80 2.30 12 51
[PAG] ng/ml
[PAG] (ng/ml)
30 40 y = -0.4305x2 + 4.5561x + 13.034

y = 0.7857x2 - 10.978x + 49.335
R = 0.59
R = 0.852 35
25
30
20 25
15 20
15
10
10
5
5
0 0
2 3 4 5 6 7 8 9 10 11 12 2 3 4 5 6 7 8 9 10 11 12
Estimate time of gestation (month)
Estimate age of gestation (month)
a) Maternal plasma b) Fetal plasma

[PAG] (ng/ml)
[PAG] (ng/ml)
11 y = 0.2572x2 - 3.0484x + 12.363 20 y = -0.47x2 + 6.3187x - 10.217

10 R = 0.97 18 R = 0.69
9 16
8 14
7
12
6
10
5
8
4
6
3
4
2
1
2
0 0
2 3 4 5 6 7 8 9 10 11 12 2 3 4 5 6 7 8 9 10 11 12
Estimate time of gestation (month) Estimate time of gestation (month)
c) Allantoic fluid d) Amniotic fluid

Figure 1. Profiles of PAG concentrations during gestation in difference samples
In bovine, PAG can be detected in maternal circulation of some cows at around Day 28 of pregnancy,
and in all cows (concentrations higher than 0.5-0.8 ng/ml) from 30 to 35 Days of pregnancy (Zoli
et al., 1992). In early and mid-gestation, concentrations increase slowly and gradually, remaining
below than 160 ng/ml till day 240 of pregnancy (Zoli et al., 1992). At one week before calving,
the bPAG1 average peak value over all cows was 1714 ng/ml in the U.S. study and 1975 ng/ml in
the European study (Dosogne et al., 1999). Around parturition, PAG concentrations increase
rapidly to reach peak values of 1 to 5µg/ml only few days before delivery (Zoli et al., 1992). The
profile of peripheral PAG concentrations is a useful indication of the feto–placental status (Patel
et al., 1997). To the best of our knowledge, this study is the first to characterize buffalo PAG
profiles during gestation, with differences kind of sampling. To conclude, our findings indicate
that PAG concentrations are correlated to the stage of gestation and that the correlation
coefficient was highest in allantoic fluid.
451
Effect of Povidone-Iodine Uterine Flushing on Insemination Success Rate

for Grazing Dairy Buffaloes
Caro B. Salces a, Gundolino P. Bajentingb, A. Casinillob and Karen S. Ciroyb

a
Center Director, Philippine Carabao Center at Ubay Stock Farm (PCC USF), Ubay, Bohol,
Philippines. bChief, farm operation, Dairy in-charge and animal record officer, respectively, Philippine
Carabao Center at Ubay Stock Farm (PCC USF), Ubay, Bohol, Philippines
*Corresponding email :cbspcc2000@yahoo.com
ABSTRACT
An operational research on evaluating the reproductive performance of dairy buffaloes with
uterine povidone-iodine infusion was conducted at the Philippine Carabao Center, Ubay Stock Farm for
the purpose of improving management protocol for artificial insemination (A.I.) in dairy buffalo cows.
Results showed that average calving interval of 12 months (28 animals) was achieved with A.I., and
uterine povidone-iodine infusion reducing the total herd calving interval to 13 months (26 animals) in
2012. Other factors such as parity number, months and year of calving had no significant effect on
calving interval.
Effects of uterine flushing with 2% povidone-iodine solution were observed in terms of reducing
days to first service and increase in pregnancy. Majority (34.8 %) of the animals came into heat at 18 to
42 days postpartum period (approximately 2nd heat cycle); while only 19.9% to 23.8% of the animals
came into heat in later heat cycles. Likewise, A.I. success rate was significantly higher (67.6%) for
animals inseminated within 42 days postpartum period compared to animals inseminated in later days
postpartum (36.3% - 47.4%). This indicated that A.I. success rate was higher when done 42 days
postpartum compared to the traditional practice of conducting A.I. 60 days postpartum.
Increasing the level of iodine for flushing to 4% solution showed further improvements in most
reproductive parameters. Return to normal condition of the uterus was shortened to 29 days postpartum
compared to 40 days for the control. Days to 1st A.I. service was reduced to 33 days postpartum
compared to 61 days for animals without flushing. A.I. success rate was increased to 75% compared to
57% and 33.3% for 2% iodine solution and control, respectively.
In summary, results of the experiments showed that uterine povidone-iodine infusion at calving
reduced uterine involution period. Reduction in uterine involution period resulted to early occurrence
of heat and early insemination postpartum. Early insemination within 42 days postpartum resulted to
higher A.I. success rate and subsequently shorter calving intervals in dairy buffaloes.
Keywords: Uterine flushing, Artificial insemination, Calving interval, Buffaloes
INTRODUCTION
Reproduction performance of buffaloes in the Philippines particularly under ranch production
system is relatively poor. Experiences at the Philippine Carabao Center at Ubay Stock Farm (PCC at
USF) where dairy and beef buffaloes were raised under ranch production system with natural breeding
showed an average calving interval of approximately 22-24 months (Salces et al., 2006).
In 2004, the center implemented the early calf weaning system at the age of 4 months and it
effectively reduced the calving interval to 18 months for breeding animals (Salces et al., 2006). A day
old weaning system for dairy buffaloes was also implemented in 2007; likewise, the system reduced
calving interval to 15 to 16 months with natural breeding.

452
Improving reproductive performance with the use of artificial insemination (A.I.) as the primary
mating system in the dairy farm was a challenge in 2009; the objective was to achieve a calving
interval of 14 months in the dairy buffaloes.
Uterine flushing with iodine solution has been used as a veterinary procedure to treat animals
with problems during parturition (Sarabia et al., 2009). The procedure is known to enhance the
recovery of the uterus and treat post breeding endometritis (Brinsko, 2001). While this technique has
been shown to improve pregnancies in problematic breeding animals the possibility of using the
technology to all cows as preventive treatment to enhance recovery and protection of the uterus from
infection for improving fertility and rebreeding has to be evaluated.

Study 1. Reproductive Performance of Dairy Buffaloes with Uterine Povidone-Iodine Solution
Infusion on Heat Occurrence and Artificial Insemination Success Rate
Purely natural breeding was implemented during the period 2006 to 2008 while artificial
insemination with clean up bull was implemented in 2009 to 2011. A teaser bull was also used as aid in
heat detection in the implementation of the AI program. Within 48 hours after birth, all newly calved
cows were flushed with 120 ml of 2% povidone-iodine solution (20 ml Povidone-Iodine 10% solution,
Betadine® + 80 ml distilled water) into the uterus and reproductive tract to cleanse the organ in
preparation for the next cycle. Uterine flushing was done using 60 ml syringe attached to an A.I. straw
sheet. Two doses of 60 ml 2% povidone-iodine solution was inserted into the uterus guided by hand
through rectal palpation. Cows calving between July 2009 and December 2011 were reproductive tract
and uterine flushed with the 2% Povidone-Iodine Solution.
Heat detection was observed from the teaser bull before and after milking in the morning (4:00
to 6:00 AM) and in the evening at 5:00 to 7:00 PM.
Calving intervals from 2007 to 2009 for purely natural breeding and calving intervals from
2010 to 2012 with A.I. and natural breeding were considered in the analysis for calving interval.
Study 2. Evaluation of Three Levels of Povidone-Iodine Solution Flushing on Uterine Involution, Heat
occurrence and Pregnancy in Dairy Buffaloes.
Thirty pregnant buffaloes were selected for the experiment and randomly allotted to a
completely randomized design (CRD) as follows: T1 – control (no uterine flushing); T2 – 2% solution
(20 ml povidone-iodine 10% solution, Betadine® + 80 ml distilled water); T3 – 4% solution (40 ml
povidone-iodine 10% solution, Betadine® + 60 ml distilled water). Uterine flushing was done once
within the first
48 hours from birth. Uterine status was recorded and monitored at 5 days interval. Data were
analysed by analysis of variance following a completely randomized design with the model: Y=µ +
Povidone-iodine level + error.

Study 1. Reproductive Performance of Dairy Buffaloes with Uterine Povidone-Iodine Solution
Infusion on Heat Occurrence and Artificial Insemination Success Rate
The average calving interval (Table 1) was 16 months with purely natural breeding (2007 -
2009) and 14.4 months with A.I. and natural breeding (2010 – 2012). Other factors affecting calving
interval such as year, months of calving, and parity number from 2007 to 2011 did not cause significant
differences on calving interval. Result of the implementation of A.I. in the dairy herd from 2009
to 2012 showed that A.I. was an effective at shortening calving interval in dairy buffaloes. The shortest
annual calving interval of 13 months from 43 cows was achieved in 2012, where a combination of A.I.
and natural breeding was implemented. An average of 12 months calving interval from A.I. (28
animals) was likewise achieved in 2012; this was a major factor in reducing the total herd calving
interval of 13 months. (Table 1).
453
Study 2. Evaluation of Three Levels of Povidone-Iodine Solution Flushing on Uterine Involution, Heat
occurrence and Pregnancy in Dairy Buffaloes
Results showed that return to normal condition of the uterus was significantly earlier in treated
animals with 29 days for 4% solution and 31 days for 2% solution and 40 days for control animals. A
normal size but hard uterus was also recorded in control and 2% solution treated animals but none in
4% solution treated animals. Average occurrence of heat postpartum was lowest at 33 days for 4%
solution treated animals; followed by 44 days for 2% solution treated, and 61 days for control. A.I.
success rate was also highest with 75% for 4% solution; followed by 2% solution (57%) and control at
33.3% A.I. success rate (Table 2).
Result of the trial explains the current status of A.I. success rates in the country and is in
agreement with the result of the first experiment. The low success rate of A.I. in buffaloes could be due
to uterine involution and possible uterine infection. Without uterine flushing uterine involution takes
longer and infection was possible, causing lower A.I. success rate as shown by 33% success rate in
control animals. The three year study consistently showed that success rate was higher when done
within 18-42 days postpartum.
In summary uterine povidone iodine infusion within 48 h after calving reduced uterine
involution period. Reduction in uterine involution period resulted to early occurrence of heat and early
insemination postpartum. Early insemination within 42 days postpartum resulted to higher A.I. success
rate and subsequently shorter calving intervals in dairy buffaloes.
REFERENCES
Bringko, S.P. 2001. How to Perform Uterine Lavage. Indication and Practical Techniques. In:
Proceeding of the Annual Convention of AAEP. Vol 47 pp 407-411.
Bozzini, G. 2012. Medicate Uterine Infusions as a mean of Achieving Higher Rates of Conception.
Google pub. May 22, 2012.
Dehligner, K. 2012. Uterine Lavage for Camelid Breeding Management. Google pub. May 22,
2012.
Salces, C.B., G.P. Bajenting ,K.S. Ciroy and O. Godinez. 2006. Effect of early weaning on calving
intervals of dairy and beef buffaloes under ranch production systemIn: Proceedings of the 43rd
Scientific Seminar and Annual Convention. Phil. Society of Animal Science. Boracay Island,
Aklan, Philippines. p.42
Salces, C.B., G.P. Bajenting and K.S. Ciroy. 2007. Effect of early weaning on calving intervals in
dairy and beef buffaloes under ranch production system. In: Proceedings of the 43rd Scientific
Seminar and Annual Convention. Phil. Society of Animal Science. Borakay Island, Aklan,
Philippines. pp. 25-26
Sarabia, A.S. 2009. Dairy Buffalo Production Hanbook. Phillippine Carabao Center. Science City of
Monuz. Pp 36-37.
454
Table 1. Calving intervals (months) of dairy buffaloes from 2007 to 2012 with 3 different breeding
methods at PCC USF dairy farm.
BREEDING 2007 2008 2009 2010 2011 2012
METHOD
Natural 15.2 (17) 15.9(19) 16.9 (26) 17.8 (26) 17.1 (23) 15(15
A.I. n/a n/a n/a 15.1 (28) 12.9 (26) 12(28)

Natural & n/a n/a n/a 15.6 (54) 14.7 (49) 13 (43)
A.I.
Table 2. Effect of uterine flushing with 3 levels of povidone-iodine solution on reproductive

performance of dairy buffaloes at PCC USF.
Levels of povidone-iodine n DPAI* DNU* Pregnancy rate
solution
B1 – control or 0% solution 9 60.9a 39a 33%
B2 – 2% solution 14 43.9 ab
31ab 57%
B3 – 4% solution 8 32.7 b
29 b
75%
DPAI – days postpartum A.I.Uterine involution period; DNU – days to normal uterus
* Means with different superscripts are significantly different, P< 0.05
455
Effects of Curcumin on Buffalo Embryonic Development In Vitro
Jiang-Hua SHANG, Chun-Yan YANG, Hai-Ying ZHENG, Jing-Qin, Jian WANG, Fen-Xiang
HUANG, Bing-Zhuang YANG and Xiang-Wei LIANG*

Nanning 530001, P.R. China.
ABSTRACT
The present study investigated the effects of curcumin on buffalo embryonic development by
incubating zygotes without or with the compound during in vitro culture (IVC). Cumulus–oocyte
complexes retrieved from antral follicles of water buffalo ovaries were matured for 20-22 hrs and
inseminated with motile buffalo sperm in Tyrode’s medium for 24 hrs. The presumed zygotes were
washed 3 times and transferred into 50 µl droplets of IVC medium (TCM 199 + 10% FBS)
supplemented with curcumin in various concentrations (0 µM, 2.5 µM, 5 µM, 10 µM and 20 µM,
respectively) and co-cultured with buffalo cumulus cells monolayer for more than 7 days to
evaluate the developmental ability of embryos (0, 2.5, 5, 10 and 20 µM groups, respectively). The
cleavage rate (CR) and blastocyst rate (BR) were assessed at 48 hrs and 168 hrs, respectively, after
fertilization (0 hrs). The results showed that there was a slight decrease in the CRs from 0 µM group
(control) to 20µM group (60.34%, 56.18%, 57.69%, 55.09% and 45.58%, respectively) but there
was no statistical difference among all groups. However, the BR in 20 µM group (9.82%) was
significantly lower (p﹤0.05) than those from 0µM to 10 µM groups (30.61%, 26.30%, 26.80% and
27.48%, respectively). Thus, we concluded that exposure of buffalo zygotes with high-dose
curcumin (20 µM) during IVC has injurious effects on embryonic development in vitro.
Keywords: buffalo, curcumin, embryo, in vitro culture (IVC)
INTRODUCTION
Curcumin, a yellow pigment of Curcuma longa, has been applied as an anti-inflammatory,
anti-oxidative and anti-carcinogenic compound (Ramsewak et al., 2000). The multiple therapeutic
effects of curcumin have manifested apoptosis-inducing, anti-proliferative and anti-carcinogenic
activities in a variety of cell lines and animals. Because of the efficacy in human tissues, curcumin
was also applied as a medicine to prevent and treat various diseases, including cancers of the colon,
breast and pancreas.
Up to now, only a few studies have investigated the potential effect of curcumin on embryo
development in vivo and/or in vitro. A study using rat as animal model showed that orally
administered curcumin had no toxic effects on fertility or pregnancy in the fed rats and no
malformations in their offspring (Ganiger et al., 2007). Another study confirmed that zebrafish
embryos treated with curcumin was led to the developmental defects include bent or hook-like tails,
spinal column curving, edema in pericardial sac, retarded yolk sac resorption, and shorter body
length (Wu et al., 2007). Moreover, recent studies showed that curcumin not only inhibits oocyte

456
maturation and disrupts spindle structure but also promotes injurious effects on in vitro fertilization
and embryonic development in mouse (Wu et al., 2007, Chen et al., 2010; Bielak-Zmijewska et al.,
2012; Chen et al., 2012; Huang et al., 2013).
These published data focused on small model animals. In our present study, we focus on
buffalo, an important big livestock, by incubating zygotes without or with 2.5, 5, 10 or 20µM
curcumin throughout the in vitro culture (IVC) to investigate the dose effects of curcumin on the
embryo development.

Chemicals
Chemicals and media were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Life
Technologies (GIBCO) (Grand Island, NY, USA), respectively, unless otherwise stated.
Oocyte maturation, fertilization and embryo culture in vitro
Ovaries of swamp buffalos and bovine were collected at a local slaughterhouse shortly after
slaughter and were transferred in physiological saline at 25 to 30 °C to the laboratory within 6 h.
Immature oocytes were retrieved from small (2 to 8 mm in diameter) antral follicles and only
Cumulus–oocyte complexes (COCs) with intact cumulus cells and evenly granulated cytoplasm
were selected for IVM in TCM 199 (GIBCO) supplemented with 10% FBS, 10 μg/mL FSH, 10
IU/mL LH, 1 μg/ml67 17 β-estradiol, 25 ng/mL EGF at 39℃, 5% CO2 in air atmosphere.
Spermatozoa were prepared from frozen-thawed semen, obtained from a Nili-Ravi bull that
was previously tested for IVF in our laboratory (Liang et al., 2007; Shang et al., 2007). An aliquot
(0.25 mL) semen was placed under 3 mL of Hepes-buffered Sperm-TALP medium supplemented
with 0.3% (w/v) BSA (Fraction V) in a conical tube for swim-up procedure. The supernatant was
collected and centrifuged at 500g for 5 min after incubation for 30 min at 39 °C. The pellet of
spermatozoa was resuspended and washed once in Fert-TALP medium, supplemented with 0.6%
(w/v) BSA (Fatty acid free), 10 ng/ml heparin and PHE (20 μM penicillamine, 10 μM hypotaurine,
0.5 μM epinephrine), and centrifuged again, then was resuspended to a final concentration (at
insemination) of 2 to 5 × 106 mL in the same medium.
At 20-22 hrs of maturation, the buffalo COCs were washed three times with Fert-TALP and
then introduced into insemination drops of 50 μL sperm suspension under mineral oil (10-15 COCs
per drop) in a humidified atmosphere of 5% CO2 in air at 39 °C.
Approximately 22 to 24 hrs after IVF, the putative zygotes were removed from Fert-TALP
and washed twice in culture medium (Hepes-buffered TCM-199 supplemented with 10% FBS) and
then randomly assigned equally into 5 groups. The grouped zygotes were washed once in culture
medium supplemented with curcumin at 0 (control), 2.5, 5, 10, and 20 μM (2.5, 5, 10, and 20 μM groups,
respectively), and then transferred into 50 μL droplets of the same medium and co-cultured with
buffalo cumulus cells monolayer for more than 7 days to assess the percentage of blastocysts. Fresh
culture medium was replaced half of volume in the drop every 48 hrs. The cleavage rate (CR) and
blastocyst rate (BR) from inseminated oocytes were assessed at 48 hrs and 168 hrs, respectively,
after fertilization (0 hrs).
The data were analyzed using one-way ANOVA of SPSS 11.5 (SPSS Inc, Chicago, IL, USA)
and are presented as the mean±SEM, with significance set at p<0.05.
457

Several researches have confirmed that curcumin exerts an adverse effect on zebrafish and
mouse embryos (Wu et al., 2007, Chen et al., 2010; Bielak-Zmijewska et al., 2012; Chen et al.,
2012; Huang et al., 2013). Chen et al. (2012) reported that high-dose (20 µM) of
curcumin-pretreated mouse oocytes display significantly decreased fertilization rates and cleavage
to the two-cell and blastocyst stages, compared to the untreated control (0µM) and low-dose (5 and
10 µM) groups, indicating that curcumin induces loss of fertilization and subsequent embryonic
development in a dose-dependent manner. Also, Huang et al. (2013) demonstrated the mouse
blastocysts treated with 24 μM of curcumin did not survive in vitro, and the implantation of the
pretreated blastocysts differed significantly between the treatment (6 and 12 μM of curcumin) and
control groups (0 μM of curcumin).
As shown in Table 1, in our study, there was a slight decrease in the cleavage rates from 0
µM group to 20µM group (60.34%, 56.18%, 57.69%, 55.09% and 45.58%, respectively); however,
there was no statistical difference (p>0.05) among all groups. However, the percentage of
blastocysts developed from inseminated oocytes in 20 µM group (9.82%) was significantly lower (p
﹤0.05) than those from 0µM to 10 µM groups (30.61%, 26.30%, 26.80% and 27.48%, respectively).
The results are in consistency with those from other reports. Thus, we concluded that exposure of
buffalo zygotes with high-dose curcumin (20 µM) during the IVC has injurious effects on
embryonic development in vitro.
ACKNOWLEDGEMENTS
This research was supported by grants from the National Natural Science Foundation of
China (31160456), the Natural Science Foundation of Guangxi, China (GuiKeZi 0991011,
2011GXSFB018045), the Scientific Project of Guangxi Bureau of Fisheries, Animal Husbandry and
Veterinary (GuiMuYuKe 1204910) and the Fundamental Research Funds for Guangxi Buffalo
Research Institute (ShuiNiuJi 0830001).
REFERENCES
Bielak-Zmijewska A, M. Sikora-Polaczek, K. Nieznanski, G. Mosieniak, A. Kolano, M.
Maleszewski, J. Styrna and E. Sikora. 2010. Curcumin disrupts meiotic and mitotic
divisions via spindle impairment and inhibition of CDK1 activity. Cell Prolif. 43(4):
354-364.
Chen, C.C., M.S. Hsieh, Y.D. Hsuuw, F.J.Huang and W. H Chan. 2010. Hazardous effects of
curcumin on mouse embryonic development through a mitochondria-dependent apoptotic
signaling pathway. Int. J. Mol. Sci. 11: 2839–2855.
Chen C. C. and W. H.Chan. 2012. Injurious effects of curcumin on maturation of mouse oocytes,
fertilization and fetal development via apoptosis. Int. J. Mol. Sci. 13: 4655-4672.
Ganiger S, H.N. Malleshappa and H. Krishnappa. 2007. A two generation reproductive toxicity
study with curcumin, turmeric yellow in Wistar rats. Food Chem. Toxicol. 45(1): 64–69.
Huang F.J., K.C. Lan, H.Y. Kang, Y.C. Liu, Y.D. Hsuuw, W.H. Chan and K.E. Huang. 2013. Effect
of curcumin on in vitro early post-implantation stages of mouse embryo development.
EUR. J. Obstet. Gyn. R. B. 166(1): 47-51.
Liang X., X. Zhang, B. Yang, M. Cheng, F. Huang, C. Pang, G. Qing, C. Liao and S. Wei.
Senatore E.M., Bella A., Presicce G.A., 2007. Pregnancy and calving rates following
transfer of in-vitro-produced river and F1 (river × swamp) buffalo (Bubalus bubalis)
458
embryos in recipients on natural oestrus or synchronized for ovulation. Reprod. Fertil. Dev.
19(5): 670–676.
Shang J.H., Y.J. Huang, X.F. Zhang, F.X. Huang and J. Qin, 2007. Effect of β-mercaptoethanol and
buffalo follicular fluid on fertilization and subsequent embryonic development of water
buffalo (Bubalus bubalis) oocytes derived from in vitro maturation. Ital. J. Anim. Sci.
6(Suppl. 2): 751-754.
Ramsewak R.S., D.L. DeWitt and M.G. Nair. 2000. Cytotoxicity, antioxidant and anti-inflammatory
activities of curcumins I–III from Curcuma longa. Phytomedicine 7(4): 303–308.
Wu J.Y., C.Y. Lin, T.W. Lin, C.W. Ken and Y.D. Wen. 2007. Curcumin affects development of
Zebrafish embryo. Biol. Pharm. Bull. 30(7): 1336-1339.
Table 1: Effects of curcumin supplemented in the IVC media on cleavage and blastocyst rates.
Curcumin (μM) Oocyte Replicates Cleavage (%) Blastocyst (%)

0 213 11 130 (60.34±14.33) 64 (30.61±12.92) a
2.5 218 11 115 (56.18±19.30) 58 (26.30±16.02) a
5 222 11 135 (57.69±14.71) 60 (26.80±13.15) a
10 217 11 116 (55.09±13.06) 61 (27.48±12.29) a
20 212 11 97 (45.58±15.00) 21 (9.82±7.42) b
p=0.307 p=0.017
a,b
Within the same parameter, values with different superscript differed significantly (p<0.05).
459
Supplementation of Epidermal Growth Factor into In Vitro Maturation Medium

Improves Fertilization Efficiency of Swamp Buffalo Oocytes
Nguyen VIET LINH, a* Van Hanh NGUYEN, a Xuan Nguyen BUI, a Nguyen Thi UOCa and Bui
Thi XUAN MINHb
a
Laboratory of Embryo technology, Institute of Biotechnology, Vietnam Academy of Science and
Technology,Hanoi, Vietnam;
b
Center of Prenatal Diagnosis, National Hospital of Obstetrics and Gynecology, Hanoi, Vietnam.
*
Corresponding e-mail: nvlinh@ibt.ac.vn
ABSTRACT
We have demonstrated that supplementation of 17β-estradiol during IVM could significantly
improve the fertilization and embryo production in swamp buffalo. In the present study, we reported
further attempt on improvement of IVM and IVF of swamp buffalo oocytes by supplementation of
epidermal growth factor into IVM medium. Cumulus-oocyte complexes were collected from follicle
of 1-5mm in diameter of ovaries of slaughtered swamp buffaloes. In vitro maturation in medium
containing 0.5 µg/ml FSH, 0.1µg/ml LH, 3 µg/ml 17β-estradiol with or without epidermal growth
factor (EGF) 15µg/ml (group 1 and group 2, respectively) was carried out in 28 hours at 38.5 0C
under 5% CO2 humidified atmosphere. In vitro fertilization was conducted with thawed semen of
swamp buffalo either separated by swim-up or Percoll procedures. In vitro culture of zygotes was in
B2 Menezo medium. Maturation rate (estimated by extrusion of the first polar body) of oocytes
cultured with EGF was significantly higher than that of oocytes cultured without EGF (68.8 ± 0.2 and
56.7 ± 0.2%, respectively). With both sperm separation methods, cleavage and blastocyst formation
rates of oocytes matured with EGF were significantly higher than those of oocytes matured without
EGF: 69.2 ± 0.4 and 19.5 ± 0.3% vs. 55.0 ± 0.7 and 13.8 ± 0.4%, respectively for swim-up; 66.4 ± 0.5
and 19.3 ± 0.3% vs. 54.6 ± 0.3 and 14.1 ± 0.2%, respectively for Percoll. These results showed that
supplementation of 15µg/ml EGF could effectively contribute to maturation and subsequent
fertilization and development of oocytes of swamp buffaloes.
Keywords: swamp buffalo, IVF, IVM, EGF, swim-up, Percoll
INTRODUCTION
Since the first buffalo calf born from in vitro fertilization of buffalo ooctyes (Madan et al.,
1991), in vitro embryo production (IVEP) has proved applicable for research and development in
buffalo, a popular domestic animal of Asia, especially of India and Southeast Asia countries.
However, due to poor superovulatory response, productivity of IVF in buffalo is still low (Suresh et
al., 2009). Many attempts have been made for improving the IVEP systems. For collecting buffalo
oocytes, aspiration and slicing have been applied to abattoir ovaries (Nandi et al., 2002).
Supplementation of substances such as buffalo serum (Chauhan et al., 1998), hormones (Samad et al.,
1998), steer serum and follicular fluid (Nandi et al., 2002) has been conducted in order to improve the
maturation rates and along with that, in vitro fertility and development of buffalo embryos. Epidermal
growth factor (EGF) has been widely used either alone or combined with other substances. In river
buffalo, EGF, combined with FSH, or insulin, transferrin and selenium (ITS) contributed to
maturation and development of buffalo oocytes after IVF (Raghu et al., 2002). Moreover, EGF
combined with IGF-1helped increasing the rates of matured oocytes, penetration and development
after IVF (Purrohit et al., 2005). However, effect of EGF on maturation and development after IVF of
swamp buffalo is not well reported. In the present study, we report the effect of EGF on swamp

460
buffalo oocyte maturation and IVF embryo development in a system previously improved by
supplementation of 3 µg/ml 17β-estradiol (Viet Linh et al., 2005).

Oocyte collection and in vitro maturation (IVM)
Ovaries were collected at a local abattoir and transported at 25 0C to the laboratory within 5
hours in physiological saline. Cumulus-oocytes complexes (COCs) were collected from follicle of 1-5
mm in diameter, washed several times in TCM 199 supplemented with 5% FBS and HEPES. COCs
were then subjected to in vitro maturation (IVM) in TCM 199 supplemented 0.5 µg/ml FSH,
0.1µg/ml LH, 3 µg/ml 17β-estradiol, 10% FBS, and either with or without epidermal growth factor
(EGF) 15µg/ml for 28 hours at 38.5 0C in humidified atmosphere. Assessment of maturation status is
carried out after removal of cumulus layers by treatment with hyaluronidase and gentle pipetting. An
oocyte is considerred matured if at the time of assessment it possesses a visible first polar body.
In vitro fertilization and in vitro culture
In vitro fertilization were carried out with frozen swamp buffalo semen (Moncada breeding
farm, Vietnam). Semen straws were thawed in a waterbath at 37 0C for 10 seconds. Straws were then
exposed to room temperature for further 20 seconds. Motile spermatozoa were separated either by
swim-up or Percoll procedure. COCs were washed in TCM 199 supplemented with FBS and HEPES.
Subsequently, groups of 10 oocytes were washed in fertilization medium (FERT), and transferred to
50µl drops of FERT supplemented with epinephrine (1µM), hypotaurine (10µM), penicilliamine
(20µM) and heparine (0.5µg/ml). In vitro fertilization of groups was carried out in 4 hours. Zygotes
were then transferred from drops of FERT into 1ml TCM 199 HEPES in a 1.5 centrifuge tube and
vortexed for 2 minutes at medium speed to remove cumulus cells. Denuded zygotes were then sorted
from the debris, washed in B2 Menezo medium. Embryos were cultured in 50µl drops of B2 Menezo
for 7 days. Number of cleaved embryos and blastocysts were examined in each group.

Effect of supplementation of EGF on in vitro maturation of buffalo oocytes is presented in
Table 1. Maturation rate (estimated by extrusion of the first polar body) of oocytes cultured with EGF
was significantly higher than that of oocytes cultured without EGF (68.8 ± 0.2 and 56.7 ± 0.2%,
respectively). Effect of EGF on subsequent development of buffalo embryos after IVF were shown in
Table 2. For swim-up sperm separation method, cleavage and blastocyst formation rates of oocytes
matured with EGF were significantly higher than those of oocytes matured without EGF: 69.2 ± 0.4
and 19.5 ± 0.3% vs. 55.0 ± 0.7 and 13.8 ± 0.4%, respectively. Similar phenomenon happened when
we fertilized buffalo oocytes with sperm prepared by Percoll protocol, cleavage and blastocyst rates
were significantly higher in treated group than that in non-treated group: 66.4 ± 0.5 and 19.3 ± 0.3%
vs. 54.6 ± 0.3 and 14.1 ± 0.2%, respectively. Sperm preparation protocols did not affect the outcome
of in vitro fertilization, in both swim-up and Percoll procedure, we obtained similar rate of cleavage
and blastocyst.
These results showed that supplementation of 15µg/ml EGF contribute to maturation and
subsequent fertilization and development of oocytes of swamp buffaloes. The results of the present
study are in accordance with those in other reports in river buffalo. Culturing river buffalo oocytes in
both complex co-culture system and modified synthetic oviduct fluid, Nandi et al. (2003) concluded
that that EGF had more beneficial effect on buffalo oocyte maturation, and embryo cleavage than
FGF. EGF is also reported to have positive effect on maturation and cleavage after IVF of river
buffalo oocytes, especially when combined with IGF-1 (Purohit et al., 2005). Combined with serum
and ITS in a complex co-culture system, EGF showed its effect on not only maturation rate but also
on blastocyst formation rate of oocytes fertilized in vitro (Raghu et al., 2002). The results obtained in
the present study showed a similarity in the effect of EGF on maturation and development of swamp
461
buffalo oocytes to those of river buffalo ones. Similarly, supplementation of hormones, in this case,
FSH, LH, and 17β-estradiol has possitive effect on swamp buffalo oocytes (Viet Linh et al., 2005).
Combination of EGF on the pre-set system in our laboratory showed further improvement of buffalo
in maturation rate and development after IVF. Appearance of bovine serum in the medium has also
contribute to oocytes for completing events in maturation, fertilization and development.
In conclusion, supplementation of 15µg/ml EGF effectively contribute to maturation and
subsequent fertilization and development of oocytes of swamp buffaloes in combination with 5 µg/ml
FSH, 0.1µg/ml LH, 3 µg/ml 17β-estradiol, and 10% FBS. The results allow further research and
development on in vitro production of swamp buffalo embryos.
ACKNOWLEDGEMENTS
Presentation of this study is supported by Vietnam National Foundation for Science and
Technology Development (Nafosted) for young researcher.
REFERENCES
Chauhan M.S., S.K. Singla, P. Palta, R.S. Manik and M.L. Madan. 1998. In vitro maturation and
fertilization, and subsequent development of buffalo (Bubalus bubalis) embryos: effects of
oocyte quality and type of serum. Reprod. Fertil. Dev. 10: 173-177.
Khan I.Q., H.A. Samad and N.U. Rehman. 1997. Quantity and quality of buffalo follicular oocytes
recovered by aspiration and scoring methods for in vitro studies. Pak. Vet. J. 17: 187-189.
Madan M.L., S.K.Singla, S. Jailkhavi and J.D. Ambrose. 1991. In vitro fertilization in buffalo and
birth of first ever IVF buffalo calf Proceedings of Third World Buffalo Congress 1991;
Varna, Bulgaria, 7: 11-17.
Nandi S., B.M. Ravindranatha, P.S.P. Gupta, H.M. Raghu and P.V. Sarma. 2003. Developmental
competence and post thaw survivability of buffalo embryos produced in vitro: effect of
growth factors in oocyte maturation medium and of embryo culture system. Theriogenology
60: 1621-1631.
Nandi S., H.M. Raghu, B.M. Ravindranatha and M.S. Chauhan. 2002. Production of buffalo (Bubalus
bubalis) embryos in vitro: premises and promises. Reprod. Domest. Anim. 37: 65- 74.
Purohit G.N., M.S. Brady and S.S. Sharma. 2005. Influence of epidermal growth factor and insulin
like growth factor 1 on nuclear maturation and fertilization of buffalo cumulus oocyte
complexes in serum free media and their subsequent development in vitro. Anim. Reprod.
Sci. 87: 229-239.
Raghu H.M., S. Nandi and S.M. Reddy. 2002. Effect of insulin, transferrin and selenium and
epidermal growth factor on development of buffalo oocytes to the blastocyst stage in vitro in
serum semi-defined media. Vet. Rec. 151: 260-265.
Samad H.A., I.Q. Khan, N.U. Rehman and N. Ahmad. 1998. The recovery, in vitro maturation and
fertilization of Nili-Ravi buffalo follicular oocytes. Asian Aust. J. Anim. Sci. 11: 491-497.
Viet Linh N., N.T. Uoc, Q.X. Huu, N.V. Hanh, N.H. Duc, N.T. Thanh, B.L. Chi, N.K. Tich, D.D.
Long, F. Rennis and B.X. Nguyen. 2005. Effect of 17β-estradiol supplementation on the in
vitro maturation and embryo production in swamp buffalo. Proceedings of the Annual
Conference of the Asian Reproductive Biotechnology Society 2005, Hanoi, Vietnam: 167.
462
Table 1: Effect of EGF on in vitro maturation of buffalo oocytes.

Group No. of oocytes examined Matured oocytes (n, %)
1 1368 941 (68.8 ± 0.2)a
2 1290 732 (56.7 ± 0.2)b
8-10 replicates were performed. Different superscripts within a column indicate significant diference
(P<0.05).
Table 2: Effect of EGF on developmental competence of buffalo oocytes after IVF

Oocytes fertized by sperms treated by Oocytes fertized by sperms treated by
Group swim-up method (n, %) Percoll method (n, %)
Cultured Cleaved Blastocyst Cultured Cleaved Blastocyst
914 258 643 187
1 1320 a a 968 a
(69.2 ± 0.4) (19.5 ± 0.3) (66.4 ± 0.5) (19.3 ± 0.3)a
665 167 754 195
2 1210 1380
(55.0 ± 0.7)b (13.8 ± 0.4)b (54.6 ± 0.3)b (14.1 ± 0.2)b
8-10 replicates were performed. Different superscripts within a column indicate significant diference
(P<0.05).
463
Effect of Improving Reproductive Management on Dairy Farm Economics
Nasim AHMAD, * Ejaz AHMAD, Zahid NASEER, Umair RIAZ and Ali HUSNAIN
Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore-Pakistan

*Corresponding e-mail: drnasim@yahoo.com
ABSTRACT
Livestock is contributing 11.2% to national GDP and 55.1% agriculture GDP. The main source
of livestock is milk, meat and sale of animal and it’s by products. Although there is plenty of data
available on economic aspects of the dairy enterprises, very limited data is available on economic
analysis of dairy enterprise in Pakistan. The objectives of the experiment were to elaborate the effect of
improving reproductive management on farm productivity, to estimate the buffalo and cow farm
sustainability and to inspect the limitations and prospects at farm. Information of three production years
(2007, 2008, and 2009) was taken from a new commercial farm in District Kasur, Punjab through their
records, direct questioning and discussion. On fortnightly farm visits, advisory and technical services
were provided related to reproduction (fixed-time AI, synchronization, reproductive ultrasonography,
semen quality evaluation of frozen thawed doses, and heat detection etc). In this study the economics of
the dairy farm was analyzed by Gross Margin Analysis. Gross margin analysis was performed by
calculating the difference between gross output and variable cost of enterprise. The gross out
comprised of the milk and animal sales, milk consumption at farm and byproducts transferred. Variable
cost like purchased feed was calculated on financial price bases and price of those things which were
farm produced such as grains, were calculated on economic prices and opportunity cost principle
Due to improve reproductive and health management the birth rate gradually improved every year from
24% in 2007 to 40% in 2009 whereas the mortality rate decreased from 4.6% to 2.5% respectively. The
highest average milk yield/ animal (2509L/year) and per animal milk yield (6.9L/d) was recorded in
2009 and the lowest milk production during 2008 (6.0 L/d). During the year of 2009 gross margin and
overall profit was high that is due to improved milk production, sale of animals and reduction of cost
on feed. However, in 2007 and 2008 net profit was low. Generally the economic performance of the
farm is encouraging. Farm losses were significantly low 1.5M PKR in 2009 as compared to 7.5M PKR
in 2007. It is highly variable business, dependent on feeding cost and reproduction etc., and has the
ability to reduce losses significantly when reproduction is enhanced. Improved feeding and
reproductive management of dairy herd proves helpful in reducing the economic losses of the dairy
farm.
Keywords: Reproductive management, gross margin analysis, farm economics
INTRODUCTION
Livestock is major contributing sector in the local and national economy where it contributes
11% share in GDP of Pakistan (Anonymous, 2012). It also plays a vital role in farm household level
income where cow and buffaloes are main livestock species majorly owned by small farmers.
However, commercial dairy farming, for the last decade is growing at an enormous rate. Dairy farming
produces substantial amount of daily cash. It also subsidizes to the upgrading of the livelihoods of poor
people that is why in many developing countries including Pakistan dairy production is becoming
increasingly important. Reproductive performance influencing the profitability of dairy herds (Plaizier
et al., 1997; Esslemont et al., 2001) and affects the amount of milk produced per cow per day of herd
life, breeding costs, rate of voluntary and involuntary culling, and the rate of genetic improvement for
traits of economic importance (Britt, 1985: Evans et al., 2006). Prolonged state of negative energy

464
balance (NEB) in the periparturient and early postpartum periods is major cause in decreasing
reproductive efficiency in dairy cows (Van Saun, 1997: Roche et. al., 2000). Reproductive management
has received increased attention in recent years as new technologies and programs have been developed
to aid dairy managers in efficiently breeding cows and heifers (Olynk and Wolf, 2008). Although there
is plenty of data available on economic aspects of the dairy enterprises, very limited data is available on
economic analysis of dairy enterprise in Pakistan. The objectives of the experiment were to elaborate
the effect of improving reproductive management on farm productivity, to estimate the buffalo and cow
farm sustainability and to inspect the limitations and prospects at farm

Research site and animals
This study was conducted in Living Dairy Chunian (Commercial Dairy Farm), in Kasur District
(30.58°N, 73.59°E) comprising three years 2007 to 2009. The farm's dairy herd is of Nili-Ravi
buffaloes and cross bred dairy cows. The cross bred cows included blood of Holstein, Jersey,
Cholistani and Sahiwal.
Research design
Information of dairy enterprise costs and revenues was directly collected from the farm records
that cover the scope of three production years. Date collection techniques were based on question
answer and conversation with manager and workers of farm. During the period under report the project
team visited the farm fortnightly. Advisory and technical services related to reproduction e.g., animal
recording, heat detection, natural breeding and artificial insemination, frozen semen quality check and
use of ultrasonography for pregnancy testing and synchronization (fixed timed A.I,CIDR, Ovsynch
protocol, PG protocol ) etc.
In this study the economics of the dairy farm was analyzed by Gross Margin Analysis, which
was the unit of analysis in assessing the economic outlook of dairy business. Gross margin analysis was
performed by calculating the difference between gross output and variable cost of enterprise. The gross
out comprised of the milk and animal sales, milk consumption at farm and byproducts transferred.
Variable cost like purchased feed was calculated on financial price bases and price of those things
which were farm produced such as grains, were calculated on economic prices and opportunity cost
principle

Indicators of production performance of Commercial Dairy in District Kasur (Table 1)
The total herd size as well as number of milking animals decreased gradually during the three
years of study due to culling. The birth rate gradually improved every year from 24% in 2007 to 40% in
2009 whereas the mortality rate decreased from 4.6% to 2.5% respectively. This indicates that
reproductive performance enhanced. Obviously this is due to improved reproductive management
practices adopted gradually at the farm advised by the project team. In 2009, highest average milk
production was recorded and per animal milk yield (6.9L/d) and the lowest milk production during
2008 (6.0 L/d). This might be due to better management of feed resources and reproductive
management.
Economic performance & enterprise viability of the farm during the study period (Table 2)
Economic consequences due to a non optimal reproductive performance of a herd consist of
extra expenditures and losses in incomes (Seegers, 2006). Returns from sale of milk remained constant
in 3 years of research. Whereas returns from the culling of animals (4,010,000 PKR) were highest in
the last year so it improved the total returns as compared to earlier years (18,062,500 PKR). Total
variable expenses (Feeds, Medicaments, Casual labor, Maintenance & repair, Consumables, Electric
465
and water, Stationary) reduced significantly in last year as compared to earlier years and it happened
due to better management of resources.
The Maximum gross margin and net gain was taken during 2009 because of increase in milk
production, sales of animas and reducing cost on feed (Table 2). However, in 2007 and 2008 it was
very low. Production cost of milk/liter was less in 2007 as compared to selling price. It made the farm
to get significant profit. However cost of production increased in 2008 due to international crisis in
business which directly affected the prices of feed ingredients. In 2007 and 2008 the gross margin and
overall income was lowest because of reduced average milk yield, less number of animal heads and
increased cost of feed. Milk production cost /liter was higher than selling price from 2007 to 2009
resulting in fewer profit margins.
The major expense on dairy farm is incurred on feed (75%). The project team services were free
and not included in the overall farm expenditure. Farm contribution regarding conduct of research trials
and activities is also not calculated in this experiment. Overall, economically farm situation was
encouraging. It is highly variable business, dependent on feeding cost and reproduction etc., and has
the ability to reduce losses significantly when some technical advisory services are provided. This
suggests that project did not enhance the profitability but reduced the losses significantly. Improved
feeding and reproductive management of dairy herd proves helpful in enhancing the economics of the
farm.
REFERENCES
Anonymous. 2012. Economic Survey of Pakistan. Finance Division, Economic Advisors Wing,
Ministry of Finance, Government of Pakistan, Islamabad. p. 29.
Britt, J. H. 1985. Enhanced reproduction and its economics implications. J. Dairy Sci. 68:1585-1592.
Esslemont, R.J., M.A. Kossaibati and J. Allcock. 2001. Economics of fertility in dairy cows. In:
Fertility in the High Producing Dairy Cow, BSAS Occasional Publication No. 26, pp. 21–29.
Evans, R.D., M. Wallace, L. Shalloo, D.J. Garrick and P. Dillon. 2006. Financial implications of recent
declines in reproduction and survival of Holstein-Friesian cows in spring-calving Irish dairy
herds. Agri. System. 89: 165-183.
Olynk, N.J. and C.A. Wolf. 2008. Economic Analysis of Reproductive Management Strategies on US
Commercial Dairy Farms. J. Dairy Sci. 91: 4082-4091.
Plaizier, J.C.B., G.J. King, J.C.M. Dekkers and K. Lissemore. 1997. Estimation of economic values of
indices for reproductive performance in dairy herds using computer simulation. J. Dairy Sci. 80:
2775–2783.
Roche, J.F., D. Mackey and M.D. Diskin. 2000, Reproductive management of postpartum cows. Anim.
Reprod. Sci. 60-61: 703-712.
Seegers, H. 2006. Economics of the reproductive performance of dairy herds. World Buiatrics
Congress. pp. 292-302.
Van Saun, R.J. 1997. Prepartum nutrition: the key to diagnosis and management of periparturient
disease. In: The Bovine Proceedings — No. 30, September 1997. pp. 33–42.
466
Table 1. Indicators of Production Performance of Commercial Dairy Farm in District Kasur

Years
Production Parameters 2007 2008 2009
Before Project Project 1st Year Project 2nd Year
Total herd size/ year, LU* 325 300 275
Number of Milking cows/year 220 200 160
Average Birth rate/year, LU (%) 80 (24) 95 (31) 110 (40)
Average mortality rate/year LU (%) 4.6 3.3 2.5
Total milk yield, L/ year 511,000 438,000 401,500
Average Milk yield / year/ head, L 2323 2190 2509
Total milk yield/ day, L 1400 1200 1100
Average milk yield/day/head, L 6.36 6.00 6.87
Total herd size/ year, LU* 325 300 275
Newly purchased Culling begins, Feeding all silage
Remarks milk animals, high feeding instead of fresh
mortality, compromised fodder
*1 LU= 1 cow, 1 bull, 0.5 replacement heifer, 0.25 calves
Source: ACA dairy farm record (unpublished) Project 1st and 2nd Year: Fortnightly visit and
technical and advisory services provided related to reproduction by the ALP Project
Table 2. Economic Performance and Farm Viability

Particulars /Returns Net expenses and returns in PKR during the period
of three years
2007 2008 2009
Milk sale (Rs.) 14,308,000 14,454,000 14,052,500
Sale of calves, bulls, cull cows (Rs.) -- 100,000 4,010,000
Total Returns 14,308,000 14,554,000 18,062,500
Feeds(concentrate, fodder, silage) 16,790,000 19,272,000 14,330,000
Medicaments 200,000 180,000 150,000
Casual labor 150,000 180,000 120,000
Maintenance & repair 100,000 100,000 100,000
Consumables 10,000 10,000 9,600
Electric and water 480,000 480,000 480,000
Stationary 5000 5500 6000
Total Variable Expenses 17,735,000 20,227500 15,195,600
Gross Margin, (Total Returns Minus -3,427,000 -5,673,500 2,866,900
Total Variable Expenses)
Fixed Expenses
Permanent labor 2,400,000 2,400,000 2,400,000
Total Fixed Expenses (Rs.) 2,000,000 2,000,000 2,000,000
Total Expenses (Rs.) 22,135,000 24,627,500 19,595,600
Net Profit (Total Returns – Total -7,827,000 -10,073,500 -1,533,100
Expenses) (Rs.)
Loss (Rs. million) 7.8 10.0 1.5
Cost of production per liter (Rs.) 43 56 49
Selling Price of milk (Rs.) 28 33 35
467
Localization of GnRH Receptors in Buffalo Cow Pituitary Gland
in Follicular and Luteal Phases
Thuchadaporn CHAIKHUNa,b, Pongsiwa SOTTHIBANDHUc and Siriwat SUADSONGa*
a
Department of Obstetric Gynecology and Reproduction, Faculty of Veterinary Science,
Chulalongkorn University, Bangkok, 10330, Thailand
b
Clinic of Obstetric Gynecology Andrology and Artificial Insemination in Domestic Animals,
Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok, 10530, Thailand
c
Department of Veterinary Anatomy, Faculty of Veterinary Medicine, Mahanakorn University of
Technology, Bangkok, 10530, Thailand
*Corresponding e-mail: ssuadsong@hotmail.com
ABSTRACT
Several studies in the efficiency of gonadotropin releasing hormone (GnRH) for stimulating ovarian
resumption and controlling ovulation in buffalo cows have had mainly positive results. However,
information on the location of GnRH receptors (GnRHR) in the pituitary gland of buffalo cow has
not yet been reported. The objective of this research was to determine the localization of GnRHR in
the pituitary gland of buffalo cow during the follicular and luteal phases of the estrous cycle. The
pituitary glands were collected from 6 buffalo cows at the slaughterhouse after the heads were
perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) into a common carotid
artery, and prepared for paraffin blocks and further processed for immunohistochemistry against
GnRHR. The phases of their estrous cycle were identified by the presence of estrous signs and the
ovarian appearances (follicular phase; n=3, luteal phase; n=3). The results showed distributions of
GnRHR located in the cytoplasm of subpopulations of cells in the pars distalis and pars intermedia,
which might be regarded as gonadotrophs. In both the follicular and luteal phases, the GnRHR
positive cells were found intensely accumulated in the proximal part of the pars distalis and pars
intermedia. In contrast, in the distal part of both areas, only a few numbers of GnRHR positive cells
were detected. This study is the first to report the distributions of GnRHR in pituitary glands of the
buffalo cows. No difference was found in this distribution between the follicular and luteal phases
of the tested cows. This study suggests that the target cells of the GnRH released by hypothalamic
neurons are in the pituitary gland. Our findings expose some of the mechanisms involved in the
action of GnRH in the hypothalamic-pituitary-ovarian axis, the understanding of which is central to
the enhancement of buffalo reproductive performance.
Keywords: localization, GnRH receptors, buffalo cow, pituitary gland
INTRODUCTION
Mammalian reproductive function is controlled by the coordination of neuroendocrine substrates,
peptide hormones and steroid hormones in the hypothalamic-pituitary-ovarian (HPO) axis.
Gonadotropin releasing hormone (GnRH), a decapeptide hormone, is the main hormone of
reproduction and it, in turn, is controlled by kisspeptin and G protein-coupled receptors (GPR54 or
kisspeptin receptors) signaling in the hypothalamus in mammals (Roseweir and Millar, 2009;
Tsukamura and Maeda, 2011; Chaikhun et al., 2013). The effects of GnRH on luteinizing hormone
(LH) and follicle stimulating hormone (FSH) synthesis and secretion appear after the interaction of
GnRH with its cognate GnRH receptors (GnRHR) in the anterior pituitary gonadotrophs (Naor,
2009). Then, luteinizing hormone (LH) and follicle stimulating hormone (FSH) influence follicular

468
development, ovulation and steroidogenesis in the ovary and these gonadal steroid hormones show
feedback regulation of GnRH secretion along the estrous cycle (Tsukamura and Maeda, 2011).
Previous studies in the efficiency of GnRH for stimulating ovarian resumption and controlling
ovulation in buffalo cows have had mainly positive results, but there are many variations in
response depending on the state of estrous cycle and the physical status of the animal (Singh et al.,
1984; Aboul-Ela et al., 1985; Pattabiraman et al.,1986; Suthikrai, 1994; Chaikhun et al., 2010).
However, fundamental information on the location of GnRHR in the pituitary gland of buffalo cows
has not yet been reported. The objective of this research was to determine the localization of
GnRHR in the pituitary gland of buffalo cows during the follicular and luteal phases of the estrous
cycle.

Sample
Six pituitary glands of buffaloes were collected from slaughter houses. The heads were perfused
with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) into a common carotid artery within
15 min after the animals died. The pituitary glands were collected and fixed in the same fixative for
24 hr. The samples were prepared for paraffin blocks. All pituitary glands were identified according
to the phase of their estrous cycle (Fischer and Bodhipaksha, 1992; Ali et al., 2003; Tienthai et al.,
2008). Follicular phase pituitary glands (n=3) were initially determined according to the buffalo’s
estrous signs such as vulva edema, cleared vaginal discharge and a more than 1 cm of diameter of
follicle present in the ovary which was detected by observation and rectal palpation before
slaughter. Then, confirmation of the estrous stage was done by means of postmortem ovarian
morphology. Luteal phase pituitary glands (n=3) were initially determined by the fact that the
buffalo did not show estrous signs such as vulva edema, cleared vaginal discharge and a protruding
corpus luteum present in the ovary which was detected by observation and rectal palpation before
slaughter. Then, confirmation was done by means of the postmortem ovarian morphology.
Immunohistochemistry (IHC)
Serial paraffin sections from each block of samples were prepared at 4 microns for GnRHR
immunohistochemical study. The sections were deparaffinized and rehydrated in a graded series of
ethyl alcohol. Antigen retrieval was done by 800 w in microwave for 10 min. Following this step,
the endogenous peroxidase activity was blocked by incubating in 1% hydrogen peroxide in
methanol for 30 min at room temperature. The non- specific binding was blocked using 10% normal
horse serum (Gibco®) for 20 min at room temperature. The sections then were incubated with the
1:100 goat polyclonal anti-human GnRH receptor antibody (catalog no. sc-8682, Santa Cruz®) at 4
°C, overnight (16 hr). After that, a biotinylated link (LSAB+System-HRP, catalog no.K0679,
Dako®) was used at room temperature for an hour. The sections then were incubated with
streptavidine horseradish peroxidase (LSAB+System-HRP, catalog no.K0679, Dako®) at room
temperature for 50 min. In the final step, 3, 3’-diaminobenzidine (DAB, catalog no. K3467,
Dako®), a chromogen, was added to visualize bound enzyme (brown colour). In each step, the
sections were washed in 10 mM phosphate buffer saline (PBS; pH 7.2). Positive control reaction
was prepared using sheep pituitary gland, and negative control reaction was conducted using PBS
instead of primary antibody application. GnRHR-immunoreactive (ir) cells were identified under
light microscope. The number of GnRHR-ir cells as minority (<30% of total cells), intermediate
(30-70% of total cells) and majority (>70% of total cells).
RESULTS
The results showed reactions of GnRHR located in the cytoplasm of subpopulations of cells in the
pars distalis and pars intermedia, which might be regarded as gonadotrophs. In both the follicular
and luteal phases, the GnRHR positive cells were found intensely accumulated in the proximal part
of the pars distalis and pars intermedia and constituted the majority of cells in these areas. In
contrast, in the distal part of both areas, only a few numbers of GnRHR positive cells were detected.
No difference was found in this distribution between the follicular and luteal phases of the tested
469
buffalo cows which were >70% GnRHR-ir cells. The comparison between the positive GnRHR
sample reactions and the negative control samples is shown in Figure 1.
DISCUSSION
This study is the first to report the distributions of GnRHR in the pituitary glands of buffalo cows.
Although successful buffalo GnRHR gene detection has been reported (Konwar and Srivastava,
2005), GnRHR localization data in buffalo has never been presented in any reliable scientific
publication or database. Generally, GnRHR is expressed on the membrane of anterior pituitary
gonadotroph, which relates to reproductive function (Rispoli and Nett, 2005). Interestingly, our
study found GnRHR-ir cells located in the cytoplasm of subpopulations of cells in not only the pars
distalis but also in the pars intermedia with an intense accumulation in the proximal part. In other
animals, it has been found only in the pars distalis (Christopher et al., 1994; Soga et al., 2005). No
difference was found in this distribution between the follicular and luteal phases of the tested
buffalo cows in this study. However, there are many studies in GnRHR density relating to both
mRNA and numbers of GnRHR (which determines their ability to respond to GnRH). Research
studies on gonadotrophs in sheep (Clarke et al., 1987; Gregg and Nett, 1989; Nett et al., 2002),
cattle (Schoenemann et al., 1985) and laboratory animals (Bauer-Dantoin et al., 1995; Yasin et al.,
1995) found that the density has been shown to increase during the follicular phase and is at its
highest point just pre-ovulation. This evidence supports the long known fact that estrogen is the
hormone regulating positive pituitary responsiveness to GnRH (Reeves et al., 1971), although the
mechanism is still unclear. In contrast, progesterone acts as a negative regulator on the numbers of
GnRHR in gonadotrophs during the luteal phase which presents the active corpus luteum (Crowder
and Nett, 1984; Brooks and McNeilly, 1994; Turzillio et al., 1998). Factors affecting GnRHR gene
expression in gonadotrophs are mainly GnRH, partially gonadal steroid hormones (estrogen and
progesterone), and growth factors (inhibin and activin) (Rispoli and Nett, 2005). In our study, the
distribution of GnRHR-ir cells was the same in both the follicular and luteal phases. This could be
because LH pulse regulation in buffalo cows might not be involved at the anterior pituitary level.
The control of LH releasing might be, instead, related to GnRH and/or kisspeptin regulation at the
hypothalamus level. This assumes that the feedback control of estrogen in the follicular phase and
progesterone in the luteal phase during the estrous cycle may also have its primary influence at the
hypothalamus level rather than at the anterior pituitary level in buffalo cows. A study on the
mutations of GnRHR in humans reported that it can cause hypogonadotropic hypogonadism leading
to LH and FSH deficiency (De Roux, 2006). Therefore, it is possible that anestrous or infertile
animals might also have this problem, which is an issue to be considered in further studies. Our
present research suggests that, in buffalo cow, the target cells of the GnRH released by
hypothalamic neurons are in the pituitary gland. These findings expose some of the mechanisms
involved in the action of GnRH in the HPO axis, the understanding of which is central to the
enhancement of buffalo reproductive performance (which might be different from other animals).
However, more studies on the mechanism of the HPO axis on pituitary responsiveness during the
different stages of the estrous cycle should be done in the future.
ACKNOWLEDGEMENT
Thank you for grant support from the Thailand Research Fund, Vet. CU. Graduate Thesis Grant.
and The 90th Anniversary of Chulalongkorn University fund (Ratchadaphiseksomphot Endowment
Fund). Thank you for the Histopathological Laboratory, the Mahanakorn Veterinary Diagnostic
Center, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok,
Thailand. For English editing and review, thank you Mr. Philippe Marcou, USA.
REFERENCES
Ali, A., A.K. Abdel-Razek, S. Abdel-Ghaffar and P.S. Glatzel. 2003. Ovarian follicular dynamics
in buffalo cows (Bubalus bubalis). Reprod. Domest. Anim. 38: 214-218.
Bauer-Dantoin, A.C., J. Weiss and J.L. Jameson. 1995. Roles of estrogen, progesterone, and
470
gonadotropin-releasing hormone (GnRH) in the control of pituitary GnRH receptor gene

expression at the time of the preovulatory gonadotropin surges. Endocrinology. 136: 1014-
1019.
Brooks, J. and A.S. McNeilly. 1994. Regulation of gonadotrophin-releasing hormone receptor
mRNA expression in the sheep. Endocrinology. 143: 175-182.
Chaikhun, T., P. Sotthibandhu and S. Suadsong. 2013. The role of kisspeptin signaling in
reproduction of ruminants. Thai J. Vet. Med. 43 (1): in press.
Chaikhun, T., T. Tharasanit, J. Rattanatep, F. De Rensis and M. Techakumphu 2010. Fertility of
swamp buffalo following the synchronization of ovulation by the sequential administration
of GnRH and PGF2alpha combined with fixed time artificial insemination. Theriogenology.
74: 1371-1376.
Clarke, I.J., J.T. Cummins, M.E. Crowder and T.M. Nett. 1987. Pituitary receptors for
gonadotropin-releasing hormone in relation to changes in pituitary and plasma luteinizing
hormone in ovariectomized-hypothalamo pituitary disconnected ewes. I. Effect of changing
frequency of gonadotropin-releasing hormone pulses. Biol. Reprod. 37: 749-754.
Crowder, M.E. and T.M. Nett. 1984. Pituitary content of gonadotropins and receptors for
gonadotropin-releasing hormone (GnRH) and hypothalamic content of GnRH during the
periovulatory period of the ewe. Endocrinology. 114: 234-239.
De Roux, N. 2006. GnRH receptor and GPR54 inactivation in isolated gonadotropic deficiency.
Best Practice & Research Clinic. Endocrin. & Met. 20(4): 515-528.
Fischer, H. and P. Bodhipaksha. 1992. Distribution ecology and adaptation. In: Buffalo production
(Ed. Tulloh, N. M. and Holmes, J. H. G.) . The Netherlands: Elsevier Science Publishers B.
V. pp. 153-169.
Gregg, D.W.and T.M. Nett. 1989. Direct effects of estradiol-17 beta on the number of
Gonadotropin releasing hormone receptors in the ovine pituitary. Biol. Reprod. 40: 288-293.
Knox, C.J., S.K. Boyd and S.A. Sower.1994. Characterization and localization of gonadotropin-
releasing hormone receptors in the adults female sea lamprey, Petromyxon marinus.
Endocrinology. 134 (1): 492-498.
Nett, T.M., A.M. Turzillo, M. Baratta and L.A. Rispoli. 2002. Pituitary effects of steroid hormones
on secretion of follicle-stimulating hormone and luteinizing hormone. Domest. Anim.
Endocrinol. 23: 33-42.
Reeves, J.J., A. Arimura and A.V. Schally. 1971. Changes in pituitary responsiveness to luteinizing
hormone-releasing hormone (LH-RH) in anestrous ewes pretreated with estradiol benzoate.
Biol. Reprod. 4: 88-92.
Rispoli, L.A. and T.M. Nett. 2005. Pituitary gonadotropin-releasing hormone (GnRH) receptor:
structure, distribution and regulation of expression. Anim. Reprod. Sci. 88: 57-74.
Schoenemann, H.M., W.D. Humphrey, M.E. Crowder, T.M. Nett and J.J. Reeves. 1985. Pituitary
luteinizing hormone-releasing hormone receptors in ovariectomized cows after challenge
with ovarian steroids. Biol. Reprod. 32: 574-583.
Soga, T., S. Ogawa, Y. Sakuma and I.S. Parhar. 2005. Localization of the three GnRH types and
GnRH receptors in the brain of cichlid fish: insights into their neuroendocrine and
neuromodulator functions. J. Comp. Neurol. 487(1): 28-41.
Tienthai, P., K. Sajjarengpong and M. Techakumphu. 2008. Estrogen receptor alpha localization in
Thai swamp buffalo oviduct during the follicular and luteal phases. Thai J. Vet. Med. 38 (4):
35-44.
Turzillo, A.M., J.A.Clapper, G.E. Moss and T.M. Nett. 1998. Regulation of ovine GnRH receptor
gene expression by progesterone and oestradiol. J. Reprod. Fertil. 113: 251-256.
Yasin, M., A.C. Dalkin, D.J.Haisenleder, J.R. Kerrigan and J.C. Marshall. 1995. Gonadotropin-
releasing hormone (GnRH) pulse pattern regulates GnRH receptor gene expression:
augmentation by estradiol. Endocrinology. 136: 1559-1564.
471
A B
C D
Figure 1. The GnRHR-ir cells (arrows) present in cytoplasm of subpopulations of cells in the pars
in B and D. Scale bar, 20 m.

distalis at low and high magnifications in A and C, respectively which compare to negative control
472
Progestin-Based Protocol for Synchronization of Follicular Wave Emergence in

Buffaloes During Summer and Winter
Lindsay Unno GIMENESa, Nélcio Antonio Tonizza de CARVALHOb, Júlia Gleyci SOARESc,
Marcos Roberto CHIARATTId, Carolina Habermann MACABELLId, Márcio Leão
FERRAZe, Diego Cavalcante de SOUZAf, Henderson AYRESg and Pietro Sampaio
BARUSELLIc
a
b
Agência Paulista de Tecnologia e Agronegócios, Registro – SP, Brazil
c
d
Departamento de Medicina Veterinária, LMMD, FZEA-USP, Pirassununga - SP, Brazil
e
Vida Reprodutiva Consultoria Animal, Cravinhos – SP, Brazil
f
Coordenadoria de Assitência Técnica Integral, Registro – SP, Brazil
g
MSDAnimal Health, São Paulo – SP, Brazil
*Corresponding e-mail: gimeneslu@fcav.unesp.br
ABSTRACT
The aim of the present study was to evaluate the effects of type of norgestomet auricular
implant (new – N or previously used during 5 days - U), season of the year (summer - S and winter
- W), and parity (12 heifers - H and 23 cows - C) on synchronization of follicular wave emergence
in buffaloes. For this purpose, 35 buffaloes were examined daily by ultrasonography until follicular
wave emergence was detected. Data were analysed by ANOVA, using PROC GLIMMIX. No
interactions were observed in none variables. Time of follicular wave emergence and number of
follicles at emergence were not affected by type of implant or season of the year. Parity also did not
influence the number of follicles at emergence. However, follicular wave emergence occurred later
in heifers than in cows. In conclusion, the previous use of a norgestomet auricular implant
independent of the season of the year does not affect the time or the number of follicles at follicular
wave emergence in buffaloes. Nevertheless, although heifers and cows had a similar number of
follicles at emergence, the time of follicular wave emergence occurs earlier in cows than in heifers.
Keywords: auricular implant, buffalo, follicular wave, progestin, synchronization
INTRODUCTION
Nowadays several hormonal protocols were developed for synchronization of follicular
wave emergence and/ or ovulation for artificial insemination, superovulation and also for ovum
pickup – in vitro embryo production in bovine and buffaloes (Baruselli et al., 2010; Baruselli et al.,
2012). This tool allows that all animals can be in the same phase of estrous cycle. An association of
progesterone/ progestin source plus an estradiol ester induces follicular atresia and a new follicular
wave emerges (reviewed in Martinez et al., 2005). The possibility of using a progestin device more
than once reduces cost of hormonal treatment. However, the efficiency of this procedure in animals
with different status (i.e. heifers vs. cows) during different seasons of the year was not yet reported.
The aim of the present study was to evaluate the effects of type of norgestomet auricular
implant, season of the year, and parity on synchronization of follicular wave emergence in
buffaloes.

Thirty five buffaloes were homogeneously assigned according to ciclicity and then grouped
by parity (12 heifers - H and 23 cows - C) to receive on random day of estrous cycle (D0) a dose of
2 mg of estradiol benzoate (Gonadiol®, MSD, Brazil). On this same day, animals were assigned to

473
receive a norgestomet auricular implant (Crestar ®, MSD, Brazil) new (N) or previously used
during 5 days (U), on summer (S; February) and winter (W; July), in a 2 x 2 x 2 factorial design.
Follicular dynamics was performed by ultrasonography (5 MHz probe, Chison®, China)
each 24 hours from D0 until follicular wave emergence was detected. Follicular wave emergence
was defined as occurring on the last day on which the retrospectively dominant follicle was 4 mm
(Ginther et al., 1997). Data were analysed by ANOVA, using PROC GLIMMIX (SAS®, Country?).

No interactions were observed in none variables (P ≥ 0.05). Results of time of follicular
wave emergence and number of follicles at emergence relative to type of implant, season of the
year, and parity are shown in Table 1.
Time of follicular wave emergence and number of follicle at emergence were not affected by
type of implant. These results corroborate with previous studies in bovine (Cerri et al., 2009) and in
buffaloes (Carvalho et al., 2007) in which reutilization of progesterone devices in protocols of fixed
time artificial insemination (FTAI) resulted in patterns of follicular development and ovulation
similar to new devices.
Both variables also were not affected by season of the year. This result was expected
because satisfactory conception rates were already reported when progestagens are used during
breeding and non-breeding seasons in buffaloes (De Rensis and López-Gatius, 2007; Baruselli et
al., 2010). It is also known that outcomes of FTAI are related to success of follicular wave
emergence synchronization.
Parity also did not influence the number of follicles at emergence. However, follicular wave
emergence occurred later in heifers than in cows (4.2 ± 0.2 vs. 3.8 ± 0.1, P = 0.03).
In conclusion, the previous use of a norgestomet auricular implant or the season of the year
do not affect the time or the number of follicles at follicular wave emergence in buffaloes.
Nevertheless, although heifers and cows had a similar number of follicles at emergence, the time of
follicular wave emergence occurs earlier in cows than in heifers.
ACKNOWLEDGEMENT
The authors thank MSD Animal Health (São Paulo - Brazil), for providing the hormones
used in this study.
REFERENCES
Baruselli, P.S., M.F. Sá Filho, R.M. Ferreira, J.N.S. Sales, L.U. Gimenes, L.M. Vieira, M.F.
Mendanha and G.A. Bó. 2012. Manipulation of follicle development to ensure optimal
oocyte quality and conception rates in cattle. Reprod. Dom. Anim. 47: 134-141.
Baruselli, P.S., L.U. Gimenes, N.A.T. Carvalho, M.F. Sá Filho and M.L. Ferraz. 2010.
Folliculogenesis applied to reproductive biotechnologies in buffaloes. In: Proceedings of
International Buffalo Conference, Nova Delhi. pp. 167-176.
Carvalho, N.A.T., E.M. Nagasaku, F. S. Vannucci, L. M. Toledo, P. S. Baruselli. 2007. Ovulation
and conception rates according intravaginal progesterone device and hCG or GnRH to
induce ovulation in buffalo during the off breeding season. Italian J. Anim. Sci. 6: 646-648.
Cerri, R.L., H.M. Rutigliano, R.G. Bruno and J.E. Santos. 2009. Progesterone concentration,
follicular development and induction of cyclicity in dairy cows receiving intravaginal
progesterone inserts. Anim. Reprod. Sci. 110: 56-70.
De Rensis and F., López-Gatius. 2007. Protocols for synchronizing estrus and ovulation in buffalo
(Bubalus bubalis): a review. Theriogenology. 67: 209-216.
Ginther, O. J., K. Kot, L.J. Kulick and M.C. Wiltbank. 1997. Emergence and deviation of follicles
during the development of follicular waves in cattle. Theriogenology. 48: 75-87.
Martínez, M.F., J.P. Kastelic, G.A. Bó, M. Caccia and R. J. Mapletoft. 2005. Effects of oestradiol
and some of its esters on gonadotrophin release and ovarian follicular dynamics in CIDR-
treated beef cattle. Anim. Reprod. Sci. 86: 37-52.
474
Table 1. Follicular wave emergence in buffaloes according to type of implant (N: new vs. U:
previously used during 5 days), season of the year (S: summer vs. W: winter), and parity (H: heifers
vs. C: cows).
Time of follicular P Value Number of follicles at P

wave emergence follicular wave Value
emergence
N (n=18) 3.9 ± 0.2 28.2 ± 2.4
Implant 0.91 0.41
U (n=17) 3.9 ± 0.1 24.2 ± 3.3
S (n=19) 3.9 ± 0.1 22.7 ± 2.5

Season 0.52 0.14
W (n=16) 3.9 ± 0.2 29.1 ± 3.2
H (n=12) 4.2 ± 0.2 28.5 ± 4.1

Parity 0.03 0.44
C (n=23) 3.8 ± 0.1 24.7 ± 2.4
475
Influence of Parity and Season of the Year on Oocyte Quality and Number in
Buffaloes
Lindsay Unno GIMENESa, Carolina Habermann MACABELLIb, Nélcio Antonio Tonizza de
CARVALHOc, Júlia Gleyci SOARESd, Márcio Leão FERRAZe, Diego Cavalcante de
SOUZAf, Henderson AYRESg, Daniel Carlino JOAQUIMh, Yeda WATANABEh, Osnir
WATANABEh, Pietro Sampaio BARUSELLId, Flavio Vieira MEIRELLESb and Marcos
Roberto CHIARATTIb
a
b
Departamento de Medicina Veterinária, LMMD, FZEA-USP, Pirassununga - SP, Brazil
c
Agência Paulista de Tecnologia e Agronegócios, Registro – SP, Brazil
d
e
Vida Reprodutiva Consultoria Animal, Cravinhos – SP, Brazil
f
Coordenadoria de Assitência Técnica Integral, Registro – SP, Brazil
g
MSDAnimal Health, São Paulo – SP, Brazil
h
WTA, Cravinhos – SP, Brazil
*Corresponding e-mail: gimeneslu@fcav.unesp.br
ABSTRACT
The aim of the present study was to evaluate the effects of season of the year (summer and
winter) and parity (heifers and cows) on oocyte quality and number in buffaloes. For this purpose,
71 buffaloes had follicular wave emergence synchronized before OPU. OPU of all follicles ≥ 2mm
was done 5 days after the beginning of the hormonal protocol, in 4 replicates (two for each season).
Data were analyzed by ANOVA using PROC GLIMMIX, in a 2 x 2 factorial arrangement of
treatments. No interactions were observed in following variables: number of follicles, number of
total and viable oocytes, recovery rate, percentage of viable oocytes, grade I oocytes, grade II
oocytes, grade III oocytes, denuded oocytes, expanded cumulus oocytes, and atretic/ degenerated
oocytes. Number of follicles visualized at OPU and recovery rate were not affected by parity or
season. Relative to parity, number of total and viable oocytes were greater in heifers than in cows,
respectively. Concerning season of the year, number of viable oocytes and viable oocyte rate were
increased in winter. In conclusion, better oocyte quality can be obtained from heifers and during
winter in buffaloes. However, the number of total oocytes seems to be more influenced by parity
than by season of the year in this species.
Keywords: heifer, cow, oocyte recovery, ovum pickup.
INTRODUCTION
Buffalo reproduction is influenced by seasonality, which in natural conditions limits
breeding, calving and milk yield to a restrict period of the year. This feature has been also described
to vary according to parity. Heifers seem to be less sensitive to changes in day-length along the year
than cows (reviewed in Terzano et al., 2012). Several strategies of management and the use of
reproductive biotechnologies have been utilized to allow a better productive performance of the
species. Therefore, the aim of the present study was to evaluate the effects of season of the year
(summer and winter) and parity (heifers and cows) on oocyte quality and number in buffaloes.

For this purpose 71 buffaloes were treated on random day of estrous cycle with 2 mg of
estradiol benzoate (Gonadiol®, MSD Animal Health, Brazil), 530µg of d-cloprostenol (Ciosin®,
MSD Animal Health, Brazil) plus a norgestomet auricular implant (Crestar®, MSD Animal Health,
Brazil) for synchronizing follicular wave emergence. OPU of all follicles ≥ 2mm was done on Day
476
5 after the beginning of the hormonal protocol, in 4 replicates (two for each season; Winter: July
and Summer: January/ February).
Before OPU, numbers of follicles in each ovary were counted by ultrasonography (5 MHz
microconvex transducer housed in a plastic vaginal probe; Aloka SSDV500, Aloka, Japan), as well
as respiratory frequency (RF), rectal temperature (RT) and cutaneous temperature (CT) of all
animals were recorded. Data were analyzed by ANOVA using PROC GLIMMIX (SAS), in a 2 x 2
factorial arrangement of treatments.

As expected, RF, RT, and CT were greater in summer than in winter (P ≥ 0.05). No
interactions were observed among variables (P ≥ 0.05). All data is summarized in Table 1 according
to mean effects. Number of follicles visualized, number of total oocytes, number of viable oocytes,
and percentage of viable oocytes is also shown in Figure 1.
Number of follicles visualized at OPU and recovery rate were not affected by parity or
season. These data corroborate with those described by Di Francesco et al. (2012), however
partially agrees with Manjunatha et al. (2009). These authors also did not observe differences in
recovery rate during peak or low breeding season, however, they reported an increase in the number
of follicles during peak.
Relative to parity, number of total and viable oocytes were greater in heifers than in cows,
respectively. These findings could be explained by the greater number of grade 2, denuded, and
degenerated/ atretic oocytes in heifers than in cows, respectively. Additionally, cows had a greater
number of expanded cumulus oocytes than heifers.
Concerning season of the year, number of viable oocytes and percentage of viable oocyte
were increased in winter, mainly due to an increase in grade III oocytes and a decrease in expanded
cumulus oocytes in this season. These findings are not in agreement with Manjunatha et al. (2009),
whose did not find any influence of season in oocyte quality, and with Di Francesco et al., (2012)
whose reported an unexpected increase in expanded and abnormal expanded oocytes during
autumn-winter. Differences in experimental conditions (i.e. nutrition or luminosity) could be related
to these divergent results.
In conclusion, better oocyte quality can be obtained from heifers and during winter in
buffaloes. However, the number of total oocytes seems to be more influenced by parity than by
season of the year in this species.
ACKNOWLEDGEMENTS
Vida Reprodutiva Consultoria Animal, WTA, MSD Animal Health, FAPESP (10/09561-7,
11/14207-0).
REFERENCES
Di Francesco, S., M.V.S. Novoa, D. Vecchio, G. Neglia, L. Boccia, G. Campanile, L. Zicarelli and
B. Gasparrini. 2012. Ovum pick-up and in vitro embryo production (OPU-IVEP) in
Mediterranean Italian buffalo performed in different seasons. Theriogenology 77: 148-154.
Manjunatha, B.M., J.P. Ravindra, P.S.P. Gupta, M. Devaraj and S. Nandi. Effect of breeding
season on in vivo oocyte recovery and embryo production in non-descriptive Indian river
buffaloes (Bubalus bubalis). 2009. Anim. Reprod. Sci. 111: 376-383.
Terzano, G.M., V.L. Barile and A. Borghese. 2012. Overview on reproductive endocrine aspects in
buffalo. J. Buffalo Sci. 1: 126-138.
477
Table 1. Effects of season of the year and parity on oocyte quality and number in buffaloes (mean
effects).
Season Parity
Winter Summer P Heifer Cow P
Follicles (n) 19.3 ± 1.2 16.2 ± 1.2 0.08 18.4 ± 1.3 16.9 ± 1.0 0.38
Total oocytes (n) 15.8 ± 1.5 12.2 ± 1.7 0.09 16.6 ± 1.9 10.9 ± 0.9 0.01
Recovery rate (%) 75.5 ± 3.8 64.4 ± 3.9 0.05 86.9 ± 6.8 64.2 ± 4.3 0.09
Viable oocytes (n) 10.3 ± 1.0 6.6 ± 1.1 < 0.01 10.0 ± 1.3 6.4 ± 0.8 0.02
Viable oocytes (%) 65.1 ± 2.3 50.5 ± 3.6 < 0.01 59.7 ± 2.9 54.7 ± 3.8 0.21
Grade I oocytes (n) 0.5 ± 0.1 0.4 ± 0.1 0.69 0.5 ± 0.2 1.5 ± 0.3 0.43
Grade II oocytes (n) 2.1 ± 0.3 1.8 ± 0.3 0.43 2.4 ± 0.3 1.5 ± 0.3 0.04
Grade III oocytes (n) 7.1 ± 0.8 3.8 ± 0.7 < 0.01 6.2 ± 0.8 4.4 ± 0.7 0.05
Denuded oocytes (n) 4.3 ± 0.7 3.9 ± 0.6 0.61 4.9 ± 0.7 3.2 ± 0.4 0.04
Expanded cumulus oocytes (n) 0.1 ± 0.1 0.5 ± 0.1 0.02 0.2 ± 0.1 0.5 ± 0.2 0.04
Atretic/ degenerated oocytes (n) 1.2 ± 0.2 1.2 ± 0.2 0.81 1.6 ± 0.3 0.9 ± 0.2 0.03
A B
Season: P = 0.09
Parity: P = 0.01 (heifer>cow)
C D
Season: P < 0.01 (winter>summer)
Season: P < 0.01 (winter>summer)
Parity: P = 0.02 (heifer>cow)
Parity: P = 0.21
Figure 1. Effects of season of the year and parity on number of follicles, number of total oocytes,
number of viable oocytes, and percentage of viable oocytes in buffaloes (all groups combined).
478
Comparison of Two Synchronization Protocols for Timed Artificial

Insemination in Acyclic Italian Mediterranean Buffalo Cows out of the
Breeding Season
Domenico VECCHIOa*, Pasquale ROSSIa, Gianluca NEGLIAa, Valentina LONGOBARDIa,

Angela SALZANOa, Giovanna BIFULCO a and Giusepppe CAMPANILEa
a
University of Study of Naples “Federico II” Faculty of Veterinary Medicine Department of
Veterinary Medicine and Animal Production Naples, Italy.
*Corresponding email d.vecchio@unina.it
ABSTRACT
The aim of the present study was to compare two synchronization protocols for timed
artificial insemination (TAI) in acyclic pluriparous buffalo cows during the non breeding season.
Two experiments were conducted to evaluate the ovarian follicular response and pregnancy rate.
The cyclic status was evaluated by two transrectal ultrasonography performed at Day -11 and Day
0. Buffaloes that in both investigations did not show the presence of a corpus luteum (CL) were
classified as acyclic. Acyclic pluriparous buffaloes (n=34) were randomly assigned to Group 1 (G1)
and Group 2 (G2), homogeneous for Days in Milk (81±27 vs 83±13, respectively in G1 and G2). In
G1 (n=17) buffaloes received 12µg of buserelin acetate i.m. (GnRH) on the first day of the
synchronization protocol (Day 0), 0.524 mg of cloprostenol (Pgf2α) on Day 7 and 12µg of buserelin
acetate i.m. on day 9 (Ovsynch-TAI). In G2 (n=17) buffaloes received a progesterone-releasing
intravaginal device containing 1.55 g of progesterone (P4) and 12µg of buserelin acetate i.m.
(GnRH) on Day 0. On Day 8 the P4 device was removed and 0.524 mg of cloprostenol (PGF2α) +
500UI of PMSG i.m. were administered. Finally, 12µg of buserelin acetate i.m. were given on Day
10. Ten animals (5/group) underwent transrectal ultrasonography of the ovaries daily, from Day 0 to
Day 11 in G1 and from Day 0 to Day 12 in G2, to determine the presence and diameter of the
follicles, the dominant follicle (DF) diameter and the ovulation rate. Subsequently, fixed TAI was
performed 20 hours after the last GnRH in all buffaloes. Ultrasonography was carried out 25 and 45
days after TAI to evaluate pregnancy rate and the incidence of late embryonic mortality (LEM). No
differences were observed between G1 and G2 in the following parameters: DF diameter (mm) on
Day 0 (9.5±1.9 vs 8.8±4.3), DF diameter (mm) on Day of Pgf2α administration (11.5±2.3 vs
10.6±1.6), DF growth rate (mm) between PGF2α and last GnRH administration (1.6±0.3 vs
1.8±1.7), DF maximum diameter (13.2±1.9 vs 13.0±2.3 mm) and ovulation rate (80% vs 80%).
However, in G2 pregnancy rate increased at 25 (29.4% vs 58.8%, respectively in G1and G2; P=
0.08) and 45 (23.5% vs 58.8%, respectively in G1and G2; P<0.05) days after TAI. In conclusion, it
was demonstrated that, despite similar results in terms of follicular dynamics, growth and ovulation
rate, the protocol G2 is the most efficient to improve fertility in buffalo out the breeding season.
Keywords: acyclic buffaloes, TAI, PMSG, non breeding season


479
Morphometric and Functional Characteristics of Buffaloes and Cows

Corpus Luteum at Different Stages of the Estrous Cycle
Luciana LEALa*, Eunice OBAb, Carla MOYA-ARAÚJOc and Nereu PRESTESb
a
Faculty of Veterinary Medicine, University of Paraná (UNIPAR), Umuarama, Paraná
State/Brazil; bDepartment of Animal Reproduction and Veterinary Radiology, FMVZ, São Paulo
State University (UNESP) - Distrito de Rubião Júnior, sem número - Botucatu/SP- 18618-000,
Brazil; cFaculty of Veterinary Medicine, Integrated College of Ourinhos (FIO), Ourinhos, São
Paulo State/Brazil.
*Corresponding e-mail: lu_s_leal@yahoo.com.br
ABSTRACT
The aims of the present study were to evaluate the morphometry of corpus luteum (CL) and
progesterone (P4) plasma concentration of 86 buffaloes (33 pregnant and 53 non-pregnant) and 95
cows (36 pregnant and 59 non-pregnant) at the moment of slaughter. Seventy CLs of buffaloes and
110 CL of cattle were analyzed. The CL classified as II and III were more common in both species
(35.7 and 41.4% for buffaloes and 43.6 and 35.5% for cows). The 29 non-pregnant buffaloes had a
total of 36 CL, being 19.4% CLI; 33.3% CL II; 27.8% CL III and 19.4% CL IV. The 51 non-
pregnant cows had a total of 71 CL, being 26.8% CL I; 47.9% CL II; 21.1% CL III and 4.2% CL
IV. The average diameters of bubaline and bovine CL were 5.2 ± 0.9 and 6.4 ± 1.8 mm (CL I); 17.6
± 2.6 and 19.8 ± 3.2 mm (CL II); 17.2 ± 2.1 and 20.0 ± 3.2 mm (CL III); 7.8 ± 1.8 and 8.7 ± 2.7
mm (CL IV), respectively. The mean plasma concentrations of P4 were 5.6 (CL I); 5.4 (CL II); 4.7
(CL III) and 0.5 (CL IV) ng/mL for buffaloes and 0.02 (CL I); 6.3 (CL II) and 6.4 (CL III) ng/mL
for cows. In both species, P4 concentration was similar between stages II and III. The results
indicated that the characterization of the CL provides important information about the status of
estrous cycle.
Keywords: Corpus luteum, Progesterone, Buffaloes, Cows
INTRODUCTION
According to Fields & Fields (1996) the corpus luteum (CL) is formed from the hyperplasia
and differentiation of granulosa and theca cells of ovulatory follicle. It is a structure recognized for
ability to synthesize and release progesterone (P4), a hormone that is closely related with the
maintenance of an appropriate environment for embryonic development and with the maintenance
of the CL itself during the period between ovulation and implantation.
The evaluation of CL provides important information about the reproductive status of
female and allows the suitability of procedures for synchronization of estrous cycle (Viana et al.,
1999).
The bubaline CL is commonly inserted into the ovarian stroma and it is generally lower than
in cows, with a maximum weight of 2.3 g and a maximum diameter of 15 mm (Roy & Mullick,
1964). Figueiredo et al. (1997) found maximum luteal diameter of 17.7 mm in Nellore cows. In the
same breed, Neves et al. (2002) reported luteal diameter of 18.4 and 18.7 mm in pregnant and non-
pregnant animals for left ovaries and 15.3 and 16.4 mm in pregnant and non-pregnant animals for
right ovaries, respectively.
According to Ireland, Murphee and Coulson (1980) there are four morphological changes
identifiable in the appearance of bovine CL after ovulation. According to these authors, the
diameters of CL in stages I, II, III and IV ranging from 5 to 15; 16-20, 16-20 and <10 mm,
respectively.
The aim of the present study was to evaluate the morphometric and functional characteristics
of buffaloes and cows CL at different stages of estrous cycle, in the moment of slaughter.

480

The ovaries were obtained from 86 Murrah x Mediterranean buffaloes (33 pregnant and 53
non-pregnant) and 95 Zebu cows (36 pregnant and 59 non-pregnant) in slaughterhouses located in
the cities Lençóis Paulista (Frigol®, distance 55 Km), Rancharia (Better Beef®, distance 318 Km)
and Cajati (Frivale®, distance 460 Km), São Paulo State, Brazil. Weigth and age were not
determined but all evaluated females were adult. The ovaries of each female were separately
collected, kept in labeled plastic bags containing heated saline solution (36ºC) added of 100
units/mL penicillin and 100 mg/mL streptomycin, and transported to the laboratory in an isothermal
box. In laboratory, the ovaries were dissected and the CL diameter was obtained with a digital
pachymeter.
In total, were analyzed 70 CL of buffaloes and 110 CL of cattle; the classification of CL
followed Ireland, Murphee and Coulson (1980): CL I (days 1 to 4 of the estrous cycle/ 5 to 15 mm
of diameter) - CL as a small red spot, not covered by the epithelium; CL II (days 5 to 10 of the
estrous cycle/ 16 to 20 mm) - CL fully formed, with vascularization visible in the periphery. When
sectioned, the peak is red or brown, while the rest is orange or yellow; CL III (days 11 to 17 of the
estrous cycle /16 to 20 mm) - CL is presented entirely orange or yellow. Vascularization is visible
on the peak; CL IV (days 18 to 20 of the estrous cycle /<10 mm) – CL in regression, light yellow or
white with no apparent vascularization.
Blood was collected directly from the jugular vein at the time of bleeding, on the slaughter
line. Blood was centrifuged at 1500 xg for 15 minutes and the plasma obtained was stored in
identified plastic tubes, maintained at -80 ºC until analysis.
The statistical analysis was performed in Department of Biostatistics, Institute of
Biosciences, UNESP/Botucatu-SP, Brazil. The results were submitted to statistical treatments of
mean, standard deviation and percentage. For the CL variable, according to the reproductive status,
was used the Mann-Whitney test, since this did not show a normal distribution. In comparison
between species, for all variables, was used the Mann-Whitney test. In all analyzes were considered
statistically significant when p <0.05.

In the present study, the CL classified as II and III were more common in both species (35.7
and 41.4% for buffaloes and 43.6 and 35.5% for bovine species, respectively, p <0 .05), as noted in
Table 1. This behavior was similar to that described by Abdoon & Kandil (2001), which determined
the days of the estrous cycle of buffaloes slaughtered using a similar classification to the CL and
obtained 16.1, 25.3, 40.2 and 18.4% for CL I, II, III and IV, respectively.
With respect to non-pregnant animals that contained CL in the ovaries, 29 buffaloes had a
total of 36 CL because seven females had two CL. Of the 36 CL, 7 (19.4%) were classified as CLI;
12 (33.3%) CL II; 10 (27.8%) CL III and 7 (19.4%) CL IV. The 51 non-pregnant cows had a total
of 71 CL, being 19 (26.8%) CL I; 34 (47.9%) CL II; 15 (21.1%) CL III and 3 (4.2%) CL IV. The
average diameters of each category of bubaline and bovine CL were 5.2 ± 0.9 (n= 7) and 6.4 ± 1.8
(n= 20) mm to CL I; 17.6 ± 2.6 (n= 25) and 19.8 ± 3.2 (n= 48) mm to CL II; 17.2 ± 2.1 (n= 29) and
20.0 ± 3.2 (n= 39) mm to CL III; 7.8 ± 1.8 (n= 9) and 8.7 ± 2.7 (n= 3) mm to CL IV, respectively.
In this study, the measures of bubaline and bovine CL in all stages corresponded to findings
by Ireland, Murphee and Coulson (1980), even considering that the researchers evaluated CL of
Hereford heifers. In 146 Hereford heifers, the percentages of CL I, II, III and IV were 20, 29, 36
and 15%, respectively.
Nascimento et al. (2003) determined the different phases of the estrous cycle in cows with
cyclic ovarian activity, using the same classification of Ireland, Murphee and Coulson (1980). The
frequency of animals showing CL in stages I, II, III and IV were 28.1, 28.1, 28.1 and 15.6%,
respectively. In addition, they measured the areas of the CL, which were 2.3 ± 0.4 cm 2 (stage I), 3.1
± 0.4 cm2 (stage II), 3.9 ± 0.4 cm2 (stage III) and 2.4 ± 0.5 cm2 (stage IV).
Regarding P4 plasma concentration were considered only females who had one CL in both
ovaries, to have a more accurate result.
481
The mean plasma concentrations of P4 were 5.6 ± 0.0 (CL I; n= 1); 5.4 ± 2.6 (CL II; n= 22);
4.7 ± 1.6 (CL III; n= 26) and 0.5 ± 0.5 (CL IV/ n= 4) ng/mL for buffaloes. The mean plasma
concentrations of P4 were 0.02 ± 0.0 (CL I; n= 1); 6.3 ± 4.2 (CL II; n= 35) and 6.4 ± 3.3 (CL III; n=
33) ng/mL for cows. In this experiment, no cow showed CL IV exclusively, always in combination
with other CL in ovaries, so P4 was not measured in these animals. In both species, P4 concentration
was similar between stages II and III (p ≥ 0.05, "t" test).
In conclusion, these results indicated that the characterization of the CL provides important
information about the reproductive status of female, allowing procedures for manipulation of the
estrous cycle more successfully.
REFERENCES
Abdoon, A.S.S. and O.M. kandil. 2001. Factors affecting number of surface ovarian follicles and
oocytes yield and quality in Egyptian buffaloes. Reprod. Nutr. Dev. 41: 71-77.
Fields, M.J. and P.A. Fields. 1996. Morphological characteristics of bovine corpus luteum during
the estrous cycle and pregnancy. Theriogenology. 45: 1295-1355.
Figueiredo, R.A., C.M. Barros, O.L. Pinheiro and J.M.P. Soler. 1997. Ovarian follicular dynamics
in Nelore breed (Bos indicus) cattle. Theriogenology. 47: 1489-1505.
Ireland, J.J., R.L. Murphee, and P.B. Coulson. 1980. Accuracy of predicting stages of bovine
estrous cycle by gross appearance of the corpus luteum. J. Dairy Sci. 63: 155-160.
Nascimento, A.A., N.L. Pinheiro, A. Sales and J.H.M. Viana. 2003. Correlação morfométrica do
ovário de fêmeas bovinas em diferentes estádios reprodutivos. Braz. J. Vet. Res. Anim. Sci.
40: 126-132.
Neves, M.M., A.P. Marques Jr. and C.V. Santan. 2002. Características de ovários de animais
zebu (Bos taurus indicus) coletados de abatedouros. Arq. Bras. Med. Vet. Zoot. 54: 651-654.
Roy, D.J. and D.N. Mullick. 1964. Endocrine functions of corpus luteum of buffaloes during estrus
cycle. Endocrinology. 75: 284-287.
Viana, J.H.M., A.M. Ferreira, W.F. Sá and L.S.A. Camargo. 1999. Características morfológicas e
funcionais do corpo lúteo durante o ciclo estral em vacas da raça Gir. Arq. Bras. Med. Vet.
Zootec., 51: 251-256.
Table 1. Distribution of CL frequency according to the classification in buffaloes and cows.

CL classification
Species Total (%)
I (%) II (%) III (%) IV (%)
Bubaline 7 (10,0)b 25 (35,7)a 29 (41,4)a 9(12,9)b 70(100,0)
Bovine 20 (18,2)b 48 (43,6)a 39 (35,5)a 3 (2,7)b 110(100,0)
p< 0,0001; Mann-Whitney test
482
Response to The First GNRH and Pregnancy Outcome in Buffaloes Underwent

Ovsynch and Fixed Timed Artificial Insemination
Gianluca NEGLIA*, Bianca GASPARRINI, Roberta CIMMINO, Gianluigi ZULLO,
Giuseppe ALBERO, Luigi ZICARELLI and Giuseppe CAMPANILE
Department of Veterinary Medicine and Animal Production, Federico II University, Via F. delpino
1, 80137 Napoli, Italy
*Corresponding email: neglia@unina.it
ABSTRACT
The peculiar reproductive characteristics of the species limit the utilization of artificial
insemination (AI) in buffalo. For this reason, in the last years a great attention has been focused on
estrus synchronization methods based on fixed timed artificial insemination (FTAI). Among these,
the Ovsynch-TAI program has been successfully applied in buffalo. The aim of this study was to
assess the response to the first GnRH, prostaglandin and second GnRH in buffaloes synchronized
by Ovsynch and subsequent pregnancy outcome. The trial was carried out on 59 buffaloes
(138.7±86 days in milk) bred in a commercial farm in the South of Italy. Ten days before the start
of the study all the animals underwent clinical and ultrasound examination, to confirm the absence
of any gross abnormalities of the genital tract. Synchronization of the estrous cycle was performed
by Ovsynch-TAI program which involved the injection of a GnRH agonist on Day 0 (12 μg of
buserelin acetate), prostaglandin on Day 7 (5 mg of Dinoprost), and a GnRH agonist again on Day 9
(12 μg). The AI was carried out 16 h after the second GnRH injection, using frozen-thawed semen
from bulls of proven fertility. The ultrasound examinations of the ovaries were performed by using
a portable Sonoace Pico equipped with a 10 MHz linear transducer adapted for transrectal
examination in large domestic animals on Day 0, 1, 7, 9, 10 and 11. All visible antral follicles were
recorded and classified into three categories, according to their size: small (diameter < 0.5 cm),
medium (diameter between 0.5 and 1 cm) and large (diameter > 1cm). The dimensions of the
dominant follicle and the corpus luteum (CL) were further registered. In order to evaluate the
characteristics of the CLs, the colour-doppler mode was activated in order to display signals for
blood flow in vessels. Pregnancy diagnosis was carried out on 35 days post-AI and the statistical
analysis was performed by ANOVA and chi-square test. It was observed that 37 buffaloes (62.7%)
ovulated after the first GnRH treatment and showed a functional corpus luteum on day 7.
Interestingly, animals that ovulated after the first GnRH injection showed a lower (P=0.08) CL area
(22.1±10.1 vs. 28.6±10.3 mm, respectively) and a larger follicle (10.5±4.3 vs. 8.5±3.0, respectively)
on day 0. A total of 54 buffaloes (91.5%) showed a functional CL on day 7, although luteolysis
occurred only in 50 subjects (84.7%). All the buffaloes (37/37) that ovulated after the first GnRH
injection underwent luteolysis. On the contrary, CLs responsive to prostaglandins were recorded
only in 59% (n=13) of the animals that did not ovulate after the first GnRH (P<0.01). The response
to the second GnRH treatment was very high, since 91.5% of the buffaloes ovulated on the day after
FTAI. The ovulation rate was 97.3% (36/37) and 81.8% (18/22) in animals that had either a positive
or negative response to the first GnRH injection, respectively. Total pregnancy rate was 59.3%
(35/59) on day 35 post-AI. Interestingly, a significantly higher (P<0.01) pregnancy rate was
observed in the animals that ovulated after the first GnRH injection (30/37=81.1%) than in those
that did not ovulate (5/22=22.3%). As expected, no pregnancies were recorded in animals that did
not respond to both the prostaglandin and second GnRH. These results suggest that the response to
the first GnRH rather than that to both prostaglandin and the second GnRH may allow to predict the
pregnancy outcome after FTAI. In conclusion, the ovulation rate after the first GnRH could be
utilized as a preliminary screening of buffaloes to be inseminated, reducing FTAI costs.
Keywords: GnRH, Fixed-time Artificial Insemination buffalo, Ovsynch, pregnancy
483
Comparison of Botu-bov and Tris as Freezing Extenders of Buffalo Sperm

Recovered from Epididymal Cauda
Aline Sousa CAMARGOS, a Eunice OBA, a Gabriel Augusto MONTEIRO, a Yamê Fabres
Robaina SANCLER-SILVA,a Mariana ZORZETTO, a Rosiara Rosária Dias MAZIERO, a
Frederico Ozanam PAPAa and Alcides Amorim RAMOSb
a
Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine
and Animal Science – UNESP, Distrito Rubião Jr. s/ n., Botucatu, SP, Brazil
b
Department of Animal Production, School of Veterinary Medicine and Animal Science – UNESP,
Fazenda Lageado, Botucatu, SP, Brazil
* Corresponding e-mail: euniceoba@fmvz.unesp.br
ABSTRACT
Valuable genetic material can be preserved by the cryopreservation of epididymal sperm.
This study evaluated the viability of pre-freezing and post-thawed sperm samples recovered from
the epididymal cauda of buffaloes. Epididymides from eight Murrah buffaloes with 18 months of
age were used. Semen samples were diluted in two different freezing extenders: Botu-bov (BB) and
Tris (TRIS). Immediately after slaughter, both testicles from each animal were collected and
transported at 4o C for six hours interval. In laboratory, the removed epididymides were flushed to
obtain sperm and the fractions were diluted in both freezing extenders (BB and TRIS). Semen doses
were analyzed before and after frozen at -196o C. BB and TRIS pre-freezing results were
38.54±22.33%b and 14.17±12.78%a for total motility (TM), 25.00±16.12a and 9.44±9.11a for
progressive motility (PM), 7.21±0.98a and 5.09±2.65a for percentage of rapid cells (RAP),
91.08±12.53b and 63.33±31.47ª for velocity of trajectory (VAP), 73.54±20.17b and 49.50±9.11ª for
linear progressive velocity (VSL), 172.21±24.55ª and 116.94±59.48ª for curvilinear velocity (VCL),
respectively (P < 0.05). BB and TRIS post-thawing results were 42.25±21.50b and 17.62±19.46a for
TM, 27.25±24.86a and 18.00±13.68a for PM, 7.35±0.98a and 6.26±1.13a for RAP, 91.42±16.86ª and
75.96±13.17ª for VAP, 67.96±12.13ª and 60.04±10.42ª for VSL, 177.54±23.53b and 141.29±24.97a
for VCL, respectively (P < 0.05). The sperm recovered from the epididymal cauda, after 6 h
storage of epididymides at 5 °C ensures sperm preservation demonstrating that the diluent Botu-bov
had higher total motility both pre- and post-freezing when compared with TRIS. Additionally, the
sperm frozen with the diluent Botu-bov showed higher values of VSL at post-thawing. These
findings may reflect in improvement of conception rates.
Keywords: semen, Bubalus bubalis, epididymal, freezing.
INTRODUCTION
Improving the productive and reproductive system will only be possible through the use of
modern technologies that enable the best use of animals genetically superior. Epididymal cauda
accumulates mature spermatozoa for further ejaculation (Jones, 1998) and allows a greater number of
recovered spermatic cells compared with semen collection using an artificial vagina. Despite the advances
in the field of cryopreservation of semen and artificial insemination in livestock production, the
conception rate of buffalo frozen semen of about 33% is still considered unsatisfactory. This may be
attributed to damage caused by the formation of intracellular and extracellular ice crystals during
cryopreservation, which causes damage to the plasma and acrosomal membrane and decreases
motility.
The harvest of sperm from the epididymal cauda has proven to be an efficient mean of
recovering viable sperm cells in several species. This technique allows preservation of valuable
genetic materials from injured or deceased animals by means of cryopreservation. Accordingly, this
technique assists the propagation of favorable characteristics from genetically superior males.
Although numerous studies on the cryopreservation of epididymal sperm of other species showed
the feasibility of this technique, studies in buffaloes are still scarce. Studies directed to the
484
cryopreservation of epididymal semen of buffalo have focused the African buffalo (Syncerus caffer)
in order to create a gene pool for the purpose of conservation of the species and also to produce
breed disease-free buffalo whom may have economic potential for farming.
This study evaluated the viability of pre-freezing and post-thawed sperm samples recovered
from the epididymal cauda of buffaloes using Botu-bov and Tris freezing extenders.

Epididymides from eight Murrah buffaloes with 18 months of age were used. Semen
samples were diluted with two different freezing extenders: Botubov (BB) and Tris (TRIS).
Immediately after slaughter, both testicles from each animal were collected and transported
o
at 4 C at maximum six hours interval. In the laboratory, the removed epididymides were flushed
with skim-milk based extender Botu-Semen in order to obtain sperm. After 15 minutes, samples
were centrifuged and the pellets were resuspended in both freezing extenders (BB and TRIS). For
cryopreservation, the straws containing sperm were maintained at 5o C for 4h, and then placed
horizontally 3 cm above liquid nitrogen level for 20 minutes before immersion into the liquid
nitrogen. Semen samples were analyzed before and after freezing.
Samples were evaluated by Computer-Assisted Semen Analysis (Hamilton Thorne HTM-
IVOS 12, Beverly, MA) to verify the percentages of total sperm motility (TM) and progressive
sperm motility (PM), velocity of trajectory (VAP; µm/s), linear progressive velocity (VSL; µm/s),
curvilinear velocity (VCL; µm/s) and % of rapid sperm (RAP). Fluorescent probes (CFDA and IP)
were used to evaluate the plasma membrane integrity (IM).
For statistical analysis, data was evaluated by ANOVA (SAS, 2012). Differences between
means were tested with Turkey’s test at 5% probability.

The epididymis stored for 6 hours at 5o C had variable sperm quality. The cold storage is
important for the epididymal transport because the distance between the farm and a specialized
buffalo reproduction center is presented as an obstacle in the preservation of epididymal sperm.
Additionally, studies in cats, dogs, goats, pigs and cattle have shown superior sperm viability if the
epididymides were stored between 4 and 5 ° C, compared with those kept at room temperature.
BB and TRIS pre-freezing results (Tab. 1 and 2) were 38.54±22.33%b and 14.17±12.78%a
for total motility, 25.00±16.12a and 9.44±9.11a for progressive motility, 7.21±0.98a and 5.09±2.65a
for percentage of rapid cells, 91.08±12.53b and 63.33±31.47ª for VAP, 73.54±20.17b and
49.50±9.11ª for VSL, 172.21±24.55ª and 116.94±59.48ª for VCL, respectively (P < 0.05).
BB and TRIS post-thawing results (Table 1 and 2) were 42.25±21.50b and 17.62±19.46a for
total motility, 27.25±24.86a and 18.00±13.68a for progressive motility, 7.35±0.98a and 6.26±1.13a
for percentage of rapid cells, 91.42±16.86ª and 75.96±13.17ª for VAP, 67.96±12.13ª and
60.04±10.42ª for VSL, 177.54±23.53b and 141.29±24.97a for VCL, respectively (P < 0.05). The
findings in the present study show that sperm recovered from the epididymal cauda, after 6 h
storage of epididymides at 5 °C ensures sperm preservation, demonstrating that the diluent Botu-
bov had higher total motility both pre and post freezing when compared with TRIS. Additionally,
the sperm frozen with the diluent Botu-bov showed higher values of VSL at post-thawing. These
findings may reflect in improvement of conception rates. Whereas the recuperation of sperm from
the epididymis is the last chance to preserve the genetic material of genetically superior animals,
techniques that improve the results of cryopreservation are of paramount importance to improve
reproductive outcomes.
REFERENCES
Jones, R. 1998. Plasma membrane structure and remodeling during sperm maturation in the
epididymis. J. Reprod. Fertil. 53: 73-84.
485
Table 1. Parameters of motility, rapid cells and plasma membrane integrity of pre-freezing (Pre)
and post-thawed (Post) sperm samples recovered from the epididymal cauda using Botubov (BB) or
Tris as freezing extenders.
Total motility Progressive motility Rapid cells Plasma membrane integrity
Groups
Pre Post Pre Post Pre Post Pre Post
BB 38.54±22.33b 42.25±21.50b 25.00±16.12a 27.25±24.86a 7.21±0.98a 7.35±0.98a 36.62±23.33a 38.58±21.27a
TRIS 14.17±12.78a 17.62±19.46a 9.44±9.11a 18.00±13.68a 5.09±2.65a 6.26±1.13a 12.61±12.18a 24.54±18.23a
Table 2. Velocity parameters of pre-freezing (Pre) and post-thawed (Post) sperm samples recovered
from the epididymal cauda using Botubov (BB) or Tris as freezing extenders.
Velocities
Groups VAP VSL VCL
Pre Post Pre Post Pre Post
BB 91.08±12.53b 91.42±16.86a 73.54±20.17b 67.96±12.13a 172.21±24.55a 177.54±23.53b
TRIS 63.33±31.47a 75.96±13.17a 49.50±9.11a 60.04±10.42a 116.94±59.48a 141.29±24.97a
486
Adding Motility Stimulants to Improve Freezing of Buffalo Sperm Recovered

from Epididymal Cauda
Aline Sousa CAMARGOS, a Eunice OBA, a Gabriel Augusto MONTEIRO, a Yamê Fabres
Robaina Sancleir SILVA, a Mariana ZORZETTO, a Rosiara Rosária Dias MAZIERO,
a
Frederico Ozanam PAPAa and Alcides Amorim RAMOSb
a
and Animal Science – UNESP, Distrito Rubião Jr. s/ n., Botucatu, SP, Brazil
b
* Corresponding e-mail: euniceoba@fmvz.unesp.br
ABSTRACT
The cryopreservation of epididymal sperm is important to preserve genetic material from
valuable buffalo bulls. This study evaluated the viability of post-thawed sperm samples recovered
from the epididymal cauda adding motility inductors. For that, were used epididymides from eight
Murrah buffaloes with 18 months of age. Semen samples were submitted to three different
conditions: (CT - control) without adding medium, (SPERM) adding Sperm Talp medium, and
(FERT) adding Fert Talp medium. Immediately after slaughter, both testicles from each animal
were collected and transported at 4o C at maximum six hours interval. In laboratory, the removed
epididymides was flushed to obtain sperm and diluted in the freezing extender. Each buffalo sperm
were divided and fractions were submitted to all conditions (CT, SPERM and FERT). Semen doses
were frozen at -196o C. CT, SPERM and FERT post-thawing results were 13.63±8.91, 38.77±8.91
and 42.83±8.91 for total motility, 7.30±8.74, 24.87±8.74 and 29.70±8.74 for progressive motility,
6.04±0.92, 6.74±0.92 and 6.93±0.92 for percentage of rapid cells (P < 0.05). In conclusion, diluted
semen supplementation with Sperm or Fert talp increases the motility of cauda epididymal sperm of
buffalo bulls.
Keywords: semen, buffalo, epididymal, freezing, motility, medium.
INTRODUCTION
The cryopreservation of epididymal sperm is important to preserve genetic material from
valuable buffalo bulls. In other species, studies revealed an improvement of epididymal sperm
quality after incubation with Sperm talp and Fert talp before freezing (Monteiro et al., 2012; 2011;
Melo et al., 2010), because these mediums can stimulate semen motility. This study aimed to
evaluate the viability of post-thawed sperm samples recovered from the epididymal cauda adding
these motility factors.

Were used epididymides from eight Murrah buffaloes with 18 months of age. Immediately
after slaughter, both testicles from each animal were collected and transported at 4 o C at maximum
six hours interval.
In laboratory, the removed epididymis was flushed with to obtain sperm and diluted in Botu-
Semen. Each buffalo sperm were divided and fractions were incubated under three different
conditions: without adding medium (CT – control; Botu-Semen), adding Sperm Talp medium
(SPERM; 80% Botu-Semen and 20% Sperm-talp), and adding Fert Talp medium (FERT; 80%
Botu-Semen and 20% Fert-talp). After 15 minutes, samples were centrifuged and the pellets were
resuspended in freezing extender Tris. For cryopreservation, the straws were maintained at 5o C/4h,
and then 20 minutes at 3 cm above liquid nitrogen before immersion.
Post-thawed samples were evaluated by Computer-Assisted Semen Analysis (Hamilton
Thorne HTM-IVOS 12, Beverly, MA) to verify % total sperm motility (MT), % progressive sperm
487
motility (MP), velocity of trajetory (VAP; µm/s), linear progressive velocity (VSL; µm/s),
curvilinear velocity (VCL; µm/s) and % rapid sperm (RAP). Fluorescent probes (CFDA and IP)
were used to evaluate % plasma membrane integrity (IM).
For statistical analysis, data were evaluated by ANOVA (SAS, 2012). Differences between
means were tested with Tukey test at 5% probability.

SPERM and FERT groups post-thawing results were significant better than CT group on
MT, MP, VAP, VSL, VCL and IM parameters. Motilities results were 13.63±8.91%, 38.77±8.91%
and 42.83±8.91% for total sperm motility, and 7.30±8.74%, 24.87±8.74% and 29.70±8.74% for
progressive sperm motility to groups CT, SPERM and FERT, respectively (P < 0.05).
Similar great results were observed on velocity parameters, with 69.87±10.64 µm/s,
85.43±10.64 µm/s and 89.47±10.64 µm/s for VAP, 52.47±9.34 µm/s, 65.87±9.34 µm/s and
72.60±9.34 µm/s for VSL, and 136.80±20.04 µm/s, 159.97±20.04 µm/s and 166.23±20.04 µm/s for
VCL to groups CT, SPERM and FERT, respectively.
An improvement was observed even in plasma membrane integrity of groups that received
motility inductors, as follows: 11.47±8.99% for CT, 36.23±8.99% for SPERM and 39.67±8.99% for
FERT. No significant differences were observed between SPERM and FERT results, both showed
good results on the parameters above.
Rapid sperm was the unique parameter evaluated that did not show significant difference
between results of the groups evaluated (6.04±0.92% for CT, 6.74±0.92% for SPERM and
6.93±0.92% for FERT).
As occurs with other species, some factors can be detrimental to sperm motility and viability
during frozen process. So, incubation with these motility stimulants is a good alternative to
increment buffalo semen parameters, and avoid low quality of semen for artificial insemination
(Monteiro et al., 2012; 2011; Melo et al., 2010).
In conclusion, diluted semen incubation with Sperm talp or Fert talp increases the quality of
cauda epididymal sperm of buffalo bulls.
REFERENCES
Monteiro, G.A., P.N. Guasti, A. S. Rocha, I. Martin, Y.F.R.S. Silva, C.P. Freitas and F.O. Papa.
2012. Effect of storage time and temperature of equine epididymis on the viability, motion
parameters, and freezability of epididymal sperm. Journal of Equine Veterinary Science 87:
901-909.
Monteiro, G.A., F.O. Papa, F.S. Zahn, J.A. Dellaqua Jr, C.M. Melo and R.R.D.Maziero. 2011.
Cryopreservation and fertility of ejaculated and epididymal stallion sperm. Animal
Reproduction Science 127: 197-201.
Melo, C.M., G.A. Monteiro, B.R. Avanzi, P.N. Guasti, M.A. Alvarenga, J.A. Dellaqua Jr, F.S. Zahn
and F.O. Papa. 2010. Advances in stallion’s epididymal sperm technology. Pferdeheilkunde
26: 48-52.
SAS Institute Inc., 2012. SAS/ETS® Software: Changes and Enhancements for Release 6.12. In:
SAS Institute Inc., Cary, NC. 112.
Table 1. Parameters of post-thawed sperm samples recovered from the epididymal cauda adding
Sperm talp, Fert talp or without stimulants (control).
Motilities Velocities Membrane
Groups Rapid cell
MT MP VAP VSL VCL integrity
CT 13.63±8.91b 7.30±8.74b 69.87±10.64b 52.47±9.34b 136.80±20.04b 6.04±0.92a 11.47±8.99b
SPERM 38.77±8.91a 24.87±8.74a 85.43±10.64a 65.87±9.34a 159.97±20.04a 6.74±0.92a 36.23±8.99a
FERT 42.83±8.91a 29.70±8.74a 89.47±10.64a 72.60±9.34a 166.23±20.04a 6.93±0.92a 39.67±8.99a
488
Effect of Testicular Thermoregulation on the Quality of Buffalo Sperm
Carlos Ramires NETO, Monteiro, G.A., Sancler-Silva, Y.F.R.; Resende, H.L., Zorzetto, M.F.,
Oba, E.
College of Veterinary Medicine and Animal Science – (UNESP) / Univ Estadual Paulista:
Department of Animal Reproduction and Veterinary Radiology. Botucatu, Sao Paulo, Brazil.
*Corresponding e-mail: carlosramiresneto@hotmail.com
ABSTRACT
The process of spermatic division and differentiation (spermatogenesis) occurs with
intratesticular temperature lower that the corporal temperature and for that is essential that the
testicular thermoregulation mechanism occurs properly. For evaluation of the scrotal surface
temperature can be used the infrared thermography or testicular sensors, besides that, can be
evaluated the blood flux in the spermatic cord through the Doppler ultrasonography. Thus, the aim
of this study is to analyze the testicular thermoregulation in adult buffaloes through scrotal
thermography and Doppler ultrasound of testicular artery and verify its effect on sperm quality. For
that were used seven healthy buffaloes, with age of 3 and 4 years, of the Murrah breed. The animals
were subjected to 3 semen collections using artificial vagina, with one day of interval. In addiction,
the retal temperature measurement (RT) with dry bulb thermometer, the measurement of scrotal
surface temperature (SST) and body surface temperature (BST) through infrared thermography and
the pulsatility (PI) and resistivity (RI) index of testicular artery by Doppler ultrasonography, were
performed using 2 distinct moments: animals previously placed to shade (M1) and animals
subjected to 4 hours of sun (M2). All parameters were compared by T test and the correlations were
performed by Pearson test using the In Stat Graph Pad 3® program. The significant level
considered was 5%. There was an increase (p<0,05) of RT, SST, SNT and RI in M2. increasing
trend was observed (0,05>p>0,01) PI and RI between M1 and M2. There was a low correlation
between SST and semen quality. The results of this study allow us to conclude that adult buffaloes
have low ability to perform body and testicular thermoregulation in situations of enviromental heat
stress. However, this low capacity of testicular temperature maintenance demonstrated no
correlation with the sperm kinetic parameters and sperm morphological defects in buffalo
spermatozoa.
Keywords: buffalo, testicular thermoregulation, thermography, Doppler ultrasonography, thermal

stress, semen.
INTRODUCTION
The testicular thermoregulation in domestic animals depends on contraction and relaxation
of dartos and cremaster muscles, sweat gland activity, heat radiation from the scrotal surface and
arteriovenous thermal exchange through the countercurrent transfer system in the pampiniform
venous plexus (Ashdown and Hancock, 1980; Setchell, 1991; Coulter and Kastelic, 1994).
Due to the fact of blood supply in the testes be naturally deficient in situations of increased
intratesticular temperature, increased cellular metabolism occurs and consequently there is a higher
need for oxygen. The low oxygen leads to cell death by triggering the process of testicular
degeneration (Blanchard et al., 1996).
In order to evaluate the efficiency of testicular thermoregulation the testicular temperature
can be measure by introducing sensors into the gonads, however, this procedure is considered
invasive and can offer a danger to the animal. Therefore, Coulter et al. (1988) evaluated the
testicular temperature using noninvasive method of infrared termograraphy and showed no
difference between this method in relation to sensors.
Furthermore, an indirect evaluation of testicular thermoregulation can be performed by
Doppler ultrasound exam of the spermatic cord, once the testicular thermoregulation is directly
489
related to blood flow in the testicular artery. Despite the high incidence of reproductive problems
related to testicular temperature control, there are few studies evaluating this temperature in cattle
(Coulter et al., 1997; Kastelic et al., 1996; Barros et al., 2009), in goats (Coulter et al., 1988;
Maloney and Mitchell, 1996; Kastelic et al., 1999; Silva et al., 2006), in humans (Gold et al., 1977;
Layfaye and Hermabessiere, 1980) and in horses (Ramires Neto et al., 2012).
Thus, the aim of this study is to analyze the testicular thermoregulation in adult buffaloes
through scrotal thermography and Doppler ultrasound of testicular artery and verify its effect on
sperm quality.

Four Murrah buffaloes aged between 4 and 7 years old were used for the study. Initially,
three ejaculates from each buffalo were collected with an interval of 2 days to eliminate possible
damaged cells from epididymal cauda and to stabilize the sperm parameters.
After the stabilization the animals were subjected to 3 semen collections using artificial
vagina, with one day of interval. The ejaculates were diluted with Tris-yolk extender at a
concentration of 50x106 spermatozoids/ml. After that the kinetic parameters were analyzed through
computerized system CASA and the morphological abnormalities by Differential Interference
Contrast Microscopy (DIC).
The retal temperature measurement (RT) with dry bulb termometre, the measurement of
scrotal surface temperature (SST) and body surface temperature (BST) through infrared
thermography (Infra CamTM by FLIR Systems Inc) and the pulsatility (PI) and resistivity (RI) index
of testicular artery by Doppler ultrasonography (My Lab5 by Esaote), were performed in a day with
high enviromental temperature (32,7 oC) using 2 distinct moments: animals previously placed to
shade (M1) and animals subjected to 4 hours of sun (M2).
The termography images were analyzed by ThetrofmaCAM Quick ReportTM software and
the ultrassonographs through Esaote software.
All parameters were compared by T test and the correlations were performed by Pearson test
using the In Stat Graph Pad 3® program. To perform the correlation was considered only the value
of variation of the parameters RT, SST, BST, PI and RI between M1 and M2.
The significant level considered was 5%.
RESULTS
There was an increase (p<0,05) of RT (37,5±0,34a vs 39,0±0,43b; M1 and M2 respectively),
SST (31,0±0,55a vs 35,2,0±1,37b; M1 and M2 respectively) and BST (33,4±3,0a vs 38,48±0,22b;
M1 e M2 respectively). Increasing trend was observed (0,05>p>0,01) in PI (0,99±0,45a vs
1,2±0,26b; M1 and M2 respectively) and in RI (0,64±0,15a vs 0,71±0,07b; M1 e M2 respectively) in
M2.
It was observed strong negative correlation between the variation of SST and BST, between
SST and TR and strong positive correlation between SST and RI. The correlation between the
variation of SST and PI was considered negative average. There was a low correlation between SST
and total sperm motility, between SST and progressive sperm motility, between SST and percentage
of rapid sperm cells, between SST and major defects of the sperm morphology and between SST
and minor defects of sperm morphology (Table 1).
DISCUSSIONS
In the present study was observed fewer 2.5 ° C in scrotal surface temperature than in body
surface temperature, which is essential for spermatogenesis occurs normally (Setchell, 1991). These
data corroborate with Ashdown and Hancock (1980) which found that in domestic animals, the
testicular temperature is lower than the body temperature.
As observed in humans (EDDY et al., 2001), in sheep (Couter et al., 1988) and in horses
(RAMIRES NETO et al., 2012), the thermographic analysis of the scrotum was demonstrated a
practical and non-invasive method for measure the scrotal surface temperature of buffaloes. I was
490
demonstrated this method has a great potential to use in the field and an option to additional exam
of the soundness of buffalo. Mainly because the measurement of scrotal surface temperature be a
good indicator of intratesticular temperature as observed by Coulter et al. (1988).
In the present study there was a significant increase in rectal and body surface temperature
after 4 hours of exposure of the buffaloes to the sun, indicating that the animals were in heat stress
in M2. These data can be explained by the observation of Das et al. (1999) that because of having a
lower density of sweat glands on the body surface the buffaloes have low ability to perform
thermoregulation.
The increase in resistivity and pulsatility index of testicular artery in M2 demonstrate that in
conditions of heat stress adult buffaloes activate its mechanisms of testicular thermoregulation.
However, besides being observed increase in scrotal surface temperature in M1 in relation to M2,
this increase was similar to the increase of body surface temperature, indicating that in the buffaloes
the testicular thermoregulatory mechanisms are not efficient, which may be a consequence of its
poor body thermoregulation, as described by Das et al. (1999).
It was observed a low correlation between testicular thermoregulation and sperm kinetic
parameters of buffaloes. This find corroborate with Fernandes et al. (2008), which evaluated the
effect of increased testicular temperature in bulls and have observed no changes in sperm kinetic
parameters of these animals after insulation.
Another finding of this study was the low correlation between the ability to maintain the
testicular temperature and the alterations in sperm morphology, disagreeing with Vogler (1991) and
Fernandes et al. (2008) which observed an increase of sperm morphology alterations after testicular
insulation in bovine and Blanchard et al. (1996) which observed an increase of alterations in sperm
morphology in stallions subjected to heat stress for prolonged periods.
CONCLUSIONS
The results of this study allow us to conclude that adult buffaloes have low ability to
perform body and testicular thermoregulation in situations of enviromental heat stress. However,
this low capacity of testicular temperature maintenance demonstrated no correlation with the sperm
kinetic parameters and sperm morphological defects in buffalo spermatozoa.
REFERENCES
Ashdown, R.R. and J.L. Hancock. 1980. Functional anatomy of male reproduction. In: E.S.E.,
Hafez, Editor, Reproduction in Farm Animals, Lea & Febiger, Philadelphia., 7–29.
Barros, C.M.Q., E. Oba, J.B. Siqueira, L.S. Leal and J.P. Kastelic. 2009. Effect of ambient
temperature on scrotal, intratesticular, and intravascular temperatures and testicular blood
flow in bulls (“Efeito da temperatura ambiente sobre as temperaturas escrotal intratesticular,
intravascular e fluxo sanguíneo testicular de touros”). Vet. e Zootec.16: 354-361.
Blanchard, T.L. and D.D. Varner. 1996. Evaluation breeding soundness in stallions - 1. The basic
evaluation. Vet. Med. 91: 54-63.
Blanchard, K.T., E.K. Allard and K. Boekelheide. 1996. Fate of germ cells in 2,5-hexanedione-
induced testicular injury. I. Apoptosis is the mechanism of germ cell death. Toxicol. Appl.
Pharmacol. 137: 141–148.
Coulter, G.H. and J.P. Kastelic. 1994. Testicular thermoregulation in bulls. Proc. 15th. Tech. Conf.
Artific. Insem. Reprod., Milwaukee, WI. National Association of Animal Breeders,
Columbia, MO, 1- 14.
Coulter, G.H., P.L. Senger and D.R.C. Bailey. 1988. Relationship of scrotal surface temperature
measured by infrared thermography to subcutaneous and deep testicular temperature in the
ram. J. Reprod. Fert. 84: 417-423.
Coulter, G.P., R.B. Cook and J.P. Kastelic. 1997. Effects of dietary energy on scrotal surface
temperature, seminal quality and sperm production in young beef bulls. J. Animal Sci. 75:
1048-1052.
491
Das, S.K., R.C. Upadhyay and M.L, Madan. 1999. Heat stress in Murrah buffalo calves. Livestock
Production Science 61: 71–78.
Eddy, A. L., L.M. Van Hoogmoed and J.R. Snyder. 2001. The role of thermography in the
management of equine lameness. Vet. J. 162: 172–181.
Fernandes, C.E, M.A.N., Dode, D. Pereira and A.E.D.F. Silva. 2008. Effects of scrotal insulation in
Nellore bulls (Bos taurus indicus) on seminal quality and its relationship with in vitro
fertilizing ability. Theriogenology 70: 1560–1568
Gold, R.H., R.M. Ehrlich, B. Samuels, A. Dowdy and R.T. Young. 1977. Scrotal thermography.
Radiology 122: 129-132.
Lafaye, C. and J. Hermabessiere. 1980. Thermographie diagnosis of bilateral varicocele. 15.
LAFAYE, C.; HERMABESSIERE, J. (1980) Thermography diagnosis of bilateral
varicocele. Ada. Themo. 5: 155-157.
Kastelic, J.P., R.B. Cook and G.H.Coulter. 1996. Insulation of the scrotal neck affects semen
quality and scrotal/testicular temperatures in bulls. Theriogenology 45: 935.
Kastelic, J.P., R.B. Cook and G.H. Coulter. 1999. Effects of Ambient Temperature and Scrotal
Fleece Cover on Scrotal and Testicular Temperatures in Rams. Can. J. Vet. Res. 63: 157-
160.
Maloney, S.K. and D. Mitchell. 1996. Regulation of ram scrotal temperature during heat exposure,
cold exposure, fever and exercise. J. Physiol. 496: 421– 430.
Ramires Neto, C., G. A. Monteiro, D. J. Z. Delfiol, E.G. Fioratti, DellÀqua, Jose Antonio, F.O.
Papa and M. A. Alvarenga. 2012. Study of testicular thermoregulation efficiency in stallion
with different ages In: 6th Internatiol Symposium on Stallion Reproduction. Viena. Journal
of Equine Veterinary Science 32: 510 – 510.
Setchell, B.P., 1991. Male reproductive organs and semen. Repro. Dom. Anim. 8: 221–249.
Silva, M.O.C., M.H.F. Bariani, D. Fernandes, A.J. Birck and H.RA. Resende, J.A. Peres, A.L.
Filadelpho. 2007. Criptorquidismo em equinos. Rev. Cient. Med. Vet. 8: 7 – 17.
Vogler, C.J., R.G. SaackeBame, J.M. Dejarnette and M.L. Mcgilliard. 1991. Effects of scrotal
insulation on viability characteristics of cryopreserved bovine semen. J. Dairy Sci. 74: 3827-
3835.
Table 1: Correlations between the variation of scrotal surface temperature (SST) and body surface
temperature (BST), between rectal temperature (RT) and the pulsatility index (PI), between the
resistivity index (RI) and total sperm motility (TM), between the progressive motility (PM) and
percentage of rapid sperm cells (RAP), between the major defects of sperm morphology (DM) and
minor defects of the sperm morphology (Dm).
BST RT PI RI TM PM RAP DM Dm
SST -0,77 -0,8 0,62 0,73 -0,07 -0,18 -0,03 -0,04 -0,08
492
Attenuation of LPS Induced Proinplammatory Gene Expression by Conjugated

Linoleic Acid (CLA) in Buffalo Granulosa Cells
Vengala Rao YENUGANTI a, Onnureddy KAIPAa and Dheer SINGH a *

a
Molecular Endocrinology Laboratory, Animal Biochemistry Division, National Dairy
Research Institute (NDRI), Karnal-132001, Haryana, India.
*Corresponding Author: drdheer.singh@gmail.com
ABSTRACT
Lipopolysaccharide (LPS) is a component of gram negative bacterial (E. coli) outer
membrane. Previous studies showed that LPS present in plasma and follicular fluid of postpartum
uterine infected animals and inducing the inflammatory response in bovine granulosa cells.
Conjugated linoleic acid (CLA) is a dietary fatty acid that has unique properties like anti-
inflammatory, anti-cancer, anti-atherogenic and anti-diabetic effects. In present study we studied
the effects of LPS on proinflammatory gene expression and CLA as anti-inflammatory agent to
attenuate LPS induced inflammatory gene expression in cultured buffalo granulosa cells under
serum free media. Granulosa cells were cultured and treated with LPS (1.0 µg/ml) for 6 h at
different time intervals and pro-inflammatory cytokines (TNFα, IL-1β and IL-6) were analyzed
using qRT- PCR. Result shown that all the three pro-inflammatory cytokines were significantly
(P≤0.01) up regulated within 2h and then decreased sharply and reached to almost normal level
within 6 h. The Conjugated linoleic acid (CLA), ligand of PPARγ, has been shown to be a potent
anti-inflammatory agent. To test, whether CLA can prevent the LPS induced pro-inflammatory
cytokines expression in granulosa cells, cells were cultured 48 hrs for basal growth, after basal
growth cells were co-treated with CLA (10 µM) and LPS (1.0 µg/ml) for 2 h followed by analysis
of expression of pro-inflammatory cytokines. Surprisingly, CLA had no effect on pro-inflammatory
cytokine expression. Further, cells were pre-treated with CLA for 24hr after basal growth and then
cells were exposed to LPS for 2 h. Result showed that cells pre-treated with CLA showed
significantly (P≤0.01) less expression of pro inflammatory cytokines when comparison to LPS
alone. In conclusion, the results of the present study showed that CLA pre-treatment can prevent the
LPS induced pro-inflammatory cytokines expression in granulosa cells in vitro. (Work supported by
Department of Biotechnology (DBT), Govt. of India.)
Keywords: Reproduction, Conjugated linoleic acid, LPS, Granulosa cells and proinflammatory
genes

493
Frozen-Thawed Epididymal Sperm Quality and the Success Rate of Artificial

Insemination in Spotted Buffaloes (Bubalus bubalis carabanensis)
Yusnizar YULNAWATIa,b,c, *, Hera MAHESHWARId, Muhammad RIZALe and Arief
BOEDIONOd
a
Graduate School, Bogor Agricultural University, IPB Darmaga Campus, Bogor, 16680, West
Java, Indonesia, bResearch Center for Biotechnology, Indonesian Institute of Sciences (LIPI), Jl.
Raya Bogor km. 46, Cibinong, 16911, West Java, Indonesia, cDepartment of Animal Breeding and
Genetics, Swedish University of Agricultural Sciences (SLU), P.O. Box 597, S-75124, Uppsala,
Sweden, dDepartment of Anatomy, Physiology and Pharmacology, Faculty of Veterinary Medicine
IPB, Jl. Agatis, IPB Darmaga Campus, Bogor, 16680, West Java, Indonesia, eDepartment of
Animal Science, Faculty of Agriculture, Lambung Mangkurat University, Jl. Jenderal Ahmad Yani
km 36 Banjarbaru, 70714, South Borneo, Indonesia
* Corresponding email: yulnawati@yahoo.com
ABSTRACT
Spotted buffalo is an exotic species in Indonesia, especially in Torajaland, South Sulawesi.
Population of this species is close to become extinct due to a high slaughtering rate of this animal in
the funeral ceremonies. The extinction risks are become worse since the farmer kept the bull with
special treatment and prevent them from mating activity. Therefore, we tried to conserve gamete by
collecting the sperm from caudal epididymides of bulls soon after slaughtering. Soya bean-based
(A) and two egg yolk-based (B and C) extenders were used in order to find out the optimal freezing
extender that could maintain the quality and fertility of post-thawed epididymal sperm. The post-
thawed progressive motility of epididymal sperm was 45, 40 and 39.2%, while livability was 64.8,
66 and 63.3% and functional membrane integrity was 65.8, 65.4 and 63% for A, B and C extenders,
respectively. In general, there were no significantly different (P>0.05) of all observed parameters in
different phospholipid-based extenders. The pregnancy rate obtained from soya bean-based
extender (37.5%) was also similar (P>0.05) to that of egg yolk-based (40%) extender. In
conclusion, soya bean-based and egg yolk-based extenders could maintain the post-thawed quality
of spotted buffalo epididymal sperm and yielded similar pregnancy rate following artificial
insemination program.
Keywords: Epididymal Sperm, Artificial Insemination, Spotted Buffalo
INTRODUCTION
Spotted buffalo (Bubalus bubalis) is an ordinary Indonesian biodiversity that has been
domesticated mostly in Torajaland, South Sulawesi since more than thousand years ago. In the
Toraja culture, the male spotted buffaloes are slaughtered during funeral ceremony. Because of that,
the cost of a fully-grown male spotted buffalo become very expensive, start from 5,000 to 35,000
USD (Toraja statistic data 2012). This situation may cause the extinction of spotted buffalo, since
farmers keep the bull with a special treatment and do not allow them to perform natural mating
activity. One possible technique that can apply to conserve spotted buffalo is artificial insemination
(AI) using sperm that collected from cauda epididymal tissues. In general, post-thawed buffalo
sperm has lower quality compared to cattle due to the differences in the lipid ratio of the
spermatozoa. This is also give negative correlation to the success rate of artificial insemination
program (Andrabi, 2009). The objective of this study was to define an appropriate extender based
on its phospholipid composition that could maintain the livability as well as fertilizing ability of
post-thawed epididymal sperm in AI program.

Epididymal sperm collection and cryopreservation process
494
Six pairs of cauda epididymis tissues were collected from six to ten years old bulls.
Epididymal sperm were collected with incision and flushing method using 0.9% (w/v) NaCl. The
quality of fresh sperm was analyzed based on its progressive motility, concentration, morphology
and livability. Sperm with >70% of progressive motility and <15% abnormal morphology were
diluted into three different extenders and grouped as A, B and C. A group was a soya-bean based
extender that has been commercially used for bovine semen. Meanwhile, B group was the tris-20%
egg yolk based extender and C group was a citrate-20% egg yolk based extender. Each group of
epididymal sperm plus extenders (semen) was packaged into 0.25 ml straw and equilibrated in 4°C
for 3 hours. Cryopreservation process started by placing the straws 10 cm above the liquid nitrogen
for 15 minutes and then the straws were plunged into the liquid nitrogen for long-term storage
before AI. Sperm quality was checked after thawing from all groups. The thawing was performed in
37°C water-bath for 30 second.
Analyzing of sperm quality
The sperm quality was analyzed at several stages, including soon after collection (fresh
sperm), post dilution, post equilibration and post-thawing. The total number of sperm was examined
using a haematocytometer and Neubauer chamber. Meanwhile, the percentage of progressive
motility was assessed in a small drop of sperm suspension between a glass slide and a coverslip at
37ºC under a light microscope. Furthermore, the sperm livability and morphology were determined
by eosin-nigrosin staining. Viable spermatozoa remained unstained while dead cells were stained
red. The functional plasma membrane integrity was determined using hypo-osmotic swelling (HOS)
assay. HOS solution was consisted of 0.73 g sodium citrate and 1.35 g fructose dissolved in 100 ml
distilled water (osmotic pressure ~190 mOsmol/Kg). To assess the plasma membrane integrity,
semen (50 µl) was mixed with of HOS solution (500 µl) and incubated for 30 minutes at 37°C
before examining under a phase contrast microscope. Spermatozoa were assessed for swelling
characterized by coiled tail indicating functional integrity of the plasma membrane. For evaluation
of sperm motility, livability and morphology, at least 200 spermatozoa were counted.
Artificial insemination
Eighteen female buffaloes were injected using double dosage of prostaglandin (PGF2) with
11 days interval to synchronize the estrous periods. Due to limited number of females that were
available to be AI recipient, we classified these animals to be inseminated within two groups only,
i.e using epididymal sperm that was diluted in soya bean-based versus tris-egg yolk-based
extenders. Estrous detection was performed 72 hours after second PGF2 injection. Females were
inseminated twice using one straw of semen from similar bull per each time. The interval time
between first and second AI was 6 hours. The females were examined for pregnancy through rectal
palpation at days-90 post-insemination.
Data Analysis
All the experiments were replicated six times using epididymal from six individual bulls.
Results were expressed as the means ± standard deviation (sd). A difference with value P<0.05 was
considered statistically significant. The data obtained were analyzed by one-way analysis of
variance for comparison between extenders with the help of Statistical Product and Software
Solutionversion-13 (SPSS Inc., Chicago, IL, USA). The data on pregnancy rates was analyzed
using Chi-square test within 5% (P<0.05) level of confidence to determine statistical significance.

The average quality of sperm concentration and other parameters were shown in Table 1. In
general, the spotted buffalo epididymal sperm quality was suitable to be processed and preserved as
frozen semen. The post-thawed quality was also similar (P>0.05) in all extender groups (Table 2).
Previous studies in some species indicated that epididymal spermatozoa are less tolerant to
cryopreservation process than ejaculate spermatozoa (Martins et al., 2009). Even though, recent
report showed a similar pregnancy rate using epididymal sperm (55.8%) versus ejaculate sperm
(53.8%) in sheep (Àlvarez et al., 2012). The decreasing of temperature that happened extremely
during cryopreservation process induces the formation of intracellular ice crystals and the osmotic
495
and chilling injury of sperm cells. Ice crystal formation caused sperm cells injured and being
damaged to the cytoplasmic fracture, affects on the cytoskeleton and genome related structures, and
can affect on sperm tail and its motile ability as well as reducing the fertility due to compromising
the integrity of plasma membrane (Andrabi et al., 2008). Cold-shock influences the
cholesterol/phospholipids ratio, content of lipids in the bilayer, degree of hydrocarbon chain
saturation and protein/ phospholipid ratio on the plasma membrane (Medeiros et al., 2002), reduces
membrane permeability to water and solutes and injures acrosomal membranes, as well as
mitochondrial sheath and axoneme (Salamon & Maxwell 2000).
The combination of tris-citric egg yolk extender has been recommended for routine use to
cryopreserve buffalo bull semen. Previous results in other species showed that tris–fructose–egg
yolk, citrate–egg yolk and lactose–egg yolk extenders showed similar ability to protect spermatozoa
against freeze–thawing damages. In the absence of egg yolk, none of the diluents used tris-, milk-
and citrate-based gave protection to spermatozoa during freeze–thawing (Andrabi 2009). Egg yolk
with above 20% of concentration will decrease the pH of extender towards the acidic side and could
also reduce the sperm motility. Muller-Schlosser et al. (2001) believed that steroid hormones and its
precursors present in egg yolk were associated with poor fertilization potential of the spermatozoa.
Previous study reported the use of soya bean-based extender in order to avoid the risk of animal
origin compound that could transmit diseases from egg yolk. Their results showed that soya bean-
based extender could maintain the sperm quality as well as egg yolk-based extenders (Martinez-
Pastor et al., 2009). Similar to our result, a comparison study of EY and soya bean effect in
epididymal sperm of African buffalo showed that there is no significantly different in post-thawed
sperm motility (Herold et al., 2004; 2006).
The pregnancy and parturition rates after AI within A and B group were 37.5% and 40%
(P>0.05), respectively (Table 3). Previous study reported that the conception rate in river buffalo
was 41.5% in egg yolk-based and 56% in soya bean-based extenders (Akhter et al., 2011). Some
problems in buffalo AI program are both from female and male sides. Silent heat phenomenon that
is common in female is caused by low hormonal level. This condition was generated the difficulties
in estrous detection, inappropriate time for insemination, and the failure of fertilization. Small
uterine body size prevent the sperm to be introduced into one uterine horn could also be a reason for
low conception rates following AI in buffalo (Sansone et al., 2000). Due to that we understand that
high quality of sperm is absolutely needed to increase the success rate of AI. Although, we optimist
that frozen-thawed epididymal sperm, either in egg yolk or soya bean-based extender, is a good
source and will be applicable to be used in AI to support the spotted buffalo conservation program.
In conclusion, both egg yolk-based and soya bean-based extenders could maintain the post-thawed
spotted buffalo epididymal sperm quality and yielded similar pregnancy rate in AI program.
REFERENCES
Akhter S, M.S. Ansari, B.A. Rakha, S.M.H. Andrabi, N. Ullah and M. Khalid. 2011. Soya-lecithin
in Extender Improves the Freezability and Fertility of Buffalo (Bubalus bubalis) Bull
Spermatozoa. Reprod. Dom. Anim. 47: 815-819.
Álvarez M, J. Tamayo-Canula, C. Martínez-Rodríguez, E. López-Urueña, S. Gomes-Alvesa, L.
Anela, L. Martínez-Pastor and P. de Paz. 2012. Specificity of the extender used for freezing ram
sperm depends of the spermatozoa source (ejaculate, electroejaculate or epididymis). Anim.
Reprod. Sci. 132: 145-154.
Andrabi S.M.H. 2009. Factors affecting the quality of cryopreserved buffalo (Bubalus bubalis) bull
spermatozoa. Reprod. Dom. Anim. 44: 552–569.
Andrabi S.M.H., M.S. Ansari, N. Ullah, M.Anwar, A. Mehmood and S. Akhter. 2008. Duck egg
yolk in extender improves the freezability of buffalo bull spermatozoa. Anim. Reprod. Sci. 104:
427–433.
Herold F.C., K. de Haas, B. Colenbrander and D. Gerber. 2006. Comparison of equilibration times
when freezing epididymal sperm from African buffalo (Syncerus caffer) using TriladylTM or
AndroMed®. Theriogenology 66: 1123-1130.
496
Herold F.C., J.E. Aurich and D. Gerber. 2004. Epididymal sperm from the African buffalo
(Syncerus caffer) can be frozen successfully with AndroMed® and with Trilady™ but the
addition of bovine seminal plasma is detrimental. Theriogenology 61: 715–724.
Martins C.F., K. Driessen, P. Melo Costa, J.O. Carvalho-Neto, R.V. de Sousa, R. Rumpf and M.N.
Dode. 2009. Recovery, cryopreservation and fertilization potential of bovine spermatozoa
obtained from epididymides stored at 5ºC by different periods of time. Anim. Reprod. Sci. 116:
50–57.
Martínez-Pastor F., F. Martínez, M. Àlvarez, A. Maroto-Morales, O. García-Alvarez, A.J. Soler, J.J.
Garde, P. de Paz and L. Anel. 2009. Cryopreservation of Iberian red-deer (Cervus elaphus
hispanicus) spermatozoa obtained by electroejaculation. Theriogenology 71: 628–638.
Medeiros C.M., F. Forell, A.T. Oliveira and J.L. Rodrigues. 2002. Current status of sperm
cryopreservation: why isn't it better? Theriogenology 57: 327-344.
Muino R., M. Fernandez and A.I. Pen. 2007. Post-thaw survival and longevity of bull spermatozoa
frozen with an egg yolk-based or two egg yolk-free extenders after an equilibration period of 18
h. Reprod. Domest. Anim. 42: 305–311.
Muller-Schlosser F., V. Aires, E. Hinsch and K.D. Hinsch. 2001. Evaluation of the quality of a new
generation of egg yolk free semen diluters for cryopreservation of bovine semen. 34 th
Conference on physiology and pathology of reproduction, Giessen.
Salamon S. and W.M. Maxwell. 2000. Storage of ram semen. Anim. Reprod. Sci. 62:77–111.
Sansone G., M.J.F. Nastri and A. Fabbrocini. 2000. Storage of buffalo (Bubalus bubalis) semen.
Anim. Reprod. Sci. 62: 55–76.
Table 1. Quality of fresh spotted buffalo epididymal spermatozoa (mean ± SD).

No Parameters Mean ± SD
1 Concentration (x106 cells/ml) 3578.33 ± 740.33
2 Progressive Motility (%) 74.17 ± 1.86
3 Livability (%) 85.02 ± 2.35
4 Abnormality (%) 7.1 ± 1.16
5 Functional membrane integrity (%) 86.22 ± 1.94
Table 2. The average quality of post-thawed epididymal sperm in different extenders.

No Group % Motility % Livability % MI*
1 A 45.00 ± 4.08 64.82 ± 1.94 65.82 ± 1.98
2 B 40.00 ± 2.89 65.99 ± 3.37 65.43 ± 4.16
3 C 39.17 ± 5.34 63.26 ± 3.95 63.03 ± 3.61
*MI: functional membrane integrity; No statistical difference was obtained (P>0.05)
Table 3. Pregnancy and parturation rates (PPR) following AI program.

No Extenders Number of cows PPR (%)
1 A (soya bean-based) 8 3 (37.5)NS
2 B (egg yolk-based) 10 4 (40) NS
Total 18 7 (100)
*No statistical difference was obtained (P>0.05)
497
Supplementation of Buffalo Follicular Fluid:

Beware of Other Sources of Steroid Hormones during Culture
Kitiya SRISAKWATTANAa* , Kriengsak TASRIPOOa, Wanvipa SUTHIKRAIa,

Supitchaya TREEBONMUANGa and Maneewan KAMONPATANAb
a
Research and Development Center for Livestock Production Technology, Faculty of Veterinary
Science, Chulalongkorn University, Henri Dunant Street, Phatumwan District, Bangkok 10330,
THAILAND
b
Professor emeritus
*Corresponding e-mail: skitiya@chula.ac.th
ABSTRACT
An attempt was made to describe the updated information about the factors to be concerned
before using of buffalo follicular fluid for supplementation in vitro of buffalo oocyte. There are not
only diameter of the follicle, the present of corpus luteum (CL), growth of follicles, stage of estrus
cycle, healthy and atretic state of the ovarian follicles. The progesterone and estradiol-17B
concentration of follicular fluid from individual laboratory have to be determined for optimization
of follicular fluid for supplementation. Fortunately, the effect is not severe because of the mixture
of fluid from various stages of oestrous cycle. It is suggested to pre -determine the progesterone and
estradiol-17β concentration of follicular fluid from different size of follicles and the present of CL,
whose ovaries, retrieved from slaughterhouse, were at unknown estrus cycle. The other natural
sources of progesterone and estradiol-17β were also discussed in this study. Therefore, the amount
of supplementation may be not as high as expected. In conclusion, optimization of follicular fluid is
suggested. This might be lead to reduce the amount of chemical steroids supplementation. Hence,
the cost of expense is reduced.
Keywords: steroid, follicular fluid, follicle, corpus luteum, buffalo, progesterone, estradiol-17β
INTRODUCTION
Supplementation of natural steroid was demonstrated in culture medium with biological
compounds has been demonstrated to be advantages for embryo development in vitro (Carolan et
al., 1995). Results, however, have been varied in optimal concentration of buffalo follicular fluid
used. Furthermore, other sources of natural steroid,apart from follicular fluid, were described.
Therefore, the amount of supplementation may be not as high as expected. The objective of this
study was to suggest some points to be concerned, in order to optimize the amount of follicular
fluid.
Various Amount Of Follicular Fluid Use For Supplementation
It is noted that the optimal amount used of follicular fluid for in vitro maturation are
varied (Table I) from 10% (Yadav et al., 1997; Tajik et al., ,2000 Abdoon,2002), 20% (Das et
al.,1996; Chauhan et al.,1997 ; Tajik et al,2000) till 100% (Gupta et al.,2001 ; Nandi et al.,2004 ).
Furthermore, the reports on follicle size are also varied (Table I). Nandi et al. (2004) showed the
better results when they used follicular fluid from small follicle (< 3 mm in diameter), whereas
Gupta et al. (2001) preferred follicle > 5 mm in diameter, Yadav et al. (1997) chose 2-12 mm in
diameter for their experiments, that was similar to Chauhan et al. (1997) who used 4-10 mm. in
diameter. One of the effects on such variability might be the level of hormonal concentration in the
follicular fluid at the time of aspiration. Our results showed that there were strongly relationship
between follicle size and the presence of corpus luteum(CL). This study suggested that follicular
498
fluid from the ovaries with CL of the follicle both size should not be suitable for IVM. Because the
high level of progesterone inhibits estrus and the ovulatory surge of LH (Hafez and Hafez, 2000).
The concentration of the progesterone in the follicular fluid is critical for oocyte maturation (Atheya
and Totey, 2002).
Our previous results(Srisakwattana et al.,2005) also suggested that the follicular fluid from
large size of follicle of the ovaries without CL is tending to preferable to use in oocyte maturation
than those from small follicles because our data showed that the content of estradiol-17β of the
large follicles was much higher than those of the small follicles at the time of aspirating. The
progesterone of the follicles with CL was higher than those from follicles without CL
(Srisakwattana et al, 2005).
Kulkarni et al. (1994) also reported the wide range of progesterone and estradiol-17β
concentration in their three sizes of follicles.
Our previously study (Srisakwattana et al.,2005) showed that as the follicular size of the
ovaries without CL increased, the progesterone and estradiol-17β levels in the follicular fluid
increased. This results was also corresponded to other’s results (Eissa et. al,1995, 1996; Hooda and
Yadav, 2002;Atheya and Totey, 2002) that as the follicular size increased, follicular fluid
concentration of progesterone and estradiol-17β significantly increase. Hooda and Yadav (2002)
also reported that the increases in estradiol-17β concentration with the increase in follicle size in
their study could be because of the cumulative effect of increasing aromatase activity per cell and
increasing cell number.
These indicated that stage of follicle development and their size and the presence of CL
were possibly among the factors that resulted in such a wide range amount of the % follicular fluid
used for in vitro oocyte maturation have been reported (Table 1).
Other Sources of Natural Steroids
Although, the concentrations of estradiol and progesterone in fetal bovine serum (FBS) are
low, however, their presence should be considered when FBS is included in culture systems
(Stubbing et al., 1989; Gordon 2003; van de Valk et al., 2010). Cumulus cells of bovine COCs
matured in vitro are able to secrete estradiol and progesterone when matured in medium
supplemented with bovine serum albumin (BSA) and gonadotrophins, and that this steroidogenesis
can be modulated by steroids (Mingoti et al.,2002) and they also reported that BSA was also
contaminated with progesterone.
It was reported that granulose cell of follicle with the presence of CL, when cultured with
FBS, showed a higher peak of secreted P4 (22.65, 29.96 ng/ml) than those of without CL (17.12,
18.6 ng/ml) (Srisakwattana et al., 2006). Acyclic buffaloes have lower concentrations of estradiol
and insulin concurrent with higher concentrations of progesterone in the follicular fluid (Khan et al.,
2012). In ovaries, the maturation of oocyte is also governed by estrogen produced by the granulosa
cells (Atheya and Totey, 2002).
Table 1. Various concentrations of buffalo follicular fluid and follicle diameter used for in vitro
maturation, fertilization and subsequent embryo development.
Reference Kumar et Nandi et Kundu et Gupta et al, Tajik et Chuahan Yadav et Shang et
s al,2007 al,2004 al,2003 2001 al,2000 et al,1997 al, 1997 al.,2007
Follicul 5% 10% 100% 10% 10% 20% 10% 10%

ar fluid 50% 20% 40%
used (%) 100%
Follicle 2-8 <3 - >3 - 4-10 2-12 2-8

diameter 3-8
(mm) >8
499
CONCLUSION
Optimization of follicular fluid, whose the ovaries were at unknown estrous cycle, is
suggested. Therefore, follicular fluid should be classified according to the size of follicles whose
oocytes are retrieved. This should help to select the more appropriate source of follicular fluid for
supplementation in order to obtain higher maturation rate and subsequent embryo development.
Apart from other factors included in follicular fluid should be aware. It is suggested that each batch
of follicular fluid should have preliminary information on progesterone and estradiol-17B
concentrations. The amount of supplementation may be not as high as expected. Therefore, the
addition of commercial steroid for supplementation might be reduced. Hence, the cost of chemical
expense will be decreased.
REFERENCES
Abdoon, A.S.S. 2002. In vitro production of buffalo embryos, problems and possibilities. In:
Proceeding of 9th International Congress on Biotechnology in Animal Reproduction ;
December2-4,2002. India pp. 33-38.
Atheya, U.K. and S.M. Totey. 2002. Steroid concentration of the buffalo (Bubalus bubalis) ovarian
follicular fluid. Buffalo J. 18(1): 137-141.
Carolan, R.C., P. Lonergan, A. Van-Langendonckt, A. Mermillod.1995. Factors affecting bovine
embryo development in synthetic oviduct fluid following oocyte maturation and fertilization
in vitro. Theriogenology 43(6):1115-1128.
Chauhan,M.S., Palta, P., Das, S.K., Katiyar, P.K. and M.L. Mada. 1997. Replacement of serum and
hormone additives with follicular fluid in the IVM medium: effects of maturation,
fertilization and subsequent development of buffalo oocytes in vitro. Theriogenology 48(3):
461-469.
Das, S.K.,Chauhan, M.S., Palta, P., Katiyar, P.K. and M.L. Madan. 1996. Replacement of fetal
bovine serum and FSH with buffalo follicular fluid in in vitro maturation of buffalo oocytes.
Theriogenology 45(1): 245.
Eissa, H.M. 1995. Concentrations of steroids and biochemical constituents in follicular fluid of
buffalo cows during different stages of the oestrous cycle. Buffalo J. 11(3): 331-340.
Eissa, H.M. 1996. Concentrations of steroids and biochemical constituents in follicular fluid of
buffalo cows during different stages of the oestrous cycle. British Vet. J. 152: 573-581.
Gordon, I. 2003. Laboratory Production of cattle Embryos. 2003. CABI Publishing, 2nd edition (I. Gordon)
Gupta, P.S.P., Nandi, S.,Ravindranatha, B. and P. Sarma. 2001. Effect of buffalo follicular fluid
alone and in combination with PMSG and M199 on in vitro buffalo oocyte maturation.
Asian-Aust. J. Anim Sci. 14(5): 693-696.
Hafez, B. and E.S.E. Hafez. 2000. Reproduction in farm animal.7th (ed). Lippincott Williams and
Wilkins
Hooda ,O.K. and P.S. Yadav. 2002. Concentration of some reproductive hormones in buffalo
follicular fluid. Indian J. Anim. Sci. 72(11): 971-972.
Khan, F.A., Das G.K., Megha, Pande, Sarkar, M., Mahapatra, R.K. and U.Shnakar. 2012.
Alterations in follicular fluid estradiol, progesterone and insulin concentrations during
ovarian acyclicity in water buffalo (Bubalus bubalis) Anim. Reprod Sci. 130:27-32
Kulkarni,B.A., Deshmukh, B.T., Katkam, R.R. and C.P. Puri. 1994. Follicular fluid steroid
hormone levels of the Indian buffalo. Buffalo J. 10(1): 71-74.
500
Kumar, D., Palta, P., Manik, R.S., Singla, S.K., and S. Chauhan. 2007. Effect of culture media and
serum supplementation on the development of in vitro fertilized buffalo embryos. Indian J.
Anim Sci. 77(8):697-701
Kundu, R.L.,Dhanda, O.P. and S. Sajjan. 2003. In vitro maturation of buffalo oocytes in diffent
biological fluids and their effect on subsequent fertilization, cleavage and blastocyst
development. Bubalus Bubalis 9(1): 56-63.
Mingoti, G. Z,Garcia, J.M. and A.A.M. Rosa-e-Silva. 2002. Steroidogenesis in cumulus cells of
bovine cumulus-oocyte-complexes matured in vitro with BSA and different concentrations
of steroids Anim. Reprod Sci. 69:175-186
Nandi, S., Rughu, H.M., Ravindramatha, B.M., Gupta, P.S. and P.V. Sarma. 2004. In vitro
development of buffalo oocytes in media-containing fluids from different size class follicles.
Reprod Domest Anim. February 1,39 (1): 33-38.
Shang, J.H., Y.J.Huang,X.F. Zhang, F.X. Huang and J. Qin. 2007. Effect of B-nercaptoethanol and
buffalo follicular fkuid on fertilization and subsequent embryonic development of
waterbuffalo (Bubalus bubalis) oocytes derived from in vitro maturation. Ital. J. Anim. Sci.
(Suppl.2):751-754.
Srisakwattana, K., Tasripoo, K., Suthikrai, W., Chethasing, S. and M. Kamonpatana. 2005.
Progesterone and estradiol-17B concentration in buffalo follicular fluid related to size of
follicle from ovaries with CL and without CL The Symposium on Advanced Biotechnology
in Breeding, Molecular Genetics and Nutrition for Increment of Cattle and Swamp 31
August-1 September,2005,Bangkok,Thailand
Srisakwattana, K., Tasripoo, K., Suthikrai, W., Chethasing, S., Ratree, J. and M. Kamonpatana.
2006. Progesterone and estradiok-17β secretion in vitro culture of isolated buffalo
granulose cell from ovaries with CL and without CL. Buffalo J. 22(2):107-120.
Stubbings, R.B.Liptrap,R.M. and P.K. Busrur. 1989. Estradiol and progesterone concentrations in
fetal bovine serum Theriogenology 31 (1); 260.
Tajik, P., Goorani-Nejad and Ghasemzadeh-Nava. 2000. In vitro maturation of water buffalo
oocytes in different concentrations of bovine and buffalo follicular fluid. Theriogenology
53: 470 (Abstr).
Van de Valk, J.,D. Brunner, K. de Smet, A. Fex Svenningsen, P. Honegger, L.E. Knudsen, T. Lindl,
J. Noraberg, A. Price, M.L. Scarino, G. Gstraunthaler. 2010. Optimization of chemically
defined cell culture media-replacing fetal bovine serum in mammalian in vitro methods.
Toxicology in Vitro 24: 1053-1063.
Yadav, P.S., Saini, Anil, Solanki, V.S., Hooda, O.K. and S.K. Jindal. 1997. Comparison of
follicular fluid and estrus serum on in vitro maturation, fertilization and development of
buffalo embryos. Int. J. Anim. Sci. 12: 193-196.
501
Use of Commercially Available Bovine Semen Sexing Agent in Buffalo:

Preliminary Report of the Effect on the Conception Rate
Vittoria Lucia BARILE, a*Corrado PACELLI, b Giuseppe DE SANTIS, a Daniela

BALDASSI, a Marco MAZZI a and Giuseppina Maria TERZANOa
a
Consiglio per la Ricerca e la Sperimentazione in Agricoltura –Animal Production Research Centre
(CRA-PCM), Monterotondo (Rome), Italy
b
Department of Animal Production Science, University of Basilicata, Potenza, Italy
*Corresponding e-mail: vittorialucia.barile@entecra.it
ABSTRACT
The aim of this work was to test the efficacy in buffalo of a post-thaw semen treatment
product, called HeifersPlusTM (HP), available on the market for the bovine. This product works by
enhancing the fertility of the X-chromosome bearing sperm and slowing the motility of the Y-
chromosome bearing one, and attempts to alter the bovine sex ratio in favour of female. In this
preliminary report we have evaluated the effect on conception rate (CR) after timed artificial
insemination (TAI) comparing the use of HP treated semen with non-treated one in buffaloes
synchcronized with Ovsynch; moreover, we have evaluated the effectiveness of delayed
insemination, testing the product either at 16h (standard protocol) or 24h (delayed protocol, as
suggest by the manufacturer) TAI after GnRH. A total of 281 buffaloes were inseminated, 137 with
HP treated semen (treated group) and 144 with no treated semen (control group). Significant
differences were found in the CR, being 27.01% in the treated group and 42.36% in the control one
(P≤0.007).The time of insemination (16h vs 24h) influenced the CR, being reduced at 24h in
respect to standard16h TAI. This was particularly evident in the control group where the CR was
50.88 and 36.78%, respectively at 16 and 24 h (P=0.09). In the treated group there was not
significant differences in the CR between the two different time of AI (22.81 and 30.00%,
respectively at 16 and 24h), however in both time of AI the observed CR was lower compared with
the value obtained in the control group with no treated semen.
Keywords: buffalo, AI, semen sexing, conception rate
INTRODUCTION
Sexing technology takes advantage of the physiological differences between X and Y
spermatozoa, favouring the presence of the desired kind of spermatozoa in the moment of
fertilization and deviating the physiological sex ratio (Seidel, 1999). Although the basic principles
controlling the sex of mammalian offspring have been known for a relatively long time, recent
application of flow cytometry sorting system led to differentiation and separation of living X and Y
chromosome-bearing spermatozoa (Garner and Seidel, 2008). However, a great disadvantage of this
approach is the high cost of the semen doses and the reduced pregnancy rates when compared with
no-sexed semen.
Recently, a post-thaw semen treatment product, called HeifersPlusTM, is available on the
market for the bovine. It works by enhancing the fertility of the X-chromosome bearing sperm and
slowing the motility of the Y-chromosome bearing one, and attempts to alter the bovine sex ratio in
favour of female. This product requires a delay in timing of insemination, therefore using Ovsynch
protocol cows have to be bred 24h after GnRH.
The objective of work was to determine the efficacy of HeifersPlusTM (HP) in buffalo. In this
preliminary report we have evaluated the effect on conception rate (CR) after timed artificial
insemination (TAI), comparing the use of HP treated semen with non-treated one in buffaloes

502
synchcronized with Ovsynch; moreover, we have evaluated the effectiveness of delayed

insemination, testing the product with TAI either at 16h (standard protocol) or 24h (as the
manufacturer’s suggested) after last GnRH injection.

The trial was carried out on 281 Italian Mediterranean buffaloes cows of different ages and
parity, starting from February until May (low breeding season for buffalo reared in Italy).
Buffaloes were divided into HeifersPlusTM (HP) (n=137) and Control (n=144) groups and
subjected to oestrus synchronization and timed artificial insemination (TAI) using Ovsynch
protocol. The treatment schedule was:150 μg GnRH on day 0 + 0.15 mg cloprostenol (PGF2
analogue) on day 7 + 150 μg GnRH on day 9. After the last GnRH injection, two different time of
TAI were used: 16h as standard protocol or 24h (as the manufacturer’s suggested). HP and Control
were equally distributed in each TAI subgroups (16h n=57 HP and 57 Control; 24h n=80 HP and
87 Control). Buffaloes were inseminated using frozen-thawed semen of proved bull. In the HP
group, after thawing, semen was added with the HeifersPlusTM and incubated in water bath at 37°C
ultrasound 28 days after TAI and confirmed at 42 days. Data were analyzed by 2 test (SAS/STAT
for 20 minutes before insemination, according to the kit instructions. The CR was assessed by
User’s Guide).

This is the first attempt to use the HeiferPlus in buffalo species. In this preliminary report
the effect of treatment on the conception rate (CR) is presented.
Significant differences were found in the overall CR of buffaloes confirmed pregnant at 42
day after TAI, between the group inseminated with semen treated with HeifersPlus (HP) and the
group Control (Table 1).The treated semen resulted to give a lower fertility compared to the control
being the CR 27.01% in the HP group and 42.36% in the Control group (P≤0.007). The embryonic
loss, assessed by ultrasound at 42 day, was very low and was equally distributed between animal
inseminated with treated and non-treated semen (data not shown); therefore the lower CR found in
HP group cannot be attributed to embryonic mortality.
The HeiferPlus is a post-thaw semen treatment product that attempted to alter the bovine sex
ratio in favour of females. The product works by enhancing the fertility of the X-chromosome
bearing sperm and slowing the motility of the Y-chromosome bearing sperm. When inseminated,
the sperm sort in the reproductive tract of the dam and the result should be more ova fertilized by
the X-chromosome bearing sperm. Although the ingredients are undisclosed, the manufacturer
published internet resources supporting its efficacy either on CR or on sex-ratio. They reported that
the CR remained either unchanged or slightly better and sex ratio ranged from 67% to 100% heifer
calves produced when HeiferPlus was used (field trial data 2006/2007; Emlab Genetic).
There are no reports on the use of HeifersPlus in species other than the bovine. The only
scientific trial found in literature, that study the effectiveness of this semen sexing agent, is by
Curry et al. (2009). The Authors reported that of the cows inseminated with control semen 48.0%
became pregnant, while 54,5% of HP cows became pregnant. In contrast with the manufacturer
data, no significant difference in sex-ratio between control and HP group was found by these
Authors. To be more precise, Curry et al. (2009) used Ovsynch protocol to synchronize the animals,
but inseminated 12h after the last GnRH that is early respect the suggestion of the manufacturer and
this earlier insemination could be the explanation of the lack in sex-ratio shift.
In this work we wanted to evaluate, in addition to the attempt to obtain more female calves,
the effectiveness of delayed insemination in CR testing the product either at 16h or 24h TAI after
last GnRH injection.
From the results of our work seems that the time of insemination (16h vs 24h) influenced the
CR, being reduced in 24h TAI respect to the standard16h TAI for the Ovsynch program (Table 1).
This was particularly evident in the Control group where the CR was 50.88 and 36.78%,
respectively at 16 and 24 h(P=0.09). In the HP group there were not significant differences in the
503
CR between the two different TAI scheduled (22.81 and 30.00%, respectively at 16 and 24h),
however in both time of AI the observed CR was lower compared with the value obtained in the
control group with no treated semen. The CR obtained in the Control utilizing the standard Ovsynch
procedure (16h TAI) shows that the animal respond positively to the oestrus synchronization
treatment being the CR similar to those reported by others Authors (Barile, 2012).
At present, the only effective method for achieving sex pre-selection before conception,
requires separation of X-chromosome from Y-chromosome bearing sperm and the flow cytometric
technology is capable of producing sexed sperm at 90% of purity. The success of the technology
depends mainly on the fertilizing capacity of the sorted semen that to date is still variable (Rath and
Johnson, 2008). Thus, also using sexed semen the CR is reduced due to the separation process and
the lower number of spermatozoa present in each sexed straw. In the bovine, where use of sexed
semen for AI is being widely commercialized, a 20% reduction in conception rates compared to
unsorted semen has been reported (Norman et al., 2010). The same reduction in CR was found in
this work using HP treated semen. Therefore a reliable method of swaying the sex ratio in favour of
female economically most convenient could be desirable in dairy industry, in bovine as in buffalo.
If the results indicate from the HeiferPlus producer company in terms of female born will be
corroborate, the product could be an inexpensive approach to the sex pre-selection of offspring,
with the advantage to be used with the semen of whatever bull.
ACKNOWLEDGEMENT
This study was supported by Italian Ministry of Agriculture, Grant no. D.M. 13459/7303/2010
(SOS ZOOT)
REFERENCES
Barile, V.L. 2012. Technologies related with the artificial insemination in buffalo. J.Buffalo Sci. 1:
139-146.
Curry E., S.L. Pratt, D.R. Lapin and J.R. Gibbon. 2009. Efficacy of a commercially available post-
thaw bovine semen sexing kit in both single-ovulating and hyperstimulated cows. Anim.
Reprod. Sci.116: 376-380.
Emlab Genetics 2009-2012 [homepage on the Internet]. Press release: December 2007. Available
from: http://www.emlabgenetics.com/
Garner, D.L. and G,E, Seidel Jr. 2008. History of commercializing sexed semen for cattle.
Norman, H.D., J.L. Hutchison and R.H. Miller. 2010. Use of sexed semen and its effect on
conception rate, calf sex, dystocia, and stillbirth of Holsteins in the United States. J. Dairy
Sci. 93: 3880-3890.
Rath, D. and L.A..Johnson. 2008. Application and commercialization of flow cytometrically sex-
sorted semen. Reprod. Dom. Anim. 43 (Suppl. 2): 338-346.
Seidel, G.E. Jr and L.A. Johnson. 1999. Sexing mammalian sperm – overview. Theriogenology 52:
1267-1272.
Table 1. Conception rate (CR) in buffaloes inseminated with HeifersPlusTM treated (HP) or control
semen in two different TAI time (16h and 24h after GnRH in Ovsynch program)
HP Control
TAI Insemin Pregnant CR Insemin Pregnant CR
No No % No No %
16h 57 13 22,81** 57 29 50,88**
24h 80 24 30,00 87 32 36,78
Total 137 37 27,01* 144 61 42,36*
**P=0,002 ;*P=0,007 (in the row)
504
Leptin and Pregnancy: Preliminary Results in Buffalo Cows (Bubalus bubalis)
Olimpia BARBATO,a * Giuseppina M. TERZANO,b Gabriele BRECCHIA,a Luca TODINI,c

Claudio CANALI,a Vittoria L. BARILE b
a
Department of Biopathological Veterinary Science, Faculty of Veterinary Medicine, University of
Perugia, Italy,
b
Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Animal Production Research Centre
(CRA-PCM), Monterotondo (Rome), Italy,
c
Department of Environmental Science, University of Camerino, Italy.
*Corresponding e-mail: olimpia.barbato@unipg.it
ABSTRACT
Leptin is a 16.4 kDa peptide hormone, encoded by the obese gene (ob) and synthesized mainly
by adipocytes, but gene expression has been found in several additional peripheral tissues such as
placenta and fetal tissues, mammary gland, brain and pituitary, stomach, skeletal muscle, duodenum,
abomasum and calf rumen. Beyond the primary role in food intake and energy expenditure, leptin has
been implicated in numerous regulatory functions, including glucose metabolism, lipid oxidation,
endocrine system, blood pressure, haematopoiesis, angiogenesis, brain and bone development, wound
healing and cell differentiation and proliferation. Recent findings suggest that leptin could be implicated
in important reproductive process such as embryonic development and implantation. Thus, the aim of
the present study was to verify whether the maternal blood concentrations of leptin may be related
to the successful establishment and maintenance of pregnancy in buffalo. Thirteen buffalo cows that
become pregnant after artificial insemination (group A) and 13 that did not conceived (group B)
were used. Blood samples were collected at day 0, 14, 23, 25, 28 and 40 from both groups, plus at
day 60 and 80 in pregnant animals (day 0 =AI) for leptin, progesterone (P4) and pregnancy-
associated glycoproteins (PAG). Starting from day 0, plasma leptin concentrations were constantly
low in all the cows that failed to conceive (3.44 ± 0.94 ng/mL), while constantly high levels were
found in animals become pregnant (19.7 ± 2.85 ng/mL). The present data appear to show a
relationship between the concentration of leptin in maternal blood and the ability of buffalo cows to
become pregnant.
Keywords: leptin, P4, PAG, pregnancy, buffalo
INTRODUCTION
Leptin is a 16-kDa hormone secreted mainly by adipocytes and plays a crucial role in
regulating food intake and energy expenditure (Camfield et al., 1995). In addition to its primary
role, regulating whole-body energy balance, it has been implicated in numerous other functions,
including glucose metabolism, lipid oxidation, endocrine system, blood pressure, haematopoiesis,
angiogenesis, brain and bone development, wound healing and cell differentiation and proliferation
(Wylie, 2011). Furthermore, the crucial role of leptin in reproductive physiology arose since its
discovery, being considered a messenger of energy stores and metabolism to the reproductive centres, in
order to allow the onset of puberty and to maintain the cyclic activity (Barash et al., 1996; Dall’Aglio et
al., 2006). Leptin has also been implicated in the regulation of ovarian and oviductal functions,
oocyte maturation and preimplantation embryo development in a variety of animal species (Smith et
al., 2002; Zerani et al., 2005). In ruminants, leptin plays a role in luteal tissue development and
progesterone production (Nicklin et al., 2007; Jessica et al., 2008, Kumar et al., 2012) as in other
species (Zerani et al., 2004).
To our knowledge, no study has been reported on correlation between leptin blood levels
and fertility in buffalo species. Therefore, the present study was performed to investigate whether
plasma leptin concentrations could be related to the successful establishment of pregnancy in
buffalo cows.

505

From a wider group of Italian Mediterranean buffalo cows, subjected to a synchronization
and artificial insemination (AI) program, 26 animals were chosen for this study;: 13 who conceived
(group A) and 13 who did not conceived (group B). Buffaloes were synchronized with a
Progesterone Releasing Intravaginal Device (PRID) inserted for 10 days (day 0=day of PRID
insertion) plus an intramuscular injection of 1000 IU of pregnant mare serum gonadotropin (PMSG)
and 0.15 mg of cloprostenol (PGF2α analogue) at day 7 and artificially inseminated at 72 and 96 h
after PRID withdrawal.
To determine leptin, P4 and PAG levels, blood samples were taken from the jugular vein in
10 mL Ethylenediaminetetracetic Acid (EDTA) tubes at day 0, 14, 23, 25, 28, 40 in all animals,
plus at day 60 and 80 in the pregnant ones (day 0 =AI). Plasma was immediately separated (3000
rpm for 10 min) and stored at - 20 °C until assayed. Pregnancy status and embryo viability
(visualization of heartbeat) were performed 25, 28 and 40 days after the AI by ultrasound scanning.
Pregnancy was then confirmed by rectal palpation at day 60 and 80 post-AI.
The leptin plasma concentration was determined by a commercially available multi-species
leptin radioimmunassay (RIA) Kit (Linco Research, Inc., USA), previously validated for buffalo
species by Terzano et al. (2003). The sensitivity of the leptin assay was 0.37 ng/mL and the intra-
and inter-assay coefficients of variation were 5.6 and 8%, respectively.
The P4 plasma concentration was assayed by RIA Kit (PROG- RIA-CT, Dia Source,
Pantec., Italy). The sensitivity of the assay was 0.08 ng/mL and the intra- and inter-assay
coefficients of variation were 7 and 11%, respectively.
Plasma PAG concentrations were determined by a RIA according to the method described
by Perenyi et al. (2002), with some modifications. The specific antibody was an anti-goat
PAG55kDa+62kDa serum (AS#706) raised in the rabbit, and the bovine PAG 67 kDa preparation
(boPAG67kDa, accession number A61232) was used as both standard and tracer (Zoli et al., 1991).
The intra- and inter-assay coefficients of variation were 2.50% and 7.80%, respectively. PAG
concentration was not assayed on day 14 since, in buffalo cow, its concentration cannot be detected
in maternal blood before day 23-24 after breeding. All determinations were performed in duplicate.
Data were analysed by ANOVA, using the GLM procedure of SAS/STAT software in the
SAS System for Windows, release 9.3 (SAS Institute, USA). A probability of 5% (P<0.05) was
established as the level for statistical significance.

In this study, it was determined the concentration of plasma leptin in pregnant and non-
pregnant buffalo cows in order to establish relationship between maternal circulating leptin and the
ability to became pregnant. Moreover, P4 and PAG determination was carried out to monitoring
hormonally the pregnancy status.
In the pregnant buffaloes (group A), PAG was detectable in the maternal circulation from
day 25 after AI; in non-pregnant buffaloes (group B), plasma PAG levels remained always below
(0.3 ng/mL. PAG plasma concentrations were significantly higher in group A than that of in group
B at day 25 (P<0.03), 28 (P<0.002) and 40 (P<0.0001). P4 profiles confirmed the pregnancy status
of group A. Significant differences were observed on day 14 (P<0.06), 23, 25 (P<0.0001), and 28
(P<0.02) between groups A and B. As shown in Figure 1, starting from day 0, leptin plasma
concentrations were always significantly higher (P<0.0001) in pregnant (19.7 ± 2.85 ng/mL) than
that of in non-pregnant buffaloes (3.44 ± 0.94 ng/mL). Within groups, there was no effect of the day
of sampling on leptin concentration, remaining the values invariable throughout the period
investigated either in pregnant or in non-pregnant.
To our knowledge, there are no reports in literature regarding leptin trend related to
pregnancy in buffalo. Studies on maternal serum leptin are reported in other species. Henson and
Castracane (2000) have reported that maternal serum leptin concentrations are greater in pregnant
than those of in non-pregnant subject in human, non-human primate and rodents and remain
elevated until parturition. In ruminants, data on the leptin trend are reported only for pregnant
506
animals. In pregnant sheep, the leptin concentration in maternal blood was found to be almost
double in middle pregnancy period, compared to the value before conceiving, and then decreased
until parturition (Ehrhardt et al., 2002). In pregnant dairy cow, circulating leptin levels values
remain more or less unvaried until middle pregnancy, rise during late pregnancy, and significantly
drop around parturition (Nikolic et al., 2003; Parola et al., 2007). Our results seem to be in
agreement with these studies demonstrating the direct involvement of leptin in pregnancy; moreover
a proper level of leptin seems to be necessary to conceive. Probably, the higher leptin levels in
pregnant cows are in relation with the better energy balance of these animals. In mouse, for
example, has been reported that adequate leptin levels are required for conception, implantation and
gestation (Malik et al., 2001).
The expression of the leptin and its receptor in hypothalamus area responsible of
Gonadotropin- Releasing - Hormone (GnRH) release, in pituitary gland, responsible for synthesis
and release of gonadotropins, in ovarian and uterus cells of many species, suggests that leptin plays
a role endocrine and autocrine/paracrine in important processes concerning reproduction (Cervero
et al., 2006). Expression of leptin and its receptor in corpus luteum (CL) during oestrus cycle in
buffalo, corresponding to the pattern of P4 secretion, suggest a positive effect of leptin on luteal
steroidogenic function in this species (Kumar et al., 2012), confirming the involvement of leptin in
the reproduction process also in ruminants.
The data of the present work, although preliminary, suggest that a relationship between
plasma leptin concentration and ability to become pregnant could be present in buffalo.
REFERENCES
Barash, I.A., C.C. Cheung, D.S. Weigle, R. Hongping, EB. Kabigting, JL. .Kuijper, D.K. Clifton
and R.A. Steiner. 1996. Leptin is a metabolic signal to the reproductive system.
Endocrinology 137: 3144-3147.
Camfield, L.A., F.J. Smith, Y. Guisez, R. Devos and Burn P. 1995. Recombinant mouse OB
protein: evidence for a peripheral signal linked adiposity and central neural networks.
Science 269: 546-549.
Cervero, A., F. Dominguez, J.A.Horcajadas, A. Quimomero, A. Pellicer and C. Simon. 2006. The
role of the leptin in reproduction. Curr. Opin. Obstet Gynecol. 18: 297-303.
Dall'Aglio, C., P. Ceccarelli, L. Pascucci, G. Brecchia and C. Boiti. 2006. Receptors for leptin and
estrogen in the subcommissural organ of rabbits are differentially modulated by fasting,
Brain Research 1124: 62-69.
Ehrhrdt, R.A., A.W. Bell and Y.R. Boisclair. 2002. Spatial and development regulation of leptin in
fetal sheep. Am. J. Physiol. Regulatory Integrative Comp. Physiol. 282: R1628-R1635.
Henson, M.C. and V.D. Castravìcane. 2000. Leptin in Pregnancy. Biol. Reprod. 63: 1219-1228.
Jessica R.W., A.K. Robin, A.A. Elizabeth, D.M. Melissa, W.O. Chad, B.F. Theresa, J.T .Erin, L.S.
Randy and R.G. Michelle. 2008. Characterization of the leptin receptor angiopoietin-1, and
fibroblast growth factor-2 in the caprine corpus luteum through out the luteal phase of the
estrus cycle. Biol. Reprod. 78: 79-113.
Kumar, L., R.P. Panda, I. Hyder, V.P. Yadav, K.V.H. Sastry, G.T. Sharma, R.K. Mahaptra, S. Bag,
S.K. Bhure, G.K. Das, A. Mitra and M. Sarkar. 2012. Expression of leptin and its receptor
in corpus luteum during estrous cycle in buffalo (Bubalus bubalis). Anim. Reprod. Sci. 135:
8-17.
Malik, N.M., N.D. Carter, J.F. Murray, R.J. Scaramuzzi, C.A. Wilson and M.J. Stock. 2001. Leptin
requirement for conception, implantation, and gestation in the mouse. Endocrinology 142:
5198-5202.
Nicklin, L.T., R.S. Robinson, P. Marsters, B.K. Campbell, G.E. Mann and M.G. Hunter. 2007.
Leptin in the bovine corpus luteum: receptors expression and effects on progesterone
production. Mol. Reprod. Dev. 74: 724-729.
507
Nikolic, J.A., M. Kulkusar, L. Katai, O. Nedic, S. Janosi and G. Huszenica 2003. Periparturient
endocrine and metabolic changes in healty cows and in cows affected by mastitis. J. Vet.
Med. series A - Physiology Pathology Clinical Medicine 50: 22-29.
Parola, R., E. Macchi, D. Fracchia, A. Sabbioni, D. Avanzi, M. Motta, P. Accornero and Baratta M.
2007. Comparison between plasma and milk levels of leptin during pregnancy and lactation
in cow, a relationship with β-lactoglobulin. J. Anim. Phis. Anim. Nutr. 240-246.
Perenyi, Z., O. Szenci, J. Sulon, , P.V. Drion and J.F. Beckers., 2002. Comparison of the ability of
three radioimmunoassays to detect pregnancy-associated glycoproteins in bovine plasma.
Reprod. Domest. Anim. 37: 100-104.
Smith, G.D., L.M. Jackson, D.L.Foster. 2002. Leptin regulation of reproductive function and
fertility. Theriogenology 57: 73-86.
Terzano, G.M., S. Bartocci, S. Terramoccia and A. Parmeggiani. 2003. Leptin level in plasma of
lactating buffaloes fed two diets with different energy and protein concentrations. Ital. J.
Anim. Sci. 2 (1): 181-183.
Wylie, A.R.G., 2011. Leptin in farms animals: whrere are we and where can we go? Animal. 5: 246-
267.
Zeran, M., C. Boiti, G. Brecchia, C. Dell’Aglio, P. Ceccarelli and A. Gobbetti 2004. Ob receptor in
rabbit ovary and leptin in vitro regulation of corpora lutea. J. Endocrin. 182: 279-288.
Zerani, M., C .Boiti, C. Dall’Aglio, L. Pascucci, M. Maranesi, G. Brecchia, C. Mariottini, G.

Guelfi, D. Zampini and A. Gobbetti. 2005. Leptin receptor expression and in vitro leptin
actions on prostaglandin release and nitric oxide synthase activity in the rabbit oviduct. J.
Endocrin. 185: 319-325.
Zoli, A.P., J.F. Beckers, P. Wouters-Ballman, J. Closset, P. Falemagne and F. Ectors, 1991.
Purification and characterization of a bovine pregnancy-associated glycoproteins. Biol.
Reprod.45: 1-10.
Figure 1. Plasma concentration of Leptin (mean ± SD) in pregnant and non-pregnant buffalo cows.
Pregnant
30
* * Not Pregnant
Leptin ng/mL
25 * * *
*
20
15
10
0
0 14 23 25 28 40 60 80
* P<0.0001
Days of pre gnancy
508
Preliminary Evidence on Effect Induced by Heiferplus after In Vitro Exposure

on Functional Parameters of Buffalo's Spermatozoa
Fiorenza MINERVINIa, Rosangela GUASTAMACCHIAb, Antonella GARBETTAa and

Vittoria Lucia BARILEb*
a
Institute of Sciences of Food Production (ISPA), National Council of Research (CNR), Via G.
Amendola 122/O, 70125 Bari (Italy)
b
Consiglio per la Ricerca e la Sperimentazione in Agricoltura-AnimalProductionResearch Centre
(CRA-PCM), Monterotondo (Rome), Italy
*Corresponding e-mail: vittorialucia.barile@entecra.it
ABSTRACT
Interest in buffalo breeding has largely increased worldwide as a dairy purpose animal.
Since in a dairy farm heifer calves are significantly more valuable than bull calves, the birth of
female animals prove to be an importantly aim. Recently, a new sexing agent (HeifersPlusTM) for
selecting X-chromosome bearing spermatozoa has been commercialized for the bovine. Up to now,
no data are available on the use of HeifersPlusTM in the artificial insemination program in buffalo and
on its influence on buffalo sperm quality after in vitro exposure. The aim of this study was to assess
the influence of HeiferPlusTM treatment on some functional parameters of frozen-thawed buffalo
spermatozoa. Semen of two Italian Mediterranean buffalo bull used in AI programme was used.
Functional parameters assessed by cytofluorimetric analysis were: sperm viability (by using
propidium iodide probe), acrosome reaction (by using PNA-FITC probe), sperm chromatin stability
(by using acridine orange probe) and basal ROS determination (by using DCFH-DA probe). The
treatment with HeiferPlusTM induced a significant (P≤0.05) reduction of sperm viability, whereas
oxidative status and sperm chromatin stability were not affected by the treatment. Concerning
acrosome reaction, the percentage of live spermatozoa at 0h and at 3h after thawing with and
without Ca2+ exposure was significantly (P≤0.05) reduced after the treatment with HeifersPlusTM. A
significant reduction of reacted spermatozoa was observed at3h after exposure with HeifersPlusTM
and Ca2+ treatment. From these data it seems that the treatment has adversely affected some of the
functional parameter of buffalo spermatozoa after in vitro exposure.
Keywords: Buffalo spermatozoa, Semen sexing agent, Flow cytometry, Sperm viability, Acrosome
reaction, Sperm chromatin stability
INTRODUCTION
At the present, attention has been focused on buffalo as a dairy purpose animal, instead of a
rustic triple-purpose one (milk, meat and drought power) and, consequently, interest in buffalo
breeding has largely increased worldwide. One of the major constraints in maximizing the
production of this species regards the reproductive efficiency that is hampered in the female buffalo
by a delayed attainment of puberty, seasonality, long postpartum anoestrus and poor oestrous
expression behaviour with consequent limitations of artificial insemination (AI) (Barile 2005).
Concerning AI, another aspect to take into account regards the cryopreservation technique utilized
for semen storage. According to some authors, the cryotolerance of buffalo spermatozoa is poor and
this has been related with the low membrane phospholipid content and its loss during freezing and
thawing procedure (Sansone et al., 2000).
Several methods have been developed over the years for the laboratory assessment of semen
functional parameters in relation to fertility potential. Among these, the application of flow
cytometry to sperm analysis has gradually increased over the last decades. The use of this technique
for sperm evaluation has provided rapid, reliable, objective results, consenting the measurement of
more specific traits such as viability, acrosome integrity, mitochondrial function, capacitation status
509
and DNA integrity (Gillan et al., 2005; Petrunkina and Harrison, 2011). Our previous work has
been showed that flow cytometry can be utilized for the assessment of different functional
parameters of frozen-thawed spermatozoa also in buffalo (Minervini et al., 2012).
Recently, a post-thaw semen treatment product, called HeifersPlusTM, is available on the
market. HeiferPlus is a new sperm sexing agent used to increase the percentage of heifer calves in
cattle. The product works by enhancing the fertility of the X-chromosome bearing (female) sperm
and slowing the motility of the Y-chromosome bearing (male) sperm. When inseminated, the sperm
sort in the reproductive tract of the dam and the result will be more ova fertilized by the X-
chromosome bearing sperm. Thus this sexing agent attempts to alter the bovine sex ratio in favour
of female. Up to now, no data are available on the use of HeiferPlus in buffalo AI program and on
its influence on buffalo sperm quality after in vitro exposure.
The aim of this study was to evaluate the effect induce by HeiferPlus on functional
parameters of frozen-thawed buffalo spermatozoa by using the following flow cytometry analysis:
sperm viability, acrosome reaction, sperm chromatin stability and basal ROS determination.

Commercially available 0.50 ml straws of frozen semen from two buffalo bulls (aged 5-8
years) with known fertility were used in this study. Straws were purchased from an Italian AI
breeding station supplying high quality genetics For each buffalo bull, two straws of the same
ejaculate were used for all flow cytometric analysis. After thawing, one straw was exposed to
HeiferPlus treatment following the Company’s instructions, the other one was used as control.
Sperm cells were diluted to a suitable concentration, depending on parameter determination
requirements, in Phenol red free Tyrode Albumin Lactate Pyruvate (TALP) Medium (Rathi et al.,
2001). For each buffalo bull at least two independent determinations were carried out for each
straw.
Sperm viability
Sperm viability was assessed by using Propidium Iodide (PI) probe that can enter the cell
and stain the nucleus when the plasma membrane is damaged, following the protocol of
Giannoccaro et al. (2010). PI, excited at 488 nm, was read with 650/13 nm bandpass emission filter
(FL-3) by using a logaritmic histogram. Viable spermatozoa with intact plasmalemma (PI-negative)
were observed in the second decade while membrane-damaged cells (PI-positive) were visible in
the fourth decade. Cell viability was expressed as percentage of PI-negative cells.
Acrosome reaction
Acrosome status, associated to sperm viability, was examined with PNA-FITC (as marker
for acrosomal leakage) and PI (as marker for cell death) probes, as described by Minervini et al.
(2012). Acrosomal status of spermatozoa was assessed in the presence or in absence of the Ca2++
ionophore A23187 after 3 h at 37°C. The emission fluorescence of PNA-FITC was read with 530/30
nm bandpass emission filter (FL1) and the emission fluorescence of PI was read with 650/13 nm
bandpass emission filter (FL3) by using a logarithmic histogram.
Sperm chromatin structure assay (SCSA)
The SCSA was performed by using metachromatic properties of the fluorescent acridine
orange following the protocol described by Minervini et al. (2012). Acridine orange emits green or
red fluorescence when it intercalates to double-stranded (native) or single-stranded (damaged)
DNA, respectively. Chromatin structure stability was evaluated on the basis of three DNA
fragmentation index (DFI) parameters, such as %-DFI, X-DFI and SD-DFI, assessed as described
by Minervini et al. (2012). The %-DFI represents the percentage of damaged spermatozoa, X-DFI
represents the degree of DNA damage and SD-DFI indicates the extent of denaturation or how far
each individual spermatozoa may deviate from the main population.
Basal ROS determination
The assessment of basal ROS in buffalo spermatozoa was performed by using 2, 7,-
dichlorodihydrofluorescein diacetate (DCFH-DA) probe following the protocol described by
Minervini et al (2010). Following stress induction, DCFH-DA is oxidized by H2O2 (or lipid
510
peroxides) to yield 2, 7,-dichlorofluorescein (DCF), with increased fluorescence. The fluorescence

of probe was read by flow cytometry with 530/30 nm bandpass emission filter (FL1) by using
logarithmic histogram.
Data were analyzed by t-test (Sigma Plot v.11; Systat Software Inc.) and data are expressed
as mean ± standard deviation. Significant differences between treatment groups were declared at
P0.05.

The effected of HeiferPlusTM treatment on buffalo spermatozoa are reported in Table 1 and
2. After thawing, buffalo spermatozoa used as control, showed sperm functional parameters in
agreement with those reported in previous work in wich a multiparametric assessment of frozen-
thawed buffalo spermatozoa using flow cytometry was performed (Minervini et al., 2012).
In semen enriched with HeiferPlusTM a significant (P≤0.05) reduction of sperm viability was
observed compared to control (5.3% in the treated semen vs 36.4 % in the control), while the sperm
chromatin stability (SCSA) and the sperm oxidative status (ROS) were not affected by the
treatment, being the values of HeiferPlusTM treated semen similar to the control (Table 1). The
sperm viability is related to the integrity of the outer membrane (plasmalemma) that is essential for
sperm reproductive functions, such as sperm metabolism, capacitation, zona pellucida binding and
acrosome reaction. Assessment of plasmalemma integrity is considered to be useful for predicting
the fertilizing ability of a sperm cell (Selvaraju et al., 2008). The reduction of viability, to intend as
loss of integrity of plasmalemma, found in this study, could predict an adverse effect of treatment
on the subsequent fertility. On the other hand, destabilization of the sperm membrane is necessary
to prepare the sperm for the acrosome reaction, and this occurs physiologically during the
capacitation process. Thus HeiferPlusTM could work by selectively capacitating X-bearing sperm.
This activity influences the functional and integrity membrane of spermatozoa, with consequent
reduction of sperm viability. Sperm that have undergone capacitation display hyperactivated
motility (De Jonge, 2005) and in this case X-bearing sperm, swimming faster, will reach the
fertilization site first respect to Y-bearing sperm.
The “pre-capacitation” of X-bearing sperm could be explained by the data concerning the
acrosome reaction (Table 2). The percentage of live spermatozoa with intact acrosome at 0 and 3h
after thawing with and without Ca2+ incubation, was significantly (P≤0.05) reduced after treatment
with HeiferPlusTM compared to control. This could indicate that a higher rate of spermatozoa was
moving towards acrosome reaction process. Ca2+ influx has been demonstrated to occur in sperm
after exposure to factors emanating from the female reproductive tract. The combination of
increased Ca2+ availability and mechanisms in place to rapidly transport calcium into sperm are
involved in the acrosome reaction stimulation (De Jonge, 2005). In fact, after 3h in vitro incubation
of spermatozoa with Ca2+, the rate of acrosome intact sperm population decreases from 55.7 to
35.2% in control and from 25.3 to 16.8% in HeiferPlusTM treated as shown in Table 2, respect the
3h after incubation without Ca2+, most likely because they underwent acrosome reaction. Indeed, a
higher rate of acrosome reacted spermatozoa was found after 3h Ca2+ exposure in both treated and
control, although reduced significantly (P<0.05) in HeiferPlusTM treated sperm compared to control.
Probably, the “pre-capacitation” effect, following HeiferPlus treatment, could stimulate a premature
acrosome reaction rendering the spermatozoa non viable. This could be the reason of the decreased
percentage of acrosome reacted assessed after 3h Ca2+ exposure, being the data referred only to live
spermatozoa.
In conclusion, from these data it seems that the treatment, among the sperm functional
parameters considered, affects viability probably by anticipating capacitation and acrosome
reaction.
REFERENCES
Andrabi S.M.H. 2009. Factor affecting the quality of cryopreserved buffalo (Bubalus bubalis) bull
511
spermatozoa. Reprod. Domest. Anim. 44: 552-569.

Bailey J.L., J.F. Bilodeau and N. Cornier. 2000. Semen cryopreservation in domestical animals : a
damaging and capacitating phenomenon. J. Androl. 21: 1-7.
Barile V.L. 2005. Improving reproductive efficiency in female buffaloes. Livest. Prod. Sci.9: 183-
194.
De Jonge. 2005. Biological basis for human capacitation. Hum. Reprod. 11: 205-214.
Evenson D.P., K.L. Larson and L.K. Jost. 2002. Sperm chromatin structure assay : its clinical use
for detecting sperm DNA fragmentation in male infertility and comparisons with other
techniques. J. Androl.23: 25-43.
Giannoccaro A., G.M. Lacalandra, A. Filannino, F. Pizzi, M. Nicassio, M.E. Dell’Aquila and F.
Minervini. 2010. Assessment of viability, chromatin structure stability, mitochondrial
function and motility of stallion fresh sperm by using objective methodologies. JCAB. 4: 34-
41.
Gillan L., G. Evans, W.M. Maxwell. 2005. Flow cytometric evaluation of sperm parameters in
relation to fertility potential. Theriogenology 63: 445-457.
Minervini F., G.M. Lacalandra, A. Filannino, A. Garbetta, M. Nicassio, M.E. Dell’Aquila and A.
Visconti. 2010. Toxic effects induced by mycotoxin fumonisin B1 on equine spermatoza:
assessment of viability, sperm chromatin structure stability, ROS production and motilità.
Tox. In vitro. 24:2072-2078.
Minervini F., R. Guastamacchia, F. Pizzi, M.E. Dell’Aquila and V.L. Barile. 2012. Assessment of
different functional parameters of frozen-thawed buffalo spermatozoa by using
cytofluorimetric determinations. Reprod. Dom. Anim. doi: 10.1111/j.1439-
0531.2012.02152.x.
Petrunkina A.M. and R.A.P. Harrison. 2011. Cytometric solutions in veterinary andrology:
developments, advantages and limitations. Citometry A. 79A:338-348.
Rathi R., B. Colenbrander, M.M. Beverm and B.M. Gadella. 2000. Evaluation of in vitro
capacitation ofstallion spermatoa. Biol. Reprod. 62: 462-470.
Sansone G., M.J. Nastri and A. Fabbrocini. 2000. Storage of buffalo (Bubalus bubalis) semen.
Anim. Reprod. Sci. 62: 55-76.
Table 1. Effect of HeiferPlus treatment on sperm functional parameters (viability, SCSA, ROS)
assessed by flow cytometry in buffalo.
Control HeiferPlus
Parameters
Sperm viability (%) 36.4 ± 17A 5.3 ± 4a
Sperm chromatin stability (SCSA)
X-DFI 235.7 ± 30 243.6 ± 36
%-DFI 17.6 ± 5 18.2 ± 5
SD-DFI 46 ± 9 56 ± 10
Basal ROS level
12.1 ± 8 8.5 ± 4
(mean fluorescence DCF-DA)
A vs a = P≤0.05
Table 2. Effect of HeiferPlus treatment on acrosome reaction in buffalo spermatozoa, assessed by

flow cytometry, after thawing (T 0) or in vitro induction (T 3h) with or without (w; w/o) Ca2+.
Acrosom Reaction Control HeiferPplus
Acrosome Acrosome Reacted Acrosome Acrosome Reacted
Intact Live Live Intact Live Live
% % % %
A a
T0 56 ± 18.5 0.05 ± 0.08 24.7 ± 6.8 0
T 3h w/oCa2+ 55.7 ± 13.4B 0.7 ± 0.8 25.3 ± 14.6b 0.9 ± 1.8
T 3h wCa2+ 35.2 ± 13.4C 10.2 ± 4.7D 16.8 ± 10.8c 1.5 ± 0.7d
A vs a; B vs b; C vs c; D vs d = P≤0.05
512
Pre-ovulatory Follicle Size, LH Peak Values and Pregnancy Induced by Three

Synchronization Treatments in Buffalo Cows, during Non Breeding Season
Giuseppina MariaTERZANO a*, Mariafrancesca MASCHIOa, Olimpia BARBATO b and

Vittoria Lucia BARILEa
a
Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Animal Production Research Centre
(CRA-PCM), Monterotondo(Rome), Italy; bDepartment of Biopathological Veterinary Science,
Faculty of Veterinary Medicine, University of Perugia, Italy
*Corresponding email: giuseppinamaria.terzano@entecra.it
ABSTRACT
Lactating buffalo cows (n=18) were assigned to 3 different synchronization treatments: group
A (n=7) treated with PRID for 10 days + 1000 IU PMSG and PGF2α (0.15mg cloprostenol) on day 7;
group B (n=6) treated as group A but PMSG and PGF2α were administered on day 10; group C
(n=5), treated with GnRH; (150μg gonadorelin) on day 0 + PGF2α on day 7 + GnRH on day 9
(Ovsynch protocol). Buffaloes were artificially inseminated two times (at 72 and 96h from PRID
removal) in group A and B and once (at 16h from the second GnRH injection) in group C. The size of
pre-ovulatory follicle was assessed by ultrasonography every 12h, starting 24h from PRID removal
(group A and B) or 12h from PGF2α injection (group C); at the same time blood samples were
collected every 4h for the measurement of plasma LH. The pre-ovulatory follicle size was larger
(P<0.05) in group A as compared to B and C groups and in ovulating as compared to non-ovulating
animals (P<0.001); in animals that became pregnant the differences tended to be significant (P<0.08)
as compared to non-pregnant ones. LH peak values were higher (P< 0.05) in group A as compared to
B and C groups and in ovulating as compared to non-ovulating animals (P<0.001). The ovulation rate
(OR) was influenced by treatments: in group A it was 100%; in group B and C the ORs were 50% and
60%, respectively. The conception rates were 71, 4%, 16, 7% and 40% in group A, B and C,
respectively. In conclusion the ovulation and conceptions rates were higher in animals of group A;
that showed also increased pre-ovulatory follicle size and LH peak values.
Keywords: buffalo cow, preovulatory follicle, LH peak, ovulation rate, conception rate, estrus
synchronization
INTRODUCTION
Buffalo shows seasonality in breeding activity and became sexually active in response to a
decreasing day length in late summer to early autumn; consequently buffaloes calving during
unfavourable season may not resume the ovarian activity until the following favourable season.
Therefore, during the non breeding season, hormonal treatments have been designed to
control both luteal and follicular functions, providing exciting possibilities for synchronization of
follicular growth and ovulation, to enable the use of timed artificial insemination (TAI). The main
utilised treatments are based on progesterone-containing devices (PRID or CIDR) along with
estradiol, pregnant mare serum gonadotrophin (PMSG) and prostaglandin (PGF2), or
gonadotrophin releasing hormone (GnRH) associated with PGF2 (Ovsynch protocol; Barile et al.
2005).
Previous studies have showed that different oestrus synchronization treatments influence
pre-ovulatory follicle size and pregnancy success in buffalo cows (Barile et al. 2007; Pandey et al.
2011) and in dairy cows (Herlihy et al. 2011)
In this study the effectiveness of each of the hormonal treatments was assessed with pre-
ovulatory follicle size, by transrectal ultrasound, and plasma LH peak concentration. In addition
ovulation and pregnancy rate per TAI were evaluated.

513

Eighteen lactating Italian Mediterranean buffalo cows were divided in three homogeneous
groups and treated with a progesterone releasing intravaginal device (PRID C.M.; CEVA Santè
Animale, Libourne, France) inserted for 10 days (day 0=day of PRID insertion) plus an i.m.
injection of 1000 IU of pregnant mare serum gonadotrophin (PMSG; Ciclogonina, Fort Dodge
Animal Health, NJ, USA)) and 0.15 μg of cloprostenol (PGF2α; Dalmazin, Fatro, Ozzano Emilia,
BO, Italy) at day 7 (Group A, n=7) or day 10 ( day of PRID removal) (Group B, n=6); Group C
(n=5) was treated with Ovsynch protocol, i.e. an i.m. injection of gonadotrophin releasing hormone
(GnRH; 150 μg gonadorelin, Enagon, Intervet, Aprilia, LT, Italy) at day 0 + PGF2α ( 150 μg
cloprostenol at day 7) + 150 µg GnRH at day 9. Buffaloes were artificially inseminated with
frozen/thawed semen of progeny testing bulls at 72 and 96 h from PRID removal in the groups A
and B and at 16 h from the 2nd GnRH injection in the group C.
To verify ovulation, on day -2, -1, 0 (day 0 = day of the 1th AI in groups A and B; day of a
single AI in group C) all buffaloes undergone, twice daily, at 12h interval, an ultrasound
examination of ovaries by using a portable ultrasound unit (Aloka SSD-500, Aloka CO. Ltd.,
Tokyo, Japan) with a 7.5 MHz linear rectal probe. The images of dominant follicles were frozen on
the screen and measurements were taken using the internal calipers on the Aloka 500V. Ovulation
was defined as the disappearance of a previously identified follicle (>10 mm diameter) from one
ultrasound examination to the next. Pregnancy status and embryo viability (heartbeat) were
performed 26 days after the first artificial insemination (AI) by ultrasound scanning and confirmed
at day 45 post-TAI.
To determine luteinizing hormone (LH), blood samples were taken from the jugular vein in 10
mL EDTA tubes at 4 hours intervals, starting from 24 h after PRID removal (groups A and B) or
from 12 h from PGF2 injection (group C). Plasma was immediately separated (3000 rpm for 10
min) and stored at -20°C until analysed. All determinations were done in duplicate. Plasma LH
concentrations were measured by an ELISA test (LH DETECT, INRA, France) suitable for this
species (Maurel et al. 1995). For buffalo plasma the recovery rate was 80% and the inter and intra
assay coefficient of variation were12.7% and 7% respectively. The sensitivity of the assay was 0.1
ng/mL.
Data were analysed by ANOVA, using the GLM procedure of SAS/STAT software in the SAS
System for Windows, release 9.3 (SAS Institute, Cary, NC, USA, 2008). The statistical model
included treatments (PRID7, PRID 10 or Ovsynch), pregnancy and ovulation as the main factors.
All values were expressed as mean ± standard deviation (sd). A probability of 5% (P≤0.05) was
established as the level for statistical significance in all analyses. The χ2 test was used for data
related to the ovulation and conception rates.
RESULTS
The diameter of the pre-ovulatory follicles (POF) ranged from 0.86 to 1.78 cm; it was
significantly larger (P<0.05) in group A as compared to B and C groups (1.60 ± 0.13 vs 1.21 ± 0.38
vs 1.25 ± 0.21 cm, respectively); it was significantly larger (P<0.05) in all the animals that ovulated
as compared to non-ovulated ones (1.53 ± 0.15 vs 0.95 ± 0.17 cm, respectively) and it was larger in
all the animals that became pregnant as compared to non-pregnant ones (1.51 ± 0.16 vs 1.26 ± 0.36
cm, respectively) (Table 1).
The LH peak was detected in 100%, 50% and 20% of the animals in group A, B and C,
respectively. LH peak mean values, expressed in µg/1, ranged from 0.30 to 9.68; they were
significantly higher (P<0.05) in group A as compared to B and C groups (5.91± 1.25 vs 2.52 ± 2.18
vs 3.32 ± 3.67, respectively), they were significantly higher (P<0.01) in all the animals that
ovulated as compared to non-ovulated ones (5.25 ± 2.23 vs 0.96 ± 0.63, respectively) and in all the
animals that became pregnant as compared to non-pregnant ones (5.00 ± 2.17 vs 3.31 ± 3.03,
respectively) (Table 1).
514
At the time of TAI, the overall ovulation rates were 100% in group A, 50% in group B and
60% in group C. The overall conception rates were 71.4%, 16.7% and 40% in group A, B and C,
respectively (Table 2).
DISCUSSIONS
This study provides more information regarding the effectiveness of synchronization
treatments in TAI protocol, in lactating buffalo, cows during the non breeding season. The results of
this trial support the hypothesis that an optimal size of pre-ovulatory follicles (POF) may improve
the pregnancy rate in synchronized buffaloes. In this study buffaloes with POF ranging between
1.40 and 1.78 cm of diameter presented higher pregnancy rate than those detected with smaller
follicles. This finding coincides with the results of Barile et al. (2007), Pandey et al. (2011), Neglia
et al. (2011) and Rahman et al. (2012), showing that a larger POF was more competent to establish
pregnancy in water buffaloes. Several reports in dairy cattle have also showed a positive association
between POF size and subsequent conception rate (Lopes et al. 2007; Perry et al. 2007). Moreover
the treatment of group A induced ovulation of larger follicles and more capable of establishing
pregnancy and can be successfully employed in the low breeding season to increase the
effectiveness of TAI programmes and to improve the fertility rate. The dosage of LH has allowed us
to detect the presence of a net preo-vulatory peak, easily appreciable in 77.8% of treated animals
(14/18).
The plasma LH peak levels during the ovulatory surge were significantly different among
groups and significantly higher in ovulated buffaloes: in B and C groups the higher standard
deviation leads to a lesser degree of synchronization respect to A group. This finding indicates that
treatments influence the magnitude of pituitary release of LH during the ovulatory surge. The LH
peak levels found in this study are within the range of plasma levels reported by Barile et al. (2005);
Roy and Prakash (2009); Terzano (2012).
It was concluded that administration of PMSG and PGF2a on day 7 during a 10 day PRID
treatment gave POF with larger size consequently improved ovulation and pregnancy rates in
buffalo cows compared with administering PMSG + PGF2a on day 10 (Barile et al. 2005) and with
Ovsynch protocol.
REFERENCES
Barile, V.L. 2005. Reproductive efficiency in female buffaloes. In: Progress in buffalo livestock,
(Ed. A. Borghese). Fao Inter-Regional Cooperative Research Network on Buffalo. pp. 77-
107.
Barile, V.L., A. Malfatti, L. Todini, O. Barbato, C. Pacelli, G.M. Terzano, S. Allegrini, M. Mazzi
and A. Borghese. 2005. LH peak and ovulation in buffalo cows treated for oestrus
synchronisation using two different hormonal schedule. Ital. J. Anim. Sci. 4 (2): 307-309.
Barile, V.L., S. Allegrini, M. Maschio, G. De Santis, G. Neglia and A. Borghese. 2006.
Prostaglandin and PMSG administration at different time during an oestrus synchronization
protocol in buffalo. Reprod. Dom. Anim. 41(4): 345(Abstr.).
Barile, V.L., G.M. Terzano, S. Allegrini, M. Maschio, M. Razzano, G. Neglia and C. Pacelli. 2007.
Relationship among preovulatory follicle, corpus luteum and progesterone in oestrus
synchronized buffaloes. Ital. Journ. Anim. Sci. 6: 663-666.
Herlihy, M.M., M.A. Crowe, M.G. Diskin and S.T. Butler. 2011. Effects of synchronization
treatments on ovarian follicular dynamics, corpus luteum growth and circulating steroid
hormone concentrations in lactating dairy cows. J. Dairy Sci. 95: 743-754.
Lopes, A.S., S.T. Butler, R.O. Gilbert and W.R. Butler. 2007. Relationship of pre-ovulatory follicle
size, estradiol concentrations and season to pregnancy outcome in dairy cows. Anim.
Reprod. Sci. 99: 34-43.
Maurel, M.C., A. Malfatti, A. Debenedetti, A. Catalano and V.L. Barile. 1995. Detection of the
preovulatory LH surge in the Buffalo by an enzyme-immunologic assay. In: Proceedings of
515
the XXX international Symposium of Società Italiana per il Progresso della Zootecnica
“Reproduction and animal breeding: advances and strategy”. Milano, Italy. pp: 405-406.
Neglia G., D. Vecchio, M. Russo, R. Di Palo, C. Pacelli, A. Comin, B. Gasparrini and G.
Campanile.2011. Efficacy of PGF2α on pre-ovulatory follicle and corpus luteum blood
flow. Reprod. Domest. Anim. 47: 26-31.
Pandey A.K., G.S. Dhaliwal, S.P.S. Ghuman and S.K. Agarwal. 2011 Impact of pre-ovulatory
follicle diameter on plasma estradiol, subsequent luteal profiles and conception rate in
buffalo (Bubalus bubalis) Anim. Reprod. Sci.123 (3–4): 169–174.
Perry G.A., M.F. Smith, A.J. Roberts, M.D. MacNeil and T.W. Geary. 2007. Relationship between
size of the ovulatory follicle and pregnancy success in beef heifers. J. Anim. Sci. 85 (3): 684-
689.
Rahman Md S., A.S. Shoag, Md M. Kamal, F.Y. Bari. and M. Shamsuddin. 2012. Preovulatory
follicular and subsequent luteal size influence pregnancy success in water buffaloes. J. of
Reprod. and Dev. 58: 219-222.
Roy K.S. and B.S. Prakash. 1999. Changes in endocrine profiles during ovsynch and ovsynch plus
norprolac treatment in Murrah buffalo heifers at hot summer season. Tropical animal health
and production. 41(4): 677-687.
Terzano G.M., V.L. Barile and Borghese. A. 2012. Overview on Reproductive Endocrine Aspects
in Buffalo. J of Buffalo Sci., 1(2): 126-138.
Table 1. Ovulatory follicle diameter and LH peak (mean ± s.d) by treatments, pregnancy and ovulation.
Classes Ovulatory follicle diameter (cm) LH peak (µg/1)

Group A (n=7) 1.60±0.13a 5.91±1.25a
Group B (n=6) 1.21±0.38b 2.52±2.18b
Group C (n=5) 1.25±0.21b 3.32±3.67ab
Ovulated (n=13) 1.53±0.5A 5.25±2.23A
Not ovulated (n=5) 0.95±0.17B 0.96±0.63B
Pregnant (n=8) 1.51±0.16 5.00±2.17
Not pregnant (n=10) 1.26±0.36 3.31±3.03
Values within columns (class per class) with a, b letters differ for P<0.05; with A, B letters differ for
P<0.001.
Table 2. Ovulation and conception rate (%) among treatment groups.

Group Number Ovulation rate % Conception rate % (n)*
of (n)
animals
A 7 100 (7/7)a 71.4 (5/7)a
B 6 50(3/6)b 16.7 (1/6) b
C 5 60 (3/5)b 40 (2/5)ab
*on day 26 after the first AI
516
Progesterone Treatment during the Periovulatory Period Decreases Embryo

Production in Superovulated Buffaloes
Júlia Gleyci SOARESa*, Nelcio Antonio Tonizza de CARVALHOb, Diego Cavalcante de
SOUZAc, José Ricardo Garla MAIOd, José Nélio Souza SALESe, Lais Mendes VIEIRAa and
Pietro Sampaio BARUSELLIa
a
Departamento de Reprodução Animal, Universidade de São Paulo, São Paulo, Brasil.
b
Unidade de Pesquisa e Desenvolvimento/Pólo Regional do Vale do Ribeira/APTA, Registro-SP;
c
CATI, Registro-SP; dOuro Fino Saúde Animal, Ribeirão Preto-SP; eDepartamento de Ciências
Veterinárias, UFPB, Areia, PB, Brasil.
*Corresponding email: juliasoares@usp.br
ABSTRACT
The present study evaluated the hypothesis that elevated progesterone during the expected
multiple ovulations could improve the embryo recovery rate in superovulated buffalo. Buffalo donors
were randomly assigned into 2 groups in a cross over experimental design: Control (C-G; n=8) and
progesterone treatment groups (P4-G; n=8) during the periovulatory period. Follicular wave emergence
was synchronized with an intravaginal progesterone (P4) device and an injection of 2 mg i.m. of
estradiol benzoate at random stage of the estrous cycle (Day 0; D0 AM). From D4 AM, all buffaloes
received 200 mg i.m. of FSH twice-daily, in 8 applications of decreasing doses. A dose of PGF2α (0.53
mg; i.m.) was given on D6 PM and D7 AM. The intravaginal P4 device was removed on D7 PM from
the C-G and on D10 PM from the P4-G. On D8 PM, all buffaloes received 25 mg i.m. of pLH.
Artificial inseminations (AIs) were carried out 12 and 24 h after the pLH treatment. The ova/embryos
were collected nonsurgically 6 days after the second AI (D14 PM). Ovarian ultrasound examinations
SAS®. On Day 8, similar number of follicles 8 mm was verified in both groups (P4-G=12.1±3.2 vs.
were performed on D0, D8 and on D14. The variables were analyzed by GLIMMIX procedure of
C-G=11.0±2.7; P=0.68). However, no structures (ova or embryos) were recovered from superovulated
buffaloes treated with P4 during the periovulatory period (P4-G=0.0±0.0) compared to control group
(C-G=1.9±0.7; P=0.03). Consequently, no transferable (0.0±0.0 vs. 1.6±0.7; P=0.04) and freezable
embryos (0.0±0.0 vs. 1.6±0.7; P=0.04) were verified in buffaloes treated with P4-G (P4-G vs. C-G,
respectively). Moreover, buffaloes from P4-G had a lower ovulation rate (13.5±4.9 vs. 71.5±16.1%;
the number of follicles  8mm on the flushing day (D14 PM) increased in P4-G compared to C-G
P=0.002) and a lower number of CLs on D14 (1.1±0.3 vs. 8.0±2.8; P=0.04) than those of C-G. Also,
(10.5±3.0 vs. 3.6±0.9; P=0.11). Results indicate that treatment with progesterone during the
periovulatory period was not effective to increase embryo production in superovulated buffaloes.
Keywords: superovulation, buffaloes, embryo, progesterone, periovulatory
INTRODUCTION
Numerous studies have been conducted to evaluate the efficiency of superovulation and embryo
transfer in buffaloes in the world (Drost et al., 1983; Zicarelli et al., 1994; Baruselli, 1994; Madan et al.,
1996; Carvalho et al. 2002; Campanile et al., 2010). The use of this technique still has limitations on this
species, mainly related to the low embryo recovery rate (1-3 viable embryos for harvest; Misra et al.,
1990; Ambrose et al., 1991; Baruselli, 1994; Zicarelli et al., 1994; Taneja et al., 1995; Madan et al., 1996;
Zicarelli et al., 2000; Baruselli et al., 2000; Carvalho et al., 2002).

517
According to Hunter (1988), the mechanisms involved in oocyte transportation (ciliary beats of
the epithelium of the oviduct and waves of contraction of the myosalpinx) are controlled by ovarian
steroids. It is known that buffaloes had lower plasmatic concentrations of estradiol than cows during the
estrous cycle (Batra et al., 1983). The low number of embryos obtained in buffalo MOET could be
attributed to high estrogen (E2) levels during the superovulation treatment, as postulated by Misra et al.
(1998). Prolonged exposure to elevated concentrations of 17-estradiol may change the intrauterine
and/or oviductal environment and, consequently, impair normal embryonic development and transport. It
is also possible that buffalos are more sensitive to high 17-estradiol levels during superstimulation
treatments than cows (Beg et al., 1997). Carvalho et al. (2011) found in histological sections, thicker
infundibulum in buffaloes compared to cattle. The authors report that this finding may explain the lower
recovery rate of embryonic structures in buffaloes subjected to MOET protocols, since the E2 can reduce
the size of the oviduct lumen for promoting smooth muscle contractions of this organ (Hunter, 1988).
The increase in the estradiol/progesterone proportion may impair the interaction between
oocytes and endossalpinge ciliate cells during the ovulations. The absence of this interaction promotes
failure of oocytes uptake because oviduct fluid flow is directed into the abdominal cavity during
ovulation (Hunter, 1988). Additionally, it was found that the epithelium of the oviduct is able to select
viable oocytes (mature or not) in mares, but if the oocyte is degenerated, it floats in the lumen of the
oviduct indicating that the oviduct is able to select oocytes (Kolle et al., 2009). The low embryos
recovery rate of buffaloes compared to cattle may be related to some failures in the process of
collection and/or transport of oocytes by the oviduct (Baruselli et al., 2000; Carvalho et al., 2007).
Accordingly, the P4 could assist in reducing the deleterious effects of high concentrations of E2, since
reduction in serum concentrations of E2 in superovulated buffaloes treated with progesterone during
concentrations stimulates the receptors  (inhibitors of contractility), causing relaxation of the

the periovulatory period was observed (Baruselli et al. 2002). Furthermore, the increase in plasma P4
ampoule-isthmus junction and allowing the transport of the ova to the isthmic portion of the oviduct
(Caschetto et al., 1979; Helm et al., 1982).
Thus, this study hypothesizes that the maintenance of P4 device during the periovulatory period
could decrease the estrogen concentrations and the proportion of estrogen/progesterone, and consequently
could decrease the contractility of the genital system, facilitate uptake of oocytes by fimbriae and increase
the embryo recovery rate in superovulated buffaloes.

Animals and experimental design
Buffalo Murrah donors were randomly assigned according to age, milk production, body
condition score, days postpartum, parity and ovarian activity (presence of corpus luteum: CL) into 2
groups in a cross over experimental design (35 d interval): Control (C-G; n=8) and progesterone
treatment groups (P4-G; n=8) during the periovulatory period. Follicular wave emergence was
synchronized with an intravaginal progesterone (P4) device (Sincrogest®, Ouro Fino Saúde Animal,
São Paulo, Brazil) and an injection of 2 mg i.m. of estradiol benzoate (Sincrodiol®, OuroFino, Brazil)
at random stage of the estrous cycle (Day 0; D0 AM). From D4 AM on, all buffaloes received 200 mg
i.m. of FSH (Foltropin-V®, Bioniche Animal Health, Canada) twice-daily, in 8 applications of
decreasing doses. A dose of PGF2α (0.53 mg i.m. sodic cloprostenol; Sincrocio®, Ouro Fino) was given
on D6 PM and D7 AM. The intravaginal P4 device was removed on D7 PM from the C-G and on D10
PM from the P4-G (the P4 device remains until 48 h after the administration of the ovulation inducer –
D10 PM). On D8 PM, all buffaloes received 25 mg i.m. of pLH (Lutropin-V®, Bioniche Animal
Health). Artificial inseminations (AIs) were carried out 12 and 24 h after the pLH treatment. The
ova/embryos were collected nonsurgically 6 days after the second AI (D14 PM). At the same day
518
buffaloes received the administration of PGF2α (0.53 mg i.m. sodic cloprostenol; Sincrocio®, Ouro
Fino).The total number of ova/embryos, unfertilized oocytes and Grade 1 (excellent or good), 2 (fair)
and 3 (poor) embryos were classified according to the IETS Manual (IETS, 1998). Grade 1 embryos
were considered to be suitable for freezing, and Grade 1 and 2 embryos were considered to be
transferable.
Ultrasonographic examinations
Transrectal ovarian ultrasound examinations (DP2200Vet Mindray, China) were performed on
D0 to verify ovarian activity (presence of CL), on D4 to verify the number of follicles recruited on the
 8 mm) and on D14 to check the superovulatory response (number of CLs). The variables were
follicular emergence ( 3mm), on D8 PM to verify the superestimulation response (number of follicles
analyzed by GLIMMIX procedure of Statistical Analysis System (SAS®).
8 mm (P4-G=12.1±3.2 vs. C-G=11.0±2.7; P=0.68). However, no structures (ova or embryo) were

At the end of the superstimulation (D8) the two treatment groups had similar number of follicles
recovered from superovulated buffaloes treated with P4 during the periovulatory period (P4-
G=0.0±0.0) compared to control group (C-G=1.9±0.7; P=0.03). Consequently, no transferable (0.0±0.0
vs. 1.6±0.7; P=0.04) and freezable embryos (0.0±0.0 vs. 1.6±0.7; P=0.04; Table 1) were verified in
buffaloes treated with P4-G (P4-G vs. C-G, respectively). There may be multiple physiological
mechanisms that result in the reduced fertility when P4 is elevated near AI. Progesterone may alter
sperm or oocyte transport by altering uterine or oviducal contractility, and thus reduces fertilization
(Hunter 2005). Furthermore, the reduced endometrial thickness with slight elevations in P4 (Souza et
al. 2011) may indicate other major effects of P4 on the uterus that could result in reduced embryo
development. Moreover, circulating P4 near AI has been shown to be detrimental to fertility in dairy
cattle, but the underlying physiological mechanisms that reduce fertility are not well understood
(Wiltbank et al., 2012).
In this study, buffaloes from P4-G had a lower ovulation rate (13.5±4.9 vs. 71.5±16.1 %;
the number of follicles  8mm on the flushing day (D14 PM) increased in P4-G compared to C-G (10.5
P=0.002) and a lower number of CLs on D14 (1.1±0.3 vs. 8.0±2.8; P=0.04) than those of C-G. Also,
± 3.0 vs. 3.6 ± 0.9; P=0.11). Gimenes et al. (2011) found that the acquisition of ovulatory capacity in
buffalo occurred when the dominant follicle reached 8.5mm in diameter. In the present study, although
there are many follicles ( 8mm) at the moment of the LH treatment, superovulated buffalo with high
P4 levels at LH treatment had a low ovulation rate. Buffalo exposed to high P4 during the periovulatory
period had low concentrations of E2 (Baruselli et al., 2002), which could negatively influence the
expression of LHR in the follicles. Studies showed significant association between LHR expression and
estradiol (Jeppesen et al., 2012). Furthermore, after the superovulatory treatment with FSH, donors
from P4-G were exposed to high circulatory P4 concentration that could reduce the LH pulsatility
(Stock and Fortune, 1993). It may have led the superovualtory follicles to the initiation of an atresia
process that could reduce the ovulatory capacity (Ginther et al., 2001).
Once the dominant follicle enters in the process of atresia, its properties are changed, among
them, the reduction in mRNA expression for gonadotropins receptors (Xu et al., 1995), both in
granulosa and theca cells (Ireland; Roche, 1983), preventing ovulation is induced. Thus, in the present
study, reduction of LHR could be occurred resulting in the decreased ovulation rate in the G-P4.
In conclusion, results of this study indicate that treatment with progesterone during the
periovulatory period was not effective to increase embryo production in superovulated buffaloes,
rejecting the initial hypothesis.
519
REFERENCES
Ambrose, J.D., S.K. Singla, S. Sailkhani, et al. 1991. Superovulation response in buffaloes (Bubalus
bubalis) to different treatment regimes of folltropin. Theriogenology 35:181.
Baruselli, P.S. 1994. Basic requiriments for artificial insemination and embryo transfer in buffaloes.
Buffalo J. 2: 53-60.
Baruselli, P.S., E.H. Madureira, J.A. Visintin, R. Porto-Filho, N.A.T. Carvalho, G. Campanile and Z.
Zicarelli. 2000. Failure of oocyte entry into oviduct in superovulated buffalo. Theriogenology
53: 491.
Baruselli, P.S., M.O. Marques, R.P. Arruda, N.A.T. Carvalho and C.A.Oliveira. 2002. Plasma estradiol
and progesterone concentrations in superovulated buffalo in presence of CIDR-B devices.
Theriogenology 57: 761.
Batra, S.K. and R.S. Pandey. 1983. LH and oestradiol-17 in blood plasma and milk during the oestrous
cycle and early pregnancy in Murrah buffaloes. Anim. Rep. Sci. 5: 247-257.
Beg, M.A., P.C. Sanwal and M.C. Yadav. 1997. Ovarian response and endocrine changes in buffalo
superovulated at midluteal and late luteal stage of the estrous cycle: a preliminary report.
Campanile, G., P.S. Baruselli, G. Neglia, D. Vecchio, B. Gasparrini, L.U. Gimenes, L. Zicarelli and
M.J. D’Occhio. 2010. Ovarian function in the buffalo and implications for embryo
development and assisted reproduction. Anim. Repro. Sci. 121: 1-11.
Carvalho, N.A.T., P.S. Baruselli, E.H. Madureira, et al. 2002. Control of ovulation subsequente to
superstimulation of follicular growth in buffalo: fertilization and embryo recovery.
Carvalho, N.A.T., F.S. Vannuci, M. DÁngelo, A.G. Gallupo, G.M. Melo, R.J. Souza, M. Nichi. L.U.
Gimenes, M.F S.á Filho, C.C.Martiz, E.S.C. Castricini and P.S. .Baruselli. 2007. Oocytes
transport across the oviduct of Murrah and Nelore cows. Ital. J. Anim. Sci. 6: 649-651.
Carvalho, N.A.T., P.P. Bombonato, M.D. Angelo and P.S. Baruselli. 2011. Anatomical and functional
characterization of the genital system of female buffaloes (Bubalus bubalis) and its implications
on multiple ovulation and embryo transfer. Rev Bras Reprod. Anim, 35: 95-103.
Caschetto, S., , B. Lindlom, N. Wiqvist and L. Wilhelmsson. 1979. Prostaglandins and the contractile
function of the human oviductal ampulla. Gynecology and Obstetric Invest 10: 212-220.
Drost, M., J.M. Wright Junior, W.S. Cripe, et al. 1983. Embryo transfer in water buffalo (Bubalus
bubalis). Theriogenology 20: 549-85.
Gimenes, L.U., N.A. Carvalho, M.F. Sá Filho, F.S. Vannucci, J.R. Torres-Júnior, H. Ayres, R.M.
Ferreira, L.A. Trinca, E.S. Sartorelli, C.M. Barrom, M.P. Beltran, G.P. Nogueira, R.J.
Mapletoft and P.S. Baruselli. 2011. Ultrasonographic and endocrine aspects of follicle
deviation, and acquisition of ovulatory capacity in buffalo (Bubalus bubalis) heifers. Anim
Reprod Sci. 123(3-4): 175-179.
Ginther., O.J, D.R. Bergfelt, M.A. Beg and K. Kot. 2001. Follicle selection in cattle: role of luteinizing
hormone. Biol. Reprod. 64(1): 197-205.
Helm, G., C. Owman, N.O. Sjoberg and B. Walles. 1982. Motor activity of the human fallopian tube in vitro
in relation to plasma concentration of oestradiol and progesterone, and the influence of
noradrenaline. J. Rep. Fert. 64: 233-242.
Hunter, R.H.F. 1988. The fallopian tubes: their role in fertility and infertility. Berlin: Springer-Verlag
112-156.
Hunter, R.H.F. 2005. The Fallopian tubes in domestic mammals: how vital is their physiological
activity? Reprod. Nutr. Dev. 45: 281–290.
Ireland, J.J and J.F. Roche. 1983. Development of nonovulatory antral follicles in heifers: changes in
steroids in follicular fluid and receptors for gonadotropins. Endocrinology 112: 150-156.
520
Jeppesen, J.V., S.G. Kristensen, M.E. Nielsen, P. Humaidan, M. Dal Canto, R. Fadini, K.T. Schmidt, E.
Ernst and C.Y. Andersen. 2012. LH-Receptor Gene Expression in Human Granulosa and
Cumulus Cells from Antral and Preovulatory Follicles. J Clin Endocrinol Metab 97(8):epub
Kolle, S., S. Dubielzig, S. Reese, A. Wehrend, P. Konig and W. Kummer. 2009. Ciliary Transport,
Gamete Interaction, and Effects of the Early Embryo in the Oviduct: Ex Vivo Analyses Using a
New Digital Videomicroscopic System in the Cow. Biology of Reproduction 81: 267–274.
Madan, M.L., S.K. Das and P. Palta. 1996. Application of reproductive technology to buffaloes. Anim.
Reprod. Sci. 42: 299-306.
Misra, A.K., B.V. Joahi, P.L. Agrawala, et al. 1990. Multiple ovulation and embryo transfer in indian
buffalo (Bubalus bubalis). Theriogenology 33: 1131-1141
Misra, A.K, R. Kasiraj, M. Mutha Rao, et al. 1998. Rate of transport and development of
preimplantation embryo in the superovulated buffalo (Bubalus bubalis). Theriogenology 50:
637-649.
Souza, A.H., A. Gu¨men, E.P.B. Silva, A.P. Cunha, J.N. Guenther, C.M. Peto, D.Z. Caraviello and
M.C. Wiltbank. 2007. Supplementation with estradiol-17 beta before the last gonadotropin-
releasing hormone injection of the Ovsynch protocol in lactating dairy cows. J. Dairy Sci. 90:
4623–4634.
Stock, A.E. and J.E. Fortune, 1993. Ovarian follicular dominance in cattle: relationship between
prolonged growth of the ovulatory follicle and endocrine parameters. Endocrinology .132(3):
1108-1114.
Taneja M., G. Singh, S.M. Totey, et al. 1995. Follicular dynamics in water buffalo superovulated in
presence or absence of a dominant follicle. Theriogenology 44: 581-597.
Wiltbank, M.C., A.H. Souza, P.D. Carvalho, R.W. Bender, A.B. Nascimento. 2012. Improving fertility
to timed artificial insemination by manipulation of circulating progesterone concentrations in
lactating dairy cattle. Reprod. Fertil. Dev. 24: 238–243.
Xu, Z., H.A. Garverick, G.W. Smith, M.F. Smith, S.A. Hamilton and R.S. Youngquist. 1995.
Expression of follicle-stimulating hormone and luteinizing hormone receptor messenger
ribonucleic acids in bovine follicles during the first follicular wave. Biol. of Rep. 53: 951-957.
Zicarelli, L., R. Boni, C. Pacelli , et al. 1994. Superovulation in italian mediterranean buffaloes using
FSH-p with diferent treatments. In: Proceedings of 4th World Buffalo Congress, São Paulo.
3:462-464.
Zicarelli, L., P.S. Baruselli, G. Campanile, , et al. 2000. Embryo recovery in buffalo with timed
ovulation and insemination subsequent to follicle superstimulation. In: Proceedings of 14th
International Congress of Animal Reproduction, Estocolmo. 2: 16-19.
Table 1. Follicular dynamics and total of structures (ova/embryo) of animals treated with a P4 device
removed on D7 PM (C-G) and on D10 PM (P4-G).
Treatment
C-G P4-G
Number of follicles 3 mm D4
(n=8) (n=8) P value
Number of follicles 8 mm D8
25.9±4.9 23.1±4.0 0.30
11.0±2.7 12.1±3.2 0.68
Total structures (ova/embryo) 1.9±0.7 0.0±0.0 0.03
Transferable 1.6±0.7 0.0±0.0 0.04
Freezable 1.6±0.7 0.0±0.0 0.04
Ovulation rate (%) 71.5±16.1 13.5±4.9 0.002
Number of follicles 8mm on D14

Number of CLs on D14 8.0±2.8 1.1±0.3 0.04
3.6 ± 0.9 10.5 ± 3.0 0.11
521
PGF2α Treatment during the Periovulatory Period Increases the Number of

Embryos Recovered from Superovulated Buffaloes
Júlia Gleyci SOARESa*, Nelcio Antonio Tonizza de CARVALHOb, Benedicto MARTINS
JÚNIORb, Diego Cavalcante de SOUZAc, José Ricardo Garla MAIOd, José Nélio SALESe,
Lais Mendes VIEIRAa and Pietro Sampaio BARUSELLIa
a
Departamento de Reprodução Animal, Universidade de São Paulo, São Paulo, Brasil.
b
Unidade de Pesquisa e Desenvolvimento/Pólo Regional do Vale do Ribeira/APTA, Registro-SP;
c
CATI, Registro-SP; dOuro Fino Saúde Animal, Ribeirão Preto-SP; eDepartmento de Ciências
Veterinárias, UFPB, Areia, PB, Brasil.
*Corresponding email: juliasoares@usp.br
ABSTRACT
The low embryo recovery rate reported in buffaloes may be related to the failure of oocytes
to enter the oviduct after superstimulation of follicular growth. We hypothesized that the use of
PGF2α during the periovulatory period stimulates the contractile activity of tissue tubal smooth
muscle, allowing portions of the oviduct fimbriae to be activated to capture the oocytes, improving
the number of embryos recovered from superovulated buffaloes. Buffalo donors were randomly
assigned into 2 groups in a cross over experimental design; control group without PGF2α injection
(C-G; n=22) and PGF2α group (PGF-G; n=22) with PGF2α injection. Follicular wave emergence
was synchronized with an intravaginal progesterone (P4) device and an injection of 2 mg i.m. of
estradiol benzoate at random stage of the estrous cycle (Day 0; D0 AM). From D4, all buffaloes
received 200 mg i.m of FSH twice-daily, in 8 applications of decreasing doses. A dose of PGF2α
(0.53 mg; i.m.) was given on D6 PM and D7 AM, and the P4 device was removed on D7 PM. On
D8 PM, 20µg of GnRH were given. Inseminations were done 12 and 24 h after the GnRH
treatment. Buffaloes from PGF-G received four extra doses of PGF2α (0.53 mg, i.m.) from D8 PM
to D10 AM 12 h apart. The ova/embryos were collected nonsurgically 6 days later (D14). The
variables were analyzed by GLIMMIX procedure of SAS®. The treatments resulted in a similar
number of follicles 8 mm on Day 8 (PGF-G=18.6±3.0 vs. C-G=17.1±2.0; P=0.80). A total number
of ova and embryos recovered was greater in superovulated buffaloes treated with PGF2α during
the periovulatory period (PGF-G=3.5±0.6) compared to control group (C-G=2.3±0.5; P=0.02).
Also, an increased number of transferable embryos was verified in treated buffaloes (PGF-
G=2.7±0.6 vs. C-G=1.8±0.5; P=0.05). Furthermore, the number of freezable embryos tended to
increase in PGF-G (PGF-G=2.6±0.6 vs. C-G=1.8±0.5; P=0.08). However, no differences were
observed in the number of degenerated embryos between the two groups (PGF-G=0.3±0.1 vs. C-
G=0.4±0.1; P=0.61). Results indicate that the administration of PGF2α during the periovulatory
period was effective to increase the number of embryos recovered from superovulated buffaloes.
Keywords: superovulation, buffaloes, embryo, PGF2α, periovulatory
INTRODUCTION
Although the advances provided by the MOET technique have revealed that buffaloes have
satisfactory responses to superovulatory treatment (Baruselli, 1997; Baruselli et al., 2000), their
embryo recovery is less efficient than cows. A low number of embryos recovered in buffaloes has
been described by several authors (Karainov, 1986; Madan, 1990; Misra et al., 1990; Ambrose et
al., 1991; Baruselli, 1994; Zicarelli et al., 1994; Taneja et al., 1995; Madan et al., 1996; Drost,
1996; Zicarelli, 1997; Zicarelli et al., 2000; Baruselli et al., 2000; Carvalho et al., 2002). According
to Baruselli et al. (2000), only 34.8% of buffalo ovulated through superstimulation of follicular
growth resulting in recovered embryonic structures; it is much lower than that found in bovine
reported by Adams (1994) who recorded rates of 63% to 80%. This low embryo recovery rate

522
reported in buffaloes may be related to the failure of oocytes to enter the oviduct after
superstimulation of follicular growth (Baruselli et al., 2000).
The α and  receptors in oviduct are highly responsive to sex steroids (Pauerstein et al. 1974),
and prostaglandins are intimately involved in controlling the rhythmic tubal contractions and
relaxations necessary for ovum transport (Riehl and Harper, 1981). When the α receptors are
when  receptors are stimulated, there is an inhibition of contractility. The ampulla smooth muscle
stimulated, there is an increase in contractility of smooth muscle in the ampulla and fimbriae, and
contractions observed following ovulation may be caused by the influx of high concentrations of
PGF2α expelled together with the follicular fluid. Conversely, inhibitors of prostaglandin synthesis
inhibit ovulation (Ainsworth et al. 1979). Thus, prostaglandin F2α (PGF2) could play an important
role in ovulation and transportation of female gametes.
In rabbits, Osada et al. (1999) verified that the subsequent administration of PGF2α (after
ovulations) restimulates the tubal tissues to commence contractile activity, and thus allows the
fimbrial portions of the tubal ampullae to capture ova actively. Therefore, we hypothesized that a
sequential administration of PGF2α during the periovulatory period stimulates the contractile
activity of tissue tubal smooth muscle, allowing portions of the oviduct fimbriae to be activated to
capture the oocytes and consequently the transport of embryos by the oviduct is improved and the
number of embryos recovered from superovulated buffaloes is increased.

Buffalo Murrah donors were randomly assigned according to age, milk production, body
condition score, days postpartum, parity and ovarian activity (presence of corpus luteum: CL) into 2
groups in a cross over experimental design (35 d interval): Control (C-G; n=22) and PGF2α (PGF-
G; n=22) during the periovulatory period. Follicular wave emergence was synchronized with an
intravaginal progesterone (P4) device (Sincrogest®, OuroFino, São Paulo, Brazil) and an injection
of 2 mg i.m. of estradiol benzoate (Sincrodiol®, OuroFino) at random stage of the estrous cycle
(Day 0; D0 AM). From D4 AM on, all buffaloes received 200 mg i.m of FSH (Foltropin-V®,
Bioniche Animal Health, Canada) twice-daily, in 8 applications of decreasing doses. A dose of
PGF2α (0.53 mg; i.m.; sodic cloprostenol; Sincrocio®, Ouro Fino) was given on D6 PM and D7
AM, and the P4 device was removed on D7 PM. On D8 PM, 20µg of GnRH (buserelin acetate;
Sincroforte®, OuroFino) were given. Artificial inseminations (AIs) were carried out 12 and 24 h
after the GnRH treatment. Buffaloes from PGF-G received four extra doses of PGF2α (0.53 mg
sodic cloprostenol; Sincrocio®, Ouro Fino) from D8 PM to D10 AM 12 h apart. The ova/embryos
were collected nonsurgically 6 days after the second AI (D14). At the same day buffaloes received
the administration of PGF2α (0.53 mg i.m. sodic cloprostenol; Sincrocio®, Ouro Fino).The total
number of ova/embryos, unfertilized oocytes and Grade 1 (excellent or good), 2 (fair) and 3 (poor)
embryos were classified according to the IETS Manual (IETS, 1998). Grade 1 embryos were
considered to be suitable for freezing, and Grade 1 and 2 embryos were considered to be
transferable.
Ultrasonographic examinations
Transrectal ovarian ultrasound examinations (DP2200Vet Mindray, China) were
performed on D0 to verify ovarian activity (presence of CL), on D4 to verify the number of follicles
(number of follicles  8 mm) and on D14 to check the superovulatory response (number of CLs).
recruited on the follicular emergence ( 3mm), on D8 PM to verify the superestimulation response
The variables were analyzed by GLIMMIX procedure of Statistical Analysis System (SAS®).
The treatments resulted in a similar number of follicles 8 mm at end of superovulatory

treatment compared to the control (Day 8; PGF-G=18.6±3.0 vs. C-G=17.1±2.0; P=0.80). However,
the number of total structures recovered (ova and embryos) was greater in superovulated buffaloes
523
treated with PGF2α during the periovulatory period (PGF-G=3.5±0.6) compared to the control (C-
G=2.3±0.5; P=0.02; Figure 1). The increase in PGF2α prior to ovulation in the follicular fluid is a
requisite to induce contractions of the follicular wall, which then ruptures, expelling the ovum and
accompanying follicular fluid containing high levels of PGF2α into the tubal ampulla (LeMaire and
Marsh, 1975). It is believed that these elevated levels of PGF2α in the follicular fluid are necessary
to enhance ovum capture by the tubal fimbria. Conversely, indomethacin (inhibitor of PG synthesis)
inhibits ovulation (Ainsworth et al., 1979). The outer linear layer and inner annular layer of the
tube's smooth muscles contain both α and β receptors, the sensitivity of which is affected cyclically
by the influence of endocrine substances. These receptors are highly responsive to sex steroids
(Pauerstein et al., 1974) and prostaglandins and are intimately involved in controlling the rhythmic
tubal contractions and relaxations necessary for ovum transport (Horton et al., 1965; Hodgson,
1976; Rajkumar et al., 1979 Riehl and Harper, 1981). In rabbits, the elevated estrogens at ovulation
stimulate the α receptors, increasing tubal contractility, and are involved in retaining the ovum in
the ampulla near the ampullar-isthmic junction (AIJ). After ovulation, the elevation in progesterone
stimulates the β receptors, causing relaxation of the AIJ, allowing transport of the ovum into the
isthmic portion of the tube (Caschetto et al., 1979; Helm et al., 1982).
In this study, increased number of transferable embryos was verified in PGF treated
buffaloes (PGF-G=2.7±0.6 vs. C-G=1.8±0.5; P=0.05). Furthermore, the number of freezable
embryos tended to increase in PGF-G compared to the control (PGF-G=2.6±0.6 vs. C-G=1.8±0.5;
P=0.08). However, no differences were observed in the number of degenerated embryos among
groups (PGF-G=0.3±0.1 vs. C-G=0.4±0.1; P=0.61). PGF2α causes an increase in tubal contractile
activity, and the subsequent administration of PGF2α restimulates the tubal tissues to commence
contractile activity, and thus allows the fimbrial portions of the tubal ampullae to capture ova
actively (Osada et al., 1999).
PGF2α increases the contractility of the oviductal smooth muscles, resulting in increased
speed of embryonic transport (Lindblom et al., 1978; Weber et al., 1991; Kissler et al., 2004).
Furthermore, Osada et al. (1999) affirmed that the high concentrations of PGF2α observed in the
follicular fluid are highly important in inducing transport of the ova from the follicle to the ampulla,
then through the tube, until they finally arrive at the uterine cavity for nidation and further
development.
In conclusion, results of this study were indicative that the administration of PGF2α during
the periovulatory period was effective to increase the number of embryos recovered from
superovulated buffaloes.
REFERENCES
Adams, G.P. 1994. Control of ovarian follicular wave dynamics in cattle: implications for
synchronization & superstimulation. Theriogenology 41: 19-24.
Ainsworth, L., B.K. Tsang, B.R. Downey, R.D. Baker, T.J. Marcus and D.T. Amstrong. 1979. Effects
of indomethacin on ovulation and luteal function in gilts. Biol. Reprod. 21: 401-412.
Ambrose, J.D., S.K. Singla and S. Sailkhani. 1991. Superovulation response in buffaloes (Bubalus
bubalis) to different treatment regimes of folltropin. Theriogenology 35: 181.
Baruselli, P.S., E.H. Madureira, J.A. Visintin, R. Porto-Filho, N.A.T. Carvalho, G. Campanile and Z.
Zicarelli. 2000. Failure of oocyte entry into oviduct in superovulated buffalo. Theriogenology
53: 491.
Baruselli, P.S. 1994. Basic requirements for artificial insemination and embryo transfer in buffaloes.
Buffalo J. 2: 53-60.
Baruselli, P.S., R.G. Mucciolo, J.A. Visintin, W.G. Viana, R.P. Arruda, E.H. Madureira, C.A.
Oliveira and J.R. Morelo-Filho. 1997. Ovarian follicular dynamics during the estrous cycle in
buffalo (Bubalus bubalis). Theriogenology 47: 1531-1547.
524
Carvalho, N.A.T., P.S. Baruselli and E.H. Madureira. 2002. Control of ovulation subsequence to
superstimulation of follicular growth in buffalo: fertilization and embryo recovery.
Caschetto, S., B. Lindlom, N. Wiqvist and L. Wilhelmsson. 1979. Prostaglandins and the contractile
function of the human oviductal ampulla. Gynecology and Obstetric Invest 10: 212-220.
Drost, M. 1996. Reproductive technology in buffaloes (Bubalus bubalis). Bulgarian J. Agric. Sci. 2:
93-102.
Helm, G., C. Owman, N.O. Sjoberg and B. Walles. 1982. Motor activity of the human fallopian tube in
vitro in relation to plasma concentration of oestradiol and progesterone, and the influence of
noradrenaline. J. Rep. Fert. 64: 233-242.
Hodgson, B.J. 1976. Effects of indomethacin, ICI 46:474 administrated during ovum transport on
fertility in rabbits. Biol. Reprod. 14: 451-457.
Horton, E.W., I.H.M. Main and C.J. Thompson. 1965. Effects of prostaglandins on the oviduct,
studied in rabbits and ewes. J. Physiol. 180: 514-538.
Karainov, C. 1986. Comparative studies on the superovulatory efect of PMSG and FSH in water
buffallo (Bubalus bubalis). Theriogenology 26: 51-9.
Kissler, S., L. Wildt, K. Schmiedehausen, J. Kohl, A. Mueller, A. Rody, A. Ahr, T. Kuwert, M.
Kaumann and E. Siebzehnruebl. 2004. Predictive value of impaired uterine transport function
assessed by negative hysterosalpingoscintigraphy (HSSG). Eur. J. Obstet. Gynecol. Reprod.
Biol. 113: 204–208.
LeMaire, W.J. and J.M. Marsh. 1975. Interrelationship between prostaglandins, cyclic AMP and
steroids in ovulation. J. Reprod. Fertil. Suppl 22: 53-74.
Lindblom, B., L. Hamberger and N. Wiqvist. 1978. Differentiated contractile effects of
prostaglandins E and F on the circular and longitudinal smooth muscle of the human oviduct.
Fertil. Steril. 30: 553–559.
Madan, M.L. 1990. Factors limiting superovulation response in embryo transfer program in buffalo.
Theriogenology 33: 280.
Madan, M.L., S.K. Das and P. Palta. 1996. Application of reproductive technology to buffaloes.
Misra, A.K., B.V. Joahi, and P.L. Agrawala. 1990. Multiple ovulation and embryo transfer in indian
buffalo (Bubalus bubalis). Theriogenology 33: 1131-41.
Osada, H., T.K. Fujii, I. Tsunoda, K. Takagi, K. Satoh, K. Kanayama and T. Endo. 1999. Fimbrial
Capture of the Ovum and Tubal Transport of the Ovum in the Rabbit, with Emphasis on the
Effects of B2-Adrenoreceptor Stimulant and Prostaglandin F2α on the Intraluminal Pressures of
the Tubal Ampullae. J. Assist. Reprod. Gen. 16: 373-379.
Pauerstein, C.J., B.J. Hodgson, B.D. Fremming and J.E. Martin. 1974. Effects of sympathetic
denervation of the rabbit oviduct on normal ovum transport and on transport modified by
estrogen and prosterone. Gynecol. Invest. 5: 121-132.
Rajkumar, K., S.K. Garg, and P.I. Sharma. 1979. Relationship between concentration of
prostaglandins E and F in the regulation of ovum transport in rabbits. Prostaglandins Med. 2:
445-454.
Riehl, R.M. and M.J.K. Harper. 1981. Changes in prostaglandin binding capacity of single oviductal
smooth muscle cells after ovulation in the rabbit. Endocrinology 109: 1011-1016.
Taneja M., G. Singh and S.M. Totey. 1995. Follicular dynamics in water buffalo superovulated in
presence or absence of a dominant follicle. Theriogenology 44: 581-97.
Weber, J.A., D.A. Freeman, D.K. Vanderwall, and G.L. Woods. 1991. Prostaglandin E2 Hastens
Oviductal Transport of Equine Embryos. Biol. Reprod. 45: 544-546.
Zicarelli, L. 1997. Reproductive seasonality in buffalo. in: Proc. Third Course on Biotechnology of
Reproduction in Buffaloes, Caserta, Italy, issue ii, 29–52.
Zicarelli, L., P.S. Baruselli and G. Campanile. 2000. Embryo recovery in buffalo with timed
ovulation and insemination subsequent to follicle superstimulation. In: Proceedings of 14th
International Congress of Animal Reproduction, Estocolmo. 2: 16-9.
525
Zicarelli, L., R. Boni and C. Pacelli C. 1994. Superovulation in italian mediterranean buffaloes
using FSH-p with different treatments. In: Proceedings of 4th World Buffalo Congress, São
Paulo. 3: 462-4.
Figure 1. Total number of structures (ova and embryo recovered), transferable, and
freezable embryos of buffalo donors (n=22) treated with sequential doses of
PGF2α during the periovulatory period.
526
Use of Different Progestagens for Ovulation Synchronization and TAI in

Buffaloes during the Non Breeding Season
Nelcio Antonio Tonizza de CARVALHO a*, Júlia Gleyci SOARESb, Everton Luís REISc,
Fernando da Silva VANNUCCIa, José Nélio Souza SALESd and Pietro Sampaio
BARUSELLIb
a
Unidade de Pesquisa e Desenvolvimento de Registro-Pólo Regional do D.S.A. do Vale do
Ribeira/APTA, Registro-SP, Brazil
b
Departamento de Reprodução Animal, VRA-FMVZ-USP, São Paulo-SP, Brazil
c
MSD Animal Health, São Paulo – SP, Brazil, dCentro de Ciências Veterinárias, Campus II-UFPB,
Areia-PB, Brazil.
*Corresponding e-mail: nelcio@apta.sp.gov.br
ABSTRACT
The aim of this study was to evaluate the effect of using two progestagens (progesterone
intravaginal device or norgestomet subcutaneous implant) in the follicular response and pregnancy
rate of two categories of buffaloes (nulliparous and multiparous) which were synchronized for
timed artificial insemination (TAI) during the non breeding season. In the study, 216 buffaloes were
randomly assigned into two groups (Group progesterone; G-P4, n=97 and Group Norgestomet; G-
Nor, n=119). At random stage of the estrous cycle (D0; PM), the buffaloes of G-P4 received an
intravaginal device containing 1.0 g of progesterone (P4) and animals of the G-Nor was inserted
with a subcutaneous implant with 3.0 mg of norgestomet. At the same moment (D0, PM) all
females received 2.0 mg i.m. of estradiol benzoate (EB). On D9 (PM), 150 μg i.m. of PGF2 (d-
cloprostenol sodic) and 400 IU of eCG were administered, followed by progestagens removal. After
48 h (D11, PM) the ovulation was induced by administration of 10µg i.m. of GnRH (buserelin
acetate). In a subset of the buffaloes (G-P4, n=22 and G-Nor, n=30), transrectal ovarian ultrasound
examinations were performed on D0 to verify ovarian activity, on D9 to check the diameter of
largest follicle and from D11 to D14 (12/12h for 60h) to verify the disappearance of the ovulatory
follicle. All animals were submitted to TAI 64 h after progestagens removal (D12, AM). Pregnancy
examinations were conducted by transrectal ultrasonography at 30 days after TAI. The variables
were analyzed using the GLIMMIX procedure of SAS®. There was no interaction between
progestagens and animal categories (P>0.05). Moreover, there were no differences (P>0.05)
9.80.4 mm), ovulatory follicle  (13.50.5 vs. 14.00.3 mm), interval between progestagen
between treatment (G-P4 and G-Nor) in the diameter () of the dominant follicle at D9 (9.80.5 vs.
and pregnancy rate [46.4% (45/97) vs. 49.6% (59/119)]. However, the ovulatory follicle  was
removal and ovulation (71.02.6 vs. 70.92.6 h), ovulation rate [86.4% (19/22) vs. 93.3% (28/30)]
greater (P<0.01) for the nulliparous compared to multiparous (14.30.2 vs. 13.00.5 mm). It was
concluded that the both progestagen types resulted in satisfactory follicular responses, ovulation and
pregnancy rates in both categories of synchronized buffaloes for TAI during the non breeding
season.
Keywords: progestagen, synchronization, buffalo, ovulation, pregnancy, timed artificial

insemination
INTRODUCTION
During the non breeding season (spring and summer), buffalo often exhibit a high anestrous
incidence, which extends the calving to conception interval and, consequently, reduces reproductive
performance (Zicarelli et al., 2007). Therefore, hormonal treatments have been designed to control
both luteal and follicular functions, providing exciting possibilities for synchronization of follicular
growth and ovulation to enable the use of timed artificial insemination (TAI) during the non

527
breeding season (Singh et al., 1988; Barile et al., 2001; Neglia et al., 2003; Baruselli et al., 2005; De
Rensis et al., 2005; Carvalho et al., 2013).
The use of a progesterone/progestin (P4) source and estradiol (E2) on the first day of TAI
program has been the most commercially utilized type of estrous synchronization protocol in South
America (Bó et al., 2003, 2007; Baruselli et al., 2004; Meneghetti et al., 2009). The association
between E2 and P4 in cattle induces follicular regression and subsequent synchronous follicular
wave emergence (Bó et al., 1995, 1996; Caccia and Bó, 1998). A common aspect among these
protocols is the insertion of an intravaginal device containing progesterone or an ear implant
containing norgestomet on day 0 (Sá Filho et al., 2010). Progesterone is an important regulator of
the frequency of pulsatile secretion of LH, and hence plays an important regulatory role in
preovulatory follicle development (Herlihy et al., 2012). Lesser concentrations of P4 in the cycle
preceding ovulation may increase the risk of inferior oocyte quality before ovulation and poor
embryo quality after fertilization (Ahmad et al., 1995; Revah and Butler, 1996).
The optimization of reproductive efficiency is one of the main factors that contribute to
improved production performance and profitability of buffalo herd. Thus, the aim of the present
study was to evaluate the effects of two progestagens (progesterone intravaginal device or
norgestomet subcutaneous implant) on follicular response and pregnancy outcomes of two
categories (nulliparous and multiparous) of buffaloes (Bubalus bubalis) synchronized for TAI
during the non breeding season.

Buffaloes (Murrah; n=216) were randomly assigned according to category, age, weight,
body condition score and ovarian activity (presence of corpus luteum: CL) into two groups: Group
progesterone (G-P4, n=97) and Group Norgestomet (G-Nor, n=119). At random stage of the estrous
cycle (D0; PM), the buffaloes of G-P4 received an intravaginal device containing 1.0 g of
progesterone (P4; DIB®, MSD Animal Health, Brazil) and animals of the G-Nor was inserted with a
subcutaneous implant with 3.0 mg of norgestomet (Crestar®, MSD Animal Health, Brazil). At the
same moment (D0, PM) all females received 2.0 mg i.m. of estradiol benzoate (EB; Gonadiol®,
MSD Animal Health, Brazil). On D9 (PM), 150 μg i.m. of an analogue of PGF2 (d-cloprostenol
sodic, Preloban®, MSD Animal Health, Brazil) and 400 IU of eCG (Novormon®, MSD Animal
Health, Brazil) were administered, followed by progestagens removal. After 48 h (D11, PM) the
ovulation was induced by administration of 10 µg i.m. of buserelin acetate (GnRH, Conceptal®,
MSD Animal Health, Brazil). All animals were submitted to TAI 64 h after progestagens removal
(D12, AM).
Ultrasonography examinations
Pregnancy examinations were conducted by transrectal ultrasonography 30 days after TAI.
In a subset of the buffaloes (G-P4, n=22 and G-Nor, n=30), transrectal ovarian ultrasound
examinations (DP2200Vet Mindray, China) were performed on D0 to verify ovarian activity
(presence of CL), on D9 to check the diameter of the largest follicle and from D11 to D14 (12/12h
for 60h) to verify the disappearance of the ovulatory follicle. Ovulation was considered to have
occurred when a large follicle, previously observed, was no longer present. The variables were
analyzed using the GLIMMIX procedure of SAS®.

There was no interaction between progestagens and animal categories (P>0.05). Moreover,
dominant follicle at D9 (9.80.5 vs. 9.80.4 mm), ovulatory follicle  (13.50.5 vs. 14.00.3 mm),
there were no differences (P>0.05) between treatment (G-P4 and G-Nor) in the diameter () of the
interval between progestagen removal and ovulation (71.02.6 vs. 70.92.6 h), ovulation rate
[86.4% (19/22) vs. 93.3% (28/30)] and pregnancy rate [46.4% (45/97) vs. 49.6% (59/119); Table 1].
528
However, the ovulatory follicle  was greater (P<0.01) for the nulliparous compared to multiparous
(14.30.2 vs. 13.00.5 mm).
Previous studies carried out in buffaloes showed satisfactory follicular response and
conception rate in synchronized buffaloes for TAI with progesterone intravaginal devices or
norgestomet ear implants during the non breeding season. Buffaloes treated with DIB® and Crestar®
showed no differences on conception rate [43.7% (14/32) vs. 50.0% (28/56) respecively; P>0.05;
follicle diameter () on D9 (9.80.7 vs. 9.50.3 mm), the ovulatory follicle  (14.10.4 vs.
Carvalho et al., 2007]. Also, in another study were found similar results (P>0.05) for dominant
14.50.3 mm), the interval GnRH administration/ovulation (29.0  3.7 vs. 30.0  1.9 h), the
ovulation rate [92.3% (12/13) vs. 94.1% (16/17)] and to the conception rate [47.7% (34/65) vs.
47,7% (34/65)] between buffaloes treated with DIB® and Crestar®, respectively (Carvalho et al.,
2011).
There was no difference on TAI conception rate of cows subjected to CIDR and Crestar
treatments (P>0.05), been 52.0% (52/100) and 42.7% (44/103), respectively (Baruselli et al., 2002).
The authors concluded that progesterone and progestogen treatments allowed acceptable conception
rates at TAI and these treatments induced higher service rates, increasing pregnancy rate at AI and
anticipating the parturition on the subsequent breeding season in lactating beef cows. Moreover, Sá
Filho et al. (2010) used intravaginal devices containing progesterone or ear implants containing
norgestomet (a progestin) and verified that TAI has successfully been utilized in Bos taurus and Bos
indicus cattle, and these programs provide an organized approach to enhance the use of AI and
improve the reproductive efficiency in beef herds.
It is concluded that both progestagens used resulted in satisfactory follicular responses,
ovulation and pregnancy rates in both categories of synchronized buffaloes for TAI during the non
breeding season.
ACKNOWLEDGEMENTS
The authors thank to MSD Animal Health for support this research and the buffalo farms:
Barra do Capinzal, Santa Eliza, Santa Helena and Várzea Grande for allowing the use of their
animals and facilities for this study.
REFERENCES
Ahmad, N., F.N. Schrick, R.L. Butcher and E.K. Inskeep, 1995. Effect of persistent follicles on
early embryonic losses in beef cows. Biol. Reprod. 52: 1129–1135.
Barile, V.L., A. Galasso, E. Marchiori, C. Pacelli, N. Montemurro and A.Borghese, 2001. Effect of
PRID treatment on conception rate in Mediterranean buffalo heifers. Livestock Prod. Sci. 68:
283–7.
Baruselli, P.S., M.O. Marques, N.A.T. Carvalho, E.H. Madureira and E.P. Campos Filho. 2002.
Efeito de diferentes protocolos de inseminação artificial em tempo fixo na eficiência
reprodutiva de vacas de corte lactantes. Rev. Bras. Rep. Anim. 26:n. 3: 218-221.
Baruselli, P.S., E.L. Reis, M.O. Marques, L.F. Nasser and G.A. Bó, 2004. The use of hormonal
treatments to improve reproductive performance of anestrous beef cattle in tropical climates.
Anim. Reprod. Sci. 82–83: 479–486.
Baruselli P.S. and N.A.T. Carvalho. Biotechnology of reproduction in buffalo (Bubalus bubalis).
2005. Rev. Bras. Rep. Anim. 29: 4–17.
Bó, G.A., G.P. Adams, R.A. Pierson and R.J. Mapletoft. 1995. Exogenous control of follicular
wave emergence in cattle. Theriogenology. 43: 31–40.
Bó, G.A., M. Caccia, M.F. Martínez and R.J. Mapletoft. 1996. Follicle wave emergence after
treatment with estradiol benzoate and CIDR vaginal devices in beef cattle. In: Proc. Int.
Congr. Anim. Reprod. 22: Sydney, Australia (abstract).
529
Bó, G.A., P.S. Baruselli and M.F. Martinez. 2003. Pattern and manipulation of follicular
development in Bos indicus cattle. Anim. Reprod. Sci. 78: 307–326.
Bó, G.A., L. Cutaia, L.C. Peres, D. Pincinato, D. Maraña and P.S. Baruselli, 2007. Technologies for
fixed-time artificial insemination and their influence on reproductive performance of Bos
indicus cattle. Soc. Reprod. Fert. Suppl. 64: 223–236.
Caccia, M. and G.A. Bó, 1998. Follicle wave emergence following treatment of CIDR-implanted
beef cowswith estradiol benzoate and progesterone. Theriogenology 49: 34 (abstract).
Carvalho, N.A.T., E.M. Nagasaku, F.S. Vannucci, L.M. Toledo and P.S. Baruselli, 2007. Uso do
DIB® e do CRESTAR® para a sincronização da ovulação e IATF em búfalas leiteiras
durante a estação reprodutiva desfavorável. In: XXI Reunião Anual da Sociedade Brasileira
de Tecnologia de Embriões, 2007, Costa do Sauípe. Acta Scientiae Veterinariae Porto
Alegre-RS: UFRGS 35. pp. 1132-1132.
Carvalho, N.A.., J.G. Soares and P.S. Baruselli, 2011. Uso do DIB e do CRESTAR para a
Sincronização da Ovulação e IATF em Novilhas Búfalas Durante a Estação Reprodutiva
Desfavorável. In: XXV Reunião Anual da Sociedade Brasileira de Tecnologia de Embriões,
Cumbuco - CE. Acta Scientiae Veterinariae. Porto Alegre - RS: UFRGS 39.
Carvalho, N.A.T., J.G. Soares, R.M. Porto Filho, L.U. Gimenes, D.C. Souza, M. Nichi, J.N. Sales,
and P.S. Baruselli, 2013. Equine chorionic gonadotropin improves the efficacy of a timed
artificial insemination protocol in buffalo during the nonbreeding season. Theriogenology
79: 423–428.
De Rensis, F., G. Ronci, P. Guarneri, B.X. Nguyen, G.A. Presicce and G. Huszenicza. 2005.
Conception rate after fixed time insemination following Ovsynch protocol with and without
progesterone supplementation in cyclic and non-cyclic Mediterranean Italian buffaloes
(Bubalus bubalis). Theriogenology 63: 1824–1831.
Herlihy, M.M., M.A. Crowe, M.G., Diskin and S.T. Butler 2012. Effects of synchronization
treatments on ovarian follicular dynamics, corpus luteum growth, and circulating steroid
hormone concentrations in lactating dairy cows. J. Dairy Sci. 95: 743–754.
Meneghetti, M., O.J. Sá Filho, R. Peres, G. Lamb and J.L.M. Vasconcelos, 2009. Fixed-time
artificial insemination with estradiol and progesterone for Bos indicus cows. I. Basis for
development of protocols. Theriogenology 72: 179–189.
Neglia, G., B., Gasparrini, R.D. Palo, C.D. Rosa, L. Zicarelli and G.Campanile, 2003. Comparison
of pregnancy rates with two estrus synchronization protocols in Italian Mediterranean
buffalo cows. Theriogenology 60: 125–133.
Revah, I. and W.R. Butler. 1996. Prolonged dominance of follicles and reduced viability of bovine
oocytes. J. Reprod. Fertil. 106: 39–47.
Sá Filho, M.F., A.M. Crespilho, J.E.P. Santos, G.A. Perry and P.S. Baruselli. 2010. Ovarian follicle
diameter at timed insemination and estrus response influences the likelihood of ovulation
and pregnancy after synchronization with progesterone or progestin-based protocols in
suckled Bos indicus cows. Anim. Reprod. Sci. 120: 23–30.
Singh, G., G.S. Dhaliwal, R.D. Sharma and R.K. Biswas. 1988. Treatment of summer anestrous
buffaloes (Bubalus bubalis) with progesterone releasing intravaginal device plus pregnant
mare serum gonadotropin. Theriogenology 29: 1201–6.
Zicarelli L. 2007. Can we consider buffalo a non precocious and hypofertile species?. Ital J Anim
Sci. 6(Suppl. 2): 143–154.
530
Table 1. Follicular dynamics and pregnancy rate of synchronized buffaloes for TAI with
progesterone intravaginal devices (G-P4) or norgestomet subcutaneous implants (G-
Nor) during the non breeding season.
Treatment P value
G-P4 G-Nor Treat Cat Int
Diameter of follicle on D9 (mm) 9.8±0.5 9.8±0.4 0.88 0.58 0.99
Diameter maximum dominant follicle
13.9±0.5 14.2±0.3 0.37 0.008 0.71
(mm)
Diameter of the ovulatory follicle (mm) 13.5±0.5 14.0±0.3 0.20 0.007 0.69
Ovulation rate (%) 86.4 (19/22) 93.3 (28/30) 0.39 0.39 0.63
Interval from device removal to ovulation
71.0±2.6 70.9±2.6 0.76 0.60 0.52
(h)
Pregnancy rate (%) 46.4 (45/97) 49.6 (59/119) 0.57 0.99 0.57
531
Use of Intravaginal Progesterone Devices during Eight or Nine Days in the

Ovulation Synchronization Protocol for TAI in Buffaloes during the Non
Breeding Season
Nelcio Antonio Tonizza de CARVALHOa*, Júlia Gleyci SOARESb, Diego Cavalcante de
SOUZAc, José Ricardo Garla MAIOd, José Nélio Souza SALESe, Benedicto MARTINS
JÚNIORf, Rodrigo Caron MACARIf and Pietro Sampaio BARUSELLIb
a
Unidade de Pesquisa e Desenvolvimento de Registro - Pólo Regional do D.S.A. do Vale do
Ribeira/APTA, Registro-SP, Brazil
b
Departamento de Reprodução Animal, VRA-FMVZ-USP, São Paulo-SP, Brazil
c
EDR/CATI, Registro-SP, Brazil
d
Ourofino Agronegócio, Cravinhos-SP, Brazil
e
Centro de Ciências Veterinárias, Campus II-UFPB, Areia-PB, Brazil
f
Fazenda Santa Eliza, Dourado-SP, Brazil.
*Corresponding author: nelcio@apta.sp.gov.br
ABSTRACT
The present study aimed to evaluate the effect of using the intravaginal progesterone device
for eight or nine days on the follicular response and pregnancy rate in lactating buffaloes
synchronized for timed artificial insemination (TAI) during non breeding season. Two hundred and
twenty buffaloes were randomly assigned according to age, parity, days postpartum, body condition
score and ovarian activity into two groups: intravaginal progesterone device for 8 days (G-8d,
n=110) or for 9 days (G-9d, n=110). At random stage of the estrous cycle (D0, PM) the buffaloes
(G-9d) received an intravaginal progesterone device (P4) used a third time and 2.0 mg i.m. of
estradiol benzoate (EB). On day 1 PM, buffaloes from G-8d received the same treatment described
above for G-9d. On D9 (PM), buffaloes received 0.53 mg i.m. of PGF2 (Cloprostenol sodic) and
400 IU of eCG, followed by P4 device removal. After 48 h (D11, PM), the ovulation was induced
by the administration of 10 µg i.m. of GnRH (buserilin acetate). In a subset of the buffaloes (G-8d,
n=12 and G-9d, n=12), transrectal ovarian ultrasound examinations were performed on D0 to verify
ovarian activity, on D9 to check the diameter of largest follicle and from D11 to D14 (12/12h for
60h) to verify the disappearance of the ovulatory follicle. All animals were submitted to TAI 64 h
after P4 removal (D12, AM). Pregnancy examinations were conducted by transrectal
ultrasonography 30 days after TAI. The variables were analyzed using the GLIMMIX procedure of
SAS®. There were no difference between experimental groups (G-8d and G-9d) for all variables
analyzed (P>0.05): diameter () of the largest follicle in D9 (9.30.7 vs. 10.30.4mm);  of the
ovulatory follicle (15.40.7 vs. 14.40.4mm); interval between P4 removal and ovulation (73.53,1
vs. 76.83.8h); ovulation rate [66.7% (8/12) vs. 83.3% (10/12)]; pregnancy rate [42.7% (47/110) vs.
50.9% (56/110)]. It was concluded that third used intravaginal P4 devices for eight or nine days
resulted in satisfactory follicular response and pregnancy rate of the synchronized lactating
buffaloes for TAI during the non breeding season. However, the 8.2% decreasing pregnancy rate in
the G-8d should be considered in future investigations.
Keywords: progesterone, synchronization, buffalo, ovulation, pregnancy, non breeding season
INTRODUCTION
The association of a progesterone source and estradiol on the first day of TAI program has
been commonly used for estrous synchronization protocol (Bó et al., 2003, 2007; Baruselli et al.,
2004; Meneghetti et al., 2009) and induces follicular regression and subsequent synchronous
follicular wave emergence (Bó et al., 1995, 1996; Caccia and Bó, 1998). Progesterone (P4) is an
important regulator of the frequency of pulsatile secretion of LH, and hence plays an important
regulatory role in preovulatory follicle development (Herlihy et al., 2012). This steroid is widely
532
used in intravaginal devices and most have in their constitution 1 to 1.9 g of P4-impregnated
silicon. P4 concentrations present in devices are sufficient to promote synchronization of ovulation
in Bos indicus (Baruselli et al., 2004). However, there is a paucity of published information
regarding the reuse of progesterone devices in buffaloes.
Reuse of intravaginal P4 devices have been reported in cows (Colazo et al., 2004;
Meneghetti et al., 2009), ewes (Ungerfeld, 2009) and goats (Oliveira et al., 2001; Carvalho et al.,
2006; Vilariño et al., 2011; Vilariño et al., 2013), without decreasing fertility rate. Goats receiving
new or reused CIDR (intravaginal progesterone device) showed similar estrous response and
pregnancy rates with second (Oliveira et al., 2001) or third uses (Nogueira et al., 2008). Therefore,
the use of a CIDR-G for 5 to 7 d could leave residual releasable P4 and the reuse of devices could
be proposed (Vilariño et al., 2011). Van Cleeff et al. (1992) reported in cattle that CIDR-B (1.9 g of
progesterone) still contained P4 after its use, with the amount dependent on the duration of
insertion. These findings have important economic implications, making insemination programs
more affordable (Vilariño et al., 2011).
Previous studies [Carvalho et al. (unpublished data)] found decreased in progesterone
circulating concentrations in buffaloes treated with twice used intravaginal P4 devices. It was also
found in animals treated with the third used device. In these animals, occurred anticipation at the
time of ovulation (Carvalho et al., 2009), and, because of this, the TAI was performed on average 4
h before ovulation, which may be resulted on a numerically lower pregnancy rates (Soares et al.,
2012). Also, Colazo et al. (2004) observed lower pregnancy rate in cattle receiving a twice-used
CIDR insert and suggested that blood P4 concentrations may not have been maintained in all cattle
throughout the 7-days protocol. Therefore, we hypothesized that the rapid decrease in P4
concentrations observed in buffaloes treated with third used intravaginal P4 devices anticipates the
ovulation and consequently decreases the pregnancy rate in buffaloes. Thus, the aim of this study
was anticipating on one day the P4 device removal used a third time, to provide adequate
progesterone concentration throughout the protocol and thus enable satisfactory pregnancy rate in
lactating buffaloes synchronized for TAI during the non breeding season.

Buffaloes (Murrah; n=220) were randomly assigned according to age, parity, days
postpartum, body condition score and ovarian activity (presence of corpus luteum: CL) into two
groups: intravaginal P4 device for 8 days (G-8d, n=110) or for 9 days (G-9d, n=110). At random
stage of the estrous cycle (D0, PM) the buffaloes (G-9d) received an intravaginal P4 device
(Sincrogest®, Ourofino Agronegócio, Brazil) used a third time and 2.0 mg i.m. of estradiol benzoate
(EB; Sincrodiol®, Ourofino Agronegócio, Brazil). On day 1 (PM), buffaloes from G-8d received the
same treatment described above for G-9d. On D9 (PM), buffaloes received 0.53 mg i.m. of PGF2
(Cloprostenol sodic, Sincrocio®, Ourofino Agronegócio, Brazil) and 400 IU of eCG (Novormon ®,
MSD Animal Health, Brazil), followed by P4 device removal. After 48 h (D11, PM), the ovulation
was induced by the administration of 10 µg i.m. of GnRH (buserilin acetate, Sincroforte®, Ourofino
Agronegócio, Brazil). All animals were submitted to TAI 64 h after P4 removal (D12, AM).
Ultrasonography examinations
In a subset of the buffaloes (G-8d, n=12 and G-9d, n=12), transrectal ovarian ultrasound
examinations (DP2200Vet Mindray, China) were performed on D0 to verify ovarian activity
(presence of CL), on D9 to check the diameter of largest follicle and from D11 to D14 (12/12h for
60h) to verify the disappearance of the ovulatory follicle. Ovulation was considered to have
occurred when a large follicle, previously observed, was no longer present. Pregnancy examinations
were conducted by transrectal ultrasonography 30 days after TAI. The variables were analyzed
using the GLIMMIX procedure of SAS®.
533

There were no difference (P>0.05) between buffaloes treated with intravaginal P4 device for
mm);  of the ovulatory follicle (15.40.7 vs. 14.40.4 mm) and the interval between P4 removal
8 or 9 days (G-8d and G-9d) for diameter () of the largest follicle in D9 (9.30.7 vs. 10.30.4
and ovulation (73.53.1 vs. 76.83.8 h). It is known that P4 is a steroid of great importance in
reproduction, being responsible for estrous cycles duration and maintenance of pregnancy
(Loonergan et al., 2011). Furthermore, P4 inhibits the LH peak, ovulation and estrous behavior
(Adams et al., 1992). According to some studies, as CIDR inserts release P4 for at least 15 days
(Macmillan et al., 1991; Macmillan and Peterson, 1993), a CIDR could potentially be used at least
twice in current estrus synchronization protocols.
In the present study, no differences were found between ovulation rate [66.7% (8/12) vs.
83.3% (10/12)] and pregnancy rate [42.7% (47/110) vs. 50.9% (56/110)]. These results
corroborated with data obtained in buffalo during the non breeding season which new intravaginal
P4 devices, used once or twice, provided satisfactory results of ovulation and pregnancy rates for
ovulation synchronization and TAI program (Soares et al., 2012). Similar pregnancy rates have
been reported after the addition of new or used P4 devices to synchronization protocols in beef
cattle (Colazo et al. 2004; Pincinato et al. 2007). Accordingly, Meneghetti et al. (2009) verified in
Bos indicus that CIDR may be used as many as 4 times with no negative effects on pregnancy rates.
Also, the reuse of P4 devices containing 1.0 g of progesterone was reported in heifers for TAI
programs using 7–8 d of treatment, which resulted in an acceptable pregnancy rate, similar to that
obtained with new devices (Bó et al., 2003).
However, in the current study, although not statistically different, there was an 8.2%
decreasing pregnancy rate in the G-8d. Colazo et al. (2004) found that pregnancy rate to TAI did
not differ between cattle synchronized with a new or once-used CIDR (P = 0.28, 57.5, 63.8, 47.9,
47.9% for one new, one once-used, one twice-used, or two twice-used CIDRs, respectively), but the
authors considered that pregnancy rate was lower in cattle synchronized with a twice-used CIDR.
Moreover, Martinez et al. (2004) verified that the use of a device with low content of P4 (used DIB)
could have resulted in lower fertility to TAI than other intravaginal devices delivering a higher
content of P4, when considered that heifers coming in estrus early as non-pregnant as previously
described. Also, was considered that if a once-used DIB is to be included in synchronization
protocol for a TAI programme only, the timing of second GnRH and AI should be brought forward
(e.g. 12 h; Martinez et al., 2012)
It was concluded that the third used intravaginal P4 devices for eight or nine days resulted in
satisfactory follicular response and pregnancy rate of the synchronized lactating buffaloes for TAI
during the non breeding season, rejecting the initial hypothesis. However, the 8.2% decreasing
pregnancy rate in the G-8d should be considered in future investigations.
ACKNOWLEDGEMENTS
The authors thanks to Ourofino Agronegócio for support this research and the buffalo farms:
Santa Eliza and Santa Helena for allowing the use of their animals and facilities for this study.
REFERENCES
Adams, G. P., R. L. Matteri and O.J. Ginther. 1992. Effect of progesterone on ovarian follicles,
emergence of follicular waves and circulating follicle-stimulating hormone in heifers.
Journal of Reproduction and Fertility. 96: 627-640.
Baruselli, P. S., E. L. Reis, , M.O. Marques, L.F. Nasser and G.A. Bó, G.A. 2004. The use of
hormonal treatments to improve reproductive performance of anestrous beef cattle in
tropical climates. Anim. Reprod. Sci. 82–83:479–486.
Bó, G. A., G. P. Adams, R. A. Pierson and R. J Mapletoft. 1995. Exogenous control of follicular
wave emergence in cattle. Theriogenology 43:31–40.
534
Bó, G. A., M. Caccia, M.F. Martínez, and R.J. Mapletoft. 1996. Follicle wave emergence after
treatment with estradiol benzoate and CIDR vaginal devices in beef cattle. In: Proc. Int.
Congr. Anim. Reprod. 22:Sydney, Australia (abstract).
Bó, G. A., P.S. Baruselli and M.F. Martinez. 2003. Pattern and manipulation of follicular
development in Bos indicus cattle. Anim. Reprod. Sci. 78:307–326.
Bó, G. A., L. Cutaia, L.C. Peres, D. Pincinato, D. Maraña and P.S. Baruselli. 2007. Technologies
for fixed-time artificial insemination and their influence on reproductive performance of Bos
indicus cattle. Soc. Reprod. Fert. Suppl. 64:223–236.
Caccia, M. and G.A. Bó. 1998. Follicle wave emergence following treatment of CIDR-B implanted
beef cows with estradiol benzoate and progesterone. Theriogenology 49:341(Abstract).
Carvalho, G. R., M.P. Palhão, J.F. Fonseca, F.N. Zambrini, H. Rovay, C.A.S. Bispo, R.R. Araújo,
and S.S. Santos. 2006. Induction of estrus in non-lactating goats with reused intravaginal
dispositives. In: 43th Reunião Anual da Sociedade Brasileira de Zootecnia, pp. 1–4.
Carvalho, N. A. T., M. Raposo, J. R. G. Maio, M. Nichi and P.S. Baruselli. 2009. Uso de
dispositivo intravaginal de progesterona (Sincrogest) para sincornização da ovulação em
búfalas na estação reprodutiva desfavorável. Anais do XVIII Congresso Brasileiro de
Reprodução Animal 391-391.
Colazo, M. G., J. P. Kastelic, P. R. Whittaker, Q. A Gavaga, R Wilde and R.J. Mapletoft. 2004.
Fertility in beef cattle given a new or previously used CIDR insert and estradiol, with or
without progesterone. Anim. Reprod. Sci. 81:25–34.
Herlihy M. M., M. A Crowe, M. G Diskin and S. T. Butler, 2012. Effects of synchronization
treatments on ovarian follicular dynamics, corpus luteum growth, and circulating steroid
hormone concentrations in lactating dairy cows. J. Dairy Sci. 95 :743–754.
Lonergan P. 2011. Influence of progesterone on oocyte quality and embryo development in cows.
Theriogenology 76:1594–601.
Macmillan, K. L. and A.J. Peterson. 1993. A new intravaginal progesterone releasing device for
cattle (CIDR-B) for oestrous synchronization, increasing pregnancy rates and the treatment
of post-partum anoestrus. Anim. Reprod. Sci. 33:1–25.
Macmillan, K. L., V. K Taufa, D. R Barnes and A.M. Day. 1991. Plasma progesterone
concentrations in heifers and cows treated with a new intravaginal device. Anim. Reprod.
Sci. 21:25–40.
Martinez, M. F., J. P Kastelic and R.J. Mapletoft. 2004. The use of estradiol and ⁄ or GnRH in a
two-dose PGF protocol for breeding management of beef heifers. Theriogenology 62:363–
372.
Martinez, M. F., G Nava, K. J Demmers, D. Tutt, M. Rodriguez Sabarrós, B. Smaill, M. Corti and J.
Juengel. 2012. Intravaginal Progesterone Devices in Synchronization Protocols for Artificial
Insemination in Beef Heifers. Reprod. Dom. Anim. 47:230–237.
Meneghetti, M., O. J. Sá Filho, R. Peres, G. Lamb and J. L. M. Vasconcelos. 2009. Fixed-time
artificial insemination with estradiol and progesterone for Bos indicus cows. I. Basis for
development of protocols. Theriogenology 72:179–189.
Nogueira, D. M., Lopes, E. S., Jr., Christilis, M., Monte, A. P. O., Martins, S. R. 2008. Fertility of
dairy goats raised in semi-arid zone of North-eastern Brazil after artificial insemination and
use of controlled internal drug release (CIDR) for up to three times for estrus
synchronization. In: 45th Reunião Anual da Sociedade Brasileira de Zootecnia, pp. 1–4.
Oliveira, M. A. L., S. I. Guido and P.F. Lima. 2001. Comparison of different protocols used to
induce and synchronize estrus cycle of Saanen goats. Small Rumin. Res. 40:149–153.
Pincinato, D., L. Cutaia, L.C. Peres, and G. A. Bó. 2007: Effect of progesterone content in a vaginal
insert on pregnancy rates in Bos indicus cross-bred beef heifers inseminated at a fixed-time.
Soc. Reprod. Fertil. Suppl 64:518.
535
Soares, J. G., N. A. T. Carvalho, D. C. Souza, J. R. G Maio, J. N. S Sales and P. S. Baruselli. 2012.

Use of intravaginal progesterone devices of first, second and third uses for the
synchronization of ovulation and TAI of lactating buffaloes during the non breeding season.
Anim. Reprod. 9:508-508.
Ungerfeld, R. 2009. The induction of oestrus in ewes during the nonbreeding season using pre-used
CIDRs and oestradiol-17β treatment. Small Rumin. Res. 84:129–131.
Van Cleeff, J., M. C. Lucy, C. J. Wilcox and W.W. Thatcher. 1992. Plasma and milk progesterone
and plasma LH in ovariectomized lactating cows with new or used controlled internal drug
release devices. Anim. Reprod. Sci. 27:91–106.
Vilariño M., E. Rubianes and A. Menchaca. 2011. Re-use of intravaginal progesterone devices
associated with the Short-term Protocol for time artificial insemination in goats.
Theriogenology 75:1195–200.
Vilariño, M., E. Rubianes and A. Menchaca. 2013. Ovarian responses and pregnancy rate with
previously used intravaginal progesterone releasing devices for fixed-time artificial
insemination in sheep. Theriogenology 79:206–210.
536
Plasma Progesterone Radioimmunoassay as a Tool to Confirm Ovarian

Cyclicity of Recipients Following Nonsurgical Transfer of Cloned Swamp
Buffalo (Bubalus bubalis Lin.) Embryo
Wanvipa SUTHIKRAI , a* Ratree JINTANA, a Sunpetch SOPHON,a Kriengsak,
TASRIPOO a Sungworn USAWANG , a Viboon MANOMAIWONG, b Kitiya
SRISAKWATTANA, a and Maneewan KAMONPATANA a
a
The Project : The Use of Nuclear Technology to Improve Reproductive Efficiency in Dairy Cattle
and Swamp Buffalo, Research and Development Centre for Livestock Production Technology,
Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand
b
Wuathong Farm, Jareontham, Wihandaeng District, Saraburi Province, 24190, Thailand
*Corresponding e-mail: swanvipa@chula.ac.th,
ABSTRACT
The purpose of this study was the used of plasma progesterone (P4) profiles for assessing
cyclic ovarian activity in ten swamp buffalo (Bubalus bubalis Lin.) recipients . Blood sampling
were collected 10 days interval and at the day of transferring embryo from jugular vein into
heparinized tubes. Plasma was separated and stored at -20 oC until P4 was determined by
radioimmunoassay (RIA). The ovary containing a palpable functional corpus luteum (CL) of
recipients was transferred non-surgically with 1-3 cloned swamp buffalo embryos at various stage.
Plasma P4 levels showed on the day of cloned embryo were transferred, the recipients were in
follicular phase, early-luteal phase, mid-luteal phase, late-luteal phase, anoestrous and ovulation
while mean + SD plasma P4 were 0.37(n=1), 0.75 + 0.06 (n=3), 1.88 + 0.54(n=11), 0.78 +
0.19(n=4), 0.04 (n=1) and 0.21 + 0.04 (n=3 ) ng/ml, respectively. And the accuracy of rectal
palpation was 78.0%. In the present study pregnant recipients with plasma P4 concentration >0.52
ng/ml and maintained beyond 80 days were found when transferred of morula, blastocyst and
blastocyst + hatched blastocyst to animals which were in late-luteal phase, mid-luteal phase, early-
and mid-luteal phase, respectively. One recipient, which received two blastocyst at mid-luteal
phase, gave birth at Day 326 to one calf on 25 October,2011. In conclusions, plasma P4 RIA could
use as a tool to confirm ovarian cyclicity of recipients as follows; 1) evaluation stage of oestrous
cycle; and 2) following up period of gestation. Therefore, these will improve successful
pregnancies after embryo transfer.
Keywords: Progesterone radioimmunoassay, recipients, oestrous cycle, cloned swamp buffalo

embryo
INTRODUCTION
It was noteworthy that cloning buffalo was more difficult than cloning other livestock, the
pregnancy rate was low and abortion commonly occurred after cloned swamp buffalo embryos
were transferred. One factor of the success of cloned swamp buffalo embryo transfer is
reproductive status of recipients. Suitability of recipients is dependent on the timing of oestrous and
the presence of a functional corpus luteum. Swamp buffalo are regarded as difficult animal for
oestrous detection therefore, the day for cloned embryo transfer cannot be based on oestrous
observation for assessment by rectal palpation. Progesterone radioimmunoassay first appeared in
buffalo research since 1976 (Kamonpatana et al., 1976) and quickly become a tool to determine
reproductive status in buffaloes. The changes in concentrations of progesterone in blood during the
oestrous cycle are similar to those in cattle, but the peak concentration is relatively less.(Dobson
and Kamonpatana, 1986; Singh et al., 2001) Progesterone concentration have been utilized for
assessing cyclic ovarian activity and for early diagnosis of pregnancy and non-pregnancy
(Kamonpatana et al.,1989; Perera, 2011).Ovarian activity and pregnancies derived from cloned
swamp buffalo embryo transfer relative to peripheral plasma progesterone of maternal
537
progesterone (P4) , have not been reported. In present study is aimed at the used of progesterone
radioimmunoassay (RIA) technique to confirm the ovarian activity of the recipients.

Experimental animals
The study was carried at the Wuathong farm, Saraburi Province in central part of Thailand,
belong to Mr. Viboon Manomaiwong during January 2011 to August 2012. Ten Swamp buffalo
were first or second calving , non-lactating healthy animals and had been rectally palpated and
found to have no abnormality of the reproductive tract were selected for this study.
Blood sampling and plasma P4 assay
Blood sampling were collected from all recipients 10 days interval and at the day of
transferring embryos from jugular vein into heparinized tubes. Plasma was separated by
centrifugation (15mins,1,500 g) and stored at -20 oC until assayed. For the determination of plasma
P4 levels RIA as described by Kamonpatana et al. (1979) were used.
Cloned swamp buffalo embryo transfer
Each recipient received 1-3 fresh cloned swamp buffalo embryos (morula, early blastocyst,
blastocyst and hatched -blastocyst). They produced by somatic cell nuclear transfer using adult ear
fibroblast cells as nucleus donors were transferred nonsurgically into the uterine horn ipsilateral to
the ovary containing a palpable functional corpus luteum.

Validation of RIA
The reliability of the method was tested in three pools of low, medium and high standard
progesterone added in the blank plasma pools. The coefficient of variation (CV) of the three pools
of internal control of the assay were 10.26% (0.1 ng/ml) 9.95% (0.5 ng/ml) and 8.60% ( 1.0 ng/ml).
The CV of inter assay in pools low, medium and high standard added were 13.1%,14.8% and 10.3%
respectively. The sensitivity of the assay was 0.03 ng/ml.
Plasma P4 profiles of ovarian cyclicity
It was found that , ten day interval concentration of plasma P4 ( January 2011 to August
2012) of ten recipients showed luteal phase which plasma ranged between 0.52-5.83 ng/ml and
follicular phase which plasma P4 ranged between 0.31-0.50 ng/ml and ovulation or anoestrous
cycle which plasma P4 were <0.3 ng/ml. At the day of cloned swamp buffalo embryos were
transferred the presence of corpus luteum in the ovaries of the recipient animals was checked by
palpation per rectum and were confirmed by levels of plasma P4. It was found that the recipients
were in anoestrous (4.35 %) , ovulation (13.04%) follicular phase (4.35%) early luteal phase
(13.04 %) mid-luteal phase (47.83 %) and late luteal phase (17.39%). The mean + SD of plasma P4
concentration were 0.04 ng/ml, 0.21 + 0.04 (0.17-0.22) ng/ml, 0.37 ng/ml, 0.75 + 0.06 (0.65- 0.84)
ng/ml, 1.88 + 0.54 (1.09-2.99)ng/ml and 0.78 + 0.19 (0.56-1.02) ng/ml. in anoestrous, ovulation,
follicular phase, early luteal phase, mid-luteal phase and late luteal phase animals respectively. This
indicate range of plasma P4 concentration with and without a corpus luteum on their ovaries were
0.65-2.99 ng/ml (n= 18) and 0.04-0.37 ng/ml (n= 5) respectively. In the present study, showed the
accuracy of diagnosing corpus luteum in the ovaries was 78.0% for rectal palpation. Other study
have reported the accuracy of diagnosing ovarian contents was 82 and 91% for rectal palpation and
plasma P4 respectively (Jainudeen et al., 1983). Pregnancy diagnosis was determined by increasing
plasma P4 (>0.5 ng/ml) at 30 days after cloned swamp buffalo embryos were transferred. It was
found that , percentage of pregnant and non-pregnant animals were 56.52 % (n=13)and 43.48 %
(n=10) respectively. Pregnancy rate was higher than early study reported by Saikhun et al. (2004)
which pregnancy rate were 40 % at 30 days after transfer of cloned embryos to swamp buffaloes.
This requires the improvement and utilization of practical and effective biotechnology. Embryonic
development in buffaloes occurs at a faster rate than in cattle and this has implications for the
earlier establishment and functionality of the corpus luteum in buffalos (Campanile et al., 2010). In
this study, the correlation between stage of embryo development and stage of oestrous cycle of
538
recipients at the day of transfer were study (Table 1).The present study has shown that pregnancy
were maintained beyond 80 days, following transfer of morula stage embryo to animals which
were in late -luteal phase , blastocyst stage were transferred to animals which were in mid- luteal
phase and blastocyst + hatched blastocyst stage were transferred to animals which were in early
and mid -luteal phase. The success of this study, was supported by one (7.7%) surviving calf was
born on Day 326 of gestation (25 October 2011).
In conclusion , plasma progesterone radioimmunoassay as a tool to confirm ovarian
cyclicity of recipients as follows;
1. To evaluate stage of oestrous cycle at the day of embryo transfer.
2. To Follow up period of gestation.
REFERENCES
Campanile, G., P.S. Baruselli, G. Neglia, D. Vecchio, B. Gasparrini, L.U. Gimenes, L. Zicarelli and
M.J.D’ Occhio. 2010. Ovarian function in the buffalo and implications for embryo
development and assisted reproduction. Anim. Reprod. Sci. 121: 1-11.
Dobson, H. and M. Kamonpatana. 1986. A review of female cattle reproduction with special
reference to comparision between buffaloes, cows and Zebu. J.Reprod.Fert. 77:1-36.
Jainudeen, M.R., W. Sherifuddin and F.B.Ahmad. 1983. Relationship of ovarian contents to
plasma progesterone concentration in the swamp buffalo (Bubalus bubalis ). Vet. Rec. 113:
369-372.
Kamonpatana, M., Y. Luvira, P. Bodhipaksha and A. Kunawongkrit. 1976. Serum progesterone,
17- Hydroxyprogesterone and 17-B Oestradiol during oestrous cycle in swamp buffalo in
Thailand. On nuclear techniques in animal production and health as related to the soil-plant
system. IAEA, Vienna: 569-578.
Kamonpatana, M., C. Pansin, S. Sophon, S. Saravasri, K. Srisakwattana and W.Suthikrai.1989.
Biotechnology based on progesterone RIA to improve reproductive efficiency in swamp
buffaloes at small farm. Buffalo J.1: 1-12.
Perera, B.M.A.O.2011. Reproductive cycles of buffalo. Anim. Reprod. Sci.124:194-199.
Saikhun, J., N. Kitiyanant, C. Songtaveesin, K. Pavasuthipaisit and Y. Kitiyanant. 2004
Development of swamp buffalo (Bubalus bubalis )embryo after pathenogenetic activation
and nuclear transfer using serum fed or starved fetal fibroblasts. Reprod. Nutr. Dev. 44: 65-
78.
Singh, B., V.D. Dixit, P. Singh, G.C. Georgie and V.P. Dixit. 2001. Plasma inhibin levels in
relation to steroids and gonadotrophins during oestrous cycle in buffalo.
Reprod.Domest.Anim. 36:163-167.
539
Table 1. Twenty three times of cloned swamp buffalo embryo transfers: Oestrous cycle
relative to recipients plasma progesterone levels on the day of cloned swamp
buffalo embryo was transferred, stage of cloned embryo and period of gestation
(days).
Stage of cloned embryos (number) Oestrous cycle Levels of P4 Days of

(ng/ml) pregnant
Morulae (3) Follicular phase 0.37 0
Morulae (2) Ovulation 0.22 60
Mid-luteal phase 1.56 0
Late-luteal phase 0.73 70
Morulae (3) Late-luteal phase 0.79 80
Early blastocyst (2) Anoestrous 0.04 0
Blastocyst (2) Ovulation 0.25 0
Blastocyst (2 Early-luteal phase 0.84 50
Blastocyst (3) Early-luteal phase 0.65 0
Blastocyst (2) Mid-luteal phase 2.99 0
Mid-luteal phase 2.58 326*
Blastocyst (1) + hatched blastocyst (1) Mid-luteal phase 1.09 80
Early-luteal phase 0.75 180
Hatched blastocyst (1) Ovulation 0.17 0
* Delivered the world ‘s first cloned swamp buffalo in Thailand, it is at 1 year and 6 months of age
at present.
540
Seasonal Effect on Oocytes Recovery Rate and Maturation Rate of Swamp

Buffalo Ovaries Collected from Slaughterhouse in Thailand
Kriengsak TASRIPOO, 1Kitiya SRISAKWATTANA1 and Sunpetch SOPHON2

1
Research and Development Centre for Livestock Production Technology, Faculty of Veterinary
Science, Chulalongkorn University, Henri Dunant street, Phatumwan, Bangkok 10330, Thailand.
2
Faculty of Veterinary Medicine, Mahanakorn University of Technology, Nong Chok, Bangkok
10530, Thailand
Corresponding e-mail: Kriengsak.T@chula.ac.th
ABSTRACT
This retrospective work was carried out to study the seasonal effects on the number of
oocyte per ovary that collected in each season for 6-years; and in vitro maturation rate of swamp
buffalo oocytes retrieved from the slaughterhouse in unknown reproductive status. In summer
(February-May), a total of 2,465 aspirated oocytes had been collected from 2,462 ovaries. In rainy
season (June-September), a total of 4,371 aspirated oocytes had been collected from 4,301 ovaries.
In winter (October-January), a total of 2,201 aspirated oocytes had been collected from 2,071
ovaries.The number of oocyte per ovary in summer, rainy and winter season, were ranging between
0.88-1.15, 0.87-1.16 and 0.92-1.33, respectively. The averages of each ratio were 1.00, 1.02 and
1.06, respectively. After in vitro maturation, the numbers of mature oocytes were 1,043, 1,976 and
879 in summer, rainy and winter season, respectively.The range of maturation rate in summer, rainy
and winter season, of six-year data, were 29.3%-57.9%, 29.1%-65.4% and 29.4%-57.6%,
respectively. The average maturation rate was 44.4%, 50.3% and 43.9% in summer, rainy and
winter season, respectively. In conclusion, our data showed that the season did not affect on the
number of oocyte per ovary and in vitro maturation rate of swamp buffalo oocytes in Thailand.
Keywords: Swamp buffalo oocyte, in vitro maturation, season, recovery rate
INTRODUCTION
Buffalo (Bubalus bubalis) has the potential superiority over cattle under the harsh climate of
tropics (Purohit et al 2003).
Reproductive efficiency is the primary factor affecting productivity and is hampered in
female buffalo by (i) inherent late maturity, (ii) poor estrous expression in summer, (iii) distinct
seasonal reproductive patterns and (iv) prolonged intercalving intervals (Raza et al., 2001), and
weak/silent oestrous signs, seasonal anoestrus, a long post-partum anoestrus period, delayed age of
puberty and low conception rates limit the productivity of buffalo (Nandi et al., (2002). The lowest
levels of sexual activity were observed during the summer months (Dobson and Kamonpatana
1986). Buffalo are usually slaughtered when they become unreproductive due to subfertility or to
poor feed conditions and both of which affected the number and quality of ovarian follicles,
thereby, contributing to lower fertility (Das et al., 1996). It was reported that high environmental
temperature had a detrimental effect on the oocyte yield, quality and developmental competence of
buffalo oocytes collected from slaughterhouse ovaries (Manjunatha et al., 2009; Amer et al., 2008).
The abattoir-derived ovaries provided a cheap, relatively easily and abundant source of oocytes
(Nandi et al., 2002; Jamil et al., 2007). The recovery of total and usable quality oocytes from
slaughter-house ovaries is low in this species (Palta and Chauhan, 1998; Purohit et al., 2003).
Oocyte quality is a key limiting factor in female fertility and a poor understanding of what
constitutes oocyte quality and the mechanism governing (Gilchrist et al., 2008).
The reports of seasonal effects on buffalo oocytes recovery rate is not consistent. There are
very few reports available on the effect of season on ratio of oocyte recovery per ovary and in vitro
maturation (IVM) in buffalo derived from slaughtered house.

541
In this study, a retrospective analysis was carried out on data produced from abattoir-derived
ovaries in in vitro embryo production lab in a 6-year period. The objective of this study was to
evaluate effects of season on oocyte recovery and in vitro maturation of buffalo oocyte derived
from slaughterhouse.

Reagents
All reagents were purchased from Sigma-Aldrich (St Louis, USA) unless otherwise stated.
Experimental design
A retrospective analysis was carried out on data recorded over entire 6 years in the in vitro
embryo production laboratory. In order to study the effects of the season we considered the
following 3 periods of the year; February-May (summer), June-September (rainy season) and
October-January (winter), according to the information from Thai Meteorological Department. Per
each collection day, we recorded the number of ovaries aspirated and total number of retrieved
oocytes.
Due to the limited source of buffalo oocytes that from slaughterhouse which are mostly poor
quality and lower number of usable oocyte, it is needed to have as many as possible for each session
of the experiment by using all the cumulus-oocyte complexes with compact cumulus cells and also
collected from ovaries with and without corpus luteum (CL) were retrieved in this study.
Collection of ovaries and oocyte recovery and in vitro maturation of oocytes
The methodology is described previously (Tasripoo et al., 2007).Briefly, buffalo cumulus
oocyte complex (COCs) were recovered by aspiration of antral follicle (3-6 mm in diameter) on
ovaries obtained from slaughterhouse. After washing , the group of 10 COCs were cultured in
TCM199 +10% buffalo follicular fluid+0.02% AU/ml FSH + 50 IU /ml hCG and I ug/ml E2 for 1-
20 h at 38.5 oC in a humidified atmosphere of 5 % CO2.
Results (MII), are expressed as mean and standard errors.

The summarized of number of ovaries and oocytes collected in each seasonal period for six
years and the average of the percentages of maturation accordingly were showed in Table 1.
The averages of each ratio were 1.00, 1.02 and 1.06, in summer, rainy and winter season,
respectively.
After in vitro maturation, the average maturation rate was 44.4%, 50.3% and 43.9% in
summer, rainy and winter season, respectively.
The number of oocytes per ovary of each season was not different among three seasons on
data recorded over entire 6 years in the in vitro embryo production laboratory
Our results corresponded to the study by Hamam et al (2001) that the average number of
buffalo oocytes per ovary, without significant variation between the four seasons. It is the quality of
buffalo oocytes, but not the recovery rate, was affected by seasonal variations. These results also
corresponded to the report by Di Francesco et al (2011) that the oocyte number recovered per ovary
and the number and incidence of good quality oocytes were not affected by season. In swamp
buffalo, the hot season affected significantly the number of oocytes collected per animal (Uoc et al.,
2007).
It was reported that variations in the oocyte yield among different studies are due to
differences in geographical location in relation to the status of animals slaughtered, season of ovary
collection, number of ovaries processed and techniques employed by different workers and criteria
for selecting ovaries at slaughterhouse might have influenced the oocyte yield in different studies
(Sharma and Loganathasamy, 2007).
Our results showed that the average of maturation rates were not different among the three
seasons of six year data. In Egypt, during winter (December, January and February), the percentage
of good oocytes increased, the maturation rate increased (Zoheir et al., 2007).
542
When buffaloes become unreproductive, they will be ready for slaughtering at any time of
the year. This might be one of the possibly reasons that the average maturation rates were not
different among three seasons of 6-year data. Aged and detrimental body condition of slaughtering
of buffaloes affected the lower yield of oocytes (Nandi et al., 2002). Furthermore, ovaries of
slaughtered animals coming from follicle in different stages of growth and atresia resulted in
heterogeneous quality of oocytes (Bhofwani et al., 2007).
These recovery effects might be clearly seen for the animal from well –management farming
and the animals were carried out for this specific research purpose. And the environmental factors
are under controlled in some extents. The higher chance for obtaining the good quality oocytes may
be from such farms, than those from small holder farms. And, by ovum pick up (OPU), the higher
quality as well as the quantity of retrieval oocytes can be easily obtained. Therefore, the success of
in vitro oocyte maturation could be obtained. OPU and oocytes recovered from abattoir-derived
ovaries are different in recovery rate due to the genetic, nutrition, environmental, and stressful
conditions (Drost, 2007).
In conclusion, our data showed that the season did not affect on the number of oocyte per
ovary and in vitro maturation rate of swamp buffalo oocytes in Thailand
REFERENCES
Amer,H.A., A.O. Hegab and S.M. Zaabat. 2008. Effects of ovarian morphology on oocyte quantity
and quality, granulose cells, in vitro maturation, and steroid hormone production in
buffaloes. Anim. Reprod. 5 (1-2): 55-62.
Bhofwani, S., H. Aim, H. Torner, W. Kanitz and R. Poehland. 2007. Selection of developmentally
competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate
after bovine nuclear transfer. Theriogenology : 341-345
Das, G.K., G.C. Jain, V.S., Solanki and V.N. Tripathi. 1996. Efficacy of various collection
methods for oocyte retrieval in buffalo. Theriogenology 46: 1403-1411.
Di Francesco, S., L. Boccia, G. Campanile, R. Di Palo, D. Vecchio, G. Neglia, L. Zicarelli and B.
Gasparrini. 2011. The effect of season on oocyte quality and developmental competence in
Italian Mediterranean buffaloes (Bubalus bubalis). Anim. Reprod. Sci. 123:4 8-53.
Dobson, H. and M. Kamonpatana. 1986. A review of female cattle reproduction with special
reference to a comparison between buffaloes, cows and zebu. J. Reprod. Fertil. 77: 1-36.
Drost, M. 2007. Advanced reproductive technology in the water buffalo. Theriogenology 68:450-
453.
Gilchrist, R.B., M. Lane and J.G. Thompson. 2008. Oocyte-secreted factors: regulators of cumulus
cell function and oocyte quality. Human Reprod. Update 14(2): 159-177.
Hamam, A.M, Karima, Karima,G.M. Mahmoud, M.F. Nawito, A.A.M. Seida and S.M.A. Nawar.
2001. Effect of the seasonal changes on the recovery, quality and maturation of buffalo
oocytes in vitro. Egyptian J. Vet Sci. 35: 123-133.
Jamil,H., H.A Samad, N.U. Rehman, Z.I. Qureshi and L.A Lodhi. 2007. In vitro maturation and
fertilization of river buffalo follicular oocytes in media supplemented with oestrus buffalo
serum and hormones. ACTA VET BRNO 76:399-404.
Manjunatha, B.M, J.P. Ravindra, P.S.P. Gupta, M. Devaraj and S. Nandi. 2009. Effect of breeding
season on in vivo oocyte recovery and embryo production in non-descriptive Indian River
buffaloes (Bubalus bubalis). Anim. Reprod. Sci.111: 376-383.
Nandi,S., H.M. Raghu, B.M. Ravindranatha and M.S. Chauhan. 2002. Production of buffalo
(Bubalus bubalis) embryos in vitro: premises and promises. Reprod. Dom. Anim. 37:65-74
Palta, P and N.S. Chauhan. 1998. Laboratory production of buffalo (Bubalus bubalis) embryos.
Reprod. Fert. Dev. 10 (5): 379-391.
Purohit, G.N., Duggal,G.P. Dadarwal, D. Kumar, D. Yadav, R.C. and S. Vyas. 2003. Reproductive
biotechnologies for improvement of buffalo: the current status. Asian-Australasian J. Anim.
Sci. 16 (7):1071-1086.
543
Raza.A., H.A. Samad, N.U. Rehman and E.U.H. Zia. 2001. Studies on in vitro maturation and
fertilization of Nili-Ravi buffalo follicular oocytes. Int J. Agri & Biol 3(4): 503-506.
Tsripoo, K., K. Srisakwattana, S. Sophon, W. Nualchuen and S. Usawang. 2007. Cloning of buffalo
fibroblast cell from donor of different ages. Buffalo J. 23(2):141-152.
Uoc, N.T., F. de. Rennis,, N.H. Duc, L.C. Bui, N.V. Hanh, N.V. Linh, N.T. Thanh, Q.X. Huu, D.D.
Long and B.X. Nguyen. 2007. Effect of season on quality of oocytes, results of in vitro
maturation, and somatic cell nuclear transfer in swamp buffalo. Reprod. Fertil. Dev. 19(1):
163-164.
Zoheir, M.A. K., A.S. Abdoon, K.F. Mahrous, M.A. Amer, M.M. Zaher, Li-Guo,Yang and E.M. El-
Nahass. 2007. Effects of season on the quality and in vitro maturation rate of Egyptian buffalo
(Bubalus bubalis) oocytes. J. Cell and Anim. Biol. 1(2):029-033.
Table 1. The relationship between the seasons and recovery rate and in vitro maturation rate of
swamp buffalo oocytes over entire 6 years of recorded data
Year Month/season Total no. of Total no. Ratio MII (%)*

collected of aspirated oocytes/ (mean +SD)
ovaries per oocytes ovary
period
1 Feb-May/summer 534 512 0.96 231(45.9+9.4)
2 Feb-May/ summer 215 207 0.96 65(34.8+13.7)
3 Feb-May/ summer 538 475 0.88 146(29.3+12.6)
4 Feb-May/ summer 635 732 1.15 303(42.3+12.1)
5 Feb-May/ summer 311 280 0.90 154(57.9+19.5)
6 Feb-May/ summer 229 259 1.13 144(56.0+14.9)
Overall 2462 2465 1.00 1043(44.4+11.4)
1 June-Sep/rain 1119 1,302 1.16 499(39.5+17.9)

2 June-Sep/rain 1030 927 0.9 246(29.1+12.6)
3 June-Sep/rain 894 915 1.02 440(47.8+13.2)
4 June-Sep/rain 658 681 1.03 461(65.4+13.2)
5 June-Sep/rain 371 347 0.93 203(58.2+13.5)
6 June-Sep/rain 229 199 0.87 127(62.+10.6)
Overall 4301 4371 1.02 1976(50.3+14.1)
1 Oct-Jan/winter 246 233 0.94 71(29.4+14.3)

2 Oct-Jan/winter 518 659 1.27 234(35.1+11. 7)
3 Oct-Jan/winter 176 235 1.33 112(46.9+7.2)
4 Oct-Jan/winter 975 902 0.92 362(40.2+10.8)
5 Oct-Jan/winter 31 35 1.13 19(54.3+0)
6 Oct-Jan/winter 125 137 1.10 81(57.6+21.5)
Overall 2071 2201 1.06 879(43.9+11.0)
*No statistical difference was obtained (P>0.05)
MII = metaphase II
544
In Vitro Embryo Production and Transfer of Bubaline Embryos Using Oocytes

Derived from Transvaginal Ultrasound-GuideFollicular Aspiration (TUFA)
Flocerfida AQUINO, a* Eufrocina ATABAY, a Edwin ATABAY, b Marlon OCAMPO, a Peregrino
DURAN, a Prudencio PEDRO, a Danilda DURAN, a Rodante DE VERAa and Libertado CRUZa
a
Reproductive Biotechnology Unit, Philippine Carabao Center, ScienceCity of Muñoz, Nueva Ecija,
Philippines
b
Philippine Carabao Center-Central Luzon State University, Science City of Muñoz, Nueva Ecija,
Philippines
*Corresponding email: floaquino@yahoo.com
ABSTRACT
Two separate studies sought to assess the effects of age (Study 1) on quantity and quality of
cumulus oocyte complexes (COCs) and to assess the in vitro embryo production (Study II) using the
Transvaginal Ultrasound-guided Follicular Aspiration (TUFA) technology. Ten (n=10) multiparous
Bulgarian Murrah buffaloes were classified into two age groups consisted of 8-12 years old (Group1)
and 13-17 years old, (Group2). Five cows were used in each age group where each cow serves as
oocyte donor cow. The collection of oocytes was done weekly for seven months. The collected COCs
were subjected to in vitro maturation, fertilization and embryos culture where bubaline embryos
produced were transferred to surrogate cows.
Results in study I showed that there were more COCs (71) collected in Group I than in Group 2
cows with 29 COCs. The collected COCs from Group I were classified into rank A, 5.63% (n=4) with
intact 3 layers cumulus cells, rank B, 2.82% (n=2) with uneven dark ooplasm but had 3 layers intact
cumulus cells, rank C, 32.39% (n=23) homogenously dark granulation of ooplasm and with
intact/compact, 1 to 2 layers cumulus cells and rank D, 59.15% (n=42) without cumulus cells. In the
older donor cows, the COCs derived were classified to rank A, 6.90% (n=2), rank B, 6.90% (n=2), rank
C, 31.03% (n=9) and rank D, 55.17% (n=16).Results showed that younger buffalo donor cows gave
significantly higher (P<0.05) number of oocytes collected (71) as against only 29 for older donor cows
In study II, the pooled TUFA derived COCs gave an in vitro fertilization rate of 40.00%. Out of this,
32.50% developed into morulae and 32.5% developed to the blastocyst stage. All the blastocysts
produced were transferred to surrogate cows where an embryo developed to full term resulting to a live
birth of a male calf. The TUFA-IVEP using oocytes from the donor cows gave a success rate of 10%.
Keywords: Bubaline embryos, oocytes, IVEP
INTRODUCTION
The In Vitro Fertilization and Embryo Transfer (IVF-ET) techniques for improving the genetic
improvement of buffaloes have been successfully demonstrated at the institutional farm and at the
farmer’s level in the Philippines. Success rate using IVF-ET technique ranges from 14 to 15% and this
efficiency is still low compared to the output of Indian and Italian researchers(Neglia et. al., (2003),Galli
et al., 2001). To strengthen the implementation of the carabao development program, the Philippine
Carabao Center is continuously in search for a practical but efficient technique to enhance the genetic
improvement program of buffaloes for milk and meat.
The use of TUFA which was originally applied in human and was first used in cattle in the early
1990s (Boni et al., 1994), has provided the breeding industry the opportunity to increase the number of
calves from donors of high genetic merits. It made available oocytes from live cows, giving the
opportunity to use germplasm of known source for embryo production. The TUFA technique in
545
combination with In Vitro embryo production (IVP) technology has been a routine procedure, and over
the years has become increasingly commercialized particularly in the cattle breeding industry. The use
of TUFA technology in buffalo started in 1993 (Boni et al., 1994) and there has been an increasing
interest in TUFA-IVP technologies for embryo production in water buffaloes (Boni et al., 1996, Neglia
et al., 2003, Techakumphu et al., 2004 and Yadav et al., 2006).
The application of TUFA technique has been carried out in this study to 1) determine the effects
of age of buffaloes on the quantity and quality of the oocytes collected from the live donor cows and 2)
monitor the In Vitro maturation (IVM), IVF, IVC and ETto surrogate dam.

Study1: Effects of age on the quantity and quality Bubaline oocytes using Transvaginal Ultrasound-
Guided Follicular Aspiration (TUFA) technology
The donor buffalo cows.
Ten (10) Bulgarian Murrah buffalo cows raised at the PCC-Gene Pool farm were used in the
study. The donor cows were assigned into groups, i.e. 8-12 years (Group 1) and 13-17 years, (Group 2),
average body weight of 600 kg and a body condition score of 3.5 to 4.() Each age group consisted of 5
cows. The experimental cows were maintained under complete confinement system of management.
The animals were raised in group in a free stall where they were given standard rations composed of
Napier soilage (40%), rice straw (40%), spent grain (7%) and supplementary concentrate pellets (13%).
Transvaginal Ultrasound-Guided Follicular Aspiration (TUFA).
Follicles from each of the ten donor cows were collected using the developed protocol on
TUFA technology. The TUFA session was done in the morning while they were restrained in a
standing position which involved the aspiration of ovarian antral follicles with diameter size > than
5mm (all antral follicle ≥? mm in diameter were collected) through the vaginal route for the collection
of oocytes. Animals were restrained in a chute while in standing position. The ovaries were positioned
by rectal palpation in front of a vaginal scanner, TUFA Probe, Model HCV 4710 MV and with the use
of an Ultrasonic Scanner Model HS-2000, Honda Electronics Co. Ltd., Japan. The probe is equipped
with a 50-cm long needle (FHK #01281350, Japan), 18g, which drew the samples into a sterile tube
under 50mm Hg permanent negative pressure with a vacuum pump (FHK, Model #4, Japan). Oocytes
were collected using Phosphate Buffered Saline solution (PBS) with 0.3% Polyvinyl Alcohol (PVA),
50 µg/mL gentamycin and 20µg/mL heparin. A clean, labeled, 50 ml collecting tube was prepared and
assigned to each of the donor cow every time the TUFA activity was conducted. After each TUFA
session, the silicon tubing was thoroughly washed with PBS to ensure that all aspirated oocytes were
collected in 50 ml collecting tube. The aspirated follicular samples were maintained at 37°C in a
portable incubator and brought to the laboratory for further processing. The contents of the tube were
washed and searched using an Encom Filter (Fujihira, Japan) under stereomicroscope (Nikon, Japan).
Searching and evaluation of follicular aspirates
The aspirates derived from follicular aspiration were brought to the laboratory. Extra
care was observed in handling and transporting in order to maintain its livability. The collected
aspirates were placed in the pre-warmed water bath with temperature set at 37o C. The contents of each
of the collecting tube were poured into a searching dish using an Encom Filter (Fujihira, Japan). The
tubes were washed thoroughly with PBS solution to ensure that there were no cumulus oocyte
complexes (COCs) left in the tube. The poured aspirates in the Emcon filter were washed thoroughly
with PBS solution to ensure that the aspirates were totally clear and free of blood. Searching of the
COCs was done under the stereomicroscope (Nikon, Japan). The number of COCs from each donor
cow was counted, recorded and were classified according to the morphology of the ooplasm and the
appearance of the cumulus cells as followed: rank A with intact 3 layers cumulus cells, rank B with
uneven dark ooplasm but had 3 layers intact cumulus cells, rank C homogenously dark granulation of
546
ooplasm and with intact/compact, 1 to 2 layers cumulus cells and rank D without cumulus cells (PCC
Reproductive Biotechnology Laboratory Classification System
Study 2:In Vitro Maturation, Fertilization and Culture of Bubaline Oocytes Derived from Transvaginal
Ultrasound-guided Follicular Aspiration.
IVM/IVF/IVC
The searched COCs were washed several times with PBS solution and were then placed in a
Falcon Petri dish, 35 X 10 mm with droplets of TCM-199 (band?), 50 µl each layered with mineral oil,(
M-8410 Sigma). which was pre-incubated in a water jacketed incubator for at least 2 hours. After
washing, the oocytes in the maturation droplets, they were finally placed in other maturation droplet
and incubated for 22-24 hours maturation period.
In vitro fertilization (IVF) of matured oocytes was done using frozen buffalo semen thawed at
37ºC for 15 seconds. The thawed semen were washed in a 15 ml centrifuge tube with a pre-incubated
modified Brackett and Oliphant medium (BO medium, Brackett and Oliphant, 1975) by centrifugation
at 800 x g for 5 minutes. After centrifugation the supernatant was discarded, leaving only about 200ul
of the sperm pellet.
Semen concentration was determined using a hemocytometer and adjusted to 4 x 106 sperm/mL
concentration with BODM. Twenty five µL of the semen suspension was added to pre-equilibrated
fertilization droplets consisting of 25ul (equal volume of IVF medium and sperm pellet) BODM
supplemented with 3mg/mL BSA and 2.5mM theophylline, to have a final concentration of 2x106
sperm/mL. The in vitro matured oocytes were transferred to the fertilization droplets. The spermatozoa
and oocytes were co-incubated for 16 to 18 hr at 38.5ºC under 5% CO2.
The preparation of the in vitro culture medium was done using the modified Synthetic
Oviductal Fluid medium (mSOF). . In vitro culture working medium was prepared by adding 30 mg of
Bovine Serum Albumin (BSA 6003) to 10ml mSOF and was again filtered. In a 35 x 10 mm Falcon
Petri dish, several microdroplets with 50ul droplets each of the IVC medium were pipetted and was
layered with mineral oil.
The prepared dish was placed in a triple gas incubator. Thereafter, the in vitro fertilized oocytes
were transferred into the IVC dish containing the mSOF microdroplets with BSA. Sperms attached to
the oocytes were totally removed before they were placed in the IVC dish. This process was done
carefully in order not to harm the oocytes. The pipette that was used to remove the sperms attached to
the fertilized oocytes was prepared with fine surface and with a small bore just good enough to remove
the sperms surrounding the fertilized oocytes. The fertilized oocytes were washed in the separate IVC
droplets and were then placed in the final culture droplets. The culture dish containing the fertilized
oocytes was incubated in a humidified incubator with a gas phase of 5% O2, 5% CO2 and 90% N2 level.
The cleavage rate was observed 24 hours post-culture. The number of embryos that developed into
blastocysts on the Day-7 of in vitro culture was observed and recorded.

Results in study 1 involving the average quantity and quality of collected oocytes in the 22
TUFA sessions in ten donor cows were summarized in Table1. There were more COCs (71) collected
in Group I than in Group 2 cows with 29 COCs. The same trend was observed where Su et al. (2009)
aspirated fewer follicles and obtained lower COC numbers in old cows than in middle-aged and young
cows. The collected COCs from Group I were classified into rank A, 5.63% (n=4) with intact 3 layers
cumulus cells, rank B, 2.82% (n=2) with uneven dark ooplasm but had 3 layers intact cumulus cells,
rank C, 32.39% (n=23) homogenously dark granulation of ooplasm and with intact/compact, 1 to 2
layers cumulus cells and rank D, 59.15% (n=42) without cumulus cells. In the older donor cows (Group
2), the COCs derived were classified to rank A, 6.90% (n=2), rank B, 6.90% (n=2), rank C, 31.03%
(n=9) and rank D, 55.17% (n=16).
547
For the 2 age groups there is no significant difference on the quality of oocytes collected but
has significant difference only on the quantity of oocytes collected with the younger age group yielded
71 oocytes compared to the older group of cows with only 29 oocytes collected.
In study 2, of the 100 COCs, 53% maturation rate was recorded after 22-24 hours maturation
period, Table 2. Twenty six (26%) of the COCs were not fertilized and 16% were degenerated. The
matured COCs (53%) that were subjected to fertilization for 24 hrs period gave a fertilization rate of
40%. Of the fertilized embryos, 13 (32.50%) developed to morula stage embryo and 13 embryos
developed further to blastocyst (32.50%) 7 days after in vitro fertilization. The blastocysts produced
were transferred to surrogate cows where an embryo developed to full term resulting to a live birth of a
male calf. These TUFA derived oocytes after IVM, IVFand ET gave a success rate of 10%.
REFERENCES
Boni, R.S., V. Di Paloand and L. Barbiere.1994. Ovum pick-up in deep anestrus buffaloes. Proc IV
World Buffalo Congress 3: 480-482
Boni, R.S., S. Roviello and L. Zicarelli. 1996. Repeated ovum pick-up in Italian Mediterranean buffalo
cow. Theriogenology 46: 899-909
Neglia G., B. Gasparini, V.C. Brienza, D. Palo, G. Campanile, G. Presicce and L. Zicarelli. 2003.
Bovine and buffalo in vitro embryo production using oocytes derived from abattoir ovaries or
collected by transvaginal follicular aspiration. Theriogenology 59: 1123-1130.
Su, L., S. Yang, X. He,X. Li,J. Ma, Y. Wang, G. Presicce and W. Ji. 2009. Effect of Donor Age on
the Developmental Competence of Bovine Oocytes Retrieved by Ovum Pick Up. Reprod.
Domest. Anim. Volumes: page of print? .47(2):184-9
Techakumphu. M., A. Promdireg, A. Na-Chiengmai and Phutikanit. 2004. Repeated oocyte pick up in
prepubertal swamp buffalo (Bubalus bubalis) calves after FSH stimulation. Theriogenology
61:705-1711
Yadav S.K., A.K. Misra, R. Sharma, S. Prasadand and H.P. Gupta. 2006. Repeated transvaginal
ultrasound-guided aspiration of oocytes (OPU) from Murrah buffaloes. Proceedings, 5th Asian
Buffalo Congress, Naning China, April 18-22.
Table 1. Average quantity and quality of collected COCs by TUFA technique in Bulgarian Murrah
buffalo cows.
Mean # of Mean #
Donor Aspirated of
Cow No # of Quality and % of collected oocytes Total Follicles Oocytes %
Age Oocytes A % B % C % D % # of follicle /Cow/ /Cow Recovery
Aspirated Session /Session Rate
8-12 5 71 4 5.63 2 2.82 23 32.39 42 59.15 110 2.35 1.39 59.0%
13-17 5 29 2 6.90 2 6.90 9 31.03 16 55.17 53 2.17 1.09 50.0%
Table 2. In vitro fertilization and embryo development of TUFA-derived COCs in Bulgarian Murrah
buffalo cows.
No of Matured Unfertilized Degenerated 2-cell Morulae Blastocyst
oocytes
53 26 16 40 13 13
100
(53.00%) (26%) (16%) (40.00%) (32.50%) (32.50%)
548
L-Carnitine Influences the Developmental Competence of Buffalo Oocytes from

Aged Donors
Marlon B. OCAMPO,*Prudencio B. PEDRO, Peregrino G. DURAN, Eufrocina P. ATABAY,

Excel Rio S. MAYLEM, Flocerfida P. AQUINO, Lerma C. OCAMPO and Libertado C. CRUZ
Philippine Carabao Center-Department of Agriculture, Science City of Munoz, Nueva Ecija,

Philippines
*Corresponding author e-mail: ocampomarlon29@yahoo.com
ABSTRACT
The oocyte developmental competence is acquired throughout folliculogenesis and is associated
with appropriate differentiation and responsiveness to the LH surge. In older/aged donors, this
developmental competence is compromised with reduced capacity to complete maturation (both
nuclear and cytoplasmic) and be fertilized. In this study, we investigated the acquisition for
developmental competence of buffalo oocytes derived from donors (10 yrs and older) by up-regulation
of beta-oxidation by the addition of fatty acid transport cofactor L-carnitine. In a dose-dependent
manner, the completion of 1st meiosis, ability to support MPN formation after sperm penetration,
cleavage and development to the blastocyst stage were examined. Results showed an improved rate on
oocyte competence when cultured with L-carnitine regardless of concentrations (5 mg/ml and 10
mg/ml) used vs. the control, though not statistically significant. Of particular interest is on the
improved total blastomere count on the blastocyst stage embryos derived from culture with L-carnitine
addition. These observations supported the potentials of including L-carnitine in the culture medium in
improving the competence of buffalo oocytes derived from aged donors. The system is going to be
useful and practical for in vitro buffalo embryo production since most of the ovaries coming from local
slaughterhouses are derived from aged and culled buffaloes.
Keywords: L-carnitine, buffalo oocytes, blastocyst

549
ART’s for in vitro Production of Buffalo Embryos (Philippine experience)
Marlon B. OCAMPO, * Prudencio B. PEDRO, Peregrino G. DURAN, Eufrocina P. ATABAY,

Excel Rio S. MAYLEM, Flocerfida P. AQUINO, Lerma C. OCAMPO and Libertado C. CRUZ
Philippine Carabao Center, Reproductive Biotechnology Unit, Science City of Munoz, Nueva Ecija,
Philippines
*Corresponding e-mail: ocampomarlon29@yahoo.com
ABSTRACT
In the Philippines, the first buffalo calf born out embryos produced in vitro was a female in
1996 (Ocampo et al., 1997). Since then, various efforts were undertaken to further improve the
production of embryos in vitro from oocytes derived from slaughterhouse-derived ovaries and/or
through ovum-pick up from genetically superior buffalo females. Most of the developments in our
laboratories were largely the results of our understanding of the oocyte maturation (both nuclear and
cytoplasmic), sperm capacitation and embryonic developments. It is the interest of this paper to share
our experiences on the development of IVM/IVF/IVC protocol that would consistently support the
acquisition of immature oocytes of developmental competence to mature, be fertilized, results to zygote
production and eventually develop to youngs offsprings. Moreover, the reasons for doing IVF in
buffalo, the efficiencies of each step and the needs for improvement, the physiological mechanisms
through which in vitro fertilization and embryonic development is accomplished will be discussed.
Keywords: Buffalo embryos, In Vitro Fertilization

550
Developmental Competence of Water Buffalo Oocytes In Vitro in

Single Base Medium-Synthetic Oviductal Fluid
Lerma C. OCAMPOa*, Danilda H. DURANa, Prudencio B. PEDROa, Flocerfida P. AQUINOa,
Eufrocina P. ATABAYa, Marlon B. OCAMPOa, E.S. MAYLEMa, P.G. DURANa and L.C. CRUZa
a
Reproductive Biotechnology Unit Philippine Carabao Center, Science City of Munoz, Nueva Ecija,
Philippines
*Corresponding e-mail: oxengurlram@yahoo.com
ABSTRACT
Mammalian oocytes ability to achieve full developmental competence up to the blastocyst stage
following in vitro fertilization requires a carefully regulated environment which is dependent on the
compositions of the medium used. Oftentimes, the variabilities observed on the results under in vitro
systems could be traced back on the differences on the media (eg., type, batches, lot) used and its
supplementation. Thus, a need for a single culture system that would support the oocytes acquisition of
competence for complete maturation (both nuclear and cytoplasmic), the resulting zygotes development
past the “cell-block” stage up to the blastocyst stage is imperative. In this study, a modified synthetic
oviductal fluid (mSOF) based medium was utilized if buffalo oocytes collected in vivo (through OPU)
and/or in vitro (from abattoir) would acquire the necessary developmental competence in the
production of blastocyst stage embryos. In both conditions, initial results showed no significant
difference on the oocytes ability for completion of maturation (both 1st and 2nd meiosis). Similarly, the
cleavage rate of the resulting zygotes remained comparable. These observations show the usefulness
and practicality of using mSOF as a base medium for buffalo oocytes acquisition of competence for
maturation and eventual embryo development in an in vitro culture system.
Keywords: buffalo oocytes, single medium, developmental competence


551
Blastocyst Formation after Intracytoplasmic Sperm Injection in Bovine and

Buffalo Oocytes Derived from Slaughter House
Prudencio B. PEDRO*, Eufrocina P. ATABAY, Edwin C. ATABAY, Flocerfida P. AQUINO,

Marlon B. OCAMPO, Excel Rio MAYLEM, and Libertado C. Cruz
Reproductive Biotechnology Laboratory, Office of the Executive Director, Philippine Carabao

Center, National Headquarters and Gene Pool, Science City of Munoz 3120 Nueva Ecija,
Philippines
*Corresponding e-mail: prudz_pedro@yahoo.com
ABSTRACT
The objective of this study is primarily to develop skills and establish efficient
intracytoplasmic sperm injection (ICSI) procedure for buffaloes. Buffalo matured oocytes side by
side with bovine oocytes derived from slaughter house were used in this study. In order to assess
the embryo development of ICSI or non-sperm injected (sham) oocytes, they were exposed to 7%
ethanol for 5 min and then transferred to TCM-199 supplemented with 10% FBS for 3h. Activated
oocytes were further cultured with the presence of CHX for 5 h and finally cultured in mSOF
medium. For cattle, the rates of cleavage, morula and blastocyst development of sperm injected
oocytes treated with the combination of alcohol and CHX were comparatively higher (78.1%,
25.0% and 19.7%, respectively) than after activation alone with 7% alcohol (65.8%, 19.5% and
14.6%, respectively) and with non treated groups (17.6%, 0% and 0%), respectively). For buffalo
sperm injected oocytes, the cleavage, morula and blastocyst rates were also higher for oocytes
treated with combination of ethanol and CHX (60%, 35% and 21%, respectively) than those of
ethanol treatment alone (29.2%, 16.6% and 11%, respectively). For the non treated oocytes, only
14% of them cleaved with no further development. The results indicate that activation treatment is
necessary for the development of buffalo and bovine oocytes after ICSI and combined treatment
with ethanol and CHX have better cleavage and blastocyst formation rates than those of ethanol
treatment alone by conventional ICSI.
Keywords: ICSI, CHX, activation, matured oocytes, blastocyst, bovine, buffalo
INTRODUCTION
Buffalo has been a good source of draft animal and is now becoming a popular source of
meat and milk in the Philippines. However, limited studies were reported on the intracytoplasmic
sperm injection (ICSI) as one of the powerful techniques that may facilitate the genetic
improvement of livestock. The first ICSI in mammals was reported by Uehara and Yanagimachi
(1976). They injected hamster or human spermatid into a hamster oocyte, and observed the
transformation of the sperm head into a male pronucleus. Since this first report, this technique has
been successfully used in mice (Palermo et. al., 1992). Many investigators have tried to improve
the efficiency of production of ICSI embryos. Artificial activation of oocytes after ICSI, however,
has been required for embryos to progress beyond the pronuclear stage and continue further
development. Ethanol has been widely used for activation of mammalian oocytes such as those of
mouse, cow etc. In order to improve the activation of bovine oocytes ICSI has been combined with
chemical activation such as ethanol, (Liang et al.,2011), calcium ionophore, (Goto et. al., 1996), or
552
by association of these stimuli with cycloheximide (CHX) (Suttner et. al., 2000) (Atabay et al.
2006) as protein synthesis inhibitor.
ICSI is potentially valuable in using very valuable semen samples most effectively which
can possibly produce superior animals.

Collection and IVM of oocytes.
Immature oocytes were collected from both buffalo and cattle ovaries aspirated from 2-8
mm follicles. Cumulus-oocyte complexes (COCs) were washed in phosphate buffered saline (PBS)
medium supplemented with 3mg BSA/ml and cultured in IVM medium (TCM 199)(Gibco
Laboratories, Grand Island, NY, USA), supplemented with 10% fetal calf serum (FCS, Gibco),
0.02unit/ml follicle stimulating hormone (FSH, Sigma), 10 ng/ml EGF(Sigma), 1µg/ml estradiol-17
ß(Sigma), and 50µg/ml gentamycin sulfate at 38.5 °C under humidified atmosphere of 5% CO 2 in
air for 22 to 24 h. Oocytes with expanded cumulus cells were freed by treatment first with PBS
containing 0.05% hyaluronidaze (type 1-S, Sigma-Aldrich) for 1-2 min and then gentle pipetting or
by vortex for 2 min.
Sperm injection, activation and in vitro culture
ICSI was performed in a 60 x 15 mm Petri dish with the micromanipulator system.
Immediately before sperm injection, buffalo or bovine motile spermatozoa from washed frozen-
thawed were immobilized by scoring the tail with the tip of the injection needle(GD-Narishige,
Japan) with an 8-10µm in diameter against the bottom of the dish. A single, motile buffalo or cattle
spermatozoon was immobilized against the bottom of the 10%PVP droplet, the tail loaded first with
a minimum volume of medium into the injection pipette and then injected into the cytoplasm of the
oocyte. An oocyte should be positioned on the holding pipette(100µm)in such a way that the first
polar body was either at the 12 or 6 o’clock position and when the injection pipette should approach
the oocyte from the 3 o’clock direction. The oocyte was gently injected from the 3 0’clock
direction aspirating small amount of cytoplasm together with sperm and returning back inside the
cytoplasm. They were activated 3 h after injection and were further activated with mSOF medium
supplemented with 10µg/ ml CHX and cultured for 5 h at 38.5ºC under 5 % CO 2, 5% O2 and 90%
N2 gas system. Embryos developed to 4-8 cell stage on the 3rd day of culture were further cultured
in mSOF supplemented with 10% FCS for 5 days under the same culture conditions.

To assess the embryo development of sperm injected (ICSI) or non-sperm injected (sham)
bovine and buffalo oocytes. When sham injected bovine oocytes were treated with both ethanol and
CHX, cleavage, morula and blastocyst rates (40.9%, 18.3% and 4.5%, respectively) were quite
similar with those of oocytes treated with only ethanol (31.5%, 15.7% and 5.3%, respectively).
However, only 10% oocytes without treatments cleaved and showed no further development (Table
1). As shown in Table 3, when buffalo sham injected oocytes were treated both ethanol and CHX, a
higher cleavage rate (36.4%) was obtained compared with that of oocytes treated with only alcohol
(21.4%), and 22.7% of them developed to the morula stage; however, no further development was
observed for the oocytes treated with only alcohol. Furthermore, oocytes without treatment showed
no development at all. In the present study, the low blastocyst development in the sham injected
oocytes may have been the proposal that oocytes activation requires either sperm entry to
cytoplasm, which provides some activation factors, or some type of stimuli, which cause
intracellular calcium increase to the requirement of sperm.
553
For cattle, the rates of cleavage, Morula and blastocyst development of sperm injected
oocytes treated with alcohol plus CHX were higher (78.1%, 25.0% and 19.7% ) (Table 2) than after
activation alone with 7% alcohol (65.9%, 19.5% and 14.6%) and with non treated groups (15.0%,
0%, 0%) respectively. For buffalo sperm injected oocytes, the result was shown in Table 4. The
cleavage, morula and blastocyst rate is comparatively higher with ethanol plus CHX (60.0%,
40.0%, and 25.0%) than ethanol alone (29.2%, 16.6%, 10.4%) respectively. For the non treated,
only 12.5% cleaved with no further development. The blastocyst yield the treated group (4.5% to
5.3%) seemed similar with those (0 to 8%) reported previously by other laboratories (Horiuchi et
al., 2002). However, buffalo blastocyst rate in the study was quite low compared with other reports
(Wei and Fukui, 2002) which indicates that failure in the early post fertilization events (i.e., from
fertilization to cleavage) is the main determinant of successful development after bovine ICSI.
Application of additional activation stimuli to the bovine ICSI oocytes has been considered
important for the decondensation of sperm heads, pronuclear formation, cleavage and embryonic
development (Rho et al., 1998.).
For buffalo sperm injected oocytes, the cleavage, morula and blastocyst rate is
comparatively higher with ethanol plus CHX (60.0%, 40.0%, and 25.0%) than ethanol alone
(29.2%, 16.6%, 10.4%) respectively. For the non treated, only 12.5% cleaved with no further
development. Our blastocyst rate (4 to 25 %) seems similar with previous reports (14 to 21%)
(Fujinami et. al., 2004) maybe due to the quite low quality of oocytes that were derived from aging
animals killed from slaughter house especially in buffaloes (Table 4). In the present study, the
blastocyst formation increased when ethanol was combined with CHX.
In summary, activation of oocytes by chemical stimuli, may affect the developmental
potential of ICSI oocytes. These factors alone or together could improve blastocyst development of
bovine or buffalo after ICSI. Further studies are needed to develop more efficient procedure that
will increase the rates of fertilization and embryonic development especially in buffaloes.
REFERENCES
Atabay, E.C., F.P. Atabay, R.V. de Vera, F.V. Mamuad and Cruz L.C. 2006. Chemical and
electrical activation of swamp buffalo (Bubalus bubalis) oocytes with or without
cycloheximide treatments. Buffalo J. 21(2): 121-130.
Fujinami, N., Y. Hosoi, H. Kato, K. Matsumoto, K. Saeki, and A. Iritani. 2004. Activation with
ethanol improves embryo development of ICSI-derived oocytes by regulation of kinetics of
MPF activity. J. Reprod. Dev. 50:171–178.
Goto, K., A. Kinoshita, Y. Nakanishi and K. Ogawa. 1996. Blastocyst formation following
Intracytoplasmic injection of in vitro derived spermatids into bovine oocytes. Human
Reprod. 11: 824-829
Horiuchi, T., C. Emuta, Y. Yamauchi, T. Oikawa, T. Numabe, and R. Yanagimachi. 2002. Birth of
normal calves after intracytoplasmic sperm injection of bovine oocytes: a methodological
approach. Theriogenology 57:1013–1024.
Liang, Y.Y., D.N. Ye, C. Laowtammathron, T. Phermthai, T. Nagai, T. Somfai and R. Parnpai.
2011. Effects of chemical activation treatment on the development of swamp buffalo
(Bubalus bubalis) oocytes matured in vitro and fertilized by intracytoplasmic sperm
injection. Reprod. Domest. Anim. 46(1): 67-73.
Palermo, G., H. Joris, P. Dovroey and A.C. Van Steirteghem. 1992. Pregnancies after
Intracytoplasmic injection of single spermatozoon into oocytes. Lancet 340: 17-18.
554
Rho, G.J., B. Wu, S. Kawarsky, S.P. Leibo, and K.J. Betteridge. 1998. Activation regimens to
prepare bovine oocytes for intracytoplasmic sperm injection. Mol. Reprod. Dev. 50:485–
492.
Suttner, R., V. Zakhartchenko, P. Stojkovic, S. Muller, R. Alberio, I. Medjugorac, G. Brem, E.
Wolf, and M. Stojkovic. 2000. Intracytoplasmic sperm injection in bovine: effects of oocyte
activation, sperm pretreatment and injection technique. Theriogenology 54: 935–948.
Table 1 . Bovine embryonic development after sham Intracytoplasmic Sperm Injection(ICSI)

injection procedure without sperm.1
Groups No. of oocytes 2-4 cell (%) Morula (%) Blastocyst(%)
cultured
Not treated 20 2(10.0)a 0(0) (0)
Alcohol alone 19 6(31.5)b 3(15.7)a 1(5.3)a
Alcohol plus CHX 22 9 (40.9)c 4(18.3)a 1(4.5)a
Value within the same column with different letters (a,b,c) differ significantly(p>0.05)
1. Matured oocytes were injected with the same ICSI procedure without sperm.
Table 2. Bovine embryonic development after ICSI with frozen-thawed spermatozoa after
injection and activation treatments.1
cultured
Not treated 20 3(15.0)a 0(0)a (0)
Alcohol alone 41 27(65.9)b 8(19.5)b 6(14.6)a
Alcohol plus CHX 96 79 (78.1)c 24(25.0)c 19(19.7)a
1. Matured oocytes were injected with ICSI procedure with sperm.
Table 3. Survival rate of buffalo oocytes after sham injection.1

cultured
Not treated 26 0(0)a 0(0)a 0(0)
Alcohol alone 56 12(21.4)b 0(0)a 0(0)
Alcohol plus CHX 44 16 (36.4)c 10(22.7)b 0(0)
1. Matured oocytes were injected without sperm by ICSI procedure
Table 4. Survival of buffalo oocytes after ICSI injection.1

cultured
Not treated 24 3(12.5)a 0(0)a 0(0)a
Alcohol alone 48 14(29.2)b 8(16.6)b 5(10.4)b
Alcohol plus CHX 80 48(60.0)c 32(40.0)c 20(25.0)c
1. Matured oocytes were injected with the presence of spermatozoa by ICSI procedure
555
Somatic Cell Nuclear Transfer as a Tool for the Multiplication of Genetically

Superior Water Buffaloes: The Philippine Initiatives
Edwin C. ATABAY1, Eufrocina P. ATABAY2, Flocerfida P. AQUINO2, Peregrino G.
DURAN2, Prudencio B. PEDRO2 and Libertado C. CRUZ2
1
Philippine Carabao Center at Central Luzon State University, Science City of Muñoz, Nueva Ecija,
2
Reproductive Biotechnology Unit, Philippine Carabao Center, Science City of Muñoz, Nueva Ecija,
Philippines, 3120
ABSTRACT
Reproductive biotechnology has been playing a significant role in facilitating genetic
improvement in water buffaloes. The Philippine Carabao Center has recently adopted the somatic cell
nuclear transfer technology to complement other existing reproductive tools for buffaloes. The present
work was conducted to develop/optimize a system for cloning through somatic cell nuclear transfer in
water buffalo, to evaluate the ovulation synchronization efficiency of the CIDAR-SYNCH-eCG
protocol, to determine the maximum time lapse where ear skin cell proliferation activity was not
affected, and to evaluate the in vivo development of reconstructed embryos by transfer to recipient
animals.
Buffalo clone embryos had been successfully produced in-vitro. The slit and squeeze
enucleation method and simultaneous fusion and activation of couplets with electrical stimuli followed
by ethanol and cycloheximide treatment improved the development of clone embryos. Subsequently,
optimum condition for culture of clone embryos was achieved with modified synthetic oviductal fluid
under a 3-gas system. The maximum time lapse where ear skin cell proliferation activity was not
reduced significantly was at 120 h post- collection. The use of CIDAR-OVSYNCH-eCG protocol
resulted in 88% ovulation response by the recipient females. The development of clone embryos in-
vivo after several transfers made (89) had been observed to be up only to early pregnancy stage, but
failed to achieve further development to term. Future researches should focus on reprogramming in
order to understand the underlying factors which can improve the efficiency of cloning to make it a
relevant and useful reproductive tool in buffalo species.
Keywords: Clone embryos, Embryo transfer, Somatic cell, Water buffaloes
INTRODUCTION
The Philippine Carabao Center (PCC) is mandated to develop and promote the Philippine
Carabao not only for draft power but more importantly for meat and milk to provide additional
income and better nutrition to the rural farming communities.
During the past several years, PCC has engaged in various reproductive techniques that
focus on harnessing the genetic potentials of superior female buffaloes. Reproductive biotechniques
applied to female buffaloes, such as in vitro embryo production, ovum pick up, embryo transfer
however, have yet to be improved several folds to be considered a practical technique at the farm
level.
Among the recent advances in animal reproduction biotechniques, the cloning technology by
Somatic Cell Nuclear Transfer (SCNT) offers huge potential after live offspring have been
produced in wide range of farm animals. This reproductive technology can significantly reduce the
generation interval and bring about desired genetic improvements at a much accelerated rate.
Moreover, buffaloes are known to have poor reproductive performance, including inefficient
ovarian response to superovulation treatment in conventional embryo transfer (Madan et al., 1996)
and low oocyte/quality and quantity for in vitro fertilization (Nandi et al., 2002). This could be due
to the smaller number of ovarian follicular population in the water buffalo ovaries compared with
domestic cattle (Ty et al., 1989; Jainudeen et al., 1993). Although live calves have been produced
from in vitro fertilized embryos, the in vitro maturation and fertilization rates of buffalo oocytes
were generally low compared with cattle. Considering the low production of preimplantation
556
embryos by superovulation and in vitro fertilization techniques in buffaloes, cloning by somatic cell
nuclear transfer is an alternative method for buffalo reproduction
In water buffaloes, the use of SCNT combined with embryo transfer can maximize the
utilization of the “super buffaloes” with high milk yield as nuclear donor for eventual multiplication
of these genetically and economically valuable livestock. Essentially, the resulting clones of
superior buffaloes could contribute to the building up of an elite nucleus for use in enhanced wide-
scale genetic improvement program that will be of benefit to thousands of carabao owners in the
country.
OBJECTIVES
The present work was conducted to develop a system for cloning through somatic cell nuclear
transfer in water buffalo, to evaluate the ovulation synchronization efficiency of the CIDAR-
SYNCH-eCG protocol, to determine the maximum time lapse where ear skin cell proliferation
activity was not affected, and to evaluate the in vivo development of reconstructed embryos by transfer
to recipient animals.

Experiment 1. Nuclear transplantation and Culture of Clonal Embryos In -Vitro
Collection and in vitro maturation of follicular oocytes
Swamp buffalo (Bubalus bubalis) ovaries were collected from a local abattoir and
transported to the laboratory in normal saline solution. Cumulus-occyte complexes (COCs) were
aspirated from small antral follicles (2-8 mm in diameter) and washed 3 times with HEPES-
buffered modified Tyrode’s medium (TALP-HEPES) [Bavister et al., 1983] supplemented with 3
mg/ml bovine serum albumin (BSA, Fraction V), 0.2 mM sodium pyruvate and 50 μg/ml
gentamicin sulfate. The COCs then cultured in HEPES-buffered TCM 199 supplemented with 10%
calf serum, 0.02 units/ml follicle stimulating hormone (FSH) 1 μg/ml estradiol-17 β, 10 ng/ml
epidermal growth factor, 0.2 mM sodium pyruvate and 50 μg/ml gentamycin sulphate. Ten to
twelve COCs were cultured for 24 h in a 50-μl droplet of maturation medium overlaid with mineral
oil under a humidified atmosphere of 5% CO2 in air at 38.5 °C.
Preparation of recipient oocytes
In-vitro matured swamp buffalo oocytes were freed with cumulus cells by vortexing for 3
min in a 1.5 ml tube containing 200 μl of TALP-HEPES with 0.1% (w/v) hyaluronidase. After the
removal of the cumulus cells, the denuded metaphase II (MII) oocytes with first polar body were
enucleated by slit and squeeze method. Only successfully enucleated oocytes were used as recipient
cytoplast for nuclear transfer.
Preparation of donor cells
Ear skin biopsies from dairy buffalo were stored at 4ºC for different time points after
collection. After storage at specified time, tissue samples were processed for primary culture. To
obtain primary cultured fibroblasts, biopsied ear skin were cultured for 10 days in Dulbecco’s
modified eagles medium (DMEM/F12) supplemented with 10% FCS and 100 IU/ml penicillin G
potassium and 100 µg/ml streptomycin sulfate under a humidified atmosphere of 5% CO 2 in air at
37°C. Percent live cells and population doubling were determined. After 8 to 10 passages, the ear
skin-derived fibroblasts were cryopreserved and stored in liquid nitrogen until use. One week before
use, frozen-thawed ear skin fibroblasts were cultured for 3 or 4 days in DMEM/F12 supplemented
with 10% FCS and 50 µg/ml gentamicin sulfate and then further cultured for 3 or 4 days in
DMEM/F12 supplemented with 0.5% FCS (serum-starvation) under a humidified atmosphere of
5% CO2 in air at 37°C and were used as nuclear donor.
Nuclear transplantation and culture of clonal embryos in vitro
After serum-starvation treatment, the nuclear donor cells were trypsinized and inserted
individually into the perivitelline space of the enucleated oocytes. The couplets were fused and
activated simultaneously with two direct current pulses of 200 V/mm for 20 µsec using an Electro
Cell Fusion (LF-100, Life Tec Co., Tokyo, Japan) between two wire electrodes overlaid with 0.3 M
557
mannitol (Kanto) solution. After 1 h, successfully fused couplets were incubated in 7% ethanol for
5 min and then transferred to a modified synthetic oviductal fluid [Takahashi et al., 1996]
supplemented with 20 amino acids, After CHX treatment, the reconstituted embryos were
thoroughly washed and then placed in the culture medium excluding CHX at 39°C in 5% CO 2, 5%
O2 and 90% N2 for 7 days. The fusion and cleavage rates were determined at 1 and 33 h after fusion,
respectively. Subsequent development up to the blastocyst stage was determined thereafter until
174 h after fusion.
Experiment 2. Transfer of Reconstructed Embryos to Recipient Cows for In Vivo Development.
Animals
Selected recipient animals were either natural estrus or synchronized following the CIDAR-
SYNCH-eCG ovulation synchronization procedure. Ovarian status of the female animals was first
ascertained for the presence of corpus luteum to ensure that the animals are cycling. On day 0,
progesterone implant (CIDAR) was inserted intravaginally and the first dose of 2 ml of Cystorelin
was injected. A week later, CIDAR implant was removed followed by injection with 2 ml PGF2-
alpha and 500 IU of eCG (Serum Gonadotropin/Chorionic Gonadotropin, 5 ml). Two days after
(day 9) PGF2 injection, the animals received the second dose of GnRH and embryo transfer was
performed on day 15. For natural estrus recipients, embryo transfer was done 5-6 days after the
observed ovulation by ultrasonography.
Embryo transfer
Buffalo clone embryos were transferred to the suitable recipient animals with corpus luteum.
One or two clone embryos were transferred into the recipient uterine horn ipsilateral to the corpus
luteum. Pregnancy diagnosis was done by ultrasound 30 days after transfer or by rectal palpation
on day 60 after embryo transfer.
Data were subjected to one way ANOVA followed by Fisher’s Protected Least Significant
Difference as a Post Hoc-Test using the StatView Software (Abacus Concepts Inc., Berkeley, CA,
USA).

Part I. In-Vitro Production of Buffalo Clonal Embryos through Somatic Cell Nuclear Transfer
Procedure
Buffalo clone embryos had been successfully produced in-vitro. The refinement of enucleation
technique by slit and squeeze method and simultaneous fusion and activation of couplets with electrical
stimuli followed by ethanol and cycloheximide treatment improved the development of clone embryos.
Subsequently, optimum condition for culture of clone embryos was achieved with modified synthetic
oviductal fluid (mSOF) under a 3-gas system. The improved developmental capacity of reconstructed
embryos from slit and squeeze method could be attributed to the absence of oocyte exposure to
ultraviolet light for confirmation of enucleation. The maximum time lapse where ear skin cell
proliferation activity was not reduced significantly was at 120 h post- collection (hpc), beyond 120 h,
significant reduction in the proportion of viable cells was observed. The post-thawing viability of the
ear skin fibroblasts cryopreserved with the alternative rapid freezing procedures was comparable
with that of the ultra-low temperature freezer. The successful freezing of buffalo ear skin fibroblasts
not only provided ready source of donor cells for buffalo cloning but also allowed cryobanking of
valuable genetic material for future production and research use.
Experiment 2. Transfer of Clone Embryos to Recipient Cows for In Vivo Development
One major factor influencing the success of embryo transfer in general is the synchrony
between the recipients’ estrus cycle and the developmental stage of the transferrable embryos. Part
of the major effort to improve the current success rate of embryo transfer in buffaloes is the
development of efficient ovulation synchronization instead of estrus in recipient animals. Results of
the initial trials showed 88% (15/17) ovulation rate in synchronized recipient animals and the range
of ovulation was 24 to 48 h after the second GnRH injection. The ovulation response (88%)
achieved in the present study is considerably higher as compared to the control group (79%) from
the standard protocol on PGF2 which was also characterized by a wide ovulation range. With the
558
CIDAR-SYNCH-eCG protocol, the ovulation response was more synchronous among the treated
recipient animals. As to the production of clone calves expected out of the Project, the development
of clone embryos in-vivo after several transfers made (89) had been observed to be up only to early
pregnancy stage, but failed to achieve further development to term. One of the major factors
attributing to this could be the incomplete reprogramming and epigenetic modifications occurring in
clone embryos/fetus themselves which contributed to the generally low efficiency rate of cloning
technique, which is found much lower in buffaloes than in other livestock species.
REFERENCES
Bavister, B.D., M.L. Leibfried and G. Lieberman. 1983. Development of preimplantation embryos
of the golden hamster in a defined culture medium. Biol. Reprod. 28: 235-247.
Jainudeen, M.R., Y. Takahashi, M. Nihayah and H. Kanagawa. 1993. In vitro maturation and
fertilization of swsamp buffalo (Bubalus bubalis) oocytes. Ani. Reprod. Sci. 31: 205-212.
Madan, M.L., S.K. Das and P. Palta. 1996. Application of reproductive technology to buffaloes. Ani.
Reprod. sci. 42: 299-306.
Nandi, S., H.M. Raghu, B.M. Ravindranatha and M.S. Chauhan. 2002. Production of buffalo
(Bubalus bubalis) embryos in vitro: premises and promises. Reprod. Dom. Ani. 37: 65-74.
Takahashi, Y., M. Hishinuma, M. Matsui, H. Tanaka and H. Kanagawa. 1996. Development of in
vitro matured/fertilized bovine embryos in a chemically defined medium: influence of oxygen
concentration in the gas atmosphere. J. Vet Med. Sci. 58: 897-902.
Ty, L.V., D. Chupin and M.A. Driancourt. 1989. Ovarian follicular population in buffaloes and
cows. Ani. Repro. Sci. 19: 171-178.
559
Improving Ovulation and Conception Rates in Oestrus Synchronized and

Artificially Inseminated Water Buffaloe Cows by Immunization Against Inhibin
Bahareldin-Ali ABDALLA a, Guang-Sheng QINb, Zheng-Zhun LIANGb, Ri-Hong GUOa, Hui LIc
and Zhen-Dan SHIc, b*
a
College of Animal Sciences, South China Agricultural University, Guangzhou 510642 China
b
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Guangxi Buffalo
Research Institute, Nanning 530000, China
c
Laboratory of Animal Breeding and Reproduction, Institute of Animal Science, Jiangsu Academy of
Agricultural Sciences, Nanjing, 210014, China
*Corresponding email: zdshi@jaas.ac.cn
ABSTRACT
This study aims to improve conception rate of water buffalo cows by immunization against inhibin.
Eleven multi-parity buffaloes cows were i.m. immunized with inhibin immunogen (2 mg of inhibin α
subunit protein in mineral oil adjuvant), and another 13 animals served as adjuvant treated controls.
Following Ovsynch treatment, inhibin-immunized cows showed stronger expression of oestrus (72% vs
30%, P< 0.05), and had more developing follicles, higher number of corpora lutea (0.9±0.1 vs 0.5±0.1,
P< 0.05), than did the control animals. In addition, 40 days after artificial insemination, the conception
rate was markedly higher in immunized group than that of in the control group (45.5% vs 15.4%,
P > 0.05). These results indicated that immunization against inhibin, through promoting development
and functions of ovarian follicles and corpora lutea (CL), apart from oocyte and embryo qualities, may
improve reproductive performance or conception in water buffalo cows.
Keywords: inhibin immunization, ovarian follicles, corpus luteum, conception rate, water buffaloes
INTRODUCTION
Water buffaloes are low in reproductive efficiency (Drost, 2007), which is a serious obstacle to
production and breed improvement. The low reproductive efficiency in the buffalo is expressed by
weaker oestrus behaviour display (Jainudeen et al., 1993; Ohashi, 1994; Gordon, 1996), longer
postpartum anoestrus period (El-Wishy, 2007), delayed puberty (Kandasamy et al., 1989) and lower
conception rate after service (Sá Filho et al., 2009), as compared with those in cattle. The poor
reproductive performance or reduced conception rate in buffaloes is associated with poor development
of the ovarian follicles (Vittoria, 1997), and poor oocyte developmental competence which, in turn,
also leads to poor embryo developmental competence (Nandi et al., 2002). Further, poorly developed
follicle will lead to formation of suboptimal corpus luteum, and support to embryo development
insufficient. Immunization against inhibin has been shown to enhance follicular development, to
improve luteal function and oocyte maturation quality and embryo developmental competence (Li et al.,
2011). Through the above aspects, it is likely that immunization against inhibin would improve
conception rate and reproductive efficiency in water buffaloes following oestrus synchronization and
artificial insemination.

Preparation of inhibin immunogen
recombinant inhibin  subunit fusion protein, as reported elsewhere (Mei et al., 2009). Placebo
The inhibin immunogen was prepared as the mineral oil emulsion containing 2.0 mg/ml
immunogen was prepared with physiological saline homogenized with mineral oil adjuvant.
560
Animals and treatments

The animal trial was carried out in late spring of May 2012 when buffalo reproductive activity was
getting low. Twenty four multi-parity (Crossbred Murrah-Swamp) buffalo cows of 4-6 years of age,
which expressed sound reproductive performance and had calved normally in the previously 6 months,
were randomly assigned into immunized (n=11) and non-treated control (n=13) groups. Both groups
of animals underwent a conventional Ovsynch hormone treatment program. On day 1 of the program
after the first i.m. injection of GnRH, immunized buffaloes were administered with a single i.m.
injection of 1 ml of the immunogen, while the control animals were given the placebo injection.
Throughout the trial, all the animals were raised in a hygienic environment, well fed and had free
access to fresh and clean water.
Detection of oestrus expression
Oestrus sign was visually observed from the day after the Ovsynch program. Expression of oestrus
behaviours were recorded for occurrence of the time and duration, and the scores recorded according to
the criteria given by Lyimo et al., (2000) and Ohashi (1994). Behavioral displays of oestrus were
further confirmed with additional observations such as swelling of vulva, mucus discharge, and present
of large follicle >9 mm through ultrasonic scanning.
AI and conception diagnosis
Within 12-24 h following observation of oestrus expression, buffaloes showing external oestrus
behaviours were artificially inseminated with approximately 4x106 sperm cells of frozen-thawed semen
from a single bull of known fertility. Pregnancy was confirmed by ultrasonography 40 days after
service for determining conception rate.
Ultrasonograph
Transrectal ultrasonography was performed using a real-time ultrasound scanner (Aloka HS-101V;
Honda, Tokyo, Japan), with a 5.MHz rectal transducer probe. The number of follicles present on the
ovaries with diameter in various size class as small (≥2- 4 mm), medium (5-9 mm) and large (≥10 mm)
were recorded on days 1 and 10 of the Ovsynch program. Additionally, on day 20 after onset of the
program, or 9 days after AI when development of CL was maximal, ultrasonography of presence and
number of corpora lutea on the ovary was carried out, to reveal ovulation rate and corpora lutea size.
Statistical analyses
One-way analysis of variance (ANOVA) was used to compare both number and size of follicles,
oestrus display, diameter of CL, between immunized and control animals. Percentages of animals
showing oestrus behavior, functional CL, and conception rate, were analysed by student t-test.
RESULTS
Oestrus behaviour expression
Higher percentage of immunized buffaloes showed intensive external signs of various oestrus
behaviours than did the controls (72±14 vs 30±13, P< 0.05) (Table 1). The oestrus behaviours occurred
earlier time in the former than in latter group of buffaloes after the Ovsynch program (19.6 h vs 15.6 h,
P< 0.05), so was the duration of display of oestrus behaviour (14.3 h vs 8.1 h, P>0.05).
Ovarian follicular development
There was no significant differences (P>0.05) between two experimental groups of animals
regarding follicles number within small, medium and large size classes on day 10 of Ovsynch program,
which tended to be higher in immunized group for the small size follicles (data not shown).
Ovulation and conception rates
Under observation on 9 days after AI, the immunized group had higher number of corpora lutea
than did the controls (0.9±0.1 vs 0.5±0.1, P> 0.05). The conception rate as checked on day 40 after AI
were numerically but, not statistically, higher in the immunized than in the control cows (45.5% vs
15.4%, P =0.115) (Table 2).
561
DISCUSSIONS
This study was carried out to test the hypothesis that immunoneutralization against inhibin could
improve oestrus expression and conception rate in water buffalo, by means of stimulating ovarian
follicular development and follicular function, enhancing oocyte maturation quality and the post-
fertilization embryonic development competence. Results obtained so far tended to support the
hypothesis, that a higher portion of the inhibin immunized buffaloes displayed intense oestrus
behaviours, ovulated and established pregnancy after artificial insemination.
Though the single immunization used in this study did not substantially increased number of
ovarian follicles of different size classes, improved follicular function was seen as more immunized
buffaloes exhibited external signs and behavioral displays, which also occurred earlier and lasted
longer. Earlier occurrence of oestrus behaviours were also observed in inhibin immunized Holstein
cows (Li et al., 2009). Since oestrus behaviours are driven to occur by rising secretions of oestradiol,
while anti-inhibin antibody could stimulate oestradiol production by granulosa cells (Jimenez-Krassel
et al., 2003), it is possible that immunized buffaloes secrete more amount of oestradiol after undergoing
Ovsynch program, in other words, had enhanced follicular function.
Besides, more inhibin immunized buffaloes ovulated, which also suggested advanced maturation
of the dominate follicles. About 50% of ovulated cows in the immunized group successively conceived
compared with only about one third in the control group. Such result indicated both oocyte quality and
the subsequent embryo development, and the condition of the reproductive tract supporting early
embryo development, must have been improved in the immunized cows. Our previous work had shown
that anti-inhibin antibody substantially improved oocyte maturation and early embryo development
quality (Li et al., 2011), which could partly account for the higher conception rate in the immunized
cows, together with the augment luteal function that should follow after inhibin immunization (Medan
et al., 2004; Li et al., 2011).
In summary, the above results obtained in the present study need to be further verified by using
larger numbers of animals, and at different levels of reproductive activities in the summer and also
autumn to winder months, and together with data of hormonal changes.
ACKNOWLEDGEMENTS
This work was supported by Guangxi Provincial Key Laboratory of Water Buffalo Genetics,
Breeding and Reproduction Open Grant SNKF-2012-04, National Science and Technology Research
and Development Grant 2011BAD19B02-6.
REFERENCES
Drost, M. 2007. Advanced Reproductive Technology in the Water Buffalo. Theriogenology 68(3):450-
453.
El-Wishy and A. B. 2007. The Postpartum Buffalo. II. Acyclicity and Anestrus. Anim. Reprod. Sci.
97(3-4):216-236.
Gordon, I. 1996. Controlled Reproduction in Cattle and Buffaloes 1st ed. CAB International,
Willingford, UK.PP.
Jainudeen, M.R. and Hafez E. S. E. 1993. Cattle and buffalo. In: Hafez, E. S. E. (Ed.)
Reproduction in Farm Animals, 6th ed. Lea and Febiger, Philadelphia, USA, pp. 315–329.
Jimenez-Krassel, F., M.E. Winn, D. Burns, J.L. Ireland, and J.J. Ireland. 2003. Evidence for a Negative
Intrafollicular Role for Inhibin in Regulation of Estradiol Production by Granulosa Cells.
Endocrinology 144(5):1876-1886.
Kandasamy, N., V. Ulaganathan, and A.R. Krishnan. 1989. Prenatal Mortality, Sex Ratio and Herd
Life of Murrah Buffaloes in Tamil Nadu. Indian. Journal. Dairy. Science 42:625-626.
562
Li, C., Y.L. Zhu, J.H. Xue, S.L. Zhang, Z. Ma, and Z.D. Shi. 2009. Immunization against Inhibin
Enhances Both Embryo Quantity and Quality in Holstein Heifers after Superovulation and
Insemination with Sex-Sorted Semen. Theriogenology. 71(6):1011-1017.
Li, D.R., G.S. Qin, Y.M. Wei, F.H. Lu, Q.S. Huang, H.S. Jiang, D.S. Shi, and Z.D. Shi. 2011.
Immunisation against Inhibin Enhances Follicular Development, Oocyte Maturation and
Superovulatory Response in Water Buffaloes. Reprod. Fertil. Dev. 23(6):788-797.
Lyimo, Z.C., M. Nielen, W. Ouweltjes, T.A. Kruip, and F.J. van Eerdenburg. 2000. Relationship
among Estradiol, Cortisol and Intensity of Estrous Behavior in Dairy Cattle. Theriogenology
53(9):1783-1795.
Medan, M.S., S. Akagi, H. Kaneko, G. Watanabe, C.G. Tsonis, and K. Taya. 2004. Effects of Re-
Immunization of Heifers against Inhibin on Hormonal Profiles and Ovulation Rate.
Reproduction. 128(4):475-482.
Mei, C., M.Y. Li, S.Q. Zhong, Y. Lei, and Z.D. Shi. 2009. Enhancing Embryo Yield in Superovulated
Holstein Heifers by Immunization against Inhibin. Reprod. Domest. Anim. 44(5):735-739.
Nandi, S., H.M. Raghu, B.M. Ravindranatha, and M.S. Chauhan. 2002. Production of Buffalo (Bubalus
Bubalis) Embryos in Vitro: Premises and Promises. Reprod Domest Anim 37(2):65-74.
Ohashi, O.M. 1994. Estrus Detection in Buffalo Cow. Buffalo. J. 10(Suppl 2): 61-64.
Sa Filho, M.F., N.A. Carvalho, L.U. Gimenes, J.R. Torres-Junior, L.F. Nasser, H. Tonhati, J.M. Garcia,
B. Gasparrini, L. Zicarelli, and P.S. Baruselli. 2009. Effect of Recombinant Bovine
Somatotropin (Bst) on Follicular Population and on in Vitro Buffalo Embryo Production. Anim.
Reprod. Sci. 113(1-4):51-59.
Vittoria, A. 1997. Third Course on Biotechnology of Reproduction in Buffaloes, Supplement. Bubalus.
bubalis. 4:15-20.
Table 1. Various scores of expressions of oestrus behaviours in immunized and control water
buffaloes (Mean ± SEM or percentage data).
Signs of oestrus Immunized Control P-value

No. of buffaloes 11 13
Onset of oestrus signs (h) 15.9±0.5 19.6±0.3 0.008
Duration of oestrus (h) 14.3±4.8 8.1±0.3 0.37
Total intensive oestrus expression (%) 7 (72) 4 (30) 0.04
Vulva sniffed (%) 8 (72.7) 3 (23) 0.01
Flehman (%) 8 (72.7) 4 (31) 0.04
Mounted by cow or bull (%) 6 (54.5) 2 (15.4) 0.04
Restlessness (%) 9 (81.8 ) 4 (31) 0.01
Mucous vaginal discharge (%) 9 (81.8) 5 (38.5) 0.03
Table 2. Number and size of corpora lutea present on the ovary on day 9 after AI in immunized and
control buffalo cows (Mean ± SEM).
Variable Immunized Control P-value

No. of buffaloes 11 13
No. of corpora lutea 0.9±0.1 0.5±0.1 0.04
Conception rate % 45.5 (5) 15.4 (2) 0.11
563
Uterine Microbial Flora of Nili-Ravi Buffalo during Estrus and its Relationship
with Pregnancy Rate in Pakistan
Sohail RAZA, a Masood RABBANI, a* Nasim AHMAD, b Ali Ahmad SHEIKH, a Khushi
MUHAMMAD, c Fareeha AKHTARa and Habib Ur REHMANa
a
University Diagnostic Lab, University of Veterinary and Animal Sciences Lahore 54000 Pakistan.
b
Department of Theriogenology, University of Veterinary and Animal Sciences Lahore 54000 Pakistan.
C
Department of Microbiology. University of Veterinary and Animal Sciences Lahore 54000 Pakistan.
*Corresponding email: mrabbani@uvas.edu.pk
ABSTRACT
Microbial flora of body plays a pivotal role in protecting body against infection and enhances
its ability to compete pathogens. Some of the microorganisms are beneficial for the uterine body and
some are detrimental. Presence of Actinomyces pyogenes and Fusobacterium necrophorum are reported
to be important pathogens causing metritis with lower conception rate and increase calving intervals. In
order to check the presence of microbes in Nili Ravi buffaloes and its affect on the pregnancy rate, a
trial was conducted on 50 buffaloes from an organized dairy farm. Samples were collected using
specially prepared devise just before artificial insemination and transferred to the lab for bacterial
identification. Two to three months later, pregnancy of the sampled animals was checked. More than
173 strains belonging to six different genera identified as Escherichia coli (E. coli), Lactobacillus spp.,
Micrococcus spp., Staphylococcus spp., Citrobacter spp. and Proteus spp. were identified. Among
these, E. coli was most prevalent (100 %) followed by S. aureus (98 %), Lactobacillus (86 %),
Micrococcus (26 %), S. epidermidis (14 %), Proteus (12 %), Citrobacter (10 %). Relationship of
bacterial species with pregnancy rate then found that 4 animals aborted in first trimester were having
significantly high number (p< 0.05) of Citrobacter species. It is concluded that E.coli, Staph. aureus, S.
epidermidis and Lactobacillus species are normal microbial flora of Nili-Ravi buffalo Proteus species
and Micrococcus species are mostly present in pregnant animals while presence of Citrobacter species
is detrimental for the pregnant animals and may lead to abortion.
Keywords: Bio-film, Uterine flora, Nili-Ravi, Pregnancy rate.
INTRODUCTION
Microbial flora of body plays a significant role in protection against infectious diseases (Reid et al.,
1998). Various bacteria can be isolated from uterine body of normal cyclic animals which includes
Escherichia coli, Staphylococcus spp., Bacillus spp., Yersinia enterocolitica, Citrobacter diversus,
Bacillus spp., Micrococcus spp. (Ahmed et al., 2007; Azawi et al., 2007; El-Jakee et al., 2008 and Gani
et al., 2008). Lactobacillus spp. proved beneficial while Actinomyces pyogenes and Fusobacterium
necrophorum causes metritis and other uterine diseases (Azawi et al., 2008). These pathogens cause
low fertility, hormonal imbalance and stoppage of ovulation (Sheldon et al., 2004). Uterine infections
in buffalo are much higher as compare to cows (Jainudeen, 1986). Keeping in view, this study was
designed to investigate microbial flora of uterus in buffalo during estrus and to evaluate their impact on
pregnancy rate.

Sampling
A total of 50 uterine fluid/secretion samples of Nili-Ravi buffaloes during estrus period were
collected using sterilized artificial insemination (AI) rod fitted with disposable syringe as per protocol

564
mentioned earlier (Williams et al., 2007). The samples were transported to University Diagnostic Lab,
University of Veterinary & Animal Sciences, Lahore, Pakistan for further processing. A complete
clinical history and management of each animal was also recorded. All animals were dewormed and
vaccinated as per schedule.
Isolation and Identification of the bacteria
Primary isolation of the bacteria was carried using blood agar. Bacterial colonies were separated
and identified biochemically following instructions to Burgey’s Manual. Bacterial confirmation was
also done using API Kits.
Pregnancy rate
Rectal palpation technique for pregnancy status of these animals was also performed after 2-3
months post insemination.
Data was tabulated using Microsoft Excel (MS Excel 2007, Microsoft Corporation, Redmond,
WA). Descriptive Statistical analysis like percentages, confidence interval and graphs were also
developed using commercially available statistical package SPSS (Version 18, for Windows; SPSS,
Chicago, IL). Statistical association of pregnancy status between the presence and absence of certain
isolated micro flora was calculated by using Fisher Exact Test using SPSS.

All uterine samples (100 %) were positive for presence of various bacteria. A total of 173 bacterial
isolates belonging to six genera such as E. coli, Lactobacillus spp., Micrococcus spp., Staphylococcus
spp., Citrobacter spp. and Proteus spp. were recovered (Table-1). The most prevalent bacterial isolates
in both pregnant and non pregnant animals were E. coli, Lactobacillus spp., and S. aureus, while
Citrobacter spp. were prevalent in aborted buffaloes. S.epidemidis, Micrococcus spp. and Proteus spp.
were prominently observed in the pregnant animals than non pregnant. Similar results were observed
by other researchers who have identified E. coli, Staphylococcus spp., Bacillus spp., Yersinia
enterocolitica, C. diversus, Bacillus spp., Micrococcus spp. from uterus (Ahmed et al., 2007; Azawi et
al., 2007; El-Jakee et al., 2008 and Gani et al., 2008).
E. coli (100%) was recovered from pregnant, non pregnant and aborted animals. E. coli is the most
commonly isolated bacteria from bovine uterus of normal and puerperal animals (William et el., 2007).
E. coli was isolated from normal pre-pubertal buffaloes (Torres et al., 1997 and El-Jakee et al. 2008).
Hanafi et al. (2008) studied that prevalence of E. coli in healthy animals was 71. 21% and in
endometric animals it was 18.75%. These studies further supported the results of current research that
E. coli present in both healthy and diseased animals. Statistical analysis reveals that there is no
significant (p=0.9) relationship in pregnancy rate with presence of E. coli in uterus.
Prevalence of S. aureus in this study was recorded 98% with no significant relationship with
pregnancy rate statistically (p=0.84). Previous findings also indicate that S. aureus, being opportunistic
organism, is the most common bacterial isolates of Nili Ravi buffaloes and usually associated with
endometritis (Usmani et al., 2000 and William et al., 2007). Prevalence of Lactobacillus spp. and
Micrococcus spp. in this study was 86% and 26%, respectively with no statistically significant
relationship with pregnancy rate (p=0.289 and p=0.117, respectively). Prevalence of both organisms
was more in pregnant animals as compare to non pregnant animals. Previous study also indicates the
high prevalence of both of these organisms in normal animals than in animals with ovarian inactivity
(Ahmad et al., 2007, Abd El-Moeez et al., 2008 and Hanafi et al., 2008). The isolation of S.
epidermidis and Proteus spp. was almost same prevalence from pregnant and non pregnant animals in
this study. Ahmad et al., 2007 concluded that S. epidermidis is mostly isolate from pregnant animals.
Proteus spp. is most prevalent organism of the normal purpeural animals. Proteus spp. is an
opportunistic contaminant of uterus not usually associated with endometritis (Jadon et al., 2005).
Prevalence of Citrobacter spp. was 10% in total samples, out of which 80% was present in aborted
565
animals and 10% in non-pregnant animals. Previous study suggested that Citrobacter spp is co-related
with the presence of severe endometrial lesions (Messier et al., 1984). Citrobacter spp. causes the
sporadic abortion in bovines which is also evident from current study as there exist a significant
relation (p=0.0001) between presence of Citrobacter spp. and pregnancy rate.
After insemination, 68% of the animals became pregnant, 24% remained non pregnant and 8%
were aborted. Reproductive disease history showed that 30 animals had encountered the reproductive
disease. Eight animals had the problem of anoestrus, out of which seven became pregnant and one
remain non pregnant. Thirteen animals had the problem of metritis, out of which 3 became non
pregnant, one aborted and 9 became pregnant. Six animals had the problem of inactive ovaries, out of
which 5 became pregnant and one remained non pregnant. Two animals had the problem of pyometra,
out of which one became non pregnant and other aborted. One non-pregnant animal showed the
problem of retained placenta. It has been observed in this study that most of the animals which had
problem of anoestus became pregnant. Results of this study show that animals which had previous
problem of metritis (25 %) were non pregnant while 7.6 % aborted and 68.4 % became pregnant. In the
animals having history of inactive ovaries, 16.6 percent became non pregnant while 83.34 % became
pregnant and the animals showing the problem of pyometra before, 50 % of them became non pregnant
and 50 % aborted. Animal which had the problem of retained placenta remained non pregnant.
CONCLUSIONS
From the current study, it was concluded that Citrobacter spp.may be linked with problems related
to reproductive system of Nili-Ravi buffalo. Furthermore anoestrus, metritis and inactive ovaries are
not fatal for the uterine health. Pyometra and retained placenta diseases are most fatal for the
reproductive health of the animals especially in Nili-Ravi buffaloes.
As this is a preliminary study for the isolation of bacteria from uterus of Nili-Ravi buffalo, there is a
need to establish metagenomic study to figure out other microbiotas which are difficult to isolate using
conventional isolation techniques and relate them with pregnancy rate of this precious animal.
REFERENCES
Abd El-Moeez, S.I., W.M. Ahmed, J. A. El-Jakee and F.R. El-Seedy. 2008. Observation on
lactobacillus spp. In the genetal tract of buffaloe-cows with emphasis on its in vitro probiotic
activity. Global Veterinaria 2(1): 15-21.
Ahmed, W.M, J.A. El-Jakee, F.R. El-Seedy, K.I. El-Ekhnawy and S.I. Abd El-Moez. 2007. Vaginal
bacterial profile in buffalo-cows in relation to ovarian activity. Global Veterinaria 1(1): 01-
08.
Azawi, O. I., S. N. Omran and J. J. Hadad. 2007. Clinical, Bacteriological and Histopathological Study
of Toxic Puerperal Metritis in Iraqi Buffalo. J. Dairy Sci. 90: 4654-4660.
Azawi, O.I., M.A. Rahawy and J.J. Hadad. 2008. Bacterial Isolates Associated with Dystocia and
Retained Placenta In Iraqi Buffaloes. Reprod. Dom. Anim. 43: 286–292.
El-Jakee, J.A., W.M. Ahmed, F.R. El-Seedy. and S.I. Abd El-Moez. 2008. Bacterial profile of the
genital tract in female buffaloes during different reproductive stages. Global. Veterin. 2 (1): 7-
14.
Gani, M.O., M.M. Amin, M.G.S. Alam, M.E.H. Kayesh, M.R. Karim, M.A. Samad and M.R.
Islam.2008. Bacterial flora associated with repeat breeding and uterine infections in dairy
cows. Bangl. J. Vet. Med. 6(1): 79-86.
Hanafi, E.M., W.M. Ahmed, S.I. Abd-El-Moez, H.H. El-Khadrawy and A.R. Abd-El-Hameed. 2008.
Effect of Clinical Endometritis on Ovarian Activity and Oxidative Stress Status in Egyptian
Buffalo-Cows. Ameri-Euras. J. Agric & Environ Sci. 4(5): 530-536.
566
Jadon, R.S., G.S.Dhaliwal, S.K. Jand. 2005. Prevalence of aerobic and anaerobic uterine bacteria
during peripartum period in normal and dystocia-affected buffaloes. Animal Rep. Sci. 88:
215–224.
Jainudeen, M. R. 1986. Reproduction in water buffalo. In: Morrow DA (ed), Current Therap in Therio.
W.B. Saunders, Philadelphia, PA, pp: 443–449.
Messier, S., R. Higgins, Y. Couture and M. Morin.1984. Comparison of Swabbing and Biopsy for
Studying the Flora of the Bovine Uterus. Can. Vet J. 25: 283-288.
Reid, B.A., A.W. Bruce and V. Sameianov. 1998. The role of Lactbacillus in preventing urogenetal
and intestinal infections. Int. Dairy J. 8: 555-562.
Sheldon, I.M. and H. Dobson 2004. Postpartum uterine health in cattle. Ani. Rep. Sci. 82-83: 295-306.
Torres, E. B., T. Nakao, T. Hiramune, M. Moriyoshi, K. Kawata and K. Nakada. 1997. Stress and
uterine bacterial flora in dairy cows clinically normal and abnormal puerperium. J Rep & Dev
43(2): 157-163.
Usmani, R.H., N. Ahmad, P. Shafiq and M.A. Mirza. 2000. Effect of subclinical uterine infection on
cervical and uterine involution, estrus activity and fertility in postpartum buffaloes.
Williams, E.J., D.P. Fischer, D.E. Noakes, G.C.W. England, A. Rycroft, H. Dobson and I.M
Sheldon.2007. The relationship between uterine pathogen growth density and ovarian
function in the postpartum dairy cow. Theriogenology 68: 549-559.
Table- 1: Prevalence of uterine microbial flora in each stage of pregnancy of Nili-Ravi buffalo.
Name of Bacteria Prevalence Total Pregnant Total Non-Pregnant Total Pregnant then
No. (%) No. / % No./ % Aborted No./ %
E. coli 50 (100) 34/68 12/24 4/8
S. aureus 49 (98) 34/69.38 11/22.44 4/8
Lactobacillus spp. 43/86 28/65.12 11/25.58 4/9.3
Micrococcus spp. 13/26 12/92.30 1/7.69 0/0
S. epidermidis 7/14 4/57.14 2/28.57 1/14.28
Proteus spp. 6/12 4/66.67 2/33.33 0/0
Citrobacter spp. 5/10 0/0 1/20 4/80
567
Reproductive Performance of Murrah Buffaloes under

Intensive Farming System in Thailand
Thuchadaporn CHAIKHUNa, Ranchuan HENGTRAKUNSINb and Jamlong

MITCHAOTHAIc*
a
b
Murrah Dairy Company Limited, Plangyao, Chachengsao 24190. Thailand
c
Clinic of Swine, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok,
10530, Thailand
*Corresponding e-mail: jmitchaothai@yahoo.com
ABSTRACT
The evaluation of reproductive performance is an important protocol for the analyzing and
planning of dairy buffalo farm production. The reproductive data of ninety-six Murrah buffaloes,
which were raised under intensive farming management in Thailand, between year 2003 and 2011
were evaluated as mean±SD and statistical analysed on reproductive indices. The results showed
that the age at first calving was 45.9 ± 5.8 months (n=22). The average calving interval was 487.5 ±
149.5 days (n=215) but the longest interval (565.3 ± 158.8 days; n=75) occured in primiparous
cows (P < 0.05) when compared with multiparous cows. The high percentages of calving cows were
mainly found in August, September and October (11.2%, 19.3% and 16.8%, respectively; n=322).
In conclusion, the Murrah buffaloes in this study, which were raised in an intensive farm
environment, had low reproductive performances which suggest that the age at first calving and the
calving interval in primiparous cows should be improved. Additionally, the results from this study
imply that the most favorable season for conception in Murrah buffaloes in Thailand is the winter
season.
Keywords: reproductive performance, intensive farming system, Murrah buffaloes
INTRODUCTION
Historically, buffalo husbandry in Thailand has involved swamp buffaloes raised by small
family units using free range grazing methods and natural spontaneous mating. Buffaloes have
primarily been used as draught animals and for meat production (Chaikhun et al., 2012). In the
present decade, only one intensive buffalo farm in Thailand produces dairy products from Murrah
and swamp buffaloes. Utilizing Murrah and swamp buffaloes for large scale, commercial dairy
production is a new industry in Thailand and the market for these products is just being developed.
The evaluation of reproductive performance is an important protocol for analysis and planning in
this newly emerging dairy buffalo farm industry. There are, however, few reports on the
reproductive performance of Murrah buffaloes in Thailand – and none done under intensive dairy
farming conditions. The objective of this study was to investigate the reproductive performance of
Murrah buffaloes in an intensive dairy farm setting.

Animals were raised under intensive farming management in Chachoengsao province,
eastern Thailand. The buffaloes were divided into 8 units depending on their status, i.e. new born
calves (1st month), calves (between 1-12 months), heifers (13-30 months), heat detection and
service cows, milking cows, late gestation period cows, male buffaloes (13-30 months) and bulls.
Feeding management was done according to a cut-and-carry or zero grassing system with
concentrated supplements. The buffaloes were fed in mangers in their barns, with different qualities
(percent of protein) and quantities, according to their respective units and milk yield (for milking

568
cows). They were checked for health status and disease annually and were vaccinated for foot and
mouth disease and hemorrhagic septicemia by veterinarians in a health monitoring program. The
buffaloes at this farm were bred by both natural mating and by artificial insemination. Reproductive
data such as birth date, date of first calving, and date of calving were collected from ninety-six
Murrah buffaloes between years 2003 and 2011. The age at first calving and calving interval were
evaluated as mean ± SD. Survival analysis was performed to detect differences in survival
probability based on calving intervals and buffalo parities at P < 0.05. Frequency of calving in each
month was calculated.

The results in our study found that the age at first calving was 45.9 ± 5.8 months (n=22)
(Table 1) which was higher than Murrah buffaloes in Brazil (39.05 ± 6.95 months) (Vasconcellos
and Tonhati, 1998) but lower than Sri Lanka (51 ± 8.6 months), South India (52.49 ± 0.34 months)
and Bangladesh (57.8 months) (Lundstrom et al., 1982; Alam and Ghosh, 1993; Prasad and Prasad,
1998). Campanile et al. (2001) suggested that nutritional management (from the time of weaning
and during the pre-pubertial period) has a particular influence on the age and body weight at first
conception in buffalo heifers. Similarly, feeding level and energy level of diet also have an effect on
growth and on body and sexual development and the onset of puberty, as stated by the authors who
did research on Murrah in India (Kaur and Arora, 1989). Bodhipaksha (2006) reported swamp
buffaloes in Surin province in northeastern Thailand had 58.4 ± 13.4 months age at first calving.
The discrepancy with the results of this current study could be the result of differences in breed and
nutritional level supplementation
The average calving interval was 487.5 ± 149.5 days or (n=215) (Table 1) which was lower
than Murrah buffaloes in Bangladesh and South India (about 545 days) (Alam and Ghosh, 1993;
Kandasamy et al., 1993). In Brazil, the calving interval (385 ± 53.4 days) is shorter than our study
(Vasconcellos and Tonhati, 1998). In the current study, when the calving interval of the first parity
buffaloes was excluded from calculation, the calving interval of the second to sixth parity was 445.8
± 126.3 days. This implied an influence of the first parity on reproductive performance. The longest
interval in our study (565.3±158.8; n=75; P < 0.05) (Figure 1) was in first parity cows which is
similar to reported calving intervals of Murrah buffaloes in India (Kandasamy et al., 1993). This
could be caused by negative energy balance, inappropriate nutritional status, and poor functioning
of the reproductive system in first parity cows as we found in our earlier study of swamp buffaloes
in the same farm (Chaikhun et al., 2012). The factors effecting the calving interval are: the
individual cow, farm management, parity, and the year and season of conception (Lundstrom et al.,
1982; Alam and Ghosh, 1993; Kandasamy et al., 1993; Abayawansa et al., 2011). In addition,
uncontrollable factors can obviously have an effect on reproductive status in intensive farming
operations such as: price and availability of feed, diseases and climate (Chaikhun et al., 2012). In
the present study, there was also a trend towards shorter calving intervals with higher parity (Figure
1); although we cannot state this definitively due to the low number of studied buffaloes at parity 5
and 6. Thus, improving reproductive performance in relation to the onset of puberty and the first
parity might lead to better reproductive performance later , in buffaloes with higher parities.
The proportions of calving cows in this study were high in August, September and October
(11.2%, 19.3% and 16.8%, respectively; n=322) while rather low proportions of calving cows were
found in March, April and May (Figure 2). This pattern was rather close to the report of Borghese et
al. (1993) who found the highest calving frequency in August-September and the lowest calving
frequency in May and June. The average gestation period of buffalos is 310 days with variations
due to maternal, fetal, genetic and environmental factors (Jainudeen and Hafez, 2000). Chaikhun et
al. (2012) have reported a gestation period of 321.4 ± 11.3 days in an intensive dairy buffalo farm in
Thailand. This suggests that the buffaloes calving in August – October started their pregnancy
between September and December, which is the start of winter season (which follows the wet
season in Thailand). Similarly, Sule et al. (2001) and Chaikhun et al. (2012) also stated that winter
appeared to be the most favorable season while summer seems to be the most unfavorable season
569
for buffalo reproduction. This is most likely related to the availability of food (especially in the wet
season) - the larger supply of grass at this time provides better nutrition, thus helping postpartum
cows to return to estrous cycle and become pregnant in the winter season (Vale et al., 1990;
Chaikhun et al., 2012).
In conclusion, the Murrah buffaloes in this study, which were raised in an intensive farm
environment, had low reproductive performances suggesting the age at first calving and the calving
interval in primiparous cows should be improved. Additionally, this study indicates that the most
favorable season for conception in Murrah buffalo in Thailand is the winter season.
ACKNOWLEDGEMENT
For English language editing and review, the authors wish to thank to Mr. Philippe Marcou
for his professional assistance.
REFERENCES
Abayawansa, W.D., S. Prabhakar, A.K. Singh and P. Brar. 2011. Effect of climatic changes on
reproductive performance of Murrah buffaloes in Punjab: A retrospective analysis. Indian J.
Anim. Sci. 81(4): NP.
Alam, M.G.S. and A. Ghosh. 1993. Reproductive patterns of rural buffaloes (Bubalus bubalis) in
Bangladesh. Buffalo Bull. 12(3): 66-69.
Bodhipaksha, P. 2006. Reproductive physiology and hormones of female swamp buffaloes. In:
International course of buffalo reproduction and reproductive biotechnology. 3rd ed. C.
Lohachit, M. Techakumphu, S. Sirivaidyapong, T. Tharasanit and O. Cheunsuang (eds).
Chulalongkorn University. Bangkok. 36-38.
Borghese, A., V.L. Barile, M.G. Terzano, G. Annichiarico, A. De Benedetti and A. Malfatti. 1993.
Anoestrous length in Italian buffalo cows. Note I. In: Proceedings of the International
Symposium "Prospect of Buffalo Production in the Mediterranean and in the Middle East",
Cairo, Egypt, 912 November 1992. EAAP Publication, Pudoc, Wageningen, n. 62: 389-392.
Campanile, G., R. Di Palo, B. Gasparrini, M.J.D. Occhio and L. Zicarelli. 2001. Effect of early
management system and subsequent diet on growth and conception in maiden buffalo heifer.
Lives. Prod. Sci. 71: 183-191.
Chaikhun, T., R. Hengtrakunsin, F. De Rensi, M. Techakumphu and S. Suadsong. 2012.
Reproductive and dairy performances of Thai swamp buffaloes under intensive farm
management. Thai J. Vet. Med. 42(1): 81-85.
Jainudeen M.R. and E.S.E Hafez. 2000. Gestation, prenatal, physiology, and parturition. In:
Reproduction in Farm Animals (7th edition, Ed. B. Hafez and E.S.E. Hafez). Lippincott
Williams & Wilkins, Maryland, USA. pp. 140-155.
Kandasamy, N., V.U. Iagaiathan and A.R. Krishnan. 1993. Non-genetic factors affecting calving
interval and dry period of Murrah buffaloes. Buffalo Bull. 12(3): 63-64.
Kaur, H. and S.P. Arora. 1989. Growth and puberty as influenced by the plan of nutrition in Murrah
buffaloes. Buffalo J. 1: 57-64.
Lundstrom, K., H. Abeygunawardenar, L.N.A. De Silva and B.M.A.O. Perera. 1982. Environmental
influence on calving interval and estimates of its repeatability in the Murrah buffalo in Sri
Lanka. Anim. Reprod. Sci. 5(2): 99-109.
Prasad, S. and R.B. Prasad. 1998. Measures of reproductive estimates in rural buffalo herds of
Meerut district of Uttar Pradesh (India). Buffalo Bull. 17(2): 27-29.
Sule, S.R., A.L. Taparia, L.S. Jain and S.P. Tailor. 2001. Reproductive status of Surti buffaloes
maintained under sub-humid conditions of Rajasthan. Indian Vet. J. 78: 1049-1051.
Vale, W.G., O.M. Ohashi, J.S. Sousay and H.F.L. Ribeiro. 1990. Studies on the reproduction of
water buffalo in the Amazon basin. In: Livestock reproduction in Latin America.
Proceedings of the Final Research Coordination Meeting. Bogotà, Colombia, Sept. 19-23,
1988. IAEA, Vienna. p 201–210.
570
Vasconcellos, B.F. and H. Tonhati. 1998. Inbreeding and its effects on some productive and
reproductive traits in a Murrah buffalo herd. J. Anim. Breeding and Genetics. 115: 299-306.
Table 1. Reproductive performances of Murrah buffaloes under intensive farm management in Thailand,
between year 2003 and 2011
Performance index Mean ± SD Number of data
Age at first calving (months) 45.9 ± 5.8 22
Calving interval (days)
Parity 1 565.3 ± 158.8 75
Parity 2 470.3 ± 157.2 54
Parity 3 444.4 ± 123.0 40
Parity 4 416.0 ± 63.6 25
Parity 5 439.0 ± 101.8 14
Parity 6 385.6 ± 32.8 7
Overall 487.5 ± 149.5 215
Figure 1. Plot of calving interval survivor functions of the buffaloes during the period of 2003 to 2011
(*Significant different at P < 0.05 from other parities)
Figure 2. Percentage of calving cows during the period of 2003 to 2011 (n = 322)
571
Fetal Loss in Dairy Buffaloes in Eastern of Thailand
Thuchadaporn CHAIKHUN,a Ranchuan HENGTRAKUNSINb and Jatuporn KAJAYSRIa*

a
b
Murrah Dairy Company Limited, Plangyao, Chachengsao 24190. Thailand
*Corresponding e-mail: jatuporn@mut.ac.th
ABSTRACT
Fetal loss can be a significant problem affecting individual animal productivity and livestock
farm production in general. The objective of this study was to evaluate fetal loss in buffaloes which
were raised in an intensive dairy buffalo farm located in Chachoengsao province, Thailand. This
retrospective study was carried out in 228 pregnant buffaloes, which were inseminated by natural
and artificial mating, between the years 2003 and 2011. Fetal loss in this study was divided into two
categories: abortion (termination of pregnancy on ≥ 90 days after mating) and stillbirth (the fetus
was born dead or the calf died within 3 days of birth). The results showed that the total fetal loss
was 7.19% (n=20/228) of which 4.32% (n=12/228) were due to abortion and 2.88% (n=8/228) to
still birth. The rate of fetal loss was overall relatively consistent although it did show a small
seasonal variation - being slightly higher in the rainy season (between June and October). In
conclusion, the fetal loss rate in this research was comparatively low and in the buffalo farming
industry generally considered in the acceptable range. However, proper farm management of
pregnant buffalo and the careful monitoring of their health can effect fetal loss and should be
considered throughout the year - especially in the rainy season.
Keywords: fetal loss, abortion, stillbirth, buffalo
INTRODUCTION
Fetal loss can be a significant problem affecting individual animal productivity and livestock
farm production in general. In dairy cattle, a fetal loss or abortion rate of 3-5% annually is
considered (Forar et al., 1996). In buffalo, most reports concern embryonic loss (Campanile and
Neglia, 2007) - there are few reports on fetal loss due to abortion and still birth. The objective of
this study was to evaluate fetal loss in buffaloes in dairy farm in Thailand.

The buffalo in both Murrah and swamp types were raised in an intensive dairy buffalo farm
located in Chachoengsao province, Thailand. The buffaloes were inseminated by natural and
artificial mating. The recorded data was carried out in 228 pregnant buffaloes between the years
2003 and 2011. Fetal loss in this retrospective study was divided into two categories: firstly,
abortion means termination of pregnancy on ≥ 90 days after mating and secondly, still birth means
the fetus was born dead or the calf died within 3 days of birth.

The results in this study showed that the total fetal loss was 7.19% (n=20/228) of which
4.32% (n=12/228) were due to abortion and 2.88% (n=8/228) to still birth. There is a report in rural
buffalo herds in India between the year 1995 and 1996 found that the abortion rate and still birth
rate were 4.04% (n=121/2,989) and 0.06% (n=2/2,989), respectively (Prasad and Prasad, 1998).
This is lower than in our study and might be due to the different data collection method used (their
study relied on memory and was based on interviews, ours used written reports) and the farming
management system applied to the test population (their study was based on conventional rural farm
settings, ours on buffaloes in an intensive farming system). In dairy cattle under intensive farming
systems in the northwestern United State was reported that the fetal loss between 31 and 260 days
572
of gestation was 10.2% a year- which was higher than our study. There are many common factors
affecting fetal loss in animals, for example; infectious diseases, genetic abnormalities, heat stress,
toxic agents and farm management (Forar et al., 1996; Chandel and Kher, 1999; Martucciello et al.,
2009). The rate of fetal loss was overall relatively consistent although it did show a small seasonal
variation- being slightly higher in the rainy season (between August and October) (Fig 1). It is
possible that in tropical areas during the rainy season, the animals suffer from heat and humid stress
(high temperature and humidity) which might induce abortion in pregnant cows. Additionally,
blood parasite diseases from blood sucking insects such as typanosomiasis could be another cause
of increased abortion and still births in the rainy season because there are a larger number of these
insects during this season (especially, the Tananidae family and Stomoxydinae family) and they are
difficult to control in buffalo farm settings (Payne et al., 1991; Lang, 2001). Farm management
factors can also affect fetal loss such as: high density of the cows in each stable/ unit, calving
environment and procedures and nutritional management.
In conclusion, the fetal loss rate in this research was comparatively low and in the buffalo
farming industry generally considered to be in the acceptable range. However, proper farm
management of pregnant buffalo and the careful monitoring of their health can affect fetal loss and
should be considered throughout the year - especially in the rainy season. Also, more descriptive
and detailed abortion records should be kept for future health monitoring reference and analysis.
ACKNOWLEDGEMENTS
For English editing and review, thank you Mr. Philippe Marcou, B.A.Philosophy/ English
Boston College, M.A. Systematic Theology/Pastoral Counseling Boston University, USA.
REFERENCES
Campanile, G. and G. Neglia. 2007. Embryonic mortality in buffalo cows. Ital. J. Anim. Sci.
6(suppl.2): 119-129.
Chandel, B.S. and H.N Kher. 1999. Isolation of Blue tongue virus from an aborted buffalo foetus
of buffalo (Bubalus bubalis) in India. Buffalo Bull. 18(1): 20-22.
Forar, A.L., J.M. Gay, D.D. Hancock and C. Gay. 1996. Fetal loss frequency in ten Holstein dairy
herds. Theriogenology 45: 1505-1513.
Lang, P.S. 2001. Studies on incidence and control of Trypanosomiasis in buffalos caused
by Trypanosome avansi steel 1885 in North Vietnam. In: Proceeding Buffalo Workshop
December 2001. Available online: http://www.mekarn.org/procbuf/pham.htm. Retrieved
date: Jan 30, 2013.
Martucciello, A., G.M. De Mia, M. Giammarioli, I. De Donato, G.G. Iovane and G. Galiero. 2009.
Detection of Bovine viral diarrhea virus from three water buffalo fetuses (Bubalus bubalis)
in southern Italy. J. Vet. Diagn. Invest. 21: 137-140.
Payne, R.C., I.P. Sukanto, D. Djauhari and T.W. Jones. 1991. Trypanosoma evansi infection in
bovine and buffalo calves in Indonesia. Vet. Parasit. 38: 253-256.
Prasad, S. and R.B Prasad. 1998. Measures of reproductive estimates in rural buffalo herds of
Meerut district of Uttar Pradesh (India). Buffalo Bull. 17(2): 27-29.
573
Figure 1. The number of fetal loss during the period of 2003- 2011.
574
Study of Frequency Distribution of Calving of River Buffaloes (Bubalus bubalis)

in Twelve Farms in Different Ecological Areas in Venezuela
C. C. Ch. Montiel M., N. Montiel-Urdaneta*, N. Berrios, N. Morillo S., J. Belandria, M. Andará,
and J. Arias
*Department of Production and Industry Animal. School of Veterinary Sciences. The University of the
*Corresponding e-mail: nsmontiel@gmail.com; nmontiel@cantv.net; Mobile: 00584143607847
Abstract
Venezuela is located in the northern hemisphere, between the Ecuador and the Tropic of
Cancer; between coordinates 00° 38' 53" and 12° 12' 00" LN; 59° 47' 50" and 73° 22' 38" LW. The
reproductive behavior of the buffaloes is characterized by a character poliestrual with marked
seasonal trend in their sexual activity, which is influenced by physiological, health, nutritional
factors, aspects of management and particularly environmental (temperature, humidity,
precipitation, evaporation, daylight hours.); the country observed two quite demarcated times with
respect to precipitation observing a bimodal behaviour, two rainy season and two periods without
precipitation. 17335 observations of the month of calving of the buffaloes corresponding to twelve
farms located in different ecological areas of Venezuela between the years 1993-2012 were used for
the study. The procedure was used for the calculation of the frequency of calving PROC FREC of
the statistical package SAS (Version 8, 2000); yielding the following results: January: 7.81%;
February: 5.37%; March: 3.24%; April: 2.57%; May: 2.73%; June: 3.89%; July: 6.75%; August:
12.44%; September: 15.10%; October: 16.18%; November: 13.04% and December 10.81%; as you
can see there are months March, April, May and June where could scarcely occur 1, 2 or 3 calving
per month; as well as not calving during the mentioned months. It is concluded that the frequency of
calving showed a marked seasonality between July to December to the concentration of the 74.32%
of calving that occurred in the year.
Keywords: buffaloes, reproduction, seasonality, calving

575
Generation of Genetically Modified Buffalo Embryos Using Multiple-Locus

Gene Targeting
Chunying Pang, Tingxian Deng, Xianwei Liang, Yingyin Zhang and Bingzhuang Yang*

*Corresponding: yangbri@126.com
ABSTRACT
The aim of the present study was to generate genetically modified (GM) buffalo embryos using
multiple-locus gene targeting and microinjection technology. To generate the GM buffalo embryos,
we constructed a multiple-locus gene targeting vector pBC1-DS2-Fat1-eGFP-DS1 and investigated
the effects of injection timing after fertilization (5-6 h, 9-10 h, 13-14 h, 17-18 h and 21-22 h) and
vector DNA concentrations (10, 50 and 100µg/mL). Results indicated that blastocysts of the 9-10 h
group had the highest eGFP expression which was significantly higher than the blastocysts of 5-6 h
and the 21-22 h groups (p<0.01) In the concentration test, the 50 µg/mL group had the highest eGFP
expression and was significantly higher than the 100 µg/mL group. In addition, PCR was used to
test the site specific integration of the ω-3 fatty acid desaturase gene (Fat1) in the GM buffalo
embryos. Sequencing results showed that the Fat1 gene has site-specific integrated to the buffalo
genome by nested-PCR test. The Fat1 gene expression in the GM buffalo embryos were tested by
qRT-PCR. The qRT-PCR results indicated that the expression was higher in the GM buffalo embryo
when compare to the controls. In conclusion, the genetically modified buffalo embryos were
obtained by multiple-locus gene targeting and microinjection technology this will provide
transplantation donator for Fat1 gene targeted cloning buffalo with embryo transfer.
Keywords: multiple-locus gene targeting, microinjection, transgenic embryo, Fat1, buffalo


576
Effect of Frozen Semen from Italian Mediterranean Buffalo on Some

Reproductive Parameters and Conception Rate Performed to Different Species
Water Buffalo in Southern China
Guangsheng QIN， Bingzhuang YANG, Zhengzhun TAN， Hui LI, Jian HUANG， Xianwei
LIANG*
Key Laboratory of Buffalo Genetics, Breeding and Reproduction technology, Ministry of Agriculture
and Guangxi, Buffalo Research Institute, Chinese Academy of Agricultural Sciences, 24-1Yongwu
Road, Nanning 530001, P.R. China.
*Corresponding email: liangbri@126.com
ABSTRACT
The aim of the present study was to determine the effect of insemination of Italian
Mediterranean buffalo by artificial insemination in China. The frozen semen of Italian
Mediterranean buffalo imported to China was used to inseminate the existing Murrah, Nili-Ravi and
local buffalo from Guangxi Buffalo Research Institute. During 3 years, 65 Murrah buffaloes, 48
Nili-Ravi buffaloes and 52 local buffaloes their ages range between 2.5 and 9 years old with natural
estrus were selected and inseminated with the Italian Mediterranean buffalo frozen semen in the
uterine horn by the rectum deep grasp. 40 days after mating, the early pregnancy diagnosis of these
inseminated animals was checked by B Mode ultrasound. The results showed that the conception
rate in cow of Murrah, Nili-Ravi and local buffalo were 45.31%, 52.08%, 48.08%, respectively.
And the average conception rate was 47.88%. Although there was no significant difference (P>0.05)
among the different buffalo species. The results suggested that it was practicable that the
introduction of Mediterranean water buffalo frozen semen to carry out hybrid combinations, to
improve the existing water buffalo species and to improve their production performance.
Keywords: Mediterranean Water Buffalo Frozen Semen; Artificial Insemination; Conception rate
INTRODUCTION
Italian Mediterranean buffalo is a genetic type used as milk production, which is known as one
of the best river buffalo with good milk performance in the world. The average milk yield in
lactation can reach to 2168 kg, even 4000-5000 kg from some of the high productive buffalos. In
Italy, buffalo milk is processed into fresh cheese that is contributed in both local and international
markets. Guangxi Buffalo Research Institute is the only institute in China which focuses on buffalo
studies. In addition, it has two types of river buffalo (Murrah and Nili-Ravi buffalo). China
imported the frozen semen of Italian Mediterranean buffalo in July of 2007 at the first time and it
was used to inseminate the existing Murrah, Nili-Ravi and local buffalo for research. This program
was supported by the fund of International technical cooperation projects and “948” Program of
Ministry of Agriculture of China. The aim of importing Italian Mediterranean buffalo frozen semen,
in one hand, was to breed a new buffalo species by selecting and cultivating inseminated animals
with the frozen semen. In another hand, the aim was to be selected the best cross-combination of
mating and to improved production performance of offspring. The present study has been designed
to determine the effect of artificial insemination with frozen semen of Italian Mediterranean buffalo
in China, as well as to provide a basis for future scientific research and clinical applications.

577

The study was conducted at Guangxi Buffalo Research Institute, China. A total of 165 water
Buffalos ages range between 2.5 and 9 years were assigned for this study, Murrah buffaloes,
Nili-Ravi buffaloes and local buffaloes was 65、48 and 52 respectively. All animals with good health
and have no reproductive disease history. The body weight ranged from 350 to 650 kg. The straw
frozen semen of Italian Mediterranean buffalo imported from Cooperativa Fecondazione Artificiale
(CoFA). The effective sperm count of straw frozen semen was above 107/ml and the spermatozoa
motilities of thawing semen were above 0.4.
Estrus detection
Estrus detection was performed to all animals visually observed by trained technician everyday.
Cows also exposed to vasectomized bulls to observe spontaneous estrus. When a cow displayed
spontaneous, overtly evident estrous-behavior, follicular development was checked by B-mode
everyday to make sure the location of dominant follicle. Ovulation was also observed every 4 to 6
hours per day. Cows were artificially inseminated until ovulation was verified based on estrous
behavior and mucus discharge.
Method of semen thawing
The frozen semen of Italian Mediterranean buffalo first was taken it out from a nitrogen canister,
and stayed in the air for a short 5 to 8 seconds, then put it on a water bath at 37.5 -39℃ for 10-15s.
The motility and modality of semen was checked by microscope, post-thaw motility of semen was
above 0.40.
Method of artificially insemination
Buffalo cows were artificially inseminated with frozen semen approximately 5×106sperm cell
of Italian Mediterranean buffalo after confirming the estrus 12- 24h.
Pregnancy diagnosis
Pregnancy diagnosis was checked on day 40 post insemination by both rectal palpation and
ultrasonography.
Data collection and statistical analyses
Incidence of estrus duration, estrous behavior signs, ovulation time, follicular development and
conception rate were recorded. Take different buffalo species as a parameter, the number of
inseminated cows and pregnant ones as results. All data were analyzed using a chi-squared test by
SAS 8.0. Difference was not significant ( P＞0.05).
RESULTS
Effect of frozen semen from Mediterranean buffalo bull on conception rate under different buffalo
species
As can be seen from table 1, 65 Murrah buffaloes, 48 Nili-Ravi buffaloes and 52 local buffaloes
with natural estrus were selected and inseminated with the Italian Mediterranean buffalo frozen
semen in the uterine horn by the rectum deep grasp during 3 years. The conception rate in artificial
insemination of Murrah, Nili-Ravi and local buffalo was 45.31%, 52.08%, 48.08%, respectively.
According to table 1, there is no significant difference on conception rate between species that
artificial inseminated with Italian Mediterranean buffalo frozen semen P＞0.05）.Moreover, the
average conception rate was 47.88%.
Effect of frozen semen from Mediterranean buffalo bull on conception rate under different buffalo
ages
In order to know whether there are different in pregnancy of AI between heifer buffalos and
multiparous buffalos.The results of heifer buffalos and multiparous buffalos pregnancy was 54.55%
and 44.44% respectively in the Table 2.The pregnancy rate per AI was higher in heifer buffalos than
multiparous buffalos, however, the difference is not significantly P＞0.05）.
Effect of ovulation time on conception rate
Comparison pregnancy rate with AI before ovulation time and after ovulation time, it was
578
found that their pregnancy rate was 43.08%, 52.56% and 28.57% when AI was done in 6-8 hours
before ovulation, 1-3 hours after ovulation and 4-6 hours after ovulation. There was an apparently
effect of ovulation time on conception rate presented in table 3, the best time for mating was in 1-3
hours after ovulation and the conception rate of it was significantly higher than that in 4-6 hours
after ovulation P 0.05）. But no difference was found in conception rate between 6-8 hours before
ovulation and 1-3 hours after ovulation P＞0.05）.
DISCUSSIONS
According to the results, the average pregnancy rate per AI was 47.88% which is slightly lower
than the result reported by Moioli (Moioli et al., 1998) 56%）, and in agreement with Zicarelli
(Zicarelli et al., 1997) 42.5%-51.1%）. This means it is practicable for the existing buffalos in
China to adapt to breeding system by submitting AI with imported Italian Mediterranean buffalo
frozen semen.
From these results we also conclude that the conception rate of Nili-Ravi buffalo was the
highest one among all inseminated cows (52.08%). However, the difference of conception rate
between species was not significant P＞0.05）, which is similar to the results reported by Cai
(1997). Cai reported that buffalos aged from 3 to 8 had the highest pregnancy rate (66.5%), buffalos
aged from 9 to 12 and above 13 had a pregnancy rate of 45.6%, 35.3% respectively. These studies
show that the pregnancy rate of AI conducted on heifer buffalos was higher than multiparous
buffalos. The main reason, which could be the uninfected uterus and tractus genitalis of heifer
animals, resulted to be beneficial to spermatiation and embryo nidation. Moreover, the multiparous
buffalos often suffer a variety of reproductive diseases. Also the less activated ovary of old aged
buffalos may lead to delayed ovulation, which can affect the conception rate in return.
The effect of ovulation time on AI is given in Table 3, which indicated that ovulation time has a
great influence on the pregnancy rate. But estrous detection is difficult in buffalo because of the
scarce behavior signs. It is also difficult to recognize buffalo cows in heat because of their
asynchronism. In addition, due to variable duration of estrus (4–64 h) (Ohashi, 1994; Baruselli,
2001) and the difficulty encountered in predicting the time of ovulation, artificial insemination
(AI) in buffaloes is limited. Therefore it is important to know the regular pattern of ovulation and
the best time for artificial insemination in buffalos. In this study, 125 estrous cycles of 85 buffalos
were monitored by B-mode (HS-101V, Japan), the average time from the start of estrous to
ovulation was 40.7 hours (19-96h), which is similar to the results of 40.8 h (Zhou, 2004; Qisheng,
2004) and 47 h (Li, 2007). The results (Wu, 2007) also showed the best time for AI to almost all
buffalos is at the 40 hours after estrous. Artificial insemination submitted at 36 to 40 hours after
estrous had a conception rate of 69.86%. On the contrary, artificial insemination performed at 24
hours before estrous or 60 hours after estrous had a lower conception rate (25.71% and 32%
respectively). The difference was significant (P<0.05).
In conclusion, artificial insemination performed to the existing Murrah, Nili-Ravi and local buffalo
in China with the imported Italian Mediterranean buffalo frozen semen improves the conception
rate. In our opinion it is practicable to carry out hybrid combination research and improve the
existing buffalo species as well as their production performance by introducing Italian
Mediterranean buffalo frozen semen.
ACKNOWLEDGEMENT
This work was supported by the fund of International technical cooperation projects
(2008DFA30320), “948” Program of Ministry of Agriculture of P.R. China (No.2006-G49) and the
Guangxi scientific research Projects (2012GXNSFDA053014, 1123005-3).
579
REFERENCES
Baruselli P.S. 2001.Control of follicular development applied to reproduction Biotechnologies in
buffalo. In: Proc I Congresso Nazionale sull’allevamento del bufalo: 128–46.
Cai, Q.Z. 1997. Effect of breed, season, age and parity on pregnancy of AI buffalo. Chinese Journal
of Veterinary Science 1：98-99.
Li, Z.Q. 2007. Resarch on artificial insemination of buffalo in breeding season [J]. Guangxi Journal
of Animal Husbandry & Veterinary Medicine 23(3): 116-117.
Moioli B.M.，F. Napolitano，S. Puppo，V.L. Barile，G.M. Terzano，A. Borghese，A. Malfatti，
A. Catalano and A.M. Pilla. 1998. Pattern of estrous，time of LH release and ovulation and
effects of time of artificial insemination in Mediterranean buffalo cows. Anim. Sci. 66：87-91.
Ohashi O.M. 1994. Estrous detection in buffalo cow. Buffalo J. (Suppl. 2):61–64.
Wu, S.，J.R. Bin，Y.X. Zhang ，Y.M. Wei，Z. Y. Wu. 2007. Research estrous characteristics and
timely insemination of Guangxi buffalo. China Herbivores 27(6)：34-35.
Zhou, Q.S. 2004. Experience of artificial insemination. Guangxi Journal of Animal Husbandry &
Veterinary Medicine 20 2）：61-64.
Zicarelli, L.，C. De Filippo，M. Francillo，C. Pacelli and E. Villa. 1997. Influence of insemination
technique and ovulation time on fertility percentage in synchronized buffaloes. Proc. Fifth
world buffalo congress， Caserta， Italy，13-16：732-73
580
Table 1. Comparison of buffalo artificial fertilization effect on different breeds.
Breed Numbers of AI Numbers of pregnancy pregnancy rate(%)
Murrah buffalo 65 29 45.31

Nili-Ravi buffalo 48 25 52.08
Local buffalo 52 25 48.08
Total 165 79 47.88
Table 2. Effect of buffalo pregnancy rate on different ages.
Age Numbers of AI Numbers of

pregnancy rate %）
pregnancy
Heifer buffalos 44 24 54.55
2.5-4years）
multiparous buffalos 72 32 44.44
(5-9years)
Table 3. Effect of artificial insemination to buffalo conception rate time on ovulation

time.
ovulation time Numbers of Numbers of pregnancy

AI pregnancy
rate %）
6-8 hours before 65 28 43.08
ovulation
1-3 hours after ovulation 78 41 52.56
4-6 hours after ovulation 21 6 28.57
581
Early Embryo Development in Buffalo
Adriana Caroprezo MORINI, *1 Carlos Eduardo AMBRÓSIO, 2João Carlos MORINI

JUNIOR, 3 Flávia Verechia Thomaz. PEREIRA,5 William Gomes VALE,1 Isadora Karolina
Freitas de SOUSA,1 Kedson Alessandri Lobo NEVES,1 Antonio Humberto Hamad
MINERVINO1 and Maria Angélica MIGLINO4
1
Institute of Biodiversity and Forests, Federal University of Western Pará, 68035-110, Santarem,
PA, Brazil 2 Faculty of Animal Science and Food Engineering, University of Sao Paulo, 13635-900,
Pirassununga, SP, Brazil 3 Faculty of Agronomy, Federal University of Mato Grosso do Sul,
79560-000, Chapadao do Sul, MS, Brazil 4 Department of Surgery, Faculty of Veterinary Medicine
& and Animal Science, University of Sao Paulo, 05508-270 Sao Paulo, SP, Brazil 5 Department of
Histology and Embryology, Sao Paulo State University, Dracena Campus, 17900-000 Dracena, SP,
Brazil
*Corresponding email : drimorini@usp.br
ABSTRACT
The aim of this study was to describe the developmental changes in the buffalo conceptuses
from 10-60 days of pregnancy using the crown rump methodology to diagnose the estimated age of
those samples. Macroscopic examinations were carried out on 96 buffalo conceptuses; embryo/fetus
was measured to determine its age. A stereomicroscope and Scanning Electron Microscopy were
used to describe macroscopic external details of embryos. Samples were photographed to be able to
identify main organs like heart, liver, gut, pharyngeal arches and lung, during the development.
Fetuses were used for description of ossification points using the Alizarine technique. The obtained
results revealed that mammal embryos until 5th weeks of gestation have too much similar
characteristics and it is difficult to distinguish the species without checking the placenta features.
Similarities between bovine and buffalo persist, except for fetal stages in which buffalos seem to
have faster development than that of bovine. In conclusion, the overall data indicated the important
characteristics that can be evaluated to verify the viability of buffalo embryos and help the
evaluations by ultrasonographic exams.
Keywords: Buffalo, Conceptus, Embryo, Morphology
INTRODUCTION
Small lots of the animals brought to Brazil and have reproduced so well that they now total
about 400,000 head and are still increasing, especially in the lower Amazon region. Buffalo meat
and milk are now sold widely in Amazon towns and villages; the meat sells as beef (Popenoe,
1984). Placentation of water buffalo was described on literature (Carvalho et al., 2006) but early
stages of implantation are not available. There is succinct information available about embryo
morphology. Information about embryo/fetuses morphology is restricted to some studies made
fourth years ago (Schmidt, 1964, Singh, 1963, Abdel-Raouf, 1968, Abdel-Raouf, 1970).
The application of reproductive techniques such as artificial insemination, in vitro fertilization and
embryo transfer in water buffalos has had success, moreover has an increase of interest in the
production in wide scale of embryos genetically improved for fast multiplication (Meena, 2006).
One recent study established the fetometry of different fetal parts and organs with accuracy in the
estimation of gestational age by ultrasonography in buffaloes. (Ali and Fahmy, 2008). To give
support for reproductive technology, and know further information about buffalo morphology, we
evaluated morphologic aspects of buffalo embryos and membranes with age between 10-60 days of
gestation.

582

The study was done after the approbation of the Bioethics Committee of the School of
Veterinary Medicine and Animal Science (FMVZ) from Sao Paulo University (USP). It were used,
96 female pregnant buffalos (Bubalus bubalis) originating from Macapa-Amapa-Brazil. clamps, and
then it were. After 48 hours of fixation perfused with 4% buffered paraformaldehyde (Sigma, St.
Louis, MO) or Bouin solution, embryo with its fetal membranes was withdrawn and transported for
processing the analyses in the Laboratory of Anatomy and Histology of the FMVZ-USP - Sao
Paulo/SP. The embryos were distributed in groups according itss estimated using the crow rump.

Embryos with less than 0.3 cm (-10 days) cannot be seeing without magnifying glass, in this
material we only can identify one long line representing the neural tube that is completely open as
in human (Sadler, 2003) and bovine (Chang, 1952). The long axis of the opaque region distinguish
cranial and caudal region. The most prominent features at this stage are the forming neural tube and
somites (figure 1A). It is possible to detect the fetal membranes amnion, allantois and yolk sac. The
allantoic membrane looks like a tail of whale or has a “T” shape (figure 1A). In bovine this early
development of the allantois was observed in a 3.92 mm embryo, with 21 days after fertilization
(Maddox-Hyttel, 2003). The length of bovine and bubaline embryos was the same, but the age was
different, remembering that our age was estimated and the bovine one was accurate. According to
the Nomina Embryologica Veterinaria (NEV) (Frewein, 1994) this stage correspond to the Stage 8
(Periodus tubi neuralis).
Embryos with 0.3cm (±10 days) of length presents macroscopically defined only two
spheres representing the heart and a long tube representing the primitive gut (figure 1B). At this
stage head and tail folds are much more proeminent, head contains three brain vesicles and the
forebrain has optic vesicles, it also can be distinguish the placodes otic, optic, olfactory and
hypophyseal (Knospe, 2002). We also identified those details also in our buffalo embryos, except
the affirmative that head and tail are more proeminent, in our samples embryos are starting its
curvature and have head, tail and body shape at proportional sizes.
Embryos with 0.5cm (±15 days) have “C” shape, as described in bovine from 20-30 days of
gestation (Assis Neto et al., 2009). It was observed three depressions that suggest the branchial arcs
(mandibular, hyoid and glossopharyngeal) those depressions become distinct to either side of the
hindbrain region, another clear depression at the same region is the otic placode depigmented. The
primitive gut represented by a tube can be distinguished into foregut, midgut and hindgut (figure
1B). Dorsal aorta and the beginning of it ramification starts head angiogenesis. There is a large
distribution of circulatory vessels (figure 1C). The nephric tissue cannot be distinguish in
pronephros, mesonephros or metanephros, but the position of the tissue can show us that we have
mesonephros expanding yet. According to NEV this stage corresponds to the Stage 9 (Periodus
pharyngealis initialis).
Embryos with 0.3/0.5cm of CR (±10/15 days) can be found in the middle of the pregnant
uterus horn and this one do not have any external characteristics that can confirm the gestation.
When incised the non-pregnant horn we did not present any remnant of fetal membranes, according
to the review of bovine development (Assis Neto et al., 2009) at this age of gestation there is fetal
membranes only into the pregnant horn. The pregnant horn have thin and elongated corion filament
for all the length of the horn as in bovine specie (Winters, 1942), the amnion leave from de medium
portion of the coeloma layer, there is loss liquid inside it. The yolk sac is elongated and has “T”
shape, each one of its extremities has around 4 cm, as in bovine with 25 days of gestation (Assis
Neto et al., 2009).
Embryos with 0.6/0.7cm of CR (±17/19 days) also present “C” shape. There are four defined
primitive structures at the branchial arcs region (mandibular arch, hyoid arch, glossopharyngeal
arch, maxilla and oral groove) the otic placode was also depigmented at this age (figure 1D). The
heart was very proeminent and situated under the head of the embryo (figure 3A). As saw in cats we
also can distinct pouches and clefts of the branchial or pharyngeal archs term that the authors used.
583
The same authors described the anterior neuropore closed; our embryos also have what we named
cranial neuropore closed. The mesonephric crest comes from the dorsal portion of the embryo
(figure 1D). There is no hindlimb or forelimb bud formation. The hepatic primordial is represented
by a portion of the foregut (figure 1D). At this period the gestational sac is only in one horn of the
uterus, there is no signal of implantation area, or even cotyledons at chorion region. The allantoic
membrane has liquid in good quantity and it is separated of chorion. The amnion contours embryo
shape and the yolk sac also is elongated with two large filaments, as in cat embryos (Knospe, 2002)
the connection between midgut and yolk sac has become constricted and surrounded by the intra
and extra-embryonic coelom. All those fetal membranes characteristics described are very similar
with bovine description (Assis Neto et al., 2009). According to NEV this stage corresponds to the
Stage 10 (Periodus pharyngealis ultima).
External features are important to characterize the phase of the embryo development and if
they are growing according to the normal develop of the specie. Those results demonstrated in our
study can help morphologists but also can be a tool to veterinarians that use techniques such as
ultrasound to verify the viability of the conceptus.
In conclusion, the overall data indicated the most important external features in buffaloes for
evaluation of embryo/fetal development, estimation of gestational age and contribution with other
techniques that use morphology data.
ACKNOWLEDGMENTS
To FAPESP (Fundação de Amparo a Pesquisa de São Paulo) for financial support; Prof Dr.
Haroldo José Ribeiro, for introduce me to the abattoir; Prof. Dr. José Roberto Kfoury Junior for
came with me at the first time that I was at Amapá and for all the staff of the abattoir for receive me
very well and collaborate with my collect process.
REFERENCES
Abdel- Raouf, M., M. A. Elnagger and M.R.F. Elbab. 1974. Development of fetal testis in buffalo.
Zeitschrift Fur Anatomie Und Entwicklungsgeschichte 144: 227-236.
Abdel -Raouf, M. and M.A. EL-Nagger. 1968: Biometry of the Egyptian buffalo foetus. U.A.R. J.
Vet. Sci. 5: 37-43.
Abdel-Raouf, M. and M.A.EL-Nagger. 1970, Further study of the biometry and development of the
Egyptian buffalo foetus. U.A.R. J. Vet. Sci. 7: 125-140.
Ali, A. 2004. Effect of gestational age and fetal position on the possibility and accuracy of
ultrasonographic fetal gender determination in dairy cattle. Reproduction in Domestic
Animals 39P: 190-194.
Ali, A., and S. Fahmy. 2008. Ultrasonographic fetometry and determination of fetal sex in buffaloes
(Bubalus bubalis). Animal Reproduction Science 106: 90-99.
Assisneto, A.C., F.T.V. Pereira, T.C. Santos, C. E. Ambrosio, R. Leiser, and M.A. Migilno 2009.
Morpho-physical Recording of Bovine Conceptus (<i>Bos indicus</i>) and Placenta from
Days 20 to 70 of Pregnancy. Reproduction in Domestic Animals.
Carvalho, A.F., K. Klisch, M.A. Migilno, F.T.V. Pereira and E. Bevilacqua. 2006. Binucleate
trophoblast giant cells in the water buffalo (<I>Bubalus bubalis</I>) placenta. Journal of
Morphology 267: 50-56.
Chang, M.C. 1952. Development of bovine blastocyst with a note on implantation. The Anatomical
Record 113: 143-161.
Frewein, J., R. E. Habel and W.O. Sack. 1994: Nomina Anatomica Veterinaria. 4th edn Nomina
Histologica, 2nd edn Nomina Embryologica Veterinaria. edn. Zurich, Ithaca, New York
WAVA.
Maddox-Hyttel, P.A., G. Vajta, I. Lewis, P. Rogers, L. Cann, H. Callesen, P. Tveden- Nyborg and
A.Trounson. 2003: Immunohistochemical and ultrastructural characterization of the initial
post-hatching development of bovine embryos. Reproduction 125: 607-623.
584
Meena, C.R.D. 2006: Development of water buffalo (Bubalus bubalis) embryos from in vitro
matured oocytes reconstructed with fetal skin fibroblast cells as donor nuclei. Animal
Reproduction Science 93: 258-267.
Popenoe, H.E.A. 1984. The Water Buffalo: New Prospects For An Underutilized Animal. Ad Hoc
Panel of the Advisory Committee on Technology Innovation Board on Science and
Technology for International Development Commission on International Relations National
Research Council, 1.
Sadler, T.W.E. 2003. Embriologia Médica - Langman. Rio de Janeiro: Guanabara Koogan.
Schmidt, K., S. EL-Sawaf and K. Fouad. 1964. The development of the gravid uterus during the
gestation period in Egyptian buffaloes. Vet. Med. J. Giza 10: 119-140.
Singh, S., O.P.S. Senger and S.N. Singh. 1963. Prenatal development of buffalo (Bos bubalis L).
Agra. Univ. J. Res. 12: 197-145.
Winters, L.M.G. and R.E. Comstock. 1942. Prenatal development of the bovine. Univ Agric Exp
Sta Minnesota Tech Bull 151: 3-50.
Figure 1: Lateral view of buffalo embryos A: Embryo with less than 0.3cm of CR (±10 days of
gestation). The extraembryonic membranes have been removed to expose the dorsal surface of the
embryo. B: Embryo with 0.3cm of CR (±15 days of gestation). C: Embryo with 0.5cm of CR (±15
days of gestation). D: Embryo with 0.7cm of CR (±19 days of gestation). Legend: al, allantois; 4°v,
fourth ventricle; am, amnion; cvf, cervical flexure; fl, forelimb bud; fg., foregut; fp, foot plate; ga,
glossopharyngeal arch; gt, genital tubercle; h, heart; ha, hyoid arch; hl, hindlimb bud; hg, hindgut;
hp, hand plate; l, liver; lp, lens placode; ma, mandibular arch; mb, midbrain; md, mandible; mes,
mesonephron; mx, maxilla; nt, neural tube; og, oral groove; opc, optic cup; ope, optic evagination;
pi, pinna; pl, phalange; rb, rib; so, somite; tb, tail bud; v, ventricle; vb, viteline bridge; ys, yolk sac.
585
Glucose, Cholesterol, Total Protein and Growth Factor Insulin-like Type I in

Follicular Fluid of River Buffaloes (Bubalus bubalis)
Carlos GALLEGO¹, Margarita PARDO², Aline CAMARGOS², Diego SOUSA², Maria

LENZ³, Alcides. RAMOS², Andre JORGE², Fernanda LADIM² and Eunice OBA²*
¹ Institute of Animal Science, San José de las Lajas. Havana. Cuba

Email: cgallego@ica.co.cu
² Estadual Paulista University "Julio de Mesquita Filho" Campus of Botucatu. Brazil
³ Federal University of Mato Grosso. Campo Grande. Mato Grosso. Brazil
*Corresponding eขmail: euniceoba@fmvz.unesp.br
ABSTRACT
In order to establish the concentrations of glucose, cholesterol, total protein and growth
factor insulin-like type I (IGF-I) in the follicular fluid, 26 Murrah breed river buffaloes, between 45
and 70 days postpartum, empty, multiparous, with average live weight of 675 ± 56 kg and average
body condition of 3.5 points on a scale of 1-5, were used in this study. The fluid was collected from
dominant follicles with diameters between 8 and 12 mm by OPU, and was not taken into account
the stage of the estrous cycle. Using this technique, the wave of follicular development was
synchronized six days prior to collection. Biochemical analysis was performed to glucose and
cholesterol through the enzymatic colorimetric method using commercial kit glicose
CHOLESTEROL GOD-PAP and CHOD-PAP (Kovalent), respectively. Determination of total
protein was carried out by using total protein commercial kit (Kovalent) Biuret method, and the
readings were performed using absorption spectrophotometry with visible light. Concentration of
IGF-I was measured by Radioimmunoassay (RIA) technique using commercial IRMA Kit IGF-I
(INMUNOTECH). Descriptive statistics were developed using the PROC MEANS procedure of
SAS (2009). Concentration of glucose (4.0 ± 0.75 mmol / L ¯¹) and IGF-I (340 ± 129.83 ng / mL ¯¹)
were higher than those reported by other authors in river buffaloes and cows, respectively.
However, cholesterol levels (0.51 ± 0.12 mmol / L ¯¹) and total protein (58.4 ± 4.43 g / L ¯¹)
behaved inferior to other studies in same species. The results indicated that there is relationship
among the nutritional aspects, diameter of follicles aspirated and productive period in the
concentration of biochemical indicators.
Keywords: biochemical profile, follicular fluid, postpartum.
INTRODUCTION
Specialized systems in milk production, have high incidence of metabolic and nutritional
problems, often not reach in a clinical sense, but that compromise productive and reproductive
processes of the dairy farms. Novel research shows the existence of complex interactions between
nutritional balance and metabolic disorders. Thus, during the prepartum diet, the metabolic changes
that accompany the onset of lactation, and body condition prepartum induce changes in serum
levels of different metabolites, which could affect the proper functioning of tissues and organs
(Hagawane et al., 2009).
Follicular liquor characteristics can be affected by physiological conditions and the state of
the follicle, such as its size, growth and degeneration. The composition of this fluid, research
arouses interest by increasing knowledge of the processes that occur during follicular development
and its metabolic and biochemical changes during postpartum productive and reproductive stages
(Khan et al., 2011). Studies by Leroy et al. (2008) demonstrated that the metabolic changes that
accompany the onset of lactation, product of negative energy balance (NEB), produce changes in
the biochemical composition of different metabolites in follicular fluid, and determine, in turn, the

586
subsequent quality of oocytes and subsequent fertility of the animals. Known concentrations of
these elements in the human follicular fluid (Leese and Lenton, 1990), cattle (Orsi et al., 2005),
goats (Herrick et al., 2006) and sheep (Nandi et al., 2007), which contributes knowledge of nutrients
and metabolic needs that enable good reproductive performance.
In buffaloes, little research carried out in this regard, resulting in limited knowledge that
could explain the physiological processes that occur during early lactation and its relationship with
existing metabolic compounds in follicular liquor. For these reasons, this study was objective, know
the levels of cholesterol, glucose, total protein and growth factor insulin-like type I in follicular
fluid of buffaloes River, between 45 and 70 days postpartum systems dual purpose.

The work was performed at the experimental dairy buffalos, Faculty of Veterinary Medicine
of the UNESP, Campus of Botucatu, Brazil. We used 26 Murrah breed river buffaloes, between 45
and 70 days postpartum, empty, multiparous and average live weight of 647 ± 56 kg. The body
condition score was measured at an average of 1-5 points, which the estimate of Houghton et al.
(1990), showed an average of 3.5 points. The management developed a semi-intensive, with a
rotational system of paddocks, with a predominance of improved grasses Brachiaria (Brachiaria
brizantha) and Guinea (Panicum maximum) was supplemented with 2 kg of concentrate with 16%
crude protein (PB).
The milking was done with support from buffalo calf, manually, once a day and between
06:00 and 07:30 in the morning. Regardless of the stage of the cycle, the fluid was obtained from
dominant follicles with diameters between 8 and 12 mm, by aspiration in vivo, for once and
according to the methodology described by Bols et al.,1996. Using the technique, the wave of
follicular development was synchronized six days prior to collection. After the extraction, samples
were transported at 4 ˚ C to the laboratory and stored at -20 ˚ C in storage tubes (Eppendorf) until
subsequent analysis.
Analytical methods for the determination of the studied elements are shown in Table 1. The
readings for glucose, total protein and cholesterol were performed using absorption
spectrophotometry with visible light (Semi Stardust Spectrophotometer CM 15). The determination
of the growth factor insulin-like type I was performed by radioimmunoassay, using a solid phase
gamma counters (Cobra II RIA). The values of biochemical elements were analyzed using
descriptive statistics, using the PROC MEANS procedure of SAS (2009).

Glucose is an important energy source for the operation of the ovary, and it is known that
the blood-permeable follicular it. Table 2 shows the mean values obtained from biochemical
constituents follicular liquor study animals. The average glucose concentration was above results
Nandi et al. (2008) (2.42 ± 0.31 mmol / L ¯¹) in animals of the same species. However, the studies
that developed Villa et al. (2009) in subfertile cows (3.8 ± 0.19 mmol / L ¯¹) and fertile (4.0 ± 0.19
mmol / L ¯¹), and showed lower values similar to ours, respectively. In general, these variations
could be related to nutritional factors, energy balance postpartum and diameter of follicles
aspirated, since the blood-follicle is more permeable to larger follicles.
The concentrations of total cholesterol and proteins play roles in ovarian physiology, as
precursors of steroid hormones and growth factors of peptide origin, respectively. Both elements
had values lower than those reported Arshad et al. (2005) from ovaries of slaughtered buffaloes
during the delivery of lower concentration. It is possible that the use of unproductive females or the
end of their lactation, when compared with production animals in our study, could justify the
concentrations found, due to mobilization to the mammary gland serum lipid and protein for milk
synthesis. The diameter of the follicles aspirated in both studies could also be a factor in the results
obtained determinate. Studies carried out by Thangaver & Nayeem (2004) demonstrated that the
dominant follicles have a lower concentration of cholesterol, which is attributable to increased
conversion capacity of this element towards the formation of steroid hormones.
587
It is known that the pituitary gonadotropins have a fundamental role in growth control and
follicular development. However, there are other factors that are directly related to the processes of
folliculogenesis. Among these, those of insulin-like growth (IGF-I) and II (IGF-II) also contribute
in a specific way and at different stages in the regulation of cell proliferation and differentiation. In
general, these growth factors, stimulate, autocrine and paracrine manner, the functions developed
follicle stimulating hormone (FSH) and luteinizing hormone (LH) in follicular population. The IGF-
I levels were found in the liquor of buffalo follicular studied showed higher concentrations than
those reported by Kinigsson et al. (2008) in serum and high producing dairy cows during peak
production. These variations may be related to the high energy demands that are required during
periods of higher milk production, which inhibit the synthesis of IGF-I in the liver.
Although the highest concentration of IGF-I in the follicular fluid of dominant follicles synthesize
in liver, and is regulated by growth hormone (GH) from the pituitary gland, other studies
demonstrate the ability to produce IGF-ovary I theca cells and in the membrane of granulosa cells,
which could contribute to an increase in the concentration of these factors in the follicular fluid,
when compared with blood circulation Khalid et al. (2000). Follicular size also contributes to
increased concentrations of these growth factors, it is known that the preovulatory follicles
containing greater amounts of IGF-I, due to a decrease in biotilization of these compounds during
the final stages of growth and follicular atresia. By taking into account the species under study, the
aspiration of follicles with diameters between 8 and 12 mm, would be correlated with high
concentrations of IGF-I learned on the job.
Prepartum nutritional deficiencies related to negative energy balance (NEB) postpartum
commit serum glucose and insulin, which causes a decrease in the levels of IGF-I in serum and
follicular liquor. In relation to other work (Spicer and Geisert, 1992), high concentrations of glucose
found in the follicular liquor could justify animals during the study period did not show a
significant negative energy balance, leading to IGF- I superior to those reported in cow milk
production. There are studies that suggest that the concentrations of these growth factors in the
follicular fluid, compared with plasma levels may be more resistant to nutritional disturbances, so
that the concentration of liver and ovarian origin may be under different control systems (Matoba et
al., 2012).
Overall, the study helped to know the values of some biochemical elements follicular fluid,
postpartum, in a model dairy buffalos. The results show concentrations that differ of other works
and domestic species, and that they depend, fundamentally, of the state nutritional prepartum and
postpartum, diameter of the aspired follicles, stage of the estrous cycle and productive period.
However, further research is recommended to evaluate specifically the relationship between the
above aspects, and the concentrations of the elements present in the follicular fluid and postpartum
productive and reproductive performance, and other important indicators of metabolic profile that
used in dairy animals.
REFERENCES
Arshad, H.M., N. Ahmad, Z. Rahman, H.A. Samad, N. Akhtar and S. Ali. 2005. Studies on some
Biochemical constituents of ovarian Follicular Fluid and Peripheral Blood in buffalo.
Pakistan Vet. J. 25: 76.
Bols, P., Vandenheede., J. Van Soom and A. Kruif. 1995. Transvaginal ovum pick-up (OPU) in the
cow: a new disposable needle guidance system. Theriogenology 43: 677.
Hagawane, S.B., S.B. Shinde and D.N. Rajguru. 2009. Haematological and Blood Biochemical
Profile in Lactating Buffaloes in and around Parbhani city. Vet. World 2: 467.
Herrick, R. L. Michelle, D. Gardner, E. Behboodi, S. Memili, Y. R. Blash, Echelard and P.L.
Houghton, R.P. Lemenager, L.A. Horstman, K.S. Hendrix, and G.E. Moss. 1990. Effects of body
composition, pre and postpartum energy level and early weaning on reproductive
performance of beef cows and preweaning calf gain. J. Anim. Sci. 68:143
588
Khan, F.A., G.K. Das, Pande, Megha, M.K. Pathak and M. Sarkar, 2011. Biochemical and
hormonal composition of follicular cysts in water buffalo (Bubalus bubalis). Animal
Reproduction Science 124: 61.
Khalid, M., W. Haresing and M. Luck. 2000. Secretion of IGF-I by ovine granulosa cell: effects of
growth hormone and follicle stimulating hormone. Anim. Reprod. Sci. 58: 261.
Kinigsson, C., G. Savoini, Govoni, Nadia., G. Invernizzi, A. Prandi, H. Kindahl and C.
Veronesi. 2008. Energy balance, leptin, NEFA and IGF-I plasma concentrations and resumption of
post partum ovarian activity in Swedish red and white breed cows. Acta Veterinaria
Scandinavica 50: 3.
Leese, A and A. Lenton. 1990. Glucose and lactate in human follicular fluid: concentrations and
interrelationships. Human Reprod. 5: 915.
Leroy, J., A. Van Soom, G. Opsomer, I. Goovaerts, and P. Bols. 2008. Mechanisms Linking
Nutrition and Reduced Oocyti and Embryo Quality in High-yielding Dairy Cows. Reprod.
Dom. Anim. 43: 623.
Matoba, S., L. O’Hara, F. Carter, A. Nelly, T. Fai, D. Rizos and P. Lonergan. 2012 The association
between metabolic parameters and oocyte quality early and late postpartum in Holstein dairy
cows. Journal of Dairy Science 95: 1257- 1266.
Nandi, S. V. Girish Kumar, M. Manjunatha and P. Gupta. 2007. The biochemical composition of
ovine follicular fluid in relation to follicle size, Dev Growth Differ. 49: 66.
Nandi, S., V. Girish Kumar, B. Manjunatha, H.S. Ramesh and P. Gupta. 2008. Follicular fluid
concentration of glucose, lactate and pyruvate in buffalo and sheep, and their effects on
culture oocytes, granulosa and cumulos cell. Theriogenology 69: 186.
Orsi, N., N. Gopichandran, H. Leese, M. Picton and E. Harris. 2005. Fluctuations in bovine ovarian
FF composition throughout the oestrous cycle. Reproduction.13:129.
Spicer, L and R. Geisert. 1992. Concentrations of insulin-like growth factor-I, estradiol and
progesterone in follicular fluido of ovarian follicles during early pregnancy in cattle.
Theriogenology
Table 1. Measurement systems and methods used for each element biochemistry.
Elements Units (SIU)* Analytical Methods

Glucose mmol/L¯¹ Glucose Oxidase/Peroxidase
Cholesterol mmol/L¯¹ Cholesterol Oxidase/Peroxidase
Total Protein g/L¯¹ Biuret
IGF-I ng/mL¯¹ Radioimmunoassay
SIU: International System of Units.
Tabla 2. Biochemical elements obtained in the ovarian follicular fluid stream buffaloes postpartum.
Elementos n Average Minimun Maximum CV (%) DE
Glucose 26 3.90 1.46 4.64 1.06 0.75

(mmol/L¯¹)
Cholesterol 26 0.51 0,26 1,16 0.97 0,12
(mmol/L¯¹)
Total Protein 26 58.4 49.2 67.6 82.7 4.43
(g/l¯¹)
IGF-I 26 340.36 129.90 619.81 38.14 129.83
(ng/mL¯¹)
589
Uterine Involution, Fluid Accumulation and Ovarian Activity after Injection of

Prostaglandin at Different Periods of Buffalo Puerperium
Aline Sousa CAMARGOSa, Eunice OBAa*, Alcides Amorim RAMOSb, João Carlos Pinheiro
FERREIRAa, Diego Gouvea SOUZAa, André Mendes JORGEb and Nélcio Tonizza
CARVALHOc
a
Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and
Animal Science – UNESP, Distrito Rubião Jr. s/ n., Botucatu, SP, Brazil
b
c
APTA, Fazenda Experimental, Rodovia Régis Bittencourt km 435, Registro, SP, Brazil
* Corresponding email: euniceoba@fmvz.unesp.br
ABSTRACT
This study aimed to evaluate the effect of chloprostenol administration, at early or
intermediary puerperium, under uterine involution, intrauterine fluid accumulation and ovarian
activity return. 30 Murrah postpartum buffaloes were randomly divided into three groups: CONT
(saline, n = 10); CLO2 (chloprostenol at days 2 and 5 postpartum, n = 10) and; CLO15
(chloprostenol at days 15 and 20 postpartum, n = 10). Gynecological exams were performed at days
2, 7, 14, 21 and 28 postpartum, when uterine involution degree (1 to 3 scale, by transrectal
palpation), intrauterine fluid accumulation (0 to 3 scale, by ultrasound exam) and ovarian activity
(B-mode ultrasound exam) were evaluated. CLO2 group presented higher uterine involution (2.00 ±
0.23, 1.66 ± 0.23, 1.58 ± 0.23 for groups CLO2, CONT and CLO15, respectively) and faster
ovarian activity return in relation to groups CONT and CLO15 (P < 0.05). Groups CLO2 and
CLO15 showed lower intrauterine fluid accumulation compared to CONT group (2.04 ± 0.20, 1.58
± 0.20, 1.92 ± 0.20 for groups CONT, CLO2 and CLO15, respectively; P < 0.05). Prostaglandin
analogue administration in postpartum buffalo benefited uterine involution, lochia expulsion and
ovarian activity return, improving reproductive efficiency in this specie.
Keywords: uterine involution, lochia, ovarian activity, chloprostenol, postpartum buffalo
INTRODUCTION
Immediately after parturition, begins the process of uterine involution, when changes occur
in the uterus of the female for the organ recover of the changes occurred during the period of
pregnancy, to finally achieve volume, size, position and reproductive capacity for a future
pregnancy (Kozicki, 1998). The hormone responsible for uterine involution process postpartum is
prostaglandin F2α (PGF2α) (Kindahl et al., 1999; Mishra and Prakash, 2005). Therefore, protocols
were developed to use PGF2α synthetic analogs postpartum, aiming to accelerate uterine involution
and reduce calving interval (Fernandes et al., 2002).
The aim of this study was to verify if the administration of synthetic prostaglandin analogue
(chloprostenol), at early or intermediate puerperium, induces effects on uterine involution,
intrauterine fluid accumulation and return of ovarian activity in postpartum buffaloes.

30 pregnant Murrah buffaloes (between 1 and 9 births), with body condition score between
3.5 and 4.0 (0 to 5 scale) and adequate health, were used. The animal feed included Brachiaria
decumbens grazing with water supply and mineral supplementation ad libitum. The study included
only those animals that showed normal parturition without retained placenta, i.e., with elimination
of fetal membranes within the first 12 hours postpartum.
After parturition, buffaloes were randomly distributed, according to partum occurrence, in
three treatment groups: CONT (control; N = 10) – administration of 2 mL of saline intramuscularly
590
(i.m.) at days two and five postpartum (day 0 = parturition); CLO2 (N = 10) – administration of 2
mL of chloprostenol (0.530 mg) i.m. at days two and five postpartum; and CLO15 (N = 10) –
administration of 2 mL of chloprostenol i.m. at days 15 and 20 postpartum.
Gynecological examinations were performed on days 2, 7, 14, 21 and 28 postpartum.
Initially, were evaluated the uterine involution degree, via rectal palpation (Grunert, 1980), as
follows: Grade 1 - low involuted uterus with its entire mass in abdominal cavity, with large
asymmetry, it is not possible to exam its entire length; Grade 2 - uterus with part of its mass in the
abdominal cavity, with asymmetry between the horns, making it possible to palpation and
examination of its entire length and; Grade 3 - almost entirely uterus in pelvic cavity, presenting
symmetry or few asymmetry.
Animals also were submitted to B-mode ultrasonography for evaluation of intrauterine fluid
accumulation, as the scale of Krueger et al. (2009), where: 0 - no fluid; 1 - between 0 and 1.5 cm; 2
- between 1.5 and 2.5 cm and; 3 - greater than 2.5 cm. In addition, B-mode was also used for
evaluation of ovarian activity postpartum, considering the presence of follicles larger than 10 mm
and/or corpus luteum in right and left ovaries.
For statistical analysis, data were subjected to ANOVA by SAS (2012), as causes of
variation the effect of treatment (CONT, CLO2 and CLO15), of days of evaluation (2, 7, 14, 21 and
28) and the treatment versus day interaction. Differences between means were tested with Tukey
test at 5% probability for all causes of variations.

Uterine involution degree was not significantly different (P > 0.05) between groups only in
gynecological examination on day 2 after parturition. From that gynecological exam, the CLO2
group had an average uterine involution degree significantly higher than the other groups (P <
0.05). CLO15 group showed the lowest degree of uterine involution in the gynecological
examination until day 14, when it presented a significant increase in involution degree. On day 28
postpartum, CLO15 showed higher average than control group. The period with faster involution of
the groups treated with chloprostenol was after the end of hormonal protocols, corresponding at
days 7 and 14 for groups CLO2 and CLO15, respectively. It has been reported a mean reduction of
4.2 days in the total period of uterine involution, after chloprostenol application on days 2 and 7
post-partum (Ferraz, 2006).
The averages of CONT, CLO2 and CLO15 groups of intrauterine fluid accumulation did not
show significant difference 2 days postpartum. On this day, it was observed the largest volume of
uterine fluid during the study period. On next evaluation (day 7), CLO2 and CLO15 groups showed
lower fluid accumulation in relation to control group. CLO2 group continued presenting significant
decrease until day 21, different of CLO15 that showed no changes between days 7 and 14. The
period of gynecological examinations coincided with lochia elimination of physiological
puerperium. The most significant decreases in uterine contents were at 7-21 and 21-28 days for
CLO2 and CLO15 groups, respectively. These periods coincide with the end of hormonal protocols
applied.
According to the results observed for uterine involution and fluid accumulation,
chloprostenol acted positively on the uterine tone, favoring elimination of lochia. It has been
reported previously that endogenous prostaglandin myotonic action is enhanced when it is
administered exogenous prostaglandin, leading to greater amplitude of uterine contractions (Ferraz,
2006). The beneficial action of chloprostenol on uterine fluid elimination of buffaloes postpartum
was previously reported by Camargos et al. (2012).
About ovarian activity, was observed presence of follicles larger than 10 mm on day 26 in
CLO2. In CONT and CLO15 groups, were not observed any ovarian activity until 28 days
postpartum. Animals that showed early resumption of ovarian activity were those who had the
faster uterine involution. There was significant difference of average uterine involution degree in
buffaloes with (2.79 ± 0.23) and without ovarian activity (1.55 ± 0.24, P < 0.05). The same occurred
591
with average intrauterine fluid accumulation degree (0.29 ± 0.19 and 2.14 ± 0.20 for ovarian
activity present and absent, respectively).
In conclusion, prostaglandin analogue administration in postpartum buffalo benefited uterine
involution, lochia expulsion and ovarian activity return, improving reproductive efficiency in this
specie.
REFERENCES
Kozicki, L. E. 1998. Aspectos fisiológicos e patológicos do puerpério em bovinos. Arch. Vet. Sci.
3: 9-19.
Krueger, L., J. Koerte and G. Tsousis. 2009. Transrectal Doppler sonography of uterine blood flow
during the first 12 weeks after parturition in healthy dairy cows. Anim. Reprod. Sci. 114:
23-31.
Mishra, D. P. and B. S., Prakash. 2005. Validation of a 13, 14-dihydro-15-keto-PGF2α enzyme
immunoassay and its application for reproductive health monitoring in postpartum
buffaloes. Anim. Reprod. Sci. 90: 85-94.
Kindhal, H., M. Bekana and K. Kask. 1999. Endocrine aspects of the uterine involution in the cow.
Reprod. Dom. Anim. 34: 199-205.
Fernandes, C. A. C., A. M. Ferreira and J. H. M. Viana. 2002. Efeito do cloprostenol sódico no pós-
parto de vacas leiteiras sobre o retorno da atividade reprodutiva. A Hora Vet. 126: 102-
105.
Grunert, E. 1980. Current therapy in theriogenology: diagnosis, treatment and prevention of
reproductive diseases in animals. W.B. Saunders, Philadelphia.
SAS Institute Inc. 2012. SAS/ETS® Software: Changes and Enhancements for Release 6.12. SAS
Institute Inc., Cary, NC. 112.
Ferraz, P. C. 2006. Efeito do cloprostenol (PGF2α) sobre o puerpério de búfalas (Bubalus bubalis)
leiteiras da raça Murrah. Master Thesis, University of Bahia State, Itapetininga, Bahia.
Camargos, A. S., E. Oba, A. A. Ramos, O. F. Sanchez and E. Padron. 2012. Effect of chloprostenol
on uterine involution and fluid accumulation of postpartum buffaloes. Anim. Reprod. 9:
999.
Table 1. Uterine involution and fluid accumulation (±SD) of buffaloes during puerperium with or
without prostaglandin injection.
*Acknowledgment to FAPESP for financial support.
592
Values of 13,14-Dihydro-15-Keto-PGF2α, Progesterone and Oestradiol After

Injection of Prostaglandin at Different Periods of Buffalo Puerperium
Aline Sousa CAMARGOSa, Eunice OBAa*, Alcides Amorim RAMOSb, João Carlos Pinheiro
FERREIRAa, Diego Gouvea SOUZAa, André Mendes JORGEb and NélcioTonizza
CARVALHOc
a
Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and
Animal Science – UNESP, Distrito Rubião Jr. s/ n., Botucatu, SP, Brazil
b
c
APTA, Fazenda Experimental, Rodovia Régis Bittencourt km 435, Registro, SP, Brazil
* Corresponding email: euniceoba@fmvz.unesp.br
ABSTRACT
Knowledge of the effectiveness of prostaglandins in uterine involution process led to the
development of protocols with its analogues in postpartum period. However, this hormone
mechanism of action is not yet fully elucidated. Thus, the objective of this study was to verify if
chloprostenol administration, at early or intermediary puerperium, can induce changes on
progesterone, PGFM and oestradiol plasma concentrations. 30 Murrah postpartum buffaloes were
randomly divided into three groups: CONT (saline, n = 10); CLO2 (chloprostenol at days 2 and 5
postpartum, n = 10) and; CLO15 (chloprostenol at days 15 and 20 postpartum, n = 10). Blood
samples were collected from jugular vein to measure progesterone, PGFM and oestradiol plasma
concentrations at days 2, 7, 14, 21 and 28 postpartum. CLO2 group presented lower progesterone
and PGFM plasma concentrations in relation to CONT and CLO15 groups (0.23 ± 0.00 and 0.32 ±
0.11, 0.19 ± 0.00 and 0.23 ± 0.11, 0.23 ± 0.00 and 0.30 ± 0.19, for groups CONT, CLO2 and
CLO15, respectively; P < 0.05). There was no significant difference in oestradiol plasma
concentration between experimental groups (P > 0.05). Prostaglandin synthetic analogue
administration induced hormonal changes in postpartum buffaloes, which can partially explain its
positive effect under reproductive function of this specie.
Keywords: PGFM, postpartum buffalo, enzymeimunoassay, radioimmunoassay
INTRODUCTION
During puerperium, there is an intense production of prostaglandin F2α (PGF2α) by
intercaruncular region of endometrial epithelial surface (Asselin et al., 1998; Skarzynski et al.,
2000) correlated to uterine involution process (Kindahl et al., 1999). Hormonal protocols based on
PGF2α analogues may be used in postpartum period, aiming to accelerate uterine involution and
consequently to reduce calving interval (Fernandes et al., 2002). However, it is unclear if the
administration of these protocols of PGF2α analogs have an effect on the production or secretion of
hormones related to reproductive function. Thus, the objective of this study was to evaluate if
administration of prostaglandin synthetic analogue (chloprostenol), at early or intermediate
puerperium, induces effects on 13,14-dihydro-15-keto-PGF2α (PGFM), progesterone and oestradiol
plasma concentrations of postpartum buffaloes.

30 pregnant Murrah buffaloes (between 1 and 9 births), with body condition score between
3.5 and 4.0 (0 to 5 scale) and adequate health, were used. The animal feed included Brachiaria
decumbens grazing with water supply and mineral supplementation ad libitum. The study included
only those animals that showed normal parturition without retained placenta, i.e., with elimination
of fetal membranes within the first 12 hours postpartum.

593
After parturition, buffaloes were randomly distributed, according to partum occurrence, in

three treatment groups: CONT (control; N = 10) – administration of 2 ml of saline intramuscularly
(i.m.) at days two and five postpartum (day 0 = parturition); CLO2 (N = 10) – administration of 2ml
of chloprostenol (0.530 mg) i.m. at days two and five postpartum; and CLO15 (N = 10) –
administration of 2 ml of chloprostenol i.m. at days 15 and 20 postpartum.
On days 2, 7, 14, 21 and 28 postpartum, blood samples were collected from jugular vein into
heparinized tubes for measurement of PGFM, oestradiol and progesterone, by the vacuum
collection system. Samples were kept in refrigerated cooler for 10 minutes and centrifuged (2500
rounds) during 15 minutes. Plasma was stored in microtubes and frozen (-20º C) for later analysis.
Oestradiol and progesterone concentrations were measured using Coat-a-Count (Diagnostics
Products Corporation®, Los Angeles, CA, USA) diagnostic kits according to the manufacturer’s
instructions and analyzed by radioimmunoassay (Packard Cobra II Auto Gamma).
PGFM measurement was performed using enzyme immunoassay diagnostic kit (DetectX 13,
14-dihydro-15-keto-PGF2α®, Arbor Assays, EUA) according to manufacturer’s instructions and read
in spectrophotometer.
For statistical analysis, data were submitted to ANOVA by SAS (2012), with causes of
variation the effect of treatment (CONT, CLO2 and CLO15), days of evaluation (2, 7, 14, 21 and
28) and treatment versus day interaction. Differences between means were submitted to Tukey test
at 5% probability for all causes of variations.

CLO2 group presented PGFM plasma concentration significantly higher than CONT and
CLO15 (P < 0.05). In all groups, significant differences were observed between days of blood
collection. Day 2 showed a greater PGFM level than day 7, as days 14 and 21 presented higher
concentrations than the 28th day postpartum. The administration of exogenous prostaglandin in the
third week after parturition induced an increase in plasma PGFM, suggesting that chloprostenol
may influence production and secretion of endogenous PGF2α in this period.
About plasma progesterone concentration, significant difference was observed only between
CONT and CLO2 (P < 0.05). All groups showed significant difference in plasma progesterone
concentration between different days (P < 0.05). In the first two weeks after parturition,
progesterone concentrations were low (up to 0.25 ng/ml for all groups. While the control group
showed a progressive increase, groups that received chloprostenol (CLO2 and CLO15) showed a
decrease in concentrations. Between 14 and 21 days, there was a significant increase in the
concentrations of CONT and CLO15 groups, reaching values in plasma progesterone concentration
above 0.4 ng/ml at day 21 postpartum. This increase was not observed in group CLO2, which
continued to present low concentrations of progesterone until day 28.
No significant difference was observed in oestradiol plasma concentrations between
treatment groups (P > 0.05). Significant difference was observed only between days of blood
collection. Oestradiol concentration increased significantly between 2 and 7 days after parturition,
coincident with decreasing concentrations of PGFM at the same period. Oestradiol decreases the
basal production of PGF2α by down-regulation of COX-mRNA (Xiao et al., 1998). However, the
same difference was not observed between days 7 and 14, when the concentration of oestradiol
remained constant. The maximum value was observed at day 21, followed by a marked decrease
within seven days.
According to the results obtained in this study, it can be concluded that administration of
postpartum protocols of exogenous prostaglandin is able to induce changes in the levels of
hormones related to reproductive function in postpartum buffaloes.
REFERENCES
Asselin, E., P. Droplet and M.A. Fortier. 1997. Cellular mechanisms involved during oxytocin-
induced prostaglandin F2 alfa production in endometrial cells in vitro: role of
cycloxygenase 2. Endocri. 138: 4798-4805.
594
Skarzynski, D.J., Y. Miyamoto and K. Okuda. 2000. Production of prostaglandin F2α by cultured
bovine endometrial cells in response to tumor necrosis factor α: cell type specificity and
intracellular mechanisms. Bio. Reprod. 62: 1116–20.
Kindhal, H., M. Bekana and K. Kask. 1999. Endocrine aspects of the uterine involution in the cow.
Reprod. Dom. Anim. 34: 199-205.
Fernandes, C. A. C., A. M. Ferreira and J. H. M. Viana. 2002. Efeito do cloprostenol sódico no pós-
parto de vacas leiteiras sobre o retorno da atividade reprodutiva. A Hora Vet. 126: 102-
105.
SAS Institute Inc. 2012. SAS/ETS® Software: Changes and Enhancements for Release 6.12. SAS
Institute Inc., Cary, NC. 112.
Xiao, C.W., J.M. Liu, J. Sirois and A.K Goff. 1998. Regulation of cyclooxygenase-2 and
prostaglandin F synthase gene expression by steroid hormones and interferon-t in bovine
endometrial cells. Endocri. 139: 2293–9.
Table 1. PGFM levels (±SD) of buffaloes during puerperium with or without prostaglandin
injection.
Table 2. Progesterone and oestradiol levels (±SD) of buffaloes during puerperium with or without
prostaglandin injection.
*Acknowledgment to FAPESP for financial support.
595
Molecular Evaluation of Developmental Competence of Oocytes Collected In Vivo

from Buffalo and Bovine Heifers during Winter and Summer
Roberta Machado FERREIRAac*, Carolina H. MACABELLIa, Nélcio Antônio Tonizza de

CARVALHOb, Júlia Gleyci SOARESc, Lindsay Unno GIMENESd, Márcio Leão FERRAZe,
Yeda Fumie WATANABEf, Osnir WATANABEg, Carlos Alberto RODRIGUESh, Laís Mendes
VIEIRAc, Flávio Vieira MEIRELLESa, Pietro Sampaio BARUSELLIc and Marcos Roberto
CHIARATTIa
a
School of Zootechnic and Food Engineering/USP, Pirassununga, SP, Brazil
b
APTA, Registro, SP, Brazil
c
School of Veterinary Medicine and Zootechnic/USP, São Paulo, SP, Brazil
d
School of Agrarian and Veterinarian Science/UNESP, Jaboticabal, SP, Brazil
e
Vida Reprodutiva Ltda, Cravinhos, SP, Brazil
f
Vitrogen, Cravinhos, SP, Brazil
g
WTA, Cravinhos, SP, Brazil
h
SAMVET, São Carlos, SP, Brazil
*Corresponding email: robertinhavet@yahoo.com.br
ABSTRACT
Buffaloes and bovines are polyestrous and seasonal or annual livestock, respectively, that show
reduced fertility during heat stress. To investigate whether reduced fertility is related to oocyte
competence in both species, immature oocytes from buffalo and bovine heifers were collected during
winter and summer and subjected to molecular analyses. In each season, heifers of both species had
their follicular wave emergence synchronized with a standard protocol (Ferreira et al., 2011). Before
being subjected to ovum pick up (OPU), cutaneous (CT; oC) and rectal (RT; oC) temperatures and
respiratory rate (RR; breaths/min) were measured. Oocytes’ RNA was extracted to evaluate the
expression of target genes related to mtDNA replication/transcription (PPARGC1A, TFAM and MT-
CO1), apoptosis (BAX and BCL2) and HS (HSP90AA1 and HSPA1AB). ACTB, HIST1H2AG and
GAPDH were initially chosen as housekeeping genes. In buffaloes, CT (35.0±0.4 vs 23.8±0.5), RT
(38.7±0.1 vs 38.0±0) and RR (21.3±1.2 vs 15.4±1.1) were higher during summer than winter.
However, in bovine heifers, RT (38.7±0.1 vs 38.6±0.1) and RR (44.8±1.5 vs 40.6±1.5) were similar in
both seasons, while CT (31.6 ±0.3 vs 30.2±0.3) was increased during summer. Reduced expression of
ACTB, HIST1H2AG and GAPDH was evidenced during summer, disqualifying them as housekeeping
genes. Similarly, the expression of all target genes was reduced during summer in oocytes of both
species. In summary, physiological responses to heat stress seem to be more intense in buffalo than
bovine heifers. However, in both species, negative effects of heat stress upon oocyte quality occur at
the molecular level and affects genes related to several biological functions.
Keywords: Buffalo, Bovine, Oocyte quality, Heat stress, Apoptosis, Gene expression
INTRODUCTION
The water buffalo (Bubalus bubalis) is a polyestrous seasonal livestock species of tropical and
sub-tropical environments (Zicarelli, 1994 ), while Holstein cattle (Bos taurus) is a polyestrous annual
livestock species evolved in Europe, and thus more sensitive to tropical conditions such as increased
temperature and humidity (Adeyemo et al., 1979; Turner, 1982; Madalena et al., 1990; Rocha et al.,
1998). However, even with such different degree of adaptation to heat conditions, it is well known that
in tropical countries heat stress is considered one of the main factors that lead to poor fertility in bovine

596
and buffalo females. High environmental temperature had a detrimental effect on the oocyte yield,
quality and developmental competence of buffalo oocytes collected from slaughterhouse ovaries
(Nandi et al., 2001). Similarly, oocytes harvested from Holstein cows during summer show reduced
ability to develop to the blastocyst stage after in vitro fertilization when compared to oocytes harvested
during winter (Rocha et al. 1998, Al-Katanani et al. 2002, Ferreira et al. 2011). Exposure of Holstein
heifers to heat stress between the onset of estrus and insemination increased the proportion of abnormal
and developmentally retarded embryos as compared to heifers maintained at thermoneutrality,
reinforcing the deleterious effect of heat stress on heifers’ oocytes competence (Putney et al. 1989).
The mammalian oocyte relies heavily on components stored in the ooplasm during oogenesis to
develop into a viable blastocyst. The components stored in the ooplasm, which include mRNAs,
proteins, organelles and energetic substrates, are critical during the earliest stages of embryonic
development, when the transcriptional activity of the embryo is limited, as reviewed by Picton et al.
(1998) and Meirelles et al. (2004). For instance, the amount of mitochondrial DNA (mtDNA) increases
sharply during oocyte growth (Cao et al. 2007, Cree et al. 2008, Wai et al. 2008) and the final amount
accumulated in fully-grown oocytes has been linked to their competence (Wai et al. 2010). Therefore,
any factor disturbing expression of genes controlling mtDNA replication during this window of
mtDNA replication may damage oocyte competence by a possible impairment of mitochondrial
function. Besides, expression of specific genes during the period the oocyte is growing may
predetermine gamete’s chance to develop to the blastocyst stage (i.e. increased BAX/BCL2 ratio), as
discussed by Gendelman and Roth (2012). Thus, it is possible that disturbing of oocyte growth is
associated to poorer embryonic developmental rates, which in turns have been related to the low
fertility during heat stress (Ferreira et al. 2011).
Based on these evidences, we hypothesized that altered expression levels of genes related to
mitochondria (PPARGC1A, NRF1, TFAM, POLG, POLG2 and MT-CO1), apoptosis (BCL2, BAX and
ITM2B) and heat stress (HSP90AA1 and HSPA1AB) are linked to the poorer competence of these
oocytes during summer heat stress.

Experimental design and sample collection
The experimental design and sample collection was conducted as described previously by
Ferreira et al. (2011). In brief, bovine (from commercial farms in Descalvado and São Pedro/SP,
Brazil) and buffalo (from an experimental farm in APTA-Registro/SP, Brazil) that were cycling, and
have never been inseminated were used. These heifers had their follicle wave emergence synchronized
based on a standard protocol (3 mg norgestomet, 2 mg estradiol benzoate and 0.530 µg sodium
cloprostenol on Day 0 and OPU on Day 5; Ferreira et al., 2011) before being subjected to OPU sessions
during the winter and the summer. Each animal was submitted only once to OPU and had their rectal
(RT; oC) and cutaneous (CT; oC) temperatures and respiration rate (RR; breaths/min) registered at that
time (Ferreira et al., 2011). Cumulus-oocyte complexes (COCs) recovered during OPU sessions were
morphologically classified based upon oocyte cytoplasm characteristics and the number of cumulus cell
layers as viable and unviable (Ferreira et al. 2011). Only viable COCs were used for molecular
analyses. These oocytes were denuded of cumulus cells by vortexing, extensively washed in PBS with
0.1% polyvinyl-pyrrolidone (PVP), and stored at -80ºC individually in 0.2 ml polystyrene PCR tubes in
1 µl of PBS with 0.1% of PVP and 1 U/µl of RNase inhibitor (RNase OUT, Invitrogen, Carlsbad, CA).
Relative quantification of mRNA amounts
The genes target used in this study were PPARGC1A, TFAM, MT-CO1, BAX, BCL2, HSP90AA1
and HSPA1AB (Kawarsky & King, 2001; Roth & Hansen, 2004; Chiaratti et al., 2010b). ACTB,
HIST1H2AG and GAPDH were initially chosen as housekeeping genes (Chiaratti et al., 2010b).
Primers and TaqMan MGB probes used for gene expression analyses of these genes were designed
using an assay by design service offered by Applied Biosystems. Before quantitative PCR
597
amplification, cDNA was pre-amplified using the TaqMan PreAmp Master Mix kit (Applied
Biosystems) according to manufacturer’s recommendations as follows: a 10 µl reaction was prepared
containing a pool of all primers used (each one at 45 nM) + 1 x TaqMan PreAmp Master Mix (Applied
Biosystems) + 4 µl of template (cDNA samples), subjected to 20 thermal cycles and stored at -20oC.
The linearity of amplification of all transcripts was determined as suggested by the manufacturer.
Quantitative PCR for relative quantification of gene-specific mRNA transcripts was done in 20 µl
reactions containing 1 x TaqMan MGB assay (consisting of specific primers and probe for a single
gene), 1 x TaqMan Gene Expression Master Mix and 2 µl of template. For each sample, pre-amplified
cDNAs were diluted 25-fold to be used as template. All gene-specific cDNAs amplified for a particular
sample were always run in the same PCR plate. The following cycling conditions were applied for
amplification: initial denaturation at 95oC for 15 min followed by 40 cycles consisting of 95oC for 15
sec and 60oC for 1 min. Probe fluorescence was read at the end of each extension step (60oC). Standard
curves were generated for each gene-specific cDNA analyzed using four 5-fold serial dilutions of
sample pools to check amplification efficiency. Since all assays showed to have high amplification
efficiency (roughly 100%), target transcript amounts in each sample were determined using the 2(-
Delta Delta C(T)) method (Livak & Schmittgen 2001). PCR products of each amplification assay were
run onto 2% agarose gels to check the specificity of the amplified fragment. When expression of a
specific transcript was not verified for a determined sample this result was excluded from further
analyses. Regarding all amplifications performed, most of transcripts did not fail to amplify or failed at
most twice.
Tests for normality of residuals and homogeneity of variances were conducted for each variable.
Data that did not fulfill the assumptions for ANOVA were transformed accordingly (log and square
root). All analyses were done by ANOVA using the GLIMMIX procedure of SAS version 9.2
(SAS/STAT, SAS Institute Inc., Cary, NC) for normal distribution. Significance was considered at P <
0.05. Values are presented as means ± standard error of the mean (S.E.M.). Analysis of gene expression
stability between winter and summer was done using qBase program based on geNorm analysis.

Although buffaloes have the ability to survive under unfavorable conditions, they are as
reproductively sensitive to heat stress as bovine. It was shown recently that the intensity of changes of
all physiological responses (i.e. RT, CT and RR) was more intense in young buffaloes than in the adults
indicating the greater susceptibility of younger buffaloes to heat stress. An opposite effect is been
observed in Holstein cattle when physiological responses are compared between heat-stressed cows and
heifers (Sartori et al., 2002). In this case, lactating Holstein cows have greater increase in RT, CT and
RR than do heifers in response to an increase in environmental temperature. Similarly, it was found
herein that buffalo heifers had increased their CT (35.0±0.4 vs 23.8±0.5), RT (38.7±0.1 vs 38.0±0) and
RR (21.3±1.2 vs 15.4±1.1) in summer compared to winter, while in bovine Holstein heifers RT
(38.7±0.1 vs 38.6±0.1) and RR (44.8±1.5 vs 40.6±1.5) were unaltered by season. In bovine, only CT
(31.6 ±0.3 vs 30.2±0.3) was increased during summer heat stress compared to winter.
Seventeen oocytes from buffalo and 32 from bovine heifers were analyzed. Reduced expression
of ACTB, HIST1H2AG and GAPDH was evidenced during summer heat stress compared to winter,
disqualifying them as housekeeping genes. Thus they were treated as extra target genes. Consequently,
further analyses of the other seven target genes were performed without normalizing them by
housekeeping as performed by Chiaratti et al. (2010). Similarly as ACTB, HIST1H2AG and GAPDH,
the expression of PPARGC1A, TFAM, MT-CO1, BAX, BCL2, HSP90AA1 and HSPA1AB transcripts
was reduced during summer heat stress in oocytes of both species. Thus, biological functions such as
mitochondrial transcription and replication, apoptosis and production of chaperones may be altered
during heat stress, disrupting oocyte quality.
598
The molecular mechanisms triggered by elevated temperatures in bovine oocytes are still not well
known. An improved knowledge of what genes and mechanisms are altered by heat stress and how it
affects reproduction may allow the development of strategies to attenuate heat stress negative effects
upon oocytes, thereby improving reproduction efficiency. The current study evidenced that the negative
effect of summer heat stress upon the quality of buffalo and bovine oocytes occurs at the molecular
level and affects at least ten genes related to several important biological functions.
ACKNOWLEDGMENTS
grindus Farm, Campestre Farm, MSD Saúde Animal and FAPESP (Process number.
2012/07510-1).
REFERENCES
Adeyemo, O., E. Heath, B. K. Adadevoh, J. Steinbach and E. A. Olaloku. 1979. Some physiological
and behavioral responses in Bos indicus and Bos taurus heifers acclimatized to the hot humid seasonal
equatorial climate. Int. J. Biometeorol 23:231-241.
Al-Katanani, Y. M., F. F. Paula-Lopes and P. J. Hansen. 2002. Effect of season and exposure to heat stress on
oocyte competence in Holstein cows. J. Dairy Sci. 85:390–396.
Cao L., H. Shitara, T. Horii, Y. Nagao, H. Imai, K. Abe, T. Hara, J. Hayashi and H. Yonekawa. 2007. The
mitochondrial bottleneck occurs without reduction of mtDNA content in female mouse germ cells. Nat.
Genet. 39:386-390.
Chiaratti, M. R., Meirelles, F. V. 2010a. Mitochondrial DNA copy number, a marker of viability for oocytes.
Biology of Reproduction. 83(1):1-2.
Chiaratti, M. R., F. F. Bressan, C. R. Ferreira, A. R. Caetano, L. C. Smith, A. E. Vercesi and F. V. Meirelles.
2010b. Embryo mitochondrial DNA depletion is reversed during early embryogenesis in cattle. Biology of
Reproduction 82:76-85.
Cree L. M., D. C. Samuels, S. C de Sousa Lopes, H. K. Rajasimha, P. Wonnapinij, J. R. Mann, H. H. Dahl and
P. F. Chinnery. 2008. A reduction of mitochondrial DNA molecules during embryogenesis explains the
rapid segregation of genotypes. Nat. Genet. 40:249-254.
Ferreira, R. M., H. Ayres, M. R. Chiaratti, M. L. Ferraz, A. B. Araújo, C. A. Rodrigues, Y. F. Watanabe, A. A.
Vireque, D. C. Joaquim, L. C. Smith, F. V. Meirelles and P. S. Baruselli. 2011. The low fertility of repeat-
breeder cows during summer heat stress is related to a low oocyte competence to develop into blastocysts.
J. Dairy Sci. 94:2383–2392.
Gendelman, M. and Z. Roth. 2012. Seasonal effect on germinal vesicle-stage bovine oocytes is further expressed
by alterations in transcript levels in the developing embryos associated with reduced developmental
competence. Biol. Reprod. 86:1-9.
Haque, N., A. Ludri, S. A. Hossain and M. Ashutosh. 2012. Comparative studies on temperature threshold for
heat shock protein 70 induction in young and adult Murrah buffaloes. J. Anim. Physiol. Anim. Nutr.
96(5):920-929.
Kawarsky, S. J. and W. A. King. 2001 Expression and localisation of heat shock protein 70 in cultured bovine
oocytes and embryos. Zygote 9:39-50.
Madalena, F. E., A. M. Lemos, R. L. Teodoro, R. T. Barbosa and J. B. N. Monteiro. 1990. Dairy production and
reproduction in Holstein–Friesian and Guzera crosses. J.Dairy Sci.73:1872–1886.
Meirelles F.V., A. R. Caetano, Y. F. Watanabe, P. Ripamonte, S. F. Carambula, G. K. Merighe and S. M. Garcia.
2004. Genome activation and developmental block in bovine embryos. Anim. Reprod. Sci. 82-83:13-20.
Nandi, S., M. S. Chauhan, P. Palta. 2001. Effect of environmental temperature on quality and developmental
competence in vitro of buffalo oocytes. Vet. Rec. 148:278–279.
Picton H., D. Briggs and R. Gosden. 1998. The molecular basis of oocyte growth and development. Mol. Cell.
Endocrinol. 145:27-37.
Putney, D. J., M. Drost and W. W. Thatcher. 1989. Influence of summer heat stress on pregnancy rates of
lactating dairy cattle following embryo transfer or artificial insemination. Theriogenology 31:765–778.
599
Rocha, A, R. D. Randel, J. R. Broussard, J. M. Lim, R. M. Blair, J. D. Roussel, R. A. Godke and W. Hansel.

1998. High environmental temperature and humidity decrease oocyte quality in Bos taurus but not in Bos
indicus cows.Theriogenology 49:657–665.
Turner, H. G. 1982. Genetic variation of rectal temperature in cows and its relationship to fertility. Animal
Production 35:401–412.
Roth, Z. and P.J. Hansen. Involvement of Apoptosis in Disruption of Developmental Competence of Bovine
Oocytes by Heat Shock During Maturation. 2004. Biology of Reproduction 71:1898–1906.
Zicarelli, L. 1994. Management under different environmental condition. Buff. J. (Suppl. 2), 17–38.
Wai T., A. Ao, X. Zhang, D. Cyr, D. Dufort and E. A. Shoubridge. 2010. The role of mitochondrial DNA copy
number in mammalian fertility. Biol Reprod 83:52-62.
Wai T., D. Teoli and E. A. Shoubridge. 2008 The mitochondrial DNA genetic bottleneck results from replication
of a subpopulation of genomes. Nat Genet 40:1484-1488.
600
The Carabao Development Program as the Cornerstone of the Livestock

Biotechnology Program in the Philippines
Eufrocina P. ATABAY 1, Edwin C. ATABAY2 and Libertado C. CRUZ1

1
Reproductive Biotechnology Unit, Philippine Carabao Center, Science City of Munoz, Nueva Ecija,
2
Philippine Carabao Center-CLSU, Science City of Munoz, Nueva Ecija, Philippines.
*
Corresponding e- mail s: bingay2003@yahoo.com
ABSTRACT
This paper highlights the expansion of the Research and Development (R&D) Program of
the Philippine Carabao Center (PCC) from water buffaloes to other livestock species. PCC has been
carrying out its mandate for almost two decades now to increase milk and meat productivity of
buffaloes aimed at improving the well-being of the farming communities. The Carabao
Development Program (CDP) is the main engine developed by PCC to carry out the mandate. R&D
is one component of the CDP which involves the use of biotechnology by various R&D disciplines.
Among these biotechnologies, reproductive technologies represent the major tool used by PCC for
genetic improvement of buffaloes in the country. In the area of breeding and genetics, recent
research efforts are geared towards genomic breeding or the use of DNA or marker assisted
technologies to complement the conventional method of genetic selection in buffaloes. Meanwhile,
research and development efforts in animal health have been intensified to enhance the Center’s
capability for disease detection, prevention and control. In the area of animal nutrition, research
initiatives on rumen biotechnology have resulted in improved rumen function and digestibility and
in increased feed utilization efficiency. On environment and biosafety, the application of bio
inoculants for efficient waste degradation has been practiced at the PCC Institutional Genepool.
Recognizing the great R&D achievements of the Center, in 2008, PCC became the lead agency in
livestock biotechnology by virtue of Administrative Order No. 9 issued by the Department of
Agriculture. This officially sealed and stamped the PCC as R&D institution for livestock
biotechnology. Essentially, the various biotechnologies developed in buffaloes by PCC are
therefore being extended and applied to other livestock species in the country.
Keywords: Philippine carabao, water buffalo, research and development, livestock biotechnology
INTRODUCTION
This paper presents the Research and Development (R&D) effort of the Philippine Carabao
Center (PCC) from water buffaloes to other livestock species. Consistent with the goal of the
Department of Agriculture-Philippines for food sufficiency, PCC has been carrying out its mandate
for almost two decades to increase milk and meat productivity of water buffaloes ultimately aimed
at improving the well-being of the rural farming communities. The Carabao Development Program
(CDP) is the main engine developed by PCC to achieve genetic improvement and increased
productivity in buffaloes. Importantly, biotechnology is an integral component of buffalo
production system which include among others, reproductive biotechnology, animal breeding and
genetics, animal health, animal nutrition, biosafety and environment, and more recently is the
application of DNA-based technology across those above-mentioned disciplines. Most recently,
with PCCs outstanding research accomplishments, the application of these technologies have been
extended to other livestock species in the country
Livestock Biotechnology Disciplines:
Reproductive Biotechnology
Among these biotechnologies, reproductive technologies represent the earliest and primarily
used by PCC for genetic improvement of buffaloes in the country. These include Artificial
Insemination and Estrus Synchronization, Semen cryopreservation, recently semen evaluation with
Computer Assisted System (CASA) all allowing maximum use of male genetics for breeding.
601
Meanwhile, in- vitro embryo production (IVEP), Ovum-Pick Up (OPU), Embryo Transfer, Somatic
Cell Nuclear Transfer, Intracytoplasmic Sperm Injection, Embryo and Oocyte Cryopreservation
technologies enable the full utilization of superior female buffaloes for breeding activities. In
addition, the Cryobanking of genetic materials (semen, embryo, oocytes and DNA, somatic cells)
has been initiated with the establishment of Cryobanking facilities at PCC. The Cryobanking
program intends to conserve various animal genetic biodiversity in the country.
Breeding and Genetics
Meanwhile, in the area of breeding and genetics, recent research efforts are geared towards
genomic breeding or the use of DNA or marker assisted technologies to complement the
conventional method of genetic selection in buffaloes/livestock. Specifically, on-going DNA or
molecular related researches are focused on selection of animals with quantitative traits (milk yields,
growth rate), breed identification, and parental/pedigree verification. More recently, research
projects on DNA-based screening genetic defects affecting livestock such as BLAD (Bovine
Leukocyte Adhesion Deficiency), Citr (Citrullinemia), CVM (Complex Vertebral Malformation)
and DUMPS (Deficiency of Uridine Monophosphate Synthase) which are of reproductive and
economic implications in livestock have been initiated. Interesting development along this area is
the PCC’s partnership with PCARRD, BAI, and the swine sector in pursuing a huge project on
DNA-based screening for genetic defects and economic traits of food animal such as the swine
species.
Animal Health
In the past few years, research and development efforts in animal health have been
intensified to enhance the Center’s capability for disease detection, prevention and control.
Molecular techniques have been developed which are complimentary to the existing conventional
cultural and serological diagnostic procedures. Diagnostic kit for rapid screening of FMD virus was
one of the earlier outputs of the Center’s engagement in molecular research. Various research works
on important diseases affecting buffaloes such as mastitis, brucellosis, trypanosomiasis, etc. have
been approached by molecular techniques. More recent efforts were on DNA-based detection and
characterization of some emerging bacterial, protozoan, viral, parasitic pathogens in livestock.
Likewise, local and international research collaborations have increased over short time primarily
for various zoonotic diseases (ex. bovine tuberculosis, leptospirosis, etc). On-going research
activities are the development of diagnostic kit for gastro-intestinal infection in swine and vaccine
production/trial for Schistosoma japonicum. The expanding research scope demonstrates the PCC’s
distinct capacity for DNA-based animal health R&D.
Animal Nutrition
In the area of animal nutrition, research initiatives on rumen biotechnology have resulted in
improved rumen function and digestibility and in increased feed utilization efficiency. One
important on-going biotechnology research is on improving the nutritive value of rice straw for
ruminant livestock by increasing its lignin degradability using selected tropical edible white rot
fungi. Moreover, studies on the supplementation with feed ingredient and compounds in the animal
diet can influence the microbial protein synthesis and improve rumen function and milk production.
It was found out that the feed augmentation with amino acids, as slow release NPN improved rumen
function, fiber degradation and enhanced microbial protein turn over in buffaloes which can also
result in higher milk fat content. Another major related research is on rumen microbiology which
deals with the production of alternative source of energy from food crops. Isolation,
characterization and preservation of rumen microbes associated with hydrolysis for cellulose
ethanol production is being conducted by PCC in collaboration and support of the Department of
Science and Technology.
Environment and Biosafety
On environment and biosafety, the application of bio inoculants for efficient waste
degradation has been practiced at the PCC Institutional Genepool. While efforts on methane gas
production from buffalo manure is a continuing effort of PCC as a part of mitigating measure
against the negative effect of animal production on the environment.
602
Pcc’s Mandate To Lead In Livestock Biotechnology

In 2008, by virtue of AO 9 of the Department of Agriculture, PCC was given additional
mandate to lead livestock biotechnology in the Department. This officially seals and stamped the
PCC as an R&D institution for livestock biotechnology, expanding its R&D function from being a
single commodity to a multi-commodity agency. Various biotechnology disciplines developed in
buffaloes are therefore being extended to other livestock such as cattle, goat, sheep and swine
species.
Technical Capability Building And Collaborations:
Parallel to the expansion of its research function, is the key requirement for an increase in
number and technical capability of PCC researchers. International research collaborations with
prestigious Universities and Research Institutions are extremely helpful and necessary to this effort
of strengthening our capacity for livestock biotechnology R&D. Similarly, active linkage with the
local research and partner institutions are tremendous support in the advancement of the national
livestock biotechnology agenda of the DA.
603
Factors Affecting the Performance of an Artificial Insemination Program in

North Coast Colombia
Jesus BERDUGO1,2*, Natalia VILLADA1, Juan ANGEL2 and Walter CARDONA-MAYA3

1
GINVER. Corporación Universitaria Remington, Medellín, Colombia
2
Colbufalos SAS, Planeta Rica, Colombia
3
Grupo Reproducción, ,Universidad de Antioquia. AA 1226 . Medellín, Colombia.
*Corresponding email: jesus.berdugo@remington.edu.co
ABSTRACT
Colombian buffalo breeders use artificial insemination (AI) with proven bulls for the
improvement programs. The aim of this work is to show the results of a natural cycle AI program.
This retrospective work was carried in North Coast, Colombia from August 2007 to March 2011.
Animals were maintained in the field, fed with grass (Brachiaria decumbens and Brachiaria
humidicola) and mineral supplementation with 6% of phosphorus adlibitum, , milked manually
once or twice a day with the aid of the calf. Results are shown in terms of pregnancy rates (PR), the
effect of season and bull were evaluated. 2127 inseminations from thirty five different bulls were
analyzed. The overall PR of the study was 46,32%. Monthly statistical differences in PR during the
evaluated period were found. Inseminations and the pregnancy rate shows a seasonal pattern during
the analyzed period, being the highest from August to January (51,98% ) and lowest from February
to June (44,04%, p value p= 0.000694). The bull have impact on PR varying from 17% to 84%
being constant during the year, some bulls obtain PR over the median during 9 or 8 months. The
results shown confirm the seasonal effect over the AI program and demonstrate the variation in
pregnancy rates with the bulls. The two mentioned factors must be taken in account to establish AI
programs, evaluation of the bulls in a different way in order to evaluate if they have defects in their
fertilizing capacity or other functional defects and the season.
Keywords: Artificial Insemination, Factors, Bull, Seasonality, biotechnologies, Colombia
INTRODUCTION
The buffalo population in Colombia has experienced a rapid growth during the last five
years as a consequence the demand for productive, efficient and reproductively adapted to
environment animals. One way to reach this objective by the use of reproductive biotechnologies
such us artificial insemination (AI), that has been used from 1976 (1).Natural AI breeding programs
have problems some of them associated to estrus detection which can be overcome using teaser
bulls, seasonality and fertility of the. The aim of this research was analyze the pregnancy rates (PR)
of a AI program according seasonality and bull effect during an extended period of five years.
MATHERIALS AND METHODS

This work were carried out in “Aguas Claras” a dairy buffalo farm, owned by Colbufalos
S.A.S, located in an ecological zone of tropical humid forest at 8° 28´ 69´´ N; 75° 16´ 54´´ O, North
Coast, Colombia, South America with an altitude 60 mosl and an annual precipitation of 1,600 mm.
The data were collected between August 2007 to March 2011.
Artificial Insemination Program: Forty-five days postpartum cows with body score above
3.0 (scale 1.0-5.0) were included in the AI program. They were distributed in groups of 50–60
females. Five epididimectomized (teaser) bulls were introduced into the female group which was
checked for estrus sings 4 times per day, 30 minutes per time, two times during each milking and
two times in the field. Insemination was performed by four different inseminators by transcervical
standard technique once 12 hours after the female no longer accept be mounted by the bull. Each
female stayed in the program during 60 days, time during which females were inseminated once or
604
twice. Inseminated females were evaluated by ultrasound and rectal palpation 30-40 and 60 days
after AI, respectively. Semen from thirty five bulls were used (32 Mediterranean and 3 Murrah).
Bulls with more than 10 inseminations were used for statistical analysis. p values p≤0.05 were
considered significant. Descriptive statistics for data are used.

During the period 2007 to 2011, two thousand one hundred and twenty seven (2127)
inseminations were performed with a general PR of 46,32% (1053/2127). PR ranged between 68%
in April and 26% in June. The overall PR of the study was 46,32%. Monthly differences in PR
during the evaluated period were found (Fig 1). Inseminations and the pregnancy rate shows a
seasonal pattern during the analyzed period, being the highest from August to January (51,98% )
and lowest from February to June (44,04%, p=0.000694), this data is confirmed by Aksoy et al,
2002. (6)
Figure 1. Variation in pregnancy rates during the year (2007-2011)
The results obtained in this paper agree with those presented previously by our research
team in 2012, there are no differences in PR (45,5% vs 46,32%) between both publications. (2) The
number of inseminations shows also a seasonal pattern. No differences in PR were found between
inseminators.
The results show differences in PR between bulls varying from 17% to 84% being constant
during the year, it means that some bulls obtain PR over the median for 9 or 8 months during the
year.
Table 1. Differences in pregnancy rates within bulls.

Times over the Times over the
Bull Bull
median median
1 2 7 1
2 2 8 2
3 8 9 2
4 5 10 7
5 7 11 1
6 9 12 6
The results confirm that PR in AI program using the natural seasonality, teaser bulls and estrus
detection by observation, as expected, are better than the these obtained using AI at fixed time FTAI
(3). It must be notice that in spite of good quality of frozen semen, there are differences in PR
perhaps related with subtle alterations of the sperm no detected using standard evaluation methods
605
As cattle semen analysis, it would be advisable to apply a set of functional tests for
buffaline semen additionally to the same standard evaluations (4) in order to identify subfertile or
infertile bulls.Usually semen used in AI programs is delivered to farm with a defined number of
motile spermaotozoa and morphology, this results shows that this information is not a “insurance”
to get pregnancies because it is possible that other aspects related fertilizing capacity and genetic
content that could affect the results of the program in terms of PR Teaser epididimectomized bulls
worked well contributing to reach the accomplished PR. Zicarrelli, et al (2) also reported increasing
in PR by using vasectomized bulls when compared with no bulls. They reported 9% of error in heat
detection with bulls in the AI program (5). Insemination performed 12 hours after the rejection of
the male by the female is a good practice to maintain pregnancy rates, especially due to the
variation in the length of estrus in buffaloes.
It has been shown fertility of the bulls, using the median as a value to express the fertility of the
bulls demonstrate that fertility of the bulls are constant during the period evaluated, high fertility
bulls are observed in spite of the different factors associated with the insemination program.
Koonjaenak, et al . demonstrate in Thailand that there are no differences in chromatin structure and
morphologically abnormal forms during seasons.
IMPLICATIONS
AI arranged to natural breeding seasonality in buffaloes involves reasonable PR, very close
to PR obtained in natural mating. Under field conditions we need to take in account the fertility of
the bulls and the season when people need to improve effiency of the program, experienced
inseminators help to maintain PR in reasonable levels. More research is needed to introduce new
semen parameters as part of the quality control of the straws to identify suitable and fertile semen.
REFERENCES
Jaramillo, G. 2000 Personal Communication
J.A. Variation in pregnancy rates in a natural artificial insemination program in water buffalo
(Bubalus bubalis) in the north coast colombia: Bull and month effect. Brasil, Ciência vol:22
fasc: 1 págs: 286 – 289, 2012
Zicarelli, L., L. Esposito, L. Carelli, G. Campanile, R. Di Palo and D. Armstrong. Effects of using
vasectomized bulls in artificial insemination practice on the reproductive efficiency of
Italian Buffalo Cows. Animal Reproduction Science, v. 47, n.3, p. 171- 180, 1997.
Andrabi, S.M.H. Factors affecting the quality of the cryopreserved buffalo (Bubalus bubalis) bull
spermatozoa. Rweprod. Domestic Anim. 44 552-659, 2009
Baruselli, P. S. Reprodução de bubalinos. Anais: I Simpósio brasileiro de bubalinocultura. Cruz das
Almas - BA, p. 117-153, 1996.
Aksoy, M., A. Kaya. Effect of seasonal conditions on oestrus occurrence and postpartum period in
Anatolian water buffaloes." Dtsch Tierarztl Wochenschr 109(9): 416-418. (2002).
Koonjaenak, S., A. Johannisson. (2007). "Seasonal variation in nuclear DNA integrity of frozen-
thawed spermatozoa from Thai AI swamp buffaloes (Bubalus bubalis)." J Vet Med A
Physiol Pathol Clin Med 54(7): 377-383.
606
First Report of an Artificial Insemination Program with Sexed Semen in North

Coast Colombia
Jesus BERDUGO1,2* and Juan ANGEL2
1
GINVER. Corporación Universitaria Remington, Medellín, Colombia
2
Colbufalos SAS Planeta Rica, Colombia
ABSTRACT
Recently, following the birth of the first buffalo calves using AI with sexed semen,
commercial interest to exploit sexing of semen in this species too is aroused. The aim of this work
is to show the results of this program in terms of pregnancy rates (PR) and female born. This
retrospective work was carried out from September 2010 to August 2012 in a farm located at
Caribbean Region of Colombia. All animals were maintained in the field, milked manually once or
twice a day with the aid of the calf, feed with grass (Brachiaria decumbens and Brachiaria
humidicola) and mineral supplementation with 6% of phosphorus adlibitum. Mediterranean sexed
semen from 7 different bulls from two different laboratories were used. Estrus detection were
performed using vasectomized bulls and females were inseminated 12 hours are the cow don’t
accept the male. 679 animals was inseminated, 279 animals were pregnant with a pregnancy rate of
39,61%, to date 111 animals was born with a 83% of concordance wih the gender of the straws. The
results obtained are lower than expected from accuracy sex sorting protocols, as expected lower
pregnancy rates were obtained when compared with non sexed semen (44.04%). No dystocia and
morphological defects were observed in the animals born and we are analyzing the real effect over
milk production. To date this is the largest series of artificial insemination with sexed semen in the
world and show the feasibility of the procedure in large scale.
Keywords: Artificial Insemination, Sexed semen, Improvement, biotechnologies
INTRODUCTION
The use of reproductive biotechnologies to produce meat and milk as part of the animal
breeding programs is mandatory in the field, one possibility is the use of the artificial insemination
(AI), recently the use of sexed semen became a reality, Artificial insemination with frozen semen
has been used in buffaloes in our country since 1976 (1), since 2002. It has been considerable
interest in insemination and two years ago (2010) sexed semen became available. First established
pregnancies in Mediterranean Italian buffaloes (Bubalus bubalis) following deposition of sexed
spermatozoa near the utero tubal junction were informed in 2005 (2) and in 2010 Lu and
coworkers report the born of calves after insemination deep in the horn (3). Sexing semen is may
cause damage of the sperm in spite of having ideal conditions such as highly fertile bulls, skilled AI
technicians, and well-managed heifers. The major cause of lowered fertility, however, likely is not
due to lowered numbers of live sperm per AI dose; the sorting process does damage sperm slightly
(4) although not nearly as much as cryopreservation this fact makes that veterinarians evaluate the
semen under field conditions, the aim this report is to show our first experience with sexed semen
in a farm of Colombia.

This work were carried out in “Aguas Claras” a dairy buffalo farm, owned by Colbufalos
SA.S, located in an ecological zone of tropical humid forest at 8° 28´ 69´´ N; 75° 16´ 54´´ O, North
Coast, Colombia, South America with an altitude 60 mosl and an annual precipitation of 1,600
mm. The data were collected between September 2010 and August 2012.
Artificial Insemination Program: Forty-five days postpartum cows with body score above 3.0 (scale
1.0-5.0) were included in the AI program. They were distributed in groups of 50–60 females. Five
607
epididimectomized (teaser) bulls were introduced into the female group which was checked for
estrus sings 4 times per day, 30 minutes per time, two times during each milking and two times in
the field. Insemination was performed by transcervical standard technique once 12 hours after the
female no longer accept be mounted by the bull. Four different inseminators inseminate the animals.
Each female stayed in the program during 60 days, time during which females were inseminated
once or twice. Inseminated females were evaluated by ultrasound machine and by rectal palpation
30-40 and 60 days after AI, respectively. Sexed semen from three Mediterranean bulls from COFA
(Central of Artificial Fecundation) were used
RESULTS
Pregnancy rates varies from 3.8% to 57% between bulls.
In terms of fecundity our results allow us to propose research projects related fertility in buffalo
specie due to the lower sperm concentration used to obtain pregnancies.
IMPLICATIONS
The use of sexed semen in farm animal as part of genetic improvement has been shown to be
feasible with variable degree of efficiency in a number of species, and proved to be economically
viable in cattle.
REFERENCES
Jaramillo, G. 2002 Personal communication.
Presicce G.A., S. Verberckmoes, E.M. Senatore, P. Klinc and D. Rath. 2005. First established
pregnancies in Mediterranean Italian buffaloes (Bubalus bubalis) following deposition of
sexed spermatozoa near the utero tubal junction. Reprod Domest Anim. 40(1): 73-5.
Lu, Y., M. Zhang, S. Lu, D. Xu, W. Huang, B. Meng, H. Xu and K. Lu. 2010. Sex-preselected
buffalo (Bubalus bubalis) calves derived from artificial insemination with sexed sperm.
Anim. Reprod. Scie. 119: 169-171.
Schenk, J.L., T.K. Suh, D.G. Cran and G.E. Seidel Jr. 1999. Cryopreservation of flow-sorted bovine
spermatozoa. Theriogenology. 52, 1375–1391.
608
Assessment Buffalo Spermatozoa Parameters in using Flow Cytometry
Jose Manuel MAYORGAa, Walter CARDONA-MAYAa and Jesus BERDUGOb *

a
Grupo Reproducción, Universidad de Antioquia, Medellin, Colombia.
b
GINVER, Corporación Universitaria Remington, Medellín, AA 1226 Colombia
ABSTRACT
There is a particular interest in describe the characteristics of semen using techniques that allow
the evaluation the sperm functionality. A straw of semen is a heterogenic population of spermatozoa
cells need to be qualified by its functional a physiological characteristics. Flow cytometry is a useful
tool that provides an accurate, objective, rapid and representative data per sample evaluation. This
methodology could have a great impact on the evaluation of buffalo sperm, especially in the artificial
insemination programs. This study was carry out by the evaluation of cryopreserved semen samples
from six buffaloes bulls of the murrah breed. Flow cytometry were used to measure membrane integrity
by SyBR14-PI, mitochondrial function by DIOC6/PI, metabolic reactive oxygen species by DCFDA
and SCSA using acridine orange. Sperm motility and viability were similar between bulls,
43.8±15.6millions/ml and 49.3±12.9% respectively. A consistent rate of sperm cells showed high
mitochondrial membrane potential 50.41±11.17% similar to the population with a plasma membrane
integrity 43.18±10.97%. The DNA fragmentation index was 5.46±14.36% and ROS production
55.46±16.15 arbitrary units. These results indicate that flow cytometry is an informative methodology
for a quick multiparametric assessment of sperm quality in buffalo. In particular, SCSA, mitochondria
potential and sperm membrane integrity resulted in sperm functional parameters sensitive enough for
the diagnosis of frozen-thawed semen fertilizing potential.
Keywords: Buffaloes, Colombia, Flow cytometry, Sperm quality
INTRODUCTION
Buffaloes are an interesting animal for meat and milk producer due to adaptability to different
environmental factors, especially in developing countries (Berdugo, 2011). In Colombia artificial
insemination programs with frozen semen are reported since 2002. The application of reproductive
technologies such us artificial insemination are new, as a consequence the application of the latest tools
to analyze the quality of the semen is mandatory, particularly functional assays. It has been reported the
buffalo semen is more vulnerable to hazards of freezing and thawing than cattle sperm (Andrabi, 2009),
as a consequence of its low membrane phospholipid content and its loss during freezing and thawing
functional assessment of the sperm are one the modern tools that andrologist has to evaluate the quality
of a sperm sample, trying to analyze the anatomy of the sperm and the potential to fertilize, more than
the quantity, morphology and motility of the cells. It includes the analysis of membrane integrity,
mitochondrial function, metabolic reactive oxygen species, DNA integrity and others test (Minervini et
al., 2012). The aim of this paper is to show the application of the flow cytometry to analyze functional
characteristics of frozen semen to be used in artificial insemination programs in Colombia.

Frozen semen from 6 murrah breed buffaloes were obtained from the University of Antioquia.
Bulls were from performance testing program of the Colombian Buffalo breeders association. 0.5 mL
straws were thawed by immersion in a water bath at 37°C for 30 seconds. Sperm concentration was
evaluated by hemocytometer method, vitality using eosin Y 0.5% and motility by direct estimation by
trained observers.
609
Reactive oxygen species measure (ROS)

The intracellular sperm ROS (H202, HO-, ONNOO-) levels were evaluated by flow cytomety
using 2’,7’-diclorofluorescein diacetate (DCFH-DA), a signal molecule, permeable, non fluorescent,
highly sensitive to intra cellular oxidation/reduction (redox) (Minervini et al., 2012).
Sperm Chromatin Structure Assay (SCSA)
Sperm chromatin structure assay was used to measure the DNA fragmentation index (%DFI), as
previously described by Evenson et al. (2013) with some modifications development in our laboratory
(Gil-Villa et al., 2009; Gil-Villa et al., 2010; Rodriguez et al., 2011). The DFI were determined by the
increased ratio of red/(red green) fluorescense.
Mitochondrial Membrane Potential (MMP)
The mitochondrial membrane potentials were measure (3,3′-dihexyloxacarbocyanine iodide
(DIOC6) staining, a cationic lipophilic and permeable carbocianine, able to release a high signal when
is incorporated by functionally mitochondria but, on pro-apoptotic cells the MMP could be disturbed
due to a change of the mitochondrial electrochemical gradient (Mayorga-Torres et al., 2012; Kadirvel
et al., 2009).
Plasma membrane integrity
The integrity of the plasma membrane was assessed with LIVE/DEAD sperm viability kit
(LIVE/DEAD Sperm Viability Kit, Molecular Probes, Eugene, OR, USA). Using this kit, one can
discriminate between intact and damaged spermatozoa. Due to its properties the SYBR-14 accumulates
inside of living cells and produce a green fluorescence and dead cells with permeable membrane are
permeable to PI and produce red fluorescence.
Data processing
All functional assays were performed by flow cytometric analysis, after acquisition data were
evaluated using WimnDi 2.9 Software (Scripps Research Institute, La Jolla, CA). Values obtained are
not compared between bulls.
RESULTS
These results show the evaluation of semen parameters of frozen-thawed semen to be used for
improvement of buffalo production in Colombia. Motility and vitality using standard/visual techniques,
compared with fluorocytometric techniques.
Sperm motility and viability were similar between the 6 bulls evaluated, 43.8±15.6 millions/m
and 49.3±12.9%, respectively. The mean value of sperm cells showed high mitochondrial membrane
potential 50.41±11.17% and plasma membrane integrity 43.18±10.97%. The DNA fragmentation index
was 5.46±14.36% and ROS production 55.46±16.15 arbitrary units. In the figure 1 we can see the
distribution of the functional parameters of each 7 one of the bulls evaluated.
610
Functional parameters
100 % Cells-High MMP
ROS production (AU)
80 % DFI
% Cells-Low PMM
60
40
20
1 2 3 4 5 6
Buffaloes
In
Figure 1. Functional parameters of Murrah frozen thawed semen.
Assessing sperm viability found relation between vital staining and obtained by performing
flow cytometry, there is a close description of the same characteristic in this population of cells.
Viability
100
Eosin Y
80
SyBR 14/IP
% Viable cells
60
40
20
0
1 2 3 4 5 6
Buffaloes
Figure 2. Vitality and plasma membrane integrity.
DISCUSSION
Data shown allow us to think that the sperm of the straws are enough for fertilization field trials
will give information that about the real fertility of each bull.
The values obtained are quit similar to those reported by Minerivini et al. (2012) in
mediterranean breed, regarding sperm viability, mitochondrial membrane potential and sperm
chromatin assay. ROS the data obtained are quite similar to those reported by Kadirvel et al. (2009) in
murrah. The data show an association between ROS production and DFI, were high levels of ROS are
related with increase of DNA fragmentation. The DFI only show a high index in the male six reaching
a 24.69% of fragmentation which also show an increase of the necrotic population cells.
611
We also observed that the percent of necrotic cells show a similar behavior with the viability,
which could lead us to think that these events are related or be describing a similar pattern.
Certainly functional evaluation of sperm give us more information related the material to obtain
pregnancies but it is quite difficult to be used for clinical decisions, many people usually ask about cut-
off values that today we are unable to propose them, specially after each step in evaluation we have less
functional sperm to fertilize, more research is needed to demonstrate the real usefulness of this
techniques in the clinical practice.
ACKNOWLEDGEMENTS
The authors want to thank Dr. Mario Fernando Ceron from University of Antioquia, Colombia
that kindly providing the semen samples evaluated in this paper. This study was supported by Estrategia de
Sosteniblidad 2012-2013 from University of Antioquia.
REFERENCES
Andrabi, S.M.H., 2009. Factors affecting the quality of the cryopreserved buffalo (Bubalus bubalis)
bull spermatozoa. Reprod Dom Anim. 44:552-659.
Berdugo, J. Historia de la aplicacion de las biotecnologias reproductivas a la cria del bufalo en
Colombia. Submited to publication Uni pluriversidad Vol 13(1): 7-13 2013
Evenson, D.P. 2013. Sperm chromatin structure assay (SCSA®). Methods Mol Biol. 927:147-64.
Gil-Villa, A.M., W. Cardona-Maya, A. Agarwal, R. Sharma, and A. Cadavid. 2010. Assessment of
sperm factors possibly involved in early recurrent pregnancy loss. Fertil Steril. 94:1465-72.
Gil-Villa, A.M., W. Cardona-Maya, A. Agarwal, R. Sharma, and A. Cadavid. 2009. Role of male factor
in early recurrent embryo loss: do antioxidants have any effect?. Fertil Steril. 92:565-71.
Kadirvel, G., S. Kumar, and A, Kumersan. 2009. Lipid peroxidation, mithocondrial membrane
potential and DNA integrity of spermatozoa in relation to intracellular reactive oxygen species
in liquid and frozen thawed buffalo semen. Anim. Reprod. Sci. 114:125-134.
Mayorga-Torres, B.J.M., W. Cardona-Maya, A. Cadavid, and M. Camargo. 2013. Evaluación de los
parámetros funcionales espermáticos en individuos infértiles normozooespérmicos. Actas Urol
Esp. (In press).
Minervini, F., R. Gusastamacchia, F. Pizzi, M. Aquilla, and V. Barile. 2012. Assesment of different
functional parameters of frozen-thawed buffalo spermatozoa by using cytofluorimetric
determinatios. Reprod Dom Anim. 10:1439-1446.
Rodriguez, E., A.M. Gil-Villa, D.C. Aguirre-Acevedo, W. Cardona-Maya, and A.P Cadavid. 2011.
Evaluación de parámetros seminales no convencionales en individuos cuyas parejas presentan
muerte embrionaria temprana recurrente: en busca de un valor de referencia. Biomedica.
31:100-107.
612
Effects of L-carnitine Supplemented in Maturation Medium on the Maturation

Rate of Swamp Buffalo Oocytes
Teewara PHONGMITR,1 Yuanyuan LIANG,1 Kanokwan SRIRATTANA,1 Kanchana
PANYAWAI,1 Nucharin SRIPUNYA,1 Chatahai TREETAMPINICH2 and Rangsun
PARNPAI1*
1
Embryo Technology and Stem Cell Research Center and School of Biotechnology, Suranaree
University of Technology, Nakhon Ratchasima 30000, Thailand
2
Department of Obstetrics and Gynecology, Faculty of Medicine Ramathibodi Hospital, Mahidol
University, Bangkok 10400, Thailand
*Corresponding Email: rangsun@g.sut.ac.th
ABSTRACT
In vitro maturation (IVM) is a first and crucial step to determine the success of in vitro
embryo production. L-carnitine enhances lipid metabolism in cells and exerts antioxidant effects as
well. Such properties are known to be beneficial for oocyte maturation. Thus, to investigate the
effects of L-carnitine during IVM on maturation rate of swamp buffalo oocytes, oocytes were
matured in IVM medium supplemented with 0.3, 0.6 and 1.2 mg/mL of L-carnitine (0.3, 0.6 and 1.2
L-carnitine groups, respectively). Oocytes matured in 0 mg/mL of L-carnitine were treated as a
control group. After IVM, oocytes were denuded by hyaluronidase and fixed with ethanol: acetic
acid (3:1) for 3 days. After that, the fixed oocytes were stained with 1% (w/v) orcein in acetic acid
for 10 min. Oocytes appearing a metaphase plate and one polar body were regarded as metaphase II
stage (MII). Supplementation of L-carnitine at 0.3 mg/mL (0.3 L-carnitine group) showed a
significantly higher MII rate than that in control group (83/123, 67.5% vs 69/120, 57.5%); however,
the rate was not significantly higher than those in 0.6 mg/mL (79/124, 63.7%) and 1.2 mg/mL
(79/123, 64.2%) groups. In conclusion, supplementation of L-carnitine during IVM could improve
the nuclear maturation rate to MII and 0.3 mg/mL was the optimal concentration.
Key words: L-carnitine, oocyte maturation, swamp buffalo
INTRODUCTION
Swamp buffalo (Bubalus bubalis) is a fundamental livestock resource in many countries,
especially Southeast Asia, providing milk, meat and labor. Although the in vitro embryo production
(IVEP) efficiency in buffalo has greatly improved over the year and offspring have been produced,
several issues still remain (Gasparrini et al., 2006). One of the major problems in this species is low
number of good quality oocytes (Totey et al., 1992; Khalil et al., 2010). The quality of oocyte is a
primary marker to predict developmental competence and quality of embryo both in vivo and in
vitro. Development of a suitable culture system for IVM of oocyte is a major component of the
IVEP procedures (Gasparrini, 2002). Interestingly, mitochondrial organization and adenosine
triphosphate (ATP) levels reflect the developmental competence of oocytes (Stojkovic et al., 2001).
Previous studies reported that L-carnitine plays a crucial role in lipid metabolism by transporting
the fatty acid into mitochondria for beta-oxidation to generate the ATP, which can enhance oocyte
maturation and embryo developmental competence in porcine (Somfai et al., 2011; Wu et al.,
2011). Supplementation of L-carnitine during oocyte maturation significantly increased lipid
metabolism and improved oocyte developmental competence in mice (Dunning et al., 2010;
Dunning et al. 2011). Moreover, L-carnitine exerts antioxidant properties by reducing the levels of
reactive oxygen species (ROS), H2O2 and increasing glutathione concentration during IVM of
porcine oocyte (Somfai et al., 2011; Wu et al., 2011). Hence, the aim of this study was to determine
the effects of L-carnitine addition to IVM medium in nuclear maturation on swamp buffalo oocytes.

613

In vitro oocyte maturation
Cumulus-oocyte complexes (COCs) were collected from 2-8 mm follicles and randomly
cultured in IVM medium [TCM199 supplemented with 10% fetal bovine serum (FBS), 0.02
AU/mL follicle stimulating hormone (FSH, Antrin®, Denka Phamaceutical Co., Kanagawa, Japan),
50 iu/mL human chorionic gonadotropin (hCG, Chorulon®, Intervet UK Ltd), and 1 µg/mL 17β
estradiol] supplemented with L-carnitine (C0158, Sigma) at 0 (control), 0.3, 0.6, and 1.2 mg/mL
(0.3, 0.6 and 1.2 L-carnitine group, respectively) at 38.5 °C under humidified atmosphere of 5%
CO2 in air for 23 h.
Evaluation of nuclear maturation
After IVM, COCs were denuded and mounted on glass slide and fixed with ethanol: acetic
acid (3:1). After 3 days fixation, oocytes were stained with 1% (w/v) orcein (Sigma) in acetic acid
for 10 min, and rinsed with glycerol: acetic acid: water (1:1:3) (Somfai et al., 2011). The nuclear
stage was evaluated under a phase-contrast microscope (Olympus, Tokyo, Japan). Oocytes
displaying a metaphase plate and one pink color polar body (PB) were regarded as at MII stage.

Effects of L-carnitine added to in vitro maturation medium on nuclear maturation rate of swamp
buffalo oocytes.
In the present study, L-carnitine supplementation to IVM medium improved nuclear
maturation of swamp buffalo oocyte (Table 1). The highest nuclear maturation rate was obtained in
the 0.3 L-carnitine group (83/123, 67.5%), which was significantly higher than that in control group
(69/120, 57.5%). This finding coincides with the results of Somfai et al. (2011), describing an
increase in maturation rate of porcine oocytes in the presence of L-carnitine. In contrast, our results
are different from that reported by Wu et al. (2011) who did not experience a significant
improvement of maturation rate by adding L-carnitine to the IVM medium. Moreover, they reported
reduced maturation rates at 2 mg/mL L-carnitine concentration. In our study, although the
maturation rate in 0.6 and 1.2 L-carnitine groups (79/124, 63.7% and 79/123, 64.2%) appeared
higher, but they were not significantly different when compared with control group (69/120, 57.5%).
The conflicting results may be due to the differences in IVM conditions and IVM procedures or
different species used.
The improvement of oocytes maturation by supplementation of L-carnitine during IVM may
be resulted from the utilization of lipid via β-oxidation to generate the ATP which is necessary for
the resumption of meiosis and cytoplasmic maturation (Ferguson & Leese, 2006). It was reported
that supplementation of L-carnitine during oocyte maturation significantly increased β-oxidation
and lipid metabolism which resulting in improving oocyte developmental competence (Dunning et
al., 2010; Dunning et al., 2011). Moreover, enhancing mitochondrial functions by using L-carnitine
improved oocyte maturation, underlining the importance of lipid metabolism for nuclear and
cytoplasmic maturation of oocytes (Somfai et al., 2011). Additionally, it was revealed that the lipid
content in bovine and porcine oocytes decreased after IVM, indicating that lipid was used as a key
energy source during maturation (Cetica et al., 2002; Sturmey and Leese, 2003; Ferguson and
Leese, 2006). This suggested that endogenous lipid metabolism plays an important role during
oocyte maturation. Besides its role in metabolism, L-carnitine acts as an antioxidant reducing the
levels of ROS in oocytes during IVM (Somfai et al., 2011; Wu et al., 2011). Incubation of oocytes
and embryos with L-carnitine was reported to reduce cytoskeleton damage and decrease the level of
apoptosis via its antioxidant action (Mansour et al., 2009). Since both lipid metabolism and redox
status are important for the maturation and further competence of oocytes, the present study
proposes that the improvement of maturation of swamp buffalo oocytes by treatment with L-
carnitine may either be due to (1) its action as an enhancer of lipid metabolism via β-oxidation to
generate the ATP which is necessary for oocyte metabolism; and/or (2) it acts as an antioxidant
(ROS scavenger) by preventing apoptotic events in oocytes (Mansour et al., 2009) through its
antioxidant effect. In conclusion, supplementations of L-carnitine in IVM medium improved the
614
nuclear maturation of buffalo oocytes. Further studies are still needed for the clarification of the
effect of L-carnitine addition on embryo development to the blastocyst stage.
REFERENCES
Cetica P., L. Pintos, G. Dalvit and M. Beconi. 2002. Activity of key enzymes involved in glucose
and triglyceride catabolism during bovine oocyte maturation in vitro. Reproduction 124:
675-681.Dunning K.R., K. Cashman, D.L. Russell, J.G. Thompson, R.J. Norman and R.L.
Robker. 2010. Beta-oxidation is essential for mouse oocyte developmental competence and
early embryo development. Biol. Reprod. 83: 909-918.
Dunning K.R., L.K. Akison, D.L. Russell, R.J. Norman and R.L. Robker. 2011. Increase beta-
oxidation and improved oocyte developmental competence in response to L-carnitine during
ovarian in vitro follicle development in mice. Biol. Reprod. 85: 548-555.
Ferguson E.M. and H.J. Leese. 2006. A potential role for triglyceride as an energy source during
bovine oocyte maturation and early embryo development. Mol. Reprod. Dev. 73: 1195-1201.
Gasparrini B. 2002. In vitro embryo production in buffalo species: state of the art. Theriogenology
57: 237-256.
Gasparrini B., L. Boccia, J. Marchandise, R.D. Palo, F. George, I. Donnay and L. Zicarelli. 2006.
Enrichment of in vitro maturation medium for buffalo (Bubalus Bubalis) oocytes with thiol
compounds: Effects of cystine on glutathione systhesis and embryo development.
Khalil W.K.B., S.S. Alam, I.A.H. Barakat, A.M. Hassan and K.F. Mahrous. 2010. Effect of in vitro
culture on the expression of genes enhanced meiotic progression in Egyptian buffalo
oocytes. J. Appl. Biosci. 32: 1977-1988.
Mansour G., H. Abdelrazik, R.K. Sharma, E. Radwan, T. Falcone and A. Agarwal. 2009. L-
carnitine supplementation reduces oocyte cytoskeleton damage and embryo apoptosis
induced by incubation in peritoneal fluid from patients with endimetriosis. Fertil. Steril. 91:
2079-2086.
Somfai T., M. Kaneda, S. Akagi, S. Watanabe, S. Haraguchi, E. Mizutani, T.Q. Dang-Nguyen, M.
Geshi, K. Kikuchi and T. Nagai. 2011. Enhancement of lipid metabolism with L-carnitine
during in vitro maturation improves nuclear maturation and cleavage ability of follicular
porcine oocytes. Reprod. Fertil. Dev. 23: 912-920.
Stojkovic M., S. Machado, P. Stojkovic, V. Zakhartchenko, P. Hutzler, P.B. Goncalves and E.
Wolf. 2001. Mitochondrial distribution and adenosine triphosphate content of bovine
oocytes before and after in vitro maturation: Correlation with morphological criteria and
developmental capacity after in vitro fertilization and culture. Biol. Reprod. 64: 904-909.
Sturmey R.G. and H.J. Leese. 2003. Energy metabolism in pig oocytes and early embryos.
Reproduction 126: 197-204.
Totey S.M., G. Singh, M. Taneja, C.H. Pawshe and G.P. Talwar. 1992. In vitro maturation,
fertilization and development of follicular oocytes from buffalo (Bubalus Bubalis). J.
Reprod. Fert. 95: 597-607.
Wu G.Q., B.Y. Jia, J.J. Li, X.W. Fu, G.B. Zhou, Y.P. Hou and S.E. Zhu. 2011. L-carnitine enhances
oocyte maturation and develpoment of parthenogenetic embryos in pig. Theriogenology 76:
785-793.
615
Table 1. Effects of L-carnitine added to in vitro maturation medium on maturation rate of swamp
buffalo oocytes.*
No. (%) of metaphase II

Groups No. of oocytes
oocytes
0 mg/mL 120 69 (57.5) b
0.3 mg/mL 123 83 (67.5) a
0.6 mg/mL 124 79 (63.7) ab
1.2 mg/mL 123 79 (64.2) ab
*7 replicates were performed.

Different superscripts within a column indicate significant differences (P<0.05, ANOVA).
A B C
D E F
Figure 1. Nuclear status of swamp buffalo oocytes after in vitro maturation.

A: GV stage, B: GVBD stage, C: Metaphage I stage, D: Metaphage II stage,
E: Anaphase I stage, F: Telophase I stage.
616
Influence of Growth Factors on Survival and Development of Swamp Buffalo

Early Antral Follicle cultured In Vitro
Kwanrudee KAEWMUNGKUNa, Kanokwan SRIRATTANAa, Kanchana PUNYAWAIa,

Nucharin SRIPUNYAa, Yuanyuan LIANGa, Siwat SANGSRITAVONGb and Rangsun
PARNPAIa*
a
Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of
Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000,
Thailand
b
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science
Park, Phahonyothin Road, Khlong Nueng, Khong Luang, Pathum Thani 12120, Thailand
*
Corresponding email: rangsun@g.sut.ac.th
ABSTRACT
Buffalo ovary has a large number of follicles but a few follicles are able to grow and
ovulate. After collecting oocytes for in vitro maturation, a lot of small follicles still remained in the
ovaries. In this study, survival and development of these remaining early antral follicles in the
ovaries were investigated after in vitro culture. Growth factors may be essentially required for
follicle development. Therefore, the aim of this study was to examine the effects of growth factors,
basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I), on in vitro culture of
swamp buffalo early antral follicles. The follicles isolated from buffalo ovaries were divided into
two groups, according to their diameters, Group I: 400-599 µm and Group II: 600-799 µm. The
follicles were embedded in collagen gel and cultured in the medium supplemented with either one
of the two growth factors or combinations of the two factors for 14 days. The diameters of follicles
were measured at 7 and 14 days of culture. Culture medium supplemented with bFGF showed
significantly the highest survival rate in all size groups of the follicles (44.8% and 32.7% in groups
I and II, respectively), except for bFGF+IGF-I group in Group I. However, when combined with
IGF-I, its promoting effect on follicular survival and development was neutralized. Follicles from
both Groups I and II cultured in medium supplemented with bFGF for 14 days (also follicles
cultured for 7 days in Group I ) tended to have increased follicle size compared to other groups;
follicles from the bFGF group showed a significantly higher increase in size compared to
bFGF+IGF-I group. IGF-I had on effects on follicular survival and development in both Groups I
and II.
In conclusion, bFGF is a suitable supplement for in vitro culture of the early antral follicles
in swamp buffalos in terms of the survival for both Groups I and II and development for Group I.
Keywords: swamp buffalo, early antral follicle, in vitro cuture, growth factor
INTRODUCTION
The assisted reproductive technologies (ARTs) such as in vitro fertilization (IVF), cloning
and intracytoplasmic sperm injection have been applied for buffalo production. Source of oocytes
using in ARTs was mainly in vitro matured (IVM) oocytes aspirated from graafian follicles. After
oocyte collection, preantral and early antral follicles still remained in the ovaries. In vitro follicle

617
culture might be a promising technique to make use of oocytes from theremaining follicles. Also the
technique could be used to study the mechanism of follicle survival and development after in vitro
culture. The first successful follicle culture was carried out in mouse by Eppig and O’Brien (1996),
with a live pup born from IVF embryos derived from primordial follicles cultured in vitro.
Moreover, Yamamoto et al. (1999) successfully produced a live calf through IVF u of IVM oocytes
obtained from in vitro cultured early antral follicles in diameter of 500-700 µm. Additionally, Gupta
and Nandi (2010) cultured riverine buffalo follicles in diameter of 40-100, 101-200, 201-300, 301-
400 and 401-500 µm; their viability rates were 76.1%, 78.1%, 85.2%, 92.5% and 92.6%,
respectively. Based on the previous report, in this study follicles in diameter of 400-599 and 600-
799 µm were used for in vitro culture. Moreover, many previous reports stated that additional
growth factors such as insulin-like growth factor-I (IGF-I) (Zhou and Zhang 2005; Sharma et al.,
2009; Gupta et al., 2010; Magalhães-Padilha et al., 2012) and basic fibroblast growth factor (bFGF)
(Nilsson et al., 2001; Zhou and Zhang, 2005; Sharma et al., 2009) and nutrients may be essentially
required for follicle development (Saha et al., 2000; Gupta et al., 2002; Zhou and Zhang, 2005;
Rajarajan et al., 2006; Sharma et al., 2009; Gupta et al., 2010). Thus, the objective of this study is to
examine the effect of growth factors (IGF-I and bFGF) on survival and development of early antral
follicles cultured in vitro in swamp buffalos.

Collection of early antral follicles
The ovaries were washed in 70% ethanol (The liquor distillery organization, Chachoengsao,
Thailand) for 1 minute followed by rinsing in 0.9% NaCl (Carlo Erba, Milano, Italy) for another 1
minute. Cortical slices excised from the ovaries were cut into pieces of 1-2 mm with a surgical
blade. The minced tissues were rinsed in 0.9% NaCl for 3 times and placed in HEPES (Sigma-
Aldrich, St. Louis, MO, USA) -buffered TCM 199 (Sigma-Aldrich) supplemented with 0.1%
collagenase type IV (Gibco BRL, Grand Island, NY, USA) with 40 units/ml DNase (Roche,
Mannheim, Germany) and then the tissues were incubated at 37ºC for 1 h. After digestion, the
follicles were isolated using fine forceps in HEPES-buffered TCM 199 supplemented with 300
IU/ml penicillin G (Sigma-Aldrich) and 0.3 mg/ml streptomycin (Sigma-Aldrich) under a
stereomicroscope. The diameters of follicles were measured with a video micrometer on a screen
connected to a CCD camera on an inverted microscope (IX71, Olympus) to categorize into 2 groups
depending on their diameters (Group I: follicles of 400-599 µm in diameter, Group II: follicles of
600-799 µm in diameter).
In vitro culture of early antral follicles
The follicles from all groups were embedded in collagen gel for in vitro culture. Briefly, a
collagen mixture was prepared by mixing a 0.3% acid collagen solution (Cellmatrix Type I-A®,
Osaka, Japan), 10 times-concentrated TCM 199, and 0.05 N sodium hydroxide solution (Carlo
Erba) at the ratio of 8:1:1 (v:v:v) on ice with additional supplementations of 22 mg/ml sodium
bicarbonate (Sigma-Aldrich) and 47.7 mg/ml HEPES. Base layer was prepared by filling 0.4 ml
collagen mixture into 4–well dish. Then the dish was placed on a warm plate at 35°C for 5 min.
Four early antral follicles were placed on the base layer. The remaining collagen mixture was
warmed at 25°C for 5 minutes. The base layer contained 4 early antral follicles was overlaid with
0.4 ml warm collagen mixture. And then the dish was incubated at 37°C for 10 min. After
gelatinization, 5 ml in vitro growth (IVG) medium, TCM 199 supplemented with 2 mM L-
618
glutamine (Sigma-Aldrich), 0.23 mM sodium pyruvate (Sigma-Aldrich), 2 mM hypoxanthine

(Sigma-Aldrich), 1% insulin-transferin-selenium (ITS) (Sigma-Aldrich: insulin 6.25 µg/ml,
transferrin 6.25 µg/ml, selenium 6.25 ng/ml), 100 IU/ml penicillin G, 0.1 mg/ml streptomycin, and
1 µg/ml FSH (Folltropin-v® Bellevi, Ontario, Canada) were added on top of the collagen gel.
Control medium was IVG medium without growth factors. Supplementation media were IVG
medium with 1) bFGF (50 ng/ml), 2) IGF-I (100 ng/ml) and 3) bFGF (50 ng/ml?) + IGF-I (100
ng/ml). The dish was cultured at 38.5°C under a humidified atmosphere with 5% CO2 in air for 14
days. Half volume of IVG medium was replaced every 2 days. The diameters of the follicles were
measured at 7 and 14 days of culture with a video micrometer on a screen connected to a CCD
camera on an inverted microscope.
Data on survival rates after in vitro culture of early antral follicles were evaluated by Chi-
square with at least 10 replications per treatments. Data on the increase in diameter of early antral
follicles were statistically analyzed by Completely Randomized Design (CRD). Analyses of
variances were performed by Statistical Analysis System (SAS Institute Inc., Cary, USA).

In Group I (ø 400-599 µm), a significantly higher rate of follicles from bFGF treated group
(44.8%) was able to survive after culture compared to the control (26.1%) and IGF-I (21.6%)
groups; however, the rate is similar to the bFGF+IGF-I group (37.3%). Increase in follicle diameter
in Group I on Day 7 was not significantly different among all groups. However, on Day 14 the
supplementation of bFGF showed a significant increase in the diameter (387.6±15.9µm) compared
to the control (228.0±10.6µm); however, the effect was neutralized when combined with bFGF
(192.1±14.3µm) and the increase was not different from the bFGF group (260.1±15.0µm) (Table 1).
In Group II (ø 600-799 µm), the survival rate of follicles from the bFGF groupt (32.7%) was
significantly higher than those of IGF-I (19.0%), bFGF+IGF-I (11.8%) and control groups (12.8%).
The supplementation of the growth factors had no effects on the increase in follicle diameter in this
group on Days 7 and 14; whereas, the follicles from bFGF+IGF-I group had a significantly lower
increase compared to the control group (Table 2).
The results showed that follicles from all groups were able to grow. The control medium had
essential hormones and chemicals for follicular development such as FSH, ITS and hypoxanthine.
From the results, follicles from both Groups I and II cultured in medium supplemented with bFGF
had the highest survival rate (except for bFGF+IGF-I group in Group I) among all groups, and
tended to have increased follicle size compared to other groups; follicles from the bFGF group
showed a significantly higher increase in size compared to bFGF+IGF-I group. This result agrees
with previous studies reporting that bFGF increased survival rate in goat preantral follicles cultured
in vitro (Zhou and Zhang, 2005), and that bFGF at a high concentration had beneficial effects on
survival rates of sheep follicles cultured in vitro (Peng et al., 2010). Moreover, bFGF could promote
follicular cell growth and development in mouse primordial and early antral follicles (Nilsson et al.,
2001). In this study, IGF-I had no effects on follicular survival and development, and this fact is not
consistent with previous reports (Sharma et al., 2009; Gupta el al., 2010); IGF-I was reported to
maintain survival rate and promote follicular development and induce antral formation in riverine
buffalo follicles (Sharma et al., 2009; Gupta el al., 2010). The reason for this discrepancy is not
clear.
619
In conclusion bFGF was the most suitable growth factor for in vitro culture of buffalo early
antral follicles. However, when combined with IGF-I, its promoting effect on follicular survival and
development was neutralized.
ACKNOWLEDGEMENTS
This study was supported by the Higher Education Research Promotion and National
Research University Project of Thailand, Office of the Higher Education Commission and
Suranaree University of Technology. K. Srirattana, S. and Y. Liang were supported by SUT
postgraduate research fellowships.
REFERENCES
Eppig, J.J. and M. J. Brien. 1996. Development in vitro of mouse oocytes from primordial follicles.
Biol. Reprod. 54: 197-207.
Gupta, P.S., S. Nandi, B. Ravindranatha and P. Sarma. 2002. In vitro culture of buffalo (Bubalus
bubalis) preantral follicles. Theriogenology 57: 1839-1854.
Gupta, P.S. and S. Nandi. 2010. Viability and growth of buffalo preantral follicles and their
corresponding oocytes in vitro: effect of growth factors and beta mercaptoethanol. Reprod.
Domest. Anim. 45: 147-154.
Magalhães-Padilha, D.M., A.B. Duarte, V.R. Araújo, M.V. Saraiva, A.P. Almeida, G.Q. Rodrigues,
M.H. Matos, C.C. Campello, J.R. Silva, M.O. Gastal, E.L. Gastal and J.R. Figueiredo.
2012. Steady-state level of Insulin-like growth factor-I (IGF-I) receptor mRNA and the
effect of IGF-I on the in vitro culture of caprine preantral follicles. Theriogenology 77:
206-213.
Nilsson, E.E., J.A. Parrott and M.K., Skinner. 2001. Basic fibroblast growth factor induces
primordial follicle development and initiates folliculogenesis. Mol. Cell. Endocrinol. 175:
123-130.
Peng, X., M. Yang, L. Wang, C. Tong and Z. Guo. 2010. In vitro culture of sheep lamb ovarian
cortical tissue in a sequential culture medium. J. Assist. Reprod. Genet. 27: 247-257.
Rajarajan, K., B.S. Roa, R. Vagdevi, G. Tamilmani, G. Arunakumari, M. Sreenu, D. Amarnath, B.R.
Naik and V.H. Rao. 2006. Effect of various growth factors on the in vitro development of
goat preantral follicles. Small. Rumin. Res. 63: 204-212.
Saha, S., M. Shimizu, M. Geshi and Y. Izaike. 2000. In vitro culture of bovine preantral follicles.
Sharma, G.T., P.K. Dubey and S.K. Meur. 2009 Survival and developmental competence of buffalo
preantral follicles using three-dimensional collagen gel culture system. Anim. Reprod. Sci.
114: 115-124.
Zhou, H and Y. Zhang. 2005. Effect of growth factor on in vitro development of caprine preantral
follicle oocytes. Anim. Reprod. Sci. 90: 265-272.
Zhou, H and Y. Zhang. 2005. Regulation of in vitro growth of preantral follicles by growth factors
in goats. Domest. Anim. Endocrinol. 28: 235-242.
620
Table 1. Increase of follicle diameter after in vitro culture in Group I (Ø 400-599 µm).
Increase in diameter (µm)

Survival rate Initial diameter (µm) (mean ± S.E.M.)
Growth factors
(%) (mean ± S.E.M.)
Day 7* Day 14
30/115
Control 532.3±8.1 165.8±9.5 228.0±10.6b
(26.1b)
52/116
bFGF 500.5±5.9 196.5±9.9 387.6±15.9a
(44.8a)
25/116
IGF-I 502.6±6.4 210.3±9.0 260.1±15.0ab
(21.6b)
44/118
bFGF+IGF-I 512.6±4.6 220.3±8.0 192.1±14.3b
(37.3ab)
*
Day 0 = the beginning of in vitro culture
a,b
Mean with different superscripts in the same column differs significantly (survival rate P<0.05,
Chi-square, increase in diameter P<0.05, CRD).
Table 2. Increase of follicle diameter after in vitro culture in Group II (Ø 600-799 µm).
Increase in diameter (µm)

Survival rate Initial diameter (µm) (mean ± S.E.M.)
Growth factors
(%) (mean ± S.E.M.)
Day 7* Day 14
14/109
Control 686.1±4.8 211.7±9.6ab 152.5±12.9ab
(12.8b)
35/107
bFGF 684.7±4.7 259.1±14.1a 230.4±13.8a
(32.7a)
20/105
IGF-I 714.3±5.8 184.8±9.7ab 101.3±14.7ab
(19.0b)
13/110
bFGF+IGF-I 697.5±5.4 107.5±5.6b 60.0±4.2b
(11.8b)
*
Day 0 = the beginning of in vitro culture
a,b
Mean with different superscripts in the same column differ significantly (survival rate P<0.05,
Chi-square, increase in diameter P<0.05, CRD).
621
Buffalo Genetics and Breeding
Genetic Parameters for Reproductive Traits of Crossbred Buffaloes from Brazil,

Estimated by Bayesian Inference
Carlos Henrique Mendes MALHADOa*, A.A. RAMOSb, P.L.S. CARNEIROa and

J.A. CARRILLOc
a
State University of Southwest Bahia, Jequié, BA, Brazil
b
Paulista State Univeristy, Botucatu, SP, Brazil
c
University of Maryland, College Park, MD, US
*corresponding email: carlosmalhado@gmail.com
ABSTRACT
The objective of the study was to estimate heritability for calving interval (CI) and age at
first calving (AFC) and also calculate repeatability for CI in buffaloes using Bayesian inference.
The Brazilian Buffaloes Genetic Improvement Program provided the database. Data consists on
information from 628 females and four different herds, born between 1980 and 2003. In order to
estimate the variance, univariate analyses were performed employing Gibbs sampler procedure
included in the MTGSAM software. The model for CI included the random effects direct additive
and permanent environment factors, and the fixed effects of contemporary groups and calving
orders. The model for AFC included the direct additive random effect and contemporary groups as a
fixed effect. The convergence diagnosis was obtained using Geweke that was implemented through
the Bayesian Output Analysis package in R software. The estimated averages were 433.2 days and
36.7months for CI and AFC, respectively. The means, medians and modes for the calculated
heritability coefficients were similar. The heritability coefficients were 0.10 and 0.42 for CI and
AFC respectively, with a posteriori marginal density that follows a normal distribution for both
traits. The repeatability for CI was 0.13. The low heritability estimated for CI indicates that the
variation in this trait is, to a large extent, influenced by environmental factors such as herd
management policies. The age at first calving has clear potential for yield improvement through
direct selection in these animals.
Keywords: age at first calving, calving interval, Gibbs sampler, heritability, repeatability
INTRODUCTION
Buffaloes are regarded as a good option to dairy breeding, because of their potential milk
production in distinct environment conditions. The profits from milk production systems are
directly influenced by female reproductive characteristics, like age at first calving and calving
interval.
REML and Bayesian methods have been applied extensively in animal breeding to estimate
covariance components and genetic parameters. When a large data set is analyzed, a priori
information tends to be subjugated by the likelihood function in the establishment of the a
posteriori distribution. In this case, the parameter estimates are close to those obtained by methods
based on likelihood functions. However, this may not be the case when the sample size is limited
because the maximum likelihood procedure only produces well-defined properties when the sample
size is sufficiently large (Gianola and Fernando, 1986). The Bayesian method is well suited for
analyzing small populations when extensive historical information is available (Carneiro et al.,
2007). The objective of the study was to estimate heritability for calving interval (CI) and age at
first calving (AFC) and also calculate repeatability for CI in buffaloes using Bayesian inference

Program provided the database. Data consists on information from 628 females and four
different herds, born between 1980 and 2003.

623
In order to estimate the variance, univariate analyses were performed employing

Gibbs sampler procedure included in the MTGSAM software as described by Van Tassell and Van
Vleck (1995). The model for CI included the random effects direct additive and permanent
environment factors, and the fixed effects of contemporary groups and calving orders. The model
for AFC included the direct additive random effect and contemporary groups as a fixed effect. The
CG contained animals of the same herd, season and year of birth or parturition. The MTGSAM uses
the Gauss–Seidel iterative method in the mixed model equation to obtain an initial value for the
fixed and random effects to be used in the Gibbs sampler. The initial numbers were arbitrarily
obtained using a single chain with 100,000 iterations. The convergence diagnosis were analyzed
through the Geweke (1992) and the burn-in, thinning and chain length were determined by Raftery
and Lewis (1992) method using the algorithm implemented in R software through the package BOA
(Bayesian Output Analysis) (Smith, 2005).

The estimated averages were 433.2 days and 36.7months for CI and AFC, respectively. The
means, medians and modes of estimates of variance components for calving interval and age at first
calving were very similar (Table 1), with exception of the permanent environment variance for CI.
This is in accordance with Carlin and Louis (2000), who reported that similar values are expected
for an a posteriori marginal density that follows a normal distribution. The means, medians and
modes for the calculated heritability coefficients for traits were similar (Figure 1 and 2), with a
posteriori marginal density that follows a normal distribution for both traits.
The heritability coefficient for AFC was high (0.42). The age at first calving has clear
potential for yield improvement through direct selection in these animals. On the other hand, the
heritability for CI was low (0.10). This is in agreement with values reported in the literature on
dairy cattle (Abdallah and McDaniel, 2000) and buffaloes (Ramos et al., 2006; Seno et al., 2010).
However, studies on buffaloes by Mahdy et al. (1999) and Cassiano et al. (2004) reported values
equal to 0.17 and 0.26, respectively. The low heritability estimated for CI indicates that the
variation in this trait is, to a large extent, influenced by environmental factors such as herd
management policies.
REFERENCES
Abdallah, J.M. and B.T McDaniel. 2000. Genetic parameters and trends of milk, fat, days open and
body weight after calving in North Carolina experimental herds. J. Dairy Scie. 83:1364-
1370.
Carlin, B.P. and T.A. Louis. 2000. Bayes and empirical bayes methods for data analysis. 2th Ed.
London: Chapman and Hall. pp. 419.
Geweke, J. 1992. Evaluating the accuracy of sampling-based approaches to the calculations of
posterior moments. In: Bayesian statistics (Ed. J.M. Bernado, J.O. Berger, A.P. Dawid,
A.F.M. Smit). Oxford University, UK.
Carneiro J.M.Jr., R.F. Euclydes, R.A. Torres and S. Lopes. 2007. Estimation of variance
components using Bayesian and frequentist inferences considering simulated data under
heterogeneity of variance. R. Bras. Zootec. 36:1539-1548.
Gianola, D. and R.L. Fernando. 1986. Bayesian methods in animal breeding theories. J. Anim. Scie.
63:217-244.
Mahdy, A.E., O.M. El-Shafie and M.S. Ayyat. 1999. Genetic study and sire values for some
economic traits in Egyptian buffaloes. Alex. J. Agric. Res. 44:15-35.
Cassiano, L.A.P., A.S. Mariante, C. McManus, J.R.F. Marques and N.A. Costa. 2004. Parâmetros
genéticos das características produtivas e reprodutivas de búfalos na Amazônia brasileira.
Pesq. Agropec. Bras. 39:451-457.
Ramos, A.A., C.H.M. Malhado, P.L.S. Carneiro, D.M.M.R. Azêvedo and H.C. Gonçalves. 2006.
Phenotypic and genetic characterization of the milk yield and calving interval in buffalo of
the Murrah breed. Pesq. Agropec. Bras. 41:1261-1267.
624
Raftery, A.E. and S. Lewis. 1992. How many iterations in the Gibbs sampler? In: Bayesian statistics
(Ed. J.M. Bernardo, J.O. Berger, A.P. Dawid). Oxford University, UK.
Seno, L.O., V.L. Cardoso, L. El Faro, R.C. Sesana, R.R. Aspilcueta-Borquis, G.M.F. de Camargo
and H. Tonhati. 2010. Genetic parameters for milk yield, age at first calving and interval
between first and second calving in milk Murrah buffaloes. Livest. Res. Rural Dev. pp. 22.
Smith, B.J. 2005. Bayesian output analysis program (BOA) for MCMC.
Van Tassel, C.P. and L.D. Van Vleck. 1995. A manual for use of MTGSAM. A set of FORTRAN
programs to apply Gibbs sampling to animal models for variance components estimation
(DRAFT). Lincoln: Department of Agriculture, Agricultural Research Service.
Table 1. Means, standard deviation (S.D.), median, mode and higher a posteriori density interval
(HPD) of the genetic parameters for calving interval (CI) and age at first calving (AFC).
Traits Parameters Mean S.D. Median Mode HPD

Low limit High limit
CI σa 2
833.19 400.52 799.55 773.02 682.70 1573.80
σ pe2
166.63 253.58 20.98 0.01 0.00 783.66
σe 2
6881.99 483.33 6861.38 6825.46 5955.52 7848.93
σp 2
7885.85 458.33 7866.47 7839.58 6991.64 8779.86
2
h 0.10 0.05 0.10 0.10 0.01 0.19
R 0.13 0.15 0.13 0.13 0.03 0.22
AFC σa 2
12.46 3.85 12.24 11.03 5.40 20.33
σ 2e 17.07 3.05 17.04 16.67 11.52 23.42
σp 2
29.53 2.26 29.41 29.14 25.15 33.86
2
h 0.41 0.11 0.42 0.42 0.20 0.63
a2 is the direct additive genetic variance component; pe2 is the permanent environment variance; e2 is the residual
(1)
variance; p2 is the phenotypic variance; h2 is the heritability; R is repeatability.
Figure 1. A Posteriori distribution of the heritability coefficient for age of first calving (AFC)
estimated by Bayesian inference.
625
Figure 2. A Posteriori distribution of the heritability coefficient for calving interval (CI) estimated
by Bayesian inference.
626
Genetic Parameters for Growth Traits of Mediterranean Buffaloes from Brazil,

Estimated By Bayesian Inference
Vanius FALLEIROa,e, E.S. SILVEIRAa, A.A. RAMOSb, P.L.S. CARNEIROc, J.A.

CARRILLOd, C.H.M. MALHADOc*
a
State University of Southwest Bahia, Itapetinga, BA, Brazil
b
c
d
University of Maryland, College Park, MD, USA
e
Federal Institute IF, Rio do Sul, SC, Brazil
*corresponding e-mail: carlosmalhado@gmail.com
ABSTRACT
Quantitative analysis of growth genetic parameters is not available for many breeds of
buffaloes making selection and breeding decisions an empirical process that lacks robustness. The
objective of this study was to estimate heritability for birth weight (BW), weight at 205 days
(W205) and 365 days (W365) of age using Bayesian inference. The Brazilian Program for Genetic
Improvement of Buffaloes provided the data. For the traits BW, W205 and W365 of Brazilian
Mediterranean buffaloes 5169, 3792 and 3883 observations have been employed for the analysis,
respectively. In order to obtain the estimates of variance, univariate analyses were conducted using
the Gibbs sampler included in the MTGSAM software. The model for BW, W205 and W365
included additive direct and maternal genetic random effects, random maternal permanent
environmental effect and contemporary group that was treated as a fixed effect. The convergence
diagnosis was performed employing Geweke, a method that uses an algorithm from the Bayesian
Output Analysis package that was implemented using R software environment. The average values
for weight traits were 37.6±4.7 kg for BW, 192.7±40.3 kg for W205 and 298.6±67.4 kg for W365.
The heritability posterior distributions for direct and maternal effects were symmetric and close to
those expected in a normal distribution. Direct heritability estimates obtained using the modes were
0.30 (BW), 0.52 (W205) and 0.54 (W365). The maternal heritability coefficient estimates were
0.31, 0.19 and 0.21 for BW, W205 and W365, respectively. Our data suggests that all growth traits
and mainly W205 and W365, have clear potential for yield improvement through direct genetic
selection.
Keywords: bubalus bubalis, Gibbs sampler, Heritability
INTRODUCTION
The knowledge of genetic parameters for traits in buffaloes is essential for the genetic
improvement of livestock and livestock development. Such knowledge allows prediction of the
intended gain from selection, either directly or indirectly, and facilitates the choice of criteria and
methods. However, quantitative analysis of growth genetic parameters is not available for many
breeds of buffaloes making selection and breeding decisions an empirical process that lacks
robustness.
The estimation of (co) variance components via Restricted Maximum Likelihood (REML)
has been the usual method in Brazil, as it can be seen in the study by Yokoo et al. (2007), among
others. Yet, the Bayesian inference via Gibbs sampling, which is one of the Markov Chain Monte
Carlo methods, is an excellent alternative for the estimation of (co) variance and genetic parameters,
since it allows accurate estimates to be obtained with greater flexibility due to the posteriori
distributions. The Bayesian process specifies distributions for each random variable of the model
and then it combines these distributions into a joint posterior distribution. The estimates of
627
parameters are obtained from the joint posterior distribution through Gibbs Sampler. In addition, the
researcher may have information from previous experiments that strongly indicate the value that a
variance component may have, and the Bayes approach allows the a priori information to be
included in the analysis.
It also allows the design of exact probability intervals for the estimates of parameters
(Faria et al., 2007). The objective of this study was to estimate heritability for birth weight (BW),
weight at 205 days (W205) and 365 days (W365) of age using Bayesian inference.

The Brazilian Program for Genetic Improvement of Buffaloes provided the data. For the
traits BW, W205 and W365 of Brazilian Mediterranean buffaloes 5169, 3792 and 3883
observations have been employed for the analysis, respectively. In order to obtain the estimates
of variance, univariate analyses were conducted using the Gibbs sampler included in the MTGSAM
software. We used models that included random genetic effects, direct and maternal, and permanent
maternal environment, and the fixed effect of contemporary group (CG). The contemporary groups
consisted of animals of the same sex, time (season and year of birth or calving) and farm of origin.
The initial number of iterations was arbitrarily obtained using a single chain of 100,000
iterations and a burn-in period of 10,000 iterations with a sampling interval of 10. The method
described by Raftery and Lewis (1992), using an algorithm implemented in the software R by
means of the Bayesian Output Analysis Package (BOA) (Smith, 2005), was applied to these data to
set burn-in, chain length and sampling interval. In addition, the BOA package was used in the
convergence diagnostic test, following the method of Geweke (1992).
Descriptive statistics (mean, median and standard deviation) and confidence interval and/or
high density were obtained using the BOA package of software R. The high-density interval
provides an interval that includes 95% of the sample, in addition to being a measure of reliability.
This interval can also be applied to non-symmetrical distributions (Hyndman, 1996).

The average values for weight traits were 37.6±4.7 kg for BW, 192.7±40.3 kg for W205 and
298.6±67.4 kg for W365. These results were slightly lower than those reported for buffaloes raised
exclusively for meat. Malhado et al. (2008) reported means of 196.0±33.5 (W205) and 301.6±58.7
(W365) for Mediterranean breed buffaloes. The values reported are higher than those for dairy
buffaloes in Jorge et al. (2005), who observed averages of 170.2±8.9 (W240) and 251.8±31.8 kg
(W365) in Murrah buffaloes.
The mode is considered the most appropriate measure of a posteriori distribution (Wright
et al., 2000) and it can be used to find the values of higher frequencies (maximum distribution).
Other measures of central tendency, such as mean and median, can also summarize a posteriori
distribution, especially if these densities are approximately symmetrical when such measurements
are similar. The posterior heritability distributions for direct and maternal effects were symmetric
and close to those expected in a normal distribution (Figure 1 to 3).
The mean (0.31), median (0.30) and mode (0.30) of estimates of heritability coefficient for
birth weight (BW) (Table 1) were greater than the values obtained through REML by Cassiano et
al. (2004) in Mediterranean (0.16) and Jafarabadi buffaloes (0.28). In the other hand the results of
this work were smaller than the ones reported by Cassiano et al. (2004) working with Carabao
(0.39) and Murrah (0.62) buffalo breeds.
Direct heritability estimates obtained using the modes were 0.52 (W205) and 0.54 (W365).
Malhado et al. (2012) reported lower modes for direct heritability on their study on Jaffarabadi
buffaloes using Bayesian inference, with values of 0.43 and 0.48 for W205 and W365, respectively.
The maternal heritability coefficient estimates were 0.31, 0.19 and 0.21 for BW, W205
and W365, respectively. Our data suggests that all growth traits and mainly W205 and W365, have
clear potential for yield improvement through direct genetic selection.
628
The genetic correlations between the direct and the maternal effects were -0.64 (W205)
and -0.83 (W365). The negative genetic correlation between the direct genetic effect and the
maternal effect represents the antagonism between the genes for growth and maternal ability.
However, the data structure may be insufficient for accurate estimation of the parameters.
According to Schaeffer (1999), obtaining good estimates requires data from multiple generations
with appropriate associations with practice.
REFERENCES
Cassiano, L.A.P., A.S. Mariante, C. McManus, J.R.F. Marques and N.A. Costa. 2004. Parâmetros
genéticos das características produtivas e reprodutivas de búfalos na Amazônia brasileira.
Pesq. Agropec. Bras. 39:451-457.
Faria, C.U., C.U. Magnabosco, A. Los Reyes, R.B. Lôbo and L.A.F. Bezerra. 2007. Inferência
bayesiana e sua aplicação na avaliação genética de bovinos da raça Nelore: Revisão
Bibliográfica. Ciênc. Anim. Bras. 8:75-86.
posterior moments. In: Bayesian statistics (Ed. J.M. Bernado, J.O. Berger, A.P. Dawid and
A.F.M. Smit). Oxford University. pp. 526.
Malhado, C.H.M., A.A. Ramos, P.L.S .Carneiro, D.M.M.R. Azevedo, J.C. Souza and R. Martins
Filho. 2008. Improvement and population structure of Mediterranean water buffaloes
raised in Brazil. Pesq. Agropec. Bras. 43: 215-220.
Malhado, C.H.M., A.C.M. Malhado, A.A. Ramos, P.L.S. Carneiro, F. Siewerdt and A. Pala. 2012.
Genetic Parameters of Some Performance Traits by Bayesian Inference for Dual Purpose
Jaffarabadi Buffaloes. Arch. Tierzucht. pp. 6.
Raftery, A.E., S. Lewis. 1992. How many iterations in the Gibbs sampler? In: Bayesian statistics 4
(Ed. J.M. Bernardo, J.O. Berger and A.P. Dawid). Oxford University Press. 763-773.
Schaeffer, L.R. 1999. Animal models-10-637-winter 99. University of Guelph, Guelph, Canada.
Van tassel, C.P., L.D. and Van Vleck. 1995. A manual for use of MTGSAM. A set of FORTRAN
programs to apply Gibbs sampling to animal models for variance components estimation,
Lincoln: Department of Agriculture, Agricultural Research Service.
Yokoo, M.J.I., L.G. Alburquerque, R.B. Lôbo, R.D. Sainz, J.M. Carneiro Júnior, L.A.F. Bezerra
and F.R.C. Araujo. 2007. Estimativas de parâmetros genéticos para altura do posterior,
peso e circunferência escrotal em bovinos da raça Nelore. R. Bras. Zootec. 36: 1761-1768.
629
Figure 1. A posteriori distributions of the heritabilities for birth weight.
Figure 2. A posteriori distributions of the heritabilities for weight at 205 days of age.
Figure 3. A posteriori distributions of the heritabilities for weight at 365 days of age.
630
(HPD) of the variance components and heritability values for weight at 205 (W205), 365 (W365)
and 550 (W550) days of age obtained using Gibbs sampler.

Low High
a2
limit limit
WB 2,13 0,61 2,09 2,05 0,93 3,27
σm 2
pe
2,10 0,54 2,09 2,09 1,04 3,16
2
p
0,23 0,19 0,19 0,06 0,01 0,63
2
6,81 0,24 6,80 6,77 6,33 7,28
2
hd 0,31 0,08 0,30 0,30 0,16 0,48
2
hm 0,30 0,07 0,30 0,31 0,16 0,45
a2
ram -0,71 0,10 -0,73 -0,75 -0,91 -0,51
W205 619,56 151,15 607,76 609,21 339,51 915,74
σm2
pe
234,65 60,36 239,95 232,57 112,82 354,61
2
e
32,71 24,34 28,79 22,02 0,02 77,71
2
p
293,84 78,49 299,53 306,79 140,92 439,00
2
1180,78 117,11 1170,48 1162,09 962,53 1407,23
hd 2 0,51 0,08 0,51 0,52 0,35 0,68
hm2 0,19 0,03 0,19 0,19 0,12 0,27
a
ram -0,61 0,10 -0,62 -0,64 -0,80 -0,38
2
W365 1449,48 390,45 1424,10 1379,22 891,22 2055,11
σm2
pe
577,37 141,44 569,11 567,59 325,14 863,88
2
e
29,26 25,05 22,63 7,70 0,38 80,23
2
p
577,54 159,52 589,31 602,42 249,56 868,11
2
2633,66 270,14 2608,93 2551,27 2145,76 3174,63
hd 2 0,54 0,06 0,54 0,54 0,40 0,67
2
hm 0,21 0,03 0,21 0,21 0,14 0,28
a is the additive genetic variance, σm2 is the maternal genetic variance, pe2 is the maternal
ram -0,80 0,06 -0,81 -0,83 -0,92 -0,68
(1) 2
permanent environmental variance, e2 is the residual variance, p2 is the phenotypic variance,
hd2 is the direct heritability, hm2 is the maternal heritability, and ram refers to the genetic correlation
between direct and maternal effects.
631
Growth Traits of Anatolian and Anatolian x Italian Crossbred Buffalo Calves

Under the Village Conditions
Özel ŞEKERDENa*
a
Mustafa Kemal Üniversitesi, Ziraat Fakültesi, Zootekni Bölümü, Antakya, Turkey
Corresponding e-mail:sekerden@mku.edu.tr
ABSTRACT
The study was carried out to compare growth performances of Anatolian and crossbred
(Anatolian x Italian) buffalo calves (53 F1, 66 Anatolian, 26 F1xAnatolian) raised at Ilıkpınar
Village of Kırıkhan District of Hatay Province.
Body measurements were determined in the period of 0-12 months of age. The effects of genotype,
sex, birth year effects on each characteristic in each age were investigated using GLM variance
analysis. The means of each characteristic in each age for each genotype were calculated. Duncan
test was used in comparison of the averages of each characteristic SPSS programme was used in
the statistical procedures.Genotype created significant variation in live weight at 1 and 6 months of
ages respectively and on almost every body measurement almost in every age.
Genotype* birth weight, genotype*sex and genotype*birth year* sex interactions were found
statistically significant in the point of view of various characteristics in various ages.
Factors had significant effects on various characteristics in various ages.
It can be said that, F1 growth was the most speedly and Anatolian buffaloes stayed behind of the
other genotypes from the point of view of all the traits.
Keywords: Buffalo, Anatolian, Italian, Body measurements
INTRODUCTION
The most part of increasing in body measurements in male and female animals comes true
until 6 months of age (Tusavara et al., 1989; Rajagopalan and Nirmalan, 1989; İzgi et al., 1992).
Literature knowledges of height at withers and body length of Egypt, Bulgaria and Anatolian
buffaloes in Table 1, the ones belong to chest girth, chest depth and shin girth in Table 2 are given.
The study was carried out to compare growth performances of Anatolian and crossbred
(Anatolian x Italian) buffalo calves (53 F1, 66 Anatolian, 26 F1xAnatolian) raised at Ilıkpınar
Village of Kırıkhan District of Hatay Province.

The material of the research was formed by as a total of 145 calves (53 F1, 66 Anatolian
(An), 26 F1xAnatolian) that were born in the period of 2003-2008 at 11 units of Ilıkpınar Village of
Kırıkhan District of Hatay Province. Various body measurements (height at withers, body length,
chest depth, chest width, chest girth, shin girth) were taken from the calves in the period of 0-12
months of age period. In addition live weight data were also taken from the animals at 1 numbered
unit.
It can be said that buffalo feeding was almost based on pasture in Ilıkpınar Village.
Genotype, sex, birth year effects on each characteristic for each age were investigated using
GLM variance analysis. Therefore the following linear simple model that variation sources which
took into consideration were included in it was used.
Yijkl = µ + Bi + Cj + Dk + eijkl ………………………………..(1) Here;

632
Yijkl : Phenotypic value belonged to investigated charecteristic (For example height at wither), µ :
General mean, Bi : i. The effect of calf genotype (i:1, 2, 3); Cj: j. Effect of sex (j: 1, 2), Dk: k.
Birth year effect (k: 1, 2, 3, 4, 5, 6); eijkl : Residue.
The means of each characteristic in each age for each genotype were calculated. Duncan
test was used in comparison of the averages of each characteristic SPSS programme was used in
the statistical procedures.

The characteristics that were effected at statistically significant from factors taken into
consideration were determinated by using the results of the variance analysis
Genotype created significant variance on live weight at only 6 months of age. But genotypic
effect was significant on almost every characteristic and in every age.
Means of Body Measurement
It can be said that F1’s growth was the most rapidly from the point of view of all the
characteristics investigated (except shin girt). However Anatolian’s stayed at the most behind of
other genotypes from the point of view of all the characteristics investigated. Sometimes (An x F1)
were> An, simetimes An were >(F1xAn), sometimes both of genotypes had similar average values
(Table 3). Genotypes could be arranged like the following according to values reached at 12 months
of age;
Shin girth: F1= (F1xAn)>An ; Chest depth: F1>(F1xAn)
Chest girth: F1> (F1xAn) Chest width: An>(F1xAn)
As a conclusion at 12 months of age; it can be said that, F1’s had the highest average values
from the point of view of live weight, body length, chest depth, chest width; similar averages to (F1
xAn) from the point of view of height at withers, chest girth; slight lower average than (F1 xAn)
from the point of view of shin girth. Anatolian’s were the most behind in the point of view live
weight, height at withers, chest girth and shin girth. (F1xAn)’s were the most behind in the point of
view of body length, chest depth and chest width.
Averages of some body measurements of the project material were compared with literature
knowledges given in Table 4 and Table 5.
It can be said that F1’s followed Egypt buffaloes almost in the point of view of all the
characteristics investigated except shin girth. At 12 months of age Egypt buffaloes were in front of
Bulgaria buffaloes and F1’s respectively in the point of view of height at withers and body length.
Egypt buffaloes were the most before in the point of view of st girth. Bulgaria buffaloes were the
most behind in the point of view of chest depth (Table 4, Table 5).
As a conclusion, in the period of until 12 months of age body structure of Bulgaria buffaloes
were higher, longer and wider but lower deep. Egypt buffaloes had huger size than F1’s, (F1xAn)’s
and Anatolia’s. Anatolian buffaloes had the smallest size in all the genotypes. The situation can be
explained that various buffalo population mentioned had different genotypes, different body
characteristics, in addition of different husbandry conditions.
REFERENCES
İzgi, N., A. Ramiz, A. Kılıç and M. Şahin. 1992. Usage possibilities of cow’s milk instead of
buffalo milk in buffalo calves raising. Afyon Buffalo Husbandry Research Institute. pp. 25.
Nigm, A.A. 1996. Characterization of the Egyptian buffalo. In: Proceedings of the 1996
International Symposium on Buffalo Resources and Production Systems, Kahire. pp. 1-8.
Peeva, T. 1996. Possibilities for reduction of the age at first calving. In: Proceedings of the 1996
International Symposium on Buffalo Resources and Production Systems, Kahire, pp. 47-50.
Rajagopalan, T.G. and G. Nirmalan. 1989. Pattern of growth of male cross-bred Surti Buffalo
Calves. Kerala J. of Vet. Sci. 20(2): 42-48.
Şekerden, Ö., M. Küçükkebapçı and A. Kopar. 2001. Growth characteristic, population structure in
point of view blood serum Tf types and relationships between growth characteristic and Tf types
633
in Anatolian buffaloes of Kocatepe Agricultural Research Institute. Journal of Atatürk Univ.

Fac. of Agric. 32(1): 67-75.
Şekerden, Ö. and I. Tapkı, 2003. Growth characteristics of Anatolian buffaloes of Hatay Province
under the village conditions. Journal of Atatürk Univ. Fac. of Agric. 34(1): 51-55.
Tusuvara, M., L.S. Jain and S.P. Tailor. 1989. Growth pattern in buffalo calves. Indian J. of Dairy
Sci. 42(4):661-665.
634
Table 1.Various body measurements of Egypt, Bulgaria and Anatolian buffaloes in various ages
(cm) (x).
Age Height at withers Body length

(Mo Sex 1 2 3 4 1 2 3 4
nth) (xx)
1 M 71.2 78.3 55.0 65.0
F 69.2 78.1 55.5 64.8
3 M 85.2 85.7 73.4 71.9
F 84.2 83.4 72.3 69.6
6 M 93.8 93.3 106.0 81.0 80.5 90.8
F 89.6 91.8 105.0 79.1 76.9 89.5
9 M 97.9 94.1 102.8 86.4 82.5 104.8
F 98.1 92.2 84.6 81.1
12 M 105.7 100.9 123.0 108.1 95.6 92.8 110.0 111.0
F 102.2 101.5 121.0 91.7 90.8 109.5
(x) 1: Şekerden et al. (2001), 2: Şekerden and Tapkı (2003), 3: Nigm (1996), 4: Peeva (1996)
(xx) M: Male, F: Female
Table 2. Literature knowledges belong to chest girth, chest depth and shin girth of Egypt, Bulgaria
and Anatolian buffaloes (cm) (x).
Age Sex Chest girth Chest depth Shin girth

(Month) (xx) 1 2 3 4 1 2 3 4 1 2 3 4
1 M 75.1 86.1 26.7 29.9 13.5 14.3
F 73.4 85.2 26.0 29.3 12.4 13.6
3 M 104.5 99.3 37.4 34.3 14.7 14.9
F 103.5 95.3 37.3 33.4 13.9 13.9
6 M 121.9 110.3 133 140.2 43.8 39.0 16.2 14.9
F 119.3 110.1 131 42.6 37.9 15.1 14.2
9 M 132.0 119.1 152.7 46.1 40.6 39.3 16.7 15.3
F 133.7 121.6 46.2 41.1 16.1 15.3
12 M 139.4 130.2 161 50.2 45.7 42.7 18.0 16.7
F 142.3 134.8 161 49.6 47.3 17.2 16.1
(x) 1: Şekerden et al. (2001), 2: Şekerden and Tapkı (2003), 3: Nigm (1996), 4: Peeva (1996)
(xx) M: Male, F: Female
Table 3. Averages of various body characteristics of genotypes.

Characteris- Age Genotype (x)
tics (month) F1 Anatolian Anatolian x F1
_ _ _ _ _ _
X ± SX X ± SX X ± SX
Live weight 1 52.8±3.15 a 47.8±1.76 a 52.6±2.92 a
(kg) 3 83.4±3.35 b 72.2±2.48 a 81.7±3.84 ab
6 112.8±3.88 b 90.8±3.21 a 114.6±5.99 b
9 140.0±4.04 ab 126.1±4.35 a 143.5±8.56 b
12 181.0±10.78 a 159.8±12.02 a 164.4±7.18 a
Height at 1 79.2±0.59 c 75.8±0.51 a 77.1±0.62 b
withers 3 88.7±0.67 b 84.1±0.71 a 87.4±1.02 b
(cm) 6 96.3±0.75 b 90.5±0.74 a 95.7±1.11 b
9 101.4±0.69 b 96.5±0.75 a 97.8±1.36 a
12 106.3±0.88 b 101.5±0.99 a 106.5±1.80 b
Body 1 69.1±0.58 c 65.6±0.60 b 63.2±1.08 a
lenght (cm) 3 79.8±0.78 c 74.4±0.75 b 71.6±1.21 a
635
6 87.9±0.95 b 80.3±0.76 a 78.3±1.24 a

9 92.8±1.06 c 87.5±0.79 b 83.3±1.54 a
12 99.6±1.15 c 94.6±1.01 b 89.7±1.49 a
Chest depth 1 30.0±0.27 b 27.4±0.33 a 27.6±0.43 a
(cm) 3 35.8±0.39 b 33.5±0.38 a 33.3±0.62 a
6 41.1±0.42 c 36.9±0.46 a 38.5±0.65 b
9 43.7±0.48 b 40.7±0.48 a 40.7±0.76 a
12 47.7±0.49 b 45.9±0.57 ab 44.2±0.74 a
Chest width 1 17.4±0.25 c 15.8±0.22 b 14.7±0.45 a
(cm) 3 20.2±0.32 b 18.8±0.31 a 18.7±0.60 a
6 22.5±0.38 b 20.3±0.36 a 20.2±0.59 a
9 24.5±0.37 b 23.3±0.37 b 21.6±0.62 a
12 26.6±0.41 b 26.1±0.59 b 23.1±0.71 a
Chest girth 1 85.5±0.98 b 81.0±0.66 a 85.4±1.19 b
(cm) 3 101.9±1.04 b 94.8±1.18 a 100.3±1.56 b
6 113.0±1.08 b 104.2±1.18 a 114.5±1.72 b
9 123.9±1.19 b 117.6±1.28 a 120.8±2.16 a
12 133.3±1.62 b 126.9±1.49 a 133.9±2.22 b
Shin 1 13.3±0.14 b 12.8±0.10 a 13.7±0.15 c
girth 3 14.4±0.15 b 13.6±0.11 a 14.8±0.20 c
(cm) 6 15.0±0.14 b 13.8±0.11 a 15.7±0.22 c
9 15.6±0.16 b 14.9±0.16 a 16.0±0.24 b
12 16.9±0.22 b 15.9±0.21 a 17.2±0.31 b
(x) Different letters in the same age group (at the same line) showed genotypes
that were different at a significant degree to each other for each characteristic.
Table 4. Comparison of genotypes [(F1, An, (F1xAn)] in the project with to each other and some
literature knowledges given (x).
Characteristics Age (month)

1 3 6
Height at F1>(F1xAn)>An>2>1 F1>(F1xAn)>An=1=2 3>F1>(F1xAn)>2>1>An
Withers
Body F1>An>2>(F1xAn)>1 F1>An>1>2=(F1xAn) 3>F1>1>An=2>(F1xAn)
Length
Chest girth F1=2=(F1xAn)>An>1 1>F1>(F1xAn)>2>An 4>3>1>(F1xAn)>F1>2=An
Chest F1>(F1xAn)=An>2>1 1>F1>2=(F1xAn)=An 1>F1>2=(F1xAn)>An

Depth
Shin girth 2>(F1xAn)=1=F1>An (F1xAn)=F1=1=2>An 1>(F1xAn)>F1>2>An
(x) 1:Şekerden et al. (2001); 2: Şekerden and Tapkı (2003), 3: Nigm (1996); 4: Peeva (1996).
Table 5. Comparison of genotypes [(F1, An, (F1xAn)] in the Project with to each other and some
literature knowledge given (x).
Characteristics Age (month)

9 2
Height at withers 4>F1>(F1xAn)=2>1 3>4>F1=(F1xAn)>2>An=1
Body length 4>F1>An>1>(F1xAn)>2 4=3>F1>An=1>2>(F1xAn)
Chest girth 4>1>F1>(F1xAn)=2>An 3>1>F1=(F1xAn)>2An
Chest depth 1>F1>2>(F1xAn)=An>4 1>F1>2>An>(F1xAn)>4
Shin girth 1>(F1xAn)>F1=2>An 1=F1xAn>F1>2>An
(x) 1:Şekerden et al. (2001); 2: Şekerden and Tapkı (2003), 3: Nigm (1996); 4: Peeva (1996).
636
Population Parameters Based on Known Pedigree Records from Brazilian

Jaffarabadi Buffaloes
Carlos Henrique Mendes MALHADOa*, P.C. FERRAZa, A.A. RAMOSb, P.L.S.

CARNEIROa, E.S. ARAGÃOa, A.C.B. BARBOSAa and J.A. CARRILLOc
a
State University of Southwest Bahia (UESB), Jequié, BA, Brazil
b
Paulista State Univeristy, Botucatu (UNESP), SP, Brazil
c
University of Maryland (UMD), College Park, MD, US
ABSTRACT
The objective of this study was to evaluate the effective number of founders and ancestors,
generation intervals and completeness of pedigree in Jaffarabadi breed buffaloes raised in Brazil.
Pedigree records of 1,272 animals born from 1966 were used. The parameters were estimated using
ENDOG, computational population genetic software. The obtained value for completeness of
pedigree was 99.5, 50.9, and 20.5 for, the first, second and third generations, respectively.
Generation interval estimates expressed in years and considering different pathways were
12.28±6.90 (sire-son), 11.55±6.07 (sire-daughter), 8.20±2.63 (dam-son) and 8.79±4.33 (dam-
daughter). The overall average generation interval was 10.17±5.43 years. The number of founders,
equivalent founders and ancestor animals that contributed for the genetic diversity in the reference
population (1059) were 136, 130 and 134, respectively. Effective number of founder (fe=8) and
ancestors (fa=7) were small, and the calculated expected inbreeding increase per generation was
4.99%. Four ancestors explained 50% of the genetic variability in the population and the major
ancestor contributed with approximately 33% of the total population genetic variation. The genetic
diversity within the current population is low as a consequence of a reduced number of ancestors.
Keywords: ancestors, founders, generation interval
INTRODUCTION
Studies using historical pedigree records have the potential to identify the factors that have
influenced the genetic history of a population (Valera et al., 2005). Moreover, some population
parameters are strongly dependent on management and mating systems, and have significant
impacts on genetic variability. In addition to productivity, cattle breeders are also concerned with
monitoring the genetic health of their livestock. An assessment of the within-population genetic
variability and population structure is necessary for the implementation of effective selection
programs and to establish appropriate regimes for the management of the genetic stock. In this
respect, genealogical assessment is an important tool to guide genetic management strategies
(Glazewska and Jezierski, 2004).
In recent years several studies have been published on population structure in different
species, including buffaloes ( Santana et al., 2011; Malhado et al., 2012; Teixeira Neto et al., 2012).
However, there are no equivalent studies for the Jaffarabadi breed (in Brazil or other countries). The
major objective of the present study was thus to evaluate the population structure of a closed
Jaffarabadi buffalo herd using pedigree analysis. The parameters used include effective number of
founders and ancestors, and generation interval.

Pedigree information was obtained of 1,272 animals of Jaffarabadi breed born from 1966
to 2002 from a closed herd from Brazil. Pedigree analysis and parameters estimates based on gene
origin probabilities were performed using the ENDOG 4.8 program (Gutierrez and Goyache, 2005).
The average generation interval of the four gametic pathways was estimated using the
following steps: sire-son, sire-daughter, dam-son and dam-daughter. Genetic history was assessed
637
by calculating the effective number of founders and the effective number of ancestors. The effective
number of founders (fe) represents the number of animals which, under random mating, would
produce the same genetic variability as that observed in the study population. This is computed as
where, qk is the probability of gene origin for ancestor k.
The effective number of ancestors (fa ) represents the minimum number of animals
(founders or non-founders) that are necessary to explain the total genetic diversity of the study
population. It is calculated in a similar way to the effective number of founders: , where
qj is the marginal contribution of ancestor j, which represents the genetic contribution made by an
ancestor that is not explained by another ancestor chosen previously. This parameter complements
the information offered by the effective number of founders by accounting for the losses of genetic
variability produced by the unbalanced use of reproductive individuals producing bottlenecks. The
founder equivalents (fg) can be defined as the number of founders that would be expected to
produce the same genetic diversity as in the population under study if the founders were equally
represented and no loss of alleles occurred (Ballou and Lacy, 1995).

Generation interval estimates for the four gametes pathways were equal 12.28±6.90 (sire-
son), 11.55±6.07 (sire-daughter), 8.20±2.63 (dam-son) and 8.79±4.33 years (dam-daughter), and the
average generation interval was 10.17±5.43 years. Teixeira Neto et al. (2012) reported generation
interval for Mediterranean buffaloes, equal the 8.79±2.29 (sire-son), 9.59±2.80 (sire-daughter), 7.42
± 3.04 (dam-son), 7.94±2.7 years (dam-daughter), with an average interval of 8.71±2.85 years.
Santana et al. (2011) also reported generation intervals lower than in our study: 7.38 (sire-son),
7.42 (sire-daughter), 6.60 (dam-son), 6.44 (dam-daughter), with an average interval of 6.89 years.
This high generation interval in the Jaffarabadi herd is consequence of several factors, among them,
the use of the main breeder (the sire with the largest number of offspring (271) who was breed from
1975 to 1991) over a prolonged period of time.
The number of founders, equivalent founders and ancestor animals contributing to the
reference population (1,059) were 136, 130 and 134, respectively (Table 1). With much smaller
values effective number of founders (fe) and ancestors (fa ) were 8 and 7, respectively. Expected
increase of inbreeding caused by the unbalanced contribution of founders was 4.99 percent. The
ratio of effective number of founder to the number of founders was 0.058, indicating a high
disequilibrium between founder contributions. This fact results from an excessive use of some
animals as breeders, especially good bulls (as is common practice in a closed herd).
Higher values were reported by Santana et al. (2011) (fe=58, fa= 35, Murrah), Malhado et
al. (2012) (fe=60, fa =36, Murrah) and by Teixeira Neto et al. (2012) (fe=71, fa =71, Mediterranean) in
buffaloes from Brazil. However, those authors used data from several buffalo herds in Brazil. A
recent study of a closed population (Zandi sheep) reported higher values for fe (86) and fa (74)
(Ghafouri-Kesbi, 2010).
The extent of the genetic bottleneck expressed by the fe/fa ratio (effective number of
founders/effective number of ancestors) was 1.14, indicating that Jaffarabadi herd also suffered a
small bottleneck effect. The fe/fa ratio is caused by a decrease in the number of breeders in any given
generation. Increasing generations will also augment the chance of a bottleneck (Ghafouri-Kesbi,
2010). Therefore, in populations with a long historical pedigree, smaller estimates of fa would be
expected.
Just five ancestors explained 4% of the genetic variability in the population and the top
ancestor contributed approximately 33% of the genetic variability (Table 2).The very small number
of individuals explaining the variability of the Jaffarabadi herd is a consequence of the reduced
effective number of ancestors, in conjunction with a larger effect of a single animal. Malhado et al.
(2012) and Teixeira Neto et al. (2012) observed 17 and 30 animals explaining 50% of the genetic
variability of the Murrah and Mediterranean breed, respectively. In a similar study on Murrah
638
buffaloes, 19 animals were found to explain 50% of the genetic diversity of the population (Santana
et al., 2011).
To our knowledge, all studies that have analyzed buffalo herds from Brazil have found that
a small number of animals contribute disproportionately to the genetic diversity of population. The
low genetic variability of buffalos in Brazil can be partly explained by the process of introduction of
the animals into the country (Malhado et al., 2012). The first imports of the Buffalo occurred in the
years 1930, 1952, 1955, 1960 and 1962, and were frequently associated with the importation of
Zebu cattle. However, it is not accurately known how many Buffalo were imported during this
period. In 1966, when the Brazilian Association of Buffalo Breeders (ABCB) was founded, there
were no more than 50 to 60 purebred individuals of either the Murrah or the Jaffarabadi breed. All
other buffalos at this time were some combination of crossbreeding of the three main breeds
(Murrah, Jaffarabadi and Mediterranean). In 1976 there were still only 26 animals registered in the
Herd Book of ABCB. Of these animals, some were used as breeders - a fact which explains why so
few founders are responsible for such a high proportion of the current genetic variability of buffalo
in Brazil.
IMPLICATIONS
The genetic variability within the current Jaffarabadi buffalo population is low as a
consequence of a very small number of animals historically contributing to herd. In addition,
immediate action should be implemented, as the introduction the new breeders, to reduce the
generation interval.
REFERENCES
Ballou, J.D. and R.C. Lacy. 1995. Identifying genetically important individuals for management of
genetic variation in pedigreed populations. In: Population management for survival and
recovery: analytical methods and strategies in small population management (Ed. J.D.
Ballou, M. Gilpin and T. J. Foose). New York: Columbia University Press. pp. 76-111.
Ghafouri-Kesbi, F. 2010. Analysis of genetic diversity in a close population of Zandi sheep using
genealogical information. J. Genet. 89:479-483.
Glazewska, I. and T. Jezierski. 2004. Pedigree analysis of Polish Arabian horses based on founder
contributions. Livest. Prod. Sci. 90:293-298.
Gutierrez, J.P. and F. Goyache. 2005. A note on ENDOG: a computer program for analysing
pedigree information. J. Anim. Breed. Genet. 122:172-176.
Malhado, C.H.M., A.C.M. Malhado, P.L.S. Carneiro, A.A. Ramos, J.A. Carrillo and A. Pala. 2012.
Population structure and genetic variability in the Murrah dairy breed of water buffalo in
Brazil accessed via pedigree analysis. Trop. Anim. Health Prod. 44:1891-1897.
Santana, M.L.Jr., R.R. Aspilcueta-Borquis, A.B. Bignardi, L.G. Albuquerque and H. Tonhati. 2011.
Population structure and effects of inbreeding on milk yield and quality of Murrah
buffaloes. J. Dairy Sci. 94:5204-5211.
Teixeira Neto, M.R., J.F. Cruz, A.A. Ramos, P.L.S. Carneiro, D.M.M.R. Azevêdo, R. Bozzi and
C.H.M. Malhado. 2012. Genetic variability in Mediterranean an buffalos evaluated by
pedigree analysis. Cienc. Rur. 42:2037-2042.
Valera, M., A. Molina, J.P. Gutierrez, J. Gomez, F. Goyache. 2005. Pedigree analysis in the
Andalusian horse: population structure, genetic variability and influence of the Carthusian
strain. Livest. Prod. Sci. 95:57-66.
639
Table 1. Parameters characterizing the probability of gene origin in the Jaffarabadi herd.
Size of population 1271
Number of animals in the population reference 1059
Base population (one or more parents unknown) 212
Number of founder animals for the reference population 136
Number of equivalent founder animals for the reference population 130
Number of ancestor animals for the reference population 134
Effective number of founder animal in the reference population (fe) 8
Effective number of ancestor animal in the reference population (fa) 7
Number of ancestors explaining 50 % variability 4
Table 2. Description of the ten major ancestors (founders or not) in the Jafarabadi herd. The table
lists the identification of the individuals, their sire and dam, their sex, contribution for ancestors, the
average relatedness (AR) and number of offspring to indicate the great influence of some animals.
Ancestral Sire Dam Sex Year of birth Contribution AR (%) Offspring

(%)
1239 0 0 M 1967 33.57 28.95 271
1204 0 0 M 1986 7.74 6.80 152
1149 0 0 M 1984 6.52 5.57 25
1205 0 0 M 1993 5.09 4.36 109
1235 0 0 F 1970 3.74 3.25 14
2123 0 0 M 1986 3.51 3.06 69
2192 1162 1148 M 1981 3.33 3.00 40
2126 1231 1233 F 1974 3.02 3.00 14
5610 0 1178 M 1995 2.73 2.40 58
2129 0 0 F 1976 2.31 2.11 14
640
Inbreeding, Average Relatedness Coefficient and Effective Population Size In

Jaffarabadi Buffaloes Raised In Brazil
Carlos Henrique Mendes MALHADOa *, P.C. FERRAZa, A.A. RAMOSB, P.L.S.
CARNEIRoc, E.S. ARAGÃOa, A.C.B. s BARBOSAa and J.A. CARRILLOc
a
State University of Southwest Bahia (UESB), Jequié, BA, Brazil
b
Paulista State Univeristy, Botucatu (UNESP), SP, Brazil
c
University of Maryland (UMD), College Park, MD, US
ABSTRACT
The major aim of this study was to evaluate the inbreeding (F), average relatedness
coefficient (AR) and effective population size (Ne) in the Jaffarabadi buffalo breed from Brazil.
Pedigree information of 1,272 animals born from 1966 was used. The effective population size was
calculated in two ways: first, computed via individual increase in inbreeding and second estimated
by individual increase in coancestry. The known generation numbers were 1.24, 1.76 and 2.64 for
complete, equivalent and maximum generation, respectively. The effective size computed via
individual increase in coancestry was small with a value of 10.82±1.29. The effective size
computed by individual increase in inbreeding (10.40±3.69) was very similar but a little smaller
than the previous reported value. The average values of F and AR for the population reference
(1,059) were 4.22 and 12.5 percent. The mean of F for inbred animals (319) was 14.0%. The F and
AR means were 5.7 and 13.3% for animals with at least 1.5 known equivalent generation and 9.3
and 15.97% for individuals having at least 2.5 equivalent generations known. It was found 78
matings between half sibs (6.14%) and 67 matings (5.27%) between parent-offspring. The
estimated inbreeding increase per generation by considering maximum generation, complete
generation and equivalent generation were 1.21%, 5.18% and 3.57%, respectively. Considering the
uncompleted pedigree, the estimated inbreeding for the reference population could be
underestimated.
Keywords: endogamy, generation, pedigree
INTRODUCTION
Inbreeding (F) can be defined as the probability that two alleles at any locus are identical
by descent, and occurs when related individuals mate (Malécot, 1948). The potentially negative
consequences of inbreeding can be a problem for domestic animals, especially where large
populations often stem from a limited number of founding individuals. This is possibly the case for
water buffalo (Bubalus bubalis) in Brazil (Malhado et al., 2012a).
The average relatedness coefficient (AR) of a founder animal indicates its genetic
contribution to the population. Hence, AR can be used as an alternative or complement to the
inbreeding coefficient, to predict the inbreeding of a population in the long term, since it takes into
account the percentage of full pedigree originated from a founder (Gutiérrez and Goyache, 2005).
Another important population parameter is the effective population size (Ne). This parameter
affects the behavior of genes under selection, influencing the variance of response to selection, the
limits of selection and the survival of populations in conservation programs in the short and long
term (Wang and Caballero, 1999). So, the major aim of this study was to evaluate the inbreeding,
average relatedness coefficient, and effective population size in the Jaffarabadi buffalo breed from
Brazil.

641

Pedigree information was obtained of 1,272 animals of Jaffarabadi breed born from 1966 to
2002, from a closed herd from Brazil. Pedigree analysis and parameters estimates based on gene
origin probabilities were performed using the ENDOG 4.8 program (Gutierrez and Goyache, 2005).
The completeness pedigree includes the description of the completeness of each ancestor in
the pedigree to the 4th parental generation. The following parameters were calculated for each
individual: 1) the number of fully traced generations; 2) the maximum number of generations
traced; 3) the equivalent complete generations for each animal in the pedigree data.
The individual inbreeding coefficient (F) was computed following Meuwissen and Luo
(1992). The change in inbreeding (∆F) were calculated as described by Falconer and Mackay and
modified by Gutierrez et al. (2009).
The average relatedness coefficient of each individual is defined as the probability that an
allele randomly chosen from the whole population in the pedigree belongs to a given animal
(Gutierrez and Goyache, 2005). The average relatedness coefficient can thus be interpreted as a
representation of the animal in the whole pedigree regardless of the knowledge of its pedigree.
The effective size was calculated in two ways: First, estimate of effective population size
( ), “called realized effective size” by Cervantes et al. (2008) was computed from , that is
computed by averaging the of the n individuals included in a given reference subpopulation, as
. Additionally, a standerd error of was computed from the standard deviation of
and the square root of the size (n) of the reference subpopulation as (Gutierrez
et al., 2008).
Second, the effective populations size ( ) was estimated from increase in coancestry for
all pairs of individuals j and k in a reference subpopulation (Cervantes et al., 2011).

Completeness level for the whole pedigree was 86.6, 44.7, and 18.2% for, respectively, the
first, second and third generations. Malhado et al. (2012b) reported level for pedigree of 76.8, 49.2
and 27.7 for first, second and third generations in the Murrah breed. Santana et al. (2011), also
studying the Murrah breed from Brazil, calculated the average pedigree for animals born within the
last 10 years. They obtained values of 76.2% for first, 66.3% for second and 57.8% for third
generation. Likewise, Teixeira Neto et al. (2012) reported percentage of known pedigree at the first,
second and third generations as 60.51, 15.27 and 2.14% in the Mediterranean breed. This shallow
pedigree for buffaloes in Brazil can be explained by relatively recent foundation of the Brazilian
Association of Buffalo Breeders (ABCB) in 1966 (Malhado et al., 2012b).
The average values of inbreeding (F) for the whole analyzed pedigree was 4.2% (319
inbreed animals). Lower values of F in buffaloes have been reported by Santana et al. (2011)
(2.14%), Malhado et al. (2012b) (1.28%) and Teixeira Neto et al. (2012) (0.34%). The average
values of F for inbreed animals was 14.0%, close to that estimated by Teixeira Neto et al. (2012)
(16%) in Mediterranean buffaloes. This high value (14.0%) of F indicates that mating among
closely related individuals was not being avoided. Indeed, from 1982 to 1990 the average of F for
inbreed animals was 25% (Figure 1). A total of 78 matings were observed between half sibs and 67
matings between parent-offspring throughout the study period. In addition, in some years (e.g.1988
and 1999) most matings were inbred (Figure 2).
The increase in inbreeding for each maximum generation (1.21%), complete generation
(5.18%) and equivalent generation (3.57%) and the relatively short knowledge of pedigree indicates
that the value for inbreeding in the population is underestimated. Recent work on Murrah Buffaloes
demonstrated an increase of approximately 2 % for the average F per each complete generation
(Malhado et al., 2012b). More generally, the computation of inbreeding coefficients depends on the
level of pedigree completeness and on the base year or population used for the calculations
642
(Boichard et al., 1997). Inbreeding levels are biased downward when pedigree information is
incomplete, and the Ne is overestimated.
The average relatedness coefficient (AR) between individuals was estimated at 12.5% and
the highest individual coefficient was 28.95%. This is considerably higher than some other recent
estimates - 3.58% (Santana et al., 2011), 2.05% (Malhado et al., 2012b) and 0.37% (Teixeira Neto
et al., 2012) - and may indicate that inbreeding could increase in the near future if remedial
measures are not implemented. One potential strategy would be to use the average relatedness
coefficient to guide for sire selection. AR can be used to estimate long-term inbreeding of a
population and to inform modifications to management practice to conserve the genetic variability
of a population (Goyache et al., 2010). A very high AR indicates that the parents of an individual
have close common ancestors, while a low average relatedness coefficient indicates that the animal
shares alleles by common descent with only a relatively small percentage of the population.
The effective size computed via individual increase in coancestry ( ) was 10.82±1.29 -
close to the effective size computed by individual increase ( ) in inbreeding (10.40±3.69). Santana
et al. (2011) also reported small effective size (40) in using data from several Murrah herds. An
effective population size of at least 50 animals is sufficient to prevent inbreeding depression – the
minimum level recommended by the FAO (2007). The effective size the Jaffarabadi breed (10) is
much lower than the critical value (50) to avoid the deleterious effects of inbreeding or the loss of
genetic diversity through genetic drift. The ratio was 1.04: close to that expected (=1) in an
idealized population. The comparison between and gives valuable information on
population structure (Gutierrez et al., 2008) since the two parameters are assumed to be measures of
the same accumulated drift process, from the foundation of the population to the present time. As
they would be asymptotically equivalent in an idealized population, the disagreement between them
is mainly caused by their differential ability to assess the effect of preferential matings.
IMPLICATIONS
Based on these findings we would strongly recommend the introduction of new reproductive
individuals, with lowest possible AR coefficients with the herd, thereby reducing the probability of
deleterious effects related to inbreeding and increasing the future genetic variability and the
effective size of the herd.
REFERENCES
Boichard, D., L. Maignel and E. Verrier. 1997. The value of using probabilities of gene origin to
measure genetic variability in a population. Genet. Sel. Evol. 29:5-23.
Cervantes, I., F. Goyache, A. Molina, M. Valera and J.P. Gutierrez. 2008 Application of individual
increase in inbreeding to estimate realized effective sizes from real pedigrees. J. Anim.
Breed. Genet. 125:301-310.
Cervantes, I., F. Goyache, A. Molina, M. Valera and J.P. Gutierrez. 2011. Estimation of effective
population size from the rate of coancestry in pedigreed populations. J. Anim. Breed.
Genet. 128:56-63.
FAO. 2007. The State of the World’s Animal Genetic Resources for Food and Agriculture (Ed. B.
Rischkowsky and D. Pilling). Rome, Italy.
Goyache, F., I, Fernández, M.A. Espinosa, L. Payeras, L., Pérez-Pardal, J.P. Gutiérrez, L.J. Royo
and I. Álvarez. 2010. Demographic and genetic analysis of the Mallorquina sheep
flockbook. Itea 106:3-14.
Gutierrez, J.P. and F. Goyache. 2005. A note on ENDOG: a computer program for analysing
pedigree information. J. Anim. Breed. Genet. 122:172-176.
Gutierrez, J.P., I. Cervantes, A. Molina, M. Valera, F. Goyache. 2008. Individual increase in
inbreeding allows estimating effective sizes from pedigrees. Genet. Sel. Evol. 40:359-378.
Gutierrez, J.P., I. Cervantes and F. Goyache. 2009. Improving the estimation of realized effective
population sizes in farm animals. J. Anim. Breed. Genet. 126:327-332.
643
Malécot, G. 1948. Les mathématiques de I`hérédité Paris: Masson et Cie.

Malhado, C.H.M., A.C.M. Malhado, P.L.S. Carneiro, A.A. Ramos, D.P. Ambrosini and A. Pala.
2012a.Inbreeding depression on production and reproduction traits of buffaloes from
Brazil. Anim. Sci. J. (In press).
Malhado, C.H.M., A.C.M. Malhado, P.L.S. Carneiro, A.A. Ramos, J.A. Carrillo and A. Pala.
2012b. Population structure and genetic variability in the Murrah dairy breed of water
buffalo in Brazil accessed via pedigree analysis. Trop. Anim. Health Prod. 44:1891-1897.
Meuwissen, T.H.E. and Z. Luo. 1992. Computing inbreeding coefficients in large populations.
Genet. Sel. Evol. 24:305-313.
Santana, M.L.Jr., R.R. Aspilcueta-Borquis, A.B. Bignardi, L.G. Albuquerque and H. Tonhati.
2011. Population structure and effects of inbreeding on milk yield and quality of Murrah
buffaloes. J. Dairy Sci. 94:5204-5211.
Teixeira Neto, M.R., J.F. Cruz, A.A. Ramos, P.L.S. Carneiro, D.M.M.R. Azevêdo and R. Bozzi.
2012. Genetic variability in Mediterranean an buffalos evaluated by pedigree analysis.
Cienc. Rur. 42:2037-2042.
Wang, J. and A. Caballero. 1999. Developments in predicting the effective size of subdivided
population. Hered. 82:212-226.
644
Figure 1. Inbreeding coefficient (F), average relatedness coefficient (AR), and average of F for
inbreed animals by year of birth.
Figure 2. Inbred animals and total number of animals born by year.
645
Genetic Parameters for Milk Yield and Lactation Length of Crossbred Buffaloes
from Brazil by Bayesian Inference
Carlos Henrique Mendes MALHADOa *, A.A. RAMOSb, P.L.S. CARNEIROa, J.C. SOUZAc
and J.A. CARRILLOd
a
b
c
Universidade Federal do Mato Grosso do Sul, Dourorados, MS,
d
University of Maryland, College Park, MD, US.
ABSTRACT
The objective of the study was to estimate heritability and repeatability for milk yield (MY)
and lactation length (LL) in buffaloes using Bayesian inference. The Brazilian genetic improvement
program of buffalo provided the data that included 628 females, from four herds, born between
1980 and 2003. In order to obtain the estimates of variance, univariate analyses were performed
with the Gibbs sampler, using the MTGSAM software. The model for MY and LL included direct
genetic additive and permanent environment as random effects, and contemporary groups, milking
frequency and calving number as fixed effects. The convergence diagnosis was performed with the
Geweke method using an algorithm implemented in R software through the package Bayesian
Output Analysis. Average for milk yield and lactation length was 1,546.1±483.8 kg and 252.3±42.5
days, respectively. The heritability coefficients were 0.31 (mode), 0.35 (mean) and 0.34 (median)
for MY and 0.11 (mode), 0.10 (mean) and 0.10 (median) for LL. The repeatability coefficient
(mode) were 0.50 and 0.15 for MY and LL, respectively. Milk yield is the only trait with clear
potential for genetic improvement by direct genetic selection. The repeatability for MY indicates
that selection based on the first lactation could contribute for an improvement in this trait.
Keywords: Gibbs sampler, heritability, repeatability
INTRODUCTION
The first imports of Buffalo (Murrah, Mediterrean and Jaffarabadi) occurred in 1930, 1952,
1955, 1960 and 1962, and were frequently associated with the importation of Zebu cattle (Malhado
et al., 2012). Recent estimations suggest that there are currently about 1.2 million buffalos
distributed across all Brazilian states (Ibge, 2012), with large percentage of crossbred animals.
There have been several studies on the genetic parameters of Murrah and Mediterranean breeds in
Brazil (Ramos et al., 2006; Malhado et al., 2008; Seno et al., 2010). However, equivalent studies are
lacking for the crossbred Buffaloes in Brazil.
REML and Bayesian methods have been applied extensively in animal breeding to
estimate covariance components and genetic parameters. The Bayes methodology provides a
solution for the finite sample size problem, because an exact a posteriori distribution exists for each
large or small data set from which inferences can then be drawn. When a large data set is analyzed,
a priori information tends to be subjugated by the likelihood function in the establishment of the a
size is sufficiently large (Gianola and Fernando, 1986). The Bayesian method is well suited for
analyzing small populations when extensive historical information is available (Carneiro et al.,
2007).
The objective of the study was to estimate heritability and repeatability for milk yield and
lactation length in crossbred buffaloes using Bayesian inference.

646

The Brazilian genetic improvement program of buffalo provided the data that included 628
females, from four herds, born between 1980 and 2003. Univariate analyses were performed with
the Gibbs sampler to obtain the estimates of variance and covariance, using the program MTGSAM
(Multiple Trait Gibbs Sampling for Animal Models) as described by Van Tassell and Van Vleck
(1995). The model for milk yield and lactation length included random effects direct additive and
permanent environment factors, and effects of contemporary groups (CG) and number of milking
(NM) and calving orders (CO). The CG contained animals of the same herd, season and year of
birth or parturition.
The MTGSAM uses the Gauss–Seidel iterative method in the mixed model equation to
obtain an initial value for the fixed and random effects to be used in the Gibbs sampler. The initial
numbers were arbitrarily obtained using a single chain with 100,000 iterations. The convergence
diagnosis were analyzed through the Geweke method (1992) and the burn-in, thinning and chain
length were determined by Raftery and Lewis (1992) method using the algorithm implemented in R
software through the package BOA (Bayesian Output Analysis) (Smith, 2005).

The means for milk yield and lactation length were 1,546.1±483.8 kg and 252.3±42.5 days,
respectively. Seno et al. (2010) reported means of 1,594.4 kg and 271.6 days for milk yield and
lactation length, respectively, in Murrah buffaloes from Brazil.
The heritability coefficients were 0.31 (mode), 0.35 (mean) and 0.34 (median) for MY,
with small discrepancy from normal distribution (Figure 1). This heritability is higher than
estimated for Murrah breed (Ramos et al., 2006; Tonhati et al., 2008; Rodrigues et al., 2010, Seno et
al., 2010). Our result indicates a high potential of genetic gain for milk yield through selection.
The heritability coefficients for lactation length (Figure 1) were 0.11 (mode), 0.10 (mean)
and 0.10 (median). Other studies have reported values comparable to the present ones: 0.10 for
Murrah buffaloes (Rodrigues et al., 2010) and 0.08 for crossbred buffaloes (Malhado et al., 2009).
Our result suggests that direct selection for this trait is unlikely to result in substantial gains.
The mean, median and mode of the repeatability coefficients were similar and equal to
0.50 and 0.15 for MY and LL (Figure 2), respectively. Other studies have reported repeatability
coefficients for MY of 0.28 Tonhati et al. (2000), 0.32 Ramos et al. (2006), 0.51 Malhado et al.
(2009) and 0.33 Rodrigues et al.(2010). Malhado et al. (2009) estimated a repeatability of 0.12 for
LL suggesting that there may be large oscillations in the duration of lactation. The repeatability for
MY indicates that selection based on the first lactation could contribute for an improvement in this
trait.
IMPLICATIONS
Milk yield is the only trait with clear potential for genetic improvement by direct genetic
selection. The repeatability for MY indicates that selection based on the first lactation could
contribute for an improvement in this trait.
REFERENCES
Carneiro, J.M.Jr., R.F. Euclydes, R.A. Torres and S. Lopes. 2007. Estimation of variance
components using Bayesian and frequentist inferences considering simulated data under
heterogeneity of variance. R. Bras. Zootec. 36:1539-1548.
Gianola, D. and R.L. Fernando. 1986. Bayesian methods in animal breeding theories. J. Anim. Scie.
63:217-244.
Malhado, C.H.M., A.A. Ramos, P.L.S. Carneiro, D.M.M.R. Azevedo, R.A.M. Affonso, D.G.
647
Pereira and J.C. Souza. 2009. Genetic parameters of reproductive and productive traits in
cross-breed water buffaloes in Brazil. Rev. Bras. Saúde Prod. Anim. 10: 830-839.
Malhado, C.H.M., A.C.M. Malhado, P.L.S. Carneiro, A.A. Ramos, J.A. Carrillo and A. Pala.
2012b. Population structure and genetic variability in the Murrah dairy breed of water
buffalo in Brazil accessed via pedigree analysis. Trop. Anim. Health Prod. 44:1891-1897.
(Ed. J.M. Bernardo J.O. Berger and A.P. Dawid). Oxford University, UK.
Rodrigues, A.R., J.R.F. Marques, C.V. Araújo, R.N.C. Camargo Júnior and L.N.S. Dias. 2010.
Estimation of genetic parameters of dairy buffaloes productive characteristics Eastern
Amazon. Arq. Bras. Med. Vet. Zootec. 62:712-717
Seno, L.O., V.L. Cardoso, L. El Faro, R.C. Sesana, R.R. Aspilcueta-Borquis, G.M.F. de Camargo
and H. Tonhati. 2010. Genetic parameters for milk yield, age at first calving and interval
between first and second calving in milk Murrah buffaloes. Livest. Res. Rural Dev. 22.
Tonhati, H., M.F.C. Muñoz, J.A. Oliveira, J.M.C. Duarte, T. Furtado and S.P. Tseimazides. 2000.
Parâmetros Genéticos para produção de leite, gordura e proteína em bubalinos. R. Bras.
Zootec. 29:2051-2056.
Tonhati, H., M.F.C. Muñoz, J.A. Oliveira, L. El Faro, A.L.F. Lima and L.G.A. 2008. Test-day milk
yield as a selection criterion for dairy buffaloes (Bubalus bubalis Artiodactyla,Bovidae).
Genet. Mol. Biol. 31:674–679
648
Figure 1. A posteriori distributions of the heritabilities for milk yield (MY) and lactation length
(LL) estimated by Bayesian inference.
Figure 2. A posteriori distributions of the repetabilities for milk yield (MY) and lactation length
(LL) estimated by Bayesian inference.
649
Genetic Correlation for Pre-Weaning and Post-Weaning Traits of

Mediterranean Buffaloes from Brazil, Estimated By Bayesian Inference
Vanius FALLEIROa,e, E.S. SILVEIRAa, A.A. RAMOSb, P.L.S. CARNEIROc, J.A.
CARRILLOd, C.H.M. MALHADOc*
a
State University of Southwest Bahia, Itapetinga, BA, Brazil
b
c
d
University of Maryland, College Park, MD, USA
e
Federal Institute IF, Rio do Sul, SC, Brazil
ABSTRACT
The aim of this study was to estimate genetic, environmental and phenotypic correlation
between birth weight (BW) and weight at 205 days age (W205), BW and weight at 365 days age
(W365) and W205-W365, using Bayesian inference. The Brazilian Program for Genetic
Improvement of Buffaloes provided the data that included 3,883 observations from Mediterranean
breed buffaloes. With the purpose to estimate variance and covariance, bivariate analyses
were performed using Gibbs sampler that is included in the MTGSAM software. The model for
BW, W205 and W365 included additive direct and maternal genetic random effects, maternal
environmental random effect and contemporary group as fixed effect. The convergence diagnosis
was achieved using Geweke, a method that uses an algorithm implemented in R software through
the package Bayesian Output Analysis. The calculated direct genetic correlations were 0.34 (BW-
W205), 0.25 (BW-W365) and 0.74 (W205-W365). The environmental correlations were 0.12, 0.11
and 0.72 between BW-W205, BW-W365 and W205-W365, respectively. The phenotypic
correlations were low for BW-W205 (0.01) and BW-W365 (0.04), differently than the obtained for
W205-W365 with a value of 0.67. The results indicate that BW trait have low genetic,
environmental and phenotypic association with the two others traits. The genetic correlation
between W205 and W365 was high and suggests that the selection for weight at around 205 days
could be beneficial to accelerate the genetic gain.
Keywords: Bubalus bubalis, Environmental Correlation, Gibbs Sampler, Phenotypic Correlation
INTRODUCTION
Buffaloes of Murrah, Mediterranean, Jabarabadi, and Carabao breeds were introduced to
Brazil at the beginning of the 20th century (Cassiano et al., 2003) and recent estimations suggest that
there are currently about 1.2 million buffalos distributed across all Brazilian states (Ibge, 2012).
On developing an animal selection program is necessary to know the factors which
influence growth as well as the genetic correlations among the weight traits since these genetic
parameters are basic elements on the establishment of guidelines for animal improvement. The
correlation between traits is an important aspect to deal with in breeding programs, because genetic
change in a given trait can affect other traits (Vencovsky and Barriga, 1992). In addition, in most
breeding programs the strategy is based on selection for different traits simultaneously and,
therefore, knowledge on the genetic correlation between traits is useful for the establishment of
selection criteria.
REML and Bayesian methods have been applied extensively in animal breeding to
estimate covariance components and genetic parameters. The Bayes methodology provides a
solution for the finite sample size problem, because an exact a posteriori distribution exists for each
large or small data set from which inferences can then be drawn. When a large data set is analysed,
a priori information tends to be subjugated by the likelihood function in the establishment of the a
650
size is sufficiently large (Gianola and Fernando, 1986).
The aim of this study was to estimate direct genetic, environmental and phenotypic
correlation between birth weight (BW) and weight at 205 days age (W205), BW and weight at 365
days age (W365) and W205-W365, using Bayesian inference.

The Brazilian Program for Genetic Improvement of Buffaloes provided the data that
included 3,883 observations from Mediterranean breed buffaloes raised in four herds.
In order to obtain the covariance and variance estimates, bivariate analyses were performed
using the Multiple Trait Gibbs Sampling for Animal Models (MTGSAM), as described by Van
Tassell and Van Vleck (1995). The model for BW, W205 and W365 included additive direct as
random effect and contemporary group as fixed effect. The contemporary groups consisted of
animals of the same sex, time (season and year of calving) and farm of origin.
The initial number of iterations was arbitrarily obtained using a single chain of 100,000
iterations and a burn-in period of 10,000 iterations with a sampling interval of 10. The method
described by Raftery and Lewis (1992), using an algorithm implemented in the software R by
means of the Bayesian Output Analysis Package (BOA) (Smith, 2005), was applied to these data to
set burn-in, chain length and sampling interval. In addition, the BOA package was used in the
convergence diagnostic test, following the method of Geweke (1992).

The direct genetic correlations for BW were 0.34 (BW-W205) and 0.25 (BW-W365) (Figure
1 and 2). The environmental correlations were 0.12 and 0.11 between BW-W205 and BW-W365,
respectively (Table 1). The phenotypic correlations were low for BW-W205 (0.01) and BW-W365
(0.04). The results indicate that BW trait have low genetic, environmental and phenotypic
association with the two others traits.
Cardoso et al. (2004) reported low genetic correlation (0.23) for such trait in a Nelore herd.
Working with buffaloes, Malhado et al. (2008) obtained, through REML, values of 0.21, 0.09 and
0.8 for genetic, phenotypic and environmental correlations, respectively.
According to Malhado et al. (2004), birth weight trait presents inconsistent distribution
since in practice the newborns are usually weighted in improper scales putting the accuracy of the
measurements at stake which quite often generates biased registers and therefore altered
distribution.
The direct genetic (Figure 3), phenotypic and environmental correlations for W205-W365
were 0.74, 0.72 and 0.67, respectively. The genetic correlation between W205 and W365 was high
and shows that part of the genes which influence W205 also influence W365 suggesting that the
selection for weight at around 205 days could be beneficial to accelerate genetic gain.
The estimates obtained in this study were lower than the reported by Malhado et al. (2012)
in the Jaffarabadi breed from Brazil. These authors estimated correlations of 0.99 and 0.88 for
genetic and phenotypic correlation, respectively.
REFERENCES
Cassiano, L.A.P., A.S. Mariante, C. McManus and J.R.F. Marques. 2003. Caracterização fenotípica
de raças bubalinas nacionais e do tipo Baio. Pesq. Agropec. Bras. 38: 1337-1342.
Cardoso, F.F., R.A. Cardelino and L.T. Campos. 2004. Componentes de (co)variância e parâmetros
genéticos de caracteres pós-desmama em bovinos da raça Angus. Revista Brasileira de
Zootecnia Viçosa 33: 313-19.
posterior moments. In: Bayesian statistics (Ed. J.M. Bernado, J.O.Berger, A.P. Dawid and
A.F.M. Smit). Oxford University, New York. pp. 526.
651
Gianola, D. and R.L. Fernando. 1986. Bayesian methods in animal breeding theory. J. Anim. Sci.
63: 217-244
IBGE. 2012. Censo demográfico. Instituto Brasileiro de Geografia e Estatística, Brasil.
Malhado, C.H.M., P.L.S. Carneiro and A.A. Ramos, J.C. Souza. 2004. Análise da distribuição de
pesagens em diferentes idades de bubalinos da raça Mediterrânea. Rev. Cient. Prod. Anim. 6:
No.2.
Malhado, C.H.M., A.A. Ramos, P.L.S.Carneiro, D.M.M.R. Azevedo, J.C. Souza and R. Martins
Filho. 2008. Improvement and population structure of Mediterranean water buffaloes raised
in Brazil. Pesq. Agropec. Bras. 43: 215-220.
Malhado, C.H.M., A.C.M. Malhado, A.A. Ramos, P.L.S. Carneiro, F. Siewerdt and A. Pala. 2012.
Genetic Parameters of Some Performance Traits by Bayesian Inference for Dual Purpose
Jaffarabadi Buffaloes. Arch. Tierzucht. 6:567-576.
Raftery, A.E., S. Lewis. 1992. How many iterations in the Gibbs sampler? In: Bayesian statistics 4
(Ed. J.M. Bernardo, J.O. Berger and A.P. Dawid). Oxford University Press.
Van tassel, C.P. and L.D. Van Vleck. 1995. A manual for use of MTGSAM. A set of FORTRAN
programs to apply Gibbs sampling to animal models for variance components estimation,
Lincoln: Department of Agriculture, Agricultural Research Service.
Vencovsky, R. and P. Barriga. 1992. Genética biométrica no fitomelhoramento. Ribeirão Preto:
SBG.
(HPD) of the genetic, environmental and phenotypic correlation for birth weight (BW) and weight
at 205 (W205) and 365 (W365) days of age obtained using Gibbs sampler.
Traits Parameters Mean S.D Median Mode HPD

Low High
limit Limit
BW-W205 Direct genetic 0.37 0.11 0.37 0.35 0.15 0.59
Environmental 0.01 0.05 0.01 0.01 -0.09 0.10
Phenotypic 0.13 0.02 0.13 0.13 0.08 0.17
BW-W365 Direct genetic 0.24 0.12 0.24 0.25 -0.07 0.46
Environmental 0.06 0.05 0.06 0.06 -0.32 0.15
Phenotypic 0.11 0.02 0.11 0.11 -0.62 0.15
W205-W365 Direct genetic 0.74 0.04 0.75 0.75 0.66 0.82
Environmental 0.67 0.03 0.67 0.67 0.61 0.73
Phenotypic 0.72 0.02 0.72 0.72 0.68 0.75
652
Figure 1. A posteriori distribution of the direct genetic correlation between birth weight and weight
at 205 days of age (BW_W205) estimated by Bayesian inference.
Figure 2. A posteriori distribution of the direct genetic correlation between birth weight and weight
at 365 days of age (BW_W365) estimated by Bayesian inference.
Figure 3. A posteriori distribution of the direct genetic correlation between weight at 205 and
weight at 365 days of age (W205_W365) estimated by Bayesian inference.
653
Productive and reproductive traits in Murrah breed from Brazil
Alcides Amorim RAMOSa, C. H.M. MALHADOb*, A.C.M. MALHADOc, P.L.S.

CARNEIROb, J.C. SOUZAd, A. PALAe
a
Universidade Estadual Paulista, Botucatu, SP, Brazil
b
Universidade Estadual do Sudoeste da Bahia, Jequié, BA, Brazil
c
Universidade Federal do Alagoas, Maceió, Al, Brazil
d
Universidade Federal do Mato Grosso do Sul, Dourorados, MS, Brazil
e
Canakkale Onsekiz Mart University, Ziraat Fak, Turkey.
ABSTRACT
Major objective of this study was to estimate heritability and genetic correlations between
milk yield (MY) and calving interval (CI) and lactation length (LL) in Murrah buffaloes using
Bayesian inference. The database used belongs to the genetic improvement program of four buffalo
herds from Brazil. To obtain the estimates of variance and covariance, bivariate analyses
were performed with the Gibbs sampler, using the program MTGSAM. The heritability coefficient
estimates were 0.28, 0.03 and 0.15 for MY, CI and LL, respectively. The genetic correlations
between MY and LL was moderate (0.48). However, the genetic correlation between MY and CI
showed large HPD regions (highest posterior density interval). Milk yield was the only trait with
clear potential for genetic improvement by direct mass selection. The genetic correlation between
MY-LL indicates that indirect selection using milk yield is a potentially beneficial strategy. The
interpretation of the estimated genetic correlation between MY-CI is difficult and could be spurious.
Keywords: genetic correlation, Gibbs sampler, heritability
INTRODUCTION
The lifetime production of a dairy cow is an indication of its utility and is influenced by key
fertility parameters such as age at first calving, calving intervals, length of each lactation and
probability of surviving from one lactation period to the next (Hossein-Zadeh, 2011). Likewise,
the economic return of buffalo milk depends of the milk production and reproductive efficiency of
animals – the latter particularly affected by calving interval (Ramos et al., 2006).
Breeding buffaloes for increased milk production and reproductive efficiency may not be
straightforward. Genetic antagonism has been reported between traits associated with reproductive
efficiency and milk production in cattle (Marti and Funk, 1994). However, these findings are by no
means universal and some researchers have reported favorable associations between reproductive
traits and milk yield in both dairy cattle (Hossein-Zadeh, 2011) and buffaloes (Ramos et al., 2006).
The typical procedure of estimation /prediction -of breeding value- is normally based on
restricted maximum likelihood/best linear unbiased prediction (REML/BLUP). However, because
REML / BLUP uses approximation and assumptions of asymptotic normality, it provides only
approximate confidence intervals for genetic parameters and variance, when the distribution of the
estimators are unknown (Resende, 2002). A potential solution is the adoption of a Bayesian
approach that allows the construction of exact probability intervals for estimates of genetic
parameters. Major objective of the present study was to estimate heritabilities and genetic
correlations associated of the milk yield (MY) with calving intervals (CI) and lactation length (LL)
in water buffaloes using Bayesian inference (BI).

Data on milk yield (2910), lactation length (2910) and calving interval (1721) were collected
from 702 Murrah buffaloes born between 1982 and 2003 of four herds from Brazil. The averages of

654
the traits were 1631.5±642.1 kg, 269.4±43.1 days and 411.0 ± 80.1 days for MY, LL and CI,
respectively.
Variance components, heritability coefficients and repeatabilities were calculated for milk
yield, lactation length and calving interval. We also estimated the genetic, environmental and
phenotypic correlations between MY-LL and MY-CI
Bivariate analyses were performed with the Gibbs sampler to obtain the estimates of variance
and covariance, using the program MTGSAM (Multiple Trait Gibbs Sampling for Animal Models)
as described by Van Tassell and Van Vleck (1995). The models included random effects direct
additive and permanent environment factors, and effects of contemporary groups (CG) and number
of milking (NM) and calving orders (CO). The CG contained animals of the same herd, season and
year of birth or parturition.
The initial numbers were arbitrarily obtained using a single chain with 150,000 iterations and
a burn-in period of a 25,000 samples was used with samples taken each 25 cycles. The convergence
diagnosis was analyzed through the Geweke method (1992) using the algorithm implemented in R
software through the package BOA (Bayesian Output Analysis) (Smith, 2005).

The means, medians and modes of estimates of variance components and genetic
parameters for milk yield and lactation length were similar (Table 1). According to Wright et al.
(2000), the modal is the most appropriate position measure for a posteriori distributions, and best
reflects the higher frequency values (maximum distribution).
The heritability estimates for milk yield was moderate (mode = 0.28), suggesting that this
traits has enough additive genetic variation to respond well to direct mass selection. The modal
estimation of heritability for lactation length was 0.15 (Table 1), which indicates small possibility of
direct selection for the trait.
The heritability for CI was zero (mode = 0.00). This is in agreement with value reported in
the literature on Buffaloes (Ramos et al. 2006). Heritability estimates of calving interval are
obtained using field data, which are subject to interference from the breeder. For example, measures
of calving interval frequently omit dams culled for low production or reproductive problems, a
strategy that can result in reduction of additive genetic variance. Additionally, this low heritability
implies that variations in these traits are, to a large extent, influenced by environmental factors such
as herd management policies. New studies with censored data can overcome this problem.
The repeatability of the traits were 0.38, 0.16 and 0.06 (modes) for MY, LL and CI,
respectively (Table 1), indicating that the selection of animals based on limited information about
the first lactation can only be used to improve milk yield.
The estimated correlations between milk yield and the other two traits were positive (Table
2). However, the genetic correlation between MY and CI showed large HPD region. Similar results
were reported by Aspilcueta-Borquis et al. (2010), who estimated the genetic correlations of milk
yield and different milk quality traits.
The correlations (genetic, phenotypic and environmental) between MY and LL were
moderate (from 0.47 to 0.52). High genetic correlations between these two traits in buffaloes have
been reported in the other studies 0.89 (Malhado et al., 2009) and 0.72 (Rodrigues et al., 2010)
using the REML method. These results indicate that selection for milk yield could promote
moderate changes in the lactation length.
The three position measures for genetic correlation of MY-CI ranged from 0.35 to 0.40.
However, the high standard deviations and large HPD complicate the interpretation of the
parameter. Ramos et al. (2006) used REML in the same set of data and estimated a negative
correlation of -0.22. Malhado et al. (2009) have also reported a negative value of -0.25.
IMPLICATIONS
Milk yield was the only trait with clear potential for genetic improvement by direct
selection. The genetic correlation between MY-LL indicated that indirect selection using milk yield
655
is a potentially beneficial strategy. The interpretation of the estimated genetic correlation between
MY-CI is difficult to interpret and could be spurious.
REFERENCES
Aspilcueta-Borqueis, R.R., F.R. Araújo Neto, F. Baldi, A.B. Bignardi, L.G. Alburquerque and H.
Tonhati. 2010. Genetic parameters for buffalo milk yield and milk quality traits using
Bayesian inference. J. Dairy Sci. 93:2195-2201.
Hoossein-Zadeh, N.G. 2011. Estimation of genetic and phenotypic relationships between age at first
calving and productive performance in Iranian Holsteins. Trop. Anim. Health Prod.
43:967–973.
Malhado, C.H.M., A.A. Ramos, P.L.S. Carneiro, D.M.M.R. Azevedo, P.R.A.M. Affonso, D.G.
Pereira and J.C. Souza, 2009. Genetic parameters of reproductive and productive traits in
cross-breed water buffaloes in Brazil. Rev. Bras. S. Prod. Anim. 10:830-839.
(Ed. J.M. Bernardo, J.O. Berger and A.P. Dawid). Oxford University, UK.
Resende, M.D.V. 2002. Biometric genetic and statitical in improvement of perennial plants,
Embrapa Informação Tecnológica. pp. 975.
Rodrigues, A.R., J.R.F. Marques, C.V. Araújo, R.N.C. Jr. Camargo and L.N.S. Dias. 2010.
Amazon. Arq. Bras. Med. Vet. Zootec. 62:712-717.
Wright, D.R., H.S. Stern and J. Berger. 2000. Comparing traditional and Bayesian analyses of
selection experiments in animal breeding. J. Agr. Biol. Environ. Stat. 5:240-256.
656
Table 1. Means, standard deviation (S.D.), median, mode and higher posteriori density interval
(HPD) of the genetic parameters for traits milk yield (MY), lactation length (LL) and
calving interval (CI).

Low limit High limit
MY σa 2
84221.70 15144.22 83824.00 83151.80 56941.68 115458.60
σ pe2
31746.39 10969.00 31443.19 32461.23 11231.70 51908.61
σe 2
184770.38 6522.83 184827.90 18529.54 171751.15 197921.10
σp 2
300738.48 11257.90 300738.48 299834.49 280006.93 323010.60
2
h 0.28 0.04 0.28 0.28 0.19 0.36
R 0.38 0.02 0.38 0.38 0.34 0.42
LL σa 2
265.64 59.23 263.75 259.85 151.78 383.11
σ pe2
33.25 33.91 21.89 7.54 2.80 105.20
σ 2e 1508.02 52.09 1507.03 1497.00 1404.36 1603.52
σp 2
1806.90 58.05 1804.81 1791.43 1697.77 1923.4
2
h 0.15 0.03 0.15 0.15 0.09 0.21
R 0.17 0.02 0.16 0.16 0.13 0.21
CI σa 2
485.15 397.79 400.87 109.75 7.97 1252.72
σ pe2
426.88 373.01 332.00 91.02 0.60 11150.84
σ 2e 14314.30 611.60 14295.27 14253.88 13144.33 15520.15
σp 2
15226.63 574.86 15217.21 15309.88 14127.33 163631.68
2
h 0.03 0.021 0.03 0.00 0.00 0.08
R 0.06 0.026 0.06 0.06 0.01 0.11
(1)
a is the direct additive genetic variance component; pe is the permanent environment variance; e is the residual
2 2 2
variance; p2 is the phenotypic variance; h2 is the heritability; R is repeatability.
Table 2. Environmental, phenotypic and genetic correlations between milk yied (MY) with
lactation length (LL) and calving interval (CI).
Correlation Traits Mean S.D. Median Mode HPD
Low High
limit limit
Genetic LL 0.47 0.11 0.47 0.48 0.23 0.69
CI 0.40 0.30 0.38 0.35 -0.07 0.99
Phenotypic LL 0.47 0.03 0.47 0.47 0.41 0.53
CI 0.08 0.03 0.08 0.08 0.03 0.14
Environmental LL 0.52 0.02 0.52 0.52 0.48 0.56
CI 0.07 0.03 0.07 0.07 0.01 0.13
(1)
S.D. is the standard deviation; HPD is a higher posteriori density interval.
657
Genomic Exploration of Pakistani Buffalo- our Black Gold
Masroor Ellahi BABAR*1, Tanveer HUSSAIN2, Abdul WAJID2, Asif NADEEM2, Waqas
Ahmad KHAN2, Ambreen FATIMA2, Rashid SAIF2, Muhammad Mudassar MANZOOR2 and
Sajjad Ali SHAH2
1
Department of Livestock Production, University of Veterinary and Animal Sciences Lahore 54000,
Pakistan.
2
Institute if Biochemistry and Biotechnology, University of Veterinary and Animal Sciences Lahore
54000, Pakistan.
*
Corresponding author: drbabar@hotmail.com
ABSTRACT
Indian subcontinent is presupposed as the center for river buffalo domestication. Pakistan is
the world second largest country in buffalo population. Recent studies on buffalo in Pakistan have
contributed to its genomics knowledge. The cytochrome b gene and D-loop of the mitochondrial
DNA have given constructive conclusion about the domestication of history of Bubalus bubalis,
confirming independent domestication between swamp and river buffaloes at geographically distant
locations. The mitochondrial sequence diversity led to conclude that having bordering area proximity
between Pakistan and India might have contributed to the initiation of domestication of the present
day river buffalo. A set of ISAG/ FAO recommended microsatellites showed considerable genetic
diversity among 5 buffalo breeds of Pakistan and found highly useful for breed, ownership and
parentage confirmation in forensic cases. The Sex-Determining Region of Y chromosome (SRY)
coding region was found highly conserved in Pakistani buffaloes and was used to construct bovine
phylogeny with other bovides conforming buffalo position in the tree. Such studies helped to
understand the genetic diversity and evolution of buffalo. Among other genetic projects recently
completed on Pakistani buffaloes also include analysis of AMP-activated protein kinase (AMPK α2,
β1, γ3) participated in the regulation of metabolic pathways in glucose and fatty acid metabolism,
glycogen metabolism, protein synthesis and meat quality. The SNPs found in these genes may be
associated with economically important traits in buffaloes. Milk production gene Pit-Oct-Unc
Domain, Class 1, Transcription Factor 1 (POU1F1) were also examined which gave useful
information in designing breeding strategies of the country and Markers Assisted Selection in future.
These results can facilitate future researchers interested in buffalo genomics in the world.
Keywords: Genetic diversity, mt DNA, Microsatellite markers, SRY genes, AMPK genes,
Cytokines, Buffalo breeds, Pakistan
INTRODUCTION
Other than cattle and other small ruminants, the buffalo is being an important species and
present superlative significance to several countries within Asia, likely to those of the Indian
subcontinent. The domestic buffaloes act as multipurpose animal for major sources such as milk
production, meat, draft power, hide and employment to the trivial farmers and landless laborers in
several countries of Asia (Hussain et al., 2009). The Asian domestic water buffalo is of two types,
river and swamp found on the difference of Karyotyping, body size, external appearance and other
biological characteristics (Kumar et al., 2007). Primarily, the distinction between these two breeds
was explicated by their divergent morphological features, however, the genetic distinctions have been
revealed subsequently in karyotyping, microsatellite markers and mitochondrial DNA (mtDNA)
patterns.
658
Pakistan encompasses 29.9 million buffalo heads, including five major breeds Nile, Ravi, Nili-Ravi,
Kundi and Aza-kheli. The importance of this species to the Pakistani dairy industry is gigantic,
providing a total of 65% milk production in the country (Khan et al., 2007).
The mitochondrial DNA is normally maternally inherited and the mitochondrial genome (mtGenome)
is not subjected to recombination, along with these two defined aspects, it is easy to carry out large
number of individual’s genotyping. The mtDNA polymorphism has found incredible convention in
the maternal lineage evolutionary studies of different species. Both the D-loop and cytochrome b
gene have been afford a momentous approaching into the domestication and past
migration/expansion history of buffaloes (Babar et al., 2011; Hussain et al., 2009; Kumar et al.,
2007; Saif et al., 2012). Although little is known about the genetics of Pakistani buffalo breeds,
however, our previous study on mitochondrial D-loop inveterate that Pakistani buffaloes are of river
type and genetic diversity through mitochondrial D-loop sequences suggested that having bordering
area proximity between Pakistan and India might have contributed to the initiation of domestication
of the present day river buffaloes. Our previous studies also confirming the independent
domestication events between river and swamp buffaloes (Babar et al., 2011; Hussain et al., 2009 and
Saif et al., 2012). Several previous studies have increasingly evident that most of the livestock
species have been domesticated more than once (Jansen et al., 2002 and Bruford et al., 2003). The
reduced median network of our previously D-loop sequences signified that Pakistani buffalo might be
a consequence of multiple domestication events.
Our previous studies have been reported on the water buffalo genetic diversity through mitochondrial
D-loop (Babar et al., 2012 and Hussain et al., 2009) and cytochrome b gene (Saif et al., 2012a and
Saif et al., 2012b) give a significant insight into the genetic structure. Along with all these
approaches, determining the genetic differentiation and relationship between breeds and species is
equally suitable through Y chromosome analysis. SRY (Sex Determining region Y), being a
candidate non-recombining Y-chromosomal gene for sex determination, has been considering as an
ideal and best molecular marker to discriminate related species, especially in single nucleotide
polymorphism (SNP) analysis. Our result found a significant polymorphism locating at the site of
202 bp of the SRY gene coding region was a Cytocine (C), but the nucleotide at this region was
identified a Guanine (G) for swamp buffalo. Our finding supported to some previous studies
suggested that this Y-linked SNP displayed type specific alleles differentiating swamp and river
buffaloes. The NJ phylogenetic tree based on Y chromosome SRY gene sequences showed a clear
divergence of river buffalo and swamp buffalo.

The microsatellite markers are recommended for the analysis of genetic variation and
relationship between populations as recommended in the MoDAD project proposed by FAO,
(Hoffmann et al., 2004). Microsatellite markers have been confirmed to be valuable markers for a
variety of purposes, such as determination of genetic variation, genome mapping, parentage
determination, cancer research and disease research (Garcia-Moreno et al., 1996). Total of 8
microsatellite markers were used for genotyping of 123 DNA samples of five buffalo breeds (Nili 25,
Ravi 28, Nili-Ravi 25, Kundi 25 and 20 Azakheli). Although our previous result of D-loop sequences
poorly differentiated between Nili-Ravi and Kundi individuals (FST = -0.0054), using 8 microsatellite
markers data grouped these two breeds into separate clusters (Fig. 1). Nili and Ravi buffalo breeds
are very close to each other in their genetic structure and they both are close to the Nili-Ravi which is
the major buffalo breed of Pakistan. Azakheli breed which is thought to be wild buffalo of Swat
region is clearly different from Nili, Ravi and Nili-Ravi breed of Pakistan.
659
Figure 1. Dendrograms Based on Nei's (1972) Genetic distance, UPGMA method, Modified from NEIGHBOR
procedure of PHYLIP V. 3.5
RESULTS AND DISSCUSIONS

To meet the demands of a rising human population in Pakistan, milk and meat production will
need to accelerate with respect to total outputs, production efficiency and product quality. To
assemble the purpose adenosine monophosphate-activated kinase (AMPK) and Pit-Oct-Unc Domain,
Class 1, Transcription Factor 1 (POU1F1) is a member of the tissue-specific POU homeobox
transcription factor family are carried out in Pakistan buffalo breeds. AMPK has a vital role in
maintaining energy balance in all eukaryotes and regulates all aspects of cellular function (Hardie,
2011). AMPK is a heterotrimeric complex made up of seven α, β and γ isoforms, a catalytic subunit
alpha and a regulatory subunit beta, in addition to the regulatory subunit γ (γ1, γ2, γ 3) (Carling,
2004). Indirectly AMPK also promotes feeding and feeding behavior (Hardie et al., 2012). As
PRKAG3 have a key function in the regulation of energy metabolism in skeletal muscle leading to an
enhanced glucose transfer and glycogen synthesis (Milan et al., 2000), so all these studied genes
might be associated with meat quality in buffalo. The aim of this study was to find out the novel
variations at the population level so this study exemplified the candidate gene approach. This is
earliest report of molecular markers for identification of traits related to energy metabolism in
Pakistani buffaloes that can be used for future selection and breeding programs. So, PRKAG3 gene
sequences can potentially be used to assure meat type and quality, and to prevent fraudulent labeling
and illegal hunting, and not to let people repent on eating religiously prohibited meat and become
allergic in some instances.
POU1F1 gene in buffalo encodes a polypeptide chain of 291 Amino acids ( ~ 33kD),
containing 6 exons and 5 introns, locating on chromosome 1. The aim of this candidate gene
approach was carried to obtain and associate polymorphism with the milk production trait. This was
the first step in Pakistani buffaloes to confirming the genetic markers for production traits.
All these reports contributed towards existing knowledge of buffalo genetics and these can help in
understanding the genetic architecture of buffaloes. The present information illustrated a
comprehensive insight into the genetics of the Pakistani buffalo breeds and it would be facilitate the
future researchers concerned with buffalo genomics in the world. The data can act as base line
information for future breeding conservation program.
REFERENCES
Jansen, T., P. Forster, M. A. Levine, H. Oelke, M. Hurles, C. Renfrew, J. Weber and K. Olek. 2002.
Mitochondrial DNA and the origins of the domestic horse. Proc Natl Acad Sci USA 2002,
99:10905-10910.
660
Bruford, M. W., D. G. Bradley and G. Luikart. 2003. DNA markers reveal the complexity of
livestock domestication. Nat Rev Genet. 4:900-910.
Hoffmann, I., P.A. Marsan, J.S.F. Barker, E.G. Cothran, O. Hanotte, J.A. Lenstra, D. Milan, S.
Weigend and H. Simianer. 2004. New MoDAD marker sets to be used in diversity studies for
the major farm animal species: recommendations of a joint ISAG/FAO working group. In:
Proceedings of 29th International Conference on Animal Genetics, Tokyo, Japan.
Carling, D. 2004. The AMP-activated protein kinase cascade–a unifying system for energy control.
Trends Biochem Sci 29, 18–24.
Babar, M. E., T. Hussain, M. Imran, M. Nagarajan and S. Kumar. 2011. Mitochondrial DNA
diversity patterns in Pakistani buffalo. Anim genet. doi:10.1111/j.1365-2052.2011.02250.x
Hussain, T., M. E. Babar, A. Nadeem, R. Jabeen and A. Ali. 2009. Phylogenetic Analysis of Kundi
Buffalo Breed of Pakistan Through Mitochondrial D-Loop Region. Pakistan J. Zool. Suppl.
Ser., No.9, pp. 341-246, 2009.
Saif, R., M. E. Babar, A. R. Awan, A. Nadeem, Abu-Saeed Hashmi and T. Hussain. 2012. DNA
fingerprinting of Pakistani buffalo breeds (Nili-Ravi, Kundi) using microsatellite and
cytochrome b gene markers. Mol Biol Rep, 39:851–856
Saif, R., M. Wasim and M. E. Babar. 2012. Molecular phylogeny of Pakistani riverine buffalo based
on genetic variability of mitochondrial cytochrome b gene. Mol Biol Rep, 39:9707–9714.
Kumar, S., M. Nagarajan, J. S. Sandhu, N. Kumar and V. Behl. 2007. Phylogeography and
domestication of Indian rive buffalo. BMC, 7:186
Kumar, S., M. Nagarajan, J. S. Sandhu, N. Kumar, V. Behl and G. Nishanth. 2007. Mitochondrial
DNA analyses of Indian water buffalo support a distinct genetic origin of river and swamp
buffalo. doi:10.1111/j.1365-2052.01602.x
661
Application of Reactions Norms in Study of Genotype Environmental

Interaction for Milk Yield of Buffaloes
Humberto TONHATI1*, Rusbel Raul ASPILCUETA-BORQUIS1, Ana Claudia de
FREITAS1, Gregório Miguel Ferreira de CAMARGO1, and Fernando BALDI1
¹
Faculty of Agrarian and Veterinary Sciences (FCAV), State University of São Paulo, Jaboticabal
(UNESP), 14884900, SP, Brazil.
*Corresponding email: tonhati@fcav.unesp.br
ABSTRACT
The aim of this study was to estimate genetic parameters for milk yield (MY) in buffaloes
using reaction norms. Model included the additive direct effect as random and contemporary group
(herd and year of birth) were included as fixed effects and cow age classes (linear) as covariables.
The animal additive direct random effect was modeled through linear Legendre polynomials on
environment gradient (EG) standardized means. Mean trends were taken into account by a linear
regression on Legendre polynomials of environmental group means. Residual variance was
modeled trough 6 heterogeneity classes (EG). These classes of residual variance was formed : EG1:
mean = 866,93 kg (621,68 kg-1011,76 kg); EG2: mean= 1193,00 kg (1011,76 kg-1251,49 kg);
EG3: mean= 1309,37 kg (1251,49 kg –1393,20 kg ); EG4: mean= 1497,59 kg (1393,20 kg–
1593,53 kg); EG5: mean =1664,78 kg (1593,53 kg –1727,32kg) e EG6: mean= 1973,85 kg
(1727,32 kg –2422,19 kg ).(Co)variance functions were estimated by restricted maximum
likelihood (REML) using the GIBBS3F90 package. The heritability estimates for MY raised as the
environmental gradient increased, varying from 0.20 to 0.40. However, in intermediate to favorable
environments, the heritability estimates obtained with Considerable genotype-environment
interaction was found for MY using reaction norms. For genetic evaluation of MY is necessary to
consider heterogeneity of variances to model the residual variance.
Keywords: random regression, Legendre, environment gradient
INTRODUCTION
In the reaction norm model (RN), the expression of a genotype in different environments is
described as a linear function of a value or environmental gradient (EG). The EG is usually defined
as the average performance of all genotypes in that environment. On the other hand, the random
regression models (RRM) allows the GxE effect to be systematically evaluated by regression of
breeding values of bulls on some environment measure where their offspring is raised. This method
allows to include information of a dependent variable in the explanatory model and has the
advantage of objectively discriminating environments as more or less favorable (Kolmodin et al.,
2002; Calus and Veerkamp, 2003). Environmental and management factors may significantly
impact the yield level and cause reranking of sires in different calving environments (Calus and
Veerkamp, 2003). The aim of this study was to verify the presence of GxE for PL270 through
reaction norms of environment, through random regression models using Bayesian approach in
buffalos raised in Brazil.
MATERIALSEMETHODS
In this study the milk yield from 1,004 first lactations of Murrah buffaloes at 270 days of
lactation (MY270) was analyzed, from 24 to 48 months of age, daughters of 203 sires, from 12
herds in the state of São Paulo and calving between 1985 to 2007. Milk yield was obtained from the
fifth day, and the first control considered until the 45th day after calving. The MY270 was
calculated by interpolation method suggested by International Committee for Animal Registry
(ICAR, 2002) and in the analysis were included animals with at least four controls. Contemporary
groups were defined as herd and calving year, applying the restriction that each group should
662
contain, at least, three animals. In all analysis, file containing 2,074 pedigree animals were used.
In the reaction norm model (RxN), the components of variance and breeding values were
regressed on the EG. In EG formation, farm production and year of birth were considered.
Subsequently, the EG with similar MY270 average were grouped into 6 classes as follows: EG1:
mean = 866.93 (621.68 to 1011.76); EG2: mean = 1,193.00 (1,011.76 to 1,251.49 ); EG3: mean =
1,309.37 (1,251.49 to 1,393.20); EG4: mean = 1,497.59 (1,393.20 to 1,593.53); EG5: mean =
1,664.78 (1,593.53 to 1,727, 32) and EG6: mean = 1,973.85 (1,727.32 to 2,422.19). This EG
grouping was made in order to obtain class groups with similar observations (Table 1). In random
regression analysis (RxN), Legendre linear polynomials (LLP) were used, regressed on MY270
average, adjusted for each EG class. The LLP were also used to model the fixed effect of the
population average tendency.
Bayesian inference was performed to obtain (co)variance components using GIBBS3F90
program (MISZTAL, 2010), in which Gibbs sampler is applied. In matrix notation the complete
model of random regression may be presented as:
y XZa e
where y is the vector of observations (MY270), β, a and e are, this order, vectors of fixed effects,
direct additive genetic coefficients and residual, X and Z are respectively the incidence matrices
related to β and a . Uniform a priori distributions were considered for fixed effects, Gaussian
constant
distributions for genetic coefficients and reversed Wishart for genetic variance components, where:
a | k a ~ N[0,(k a  A)]
k a | Sa ,va ~ IW [Sa va , va ]
R | Sr ,vr ~ IW [Sr vr , vr ]
where ka is the matrix of (co)variances between the random regression coefficients for the genetic
effect; A is the additive relationship matrix. A 1,500,000 cycles length chain was established, with
an initial drop of 150,000; and sampling at each 100, totaling 13,500 samples for further analysis.
After this step, descriptive statistics and high-density interval of each estimated parameter
(covariance function and residual variances) were obtained.

The MY270 average increased as environmental conditions were more favorable. There was
greater variation for MY270 in EG1 e EG6 environment according to EG2-GA5 environments
(Table 1).
The inclusion of indices from environmental variables within farms or production systems
showed that animals had different behaviors for each environment and management.
The additive genetic variance ranged from 36,562.76 (kg2) until 100,426.38 (kg2) and as
environmental conditions were more favorable, the genetic variance increased. Probably, in less
favorable environments (EG1), animals with high and low yields had similar performance, due to
the higher level of environmental restriction, and consequently the genetic variability was lower. In
dairy cattle, the genetic variances tended to increase as conditions or environmental gradients
changed. Consequently, when the (co)variance change over the environments, the genetic
correlation characteristics may change (Kolmodin et al., 2002).
EG- environmental gradient, Nº- number of animals per EG, SD- Standard Deviation, CV-
Coefficient of variation, Min- Minimum, Max- Maximum.
The residual variances increased as phenotypic and environmental conditions were more
favorable, presenting similar trends ranging from 59,553.71 (kg2) until 144,667.81 (kg2) and from
96,116.47 (kg2) until 245,094.19 (kg2), respectively.
The heritability ranged from 0.21 to 0.4. In buffaloes populations, the heritability to MY270
ranged from 0.14 to 0.20 (Rosati and Van Vleck, 2002; Tonhati et al., 2004). Kolmodin et al. (2002)
663
reported that in uni-characteristic RRM the characteristics heritability would tend to increase as
yield levels increased, which agrees with this study. Estimates of genetic correlations between
different EGs were higher as the EG were closer or more similar.
CONCLUSIONS
The reaction norm modeling using Bayesian approaches properly estimated the population
parameters according to values expected for the specie. Based on the criteria used to define the
environmental gradient, it can be inferred the presence of genotype-environment interaction for
milk production at 270 days of lactation.
REFERENCES
Calus, M. and Veerkamp, R. 2003. Estimation of environmental sensitivity of genetic merit for milk
production traits using a random regression model. J. Dairy Sci. 86: 3756-3764.
ICAR 2002 Section 2.4 Guidelines for buffalo milk recording for low to medium and medium to
high input production systems. In: International agreement of recording practices.
www.icar.org. Accessed in 20 June 2012.
Kolmodin, R. Stramberg, E. Madsen, P. 2002. Genotype by environment interaction in Nordic dairy
cattle studied using reaction norms. A. Agric Scandinavia, Norway, v.52, p.11.
Misztal, I. BLUPF90 family of programs (2010). Available in: http://nce.ads.uga.edu/~ignacy/newp
rograms.html. Accessed in 06 July 2012.
Rosati, A. and Van Vleck, L. D. 2002. Estimation of genetic parameters for milk, fat, protein and
mozzarella cheese production in Italian river buffalo Bubalus bubalis population. Livest. Prod.
Sci. 74:185-190.
Tonhati, H., Muñoz, F., Duarte, J., Reichert. R., Oliveira, R. and Lima, A. 2004. Estimates of
correction factors for lactation length and genetic parameters for milk yield in buffaloes.
Arquivo Brasileiro de Medicina Veterinária e Zootecnia 251-257.
664
Table 1. Number of observations and descriptive statistics for the MY270 (kg) according to the
environmental gradient (EG).
EG Nº Average (kg) SD CV Min Max

1 210 866.93 120.58 13.91 621.68 1,011.76
2 243 1,193.00 64.05 5.37 1,023.03 1,251. 49
3 172 1,309.37 38.81 2.96 1,259.88 1,379.05
4 215 1,497.59 69.68 4.65 1,393.21 1,593.54
5 224 1,664.78 43.99 2.64 1,605.16 1,727. 32
6 211 1,973.85 183.63 9.30 1,728.42 2,422.19
665
High conservation of SRY gene in Buffalo compared to other Bovids

Tanveer HUSSAINa,b*, Muhammad MUDASSAR MANZOORa§, Abdul WAJIDa, Asif
NADEEMa, Akhtar ALIa, Sajjad ALI SHAHa, Kamran ABBASa, Ahmad NAWAZa, Marcos DE
DONATOb,d and Masroor ELLAHI BABARe
a
Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences Lahore
54000, Pakistan
b
Department of Animal Science, Cornell University, Ithaca, NY, USA
c
Lasbela University of Agriculture, Water and Marine Sciences, Utthal, Balochistan, Pakistan
d
IIBCA, Universidad de Oriente, Cumana, Venezuela
e
Department of Livestock production, University of Veterinary and Animal Sciences Lahore 54000,
Pakistan
§
Equally contributed to this manuscript.
*Corresponding email: tanveer.hussain@uvas.edu.pk
ABSTRACT
The aim of the present study was to determine the whole nucleotide sequence of the ORF (open
reading frame) of the sex determining region Y (SRY) in two Pakistani buffalo breeds Kundi and Nili-
Ravi. The SRY gene is responsible for testis determination in mammals. A total of 50 males
representing both breeds were genotyped, found two polymorphic sites identified represent
transversional substitutions and common in both breeds. One polymorphism locating at the site of 202
bp of the SRY gene coding region was a Cytocine (C), but the nucleotide at this region was identified a
Guanine (G) for swamp buffalo, while other transversional substitutions (A-T) was also identified in
both breeds locating at the site of 470 bp of the SRY gene coding region. Our finding suggested that this
Y-linked SNP displayed type specific alleles and used as useful marker for differentiating domestic
buffaloes. Y-chromosome variation has established to be a significant genetic marker for
phylogeography of bovinae family from a male perspective.
Keywords: SRY gene, Polymorphism, Buffalo, Pakistan
INTRODUCTION
The domestic water buffalo is a species of great economic potential, especially in developing
countries. It is a major source of milk and meat. Pakistan is ranked second among the countries rich in
water buffalo (river type) genetic resources in the world, having 31.7 million heads (GOP, 2010-11).
The annual milk and meat productions of Pakistan from water buffalo are nearly 21.622 and 0.738
million tons respectively (FAO, 2009-2010). Azakheli Kundi, Nili, Ravi and Nili-Ravi are five buffalo
breeds present throughout Pakistan (Hussain et al., 2009), of which Nili-Ravi and Kundi buffalo breeds
are considered to be best native riverine type breeds. The assessment of genetic diversity is an
important tool for executing the genetic resources conservation and breeding plans, which is yet to be
characterized completely within these Pakistani buffalo breeds and other species of bovine tribe.
Many previous studies have been reported on the water buffalo genetic diversity. A variety of
genetic markers, including mitochondrial DNA (Verkaar et al., 2004b; Kumar et al., 2007; Lei et al.,
2007), Y chromosome (Cheng et al., 2001; Zhang et al., 2006; Yindee et al., 2010) and autosomal DNA
microsatellite markers (Backer et al., 1997; Kumar et al., 2006; Zhang et al., 2007), all demonstrated
the huge genetic variation among species. Along with other approaches, Y chromosome analysis is
equally suitable for determining the genetic differentiation and relationship between the breeds and
species. SRY, being a candidate non-recombining Y-chromosomal gene for sex determination, has been
utilizing as an ideal molecular marker to differentiate related species, especially in single nucleotide
666
polymorphism (SNP) analysis. A number of studies have been reported on bovine tribe species using
Y-chromosomal SRY gene including buffalo (Nijman et al., 2008; Yindee et al., 2010), cattle (Edwards
et al., 2011; Perez-Pardal et al., 2009), goat (Prashant et al., 2009) and sheep (Meadows et al., 2004).
Here we assess the genetic variability in the 690 bp coding region of SRY gene in Pakistani buffalo
breeds, Nili Ravi and Kundi, and reveal their phylogenetic relationship with other bovine species by
constructing a comprehensive bovine Y-chromosomal phylogeny.

A total of 50 blood samples were collected from the two significant buffalo breeds of Pakistan.
Twenty-five samples each from unrelated male buffaloes Kundi and Nilli-Ravi breeds with typical
phenotypic features was obtained by puncturing the jugular vein and DNA was isolated using the
extraction method of Sambrook and Russel (2001).
To amplify the complete SRY gene, two set of primers SRY-1 forward (TTGCACTAA-
GTCAGTCTGTGGTAA), SRY-1 reverse (TCGGGTTGCATAGTATTGAAGA) and SRY-2 forward
(ACTAGCCATACACCGAGACAAA), SRY-2 reverse (GGACCAGTTATATTGGA-AAGTCTG)
were designed to amplify a segment of 690 bp coding region of domestic buffalo SRY gene. PCR
reaction was carried out in a final volume of 25 μL containing 50 ng of genomic DNA, 2 mM Mgcl2,
25 μM each dNTPs, 10 μM of each primer, 5 unit of Taq DNA polymerase. The reaction mixture was
cycled in a thermocycler using the following reaction conditions: 95 ºC (4 min), 35 cycles of 95 ºC (30
s), 54 ºC (45 s), 72 ºC (30 s) followed by a final step at 72 ºC (10 min). The PCR products were
sequenced on the ABI 3130XL DNA sequencer using the BigDyeTM Terminator Cycle Sequencing Kit
(Applied Biosystem USA). Both forward and reverse primers were applied to sequence.
The sequences of complete SRY gene were aligned against reference sequence by using BioEdit
7.0.9.0 (Hall, 1999). The pairwise comparisons of observed sequence differences, number of transition
and transversion and nucleotide composition by codon position were analyzed using the computer
program MEGA version 5 (Kumar et al., 2011). Sequence variation sites/polymorphism, diversity
analysis were performed in DnaSP 5.1 (Librado and Rozas, 2009). Using the computer program
MEGA 5, a Neighbor-Joining (NJ) phylogenetic tree based on two parameter models were
reconstructed to carry out phylogenetic analysis. For complete analysis of SRY gene, sequences from
GenBank Water Buffalo FJ546413, DQ336535; Swamp Buffalo GQ2593331,DQ1197747; Afirican
Buffalo DQ336534; Bison bonasus DQ336533,AY079142; Bos fontalis DQ336530; Bos gaurus
DQ336529; Bos grunniens AY079144, DQ336531; Bos indicus EU233301, EU233304; Bos javanicus
AY079146; Bos Taurus EU233320, EU294189; Ovis aries Z30265; Ovis Canadensis HQ384295;
Capra hircus EU581862, JN561345; Moschus berezovskii AY357218; Odocoileus virginianus
DQ888702; Rangifer tarandus AB247629; Alces alces cameloides AY148965; Capreolus capreolus
DQ888700; Cervus elaphus scoticus DQ888695.

Sequence analysis of SRY gene
A complete 690 bp long coding region of SRY gene of two Pakistani buffalo breeds, Kundi and
Nilli-Ravi were successfully sequenced. The complete ORF of SRY gene is identical with those of
swamp buffalo and cattle, encoding 229 amino acids. Overall two polymorphic sites are identified
represent transversional substitutions and common in both breeds. With complete concurrence, one
nucleotide locating at the site of 202 bp of the SRY gene coding region was a Cytocine (C), but the
nucleotide at this region was identified a Guanine (G) for swamp buffalo in previous studies (Zhang et
al., 2006), which led to a missense substitution of Glycine instead of Arginine. Other transversional
substitutions (A-T) was also identified in both breeds locating at the site of 470 bp of the SRY gene
coding region and no insertions/deletions were identified. We also further investigated that the two
667
different alleles showed at the site of 202 bp of the SRY gene coding region, seem type specific Y-
liked alleles in domestic buffalo differentiating rivr buffalo and swamp buffalo.
Phylogenetic analysis
Various molecular genetic studies documented on the origin of the domestic water buffalo have
indicated a clear separation by mitochondrial DNA (mtDNA) cytochrome b gene and displacement
loop, autosomal microsatellites and coding genes (Baker et al., 2007; Zhang et al., 2007). Y-
chromosome variation has established to be a significant genetic marker for phylogeography of bovinae
family. The NJ phylogenetic tree (Figure 1) based on Y chromosome SRY gene sequences showed a
clear divergence of river buffalo and swamp buffalo. The phylogenetic tree showed that haplotypes of
both breeds cluster with each other and also with other river buffaloes and swamp buffalo made
separate clade. Less divergence of SRY in Bovidae suggested a much closed genetic relationship
between Bos indicus, Bos taurus, Bos grunniens, Bos frontalis, Bos gaurus, and Bos javanicus.
REFERENCES
Barker J.S., S.S. Moore, D.J. Hetzel, D. Evans, S.G. Tan and K. Byrne. 1997. Genetic diversity of
Asian water buffalo (Bubalus bubalis): microsatellite variation and a comparison with protein-
coding loci. Anim. Genet. 28: 103-115.
Economic Survey. 2010-11. Economic Advisor Wing, Finance Division, Govt. of Pakistan, Islamabad,
Pakistan.
Edwards, C.J., C. Ginja, J. Kantanen, L. Pe´rez-Pardal and A. Tresset. 2011. Dual Origins of Dairy
Cattle Farming – Evidence from a Comprehensive Survey of European Y-Chromosomal
Variation. PLoS ONE 6(1): e15922.
FAO. 2010. FAOSTAT.
Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for
Windows 95/98/NT. Nucleic Acids Symposium Series 41(41): 95-98
Hussain, T., M.E. Babar, A. Nadeem, R. Jabeen and A. Ali. 2009. Phylogenetic Analysis of Kundi
Buffalo Breed of Pakistan through Mitochondrial D-Loop Region. Pak. J. Zool. 9: 341-246.
Peter, J.N. 2004. American Indian mtDNA and Y Chromosome Studies: The Current State of the Field.
Westminster, International Institute for Indigenous Resource Management.
Kumar, S., M. Nagarajan, J.S. Sandhu, N. Kumar, V. Behl and G. Nishanth. 2007. Mitochondrial DNA
analyses of Indian water buffalo support a distinct genetic origin of river and swamp buffalo.
Anim. Genet. 38: 227-232.
Librado, P. and J. Rozas. 2009. DnaSP v5: A software for comprehensive analysis of DNA
polymorphism data. Bioinformatics 25: 1451-1452.
Lei, C.Z., W. Zhang and H. Chen. 2007. Independent maternal origin of Chinese swamp buffalo
(Bubalus bubalis). Anim. Genet. 38: 97-102.
Meadows, J.R.S., R.J. Hawken and J.W. Kijas. 2004. Nucleotide diversity on the ovine Y chromosome.
International Society for Animal Genetics. Anim. Genet. 35: 379-385.
Moioli, B., A. Georgoudis, F. Napolitano, G. Catillo, E. Giubilei, C. Ligda and M. Hassanane. 2001.
Genetic diversity between Italian, Greek and Egyptian buffalo. Livest. Prod. Sci. 70: 203-211.
Nijman, I.J., C.J. Dick, B. Van, M. Lisette, M. Van Cann, M. Yindee, E. Cuppen and J.A. Lenstra.
2008. Phylogeny of Y chromosomes from bovine species. Cladistics 24 (5): 723-726.
Perez-Pardal, L., L.J. Royo, A. Beja-Pereira., I. Curik and A.Traore. 2009. Y specific microsatellites
reveal an African subfamily in taurine (Bos taurus) cattle. Anim. Genet. 41: 232-241.
Prashant, D.S. Gour, P.P. Dubey, A. Jain, D.K. Nanda, B.K. Joshi and D. Kumar. 2009. Complete
nucleotide sequencing, SNP identification and characterization of SRY gene in Indian
Sangamneri goat. Afri. J. Biotech. 8 (13): 2939-2942.
Sambrook J. and D.W. Russell. 2001. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring
Harbour, Cold Spring Laboratory Press, NY.
668
Singh, J., A.S. Nanda and G.P. Adams. 2000. The reproductive pattern and efficiency of female
buffaloes. Anim. Repro. Sci. 60-61:593-604.
Sneath, I.H.A. and R.R. Sokal. 1973. Numerical Taxonomy. W. H. Freeman And Company, San
Francisco.
Verkaar, E.L., C. Zijlstra, E.M. van 't Veld, K. Boutaga, D.C. van Boxtel and J.A. Lenstra. 2004.
Organization and concerted evolution of the ampliconic Y-chromosomal TSPY genes from cattle.
Genomics 84(3): 468-474.
Verkaar, E.L.C., M. Beeke, E. Hanekamp, I.J. Nijman and J.A. Lenstra. 2004. Maternal and paternal
lineages in interbreeding bovine species. Is wisent a hybrid species? Mol. Biol. Evol. 21:1165-
1170.
Yindee, M., B.H. Vlamings, W. Wajjwalku, M. Techakumphu, C. Lohachit, S. Sirivaidyapong, C.
Thitaram, A.A.A.W.K. Amarasinghe, P.A.B.D.A. Alexander, B. Colenbrander and J.A. Lenstra.
2010. Y-chromosomal variation confirms independent domestications of swamp and river
buffalo. Anim. Genet. 41: 433-435.
Zhang, Y., D. Sun, Y. Yu and Y. Zhang. 2006. A Y-linked SNP in SRY Gene Differentiates Chinese
Indigenous Swamp Buffalo and Introduced River Buffalo. Asian-Aust. J. Anim. Sci. 19. (9): 1240-
1244.
Zhang, Y., D. Sun, Y. Yu and Y. Zhang. 2007. Genetic diversity and differentiation of Chinese
domestic buffalo based on 30 microsatellite markers. Anim.Genet. 38: 569-575.
669
Figure1. NJ phylogenetic tree representing phylogenetic relationship between buffalo and other bovids
based on Y-chromosome SRY coding sequence.
670
The Swamp Buffalo: Domestication, Dispersal, and Genetic Differentiation

Yi ZHANGa*, J.S.F. BARKERb, D. VANKANc and Yuan ZHANGa
a
Department of Animal Genetics and Breeding, College of Animal Science and Technology, China
Agricultural University, Beijing 100193, China
b
School of Environmental and Rural Science, University of New England, Armidale, NSW 2351,
Australia
c
The School of Veterinary Science, The University of Queensland, Gatton Campus QLD 4343,
Australia
*Corresponding email: yizhang@cau.edu.cn
ABSTRACT
Water buffalo is an important livestock species in Asia as well as in the world. The swamp type
buffalo, found throughout southeast Asia, from Assam and Nepal in the west to the Yangtse valley
of China, is traditionally reared to produce draft power and meat. In this study, microsatellite
markers were analyzed to determine genetic origin and population relationships of swamp buffalo
in China and south-east Asia. Results showed that populations in south-east Asia and southwest of
China had highest level of genetic variability. Differentiation among the Chinese swamp
populations was much less than among the southeast Asian. Relationships among the swamp
populations (DA genetic distances and STRUCTURE analyses) show the southeast Asian
populations separated into two groups by the Chinese populations. Given these relationships and the
patterns of genetic variability, we postulate that the swamp buffalo was domesticated in the region
of the far south of China, northern Thailand and Indochina. Following domestication, it spread
south through peninsular Malaysia to Sumatra, Java and Sulawesi, and north through China, and
then to Taiwan, the Philippines and Borneo.
Keywords: Bubalus arnee, microsatellite, genetic diversity, genetic relationship
INTRODUCTION
The domestic buffalo, Bubalus bubalis, are reared in tropic and sub-tropic regions as
multi-purpose agricultural animals producing milk, meat and draft power (Cockrill, 1974).
According to FAO (2011), there are 195 million buffalo in over 40 countries on all five continents,
with about 97% found in Asia. Two types of domestic buffalo exist: swamp type and river type.
They have distinct morphology and distributions as well as marked genetic divergence.
Knowledge of population history and genetic variability is of interest in terms of breeding
program design and optimal utilization of genetic resources. Our previous studies of genetic
diversity have been primarily of swamp buffalo populations in south-east Asia (Barker et al., 1997)
and China (Zhang et al., 2007), and Nepalese populations of wild buffalo and domestic river buffalo
(Flamand et al., 2003). By combining these three previously published data sets, our interest is in
further understanding the relationships among the populations and their demographic history, and
possibly the origin and domestication of the swamp type of water buffalo.

Genotypes
We have used data from three of our published studies on microsatellite genetic variation in

671
water buffalo populations – Barker et al. (1997): 21 loci in 11 south-east Asian and Australian
(hereafter SE Asian) populations (eight swamp, three river), Zhang et al. (2007): 30 loci in 20
Chinese populations (18 swamp, two river) and Flamand et al. (2003): 10 loci in three Nepalese
populations (wild, domestic river and hybrid) (Fig.1). For the first two data sets, 20 loci were in
common, and the 10 used for the Nepalese populations were included in these. DNA had been
retained for the Nepal samples, and the other 10 loci were genotyped in the same laboratory as for
the first 10. As the initial genotyping had been done in three different laboratories, we used two
approaches to standardise genotype scoring. We compared allele frequency distributions, separately
for swamp and river buffalo, for each locus. We genotyped a selected set of 30 samples from the
Chinese populations in the laboratory where the Nepal samples were assayed, and using the same
protocols. The final data set includes genotypes of 34 populations at 18 loci.
Statistical methods
The allelic richness for each population was estimated using FSTAT2.9.3 (Goudet, 2001).
Population differentiation measures, F statistics and their significance, were determined using
FSTAT Version 2.9.3 (Goudet, 2001). Genetic relationships among the populations were studied
using two clustering methods: a neighbour-joining tree based on DA Genetic distances using
POPTREE2 program (Takezaki et al., 2010) and a model-based clustering using the Structure
program (Pritchard et al., 2000).
RESULTS
Genetic variability
Expected heterozygosity varied from 0.529 to 0.651 in Chinese buffalo and from 0.353 to 0.589
in SE Asian buffalo. Allelic richness ranged from 3.696 to 5.25 in Chinese buffalo and from 2.373
to 4.682 in SE Asian buffalo. The mean numbers of alleles per locus were significantly different
among all populations (P < 0.0001), among the SE Asian populations (P < 0.0001) and among the
river (including Nepal) populations (P = 0.013), but not among the Chinese swamp populations.
Differences among the SE Asian populations are due primarily to lower values for Australia, Sabah
and Sarawak.
Population differentiation and relationships
Pair-wise estimates of FST emphasise the extent of population differentiation, with only 33
non-significant out of 561 tests. Differentiation among the Chinese swamp populations (mean FST =
0.0240.017) is much less than among the SE Asian (0.1650.100). Within China, differentiation is
primarily due to the DH population.
The dendrogram (Fig. 2a) indicates the clear separation of the swamp and river populations,
and of the Nepal wild among the latter. Relationships among the swamp populations are unexpected,
with the SE Asian populations separated into two groups by the Chinese populations (except DH):
Musuan, Sabah and Sarawak are most closely related to the eastern Chinese populations (FA and
XF), and the remaining SE Asian populations are most closely related to the south-western (XL, DD
and DC). Among the Chinese populations, DH is an outlier, nearest to the river populations.
Structure analysis at K=2 completely separated the river and swamp buffalo populations. The
Chinese DH swamp population had the lowest Q value (0.917) among the swamp, and Nepal wild
(0.988) among the river populations. The average inferred membership coefficients (Q) for each
population are shown for K = 4 and K = 8 (Fig. 2b).
DISCUSSIONS
Archaeological, anatomical and historical evidence supports the contention that both river and
swamp domestic buffalo (B. bubalis) are descended from B. arnee (Cockrill, 1984), and genetic
evidence clearly points to independent domestications of the two types. However, the time and
672
place of domestication of the swamp type is very uncertain, and still subject to debate.
A consistent finding in studies of domestic animals with all molecular markers is that genetic
variability decreases with increasing geographical distance from the centre of domestication
(Groeneveld et al., 2010). Among the swamp populations in current study, genetic variability (both
He and allelic richness) is highest for DH (possibly inflated by some river/wild ancestry) and Surin,
with Trengganu, DC, XL and DD showing next highest variability for one or other criterion. Even
higher variability (He = 0.637 – 0.677 over all populations, 0.607 for the seven loci in common with
ours) has been recorded (Berthouly et al., 2010) for populations in the northern Vietnam province.
As for Chinese swamp populations, genetic variability (both He and allelic richness) significantly
decreases from southwest to northeast along the southern transect: regression coefficients
-0.0061±0.0018, P<0.05, and -0.0530±0.0147, P<0.05 respectively. Genetic variability also
decreases from Surin south through Trengganu to Bogor and Sulawesi. Sabah, Sarawak and
Australia show the lowest levels of genetic variability. Together, these results point to the
domestication centre for swamp buffalo in a region encompassing the far south of China, and
northern Thailand and Indochina.
Given the clear separation of the SE Asian populations into two groups (Fig. 2a), it can be
postulated that following domestication, swamp buffalo spread south through peninsular Malaysia
to the islands of Indonesia (Sumatra, Java and Sulawesi), and north/northeast into central China and
then through an eastern island route via Taiwan to the Philippines and Borneo.
ACKNOWLEDGEMENTS
This manuscript is modified from a paper that we have been published in Animal Genetics.
This work was partially supported by National Natural Scientific Foundation of China (30901015).
REFERENCES
Barker, J.S.F., S.S. Moore, D.J.S. Hetzel, D. Evans, S.G. Tan and K. Byrne. 1997. Genetic diversity
of Asian water buffalo (Bubalus bubalis): microsatellite variation and a comparison with
protein-coding loci. Anim. Genet. 28: 103-115.
Berthouly, C., X. Rognon, T. Nhu Van, A. Berthouly, H. Thanh Hoang, B. Bed’hom, D. Laloë, C.
Vu Chi and E. Verrier, J.-C. Maillard. 2010. Genetic and morphometric characterization of a
local Vietnamese swamp buffalo population. J. Anim Breed. Genet. 127: 74-84.
Cockrill, W.R. 1974. The Husbandry and Health of the Domestic Buffalo, Rome.
Cockrill, W.R. 1984. Water buffalo. In: Evolution of Domesticated Animals (Ed. I.L. Mason).
Longman, London. pp. 52-63.
Flamand, J.R.B., D. Vankan, K.P. Gairhe, H. Duong and J.S.F. Barker. 2003. Genetic identification
of wild Asian water buffalo in Nepal. Anim. Conserv. 6: 265-270.
Groeneveld, L.F., J.A. Lenstra, H. Eding, M.A. Toro, B. Scherf, D. Pilling, R. Negrini, E.K. Finlay,
H. Jianlin, E. Groeneveld and S. Weigend. 2010. Genetic diversity in farm animals – a
review. Anim. Genet. 41(1): 6-31.
Goudet, J. 2001. FSTAT, a program to estimate and test gene diversities and fixation indices
(version 2.9.3).
Pritchard, J.K., M. Stephens, P. Donnelly. 2000. Inference of population structure using multilocus
genotype data. Genetics 155; 945-959.
Takezaki, N., M. Nei and K. Tamura. 2010. POPTREE2: Software for constructing population trees
from allele frequency data and computing other population statistics with Windows interface.
Mol. Biol. Evol. 27: 747-752.
Zhang, Y., D. Sun, Y. Yu and Y. Zhang. 2007. Genetic diversity and differentiation of Chinese
domestic buffalo based on 30 microsatellite markers. Anim. Genet. 38: 569-575.
673
Figure 1. Map showing the locations of all sampled populations (  SE Asian populations, 
Chinese populations, Nepal populations). Underlined locality names indicate river type
populations.
Figure 2. Dendrogram of relationships among all populations using DA genetic distances (a) and
STRUCTURE analysis with inferred membership coefficients for each population (b).
674
Single Nucleotide Variations in the Buffalo Kappa-Casein Gene (CSN3)
Nedenia B STAFUZZAa, Kelli J KOCHANb, Penny K RIGGSb and M Elisabete J

AMARALa*
a
Department of Biology, Sao Paulo State University (UNESP/IBILCE), Sao Jose do Rio Preto-SP,
15054-000, Brazil
b
Department of Animal Science, Texas A&M University, College Station-TX, 77843, USA
*Corresponding email: eamaral@ibilce.unesp.br
ABSTRACT
Caseins are the major milk proteins associated with lactation performance, milk composition
and cheese yield efficiency, representing around 80% of the total amount of proteins found in milk.
Among the caseins, kappa-casein is the protein that stabilizes micelle structure during milk
coagulation process and being used during the cheese production. The kappa-casein gene (CSN3)
has been previously mapped to buffalo chromosome 7 using a radiation hybrid panel and a
comparative map was established using the sequence from bovine chromosome 6. The molecular
structure of this gene has also been established in river buffalo, with a total length of 13,191 bp
(GenBank: AM900443.1) and containing five exons. In this study we searched for single nucleotide
variations in specific regions of the CSN3 gene in three animals representing the Murrah breed.
Sequencing reactions were performed using ABI3730xl sequencer. The “primer walking” method
was used to span the 5’-UTR and intron 2 regions of the gene, for which ten primer pairs were
designed using Oligo 6 software. BLAST tool was used to verify the primers specificity. DNA
sequences assemblies from all three animals were performed with Sequencher® software 4.1, while
multiple alignments were performed using Clustal W software to identify single nucleotide
variations. The sequencing revealed a total of 19 single nucleotide variations with 13 located in the
upstream regulatory region of the gene (5’-UTR) and six on intron 2. These variations can be
validated using commercial populations segregating specific economic traits.
Keywords: Bubalus bubalis, DNA sequencing, Kappa-casein, Murrah
INTRODUCTION
Milk derived from dairying species contains four proteins classified as caseins (alpha S1-, alpha
S2-, beta- and kappa-casein), representing 80% of the total amount of milk proteins. Particularly,
the kappa-casein protein plays an important role in the formation, stabilization and aggregation of
the protein micelles, during the cheese production process (Hurley, 2012). The kappa-casein gene
(CSN3) has been previously mapped to buffalo chromosome 7 and a comparative map was
established with bovine chromosome 6, which is homolog to buffalo chromosome 7 (Goldammer et
al., 2007).
Analysis of the CSN3 gene sequence in different mammalian species revealed a high
conserved exon-intron structure (Rijnkels, 2002). In river buffalo, this gene is organized in five
exons containing 64 bp, 62 bp, 33 bp, 517 bp and 172 bp of size respectively, and four introns with
2,437 bp, 5,906 bp, 2,109 bp and 1,859 bp of size respectively (Figure 1.A). The 5’ untranslated
region (5’-UTR) has a total of 2,506 nucleotides of length, while the 3’-UTR has 2,065 nucleotides
(GenBank: AM900443.1).
Genetic polymorphisms of CSN3 gene have been previously described in cow (Szymanowska
et al., 2004; Robitaille et al., 2005; Ju et al., 2011; Huang et al., 2012 ), sheep (Feligini et al., 2005),
and goat (Kiplagat et al., 2010; Dagnachew et al., 2011), showing differences in nucleotides content
in coding and non-coding regions. In buffalo, there are few studies describing genetic variations on
CSN3 gene, which have been found mainly in coding regions (Mitra et al., 1998; Otaviano et al.,
2005; Abassi et al., 2009). In this study, we searched for single nucleotide variations in two non-

675
coding regions of the CSN3 gene (5’-UTR and intron 2) using three animals representing the
Murrah breed.

Blood samples were collected from three females Murrah buffaloes using vacuum tubes
treated with 15% EDTA as anticoagulant. Genomic DNA was extracted from leukocytes using
standard phenol/chloroform and ethanol precipitation procedures as described by Sambrook et al.
(1989).
PCR reactions were performed in 50-µl total volume containing 50 ng DNA, 1.5 mM MgCl2,
10mM Tris-HCl, 50 mM KCl, 0.2 mM dNTP, 10 pmol of forward and reverse primers and 0.5 U of
AmpliTaq Gold polymerase (Life TechnologiesTM). The amplifications were carried out under the
following conditions: initial denaturation at 95 °C for 10 min; 35 cycles of 95 °C for 30 s, annealing
temperature (Table 1) for 30 s and 72 °C for 30 s; and a final extension at 72 °C for 7 min. The PCR
products were visualized on 2% agarose gels in 1% TBE buffer and stained with ethidium bromide.
PCR products were purified with PSIΨClone Kit (Princeton SeparationsTM) and used as
templates in cycle sequencing with the Bid Dye Terminator v.3.1 kit (Life TechnologiesTM).
Sequencing reactions were performed using an ABI3730xl capillary sequencer (Life
TechnologiesTM). The primer walking method was used to span the 5’UTR to intron 2 region of the
gene, for which ten primer pairs were designed using Oligo Primer Analysis software version 6
(www.oligo.net/). All designed primers were analyzed by basic local alignment search tool
(BLAST) analysis (www.ncbi.nlm.nih.gov/BLAST) to ensure their respective homology over
buffalo genome.
The quality of the DNA sequences and the assemblies were performed with Sequencher®
software 4.1 (www.genecodes.com), while multiple alignments were performed using the Clustal W
software (www.clustal.org) to identify single nucleotide variations when compared with the buffalo
CSN3 reference sequence (GenBank: AM900443.1).

A total of 4,366 nucleotides of the CSN3 gene were sequenced from three Murrah females,
including 2,506 bp from 5’-UTR, 57 bp from exon 2 and 1,803 bp from intron 2 (Figure 1.B).
Multiple sequence alignment analysis from the gene sequence from the three females revealed
a total of 19 single nucleotide variations when compared with the buffalo CSN3 gene reference
sequence (GenBank: AM900443.1). Thirteen variations were located in the 5’-UTR region and six
in the intron 2 (Table 2).
BLAST analyses were also performed using both bovine CSN3 gene sequences
corresponding to allele A (GenBank: AY380228.1) and allele B (GenBank: AY380229.1), which
reveals 168 variations between buffalo and bovine including 102 variations in the 5’-UTR and 66
on intron 2.
Single nucleotide variations in non-coding regions on the CSN3 gene were also observed in
cattle by Huang et al. (2012). In their study, they described 13 single nucleotide variations
associated with kappa-casein concentration in milk produced by Holstein breed. The variations
found in cattle were different from those observed in our study with buffaloes.
Nucleotide variations located within potential regulatory sequences, like 5'-UTRs and introns
suggests possible influence on the binding recognition sites of transcription factors related with the
buffalo CSN3 gene expression. The 19 single nucleotide variations found in this study can be used
as potential candidate markers for additional association studies using commercial population of
different buffalo breeds.
REFERENCES
Abassi, B., J. Fayazi, M.T. Beigi Nasiri, H.A. Roshanfekr, Kh. Mirzadeh and A.S. Sadr. 2009.
Analysis of kappa casein gene polymorphism by PCR-RFLP in buffalo population in
Khouzestan province. Res. J. Biol. Sci. 4:1073-1075.
676
Dagnachew, B.S., G. Thaller, S. Lien and T. Ådnøy. 2011. Casein SNP in Norwegian goats:
additive and dominance effects on milk composition and quality. Genet. Sel. Evol. 43:31.
Feligini, M., S. Vlaco, V.C. Curik, P. Parma, G. Greppi and G. Enne. 2005. A single nucleotide
polymorphism in the sheep kappa-casein coding region. J. Dairy Res. 72:317-321.
Goldammer, T., R. Weikard, M.N. Miziara, R.M. Brunner, R. Agarwala, A.A. Schäffer, J.E.
Womack and M.E.J. Amaral. 2007. A radiation hybrid map of river buffalo (Bubalus
bubalis) chromosome 7 and comparative mapping to the cattle and human genomes.
Cytogenet. Genome Res. 119:235-241.
Huang, W., F. Peñagaricano, K.R. Ahmad, J.A. Lucey, K.A. Weigel and H. Khatib. 2012.
Association between milk protein gene variants and protein composition traits in dairy
cattle. J. Dairy Sci. 95:440-449.
Hurley, W. L. 2012. Milk Protein. 1st Ed. InTech, Rijeka, Croatia.
Ju, Z., J. Huang, Q. Li, H. Wang, J. Zhong and C. Wang. 2011. The polymorphisms of k-casein
gene and their associations with milk production traits and expression analysis in Chinese
Holstein cattle. Afr. J. Biotechnol. 10:13368-13375.
Kiplagat, S.K., M. Agaba, I.S. Kosgey, M. Okeyo, D. Indetie, O. Hanotte and M.K. Limo. 2010.
Genetic polymorphism of kappa-casein gene in indigenous Eastern Africa goat
populations. Int. J. Genet. Mol. Biol. 2:001-005.
Mitra, A., P. Schlee, I. Krause, J. Blusch, T. Werner, C.R. Balakrishnan and F. Pirchner. 1998.
Kappa-casein polymorphisms in Indian dairy cattle and buffalo: a new genetic variant in
buffalo. Anim. Biotechnol. 9:81-87.
Otaviano, A.R., H. Tonhati, J.A.D. Sena and M.F.C. Muñoz. 2005. Kappa-casein gene study with
molecular markers in female buffaloes (Bubalus bubalis). Genet. Mol. Biol. 28:237-241.
Rijnkels, M. 2002. Multispecies comparison of the casein gene loci and evolution of casein gene
family. J. Mammary Gland Biol Neoplasia 7:327-345.
Robitaille, G., M. Britten, J. Morisset and D. Petitclerc. 2005. Polymorphism in the bovine κ-casein
(CSN3) gene and the 5’-flanking region: sequence analysis of CSN3 A and B alleles.
Anim. Genet. 36:184-185.
Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd Ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Szymanowska, M., E. Siadkowska, M. Lukaszewicz and L. Zwierzchowski. 2004. Association of
nucleotide-sequence polymorphism in the 5’-flanking regions of bovine casein genes with
casein content in cow’s milk. Lait 84:579-590.
677
Table 1. Primers pairs designed using Oligo Primer Analysis software and used for the sequencing
of 4,366 bp region of the buffalo CSN3 gene by primer walking method.
Primer name Primers sequences (5'-3') Length Annealing temperature

CSN3_1 F: GCATTCCATTAACCGAGACTGA 598 bp 66ºC
R: ATTAAGGGATCAATCTGGATTATGCT
CSN3_2 F: CAAGACAGTGAATCTATTCCTACAGAC 498 bp 64ºC
R: CTTGATGATTACAATACAGTACAGTGA
CSN3_3 F: CTACAATCTTAAAAGGTAGCCAGT 839 bp 64ºC
R: ATCTGCTTCTCCAGTAATCATAA
CSN3_4 F: CTACCCTATACTTGAGTCTTTAGAA 733 bp 58ºC
R: GCTTATTAAGGTGAATTGGTC
CSN3_5 F: GCAAGTCCCACCTATATGCTATGTC 519 bp 64ºC
R: GGTTAATGCCAGGATAGTCACAACT
CSN3_6 F: CATTGCTCCAAGTCACTCCTA 755 bp 64ºC
R: GCACTTATTTGCCAACAGTAAGA
CSN3_7 F: CCATAGAGGAAGATCAGAA 817 bp 58ºC
R: TCAGCCATTGGTATGTTAT
CSN3_8 F: CCATGTGTAAAATTAGATAGCCAGT 726 bp 64ºC
R: GAGGTACAGGTCAGCAGTTGATT
CSN3_9 F: CAAGTCTGGTTCAGTCTCTT 568 bp 60ºC
R: GGTATTCCCATCTCTTTATGA
CSN3_10 F: CACCCTTCTACCATCTTGCT 569 bp 60ºC
R: GGATGCCATGATCTTCGT
Table 2. Single nucleotide variations in 5’-UTR and intron 2 of the buffalo CSN3 gene sequenced
in this study.
5’ Untranslated Region Intron 2

CSN3 gene CSN3 gene CSN3 gene
position Single Position Single Position Single
nucleotide nucleotide nucleotide
(GenBank: (GenBank: (GenBank:
variation variation variation
AM900443.1) AM900443.1) AM900443.1)
324 T/G 929 G/A 2,788 G/A
350 -/G 930 A/G 2,801 G/A
583 G/A 1,083 G/A 3,133 G/C
918 A/C 1,207 A/G 3,959 A/G
920 C/A 1,276 C/T 4,020 T/C
921 A/C 1,278 C/- 4,264 T/-
922 C/A
678
Figure 1. A. Schematic representation of buffalo CSN3 gene based on the sequence information
available in GenBank (AM900443.1). Diagonally hatched blocks indicate 5’- and 3’-UTR, white
block indicate signal peptide encoding sequences and black blocks represent mature peptide
encoding exons. Numbers above blocks and lines indicate exon and intron sizes respectively. B.
Schematic representation of 4,366 nucleotides from buffalo CSN3 gene sequenced in this study,
which spans the 5’-UTR and intron 2 regions of the gene.
679
Allelic Variant Analysis of the DRB3 Gene by PCR-RFLP in Mediterranean

Buffaloes Breed
Lucas Matheus OLIVATTOa, Nedenia Bonvino STAFUZZAa, Bruna Cristina Machado
NARESSIa, Humberto TONHATIb and Maria Elisabete Jorge AMARALa*
a
Department of Biology, UNESP-São Paulo State University, IBILCE, São José do Rio Preto, SP
15054-000, Brazil.
b
Department of Zootecnia, UNESP-São Paulo State Univesity, FCAV, Jaboticabal, SP 14884-900,
Brazil.
*Corresponding e-mail: eamaral@ibilce.unesp.br
ABSTRACT
The DRB3 gene is a member of the mammalian major histocompatibility complex (MHC)
class II cluster. Allelic variants of this gene have been associated with bovine leukemia virus
infection and mastitis in cattle. In this study we searched for allelic variants in the exon 2 of the
DRB3 gene using the method PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment
Length Polymorphism). DNA samples from 24 Mediterranean buffaloes were extracted from
follicle bulb. For PCR amplification of the exon 2 we used one cattle derived primer pair previously
described in the literature and successfully used to generate PCR products with buffalo DNA,
including the breeds Murrah, Nili-Ravi, Surti, Jafarabadi and Carabao. The expected PCR product
size with buffalo DNA was 304 bp, which was digested with three restriction enzymes, RsaI
(restriction site: GT↓AC), PstI (restriction site: CTGCA↓G) and HaeIII (restriction site: GG↓CC).
The results showed six polymorphic patterns with the enzyme RsaI: RsaI-A (237+67 bp), RsaI-B
(183+121 bp), RsaI-C (114+93+67+30 bp), RsaI-D (144+93+67 bp), RsaI-E (144+67+54+39 bp)
and RsaI-F (114+67+54+39+30 bp); four polymorphic patterns with the enzyme HaeIII: HaeIII-A
(222+82 bp), HaeIII-B (170+82+52 bp), HaeIII-C (193+82+29 bp) and HaeIII-D (170+134 bp) and
only one polymorphic pattern with the enzyme PstI: PstI-A (230+68+6 bp). The digestion with the
enzyme HaeIII generated eight genotypes within the population, where 17 animals were classified
as heterozygotes, with the following genotypes: HaeIII-A/HaeIII-D in five animals, HaeIII-
B/HaeIII-D in five animals, HaeIII-A/HaeIII-B in three animals, HaeIII-A/HaeIII-C in two animals
and HaeIII-C/HaeIII-D observed in two animals. The RsaI restriction enzyme also generated eight
genotypes, where 15 animals were heterozygotes. The most frequent genotype observed was RsaI-A
/RsaI-B found in six animals. The PstI restriction enzyme generated one heterozygote genotype –
PstI-A/304 bp – observed in three animals, and one homozygote genotype (304/304 bp) observed in
21 animals. This study provides some insight about the allelic variants observed in one population
of Mediterranean buffalo breed in Brazil. The variant analysis showed low genetic variability
among the individuals, possibly due to a high degree of inbreeding in the population. These facts
are concerning, since the MHC cluster genes are the most polymorphic genes of the mammalian
genome.
Keywords: Bubalus bubalis, MHC, PCR-RFLP, polymorphism, restriction enzyme


680
Cattle-Derived Microsatellite Markers to Study the Water Buffalo Genome
Nedenia B STAFUZZA,a and M Elisabete J AMARALa*

a
Department of Biology, Sao Paulo State University (UNESP/IBILCE), Sao Jose do Rio Preto-SP
15054-000, Brazil
*Corresponding email: eamaral@ibilce.unesp.br
ABSTRACT
Microsatellites are well-known DNA markers used in a variety of studies such as genome
mapping, genetic diversity analysis, genetic conservation and phylogenetic studies. Although
microsatellites are important markers, their development and characterization demands extensive
time and high cost. Thus, before new markers are developed for a particular species, it is
worthwhile to test the available markers from related species. In the present study, we evaluate
cattle-derived microsatellite markers for genetic studies of water buffalo. Eighty-five percents of a
total of 120 microsatellite markers were optimized using buffalo DNA (Bubalus bubalis). The
results showed in this paper were also deposited in the National Center for Biological Information
database (NCBI) (ProbeDB and UniSTS) for use in population genetic studies of buffalo by the
scientific community. The use of heterologous primers significantly reduces the cost of developing
specific markers for buffalo, providing a useful short cut for the genetic population analysis and
gene mapping studies.
Keywords: Bubalus bubalis, Microsatellites, PCR primers, Water buffalo
INTRODUCTION
Microsatellite markers are short DNA sequences found in all prokaryotic and eukaryotic
genomes considered as tandem repeats of 1-6 nucleotides. The high degree of polymorphism, the
frequency of occurrence in the population, and the random distribution across the genome makes
these markers suitable for various analyses, e.g. genome mapping, population genetics, conservation
and phylogenetic studies (Baratii et al., 2001). Although microsatellites are powerful DNA markers,
their identification as novel markers requires technical expertise and their molecular
characterization is quite labor intensive, costly and traditionally requires screening of genomic
libraries with appropriate probes (Zane et al., 2002). The repeat-flanking sequences of a
microsatellite locus are often conserved between related species making possible their use in cross-
species by PCR amplification.
The genome of the bovine, the main livestock species in the world has several genomic tools
and informatics resources available, including whole genome maps (International Bovine BAC
Mapping Consortium, 2007; Arias et al., 2009) and sequencing (Bovine Genome Sequencing and
Analysis Consortium, 2009). More than four thousand microsatellite markers have been developed
for cattle (Barendse et al., 1994; Bishop et al., 1994; Ihara et al., 2004), which have been used in
studies involving related species, such as sheep (Ellegren et al., 1997; de Gortari et al., 1997),
african buffalo (van Hooft et al., 1999), goat (Vaiman et al., 1996; Kim et al., 2004), and water
buffalo (Arora et al., 2004; Kumar et al., 2006).
Taking advantage of the buffalo-bovine chromosome homologies and the extensive marker
resources available for cattle, the goal of this study was to evaluate a set of cattle-derived
microsatellite markers for the amplification of orthologous loci in the water buffalo genome.

A total of 120 microsatellite markers were selected from available literature. The PCR
reactions were performed in a MJ Research PTC-200 thermocycler with thermal gradient software.
Bovine DNA samples were included as controls in each amplification experiment since the PCR
primers were cattle-derived. PCR mixtures included: 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM
681
KCl, 10 mM dNTPs, 0.2 mM each primer, 0.5 unit of AmpliTaq Gold polymerase (Life
TechnologiesTM) and 50 ng DNA in a 10 µl reactions volume. The PCR conditions were as follows:
initial denaturation at 94°C for 10 min, followed by 35 cycles at 94°C for 30 sec (denaturation), 50
to 65°C for 30 sec (annealing depending on the individual microsatellite marker), extension at 72°C
for 30 sec and a final extension at 72°C for 7 min. The PCR products were electrophoresed through
2% agarose gels in 1X TBE buffer containing ethidium bromide and photographed under UV light.

In this study, 85% of cattle-derived microsatellite markers produced robust PCR products
with buffalo DNA. Although these types of markers have been used in other species of Bovidae
family, the percentage of cattle-derived microsatellites that could be used in buffalo genome was
higher than that reported for sheep (40%) and goat (34%) (Vaiman et al., 1996; de Gortari et al.,
1997). This observed high percentage could be explained by the closer genetic relation between
cattle and buffalo since both belong to the subfamily Bovinae.
The references for each microsatellite marker and the PCR annealing temperatures used in
this study are listed in Table 1. Additionally, this information is publicly available at the National
Center for Biological Information (NCBI) databases ProbeDB (http://www.ncbi.nlm.nih.gov/
genome/probe/) and UniSTS (http://www.ncbi.nlm.nih.gov/sites/entrez?db=unists).
The water buffalo is an important livestock species for countries outside Europe and United
States, including most countries with less financial resources for livestock research. Brazil is the
pioneer leader in buffalo genome research with the development of genomic tools for whole
genome mapping of water buffalo (Amaral et al., 2007; Stafuzza et al., 2012). The use of
heterologous primers significantly reduces the cost of developing specific markers for buffalo,
providing a useful short cut for the genetic population analysis and gene mapping studies.
REFERENCES
Amaral, M.E.J., K.E. Owens, J.S. Elliott, C. Fickey, A.A. Schäffer, R. Agarwala and J.E. Womack.
2007. Construction of a river buffalo (Bubalus bubalis) whole-genome radiation hybrid
panel and preliminary RH mapping of chromosomes 3 and 10. Anim. Genet. 38: 311-314.
Arias, J.A., M. Keehan, P. Fisher, W. Coppieters and R. Spelman. 2009. A high density linkage
map of the bovine genome. BMC Genet. 10:18.
Arora, B.R., B.D. Lakhchaura, R.B. Prasad, M.S. Tantia and R.K. Vijh. 2004. Genetic diversity
analysis of two buffalo populations of northern India using microsatellite markers. J. Anim.
Breed. Genet. 121:111-118.
Baratii, M., A. Alberti, M. Groenen, T. Veenendaal and F.D. Fulgheri. 2001. Polymorphic
microsatellites developed by cross-species amplifications in common pheasant breeds. Anim.
Genet. 32:222-225.
Barendse, W., S.M. Armitage, L.M. Kossarek, A. Shalom, B.W. Kirkpatrick, A.M. Ryan, D.
Clayton, L. Li, H.L. Neibergs, N. Zhang, W.M. Grosse, J. Weiss, P. Creighton, F.
McCarthy, M. Ron, A.J. Teale, R. Fries, R.A. McGraw, S.S. Moore, M. Georges, M. Soller,
J.E. Womack and D.J.S. Hetzel. 1994. A genetic linkage map of the bovine genome. Nat.
Genet. 6:227-235.
Bishop, M.D., S.M. Kappes, J.W. Keele, R.T. Stone, S.L.F. Sunden, G.A. Hawkins, S.S. Toldo, R.
Fries, M.D. Grosz, J. Yoo and C.W. Beattie. 1994. A genetic linkage map for cattle.
Genetics 136:619-639.
Bovine Genome Sequencing and Analysis Consortium. 2009. The genome sequence of taurine
cattle: a window to ruminant biology and evolution. Science 324:522-528.
De Gortari, M.J., B.A. Freking, S.M. Kappes, K.A. Leymaster, A.M. Crawford, R.T. Stone and
C.W. Beattie. 1997. Extensive genomic conservation of cattle microsatellite heterozygosity
in sheep. Anim. Genet. 28:274-290.
682
Ellegren, H., S. Moore, N. Robinson, K. Byrne, W. Ward and B.C. Sheldon. 1997. Microsatellite
evolution - A reciprocal study of repeat lengths at homologous loci in cattle and sheep. Mol.
Biol. Evol. 14:854-860.
Ferretti, L., B.G.D. Urquhart, A. Eggen, I. OIsaker, B. Harlizius, B. Castiglioni, A. Mezzelani, S.S.
Toldo, U. Thieven, Y. Zhang, A.L G. Morgan, V.M. Teres, M. Schwerin, I. Martin-Burriel,
B.P. Chowdhary, G. Erhardt, I.J. Nijman, E.P. Cribiu, W. Barendse, H. Leveziei, R. Fries
and J.L. Williams. 1997. Cosmid-derived markers anchoring the bovine genetic map to the
physical map. Mamm.Genome. 8:29-36.
Ihara, N., A. Takasuga, K. Mizoshita, H. Takeda, M. Sugimoto, Y. Mizoguchi, T. Hirano, T. Itoh,
T. Watanable, K.M. Reed, W.M. Snelling, S.M. Kappes, C.W. Beattie, G.L. Bennett and Y.
Sugimoto. 2004. A comprehensive genetic map of the cattle genome based on 3802
microsatellites. Genome Res. 14:1987-1998.
International Bovine BAC Mapping Consortium. 2007. A physical map of the bovine genome.
Genome Biol. 8:1-17.
Itoh, T., T. Watanabe, N. Ihara, P. Mariani, C.W. Beattie, Y. Sugimoto and A. Takasuga. 2005. A
comprehensive radiation hybrid map of the bovine genome comprising 5593 loci. Genomics
85:413-424.
Kappes, S.M., J.W. Keele, R.T. Stone, R.A. McGraw, T.S. Sonstegard, T.P.L. Smith, N.L. Lopez-
Corrales and C.W. Beattie. 1997. A second-generation linkage map of the bovine genome.
Genome Res. 7:235-249.
Kim, K.S., M.S. Min, J.H. An and H. Lee. 2004. Cross-species amplification of Bovidae
microsatellites and low diversity of the endangered Korean goral. J. Hered. 95:521-525.
Kossarek, L.M., W.M. Grosse, O. Finlay, W. Barendse, D.J.S. Hetzel and R.A. McGraw. 1994a.
Rapid communication: bovine dinucleotide repeat polymorphism RM209. J. Anim. Sci.
72:528-528.
Kossarek, L.M., W.M. Grosse, O. Finlay, A.B. Dietz, J.E. Womack and R.A. McGraw. 1994b.
Rapid communication: bovine dinucleotide repeat polymorphism RM095. J. Anim. Sci.
72:254-254.
Kumar, S., J. Gupta, N. Kumar, K. Dikshit, N. Navani, P. Jain and M. Nagarajan. 2006. Genetic
variation and relationships among eight Indian riverine buffalo breeds. Mol. Ecol. 15:593-
600.
McGraw, R.A., W.M. Grosse, S.M. Kappes, C.W. Beattie and R.T. Stone. 1997. Thirty-four bovine
microsatellite markers. Anim. Genet. 28:66-68.
Mezzelani, A., Y. Zhang, L. Redaelli, B. Castiglioni, P. Leone, J.L. Williams, S.S. Toldo, G.
Wigger, R. Friesand L. Ferretti. 1995. Chromosomal localization and molecular
characterization of 53 cosmid-derived bovine microsatellites. Mamm.Genome. 6:629-635.
Oraby, H.A., S.M. El Nahas, H.A. De Hondt, A. El Ghort and M.F.A. Samad. 1998. Assignment of
PCR markers to river buffalo chromosomes. Genet. Sel. Evol. 30:71-78.
Ponce De Leon, F.A., S. Ambady, G.A. Hawkins, S.M. Kappes, M.D. Bishop, J.M. Robl and C.W.
Beattie. 1996. Development of a bovine X chromosome linkage group and painting probes
to assess cattle, sheep, and goat X chromosome segment homologies. Proc. Natl. Acad. Sci.
USA 93:3450-3454.
Smith, T.P.L., L.J. Alexander, T.S. Sonstegard, J. Yoo, C.W. Beattie and M.F. Broom. 1996.
Construction and characterization of a large insert bovine YAC library with five-fold
genomic coverage. Mamm. Genome. 7:155-156.
Stafuzza, N.B., C.A. Abbey, C.A. Gill, J.E. Womack and M.E.J. Amaral. 2012. Construction and
preliminary characterization of a river buffalo bacterial artificial chromosome library. Genet.
Mol. Res. 11:3013-3019.
Stone, R.T., J.C. Pulido, G.M. Duyk, S.M. Kappes, J.W. Keele and C.W. Beattie. 1995. A small-
insert bovine genomic library highly enriched for microsatellite repeat sequences. Mamm.
Genome. 6:714-724.
683
Vaiman, D., D. Mercier, K. Moazami-Goudarzi, A. Eggen, R. Ciampolini, A. Lépingle, R. Velmala,

J. Kaukinen, S.L. Varvio, P. Martin, H. Levéziel and G. Guérin. 1994. A set of 99 cattle
microsatellites: characterization, synteny mapping, and polymorphism. Mamm.Genome.
5:288-297.
Vaiman, D., L. Schibler, F. Bourgeois, A. Oustry, Y. Amigues and E.P. Cribiu. 1996. A genetic
linkage map of the male goat genome. Genetics 144:279-305.
Van Hooft, W.F., O. Hanotte, P.W. Wenink, A.F. Groen, Y. Sugimoto, H.H.T. Prins and A. Teale.
1999. Applicability of bovine microsatellite markers for population genetic studies on
African buffalo (Syncerus caffer). Anim. Genet. 30:214-220.
Zane, L., L. Bargelloni and T. Patarnello. 2002. Strategies for microsatellite isolation: a review.
Mol. Ecol. 11:1-16.
684
Table 1. Summarized information about the cattle-derived microsatellite markers used for the
amplification of orthologous loci in the water buffalo genome.
Marker T* UniSTS Marker T* UniSTS Marker T* UniST Marker T* UniSTS

BL10981 65°C 251324 BMS20752 57°C 66478 CSSM0362 65°C S
250971 MB1012 65°C 250773
BL222 56°C 251054 BMS21522 61°C 16531 CSSM0372 58°C 250972 RM0192 59°C 251183
BL412 65°C 250736 BMS21962 56°C 34202 CSSM0476 56°C 519102 RM09510 56°C 250928
2
BM121 57°C 73368 BMS22272 60°C 2305 CSSM0542 63°C 250977 RM20911 65°C 250931
BM13122 54°C 30885 BMS22632 65°C 2985 ETH122 56°C 250760 RM3212 57°C 519109
BM18562 65°C 53060 2
BMS2377 56°C 2641 FCB1932 56°C 250722 RM35012 65°C 75313
BM18642 58°C 519391 BMS25032 60ºC 79334 HUJ6142 65°C 250838 RM3722 57°C 77624
BM25042 57°C 26344 BMS25922 59°C 44531 IDVGA-297 59°C 251444 SRC2592 54ºC 251196
BM3102 58ºC 1373 BMS26292 58ºC 66397 IDVGA-477 56°C 251745 SRC2762 57°C 251197
2
BM4208 60°C 65174 BMS26584 65°C 66668 IDVGA-527 60°C 251460 TGLA102 58ºC 250985
BM60002 56°C 7843 BMS28472 60ºC 2618 IDVGA-532 65°C 251461 TGLA1792 65°C 251232
2 2
BM6438 63°C 79562 BMS4030 65°C 74568 ILSTS0142 58ºC 250882 TGLA3254 59°C 250950
BM65062 63°C 47201 BMS4173 64°C 75699 ILSTS0174 54°C 250806 TGLA492 56°C 239049
BM65262 65°C 28213 BMS5002 63°C 519394 ILSTS0762 58°C 251349 TGLA574 56°C 250987
BM81292 56°C 30644 BMS5013 56°C 14607 INRA0412 50°C 251127 TGLA732 54°C 250988
BMC50122 56°C 69365 BMS5272 60°C 65219 INRA1208 64°C 251161 TGLA95 55°C 251314
BMS10012 65°C 44386 BMS5552 55°C 51137 INRA1342 59°C 250829 URB0372 57°C 250818
BMS11482 54°C 76263 BMS6782 56°C 8923 INRA2019 63°C 251682 UWCA72 61°C 250762
BMS12342 54°C 24548 2
BMS817 57°C 73593 INRA308 59°C 250757 XBM1113 60°C 251008
BMS12902 56°C 80216 BMS8352 60°C 65587 MAF452 58°C 250702 XBM242 59°C 251009
BMS15912 58°C 42922 BMS8362 56°C 41471 MB0052 58°C 250849 XBM3612 56°C 251341
BMS18203 56°C 23799 BP22 56°C 250710 MB0662 60ºC 250888 XBM713 65°C 251010
BMS19092 54°C 10927 CSSM0065 58°C 250965 MB0992 65°C 250771 XBM732 61°C 251004
BMS19202 56°C 66399 CSSM0194 59°C 251074
*T= annealing temperature in water buffalo; 1Smith et al., 1996; 2Ihara et al., 2004; 3Stone et al.,
1995; 4Kappes et al., 1997; 5Itoh et al., 2005; 6Oraby et al., 1998; 7Mezzelani et al., 1995; 8Vaiman
et al., 1994; 9Ferretti et al., 1997; 10Kossarek et al.,1994b; 11Kossarek et al.,1994a; 12McGraw et al.,
1997; 13Ponce de Leon et al., 1996.
685
Genetic Estimates for Productive Parameters in Buffaloes in Different Ecological

Areas in Venezuela
Nestor Simon Montiel URDANETA *, C. C.Ch. MONTIEL M., N. BERRIOS, N. S. MORILLO,

J. BELANDRIA, M. ANDARÁ and J. ARIAS
* Department of Production and Industry Animal. School of Veterinary Sciences. The University of the
* Corresponding e-mail: nsmontiel@gmail.com; nmontiel@cantv.net
ABSTRACT
Used the production records of 12 farms located in different ecological zones of Venezuela
from 1992 to 2012 (32552 lactations). Repeatability, heritability and variance components were
estimated for the milk yield, fat and protein percentages. These estimates were carried out through
animal model of unique feature using the DFREML software. The farm was regarded as fixed in the
model effect. The characteristics of milk yield, age at first calving were used as a covariate. The
additive genetic and environmental effects were also included in the model. The means (± SD) values
for the milk yield, fat and protein percentages were: 1545.23 ± 665.57 kg; 7.15 ± 0.85%; and 4.69 ±
0.18%, respectively. Heritabilities (± SE) for the milk yield, fat and protein percentages were: 0.089 ±
0.09; 0.049 ± 0.06; and 0.0002 respectively. For repeatability estimates were: 0.298; 0.052 and 0.0003
respectively. These values should be taken as the reference for future genetic evaluations of other
farms; also you should continue emphasis on the quality of the milk production records at the level of
farms.
Keywords: buffaloes, animal model, genetic estimates


686
Index of Heritage's Weight at Birth and Effects of Some Environmental Factors on

Buffalo Calf Using the Animal Model
Nestor Simon Montiel URDANETA *; C. C.Ch. MONTIEL M.; N. BERRIOS; N. S. MORILLO;

J. BELANDRIA; M. ANDARÁ, and J. ARIAS.
*Department of Production and Industry Animal. School of Veterinary Sciences. The University of the
*Corresponding e-mail: nsmontiel@gmail.com; nmontiel@cantv.net
ABSTRACT
With the finaliadad to estimate the heritability (h²) and the effect of some environmental factors
affecting weight at birth of buffalo calf, 13212 records of calving of 12 farms between the years 1993-
2012 were used. Use a mixed model that included the fixed effects of year of birth (A), month of birth
(M), sex (S) and number of calving (N) and interactions N*A; N*M and N*S; as well as the random
effect of the father (P). The models used were: maximum likelihood Residual free of derivatives
(DFREML); Maximum Residual likelihood (REML) with the procedure PROC MIXED and method III
of Henderson with the program of minimum squares ordinary of mixed models and maximum
likelihood (LSMLMW). The average weight was 38.95 kg ± 0.52; with a range of 17 to 55 kg; the
weight of the males was 38.44 kg ± 6.37; and females 37.25 kg ± 4.52; the percentage of males born in
the period 1993-2012 was: 54.39% (7187) and 45,60% (6025) for females. The estimate of h² was
DFREML 0.35 ± 0.16; REML 0.34 and M.III 0.33 ± 0.11; N*A; N*M and N*S affected the weight at
birth (P <. 05). The value of the estimated heritability can be considered intermediate and it should be
taken into account in future programmes of selection of the studied herds.
Keywords: weight at birth, heritability, buffalo calf, animal model.


687
Genetic Parameters for Productive and Reproductive Traits for Milk Buffalo
in Brazil
Camila da Costa BARROSa*, Rusbel Raul ASPILCUETA-BORQUISb, Humberto

TONHATIb and Angelina Bossi FRAGAa
a
Federal University of Alagoas, Brazil
b
Department of Animal Science, São Paulo State University, Jaboticabal, SP, Brazil
Corresponding email: camila.barros@live.com
ABSTRACT
Knowing the genetic parameters of productive and reproductive traits in milking buffaloes is
essential for planning and implementing of a program genetic selection. In Brazil, this information
is still scarce. The objective of this study was to verify the existence of genetic variability in milk
yield of buffaloes and their constituents, and reproductive traits for the possibility of application of
the selection. A total of 9,318 lactations records from 3,061 cows were used to estimate
heritabilities for milk yield (MY), fat percentage (%F), protein percentage (%P), length of lactation
(LL), age of first calving (AFC) and calving interval (CI) and the genetic correlations among traits
MY, %F and %P. The (co)variance components were estimated using multiple-trait analysis by
Bayesian inference method, applying an animal model, through Gibbs sampling. The model
included the fixed effects of contemporary groups (herd-year and calving season), number of
milking (2 levels), and age of cow at calving as (co)variable (quadratic and linear effect). The
additive genetic, permanent environmental, and residual effects were included as random effects in
the model. Estimated heritability values for MY, %F, %P, LL, AFC and CI were 0.24, 0.34, 0.40,
0.09, 0.16 and 0.05, respectively. The genetic correlation estimates among MY and %F, MY and
%P and %F and %P were -0.29, -0.18 and 0.25, respectively. The production of milk and its
constituents showed enough genetic variation to respond to a selection program. Negative estimates
of genetic correlations between milk production and its components suggest that selection entails a
reduction in the other.
Keywords: genetic correlation, heritability, milk yield, milk buffalo
INTRODUCTION
There are approximately 1.277.199 buffaloes in Brazil (IBGE, 2011). This number
continuous to increase, for instance, between 2010 and 2011 years, the buffalo herd expanded 7.8%
per year, exceeding the bovine herd, which reached 1.6% per year. This fact may be associated with
increased consumer demand for products of buffalo. Among them stands out the mozzarella cheese,
which has been used in the culinary because of its flavor and its organoleptic qualities. However,
although with substantial growth, there is a need to increase production rates of Brazilian buffalo
herd. Moreover, for the improvement of production levels is essential knowing the genetic
parameters of productive and reproductive traits in milk buffaloes for planning and implementing of
a program genetic selection. In Brazil, this information is still scarce. The objective of this study
was assessed the genetic variability for milk yield and their constituents, and reproductive traits in a
Buffalo population in Brazil for the possibility of application of the selection.

A total of 9,318 lactations records from 3,061 cows were used. The animals were born
between the years 1986 and 2009 at farms of São Paulo State, and Rio Grande do Norte State. The
studied traits were milk yield (MY), fat percentage (%F), protein percentage (%P), lactation length
(LL), calving interval (CI) and age at first calving (AFC). The data structure is presented in Table 1.
Lactation records less than 90 days, age at first calving over 60 months and calving interval greater
than 650 days were deleted. There were defined two seasons of calving and birth: (i) rainy season -
688
which included the months from April to September in the herd located in the state of Rio Grande
do Norte and the months between October and March, in herds located in the State of São Paulo and
(ii) The dry season- included the months from October to March, in the herd located in the state of
Rio Grande do Norte and from April to September in herds located in the State of São Paulo.
Contemporary group (CG) were consisting of herd-year-season of birth for AFC, and herd-year-
season of calving for MY, % F, % P, LL and CI. The covariance components were estimated by
Bayesian inference in multi-trait analyses with the GIBBS2F90 software (Misztal, 2007). The
model of analysis included fixed effects of CG, number of milking (1 or 2), and the covariable cow
age at calving (linear and quadratic effects). Additive genetic and permanent environmental effects
were included as random effects. In matrix notation, the model used to analyze can be represent as:
y = Xβ + Za + Wp + e
in which y is a vector of observed traits; X is the incidence matrix of fixed effects; β is a vector of
fixed effects (CG, number of milking, and the covariable cow age at calving as linear and quadratic
effects); Z is the incidence matrix of additive genetic random effects; a is a vector of additive
genetic random effects; W is the incidence matrix of permanent environmental random effect; p is a
vector of permanent environmental random effects; and e is a vector of random error effects.
Uniform a priori distribution was defined for fixed effects (β). Gaussian and inverted Wishart
distributions were defined as a priori distributions for random effects and (co)variance components,
respectively.
β ∝ constante;
a |G ~ MVN[0,(G  A)];
p | P ~ MVN[0,(P  In )];
G | Sg ,vg ~ IW[Sg vg ,vg ];
P | Sp ,vp ~ IW[Sp vp ,vp ];
R | Sr ,vr ~ IW[Sr vr ,vr ];
permanent environmental effects, residual, and identity, respectively;  is the Kronecker product;
Where A, G, P, R, and In are the matrices of relationship, (co)variances of additive genetic effects,
and Sg and vg; Sp and vp; Sr and vr are the values of a priori and degree of freedom for additive
genetic, permanent environmental, and residual (co)variances, respectively. A total of one million
samples were generated in the analyses and a burn-in period of 100,000 samples was used with
samples taken each 100 cycles. The convergence was verified using the Gibbanal program.

The heritability estimates are presented in Table 2. Heritability for milk yield was moderate
(0.24), suggesting the existence of enough genetic variability to respond to selection programs.
Results similar to those found in this study were observed in the literature, which ranged from 0.20
to 0.25 (Tonhati et al. 2000a; Malhado et al., 2007; Rodrigues et al., 2010; Seno et al., 2010).
Conversely, Rosati and Van Vleck (2002) obtained heritability estimate of 0.14 for the MY with
buffaloes from Italy. These differences are probably not only the differences between regions, but
also because of genetic differences among herds. The heritability for the percentages of fat and
protein were moderate, 0.34 and 0.40, respectively. These results suggest that both traits would
respond well to a selection program. Aspilcueta-Borquis et al. (2010) reported similar estimates of
heritability for %F and %P, which were 0.32 and 0.39, respectively. Lower heritability for
percentages of fat and protein were recorded by Tonhati et al. (2000a) and Rosati and Van Vleck
(2002). The high genetic variability in this study among animals is in part of due they had not been
intensely selected. Nevertheless, there is a big difference between management the herds, causing
low heritability estimates, being largely resulting environmental effects.
The heritability estimated for length of lactation (0.09) was similar to those reported by Aziz
et al. (2001), Malhado et al. (2009) and Rodrigues et al. (2010), whose values ranging between 0.08
and 0.09. The low heritability value of LL indicates that this trait is influenced largely by non-
genetic factors, it is possible to obtain better performance through proper management in the herd.
689
The heritability estimated for calving interval was 0.05. The value obtained in this study is
in agreement with results previously reported in the literature whose ranged from 0.00 to 0.07 (Aziz
et al., 2001; Cassiano et al., 2004; Ramos et al. , 2006; Malhado et al., 2009). The results indicate
that improvement in management is more efficient than selection.
The heritability for age at first calving was 0.16. This value was higher than the recorded
value (0.07) by Seno et al. (2010). However, less than the values obtained by Tonhati et al. (2000b),
Cassiano et al. (2004) and Malhado et al. (2009), which were 0.20, 0.24 and 0.41, respectively.
Therefore the trait AFC would respond well to a selection program.
The estimates of genetic correlations between milk yield and percentages of fat and protein
were -0.29 and -0.18, respectively. These values are in agreement with the values obtained in the
literature (Tonhati et al., 2000a; Rosati and Van Vleck, 2002). These results indicate that when
applying the selection to increase milk production, there is a decrease in the percentage of fat and
milk protein. However, the genetic correlation between %F and %P was 0.25, in agreement with
values obtained by Tonhati et al. (2000a), Rosati and Van Vleck (2002) and Aspilcueta-Borquis et
al. (2010). Therefore, the direct selection to increase %G in milk would also increase %P.
REFERENCES
Aspilcueta-Borquis, R.R., F.R. Araujo Neto, F. Baldi, A.B. Bignard and L.G. Albuquerque. 2010.
Genetic parameters for buffalo milk yield and milk quality traits using Bayesian inference. J.
Dairy Sci. 93:2195-2201.
Aziz, M.A., S.J. Schoeman, G.F. Jordaan, O.M. El-Chafie and A.T. Mahdy. 2001. Genetic and
phenotypic variation of some reproductive traits in Egyptian buffalo. S. Afr. J. Anim. Sci.
3:195-199.
Cassiano, L.A.P., A.S. Mariante, C. McManus, J.F.R Marques and N.A. Costa. 2004. Genetic
parameters of production and reproduction traits of buffaloes in the Brazilian Amazon. Pesq.
agropec. bras. 39:451-457.
IBGE. 2011. Brazilian Institute of Geograph and Statistics.
Malhado, C.H.M., A.A. Ramos, P.L.S. Carneiro, J.C. Souza and A. Piccinin. 2007. Genetic and
phenotypic parameters for milk production of Murrah buffaloes. R. Bras. Zootec. 36:376-
379.
Malhado, C.H.M., A.A. Ramos, P.L.S. Carneiro, D.M.M.R. Azevedo, P.R.A.M. Affonso, D.G.
Pereira and J.C. Souza. 2009. Genetic parameters of reproductive and productive traits in
cross-breed water buffaloes in Brazil. Rev. Bras. Saúde Prod. An. 10:830-839.
Misztal, I. 2007. BLUPF90 family of programs. http://nce.ads.uga.edu/~ignacy/newprograms.html.
Ramos, A.A., C.H.M. Malhado, P.L.S. Carneiro, H.C. Gonçalves and D.M.M.R. Azevedo. 2006.
the Murrah breed. Pesq. agropec. bras. 41:1261-1267.
Rodrigues, A.E., J.R.F. Marques, C.V. Araujo, R.N.C. Camargo Junior and L.N.S. Dias. 2010.
Amazon. Arq. Bras. Med. Vet. Zootec. 62:712-717.
Rosati, A. and L.D. Van Vleck. 2002. Estimation of genetic parameters for milk, fat, protein and
mozzarella cheese production for the Italian river buffalo Bubalus bubalis population.
Livestock Production Science. 74:185–190.
Seno, L., V.L. Cardoso El, L. Faro, R.C. Sesana, R.R. Aspilcueta-Borquis, G.M.F. de Camargo and
H. Tonhati. 2010. Genetic parameters for milk yield, age at first calving and interval
between first and second calving in milk Murrah buffaloes. LRRD. (In press).
Tonhati, H., M.F.C. Muñoz, J.A. Oliveira, J.M.C. Duarte, T.P. Furtado and S.P. Tseimazides.
2000a. Genetic Parameters of Milk Production, Fat and Protein Contents in Buffalo Milk.
Rev. bras. zootec. 6:2051-2056.
Tonhati, H., F.B.Vasconcellos and L.G. Albuquerque. 2000b. Genetic aspects of productive and
reproductive traits in a Murrah buffalo herd in São Paulo, Brazil. J. Anim. Breed. Genet.
331-336.
690
Table 1. Summary of data structure and descriptive statistics for milk yield (MY), fat percentage
(%F), protein percentage (%P), length of lactation (LL), calving interval (CI) and age of first
calving (AFC).
MY %F %P LL CI AFC
(Kg) (days) (days) (months)
Number of records 9,318 2,877 2,872 9,318 5,672 2,389
Number of cows 3,061 1,281 1,281 3,061 2,054 2,389
Number of 161 161 161 161 161 178
contemporary
groups
Mean 1,872.75 6.55 4.26 258.83 422.76 37.57
Standard-deviation 637.59 1.00 0.28 58.87 73.16 5.73
CV% 34.04 15.27 6.61 22.74 17.30 15.25
Table 2. Heritability estimates values and standard-deviation (SD) for Milk yield (MY), fat
percentage (%F), protein percentage (%P), length of lactation (LL), calving interval (CI) and age of
first calving (AFC).
h2 SD
MY 0.24 0.02
%F 0.34 0.05
%P 0.40 0.05
LL 0.09 0.01
CI 0.05 0.01
AFC 0.16 0.03
691
Genetic Variants of POU1F1 gene in Azakheli buffalo breed of Pakistan
Asif Nadeema*, Rashid Majeeda, Masroor Ellahi Babarb, Tahir Yaquba, Maryam Javeda,
Tanveer Hussaina and Ahmad Nawaz Khosac
a
Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences Lahore,
Pakistan.
b
Faculty of Animal Production & Technology, University of Veterinary and Animal Sciences
Lahore, Pakistan.
c
Faculty of Veterinary and Animal Sciences, Lasbela University of Agriculture, water and Marine
Sciences, Balochistan, Pakistan.
Corresponding Email: asif_cemb@hotmail.com
ABSTRACT
POU1F1 encodes a pituitary-specific transcription factor which is involved in pituitary
development and regulating the hormone expression in animals. In present study, POU1F1 gene
was screened for polymorphic sites in Azakheli buffalo breed of Pakistan. Based on six pairs of
primer sequences, DNA sequencing and Blast analysis, 15 single nucleotide polymorphisms were
identified, from which 5 polymorphisms were in exonic and remaining were in intronic region of
gene. Polymorphism in exon of gene showed change in amino acid, two synonymous and three non-
synonymous. Synonymous, valine to valine; serine to serine and non-synonymous, proline to
leucine; leucine to serine; methionine to threonine were observed. Genotype and allele frequencies
of identified polymorphism of POU1F1 gene were also calculated. This is the first report toward
genetic screening of this gene at molecular level in Azakheli buffalo breed of Pakistan. Identified
novel polymorphisms can be used for association with different traits.
Keywords: Genetic variants, POU1F1 Gene, Azakheli and Buffalo
INTRODUCTION
Azakheli is the buffalo breed of Pakistan and mainly localized in the Swat valley of Khyber
Pakhtunkhwa (Khan, 2003). This buffalo breed has comparatively small stature which makes it
suitable for the hilly area. Azakheli has very good ability to convert low grade feed stuff to milk and
meat (Babar et al., 2009).
Pit-Oct-Unc Domain, Class 1, Transcription Factor 1 (POU1F1) encodes a pituitary-specific
transcription factor which involved in pituitary development and regulating the hormone expression
in animals. This factor is a member of the Pit-Oct-Unc (POU) family of transcription factors that
are involved in regulating the hormonal expression (Cohen et al., 1996). Giving the importance of
this gene in the control of gene expression of important hormones, it is a good candidate gene for
marker assisted selection (Lan et al., 2009). Several mutations in POU1F1 gene were associated
with body weight, milk yield, milk proteins, fat yields and litter size in bovine and caprine. No
previous studies have investigated the polymorphisms of this gene in Pakistani buffalo breed.
The aim of our research was to study the POU1F1 gene nucleotide sequence to highlight
possible polymorphisms in Azakheli buffalo using sequencing approaches.

Blood samples of true representative individuals of Azakheli buffalo were collected from the
Jugular vein of each selected animal into vacutainer blood collection tubes containing 200 µL
anticoagulant i.e. Ethylenediaminetetraacetic acid (0.5 M EDTA). The field blood samples were
placed in ice immediately after their collection and brought to the laboratory and stored temporarily
in freezer at -20°C before DNA extraction. Inorganic DNA extraction method was performed on
each of the blood samples (Sambrook and Russell., 2001). For the quantification of DNA, 0.8 %
692
agarose gel was used. Gel was visualized under UV light in gel documentation system (Bio-Rad)
and quantity was estimated.
Primers were designed from Gene Bank Accession No. NC_007299.4 whole genome
shotgun sequence by using software Primer3 (Table 1). The PCR reaction mixture composition
were followed as described by Maryam et al. (2012). After the amplification, PCR products were
precipitated for sequencing on ABI genetic analyzer 3130XL. All the sequences were aligned with
the help of online software blast2 sequence (http/www.ncbi.nlm.nih.com). SNPs were identified
from the aligned sequences. Frequencies were computed with the use of POPGENE (version 1.31)
software package (Yeh et al., 1999).

Based on six pairs of primer sequences, DNA sequencing and blast anaylsis revealed fifteen
novel polymorphisms which were firstly identified in Pakistani Buffalo. Briefly, ten novel
polymorphisms in intronic and five in exonic region were identified (Table 2). Polymorphisms in
exon of gene showed change in amino acid, two synonymous and three non-synonymous.
Synonymous, valine to valine; Serine to serine and non-synonymous, proline to leucine; leucine to
serine; methionine to threonine are shown in Table 3. Genotype and allele frequencies of identified
polymorphism of POU1F1 gene are given in Table 4. Genotype frequency and allele frequency
(CC=80%, C=85%) at position 35755942 was higher as compared to other identified
polymorphism.
In bovine several mutations in POU1F1 gene were associated with body weight, milk proteins
and fat yields (Renaville et al., 1997). Mutations found in the exons showed a positive association
with milk yield, quality and growth traits (Renaville et al., 1997, Huang et al., 2008). Several
genetic variations were also reported in caprine POU1F1 gene (Lan et al., 2007a&b), and
associated with milk yield, litter size and body weight (Lan et al., 2007c). Song et al. (2005) also
reported the polymorphisms and its associations with growth performance in the intron of the swine
POU1F1 gene. This is the first report toward genetic screening of this gene at molecular level in
Azakheli buffalo breed of Pakistan. Identified novel polymorphism can be used for association with
different traits.
REFERENCES
Babar, M. E., T. Hussain, A. Nadeem, R. Jabeen and M. Javed. 2009. Genetic Characterization of
Azakheli Buffalo Breed of Pakistan using Microsatellite DNA Markers. Pakistan J.
Zoology. 9: 361-366.
Cohen L.E., F.E. Wondisford and S. Radovick. 1996. Role of Pit-1 in the gene expression of growth
hormone, prolactin and thyrotropin. Endocrinol Metab Clin North Am. 25: 523–540.
Huang W, C. Maltecca and H. Khatib. 2008. A proline-to-histidine mutation in POU1F1 is
associated with production traits in dairy cattle. Anim Genet 39:554–557
Khan, M. S., 2003. Azi-Kheli buffalo – an important genetic resource. Buffalo Newsl., 18: 3.
Lan XY, C.Y. Pan, H. Chen, C.L. Zhang, J.Y. Li, M. Zhao, C.Z. Lei, A.L. Zhang and L. Zhang.
2007b. An AluI PCR-RFLP detecting a silent allele at the goat POU1F1 locus and its
association with production traits. Small Rumin Res 73:8–12
Lan XY, C.Y. Pan, H. Chen, C.Z. Lei, L.S. Hua, X.B. Yang, G.Y. Qiu, R.F. Zhang and Y.Z. Lun.
2007c. DdeI polymorphism in coding region of goat POU1F1 gene and its association with
production traits. Asian Australas J Anim 20:1342–1348
Lan XY, C.Y. Pan, H. Chen and C.Z. Lei. 2007a. A DdeI PCR-RFLP detecting genetic variation of
goat POU1F1 gene. Can J Anim Sci 87:13–14
693
Lan XY, M.J. Li, H. Chen, L.Z. Zhang, Y.J. Jing, T.B. Wie, G. Ren, X. Wang, X.T. Fang, C.L.
Zhang and C.Z. Lei. 2009. Analysis of caprine pituitary specific transcription factor-1 gene
polymorphism in indigenous Chinese goats. Mol Biol Rep 36:705–709
Maryam, J., M. E. Babar, A. Nadeem and T. Hussain. 2012. Genetic variants in Interferon Gamma
(IFN-γ) gene are associated with resistance against ticks in Bos taurus and Bos indicus. Mol.
Bio. Rep. 39(4):4565-4570.
Renaville R, N. Gengler, E. Vrech, A. Prandi, S. Massart, C. Corradini, C. Bertozzi, F. Mortiaux,
A. Burny and D. Portetelle. 1997. PIT-1 gene polymorphism, milk yield, and conformation
traits for Italian Holstein-Friesian bulls. J Dairy Sci 80:3431–3438
Sambrook J. and D.W. Russell. 2001. Molecular Cloning: A laboratory Manual, 3rd edn. Cold
spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA.
Song, C., Bo Gao, Y. Teng, X. Wang, Z. Wang, Q. Li, H. Mi, R. Jing and J. Mao. 2005. MspI
polymorphisms in the 3rd intron of the swine POU1F1 gene and their associations with
growth performance. J Appl Genet 46(3): 285-289
Yeh, F., C. Boyle, T. Rongcai, Z.Y. Ye and J. M. xian, 1999. POPGENE, Version 1.31. A
Microsoft window based free ware for population genetic analysis. University of Alberta,
Edmonton.
Table 1. List of Primers of POU1F1 Gene.
Primer Name Primer Sequence

POU F1 GGTAGTGAGATTTGAAACGGC
POU R1 GCTTTGAATCTTGCTAGTTTAG
POU F2 CCAAACTCCTAAATGTTTGTGC
POU R2 GAGTTTGGGGACGCTTGGATGC
POU F3 GGGCCAAAATTTTCCATGTATC
POU R3 GCTGTGGGACACACAACTCCG
POU F4 GCAAAACTGAAGCTCATGGC
POU R4 GGTTTCCCTCAGTTGAATTTG
POU F5 GATTTGATAGCATTTAGAGC
POU R5 GTATTGTTTGATTCAAGAACAG
POU F6 CATCTCCCTTCTTTCCTGCC
POU R6 CCCATAAAGAATATCACCAG
694
Table 2. List of identified SNPs in POU1F1 Gene.
SNP ID SNP Position Change in Nucleotide Region

POU1 35753434 A>G Intronic
POU2 35763391 T>C Intronic
POU3 35763354 T>C Exonic
POU4 35763324 G>C Exonic
POU5 35763297 A>C Intronic
POU7 35763282 G>A Intronic
POU8 35763245 G>C Intronic
POU9 35756152 C>T Exonic
POU12 35755987 C>T Intronic
POU13 35755946 C>A Intronic
POU15 35752836 T>A Intronic
Table 3. Change in Amino Acid of Exonic SNPs.
Change in
SNP ID SNP Position Change in Amino Acid
Codon
Valine to Valine
POU3 35763354 GUU>GUC
(Synonymous)
Serine to Serine
POU4 35763324 UCG>UCC (Synonymous)
Proline to Leucine
POU9 35756152 CCC>CUC
(Non-Synonymous)
Leucine to Serine
POU10 35756107 UUG>UCG
(Non-Synonymous)
Methionine to Threonine
POU11 35756086 AUG>ACG
(Non-Synonymous)
695
Table 4. Genotype and Allele Frequencies of POU1F1 Gene.
SNP
SNP Position Genotype Frequency Allele Frequency
ID
AA AG GG A G
POU1 35753434
0.1750 0.1000 0.7250 0.2250 0.7750
CC CT TT C T
POU2 35763391
0.6500 0.1500 0.2000 0.7250 0.2750
CC CT TT C T
POU3 35763354
0.7500 0.1000 0.1500 0.8000 0.2000
CC CG GG C G
POU4 35763324
0.7000 0.1000 0.2000 0.7500 0.2500
AA AC CC A C
POU5 35763297
0.2500 0.1250 0.6250 0.3125 0.6875
CC CT TT C T
POU6 35763283
0.3750 0.2500 0.3750 0.5000 0.5000
AA AG GG A G
POU7 35763282
0.5000 0.2000 0.3000 0.6000 0.4000
CC CG GG C G
POU8 35763245
0.7500 0.1750 0.0750 0.8375 0.1625
CC CT TT C T
POU9 35756152
0.3000 0.2500 0.4500 0.4250 0.5750
CC CT TT C T
POU10 35756107
0.5000 0.2000 0.3000 0.6000 0.4000
CC CT TT C T
POU11 35756086
0.3500 0.3000 0.3500 0.5000 0.5000
CC CT TT C T
POU12 35755987
0.3500 0.2000 0.4500 0.4500 0.5500
AA AC CC A C
POU13 35755946
0.7000 0.1000 0.2000 0.7500 0.2500
CC CT TT C T
POU14 35755942
0.8000 0.1000 0.1000 0.8500 0.1500
AA AT TT A T
POU15 35752836
0.7500 0.1500 0.1000 0.8250 0.1750
696
Single Nucleotide Polymorphisms in Bovine CYP11B1 Gene in Nili Ravi Buffalo

Breed of Pakistan
Maryam Javeda*, Asif Nadeema, Masroor Ellahi Babarb And Sidra Manzoora
a
54000, Pakistan.
b
Lahore 54000, Pakistan.
Corresponding Email: maryam.javed@uvas.edu.pk
ABSTRACT
Cytochrome P450, family 11, subfamily b, polypeptide 1 (CYP11B1) gene belongs to a family
of genes called CYP (cytochrome P450). Bovine CYP11B1 gene is located at chromosome 14,
consists of eight introns and nine exons. This gene provides instructions for making an enzyme
called 11β-hydroxylase that catalyzes both the 11β and 18-hydroxylation of corticosteroids in
bovine. In some species, the CYP11B1 gene has developed into distinct isoforms, whereas in pig,
sheep and cattle functional unity is conserved. Keeping in view the importance of this gene, a
research work was planned to identify the polymorphism in local buffalo breed of Pakistan. Total 7
single nucleotide polymorphisms were identified in exon 4 and 5 of this gene. This is a first report
toward genetic screening of CYP11B1 gene at molecular level in Nili Ravi buffalo. The present
study will provide a better selection of this candidate gene. It will further help us in developing
association of SNPs in buffalo population.
Keywords: SNPs, CYP11B1 Gene, Nili Ravi, Buffalo
INTRODUCTION
Pakistan has a variety of animal genetic resources and diversity both at phenotypic and
genetic levels. According to economic survey of Pakistan (2009-10), the national herd consists of
30.8 million buffaloes, which are contributing almost 68% of total milk produced in the region
(Khan et al., 2008). The total milk produce is insufficient human demands. No doubt, milk
production has shown an increasing trend over last several years, but this increase in milk
production is due to increase in total number of milk producing animals not per animal production.
The production per animal is less due to low genetic potential of our animals (Bilal and Sajjad
2005).
The recent development in molecular biotechnology and genomic knowledge for various
species has made this possible to understand the role of different candidate genes affecting these
traits (Falconer and Mackay 1996, Awad et al., 2010). Identification of SNPs (single nucleotide
polymorphisms) in the gene may be a promising strategy to clearly understand the physiological
background of quantity and quality of milk synthesis.
Keeping in view the above facts, a research work was planned to identify the polymorphism
in cytochrome P450, family 11, subfamily B (CYP11B1) gene and its relation with milk yield. The
bovine CYP11B1 gene is positioned in chromosomal region BTA14q12 (Kaupe et al., 2004). More
precisely, the CYP11B1 gene is located from 1,302,902 bp to 1,310,580 bp on chromosome 14. Its
reference sequence length is about 7.679 kilobase pairs(http://www.ncbi.nlm.nih.gov/gene/282422)
It constitute of eight introns and nine exons (Lisurek and Bernhardt 2004). This CYP11B1 gene is a
functional and positional candidate gene for the QTL related to fat metabolism, milk yield, protein
yield and fat content (Kaupe et al., 2007).
Keeping in view the importance of this gene, a research work was planned to identify the
polymorphism in local buffalo breed of Pakistan. Total 7 SNPs were identified in exon number 4
and 5 of this gene. This is a first report towards genetic screening of CYP11B1 gene at molecular
level in Nili Ravi buffalo.

697

CYP11B1 is an important candidate gene associated with milk production trait in mammals.
The present study was conducted for genetic characterization of CYP11B1 gene in Nili-Ravi buffalo
breed of Pakistan. Unrelated animals with typical phenotypic features known for a given breed were
selected from several respective breeding tracts. Blood samples were collected from the jugular vein
of cattle by 10 ml disposable syringes and preserved in 50 ml storage tubes having 400 mL 0.5 M
Ethylenediamine tetra-acetic acid (EDTA) as anticoagulant.
For PCR amplification, genomic DNA was extracted from blood samples using inorganic
method (Sambrook and Russel 2001). DNA quantification was carried out using 0.8% Agarose gel.
Using Exon Prime Intronic Centre (EPIC) technique two sets of primer were designed for cattle
(Gene Bank Accession no. NC_007312.5, Bos Taurus chromosome # 14, whole genome shotgun
sequence) by “Primer3” software (http/www.primer3.com). All forward and reverse primers were
amplified on 35 DNA samples of Nili Ravi. After the precipitation, PCR products were sequenced.
Sequencing results were analyzed with BioEdit software
(http://www.mbio.ncsu.edu/bioedit/bioedit.html). Each and every nucleotide position that showed
no alignment with reference sequence was studied for SNPs.
Allelic and genotypic frequencies for the observed SNPs were calculated using
bioinformatics software with the use of POPGENE (version 1.31) software package (Yeh et al.,
1999).

In this study, we identified 7 SNPs in CYP11B1 gene in Nili Ravi Buffalo breed of Pakistan.
To search for polymorphic sites in the gene, DNA was extracted from a wide range of buffalo.
Using the sequencing approach, 7 SNP in an exonic region were identified. From these SNPs, one is
silent polymorphism and not changing the amino acid. Remainings are significant in the sense that
causing change in amino acid sequence which ultimately can affect the structure as well as function
of the protein.
List of identified polymorphism, position of mRNA and amino acid, change in codon and
amino acid are given in Table-1. Results of polymorphism show that there is change in amino acids.
Leucine has changed to phenylalanine and both amino acids are nonpolar and neutral. Arginine is
basic polar and positively charged and changed to glutamine which is polar and neutral. Methionine
and alanine are nonpolar and neutral and has changed to threonine, asparagine and arginine which
are polar and neutral in nature. This change in nature of amino acid will definitely influence the
nature of protein. Genotypic and allelic frequencies of identified SNPs in CYP11B1 gene are given
in table-2.
The bovine CYP11B1 gene is located on chromosome 14 near marker ILSTS039 which is
associated with milk traits (Looft et al., 2001, Kühn et al., 2004). This makes the CYP11B1 gene a
functional and positional candidate gene for milk production traits. Secondly CYP11B1 persuade
the production of cortisol, androgen function and ultimately the proliferation of milk gland cells
(Brettes and Mathelin 2008). Cortisol is one of the principal hormones involved in lipogenesis and
lipolysis. Boleckova et al. (2012) and Mai et al. (2010) reported the positive associations of
CYP11B1 polymorphism with milk production and composition traits. Kaupe et al. (2007) reported
the joint analysis of the influence of CYP11B1 and DGAT1 genetic variation on milk production,
somatic cell score, conformation, reproduction, and productive lifespan in German Holstein cattle.
The identified SNPs in the current study can be used as genetic marker for different traits.
Besides the identification of SNPs in the present study, gene expression patterns dependent review
will help to understand the molecular mechanisms of this gene to have role in production traits in
buffaloes.
698
REFERENCES
Awad, A., I. Russ, R. Emmerling, M. Förster and I. Medugorac. 2010. Confirmation and refinement
of a QTL on BTA5 affecting milk production traits in the Fleckvieh dual purpose cattle
breed. Ani Genet. 41:1-11.
Bilal M.Q. and M.S. Sajid. 2005. Meeting milk demand (The only way is to modernize dairy
farming). The Nation.P-26.
Boleckova, J., J. Matejickova, M. Stipkova, J. Sefrova, M. Krejcova and L. Barton. 2012. The
association of five polymorphisms with milkproduction traits in Czech Fleckvieh cattle. J
Anim Sci. 57 (2): 45–53.
Brettes J.P and C. Mathelin. 2008. Effet dual des androgènes sur la glande mammaire. Bulletin du
Cancer (Paris), 95, 495–502
Falconer D.S and TFC. Mackay. 1996. Introduction to quantitative genetics. Fourth edition, Pearson
education limited.
Kaupe, B., H. Brandt, E-M. Prinzenberg and G. Erhardt. 2007. Joint analysis of the influence of
CYP11B1 and DGAT1 genetic variation on milk production, somatic cell score,
conformation, reproduction, and productive lifespan in German Holstein cattle. J Anim Sci.
85:11-21.
Kaupe, B., S. Kollers, R. Fries and G. Erhardt. 2004. Mapping of CYP11B1 and a putative CYHR1
paralogous gene to bovine chromosome 14 by FISH. Anim. Genet. 35:36.
Khan, M.S., ZA. Rehman, AM. Khan and S. Ahmad. 2008. Genetic resources and diversity in
Pakistani cattle. Pak Vet J. 28(2): 95-102.
Kühn, C., G. Thaller, A. Winter, ORP. Bininda-Emonds, B. Kaupe, G. Erhardt, J. Bennewitz, M.
Schwerin and R. Fries. 2004. Evidence for multiple alleles at the DGAT1 locus better
explains a quantitative trait locus with major effect on milk fat content in
cattle. Genetics. 167:1873–1881.
Lisurek, M and R. Bernhardt. 2004. Modulation of aldosterone and cortisol synthesis on the
molecular level.Mol Cell Endocrinol. 27:149–159.
Looft, C., N. Reinsch, C. Karall-Albrecht, S. Paul, M. Brink, H. Thomsen, G. Brockman, C. Khun,
M. Schwerin and E. Kalm. 2001. A mammary gland EST showing linkage disequilibrium to
a milk production QTL on bovine chromosome 14. Mamm. Genome. 12(8):646-650.
Mai, MD., G. Sahana, FB. Christiansen and B. Guldbrandtsen. 2010. A genome-wide association
study for milk production traits in Danish Jersey cattle using a 50K single nucleotide
polymorphism chip1. J Anim Sci. 88(11):3522-3528.
Sambrook, J. and D. W. Russell. 2001. Molecular Cloning: A laboratory Manual, 3 rd edn. Cold
Yeh, F., C. Boyle, T. Rongcai, Z.Y. Ye and J. M. xian, 1999. POPGENE, Version 1.31. A
Microsoft window based free ware for population genetic analysis. University of Alberta,
Edmonton.
Table 1. Identified Polymorphism and change in amino acid.
Sr. Change in
SNP ID mRNA # Amino acid # Change in Amino acid
# Codon
1 CYP1 690 230 TTG > TTC Leucine to phenylalanine
2 CYP2 699 233 ACC > ACT Threonine to Threonine
3 CYP3 734 245 CGG > CAG Arginine to Glutamine
4 CYP4 752 251 ATG > ACG Methionine to threonine
5 CYP5 772 258 GAC > AAC Alanine to Asparagine
6 CYP6 773 258 ACC > AAC Alanine to Asparagine
7 CYP7 893 298 ATG > AGG Methionine to arginine
699
Table 2. Genotypic and allelic frequency of identified SNPs.
SNP ID Genetypic Frequency Allelic Frequency

AA AB BB A B
CYP1 0.6600 0.1500 0.1900 0.7250 0.2750
CYP2 0.5954 0.1363 0.2683 0.6585 0.3415
CYP3 0.7500 0.1000 0.1500 0.8000 0.2000
CYP4 0.1951 0.1707 0.6341 0.2805 0.7195
CYP5 0.5000 0.2000 0.3000 0.6000 0.4000
CYP6 0.4300 0.1200 0.4500 0.3300 0.6700
CYP7 0.6300 0.1500 0.2200 0.6585 0.3415
700
Identification of Single Nucleotide Polymorphisms in OLR1 Gene in Nili Ravi

Buffalo
Maryam Javed1*, Masroor Ellahi Babar2, Asif Nadeem1, Tahir Yaqub1 And Tanveer
Hussain1
1
54000, Pakistan.
2
ABSTRACT
The Oxidized low density lipoprotein receptor (OLR1) gene encodes a vascular endothelial
cell-surface receptor OLR1 protein that binds, internalizes and degrades the oxidized forms of low-
density lipoproteins. Research project was planned for the identification of polymorphism in coding
regions of OLR1 gene in local Buffalo. Unrelated animals of Nili Ravi buffalo with typical
phenotypic features were selected for blood samples. Blood samples, from different Government
livestock farms, were collected from the jugular vein into ethylenediamine tetra-acetic acid
containing tubes. DNA was extracted by organic method using phenol-chloroform-isomyl alcohol.
Quantification of the DNA samples was done with the help of Spectrophotometer. Nine set of
Primers were designed by using Primer3 software. PCR amplification was done and product was
checked by gel electrophoresis. Ethanol precipitation was performed to make PCR product free of
any unused dNTPs and primers. Purified products were sequenced. For data analysis, Chromas
software was used along with sequence-Alignment program available at NCBI. Results were
analyzed by using the different statistical tools and subjected to comparison with the existing data
on NCBI. Fifteen polymorphisms in Nili Ravi buffalo were identified in exonic regions of the gene,
out of which five were silent. Estimation of genotypic and allele frequencies provided additional
information regarding frequency in selected population. These findings of OLR1 gene could be
used for correlation of identified polymorphism with different traits of animals. The aim of research
was to identify gene that underlie the genetic variation in local buffalo breed of Pakistan.
Keywords: Polymorphisms, OLR1 Gene, Nili Ravi, Buffalo
INTRODUCTION
The water buffalo (Bubalus bubalis) is an important dairy animal in the Pakistan, as about
68 % of the milk in Pakistan being produced by buffaloes, followed by cattle (27%) and
sheep/goat/camel (5%). Due to high fat contents of buffalo milk, it is the most preferred dairy
species in Pakistan. Economically, fat is the second most important component in milk, after
proteins (Naslund et al., 2008). Collective effect of multiple genes and environmental factors
determined milk fat percentage that is a quantitative trait (Afzal. 2010).
A vascular endothelial cell surface receptor Oxidized low density lipoprotein receptor
(OLR1) protein encodes by Oxidized low density lipoprotein receptor (OLR1) gene that binds,
internalizes and degrades the oxidized forms of low-density lipoproteins (OLDL), (Nagase et al.,
2000) has been recognized as a functional and positional candidate gene for milk fat percentage and
milk fat yield (Khatib et al., 2006). This gene is located on the bovine chromosome number 5 and
organized in 6 exons. The total length of this gene is 11306 bp.
In present study, genetic characterization of all six exons of this gene has been done in Nili-
Ravi buffalo to identify some novel Single Nucleotide Polymorphisms. For this purpose sequencing
analysis of blood samples from Nili-Ravi animals (n=48) revealed a total of 15 polymorphisms in
701
different exons of the gene. Allelic frequency and genotypic frequency has indicated their
significance in the population.

The present study was conducted to genetically characterize the OLR1 gene in Nili-Ravi
Buffalo. The work was carried out at Molecular Biology and Genomics Lab, Institute of
Biochemistry & Biotechnology, University of Veterinary and Animal Sciences Lahore.
Unrelated animals (different families having no blood relation up to almost six
generations) with typical phenotypic features known for a given breed were selected from several
respective breeding tracts and from the Government livestock farms. Ten ml blood was collected
aseptically from the Jugular vein of each selected animals into 50 ml Falcon tubes containing 200 µl
anticoagulant i.e. Ethylenediamine tetra-acetic acid (0.5 M EDTA). Organic extraction method by
Phenol-Chloroform-Isoamylalcohol (PCI) was performed on each of the blood sample. Primers
were designed (GeneBank Accession no. NC_007303.4 Bos taurus, whole genome shotgun
sequence) by using software Primer3 (http://frodo.wi.mit.edu/). Amplication of these primers was
done using Polymerase Chain Reaction. After the precipitation of PCR products were sequenced.
Multiple amplifications from the original samples were done to minimize the possibility of
identifying PCR-induced mutations. Sequencing was done on the principle of Sanger Chain
Termination.
All the sequences were aligned with the help of software blast2sequence (Tatusova et al.,
1999). 15 Single Nucleotide Polymorphism (SNPs) was detected from the aligned sequences.
Allelic frequencies were calculated from the observed SNPs using POPGENE version 1.31
(http://www.ualberta.ca/~fyeh).

In this study, we identified 15 Single Nucleotide Polymorphisms in OLR1 Gene in Nili Ravi
Buffalo breed of Pakistan. To search for polymorphic sites in the gene, DNA was extracted from a
wide range of buffalo. Using the sequencing approach, 15 SNP in exonic region were identified.
Identified polymorphic sites in the gene with SNPs position, change in nucleotide, transition or
transversion and amino acid conversion are presented in table-1.
Out of total fifteen, eight SNPs were Transitions (Purine to Purine or Pyrimidine to
Pyrimidine) and seven were Transversions. From these SNPs, five are silent polymorphism and not
changing the amino acid. Remainings are significant in the sense that causing change in amino acid
sequence which ultimately can affect the structure as well as function of the protein.
From statistical analysis done by using POPGENE, genotypic and allelic distribution of each
polymorphism was calculated. These values have been given in the table-2.
Estimation of genotype and allele frequencies of OLR1 in buffalo provided additional
information regarding frequency in selected population. OLR1 is a candidate gene affecting milk
composition traits. Khatib et al. (2006 & 2007) associated the SNPs with milk fat traits in cattle
with a significant increase in fat yield and fat percentage. Mango et al. (2003) and Chen et al.
(2003) reported the SNPs in 3′UTR and associated with acute myocardial infarction and with
coronary artery disease respectively. Moreover, it was also reported that 3′UTR sequences are
involved in the regulation of gene expression and can control stability of mRNA, polyadenylation,
rates of translation, nuclear transport, and gene silencing (Conne et al., 2000). It is worth
mentioning that SNP in human gene were identified and associated with Alzheimer’s disease.
(Luedecking-Zimmer et al., 2002, Lambert et al., 2003). Wang et al. (2012) reported the association
of an OLR1 polymorphism with milk production traits in the Israeli Holstein population. Results of
the present study showed that findings of OLR1 gene could be used for correlation of identified
polymorphism with different traits of animals.
REFERENCES
Afzal, M. 2010. Re-designing smallholder dairy production in Pakistan. Pak. Vet. J. 3:187-190.
702
Chen, Q., S. E. Reis, C. Kammerer, W. Y. Craig, S. E. LaPierre, E. L. Zimmer, D. M. McNamara,

D. F. Pauly, B. Sharaf, R. Holubkov, C. N. Bairey Merz, G. Sopko, F. Bontempo and M. I.
Kamboh. 2003. Genetic variation in lectin-like oxidized low-density lipoprotein receptor 1
(LOX1) gene and the risk of coronary artery disease. Circulation 107:3146–3151.
Conne, B., A. Stutz and J. D. Vassalli. 2000. The 3′ untranslated region of messenger RNA: A
molecular ‘hotspot’ for pathology? Nat. Med. 6:637–641.
Khatib, H., G.J.M. Rosa, K. Weigel, F. Schiavini, E. Santus and A. Bagnato. 2007.
Additionalsupportforanassociation between OLR1 and milk fat traits in cattle. Anim Genet
38: 308–310
Khatib, H., S.D. Leonard, V. Schutzkus, W. Luo and Y.M. Chang (2006). Association of the OLR1
Gene with Milk Composition in Holstein. Dairy Cattle. J. Dairy Sci. 89:1753–1760.
Lambert, J. C., E. Luedecking-Zimmer, S. Merrot, A. Hayes, U. Thaker, P. Desai, A. Houzet, X.

Hermant, D. Cottel, A. Pritchard, T. Iwatsubo, F. Pasquier, B. Frigard, P. M. Conneally, M.
C. Chartier-Harlin, S. T. DeKosky, C. Lendon, D. Mann, M. I. Kamboh and P. Amouyel.
2003. Association of 3′-UTR polymorphisms of the oxidised LDL receptor 1 (OLR1) gene
with Alzheimer’s disease. J. Med. Genet. 40:424–430.
Luedecking-Zimmer, E., S. T. DeKosky, Q. Chen, M. M. Barmada and M. I. Kamboh. 2002.

Investigation of oxidized LDL-receptor 1 (OLR1) as the candidate gene for Alzheimer’s
disease on chromosome 12. Hum. Genet. 111:443–451.
Mango, R., F. Clementi, P. Borgiani, G. B. Forleo, M. Federici, G. Contino, E. Giardina, L. Garza,

I. E. Fahdi, R. Lauro, J. L. Mehta, G. Novelli and F. Romeo. 2003. Association of single
nucleotide polymorphisms in the oxidised LDL receptor 1 (OLR1) gene in patients with
acute myocardial infarction. J. Med. Genet. 40:933–936.
Nagase, M., S. Kaname, T. Nagase, G. Wang, K. Ando, T. Sawamura and T. Fujita. 2000.
Expression of LOX-1, an Oxidized Low-Density Lipoprotein Receptor, in Experimental
Hypertensive Glomerulosclerosis. J. Am. Soc. Nephrol. 11: 1826–1836.
Naslund, J., W.F. Fikse, G.R. Pielberg and A. Lunden. 2008. Frequency and Effect of the Bovine
Acyl-CoA: Diacylglycerol Acyltransferase 1 (DGAT1) K232A Polymorphism in Swedish
Dairy Cattle. J. Dairy Sci. 5:2127–2134.
Tatusova, T. A., I. K. Mizrachi and J.A. Ostell. 1999. Complete genomes in WWW Entrez: data
representation and analysis. Bioinformatic. 15:536-543.
Wang, X., F. Peñagaricano, R. Tal-Stein, E. Lipkin and H. Khatib. 2012. Association of an OLR1
polymorphism with milk production traits in the Israeli Holstein population. J Dairy Sci.
95(3):1565-7.
703
Table 1. Polymorphic sites detected in the OLR1 gene
Sr. No SNPs Position SNP Change Transition/ Amino Acid

Nucleotide with Transversion Conversion
1 107080902 C A Transversion Proline to Histidine
2 107080942 C T Transition Glycine
3 107084380 C A Transversion Serine to Tyrosine
4 107084386 C G Transversion Alanine to Glycine
5 107084396 A C Transversion Leucine
6 107084402 A C Transversion Lycine to Glutamine
7 107084454 T C Transition Leucine to Serine
8 107084484 A C Transversion Glutamine to Arginine
9 107084569 C T Transition Histidine
10 107089859 T C Transition Serint to Proline
11 107090332 T C Transition Asparagine
12 107090355 T C Transition Glycine
13 107090986 G A Transition Arginine to Lycine
14 107091007 G A Transition Arginine to Histidine
15 107091125 G C Transversion Arginine to Threonine
704
Table 2. Genotypic and allelic frequency of identified SNPs of OLR1 gene
Polymorphisms Genetypic Frequency Allelic Frequency

AA AB BB A B
107080902 C →A 0.6341 0.1463 0.2195 0.7073 0.2927
107080942 C →T 0.4400 0.4500 0.1100 0.6650 0.3450
107084380 C→ A 0.120 0.430 0.4500 0.3300 0.6700
107084386 C→ G 0.6500 0.1500 0.2000 0.7250 0.2750
107084396 A →C 0.1750 0.1000 0.7250 0.2250 0.7750
107084402 A→ C 0.6500 0.1500 0.2000 0.7250 0.2750
107084454 T→ C 0.7500 0.1000 0.1500 0.8000 0.2000
107084484 A→C 0.7000 0.1000 0.2000 0.7500 0.2500
107084569 C→T 0.5854 0.1463 0.2683 0.6585 0.3415
107089859 T→C 0.3750 0.2500 0.3750 0.5000 0.5000
107090332 T→C 0.5000 0.2000 0.3000 0.6000 0.4000
107090355 T→C 0.8000 0.1000 0.1000 0.8500 0.1500

107090986 G→A 0.120 0.430 0.4500 0.3300 0.6700
107091007 G→A 0.6829 0.0976 0.2195 0.7317 0.2683
107091125 G→C 0.1951 0.1707 0.6341 0.2805 0.7195
705
POU1 Transcription Factor 1 DNA Polymorphism in Nili Ravi Buffalo
Rashid MAJEEDa, Asif NADEEMa, Masroor ELLAHI BABARb, Maryam JAVEDa, Sadia
MUNIRa and Asma MANZOORa
a
54000, Pakistan.
b
*Corresponding Email: asif_cemb@hotmail.com
ABSTRACT
Pit-Oct-Unc Domain, Class 1, Transcription Factor 1 is a member of the tissue-specific POU
homeobox transcription factor family that is found in all mammals. This factor is a member of the
Pit-Oct-Unc (POU) family of transcription factors that are involved in regulating the hormonal
expression in animal growth and development. POU1F1 encodes a pituitary-specific transcription
factor which is involved in pituitary development and regulating the hormone expression in
animals. This gene is located on BTA1q21–22 and consists of 15952 base pair which has 6 exons
and 5 introns and encodes a 33kDa protien. In present study POU1F1 gene was screened for
polymorphic sites in Nili Ravi buffalo breed. Total 19 Single Nucleotide Polymorphism were
found, from which 4 were exonic and 15 were intronic. This is a first report toward genetic
screening of POU1F1 gene at molecular level in Nili Ravi buffalo. No work has been reported on
this gene in buffalo. In this study we identified a new set of Single Nucleotide Polymorphism which
are useful for association studies.
Keywords: Polymorphisms, POU1F1 Gene, Nili Ravi, Buffalo
INTRODUCTION
Livestock sector, as a whole, plays a crucial role in the rural economy of Pakistan and within
this sector, milk is the largest and single most important commodity (Garcia et al. 2003). In buffalo
milk production, Pakistan stands second in the world (Bilal et al. 2008). In Pakistan major sources
of milk is buffalo and cattle, but buffalo is playing a leading role in the national economy by
producing milk, meat and drought power. Out of total milk produced in the country, buffalo
contributes about 68 %, cattle (27%) and sheep/goat/camel (5%). Nili Ravi is very popular buffalo
breed of Punjab and has a massive body which is wedge shaped and the color of this breed is black
but often has white markings on the muzzle, lower parts of the legs, fore head and switch of the tail.
Due to these distinctive markings, animals of this breed are often called as Punjkalian. Their horns
are small and curly, and have wall eye and a large, strong udder.
Pit-Oct-Unc Domain, Class 1, Transcription Factor 1 (POU1F1) encodes a pituitary-specific
transcription factor which is involved in pituitary development and regulating the hormone
expression in animals. This factor is a member of the Pit-Oct-Unc (POU) family of transcription
factors that are involved in regulating the hormonal expression in animal growth and development
(Cohen et al. 1996). This gene is located on the buffalo chromosome no. 1, and consists of 15952
base pair which has 6 exons and 5 introns which encodes a 33kDa protien. POU1F1 transcription
factor protein has two important regions for the transcriptional regulation of target promoters
(Bastos et al. 2006). No previous studies have investigated the SNPs in local buffalo breed, that
being so, the aim of current study was to reveal the SNPs of POU1F1 gene in Pakistani buffalo
using sequencing approaches.

706

Blood samples from true representative individuals of Nili Ravi breed of buffalo were
collected from different Government livestock farms. Ten mL blood was collected, aseptically from
the Jugular vein of each selected buffalo into 50 mL Falcon tubes containing 200 µL anticoagulant
i.e. Ethylenediamide tetra-acetic acid (0.5 M EDTA). Inorganic DNA extraction method was
performed on each of the blood samples (Sambrook and. Russell., 2001). For the quantification and
quality of DNA, 0.8 % Agarose gel was used. Gel was visualized under UV light in Gel
documentation system (Bio-Rad) and quality and quantity was estimated. DNA would appear as
single compact pink fluorescent band free from degradation. On the basis of gel electrophoresis all
samples were brought at same concentration level i.e.50 ng/uL. Six set of Primers were designed
from Bubalus bubalis POU1F1 gene (Gene Bank Accession No. NC_007299 whole genome
shotgun sequence) by using web based software Primer3 (Table-1).
PCR Optimization Multiple options for the PCR reaction mix and PCR cyclic conditions
were tried with the objectives to get maximum amplification and using minimum volume of
chemicals. PCR Amplification was confirmed by Agarose Gel Electrophoresis. After the
amplification of the desired portion of the DNA, PCR products were precipitated and loaded on 1 %
gel to examine its quality. After the precipitation, PCR products were sequenced. Multiple
amplifications from the original samples were done to minimize the possibility of identifying PCR-
induced mutations.
All the sequences were aligned with the help of online software blast2 sequence
(http/www.ncbi.nlm.nih.com) (Tatusova and Madden. 1999). Single Nucleotide Polymorphism
(SNPs) were identified from the aligned sequences. Frequencies were calculated by POPGENE.

The results showed total 19 polymorphic sites in the POU1F1 gene in Nili Ravi buffalo. List of
identified polymorphism with change in nucleotide, position, genotype and allelic frequency are
given in table-2. At position 35763354, an exonic type of SNP was found in which T was replaced
with C resulting in codon changing from GUU to GUC. With the change in the sequence of codon,
amino acid was not changed as both of the codons GUU and GUC code for the same amino acid
i.e. Valine (V) that’s why this is a Synonymous type of mutation. POU6, at position 35763324, an
exonic type of SNP was found in which G was replaced with C, resulting in codon changing from
UCG to UCC. With the change in the sequence of codon, amino acid was not changed as both of
the codons UCG and UCC code for the same amino acid i.e. Serine (S) that’s why this is a
Synonymous type of mutation.
SNP POU9, at position 35756152 of the POU1F1 gene, an exonic type of SNP was found
commonly in both of the Nili Ravi and Azakheli Buffalo in which C was replaced with T resulting
in codon changing from CCC to CUC. With the change in the sequence of codon, amino acid also
changed from Proline (P) to Leucine (L) as codon CCC codes for Proline and CUC codes for
Leucine. So this is a non-synonymous type of mutation. SNP POU15, an exonic type of SNP was
found in which T was replaced with C, resulting in codon changing from GUU to GUC. With the
change in the sequence of codon, amino acid was not changed as both of the codons GUU and GUC
code for the same amino acid i.e. Valine (V) that’s why this is a Synonymous type of mutation.
Several polymorphisms in POU1F1 gene were identified associated with milk yield, Milk
quality, body weight, milk proteins, fat yields, and growth traits in cattle (Renaville et al., 1997;
Renaville et al., 2007 and Huang et al., 2008). Several genetic variations were also reported in
caprine POU1F1 gene and associated with milk yield, litter size in goat and body weight (Lan et al.,
2007a,b,c). Various Polymorphisms in POU1F1 gene were also associated with growth
performance of the swine (Song et al. 2005). Thus POU1F1 is a potential major gene or marker for
production and growth traits.
707
REFERENCES
Bilal, M.Q., B.B. Khan and G. Muhammad. 2008. Dairy Industry in Pakistan. In: “Health and
Husbandry of Dairy Animals.” Edited by BB Khan, Department of Livestock Management,
University of Agriculture, Faisalabad, Pakistan.
Cohen, L.E., F.E. Wondisford and S. Radovick. 1996. Role of Pit-1 in the gene expression of
growth hormone, prolactin and thyrotropin. Endocrinol Metab Clin North Am. 25: 523–540.
Garcia, B., F. Farnir, L. Karim, N. Cambisano, J. Kim, A. Kvasz, M. Mni, P. Simon, J.M. Frere,
W. Coppieters and M. Geoges. 2003. Genetics and functional confirmation of the causality
of the DGAT1 K232A quantitative trait nucleotide in affecting milk yield and composition.
PNAS. 101:2398-2403.
Huang W, C. Maltecca and H. Khatib. 2008. A proline-to-histidine mutation in POU1F1 is
associated with production traits in dairy cattle. Anim Genet 39:554–557
Lan XY, C.Y. Pan, H. Chen, C.L. Zhang, J.Y. Li, M. Zhao, C.Z. Lei, A.L. Zhang and L. Zhang.
2007b. An AluI PCR-RFLP detecting a silent allele at the goat POU1F1 locus and its
association with production traits. Small Rumin Res 73:8–12
Lan XY, C.Y. Pan, H. Chen, C.Z. Lei, L.S. Hua, X.B. Yang, G.Y. Qiu, R.F. Zhang and Y.Z. Lun.
2007c. DdeI polymorphism in coding region of goat POU1F1 gene and its association with
production traits. Asian Australas J Anim 20:1342–1348
Lan XY, C.Y. Pan, H. Chen and C.Z. Lei. 2007a. A DdeI PCR-RFLP detecting genetic variation of
goat POU1F1 gene. Can J Anim Sci 87:13–14
Renaville R, N. Gengler, E. Vrech, A. Prandi, S. Massart, C. Corradini, C. Bertozzi, F. Mortiaux,
A. Burny and D. Portetelle. 1997. PIT-1 gene polymorphism, milk yield, and conformation
traits for Italian Holstein-Friesian bulls. J Dairy Sci 80:3431–3438
Sambrook J. and D.W. Russell. 2001. Molecular Cloning: A laboratory Manual, 3rd edn. Cold
Tatusova T.A. and T.L. Madden. 1999. "Blast 2 sequences a new tool for comparing protein and
nucleotide sequences". FEMS Microbiol. Lett.174:247-250.
Table 1. List of Primers.
Sr. No. Primer Name Primer Sequence Product

(5’-3’) Size
POU F1 CCTGCAGTATAAATACCAG
1 548bp
POU R1 CAGACTCACTGTTTACTTG
POU F2 GGGAAAAATGTCAACCCCT
2 474bp
POU R2 GAAGGCGTTACAGATCAGG
POU F3 TAGAACTGAGACTGGCTGTC
3 424bp
POU R3 GTCTTAGACTTCTGAGCAGC
POU F4 TGGCAGATGTTCCTATCTGA
4 579bp
POU R4 GGCCTTGCTTTTCTTCATAG
POU F5 GCTGGCTAAAGAGCAAGATT
5 580bp
POU R5 CTTGCAGAGCAAAACTGTTC
POU F6 GATTTTGATGGGCCTAAACC
6 624bp
POU R6 CAGCCTTTGGGAAAAGAATC
708
Table 2. Genotype and Allele Frequency of Identified Polymorphisms.
Sr. SNP Change in SNP Genotype Frequency Allele

No. ID Nucleotide Position Frequency
POU A>T AA AT TT A T
1 1 35766765
0.1707 0.0976 0.7317 0.2195 0.7805
POU AA AG GG A G
2 2 G>A 35766681
0.7561 0.1220 0.1220 0.8171 0.1829
POU A>G AA AT TT A T
3 3 35753434
0.1707 0.0976 0.7317 0.2195 0.7805
POU CC CT TT C T
4 4 T>C 35763391
0.6341 0.1463 0.2195 0.7073 0.2927
POU CC CT TT C T
5 5 T>C 35763354
0.5854 0.1463 0.2683 0.6585 0.3415
POU G>C CC CG GG C G
6 6 35763324
0.6829 0.0976 0.2195 0.7317 0.2683
POU CC CT TT C T
7 7 T>C 35763283
0.5366 0.1463 0.3171 0.6098 0.3902
POU T>C CC CT TT C T
8 8 35763255
0.5610 0.1220 0.3171 0.6220 0.3780
POU CC CT TT C T
9 9 C>T 35756152
0.0976 0.1707 0.7317 0.1829 0.8171
POU CC CT TT C T
10 10 T>C 35755942
0.6341 0.1707 0.1951 0.7195 0.2805
POU T>A AA AT TT A T
11 11 35753683
0.5854 0.1463 0.2683 0.6585 0.3415
POU CC CT TT C T
12 12 C>T 35753640
0.2195 0.1463 0.6341 0.2927 0.7073
POU C>G CC CG GG C G
13 13 35753588
0.2683 0.1463 0.5854 0.3415 0.6585
POU AA AG GG A G
14 14 A>G 35753575
0.1951 0.1707 0.6341 0.2805 0.7195
POU CC CT TT C T
15 15 T>C 35753544
0.5610 0.1463 0.2927 0.6341 0.3659
A>G AA AG GG A G
16 POU 35753326
16 0.2927 0.0976 0.6098 0.3415 0.6585
AA AT TT A T
17 POU T>A 35752836
17 0.6341 0.1463 0.2195 0.7073 0.2927
G>T GG GT TT G T
18 POU 35751213
18 0.1951 0.1707 0.6341 0.2805 0.7195
CC CG GG C G
19 POU G>C 35751223
19 0.1463 0.7805 0.0732 0.5366 0.4634
709
Bovine Stearoyl-CoA Desaturase Gene Polymorphism in Nili Ravi Buffalo
Maryam JAVEDa*, Asif NADEEM a and Masroor ELLAHI BABARb

a
54000, Pakistan.
b
ABSTRACT
Polymorphism of the gene has an effect on expression and regulation of gene transcription
or the amino acid sequence of the gene product. The associations between polymorphisms in
numerous genes have already been tested in the several studies but as for the bovine stearoyl-CoA
desaturase (SCD) gene, the studies on single nucleotide polymorphisms (SNPs) are limited. Bovine
SCD has been localized to chromosome 26q21 and is approximately 17 kb, comprises six exons and
five introns. In present study SCD gene was screened for polymorphic sites in Nili Ravi buffalo
breed. Total 31 SNPs were found in exonic, intronic and UTR region. In this study we identified a
new set of SNP useful for association studies.
Keywords: Polymorphisms, SCD Gene, Nili Ravi, Buffalo
INTRODUCTION
Bovine database of candidate genes and genetic markers for different traits have been
developed to provide an integral research tool to study lactation. The database contains 943 genes
and genetic markers involved in mammary gland development and function (Ogorevc et al., 2009)
but polymorphism in stearoyl coenzyme A desaturase (SCD) gene and dairy traits such as milk
production have already tested in several independent studies.
It is reported by Mele et al. (2007) that single nucleotide polymorphism in SCD gene is
associated with significant influence on the milk fat composition in dairy cattle. Stearoyl-Coenzyme
A desaturase is an important lipogenic enzyme that catalyzes the biosynthesis of monounsaturated
fatty acids (MUFA) from their saturated counterparts as well as the synthesis of conjugated linoleic
acid (CLA) in mammary gland and adipose tissues of ruminant animals (Ntambi et al., 2002). CLA
has received wide attention in recent years because of its potential in protecting against cancer,
atherogenesis and diabetes (Dhiman et al., 2005).
Keeping in view the importance of this gene, research work was planned to identify the
polymorphism in SCD gene in Nili-Ravi buffalo breed of Pakistan.

Unrelated animals with typical phenotypic features known for a given breed were selected
from several respective breeding tracts and from the Government livestock farms. Ten ml blood
was collected aseptically from the Jugular vein of each selected animals into 50 ml Falcon tubes
containing 200 µl anticoagulant i.e. Ethylenediamine tetra-acetic acid (0.5 M EDTA).
Organic extraction method by Phenol-Chloroform-Isoamylalcohol (PCI) was performed
on each of the blood sample. Primers were designed (GeneBank Accession no. AY241932) by
using software Primer3 (http://frodo.wi.mit.edu/). Amplication of these primers was done using
Polymerase Chain Reaction. After the precipitation of PCR, products were sequenced. Sequencing
was done on the principle of Sanger Chain Termination.

710
All the sequences were aligned with the help of software blast2sequence (Tatusova et al.,
1999). Single Nucleotide Polymorphism (SNPs) was detected from the aligned sequences.

Using the sequencing approach, analysis of SCD gene, in Nili Ravi buffalo breed of Pakistan,
revealed 31 polymorphisms. Seventeen SNPs in exonic, eight in intronic and six SNPs in 5’UTR
region were reported. SNP ID, change in nucleotide, transition/ transversion, exonic/ intronic
bifurcation are presented in table-1.
These SNPs can be associated with different traits in buffalo specially milk fat% and milk fat
yield. Lock and Gransworthy (2002) reported that concentration of unsaturated fatty acids in milk
fat may be affected by the activity of SCD, which suggested that SCD genotype can also affect
CLA concentration in fat of milk. The SCD gene is a key component in the leptin-signaling
pathway and it is most important leptin-regulated gene (Cohen et al., 2002). Taniguchi et al. (2004)
studied the full length of bovine SCD gene and found three single nucleotide polymorphisms
(SNPs). In 2006, Man et al. reported that stearoyl-coenzyme A desaturase (SCD is involved in
triglycerides (monounsaturated fatty acids) synthesis by confocal microscopy,
coimmunoprecipitation, and fluorescence energy transfer using HeLa cells. Alim et al. 2012
reported that stearoly-CoA desaturase is a multifunctional complex enzyme which is important in
biosynthesis of fatty acids. Another study indicated that SNP of SCD gene is associated with
myristoleic acid concentration in milk (Maharani et al., 2012). The stearoly CoA desaturase (SCD1)
and Diacylglycerol-acyl transferase 1(DGAT1) genes in cattle are involved in endogenous synthesis
of Intramuscular fat which is key parameter for evaluation of nutritional quality of beef (Wu et al.,
2012).
The results suggest that SCD is a candidate gene that influences milk production traits, and
provides potentially useful information for in dairy breeding programs. The identified
polymorphisms could be potential genetic markers to improve the production performance of
buffalo breed of Pakistan.
REFERENCES
Alim M. A., Y. P. Fan, X.P. Wu, Y. Xie, Y. Zhang, S.L. Zhang, D. X. Sun, Y. Zhang, Q. Zhang, L.
Liu and G. Guo. 2012. Genetic effects of Stearoyl-coenzyme A desaturase (SCD)
polymorphism on milk production traits in Chinese dairy population. Mol. Biol. Rep. 39:
8733-8740.
Dhiman T. R., S.H. Nam and A.L. Ure. 2005. Factors affecting conjugated linoleic acid content in
milk and meat. Critical reviews in Food science and Nutrition. 45: 463-482.
Lock A. L and P. C. Gransworthy. 2002. Independent effects of dietary linoleic and linolenic fatty
acids on the conjugated linoleic acid content of cow’s milk. Anim. Sci. 74: 163-176.
Maharani D., Y. Jung, W.Y. Jung, C. Jo, S.H. Ryoo, S.H. Lee, S.H. Yeon and J.H. Lee. 2012.
Association of five candidate genes with fatty acid composition in Korean cattle. Mol Biol
Rep 39: 6113-6121.
Mele M., G. Conte, B. Castiglioni, Chessa, N. P. Macciottta, A. Serra, A. Buccioni, G. Pagnacco
and P. Secchiari. 2007. Stearoyl coenzyme A desaturase gene polymorphism and milk fatty
acid composition in Italian Holstein. J Dairy Sci. 90(9):4458-4465.
Ntambi J. M., M. Miyazaki, J. P. Stoehr, H. Lan, C. Kendziorski, B. S. Yandel, Y. Song, P. Cohen,
J. M. Friedman and A. D. Attie. 2002. Loss of stearoyl coenzyme A desaturase-1 function
protects mice against adiposity. PNAS. 99:11482-11486.
Ogorevc, J., T. Kunej, A. Razpet and P. Dovc. 2009. Database of cattle candidate genes markers for
milk production and mastitis. Animal Genetics. 40: 832-851
Taniguchi, M., T. Utsugi, K. Oyama, H. Mannen, M. Kobayashi, Y. Tanable, A. Ogino and S.
Tsuji. 2004. Genotype of stearoyl coA desaturase is associated with fatty acid composition
in Japanese black cattle. Mamm. Genome 15: 142-148.
711
Tatusova, T. A., I. K. Mizrachi and J.A. Ostell. 1999. Complete genomes in WWW Entrez: data
representation and analysis. Bioinformatics. 15:536-543.
Man, W.C., M. Miyazaki, K. Chu and J. Ntambi. 2006. Colocalization of SCD1 and DGAT2:
implying preference for endogenous monounsaturated fatty acids in triglycerides synthesis.
J Lipid Res. 47(9):1928-39
Wu, X. X., Z. P. Yang, X. K. Shi, J. Y. Li, D. J. Ji, Y. J. Mao, L. L. Chang and H. J. Gao. 2012.
Association of SCD1 and DGAT1 SNPs with the intramuscular fat traits in Chinese
Simmental cattle and their distribution in eight Chinese cattle breeds. Mol. Biol Rep. 39:
1065-1071.
712
Table 1. Polymorphic sites detected in the SCD1 gene.
Sr. SNP ID Query Change in Transition/ Exonic/

N0. No. nucleotide Transversion Intronic
1 SCD1 1908 T→C Transition 5’UTR

2 SCD 2 1916 C→G Transversion 5’UTR
3 SCD 3 2014 T→C Transition 5’UTR
4 SCD 4 2015 G→T Transversion 5’UTR
5 SCD 5 2020 C→G Transversion 5’UTR
6 SCD 6 2096 C→T Transition 5’UTR
7 SCD 7 2648 G→A Transition Exonic
8 SCD 8 2689 T→C Transition Exonic
9 SCD 9 2779 C→T Transition Exonic
10 SCD 10 2844 A→C Transversion Exonic
11 SCD 11 2873 T→C Transition Intronic
12 SCD 12 6596 G→C Transversion Intronic
13 SCD 13 6631 T→C Transition Intronic
14 SCD 14 6689 C→A Transversion Intronic
17 SCD 17 6855 G→A Transition Intronic
18 SCD 18 6858 G→A Transition Intronic
19 SCD 19 8287 C→T Transition Intronic
22 SCD 22 8453 A→T Transversion Exonic
26 SCD 26 10080 C→G Transversion Intronic
27 SCD 27 10153 A→G Transition Exonic
713
Genetic Parameter Estimates for Milk Yield and Lactation Length in Buffalo
Yenny GARCÍAa*, Luis M. FRAGAa, Humberto TONHATIb, Daniel ABREUb, Raúl

ASPILCUETAb, Arelis HERNÁNDEZa, Eulogio PADRÓNc, Gladys GUZMÁNa, Marta
MORAa and Dayron QUIÑONEZa
a
Institut of Animal Science. Carr. Central km 471/2. San José de las Lajas. Havana province, Cuba
b
Faculdade de CiênciasAgrárias e Veterinárias, Universidade Estadual Paulista, Jaboticabal
(UNESP), Brasil
c
Genetic Enterprise “El Cangre”. Carr. “El Cangre” km. 6½ Güines, Havana province, Cuba
*
Corresponding email: ygorta@ica.co.cu
ABSTRACT
The objective of this study was to estimate genetic parameters for milk yield at 244 days and
lactation length in graded buffalo cows at the “El Cangre Cattle Genetic Enterprise. Data were
gathered from 2575 lactations, 1377 buffalo cows, 37 milking units and between 2002- 2009
calving years. It was employed the Restricted Maximum Likelihood method (REML) for
estimating (co)variance components with multi trait model. Average of milk yield at 244 days and
lactation length were 864 kg and 240 days, respectively. Heritability was 0.15 for milk yield and
0.13 for lactation length. Genetic correlation between these traits was 0.63. It was concluded that it
is necessary to intensify selection and to increase control of the information of the genetic herds to
obtain high precision in the estimates and therefore, obtain bigger genetic progress in of this species
in our country.
Keywords: buffalo, genetic parameters, milk yield
INTRODUCTION
The bubalinoculture, is positioned at the moment like a promissory cattle alternative under
the difficult conditions of economic crisis of countries of Latin America and the Caribbean (Patiño,
2009). In the last decade, diverse countries have carried out actions to develope selection programs
with the purpose of identify and disseminate into herds, tested individuals as superiors in
characteristics of economic importance (Rosati and Van Vleck, 2002; Tonhati et al., 2006).
It is important to have present heritabilities of different characteristics to select and by this way,
maximize the perspective answer. For example, milk yield is characterized to present low to
medium heritability estimates (0.14 to 0.30) (Hurtado-Lugo et al., 2006; Tonhati et al., 2008;
Malhado et al., 2009; Aspilcueta-Borquis et al., 2010; Rodríguez et al., 2010).
In our country has not been yet carried studies on genetic parameters estimation in
buffaloes. Therefore the objective of this work was to estimate genetic parameters for milk yield
(MY244) and lactation length (LL) in buffalo cows in Cuba.

The study was developed at “El Cangre” Genetic Enterprise in Mayabeque. Data was
gathered from 2575 first lactations records, 1377 buffalo graded cows, 37 milking units and 2002
to 2009 calving years. For data proccesing was used SAS (Statistical Analysis System) (2007). In
model to parameter estimated were included the effects resulting significant (P <0,05) when proc
MIXED was initially evaluated.
It was employed Restricted Maximum Likelihood method (REML) for estimating (co)
variance components with a multi trait model, developed inside the remlf90 (Misztal 2001).
Variance components were estimated by using animal model for milk yield and lactation length.
Model included in both traits evaluated, as fixed effect: contemporary groups and random direct

714
additive and residual effect. Contemporary groups were defined by herd, year of test day and test
semester (1: January-June and 2: July-December).
The animal model can be represented as:
y = Xβ + Zµ + ε
Where: y is the vector of observations (MY244 and LL); β is the vector of fixed effects; µ is the
vector of additive genetic random effects; X and Z are incidence matrices relating to β and µ,
respectively; ε is the vector of residual effects.

Number of observations, means, standard deviation and coefficients of variation of the milk
yield and length lactation are shown in the table 1. A high variability was observed according to the
deviations standard and the variation coefficients. Mean for Milk yield was 864±345 kg this value
was superior to that informed by other authors in Bufallypso graded in our country (Fraga et al.,
2007; Méndez & Fraga, 2009; García et al., 2010). However, lower than those referred by various
authors in other breeds and countries: Murrah in Brazil (Ramos et al., 2006; Tonhati et al., 2008;
Malhado et al., 2009; Aspilcueta et al., 2010). in Italy, in the Mediterranean breed (Rosati and Van
Vleck, 2002) and in Colombia with buffalo graded cows (Hurtado-Lugo et al., 2006).These results
could be influenced by differences in genetic quality of animals, as a consequence of advanced
programs of genetic improvement and carried out in those countries for decades. Mean observed for
lactation length was 240±51 days, similar result was informed by Mitat (2008) and Méndez & Fraga
(2009) in this same genotype and lower to the informed in Brazil and Italy (Rosati and Van Vleck
2002; Aspilcueta et al., 2010). Heritability for milk yield was 0.15±0.04. Similar results were found
in bovine of different breeds by Gonzales-Peña (2006) and Hernández et al. (2011) with estimates
of 0.15 in both cases. Greater values of heritabilities were obtained by Tonhati et al. (2008) and
Rodríguez et al. (2010) with 0.24 and 0.25, respectively and similar to the informed by Hurtado-
Lugo et al. (2006) and Malhado et al. (2009) of 0.16 and 0.17, respectively with graded buffalo in
Colombia and with the Murrah breed in Brazil. Results found in this study can be a consequence of
the low level of genetic selection carried out on buffalo cows, since during the years exploiting this
species in the country, the primary objective has been the herd growth. It is necessary to intensify
the selection and to increase control of information on breeding herds to obtain greater precision on
parameter estimations and afterwards, to obtain better genetic progress into this species in Cuba.
For length lactation heritability estimation was of 0.13±0.06, which was superior to that found by
Hernández et al. (2011) of 0.05 in cattle in our country and that of Malhado et al. (2009) and
Rodríguez et al. (2010) of 0.08 in both cases in buffaloes in Brazil, as well as, inferior to studies
carried out at the India by Taylon and Jain (1987) whose reported a heritability estimate of 0,26 for
this trait. Genetic correlation among these traits trait was 0.63, value considered as high and
positive it was inferior to that observed by Tonhati et al. (2008) and Rodríguez et al. (2010) of 0,90
and 0,96, respectively. Selection for milk yield would propitiate the increase of the lactation
duration.
REFERENCES
Aspilcueta, R., A.B. Bignardi, L. Seno, G. Camargo, M. Muñoz-Berrocal, L. Albuquerque, R. Di
Palo and H. Tonhati. 2010. Genetic parameters for milk yield analyzed by test-day models in
Murrah buffaloes in Brazil. Italian J Anim Sci. 9(34):179-182.
Fraga, L.M., Gutiérrez, Maritza, O. Fundora, Mora, Marta, González and E. María. 2007.
Resultados preliminares de la capacidad de producción lechera del búfalo de río (Bufalipso) en
una unidad con ordeño mecánico. Revista Cubana de Ciencia Agrícola 41(2): 131-133.
García, Y., L.M. Fraga, E. Padrón, N. Rodríguez, A. Alcides, G. Guzmán and M. Mora. 2010.
Genetic Studies on Water Buffalo Species (Bubalus bubalis) in Cuba. 9th World Buffalo
Congress, Argentina.
715
González-Peña, D. 2006. Evaluación genética del ganado Siboney de Cuba empleando la

producción en el día de control bajo un modelo de regresión aleatoria. Tesis para optar por el
grado de Doctor en Ciencias Veterinarias. Centro de Investigación para el Mejoramiento
Animal, Ministerio de la Agricultura, Cuba.
Hernández, A., R. Ponce de León. S.M. García, G. Guzmán and M. Mora. 2011. Evaluación
genética del bovino lechero Mambí de Cuba. Revista Cubana de Ciencia Agrícola 45(4): 355-
359.
Hurtado-Lugo, N., M.F. Cerón-Muñoz and A. Gutiérrez-Valencia. 2006. Estimación de parámetros
genéticos para la producción de leche en el día del control en búfalos de la Costa Atlántica
Colombiana. LRRD. (In press).
Malhado, C.H., A. Ramos, P. Carneiro, D. Azevedo, P. Affonso, D. Pereira and J. Souza. 2009.
Estimativas de parâmetros genéticos para características reprodutivas e produtivas de búfalas
mestiças no Brasil. Rev. Bras. Saúde Prod. An. 10(4): 830-839.
Méndez, M. and L.M. Fraga. 2009. Factores no genéticos en la producción lechera de las búfalas
Bubalus bubalis en la provincia Granma, Cuba. Revista Cubana de Ciencia Agrícola.
43(4):239-244.
Mitat, A. 2008. La producción de leche en el día de control para la selección de búfalas en Cuba.
Tesis presentada en opción al grado científico de Doctor en Ciencia Veterinarias, IIPP and
CIMA, Ciudad de la Habana, Cuba.
Patiño, E. and Maria. 2009. Leche de búfala versus leche de vaca. Perulactea 211(Abstr.).
Ramos, A.A., C.H. Malhado, P.L.S. Carneiro, J.C. Souza, A. Piccinin and F. Ferraz. 2006.
Distribuição uni e bivariada da produção deleite e do intervalo de partos em bubalinosda raça
murrah no Brasil. Memorias. In: Proceeding of III Simposio Búfalos de las Américas,
Medellín, Colombia. pp. 27-39.
Rodrigues A.E., J.R.F. Marques, C.V. Araújo and R.N.C. Camargo. 2010. Estimação de
parâmetros genéticos para características produtivas em búfalos na Amazônia Oriental. Arq.
Bras. Med. Vet. Zootec. 62: 712-717.
Rosati, A. and L.D. Van Vleck. 2002. Estimation of genetic parameters for milk, fat, protein and
mozzarella cheese production in the Italian river buffalo Bubalus bubalis population. Livestock
Production Science 74: 185-190.
SAS. 2007. SAS User’s guide: Statistics. Version 9.1.3. SAS Institute, INC, Cary, N.C., USA.
Taylor, S.P. and L.S. Jain. 1987. Genetic studies on production traits medium sized buffaloes.
Indian J. Anim. Sci. 57: 771-774.
Tonhati H, M. Cerón-Muñoz, A.J. de Oliveira, L. El Faro, A.L. Ferreira Lima and L. Galvão de
Albuquerque. 2008. Test-day milk yield as a selection criterion for dairy buffaloes (Bubalus
bubalis Artiodactyla, Bovidae). Genet. Mol. Biol. 31(3):674-679.
Tonhati, H., G. Mendoza, R. Sesana and S.A.A. Alburquerque. 2006. Programa de mejoramiento
del búfalo lechero en Brasil Memorias. In: Proceeding of III Simposio Búfalos de las
Américas. Medellín, Colombia. pp.123-130.
Table 1. Number of observations, means (kg), standard error and variation coefficient (%) of the
evaluated productive characteristics.
Traits # observations Means(kg) SE CV (%)

MY244 2575 860.2 345.3 31.9
LL 2575 240 51 18.9
716
Genetic Parameters and Trends for Weaning Weight and Calving Interval
of Department of Livestock Development Swamp Buffalo
Nikorn SANGHUAYPHRAIa*, Sansak NAKAVISUTa, Chayut DONGPALETUMb,
Goonlaphat PHOTHIKANITc and Sirisook SUPANUNc
a
Bureau of Animal Husbandry and Genetic Improvement, Department of Livestock Development,
Ministry of Agriculture and Cooperatives, Bangkok 10400, Thailand
b
Phayao Research and Testing Station, Phayao 56000, Thailand
c
Lamphayaklang Livestock Breeding and Research Center, Lopburee 15190, Thailand
*Corresponding email: nikornasa@hotmail.com
ABSTRACT
The purpose of this study was to estimate the variance components and genetic parameters
for prediction of breeding values and genetic trends of weaning weight (WW, 8 months) and
calving interval (CI) in DLD swamp buffalo. The variance components and genetic parameters were
estimated by using single trait mixed animal model analysis from 4,950 records of DLD swamp
buffaloes in 10 government breeding herds (1997 – 2011) by Restricted Maximum Likelihood
(REML) procedure. Breeding values were estimated by Best Linear Unbiased Prediction (BLUP)
method with BLUPF90-BeefPAC 2.4 program. Growth trait was weaning weight and fertility trait
was calving interval. The estimates of direct heritability (h2), maternal heritability (m2) and maternal
permanent environment effect (c2) of WW were 0.41, 0.18 and 0.12, respectively. The heritability
(h2) of CI was 0.08. Genetic trends of male and female WW traits were increased by 0.220 and
0.234 kg/year and CI trait was decreased by 0.57 days/year.
Keywords: variance components, genetic parameter, genetic trend, swamp buffalo
INTRODUCTION
Swamp buffalo has been raised as a part of Thai agriculture at small farmer level for draft
power and meat production. At present, buffalo population in Thailand is about 1.24 million
heads. 74 % of the buffalo in Thailand predominantly is distributed in the northeast of the country.
There are approximately 277,977 families raised buffalo and 85 % of those live in the northeast.
Buffalo provide less than 5 % of the farm power for small farmer (DLD, 2012). However, the
population has considerably decreased in number with a negative annual growth rate due to many
factors. The production system of swamp buffalo under the village householders is traditionally to
keep animals in their own-built housing in the backyard as a part of their family. Numbers of
buffalo per household is approximately 3.25 females. Buffalo has the capability of utilizing
agricultural waste products or low quality feed. Buffalo is typically fed on available native grasses,
crop residues, rice straw and rice stubble or any substantial sources of available roughage. When the
cropping season begins during rainy season, buffalo is normally tied up and fed only rice straw,
nevertheless growth can still be obtained. Most of the farmers do not keep buffalo bulls but rather
share them with their neighbors. Buffalo breeding in the village is generally random mating.
Artificial insemination is very limited due to the lack of accurate heat detection and the distances
from AI Centers (Virakul et al., 2006).
Genetic improvement program for Buffalo in Thailand was established by Department of
Livestock Development (DLD) in 1979. Selection based on growth performance testing program
was used as a tool to define the superior animals to establish elite herd. A closed nucleus herd has
been set up at Surin Livestock Breeding Center. Buffalo recording system has been started since
then. The growth data recorded are weights at birth, at 240 days old and at later ages of every six
months.
New technology of genetic evaluation has been used since 1996 when Australian Centre for
International Agricultural Research (ACIAR) funded a project in Thailand. The BREEDPLAN
717
program supported by Agricultural Business Research Institute, University of New England,

Australia has enabled genetic evaluation procedure. The historic data of the previous records about
the buffalo performance test program from Department of Livestock Development (DLD) breeding
stations were loaded into a customized database and analyzed in a full animal model using Best
Linear Unbiased Prediction methodology (BLUP). The estimated breeding values (EBV) of each
animal are being used as a part of the selection criteria for the elite breeding program for swamp
buffalo. The database is also used to maintain genetic diversity within the population of Thai
swamp buffalo and encourage in-situ genetic conservation of this species (Allen, 1998).
The purpose of this study was to estimate variance components and genetic parameters for
prediction of breeding values and genetic trends of weaning weight and calving interval in DLD
swamp buffaloes.

Data used in the study
The dataset of swamp buffalo used in this study was collected from 10 DLD breeding
stations and centers comprising 4,950 records from 1997 to 2011, where the farm management
practices followed the action plan and guideline of the bureau of animal husbandry and genetic
improvement, DLD. The records included pedigree information, body weight at 8 months of age
(WW, weaning weight) and calving interval (CI) as shown in Table 1.
Data Analysis
Variance component estimations was separately performed for each trait using Restricted
Maximum Likelihood procedure (REML; Patterson and Thompson, 1971) in BLUPF90-BeefPAC
2.4 (Duangjinda et al., 2005). Fixed effects fitted in the animal mixed model included sex,
contemporary group, weighing date, age of dam when giving birth. The estimated variance
components were used for calculation of direct heritability; h2, maternal heritability; m2, maternal
permanent environment ratio; c2 ( h 2  σ a ; m2   m ; c 2   c ), respectively.
2 2 2
σ 2p  p2  p2
y  Xb  Za  ZmWc  e
Statistical Models for analysis of each trait Mrode (1996) are as follows;
y  Xb  Za  e
(1 )
(2 )
where y = vector of observations, b = vector of fixed effects, a = vector of direct genetic
effects, m = vector of maternal genetic effects, c = vector of maternal permanent environmental
effects, e = vector of residual errors, X, Z,W = Incident matrix fixed, direct genetic and maternal
permanent environment effect,  a2 = additive variance,  m2 = maternal variance,  a m = additive and
maternal covariance,  c2 = maternal permanent environment variance,  e2 = residual variance or
error variance , 1 = WW and 2 = CI
Variance components estimated from this study were used to calculate genetic parameters
and breeding values as well as genetic trends of studied traits.

Estimation of genetic parameters for DLD swamp buffalo
The estimates of genetic parameters for weaning weigh h2, m2 and c2 were 0.41, 0.18 and
0.12, respectively. The heritability estimate of 0.41 from this study was higher than the estimate of
0.26 reported by Meyer et al. (2000). Results from this study indicated that weaning weight of
swamp buffaloes is under the control of the additive genetic effects inherited from their parents,
partly from the maternal genetic effects and permanent environmental effects of the dams.
Maternal effects involved maternal ability in terms of quality and quantity of milk available for
buffalo calves before weaning which is due to the genetic make-up of the dam and the permanent
environmental effect of the dam.
718
Heritability for calving interval was estimated at 0.08 in this study which was a slightly
higher than the estimate of 0.07 by Kongsook et al. (2006) and close to the value estimated in
Indian diary buffalos (0.07) reported by Dahama (1995).
The results clearly showed that higher level of additive genetic effect control on growth trait
than on the reproduction traits, therefore, the genetic improvement of growth traits can result in a
faster rate of response than that for reproduction traits. Fertility is of lowly heritable traits indicating
high effect of environment is involved in the traits. Genetic improvement can be achieved more
slowly from lowly heritable traits. However, the variability of the genetic parameter estimated in
different studies is probably due to the differences in population genetic and structure, environment
and variance component estimation procedure (Sivarajasingam et al., 1998).
Genetic trends of DLD swamp buffaloes
Weaning weight of DLD swamp buffaloes had a clear tendency to linearly increase with the
estimate rates of 0.22 kg per in males and 0.23 kg per year in females (Figure 1). Genetic selection
based on estimated breeding values (EBV) is suitable. However, to obtain accurate EBV with high
accuracy appropriate phenotypic data recording is necessarily required.
Calving interval is one of the fertility traits that have low heritability which was estimated at
8% in this study. Slow progress in genetic improvement of this trait can be expected despite the
necessity to genetically improve the trait from selection based on EBV from current generation to
the next. Figure 2 shows the genetic improvement of calving interval with a decrease averaged at
0.57 days per year in the calving interval trait.
Genetic improvement of Thai swamp buffalo program has been set up since 1977, where
selection of the replacing animals was made through growth performance. Buffalo calves which had
high growth rate were selected from the herds and tested at central testing station. Selection was
made through their performances to establish the elite herds. The trends of genetic responses have
substantial increased according to the replacement buffaloes were selected by estimated breeding
values from BLUP animal model analysis. That approach has led to the increased likelihood of co-
selection of related animals. Increases of genetic responses of all traits were because of the intense
selection of the small number of parents in the current generation and maintenance of genetic
variation of the herd. In addition, the changes in the structure of breeding program such as the
reduction of generation interval by shortening the time use of sires and dams in breeding herd as
well as increasing selection intensity.
Moreover, proper management plan, proper feeding, early weaning can help maintain good
health of buffalo dams and recover quickly after birth, reduce time from birth to estrous and have
high conception rates contributing to reduction of calving interval.
REFERENCES
Allen, J. 1998. Breedplan version 4.1 explanation (21 June). ABRI. Armidale, NSW, Australia.
Department of Livestock Development (DLD). 2012. Statistical Yearbook, Bangkok,
Thailand. http://www.dld.go.th/ict/yearly/yearly43/yearly43.html. Accessed in 2
December12.
Duangjinda, M., I. Misztal and S. Tsuruta. 2005. BlupF90–PCPAK version 2.5 Khon Kaen
University and the University of Georgia.
Dahama, R.S. 1995. Genetic Analysis of Reproductive Traits in Buffaloes. Ind. J.of Dai. Sci..
48:317-322.
Kongsook, S., C. Chocksawat and A. Na-Chiangmai. 2006. Genetic parameter of fertility traits
of Thai swamp buffalo. Proceedings of Animal Breeding and Farm Management, Animal
Husbandry Division, Department of Livestock Development. Bangkok, Thailand. 183-189.
Meyer, K., H.U. Graser and A. Na-Chiangmai. 2000. Estimates of genetic parameters for
growth and skeletal measurements in Thai swamp buffalo. J. Anim. Sci. 70:399-406.
Mrode, R.A. 1996. Linear Model for the Prediction of Animal Breeding Values. CAB
International, Wallingford UK.
Sivarajasingam, S., B. Kinghorn and J. Van Der Werf. 1998. Animal Breeding and Genetics for
719
the Tropics. Handbook for course held in conjunction with the 6th World Congress on
Genetic Applied to Livestock Production. University of New England, NSW.
Virakul, P., C. Lohachit, M. Techakumphu, S. Sirivaidyapong, T. Tharasanit, O. Cheunsuang.
2006. Conception gestation andparturition in swamp buffalo. In: International Course of
Buffalo Reproduction andReproductive Biotechnology. 3rd ed. Chulalongkorn University.
Bangkok. 62-67.
720
Table 1. Data Characteristics of DLD swamp buffalo.
Traits N Means Std

weaning weight(8 month), WW (kg) 4,950 166.12 33.49
calving interval, CI (day) 772 534 107
Table 2. Variance Component and genetic parameters on weaning weight and calving interval
traits of DLD swamp buffalo using REML.
Variance Component and genetic parameters

Traits
σ2 a σ2m σam σ2 c σ2 e σ2 p h2 m2 c2
WW 308 135 -150 94.6 295 742.6 0.41 0.18 0.12
CI 1000 - - - 11282 12182 0.08 - -
721
EBV (Kg)
4
y = 0.22x + 0.2373
3.5 R² = 0.795
2.5 y = 0.2337x - 0.1818

R² = 0.8938
2
1.5
WW(male)
1 WW(female)
Linear (WW(male))
0.5
Linear (WW(female))
0
-0.5
year
Figure 1. Genetic trend of weaning weight of DLD swamp buffalo in 1997 – 2011.
EBV (day)
0.00
-2.00
-4.00
-6.00
y = -0.5662x - 2.052
-8.00 R² = 0.8003
CI
Linear (CI)
-10.00
-12.00
-14.00
year
Figure 2. Genetic trend of calving interval of DLD swamp buffalo in 1997 – 2011.
722
Genetic Polymorphism in Caspase Activating Recruitment Domain 15 and its

Association with Incidence of Mastitis in Murrah Buffalo
Indrasen CHAUHAN, Archana VERMA*, Ishwar Dayal GUPTA and Gokul S. SONAWANE
Dairy Cattle Breeding Division, National Dairy Research Institute, Karnal-132001, India.
*Corresponding email: archana.ndri@gmail.com
ABSTRACT
Caspase Activating Recruitment Domain 15 (CARD15) gene is involved in innate immunity
and has been identified as one of the candidates for disease resistance. The gene has been mapped in
Bovine (BTA18), Human (HSA16) and Mouse (MMU8). Bovine CARD15 gene spans ~30 kb and is
comprised of 12 exons, 11 of which are coding. Present study was undertaken with the objectives to
identify polymorphism in CARD15 gene coding region and to analyze association between genetic
variants with incidence of mastitis in Murrah buffalo. Genomic DNA isolated from whole blood of 150
lactating Murrah buffaloes was amplified using fifteen sets of the primers to cover complete coding
region of the gene and the amplicons were subjected to RFLP analysis using BsaH1, PvuII and SacI
restriction enzymes (REs). BsaHI-RFLP revealed polymorphism in the 824 bp fragment of primer 12.3
exhibiting AA, AB and BB genotypes. SacI RFLP exhibited AB and BB genotypes for primers 1 and
12.1, whereas monomorphic BB genotype for primer 4.1. RFLP patterns using PvuII RE exhibited BB
genotype for contig 2, AB and BB for contig 4.2, 4.3 and 5-6 and AA, AB and BB genotypes for contig
12.3. The frequencies for A and B alleles varied from 0.04 to 0.64 and 0.36 to 0.96 respectively
indicating B allele to be more prevalent in the population studied. Statistical analysis indicate a
significant association between incidence of mastitis and genotypes of P12.3-PvuII (P<0.06). Buffaloes
with AA genotype were with lower incidence of mastitis in comparison to those with AB and BB
genotypes.
Keywords: CARD 15, PCR-RFLP, Mastitis, Murrah buffalo
INTRODUCTION
Bovine Mastitis is the most common and devastating disease of dairy animals caused due to
invasion of the mammary gland by several species of bacteria resulting in an inflammatory response. In
response to pathogen infiltration, the vertebrate immune system has evolved multiple defense systems,
which can be broadly classified into innate and adaptive immunity, to repel and kill the invasive
microbe. The innate immune response is the first line of defense against invading organisms and is
directly related to acute and chronic inflammation of the mammary gland (Rainard and Riollet, 2006)
The immunocytes have receptors called Pattern Recognition Receptors (PRRs), which recognize
specific Pathogen Associated Molecular Patterns (PAMPs). The interaction between PRR and PAMP
stimulates extra cellular complement pathway as well as intracellular signaling pathways culminating
in inflammatory responses. Pattern recognition receptors such as CD14, toll-like receptor (TLR) 4, and
caspase-recruitment domain (CARD) 15 bind bacterial lipopolysaccharide and peptidoglycan, and their
activation leads to production of inflammatory cytokines. Caspase Activating recruitment Domain15
(CARD15) gene is involved in innate immunity and has been identified as one of the candidate gene
for disease resistance.
Bovine CARD15 gene is located on BTA18, spans ~30 kb and is comprised of 12 exons, 11 of
which are coding. Association of polymorphic variants of CARD 15 gene with diseases like Crohn’s
disease, Blau syndrome and Early-Onset Sarcoidosis (Hugot et al., 2001; Kanazawa et al., 2005) have
been reported in human. But studies in animals are scanty. Taylor (2004) has identified thirty-six single

723
nucleotide polymorphisms (SNPs) including 26 within the gene transcript and Pant et al. (2007) have
reported four SNPs including one in intron 10 and three in the exon 12 , which have been associated
with increased Somatic Cell Score (SCS) in Holstein Friesian. However, no such study has been carried
out in buffaloes. Therefore, present study aimed to explore polymorphism and to associate genetic
variants of CARD 15 gene with incidence of mastitis in Indian Murrah buffalo.

DNA extraction
One hundred and fifty randomly selected lactating Murrah buffaloes maintained at the cattle
yard of National Dairy Research institute, Karnal, Haryana have been included in the present study.
Ten ml of whole blood from jugular vein were collected in sodium EDTA vacuutainer tubes. Genomic
DNA was extracted from blood samples by phenol chloroform method (Sambrook and Russell, 2001)
and stored at -20ºC. Quality of the DNA was checked by agarose gel electrophoresis and the quantity
was estimated by UV spectrophotometer method by reading absorbance at A260/A280 nm.
PCR amplification
Fifteen sets of forward and reverse gene-specific oligonucleotide primers were designed using
primer 3 software based on the Bos taurus sequence available in the NCBI-Genbank Accession No:
AY518748.1. The sequence of oligonucleotide primers their respective annealing temperature and PCR
product size are listed in the Table 1. The PCR amplification was performed in programmed Thermal
cycler (PTC 200 MJ Research) in a final volume of 25 µl, which contains 3 µl genomic DNA (50ng/
µl), 0.6 µl of each primers (100 pM/ µl), 0.025 µl MgCl2 (15mM), 0.5 µl d NTPs(10mM), 2.5 µl of
10x buffer, 0.25µl of Taq DNA polymerase (5U/µl, Invitrogen, USA). The PCR reaction mixture was
incubated in thermal cycler initially at 95º C for 5 minutes followed by 34 cycles of 95ºC for 30
seconds, specific annealing temperature of each primer (Table 1) for 30 seconds, 72ºC for 30 seconds
and a final extension of 72ºC for 10 minutes. The amplified PCR products were checked by
electrophoresis on 1.5% agarose gel in 1xTBE along with 100 bp DNA marker and ethidium bromide
(1µl/100ml). staining. The amplified products were visualized, size estimated and documented under
Gel Documentation system.
PCR-RFLP
Amplicons were subjected to restriction digestion to identify polymorphism. BsaH1, PvuII and
SacI restriction enzymes (REs) were selected using NEB cutter V 2.0 by submitting Bos taurus
reference sequences (Accession number AY 518748.1). The RE digestion was set up with10 µl PCR
product, 2 µl buffer and 0.3l SacI (10U/µl) or 0.21l PvuII (20U/ µl) or BsaHI (10U/ µl ) and HPLC
water to make up the reaction volume to 20 µl and the mixture was incubated at 37 ºC for four hours.
The restriction fragments were resolved on 2.5% agarose gel, visualized in UV Transilluminator and
photographed with gel documentation system (DNR Minibis, Israel). The band patterns were scored to
assign specific genotype viz. homozygote AA, BB and heterozygote AB and the respective genotypic
and gene frequencies were calculated. The variants obtained by PCR-RFLP were also custom
sequenced (Chromous biotech, India) and multiple sequence alignment was done with Bio-Edit
software.
Association analysis with incidence of mastitis
For carrying out association studies, the data on incidence of mastitis in Murrah buffaloes were
recorded from the treatment register of Animal Health Complex, NDRI, Karnal for a period of 9 years
(2003 to 2011). The animals were grouped as mastitis affected (once or more than once) and never
affected at least upto fourth lactation. Within each group animals were assigned CARD15 genotypes
obtained by PCR-RFLP analysis. Association of gene variants with mastitis affected animals and not
affected animals were calculated using χ2 test (Snedecor and Cochran, 1994).
724

Fifteen sets of primers yielded overlapping products, which were custom sequenced. The
nucleotide sequence data were submitted to NCBI Genbank and obtained Accession no. HQ652504 for
the complete coding sequences of CARD15 gene of Murrah buffalo.
CARD15-BsaHI RFLP analysis revealed monomorphic patterns for P2, P4.1 and polymorphism
in P12.3, BB genotype being the most frequent and AB, the least. Other contigs did not reveal
restriction site with BsaHI enzyme. PvuII had restriction site for six primers of CARD 15 gene. P2 and
P4.3 exhibited monomorphism with BB genotypes, whereas P4.1, P4.2, P5.6 and P12.3 exhibited
polymorphism having AB and BB genotypes. Furthermore, the homozygote occurred with higher
frequency than the heterozygote. As expected the B allele was more frequent as compared to A allele.
In case of P12.3 –PvuII three types of genotypes were differentiated. It was the only primer in which
heterozygote had the highest frequency. Also the A allele had the higher frequency than B allele. SacI
revealed restriction sites in P1, P4.1 and P12.1.In P4.1 exhibited monomorphic patterns. P1 and P12.1
revealed AB and BB genotypes. The BB genotype proportioned higher than AB genotype. The
frequencies for A and B alleles in varied from 0.04 to 0.64 and 0.36 to 0.96 respectively indicating B
allele to be more prevalent in the population studied (Table 2).
Statistical analysis indicate a significant association between incidence of mastitis and
genotypes of P12.3-PvuII (P<0.06). It was further explored that BB genotype 69.2% affected animals
followed by AB (63.1%) and least affected are exhibited by AA genotype (44.6%), where overall
56.67% of the animals have suffered from mastitis at least once in their productive life. Thus it can be
concluded that buffaloes with AA genotypes were less susceptible to mastitis as compared to those
with AB and BB genotypes. There is no any report available in the literature pertaining to PCR-RFLP
analysis of CARD15 gene in Murrah buffaloes or cattle to discuss the genotypes, genotypic and allelic
frequencies. However, some reports in cattle are worth mentioning here.
Taylor (2004) was the first to explore association of this gene with any disease (Johne’s disease
or paratuberculosis) in cattle. But no association was found between CARD15 and Johne’s disease
attributable to small sample size. This association study was replicated by Pinedo et al. (2009) in a
breed panel consisting of Holstein, Jersey and Brahman–Angus crosses by using three single
nucleotide polymorphisms (E2[-32], 2197/C733R and 3020/Q1007L) earlier reported by Taylor et al.
(2006). Pant et al. (2007) reported significant association of a SNP 3020A>T with mastitis, somatic cell
score, supporting the role of bovine NOD2/CARD15 gene in susceptibility to infections and production
traits milk yield, protein yield in Canadian Holsteins. In the same study, another SNP 4500A>C was
reported to be associated with milk yield, fat yield and protein yield. Soumya (2011) reported
polymorphic patterns using SacI and PvuII-RFLP in Sahiwal cattle but association with incidence of
mastitis was non-significant. Since present study revealed significant association between PvuII-RFLP
for contig 12.3 and incidence of clinical mastitis, therefore, it is proposed to validate this finding
further and utilize this information for marker assisted selection of buffaloes for less susceptiblity to
mastitis.
REFERENCES
Hugot, J.P., H. Zouali and S. Lesage. 2003. Lessons to be learned from the NOD2 gene in Crohn’s
disease. Eur. J. Gastroenterol Hepatol. 15: 593-597.
Kanazawa, N., I. Okafuji, N. Kambe, R. Nishikomori, M. Nakata-Hizume, S. Nagai, A. Fuji, T. Yuasa,
A. Manki, Y. Sakurai, M. Nakajima, H. Kobayashi, T.D. Kanneganti, M. Lamkanfi, and G.
Nunez. 2007. Intracellular NOD-like receptors in host defense and disease. Immunity 27: 549-
559.
725
Pant, S.D., F.S. Schenkel, I. Leyva-Baca, B.S. Sharma and N.A. Karrow. 2007. Identification of single
nucleotide polymorphisms in bovine CARD15 and their associations with health and production
traits in Canadian Holsteins. BMC Genomics 8: 421.
Pinedo, P.J., C.D. Buergelt, G.A. Donovan, P. Melendez, L. Morel, R. Wud, T.Y. Langaee and D.O.
Rae. 2009. Association between CARD15/NOD2 gene polymorphisms and paratuberculosis
infection in cattle. Veterinary Microbiology 134:346–352.
Rainard, P. and C. Riollet. 2006. Innate immunity of the bovine mammary gland. Veterinary Research
37:369–400.
Sambrook, J. and D. W. Russell. 2001. Preparation and analysis of eukaryotic DNA. In: Molecular
Cloning: A Laboratory Manual. 3rd Edition. Cold Spring Harbor Laboratory Press, New York:
6.1-6.62.
Soumya, N.P. 2011. CARD15 gene polymorphism and its association with mastitis in Sahiwal cattle.
Ph.D. Thesis, Deemed University, India.
Snedecor, G.W. and W.G. Cochran. 1994. Statistical methods. 8th Ed. Oxford and IBH Publication,
Kolkata.
Taylor, K.H. 2004. Genetic analyses of bovine CARD15, a putative disease resistance gene. Ph.D.
Thesis, the Office of Graduate Studies of Texas A&M University.
Taylor, K., J. Taylor, S. White and J.E. Womack. 2006. Identification of genetic variation and putative
regulatory regions in bovine CARD15. Mammalian Genome 17: 892–901.
726
Table 1. Sequence of the primers designed for amplification of twelve exons of CARD 15 gene of
Indian Murrah Buffalo.
Primer* Sequence Ta(ºC) Product Size (bp)

P1 F 5’-TGGAGTTCCTCTTCAGTTCC-3’ 53
252
R 5’-ACACTGTGTGCTGTCACCTC-3’
P2 F 5’-CAGACTTGCCCTCTCCACTA-3’ 52
500
R 5’-CAGTTTCGTACTGGCTGATG-3’
P3 F 5’-GTGTTCACTTCACCACCTGA-3’
52 246
R 5’-GTAGAAGAAAGGCAGCCAAC-3’
P4.1 F 5’-GGTCCCATTTTCACATGGT-3’
57.7 782
R 5’-GCTTCCTCA GGT ACA GTT CG-3’
P4.2 F 5’-GAA CTCAGC CTC AAG GGC TTCT-3’
62.7 705
R 5’-CCC TGTGAT CTG GAG GTT GTGC-3’
P4.3 F 5’-CATCTCTTCCAAGATCACAGG-3’
53 600
R 5’-ATCACCCAGCATCACTCA-3’
P5-6 F 5’-CTCATTGGCTCTGTGTTCAG-3’
53.5 459
R 5’-CGAAAGTCTCCAGGAGTAGG-3
P7 F 5’-GCCCTCTGTCTCAGTCTCTC-3’
53 209
R 5’-GGCTGAAGAATTGGTTTCAC-3’
P8 F 5’-GTGTCTCGAACACAAAGCAG-3’
52 162
R 5’-GGACGAACTCTGAGGCTTAC-3’
P9 F 5’-CATTTTGCCCTTCTTGAGTT-3’
52 247
R 5’-ACACACACATCAGCTTCCAC-3’
P10 F 5’-CAGTCAACTGGGTTTTCTCC-3’
53 179
R 5’-CAGAAACATCCCCTCTCAAG-3’
P11 F 5’-AAAACCAAGCATCCTCAAAG-3’
53 211
R 5’-AGTTCCTCTCTCCCTTCACC-3’
P12.1 F 5’-CTCTGGTCCAGTCCTCACTC-3’
53 810
R 5’-AGTTGCTGGGCAGTCATATT-3’
P12.2 F 5’-GCTACAACTCACCCTGCTCT-3’
52 786
R 5’-CTGAAGCCAAATGTGTTTTG-3’
P12.3 F 5’-TGTGACCAAACACAATGAAG-3’
53 824
R 5’-AAGGACCACAGAGACCAGAC-3’
* Primers have been designed using Primer 3 software
727
Table 2. Genotypic and Allelic Frequencies of CARD15 PCR-RFLP in Murrah Buffaloes*.

Primer-RE Genotypic Frequency Allelic Frequency
combination
AA AB BB A B
P1-SacI 0.00 (00) 0.31 (47) 0.69 (103) 0.17 0.83
P4.1-PvuII 0.00 (00) 0.29 (44) 0.71 (106) 0.14 0.86
P4.2-PvuII 0.00 (00) 0.36 (54) 0.64 (96) 0.18 0.82
P4.3PvuII 0.00 (00) 0.09 (14) 0.91 (136) 0.04 0.96
P5.6-PvuII 0.00 (00) 0.22 (34) 0.78 (116) 0.11 0.89
P12.1-SacI 0.00 (00) 0.12 (19) 0.88 (131) 0.06 0.94
P12.3-BsaHI 0.11 (16) 0.05 (08) 0.84 (126) 0.14 0.86
P12.3-PvuII 0.37 (56) 0.54 (81) 0.09 (13) 0.64 0.36

*Number of animals exhibiting a particular genotype have been indicated in parenthesis
728
Genetic Variability in Production and Immune Function Genes Associated with

Production Traits and Incidence of Mastitis in Indian Murrah Buffalo
Archana VERMA*, Ishwar Dayal GUPTA and Ravinder Singh GANDHI
Dairy Cattle Breeding Division, National Dairy Research Institute, Karnal-132001, India.
*Corresponding email: archana4wbc2013@gmail.com
ABSTRACT
Identification of genes of economically important traits and analysis of their polymorphic variants,
whose products are responsible for a particular phenotype, may play a key role in assessing the genetic
potential for selection of an animal. The polymorphic variants of several genes have been shown to
affect the production, reproduction performance and disease resistance in dairy animals. This paper
elaborates the detection of genetic polymorphisms in such candidate genes like bovine growth
hormone (bGH), kappa casein(CSN3), beta-lactoglobulin(b-lg), Pit-1, leptin (LEP), lactoferrin(Lf),
interleukin 8(IL8), toll-like receptor 4 (TLR4) and establish their association with milk production, fat
and protein %, body weight gain, conception rate and incidence of mastitis. Genomic DNA was
extracted from whole blood of 150 Murrah buffalo females and amplicons were obtained using
specific primers for the genes. Characterization of specific genes was done by custom DNA
sequencing. Polymorphism was detected by subjecting the amplicons to PCR-SSCP (LEP and Lf) and
PCR-RFLP (bGH, CSN3, b-lg, Pit-1, LEP, IL 8, TLR 4 with a battery of restriction enzymes) analysis.
DNA sequences of each gene variant were subjected to Clustal W alignment and variations observed,
which revealed a varying degree of variability exhibiting monomorphic as well as polymorphic
patterns. Statistical analysis carried out to assess the level of significance for the association of genetic
variation with production performance and disease resistance. Since buffalo is less explored specie
from molecular genetics point of view, combining information on phenotypic data with molecular data
could be the most powerful tool marker assisted selection (MAS) for breed improvement.
Keywords: DNA Polymorphism, Murrah buffalo, production, disease resistance genes


729
Genotyping and Molecular Characterization of NRAMP1/-2 Genes as Location of

Markers for Resistance and/or Susceptibility to Mycobacterium bovis in Swamp
and Riverine Type Water Buffaloes
Claro N. MINGALA*, Lawrence P. BELOTINDOS, Nancy S. ABES and Libertado C. CRUZ
Philippine Carabao Center National Headquarter and Gene Pool, Science City of Munoz 3120, Nueva
Ecija, Philippines
*Corresponding email: cnmingala@hotmail.com
ABSTRACT
Natural resistance-associated macrophage proteins (NRAMPs) have been associated to disease
resistance across animal species. It has critical role in innate immunity and influence in adaptive
immunity. This study investigated the contribution of NRAMP1 and NRAMP2 gene to the resistance or
susceptibility of swamp and riverine buffalo to Mycobacterium bovis infection. Animals were tested
for TB by single intradermal tuberculin test (SITT) using Bovine antigen. Reactors to SITT were
subjected to comparative intradermal tuberculin test (CITT). Blood samples were collected from the
reactors then subjected to DNA extraction to isolate the NRAMP1 and NRAMP2 genes. Isolated
NRAMP genes were then examined by single strand conformational polymorphism (SSCP) assay. The
3’UTR were sequenced and then aligned. The SSCP result showed that among the reactor animals to
intradermal tuberculin test, four conformational patterns were observed in the 3’UTR of the NRAMP1
gene while two were observed in the 3’UTR of the NRAMP2 gene. SSCP showed that the frequency
of four-band pattern were mostly from the reactor animals (66.41%). Sequence alignment clearly
established the nucleotide polymorphisms between the conformational patterns. These polymorphisms
suggested as a potential markers for resistance or susceptibility to Mycobacterium infection. Allelic
patterns will be very useful in future breeding plan for the selection of resistant animals.
Keywords: NRAMP, Water buffalo, Mycobacterium, Disease resistance
INTRODUCTION
Natural resistance-associated macrophage proteins (NRAMPs) have been associated to disease
resistance across animal species. It has critical role in innate immunity and influence in adaptive
immunity. NRAMP1/-2 genes were isolated from a murine BCG candidate gene and were designated
as natural-resistance associated macrophage protein gene. These genes were found during genetic
studies of mice to mediate antimicrobial activity of macrophages against intracellular parasites early
during infection. NRAMP1/-2 genes have been recognized as important for their role in infection
resistance in several other species, in addition to mice. NRAMP1/-2 homologues, variants and
polymorphisms have also been investigated in humans and bovines.
Based on previous studies, there are particular sequences of NRAMPs which correlate with
resistance or susceptibility to various diseases such as Brucella abortus (Borriello et al., 2006),
Mycobacterium sp. (Bradley et al., 1979; Gros et al., 1981) and Salmonella sp. (Lissner et al., 1983) in
cattle. Considering the potential application of a suitable genetic marker for resistance in buffaloes
against these various diseases and the scarcity of studies supporting the use of the polymorphisms at
the 3’UTR (GT)n microsatellite as resistance markers, the aim of this study is to investigate the
frequency of NRAMP alleles in swamp and riverine buffaloes and to determine the role of NRAMP
genes in resistance and susceptibility to infection with Mycobacterium bovis.

730

Intradermal tuberculin tests
Single intradermal tuberculin test (SITT) was performed on each animal by injecting 0.1ml of
bovine tuberculin purified protein derivative (PPD) using a standard tuberculin syringe. Prior to
inoculation into the right caudal fold, skin thickness was measured with 0.5 mm precision calipers.
After 72 h, fold thickness was again measured, and the increase was recorded. In all the tests, an
increase of greater than 3mm was considered positive.
After 60 days, all reactor animals to SITT were subjected to comparative intradermal tuberculin
test (CITT) using both avian and bovine purified protein derivatives (PPD). Intradermal injections of
0.1 mL (20000 IU/ mL) bovine PPD and 0.1 mL (20000 IU/ mL) avian PPD were administered
separately in each caudal fold, after having measured and recorded the fold thickness with the caliper.
Caudal fold thickness was measured again at both injection sites after 72 hrs. The reaction at each site
was derived by measuring the difference of the fold thickness before and 72 hrs. after the injection. An
animal was considered reactor to CITT if the difference between the bovine and the avian reaction was
greater than 3 mm.
Blood sample collection and DNA extraction
A total of 1,486 water buffalo blood samples were collected from the CITT reactors and non-
reactors via the jugular vein. These blood samples were collected in various places in the Philippines.
Blood was collected in sterile tubes with anti-coagulant (lithium heparin) and maintained at 4oC until
arrival at the laboratory. DNA was extracted from whole blood samples using DNA extraction kit
(Promega, USA) according to the manufacturer’s instructions. The extracted DNA (50-100 µg/ml) was
stored at 4oC until used.
Amplification of NRAMP 1/-2 DNA
A part of the 3’UTR region of both NRAMP 1/-2 genes were amplified using two pairs of
primers designed specifically for each gene. NRAMP 1 forward primer was designed from coding
region to amplified the start of 3’UTR region:(NRAMP 1: F – 5’ AAG AGG ATC AGG AGA AGG
GGA 3’, R – 5’ CTG GAA CTC ACG TTG GCC A 3’; NRAMP 2: F – 5’ AGG TAG CCA TCA GAG
CCA GTG TGT – 3’, R – 5’ GGA GGA AGG GGA AGG TTT CTC CA – 3’). The forward primer for
NRAMP 1 was part of the gene coding region. Five µl of DNA (50-100 µg/ml) from each sample was
added to the PCR reaction mixture containing 10.3 μl DDW, 2.0 μl 10x PCR Buffer (10mM Tris-HCl
(pH9.0), Takara, Japan), 1.6 μl dNTPs (2.5 mM each, Takara, Japan), 0.5 μl each of 10 pmol of each
primers, 0.1 μl of 5U rTaq polymerase (Takara, Japan) and 5.0 μl of DNA (50-100 µg/ml) template.
Thermocycler was used to amplify the target gene with initial denaturation at 94oC for 30 sec,
34 cycles of denaturation step at 94oC for 30 sec, annealing step at 60oC for 45 sec, and extension step
at 72oC for 1 min and a final extension step at 72oC for 7 min. The amplified PCR products were
visualized in 2 % agarose gel and photographed under short-UV wave illumination. An approximately
180 bp product was predicted for NRAMP 1 and 784bp for NRAMP 2.
DNA Sequencing and Alignment Analyses
The amplified bands corresponding to the desired size of NRAMP 1 and NRAMP 2 were
excised from the gel and purified using the Wizard SV Gel and PCR Clean Up System (Promega,
USA). The purified DNA fragments were ligated into the pGEM-T easy vector (Promega, USA), and
transformed into a competent E. coli JM109. In each DNA analysis, 8–10 plasmid clones were
sequenced. Positive clones were selected for DNA sequencing which was done in Korea and Japan.
Nucleotide sequences were aligned and analyzed using the online program MultAlin (Corpet, 1988) for
multiple sequence alignment.
731
RESULTS
Intradermal tuberculin test in water buffaloes
Out of 1,486 water buffaloes tested, 208 reacted to bovine antigen SITT (14.00%) and 10
reacted to bovine-avian antigens CITT (0.67%). From the 208 animals reacted to bovine antigen, 190
were riverine buffaloes and 18 were swamp buffaloes, these reactors to bovine tuberculin test were
used for genotyping and molecular characterization of NRAMP 1/-2 genes as location of markers for
resistance and/or susceptibility to Mycobacterium bovis in swamp and riverine water buffaloes.
Molecular Detection of NRAMP 1/-2 Gene and Single-Stranded Conformational Polymorphism

(SSCP) Analysis among Water Buffalo Reactors to SITT and CITT
From the 190 riverine buffalo reactors, 131 contain NRAMP 1 gene (92.26%) while 32 contain
NRAMP 2 gene (15.35%). All swamp buffaloes exhibited NRAMP 1/-2 genes. Water buffaloes that
were positive to NRAMP 1/-2 gene were further analyzed using SSCP analysis. Out of 131 riverine
buffalo reactors that positive to NRAMP 1, 106 were positive to SSCP; 2 had single band (1.53%), 15
had double bands (11.54%), 2 had triple bands (1.53%) and, 87 had four bands (66.41%). Riverine
buffalo reactors to CITT, one had double bands (11.11%) and 4 had four bands (44.44%). Most of
swamp buffalo reactors to bovine antigen that exhibited NRAMP 1 gene had four bands (17 out of 18)
while all NRAMP 2 genes of swamp buffaloes had double bands. The SSCP analysis frequency of
NRAMP 2 gene of riverine buffaloes showed that out of 32 riverine buffalo reactors to SITT, 19 had
single band (59.38%), three had double band (9.38%). Riverine buffalo reactors to CITT showed only
one with double (3.13%).
Nucleotide Sequence Alignment of NRAMP1/-2 UTR

The sequences of the four different conformational patterns generated by the SSCP of NRAMP
1 3’UTR (174bp) were aligned with other three outlgoup, Bubalus arnee (U27105.1), Bos Taurus
(NRAMP1: DQ848779.1; NRAMP2: NM_001101103.1) and Bison bison (U39614.1). Four
nucleotide polymorphisms were observed in the NRAMP 1 3’UTR four-band pattern among the other
four conformational patterns. On the other hand, two nucleotide polymorphisms were observed in the
NRAMP 1 3’UTR single-band pattern and one polymorphism in each of the two- and three-band
patterns. A six-nucleotide (GTGTGT) deletion was also observed among the water buffalo NRAMP 1
3’UTR sequences at position 120 – 125 compared to B. arnee and B. taurus. However, this deletion
was similar to that of the B. bison sequence. The sequences of the two different conformational
patterns of NRAMP 2 3’UTR (779 bp) were aligned with Bos taurus sequence as the outgroup. Forty
nucleotide polymorphisms were observed between the two conformational patterns. Nucleotide
deletions appeared in two sites of the sequence, position 299-302 with two nucleotides deletion and in
position 757 with one nucleotide deletion. These deletions were in comparison to B. taurus as the
outgroup.
DISCUSSION
The present study was performed to investigate a probable contribution of NRAMP 1 and
NRAMP 2 genes in response to Mycobacterium infection particularly in water buffaloes. The relative
differences in singled-stranded DNA migrations revealed mutations and/or insertion among water
buffaloes reactor to tuberculin skin test. This provides the first evidence of the genetic variability of
the NRAMP 3’UTR region within the swamp and riverine buffaloes breeds. The SSCP patterns clearly
showed that polymorphism in the 3’UTR region may act as an enhancer or silencer for the defense
mechanism of Brucella abortus intracellular pathogens (Ranja et al., 2011; Tuggle, et al., 2005;
Felmlee et al., 1995). The immune function and the production performance had also been shown (Wu
et al., 2008).
732
Polymorphism of swamp and riverine buffalo NRAMP 3’UTR region as a result of the SSCP
analysis may correlate to resistant or susceptible of buffaloes to pathogens. This point mutation or
insertion can be used further as SNP markers which could be helpful to breeders for future association
studies, selecting superior germplasm and conservation strategies. The study will augment the
information available and will be useful in further studies to determine the role of the NRAMP gene in
disease resistance and for selection of bovine tuberculosis resistant animals that may be useful for
establishing a possible association with productive parameters.
The demonstration of obvious occurrence of polymorphisms in the NRAMP 1 and NRAMP 2
3’UTR regions suggests transcription differences in immune function to either resistance/tolerance or
susceptibility to Mycobacterium infection. Further studies are suggested to elucidate the influence of
these polymorphisms discriminating a recovered to the currently infected hosts.
The result of this study supports the assessment of these polymorphisms as potential markers
for resistance or susceptibility to Mycobacterium infection. Therefore, the findings about the allelic
patterns comparing the reactor and non-reactor water buffaloes may be very useful in future breeding
plan for the selection of resistant animals.
REFERENCES
Borriello, G., R. Capparelli, M. Bianco, D. Fenizia, F. Alfano, F. Capuano, D. Ercolini, A. Parisi, S.
Roperto and D. Iannelli. 2006. Genetic resistance to Brucella abortus in the water buffalo
(Bubalus bubalis). Infect. Immun. 74: 2115-2120.
Borriello, G., R. Capparelli, M. Bianco, D. Fenizia, F. Alfano, F. Capuano, D. Ercolini, A. Parisi, S.
Roperto and D. Iannelli. 2006. Genetic resistance to Brucella abortus in the water buffalo
(Bubalus bubalis). Infect. Immun. 74: 2115-2120.
Bradley, D.J., B.A. Taylor, J. Blackwell, E.P. Evans and J. Freeman. 1979. Regulation of Leishmania
populations within the host. III. Mapping of the locus controlling susceptibility to visceral
leishmaniasis in the mouse. Clin. Exp. Immunol. 37: 7-14.
Corpet, F. 1988. Multiple sequence alignment with hierarchical clustering. Nucl. Acids Res. 16:
10881-10890.
Felmlee, T.A., Q. Liu, A.C. Whelen, D. Williams, S.S. Sommer and D.H. Persing. 1995. Genotypic
detection of Mycobacterium tuberculosis rifampin resistance: comparison of single-strand
conformation polymorphism and dideoxy fingerprinting. J. Clin. Microbiol. 33: 1617-1623.
Gros, P., E. Skamene and A. Forget. 1981. Genetic control of natural resistance to Mycobacterium
bovis (BCG) in mice. J. Immunol. 127: 2417-2421.
Lissner, C.R., R.N. Swanson and A.D. O'Brien. 1983. Genetic control of the innate resistance of mice
to Salmonella typhimurium: expression of the Ity gene in peritoneal and splenic macrophages
isolated in vitro. J. Immunol. 131: 3006-3013.
Ranjan, R., C.D. Bhong, K.N. Chavan, S.N.S. Parmar and C.G. Joshi. 2011. Polymorphism in 3’UTR
region of Slc11a1 gene in Indian breeds of cattle. Vet. Arhiv. 81: 349-357.
Tuggle, C.K., L. Marklund, T.J. Stable, M.A. Mellencamp and A. Stumbaugh. 2005. Genetic markers
for screening animals for improved disease resistance (NRAMP). United States Patent,
6844159B2. http://ddr.nal.usda.gov/handle/10113/6983.
Wu, H., D. Cheng and L. Wang. 2008. Association of polymorphism of Nramp1 gene with immune
function and production performance of large white pig. J. Genet. Genomics 35: 91-98.
733
Buffalo Genetic Improvement Programme in Nepal-Current Status and Future

Prospects
Bhola Shankar SHRESTHAa, Lok Nath PAUDELb, Netra Pradad OSTIc and Babu Kaji
PANTAd
a
Bovine Research Programme, NARC, Nepal
b
Livestock Production Directorate, DLS, Nepal
c
Animal Nutrition Division, NARC, Nepal
d
National Dairy Development Board, Nepal
*Corresponding email: bsshrestha@yahoo.com
ABSTRACT
Livestock is an integral and important component of mixed farming system in Nepal.
Buffalo is the most important livestock species contributing highest share in the livestock sector
GDP. Buffalo alone contributes for 70% and 60% of national annual milk and meat production
respectively. Nearly half of the households of the country keep buffaloes primarily for milk and
meat and also for manure, hide, traction and ploughing agricultural land. Three native buffalo
breeds, all riverine type have been identified and their production performance been documented.
Selective breeding for genetic improvement of native breed in some potential pockets have been
recommended owing to the variability in the performance of indigenous buffaloes. Murrah is the
important buffalo breed introduced from India for upgrading indigenous buffaloes and that has led
to crossbred buffalo population in the major dairy pocket areas of the country. Considering the
importance of buffaloes, the Department of Livestock Services and Nepal Agricultural Research
Council have jointly initiated the Buffalo Genetic Improvement Project and is being implemented in
10 districts along with 2 government buffalo farms since mid of 2010. Around 800 farm families
owning buffaloes have been registered with around 1800 milking buffaloes for Pedigree and
Performance Recording Scheme (PPRS). Records on monthly milk yield, milk content analysis,
breeding and reproduction and health are being regularly taken and data are being managed on
Microsoft Access Database specifically developed for this purpose. The process and status of
implementation, challenges faced and way forward for genetic improvement of buffaloes in the
country have been discussed in this paper.
Keywords: Buffaloes, Genetic improvement, Murrah, Pedigree and Performance Recording

Scheme
INTRODUCTION
Buffalo is the most important livestock species among domesticated animals in Nepal in
terms of its contribution in national meat and milk production, share in livestock sector gross
domestic production (LGDP) and food and nutrition security at household level. The species alone
produces about 70% and 60% of the national milk and meat production annually whereas
contributes around 53% in the LGDP of the country. Nearly half of the households in the country
keep buffaloes particularly for milk, meat, manure, traction power and hides. Most people in Nepal
prefer buffalo milk over cattle due to its appealing white color and richness in fat and solid not fat
(SNF) content which fetches higher price per liter of milk.
Nepal is one of the countries in Asia having highest livestock population density per unit of
cultivated land, but still the country is not self-sufficient in fulfilling its internal demand for
livestock products particularly meat and milk. Consequently live animals and livestock products

734
worth billions of rupees are being imported annually in the country. This is because of the
cumulative effects of poor genetic potential or inability to tap the existing potential of most of the
livestock species including buffaloes that further aggravated by the overall poor feeding, breeding
and health management. Thus, it has been felt extremely essential to have sustainable and systematic
buffalo genetic improvement programme to cater the national demand especially of milk and meat
in Nepal.
Buffalo Population Distribution and Trend in Nepal

The estimated buffalo population in the country is 4.99 million which are found across all
agro-ecological regions of the country (Table 1), though the distribution is highest in the hill region
(52.4% of the total population) and lowest in the mountain region (8.6%). Development region-
wise, the buffalo population is highest in central (25.6%) and western development regions (25.2%)
and lowest in far western development region (10.6%)
The district-wise buffalo population distribution is presented as Figure 1. Some districts have
buffalo population of more than 120,000 heads, whereas there are some mountain districts which
possess less than hundred heads. In general, districts in Terai and hill regions have more buffalo
population, whereas mountain districts have the least. Manang and Mustang districts, also known as
the districts beyond the Himalayas have almost no buffalo population.
During the last 10 years (2001-2011) period, both total buffalo population and milking
buffalo heads increased steadily (Figure 2). The average annual growth rate in total buffalo
population was 3.77 percent, and similar increment in the number of milking animals was also
observed. The proportion of milking buffaloes to total buffalo has been found to be 25.8%
Despite steady growth of buffalo population and total buffalo milk and meat production, the
productivity (milk and meat) has not been increased tangibly during the last decade (Figure 3 and 4).
Both milk and meat productivity remained more or less static during the same period. The absolute
increase in buffalo population is further putting pressure on limited feeding resources; hence
increasing the productivity of buffalo is even more important.
Buffalo Breeds
Three native buffalo breeds, namely Lime, Parkote and Gaddi have been identified and
characterized in Nepal. Gaddi buffaloes are localized in the far western hills of Nepal and somewhat
resembles Murrah breed, whereas Lime and Parkote are found scattered throughout the hills and
Terai region of the country. Lime buffaloes resemble to Carabao in phenotypic appearance, but it
has been confirmed as riverine type with 2n=50 chromosomes (Rasali et al., 1998a). The remaining
two Parkote and Gaddi are also riverine type. Due to indiscriminant breeding, intermediate between
Lime and Parkote are also found in the greater number. It has been also reported that population of
Gaddi buffalo is declining in recent days.
Indigenous buffaloes of Nepal have the ability to adapt across different agro-ecological
zones, exist in low plain of nutritional regime, possess efficient forage digestion ability and cold
tolerance, and have relatively smaller body size. Therefore, they are highly suitable to thrive on
narrow and steep slope of the Hills and Mountains of the country.
Murrah is the primary breed of buffalo introduced in the country to increase milk production
especially in the dairy pocket areas of the country. Both milking buffaloes and breeding bulls have
been introduced from India. Crossbred Murrah buffaloes (various blood levels) are found in low
hills and Terai regions particularly in the areas with milk marketing potentials. Government
breeding policy is to upgrade indigenous buffaloes without any restriction to Murrah blood level in
the Terai region. The coverage of AI in buffaloes is quite low (<5% of the total breedable
population) compared to AI in cattle, whereas distribution of Murrah breeding bulls have been the
major focus of the government for cross breeding. Besides Murrah, some Surti and Jafrabadi, both
735
Indian breeds have also been introduced in the country directly by the farmers, but the numbers are
very limited. Semen of Nili Ravi buffalo was also introduced once, but there virtually exist any Nili
Ravi crossbred buffaloes in the country. It has been estimated that around 1/4th of the total buffalo
population in the country are crossbreds of Murrah and a few of them are the pure Murrah (DLS,
2012).
Existing Buffalo Production System

The buffalo production in Nepal can be broadly categorized into following four systems:
1. Subsistence buffalo production across the country
2. Buffalo production with seasonal migration in the high hills and Terai
3. Smallholder dairying in peri urban/ urban areas
4. Semi/commercial buffalo production in major dairy pocket areas
Majority of the rural households keeping 1-2 milking buffaloes primarily for fulfilling the
household need for milk across the country comes under subsistence buffalo production. The surplus
milk is either converted into ghee (clarified butter) or sold in the villages if opportunity for selling
exists. Ghee is mostly home consumed but the surplus is sold in the nearby market. The buffaloes
under this system are mostly kept under stall feeding with cut and carry practices but grazing in the
roadside, nearby forest or fallow cropland is also practiced. The system is generally low input and
low output oriented.
In some parts of Terai, large number of indigenous buffaloes (about 25 or more) are owned
by some farmers, are kept under minimum inputs and seasonally migrated to other places from the
farmers’ homestead in search of feeding resources. Production level under this system is generally
poor. Similar system, but with less number of buffaloes kept in the high hill regions of the country
particularly in the mid and far western region are also seasonally migrated during wet summer
months.
In the peri-urban as well as some urban areas, smallholder buffalo keeping has largely
emerged in the country with opportunity to selling milk. Generally Murrah or crossbred buffaloes
are kept, number of milking animals per farmer varies from 1-5 and milk is sold either directly to
the consumers or to the dairy industries though milk producers’ cooperatives. Buffaloes under this
system are kept under stall feeding system, sometimes as mixed herd with cattle. Disposal of male
calves at early stage for saving milk is prevalent in some parts of the country. Furthermore, buying
buffaloes in early stage of lactation, keeping throughout the lactation period and selling after
cessation of lactation is practiced in urban areas under this system.
Medium to large scale commercial or semi commercial buffalo farming is gradually
emerging in the major dairy pocket areas, but such commercial buffalo herds are minimal as
compared to the emerging commercial large dairy cattle herd in the country.
Potential and Performances of Indigenous and Exotic Buffaloes

Lactation Performances
The lactation performances of indigenous buffaloes have been reported by several authors
particularly through performance recording from farmers’ herds particularly from western hills of
Nepal (Joshi et al., 1992; Rasali, 1998; Amatya et al., 2000; Shrestha et al., 2005; Paudel et al.,
2012) conducted at different years. The mean lactation yield reported by these authors varied from
874 to 1433 lit, but there has been no significant difference between yield of Lime and Parkote
buffaloes. Location variations within indigenous buffaloes have also been reported by Shrestha et al.
(2005) which has been further confirmed by study of Paudel et al. (2012). Within indigenous breeds,
the lactation yield varied greatly (Figure 5) and selective breeding within indigenous buffaloes for
genetic improvement have been recommended (Shrestha et al., 2005).
736
The milk yield and milk content of indigenous and crossbred buffaloes from two western hill
districts, namely Gulmi and Arghakhanchi has been presented in Table 2. The lactation milk yield of
Murrah crossbred has been found to be significantly (p<0.001) higher than those of yield of Lime
and Parkote buffaloes, whereas fat content of milk from indigenous Lime and Parkote buffaloes was
significantly higher compared to milk from Murrah crossbred (Paudel et al., 2012). The SNF content
of milk didn’t vary between breeds, but total solid content was again higher in the milk from
indigenous buffaloes compared to the milk from crossbred.
The average daily milk yield of Gaddi buffaloes, another indigenous buffalo breed localized
in far western hill districts, for the first three months after calving was 4.69 lit from two teats and
4.62 lit in next three months from three teats. The lactation length varied from 14 to 22 months
(Pokharel and Tiwary, 2007).
In another study, Rasali et al. (1998) had reported that the lactation yield of buffaloes
increased with the increasing level of Murrah blood. The Murrah crossbred buffaloes with 25-49%,
50-74% and >75% Murrah blood level produced 1013 lit, 1190 lit and 1440 lit of milk per lactation
respectively under farmers’ management condition.
The average lactation yield of Murrah buffaloes in Livestock Development Farm, a
government owned farm has been found to be 1543 lit in a standard lactation of 305 days. The
lactation performance of buffaloes in their 1st, 2nd, 3rd, 4th and 5th lactations were 1279±288 lit,
1556±318 lit, 1520±305 lit, 1562±283 lit and 1578±192 lit., respectively in 305 days (LDF,
2064/65).
In Chitwan (inner Terai), Kolachhapati et al., (1993) found the average lactation milk yield
of crossbred buffaloes to be 1927.95±76.618 lit in 10.42 months. Crossbred buffalo had nearly 50%
higher milk yield and the age at 1st calving was shorter (4.69±0.07 years) when compared to local
buffaloes (4.91±0.08 years).
In another study from eastern Terai region of Nepal with a limited number of animals under
recording, performances of Murrah/ crossbred buffaloes have been reported to be better (Table 3).
More than 60% of the animals under recording had lactation yield of more than 2100.0 lit
Reproductive Performance
The mean age at first calving of indigenous buffalo was 4.5 years which was not
significantly different between Lime and Parkote buffaloes (Shrestha et al., 2005) though it was
slightly higher (4.56 years) for Lime buffaloes as compared to that of Parkote (4.48 years) and
Murrah cross buffaloes (4.36 years). There was significant variation (P<0.001) in age at first calving
among buffaloes of different sites indicating influence of management differences. Significant effect
of altitude on age at first calving in buffalo has been reported by Rasali (1996). The age at first
calving in indigenous buffaloes has been reported to be 53.8 months under stall-feeding system and
59.7 moths under grazing management (Joshi et al., 2001). The authors have also reported that about
5% of the buffalo populations in the western hills calve for the first time within age of 40 months
and 75% within age of 59 months.
The average calving interval of buffaloes was 600 days (20 months). The calving interval
was neither significantly different for different buffalo breeds nor for the buffaloes at different sites.
The average calving interval of Murrah cross buffalo was shorter (545 days) as compared to those of
Lime (600 days) and Parkote (604 days) buffaloes (Shrestha et al., 2005).
Initiation of Buffalo Genetic Improvement Programme

To cater the increasing demand for milk in the formal market, government of Nepal adopted
the policy of cross breeding indigenous buffaloes with Murrah bulls. The Department of Livestock
Services through its network at district and village level initiated Murrah bull distribution
programme for cross breeding. The government also imported Murrah buffaloes from India whereas
inflow of these buffaloes also took place from the farmers initiatives from the open boarder. The
737
government policy has been upgrading of indigenous buffaloes to unrestricted blood level of Murrah
particularly in Terai and low hills with ample milk marketing opportunities. Government farms were
established as the nucleus farms to supply breeding bulls to the farming communities. However,
system of distributing breeding bulls of known genetic worth did not commence in the country till
the recent past, which actually represents half of the herd. External appearance and availability (only
the phenotypes of the animals) were the criteria for distributing breeding bulls. Thus the efforts lag
behind from the expected and potential improvement in buffalo productivity.
Later on, recording system particularly for indigenous and crossbred buffaloes at farmers
herd initiated from the research centers depicted the potentiality and variability with
recommendation for selective breeding within indigenous breeds in potential pockets for genetic
improvement. However, such intermittent recording could not lead to concrete buffalo genetic
improvement programme in the country.
Pedigree and Performance Recording Scheme (PPRS) for genetic improvement of dairy
cattle initiated jointly by Nepal Agricultural Research Council (NARC) and Department of
Livestock Services (DLS) in the major dairy pocket areas from July 2008 with technical and
financial assistance of FAO under Technical Cooperation Programme (TCP). The project has been
able to identify potential bull mothers with high estimated breeding values (EBVs) for milk and
valuable solid production. The male calves born from these potential bull mothers inseminated with
imported proven bull semen are being utilized for semen collection and wider scale use for
dissemination of genetic progress throughout the country.
Inspired by the positive impact of dairy cattle genetic improvement programme (DCIP), the
Department of Livestock Services in collaboration with NARC has started buffalo genetic
improvement programme (BGIP) for genetic improvement of buffaloes since July 2010.
Implementation Status of Buffalo Genetic Improvement Programme

The buffalo genetic improvement programme has been started in 10 districts (9 in Terai and
1 in hill region) along with two government owned farms. Series of discussion with stakeholders at
district level took place before selecting a total of 850 herds (farmers) registration for pedigree and
performance recording scheme (PPRS) under buffalo genetic improvement programme (herd
registration). All buffaloes under these registered herds have been uniquely identified with year tags
and information such as date of birth, sire and dam breeds and ID (if available) have been compiled
(base animal registration). In each cluster, data recorder and AI technicians have been identified and
assigned for the effective implementation of the PPRS. Milk analyzers, district supervisors and the
coordinators were also assigned in each district from the respective staff of the district livestock
services office (DLSO). These recorders, inseminators, lab technicians, supervisors and coordinators
have been intensively trained on data recording, inseminating, milk analyzing, reporting,
supervising and monitoring of the PPRS works.
The recorders collect monthly production records from each registered herds. Milk
production record is taken once in a month in the evening and next morning of each individual
lactating buffaloes. Along with yield, the recorders take composite 40ml milk sample (20ml in
evening and 20 ml in the morning) and send it with production record to the Lab where it is
analyzed for fat, protein, SNF content and conductivity by lacto-scan machine. The recorders also
take the record on events such as AI or natural services, calving, culling or death of animals,
diseases and treatment and movement of animals that have occurred during the past one month. The
monthly record format filled by the recorder and supplemented by the Lab with milk content
analysis is monthly sent to the Central Data Management Unit (CPMU) at Animal Breeding
Division, NARC. These data are managed in an access database specifically developed for this
purpose (Figure 6). Farmers are provided with the periodic feedback as well as immediately when
738
the conductivity of milk of individual buffalo crosses 6, informing the farmers that the animal might
have been infected with sub clinical mastitis and to take the necessary measures to control.
The data thus obtained are analyzed and buffaloes with highest estimated breeding values in
term of lactation milk production as well as total valuable solid (fat + protein) produced are
identified. These animals are inseminated with the imported frozen semen (imported from India) and
male calves born from these planned mating are envisioned as future breeding bulls that would be
extensively utilized for semen collection and wider scale dissemination.
Constraints for Buffalo Genetic Improvement Programme

Some of the constraints (managerial and technical) that are hindering the buffalo genetic
improvement programme in the country are:
1. Extremely small and scattered herd - increases recording cost
2. Disposal of buffaloes at the end of lactation
3. Lower AI success rate and low rate of adoption
4. Difficult to convince the farmers on importance of recording for genetic improvement
5. Early milking in the morning- recording inconvenience
6. Greater age at first calving and calving interval
7. Farmers’ perception- slow process
8. Limited manpower and infrastructure
9. Long term nature- lack of commitment from the related stakeholders
10. Discontinuity of work- selective breeding in indigenous buffaloes- though potential exists
not initiated properly
11. Lack of assurance of fund for long term project
Initiation of 'Improving Nutrition and Productivity of Buffaloes to Adapt to the Impacts of

Climate Change in Nepal'
Realizing the very high importance of buffaloes in Nepal but there exist some limitations for
the improvement of their production and productivity in the context of climate change, Michigan
State University (MSU), in collaboration with NARC, Agriculture and Forest University (AFU) and
DLS in Nepal has been conducting collaborative research on forage crop cultivation, feeding, and
reproductive management with the goal to enhance the productivity of buffaloes through improved
feeding and reproduction strategies and enhance the ability of smallholder buffalo farmers to adapt
to the consequences of climate change. The project has selected three pilot districts: Chitwan,
Tanahun and Gorkha of the Gandaki river basin (GRB). These research sites will serve as platforms
for applied research, farmer training, outreach and technology transfer. The project will collaborate
with the ongoing BGIP jointly funded by the Government of Nepal (GoN) and USAID. The main
objective of this collaborative research project is to study the impacts of climate change on forage
cultivation, feed and forage availability, and reproductive efficiency in buffaloes in Nepal.
The specific objectives of this project are listed follows:
1. Determine climate change characteristics in Nepal that specifically affect forage production,
quality, species composition, and current forage cultivation practices to develop improved
practices to increase production and productivity of forage crops in the face of climate change;
2. Determine existing buffalo rearing practices in the GRB region of Nepal including feeding,
breeding, housing, and animal health conditions, and record socio-economic and
demographic features of buffalo rearing households in relation to climate related threats;
3. Determine factors related to buffalo rearing in relation to vulnerability to climate change and
identify major risk factors for future consideration, focusing on the mid-hills of Nepal;
4. Develop low cost feeding packages for different growth stages of buffaloes using locally
available nonconventional feed stuffs, and
739
5. Develop reproduction management strategies to improve heat detection and AI techniques to

improve conception rates and reduce calving intervals.
It has been envisaged that this collaborative research support project in buffalo productivity
improvement will be a milestone in the changing context of global warming and climate change in
Nepal.
 Implementation of selective breeding programme for genetic improvement of indigenous

The Way Forward
buffaloes-PPRS need to be initiated in some potential pockets of indigenous buffaloes in the
 More specific research on buffalo productivity improvement in the context of climate change
country
 Medium to long term strategies need to be developed based on buffalo breeding policy
 Coordinated approach from all concerned stakeholders (research, extension, education, dairy
farmers’ cooperatives and dairy industries)
 Consolidated efforts for continuity of PPRS in buffalo genetic improvement
REFERENCES
ABD. 2065/66. Annual Report, Animal Breeding Division, NARC, Khumaltar, Lalitpur, Nepal.
Amatya, N., D.P. Rasali and R.S. Rana. 2000. Evaluation of phenotypic and production
characteristics of indigenous buffalo types in the western hills of Nepal. Lumle Technical
Paper No 2000/1. Kaski, Nepal. Agriculture Research Station, Lumle.
DLS. 2012. Annual Report, Department of Livestock Services, Harihar Bhawan, Lalitpur, Nepal.
FAOSTAT. 2013 at http://faostat.fao.org/site/573/default.aspx#ancor viewed on Jan 25th at 11: 50.
Joshi B.R., H.D. Joshi and B.S. Shrestha. 2001. Bovine infertility: Studies on normal fertility status
and the factors associated with bovine infertility in the hills of Nepal. Lumle Technical
Paper No. 2001/4. Kaski, Nepal. Agriculture Research Station, Lumle.
Joshi B.R., R.K. Kadariya, N.P.S Karki and D.B. Gurung. 1992. Milk production of local and 50%
Murrah crossbred buffaloes under farmers' traditional management in the western hills of
Nepal. Working Paper No. 92/16 Kaski, Nepal. Lumle Agriculture Research Center.
Kolachhapati, M. R., N. R. Devkota and D. B. Nepali. 1993. Performance Evaluation of Farmers'
Buffalo Herd in Chitwan, Nepal. J. Inst. Agric. Anim. Sci. 14: 97-101.
LDF. 2064/65. Annual Report, Livestock Development Farm, Pokhara, Kaski, Nepal
MoAD. 2012. Ministry of Agricultural Development Website at
http://www.moad.gov.np/content.php?id=234
Paudel, L.N., U.ter Meulen, C. wollny, H. Dahal and M. Gauly. 2012. Comparative analysis of the
production performance of indigenous and Murrah cross breeds of buffaloes in mid hills of
Nepal. In: Proceeding of the 6th National Animal Science Convention. Nepal Animal
Science Association. 25-26 Sept, 2011, pp 164-176.
Pokharel, P.K. and M.R. Tiwary. 2007. Gaddi Buffalo: An indigenous breed of far western Nepal.
Ital. J. Anim. Sci. 6(Suppl.2): 1230-1233.
Rasali, D.P. 1996. Effect of altitude, management system and parity on the production performance
of monsoon calving indigenous hill buffaloes under farmers' management in the western
hills. LARC Seminar Paper No. 96/28.Kaski, Nepal. Lumle Agriculture Research Center.
Rasali, D.P. 1998. Present status of indigenous buffalo genetic resources in the western hills of
Nepal. Proceeding of the Fourth Global Conference on Conservation of Domestic Animal
Genetic Resources. NARC and RBI. pp 165-167.
740
Rasali, D.P., D.B. Gurung and E.R Yadav. 1998b. Performance recording of lactating local and
crossbred cows and buffaloes of various exotic blood levels under farmers' management in
the western hills – 1995-97. LARC working paper No. 98/39.
Rasali, D.P., H.D. Joshi, R.K. Patel and A.H Harding. 1998a. Phenotypic clusters and karyotypes of
indigenous buffaloes in the western hills of Nepal. Lumle Technical Paper No. 98/2. Kaski,
Nepal; Agriculture Research Station, Lumle.
Shrestha, B.S., N. Amatya, R.M. Singh, P.K. Jha, B.R. Acharya and D.B. Gurung. 2005. Production
performances of indigenous buffaloes in the western hills of Nepal. Nepal J. Sci. Tech. 6:
121-127.
Table 1. Buffalo Population by Development and Ecological Regions (2010/11) of Nepal.
Ecological Development Regions

Regions Eastern Central Western Mid-western Far-western Total
Mountain 146,682 148,047 88 34,273 99,185 428,275
(8.6%)
Hills 427,286 644,691 934,358 404,927 205,464 2,616,726
(52.4%)
Terai 587,724 486,383 325,513 324,924 224,104 1,948,648
(39%)
Total 1,161,692 1,279,121 1,259,959 764,124 528,753 4,993,649
(23.3%) (25.6%) (25.2%) (15.3% (10.6%)
Source: (MOAC, 2012)
Table 2. Lactation performance of indigenous and crossbred buffaloes.
Breeds Lactation Yield Fat Content (%) SNF Content (%) Total Solid (%)
(mean± SE) (mean± SE) (mean± SE) (mean± SE)
Lime 1418.48 ± 45.56 8.04 ± 0.069 12.39 ± 0.077 20.57 ± 0.083
Parkote 1433.39 ± 45.98 8.17 ± 0.079 12.25 ± 0.082 20.48 ± 0.124
Murrah cross 1726.11 ± 45.89 6.83 ± 0.056 12.31 ± 0.056 19.20 ± 0.099
Source: Paudel et al., 2012
Table 3. Lactation Milk Yield of Murrah Buffaloes in Dhanusha District.
Lactation Yield Number of Buffaloes %age

>2400 lit 34 31.8%
>2100 and <2400 lit 34 31.8%
>1800 and <2100 lit 26 24.3%
<1800 lit 13 12.1%
Total 107 100%
Source: ABD (2065/66)
741
Figure 1. District wise buffalo population distribution.
Figure 2. Trend of total and milking buffaloes in last decade. (Source: FAOSTAT, 2013)
742
Figure 3. Annual buffalo milk production and Figure 4. Annual buffalo meat production
productivity. and productivity.
Source: FAOSTAT (2013)
Figure 5. Variation in the lactation performance of indigenous buffaloes. (Source: Shrestha et al.,
2005)
Figure 6. Data management system for buffalo genetic improvement programme in Nepal.
743
Optimization of the Transfection Efficiency of Buffalo Fetal Fibroblasts Cells

Using Liposome
Tingxian DENG, Chunying PANG, Yingyin ZHANG, Bingzhuang YANG, Xianwei LIANG *
* Corresponding email: liangbri@126.com
ABSTRACT
Transfection is the process of deliberately introducing nucleic acids into cell and it is a
powerful tool for gene function (gene expression and regulation, signaling pathway and drugs
screening), gene therapy and transgenic animal research, etc. This study was designed to explore the
transfection efficiency of buffalo fetal fibroblasts cells that provide a feasible method for getting a
large number of transgenetic buffalo cloning cells. In the study, the exogenous DNA was transfected
into Buffalo Fetal Fibroblasts using lipofectamine LTX. And then the impacts of liposome amount,
plasmid amount and size, transfection time course, initial plated density and different promoter used
on transfection efficiency were explored. It was indicated that 4 μg pmaxGFP plasmid DNA with 4
μL liposome resulted in the highest transfection efficiency and its Cell activity was up to 98% after
6 h transfection. In conclusion, these results will be used as a good foundation in production of
transgenic cloned buffalo.
Keywords: liposome, fibroblasts, transfection, Buffalo


744
The Construct of Fat-1 Gene Targeted Buffalo Kidney Fibroblast Cell
Yingyin ZHANG, Chunying PANG, Tingxian DENG, Bingzhuang YANG and Xianwei LIANG
* Corresponding author: liangbri@126.com
ABSTRACT
Transgenic animal models expressing polyunsaturated fatty acid genes have been widely used
to improve milk quality, which have some significant meaning for further promoting buffalo dairy
production. Previouse studies have shown that the ω-3 fatty acid desaturase gene (Fat1) could
coordinate the proportion of ω-6PUFAs/ω-3PUFAs to improve the contents of ω-3PUFAs, which
provide an effective method for promoting the buffalo dairy products quality. In this study, an
investigation designed to knock into the Fat-1 gene by multiple-locus gene targeting in buffalo
kidney fibroblast cells was performed. The buffalo multiple-locus gene targeting vector
pBC1-DS1-Fat1-EGFP-DS2 was successfully constructed. eGFP (enhanced Green Fluorescent
Protein, eGFP) expressing cells were obtained by electroporation, screened and purification by
cloning ring method The eGFP expressing cellswere confirmed by PCR and sequencing. After
growing into cell clones that will provide nucleus transplantation donator for Fat-1 gene targeted
somatic cloning buffalo.
Keywords: multiple-locus gene targeting, Fat-1, Kidney Fibroblast Cell, Buffalo

745
Multiple-trait genomic evaluation for milk yield and milk quality traits using
genomic and phenotypic data in buffalo in Brazil1
Humberto TONHATI1, Rusbel Raul ASPILCUETA-BORQUIS1, Ana Claudia de
FREITAS, Gregório Miguel Ferreira DE CAMARGO1, Fernando BALDI1
1
FAPESP, CAPES, CNPq– Financial support
1
Universityof São Paulo State- Departamento de Zootecnia da Faculdade de Ciências Agrárias e
Veterinárias, FCAV/UNESP, 14884-000 - Jaboticabal, SP, Brazil.
*Corresponding email: tonhati@fcav.unesp.br Tel/Fax: 551632092678
ABSTRACT
The use of markers distributed all long the genome may increase the accuracy of the
predicted additive genetic value of young animals that are candidates to be selected as
reproducers. In commercial herds, due to the cost of genotyping, only some animals are
genotyped and procedures, divided in two or three steps, are done in order to include these
genomic data in genetic evaluation. However, genomic evaluation may be calculated using one
unified step that combines phenotypic data, pedigree and genomics. The aim of the study was to
compare a multiple-trait model using only pedigree information with another using pedigree and
genomic data. In this study, 9,318 lactations from 3061 buffaloes were used, 384 buffaloes were
genotyped using a Illumina bovine chip (Illumina Infinium® bovineHD BeadChip). Seven traits
were analyzed milk yield (MY), fat yield (FY), protein yield (PY), lactose yield (LY), fat
percentage (F%), protein percentage (P%) and somatic cell score (SCSt). Two analyses were
done: one using phenotypic and pedigree information (matrix A) and in the other using a matrix
based in pedigree and genomic information (one step, matrix H). The (co)variance components
were estimated using multiple-trait analysis by Bayesian inference method, applying an animal
model, through Gibbs sampling. The model included the fixed effects of contemporary groups
(herd-year-calving season), number of milking (2 levels), and age of buffalo at calving as
(co)variable (quadratic and linear effect). The additive genetic, permanent environmental, and
residual effects were included as random effects in the model. The heritability estimates using
matrix A were 0.25, 0.22, 0.26, 0.17, 0.37, 0.42 and 0.26 and using matrix H were 0.25, 0.24,
0.26, 0.18, 0.38, 0.46 and 0.26 for MY, FY, PY, LY, %F, %P and SCCt, respectively. The
estimates of the additive genetic effect for the traits were similar in both analyses, but the
accuracy were bigger using matrix H (superior to 15% for traits studied). The heritability
estimates were moderated indicating genetic gain under selection. The use of genomic
information in the analyses increases the accuracy. It permits a better estimation of the additive
genetic value of the animals.
Keywords: accuracy, genomics, milk quality
INTRODUCTION
The animal breeding is done based on phenotypic and pedigree data. With the advances
in this area due to the informatics and statistics, unbiased estimators of the additive genetic
values of animals raised in different environments were developed, as well as, the possibility to
estimate a lot of parameters using a big quantity of data (Brito, 2011).
The introduction of molecular genetic techniques permits studies of identification,
characterization and genetic mapping. The use of molecular markers brings more security, speed
and efficiency to the studies. The use of these techniques with the methodologies of quantitative
genetics and conduction of segregating population will permit the creation and development of
new manners to estimate the additive genetic effect (Beredet et al, 1997).

746
The genome sequence of many species generated lots of information that allied to
methodology evolution permitted the identification of many molecular genetic markers. The
genomics advances, the genome sequence of many species, including cattle, and the high density
SNP mapping permitted the inclusion of genomic information in genetic evaluation programs
(Brito, 2011).
The genomic selection uses simultaneously thousand of molecular markers distributed all
long the genome. It will permit the increase in accuracy in the prediction of additive genetic
values of low heritability traits and/or traits in traits that are difficult to be measured. So, the aim
of the study was to compare the estimates of genetic parameters and accuracies using different
methodologies (pedigree matrix x pedigree+genomic matrix) for milk production traits in
buffaloes.

In the present study, 9,318 lactations from 3061 buffaloes daughters of 114 sires were
used. The buffaloes calved from 1996 to 2010 and were raised in 10 different herd from São
Paulo state, Brazil. For genomics analyses, 384 buffaloes were genotyped using the chip
Illumina Infinium® bovine HD Bead Chip.
Among the 688,593 bovine SNPs that were genotyped successfully, only 16,580 markers
were polymorphic and presented minor allele frequency greater than 0.05, enabling their use in
association studies and genomic selection. After filtering the data, the number of markers with
fixed alleles was 26,042 SNPs. There were a large number of markers with minor allele
frequency smaller than 0.05 (645,971 SNPs), not including fixed alleles. The call rate for all
samples, which indicates the quality of the genotyping, varied from 54 to 90% (mean: 85%).
Usually, with bovine samples, call rates greater than 98% are expected. The low call rate
obtained in the present study is likely an indicator that some animals had differences in
hybridization of the samples with the primers, thus leading to possible problems in amplifying
the markers. After, the markers obtained were related with the relative matrix.
The lactations were truncated at 305 days, because only 12% of the buffaloes had
superior lactation duration. The milk yield was obtained from the fifth lactation day. The first
record was considered until the 45th day after calving.
The trait studied were milk yield (MY), fat yield (FY), protein yield (PY), lactose yield
(LY), fat percentage (F%), protein percentage (P%) and somatic cell score (SCSt) per lactation.
The somatic cell count does not present a normal distribution, so it is transformed in a
logarithmic scale (SCSt), SCSt = (log2(CCS/100.000))+3, proposed by Dabdoub e Shook (1984).
The lactations with less than 90 days were not considered. The ages at calving varied
from 2 to 8 years. There are two calving seasons: one from October to March and the other from
April to September. The contemporary group was composed by herd-year-calving season, each
contemporary group need the have at least 4 animals. The pedigree archive has 13,644 animals in
relative matrix. The general data structure is presented in Table 1. In order to obtain the
(co)variance components a multiple-trait analysis , in a Bayesiana context, using GIBBS2F90
program (Misztal et al., 2011) in which is possible to use a relative-genomic matrix. In the
animal model, were considered the fixed effects of contemporary group, number of milkings, age
of the buffalo at calving (linear and quadratic effect) as covariable and random effect of additive
genetic, permanent environment and residual. The matrix notation is:
y  X  Za  Wp  e
Where β, a, p, e are the random effects for additive genetic, permanent environment and residual
respectively; X, Z and W are the incidence matrixes related to the random effects for additive
genetic and permanent environment respectively; “a priori” β uniform distribution was
747
established; for random effects, Gaussian distributions and for (co)variance components, it was
used an inverted Wishart distribution.
b  cons tan te a | G ~ NMV[0, (G  A*)]

p | P ~ NMV[0, ( P  I n )] G | S g , vg ~ IW[ S g vg , vg ]
P | S p , v p ~ IW[ S p v p , v p ] R | Sr , vr ~ IW[Sr vr , vr ]
residual and identity, respectively;  is the operator of the Kronecker product, where Sg and vg;
Where G, P, R, In are the (co)variance matrixes of the additive genetic, permanent environment,
Sp and vp; Sr and vr are the values “a priori” of freedom degree for (co)variances of additive
genetic, permanent environment and residual, respectively. The matrix A* is the relative matrix
done by pedigree (A) or matrix of animals with pedigree and genotyped animals (H).

The heritability estimates for the traits (Table 1) were similar for both models (matrix A
and matrix H). These estimates were similar the ones estimated by Aspilcueta et al (2010).
However, the standard deviations are lower when matrix H is used. It may be caused because of
the better structure of animals’ relation in relative matrix (Hayes e Goddard, 2010).
For all the traits, the accuracy of prediction of the additive genetic values was higher
when matrix H was used (Table 1). The use of a multiple-trait analyses, using matrix H, is
incremented by 16.67%, 15.52%, 18.97%, 16.39%, 17.74%, 20.97% and 21,05% for MY, FY,
LY, PY, G%, P% and SCSt respectively.
Table 1 – Means and standard deviations (SD) of the accuracies of prediction of the genetic
values and heritability estimates (h2) for milk yield (MY), fat yield (FY), protein yield (PY), lactose
yield (LY), fat percentage (F%), protein percentage (P%) and somatic cell score (SCSt) using matrix A
and H in buffaloes.
Traits Matrix A Matrix H
Mean ± SD h2 ± DP Mean ± DP h2 ± DP
MY 0.66 ± 0.16 0.25 ± 0.04 0.77 ± 0.09 0.25 ± 0.03
FY 0.58 ± 0.15 0.22 ± 0.08 0.67 ± 0.11 0.24 ± 0. 04
PY 0.58 ± 0.16 0.26 ± 0.09 0.69 ± 0.11 0.26 ± 0.05
LY 0.59 ± 0.15 0.17 ± 0.09 0.70 ± 0.10 0.18 ± 0.04
%F 0.61 ± 0.14 0.37 ± 0.09 0.71 ± 0.09 0.38 ± 0.04
%P 0.62 ± 0.13 0.42 ± 0.08 0.73 ± 0.09 0.46 ± 0.04
SCCt 0.57 ± 0.19 0.26 ± 0.09 0.69 ± 0.13 0.26 ± 0.05
Matrix A, estimates using relative matrix
Matrix H, estimates using matrix (pedigree+genomics)
In Table 2, the estimates for genetic correlations among the traits using matrix A and H are
presented. They were similar, except for somatic cells that are low, but in different direction.
Estimates of genetic correlations of SCSt in buffaloes are scarce. Aspilcueta et al. (2010) related
that genetic correlations are negative among milk yield, fat and protein percentage and SCSt.
However, studies with dairy cattle, the genetic correlation is low but positive among the milk
yield and fat and protein percentage with SCSt, similar to the ones found in this study when
genomic information is used. The selection to increase the milk yield, increase the somatic cells
count.
748
Table 2. Means of the “a posteriori” distributions of the genetic correlations for milk yield (MY),
fat yield (FY), protein yield (PY), lactose yield (LY), fat percentage (F%), protein percentage (P%) and
somatic cell score (SCSt) using matrix A (under the diagonal) and H (above the diagonal) in buffaloes.
MY FY PY LY %F %P SCCt
MY 0.78 0.94 0.66 -0.35 -0.16 0.10
FY 0.73 0.74 0.10 0.38 0.11 0.05
PY 0.93 0.64 0.08 0.23 0.42 0.09
LY 0.68 0.12 0.12 0.13 0.11 0.12
%F -0.37 0.39 0.21 0.11 0.33 0.18
%P -0.21 0.12 0.49 0.13 0.29 0.17
SCCt -0.10 -0.11 -0.12 -0.15 -0.08 -0.10
Matrix A. estimates using relative matrix
Matrix H. estimates using matrix (pedigree+genomics)
CONCLUSIONS
The heritability estimates for all the traits were moderated. It permits genetic gain under
selection. However, genetic correlations among milk yield and fat, protein percentage and
somatic cell score are not desired. The selection to increase milk yield, may reduce the others.
Selection index based on economic values are necessary to ponder the traits. The use of genomic
data increased the accuracy of heritability estimates and the accuracy of prediction of the genetic
values due to a better relative structure.
REFERENCES
Aspilcueta, R. B., F. R.Araujo Neto, F. Baldi, A. B. Bignardi, L. G. Albuquerque and H.
Tonhati. 2010a. Genetic parameters for buffalo milk yield and milk quality traits using
bayesian inference. J. DairySci.93:2195-2201.
Bered, F., J. F. Barbosa Neto and F. I. F. Carvalho. Marcadores moleculares e sua utilização no
melhoramento genético de plantas. Ciência Rural, Santa Maria, v.27, n.3, p. 513-520,
1997.
Brito, F. V. J. BrancciniNeto, M. Sargolzaei, J.A. Cobuci and F.S. Schenkel. Accuracy of
genomic selection in simulated populations mimicking the extent of linkage
disequilibrium in beef cattle. BMC Genetics, London, v.12, n. 80, 2011.Dabdoub, S. A.
M.; Shook, G. E. Phenotypic relations among milk yield, somatic count cells, and
mastitis.Journal of Dairy Science, v. 67, p. 163-164, suplemento 1, 1994.
Hayes, B., M. Goddard. Genome-wide association and genomic selection in animal
breeding.Genome, Ottawa, v.53, p. 876–883, 2010.
Misztal, I. and Aguilar, I.; Tsuruta, S.; Legarra, A. 2011. Using the BLUPF90 family of
programs for genomics.Disponível em: <http://nce.ads.uga.edu/~ignacy/genomic-
blupf90/>. Acessoem: 23 ago. 2012.
749
Preliminary Results on the Growth Performance of F1 Mediterranean Buffalo

Offspring Crossed In China
Guangsheng QIN, Chunyan YANG, Zhengzhun TAN, Jian HUANG, Hui LI, Xianwei
LIANG , Bingzhuang YANG *
*Corresponding email: yangbz@163.com
ABSTRACT
This study was carried out to determine the climate adaptation and growth performance for
Mediterranean buffalo F1 in China, especially in Guangxi region. 53 Mediterranean crossbred
buffalo of early stage (0-12 months) were selected and the growth performance such as body
weight, body length, body height, chest circumference and the abdominal circumference, as well as
the physiological and biochemical parameters were measured monthly. The results showed that the
rectal temperature, respiratory frequency, heart rate and blood biochemical parameters were no
significant difference between Mediterranean and Mediterranean F1 (Mediterranean buffalo frozen
semen crossbred with Murrah, Nili-Ravi and local buffalo) (P> 0.05). There are also no significant
differenence(P> 0.05) among the rectal temperature, respiratory rate, heart rate and blood
biochemical parameters and birth body weight for Mediterranean buffalo F1, Murrah and Nili
buffalo which introduced into China 70 years ago. The growth rate of one month-old Murrah
F1(crossbred with local buffalo) and Nili-Ravi F1(crossbred with local buffalo) were higher than
Mediterranean F1 (crossbred with local buffalo), but the difference were not significant (P> 0.05).
In addition, the growth rate between Mediterranean * Murrah F1 and Mediterranean* Nili-Ravi F1
during 0-12 months of age were basically the same. The body height, body length, chest
circumference and abdominal circumference had the same trends for 1-12 months of Mediterranean
* Murrah F1, Mediterranean* Nili-Ravi F1 and Mediterranean* Guangxi Local buffalo F1.
Therefore, the Mediterranean buffalo introduced into China, were not only able to fully adapt to the
South of China, especially Guangxi environmental climatic conditions, but also showed good
growth performance.
Keywords: Mediterranean buffalo F1, growth performance, blood biochemical parameters,

physiological parameters
INTRODUCTION
We used Indian Murrah and Nili-Ravi as male parent and crossed with Chinese local
buffaloes,therefore, the production of meat and milk had been improved in the past 30 years. The
Italian Mediterranean buffalo is one of good milk breed of River Buffalo in Europe. It has high milk
performance producing more than 5000 kg/lactation 270 days (Antonio Borghese, et al, 2006). And
a large number of bulls have been submitted to performance and progeny tests and a millions of
semen doses from bulls of proven high genetic value are available for artificial insemination in Italy
and in the world. In order to improve the performance of milk and meat of Guangxi local buffalo, a
millions of doses frozen semen of Mediterranean buffalo were introduced into GuangXi Buffalo
750
Research Institute, and the effect of frozen semen of Mediterranean buffalo AI showed good results
in Guangxi (Qin, et al, 2010). The first case of the Mediterranean-Murrah (MMF1),
Mediterranean-Nili-Ravi (MNF1) and the Mediterranean -the local (MLF1) crossbred buffalo were
born in Guangxi and China. Up to now, there were more than 53 Mediterranean crossbred
buffaloes. The parameters of their growth performance such as body weight, body length, body
height, chest circumference and the abdominal circumference, as well as the physiological and
biochemical substance of those 53 Mediterranean crossbred buffaloes were measured.

All the F1 buffaloes which were Mediterranean buffalo frozen semen crossed with Murrah,
Nili-Ravi and Chinese swamp buffalo were born in Guangxi Buffalo Research Institute Buffalo
Farm. The parameters of their growth performance as well as the physiological and biochemical
substance were measured. The data were collected monthly from birth to 12 months of age. Body
temperature was measured with a thermometer inside the rectum. The vein blood samples for
experimental buffalo were collected individually and were measured using conventional methods.
All the data were analyzed using SPSS 17.0.

3.1 Body temperature, respiration rate and pulse rate
Body temperature, respiration rate and pulse rate of MMF1, MNF1 and MLF1 were
determined each month. The results showed that there was no significant difference (P> 0.05)
among the gender, age and breed. The rectal temperature was 36.5-38.5 ° C, respiratory rate was
10-30 beats / min and pulse rate was 30-50 beats / min. These results were similar to the findings of
Zhang et al ( 2000) in Murrah and Nili- ravi buffaloes. The rectal temperature of these buffalo was
36.1 ℃ -38.5 ℃ (average 37.8℃).The respiratory rate was 9-13 times / min. And pulse rate
ranged in 45-50 beats / min. In the term of the rectal temperature, respiratory rate and pulse rate in
the experimental animals, the daytime of those value were higher than that of nightime and during
afternoon of those value were higher than that of in the morning time, which was also consistent
with previous researchers (Zhang, 2000).
3.2 Body weight
The body weight of 0-12 month was measured among MMF1, MNF1and MLF1
buffalo.( Figure 1), the results showed that there were no significant difference (P> 0.05) in birth
body weight among three breeds of hybrid buffalo. The body weight ranged from 33 kg to 45 kg.
The monthly growth rate of the MMF1 buffalo and MNF1 buffalo were higher than MLF1,
but the difference were not significant (P> 0.05).Whereas the growth rate of 0-12 months of age
between MMF1 buffalo and MNF1 buffalo was not significant. Compared with Murrah F1 buffalo,
Nili-Ravi F1 buffalo and local breed (such as Binhu breed, Dehong breed, Guizhou breed, Shanghai
breed, data from Zhang , 2000), there were no difference in three crossbred Mediterranean buffalo
for newborn body weight and one year age body weight And the birth body weight of
Mediterranean hybrid buffalo was slightly higher than that of the Murrah and F1, Nili-Ravi and F1
buffalo, but the difference was not significant (P> 0.05). However the 12-month-old Mediterranean
crossbred buffalo body weight (309-325 kg) (P <0.05) were significantly higher than that of
Murrah, Nili-Ravi and their crossbred buffalo with Guangxi local swamp type buffalo (211.0-277.0
kg). Those results showed that the Mediterranean buffalo introduced into China, not only could
751
fully adapt to the environment and climate conditions of the South of China, especially in Guangxi,
but also showed good growth performance..
3.3 Body sizes
The body sizes (such as body height, body length, chest circumference and abdominal
circumference indicators) of three hybrid buffalo were measured. From the Figure 2, 3, 4 and 5, the
results showed that MMF1, MNF1and MLF1 buffalo had similar growth trend of body sizes in 1-12
month stage. It can be seen that the growth rate of the MLF1 after 10 months of age was slower
than one of MMF1 and MNF1 buffalo. It may be caused by crossing with different breeds. Murrah
and Nili-Ravi buffalo grew faster than the local buffalo, which led to the growth of the crossbred
was different after hybridization with the Mediterranean water buffalo.
3.4 Biochemical parameters
As can be seen from Table 1, there was no significant difference in 12-month-old of blood
biochemical parameters among MMF1, MNF1and MLF1 buffalo. The results of the determination
of the white blood cells, red blood cells, hemoglobin were similar with the result of the local buffalo
(Mao et al, 1974 ;Lin et al,1981 ) The biochemical parameters also were similar with the data of
Murrah and Nili-Ravi hybrid buffalo with 12 months of age ( Yang et al,2005 ).But the results were
different from Xilin buffalo(Li et al, 2006 ). It may be related with the buffalo breed or
environmental factors. The blood potassium, sodium, chloride, calcium, phosphorus and other
mineral elements in Mediterranean hybrid buffalo were similar with the results of 12-15 months old
Murrah and Nili-Ravi cross-breed buffalo in previous study (Liang, et al, 2007) These data in our
adaptability to provide a scientific basis for further study of the Mediterranean hybrid buffalo, and
also lay the foundation for further study of the production performance of the Mediterranean water
buffalo.
ACKNOWLEDGEMENT
This work was supported by the International technical cooperation projects 2008DFA30320 ,
“948” Program of Ministry of Agriculture of P.R. China (No.2006-G49) and the Guangxi scientific
research Projects (2012GXNSFDA053014, 1123005-3.
REFERENCES
Borghese, A. and Mazzi. Buffalo Production and Research. 2006.FAO regional office for Europe
inter regional cooperative research network on buffalo ,Rome, Italy：1- 39.
Qin, G.S., B.Z. Yang，Z.Z. Tan，H. Li, J. Huang and X.W. Liang. 2010. Effect on Artificial
Insemination of Italian Mediterranean buffalo. China Cattle Science. 26(5):33-35.
Zhang, C.X. Editor. 2000. China Buffalo Science. Guangxi scientist Technology Press, Nanning.
Mao, Z. and Y.G. Zhang. 1974. Measurement of several main blood physiological indexes to
Jiangsu province buffalo. Animal Husbandry & Veterinary Medicine. 3: 30-31.
Lin, Y. 1981.Calves buffalo physiological index determination of Binhu buffalo in Hubei. Hubei
Journal of Animal Husbandry & Veterinary Medicine. 4：8-11.
Yang, B.Z., X.W. Liang, Q.Y. Wen, C.X. Zou, G.S. Qin and K.Liang. 2005. Measurement on main
blood physiological and biochemical index of Hybrid buffalo in different growth stage.
China Herbivores. (4)：23-25．
Li. D.F., R.Y. Yao, Z.F. Xu，S.K. Guo and H.Y.Qin. 2006. Measurement on 27 blood biochemical
indicators of Xilin buffalo: 175-177. The fifth Asian buffalo conference, Central
Compilation & Translation Press.
752
Liang，K., C.X. Zou, X.W. Liang, G．S. Qin, B.Z. Yang and Q.Y.Wen. 2007. Measurement on
blood biochemical and blood of heifer buffalo. Guangxi Journal of Animal Husbandry &
Veterinary Medicine. 23(3): 106-108.
350
Body Wei ght ( k g)
Body hei ght ( c m)

140 MMF1
300 MMF1 MNF1
MNF1 120
250 MLF1
MLF1 100
200
80
150
60
100
40
50
20
0
0
0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12
mont h mont h
Figure 1 body weight for MNF1,MMF1and Figure 2 body height for MNF1,MMF1and
MLF1 buffalo (0-12 months) bar chart MLF1 buffalo (0-12 months)
Bo d y h e i g h t ( c m)
140 MMF1
200
Ches t c i r c umf er enc e( c m)
MNF1 MMF1
120 MNF1
MLF1 150
100 MLF1
80 100
60
50
40
20 0
0 1 2 3 4 5 6 7 8 9 10 11 12
0
mont h
0 1 2 3 4 5 6 7 8 9 10 11 12
mont h
Figure 4. chest circumference for MNF1, MMF1
Figure 3. body length for MNF1,MMF1 and
and MLF1 buffalo (0-12 months)
MLF1 buffalo (0-12 months)
f erence(cm
)
250
MMF1
MNF1
i nal
200
Abdom
MLF1
ci rcum
150
100
50
0
0 1 2 3 4 5 6 7 8 9 10 11 12
mont h
Figure 5. abdominal circumference for MNF1, MMF1and MLF1 buffalo (0-12 months)
753
Table 1. The blood biochemical parameters for the MNF1,MMF1and MLF1 of 12 month buffalo.
K Na Cl Ca P) Fe WBC PLT RBC HGB
×109/L ×1012/L
Breed
mmol/l mmol/l g/L
MNF1 4．16 142．5 95．4 2．25 2．63 26．1 10.4 778 4.52 134
MMF1 3．98 141．5 93．1 2．29 2．12 26．5 9.9 474 4.42 124
MLF1 4．43 140．3 94．5 2．12 2．42 22．3 8.6 219 3.98 111
754
Karyotype Analysis of Mediterranean Buffalo and Its Hybrids
Fenxiang HUANG, Bingzhuang YANG, ZhengZhun TAN, Jian HUANG, Hui LI, Chunying
PANG, Jianghua SHANG and Xianwei LIANG*

*Corresponding email：liangbri@126.com
ABSTRACT
In the present study, chromosome specimens were prepared by culture of peripheral blood
lymphocytes, and karyotypes were analyzed routinely from different varieties of Mediterranean
buffalo (M), F1 of Mediterranean buffalo × Murrah buffalo (MM), F1 of Mediterranean buffalo ×
Nili-Ravi buffalo (MN) and F1 of Mediterranean buffalo× Guangxi native swamp buffalo (MG).
The results were shown that the chromosomes of M, MM and MN are 2n=50, which are in
consistence with those of river buffaloes of Murrah and Nili-Ravi reported by other researchers.
However, the chromosomes of MG are 2n = 49, which are disagree with 2n=48 of swamp buffalo
and 2n=50 of riverine buffalo. These results confirmed that Mediterranean buffalo belongs to the
riverine subspecies as the same as Murrah and Nili-Ravi instead of the swamp subspecies as the
Guangxi native buffalo.
Keywords: Mediterranean buffalo, hybrid, chromosome, karyotype
INTRODUCTION
Italian Mediterranean buffalo is one of the best milk buffalo breed in the world and
well-known for the high yield and good quality in milk, with an average lactation milk yield of
2168 kg, even 4000-5000kg from some excellent individuals. The national buffalo breeding farm in
Guangxi Buffalo Research Institute (GXBRI) has reared two exotic river breeds, Murrah and
Nili-Ravi for several decades to explore and implement the Chinese native swamp buffalo genetic
improvement since 1970s. Recently, GXBRI purchased several thousand semen straws of
Mediterranean buffalo, and the third river breed from Italy so that more exotic bloodlines can be
involved in the crossbreeding and improvement in buffalo in China.
In china, the studies on karyotypes of swamp buffalo, riverine Murrah and Nili-Ravi buffalo
have been reported since 1991 (Huang et al., 1991; Huang et al., 2003). But there is no report on
that of Mediterranean buffalo and its hybrids crossbred with Murrah, Nili-Ravi and Chinese native
buffalo. In the present study, the karyotypes were prepared from different varieties of Mediterranean
buffalo (M), F1 of Mediterranean buffalo × Murrah buffalo (MM), F1 of Mediterranean buffalo ×
Nili-Ravi buffalo (MN) and F1 of Mediterranean buffalo× Guangxi native swamp buffalo (MG) to
analyze preliminarily its characteristic of M and its hybrids, which is of great significance for
enriching basic buffalo cytogenetic information and providing a theoretical basis for the cultivation
of new dairy buffalo varieties in China.

755

Chemicals
Chemicals and media were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Life
Technologies (GIBCO) (Grand Island, NY, USA), respectively unless otherwise stated.
Materials
Blood samples from 6 of M, 8 of MM, 5 of MN and 7 of MG raised in the national buffalo
breeding farm in GXBRI were used to study the normal chromosomal pattern. About 5 ml of
peripheral blood from the jugular vein of each animal was drawn aseptically in disposable syringe
containing 0.1ml sodium heparin.
Preparation of chromosome samples
The procedure of lymphocytes culture and chromosome sample preparation was modified
slightly according to Huang et al. (1991) and Ali et al. (2001). Briefly, 0.7 ml blood sample from
each animal was mixed gently with 7 ml RPMI 1640 (supplemented with 100 μg/ml streptomycin,
100 IU/ml penicillin and 50 μg/ml heparin) and 0.1 ml PHA (1mg/ml) in a T50 flask (Jet Biofil),
and then incubated at 38 oC for 68-72 h. Colchicine was added to a final concentration of 0.06
μg/ml 3 h prior to harvesting. The mixture was transferred into a 15 ml conical tube and centrifuged
for 8 min at 2000 rpm. The pellet of cells was resuspended and swelled in hypotonic (0.075 M KCI)
for 25 min at 38 oC, then fixed 2 times for 15 min each time in methanol-acetic acid (3:1). After
fixation, several drops of the cell suspension were dropped from a distance of 10-15 ml on each
slide which had been immersed into ice water prior to use.
After the slides were dipped in coupling jar containing 1:19 of Giemsa stain diluted in PBS
(pH 6.8) for 15-20 min, they were rinsed thoroughly in tap water gently and allowed to dry in air,
and then examined under a microscope (Nikon).

Statistics of the chromosome number
The mounted slides were scanned at low power (100×) and the well-scattered, appropriate in
length and clearly visible metaphases were selected to photomicrograph by digital CCD under oil
immersion (1000×). The homologous chromosomes in the digital photograph were cut and
rearranged in pairs by Photoshop in their descending order of size according to Iannuzzi et al.
(1990). 50 of metaphases for each animal were analyzed and counted the number of chromosome.
Karyotype analysis of Mediterranean buffalo and its hybrids
As shown in Figure 1, the chromosome number of M is 2n = 50, including 48 of autosomes
(in which 6 are submetacentric, 4 are metacentric and 38 are telocentric) and 2 of sex chromosomes
(male: XY vs female: XX). The X is the longest telocentric chromosome, and the Y is also a
telocentric chromosome but quite shorter than the X. The karyotypes of MM (Figure 2) and MN
(Figure 3) are identical to that of M. The results confirmed that Mediterranean buffalo belongs to
the riverine subspecies as the same as Murrah and Nili-Ravi published by other researches (Iannuzzi
et al., 1990; Huang et al., 1991; Ali et al., 2003). However, the chromosomes of MG are 2n = 49 (as
shown in Figure 4), including 47 of autosomes (in which 5 are submetacentric, 5 are metacentric
and 37 are telocentric) and 2 of sex chromosomes, which are similar to those of M, MM and MG..
Within 5 metacentric chromosomes, 4 can be paired while the longest one can not be paired.
Similarly, 1of 5 submetacentric chromosomes and 1 of 37 telocentric chromosomes can not be
paired, respectively. The karyotype of MG is disagree with 2n=48 of swamp buffalo and 2n=50 of
756
riverine buffalo. The result indicated MG is a different variety.

Our previous study and other reported results have demonstrated that the 3 unpaired
autosomes in the karyotype of 2n = 49 buffalo crossbred river buffalo with swamp buffalo are
homologous and can be matched between chromosome 4 and 9 from river buffalo and chromosome
1 from swamp buffalo (Huang et al., 2003; Michelizzi et al., 2010), which is consistence with the
result of MG and is shown as Chromosome 1 in Figure 4.
ACKNOWLEDGEMENT
This research was supported by grants from the International Cooperation Project from the
Ministry of Science & Technology of China (2008DFA30320), Guangxi Scientific and
Technological Project (1123005-1) and the Fundamental Research Funds for Guangxi Buffalo
Research Institute (ShuiNiuJi 1101010).
REFERENCES
Ali S., Z. Ahmad, Mohiuddin, P. Akhtar and M. Ashfaq. 2001. Studies on the karyotype of the
Nili-Ravi buffalo. Pakistan Vet. J. 21(2): 72-76.
Huang Y. and Y. Liu. 1991. Chromosomes of triple crossbred buffaloes and their parental and filial
generations. Buffalo J. 2: 181-187.
Huang Y. J., J. H. Shang, M. M. Liang, X. F. Zhang and F. X. Huang. 2003 Studies of chromosomal
heredity and fertility of progenies (2n=49) crossed between river and swamp buffalo.
Hereditas (Beijing). 25(2): 155-159.
Iannnuzzi L., G. P. Di Meo, A. Perucatti and L. Ferrara, 1990.The high resolution G- and R-banding
pattern in chromosomes of river buffalo (Bubalus bubalis L.). Hereditas. 112: 209-215.
Michelizzi V. N., M. V. Dodson, Z. Pan., M. E. Amaral, M. E. J. Amaral, J. J. Michal, D. J. McLean,
J. E. Womack and Z. Jiang. 2010. Water buffalo genome science comes of age. Int. J. Biol.
Sci. 6(4): 333-349.
757
Figure 1. karyotype of Mediterranean buffalo (M) female.
Figure 2. karyotype of F1 of Mediterranean buffalo × Murrah buffalo (MM) female.
758
Figure 3. karyotype of F1 of Mediterranean buffalo × Nili-Ravi buffalo (MN) female.
Figure 4. karyotype of F1 of Mediterranean buffalo× Guangxi local swamp buffalo (MG) male.
759
Estimation of Genetic Parameters for Growth Traits of Three Genotypes of Water

Buffalo Bulls Raised on a Ranching Operation
Agapita J. SALCESa, Gundolino P. BAJENTINGb and Caro B. SALCESb*
a
Animal and Dairy Sciences Cluster, University of the Philippines Los Baños,Colege, 4031, Laguna,
Philippines
b
Philippine Carabao Center Ubay Stock Farm, Ubay, Bohol, 6315Philippines
*Corresponding email: ajsalces@yahoo.com
ABSTRACT
Bulls from three water buffalo genotypes, Philippine Carabao, Bulgarian Murrah buffalo, and
US Murrah buffalo were evaluated from 2002 to 2012 in order to estimate the genetic parameters for
growth traits. A total of 1075 observations for birth weight, yearling weight, 2 year old weight and 3
year old weight were analyzed for variance and covariance components using Multi Trait Derivative
Free Restricted Maximum Likelihood (MTDFREML) by Boldman et al., 1993. For the three genotypes
all the growth traits studied showed medium heritability estimate ranged from 0.40 to 0.50. Genetic
correlations among traits were all medium and positive which suggest that birth weight could be used
as an indicator for early selection for growth considering its positive genetic correlations with
succeeding growth traits like yearling weight, 2 year old weight and 3 year old weight.
Key words: genetic parameters, growth traits, water buffalo
INTRODUCTION
Water buffalo is a multipurpose animal for meat, milk and draft power. It is one of the richest
genetic resource in Asia and is an important component in agriculture and lifestyle. Very little
selection has been done to improve the performance of buffaloes. Phenotypic selection will not be
enough to fulfill the growing need of meat, milk and draft power in sustainable farming system.
The water buffalo provides a major source of meat but it has always been considered inferior to
beef and cheap source of meat by-products. Recent studies have shown that buffalo meat is similar to
beef and can be considered as high quality meat. Its meat is lean, lower cholesterol and has excellent
blending quality for production of processed products like corn beef, hot dogs and sausages. At present,
buffalo meat is considered tender buff in Australia, black gold in Argentina and possibly Nueva beef in
the Philippines. Buffalo steaks have been rated higher than beef steaks in some taste tests in Australia,
Malaysia, Venezuela and Trinidad. Innovative meat products that will be developed may find niche
markets all over the world.

760
The Center at Ubay Stock Farm (PCC at USF) operating under a ranch production system is
tasked for breeding and selection of Murrah Buffaloes (USMB) imported from the United States in
1993. These animals had been selected for meat production. Also, it raises other genetic groups like
Bulgarian Murrah buffalo and Philippine Carabao. The methods for evaluating genetic improvement in
a herd are selection experiments in a constant environment, divergent selection, replication of the same
genetic material in successive generations and analysis of field records (Hill, 1972a; Hill, 1972b).
Evaluation of different genotypes of buffalo will provide the basis for designing breeding plan and
measuring genetic progress in the future hence this study.

Three genotypes involved in the study namely, Philippine Carabao (PC), Bulgarian Murrah
buffalo (BMB)and U.S. Murrah buffalo (USMB) bulls. These animals were raised under ranch
operation. Weight of the animals were taken at birth and every 3 months thereafter until 3 years of age
and data in 2002 to 2012 were used for analysis.
Preliminary data analysis was conducted to determine which among the fixed effects will be
included in the final model for genetic parameter estimation using SAS v9.1 GLM Procedures. The
model followed was :
Yijk = µ + αi + βj + b(Dijk) + εijk
Where
Yijk = observation on trait considered for growth of kth individual of jth genotype in ith year
αi = ith year of birth, i = 1,2,3,4,5,6,7,8,9,10
βj = jth genotype j= 1,2,3
b = regression of days on taking observation ijk to the observed values to adjust for a fixed day
(12 months (yearling weight), 24 months (2 year old weight) & 36 months 3 year old
weight))
Dijk = age of dam as covariate in days when the observations were taken
εijk = error on traits considered for growth of kth individual individual of jth genotype in ith
year
A total of 1075 observations for birth weight, yearling weight, 2 year old weight and 3 year old
weight were analyzed for variance and covariance components using Multi Trait Derivative Free
Restricted Maximum Likelihood (MTDFREML) by Boldman et al. (1993).
To compute for the genetic parameters i. e. heritability, genetic and phenotypic correlations, the
model used in the analysis were as follows :
y = Xb + Zu + e
y = vector of observations for trait (Birth weight, yearling weight, 2 year old weight & 3 year
old weight))
X = incidence matrix relating to fixed effects (year of birth, genotype)
b = unknown vector of fixed effects
Z = incidence matrix relating to random effect (animal)
u = unknown vector of random effect
e = vector of random residual error
Variance and covariance components were estimated using Multi Trait Derivative Free Restricted
Maximum Likelihood (MTDFREML) by Boldman et al. (1993).
761

Least Square Means of body weight
The body weight estimates for the three genotypes namely Philippine Carabao, Bulgarian
Murrah buffalo and U.S. Murrah buffalo at all ages were significant (P<0.05) (Table 1).
The average birth weight 32.19kg of Phil. Carabao was significantly lower than the birth
weights of Bulgarian Murrah buffalo and U.S. Murrah buffalo. These findings were slightly higher
than the report of Shrestha (1992).
The yearling weight of Phil. Carabao, 176.20 kg was significantly lower than Bulgarian
Murrah buffalo and U.S. Murrah buffalo. Although U.S. Murrah buffalo was significantly lower than
Murrah buffalo.
The 2 year old body weight at 275.15 kg of Phil. Carabao is significantly lower than both genic
groups. The same trend in weight with birth weight was followed.
The 3 year old body weight at 360.55 kg of Phil. Carabao was significantly the lowest among
the 3 genetic groups.
Genetic Parameter Estimates
Heritability estimates of body weight at birth 0.40 ± 0.25 of Phil. Carabao was higher than the
estimates of Shrama et al. (1976) but lower than Shrestha (1992) which was 0.65 (Table 2). For both
genetic groups, Bulgarian and U.S. Murrah buffalo estimate of 0.42 and 0.45 respectively. For yearling
weight, heritability range of 0.41 to 0.48 were higher than estimates of Nautial and Bhat (1979) 0.39.
For 2 year old body weight heritability estimate range of 0.40 to 0.50 for the 3 genetic groups were
the same with the values reported by Nautial and Bhat (1979). The estimate of heritability at 3 year old
body weight was 0.47 to 0.50 which were lower than the findings of Shrestha (1992) 0.69. Relatively
higher standard error obtained in this study was due to the low number of observation to estimate the
genetic parameters.
The phenotypic correlation of body weight at birth, yearling weight, 2 year old weight and 3
year old weight is presented in Table 3. Phenotypic correlation across the genetic groups were high
(0.70 – 0.89) indicating very high correlation between birth weight and the succeeding weights in bulls.
Table 4 presents the genetic correlations of body weight of different genetic groups at different
ages of growth. At birth to yearling weight 0.40 to 0.56, medium and positive genetic correlation for
all the 3 genetic groups indicate that birth weight in bulls positively correlated with succeeding weights
until 3 year old weight.
REFERENCES
Boldman, K.D., L.A. Kriese, L.D. Vleck and S.D. Kachman. 1993. U.S. Department of Agricultural
Research Service.
Castillo, A.C., E.C. Macalandag, F.A. Moog and C.B. Salces. 1988. Phil. Jour. Vet. Anim. Sci. 14
(34): 11-24.
Hill, W.G. 1972a. Anim. Breed. 40 : 1-5. (Abstr.).
Hill, W.G., 1972b. Anim. Breed. 40 : 193 - 212. (Abstr.).
Nautial, L.P. and P.N. Bhat. 1979. Effect of various factors on body weight in Indian Buffaloes. Indian
J. Anim. Scie. 49 (12): 979-983.
PCRDC. 1987. Annual Report.
Salces, C.B., D.P. Baldos, C.C. Sevilla and B.A. Oliveros. 1990. Phil. Jour. Vet. Anim. Sci. 15: 11-24.
SAS Institute Inc. 2000. SAS/STAT User’s guide : Version 5. SAS Institute Inc., Cary, NC.
762
Table 1. Least square means of body weight (kg) of different genotypes at different ages of growth
from birth to three years of age.
AGE, years GENETIC GROUP

Philippine Carabao Bulgarian Murrah U.S. Murrah Buffalo
Buffalo
Birth 32.19a 34.70b 35.10b
1 176.20 a 232.50 b 225.60c
a b
2 275.15 360.50 363.57b
3 360.55 a 435.75 b 469.58c
Any two means in a horizontal line having a common letter are not significantly different (P>0.05).
Table 2. Estimates of heritability and standard error (S.E.) of body weight of different genotypes at
different ages of growth from birth to three years of age.
AGE, years GENETIC GROUP

Philippine Carabao Bulgarian Murrah U.S. Murrah Buffalo
Buffalo
Birth 0.40 ± 0.20 0.42 ± 0.25 0.45 ± 0.32
1 0.45 ± 0.32 0.41 ± 0.28 0.48 ± 0.40
2 0.50 ± 0.28 0.49 ± 0.35 0.40 ± 0.25
3 0.50 ± 0.35 0.50 ± 0.40 0.47 ± 0.35
Table 3. Phenotypic correlations of body weight of body weight of different genotypes at different
ages of growth from birth to three years of age.
AGE, GENETIC GROUP

years
Philippine Carabao Bulgarian Murrah Buffalo U.S. Murrah Buffalo
1 2 3 1 2 3 1 2 3
Birth 0.80 0.85 0.70 0.86 0.78 0.79 0.85 0.78 0.77
1 0.72 0.88 0.76 0.86 0.89 0.85
2 0.70 0.79 0.87
Table 4. Genetic correlations of body weight of body weight of different genotypes at different ages of
growth from birth to three years of age.
AGE, GENETIC GROUP

years
Philippine Carabao Bulgarian Murrah Buffalo U.S. Murrah Buffalo
1 2 3 1 2 3 1 2 3
Birth 0.40 0.45 0.50 0.56 0.48 0.49 0.45 0.58 0.47
1 0.52 0.58 0.46 0.56 0.49 0.55
2 0.60 0.49 0.57
763
Some Environmental Factors Affecting Performance Traits in Registered Nili

Ravi Buffalo Population at Field Area of Pakistan
Manzoor AHMADa, Khalid JAVEDb, Waseem SHAHZADc, Fayyaz MEHMOODc and
Muhammad Hanif KHANd
a
Buffalo Research Institute, Pattoki District Kasur, bUniversity of Veterinary and Animal Sciences,
Lahore, cLivestock Production Research Institute, Bahadurnagar, Okara, dLivestock and Dairy
Development Board, Punjab, Pakistan.
*Corresponding email: amanzoor.5@gmail.com.
ABSTRACT
Data on 12334 performance records of 9577 Nili-Ravi buffaloes for the parities 1 to 6 in
the progeny testing program (PTP) of field areas of 10 districts i.e. Gujrat, Gujranwala,
Hafizabad, Kasur, M.B. Din, Bahawalnagar, Faisalabad, Pakpattan, T.T. Singh and Vehari
under Buffalo Research Institute, (BRI) Pattoki District Kasur, Punjab, Pakistan during the
period 2006 to 2010 were utilized to study some environmental factors affecting the
performance traits i.e. lactation length, 305-day milk yield and total lactation yield The data
were analyzed by test day model using Statistical Analysis System (SAS 9.3), 2011 for fixed
effects. The phenotypic correlations among various performance traits were also estimated. The
least squares means for lactation length were 246.3±1.2 days. The 305-day milk yield and total
lactation yield were averagely1735.3±8.1 and 1910.2±10.4 kg, respectively. The number of test
days ranged from 2 to 10 for 305-day milk yield while it was 2 to 22 for total lactation yield.
The phenotypic correlation among these performance traits ranged from 0.75 to 0.95. The
regressions of performance traits on lactation length were also estimated. The effect of district,
year and season of calving showed highly significant (P<0.01) variation for all the three
performance traits while the effect of parity was highly significant (P<0.01) for 305-day milk
yield and total lactation yield but non-significant for lactation length, respectively. The
improvement in milk production could be achieved through efficient breeding strategy and
improvement in nutrition and other management practices.
Keywords: buffaloes, lactation length, test day milk yield, total milk yield, 305-day milk yield
INTRODUCTION
In Pakistan there are 31.7 million heads of buffaloes, which play a key role in the rural
economy of the country. Buffaloes provide more than 60 percent of milk consumed in the country
(Anonymous, 2011). The Nili-Ravi is well known for its superior dairy qualities among the world
buffalo breeds (Ahmad, 2007). For the genetic improvement the Buffalo Research Institute has
launched one of the largest buffalo progeny testing program encompassing three Buffalo Farms
(LES, Bhunikey, Haroonabad, and ChakKatora), 2 Military Buffalo Farms (Khayber and Punjnad)
and in registered buffaloes of the farmers and breeders in 10 districts; Kasur, Gujrat, Gujranwala,
M. B. Din, Hafizabad, Faisalabad, T.T. Singh, Pakpattan, Vehari and Bahawalnagar. It is the only
way to guaranty the desired genetic transmission in the progeny in terms of milk or beef
(Anonymous, 2007). The main objective of this study was to estimate some environmental factors
affecting production traits in registered Nili-Ravi buffalo population in the field area. The
information so generated will be used for the enhancement of milk production in the said breed.

The data set varied from 1st to 6th parity on 12334 performance records of 9577 Nili-Ravi
buffaloes calved during 2006 to 2010 of PTP area in 10 districts i.e. Gujrat, Gujranwala, Hafizabad,

764
Kasur, M.B. Din, Bahawalnagar, Faisalabad, Pakpattan, T.T. Singh and Vehari under Buffalo
Research Institute, (BRI) Pattoki District Kasur, Punjab, Pakistan were utilized to study some
environmental factors affecting the performance traits i.e. lactation length (LL) , 305-day milk yield
(305DLMY) and total lactation yield (TLMY). The data were analyzed by test day model using
Statistical Analysis System (SAS 9.3), 2011 for fixed effects. The phenotypic correlations among
various performance traits were also estimated. Abnormal lactations for any recorded reason were
not utilized in the analysis. Lactations having less than two test days (60 days) were also excluded.
The total number of records eliminated due to editing was 240, which was less than 2 percent of the
total. The test day (TD) information was stored in the data file containing buffalo identification
number, dates of birth / registration, calving, drying, lactation number (parity), number of test days
(months), lactation length, 305 day milk yield and total lactation milk yield. The data were then
analyzed by test day model (Bilal, 2007) to estimate the fixed effects for performance traits.

The means and coefficients of variation for various production traits in Nili Ravi buffalo are
presented in Table I. The coefficients of variation for different traits ranged from 51.7 to 60.4
percent. The comparison of the values for coefficients of variation revealed that TLMY had large
variability (60.4%), followed by LL and 305 DLMY. The 305 DLMY had least variability (51.7
%). The 305 DLMY ranged from 76 to 6667 kg with an average of 1735.3 kg (±896.3 SD). The
mean TLMY was 1910.2±1153.4 with a range from 76 to 6719 kg. The average LL was 246.3 days
with a standard deviation of 127.5. The range was 61 to 671 days. In these traits the distribution was
not normal. The distribution is skewed to the left for all these performance traits.
Environmental effects
The least square analysis of variance (Table II) showed the effects of district, year and
season of calving which indicated highly significant (P<0.01) variation for all the three performance
traits. However, the effect of parity was highly significant (P<0.01) for 305DLMY and TLMY only
while non-significant for LL, respectively. The results of the present study are in line with the
results reported by Javed et al. (2001) and Ahmad et al. (2009). On the other hand findings of some
other workers are not in line with the results of present study; Ahmad et al. (2001) and Ahmad et al.
(2003). The variation might be due to the difference in the breeds, herds, size of data sets, methods
of estimation, the level of productivity and even the periods of time for this particular trait.
Lactation length
The overall mean of population for lactation length (LL) was 246.3±1.2 days. The least
squares means (LSM) of LL for different districts, year, season of calving and lactation number
(parity) showed that maximum (267.4±3.4 days) LL was observed in district Faisalabad while
minimum (143.7±5.6 days) was found in district Pakpattan. The LSM of LL among other districts
also varied significantly. The buffaloes calved during the year 2007 had highest (262.6±2.0 days)
LL while those calved during 2010 had lowest (100.2±7.4 days) LL. The animals calved during hot
dry season had maximum (212.9±3.3 days) LL while those calved during winter season had
minimum (185.1±2.7 days) LL. In the fifth lactation the buffaloes showed longest (201.4±4.0 days)
parity while in the first lactation shortest (193.4±2.8 days) parity was observed. The findings of the
present study are partially in agreement with the findings of Ahmad et al. (2003).
305-day lactation milk yield

The overall mean of population for 305-day milk yield (305 DLMY) was 1735.3±8.1. The
estimates were lower than some earlier reports (Ahmad, 2007; Ahmad et al., 2008), the reason
being that in the present study mostly the available single parity lactation records are included in the
analysis, while the previous studies were based on lactation records of all parities. The LSM for
305DLMYshowed that maximum (2064.6±22.9 kg) 305DLMYwas observed in district Faisalabad
while minimum (981.3±39.7 kg) was found in district Pakpattan. The LSMof 305DLMYamong
765
other districts also varied significantly. The buffaloes calved during the year 2007 had highest
(1771.9±14.1 kg) lactation yield while those calved during 2010 had lowest (977.5±52.2) lactation
yield. The animals calved during hot dry season had maximum (1575.8±23.3kg) lactation yield
while those calved during winter season had minimum (1311.7±19.0 kg) lactation yield. In the 6th
lactation the buffaloes showed maximum (1514.5±35.2 kg) yield while in the first lactation lowest
(1347.2±19.4 kg) yield was observed. The results of the present study are partially sported by
Ahmad et al. (2008) and Ahmad et al. (2009). The regression of 305DLMYon lactation length was
5.2±0.04 kg showing an increase in 305DLMYof 5.2±0.04 kg with each day increase in lactation
length. Ahmad et el. (2003) also reported similar results in their study on Nili Ravi buffalo kept at
Livestock Experiment Station Bahadurnagar, Okara.
Total lactation milk yield

The overall populations mean for total lactation milk yield (TLMY) was 1910.2±10.4 kg.
The estimates were lower than some earlier reports (Ahmad, 2007; Ahmad et al., 2008). The LSM
for TLMY showed that maximum (2363.9±29.5 kg) TLMY was observed in district Faisalabad
while minimum (972.4±51.1 kg) was found in district Pakpattan. The significant variation for this
trait among other districts was also noted. The buffaloes calved during the year 2007 had highest
(1991.9±18.1 kg) TLMY while those calved during 2010 had lowest (941.9±67.1 kg) TLMY. The
variation in TLMY during different years reflects the level of management as well as environmental
effects like temperature, rain fall, availability of feed and fodder etc. The animals calved during hot
dry season had maximum (1658.8±30.0 kg) TLMY, followed by 1645.8±23.7, 1514.0±33.8 and
1502.2±27.1 during hot humid, spring and autumn seasons, respectively. The buffaloes calved
during winter season had minimum (1386.7±24.4 kg) TLMY. In the 2nd lactation the buffaloes
showed maximum (1585.4±24.5 kg) yield while in the first lactation lowest (1444.9±24.9 kg) yield
was observed. The LSM among other parities showed non-significant variation. The low production
in the first parity was due to non-function of all secretary tissues of the udder in early ages which
become fully functional in the latter parities. The results are supported by the findings of Ahmad et
al. (2001) and Ahmad et al. (2008).The regression of TLMY on LL was 7.8±0.04 kg, showing an
increase in TLMY of 7.8±0.04 kg with each day increase in LL. Ahmad et el. (2003) also reported
similar results in their study on Nili Ravi buffalo kept at Livestock Experiment Station
Bahadurnagar, Okara.
Correlations
The phenotypic correlation of lactation length with 305-day milk yield and total lactation
milk yield were 0.75 and 0.87, respectively. Which were found highly significant (P<0.01).
Similarly the correlation of 305-day milk yield with total milk yield was 0.95, which was also
highly significant (P<0.01). The findings of the present study are in line with the findings of Ahmad
(1999) and Ahmad et. al. (2001). Ahmad et al. (2001) analyzed the large data set of dairy cattle in
Pakistan and found that the phenotypic correlation of lactation length with 305-day milk yield and
total lactation milk yield as 0.63 and 0.74. In the same study the correlation of 305-day milk yield
with total lactation milk yield was 0.95. The correlations was also found positive and highly
significant (P<0.01).
CONCLUSIONS
In short the estimates of performance traits for this study were lower than some earlier
reports (Ahmad, 2007; Ahmad et. al. 2008). The improvement in milk production could be
achieved through efficient breeding strategy and improvement in nutrition and other
management practices.
REFERENCES
Ahmad, M.1999. Genetic evaluation of native and crossbred dairy cattle in Pakistan. Ph.D. Thesis,
University of New England, Armidale, NSW, Australia.
766
Ahmad, M. 2007. Estimated breeding values and genetic trend for milk yield in Nili Ravi buffaloes.
Italian J. Anim. Sci. 2: 393-396.
Ahmad, M., A. Parveen, A. Ghaffar and M. M. Aziz. 2008. Estimated breeding values and genetic
trend for 305-day milk yield in buffalo herd at LES Chak Katora. Pak. J. Agri. Sci. 45: 212-
214.
Ahmad, M., J.H.J. van der Werf and K. Javed. 2001. Genetic and phenotypic correlations for some
economic traits in dairy cattle. Pak. Vet. J. 21: 81-86.
Ahmad, M., M. Ahmad and N. A. Naz. 2003. Environmental and genetic factors affecting first
lactation milk yield, peak milk yield and persistency of lactation in Nili-Ravi buffaloes. Pak.
J. Vet. Res. 1: 20-24.
Ahmad, M., M. S. Azhar and M. M. Aziz. 2009. Performance of buffalo population using test day
milk yield in progeny testing program areas of district Gujranwala. J. Anim. Pl. Sci. 19: 10-
12.
Ahmad, M., R. Hussain and K. Javed. 2001. Effect of some environmental factors on lactation
curve total milk yield, peak yield and persistency of lactation in crossbred cows. J. Anim.Pl.
Sci. 11: 147-149.
Anonymous. 2007. First Annual Report (2005-06 to 2006-07). Buffalo Research Institute, Pattoki
District Kasur. pp-29.
Anonymous. 2011. Economic Survey, Economic Affairs Division, Govt. of Pakistan, Islamabad.
Bilal, G. 2007.Estimation of breeding values for Sahiwal cattle by using test day milk yield. M.Sc.
Thesis, Deptt. Anim. Breed.Genet. Univ. Agri. Faisalabad, Pakistan.
Javed, K., M. Afzal, M. Ahmad and R. Hussain. 2001. Genetic studies on Cholistani cows: I.
Production traits. J. Anim. Pl. Sci. 11: 48-52.
SAS, 2011. New release SAS 9.3 for Microsoft Windows : Cary, NC, USA.
Table 1. Means, standard deviation (SD) and coefficient of variation (C.V.) for various
performance traits in Nili Ravi buffaloes.
Traits No. of obs. Mean ± SD Range C.V. (%)
Lactation length (days) 12334 246.3±127.5 61 - 671 51.74
305-day milk yield (kg) 12334 1735.27±896.3 76 - 6667 51.69
Total lactation milk yield (kg) 12334 1910.17±1153.4 76 - 6719 60.40
Table 2. Analysis of variance (F-ratio) for various performance traits in Nili Ravi buffaloes.
Source of variation D.F. Lactation length 305-day milk Total lactation milk
yield yield
District 9 153.6** 162.7** 171.7**
Year of calving 4 197.0** 137.7** 139.2**
Season of calving 4 30.6** 44.7** 29.9**
Parity 5 1.7NS 11.6** 5.9**
Residual 12311
**P<0.01; NS = Non-significant
767
Genetic Parameters for Milk Yield and Milk Component Traits Estimated
from Test Day and 305D Lactation Records of Philippine Dairy Buffaloes
Ester B. FLORES a, Brian P. KINGHORN a and Julius VAN DER WERFa

a
School of Environmental and Rural Sciences, University of New England, Armidale NSW Australia
*Corresponding email: eflores@myune.edu.au
ABSTRACT
Genetic parameters were estimated from test day and 305D Lactation records of multi-parity
Philippine dairy buffaloes by random regression and repeatability models. There were 16,901 test
day records from 2,303 lactations of 1st – 3rd parity cows from 1997 to 2012. Traits included milk,
fat and protein yields, fat percentage and protein percentage. Varying orders of Legendre
polynomials were combined with the Wilmink’s function and were used in random regression.
Heritabilities for lactation yields estimated from 305D records using a multivariate
repeatability model were moderate; 0.25, 0.14, 0.19, 0.28 and 0.20 for milk yield, fat yield, protein
yield, fat percentage and protein percentage traits, respectively. Heritabilities based on test day
records using a multivariate repeatability model were lower but so were the standard errors.
Heritabilities for individual test days of first parity cows estimated based on random regression
models were higher than those obtained using repeatability models, ranging from 0.14 – 0.27 for
milk yield; 0.11 – 0.14 for fat yield; 0.11 – 0.18, for protein yield; 0.12 – 0.33 for fat percentage and
0.05 – 0.29 for protein percentage traits. Genetic correlations between yield traits were highly
positive whereas it was negative and moderate to high between milk yield and percentage traits.
Thebest random regression model fitted a Wilmink’s function for milk yield and a first order
Legendre polynomial for milk component traits for random additive genetic and permanent
environment effects. This model provides parameters suitable for selecting cows for milk
production and persistency.
Keywords: Random regression, Genetic parameters, Milk yield, Dairy buffaloes

INTRODUCTION
In the livestock industries, the commonly used model for breeding value estimation is the
animal model. Milk production traits that have been included in genetic evaluation include milk, fat
and protein yield, milk fat percentage and protein percentage. Daily yields are summed up over the
entire lactation to a single measure and standardized to 305-day (305D) length. However, since only
a single measure is taken for the entire lactation, there is averaging of systematic effects that vary
over the entire lactation. Genetic evaluations have shifted to the use of test day measures directly as
systemic environmental effects are accounted more accurately on each test day. Test day (TD)
yields can be used directly in a model wherein each record from the same lactation is considered asa
different trait or as repeated measures of the same trait. Random regression (RR) test day models
involve the regression of merit on days in lactation to account for variation between cows in their
performance across the lactation trajectory. This allows an individual cow’s lactation curve to
deviate from the average, making it possible to select for lactation persistency (Jamrozik et al.,
1997). Functions frequently used in various studies to describe the shape of the lactation curve
include Woods’s model, Legendre polynomials (Guo et al., 2002) and Wilmink’s function
(Schaeffer et al., 2000). The fit of RR models was reported to be better with higher order
polynomial functions but increases further the number of parameters to be estimated. Furthermore,
the use of higher order polynomials often have “end-of-range” problems resulting in erratic and
extreme estimates of variance and genetic parameters (Meyer, 2005).
While a number of studies regarding estimation of genetic parameters for milk production
traits have been reported in dairy buffaloes, there is very little information regarding the use of RR
models for genetic evaluation. Sesana et al. (2010) estimated genetic parameters for dairy buffaloes
768
by random regression using Legendre polynomials on days in lactation and reported higher values
for additive genetic variance at the beginning of lactation and negative genetic correlations between
test days in early and mid to late lactation. The latter could be an indication of “end-of-range”
problem which might be remedied by using Wilmink’s function. The objective of this study is to
compare various random regression models for estimating genetic parameters for milk production
traits in Philippine dairy buffaloes. Such models can also provide breeding values for persistency, a
problem in dairy buffaloes often observed as short lactation period (Tonhati et al., 2008). Random
regression models for test days are compared with repeatability models and also with models based
on 305D lactation records to obtain a comprehensive set of genetic parameters for milk production
traits to develop appropriate models for genetic evaluation.
Lactation records of 2,303 first to third parity dairy buffalo cows with 16,901 TD measures
from 1997 to 2012 from nine (9) herds were available for the study. The 305D lactation records
were calculated by single trait prediction based on a method by Flores et al. (2013). For individual
cow lactations, a maximum of 10 test day records were used.
Various mixed models for 305D or TD records were used to estimate genetic parameters by
multi-trait analyses. Multi-trait analysis was between milk, fat and protein yield traits and another
between milk yield, fat% and protein% traits. The model was: , where y is the
vector of milk, fat, protein, fat% or protein% observations; b is the vector of environmental fixed
effects, herd-year-season (HYS) of calving or herd-test-day (HTD); u is the vector of random
additive genetic effects; pe is the vector of permanent environmental effects; e is the vector of
random residual effects, while X and Z refers to incidence matrices for fixed and random effects,
respectively.
First lactation TD records were used directly in a random regression model to estimate
heritability at different days in milk (DIM) in a given lactation. The RR model is given as:
where yijkl is the test day record l of
cow j made on DIMjl of lactation; HTDi is the fixed effect of herd-test date i; eijkl is random residual
effect; ßkm, αjm, and pejm are fixed regression on days in milk (DIM) within sub-class k age-season
of calving, random additive genetic and random permanent environment regression of mth order on
days in milk, respectively. The Wilmink’s function (Wil) and Legendre polynomial (Legm) of
varying orders describe the shape of the lactation curve. For Wilmink’s function, let
whereas for Legendre polynomial, let
. The
days in milk were standardized from -1 to 1 for all Legendre polynomial functions. Residual
variances were allowed to vary for each of the ten (10) TD periods in a lactation but residual
covariance between TD periods were assumed to be zero. For bivariate analyses, residual
covariances between traits in the same TD period were allowed to vary. Bivariate analyses were
done between milk yield and with each of the milk component traits. Various combination of
Wilmink’s function and Legm of varying order of fit were used for the fixed and random regression
coefficient estimation. For all models, the F1/F2 format describes the combination of functions for α
(F1) and pe (F2) effect respectively. Average Information Residual Maximum Likelihood
(ASREML) software (Gilmour et al., 2009) was used for variance component estimation.
Random α and pe regression coefficients were used to build the covariance matrix for
different days in milk along the lactation period (Jamrozik et al., 1997). Heritabilities for a
particular DIM i in lactation were calculated by dividing the estimated genetic variance with the
sum of genetic, permanent environment and appropriate residual variances also for that particular
DIM. Different models were compared based on heritability, log likelihood, goodness of fit,
Akaike’s Information Criterion (AIC) and Schwarz’ Bayesian Information Criterion (BIC).
Goodness of fit for the different random regression models were tested using chi-square (X2m-p) test
for the weighted residual sums of squares (van der Werf et al., 1998): The lower value for both AIC
and BIC indicates best fitting model.
769

There were no significant differences in estimates of heritability for the five traits with
running a multivariate analysis of first parity records only compared with the use of multi-parity
records (Table 1). Exceptions were the estimates obtained for fat% and protein% traits from using
first parity 305D lactation records only (305DL1 model) where the heritabilities were significantly
higher compared with the use of multi-parity records (305DL13 model). For all traits, the use of
multi-parity records lowered the standard error (se) of heritability. A very high positive genetic
correlation was observed between yield traits for all 305D and test day models, whereas it was
moderate but negative between milk yield and fat/protein percentage traits. Because of the high
genetic correlation between yield traits, it may be better to do multi-trait analysis of milk yield, fat
and protein percentage to improve milk yield and quality.
Table 1. Heritability and genetic correlation estimates by running multi‐trait repeatability model on milk and milk component traits
of Philippine dairy buffaloes
Trait 305DL1 model 305DL13 model TDL1 model TDL13 model
2 2 2 2
h ±se rG h ±se rG h ±se rG h ±se rG
Mil k yi el d 0.23 ±0.09 ‐ 0.25±0.05 ‐ 0.12±0.05 ‐ 0.15±0.03 ‐
Mil k fa t yiel d 0.10 ±0.08 0.94 0.14±0.05 0.99 0.05±0.04 0.97 0.08±0.03 0.99
Protei n yi el d 0.16 ±0.09 0.98 0.19±0.05 0.99 0.10±0.05 0.96 0.09±0.03 0.97
Mil k fa t percenta ge 0.34 ±0.13 ‐0.97 0.28±0.06 ‐0.96 0.05±0.03 ‐0.74 0.05±0.02 ‐0.83
Mil k protei n percenta ge 0.50 ±0.14 ‐0.60 0.20±0.08 ‐0.67 0.04±0.03 ‐0.58 0.05±0.03 ‐0.70
305DL 1 mo del -first lactatio n 305D reco rds were used; 305DL 13 mo del - first to third lactatio n 305D reco rds were used wherein reco rds fro m different
parities are co nsidered repeated measures o f the same trait; TDL 1 mo del - test day reco rds o f first parity co ws were used wherein reco rds in the same
lactatio n are co nsidered repeated measures o f the same trait; TDL 13 mo del - test day reco rds o f first to third parity co ws were used wherein reco rds fro m
different parities are co nsidered repeated measures o f the same trait. r G = genetic co rrelatio n between milk yield and each o f the fo ur milk co mpo nent traits.
Wald F statistics were higher for models that were fitted with the Wilmink’s function
compared with Legm for fixed regression given the same random regression functions. Generally,
models with more parameters (Wil/Leg3, Leg2/Leg3 and Leg1/Leg3) have better AIC and BIC
values (Table 2) but may not necessarily have better fit as indicated by bigger Goodness-of-fit
statistics. Higher genetic variance in early lactation was observed for models fitted with Wil for α
effects and Legm for pe effects (Fig. 1). The use of 2nd order Leg for both α and pe effects had
higher variance in the beginning of lactation, similar to the result of Sesana et al. (2010).
Table 2. Measures of goodness of fit of various RR models applied on 1st parity TD records of dairy buffalo cows
1
Regres s i on functi on No. of ra ndom Goodnes s Log 3 3
Model 2 AIC BIC
a pe Fi xed effect pa ra meters of fi t s ta ti s ti c Li kel i hood
M3 Leg2 Leg2 Leg2 12 0.43 ‐5843.8 11712 11733
M5 Leg1 Leg3 Leg3 13 0.46 ‐5716.0 11458 11481
M6 Wi l Wi l Leg3 12 0.81 ‐5677.8 11380 11401
Leg2/Leg2 Leg2 Leg2 Wi l 12 0.27 ‐5578.0 11180 11202
Wi l /Wi l Wi l Wi l Wi l 12 0.34 ‐5573.2 11170 11192
Wi l /Leg3 Wi l Leg3 Wi l 16 2.09 ‐5544.7 11121 11150
1 Leg1 - first o rder Legendre po lyno mial; Leg2 - 2nd o rder Legendre po lyno mial; Leg3 - 3rd o rder Legendre po lyno mial; Wil - Wilmink's functio n.
2 Go o dness o f fit statistic, ê = g - ĝ, where g is vecto r o f half sto red elements o f pre-estimated co variance matrix G where test day reco rds were
subjected to multi-trait analysis and ĝ is vecto r o f expected value based o n RR fitted to milk test day reco rds. 3 A IC - A kaike's info rmatio n criterio n, B IC
- Schwarz' B ayesian info rmatio n criterio n
Model Leg1/Leg3 had overall lower variance across lactation and may indicate inability to model
the lactation curve sufficiently. The Wil/Wil model, fitted with Wilmink’s function for random α
and pe coefficient estimation did not show extreme values in early and late lactation. Heritability
770
estimates obtained with the Wil/Wil model were also more consistent throughout the lactation
period (Fig. 2) and may be better fitting model for milk yield trait. As expected, heritabilities
obtained by the Wil/Leg3 model were significantly higher than TDL1 and lower order random
regression models.
Fig. 1. Additive genetic variance estimated from first parity Figure 2. Estimates of heritability for first parity milk yield
milk test day records by random regression model trait by random regression model
1.20
0.28
1.00
0.25
Variance, kg 2
0.80 0.22
Heritabiliity
Wil/Wil Wil/Wil
0.60 0.19
Wil/Leg3 Wil/Leg3
0.16
0.40 Leg1/Leg3 Leg1/Leg3
0.13
0.20 Leg2/Leg2 Leg2/Leg2
0.10
0.00 0.07
15 45 75 105 135 165 195 225 255 285 315 30 60 90 120 150 180 210 240 270 300
Days in milk Days in Milk
Wil/Wil ‐ RR model using Wilmink's function for additive genetic (a) and permanent environment (pe) effect, Leg1/Leg3 ‐ model using first order and 3rd order LP for a and pe effect
respectively, Leg2/Leg2 ‐ model using 2nd order LP for a and pe effect. Wil/Leg3 ‐ model using Wilmink's function and 3rd order LP for a and pe effect respectively. All models utilize
Wilmink's function for the fixed regression component.
Two-trait analysis between milk yield and the milk component traits revealed that Wil-
Leg1/Wil-Leg1, a model utilizing Wilmink’s function for milk yield and 1st order LP for milk
component traits to estimate both random α and pe coefficients was the better model. Genetic
variances for milk yield throughout the lactation period was similar when run together with any one
of the milk component traits in a bivariate analysis. Heritability estimates at different DIM were
similar to running a single trait analysis (Table 3). Genetic correlations between traits were also
high, consistent with TDL1 model especially at DIM90. The genetic variances for milk component
traits may change in the future with more records, but these estimates suggest making genetic
progress for milk yield and milk quality is possible.
Table 3. Genetic correlations and heritabilities of milk and milk component traits at different stages of lactation estimated in a two‐trait analysis by
1
random regression model from first parity test day records of Philippine dairy buffalo cows
Milk a nd fa t yield Milk yield and fat% Milk and protein yield Milk yield a nd protein%
2 2 2 2 2 2 2 2
DIM rG(MY‐FY) h FY h MY rG(MY‐FY) h FY h MY rG(MY‐F%) h F% h MY rG(MY‐F%) h F% h MY
30 0.93 0.11 0.20 ‐0.57 0.12 0.18 0.98 0.11 0.14 ‐0.83 0.05 0.21
90 0.98 0.12 0.22 ‐0.67 0.15 0.19 0.99 0.17 0.20 ‐0.87 0.17 0.22
180 0.97 0.12 0.24 ‐0.86 0.17 0.20 0.97 0.17 0.21 ‐0.74 0.17 0.21
240 0.92 0.11 0.23 ‐0.87 0.26 0.19 0.95 0.15 0.20 ‐0.71 0.23 0.22
300 0.85 0.12 0.24 ‐0.90 0.33 0.19 0.88 0.15 0.20 ‐0.74 0.25 0.27
1 Fo r all bi-variate analysis,the mo del used was Wil-Leg1/Wil-Leg1 where, A dditive genetic effect: Trait 1= Wilmink's functio n - Trait 2 = 1st o rder Legendre po lyno mial/P ermanent
enviro nment effect: Trait1 = Wilmink's functio n - Trait 2 = 1st o rder Legendre po lyno mial. Fo r each bi-variate analysis, trait 1is milk yield and trait 2 is any o ne o f the milk co mpo nent
trait
REFERENCES
Flores, E. B., Kinghorn, B. P. and J.H.J. van der Werf. 2013. Predicting lactation yields in dairy
buffaloes by interpolation and multiple trait prediction. Livest. Sci., 151, 97-107
Gilmour, A. R., B. J. Gogel, B. R. Cullis and R. Thompson. 2009. ASReml User Guide Release 3.0.
Hemel Hempstead, HP1 1ES, UK: VSN International Ltd.
Guo, Z., M. Lund, P. Madsen, I. Korsgaard and J. Jensen. 2002. Genetic Parameter estimation for
milk yield over multiple parities and various lengths of lactation in Danish Jerseys by
random regression models. J Dairy Sci, 85, 1596 - 1606.
Jamrozik, J., L. R. Schaeffer and J. C. M. Dekkers. 1997. Genetic Evaluation of Dairy Cattle Using
Test Day Yields and Random Regression Model. J Dairy Sci, 80, 1217 - 1226.
Meyer, K. 2005. Random regression analyses using B-splines to model growth of Australian Angus
cattle. Genet. Sel. Evo., 37(6), 473
771
Sesana, R., A. Bignardi, R. Borquis, L. El Faro, F. Baldi, L. Albuquerque and H. Tonhati. 2010.
Random regression models to estimate genetic parameters for test-day milk yield in
Brazilian Murrah buffaloes. J Anim Breed Genet, 127(5), 369-376
Tonhati, H., M. Cerón-Muñoz, J. Ademir de Oliveira, L. El Faro, A. L. F Lima and L.G.
Albuquerque. 2008. Test-day milk yield as a selection criterion for dairy buffaloes. (Bubalus
bubalis Artiodactyla, Bovidae). Genet.Mol. Bio., 31(3), 674 - 679.
Van der Werf, J. H. J., M. E. Goddard and K. Meyer. 1998. The Use of covariance functions and
random regressions in test day models. J Dairy Sci, 81, 3300 - 3308.
772
Improvement, Utilization and Conservation of Buffaloes under New Legislation of

Pakistan
Muhammad Nawaz SAEED,a*Tasneem AKHTAR,a Muhammad Sajjad KHANb and Shaukat Ali
BHATTIc
a
Livestock and Dairy Development Department Government of Punjab, bDepartment of Animal
Breeding and Genetics University of Agriculture Faisalabad, cInstitute of Animal Nutrition and Feed
Technology University of Agriculture Faisalabad Pakistan.
*Corresponding email: saeedkahlon@aol.com
ABSTRACT
Buffalo is the main stay for millions of small farmers and is major source of fresh milk for most
of the big cities throughout Pakistan. Contribution of buffalo to the national milk and meat supply is
about 65% and 50%, respectively. In the recent past, many steps have been taken for the development
of buffalo in the country, however, recent constitutional amendment to allow agriculture to be a
provincial subject, necessitated new laws. ‘Punjab livestock breeding act 2012’ is, therefore, being
passed to legislate improved utilization of buffaloes and other species. Similar acts are likely to be
floated by the other provinces and are expected to put a halt to indiscriminate breeding of indigenous
species. Under the act, a livestock breeding services authority will be established to regulate the
livestock breeding services and will regulate the development and conservation of indigenous livestock
resources. The Nili and Ravi breeds of buffaloes are, therefore, likely to get their due recognition along
with Nili-Ravi; the main buffalo breed of Punjab. The standards for selection of animals, for
semen/embryo and semen production facilities should be available in a year. Breed associations are
given a due role in breed development and promotions as they will be able to maintain a herdbook and
will contribute in pedigree verification in the breed improvement programs. Five years down the road,
sale of purebred animals of a breed will also be controlled by these associations. An important segment
incorporated in the law is encouragement of promotional activities for conservation of near threatened
breeds. While making such laws is a step in the right direction, capacity building of different
stakeholders is expected to be a major activity in the near future. Presence of a regulatory framework is
expected to improve buffalo utilization and conservation in Pakistan.
Keywords: Legislations, , conservation, breed associations, Punjab


773
Genetic Parameters for Test-Day Fat Yield Estimated by Random Regression

Models in Dairy Buffaloes using Bayesian Inference
Lais Costa BRITO,a* Rusbel Raul ASPILCUETA BORQUISb, Humberto TONHATIb,
Robledo de Almeida TORRESa
a
Department of Animal Science, Federal University of Viçosa, Viçosa 36570-000, Minas Gerais,
Brazil, bDepartment of Animal Science, São Paulo State University (FCAV-UNESP), Jaboticabal
14884-900, São Paulo, Brazil.
*Corresponding e-mail: laiscostabrito@gmail.com
ABSTRACT
This study modeled variations in test-day fat yield of first lactation of Buffaloes cows by
random regression model (RRM) fitted by Legendre orthogonal polynomials (LOP) compared to 3
alternatives models fitting B-splines. A total of 10691 monthly test-day fat yield records of 1388
first lactations from buffaloes of the Murrah breed born between 1985 and 2007 from 12 herds in
the state of São Paulo, Brazil, were used. The fixed effects common for all models were the
contemporary group, defined as the herd-year-month or herd-year and calving season of the test
day, numbers of milkings per day (two levels), the covariable dam age at calving (linear and
quadratic effects) and the average trend of fat yield was modeled by quartic LOP or while using b-
splines, cubic LOP. Estimates of (co)variance components were obtained by a Bayesian framework,
applying an animal model, through Gibbs Sampling. The residual variances were grouped in ten
classes. The random additive genetic and permanent environmental effects were modeled by cubic
and quadratic Legendre orthogonal polynomials, respectively, or using linear b-spline functions
with 3 to 6 knots. The heritability estimates were moderate (0.24±0.04), ranged from 0.17 to 0.4.
Heritability estimates increased at begin and the end of lactations. According to the deviance
information criteria (DIC), the best overall performance for both the additive genetic and permanent
environmental effects for fat production was that with three knots located at 5th, 60th, 305th days of
lactation. The model which considered Legendre orthogonal polynomials were the worst model. B-
spline functions could be applied as an alternative to Legendre polynomials to model covariance
functions to test-day fat production.
Keywords: B-spline, Legendre polynomial, Gibbs sampler, genetic parameters


774
Development of Multiplex PCR for Sexing Buffalo Embryos
Wisut NUALCHUENa* Kriengsak TASRIPOO a, Kitiya SRISAKWATTANA a, Supitchaya

TREEBONMUANGa, Sangwon USAWANGa and Mongkol TECHAKUMPHUa
a
Research and Development Center for Livestock Production Technology, Department of
Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn
University, Phathumwan, Bangkok 10330, Thailand
*
Corresponding email: nualchuen@hotmail.com
ABSTRACT
The objective of this study was to identify the sex of in vitro produced buffalo embryos
using an efficient polymerase chain reaction (PCR) assay. Buffalo oocytes collected from
slaughtered animals were in vitro matured, fertilized and cultured for development until morula and
blastocyst stage. The embryos were washed twice in phosphate buffered saline (PBS) and once in
1X PCR buffer then each embryo was placed separately in 20 µl 1X PCR buffer and stored at -20°C
for DNA extraction and PCR assay. A pair of bovine satellite primers common to both male and
female and a pair of male-specific primers (BRY.1) which targeted male-specific sequence in the
buffalo DNA were used in PCR assay. The assay was carried out on whole blood samples, collected
from adult 10 male and 10 female total 20 buffaloes and 50 morula, 50 blastocyst in vitro fertilized
buffalo embryos. When DNA samples from blood were amplified, the sex was determined by PCR
always corresponded to the anatomical sex. The results are females representing only the 216 bp
while males representing both 300 and 216 bp. The percentage of sex determination is 20 out of 20
(100%) accurate for blood samples. The morula and blastocyst stage sex determination of 44 out of
50 (88%) and 46 out of 50 (92%) respectively, were successfully determination using the multiplex
PCR assay. In conclusion, this multiplex PCR assay could be used as a reliable and efficient tool for
sex determination of in vitro fertilized buffalo embryos satisfactory.
Keywords: Sexing, Embryo, Buffalo, Multiplex PCR
INTRODUCTION
Sex predetermination of offspring in agriculturally important species has an immense
potential in the livestock industry. With the improvement of in vitro fertilization (IVF), in vitro
culture (IVC) and embryo manipulation techniques, sexing of preimplantation embryos can assist
the dairy producer in managing his resources more effectively (Shea, 1999). Gender preselection
has a clear application in several fields. Not only is it an important productive tool because it allows
the adjustment of offspring gender to market demands, but it also contributes to minimizing gender-
linked genetic diseases and might restore a balanced male-female ratio in endangered species. The
development of molecular techniques, the need for quick, reliable, easy to perform method for sex
determination can be accomplished using PCR (Chrenek et al., 2001). However, the lower
reproductive efficiency of domestic buffaloes is a major impediment to proper use of buffaloes for
milk and meat production worldwide. The most efficient techniques to improve the buffalo
production are artificial insemination (AI), in vitro fertilization and embryo transfer (ET). These
techniques were tried to raise the efficiency of artificial breeding of native buffaloes. Selection of
the sex of offspring of such species might have a tremendous impact on buffalo dairy industry.
The PCR method has been standardized to determine sex. It is known that autosomal and
sex chromosome specific sequences of Bovidae are highly conserved during evolution, bovine Y-
chromosome sequences, BRY.1 was found to be repeated exclusively on the Y- chromosome of
775
cattle, sheep and goats (Matthews and Reed, 1991). The major livestock animals, cattle, buffalo,
sheep and goat belong to the family Bovidae (Appa Rao et al., 1993). Appa Rao and Totey (1992)
reported that it is possible to identify the sex of buffalo embryos using bovine Y-chromosome
specific primer by PCR. The aim of this preliminary study was to apply the PCR assay, as a fast,
efficient and low cost method, to determine the sex of in vitro fertilization produced buffalo
embryos.

All chemicals used in this study were from Sigma Aldrich (St. Louis, MO, USA), except
where otherwise indicated. All reagents were tested for cell culture quality.
In vitro maturation
The procedure of in vitro maturation (IVM) was modified from Tasripoo et al. (2006). the
buffalo ovaries were obtained from a local slaughterhouse and transported to the laboratory within
4 h in 0.9% (w/v) normal saline at 28-30°C. The oocytes were aspirated from 2 to 6 mm antral
follicles with an 18-gauge needle attached to a 10 mL disposable syringe. Oocytes were
morphologically selected under stereomicroscope. Only cumulus-oocyte complexes (COCs) with
homogenous ooplasm were selected for in vitro maturation and were washed four times in tissue
culture medium199 (TCM199) plus 10% fetal bovine serum (FBS). Groups of 10 COCs were
cultured in 50 µl droplets of TCM 199 supplemented with 10% follicular fluid, 0.02 AU/mL follicle
stimulating hormone (FSH), 50 IU/mL hCG (Chorulon®Intervet, USA) and 1 µg/mL 17β estradiol.
In vitro maturation of COCs was performed for 19-20 h at 38.5°C in a humidified atmosphere of
5% CO2 in air.
In vitro fertilization and in vitro embryo culture
The procedure of in vitro fertilization (IVF) and in vitro embryo culture (IVC) was modified
from Tasripoo et al. (2006). One straw of frozen semen of swamp buffalo (0.25 mL per straw) were
thawed at 37°C for 30 sec and then submitted to swim-up procedure in 1 mL of sperm tyrode lactate
medium (SPTL). The upper part was pipette to spin at 500 rpm for 5 min in SPTL 15 mL/tube. The
pellet was resuspended with SPTL containing 10 µg/mL of heparin in order to obtain 106
sperm/mL. The dilution of sperm in 50 µl per drop was incubated under 5% CO2 in humidified air
at 38.5°C for 15 min before matured oocytes was transferred to the drop. The oocytes were allowed
to contact with sperm for 18 h. The oocytes in each drop were transferred to 50 µl drop of synthetic
oviduct fluid (SOF) +1% FBS and was incubated in a humidified atmosphere of 5% CO2, 5% O2
and 90% N2 for 48 h. The 8-cell stage embryos were transferred to cultured in SOF supplemented
with 5% FBS and co-cultured with buffalo oviductal cell. On day 7 of cultured for development
until morula and blastocyst stage. The embryos were washed twice in PBS and once in 1X PCR
buffer then each embryo was placed separately in 20 µl 1X PCR buffer and stored at -20°C for
DNA extraction and PCR (Samah et al., 2009).
Extraction of Genomic DNA from Blood and embryos
Whole blood from ten male and ten female adult buffaloes were collected in heparinized
vaccutainer tubes for optimization of the PCR conditions. Genomic DNA was extracted from the
blood by Genomic DNA mini kit (Geneaid Biotech Ltd., Taiwan). Finally, DNA concentration was
adjusted to 100 ng/mL by measuring the OD at 260 nm. Extraction of DNA from embryos, total
number of 50 morula and 50 blastocyst IVF produced buffalo embryos was used. Embryos were
taken out of deep freeze and the volume was adjusted to 20 µl by adding 1X PCR buffer, then 4 µl
proteinase K was added to each embryo and the reaction. All embryos were incubated at 50°C for
18 h and a deactivation step for proteinase K was carried out at 95°C for 10 min (Appa Rao et al.,
1993).
776
Primers and PCR Amplification

The primers used in the PCR reaction are two pairs of primers were designed. We are used
primers derived from bovine Y-chromosome specific sequences for sex selection in buffalo
embryos. The first pair is gender-neutral primers which targeted bovine satellite sequences common
to both male and female. Sat I (5’-TGG AAG CAA AGA ACC CCG CT -3’) and Sat II (5’- TCG
TGA GAAACC GCA CAC TG -3’) (Plucienrriczak et al., 1982; Manna et al., 2003). It amplified
216 bp. The other primer pair is male-specific primers. BRY. l I (5’- GGA TCC GAG ACA CAG
AAC AGG -3’) and BRY.1 II (5’- GCT AAT CCA TCC ATC CTA TAG -3’) (Reed et al., 1989),
which targeted male-specific sequence. It amplified 300 bp DNA fragment in males only.
The multiplex PCR amplification was carried out in a total of 25 µl reaction volume. The
PCR components concentration were optimized to be 1X PCR buffer, 1.5 mM MgCl, 0.5 mM
dNTPs and 12.5 mol of each primer, 1 unit of Taq polymerase and either 100 ng of genomic DNA
or 5 µl of embryonic lysate. The amplification cycles were carried out in a Thermocycler. Reaction
conditions were also optimized to be 95°C for 3 min as initial denaturation followed, by 35 cycles
of 94°C for 45 sec, 58°C for 1 min and 72°C for 1 min. A final extension step at 72°C for 10 min
was followed. Positive male DNA isolated from buffalo whole blood and negative control (no
template) were included in each PCR run to ensure no cross contamination or amplification failure
due to presence of inhibitors. All samples were repeated twice to ensure reproducibility of the PCR
test 2l6 bp and 300 bp.
Agarose Gel Electrophoresis
Amplification products were electrophoresed in 2% agarose gel containing 1XTBE (tris
borate EDTA) at 80 volts for 60 min and visualized under ultraviolet light. To assure that the
amplification products were of the expected size, a 50 bp DNA ladder was run simultaneously as a
marker.

A multiplex PCR using both the gender-neutral and the male-specific primers was conducted
on the genomic DNA to detect the sex. The results of PCR of all blood samples confirmed the
anatomical sex of the adult animal. Male animals amplified both 300 and 216 bp DNA fragments
while females amplified only the 216 bp DNA fragment as shown in figure 1. The examined
animals confirmed the reliability and specificity of the PCR sexing method. Sex determination of
buffalo embryos, PCR was conducted on crude DNA obtained from embryo samples. The results
are females representing only the 216 bp while males representing both 300 and 216 bp DNA
fragment as shown in figure 2. The percentage of sex determination is 20 out of 20 (100%), 44 out
of 50 (88%) and 46 out of 50 (92%) accurate for blood samples, morula and blastocyst respectively.
The male ratio was observed to be 47.72% and 52.17% for morula and blastocyst respectively
(Table 1).
The results of PCR of blood samples were in complete agreement with the anatomical sex
and thus confirming the reliability of the sexing method. Then, PCR reactions were carried out on
each IVF produced buffalo embryos. Our results also showed that bovine primers (satellite and
BRY.1) could be used for sex determination successfully of Thai swamp bufflo in this study. The
efficiency rates of our multiplex PCR method was 100% accuracy for blood sample sex
determination. In conclusion, our multiplex PCR assay could be used as a reliable and efficient tool
for sex determination of IVF produced buffalo embryos. Although this study was preliminary the
optimisation of this method for embryo sex determination, with an improvement in embryo transfer,
may rapidly improve the genetic selection of buffaloes.
777
REFERENCES
Appa Rao, K.B.C. and S.M. Totey. 1992. Sex determination in sheep and goats using bovine Y-
chromosome specific primers via polymerase chain reaction: Potential for embryo sexing.
Indian J1. Exp. Biol. 30:775-777.
Appa Rao, K.B.C., C.H. Pawshe and S.M. Totey, 1993. Sex determination of in vitro developed
buffalo (Bubalus bubalis) embryos by DNA amplification. Mol. Reprod. Dev. 36:291-296.
Chrenek, P., L. Boulanger, Y. Heyman, P. Uhrin, I. Laurincik, I. Bulla and I.P. Renard. 2001.
Sexing and multiple genotype analysis from a single cell of bovine embryo. Theriogenology
55:1071-1081.
Manna, L., G. Neglia, M. Marino, B. Gaspanin, R. Di Palo and L. Zicarelli. 2003. Sex
determination of buffalo embryos (Bubalus bubalis) by polymerase chain reaction. Zygot.
11:17-22.
Matthews, M.E. and K.C. Reed. 1991. A DNA sequences that is present in both sexes of
Artiodactyla is repeated on the Y-chromosome of cattle, sheep and goat. Cytogenet. Cell.
Genet. 56:40-44.
Plucienrriczak, A., I. Skowronski and I. Iaworski. 1982. Nucleotide sequence of bovine 1.715
satellite DNA and its relation to other bovine satellite sequences. J. Mol. Biol. 158:293-304.
Reed, K.C., K.I. Matthaei, D.A. Mann, S. Beaton and M.A. Matthews. 1989. Determination of
genetic sex in ruminants using Y-chromosome specific polynucleotides. Patent Cooperation
Treaty. pp. 121.
Shea, B.F. 1999. Determining the sex of bovine embryos using polymerase chain reaction results: A
six-year retrospective study. Theriogenology 51:841-854.
Tasripoo, K., K. Srisakwattana, W. Suthikrai, S.Treebonmuang and M. Kamonpatana 2006.
Potential use of buffalo follicular fluid for in vitro maturation supplementation of buffalo
oocytes. In: Proceedings of the 5th Asian Buffalo Congress, Nanning, China. pp. 610-615.
Table 1. Efficiency of sex determination by multiplex PCR of buffalo blood sample, morula and
blastocyst stage embryos.
Sample N Sexed % Sexed Male Female Sex ratio % of

of sexing male/female males
Blood 20 20 100 10 10 1.00 50.00
Morula 50 44 88 21 23 0.91 47.72
Blastocyst 50 46 92 24 22 1.09 52.17
Figure 1. Agarose gel electrophoresis of multiplex PCR products amplified from buffalo genomic
DNA isolated from blood. Lane1(M) 50 bp ladder DNA size marker, lane 2, 3 are females
representing only the 216 bp while lanes 4, 5 and 6 are males representing both 300 and 216 bp.
778
Figure 2. Agarose gel electrophoresis of multiplex PCR products amplified from IVF produced
buffalo embryos. Lane1(M) 50 bp ladder DNA size marker, lane 2, 3 are females representing only
the 216 bp while lanes 4, 5 and 6 are males representing both 300 and 216 bp.
779
Studies on Linear Type Traits and Morphometric Measurements in Nili Ravi

Buffaloes of Pakistan
*Khalid JAVED, Riaz Hussain MIRZA, Muhammad ABDULLAH and Talat Naseer
PASHA Maqsood AKHTAR
Department of Livestock Production, University of Veterinary and Animal Sciences, Lahore

Pakistan
*Corresponding email: khalidjaved@uvas.edu.pk
ABSTRACT
Physical appearance of most of cattle breeds in different parts of the world has been
studied extensively but such type of information in buffaloes is very rare especially the concept of
linear type scoring in dairy buffaloes is very recent. The present study was designed to develop
some linear scoring system for Nili Ravi buffaloes in Pakistan using the ICAR, 2010 guide lines
developed for cattle breeds. Nili Ravi buffaloes maintained at 5 Institutional herds (Pattoki, Chack
Katora, Haroonabad, Khushab, Rakh Ghulaman) in Punjab and few private breeder’s farms were
utilized in the present study. Body measurements were recorded in centimeters and the linear type
traits were scored on 1-9 scale per guidelines of the ICAR. A total of 1180 records on some linear
type traits and body measurements were generated over a scoring period of 2 years. PROC
MIXED procedures under SAS (2003) and LSD under SPSS program were used to analyse
data.(SPSS, 2004). Mean score for height at sacrum (135.766±4.401cm), for bone structure
(5.344±1.787), for dairy form (5.619±1.203), for horn diameter (18.646±2.059 cm), for ear
length (29.5±2.118 cm), for tail length (103.515±12.551 cm), for rump length (43.516±2.582 cm)
and for average score day milk yield 6.85±2.19 kg were recorded. Height at sacrum (0.26) and
ear length (0.163) and rump length (0.158) were positively and significantly correlated with score
day milk yield. Bone structure was found to be negatively correlated (-0.219) with score day milk
yield and it was highly significant. Dairy form, horn diameter and tail length were not correlated
with score day milk yield. The results of the present study indicate that most of the linear type
traits and body measurements in Nili Ravi buffaloes fall under the intermediate value when
compared with other buffalo breeds. All the traits had variation among the herds. Positive
correlation between milk yield and other traits like height at sacrum, ear length and rump length
need further investigations to reach at some conclusion to include these traits in selection strategy
for improvement in milk yield.
Keywords: Linear type traits, morphometric measurements, Nili Ravi buffaloes
INTRODUCTION
Physical appearance of most of cattle breeds in different parts of the world has been
studied extensively but such type of information in buffaloes is very rare especially the concept of
linear type scoring in dairy buffaloes is very recent. Only some basic type of information related
to few body measurements in buffaloes is available. Linear type traits are the basis of all modern
type classification systems and the foundation of all systems for describing the dairy animals.
Animals of desirable type and conformation generally produce and reproduce for a longer period
of time. The dairy farmers use type classification as a management tool in breeding and selection
decisions. Type traits are very important because of their relationship with performance traits of
dairy cattle. Keeping in view the importance of linear classification in dairy cattle, the present
study was designed to develop some linear scoring system for Nili Ravi buffaloes in Pakistan
using the guide lines developed for cattle breeds (ICAR, 2010)

780
Nili Ravi buffalo herds maintained at 5 Institutional herds in Punjab and few private
breeders’ farms were utilized in the present study. The data included 227, 297, 172, 165, 269 and
50 recrds on Pattoki, Chack Katora, Haroonabad, Khushab, Rakh Ghulaman and private breeders,
respectively. Body measurements wre recorded in centimeters scale and the linear type traits were
recorded on a scale of 1-9 as per guidelines of the ICAR (2010). A total of 1180 records on some
linear type traits and body measurements were generated over a scoring period of 2 years.
Variation in these type traits and body measurements due to differences in herds was
studied using PROC MIXED procedures under SAS(2003) and the correlation of these traits with
score day milk yield and least square means were compred using LSD test and the pearson
correlationsmethod using SPSS program.(SPSS, 2004)

Mean score for height at sacrum was 135.766±4.401 cm. Similar findings have been
reported in Murrah (Andrea et al., 2010) Italian (Campanile et al., 2003) and Mediterranean
buffaloes {Negretti et al., 2008). Bone structure was scored as 5.344±1.787 measured on a linear
scale of 1-9 (ICAR, 2010 ). In Danish Holstein an average score of 5.31±1.51 has been reported
(Laursen et al., 2009) whereas a higher score of 6.69 ±0.84 for same breed (Lassen and Mark,
2008) and 7.57 ± 0.75 in Jersey cows (Boelling et al., 2001) has been reported. Dairy form was
recorded on a linear scale scale of 1-9 and its mean value was found as 5.619±1.203. Horn
diameter at the base of the horn was measured as 18.646±2.059 cm. Khan et al. (2009) in Azi
Khale buffalo has reported horn mid circumference as 20.1 ± 0.28 cm where as in Swamp
buffaloes a higher value (29.17 ± 0.49 cm) has been recorded (Kalita et al., 2010). The
differences in horn measurements are mainly due to genotype as well as environmental factors.
Ear length has been found as 29.5±2.118 cm in the present astudy. A simar value of 29.3±0.1 cm
has been reported in Banni buffaloes (Mishra et al., 2009) whereas Khan et al. (2009) in Azi khale
buffalo has reported ear length of 21.3± 0.25 cm in Azi Khale buffaloes. Average score for tail
length was found as 103.515±12.551 cm in the current study. In an earlier study Cockrill (1974)
has reported average tail length 49 inches (125cm) in Nili and 47.5 inches (121 cm ) in Ravi
breeds.
Rump length (43.516±2.582 cm) and average score day milk yield (6.85±2.19 kg) have
been recorded in the present study Comparable findings for rump length have been reported in
Azi Khale (Khan et al., 2009), Italian (Campanile et al., 2003), Tarai (Prasad and Kumar, 2005),
Mediterranean buffaloes (Negretti et al., 2008) and Murrah (Andrea et al., 2010). Various
researchers have reported different average daily milk in differentbbreeds and climatic conditions.
Most of the traits under study varied among different herds. These differences might be due to
hereditary as well as environmental / managemental factors. Herd effect was found to be highly
significant for dairy form and its score ranges from 4.3±1.07 in herd 1 to 6.43±1.04 in herd 6.
Horn diameter was significantly different in different herds and its score ranges from 17.88±2.12
cm in herd 1 to 19.02±1.88 cm in herd 2. Berthouly et al. (2010) has reported a significant effect
of herd on horn diameter in Vietnamese Swamp buffaloes. Average score for ear length ranges
from 28.5±1.95 cm in herd 5 to 30.26±1.58 cm in herd 6 and this variation was found to be
significant. In Chilika buffaloes, Patro et al. (2003) has reported a significant effect of herd on ear
length. Khan (2009) has reported a significant effect of herd on ear length in Sahiwal cows. Tail
length varied significantly across herds and its value ranges from 99.52±14.05 cm in herd 4 to
107.5±14.9 cm in herd 6. Patro et al. (2003) has reported a significant effect of herd on tail length
in Chilika buffaloes, Average score for rump length varies from 42.5±2.03 cm in herd 5 to
45.12±1.89 cm in herd 6 and this was a significant difference. Lin et al, 1983 has reported a
781
highly significant effect of herd on rump length in Holstein & ayreshire line. Khan (2009) has
reported a significant effect of herd on rump length in Sahiwal cows. Score day milk yield varies
across herds and it ranges from 5.37±1.58 kg in herd 5 to 7.88±1.93 kg in herd 1. I n a study on
factors affecting performance of Nili Ravi buffaloes in Pakisten, Cady et al. (1983) has reported a
highly significant effect of herd on lactation milk yield
Correlation of type traits and body measurements with score day milk yield
Height at sacrum was positively and significantly correlated (0.26) with score day milk
yield. Similar reports are available in literature for different breeds of cattle (Guernsey, 0.24;
Holstein, 0.29) as reported by Cruickshank and Weigel (2002) and Haas et al. (2007),
respectively. Bone structure was found to be negatively correlated (-0.219) with score day milk
yield being highly significant. This relationship shows that buffaloes with broader and thicker
bones of the hind legs especially cannon bone produce less milk. Further studies are needed to
verify this relationship. Dairy form was found to be not correlated with score day milk yiled
(0.04, non significant). Whereas most of the workers has reported a very high correlation (0.59) of
dairy form with milk yield (Harris, 1992 in Guernsey cows), 0.25 in Holstein grade and 0.29 in
Holstein registered (Short and Lawlor, 1992). The results of the cuurent study do not agree with
most of the reports available in the literature and the reason might be very small number of
records (452). However ear length and tail length was positively and significantly correlated
(0.163; 0.02) with milk yield. The probable reason of this correlation is that size of ear is more in
latter parity buffaloes who have more milk yiled than first calvers. Khan (2009) has reported a
positive correlation of rump length and tail length with score day milk yield in Sahiwal cows.
CONCLUSIONS
The most of the linear type traits and body measurements in Nili Ravi buffaloes fall under
the intermediate value when compared with other buffalo breeds. The effect of variation due to
herd was found significant for all traits. This variation can be minimized through providing better
management and recording practices. Some of the traits have been foud positively correlated with
milk yield such as height at sacrum, ear length and rump length but these relationships need
further investigation so that selection for better milk yield can be made on the basis of these traits.
REFERENCES
Andrea M V, R .S. Cerqueira, C.R. Marcondes, C.M. Macedo junior, D.R. Santos and K.N.
Oliveira. 2010. Correlations between linear measurements and milk production in Murrah
Buffaloes. Proceedings of the 9th World Buffalo congress, Argentina. 351-353
Berthouly C, X. Rognon, T. Nhu Van, A. Berthouly, H. Thanh Hoang, B. BedHom, D. Laloe, C.
Vu Chi, E. Verrier and J.C. Maillard. 2010. Genetic and morphometric characterization of
a local Vietnamese swamp buffalo population. J. Anim. Breed. & Genet. 127 (1): 74-84.
Boelling D, P. Madsen and J. Jensen. 2001. Genetic parameters of foot and leg traits in fture AI
bulls. Acta. Agric. Scand. Sect. A, Anim Sci.51:122-128
Campanile G, R. Dil Palo, C. De Rosa, V. Peretti, L. Amante, F. Ciotola and A. Coletta. 2003.
Preliminary results on Mediterranean Italian buffalo morfometry. Ital J Anim Sci. Vol.
2 (Suppl. 1) 337-339.
Cockrill W R. 1974.The husbandry and health of the domestic buffalo, FAO, Rome, Italy.
Cruickshank J.K,, A. Weigel, M.R. Dentine and B.W. Kirkpatrick. 2002. Indirect prediction of
herd life in Guernsey dairy cattle. J Dairy Sci. 85:1307–1313.
Harris B.L., A.E. Freeman and E. Metzger. 1992. Genetic and phenotypic parameters for type
and production in Guernsey dairy cows. J. Dairy Sci. 75(4): 1147-1153
782
Haas, Y de, L.L.G. Janss, H.N. Kadarmideen. 2007. Genetic and phenotypic parameters for
conformation and yield traits in three Swiss dairy cattle breeds. J. Anim. Breed. & Genet.
124 (1): 12-19.
ICAR, 2010. International Committee for Animal Recording , ICAR conformation working
group, version 4, May 2010.
Kalita R, A. Dandapat, B.D.C. Kamal, G.C. Das and R.N. Goswami. 2010. Conformation traits
of Swamp buffalo of Assam at different age groups.Indian J. anim. Res. 44(4): 300-302
Khan S.A,, M.S. Khan, Y. Muhammad, S. Sultan, F. Muhammad, S.A. Khan and G. Jabbar.
2009. On-Farm performance of Azi Kheli buffalo. Pak. J. Zool. Suppl.ser. No 9:209-212
Khan M.A. 2009. Characterization of Sahiwal cattle for linear type traits. Ph.D. thesis, University
of Agriculture, Faisalabad, Pakistan.
Lassen J. and T. Mark. 2008. Genotype by housing interaction for conformation and workability
traits in Danish Holsteins. J. Dairy Sci. 91:4424-4428.
Laursen, M. V, D. Boelling and T. Mark. 2009. Genetic parameters for claw and leg health, foot
and leg conformation, and locomotion in Danish Holsteins. J. Dairy Sci. 92:1770-1777.
Lin, C.Y., T.R. Batra, A.J. Mcallister, J.P.F. Darisse, A.J. Lee, G.L. Roy, J.A. Vesely and K.A.
Winter. 1983. Differences between sire groups in body measurements and body weight
changes of lactating cows. Can. J. Anim. Sci. 63:27-37
Mishra, B.P., K.P. Singh, C.B. Chavan, D.K. Sadana , R.S. Kataria, P. Kathiravan and S.P.S.
Ahlawat. 2009. Characterization of Banni buffalo of Western India. AGRI 44:77-86
Negretti, P., G. Bianconi, S. Bartocci, S. Terramoccia and M. Verna. 2008. Determination of live
weight and body condition score in lactating Mediterranean buffalo by Visual Image
Analysis: a Review article. Lives. Sci. 113 (1):1-7.
Patro, B.N., P.K. Mishra and P.K. Rao. 2003. Chilika buffaloes in Orissa: a unique germplasm.
AGRI 2003, 33: 73-79.
Prasad, R.B. and S. Kumar. 2005. Penotypic characterization of Tarai buffalo breed. Buffalo
newsletter 21:9-10
SAS Instt. Inc.2010. SAS/STAT user’s gude, Release 9.1.Carry, North Carolina, USA
Short T H, Lawlor T J, 1992. Genetic parameters of conformation traits, milk yield, and herd
life in Holsteins. J. Dairy Sci. 75:1987-1998.
SPSS Inc. 2004.SPSS for Windows release 13.0, Chicago, USA
783
Identification of Amino Acid Substitutions and Structural Model

Prediction of POU1F1 Gene in Azakheli Buffalo Breed of Pakistan
Asif Nadeema*, Maryam Javeda and Masroor Ellahi Babarb
a
Institute of Biochemistry and Biotechnology, bFaculty of Animal Production & Technology,
University of Veterinary and Animal Sciences Lahore 54000, Pakistan.
*Corresponding Email: asif_cemb@hotmail.com
ABSTRACT
POU class 1 homeobox 1 (POU1F1) is a member of the tissue-specific POU-containing
transcription factor family and is expressed in mammalian pituitary gland. This gene is known to
control the transcription of prolactin (PRL) gene which is associated with different production traits
in dairy animals. To evaluate the effect of this gene on different traits, a study was conducted using
30 animals of Azakheli buffalo breed of Pakistan. After DNA extraction, PCR and sequencing
analysis were carried out to examine the exons. After analysis of sequences, three amino acid
substitutions were found. First SNP in exonic region of the gene was a change of amino acid from
proline to leucine, second change was a leucine to serine and third was metheonine to threonine
substitution. Proline to leucine change was polar to polar conversion but remaining two were a
conversion of polar into non-polar amino acid. Using amino acid information structural model for
protein was predicted.
Keywords: amino acid substitutions, protein model, Azakheli buffalo
INTRODUCTION
The POU1F1 gene (also named PIT-1 or GHF-1) is a member of the POU homeodomain
family of transcription factors (Bodner et al., 1988; Ingraham et al., 1988) expressed in the pituitary
and its expression is vital for the normal differentiation, development and survival of three
adenohypophysis cell types, thyrotrophs, somatotrophs and lactotrophs (Li et al., 1990; Simmons et
al., 1990). It is also important for the proper expression of growth hormone (GH), prolactin (PRL)
(Nelson et al., 1988), thyroid-stimulating hormone (TSH) (Li et al., 1990) and POU1F1 gene itself
(Chen et al., 1990; McCormick et al., 1990).
According to Anfinsen’s (1973) thermodynamic hypothesis, proteins are not assembled into
their native structures by a biological process. Protein folding is a purely physical process that
depends only on the specific amino acid sequence of the protein and the surrounding solvent
(Anfinsen, 1973). This suggests that one should be able to predict, at least theoretically, the three-
dimensional (3D) conformation of a protein from its sequence alone.
Protein structure determination is an important area of research in molecular biology and
structural genomics. Understanding the proteins structure will shed light on protein function and
active sites which will facilitate the site-directed mutagenesis studies.

784

Inorganic DNA extraction method (Sambrook and. Russell., 2001) was performed on blood
samples of Azakheli buffalo. Primers were designed from Gene Bank Accession No. NC_007299.4
whole genome shotgun sequence by using software Primer3 (http://frodo.wi.mit.edu/). PCR
reaction mixture composition were followed as described by Maryam et al (2012) After the
amplification, PCR products were precipitated for sequencing on ABI genetic analyzer 3130XL. All
the sequences were aligned with the help of online software blast2 sequence
(http/www.ncbi.nlm.nih.com) and polymorphisms were identified. For protein modelling, the
Swiss-Model Workspace (Arnold et al., 2006) was accessed on the Web
(http://swissmodel.expasy.org/workspace/ ). Values of QMEAN score (Benkert et al., 2011) and bit-
score (Chen et al., 2009) of substitutions protein was presented.

DNA sequencing and Blast analysis revealed three novel polymorphisms in exonic region were
identified (Table-1). The Swiss-Model Workspace was used for protein structure homology
modelling. Structure of substitutions protein is presented in fig-1. E Value of standard protein was
2.71e-59 while E value of substitutions protein was 5.65342e-59. QMEAN Z-Score was -0.923 (fig-
3). Z-score of C_beta interaction energy and all-atom pairwise energy was 1.31 and 2.71. bit-score
values of substitutions protein was 344.0 (fig-2). Sequence Identity was 84.828%.
The E-value shows the significant score. In the Swiss Model Workspace the QMEAN4 score is
to evaluate the generated models. The global QMEAN4 scoring function ( Benkert et al. 2008) is a
linear combination of four structural descriptors using statistical potentials: The local geometry is
analysed by a torsion angle potential over three consecutive amino acids. Two distance-dependent
interaction potentials are used to assess long-range interactions: the first is a residue-level
implementation based on C-beta atoms only and the second an all-atom potential which is able to
capture more details of the model.
Sequence identity above 30% is a relatively good predictor of the expected accuracy because
the deviation from the least-squares curve relating sequence identity to the accuracy is relatively
small.
Three-dimensional protein structure is very useful sources of information for the functional
annotation of protein molecules. These structures are best determined by experimental methods
such as X-ray crystallography and nuclear magnetic resonance spectroscopy. However, the
experimental methods cannot always be applied. In such cases, prediction of the protein structure by
computational methods can frequently result in a useful model.
E-value of sequence containing substitutions reflects that these polymorphisms can further be used
for investigating the role of this gene in different traits in buffalo.
REFERENCES
Anfinsen C.B. 1973. Principles that govern the folding of protein chains. Science, 181(96), 223-
230.
Arnold K., L. Bordoli, J. Kopp and T. Schwede. 2006. The SWISS-MODEL workspace: a web-
based environment for protein structure homology modeling. Bioinformatics, 22(2), 195-
201.
Benkert P, M. Biasini and T. Schwede. 2011. "Toward the estimation of the absolute quality of
individual protein structure models." Bioinformatics, 27(3):343-50.
785
Bodner M., J.L. Castrillo, L.E. Theill, T. Deerinck, M. Ellisman and M. Karin. 1988. The pituitary-
specific transcription factor GHF-1 is a homeobox-containing protein. Cell 55:505–518
Chen C.C., Hwang JK, Yang JM: (PS)2-v2: template-based protein structure prediction server.
BMC Bioinformatics 2009, 10:366
Chen R, H. Ingraham, M.N. Treacy, V.R. Albert, L. Wilson and M.G. Rosenfeld. 1990.
Autoregulation of PIT-1 gene expression mediated by two cis-active promoter elements.
Nature. 346:583–586
Ingraham H., R. Chen, H.J. Mangalam, H.P. Elsholtz, S.E. Flynn, C.R. Lin, D.M. Simmons, L.
Swanson and M.G. Rosenfeld. 1988. A tissuespecific transcription factor containing a
homeodomain specifies a pituitary phenotype. Cell. 55:519–529
Li S., Crenshaw EB, E.J. Rawson, D.M. Simmons, L.W. Swanson and M.G. Rosenfeld. 1990.
Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-
domain gene PIT-1. Nature. 347:528–533
Maryam, J., M. E. Babar, A. Nadeem and T. Hussain. 2012. Genetic variants in Interferon Gamma
(IFN-γ) gene are associated with resistance against ticks in Bos taurus and Bos indicus. Mol.
Bio. Rep. 39(4):4565-4570.
McCormick A., H. Brady, L.E. Theill and M. Karin. 1990. Regulation of the pituitary-specific
homeobox gene POU1F1 by cell-autonomous and environmental cues. Nature 345:829–832
Nelson C., V.R. Albert, H.P. Elsholtz, L.I.W. Lu and M.G. Rosenfeld. 1988. Activation of cell-
specific expression of rat growth hormone and prolactin genes by a common transcription
factor. Science 239:1400–1405
Sambrook J and D.W. Russell. 2001. Molecular Cloning: A laboratory Manual, 3rd edn. Cold
Simmons D.M., J.W. Voss, H.A. Ingraham, J.M. Holloway, R.S. Broide, M.G. Rosenfeld and L.W.
Swanson. 1990. Pituitary cell phenotypes involve cell-specific Pit-1 mRNA translation and
synergistic interactions with other classes of transcription factors. Genes Dev 4:695–711
Figure 1. Structure of substitutions Protein.
786
Figure 2. Score components.
Figure 3. Estimated absolute model quality.
Table 1. List of identified Polymorphisms.

Sr. # Change in Amino acid Change in Codon Change in Amino acid
Nucleotide Position
1 C>T 92 CCC>CUC Proline to Leucine
2 T>C 117 UUG>UCG Leucine to Serine
3 T>C 124 AUG>ACG Methionine to Threonine
787
Polymorphisms in Osteopontin Gene in Amazon Buffaloes
Sebastião Tavares ROLIM FILHOa,b*, Haroldo Francisco Lobato RIBEIROa,b, Gregório

Miguel Ferreira de CAMARGOc, Diercles Francisco CARDOSOc, Raul Rusbel
ASPILCUETA-BORQUISc, Humberto TONHATIc, Kim de Borborema NUNESb, William
Gomes VALEb,d, Keitiane Colares de SOUSAa, Ellen Yasmin Eguchi MESQUITAb, José Luiz
BOARETTOa and Gisélia de Lourdes Cardoso de ALCÂNTARAa
a
Universidade Federal Rural da Amazônia, Belém, Brazil; bCurso de Pós Graduacão em Ciência
Animal, Universidade Federal do Pará, Belém, Brazil; cUniversidade Estadual Paulista,
Jaboticabal, Brazil; dUniversidade Federal Oeste do Pará, Santarém, Brazil
*Corresponding email:sebastiaorolim@hotmail.com
ABSTRACT
The aim of this work was to identify polymorphisms in the osteopontin gene. It was used in
this experiment 306 male buffaloes, older than 18 months, bred in two farms, one in the State of
Amapá and the other farm in the State of Pará. There was identified three SNP polymorphisms for
the region amplified by the primer OS4 (5ùpstream) and four SNP polymorphisms for the region
amplified by the primer OS9 (exon 5 to exon 6). The polymorphisms were in positions 1478, 1513
and 1611 in the region amplified by OS4 and positions 6690, 6737, 6925 and 6952 in the region
amplified by OS9. These data indicate that the osteopontin gene is important because it can have a
substantial influence on the reproductive traits of male buffaloes.
Keywords: SNP, reproductive traits, Bubalus bubalis
INTRODUCTION
Several researches have been recently performed in order to find some candidate genes that
could be used to select productive and reproductive traits in buffaloes (Gil et al., 2009; Clempson et
al., 2011; Silva, 2011). It was found a great amount of several proteins, mainly osteopontin, which
was presented in seminal plasma of buffaloes, and being considerably presented in high fertility
animals when compared to low fertility ones. The osteopontin gene was described in buffaloes by
Tantia et al. (2008) and according Cancel et al. (1997) it is a protein involved in various biological
events and presented in different tissues, for example, bovine seminal plasma.
Therefore, the aim of this work was to verify the existence of polymorphisms in the gene for
osteopontin in male buffaloes extensively bred in the Amazon region.

Animals
Three hundred and six bulls were evaluated, all of them with body condition score (BCS)
above 3, evaluated according Vale (2005), and ages ranging from 18 months to 60 months. From
the animals evaluated (n=306) only 226 animals (n=226) had ejaculated good quality samples.
There were also collected hair follicles from all animals, but only 226 animals amplified the desired
DNA fragment.
DNA extraction
and then it was quickly centrifuged. After that, there was added 500 μL of solution TE-Tween (50
Approximately 40 hair follicles / animal were deposited in a microcentrifuge tube (1.5 ml)
mM Tris, 1 mM EDTA, 0.5% Tween 20) in each sample, followed by incubation bath at 65oC for
1.5 hour with periodic agitation. Next, it was added 2 μ L proteinase K / tube (600 μ g / μ L) and
incubated at 55oC for 6 hours with periodic agitation. And finally the samples were incubated at 37o
C overnight.

788
Afterwards it was added one volume of PCI (phenol-chloroform-isoamyl alcohol) to 1

volume of sample and the tube was vigorously shaken for 10 seconds in an automatic shaker
machine. Subsequently, the tube was centrifuged for 10 minutes at 12,000 rpm at 23° C and the
μL. Then was done DNA precipitation with 1/10 sample volume of sodium acetate, 0.3 M
supernatant was transferred to a new tube. The end of this phase volume was approximately 300
(approximately 30 μ L) and cold absolute ethanol (approximately 1 mL). After mixing by inversion,
the tubes were placed in a freezer at -80° C for 1 hour and subsequently centrifuged at 4oC for 25
dried at room temperature, and then stored in 100 μ L TE (10:1). After the extractions, the DNA
minutes at 12,000 rpm. The supernatant was discarded and the remaining DNA was completely
samples were subjected to electrophoresis on agarose gel (0.8%) in 1X TBE buffer (89 mM Tris-
HCl, 2.5 mM EDTA and 89 mM Boric Acid, pH 8.3) with gelred (4μL/mL) to 100V for
approximately 50 minutes.
Verification of the quantity and quality of DNA obtained
The quantity and quality verification of the obtained material was done throughout the
spectrophotometer (Nanodrop 1000, Thermo Scientific, USA, 2008). The quantification was based
Thus, concentration is measured by the relation 1 OD260 = 50 μ g / ml DNA. Moreover, the quality
on absorbance arises from the fact that DNA has a peak absorbance of light at 260 nm in length.
was measured by the absorbance ratio A260/A280. The proteins showed peak absorbance of 280nm
and this may be possible due to contaminants of DNA solution. Therefore, it is expected that the
After finding these parameters, the stock solution was diluted to 70 μ g / mL solution in use. And
A260/A280 ratio is between 1.8 and 2.0. A ratio of less than 1.8 suggests protein contamination.
then both were frozen in a freezer.

The primers used
The OPN protein gene has about 9 kb and consists of seven exons with 72 bp, 68 bp, 39 bp,
81 bp, 42 bp, 306 bp and 727 bp (exons 1, 2, 3, 4, 5, 6 and 7 respectively) and six introns of 1086
bp, 118 bp, 2478 bp, 492 bp, 771 bp and 699pb (introns 1, 2, 3, 4, 5 and 6, respectively). The
coding region of the protein is in exons 2, 3, 4, 5, 6 and 7 (Tantia et al., 2006).
The primer pairs used were the OS4 which amplifies a region of 5'Upstream 508pb and OS9 that
amplifies a region of exon 5 to exon 6 of 890pb according Tantia et al. (2008).
Reaction Amplification of DNA by PCR (Polymerase Chain Reaction)
PCR (Polymerase chain reaction) reactions in a final volume of 15 μL containing 1.5 μ L DNA (70
The samples of genomic DNA are specified by each regions of primers that are amplified by
ng), 1.5 μ L of each primer (15 pM), 6.5 μ L of 2X GoTaq Colorless Master Miss Taq DNA
Polymerase and 4.0 water (nuclease free). The primers used were OS4
(5'CAGTAACCCTGCTCGGTCAT3 'and 5' AGCACTGACTTCCAG CATCC3 ') that amplified a
region of 508pb (nucleotide 1289 to nucleotide 1796) and OS9
(5'CCTCTGAGGAAACTGATGACAA3' and 5'AGAGTTGACGTCCTGGCTGT3 ') that amplified
a region of 890pb (nucleotide 6513-7402) followed what was described by Tantia et al. (2008).
Amplification cycles followed Mastercycler Gradient thermocycler programming ® 5331
Eppendorff, Germany, 2005. It was made a gradient PCR in order to identify optimal annealing
temperature of the primers that was 57 ° C for both pairs. The cycle was repeated from the second
removed from thermal cycler. After amplification, an aliquot of 3 μ l of each sample was diluted
to the fourth steps for 35 times. After the fifth step, the samples were kept at 4 ° C until they were
with 2 μ L of running buffer (4μL/mL of gel red) and were subjected to electrophoresis on agarose
gel (1.5%) in TBE buffer 1 X (89 mM Tris-HCl, 2.5 mM EDTA and 89 mM Boric Acid, pH 8.3) at
90 V for approximately 50 minutes. The display was made in UV light; the gel was submitted to
photodocumentation in Gel-Doc apparatus (Bio-Rad) and analyzed with the software Image
analysis of Kodak, to evaluate the efficiency of PCR depending on the size of the amplified
fragment.
Sequencing
789
The PCR products were amplified again to a final volume of 20 μL then subjected to
purification according to the protocol recommended by the kit Wizard SV Gel and PCR Clean-Up
System, Promega, USA. The PCR product was sequenced from both primers (forward and reverse)
using the chain termination technique for dideoxinucleotídeos (ddNTPs), using the ABI PRISM
BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) on an ABI 3730
XL automated sequencer (Applied Biosystems) in outsourced by the laboratory of Biochemistry and
Molecular Biology, Department of Technology FCAV. For the analysis and identification of
polymorphisms, the sequences obtained were analyzed and visualized with the programs
CodonCode Aligner.

PCR technique
Both sets of primers amplified the specific regions of the osteopontin gene (Figures 1 and 2).
Every size corresponds to the sizes given above. It was carried out a reaction in a thermal cycler
gradient and both had their primers optimized annealing temperature to 57 º C.
Sequencing
There were Identified three polymorphisms SNP type for the region amplified by primer
OS4 (5 'UTR) gene osteopontin through the sequencing of the 132 animals and 4 polymorphisms
SNP type for the region amplified by primer OS9 (exon 5 to exon 6) through sequencing of 176
animals. The polymorphisms were at positions 1478, 1513 and 1611 in the region amplified by OS4
and at positions 6690, 6737, 6925 and 6952 in the region amplified by the OS9 (Table 1). These
polymorphisms are described for osteopontin gene in buffaloes, and some of them were described
for the first time in buffaloes. The sequences and polymorphisms were submitted to the NCBI
(National Center for Biotechnology Information). Tantia et al. (2008) who had described for the
first time osteopontin gene SNPs in buffaloes, observed six SNPs, five in intronic regions and a
region upstream. SNPs in the intronic regions were first described, since the SNP in the region
upstream (indel) was reported by the same Schnabel et al. (2005) in B. taurus. Therefore in the
present study it was observed that two polymorphisms were also described by Tantia et al. (2008)
located in regions 1478 and 6925 bp gene osteopontin, however SNPs located in regions 1513,
1611, 6690, 6737, 6925pb are the first described. The osteopontin gene has several polymorphisms
like SNPs that can be used to assess reproductive traits of buffaloes.
REFERENCES
Cancel, A.M., D.A. Chapman and G.J. Killian. 1997. Osteopontin is the 55-kilodalton fertility-
associated protein in holstein bull seminal plasma. Biol. Reprod. 57:1293-1301.
Clempson, A.M., G.E. Pollot, J.S. Brickell, N.E. Bourne, N. Munce and D.C. Wathes. 2011.
Polymorphisms in the autosomal genes for mitochondrial function TFAM and UCP2 are
associated with performance and longevity in dairy cows. Animal. 5 (9); 1335-1343.
Gil, F.F.M., F.R.P. Souza, G. Stefani, H. Tonhati and L.G. Albuquerque. 2009. Caracterização de
polimorfismo no éxon 1 do gene da grelina em búfalos da raça murrah (Bubalus bubalis). In:
Proceedings 55º Congresso Brasileiro de Genética. Águas de Lindóia, São Paulo, Brasil. p.
192.
Schnabel, R.D., J. Kim, M.S. Ashwell, T.S. Sonstegard, C.P.V. Tassell, E.E. Connor and J.F.
Taylor. 2005. Fine-mapping milk production quantitative trait loci on BTA6: Analysis of the
bovine osteopontin gene. Proceedings of the National Academy of Sciences of the United
States of America. 102 (19): 6896–6901.
Silva, M.M. 2011. Proteínas do plasma seminal de touros Bos indicus e associações com parâmetros
seminais. M.Sc. Thesis. Universidade Federal do Ceará, Fortaleza, Ceará, Brasil.
Tantia, M.S., R.K. Vijh, B.P. Mishra, B. Mishra, S.T. Bharani Kumar and M. Sodhi. 2006. DGAT1
and ABCG2 polymorphism in Indian cattle (Bos indicus) and buffalo (Bubalus bubalis)
breeds. BMC Veterinary Research. 32 (2): 1746- 6148.
790
Tantia, M.S., B. Mishra, S.T. Bharani Kumar, B.P. Mishra, R.S. Kataria, M. Mukesh and R.K. Vijh.
2008. Characterization of Osteopontin gene of Bubalus bubalis. Animal. 7(2): 987–990.
Vale, W. G. 2005. The challenges and constrains for buffalo husbandry in world: dealing with
pathology hereditary problems. In: Proceedings Congrezzo Nazionale sullállevamento del
Bufalo, 3, Capaccio-Paestum, Italia. 1. p.20-30.
Figure 1. The fragments of 508 bp amplified by PCR using specific primers (OS4) for the region
5'UTR osteopontin gene in buffalos.
Figure 2. The fragments of 890 bp amplified by PCR using specific primers (OS9) for the region of
exon 5-6 of osteopontin gene in buffalos.
Figure 3. The output from sequential analysis of three animals with the program CodonCode
Aligner example of a SNP identified. The first sequence refers to an animal
heterozygous A / G (two peaks), the second refers to an animal homozygous A / A, and
the last to an animal homozygous GG.
Table 1. Indication of position, region and replacement of SNPs identified and accession number of
the sequences deposited in NCBI.
Localization (pb) primer Region SNP Access number to NCBI
1478 OS4 5’UTR T/C JQ613157
1513 OS4 5’UTR A/G JQ613157
1611 OS4 5’UTR A/T JQ613157
6690 OS9 Íntron 5 A/T JQ613157
6737 OS9 Íntron 5 A/G JQ613157
6925 OS9 Íntron 5 A/G JQ613157
6952 OS9 Íntron 5 C/T JQ613157
791
Relationship between Body Measurements and Milk Production in Nili-Ravi

Buffaloes Maintained at Commercial Farms in Peri-Urban Vicinity of Lahore
Nisar AHMADa*, Muhammad ABDULLAHa, Khalid JAVEDa, Muhammad Sulman

KHALIDa, Masroor Ellahi BABARa, Umair YOUNASa and NASRULLAHb
a
Department of Livestock Production, Faculty of Animal Production and Technology, University of
Veterinary and Animal Sciences, Lahore, Punjab, Pakistan.
bDepartment of Livestock Management, Faculty of Veterinary Sciences, Lasbela University of
Agriculture, Water and Marine Sciences, Uthal, Balochistan, Pakistan
*Corresponding author e-mail: nisarahmad@uvas.edu.pk
ABSTRACT
The aim of this study was to correlate the milk yield of Nili-Ravi buffalo with
morphometric measurements of buffalo with milk production. Data related to average heart girth
(HG), body length (BL), body height (BH), pin to pin distance (PP), hook to hook (HH) and body depth
(BD) were collected from Nili-Ravi buffaloes (n= 200) after random selection from commercial
dairy herds in peri-urban areas of the Lahore city. The buffaloes were selected at random basis in a
range from first to sixth parity. Theresult of this study showed that Nili-Ravi buffaloes had the
average 203.2±11.0 cm for HG, 147.3 ±7.2 cm for BL, 140.2±7.2 cm for BH, 30.2±3.7cm for PP,
56.9±4.5 cm for HH and 85.8±5.8 cm for BD, respectively. Milk production of these selected
animals ranged between 4 to 22 liters with an average of 12.3±34 liters per day. Positive significant
(P<0.05) phenotypic correlation was seen among milk yield and various body measurements that
considerably implies a that there is strong impact of measuring traits on milk yield. The present
study will be helpful as a selection tool to enhance and evaluate the production potential by setting
standards of Nili-Ravi buffalo breed.
Keywords: Nili-Ravi buffalo, milk production, body size, phenotypic correlation
INTRODUCTION
Pakistan is facing the milk shortage and buffalo farmers are receiving less income. This all
is due to poor selection criteria, let pass managemental systems prevailing in country, coming
through traditions developed by their intimates and lesser fodder production. So the genetic
potential of the animal would appropriately be judged and selection would be made in positive
direction. The body structure of milking animals is not only important to demonstrate the physical
beauty of dairy animals but also for their high milk productivity. Ugur (2005) found significant
relationship between body measurements of dairy animals and milk production. Bardakcioglu et al.
(2011) reported that Holstein cows with higher chest girth and wither height had more milk yield.
Lin et al. (1987) determined the positive correlations between few body measurements and milk
yield in different genotypes of Holstein cattle. Bardakcioglu et al. (2004) found that Holstein cow
with more body weight (BW) and body height (BH) produced more milk when compared to the
animals of lower wither height and body weight. There is immense need to develop certain
standards for such traits in the prime dairy animals of our country as no work has been done in
respect of this important selection criterion.

792

Study location
The present study was conducted in the peri-urban areas of Lahore district which is the
home tract and primary rearing area of Nili-Ravi buffaloes. Nili-Ravi buffaloes (n=200) were
selected for this study randomly. The various body dimensions along with milk yield were noted.
The animals from first to the sixth parity were selected. Animals were further grouped according to
the stage of lactation at the time of study being conducted.
Body dimensions
Various body measurements were recorded like body length (BL) which was taken as
distance from point of shoulder at one side of animal body moved along the animal’s body in
transectional/skewed direction up to the pin bone. Body height (BH) was measured as distance
from the withers of the animal to the surface of the platform while keeping animal in immobile
state. Heart girth (HG) was measured by taking the measurement of the circumference of the chest
with a tape rule. Pin to pin (PP) distance was measured as distance straight between two pin bones of
each side of animal’s body. Hook to hook (HH) distance was measured between two hook bones of
each side of animal’s body using measuring tape. Body depth (BD) of the animal was measured by
subtracting the two different measurements i.e. height at top line and height at bottom line.
Whereas, height at top line was measured by placing the side hand of the measuring scale/rod on
the ventral region of the animal at withers. The distance from the floor in straight direction was
measured in centimeters. And height at bottom line was taken by placing the side hand of the
measuring scale/rod on the dorsal region of the animal perpendicular to the place from where
height at top line was measured before.
Milk Yield
The milk production from each animal was noted on the day when its different
measurements were taken. Animals were hand milked with steel bucket underneath. Thereafter
milk yield was calculated.
The mean and standard error values of different body measurements were worked out. The
Pearson’s Correlation between the different measurements, lactation stage, parity and milk contents
was determined (Steel et al., 1997).
The animals which were calved hundred days before or less than hundred days were
included in this stage. 47 out of 200 animals were found in this stage. The animals which calved
beyond last hundred to two hundred days were included in this stage i.e. 76 animals out of 200 and
animals with last calving date passed beyond 200 days were also included in this stage. Mostly
animals were observed in this stage during the study i.e. 77% animals. More than two third animals
were found in mid or in late lactation stage implying that their peak production time has been
passed months before. Also, that in coming couple of months, calving season of the Nili-Ravi
buffaloes has to be started, showing that buffalo follows the seasonality behavior of calving. These
findings are in accordance with the findings of Hassan et al. (2010). The results of this study
showed that in commercially reared Nili-Ravi Buffaloes, the average HG was 203.2±11.0 cm, BL
147.3 ±7.2 cm, BH 140.2±7.2 cm, PP 30.2±3.7cm, HH 56.9±4.5cm and BD 85.8±5.8cm,
respectively (table-3). The relationship of BL with HG, PP and HH was found highly positive and
significant (P<0.01). Heart girth was found to have high positive and significant (P<0.01)
correlation with age, BW, parity and MP. This result coincides with the findings of Bardaakcioglu
793
et al. (2011) and Sieber et al. (1988). The findings of Ugur (2005) also suggested the significant
(P>0.05) relationship of body conformation traits like HG, BL, BH and chest depth with MP.
Distance between two hook bones also has highly positive significant association with PP and HG.
Among the body measurements, only the HG was significantly related with BD. whereas,
association among other body measurements was found weak and non-significant (table-4). Heart
girth was found to have high positive and significant correlation with age, BW, parity and MP at
level P<0.01. Body length and distance between two hook bones of animal body had high positive
and significant (P<0.01) association with BW, lactation and MP. Body length was also positively
and significantly related with age at level P<0.05. Body depth has positive relation and was
significant (P<0.05) with BW and parity. Kuczaj et al. (2000) found significant correlation between
chest depth with MP. Body height also showed positive and significant (P<0.05) correlation with
parity. The only high negative and significant (P<0.01) correlation was found between lactation
stage of animal with it’s hear girth (table-5).
REFERENCES
Bardakcioglu, H. E., S. Sekkin and H. D. Toplu. 2011. Relationship between some teat and body
measurements of Holstein cows and sub-clinical mastitis and milk yield. J. Anim. Vet. Adv.
10(13): 1735-1737.
Bardakcioglu, H. E., M. K. Turkyilmaz and A. Nazligul. 2004. The relationship between milk
production and some udder and body measurements in Holstein cow. Indian Vet. J. 81: 67-
71.
Hassan, F., M. S. Khan, M. S. Rehman, M. Sarwar and S. A. Bhatti. 2007. Seasonality of calving in
Nili-Ravi buffaloes, purebred Sahiwal and crossbred cattle in Pakistan. Italian J. Anim. Sci.
6(2):1298-1301.
Kuczaj, M., W. Kruszyński, E. Pawlina and J. Akińcza. 2000. Relations between milk performance
and udder dimensions of black-white cows imported from Holland. Elect. J. Pol. Agri. Uni.
3(2):1.
Lin, C. Y., A. J. Lee, A. J. Mcallister, T. R. Batra. G. L. Roy. J. A. Vesely. J. M. Wauthy and K.A.
Winter. 1987. Intercorrelations among milk production traits and body and udder
measurements in Holstein heifers. J. Dairy. Sci. 70: 2385-2393.
Sieber, M., A. E. Freeman and D. H. Kelley. 1988. Relationships between body measurements,
body weight, and productivity in Holstein dairy cows. J. Dairy. Sci. 71: 3437-3445.
Steel, R. G. D., J. H. Torrie and D. A. Dickey. 1997. Principles and Procedures of Statistics 3rd Ed.
McGraw-Hill Book Co. Inc., New York, U.S.A.
Ugur, F. 2005. Relationships between body measurement of dairy calves at six month of ages and
age at first calving and milk production. J. Cent. Europ. Agri. 6(2): 191-194.
Table 1. Mean ± standard deviations of age and body weight belonging to different parties.
Body Weight
Order of Lactation No. of Animals Age (Years) (Kgs)
Lactation No. 1 4 5.4±0.3 443.8±12.8
Lactation No. 2 32 7.4±0.8 486.7±62.9
Lactation No. 3 63 9.5±1.2 544.4±83
Lactation No. 4 53 11.6±1.4 584±81.9
Lactation No. 5 34 13.0±0.8 594.4±64.8
Lactation No. 6 14 13.85±0.74 592.8±65.3
794
Table 2. Mean± S.D, C.V and range of various production parameters.
Variables No. of Animals Mean ± S.D C.V (%) Range

Age 200 10.5±2.3 22.7 (4.5-15)
Body Weight (kgs) 200 554.5±84.2 15.1 (350-700)
Lactation No. 200 3.6±1.2 36.13 (1-6)
Milk Production (Liters) 200 12.3±3.4 28.2 (4-22)
S.D=standard deviation C.V=coefficient of variations
Table 3. Mean with S.D, C.V and range of various Body Measurements.
Variables No. of Animals Mean(cm) ± S.D C.V (%) Range
(cm)
Heart Girth 200 203.2±11.0 5.4 172-227
Body Length 200 147.3 ±7.2 4.8 123-173
Body Height 200 140.2±7.2 4.3 128-150
Pin to Pin distance 200 30.2±3.7 12.3 15.5-39
Hook to Hook distance 200 56.9±4.5 7.86 36.5-73
Body Depth 200 85.8±5.8 6.8 72-100
S.D=standard deviation C.V=coefficient of variations
Table 4. Phenotypic correlations among body measurements.

HG BL BH PP HH BD
HG 1 0.416** 0.128 0.073 0.333** 0.175*
BL 1 0.102 0.206** 0.282** 0.118
BH 1 0.045 0.049 0.039
PP 1 0.597** -0.078
HH 1 0.006
BD 1
* Significant at P< 0.05 ** Significant at P< 0.01
HG= heart girth, BL= body length, BH= body height, PP= pin to pin distance, HH= hook to hook distance,
BD= body depth
Table 5. Phenotypic correlations between body measurements and age, BW, LN & MP.
AGE BW LN LS MP
HG 0.233** 0.664** 0.479** -0.198** 0.636**
BL 0.144* 0.362** 0.318** -0.101 0.289**
BH 0.095 0.127 0.172* 0.027 0.203**
PP 0.040 0.131 0.087 -0.043 0.059
HH 0.103 0.328** 0.196** -0.064 0.358**
BD 0.081 0.163* 0.146* -0.010 0.132
HG= heart girth, BL= body length, BH= body height, PP= pin to pin distance, HH= hook to hook distance,
BD= body depth, BW= body weight, LN= lactation no., LS= lactation stage and BCS= body condition score
795
Buffalo Milk Transcriptomics
Sanjana KURUPPATH,a Amit KUMAR,a Vengama Naidu MODEPALLI,a Ngo Khanh

PHUONG,b Sally Louis GRASc and Christophe LEFEVREa*
a
Centre for Biotechnology and Interdisciplinary Sciences, Deakin University, Australia
b
National Institute of Hygiene and Epidemiolog, Hanoi, Vietnam
c
VBio21 Institute, Melbourne University, Australia
*Corresponding email: clefevre@deakin.edu.au
ABSTRACT
We have used high throughput sequencing technology to investigate the nucleic acid content of
buffalo milk. Buffalo milk contains cells from which messenger RNA can be isolated for transcriptome
sequencing and, relatively large amount of extracellular small RNAs including significant amounts of
micro-RNA (miRNA). The transcriptome of cells isolated from buffalo milk or colostrum contains all
major milk protein transcripts at relatively high levels, indicating that milk cells encompass a
significant proportion of mammary epithelial cells, and milk cell transcriptomics provides insight onto
the lactating mammary gland transcriptome. Skim milk from both colostrum and milk contain
significant concentrations of small RNA (> 200 ng/ml) with a large proportion of miRNA, a class of
regulatory molecules that have been proposed as powerful markers of milk and milk product quality
and, potential signalling molecules. Over 300 putative miRNAs were identified and quantified by
RNA sequencing. Comparisons with similar data from other mammals (cow, pig, human, and wallaby)
are revealing conserved or lineage-specific milk miRNAs together with differential expression between
animals and during lactation. We have also discovered that a significant proportion of these miRNAs
may be packed inside exosomes, suggesting a general mechanism of regulatory miRNA transmission to
the infant through a new milk exosome pathway. These exciting results will assist investigating the full
role of milk miRNA molecules by comparative lactation biology and the development of new uses for
these promissing biomarkers to assess lactation and milk quality.
Keywords: lactation, markers, milk, miRNA, transcriptome
INTRODUCTION
Recent studies have reported transcriptome analysis of mRNA isolated from milk cells or milk fat
globules and the presence of small RNA in bovine, human and other milk. The identification of high
levels of a large number of miRNAs in the milk of eutherian mammals (human, mouse, cow and pig )
(Chen et al., 2010; Hato et al., 2010; Kosaka et al., 2010; Gu et al., 2012; Zhou et al., 2012) and the
recent observation that exogenous miRNAs consumed from plant foods may directly influence gene
expression in animals (Zhang et al., 2012), raise new questions about the use of miRNAs as biomarkers
and their putative role in mammary gland physiology together with their potential effects on the young
(Kumar et al., 2012). Here, we explore buffalo milk RNA content, showing that RNA isolated from
colostrum cells contains high level of milk protein transcripts revealing in part the mammary epithelial
cell transcripome and, that buffalo colostrum and milk contain a large population of miRNA with
similarity and differences in composition with the milk of other mammals.

Milk fractionation
Buffalo milk and colostrum were collected at the Shaw River Dairy in the state of Victoria,
Australia. Approximately 2 to 4 litres of milk or colostrum were collected from different animals at the
farm, stored on ice and brought to the lab (3 to 12 hours). The samples were filtered through a large
796
gauge (150 μm) to remove hair and impurities and subjected to centrifugation at 2,000 g for 15 minutes
at 4°C to pellet cells and separate fat and skim milk fractions. Cell pellets were washed 2-3 times in
PBS before RNA extraction. Fractions were stored at -80°C. A purified exosome fraction was prepared
with ExoQuickTM solution (SBI-System Biosciences) following manufacturer’s instructions.
RNA preparation and sequencing
Cellular RNA was prepared with the RNeasy minikit (Qiagen, Sydney, Australia) following
manufacturer’s instructions. Total skim milk RNA extractions were performed with the Ambion
miRNA Isolation Kit (Life Technologies), according to manufacturer instruction for body fluids. RNA
quality and quantity were evaluated on the Agilent Bioanalyser. RNA sequencing (RNA-seq
quantification procedure) was contracted from BGI, Shenzen. About 10 to 20 Million reads (50
nucleotide maximum read length), corresponding to over 2 Gbytes of data were obtained for each
sample. Poly-A selection or size selection of fragments below 40 nucleotides were applied before
mRNA or small RNA sequencing, respectively.
Bioinformatics analysis
Because an annotated genome sequence of buffalo is not yet available, read mapping was
performed against the closely related bovine genome reference (version UMD3.1, Simin et al., 2009)
using Bowtie2 (Lanmead and Salzberg, 2012) for small RNA and Tophat (Trapnell et al., 2009) for
transcriptome data. Cufflink (Trapnell et al., 2010) and SeqMonk software
(www.bioinformatics.bbsrc.ac.uk/projects/seqmonk, RPKM quantification pipeline for RNA-seq
function) were used for transcriptome quantification and sequence annotation. Alignment details were
also visualized and investigated further in SeqMonk. DSAP (Deep Sequencing Small RNA Analysis
Pipeline) (Huang et al., 2010) was used for comparative miRNA annotation against the miRBase
miRNA reference database (Griffiths-Jones, 2006). Functional cluster analysis was done with the
online tool DAVID (Huang da et al., 2009a,b).
RESULTS
The colostrum cell transcriptome contains significant levels of mammary epithelial mRNA.
About 25-40 Million cells were recovered by centrifugation from 2 litres of colostrum, leading to
0.5 to 4 ug of total RNA after purification. RNA sequencing was performed on a colostrum sample.
From a total of 7.457.316 reads, 5.472.416 reads could be mapped onto the publicly available bovine
genome sequence (version UMD3.1) and 45% of the reads mapped into 16117 known gene transcripts.
Annotation against the known genes of the bovine genome identified 16117 gene transcripts with
expression values above 1 RPKM (Read per Kilobase per Million). Table 1 shows the results for the
most highly expressed genes; Kappa-casein, Beta-lactoglobulin, Beta-casein, actin, Alpha-S1-casein,
Alpha-S2-casein and Alpha-lactalbumin (20% of total reads). Ribosomal proteins were enriched.
A number of unannotated genes can also be newly identified in colostrum, including non-coding RNA.
However, the data did not show high expression of miRNA genes, including miRNA sequences
identified below at high concentration in milk. Functional analysis of highly expressed genes identified
ribosomal function and translation, cytoskeleton, cell mobility, membrane vesicles, angiogenesis, milk
and mammary gland and S100 calcium proteins as enriched functionalities, confirming the
predominance of exfoliated mammary epithelial in the colostrum of healthy animals. Less significant
clusters (stress response, myofibril assembly, apoptosis, leukocyte migration, positive regulation of
biosynthesis, respiration, skin development, antigen presentation and oestrogen response) suggest the
presence of other cell types (myoepithelial, skin and immune cells). These results show how
quantification of gene expression by milk cells transcriptomics provides a useful non-invasive
methodology to analyse gene expression during lactation in buffalo, revealing the expression of new
and known genes, and allowing comparison with data from other sources or other species.
Colostrum and milk contain significant amount of small RNA including large amount of miRNA.
We estimated skim milk RNA concentration to range 0.1 to 1 μg/ ml, similar to other milk
797
(Maningat et al., 2009), with slightly higher concentration in colostrum, with micro-RNA (18-26 nt)
representing over 50% of total RNA. Small RNA sequencing of colostrum and milk a variety of small
RNAs and RNA fragments from different origins; tRNA, miRNA, gene transcript, rRNA, snoRNA,
snRNA and miscellaneous RNA. Remarkably, a large number of miRNAs are found at high
concentration followed by a number of tRNAs, a miscellaneous RNA as well as two genomic regions
of unknown function, likely to represent novel miRNA species (table 2). In total, 314 and 294 putative
miRNA sequences were identified in buffalo colostrum and milk respectively, accounting for 30% of
all mapped reads from colostrum and 44% in milk, indicating highest relative abundance in milk than
in colostrum. For example the 10 most abundant miRNA represent one fifth (18%) and one third (33%)
of all mapped reads from colostrum and milk respectively. MiR-148a and miR-30a are the most
abundant in both colostrum and milk. However, while these miRNA are detected at similar abundance
in milk (10% of mapped reads), in the colostrum miR-30a is 2.8 fold more abundant than miR-148a.
Most of the high concentration miRNAs are found at comparable levels in colostrum and milk (miR-
30a, miR-191, miR-141, miR-181a-1, miR-22, let-7a-1, miR-182, let-7a-2, miR-181a-2, miR-21),
others are apparently enriched in either milk (miR-148a, miR-375, let-7f-2, miR-143) or colostrum
(miR-27b, miR-186, miR-26a-2, miR-26c). One novel putative miRNA-like sequence was also
enriched in milk. While most miRNA enrichment ratios in colostrum are around ten fold and do not
exceed 27-fold, some milk miRNA are apparently highly (200-450 fold) enriched in milk (miR-375,
miR-411 and miR-381) (table 3). The new putative miR-like sequence was also enriched 19-fold in
milk. These results show the differential abundance of discrete miRNA species and additional
experiments will be necessary to infer the variability between colostrum and milk samples from
different animals and confirm the identities of colostrum or milk enriched miRNA sequences.
Nevertheless, it is clear that milk is a rich source of information on miRNA, providing numerous
markers to assess lactation and new potential milk functionalities associated with miRNA.
Exosome small RNA profiling
A number of publications have recently reported that milk miRNA may be packaged inside
exosomes, conferring stability and resistance to RNAse activity (Chen et al., 2010; Kosaka et al.,
2010). In order to evaluate the contribution of the exosome fraction to colostrum RNA content, we
purified exosome from the skim milk using the ExoQuickTM reagent. The RNA-seq profile of exosome
was very similar to the profile from total skim colostrum. Amongst the 235 most highly represented
overlapping contigs, the majority (133) had quantification within 2-fold change and all contig had
differential quantification below 5-fold, suggesting that there is no highly significant difference
between small RNA prepared from ExoQuick exosome and skim milk, with only a modest enrichment
(3-fold) of small RNAs, such as tRNA, and selective miRNAs and a modest (2-3 fold) under
representation of mRNA sequences in exosomes. It is not clear whether this small difference is due to
technical variation only or means that small RNA is concentrated in exosomes because ExoQuick may
not allow a sufficient purification of exosomes. In future experiments the purification and
characterization of milk exosome and exosome RNA will need to address this using alternative
separation and purification methods such as centrifugation and biochemical analysis.
Comparative analysis of milk miRNAs
In order to identify conserved or specific milk miRNA, a number of small RNA sequencing
datasets were retrieved from the publicly available Gene Expression Omnibus Database GEO (Barrett
et al., 2011), including data on human and pig milk (Gu et al., 2012; Zhou et al., 2012). These data
were re-processed using the online miRNA analysis pipeline for comparative analysis DSAP (Huang et
al., 2010), together with data that we generated on buffalo and one peak lactation milk sample from a
marsupial, the tammar wallaby. Unfortunately bovine milk RNA data (Hata et al., 2010) were not
publicly available for direct comparison. Table 4 presents the quantification results (expressed in
percentage of total miRNA) of the most important miRNA identified. It can be seen that miR-148a is
consistently present at the highest concentration in the milk of all the species. However, while some
798
miRNAs are found at comparable levels in all species (for example miR-30a, mir101, miR-3596d),
others are highly enriched in particular species, with miR-181a and miR-27b apparently enriched in
wallaby milk while miR-486, miR-185 and miR-103 are enriched in pig, miR-30b in humans or miR-
423 in buffalo. It should also be noted that, when duplicate samples are available (pig and human) the
abundance of particular miRNA may vary significantly between individuals, for example miR-101, let-
7, miR-21 and miR-30b in human. Although the cause of such variation (technical, genetic or
physiologic) remains to be established, the results confirm high abundance of miR-148a in mammalian
milk and suggest additional universal milk miRNA markers. They also highlight potential differences
between milk miRNA profiles from different species, and the requirement for additional experiments to
control further for technical, genetic and physiological factors affecting milk miRNA content.
DISCUSSIONS
In this paper we have presented preliminary results on the transcriptome analysis of buffalo milk.
We have shown that milk cells transcriptome sequencing predominantly shows a mammary gland
epithelium transcription profile and suggests the presence of other cell types. Thus, transcriptome
profiling provides a non invasive methodology to investigate lactation in buffalo to characterise
changes in gene expression associated with the transition between colostrum and milk synthesis. We
have also shown that colostrum and milk contains a relatively large amount of small RNA, with a large
proportion of miRNA with over 300 known and novel miRNA identified in buffalo colostrum and
milk. Buffalo colostrum and milk miRNA could be secreted in exosomes, although this needs to be
confirmed. Surprisingly, despite high level of milk protein transcripts in the transcriptome of colostrum
cells, the expression of miRNA precursors could not be identified in cell data.
miRNA sequences could be developed into universal milk miRNA markers while other miRNAs
may be found at variable levels in the milk of different species. These observations suggest that milk
transcriptomics and miRNA analysis provides an alternative method to evaluate milk quality and
analyse milk origin as well as lactation physiology. One limitation of the study is the reliance on the
bovine reference genome sequence for annotation and access to the buffalo genome sequence would
benefit the accuracy of the analysis in the future. The results highlight how further experiments are
needed to fully characterise the biogenesis and identify factors influencing milk miRNA composition.
Nevertheless the present report illustrates how milk transcriptomics, including the integration of cell
transcriptome and milk miRNA data within a comparative framework, has the potential to allow the
development of new methodology to analyse lactation in greater details, while providing new quality
biomarkers and novel functional miRNA candidates potentially regulating the development of the
young during lactation, or carrying additional health benefits associated with the consumption of milk.
ACKNOWLEDGMENTS
The authors would like to thank Shaw River Dairy for kindly providing milk samples. Funding
was provided by Rural Industries Research and Development Corporation (RIRDC) in Australia
(Project PRJ-005364).
REFERENCES
Chen, X., C. Gao, H. Li, L. Huang, Q. Sun, Y. Dong, C. Tian, S. Gao, H. Dong, D. Guan, X. Hu, S.
Zhao, L. Li, L. Zhu, Q. Yan, J. Zhang, K. Zen and C.Y. Zhang. 2010. Identification and
characterization of microRNAs in raw milk during different periods of lactation, commercial
fluid, and powdered milk products. Cell Res. 20(10): 1128-37.
Hata, T, K. Murakami, H. Nakatani, Y. Yamamoto, T. Matsuda and N. Aoki. 2010. Isolation of bovine
milk-derived microvesicles carrying mRNAs and microRNAs. Biochem. Biophys. Res.
Commun. 396(2): 528-33
799
Kosaka, N., H. Izumi, K. Sekine and T. Ochiya. 2010. MicroRNA as a new immune-regulatory agent
in breast milk. Silence 1(1): p. 7.
Zhou, Q., M. Li, X. Wang, Q. Li, T. Wang, Q. Zhu, X. Zhou, X. Wang, X. Gao and X. Li. Immune-
related MicroRNAs are Abundant in Breast Milk Exosomes. Int. J. Biol. Sci. 2012. 8(1): 118-
23.
Gu, Y., M. Li, T. Wang, Y. Liang, Z. Zhong, X. Wang, Q. Zhou, L. Chen, Q. Lang, Z. He, X. Chen, J.
Gong, X. Gao, X. Li and X. Lv. 2012. Lactation-related microRNA expression profiles of
porcine breast milk exosomes. PLoS One 7(8): pp. e43691.
Zhang, L., D. Hou, X. Chen, D. Li, L. Zhu, Y. Zhang, J. Li, Z. Bian, X. Liang, X. Cai, Y. Yin, C.
Wang, T. Zhang, D. Zhu, D. Zhang, J. Xu, Q. Chen, Y. Ba, J. Liu, Q. Wang, J. Chen, J. Wang,
M. Wang, Q. Zhang, J. Zhang, K. Zena and C.Y. Zhang. 2012. Exogenous plant MIR168a
specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by
microRNA. Cell Res. 22(1): 107-26.
Kumar, A., L. Buscara, S. Kuruppath, K.P. Ngo, R.K. Nicholas and C. Lefèvre. 2012.
The emerging role of micro-RNAs in the lactation process. In: Lactation Natural Processes (Ed.
M. Lisa, R. Cruz, C. Douglas and O. Gutierrez). Physiological Responses and Role in
Maternity, Nova publishing, Series: Obstetrics and Gynecology Advances.
Aleksey, V.Z., L.D. Arthur, L. Florea, D.R. Kelley, M.C. Schatz, D. Puiu, F. Hanrahan, G. Pertea, C.P.
Van Tassell, T.S. Sonstegard, G. Marçais, M. Roberts, P. Subramanian, J.A. Yorke and S.L.
Salzberg. 2012. A whole-genome assembly of the domestic cow, Bos taurus. Genome Biol.
2009. 10(4): pp. R42.
Langmead, B. and S.L. Salzberg. Fast gapped-read alignment with Bowtie 2. Nat Methods. 9(4): 357-
359.
Trapnell, C., L. Pachter and S.L. Salzberg. 2009. TopHat: discovering splice junctions with RNA-Seq.
Bioinformatics 25(9): 1105-1111.
Trapnell, C., B.A. Williams, G. Pertea, A. Mortazavi, G. Kwan, M.J. van Baren, S.L. Salzberg, B.J.
Wold and L. Pachter. 2010. Transcript assembly and quantification by RNA-Seq reveals
unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol.
28(5): 511-515.
Huang, P.J., Y.C. Liu, C.C. Lee, W.C. Lin, R.R. Gan, P.C. Lyu and P. Tang. 2010.
DSAP: deep-sequencing small RNA analysis pipeline. Nucleic Acids Res. 38: W385-W391.
Griffiths-Jones, S. 2006. miRBase: the microRNA sequence database. Methods Mol. Biol. 342: 129-
138.
Huang da, W., B.T. Sherman and R.A. Lempicki. 2009. Systematic and integrative analysis of large
gene lists using DAVID bioinformatics resources. Nat Protoc. 4(1): 44-57.
Huang da, W., B.T. Sherman and R.A. Lempicki. 2009. Bioinformatics enrichment tools: paths toward
the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37(1): 1-13.
Maningat, P.D., P. Sen, M. Rijnkels, A.L. Sunehag, D.L. Hadsell, M. Bray and M.W. Haymond. 2009.
Gene expression in the human mammary epithelium during lactation: the milk fat globule
transcriptome. Physiol. Genomics. 37(1): 12-22.
Barrett, T,, D.B. Troup, S.E. Wilhite, P. Ledoux, C. Evangelista, I.F. Kim, M. Tomashevsky, K.A.
Marshall, K.H. Phillippy, P.M. Sherman, R.N. Muertter, M. Holko, O. Ayanbule, A. Yefanov
and A. Soboleva. 2011. NCBI GEO: archive for functional genomics data sets--10 years on.
Nucleic Acids Res. 39: D1005-10.
800
Table 1. Estimated gene expression value (RPKM) of the top 40 highly expressed genes in buffalo
colostrum cells. Annotation based on known genes.
___________________________________________________________________________________
____________
Feature RPKM Description
___________________________________________________________________________________
____________
A3FJ56_BOVIN 25003.654 kappa-casein precursor
E7E1Q6_BOVIN 24943.867 beta-lactoglobulin precursor
CASB_BOVIN 23101.137 Beta-casein
CASA1_BOVIN 15518.425 Alpha-S1-caseinAntioxidant peptide
ACTG_BOVIN 8668.58 Actin, cytoplasmic 2Actin
CASA2_BOVIN 8300.328 Alpha-S2-caseinCasocidin-1
LALBA_BOVIN 6650.6323 Alpha-lactalbumin
GLCM1_BOVIN 5580.567 Glycosylation-dependent cell adhesion mol. 1
LOC100138493 5008.327 No description
Q9TTW4_BOVIN 3994.985 Bos taurus actin, beta (ACTB), mRNA.
A1L586_BOVIN 2062.3418 Cell division cycle 2-like 1 (PITSLRE proteins)
FTH1 1980.6294 No description (ferritin)
RS8_BOVIN 1926.653 40S ribosomal protein S8
CLD4_BOVIN 1896.1378 Claudin-4
TCTP_BOVIN 1803.7678 Translationally-controlled tumor protein
FRIH_BOVIN 1659.6805 Ferritin heavy chain
F1MDN4_BOVIN 1472.7244 60S acidic ribosomal protein P0
F1MSH2_BOVIN 1440.4453 40S ribosomal protein S17
A6QLZ0_BOVIN 1318.026 galectin-3
RLA1_BOVIN 1304.2863 60S acidic ribosomal protein P1
RSSA_BOVIN 1289.9564 40S ribosomal protein SA
SAT1_BOVIN 1257.0404 Diamine acetyltransferase 1
K1C19_BOVIN 1199.5497 Keratin, type I cytoskeletal 19
F1N3A1_BOVIN 1182.7985 thrombospondin-1 precursor
GBLP_BOVIN 1101.0466 Guanine nucleotide-binding protein,beta-2-like 1
UBB_BOVIN 1087.1433 Polyubiquitin-BUbiquitin
S10A2_BOVIN 998.4555 Protein S100-A2
RL23_BOVIN 997.4421 60S ribosomal protein L23
F1MXZ1_BOVIN 995.5218 cyclic AMP-dependent transcription factor ATF-4
Q6KDN8_BOVIN 986.4321 Heat shock 70kDa protein 5
FABPH_BOVIN 986.3396 Fatty acid-binding protein, heart
MYL6_BOVIN 955.821 Myosin light polypeptide 6
RL13A_BOVIN 949.2817 60S ribosomal protein L13a
F1MI46_BOVIN 917.4327 osteopontin precursor
___________________________________________________________________________________
____________
801
Table 2. Annotation and estimated concentration value (Read per million mapped read) of the most
abundant small RNA species in buffalo colostrum and milk whey fractions, showing the predominance
of miRNA sequences in small RNA sequencing data.
Feature colostrum milk

bta-miR-148a 24443.84 118233.15
bta-miR-30a 69152.28 105874.38
bta-miR-375 146.55 29746.38
bta-miR-27b 24243.83 5594.37
bta-miR-191 20458.34 16623.43
bta-let-7f-2 5910.77 16416.83
bta-miR-141 15563.88 14058.34
miR-like 606.06 11735.43
bta-miR-186 11394.07 3798.19
bta-miR-181a-1 10255.10 5582.12
tRNA 10020.96 0.68
bta-miR-26a-2 9965.26 1829.36
tRNA 9927.73 59.15
bta-miR-22 6816.38 8447.21
tRNA 1726.28 8436.12
tRNA 7270.51 0.85
bta-miR-26c 6878.14 1773.96
miR-like 6746.99 6393.33
tRNA 6682.40 575.44
bta-let-7a-1 3600.00 6494.77
tRNA 1131.36 6184.01
bta-miR-182 4935.77 5915.77
misc_RNA 5910.13 79.54
tRNA 1202.44 5716.02
tRNA 5566.68 71.52
tRNA 1171.30 5549.37
bta-let-7a-2 3293.71 5545.06
bta-miR-143 657.46 5391.12
bta-miR-181a-2 5329.26 3037.49
bta-miR-21 5266.73 3371.22
tRNA 986.47 5110.74
Table 3a. List of colostrum enriched miRNA above 500 mapped read per million. Sorted by decreasing
abundance in colostrum.
colostrum milk Ratio C/M
bta-miR-27b 24243.828 5594.369 4.333612602

bta-miR-26a-2 9965.256 1829.3616 5.447395419
802
bta-miR-99a 1592.4901 300.1166 5.306237976

bta-miR-183 1441.9 108.89174 13.24159206
bta-miR-19b-2 1175.0803 118.11955 9.948228723
bta-miR-200a 1057.6003 262.68402 4.026131091
bta-miR-23b 958.7738 35.10548 27.3112289
bta-miR-30b 834.5939 103.40785 8.070895005
bta-miR-93 754.83295 62.455097 12.08601037
bta-miR-378 647.4084 95.603294 6.771821063
bta-miR-19b 568.63574 60.080967 9.464490477
Table 3b. List of milk enriched miRNA above 500 mapped read per million. Sorted by decreasing
abundance in milk.
col milk Ratio M/C
bta-miR-148a 24443.84 118233.15 4.836930286

bta-miR-375 146.54626 29746.377 202.9828465
miR-like 606.05585 11735.431 19.36361311
bta-miR-143 657.4562 5391.1196 8.199967694
bta-miR-486 98.79269 2592.2058 26.23884217
bta-miR-10b 35.121883 2096.867 59.70257916
bta-miR-411 5.1458964 1136.221 220.8013749
bta-miR-381 1.9912229 901.4174 452.6953763
bta-miR-340 113.21476 602.1967 5.319065288
803
Table 4. Comparative abundance of milk miRNA (% of total miRNA content, ‘-‘ when <0.1%) in
wallaby (t3: late lactation), Buffalo (bce: colostrum exosome, bc: colostrum or b: milk), human (4
subjects) and pig (lactation day 0 to 27) milk.
mir wal buffalo Human pig

t3 bce bc bm H1 h2 h5 h4 p0a p0b p3 p7 p14 p21 p28
148a 22.9 26.8 11.3 30 35.1 27.5 25.7 36.5 33.6 31.6 36.6 65.4 33.1 57.8 29.5
30a-5p 6.5 15.9 20.5 28.3 5.1 1.2 4.3 1.2 6.2 6 4.8 6.4 6.2 8.8 5.9
181a 20.1 6.3 8.2 3.9 0.6 0.5 1.3 0.7 1.3 1.3 0.9 1.5 1.3 1.8 1.2
486-5p - - - - - - - - 8.2 10.2 14.3 0.9 8.4 1.4 8.9
185 14.2 - 0.1 6.2 3.3 1.6 0.9 1.3 0.5 0.5 0.5 0.7 0.7 0.7 0.5
103 - - - - - - - - 5.7 6 13.7 1.3 6.1 1.6 5
101 0.2 2.9 2.4 3 1.5 10 2.2 4.1 0.7 0.7 0.7 0.2 0.6 0.2 0.7
23a 0.1 0.6 1.2 1.6 5.8 1 9.2 2.3 0.7 0.7 0.5 0.3 0.7 0.3 0.7
24 7 8.9 7.8 2.2 0.2 1.1 0.2 0.5 0.8 0.8 0.7 1 0.9 1 0.8
let-7 0.1 4 2.4 2.1 1.2 8.4 1.2 4 1.5 1.5 1.5 0.7 1.4 0.8 1.5
200a 0.2 0.4 0.3 - 1.3 1.4 7.9 5.9 0.6 0.7 0.5 0.3 0.6 0.3 0.7
3600 1.1 3.8 7.3 0.9 0.7 0.4 1.8 1 0.6 0.7 0.4 0.3 0.6 0.4 0.7
192 0.5 0.1 0.3 - 6.6 2.2 2.7 2.1 0.2 0.2 0.2 0.2 0.2 0.2 0.2
21 0.1 - - - 2.4 6.2 1.9 1.6 0.1 0.1 0.1 - 0.1 - 0.1
30b-3p - - - - 1.3 6.2 1.2 1.4 - - - - - - -
3596d 1.1 1.6 2.4 0.8 2.6 1.5 2.3 4 5.7 6 3.6 1.5 5.4 2 6.2
27b 5.3 0.1 0.1 0.7 - - - - - - - - - - -
30b 0.9 0.1 0.1 0.9 0.6 0.3 0.1 0.3 2.7 2.6 1.6 0.7 2.7 1 4.9
191 0.1 1.6 4.3 0.1 0.2 0.1 0.3 0.1 0.9 0.8 0.7 0.8 0.9 0.7 0.8
3596a - - - - 2 0.3 4 1.9 0.4 0.4 0.2 0.1 0.4 0.1 0.4
423-5p 0.6 3.2 3.4 2.4 - - 0.1 - 0.1 0.1 - 0.1 - 0.2 0.1
146b 0.4 1.3 3.3 0.6 - - - - - 0.1 - - - - -
804
Compare of Phenotypic Variation with the Polymorphism of One Coagulation

Related Gene Locus in Buffalo Kappa-Casein
Bo LINa, Daxi RENb, Bingzhuang YANGa, Ling LIa, Tang YANa, Haoru LONGa and
Qingkun ZENGb*
a
b
Institute of Dairy Science, College of Animal Sciences, Zhejiang University, Hangzhou 310029, P.
R. China.
*Corresponding email: zengqk@yahoo.com.cn
ABSTRACT
Kappa-casein (κ-CN) is a protein which highly correlated with coagulation property of dairy
cow milk, while its function was influenced by its genetic polymorphism. The phenotypic variation
of kappa-casein from buffalo was analysized by High Performance Liquid Chromatography
(HPLC), and genetic polymorphism of kappa-casein was also determined the by Polymerase Chain
Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) in this study, aimed to research
the relationship between buffalo κ-CN genetic polymorphism and its phenotypic variation. The milk
proteins from 98 individuals of water buffalo without consider their breed were analyzed by HPLC,
then blood DNA was extracted to determine polymorphism of one polymorphic locus in κ-CN gene
which generally been recognized to related with milk coagulation property. The results showed
three obvious κ-CN phenotypes can be screened by HPLC, the frequencies of BB, BC and AB were
76.5%, 6.1% and 17.3%, respectively. Three genotype, AA, BB and AB were also screened from the
researched κ-CN gene polymorphic locus, frequencies of each genotype were 4.1%, 26.5% and
69.4%, respectively, and the allele frequencies of A and B were 17.3% and 82.7%. Compare the
genetic polymorphism of κ-CN with its phenotypic variation showed there were no corresponding
relationships between κ-CN genotypes with its phenotypes, all the three genotypes can be found in
BB phenotypic samples, while they also can be found in AB phenotypic samples. The results
indicated the researched polymorphic locus of κ-CN gene in this study would not the main locus
influencing phenotype of buffalo κ-CN, suggested effect of this locus on milk coagulation would be
exerted by influencing other characteristics of κ-CN while not HPLC detectable variation.
Keywords: buffalo, kappa-casein; gene polymorphism; phenotypic variation; relationship
INTRODUCTION
Buffalo milk is particularly suitable for Mozzarella manufacture due to its good rennet
coagulation property (RCP), white color and high total solids. It has been reported that the good
RCP of buffalo milk was related with buffalo κ-CN genotype, as κ-CN is an essential protein in
renneting, and genotype of buffalo κ-CN mainly belong to BB genotype which is a genotype been
reported to beneficial for milk rennet coagulation (Patil et al. 2003; Ren et al. 2013). Although most
of studies researched on buffalo κ-CN genetic polymorphism concluded this gene was
monomorphic, and just has BB genotype, analysis of buffalo milk protein by RP-HPLC indicated
there are at least two κ-CN phenotypes existed in buffalo milk (Feligini et al. 2009; Bonfatti et al.
2012), besides, genetic sequence of κ-CN also find there were polymorphisms existed in this gene
805
(Masina et al. 2007). The difference of protein phenotype was mainly determined by genotype
indicated there would be some genetic mutations in κ-CN to induce the polymorphism of
phenotypes. Although there are several genetic polymorphism locus on κ-CN have been reported
(Masina et al. 2007), which is the important locus influencing protein polymorphism still unclear.
Until recently, the widely researched κ-CN polymorphism point which reported can affect dairy
cow milk coagulation prosperity located in exon 4 of κ-CN gene (Ikonen et al. 2004; Hallén et al.
2008), furthermore, the genetic polymorphisms were also only found in exon 4 of buffalo κ-CN
gene (Masina et al. 2007). Therefore, this study was conducted to research effect of one of the
widely researched genetic locus in buffalo κ-CN gene (located in exon 4) which has been reported
to a locus can influence milk coagulation properties on κ-CN phenotypes.

Milk and blood sampling and process
A total of 98 individual milk and blood samples of water buffaloes were collected in September
2012 without considering the breed, the sampled buffalo including Murrah, Nili-Ravi, crossbred of
Murrah and Nili-Ravi, crossbred breed of river type buffalo and Chinese local swamp type buffalo.
Animals were reared in dairy buffalo farm of Guangxi Buffalo Research Institute, The Chinese
Academy of Agricultural Sciences (Nanning, south of China). All cows were given same feed and
management, housed in loose housing systems, fed according to standard practice, and milked twice
a day. The milk and blood samples were immediately placed on ice after collection and transferred
to the laboratory, milk samples were aliquoted, skimmed (centrifuged for 30 min at 3000g at 4℃)
and preserved at -40 ℃ until RP-HPLC analysis. Blood samples was stored in -80 ℃ until DNA
extraction.
RP-HPLC assessment of milk protein
The method used for pretreatment of buffalo milk samples and separation of buffalo milk
protein fractions was in accord with method reported in Bonfatti et al. (2013) rigidly. The same
equipments, an Agilent 1100 Series chromatograph (Agilent Technologies, USA) and a column
column C8 (Zorbax 300SB-C8 RP, Agilent Technologies), as Bonfatti et al. (2013) were also used.
The agent, sample pretreatment procedures, model and situation of RP-HPLC equipment and
gradient elution condition used in this study were almost same as research conducted by Bonfatti et
al. (2013). The each chromatographic peak on the chromatograms corresponded milk protein was
identified in accordance with results of Feligini et al.(2009) and Bonfatti (2013).
Blood DNA extraction and PCR amplification
Buffalo blood DNA was isolated by using QIAamp DNA Blood Mini Kit (QIAGEN, Germany),
DNA purity was determined by measure absorbance ratio of 260/280 nm. The primers κ-CN-F (5’-
GCCCAAATTCTTCAATGGCAAG-3’) and κ-CN-R (5’-CTGCGTTGTCTTCTTTGATG -3’) were
used to amply a fragment with a length of 334bp in exon 4 of κ-CN gene. All the reactions were
carried out in 50 µl reaction tubes. Each single PCR amplification reaction (20µl) contained 2
μL,10× buffer, 2 μL 2.5 mM dNTP mixture, 1 μL each primer (forward and reverse), 0.25 μL Taq
polymerase (5 U/ μL), 11.75 μL nuclease free water, and 2 μL DNA template (50 ng/μL). The PCR
conditions used were as follows: 94 °C for 3 min (initial denaturation) and then 35 cycles of the
sequence 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s, a final primer extension was carried out
at 72 °C for 10 min. Finally the samples were cooled to 4 °C until retrieved.
PCR-RFLP analysis
For genotyping, the PCR product of κ -casein was digested with HindIII, PCR product were
806
subjected to digestion by HindIII in a total volume of 25 μL (15μL reaction solution, 3.5 μL enzyme
buffers, 2 μL enzyme, and 4.5 μL water) and incubated at 37℃ for overnight. The enzyme digested
products were run on a 2% agarose gel with the 5000bp DNA marker (MBI Fermentas) and
electrophoresis results was visualized by UV transilluminator.
Corresponding relationship between κ-CN genotype and phenotype
After genotypic and phenotypic information of samples were obtained, the samples were
separated into three different groups according to their genotype, then the κ-CN phenotypic variants
proportion in each group were calculated, aimed to see whether there are corresponding relationship
between the κ-CN genotype and phenotype.

Kappa-casein gene polymorphisms
In this study, genotype of κ-CN was judged by the gene fragments composition which was
generated by using restriction enzyme HindIII to digest the 334bp of PCR product. Only one 334bp
fragment will be generated in case of AA genotype; two fragments, 230bp and 104bp, in case of BB;
three fagements, 334bp, 230 bp and 104 bp in case of AB genotype. The electrophoretogram of
restriction enzyme fragment from different κ-CN genotypes was showed in fig.1. Results showed
all the three genotypes of κ-CN were found in this polymorphic locus, and the frequency of each
genotype were 4.1%, 26.5% and 69.4%, respectively, and the allele frequencies of A and B were
17.4% and 82.6%. Compare with most of other studies which reported there one BB genotype
existed in buffalo κ-CN (Gangaraj et al. 2008; Riaz et al. 2008; Abdel et al. 2009) , our study found
there genotypes existed at the polymorphic locus in exon 4, though BB genotype occupied largest
proportion. Gouda et al. (2013) also found there were BB and AB genotype existed in buffalo κ-CN
gene, while AA was not detected, while our study showed AA type was existed although with small
proportion (4.1%). The reason would be due to the sample in our study comes from different breeds
of buffalo, including pure river type buffalo and the crossbred of river type buffalo with swamp
buffalo. Most of the AB genotype and all of the genotype AA appeared in crossbred buffalos, while
just several AB and none of AA genotype were found in rive type buffalo. To our knowledge, there
still have no thoroughly research on swamp type buffalo κ-CN genotype type until now on, our
study here indicated research into swamp type buffalo milk protein gene polymorphism is
meaningful for use of crossbred of rive and swamp buffalo as dairy buffalo.
Kappa-casein protein phonotypic variants
Kappa-caseins were found to be heteromorphic in buffalo milk samples, and three phenotypic
variants ofκ-casein, been named BB, AB and BC, can be separated from buffalo milk protein (Fig.
2), he frequencies of BB, BC and AB were 76.5%, 6.1% and 17.3%, respectively. Although it was
hard to calibrate the milk protein peak due to buffalo milk proteins standards are not available in
this study, we can determine the peak corresponded protein by contrasting our results with the
results of Bonfatti et al. (2013), because our RH-HPLC assay was carried out in according with
method of Bonfatti et al. (2013) rigidly. The B and A variant of κ-caseins screed from this study was
corresponded well with X1 and X2 variants in results of Bonfatti et al. (2013). The C variant of
κ-casein was judged from result of Feligini et al. (2009), in their results, three variants of κ-casein
also been separated from buffalo milk, and one variants corresponded chromatographic peak
located before αs2-CN, the other two variants peaks located between αs2-CN and αs1-CN, which
were well consisted with our results. Compare with most reports said that the κ-CN gene
monomorphic gene, the κ-CN protein phenotype appeared different variants was inconsistent with
those conclusions. Due to protein is the ultimate functional executor of gene, exist of κ-CN variants
807
in buffalo indicated the milk processing properties would be influenced, however, find out the main
factor which inducing the variation in protein was the only way elucidate the root reason of
difference in milk processing prosperities.
Relationship between kappa-casein gene polymorphisms and phonotypic variants
In order to elucidate whether the difference of κ-CN protein in this study was induced by the
mutation of the gene locus (located in exon 4) which widely reported to a main locus influencing
dairy cow milk coagulation property (Ikonen et al. 1997, 1999; Hallén et al. 2008) or not, we
compared the corresponding relationship between κ-CN genotype and phonotypic variants, results
was showed in table 1. As we can see from table 1, all the three phenotypes (BB, AB, BC) can be
found in BB genotypic samples, they also can be found in AB genotypic samples, and BB and AB
phenotypes also can be found in AA genotypic samples. The results indicated there were no
corresponding relationships between κ-CN genotypes on this locus with its protein phenotypes.
Masina et al. (2007) revealed there were three polymorphisms in exon 4 ofκ-CN, two of them could
influence gene expression and lead to change of protein function, however, the polymorphic locus
of κ-CN which widely been reported to affect milk coagulation properties would not the detrimental
factor induce κ-CN variants detected by HPLC. Except genetic sequence which is the most
important factor, protein phenotype can be influenced by several factors. Among them,
posttranslational modifications, such as phosphorylation and glycosylation also can produce
different κ-CN isoforms (Holland et al. 2006; Jensen et al. 2012). Therefore, further studies,
including genetic and proteomic, were need to find out factors inducing different κ-CN variants.
Although many studies showed κ-casein in buffalo was monomorphic and association of milk
processing properties with buffalo casein genotypes cannot be researched (Gangaraj et al. 2008;
Ren et al. 2011), our study found the polymorphism not only existed in κ-CN gene, but also existed
in κ-CN protein, especially in the buffalo hybridized from river type and swamp buffalo, therefore,
effects of genetic or phenotypic variation on milk coagulation prosperity should be researched in
future.
ACKNOWLEDGEMENT
This work was supported by National Natural Science Foundation of China (31260384) and
Basic Scientific Research Fund of Buffalo Research Institute, CAAS(12050007), the authors here
would like to appreciate Dr. Ren Daxi who work in Zhejiang University help us on milk protein
RP-HPLC analysis.
REFERENCES
Abdel D. A.M.H., Mahmoud. Gh.M. Karima, M.F. Nawito., M.M. Ayoub and S. F. Darwish. 2009.
Genotyping of kappa-casein gene in Egyptian buffalo bulls. Livest. Sci. 122(2): 286-289.
Bonfatti. V., M. Giantin., R. Rostellato, M. DacastoB and P. Carnier. 2013. Separation and
quantification of water buffalo milk protein fractions and genetic variants by RP-HPLC. Food.
Chem. 136:364–367
Feligini. M. I. Bonizzi, J.N. Buffoni, G. Cosenza and L. Ramunno. 2009. K-Caseins in Water
Buffalo Milk by Reverse Phase-High Performance Liquid Chromatography and Mass
Spectrometry. J. Agric. Food Chem. 57: 2988–2992.
Gangaraj. D.R., S. Shetty, M.G. Govindaiah, C.S. Nagaraja, S.M. Byregowda and M.R. Jayashankar.
2008. Molecular characterization of kappa-casein gene in buffaloes. ScienceAsia. 34: 435–439.
Gouda. E. M., M.K. Galal and S.A. Abdelaziz. 2013. Genetic Variants and Allele Frequencies of
Kappa Casein in Egyptian Cattle and Buffalo Using PCR-RFLP. J. Agric. Sci. 5(2):197-203.
808
Hallén, E., A. Wedholm, A. Andrén, and A. Lundén. 2008. Effect of β-casein, κ-casein and
β-lactoglobulin genotypes on concentration of milk protein variants. J. Anim. Breed. Genet.
125:119–129.
Ikonen, T., S. Morri, A. M. Tyriseva, O. Ruottinen, and M. Ojala. 2004. Genetic and phenotypic
correlations between milk coagulation properties, milk production traits, somatic cell count,
casein content and pH of milk. J. Dairy Sci. 87:458–467.
Jensen. H. B., J. W. Holland, N. A. Poulsen and L.B. Larsen. 2012. Milk protein genetic variants
and isoforms identified in bovine milk representing extremes in coagulation properties. J.
Dairy Sci. 95:2891–2903.
Masina. P., A. Rando, P. Di Gregorio, G. Cosenza and A. Mancusi. 2007.Water buffalo kappa-casein
gene sequence. Ital .J.anim.Sci. 6 (Suppl. 2):353-355.
Mitra A, P. Schlee, I. Krause, Blusch J, Werner T, Balakrishnan CR, Pirchner F.1998. Kappa casein
polymorphisms in Indian dairy cattle and buffalo: a new genetic variant in buffalo. Anim
Biotechnol 9, 81–7.
Patil, M. R., Borkhatriya, V. N., Boghra, V. R., & Sharma and R. S. 2003. Effect of bovine milk
kappa-casein genetic polymorphs on curd characteristics during cheddar cheese manufacture. J.
Food Sci. Tech. 40: 582-586.
Ren D.X, B. Chen, Y.L. Chen, S. Miao and J.X. Liu. 2013.The effects of κ-casein polymorphism on
the texture and functional properties of mozzarella cheese. http://dx.doi.org/10.1016/j.idairyj.
Ren D., S. Miao, Y. Chen, C. Zou, X. Liang and J. Liu. Genotyping of the k-casein and
β-lactoglobulin genes in Chinese Holstein, Jersey and water buffalo by PCR-RFLP. J. genetic,
2011, 90(1), 1-5.
Riaz.M.N., N.A. Malik., F. Nasreen and J.A. Qureshi. Molecular marker assisted study of
kappa-casein gene in Nili-Ravi (buffalo) breed of Pakistan. Pakistan Vet. J. 28(3): 103-106.
1 2 3 4 5 6 7 8 9 10 M
500bp
250bp
100bp
Figure. 1. Electrophoretogram of restriction enzyme fragment from different κ-CN genotypes.

Lan1-4, 7-9 are BB genotypes; lan 5 is AA genotype, lan 6 and 10 are AB genotype. M is DNA
marker.
809
Κ-CN(B) as2-CN as2-CN β-CN α-LA β-LG
Κ-CN(B) Κ-CN(A)
2
Κ-CN(B) Κ-CN(C)
3
Figure. 2. RP-HPLC chromatograms of buffalo milk with different κ-CN phenotypic variants
(κ-CN A, B or C) obtained using the optimized elution condition reported by Bonfatti et al. (2013).
1: BB phenotype; 2: AB phenotype; 3: BC phenotype.
Table 1. The κ-CN phenotypic variants proportion in different genotypic groups.
Genotypic groups phenotypic variants proportion

bb 75.0%
aa ab 25.0%
bc 0.0%
bb 79.1%
bb ab 14.9%
bc 6.0%
bb 70.4%
ab ab 22.2%
bc 7.4%
810
Comparative Proteomic Analysis of the Changes of Milk Protein Associated with

Different Breeds of Buffalo
Daxi RENa, Bo LINb, Shan-shanLIa, YouliangCHENa and QingkunZENGb*
a
Institute of Dairy Science, College of Animal Sciences, Zhejiang University, Hangzhou 310029, P.
R. China, bKey Laboratory of Buffalo Genetics, Breeding and Reproduction technology, Ministry of
*Corresponding email: zengqk@yahoo.com.cn
ABSTRACT
The aim of this research was to compare the effect of crossbreed between Swamp buffalo and
River buffalo on milk protein content, composition and proteomic. 156 milk samples were collected
from Guangxi Water Buffalo Institute, include 40 Nili-Ravi (N), 34 Murrah (M), 36 N-M
crossbreed, 46 crossbreed from N with local swamp buffalo and M with local swamp buffalo (Z).
The result of protein content showed that the crossbreed (Z) has the highest content of protein
(4.46%), significantly higher than the M (4.40%), N-M (4.16%) and N (3.76%). The protein
composition of buffalo milk was determined by RP-HPLC. Among the 4 groups, notable difference
(p<0.05) was found at κ-casein and α-lactalbumin. The Z milk has the highest content of κ-casein
(9.14%), while the content was lowest in N-M milk (7.86%); however, opposite result was found in
α-lactalbumin, the Z milk content was the lowest (6.79%), while the highest was found in M milk
(7.60%). The comparative proteomic analysis among the four groups was also done by 2D-PAGE,
and the identification of milk protein was followed MALDI-TOF-MS and confirmed by
Western-blot. 24 different points were found among them, most of points were precursor from
different kind of casein. Some interesting points were found in PIGR-protein, κ-casein, β-casein and
αs1-casein, which could be related with crossbreeding. The result of this study indicating that
crossbreeding between swamp buffalo and river buffalo has notable relationship with protein
content, composition and proteomic.
Keywords: swamp buffalo, river buffalo; crossbreed; comparative proteomic


811
Productive and Reproductive Parameters in Buffaloes and Cows in a Farm

Located In Tropical Dry Forest in Venezuela
C. C. Ch. M. Montiel, N. Montiel-Urdaneta*, R. Rincón, N. Berrios, N. S. Morillo,

J. Belandria, M. Andará and J. Arias
*Department of Production and Industry Animal. School of Veterinary Sciences. The University of
the Zulia. Maracaibo, Zulia State. Venezuela.
* Corresponding e-mail: nsmontiel@gmail.com; nmontiel@cantv.net; Mobile: 00584143607847
ABSTRACT
We evaluated the productive and reproductive performance of buffaloes and cows in a
farm of the municipality Rosario of Perijá, State Zulia; under the same conditions: food, health,
etc. The study covered the years 1990-2012 and 2004-2012 for cows and buffaloes, respectively.
An analysis of variance was carried out considering year of calving and calving season as fixed
effects for the variables: length of lactation (DL); milk yield (PL); dry days (DS); production
calving (PP); age at first service (EPS); age at first calving (EPP); calving to conception (PC);
calving interval (IP); inseminations per conception (IC) and ride per conception (MC). Significant
difference was observed in all parameters (P<0.001) except IP was slightly higher in the buffaloes
(P>0.05). It is important to highlight EPS; PPE and PC in favor of the buffaloes.
Parameters Obs. Buffaloes Buffaloes Obs. Cows Cows

DL (days) 634 282.47±72.90a 4119 289.20 ± 80.75b
PL (kg) 619 1302.12±375.48a 4100 1416.33 ± 556.4b
DS (days) 500 143.88±81.27a 3577 136.34 ± 90.76b
PP (kg) 484 3.10±0.88a 3562 3.40 ± 1.15b
EPS (months) 117 26.61±4a 249 34.95 ± 6.10b
EPP (months) 117 38.12±4.84a 250 44.52 ± 6.15b
G (days) 759 311.26±10.33 4595 281.01 ± 15.64
PC (days) 507 114.97±92.89a 3895 142.89 ± 99.66b
IP (days) 500 430.48±98.54 ns 3867 427.11 ± 103.44 ns
IC (n) 185 1.48±0.78a 240 1.40 ± 0.64b
MC (n) 666 1.15±0.53a 4425 1.27 ± 0.62b
Significant difference (P<0.001), denoted by different letters in the same row
Keywords: cow; buffalo; tropical dry forest; productive and reproductive parameters

812
Productive and Reproductive Parameters in Buffaloes and Cows in a Farm

Located in Tropical Humid Forest in Venezuela
C. C. Ch. Montiel, N. Montiel-Urdaneta*, H. Hernández, N. Berrios, N. S. Morillo,

J. Belandria, M. Andará and J. Arias
*Department of Production and Industry Animal. School of Veterinary Sciences. The University of
the Zulia. Maracaibo, Zulia State. Venezuela.
* Corresponding e-mail: nsmontiel@gmail.com; nmontiel@cantv.net; Mobile: 00584143607847
ABSTRACT
We evaluated the productive and reproductive performance of a farm of buffaloes and cows
in the municipality San José of Perijá, State Zulia; under the same conditions: food, health, etc. The
study covered the years 1985-2012; 2001-2012 for cows and buffaloes, respectively. An analysis of
variance was carried out considering year of calving and time of calving as fixed effects for the
variables: length of lactation (DL); milk yield (PL); dry days (DS); production calving (PP); age
first service (EPS); age first calving (PPE); calving conception (PC); calving interval (IP);
insemination conception (IC) and ride per conception (MC). Significant difference was observed
(P<0.001) in all parameters evaluated less IP. It is important to highlight EPS; PPE and PC in favor
of the buffaloes; also the very good PL in buffaloes scarcely surpassed by the cows with 328.95 kg;
despite having the herd of cows 27 years of adaptation in the area.
Parameter Obs. Buffaloes Buffaloes Obs. Cows Cows

DL (days) 817 311.47±77.02a 4654 332.77±84.52b
PL (kg) 815 1676.57±528.28a 4660 2005.52±749.22b
DS (days) 745 119.48±95.47a 4191 97.39±75.03b
PP (kg) 703 4.04±1.19a 4174 4.74±1.58b
EPS (months) 153 22.99±3.72a 1341 30.14±7.79b
EPP (months) 155 33.80±4.07a 1361 40.13±7.60b
G (days) 1176 309.25±11.33 7237 283.17±11.95
PC (days) 730 124.36±109.8a 5327 150.25±100.29b
IP (months) 758 435.18±119.06ns 5225 434.79±99.40ns
IC (n) 95 1.27±0.55a 5370 1.59±0.97b
MC (n) 1126 1.07±0.29a 2954 1.24±0.68b
Significant difference (P<0.001) different letters in the same row
Keywords: cow; buffalo; tropical subhumid forest; productive and reproductive parameters

813
Microsatellite Analysis of Thai Swamp Buffalo Cloned Calf

Derived from Ear Fibroblasts
Supitchaya TREEBONMUANGa*, Wisut NUALCHUENAa, Kitiy SRISAKWATTANAa,

Kriengsak TASRIPOOa and Duangsmorn SUWATTANAb
a
Research and Development Centre for Livestock Production Technology, Faculty of Veterinary
Science, Chulalongkorn University, Henri Dunant Street, Phathumwan, Bangkok 10330, Thailand,
b
Department of Animal Husbandry, Faculty of Veterinary Science, Chulalongkorn University,
Henri Dunant Road, Bangkok 10330,Thailand.
*Corresponding: e-mail: Chetasing@yahoo.com
ABSTRACT
A study was conducted to identify whether the cloned buffalo calf derived from donor ear
fibroblasts used for somatic cell nuclear transfer (SCNT). Genomic DNA was isolated from the
blood of the cloned buffalo, surrogate recipient buffalo , random buffalo and the trypsinzed
donor cells. The isolated genomic DNA samples were used for microsatellite assay using 12
microsatellite markers labeled with one of the fluorescent dyes FAM, Yakima Yellow, ATTO550
and ATTO565. DNA fragments were detected on a vertical electrophoresis, 7% polyacrylamide
gel (7% PAGE) in a single reaction and visualized by Ethidium-Bromide and used for
determination the sex of each samples. For confirmation, fragments analysis of 12 multiplex sets of
PCR reaction was performed by capillary electrophoresis (CE) on automated sequencer (ABI 3130
instruments). Comparison of the twelve microsatellites of alleles in the cloned buffalo calf with
donor cell and surrogate mother indicated there was 100% identity to donor cells and was different
from the recipient females and random buffalo. This study shows that the calf was indeed a clone
derived from the donor ear fibroblast cells used.
Keywords: swamp buffalo, Thailand’s cloned buffalo, microsatellites marker, fragment analysis,
genetic identity
INTRODUCTION
Microsatellites are short, tandem repeated sequences of DNA (also called short tandem
repeats, STRs) and are found in the genome of most species studied so far (King et al.,2006). They
are found evenly distributed along the chromosome in a eukaryotic organism; mostly within the non-
coding sequences of the DNA (King et al., 2006).The cloning of buffaloes has a special significance
in the genetic improvement of Buffalos. Buffaloes are important domestic animals that are distributed
in the tropical and subtropical regions, providing a high quality of milk, meat, and work power (Shi
et al., 2007.). The first buffalo somatic cell nuclear transfer (SCNT) offspring were reported by Shi et
al.2007. In Thailand, the first swamp buffalo (Bubalus Bubalis) cloned calf derived from donor cells
was taken from an adult somatic cells have been produced successfully since 2011 .Donor cells was
taken from ear fibroblast of an adult male swamp buffalo with a special unique characteristic Thai
swamp buffalo was selected as donor cell (Tasripoo et al., 2011). These markers can be used in
characterization of species populations, genetic diversity, population studies (Van et al., 2007) and
parentage control. Parentage analysis was perform on cloned calves, surrogate buffalo and nuclear
donor cells to compare the genetic diversity of the calf with that of the donor cells used for SCNT in
buffalo as previous report (Shi et al., 2007). Thus, the aim of the present study is to employ some
microsatellite markers to identify the Thai swamp buffalo cloned calf derived from donor ear
fibroblasts.

814

Genomic DNA was isolated from the blood of the cloned buffalo, surrogate recipients
buffalo and random buffalo. The donor ear fibroblasts cells were trypsinized before extraction. The
genomic DNA was extracted by using a commercial kit (QiAmp® DNA Mini kit) according to the
manufacturer instruction. DNAs were assessed and quantified using 2 % agarose gel electrophoresis
before subjected to polymerase chain reaction (PCR) using each primer of selected microsatellite
markers (ITSLT033, ITSLT006 CSSM022, CSSM038, ETH121, CSSM043, CSSM057, CSSM029,
BC1013, CSSM019, BM1818 and CSSM041; M1-M12, respectively). The microsatellites used in
this study were recommended by the International Society of Animal Genetics (ISAG/FAO) labeled
with fluorescent dye (FAM, Yakima Yellow, ATTO550 and ATTO565). The reaction were
according to the following cycling profile: The PCR cycle was primary denaturation at 95°C for 5
min, denaturation (95°C for 30 sec.), annealing at 60° C for 30 sec, and an extension at 72°C for 30
sec, and following by final extension cycle at 72°C for 30 min ,(Treebonmuang et al., 2010).The
success of the PCR was detected by running horizontally of the PCR product on 2% agarose gel
electrophoresis to assess their quality and quantity. DNA fragments were detected on a vertical
electrophoresis that determined for each marker separately to amplified products in a single
reaction, on 7% polyacrylamide (7% PAGE). After electrophoresis at 60 volt for 16 hr, gel (size
20x20 cm) was stained with Ethidium Bromide (Et-Br) (10 ng/ml) for 50 min. Then DNA
fragments were visualized by UV transillumination and analysis by Photo-Capt software (Doc-print
II). The 12 multiplex sets of PCR reactions per sample were set up for the amplification from
capillary electrophoresis (CE). One primer of each pair was synthesized with a fluorescent dye on
the 5’ end to allowed detection and sizing of fragments on Applied Biosystems 3130 Series Genetic
Analyzers Using the new Report Manager feature in Gene Mapper ® Software v3.7 (Applied Bio-
systems, USA).

In this study represents to confirm the cloned swamp buffalo were genetically identical by
microsatellite analysis of genomic DNA from the donor ear fibroblasts used for SCNT. In Figure 1
that shown a good of isolated DNAs were assessed quality and quantity by 2% agarose gel. The
genomic DNA isolated from blood and cell culture samples was successfully amplified at 12
microsatellite loci. The amplified products, analyzed by 2 % agarose gel electrophoresis, in Figure 2
were found homozygous of all alleles. Subsequently, they were loaded on a high resolution 7%
PAGE used to determine the amplified fragment length, a special unique characteristic Thai swamp
buffalo calf (S) was selected as donor cell (D) ( Figure 3). In Figure 4, a representative showing
multiplex microsatellite data and comparison of allele size of the cloned buffalo, donor cell line and
other samples, allele band to confirm the genetic identity of cloned buffalo could be compare with
DNA sample from it donor cell line, showing that the calf was indeed a cloned from the donor cells
used. Automated fluorescence technologies based capillary electrophoresis (CE) are widely used for
detection and size determination of PCR products (Suwattana et al.,2010, Bhuyan et al.,2010), and
make it possible to run multiplex PCRs by using primer pairs that are differently labeled with
fluorescent dye (Jain et al.,2010). In this study the fluorescent dyes are FAM, Yakima Yellow,
ATTO550 and ATTO565 (www.eurofinsdna.com). All of the 12 microsatellite markers observed
were similar between the calf and the donor cell line shown that in Table 1. By gender analysis
(Nualchuen et al., 2010) and DNA sequencing indicate that the cloned calf is male. In conclusion,
DNA profile of cloned buffalo was 100% similarly to that of donor cell line (ear fibroblast). These
results suggest that the 12 microsatellite markers can be used successfully for identification of
cloned buffalo.
815
REFERENCES
Bhuyan D.K., M.L. Sangwan, V.C. Golr and R.K. Sethi. 2010. Studies on DNA
fingerprinting in Murrah buffaloes using microsatellite markers. Indian J Biotech. 9:367-
370.
FAO. 2011.Molecular genetic characterization of animal genetic resources. FAO Animal
Production and Health Guidelines. No.9.Rome (p70-71).
ISAG/FAO Standing Committee. (2004). Secondary Guidelines for Development of
National Animal Genetic Resources Management Plans. Measurement of Domestic Animal
Diversity (Mo-DAD) Recommended Microsatellite Markers. Recommendations of Joint
ISAG/FAO Standing Committee http://dad.fao.org/
Jain N., P. Muraleedharan, G. Rao, C. Reddy and Reedy S.K. 2011. Comparative evaluation
of whole genome amplification methods in water buffalo frozen semen. Indian J Biotech.
10:39-44.
King R C., R.D. Stansfiedl and P.K. Mulligan. 2006. A Dictionary of Genetics,7 edn. Oxford
University Press Inc, USA, New York.
Multiplex microsatellite analysis with 5’ Dye-Labeled marker sets using FAM, YAKIMA,
YELLOW, ATTO 550, ATTI 565 and ATTO 633 on an ABI 3130XL GENETIC
ANALYZER. (www.eurofinsdna.com)
Nualchuen W., K. Tasripoo, K.Srisakwattana, S. Treebonmuang, S. Usawang S. and
M. Techakumphu. 2010. Development of Mehtodology for sexing bovine and goat
using polymerase chain reaction. The ICVS 35th Thai Veterinary Medical association
International conference on Veterinary Science 2010. 2-5 November 2010, pp 220.
Shi D., F. Lu, Y. Wei, K. Cui, S. Yang, J. Wei and Q. Liu. 2007. Buffalo (Bubalus Bubalis)
Cloned by Nuclear Transfer of Somatic Cells. Biol Reprod. 77:285-291.
Suwattana D., J. Jirasupphachok, S. Kanchanapangka and W. Koykul. 2010. Tetranucleotide
microsatellite markers for molecular testing in Thai domestic elephants ( Elephas
maximus indicus). Thai J Med. 40(4):405-409.
Tasripoo K., W. Suthikrai, S. Sophon, R. Jintana, K. Srisakwattana, W. Nualchuen , S.
Usawang, M. Kamonpatana, M. Techakumphu and A. Bintvihok. 2011. First swamp
buffalo cloned calf produced from adult ear fibroblast cells.(under submission).
Treebonmuang S., W. Nualchuen, K. Srisakwattana, J. Jirasupphachok and D. Suwattana.
2010. Characterization of microsatellite polymorphism in Thai domestic buffaloes. In
Proceeding of the ICVS 35th Thai Veterinary Medical association International conference
on Veterinary Science. pp. 221(abstr.)
Van Eenennaam A L., R.L. Weaber, D.L. Drake, M.C.T. Penedo, R.L. Quaas, D. Garrick
and E.J. Pollak. 2007. DNA-based paternity analysis and genetic evaluation in a large,
ommercial cattle ranch setting. J Anim Sci 85:3159-3169.
Figure 1: DNAs were assessed and quantified using

2% agarose gel electophoresis. A good genomic
DNA isolate from the blood of surrogate recipients
buffalo (a) and the cloned buffalo (b) , The donor
swamp buffalo from ear fibroblast (c) and random
buffalo (d)
816
Figure 2: The representative amplify PCR product

separated by 2 % agarose gel electrophoresis.
M: marker 100 bp, M1: ILSTS006, M2: ILSTS006
M3: CSSM022 , M4: CSSM038, M5: ETH121
and M6: CSSM043. The results found that all
microsatellite alleles were found homozygous
(arrow).
M5 M6 Figure 3: The representative microsatellite allele results

of M5 and M6 that separate by 7% PAGE. M:
marker 100 bp, R: recipient mother, S:clone calf,
D: donor cell line and C: random buffalo. It was
found that the swamp buffalo calf (S) was selected
as donor cell (D).
M R S D C R S D C
Table 1. Comparison of 12 microsatellite analysis of buffalo cloned calf and donor cells.
Microsatellite loci *
Samples
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12
Surrogate
mother 153/155 278/278 209/209 171/171 183/183 254/254 112/118 177/177 212/218 153/158 260/274 133/141
Donor cell 151/155 279/279 206/208 173/181 183/187 251/254 118/123 177/191 212/214 155/162 271/274 141/146
Cloned calf 151/155 279/279 206/208 173/181 183/187 251/254 118/123 177/191 212/214 155/162 271/274 141/146
Random 140/144 272/275 209/209 181/187 185/185 250/250 118/120 183/186 218/226 137/142 269/269 145/147
sample
* M1:ILSTS003, M2: ILSTS006, M3: CSSM022, M4: CSSM038, M5:ETH121, M6:CSSM043, M7:CSSM057, M8:CSSM029, M9:BC1013,
M10:CSSM019, M11: BM1818, CSSM041.
817
Figure 4. Representation of amplification profile of primer M1-M6 in Capillary Electrophoresis (CE),

showing that the calf was indeed a cloned from the donor cells used. From the top to bottom
showing peaks of the recipient genomic DNA (4a), clone buffalo genomic DNA (4b), somatic cell
nuclear donor (4c) and random genomic DNA (4d) respectively. The Y-axis indicates the peak
heights; the x-axis indicates DNA size and the number in the boxes indicates the allele size of each
peak.
818
The Comparison of Some Reproductive Traits of Anatolian and F1 Crossbred

(Anatolian X Italian) Buffalo under Village Conditions In Turkey
Özel ŞEKERDEN
Department of Animal Science, Faculty of Agriculture, Mustafa Kemal University, Antakya

*Corresponding e-mail: sekerden@mku.edu.tr
ABSTRACT
This study compared reproductive traits of Anatolian and Anatolian x Italian F1 crossbred
buffaloes in Ilıkpınar Village of Kırıkhan District of Hatay Province, Turkey. Previous studies of
the same genotypes compared growth characteristics, and milk yield and somatic cell numbers in
milk, and this present study has dealt with some reproductive traits.
The study material involves various breeding records of Anatolian and Anatolian x Italian
crossbred female buffaloes. The buffalos in various lactation orders were from two units
representing buffalo herds of Ilıkpınar Village. The records cover periods: 2001–2011 and 2003–
2011, respectively, for Anatolian and crossbred buffaloes. The numbers of Anatolian and F1
crossbred buffaloes in terms of trait and genotype were respectively 12 and 10 for first calving age;
87 and 21 for calving interval; and 20 and 5 for gestation period.
The effects of genotype and calving year on examined features were determined by GLM
variance analysis and mean values were calculated using SPSS Program. It was concluded that there
was no significant difference between Anatolian and Anatolian x Italian F1 crossbreeds in terms of
the examined reproductive traits.
Keywords: Buffalo, Anatolian, Italian, Crossbred, Reproductive Traits
INTRODUCTION
Buffalo have low heredity of reproductive traits, demonstrating that such traits are affected
by many environmental factors. Therefore, reproductive traits in buffalo show significant variance
(Ramos et al., 2006; Jabalkandi, 2010).
Aziz et al. (2001) determined the significance (P<0.01) of the effect of calving year on
calving interval in Egyptian buffalo. However, Prakash et al. (1989) on Murrah buffaloes and Afifi
et al. (1992) on Egyptian buffaloes reported that this effect was not significant.
Feeding and herd management are the most important environmental factors affecting the
reproductive yield of buffalo. In case of insufficient energy intake, sexual maturity is delayed and
conception rate decreases; in case of protein deficiency in rations, appetite reduces, and therefore
sexual maturity is delayed and the days open increases (Schingoethe et al., 1988).
The first calving age was reported as 1075 days in Anatolian buffalo (İzgi and Asker 1988).
In a study, Fooda et al. (2011) reported the first calving age of Egyptian buffalo as 29 and 31
months in 1st and 2nd farms; of Egypt x Italian crossbreds as 27 months and 31 months in 1st and 2nd
farms.
Calving interval was 470.4± 19.0, 423.0±21.5 and 564.6±98.5 days in 1st, 2nd and 3rd calving
interval orders respectively in Anatolian buffalo (Uslu, 1970); 437.2 days in Romanian buffalo
(Velea, 1991); 512.7±9.4 and 538.3±11.4 days in Murrah and Surti buffalo respectively in India
(Neog et al., 1991); 395 days and 418 days in 1st and 2nd farms of Egyptian buffalo, and 429 and
433 days in 1st and 2nd farms of F1 by Fooda et al. (2011).
Gestation period is cited 320±1.20 days in Anatolian buffalo by Uslu (1970); 308 days in
Bangladesh buffalo by Faruque (1995); 307-316 days in Egyptian buffalo by Metry (1996); 317
days and 315 days in 1st and 2nd farms of Egyptian buffalo and 314 days and 313 days in 1st and 2nd
farms of Egyptian x Italian crossbred F1s by Fooda et al. (2011).

819
In a comparison of reproductive traits of Egyptian buffalo and Egyptian x Italian crossbred

F1 buffalo in 2 different farms, Fooda et al. (2011) reported that all reproductive traits (calving
interval, service period, the days open), except the first calving age in crossbred buffalo, are greater
than the ones of Egyptian buffalo and the reproductive traits of Egyptian buffalo are better than
Italian x Egyptian F1 crossbred buffalo.
In previous studies (Şekerden, 2010 and Şekerden, 2011) growth characteristic and milk
yield and somatic cell numbers in milk of Anatolian and Anatolian x Italian crossbreds were
compared in Ilıkpınar Village buffalo herd. This present study was conducted in village conditions
in Turkey in order to investigate whether reproductive performances of Anatolian buffaloes can be
improved or not by crossing with Italian buffaloes.

The study was conducted in 2 farms representing buffalo herds of Kırıkhan Ilıkpınar Village
of Hatay Province. The study material consists of various breeding records of Anatolian and
Anatolian x Italian crossbred buffalo in various lactation orders. The records cover the periods
2001–2011 for Anatolian and 2003–2011 for F1 buffalo.
Ilıkpınar Village has an appearance like a big buffalo farm with approximately 150
breedable buffalo. Almost all feeding is based on the grazing land of village. In one of the study
units, a limited amount of silage is produced and additional feed is given after the return from
pasture. In the other farm, the feeding level is lower. Births generally occur during March and April.
Breeding records of Anatolian buffaloes have been kept by the author since 1996.
Inseminations were started in April, 2002, within the scope of an Anatolian x Italian crossbreeding
Project, and the first crossbred calves were born in 2003. Since the animals studied were from the
village herd, the animals were artificially inseminated after their oestrus was synchronized.
Therefore, gestation periods could be calculated only in Anatolian and F1 buffaloes within the
Project. The evaluation was made after being combined the data of some years that, there were a
few data, with others for each trait.
The effects of genotype and calving year on the examined features were examined through
GLM (General Linear Model) analysis of variance (ANOVA). To that end, a simple linear model
(Equation 1) including variance sources considered for each feature was used;
Yijm= µ + Gi + Cyearj + (G*Cyear)ij + eijm . . . . . .. .. . . . . . . . . . . (1)
Where; Yijm: Examined feature (for example gestation period), µ: General average, G:
Genotype effect (i: Anatolian, F1), Cyear: The effect of calving year (j: 1, 2, 3, 4, 5), (G*Cyear)ij:
The interaction between genotype and calving year, eijm: Error term.
Averages were calculated for the examined features for each genotype. SPSS was used in all
statistical analyses.

Table 1 shows ANOVA results of various features and Table 2 shows averages of
investigated characteristics.
The effect of genotype was non-significant for 3 of the examined features. Calving year is
related to significant variance in calving interval and gestation period. The significant effect of
calving year may be due to climatic changes that occurred during the 10-year period of the study.
Since the feeding depends on grazing areas in village, the change in feeding system is an anticipated
outcome. On the other hand, feeding is one of the most influential environmental factors on
reproductive traits (Schingoethe et al., 1988; Perera, 2011). While some studies in the literature
reported that calving year significantly affects calving interval (Aziz et al., 2001), others found that
this effect was insignificant (Prakash et al., 1989; Afifi et al., 1992).
820
Table 1. Analysis of variance of calving interval, first calving age and gestation period.
Variance Source Calving interval First calving age Gestation period
f.d. F Signif f.d. F Signi f.d. F Signif

(x) icance fican icanc
ce e
Genotype 1 0.368 1 0.696 0.416 1 0.150 0.703
Calving year 4 2.659* 0.037 2 1.004 0.387 2 2.815* 0.084
Genotype*Calving 1 0.774 0.381 1 0.535 0.474 1 0.218 0.645
year
Error 101 17 20
Total f.d. 108 22 25
The average of error 21164.949 18743.881 77.143
square
R2 0.116 0.224 0.221
*P <0.05, Coefficient determination of model, (x) f.d. freedom degree
Table 2. Average calving interval, first calving age and gestation period (days).
Environmental Subgroup Calving interval First calving age Gestation period

factor
- - - - - -
N X ± SX N X ± SX N X ± SX
Genotype Anatolian 87 599.2±15.27 12 1210.4±35.38 20 313.3±2.06
F1 21 545.2±38.5 10 1126.4±44.37 5 314.0±4.21
While the averages of calving interval, first calving age and gestation period are shorter in
F1s (Table 2), the differences between Anatolian and F1s for these features are not significant (Table
1). It may be stated that average gestation period is the same in both genotypes (Table 2).
The average calving interval determined for the Anatolian breed is longer than reported in both
other studies in the literature (Uslu, 1970), and those reported for various breeds in different
countries. The average calving interval determined in this present study for F1 crossbreds is close to
the result of one study in the literature (Neog et al., 1991, Murrah and Surti breeds), a little shorter
than the value of 3rd calving order given for Anatolian breed in the literature (Uslu, 1970), and
longer than other results.
The first calving age average calculated for the Anatolian breed is older than the results of some
studies (Uslu, 1970; Afifi et al., 1991; Fooda et al., 2011). The first calving age average calculated
for F1 genotype is smaller than the results of some studies (Afifi et al., 1992), greater than the result
of one study (Fooda et al., 2011), and similar with the results of some studies (Uslu, 1970; Metry,
1996) in literature.
The averages of gestation period in Anatolian and F1 are similar in this study. The gestation period
determined for Anatolian x Italian F1 crossbreds is the same as that determined for Egyptian x
Italian F1 crossbreds by Fooda et al. (2011); the gestation period average determined for the
Anatolian breed is slightly shorter than that determined for Egyptian buffalo by Fooda et al. (2011).
It would be expected that the averages found in this present study for reproductive traits would
differ from the results of most studies in the literature. This variation is due to genetic diversity and
different environmental conditions, such as feeding and herd management (Ramos et al., 2006;
Jabalkandi, 2010; Perera, 2011). This outcome may be explained because the present study was
conducted in village conditions, in which feeding was almost entirely dependent on grazing lands,
whereas the other studies in the literature were conducted in different countries and regions, in
821
different research herds and under different climatic conditions. Thus, the previous results for the
Anatolian breed (Uslu, 1970) were obtained from a study conducted at the Afyon Buffalo Research
Institute.
In conclusion, the present study found no significant difference between Anatolian breed and
Anatolian x Italian F1 crossbreds in terms of the examined reproductive traits.
REFERENCES
Afifi, E.A., M.H. Khalil, L.H. Bedeir and S.M. Zeidan. 1992. Genetic analysis of reproductive traits
in Egyptian buffaloes. Egypt J. Anim. Prod. 29(2): 139-154.
Aziz, M.A., S.J. Schoeman, G.F. Jordan, O.M. El-Chafie and A.T. Mahdy. 2001. Genetic and
phenotypic variation of some reproductive traits in Egyptian buffalo. South African J. of
Anim. Sci. 31 (3): 195-199.
Faruque, O. 1995. Indigenous buffaloes in the coastal area of Bangladesh. Buffalo Newsletter, 4: 3-
5.
Fooda, T.A., A.R. Elbeltagi, R. Laila, H. Set El-habaeib and S. Awad. 2011. Evaluated of Egyptian
buffaloes crossing with Italian buffaloes for reproductive traits. J. of American Sci. 7 (7):
209-213.
İzgi, A.N. and R. Asker. 1988. Effects of birth season and first calving age on lactation length and
milk yield in buffaloes. Buffalo Research Inst. Publ. 19.
Jabalkandi, A., Gh. Manafiaza and S. Razzagzadeh. 2010. Effect of supplemented ration on some
reproductive traits in Azeri buffaloes of Iran. Italian J. of Anim. Sci. 14: 15-16.
Metry, G.H. 1996. The main dairy animal in Egypt. Academy of Scientific and Technology: 39 pp.
Neog, P.K., D. Das and R.N. Goswami. 1991. Performance of Surti and Murrah buffaloes under the
agro- climatic conditions of Assam. J. of the Assam Veterinary Council (1991) 1: 55-57,
Perera, B.M. 2011. Reproductive cycles of buffalo. Anim. Reprod. Sci. 124(3-4):194-199.
Prakash, A., V.N. Tripathi and S.S. Tomer. 1989. Genetic analysis of reproductive traits of Murrah
buffaloes. Indian J. Dairy Sci. 42: 426-430.
Ramos, A.A., C.H.M. Malhado and P.L.S. Carneiro. 2006. Caracterizaçao fenotipica e genetica da
produçao de leite e do intervalo entre partos em bubalinos da Raça Murrah. Pesquisa
Agropecuaria Brasileira 41 (8): 1261-1267.
Schingoethe, D., F.M. Byers and G.T. Schelling. 1988.. Nutrient needs during critical periods of the
life cycle. The Ruminant Animal Digestive Physiology and Nutrition: 421-447.
Şekerden, Ö. 2010. Growth traits of Anatolian and Anatolian x Italian crossbred buffalo calves and
effects of genotype, sex and birth year on growth traits. Hayvansal Üretim 51(2): 34-43
Şekerden, Ö. 2011. actors affecting somatic cell counts and theit relations with milk and milk
constituent yield in Anatolian and Anatolian xItalian F1 crossbred buffaloes. Hayvansal
Üretim 52(1): 9-16.
Uslu, N.T. 1970. Comparative researchs on various characteristics and milk yield of Afyon Region
buffaloes under the village condition. Doctorate thesis, Birlik Press, Bornova, Turkey.
Velea, C., I. Bud, G. Muresan, V. David, M. Vomir, C. Cristea and L. Elisei. 1991. The main milk
traits of Romanian buffaloes breed. Third World Buffalo Congress, Varna, Bulgaria, May
1991, Sofia, Bulgaria, Agricultural Academy, Proceedings, II. Pp. 494-499.
822
Buffalo Nutrition and Feeding
Minerals Status of Soil, Fodder and in Lactating Nili-Ravi Buffaloes in Irrigated

Agro-ecological Zone of Punjab, Pakistan
Muhammad Saleem AKHTAR*, Allah BACHAYAc, Laeeq Akbar LODHIa, Abdul Asim
FAROOQ, Muhammd Mazhar AYAZ, Saeed MURTAZA, Muhammad ARSHAD, Irtaza
HUSSAIN, Abdul BASIT, Ijaz AHMADa, Mushtaq HUSSAINb and Zahida TASAWARb
Faculty of Veterinary Sciences, Bahauddin Zakariya University, Multan, Pakistan, aUniversity of

Agriculture, Faisalabad, Pakistan, bInstitute of Pure and Applied Biology, Bahauddin Zakariya
University, Multan, Pakistan, cDepartment of Animal Sciences, Allama Iqbal Open University,
Islamabad
*Corresponding email: drsaleem46@hotmail.com
ABSTRACT
The current study was designed to evaluate the macro and trace mineral profile in soil, fodder
and in buffaloes of irrigated agro-ecological zone. 60 soil and 60 fodder samples were collected
from Block-1 (Tehsil Dera Ghazi Khan) and Block-2 (Tehsil Taunsa Sharif), whereas, in each
Block, 60 blood samples were collected randomly from lactating buffalo. The concentrations of
calcium, magnesium, copper, iron and zinc were quantified with the help of atomic absorption
spectrophotometer whereas sodium and potassium were measured by a flame photometer. There
was non-significant (P>0.05) difference in soil and fodder macro (calcium, magnesium, sodium,
potassium) and trace mineral (copper, iron, zinc) concentrations between block-1 and 2. There was
non-significant (P>0.05) difference in calcium, magnesium and sodium concentrations whereas
significant (P<0.05) differences were observed for potassium concentrations in buffaloes of block-1
and block-2. There was non-significant (P>0.05) difference in copper, iron and zinc concentrations
in buffaloes of block-1 and block-2. In block-1 and block-2, all buffaloes were deficient for zinc. It
was concluded that buffaloes raised in the study area were lacking in some minerals, and for
optimal production these minerals must be supplemented.
Keywords: Macro minerals, Trace minerals, Buffaloes, Fodder, Soil
INTRODUCTION
Livestock, especially ruminants, survive on plant nutrients, the quality and quantity of which
depend to some extent on soil type, level of fertilization and irrigation. Certain major nutrients of
plants such as protein, carbohydrates and lipid do not vary much because they are mostly genetical
parameters and species specific, but mineral contents may vary to a variable extent with reference
to the soil. Macro and micro mineral play vital role for numerous metabolic functions and their
deficiency affects the normal production. In cattle, mineral availability mainly influenced by
production system, feeding practices and environment (Singh and Bohra, 2005).
The mineral contents of fodder depend on an intricate interrelationship of soil, climate,
topography, plant species, its age and yield, pasture or fodder management and cropping patterns
(McDowell and Arthington, 2005). In the process of intensive farming practices, soils from all over
the country are getting depleted for one or more mineral element resulting in imbalances of mineral
elements in soil, plants and animals. Different diseases caused by mineral deficiency and toxicity in
livestock have existed for ages in many countries including Pakistan. Previous studies have
described the composition of forage frequently used for nourishing dairy cattle (Khan et al., 2006;
Fardous et al., 2010); but, the mineral content of the fodders has not been studied in detail. Limited
research work conducted in rural areas has suggested mineral inadequacies in the soil and fodder.
To know the mineral demands of ruminants, it is imperative to know the mineral levels in soil and
forage (Pereira et al., 1997). Keeping in view the changing agro-climatic conditions of southern

824
Punjab, current study was designed to evaluate the macro and trace mineral status in soil, fodder
and in buffaloes of irrigated agro-ecological zone of Punjab, Pakistan.

The present study was conducted at District Dera Ghazi Khan, which is located in irrigated
agro-ecological zone of Punjab, Pakistan. For collection of soil, fodder and blood samples from
Nili-Ravi buffaloes, renowned agriculture villages were selected in two Tehsils namely, Tehsil
Dera Ghazi Khan (Block-1) and Tehsil Taunsa Sharif (Block-2). The sample collection was done
during November-December 2011.
Sample Collection and Preparation:
From each Block, a total of 60 soil and 60 fodder samples were collected. Soil samples were
collected at 60 different sites in each Block by simple random sampling. Composite fodder samples
were collected randomly at 60 different sites in each Block. In each Block, 60 blood samples were
collected randomly from lactating buffaloes. Lactating buffaloes ranged age from 3-10 years with 2
to 6 parity. The collected serum was retained in vials, branded and were stored at -20˚ C till
analyzed.
Soil samples were subjected to extraction by following Ammonium bicarbonate diethylene
triamine penta acetic acid (ABDTPA) (Soltanpour, 1985) whereas for fodder and serum mineral
determination, samples were digested as the method described by Richard (1968). The
concentration of macro minerals (Calcium, Magnesium) and trace minerals (Copper, Iron, Zinc)
were quantified with the help of atomic absorption spectrophotometer (Perkin Elmer, Analyst 200,
France) whereas sodium and potassium were measured by a flame photometer (PFP7, Jenway,
UK).
Statistical Analysis:
The mean (± SE) for macro and trace minerals in soil, fodder and serum of Block-1 were
compared with mean values of soil, fodder and serum of Block-2 by Z-test (Steel et al., 2006).
RESULTS
There was non-significant (P>0.05) difference in fodder calcium, magnesium, sodium and
potassium concentrations between block-1 and 2. Similarly, non-significant (P>0.05) differences
were observed for fodder copper, iron and zinc concentrations between block-1 and 2 (Table-1).
Between block-1 and 2, there was non-significant difference for soil calcium, magnesium, sodium
and potassium concentrations. Consequently, between block-1 and 2, there was non-significant
difference in soil copper, iron and zinc concentrations (Table-2).
There was non-significant (P>0.05) difference in calcium, magnesium and sodium
concentrations in buffaloes of block-1 and block-2, whereas significant (P<0.05) differences were
observed for potassium concentrations in buffaloes of block-1 and block-2. There was non-
significant (P>0.05) difference in copper, iron and zinc concentrations in buffaloes of block-1 and
block-2. In block-1 and block-2, all buffaloes were deficient for zinc (Table-3).
DISCUSSION
The findings of the current study indicated that the soils were lacking in various evaluated
macro minerals (magnesium and potassium) in block-1 and block-2. Wide-ranging variations in
soil mineral concentrations have been reported by many other workers (Khalili et al., 1993; Pereira
et al., 1997; Sharma et al., 2003; Ndebele et al., 2005). Different aspects have been ascribed
responsible for variations in the mineral concentrations in soil of the plain areas. The soil of Dera
Ghazi Khan is sandy clay loam in nature with slightly alkaline in reaction and deficiency of
minerals in soil may be due to increased crop yield which removes minerals from the soil
(McDowell and Arthington, 2005). In the present study, the concentrations of macro and trace
minerals in the fodders of two blocks were above the critical level (Table 2). Calcium is important
in decreasing the acidity of soil and as well as it is used as a key nutrient for normal plant
825
development. In the present study, soil calcium ranged from 110.4 to 134.9 ppm, and there was
non-significant difference between blocks. The soil calcium concentrations in both the blocks were
above the critical level of 71 ppm. Adams and Hartzog (1960) described that higher levels of
calcium in soil could increase calcium concentrations in the fodder. Likewise, in the present study,
all fodder samples had higher calcium levels than the critical level reported for the fodder
(McDowell and Conrad 1977). In current study, the magnesium, sodium and potassium
concentrations in soils of block-1 and block-2 were below critical levels; consequently
concentrations of these minerals in fodder samples were higher than the critical level. These
findings are in antagonistic to Ashraf et al. (2006) who detected lower potassium levels in fodder
grown-up in soils having higher potassium concentrations. In the present study; copper, iron and
zinc concentrations in soil and fodder of block-1 and block-2 were above the critical levels.
In both the blocks serum magnesium, potassium and zinc concentrations were lower than
normal. The mean concentrations of calcium in the present study are within normal range and are in
agreement with Yadav et al. (1998) and Das et al. (2004). The results of present study for sodium
are also in agreement with Khan et al. (2008). Sodium concentrations are also in normal ranges in
both the blocks and it may possibly be by reason of feeding of common salt in surplus amount to
the buffaloes as it is the unchanging exercise in this area. Feeding of common salt to animals at the
rate of 100–150 g of salt/animal/ day is also reported in India by Kumaresan et al. (2008). Copper
concentrations in the serum of buffaloes observed in present study are in agreement with Bedi and
Khan (1984). However, all buffaloes involved in current study were deficient in zinc, and these
findings corroborate with Yadav and Khirwar (2000). Compared to our study, high concentrations
of zinc have also been reported (Bedi and Khan 1984; Das et al., 2004).
Results of current study conclude that buffaloes raised in study area were lacking in some
minerals, and for optimal production these minerals must be supplemented in diet.
REFERENCES
Adams, F. and D. L. Hartzog. 1960. The nature of yields responses of florunner peanuts to lime.
Peanut Sci. 7:120–123.
Ashraf, M. Y., A. Khan, M. Ashraf and S. Zafar. 2006. Studies on transfer of mineral nutrients
from
feed, water, soil and plants to buffaloes under arid environments. J. Arid Environ. 65:632-
643.
Bedi, S. P. S. and S. A. Khan. 1984. Trace element status of soil, fodder and animals in Bijnore
district of Uttar Pradesh. Ind. J. Anim. Sci. 54:570-574.
Das A, T. K. Ghosh and S. Haldar. 2004. Status of major and trace elements in grazing cattle and
buffaloes of red laterite and new alluvial zone of West Bengal. Ind. J. Anim. Sci. 74:285-
291.
Fardous, A., G. Sumaira, A. S. Zahid, A. Kafeel, Z. I. Khan, M. Ibrahim, A. E. W. Ahmad, S. Ullah
and E.E. Valeem. 2010. Sodium, potassium and magnesium dynamics in soil, plant,
animal continuum. Pak. J. Bot. 42:2411-2421.
Khalili, M., E. Lindgren and T. Varvikko. 1993. A survey of mineral status of soil, feeds and cattle
in the Selale Ethiopian highlands. II. Trace elements. Trop. Anim. Health Prod. 25:193-
201.
Khan, Z. I., M. Danish and K. Ahmad. 2008. Macromineral profile in forage, blood plasma and
urine of grazing buffaloes with respect to seasonal variation. Buff. Bullet. 27:173-176.
Khan, Z. I., A. Hussain, M. Ashraf and L. R. McDowell. 2006. Mineral status of soils and forages
in
southwestern Punjab-Pakistan: Trace-minerals. Asian-Australian J. Anim. Sci. 19:1139 –
1147.
Kumaresan, A., P. P. Prabhakaran, K. M. Bujarbaruah, K. A. Pathak, B. Chhetri and S. K. Ahmed.
2008. Reproductive performance of crossbred dairy cows reared under traditional low
826
input production system in the eastern Himalayas. Trop. Anim. Health Prod.
10.1007/s11250-008-9155-0.
McDowell, L. R. and J. D. Arthington. 2005. Minerals for Grazing Ruminants in Tropical Regions.
Extension Bulletin Animal Science Department, University of Florida.
McDowell, L. R. and J. H. Conrad. 1977. Trace mineral nutrition in Latin America. World Anim.
Rev. 24:24.
Ndebele. N., J. P. Mtimuni, I. D. T. Mpofu, S. Makuza and P. Mumba. 2005. The status of selected
minerals in soil, forage and beef cattle tissues in a semi-arid region of Zimbabwe. Trop. Anim.
Health Prod. 37:381–393.
Pereira, J. V., L. R. McDowell, J. H. Conrad, N. Wilkinson and F. Martin. 1997. Mineral status of
soils, forages and cattle in Nicaragua. I. Trace minerals. Rev. Fac. Agron., 14:73–89.
Richard L A. 1968. Diagnosis and improvements of saline and alkaline soils. (1st Ed.). Agri.
Handbook
No. 60, IBH Publishing Co., New Delhi, India.
Sharma, M. C., C. Joshi and S. Gupta. 2003. Prevalence of mineral deficiency in soils, plants and
cattle of certain districts of Uttarpredesh. Ind. J. Vet. Med., 23:4–8.
Singh, V. and B. Bohra. 2005. Livestock feed resources and feeding practices in hill farming
system—a review. Ind. J. Anim. Sci. 75:121-127.
Soltanpour, P. N. 1985. Use of AB-DTPA soil test to evaluate elemental availability and toxicity.
Comm. Soil Sci. Plant Anal. 16:323-330.
biometrical approach. 3rd Ed., McGraw Hill Co. New York, USA.
Yadav, P. S., A. B. Mandal, V. Kapoor, K. R. Sunaria and N. S. Mann, 1998. Mineral status of
cows and buffalos in Rewari district of Haryana. Ind. J. Anim. Sci. 68:1059-1061.
Yadav, S. and S. S. Khirwar. 2000. Soil-plant-animal relationship of zinc in milch buffaloes of
Jind district in Haryana. Ind. J. Anim. Sci. 70:965-967.
Table 1. Mean (±SE) concentrations of different minerals in soil of Block-1 & Block-2.
Critical
Mineral Concentrations Block-1 Block-2
(CC)
Calcium (ppm) 71 122.34 ± 0.89 a 123.31 ± 0.86 a
Magnesium (ppm) 9.10 9.02 ± 0.33 a 9.05 ± 0.29 a
a
Sodium (ppm) - 0.04 ± 0.001 0.04 ± 0.001 a
Potassium (ppm) <37 0.14 ± 0.005 a 0.13± 0.005 a
a
Copper (ppm) 1 1.39 ± 0.19 1.44 ± 0.18 a
Iron (ppm) 20 28.06 ± 2.16 a 26.26 ± 2.11 a
a
Zinc (ppm) 1.50 1.87 ± 0.15 1.91 ± 0.16 a
Values sharing similar superscripts differed non-significantly
827
Table 2. Mean (±SE) concentrations of different minerals in fodder of Block-1 & Block-2.
Critical
Mineral Concentrations Block-1 Block-2
(CC)
Calcium (%) 0.30 0.47 ± 0.04 a 0.45 ± 0.02 a
a
Magnesium (%) 0.12 0.15 ± 0.01 0.17 ± 0.01 a
Sodium (%) <0.08 0.40 ± 0.02 a 0.42 ± 0.02 a
a
Potassium (%) <0.25 0.83 ± 0.01 0.87 ± 0.01 a
Copper (ppm) 10 19.28 ± 2.66 a 18.98 ± 3.32 a
a
Iron (ppm) 30 419.54 ± 41.22 422.13 ± 38.67 a
a
Zinc (ppm) 30 31.14 ± 2.53 30.94 ± 2.37 a
Table 3. Mean (±SE) concentrations of different minerals in serum of buffaloes of Block-1 &
Block-2.
Mineral Block-1 Block-2

Calcium (mmol/l) 2.16 ± 0.05 a 2.15 ± 0.04 a
Magnesium (mmol/l) 0.19 ± 0.01 a 0.21 ± 0.01 a
Sodium (mmol/l) 144.06 ± 1.72 a 142.5 ± 1.56 a
Potassium (mmol/l) 2.68 ± 0.26a 1.99 ± 0.19b
Copper (µmol/l) 12.12 ± 1.51 a 12.69 ± 1.09 a
Iron (µmol/l) 42.19 ± 3.12 a 41.95 ± 2.32a
Zinc (µmol/l) 10.11 ± 1.91 a 9.91 ± 1.05 a
828
Digestibility and Performance of Buffalo Fed Total Mixed Ration with

Different Levels of Citric Waste
Suthipong URIYAPONGSON*, Chalong WACHARAPAKORN, Chainarong
NAVANUKRAW, Chirasak PHOEMCHALARD, Julakorn PANATUK and Wechasit
TOBURAN
Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand

*Corresponding email: suthipng@kku.ac.th
ABSTRACT
The experiment was conducted to determine the utilization of citric waste (CW) in total
mixed ration (TMR) for buffaloes. Sixteen swamp buffaloes (200.9 ± 3.1 Kg body weight) were
randomly allotted into 4 treatments, 4 replications (one buffalo/one replication) according to the
Completely Randomize Design Experiment (CRD). TMR diets were formulated to contain 14%
crude protein and 2.6 Mcal/kg energy according to the requirement of buffaloes. The CW was
substituted for cassava in TMR at 0, 10, 20 and 30%, respectively. The buffaloes were raised in
individual cages for 2 months feeding trial. Growth and feed intake (FI) were recorded and
determined for FI, average daily gain (ADG) and feed per gain (FCR). Feces were also recorded
and collected to determine digestion coefficient using acid insoluble ash (AIA) marker. The results
showed that the substitute of CW had no effect on ADG, FI and FCR of buffaloes (P>0.05). The
digestion coefficient of dry matter, protein, and NDF were not different (P>0.05) among diets.
Diet with 10% CW had the highest digestibility (P>0.01). The digestibility of ADF was decreased
with the higher level of CW.
Keywords: digestibility, waste product, citric plant, buffalo
INTRODUCTION
Buffaloes are the important domestic animal, especially for small holder in the Northeast
of Thailand. The numbers of buffaloes are rapidly decreased every year. To increase efficiency of
buffalo production, several factors need to be improved especially the diets. The good quality diet
can increase the efficiency of production such as weight gain, carcass quality and meat production.
Waste products from cassava are cassava peel and cassava pulp which account for 3 and 6 % of
fresh weight respectively (Rakshit, 2003). Both waste had high production but low cost (0.50 Baht
to 1.5 Baht/Kg) and can be used for citric acid production by mixed with rice brand and inoculated
with Aspergillus niger for 7 days. There is a lot of residue left after citric was harvested. These
residues have high acidity but still have some protein (2.98%) which can be used for animal feed.
Previous research showed that CW can be used in cattle diet up to 16 % without any effect on
feedlot performance. According to several researches, buffalo can use low quality roughage and
high lignin feed ingredients than cattle. Then citric waste (CW) should be able to use as the feed
ingredient in concentrate diet for buffalo. If CW can be used more in buffalo diet, it will be useful
not only to reduce feed cost for buffalo production but also reduce waste from citric plant. The
objective of this experiment is to study on digestibility of diet with several levels of CW in
buffaloes.
Citric acid is an organic acid used in several industries such as food, beverage, medicine
and cosmetics. Several crop residues can be used for citric production such as corn cob and
cassava waste (Hossain et al., 1983). At least 2 plants in Thailand used cassava waste for citric
acid production. One plant in Kalasin uses cassava pulp as the major sources in Solid State
Fermentation process (SSF). The second plant in Samutsongkarm uses cassava chip as the major
sources in the Submerge Fermentation process (SF). The SSF is more popular process due to more
citric production and less waste (Pandey and Soccol, 1998). With an appropriate water (60 %

829
moisture) and fermentation period, there will be a production of citric 26-27 g/100 of substrates
(Grewal and Kalra, 1995; Prado et al., 2005). Uriyapongson et al. (2006) reported that waste
product was high in moisture (77.63 %), acid (pH 4), fiber and lignin and 3-5% protein available.
Uriyapongson et al. (2006) and Prapunsil (2008) reported that CW had NDF, ADF and
acid detergent lignin (ADL) at 86.13, 68.17 and 19.74 % of dry matte respectively. This high fiber
waste can be used at low level in non-ruminant. Dnupatra and Todsapon (2007) used CW to
replace cassava at 0, 5 and 10% in goat diet. They concluded that, concentrate intake of the goat
was lower due to high acid of CW and recommend to supplement not more than 5 % in
concentrate diet for weaning goat. Peeraporn et al. (2008) used CW and rice straw in the total mix
ration (TMR) for dairy cattle. The ratio of roughage to concentrate was 40 to 60 and levels of CW
in TMR were 0, 10, 20 and 30 %, respectively. The results showed that feed intake was higher due
to more fiber in rice straw than in CW. Milk production (4 % fat collected milk) increased directly
to the level of CW while milk fat and milk protein were directly decreased. This was due to rice
straw was the better precursor for VFA production than CW. However, used 30% CW with rice
straw reduce feed cost and made more profit to farmers. Prapunsil (2008) used CW in concentrate
diet at 0, 10, 20 and 30% in 162 Kg. Brahman X Native crossbred cattle. The result showed that
cattle fed 10 % CW had the highest feed intake and weight gain. More CW in the diet reduced
digestibility of the cattle because of the higher fiber (68.17 %). He concluded that CW can be used
up to 10 % without any effect on feedlot performance and carcass characteristics.

Sixteen culled water buffaloes (200.9±3.1 kg. body weight) were randomly allotted into 4
treatments and 4 replications according to Completely Randomized Design experiment. All
buffaloes were fed daily with total mix ration (TMR) at 3 % of body weight while water was
available at all times. The TMR diets were formulated to provide 14% crude protein and 2.67
Mcal/kgDM gross energy according to NRC (1984) as showed in Table 1. The CW was
supplemented for cassava at 0, 10, 20 and 30% in the TMR. Feed intake was daily recorded. Feed
and feces samples were collected to determine for dry matter (DM) crude protein (CP), ash and
ether extract, EE) according to procedure of AOAC (1985). Neutral detergent fiber (NDF) and
acid detergent fiber (ADF) were determined according to procedure of Goering and Van Soest
(1970). Acid insoluble ash (AIA) was determined according to procedure of Van Keulen and
Young (1977) and used as an indicator to evaluate digestion coefficient. The digestion trial was
run for 2 months feeding period. All data were determined for Analysis of variance. Treatment
means were compared using Duncan’s New Multiple Range Test (SAS, 1990).

The chemical composition of experimental diets showed higher NDF and ADF when
higher percentage of CW was supplemented (Table 2). Supplementation of CW had no effect on
feed intake final weight, weight gain and feed conversion ratio (P>0.05) as showed in Table 3.
Weight gain buffalo was 0.44 kg/day and buffaloes from T1 showed the higher weight gain
compared to other treatments. Weight gain from this experiment was lower than those reported by
Wanapat and Wachirapakorn (1990) in buffalo fed 500 g concentrated per day and gain 0.8
kg/day. Uriyapongson et al. (2003) also showed higher weight gain (approximately 0.8 kg/day)
when used broken rice substituted for corn in culled buffaloes. Prapunsil et al. (2008) concluded
that cattle had more FCR when used more CW in the concentrate diets.
Digestion coefficient using AIA as an indicator showed that buffaloes had similar
digestibility of DM, protein (CP), crude fiber and NDF (P>0.05) as showed in Table 4. However,
digestibility of OM of buffaloes fed 10% CW was higher than other treatments. The digestibility
of ADF decrease lower as the level of CW increased. Prapunsil et al. (2008) studied in beef cattle
also showed that digestibility of protein was higher in 10% CW than without CW. The higher
level of CW reduced digestibility of ADF and feed intake in cattle. Therefore, utilization of CW to
supplement for cassava had no effect on feed intake, weight gain, feed conversion ratio in
830
buffaloes (P>0.05). The digestion coefficient of nutrient (DM, CP and NDF) were similar among
treatments but digestibility of OM in diet with 10% CW had the highest value (P<0.01) while
digestibility of ADF in non-supplemented group had the highest value (P<0.01).
REFERENCES
AOAC. 1995. Official method of analysis. 18th edn. Association of Official Analytical Chemists,
Arlington, Virgia.
Danupatra, C. and T. Moonanee. 2007. Study on citric waste supplementation for cassava in
weaning goat. Special problem, Department of Animal Science, Faculty of Agriculture,
Khon Kaen University, Thailand.
Goering, H.K. and P.J. Van Soest. 1970. Forage Fiber Analysis: ARS Hanbook, Department of
Agriculture, Washington. DC.
Grewal, H.S. and K.L. Kalra. 1995. Fungal production of citric acid. Biotech. Adv. 13: 209-234.
Hossain, M., J.D. Brooks, and I.S. Maddox. 1983. Production of citric acid from whey peament by
fermentation using Aspergillus niger. Newzealand J. Dairy Sci. 19: 161-168.
NRC. 1984. Nutrient Requirements of Beef Cattle. 6th Revised Ed. National Research Council,
National Academy Press, Washington, DC. pp. 90.
Pandey, A. and C.R. Soccol. 1998. Bioconversion of biomass: a case study of lignocellulose
bioconversions in solid-state fermentation. Brazilian Arch Biol. Technol. 41: 379-390.
Peraporn, S., C. Wacharapakorn, N. Sornsongnern and S. Saetaew. 2008. Effect of citric waste and
rice straw in total mixed ration for dairy cattle. The fourth Animal Science conference,
Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand.
Prado, F.C., L.P.S. Vandenberghe, A.L. Woiciechowski, J.A. Rodrigues-Leon and C.R. Soccal.
2005. Citric acid production by solid-state fermentation on a semi-pilot scale using
different percentage of treated cassava bagasse. Bra. J. Chem. Eng. 22: 547-555.
Prapunsil, T. 2008. Effect of citric waste in concentrate diet on feed intake growth performance
carcass characteristics and meat quality of beef cattle. Master Thesis, Department of
Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand.
Rakshint, S.K. 2003. Recent trends in cassava starch production and application. Bioresearch
Technol. 67:47-52.
SAS Institute Inc. 1990. SAS/STAT User’s guide. SAS Institute Inc., Cary, USA.
Van Keulen, J. and B.A. Young. 1977. Evaluation of acid- insoluble ash as a natural marker
in ruminant digestibility studies. J. Anim. Sci. 40: 281-287.
Wanapat, M. and C. Wachirapakorn. 1990. Utilization of roughage and concentrate by feedlot
awamp buffalo (Bubalus bubalis). Asian-Aust. J. Anim. Sci. 3:195-204.
831
Table 1. Feed ingredient of total mixed ration.
Total Mixed Ration

Feed ingredient (%)
T1 T2 T3 T4
Citric waste 0 10 20 30
Cassava 40 30 20 10
Rice straw 30 30 30 30
Soybean meal (44%CP) 12.1 12 12.2 12.5
Rice bran 2.3 2.05 1.5 1
Palm oil meal 3.2 3.2 3.2 3.2
Palm oil 2 2.4 2.8 3.1
Molasses 5 5 5 5
Di-calcium 1 1 1 1
Mineral 1 1 1 1
Salt 0.5 0.5 0.5 0.5
Urea 2.1 2.05 2 1.9
Lime 0.5 0.5 0.5 0.5
Sulfur 0.3 0.3 0.3 0.3
Total 100 100 100 100
Price / kg (Baht) 5.7 5.2 4.8 4.4
Table 2. Nutrient composition of Total Mixed Ration.
Nutrient % T1 T2 T3 T4
Dry matter (DM), % 88.37 88.80 87.33 86.47
% Dry matter
Organic matter (OM) 90.25 89.54 88.23 86.24
Ash 9.74 10.45 11.76 13.75
Crude protein 14.32 14.74 14.56 14.16
Ether extract (EE) 2.34 3.00 3.93 3.19
NDF 31.96 37.94 41.87 47.58
ADF 27.38 28.53 31.66 36.25
Total Energy (GE), Mcal/kg 3.80 3.85 3.80 3.80
Note T1, T2, T3, T4 means diet with CW at 0, 10, 20 and 30 %
Table 3. Effect of citric waste in concentrate diet on growth performance in buffaloes.
Treatments
Characteristics SEM P-value
T1 T2 T3 T4
Initial weight (kg) 207.00 191.25 207.00 198.67 5.78 0.22
Final weight (kg) 239.75 220.50 229.50 221.67 8.42 0.40
Weight gain (kg) 0.55 0.49 0.38 0.37 0.05 0.17
Feed intake (kg/d) 5.94 5.14 4.56 4.82 0.43 0.19
Feed conversion ratio 11.07 11.11 12.04 13.56 1.39 0.64
832
Table 4. Digestibility of nutrient in buffaloes fed different level of CW.
Treatment
Characteristics SEM P-value
T1 T2 T3 T4
Dry matter digestibility (%) 54.11 60.44 54.60 53.23 2.11 0.21
Organic matter digestibility (%) 63.65b 65.42a 63.84b 62.00c 0.17 <0.01
Digestibility of protein (%) 67.23 70.55 68.58 67.13 1.00 0.19
Digestibility of NDF (%) 53.61 51.85 51.05 50.81 1.50 0.59
a b
Digestibility of ADF (%) 52.62 50.31 49.66b 47.33c 0.57 0.01
abc
within row were difference (p<0.05)
833
Milk Yield Response of Bypass Protein Feeding (Soybean Meals) in

Dairy Animals
Netra Prasad OSTI a*, Pulkit Mandala, Bhola S.a and Shresthab
a
Animal Nutrition Division, National Animal Science Research Institute (NASRI)
b
Bovine Research Program, Khumaltar Lalitpur Nepal
*Corresponding email: n_osti@yahoo.com
ABSTRACT
Samples of protein rich ingredients were collected from different places of Kathmandu,
Bhaktapur and Lalitpur districts of Nepal and analyzed for dry matter (DM) and crude protein
(CP) content. Eighteen dairy animals (cows) were randomly assigned to full fat soybean meal
(grain) thereafter de-oiled soybean meal with untreated and heat treatment on three level of
feeding (0.5, 1.0, and 1.5 kg/animal/day). Before starting the trial, animals were kept in
adaptation period for 7 days with same feeding regime. In the feeding regime, ration was
calculated based on DM intake as 3 percent of body weight of animals. Of the total DM
allowance, 1/3 portion was from concentrate and 2/3 portion from roughages. Roughages were
also divided in to 1/3 portion as green and 2/3 as dry (rice straw) on dry matter basis. For those
animals producing above 10 kg milk, additional one kg concentrate was given for every 2 kg
milk production. Among 11 grain legumes analyzed, soybean (Glycine max) grain had the
highest percent CP (42.27%) followed by broad bean (Vicia faba), black gram (Vigna radiate
var mungo) and the lowest were found in cow pea (Vigna unauiculata) on dry matter basis. In
the feeding trials, the mean milk yield was found 10 kg per day per lactating animal which was
not different among the forms of soybean and levels of feeding but significantly different
(p<0.01) between before and after bypass protein feeding to the animals. Milk yield was low (8
kg/d/head) before bypass protein (BP) feeding, significantly higher during BP feeding (10.0
kg/animal/day) and again dropped down (8 kg/animal/day) when BP was withdrawn. In the
buffalo growing areas bypass protein could play important role to maintain milk production
during the lean period and this type of research should be emphasized in the programs of buffalo
growing areas.
Keywords: Protein rich ingredients, bypass protein, animal nutrition
INTRODUCTION
Feed is the main component to improve animal production. At present about 36 percent
feed deficit in the country and whatever available are low in quality, less digestible due to higher
fiber content like rice, wheat and pulses straws, sugarcane bagas etc (Upreti and Shrestha 2006).
These feed resources are deficient in one or other nutrients (energy and protein) which results
low absorption of nutrients, wastage for transportation time and storage problems. Now a day’s
people trying to adopt high yielding dairy animals, especially near to urban and market
assessable areas Chitwan, Pokhara, eastern Terai and road side areas. Large numbers of dairy
animals are brought to farm for milk production purposes from local collection and transported
from aboard. Existing stock are also upgraded through artificial insemination, embryo transfer
technology. Pace of breeding approach are not sufficiently upgraded with feeding and
management technologies which are seen in farmers filed around the milk production pocket
areas and there are not sufficient feeding resources for added stock and whatever available are
not in good quality.
In high yielding dairy animals, the capacity of rumen do not proportionately meet the
nutrients requirement of her milk production level to hold roughages and concentrate in the
rumen. And the good quality protein (true protein) degrade in rumen by microbes (bacteria and

834
protozoa), after come to in small intestine the amount she intake will ultimately reduce by
degradation in rumen therefore the efficiency of good quality protein will decrease.
Research works has been carried out in conventional protein resources by NARC and
other international research organization (McDONALD et al 1995) but limited research works
on use of rumen protected protein (by pass protein) available, in this regards Garg et al (2005)
reported that the degree of protein protection in formaldehyde treated rapeseed meal was 76.5
compared to an equivalent value of 36.3 percent in untreated meal. Some work has been done in
this line (ILRI, 2004) in African countries showed beneficial effect on milk production by using
rumen protected protein sources. For example soybean can be treated with heat and 55 to 65
percent of protein can be made by pass protein which ultimately protected degradation of lysine
and methionine from rumen microorganisms. Gross chemical analysis of protein sources found
in the country showed higher level of crude protein (Animal Nutrition Division’s reports) but
studies on this line are very limited. For better utilization of good quality protein sources, there
should be alteration of protein where rumen bacteria and protozoa may not attack on protein sources
and make available in small intestine in other word the amount of ammonia produced in the rumen
from various protein sources is inversely correlated with the N2 retention. Therefore high milk
yielding dairy animal need good quality feeding resources, which are not likely to degrade in the
rumen and directly go to abomasums and absorb in small intestine (bypass). Some feed
ingredients are high bypass value (cotton seed cake 55) and some are low, low value ingredients
could be altered their protein by chemically or heat treatments.

Sample collection and analysis
Samples of protein rich ingredients (table 1) Vigna radiata var mungo (Black gram-
Mas), Vicia feba ( Broad bean (Bakulla), Vigna unquiculata (Cow pea-Bodi), Pisum sativum
(Field pea-Kerau), Dolichos biflorus (Horse bean-Gahat), Phaseolus vulgaris (Kidney bean-
Rajma), Lens culinaris (Lentil-Musuro), Vigna radiata var aereu (Mung bean-Mung), Brassica
campestris (Mustard cake), Vigna umbellate (Rice bean-Masyam), Glycine max (Soybean-
Bhatmas), Soybean cake (Glycine max), Til cake (Sesamum indicum) and White bean(Seto simi)
were collected from various location of Nepal and analyses for dry matter (DM) and crude
protein (CP) using AOAC (proximate analysis) method (AOAC 1990).
Feeding trials and data analysis
Eighteen dairy animals (cattle) were randomly assigned to full fat soybean meal (grain)
for 15 days there after de-oiled soybean meal for 15 days with untreated and heat treatment
(table 2) on three level of feeding (0.5, 1.0, and 1.5 kg/animal/day). Before starting the trial
animals were adopted for 7 days with same feeding regime. Ration was calculated on 3 percent
of body weight as dry matter requirement. Of the total ration, 1/3 portion concentrate and 2/3
portion roughages. Roughages were also divided in to 1/3 portion green and 2/3 dry (rice straw)
and those animals producing above 10 kg milk additional one kg concentrate was also given for
every 2 kg. Data obtained in the trials were subjected to general linear model analysis in
computer, comparison were made with paired t-test (Minitab, 95).

Proximate composition
Proximate composition of protein rich ingredients, on dry matter basis (grains and cakes)
are presented in table 1. Among the grains, soybean (Glycine max) grain contain higher percent
(42.27) crude protein (CP) fallowed by broad bean (28.12) (Vicia faba), black gram (24.57)
(Vigna radiate var mungo) and the lowest was found in cow pea (18.93) (Vigna unauiculata) on
dry matter basis. In the cakes, soybean cakes contained higher (44.1%) crude protein followed
by Til (Sesamum indicum) (40.63%) and mustard (Brassica campestris) cakes (38.255).
Milk yield response of soybean
835
Table 2 present the milk yield response of different forms of soybean meals and level of
feeding. The mean milk yield was found 10 kg per day per lactating animals which was not
different among the forms of soybean and levels of feeding but significantly different from
before and after bypass protein feeding to the animals. Milk yield was low (8 kg/d/h) before
bypass protein feeding (figure 1) and when bypass protein was withdrawn (remove) from the
feeding regime; the milk yield was sharply declined (8 kg/d/h). The heat treatment in the full fat
soybean (grain) meal was found pronounce (effective) as compared with de-oiled soybean meal
on milk yield of dairy animals, this may be possibility of heat and pressure when oil extraction
from the soybean grain to make de-oiled soybean meal (figure 1) make protein protection in de-
oiled soybean meal. In this regards, Suresh et al (2011) reported that rapeseed meal (Brassica
campestris) undegradable protein (UDP) was beneficial to increase milk production in dairy
animals up to 571 g UDP/animal/day. Similarly, Garg et al (2003) reported that when 1 kg
additional rumen protected sunflower (Helianthus annuus) meal was given to lactating dairy
buffalo there was significantly increase in milk yield and fat percent in the milk.
CONCLUSION
Grain legumes are rich source of protein; mustard cake, til cake and soybean cake are
available in Nepal to feed livestock. Crude protein analysis show soybean meal contain the
highest crude protein content and from the feeding trial increasing trend in milk production was
observed while supplementing soybean meal therefore this legume is the best legume for feeding
animals and use as bypass protein source for dairy animals. In the buffalo growing areas bypass
protein could play important role to maintain milk production during the lean period and this
type of research should be emphasized in buffalo production areas.
REFERENCES
Association of Official Analytical Chemists (AOAC). 1990. Official methods of analysis 13th ed.
Association of Official Analytical Chemists (AOAC) Washington D.C.
Banerjee, G.C. 1991. A text book of animal husbandry 7th Edition, Oxford and IBH Publishing.
FAO. 2012. Balanced feeding for improvising livestock Productivity - Increase in milk
production and nutrient use efficiency and decrease in methane emission, by T.K. Walli,
M.R. Garg and Harindra P.S. Makkar. FAO Animal Production and Health Paper No.
172. Rome, Italy.
Garg, M. R., P. L. Sherasia, B. M. Bhandari, S. K. Gulati and T. W. Scott. 2005. Effect of
feeding rumen protected protein on milk production in low yielding crossbred cows.
Anim. Nut. and Feed Tech. 5 (1):1-8
Garg, M.R., P.L. Sherasia, B.M. Bhandari, S.K. Gulati and T.W. Scott 2003. Effect of feeding
rumen protected protein on milk production in lactating buffaloes. Anim. Nut. and Feed
Tech. 3 (2):151-157. http://www.freepatentsonline.com/5225230.html
International Livestock Research Institute (ILRI), Annual Report 2004.
McDONALD, P., R.A. Edwards, J.F.D. Greenhalgh and C.A. Morgan. 1995. Roots, Tubers and
related by-products. Animal Nutrition. Fifth edition. Addison-Wesley. Pp 481-488.
Minitab Windows. 1995. Minitab Statistical Software version 13.20.
Suresh, K.P., Raghavendra Bhatta, S. Mandal and K.T. Sampath 2011. Effect of bypass protein
on milk yield in Indian cattle-A meta-analysis. Anim. Nut. and Feed Sci. 11 (1): 19-26.
Upreti, C.R. and B.K. Shrestha 2006. Nutrient contents of feeds and fodder in Nepal. Animal
Nutrition Division (NARC). pp144.
836
Table 1. Proximate composition of protein rich ingredients.
S/N Sample name Botanical name DM% CP%

Grains
1 Black gram (Mas) Vigna radiata var mungo 90.41 24.57
2 Broad bean (Bakulla) Vicia faba 90.85 28.12
3 Cow pea (Bodi) Vigna unquiculata 88.26 18.93
4 Field pea( Kerao) Pisum sativum 88.68 22.47
5 Horse bean (Gahat) Dolichos biflorus 89.12 23.99
6 Kidney bean (Rajma) Phaseolus vulgaris 89.65 22.34
7 Lentil (Musuro) Lens culinaris 89.93 22.71
8 Mung bean (Mung) Vigna radiata var aereus 89.01 20.82
9 White bean (Seto simi) NA 91.18 19.28
10 Rice bean (Masyam) Vigna umbellata 88.15 21.34
11 Soybean (Bhatmas) Glycine max 88.89 42.27
Cakes
12 Mustard cake Brassica campestris 91.16 38.25
13 Soybean cake Glycine max 89.13 44.10
14 Til cake Sesamum indicum 88.08 40.63
Mean 89.39 29.01
P-value 0.76 0.00
F-value 0.67 8.14
837
Table 2. Milk yield response from heat treated and untreated soybeans (bypass protein).
Milk yield difference

Milk yield (kg/d/h) (kg/d/h)
Soy- Post- During- TRT- Incre
treatme Soy-meal- TRT TRT initial TRT-Post ment
Soy-type nt* (kg) Initial (A (B) (C) (C-A) (C-B) %
Full fat
soybean meal 1 0.50 7.85 8.83 9.09 1.24 0.26 13.68
,, 1 1.00 8.76 8.34 9.75 0.99 1.40 10.16
,, 1 1.50 7.98 8.49 8.71 0.73 0.22 8.38
,, 2 0.50 9.95 9.02 10.88 0.94 1.87 11.78
,, 2 1.00 8.63 8.97 9.51 0.89 0.55 18.35
,, 2 1.50 10.69 10.02 12.05 1.36 2.03 10.15
De-oiled
soybean meal 1 0.50 8.13 8.83 9.96 1.83 1.13 9.31
,, 1 1.00 9.00 8.34 10.01 1.02 1.67 11.78
,, 1 1.50 8.21 8.49 9.68 1.46 1.19 20.86
,, 2 0.50 10.15 9.02 10.23 0.08 1.21 0.76
,, 2 1.00 8.86 8.97 9.07 0.21 0.10 2.29
,, 2 1.50 9.71 9.18 10.94 1.22 1.75 11.17
Mean (full fat soybean meal) 8.98 8.94 10.00 1.02 1.05 10.20
Mean (de-oiled soybean meal) 9.01 8.81 9.98 0.97 1.17 9.71
Mean (untreated) 8.53 8.67 9.71 1.18 1.04 12.15
Mean (heat treated) 8.97 8.92 9.89 0.92 0.97 9.30
Mean (level of feeding) 0.50 kg 9.03 9.00 9.90 0.87 0.90 8.78
1.00kg 8.81 8.83 9.88 1.07 1.05 10.86
1.5 kg 8.42 8.56 9.62 1.20 1.06 12.50
Grand mean 8.99 8.87 9.99 1.00 1.11 10.69
*Soy-treatment 1= untreated, 2= heat treated
838
Figure 1. Milk yield response of dairy animals with bypass protein feeding.
Milk Yield Response of Bypass Protein
12.00
10.00
8.00
Milk Yield (kg/d/h)
Full fat soybean meal

Deoiled soybean
meal
6.00
Before BP
Feeding After BP
4.00 Feeding
During Bypass Protein (BP) Feeding
2.00
0.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Days in T rial
Untreated Heat treated
839
Body Composition and Net Energy Requirements for Maintenance

of Non-Castrated Water Buffaloes (Bubalus bubalis)
André Mendes JORGEa*, José Antonio de FREITASb, Carlos Augusto de Alencar
FONTESc, André Michel de CASTILHOSd, José Nicolau Próspero PUOLI FILHOd,
Rafael Silvio Bonilha PINHEIROe, Caroline de Lima FRANCISCOd, Maurícia
BRANDÃO DA SILVAd, Isabela de Mello PADOVANd and Alexandre COMINOTTEd
a
Animal Production Department, School of Veterinary Medicine and Animal Science, Univ
Estadual Paulista UNESP, 18618-000, Botucatu, Sao Paulo, Brazil. CNPq Researcher.
b
Univ Federal do Paraná UFPR, 85950-000, Palotina, Paraná, Brazil.
c
Univ Estadual do Norte Fluminense UENF, 28015-620, Campos dos Goytacazes, Rio de
Janeiro,Brazil.
d
School of Veterinary Medicine and Animal Science, Univ Estadual Paulista UNESP, 18618-
000, Botucatu, Sao Paulo, Brazil.
e
Biology and Animal Science Department, Faculty of Engineering of Ilha Solteira, Univ
Estadual Paulista UNESP,15385-000, Ilha Solteria, Sao Paulo, Brazil.
* Corresponding e-mail: andrejorge@fmvz.unesp.br
ABSTRACT
The research aimed to estimate body contents of protein and energy and net
requirements of energy for maintenance of buffaloes, slaughtered at different stages of
maturity. There were used 14 Mediterranean intact males with initial average body weight
of 352.2 ± 24.3 kg and average age of 24 months. The animais were randomly divided
into four experimental groups. One group was designed to slaughter at the beginning of the
experimental period (IS). The animals of another group were restricting fed, receiving,
individually, levels of protein and energy 15% above maintenance (RF). The animals of the two
remaining groups were individually fed ad libitum (SW450 and SW500) to reach weights
corresponding to 100 and 110 percent of the mature weight of the buffalo cows
(respectively 450 and 550 kg). The ration contained ground-corn cobs, soybean meal, urea,
minerals, and signal-grass (Brachiaria decumbens) hay, with a concentrate: roughage ratio
of 50:50 and 13% of crude protein on a dry matter basis. To estimate changes in body
composition inside the range of weights included in the trial, linear regression equations of log
protein (kg), fat (kg) and energy (Mcal) as a function of log empty-body-weight (EBW),
in kg, were fitted. Energy requirements for maintenance were obtained as estimated heat
production at zero level of energy intake. Buffaloes submitted to fattening in feedlot presented
early body fat deposition, and had with the same live weight lower protein content and higher
fat content and energy per unit weight than european-zebu crossbred cattle.
Keywords: energy, fat, maintenance, protein, stage of maturity
INTRODUCTION
The energy requirement for maintenance is defined as the amount of energy consumed
which results in no loss or gain of energy by the body tissues of the animal. The energy costs of
maintenance include body temperature regulation, the essential metabolic processes and
physical activity, and account for 70% of total energy requirements of mature beef cows (NRC,
1996).
Lofgreen and Garret (1968) cited that the net energy requirements for maintenance,
according corresponding to the heat production of the animal fasting. When energy intake is
zero, the caloric increment is also zero and net requirements for maintenance correspond to the
basal metabolism and the volunteer activities of the animal. Heat production fasting was
840
estimated by the same authors by regression were considered several levels of energy intake,
obtaining the estimate of 77 kcal/kgBW0.75 for steers and heifers.
The energy requirements for maintenance vary with age, sex, body composition and
nutritional status of the animal (Coelho da Silva and Leão. 1979), being also influenced by
volume and location of body fat deposits, weight, the nutritional level, physical activity and the
environment (Ferrel and Jenkins, 1983; Solis et al., 1988).
In Brazilian conditions, there are few works with buffaloes, particularly on body
composition and nutrient requirements of the different genetic groups, under the most different
feeding conditions. Therefore this study aimed to evaluate body composition and net energy
requirements for maintenance of non-castrated buffaloes finished in feedlot.

Fourteen crossbred Mediterranean buffalo bulls, with an average age of 24 months and
body weight (BW) of 352.2 ± 24.3 kg were used and randomly assigned into four treatments:
initial slaughter or reference (IS), restricted feeding (RF) and two treatments where they were
fed ad libitum (SW450 and SW500) until they reach slaughter weights of 450 and 500 kg,
respectively . In the IS and RF treatments were allocated 3 animals each and SW450 and
SW500 treatments, 8 animals, four in each slaughter weight.
After an adaptation period of 60 days, all animals received the same treatment, were
slaughtered IS group, serving as a reference in the study of initial body animals. SW450 and
SW500 animals received during the experimental period, balanced diet ad libitum, formulated
according to NRC (1984) for daily live weight gain of 1.1 kg, while the RF animals received the
same diet in limited quantities in order to ingest protein and energy levels 15% above the
maintenance requirements. The forage: concentrate ratio in the diet was 1:1.
The animals received feed individually twice a day (morning and afternoon). The
quantities fed to the animals RF were adjusted according to the body weights taken every 28
days, while the animals SW450 and SW500 were adjusted so as to maintain the leftovers
between 5 and 10% of the offered. The quantities of feed and the leftovers were recorded daily,
and collected feed samples and leftovers individually once a week. SW450 and SW500 animals
were slaughtered as they reached the predetermined weights of 450 and 500 kg As was
slaughtered an SW450 animal group was also slaughtered an RF animal group closest to him as
to body weight, at the beginning of the experiment.
Prior to slaughter, the animals were fasted for 16 hours. Of each slaughtered animal was
weighed and samples were collected from the head, hide, feet, rumen, reticulum, omasum,
abomasum, large intestine, small intestine, mesentery, internal fat, heart, kidneys, liver, spleen,
lung, tongue , blood, esophagus, trachea, and reproductive tract. The two half carcasses were
weighed on the day of slaughter and later cooling at -5 ° C and 18 hours. After this period, a
representative sample of the left carcass corresponding to 9th-11th rib section according to
Hankins and Howe (1946) procedure to determine physical carcass composition.
The empty body weight (EBW) of the animals was determined by the sum of
carcass weight, blood, head, hide, feet, tail, viscera and organs. The relationship obtained
between EBW and BW of the animals reference (IS) was used to estimate the in itial EBW
of the remaining animals.
Body fat content, crude protein, water and mineral were made as described by
Freitas et al. (2000). Body energy content (BEC) was made from the protein and fat
contents and their caloric equivalents, according to the ARC (1980) equation: BEC (Mcal) =
(5.6405*X + 9.3929*Y), wherein X = body protein and Y = body fat.
Prediction of net energy content, protein and fat retained in the body was performed
by fitting regression equations of the logarithm of body energy content, fat and
protein as a function of logarithm of EBW, according to ARC (1980) by model: Y i =
µ + b i * X i +e ij wherein Y i = logarithm of the total energy content (Mcal), protein (kg)
or fat in empty body weight of the animal i; µ = mean effect (intercept); b i =
841
regression coefficient of the logarithm of the energy and protein content in the
logarithm of EBW; X i = Logarithm of empty body weight the animal i; e = random
error, assumed normally distributed with mean zero and variance σ2. The analysis of
variance was used the F-test at 5% probability.
The net energy for maintenance (El m ) were determined by regression of the
logarithm of the heat production as a function of metabolizable energy intake in
Kcal per day and per unit of metabolic weight (MEI/day/BW 0.75 ), extrapolating the
equation for the zero level of ME intake, according to Lofgreen and Garret (1968).

The body fat content can be expressed by the following equation: Log kg Body Fat = -
3.23810 + 1.98157*EBW.
The body fat content increased with increasing body weight similar to ARC (1980).
The results of this study (Table 1) indicate an increase in body fat content from 150.8
to 224.7 g / kg EBW for buffaloes as the EBW rises from 350 to 500 Kg These results
are consistent with the results found by the ARC (1980), where we observed the
elevation of body fat content with increasing body weight empty.
Energy content can be expressed by equation:

Log body energy content = - 0.243050 +1.5115700*EBW
The body energy content increased as the EBW increase (2.54 to 3.13 Mcal / kg
EBW between 350-550 Kg range weight), similar to body fat content,.
Protein content can be expressed by equation: (Table 2)

Log body protein content = 0.347356 + 0.567323*EBW
It was observed that the body protein content increased with increasing EBW,
but the concentration of protein (g / kg of EBW ) declined with increasing EBW,
which is consistent with ARC (1980). This is due to the slowdown in growth and
greater muscle fat accumulation, with increased body weight.
Log Heat Production = 1.17678 + 0.0025921*MEI (r2 = 0.99)
Extrapolating equation to zero intake of metabolizable energy (MEI), the net requirement of
maintenance was 56.18 kcal/kg BW 0.75
The energy requirement for maintenance of the present study was similar to that found
by Paulino et al. (1999) with four zebu breeds in feedlot and 20% lower than NRC
(1996) for beef cattle.
The data of body composition and nutritional requirements of buffaloes under tropical
conditions allow more accurate feed formulation, as currently performed are based on
standards with beef cattle, different foods and temperate climate.
REFERENCES
Agricultural Research Council - A.R.C. 1980. The Nutrient Rcquirements of Farm
Livestock. England, Commonwealth Agricultural Bureaux, 1980. 350p.
Coelho da Silva, J.F. and M.I. Leão. 1979. Fundamentos de nutrição dos ruminantes.
Piracicaba. Livroceres, 380p.
Ferrell, C.L. and T.G. Jenkins. 1983. Energy utilization by mature, non-pregnant, non-
lactating cows of different types. J. Anim. Sci.,v.58, n.1, p.234-243.
Freitas, J.A., C.A.A. Fontes, J.E. Soares, A.M. Jorge and L.H. Castilho-Estrada. 2000.
Composição corporal e exigência de energia para mantença de bovinos (zebuínos e
842
mestiços) e bubalinos não castrados, em confinamento. Arq. Ciên. Vet. Zool.

UNIPAR, v.3, n.1, p.19-29.
Hankins, O.G. and P.E. Howe. 1946. Estimation of The Composition of beef carcass cuts.
s.e.. USDA. (Technical bulletin. 926).
Lofgreen, G.P. and W.N. Garret, 1968. A system for expressing net energy requirements
and feed values for growing and fìnishing bcef cattel. J. Anim. Sci.. v.27, n.3,
p.793-806.
National Research Council - N.R.C. 1984. Subcommittee of Beef Cattle Nutrition. Nutrient
Requirements of Beef Cattle. 6th. ed, Washington, D.C., 90p.
National Research Council - N.R.C. 1996. Subcommittee of Beef Cattle Nutrition. Nutrient
requirements of beef cattle, 7th. ed. Washington, D.C. 242 p,
Paulino, M.F., C.A.A. Fontes, A.M. Jorge, J.C. Pereira and P. Gomes Júnior. 1999. Exigências
de Energia para Mantença de Bovinos Zebuínos Não-Castrados em Confinamento1
Rev. bras. zootec., v.28, n.3, p.621-626.
Solis, J.C., F.M. Byers, G.T. Sciielling, C.R. Long and L.W. Greene. 1988. Maintenance
requirements and energetic efficiency of cows of different breeds types. Anim. Sci.,
v.66, n.3, p.764-773.
Table 1. Fat and energy content in the empty body weight of non -castrated
Mediterranean buffaloes finished in feedlot .
Body Empty Body Body Fat Content Energy Content

Weight (Kg) Weight (Kg) (g/kg EBW) (Mcal / Kg EBW)
350 289.7 150.8 2.54

400 338.1 175.8 2.75
450 386.5 200.1 2.95
500 434.8 224.7 3.13
Table 2. Body protein content in the empty body weight of non-castrated crossbred
Mediterranean buffaloes finished in feedlot .
Body Weight EBW Protein Content Protein Total

(kg) (kg) (g/kg EBW) (kg)
350 289,7 190 54,47
400 338,1 180 60,55
450 386,5 170 65,33
500 434,8 160 69,85
843
Allometry of Organs and the Gastrintestinal Tract from Water Buffaloes

(Bubalus bubalis) Finished in Feedlot
André Mendes JORGEa*, Rafael Silvio Bonilha PINHEIROb, Maurícia BRANDÃO DA
SILVAc, Caroline de Lima FRANCISCOc, André Michel de CASTILHOSc, Dayane Cristina
RIVAROLIc, Carolina Cesarino COUTINHOc and José Nicolau Próspero PUOLI FILHOc
a
Estadual Paulista UNESP, 18618-000, Botucatu, Sao Paulo, Brazil. CNPq Researcher. CAPES-PAEX
b
Biology and Animal Science Department, Faculty of Engineering of Ilha Solteira, Univ Estadual
Paulista UNESP,15385-000, Ilha Solteira, Sao Paulo, Brazil.
c
Estadual Paulista UNESP, 18618-000, Botucatu, Sao Paulo, Brazil.
* Corresponding email: andrejorge@fmvz.unesp.br
ABSTRACT
The objective of present study was to evaluate the relative growth of organs and viscera
from water buffaloes. Fifteen Mediterranean intact males, averaging, 356.7 kg initial live weight
and twenty four months of age, were used. The animals were ramdomly assigned into three groups
(categories). One group was ramdomly assigned to immediate slaughter (AR), the rest two groups
were full-fed a ration containing 50% concentrate, dry matter basis until reaching the slaughter
weights of 450 and 500kg, respectively. At slaughter the empty body weight was determined and
the weights of head, feet, leather, lungs, heart, liver, spleen, rumen-reticulum, omasum, abomasum,
small intestine, large intestine were recorded. Regression equations of log weight of organs and
viscera as a function of log empty-body-weight (EBW), were fitted. All body components studied,
with exception of liver and spleen, developed slower than in relation to EBW.
Keywords: abomasum, large intestine, omasum, relative growth, rumen reticulum, small intestine
INTRODUCTION
The Brazilian marketing of beef is done through the carcass weight (yield), with direct
influence of the weights of organs and viscera (Jorge et al., 2003). Differences exist between
species and genotypes for the development model or rate of formation of organs and tissues which
constitute the body mass (Black, 1989; Jorge et al., 1999; Jorge and Fontes, 2001). The
development rate can also be affected by age, breed, sex, nutritional status and other environmental
causes (Coleman et al. 1993; Patterson et al. 1995; Almeida et al, 2001).
The study of the relative development of organs and tissues can be accomplished according
to various models and the allometric coefficient is used in order to know the ratio of the growth rate
of a particular part opposite the whole (Robelin et al. 1974). The results with European breed
animals are not necessarily applicable to Zebu and crossbred, or water buffaloes.
In Brazilian conditions, there are few works with buffaloes, particularly on the allometric
study of the different components of the body, it is necessary to study the behavior of the different
genetic groups, under the most different feeding conditions. Therefore this study aimed to evaluate
the relative growth of organs and viscera of water buffaloes finished in feedlot and slaughtered at
two stages of physiological maturity (slaughter weight).

The experiment was carried out at School of Veterinary Medicine and Animal Science, Sao
Paulo State University, Botucatu campus. Fifteen non-castrated Mediterranean buffaloes with
average age of 24 months and body weight (BW) 356.7 kg were used.
Animals were kept in feedlot and randomly assigned in individual stalls with concreted total
area of 30 m2. The animals were randomly allocated allocated divided into three experimental
844
groups (categories) with 5 animals each: initial slaughter or reference (IS), and "ad libitum" feeding
groups or the desire to reach slaughter weights fixed (SL450 and SL500 categories) of 450 and 500
kg BW, respectively.
Before the adaptation period, the animals were weighed, identified with earrings, treated
against endo and ectoparasites and received 1,500,000 IU vitamin A injection. The adaptation
period lasted 60 days, when the three categories animals received feed used during the experimental
period. After the adaptation period, the IS group animals were slaughtered serving as a reference in
the study of initial body composition of SL450 and SL500 category animal. The experimental
period lasted not pre-set, since the animals were slaughtered when they reached pre-set weights of
450 kg or 500 kg BW, the animals were weighed every 28 days and, as the animal approached to
slaughter weight preset, was weighing at shorter intervals to be slaughtered near the weight
specified.
Prior to slaughter, the animals had a 16 hour period of fasting. The slaughter was carried out
through concussion and posterior section of the jugular vein. At slaughter were weighed separately:
head, feet, leather, rumen-reticulum, omasum, abomasum, small intestine, large intestine, internal
fat, liver, heart, kidneys, spleen, lungs, tongue, blood, mesenteric, tail, esophagus, trachea, and
reproductive tract, weighed together. The carcass was divided into two halves and these weighed
individually.
The empty body weight (EBW) of IS animals was determined by the sum of carcass weight,
blood, head, feet, skin, tail and viscera. Specific relationships between EBW and BW were
determined. The value obtained was used to estimate the initial EBW of remaining experimental
animals. The final EBW of these animals was determined similarly to that obtained from IS
animals, during slaughter. The relationship observed for IS animals between EBW and carcass
weight was used to estimate the initial carcass weight of the animals from remaining categories
(SL450 and SL500). After the slaughter of IS animals, animals in categories SL450 and SL500
were fed with Brachiaria (Brachiaria decumbens Stapf.) hay, soybean meal, ground corn and cobs,
urea, calcium phosphate, limestone, salt and mineral mixture. The ration formulation followed the
NRC (1984, 1996) standards to enable daily weight gain of 1.1 kg were maintained at ratios of
concentrate: roughage close to 1:1 on dry matter (DM) basis. The concentrate consisted of 76.5%
ground corn, 20.1% soybean meal, 1.6% urea and 1.8% mineral mixture and the diet has a
following composition: dry matter (DM) = 88.5%, crude protein (CP) = 12.8%, metabolizable
energy (ME) = 2.4 Mcal/kg and macro minerals.
Statistical analyzes were performed using the GLM (General Linear Model) statistical
organ and viscera as a function of the logarithm of empty body weight, according the model Yij = 
package (SAS, 1994). We adopted the regression equation of the logarithm of the weight of each
weight of animal j and breed i;  = mean effect (intercept), bi = regression coefficient of the
+ bi Xij + eij, where: Yij = logarithm of the total weight of organs and viscera (kg) in the empty body
logarithm of the weight of organs and viscera (kg) as a function of the logarithm of the empty body
assumed normally distributed with mean zero and variance 2.To verify the hypothesis b = 1, was
weight; Xij = logarithm of empty body weight of the animal j from breed i, eij = random error,
used the "t" test (Student) (P <0.05). If b = 1, the growth was isogonic, indicating that the growth
rate of "X" and "Y" were similar in the range considered. When b ≠ 1, the growth was heterogonic,
being early if b <1 and later if b> 1.

The regression or allometric coefficients (β) to the equations of Table 1, except for the liver
and spleen were less than 1, revealing negative allometry negative, i.e., the intensities of developing
these body parts were inferior to the empty weight, denoting that are early growth. The results of
this study agree with the authors that worked with cattle, and possibly reflect the earlier maturity of
these parts considered in relation to the development of other tissues, such as muscle and adipose
especially in later maturity (Robelin et al. 1974; Jorge and Fontes, 2001). Leather has coefficient
845
isometric or slightly negative, agreeing with the approximate value of 0.94 in this study, although
the authors report that there may be variations depending on the breed (Berg and Butterfield, 1976).
The regression coefficients for liver (β = 1.03) and spleen (β = 1.13), showed, respectively,
the intensity of growth equal and superior to the EBW. In relation to other vital organs such as the
heart (β = 0.53) and lungs (β = 0.50), liver and spleen of buffaloes in this study were considered late
maturity, which corroborates with Jorge and Fontes (2001) observations with four Zebu genetic
groups, slaughtered at different stages of maturity. These results also confirm in buffaloes, the
observations of Berg and Butterfield (1976) working with cattle, that these bodies have priority in
the use of nutrients and have greater growth in an earlier stage of the animal's life.
The regression coefficients (β) of the equations for the gastrointestinal tract components were
smaller than 1 (ranging from 0.51 to 0.89), revealing negative allometry, i.e., the intensities of
developing these body parts were inferior to the empty body weight. These results are in agreement
with results in cattle (Robelin et al., 1974) and with sheep (Black, 1989; Osorio et al. 1994) and
reflects the earlier maturity of parts considered in the development of muscle tissue and especially
adipose tissue, later maturity.
It is important to emphasize Black (1989) assertion, according to which the growth of organs
such as liver, kidneys and digestive tract, involves rapid changes in weight when animals receive
diet above maintenance level, and presents remarkable atrophy when receiving below the
maintenance level. In the present study, buffaloes were fed "ad libitum" without food restriction.
Similar results from buffaloes in the present study buffaloes was found in a study with four
Zebu genetic groups (Gyr, Guzerat, Nelore and Mocho Tabapuã) slaughtered at different stages of
physiological maturity (Jorge and Fontes, 2001). These authors found allometric coefficients for
rumen-reticulum, omasum, abomasum, small intestine and large intestine of 0.62, 0.84, 0.88, 0.77
and 0.59, respectively.
In this experimental conditions the gastrointestinal tract components and other internal
organs, except the liver and spleen from Mediterranean buffaloes, finished in feedlot showed lower
growth impetus in relation to empty body weight.
REFERENCES
Almeida, M.I.V., C.A.A. Fontes, F.Q. Almeida, S.C.V. Valadares Filho and R.F. Guimarães. 2001.
Avaliação do crescimento de tecidos e órgãos de novilhos mestiços Holandês-Gir durante o
ganho compensatório. 1. Carcaça. Braz. J. Anim. Sci. 30:526-534.
Berg, R.T. and R.M. Butterfield. 1976. New concepts of cattle growth. New York, Sydney
University, 240p.
Black, L.L. 1989. Crecimiento y desarrollo de corderos. In: Haresign, W. Producción ovina. México
: AGT, p.23-62.
Coleman, S. W., Evans, B.C. and J.J. Guenther. 1993. Body and carcass composition of angus and
charolais steers as affected by age and nutrition. J. Anim. Sci. 71:86-95.
Jorge, A.M., C.A.A. Fontes and M.F. Paulino. 1999. Tamanho relativo dos órgãos internos de
zebuínos sob alimentação restrita e Ad libitum. Braz. J. Anim. Sci. 28:374-380.
Jorge, A.M. and C.A.A. Fontes. 2001. Desenvolvimento relativo das partes do corpo de zebuínos
de quatro raças. Cienc. Rural 31:857-861.
Jorge, A.M., C.A.A. Fontes and R.C. Cervieri. 2003. Crescimento Relativo e Composição do
Ganho de Tecidos da Carcaça de Zebuínos de Quatro Raças. Braz. J. Anim. Sci. 32:986-
991.
National Research Council - NRC. Nutrient requeriments of beef cattle. 1984. 6.ed. Washington,
D.C., 90p.
National Research Council - NRC. Nutrient requeriments of beef cattle. 1996. 7.ed. Washington,
D.C., 242p.
846
Osorio, J.C. da S., F.Siewerdt, and M.T.M. Osório. 1994. Desenvolvimento alométrico das regiões
corporais em ovinos. In: Proceedings of Annual Meeting of Brazilian Society of Animal
Science, 31, Maringá: SBZ, p.240.
Patterson, D.C., R.W. Steen and D.J. Kilpatrick. 1995. Growth and development in beef cattle. 1.
Direct and residual effect of plane of nutrition during early life on components of gain and
food efficiency. J. Agric. Sci. 124:91-100.
Robelin, J., Y. Geay and C. Béranger. 1974. Croissance relative des différentes tissus, organes at
régions corporelles dês taurillons frisons, durant la phase d’engraissement de 9 a 15 mois.
Ann. Zoot. 23:313-323.
SAS. 1994. User's Manual, Statistical Analyses System Institute, Cary, NC. 588p.
Table 1. Parameters of the regression equations of the logarithm of the weight of the body parts
(kg) in the logarithm of empty body weight (EBW) in Mediterranean buffaloes.
Parameters of regression equations

Components Intercept Regression coefficient (β) r2
Head 0.165989 0.613439 0.35**
Feet -0.278384 0.570634 0.54**
Leather -0.427283 0.937510 0.64**
Heart -1.355763 0.531350 0.38**
Liver -1.925470 1.029580 0.66**
Spleen -2.348010 1.130299 0.66**
Lungs -1.178830 0.500299 0.54**
**P<0.01
Table 2. Parameters of the regression equations of the logarithm of the weight of visceras (kg) as a
function of the logarithm of empty body weight (EBW) in Meidterranean buffaloes.
Parameters of regression equations

Components Intercept Regression r2
coefficient (β)
Rumen-reticulum -0.738555 0.650795 0.45**
Omasum -1.749094 0.853810 0.32**
Abomasum -2.295323 0.890933 0.57**
Small intestine -1.846523 0.867650 0.53**
Large intestine -1.057916 0.512335 0.34**
1
Stomach -0.741026 0.649298 0.58**
2
Intestines -0.910269 0.692980 0.58**
3
Gastrintestinal tract -0.509543 0.646394 0.63**
1 2
**P<0.01); Estomach = Rumen-reticulum + Omasum + Abomasum; Intestinos = Smal intestine
+ Large intestine; 3 Gastrintestinal tract = Stomach + Intestines
847
Distribution of beta-catenin in colon precarcinomatous lesions of rats fed with

functional buffalo milk.
María CATUOGNO, Gabriela RAMÍREZ, María MONTENEGRO, Marcial SÁNCHEZ

NEGRETTE
Cátedra de Patología General y Sistemática. Facultad de Ciencias Veterinarias. UNNE. Sargento

Cabral 2139. Corrientes Argentina.
* Corresponding email: patgral@vet.unne.edu.ar
ABSTRACT
The aim of this study was to determine the distribution of beta-catenin in cells of Displastic
Lesions (DL) in rats fed with buffalo milk. Milk of 8 fish oil supplemented Murrah and crosses
Murrah x Mediterránea buffaloes was used. The supplementation was carried out to get functional
milk with higher concentration in CLA and omega-3. This milk was administered to 42 rats to
evaluate the effect on the distribution of beta-catenin in DL. The DL were induced by the
cancerigenous 1,2- dimethylhydrazine (DMH). The immunohistochemistry technique was
performed to detect DL beta-catenin. Colonic epithelial cells of a normal intestinal mucosa clearly
displayed beta-catenin membranous expression. In superficial cells the protein could be identified
also in the cytoplasm but with weaker membranous stain. In the cells which were positioned deep in
the mucosa are observed a lack of protein in the cytoplasm remaining only in cytoplasmic
membrane so the cytoplasm was negative to the presence of beta-catenin. The enterocytes located
on muscular mucous showed a weaker membranous stain compared with superficial cell
membranes. Beta-catenin in the nucleous of normal cells was not observed. In DL of buffalo milk
fed rats the beta-catenin distribution substantially changed according to severity of DL. We
conclude that distribution of beta-catenin in colon DL of rats fed with functional buffalo milk, is
affected according to cell differentiation. These findings could be useful in detection of
precarcinomatous lesions in early stage.
Keywords: Beta-catenin – Buffalo milk – dysplastic lesions
INTRODUCTION
Beta-catenin is a protein that originally was found complexed with E-cadherin, α-catenin,
and -catenin (Ozawa et al., 1989); its NH2- terminal region appears to be necessary for cell-cell
adhesion (Oyama et al., 1994). When beta-catenin or APC genes are mutated or Wnt signaling
pathway is activated, beta-catenin accumulates in the cytosol, binds protein of the T-cell factor
family of transcriptions factors and moves to the nucleous (Mann et al., 1999). On the other hand,
the Aberrant Cript Foci (ACF) are considered an intermediate biomarker usefull in colon cancer,
such lesions are widely used to detect promoter and suppressor substances. The ACF were
considered by Bird (1987) as preneoplastic changes of colon cancer based on its cellular, molecular
and morphological features (Bird, 1987; Bird, 1995). The ACF are thought to be premalignant
lesions. Dysplasia is defined as a “precarcinomatous change” and as an “unequivocal neoplastic
alteration of the colonic epithelium” (Riddell et al., 1983). It has been described that the Beta-
catenin Accumulate Cripts are used as a new biomarker in rat colonic carcinogenesis because they
are strongly predisposed to colon cancer (Yamada et al., 2000; Yamada et al., 2001). In addition to
oncogenic beta-catenin protein accumulation, those lesions frequently hide mutations in beta-
848
catenin gene, which are involved in colon cancer development (Takahashi et al., 1999; Harada et
al., 1999). The aberrant beta-catenin expression in colonic tumors and the findings of beta-catenin
mutations in small adenomas suggest that beta-catenin alterations are early events in human
colorectal carcinogenesis. Normal colonic ephitelial cells which are surrounding human ACF
display strong membranous expression of beta-catenin and lack of cytoplasmic and nuclear
expression. Cytoplasmic expression of beta-catenin was seen in 25 of 46 ACF with dysplasia. In
ACF with dysplasia, reduced membranous expression of beta-catenin was associated with increased
nuclear and cytoplasmic expression. The membranous expression of beta-catenin was reduced and
the cytoplasmic and nuclear expression increased in ACF according to their degree of dysplasia.
These data suggest that ACF and their aberrant expression of beta-catenin play a role in colon
tumorigenesis (Hao et al., 2001). Epidemiologic studies suggest that 35% of death because of
cancer, are due to determinate kind of diet (Doll, 1992). Because of that, prevention through diet
intervention is an easy and logical approach for cancer treatment and prevention (Keenan et al.,
1998). Many investigators have demonstrated experimental colonic carcinogenesis to be inhibited
feeding rats with a fish oil high concentration diet (Reddy et al, 1986; Deschner et al, 1990) or with
an omega-3 fatty acid supplementation (Minoura et al, 1998). On the other hand, it has been proved
that buffalo milk can be improved on its Conjugated Linoleic Acid (CLA) and omega-3 fatty acid
basal concentration through a strategic supplementation with fish oil, which step up CLA and
omega-3 basal levels. Milk and meat of ruminants are the main sources of CLA. The isomers of
CLA are able to promote several beneficial effects for the organism such as anti-cancerigenous
effects. (Patiño et al, 2010; Patiño et al, 2012)

This study was performed with samples obtained from a proyect (PICTO-UNNE 2007-
00119) developed in our laboratory. Dysplastic lesions were obtained in rats fed with functional
milk from fish and sunflower supplemented buffaloes. Dysplastic lesions were classified according
to severity of dysplasia in mild, moderate and severe following the criterion gave by Siu y col.
(1997). We obtained: Group 5: 57 Dysplastic lesions (Mild: 51; Moderated: 6), Group 6: 35
Dysplastic lesions (Mild: 34; Moderated: 1); Group 7: 18 Dysplastic lesions (Mild: 17; Moderated:
1); y Group 8: 22 Dysplastic lesions (Mild: 21; Moderated: 1). After classified and count dysplastic
lesions stained with Hematoxilin and eosin, 4 serial samples were made for each dysplastic lesion
found previously. The samples were put on microscope slides pre-treated with Poly-l-Lisyne. Then
the technique was performed with the following steps: Deparaffinized and rehydratation. Antigen
retrieval by heating sections in citrate buffer (pH 6) inside a microwave oven. Endogenous
peroxidase activity was inhibited by incubation with 0, 3 % hydrogen peroxidase in methanol.
Blocking non specific staining with normal goat serum diluted 1/20 in PBS (pH 7.4). Incubation
with primary antibody: Sections were incubated in humidified chamber for 10 hours at 4 ºC with
Purified Mouse monoclonal anti beta-catenin antibody Transduction Laboratories. The working
solution was diluted 1/100. Incubation with biotinylated secondary antibody: Polyclonal anti IgG
mouse biotin conjugated. Code AM-B1. This antibody was produced in Laboratorio de
Endocrinología y Tumores Hormonodependientes de la Facultad de Bioquímica y Ciencias
Biológicas de la UNL. The working solution was diluted 1/100. The Incubation was made in
humidified chamber for 30 minutes at 25 ºC. Incubation with ExtrAvidin ™- Peroxidase (Buffered
aqueous solution) from Sigma-Aldrich. Working solution was diluted 1/200 in PBS. The sections
were incubated in humidified chamber for 30 minutes at 25ºC. Cromogen: was used in a working
solution of 0,05% diamnobenzidine tetrahydrocloride + 0,015 % hydrogen peroxidase in PBS.
849
Samples were incubated for 2 ½ minutes at 25 ºC. Dehydration and mountage of the sections. Once
the samples were stained by immunohistochemistry technique, the microscope evaluation was made
according to: Differences between lesions and normal surrounding ephitelium respect the amount of
beta-catenin protein (Intense stain, higher amount of protein); distribution of beta-catenin in
cytoplasm or nucleous; possibility to watch differences between mild, moderated and severe
dysplastic foci related to the kind of milk provided to the experimental animals; all the analyses
were made having the normal surrounding epithelium to dysplastic lesions as an internal control.
RESULTS AND CONCLUTIONS

The normal epithelial colonic cells clearly display prevalence of beta-catenin membranous
expression. In more differenciated superficial cells can be identified beta-catenin in cytoplasm also
with a lighter stain than the observed in membrane. It does not occur the same thing in calls which
are deeper in the mucosa. Those cells progressively loose protein in the cytoplasm staying only in
the membrane so the cytoplasm results negative for the presence of beta-catenin. The enterocytes
which are located over muscular mucosa have a lighter membranous stain than the membranes of
superficial cells. Beta-catenin is not observed inside nucleous of normal cells.
Mild Dysplasias
The distribution of beta-catenin in dysplastic lesions is modified according severity of the dysplasia
variation. In dysplastic lesions classified as mild with HE stain, the distribution of beta-catenin
protein does not display the irregularity observed in moderated dysplastic lesions. Mild lesions only
display a membranous distribution similar to normal glands except for the accumulation in
cytoplasm (in deeper cells) which was negative in normal cells.
Moderated Dysplasias
In moderated dysplastic lesions a clear accumulation of protein in dysplastic glands is seen if is
compared with surrounding normal cells. In a higher magnification can be seen a strong
membranous stain, more intense in dysplastic gland cells than in normal cells, besides can be
observed a beta-catenin accumulation in nucleous of dysplastic gland cells.
Severe Dysplasias
Dysplastic lesions found in all of the experimental animals were classified as mild and moderated.
No lesions of the severe kind and in situ carcinomas were seen, so there is no data about the
distribution of beta-catenin in those lesions.
We conclude the distribution of beta-catenin in DL induced by DMH in functional buffalo milk fed
rats is affected according to cell differentiation, which is seen in DL presented in human beings
(Hao et al., 2001). In well differentiated cells, beta-catenin are located in membrane to play its role
in the cell to cell adherence, meanwhile, in less differentiated cells, membranous expression is
reduced. This fact is associated a higher expression in cytoplasm and later in the nucleous as the
severity of dysplasia is growing. These findings could help in the process of early precarcinomatous
lesions detection.
References
Bird, R. P. 1987. Observation and quantification of abberrant crypts in the murine colon trated with
a colon carcinogen: Preliminary findings. Cancer Lett; 37: 147-151.
Bird, R. P. 1995. Role of aberrant crypt foci in understanding the patogénesis of colon cancer.
Cancer Res. 93: 55-71
850
Deschner, E. E., J. S. Lytle, G. Wong, J. F. Ruperto and H. S. Newmark. 1990. The effect of dietry
omega-3 fatty acids (fish oil) on azoximethanol-induced focal areas of dysplasia and colon
tumor incidence. Cancer 66: 2350-2356.
DOLL, R. 1992. The lessons of life: keynote address to the nutrition and cancer conference. Cancer
Res. 52:2024-2029.
Hao, X. P., T. G. Pretlow, S. Rao and T. P. Pretlow. 2001. β-Catenin expression is altered in human
colonic aberrant cript foci. Cancer Research 61: 8085-8088.
Harada, N., Y. Tamai, T. Ishikawa, B. Sauer, K. Takaku, M. Oshima and M. M. Taketo. 1999.
Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene.
EMBO J. 18: 5931-5942.
Keenan, K. P., P. Laroque and R. Dixit. 1998. Need for dietary control by caloric restriction in
rodent toxicology and carcinogenicity studies. J Toxicol Environ Health B. 1: 135-148.
Mann, B., M. Gelos, A. Siedow, M. L. Hanski, A. Gratchev, M. Ilyas, W. F. Bodmer, M. P. Moyer,
E. O. Riecken, H. J. Buhr and C. Hanski. 1999. Target genes of β-catenin – T cell-
factor/lymphoid-enhancer-factor signaling in human colorrectal carcinomas. Proc Natl
Acad Sci USA 96:1603-1608.
Minoura, T., T. Takata, M. Sakaguchi, H. Takada, M. Yamamura, K. Hioki and M. Yamamoto.
1998. Effect of dietary eicosapentaenoic acid on azoximethane induced colon carcinogenesis
in rats. Cancer res 48: 4790-4794.
Oyama, T., I. Kanai, A. Ochiai, S. Akimoto, T. Oda, K. Yanagihara, A. Nagafuchi, S. Tsukita, S.
Shibamoto, F. Ito, M. Takeichi, H. Matsuda and S. Hirohashi. 1994. A truncated β-catenin
disrupts the interaction between E-cadherin and α-catenin: a cause of loss of intercellular
adhesiveness in human cancer cell lines. Cancer Res 54: 6282-6287.
Ozawa, M., H. Baribault and R. Kemler. 1989. The cytoplasmic domain of the cell adhesion
molecule uvomorulin associates with three independent proteins structurally related in
different species. EMBO J 8: 1711-1717.
Patiño, E.M., M. A. Judis, S. C. Guanziroli, M. Sánchez Negrette, D. O. Pochon, J. F. Cedrés, M.
M. Doval, A. M. Romero, G. A. Crudeli and G. Rebak. 2010. Conjugated linoleic acid and
omega 6 and 3 in buffalo (bubalus bubalis) milk in Corrientes, Argentina. Rev. Vet. 21:244-
248.
Patiño, E.M., M. A. Judis J, M. Sánchez Negrette, D. O. Pochon, J. F. Cedrés, G. Rebak, A. M.
Romero, M. M. Doval and G. A. Crudeli. 2012. Influence of fish oil in the concentration of
conjugated linoleic acid and omega 6 and 3 in buffalo milk. Arq Bras Med Vet Zootec. Vol
64 (2):427-433
Reddy, B. S. and H. Marayama.1986. Effects of dietary fish oil on azoximethane induced colon
carcinogenesis in male F344 rats. Cancer Res 46:3367-3370.
Riddell, R.H., H. Goldman, D. F. Ransohoff, H. D. Appelman, C. M. Fenoglio, R. C. Haggitt, C.
Ahren, P. Correa, S. R. Hamilton, B. C. Morson, S. C. Sommers and J. H. Yardley, 1983.
Dysplasia in inflammatory bowel disease: standardized classification with provisional
clinical applications. Hum Pathol. 14: 931-966.
Takahashi, M., K. Fukuda, T. Sugimura and K. Wakabayashi. 1999. Beta-catenin is frequently
mutated and demonstrates altered cellular location in azoxymethane-induced rat colon
tumors. Cancer Res. 58:42-46.
Yamada, Y., N. Yoshimi, M. Hirose, K. Kawabata, K. Matsunaga, M. Shimizu, A. Hara and H.
Mori. 2000. Frequent β-catenin gene mutations and accumulations of the proteína in the
851
putative preneoplastic lesions lacking macroscopic abberrant crypt foci appearance in rat
colon carcinogenesis. Cancer Res 60:3323-3327.
Yamada, Y., N. Yoshimi, Y. Hirose, K. Matsunaga, M. Katayama, K. Sakata, M. Shimizu, T. Kuno
and H. Mori. 2001. Sequential analysis of morphological and biological properties of beta-
catenin accumulated crypts, provable premalignant lesions independent of aberrant crypt
foci in rat colon carcinogenesis. Cancer Res. 61: 1874-1878.
852
Anticancer Effects of Bubaline Functional Milk with Higher Concentration of

Conjugated Linoleic Acid and Omega-3 Fatty Acids
Gabriela Verónica RAMIREZ, Gabriela Inés VILLORDO, María de las ANGUSTIAS
MONTENEGRO, María Silvia CATUOGNO and Marcial sanchez NEGRETTE
Cátedra de Patología General y Sistemática. Facultad de Ciencias Veterinarias- UNNE- Sargento

Cabral 2139- Corrientes (3400), Argentina
*Corresponding email: patgral@vet.unne.edu.ar
ABSTRACT
The fat of ruminants products (milk, meat) in many cases is considered harmful to health
because contains saturated fatty acids, but in recent years it been found that some components such
as the conjugated linoleic acid (CLA ) and Omega-3 fatty acids, have many beneficial properties,
among which stands out its anticancer properties. Many factors may influence the increase of the
Omega-3 fatty acids and CLA in milk, but the food is a preponderant factor, therefore the
manipulation of the animal diet to increase the concentration of these acids in milk has gained
prominence in recent years.
Many investigations were conducted supplementing the diet of animals with various substances,
including fish oil, to obtain a functional food. The effect of functional buffalo milk obtained from
animals that were supplemented with fish oil, was evaluated in rats which induced colon cancer
through subcutaneous injections of the carcinogen 1,2-dimethylhydrazine (DMH). Twenty
multiparous buffaloes breeds Murrah and crossbred Murrah x Mediterranean, where randomly
divided into two lots, control and experiment: the lot control was fed exclusively with natural
pasture and the experimental lot received, also to natural pasture, supplementation with fish oil for
35 days. The biochemical analysis of milk indicated at the end of the experiment an increase of
86% in CLA and 48% in fatty acids omega-3 in the experimental lot compared to the control lot.
The rats (n = 72) were randomly divided into lots that received water or milk, with or
without treatment with the carcinogen, for 123 and 240 days. At the end of the experiment
parameters were performed in each animal the macro and microscopic analysis of the large bowel,
determining the presence, number and location of tumors and precancerous lesions called
dysplastic crypt foci (DCF).
The results showed in rats that received functional milk for 123 days fewer tumors (20%) and DCF
(70.83%) compared with rats that received water. Respect to lot that received functional milk
during 240 days showed a diminution of tumors (77.78%) and DCF (88.89%).The results obtained
allow inferring that the supply with fish oil to the feeding of buffalo, can increase levels of
beneficial fatty acids, such as omega-3 fatty acids and CLA, helping to enhance the anticancer
effect of milk, which was evaluated in this experimental colon cancer biological model.
Keywords: Functional milk, Colon cancer, Omega-3 fatty acids, Conjugated linoleic acid, Fish oil
INTRODUCTION
The etiology of colon cancer is complex and involves genetic and environmental factors,
eating habits especially. The relationship between colon cancer and dietary components (fats,
fibers, vegetables, vitamins and other elements), have been evaluated in epidemiological and
experimental studies. Epidemiological studies in different populations and experimental animals
support the hypothesis that the amount and type of dietary fat and fatty acid composition are one of
the factors in colon carcinogenesis (Roynette et al., 2004). It has been found that bovine milk
contains many potential components to inhibit the process of carcinogenesis. These components are
sphingomyelin, conjugated linoleic acid (CLA), butyric acid, vitamin A and D (Parodi, 1996).
Moreover, numerous investigations have determined that buffalo milk also has a high value,
because it has more total solids, fat, protein and lactose than bovine milk (Patiño, 2003).
853
The purpose of this report was to determine the anticancer effect of functional buffalo milk
obtained from animals that were supplemented with fish oil on the development of tumors and pre-
neoplastic lesions called dysplastic crypt foci (DCF) in the colon of rats treated with the carcinogen
1,2-dimethylhydrazine (DMH ).

Animals and Experimental Plan
Seventy-two, male Wistar rats were purchased at age 6 weeks (160g) from Centro de
Experimentaciones Biológicas y Bioterio de la Facultad de Ciencias Veterinarias de la Universidad
Nacional del Litoral (Santa Fe, Argentina) and housed at individual cages in a temperature–
controlled room at 21ºC. The animals were given a nutritionally adequate diet and randomly
divided into two main groups and sub–groups at the following manner: Control group (without
DMH treatment) formed by: Sub-group-1 and Sub-group-2 with 9 animals each, which received
water for 123 and 240 days respectively; Sub-group-3 and Sub-group-4 with 6 animals each which
received milk for 123 and 240 days respectively. Experimental group (animals inoculated with
DMH). These animals were housed in a separate room from the control group, formed by: Sub-
group-5 and Sub-group-6 with 15 animals each, which received water for 123 and 240 days
respectively; Sub-group-7 and Sub-group-8 with 6 animals each which received milk for 123 and
240 days respectively.
Supplementation with fish oil
Were used twenty multiparous buffaloes of Murrah bread and half-bread Murrah x
Mediterranean fed with natural pasture. The natural pasture was comprised mostly for species like
Andropogon lateralis, A. sellononaus, Cynodon dactylon, Elionorus sp., Paspalum notatum, P.
almun chase, Sorghastrum agrostoides, Desmodium canum and Shylosanthes macrosona. The
buffaloes received a daily supplement of 2 kg of milled corn by animal and 140 ml of fish oil. For
supplementation, each animal entered in an individual feeder, for the purpose of meeting with the
assumption of independence. The test itself was performed during 35 days. The milk samples were
obtained at days 1 and 35 during the milking routine, collected in disposable containers, frozen to -
20°C and packaged in boxes of polyurethane until arrival at the lab.
Milk administration
Milk administration to rats started one week before the first DMH injection and maintained
for 123 and 240 days, according to the experimental plan.
Treatment with 1.2 dimethilhidrazina (DMH)
Intestinal tumors were induced by 1,2-dimethylhydrazine (DMH) given subcutaneously as
5 weekly doses at 20mg/kg body weight. The carcinogen (DMH) solution was prepared just before
its administration. DMH solution use for injection comprised 400 mg of DMH dissolved in 100 ml
of sterile distilled water containing 37 mg of ethylene-diamine tetraacetic (EDTA) and the pH was
raised to 6.5 with sodium hydroxide. The animals for vehicle treatment were given the same
volume of EDTA, pH 6.5.
At the end of the experiment, all rats were sacrificed. The large intestine was removed, cut
open longitudinally and fixed with 10% buffered formalin for 24 hours. For macroscopic study
determined the number, size and location of tumors in anatomical segments of intestine (rectum,
distal colon, proximal colon and cecum). The tumors were grossly classified into: Polypoid type
growth (pedunculated lesions and sessile or broad-based lesions), and Non-polypoid type growth
without intramucosal protuberant growth (flat tumors with plaque-shaped or ulcerative-infiltrative
carcinomas). To study the size, tumors were grouped into five categories or ranges: from 1 to 5mm,
6 to 10mm, 11 to 15mm, 16 to 20mm and more than 20 mm.
For microscopic study, the samples were processed according to the Swiss-roll technique (Rubio et
al., 1986). Histological sections 5 µm thick were prepared and stained with hematoxylin and eosin
to detect, locate and characterize the DCF.
854
RESULTS
Rats consumed buffalo milk daily without showing rejection. All animals were adapted to
the conditions of the biotery and were weight increased over the experience. No changes were
observed physical or behavioral changes in any of the rats treated with the drug carcinogenic.
Gross and histopathologic examinations
No histopathologic change was found in the intestine of control groups rats (with water or
milk administration) that had not been injected with carcinogen.
The autopsies revealed a decreased number of tumors (20%) in Group 2 rats (Experimental) that
receiving buffalo milk (Sub-group 7), comparing with the Sub-group 5 that received only water (4
vs. 5). Rats of Sub-group 8, who received milk for a longer period, it also reduced the number of
tumors (77.78%) compared with the Sub-group 6 that received only water (2 vs. 9) (Table 1).
In the four sub-group of both experimental groups, the biggest frequency of tumors corresponded
to the sessile type, and in second place to the fat type. No ulcerative-infiltrative carcinomas were
found. The intestinal tumors were more commonly found in the distal colon, proximal colon and
cecum, without significant difference in both experimental groups. As for the size of the tumors,
the Sub-group 5 and Sub-group 7 stayed in the range of 0-5 mm, without significant differences,
while in the Sub-group 6 a tumor was observed in range of 11-15 mm and one tumor in range of 6-
10 mm in the Sub-group 8.
With regard to the DCF, a significant decrease was observed (70, 83%) in the Sub-group 7
compared with the Sub-group 5 (7 vs. 24) (Table 2). In the Sub-group 8 were also observed a
decrease of the number of DCF (77, 78%) with regard to the Sub-group 6 (2 vs. 9).
Results of histology examination demonstrate that, in both periods of experimentation (123 and 240
days), ) the rat that receiving buffalo milk supplemented with fish oil, decreased the tumor
incidence and the number of DCF of the colon in a model of experimental carcinogenesis of the
large intestine.
DISCUSSIONS
Several studies demonstrated that the cow milk and their components reduced tumor
development in several models of experimental carcinogenesis using rats and mice. Catuogno et al.
(2006) demonstrated that the supplementation with butyric acid, a normal component of the milk,
during 26 weeks a marked decreased the DCF in the large colon of rats injected with the carcinogen
DMH. In other experience, rats that received skim milk during 14 weeks, was observed lower
number of tumors induced experimentally (Sánchez Negrette et al., 2005).
In our experience, we found that who consumed buffalo milk showed significant reduction in
tumor growth and dysplastic crypt foci.
These results agree with other research in which dairy cattle diets were used (Montenegro et
al., 2002; Negrette Sánchez et al., 2005), so we can conclude that the buffalo milk is a high value
functional food due to its components with the potential to decreased the process of carcinogenesis.
REFERENCES
Catuogno, M.S., M.A. Montenegro and M. Sanchez Negrette. 2006. Disminución del desarrollo de
Focos de Criptas Displásicas en el colon de ratas suplementadas con ácido butírico.
Comunicaciones Científicas, Tecnológicas Universidad Nacional del Nordeste.
Montenegro, M.A., M.S. Catuogno, F.D. Lizarraga and M. Sánchez Negrette. 2002. Efectos de
dietas
lácteas sobre la carcinogenesis experimental del intestine grueso inducido con 1, 2-
dimetilhidrazina en ratas, Comunicaciones Científicas y Tecnológicas.
Parodi, P.W. 1996. Milk fat components: posible chemopreventive agents for cancer and other
diseases. Australian J. Dairy Tech. 5: 24-32.
Patiño, E.M., F.I. Mendez, E.L. Faisal, J.F. Cedres, L.G. Gomez and M.C. Guanziroli Stefani.
2003.
Buffalo Milk Composition of Murrah and Half-Breed Murrah x Mediterraneo in Corrientes,
855
Argentina. Buffalo Newsletter 18: 8-10.

Roynette, E.C., P.C. Calder, Y.M. Dupertis and C. Pichard. 2004. N-3 polyunsaturatedfatty acids
and
colon cancer prevention. Clin Nutr. 23:139-151.
Rubio, C.A., G. Nylander, M. Sveander, A. Duvander and M.L. Alun. 1986. Minimal invasive
carcinoma of the colon in rats. A.J. Pathol. 123: 161-165.
Sánchez Negrette, M., M.A. Montenegro, W.J. Lértora, M.S. Catuogno. 2005. Disminución del
número de tumors intestinales inducidos por 1,2-dimethilhidrazina en ratas alimentadas con
leche descremada. Rev. vet. 16(1): 28-31.
Table 1. Frequency and location of tumors in the segments of the large bowel in both experimental
groups.
Sub-group 5 Sub-group 6 Sub-group 7 Sub-group 8

Location n=15 n=6 n=15 n=6
(DMH + water (DMH + water (DMH + milk (DMH + milk
123 days) 240 days) 123 days) 240 days)
Rectum 0 0 0 0
Distal colon 4 8 3 2
Proximal colon 1 1 1 0
Cecum 0 0 0 0
Total 5 9 4 2
Table 2. Frequency and location of DCF in the segments of the large bowel in both experimental
groups.
Sub-group 5 Sub-group 6 Sub-group 7 Sub-group 4

Location n=15 n=6 n=15 n=6
(DMH + water (DMH + water (DMH + water (DMH + water
123 days) 240 days) 123 days) 240 days)
Rectum 0 0 0 0
Distal colon 16 5 8 1
Proximal colon 7 2 1 0
Cecum 1 0 0 0
Total 24 7 9 1
856
Virtues of the Milk from Water Buffalo

Muhammad YOUNASa, Kashif ISHAQb, Muhammad YAQOOBa and Tanveer AHMADb
a
Department of Livestock Management, University of Agriculture, Faisalabad-38040, Pakistan
b
Department of Livestock Production & Management, Pir Mehr Ali Shah Arid Agriculture University,
Rawalpindi, Pakistan
ABSTRACT
The water buffalo (Bos bubalus bubalis) of Pakistan has descended from the wild Asian water
buffalo (Bubalis arnee) which is a prime dairy animal in the country. Two famous breeds of Nili-Ravi
and Kundi play an important role in food security and acts as a safety bank for poor villagers. About 33
million buffaloes are available which contributes 65 % of the total milk production and half of the total
beef in the country. The water buffaloes found in Pakistan are among the best milch buffaloes of the
world but this specie has received very little attention as far as research and development is concerned.
The buffalo milk is protein rich, creamy in taste, with much lower levels of cholesterol and possessing
many virtues and qualities not found in other milks. These factors make a wide acceptability of the
buffalo milk all over the country. Buffalo milk is liked because of its taste and smooth texture. It’s
healthier with 58 % more cream, 40 % more protein and 43 % less cholesterol than cow’s milk. Buffalo
milk can produce real white cheese. Value addition on water buffalo milk is also expected to get more
attention in the days to come. Due to higher butterfat content water buffalo milk is converted into ghee
production in many Asian countries. Due to its rich taste, white cheese and better food health, water
buffalo milk has attained importance and has created a lot of fans and farms in western hemisphere. This
paper will dwell in detail the constituents, properties and intrinsic worth of the water buffalo milk,
factors in making this milk popular and widely acceptable and would make a particular comparison with
that of cow’s milk.
Keywords: water buffalo, milk production, virtue, constituents, food health and taste.
INTRODUCTION
The domestic buffalo is descended from the wild Asian water buffalo (Bubalis arnee), which is
now an endangered species (Rodrigues, et al. 2006). The domestic Asian water buffalo (Bubalus
bubalis) is a large bovine animal, frequently used as livestock in Asia, and also widely in South
America, southern Europe, North America and other parts of the world. There are 195 million water
buffaloes in the world and about 97 % of them (approx 190 million) are in Asia. Buffalo milk is
approximately 13% (93 MT) of world total milk production. The world top five buffalo keeping
countries and buff-milk producing countries are presented in Figure 1 and 2.
Water buffalo was domesticated 5,000 years back in Indus civilization as depicted in the buffalo
seal evacuated from Mohenjo-Daro inscription and have become an economically important ruminant
animal of this area whose economy is largely contributed by the livestock sector with 11.5% share to the
national GDP. Pakistan possesses 33 M heads of buffaloes out of which 39% are Nili-Ravi, 23 % are
Kundi and 38 % are non-descript and others (Figure 3). Pakistan ranked 3rd in the world milk producing
countries whereas total milk produced was 38.7 MT (Pakistan Economic Survey, 2011-12; ACO, 2006).
The buff-milk was estimated as 23 MT (FAOSTAT, 2011).
Out of top five buff-milk producing countries in world, Pakistan is the only countries where the
buffalo is dominant contributor to national milk production with prominent share of 65 %. The value of
buff-milk was estimated 8.6 billion $ during 2011 in Pakistan that makes this commodity as most
valuable produced in the country (FAOSTAT, 2011). This scenario shows the importance of this black

857
gold for Pakistan. This paper intended to enlighten the attributes of buff-milk in general with particular
reference to Pakistan.
Buff-milk in Pakistani Society
The total population of the country is estimated as 179.95 M in which 63% resides in rural areas.
Agricultural population is estimated approximately 41% (75 M) with total labour force of 66.89 M with
38% of it is engaged in agriculture (FAOSTAT, 2013). It was estimated that 30-35 M people are
engaged in livestock related activities and generates 30-40% of their total income from livestock and its
related enterprises (Riaz, 2008). Bilal et al. (2006) described that buffalo as a prestigious possession of
the family and furthermore the number of buffaloes kept by farmers determined the wealth and status in
the society. Sharif et al (2003) suggest that 80% of buff-milk is produced in rural areas, another 15% in
peri-urban areas and about 5% is produced in cities. Mostly the share of milk comes from Punjab
province where buff-milk is 65% of total milk production. Recently, Hagmann (2012) reported that
keeping buffalo is the most common but not always the most important source of income in peri-urban
areas of Faisalabad. He further reported that most of the farmers were small scale subsistence and were
landless (62%). Buffaloes were the preferred dairy animals, accounting for 87% (SD 18.4), 82% (SD
17.1) and 76% (SD 19.3) of the dairy animals of poor, well off and rich households (P<0.05). He
concluded that Nili-Ravi was most prevalent in the study area which is considered the best dairy breed
in Pakistan. The most common source of income was milk sale.
Buff-Milk and its Economics
The buffalo milk is more economical than cow milk. Suresh et al. (2009) comparatively studied
the economics of milk production from buffalo and cow in Karnal district of Haryana, India. They found
that the buffalo was more profitable as compared to that of cow. The gross income (Rs. 22249.52) and
net income (Rs. 3720.28) per buffalo per annum was significantly higher as compared to (Rs. 17498)
and (Rs. 2028) per cow per annum respectively (Table 1). While working on public farm, Ahmad
(2002) found cross bred cattle gives more return that indigenous cow and buffalo until maturity. In
another study, Ahmad (2004) concluded that the Nili-Ravi buffalo was more efficient for milk
production and for income generation than Sahiwal cow at Livestock Experiment Station,
Bahadurnagar. Socio-economics of dairy buffalo enterprises was analyzed by Günlü et al. (2010) in
Afyonkarahisar province in Turkey. They concluded that financial rantability and economic rantability
were -6.08% and -5.69% in the economic analysis of enterprises while cost/benefit ratio was 0.92. In
other studies, Singh et al. (2006) while working on economic analysis in tribal areas of Udaipur and
Jamal et al. (2003) studying on land holding, herd status and economics of buffalo milk in Peshawar
found otherwise trends with good economic output from dairy buffaloes. Shah et al. (2009) reported that
the average cost benefit ratio in the Jhang District (Central Punjab, Pakistan) was 1:1.82 that further
broken in three categories were 1:1.91, 1:1.68 and 1:1.87 for large, medium and small farmers
respectively. The cost benefits ratio of large farmers was higher than the medium and small farmers.
The development of the industrial sector is imperative for enhancing of the buff-milk utility in
Pakistan. The current situation is not much conducive for the improvement needed in near future and it
is becoming worsened due to lack of investors, abundance of subsistence farmers (resource poor and
landless buffalo owners) and un-willingness of the high ups. Burki et al. (2006) comprehensively
described the price trends of milk production and future forecasts using ARIMA model. They concluded
that the gap between nominal and real price of fresh and UHT is decreasing and this will result in the
shutting down more milk plants in Pakistan. The economics of the buff-milk production varies area to
area and principally depends on the art of management and could be improved by the investing sincere
efforts for the development of dairy industry that will ultimately be beneficial for buffaloes as well as
pro buffalo keepers.
858
Marketing of Buff-Milk
Buff-milk market is settled as seasonal in Pakistan. The farmers have adopted this to get the
benefit of fodder availability in pre-winter and furthermore to avoid the complication of heat stress to
which this creature is more vulnerable (Younas et al. 2012). The buff-milk is surplus in winter while the
cow milk is available adequately during the pre-summer period that is in April-May. On other hand, the
milk consumption is low during the winter and is at its peak during the summer due to more consumer
demand for the products such as buttermilk, yogurt, and ice cream (Tariq et al. 2008).
Over 95-97% of the milk produced is marketed through informal agents and only 3-5% of total
milk by formal channels (Zia, 2006). Unfortunately the buff-milk market is poor which is mostly
concentrated in South Asian countries. In Pakistan, the market is dominated by the middlemen and the
resource constraint farmers fail to get the benefit of the products due to less access to potential market
and lack of cold chains (Tariq et al. 2008). The buff-milk is produced in the pockets where the inputs
especially the fodder is available in bulk and the land under fodders has less competition with the cash
crops. As most of the buffalo keepers are subsistence farmers and they cannot sale milk directly to
processing industry without the middlemen who provides the services of vehicle for trading the milk to
the processors. There is fare potential in the buff-milk market and that is still challenge to crop this
which is evident as Sadia (2011) reported that milk produced in country was 28% cheaper than the
world market. Minimizing the role of middleman, provision of subsidies and better means to access the
potential market could result in better situation.
Usage of Buff-milk
Whole Fresh Milk
Unlike cow milk, Buff-milk is less processed. In Pakistan, 90% of the milk used as fresh without
any value addition (Zia, 2006). The most common processed milk is UHT in the country. The top buff-
milk producing counties are given in Figure 2.
Dairy Products from Buff-milk
High fat contents in buff-milk show its possibility for producing the milk cream products
(Fernandes et al. 2007). The common products made are butter, ghee, cream, ice-cream and cheese.
Butter
Butter is a fatty product where the watery phase is dispersed in the oily phase, forming a
water/oil emulsion (Bylund, 1995). According to FAOSTAT (2011) more than 80% of the world buff-
milk butter was produced in India. Buff-milk butter was 16% of the total butter produced in the world.
The work of Fernandes et al. (2007) shows that the buff-butter has a good acceptance on local market
except the color that could be improved by using a dye. In South Asian countries the buff butter which is
white in color has more acceptances and is used for sweets and dehydrated milk products. The official
data about the butter production is not available about Pakistan; however it is commonly produced in the
rural life for domestic use only especially in winter. However, most of butter is used to transform into
ghee.
Ghee
Ghee is the most commonly used in the Asian countries especially in Indo-Pak subcontinent.
Buff-milk ghee is produced in more quantity than that of cow milk ghee. About 99% of the Buff-milk
ghee is produced in India and Pakistan (FAOSTAT, 2011) may be due to longer shelf life than butter.
The ghee from water buffalo milk has a bigger grain size than ghee from cow milk. It is produced by
heat treatment of the butter in conventional methods.
Fermented milk products
It includes products like Dahi, Yoghurt and Chakka etc. Saudi Arabia is top yoghurt producing
country in the word (FAOSTAT, 2011). Ironically the authentic data about Dahi or yoghurt production
from buff-milk is not available. However, it can be commonly observed that the Dahi produced by
unconventional means from buff-milk is available for sale in the markets of India and Pakistan. The
859
work of Ahmad et al. (2008) concludes that buffalo milk had higher concentrations of protein, fat, ash
and lactose than cow milk. The casein micelles from buffalo milk were more mineralized and less
hydrated than their counterpart’s cow milk. However, due to difference in buffering capacity, the
acidification process requires some changes. Low fat buffalo milk yoghurt are a rich source of nutrients
and are nutritionally preferable to cows’ milk yogurts (Han, et al. 2012). Buttermilk or Lassi is also
commonly used in Pakistan during summer.
Cheese
Egypt is the leading buff-milk cheese producing country in the world followed by Italy and
China. About 90% of the buff-milk cheese is produced in the Africa. Soft cheese varieties are
Mozzarella in Italy, Karish, Mish and Domiati in Egypt, Madhfor in Iraq, Alghab in Syria, Vladeasa in
Romania; the semi-hard cheese Beyaz Peyneri is made in Turkey; hard cheeses include Braila in
Romania, Rahss in Egypt, White brine in Bulgaria and Akkawi in Syria. Mozzarella cheese is trade
mark buff-milk soft cheese that got the Protected Denomination of Origin (PDO) status under European
Union regulations. However, regarding the amount of cheese production, cow dominates the buff-milk.
Other varieties are Camembert, Gouda, Camembert cheese and Stracchino like cheese have been
produced (Addeo et al. 2007).
Other products
Heat-concentrated milk products in Pakistan include paneer, khoa, rabri, kheer and basundi and
many sweets like klakand, chum-chum etc. The whey is used for making Ricotta and Mascarpone in
Italy, and Alkarish in Syria and Egypt. Proteins of buffalo milk, particularly the whey proteins, are more
resistant to heat denaturation as compared to the cow milk proteins.
Physicochemical and Nutritional Comparison
Buff-milk is white and beautifully smooth. The buff-milk is more nutritious than cow milk. It
has less cholesterol value than cow so it can be more popular in health conscious markets. In addition to
the significant cholesterol and calcium benefits, Buffalo Milk is also a rich source of iron, phosphorus,
vitamin A and of course protein. Buffalo Milk also contains high levels of the natural antioxidant
tocopherol. It does not develop the allergy as in other case is CMA (cow's milk allergy). Buff-milk has
higher protein than cow and its protein is more resistant to heat denaturation. There is 40-50% higher fat
content in the milk. To produce 1 kg of cheese, only 5 kg of the buff-milk is required as compare to 8 kg
cow milk. Cow milk required 14 kg milk to produce 1 kg butter as compare to buff-milk that is only 10
kg (Khan & Iqbal, 2009). The buff-milk has higher immunoglobulin, lactoferrin. Some of the marvelous
facts about the buff milk as compared to that of cow’s milk are summarized in Table 2.
CONCLUSION
The buff-milk is more economical and nutrition rich than cow milk. There is a need to formulate
the precise strategy to improve the industrial sector in buffalo keeping countries and to cater the
increasing demand of milk in other parts of world. Many more attributes need attention of the
researchers to be exploited in various buffalo producing countries like Pakistan. Much more attention is
needed on this specie and the widely accepted valuable milk in the custodian countries of the buffalo.
REFERENCES
ACO. 2006. Livestock Census 2006. Government of Pakistan. Statistics Division, Agriculture Census
Organization, Gurumangat Road, Gulberg-III, Lahore, Pakistan.
Addeo, F., V. Alloisio, L. Chianese, V. Alloisio. 2007. Tradition and innovation in the water buffalo
dairy products. Ital. J. Anim. Sci. 6: 51-57.
860
Ahmad S., I. Gaucher, F. Rousseau, E. Beaucher, M. Piot, J.F. Grongnet, F. Gaucheron. 2008. Effects of
acidification on physico-chemical characteristics of buffalo milk: A comparison with cow’s
milk. Food Chem.106: 11–17.
Ahmad, M. 2002. Economics of rearing of Buffalo, Sahiwal and Crossbred heifers up to maturity. Int. J.
Agri. & Bio. 4:153-155.
Ahmad, M. 2004. Comparison of income from Nili-Ravi buffalo and Sahiwal cattle herds of Livestock
Experiment Station, Bahadurnagar. Int. J. Agri. & Bio.6: 213-214.
Ahmad, S. 2010. Understanding of the molecular changes in casein micelles of buffalo milk as a
function of physico-chemical conditions: a comparison with cow milk. PhD Thesis, Agro
campus Ouest-INRA, France.
Ahmad, S. F.M. Anjum, N. Huma, A. Sameen, T. Zahoor. 2013. Composition and physico-chemical
characteristics of buffalo milk with particular emphasis on lipids, proteins, minerals, enzymes
and vitamins. J. Anim. & Plant Sci. 23(1 Suppl.): 62-74.
Asif, M., and U, Sumaira. 2010. A comparative study on the physicochemical parameters of milk
samples collected from Buffalo, Cow, Goat and Sheep of Gujrat, Pakistan. Pak. J. Nutri. 9:
1192-1197.
Benincasa, C., J. Lewis, G. Sindona, A. Tagarelli. 2008. The use of multi element profiling to
differentiate between cow and buffalo milk. Food Chem.110: 257-262.
Bilal, M.Q., M. Suleman, A. Raziq. 2006. Buffalo: Black gold of Pakistan. Lives. Res. Rur. Dev. 18:(9).
Burki, A.A., M.A. Khan, F. Bari. 2004. The State of Pakistan’s Dairy Sector: An Assessment. The Pak.
Devel. Rev. 43: 149-174.
Bylund, G. 1995. Dairy Processing Handbook. Tetra Pak Processing Systems, Lund, Sweden. pp. 263-
278.
Eckles, C.H., B.C. Willes, M. Harold. 2001. Milk and milk products. New Dehli.
FAOSTAT. 2011. Food and Agriculture Organization of the United Nations. http://faostat. fao.org /site
/569/default.aspx#ancor.
FAOSTAT. 2013. Food and Agriculture Organization of the United Nations. http://faostat. fao.org /site
/569/default.aspx#ancor.
Fernandes, S.A.A., A. de L.S. Ramos, E.M. Ramos, D.V.S. Veras, F.R. Pinheiro, S.L. Neto, L.A.
Requião, T.S. Amui, J.C.S. Carneiro, S.V. Matarazzo. 2007. Sensory evaluation of buffalo
butter. Ital. J. Anim. Sci. 6: 1140-1142.
Günlü, A., H. Çiçek, M. Tandoğan. 2010. Socio-economic analysis of dairy buffalo enterprises in
Afyonkarahisar province in Turkey. J. Food, Agri. & Envir. 8: 689-691.
Hagmann, J. 2012. Opportunities and constraints of peri-urban buffalo and dairy cattle systems in
Faisalabad, Punjab, Pakistan. ICDD WORK. PAPERS. 2: 6-59.
Han, X., L.L. Frank, L. Zhang, M.R. Guo. 2012. Open access chemical composition of water buffalo
milk and its low-fat symbiotic yogurt development. Funct. Foods in Health & Dise. 4:86-106.
Jamal, S., M. Syed, M. Farooq, S. K. Hamid. 2003. Land holding, herd status and economics of buffalo
milk production at commercial dairy farmers in Peshawar Division. J. Anim. Vet. Adv. 1:4-11.
Khan, B.B. and A. Iqbal. 2009. The Water Buffalo: An underutilized source of milk & meat: A review.
Pak. J. Zool. Suppl. 9:517-521.
Menard, O., S. Ahmad, F. Rousseau, V. Briard-Bion, F. Gaucheron, C. Lopez. 2010. Buffalo vs. cow
milk fat globules: Size distribution, zeta-potential, compositions in total fatty acids and in polar
lipids from the milk fat globule membrane. Food Chem. 120:544-551.
Pakistan Economic Survey. 2011-12. Government of Pakistan, Economic Advisor’s Wing, Finance
Division, Islamabad, Pakistan.
Riaz, K. 2008. A Case Study of Milk Processing: The Idara-e-Kissan Cooperative. The Lhr. J. Eco.13:
87-128.
861
Rodrigues, A.S.L., J.D. Pilgrim, J.F. Lamoreux, M. Hoffmann, T.M. Brooks. 2006. The value of the
IUCN Red List for conservation. Trends Ecol. & Evol. 21:71-76.
Sadia, H. 2011. Pakistan- milk production fact sheet. Dairy Report, 2011, IFCN. pp:136.
Shah, A., A. Saboor, S. Ahmad. 2009. An estimation of cost of milk production in Pakistan: A
microeconomic approach. Sarhad J. Agric. 25:141-146.
Sharif, M., W. Malik, N.I. Hashmi, U. Farooq. 2003. “Action Plan for Livestock Marketing Systems in
Pakistan,” Social Sciences Division. Pak. Agri. Res. Coun., Islamabad.
Singh, R.N., A.K. Chauhan, S. P. Sharma. 2006. Economic analysis of milk production in tribal area of
Udaipur. Indian J. Dairy Sci. 59:328-336.
Suresh, R., R.S. Tripathi, A. Solanki. 2009. Comparative economics of buffalo and cow milk production
in Karnal district of Haryana. Indian J. Anim. Res. 43: 224-225.
Tariq, M., M. 1. Mustafa, A. Iqbal, H. Nawaz. 2008. Milk marketing and value chain constraints. Pak. J.
Agri. Sci. 45:195-200.
Younas, M., K. Ishaq, I. Ali. 2012. Effect of Climate Change on Livestock Production in Pakistan.
Proc. 2nd Internat. Sem. Anim. Indus., Jakarta, 5-6 July. pp 554-561.
Zia, U. 2006. Analysis of Milk Marketing Chain, Pakistan. FAO Food Laws Manual.
862
Table 1: Comparative Costs and Returns of Buffalo and Cow Milk Production
(Rs. / annum / animal)
Particulars Buffalo Cow % of Difference
Green Fodder 5869.52 5347.64 8.89
Dry Fodder 2372.67 1958.73 17.45
Concentrates 2402.95 2022.05 15.85
Human labour charges 3710.83 3048.58 17.85
Miscellaneous expenses 463.5 279.55 39.69
Interest on working capital 592.78 506.26 14.6
Variable cost 14819.47 12656.55 14.6
Fixed cost 3116.99 2306.75 25.99
Total cost (Variable + Fixed) 18529.24 15469.56 16.51
Gross return 22249.52 17498.19 21.35
Net return 3720.28 2028.63 45.7
Family labour income 7431.11 5077.21 31.68
Benefit-cost ration 1.2 1.13 5.83
Green Fodder 5869.52 5347.64 8.89
Dry Fodder 2372.67 1958.73 17.45
Suresh et al. 2009
Table 2. Comparison of Buffalo and cow milk.

Parameters Buffalo Cow Reference
Acidity (%) 0.21±0.03 0.17±0.02 Asif & Sumara (2010)
0.13 0.15 Bilal et al. (2006)
Acidity (D°) 16.2 ---- Ahmad et al. (2010)
Ash (g/kg) 8.4 ± 0.2 7.7 ± 0.1 Ahmad et al. (2008)
Average size of fat globules, micron 5.01 3.85 Bilal et al. (2006)
5.00 --- Ahmad et al. (2008)
Butterfat (% age) 7.97±0.44 4.00±0.43 Asif & Sumara (2010)
Fat (g/kg) 70 ± 6 41 ± 1 Ahmad et al. (2008)
80 39 Bilal et al. (2006)
71 -- Han et al. (2012)
Calcium (IU) 195 120 Bilal et al. (2006)
Total Calcium (m/M) 47.1 ± 1.2 30.5 ± 0.8 Ahmad et al. (2008)
Cholesterol (mg/100gm) 8 14 Bilal et al. (2006)
6 31 Khan & Iqbal (2009)
Color White Creamy Bilal et al. (2006)
Color under UV light Greenish yellow Pale bluish Bilal et al. (2006)
Milk Protein 4.01±0.23 2.98±0.32 Asif & Sumara (2010)
4.50 3.20 Bilal et al. (2006)
4.6 -- Menard et al. (2010)
5.0 -- Han et al. (2012)
4.4 3.5 Ahmad et al. (2008)
Density 1.033±0.002 1.029±0.001 Asif & Sumara (2010)
1.02 1.02 Bilal et al. (2006)
Dry matter (g/kg) 174.5 ± 8.2 136.7 ± 10.8 Ahmad et al. (2008)
Energy (Kcal) 110 66 Eckles et al. (2001)
Energy (K J) 412 …. Ahmad et al. (2013)
463 275 Bilal et al. (2006)
Fatty Acids (saturated) % wt. 70.8 -- Menard et al. (2010)
g/100 gm 4.2 2.4 Eckles et al. (2001)
Cont…
Fatty Acids (unsaturated) % wt. 29.2 -- Menard et al. (2010)
g/100 gm 1.9 1.2 Eckles et al. (2001)
Freezing point depression 0.560 0.570 Bilal et al. (2006)
Freezing point (°C) -0.526 -- Ahmad et al. (2010)
863
Table 2. Comparison of Buffalo and cow milk (Continue)

Iron (micro gram/100 mL) 42-152 -- Ahmad et al. (2013)
Iron (µg/kg) 301 ±18 325 ± 13 Benincasa et al. (2008)
Lactose (%) 5.41±0.54 4.51±0.38 Asif & Sumara (2010)
Lactose (g/Kg) 52.1 ± 1.1 48.0 ± 0.1 Ahmad et al. (2008)
No. fat globules, million/ mm3 3.2 2.96 Eckles et al. (2001)
3.2 --- Ahmad et al. (2013)
Nutritive value No difference No difference Bilal et al. (2006)
pH 6.81 ± 0.06 6.76 ± 0.04 Ahmad et al. (2008)
6.7 6.6 Bilal et al. (2006)
Phosphorus (mM) 27.7 ± 1.4 19.2 ± 1.0 Ahmad et al. (2008)
Protein Efficiency Ratio (PER) 2.75 2.49 Khan & Iqbal (2009)
Refractive index 1.345 1.33 Bilal et al. (2006)
1.346 - 1.353 1.345-1.348 Ahmad et al. (2013)
Specific refractive index 0.206 0.205 Eckles et al. (2001)
Surface tension (dynes/cm) 55.4 55.9 Eckles et al. (2001)
55.4 -- Ahmad et al. (2008)
Total Cl (mM) 16.6 ± 0.8 21.8 ± 1.0 Ahmad et al. (2008)
Total K (mM) 28.7 ± 0.7 42.0 ± 1.0 Ahmad et al. (2008)
Total Mg (mM) 7.3 ± 0.2 4.6 ± 0.1 Ahmad et al. (2008)
Total Na (mM) 20.3 ± 0.5 17.5 ± 0.4 Ahmad et al. (2008)
Total Pi (mM) 27.7 ± 1.4 19.2 ± 1.0 Ahmad et al. (2008)
Total Solids 18.45± 0.85 12.94±0.97 Asif & Sumara (2010)
Viscosity 2.04 1.86 Bilal et al. (2006)
Figure 1. Top Buffalo keeping countries.
FAOSTAT (2011)
864
Figure 2. Top Buffalo milk producing countries.
FAOSTAT (2011)
Figure 3. Break up of Buffalo breeds in Pakistan.
ACO (2006)
865
Improving Oat Grass Silage Quality through using Exogenous Enzyme in

Cannulated Buffalo Bulls
Mahr-un-NISA a*, Abdur REHMAN a, Muhammad Aasif SHAZADa, Muhammad

SARWARa, Osman Ahmad KHANa, Abubakar SUFYAN b and Tahir MAHMOOD a
a
Institute of Animal Nutrition and Feed Technology, University of Agriculture, Faisalabad,
Pakistan.
b
Faculty of veterinary Science Bahauddin Zakariya University, Multan, Pakistan
*Corresponding Email: linknisa@gmail.com
ABSTRACT
This experiment was conducted to evaluate the chemical composition and digestion
kinetics of oat grass (Avena sativa L.) silage with or without exogenous fibrolytic enzymes.
The oat grass was harvested after 50 days of sowing and was ensiled with 0, 1, 2, and 3 g
enzyme /kg grass dry matter (DM) for 21 d in completely randomized design. Diets were
designated as EO, E1, E2 and E3. A linear increase (P<0.05) in oat grass silage crude protein
(CP) and true protein (TP) was observed but a reduction (P<0.05) in neutral detergent fiber
(NDF) and acid detergent fiber (ADF) was noticed with increasing enzyme level. The DM
and OM losses remained unaffected (P>0.05) across all enzyme levels. A linear decrease
(P<0.05) in CP and TP losses was noticed in silage treated with increasing enzyme level.
Increasing enzyme level caused a linear decrease (P<0.05) in pH during 1st, 2nd and 3rd week
of ensilation. In situ, the enzyme treatment did not affect (P>0.05) the extent of digestion and
lag time of DM, CP, NDF and ADF and digestibility of CP, NDF and ADF and rate of DM,
NDF and ADF digestion. Results indicate that enzyme application at the time of ensilation
can reduce the nutrient losses and fiber fractions of oat grass silage without affecting its
digestibility.
Keywords: Oat grass, Silage, Exogenous enzyme, Buffalo Bull, Digestibility
INTRODUCTION
The conservation of fodder as silage will not only ensure its quality and a year around
constant supply but it will also make livestock productivity cost effective. Early achieved
anaerobic conditions and rapid decline in pH can minimize the nutrient losses by reducing
respiration and prolonged fermentation (Charmley, 2001). The usage of fibrolytic enzymes
while ensiling fodder may help achieve rapid pH reduction. They hydrolyze fiber contents
into reducing sugars (Sheperd and Kung 1996a) and thus increase the fermentation rate by
providing epiphytic lactic acid producing bacteria with readily fermentable carbohydrates
(Stokes,1992). The scientific evidence regarding the influence of exogenous enzymes on the
silage quality is limited. Therefore, the present study was planned to examine the nutrient
composition and digestion kinetics of oat grass ensiled with or without fibrolytic enzymes.

Fifty days old grass was wilted for three days to attain 60% moisture. Oat grass
was chopped and mixed with mixer. Commercial cellulase+hemicellulase mixture of enzyme
(Allzyme®, an Aspergillus nigar product by Alltech) was used as an inoculant to ensile oat grass.
The enzyme mixture at the rate of 0 (E0), 1 (E1), 2 (E2) and 3 (E3) g of enzyme /kg of DM and
molasses (2%) were dissolved in water and the solution was sprayed on chopped oat grass at the
time of ensilation. Treated oat grass was then ensiled in 36 (9 for each enzyme level) laboratory
silos (transparent thick, 40×20 cm polyethylene bags of 2 kg capacity). These silos were

866
pressed for air exclusion and sealed to achieve the anaerobic conditions and were ensiled for
21d at room temperature (Sarwar et al., 2006). Triplicate silos for each treatment were
opened at 7th and 14th day of ensilation to determine the silage pH. After 21d the remaining
silos were opened and samples from each silo was collected and analyzed to determine
silage OM, DM, TP and CP (AOAC. 1990) NDF and ADF (Van Soest et al., 1991)
Four ruminally cannulated Nili Ravi buffalo bulls (400+20 Kg) were used in 4×4 Latin
square design to determine the digestion kinetics of E0, E1, E2 and E3 diets. Four test diets placed
in the rumen of 4 bulls alternatively during four periods of 5 d each. Nylon bags measuring 10×23
cm with an average pore size of 50 µm were used. 10 g sample, in triplicate bags were
incubated in the rumen for 0, 1, 2, 4, 6, 10, 16, 24, 36, 48 and 96 h, in reverse order and
were removed all at the same time (Sarwar et al., 2004). After removal from the rumen,
bags were washed in running tap water until the rinse was clear. The bags were then dried
in a forced air oven at 55°C for 48 h for the determination of lag time, rate and extent of
disappearance of DM, CP, NDF and ADF of enzymatic treated silage. The lag time was
calculated according to method described by Mertens and Loften (1980) Silage–pH was
determined by using pH-mV meter (HM-21P, TOA Corporation, Tokyo, Japan).
The data collected for each parameter was subjected to analysis of variance technique
using multivariate analysis in General Linear Model option of SPSS 17.0 (SPSS Inc.,
Chicago, IL, USA). In case of significance (P<0.05) Duncan’s New Multiple Range Test was
applied to separate the means.

A linear increase (P<0.05) in oat grass silage crude protein (CP) and true protein (TP)
was observed but a reduction (P<0.05) in neutral detergent fiber (NDF) and acid detergent
fiber (ADF) was noticed with increasing enzyme level (table-1). This increase in CP and TP
may be due to decrease in pH which might have reduced the proteolytic activity in silage.
Reduction in NDF and ADF contents of silage can be ascribed to the conversion of some
fiber fraction to the reducing sugars by the enzymes (Kung et al., 2003).
The highest pH decrease was observed in E3 which was 56% higher than that
observed in E0 (table2). This decrease in pH in concordance with findings of other
researchers (Sun et al., 2009; Dean et al., 2005; Colombatto et al., 2004; Adogla-Bessa et al.;
1999), who reported less pH in enzyme treated silages than untreated silages.
In-situ DM digestibility was different (P<0.05) across all treatments but CP, NDF and
ADF digestibility remained unchanged (Table.2). Similar findings were reported by other
researchers (Kozelov et al., 2008; Mandebvu et al., 1999). Enzyme application must have
reduced lag time but it did not and may be simply that released sugars from the fiber might
have been utilized by lactic acid producing bacteria in silo (Kung et al., 2003). This explains
why enzymes had no effect on digestion kinetics in this study.
REFERENCES
Adogla-bessa, t., e. Owen and a.t. Adesogan. 1999. Ensiling whole crop wheat with
cellulase+hemicellulase based enzymes: 3. Comparing effects of urea or enzyme
treatment on forage composition and stability. Anim. Feed sci. Tech. 82: 51-61.
AOAC. 1990. Association of Official Analytical Chemists International. Official Methods of
Analysis, 15th Ed. Arlington, VA, USA.
Charmley, E. 2001. Towards improved silage quality: A review. Can. J. Anim. Sci. 81:157-
168.
Colombatto, D., F.L. Mould, M.K. Bhat and E. Owen. 2004. Influence of exogenous
fibrolytic enzyme level and incubation pH on the in vitro ruminal fermentation of
alfalfa stems. Anim. Feed Sci. Tech. 137: 150-162.
867
Dean, D.B., A.T. Adesogan, N. Krueger and R.C. Littell. 2005. Effect of fibrolytic enzymes
on the fermentation characteristics, aerobic stability, and digestibility of bermudagrass
silage. J. Dairy Sci. 88: 994-1003.
Kozelov, L.K., F. Iliev, A.N. Hristov, S. Zaman and T. A. McAllister. 2008. Effect of
fibrolytic enzymes and an inoculant on in vitro degradability and gas production of
low dry matter alfalfa silage. J. Sci. Food Agric. 88:2568–2575.
Kung Jr L., M.R. Stokes and C.J. Lin. 2003. Silage additives, in Silage Science and
Technology, ed. by Buxton DR, Muck RE and Harrison JH. American Society of
Agronomy, Madison, WI. Pp. 305-360.
Mandebvu, P., J.W. West, M.A. Froetschel, R.D. Hatfieldc, R.N. Gates and G.M. Hill. 1999.
Effect of enzyme or microbial treatment of Bermuda grass forages before ensiling on
cell wall composition, end products of silage fermentation and in situ digestion
kinetics. Anim. Feed Sci. Tech. 77: 317-329.
Mertens, D.R. and J.R. Loften. 1980. The effect of starch on forage fiber digestion
kinetics in vitro. J. Dairy Sci. 63:1437-1446.
Sarwar, M., M. Nisa, Z. Hassan and M.A. Shahzad. 2006. Influence of urea molasses treated
wheat straw fermented with cattle manure on chemical composition and feeding value
for growing buffalo calves. Livestock Sci. 105: 151-161.
Sarwar, M., M.A. Khan and M. Nisa. 2004. Effect of organic acids or fermentable
carbohydrates on digestibility and nitrogen utilization of urea treated wheat straw in
buffalo bulls. Aust. J. Agric. Res. 55:235-240.
Sheperd, A.C. and L. Kung. 1996a. Effects of an enzyme additive on composition of corn
silage ensiled at various stages of maturity. J. Dairy Sci. 79:1767-1773.
Stokes, M. R. 1992. Effects of an enzyme mixture, an inoculant, and their interaction on
silage fermentation and dairy production. J. Dairy Sci. 75:764-773.
Sun, Z.H., S.M. Liu, G.O. Tayo, S.X. Tang, Z.L. Tan, B. Lin, Z.X. He, X.F. Hang, Z.S. Zhou
and M. Wang. 2009. Effects of cellulase or lactic acid bacteria on silage fermentation
and in vitro gas production of several morphological fractions of maize stover. Anim.
Feed Sci. Tech. 152: 219–231.
Van Soest, P.J., J.B. Robertson and B.A. Lewis. 1991. Methods for dietary fiber, neutral
detergent fiber, and non-starch polysaccharides in relation to animal nutrition. J.
Dairy Sci. 74: 3583-3597.
Table 1. Nutrient composition of oat grass silage after 21 d of ensilation.
Treatments1 Probabilities2
SE
Items (g/Kg) E0 E1 E2 E3 L Q
Dry matter 288 286 288 289 1.1 NS NS
Organic matter 902 896 892 900 2.7 NS NS
Crude protein 114.5b 127.2 a
128.7a 129a 1.6 * NS
True protein 98.4b 105a 107.2a 108.6a 0.7 * NS
Neutral detergent fiber 368a 343 b
330.7bc 324c 2 * NS
Acid detergent fiber 208.3a 185.3b 186.3b 181.7b 2.5 * NS
1
E0, E1, E2 and E3 represent oat grass ensiled with enzyme at the rate of 0, 1, 2 and 3g/Kg of
dry matter, respectively.
2
L= Linear and Q= quadratic responses towards increasing enzyme level. NS= Non-
significant (P>0.05) and *= significant (P<0.05).SE= Standard error. a, b, c Means sharing
different superscripts differ significantly (P<0.05).
868
Table 2. Effect of increasing level of enzyme application on change in oat grass silage pH.
pH change SE
E0 E1 E2 E3 L Q
1st week -1.4b -2.08 a
-2.1a -2.16a 0.02 * NS
2nd week -0.15 -0.03 -0.05 -0.05 0.028 NS NS
3rd week -0.47 -0.27 -0.24 -0.22 0.039 * NS
Overall3 -1.66b -2.19 a
-2.21a -2.27a 0.053 * NS
a, b, c
Means sharing different superscripts differ significantly (P<0.05).
1
2
L= Linear and Q= quadratic responses towards increasing enzyme level.
3
Averaged pH at 1st, 2nd and 3rd week of ensilation. NS= Non-significant (P>0.05) and *=
significant (P<0.05). SE= Standard error.
869
Table 3. Effect of increasing level of enzyme application on DM, CP, NDF and ADF
digestion kinetics of oat grass silage.
DM SE
E0 E1 E2 E3 L Q
3
Digestibility (%) 79.5a 78.25 ab
76.4c 77.45bc 0.21 * NS
Digestible fraction4 228.9a 223.7b 220b 223.8b 0.60 * *
Extent of digestion3 (%) 80.5 80.5 78.8 80.2 0.40 NS NS
Potentially digestible fraction4 231.8 230.2 226.8 231.9 1.27 NS NS
Lag time (h) 1.45 1.44 1.42 1.45 0.01 NS NS
Digestion rate (%/ h) 5.22 5.27 5.26 5.23 0.48 NS NS
Crude Protein
Digestibility3 (%) 89.1 88.6 89.3 89.8 0.28 NS NS
Digestible fraction4 102c 112.7b 115.6a 115.3ab 0.36 * *
Potentially digestible fraction4 106.2b 118.3a 119.6a 120.1a 0.27 * *
Lag time (h) 0.89 0.91 0.90 0.90 0.01 NS NS
Digestion rate (%/ h) 5.08b 5.08b 5.21a 5.15ab 0.01 * NS
NDF
Digestibility3 (%) 53.6 53.2 53.1 53.8 0.25 NS NS
Digestible fraction4 197.4a 182.5b 175.6c 174.2c 0.86 * *
Potentially digestible fraction4 225.6a 207.1b 199.1bc 195.4c 1.0 * *
Lag time (h) 1.94 1.93 1.92 1.94 0.01 NS NS
ADF
Digestibility3 (%) 43.6 45 45.4 46.3 0.4 NS NS
Digestible fraction4 90.7a 83.5b 84.7b 83b 0.7 * NS
Potentially digestible fraction4 110.6a 97.6b 97.3b 96.5b 0.5 * *
Lag time (h) 2.13 2.10 2.05 2.07 0.03 NS NS
1
2
L= Linear and Q= quadratic responses towards increasing enzyme level. 3 Digestibility and
extent of digestion were determined after 48 and 96 hours of ruminal incubation, respectively.
4
Fraction (g/Kg dry matter) remaining at 0 h of incubation. NS= Non-significant (P>0.05)
and *= significant (P<0.05). SE= Standard error.
a, b,c
Means sharing different superscripts differ significantly (P<0.05).
870
Effects of Roughage Types on Feed Intake, and Nutrient Digestibilities in

Swamp Buffaloes (Bubalus bubalis)
Walailuck KAEWWONGSAa*, Chalong WACHIRAPAKORNb, Metha WANAPATb and
Ngarmnit NONTASOc
a
Program in Animal Production Technology, Faculty of Technology, Udon Thani Rajabhat
University, Udon Thani, 41000, Thailand
b
Department of Animal Science, Khon Kaen University, 40002, Thailand
c
Department of Microbiology, Khon Kaen University, 40002, Thailand
*Corresponding Author E-mail: kaewwongsa@hotmail.co.th
ABSTRACT
The experiment was carried out to study the effects of roughage types on feed intake and
nutrient digestibilities in swamp buffaloes (Bubalus bubalis). Three rumen-fistulated buffaloes,
about 5 years of age with average liveweight of 500+56 kg, were allotted to receive one of the three
roughage sources according to a 3x3 Latin square design with extra period. The dietary treatments
were as follows: rice straw (RS), urea-treated rice straw (UTS) and cassava hay (CH). Buffaloes
were offered roughage ad libitum and accessed to clean water freely. Four experimental periods
were employed and each period lasted for 21 days. In each period, the animals were adjusted for 14
days to feed and voluntary intake was measured then followed by total collection method during the
last 7 days. The results showed that voluntary roughage intake of buffalo fed UTS (2.05 %BW) was
slightly higher (P>0.05) than those in buffalo fed CH and RS (1.94 and 1.68 %BW). Apparent
digestibilities of dry matter, organic matter and neutral-detergent fiber in buffalo fed UTS (64.6,
69.8, and 70.1 %) were significantly higher (P<0.05) than those buffaloes fed CH (60.9, 62.6, and
59.7 %) and fed RS (51.1, 65.4, and 55.6 %). Acid-detergent fiber digestibility was not different
(P>0.05) among treatments. Crude protein digestibility of buffalo fed CH (74.0 %) was
significantly higher (P<0.05) than those buffalo fed UTS (44.1 %) or RS (28.1 %). However,
further study to investigate the optimal feeding levels of feed in terms of efficiency of rumen
fermentation and rumen microorganisms and productive especially in swamp buffaloes should be
conducted.
Keywords: swamp buffaloes, roughage, feed intake, digestibilities
INTRODUCTION
In the tropics, likewise in Thailand, buffaloes and cattle are raised as an integral part of the
crop production system, especially where rice is the main commodity (Chantalakana, 2001). Swamp
buffaloes are fed on low-quality roughages, agricultural crop-residues, and industrial by-products,
which basically contain high levels of cellulose, hemi-cellulose and lignin, as well as low levels of
fermentable carbohydrates and poor-quality protein. However, crop residues are an available feed
resource in local areas from crop cultivation and are a very important source of roughages for
ruminants. These diets result in low performance, productivity and poor health due to their low
quality, because rice straw is low in available energy, protein and vitamin, has an imbalance of
essential minerals, and contains a large pool of structural carbohydrates (Wanapat, 1999). Efficient
utilization of roughages depends on the availability of nutrients needed by both rumen microbes and
by the animal with the ultimate aim of maximizing feed intake and performance (Preston and Leng
1987). Recent research on locally available feed resources such as crop residues, and industrial by
products, dietary addition of micronutrients, feed technology, use of performance modifiers and use
of ruminally protected fat and protein sources, have shown a significant potential to improve
growth, milk yield and reproductive performance of buffaloes (Sarwar et al., 2009). The following
study aimed at comparing the effect of different sources of roughage on feed intake and digestibility
in swamp buffaloes.
871

Experimental design and treatments
Three rumen-fistulated swamp buffaloes, 5-7 years old, were randomly assigned to three
dietary treatments according to a 33 Latin Square design with extra-period (Lucas, 1957) with
periods of 21 days on each of the following diets:
T1 RS = untreated rice straw;
T2 UTS= urea-treated (5%) rice straw;
T3 CH = cassava hay.
All the buffaloes were drenched with an anthelmintic and injected with vitamins A, D and E
prior to commencing the experiment. They were kept in individual stalls.
Sampling methods
Urea-treated rice straw was prepared by pouring a urea solution (5 kg of urea in 100 kg of
water) over a stack of straw and covering with a plastic sheet for 10 days before feeding to the
animals. Cassava foliage was post harvested, and was chopped and then sun-dried for 1-2 days to
obtain cassava hay. All the roughages were offered ad libitum with fresh feed offered in the
morning and afternoon. Feed intakes were measured daily during each three-week experimental
trial. Rectal samples of feces were taken at the end of each period and analyzed for acid-insoluble-
ash (AIA) as an internal indicator to estimate digestibility of DM and OM (Van Keulen and Young,
1977). Feeds were analyzed for DM, ash, CP, NDF and ADF by standard methods (Goering and
Van Soest, 1970; AOAC, 1985).
Statistical methods
All data were subjected to analysis of variance using Proc ANOVA (SAS, 1996) and
treatment means were statistically compared by Duncan’s New Multiple Range Test (Steel &
Torrie, 1980).

The results showed that voluntary roughage intake of buffalo fed UTS (2.05 %BW) was
slightly higher (P>0.05) than those in buffalo fed CH and RS (1.94 and 1.68 %BW). Apparent
digestibilities of dry matter, organic matter and neutral-detergent fiber in buffalo fed UTS (64.6,
69.8, and 70.1 %) were significantly higher (P<0.05) than those buffaloes fed CH (60.9, 62.6, and
59.7 %) and fed RS (51.1, 65.4, and 55.6 %). Acid-detergent fiber digestibility was not different
(P>0.05) among treatments. Crude protein digestibility of buffalo fed CH (74.0 %) was
significantly higher (P<0.05) than those buffalo fed UTS (44.1 %) or RS (28.1 %). This result is
similar to the data from Basra et al. (2003) who showed that daily DM intake for Nili-ravi buffalo
calves was affected by increasing CP content in the diet. The suggested mechanism underline this
effect is that high protein intake increases microbial fermentation in the rumen, which improves
digestion of nutrient and also increases feed intake (Granum et al., 2007).
CONCLUSIONS
This study confirmed that urea-treated rice straw and cassava hay were good roughage
quality with high nutritive value, increased voluntary roughage intake and increased digestibility of
nutrient for use as a roughage source for swamp buffaloes production.
ACKNOWLEDGEMENTS
Appreciation is extended to Udon Thani Rajabhat University, Thailand for facilities and
financially supporting this research. We also thank the Research Station farm crew for animal care,
harvest and storage of the roughage used in these experiments and Department of Animal Science,
Khon Kaen University, Thailand are acknowledges for technical assistance.
REFERENCES
AOAC. 1985. Official Methods of Analysis. Association of Official Analytical Chemists.
Washington, D.C.
872
Basra M.J., M. Nisa, M.A. Khan, M. Riaz, N.A. Tuqeer and M.N. Saeed. 2003. Nili-ravi buffalo I
Energy and protein requirements of 6-9 months old calves. Int. J. Agri. Biol. 5: 377-379.
Chantalakhana C. 2001. Water buffalo: valuable asset of the poor but disappearing. In: Proc. of a
workshop on water buffaloes for food security and sustainable rural development. Thailand.
Goering H.K. and P.J. Van Soest. 1970. Forage fiber analysis (apparatus, reagents, procedures and
some applications). Agric. Handbook. N. 397. ARS, USDA, Washington, D.C.
Granum G., M. Wanapat, P. Pakdee, C. Wachirapakorn and W. Toburan. 2007. A comparative study
on the effect of cassava hay supplementation in swamp buffaloes (Bubalus bubalis) and cattle
(Bos indicus). Asian-Aust. J. Anim. Sci. 20: 1389-1396.
Lucas H.L. 1957. Extra-period latin-square change-over designs. J. Dairy Sci. 40: 225.
Preston T.R. and R.A. Leng. 1987. Matching ruminant production systems with available resources
in the tropics and sub-tropics, 245 p. Penambul Books Armidale, NSW, Australia.
Sarwar M., M.A. Khan, M. Nisa, S.A. Bhatti and M.A. Shahzad. 2009. Nutritional management for
buffalo production. Asian-Aust. J. Anim. Sci. 22: 1060-1068.
SAS., 1996. User’s Guide Statistics version 6. Edition SAS. Inst itiate Inc.Cary, NC, USA, 429 p.
Van Keulen J. and B.A. Young. 1977. Evaluation of acid insoluble ash as a neutral marker in
ruminant digestibility studies. J. Anim. Sci. 44: 282.
Wanapat M. 1999. Feeding of Ruminants in the Tropics Based on Local Feed Resources. Khon
Kaen Publishing Comp. Ltd., Khon Kaen, Thailand, 236 pp.
Table 1. Chemical composition of dietary treatments.*
Composition
Roughage sources DM Ash OM CP NDF ADF
-----------------------%DM---------------------------
Rice straw (RS) 87.0 14.1 85.9 4.05 81.7 52.0
Urea-treated rice straw (UTS) 52.0 18.1 81.9 8.24 78.9 59.5
Cassava hay (CH) 87.0 12.8 87.2 20.0 58.4 44.5
*DM = dry matter; OM = organic matter; CP = crude protein; NDF = neutral-detergent fiber;
ADF = acid-detergent fiber
Table 2. Effect of roughage sources on voluntary feed intake and nutrient digestibilities in swamp
Buffaloes.*
Roughage sources
Items SEM
RS UTS CH
Roughage intake
kg/day 8.08 9.83 9.43 0.68
%BW 1.68 2.05 1.94 0.15
g/kg BW0.75 78.8 95.9 91.3 6.95
Digestibility, %
DM 51.1a 64.6b 60.9b 2.09
OM 56.4a 69.8b 62.6ab 2.22
CP 28.1a 44.1a 74.0b 7.11
NDF 55.6a 70.1b 59.7a 2.47
ADF 52.2 63.9 55.1 4.78
*a, b Mean in the same row with different superscripts differ (P<0.05), RS = rice straw, UTS = urea-
treated rice straw, CH = cassava hay, SEM = standard error of the means. DM = dry matter; OM =
organic matter; CP = crude protein; NDF = neutral-detergent fiber; ADF = acid-detergent fiber.
873
Comparative Performance of Calves Fed Milk and/or Milk Replacer

Supplemented with Calf Starter up to Weaning Age in Nili-Ravi Buffaloes
Muhammad ABDULLAHa*, Zeeshan Muhammad IQBALa, Muhammad SAADULLAHa,
Ahsan-ul-HAQUEb, Khalid JAVEDa, Makhdoom Abdul JABBARc and Aasim TAUSEEFb
a
Department of Livestock Production, University of Veterinary and Animal Sciences, Lahore,
Pakistan
b
Buffalo Research Institute, Pattoki, Distt. Kasur, Pakistan
c
Punjab Agricultural Research Board, Lahore, Pakistan
*Corresponding email: mabdullah@uvas.edu.pk
ABSTRACT
Buffalo calves are mostly deprived of milk due to premium prices of buffalo milk. The
experiment was designed to determine the effect of whole milk, milk replacer and whole milk cum
milk replacer on dry matter intake, average daily gain, and feed efficiency in Nili Ravi buffalo
calves. Thirty six newborn female buffalo calves were randomly divided into three treatments A
(Whole milk), B (50% whole milk & 50% milk replacer) & C (milk replacer). All the calves were
offered calf starter (19% CP) from 20 to 120 day of age and free access to drinking water. Green
fodder was also offered to all the treatment calves from 60 to 120 day of age. Average daily dry
matter intake in treatment A, B and C was 1555.55±221.280, 1488.67±157.65 and 1459.04±172.19
g, respectively, the difference being non significant (P>0.05). The average daily weight gain of
treatment A, B and C was 457.38±110.13, 426.67±78.70 and 362.22±107.83 g, respectively. There
was a significant (P<0.05) difference between the weight gain of treatment A and C while there
was non-significant (P>0.05) difference between the weight gain of treatment A&B and B&C. The
mean values for FCR in all the three treatments (A, B and C) were 3.49±0.56, 3.560.50± and
4.30±1.24, respectively. The feed conversion ratio (FCR) of treatment A and B was better than
treatment C. It can be concluded from this study that 50% whole milk can be replaced with milk
replacer in daily milk allowance of Nili Ravi calves without effecting dry matter intake, growth rate
and feed efficiency.
Keywords: whole milk, milk replacer, feed efficiency, weight gain, dry matter intake
INTRODUCTION
Buffalo has innate ability to produce milk having high milk fat contents ranging from 6-
8.5%. Due to higher milk fat contents, its milk is preferred over cow milk and fetches better price
(Sarwar et al., 2002, Khan et al., 2008). Under current husbandry conditions, neonatal calves are
often affected by lack of milk feeding due to higher buffalo milk price in market. This is the main
cause of mortality and delayed puberty. Successful calf health and growth depends on the
combination of many factors related to management, nutrition and health of neonate (Heinrichs et
al., 1995). The use of milk replacer for feeding dairy calves saves milk for human utilization and
sold to secure financial consideration (Bamn, 2002). Heifers which grow rapidly achieve puberty at
a younger age and become productive earlier. Use of milk replacer instead of fresh milk is an
alternate way to accelerate gain. The effect of composition, amount and feeding method of milk
replacer to new born calves is evident on their health, performance and behaviour (Brown et al.,
2005; Khan et al., 2007). Milk replacer is a good source of liquid feed for calves. It is often very
economical than whole milk and in many conditions is more easily handled by labour (Heinrichs et
al., 1995). Conventional milk replacer contains animal fat and whey protein. This nutrient
imbalance is one of the main reason for reduced growth in milk replacer fed calves as compared
with those raised on whole milk feeding (Quigley et al., 2006). Furthermore, higher protein
874
contents in milk may severely affect liver and kidney functions (Khan et al., 2007; Lohakare et al.,
2006).
The overall goal of this trial was to determine the effect of feeding fresh milk, fresh milk +
milk replacer and milk replacer on the performance of Nili-Ravi buffalo heifer calves.

The experiment was conducted at Buffalo Research Institute and University of Veterinary
and Animal Sciences, Ravi Campus Pattoki. Thirty six Nili Ravi buffalo heifer calves were used in
the experiment. The calves were kept in individual pens (1 X 2 meter) provided with wheat straw
as bedding material. The calves had free access to calf starter and fresh water. The calves were
given colostrums for three days and then whole milk @ 10 % during adjustment period of fifteen
days and then randomly divided into three experimental treatments (A, B and C) under completely
randomized design. The animals in treatment A were offered whole milk @ 10% of their body
weight, those in treatment B were fed 50% whole milk : 50% milk replacer (0.166 Kg dry milk
replacer was mixed in 1 lit of warm water (60 oC) @ 10% of their body weight, while the animals
on treatment C were given milk replacer (0.166 Kg dry milk replacer mixed in 1 lit of warm water
(60 oC) @ 10% of their body weight. Calves on all the treatments were given calf starter and green
fodder ad lib. The composition of commercial milk replacer and calf starter is given in Table 1.
Table 1. Chemical composition of milk replacer and calf starter (% dry matter).
Ingredient Milk Replacer Calf Starter Ration

Dry matter 95.0 90.0
Crude protein 23.5 19.0
Fat 9.0 4.0
Ash 7.5 9.0
Metabolizable energy M/cal/Kg 3300 2800
Liquid diet was offered through bucket (3.5 L capacity) fitted with soft rubber nipple by
adopting two time feeding regimen (6:00 am & 6:00 pm). Steel bucket was attached to an iron rod
at the front of individual pen at 75 cm above the floor. The bucket was washed using detergent
(bioguard) after each feeding and dried in sun light. All the calves received liquid diet for the first 8
weeks @ 10% of their body weight and then decreased @ 1 % decline every week up to weaning at
120th day of the experiment. Green fodder was offered after eight weeks as decline in the liquid
feed started. Feed refusal was recorded on daily basis to calculate the individually intake of calf
starter and green fodder.
All animals were weighed at the start of experiment and then at weekly interval. Samples of
milk and milk replacer were taken fortnightly and those of calf starter and green fodder monthly for
determination of dry matter by the method of AOAC (2000). Feed conversion ratio was calculated
as the ratio of dry matter intake to live weight gain (Lamb, 2009). All the animals were vaccinated
against FMD and HS at 1 month of age while deworming was done at 25 day of age. Each animal
was observed daily for any change in behavior and abnormality and treated by veterinarian at the
farm accordingly.
Data collected were analyzed using ANOVA technique using Completely Randomized
Design by using SAS 9.1. Differences among treatment means was tested through LSD test (Steel
et al, 1997).
875
RESULTS
The performance parameters of buffalo calves fed either milk, milk replacer or the blend are
shown in Table 2.
Table 2. Performance of Nili-Ravi buffalo calves fed milk and/or milk replacer.
Treatment B
Treatment A
Parameters (50% WM+ Treatment C (MR)
(WM)
50%MR)
Total DMI (g) 1555.55a±221.28 1488.67a±157.65 1459.04a±172.18
Average Daily Gain (g) 457.38a±110.13 426.67ab±78.70 362.22b±107.83
FCR 3.4998b±0.56 3.5576b±0.50 4.304a±1.24
WM= Whole milk, MR= Milk replacer, DMI= Dry matter intake, FCR=Feed conversion ratio
The least square means of total dry matter (Milk, concentrate and fodder) intake were not
different (P>0.05) among all the treatments. There was a slightly increase of dry matter intake in
calve raised on whole milk. This might be due to higher fat contents of whole milk. The average
daily gain was higher (P<0.05) in whole milk group than milk replacer group, while it was similar
(P>0.05) between whole milk and 50% whole milk + 50% milk replacer group. There was also
non-significant (P>0.05) difference in 50% whole milk + 50% milk replacer and milk replacer
group. The difference between Treatment A and C might be due to more DMI in treatment A than
that in C. The FCR is a useful tool to evaluate the effects of diet quality, environment and
management practices on production efficiency in growing calves. The FCR was better (P<0.05) in
treatment A than that in treatment C, While it was similar (P>0.05) between treatments A & B and
B & C. The milk replacer used in this trial consisted of ingredients being 65% from animal and
35% from vegetable source. The improved FCR in treatment A may be due to better palatability of
whole milk as animals source ingredients are are more palatable than vegetable source.
DISCUSSION
The findings of non-significant difference (P>0.05) in the dry matter intake of the three
treatments viz: A, B and C are similar to those observed by Hill et al., 2008a. They stated that dry
matter intake was not affected by the use of more fat in liquid diet. The average daily gain of calves
raised on treatment A, B and C liquid diets was 457.38, 426.67 and 362.22 gm/day. These findings
are in line with the finding of Hill et al., 2008b who observed average daily gain of calves were
0.437 kg/day, 0.380kg/day and 0.375 kg/day raised on different liquid diets. The findings of
average daily gain are in line with the finding of Hill et al., 2008a who observed the average daily
gain of calves on milk replacer was 368gm/day. The findings of FCR for treatment A, B and C
were 3.4, 3.5 and 4.3, respectively. These findings were same as observed by Lee et al., (2008).
They stated that the FCR of calves on high protein diet was 3.9 and on high energy diet was 4.2.
Implications
The above research showed that Nili Ravi buffalo calves gained more on whole milk and
50% whole milk + 50% milk replacer diet. There is non-significant difference in the average daily
gain of calves raised on these treatments. So, It can be concluded from this study that 50% whole
milk can be replaced with milk replacer in daily milk allowance of the Nili Ravi calves without
effecting the growth rate and dry mater intake, with better feed conversion ratio.
876
REFERENCES
Association of Official Analytical Chemists (AOAC) (2000). Official Methods of Analysis of
Association of Analytical Chemists international, 17th ed. Horwitz, W. (ed). Vol I
and II. AOAC International Publs, Maryland USA. Ch. 45: 12 - 20.
BAMN Bovine Alliance on Management and Nutrition (2002). A guide to modern calf milk
replacer. Contact information: AFIA Jim Rydell, 1501 Wilson Blvd., Suite 1100. Arlington,
Brown E. G., M. J. Vandehaar, K. M. Daniels, J. S. Liesman, L. T. Chapin, D. H. Heisler and N. M.
S. Weber. 2005. Effects of increasing energy and protein intake on body growth and carcass
composition of heifer calves. J. Dairy Sci. 88:585–594.
Heinrichs A. J., S. J. Wells and W. C. Losinger. 1995. A study of the use of milk replacers for dairy
calves in the United States. J. Dairy Sci. 78(12): 2831-2837.
Hill S. R., K. F. Knowlton, K. M. Daniels, R. E. James, R. E. Pearson, A. V. Capuco and R. M.
Akers. 2008a. Effects of milk replacer composition on growth, body composition and
nutrient excretion in preweaned Holstein heifers. J. Dairy. Sci. 91(8): 3145-3155.
Hill T. M., H. G. Bateman, J. M. Aldrich, PAS and R. L. Schlotterbeck. 2008b. Effect of
consistency of nutrient intake from milk and milk replacer on dairy calf performance. The
Prof. Anim. Scient. 24:85-92.
Khan, M. A., H. J. Lee, W. S. Lee, H. S. Kim, S. B. Kim, K. S. Ki, J. K. Ha, H. G. Lee and Y. J.
Choi. 2007. Pre- and post-weaning performance of Holstein female calves fed milk through
step-down and conventional methods. J. Dairy Sci. 90: 876-885
Khan, S., M. S. Qureshi, N. Ahmad, M. Amjed, F. R. Durrani and M. Younas. 2008. Effect of
pregnancy on lactation milk value in dairy buffaloes Asian-Aust. J. Anim. Sci. 21:523-531.
Lamb G.C. 2009. Feed efficiency in cow. Florida beef cattle short course. 35-42
Lohakare, J. D., A. K. Pattanaik and S. A. Khan. 2006. Effect of dietary protein levels on the
performance, nutrient balances, metabolic profile and thyroid hormones of crossbred
calves. Asian-Aust. J. Anim. Sci. 19:1588-1596.
Lohakare, J. D., A. K. Pattanaik and S. A. Khan. 2006. Effect of dietary protein levels on the
performance, nutrient balances, metabolic profile and thyroid hormones of crossbred
calves. Asian-Aust. J. Anim. Sci. 19:1588-1596.
Quigley J. D., T. A. Wolfe and T. H. Elsasser. 2006. Effects of additional milk replacer feeding on
calf health, growth, and selected blood metabolites in calves. Ameri. Dairy Sci. Associ.
89(1): 207–216.
Sarwar, M., M. A. Khan, M. Nisa and Z. Iqbal. 2002a. Dairy industry in Pakistan: A Scenario.
International J. Agric. Biol. 3: 420-428.
Steel R. G. D., J. H. Torrie, and D. A. Dickey. 1997. Principles and procedures of statistics: A
Biometrial Approach. 3rd Ed. Mc.Graw Hill Book Co. Inc., NewYork, USA. pp. 481.
877
Changes in Molecular Diversity of Rumen Methanogens in

Buffalo and Cattle in Response to Dietary Tannin
Gondelina A. RADOVANa, Cesar C. SEVILLAb, Severino S. CAPITANb, Renato SA. VEGAb,
Antonio J. Alcantarac and Medino Gideun N. YEBRONb
a
College of Agriculture, Southern Luzon State University, Lucban, Quezon, Philippines, bAnimal and
Dairy Sciences Cluster, University of the Philippines at Los Baños, College, Laguna, Philippines,
c
School of Environmental Science and Management, University of the Philippines at Los Baños,
College, Laguna Philippines.
*Corresponding email: gar_328@yahoo.com.ph
ABSTRACT
The changes in molecular diversity of rumen methanogens in buffalo and cattle fed tannin – containing
banana leaves or supplemented with commercial tannin extract was assessed using polymerase chain
reaction – denaturing gradient gel electrophoresis (PCR-DGGE). Primer set 0357 F-GC and 0691 R
was used to amplify the methanogenic archaeal community of the rumen. A total of 26 DNA fragments
were excised from DGGE gels and their nucleotide sequences were successfully determined. PCR-
DGGE band profile and nucleotide sequence analysis revealed that buffalo harbors fewer methanogens
than cattle. Methanogen resembling Methanobrevibacter sp. YE288 is the predominant methanogen in
buffalo while Methanobrevibacter thaueri strain CW and Methanobrevibacter millerae strain ZA-10
are the predominant ones in cattle. Feeding of tannin – containing banana leaves remarkably altered
rumen methanogen composition of both buffalo and cattle more than commercial tannin extract
supplementation. Furthermore, feeding of tannin – containing banana leaves to cattle significantly
increased blood urea nitrogen (BUN) but decreased rumen fluid ammonia nitrogen (RF NH3-N) level
suggesting a protective effect of tannin on feed protein and potential inhibitory effect on methanogens
by limiting the supply of available hydrogen from ammonia. Hence, feeding of tannin-containing
banana leaves is recommended as a mitigating measure against methane emission to the environment
and as part of a practical feeding strategy for ruminant production.
Keywords: banana leaves, buffalo, cattle, methanogens, tannin

INTRODUCTION
Enteric methane emission from ruminants enhances greenhouse gas build up in the atmosphere. It is
implicated to global warming as well as low animal productivity due to a 2-12% loss of gross energy
from the diet (Pen et al., 2008). Yet methanogenesis provides a means of hydrogen disposal as a
prerequisite for efficient ruminal fermentation. Previous studies suggested that feeding of tanniferous
plants or purified tannin extract to ruminants could be a potential strategy to mitigate methane
production and reduce the environmental footprints of ruminant livestock sector (Hess et al., 2006, and
Jouany and Morgavi, 2007). Banana plant is rich in tannin and is widely cultivated all over the world.
It leaves behind high level of residual biomass after harvesting which could be transformed into useful
feed resources. Hence, this study explored the potential of banana leaves as natural rumen modifying
agent of buffalo and cattle. It further examined the changes in molecular diversity of methanogens and
nitrogen balance in response to dietary tannin.

878
MATERIALS AND METHOD

Experimental Design, Animals and Treatments
Three ruminally fistulated buffalo and cattle with mean body weight of 400.00 + 45.00 and
390.00 + 49.24 kg, respectively were allocated to 3 dietary treatments for 3 repeated 21-day period in a
3 x 3 Latin square design. The treatments consisted of Treatment 1 – 70% napier + 30% concentrate
without tannin, Treatment 2 – 70% banana leaves + 30% concentrate, and Treatment 3 – 70% napier +
30% concentrate with commercial tannin extract. The experimental diets were fed twice daily at 2.5%
of the body weight of animals on a dry matter basis.
DNA Extraction and Polymerase Chain Reaction (PCR) Amplification
Rumen fluid sample was collected from each animal at the start and termination of each 21-day trial.
The DNA was extracted following the modified method of Yu and Morrison (2004) and Sharma et al.
(2003). Methanogenic archaeal 16S rDNA was amplified using primer set 0357 F-GC and 0691 R as
described by Watanabe et al. (2004). PCR was carried out using a total volume of 30 µL containing 0.5
µM of each primer, 1x buffer (Vivantis, USA), 0.2 mM dNTP, 1.5 mM MgCl2, 1 U Taq DNA
polymerase (Vivantis, USA), and 50 ng of DNA template. PCR amplification was initiated by a
denaturation at 95°C for 5 minutes followed by 30 cycles of denaturation at 94°C for 1 minute,
annealing at 55°C for 1 minute, extension at 72°C for 2 minutes, and a final extension at 72°C for 5
minutes in a C 1000 Bio-Rad Thermal Cycler (Berkeley, Ca, USA). Amplification of PCR products
was verified through electrophoresis in 1% agarose gel.
Denaturing Gradient Gel Electrophoresis (DGGE)
PCR product was resolved on 8% (w/v) polyacrylamide gel containing 30-60% gradient denaturants.
Gels were placed in a DCode™ Universal Mutation Detection System (Bio Rad, Berkely, Ca, USA)
for electrophoresis using 0.5X TAE (20 mM Tris; pH 7.4, 10 mM sodium acetate, 0.5 M EDTA)
running buffer heated to 60°C. Electrophoresis conditions for all gels were run for 16 hours at 100 V.
Gels were stained with (0.5 µg/mL) ethidium bromide (Promega Madison, Ca), and photographed
under UV.
Sequence Analysis of PCR-DGGE Fragments
A total of 27 distinct bands were excised aseptically from DGGE gels and DNA was extracted. From
this, 1 µL was used as template for PCR amplification using 0357 F, without the GC clamp and 0691 R
primer set. PCR was conducted as that in 00357 F-GC and 0691 R condition. The resulting unpurified
PCR products were sent directly to Macrogen Inc., South Korea for sequencing. Sequences of
methanogens were compared with available data listed in the GenBank nucleotide sequence database.
The BLAST (Basic Local Alignment Search Tool) search option of the National Center for
Biotechnology Information (NCBI) at http://www.ncbi.nlm.nih.gov was used (Altschul et al., 1997).
Nitrogen Balance Measurement
Nitrogen balance determination was carried out using simple relationship for nutrient balance research
in mammals as adopted from Maynard and Loosli (1969). Nitrogen content of feeds, orts, feces, urine,
and rumen fluid was determined following the AOAC procedure (1990). Blood urea nitrogen was
determined using a commercial diagnostic kit following a fully enzymatic method.
All data for the nitrogen balance were subjected to analysis of variance (ANOVA) using the General
Linear Model (GLM) procedure of Statistical Analysis System Institute (SAS, 1997). Mean
comparisons were made using Tukey’s test.

Changes in the Molecular Diversity of Methanogens
PCR-DGGE band profile (Fig. 1) and sequence analysis demonstrated higher molecular
diversity of methanogen in cattle compared to buffalo. Methanogen composition of buffalo consisted
of 3 different Methanobrevibacter species and 1 uncultured Methanobacteriaceae archaeon clone.
879
Methanogens characteristic of Methanobrevibacter sp. YE288 is the most dominant while methanogen
corresponding to Methanobrevibacter wolinii strain SH is the least dominant in buffalo. On the other
hand, methanogen composition of cattle represented 5 different Methanobrevibacter species, 1
uncultured Methanobrevibacter sp. clone, and 1 uncultured rumen archaeon clone. Methanogens
identified to Methanobrevibacter thaueri strain CW and Methanobrevibacter millerae strain ZA-10
were the predominant ones while the least dominant was methanogen-like Methanobrevibacter sp.
WBY1 in cattle. Feeding of tannin – rich banana leaves remarkably altered the methanogen
composition of both buffalo and cattle more than commercial tannin extract supplementation in the diet
(Fig. 1). Tavendale et al., (2005) reported that the disappearance of methanogens could have resulted
from either a direct effect on ruminal methanogens or an indirect effect on hydrogen production due to
lower feed degradation. While tannin in banana leaves which is substrate-bound, commercial tannin
extract in treatment 3 is in powder form and is mixed in the readily fermentable concentrate diet hence,
the possibility that it might not have remained long in the rumen to produce any observable effect on
methanogens. On the other hand, banana leaves had lower degradation (Table 1) hence could exert
prolonged adverse effect on rumen methanogens.
Nitrogen Balance
Daily nitrogen intake, fecal and urinary losses, nitrogen balance, nitrogen absorbed, and
nitrogen digested were not significantly affected (P>0.05) by dietary tannin (Table 1). However,
feeding of tannin – rich banana leaves significantly increased (P<0.05) daily blood urea nitrogen and
decreased (P<0.05) rumen fluid ammonia nitrogen in cattle suggesting a protective effect of condensed
tannin and possible inhibitory effect of ammonia on rumen methanogens due to limited availability of
hydrogen.
REFERENCES
Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Schang, Z. Zhang, W. Miller and D. J. Lipman. 1997.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Research. 25: 3389-3402.
Association of Official Analytical Chemists. 1990. Official Methods of Chemical Analysis. 15th ed.
Washington DC, volume 1.
Hess, H. D., T. T. Tieman, F. Noto, J. E. Carulla and M. Kreuzer. 2006. Strategic use of tannins as
means to limit methane emission from ruminant livestock. International Congress Series 1293.
164-167.
Jouany, J. P. and D. P. Morgavi. 2007. Use of ‘natural’ products as alternatives to antibiotic feed
additives in ruminant production. Animal. 1(10): 1443-1466.
Maynard, L. A. and J. K. Loosli. 1969. Animal Nutrition. McGraw-Hill, New York. 361-362, 472-473.
Pen, B., C. Sar, B. Mwenya and J. Takahashi. 2008. Effects of Quillaja saponaria extract alone or in
combination with Yucca schidigera extract on ruminal fermentation and methanogenesis in
vitro. Anim. Sci. J. 79: 193-199.
SAS. 1997. Statistical Analysis System Institute Inc., Cary, NC, USA.
Sharma, R., S. Jacob John, D. M. Damgaard and T. A. McAllister. 2003. Extraction of PCR-quality
plant and microbial DNA from total rumen contents. BioTechniques. 34(1): 92-97.
Tavendale, M. H., L. P. Meagher, D. Pacheco, N. Walker, G. T. Attwood and S. Sivakumaran. 2005.
Methane production from in vitro rumen incubatios with Lotus pedunclatus and Medicago
sativa, and effects of extractable condensed tannin fractions on methanogenesis. Anim. Feed
Science and Technology. 123-124:403-419.
Watanabe, T, S. Asakawa, A. Nakamura, K. Nagaoka and M. Kimura. 2004. DGGE method for
analyzing 16S rDNA of methanogenic archaeal community in paddy rice field. FEMS
Microbiology Letters. 232: 153-163.
880
Yu, Z. and M.Morrison. 2004. Improved extraction of PCR-quality community DNA from digesta and
fecal samples. BioTechniques. 36(5): 808-812.
Figure1. DGGE band patterns of methanogenic archaeal communities obtained from rumen fluid of buffalo and cattle ,
namely: (1) Methanobrevibacter sp. YE288, (2) Uncultured archaeon clone TUM-dGArc-GF1-20, (3) Methanobrevibacter
millerae strain ZA-10, (4) Methanobrevibacter thaueri strain CW, (5) Methanobrevibacter thaueri strain CW, (6)
Methanobrevibacter sp. YE288, (7) Methanobrevibacter sp. YE288, (8) Methanobrevibacter thaueri strain CW, (9)
Methanobrevibacter sp. YE288, (10) Methanobrevibacter sp. YE304, (11) Uncultured Methanobrevibacter sp. clone M10,
(12) Methanobrevibacter sp. YE304, (13) Methanobrevibacter wolinii strain SH, (14) Methanobrevibacter sp. YE288, (15)
Methanobrevibacter millerae strain ZA-10, (16) Methanobrevibacter thaueri strain CW, (17) Methanobrevibacter millerae
strain ZA-10, (18) Methanobrevibacter thaueri strain CW, (19) Methanobrevibacter millerae strain ZA-10, (20)
Methanobrevibacter sp. YE304, (21) Methanobrevibacter sp. WBY1, (22) with no sequence similarity, (23) Uncultured
Methanobacteriacea archaeon clone KR-HOI-105, (24) Methanobrevibacter sp. YE304, (25) Uncultured rumen archaeon
clone ASOL-95, and (26) Methanobrevibacter sp.YE288 (inverted color).
881
Table 1. Mean + SEM nitrogen balance, blood urea nitrogen and ruminal ammonia nitrogen
concentration of buffalo and cattle
Treatment
Parameter1 1 2 3
N intake, g/d
Buffalo 463.54 + 8.44 210.24 + 12.66 468.32 + 94.49
Cattle 486.22 + 46.05 278.55 + 96.55 397. 45 + 114.24
Fecal N, g/d
Buffalo 110.86 + 13.71 93.51 + 8.97 105.23 + 5.73
Cattle 70.04 + 8.05 61.81 + 17.37 65.86 + 19.99
Urinary N, g/d
Buffalo 132.81 + 35.32 206.61 + 78.92 672.67 + 604.17
Cattle 120.97 + 73.47 487.55 + 308.67 764.80 + 673.29
N balance, g/d
Buffalo 219.87 + 56.51 -89.87 + 97.21 -309.65 + 672.15
Cattle 295.20 + 120.46 -270.80 + 378.08 -433.21+ 758.85
N absorbed, g/d
Buffalo 352.67 + 21.87 116.73 + 20.80 363.09 + 89.55
Cattle 416.17 + 47.05 216.74 + 79.33 331.59 + 118.40
N digestibility, %
Buffalo 75.97 + 3.31 54.73 + 6.90 76.13 + 0.77
Cattle 85.25 + 2.40 76.34+ 2.70 80.79 + 6.18
BUN, mmol/L
Buffalo 5.77 + 1.07 6.80 + 0.72 6.43 + 0.77
Cattle 5.70 + 0.44b 7.00 + 0.40a 5.90 + 0.17b
RF NH3-N, mg/L
Buffalo 16.22 + 2.45 10.70 + 5.85 8.26 + 3.80
Cattle 48.07 + 8.41a 21.08 + 12.56c 31.97 + 4.22b
1/
Means with different superscript within rows are significant (P<0.05)
882
Comparative Utilization of Different Types of Roughage in Thai Swamp Buffalo

and Thai Brahman Cattle Based on In Vivo Nutrient Utilization, Nitrogen
Balance and Purine Derivatives Excretion in the Urine
Thongsuk JETANAa*, Cherapat VONGPIPATANAb, Sunpetch SOPHONc
and Anong BINTAVIHOKa
a
Science, Chulalongkorn University, Henri Dunant street, Phathumwan, Bangkok 10330, Thailand,
b
Feed and Forage Analysis Section, Animal Nutrition Division, Department of Livestock, Ministry
of Agriculture and Cooperation, Phathumthani 12000, Thailand,
c
Department of Animal Husbandry, Faculty of Veterinary Science, Mahanarkorn University, 51
Chueamsamphan Road, Khrtumrai, Khet NongChok, Bangkok 10530, Thailand
*Corresponding E-mail: Thongsuk.J@Chula.ac.th
ABSTRACT
The current study required comparing the utilization of different roughages; pangola hay
(PH), rice straw (RS) and ammoniated rice straw (RSNH4OH) between Thai swamp buffaloes
(TSB) and Thai Brahman cattle (TBC) based on their in vivo nutrient digestion, N-balance and
purine derivatives (PD) excretion in the urine. Intake of PH was lower (P<0.05) in TBC than those
in TSB, in the contrast with intake of RSNH4OH was higher (P<0.05) in TBC than those in TSB,
but intake of RS was not different between both animal species. Nutrient digestions in both animal
species were similar in those fed PH, RS and RSNH4OH, except for NDF digestion in RSNH4OH
was higher (P<0.05) in TBC than that in TSB. N balance was generally greater (P<0.05) in TSB
than that in TBC when animals were fed PH and RS, but it was greater (P<0.05) in TBC than that in
TSB when animals were fed RSNH4OH. Urinary PD excretion was always lower (P<0.05) in TSB
than that in TBC when fed all roughages. This study showed that there is slight difference in using
various types of roughage between animal species when their qualities have been changed. Low
quality of RS may be due to less N and energy source to meet the nutrient requirement of rumen
microbes, therefore reducing palatability and decreasing intake of RS when compared to PH and
RSNH4OH. Ammoniated rice straw not only increased feed intake, but also enhanced NDF
digestion in TBC, but it did not affect in TSB. This may be due to difference in N utilization
between animal species. The study concluded that PH can be used well in both animal species, but
RSNH4OH is only responsive to cattle and not to buffaloes. The difference on purine derivatives
excretion in the urine between two animal species is confirmed.
Keywords: ammoniated rice straw, Brahman cattle, pangola hay, rice straw, swamp buffaloes
INTRODUCTION
It has been attempted to improve RS by several means: i) physical methods, ii) chemical and
biological treatments and iii) biotechnology. All technologies aim to solve the factor of nutritional
constraints of RS, especially, low N and high ash (silica) content (Van Soest 2006) when RS is used
as a ruminant feed. Nevertheless these technologies are difficult to be adopted to smallholder
farming, particularly in Thailand. However, it demonstrated that urea-ammonia treatment of straw
was possibly successfully transferred to the farmers in China (Ørskov 2010). In Thailand, not only
supplementation with home concentrates or commercial concentrates together with the protein-rich
forages is used to enhance the low quality diets, but selection of several improved grasses are also
introduced to farmers. Pangola grass (Digitaria decumbens) [PH] is among the type of grasses,
being planted in Thailand; this grass has fairly nutritive values and is suitable to make hay and can
be kept for a long time.
Thus, the study was conducted to compare utilization of PH, RS and RSNH4OH between
883
Thai swamp buffaloes (TSB) and Thai Brahman cattle (TBC) based on their in vivo nutrient
digestion, N-balance and purine derivatives excretion in the urine.

Experiment 1
Four male TSB (mean initial weight 3041.6 kg, 24-30 month) and four male TBC (mean
initial weight 2202.5 kg, 18-24 month), were fed daily ad libitum three times a day (07.00 h, 12.00
h and 17.00 h) with PH. The PH, contained 893 g DM/kg DM, it contained 897 g OM, 14.5 g N
(9.03% protein), 806 g NDF and 8.06 MJ/kg DM of metabolizable energy (ME). The experiments
were carried out in a 21-day experimentation; it comprises 14-day dietary adaptation and 7-day
experimentation. On days 1 and 21 of experimentation, the animals were weighed before the
morning feeding (06.00 h) and the animals were transferred (2 days before the collection period)
into individual metabolic crates fitted with containers for urine and faeces collection. Drinking
water was freely available at all time throughout the experimentation. The studies carried out were
in vivo digestion of nutrient, N balance and PD excretion in the urine. Samples of feed, total faeces
weighed and ten percent representative aliquots were collected daily and stored at -20 C. At the
and urine excretion and feed refusals were collected for 7 days (day 15-21) from each animal, then
end of each sampling period, samples from each animal were bulked, and then oven dried at 65 C,
for 72 h, prior to DM, ash and neutral detergent fibre (NDF, Van Soest et al. 1991) analysis. Daily
total urine output was collected into a plastic bag containing 100 ml of 20% (v/v) H2SO4, in order to
maintain a final pH below 3. The urine was collected at 24-hour intervals for 7 days (days 15-21).
was diluted 5 times, then stored at –20 C prior to determination of total N (AOAC 2000 method
The volume of acidified urine was immediately recorded, sub-samples taken in duplicates 20 ml
no.995.04) and PD (IAEA 1997).

Experiments 2 and 3
Animals in the experiments 2, 3 used that same animals as in the experiment 1 were
continuously used in these studies. The experimental procedures of the studies were the same
procedures as described in the experiment 1 except for diets which were changed to RS and
RSNH4OH in the experiments 2 and 3, respectively. The RS contained 887 g DM/kg and based on a
DM basis/kg, it contained 920 g OM, 8.4 g N (5.25% protein), 721g NDF and 6.27 MJ/kg DM of
ME. The RSNH4OH, contained 888 g DM/kg and based on a DM basis/kg, it contained 804 g OM,
11.3 g N (7.06% protein), 693 g NDF and 6.22 MJ/kg DM of ME. Duration of each experiment was
commenced, each animal was daily fed ad libitum with RS and supplemented with 2 kg of
concentrates (20% CP) for one month.
The means of each parameter were established in accordance with the Analysis of Variance
(ANOVA) using the procedures of the Statistical Analysis System Institute (SAS 1998). The
differences between the means were compared by the least significant difference method (LSD).

The average body weight was generally higher (P<0.01) in TSB than that in TBC (Table 1).
In the experiment 1, the intakes of DM, OM, NDF in PH were higher (P<0.01) in TSB than those in
TBC. The digestion DM, OM and NDF of PH in both animal species were similar. In experiment 2,
the intakes of DM, OM, NDF and digestions in both animal species were not different when fed
RS. In experiment 3, the intakes of DM, OM, NDF of RSNH4OH were lower (P<0.01) in TSB than
those in TBC. Intake of DM enhanced in TSB fed PH (22%) and RSNH4OH (21%) when those
compared with animals fed RS. However, RSNH4OH intake was higher (31%) in TBC than that in
TSB, and higher in TBC when compared to be fed PH (57%) and RS (51%). The intake of RS in
animals is lower than that of PH and RSNH4OH, it may be due to N content in RS being possibly
low, so that it does not meet the requirement of microbes in the rumen. This was in agreement with
Jetana et al. (2009), who reported that the intakes of DM, OM increased in buffaloes, when the N
884
contents in diet increased, Similarly reports of Ghebrehiwet et al. (1988) and Van Sovest (2006),
they demonstrated that urea or ammonia treated RS improved DM intake in animals.
There was no difference of in vivo digestion of DM and OM when both animal species fed RS
and RSNH4OH as a basal diet (0.49-0.53). Although NH4OH treated RS increasing N level (35%)
in RSNH4OH, therefore only NDF digestion of RSNH4OH in TBC (0.55) was higher than that in
TSB (0.46). This may be, i) RSNH4OH is smaller amounts of some microbial growth factors
particularly peptides, amino acids and essential volatile fatty acids etc., from rumen fermentation
which some rumen microbes solemnly require for fermentation in the rumen (Gorosito et al., 1985,
Merry et al., 1990), ii) animal species difference, therefore, RSNH4OH is more responsive to cattle
than to buffaloes, and iii) the N content (ammonia toxicity) is too high to be suitable in diets for
buffaloes, as buffaloes have a greater capacity for N recycle than cattle (Abdullah et al., 1992). So
far, nutrients digestion was higher in both animal species fed PH (0.58-0.61) than those fed RS
(0.49-0.51), but the difference in nutrients digestion of RS and PH between in animal species was
not found. However, this study indicated that RSNH4OH dramatically enhanced fibre degradation
particularly in cattle, it is possibly that treatment with NH4OH is dissolving or stimulating the
cracking-off the cuticle wax silica layer of RS (Wang et al., 2007).
In experiment 1, the N digestion in both animal species fed PH was similar. N intakes, N in
urine, faecal-N and N balance were higher (P<0.01) in TSB when compared to those in TBC (Table
1). Experiment 2, the N intakes, faecal-N and N digestion were similar in both animal species fed
RS. Urine-N was higher (P<0.01) in TBC when compared to that with TSB, but N balance was
lower (P<0.01) in TBC than that in TSB (Table 1). Experiment 3, the N intake, urine-N, faecal-N
and N balance were higher (P<0.01) in TBC than those in TSB when those fed with RSNH4OH. N-
balance was greater in TBC fed RSNH4OH than that in TSB when compared to animals fed PH and
RS. This may imply that the N-balance in TBC increased when RS was treated with ammonium
hydroxide, but N-balances generally were higher in TSB than that in TBC when those were fed PH
and RS. It is in agreement with the results of Abdullah et al. (1992), who reported that swamp
buffaloes have a greater capacity for N conservation than Kadah-Kelantan cattle. However, N-
digestion was not different when animals fed different roughages.
All types of roughage did not affect the urinary PD excretion in TSB (212-251 mmol/kg
BW0.75 d-1), but TBC showed that PD excretion in the urine was higher in animals which consumed
PH (792 mmol/kg BW0.75 d-1) compared with RS and RSNH4OH (557 and 577 mmol/kg BW0.75 d-1,
respectively). The lower excretion rate of PD output in TSB when compared with TBC, which was
recorded in the present study, is in agreement with Jetana et al. (2009) who reported a difference in
the rate of PD excretion in the urine between TBC and TSB. The low recovery of PD is present in
the urine of swamp buffaloes, it is possible that its directly related to the low of renal clearance rate
of plasma PD into the urine (Jetana et al., 2006) and to the lower glomerular filtration rates (GFR)
in buffaloes than those in cattle (Norton et al., 1979). In addition to Vo & Ørskov (2006)
demonstrated that the difference in urinary PD excretion occurs only after rumen development.
CONCLUSIONS
The study concluded that PH can be used well in both animal species, but RSNH4OH is only
responsive to cattle and not to buffaloes. The difference on purine derivatives excretion in the urine
between two animal species is confirmed.
REFERENCES
Abdullah, N., N.J. Nolan, M. Mahyuddin and S. Jalaludin. 1992. Digestion and nitrogen
conservation in cattle and buffaloes given rice straw with or without molasses. J. Agric.
Sci. (Cambridge). 119: 255-263
Association of Official Analysis Chemists. 2000. Official methods for analysis of the association
official agriculture chemists. 17th ed. AOAC, Washington, D.C.: Association official
agriculture chemists.
885
Ghebrehiwet, T., N.M. Ibrahim and J.B. Schiere. 1988. Response of growing bulls to diets
containing untreated of urea or urea-treated rice straw with rice bran supplementation. Biol.
Wast. 25: 269-280
Gorosito, A. R., J. B. Russell and P. J. Van Soest. 1985. Effect of carbon-a carbon-5 volatile fatty
acids and digestion of plant cell wall in vitro. J. Dairy Sci. 68: 840-847
International Atomic Energy Agency IAEA-Tecdoc-495. 1997. “Estimation of rumen microbial
protein production from purine derivatives in the urine.” A laboratory manual for the
FAO/IAEA Co-ordinated Research programme on development, standardization and
validation of nuclear based technologies for measuring microbial protein supply in
ruminant livestocks for improving productivity, Vienna, Austria.
Jetana T., S. Usawang, S. Thongruay, C. Vongpipatana and S. Sophon. 2009a. The Effects of
concentrate added to pineapple (Ananas Comosus linn.Mer.) waste silage in differing ratios
to form complete diets, on digestion, excretion of urinary purine derivatives and blood
metabolites in growing, male. Thai swamp buffalo. Trop. Anim. Health Prod., 41: 449-
459
Jetana T., S. Usawang, S. Thongruay, K. Tasripoo, W. Suthikrai, R. Jintana, C. Vongpipatana, S.
Kitsamraj and S. Sophon. 2006. Primary study of renal clearance of plasma allantoin in
Thai swamp buffaloes (Bubalus Bubalis). The 12th AAAP Anima Science Congress 2006,
Bexco Busan, Korea, September 18-22, 2006.
Jetana, T., W. Suthikrai, S. Usawang, S. Kijsamraj and S. Sophon. 2009b. The comparative study
digestion and metabolism of nitrogen and purine derivatives in male. Thai swamp buffalo
and Thai Brahman cattle. Anim. Sci. J. 80, 130-139
Merry, R. J., A.B. McAllan and R.H. Smith. 1990. In vitro continuous culture studies on the effect
of nitrogen source on rumen microbial growth and fibre digestion. Anim. Feed Sci. Tech.
30: 55-64.
Norton, B.W., J. B. Moran and J. V. Nolan.1979. Nitrogen metabolism in Brahman cross, buffalo,
banteng and Shorthorn steers fed on low quality roughages. Aust. J. Agri. Res. 30: 341-351
Ørskov, E.R. 2010. Use of urea for ammoniation of straw in China (Message no. 1) FAO e-mail
conference entitled ‘Successes and failures with animal nutrition practices and
technologies in developing countries: A synthesis of an FAO e-conference’ 1-30
September 2010.
SAS, 1998. User’s Guide: Statistics, Version 6.12, SAS Institute Inc., and Cary NC.
Van Soest, P.J. 2006. Review: rice straw, the role of silica and treatments to improve quality.
Anim. Feed Sci. Tech. 130: 137-171
Van Soest, P.J., J.B. Robertson, and B.A. Lewis. 1991. Methods for dietary fiber, neutral detergent
fiber and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74: 3583-
3597
Vo T.K.T. and E. R. Ørskov. 2006. Causes of difference in urinary excretion of purine derivatives
in buffaloes and cattle. Anim. Sci. 82: 355–358
Wang, J. K., J.X. Liu, J.Y. Li, Y.M. Wu and J.A. Ye. 2007. Histological and rumen degradation
changes of rice straw stem epidermis as influenced by chemical pretreatment. Anim. Feed
Sci. Tech. 136: 51-62
886
Table 1. Intakes of nutrient, the coefficients of in vivo digestions, N-balance and N digestion in Thai swamp buffaloes and Thai Brahman cattle fed ad
libitum PH, RS and RSNH4OH.
Experiment 1 Experiment 2 Experiment 3

PH SEM P-value RS SEM P-value RSNH4OH SEM P-value
Buffalo Cattle Buffalo Cattle Buffalo Cattle
Numbers of animals 4 4 4 4 4 4
Body weight 304 a 220 b 10.6 0.01 327 a 242 b 11.6 0.01 333a 246b 12.5 0.01
a a a
Metabolic body weight 72.7 57.3b 2.08 0.01 77.0 61.1b 2.21 0.01 78.0 62.0b 2.37 0.01
Intakes
DM (g/ BW0.75d-1) 59.7a 49.1b 6.97 0.01 48.8 51.0 3.81 >0.05 59.0 b 77.1 a 4.69 0.01
0.75 -1 a
OM (g/ BW d ) 52.6 45.2b 5.25 0.01 42.0 43.9 3.28 >0.05 50.7 b 66.3 a 4.03 0.01
NDF (g/BW0.75d-1) 48.7 a 40.1b 4.87 0.01 36.5 38.1 2.85 >0.05 40.6b 53.1a 3.23 0.01
The coefficient of in vivo digestion
DM 0.61 0.58 0.05 >0.05 0.49 0.50 0.08 >0.05 0.50 0.51 0.03 >0.05
OM 0.66 0.64 0.05 >0.05 0.51 0.51 0.09 >0.05 0.53 0.53 0.03 >0.05
NDF 0.66 0.64 0.05 >0.05 0.48 0.50 0.08 >0.05 0.46b 0.55a 0.05 0.01
0.75 -1
887
N-balance (mg/BW d )
N intakes 865 a 711b 86.4 0.01 440 459 37.0 >0.05 1240b 1622a 98.6 0.01
a
N in urine 92.4 47.7b 20.9 0.01 69.7b 196a 27.5 0.01 76.3 b 124 a 37.5 0.02
N in faeces 424 a 359b 43.6 0.01 248 271 44.3 >0.05 551 b 767 a 55.7 0.01
a
N-balance 25.4 17.5b 5.31 0.01 122 a -6.76b 78.3 0.01 613 b 730 a 67.6 0.01
-1
N digestion (g/kg d ) 498 472 66.9 >0.05 435 402 54.1 >0.05 552 525 33.8 0.15
PD excretion in the urine (mmol/kg BW0.75 d-1)
Allantoin 217 b 782a 5.52 0.01 191b 492a 7.65 0.01 188b 527a 6.08 0.01
Uric acid 32.0a 10.8b 1.26 0.01 60.3b 64.1a 1.98 >0.05 23.3b 50.1a 1.42 0.01
b
Total PD 249 792a 5.79 0.01 251b 557a 9.16 0.01 212b 577a 6.75 0.01
Values within the same row with the different superscripts are significantly (P<0.05) different.
SEM : Standard error of mean, PH : pangola hay, RS : rice straw, RSNH4OH : ammoniated rice straw, BW : body weight, BW0.75 : metabolic body weight,
d-1 : per day, DM : dry matter, OM : organic matter, NDF : neutral detergent fibre, N : nitrogen, PD : Purine derivatives
454 GS FLX Pyrosequencing Reveals Rumen Bacterial Diversity of Chinese

Water Buffalo
Chengjian YANG, Bingzhuang YANG, Caixia ZOU, Xin LIANG, Shengju WEI, Shulu LI,
Xianwei LIANG* and Hua LUO

24-1Yongwu Road, Nanning 530001, P. R. China
ABSTRACT
In this study, high-throughput 16S rRNA gene-based pyrosequencing was used to investigate
the rumen bacterial communities of Chinese water buffalo (swamp buffalo). Twelve female
Chinese water buffaloes (Xilin buffalo, Haizi buffalo, Dehong buffalo, Dechan buffalo, three of
each type) were used and given feed twice a day with forage ad libitum and four kg concentrate.
Samples were taken from each water buffalo before morning feeding by stomach tube attached to
hand air pump. Characterization of bacteria was achieved using V1-V3 hypervariable regions of
the 16S rRNA gene by a 454 GS FLX PLUS system on twelve samples. Results showed that 21,
21, 20 and 18 bacterial phyla were identified in the ruminal microbiota of Xilin buffalo, Haizi
buffalo, Dehong buffalo and Dechan buffalo, respectively. For whatever the buffalo type, rumen
bacterial community was dominated by Bacteroidetes, Firmicutes and Proteobacteria . There were
227 genera of bacterial populations commonly shared by all twelve samples, including genera of
Anaerovibrio, Bacteroides, Butyrivibrio, Lachnospiraceae, Pseudobutyrivibrio etc., indicating that
there is a core microbial community in the microbial populations of swamp water buffaloes fed
high forage diet. Lachnospiraceae, Prevotellaceae, Ruminococcaceae, Flavobacteriaceae,
Veillonellaceae families were highly present and were clearly affected by buffalo types. The
highest abundance of Prevotellaceae and the lowest abundance of Flavobacteriaceae were found
in Haizi buffalo. The highest abundance of Lachnospiraceae and the lowest abundance of
Veillonellaceae were found in Dechan buffalo. The highest abundance of Ruminococcaceae was
found in Dehong buffalo. In conclusion, results of this study provided insights into the bacterial
community structure and diversity of water buffalo and the bacterial taxa of Chinese water buffalo
(swamp buffalo) appear to be phylogenetically related.
Keywords: Chinese water buffalo, bacterial diversity, rumen, 454 GS FLX pyrosequencing

888
Rumen Bacterial Diversity of Murrah and Nili-Rivi Buffalo (Bubalus bubalis)

Assessed by 454 GS FLX Pyrosequencing
Chengjian YANG, Caixia ZOU, Xin LIANG, Shengju WEI, Shulu LI, Xianwei LIANG*,
Bingzhuang YANG and Hua LUO

24-1Yongwu Road, Nanning 530001, P. R. China
ABSTRACT
the rumen bacterial communities of Murrah and Nili-Rivi water buffaloes fed high forage diet. Six
adult female Murrah and Nili-Rivi water buffaloes (three of each type) were used and given feed
twice a day with forage ad libitum and four kg concentrate. Samples were taken from each water
buffalo before morning feeding by stomach tube attached to hand air pump. Characterization of
bacteria was achieved using V1-V3 hypervariable regions of the 16S rRNA gene by a 454 GS
FLX PLUS system on six samples. Results showed that 21 and 25 different bacterial phyla were
identified in the ruiminal microbiota of Murrah and Nili-Rivi water buffaloes, respectively. For
whatever the buffalo type, rumen bacterial community was dominated by Firmicutes,
Bacteroidetes and Proteobacteria. There were 296 genera of bacterial populations commonly
shared by all six samples, including genera of Prevotella, Butyrivibrio, Ruminococcaceae,
Ruminococcus, Enterobacter, Lactobacillus, Bacteroides, Xylanibacter etc., indicating that there is
a core microbial community in the microbial populations of water buffaloes fed high forage diet.
Lachnospiraceae, Prevotellaceae, Ruminococcaceae, Spirochaetaceae, Rikenellaceae,
Veillonellaceae families were highly present and were clearly affected by buffalo type. The highest
abundance of Lachnospiraceae, Prevotellaceae, Ruminococcaceae and the lowest abundance of
Rikenellaceae and Spirochaetaceae were found in Murrah water buffalo. In conclusion, results of
this study provided insights into the bacterial community structure and diversity of water buffalo
and the bacterial taxa of river buffalo (Murrah and Nili-Rivi buffalo) appear to be phylogenetically
related.
Keywords: water buffalo, bacterial diversity, rumen, 454 GS FLX pyrosequencing


889
Novel Methods to Improve the Nutritive Value of Low Quality Roughages for
Nili Ravi Buffalo Calves
Faisal SHAHZADa, Abdul Shakoor CHAUDHRY a, Muhammad ABDULLAHb, Jalees
Ahmad BHATTIb, Makhdoom Abdul JABBARc, Khalid JAVEDd and Abdul REHMANe
a
School of Agriculture, Food & Rural Development, Newcastle University, UK,
b
Department of Livestock Production, University of Veterinary and Animal Sciences, Lahore
c
Department of Animal Nutrition, University of Veterinary and Animal Sciences, Lahore, Pakistan
d
Department of Animal Breeding and Genetics, University of Veterinary and Animal Sciences,
Lahore, Pakistan
e
Division of Animal Nutrition, Buffalo Research Institute, Pattoki, Pakistan
Corresponding email: faisalshehzad76@yahoo.com
ABSTRACT
Raising buffalo male calves for fattening purpose in Pakistan is a challenge due to shortage
of quality forages and high cost of compound feeds. Although abundant quantity of forages such as
cereal straws is available, their utilization to fatten buffalo calves is limited due to their low
digestibility and intake. The feeding value of cereal straws including wheat straw can be improved
through various physical, chemical and biotechnological methods. Amongst these, biotechnological
methods are drawing more attention being simple and environment friendly. These methods can use
most appropriate microbes that are able to grow on moist substrates under aerobic conditions by
Solid State Fermentation (SSF). The SSF is an advantageous method to degrade lignin in order to
improve digestibility of highly lignified straws. The most important step to produce the quality
fungal biomass by SSF is to optimize and standardize ionic concentration of their growing media
and then evaluate fungal biomass by conducting laboratory and in vivo studies. It can improve the
nutritive value of low quality forages by increasing their protein and essential amino acid contents.
These SSF forages can be used as a feed to raise or fatten Nili Ravi Buffalo calves. However, the
suitability of including SSF products in a complete diet must be assessed on a small scale before
their routine use to fatten Nili Ravi buffalo calves. The SSF technology may offer a novel way of
upgrading fibrous feeds at a farmer level in order to reduce feed cost and environmental pollution.
Keywords: Wheat straw, solid state fermentation, Nili Ravi buffalo
INTRODUCTION
Pakistan ranks 2nd largest country in terms of its buffalo population including the strength of
both young male and female stock (11 million). This is indicative of a huge potential available
however, not been exploited yet. These animals are well adapted to hot and humid conditions,
tolerant to tropical diseases and efficient converters of low quality roughages into milk and meat.
Despite of all these characteristics, per head animal production in the country is very low. This may
be attributed to the poor availability of high quality feedstuffs and abundant less digestible dry
roughages which may be considered major constraints in the development of livestock sector in
Pakistan (Shahzad et al., 2009). Among the available feed resources, crop residues are emerging as
a dominant feed resource for sustainable crop-livestock systems. The feeding value of the poor
quality crop residues mainly wheat straw can be improved through various physical, chemical and
biotechnological methods. Biotechnological method to improve the quality of straw is drawing
much attention lately due to its specificity and simplicity. Biotechnological approaches have now
identified the most appropriate microbes (fungi) with the ability to grow on moist substrate and
under aerobic condition by Solid State Fermentation (SSF). The SSF is an advantageous method to
degrade lignin and improve the digestibility. Fungi grown under these conditions not only bring
better ligninolysis but also improve its digestibility by enhancing the accessibility of hemi-
cellulose. Furthermore, growth of fungal mycelium contributes in increasing the total protein
890
content of the feed (Fazaeli, 2007). By using the crop residue resources as substrate to explore the
large scale industry of various agricultural wastes to produce biomass gives an immediate solution
to fulfill the protein deficiency gap in Pakistan. Furthermore, it may change the traditional way of
feeding management of the subsistence livestock farmers through biotechnological intervention and
poor farmers may be able to effectively adopt the new technology to feed the animals.
Availability of Wheat Straw
Globally, amongst the cereal crops, wheat is one of the most important crop in the world,
providing 20% of humanity’s dietary energy supply and serving as the main source of protein in
developing nations. There was around 647.3 M t wheat produced annually in 2012 in the world,
52% of which was sourced from the China, India, United state, Russia and France. However
Pakistan contributes 20.9 and 23.5 million ton wheat production from year of 2008 to 2012,
respectively. On the basis of yearly world production of cereal grains i.e. 1210 million metric tons;
straw production can be projected to be about 1575 million metric tons. Out of this 1/3rd is wheat
straw. It contains 25-45% cellulose, 20-30% hemicelluloses and 10-15% lignin.

Major Steps to Improve Nutritive Value Of Wheat Straw By White Rot Fungi
Different white rot fungi have been tested on cereals and it’s by products to enrich their
nutritive value. However, lengthy fermentation/incubation periods of the white rot fungi for its
optimization and standardization of the growth conditions is one of the major constraints in its
adoption. Arachniotus species is one of the fast growing fungi among white rot fungi. Hence it may
be tested on the different cereals straw to check its efficacy in terms of enrichment of nutritive value
and its biological evaluation for Nili Ravi buffaloes. Its application mostly requires two following
vital steps:
Optimization and standardization of the growth conditions of white rot fungi
White rot fungi e.g. Arachniotus species and some chemical substances such as urea, HCL
and CaOH- has been used as pretreatments to crop residues, in order to use such improved materials
as an available animal feeds. Up gradation of nutritive value of crop residues like wheat straw by
SSF technology needs optimization of the ionic concentration to get the maximum nutritive value of
fungal biomass. Some workers used only Arachniotus sp. and subjected in increase the fungal
biomass protein using rice polishing as a substrate at fermentation time (72 h), C:N ratio (10:1), 1%
molasses supplementation at (48 h) and corn steep liquor (0.5%). The true protein of rice polishing
was increased from 10.93 to 17.13%. Application of mix culture e.g. Arachniotus sp. and Candida
utilis as fermenting agents on corn stover under SSF resulted in increase fungal biomass at 35oC for
3 days, ionic concentrations e.g. 0.005% MgSO4:7H2O; 0.0075% CaCl2; 0.01% KH2PO4, C:N ratio
of (30:1), 1% molasses which increased crude protein (CP) from 5.46 to 23.51%. In Koji fermenter,
fungal treated wheat and chick pea straw resulted in nutritionally superior combination involving
chick pea straw; 3% urea level and 30 days incubation period. The biomass contained CP of
30.13%. It was observed that on large-scale biomass production, there was more need of
optimization of conditions like agitation, pH and supply of oxygen. Numan and Abdunnasir (2009)
investigated biodegradation of fungal treated wheat straw through SSF. Carbon rate of substrate
was decreased, whereas, nitrogen and sulphur were increased by fungal fermentation. Fermented
wheat straw enriched with 6.48% CP as compared to untreated wheat straw. It means that ionic
concentration for the growth condition of white rot fungi is utmost important step to get the
optimum nutritive value and CP may increase more than 100 % percent by optimization of these
conditions.
Application of solid state fermentation technology
The SSF offers a low-cost alternative for producing cellulases using natural polymers
derived from agro industrial residues. It is defined as a discrete solid phase in which
microorganisms grow on the surface of moist particles as well as inside and between them.
Microbial pretreatment under SSF cultivation has the potential to be a low cost, environment
friendly alternative to other e.g. physical and chemical approaches (Shi et al., 2008). It was
891
observed that mostly 30-100% CP content might be increased for the conversion of the wheat straw
into value added ruminant feed using white rot fungus under SSF technology. Various comparative
studies were carried out between chemical and microbiological treatments on the wheat and rice
straw and depicted that microbial treatment was better than others due to release of more amount of
reducing sugars. The fungal treatment of sorghum stover under SSF resulted an increase in CP
contents from 2.54% for untreated to 4.51% and 4.59% for Pleurotus ostreatus and Pleurotus sajor
pulmonarius respectively, hence this technology has a great potential to use for ruminant nutrition
(Akinfemi et al., 2010).
RESULT AND DISCUSSION

Utilization of Fungal Biomass And Its Effect On Productive Traits In The Animals
In Pakistan among the feeding practices of livestock, crop residues contribute 35% and 50%
in rural commercial and subsistence production system respectively. For this purpose utilization of
fungal biomass as a part of diet and its effect on productive traits is conferred here.
Biomass as a part of the complete diet
Utilization of wheat straw with white rot fungus as an animal feed was studied by several
workers. The results revealed that daily gain and dry matter intake were significantly higher in the
animals fed fermented diet than non fermented. These results were in agreement with Omer et al.,
(2012) and Fazaeli et al., (2002). They concluded that feeding of biological treated roughage diet
improved animal performance as compared with un-treated. Fungal treatment of agricultural by-
products can offer unconventional animal feed which is economical and environment friendly
without any negative effects on animal health. (Abdelhamid et al., 2009). Hence utilization of
fungal biomass based diet may be checked in Nili Ravi buffalo calves to improve the growth rate of
animals.
Effect of fungal biomass on productive traits e.g. feed intake, dry matter intake, feed efficiency, feed
conversion and body weight
Main idea of the biological treatment is to increase the nutritive value in term of substrate
and production in term of growth rates in Nili Ravi buffalo calves. Feeding of fungal treated wheat
straw as a part of total mixed ration resulted in significantly (P<0.05) higher dry matter intake, DM
digestibility and the average body weight gain. (Fazaeli et al., 2002). The fungus plus soybean meal
treatment led to the higher final animal live body weight, growth rate, feed consumption and the
feed conversion (Abdelhamid et al., 2009). Many scientists observed that Murrah buffalo calves
improved their body weight by 20.6% and 29.7% after feeding diets supplemented with
Orpinomyces sp. and Piromyces sp. respectively. All productive traits may be evaluated in Nili
Ravi buffalo calves to check the growth performance by feeding of varying levels of fungal treated
diets so that an optimum level of fungal treated diet may be recommended in Nili Ravi buffalo
along with fungal based total mixed ration to provide the optimum nutrient continuously during
their production stage.
Effect of varying level of fungal biomass on the digestibility
Digestibility is simply a measure of the availability of nutrients. When digestibility is
combined with intake data, one can make an accurate prediction of overall nutritive value. Low or
poor quality roughages are usually characterized by slower ruminal digestion, longer retention
times, delayed clearance from the ruminal compartments and slow rate of passage, creating dietary
fill of the reticulorumen. Solid state fermentation of the wheat straw by the fungi significantly
increased the digestibility of dry matter from 28.1 to 37 % as well as the OM from 28 to 37 %
respectively. Lignin bonds with heavy cellulose components of cell wall and through covalent
linkages and physical binding prevents accessibility and biodegradation of the straw carbohydrates
by cellolytic and hemicellolytic micro organisms.The improvement in the digestibility could be the
result of solublization of the structural polymers by fungi. This made it more accessible to the
rumen microorganisms. However the ability of the fungi to improve the digestibility of the straw
could be different. Increase of dry matter digestibility of fermented wheat straw with Pleurotis
fungi have been reported from 15 to 46 %. Besides the culturing conditions the ability of the
892
various strains of the fungi in cell wall degradation and digestibility improvement of the wheat
straw may be different. Inclusion of the treated straw at different levels of 0, 10, 20, and 30% in the
diet of Holstein did not affect the digestibility of nutrients, except for the acid detergent fiber that
was significantly (P<0.05) reduced in the diet contained 30% treated straw (Fazaeli et al., 2002).
Many workers reported an increase of in vivo digestibility, voluntary intake and nutritive value
index in treated wheat straw with Pleurotus fungi, but the straw obtained after mushroom
harvesting as spent wheat straw did not influence these parameters significantly as observed in the
case of untreated straw when fed to steers. It appears that the changes in nutritive value of straw
may be related to the duration of fermentation of straw by fungi and stage of harvesting. (Fazaeli,
2007). It’s worthy to note that the improving digestibility of nutrients lead to improvement in
nutritive value of diets. In a study conducted by Akinfemi et al. (2010) on the enhancement of the
nutritive value of crop residues with white rot fungi showed an increase in estimated organic matter
digestibility from 42.99 to 57.75%. Hence the digestibility of fungal treated diets in Nili Ravi
buffaloes along with the exact mechanism of lignin degraradation might be planned/focused due to
inadequate information.
CONCLUSION
These SSF forages can be used as a feed for fattening of Nili Ravi Buffalo calves. However,
the suitability of these products in a complete diet must be assessed on a small scale. The SSF
technology may offer a novel way of upgrading fibrous feeds at a farmer level in order to reduce
feed cost and environmental pollution as shown in figure 1. The least but the last, Pakistan has the
potential and capacity to make Single cell protein at industrial level and promising the new era of
future research at microbial level.
Figure 1. Schematic diagram showing novel method pathway to improve the nutritive value of low
quality roughages
Low cost
Biotechnological Methods + SSF Technology= Biological feed Environment friendly
High quality
REFERENCES
Abdelhamid, A. M., S. M. Bassuny, A. A. Abd. El-Aziz and M. Y. S. A. Ibrahim. 2009. Evaluation
of biological treatments for agricultural by-products in ruminants feeding III- growth of
lambs. J. Agri. Sci. 34:6251–6259.
Akinfemi, A., O. A. Adu and F. Doherty. 2010. Conversion of sorghum stover into animal feed
with white-rot fungi: Pleurotus ostreatus and Pleurotus pulmonarius. Afr. J. Biotech.
9:1706-1712.
Fazaeli, H., Z. A. Jelan, H. Mahmodzadeh, J. B. Liang, A. Azizi and A. Osman. 2002. Effect of
fungal treated wheat straw on the diet of lactating cows. Asian Aust. J. Anim. Sci. 15:1573-
1578.
Fazaeli, H. 2007. Nutritive value index of treated wheat straw with Pleurotus fungi. Biotechnol.
Anim. Husban. 23:169–180.
Numan, Y and A. Yildiz. 2009. The effect of Pleurotus eryngii on rice bran and wheat straw under
the solid-state bioconversion for ruminant feed. J. Anim. & Plant Sci. 5:475–482.
Omer, H. A. A., F. A. F. Ali and M. Sawsan. 2012. Gad replacement of clover hay by biologically
treated corn stalks in growing sheep rations. J. Agri. Sci. 4(2):257-268
Shahzad, M. A., M. Sarwar, M. Nisa, A. Iqbal and M. Riaz. 2009. Feed consumption and weight
gain of growing buffalo calves as influenced by feeding fermentable energy source in
corncobs based diet. Pak. J. Zool. 9:707-710.
Shi, J., M. S. Chinn and R. R. Sharma-Shivappa. 2008. Microbial pretreatment of cotton stalks by
solid state cultivation of Phanerochaete chrysosporium. Biores. Technol. 99:6556–6564.
893
Effect of Cysteamine Hydrochloride on In Vitro Methane Emission in

Water Buffalo
Caixia ZOUa, Yali HUANGb, Tianshui LUC, Bingzhuang YANGa, Xianwei LIANGa*,
Chengjian YANG a and Zhongsehng XIAb
a
24-1Yongwu Road, Nanning 530001, P.R. China. b College of Animal Science and Technology,
Guangxi University, Nanning 530005, P.R. China, C Nanjing Agricultural University, Nanjing
Jiangsu 210095 , P.R. China
ABSTRACT
Cysteamine (β-mercapto-ethylamine) has been used as a cystine depleting agent under its
hydrochloride formulation for more than 20 years. In our previous studies, supplement
Cysteamine hydrochloride (CSH) could increase the Conjugated linoleic acids content in water
buffalo milk, but we do not know whether CSH could reduce the methane production. Thus, the
aim of this present study was to evaluate the effect of Cysteamine hydrochloride (CSH) on in vitro
methane emission in water buffalo. In vitro fermentations were conducted in 180-ml serum bottles
as described by Theodorou et al (1994). CSH were supplemented at levels of 0%, 0.2%, 0.4%,
0.6%, 0.8% and 1.0% based on the concentrate (DM basis). The oven-dried substrate was
comprised mainly by maize grain (25%), soybean meal (7.5%), elephant grass (26.3%), distiller’s
malt brewers (22.5%) and cassava pulps (18.75%), the concentrate to forage ratio was 32.5:67.5.
The incubation time was 72 h, methane production was measured at 6 h, 12 h, 24 h incubation
time. After 24 h incubation, rumen fluid was used to measure NH3-N concentration and VFA
composition. Methane production at 6 h, 12 h, 24 h and butyrate at 24 h were decreased by
supplementation of CSH, especially the CSH levels higher than 0.4%, while butyrate increased
significantly (P<0.05). There were no significant difference existed in total gas production (GP),
GP from soluble fraction, GP from insoluble fraction, Potential GP and GP rate constant (h-1), final
NH3-N concentration, acetate, propionate in each group (P>0.05). Therefore, supplementation of
CSH at 0.4% level could decrease the in vitro rumen methane production, while has no significant
effects on other fermentation parameters. We can conclude that CSH would be a potential methane
inhibitor, but its functional mechanisms need to be further researched.
Keywords: Cysteamine hydrochloride, Methane emission, Water buffalo, In vitro fermentations


894
Evaluation of Nutritive Value of Agricultural by-products and Industrial

by-products for Buffalo by Cornell Net Carbohydrate and Protein System
Junhua ZHOU ab, Caixia ZOUa*, Bing-zhuang YANGa, Xian-wei LIANGa, Sheng-ju WEIa,
Xin LIANGa, Shulu LI a, Youjing ZOUb* and Chenjian YANGa
a
Nanning 530001, China.
b
College of Animal Science and Technology, Guangxi University, Nanning530001, China.
*Corresponding Email:Caixiazou2002@hotmail.com, yjzou@gxu.edu.cn
ABSTRACT
In order to reveal characteristics of carbohydrate and protein fractions in agricultural
by-products and industrial by-products for buffalo, six kinds of samples were studied by the Cornell
net carbohydrate and protein system (CNCPS).Common nutritive values of the samples were
determined. The CNCPS carbohydrate fractionations, i.e. sugars(CA), starch and soluble fiber
(CB1), available neutral detergent fiber (CB2), unavailable neutral detergent fiber (CC) and CNCPS
protein fractionations, i.e. rapidly rumen degradable true protein (PB1), intermediately rumen
degradable true protein (PB2),slowly rumen degradable true protein (PB3), undegradable crude
protein (PC) and nonprotein nitrogen (PA) contents, were calculated using formulas in CNCPS. The
results showed that characteristics of CNCPS carbohydrate fractions and CNCPS protein fractions
of the one sample were different. CNCPS fully and objectively reflected the nutritive characteristics
of agricultural by-products and industrial by-products, the results provide data basis for utilization
of agricultural by-products and industrial by-products, optimization of buffalo ration.
Keywords: Buffalo, Agricultural by-products, Industrial by-products, CNCPS, Nutritive value
INTRODUCTION
The CNCPS is a feed evaluation system developed based on principles in rumen function,
microbial growth, feed digestion and passage, the chemical composition of the rations and animal
physiology. So CNCPS can specifically and truthfully reflect the feed utilization for animals and
provide the foundation parameter for the ration use (Fox et al., 1995; Pichard et al., 1977). The
objectives of this study were to reflect the nutritive characteristics of the six kinds of roughages for
buffalo and provide data basis for utilization of them and optimization of buffalo ration.

The samples were dried in an oven at 105°C for 6h, equilibrated in a desiccator, milled
through a 1mm screen and stored for later analysis. All of the samples were analyzed for dry matter
(DM), crude protein (CP), EE, ASH according to AOCO (1980). The neutral detergent fiber (NDF)
and the acid detergent fiber (ADF) were determined using the method of Van Soest et al. (1991).
The neutral detergent insoluble crude protein (NDICP) which is the CP in neutral detergent fiber
was determined after NDF determination according to the method of Van Soest et al. (1981). The
acid detergent insoluble crude protein (ADICP) which is the CP in acid detergent fiber was
determined after ADF determination according to the method of Van Soest et al. (1981). The lignin
was determined using the method of Van Soest et al. (1981). The soluble crude protein (SCP) was
895
determined using the method of Krishnamoorthy et al (1983). Starch content was determined by
enzymatic hydrolysis with amyloglucosidase (McClearyetal et al., 1994).The CNCPS nitrogenous
fraction content and carbohydrate fraction content of the roughages were calculated using the
method of Sniffen et al. (1992).

The highest content of PA in the industrial by-products was Brewer’s grain，which was
19.65%，19.74% higher than Cassava waste, Pineapple peel in terms of PB2, Pineapple peel was the
highest (56.92%). Rice straw had the very highest value of PA (30.72%)in agricultural by-products.
The highest content of CP in the industrial by-products was Brewer’s grain (Table 1).
According to the CNCPS, carbohydrates are divided into fiber carbohydrates (FC) and
non-fiber carbohydrates (NFC).The NFC is further divided into fractions CB1 and CA.The CB1
fraction represents soluble fiber and starch. The CA fraction represents the rapidly fermented water
soluble CHO fraction. The FC is further divided into fractions CB2, CC. Table 2 shows that
concentration of CHO in cassava waste was the highest in the six roughages, and the CC
concentration was the lowest, considering to other carbohydrate fractionations, the CNCPS
carbohydrate fractionations of cassava waste was the best in the industrial by-products. The
concentration of CHO in corn clothing was 1.09%, 8.53% higher than corn straw, rice straw ,then
CC was 7.10%, 6.08% lower than corn straw, rice straw, considering to other carbohydrate
fractionations, the CNCPS carbohydrate fractionations of corn clothing was the best in the
agricultural by-products (Table 2).
The crude protein was partitioned into five fractions as proposed by the CNCPS (Sniffen et
al., 1992; Chalupa et al.,1994). There has been described as: unavailable fraction C, which is the
acid detergent-insoluble nitrogen (ADIN);fraction A contains NPN ;the B fraction contains the true
protein, which based on rates of protein degradation in the rumen is further subdivided into
rapidly(B1),intermediate(B2)and slowly(B3) degradable fractions.PA has a high nutritional value
for ruminant, but has low valent of use for single – stomached animal. PC contains proteins
associated with lignin, tannin-protein complexes, which is not degradable in the rumen and is
indigestible in the intestine (Krishnamoorthy et al., 1982). Considering to the CNCPS protein
fractions, in the industrial by-products, brewer’s grain was the best, pineapple peel was the second
and cassava waste was the worst; with respect to agricultural by-products, rice straw was the worst.
CONCLUSION
Twenty three indications of CNCPS were conducted to characterize the carbohydrate and
protein fractions of agricultural by-products and industrial by-products to buffalo. Then, CNCPS
could measure more nutrient indices of feedstuff and reflect their digesting and absorbing
metabolism in buffalo fully, providing date basis for utilization of roughages and optimization of
buffalo ration.
ACKNOWLEDGEMENTS
Part of funding for this study was provided by Guangxi Science Foundation, China (Grant
no. 0832071; 0640033; 1123005-2).
REFERENCES
AOAC. 1990. Official Methods of Analysis. 15th edn. Association of Official Analytical Chemists,
Arlington, Virginia.
896
Chalupa, W., Sniffen, C. J., Cole, D. J. A. (Eds), Recent Advances in Animal Nutrition. Nottingham
University Press, Nottingham. pp. 265-275.
Fox, D. G., Barry, M .C., Pitt, R. E.,1995. Application of the Cornell net carbohydrate and protein
model for cattle consuming forage. J. Anim. Sci. 73:267-277.
Krishnamoorthy, U., Sniffen , C. J., Stern, M. D. 1983. Evaluation of mathmatical model of
digestion and an in vito simulation of rumen proteolysis to estimate the rumen undegraded
nitrogen content of feedstuffs. Bri. J. Nutr. 50:555-565.
Krishnamoorthy, U., Muscato, T. V., Sniffen, C. J., Van Soest, P. J. 1982. Nitrogen fractions in
selected feedstuffs. J. Dairy Sci. 65:217-225
McCleary, B.V., Solah, V., Gibson, T. S. 1994. Quantitative measurement of total starch in cereal
flours and products. J. Cereal Sci. 20:51-58.
Sniffen, C. J., O’Connor, J. D., Van Soest, P. J., Fox, D. G., Russell, J. B. 1992. A net carbohydrate
and protein system for evaluating cattle dietsII Carbohydrate and protein availability. J.
Anim. Sci. 70:3562-3577.
Van Soest, P. J., Robertson, J. B., Lewis, B.A. 1991. Methods for dietary fiber, neutral detergent
fiber, and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci.
74:3583-3597.
897
Table 1. The chemical composition of common roughages for Guangxi water buffalo.
DM basis Percentage in CP
Items
DM EE CP NDF ADF ASH NPN SCP LIGNIN STARCH NDICP ADICP
Industrial by-products
Cassava waste 86.68 0.51 3.80 45.01 20.25 2.04 8.76 12.58 7.37 35.30 2.29 1.05
Brewer’s grain 89.88 10.11 37.33 46.29 20.58 4.14 41.41 63.24 3.27 4.54 2.14 3.26
Pineapple peel 87.54 1.59 7.67 64.31 28.01 5.96 28.67 30.94 8.13 19.01 12.14 0.76
Agricultural by-products
Corn straw 89.20 1.82 8.34 59.56 34.00 8.61 16.89 28.34 11.85 3.64 1.01 0.36
Rice straw 88.99 2.04 5.43 69.88 47.04 13.39 30.72 38.09 10.57 3.15 51.67 0.77
Corn clothing 89.72 5.21 3.69 78.78 38.45 4.54 12.60 26.56 9.72 8.54 8.17 3.92
DM=dry matter; EE=ether extract;ASH= the mineral;NDF=neutral detergent fiber;ADF=acid detergent fiber;CP=crude protein;NPN=non-protein
nitrogen;SCP=soluble crude protein;ADICP=acid detergent insoluble crude protein;NDICP=neutral detergent insoluble crude protein.
898
Table 2. The CNCPS carbohatrate fractionations of commen roughages for Guangxi water buffalo.
Items CHO/(%DM) CA/(%CHO) CB1/(%CHO) CB2/(%CHO) NFC/(%CHO) CC/(%CHO)

Cassava waste 93.65 48.17 37.70 19.68 58.31 18.88
Brewer’s grain 48.42 25.95 3.39 58.46 24.88 6.23
Pineapple peel 84.78 4.01 22.43 50.64 22.39 23.01
Corn straw 80.12 4.14 4.55 50.40 8.58 35.49
Rice straw 73.58 1.11 4.29 54.62 4.85 34.47
Corn clothing 82.11 1.38 10.40 61.26 6.29 28.39
CHO=total carbohydrate;CA=rapidly fermented water soluble CHO fraction;CB1= the starch and soluble fiber;CB2=solube fiber;NFC=Non-fiber
carbohydrate;CC=indigestible fiber.
Table 3. The CNCPS protein fractionations of commen roughages for Guangxi water buffalo.
Items PA/ (%CP) PB1/ (%CP) PB2/ (%CP) PB3/ (%CP) PC/ (%CP)
Cassava waste 8.76 3.81 45.58 0.79 1.05
Brewer’s grain 28.41 1.83 5.72 56.88 1.16
Pineapple peel 8.67 2.27 56.92 11.38 0.76
Corn straw 16.89 11.45 70.66 0.64 0.36
Rice straw 30.72 7.37 10.24 0.90 3.77
Corn clothing 12.60 13.96 65.27 4.26 0.92
PA=non-protein nitrogen;PB1=rapidly rumen degradable crude protein;PB2=intermediately rumen degradable crude protein;PB3=slowly rumen
degradable crude protein;PC=bound crude protein
899
Effects of Beer Lees and Cassava Residues Respectively Substituting for

Soybean Meal and Grassiness on Milk Performance in
Lactating Water Buffalo
Yali HUANG ab, Caixia ZOUa*, Zhongsheng XIAb*, Bingzhuang YANGa, Xianwei LIANGa,
Shengju WEI a, Shulu LI a, Xin LIANGa and Chengjian YANGa
a
Key Laboratory of Water buffalo Genetics, Breeding and Reproduction technology, Ministry of
Agriculture and Guangxi, Water buffalo Research Institute, Chinese Academy of Agricultural
Sciences, 24-1Yongwu Road, Nanning 530001, P.R. China.
b
College of Animal Science and Technology, Guangxi University, Nanning 530005, P. R.
China.
*Corresponding email: caixiazou2002@hotmail.com, zsxia@gxu.edu.cn.
ABSTRACT
Objective of this study was to investigate the effect of beer lees and cassava residues
respectively substituting for soybean meal and grassiness on milk performance in lactating
water buffalo. Sixteen healthy water buffaloes of early lactation were selected and randomly
divided into two groups (8 each) on the base of species, parity and milk yield. Water buffaloes
in control group were fed with a basal diet consisting of concentrate mixture, beer lees, cassava
residues and grassiness as per requirements. Water buffaloes in treatment group were fed with
the diet of beer lees substituting for soybean meal at ratios of 50% (DM basis)and cassava
residues substituting for grassiness at ratios of 12.5% (DM basis). The study was conducted for
56 days after the adaptation period of 2 weeks. There was no significant effect on milk yield
and feed intake of lactating water buffalo in treatment group. The milk protein, milk fat, total
solids and solid not fat were higher (P<0.05) by 5.72%, 11.78%, 6.85% and 4.11% in treatment
group than control group, respectively. The contents of C16:0and C16:1 in milk fat were higher
(P<0.05) by 28.96% and 30.84% in treatment group than control group, respectively. There was
an increase in the contents of C18:0, C18:1and cis-9 trans-11 Conjugated Linoleic Acid in milk
fat of treatment group. In summary, there was no significant effect on milk performance of
lactating water buffalo in treatment group of beer lees and cassava residues respectively
substituting for soybean meal and grassiness, whereas milk quality was improved.
Key words: beer lees, cassava residues, lactating water buffalo, milk performance
INTRODUCTION
The crude protein content is high in beer lees. It is great significance to increase social
and economic benefits, and promote animal husbandry by full developing and utilizing beer
lees. The abandoned cassava residues were reasonably developed and utilized, it can be a better
ruminant feed through improving palatability. Therefore, its appropriate utilization makes great
sense to animal husbandry and feed industry. The supplementation of fresh beer lees (3.5 kg/d)
to high producing cows increased milk yield and extended the milk peak periods (Yao,2001).
Feeding silage cassava residues to crossbred cattle gained weight 0.245kg/d, the average of
daily weight was higher (P<0.01) in silage cassava residues group than that of ammoniated
straw group (Cai et al., 2007). Substitution of soybean meal with beer lees at ratios of 0 to 50%

900
has no negative effect on rumen fermentation characteristic and methanogenesis in vitro, but it
did not carry out animal trials (Huang et al., 2012). According to the nutrient characteristics of
high crude protein in beer lees, the study was to investigate the effects of beer lees and cassava
residues respectively substituting for soybean meal and grassiness by using animal trials on
milk performance in lactating water buffalo, which promoted the development of water buffalo
industry.

Selection and management of animals
Sixteen healthy water buffaloes of early lactation (45-65days calving) were selected.
Water buffaloes were milked twice a day at 6:00 and15:00 h. Water buffaloes were fed
roughage firstly, then were fed concentrate at the milking time.
Experimental design
The single factor random grouping test design was used in the experiment. Sixteen
healthy water buffaloes were selected and randomly divided into control group and treatment
group (8 each) on the base of species, parity and milk yield. Water buffaloes of control group
were offered concentrate (5 kg/d) and roughage (grassiness, 15 kg/d; beer lees, 10 kg/d; cassava
residues, 5 kg/d). Water buffaloes of treatment group were offered concentrate (4.85 kg/d) and
roughage (grassiness, 13.01 kg/d; beer lees, 10.58 kg/d; cassava residues, 6.99 kg/d). The study
was conducted for 56 days after the adaptation period of 2 weeks.
Collection and analysis of milk samples
Feed intake of water buffaloes was recorded every day during the trial. Milk yield of
every water buffalo was recorded daily at each milking during the trial. Milk samples of every
water buffalo were collected in the third day (at 09:00 and 17:00h) of a week. 50 ml milk
sample was analyzed for milk protein, milk fat, solid not fat content, milk lactose and total
solids by Milk Analyzer, FOSS MSC FT - 120 (Danmark). Another part of 50 ml milk sample
was collected and analyzed for the contents of various fatty acids in milk fat. The pretreatment
method of milk sample was used according to Wang et al. (2006).
Statistical analysis of the data was calculated with Excel statistical software and
performed using SPSS package programme (SPSS 16.0 software for windows), and multiple
comparison was carried out by Duncan’s. Data were analyzed by one-way analysis of variance.
Significant was declared at P<0.05, and the results were given as mean± SE.

The effects of beer lees and cassava residues respectively substituting for soybean meal
and grassiness on feed intake and milk yield of lactating water buffalo
Feed intake and milk yield of two groups in lactating water buffalo were presented in
Table 1. There was no significant effect on feed intake and milk yield of lactating water buffalo
in two groups (P>0.05).Compared with control group, day milk production of every water
buffalo was reduced 0.15 kg/d in treatment group. This finding coincides with the results of
Zhao et al. (2010), reporting that the milk yield was significantly higher in the group of the
high level of protein fodder than that group of the low level of protein fodder. Feeding the diet
of substitution of concentrate with dry beer lees at ratios of 20% to lactating cows, milk yield
was reduced by 1.6% in the group of feeding dry beer lees than control group, the difference
901
was no significant (P>0.05) (Xue, 2004). Feeding the mixed fodder to cows, the ratio of
concentrate fodder was decreased, so did the protein content and energy level in fodder, which
lead to the lower milk production (Guo et al, 2011). Compared with control group, the level of
protein and energy was slightly lower in treatment group, so milk yield was slightly lower in
treatment group than control group, but the difference was no significant (P>0.05). The results
of the present study are in agreement with those of Zhao et al. (2010), Xue (2004) and Guo et
al.(2011).
The effects of beer lees and cassava residues respectively substituting for soybean meal
and grassiness on the milk composition and fatty acids in milk fat of lactating water buffalo.
Milk composition of two groups in lactating water buffalo was presented in Table 2.
The milk protein, milk fat, total solids and solid not fat were higher (P<0.05) by
5.72%,11.78%,6.85% and 4.11% in treatment group than control group, respectively. There was
no significant effect on milk lactose in two groups (P>0.05). Compared with control group,
milk lactose was fell 1.66% in treatment group. The milk composition and quality was
improved in treatment group diet. Feeding beer lees to Holstein cows and Wenzhou water
buffalo, milk fat was increased by 4.95% and 6.89% than control group, respectively(Chen,
2011). Milk fat was increased by increasing the content of neutral detergent fiber of diet (Wang
et al, 2007). During the experiment periods, the content of crude fiber was increased when beer
lees substituting soybean meal, so milk fat was also increased, which are in agreement with
those of Chen (2011) and Wang et al.(2007).Wang et al. (2010)reported that soybean hulls, feed
jujube and fresh beer lees were added to the diets of Holstein dairy cows, milk protein was
significantly increased by adding beer lees in diets. Sun et al. (2005) reported that the average
of milk fat and milk protein was higher in the cows of mid to late lactation later which fed beer
lees, while solid not fat was slightly lower in treatment group than control group. The results of
the present study were in agreement with those of Wang et al. (2010) and Sun et al. (2005).
Fatty acids in milk fat of two groups in lactating water buffalo was presented in Table 3.
The contents of C16:0 and C16:1 in milk fat were higher (P<0.05) by 28.96% and 30.84% in
treatment group than control group, respectively. The contents of C18:0, C18:1 and cis-9,
trans-11 Conjugated Linoleic Acid in milk fat were increased by 20.02%,19.52% and 15.54%
in treatment group than control group, respectively. Chouinard (1998) reported that the level of
the fodder cellulose had a significant effect on the content of Conjugated Linoleic Acid in milk
fat. The level of the fodder cellulose was insufficient which would reduce the rumen pH value.
It influenced the normal rumen fermentation and the synthesis of Conjugated Linoleic Acid.
The results of the present study showed that the contents of various fatty acids was higher in
treatment group than control group. The content of fatty acids in milk fat was greatly improved
by feeding the experimental fodder.
CONCLUSIONS
The results of the present study showed that there was no significant effect on milk
yield and feed intake of lactating water buffalo in the treatment diet. However milk protein,
milk fat, total solids, solid not fat contents, C16:0 and C16:1 in milk fat were significantly
higher in it. It was feasible that the diet of beer lees substituting for soybean meal at ratios of
50% and cassava residues substituting for grassiness at ratios of 12.5% fed to lactating water
buffalo.
902
ACKNOWLEDGEMENTS
Part of funding for this study was provided by Guangxi Science Foundation, China
(Grant no. 0832071; 0640033; 1010019-24; 1123005-2).
REFERENCES
Cai, Y. Q. and W.Q. Yang. 2007. The experiment of gaining weight on feeding silage cassava
residues to catalo. J. Guangdong J. Anim. and Vet. Sci. 32(1): 51-52.
Chouinard, P. Y. 1998. Effect of dietary manipulation on milk conjugated linoleic acid
concentrations. J. Dairy Science. 81,supp1.1233.
Guo, D. S. and X.L. Peng. 2011. Effect of the diet of different concentrate and roughage mixed
on milk performance and milk quality in cows. J . Southwest China J. Agri. Sci. 1:
297-300.
Huang, Y. L., C.X. Zou and L.Y. Huang. 2012. Partial substitution of soybean meal with beer
lees: influence on Rumen Fermentation Characteristics and Methanogenesis of water
buffalo in vitro. J . Chinese J. Anim. Nut. 24(3): 563-570.
Table 1. Feed intake and milk yield of two groups in lactating water buffalo (kg/d).
Parameter Control Treatment

Feed intake 11.29±0.04a 11.12±0.05a
Milk yield 8.09±0.70a 7.94±0.75a
In the same row, values with different small letter superscripts mean significant difference
(P<0.05), while with same letter superscripts mean no significant difference (P >0.05). The
same as below.
Table 2. Milk composition of two groups in lactating water buffalo (%).
Milk composition Control Treatment

Milk protein 4.37±0.06a 4.62±0.08b
Milk fat 8.15±0.33a 9.11±0.33b
Total solids 18.54±0.37a 19.81±0.40b
Solid-not fat 10.23±0.06a 10.65±0.08b
Milk lactose 4.89±0.03a 4.81±0.04a
Table 3. Fatty acids in milk fat of two groups in lactating water buffalo (mg Fatty acids /100g
milk).
Fatty acids Control Treatment

C16:0 529.48±32.27a 682.80±55.05b
C16:1 38.52±2.56a 50.40±4.36 b
C18:0 190.45±14.16a 228.58±15.90a
C18:1 590.47±35.56a 705.71±47.33a
cis-9,trans-11CLA 24.13±1.38 a 27.88±1.99 a
903
Graded Replacement of Corn Grain by Wheat Grain in Sahiwal Calves:

Influence on Nutrients Intake, Digestibility, Blood Metabolites and Growth
Performance
Muhammad Aasif SHAHZAD*, Muhammad AFZAAL, Muhammad SHARIF,

Muhammad Saif ur REHMAN1, Muhammad SARWAR, Nasir Ali TAUQIR, Muhammad
RIAZ2
Institute of Animal Nutrition and Feed Technology, University of Agriculture,Faisalabad-

38040, Pakistan
1
Department of Animal Breeding and Genetics, University of Agriculture, Faisalabad-38040,
Pakistan
2
Department of Livestock Management, University of Agriculture, Faisalabad-38040, Pakistan
*Corresponding author: draasifshah@uaf.edu.pk
ABSTRACT
The study was planned to examine the effects of replacing corn grains with wheat grains
on nutrient intake, nutrient digestibility, blood metabolites and growth performance of growing
female Sahiwal calves fed on berseem (Trifolium alexandrinum) based diet. Twelve calves of
six to eight months of age were randomly divided into three groups, four calves in each group,
in a randomized complete block design. Three isocaloric and isonitrogenous diets were
formulated consisting of 65% berseem hay and corn contents were gradually replaced by wheat
at the rate of 0, 10 and 20% and were denoted as HC (high corn), MCMW (medium corn
medium wheat) and HW (high wheat), respectively. Experiment lasted for 85 days; first 20 days
served as adaptation period. Findings revealed that nutrient intake remained unaltered (P>0.05)
in calves fed HC, MCMW and HW diets. However nutrient intake tended to decrease with
increase (as percentage of body weight) of wheat grains in the diet. Digestibility of dry matter
(DM) and crude protein (CP) didn’t differ (P>0.05) across all treatments. Maximum and
minimum digestibilities (P<0.05) of fiber fractions (NDF and ADF) were noticed in calves fed
HC and HW diets, respectively. Blood glucose, urea nitrogen, blood cholesterol and creatinine
concentrations didn’t change (P>0.05) across all diets. Weight gain and feed efficiency also
remained unaltered (P>0.05) in calves fed HC, MCMW and HW diets. The findings of the
present study imply that replacement of corn grains by wheat grains, when replaced upto 20%
of feed on DM basis, didn’t influence the DM intake and digestibility, blood metabolites and
growth performance of Sahiwal calves.
Keywords: Energy source, corn / wheat grains, blood metabolites, growth, Sahiwal calves
INTRODUCTION
Considering differences in rate of starch fermentation among cereals for feed formulation
can help to improve synchronization of nutrients (energy and protein) at ruminal level which can
accelerate growth of rumen microbes and thereby nutrient digestibilities. Lower degradation of
fiber and cell wall components has been reported in ruminants fed on readily fermentable grains
than those fed slow fermenting grains (Overton et al., 1995). Rapidly fermenting grains may
reduce ruminal pH and thus constrain cellulolytic bacteria which intern impairs fiber digestion
and thus animal growth (Orskov and Fraser, 1975). Influence of altering type of cereal grains on

904
nutrient digestion and milk yield has been extensively studies for lactating cows. However,
implication of same findings on growing calves where rumen is in the process of development
doesn’t seem to be a wise act. Therefore, the present study has been planned to examine the
effects of replacing corn grains with wheat grains on the nutrient intake, digestibility of nutrient,
blood metabolites and weight gain in female sahiwal calves.

Twelve female sahiwal calves of six to eight month age were used in Randomized
Complete Block Design to evaluate the effect of replacing corn grains with wheat grains on the
growth performance. Three isocaloric (ME: 2.45 Mcal/kg) and isonitrogenous (16% CP) diets
were formulated such that corn grains were gradually replaced by wheat grains at the rate of 0
(high corn), 10 (medium corn medium wheat) and 20% (high wheat) and were denoted as HC,
MCMW and HW diets, respectively. Feed offered and refusal were weighed to calculate intake
of dry matter (DM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber
(ADF). Calves were weighed weekly before morning feeding. Nutrients digestibilities were
determined by total collection method (Shahzad et al., 2007). Feed and fecal samples were
analyzed for DM, nitrogen (AOAC. 1990) and NDF and ADF following the procedure of Van
Soest et al. (1991). Blood samples were collected at 3, 6 and 9 hours post feeding to analyse
blood glucose (Davies et al., 2007), blood urea nitrogen (BUN; Bull et al., 1991), cholesterol and
creatinine concentrations (Meyer, 1996). The data collected for each parameter were subjected to
analysis of variance technique under Randomized Complete Block Design, in case of
significance pairwise comparison was performed (Steel et al., 1996).
RESULTS
The results of average daily feed intake were found to be non-significant (P>0.05) among
the calves receiving HC, MCMW and HW diets (Table 1). However, calves fed on HC diet
tended to ingest higher feed than those fed on MCMW and HW diets. Digestibility of DM
remained unchanged (P>0.05) in calves fed HC, MCMW, and HW diets (Table 1). However,
NDF digestibility differed significantly (P<0.05) among all the treatments. Concentration of
glucose did not differ (P>0.05) because of gradual replacement of corn grains by wheat grains in
the calves (Table 1). At 3, 6 and 9 hours post feeding, the highest value of glucose concentration
was found in calves fed HW diet. Different experimental diets didn’t influence the BUN, blood
cholesterol and creatinine concentrations at 3, 6 and 9 hours post feeding. Weight gain and final
weight in calves fed on different rations remained unaltered (P>0.05;Table 1).
DISCUSSION
The findings of the present study are in agreement with the results of Gulmez and
Turkmen (2007) who found non-significant results of DM intake in lactating cows when dietary
corn grains were gradually replaced by wheat grains. Krause and Combs (2003) found decreased
intake of DM in the lactating cows when the readily fermentable carbohydrate sources were used
in the diet. Reducing tendency in DMI might be attributed to the fact that wheat compared to
corn grains, is more rapidly fermented in the rumen. Khan et al. (2008) observed non-significant
results of DM, CP and NDF digestibilities in calves fed different cereal grains while in present
results NDF digestibility was lower in the calved receiving wheat grains diet. One possible
reason may be that in their study the NDF contents were only 15% while in our study it was
about 38% NDF. Another plausible explanation of significant effect of replacing corn by wheat
905
grains on NDF digestibility might be attributed to higher fermentable starch contents of wheat
grains. This might have reduced rumen pH through increased volatile fatty acid production.
Decreased rumen pH decreases cellulolytic bacterial count and increases amylolytic bacterial
population due to which fiber degradation is decreased. The findings of unaltered blood glucose,
BUN, cholesterol and creatinine concentrations are in line with those reported by other
researcher (Gozho and Mutsvangwa, 2008 ; Zhang et al., 2010). In the present study, slightly
decreased weight gain in calves fed wheat grains might be because of low feed intake. Khan et
al. (2007) also recorded higher weight gain in calves fed on corn grains than those fed on wheat
grains. They speculated that higher weight in corn receiving calves may be attributed to the
better rumen development because gradual fermentation of corn grains might have imparted
positive effects on rumen development which might have been lacking aspect in rumen of calves
fed wheat grains as wheat is more rapidly fermented.
REFERENCES
AOAC. 1990. Association of Official Analytical Chemists. Official Methods of Analysis, 15th
Ed. Arlington, VA, USA.
Bull, R. C., D.O.Everson, D.P. Olson, K.W. Kelly, S. Curtis and G.Tzou.1991. Concentration of
serum constitutents in cpld-stressed calves from heifers and inadequate protein and (or)
energy. J. Anim. Sci. 69:853-863.
Davies, H. L., T.F.Robinson, B.L. Roeder, M.E. Sharp, N.P.Johnston, A.C. Christensen and
G.B.Schaalje. 2007. Digestibility, nitrogen balance, and blood metabolites in Llama (Lama
glama) and alpaca (lama pacos) fed barley or barley alfalfa diets. Small Rumin. Res.73:1-7.
Gozho, G.N. and T. Mutsvangwa. 2008. Influence of carbohydrate source on ruminal
fermentation characteristics, performance and microbial protein synthesis in dairy cows. J.
Dairy Sci. 91,2726–2735.
Khan, M.A., H.J. Lee, W.S.Lee , H.S.Kim, S.B.Kim, K.S.Ki, S.J.Park, J.K.Ha and Y.J.
Choi.2007. Starch source evaluation in calf starter: I. Feed consumption, body weight gain,
structural growth and blood metabolites in Holstein calves. J. Dairy Sci. 90:5259–5268.
Khan, M.A., H.J.Lee, W.S.Lee, H.S. Kim, S.B.Kim, S.B. Park, K.S. Baek, J.K. Ha and
Y.J.Choi.2008. Starch source evaluation in calf starter: II. Ruminal parameters, rumen
development, nutrient digestibilities, and nitrogen utilization in Holstein calves. J. Dairy Sci.
91:1140–1149.
Krause, K.M and D.K.Combs. 2003. Effects of forage particle size, forage source and grain
fermentability on performance and ruminal pH in midlactation cows. J. Dairy Sci. 86:1382-
1397.
Meyer, H. H., A. Abdulkhaliq, S.L. Davis, J. Thompson, R. Nabioullin and N.E. Forsberg.1996.
Effects of the callipyge phenotype on serum creatinine, total cholesterol, low-density
lipoproteins, very-low-density lipoproteins, high-density lipoproteins, and triacylglycerol in
growing lambs. J. Anim. Sci. 74:1548-1552.
Orskov, E.R and C. Fraser.1975. The effect of processing of barley based supplements on rumen
pH, rate of digestion and voluntary intake in sheep. Br. J. Nutr. 34:493-500.
Overton, T.R., M.R.Cameron, J.P.Elliot, J.H. Clark and D.R.Nelson.1995. Ruminal fermentation
and passage of nutrients to the duodenum of lactating cows fed mixtures of corn and barley.
J. Dairy Sci. 78:1981-1998.
906
Shahzad, M.A, M.Sarwar and M.Nisa.2007. Nutrient intake, acid base status and growth
performance of growing buffalo male calves fed varying level of dietary cation anion
difference. Livest. Sci.111:136-143.
Van Soest, P.J., J.B.Robertson and B.A. Lewis.1991. Methods for dietary fiber, neutral detergent
fiber and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74: 3583-
3597.
Zhang,Y.Q., D.C. He and Q.X. Meng. 2010. Effect of a mixture of steam-flaked corn and
soybeans on health, growth, and selected blood metabolism of Holstein calves. J. Dairy Sci.
93:2271-2279.
Table 1. Effect of gradual replacing corn by wheat grains on nutrients intake,

digestibilities, blood metabolites and growth performance of calves
Experimental Diets
Items 1 SE
HC MCMW2 HW3
Nutrients intake, Kg/d
Dry matter 1.85 1.67 1.56 0.11
Crude protein 0.28 0.24 0.23 0.02
Neutral Detergent fiber 0.62 0.51 0.47 0.05
Acid Detergent fiber 0.46 0.37 0.34 0.04
Nutrients digestibilities,%
Dry matter 71.80 68.30 71.65 0.87
Crude protein 76.90 73.50 74.60 0.82
Neutral Detergent fiber 67.09a 59.14b 58.38b 1.30
Acid Detergent fiber 66.40a 59.20b 54.88b 1.26
Blood metabolites, mg/dL
3-Hour Post Feeding
Glucose 62.0 63.33 64.33 1.88
Blood Urea Nitrogen 16.50 15.66 16.53 0.57
Cholesterol 82.0 76.0 88.50 6.53
Creatinine 0.73 0.86 0.76 0.06
6-Hour Post Feeding
Glucose 57.00 57.67 59.00 1.66
Cholesterol 85.67 78.00 87.33 6.36
Creatinine 0.77 0.73 0.77 0.05
9-Hour Post Feeding
Glucose 52.66 52.0 54.33 1.52
Cholesterol 83.33 79.00 86.00 6.94
Creatinine 0.73 0.83 0.80 0.07
Growth performance, Kg
Initial Weight 57.25 54.75 57.00 1.40
Final Weight 91.25 87.25 88.50 2.32
Weight gain 34.00 32.50 31.50 1.28
Average Daily Gain 0.40 0.38 0.37 0.01
Gain/Feed ratio 0.18 0.19 0.19 0.02
Means in a row with different superscripts differ significantly (P < 0.05)
1
Diets HC (High Corn) contained 20% corn and 0% wheat; MCMW (Medium Corn Medium Wheat)
contained 10% Corn and 10% Wheat ; HW (High Wheat) contained 0% Corn and 20% Wheat.
907
Response and Ruminal Characteristics of Buffalo Bulls Fed Urea-molasses

Treated Wheat Straw Inoculated with Rumen Digesta
Muhammad SARWAR, Muhammad Aasif SHAHZAD*, Mahr-un-NISA and Nasir Ali
TAUQIR
Institute of Animal Nutrition and Feed Technology, University of Agriculture, Faisalabad-38040,

Pakistan
*Corresponding email: draasifshah@uaf.edu.pk
ABSTRACT
The study was aimed to examine the influence of urea-molasses treated wheat straw
inoculated with rumen digesta (RD) on nutrient digestibility, nitrogen (N) balance and ruminal
characteristics of ruminally cannulated buffalo bulls (400+20 kg) in a 4×4 Latin Square Design.
Four isocaloric and isonitrogenous diets were formulated. Experimental diets contained 60, 70 and
80% fermented wheat straw (FWS; 4% urea, 4% molasses and 20% RD incubated for 30 d) and
were denoted as FWS60, FWS70 and FWS80 diets, respectively. The control diet (C) contained
50% untreated wheat straw. Bulls were fed at ad libitum. The study lasted for twelve weeks. Dry
matter, crude protein and neutral detergent fiber intake and digestibilities increased in bulls fed
FWS60 diet compared to those fed C, FWS70 and FWS80 diets and similar observations were
noticed for N balance. Higher blood urea N was noticed in bulls fed FWS60 diet than those fed C,
FWS70 and FWS80 diets. Bulls fed FWS60 diet had higher ruminal NH3-N concentrations and
rumen pH at 3, 6 and 9 h post feeding than those fed C, FWS70, FWS8 and C diets. The study
outcome imply that urea molasses treated wheat straw ensiled with 20% RD for a period of 30
days can be included upto 60% in the diet of ruminants without any detrimental effects on rumen
characteristics, rather it improved nutrients intake, digestibilities and N balance.
Keywords: rumen digesta, wheat straw treatment, rumen characteristics
INTRODUCTION
Fibrous crop residue constitutes major part of ruminant feed in developing countries like
Pakistan. However, its utilization is hindered because of its high neutral detergent fiber and low
concentration of required nutrients (proteins and fermentable carbohydrates). High proportion of
indigestible fibrous stuff is considered responsible for its longer stay in the rumen which lowers
the intake and impedes the ruminant productivity (Sarwar et al., 2011). On the other hand, to
narrow down the gap between nutrient availability and demand, efforts are being made to improve
nutritive value of abundantly available fibrous feedstuff and to explore the non-conventional feed
resources for ruminant feeding (Sarwar et al., 2006). In this regard, slaughter house waste like
rumen digesta is considered an unwanted and is indeed a potential cause of water pollution as it
often remains untreated and enters local rivers and water sources. The anaerobic degradation of
waste water generates methane and carbon dioxide which leads to environmental pollution. These
contents can be a good source of water soluble vitamins, crude protein, minerals and
microorganisms. The rumen contents of slaughtered animals can be used as an inoculant for
ensiling urea treated wheat straw mixed with rapidly fermentable source of carbohydrates. In
above context, the present project was planned to examine the influence of varying level of urea-
molasses treated wheat straw ensiled with rumen digesta (RD) in cannulated buffalo bulls.
Four ruminally cannulated Nili-Ravi buffalo bulls (400+20 kg) were used in 4 × 4 Latin
Square Design to evaluate the influence of different levels of fermented wheat straw (FWS) on
ruminal characteristics, nutrient digestibility, nitrogen (N) balance and blood urea N (BUN). The
FWS was prepared by treating straw with urea and molasses (each at the rate of 4%) ensiled with
908
20% RD for a period of 30 days. Four isocaloric and isonitrogenous diets were formulated.
Experimental diets contained 60, 70 and 80% fermented wheat straw (FWS; 4% urea, 4%
molasses and 20%RD incubated for 30 d) and were denoted as FWS60, FWS70 and FWS80 diets,
respectively (Table 1). The control diet (C) contained 50% untreated wheat straw (Table 1). Bulls
were individually housed and were fed at ad libitum. Experiment lasted for twelve weeks; on each
rotation, first two and third week served as adaptation and collection periods, respectively. Dry
matter (DM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF)
intake were recorded. Nutrients digestibilities (DM, CP, NDF and ADF) were determined by
using total collection method (Shahzad et al., 2009). Ruminal samples were taken from four
different locations in the rumen at 3, 6, 9 and 12 h after morning feeding for determination of pH
and NH3-N. The BUN concentration was also determined (Bull et al.,1991). Feed offered, orts and
fecal samples were analyzed for DM,CP, NDF, ADF and ash contents (AOAC,1990). Fiber
fractions (NDF and ADF) were also analyzed by using procedure described by Van Soest et al.
(1991). Data were analyzed as 44 LSD using the GLM procedure of SAS (1988). The sum of
squares of the model was separated into animal and treatment effects. When treatment effects
were detected, means were separated by Duncan’s multiple range test (Steel and Torrie, 1984).
RESULTS AND DISCUESSION

Nutrients intake, digestibilities and N balance
Intake and digestibilities of nutrients (DM, CP, NDF and ADF) increased in bulls fed
FWS60 diet compared to those fed C, FWS70 and FWS80 diets (Table 2). However, DM
digestibility remained unaltered in bulls fed C and FWS80 diets. Similar trend was followed by
other nutrients (Table 2). The N balance was higher in bulls fed FWS60 diet than those fed C,
FWS70 and FWS80 diets. Higher blood urea N was noticed in bulls fed FWS60 diet compared to
those fed C, FWS70 and FWS80 diets (Table 2).
Increased DM intake (DMI) in bulls fed FWS60 diet might be attributed to breakage of
linkage between hemicellulose and lignin, making the hemicellulose fraction partially soluble that
is highly digestible by ruminal microbes (Dass et al., 2000). Similar findings have been reported
by Cross et al. (1979) who observed higher DMI in steers when WS was ensiled with broiler litter.
Higher DM digestibility by bulls fed on FWS60 diet might be attributed to a synchrony between
availability of carbon skeleton and N units which might have enhanced ruminal microbial
activities leading to increased DM degradation. Another plausible explanation might be that
during incubation, breakage of linkage between hemicellulose and lignin in FWS60 diet might
have made hemicellulose fraction partially soluble that is highly digestible by ruminal microbes
which resultantly increased DM digestibility (Dass et al., 2000). Furthermore, higher nutrient
digestibilities in bulls fed FWS60 diet might be because of better ruminal fermentation due to
adequate coupling of energy (carbon skeleton) and protein (nitrogen) contents which might have
not been maintained in bulls fed on FWS70 and FWS80 diets. Significant increase in N balance in
bulls fed FWS60 diet might be because of increased microbial multiplication and higher enzyme
production leading to better utilization of dietary protein contents. Similar observations have been
observed by Sarwar et al (2011) who reported high N balance in growing calves when FWS was
included in diet upto 35%. Plausible reason for higher blood urea N concentration in bulls fed
FWS60 diet might be attributed to higher ruminal ammonia N reflecting higher dry matter
degradation because of enhanced microbial fermentation. Strong association between ruminal
NH3–N and blood urea N concentrations is well documented (Javaid et al., 2011)
Ruminal characteristics (ruminal NH3-N & pH)
Ruminal NH3-N concentrations at 3, 6 and 9 h post feeding significantly differed in bulls fed
varying levels of FWS diet (Table 2). Higher ammonia N was noticed in bulls fed FWS60 diet at
3, 6 and 9 h post feeding. However, NH3-N didn’t differ at 12 h in bulls fed on experimental diets.
Similar trend was noticed for rumen pH. Rumen pH remained higher at 3, 6 and 9 h post feeding
in bulls fed FWS60 diet that those fed C, FWS70, FWS80 and C diets (Table 2).
909
High ruminal NH3-N in bulls fed FWS60 diet at 3, 6 and 9 h post feeding indicated a continual
release of NH3-N from FWS due to gradual release of fiber bound-N from urea treated straw.
Similar results were observed by Sarwar et al. (2004) who stated that urea-N was fixed in the
matrices of cell wall, when urea treated WS was ensiled with fermentable energy source, N was
released slowly in the rumen so that ruminal NH3-N concentration remained high even 9 h post-
parandially. High ruminal NH3-N concentration at 3 h for bulls fed FWS60 might reflect reduced
conversion of NH3-N into bacterial proteins due to rapid release of NH3-N by urea hydrolysis.
Ruminal pH reflects a balance between ruminal volatile fatty acids and NH3-N concentration (Mir
et al., 1980). Higher ruminal NH3-N concentration increased ruminal pH in bulls fed diets
containing FWS60 diet. Lower ruminal ammonia N in bulls fed on FWS70 and FWS80 diets
might be attributed to gradual decrease in readily fermentable feed components, when respective
proportion of FWS increased in experimental diets.
ACKNOWLEDGEMENT
Authors highly appreciate the financial assistance provided by Pakistan Science Foundation
{(PSF Grant No/Project No. PSF/NSLP/P-AU (78)}, Islamabad without which execution of the
study might have not been possible.
REFERENCES
AOAC, 1990. Official Methods of Analysis15th Ed. Arlington, Virginia, USA.
Bull, R.C., D.O.Everson, D.P. Olson, K.W. Kelly, S. Curtis and G.Tzou.1991. Concentration of
serum constituents in cold stressed calves from heifers and inadequate protein and (or) energy.
J. Anim. Sci. 69: 853–863.
Cross, D. L., G. C. Skelly, C.S. Thompson and B.F. Jenny.1979. Efficiency of broiler litter silage
for beef steers. J. Anim. Sci. 47: 544-551.
Dass, R.S., U. R. Mehra and A.K, Verma. 2000. Nitrogen fixation and in situ dry matter and fibre
constituents disappearance of wheat straw treated with urea and boric acid in Murrah
buffaloes. Asian-Aust. J. Anim. Sci. 13:1133-1136.
Javaid, A., M.A. Shahzad, M.Nisa and M.Sarwar.2011. Ruminal dynamics of ad libitum feeding
in buffalo bulls receiving different level of rumen degradable protein. Lives. Sci.135: 98–102.
Mir, F. A., A. Afzal and A. H. Gillani. 1980. Effect of urea nitrogen on rumen microflora in
buffalo bulls fed sugarcane pith as a roughage source. J. Anim. Sci. 2:35-42.
Sarwar, M., J. L. Firkins and M. L. Estridge. 1992. Effect of varying forage and concentrate
carbohydrates on nutrient digestibilities and milk production by dairy cows. J. Dairy Sci.
75:1533-1541.
Sarwar, M., M.A.Shahzad, M.Nisa, D. Afzal, M. Sharif and H.A.Saddiqi.2011. Feeding value of
urea molasses treated wheat straw ensiled with fresh cattle manure for growing crossbred
cattle calves. Trop. Anim. Health and Prod. 43:543-548.
Sarwar, M., M.A., Khan and M.Nisa. 2004. Effect of organic acid on fermentable carbohydrates
on digestibility and nitrogen utilization of urea treated wheat straw in buffalo bulls. Aust. J.
Agric. Res. 55: 229-233.
Shahzad, M. A., M. Sarwar, M. Aqile, M. Nisa, K. Mahmood and M. S. Khan. 2009. Impact of
stage of maize fodder harvest on chemical composition, nutrient digestibilities and nitrogen
balance in buffalo bulls. Pak. J. Zool. 9:717-720.
Steel, R. G. D. and J. H. Torrie. 1984. Principles and Procedures of Statistics: A Biometrical
Approach’. 2nd Ed. (McGraw-Hill Book Company, New York, USA)
Van Soest, P.J., H.B.Robertson and B.A. Lewis.1991. Methods of dietary fiber, NDF and non-
starch polysaccharides in relation to animal material. J. Dairy Sci. 74: 3583–3597.
910
Table 1. Ingredient and chemical composition (%) of experimental rations.

Experimental diets1
Ingredients
C FWS 60 FWS 70 FWS 80
Treated wheat straw 0.0 60.0 70.0 80.0
Untreated wheat straw 50.0 0.0 0.0 0.0
Maize 4.0 10.0 3.0 3.0
Wheat bran 5.0 16.0 13.0 3.0
Rice polishing 15.0 5.0 5.0 5.0
Cotton seed cake 5.0 4.0 4.0 3.0
Maize gluten meal 60% 4.5 0.0 0.0 1.0
Canola meal 10.0 0.0 0.0 0.0
Cane molasses 4.0 4.0 4.0 4.0
Mineral mixer 1.5 0.5 0.5 0.5
Di-calcium phosphate 1.0 0.5 0.5 0.5
Total 100 100 100 100
Chemical Composition
Crude protein, % 11.98 11.94 11.97 11.94
Metabolizable energy, Kcal/kg 2.44 2.5 2.48 2.47
Neutral detergent fiber, % 49.02 43.43 44.49 45.04
Acid detergent fiber,% 29.93 22.93 23.03 24.26
Ash % 10.08 11.06 11.58 12.15
1
Control diet contained 50% untreated wheat straw and 50% concentrate while WS60, FWS70 and FWS80 diets
contained 60, 70 and 80% FWS, respectively. FWS was treated with urea (4%), molasses (4%) and rumen digesta
(20%), respectively
911
Table 2. Influence of varying levels of fermented wheat straw on nutrients intake, digestibilities,
nitrogen balance and ruminal characteristics in bulls.
Experimental diets1
Parameters C FWS 60 FWS 70 FWS 80 SE
Nutrients intake, Kg/d
Dry matter 7.81b 9.52a 8.65b 8.15c 0.43
b a b b
Crude protein 0.95 1.14 1.03 0.97 0.12
b a b b
Neutral Detergent fiber 3.83 4.13 3.85 3.67 0.34
Acid Detergent fiber 2.34a 2.18b 1.99ab 1.98ab 0.35
Nutrients digestibilities, %
Dry matter 45.75c 55.94a 50.71b 48.25bc 0.94
b a b b
Crude protein 65.71 72.32 66.34 65.10 0.69
Neutral Detergent fiber 40.85c 51.56a 45.47b 42.01c 0.387
Acid Detergent fiber 40.85c 51.56a 45.47b 42.01c 0.387
Nitrogen balance and blood urea N
Nitrogen balance, g/d 13.3c 32.9a 21.04b 19.25bc 2.14
b a b b
Blood urea N, mg/dl 20.67 23.21 19.79 19.67 1.96
Ruminal characteristics
3-hr
Rumen ammonia N, mg/dl 17.37b 21.63a 18.65b 17.65b 1.54
b a b b
Rumen pH 6.36 6.56 6.32 6.37 0.02
6-hr
b
Rumen ammonia N, mg/dl 16.31 19.66a 17.01b 16.53b 0.57
Rumen pH 6.31b 6.54a 6.37b 6.32b 0.01
9-hr
Rumen ammonia N, mg/dl 15.52b 18.23a 16.11b 15.32b 0.75
Rumen pH 6.42b 6.49a 6.41b 6.44b 0.01
12-hr
b
Rumen ammonia N, mg/dl 15.32 18.01a 15.79b 15.32b 0.94
Rumen pH 6.47 6.45 6.35 6.31 0.01
Means bearing different superscripts in the same row differ significantly (P<0.05)
1
Control diet contained 50% untreated wheat straw and 50% concentrate while WS60, FWS70 and FWS80 diets
contained 60, 70 and 80% FWS, respectively. FWS was treated with urea (4%), molasses (4%) and rumen digesta
(20%), respectively
912
Response of Sahiwal Heifers Receiving Maize Fodder with Supplementation of

Urea Molasses Block
Nasir Ali TAUQIR1, Muhammad Aasif SHAHZAD*1, Makdum Abdul JABBAR2,

Muhammad SARWAR1, Mahr-un-NISA1,Fayyaz AHMAD3 and Iftikhar AHMAD3
1
Pakistan
2
University of Veterinary and Animal Sciences, Lahore, Pakistan
3
Buffalo Research Institute, Pattoki District Kasur, Pakistan
*Correspondence: aasifshah9@hotmail.com
ABSTRACT
A 73 day (d) study was undertaken using 20 Sahiwal heifers to investigate the effect of
supplementing maize fodder with urea molasses block (UMB) on their feed intake and weight
gain in a complete randomized design. Four feeding regimes were randomly allotted to four
groups of animals, 5 animals in each. Animals in group A were offered maize fodder ad libitum
without UMB (control), while the animals in Group B, C and D were offered green fodder at 100,
75 and 50% of their requirement with ad libitum UMB, respectively. Dry matter (DM) intake was
higher in animals fed maize fodder ad lib. However, UMB supplementation significantly (p<0.05)
increased feed and water intake in Sahiwal heifers, however, DM digestibility remained unaltered.
Daily weight gain was significantly (p<0.05) higher for heifers in Group B than those in Groups A
and C. Blood urea nitrogen concentration was higher after 3 h of feeding in animals fed the UMB
and remained elevated up to 9 h post feeding. The study revealed that UMB supplementation with
maize fodder significantly (p<0.05) increased intake of the green fodder and growth rate in
Sahiwal heifers.
Keywords: Maize fodder, UMB, nutrient utilization, sahiwal heifer
INTRODUCTION
In developing countries ruminant are commonly fed low quality crop residues such as
wheat straw, rice straw, maize and sorghum stovers etc. Maize and sorghum are available as green
fodder crops for feeding during the summer but these are generally deficient in protein, minerals
and vitamins. These nutrient deficiencies are exacerbated during the dry season when little
available forage is low in quality and results in weight loss, low birth weights, lowered resistance
to disease, and reduced animal performance (Onwuka et al., 1989). This calls for a reasonable
level of supplementation, with particular emphasis on the energy, protein and minerals contents to
maintain animals during this period. Molasses and urea are known to contain available energy and
nitrogen, respectively, and are being used in livestock feeding (Preston and Leng, 1990).
Supplementation of mixture of urea and molasses can increase the intake of poor quality forages
up to 40% (Badurden et al., 1994) and improve energy balance of the animals (Srinivas & Gupta,
1997). However, limited information is available in the literature on the effect of urea molasses
block (UMB) feeding on the nutrient intake and performance of heifers especially during dry
season feeding. Therefore, the present study was carried out to explore the effect of
supplementation of UMB with green fodder on feed intake, nutrient digestion and growth of
Sahiwal heifers during fodder scarcity periods.

For the present study 20 Sahiwal heifers of similar age (18-20 months) and body weight
(100±15 kg) were randomly divided into four groups with five animals in each group. Animals in
group A were offered green fodder ad libitum without UMB (control), while the animals in

913
Groups B, C and D were offered green fodder at 100, 75 and 50% of their requirement with ad
libitum UMB. The composition of the urea molasses blocks is given in Table1. The DM, Crude
protein (CP), Metabolizable energy, neutral detergent fiber and ash contents of maize fodder were
22%, 9.0%,1.8 Mcal/Kg, 65% and 8.6%, respectively. Feed intake was recorded and samples of
feed and refusals were collected daily and analyzed for proximate analysis (AOAC, 1995). The
animals were weighed before morning feeding at the start of study and at fortnightly intervals
thereafter.
At the end of feeding study, a digestibility trial involving total collection of urine and feces
was undertaken to determine the digestibility of nutrients in the diet according to procedure
described by (Williams et al., 1984). Feed, orts and fecal samples were analyzed for dry matter
(DM), nitrogen (N) and organic matter (OM) using their respective methods described by AOAC
(1990), while NDF, ADF, and ADL were determined by the methods of Van Soest et al. (1991).
Blood samples were collected on first day of digestibility trial at 3, 6, 9 and 12 h post feeding to
determine blood urea nitrogen concentration (Tabacco et al., 1979).
Data obtained during the course of study was statistically analyzed using complete
randomized design through Minitab computer software (Stanisiewski et al., 1994). Duncan’s
Multiple Range Test was applied to compare the difference between the means in case of
significant differences
(Steel et al., 1997).

Feed intake
Average dry matter intake (DMI) was 5.87, 6.36, 3.91 and 2.99 kg/d by animals in Groups
A, B, C and D, respectively (Table 2). The DMI was significantly (p<0.05) higher (8.4%) in
heifers fed UMB along with green fodder ad lib as compared to other groups. Intake of CP also
showed a similar trend. It may be due to enhanced DMI because of supplemented UMB. Water
intake was significantly higher in animals fed UMB as compared to control. The trend of DMI
was also curvilinear (p<0.01) among all treatments (Table 2). Supplementary NPN through UBM
might have enhanced the fibrolytic activity of rumen microbes that improved the clearance of
digesta from the rumen and ultimately enhanced feed intake. Knox and Steel (1999) reported that
sheep offered the urea-supplemented diet ate significantly more feed than those offered the non-
urea diet.
Intake of UMB
Intake of UMB was 0.958, 1.10 and 1.22 kg/d in animals of group B, C and D (Table 2).
The consumption of UMB increased significantly (p<0.05) as the supply of green fodder was
reduced. The UMB intake also showed curvilinear (p<0.01) trend among all treatments (Table 2).
Daily consumption of UMB in calves varying from 150-300 g/d has been previously reported by
Garcia and Restrepo (1995). Such variation in UMB intake in animals has been related to
composition and texture of blocks and the eating pattern of the animals (Habib et al., 1991).
Similarly, Faizi et al. (2004) reported that calves consumed 496g UMB daily when they were fed
maize stover fodder.
Nutrient digestibility and water consumption
Digestibility of DM did not exhibit any treatment effect (Table 2). The CP digestibility
was similar for animals in groups A, C and D. While it was the highest by animals in group B
compared to those in other three groups. There was a linear decrease in CP digestibility with the
decrease in fodder supply. It has been well established that the digestibility of DM decreases as
feed intake increases or vice versa Van Soest (1994). The results of the present study are also in
agreement with that of Srinivas and Gupta (1997) who reported that digestibility of neither
proximate principles nor cell wall constituents was deviated on supplementation of UMB licks.
Water consumption by animals in group A, B C and D was 4.20, 6.80, 7.50 and 8.20 L/d,
respectively (Table 2). Water intake increased significantly (p>0.05) when animals were given
access to UMB. Animals receiving UMB consumed more water compared to the control and
914
there was a positive correlation between dietary nitrogen intake and water consumption as
reported by Faizi et al. (2004).
Weight gain
The heifers in group B had significantly (p<0.05) higher weight gain (0.77kg/d) followed
by those in groups A (0.58 kg/d), C (0.55 kg/d) and D (0.37 kg/d), while there was non-significant
difference of weight gain of animals in groups A and C. In groups C and D fodder was reduced to
25 and 50%, but the intake of UMB was not proportionally increased. Hence, the weight gain in
groups C and D was lower than groups A and B. The results of current study have supported the
earlier findings of Iqbal et al. (1994) and Rafique et al. (2000) who found that supplementation of
basal feed with UMB increased the growth rate in calves. However, the growth of calves in the
present study was higher than that reported by Iqbal et al. (1994) in buffalo calves (545 g/d versus
770 g/d). It may be attributed to the better quality of basal diet.
Feed conversion efficiency
Feed conversion efficiency (FCE) by animals in groups A, B, C and D were 10.16, 8.27,
7.14 and 8.07 kg for every kg gain in weight, respectively during the course of study (Table-3).
The heifers in group A had significantly (P<0.05) higher FCE than those fed B,C and D while
there was non-significant difference in FCE of animals in groups B,C and D. Although these
results were non-significant but better FCE was observed in animals of group B having 50%
fodder with UMB. The results of current study have supported the earlier findings of Iqbal et al.
(1994) and Rafique et al. (2000) who found that supplementation of basal feed with UMB
increased the growth rate in calves and ultimately better FCE.
CONCLUSION
Supplementation of UMB not only accelerated feed intake but growth in Sahiwal heifers
too. So, UMB could be used in fodder scarcity periods or with low quality fodders. But UMB is
not the substitute of fodder in principle.
REFERENCES
AOAC.1995.Official Methods of Analysis. 16th edn. Association of Official Analytical Chemists,
Washington, D.C.
AOAC.1990. Official Methods of Analysis. 15th Ed. Association of Official Analytical Chemists,
Badurdeen, A.M., M.N.M.Ibrahim and S. S. E. Ranawana.1994. Methods to improve utilization of
rice straw. Effect of urea ammonia treatment and urea molasses block supplementation on
intake, digestibility, rumen and blood parameters. Asian Aust. J. Anim. Sci. 7: 363-372.
Faizi, M.U., M. M. Siddiqui and G. Habib.2004. Effect of urea molasses block supplementation
on nutrient digestibility and intake of ammoniated maize stovers in cow calves. Pak. Vet. J.
24: 76-79.
Gracia, L.D and J. I. R. Restrepo.1995. Multi-Nutrient Block Handbook. (Better Farming Series
45), FAO, Rome. 28 p.
Habib, G.S., B. A. Shah, G. Wahidullah and J. Ghafranullah.1991. The importance of urea
molasses blocks as a bypass protein in animal production. The situation in Pakistan. In:
Isotope and Related Techniques in Animal Production and Health. IAEA, Vienna, Austria.
pp. 133-144.
Iqbal, T., I. Haq and M. A. Jabbar.1994. Comparative efficiency of urea molasses blocks and urea
treated wheat straw in fattening ration of male buffalo calves. 15th Annual Report, LPRI,
Bahadurnagar, Okara. 64 p.
Knox, M.R and J. W. Steel.1999. The effect of urea supplementation on production and
parasitological responses of sheep infected with Haemonchus contortus and
Trichostrongylus colubriformis. Vet. Parasit.83: 123-35.
915
Onwuka, C.F.I., A. O. Akinsoyinu and O. O. Tewe.1989. Feed value of some Nigerian browse
species: Chemical composition and in vitro digestibility of leaves. E. Afr. Agric. For. J. 54:
157-163.
Preston, T.R and R.A. Leng.1987. Matching Ruminant Production Systems with Available
Resources in the Tropics and Sub-Tropics. Penambul Books, Armidale.
Rafique, K., M. Mustafa, M. A. Awal and M. M. Hussain. 2000. Effect of medicated licks on the
performance of indigenous dairy cows of Bangladesh. Asian Aust. J. Anim. Sci. 1: 774-780
Srinivas, B and B. N. Gupta.1997. Urea molasses mineral block lick supplementation for milk
production in crossbred cows. Asian Aust. J. Anim. Sci. 19: 47-53.
Stanisiewski, E.P., J. F. McAllister, K. A. Ash, V. N. Taylor, D. D. Kratzer and J.W. Lauderdale.
1994. Minitab (release 9.2) Minitab Inc., Clecome Limited USA.
Steel, R. G. D., J. H. Torrie and D. A. Dickey.1997. Principles and Procedures of Statistics. A
Biochemical Approach 3rd edn. McGraw Hill Book Co. Inc., New York.
Tabacco, A., F. Meiattini, E. Moda and P. Tarli.1979. Simplified enzymatic/ colorimetric serum
urea nitrogen determination. Clin Chem. 25: 336-339.
Van-Soest, P.J., J. B. Robertson and B. A. Lewis.1991. Methods for dietary fiber, neutral
detergent fiber and non-starch polysaccharides in relation to animal nutrition. J. Dairy
Sci.74: 3583-97.
Williams, P. E. V., G. M. Innes, A. Brewer.1984. Ammonia treatment of straws via the hydrolysis
of urea. 1. Additions of soybean (urease), sodium hydroxide and molasses; effects on the
digestibility of urea treated straw. Anim. Feed Sci. and Technol. 11: 115-124.
Table 1. Ingredients and chemical composition of UMB.

Ingredients Percentage
Rice Polishing 14.0
Canola Meal 15.0
Maize gluten 30% 15.0
Cane Molasses 34.0
Urea 8.0
Calcium sulfate 6.0
Mineral Mixture 5.0
Calcium Oxide 2.0
Table Salt 1.0
Chemical composition, %
Dry matter 86.20
Crude Protein 35.82
Metabolizable Energy, Mcal/ kg 2.27
Neutral detergent fiber 14.55
916
Table 2. Nutrient intake, digestibilities, nitrogen balance and growth performance of Sahiwal
heifers fed different feeding regimes with or without UMB.
PARAMETERS Treatments SEM L Q C

A B C D
Nutrients intake
Fodder intake, Kg/d 30.75a 33.32a 20.46b 15.68c 1.70 0.00 0.00 0.00
UMB intake, Kg/d 0.00c 0.96b 1.10ab 1.22a 0.11 0.00 0.00 0.00
b a c d
Total Dry matter intake, Kg/d 5.87 6.36 3.91 2.99 0.32 0.00 0.00 0.00
d a b c
Crude protein intake, g/d 601.40 991.80 812.20 739.80 33.43 0.01 0.00 0.00
Water intake, lit./d 4.20b 6.80a 7.50a 8.20a 0.39 0.00 0.02 0.28
Nutrients digestibilities, %
Dry matter 61.65 62.00 61.65 63.35 0.28 0.05 0.20 0.25
ab a ab b
Crude protein 77.06 79.60 77.20 76.50 0.41 0.20 0.03 0.04
Nitrogen balance and blood urea nitrogen
Fecal N, g/d 31.36b 37.86a 31.68b 28.16b 1.00 0.02 0.00 0.02
Urine N, g/d 66.00c 110.00a 97.60b 90.80b 4.01 0.00 0.00 0.00
N balance, g/d -1.14b 10.83a 0.67b -0.59b 1.34 0.25 0.00 0.00
BUN at 3-hr, mg/dL 13.48d 36.48c 42.00b 49.00a 3.06 0.00 0.00 0.00
BUN at 6-hr, mg/Dl 13.00d 28.54c 38.00b 49.50a 3.07 0.00 0.00 0.00
BUN at 9-hr, mg/dL 12.00d 25.50c 45.50b 51.50a 3.62 0.00 0.00 0.00
Weight gain and feed efficiency
Daily gain, Kg/d 0.58b 0.77a 0.55b 0.37c 0.03 0.00 0.00 0.00
b a b c
Total gain, Kg/d 42.60 56.20 40.00 27.20 2.47 0.00 0.00 0.00
a b b b
FCE 10.16 8.27 7.14 8.07 0.31 0.00 0.00 0.48
Values within rows with varying superscripts are significantly difference (P<0.05)
917
Performance of Nili Ravi Buffalo Calves Fed Urea-Corn Steep Liquor Treated
Corn Cobs
Nasir Ali TAUQIR, Muhammad Aasif SHAHZAD*, Yasir MAHMOOD, Muhammad

SHARIF, Mahr-un-NISA and Muhammad SARWAR
Institute of Animal Nutrition and Feed Technology, University of Agriculture, Faisalabad-38040,

Pakistan
*Corresponding: draasifshah@uaf.edu.pk
ABSTRACT
The study was aimed to examine the influence of urea corn steep liquor (CSL) treated corn
cobs feeding on nutrients intake, digestibilities, nitrogen (N) balance and growth performance of
growing male buffalo calves. Twenty calves of similar age (365 ± 15days) and body weight
(215±5 Kg) were divided into four groups, five calves in each group, according to completely
randomized design. Four isocaloric and isonitrogenous rations were formulated. Control
ration contained 30% urea treated corncobs ensiled without CSL while rest of the 70% was
concentrate. Other three rations contained 30, 40 and 50% urea treated corncobs ensiled with CSL
and the rest of the proportion was concentrate. Study lasted for 100 days including first 10 days as
adaptation period. Higher dry matter (DM) intake was observed in calves fed 30 and 40% urea
treated corn cobs ensiled with or without CSL. Similar results were noticed for crude
protein and neutral detergent fiber (NDF). Blood urea N was significantly higher in
buffalo claves received urea and corn steep liquor treated rations as compared to
urea treated ration only. The highest daily weight gain was observed in buffalo calves fed
rations containing 70% urea - CSL treated corncobs as compared to those fed other three rations.
Rate of disappearance of DM and NDF decreased linearly with increasing levels of urea CSL
treated corncobs in experimental rations. It was concluded that urea-CSL treated corn cobs can
replace concentrate up to 50% in the ration of growing male buffalo calves, without any harmful
effects on their health and performance.
Keywords: Corn-cobs, CSL treatment, nutrient utilization, buffalo calves
INTRODUCTION
Crop residues are the major feed resources for ruminants in South East Asia and other
developing countries. But crop residues have poor nutritive value (low cp, high lignin content),
characterized by poor digestibility and hence their intake is also low (Sarwar et al., 1994).
Replacement of concentrates with cheaper agro industrial by-products can increase profitability
and reduce demand for cereal grain. Out of crop residues, corncobs are of low feeding value, high
lignifications, low fermentable carbohydrates, low protein contents and minerals imbalance
(Sarwar et al., 2004).
By the method of ammoniation feeding value of crop residues is increased by adding up of
nitrogen (N) and swelling solubilization of hemi cellulose fraction (Saenger et al., 1982). For
ammonia fixation in fibrous crop residues corn steep liquor (CSL) was successfully used (Sarwar
et al., 2003). Corn steep liquor not only contains soluble carbohydrates which improve
fermentation but its acidic pH can also help in fixing the excessive ammonia.
As compared to wheat straws, corn cob have porous structure, urea CSL treatment of corn
cobs could be more useful in enhancing its nutritive value. Hence the current experiment was
conducted to explore nutritive value of corncobs ensiled with urea-CSL for Nili Ravi buffalo
calves.

918
MATERIAL AND METHOD

The study was conducted in two phases; one was in situ nutritional evaluation trial while
the second was performance evaluation of urea CSL treated corncobs in Nili-Ravi buffalo calves.
Two ruminally cannulated Nili Ravi buffalo bulls were fed urea–corn steep liquor treated corn
cobs @ 1.5% of their body weight. Nylon bags measuring 10x23cm, with pore size of 50µm, were
used to evaluate the rate and extent of DM and NDF disappearance. For each time point, 5gram
sample of rations containing Urea or Uea plus CSL treated corncobs was weighed into bags
separately. These bags were exposed to ruminal fermentation for 1, 2, 4, 6, 10, 16, 24, 36, 48 and
96 hours. Rate and extent disappearance and lag time were determined for each incubation period
individually according to Mertens and Loften (1980).
In the second phase crushed Corncobs were ensiled in bulk with 4% urea and 5% CSL
moisture content was maintained at 50% of corncobs DM. Twenty male buffalo calves of similar
age (I year ±15 days) were divided into four groups (five calves in each group) according to
completely randomized design. Four iso-caloric and iso-nitrogenous diets were formulated using
NRC (2001) standards for energy and protein. Control ration contained 30% urea treated corncobs
ensiled without CSL along with 70% concentrate. Other three rations were containing 30, 40 and
50% urea treated corncobs ensiled with CSL. The rest of the proportion of rations was based on
concentrate. Animals were fed for 100 days individually, weighed at start of the experiment and
fortnight there after. Feed offered and refusals were recorded on daily basis. During last week of
the study, a digestibility trail was conducted using acid insoluble ash as digestibility marker (Van
Keulen and Young, 1977) Data was analyzed according to Completely Randomized Design (Steel
et al., 1997).
RESULTS
Lag time was the lowest (p<0.05) in buffalo calves fed UCSLT30 ration followed by those
fed UT30, UCSLT40 and was the highest (p<0.05) in buffalo calves fed UCSLT50 rations
respectively. However, the results posed curvilinear response for DM lag time against increasing
levels of urea CSL treated corncobs in the diets of buffalo calves. Rate of DM disappearance did
not show any treatment effect while rate and extent of NDF digestion linearly decreased by
increasing urea-corn steep liquor treated corncobs level (Table 1).
Dry matter intake (DMI) was significantly higher in buffalo calves fed UT30, UCSLT30
and UCSLT40 as compared to those fed UCSLT50 ration. However, the results were similar in
buffalo calves fed UT30, UCSLT30 and UCSLT40 rations, respectively. Intake of CP and NDF
represented linear and quadratic trends across all treatments. Highest (p<0.05) DM digestibilities
were observed in buffalo calves fed UCSLT 30 followed by those fed UCSLT 40 and the lowest
(p<0.05) was observed in those fed UCSLT 50 ration. Dry matter, CP, NDF and ADF
digestibilities were decreased in buffalo calves fed all the experimental rations in response to
increasing urea-corn steep liquor level in their diets. The overall trend was curvilinear in response
to urea CSL treated corncobs inclusion. Similar trend was also observed in CP, NDF and ADF
digestibilities. Highest (16.1mg /dL) BUN was observed in buffalo calves fed UCSLT50 and was
the lowest (13.2mg /dL) in those fed UCSLT30 ration. A quadratic increase in BUN was observed
in response to increasing urea CSL treated corncobs in ratios. Blood Glucose level remained
unaltered across all the treatments (Table 2)
Significantly (p<0.05) higher weight gain (116.8kg) was observed in buffalo calves fed
UCSLT 30 ration followed by those fed UCSLT 40 and UCSLT 50 rations respectively.
However, the results were similar across those fed UCSLT 30 and UT30, respectively. The results
of weight gain represented linear effect in response to increasing urea CSL treated corncobs in the
rations of buffalo calves. However, feed conversion efficiency did not show any treatment effect
(Table 2).
919
DISCUSSION
Highest lag time was observed in UCSLT 50 which might be due to more fiber portion in
the diet. The results were supported by the findings of Nisa, et al. (2004a) who reported that this
might be due to reduced forage to concentrate ration. The lowest digestibility and extent of
digestion was observed in UCSLT50 ration as compared to UCSLT30 ration that might be due
increasing fibrous portion and decreasing concentrate in the diet. Orden et al. (2001) reported that
use of urea treated ration considerably improved the extent and rate of NDF degradability. This
may be attributed to alterations in cell wall structure mainly to solubility of hemicellulose
(Ibrahim et al., 1989).
Intake of DM and digestible DM was decreased linearly with increasing urea CSL treated
corncobs levels in the rations. Present results have supported the findings of Johri et al. (1982) and
Singh and Kishan (1994) who accomplished that DMI was low due to slow permission of feed
from the rumen and passage through the digestive tract. Dry matter and fiber digestibility linearly
decreased in response to increasing levels of urea-CSL treated corncobs in the rations of buffalo
calves. These results have supported the findings of Rath et al. (2001). Similar trend of crude
protein digestibility was observed due to closely bound N in the ammoniated straw (Sundstol,
1984; Hvelpund. 1989). Jordan et al. (1983) explained that BUN might be increased due high
NPN levels in the diets. Daily weight gain was improved significantly in buffalo calves fed urea-
CSL treated corncobs rations as compared to those fed ration containing corncobs treated with
only urea. Sarwar et al. (1994) explained that it might be due to accessibility of more energy
intake and constant accessibility of NH3 at ruminal level.
REFERENCES
Ibrahim, M. N. M., S. Tamminga and G. Zemmelink. 1989. Effect of urea treatment on rumen
degradation characteristics of rice straw. Anim. Feed Sci. Technol. 24: 83.
Johri, C. B., S. K. Ranjhan and N. N. Pathak. 1982. Effect of different levels of molasses
substitution on utilization of urea impregnated wheat straw by male buffalo calves. Indian J.
Anim. Sci. 52:12.
Jordan, E. R., T. E. Chapman, D. W. Holtan and L. V. Swanson. 1983. Relationship of dietary
crude protein to composition of uterine secretions and blood in high-producing dairy cows.
J. Dairy Sci. 66:1854-1862.
Mertens, D. R. and J. R. Loften. 1980. The effect of starch on forage fiber digestion kinetics in
vitro. J. Dairy Sci., 63: 1437.
National Research Council. 2001.Nutrient Requirements of Dairy Cattle. 6thRev. Ed. National
Academy of Sciences, Washington, D.C.
Nisa, M., M. Sarwar and M. A. Khan. 2004a. Influence of ad libitum feeding of urea treated
wheat straw with or without corn steep liquor on intake, in situ digestion kinetics, nitrogen
metabolism and nutrient digestion in ruminally cannulated buffalo bulls. Austr. J. Agric.
Reasearch. 1:229.
Orden, E. R., K. Yamaki, T. Ichnohe and T. Fujihara, 2001. Feeding value of ammoniated rice
straw supplemented with rice bran in sheep: II In situ rumen degradation of untreated and
ammonia treated rice straw. Asian-Aust. J. Anim. Sci. 13: 906.
Rath, S., A. K. Verma, P. Singh, R. S. Dass and U. R. Mehra. 2001. Performance of growing
lambs fed urea ammoniated and urea supplemented wheat straw based diets. Asian-Austral.
J. Anim. Sci. 14:1078.
Saenger, P. F., R. P. Lemenager and K. S. Hendrix, 1982. Anhydrous ammonia treated of corn
stover and its effects on digestibility, intake and performance of beef cattle. J. Anim. Sci.,
54: 419.
Sarwar, M., M. A. Iqbal, C. S. Ali and T. Khaliq. 1994. Growth performance of buffalo male
calves as affected by using cowpeas and soybean seeds as a source of urease during urea
treated wheat straw ensiling process. Egyptian J. Anim.
920
Sarwar, M., M. A. Khan and M. Nisa, 2003. Effect of urea treated wheat straw ensiled with
organic acids or fermentable carbohydrates on ruminal parameters, digestion kinetics,
digestibility and nitrogen metabolism in Nili Ravi Buffalo bulls fed restricted diets. Asian-
Aust. J. Anim. Sci.
Sarwar, M., M. A. Khan and M. Nisa, 2004. Effect of organic acids or fermentable carbohydrates
on nitrogen fixation and chemical composition of urea treated wheat straw. Asian-Aust. J.
Anim. Sci. 1: 98.
Singh, P. and J. Kishan. 1994. Effect of mode of urea supplementation on nutrient utilization
from straw based diet by buffalo calves. Indian J. Anim. Sci.64: 163.
Steel, R. G. D., J. H. Torrie and D. A. Dickey. 1997. Principles and procedures of statistics. a
biometric approach 3rd Ed. McGraw Hill Book Co., New York, USA.
Sundstol, F. 1981. Methods for treatment of low quality roughages. In: J.A. Kategile, A.N. said
and F. Sundstol (Ed), utilization of low quality roughages in Africa. Aun-Agri. Dev. Report
1, Aas, Norway. Pp. 61-80.
Van Keulen, J. and B.A. Young. 1977. Evaluation of acid-insoluble ash as a natural marker in
ruminant digestibility studies J. Anim. Sci. 44:282-287.
921
Table 1. Effect of urea-corn steep liquor treated corn cobs on in- situ dry matter (DM) and Neutral Detergent Fiber (NDF) digestion
Diets1 Probabilities2
Items SE
UT 30 UCSLT30 UCSLT40 UCSLT50 L Q
Dry matter Lag time (h) 1.56b 1.46d 1.52c 1.63a 0.003 * *
Rate of DM Disappearance (%/ h) 4.525 4.625 4.6 4.55 0.029 NS NS
DM Digestibility (%) 63c 70.97a 66.10b 62.4c 0.371 * NS
Extent of DM Digestion (%) 70.86c 78.83a 73.95b 69.10d 0.098 * NS

922
kinetics
NDF Lag time (h) 1.81b 1.73c 1.82b 1.92a 0.01 * NS
Rate of NDF Disappearance (%/ h) 4.63a 4.41b 4.33b 4.27b 0.045 * NS
NDF Digestibility (%) 54.625b 60.22a 54.74b 47.20c 0.282 * NS

Extent of NDF Digestion (%) 61.30c 68.34a 62.20b 55.10d 0.21 * NS
1
UT 30 and UCSLT 40 refer to diets having 30 and 40% urea treated corn cobs and urea-corn steep liquor treated corn cobs, respectively.
SE= standard error. 2Digestibility and extent of digestion was determined after 48 and 96 hour of ruminal incubation. abcdMeans in same
row sharing different superscripts differ significantly (P<0.05). NS= non-significant (P>0.05) and *= significant (P<0.05).
Table 2. Effect of urea-corn steep liquor treated corn cobs on nutrients intake, digestibilities,BUN, and weight gain in buffalo bulls1UT
30 represents the diet having 30% urea treated corn cobs while UCSLT 30, UCSLT 40 and UCSLT 50 refer to diets having 30, 40 and
Diets1 Probabilities2
Intake (Kg/ day) SE
UT 30 UCSLT 30 UCSLT 40 UCSLT 50 L Q
Dry Matter 6.3ab 6.6a 6.2ab 6.1b 0.06 NS NS
Crude Protein 0.92b 0.97a 0.91b 0.89b 0.007 * *
Neutral Detergent Fiber 2.9b 3.1ab 3.1ab 3.2a 0.028 * *
Acid Detergent Fiber 1.6c 1.6c 1.8b 1.9a 0.013 * NS
Dry Matter Digestibility 65.3bc 72.3a 67.1b 63.6c 0.248 * *
923
Crude Protein Digestibility 75.8b 81.4a 74.1bc 71.8c 0.392 * *
Neutral Detergent Fiber Digestibility 55.1b 60.4a 56.7b 48.3c 0.284 * *
Acid Detergent Fiber Digestibility 42.6b 49.2a 43.1b 40.3b 0.451 * *
Daily Weight Gain (Kg) 1.1ab 1.21a 1.07b 1.02b 0.02 * NS
Feed Conversion Efficiency 5.7 5.4 5.8 6.01 0.113 NS NS
Blood Urea Nitrogen, mg/dL 15.3a 13.2b 15.7a 16.1a 0.195 NS *
Blood Glucose, mg/dL 88.2 99.5 86.4 87.8 3.716 NS NS
50% urea-corn steep liquor treated corn cobs, respectively. 2L= linear and Q= quadratic response for increasing level of UCSLT. SE=
standard error. abcMeans in same row sharing different superscripts differ significantly (P<0.05). NS= non-significant (P>0.05) and *=
significant (P<0.05).
Estimation of In vitro Methane Production in Buffalo and Cow
Serena CALABRÒ*, Federico INFASCELLI, Raffaella TUDISCO, Nadia MUSCO, Micaela

GROSSI, Giovanni MONASTRA, Monica I. CUTRIGNELLI
Department of Veterinary Medicine and Animal Production, University of Napoli Federico II, Via
F. Delpino 1 80137, Napoli, Italy
*Corresponding email: serena.calabro@unina.it
ABSTRACT
The greenhouse effect of methane (CH4) has been considered high compared to the other
green house gases (GHG), thought to be around 20 times that of carbon dioxide. The methane
production from enteric fermentation in animal agriculture contributes around 20% of the total
global methane. Several studies on methane production from ruminants have been effected but
few is known on buffalo, even if buffalo is considered potentially the most serious methane
contributor for its better utilization of low quality feedstuffs than cattle This study was carried
out to compare the methane production by buffalo and bovine by the in vitro gas production
technique, using three forages (corn silage, grass silage, wheat straw). As inocula rumen fluid
collected from fistulated buffalo and bovine were used. Acetic, propionic and butyric acids,
produced after 120 hours of incubation, were utilised to estimate CH4 production. The total gas
and the total volatile fatty acids production were consistently higher for bovine compared with
buffalo (OMCV: 336 vs. 276 ml/g, P<0.001 and tVFA: 88.5 vs. 81.8 mM/g, P<0.01 in dairy cow
and buffalo, respectively), although organic matter degradability was the same for both inocula
(dOM: 70.0 vs. 69.3 %). The average CH4 produced in vitro with buffalo inoculum was always
lower, either when related to the degraded organic matter (75.1 vs. 97.1 ml/g; P<0.001), or when
reported as % of total gas (18.8 vs. 20.3).
Keywords: ruminant, volatile fatty acids, degradability, stoichiometric calculation.
INTRODUCTION
The greenhouse effect of methane (CH4) is considered higher compared to the other gases,
thought to be 23 times that of carbon dioxide (IPCC, 1997). The methane production from enteric
fermentation in animal agriculture contributes around 20% of the total global methane (Moss et
al., 2000). Methanogenesis in the rumen is an essential metabolic process to maintain steady state
fermentation as this scavenges the molecular hydrogen generated during fermentation. Methane is
a natural by-product of ruminant digestion process acting as hydrogen sink. However, the
methanogenesis is influences by the rumen microbial activity, thus its production depends upon
animal species, age, management, and, mainly, by the quality and quantity of feedstuffs
administered to the animals. Many studies on methane production from ruminants (i.e. dairy and
beef cattle, goats, sheep), has been carried on (Ellis et al., 2007; Lockyer and Champion, 2001,
Bhatta et al., 2008). However, such study on buffalo is still limited, because the high cost of
equipment for measuring methane production in some location where buffalo are mainly raised.
As buffaloes are known to utilize low quality feedstuffs better than cattle (Calabrò et al., 2004;
Calabrò et al., 2008), they are considered potentially the most serious methane contributors
(Devendra et al., 1992). Direct quantification of CH4 produced by animals requires complex
equipment, is labour intensive, time consuming and expensive. An in vitro gas production
technique would offer an alternative, allowing several diets and diet combinations to be evaluated
simultaneously. Using this technique, in vitro CH4 production can be directly measured or
accurately estimated (Guglielmelli et al., 2011; Getachew et al., 2005). The objective of the
investigation was to compare the methane production by dairy buffalo and bovine cow estimated
using in vitro gas production parameters.

924

Corn silage (CS), grass silage (GS) and wheat straw (WS) dried and ground to pass a 1mm
screen were used as substrates in the experiment. The chemical composition of the substrates was
determined according to standard procedures (AOAC, 2000). Two buffalo (Bubalus bubalis) and
two bovine (Bos taurus) dry dairy cows, fitted with a rumen fistula, were used as donor animals to
provide the microbial inoculum. Animals were fed ad libitum twice daily the same diet consisted
of beet pulp, concentrate and oat hay. In vitro fermentation kinetics of the feedstuffs was
determined with the gas production technique according to the method described by Calabrò et al.
(2004). Gas production was recorded 18 times, during 120 hours of fermentation using a manual
pressure transducer (Cole and Parmer Instrument Co, Illinois, USA). At this time the fermentation
was stopped and degraded organic matter (%, dOM) was determined by filtering and burning at
550°C the OM residual. A liquid sample was analysed for volatile fatty acid (mM/g, tVFA) with a
gas chromatograph (ThermoQuest 8000top Italia SpA, Rodano, Milan, Italy; fused silica capillary
column 30 m, 0·25 mm ID, 0·25 µm film thickness), using an external standard solution composed
of acetic, propionic, butyric, isobutyric, valeric and isovaleric acids, as described by Calabrò et al.
(2006). Calculations were made to determine the gas cumulative volume per gram of organic
matter incubated (ml/g, OMCV). Acetic, propionic and butyric acids (mmol) were utilised to
estimate CH4 production according to the following equations (Van Soest, 1994): CO2 (mmol) =
Acetic/2 + Propionic/4 + 3 Butyric/2 and CH4 (mmol) = Acetic + 2 Butyric - CO2; in addition,
CH4 was also calculated as % of total gas produced at 120 h and as ml per gram of degraded OM.
The data obtained was analysed statistically using the analysis of variance (Proc GLM SAS, 2000)
using animal species (buffalo and cow) and substrate (corn silage, grass silage and wheat straw) as
main factors.

The chemical composition of the substrates is reported in Table 1. As expected, corn silage
showed a high nutritive value due to its high starch content (33.1 % DM) and low level of
structural carbohydrate (NDF: 41.2 %DM) few lignified (ADL: 2.39 % DM). On the other hand,
wheat straw showed very high cell wall content (NDF: 78.5 % DM) very rich in lignin (6.99 %
DM). Grass silage had intermediate values for all the carbohydrates fractions and the highest
content of crude protein (9.44 % DM). As results of the in vitro fermentation (Table 2), the total
gas production and the total volatile fatty acids production were consistently higher for bovine
compared with buffalo (mean values of OMCV: 336 vs. 276 ml/g, P<0.001 and tVFA: 88.2 vs.
81.7 mM/g, P<0.01 in bovine and buffalo dairy cow, respectively), although OM degradability
was the same for both inocula (mean values dOM: 70.0 vs. 69.3 %). However, for these
parameters (and the single acids as well) both inocula ranged the three forages similarly
(CS>GS>WS) and according to the chemical composition. Concerning the green house gas
production, the in vitro data appear quite interesting. Methane production with buffalo inoculum
was always lower compared to bovine inoculum, either when related to degraded organic matter
(mean values: 75.1 vs. 97.1 ml/g, P<0.001), or when reported as % of total gas (mean values: 18.9
vs. 20.3, for buffalo and bovine inoculum, respectively). These data agree with those reported by
other authors. Also Kawashima et al. (2006) found no significant differences between Brahman
cattle and swamp buffalo in methane production on the basis of DM intake. However, according to
Blümmel et al. (2005), OM degradation negatively affects CH4 emission; indeed, wheat straw,
characterized by high structural glucides of low quality, had the highest CH4 production (either
reported as ml/g dOM or % of total gas) compared to the other two forages, even if the differences
were not statistically significant. This result was found in both species. Also Moss et al. (2000)
indicate fibre digestibility as one of the main source of methane in the rumen. Surely, the morpho-
functional differences between buffalo and cow in terms of rumen capacity, motility, and retention
time of the ruminal contents, microbial activity and thus digestion influenced the methane
production in the rumen. Further data need on methane production from buffalo, either in vitro
(better measuring than estimating methane) or in vivo (in different condition of feeding
925
managements using several substrates with different carbohydrate content) in order to establish
accurate figures of methane emission in this ruminant specie.
REFERENCES
A.O.A.C. 2000. Official methods of analysis (17th ed.). Association of Official Analytical
Chemists, Inc., Arlington, V.A., USA.
Bhatta, R., O. Enishi, N. Takusari, K. Higuchi, I. Nonaka and M. Kurihara. 2008. Diet effects on
methane production by goats and a comparison between measurement methodologies. J.
Agric. Sci. 146:705-715.
Blümmel, M., D.I. Givens and A.R. Moss. 2005. Comparison of methane produced by straw fed
sheep in open-circuit respiration with methane predicted by an in vitro gas producer. Anim.
Feed Sci. Tech. 123-124:379-390.
Calabrò, S., F. Carone, M.I. Cutrignelli, S. D’Urso, G. Piccolo, R. Tudisco, G. Angelino and F.
Infascelli. 2006. The effect of haymaking on the neutral detergent soluble fraction of two
intercropped forages cut at different growth stages. Ital. J. Anim. Sci. 4:327-339.
Calabrò, S., G. Moniello, V. Piccolo, F. Bovera, F. Infascelli, R. Tudisco and M.I. Cutrignelli.
2008. Rumen fermentation and degradability in buffalo and cattle using the in vitro gas
production technique. J. Anim. Physiol. Anim. Nutr. 92:356-362.
Calabrò S., W.A. Williams, V. Piccolo, F. Infascelli and S. Tamminga. 2004. A comparison
between buffalo and cow rumen fluids in terms of the in vitro fermentation characteristics of
three fibrous feedstuffs. J. Sci. Food Agric. 84:645-652.
Devendra, C. 1992. Nutrition of Swamp Buffalo, in: Buffalo Production, World Animal Science,
C6. (N.M. Tulloh, and J.H.G. Holmes, Eds). Elsevier. Tokyo.
Ellis J.L., E. Kebreab, N.E. Odongo, B.W. McBride, E.K. Okine and J. France. 2007. Prediction
of methane production from dairy and beef cattle. J. Dairy Sci. 90:3456–3467.
Getachew, G., P.H. Robinson, E.J. DePeters, S.J. Taylor, D.D. Gisi, G.E. Higginbotham and T.J.
Riordan. 2005. Methane production from commercial dairy rations estimated using an in
vitro gas technique. Anim. Feed Sci. Techn. 123–124:391–402.
Guglielmelli, A., S. Calabrò, R. Primi, F. Carone, M.I. Cutrignelli, R. Tudisco, G. Piccolo, B.
Ronchi and P.P. Danieli. 2011. In vitro fermentation patterns and methane production of
sainfoin (Onobrychis viciifolia Scop.) hay with different condensed tannin contents. Grass
Forage Sci. 66:488–500.
IPCC (1997). Revised 1996 IPCC Guidelines for National Greenhouse Inventories. Houghton J.T.,
Meira Filho L.G., Lim B., Tréanton K., Mamaty I., Bonduki Y., Griggs D.J. Callander B.A.
(Eds). Intergovernmental Panel on Climate Change (IPCC), IPCC/OECD/IEA, Paris, France.
Kawashima, T., W. Sumamal, P. Pholsen, R. Chaithiang and M. Kurihara. 2006. Comparative
study on energy and nitrogen metabolisms between brahman cattle and swamp buffalo fed
with low quality diet. JARQ 40 (2):183–188.
Lockyer, D.R. and R.A. Champion. 2001. Methane production by sheep in relation to temporal
changes in grazing behaviour. Agriculture, Ecosystems and Environment 86:237–246.
Moss, A.R., J.P. Jouany and J. Newbold. 2000. Methane production by ruminants: its contribution
to global warming. Ann. Zootech. 49:231–253.
SAS\STAT 2000. User’s Guide (Release 6.03). SAS Inst. Inc., Cary, NC.
Van Soest, P.J. 1994. Nutritional ecology of the ruminant. Oregon: B&O Books Inc.
926
Table 1. Chemical composition of the forages

Sample CS GS WS
Dry matter, % 94.6 92.7 92.8
Ether extract, % DM 2.83 2.12 0.69
Crude protein, % DM 7.57 9.44 3.24
ADF, % DM 23.2 32.3 49.4
NDF, % DM 41.2 53.1 78.5
ADL, % DM 2.39 3.17 6.99
Starch, % DM 33.1 - -
Ash, % DM 5.24 13.3 11.0
ADF = acid detergent fibre; NDF = neutral detergent fibre; ADL = acid detergent lignin.
CS: corn silage; GS: grass silage, WS: wheat straw.
Table 2. In vitro fermentation characteristics of the forages with both inocula

Buffalo Bovine Significance
CS GS WS CS GS WS MSE Inoc Sub S*I
dOM, % 77.4 72.4 58.2 79.4 74.1 56.3 5.57 NS *** NS
OMCV, ml/g iOM 302 290 236 393 339 275 675 *** *** NS
Acetic acid, mM/g 39.0 36.8 33.7 29.6 27.8 23.4 5.38 *** * NS
Propionic acid, mM/g 17.3 14.6 12.0 11.4 9.36 7.20 0.91 *** ** NS
Butyric acid, mM/g 4.56 4.51 3.25 6.20 4.62 2.78 0.16 NS *** *
tVFA, mM/g 85.2 87.3 72.5 101 91.4 72.3 27.4 NS ** NS
CH4, ml 37.3 36.4 33.1 32.2 29.7 24.1 5.32 *** * NS
CH4, ml/g dOM 65.9 76.5 83.0 89.7 98.5 103 20.0 *** ** NS
CH4, % total gas 17.0 19.1 20.47 18.3 21.5 21.1 2.16 NS NS NS
dOM: degraded organic matter; OMCV: cumulative volume of gas related to incubated OM; tVFA:
total volatile fatty acids; CH4: methane. CS: corn silage; GS: grass silage, WS: wheat straw.
MSE: mean square error. *, **, *** and NS: P>0.05, P<0.01, P<0.001 and not significant.
927
Chemical Composition, Rumen Fermentation Kinetics, Digestibility and Energy

Value of Cassava Leaves Hay at Different Storage Times
Patricia. M. Guimaraes BEELEN, D. F. SANTOS, I. A. M. A. TEIXEIRA, P. L. AMORIM,

W. B. Lira JUNIOR, T. A. PAULA, R. N. BEELEN, A. B. FRAGA
Alagoas Federal University, Maceió, Alagoas, Brazil. São Paulo State University, Jaboticabal, São
Paulo, Brazil.
*Corresponding email: patriciabeelen@gmail.com
ABSTRACT
Cassava leaves have been widely used as a protein source for ruminants in the tropics.
However, these leaves contain high level of hydro-cyanic acid (HCN) and condensed tannins (CT).
There are evidences that making hay can eliminate more than 90% of HCN and that long-term
storage can reduce CT levels. A complete randomized design with four replicates was conducted to
determine the effect of different storage times (0-control, 60, 90 and 120 days) on chemical
composition, in vitro rumen fermentation kinetics, digestibility and energy value of cassava leaves
hay. Treatments were compared by analyzing variables using the GLM procedure (SAS 9.1, SAS
Institute, Inc., Cary, NC). Crude protein (CP) and ether extract (EE) of the cassava hay were not
affected (P > 0.05) by storage time (17.7% and 3.0%, respectively). Neutral detergent fiber, acid
detergent fiber, total carbohydrate and non-fiber carbohydrate were not affected either (P>0.05) by
storage time (47.5, 32.6, 72.3 and 25.8% respectively). However, other parameters were influenced.
CT was lower (P<0.05) in hay after 120 days of storage compared with control (1.75% versus
3.75%, respectively). Lignin and insoluble nitrogen in neutral detergent, analyzed without sodium
sulfite, were higher (P<0.01) after 120 days of storage, compared with the control (11.22 versus
13.57 and 1.65 versus 3.81% respectively). This suggests that the CT has bound to the fiber or CP
and became inactive. Consequently, the in vitro digestibility of organic matter (50.36%), total
digestible nutrients (44.79%) and energy (1.61 Mcal/KgMS), obtained from gas production data at
72 h of incubation, has increased (P<0.05) with storage times (56.83%, 51.53% and 1.86
Mcal/KgMS, respectively). The chemical composition and fermentative characteristics of cassava
hay suffered variations during the storage period. The best values were obtained after 90 days of
storage. This is probably due to the reduction in condensed tannins.
Keywords: by products, Manihot esculenta, proanthocyanin


928
Effects of Substituting Beer Lees and Cassava Residues Respectively for

Buffalo Dietary Soybean Meal and Grassiness on
Rumen Fermentation In Vitro
Zhongsheng XIA a, Yali HUANG ac, Caixia ZOUa*, Zhihui CHEN a, Xianwei LIANG b,
Shengju WEI b, Xin LIANGb and Shulu LIb
a
College of Animal Science and Technology, Guangxi University, Nanning 530004,
P.R.China;
b
Agriculture and Guangxi, Buffalo Research Institute, Chinese Academy of Agricultural
Sciences, 24-1Yongwu Road, Nanning 530001, P.R. China.3Guangxi agricultural vocational
and technical college, Nanning, 530007,P.R China.
*Corresponding Email: caixiazou2002@hotmail.com
ABSTRACT
The present study was to investigate the effects of substituting beer lees and cassava
residues respectively for buffalo dietary soybean meal and grassiness on rumen fermentation
by using in vitro gas production. The rumen fermentation cultivated in vitro by Mauricio
reading pressure system (RPT). The experiment diets were divided into 8 groups by 2×2×2
factorial design. Substitute 25% and 50%(DM) beer lees for equivalent soybean meal of the
concentrate of dietary fermentative substrate, substituting 12.5% and 25%(DM) cassava
residues and fermentative cassava residues for dietary grassiness as forages respectively, and
in addition a control group(15% soybean meal and 30% grassiness included in the diet) was
set. The results showed that: (1)group 4 (beer lees substitute of 25% for soybean meal and
cassava residues substitute of 25% for grassiness) produced most gases in 72 h;(2)the control
group has the highest ME and most DOM, but no significant difference compared with group
4(P>0.05);(3) group 5(beer lees substitute of 50% for soybean meal and cassava residues
substitute of 12.5% for grassiness) has the highest MCP, but no significant difference
compared with control group, group 4 or group 6(P>0.05); (4) group 4 has the highest NH3-N,
but no significant difference compared with the control (P>0.05); (5) group 5 has the highest
TVFO, and there was significant difference between group 5 and the control (P<0.05). In
summary, according to the characteristics of buffalo rumen fermentation in vitro, group 4 is
the most suitable diet for buffalo.
Keywords: Beer Lees, Cassava Residues, buffalo, rumen fermentation, methane
INTRODUCTION
There is high content of crude protein in beer lees. With the rapid development of feed
industry, it is of great significance to decrease urban pollution, increase social and economic
benefits, and promote livestock production that develop and utilize beer lees in diets. Cassava
residues with simple component of lignocellulose, are hard to been digested and poor
nutrition and palatability. Its feed intake is low by direct feeding animals, but cassava
residues can be a better ruminant feed through improve palatability. Therefore, its appropriate

929
utilization makes great sense to animal production and feed industry.

At present, many workers have reported beer lees and cassava residues’ feeding in
cattle. One report said high-yield cows can increase milk production and extend the milk
peak by supplementing 3.5 kg/d fresh beer lees (Yao.2001). Sun et al(2005) has reported that
cows in mid and late lactation fed beer lees can slow the milk production declining and
increase economic benefits. Each cow can increase net profit in 0.424 RMB yuan than that
without feeding beer lees. Cai et al (2007) has reported that hybridized cattle fed silage
cassava residues can gain weight 0.245kg/d, compared with the cattle fed ammoniated straw,
which shows the most significant differences (P<0.01) .
According to the beer lees’ nutrient characteristics of high crude protein, and cassava
residues with EM bacterium fermentative treatment, which have better palatability and
nutrient value, this experiment was to investigate the effects of substituting beer lees for
different proportions soybean meal by using in vitro gas production and substituting cassava
residues with EM bacterium fermentative treatment for grassiness. This experiment was to
utilize beer lees and cassava residues appropriately by different means, so as to provide a
theoretical reference and technical support for cattle production, and promote the
development of buffalo industry.

Experiment Materials
Beer lees, cassava residues, fermentative cassava residues，soybean meal, maize,
grassiness, silage maize and American alfalfa are all from Guangxi Buffalo Research
Institute , all of which are used for feed in vitro rumen fermentation after drying in 65°C and
crushing through 40-mesh screen. Table 1 shows the forage conventional nutrient
composition.
Experiment Design
The experiment diets were designed by 2×2×2 factors (3 factors and 2 levels):
concentrated feed has 1 factor and 2 levels, and beer lees are substituted 25% and 50% (DM)
respectively for soybean meal in the fermentative substrate; roughage has 2 factors each 2
levels (2×2), and cassava residues and fermentative cassava residues with 12.5% and
25%(DM) respectively substituted grassiness in the fermentative substrate. In addition a
control group was set, and consisted of 15% soybean meal, 25%maize, 30% grassiness, and
30% silage maize (Table 2). Through the effects of fermentation parameter in vitro, this
experiment was to investigate the most appropriate diet composition, in which beer lees
substituted for soybean meal and grassiness was substituted by cassava residues or
fermentative cassava residues. Meanwhile a standard hay group of American alfalfa and
empty control group were set up.
Experiment methods
Culture System in vitro
Rumen fermentation in vitro was cultured by pressure reading gas production
technique in vitro Mauricio et al (1999) RPT system, and made up artificial anaerobic rumen
buffer as Theodorou et al (1994) method. The main part of culture device in vitro is
homeothermic bain-marie shaker made in Germany, whose oscillating frequency and
temperature can be regulated. The fermentation time is 72h, and the fermentative substrate is
930
750mg (DM).
Animals provided rumen fluid and Feeding Management
Three buffaloes averaging 400±5 kg in body weight, provide rumen fluid, which were
installed permanent rumen fistula. Buffalo’s diets were based on buffalo practical feeding
formulation in Guangxi Buffalo Research Institute, which dietary nutrient levels: lactic net
energy 6.44MJ/kg, crude protein 13.72%, calcium 0.72%,phosphorus
0.48%.Concentrate-roughage ratio was 4:6(DM) and grassiness as forage. Donor buffaloes
were tethered and fed twice a day, normal light and free drink.
Items measures and methods
Measures of Gas Production Quantity and Parameter
According to GP = a + b (1−exp-ct)(φRskov et al,1979; Blümmel et al, 1993), using
Fit Curve software, value a, b, and c can be calculated. When GP for gas production quantity
(mL/g); a for high speed part of gas production quantity (mL/g); b for low speed part of gas
production quantity (mL/g); a+b for potential gas production quantity (mL/g)；c for gas
production rate (mL/h); t for fermentation time (h).
According to Theodorou et al (1999) method, gas production quantity was measured
by using pressure sensor to read the pressure in the bottle at each time cultured as
2,4,6,9,12,24,36,48,72 h, then outgassing. Gas production quantity was calculated according
to the formula:
GPt=(Pt-Pt empty)×(V0-100)/(101.3×W).
When GPt for gas production quantity at time t (mL/g); Ptempty for gas production
pressure of control group at time t (mPa); Pt for pressure read at time t (mPa); V0 for the
volume of the bottle (mL)；W for sample dry matter weight (g) ；101.3 for atmosphere
standard (mPa).) The total gas production of whole process was the sum of gas production at
all time points.
Measure of digestible organic matter(DOM) and metabolic energy(ME)
When 200mg (DM) per unit as substrate fermenting in vitro, DOM was calculated
basing on gas production after 24h culture. The formula is as follow:
DOM=(7.65±0.062)×GP24h+(353±0.59) (Menke et al (1979))
(DOM: g/kg ， GP24h: mL/g). ME was calculated basing on the formula:
ME=-0.20+0.1410×DO (Menke and Steingass)[9] (DO for digestibility of organic matter: %,
DO=17.04+1.1085GP24h).
NH3-N Measure
After 24h fermentation in vitro, taking 5mL culture solution, supernatant liquid was
taken after 10min 3000×g centrifugation. According to Searle(1984) and Feng(1993)
improved methods, standard curve and optical density values, the concentration of NH3-N
was calculated by using ammonium chloride as standard, UV-Vis Spectrophotometer(PE
Lambda 35 style) made in USA, colorimetry under 700nm wavelength.
Microbial protein (MCP) Measure
After 24h fermentation in vitro, taking 8mL culture solution, the MCP production was
measured by purine method. The treated test liquid was contrast with 0.5mol/L HCl, using
UV-Vis Spectrophotometer (PE Lambda 35 style) made in USA, colorimetry under 260nm
wavelength, and the RNA measured value was calculated based on standard curve and optical
density values. MCP production was calculated by the following formula:
931
MCP(mg/mL)=[( RNA measured value(mg/mL)×RNA nitrogen content)/ RNA nitrogen

content in bacterial nitrogen]×dilution multiple×6.25(Menke et al,1988) . (RNA nitrogen
content in Bacterial nitrogen:10%, RNA nitrogen content:17.83% ).
Volatile fatty acid (VFA) Measure
After 24h fermentation in vitro, 1mL supernatant liquid was taken and added into
8.2% metaphosphoric acid as same volume. After 10min 2000×g centrifugation at low
temperature, supernatant liquid was taken and added into internal standards crotonic acid.
The contents of propionic acid, acetic acid and butyric acid were analyzed by using Agilent
7890A style gas chromatograph and capillary column (30 m × 0.25 mm ×0.25 um).
Data and statistics
Experimental data was processed by EXCEL software, and variance analysis and
Duncan’s multiple comparison with SPSS16.0 software. The means were considered
significantly different when P<0.05, and the data was represented as average value ± standard
deviation.
RESULTS
The effects of different diets on gas production accumulated 72h, metabolic energy，
digestible organic matter, gas production parameters
According table 3, gas production accumulated 72h in group 4 and group 2 shows no
significant difference compared with control group(P>0.05) which has significant difference
compared with others (P<0.05). The control group has the most metabolic energy and
digestible organic matters and there were no significant difference compared with Group 2
and group 4(P>0.05), but significantly more than others (P<0.05).
According to Table 4 which shows the effect results of different concentrate-roughage
groups on gas production parameter. Group 7 has the most high speed gas production a. while
group 4 has most low speed gas production b, followed by control group, group 2, 5, 1, 7, 3, 6
and 8. Group 4 has the most potential gas production a+b, while group 1 and 3 have the most
gas production rate constant c, followed by group 2, 4, and 6, but control group, group 5, 7,
and 8 as least groups. Gas production rate constant c shows no significant difference among
all groups.
The effects of different diets on microbial protein and ammonia nitrogen
According to Table 5, group 5 has the most microbial protein content, while group 4,
5, and 6 show no significant difference compared with control group(P>0.05), but
significantly different with others(P<0.05). Group 4 has the most ammoniac nitrogen content,
followed by control group, group 2, 1, 3, 5, 6, 7, and 8 which is only significant less than
group 4(P<0.05).
The effects of different diet on VFA
According to Table 6, group 5 has the most acetic acid content，while control group
shows no significant difference compared with group 3 and 8 (P>0.05), but significant more
than others(P<0.05). Group 7 has the most propionic acid content，while control group is
significant less than group 5, 6 and 7 (P>0.05), but did not with others(P<0.05). Group 7 has
the most butyric acid content and significant higher than the others(P<0.05). Group 5 has the
most total acid content which shows no significant difference among each group except
Group 8(P>0.05). Group 4 has the highest ratio between Acetic and propionic acid.
932
DISCUSSION
The effects of Beer Lees substituting for different ratio soybean meal on gas production
Accumulated 72h, ME，DOM and gas production parameters
During a certain culture period, gas production can reflect the utilization degree of
rumen microbe on fermentation substrate and its nutrient value. Yi et al (2009) has reported
that substituting the powder of Broccoli stems and leaves for different ratio of soybean by
using gas production method in vitro, gas production Accumulated 72h would decrease with
the increasing level of powder of Broccoli stem and leaf. This experiment results show that
gas production decrease with the increasing level of beer lees, which are as the same as the
formers’ researches. So did the same results in metabolic energy and digestible organic matter.
Group 4 has the most potential gas production a+b and low speed gas production b, and
Group 5 on the third place. Gas production rate constant c has no significant difference
among all groups.
The effects of different experimental diets on NH3-N and MCP
NH3-N is an important material of microbe composing mycoprotein in culture system.
Its primary condition is maintaining the most appropriate NH3-N concentration of rumen
fluid which would effect the microbial growth and reproduction. Xia et al (2009) has reported
that NH3-N concentrations of goats rumens in DDGS diets group show significant difference
compared with soybean meal group, rapeseed meal group and cottonseed meal group, which
relates to the protein content of DDGS lower than the other three. This experiment results
show that the ammonic nitrogen concentration decreases with the increasing level of beer lees
substituting for soybean meal, which related to the protein content of soybean meal higher
than beer lees. This experiment results are as the same as above researches. The most
important nitrogen source provider of ruminant is microbial protein. From this experiment,
microbial protein shows no significant difference among group 4, 5 and 6(P>0.05).
The effects of of different experimental diets on VFA production
VFA, produced from carbohydrates fermentation in rumen, can be not only the
material composing body fat and milk fat, but also the energy source of ruminant. The ratios
among acetic acid, propionic acid and butyric acid were affected by both animals feed intake
level and dietary composition. Yi et al (2009) reported substituting the powder of Broccoli
stems and leaves for different ratio of soybean by using gas production method in vitro，said
that the total VFA content decreased with the increasing ratio of powder of broccoli stems and
leaves, but the concentrations of acetic acid, propionic acid and butyric acid show no
significant difference. From this experiment, group 5 has the most total acid, and there is no
significant difference among group 1 to 7(P>0.05).
CONCLUSION
(1) Group 4(diets substituting beer lees for 25% soybean meal and substituting
fermentative cassava residues for 25% grassiness) has most gas production accumulated in
72h and NH3-N; more DOM,ME and MCP; mid-level VFA in each time point.
(2) Group 5(diets substituting beer lees for 50% soybean meal and substituting
cassava residues for 12.5% grassiness) has the most MCP and total VFA, and no significant
difference compared with group 4 on NH3-N, VFA in each time point.
933
(3) Group 4 is the most appropriate combination, and followed by Group 5.
ACKNOWLEDGEMENTS
Part of funding for this study was provided by Guangxi Science Foundation, China
(Grant no. 0832071; 0640033; 1010019-24;1123005-2).
REFERENCES
BLÜMMEL M and E.R. φRSKOV. 1993.Comparison of gas production and nylon dag
degradability of roughages in predicting feed intake in cattle. Anim. Feed Sci. and
Tech. 40:109-119.
Y. CAI and W. YANG. 2007.The effect of feeding silage cassava residues on weight of
hybrid cows. Guangdong J. Anim. Vet. Sci. 32(1): 51-52．
FENG Z. and M. GAO. 1993. The improvement of determining ammonia nitrogen content of
rumen liquid by colorimetric method. Inner Mongolian J. Anim. Sci. and Prod.
(4):40-41.
MAURICIO R M, F.L. MOULD and M.S. DHANOA. 1999. A semiautomated in vitro gas
production technique for ruminant feedstuff evaluation. Anim. Feed Sci. and Tech. 79:
321-330.
MENKEK.H., L. RAAB and H.STEINGASS. 1979.The estimation of the digestibility and
metabolizable energy content of ruminant feeding stuffs from the gas production
when they are incubated with rumen liquor in vitro. Cambrigde University Press:
217-222.
MENKE K.H. and H. STEINGASS. 1988.Estimation of the energetic feed value obtained
from chemical analysis and in vitro gas production using rumen fluid. Anim. Research.
Development, 28:7-55.
φRSKOV E R, and I. MCDONAL. 1979.The estimation of protein degradability in the rumen
from incubationasurements weighed according to rate of passage. Agri. Sci.
92:499-503.
SEARLE L. 1984.The berthelot or indophenol reaction and its use in the analytical chemistry
of nitrogen:a review. Analyst, 109: 549-568.
SHEN M. 2006. The Effect of Different Forage Quality on Rumen Fermentation and
Microflora of Sheep. Master's degree thesis, Inner Mongolia Agricultural University,
Huhehaote,China
SUN G., M. ZHANG and Y. Hong. 2005. Effect of Brewage Grain Feeding Middle-anaphase
Milk Cattle [J]. Guangdong Feed. 14(2):42.
Theodorou M , B. A. Williams and M.S. DHANOA. 1994.A simple gas production method
using a pressure transducer to determine the fermentation kinetics of ruminant feed.
Anim. Feed Sci. and Tech. 48: 185-197.
XIA Na and G. ZHAO. 2009. Effect of Different Protein Dietary on Goat Rumen
Fermentation and the Synthesis of Microprotein. China Anim. Hus. and Vet. Med. 36
(7): 11-14.
YAO J. 2001 .Animal Nutrition and feed．Beijing: China Agriculture Press．
YI X. 2009. Evaluating the feasibility of broccoli residues used as feed source in ruminant
diets .Master's degree thesis, Zhejiang University, Hongzhou, China.
934
ZINN R A and F.N. OWENS F N. 1984. A rapid procedure for purine measurement and it’s
use for estimating net ruminal protein synthesis109:549-568.
Table 1. Conventional nutrients component in 6 roughages (air dry basis) %.
Beer Silage American Cassava Fermentative cassava

Grassiness
Items lees maize alfalfa residues residues
DM 25.82 23.03 18.76 82.88 20.49 31.78
CP 30.41 10.83 6.11 14.17 2.19 2.18
CF 9.07 25.16 31.99 27.47 12.97 10.62
NDF 62.60 60.97 64.12 45.89 33.25 31.75
ADF 13.55 32.02 36.80 33.88 15.55 14.86
Ca 0.31 0.48 0.53 1.31 0.45 0.40
P 0.53 0.18 0.21 0.19 0.04 0.03
Table 2. Composition of experiment diets (%).
Control group group group group group group group group

group 1 2 3 4 5 6 7 8
Maize 25 25 25 25 25 25 25 25 25
Soybean meal 15 11.25 11.25 11.25 11.25 7.5 7.5 7.5 7.5
Beer lees 0 3.75 3.75 3.75 3.75 7.5 7.5 7.5 7.5
Silage maize 30 30 30 30 30 30 30 30 30
Grassiness 30 26.25 22.5 26.25 22.5 26.25 22.5 26.25 22.5
Cassava Residues 0 3.75 7.5 0 0 3.75 7.5 0 0
Fermentative
0 0 0 3.75 7.5 0 0 3.75 7.5
cassava residues
935
Table 3. Accumulation gas production of 72 h,ME and DOM of the different experimental
diets.
Items gas production Digestible organic metabolic energy

accumulated 72h (mL/g) matter (g/kg) (MJ/kg)
Control 156.41±0.36 de 583.08±13.17d 6.90±0.27d
Group 1 143.56±0.22 bc 558.77±3.75bc 6.41±0.08bc
Group 2 149.78±1.69cd 572.69±1.92cd 6.69±0.04 cd
Group 3 141.02±2.54 b 556.79±2.52abc 6.37±0.05 abc
Group 4 158.36±2.60 e 579.92±2.11 d 6.84±0.04 d
Group 5 141.81±2.81 bc 544.30±5.66 ab 6.11±0.12 ab
Group 6 136.81±3.05 ab 547.12±4.78ab 6.17±0.10 ab
Group 7 142.48±1.39 bc 549.95±1.74 ab 6.23±0.04ab
Group 8 131.52±3.84 a 538.36±4.01 a 5.99±0.08 a
In the same column , mean value with different superscripts letter indicate significant
difference(P< 0.05) , while with same superscripts letter indicate no significant difference
(P >0.05) . The same as below
Table 4. Gas production parameters of the the different experimental diets.
Items a (mL/g) b (mL/g) a+b (mL/g) c (mL/h)

Control -2.28 157.52 155.24 0.04
Group 1 -2.35 142.91 140.56 0.06
Group 2 -3.68 152.72 149.04 0.05
Group 3 -2.67 141.42 138.75 0.06
Group 4 -3.13 160.46 157.33 0.05
Group 5 -3.26 143.09 139.83 0.04
Group 6 -3.62 139.47 135.85 0.05
Group 7 -2.17 142.66 140.49 0.04
Group 8 -2.98 132.73 129.75 0.04
936
Table 5. Microbial protein and ammonia nitrogen of the different experimental diets.
Items microbial protein ammonia nitrogen

(mg/mL) (mg/100mL )
Control 9.01±0.15de 8.70±0.64ab
Group 1 6.94±0.02a 8.51±0.82 ab
Group 2 8.01±0.20 bc 8.55±0.40 ab
Group 3 8.42±0.23cd 8.28±0.08ab
Group 4 9.08±0.05e 8.81±0.49b
Group 5 9.38±0.22e 7.81±0.37 ab
Group 6 8.98±0.26de 7.66±0.32 ab
Group 7 7.57±0.30 b 7.61±0.56 ab
Group 8 7.61±0.18 b 7.07±0.33 a
Table 6. Volatile fatty acid of the different experimental diets.
Items Acetic acid Propionic Butyric acid Total volatile Acetic acid/
(mMol) acid (mMol) (mMol) fatty acids Propionic
(mMol) acid
Control 20.25±0.29a 7.14±0.05a 5.66±0.11abc 33.06±0.44a 2.83±0.02ab
Group 1 23.40±0.34c 7.27±0.10 ab 5.81±0.17c 36.48±0.47 bc 3.22±0.01e
Group 2 22.47±0.57 bc 7.13±0.26 a 5.6±0.19 abc 35.27±1.01 abc 3.16±0.04de
Group 3 21.31±0.09 ab 6.98±0.08 a 5.18±0.14 a 33.46±0.15 a 3.06±0.05cd
Group 4 22.86±0.61bc 7.10±0.09 a 5.22±0.11 ab 35.18±0.76 abc 3.22±0.06e
Group 5 24.02±0.95c 7.97±0.47 bc 5.83±0.22c 37.81±1.64c 3.02±0.07cd
Group 6 22.48±0.87bc 7.73±0.10abc 6.34±0.08d 36.55±0.88 bc 2.91±0.08bc
Group 7 22.58±0.32 bc 8.30±0.21c 5.73±0.12bc 36.60±0.54 bc 2.72±0.04 a
Group 8 20.93±0.81 ab 7.5±0.31 ab 5.5±0.23 abc 33.99±1.35 ab 2.78±0.01 ab
937
Nutritive Value and In Situ Digestion Kinetics of Some Leguminous and Non-
leguminous Fodder Baled Silages in Buffalo Bulls
Nawaz SAEED1, Nasir Ali TAUQIR, Muhammad Aasif SHAHZAD*, Muhammad
SARWAR, Mahr-un-NISA and Muhammad Saif ur REHMAN2

Pakistan.
1
Livestock and Dairy Development Department, Government of Punjab Lahore, Pakistan.
2
Department of Animal Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan.
*Correspondence: draasifshah@uaf.edu.pk
ABSTRACT
The study was carried out for nutritional and biological evaluation of baled silage of various
leguminous and non-leguminous fodders in cannulated Nili-Ravi buffalo bulls. Lucerne, Berseem
and Oat fodders were harvested manually and chopped with a locally manufactured chopper
separately. Dry matter (DM) of each fodder was made 30% by mixing appropriate quantity of
wheat straw. Cane molasses was added in each fodder at 2% of fodder DM as a source of soluble
carbohydrates. Bales were prepared in a hydraulic straw baler and were then wrapped with
polyethylene sheet, tied and sealed with a manual sealer machine to attain anaerobic conditions.
After 30 days of fermentation period, the bales were opened and pH & lactic acid contents were
measured immediately. The silages were also analyzed for proximate compositions and fiber
fractions through standard procedures. The pH, lactic acid concentration, DM, crude protein (CP)
and fiber fractions of all the fodders were within desirable rage of good quality silage. Ruminal DM
and neutral detergent fiber (NDF), degradability, lag time, rate and the extent of digestion of
Berseem, Lucerne and their silage were similar. The NDF digestibility and extent of DM and NDF
digestion of oat was significantly (p<0.05) higher than that of its silage. The results of the present
study indicated that leguminous and non-leguminous fodders ensiled in bales with 2% molasses
have better nutritive value for buffaloes and the same may be propagated for sustainable
availability of fodder round the year.
Keywords: baled silage, lucerne, berseem, oat, In situ digestion kinetics
INTRODUCTION
A consistent supply of quality forages in sufficient amount is considered essential for cost-
effective livestock production in any agriculture based county of the world (Tauqir et al. 2007a,b).
However, its sustainable supply is threatened by multiple factors in developing countries like
Pakistan. In urban and peri-urban areas, tons of fresh fodder is required daily for dairy and beef
animals. The fodder cannot be stored as such and the dairymen have to purchase it daily. Similarly,
transportation of fresh green fodder to big cities and peri-urban areas is a further setback. In adverse
environmental conditions and rainy seasons, fodder availability and transportation is almost
blocked that leads to sudden drop in animal productivity. Moreover, the landless commercial
farming community cannot afford construction of silos and fodder storages. This situation calls for
some appropriate technology to overcome these bottlenecks in consistent fodder availability.
Hence, the current study was an attempt to address the issue. The present study was aimed to
evaluate the nutritive and economical significance of baled silage of leguminous and non-
leguminous fodders and their utilization in fistulated buffalo bulls.

The fodders (Lucerne, Berseem and Oat) were harvested at bloom stage, chopped (½ inch or
14mm) and while ensiling to maintain moisture concentration wheat straw was used and cane

938
molasses was also added at the rate of 2% on DM basis as soluble carbohydrates source. The
mixture of fodder, wheat straw and molasses was then pressed at 600-800 psi pressure in a locally
manufactured hydraulic straw baler to make the bales by wrapping the polythene sheath for 30
days. After opening bales, pH and lactic acid contents were determined immediately (Baker and
Summerson 1961). The samples of silage and fodder were also analyzed for DM, CP and total ash
using method described by AOAC, (1990), NDF, ADF, hemicellulose, cellulose and ADL by
methods of Van Soest et al. (1991). Three ruminally cannulated Nili-Ravi buffalo bulls were used
for this study. Ten days were given as adaptation period to the diet at the start of experiment
followed by 4 days of incubation period for the in situ nylon bags. The Lucerne, Berseem and Oat
fodders and their silages were dried in forced air oven and digestion coefficient of DM and NDF
was calculated at 48 hours of incubation. Rate of disappearance, lag time and extent of digestion of
DM and NDF of Lucerne, Berseem and Oat fodders and their silages were determined by the
methods illustrated by Sarwar et al. (1991).
The data collected on different parameters (DM and NDF Degradability, Lag, Rate and
Extent of degradation) during in situ trial and were analyzed according to completely randomized
design using the GLM procedure of SAS (1988). Differences among means were separated using
the Duncan’s Multiple Range Test (Snedecor and Cochran 1980).
RESULTS AND DISCUESSION

Chemical composition of Lucerne, Berseem, Oat fodders and their Silages
Chemical composition (on DM basis) of Lucerne, Berseem and Oat harvested at one-tenth
bloom stage, wheat straw and cane molasses is presented in Table 1, while Chemical composition
of Lucerne, Berseem and Oat silages (ensiled separately in bales at 30% DM with cane molasses at
2% of fodder DM) is given in table 2. The pH, lactic acid concentration, DM, CP and fiber
fractions (NDF, ADF, ADL, Hemicellulose and Cellulose) of Lucerne, Berseem and Oat silages
were within desirable rage of good quality silages.
The chemical composition, fiber fractions, pH and lactic acid concentration of all the
ensiled fodders were within desirable rage of good quality silages (Nisa et al., 2008 ; Tauqir,
2010). Molasses addition to the fodders before ensilation promoted the production of lactic acid
(Yahaya et al. 2001) that led to rapid pH drop of the ensiled medium (Leibensperger and Pitt
1988). Addition of molasses improved the fermentable carbohydrate content of the fodder thus
enhancing the growth of lactic acid producing bacteria that converted fermentable sugars in to
lactic acid and lowered final pH of the medium to terminate the microbial activity (Yahaya et al.
2001).
Digestion kinetics of fodders and their Silages
The DM and NDF degradability, lag time and extent of digestion of Berseem and its silage
didn’t differ significantly and the same was true for DM digestion kinetics of Lucerne fodder and
its silage (Table 2). The NDF degradability of Lucerne was significantly (p<0.05) higher than that
of its silage. The ensilation of oat did not affect DM degradability, lag and rate of DM degradation
in situ. However, the extent of DM degradation was significantly (p<0.05) higher than its silage
(Table 2).
Depression in silage degradability has been reported by various researchers (Rook and
Thomas 1982; Garcia et al. 1989) who explained that during ensiling process loss of readily
degradable carbohydrate contents by lactic acid producing bacteria was the major reason which
might have declined degradability of its silage. Hence reduction in degradability of silage is a
reflection of extensive loss of soluble carbohydrates due to microbial fermentation (Ruiz et al.
1992). In contrast Nadeau et al. (1996) reported that ensilation of fodders increased DM and NDF
degradability resulting from improvement in the ruminal environment by readily fermentable cell
wall substrate for cellulotytic bacteria. Slight drop in degradability of Berseem and Lucerne silages
in the present study reflected petite mutilation of soluble carbohydrates when ensiled in bales.
939
REFERENCES
AOAC. 1990.Official Methods of Analysis. (15th Ed.). Association of Official Analytical Chemists.
Arlington, Virginia, USA.
Baker, S. B and W. H. Summerson. 1961. The calorimetric determination of lactic acid in
biological materials. J. Biolog. Chem. 138: 535-543.
Garcia, A. D., W. G. Olson, D. E. Otterby, J. G. Linn and W. P. Hansen. 1989. Effect of
temperature, moisture and aeration on fermentation of alfalfa silage. J. Dairy Sci. 72: 93-97.
Leibensperger, R.Y. and R. E. Pitt. 1988. Modeling the effects of formic acid and molasses on
ensilage. J. Dairy Sci. 71:220-1228.
Nadeau, E. M., G. D. R. Buxton, E. Lindgren and P. Lingvall.1996. Kinetics of cell-wall digestion
of orchard grass and alfalfa silages treated with cellulase and formic acid. J. Dairy Sci. 79:
2207-2215.
Nisa, M., M. A. Shahzad, M. Sarwar and N. A. Tauqir. 2008. Influence of additives and
fermentation periods on silage characteristics, chemical composition, and in situ digestion
kinetics of Jambo silage and its fodder in nili buffalo bulls. Turk. J. Vet. Anim. Sci. 32: 67-
72.
Rook, J. A. F. and P. C. Thomas. 1982. Silage for milk production. National Institute of Research
in Dairying, Reading, England. ISBN 0-7084-0166-X.
Ruiz, T. M., W. K. Sanchez, C. R. Straples and L. E. Sollenberger. 1992. Comparison of “Mott”
dwarf elephant grass silage and corn silage for lactating dairy cows. J. Dairy Sci. 75:533-543.
Sarwar, M., J. L. Firkins and M. Eastridge.1991.Effect of replacing neutral detergent fiber of
forage with soy hulls and corn gluten feed for dairy heifers. J. Dairy Sci. 74:1006-1012.
SAS User’s Guide: Statistics, Version 7 Edition., SAS Inst., Inc., Cary, NC. 1998.
Snedecor, G.W. and W. G. Cochran. 1980. Statistical Mehods. 6th Ed. Iowa State Univ. Press,
Ames, IA.
Tauqir, N. A., M. Sarwar, M. A. Jabbar and S. Mahmood. 2009. Nutritive value of jumbo grass
(sorghum bicolour sorghum sudanefe) silage in lactating nili-ravi buffaloes. Pak. Vet. J. 29:1-
11.
Tauqir, N.A., M. A. Khan, M. Sarwar, M.Nisa, C. S. Ali, W. S. Lee, H. J. Lee and H. S. Kim.
2007a. Feeding value of jambo grass silage and mott grass silage for lactating nili buffaloes.
Asian-Aust. J. Anim. Sci. 20:523-528.
Tauqir, N.A., M. A. Khan, M. Sarwar, M. Nisa, W. S. Lee, H. J. Lee and H. S. Kim. 2007b.
Influence of varying dm and molasses levels on chemical composition of berseem and lucerne
silage characteristics and their in situ digestion kinetics in nili buffalo bulls. Asian-Aust. J.
Anim. Sci. 20, 887-893.
Van Soest, P. J., H. B. Robertson and B. A. Lewis. 1991. Method of dietary fiber and non-starch
polysaccharides in relation to animal material. J. Dairy Sci. 74: 3583-3593.
Yahaya, M.S., A. Kimura, J. Harai, H. V. Nguyen, M. Kawai, J. Takahashi and S. Matsuoka. 2001.
Evaluation of structural carbohydrates losses and digestibility in alfalfa and orchard grass
during ensiling. Asian Aust. J. Anim. Sci. 14: 1701-1709.
940
Table 1. Chemical composition of fodders, silages1, wheat straw and cane molasses.
Luc. Oat Wheat Cane

Parameters Lucerne Berseem Ber.Silage Oat Silage
Silage straw molasses
Dry matter 19.0 28.9 18.5 29.5 16.5 28.8 92.0 68.0
Crude protein 18.0 15.5 17.4 14.8 9.96 8.75 3.00 ---
NDF2 47.0 50.4 46.5 49.3 70.5 57.8 72.5 ---
3
ADF 30.0 31.9 28.6 31.2 38.0 34.0 48.2 ---
Hemicellulose 17.0 18.4 17.9 18.1 32.5 23.8 24.3 ---
Cellulose 21.6 23.6 21.5 23.9 33.8 29.5 40.2 ---
ADL4 8.40 8.29 7.12 7.30 4.20 4.54 8.00 ---
Ash 10.80 10.50 11.00 11.0 11.8 11.85 7.21 10.30
pH --- 4.60 --- 4.50 --- 4.15 --- ---
Lactic acid --- 3.40 --- 3.55 --- 3.80 --- ---
1
Fodders were ensiled at 30% DM with cane molasses at 2% of fodder DM, 2Neutral detergent fiber, 3Acid
detergent fiber, 4Acid detergent lignin
Table 2. Comparative in situ dry matter and neutral detergent fiber digestion kinetics of berseem,
lucerne, oat and their silages1.
Ber. Luc. Oat Oat SE

Parameters Berseem SE Lucerne SE
silage silage silage
Dry matter
Degradability1, % 68.68 68.59 0.31 70.77 70.66 0.31 65.0 64.8 0.31
Lag, hour 1.98 1.99 0.03 1.93 1.93 0.08 1.38 1.40 0.03
Rate of
degradation, % 4.24 4.23 0.12 4.27 4.26 0.04 3.63 3.68 0.12
hour-1
Extent3, % 72.86 72.85 0.01 71.81 71.92 1.35 70.0a 68.9b 0.01
Neutral detergent fiber
Degradability2, % 56.1 55.8 0.30 57.3a 56.0b 0.37 50.5a 48.0b 0.30
Lag, hour 1.98 1.98 0.02 1.98 1.98 0.04 1.70b 1.81a 0.02
Rate of
degradation, % 3.86 3.56 0.30 3.69 3.73 0.32 3.41 3.32 0.30
-1
hour
Extent3, % 62.0 62.2 0.30 63.4 63.7 0.31 67.0a 64.8b 0.30
Means within row bearing different superscripts differ significantly (p<0.05)
1
Fodders were ensiled at 30% DM with cane molasses at 2% of fodder DM, 2Degradability was determined
at 48 hours of incubation, 3Extent of digestion was determined at 96 hours of incubation.
941
Potential Benefits from the Utilization of Some Natural Feed Resources in

Thai Swamp Buffaloes
Thongsuk JETANA and Anong BINTBIHOK
Science, Chulalongkorn University, Henri Dunant street, Phathumwan, Bangkok 10330, Thailand,
*Corresponding E-mail: Thongsuk.J@Chula.ac.th
ABSTRACT
This paper aims to comply from studies of using some natural feed resources (NFRs) in
several experiments for Thai swamp buffaloes, which were conducted by the Research and
Development Center for Livestock Production Technology, Faculty of Veterinary Science,
Chulalongkorn University, Thailand, during 2002-2012. Most of NFRs are interesting, only some of
available NFRs are selected to investigate whether they are suitable to use as feeds for swamp
buffaloes. In general NFRs used as feed for ruminants may be simply divided into two types; i) as a
roughage and ii) as a supplement. Pineapple waste (PW), rice straw (RS), pangola hay (PH), Ruzi
grass (RG) and Vetivar grass (VG) were used as roughage diets, whilst leaves of luecaena, cassia,
and mulberry, rain tree pods (RTP) and cassava chip (CSC) were used as supplement diets in this
review. The results indicated that differences in using NFRs for swamp buffaloes are noticed. PW,
PH and RG are suitable as a basal diet, while RS, VG and cassia leaves need further study to
improve their quality. Only appropriate proportion of leucaena in diets fed to buffaloes, it enhances
intake, digestion, N balance and ruminal microbial production. The treatments of leucaena to
inactive of tannins may not necessary for animals fed leucaena forage couple with RTP. However,
the supplementary diets containing high starch and high sugar in CSC and RTP, depressed fibrous
diet digestion in swamp buffaloes. The supplementary diet with RTP increased ruminal microbial
yields in animals. The high starch but low N content in CSC when this material is used as a
feedstuff in ruminants, urea or non protein nitrogen is required to fulfill N sources. Mulberry leaves
can be absolutely used as a supplement in ruminants as they are without anti-nutritional factors and
toxic compounds. Subsequently, the supplementary diet with mulberry leaves increases fibrous diet
digestion and ruminal microbial yields. Several studies demonstrated the approach to use NFRs as
the alternative feeds; roughage and supplement, to improve the quality of feeds in swamp buffaloes.
The practical implication of this review is that it would be benefit the smallholder farmers to use
NFRs because they do not only improve quality of feeds but also reduce cost of feed.
Keywords: cassava chip, cassia, luecaena, mulberry, pangola hay, pineapple waste, rain tree pod,
rice straw, swamp buffaloes, vetivar grass
INTRODUCTION
There are widely natural feed resources (NFRs) in tropical countries, particularly in Thailand,
which they are required to investigate whether they are suitable to use as feeds for ruminants. In
general, feeds used in ruminants may be simplify divided into two types; i) as a roughage and ii) as
a supplement diet. The most important agricultural by-product for cattle and buffaloes is rice straw
[(Oryza sativa var.) RS], the annual production is approximately 24.7 million tons in Thailand.
Although, many means have been attempted not only to improve low quality of agricultural by-
products, but new feed resources such as other agricultural by-products and the multiple proposed
trees are also seeking for, including selection of several improved grasses and local plants in order
to improve the quality of buffalo’s feeds. The pineapple waste (Anasa comosus Linn. Mer.) from
factory, Vetiver grass (Vetiveria nemoralis A. Camus), Pangola grass (Digitaria decumbens) and
Ruzi grass (Brachiaria ruziziensis) were selected to study as roughages, on the other hand leaves of
leucaena (Leucaena leucocephala), cassica (Cassia siamea Lam. Fabaceae), mulberry (Morus
alba), rain tree pods (Samanea Saman) and cassava chip [Manihot esculenta (L.) Crantz] were
selected to study as supplement diets in several experiments of swamp buffaloes
942
The objective of this review is to report crude protein (CP) composition, digestiblility and
metabolsable energy for ruminant (MEr) content in some natural feed resources (NFRs) which are
used as roughage and supplement in swamp buffaloes and their possibility feeding potential is
evaluted.
1. Some NFRs are used as a roughage in swamp buffaloes

Rice straw : Rice straw has generally been considered as waste product, it has limited nutritive
value [4.7%CP, 48% organic matter digestibility (OMD) and metabolsable energy for ruminant
(MEr) 6.3 MJ/kg DM: OMD and MEr were calculated according to Menke et al. (1979)]. Therefore
it should be used only as replacement for part of the forage in a ration or in dry season. It should not
be used as a complete ration. A study of feeding rice straw has shown better response than that
feeding ammoniated RS (RSNH4OH) [Figure 1]. Fibre digestion in swamp buffaloes fed only rice
straw has usually occurred poorer than that fed only ruzi grass, pangola hay, pineapple waste
(Figure 1). It is surprising that none difference of nutrients digestion of RS and RSNH4OH in
swamp buffaloes was detected, it is probably because i) buffalo is better in N recycling and ii)
RSNH4OH is too high N to suitable for buffaloes.
Pineapple waste : Pineapple waste (PW) is potentially useful in Thailand. Pineapple peel from
canning factories has heen widely used for dairy cattle in southern Thailand while the by-products
of stem and root of pineapple used for cosmetic industries can also used as feedstuff because it
contains high potentially digestible fibre (6.0%CP, 77%OMD, MEr 6.5 MJ/kgDM) and is aslo
cheaply avaiable (Jetana et al. 2004). Based on a previous study it was suggested that the optimum
proprotion of concentrates to PW was 60:40 (Jetana et al. 2009). It was shown that PW has the
highest of nutrients digestion compared to other roughages (Figure 1), it may be that the particle
size of the industry processed pineapple waste is very fine and could be easily attached and utilized
by microorganisms.
Vetiver grass : Vetiver grass can generally be classified into 2 types, i) Vetiveria zizanioides Nash
and ii) Vetiveria nemoralis A. Camus. Vetiver grass (Vetiveria nemoralis A. Camus) was used to
study as roughage in swamp buffaloes. Though, the nutritive value is rather high (7.1%CP,
48%OMD, MEr 6.4 MJ/kgDM), but the digestibility of Vetiver grass was similar to that of rice
straw (Figure 1). It is probably because the structure of this grass is hard, rough and shape, therefore
these charecteristics will disturb the gastric tract, then outflow rate is very fast (Jetana et al. 2008).
In case of Vetiver grass is required to use as rouahage, further study must should be the
improvement of its quality, in particulary the structure of grass.
Pangola hay : Pangola grass (PH) is among the type of grasses, being planted in Thailand, this grass
has fairly nutritive values (6.0-9.0%CP, 60%OMD, MEr 8.1 MJ/kgDM) and is suitable to make hay
and can be kept for a long time. It was shown that fibre digestion in PH is similar to that in PW
(Figure 1).
Ruzi grass : Ruzi grasses also had high nutritive values (8-16%CP, 71%OMD, MEr 9.8 MJ/kgDM)
as same as the selected grasses, but they are high in lignin content (8-10%), consequently the
digestibility is low (Figure 1). Even though Ruzi grass can grow well in Thailand, but it should be
considerably utilized for small ruminants, as photosensitization and high oxalate may occur when
Ruzi grass is fed particularly in sheep, goats and calf (Hare and Phaikew 1997).
2. Some NFRs used as a supplementary diet in swamp buffaloes

Leucaena : A short feeding study in swamp buffaloes, none of the animals showed any visible
toxicity symptom during the period of leucaena addition to the diets was fed. The results however
indicated that only appropriate proportion of leucaena to ruzi grass was fed to buffaloes; it enhanced
intakes, digestion, N balance, ruminal microbial production (Jetana et al. 2012a). Although
leucaena is high in nutritive values (25%CP, 70%OMD, MEr 10 MJ/kgDM), a problem of leucaena
is high in tannin content (2-6%), it should not be overlooked. There are however two ways which
have been attempted to be deactivated tannins and other secondary compounds in leucaena; i) the
addition of chemicals with a high affinity for tannins, such as polyvinylpyrrolidone (PVP) and
polyethylene glycol (PEG), a synthetic polymer to which tannins have a greater binding affinity
than protein (Waghorn et al. 1994) and ii) the use of alkaline treatments, such as NaOH solution
943
(Jetana et al. 2011). However, the forms of untreated leucaena combined with rain tree pod fed
swamp buffaloes, have not shown only enhancing microbial production supply into small intestine
but also increasing fiber digestion and N balance (Figure 2).
Figure 1. Digestion of nutrients in swamp buffaloes fed ad libitum natural feed resources as a basal
diet.
Cassia siamea: Cassia siamea is one of the vegetable trees most cultivated in Southeast
Asia. This plant has great nutritional (20-22%CP, 60-70%OMD, MEr 9.0 MJ/kgDM) and medical
values. The use of Cassia siamea leaves supplemented in swamp buffaloes has resulted in better
fibre digestion than the other supplements containing similar protein such as leucaena, and
mulberry leaves (Figure 2). Consequence, the Cassia siamea is considered to be an additional
protein source to improve the efficiency of microbial growth in the rumen. Further study, this
material must be defined anti-nutritional factors and toxic compounds before it is practically used as
feedstuff (Jetana et al. 2010).
Mulberry: Mulberry leaves are also highly palatable and digestible (70-80%OMD, MEr 10-12
MJ/kgDM) to ruminants and can also be used for monogastrics and human beings. Protein content
in leaves and young stems, with a good essential amino acid profile, varies from 18-22% depending
on variety and age. Mulberry leaves can be undoubtedly used as a supplement in ruminants as they
are without anti-nutritional factors and toxic compounds. Subsequently, the supplementary diet with
mulberry leaves increases fibrous diet digestion when compared with RTP supplementation (Figure
2) and ruminal microbial yields (Jetana et al. 2010).
Rain Tree Pod: The rain tree is a tropical legume. It contained 766 g DM/kg as fresh-basis
and it contained (g/kgDM), 87.3 g ash, 29.4 g nitrogen, 313 g neutral detergent fibre, 287 g acid
detergent fibre, 52 g acid detergent lignin, 182 g total sugar, 84 g sucrose, 50 g phenolic compound,
35 g tannins, 11 g condensed tannins. As this material containing high total sugar and crude
protein, it is probably suitable to be used as a feed (Jetana et al. 2011). A study demonstrated that
the high sugar and protein content in the rain tree pod is not only advantage to the increase in the
efficiency of microbial growth in the rumen of buffaloes (Jetana et al. 2011) but also improves the
quality of milk in dairy goats (Jetana et al. 2012b). However, supplementation with RTP
containing high sugar always depresses pH in the rumen; therefore fibre digestion decreases (Figure
2). The rain tree pod can also be substituted for molasses/sugars when required for producing a bio-
extract from some fruits/vegetable/herbs in order to save cost of using sugar and molasses.
944
Figure 2. Digestion of nutrients in swamp buffaloes fed rice straw as a basal diet and
supplemented with different natural feed resources.
Cassava chip : Cassava chip (CSC) contains high level of readily soluble carbohydrates (700
g starch/kgDM) but low N content (3%CP, 80%OMD, MEr 9.4 MJ/kgDM) and is highly
degradable in the rumen comparing with other energy sources. Urea, therefore, a highly rumen
degradable non protein-nitrogen, always used as an N source when CSC is added in ruminant feed.
Swamp buffaloes fed pangola hay and supplemented with CSC+3%Urea increased DM and OM
digestions but decreased NDF digestion (Figure 3). It may be due to i) high starch content and ii)
small particle size and structure of starch easily degradable and rapid fermentation in the rumen.
Figure 3. Digestion of nutrients in swamp buffaloes fed pangola hay as a basal diet and
supplemented with different natural feed resources.
CONCLUSIONS
Several studies demonstrated the approach to use NFRs as the alternative feeds; roughage
and supplement, to improve the quality of feeds in swamp buffaloes. The practical implication of
this review is that it would be benefit to the smallholder farmers to use NFRs because it is not only
to improve quality of feeds but also to reduce cost of feed.
REFERENCES
Hare M.D. and P. Phaikew. 1997. Forage Seed Production in Northeast Thailand. In: Loch, D.S.
and Ferguson, J.E. (eds.) Forage seed production 2, Tropical and subtropical species. (CABI
Publishing) pp. 435–443.
Jetana, T., W. Suthikrai, S. Usawang, S. Thongruay, K. Thasripu, R. Jintana, C. Vongpipatana and
S. Sophon. 2004. Effects of urea-N and protein in complete pineapple waste (Ananas comosus
Linn. Mer.) Based- diet fed to Thai swamp buffaloes (Bubalus bubalis) on rumen microbial
production, some digestive and blood metabolite parameters. In: The symposium on
945
biotechnology in breeding and nutrition of cattle and swamp buffalo, Faculty of veterinary
science, Chulalongkorn university, 23-24 August 2004 pp. 35-53.
Jetana, T., S. Usawang, C.Vongpipatana, S. Thongruay and S. Sophon. 2008. Effects of
replacement of leucaena (Leucaena leucocephala) with rain tree pod (Samanea saman) as a
protein-rich supplement for cattle production. In: Proceedings of the 46th Kasetsart University
Annual Conference, Kasetsart, and 29 January - 1 February, 2008. Subject: Animals and
veterinary medicine, 2008, pp. 39-45.
Jetana T., S. Usawang, S. Thongruay, C. Vongpipatana and S. Sophon. 2009. The Effects of
concentrate added to pineapple (Ananas Comosus linn.Mer.) waste silage in differing ratios to
form complete diets, on digestion, excretion of urinary purine derivatives and blood
metabolites in growing, male. Thai swamp buffalo. Trop. Anim. Health Prod. 41: 449-459.
Jetana, T., S. Usawang, S.Thongruay, C. Vongpipatana and S. Sophon. 2010. Apparent
digestibility, nitrogen balance, ruminal microbial nitrogen production and blood metabolites
in Thai Brahman cattle fed a basal diet of rice straw and supplemented with some tropical
protein-rich trees. Asian-Aust. J. Anim. Sci. 23: 465-474.
Jetana T., C. Vongpipatana, S. Usawang and S. Thongruay. 2011. The use of tropical protein-rich
leaves as supplements to Thai swamp buffalo receiving a basal diet of rice straw and treated
leucaena (Leucaena leucocephala). Trop. Anim. Health Prod. 43: 57-67.
Jetana T., S. Thongruay, S. Uswang and R. Hengtrakulsin. 2012a. A comparative study on
mimosine, 3,4-dihydroxy pyridone (3,4-DHP) and 2,3-dihydroxy pyridone (2,3-DHP), purine
derivatives (PD) excretion in the urine, thyroid hormone and blood metabolites profiles of
Thai swamp buffalo (Bubalus bubalis) and Murrah buffalo (Bubalus bubalis) Trop. Anim.
Health Prod. 44: 887-897.
Jetana, T., S. Usawang and M. Techakampu. 2012b. The use of rain tree pods as feed supplement
dairy goats. In Proceedings of the 1st Asia Dairy Goat Conference, Rasedee Abdullah, M.
Ariff Omar, M. Ali Rajion, A. Razak Alimon, Liang Juan Boo and Haw Ah Kam (Eds.) Corus
Hotel, Kuala Lumpur, Malaysia 9-12 April 2012, pages 79-83.
Menke K.H., L. Raab, A. Salewiski, H. Steigaβ, D. Fritz and W. Schneider. 1979. The estimation of
the digestibility and metabolisable energy content of ruminant feedstuffs from the gas
production when they are incubated with rumen liquor in vitro. J. Agric.Sci., (Camb.). 93:
217–222.
Waghorn, G. C., I. D. Shelton and W. C. McNabb. 1994. Effects of condensed tannins in Lotus
pedunculatus on its nutritive value for sheep. 1. Non-nitrogenous aspects. J. Agric.Sci.,
(Camb.). 123: 99-107.
946
Effects of Lasia spinosa Thw. and Season on Plasma Leptin and Glucose of
Weaned Female Murrah X Swamp buffalo Calves
Ratree JINTANAa*, Wanvipa SUTHIKRAI a, Sunpetch SOPHONa, Runchuan
HENGTRAKULSINb, Sungworn USAWANGa and Maneewan KAMONPATANAa
a
The Project : The Use of Nuclear Technology to Improve Reproductive Efficiency in Dairy Cattle
and Swamp Buffalo, Research and Development Centre for Livestock Production Technology,
Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand.
b
Murrah farm, Nong-mai-kan, Plang-yao District, Chachoengsao Province, 24190,Thailand.
*Corresponding email: jratree@chula.ac.th
ABTRACT
The effects of Lasia spinosa Thw. and season on plasma leptin and glucose were studied in
six weaned female murrah x swamp buffalo crossbreed calves. All animals were randomly assigned
by age into control group (n=3) and treatment group (n= 3). The treatment group was fed with an
additional 30 g of dry powder of L. spinosa /animal /day to the concentrate in the morning every
day. Radioimmunoassay method was used to determine plasma leptin while plasma glucose was
determined by liquicolor GOD-PAP method. It was found that means + SD of leptin were 4.69 +
1.37, 7.01 + 2.03 and 6.94 + 3.44 ng/mL in winter, summer and rainy season respectively. The
treatment with L. spinosa tended to reduce plasma leptin in winter and rainy season and moderately
increase in summer. In control animals, means + SD of plasma glucose were 58.50+9.22 , 65.50 +
8.17 and 66.53+ 5.36 mg/dl in winter, summer and rainy season respectively while in treatment
animal means + SD of plasma glucose were 62.19 + 6.7, 68.03 + 6.05 and 64.89 + 4.55 mg/dl in
winter summer and rainy season respectively. Glucose / leptin ratio was elevated from 12 to 17 and
10 to 11 in winter and rainy season respectively. Reduction of glucose/ leptin ratio was 9 of both
control and treatment in summer.In conclusions, plasma leptins were lower in winter than in
summer and rainy season while their plasma glucoses were not different. The treatment tended to
reduce plasma leptin in winter and rainy season and modulately increase in summer. The plasma
glucose /leptin ratios were elevated in winter while they were not different in summer and rainy
season. Suggesting for future research is that more number of animals and plasma insulin should be
determined.
Keywords: Glucose, Lasia spinosa Thw, leptin, season,weaned murrah x swamp buffalo
crossbreed calves
INTRODUCTION
Unlike other dairy animals the buffalo presents the famer with problem of growth and late maturity
and summer subfertility. Development and attainment of puberty are determined primarily by
nutrition from the time of weaning (Camparnile et al., 2001). In Thailand, it was reported that
Lasia spinosa thw. being used as internal veterinary medicine as growth promoter for enhance
growth rate and improve feed efficiency in cattle and buffalo (Buranamanus, P. 2001). The
effective dose of L. spinosa Thw. is 30 g dry weight /buffalo/day as a practical use by the farmers
does help growth performance. In Thailand, higher average daily gain of swamp buffalo calves
were found in winter (Jan-Feb) than summer (Mar-May) and rainy (Jun-Jul) season which average
daily gain were 579.5, 554.4 and 552.7 g/d respectively (Luengcharatsuriya et al., 2007) . In buffalo
, leptin hormone play an important role in regulating fat metabolism and growing (Di Palo et al
2005). In addition, glucose to leptin ratios were used as a new diagnostic marker in patients with
diabetes mellitus in human ( Baban et al 2010). Therefore, attempts were made to study the effects
of L. spinosa thw. on enhancing growth rate of weaned buffalo in relation to the change of plasma
leptin and glucose of weaned female murrah x mwamp buffaloes in winter summer and rainy
season.
947

Experimental animals
The study was carried out at the Murrah farm, Chachoengsao Province in the eastern part of
Thailand, belonging to Mrs. Runchuan Hengtrakulsin during Jan-July 2006. Six murrah x swamp
buffaloes between 9 -12 months old and 115 – 156 kg body weight were selected for this study.
All animals were randomly assigned by age into two groups, control group (n=3) and treatment
group (n= 3). The treatment group was fed with an additional 30 g. of dry powder L. spinosa
Thw./animal /day to concentrate everyday in the morning (7.00 am.) for 7 months during late
winter ( Jan-Feb) summer (Mar-May) and early rainy season(Jun-Jul).
Source of plants
The whole plants (leaves, rhizome and root) of L. spinosa Thw. were collected from
Suphanburi province (Middle part of Thailand) and authenticated by comparison with the
herbarium specimens from Department of Botany, Faculty of Science, Chulalongkorn University,
Thailand.
Preparation of L. spinosa Thw .for feeding the animals
The fresh plants of L. spinosa Thw. were blended and dried at 40 oC in hot air oven
(moisture 10-13 %) and girded as powder then weighing in the amounts of 30 g/animal/day. It was
then mixed in concentrate which contained soy source meal, cassava and cassava leaves and fed
to the animals in the morning (7.00am.)
Blood collection
Plasma sample was collected at 9.00 am. every 10 days during January – July . All plasma
samples were determined for leptin and glucose.
Plasma glucose and Leptin analysis
All plasma samples were assayed for glucose by the glucose liquicolor GOD-PAP Method
(Human Gesellschaft fur Biochemica und Diagnostica MbH,Germany). Plasma concentration of
leptin was determined using a multispecies RIA kit (LINCO) Research Inc, St. Louis Mo. USA.
(Camparnile et al 2001and Delavaud et al 2002). The sensitivity of the leptin assay was 2.8 ng/mL
and the intra- and inter- assay coefficients of variation were 3.66 and 9.89 % respectively.

Effects of L.spinosa Thw. on plasma Leptin and glucose levels.
Circulated concentrations of leptin in control animals were 4.69+1.37,ng/mL 7.01+2.03
ng/mL and 6.94+3.44 ng/mL in winter summer and rainy season respectively (Table 1). a
Plasma leptin concentration of animals on 30 g of dry powder of L. spinosa /animal/day group
were 3.73+ 1.62ng/mL, 7.73+2.08 ng/mL and 6.14+3.21 ng/mL in winter summer and rainy season
respectively. These were affected by different season as shown by means + SD plasma leptin of
control animals in winter which were higher than in rainy season and summer as well as in treated
animals. These occurrences also have been reported in mature ovariectomized cows, serum
concentrations of leptin increased by 34% from early winter to the summer solstice and remained
unchanged throughout the remainder of the year (Garcia et al 2002). It was found that the
treatment with 30 g of dry powder of L. spinosa /animal /day could reduce plasma leptin in
buffalos in winter and rainy season and it moderately increased in summer season compared to the
control animals (Table 1). Plasma leptin is positively related to feeding level in adult cattle and that
an effect of meal intake could be mediated by glucose and (or) ketone bodies. (Delavaud et al
2002). In buffalo, heifers fed the high energy diet had greater circulating concentrations of
metabolic substrates (glucose, total cholesterol and HDL cholesterol) and metabolic hormones
(insulin, glucagon, leptin and T3). Thus plasma glucose concentrations were measured to evaluate
the effects of L. spinosa Thw. Similar results were found in our study, greater glucose and plasma
leptin in treated animals in summer. The reduction in plasma glucose levels correlated to lower
plasma leptin were found in animals in rainy season. In buffalo, a reduction in plasma leptin was
due to low energy diet feeding (Campanile et al 2010). In addition, it was also reported in cattle
948
with under nutrition (Ahima and Flier,2000). The declines in plasma leptin with higher plasma
glucose were found in treated animals in summer season. This is in contrast to other studies
(Delavaud et al.,2002). In the present study, the plasma glucose concentrations were not different in
both control ( 58.50-66.53 mg/dl) and treatment (62.19-68.03 mg/dl) groups. This may be
demonstrated that the glucose clearance rate was greater in heifer calves (Depew et al 1998) which
was unaffected by treatment with L.spinosa. An interest observation was average daily gains of
swamp buffalo which were 579.5, 554.4 and 552.7 g/d in winter, summer and rainy season
respectively. Association to our finding that plasma glucose / leptin ratio was greater in winter than
in summer and rainy season both in control and treatment animals.
These data provide evidence for specific changes in control and the treatment with
30 g of dry powder of L. spinosa /animal /day in winter, summer and rainy season of six female
weaned murrah x swamp buffalo crossbreed calves. The levels of plasma leptin were 4.69, 7.01 and
6.94 in winter, summer and rainy season respectively. Plasma leptin was decreased in winter and
rainy season but was moderately increased in summer. While plasma glucose/ leptin ratios were
elevated in winter, the reduction was found in summer and rainy season. The knowledge from this
point of views was convincing for further study to identify the glucose/leptin ratio as an indicator
for prediction of growth rate in buffalo and plasma insulin should also be studied.
REFERENCES
Ahima S.R. and J.S. Flier 2000. Leptin Annu.Rew.Physiol.62: 413- 437.
Baban R.S., K. A. K. Kasar and I.N. Al-Karawi. 2010. Fasting glucose to ratio as a new diagnostic
marker in patients with diabetes mellitus. Oman Medical Journal 25:269-275.
Block S.S., J.M. Smith, R.A. Ehrhardt, M.C. Diaz, R.P. Rhoads, M.E.Van Amburgh, and Y.R.
Boisclair.2003. Nutritional and developmental regulation of plasma leptin in dairy cattle.
J.Dairy Sci. 86:3206-3214.
Campanile G., P.S. Baruselli, D. Vecchio, A. Prandi, G. Neglia, N.A.T. Carvalho J.N.S. Sales, B.
Gasparrini and M.J. D’Occhio. 2010.Growth, metabolic status and ovarian function in
buffalo (Bubalus bubalis) heifers fed a low energy or high energy diet. Anim.l Reprod. Sci.
122: 74-81.
Delavaud C., A. Ferlay, Y. Faulconnier, F. Boequier, G. Kann and Y. Chilliard.2002.Plasma leptin
concentration in adult cattle: Effects of breed, adiposity, feeding level, and meal intake.
J.Anim.Sci. 80:1317-1328.
Depew C.L., L.D. Bunting, J.M. Fernandez, D.L. Thompson and R.W. Adkinson 1998
Performance and metabolic response of young daily dairy calves fed diets supplement with
chromium tripicolinate J.of Dairy.Sci. 11: 2916-2923.
Di Palo R., G. Campanile, A. Prandi, P.S. Baruselli, D. Vecchio, N.A.T. Carvalho and Zicarrelli.
2005. Plasma leptin levels in murrah buffalo heifers fed diet with two different energy
levels. Ita.J. Anim. Sci.vol. 4(supp. 2)301-303.
Garcia M.R., M. Amstalden, S.W. Williams, R.L. Stanko, C.D.Morrison, D.H. Keisler,
S.E. Nizielskit and G.L.Williams. 2002. Serum leptin and its adipose gene expression during
pubertal development, the estrous cycle and different seasons in cattle. J.Anim.Sci. 80:2158-
2167.
Luengcharatsuriya K., C. chumcheen and A. Na-Chiangmai. 2007. Factors Affecting on calving
interval of buffalo dams average daily gain of calves. htttp//www.dld.go.th/research
/AHD/2550.
Buranamanus, P. 2001. Thai local herbs for buffalo production. 1st edition , Thai religion
Publishing, Bangkok, Thailand.
949
Table 1. Mean +SD plasma leptin and glucose in winter, summer and rainy season and glucose /
leptin ratio of weaned female murrah x swamp buffalo in control animals and the treatment
animals which was fed with an additional 30 g. of dry powder L. spinosa Thw./animal /day.
Control Orally treatment with L.spinosa

Thw.
Mean + Ratio Mean + Ratio
SD (Glucose/leptin) SD (Glucose/leptin)
Winter (n=15)
Plasma leptin 4.69 + 12 3.73 + 17
(ng/mL) 1.37 1.62
Plasma glucose 58.50 + 62.19 +
(mg/dl) 9.22 6.70
Summer (n=27)
Plasma leptin 7.01 + 9 7.73 + 9
(ng/mL) 2.00 2.08
(mg/dl) 8.17 6.05
Rainny (n=15)
Plasma leptin 6.94 + 10 6.14 + 11
(ng/mL) 3.44 3.21
(mg/dl) 5.36 4.55
950
Rumen Bacterial Diversity of Water Buffalo (Bubalus bubalis) as Influenced by

Concentrate Levels
Chengjian YANG, Caixia ZOU, Xin LIANG, Shengju WEI, Shulu LI, Xianwei LIANG*,
Bingzhuang YANG and Hua LUO

ABSTRACT
the rumen bacterial communities of water buffaloes fed different levels of concentrate. Six adult
female water buffaloes were divided into two groups according to concentrate levels. Accordingly
concentrate levels were assigned as low concentrate level (1.5 kg per day), and high concentrate
level (2.5 kg per day), with forage ad libitum. Samples were taken from each water buffalo before
morning feeding by stomach tube attached to hand air pump. Characterization of bacteria was
achieved using V1-V3 hypervariable regions of the 16S rRNA gene by a 454 GS FLX PLUS
system on six samples. Results showed that 21 and 25 different bacterial phyla were identified in
the ruminal microbiota of water buffaloes fed low concentrate level and highconcentrate level,
respectively. For whatever the contrite level, rumen bacterial community was dominated by
Firmicutes and Bacteroidetes. There were 113 genera of bacterial populations commonly shared
by all six samples, including genera of Lachnospiraceae, Ruminococcaceae, Prevotella,
Butyrivibrio, Succiniclasticum etc., indicating that there is a core microbial community in the
microbial populations of water buffaloes. Lachnospiraceae, Prevotellaceae, Ruminococcaceae
families were highly present and were clearly affected by concentrate levels. The higher
abundance of Lachnospiraceae and the lower abundance of Ruminococcaceae and Veillonellaceae
were found in high concentrate level group. In conclusion, results of this study provided insights
into the bacterial community structure and diversity of water buffalo and the bacterial taxa of
water buffalo appear to be diet related.
Keywords: water buffalo, bacterial diversity, rumen, 454 GS FLX pyrosequencing

951
Effect of Rumen-Protected Methionine and Reduced Crude Protein in Lactating

Mediterranean Buffaloes Diet
Giacomo CONTÒ a, Stefano TERRAMOCCIAa, Francesca CARFÌ a, Marco MAZZI a, Carlo

BOSELLIc, Sabrina Di GIOVANNIa, Antonella CHIARIOTTIa, S.A. HUWSb and Vilma
PACEa
a
Agriculture Research Council (CRA), a Animal Production Research Centre (PCM), 00015,
Monterotondo, Roma, Italy
b
Institute of Grassland and Environmental Research (Ibers), Aberystwyth University, Penglais
Campus SY23 3DA, UK.
c
IZS, Animal Prophylaxis Research Institute for Lazio and Toscana Regions, Via Appia Nuova,
1411, 00178 (Rome) Italy
*Corresponding email: antonella.chiariotti@entecra.it
ABSTRACT
Sixteen Mediterranean homogenous lactating buffaloes were divided into low protein (LP)
and high protein (HP) groups of eight animals each. Groups were fed for 120 days two isoenergetic
diets (0.90 Milk FU/kg DM) with different crude protein (CP) contents and rumen protected
methionine (RPM) supplement. Milk yield and quality were determined. Faecal, blood, and urine
samples were collected for the detection of urea and insulin in plasma; total-N, urea-N, and
creatinine in urine; and total-N and N-digestibility in the faeces. To investigate rumen microbial
diversity four cannulated Mediterranean buffalo cows were fed LP and HP diets according to a
cross over design. Rumen samples, were analyzed for pH, microbial population quantity and
diversity using classical and molecular techniques including DGGE and Q-PCR. Milk yield,
protein, casein and fat content were similar in the two groups. Milk urea level was significantly
lower in LP group. Both urea level in plasma and urea-N in urine were significantly higher in the
HP. The total organic nitrogen excreted detected in urine and manure was higher in the HP group.
The protein level reduction in diet supplemented with RPM seems to negatively affect the growth
fungi and protozoa and methane production can be reduced by reducing rumen ciliate protozoa.
Moreover these results indicate that the use of a low CP, RPM supplemented diet did not negatively
affect milk production or quality, and reduced the nitrogen quantity excreted with urine and faeces,
thus contributing to reduce the impact of buffalo herds on the environment.
Keywords: Mediterranean buffaloes, Rumen-Protected Methionine, rumen microbial diversity
INTRODUCTION
A common approach for lowering nitrogen emissions from dairy cows is lowering the crude
protein quantity as well as supplementation with rumen-protected amino acids in the diet.
Methionine is the main limiting amino acid in lactating cows. Moreover research on the nutrition of
the buffalo is limited compared to bovines, and cannot automatically be extended, mainly because
of marked dissimilarities either at the anatomic and physiological level, or ingestion capacities and
feeds digestion, or ruminal populations and metabolism (Puppo et al. 2002). Buffalo has a
significantly better capacity to utilize low energy and protein feeds; therefore, the dairy cows diets
may provide to buffaloes an unnecessary amount of nutrients and causing pollutant effects
(Campanile et al. 2010). The aim of the present work was to determine, in a Mediterranean buffalo
herd, whether a moderate reduction in the crude protein content of a diet integrated with RPM
would either affect milk production and quality, or metabolic conditions and nitrogen excretions or
rumen microbial variations with respect to an isoenergetic control diet with higher protein content.

952

Sixteen Mediterranean homogenous lactating buffaloes cows (Bubalus bubalis L.) were
divided into low protein (LP) and high protein (HP) groups of eight animals each, milked twice a
day for a four months period. Body condition score (BCS) was determined utilizing the scale of
Wagner et al., (1988) modified for the buffalo species by Campanile et al. (1998). The animals were
fed two isoenergetic diets (0.90 Milk FU/kg DM), administered once a day (table 1). Four
cannulated Mediterranean buffalo cows were fed the same diets, ad libitum, for a three months
period, according to a cross over design.
Milk yield, individual samples of milk, blood, faeces and urine were collected monthly form
each animal. Standard chemical analyses of forages and meals, according to Association of Official
Agricultural Chemists (AOAC, 1980), are shown in Table 1. Milk samples were subjected to the
following analyses: protein and fat content, milk urea (MU) and casein. Mozzarella cheese daily
yield was calculated by Altiero et al. (1989) equation. Faeces were analysed both for total organic
nitrogen and insoluble ash in HCI according to the method of Van Keulen and Young (1977). On
urine samples creatinine concentration, urea nitrogen, and total organic nitrogen were determined.
Blood samples were analysed both for urea determination (BU) and insulin content. Whole rumen
content samples, after 15d diet adaptation and before morning feeding, were analysed both for pH
and microbial population and diversity, using either direct count and cultivation techniques or
molecular techniques including DGGE and Q-PCR. DNA was extracted (BIO101 Fast DNA SPIN
Kit for Soil) and PCR was performed as reported by Huws et al. (2007). Bacterial DNA amplicons
were loaded in equal concentrations onto a 6% polyacrylamide gel with a 35–60% denaturing
gradient, protozoal DNA amplicons were loaded in onto a 8% polyacrylamide gel with a 30–50%
denaturing gradient. Electrophoresis was performed as described by Huws et al. (2007). Absolute
quantification of total eubacteria content, Prevotella spp. was performed by Q-PCR. Statistical
analysis was performed using the GLM procedure (SAS, 2001).

Milk parameters
No significant difference was detected in daily milk yield and quality (Table 2). As
expected, the MU content was higher in the HP group compared to the LP group. No significant
difference was detected in the daily milk yield as reported by Benefield et al. (2009). Campanile et
al. (1998) also found a rapid decrease in the urea level in buffalo milk as a consequence of sharp
decreased dietary CP content, followed by a tendency towards re-equilibration of the values during
the lactation period because buffalo cow adapts itself to lack of protein easier than dairy cow.
Blood parameters
The overall means of BU in LP and HP groups were significantly different (Table 2), while
insulin levels showed the opposite trend and increased with the progress of lactation, similar to what
was found by Accorsi et al. (2005) in dairy cows. Buffaloes utilize ingested nitrogen more
efficiently than cattle, thanks to the rumen microorganisms that have a better capacity either to
synthesize protein from non-protein nitrogen, or to recycle urea in the digestive tract, resulting in
buffaloes being better able to adapt to protein-deficient diets.
Nitrogen excretion
The total urinary nitrogen was always higher in the animals fed with the HP diet compared
to those of the LP group. The same tendency was found for the urea N, with significant differences
in the overall mean (Table 3). Leonardi et al. (2003) noted that RPM supplementation did not
influence the quantity of urine excreted or the urinary or faecal excretion of nitrogen, while
increased CP quantity in the diet caused increased nitrogen concentrations both in the urine and the
faeces. The total nitrogen content of the faeces and the apparent N digestibility were always higher
in HP group, but not significantly (Table 3). Broderick et al. (2008) found higher apparent N
digestibility in dairy cows fed diets with higher protein content. Taking into account the total
manure N, it emerges clearly that the quantity of nitrogen excreted was significantly higher in the
HP group with respect to the LP group.
953
Rumen microflora
No statistical differences were detected in bacterial density both by classical (cfu/ml) and
QPCR technique (16S rDNA) (Table 4), but fungi and protozoa were significantly lower in LP
group. Methanogens associated with ciliate protozoa account for 9-25% of methanogenesis in
rumen fluid and methane production can be reduced by reducing rumen ciliate protozoa (Morgavi et
al. 2010). However, DGGE-ciliate 18S rDNA-based dendograms did not show any clustering by
diet when based either on the Dice and on the Pearson algorithm.
CONCLUSIONS
The reduction of CP quantity in the diet with RPM in lactating buffaloes diet did not
significantly modify milk yield, quality, or potential for cheese-making, for which buffalo milk in
Italy is exclusively earmarked. Moreover it significantly lowered the nitrogen quantity released into
the environment. Nevertheless diet supplemented with RPM seems to negatively affect fungi and
protozoa growth, and methane production can be reduced by reducing rumen ciliate protozoa. These
results suggest that it is possible to reduce the protein level in buffaloes’ diets through the addition
of rumen-protected methionine, leading to a reduction of the environmental impact.
REFERENCES
Accorsi, P.A., N. Govoni, R. Gaiani, C. Pezzi, C. Serena and C. Tamani. 2005. Anim. 40, 217-223.
Altiero, V., L. Moio and F. Addeo. 1989. Sci. Tecn. Latt-Cas. 40, 425-433
Benefield, B.C., R.A. Patton, Stevenson and T.R. Overton. 2009. J. Dairy Sci. 92, 4448-4455.
Broderick, G.A., M. J. Stevenson, M.J. Patton, R.A. Lobos, N.E. Olmos and J.J. Colmenero. 2008.
J. Dairy Sci. 91, 1092-1102.
Campanile, G., C. De Filippo, R. Di Palo, W. Taccone and L. Zicarelli. 1998. Livest. Prod. Sci. 55,
135-143.
Campanile, G., G. Neglia, D. Vecchio, R. Di Palo, B. Gasparrini and L. Zicarelli. 2010. CAB
Reviews: Perspective in Agriculture, Veterinary Science, Nutrition and Natural Resources 5,
1-8.
Huws, S.A., J.E. Edwards, E.J. Kim and N.D. Scollan. 2007. J. Microb. Meth. 70, 565–569
Leonardi, C., Stevenson, M., Armentano, L.E., 2003. J. Dairy Sci. 86, 4033–4042.
Morgavi, D.P., E. Forano, C. Martin and C. J. Newbold. 2010. Animal. 4,1024-1036
Puppo, S., S. Bartocci, S. Terramoccia and F. Grandoni. Amici, A., 2002. Anim. Sci. 75, 323-329.
Van Keulen, J. and B.A. Young. 1977. J. Anim. Sci. 44, 282-287.
Wagner, J. J., K.S. Lusby, J.W. Oltjen, J. Rakestnrw, R.P. Wettemann and L.E. Walters. 1988. J.
Anim. Sci. 66, 603-612.
954
Table 1. Chemical composition (g/kg DM) and net energy (Milk FU/kg DM) of feeds utilized in
experimental diets.
DMa CP EE NFC Ash NDF ADF Lignin NE

Feedstuffs
Maize silage 314.9 75.9 20.3 313.4 64.2 526.2 305.2 37.3 0.85
Lucerne hay 2nd cut 871.9 173 11.7 255.1 86.9 473.3 388.8 90.0 0.67
Soybean meal 896.5 479.9 4.4 277.7 70.7 167.3 90.4 24.8 1.15
Corn meal 880.3 88.2 30.7 743.4 14.9 122.8 39.0 16.4 1.24
Vitamin/mineral mixb 962.0 105.8 72.5 137.4 463 221.8 100.7 42.0 0.64
Diets
LP 632.5 142.4 19.5 370.9 69.6 397.6 253.2 46.0 0.90
HP 633.1 156.1 18.6 354.6 71.5 399.2 255.0 46.3 0.90
a
DM=dry matter; CP=crude protein; EE=ether extract; NFC=non fibrous carbohydrates = [1000-(Ash+CP+EE+NDF)];
NDF=neutral-detergent fibre; ADF=acid-detergent fibre; Lignin = lignin determined by sulfuric acid method; NE=net
energy.bVitamin/mineral mix contains per kg of DM: vit.A 500000 UI, vit.D 50000 UI, vit.E 600mg, vit.PP 3000mg,
Ca 38%, Fe 1600mg, Cu 300mg, Zn 3000mg, Mn 1500mg, I 7.00mg, Co 5.00mg, Se 6.00mg.
Table 2. Yield and chemical composition of milk and blood parameters.
Item LP HP
I II III IV Means I II III IV Means SEM group
Milk yield (kg/d) 9.16 8.57 7.01 5.92 7.63 9.78 8.24 6.89 5.49 7.69 2.23 ns
Milk fat (g/kg) 82.06 91.06 97.75 99.75 92.81 88.5 96.38 94.93 98.93 94.53 8.60 ns
Milk protein (g/kg) 46.08 44.45 47.09 47.30 46.21 44.72 48.12 49.13 48.12 47.42 2.90 ns
Yield mozzarella
2.28 2.18 1.99 1.65 2.01 2.49 2.28 1.92 1.52 2.07 0.53 ns
cheese (kg/d)a
Milk urea (mg/100ml) 39.76 32.35 26.44 45.28 35.53 48.38 37.11 31.41 50.67 41.09 6.88 **
Casein (g/kg) 37.98 36.26 38.82 39.50 38.00 37.22 38.64 41.01 40.10 39.10 2.58 ns
Blood urea (mM/l) 5.72 6.22 5.28 6.12 5.88 7.65 7.72 6.10 6.78 7.01 0.71 ***
Blood insulin (µg/l) 0.82 0.84 0.99 1.19 0.98 0.73 0.84 1.01 1.13 0.91 0.35 ns
a
Mozzarella cheese kg = Milk kg * [3.5 * milk protein (%) + 1.23 * milk fat (%) - 0.88] / 100;
** P<0.01;*** P<0.001.
Table 3. Nitrogen excretion from urine and faeces and apparent N digestibility.
Item LP HP
I II III IV Means I II III IV Means SEM group
Urine
Total N (g/d) 190.8 158.9 157.7 219.8 179.2 211.4 202.9 186.5 232.5 207.5 34.7 **
Urea N (g/d) 112.3 132.2 104.9 186.9 135.2 145.7 168.4 132.6 192.3 156.4 27.1 **
Faeces
Total N (g/d) 106.2 147.8 162.8 118.7 135.5 128.7 152.6 178.5 148.5 151.0 41.7 ns
N Digestibility
67.2 55.7 62.3 55.9 59.9 70.1 63.9 71.6 64.0 67.7 12.3 ns
(%)
Total manure
296.9 305.9 320.5 338.4 314.6 340.0 355.6 365.0 380.9 358.6 17.4 ***
N (g/d)
** P<0.01.; *** P<0.001.
955
Table 4. Effect of diet on rumen populations.
group pH PZ F TVB CB P Total eubacteria Prevotella spp
CFU/ml log10 pg g-1 DM
HP 6.6 1,7 .108* 2,3.107* 2,6.1011 2,2.109 3,0.108 8.04 2.65
LP 6.6 7,2 .107* 1,2.107* 4,6.1011 3,0.109 3,2.108 7.83 2.77
* P<0.05; PZ (protozoa), F (fungi), TVB (total viable bacteria), C (cellulolytic bacteria), P (proteolytic bacteria)
956
Effect of Varying Levels of NDF on Voluntary Intake, Nutrients Digestibility for

Nili Ravi Buffalo Heifers
Saeed AHMEDa*, Makhdoom Abdul JABBARa, Anjum KHALIQUEa, Khalid JAVEDb,

Muhammad ABDULLAHb and Faisal SHAHZADb
a
Department of Animal Nutrition, University of Veterinary & Animal Sciences, Lahore,
Pakistan,bDepartment of Livestock Production, University of Veterinary & Animal Sciences,
Lahore, Pakistan
*Corresponding email: saeed.ahmed@uvas.edu.pk
ABSTRACT
This study was conducted to determine the full advantage of Neutral Detergent Fiber (NDF)
focusing on its quality to ensure maximum dry matter (DM) intake in Nili Ravi buffalo heifers. For
this purpose, effect of varying levels of NDF on nutrient intake, nutrient digestibility, in situ DM &
NDF digestibility and weight gain were carried out in Nili Ravi buffalo heifers for 90 day at Buffalo
Research Institute Pattoki, Pakistan. Five diets A, B, C, D, E contained different levels (23%, 28%,
33%, 38% and 43 %) of NDF contents, respectively were prepared. Results of this study showed
significant difference in nutrient intake e.g. crude protein (CP), DM and NDF intake and resulted
higher in heifer group of diet D than others. The nutrient digestibility e.g. DM and NDF were
significantly different among treatment groups and higher in the diet B and D than others. In situ
DM and NDF digestibility were significantly highest in the group B and C as compared to others.
The live weights were significant different among the buffalo heifers offered 5 diets varying in
NDF concentration. The live weight gain per day was found highest in the group fed on diet D than
others. It was concluded that 38% level of NDF in diets improved growth performance in Nili Ravi
buffalo heifers.
Keywords: Nili Ravi buffaloes, NDF, Voluntary intake, NDF digestibility, weight gain
INTRODUCTION
There is tremendous growth in the livestock population (4.04 %) contributing 55% share of
agriculture value added and 11.6 % of total national GDP. But the fodder production on total
cultivated land has been reduced to 12.6 % (Anwar et al., 2012). Fodder shortage, insufficient feed
resources and poor feeding practices are one of the main reasons for the poor production of animals
which lead to imbalanced availability of nutrients for animals. Comparable to the cattle, the buffalo
has capability to digest the low quality roughages (high NDF) more efficiently for better growth.
Particularly, NDF (cellulose, hemicellulose and lignin) increases rate of passage and provide
intestinal bulk. In order to take full advantage of NDF, it is necessary to focus on its quality and
particle size to ensure maximum dry matter intake (Jane, 2008). The heifers and dry cows can
consume the forages that may be having lower value in neutral detergent fiber digestibility. Such
animals do not have high energy demands and low neutral detergent fiber digestibility forages can
work well into their management system. The objective of this study was to determine the optimum
level of NDF on voluntary intake and nutrients digestibility in Nili Ravi buffalo heifers.
The present research work was conducted to assess the optimum fiber requirements in Nili
Ravi buffalo heifers at Buffalo Research Institute Pattoki (BRI), Pakistan. Twenty five Nili Ravi
buffalo heifers having age 18-20 months, body weight (200 ± 20 kg) were selected and randomly
divided into 5 groups in a completely randomized design. Five isocaloric and isonitrogenous wheat
straw based total mixed rations (TMR) with different levels of NDF were prepared and fed for 90

957
days. Daily feed intake and weekly body weight observations were recorded. Collected fecal
samples were chemically analyzed to calculate DM, CP (AOAC, 2000), NDF, ADF (Van Soest et
al., 1991) and digestibility (Marghazani et al., 1999). Similarly three adult fistulated Nili-Ravi
buffalo steer (average body weight 425±23 kg, ruminally fistulated) were used to determine in situ
DM and NDF digestibility using the in situ technique with three replicates per experimental Total
Mixed ration (TMR) for 48 Hours (Hoffman and Bauman, 2003). Data was statistically evaluated
through Analysis of Variance technique in completely randomized design (Steel et al., 1997) using
statistical analysis software (SAS, 1997) and comparison of means were determined with the
Duncan’s Multiple Range Test (Duncan, 1955)

Nutrients intake
Feed intake in animals is affected by a number of factors like physical form of feed, levels
of nutrients and fiber contents. Maximum intake of dry matter (9.95±0.08 kg/day) was observed in
the group fed on diet D which contained 38% NDF level whereas minimum intake
(6.23±0.023kg/day) was observed in the group fed on diet A (Table 1), Similarly, in the group B &
C; DM intake increased further which was 6.76±0.076 Kg/day and 8.58±0.082 kg/day respectively.
However, decline trend in DM intake was noted in animals fed on diet E which contained highest
level of NDF as compared to all other dietary groups. Increasing trend of dry matter intake up to
Group D is due to increase in NDF level for the provision of optimum environment for the rumen
micro flora. But the reduction in dry matter intake in the group E may be due to the rumen fill effect
due to high NDF level. These results are in accordance with the findings of Van Soest (1994) and
Forbes (1995) who reported that ruminant’s reticulo-rumen volume determines the potential
physical intake of forages. The intake of NDF and CP was maximum in the group D i.e.
3.77kg/day, 1.19Kg/day and minimum in the group A i.e., 1.42kg/day, 0.74 kg/day respectively. A
significant difference (P<0.001) was observed among treatment groups. Similarly, same findinding
were also obtained by Arelovich et al. (2008) who investigated the relationship between NDF (% of
dietary dry matter) and intake. They also found that total DM decreased sharply in the dairy cattle
as NDF concentration increased from 22.5 to 45.8 % dietary NDF.
Nutrient Digestibility
Dry matter digestibility was higher (69.3%) in groups B as compared to others. This may be
due to increase in dietary NDF level which enhanced the scratching effect of fiber in
gastrointestinal tract (Sandoval et.al., 1987). Reduction in dry matter digestibility in the groups C,
D and E, may be due to high level of ADF contents, because the major part of ADF is non-
digestible due to high lignin contents. These results are also in line with the findings of Tjardes et
al. (2002) and concluded that high fiber treatment reduced total tract digestibility of DM. Basically
the lignin is correlated with the digestibility of hemicelluse and organic matter as described by
sullivan (1966). However CP digestibility was non-significant among the treatment groups which
may be due to is nitrogenous composition of the ration. The digestibility of NDF was maximum in
the group fed on diet B comprising of 28% NDF level and reduced with increase in NDF levels as
in groups C, D and E. These results are in line with the findings of Van Soest (1973) who reported
that reduced digestibility with increase NDF contents is attributed to decreased digestibility of
fibrous components.
In situ dry matter and NDF digestibility
Results showed significance difference among the treatment groups. In situ dry matter
digestibility was maximum in the group B fed on 28% NDF based diet and minimum in the group
fed on diet E. The NDF digestibility was maximum in the group fed on diet B & C and reduced
with the increase in NDF levels such as in the groups D and E. This may be due to optimum
environment in the rumen in group B & C. These results are in line with the findings of Beck et al.
(2007) who compiled a report on in situ dry matter degradability of the hays and concluded a
decreased in the extent of degradation of dry matter and neutral detergent fiber of hays with
increase in NDF levels. Similar report was also published by NRC (2001), as NDF contents
958
increased the lignin contents were also increased, lignification within plant material is negatively
associated with NDF digestibility.
Weight gain
Weight gain is good indicator of performance of animals on particular diets. Buffalo heifers
on all the diets gained weight. Comparatively high weight gain was on diet D due to high DM
intake. As the NDF level was further increased (as in the case of diet E with 43% NDF level) the
weight gain was reduced. These were due to low intake of DM. These results are in line with the
findings of Galyean and Defoor (2003) who reported that increasing the forage NDF level reduced
cattle performance in respect of weight gain. Similar findings were also reported by Tjardes et al
(2002) who conducted an experiment on Holstein steers and offered high and low fiber diets. They
concluded that Holstein steers consuming low fiber diets had greater efficiency of gain (1.25kg/day)
than those receiving high fiber diets (1.03kg/day).
CONCLUSION
Application of the TMR technology to raise the heifers to get the optimum performance in
terms of weight gain and feed efficiency was subjected in this trial in Pakistan. It was concluded
that 38% level of NDF in Nili Ravi buffalo heifers to get the optimum growth performance would
be a land mark in the sub-tropical climate of Pakistan.
ACKNOWLEDGEMENT
The author wishes to express his sincere appreciation to Higher Education Commission,
Pakistan for provision of funding for this project at BRI Pattoki, Pakistan.
REFERENCES
Anwar M., M. Akmal, A. Shah, M. Asim and R. Gohar. 2012. Growth and yield comparison of
perennial grasses as ranfed fodder production. Pak. J. Bot. 44(2): 547-552.
Arelovich. H. M., C. S. Abney, J. A. Vizcarra and M. L. Galyean. 2008. Effects of Dietary Neutral
Detergent Fiber on Intake of Dry Matter and Net Energy by Dairy and Beef Cattle: The
Professional Animal Scientists. 24:375-383.
AOAC. 2000. Official methods of analysis, 17th Ed., Association of official analytical chemists,
Washington, DC, USA.
Beck, P. A., S. Hutchison, C. B. Stewart, J. D. Shockey and S. A. Gunter. 2007. Effect of crabgrass
hay harvest interval on forage quality and performance of growing calves fed mixed diets. J.
Anim. Sci. 85(2):527-35.
Duncan, D. B. 1955.Multiple range and multiple F test. Biometrics.11:1-42
Forbes, J. M. 1995. Voluntary food intake and diet selection in farm animals. CAB International.
Oxon, UK. 2, 3, 4 and 9.
Galyean, M. L. and P. J. Defoor. 2003. Effects of roughage source and level on intake by feedlot
cattle. J. Anim. Sci. 81(2):8-16.
Hoffman, P. C. and L. M. Bauman. 2003. Strategies to improve milk yield of lactating dairy cows
fed red clover silage. J. Anim. Sci. 19:178-187.
Jane, A. P. 2008. Fiber in Beef Cattle Diets. Mississippi State University Publication 2489.
Marghazani, I. B., G. Habib, M. M. Siddiqui. 1999. Nitrogen retention and nutrients digestibility in
sheep given a basal diet of Sorghum hay supplemented with protein of varying
degradabilities. Sarhad J. Agric.15:381-386.
National Research Council. 2001. Nutrient Requirements of Dairy Cattle. 7th ed. Natl. Acad. Press.
Washington. DC. SAS Institue Inc. 2004. SAS System for mixed models. SAS Institue
Inc., Cary, NC.
Steel, R. G. D., J. H. Torrie and D. A. Dickey. 1997. Principles and Proceduresof Statistics. A
biometricalapproach. 3rd Ed., McGraw Hill Book Co.,New York, USA
959
Sandoval, R. A. T. K. Nielsen and P. H. Sorensen. 1987. Effects of fibre on nutrient digestion and
time of passage in growing pigs. Acta Agriculturæ Scandinavica 37: 367-373.
Tjardes. K. E., D. D. Buskirk, M. S. Allen, R. J. Templman and S. R. Rust. 2002. Neutral detergent
fiber concentration in corn silage influences dry matter intake, diet digestibility, and
performance of Angus and Holstein steers. J. Anim. Sci. 80:841-846.
Van Soest, P. J. 1973. Revised estimates of the net energy values of feeds. Proc.Cornell
Nutr.Conf, NY. U.S.A
Van Soest P J. 1991. Nutritional ecology of the ruminant. 2nd edition. Cornell.
Van Soest P J. 1994. Nutritional ecology of the ruminant. 2nd edition. Cornell.
Table 1. Mean (±S.E) of nutrients intake, nutrient digestibility, in-situ digestibility and weight gain
in Nili Ravi buffalo heifers at different NDF levels.
Groups A (23%) B (28%) C (33%) D (38%) E (43%) P≤0.05

(NDF%)
Nutrient Intake (Kg/day)
DM 6.23e±0.023 6.76d±0.076 8.58b±0.082 9.95a±0.089 7.78c±0.056 <0.001
CP 0.74d±0.024 0.811cd±0.03 1.02b±0.036 1.19a±0.050 0.936bc±0.038 <0.001
NDF 1.42e±0.04 1.88d ±0.13 2.82c ±0.10 3.77a±0.16 3.25b±0.09 <0.001
Nutrients digestibility %
DM 62.76b±0.49 69.38a±2.61 61.13b±0.94 59.09b±1.81 61.73b± 1.20 <0.05
CP 72.16±0.6 71.1±1.15 72.16±0.92 71.76±0.90 72.16± 0.44 NS
NDF 58.36b±0.36 62.6a±0.32 56.3c±0.65 56.06c±0.54 52.53d±0..26 <0.001
In situ dry matter and NDF digestibility %
DM 67.65c±2.73 78.16a±0.52 74.96ab±1.46 69.49bc±0.62 65.80c±2.98 <0.01
NDF 57.5d±0.60 67.8a ± 0.6 67.16a ± 0.73 64.63b±0.44 61.26c±0.69 <0.001
Weight Gain
Initial 235 ± 6.89 218 ± 2.54 225 ± 5.00 228 ± 6.63 224 ± 6.78 NS
Final 316bc ± 9.24 313c ± 9.05 335abc 358a ± 6.04 344ab ± 6.0 <0.05
±13.96
Gain(Kg/day) 0.91c ± 1.09 ± 0.01 1.23bc ± 0.10
bc
1.44a ± 0.03 1.33ab ± 0.06 <0.01
0.03
Change % 35.26c±1.30 45.46bc±4.70 49.88ab ± 57.49a ± 2.59 54.17ab ± 3.99 <0.01
3.82
*Means with different superscripts within same row are significantly different (P<0.05); Sig. =
significance NDF= neutral detergent fiber; ADF= acid detergent fiber; DM= dry matter; CP= crude
protein
960
Effects of Augmented Feeding with By-passed Amino Acid and Slow-released Non-
Protein Nitrogen Supplements on Milk Peak, Lactation Persistency and
Post-partum Reproductive Performance of Brazilian Buffaloes
Daniel Lopez AQUINOa, Mike Venturina DEL ROSARIOa, King Frhel VERGARAa and
Libertado Concepcion CRUZa
a
Philippine Carabao Center (PCCl, Science City of Munoz, Nueva Ecija, Philippines. 3120
*Corresponding email: dalla_1358@yahoo.com
ABSTRACT
Twenty five (25) pregnant and primiparous imported Brazilian buffaloes were allotted to five
dietary treatments namely; without (T1) or with (T2) augmented feeding plus supplementary by-passed
amino acid (T3); slow-released NPN (T4) and its combination (T5) to assess their milk peak, lactation
persistency and post-partum reproduction under intensive system of management.. The cows were
assigned in a randomized complete block design with five cows per treatment and each cow serves as
individual replicate. The dairy buffalo ration composed of corn silage (67.3%) and rice straw (9.5%)
and dairy concentrate pellets (23.2%). The supplementary concentrates, by-passed amino acid and
slow-released NPN were given at the rate of 0.5 kg/kg milk production, 100 grams and 50
grams/hd/day, respectively; for six months of lactation. The feed intake, nutrients utilization and
digestibility were also evaluated. Results indicated that augmented feeding alone or its combinations
with supplementary by-passed amino acid and slow released NPN gave significantly higher (P<0.05)
peak milk of 12 kg/d and 12.5 kg/d, respectively; than the cows without augmented feeding,
augmented feeding plus bypassed amino acid and slow released NPN. The milk peak was recorded at
68 (T1) and 71 days (T5) lactation periods of the cows. There were no significant differences of the
dietary treatments on the average lactation persistency of the cows but the data gathered (91.8%) was
closer to the reported ideal lactation persistency of 95%. No significant differences were also observed
on the post-partum reproductive performance, feed intake, digestion coefficients and the feed cost to
produce a kilogram milk of the cows however, at 146 days service period using artificial insemination,
80% of the buffaloes were already confirmed pregnant.
Keywords: Augmented feeding, by-passed amino acid, slow-released non-protein nitrogen, post-
partum reproduction
INTRODUCTION
The peak of milk production is an important indicator in developing appropriate nutrition for
dairy cows. Peak yield is achieved when the cow reaches the highest milk yield during its entire
lactation period. Normally, milk peak is exhibited at 45 - 90 days in milk (DIM), followed by the
decline in milk yield over time until the animal completely dry’s-off, (Anwar, 2009). For every
kilogram increase at milk peak, a total forecast of 200 to 250 kilograms additional milk is expected per
lactation of a cow, (Khols, 2002). Persistency is the rate of decline in the milk yield following the peak
of production of the cow. It is express in percentage unit by dividing the current monthly milk
production by the last month's milk yield of the cow. The ideal persistency of lactation of a cow is 95%
of the previous month, (Drackley, 1999). After attaining the peak-milk, the milk yield gradually drops
by 0.2%/day during the first calving and about 0.3% decline/day in the succeeding lactations, (Capuco
et al., 2003). Feeding by-passed protein or post-ruminal infusion of Methionine and Lysine to dairy
cows increased milk yield, % crude protein (3.15%) and milk fat (3.88%) contents, Socha et al., (1994b).

961
Utilization of slow-release NPN supplement to dairy buffaloes led to improvements in milk production (1.0
kg/day) and milk fat contents. In this study, the peak-milk, lactation persistency and post-partum reproductive
performance of Brazilian dairy buffaloes were evaluated when these were subjected to challenge feeding with
by-pass amino acid and or its combination with slow release NPN as sources of rumen undegradable nitrogen
(RUN) and rumen degradable nitrogen (RDN) supplements.

Twenty five (25) pregnant and primiparous Brazilian buffaloes were allotted to five dietary
treatments namely; without (T1) or with (T2) augmented feeding (AF) plus supplementary by-passed
amino acid (BPAA), (T3); slow-released non-protein nitrogen (SR-NPN), (T4) and its combination
(T5). The cows which on their last 2 months of pregnancy were assigned in a randomized complete
block design with five cows per treatment and each cow serves as individual replicate.
Feeding management
The dairy buffaloes were raised in an individual stall equipped with feed bunk and automatic
waterer. The basal roughage which composed of corn silage and rice straw were offered ad libitum.
Challenge feeding was carried-out from calving and onwards until the milk peak was achieved at
approximately120 days in milk, by giving 0.5 kg dairy concentrate pellet for every kg increased in
milk yield. Supplementary by-passed amino acid and slow-released NPN, were offered at 100 grams
and 50 grams/hd/day, respectively.
Milking management
The lactating cows were milked twice a day using a 2 x 6 tandem type milking parlor. The
morning milking starts at 4:00AM and 3:00PM for the afternoon milking. The daily milk production of
the cows was recorded and monthly milk samples were collected and analyzed for nutrient
composition and somatic cell counts using milko-scan and somatic cell counter.
Breeding management
The buffalo cows were monitored for their post-partum estrus, service period and conception
rate started one month after calving. Estrus observation was done using a vasectomized bull with a
chin ball. The cows that were observed in heat were artificially inseminated, (AI). Pregnancy diagnosis
is done through rectal palpation at 45-60 days after AI. The cows that do not conceived after 3
inseminations were subjected to natural mating by a cleaned-up bull.
Health management
The health management program at the farm was properly instituted such as vaccination against
Hemorraghic Septicemia and Sura. Monitoring the incidence of mastitis using CMT and somatic cell
analysis among cows was regularly done at the farm.

Effects of dietary treatments on the changes in body weight of dairy buffaloes
Immediately after calving, all the dairy buffaloes exhibited an average body weight (BW) loss
of 32.1 kg. The cows in T4 and T2 registered the lowest BW of 461.2 and 472.2 kg, respectively, The
low BW of the cows was attributed to the highest BW loss of 45 kg (T4) and 37.2kg (T2), after calving,
Table 1. The effects of augmented feeding with BPAA and SR-NPN was noticed when the decline in
BW of the cows was compensated and gained in weights were observed as the lactation period
progresses, Figure 1. Daily gained in weights were expected because the cows were all primiparous
and had not attained yet their mature weights (500-550 kg). The highest gainers were recorded by cows
in T2 and T5 with 0.45 and 0.47 kg ADG, respectively. Augmented feeding in combinations with
BPAA and SR-NPN (T5) provided adequate supply of energy and protein in the form of rumen
degradable nitrogen from the SRNPN and rumen undegradable amino acid from the BPAA that led to
more tissue and fat deposition among cows causing higher ADG.
962
Effects of dietary treatments on milk production, milk peak, persistency and milk quality
The dairy buffaloes subjected to AF alone and AF+BPAA+SRNPN had the highest 305 days
adjusted total milk yield of 1,433.2 kg, and 1,586.4 kg, respectively, Table 3. These treatments had
significantly higher (P>05) adjusted total milk production compared to the buffaloes in the control
group (1,120.9kg) and those cows subjected to AF+RNPN, (1,225.8kg) and AF +BPAA (1,280.6 kg).
Buffaloes given AF plus SRNPN had the highest milk peak of 12.5 kg/day and this was observed at 66
days in milk. No significant differences in milk peak were observed among the buffaloes assigned in
AF alone, AF plus BPAA, SRNPN or its combinations but when compared with the control cows,
these had significantly higher milk peak. The control cows had milk peak of only 8 kg/day recorded at
78 days in milk. In this study, the average milk peak of the cows was on the 74 DIM which is
consistent with the report of Anwar, (2009) that milk peak in buffaloes is exhibited during 45 – 90
days milk. In terms of persistency of lactation there were no significant differences among the dairy
buffaloes assigned in the five dietary treatments evaluated. The observed lactation persistency of 92%
was in agreement and was very close to the reported ideal lactation persistency of 95% in buffaloes,
(Drackley, 1999).
On milk quality, the animals given challenged feeding with supplementary BPAA had
significantly higher (P>.05) milk fat (9.72%) and total solids (18.98%) contents compared to the rest of
the treatments, Table 4. Milk protein, lactose and SNF were not significantly affected by other dietary
treatments including the control group. The highest somatic cell counts (SCC) of 151.803/ml of milk
was observed from buffaloes given AF +SRNPN. It can be noted that the gathered SCC values were
acceptable and has passed the SCC standards (20003 /ml) set for buffalo milk.
Effect on post partum reproduction of the buffaloes
There were no significant differences on the effects of AF plus BPAA and SRNPN
supplements on the post-partum reproduction in terms of service period and conception rates of the
cows. The earliest post-partum estrus of the experimental buffaloes was observed at 47 days, however;
with an average of 146 days service period, 80% of the post-partum buffaloes were already confirmed
pregnant through AI, Table 5.
Effect of AF plus BPAA and SR-NPN on feed intake of dairy buffaloes
There were no significant differences observed on the daily feed intake and feed intake as
percent of the body weight of the dairy buffaloes, Table2. On as fed basis, the control buffaloes had the
highest intake of 4.9% of the BW but this has no significant differences when compared to other
dietary treatments. The corn silage, rice straw and dairy concentrate pellets represent 67.3, 9.5 and 23.
2 %, respectively, of the total ration of the dairy buffaloes.
REFERENCES
Alexiev, A. 1998. The Water Buffalo. Sofia, Bulgaria: St. Kliment Ohridski University Press.163 p.
Anwar, R.M., P.J. Cain, P. Rowinson, M.S. Khan, M. Abdullah and M.E. Babar. 2009. Factors
Affecting the Shape of the Lactation Curve in Nili-Ravi Buffaloes in Pakistan Pakistan J.
Zool. Suppl. Ser., No.9, pp. 201-207.
Capuco, A.V.1, S.E. Ellis, S.A. Hale, E. Long, R.A. Erdman, N, X. Zhao, and M.J. Paape.
2003.Lactation persistency: Insights from mammary cell proliferation studies. J. Anim. Sci.
2003. 81(Suppl. 3):18–31.
Drackly, J.K. 2002. Nutritional Management for Transition Dairy Cows, Department of Animal
Sciences, University of Illinois, Urbana, IL 61801.
Habib, G. 2009. Nutritional Management Strategies to Improve Milk Production in Buffaloes. Pakistan
J. Zool. Suppl. Ser., No.9, pp. 533-544.
Kohls, D. 2002. Feed and Nutrition Consultant Form-A-Feed, Inc. and TechMix, LLC
963
Leonards, C. 2008.COLUMNS. Dairy Herd Management Specialist for UVM Extension based in the
Newport, Vt.
Russell, J.B. and H.B. Strobel.1989. Effect of ionophores on ruminal fermentation. Appl. Environ.
Microbiol. 55, 1–6.
Satter, L. D. and R.R. Roffler. 1977. Protein requirement and non-protein nitrogen utilization of
Ruminant. Trop. Anim. Prod. 1977 2:31
Vanbaale, M.J. 2005. Impact of increased milking frequency in early lactation and its effect on
lactation persistency with and without rbST. Proceedings of the 7th Western Dairy Management
Conference, March 9-11, 2005.
Table1. Effects of AF plus BPAA and SRNPN on body weight of cows before and after calving.
Pre-calving Post-calving BW Lost,

Treatment N
BW, kg BW, kg kg
W/o augmented feeding (control) 5 514.2 486.2 28.0
Augmented Feeding (AF) 5 509.4 472.2 37.2
AF + Bypassed Amino Acid (BPAA) 5 505.6 479.2 26.4
AF + Slow Release-NPN (SR-NPN) 5 506.2 461.2 45.0
AF + BPAA + SR-NPN 5 514.6 490.6 24.0
Mean 510.0 477.9 32.1
Table2. Feed consumption and feed intake as % of body weight.

Corn Rice Dairy Total Feed
TREATMENT Silage, Straw, Conc., intake, Intake,
kg kg kg kg % BW
Control 20.30 2.61 4.61 27.53 4.9
Augmented feeding (AF) 17.81 2.04 6.61 26.47 4.3
AF + BPAA 16.89 2.22 6.36 25.47 4.3
AF + SR-NPN 15.91 3.04 6.31 25.26 4.4
AF + BPAA + SR-NPN 17.56 2.54 6.62 26.71 4.3
Mean 17.70 2.50 6.10 26.29 4.4
Table3. Effects of AF with BPAA and SRNPN on peak milk and persistency of lactation of cows.
%
Peak- milk, Average Adj. 305d milk
Treatment Lactation
kg/d peak-day yield, kg
Persistency
W/o augmented feeding 08.4a 78 1,120.9a 93.6
With Augmented Feeding 12.0b 68 1,433.2b 91.5
AF + BPAA 10.6ab 89 1,280.6a 91.0
AF + SR-NPN 12.5b 66 1,225.8a 91.5
AF + BPAA + SRNPN 10.0ab 71 1,586.4b 91.6
Mean 10.7 74.4 1,329.4 91.8
964
Table4. Effects of AF with BPAA and SR-NPN on milk composition of the buffalo milk.
% % SCC,
Treatment % Fat % CP % TS
Lactose SNF ‘000/ml
Control 7.04a 4.33 4.63 16.15a 8.99 52.18a
Augmented Feeding 6.1a 4.69 4.78 16.29a 9.51 82.15ab
AF + BPAA 9.72b 5.00 4.36 18.98b 9.20 91.01ab
AF + SR-NPN 6.79a 4.41 4.34 15.63a 8.84 151.83c
AF + BPAA + SR-NPN 7.34a 4.70 4.65 16.34a 9.24 70.36ab
Table5. Effects of AF plus BPAA and SR-NPN on the post-partum reproduction of buffaloes.
Service Conception No. of Services/

Treatment
Period, days rate, % Conception
Control 167 80 1.5
Augmented feeding (AF) 142 80 2.0
AF + Bypass amino acid (BPAA) 161 80 2.3
AF + Slow Released NPN (SRNPN) 152 80 2.0
AF + BPAA + SRNPN 106 80 2.25
Mean 146 80 2.0
Figure 1. Effects of AF with BPAA and SR-NPN on monthly body weights of dairy buffaloes.
Figure 1. Monthly weights of the cows
700
600
T1
Body weight, kg
500
400 T2
300 T3
200 T4
100 T5
0
BC AC 1 2 3 4 5 6 7 8 9 10
Month
BC- Before calving; AC - after calving
Figure 2. Effects of AF with BPAA and SR-NPN on monthly milk production of buffaloes.
965
Effect of Protein on Microbial Protein Synthesis and Productive Performances

of Thai Swamp Buffalo (Bubalus bubalis)
Pramote PAENGKOUMa, P. TATSAPONGa, O. PIMPAb, M.D. HAREc and
S. PAENGKOUMd
a
Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima,
30000, Thailand, bFaculty of Technology and Management, Prince of Songkla University, Surat
Thani Campus, Surat Thani, 84100, Thailand, cFaculty of Agriculture, Ubon Ratchathani
University, Ubon Ratchathani, 34190, Thailand, dFaculty of Science and Technology, Agriculture,
Nakhon Ratchasima Rajabhat University, Nakhon Ratchasima, 30000, Thailand
*Corresponding email: promote@sut.ac.th
ABSTRACT
The experiment was conducted to investigate the effect of dietary crude protein on nitrogen
utilization, nutrient digestibility and protein requirement for maintenance and growth of swamp
buffalo calves. Sixteen growing swamp male buffaloes with 18 to 24 months old (average initial
weight 233 ± 25 kg) were used. The calves were assigned to completely randomized block design.
In terms of treatment, crude protein (CP) for maintenance (M) levels in the diets were M, 1.4M,
1.8M and 2.2M of dry matter (DM) and all diets were isocaloric (0.5 kg, expected body weight gain
of calves of ME for maintenance). Average daily gain (ADG) of calves were clearly increased
(p<0.05) with increasing CP content in diets. Increasing the levels of dietary protein was
significantly alter (p<0.05) intake. The relationship between ADG (g ADG kg-1 W0.75) and N intake
(NI, g kg-1 W0.75) in swamp buffalo was nitrogen intake (NI) = 0.073ADG - 0.866 (R2=0.577;
p<0.01). Present findings suggested that nitrogen requirements for maintenance and growth of
growing Thai swamp buffalo were 0.87 g N or 5.44 g CP kg-1 BW0.75 and 0.073 g N or 0.46 g CP
per g ADG kg-1 W0.75.
Keywords: protein, microbial protein synthesis, buffalo, rumen
INTRODUCTION
Swamp buffaloes (Bubalus bubalis) are used for multiple purposes as draft power, means of
transportation, capital, credit, meat, milk, social value, hides, and source of organic fertilizer for
seasonal cropping (Wanapat and Rowlinson, 2009). Buffaloes are being preferred over cattle
because of their superior quality of milk, better efficiency in utilization of nutrient from poor-
quality fibrous tropical feeds and relatively better disease resistance and adaptability to tropical
climates (Paul and Patil, 2007). Normally, the nutrition needs of the buffalo probably differ from
type of breeds found in temperate countries because of difference in genetic make-up, mature body
size, growth rate, composition of body tissue, and quality of feed and climatic conditions (Kearl,
1982). Hence, this study was designed to determine protein requirements for maintenance and
growth of growing male Thai swamp buffalo calves through a feeding trial conducted under a
tropical environment.

Sixteen growing male swamp buffalo (Bubalus bubalis) (18-24 months of age and 233 ± 25
kg initial weight) were used in a completely randomized block design. Animals were divided into 4
groups according to body weight and were assigned to treatments randomly within groups to
evaluate the response to dietary protein on animal performances. Four diets were formulated to
contain 4 level of crude protein for maintenance (M, 1.4M, 1.8M and 2.2M) derived from
Tatsapong et al. (2009). The energy contents of the diets were formulated to contain a
metabolisable energy requirement equivalent for 0.5 kg, expected body weight gain of calves
derived from Kearl (1982).
966
The experimental period was 90 days and 7 days for collection period, which it was started
after fed animal 45 days of the experiment. Urine was diluted five times with distilled water and
mixed thoroughly and stored at -20 ๐C for later analysis for PD and creatinine according to the
technique of Balcells et al. (1992). Purine derivatives and creatinine concentrate in urine were
analyzed using HPLC, C18 reversed phase column with a UV detector wave length 205 nm
(Hewlett-Packard HPLC system HP series 1100, Agilent series 1100). Feed, orts and fecal sample
were dried at 60 ๐C and analyzed for proximate principles (AOAC, 1985) and fibre analysis was
determined by methods supported by Van Soest et al. (1991).
All data were analyzed by PROC GLM procedure (SAS, 1996). Duncan’s New Multiple
Rang Test and Orthogonal Contrast Analysis (Steel and Torie, 1980) were used to compare
treatment means.

Average final weight and ADG of calves fed with differences level of CP were significantly
different among treatments (Table 1). Average daily gain (ADG) throughout the 90 d of the
experiment increased with increasing protein content in the diet (-0.05, 0.16, 0.29 and 0.51 kg/d of
M, 1.4M, 1.8M and 2.2M, respectively). The result of the present study is similar to a report of
Chumpawadee et al. (2009) who found that ADG of Thai-indigenous yearling heifers increased by
level (6 to 12%) of dietary CP. This result disagrees with the work of Devant et al. (2000) who
summarized that increasing CP content of the diets (14 to 17%CP) were not affected ADG of
crossbred heifers.
Daily DM, OM, CP and TDN intake and feed efficiency (ADG/DMI) were affected by
increasing dietary protein content (Table 1). The current results demonstrated that increasing CP
content in the diets increased DM intake of calves. These observations disagreed with Basra et al.
(2003), and Chantiratikul et al. (2009) who demonstrated non-significant differences in daily DM
intake and feed efficiency of calves fed different CP level in the diet. Therefore, the CP intake (g
kg-1 W0.75) was 3.55, 5.42, 7.34 and 9.44 with ratios 1, 1.4, 2.8 and 2.2 fold of CP for maintenance,
respectively (Table 1). This result is in agreement with previous studies in growing Nili-ravi buffalo
(Basra et al. 2003), in growing indigenous heifers (Chantiratikul et al., 2009; Chumpawadee et al.,
2009), in Korean black goats (Hwangbo et al. (2009), in yearling indigenous Thai native cattle
(Paengkoum and Tatsapong, 2009) and in growing Thai swamp buffalo calves (Tatsapong et al.,
2010) that CP or nitrogen intake was sharply affected by dietary protein concentration. It has been
reported that increasing CP intake can increase TDN, OM and DM intakes. The suggested
mechanism underling this effect is that higher protein increased microbial fermentation in the
rumen, which would improve nutrients digestion (Granum et al., 2007).
Microbial N supply (g N/d) and microbial efficiency in terms of microbial N g /kg DOMR
and g /kg DMI) increased linearly (P<0.01), but in terms of microbial N g/g CPI did not significant
different (P>0.05) with increasing protein content. This data is in agreement with the work of
Paengkoum et al. (2006) who reported that microbial N synthesis increased with increasing urea
addition from 10 to 30 g/kg OPF. In contrast, Devant et al. (2000) found that microbial protein
synthesis was not affected by protein concentration and degradability but increased with age.
However, microbial protein production may be improved by balancing the overall daily ratio of
ruminally available energy and N intake in the diet (Chumpawadee et al., 2009) and microbial yield
in the rumen depends on many factors such as the availability of carbohydrates and N in rumen,
ruminal pH, physiological effects, sources and levels of N components and stabilizing ruminal
fermentation (McDonald et al., 1995).
CONCLUSIONS
Based on this study, it can be concluded that increasing dietary protein significantly
increased (P<0.05) growth rate, nutrients intake, and microbial N supply to duodenal of growing
Thai swamp buffalo. Protein requirements for maintenance and growth of growing swamp buffalo
967
calves are 5.44 g CP kg-1 W 0.75 and 0.46 g CP per g ADG. This result indicated that CP
requirement for maintenance of buffalo male calves is similar to that recommended by Kearl
(1982), but CP requirement for growth of growing swamp buffalo male is slightly lower than that
recommended by Kearl (1982).
ACKNOWLEDGEMENTS
The authors would like to cordially thank the Office of the Higher Education Commission,
Thailand, Department of livestock development (DLD) of Thailand, and Suranaree University of
Technology for providing laboratory facilities and experimental sites of research, respectively.
REFERENCES
AOAC (Association of Official Analytical Chemists). 1990. Official Methods of Analysis of
Association of Analytical Chemistry. 15th Edn. Arlington, Virginia, pp: 1298.
Balcells, J., J.A. Guada, J.M. Peiro and D.S. Parker. 1992. Simultaneous determination of
allantoin and oxypurines in biological fluids by high-performance liquid chromatography.
Journal of Chromatography. 575: 153-157.
Basra, M.J., M. Nisa, M.A. Khan, M. Riaz, N.A. Tuqeer and M.N. Saeed. 2003. Nili-Ravi buffalo
I. Energy and protein requirements of 6-9 months old calves. International Journal of
Agriculture & Biology. 5: 377-379.
Chantiratikul, A., S. Chumpawadee, W. Kanchanamayoon and P. Chantriratikul. 2009. Effect of
dietary protein on nutrient digestibility and nitrogen metabolism in Thai-Indigenous heifers.
Journal of Animal and Veterinary Advances. 8: 297-300.
Chumpawadee, S., A. Chantiratikul, V. Rattanphun, C. Prasert and K. Koobkaew. 2009. Effect of
dietary crude protein levels on nutrient digestibility, ruminal fermentation and growth rate in
Thai-Indigenous yearling heifers. Journal of Animal and Veterinary Advances. 8: 1131-
1136.
Devant, M., A. Ferret, J. Gasa, S. Calsamiglia and R. Casals. 2000. Effects of protein concentration
and degradability on performance, ruminal fermentation, and nitrogen metabolism in rapidly
growing heifers fed high-concentrate diets from 100 to 230 kg body weight. Journal of
Animal Science. 78: 1667-1676.
Granum, G., M. Wanapat, P. Pakdee, C. Wachirapakorn and W. Toburan. 2007. A comparative
study on the effect of cassava hay supplementation in Swamp buffaloes (Bubalus bubalis)
and Cattle (Bos indicus). Asian-Australasian Journal Animal Science. 20: 1389-1396.
Kearl. L.C. 1982: Nutrient requirements of ruminants in developing countries. International
feedstuffs institute, Utah Agricultural experiment station Utah State University Logan, Utah
USA. 149 pp.
Mcdonald, P., R.A. Edwards and J.E.D. Greenhalgh. 1995. Animal Nutrition, 5th edition. Perntice
Hall, 693p.
Paengkoum, P., J.B. Liang, Z.A. Jelan and M. Basery. 2006. Utilization of steam-treated oil palm
fronds in growing goast: 1. Supplementation with dietary urea. Asian-Australasian Journal
Animal Science. 19:1305-1313.
Paengkoum, P and P. Tatsapong. 2009. Effect of different levels of protein on feed intake,
digestibility and growth rate of Thai native beef cattle fed Pangola grass as roughages. In
Japan International Research Center for Agricultural Sciences (JIRCAS) Working Report
No. 64 : Establishment of a Feeding Standard of Beef Cattle and a Feed Database for the
Indochiness Peninsula. Edited by Shuichi Oshio, Makoto Otsuka and Kritapon Sommart.
Tsukuba, Ibaraki, Japan. pp. 76-78.
Paul, S.S. and N.V. Patil. 2007. Energy and protein requirements of growing Nili-ravi buffalo
heifers in tropical environment. Journal of the Science Food and Agriculture. 87: 2286-
2293.
SAS, 1996: SAS User’s Guide: Statistics. 14th Edn. Version 6. SAS Institute. Inc., Cary, NC,
USA.
968
Steel, R.J. and J.H. Torrie.1980. Principles and Procedures of Statistic a Biometereal Approach.
2nd Edn. McGrow-Hill. New York, USA.
Tatsapong, P., P. Paengkoum, O. Pimpa and M.D. Hare. 2010. Effect of dietary protein level on
nitrogen metabolism and protein requirements for maintenance of growing Thai buffalo
(Bubalud bubalis) calves. Journal of Animal and Veterinary Advances. 9(8): 1216-1222.
Van Soest, P.J., J.B. Robertson and B.A. Lewis. 1991. Methods for dietary fiber, neutral detergent
fiber and non-startch polysaccharide in relation to animal nutrition. Journal of Dairy
Science.74: 3583-3589.
Wanapat, M and P. Rowlinson. 2009. Nutrition and feeding of swamp buffalo: feed resources and
rumen approach. http://home.kku.ac.th/metha/Nutrition%20and%20Feeding%20of%20
Swamp%20Buffalo.pdf.
Table 1. Effect of dietary protein on average daily gain (ADG) and nutrients intake of swamp
buffalo.
Dietary Crude protein (maintenance) Contrast

Items M 1.4M 1.8M 2.2M SEM L Q C
Initial weight (kg) 233 234 234 232 6.74 ns ns ns
Final weight (kg) 229 248 260 279 8.50 * ns ns
ADG (kg d-1) -0.05 c
0.16 b
0.29 b
0.51a 0.05 ** ns ns
DMI (kg d-1) 4.17c 4.97b 5.25ab 5.60a 0.16 ** ns ns
DMI(g kg-1 W 0.75) 70.43 c
81.25 b
84.13 ab
87.67a 1.94 ** ns ns
CPI (g kg-1 W 0.75) 3.55d 5.42c 7.34b 9.44a 0.21 ** ns ns
TDNI (g kg-1 W 0.75) 43.57 b
50.93 a
50.49 a
52.51a 1.31 ** ns ns
ADG/DMI g/kg) -14.20 c 31.89 b 54.49 b 92.07 a 9.91 ** ns ns
b a a
ADG/CPI (g/g) -0.28 0.48 0.62 0.86 a 0.17 ** ns ns
a, b, c, d
Values on the same row under each main effect with different superscript differ significantly (p<0.05);
ADG=Average daily gain; DMI=Dry matter intake; CPI=Crude protein intake; TDN=Total digestibility nutrient ;
SEM= standard Error of Means; ns= not significantly different (p>0.05); L, Q, C=linear, Quadratic and Cubic effects of
difference crude protein levels ; ** = significantly different (p<0.01)
Table 2. Microbial N flow at duodenum of swamp buffalo calves.
Dietary Crude protein (maintenance) Contrast

Items M 1.4M 1.8M 2.2M SEM L Q C
Microbial N supply
g N/d 92.80c 151.86b 189.44b 286.97a 17.2 ** ns ns
g N/kg DOMR 1 58.60c 75.76bc 97.33b 140.38a 8.15 ** ns ns
g N/kg DMI 22.29c 30.67bc 36.29b 51.06 a 3.16 ** ns ns
g N/g CPI 0.44 0.47 0.42 0.47 0.04 ns ns ns
a, b, c, d
Values on the same row under each main effect with different superscript differ significantly (p<0.05); SEM=
standard Error of Means; ns= not significantly different (p>0.05); L, Q, C=linear, Quadratic and Cubic effects of
difference crude protein levels ; * = significantly different (p<0.05) ; ** = significantly different (p<0.01)
1
DOMR=digestible OM fermented in the rumen
969
Effect of Protein Level and Urea Source in Concentrate on Feed Intake and
Rumen Ecology in Swamp Buffalo Fed Rice Straw
Sungchhang KANGa, Metha WANAPATa* and Peter ROWLINSONb

a
Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal
Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand.
b
School of Agriculture, Food and Rural development, Newcastle University, NE17RU, UK
*
Corresponding email: metha@kku.ac.th
ABSTRACT
Four, Thai-fitulated native swamp buffaloes were randomly assigned according to a 2×2
Factorial arrangement in a 4×4 Latin square design to assess the effect of protein level and urea on
feed intake, rumen ecology and microbial populations. The treatments were as follows: Concentrate
containing protein at 12% CP (soybean meal) (T1); 16% CP (soybean meal) (T2); 12% CP (urea)
(T3); and 16% CP (urea) (T4), respectively. All animals were fed concentrate at 1% of BW and
untreated rice straw ad libitum. The results have revealed that both rice straw and total DM intake
were significant increased (P<0.05) by urea as a protein source in concentrate as compared to
soybean meal. In contrast, there were no effects of protein level and urea on ruminal pH, while
concentration of ruminal NH3-N and blood urea-nitrogen were rather high in the treatment with
16% CP concentrate in both soybean meal and urea as protein sources. However, urea as a protein
source showed higher result of ruminal NH3-N. In addition, rumen microorganism population such
as bacteria, fungi zoospores and protozoa were affected by different level of protein in concentrate
and the highest were found in 16% CP concentrate (T2 and T4). Moreover, viable bacteria were
significantly increased with higher level of protein in concentrate (16% CP), except for amylolytic
bacteria. Based on this study, it could be concluded that protein level in concentrate affected on feed
intake and rumen ecology and the use of urea as protein source have shown better results as
compared to soybean meal. Therefore, urea (4% in concentrate) could be used as a protein (NPN)
source in swamp buffalo fed on untreated rice straw satisfactorily.
Keywords: protein level, urea, soybean meal, rumen ecology, swamp buffalo, rice straw
INTRODUCTION
Low intake and poor utilization of this feedstuff can be partly attributed to an inefficient
rumen ecosystem and an imbalance in the products of rumen fermentation (Bird, 1999). Providing
supplementations with a high concentration of true protein to ruminants fed low-quality roughage
stimulates roughage intake, digestion, and performance. The plant protein sources i.e. soybean meal
are becoming expensive due to their short supply and, therefore, use of non protein nitrogen (NPN)
is imperative in animal diets. As reported, substituting NPN such as urea has been shown to
increase voluntary feed intake (Huntingto & Archibeque, 1999), which is generally attributed to an
improvement of nutrients digestibility and an increase passage from the rumen. The utilization of
NPN by ruminants is often less efficient than the utilization of natural protein supplements. It has
been theorized that synchronization of ruminal ammonia and energy availability will result in
improved efficiency of NPN utilization and animal performance. Cassava root contains high levels
of energy and has been used as a source of readily fermentable energy in ruminant rations
(Wanapat, 2003). One strategy for using high degradable carbohydrates is to use in combination
with readily available NPN sources such as urea. Urea is commonly used as N source when highly
soluble carbohydrates are fed and maintained. However, there is limitation of data on the optimum
level of NPN cooperated to the protein level in the concentrate mixture in swamp buffaloes.
Therefore, this study was conducted to investigate the effect of protein level and urea source as
NPN in concentrate mixture on feed intake and rumen ecology in swamp buffaloes fed on rice straw
based.
970

Four, Thai – rumen fistulated swamp buffaloes male (Bubalus bubalis), about 4 years old
with 360 ± 18 kg liveweight, were assigned according to a 2 x 2 factorial arrangement in a 4 x 4
Latin square design to receive dietary treatments. The treatments were as follows: Concentrate
containing protein at 12% CP (soybean meal) (T1); 16% CP (soybean meal) (T2); 12% CP (urea)
(T3); and 16% CP (urea) (T4), respectively. All animals were fed with rice straw ad libitum as
concentrate mixtures were supplemented at 1% of BW. Feeds offered and refusal were recorded
daily for DMI measurement and were sampled twice during the last 7 days of each period for
analyze of DM, Ash, CP (AOAC, 1990) and NDF, ADF (Goering & Van Soest, 1970). At the end
of each period, rumen fluid was sampled at 0, 2, 4, and 6 h post feeding as pH and temperature were
measured immediately. Fluid samples were then used for analysis of NH3-N by the hypochlorite-
phenol procedure (Beecher & Whitton, 1978). The total direction counts of bacteria, protozoa and
fungal zoospores were done according to the method of Galyean (1989) while the viable of bacteria
groups (cellulolytic, proteolytic, amylolytic) and total viable count bacteria were used the roll-tube
technique described by Hungate (1969). A blood sample was sampled for BUN analyzes (Roseler et
al, 1993). All data were subjected to ANOVA for a 4 x 4 Latin square design with 2 x 2 factorial
arrangements of treatments using the General Linear Models (GLM) procedures of the Statistical
Analysis System Institute (SAS, 1998).

Table 1 presents the data on the feed ingredients and chemical composition of the
concentrate mixtures and rice straw. All concentrate were formulated by using the local available
feed resources, but different in terms of protein level (12% and 16% CP) and protein source
(soybean meal and urea). All concentrates were well consumed by all buffaloes. On the other hand,
rice straw is a kind of low quality roughage source with low in CP and high in structural
carbohydrate (NDF and ADF). The results on DM feed intake are showed in table 2. Rice straw and
total feed intake were significant different among all treatments (P<0.05) while all animals
consumed similar concentrate mixtures. Straw and total intakes were showed higher in the
treatments with higher level of CP containing in the concentrate and the highest was in the
treatment with urea source. These results agreed with Chen et al. (2010) who indicated DMI were
improved with increasing protein level and urea were used as NPN showed higher intake.
The data on rumen ecology and microorganism affected by protein level and urea source are
presented in table 3. There were no differences on ruminal pH while temperature was increased as
buffaloes were supplemented with concentrate containing higher protein level. Ruminal NH3-N and
BUN were showed increased in the treatments with higher level of CP; however, urea source
contained in concentrate revealed higher results than using soybean meal (P<0.05). This was similar
to the result of Chen et al. (2010) and Devant et al., (2000) who suggested that ruminal NH3-N
increased with increasing level of dietary protein especially at 4 h post feeding. Moreover, Khampa
et al. (2006) reported that using high urea level in concentrate diet showed high ruminal of NH3-N
as this was agreed to the present study. Microorganism (bacteria, fungi, protozoa) were increased by
supplemented with higher level of CP in concentrate. However, there was no difference between
urea and soybean meal source, expect protozoa. This was consistency to the finding of Khampa et
al. (2009) who revealed that fungal zoospores, protozoa and total bacteria direct count were
significantly different and had higher number in the animals fed higher protein level diet. In
addition, amylolytic bacteria were no changed among treatments while proteolytic, cellulolytic and
total viable bacteria were different. Proteolytic bacteria was found higher in the treatments with
higher level of protein while total viable bacteria were affected and showed higher result by urea
source contained in concentrate mixture (P<0.05). On the other hand, protein level and source of
protein in concentrate affected on the population of cellulolytic bacteria and the higher level of
protein and urea source revealed higher result than lower level of protein and soybean meal source
contained in concentrate.
971
Based on this experiment, it could be concluded that supplementation of protein level in

concentrate containing urea as nitrogen source could improve feed intake, rumen ecology and
ruminal microbes. These results suggested that formulated concentrate mixture containing 16% CP
with 4% urea as nitrogen source showed no altering feed intake, rumen ecology and blood
metabolites in swamp buffaloes and could be used as protein source replaced soybean meal.
ACKNOWLEDGEMENTS
The most sincerely thanks are to Tropical Feed Resources Research and Development
Centre (TROFREC) and Khon Kaen University (KKU) for their kindly support on research fund
and facility, especially supporting the first author for the Ph. D studying.
REFERENCES
AOAC. 1990. Official methods of analysis. 15th edn. Association of official Analytical Chemists,
Beecher, G.R. and B.K. Whitton. 1978. Ammonia determination: reagents modification and
interfering compounds. Anim. Biochem. 36: 243.
Bird, S.H., J.B. Rowe, M. Choct, S. Stachiw, P. Tler and R.D. Thompson. 1999. In vitro
fermentation of grain and enzymatic digestion of cereal starch. Recent Advances in Animal
Nutrition in Australia. 12: 53-61.
Chen, S., P. Paengkoun, X. Xia and P. Na-Lumpang. 2010. Effects of dietary protein on ruminal
fermentation, nitrogen utilization and crude protein maintenance in growing Thai-indigenous
beef cattle fed rice straw as roughage. J. Anim. Vet. Adv. 9(18): 2396-2010.
Devant, M., A. Ferret, J. Gasa, S. Calsaminglia and R. Casals. 2000. Effects of protein
concentration and degradability on performance, ruminal fermentation and nitrogen metabolism
in rapidly growing heifers fed high-concentrate diets from 100-230 kg body weight. J. Anim.
Sci. 78: 1667-1676.
Galyean, M. 1989. Laboratory procedure in animal nutrition research. Department of animal and
Range Science. New Mexico State University, USA.
Goering, H.K. and P.J. Van Soest. 1970. Forage Fiber Analysis (Apparatus, Reagent, Procedures
and some Application) Agric. Handbook No. 379. ARS, USDA, Washington, DC.
Hungate, R.E. 1969. A Role Tube Method for Cultivation of strict Anaerobes. Method in
Microbiology. (Eds., J.R. Norris and D. W. Ribbons). New York, Academic. NY. 313.
Huntington, G.B. and S.L. Archibeque.1999. Practical aspects of urea and ammonia metabolism in
ruminants. Proc. of the American Soc. of Anim. Sci. pp. 1-11.
Khampa, S., M. Wanapat, C. Wachirapakorn, N. Nontaso and M. Wattiaux. 2006. Effects of urea
level and sodium DL-malate in concentrate containing high cassava chip on ruminal
fermentation efficiency, microbial protein synthesis in lactating dairy cows raised under
tropical condition. Asian-Aust. J. Anim. Sci. 19(6): 837-844.
Khampa, S., P. Chaowarat, R. Singhalert and M. Wanapat. 2009. Effects of protein level in
concentrate and urea-treated corn silage on rumen ecology and milk production in lactating
dairy cows. Pak. J. Nutr. 8 (5): 588-591.
Roseler, D.K., J.D. Ferguson, C.J. Sniffen and J. Herremra. 1993. Dietary protein degradability effects
on plasma and milk urea nitrogen and non-protein in Holstein cows. Dairy Sci. 76: 525-534.
SAS. 1998. User's Guide: Statistic, Version 6, 12th Edition. SAS Inst. Inc., Cary, NC.
Wanapat, M. 2003. Manipulation of cassava cultivation and utilization to improve protein to energy
biomass for livestock feeding in the tropics. Asian-Aust. J. Anim. Sci. 16:463-472.
972
Table 1. Ingredients and chemical composition of the experimental diets.
SBM Urea Rice straw

Items
12% 16% 12% 16%
Ingredient
Cassava chip 60.0 60.0 63.0 65.0
Rice bran 16.0 5.0 29.0 25.0
Soybean meal 20.0 31.0 0.0 0.0
Urea 0.0 0.0 2.0 4.0
Molasses 2.0 2.0 4.0 4.0
Sulphur 1.0 1.0 1.0 1.0
Mineral mixture 0.5 0.5 0.5 0.5
Salt 0.5 0.5 0.5 0.5
Chemical composition
Dry matter 88.0 88.1 85.8 84.0 92.5
Organic matter 93.1 93.5 90.8 89.2 87.2
Crude protein 12.5 16.0 12.0 16.7 2.1
Neutral detergent fiber 14.3 12.3 15.9 14.8 77.0
Acid detergent fiber 7.9 7.0 8.4 7.8 56.0
Ash 6.9 6.5 9.2 10.8 12.8
Table 2. Effect of protein level and urea on dry mater feed intake.
Treatments Interaction
Items
SBM Urea
SEM PL PS PL*PS
Protein level 12% 16% 12% 16%
DM Intake
Rice straw intake
kg/d 5.02 5.12 5.16 5.45 0.09 0.08 * NS
%BW 1.57 1.61 1.62 1.71 0.04 NS * NS
0.75
g/kg BW 66.3 67.8 68.4 72.2 1.45 NS * NS
Concentrate Intake
kg/d 3.12 3.10 3.10 3.13 0.05 NS NS NS
%BW 0.98 0.98 0.97 0.99 0.01 NS NS NS
0.75
g/kg BW 44.3 41.3 40.9 41.6 0.55 NS NS NS
Total Intake
kg/d 8.14 8.22 8.25 8.57 0.11 NS * NS
%BW 2.54 2.58 2.58 2.69 0.04 NS 0.09 NS
g/kg BW0.75 107.6 109.1 109.3 113.8 1.55 NS * NS
973
Table 3. Effect of protein level and urea on rumen microbe.
Treatments Interaction
Items
SBM Urea
SEM PL PS PL*PS
Protein level 12% 16% 12% 16%
Ruminal pH 6.61 6.59 6.73 6.58 0.05 NS NS NS
Temperature, oC 39.1 39.3 39.2 39.5 0.08 * NS NS
NH3-N, mg% 9.8 13.8 14.7 16.2 0.78 ** * NS
BUN, mg/% 12.3 16.5 11.7 16.7 0.90 ** NS NS
Direct count x cell/ml
Bacteria (x 109) 9.46 13.00 9.89 14.29 0.50 *** NS NS
Protozoa (x 105) 9.38 11.50 11.19 13.38 0.64 * * NS
Fungi (x 105) 20.44 25.47 21.85 26.57 1.38 * NS NS
Roll-tube technique, CFU/ml
Amylolytic (x 108) 1.20 1.39 1.59 2.03 0.28 NS NS NS
Proteolytic (x 108) 4.08 6.52 4.84 6.78 0.49 ** NS NS
Cellulolytic (x 108) 16.24 19.45 20.68 26.88 1.63 * * NS
Total viable bacteria (x 1011) 12.28 13.47 17.12 20.43 1.95 NS * NS
974
Effect of Dried Leucaena Leaf Supplementation on Rumen Ecology, Nutrient

Digestibility and Urinary Excretion of 2,3-Dihydroxy Pyridone (2,3-DHP)
and 3,4-Dihydroxy Pyridone (3,4-DHP) in Swamp Buffaloes
Kampanat PHESATCHAa*, Metha WANAPATa* and Chris MCSWEENEYb

a
Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal
Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand, bCSIRO
Livestock Industries, Queensland Bioscience Precinct, St Lucia, QLD, Australia.
*Corresponding email: metha@kku.ac.th
ABSTRACT
The experiment was conducted to determine the effect of dried Leucaena leaf
supplementation on rumen ecology, nutrient digestibility and urinary excretion of 2,3-dihydroxy
pyridone (2,3-DHP) and 3,4-dihydroxy pyridone (3,4-DHP) in swamp buffaloes. Four rumen
fistulated swamp buffaloes were assigned according to a cross-over design to receive 3.0 kg/hd/d
dried Leucaena leaf supplementation. Swamp buffaloes were fed with rice straw ad libitum and dried
Leucaena leaf was supplemented at 3.0 kg/hd/d. Apparent digestibility of CP were increased (P<
0.05) in buffaloes supplemented with Leucaena leaf. The average values of BUN were 1.26, 13.08
mg/dl and NH3 were 8.9, 16.1 mg/dl in control and in the supplemented group, respectively.
Moreover, there were differences in total volatile fatty acid (TVFA) in Leucaena leaf
supplemented than in the control group. The efficiency of microbial protein synthesis (EMPS)
were increased by Leucaena leaf supplemented group. Population sizes of the target total bacteria,
predominant cellulolytic bacteria in the rumen were influenced by dietary treatments (P<0.05), as
enumerated by real-time PCR assays. The strain of Synnergistes jonesii was pesented in swamp
buffaloes fed supplemented with Leucaena leaf. Moreover, total DHP concentration was related to
Leucaena and mimosine intake. Based on this study, it could be concluded that supplementation of
dried Leucaena leaf at 3 kg/hd/d could be a good protein source for swamp buffaloes to improve
nutrient digestibility, rumen fermentation and microbial protein synthesis.
Keywords: Leucaena leucocephala, Synnergistes jonesii, Rice straw, Purine derivatives, Swamp
Buffalo, Dihydroxypyridone (DHP), Mimosine
INTRODUCTION
Leucaena (Leucaena leucocephala) is particulary valuable as a fodder for animal
production due to its high protein, palatability and digestibility. Leucaena continues to be one of
the most productive multipurpose tree legumes available to tropical agriculture, yielding very high
quality forage for ruminant production (Shelton & Dalzell, 2007). Leucaena has been used as a
protein supplement for ruminants. However, its use has been limited by the presence of mimosine.
Mimosine is readily degraded by plant and rumen microbe enzymes to 3-hydroxy-4(1H)-pyridone
(3,4-DHP) (Hegarty et al. 1964) and then, if the bacterium Synergistes jonesii is present, to
harmless by-products (Jones & Megarrity1986). The analysis of presence of DHP by using HPLC
most practical approach because it is confirms the presence of Synnergistes jonesii and its
effectiveness in degrading DHP when ruminant fed high level of Leucaena. Therefore, the
objective of this study were to investigate the effect of Luecaena leaf supplementation at 3
kg/hd/d on rumen ecology, nutrient digestibility and urinary excretion of 2,3-dihydroxy pyridone
(2,3-DHP) and 3,4-dihydroxy pyridone (3,4-DHP) in swamp buffaloes.

Four, Thai–rumen fistulated swamp buffalo (356 ± 26 kg of BW) were assigned according
to a cross-over design. The study was conducted over 3 weeks during which time animals received

975
a concentrate mixture 0.1% of BW, while rice straw was offered ad libitum without dried
Leucaena leaf (period 1) and then supplementation with Leucaena leaf at 3.0 kg/h/d (period 2).
Measurements of feed intake and collections of feed, refusals, feces, rumen fluid and blood were
collected during the 21 days of each period. Rumen fluid, blood samples were collected at 0, 2, 4
and 6 h post feeding. Feeds, refusals and fecal samples were analyzed using the standard methods
of AOAC (1990) for dry matter (DM), ash and acid detergent fiber (ADF). Neutral detergent fiber
(NDF) in samples was estimated according to Van Soest et al. (1991). Rumen fluid was
immediately measured for pH using a portable pH meter (HANNA, instruments HI 8424
microcomputer, Singapore) and NH3-N by Kjeltech Auto 1030 Analyzer. Volatile fatty acids were
analyzed using High Performance Liquid Chromatography (HPLC) according to Samuel et al.
(1997) and for blood urea nitrogen (BUN) according to the method of Crocker (1967). Mimosine,
3,4-DHP and 2,3-DHP in urine was determined by high performance liquid chromatography using
μBondapak C18 Column (3.9×300 mm 10μ, Water Corporation, USA) (Lowry et al. 1985).
Rumen fluid was determined for group of bacteria (amylolytic, cellulolytic and proteolytic) were
measured using roll-tube technique (Hungate, 1969) and determined direct count of bacteria,
protozoa and fungi by using the methods of Galyean (1989) by a haemacytometer (Boeco,
Singapore). Total bacteria, predominant cellulolytic bacteria and methanogenic bacteria
populations were determined by using real-time PCR according to Yu et al. (2005). Moreover, the
strain of Synnergistes jonesii was determined by using melecular polymerase chain reaction of
rumen fluid for presence of Synnergistes jonesii DNA (McSweensy et al.1993)
Statistical analyses of each parameter measures in this study were analyzed using
variance with differences determined by the method of Least Significant Difference at the 5% (P <
0.05) and 1% (P < 0.01) levels using the procedures of the Statistical Analysis System Institue
(SAS, 1998)

The data of daily feed intakes and nutrient digestibility are presented in Table 1. The intake
of rice straw was not decreased when supplemented with dried Leucaena leaf. The result has
shown that supplementation of Leucaena leaf could improve apparent digestibility, the results
were similar with Jetana et al. (2012). The NH3-N and blood urea nitrogen (BUN) concentration in
the rumen were increased when supplemented with Leucaena leaf (Table 2). Increasing ruminal
NH3-N could provide ruminal nitrogen source for microbial protein synthesis and rumen fiber
digestion (Hoover and Stokes, 1991). The total VFAs concentration and value of propionate (C3)
in supplemented dried Leucaena leaf group has shown higher (P< 0.05) than in control. Population
of total bacteria and proteolytic bacteria were significantly different (p<0.05) among treatments.
All supplemented group showed higher populations of proteolytic and total bacteria than the
control period except protozoal population. Microbial population studies by real-time PCR are
shown in Table 3. It was found that total bacteria was increased by Leucaena leaf
supplementation. However, three predominant cellulolytic bacteria (F. succinogen, R. flavefaciens,
R. albus) were not significantly different among treatments. The result of this study was similar to
the finding of Wanapat and Cherdthong (2009), who studied rumen cellulolytic bacteria
population using Real-time PCR. They found that the population of F. succinogenes was more
abundant than R. albus. Purine derivative excretion of swamp buffaloes in this experiment ranged
from 18.5 to 35.1 (mmol/d). Leucaena supplementation group showed higher efficiency of
microbial protein synthesis than the control period (Table 4). Moreover, this study was found the
strain of Synnergistes jonesii by using melecular polymerase chain reaction. the finding of
Synnergistes jonesii, the rumen bacteria capable of degrading 2,3-DHP and 3,4-DHP in swamp
buffaloes fed on Leucaena. This result indicates the presence of Synnergistes jonesii in swamp
buffalo and the potential use of high level of Leucaena. The result was confirmed by relatively
low concentration of urinary 2,3-DHP and 3,4-DHP.
976
CONCLUSION
Based on this study, it could be concluded that supplementation of Leucaena leaf at 3
kg/hd/d could be a good protein source for swamp buffalo to improve nutrient digestibility, rumen
ecolgogy microbial protein synthesis.
ACKNOWLEDGEMENT
The authors would like to express their most sincere thanks to all who have assisted and
supported the research in this study, particularly the Tropical Feed Resources Research and
Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon
Kaen University, Khon Kaen, Thailand, the Royal Golden Jubilee Ph. D. Scholarship Program.
Moreover, the authors would like to thanks to Commonwealth Scientific and Industrial Research
Organisation (CSIRO).
REFERENCES
AOAC, 1990. Official Methods of Analyses, 15th ed. Association of Official Analytical Chemists.
Arlington, VA.
Crocker, C. L. 1967. Rapid determination of urea-nitrogen in serum or plasma without
deproteinization. Am. J. Med. Technol. 33:361-365.
Galyean, M. 1989. Laboratory procedure in animal nutrition research. Department of Animal and
Range Sciences, New Mexico State University, U.S.A
Hegarty, M.P., P.G. Schinckel and R.D. Court. 1964. Reaction of sheep toconsumption of
Leucaena glauca Benth and to its toxic principlemimosine. Aus. J. Agri. Res.15: 153–167.
Jones, R.J. and R.G. Megarrity, 1986. Successful transfer of DHP-degrading bacteria from
Hawaiian goats to Australian ruminants to overcome the toxicity of Leucaena. Aus.
Vet. J. 63, 259–262
Jetana, S., T. Sirima, S. Sawong and H. runchuan. 2012. A comparatiove study on mimosine, 3,4-
dihiydroxy pyridine (3,4-DHP) and 2,3-dihydroxy pyridine (2,3-DHP), purine derivative
(PD) excretion in the urine, thyroid hormone and blood metabolites profiles ofThai swamp
Buffalo (Bubalus bubalis) and Murrah buffalo (Bubalus bubalis). Trop Anim Health Prod.
44: 887-897
Lowry, J.B., B. Tangendjaja, and M.W. Cook 1985. Measurement of mimosine and its metabolites
in biological material, J. Sci. of Food and Agriculture, 36, 799–807
McSweeney, C.S., R.I. Mackie, A.A. Odenyo and D.A. Stahl. 1993. Development of an
oligonucleotide probe targeting 16S rRNA and its application for detection and
quantitation of the ruminal bacterium Synergistes jonesii in amixed-population
chemostat.Appl. Environ. Microbiol. 59, 1607–1612.
SAS., 1996. SAS User’s Guide: Statistics Version, 6.06 Edition. SAS Institute Inc., Cary, NC.
Samuel, M., S, Sagathewan, J. Thomus, and G. Mathen. 1997. An HPLC method for estimation of
volatile fatty acids of rumen fluid. Indian. J. Anim. Sci. 67: 805-807.
Yu, Z., F. C. Michel, Jr. G. Hansen, T. Wittum, and M. Morrison. 2005. Development and
application of real-time PCR assays for quantification of genes encoding tetracycline
resistance. Appl. Environ. Microbiol. 71: 6926-6933.
Wanapat, M., and A. Cherdthong. 2009. Use of Real-time PCR technique in studying rumen
cellulolytic bacteria population as affected by level of roughage in swamp buffalo.
977
Table 1. Effect of dried Leucaena leaf supplementation on voluntary feed intake and nutrient
digestibility.
Items T1 T2 SEM P value

DM Intake
Rice straw Intake , d-1
kg 6.3 6.4 0.08 ns
% BW 1.8 1.8 0.01 ns
g kg-1BW0.75 77.9 80.3 0.67 ns
-1
Concentrate Intake, d
kg 0.32 0.32 0.05 ns
% BW 0.1 0.1 0.01 ns
g kg-1BW0.75 3.9 3.9 0.66 ns
Total Intake, d-1
kg 6.7 10.0 0.05 *
% BW 1.9 2.8 0.01 *
-1 0.75
g kg BW 81.9 122.8 0.66 *
Apparent digestibility,%
Dry matter 63.0 65.6 0.43 ns
Organic matter 65.1 66.4 0.08 ns
Crude protein 39.3 62.5 0.01 *
Neutral detergent fiber 61.2 65.1 0.67 ns
Acid detergent fiber 60.3 64.2 0.51 ns
*
P<0.05, ns = non-significant
T1 = control (no supplementation), T2 = supplementation with dried Leucaena leaf at 3.0 kg/h/d
Table 2. Effect of dried Leucaena leaf supplementation on blood urea nitrogen, ruminal pH,
temperature, NH3-N and VFA concentration.

Blood urea nitrogen, mg/dL 1.26 13.08 0.23 **
Ruminal pH, 6.4 6.4 0.03 ns
Ruminal temperature, ˚C 38.4 39.3 0.16 ns
NH3-N, mg/dL 8.9 16.1 1.06 **
Molar proportion of VFA
(mol/ 100 mol)
Total VFA, % 81.2 93.5 2.34 *
Acetic acid, % 60.6 63.6 1.69 ns
Propionic acid, % 12.2 20.3 2.28 *
Butyric acid, % 8.4 9.5 0.76 *
ns = non-significant, * P< 0.05, ** P< 0.01
978
Table 3. Effect of dried Leucaena leaf supplementation on microbial population.
Item T1 T2 SEM P value
Viable bacteria (Roll-tube technique)

Total viable bacteria, x108 cfu∙ml-1 6.7 11.4 0.39 *
Cellulolytic bacteria, x107 cfu∙ml-1 3.8 6.4 0.47 *
Proteolytic bacteria, x107 cfu∙ml-1 2.2 4.2 0.38 *
Amylolytic bacteria, x106 cfu∙ml-1 1.9 2.8 0.40 ns
Ruminal microbe x cell/mol
Bacteria , x109 2.8 3.5 0.44 ns
Protozoa, x105 7.6 4.5 0.11 *
Fungi, x105 2.3 2.4 0.15 ns
Microbial population
Real time PCR technique,
copies/ml of rumen content
Total bacteria, ×1010 6.72 8.47 0.11 *
F. succinogene, ×1010 5.22 5.74 0.15 ns
R. flavafaciens, ×1010 4.91 5.33 1.42 ns
R. albus, ×1010 6.42 6.66 1.34 ns
Methanogens, ×108 5.10 4.82 0.79 ns
Table 4. Effect of dried Leucaena leaf supplementation on microbial protein synthesis, urinary
mimosine and DHP excretion in swamp buffalo.
Microbial protein synthesis

PD excreted, mmol∙d-1 18.5 35.1 5.34 **
PD absroped, mmol∙d -1
66.4 78.9 11.43 *
Microbial nitrogen supply, gN∙d-1 55.5 97.3 8.30 *
EMPS, gN∙kg OMDR -1
20.9 37.8 6.25 *
Urinary mimosine and DHP excretion
Mimosine, μg/mL 0 3.4 1.42 ns
3,4-DHP, μg/mL 0 183.1 9.45 *
2,3-DHP, μg/mL 0 0 0 ns
Mimosine+DHP, μg/mL 0 186.5 8.65 *
979
Effects of Eucalyptus Crude Oils Supplementation on Nutrients Digestibility of

Swamp Buffaloes
Nguyen The THAOa and Metha WANAPATa*
a
Tropical Feed Resources Research and Development Center, (TROFREC), Department of Animal
Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand
* Corresponding email: metha@kku.ac.th
ABSTRACT
This study was conducted to investigate the effects of Eucalyptus (E. Camaldulensis) crude
oils (EuO) supplementation on voluntary feed intake and rumen fermentation characteristics of
swamp buffaloes. Four ruminally fistulated swamp buffaloes with initial body weight of 321± 23 kg
were randomly assigned according to a 2x2 factorial arrangement in a 4 x 4 Latin square design.
The dietary treatments were T1) untreated rice straw (RS) as roughage source without EuO
supplementation; T2) UTRS with 2 ml of EuO/hd/d; T3) urea-treated (3%) rice straw (UTRS)
without EuO supplementation and T4) UTRS with 2 ml of EuO/hd/d. Experimental animals were
kept in individual pens, concentrates were offered at 0.3%BW and roughage were fed ad libitum.
The results revealed that voluntary feed intake were improved (P<0.001) by UTRS. Apparent
digestibility of OM and NDF were also increased (P<0.01 and P<0.001, respectively) by UTRS
whereas DM, CP and ADF were not affected by neither roughage source nor EuO supplementation.
Ruminal pH, temperature and ruminal ammonia nitrogen concentrations were not significantly
affected; however, buffaloes fed with UTRS resulted in significantly higher concentration of blood
urea nitrogen (P<0.001). The present findings showed that the amylolytic and cellulolytic bacteria
population were increased (P<0.001) by UTRS. However, total bacteria, proteolytic, amylolytic and
cellulolytic bacteria population were not affected by EuO supplementation. Based on these findings,
it is suggested that EuO could be used as feed additive to modify the rumen fermentation.
Keywords: Eucalyptus oils, rumen fermentation, nutrients digestibility, swamp buffalo
INTRODUCTION
Essential oils, which are extracted from plants by steam distillation, are known to have
antimicrobial effects due to their ability to modify cell permeability in microbes (Helander et al.,
1998). These substances have also been proposed to be modifiers of rumen fermentation due to
their toxicity to some unfavorable strains of rumen bacteria, such as methanogen (Wallace, 2004).
Eucalyptus oils also well known as a traditional medicine with several biological activities such as
bacteriostatic, fungistatic, anti- protozoa, anti-inflammatory and could be modifying ruminal
fermentation characteristic and methane mitigration (Sallam et al. 2009, Araujo et al. 2009).
However, there are very few experimental data on effects of eucalyptus oils on rumen microbial
fermentation and nutrients digestibility patterns, therefore, this study was undertaken to investigate
the effects of eucalyptus oils supplementation on rumen fermentation, nutrients digestibility in
swamp buffaloes fed with urea treated and non-treated rice straw.

Briefly, EuO extraction procedure was based on a water distillation process. Approximately
100 kg of Eucalyptus leaves were collected and put into 300 liter steel barrel with 100 liter of water.
The barrel was then covered tightly with a lid which was connected with a pipe and cooling coil in a
cooling device. At the end of cooling device, a plastic bottle was used for collecting aromatic fluid.
The barrel was then boiled for approximately 5 hours, and essential oils were then collected by
separating the oils float on the aromatic fluid.
Four ruminal fistulated buffaloes with initial body weight (BW) of 420 ± 15 kg were
randomly assigned to receive four dietary treatments according to a 2 x2 factorial arrangement in a
980
4 × 4 Latin square design. The dietary treatments were as follow: The dietary treatments were T1)
untreated rice straw (RS) as roughage source without EuO supplementation; T2) UTRS with 2 ml
of EuO/hd/d; T3) urea-treated (3%) rice straw (UTRS) without EuO supplementation and T4)
UTRS with 2 ml of EuO/hd/d. Concentrate (14.2% CP) was fed daily to animals at 0.3% of
BW/day, and rice straw was offered ad libitum. All experimental animals were kept in individual
pens with clean fresh water and mineral blocks were available at all times.
The experiment was conducted for four periods and each lasted for 21 days. During the first
14 days, all animals were kept in the pen for voluntary feed intake recorded, whereas during the last
7 days, they were moved to metabolism crates for total urine and fecal collection.
Feeds and fecal samples were collected by total collection of each individual buffaloes during the
last 7 days of each period at the morning and afternoon feeding. Feeds and refusals samples
analyzed for dry matter (DM), ash and crude protein (CP) content (AOAC, 1995). At the end of
each period, rumen fluid samples were collected immediately post feeding at 0, 2, 4 and 6 h.
Rumen fluid was immediately measured for pH and temperature by using a portable pH temperature
meter. Ruminal ammonia nitrogen (NH3-N) analyzed by micro Kjeldahl methods (AOAC, 1995).
Total viable bacteria, cellulolytic, proteolytic and amylolytic was measured using roll-tube
technique (Hungate, 1969).
All data from the experiment were statistically analyzed as a 2x2 factorial arrangement in a
4x4 Latin square design using the General Linear Model (GLM) procedure SAS (1996.) Difference
between treatment means were determined by Duncan’s New Multiple Rang Test (DMRT) with
P<0.05 were accepted as representing statistically significant differences.

Effect of eucalyptus oil supplementation on feed intake and nutrient digestibility
Results of this study indicate that supplementing swamp buffaloes with EuO had no effect
on DM intake (Table 1). Little information is available on the effect of essential oils on feed intake
in ruminants. Benchaar et al. (2007) observed no change in DM intake when lactating dairy cows
were supplemented with mixed essential oil (Crina ruminants) at a dose of 750 mg/d. Intake of
roughage was significantly increased (P<0.001) in treatments with 3% urea treated rice straw. This
result was agreed with Wanapat et al. (2009) who reported that treatment of rice straw with urea
could increase dry matter intake in dairy cows.
There was no interaction between EuO addition and source of roughage for DM, CP and
ADF digestibility. Apparent digestibilities of OM and NDF were not influenced by EuO
supplementation, which agrees with the result of Benchaar et al (2007) who reported no change in
apparent total tract digestibility in lactating dairy cows supplemented with 750 mg/d of mixed
essential oils. However, higher digestibilities of OM and NDF were found in buffaloes fed with
urea treated rice straw. This would agree with the result reported by Wanapat et al. (2009). The
alkaline agents can chemically break the ester bonds between lignin and hemicellulose and
cellulose, and physically make structure fibers swollen. These effects enable rumen microbes to
attack the structural carbohydrates more easily therefore, increasing digestibility (Fadel Elseed et al.
2003).
Effect of eucalyptus oils on ruminal parameters and microbial counts

There was no interaction between EuO addition and roughage sources for ruminal
parameters (Table 2) and those values were in normal range which reported by Wanapat and Pimpa
(1999). However, BUN values were affected and higher (P<0.001) in treatments with 3% urea
treated rice straw than untreated rice straw treatments.
Data on the effect of essential oils on ruminant microbial populations are scarce. In the
present study, total viable bacteria, cellulolytic bacteria, proteolytic bacteria, and amylolytic
bacteria population were not affected by EuO supplementation in the diet. In agreement with our
findings, Wallace et al. (2002); Benchaar et al. (2007) reported that no change in bacteria count
when fed sheep and dairy cows with mixed essential oils, respectively. When compare with
981
buffaloes fed untreated rice straw-based diets, those fed urea treated-based diets had higher
(P<0.001) ruminal counts of amylolytic and cellulolytic bacteria groups; however, total viable
bacteria and proteolytic bacteria counts were unaffected by the sources of roughage.
IMPLICATIONS
Based on this study it could be concluded that supplementation of Eucalyptus oils at 2
ml/h/d on swamp buffaloes showed slightly improve on feed intake, nutrient digestibility. Further
researches are required to investigate effect of eucalyptus oils on methane mitigation.
ACKNOWLEDGMENTS
Tropical Feed Resources Research and Development Center (TROFREC), Department of
Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand and the Vietnam
International Education Development (VIED), Ministry of Education and Training, Vietnam are
gratefully acknowledged for the use of research facilities and financial support, respectively.
REFERENCES
AOAC. 1995. Official Methods of Analyses, 15th ed. Association of Official Analytical Chemists,
Arlington, VA.
Benchaar, C., H. V. Petit, R. Berthiaume, D. R. Ouellet, J. Chiquette and P. Y. Chouinard, 2007.
Effects of essential oils on digestion, ruminal fermentation, rumen microbial populations,
milk production, and milk composition in dairy cows fed alfalfa silage or corn silage. J.
Dairy Sci. 90:886–897.
Fadel-Elseed, A. M. A., J. Sekine, M. Hishinuma and K. Hamana, 2003. Effects of ammonia, urea
plus calcium hydroxide and animal urine treatments on chemical composition and in sacco
degradability of rice straw. Asian–Austral. J. Anim. Sci. 16, 368–373.
Helander, I. M., H. L. Alakomi, K. Latva-Kala, T. Mattila-Sandholm, I. Mol, E.J. Smid, L. G.
Gorris and A. von Wright. 1998. Characterization of the action of selective essential oil
components on gram-negative bacteria. J. Agric. Food Chem. 46: 3590-3595.
Hungate, R. E., 1969. In: Norris, J. R. and D. W. Ribbons (Eds.) A roll tube method for cultivation
of strict anaerobes. Method in Microbiology. Academic, New York, NY, p 313.
Wallace, R. J. 2004. Antimicrobial properties of plant secondary metabolites. Proc. Nutr. Soc.
63:621–629.
Wallace, R. J., N. R. McEwan, F. M. McIntosh, B. Teferedegne and C. J. Newbold. 2002. Natural
products as manipulators of rumen fermentation. Asian-Australas. J. Anim. Sci. 10:1458–
1468.
Wanapat, M. and O. Pimpa, 1999. Effect of ruminal ammonia nitrogen levels on ruminal
fermentation, purine derivatives, digestibility and rice straw intake in swamp buffaloes.
Asian-Aust. J. Anim. Sci. 12: 904-907.
Wanapat, M., S. Polyorach, K. Boonnop, C. Mapato and A. Cherdthong, 2009. Effect of treating
rice straw with urea or urea and calcium hydroxide upon in takem digestibility rumen
fermentation and milk yield of dairy cows. Livest. Sci. 125, 238-243.
982
Table 1. Effects of source of roughage and Eucalyptus supplementation on voluntary feed intake
and nutrients digestibility.
Items RS UTRS Contrasts

- EuO + 2ml EuO - EuO + 2ml EuO SEM RS EuO RS x EuO
Total intake
(kg/hd/d) 6.1 5.8 8.7 8.5 0.26 *** NS NS
Roughage intake
(g/h/d) 5.1 4.8 7.7 7.6 0.27 *** NS NS
Apparent digestibility (%)
DM 59.1 60.0 63.6 65.5 2.45 NS NS NS
a ab b b
OM 61.5 63.2 66.6 67.3 1.74 ** NS NS
CP 51.0 54.5 56.2 56.6 4.16 NS NS NS
a ab bc c
NDF 55.1 60.1 64.1 67.4 1.94 *** NS 0.07
ADF 47.5 49.2 52.6 51.6 2.88 NS NS NS
a, b, c
Values in the same row with different superscripts differ, ** p<0.01, *** p<0.0001, NS: Non-
significant, SEM: Standard error of mean, RS: rice straw, UTRS: Urea-treated (3%) rice straw, EO:
Eucalyptus oil, DM: dry matter, OM: organic matter, CP: crude protein; NDF: Neutral detergent
fiber, ADF: acid detergent fiber,
Table 2. Effects of roughage sources and Eucalyptus oils supplementation on ruminal parameters
and microbial population.
RS UTRS Contrasts
Items - EuO + 2ml EuO - EuO + 2ml EuO SEM RS EuO RS x EuO
o
Ruminal Temp., C 38.6 38.6 39.5 39.1 0.26 NS NS NS
pH 6.3 6.3 6.0 6.2 0.10 NS NS NS
NH3-N, mg/dl 11.9 10.7 16.8 19.4 2.43 NS NS NS
BUN, mg/dl 8.1 8.4 13.6 17.6 1.64 *** NS NS
Viable bacteria, cell/ml
Total x 108 1.94a 2.44a 4.03b 2.26a 0.455 0.07 NS *
7
Proteolytic x10 1.34 2.06 3.02 2.67 0.045 0.09 NS NS
Amylolytic x 107 2.93a 1.88ab 3.47ab 4.67b 0.575 *** NS NS
7 a a b b
Cellulolytic x 10 1.68 2.06 5.44 4.40 0.525 *** NS NS
a, b, c
Values in the same row with different superscripts differ, ** p<0.01, *** p<0.0001, NS: Non-
significant, SEM: Standard error of mean, RS: rice straw, UTRS: Urea-treated (3%) rice straw, EO:
Eucalyptus oil.
983
Influence of Urea-Calcium Mixture in High-Quality Feed Block on Ruminal

Fermentation in Swamp Buffalo
Anusorn CHERDTHONG*, Metha WANAPAT, Sayan KHANTHARIN, Waroon KHOTA,

Gasama TANGMUTTHAPATTHARAKUN, Kampanat PHESATCHA, Suban FOIKLANG
and Sung Chhang KANG
Tropical Feed Resources Research and Development Center (TROFREC),

Department of Animal Science, Faculty of Agriculture, Khon Kaen University,
Khon Kaen 40002, Thailand.
*Corresponding email: anusornc@kku.ac.th
ABSTRACT
The effect of levels of urea calcium sulphate mixture (U-cas) in high-quality feed block
(HQFB) on ruminal digestibility of nutrients, fermentation end-products and kinetics of gas
production in rumen fluid of swamp buffalo by using in vitro gas production techniques were
investigated. The dietary treatments were 7 levels of U-cas supplementation in HQFB at 0, 3, 6, 9,
12, 15 and 18%. The result revealed that gas production from soluble fractions (a) and gas
production from the insoluble fraction (b) were not changed (P>0.05), while gas production rate
constants for the insoluble fraction (c) and potential extent of gas production (a+b) were linearly
increased when increasing level of U-cas in HQFB (P<0.05). The c value was highest at 0.09 ml/h
when supplementation 18% of U-cas in the HQFB. The cumulative gas production (96 h) was
significantly different among treatments and was linearly highest when HQFB contained of 18%
U-cas (102.3 ml/0.5 g DM substrate). The in vitro dry matter degradability (IVDMD), in vitro
organic matter degradability (IVOMD), true digestibility and microbial mass were altered by
treatments (P<0.01) and were greatest at 18% of U-cas supplementation. The NH3–N
concentration were highest when urea was supplemented in HQFB while concentrations tended to
be reduced with increasing level of U-cas (P<0.05). The finding suggests that the supplementation
of U-cas in HQFB resulted in improved in vitro kinetics gas production, rumen fermentation,
microbial mass and digestibility as well as could control the rate of N degradation in the rumen
and leading to a slow rate of NH3-N released.
Keywords: Ammonia-nitrogen, buffalo, feed block, gas production technique, in vitro

digestibility, slow-release urea
INTRODUCTION
High-quality feed block (HQFB) have been used as strategic supplements for ruminants
and have been developed to contain local feed ingredients particularly those from different energy
sources (e.g. molasses, rice bran), essential minerals (S, Na, P) and NPN source or urea (Foiklang
et al., 2011). Use of urea is attractive in ruminant diets because of its low cost, with high rumen
degradability (Wanapat, 2009). However, the amount of urea that can be used in diets is limited
due to its rapid hydrolysis to NH3 in the rumen by microbial enzymes, resulting in its
accumulation in the rumen and absorption though the rumen wall (Cherdthong et al., 2011a). Urea
calcium sulphate mixtures (U-cas), ruminal slow urea release properties, have been achieved by
using urea binding to substrates such as calcium sulphate to control its release rate (Cherdthong et
al., 2011a). Cherdthong et al. (2011b) reported that supplementation of U-cas in the concentrate
diets were shown to reduce ruminal NH3 concentrations, improve feed intake, nutrient
digestibility, the cellulolytic bacterial population, as well as milk yield in ruminants.
Therefore, the aim of this study was designed to determine effect of levels of U-cas in
HQFB on ruminal digestibility of nutrients, fermentation end-products and kinetics of gas
production by using in vitro gas production techniques.

984

Seven high-quality feed blocks (HQFB) were formulated and the experimental design was
a completely randomized design (CRD). The dietary treatments were 7 levels of urea calcium
sulphate mixture (U-cas; 0, 3, 6, 9, 12, 15 and 18%) in HQFB. U-cas products were prepared
according to Cherdthong et al. (2011a). The amounts of ingredients for producing HQFB were
shown in Table 1. All ingredients were mixed well together and then were pressed into blocks
(Foiklang et al., 2011). The sample of HQFB, roughage and concentrate were dried at 60ºC, then
ground to pass a 1-mm sieve (Cyclotech Mill, Tecator, Sweden) and used for chemical analysis
and in the in vitro gas test.
Two male, rumen-fistulated swamp buffaloes with an initial body weight of 350 50 kg)
were used as rumen fluid donors. Swamp buffalo rumen fluid was collected and was prepared for
artificial saliva was done according to Menke and Steingass (1988). The 70:30 roughage and
concentrate ratio were used as substrates at 0.47 g with 0.03 g of respective HQFB and samples of
0.5 mg were weighed into 50 ml serum bottles. For each treatment, five replications were
prepared. Ruminal fluid from each animal was mixed with the artificial saliva solution of Menke
and Steingass (1988) in a proportion 2:1 (ml/ml) at 39 ºC under continuous flushing with CO2 and
40 ml of rumen inoculum mixture were added into each bottle under CO2 flushing.
During the incubation, data of gas production was measured immediately after incubation
at 0, 0.5, 1, 2, 4, 6, 8, 12, 18, 24, 48, 72 and 96 h by using a pressure transducer and a calibrated
syringe. Cumulative gas production data were fitted to the model of Ørskov and McDonald (1979)
as follows: y = a+b (1-e-ct). Inoculum ruminal fluid was sampled at 0, 2, 4, 6, 12 and 24 h post
inoculations were analysed for ammonia- nitrogen (NH3-N), in vitro dry matter degradability
(IVDMD), in vitro organic matter degradability (IVOMD), in vitro true digestibility and microbial
mass by using standard method.
All data from the experiment were analyzed as a completely randomized design using the
GLM procedure of SAS (1998).

Table 1 presents the chemical compositions of HQFB and crude protein (CP) contents for
HQFB products were ranged from 34.8 to 35.5% and were similar to those reported by Foiklang et
al. (2011). Urea was replaced by U-cas up to 100% in HQFB (18% of U-cas) and, therefore,
relatively more slowly release to ammonia than urea and could potentially be used more
efficiently by rumen microorganisms (Cherdthong et al., 2011a,b). The gas kinetics and
cumulative gas production of substrates studied are presented in Table 2. Gas production from
soluble fractions (a) and gas production from the insoluble fraction (b) were not changed (P>0.05),
while gas production rate constants for the insoluble fraction (c) and potential extent of gas
production (a+b) were linearly increased when increasing level of U-cas in HQFB (P<0.05). The c
value was highest at 0.09 ml/h when supplementation 18% of U-cas in the HQFB. In vitro
cumulative gas production techniques were developed to predict fermentation of ruminant
feedstuffs. Similarly, cumulative gas production (96 h) was significantly different among
treatments and was linearly highest when HQFB contained of 18% U-cas (102.3 ml/0.5 g DM
substrate). The in vitro gas production technique has a remarkable boost and data on fermentation
kinetics of numerous feeds are available (Infascelli et al., 2005). Under this study, improved
performance of kinetics gas could be due attributed to fermentable energy as molasses and NPN
source from 18% U-cas in HQFB, may have provided on a continuous NH3-N basis, additional
and essential nutrients needed for rumen microbes. These results were similar to our previous
work by Cherdthong et al. (2011a), which supplemented U-cas with cassava chip as an energy
source in concentrate diets results in an increased c and a+ b value of the inoculums as well as
cumulative gas production. This will indicate the availability of readily fermentable materials as a
ready energy and protein sources which will stimulate the activity of the rumen microorganisms
which in turn would accelerate the production of gas volumes.
985
The in vitro dry matter degradability (IVDMD), in vitro organic matter degradability
(IVOMD), true digestibility and microbial mass were altered by treatments (P<0.01) and were
greatest at 18% of U-cas supplementation. As the result, high gas production in 18% of U-cas
indicated high digestibility of substrates. Moreover, higher in vitro true digestibility reflects higher
microbial biomass, the result was also found in 18% of U-cas supplementation in HQFB (Table
2). This could possibly be that U-cas was more slowly release to ammonia than urea and could
potentially be used more efficiently by rumen microorganisms. These results were in agreement
with Cherdthong et al. (2011a), who reported that supplementation of urea calcium mixture
product as a slow release NPN source in concentrate diet could improve digestibility and
microbial mass in in vitro experiment. Moreover, the digestibility of fiber and cellulolytic bacterial
population were enhanced when dairy cows or beef cattle fed with U-cas (Cherdthong et al.,
2011b).
The NH3–N concentration were highest when urea was supplemented in HQFB while
concentrations tended to be reduced with increasing level of U-cas (P<0.05). This could be due to
U-cas that could control the rate of N degradation in the rumen and leading to a slow rate of NH 3-
N released when compared with 100% of urea in HQFB. In agreement with these observations,
Cherdthong et al. (2011a,b) reported that supplementation of U-cas as slow-release urea in
concentrate diet reduces the rapidity of ammonia release in the rumen without affecting other
ruminal fermentation parameters.
Based on the results of this experiment, supplementation of U-cas supplementation at 18%
DM in high quality feed block resulted in improved in vitro kinetics gas production, rumen
fermentation, microbial mass and digestibility. Moreover, U-cas could control the rate of N
degradation in the rumen and leading to a slow rate of NH3-N released. However, in in vivo study
in order to improve production efficiency of ruminant animals still warrant further research.
ACKNOWLEDGMENTS
The authors would like to express our sincere thanks to the Tropical Feed Resources
Research and Development Center (TROFREC), Department of Animal Science, Faculty of
Agriculture, Khon Kaen University, Thailand and Thailand Research Fund (TRF) and Office of
the Commission on Higher Education (CHE) through the Research Grant for New Scholar
(Contract no. MRG5580077) for providing financial support for the research and the use of the
research facilities.
REFERENCES
Cherdthong, A., M. Wanapat and C. Wachirapakorn. 2011a. Influence of urea-calcium mixtures as
rumen slow-release feed on in vitro fermentation using gas production technique. Ach. Anim.
Nutr. 65: 242-254.
Cherdthong, A., M. Wanapat and C. Wachirapakorn. 2011b. Influence of urea calcium mixture
supplementation on ruminal fermentation characteristics of beef cattle fed on concentrates
containing high levels of cassava chips and rice straw. Anim. Feed Sci. Technol.163: 43–51.
Foiklang, S., M. Wanapat and W. Toburan. 2011. Effects of various plant protein sources in high-
quality feed block on feed intake, rumen fermentation, and microbial population in swamp
buffalo. Trop. Anim. Health Prod. 43: 1517-1524.
Infascelli, F., F. Bovera, G. Piccolo, S. D’Urso, F. Zicarelli and M.I.Cutrignelli. 2005. Gas
production and organic matter degradability of diets for buffaloes. Italian J. Anim. Sci. 4
(Suppl. 2): 316-318.
Menke, K.H. and H. Steingass. 1988. Estimation of the energetic feed value obtained from
rskov, E.R. and I. McDonald. 1979. The estimation of protein degradability in the rumen from
chemical analysis and gas production using rumen fluid. Anim. Res. Dev. 28: 7-55.
incubation measurements weighted according to rate of passage. J. Agric. Sci. 92: 499–503.
SAS. 1998. User's Guide: Statistic, Version 6, 12th Edition. SAS Inst. Inc., Cary, NC.
986
Wanapat, M. 2009. Potential uses of local feed resources for ruminants. Trop. Anim. Health Prod.
41: 1035–1049.
Table 1. Ingredients and chemical compositions of high-quality feed block (HQFB) were used
an in vitro experiment.
% of urea-calcium mixture (UCM) in HQFB
Items
0 3 6 9 12 15 18
Ingredients, kg DM --------------------------------%DM-------------------------------------
Rice bran 30.0 30.0 30.0 30.0 30.0 30.0 30.0
Molasses 42.5 41.0 40.0 39.5 39.0 38.0 38.0
Urea 10.5 9.0 7.0 5.5 3.5 2.0 0.0
UCM 0.0 3.0 6.0 9.0 12.0 15.0 18.0
Cement 12.0 12.0 12.0 12.0 11.5 11.0 10.0
Sulfur 1.5 1.5 1.5 1.0 1.0 1.0 1.0
Mineral premix 1.5 1.5 1.5 1.0 1.0 1.0 1.0
Tallow 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Dry matter 74.2 74.0 73.9 73.6 73.3 73.1 73.0
--------------------------------%DM---------------------------------------
Organic matter 76.5 76.3 75.1 74.9 76.6 75.3 75.7
Crude protein 34.9 35.2 35.5 35.4 34.8 35.3 35.5
Ash 23.5 23.7 24.9 25.1 23.4 24.7 24.3
Neutral detergent fiber 14.2 15.6 15.4 14.5 15.8 14.9 14.6
Acid detergent fiber 8.2 8.6 8.5 9.0 9.2 8.3 9.4
Table 2. The effect of levels of urea-calcium mixture (UCM) in high-quality feed block
(HQFB) on gas kinetics, ruminal fermentation and digestibility.
% of UCM in HQFB P-
Items SEM
0 3 6 9 12 15 18 value
Gas kinetics
A -2.3 -2.1 -2.4 -2.6 -2.2 -2.4 -2.3 2.1 ns
B 103.1 108.7 110.2 109.9 109.7 110.8 112.4 7.2 ns
C 0.03 0.03 0.06 0.07 0.07 0.06 0.09 0.03 *
a+b 100.1 103.4 106.8 107.4 107.4 109.9 110.2 2.5 **
Gas volume, ml 93.4 93.2 95.5 95.6 94.3 97.8 102.3 2.4 *
In vitro degradability, %
IVDMD 55.3 55.9 57.1 59.4 60.7 62.6 62.6 1.5 *
IVOMD 57.3 57.9 59.0 60.7 61.9 63.6 64.4 2.1 *
True digestibility, % 57.4 58.9 59.1 62.1 62 65.4 65.7 1.5 **
Microbial mass, mg 18.7 18.9 19.0 19.0 22.2 22.8 25.6 0.4 *
NH3-N, mg%
0 h incubation 18.2 16.7 16.3 15.6 14.2 14.5 13.3 3.5 ns
2 h incubation 24.4 21.2 22.8 20.5 18.9 18.1 16.2 2.1 *
4 h incubation 29.5 25.6 24.5 24.5 22.3 19.8 18.1 1.4 *
6 h incubation 27.7 24.1 23.4 22.0 21.1 17.2 16.5 2.3 *
Mean 25.0 21.9 21.8 20.7 19.1 17.4 16.0 1.2 *
*p < 0.05, **p < 0.01, ns = non- significant differences.
987
Nutritional status of some trace minerals of water buffaloes in Egypt
a
Maha Mohamed HADY
a
Professor and Head Department of Nutrition and Clinical Nutrition, Faculty of Veterinary
Medicine, Cairo University, Egypt, Postal code112411,
*Corresponding email: mhhady@yahoo.com
ABSTRACT
The domesticated water buffalo (Bubalus bubalis ) is very important animal in Egypt as
it serves as dual purpose animal providing meat and milk for human and as draft animal.
Despite the economical importance of buffaloes in Egypt, they are subjected to different
environmental, managemental and nutritional stressors which negatively affect their
performance. A study was conducted to evaluate the nutritional status of some trace-minerals
(copper, iron &zinc) of male buffalo calves in relation to their contents in the commonly
available feedstuffs in Mid-Delta district of Egypt. Fifty blood samples were collected from
apparently healthy male buffalo calves with an average age ranged between 5 to8 months
during the winter season from several private farms at the designated district of the study. The
present study indicated that the commonly cultivated feedstuffs in the Mid- Delta district of
Egypt have critical levels of copper and zinc reflected in the marginal levels of such minerals
in the serum of the examined male buffalo calves which exhibited silent symptoms except of
low gain. Iron content in the available feedstuffs supplied surplus Fe, so that the animals did
not exhibiting any symptoms of iron deficiency. In conclusion, the levels of the copper and
zinc in the Egyptian feedstuffs cultivated in the district of the study should be investigated on
soil basis so mineral supplements must to be added. Moreover, it seemed that the
requirements of buffalo calves for these minerals are less than expected and buffaloes adapted
well with such marginal deficiencies in feedstuffs.
Keywords: Buffalo, Egypt, Copper, Iron, Zinc, Feedstuffs
INTRODUCTION
The domesticated water buffaloes (Bubalus bubalis) account for 170million in the world
(FAO, 2004), with 97% in Asia and 2% in Africa mainly Egypt. There are two general types;
the Swamp buffalo and River buffaloes. In Egypt, River buffaloes play an important role in
the rural economy as suppliers of milk and draft power. Despite the economical importance of
buffaloes in Egypt, they are subjected to different environmental, managemental and
nutritional stressors which negatively affect their performance. Essential trace minerals such
as copper, iron, and zinc are of utmost importance in regulating animals metabolism and their
deficiencies cause great economical losses in animal production. Copper is essential for
osteogenesis, hematopoiesis and mylination of nerve cells. Cu deficiency is the most common
micro-mineral deficiency for the grazing livestock worldwide. Cu deficiency exhibits
different symptoms ranged from anemia, retard growth, diarrhea, loss of hair growth and
pigment, long bone affection to silent infertility (McDowell, 1997). In many species, hidden
(subclinical) copper deficiency is far less dramatic, but economically very important as it
effects on live weight gain, especially cattle. Iron is a component of heme compounds, and it
enters in the several enzyme systems regulating body metabolism. Commonly, Fe deficiency
is greatly related to great morbidity and mortality associated with depressed immunity
988
(MÖllerberg and Moreno- Lopez, 1975).Zinc is a component of many metallo- enzymes such,
which play a great role in the metabolism of protein, energy and nucleic acids
(Underwood,1997). Blood measures are frequently used in assessment the nutritional status
of trace minerals but several limitations as species, breed, sex, age malnutrition, illness,
reproductive status, and physiological variations, can affect the serum chemistry values
(Claypool et al., 1975). Blood mineral contents in buffaloes have been reported to differ from
area to area within the same country, in Pakistan, (Akhtar et al., 2011), in Egypt, (Hady, 1984)
and In Iran (Tajik et al., 2010). A large number of factors such as, the mineral contents of
plants have been reported to vary with variation in chemical composition of soil which is
largely affected by the climatic conditions, especially temperature, rain fall and drainage of
water (McDowell and Arthington, 2005). Generally, in Egypt only fragmentary studies (Hady
1984 &1986 ; Ahmed et al.,2009) on the micro mineral status of the Egyptian water buffaloes
at certain province under certain farming system showed critical levels, while there is no
available data for the mid- Delta District . Meanwhile, the sub-clinical deficiencies without
manifestation of specific deficiency symptoms occur in this district adversely affecting the
growth, health, fertility and productivity of buffaloes. Unfortunately, the trace- elements
requirement for buffaloes is much derived from that of cattle which limits the buffaloes'
genetic potentials Therefore, the current study was undertaken to give recent data concerning
the copper, iron, and zinc status of the Egyptian water Buffaloes (Bubalus bubalis) raised in
mid-Delta district of Egypt in relation to their contents in the commonly available feedstuff in
such district so as to recommend different mineral formulas to improve buffaloes productive
and reproductive merits.

Animals and Blood sampling:
Fifty blood samples were collected from fifty apparently healthy male buffalo calves
with an average age ranged between 5 to 8 months during the winter season (November-
February) from different private farms in the area of the Mid- Delta of Egypt. Serum samples
were separated and stored at -20oC until analysis.
Ration:
The animals were fed mainly for at least a period of 70 days on whole buffalo milk
then subjected to different feeding protocols according to the farm strategy depended mainly
on green berseem (Trifolium Alexandrinum ) as it is the commonly available during winter
season plus some concentrate mixture and wheat straw. The commercial concentrate mixture
in this district was supplied by certain company and consisted of; undecrticated cotton seed
cake; course wheat bran; crushed corn; rice kernel and polish and salt. Three samples were
collected weekly from each feedstuff (n=12) during one month- period of sampling. Feed
samples were dried, ashed and the HCl-insoluble ash was determined according to Marshal
(1993).
Analysis of trace minerals in serum and ration samples:
Copper, iron and zinc were determined by atomic absorption spectrophotometer (AA-
640, Shmadzu Co., Ltd, Japan). The preparation of ration for trace mineral determination was
done according to Hady (1986).
Statistical analysis:
The data were analyzed statistically according to Sndecor and Corchan(1982).

The study of blood constituents can provide valuable information about the general
health condition of an animal and therefore, can be used for evaluation the health status of the
animal. The results of mean serum levels of copper, iron, and zinc of the male buffalo calves
during winter season in Egypt are presented in table1. The results revealed that copper, iron
and zinc serum levels (μg/dl) were ranged from 22.5 to 55.7; 63.1 to 85.9; and from 39.2 to
989
55.4, respectively. The mean serum values of copper, iron and zinc demonstrated in this study
were less than that previously reported before by the same author (1984 & 1986) for male
Egyptian buffaloes and by Ahmed et al. (2009) in normal buffalo cows. The serum
concentration of measured trace elements for water buffaloes in current study were somewhat
different from the previously reported ranges for water buffalo and other ruminants, including
sheep, goat and cattle (Hady 1986, Khan ,2003 & Pasha et al., 2010). The differences between
water buffalo and other ruminants in the serum concentration of micro -elements may be
regarded as physiological peculiarities due to their adaptation to environmental conditions and
poor feeding resources. Moreover, the reported levels for Cu, Fe and Zn in the current study
were less than that reported in Iranian male Buffaloes (Tajik et al., 2010) and in different
areas in Pakistan (Akhtar et al.,2011). Surprisingly, the mean serum Cu, Fe and Zn levels in
the present study were less than the critical levels designated by McDowell et al. (1997) but
no detected clinical signs were manifested on the examined buffalo calves except for the low
gain which emphasis much adaptation of the Egyptian buffaloes to low quality feedstuffs in
such district.
The mean iron and zinc serum levels in the buffalo calves was lower than the values
that previously recorded in Egypt (74.2&48.7vs.85.4&81.2μg/dl, respectively) by Hady
(1986), but for the different geographic districts in Egypt which might be related to the
interrelationship between the trace minerals content in the soil, plant and animal.
The results of average total ash content, copper, iron, and zinc in different investigated
feedstuffs are presented in table 2. The average total ash content (%) of different Egyptian
feedstuffs revealed that berseem exhibited the highest % ash followed by wheat straw, while
the concentrate mixture was the least. Comparable results were previously reported for
Egyptian feedstuffs (Hady, 1984& 1986; Soltan, 1996). Egyptian berseem (Trifolium
Alexandrinum) surpassed wheat straw and concentrate mixture in the total ash, copper, iron
and zinc contents. Copper content (mg/kg) in the examined feedstuffs (Table 2) revealed that
copper content was almost four times more in berseem (16.7) as for wheat straw (4.5) and one
and half time more than in the concentrate mixture (10.6). The aforementioned results are less
than the results of Hady (1984& 1986) and Soltan et al., (1996) but covering different
geographic regions. Generally, the copper content of the different Egyptian feedstuffs was
ranged between 4.5 and 16.7 mg/kg confirming the adequacy of copper to satisfy the animal's
requirement which on average is10 mg/kg established by Anon (1996 & 2006), and more than
the critical level reported by NRC (1984). However, the adequate Cu level in the Egyptian
feedstuff was not reflected on the Cu serum levels of the male buffalo calves of this study
which showed no clinical signs of Hypocuprosis but low gain. This Cu concentration could be
adequate provided if there is no Molybdenum, which often antagonistically reduces Cu
concentration (Underwood, 1981).Moreover, it is to be suggested that male buffalo calves (5-
8 month) might have more copper requirement than the recommended levels for cows and
should be above the critical level.
Iron content of berseem, wheat straw and concentrate mixture was surplus as
compared to the estimate of other micro- minerals (Cu & Zn). The Egyptian berseem recorded
the highest Fe value, followed by wheat straw then concentrates mixture (204.9, 169.2&
101.8 mg/kg, respectively). The iron content of the Egyptian berseem was comparable to the
values reported previously by Soltan (1996). In this study, the overall available feedstuffs
supplied optional extra Fe, so that the animals did not exhibiting any symptoms of Fe
deficiency, nevertheless, marginal levels of Fe in serum were recorded that is perhaps due to
the possible interactions between Cu, Zn and Fe metabolisms. The results of Zn content
(mg/kg) of berseem, wheat straw and concentrate mixture (Table 2) revealed that berseem
contained the highest Zn content (65.3) followed by concentrate mixture (35.4) then wheat
straw (17.9). In general, berseem cultivated in Egypt is capable to cover solely the Zn
requirement of animal's as the critical Zn level of forages is around 30mg/kg as shown by
Viets and Lindasy (1973). Therefore, overall average Zn level of Egyptian feedstuffs
990
examined in the current study is more than ruminant maintenance requirement estimated by
(McDowell and Arthington, 2005).
REFRENCES
Ahmed, W.M., H.H. ElKhadrawy, M. H. Emtenan, R. Amal. A. El. Hameed and H.A. Sabra.
2009. Effect of Copper Deficiency on Ovarian Activity in Egyptian buffalo-
cows.World J.Zoology.4 (1):01-05.
AKhtar, M.S., A.S. Farooq, C. MazharAyaz, H. Maqbool., H. Mushtaq and Z. Chaudhary.
2011.Trace mineral status of Nili- Ravi buffaloes in Tehsil Pattoki of district
Kastour,Pakistan.International J of Engineering,Science and Metallurgy,1(2):290-292.
Claypool, D.W., F.W. Adams, H.W. Pendell, N.A. Hartman and J.F. Bone. 1975. Relatioship
between the level of copper in the blood plasma and liver of cattle. J Anim Sci. 41:
911-914.
Hady, Maha.M. 1984.Micro- Mineral status of water Buffaloes in Egypt. Master Thesis
.Faculty of Veterinary Medicine, Cairo University. Egypt.
Hady,Maha.M. 1986.Role of micro elements in nutrition of water buffalo and its relation to
production and animal health.Ph.D.Thesis,Faculty of veterinary Medicine,Cairo
University,Egypt.
Khan, Z.I. 2003. Effect of seasonal variation on the availability of macro-and micro, nutrients
to animals (sheep and goats) through forage from soil. Ph.D Thesis Univ.
Marshal, R.T. 1993. Standard Methods for Examination of Dairy Products. American
PublicHealth Association Report, Washington, USA.
McDowell, L.R. (1997). Minerals for Grazing Ruminants in Tropical Regions, 3rd ed.
Bulletin. pp.81.
McDowell, L.R. and J.D.Arthington.2005.Minerals for grazing ruminants in topical regions of
Florida, IFAS, Ganesville.
Möllerberg L, Moreno-LópezJ. 1975.The response of normal and iron anemic calves to nasal
infection with an attenuated strain of parainfluenza-3virus.Acta Vet.Scand.16(2):186-
96.
NRC 1984. Nutrient Requirements of Domestic animals, No. 4. Nutrient requirements of
BeefCattle. 6th edition. National Research Council,Washington DC.
Pasha,T.N., U. Farooq, M.Z. Khan, Y.A. Ditta, M. Ilyas and H. Ahmed. 2010. profile of
selected trace elements in soil, forage and growing buffalo calves in rice grown region
of PunJab, Pakistan.Proceeding 9th World Buffalo congresspp685-88.
Snedcor, G.W. and W.G. Cochran, 1982. Statistical Methods. 7th Edn., The Iowa State
University Press, Ames, Iowa, USA., pp: 507.
Soltan, M. A. 1996. Mineral status of the camel (Camelus Dromedarious) in relation to
nutrition and age. Ph. D. Thesis. Presented to faculty of Veterinary Medicine,
Alexandria University, Egypt.
Tajik, J., Nazifi, S., Izadneshan, M. and S.M. Naghib. 2010. Evaluation of trace elements
serum concentrations and their correlation together, and with thyroid hormones in
water Buffalo (Bulbalus bulbalis).
Underwood, E.J. 1977. Trace elements in human and animal nutrition. Academic Press, New
York. Fourth Edition 1977. pp 545.
Viets, F.G. and W.L. Lindasy. 1973.Testing soil zinc, copper, manganese and iron. In: Soil
testing and plant analysis.pp153-172. Soil Sci .Soc. Am. Inc. Madison.WI.
.
991
Table 1. Serum levels of copper, iron, and zinc (μg/dl) of male buffalo calves in the Mid-
Delta of Egypt
Parameter Minimum Maximum Mean ± SD
Copper 22.5 55.7 30.5 ± 4.22
Iron 63.1 85.9 74.2 ± 6.32
Zinc 39.2 55.4 48.7± 7.56
Table 2. Average* total ash content (%), copper, iron, selenium and zinc in different
feedstuffs** (mg/kg)
Feedstuff Total ash % Copper Iron Zinc

Berseem 16.8± 1.70 16.7± 4.35 204.9 ± 21.8.5 65.3 ±6.30
Wheat 14.3± 3.80 4..5 ± 1.90 169.2 ± 33.8 17.9 ± 1.77

straw
Concentrate 9..9± 0..90 10.6 ± 2..60 101.8 ± 17..90 35.4 ± 4..30
mixture
*Average = mean± SD
**On dry matter basis.
992
Effect of Roughage Sources and Fibrolytic Enzyme Supplementation on

Nutrient Digestion and Rumen Fermentation in Buffaloes
Chalermpon YUANGKLANGa, Kraisit VASUPENa, S. BUREENOKa,

S.WONGSUTHAVASa, A.C. BEYNENa,e, Chalong WACHIRAPAKORNb, Pramote
Paengkoumc, S. Paengkoumd, M. Phonvisaya and T. Vorlaphima
a
Department of Animal Science, Faculty of Natural Resources, Rajamangala University of
Technology Isan, Sakon Nakhon Campus, Phangkhon, Sakon Nakhon, 47160 Thailand
b
Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 4002
Thailand
c
School of Animal Production Technology, Institute of Agricultural Technology, Suranaree
University of Technology, Muang, Nachon Ratchasima 30000 Thailand
d
Program in Agriculture, Faculty of Science and Technology, Nachonratchasima Rajabhat
University, Muang, Nachon Ratchasima 30000 Thailand
e
Department of Animal Production, College of Food and Agricultural Sciences, King Saud
University, Riyadh 11451, Kingdom of Saudi Arabia
*Corresponding email: chayua@hotmail.com
ABSTRACT
The objective of present experiment was aimed to investigate the effect of roughage sources and
fibrolytic enzyme supplementation on feed intake, nutrient digestion and rumen fermentation. Four rumen
fistulated buffalo bulls about 3.7 years old with average initial BW of 430 + 25 kg were used in a 2x2 Latin
square design. Buffaloes were received rice straw (RS) or urea-treated rice straw (UTS) with or without
fibrolytic supplementation. Fibrolytic enzyme supplementation was added on top of concentrate diet.
Concentrate diet was offered at 1.5%BW. Roughage was fully supplied. Roughage and total intakes
expressed as kgDM/d, %BW and g/kgBW0.75 were significantly difference (P<0.05) among treatments.
Rumen parameters were altered by the roughage sources. Buffalo fed UTS was better in rumen fermentation
end products than in buffalo fed RS. Enzyme supplementation was increased (P<0.05) both total and
roughage intakes. Concentrate intake was similar among treatments. Based on the experiment data, it can be
concluded that supplemental fibrolytic enzyme in buffalo diet improves feed intake, nutrient digestion and
rumen fermentation end products particularly when roughage source is a low quality roughage.
Keywords: Rice straw, Urea-treated Rice Straw, Fibrolytic Enzyme, Nutrient Digestion, Rumen
Fermentation
INTRODUCTION
It is well known that rice straw is a main roughage source for ruminant animals including
buffalo especially in the developing countries. Thailand is a country where rice production is the
main crop production and the by-products from rice production is rice straw which is available for
ruminant animals. However, it is well recognized that rice straw contains low crude protein content,
high lignin content and low digestibility (Hart and Wanapt, 1992). There are many researchers
attempt to improve the quality of rice straw by many methods such as urea treatment (Wanapat,
1999), NaOH (Khejornsart and Wanapat, 2010), alkali treatment (Wanapat et al., 1985) and ensiling
(Yuangklang et al., 1994). Urea treatment is a conceivable method to increase the nutritive value
and digestibility of rice straw (Wanapat and Pimpa, 1999). Fibrolytic enzyme is an exogenous
enzyme which has been intensively investigated to improve fiber digestibility of forages.
Beuchemin et al. (1995; 2003) reported that fibrolytic enzyme supplementation improve fiber
digestibility of forage diet. Similar resulted with Krause et al. (1998) found that enzyme
supplementation increased ADF digestibility when added to a high concentrate diet. Colombatto et
993
al. (2003) demonstrated that enzyme supplementation increased neutral detergent fiber digestibility.
Shekhar et al. (2010) who found that fibrolytic enzyme supplementation increases milk production
in dairy buffalo. In accordance with Hristov et al. (2000) found that fibrolytic enzyme
supplementation increases ruminal and intestinal nutrient digestibility. Tang et al. (2008) studied the
effect of yeast culture and fibrolytic enzyme supplementation improves in vitro gas production of
cereal straw. Khanh et al. (2012) found that fibrolytic enzyme supplementation in fermented total
mixed ration (FTMR) did not improve fiber digestion in dairy cows, but fibrolytic enzyme
supplementation in total mixed ration (TMR) did increase fiber digestion. The objective of present
experiment was aimed to investigate the effect of roughage sources and fibrolytic enzyme
supplementation on feed intake, nutrient digestion and rumen fermentation in buffalo.
Four rumen fistulated buffalo bulls about 3.7 years old with average initial BW of 430 + 25
kg were used in a 2x2 Latin square design. Buffaloes were received rice straw (RS) or urea-treated
rice straw (UTS) with or without fibrolytic enzyme supplementation. Fibrolytic enzyme
supplementation was added on top of concentrate diet. Concentrate diet was offered at 1.5BW.
Concentrate diet was consisted of 57.0%cassava chip, 7.8%soybean meal, 11.4%whole cottonseed,
13.0%rice bran, 8.0%molasses, 2.0%tallow, 1.4%urea, 0.5%dicalcium phosphate, 0.5%premix and
0.2%sulfur.The ingredients of concentrate diet were demonstrated in Table 1. Roughage was fully
supplied. Animals were housed in individual pens and they were moved to the metabolic crates for
total collection. During the last 7 day of each period, total feces samples were quantitatively
collected and weighed. Feces samples were analyzed for dry matter, ash, crude protein (AOAC,
1990) and neutral detergent fiber (NDF) and acid detergent fiber (ADF) (Van Soest et al., 1991). On
the last day of each period, rumen samples were collected at 0 and 4 hours post morning feeding.
Rumen samples were immediately measured for pH and then pH values were recorded. Rumen
samples were prepared for ammonia nitrogen determination (Bremner and Keeney, 1965) and total
microbial count (Galyean, 1989).
design using the PROC MIXED (SAS, 1996) according to the following model : ijk=
All data were statistically analyzed as a 22 factorial arrangement in a 44 Latin square
+i+j+k+l+kl+ijkl, where ijk = represents of observation from animals,  = overall mean, I

= fixed effect of period (i = 1-4), j = random effect of animal (i = 1-4), k = fixed effect of factor A
(A = roughage sources, i = 1-2), l = fixed effect of factor B (B = enzyme supplementation, j = 1-2),
kl = fixed effect of interaction and ijk = random residual. Significant differences between
treatments were determined using Duncan’s New Multiple Range Test (DMRT) (Steel and Torrie,
1980).
994
Table 1. Chemical compositions of experimental diet.
Items Rice straw (RS) Urea-treated rice straw (UTS) concentrate

1
DM (%) 92.6 53.8 90.33
-------------------------- % of dry matter --------------------------

OM 89.55 88.78 93.3
CP 2.95 8.12 12.22
NDF 73.33 71.56 22.78
ADF 43.76 43.96 13.39
Ash 10.45 11.28 6.67
1
DM = dry matter; OM = organic matter; CP = crude protein; NDF = neutral detergent fiber; ADF = acid detergent fiber

Feed intake and nutrient digestion
The chemical compositions of concentrate diet, rice straw and urea-treated rice straw are
shown in Table 1. Urea-treated rice straw contained 8.12%crude protein. Yuangklang et al. (2010)
reported urea-treated rice straw contained 5.86%crude protein. Concentrate diet was formulated to
meet nutrient requirement of buffalo according to Kearl (1982). It was clearly showed that buffalo
fed RS was lower roughage intake than buffalo fed UTS (P<0.05). Enzyme supplementation was
not influenced roughage intake (P>0.05). There was no interaction between roughage source and
enzyme supplementation. Concentrate intake was offered at restricted feeding to ensure the rumen
fermentation process. Total intake was significantly different among treatments. Buffalo fed UTS
was lower in total intake than buffalo fed RS. Digestibility of DM, OM, NDF and ADF were
significantly different among treatments (P<0.05). Buffalo fed RS was higher in DM digestibility
than buffalo fed UTS, irrespective of fibrolytic enzyme supplementation.
995
Table 2. Feed intake and nutrient digestion of buffalo fed RS or UTS with or without fibrolytic
enzyme supplementation.
1
RS UTS P-value
Items - + - + SEM Ro En Ro*En
Roughage intake,
g/d 4.35b 4.48b 6.69a 6.59a 0.08 * ns ns
%BW 1.07b 1.08b 1.58a 1.63a 0.02 * ns ns
g/kgBW0.75 47.91b 48.62b 71.72a 73.45a 0.52 * ns ns
Concentrate intake,
g/d 6.13 6.24 6.35 6.06 0.05 * ns ns
%BW 1.50 1.50 1.50 1.50 - - - -
g/kgBW0.75 64.73 67.75 68.03 67.64 0.13 ns ns ns
Total intake,
kg/d 10.48b 10.72b 13.05a 12.65a 0.09 * ns ns
%BW 2.56b 2.58b 3.08a 3.13a 0.01 * ns ns
g/kgBW0.75 115.4b 116.4b 139.8a 140.4a 0.51 * ns *
Digestion, % of intake
DM 56.65c 59.34b 65.33a 66.89a 0.56 * * *
OM 58.88c 64.38b 67.76ab 69.23a 0.45 * * *
NDF 51.18c 54.23b 58.36ab 62.45a 0.61 * * *
ADF 48.32c 52.66b 55.90ab 59.23a 0.32 * * *

1
UTS = urea-treated rice straw; RS = rice straw; abMeans within a row with unlike superscripts differ (P<0.05).
Enzyme supplementation increased DM digestibility when compared with non-enzyme

supplementation. Similar with Histov et al. (2000) found that enzyme supplementation improved
nutrient digestion both in rumen and intestinal sections. Khanh et al. (2012) demonstrated that
fibrolytic enzyme supplementation in TMR did improve nutrient digestion in dairy cows. Rumen
pH in buffalo fed RS was higher than in buffalo fed UTS. There was no influenced of enzyme
supplementation on pH. The optimal level of ruminal pH was range from 6.0-7.0 (Hungate, 1967).
Concentration of NH3-N in buffalo fed RS was lower than in buffalo fed UTS. Similar result
reported by Wanapat and Pimpa (1999) demonstrated that rumen NH3 at 17.5 mg% gave optimum
rumen fermentation and maximum feed intake and digestibility of rice straw in buffalo. Total
bacteria count in buffalo fed UTS was higher than in buffalo fed RS. On the other hand, total
protozoa count in buffalo fed UTS was lower than in buffalo fed RS.
996
Table 3. Rumen pH, NH3-N and total microbial count of buffalo fed RS or UTS with or without
fibrolytic enzyme supplementation.
1
RS UTS P-value
Items - + - + SEM Ro En Ro*En
Rumen pH 6.88a 6.85a 6.52b 6.53b 0.01 * ns ns
NH3-N, mg% 12.42b 12.29b 16.73a 16.77a 0.24 * ns ns
Total bacteria count, 1010 cell/ml 5.73b 5.81b 7.15a 7.51a 0.13 * ns ns
Total protozoa count, 105 cell/ml 12.88a 12.50a 11.10b 11.00b 0.20 * ns ns
1
UTS = urea-treated rice straw; RS = rice straw; abMeans within a row with unlike superscripts differ (P<0.05).
Based on the experiment data, it can be concluded that supplemental fibrolytic enzyme in buffalo
diet improves feed intake, nutrient digestion and rumen fermentation end products particularly when
roughage source is a low quality roughage.
ACKNOWLEDGMENT
The authors would like to express their sincere thanks for Department of Animal Science, Faculty of
Natural Resources, Rajamangala University of Technology Isan, Sakon Nakhon campus for facilities
support.
REFERENCES
AOAC (Association of Official Analytical Chemist). 1990. Official Methods of Analysis. 15th ed. AOAC,
Washington, DC.
Bremner, J.M. and D.R. Keeney. 1965. Steam distillation methods of determination of ammonium, nitrate
and nitrite. Anal. Chem. Acta. 32:218.
Goering, H.K. and P.J. Van Soest. 1970. Forage Fiber Analysis (Apparatus, Reagent, Procedures and some
Application). Agric. Handbook. No. 397, ARS, USDA, Washington D.C.
Hart, F.J. and M. Wanapat. 1992. Physiology of digestion of urea-treated rice straw in swamp buffaloes.
Asian-Aust. J. Anim. Sci. 5:617.
SAS. 1996. SAS/STAT User’s Guide (Release 6.12). SAS Inst. Inc.,Cary, NC.
Sundstol, F., N.A. Said and J. Arnason. 1979. Factors influencing the effect of chemical treatment on the
nutritive value of straw. Acta Agric. Scand. 29:179.
Wanapat, M. and O. Pimpa. 1999. Effect of ruminal NH3-N levels on ruminal fermentation, purine
derivatives, digestibility and rice straw intake in swamp buffaloes. Asian-Aust. J. Anim. Sci. 12:904.
Wanapat, M., F. Sundstol and T.H. Garmo. 1985. A comparison of alkali treatment methods to improve the
nutritive value of straw. I. Digestibility and metabolizability. Anim. Feed Sci. Technol. 12:295.
Wanapat, M., S. Polyorach, K. Boonnop, C. Mapato and A. Cherdthong. 2009. Effects of treating rice straw
with urea or urea and calcium hydroxide upon intake, digestibility, rumen fermentation and milk yield
of dairy cows. Livestock Science. 125:238.
Yuangklang, C., C. Wongnen, C. Patiphan, J. Khotsakdee, T. Kandee, K. Vasupen, S. Bureenok, S.
Wongsuthavas, A. Alhaidary, H.E. Mohamed and A.C. Beynen. 2010. Rumen Fermentation in Beef
and Buffalo Steers Fed Native or Treated Rice Straw. Journal of Animal and Veterinary Advances, 9:
3011-3015.
Zaman, M.S., and E. Owen. 1990. Effect of calcium hydroxide or urea treatment of barley straw on intake
and digestibility in sheep. Small Rumin. Res. 3:237.
997
Buffalo Health
Antigen Based Detection of Cystic Echinococcosis in Buffaloes Using ELISA and

Dot – EIA
Arumugam SANGARAN* and Lalitha JOHN
Department of Veterinary Parasitology, Madras Veterinary College, Tamilnadu Veterinary and

Animal Sciences University, Chennai-600 007, India
*Corresponding email: sangaranvet@gmail.com
ABSTRACT
Cystic echinococcosis is caused by the larval stage of the dog tapeworm, Echinococcus
granulosus. The disease is recognized as one of the world’s major zoonoses affecting human beings
and their domestic animals apart from its economic and public health importance. Development of
the cysts in the intermediate host such as buffaloes occurs in the lungs, liver and other organs. The
condition is typically a chronic infection with viable cysts pertaining in many instances throughout
the life of the affected intermediate host. Serological tests rely mostly on the detection of serum
antibodies which may vary in their sensitivity and specificity with occurrence of false negatives and
false positives. In such instances, diagnosis based on detection of antigen is reliable with better
sensitivity and specificity. In this study, detection of circulating antigen in the diagnosis of cystic
echinococcosis in buffaloes was done using Enzyme linked immunosorbent assay (ELISA) and Dot
– Enzyme immunoassay (Dot – EIA). The sensitivity and specificity were determined as 89% and
92% respectively, whereas those of Dot-EIA were determined as 94% and 96%.
Keywords: antigen detection, buffaloes, cystic echinococcosis, ELISA, Dot-EIA
INTRODUCTION
Cystic echinococcosis, a zoonotic disease of man and animals is caused by the larval stage
(metacestodes) of the dog cestode, Echinococcus granulosus, the life cycle involving two
mammalian hosts. Definitive hosts are dogs in whose intestine the adult worms occur. Intermediate
hosts are herbivores and omnivores wherein the development of the cysts occurs in liver, lungs and
other organs. Accidental infection of man occurs during natural transmission of the parasite
between the canid definitive hosts and domestic livestock intermediate hosts. In the middle east, the
prevalence of cystic echinococcosis is high in man ( El-Muhtaseb, 1984) and in sheep, goats, cattle
and camels (Al-Yaman et al., 1985). Irrespective of the host species infected, cysts occur mostly in
liver and lungs with varying degrees of involvement of one or other of these organs (Schantz,
1972). The disease remains asymptomatic in many cases and in livestock intermediate hosts like
buffaloes, the cysts are seen only on post mortem. Immunodiagnostic tests for cystic echinococcosis
rely on the detection of serum antibodies and vary in their sensitivity and specificity with false
negatives (Craig and Rickard, 1981). To overcome such problem, the detection of circulating
antigen rather than antibodies could be very useful in the immunodiagnosis of cystic echinococcosis
particularly if circulating antigen is present in the false negatives (Craig and Nelson, 1984). Hence,
in the present study, detection of antigen in the diagnosis of cystic echinococcosis in buffaloes using
ELISA and Dot-EIA was carried out to find out the sensitivity and specificity of these two assays.

Sera from buffaloes
Blood samples were collected from buffaloes at the time of slaughter at the rate of 10ml
blood per buffalo in 30ml test tubes and allowed to clot. The blood samples were refrigerated
overnight and the sera separated, centrifuged at 2000 rpm for 15 minutes. The clean sera was
pipetted into sterile 5 ml plastic vials, preserved by adding merthiolate solution to a final
concentration of 1:10,000 and stored at -20°C.
999
Collection of hydatid cysts and preparation of Hydatid Fluid Antigen (HFA)

Hydatid cysts were collected from sheep and buffaloes from the slaughter house at the time
of slaughter. Cysts were brought to the laboratory immediately and were washed with sterile normal
saline. The hydatid fluid from the clean cysts was aspirated using a 20 ml glass syringe, transferred
into a glass container and allowed to stand for few hours. The hydatid fluid was examined
microscopically to ascertain the presence (fertile) or absence (sterile) of scolices so as to assess the
fertility status of the cysts. The supernatant fluid fluid from the fertile cyst was carefully aspirated
and dialysed in dialysis membrane bag against Polyethylene glycol 6000 (Carbowax) for one hour
to concentrate the fluid to half of its original volume. The dialysed HFA was stored in 5 ml
quantities in 10 ml sterile plastic screw cap vials with merthiolate at 1:10,000 concentration as
preservative at -20°C.
Estimation of Protein content of the Hydatid Fluid Antigen
The protein content of the dialysed hydatid fluid antigen was estimated by the method of
Lowry et al., (1951).
Preparation of hyperimmune serum against dialysed hydatid fluid antigen
Three healthy rabbits were hyperimmunised with three intramuscular injections of 2ml of
dialysed hydatid fluid antigen with Freund’s adjuant (Sigma, USA) at an interval of 14 days. The
first injection of DHF antigen was with Freund’s Complete Adjuant (FCA) and the second and third
injections were given with Freund’s Incomplete Adjuant (FIA). The rabbits were bled by cardiac
puncture 8-10 days after the third injection. About 5 ml of blood was collected from each rabbit and
hyperimmune serum was separated, centrifuged at 3000 rpm for 15 minutes, preserved with
merthiolate solution in 2 ml aliquots in 5 ml sterile plastic screw cap vials and stored frozen at -
20°C.
Enzyme linked immunosorbent assay (ELISA)
Enzyme linked immunosorbent assay (ELISA) of collected sera samples was performed
using hyperimmune serum raised in rabbits against dialysed hydatid fluid antigen. ELISA procedure
as recommended by Judson et al., (1985) for detection of circulating antigen was followed with
modifications. In this assay, any absorbance value which was twice and above the absorbance value
of the negative serum was considered as positive.
Dot – Enzyme Immunoassay (Dot-EIA)
Dot – Enzyme Immunoassay (Dot-EIA) of collected serum samples was performed using
the hyperimmune serum raised in rabbits against dialysed hydatid fluid antigen. The assay was
carried out as per the method of Romia et al., (1992) for detection of circulating hydatid antigens
with certain modifications. In this assay, an appearance of brown dot at the site of application of the
sample was considered positive.

In the present study, for detection of circulating antigen for cystic echinococcosis, blood
samples from 200 buffaloes (85 samples from hydatid positive buffaloes confirmed on slaughter
and 115 samples from buffaloes without visible hydatid cysts) were collected at the time of
slaughter and the sera separated for use in ELISA and Dot-EIA. The assays were performed using
hyperimmune serum raised in rabbits against dialysed hydatid fluid antigen.
Estimation of Protein content of the Hydatid Fluid Antigen
The protein content of the dialysed fertile hydatid cyst fluid from sheep and buffaloes was
found to be 14mg per ml and 9mg per ml of dialysed fertile hydatid cyst fluid respectively. Gatne et
al., (1990) estimated the protein content of hydatid fluid and observed the protein content ranged
from 16 to 20mg per ml of the fluid.
Enzyme linked immunosorbent assay (ELISA)
Seventy six buffaloes were detected positive out of 85 sera samples from hydatid positive
buffaloes using hyperimmune serum against dialysed hydatid fluid antigen by ELISA. From 115
sera samples from buffaloes without any visible hydatid cysts, 9 sera samples were detected false
positive. By ELISA, the sensitivity and specificity of the assay in detecting antigen for cystic
1000
echinococcosis were determined as 89% and 92% respectively. Craig and Nelson (1984) utilized
ELISA and reported the test to be 85%sensitive, the results in the present study coincides with the
earlier report. Similarly, Moosa and Abdel Hafez(1994) reported Circulating antigen detection
using ELISA, also decreased the number of false negatives and was more sensitive.The differences
in the sensitivity and specificity of the assay may be attributed to low levels of circulating
antigen/immune complexes in natural infection of false negative animals, whereas the false
positivity could be due to cross reacting circulating antigens of other metacestodes.
Dot – Enzyme Immunoassay (Dot-EIA)
By Dot-EIA, 80 of the 85 sera samples from buffaloes with hydatid cysts proved positive.
Four sera samples from 115 buffaloes with no visible hydatid cysts were detected false positive
using the hyperimmune serum against dialysed hydatid fluid antigen. A sensitivity of 94% and a
specificity of 96% were observed in Dot-EIA in detecting antigen in the diagnosis of cystic
echinococcosis. Romia et al., (1992) reported circulating antigen detection to be 86 per cent
sensitive by Dot –EIA and the low sensitivity was attributed to small amounts of circulating
antigens and immune complex formation. The increased sensitivity and specificity as observed in
the present study could be due to high levels of circulating antigens which in turn reduces the
number of false negatives and false positives.
REFERENCES
Al Yaman, F.M., L. Assaf, N. Hailat and S.K. Abdel Hafez.1985. Prevalence of hydatidosis in
slaughtered animals from North Jordan. Ann.Trop.Med.Parasitol. 79: 501-506.
Craig, P.S. and M.D. Rickard. 1981. Studies on the specific immunodiagnosis of larval cestode
infections of cattle and sheep using antigens purified by affinity chromatography in an
enzyme linked immunosorbent assay (ELISA). Int.J.Parasitol. 11: 441-449.
Craig, P.S. and G.S. Nelson. 1984. The detection of circulating antigen in human hydatid disease.
Ann.Trop.Med.Parasitol. 78:219-227.
El-Muhtaseb, H.H.1984. Surgical management of hydatid cysts of the liver: retrospective study of
75 cases. Jordan Med.J., 18:35-46.
Gatne, M.L., V.S. Narasapur, V.S. Deshpande, S.M. Niphadkar. 1990. Protein content
andelectrophoretic patterns of hydatid fluid. Indian Vet.J. 67: 169-170.
Judson, D.G., J.B. Dixon, M.J. Clarkson and J. Pritchard. 1985. Ovine hydatidosis: Some
immunological characteristics of the seronegative host. Parasitology 91:349-357.
Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall. 1951. Protein determination using
folin-ciocalteau reagent. J.Biol.Chem. 193:265-275.
Moosa, R.A. and S.K. Abdel Hafez. 1994. Serodiagnosis and sero epidemiology of human
unilocular hydatidosis in Jordan. Parasitol.Res. 80: 664-671.
Romia, S.A., M.E. Yousuf, A.E. Handoussa, H.M. Rizk and S.M. Sallam. 1992. Dot-ELISA as
a diagnostic test in hydatid disease. J.Egypt.Soc.Parasitol. 22: 603-610.
Schantz, P.M. 1972. Hydatidosis: Magnitude del problemyt perspectivase de control. Biol.of Sanit-
Panam. 74: 187-197 [Schwabe, C.W.1986. Current issues of hydatid disease: a zoonosis of
increasing importance in R.C.A.Thompson The Biology of Echinococcus and hydatid
disease. George Allen and Unwin, London, pp. 81-113.
1001
Comparative Efficacy of Enrofloxacin and Oxytetracycline as Systemic Dry

Period Therapy for the Control of Bubaline Mastitis
Muhammad Kashifa*, Tanveer Ahmadb, Abdul Shakoora, Muhammad Younus Ranac, Mian
Muhammad Awaisc, Syed Aun Muhmmada, Arfan Yousafd, Muhammad Yaqoobb, Zafar
Iqbale, Amar Nasira and Adnan Khana
a
Department of Clinical Studies, College of Veterinary and Animal Sciences, Jhang, Pakistan;
b
University of Agriculture, Faisalabad, Pakistan;
c
Department of Pathobiology, College of Veterinary and Animal Sciences, Jhang, Pakistan;
d
PMAS-Arid Agriculture University, Rawalpindi, Pakistan; eDepartment of Basic Sciences, College
of Veterinary and Animal Sciences, Jhang, Pakistan
*Corresponding Author: muhammad.kashif@uvas.edu.pk
ABSTRACT
This study was conducted to compare the efficacy of systemically administered
Enrofloxacin and Oxytetracycline in buffalo during dry period as a measure for effective control of
mastitis. A total of twenty seven dry pregnant buffaloes were selected and divided into three equal
groups G1, G2 and G3. The animals of group G1 were treated with enrofloxacin 2.5 mg/kg (IM) at
14 days and 7 days prior to expected date of parturition, group G2 was treated with
Oxytetracycline-HCl 11 mg/kg (IM) at 14 days and 7 days prior to the expected date of parturition
and group G3 was kept as non-medicated control (G3). Mammary secretions from all group were
collected aseptically 14 days prior to expected calving and milk samples from each quarter
aseptically collected at day 7 and 14 post calving. The efficacy of treatments was evaluated through
the prevalence of mastitic pathogens before and after calving and bacteriological cure rate. Post-
calving prevalence of mastitic pathogens after systemic dry period therapy with enrofloxacin and
oxytetracycline was lower than control group. The cure rate of infected quarters with enrofloxacin
(91.67%) and oxytetracycline (70%) was significantly higher than that of control (21.43%) (P<
0.05); however, there was no significant difference between enrofloxacin and oxytetracycline
treated groups (P> 0.05). It was concluded that dry period therapy with antibiotics especially
enrofloxacin helped in eliminating the existing intramammary infections and preventing new
intramammary infections. It may be adopted as an integral part of management to bring this disease
under control.
Keywords: Bubaline mastitis, dry period therapy, enrofloxacin, oxytetracycline
INTRODUCTION
Mastitis is one of the most economically important diseases of milk producing animals that
cause the changes in glandular tissues affecting both the quantity and quality of milk (Ullah et al.,
2005). Different strategies can be opted to avoid this problem and dry period therapy is considered
as an essential part of mastitis control program (MCP). Dry cow therapy eliminates approximately
70% to 98% existing intra-mammary infections and prevents almost 50% to 75% new intra-
mammary infections as a fundamental part of a successful MCP (Janosi and Huszenicaza, 2001;
Petzer et al., 2009). The intra-mammary route is considered as the route of choice for delivery of
dry period therapy for high absorption of drug into the udder region but at the same time there may
be high risk of both physiological and anatomical damage to the streak canal and inoculation of
organisms at the time of infusion (Bradley and Huxley, 2003). On the other hand, systemic dry
period therapy has many advantages including better distribution of drug in the udder tissue which
may lead to better cure of intra-mammary infections and avoidance of new infections which is
possible risk at the time of administration of intramammary infusion (Ziv, 1980; Boddie and
Nickerson, 1986). Systemic administration could simplify dry cow therapy routine. Systemic
administration of antibiotics some weeks before parturition is very effective treatment for intra-
1002
mammary infections, and is advisable for practice in the field conditions (Zecconi et al., 1999;
Oliver et al., 2003). The study was conducted to evaluate the comparative efficacy of enrofloxacin
and oxytetracycline as systemic dry period therapy in the control of bubaline mastitis with an
objective to find out a better antibiotic for dry period therapy to control the bubaline mastitis,
preventing new intramammary infections and eliminating the existing intramammary infections.

Experimental Design
Dry pregnant buffaloes (n=27) were randomly selected from different livestock farms to
divided into three equal groups viz. G1, G2 and G3. The samples of mammary secretions were
collected aseptically 14 days prior to the expected calving time for isolation and identification of
prevalent mastitis pathogens. Methodology adopted was in accordance with the guidelines of
National Mastitis Council (Anonymous, 1990). Each teat end was scrubbed vigorously with cotton
gauze soaked with 70 percent ethyl alcohol. Immediately after samples collections from all groups
teat dipping was done using iodophores to seal the teat ends (Hovareshti, et al, 2007). Just after the
collection of mammary secretions, antibiotic treatments were given to the animals as follows:
Group G1 = Pregnant buffaloes (n=9) were intramuscularly administered with enrofloxacin
(Inj. Encure10TM; Nawan Laboratories, Pakistan) 2.5 mg/kg on day 14th and 7th prior to the expected
date of parturition
Group G2 = Pregnant buffaloes (n=9) were intramuscularly administered with
oxytetracycline-HCl (Inj. Oxy-KakTM LA; Kaksian, Pakistan) 11.0 mg/kg on day 14th and 7th prior
to the expected date of parturition
Group G3 = Non-medicated pregnant buffaloes (n=9)
The milk samples (10 mL) from each quarter of all groups were collected aseptically in
sterile glass vials at day 7th and 14th post calving and shifted to the Microbiology Laboratory,
College of Veterinary and Animal Sciences, Jhang for isolation and identification of prevalent
mastitis pathogens (Hogan et al., 1999).
Bacteriological Examination
Pre-and post-calving milk samples were processed for bacteriological examination. The
procedure described by National Mastitis Council Inc., USA (Anonymous, 1990) was followed for
culturing the samples and identification of mastitis pathogens. Briefly, milk samples were shaken
gently to get a uniform dispersion of the pathogens. Using a platinum-rhodium loop, 0.01 ml of
milk sample was streaked on blood agar and incubated at 37°C for 48 hours. A quarter was
considered to be infected if 5 or more similar colonies were present on plate (Roberson et al., 1988).
Absence of the bacterial colony in the cultured samples, collected at day 7th and 14th post-calving,
was interpreted as a bacteriological cure.
The cultural and morphological characteristics of primary bacterial growth were studied by
examination of colony characteristics and preparation of smears from different colonies. These
smears were stained with Gram’s staining method and examined under the microscope. The primary
growths were purified by frequent sub culturing on selective and differential media. Each of the
isolate was identified on the basis of cultural and morphological characteristics, motility, hemolytic
and biochemical properties as described earlier (Cruickshank et al., 1975). The genus of bacteria
was determined on the basis of colony morphology, gram staining, hemolytic pattern and
biochemical tests. (Anonymous, 1990).
Percent prevalence of mastitis was calculated in all groups. The cure rate of infected
quarters among groups was calculated by using chi square test comparing treated groups and the
control. All groups were compared with each other using two proportional Z-tests. All the values
were considered significant at P<0.05.
1003
RESULTS
Prevalence of mastitic pathogens in mammary secretions on day 14th pre-calving
Pre-calving analysis of mammary secretions revealed the presence of five different mastitic
pathogens in the samples under study viz. Staphylococcus aureus, Streptococcus aglactiae,
Escherechia coli, Coagulase negative staphylococci (CNS) and Corynebacterium spp. The percent
prevalence of these bacteria in different quarters of animals in all groups is shown in Table 1.
Prevalence of mastitic pathogens in milk samples on day 7th post-calving
Bacteriological examination showed that in group G1, out of 36 milk samples collected from
the individual quarters of 9 animals, only one sample was positive for Coagulase negative
staphylococci with an overall prevalence rate of 2.78% of mastitic pathogens in group G1. Similarly
only one sample was found positive in group G2 but the pathogen was Staphylococcus aureus. On
the other hand, in non-medicated control group, all the mastitic pathogens identified in pre-calving
secretions were found in milk secretions at day 7th post-calving except E. coli and Corynebacterium
spp. The overall prevalence of mastitic pathogens in control group was 30.56% (Table 1).
Prevalence of mastitic pathogens in milk samples on day 14th post-calving
On day 14th post-calving, not a single sample was found positive for the presence of mastitic
pathogens in group G1 administered with Enrofloxacin; whereas, in G2 administered with
Oxytetracycline, 2 samples were positive for S. aureus, 1 sample for S. aglactiae and 1 sample for
CNS with an overall prevalence of 11.11%. In group G3, a similar trend was observed as on day 7th
post calving but the overall prevalence rate was much higher 41.67% (Table 1).
Postpartum cure rate of infected quarters
Post-calving cure rate of infected quarters at day 14th was 91.67% (cured/infected: 11/12)
when treated with enrofloxacin (group G1); whereas, in animals administered with Oxytetracycline
(group G2), this rate was 70% (cured/infected: 7/10). On the other hand, 21.43 % cure was also
recorded in non-medicated control (group G3) that may be regarded as spontaneous cure. Statistical
analysis revealed that difference in cure rates among medicated and control groups was
significantly different (P< 0.05); whereas, among the treated groups, this difference was statistically
non-significant (P> 0.05) (Table 2).
DISCUSSIONS
In the present study, comparative efficacy of enrofloxacin and oxytetracycline was studied
in acquisition of better choice for dry period therapy in buffaloes for mastitis control. Five different
mastitic pathogens viz. S. aureus, S. aglactiae, E. coli, CNS and Corynebacterium spp. were found
in the mammary secretions of selected buffaloes. Earlier, different species of bacteria responsible
for bubaline mastitis including S. aureus, S. aglactiae, CNS, A. pyogenese, corynebacterium and
coliform spp. have been screened from buffaloes in different parts of the world (Trabla and
Canavesio, 2003; Hovareshti et al., 2007).
On day 7th post-calving, a significantly lower prevalence rate of mastitic pathogens was detected
in the animals of two groups (G1 and G2) medicated with antibiotics during dry period as compared
to those of untreated group (G3); whereas, no significant difference was detected between the
medicated groups. On day 14th prevalence rate of group G2 increased to 11.11%, in contrast, the
14th day prevalence rate of group G1 was dropped to zero; whereas, in untreated control group (G3),
this prevalence rate increased to a much higher level (41.67%). On the whole, in this study
enrofloxacin demonstrated better results as than oxytetracycline. This enhanced activity of
enrofloxacin might be ascribed to with its large distribution volume, long half life and better activity
against the pathogen involved (Soback et al., 1990). Hovareshti et al.( 2007) concluded that a higher
efficacy of commercially available dry cow preparations as compared to intramuscular injections of
tylosin and enrofloxacin but observed no difference in the tylosin or enrofloxacin antibiotic therapy
for the control of mastitis. Although, dry cow preparations (intramammary tubes) give better results
but in large dairy herds, it is almost impractical, tedious, dangerous and not expectable by the
farmers (Shpigel et al., 2006). Additionally, this may introduce environmental bacteria and fungi
into the quarters by unsanitary manipulation of udder. Moreover, a lower risk level of antibiotic
1004
residue has also been demonstrated in systemic treatment as compared to intramammary infusions
(Hovareshti et al., 2007). Systemic dry period therapy using different antibiotics had shown
inconsistent results (Soback et al. 1990; Erskine et al. 1994; Smith and Hogan, 1998; Nickerson et
al. 1999; Zecconi et al. 1999). In contrast to our findings, Shpigel et al. (2006) observed very low
cure rate after systemic cefquinome treatment that was comparable to the spontaneous cure rate
observed in untreated controls. The unfavorable results of the cefquinome systemic dry period
therapy might reflect inadequate pharmacokinetic properties of the drug regarding poor udder
penetration in subclinical mastitis and short antimicrobial effect.
Erskine et al. (1994) evaluated the efficacy of intramuscular oxytetracycline against S. aureus
induced mastitis as a dry period treatment at drying off and found a low level of protection against
S. aureus as compared to this study. This variation might be due to difference in the pre-calving
medication time. Dry period length may be long and animal regain infections during dry period.
Post-calving maximum cure rate of infected quarters (91.67%) was recorded in the buffaloes of
group G1 injected with enrofloxacin followed by those administered with oxytetracycline (G2)
(70%). Control group (G3) also showed 21% cure rate that might be regarded as spontaneous cure.
Cure rates against individual microbes were higher in enrofloxacin treated buffaloes (G1) as than
those of treated (G2) and control groups. In G1, 100% cure was observed against S. aureus, S.
agalactiae, E. coli and corynebacterium spp.; whereas, for coagulase negative staphylococci cure
rate was 66.66%.
Similar findings have been reported by Petzer et al. (2009) who used intramammary prepapartion
containing cephalexin 250 mg and neomycin sulphate 250 mg. The cure rates in this study were
94.4 % for S. aureus, 100% for S. agalactiae and S. dysgalactiae, 78.1% for CNS and 100% for the
other minor pathogens.
In the present study, four new infections (11.11%) were also occurred in the control group but
no new infections appeared in the treated groups (G2 and G3). Natzke (1971) also reported the
development of new infections during the dry period without dry period therapy. In some previous
studies, new intramammary infections rates have been reported ranging from 13.10% to 34% during
the dry period (Osteras et al., 1991; Schukken, et al., 1993; Williamson et al., 1995).
In conclusion, the systemic dry period therapy using enrofloxacin is probably very much
effective to clear the existing intramammary infections and preventing new intramammary
infections. It should be adopted as an integral part of mastitis management to bring this disease
under control.
REFERENCES
Anonymous, 1990. Microbiological procedures for the diagnosis of bovine udder infections,
National Mastitis council Inc., USA 1840 Wilson Boulevard Arlington V.A. 22201, USA.
Bradley, A.J. and J.N. Huxley. 2003. A rational approach to dry cow therapy ii - making logical
treatment decisions. In Practice. 25(1):12-17.
Boddie, R.L. and S.C. Nickerson. 1986. Dry cow therapy: effects of method of drug administration
occurrences of Intra-mammary infection. J. Dairy Sci. 69: 253-257.
Cruickshank, R., J.P. Marman and R.H.A. Swain. 1975. Medical Microbiology 12th Ed., Vol.,
Churchill Living Stone, Edinburgh. pp. 236-244.
Erskine, R.J., P.C. Bartlett, P.C. Crawshaw and D.M. Gombas. 1994. Efficacy of intramuscular
oxytetracycline as a dry cow treatment for Staphylococcus aureus mastitis. J. Dairy Sci. 77:
3347-3353.
Hogan, J.S., R. Gonzalez, R.J. Harmon, S.C. Nickerson, S.P. Oliver, J.W. Pankey and K.L. Smith.
1999. Laboratory Handbook on Bovine Mastitis. Madison, WI, USA: National Mastitis
Council.
Hovareshti, P., M. Bolourchi and A.H. Tabatabayi. 2007. Comparison of the effect of systemic and
local antibacterial therapy to control staphylococcal intramammary infection in prepartum
heifers. J. Vet. Res. 62: 7-9.
1005
Janosi, S.Z. and G. Huszenicaza. 2001. The use of dry cow therapy in control of bovine mastitis.
Vet. Med.-Czech. 46: 55-60.
Natzke, R.P., R.W. Everrtt and D.R. Bray. 1971. Effect of drying off practices on mastitis infection.
J. Dairy Sci. 58: 1818-1827.
Nickerson, S.C., W.E. Owens, L.K. Fox, C.C. Scheifinger, T.R. Shryock and T.E. Spike. 1999.
Comparison of tilmicosin and cephapirin as therapeutics for Staphylococcus aureus mastitis
at dry off. J. Dairy Sci. 82: 696–703.
Oliver, S.P., M.J. Lewis, B.E. Gillespie, H.H. Dowlen, E.C. Jaenicke and R.K. Roberts. 2003.
Prepartum antibiotic treatment of heifers: Milk production, milk quality and economic
benefit. J. Dairy Sci. 86: 1187-1193.
Osteras, O., J. Aursjo, G. Gjul and A. Jorstad. 1991. Effect of dry cow therapy on subclinical
mastitis. J. Vet. Med. 41: 529-540.
Petzer, I.M., D.C. Lourens, J.C. Watermeyer, G.H. Rautenbach and P. Thompson. 2009.
Intramammary infection rate during the dry period in cows that received blanket dry cow
therapy: efficacy of 6 different dry-cow intra-mammary antimicrobial products. J. South
African Vet. Assoc. 1: 23-30.
Roberson, J.R., L.K. Fox, D.D. Hancock, J.M. Gay and T.E. Besser. 1988. Source of intramammary
infections from Staphylococcus aureus in dairy heifers at first parturition. J. Dairy Sci. 81:
687-693.
Schukken Y.H., J. Vanvliet , D.Vandeer and F.J. Grommers. 1993. A randomized trial on dry cow
antibiotics influention in low somatic cell count herd. J. Dairy Sci. 76: 2925-2930.
Shpigel, N.Y., P.H. Kass and A. Saran. 2006. A comparative randomized field trial on
intramammary and intramuscular dry cow antibiotic treatment of subclinical Staphylococcus
aureus mastitis in dairy cows. J. Vet. Med. 53: 418–422.
Smith, K.L. and J.S. Hogan. 1998. Epidemiology of mastitis. Proceedings of the 3rd IDF
international mastitis seminar, Vol-2, Tel-Aviv, Session 6, pp.3-12.
Soback, S., G. Ziv, M. Winkler and A. Saran. 1990. Systemic dry cow therapy- a preliminary
report. Staphylococcus aureus mastitis at dry-off. J. Dairy Sci. 82: 696–703.
Tarabla, H. and V. Canavesio. 2003. Prevalence of intramammary infections by major pathogens at
parturition in dairy cows after intramuscular antibiotic therapy at drying-off: A preliminary
report. J. Dairy Res. 70: 233-235.
Ullah, S., M.Q. Bilal, Zia-ur-Rehman, G. Muhammad and S.U. Rehman. 2005. The effect of
severity of mastitis on protein and fat contents of buffalo milk. Pakistan Vet. J. 25:1-4.
Williamson J.H., M.W. Woolford and A.M. Day. 1995. The prophylactic effect of a dry cow
antibiotic against Streptococcus uberis. N Z. Vet. J. 43: 228-234.
Zecconi, A., R. Piccinini and C.P.B. Guarni. 1999. Tylosin in cows in the dry period. In
Proceedings of the National Mastitis Council, Arlington, USA, pp. 237-238.
Ziv, G. 1980. Drug selection and use in mastitis: Systemic vs. local therapy. J. Am. Vet. Med Assoc.
176: 1109-1115.
1006
Table 1. Pre- and post-calving prevalence (%) of mastitic pathogens from individual quarters
(n=9×4=36, each group) of pregnant buffaloes.
Group
G1 G2 G3
Seco
First nd First Second First Second
Micro- 14 days 14 days 14 days
sampl samp sampl sample sample sample
organism before before before
e at le at e at at day at day at day
calving calving calving
day 7 day day 7 14 7 14
14
N N N N N N N N N
% % % % % % % % %
o. o. o. o. o. o. o. o. o.
Staphyloc
13. 16. 2. 5.5 22. 16. 19.
occus 5 0 0 0 0 6 1 2 8 6 7
89 67 78 6 22 67 44
aureus
Streptococ
5.5 2.7 2.7 5.5 5.5 8.3
cus 2 0 0 0 0 1 0 0 1 2 2 3
6 8 8 6 6 3
agalactiae
2.7 2.7 2.7 5.5

E. coli 1 0 0 0 0 1 0 0 0 0 1 0 0 2
8 8 8 6
Coagulase
Negative 8.3 2. 5.5 2.7 8.3 8.3 5.5
3 1 0 0 2 0 0 1 3 3 2
Straphyloc 3 78 6 8 3 3 6
occi
Corynebac
2.7 0.0 2.7
terium 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1
8 0 8
spp.
1 33. 2. 1 27. 2. 11. 1 38. 1 30. 1 41.

Total 1 0 0 1 4
2 33 78 0 78 78 11 4 89 1 56 5 67
G1=Enrofloxacin; G2=Oxyteracycline; G3=Non- medicated control
1007
Table 2. Day 14th post calving quarter based cure rate.
Groups Total Number of Percentage Number Cure Rate

number of quarter of (%)
quarter infected Quarter
before Cured
treatment
36 12 33.33 11 91.67
G1
36 10 27.78 7 70.00
G2
36 14 38.89 3 21.43*
G3
*= Significantly different (P>0.05).
1008
Incidence and Organ Wise Involvement of Hydatidosis In Buffaloes
Arumugam SANGARAN*1and Lalitha JOHN
Department of Veterinary Parasitology, Madras Veterinary College,

Tamilnadu Veterinary and Animal Sciences University,
Chennai-600 007, India.
*Corresponding email: sangaranvet@gmail.com
ABSTRACT
Hydatidosis is a condition caused by the bladder worm stage of the dog tapeworm,
Echinococcus granulosus in herbivores such as cattle, buffalo, and sheep. The rural population
especially in underdeveloped countries is at a higher risk of acquiring hydatidosis because of close
proximity with domestic and wild animals. The condition occurs in herbivorous intermediate hosts
as a result of accidental ingestion of the eggs of the parasite with contaminated feed and water apart
from grazing on contaminated pasture with the eggs as well. The hydatid cysts in such infected
animals are normally observed in lungs, livers and other viscera depending on the number of
ingestion of the eggs of the tapeworm. Hydatidosis in animals results in significant economic loss to
the meat industry through condemnation of the infected organs, livers, lungs and other organs apart
from reduced quality of meat, milk and wool. The incidence of hydatidosis in animals and human
beings in Chennai is found to vary from 1% to 13%, with more incidence in herbivorous animals.
Considering the economic significance of the disease, a study was undertaken to know the
incidence of hydatidosis in buffaloes at slaughter in the corporation slaughter house, Chennai by
inspection of the carcass and viscera of the slaughtered buffaloes. The present study indicated that
out of 847 buffaloes observed during slaughter, 85 had hydatid cysts giving an incidence of 10 per
cent. Lungs accounted for 52 (61.18%), livers 24 (28.23%), spleen 1 (1.18%) and the involvement
of both lungs and liver was observed in 8 (9.41%) of the 85 buffaloes with hydatid cysts.
Keywords: buffaloes, hydatidosis, incidence, organ wise distribution
INTRODUCTION
Hydatidosis is caused by the cystic larval stage of Echinococcus granulosus, the dog
tapeworm and the disease is recognized as one of the world’s major zoonoses affecting human
beings and their domestic livestock. It is a disease of both economic and public health significance
owing to the condemnation of the affected organs like lungs, livers, spleen, kidneys and other
viscera in slaughtered food animals such as cattle, buffaloes, sheep etc., apart from its affection in
human beings by accidental ingestion of the eggs of the tapeworm by playing with the infected dog
or through contamination of food and water with the eggs of the parasite. Incidence of hydatidosis
in this part has been reported earlier by Sundaram and Natarajan (1960) by examination of animals
slaughtered at Madras in India. Hydatid disease in the intermediate host like buffaloes is typically a
chronic parasitic infection with viable cysts persisting in many instances throughout the life of the
affected intermediate hosts. It is a zoonosis found in most pastoral areas of the world (Al Yaman et
al., 1985). In most Mediterranean countries, the disease is hyper endemic in sheep, goats, camels
and donkeys. New foci of infection and region of endemicity have recently been recognized and
there is increasing evidence of the causative agents extending their range into areas previously
considered to have been free of infection. Considering the economic importance of the disease in
buffaloes which are slaughtered in large numbers for human consumption, the present study was
undertaken to know the incidence of hydatidosis in buffaloes in Chennai.

The incidence of hydatidosis in food animals, meant for human consumption such as
buffaloes was observed at the time of slaughter by inspecting the carcasses and viscera of the
1009
slaughtered buffaloes for the presence of hydatid cysts particularly in organs like lungs, liver,
spleen, kidneys etc. The hydatid cysts in various organs were collected from the slaughtered
buffaloes and brought to the laboratory. The hydatid cysts were examined microscopically to
ascertain whether fertile or sterile cysts based on the presence or absence of protoscolices. The
organ wise affection by hydatid cysts was also recorded so as to know the incidence in different
organs and viscera of the slaughtered buffaloes

A total of 847 buffaloes were observed during slaughter, out of which 85 had hydatid cysts
in various organs, giving an incidence of 10%. With regard to organ wise involvement, lungs
accounted for 52 (61.18%), livers 24 (28.23%), spleen 1 (1.18%) and the involvement of both lungs
and liver was observed in 8 (9.41%) of the 85 buffaloes with hydatid cysts.
The incidence of hydatidosis in buffaloes was reported to vary from 7% to 12% (Deka and
Gaur, 1990), 13.5% (Sangaran, 1994), 19.22% (Koshy, 1984), 34.88% (Hussain et al., 1992) and it
was reported to be as high as 48% by Singh and Dhar, 1988. The finding in the present study was
found to be 10% which is similar to the findings of Deka and Gaur (1990) and Sangaran (2010).
Organ wise involvement in the present study revealed that lungs were found to be more
frequent targets (61.18%) in buffaloes, which is in accordance with the findings of Pillai et al.,
(1986), who had also reported that lungs were more commonly affected with hydatid cysts than
liver. Sundaram and Natarajan (1960) had reported that lungs were more frequently involved (58%)
as compared to liver, and spleen was affected less frequently (2.7 per cent). The findings in this
study correlated well with the reports made by earlier workers.
REFERENCES
Al Yaman, F.M., L. Assaf, N. Hailat and S.K. Abdel Hafez. 1985. Prevalence of hydatidosis in
slaughtered animals from North Jordan. Ann.Trop.Med.Parasitol. 79:501-506.
Deka, D.K. and S.N.S. Gaur. 1990. Epidemiology of hydatidosis in buffaloes in western parts of
Uttar Pradesh. J.Vet.Parasitol. 4:49-53.
Hussain, A., A. Maqbool, S. Hussain, M. Athar, A. Shakoor and M.K. Amin. 1992. Studies on
prevalence and organ specificity of hydatidosis in ruminants slaughtered at Karachi and
Faisalabad abattoir, Pakistan. Indian J. Dairy Sci. 45:454-456.
Koshy, T.J. 1984. Taenid infections in dogs. Ph.D. Thesis. Tamil Nadu Agricultural University,
Coimbatore, India.
Pillai, J., K. Narayana, P.L. Rao and S. Rao. 1986. A study on the prevalence of hydatidosis in
sheep and goats at Tirupati municipal slaughter house. Indian J. Public Hlth. 30:160-165.
Sangaran, A. 1994. Immunodiagnosis of hydatidosis in some food animals and human beings.
M.V.Sc. Thesis. Tamil Nadu Veterinary and Animal Sciences University, Chennai, India.
Sangaran, A. and Lalitha John. 2010. Incidence of cystic echinococcosis in buffaloes slaughtered at
Chennai. J. Vet. Parasitol. 24:93-94.
Singh, B.P. and D.N. Dhar. 1988. Echinococcus granulosus in animals in Northern India. Vet.
Parasitol. 28: 261-266
Sundaram, R.H. and R. Natarajan. 1960. A Study on the incidence of hydatid disease in cattle in the
city of Madras. Indian Vet. J. 37: 19-24.
1010
Emphysematous Necrotic Skin Disease (Patakha): A Newly Emerging Disease in

Buffaloes (Bubalus Bubalis)
Abdul SHAKOOR1, Sayyed Aun MUHAMMD1, Muhammad YOUNAS2, Muhammad

KASHIF1, Usamn WAHEED2, Asghar HUSSAIN3, Muhammad Raza HAMEED2 and Masroor
Ellahi BABER4
1
Department of Clinical Studies, 2Department of Pathobiology, 3Department of Animal Sciences,
College of Veterinary and Animal Sciences, Jhang-Pakistan. 4Institute of Biochemistry and
Biotechnology, University of Veterinary and Animal Sciences, Lahore-Pakistan.
*Correspondence author: aunmuhammd@uvas.edu.pk
ABSTRACT
Buffalo is the premier dairy animal in Pakistan and contributes >75% milk to be consumed
all over the country. Naturally like other animals, it is prone to be affected with a great variety of
diseases. Of those, a newly emerging disease has been coming into observation for the last 10
years, named as mixed clostridial infection (PATAKHA in common parlance). A total of 30 such
cases (lactating buffaloes=08, pregnant dry buffaloes =04, Non pregnant buffalo heifers = 08 and
pregnant buffalo heifers=10) were presented at Veterinary Teaching Hospital, Department of
Clinical Studies, CVAS, Jhang during the summer months (May – July 2010-2012). Mid
laterovetnral abdominal area was affected. There was raised puffy, painful skin of variable size (10-
21 inches wide and 1 inch deep) having bursting point in the center exposing the underlying
subcutaneous tissues along with white strings intermingled with each other and tightly adhered with
superficial musculature. Digital manipulation compelled the foamy watery exudates coming out.
There was tennis ball size pockets containing gas on the margins of lesions and crepitation was
pronounced on digital manipulation. Exudates smear indicated Gram’s positive bacilli and typical
clinical signs gave the clue of being clostridial infection. Debridement preceded by daily
chloroform application and producing aerobiasis by the upper lying dry necrotic skin followed by
Hydrogen peroxide spilling expedited the healing of the wound. It was washed thoroughly with
Normal Saline and applied with Margosa oil (Neem) daily till complete healing (10-15 days).
Injection Penicill-40 ® (Benzyl and Procain Penicillin 40 lac iu, Star Laboratories, Pvt. Ltd,
Pakistan) was given intramuscular daily for 7 days besides antiseptic dressing with Tr. Iodine
preceded by vigorous freshening and washing. All animals recovered completely without eliciting
any mortality. Four lactating buffaloes were found to be effected twice time after a year apart
interval. Keeping in view the exigency of situation, research workers should focus their attention
towards this emerging problem affecting the black gold of Pakistan (Buffalo) to seek its both
therapeutic and prophylactive solution.
Keywords: anaerobiasis, Emphysemated, Buffalo, Margosa oil, Necrotic
INTRODUCTION
Pakistan is an agricultural country and livestock is the back bone of agricultural economy.
The contribution of agricultural sector is 12 percent in the GDP. Of it, livestock contributes
approximately 55.1 percent to the agricultural value added and 11.6 percent to national GDP. Its importance
may be realized from this fact that 30-35 million populations is engaged in livestock raising and derives 46
percent income from it (Annon. 2011-12). Buffalo is the premier dairy animal and its current population

1011
stands at 32.7 million heads which produce more than 75% milk to be used all over the country (Sarwar,
2002). This animal is affected with a variety of diseases naturally like viral, bacterial, parasitic,
metabolic and deficiency diseases etc. (Chakrabarti, 2011). Of these, one has been coming into
observation commonly named in common parlance “Patakha” and technically called clostredial
infection. During the last three years (2010-2012) in summer months (May–July), a total of 30 cases
were recorded and treated successfully at Veterinary Teaching hospital, College of Veterinary and
Animal Sciences, Jhang (sub-campus of University Veterinary and Animal Sciences, Lahore),
Punjab province. Pakistan.
MATERIALS & METHODS

History: Complete history was taken about the animals brought to get treated. It indicated
appearance of swelling affecting latero-ventral abdominal area a week ago, spreading and increasing
in its circumference, painful and tenderness, self bursting in the centre.
Clinical Examination: Close clinical examination indicated that latero-ventral abdominal area was
raised & elevated of variable size (10-21 inches wide and one inch deep), high rise of body temp.
(104-106F), anorexia, decreased milk production, ruptured skin in the centre of lesion exposing the
putrified subcutaneous tissue, ventral oedema anterior to udder upto the umbilical region, sub-
cutaneous dark fibrous tissue texture partially affecting the superficial part of external abdominal
oblique muscle, inflammatory exudate having bubbles, elevated painful tennis ball sized pockets near
the margins of the affected area, painful edges, insensitive & hard upper lining skin of wound,
harboring of flies, bad smell emanating from wound indicating myiasis that cleared the s/c necrosed
tissue partially. Restlessness, frequents efforts to lick the wound. Digital manipulation elicited the
surf-mixed water like foamy exudates (Plate 1-2)
Diagnosis: Depending upon the presence of effevervascent and bubbling exudate, it was diagnosed
tentatively as clostridal infection and staining of exudate showed the Gram positive bacilli (Quinn et
al., 2010; Radostits et al., 2007). To indicate the presence of various species, Schaeffer-Fulton
method (spore staining technique) was used to locate the positions of spores in the bacilli present in
the impression smear prepared from the lesions of the afflicted animals.
Treatment: Necrosed skin was cut off to visualize the underlying tissue followed by spilling of
Chloroform to cause numbness. Having opened the puffy gaseous pockets near the margins of the
wound, Hydrogen per oxide (Spectrum laboratories, Pvt., Ltd, Lahore, Pakistan) was dribbled over
the wound to cause aerobasis and removal of upperlying necrotic and gangrenous dead tissues along
with vigorous debridement daily followed by thorough washing with normal saline. Drying the
wound, Tincture iodine (Prime Laboratories, Pvt., Ltd, Lahore, Pakistan) was painted and 15 minutes
later Margosa oil (Azadrachta indica) was also anointed. Penicillin injection (Penicill-40 ®, Star
Laboratories, Pvt., Ltd, Lahore, Pakistan containing Benzyl-10,00,000 and Procain- 30,00,000 i.u.
Penicillin) was administered daily for seven days while antiseptic dressing was continued till the
complete healing of wound.
RESULTS and DISCUSSION

A total of 30 Buffaloes affected with emphysemated necrotic skin disease were treated at
Veterinary teaching Hospital, Department of Clinical studies, College of Veterinary and Animal
sciences, Jhang, Punjab Provence, Pakistan. Of these, 17 animals had the lesion on left side and 13 on
right side while 10 animals had recrudescence after a year apart on both left (n=4) and right (n=6)
side affecting latero ventral aspect of the abdomen. This bursting may be ascribing to the production
of gas by the invader organisms causing maximum pressure at that point. The putrefaction of s/c &
superficial muscular tissue may be due to the production of necrotizing toxins and spreading factors
of causative organisms, which find an anaerobic environment beneath the skin.
1012
The dependant oedema developing surrounding the lesion especially on the ventrum of the
abdomin is ascribed to the rush of blood towards that side in response to the production of toxins by
the anaerobic organisms.this disease has resemblance with wound developed in maglignant oedema
affecting cattle, sheep, goat and is caused by clostridium septicum primarily along with the
association of other clostridia like Cl. chauvei, Cl. novyi, Cl. perfrinmens and Cl. sordelli. its onset is
sudden (12-42 hours) and causes mortality in 1-2 days while Cl. chauvoei causes blackleg in which
deep musculature are affected (Quinn et al., 2010) This has some resemblance with malignant
oedema but that is very acute & fatal (Chakrabarti, 2011) but none of the case proved fatal in this
study and recovered all.
Daily debridement along with Hydrogen peroxide (Spectrum laboratories, Pvt., Ltd., Lahore,
Pakistan) spilling locally proved its worth in removing the necrotic tissue being an oxidizing agent
(Tyagi and Singh, 1999; kumar, 2004) and application of Tr. Iodine enhanced the pace of granulation
tissue formation to a bridge the gap. This may be ascribe to the slightly irritant antiseptic
characteristic of Tr. Iodine in inciting the granulation development (Harari, 1996; Kumar, 2004) and
was followed by the Neem oil anointment which kept the flies away from the wound besides being
anti-maggots, antiviral and antifungal properties (Vanugopalan, 2000; Awan, 2010 and Multani,
2010). It was interesting to note that all animals were found infected on only one side of the
abdomen on latero-ventral aspect either on left or right side at a time. There was reoccurrence of
infection at the same site in the ensuing years in 10 animals (Table 1).
Appearance of the lesion at specific site (latero ventro aspect) may be ascribed to minute
punctured wound letting the clostridial organisms into the subcutaneous tissues to grow in less
oxygen tension. This may also be due to the instinctive sitting posture of buffaloes on the ground
impregnated with a host of blunt object i.e. pebbles, small brickbats, hard lumps of clay causing
contusion of specific site where wandering spores of clostridium organisms in circulation find
conducive site to multiply for producing necrotizing toxins followed by the typical lesion containing
gas subcutaneously.
In the present study isolation of etiological agent/ agents could not be made possible but
presence of Gram positive bacilli in exudates and good response to Penicillin therapy strengthened
the conviction of being infected with a host of clostridial microorganisms. It is worth mentioning to
note that all affected animals became perfectly all right within 10-15 days after the commencement of
the treatment. There is a dire need of the hour that a thorough investigation be made about both
etiological agents along with effective therapeutic and preventive measures to save the black gold of
Pakistan (Buffaloes) from this newly emerging malady.
1013
Table 1. Sketch of information regarding the location of wounds in emphysematous necrotic skin
disease (Patakha) affected buffaloes and its recurrence.
Latero-
ventral Repetition / Summer Recovery
Animals No abdomin recurrence Season Period
al sites (2010-12) (Days)
*L *R *L *R Total
Lactating 8 4 4 2 2 4 May-July 10-15
Buffaloes
Pregnant 4 2 2 - 1 1 May-July 10-15
Dry
Buffaloes
Non 8 5 3 1 1 2 May-July 10-15
Pregnant
Buffalo
Heifers
Pregnant 10 6 4 1 2 3 May-July 10-15
Buffalo
Heifers
Total 30 17 13 4 6 10
*L =Left side, * R = Right side
REFERENCES
Anonymous, 2011-12. Economic survey of Pakistan, Ministry of Finance, Statistical Division,
Government of Pakistan, Islamabad.
Awan, M. H., 2010. Kitab-ul-Mufradat., Sheikh Guulam Ali & sons Pvt., Ltd., Publishers, Chowk
Anarkali, Lahore, Pakistan.Pp 501.
Cappuccino J. G. and N. Shermanpage, 2009. Microbiology: a laboratory manual. 7 th ed, Pearson
Education, Inc. and Dorling Kindersley Publishing, Inc Pvt. Ltd, Dehli, India. Pp83-85.
Chakrabarti, A, 2011. A Text Book of Preventive Veterinary Medicine. 4th Ed. Kalyani Publishers.
New Delhi, India.
Harari, J., 1996. Small Animal Surgery. Williams and Wilkins. Rose Tree Corporate Center Building
Two 1400 N. Provence Road Media, PA 19063 USA.
Kumar, A., 2004. Veterinary Surgical Techniques. 2nd Ed. Vikas Publishing House Pvt Ltd. New
Delhi, India.
Multani, H.C., 2010. Taj-ul-Akakir-herbs of Indo-Pak and their strange wonderful Effects. Vol.1
Sheikh Muhammad Bashir & Sons Publishers, Chowk urdu bazaar, Lahore, Pakistan. Pp 164.
Quinn, P.J., B.K. Markey, M.E. Carter, W.J. Donnelly and F.C. Leonard, 2010. Clostridium species
In Veterinary Microbiology and Microbial Disease, Blackwell Science Ltd, 9600 Garsington
Road, Oxford OX4 2DQ, UK. Pp. 84.
Radostits, O. M., C. C. Gay, D. C. Blood, and K. W. Hinchcliff, 2007. Veterinary Medicine-A
textbook of the Diseases of cattle, Sheep, Pigs, Goats, and Horses. 10th Ed. W. B. Saunders
Co., Philadelphia, USA.
Sarwar, M., M. A. Khan, Mahr-un-Nisa and Z. Iqbal, 2002. Dairy Industry in Pakistan: a scenario.
Intl. J. Agric. Biol. 4: 420-428.
1014
Tyagi, R.P.S and J. Singh, 1999. Ruminant Surgery 1st Ed. CBS Publishers and Distributor 4596/1 A,
11-Daryaganj, New Delhi-110002. India.
Venugopalan, A. 2000. Essentials of Veterinary Surgery. 5th Ed. Oxford & IBH Publishing Co. Pvt.,
Ltd. New Delhi. India.
Plate 1-2: Typical Lesions of Emphysematous

Necrotic Skin Disease (Patakha) on letro-
ventral aspect of abdomen in Buffaloes
1015
Comparative Efficacy of Three Indigenous Plants (Fumaria Parviflora,

Artemesia Maritima & Swertia Chirata) Alone or in Combination for The
Treatment of Toxaemia
Adul SHAKOOR, Sayyed AUN MUHAMMAD*, Tariq ABBAS, Muhammad KASHIF,
Asghar HUSSAIN, Muhammad RAZA HAMEED and Usman WAHEED
College of Veterinary and Animal Sciences, Jhang. Sub-campus University of Veterinary and
Animal Sciences, Lahore-Pakistan
*correspondence author: aunmuhammad@uvas.edu.pk
ABSTRACT
Three medicinal plants (Fumaria parviflora, Artemesia maritima, Swertia chirata) were used
at dose rate of 100 gm animal for 7 days singly or in combination for the treatment of toxaemia
(Zaharabad) in buffaloes. The disease scoring was made on the extent of severity as 1st (+), 2nd (++)
& 3rd (+++) degree toxaemia. First degree toxaemia disappeared within 3 days when used singly
while their blend cured it within 2 days. Second degree relieved on the 5th day within all groups
with the exception of their blend which gave recovery within 2 days. Third degree toxaemia
disappeared on the 5th day except in the group treated with their mixture which became normal on
the 4th day. In short, it was concluded that all these plants were effective and took longer time but
their blend shortened the time of recovery. All these plants proved their worth as best anti-toxaemic
agents as was considered by the folk.
Keywords: buffalo, indigenous plants, toxemia


1016
In Vivo Comparison of Specific Activity of Egg Yolk Immunoglobulins (IgY) and

Antibiotic against Staphylococcus aureus Causing Mastitis in
Buffaloes (Bubalus bubalis)
Muhammad JUNAID IQBALa, Tanveer AHMADa*, Arfan Yousafc, Sajjad-Ur- RAHMANb,
Muhammad Saqiba, Muhammad Nadeemc and Ghulam MUHAMMADa
a
Department of Clinical Medicine and Surgery, Agriculture University, Faisalabad, Pakistan.
b
Institute of Microbiology, Agriculture University, Faisalabad, Pakistan, cDepartment of Clinical
Medicine and Surgery, Arid Agriculture University, Rawalpindi
*Corresponding Email: ahmadt@edu.uaf.pk
ABSTRACT
Thirty White Leghorn hens were divided into 3 groups. Inactivated S. aureus was
administered to groups H1, H2 and H3 at the dose rate of 106 CFU/ml, 109 CFU/ml and 1012
CFU/ml on days 0, 7 and 21, respectively. Antibody titers were determined by IHA. On day 7 to 21
post inoculation of hens with bacterin-toxoid, the antibody titers of groups H1, H2 and H3 were
minimum 64, 78.8 and 97 and maximum GMT of 128, 157.7 and 194, respectively. The protein
content of egg yolk immunoglobulins was 7.05mg/ml estimated from bovine serum albumin
standard curve. For in vivo evaluation, 40 S. aureus mastitic buffaloes were selected on basis of
SFMT and divided into 4 groups (Group A and B = clinicaly S. aureus mastitis C and D =
subclinicaly S. aureus mastitis).Groups A and B received intramammary infusions of egg yolk
antibodies and antibiotic at the dose rate of 10 ml/affected quarter, respectively. Similarly, groups C
and D received the same treatment. Evaluation criteria include microbiological cure rate, clinical
cure rate, SFMT and milk yield. The milk yield of 90% and 40% buffaloes was found increased in
the groups that received egg yolk antibodies and antibiotic, respectively. Similarly, clinical and
microbiological cures rates were 50% better in the egg yolk treated buffaloes than antibiotic treated
buffalo groups. On basis of SFMT, 80% and 40% buffaloes recovered from mastitis in the egg yolk
treated and antibiotic treated buffalo groups, respectively.
Keywords: alternative treatment, buffalo, mastitis, egg yolk immunoglobulins
INTRODUCTION
Bovine mastitis ultimately results in huge economical threat to farmer because he has to
spend a lot of money on the replacement of diseased animal as well as on treatment of diseased
buffalo (Singh et al., 1998). Among bacterial factors Staphylococcus aureus (S. aureus),
Streptococcus agalactiae, E.coli etc are mainly responsible for mastitis but S. aureus is on the top in
Pakistan. Staphylococcus aureus is highly resistant to commonly used antibiotics (Gill et al., 2006).
Recent researches have concluded that specific egg yolk immunoglobulins have the ability to tackle
infections both as preventive and therapeutic agent. Somatic cell count can be greatly decreased by
the use of egg yolk immunoglobulins as a therapeutic agent for treatment of S. aureus mastitis in
dairy cows (Coleman, 1996).Therefore, Present study has been planed for In Vivo Comparison of
Specific Activity of Egg Yolk Immunoglobulins (IgY) and Antibiotic against Staphylococcus
aureus Causing Mastitis in Buffaloes (Bubalus bubalis)

Milk samples from sub clinically mastitic buffaloes were collected by following the
procedure as per described by National Mastitis Council (1990). The buffaloes were selected on the
basis of Surf Field Mastitis Test (SFMT) as per described by Muhammad et al., (2010). The growth
from blood agar was morphologically and biochemically observed and subjected to biochemical
tests for identification of S. aureus. The colonies with egg fried appearance and α or β haemolysis
were identified as S. aureus morphologically and were further subjected to catalase, coagulase tests
1017
as well as Gram staining. Bacterial concentrations for bacterial toxoid were adjusted to 106
CFU/ml, 109 CFU/ml, 1012 CFU/ml using Breed Smear method (Awan and Rehman, 2005). A year
old producing white leg horn hens were procured from local farm with history of proper
vaccination. The birds were divided into 3 equal groups. The hens were offered recommended
ration and kept under optimal conditions in the experimental sheds of department of Clinical
Medicine and Surgery. The hens were inoculated with aluminium hydroxide bacterin toxoid. Eggs
from inoculating hens were collected in maximum aseptic conditions starting after 7th day of 1st
inoculation and were stored at 4Ċ in refrigerator until used for separation of egg yolk
immunoglobulins (Zhen et al., 2008).The immunoglobulins were isolated with the method
described by ; Devi et al., (2006). Antibody titer of egg yolk immunoglobulins (IgY) was measured
through indirect haemaglutination test ( Awan and Rehman et al., 2005).
In Vivo trials
Milk samples from suspected mastitis buffalo on the basis of Surf Field Mastitis Test were
inoculated on blood agar for the isolation of S. aureus colonies. Forty S. aureus mastitic buffaloes
were divided into four equal groups (A, B, C and D). Group A and B contained clinical mastitic
buffaloes while group C and D comprised of sub clinical mastitic buffaloes. The buffaloes showing
signs of clinical mastitis with presence of milk clots and high somatic cell count were put in clinical
mastitc group A and B. The buffaloes showing no clinical signs of mastitis but were positive for
SFMT were put in sub clinical groups C and D (Zhen et al., 2008).Egg yolk immunoglobulins at the
dose rate of 20 mg/ml were administered to the group A. While group B was given intramammary
infusion of an antibiotic 5 gms (Penbiotic® Nawan) at the dose rate of 10 ml for six days. Similarly
group C of subclinical mastitic buffaloes was given egg yolk immunoglobulins treatment and group
D of sub clinically mastitic buffaloes was given antibiotic treatment (5 gm of Nawan® Company) at
the dose rate of 10 ml/ each quarter. The treatment remained continued for 6 days. The
intramammary infusion was given with the help of branula in maximum aseptic conditions. The
experiment was performed in morning post milking (Zhen et al., 2008). Evaluation criteria for the
in vivo efficacy of antibiotic and egg yolk immunoglobulins.Surf Field Mastitis Test, Clinical cure
rate, Microbiological cure rate, Comparison of milk quantity before and after treatment

The values of cumulative mean titers were maximum (241.70) in serum of hens of H3 group
and minimum 147.76 in hens of group of H1 as per Table 1
Comparison of milk yield of S. aureus mastitic buffaloes treated with egg yolk immunoglobulins and
antibiotic
The findings of current study have revealed that trend of increase in milk yield in both
clinicaly and subclinicaly S. aureus mastitic buffaloes was more prominent in buffaloes treated with
egg yolk antibodies than that of antibiotic treated buffaloes. Approximately, 90 % buffaloes showed
increase in milk yield after the administration of egg yolk antibodies to group A. only 30-4-%
buffaloes produced more milk in group B after the administration of egg yolk antibodies. The group
C and D (sub clinically mastitic buffaloes) also showed the similar results as 90% of buffaloes
showed increase in their milk yield after therapeutic use of egg yolk antibodies. The success rate
was 50% in buffaloes treated with antibiotic. Overall, 90% buffaloes produced more milk after the
therapeutic application of specific egg yolk antibodies of egg yolk antibodies. Around 30-40%
buffaloes showed higher milk production after the administration of antibiotic. The success rate was
higher in subclinicaly mastitic buffaloes (95%) than clinicaly mastitic buffaloes (80%) in the egg
yolk treated buffaloes. Approximately, 70% and 57% clinicaly and subclinicaly mastitic buffaloes
produced more milk after receiving antibiotic. Zhen et al., (2008) reported that therapeutic efficacy
of egg yolk immunoglobulins was better than antibiotic. The research conducted by Zhen et al.,
(2008) have revealed that 85% cattle produced more milk after the therapeutic application of egg
yolk immunoglobulins. The present research revealed better results than findings of Zhen et al.,
(2008) .The better results of current study were most probably due to more resistance in buffalo
against mastitis, difference in lactation periods, age and species.
1018
Effects of egg yolk immunoglobulins treatment on the clinical based cure rates of clinical and
subclinicaly S. areus mastitic buffaloes
The results of current study clearly depicts that clinical cure rate is better in the groups that
received egg yolk antibodies (A and C) than groups that received antibiotic (B and D) while minor
reduction in the inflammation of udder was recorded in the groups that received antibiotic. In the
present study, 95% of immunoglobulins treated buffaloes showed clinical recovery but only 30-
40% buffaloes showed clinical recovery in antibiotic treated buffaloes. Zhen et al., (2008)
concluded that clinical recovery rates from mastitis were 80% and 50% for immunoglobulins
treated and antibiotic treated cattle, respectively. The findings of current study were better than that
of Zhen et al., (2008). The difference in results was most probably due to difference of species,
season, lactation periods of both studies etc.
Effect of treatment on Surf Field Mastitis Test (SFMT) score trialed on clinical and subclinical
mastitic buffaloes
The findings of present study clearly depicts that 90% buffaloes were negative for SFMT in
the groups that received egg yolk antibodies (A and C). While only 60% buffaloes were negative
for SFMT in the antibiotic treated buffaloes (B and D). Zhen et al., (2009) concluded after his study
in cattle that 80% and 40% cattle were negative for California Mastitis Test (CMT) in the groups
that received egg yolk antibodies and antibiotic, respectively. The results of current research are
better than that of Zhen et al., (2008). The differences in the results were most probably due to
difference of species, season, lactation periods etc.
Effect of treatment with egg yolk antibodies and antibiotic on microbiological based cure rate of
buffaloes suffering from clinical and subclinical S. aureus mastitis
The present study concluded that 80% milk of buffaloes of groups A and C (treated with egg
yolk immunoglobulins) showed no bacterial (S. aureus) growth on blood agar. On the other hand,
80% milk samples of buffaloes treated with antibiotic (B and D) were still showing bacterial growth
on blood agar. Zhen et al., (2008) concluded that microbiological cure rate was 80% and 45% in
cattle treated with egg yolk antibodies and antibiotic, respectively. The results of current study are
better than that of Zhen et al., (2008). The difference in the results was most probably due to
difference of species, season, lactation periods etc. As per findings egg yolk immunoglobulins have
shown better results than antibiotic on the basis of clinical trials. Therefore, egg yolk antibodies
may be used as an alternative of antibiotics.
REFERENCES
Awan, J.A., and S.U. Rehman. 2005. Microbiology Mannual. Unitech Communication, Faisalabad,
Pakistan. 19-21.
Coleman, M.A. 1996. Oral administration of chicken egg yolk immunoglobulins to lower somatic
cell count in the milk of lactating ruminants. US Patent. 5: 585-598.
Devi, C., M.V. Bai, A.V. Lal, P.R. Umashankar and L. Krishnan. 2006. An improved method for
isolation of anti-viper venom antibodies from chicken egg yolk. J. Biochem. Meth. 51: 129-
138.
Gill, J.J., J.C. Pacon, M. Griffith and M.W. Sabour. 2006. Efficacy and pharmokinetics of
bacteriophage therapy in treatment of subclinical Staphylococcus aureus mastitis in lactating
dairy cattle. Anti. A. Chemoth. 50: 2912-2918.
Muhammad, G., A. Naureen, M.N. Asi, M. Saqib and F.U. Rehman. 2010. Evaluation of 3% surf
solution (Surf Field Mastitis Test) for the diagnosis of subclinical bovine and bubaline
mastitis. Trop. Anim. H. Pro. 3: 457-467.
Oldham, E.R. and M.J. Daley. 1991. Lysostaphin: Use of recombinant bacterial enzyme as a
mastitis therapeutic agent. J. Dair. Sci. 74: 4175-4182.
Rehman, S., M. Ather, A. Shakoor, G. Muhammad and A.A. Butt. 2005. Standardization of Indirect
Haemaglutination Test for titration of antibodies against Staphylococcus aureus,
Streptococcus agalactiae and Escherichia coli isolated from bubline mastitis. Int. J. Agri.
Bio. 3: 441-444.
1019
Singh, R., K.B. Singh, S.S. Sudan and R. Singh. 1998. Effect of subclinical and clinical mastitis on
milk composition in crossbred dairy cow. Int. Vet. J. 75: 462-465.
Zhen, Y., L. Jin, J. Guo, X. Li, Y. Lu, J. Chen and Y. Xu. 2008. Characterization of specific egg
yolk immunoglobulins (IgY) against mastitis causing E. coli. Vet. Microbiol. 130: 126-133.
Table 1. Cumulative mean titers of groups H1, H2 and H3 of layer hens.
Groups of hens Cumulative mean titers (CMT)

Group H1 147.76
Group H2 208.96
Group H3 241.70
1020
Antibiogram Analysis of Staphylococcus aureus Isolated from Mastitic Milk

Samples of Buffaloes in District Bhimber Azad Kashmir
Abid HUSSAIN,a* Mansur-ud-Din AHMED,a Muhammad Hassan MUSHTAQ,a Muhammad

Sarwar KHAN,b Muhammad Ather KHAN, a Muhammad NISAR a Naveed SABIR c and
Shahzad Akbar KHANc,
a
Department of Epidemiology & Public Health University of Veterinary & Animal Sciences Lahore
b
Department of Clinical Medicine, University of Veterinary & Animal Sciences Lahore
c
Faculty of Veterinary & Animal Sciences, The University of Poonch Rawalakot Azad Jammu &
Kashmir
*Corresponding author: mughal_161@yahoo.com
ABSTRACT
The present study was designed to investigate the disease characteristics of Staphylococcus
aureus mastitis in buffaloes and antibiotic susceptibility of isolates. Visual inspection and palpation
indicated as; hind quarters were asymmetric (n=2), Clinical symptoms (n=2), Udder and teat’s wounds
(n=2), Scar tissue (n=1), Warts (teat) (n=1) and all others parameters were normal. A total of 300 milk
samples (60 clinical and 240 subclinical) from mastitic buffaloes were collected from different
Government and private livestock farms in and around district of Bhimber. These samples were
subjected to antibiotic susceptibility testing. One hundred eighty isolates of Staphylococci were
obtained on the basis of growth on staph.110 medium, colony morphology and haemolytic pattern on
5% sheep blood agar plates. All the isolates were Gram positive and catalase positive. Of these, 69
were Slide Coagulase test, Tube Coagulase test and Staphytect plus tested positive and the remaining of
111 samples were negative. Those 111 samples were identified as Staphylococcus aureus (Coagulase
positive Staphylococci) and the remainders were coagulase negative Staphylococci. All these 69
isolates of Staphylococcus aureus were subjected to antibiotic sensitivity testing against the most
commonly used antibiotic in mastitis (i.e. enroflocaxin, ciprofloxacin, chlormapehnicol,
amoxicillin,ampicillin, gentamycin, novobiocin, oxytetracycline and cotrimaxazole). The antibiogram
study of these antibiotics in the form of their percentage of sensitivity was as co-trimaxazole (90%),
oxytetracycline (90.65%), amoxicillin (76.95%), gentamycin (76.95%), ampicillin (72.60%),
ciprofloxacin (80.60%), chloramphenicol (82.60%), enrofloxacin (69.56%) and novobiocin (70.86%).
Keywords: antibiogram, buffalo, mastitis, milk
INTRODUCTION
Pakistan is an agricultural country and livestock is its important sub-sector, which contributes
55.1% of total agricultural value added and about 11.5% of the total GDP. The importance of livestock
may be realized from the fact that 30-35 million rural populations is engaged in livestock raising and
deriving their 30-40% income from it (Anonymous, 2011). Of the livestock, buffalo is the mainstay of
dairy industry and contributing 75% of milk to be consumed all over the country (Sarwar et al., 2002).
The Population of buffalo in Pakistan is 31.7 Millions. It is estimated that the country produces 46

1021
billion liters of milk per year, whose value is more than that of the combined value of wheat and cotton
(Anonymous, 2011).
There are many diseases affecting the milk yield of buffaloes. Of these, mastitis is at the top.
Mastitis is one of the most important health problems of dairy animals (Ali, 2009). It is the
inflammation of the parenchyma of the mammary gland regardless of the cause and is characterized by
physical and chemical changes in the milk and pathological changes in the glandular tissue.
Inflammation may be caused by many types of injuries including infectious agents, their toxins,
physical trauma or chemical irritants. Mastitis occurs in two forms i.e. clinical and subclinical. Clinical
mastitis is recognized by abnormal milk, gland’s swelling and illness of the affected animal.
Subclinical mastitis is recognized by apparently normal milk with an increase in somatic cell count
(Radostits et al., 2000). Subclinical mastitis is 15-40 times more common than the clinical mastitis and
causes the greatest overall losses in most dairy herds. Subclinical mastitis is one of the major infections
of cattle and buffalo playing havoc for economic loss in dairy farming and the incidence is increasing
day by day (Ali et al., 2011).
Mastitis is one of the most devastating diseases in the dairy industry. Economic consequences of
mastitis, clinical or sub-clinical, include reduced milk yield, poorer quality milk, increased culling rate
and increased cost of veterinary services and medicine. Dairy farmers are not always aware of the best
practices to control mastitis. Besides bacterial infection, there are many risk factors associated with
mastitis. The disease cannot be eradicated but can be reduced to low levels by good management
(Rahman et al., 2009). This disease is associated with a decrease in milk production, deterioration of
milk quality, increased labour cost and culling. In Pakistan, statistics of current losses due to this
disease are not available.
Mastitis is caused by a wide variety of microorganisms including bacteria, fungi, yeast and
mycoplasma etc. However, the bacteria are the most frequent pathogens associated with this disease.
Of these, mastitis is mainly caused by Staphylococcus aureus, Streptococcus agalactiae, Streptococcus
dysagalactiae, Streptococcus uberis, Escherichia coli and Corynebacterium pyogenes (Radostits et al.,
2000). Staphylococcus aureus is held responsible for causing 50% cases of mastitis in buffaloes as per
studies made thus for in Pakistan starting from 1966 to 2002 (Shakoor, 2006). It causes pockets in the
depth of udder surrounded by fibrous tissue where antibiotics cannot gain access.
In Pakistan, the losses due to mastitis might be higher, because the mastitis prevention practices like
teat dipping and dry period antibiotic therapy are not in practice (Arshad, 1999). A lot of literature is
available about the risk factors of mastitis in cattle (Doherr et al., 2007; Rahman et al., 2009), but the
information about this aspect is rear in buffaloes.The epidemiology of clinical mastitis due to
Escherichia coli or Staphylococcus aureus are distinctly different. Escherichia coli is regarded as an
environmental pathogen and Staphylococcus. aureus is regarded as a contagious pathogen in that the
main source of intramammary infections is the udder flora. Risk factors that were offered to a
multivariate Poisson regression model included general management, housing, cleaning procedures,
cow and cubicle cleanliness, feeds and feeding, dry cow management, milking procedures, machine
milking, disease prevention, and milk production. Some differences in epidemiology between E. coli
and S. aureus were observed (Ali, 2009).
In principle, antibiotic sensitivity against the available chemotherapeutic agents in a region or locality
should be done from time to time to enable the veterinarians to prescribe the most suitable antibiotics
against the prevailing diseases. To gain the maximum cure rate in the treatment of mastitis, the result of
an antibiotic sensitivity testing should constitute the basis of antibiotics selection. There is a paucity of
information about the comparative antibiotics susceptibility profiles of Staphylococcus aureus isolated
from buffaloes. Keeping in view this scenario, this study was designed with the following objectives:

Study area and sampling Procedure
1022
Study area was consisting of a district of Bhimber Azad Kashmir. The different dairy farms
were selected randomly and each farm was considered as a cluster. Sample size was consisting of 300
lactating buffaloes. All lactating buffaloes with in a herd were examined by cluster sampling
technique. All lactating buffaloes were selected as study unit and all dairy procedures were included in
the epidemiological survey. Duration of study was one year.
Inclusion and Exclusion Criteria
Only lactating buffaloes were included in the study. Those were excluded from the study which
was heifers, pregnant buffaloes and ones in the dry period.
Diagnosis of Mastitis in Dairy Buffalo
Clinical mastitis diagnosis was based on signs and symptoms. Subclinical mastitis diagnosis
was based on Surf Field Mastitis Test (Muhammad et al. 1995).
Surf Field Mastitis Test
A total of 1000 quarters milk samples from 250 buffaloes were screened for detection of sub-
clinical mastitis by Surf Field Mastitis Test (Muhammad et al., 1995) and 300 quarter’s milk samples
were collected from mastitic buffaloes on the basis of Surf Field Mastitis Test and clinical signs and
symptoms of mastitis.
Collection and Transportation of milk samples
The guidelines of National Mastitis Council were followed in collecting the milk samples for
culturing and isolating of Staphylococcus aureus from the cases of mastitic buffaloes. (National
Mastitis Council, Inc., 1990). A 300 quarters’ mastitic milk samples (60 clinical and 240 subclinical)
were collected from buffaloes, aseptically in sterilized screw capped vials from clinical and subclinical
cases of mastitic buffaloes from different private livestock farms in and around the Bhimber Azad
Kashmir. The collected milk samples were transported by placing them on the crushed ice in a thermos
to the Microbiology Laboratory in the Department of Epidemiology and Public Health, University of
Veterinary and Animal Sciences Lahore, to culture milk samples for the isolation and further studies of
Staphylococcus aureus. Standard procedures for collection, handling and transportation of milk
samples and culturing of microorganisms were followed as per recommendations of Ali et al.
(2008).Diseases characteristics of Staphylococcus aureus mastitis were inspected as described my klass
et al. (2004).
Culturing of Milk Samples on Staph. 110 Medium and Blood Agar Medium
Media for the isolation of Staphylococcal species Staph. 110 medium was used as a selective. It
was prepared as described by Cruickshank et al. (1975). The 5% sheep blood agar was used for
checking the hemolytic properties of Staphylococcal species and was prepared by using the procedure
described by National Mastitis Council Inc., USA. (1990). Procedures described by Ali et al. (2008)
were followed for culturing the milk samples and identification of mastitis pathogens.
Isolation and Identification Staphylococcus aureus
Following biochemical test were performed for the identication and confirmation of
Staphylococcus aureus. Gram,s staining, Calatase tast, Coagulase test and Staphtect plus (Latex Slide
Agglutination Test). Staphtect plus (Oxoid, Basingstoke Hampshire, UK) is a Latex Slide
Agglutination test for the identification of Staphylococcus aureus by detection of clumping factor,
protein A and certain polysaccharides found in Staphylococcus aureus.
Antibiogram of Staphylococcus aureus Isolated from Mastitic Buffaloes
Antibiogram analysis which is called in vitro antibiotic susceptibility of Staphylococcus aureus
to 9 antibiotics (ampicillin, amoxicillin, oxytetracycline, chloramphenicol, enrofloxacin, ciprofloxacin,
cotrimaxazole, gentamycin and novobiocin) was determined by using disk diffusion method. To
standardize the inoculum density for a susceptibility testing, Barium Sulphate Turbidity standard,
equivalent to a 0.5 McFarland standard, was used. A Barium Sulphate 0.5 McFarland standard was
prepared. Antibiotic susceptibility was done according to the standards of National Committee for
Clinical Laboratory Standards (NCCLS), now called Clinical Laboratory Standards Institute (CLSI,
1023
2005). Staphylococcus aureus ATCC 25923 (American Type Culture Collection, Rockville, Maryland,
USA) was used as the sensitive quality control organism.

Screening of Dairy Buffaloes
A total of 1000 quarters milk samples from 250 buffaloes were screened by Surf Field Mastitis
Test for sub-clinical mastitis. Of these, 240 samples were positive to Surf Field Mastitis Test. On the
other hand, 60 mastitic quarters clinical were collected from the mastitic buffaloes. A total of 300 milk
samples were collected from mastitic buffaloes from private livestock farms from the surroundings of
Bhimber Azad Kashmir. Mastitic buffaloes milk samples were selected on the basis of clinical sign and
symptom (n=60) and other (n=240) samples were selected on the basis of Surf Field Mastitis Test
(SFMT). Surf Field Mastitis Test was used for the detection of sub clinical mastitis.
Disease Characteristics of Staphylococcus aureus Mastitis
Total of 300 quarters’ milk samples were collected from clinical cases of mastitic buffaloes
from private livestock farms. Of these quarters, Staphylococcus aureus was isolated from two quarters
and disease characteristic of these two isolates of Staphylococcus aureus from the clinical cases of
mastitis were observed by visual inspection and palpation. Both hind quarters were asymmetric (n=2),
Clinical symptoms (n=2), Udder and teat wounds (n=2), one Scar tissue (n=1), one having warts on teat
and all others parameters were normal.
Culturing of Milk Samples on Staph. 110 Medium and Blood Agar Medium
i) Staph.110 medium
The mastitic milk samples were cultured on Staph. 110 medium (Difco Laboratories, Detroit,
Michigan, USA) and were incubated at 37°C for 24-48 hours. Of 300 samples, 180 grow on this
medium and remaining 120 did not grow.The colonies on Staph. 110 plates showed smooth, circular,
moist, creamy or golden yellow appearance.
ii) Blood agar medium
The same 180 samples grown on Staph. 110 medium were further streaked on blood agar for
checking the haemolytic pattern of Staphylococcus aureus. All these were haemolytic. The colonies of
Staphylococcus aureus on blood agar were circular, 1-2 mm in diameter, raised, convex having entire
margins and yellow or creamy gold in colour (Table-1). After having pure cultures, different tests were
conducted for the identification of isolates.
Staphytect plus (Latex Slide Agglutination Test)
Out of 180 isolates of staphylococci, 69 were confirmed by Staphytect plus kit as coagulase
positive Staphylococci (Staphyloccus aureus) remaining 111 were considered as coagulase negative.
Antibiotic Susceptibility Profiles of Staphylococcus aureus
The antibiogram study of nine antibiotics in the form of their percent sensitivity was as; co-
trimaxazole (90%), oxytetracycline (90.65%), amoxicillin (76.95%), gentamycin (76.95%), ampicillin
(72.60%), ciprofloxacin (80.60%), chloramphenicol (82.60%), enrofloxacin (69.56%) and novobiocin
(70.86%). In this study disease characteristics of Staphylococcus aureus from clinical mastitis was
observed as, hind quarters were asymmetric (n=2), Clinical symptoms (n=2), Udder and teat wounds
(n=2), one Scar tissue (n=1), one having warts on teat and all others parameters were normal. These
wounds may be ascribed to the presence of Staphylococcus aureus infection because these harbour the
skin microflora especially Staphylococcus aureus. The clinical mastitis is also sometimes caused by
Staphylococcus aureus (Sears, 1993). These findings are also in line with the findings of Wang et al.
1024
(2007) who also recorded certain clinical features in cows like swollen or hard udders along with others
but these were not correlated with Staphylococcus aureus infection and were common.
From 60 milk samples of clinical mastitis, only ten yielded growth on Staph.110 medium. This
may be ascribed to instantaneous use of antibiotics in the animals by the owners themselves. Of 240
milk samples of subclinical mastitis, only 170 grew on both Staph.110 medium and blood agar,
indicating the nature of organism as haemolytic Staphylococcus aureus. This lack of growth on the
media may be due to indiscriminate use of antibiotics at the farm by the owner as a remedial measure
just at the outset. This practice is quite common at ours. All the isolated Staphylococci produced were
gram, catalase and coagulase positive. In addition to it, among all these isolates 69 were positive and
111 were negative confirmed by Staphytect plus (Latex Slide Agglutination Test). A total of 69 isolates
of Staphylococcus aureus from mastitis were evaluated through disc diffusion method for antibiotic
susceptibility testing. These are amoxicillin, ampicillin, gentamycin, enrofloxacin, cipfloxacin,
novabiocin, co-trimexazole, oxytetracycline and chloramphenicol. A reference strain of Staphylococcus
aureus (ATCC 25923) was also used as a sensitivity control. Using disk diffusion method on Mueller-
Hinton medium, inhibitory zones around the disk were recorded and results were interpreted in
percentage.
For Staphylococcus aureus, the tested chemotherapeutic agents, co-trimaxazole (90%),
oxytetracycline (90.65), amoxicillin (76.95), gentamycin (76.95) ampicillin (72.60), ciprofloxacin
(80.60), chloramphenicol (82.60), enrofloxacin (69.56) and novabiocin (70.86) showed sensitivity. It
was contrary to the findings of Zahid (2004) who found gentamycin as the drug of choice on the basis
of drug sensitivity for the treatment of clinical mastitis in buffaloes, also reported that Staphylococcus
aureus deriving from cattle was sensitive to oxytetracycline. On the other hand, Rashid (2001)
concluded gentamycin sensitive 62% against Staphylococcus aureus mastitis in buffaloes. Conducting
similar studies, Fazal–ur-Rehman (1995) concluded that gentamycin, chloramphenicol, cotrimaxazole,
amoxicillin and oxytetracycline showed an in vitro efficiency over 90 percent. Novobiocin had
efficiency between 80 to 90 percent and ampicillin efficiency was less than 80 percent. Rossetti (1993)
found that Staphylococcus aureus, the most commonly isolated organism from mastitis was 100
percent sensitive to gentamycin, chloramphenicol and cotrimaxazole and were one percent resistant to
oxytetracycline. The results of this study do not correspond to the findings of present study. Hodges et
al. (1984) also observed a similar pattern of sensitivity while conducting studies on bovine mastitis in
Newzealand, Staphylococcus aureus were 80-90% sensitive to oxytetracycline and novobiocin.
Chanda et al. (1989) reported that gentamycin was the most effective antibiotic for Staphylococci
followed by ampicillin, oxytetracycline, chloramphenicol. Khan et al. (2005) drew a conclusion that the
antibiogram analysis of gentamycin, ciprofloxacin, chloramphenicol, cotrimaxazole showed more than
90 percent sensitivity to Staphylococcus aureus derived from buffaloes and cows. Ramachandraiah et
al. (1990) made a study about antibiotic susceptibility in India and concluded that gentamycin and
chloramphenicol were 100 percent effective followed by cotrimaxazole (83.3%), pencillin (83.3%) and
oxytetracycline (66.6%) against Staphylococcus aureus mastitis. Saxena et al. (1993) construed that
Staphylococci derived from bovine mastitis were highly sensitive to gentamycin. Having conducted a
similar study, Erskine et al. (2002) made a conclusion that cotrimaxazole (99.4%), gentamycin
(98.9%), oxytetracycline (91.5%) and ampicillin (50.4%) were sensitive to various species of
Staphylococcus aureus deriving from cattle. Chaudhry and Azam (1995) made a similar study about
various species of Staphylococcus aureus deriving from buffaloes and concluded that gentamycin
(62.94%), chloramphenicol (34.41%), oxytetracycline (28.25%), cotrimaxazole (12.35%) and
ampicillin (2.43%) were sensitive.
Similar study was made by Lafi and Hailat (1998) in buffaloes and they concluded that
cotrimaxazole (75.67%), amoxicillin (72.29%), chloramphenicol (70.20%), gentamycin (70.20%),
oxytetracycline (70.20%) and ampicillin (62.16%) showed sensitivity against Staphylococcus aureus.
1025
Similarly (de Oliveira et al., 2000).made an antibiotic susceptibility testing in buffaloes and cattle and
they concluded that gentamycin and enrofloxacin were the most effective drugs against Staphylococcus
aureus. The intermittent changing pattern of antibiotic susceptibility against Staphylococcus aureus
may be ascribed to the extent of different antibiotics to be used from locality to locality. This fact has
made the co-trimaxazole as the most sensitive chemotherapeutic agent in this study. So it is mandatory
that antibiogram study be made from time to time in a locality to be on the look out of the most
effective drugs against the prevailing mastitogens i.e bacteria.
REFERENCES
Ali, L. 2009. Epidemiology of mastitis in dairy buffalo and cow in Tehsil Samundri of district
Faisalabad. Ph.D. thesis Department of Clinical Medicine and Surgery, University of
Agriculture, Faisalabad, Pakistan.
Ali, L., G. Muhammad, M. Arshad, M. Saqib and I.J. Hassan. 2008. Bacteriology of mastitis in
buffaloes in Tehsil Samundri of district Faisalabad, Pakistan. Pak Vet J. 28 (1): 31-33.
Ali, M.A., M.D. Ahmad, K. Muhammad and A.A. Anjum. 2011. Prevalence of subclinical mastitis in
dairy buffaloes of Punjab. Journal of Animal & Plant Sci. 21(3): 477-480.
Anonymous, 2011. Economic Survey of Pakistan 2010-2011. Finance Division, Economic Advisor’s
Wing. Government of Pakistan, Islamabad.
Arshad, M. F.K. Qamar, M. Siddique and S.T. Sindhu. 1999. Studies on some epidemiological aspects
of bovine mastitis. In: Proc. National Seminar on Epidemiology of Livestock and Poultry
Diseases. 19-20 January, 1995. Collage of Veterinary Sciences, Lahore. P. 16-17.
Aziz, M. A., M.A. Ghani and M.T. Muhammad. 1997. Studies on Staphylococcal subclinical bovine
mastitis with special reference to treatment by pencillin, Mardadal combination (Masticillin
M). J. Egyptian Vet. Med. Assoc. 36: 69-78.
Chanda, A., C.R. Roy, P.K. Banerjee and C. Ghha. 1989. Studies on incidence of bovine mastitis, its
diagnosis, etiology and in vitro sensitivity of the isolated pathogens. Indian Vet. J. 66: 277-
282.
Chaudhary, N.A. and B.B. Khan. 1978. Estimation of economic losses due to animal diseases in
Punjab (Final report of research project). Univ. Agri. Faisalabad.
Chaudhary, T.M. and M.W. Azam. 1995. Sensitivity of various organisms isolated from clinical cases
of mastitis in cattle, buffaloes and goats. Pakistan Vet. J. 1: 74-76.
Cheng, G.B., Z. Feng, D. Tian, H.C. Chen and A.Z. Guo, 2007. Isolation, identification and analysis of
drug resistance of mastitis-causing pathogens from Hubei. China Dairy Cattle 7:35-38 (in
Chinese).
Clinical Laboratory Standards Institute (CLSI). 2005. Performance Standards for Antimicrobial
Susceptibility testing. CLSI Approved Standards M100-S15. Clinical and Laboratory Standards
Institute, Wayne, PA. CDC. USA.
Cruickshank, R., J.P. Duguid, B.P. Marmion and R.H.A. Swain. 1975. Medical Microbiology. 12th
ed. Vol. 11. Churchill Livingstone, USA. pp. 356-360.
De Oliveira, A.P., J.L. Watts, S.A., Salmon and F.M. Aarestrup. 2000. Antimicrobial susceptibility of
Staphylococcus aureus isolated from bovine mastitis in Europe and the United States. J. Dairy
Sci. 83(4):855-862.
Doherr, M.G., M. Roesch, W. Schaeren, M. Schallibaum and J.W. Blum. 2007. Risk factors associated
with subclinical mastitis in dairy cows on Swiss organic and conventional production system
farms. Veterinari Medicina 52 (11): 487–495.
Erskine, R.J., R.D. Walker, C.A. Bolin, P.C. Bartiett and D.G. White. 2002. Trends in antibacterial
susceptibility of mastitis pathogens during a seven-year period. J. Dairy Sci. 85:1111-1118.
1026
Fazal -ur-Rehman. 1995. Studies on: (1) Evaluation of Surf Field Mastitis Test for the detection of
sub-clinical mastitis in buffaloes and cattle and (2) antibiotics susceptibility of the pathogens.
M.Sc. thesis, Dept. Clinical Medicine and Surgery. Univ. Agri. Faisalabad.
Finland. 2001. Prevalence, distribution of bacteria, and antimicrobial resistance. J. Dairy Sci. 87(8):
2433-2441.
Hodges, R.T., Y.S. Jones and J.T. Holland. 1984. Characterization of Staphylococci with clinical and
sub-clinical bovine mastitis. Newzealand Vet. J. 32: 141-145.
Iqbal, M., M.A. Khan, B. Daraz and U. Saddique. 2004. Bacteriology of mastitic milk and in vitro
antibiogram of the isolates. Pakistan Vet. J. 24: 161-164.
Khan, A.Z., G. Muhammad, T. Ali and A. Khan. 2005. Prevalence of mastitis in cross bred cows and
buffaloes and antibiotics sensitivity profiles of isolates. Pakistan Vet. J. 26: 74-76.
Klaas IC, Enevoldsen C, Vaarst M, Houe H. 2004. Systematic clinical examinations for identification
of latent udder health types in Danish dairy herds. J. Vet. Sci. 87: 1217-1228.
Lafi, S.Q. and N.Q. Hailat. 1998. Incidence and antibiotic sensitivity of bacteria causing bovine and
ovine mastitis in Jordan. Pakistan Vet. J. 18: 88-94.
Muhammad G, M. Athar A. Shakoor M.Z. Khan, Fazal-ur-Rehman and M.T. Ahmad. 1995. Surf Field
Mastitis Test: An inexpensive new tool for evaluation of wholesomeness of fresh milk.
Pakistan J. Food Sci. 3: 91-93.
National Committee for Clinical Laboratory Standards, 1990. Performance standard for antimicrobial
disc susceptibility test. 4th ed. Approved standard M2-A3. National Committee for Clinical
Laboratory Standards, Villanova pathogens, USA.
National Mastitis Council, Inc., 1990. Microbiological procedures for the diagnosis of bovine udder
infection. National Mastitis Council, Inc. Wilson Boulevard Arlington, Virginia, USA.
Radostits, O.M., C.C. Gay, D.C. Blood and K.W.Hinchcliff. 2000. Veterinary Medicine: A Textbook
of the Diseases of Cattle, Sheep, Pigs, Goats and Horses. 9th Ed. W. B. Saunders Co.,
Philadelphia, USA. p. 603-610.
Rahman, A, M.M.U. Bhuiyan, M.M. Kamal and M.Shamsuddin. 2009. Prevalence and risk factors of
mastitis in dairy cows. The Bangladesh Veterinarian. 26(2): 54 – 60.
Rossetti, C.A. 1993. Prevalence of subclinical mastitis caused by Staphylococcus aureus in buenos
aires dairy area and its susceptibility to antibiotics. Veterinaria argentina 10: 323-326.
Sarwar, M., M.A. Khan, Mahr-un-Nisa and I. Zafar. 2002. Dairy industry in Pakistan: a scenario.
International J Agri Biol. 4:420-428.
Saxena, R.K., G.N. Dutta, P. Borah and J. Buragohain. 1993. Drug susceptibility and treatment of
bovine sub-clinical mastitis. Indian Vet. J. 79: 201-203.
Shakoor, A. 2006. Preparation and evaluation of Staphylococcus aureus vaccines for the control of
mastitis in dairy buffaloes (Bubalus bubalis). Ph.D. Dissertation, Deptt. Of Vet. Clinical
Medicines and Surgery, Univ. of Agri., Faisalabad, Pakistan.
Wang, B., M.C. Liu, J.Z. Sheng and J.Z. Shen. 2007. Resistance survey of Staphylococcus aureus
isolated from bovine mastitis in Hohhot. Chin. J. Vet. Med. 43(3):30-32 (in Chinese).
Zahid, I.A. 2004. Studies on comparative incidence of subclinical and clinical mastitis and in vitro
antibiotic susceptibility of isolates from Holstein-Friesian, Jersey cows and buffaloes. Pak. Vet.
J. 24: 76-81.
1027
Table 1. Results of cultural and morphological characteristics of Staphylococcus aureus isolates.
Growth Pigment
Growth on production on
Staph. 110 on Staph. 110 Bacterial spp.
Morphology No. of isolates
medium medium and (tentative)
(colonies) Blood agar blood agar
(colonies) plates
Haemolytic (α,
β, αβ), Gram positive
Golden yellow, pinhead, cocci, grape
circular raised, rasied, convex, Golden yellow, like structure, Staphylococcus
180
1-2mm creamy gold irregularly species.
1-2mm
diameter arranged,
diameter
deeply stained
Table 2. Comparative biochemical characteristics of Staphylococcus aureus isolated from mastitic

buffaloes.
Tube
Gram’s staining Catalase Slide Coagulase test Staphytect plus
Coagulase test
Species
+ve -ve +ve -ve +ve -ve +ve -ve +ve -ve
Staphylococcus
180 - 180 - 180 - 180 - 69 111
aureus
1028
Molecular Identification of Brucella Abortus Bv5 and Strain 19 in Water

Buffaloes (Bubalus Bubalis) in Northeast Argentina
Diana MARTINEZa*, Carolina THOMPSONb, Ana RUSSOc, Roberto JACOBOa and Susana
TORIONI de ECHAIDEb
a
Cátedra de Enfermedades Infecciosas, Facultad de Ciencias Veterinarias- UNNE. Sgto. Cabral
2189, 3400-Corrientes.bINTA, EEA, Rafaela Ruta 34 km 227, 2300-Rafaela, Santa Fe.c Facultad de
Recursos Naturales-UNAF, CEDIVEF, Ruta Nac. 11 km 1164, 3600-Formosa.
*Correspondig author e-mail: demartinez@vet.unne.edu.ar
ABSTRACT
Buffalo (Bubalus bubalis) populations are spread across northern Argentina, and they share
their habitat with bovines. Both species are susceptible to brucellosis, and they are under a National
Plan of Control and Eradication. To characterize the Brucella spp. that infects buffaloes, the blood
of 35 animals that tested positive to brucellosis by a complement fixation test was collected. DNA
was obtained and analyzed by polymerase chain reaction using different molecular markers. The
genera, species, and biovars of Brucella were established by analyzing specific regions of the genes
omp31, eri, alkB, and omp2ab. Brucella spp. was identified in 15 of 35 tested buffaloes. The
product of the omp31 gene identified the genera. The detection of two fragments of 297 bp and/or
1000 bp from the eri gene confirmed the presence of B. abortus S19 and wild-type B. abortus. The
amplification of the alkB gene allowed the identification of B. abortus biovars characterized by
fragments of 498 bp (bv1, bv2, or bv4). The simultaneous amplification of 498 bp (alkB) and 1000
bp (eri) products suggested the presence of B. abortus bv1, which is highly prevalent in the cattle of
Argentina. Fragments of 827 bp and 857 bp were amplified from the omp2ab gene, and their
sequences showed 100% identity with B. melitensis and B. abortus bv5 (GenBank). However, the
721 bp product (alkB) specific for B. melitensis could not be amplified. This is the first report
indicating the presence of B. abortus bv5 in Latin America.
Keywords: brucellosis, water buffalo, molecular typification, Brucella abortus bv 5
INTRODUCTION
Buffaloes (B. bubalis) were introduced in northern Argentina in the first decade of the 20th
century. The population is currently expanding, with more than 100,000 individuals that frequently
share a habitat with bovines (Asociación Argentina de Criadores de Búfalos, 2006). Both species
are susceptible to brucellosis, a zoonotic disease primarily caused by B. abortus and responsible for
economic losses estimated at more than US$ 60,000,000 per year (García-Carrillo & Lucero, 1993).
Since 2005, buffaloes have been included in the National Plan of Control and Eradication of bovine
brucellosis based on the vaccination of female calves with B. abortus strain 19 (S19), serological
diagnosis, and the slaughter of reactors (SENASA, 2005). The aim of this work was to identify and
characterize Brucella spp. in buffaloes from NE Argentina using molecular markers.

Buffalo samples
Thirty-five buffaloes that tested seropositive for brucellosis in a complement fixation test
were selected from herds located in the provinces of Corrientes and Formosa in northeast
Argentina. Blood samples were obtained from each buffalo and stored at -20°C until use. Genomic
DNA (gDNA) was extracted using the standard phenol-chloroform-isoamyl alcohol method.
Polymerase chain reaction (PCR)
Different sets of primers were used to amplify Brucella gene fragments by PCR to
discriminate species and biovars from blood samples (Table 1). The PCR mix included 0.2 mM
dNTPs, 2.2 mM MgCl2, 1.25 U GoTaq polymerase (Promega), 0.8 mM primers, and 5 µl (0.05-0.1
1029
µg/µl) of DNA template. The PCR amplifications were started with a touch down at 64°C. The
initial reaction was 94°C for 2 min, followed by 40 cycles at a variable hybridization temperature
for 1 min, and 72°C 1 min. The final extension was performed at 72°C for 7 min. The hybridization
temperature decreased by 1°C per cycle, from 64°C to 61°C during the first four cycles, and was
maintained at 60°C for the remaining 35 cycles. All products were electrophoresed in 1.5% agarose
gels, stained with 0.001 M ethidium bromide, and visualized by UV light. A wild-type (WT) B.
abortus RN1 bv 1 and/or the vaccine strains S19 or RB51 were used as positive controls. PCR
reagents without DNA were included as negative controls.
Polymorphism analyses
To assess the polymorphism among Brucella strains, PCR products were cloned into the
expression vector pGEM-T easy, following the manufacturer’s instructions (Promega). Sequence
alignments were performed using Clustal W-BioEdit® (Hall, 1999). The generated nucleotide
sequences were compared with other Brucella sequences stored in GenBank using a Blast analysis.
RESULTS
DNA from B. abortus was detected in the blood of 15 of 35 buffaloes analyzed. Fragments
of 223 bp from omp31 were amplified from the blood of some buffaloes that were seropositive for
brucellosis. The sequences showed 100% identity with the equivalent Brucella spp. gene stored in
GenBank. An eri gene analysis identified sequences of 297 bp and 1000 bp that were specific for B.
abortus S19 and WT Brucella spp., respectively. They were detected either as independent
fragments or simultaneously (Fig. 1), and their sequences were confirmed in a Blast analysis.
B. abortus (bv1, bv2 or bv4) was identified by amplifying a 498 bp fragment from the alkB
gene. The amplification of omp2ab using two pairs of primers showed single or double DNA
fragments between 600 and 900 bp. Sequence analyses showed that the single bands of 827 bp (data
not shown) and 857 bp (Fig. 2), amplified with DSF-DSR and DSF-DSR2, respectively, were 100%
identical to those found in GenBank for B. melitensis and B. abortus bv5, respectively. Using the
primers IS711-B. MEL(alkB), the 721 bp fragment of B. melitensis was not amplified from these
buffalo samples, but a fragment of 180 bp common to B. abortus bv1, S19 and RB51 was generated
instead. The omp2ab gene includes an insertion of 138 bp missing in the reference strain 2308 bv1
de B. abortus.
DISCUSSIONS
B. abortus S19 and WT strains were identified in buffaloes using different molecular
markers. Detection of the vaccine strain was expected because the persistence of DNA from B.
abortus S19 has been reported more than one year after the vaccination of bovines in NE Argentina,
independent of the detection of antibodies (Draghi et al., 2010). Unlike B. abortus S19, which was
identified by the eri gene (297 bp), identification of the WT strains required the amplification and
sequencing of at least two genes to define the species and biovar. Because B. abortus S19 is also
bv1, the simultaneous amplification of 498 bp (alkB) and 1000 bp (eri) fragments isolated from
some buffaloes suggested the presence of the WT B. abortus bv1, which is highly prevalent in the
cattle of Argentina (Lucero et al., 2008). The identity of B. abortus bv5 was established by the
amplification of two fragments of 827 bp and 857 bp from the omp2ab gene (also common to B.
melitensis). Notably, in these buffalo samples the expected 721 bp fragment of the alkB gene for
B.melitensis was not amplified and another fragment of approximately 180 bp that was identical to
those for B. abortus bv1, S19, and RB51 was obtained instead. B. abortus bv5 was often detected in
buffaloes from Formosa simultaneously with S19, although only the latter was identified in
Corrientes. This is the first report indicating the presence of B. abortus bv5 in Latin America. The
epidemiological relevance of this strain in NE Argentina is unknown and must be investigated.
REFERENCES
Asociación Argentina de Criadores de Búfalos. 2006. Razas y difusión: Difusión del búfalo en
Argentina. http://www.bufalos.org.ar/difusion.php.
1030
Baily, G.G., J.B. Krahn, B.S. Drasar and N.G. Stoker. 1992. Detection of Brucella melitensis and
Brucella abortus by DNA amplification. J. Trop. Med. Hyg. 95: 271-275.
Bricker, B. and S. Halling. 1994. Differentiation of Brucella abortus bv. 1, 2 and 4, Brucella
melitensis, Brucella ovis, and Brucella suis bv1 by PCR. J. Clin. Microbiol. 32: 2660-2666.
Bricker, B. and S. Halling. 1995. Enhancement of the Brucella AMOS PCR assay for
differentiation of Brucella abortus vaccine strains S19 and RB51. J. Clin. Microbiol.33:
1640-1642.
Draghi, M.G., L.E. Samartino, S. Torioni de Echaide, S. Conde, E. Piazza, M. Schust, G. M. Biotti.
and Y.N. Aguirre. 2010. Persistencia de anticuerpos séricos en bovinos Hereford, 3/8
Hereford y 5/8 Cebú vacunados con Brucella abortus cepa 19. Rev. Med. Vet. 91: 52-58.
García-Carrillo, C. and N. Lucero. 1993. Enfermedades de los bovinos. Rev. Med. Vet. 2: 16-27.
Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program
for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41: 95-98.
Leal-Klevezas, D.S., A. López-Merino and J.P. Martínez-Soriano. 1995. Molecular detection of
Brucella spp.: Rapid identification of B. abortus biovar 1 using PCR. Arch. Med. Res. 26:
263-267.
Lucero, N.E., S.M. Ayala, G.I. Escobar and N.R. Jacob. 2008. Brucella isolated in humans and
animals in Latin America from 1968 to 2006. Epidemiol. Infect. 136: 496-503.
OIE. 2012. Bovine brucellosis. In: Manual of Diagnosis tests and vaccines for terrestrial Animals,
7nd Ed., Paris: OIE biological Standard Commission (Chapter 2.4.3), 1: 616-650.
Sangari, F., J.M. García-Lobo and J. Agüero. 1994. The Brucella abortus vaccine strain B19 carries
a deletion in the erythritol catabolic genes. FEMS Microbiol. Lett. 121: 337-342.
Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA). 2005. Programa de control y
erradicación de la brucelosis bovina en el país. Resolución Nacional No 115/1999, y
725/2005, 17 pp.
1031
Table 1. Oligonucleotides used to identify Brucella species and biovars.
Target Fragment
Primers Sequences (5’-3’) Specificity Source
gene size (pb)
B4 TGG CTC GGT TGC CAA TAT CAA Brucella
Omp31 223 Baily et al. (1992)
B5 CGC GCT TGC CTT TCA GGT CTG spp.
DSF GCG CTC AGG CTG CCG ACG CAA Leal Klevezas et al.
Brucella
Omp2 758-858 (1995a), *ad hoc
DSR2* TTG CCT TTT CGG GGG CAA TGA spp.
(1999)
B. abortus
DSF GCG CTC AGG CTG CCG ACG CAA 689 y 804
bv1 Leal Klevezas et al.
Omp2
Brucella (1995)ª
DSR ACC CCA GAC AGC CCA A 800 ó 830
spp.
IS711 TGC CGA TCA CTT AAG GGC CTT CAT B. abortus
Bricker and
alkB bv1,bv2, 498
416 TGC CGA TCA CTT AAG GGC CTT CAT Halling, (1994)
bv4
IS711 TGC CGA TCA CTT AAG GGC CTT CAT B. Bricker and
alkB 731
B. MEL AAA TCG CGT CCT TGC TGG TCT GA melitensis Halling, (1994)
Brucella
ERI 1 GCG CCG CGA AGA ACT TAT CAA 1000 Bricker et al. (1995)
spp.
eri
OLIGO B. abortus Sangari and
CGC CAT GTT AGC GGC GGT GA 297
2 S19 Agüero, (1994)
Figure 1. DNA fragments of 297 bp and 1000 bp from the eri gene amplified from blood of
brucellosis seropositive buffaloes, using primers ERI 1-OLIGO 2.
1 2 3 4 5 6 7 8 9 10
1000 bp
600 bp
297 bp
Lanes (L). L1 to L6, buffalo samples; L7, B. abortus bv 1; L8, B. abortus S19; L9, negative control;
L10, 100 bp molecular marker (Invitrogen®).
Figure 2. DNA fragments from the omp2ab gene amplified from blood of brucellosis seropositive
buffaloes, using primers DSF- DSR.
1 2 3 4 5 6
857 bp
600 bp
Lanes (L). L1 to L3, buffalo samples; L4, B. abortus RN1 bv1; L5, negative control; L6, 100 bp
molecular marker (Invitrogen®).
1032
Efficacy Analysis of Parasitic Integrated Control in Buffaloes

E. BASTIANETTO a, M. BRITOa, D. OLIVEIRAa, V. VENEZIANOb, R. LEITEa.
a
Escola de Veterinária, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte, MG,
Brazil
b
Escola di Medicina Veterinaria, Università di Napole Federico II, Italy.
*Corresponding email: ebastianetto@yahoo.com.br
ABSTRACT
Parasitic infestations are responsible for economic losses to buffalo farms. Using the
scientific support of epidemiological and parasitic studies in this area, the present research aimed to
evaluate the efficacy of an integrated control of parasitic disease purposed. The parasitic integrated
control provides a schedule for optimize the drugs for parasitic control to reduce the number of
treatments, reduce costs and the parasitic infestation impact in animal productions. In the
experiment was used a total population of buffaloes reared in a farm located at Minas Gerais State,
Brazil. The in the same day the calves, growing males and non lactation animals were treated with
doramectin (200 mcg/ kg BW) and the lactation animals were treated with alfa-ciano-3-
fenoxibenzil-2, 2-dimetil-3-(2,2-diclorovinil)-ciclopropano carboxilaton solution. Calves positive to
Eimeria bareillyi and Eimeria bovis oocysts were treated with toltrazuril. The lice population was
eradicated and the average numbers of trichostrongylid eggs and Eimeria sp. oocysts were
substantially reduced to acceptable account.
Keywords: buffaloes, parasitic treatment, Trichostrongylid, Eimeria sp., H. tuberculatus
INTRODUCTION
Using the epidemiological information of parasitological infections in buffaloes (Lau, 1993;
Bastianetto and Leite, 2005; Bastianetto et al., 2012) is possible to reduce the numbers of parasitic
treatments to improve sanity, reduce the selection of helminth resistance to parasitic drugs and costs
with animals medications. The parturition concentration, reproduction seasonality, results in more
non lactation animals at the end of year in Brazilian buffalo herds and this allows the utilization of
the parasitic integrated control in the different animal production categories. The high efficacy of
macrocyclic lactones to control gastrointestinal helminthes and lice (Haematopinus tuberculatus)
(Bastianetto and Leite, 2005; Bastianetto et al., 2012) favor the utilization of only drug to control
both parasitic groups. The aim of this study was evaluate the efficacy of the parasitic integrated
control after a complete parasitic diagnostic in all production categories.

This study was realized using a group of 40 buffaloes composed of adult females (n = 11),
sire (n = 1), calves (n = 10) and growing males (n = 18) raised in Fazenda Modelo da UFMG,
located in Pedro Leopoldo, Minas Gerais, Brazil. Initially they were performed a coprological tests
(Whitlock, 1948; Ueno and Gonçalves,1998) and clinical exams to identify the gastrointestinal and
ectoparasite presents to select the better drug to use. After the diagnostic of trichostrongylid eggs,
Eimeria sp. oocysts and H. tuberculatus the animals of each category were treated with doramection
or toltrazuril according to the follow schedule: in the same day (D1) the calves, growing males and
non lactation animals were treated with doramectin (200 mcg/ kg BW) and the lactation animals
were treated with topical application of alfa-ciano-3-fenoxibenzil-2, 2-dimetil-3-(2,2-diclorovinil)-
ciclopropano carboxilaton solution at Days 1, 15, 30 and 45.
Calves positive to Eimeria sp. were treated with a single dose of toltrazuril (15mg/kg BW). The
efficacy of the control purposed was checked once weekly during the follow 60 days.

1033

All buffaloes were positive to Haematopinus tuberculatus, the calves and heifers positive to
trichostrongylid worms (mean of EPG = 192, 30) and few calves (n=2) were also positive to
Eimeria bayrellii and Eimeria bovis oocysts (mean = 350, 250). The lice population was eradicated
and the average numbers of trichostrongylid eggs and Eimeria sp. oocysts were substantially
reduced to acceptable account (less than 100 EPG). In buffaloes herds, the utilization of
epidemiological knowledge of buffalo parasites associated to the adequate parasitic diagnostic
allows the utilization of the parasitic integrated control and attend the purpose of this parasitological
treatment schedule with all the benefits. Considering the buffaloes rustic, animals reared in tropical
conditions with a good nutrition managements, it is possible to eliminate all parasitic treatments
after the 24 month of age. To attend this goal is also necessary use adequate physical division of
different animal production categories and periodically use coprological and clinical exams to
monitor parasitic infestation levels.
IMPLICATIONS
The adoption of correct parasitic control in buffalo herds enables increase productivity of
meat and milk with reduced utilization of antiparasitic drugs. Besides the reduction of treatments
costs and resistance to antiparasitic drugs, it is also necessary to preserve human health.
REFERENCES
Bastianetto, E. and R.C. Leite. 2005 Control of the louse (Haematopinus tuberculatus) in herds of
water buffalo (Bubalus bubalis) raised for milk and meat. Rev Bras Reprod Anim, Belo
Horizonte. 29: 118-121.
Bastianetto, E. Costa, J.O. Guimarães, M. P. Fritas, C.M.V. Lana, A.M.Q. and R.C. Leite. 2012.
Population Dynamic, Anthelmintic Treatments and the Influence of Helminth Infections on
Weight Gain in Water Buffalo Calves (Bubalus Bubalis). J. Buf. Sci. 1: 5-12.
Lau, H.D. 1993. Helmintoses gastrintestinais dos bufalinos no estado do Pará: epidemiologia e
controle Belém:Embrapa CPATU, 38 p.
Ueno, H. AND P. C.Gonçalves. 1998. Manual para diagnóstico da helmintoses de ruminantes. 4 Ed,
p. 72.
FINANCIAL SUPPORT: INCT PECUARIA (573899/2008-8 – INCT), CNPq, FAPEMIG.
1034
Detection of Toxin Genes by PCR in Clostridium perfringens Isolates Collected

from Water Buffaloes (Bubalus bubalis) Affected by Lethal Enterotoxemia
Esterina DE CARLOa*, Alessandra MARTUCCIELLOa, Mariagabriella LUCIBELLIb,
CLEMENTINA AURIEMMAb, Giorgia BORRIELLOb, Maria MUTOa, Achille GUARINOc
and Giorgio GALIEROb
a
Istituto Zooprofilattico Sperimentale del Mezzogiorno, Sezione Diagnostica di Salerno-Centro di
Referenza Nazionale sull’igiene e le tecnologie dell’allevamento e delle produzioni bufaline, Italy,
b
Istituto Zooprofilattico Sperimentale del Mezzogiorno. Dipartimento di Sanità Animale, Portici,
Italy, cIstituto Zooprofilattico Sperimentale del Mezzogiorno, Direzione, Portici, Italy.
*Corresponding email: esterina.decarlo@cert.izsmportici.it
Abstract
The production of toxins by Clostridium perfringens (C. perfringens) in the intestinal tract is one of
the major causes of enteritis and enterotoxemia in livestock species. In the present study, 166 C.
perfringens strains isolated from 176 water buffalo calves which had died with clinical enteric
symptoms were tested for the presence of genes encoding alpha, beta, beta2, enterotoxin, epsilon
gene coding for the -toxin appeared highly frequent, being detected in 130 out of 166 isolates
and iota toxins (cpa, cpb, cpb2, cpe, etx and iap) by polymerase chain reaction (PCR) assay. The
(88%). Out of the 130 -toxin-positive isolates only in 12 (7.2%) was the gene coding for the
βtoxin detected; in 2 (1.2%) the gene coding for the enterotoxin (cpe) was detected, and in 3
(1.8%) the gene coding for the ε-toxin (etx) was detected. The cases clustered seasonally at the end
and the beginning of the year (November, December, February and March). These results confirm
the role of toxin-producing C. perfringens in lethal enteritis in water buffalo and highlight the need
for proper vaccination schedules combined with controlled feeding programs and good management
practices for the effective control of enterotoxemia in the herd.
Keywords: Clostridium (C.) perfringens, Bubalus bubalis, toxins, PCR
Introduction
The C. perfringens species is an anaerobic, gram-positive, spore-forming bacillus, which can be
differentiated into five toxinotypes (types A to E) on the basis of their production of four major
toxins (alpha, beta, epsilon and iota). In addition to these “typing” toxins, other toxins may be
directly involved in the pathogenesis of C. perfringens infections, including the beta2 toxin
described in 1997 (Gilbert et al., 1997). The beta2 toxin gene can be found in all types of C.
perfringens, as it is coded for by a plasmid. C. perfringens is widespread in nature and can be found
as a normal component of the intestinal tract of humans and animals. Infections due to C.
perfringens cause various symptoms, including necrotic enteritis, hemorrhagic enteritis and acute
enterotoxemia (Niilo, 1980; Petit et al., 1999). Enterotoxemia in water buffalo is mostly caused by
C. perfringens toxinotypes A, B and D (Worrall et al., 1987; Galiero and Consalvo, 1993). Infected
water buffalo calves usually die after a peracute course without clear clinical signs, although some
individuals can exhibit tympany, colic, opisthotonus and convulsions.
The aims of this study were (i) to investigate the presence of C. perfringens in water buffalo calves
affected by lethal enterotoxemia and (ii) to characterize the strains isolated according to the toxins
produced.
Materials and methods

Samples of gastrointestinal contents from 176 water buffalo calves were obtained from necroscopic
examination. The calves came from 50 herds and 2-5 samples per herd were analysed between July
1035
2008 and July 2012. The animals included in the study were 30-50 days old, and had all died
suddenly after exhibiting tympany and opisthotonus. Each sample was cultured on blood agar plates
containing 5% sheep blood cells (Oxoid) at 37°C under anaerobic conditions for 48 hours (Quinn et
al., 1994). Species typing was carried out by means of biochemical tests (Vitek 2 Biomereux).
DNA was extracted from bacterial cultures isolated on blood agar by boiling. The DNA extracted
was tested for the presence of the genes cpa, cpb, cpb2, cpe, etx, and iap by means of conventional
PCR in accordance with a modification of the method described by Gurjar (Gurjar et al., 2008).
mixture of 25l containing 1X Hot Start Master Mix (Qiagen), 1X Q solution and 0.6 M of each
Briefly, a simplex PCR was set up in order to detect the presence of each gene by utilizing a
of the primers listed in Table 1. The thermal profile utilized involved activation for 15 min at 95°C,
followed by 45 cycles of 30 sec at 95°C, 1 min at 55°C and an extension phase of 1 min at 55°C.
Visualization of the amplification products of the expected dimensions was corroborated by means
of automated electrophoresis (Qiaxcel, Qiagen)
Results and discussion

Bacteriological examination of faeces from 176 water buffalo calves affected by gastroenteritis with
characterization of the bacterial isolates collected showed a high incidence of -toxin-producing C.

a lethal outcome exhibited the presence of C. perfringens in 94.3% of cases (166/176). Molecular
perfringens. Indeed, the cpa gene, which codes for the -toxin, was detected in 130 out of 166
isolates of C. perfringens (88%). Out of the 130 -toxin-positive isolates, only in 12 (7.2%) was the
gene coding for the βtoxin detected; in 2 (1.2%) the gene coding for the enterotoxin (cpe) was
detected and in 3 (1.8) the gene coding for the ε-toxin (etx) was detected (Table 2). The cases
clustered seasonally at the end and the beginning of the year (November, December, February and
March). These results confirm the leading role of toxin-producing C. perfringens in lethal
enterotoxemia in water buffalo, and show that, in this animal species, this disease appears to be
mostly, but not only, associated with the toxinotype A. Although high, the prevalence of cpb2 found
in the C. perfringens type A strain isolated from the samples examined was substantially lower than
in other species (Bueschel et al., 2003). No cpb2-positive strains were found among the 3 type D
isolates.
Therefore, the correct identification of C. perfringens pathovars is critical for epidemiological
studies and for the development of effective measures. The widespread presence of this pathogen in
the environment highlights the need for proper vaccination schedules combined with controlled
feeding programs and good management practices for the effective control of enterotoxemia in
water buffalo herds.
References
Bueschel D. M., B. H. Jost, S. J. Billington, H. T Trinh., J. G. Songer. 2003. Prevalence of cpb2,
encoding beta2 toxin, in Clostridium perfringens field isolated: correlation of genotype with
phenotype. Vet. Microbiol.: 94(2): 121-129.
Galiero G., F. Consalvo. 1993. Indagine sulla presenza e diffusione delle malattie infettive e
parassitarie tra vitelli bufalini in aziende da latte. Sel.Vet. 34:1055-1063
Gilbert M., C. Jolivet-Reynaud, M. R. Popoff. 1997. Beta2 toxin, a novel toxin produced by
Clostridium perfringens. Gene 203: 65-73.
Gurjar A. A., N. V. Hegde, B. C. Love, B. M. Jayarao. 2008. Real-time multiplex PCR assay for
rapid detection and toxintyping of Clostridium perfringens toxin producing strains in feces
of dairy cattle. Mol Cell Probes. 22(2):90-5. Epub 2007 Aug 19.
Niilo L. 1980. Clostridium perfringens in Animal Disease: A Revieuw of Cuurent Knowledge.
Can. Vet. J. 21: 141-148.
Petit L., M. Gibert, M. R. Popoff. 1999. Clostridium perfringens: toxinotype and genotype. Trend
in Microbiology 7 (3): 104-110.
1036
Quinn P.J., M.E. Carter, B. Markey, G. R. Carter. 1994. Clinical Veterinary Microbiology, Mosby-
Year Book Europe Limited, London 1994. pp.191-195.
Worrall E. E., L. Natalia, P. Ronohardjo, S. Partoutomo, Tarmudji. 1987. Enterotoxaemia in water
buffaloes caused by Clostridium perfringens type A. Vet Rec. 121(12):278-9.
Table 1. DNA sequences investigated.
Length of
Toxin Target Nucleotide sequence (5’-3’) amplification
product (bp)
-toxin
cpaF TGCACTATTTTGGAGATATAGATAC
cpaR CTGCTGTGTTTATTTTATACTGTTC 116
toxin
cpb2F TAACACCATCATTTAGAACTCAAG
cpb2R CTATCAGAATATGTTTGTGGATAAAC 90
-toxin
cpbF ATTTCATTAGTTATAGTTAGTTCAC
cpbR TTATAGTAGTAGTTTTGCCTATATC 93
-toxin
etxF TTAACTAATGATACTCAACAAGAAC
etxR GTTTCATTAAAAGGAACAGTAAAC 145
cpeF AACTATAGGAGAACAAAATACAATAG
enterotoxin cpeR TGCATAAACCTTATAATATACATATTC 84
-toxin
iapF CAAGATGGATTTAAGGATGTTTC
iapR TTTTGGTAATTTCAAATGTATAAGTAG 88
Table 2. Result of the major lethal toxins of Clostridium perfringens isolated.
Number Type Toxins produced

isolated Alpha Beta1 Beta2 Epsilon Iota Enterotoxin
115 A + - - - - -
11 A + - + - - -
1 A + - + - - +
2 D + - - + - -
1 D + - - + - +
1037
BUN and Total Protein levels of Buffalo Population in Udonthani Province of

Thailand
Chanachai BOONPERM* a, Cholawit YUWACHITb , Tanom TATHONGc,
Somkiat TOOJINDAd,
a
Program in Animal Production Technology, Faculty of Technology, Udonthani Rajabhat
University, Udonthani 4100,0 Thailand
b
Program in Veterinary Technology, Faculty of Technology, Udonthani Rajabhat University,
Udonthani 41000 Thailand
c
Program in Agro-Industry, Faculty of Agriculture and Technology, NakhonPhanom University
48000, Thailand
d
Khumpawapi Livestock Office, Khumpawapi, Udontthani 41110, Thailand
*Corresponding Email: cboonperm@gmail.com
ABSTRACT
The aim of this research was to study on BBP value (Biochemical Blood Parameters) and
healthy management model of Thai buffalo (Bubalus bubalis) in Udonthani Province, Thailand. The
animal used buffalo female at more than two years old and had at least one baby. The experimental
was sampling BUN value and total protein, TP at 150 head in Kumpawapee Districts, Udonthani
Province. The result showed that the healthy management model follow up by advice of
Development of Livestock with health management of management program for the year and
showed that BBP value of buffalo had 6.74 mg/dl and 7.10 g/dl respectively, However; all data was
stay in standard value.
Keywords: blood urea nitrogen, total protein
INTRODUCTION
A buffalo population of Thailand had trend reduce, because; economics system and social
was rapid expansion, city social expansion, farmer had debt burden by agriculture system. All of
these factors were effect on buffalo population to reduce and less land tenure. The present had many
machine in all farmer for compensated labor of buffalo. Udonthani province is located in the north
east, NE of Thailand and all area had buffaloes population is second level of NE. The household
population about 13,137, them had buffalos 46,833 head consist with 15,299 male, 21,643 calf and
heifer 9,891. The first parity on purpose of buffalos production in Udonthani was for sale in the
local market in traditional production most of holder were fed the buffalo by rice straw, Si-jae
village is located in Kumphawapi district. Udonthani province had a pilot project in Behind Bhudda
project under cover by royal project under the program on extension in the development of buffalo
farming. In terms of promoting the breeding of bull by the best semen with good quality health
services by DLD staff. A distribution of mineral block and promote knowledge about the buffalo
correctly technical farmers respectively (DLD, 2013).
However; buffalo production will success by consider appropriateness of the roughage,
concentrate, management and household. The buffalo can use low quality roughage as well better
than other ruminant. The healthy management programs was requirement and important for
assessing on nutritional state and base on buffalo health. The advantages of the modern animal were
the results indicate that the nutritional integrity. which fed by good quality and reasonable health
and can indicate the overall health of the animals. Such as the Liver, Kidney and the others as well
as to indicate the exposure conditions that affect the health of buffalo (Junlun, 2013).
The model of healthy management and hematology in buffalo in certain had the purpose of
this study for used as a basis for identifying buffalo production. The health of the buffalo population
in Si-jae village, Khumphawapi District, Udonthani Province, Thailand

1038
MATTERIALS AND METHODS

Animal: Hundred and fifty swamp buffalo (Bubalus bubalis ) at two year old and first parity
were sampling and calculated by G-power programing (Suk-oun, 2011). The buffalo raised in
public grass land and ad libitum with rice straw and water.
Data collection: The experimental design and data analysis were collected type of roughage
and stocking health program, vaccinations and deworming. Blood collection 10 ml form jugular
vein by MONO vet tube according to Junlun (2013) and then centrifuge at 1200 rpm for separation
serum. The serum analyzed total protein, Blood Urea Nitrogen, BUN (mg / dl) and total protein
determination (mg / dl) with BS-400 (Mindray).
Data analysis: data analyzed by SPSS program version 17 (KKU license, 2008).

The result showed that BUN value and total protein value had 0.74 mg/dl (4-14 ±2.33
mg/dl) , TP 7.10 g/dl (5.9-8.0±0.56 g/dl) according to Sil (1992) reported in cattle clave on rice
straw based ration. BUN value found on dietary treatment are with in normal physiological value of
10.30 mg/100 ml for buffalo, Total protein value 5.9-8.0 g/dl observed in present study are higher
than the value reported by Sil (1992) in buffalo and slightly lower than the value 7.75 g. reported by
Singh (1991) in buffalo fed rice straw –based ration, However, value are close to the normal range
of 6.75 -7.82 g/100ml as reported by Gupta et al (1988) Significantly higher values for serum
protein in animals fed on ammoniated rice straw than untreated straw fed group has been observed
(Sato et al., 1988;Mishra, 1991). Significantly (P < 0:05) higher serum protein in buffalo
supplemented with Fish meal might be due to differences in quality and quantity of protein
available forabsorption at the intestinal level, differences in the rate of uptake of amino acids which
are due to the changes in hormonal levels to favour anabolic processes (Walli, 1993; Kaufmann and
Luping, 1982), Pattanaik et al. (1999) observed no significant effect on BUN and TP in buffalo fed
green oat supplemented this might be due to the absence urea in buffalo feed. The height of BUN
value may be attribute to season in Thailand.
ACKNOWLEDGEMENT
The authors gratefully thank Faculty of Technology UDRU for financial supported. Khumpawapi
Livestock Office for all supported in this experiment
REFERENCES
Chantalukana, C., P. Skunmaun. 2002. Sustainable small holder animal system. Kasetsart
University press, Bangkok. 302p.
Jun-lun, A. 2010. Buffalo healh management Faculty of veterinary science, Khon Khean
University.
Suk-Oun, P. 2010. Experimental and study design in veterinary science. Faculty of vete – rinary
science, Khon Khean University.
Sil, B. 1992. Comparative studies on feeding of different conventional proteins in urea containing
diet on performance of crossbred calves. M.V.Sc. Thesis, Deemed University, IVRI,
Izatnagar, India.
Singh, P. 1991. Utilisation of nutrients from straw as affected by mode of NPN supplementation.
Ph.D. Thesis, Deemed University, IVRI, Izatnagar, India.
Wannapat, M. and R. Pilajun. 2000. Effect of energy source and urea levels in concentrate for
swamp buffalo.. Department of Animal Science, Faculty of Agriculture, Khon khean
University. In Proc. the 3th Animal science congress.January 23: 2000. 215-226p.
Wanapat, M., F. Sundstol, J.M.R. Hall. 1986. A comparison of alkali treatment methods to improve
the nutritive value of straw. II. In sacco and in vitro degradation relative to in vivo
digestibility. Anim. Feed Sci. Technol., 14 (1986), 125 p.
1039
Wora-anu, S., M., Wannapat, C.Wachirapakorn and N. Notaso. 2000. Effect of Roughage to
concentration ratio on ruminal ecology and voluntary feed intake in cattle and swamp
buffaloes fed on urea treated rice straw. In Proc.the 19th Congress of the Asian-Australasian
of Animal Production Societies and 23th Biemial Conference of the Australian Society of
Animal Production, University of New South Wales, Sydney Australia, July 3-7,2000, Vol
13:236.
Walli, T.K. 1993. Critical assessment of rumen escape protein feeding systems and its importance
in practical rations. In: Compedium I (Review papers) at the Proceedings of the 6th Animal
Nutrition Research Workers Conference, 13-16 September, 1993. OUAT, Bhubaneshwar,
India.
Table 1: Blood parameter in Thai native buffalo 150 on traditional production

Item Unit value SD
Blood urea nitrogen, BUN mg/dl 6.74 2.33
Total protein g/dl 7.10 0.56
Figure1: Frequency of BUN and TP
1040
Prevalence and Molecular Diagnosis of Staphylococcus aureus Subclinical

Mastitis in Lactating Nili-Ravi Buffaloes (Bubalus Bubalis) at Livestock
Experiment Station, Bahadurnagar, Okara, Pakistan
Waseem SHAHZADa*, Muhammad ALTAFb, Manzoor AHMADa, Rashid MUNIRc,
Muhammad Talal AMINd, Mohammad Sarwar KHANb, Mohammad Sharif SAGARa,
Mohammad Athar KHANb, Mohammad AVAISb, Ghulam AKBARa and Fayyaz
MEHMOODa
a
Livestock Production Research Institute, Bahadurnagar, Okara, Punjab, Pakistan
b
Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore, Pakistan
c
Foot & Mouth Disease Research Center, Lahore Cantt., Pakistan
d
Department of Microbiology & Molecular genetics, University of the Punjab, Lahore, Pakistan
*Corresponding email: waseem1971@hotmail.com
ABSTRACT
Subclinical mastitis is 3 to 40 times more common than clinical mastitis and causes great
losses in the dairy herds. Staphylococcus aureus (S. aureus) is one of the most common causative
agents of subclinical bovine mastitis in dairy cattle worldwide and it has been observed that up to
50 - 100% of herds are infected with this pathogen. To determine prevalence and compare bacterial
culture methods and polymerase chain reaction (PCR) for diagnosis of subclinical mastitis caused
by S. aureus, milk samples from 164 apparently mastitis free Nili-Ravi buffaloes were collected
from lactating herd at Livestock Experiment Station, Bahadurnagar Okara. These samples were
initially tested by using subclinical mastitis detection kit “CMT Test” and eighteen milk samples
(10.97%) were found positive for subclinical mastitis by using this kit. To compare cultural
technique with PCR, all these 164 milk samples were cultured on blood agar and the bacteria were
identified by standard methods. Ten out of 164 samples showed cultural growth of the organism,
thus showing prevalence 6.09 % (10 /164) of S. aureus by using conventional culturing technique.
DNA was extracted from all 164 milk samples as well as from samples cultured in broth & blood
agar plate colonies. All these were then subjected to PCR test with primers STAA-AUI and STAA-
AVII, for a 420 bp amplicon specific for S. aureus. Thirty two (19.51 %) out of 164 milk
samples,18 broth culturing and 10 bacterial colonies were found positive for S. aureus by PCR. The
results of this study indicate that PCR is sensitive and specific for diagnosis of S. aureus subclinical
mastitis and can detect this pathogen in milk samples within few hours in Nili-Ravi buffaloes.
Keywords: Nili-Ravi buffalo, PCR, Staphylococcus aureus, subclinical mastitis,
INTRODUCTION
In Pakistan, livestock contributed approximately 55.1 percent of the agriculture value added
and 11.6 percent to the national GDP during 2011-2012 (Anonymous, 2012). The population of
buffalo has been estimated as 32.7 million (M) in Pakistan and it yielded 29.565 M tons of milk,
1.769 M tons of beef and 6.842 M No’s hides during 2011-2012 (Anonymous, 2012). Punjab
province is the homeland of Nili-Ravi buffalo, a breed that represents 65 % of the buffalo
population in the country and Pakistan is the second largest buffalo milk producing (22.27 M Tons)
country in the world (Shahzad et al., 2010).
Mastitis is one of the common problems in buffalo and field surveys of major livestock
diseases in Pakistan have ranked mastitis as number one disease in dairy animals (Khan et al.,
1991). In many countries including Pakistan, Staphylococcus aureus is the most significant
pathogen causing intra-mammary infection in dairy animals (Kuang et al., 2009; Ali et al., 2011). In
Nili-Ravi buffaloes, mastitis shortens lactation period of each animal by 57 days on an average and
reduces 438 kg of milk per lactation (Cady et al., 1983). In addition, mastitis impairs the quality of
milk and milk products (Ullah, 2004). Furthermore, the presence of S. aureus in milk may present a
1041
degree of risk to consumer due to the organism’s ability to produce enterotoxins and a toxic shock
syndrome, "toxin-1" which causes serious food poisoning (Ghorbanpoor et al., 2007).
Currently, the method of identification of the mammary gland pathogens is "in vitro"
culture, which provides the “gold standard”; however, there are several disadvantages associated
with bacterial culture as microbiological culture of milk is time consuming and species
identification by standard biochemical methods requires more than 48 hours to be completed
(Ghorbanpoor et al., 2007).
Due to above mentioned limitations of cultural methods, this study was initiated to
investigate the prevalence of S. aureus through a molecular diagnostic technique, PCR and to
compare it with bacterial cultural technique in Nili-Ravi buffaloes at Livestock Experiment Station
(LES), Bahadurnagar, Okara, Pakistan.

A total No. of 164 milk samples were collected from apparently mastitis free lactating Nili-
Ravi buffaloes being maintained at LES, Bahadurnagar, Okara, during October, 2012. These
samples were tested initially, for subclinical mastitis by using subclinical mastitis detection kit
“CMT-Test” (Cat. No. 170361, Bovi-Vet, Kruuse, Denmark). All these samples were then cultured
on Nutrient broth and were incubated for 24hrs at 37°C. These enriched samples were cultured on
blood agar and were incubated for 48 h at 37 °C. Pure bacterial cultures were identified on the basis
of cultural, morphological and biochemical characteristics (John, 2000).
DNA extraction and polymerase chain reaction
DNA was extracted directly from one ml of each of 164 collected milk samples, one ml of
each of nutrient broth cultures and colonies of each cultured sample. They were subjected to the
extraction of total genomic DNA using a commercially available DNA isolation reagent
(TRIREAGENT®, Molecular Research Center, Ohio, USA). The nucleic acid was extracted also
from known mastitis negative milk samples (20 healthy buffaloes). DNA concentration was
calculated spectrophotometrically at 260 & 280 ηm and the number of genomic copies was
determined by using Minerva Biolabs kit “VenorGeM®”. The primer pair consisted of a forward
STAA- AUI primer (5´-TCTTCAGAAGATGCGGAATA-3´) and a reverse primer STAA-AUII
(5´-TAAGTCAAACGTTAACATACG-3´) was S. aureus-specific (Forsman et al., 1997) and used
for the amplification of a 420-bp fragment from S. aureus genomic DNA. All PCR amplification
reactions, including control negative samples, were carried out in a final volume of 25 µl containing
4.8 µl lysate or 2 ng purified DNA as DNA template, 12.5 µl commercially available PCR master
mix (Pyro Start™ Fast PCR Master Mix- 2X, Fermentas, Ontario, Canada), 2 U Taq DNA
polymerase (Thermoscientific, California, USA) and 2 mM MgCl2 (Thermo scientific, California,
USA). The primers were used at a concentration of 50 ρmol µl-1.
PCR was carried out in a DNA thermal cycler (PEQLAB Biotechnologie, GmbH, Erlangen,
Deutschland) using following programs: 94 °C for 2 min., followed by 40 cycles at 94 °C for 30
sec., 55 °C for 30 sec. and 72 °C for 30 sec., with a final extension step of 72 °C for 5 min
(Forsman et al., 1997). Ten microliters of the PCR products admixed with 6X DNA loading dye ™
(Fermentas, Ontario, Canada) were sized by electrophoresis on 1% agarose gel (Gene choice ®,
Maryland, USA) (1h at 90 V) with 100-bp ladder (Fermentas, Ontario, Canada) as size marker. The
gels were stained with ethidium bromide (HP 47.1, ROTH, Karisruhe) (250 µg/ml @ one
drop/25ml of 1% agarose gel) and analyzed in a UV trans-illuminator (Dolphin -Doc, Wealtec, NV,
USA) for visualization of PCR products.
RESULTS
Out of 164 samples collected from Nili-Ravi buffaloes with no clinical signs of mastitis, 18
milk samples (10.97 %) from different quarters of different buffaloes were found positive for
subclinical mastitis by using subclinical mastitis detection kit “CMT Test”. Ten out of 164 samples
showed cultural growth of S. aureus when subjected to selective blood agar which were positive for
1042
coagulase, DNAase production and mannitol fermentation test thus confirming S. aureus through
standard bacterial culture method with a prevalence of subclinical mastitis 6.09 % (10/164).
Data recorded for extracted DNA concentration by spectrophotometric analysis showed
optical density values of 1.88 and 0.84 at 260 ηm and 280 ηm wavelengths, respectively. The 420-
bp fragment was generated in positive samples tested with STAA- AUI and STAA-AUII primers
(Fig 1) specific for S. aureus. No such amplicon was detected in control negative samples. Thirty
two (19.51 %) out of 164 milk samples, 18 (10.97 %) out of 164 broth cultures and 10 (6.09 %) out
of blood agar plate colonies were found positive for S. aureus with PCR technique. All samples
positive by culture method were also positive by PCR, whereas, out of the 164 direct milk samples,
PCR detected 22 more samples as positive for S. aureus mastitis in comparison to culture method.
Since p-value (0.0001< α = 0.05) in the statistical test-statistic (z-test; z = 3.6354) lies in the critical
region, hence there was significant difference between the performance of PCR technique &
bacterial culture technique for the diagnosis of S. aureus in subclinical mastitis in Nili-Ravi
buffaloes.
DISCUSSIONS
Several studies in Pakistan revealed that S. aureus is endemic and major cause of clinical
and subclinical mastitis in cattle and buffaloes (Ali et al., 2011). Throughout the country, there has
been no previous report on molecular diagnosis of this organism. This is probably the first
molecular diagnostic report about the prevalence of S. aureus subclinical mastitis in Nili-Ravi
buffaloes in Pakistan. Ali et al. (2011) reported 44 % (264/600) prevalence of sub-clinical mastitis
in lactating buffaloes in four different districts of Punjab, Pakistan. This overall prevalence
percentage was detected by White Side Test, a test that did not identify the causative agent. The
findings of the present study are not in agreement with the previous study as 10.97 % prevalence of
subclinical mastitis has been detected by a similar type of technique (CMT Test Kit). The
discrepancy in results of two studies might be due to different areas of studies, difference in
managemental conditions being practiced at the farms, seasonal variation, and therapeutic practices.
Similarly a prevalence of 26.92 % of S. aureus (Ali et al., 2011) subclinical mastitis detected by
culture technique was found in previous study as compared to 6.09 % prevalence of S. aureus by
culture technique in the present study. This variation may be due to a better veterinary coverage at
the farm in present study along with other factors of variation as described earlier. Molecular
methods, like PCR have been used successfully for the identification of mastitis pathogens in many
countries (Shome et al., 2011; Ajitkumar et al., 2012). The results of the present study are in
agreement with the findings of Kim et al. (2001) who compared bacteriological technique with PCR
in detection of S. aureus in milk samples taken from experimentally infected cows and found PCR
to be sensitive method for S. aureus diagnosis. During present study, a higher prevalence of S.
aureus 19.51 % (32/164) was found in direct milk samples through PCR as compared to lower
prevalence of S. aureus 6.09 % (10/164) by culture technique. The culture technique requires live
bacteria in milk sample whereas molecular technique is not affected by dead bacteria. The probable
reasons for the detection of additional 22 S. aureus by PCR might be due to presence of nonviable
particular bacteria, which did not interfere its detection by PCR technology. Thus, in culture method
which is referred as "gold standard", non-isolation may not reflect the true absence of particular
bacteria especially when mixed cultures are handled.
Since most of the studies in Pakistan revealed that S. aureus is the major pathogen isolated
from clinical and subclinical mastitis in cattle and buffalo, hence correct species identification is
important for mastitis treatment, prevention, control and epidemiological investigations, as well as
in understanding of the significance of infections caused by S. aureus. Furthermore, mastitis was
diagnosed through field test or culture technique in earlier studies. In this study, a molecular
technique (PCR) was used, which is relatively a newer and advanced technique. This molecular
technique would prove to be an adequate tool for the early identification of the most common
mastitis pathogens as well as continuous monitoring of herd health in organized dairy sector in
developing countries like Pakistan. In conclusion, the developed PCR assay can be used for rapid,
1043
sensitive, specific and reliable identification of S. aureus subclinical mastitis, directly from raw
milk to reduce the economic losses caused by S. aureus subclinical mastitis and to increase milk
production in Pakistan.
REFERENCES
Ajitkumar, P., H.W. Barkema and J.D. Buck. 2012. Rapid identification of bovine mastitis
pathogens by high-resolution melt analysis of 16S rDNA sequences. Vet. Microbiol. 155:
332–340.
Ali, M.A., M.D. Ahmad, K. Muhammad and A.A. Anjum. 2011. Prevalence of sub-clinical mastitis
in dairy buffaloes of Punjab, Pakistan. J. Anim. Plant Sci. 2: 477-480.
Anonymous. 2012. Economic Survey of Pakistan. Finance Division, Economic Advisors Wing,
Ministry of Finance, Government of Pakistan, Islamabad. Chapter 2: pp. 29-30.
Cady, R.A., S.K. Shah, E.C. Schermerhorn and R.E. McDowell. 1983. Factors affecting
performance of Nili-Ravi buffaloes in Pakistan. J. Dairy Sci. 66: 578-586.
Forsman, A., P.A. Tilsala-Timisjärvi and T. Alatossava. 1997. Identification of staphylococcal and
streptococcal causes of bovine mastitis using 16S- 23S rRNA spacer regions. Microbiol.
143:3491-3500.
Ghorbanpoor, M., M. Seyfiabad shapouri, H. Moatamedi, M. Jamshidian and S. Gooraninejad.
2007. Comparison of PCR and bacterial culture methods for diagnosis of dairy cattle's
subclinical mastitis caused by Staphylococcus aureus. J Vet Res. 62: 87-91.
John, G.H. 2000. Bergey’s manual of determinative bacteriology, Actinomycetales, 9th ed.,
Williamsand Wilkins, Baltimore.
Kim, C.H., M. Khan, D.E. Morin, W.L. Hurley, D.N. Tripathy, Jr. M. Kehrli, A.O. Oluoch and I.
Kakoma. 2001. Optimization of the PCR for detection of Staphylococcus aureus nuc gene in
bovine milk. J. Dairy Sci. 84: 74-83.
Khan, M.A., M. Ajmal, M. Yamin, M.S. Khan and M.A. Athar. 1991. Epidemiological and
economical based ranking order of buffalo and cattle diseases through active surveillance
system. Pakistan. J. Livestock Res. 1: 38-43.
Khan, M.Z. and A. Khan. 2006. Basic Facts of Mastitis in Dairy animals: A Review. Pak. Vet. J.
204-208.
Kuang, Y., K. Tani, A.J. Synnott, K. Ohshima, H. Higuchi, H. Nagahata, and Y. Tanji. 2009.
Characterization of bacterial population of raw milk from bovine mastitis by culture
-independent PCR–DGGE method. Biochem. Eng. J. 45: 76-81.
Muhammad, G., A. Naureen, M.N. Asi, M. Saqib and Fazal-ur-Rehman. 2010. Evaluation of a 3%
surf solution (surf field mastitis test) for the diagnosis of subclinical bovine and bubaline
mastitis. Trop Anim Health Prod. 42: 457–464.
Radostits, O.R., D.C. Blood and C.C. Gay. 2007. Mastitis. Veterinary Medicine: A textbook of the
diseases of cattle, horses, sheep, pigs and goats, 9th Ed., Bailer tindall, London, pp: 563-
614.
Shahzad, W., R. Munir, M.S. Khan, M.D. Ahmad, M. Ijaz, A. Ahmad and M. Iqbal. 2010.
Prevalence and molecular diagnosis of Trypanosoma evansi in Nili-Ravi buffalo (Bubalus
bubalis) in different districts of Punjab (Pakistan). Trop Anim Health Prod. 42: 1597-1599.
Shome, B.R., S.D. Mitra, M. Bhuvana, N. Krithiga, D. Velu, R. Shome, S. Isloor, S.B. Barbuddhe
and H. Rahman. 2011. Multiplex PCR assay for species identification of bovine mastitis
pathogens. J. Appl. Microbiol. 111: 1349-1356.
Ullah, S. 2004. Effect of mastitis on milk composition in buffaloes under field conditions. M.Sc
(Hons.) Thesis, Deptt. Vet. CMS, UAF, Pakistan.
1044
420 bp
Fig. 1 Confirmation of S. aureus from subclinical mastitis milk

samples by PCR by amplification of six field samples
with primer pair STAA- AUI and STAA-AUII at 420-bp.
Lane M: Molecular Ladder; Lanes 1, 2, 3, 4, 5, 6: test
samples + ve for S. aureus; Lane 7: Negative control
1045
Prevalence, Molecular Diagnosis and Treatment of Field Isolates of Toxogenic

Pasteuralla multocida in a Hemorrhagic Septicemia Outbreak in Nili-Ravi Buffalo
Calves at Livestock Experiment Station, Bahadurnagar, Okara, Pakistan
Waseem SHAHZADa*, Rashid MUNIRb, Mohammad ASIFa, Mohammad Sharif SAGARa,

Mohammad ALTAFc, Waqar ASLAMa, Ghulam AKBARa and Fayyaz MEHMOODa
a
Livestock Production Research Institute, Bahadurnagar, Okara, Punjab, Pakistan,
b
Foot & Mouth Disease Research Center, Lahore Cantt., Pakistan,
c
Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore, Pakistan
*Corresponding email: waseem1971@hotmail.com
ABSTRACT
Hemorrhagic septicemia (HS) caused by Pasteurella multocida (P. multocida) is a highly fatal
and economically devastating bacterial disease of cattle and water buffalo in Asia, Africa and Middle
East with highest incidence in South East Asia. P. multocida causes high mortality (up to 52 %) in
several parts of Pakistan. An outbreak of HS occurred in Nili-Ravi buffalo calves, maintained at
Livestock Experiment Station, Bahadurnagar, Okara, Pakistan during summer season of July, 2011. A
total number of 80 mouth swab samples from Nili- Ravi buffalo calves were screened for the presence
of toxogenic P. multocida type and strain by Polymerase Chain Reaction (PCR). Out of these 80
animals, 15 died (18.75%). Among the dead calves, nine (60%) belonged to age group between 9- 12
months, followed by four (26.67%) animals belonged to 6-9 months and two (13.34%) calves of age
group above one year. About 460-bp fragment specific for P. multocida subspecies (subsp. multocida,
subsp. gallicida, and subsp. septica) was generated with primer pairs KMTISP6 & KMTIT7 in all
culturally positive samples. Similarly a product of ~590 bp specific for species and type was amplified
by using pairs KTSP61 and KTT72 in all of the samples. The 846 bp fragments were generated in all
toxogenic P. multocida positive samples tested with toxA primers. Course of the disease was nearly
twelve days. Mortality significantly peaked at 4th days where it was 26.7 %. This is almost the first
molecular diagnostic report about the prevalence of toxogenic P. multocida field isolates causing the HS
disease in Nili-Ravi Buffalo calves at LES, Bahadurnagar, Okara, Pakistan.
Keywords: Hemorrhagic septicemia, Nili-Ravi buffalo calves, Pasteurella multocida, PCR
INTRODUCTION
The buffalo population in Pakistan has been reported as 32.7 million (Anonymous 2012) and is
the second largest buffalo milk producing (22.27 M Tons) country in the world (Shahzad et al., 2010).
Punjab province is the homeland of the Nili Ravi buffalo, a breed that represents 65 % of the buffalo
population in the country (Shahzad et al., 2010).Hemorrhagic septicemia (HS) caused by Pasteurella
multocida (P. multocida) is highly fatal and economically devastating bacterial disease of cattle and
water buffalo in Asia, Africa and Middle East with highest incidence in South East Asia (OIE, 2008).
Serotypes B: 2, B: 2, 5 and B: 5 of the organism (Carter and Heddleston system) cause HS in animals
kept at different regions of Asia (Ranjan, et al., 2011). Different studies indicate that P. multocida
causes high mortality (up to 52 %) in several parts of Pakistan (Khan et al., 2011). The disease is more
prevalent with highest mortality in buffalo calves of age 6-12 months (Farooq et al., 2011).
HS is responsible for great economic losses. In one study it was calculated that the disease
caused a loss of more than 2.170 billion Pak Rupees (PKR) per annum only in the Punjab province of
Pakistan (Anonymous, 1996). Similarly Zahoor et al. (2007) has reported 0.2 million PKR average
losses per month at Landhi cattle colony Karachi due to HS.

1046
An outbreak of HS occurred in Nili-Ravi buffalo calves, maintained at Livestock Experiment

Station, Bahadurnagar, Okara during summer season of July, 2011. This study was initiated to
investigate the species, type-specific identification (Carter and Heddleston system) and toxic/non-toxic
classification of P. multocida from this H.S outbreak through polymerase chain reaction (PCR) and its
antibiotic treatment.

Outbreak was recorded in Nili-Ravi buffalo calves of different age groups with either sex. Mouth
samples were collected from all calves showing signs and symptoms of the disease. These swabs were
cultured separately in Tryptone Soya broth (5ml) at 37°C for 24 hours and then part of this culture was
sub-cultured on agar plates for another 24 h at the same temperature whereas the remaining part was
used for PCR. Smears were prepared, Gram stained and examined for presence of P. multocida which
was further characterized by media as well as through biochemical tests. A total of 80 mouth swab
samples were screened for the presence of P. multocida type and strain by PCR.
For PCR amplification, three hundred micro-liters of these inoculums were subjected for the
extraction of total genomic DNA using a commercially available DNA isolation reagent
(TRIREAGENT®, Ohio, USA). The study also included known control negative mouth samples of
twenty healthy buffaloes maintained seprately. DNA concentration was determined
spectrophotometrically at 260 ηm and 280 ηm and the number of genomic copies was determined by
using Minerva Biolabs kit “VenorGeM®”. The primer pair KMTISP6 (5´-
GCTGTAAACGAACTCGCCAC-´3) & KMTIT7 (5´- ATCCGCTATTTACCAGTGG-´3) as described
by Townsend et al. (1998) were used to amplify a product of approximately 460- bp specific for three
subspecies of P. multocida (subsp. multocida, subsp. gallicida, and subsp. septica). Then PCR
amplification was performed with primer pair KTSP61 (5´- ATCCGCTAACACACTCTC-´3) & KTT72
(5´- AGGCTCGTTTGGATTATGAAG-´3) (Townsend et al.,1998) on the same P. multocida genomic
DNA which produce a product of approximately 590-bp specific for type B isolates of P. multocida
(types B: 2, B: 5 and B: 2, 5). The primer pair toxA consisted of a forward primer (5´-
CTTAGATGAGCGACAAGG-3) and a reverse primer toxA (5´- GAATGCCACACCTCTATAG-´3) as
described by Lichtensteiger et al., 1996 were used for the amplification of a 846-bp fragment from a
gene toxA for the identification of toxogenic P. multocida.
All PCR amplification reactions, including control negative samples, were carried out in a final
volume of 25 µl containing 4.5 µl lysate or 2 ηg purified DNA as DNA template and 12.5 µl
commercially available PCR master mix (PyroStart ™ Fast PCR Master Mix-2 X, Fermentas, Ontario,
Canada). The primers were used at a concentration of 10 ρmol µl-1. PCR was carried out in a DNA
thermal cycler (PEQLAB Biotechnologie, GmbH, Erlangen, Deutschland) with following parameters for
P. multocida species and type specific identification with an initial denaturation at 95 °C for 4 min.,
followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, extension at 72
°C for 1 min. and final extension at 72 °C for 9 min. (Townsend et al. 1998). For toxogenic P. multocida
the parameters were 40 cycles of denaturing at 94 °C for 30 s, annealing at 55 °C for 30 s, and extending
at 72 °C for 30 s (Lichtensteiger et al., 1996).
Ten microliters of the PCR products admixed with 6X DNA loading dye ™ (Fermentas, Ontario,
Canada) were sized by electrophoresis on a 1% agarose gel (Gene choice ®, Maryland, USA) (1h at 90
V) with 1-kb or 100 bp ladders (Fermentas, Ontario, Canada) as size marker. The gels were stained with
ethidium bromide (HP 47.1, ROTH, Karisruhe) (250 µg/ml @ one drop/25ml of 1% agarose gel) and
analyzed in a UV trans-illuminator (Dolphin -Doc, Wealtec, NV, USA) for visualization of PCR
products.
Chemotherapeutic study
Each of the isolate was tested for sensitivity against 8 different antibiotics such as kanamycin,
gentamicin, ceftiofur hydrochloride, amoxicillin, ciprofloxacin, enrofloxacin, tetracycline and
1047
sulfadiazine as described by Naz et al., 2012. The drug with highest zone of inhibition was selected for
the treatment of disease and was observed for disappearance of clinical signs in treated animals.
RESULTS
Out of 80 animals, 15 died (18.75%). Among the dead calves, nine (60%) belonged to age group
between 9- 12 months, followed by four (26.67%) animals belonged to 6-9 months and two (13.34%)
calves of age group above one year. Comparing death rate between various age groups, the calculated
(χ2) statistic value (=9.81) has P-value (=0.0203**) at α: 0.05, therefore the mortality due to the disease
was significantly different between various age-groups. Among the dead animals 60% were male and 40
% were female calves. The calculated (χ2) statistic value (=0.42) has P-value (=0.8120) at α: 0.05 hence,
the death ratio was homogeneous by gender. While comparing the same age groups between male and
female calves, the (χ2) statistic value (=3.54) with P-value (=0.170) at α: 0.05 which means there was no
significant difference in the death rate of same age group opposite sex. Course of the disease was nearly
twelve days. Mortality significantly peaked at 4th days where it was 26.7 %.
Veterinary intervention was successful only when instituted at early stages of the disease. The
clinical efficacy of various antibiotics was guaged in terms of abatement of clinical signs of the disease.
The ciprofloxacin, ceftiofur hydrochloride + Tylocine proved to be most efficacious antibiotics. The
ancillary treatment included the use of corticosteroids, non-steroids anti-inflammatory/antipyretic drugs.
Tracheotomy, cold water bathing twice a day and segregation of affected animals was also helpful in
reducing the mortality.
Data recorded for extracted DNA concentration by spectrophotometric analysis showed optical
density values of 1.86 and 0.89 at 260 and 280 ηm wavelengths, respectively. About 460-bp fragment
specific for three subspecies of P. multocida (subsp. multocida, subsp. gallicida, and subsp. septica) was
generated with primer pairs KMTISP6 & KMTIT7 in all culturally positive samples. Similarly a product
of approximately 590 bp specific for type B:2, B:2,5 and B:5 was amplified and detected by using pairs
KTSP61 and KTT72 in all of the samples. The 846 bp fragments were generated in all toxogenic P.
multocida positive samples tested with toxA primers (Fig. I). No above mentioned amplicons were
detected in control negative samples.
DISCUSSIONS
Several studies in Pakistan revealed that P. multocida is endemic in cattle, buffalo and camel
(Farooq et al., 2011; Khan et al., 2011). Throughout the country, there has been no molecular diagnostic
report of cattle/ buffalo being infested with P. multocida. This is the first molecular diagnostic report
about the prevalence of toxogenic P. multocida in an outbreak in Nili-Ravi buffalo calves at LES,
Bahadurnagar, Okara, Pakistan. Previously in this country the P. multocida infections in cattle and
buffalo were detected through conventional techniques like culturing and serological tests. These tests
have their own limitations like lengthy procedures, less sensitivity especially the serological methods,
and inability to differentiate different P. multocida strains.
During the study a ~846-bp fragment was amplified from all field outbreak toxogenic originated
P. multocida genomic DNA by using primers as described by Lichtensteiger et al., 1996 by amplifying
the toxA gene. Since toxogenic and non-toxogenic P. multocida do not differ on diagnostic biochemical
reactions or morphology and additional testing of lab isolates is required to differentiate toxogenic and
non-toxogenic strains by applying in vitro and in vivo methods. However, culture isolation, species
identification and toxin testing of P. multocida is time consuming and costly. The confirmation of
toxogenic P. multocida by applying the molecular diagnostic tool such as PCR is a rapid and valid assay
and it facilitate the rapid clinical diagnosis and prompt therapy. Since the H.S disease is traditionally
identified on clinical signs and symptoms in the field outbreaks, application of this modern diagnostic
1048
toll will be helpful in early diagnosis of H.S disease as well as for epidemiological studies on toxogenic
P. multocida in developing countries like Pakistan.
In another PCR, the primer pair KMTISP6 and KMTIT7 (Townsend et al., 1998) amplified a
product of approximately ~ 460-bp from all field HS outbreak isolates which is unique to all P.
multocida isolates. The findings of this also corresponds to the findings of Kumar et al. (2009) who has
also detected a 460-bp fragment in the P. multocida field samples by applying PCR collected from
buffaloes in India. The same DNAs were then amplified with primers KTT72 & KTSP61 (Townsend et
al., 1998) which specifically amplified a DNA fragment at 590-bp from HS-causing type B isolates of P.
multocida i.e B:2, B:5, and B:2,5 in all the samples. This P. multocida specific PCR assay has provided
rapid species identification without relying on phenotypic differentiation, which otherwise upto 2 weeks
before definitive bio-type results are obtained. However discrimination of the B serotype requires
additional hybridization analysis but this PCR is helpful to identify type B P. multocida that causes HS
(Types B:2, B:5 and B:2,5). So PCR applied during this study has rapidly confirmed the field diagnosis
of HS without the need to obtain pure culture and perform extensive biochemical tests.
The P. multocida targeted the young buffalo calves (18.75 % mortality). The findings of this
study are in accordance with the findings as described by Farooq et al., 2011. This high mortality in
young buffalo calves could be attributed to its acute and sometimes per acute nature of the disease which
does not allow treating the animals. Other factors could be lower antibodies level against the invading P.
multocida due to use of alum precipitated vaccine in affected calves which provides protection against
for short duration.(Affected calves were vaccinated with alum precipitated HS vaccine two months
back). Stress factors such as hot, humid rainy weather, blood protozoan parasitic diseases such as
theileiriosis, babesiosis, worms infestation could be the possible reasons of high mortality in young
calves.
REFERENCES
Anonymous, 1996. Economic analysis and survey planning. In: Epidemiology survey, Punjab. Pak.
German Bilateral Technical Cooperation, Directorate of Planning and Evaluation, Department of
Livestock and Dairy Development, Punjab, Pakistan, pp: 4-5.
Anonymous, 2012. Economic Survey of Pakistan. Finance Division, Economic Advisors Wing,
Ministry of Finance, Government of Pakistan, Islamabad. Chapter 2, pp. 29-30.
Farooq, U., Z. Saeed, M.A. Khan, I. Ali and M.F. Qamar. 2011. Sero-surveillance of hemorrhagic
septicaemia in buffaloes and cattle in southern Punjab, Pakistan. Pak. Vet. J. 31: 254-256.
Khan, A., M.K. Saleemi, M.Z. Khan, S.T. Gul, M. Irfan, and M.S. Qamar. 2011. Hemorrhagic
septicaemia in buffalo (Bubalus bubalis) calves under sub-tropical conditions in Pakistan. Pak. J.
Zool. 43: 295-302.
Kumar, P., V.P. Singh, R.K. Agrawal and S. Singh. 2009. Identification of Pasteurella multocida
isolates of ruminant origin using polymerase chain reaction and their antibiogram study. Trop.
Anim. Health Prod. 41: 573-578.
Lichtensteiger, C.A., S.M. Steenbergen, R.M. Lee, D.D. Polson and E.R. Vimr. 1996. Direct PCR
analysis for toxogenic Pasteurella multocida. J. Clinical Micro. 34: 3035-3039.
Naz, S., A. Hanif, A. Maqbool, S. Ahmed and K. Muhammad. 2012. Isolation, characterization and
monitoring of antibiotic resistance in Pasteurella multocida isolates from buffalo (Bubalus
bubalis) herds around Lahore. J. Anim. Plant Sci. 22 (Sup 3): 242-245.
O.I.E. 2008. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Vol. II. Chapter 2.4.12.
Office De International Epizootics, Paris, France. PP. 739-740.
Ranjan, R., S.K. Panda, A.P. Acharya, A.P. Singh and M.K. Gupta. 2011. Molecular diagnosis of
Haemorrhagic septicemia - A review. Vet. World. 4: 189-192.
Shahzad, W., R. Munir, M.S. Khan, M.D. Ahmad, M. Ijaz, A. Ahmad and M. Iqbal. 2010. Prevalence
and molecular diagnosis of Trypanosoma evansi in Nili-Ravi buffalo (Bubalus bubalus) in
1049
different districts of Punjab (Pakistan). Trop. Anim. Health Prod. 42: 1597-1599.
Townsend, K.M., A.J. Frost, C.W. Lee, J.M. Papadimitriou and H.J.S. Dawkins. 1998.
Development of PCR assays for species-and-type-specific identification of Pasteurella
multocida isolates. J. Clin. Microbiol. 36:1096-1100.
Zahur, A.B., U. Farooq, M. Hussain, S.H. Hashmi and R. Muneer. 2007. Respiratory syndrome: A
major threat to the livestock farmers and its economic impact. Pak. Vet. J. 27: 189-193.
590 bp
Fig. 1 Confirmation of toxogenic P. multocida from field samples by PCR by amplification of three
field samples with toxA primers at 590 bp. Lane M = Molecular Ladder, Lanes 1,2,3 test samples
positive for toxogenic P. multocida. Lane 4, negative control.
1050
Management of Khari disease Syndrome with Pentasulfates Supplementation in

Lactating Buffaloes
Doj RAJ KHANAL*, Anthony P KNIGHT1, Bhoj RAJ JOSHI2 and Hem RAJ AWASTHI3
Animal Health Research Division, Nepal Agricultural Research Council, Khumaltar, Lalitpur
1
Colorado State University, Colorado, USA
2
National Animal Science Research Institute (NASRI), NARC
3
District Livestock Services Office, Doti, Nepal
*Corresponding email: drkhanal7@gmail.com
ABSTRACT
The present study summarizes the findings of field investigation and response trial of
pentasulfates supplementation to Khari disease syndrome affected buffaloes of far western Nepal.
Khari disease syndrome is characterized by severe debilitation in lactating buffaloes of Baitadi and
Darchula districts which showed devastating effects in lactating buffaloes since last three decades.
Several years of field investigation indicated that Khari syndrome is the manifestation of the
cumulative effects of malnutrition, parasitic infection, chronic selenium intoxication,
hypophosphatemia, ingestion of toxic forages, and housing of buffaloes in defective shed without
ventilation in the areas having higher ground radiation. Various response trials were initiated in the
past but with limited success.
Adopting multipronged approaches of field and laboratory investigations over a period of
four years (2008-2012), out of 717 households visited in Khari disease affected areas, pentasulfates
(PS) was supplemented in 592 lactating buffaloes with body condition score < 2 out of 5 daily with
30-40 gm for a month during winter along with single dose of deworming medication
(oxyclozanide). PS supplementation in buffaloes with early to mid-stages of Khari disease
syndrome over four years period in different locations of Darchula and Baitadi showed 70-96%%
recovery with dramatic improvement.
In conclusion, promising effect of PS along with oxyclozanide medication on buffaloes
affected with Khari disease syndrome was obtained compared with buffaloes receiving only
oxyclozanide and those not receiving any medication, indicating the superiority of the PS
supplementation in ameliorating the Khari disease syndrome.
Keywords: lactating buffalo, Khari disease syndrome, pentasulfates, deworming practices
INTRODUCTION
Water buffalo (Bubalus bubalis) is the most important livestock commodity supporting rural
livelihoods in Nepal with the highest share in Agricultural Gross Domestic Product (AGDP) after
rice. Although the population of buffaloes is around 5.1 million compared to 7.2 million cattle, the
total contribution of buffalo to milk and meat production are 70% and 65%, respectively (ABPSD,
2012), clearly demonstrating the importance of buffalo to the Nepalese agricultural economy. The
livelihood and rural economy in two hill districts of far western Nepal-Darchula and Baitadi were
hard hit by Khari disease in lactating buffaloes since last three decades with prevalence ranging
from 34.7% to 91% (Ratala et al., 1992; Khanal et al., 2008-2009; Awasthi, 2008).
Khari disease syndrome, a chronic debilitating disease of lactating buffaloes that has been
reported from the far western Nepal (Ratala et al., 1992) is characterized by hoof and skeleton
deformities and appearance of chalky powder in the hooves (Figs.1 & 2). The word "Khari" has
been coined locally to describe the chalky powder coming from the affected hooves. Khari disease
syndrome is commonly seen during the dry, winter season (from December to May) and it subsides
once the rainy season starts, and lush green pastures become available. Stall-fed lactating buffaloes
are more severely affected than those allowed to graze. Furthermore, the affected buffalo improve
once allowed for outdoor grazing. Malnourished buffaloes are more prone to develop Khari disease
1051
syndrome than the well-fed ones. Mineral supplementation and ivermectin therapy for parasitism
result in some improvement. Khari disease symptoms also reportedly subside during pregnancy.
The Khari syndrome is not infectious or transmissible as is demonstrated by the fact that calves,
heifers and males living together in the same herd do not the disease (Singh et al., 1997). Affected
animals show progressive emaciation followed by general inability to stand and walk. Animals die
from severe debilitation after 2-3 lactations. Affected animals do not exhibit any other systemic
signs or fever.

Field visits and response trial with Pentasulfates (PS)
In coordination with the District Livestock Service Office (DLSO), a multidisciplinary team
comprising a senior professor from the Department of Clinical Sciences, Colorado State University,
veterinarians from the Department of Livestock Services and a senior scientist from Animal Health
Research Division (AHRD) along with the support staffs of DLSO and AHRD visited 717
households of Khari disease affected areas of Darchula and Baitadi districts were visited during the
Year 2006 to 2012. In Years 2006-2008, several biological samples - sera, hair, chalky powders,
soil and forages were tested (Khanal et al., 2008/2009). Based on laboratory findings and clinical
pictures of affected buffaloes, treatment for chronic Selenium toxicosis was initiated with
supplementation of pentasulphates @ 30-40 gm daily along with jaggery. Just prior to PS
supplementation, affected animals were dewormed with 3-4 boluses of oxyclozanide.
Pentasulfates and oxyclozanide were distributed to 592 buffaloes (distributed over the
period of four years: 2008-2012); only deworming medication - oxyclozanide to 41 buffaloes and
five buffaloes did not receive any treatment. Response of PS in buffaloes was assessed based on the
observation and reporting by the invigilator until three months or feedback received from farmers to
the DLSO staffs and direct observation on the subsequent visits by the multidisciplinary team. Due
to geographical remoteness and difficulty in accessing each and every households provided with PS,
only 334 farmers households were followed-up.

Laboratory investigation of different biological samples revealed high levels of selenium in
the soil (2.49 mg/Kg, max.), forages (4.37 mg/Kg, max.), water (0.032 mg/Kg, max), blood (1.65
mg/Kg, max.), hair (2.92 mg/Kg, max) and chalky powder from the affected buffalo hooves (3.63
mg/Kg, max.) (Khanal et al., 2009-2011). When Se specific antidotes, Pentasulfates were
supplemented to Khari affected buffaloes over 4 year's period from 2008 to 2012, dramatic
improvement resulted in 70% to 96% (with an average of 77.84%) of the Khari affected animals
(Table 1). Whereas in buffaloes receiving only deworming medication, the deterioration of Khari
disease syndrome was not faster while in buffaloes not receiving any medication, the progression of
Khari disease syndrome was faster and severe. In the first year, not all farmers were able to feed the
complete dose of PS due to refusal of PS by some buffaloes and its unpalatable nature while in
second and third year, most of the farmers attempted to feed complete dose of PS which resulted in
dramatic recovery (Fig. 3).
1052
Figure 1. Showing Khari affected Figure 2. Showing chalky material from

recumbent buffalo the hoof
Figure 3. Showing the response of Pentasulphates in a buffalo prior to PS

(left) and post PS supplementation (right)
Biochemical parameters showed significantly decreased (p<0.01) serum phosphorus levels
(2.7 ± 0.23 mg/dl vs. 4.3± 0.25 mg/dl) and hypoproteinemia (8.1 ± 0.24 mg/dl vs. 8.41 ± 0.47
mg/dl) in Khari affected buffaloes compared to normal controls. Likewise, Khari affected animals
had significantly (p<0.01) elevated levels of serum creatinine (3.9 ± 0.27 mg/dl vs. 1.42 ± 0.06
mg/dl) and alkaline phosphatase (p<0.05) levels (268.25 ± 43.43 U/L vs. 152.72 ± 26.39 U/L). The
elevated levels of creatinine were consistent with post-mortem findings of severe kidney damage in
the terminal cases of Khari disease syndrome. Since oak feeding is quite common in most of the
Khari affected areas, elevated levels of creatinine may be attributed to the nephrotoxic effects of
gallotannins present in the oak leaves. It is not clear whether the oaks present in that areas have
some nexus with climate change. No climatic data have been available in relation to toxic effects of
oaks present in different locations. A comparative study on oaks of different geographical regions in
relation to seasonality would shed more lights on oak toxicity in buffaloes. Elevation of serum
alkaline phosphatase in Khari affected animals may also indicate degenerative changes occurring in
the bone tissues, liver or renal tubules.
Field visits to Khari disease affected areas revealed up to 91% prevalence in some locations
(Awasthi, 2008) in contrast to a previous report of 34.7% by other investigators (Ratala et al.,
1992). Lactating buffaloes were found more susceptible to Khari disease than non-lactating or male
animals. Similarly, buffalo calves and pregnant buffaloes and cattle were not affected by the Khari
disease syndrome. Cattle and goats on the same premises were unaffected. Among lactating
buffaloes, the peak prevalence was in the 3rd lactation and between 4-9 years of age. Khari affected
buffaloes usually had a body condition score (BCS) less than 2 out 5, indicating malnutrition is a
major contributor to the etiology of this disease.
1053
Table 1: Response of pentasulfates in Khari affected buffaloes

Place and Year Number of buffaloes Number of buffaloes % Improved
supplemented with PS improved after PS
supplementation
Baitadi, 2008/2009 40 28 70.0
Baitadi, 2009/2010 203 151 74.3
Darchula 2010/2011 41 33 80.48
Darchula, 20012 50 48 96.0
Total 334 260 Average: 77.84
The prevalence of Khari syndrome in stall-fed buffaloes was 77%, followed by 14.27% in
semi stall fed and (8.73%) in grazing buffalo (Awasthi, 2008). Furthermore, he found that Khari
affected buffaloes had a very high incidence of internal parasitic infestations; 70% trematodes
followed by 21% nematodes and 11% cestodes out of 25 faecal samples tested. Due to ignorance
and poverty, very few farmers were aware of the need to deworm their animals regularly. It has
been observed that some control buffaloes receiving regular deworming medication and mineral
supplements with BCS >2 did not develop Khari syndrome.
The fact that Khari affected buffaloes once allowed to graze outdoors recover, and that the
prevalence of Khari syndrome among grazing buffaloes is much less, indicates the possible
involvement of ground radiation in the development of Khari syndrome in malnourished lactating
buffaloes. It was observed that places with higher indoor radiation had higher prevalence of Khari
disease in general (Khanal and Thakur, 2009). Controlled studies with different dose rates of
radiation exposure in experimental buffaloes are needed to rule out the role radiation plays in the
Khari syndrome.
The increment in positive response of PS supplementation for managing early to mid-stages
of Khari disease syndrome from 70% in the first year to 96% in the year 2012 (Khanal et al., 2009-
2011; Chaudhary, 2012)was implicated for two reasons: i) reluctance of farmers to feed PS in the
first year due to its unappealing appearance and unpalatable nature and, ii) multiplier effects of
beneficial properties of PS in the neighbourhoods and two subsequent field visits by the senior
faculty from US university and his support for joining hand with PS supplementation and advice to
avoid oak feeding which has created a positive mental effects among the farmers and the local
staffs. Due to which the uptake rate in the year 2012 was highly remarkable with a success rate of
96% in Mallikarjun VDC of Darchula. Now, the development agencies and local government have
to direct more resources on twice yearly deworming and PS supplementation during the winter for
managing the Khari disease syndrome at bay.
ACKNOWLEDGMENTS
The authors would like to thank USEF (Fulbright Commission) for granting Fulbright
Fellowship to the first and second authors for helping to find solution to Khari disease. Staffs of
DLSO Baitadi and Darchula and staffs of AHRD deserve special thanks for their technical support.
REFERENCES
ABPSD. 2012. Agri-Business Promotion and Statistics Division, GoN, MoAD, Nepal.
Awasthi, H.R. 2008. Epidemiological and Clinico-pathological study on Khari disease in Baitadi
District of Nepal. Thesis, IAAS, Tribhuvan University, Nepal.
Chaudhary, N. 2012. Effect of Pentasulfates in Khari affected buffaloes of Shankarpur and
Mallikarjun VDC of Darchula District. Mini-Thesis, HICAST, Purvanchal University,
Nepal.
Khanal, D.R., H. Awasthi, B.L. Kunwar, K. Bhatta, D.B. Parajuli and B.R. Shah. 2008-2009.
Epidemiological study and development of control measures on Khari disease in buffaloes
of Darchula and Baitadi Districts. In: Annual Reports of Animal Health Research Division,
Nepal.
1054
Khanal, D.R. and R.P. Thakur. 2009. Khari disease: Current status and future strategy. In:
Proceedings of the National Workshop on Nepalese Dairy Strategy, Organized by
Department of Livestock Services and Community Livestock Development (CLDP),
Harihar Bhawan, Nepal, pp. 29-35.
Khanal, D.R., B.L. Kunwar and A.P. Knight. 2009-2011. Epidemiological study and development
of control measures on Khari disease in buffaloes of Darchula and Baitadi Districts. In:
Annual Reports of Animal Health Research Division, pp. 5-8.
Ratala, D.R., P. Manandhar, and G.R. Pant. 1992. Preliminary study of Khari disease of buffalo in
Baitadi district. Bulletin of Veterinary Science & Animal Husbandr Nepal, 19-20: 1990-
1992.
Singh, U.M., D.R. Khanal and D. Chaudary. 1997. Study of Khari disease in Baitadi District. In:
Annual Report of Animal Health Research Division, Kathmandu, pp. 5-7.
1055
Isolation of Mycoplasma capricolum subspecies capricolum from Dairy Buffalo

(Bubalus bubalis)
Esterina DE CARLO a*, Alessandra MARTUCCIELLOa, Immacolata DE DONATO a,
Domenico ALFANOa, Anna CERRONE b, Achille GUARINOc, Grazia CILLARAd and
Sebastiana TOLAd
a
Referenza Nazionale sull’igiene e le tecnologie dell’allevamento e delle produzioni bufaline
b
Istituto Zooprofilattico Sperimentale del Mezzogiorno. Dipartimento di Sanità Animale, Napoli
Italy, cIstituto Zooprofilattico Sperimentale del Mezzogiorno, Direzione, Napoli, Italy
d
Istituto Zooprofilattico Sperimentale della Sardegna. Dipartimento Produzioni, Laboratorio
Ricerca e Sviluppo, Sassari, Italy
ABSTRACT
This study describes the results of an investigation undertaken to determine the cause of an
outbreak of mastitis in water buffalo (Bubalus bubalis) from a dairy farm (73 buffaloes and 81
cows) located in southern Italy. The outbreak began in May 2012. A total of 20 buffalo milk and 60
cow milk samples were examined. Bacteriological analysis of the milk showed the presence of
Staphylococcus aureus and Streptoccoccus group B. In 4 milk samples, collected from buffaloes
with clinical mastitis and negative for other pathogens, Mycoplasma spp was isolated. Each isolate
was identified by means of PCR assays using M. agalactiae, M. bovis, M. putrefaciens, M.
mycoides subsp. capri, M. capricolum subsp. capricolum, and M. mycoides cluster as reference
strains. PCR confirmed the identity of the mycoplasmas as Mycoplasma capricolum subspecies
capricolum (M. capricolum) in 4 of 80 samples of milk samples examined. The PCR result was
confirmed by means of DNA sequencing. The nucleotide sequence was compared with the
sequences in the GenBank database by using the BLASTN local alignment search tool. All bovine
milk samples proved negative for the mycoplasma. The entry and transmission of M. capricolum
may be explained by the presence of a group of goats reared on the farm. However, further studies
are necessary to define both the role of buffaloes as a reservoir for M. capricolum and the relevance
of this pathogen in buffalo mycoplasmosis.
Keywords: Mycoplasma capricolum subspecies capricolum, Bubalus bubalis, PCR, mastitis
INTRODUCTION
Buffalo rearing is a major economic resource in a wide area of central and southern Italy,
where it has largely replaced cattle rearing for the production of the milk used to make typical
mozzarella cheese. The prophylaxis and treatment of diseases of the mammary apparatus of these
animals are therefore essential to the success of this economic activity. It has long been known that
one of the causes of mastitis in buffaloes is Mycoplasma spp. (Rahman et al., 1981). The present
study describes the results of an investigation undertaken to determine the cause of mastitis in water
buffalo (Bubalus bubalis) from a dairy farm (73 buffaloes and 81 cow) located in southern Italy.

In May 2012, a mixed herd of 154 cows and water buffalo with mastitis on a dairy farm
located in southern Italy was investigated. A total of 20 buffalo milk and 60 cow milk samples were
taken.
Bacterial identification
Each mycoplasma isolate was grown at 37°C in modified Hayflick medium containing 10% horse
serum. Viable cell numbers were determined by means of the last positive dilution in liquid culture,
according to standard procedures (Rodwell et al., 1983). Cells were harvested from logarithmic-
1056
phase broth cultures by centrifugation at 20,000 x g for 30 min, and washed twice with phosphate
buffered saline (PBS, 0.1 M, pH 7.4).
Molecular assay
The final pellet was resuspended in one-tenth of the original culture volume and either used
immediately for DNA isolation or stored at -80°C. DNA was extracted and purified in accordance
with the procedure described by Ausubel et al. (1993). All isolates were identified by PCR assay.
The oligonucleotide primers used are indicated in Table 1.
Amplicons (10 µl) were examined by electrophoresis in 1.5% agar gels, stained with ethidium
bromide and examined under a UV lamp in an ImageMaster VDS-CL (Amersham/Pharmacia
Biotech). The PCR product was first purified with the Montage PCR kit (Millipore) and then sent to
BMR Genomics (http://www.bmr-genomics.it/bmr_it/BMR_home.html) for DNA sequencing. The
result was processed into sequence data by means of the Sequence Scanner Software (Applied
Biosystems). The nucleotide sequence was compared with the sequences in the GenBank database
by using the BLASTN local alignment search tool.
RESULTS AND CONCLUSIONS

Bacteriological investigation of the milk showed the presence of Staphylococcus aureus and
Streptoccoccus group B in buffaloes and bovine samples. All bovine milk samples proved negative
for mycoplasma. In 4 milk samples, collected from four buffaloes with clinical mastitis and
negative for other pathogens, Mycoplasma spp was isolated. PCR confirmed the identity of the
mycoplasmas as Mycoplasma capricolum subspecies capricolum (M. capricolum subspecies
capricolum).
While M. capricolum subspecies capricolum is primarily a goat pathogen, it has also been
encountered in sheep, cows, alpine ibex (Capra ibex, ibex) and Vaal rhebok (Pelea capreolus)
(DaMassa et al., 1992). This is the first case of isolation in buffalo. In goats, M. capricolum is
highly destructive, causing septicaemia, keratoconjunctivitis, polyarthritis, pneumonia, mastitis and
occasionally abortion. In the present study, however, the four buffaloes from which the M.
capricolum strains were isolated only presented signs of mastitis, with a slight reduction in
mammary volume and a sudden fall in milk production. Ocular lesions, arthritis and pneumonia
were not observed.
Stress is a predisposing factor for mycoplasmosis. However, stress factors were not evident
in this outbreak. It should, however, be pointed out that the cows and buffaloes were reared together
on the same farm and that the same zootechnic practices were used, although each species requires
suitably different management.
The source of this infection is not known. However, three years before the outbreak, four
goats were reared on this farm. Today, only one of these goats remains; bacteriological analysis of a
milk sample taken from this animal proved negative.
Further studies are necessary to define both the role of buffaloes as a reservoir for M.
capricolum subspecies capricolum and the relevance of this pathogen in buffalo mycoplasmosis.
REFERENCES
Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl.
1993. Current protocols in molecular biology. Wiley Interscience, New York.
DaMassa A.J., P.S. Wakenell and D.L. Brooks. 1992. Mycoplasmas of goats and sheep. J. Vet.
Diagn. Invest. 4: 101-113.
Foddai A., G. Idini, M. Fusco, N. Rosa, C. De la Fe, S. Zinellu, L. Corona, S. Tola. 2005. Rapid
differential diagnosis of Mycoplasma agalactiae and Mycoplasma bovis based on a
multiplex-PCR and a PCR-RFLP. Molecular and Cellular Probes 19: 207-212.
Hotzel, H., K. Sachse and H. Pfutzner. 1996. A PCR scheme for differentiation of organisms
belonging to the Mycoplasma mycoides cluster. Vet. Microbiol. 49:31-43
1057
Manso-Silvan, L., X. Perrier and F. Thiaucourt. 2007. Phylogeny of the Mycoplasma mycoides
cluster based on analysis of five conserved protein-coding sequences and possible
implications for the taxonomy of the group. Int. J. Syst. Evol. Microbiol. 57:2247-2258.
Monnerat, M.P., F. Thiaucourt, J. B. Poveda, J. Nicolet, J. Frey. 1999. Genetic and serological
analysis of lipoprotein LppA in Mycoplasma mycoides subsp. mycoides LC and
Mycoplasma mycoides subsp. capri. Clin. Diagn. Lab. Immunol. 6:224-230.
Peyraud, A., S. Woubit, J.B. Poveda, C. De la Fe, P. Mercier and F. Thiaucourt. 2003. A specific
PCR for the detection of Mycoplasma putrefaciens, one of the agents of the contagious
agalactia syndrome of goats. Molecular and Cellular Probes 17: 289-294.
Rahman H., K. K. Baxi, S. N. Sharma. 1981. Isolation of Mycoplasma from a buffalo (Bubalus
bubalis) with Mastitis. Zbl. Ver. Med. B. 28: 585-586.
Rodwell, A.W. and R.F. Whitcomb. 1983. Methods for direct and indirect measurement of
Mycoplasma growth. In Razin S. and J. Tully (eds). Methods in Mycoplasmology:
Mycoplasma characterization. Vol I. New York: Academic Press, pp 185-196.
Tola, S., G. Idini, D. Manunta, G. Galleri, A. Angioi, A.M. Rocchigiani and G. Leori. 1996. Rapid
and specific detection of Mycoplasma agalactiae by polymerase chain reaction. Vet.
Microbiol. 50:77-84.
Table 1. DNA sequences used in the study.
Fragment
Species Sequence Reference
size (bp)
M. FS1:5’-AAAGGTGCTTGAGAAATGGC-3’ Tola et al.,
375
agalactiae FS2: 5’-GTTGCAGAAGAAAGTCCAATCA-3’ 1996
mb-mp1F: 5’-TATTGGATCAACTGCTGGAT-3’ Foddai et
M. bovis 447
mb-mp1R: 5’-AGATGCTCCACTTATCTTAG-3’ al., 2005
Mput1:5’-
AAATTGTTGAAAAATTAGCGCGAC-3’
M. Peyraud et
Mput2: 5’- 316
putrefaciens al., 2003
CATATCATCAACTAGATTAATAGTAGCACC-
3’
M. Hotzel et
P4: 5’-ACTGAGCAATTCCTCTT-3’
mycoides 220 al.
P6: 5’-TTAATAAGTCTCTATATGAAT-3’
subsp. capri 1996
M.
capricolum Mccpl1-F: 5’-AGACCCAAATAAGCCATCCA-3’ Monnerat
1356
subsp. Mccpl1-R: 5’-CTTTCACCGCTTGTTCAATG-3’ et al., 1999
capricolum
FusA-F: 5’- Manso-
M.
TGAAATTTTTAGATGGTGGAGAA-3’ Silvan et
mycoides 781
FusA-R: 5’- al.,
cluster
GGTAATTTAATAGTTTCACGATATGAA-3’ 2007
1058
Bubaline Herpesvirus 1 Associated with Abortion in a Mediterranean Water

Buffalo
Maria Grazia AMOROSOa, Federica CORRADOa, Esterina DE CARLOb, Maria Gabriella
LUCIBELLIa, Alessandra MARTUCCIELLOb and Giorgio GALIEROa*
a
Istituto Zooprofilattico Sperimentale del Mezzogiorno Via Salute, 2 80055 Portici, Italy,
b
Referenza Nazionale sull’igiene e le tecnologie dell’allevamento e delle produzioni bufaline, via
delle Calabrie, 27 84132 Salerno, Italy.
*Corresponding email: giorgio.galiero@cert.izsmportici.it
ABSTRACT
The current study describes the first detection of a field strain of BuHV-1 in a water buffalo
foetus in Europe. The study was carried out by analysing samples of aborted buffalo foetus
collected from water buffalo farms in southern Italy during a one year period of routine state
surveillance. Foetus tissues were investigated for the presence of pathogens typically involved in
abortion. In all samples analysed, the presence of a single pathogen was observed with no cases of
co-infection. One foetus, positive for herpesvirus, was further investigated to typify the virus
identified. In particular, we characterised the herpesvirus by sequencing the gE gene (US8) and we
carried out phylogenetic analysis to assess the relationship between our isolate and other ruminant
Alphaherpesviruses. The herpesvirus discovered in this research, that we called BuHV-1
Mediterranean isolate, showed the highest degree of homology with BuHV-1 strain b6. To our
knowledge, the current study represents the first survey of BuHV-1 in buffalo abortive tissues
suggesting its involvement in an abortion episode. Genetic analysis demonstrated that the virus
isolated is closely related to, but genetically distinct from the BuHV-1 b6. These data suggest the
presence of a BuHV-1 subtype typically found in Mediterranean water buffalo and/or the existence
of a more virulent strain, possibly associated with abortion.
Keywords: Foetus, Bubaline herpesvirus 1, Glycoprotein E, Phylogenetic analysis.
INTRODUCTION
Bubaline herpesvirus 1 (BuHV-1) is a virus antigenically and genetically related to bovine
herpesvirus 1 (BoHV-1) (Thiry et al., 2007). BoHV-1 is considered responsible of a wide range of
clinical syndromes in cattle (rhinotracheitis, pustular vulvovaginitis, encephalitis and abortion)
(Nandi et al., 2009) and BuHV1 has been related only to subclinical disease in water buffalo (St.
George and Philpott, 1972; Thiry et al., 2007; Scicluna et al., 2010). Water buffalo serum positive
to herpesvirus is widely documented by serological surveys (Peshev and Christova, 2000, De Carlo
et al., 2004; Scicluna et al., 2007; Scicluna et al., 2010), however, viral isolation has only been
described in Australia (St. George and Philpott, 1972) from the prepuce of buffalo, and many years
later from water buffalo in southern Italy after pharmacological reactivation (De Carlo et al., 2004).

The study was carried out by analysing 22 samples of buffalo foetus collected in four water
buffalo farms in southern Italy during one year routine analysis. Samples were investigated for the
presence of pathogens usually involved in abortion. Analysis were carried out on liver, brain, fourth
stomach, kidney, lung and placenta. Bacterial abortive agents were investigated by microbiological
methods (Quinn et al., 2011). Chlamydophila spp., Coxiella burnetii, Leptospira spp., bovine viral
diarrhoea virus (BVDV), Neospora caninum., Toxoplasma gondii was investigated by polymerase
chain reaction (PCR) using previously described protocols (Ossewaarde and Meijer, 1999, Perugini
et al., 2009, Marianelli et al., 2007, Martucciello et al., 2009, Magnino et al., 2000).

1059
Occurrence of herpesvirus was investigated by a PCR protocol able to detect a broad range of
herpesvirus species (van Devanter et al., 1996). In all samples analysed, the presence of a single
pathogen was observed with no cases of co-infection. In particular, six foetuses were positive for C.
burnetii, six for N. caninum, five for Chlamydophila spp., one for BVDV, one for B. subtilis, one
for Salmonella spp., one for Proteus spp., and one for herpesvirus. Herpesvirus DNA was recovered
from the placenta and lung from the foetus, while other tissues were negative.
The mother of the foetus carrying BuHV-1 was a pluriparous, lactating and in the fourth month
of pregnancy. As indicated by the attending veterinarian, the animal was clinically normal before
the abortion with only a slightly elevated body temperature for 48 hours after the abortion. During
abortion there were no evident signs of gross pathological lesions, only exudate in the pelvic cavity.
The animal belonged to a farm of 460 animals (350 adults) with a yearly percentage of abortion of
around 2%. All foetuses recovered from this herd in the last five years had been analysed (for all the
possible causes of abortion) by our laboratories and this case was the first case of herpesvirus
identification. The animals on the farm had been vaccinated only once (seven years before the
episode of abortion here described), against infectious bovine rhinotracheitis (IBR) and with a
BoHV-1 gE deleted vaccine.
The herpesvirus identified in the foetus was further characterised by sequencing the gE gene
(US8). This gene was chosen since its presence can differentiate wild-type herpesviruses from the
vaccination strain, which lacks gE (Thiry et al., 2007). Viral DNA was first amplified as indicated
by Ros and Belak (1999) with some modifications. In particular, DNA underwent two successive
rounds of PCR with the following primers: buHV-gEF1: (5’-CGAGACGTGCATCTTCCACC-3’)
and buHV-gER1: (5’-GGCTCGTTGGTCGGC-3’). The reaction mix consisted of: 100 ng DNA ,
15 pmol each primer, 2 mM MgCl2, 10% DMSO , 0.2 mM each dNTP, 1 U Taq gold (Roche) , 1×
buffer (Roche) . The thermal profile consisted of 95 °C for 10 min, 35 cycles of: 95 °C for 1 min,
60 °C for 1 min, 72 °C for 1 min and a final elongation step of 72 °C for 7 min. PCR product (1 µl)
was re-amplified using the protocol described above only varying annealing temperature (62 °C
instead of 60 °C). Amplicons were purified using a Qiaquick purification kit (Qiagen) and bi-
directionally sequenced using Big Dye Terminator cycle sequencing kit (ver. 3.1; Applied
Biosystems) following the manufacturer’s instructions. After dye purification and formamide
denaturation, sequenced samples were analysed by capillary electrophoresis (3130 Genetic
Analyzer; Applied Biosystems). Sequences were analysed using the BioEdit software (BioEdit
Sequence Alignment Editor version 7.0.5.2, Ibis Therapeutics; Carlsbad, California, USA) and the
CLUSTALW alignment method. BuHV-1 and BoHV-1 reference strains were used as positive
controls.
RESULTS
The multiple nucleotide sequence alignments against the US8 gE sequence, the herpesvirus
identified in this study called BuHV-1 Mediterranean isolate (BuHV-1med, GenBank accession
number KC202807), displayed 99% identity (601 out of 609 nucleotides) with the gE gene of the b6
strain of BuHV-1 (Thiry et al., 2007; GenBank accession number: EF624469.1). BuHV-1med also
displayed 97% identity (592/609 identity and 6/609 gaps) with BoHV-5 gE (GenBank accession
number: EF624468.1) and 87% identity (531/609 identity and 12/609 gaps) with BoHV-1 gE
(GenBank accession number: EF624466.1). Amino-acid alignment of gE showed that BuHV-1med
displayed two amino-acid substitutions compared with BuHV-1b6. One substitution was
conservative to an amino-acid of the same charge (Ala→Val), the second substitution was non
conservative from a non polar to a polar amino-acid (Ala→Ser). The gE sequence of controls
matched with 100% identity to those deposited in GenBank. Phylogenetic analysis based on the
US8 gE gene was carried out to assess the relationship between BuHV-1med and other ruminant
Alphaherpesviruses and showed the highest degree of homology with BuHV-1 (Fig. 1).
1060
DISCUSSIONS
To our knowledge, the current study is the first report of the first survey of BuHV-1 in
buffalo abortive tissues suggesting its involvement in an abortion episode. This finding,
corroborated by the absence of any other abortive pathogen in the sample, is in contrast with the
existing literature that suggests bubaline herpesvirus only causes mild clinical respiratory symptoms
(St. George and Philpott, 1972; Thiry et al., 2007; Scicluna et al., 2010). Genetic analysis
demonstrated that the virus isolated is closely related to, but genetically distinct from the BuHV-1
b6 strain. Indeed, as shown in Fig. 1, the gE sequence of BuHV-1med differed from that other
BuHV-1 isolates by eight nucleotides (Thiry et al., 2007). These data suggest that the presence of a
BuHV-1 subtype typically found in Mediterranean water buffalo and/or the existence of a more
virulent strain, may be associated with abortion. However, since no histopathologic investigation
was performed on the foetal tissues, it is difficult to definitively link the presence of BuHV-1 to the
cause of abortion and further studies, including molecular and histopathological analyses and virus
isolation studies are needed to confirm this link.
REFERENCES
De Carlo, E., G.N. Re, R. Letteriello, V. Del Vecchio, M.P. Giordanelli, S. Magnino, M. Fabbi, C.
Bazzocchi, C. Bandi, G. Galiero. 2004. Molecular characterisation of a field strain of
bubaline herpesvirus isolated from buffaloes (Bubalus bubalis) after pharmacological
reactivation. The Veterinary record 154: 171-174.
Magnino, S., C.Bandi, P.G. Vigo, M. Fabbi, M. Colombo, N. Colombo, C. Genchi. 2000.
Diagnostica della Neosporosi bovina nel nord Italia. Large Animal Review 3: 25-29.
Marianelli, C., M. Tarantino, S. Astarita, A. Martucciello, F. Capuano, G. Galiero. 2007. Molecular
detection of Leptospira species in aborted fetuses of water buffalo. Veterinary Records 1:
161: 310-2.
Martucciello, A., G.M. De Mia, M. Giammarioli, I. De Donato, G. Iovane, G. Galiero. 2009.
Detection of Bovine viral diarrhea virus from three water buffalo fetuses (Bubalus bubalis)
in southern Italy. Journal of Veterinary Diagnostic Investigation 21: 137-40.
Nandi, S., M. Kumar, M. Manohar , R.S. Chauhan. 2009. Bovine herpes virus infections in cattle.
Animal health research reviews / Conference of Research Workers in Animal Diseases 10,
85-98.
Ossewaarde, J.M. and A. Meijer. 1999. Molecular evidence for the existence of additional
members of the order Chlamydiales. Microbiology 145: 411-417.
Perugini, A.G., F. Capuano, A. Esposito, C. Marianelli, A. Martucciello, G. Iovane, G. Galiero.
2009. Detection of Coxiella burnetii in buffaloes aborted fetuses by IS111 DNA
amplification: a preliminary report. Research in Veterinary Science 87: 189-91.
Peshev, R., L. Christova. 2000. Study of bovine herpes virus 1 spreading among buffalo herds in
Bulgaria. Acta virologica 44: 229-230.
Quinn, P.J., B.K. Markey, F.C. Leonard, E.S. FitzPatrick, S. Fanning, P.J. Hartigan. 2011.
Veterinary Microbiology and Microbial Disease Ed. Wiley-Blackwell.
Ros, C. and S. Belak. 1999. Studies of genetic relationships between bovine, caprine, cervine, and
rangiferine alphaherpesviruses and improved molecular methods for virus detection and
identification. Journal of clinical microbiology 37: 1247-1253.
Scicluna M.T., G. Saralli, G. Bruni, M. Sala, C. Cocumelli, D. Caciolo, R.U. Condoleo and G.L.
Autorino. 2007. Epidemiological situation of Herpesvirus infections in buffalo herds:
Bubaline Herpesvirus1 or Bovine Herpesvirus1? Italian Journal of Animal Science 6: 845-
849.
Scicluna, M.T., A. Caprioli, G. Saralli, G. Manna, A. Barone, A. Cersini, G. Cardeti, R.U.
Condoleo and G.L. Autorino. 2010. Should the domestic buffalo (Bubalus bubalis) be
considered in the epidemiology of Bovine Herpesvirus 1 infection? Veterinary microbiology
143: 81-88.
1061
St George, T.D. and M. Philpott. 1972. Isolation of infectious bovine rhinotracheitis virus from the
prepuce of water buffalo bulls in Australia. Australian Veterinary Journal 48, 126.
Thiry, J., F. Widen, F. Gregoire, A. Linden, S. Belak and E. Thiry. 2007. Isolation and
characterisation of a ruminant alphaherpesvirus closely related to bovine herpesvirus 1 in a
free-ranging red deer. BMC veterinary research 3: 26.
VanDevanter, D.R., P. Warrener, L. Bennett, E.R. Schultz, S. Coulter, R.L. Garber and T.M. Rose.
1996. Detection and analysis of diverse herpesviral species by consensus primer PCR. Journal of
clinical microbiology 34: 1666-1671.
Figure 1: Phylogenetic tree (neighbor-joining method) based on the US8 gE gene, showing
relationships between BuHVmed and other ruminant Alphaherpesviruses. The Suid herpesvirus
type 1 (SuHV-1) Becker strain gE gene was used as an outgroup. Numbers at the nodes indicate the
bootstrap confidence values obtained after 1000 replicates.
1062
Doramectin Resistance in Helminths From Buffaloes

Euardo BASTIANETTOa, Luciana ANDRADE a, Bruno BRASILa, Ronaldo NUNESa,
Marcela DRUMMONDa, Mayara BRITOa, Denise OLIVEIRAa and Romário LEITEa
a
Brazil
ABSTRACT
The helminth resistance to antiparasitic drugs has a huge impact in livestock production. In
bovines, helminth resistance to doramectin is reported since 2002 however there is no report in
buffaloes. In a routine feces examination to evaluate the efficacy of a parasitic control program
using doramectin (200 mcg /kg of body weight) in a buffalo farm, one animal was found positive to
trichostrongylid eggs after seven days post treatment. To confirm the observed resistance the animal
was treated once again and the feces were collected and analyzed at day 7 and 14. The resistant
worms were then identified by complementary exams. This observation invites all buffalo farmers
and technicians to be alerted for the use of correct practices to helminth control aiming to reduce the
frequency of anthelmintic resistance in buffalo population. Practices to reduce the calve mortality
by worms are currently used, but according to genetic selection and high production levels, it
becomes necessary to be attempted also for the subclinical parasitic infection and its control.
Keywords: helminth resistance, diagnostic, doramectina, Haemonchus spp., Ostertagia

(Teladorsagia) circuncincta
INTRODUCTION
Buffaloes are susceptible to infection by a large number of gastrointestinal helminths (Costa
et al., 1986) that can cause mortality and decrease production in livestock (Bastianetto, 2012).
Despite the knowledge about the impact of these parasites on the productivity, the actions required
for the choice of antiparasitic drugs and monitoring of treatment efficacy has not been used properly
in buffalo herds. Frequent use of the same basis of anthelmintic drugs in whole herd limits genetic
variability of parasitic population and favors the selection of resistant helminths (Paiva et al., 2001,
Souza et al., 2004). Furthermore, although there are several reports on helminth drug-resistance in
cattle, sheep, horses, (Molento, 2005, Rangel et al., 2003, Costa et al., 2011), in buffaloes, no
measure has been used to verify the efficacy of anthelmintic treatments performed. In order to
monitor the reduction of EPG in faeces of buffalo (Bubalus bubalis) treated with doramectin,
coprological tests were conducted post treatment and it was possible to identify a low efficacy of
the drug on the population of parasites present in an animal.

The study was conducted using 28 buffaloes aged between seven and twenty months, raised in
Fazenda Modelo da UFMG, located in Pedro Leopoldo, Minas Gerais, Brazil. Buffaloes were
raised on pasture with predominance of Brachiaria spp. where it had been raised cattle (Bos taurus
and Bos taurus indicus) in the previous year.
The animals were treated with Doramectin (200 mcg/kg of body weight) on 9/13/2012. After seven
and fourteen days, coprological tests were performed for the detection of eggs in faeces through the
technique of Whitlock (1948) (Ueno and Gonçalves, 1998) in the Laboratório de Endo-
Ectoparasitoses da Escola de Veterinária da UFMG. The coprological tests confirmed the presence
of helminths resistant to Doramectin. The animal positive for Trichostrongylid eggs was retreated
with the same drug and dosage and faeces were again analyzed seven days after the second
treatment. The results remained positive for helminth eggs. The resistant worms were then

1063
identified by complementary exams; fecal culture, Baermann, larvae morphological examination

(Skarjabin, 1954, Ueno and Gonçalves, 1998) and DNA sequencing (Brasil et al., 2012).

The average count of eggs in the faeces of one animal (EPG = 100) at seven and fourteen
days after the treatment confirmed the existence of gastrointestinal helminths resistant to
Doramectin in the buffalo herd evaluated. Despite the low EPG, additional tests were performed to
confirm the drug resistance findings. The resistant helminths identified belong to the genera
Haemonchus spp. and the species Ostertagia (Teladorsagia) circuncincta which are highly
pathogenic for the infected animals.
It must be considered that the buffaloes were being created in an environment that previously
attended cattle, and that identified parasites are not specific to buffaloes. It is possible that
doramectin resistant helminths remained in the environment by the adoption of inadequate sanitary
management with bovines. Sheep were also bred in the same farm, and the pasture contamination
by helminths larvae might also have occurred by other animal´s movement or tractors tires
(Greeve, 1985).
In order to achieve success in the control of helminths in buffaloes it is necessary to adopt
nutrition and good management practices associated with the use of anti-helminthic drugs. This
actions will contribute to the control of infections, acquired immune responses and might decrease
the need for frequentl antiparasitic treatment. Bastianetto (2006) evaluated the effectiveness of
different schemes of deworming and there was no difference between the average score of
helminth eggs in faeces and also weight gain among animals treated and not treated when they were
not properly fed.
In Brazil and in other tropical countries, in specific situations, it is occurring replacement of
bovine for buffalo herds. In this way it is necessary the adoption of sanitary actions against
helminth population in the environment to prevent infection of buffalo herds with resistant
helminths.
IMPLICATIONS
It is necessary to adopt an adequate helminthic control in buffaloes in order to reduce the
frequency of resistant worms and insure the potential productivity of buffaloes in tropical areas.
REFERENCES
Bastianetto, E. 2006. Disseratação (mestrado) – Universidade Federal de Minas Gerais, Escola de
Veterinária.
Bastianetto, E., J.O. Costa, M.P. Guimarães, C.V.M. Freitas, A.M.Q. Lana, and R.C. Leite. 2012,
Population dynamic, anthelmintic treatments and the influence of helminth infections on
weight gain in water buffalo calves (Bubalus Bubalis). J. of Buffalo Science 1:5-12.
Brasil, B. S. A. F., R. L. Nunes and E. Bastianetto. 2012. Genetic diversity patterns of Haemonchus
placei and Haemonchus contortus populations isolated from domestic ruminants in Brazil.
International J. for Parasitology. 42: 469- 479.
Costa, H.M.A., A.C.R. Leite, M.P. Guimarães and, W.S. Lima. 1986. Distribuição de helmintos
parasitos de animais domésticos no Brasil. Arq. Bras. Med. Vet Zoot. 38 465-487.
Costa, M.S.V.L.F., R.N. Araújo, A.J.L.F. Costa, R.F. Simões, and W.S. Lima. 2011. Anthelmintic
resistance in a dairy cattle farm in the State of Minas Gerais Rev. Bras. Parasitol. Vet.
Jaboticabal. 20: 115-120.
Greeve, J.H. 1985. Means of dissemination of parasite. In: Parasite, Pest and Predators (Ed. S. M.
Gaffers, W. E. Howard and R. E. Marash). Amsterdam, Elsevier, cap 3. pp. 29 – 47.
Paiva, F., M.O. Sato, A.H. Acuña, J.R. Jensen and M.C.R.V. Bressan. 2001. Resistência a
ivermectina constatada em Haemonchus placei e Cooperia punctata em bovinos. Hora Vet.
120: 29-34.
1064
Rangel, V.B., R.C. Leite, P.R. Oliveira and E.J. Santos JR. 2005. Resistance of Cooperia spp. and
Haemonchus spp. to avermectins in beef cattle Arq. Bras. Med. Vet. Zootec. 57: 186-190.
Skrjabin, K. L., N. P. Shikobalova and R. S. Shul’ts. 1954. Essentials of nematodology. Moscow:
The Academy of Sciences of the USSR.
Souza, A.P. 2004. Controle integrado das principais parasitoses de bovinos. Rev. Bras Parasitol.
Vet. 13: 72-79.
Ueno, H. and P.C. Gonçalves. 1998. Manual para diagnóstico da helmintoses de ruminantes. 4 Ed ,
1998, pp. 72.
1065
Breeding Techniques, Welfare and Mammary Gland Pathologies in Buffalo
Esterina DE CARLO,a Domenico VECCHIO,b Alessandra MARTUCCIELLO,a Roberta

VECCHIO,a Anna BALESTRIERI,b Carlo GRASSI,c Bianca GASPARRINI*b
a
delle Calabrie, 27 84132 Salerno, Italy
b
DISCIZIA, Faculty of Veterinary Medicine, Federico II University,via Delpino 1, Naple, Italy,
c
Veterinary freelancer
*Corresponding email: bianca.gasparrini@unina.it
ABSTRACT
The aim of this study was to verify the effect of bunks on the health, welfare and
productivity of the buffalo, a species that is characterized by high genetic and behavioural
variability and in which hierarchic phenomena are particularly evident. The trial was conducted for
a period of 120 days and involved 96 buffaloes bred in the Salerno province. The animals were
divided into two homogeneous groups according to days in milk, body condition score (BCS) and
milk production (both quality and quantity): buffaloes in Group 1 were maintained on cement pads
equipped with foam rubber bunks, while those in Group 2 were allocated to similar cement pads
without bunks. Monthly, milk production was recorded and individual milk samples were
collected, to evaluate fat and protein contents and somatic cell counts. Finally, after 2, 3 and 4
months, milk samples were sterilely collected for total bacterial counting. Each month, milk
samples underwent bacteriological analyses to detect the presence of mastitis-causing agents. At the
same time-points, the well-being of the animals was assessed through determination of the main
parameters of innate immunity, which are indicators of well-being (bactericidal capacity,
haemolytic complement, haptoglobin, serum lysozyme). Statistical analysis was performed by
means of chi-square test and ANOVA.
The results showed that mismanagement can reduce the positive effects of using structures
that can improve animal welfare.
Keywords: animal welfare, Bubalus bubalis, bunks, mastitis.
INTRODUCTION
In technologically advanced buffalo farms, shed requirements have been adapted to animal
requirements, in order to comply with regulations on hygiene and waste management. For this
reason, a high incidence of pathologies due to intensive breeding has arisen, affecting welfare and
production in this species. A typical example of this is the utilization of bunks, which improve
waste management but reduce the space available for each animal. The utilization of these
structures in dairy cattle is considered to reduce the incidence of udder pathologies. Among these,
mastitis, an inflammation of one or more udder quarters, can be considered the most common
disease in dairy breeding. In this study, we evaluated i) the effect of using bunks on the general
welfare of a group of dairy buffaloes (group 1) in comparison with a group of buffaloes for which
no bunks were provided (group 2), and ii) the quality and quantity of milk production and the
incidence of mastitis in both groups.

The study was carried out on an intensive dairy farm where welfare conditions were very
good: 19 m2/head, showers activated during the hottest hours of the day in summer, full roofing in
cows were selected; at the beginning of the study, the mean time since calving was 36  30 days.
winter and partial in summer, paddocks equipped with scrapers. A total of 96 multiparous buffalo
The animals were divided into two homogeneous groups according to days in milk, body condition
1066
score (BCS) and milk production (both quality and quantity): buffaloes in Group 1 were maintained
on cement pads equipped with foam rubber bunks, while those in Group 2 were allocated to similar
cement pads without bunks. The stalls equipped with bunks were not fitted with side-rails. At the
time of selection, the animals underwent blood tests (haemochromocytometric examination,
biochemical profile) to assess their state of health; immune tests were also carried out to determine
the levels of parameters of innate immunity, which are indicators of well-being (haemolytic
complement, lysozyme, bactericidal capacity and haptoglobin). Only animals displaying values that
were appropriate for the species, age and productive period were included in the study. Throughout
the trial period, the buffaloes were fed on rations of silomais, hay and commercially available
concentrates, in order to provide about 17 kg of dry bulk with a concentration of 15% raw proteins,
6% fats, 40% NDF and 0.91 UFL in energy. The trial was performed over a period of 120 days.
Monthly, milk production was recorded and individual milk samples were collected, to evaluate fat
and protein contents and somatic cell counts. Finally, after 2, 3 and 4 months, milk samples were
sterilely collected for total bacterial counting. Milk samples for bacteriological analyses were taken
from each animal 6, 30 and 60 days after calving; at the same time-points, blood samples were
taken in order to plot the trend in the immune parameters investigated.
At the beginning and end of the study, the BCS was individually evaluated by adapting the score for
beef cattle to the buffalo species (scale 1 – 9).
The qualitative analyses of milk were performed by means of an automatic FT2 (Foss)
apparatus and by applying the FIL-IDF 141C:2000 infrared technique. Somatic cells were counted
by a Fossomatic (Foss) apparatus by means of the UNI EN ISO 13366-2:2007 technique for
electronic optical fluorometric counters. The total bacterial load was determined by means of the
UNI EN ISO 4833:2004 horizontal method for microorganism counting, a colony-counting
technique at 30°C. The bacteriological analyses of milk were carried out in accordance with the
laboratory's internal method: samples were taken in sterile conditions from the four quarters of the
udders and cultured on Baird Parker agar, Sheep Blood agar, MacConkey agar, Sabouraud agar,
Hayflick medium containing 10% horse serum and PIM agar. Plates were incubated aerobically at
37°C ± 1°C for 24 to 48 hours. (Quinn et al. 1994).
The following parameters of clinical immunology were evaluated: haemolytic complement,
lysozyme, bactericidal capacity and haptoglobin. The haptoglobin assay is based on the difference
between the peroxidase activity of free haemoglobin and that of haptoglobin-bound haemoglobin in
an acidic environment. The peroxidase activity of bound haemoglobin is directly proportional to
the amount of haptoglobin present in the sample. The sera examined were analysed by means of the
Phase Haptoglobin Colorimetric Assay kit (Tridelta Development, Ireland). Semiquantitative
titration of the haemolytic complement is based on the quantification of the lytic activity of the
serum towards rabbit erythrocytes (Barta et al.,1993) Sera are diluted with a suspension of rabbit
erythrocytes and incubated. After centrifugation, the supernatant is recovered and read at λ 550 nm.
The lysozyme present in the sample is titrated by placing the serum in contact with Micrococcus
lysodeikticus incorporated into an agar gel and evaluating the lysis ring of the microorganism
around the deposition well of the sample (Osserman et al., 1966). The concentration of the
lysozyme is proportional to the diameter of the clarification ring around the well and is established
on the basis of a standard curve obtained from the incubation of known concentrations of lysozyme.
To determine the bactericidal capacity of the serum, the serum is placed in contact with a known
quantity of E. coli. Evaluation of the capacity of the serum to inhibit the growth of the germ is
based on the variation in the turbidity of the culture wells in the presence/absence of the sample, as
assessed through spectrophotometric reading the optical density (Amadori et al., 1997; Amadori et
al., 2002).

No statistical differences emerged with regard to qualitative and quantitative milk
production or somatic cell count. (Figures 1,2,3,4). Data recorded throughout the experimental
period showed a normal trend of the lactation curve. The hygiene characteristics of the udders were
1067
optimal in both experimental groups and the somatic cell count was lower than 300,000/ml
throughout the period, with the exception of the sample collected after 117 days in Group 1, which
showed a slightly higher value. BCS at the start (6.7 and 6.6 in Groups 1 and 2, respectively) and at
the end of the trial (7.5 and 7.6 in Groups 1 and 2, respectively) was similar in the two groups.
However, since an increase of 1 point in BCS is equivalent to an increase of about 25 kg of live
body weight, the increase in live weight was 20 and 25 kg in buffaloes in Groups 1 and 2,
respectively. Regarding udder health, a higher isolation of pathogenic bacteria (Staphylococcus
aureus and Streptococcus agalactiae) was recorded in Group 1 than in Group 2 (44.4% vs. 31.2;
P<0.05), and a peak in this trend was observed at the end of the trial (64.6% vs. 37.5%; P<0.05)
(Table 1).
The animals selected displayed very good values of the parameters of innate immunity at the
time of allocation to the experimental groups. During the study period, however, group 1 animals
displayed high levels of serum lysozyme, indicating the presence of an inflammatory phenomenon.
By contrast, the levels of haemolytic complement proved to be better in group 1, indicating a better
well-being. Differences in the levels of haptoglobin and bactericidal capacity were not significant
(De Carlo et al. 2011) (Table 2).
Visual inspection of the bunks used revealed varying degrees of depression, depending on
whether all or only a few of the animals were in the stalls. Indeed, as the bunks were made up of
undivided stretches of foam rubber (i.e. not a single piece for each animal), whenever one animals
stood up, the foam rubber in the adjacent occupied stall would form a hollow, in which biological
fluids would collect. This same dairy farm also makes use of separate bunks for individual animals.
Observation of these bunks revealed that this hollowing and the consequent collection of fluids did
not occur. The use of individual bunks is therefore to be recommended, in order to avoid marring
the positive effects of using structures that can improve animal welfare.
REFERENCES
Amadori M., I. Archetti, M. Frasnelli, M. Bagni, E. Olzi, G. Caronna and M. Lanteri. 1997. An
immunological approach to the evaluation of welfare in Holstein Frisian cattle”. J. Vet. Med.
B 44:321-327.
Amadori M., I.L. Archetti, M.M. Mondelli and M. Fazia. 2002. La valutazione del benessere
animale. In: Quaderni Fondazione Iniziative Zooprofilattiche e Zootecniche. 51: 51-54.
Barta V. and O. Barta. 1993. Testing of Hemolytic Complement and its components. 1993. Vet. Cl.
Imm. Lab., bar-Lab, Blacksburg, USA.
De Carlo E., A. Martucciello, L. Schiavo, R. Vecchio, P. Palermo, M. Amadori. 2011. Environment
and innate immunity in water buffaloes. In: Proceedings of 2011 Joint Annual
Meeting.Riccione (IT) Ed. Minerva Medica.. pp.47.
Osserman E.F. and D.P. Lawlor. 1966. Serum and urinary Lysozyme (murimidase) in monocytic
and monomyelocytic leukemia. J. Exp. Med. 124:921-952.
Quinn P.J., M.E. Carter, B. Markey, G.R. Carter. Mastitis. In: Clinical Veterinary Microbiology.
(Ed. Wolfe). Mosby-Year Book Europe, London. pp 327-344.
1068
Table 1: Isolation of bacteria in the two groups
Days since S. aureus Group S. aureus S. agalactiae Group S. agalactiae

calving 1 Group 2 1 Group 2
6 5 4 3 0
30 5 6 7 3
60 11 6 5 4
Table 2: Values of innate immunity in the two groups
Days since Haptoglobin Haemolytic Bactericidal Lysozyme

calving complement capacity
Gr.1 Gr2 Gr.1 Gr2 Gr.1 Gr2 Gr 1 Gr.2
6 1.45 1.91 81.89 78.18 93.33 93.42 5.91 4.23
30 1.32 1.34 55.19 54.72 94.00 94.45 5.98 4.87
60 1.01 1.11 80.35 69.26 92.58 92.52 2.35 2.52
Mean 1.14 mg/ml  30 86.63 % 4 µg/ml
lactation C’H50/150 µl
value
Figure 1 Daily dairy production (kg) in group 1 and group 2
Figure 2 Fat concentration (%) in milk of group 1 and group 2
1069
Figure 3 Protein concentration (%) in milk of group 1 and group 2
Figure 4 Somatic Cell count in group 1 and group 2
1070
Comparison between Two Gamma-Ifn Assays and Intradermal Tuberculin Test

for the Diagnosis of Tuberculosis in Water Buffalo (Bubalus Bubalis)
Lorena SCHIAVO, a Philippe POURQUIER,b Alessandra MARTUCCIELLO,a Laura

OLAGNON,b Damiano GIOIA,c Anna VISCITO,a Debora COZZA,d Esterina DE CARLO a*
a
delle Calabrie, 27 84132 Salerno, Italy
b
IDVET, 167 rue Mehdi Ben Barka, 34070 Montpellier, France
c
ASL Salerno ambito 1 distretto 2 Sant’Egidio Montalbino, Salerno, Italy
d
Istituto Zooprofilattico Sperimentale del Mezzogiorno, Dipartimento di Sanità Animale, Portici,
Italy
ABSTRACT
Control of tubercolosis (TB) in buffaloes is currently based on slaughter of animals deemed
positive on the basis of tubercolin testing. This test presents some disadvantages such as the need of
holding animals for 72 h and some difficulties depending on the interpretation and the site of the
PPD inoculation. Therefore, the purpose of this preliminary study was to evaluate the use of the
gamma-Interferon (γ-IFN) assay as a confirmatory test under field condition in South of Italy.
Animals from herds of known tubercolosis-positive status and from herds of known tubercolosis-
negative status were tested in the last three years using the Intradermal Cervical Tuberculin Test
(ICTT) and both the Prionics and IDVET γ -IFN test. Different antigens (PPDS, ESAT-6, CFP10)
were used in the vitro stimulation of blood lymphocytes of TB positive and negative animals to
evaluate which reagent give the best results. The samples related to animals from herd TB free were
negative for ICTT and for stimulation with recombinant proteins, while two samples reacted
positively after stimulation with bovine PPD, demonstrating the specificity of improvement through
the use of recombinant antigens. The results obtained in positive herds over the years demonstrate
the ability of the γ -IFN test to make early TB diagnosis compared to ICTT. The comparison
between results of Prionics and IDVET tests performed on samples from infected herds, show a
good correlation between the two kits.
Keywords: Tuberculosis, Bubalus bubalis, gamma-Interferon
INTRODUCTION
Bovine tubercolosis (TB) is a chronic, infectious and contagious disease caused by
Mycobacterium bovis (M. bovis), a member of the M. tuberculosis complex (MtbC) (Smith et al.,
2006). While the main host is cattle (Bos Taurus), other domestic and wild mammals, including
water buffalo (Bubalus bubalis), can also be infected. In Italy, programs for the eradication of
bovine and buffalo tuberculosis (D.M. n.592, 15 December, 1995, D. Lvo n.196, 22 May, 1999) are
limited to the use of one official test, the Single Intradermal Cervical Tuberculin Test (SICTT), and
the slaughter of positive animals, with postmortem inspection and culture isolation of M. bovis from
samples taken at the slaughterhouse. To our knowledge, no previous studies have been reported in
which two diagnostic tests have been carried out to support efforts to eradicate tuberculosis in
buffalo. The aim of the present study was therefore to evaluate the efficacy of the SICTT and the γ-
IFN assay in diagnosing tuberculosis in buffalo and their potential value in eradicating the disease
from the herds in which more than one examination is conducted. In addition, these techniques were
compared with the results of the examination of carcasses at the slaughterhouse.
The use of the gamma-Interferon (γ-IFN) test to diagnose tuberculosis is to be
recommended; indeed, in a population of cattle/buffalo, there are more infected animals and than
sick animals, on account of the persistence of the disease. SICTT is a very good means of screening
1071
and must still be regarded as the reference test. On its own, however, it only provides retrospective
evidence of exposure to the infection. Its weakness lies in the manual skill it requires and in the
correct reading and interpretation of its results, on which the reliability of the test depends. Indeed,
this diagnostic technique displays drawbacks due both to the subjectivity of interpreting the result
and to false reactions caused by phenomena of hyper-reactivity and para-hetero-allergy. The γ-IFN
test is an in vita diagnostic technique based on the same principle as SICTT. However, it is carried
out in vitro and, as demonstrated by studies conducted by other authors (Cagiola et al., 2007; Wood
et al., 2001; Dondo et al., 1996; Cagiola et al., 2004), has proved to be extremely useful as a
support for the official test.

Experimentation was conducted on animals aged more than 42 days from two infected herds
and from one herd officially deemed uninfected by tuberculosis. The tests carried out were the
SICTT and γ -IFN tests (OIE Manual, 2000 IV ed., Cagiola et al., 2003); several blood samples
were taken during the same year and, with regard to the animals from the infected herds, sampling
was repeated after 1 and 2 years. The γ -IFN test was performed by means of BOVIGAM®
(Prionics AG, Schlieren-Zurich, Switzerland) kits and, on 125 samples, also ID Screen® Ruminant
IFN-γ (ID.vet, Montpellier, France) kits, in accordance with the respective manufacturers’
instructions. In the sensitization phase, 1 ml of each blood sample was stimulated with 66 µl of
Bovigam Bovine PPD at a concentration of 0.3 mg/ml and 66 µl of Bovigam Avian PPD at a
concentration of 0.3 mg/ml (Prionics AG, Schlieren-Zurich, Switzerland), in addition to 66 µl of
Recombinant Antigens ESAT6 and CFP10 (IZSLER, Brescia, Italy) premixed and utilized at a final
concentration of 4 µg/ml each. On 226 samples, sensitization was also carried out with Italian
tuberculins (Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Perugia, Italy) by
stimulating 1 ml of blood with 100 µl of bovine PPD with an activity of 5000 UI/ml and with 100
µl of avian PPD with an activity of 2500 UI/ml. The animals that tested positive on SICTT and/or γ
–IFN were slaughtered and their carcasses underwent pathology examination and, whenever
necessary, bacteriological analysis.

The blood samples from the officially TB-free animals all proved negative on SICTT and on
γ –IFN when this latter involved the use of Recombinant Antigens in the phase of in vitro
lymphocyte stimulation. By contrast, 2 samples proved positive on γ –IFN when the test was
performed after stimulation with Bovigam Bovine PPD (Table 1). This demonstrates that the γ-IFN
test also displays fairly good specificity in the buffalo species, and that this specificity is enhanced
by the use of the recombinant proteins ESAT6/CFP10, as described by other authors (Cagiola et al.,
2007; Buddle et al., 2001).
The results obtained from the two infected herds considered (A and B) are reported in tables
2 and 3, respectively. In herd A, three samples were taken in 2010 and one in 2012. The results
obtained indicate that a diagnosis of tuberculosis can be made earlier when the γ -IFN test is used
2010 tested positive on SICTT, while 58 samples did in 2012. By contrast, the -IFN test still
than when SICTT is used. Indeed, none of the samples taken during the last sampling session in
indicated positivity in the last samples taken in 2010.
match those illustrated above. The positivity values yielded by the -IFN test following stimulation
In herd B, one sample was taken in 2011 and three were taken in 2012. The results obtained
of the sample with avian PPD suggest that, for the buffalo species, the Comparative Intradermal
Tuberculin Test should be carried out. Moreover, the results concerning the use of the Italian PPD,
whenever it was possible to use these, indicate greater sensitivity of the test. Table 4 reports the
data on 125 samples from animals belonging to infected herds. These samples were analyzed by
means of BOVIGAM® and ID Screen® Ruminant IFN-γ kits. Comparison of the results obtained
reveals a good correlation between the two kits.
1072
In conclusion, while the -IFN assay provides higher sensitivity than the SICTT, the highest
figures were obtained when both techniques were applied together, as has previously emerged from
studies conducted on cattle (Llamazares et al., 1999). Moreover, our experience suggests that the
early use of both techniques in infected herds could greatly reduce the time required to eradicate the
disease.
REFERENCES
Buddle, B. M., T.J. Ryan, J.M. Pollock, P. Andersen and G.W. de Lisle. 2001. Use of ESAT-6 in
the interferon- test for diagnosis of bovine tuberculosis following skin testing. Vet.
Microbiol. 80: 37-46.
Cagiola, M., G. Severi, S. Scorcelletti, M.B. Boniotti, E. Scoccia, M. Bugatti, M. Menichelli, K.
Forti, P. Mazzone, S. Gavaudan, A. De Giuseppe, A. Duranti and C. Maresca. 2007.
Valutazione della specificità e sensibilità di antigeni secretori precoci e ricombinanti nel test
ELISA gamma-interferon. In: Proceedings of 14th Congresso Nazionale S.I.Di.L.V., Rome,
Italy.
Cagiola, M., F. Feliziani, G. Severi, P. Pasquali and D. Rutili. 2004. Analysis of possible factors
affecting the specificity of the gamma-interferon test in tuberculosis – Free cattle herds.
Clinical and Diagnostic Laboratory Immunology Sep. 11 (5):952-6.
Cagiola, M., F. Feliziani, G. Severi, M. Menichelli, P. Pasquali, D. Rutili. 2003. Impiego del
gamma-interferon negli allevamenti bovini in Umbria. In: Proceedings of 5th Congresso
Nazionale S.I.Di.L.V., Pisa, Italy. 75-76.
Decreto Legislativo del 22 maggio 1999 n.196. Attuazione della direttiva 97/12/CE che modifica e
aggiorna la direttiva 64/432/CEE relative ai problemi di polizia sanitaria in materia di
scambi intracomunitari di animali della specie bovina e suina.
Decreto Ministeriale del 15 dicembre 1995, n. 592. Regolamento concernente il piano nazionale per
la eradicazione della tubercolosi negli allevamenti bovini e bufalini. (G.U. Serie Generale, n.
125 del 30 maggio 1996).
G. Porta, P. Banchio, G. Marmo.1996. La prova del -interferone per la diagnosi della

Dondo, A., M. Goria, G. Moda, L. Cesano, A. Garanzini, M. Giammartino, G. Minola, E. Morioni,
tubercolosi bovina: determinazione della sensibilità e della specificità in prove di campo.

Medicina Veterinaria Preventiva 13: 14-18.
González Llamazares, O.R., C.B. Gutiérrez Martín, D. Alvarez Nistal, V.A. de la Puente Redondo,
L. Domínguez Rodríguez, E. F. Rodríguez Ferri. 1999. Field evaluation of the single
intradermal cervical tuberculin test and the interferon-γ assay for detection and eradication
of bovine tuberculosis in Spain. Vet. Microbiol. 70: 55-66.
OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2000. Bovine Tuberculosis.
Office International Des Epizooties, Paris, France
Smith, N.H., S.V. Gordon, R. de la Rua-Domenech, R.S. Clifton-Hadley, R.G. Hewinson. 2006.
Bottlenecks and broomsticks: the molecular evolution of M. bovis. Nat. Rev. Microbiol. 4:
670-681.
Wood, P.R. and S.L. Jones. 2001. BOVIGAM: an in vitro cellular diagnostic test for bovine
tuberculosis. Tuberculosis 147-155.
Table 1: Results of the tests carried out on 66 animals from the officially uninfected herd
(bov: bovine; avi: avian; aus: Australian)
Test Negative Positive

N. % N. %
SICTT 66 100% - -
PPD bov aus 64 97% 2 3%
PPD avi aus 66 100% - -
ESAT6/CFP10 66 100% - -
1073
Table 2: Infected herd A (*: 2010; a: 1st sample; b: 2nd sample; c: 3rd sample; # : 2012; SICTT:
Single Intradermal Tuberculin Test; Bov: bovine; Avi: avian; aus: Australian; Rec: recombinant
antigen; -- : data not available)
N° animals SICTT Bov aus Avi aus Rec Lesioned

positive positive positive positive
159*a 7 26 3 -- 6
110*b 0 23 0 14 --
148*c 0 31 4 -- --
87# 58 50 1 34 11
#
Table 3: Infected herd B (*: 2011; : 2012; a: 1st sample; b: 2nd sample; c: 3rd sample; ita: Italian; --
: data not available)
N° SICTT Bov aus Avi aus Rec Bov ita Avi ita Lesioned
capi positive positive positive positive positive positive
234* 0 72 3 45 -- -- --
362#a 72 44 46 25 -- -- 13
249#b 15 22 34 7/160 0/89 7/89 3
137#c 25 2 11 -- 6 13 0
Table 4: Comparison between the results obtained on 125 samples analyzed by means of the
BOVIGAM® and ID Screen® kits (bov: bovine; aus: Australian)
PPD bov aus PPD bov aus

BOVIGAM ID VET
Positive positive
PPD bov aus
BOVIGAM 10 8
positive
PPD bov aus
ID VET 8 13
positive
1074
Carrier Status of Foot and Mouth Disease in Ruminants through Reverse

Transcription Polymerase Chain Reaction
Mansur-ud-Din AHMADa, Muhammad USMANa, Aftab Ahmad ANJUMb, Shakera
SADIQa, Abdul REHMANa and Muhammad Hassan MUSHTAQ*a
a
Epidemiology and Public Health, University of Veterinary and Animal Sciences, Lahore,
Pakistan
b
Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan
*Corresponding email: hassan.mushtaq@uvas.edu.pk
ABSTRACT
Foot and mouth disease (FMD) is highly infectious disease of cattle, buffalo, sheep and
goats. It is caused by genus Aphthovirus of Picornaviradae family. FMDV is RNA virus having
seven serotypes A, O, C, Asia 1, SAT1, SAT2 and SAT3. Serotypes A, O, C and Asia1 are
endemic in Pakistan and causes high economic losses to livestock industry .So priority is to apply
quick and efficient methods for detection of FMDV infection and to limit the spread of outbreaks
of the disease. Although CFT, VNT and ELISA are already being used for the diagnosis of FMDV
in Pakistan but these diagnostic techniques are time consuming and their specificity and sensitivity
is low. RT-PCR for the identification of FMDV is very much sensitive and specific, can be done
within three hours after receiving of samples to the laboratory. Foot and mouth disease (FMD) in
adult sheep and goats is frequently mild or unapparent, but can cause high mortality in young
animals. The outbreaks of FMD in 1999 in Morocco, in 2001 in the United Kingdom & in 2007 in
Cyprus has highlighted the importance of sheep in the epidemiology of the disease, although there
have been numerous examples in the past where small ruminants have been responsible for the
introduction of FMD into previously disease-free countries. The difficulty in making a clinical
diagnosis should encourage the development of more rapid screening tests to assist in future
control programs. In Pakistan, no study has been conducted to depict the role of small ruminants
in the epidemiology and transmission of FMD virus to the large ruminants. Keeping in view this
neglected area of research, present study is planned to apply the sensitive and economical RT-PCR
technique for the rapid detection of carrier status of FMD virus in ruminants; and to highlight the
importance and need of vaccination to small ruminants against FMD virus in order to control
outbreaks of the disease and transmission to the large ruminants population.
Keywords: FMD, carrier status, RT PCR, Pakistan


1075
The Metaphylactic Efficacy of Toltrazuril (baycox® bovis - bayer) and Diclazuril

(vecoxan® - esteve veterinaria) in Natural Infections of Eimeria spp. in Buffalo
Calves: A Multicenter Trial in Southern Italy
Antonio BOSCO a, Laura RINALDI a, Maria Elena MORGOGLIONE a, Mirella
SANTANIELLO a, Giovanna CAPPELLI a, Ida GUARIGLIAa, Lucio NISOLIb and Giuseppe
CRINGOLIa
a
Department of Veterinary Medicine and Animal Productions, University of Naples Federico II,
Regional Center for Monitoring Parasitic Infections (CREMOPAR, Regione Campania), Naples,
Italy ; bBayer S.p.A.
ABSTRACT
The aim of the present multicenter trial was to evaluate the efficacy of toltrazuril (Baycox®
Bovis, oral suspension, 50 mg/ml) and diclazuril (Vecoxan® oral suspension, 2.5 mg/ml) for the
control of coccidiosis by Eimeria spp. in water buffaloes in order to improve the performances of
calves reared either in individual boxes or in multiple boxes. The study was conducted in 5 buffalo
farms with a known history of coccidiosis; the farms were divided into two typology (A and B) of
management system. On each farm, 36 calves aged 5 weeks, were divided at random into three
similar groups of 12 calves each. One group was treated with toltrazuril (BAY) at the dose of 15
mg/kg, the second group was treated with diclazuril (VEC) at the dose of 1 mg/kg and the third
group was remained as untreated control group (CONT). In the three buffalo farms of Typology A
the calves of the groups BAY and VEC were treated only once (at the seventh/eighth week of age);
whereas, in the two buffalo farms of typology B the calves of the treated groups were treated twice
(once at the fifth and once at the seventh/eighth week of age). In the 5 buffalo farms the average
oocyst excretion decreased significantly in both the treated groups (BAY and VEC) compared to
the CONT groups, however the BAY groups showed intensities significantly lower than the VEC
groups. The weight gains recorded fortnightly were significantly higher in the BAY groups
compared to the VEC and the CONT groups which differed slightly.
Keywords: Eimeria spp., water buffalo, toltrazuril
INTRODUCTION
In Italy, the progressive transformation of water buffalo farms (intensive breeding
techniques and constant supplies of concentrated and/or stored forages), together with the regular
use of anthelmintic treatments, has strongly contributed to the decrease in helminth infections and
to the concurrent increase in parasites having direct transmission from host to host, such the
coccidian protozoa of the genus Eimeria (Cringoli et al., 2009). In buffaloes, these infections can be
produced by several Eimeria species, having different pathogenicity. In buffalo farms of central-
southern Italy, prevalence values of coccidiosis can reach 95% on the farms and 50% on the
animals and 7 species of Eimeria have been described so far, namely E. bareillyi, E. zuernii, E.
ellipsoidalis, E. subspherica, E. auburnensis, E. bovis and E. pellita (Cringoli et al., 1996; Fusco et
al., , 1997; Cringoli et al., 2009; Rinaldi et al., 2009. Clinical and subclinical coccidiosis may
impair growth performance in buffalo calves. Therefore, the control of Eimeria infections is of
fundamental importance for the health, the welfare and the productions of buffalo calvesThe aim of
the present multicenter trial was to evaluate the parasitological (parasite burden reduction) and
zootechnical (weight gain) effect of treatments with toltrazuril (Baycox® Bovis, oral suspension, 50
mg/ml) and diclazuril (Vecoxan® oral suspension, 2.5 mg/ml) for the control of coccidiosis by
Eimeria spp. in naturally infected water buffalo calves in southern Italy.

1076

A multicenter trial was conducted from October 2011 to October 2012 in 5 buffalo farms
located in the Salerno province of southern Italy; these farms, with a known history of coccidiosis,
were divided into two typology (A and B) based on management system. In 3 farms, the buffalo
calves were bred in individual boxes from the birth to the seventh/eighth week of age and then
transferred to the ground in multiple boxes (Typology A); in the other 2 farms, the calves were
bred on the ground in multiple boxes from the birth (Typology B). On each farm, 36 calves (aged 5
weeks, only farm 1 typology A in aged 8 weeks) were divided at random into three groups of 12
calves each, similar for age, weight and number of Eimeria oocysts per gram of faeces (OPG). One
group was treated with toltrazuril (group BAY) at the dose of 15 mg/kg, the second group was
treated with diclazuril (group VEC) at the dose of 1 mg/kg, and the third group was remained as
untreated control group (group CONT). The two drugs, namely toltrazuril (Baycox® Bovis, oral
suspension, 50 mg/ml) and diclazuril (Vecoxan® oral suspension, 2.5 mg/ml), were administered by
adding them in the ration of milk taken by the buffalo calves in the morning. Two strategic
anticoccidial schemes were used based on the typology of the farm. Specifically, in the buffalo
farms of Typology A the calves of the groups BAY and VEC were treated only once, with the
respective drug, at the seventh/eighth week of age (i.e. soon before the grounding of the animals). In
the buffalo farms of typology B, the calves of the groups BAY and VEC were treated twice, with
the respective drug, once at the fifth and once at the seventh/eighth week of age. Parasitological
analysis were performed weekly on each buffalo from the fifth to the twentieth week of age.
Individual faecal oocyst counts were carried out by the FLOTAC double technique, with a
sensitivity of 2 oocysts per gram of faeces (OPG), using the Sheather’s sugar solution (specific
gravity = 1.200) (Cringoli et al., 2010). In order to sporulate and identify the Eimeria spp. oocysts,
the faecal samples were diluted using a 2.5% potassium dichromate solution, then stored in wide-
surfaced containers and kept at 26-28 °C; the samples were oxygened several times a day (de
Noronha et al., 2009). In addition, on each experimental animal, clinical signs and body weight
were recorded every two weeks.

The species of Eimeria found in the studied animals were E. ellipsoidalis, E. bovis, E.
zuernii, E. subspherica and E. bareillyi (Table 1). In the 5 buffalo farms the average oocyst
excretion decreased significantly in both the treated groups (BAY and VEC groups) compared to
the CONT group, however the BAY groups showed intensities (OPG) significantly lower than the
VEC groups (Graphic 1, 2, 3, 4, and 5). The weight gains recorded fortnightly were significantly
higher in the BAY groups compared to the VEC and the CONT groups which differed slightly
(Table 2). The majority of the treated calves did not present clinical signs but some of these animals
had mild diarrhea that manifested by soiling of tail and hind quarter, loss of appetite and
dehydration. The clinical signs were ameliorated at different periods after the onset of treatment
with the two drugs showing a complete remission of symptoms in the groups treated with
toltrazuril. In the untreated control group coccidiosis remained subclinical in most cases.
The probability for clinical disease increases with the infection pressure in the environment
and may further increase under conditions that impose stress on the calves (Marshall, 1998;
Joachim, 2002), e.g. transport, allocation to new group, inadequate feeding, other
infectious/parasitic diseases (Bohrmann, 1991). Therefore, a metaphylactic approach is advised
(especially before a stress phenomenon) through the treatment of all exposed calves before an
expected outbreak of coccidiosis. This strategic approach is the most promising tool for efficient
control as it is expected to prevent the economical losses associated with clinical disease as well as
the losses associated with subclinical coccidiosis. Strategic application of drugs should also aim at
the reduction of environmental contamination with the infectious parasitic stages thus limiting the
infection pressure to an acceptable low level. The considerable reduction of faecal oocyst shedding
observed in the calves treated, especially those treated with toltrazuril, demonstrated that the drugs
used are suitable for such approaches if applied early enough, i.e. before the onset of massive
1077
oocyst excretion. It has been assumed that from an economical point of view, subclinical
coccidiosis exceeds the economic losses caused by severe lethal coccidiosis (Fox, 1985). This
assumption is supported by the results from our study. During the 20 weeks (i.e. the study period),
the calves gained weight in all groups and at all trial sites, however at a quite diverse level. This
observation can be attributed to the different farm management system. In all five farms under
study, the calves of the groups BAY exceed those of the groups VEC in growth by 2.6–6.1 kg and
those of groups CONT by 5.3–8.1 kg at the end of the observation period. Similar findings were
also obtained in the study by Mohamed et al. (2006) in buffaloes experimentally infected by
coccidia; in this study toltrazuril was more effective than other drugs in suppressing the Eimeria
oocyst production and also as regard to the weight gain of the animals. Clinical and subclinical
coccidiosis may impair growth performance in buffalo calves that are bred in individual boxes from
the birth and then transferred to the ground and those that are bred on the ground in multiple boxes
from the birth. The Eimeria infection in buffalo calves naturally exposed was more susceptible to
metaphylactic treatment with toltrazuril that with diclazuril exhibiting a better growth response. In
conclusion, in the 5 buffalo farms the average oocyst excretion decreased significantly in both the
treated groups (BAY and VEC) compared to the CONT groups, however the BAY groups showed
intensities significantly lower than the VEC groups. The weight gains recorded fortnightly were
significantly higher in the BAY groups compared to the VEC and the CONT groups which differed
slightly. This multicenter trial demonstrates the good efficacy of toltrazuril administered orally to
buffalo calves in subclinical natural Eimeria infections in farms of southern Italy.
ACKNOWLEDGEMENTS
The Authors would like to express sincere appreciation to Dionisio Del Grosso, Michele Del
Vecchio, Mario Parrilla for the technical collaboration in the field.
REFERENCES
Bohrmann R. 1991. Treatment with toltrazuril in a natural outbreak of coccidiosis in calves. Dtsch.
tiera¨rztl. Wschr. 98: 325–364.
Daugschies, A. and M. Najdrowski. 2005. Eimeriosis in cattle: current understanding. J. Vet. Med.
B Infect. Dis. Vet. Public Heath 52: 417-427.
Daugschies A., J. Agneessens, L. Goossens, H.H. Mengel and P. Veys. 2007. The effect of a
metaphylactic treatment with diclazuril (Vecoxan®) on the oocyst excretion and growth
performance of calves exposed to a natural Eimeria infection. Vet Parasitology 149: 199-
206.
Fox J.E. 1978. Bovine coccidiosis. Mod. Vet. Pract. 59: 599–603.
Fox J.E. 1985. Coccidiosis in cattle. Mod. Vet. Pract. 66: 113–116.
Fusco G., A. Guarino, A. Merola, V. Veneziano and G. Cringoli. 1997. Natural diffusion of Eimeria
Spp. In buffalo calves. Proceeding of the 5th World Buffalo Congress 569-573.
de Noronha A.C., W.A. Starke-Buzetti and D.W. Duszynski. 2009. Eimeria spp. in Brazilian water
buffalo. J Parasitolol 95:231-4.
Cringoli G, Guarino A, Fusco G, Merola A, Negri L, Veneziano V, Bani A, Fenizia D. 1996.
Diffusion of Eimeria spp. in buffalo breeding farms in Southern Italy and epidemiologic role
of pregnant/postpartum buffaloes. Proceedings of the 2nd Asian Buff Ass Congress: 165-
173.
Cringoli G. 2006. FLOTAC, a novel apparatus for multivalent faecal egg count technique.
Parassitologia 48: 381-384.
Cringoli G, V. Musella, MP Maurelli, ME Morgoglione, A Santaniello, R Condoleo, I Guariglia, L
Rinaldi. 2009. Helminths and arthropoda in buffalo farms from the Lazio region (Italy). Vet
Res Comm 33: 129-131.
Cringoli, G. L., Rinaldi, M.P. Maurelli and J. Utzinger. 2010. FLOTAC: new multivalent technique
for qualitative and quantitative copromicroscopic diagnosis of parasites in animals and
humans. Nat Prot 5: 503-515.
1078
Guarino, A., G. Fusco, A. Bani, V. Veneziano and G. Cringoli. 1997. Eimeria Spp. In buffalo
breeding farms in southern Italy. Proceeding of the 5th World Buffalo Congress 565-567.
Joachim, A. 2002. Kokzidiose gibt es auch bei Ka¨lbern. Dlz 8: 92–94.
Marshall, R.N., J. Catchpole, J.A. Green and K.A. Webster. 1998. Bovine coccidiosis in calves
following turnout. Vet. Rec. 143:366–367.
Rinaldi L, V. Musella, V. Veneziano, R.U. Condoleo and G. Cringoli. 2009. Helmintic infections in
water buffaloes on Italian farms: a spatial analysis. Geospat Health 3: 233-239.
Table 1. Prevalence of species of Eimeria in the 5 experimental buffalo farms, southern Italy.
FARM TYPOLOGY E. ellipsoidalis E. subspherica E. bovis E. zuernii E. bareillyi
1 A 70% 10% 8% 2% 10%
2 A 11% 28% 25% 36% 0%
3 A 5% 22% 36% 13% 24%
4 B 45% 16% 20% 11% 8%
5 B 27% 13% 14% 31% 15%
Table 2. Weight increase of buffalo calves belonging the groups treated (BAY and VEC
froups) compared with the control group (CONT group).
weight increase
FARM TYPOLOGY
GROUP BAY GROUP VEC
1 A +5.8 kg +1.7 kg
2 A +8.1 kg +2 kg
3 A +6.4 kg +3.1 kg
4 B +6.6 kg +3 kg
5 B +5.4 kg +2.3 kg
Graph 1. Dynamic of the elimination of Eimeria oocysts in three experimental groups during the
course of the trial of Farm 1 – Typology A
1079
course of the trial of Farm 2 - Typology A.
Graph 3.Dynamic of the elimination of Eimeria oocysts in three experimental groups during the
course of the trial of Farm 3 - Typology A.
1080
course of the trial of Farm 4 - Typology B.
course of the trial of Farm 5 – Typology B.
1081
Gastrointestinal Parasitic Diseases of Buffaloes and Implications of

Climate Change for these Diseases in Nepal
Bhoj Raj JOSHIP* and Shubh Narayan MAHATO
National Animal Science Research Institute, Nepal Agricultural Research Council, Kathmandu,
Nepal, Pakhribas Agriculture Centre, Dhankuta, Nepal (Now: Heifer International Nepal)
*Corresponding email: bhoj.joshi@yahoo.com
ABSTRACT
Buffaloes constitute the most important farm livestock species in Nepal contributing the
most among the farm livestock to the household and national economy in terms of milk, meat,
manure and farm power. In Nepal, buffaloes are reared from tropical plains of Terai to the
temperate climate of high altitude Himalayas, which perhaps are the highest altitudes of the world
for buffalo raising. Buffalo production system in Nepal is mostly under sedentary management
system in small herds, mostly tethered in the farm household and fed on crop by-products, except in
the high altitude regions, where larger herds are reared under seasonal migration to the high
Himalayan pastures during summer months. Gastrointestinal parasitic diseases, particularly
fasciolosis, paramphistomosis and ascariasis (in calves) constitute the most prevalent and important
diseases under the sedentary management system, while the study in migratory buffaloes has not
been conducted. The point prevalence of fasciolosis and paramphistomosis in Nepal is high (more
than 50-60 percent) throughout the plains and mid hill regions of the country and even in the higher
altitudes, where forage resources are bought from the lower valleys causing significant production
losses in terms of milk yield, body weight and fertility. Similarly, the prevalence of ascariasis in
young calves has been recorded to be more than fifty percent. Fasciolosis and paramphistomosis of
buffaloes in Nepal are mainly dependent upon grazing on harvested rice fields and feeding on rice
straw, thus directly and closely associated with rice cultivation, which is practiced in the plains and
at lower elevations of the hills. As the rice fields provide an ideal and conducive environment for
parasite transmission, with the global warming, rice cultivation might be possible even at higher
elevations, thus increasing the incidences of these diseases at higher regions as well. This paper
discusses these issues and highlights the strategies for the management of intestinal parasitic
diseases of buffaloes.
Keywords: buffaloes, management system, gastrointestinal, parasitic diseases, climate change
INTRODUCTION
In Nepal, buffaloes are the most important livestock commodity having an estimated
population of 4.8 million heads (MOAD, 2011), providing food (milk and meat), manure, power for
agricultural operations and hides, bones, hairs for the industries. Buffaloes are reared by about 48.5
% of the households in the country from low plains to the high alpine pastures of Himalayan
mountains for summer grazing, which perhaps represent the highest place on earth for buffalo
rearing. Buffaloes rank the first among the farm livestock species for their contribution in terms of
milk, meat, farm power and hides (Singh and Chapagain, 1998). Buffaloes contribute about 70
percent of total milk and 64 percent of meat produced in the country confirming their importance
among the farm livestock species.
Parasitic diseases constitute the important most problem of buffaloes reared throughout the
country. Fasciolosis, paramphistomosis and ascariasis are among the major gastrointestinal parasites
of buffaloes recorded in the country. The most commonly observed disease condition due to
Fasciola infection is the chronic fasciolosis leading losses in milk and meat production, poor
appetite, body condition and fertility. The other noticeable symptom observed is soil eating by the
infected buffaloes thus the disease condition being named as Mate (soil eating) by the farmers. The
disease condition due to paramphistomiasis alone has not been studied, however, under the field
1082
conditions; the infection of Paramphistomes is almost invariably mixed with Fasciola infection,
thus, all disease symptoms are attributed to Fasciola infection. The ascariasis infection is primarily
limited to young calves and is the only parasite which is obvious to the farmers, thus several
traditional medications have been practiced in different parts of the country. The commonly
observed symptoms of ascariasis in calves are ill-thriftiness; poor growth, pot bellied and death,
though, exact data on mortality are not available. Research studies on gastrointestinal parasites of
buffaloes were mostly conducted for fasciolosis with few studies on paramphistomiasis and
ascariasis. This paper presents the current state of knowledge on these diseases in the country and
suggests the future scenario in light of the global warming and climate change.

Epidemiological studies
Prevalence studies
Prevalence studies were carried out in different parts of the country by examining the faecal
samples collected randomly for the presence of Fasciola and Paramphistome eggs mostly by
sedimentation method (MAFF, 1984), except Mahato (1993), who used differential flotation
method (MAFF, 1984) using saturated ZnSo4 solution for flotation of eggs. These studies were
further supported by examination of the livers of slaughtered buffaloes during different months of
the year at different locations of the country for evaluating the Fasciola burden and the
developmental stage of flukes in the slaughtered buffaloes. Prevalence of ascariasis was also carried
out by collection and examination of faeces by flotation method (MAFF, 1984) of new-borne
buffalo calves at regular intervals from 15 day old to six months.
Snail habitat survey
Snail habitat and ecology was studied by monthly visit of the selected habitats in the hills
and terai to record the snail population density, dynamics and distribution. The population density
was expressed as per 5 man minute. The collected snails were preserved either in 10% v/v formalin
or 70% v/v ethanol before laboratory examination.
Evaluation of rice straw as a source of metacercaria
Rice straw, which constitute the most important forage source for buffaloes was
experimentally evaluated for contamination of Fasciola metacercaria by feeding of rice-straw for a
period of two to five months in indoor housed buffalo calves with a control group fed on tree fodder
followed by slaughter adopting standard humane procedures and examination of the livers (MAFF,
1984) to detect the presence of Fasciola in the slaughtered animals. The recovered flukes were
collected and examined for their size and shape.
Parasite studies
Identification of the species of Fasciola was carried out in the adult Fasciola parasite
specimens collected from the slaughtered buffaloes by morphometric measurements. Representative
samples of the identified parasites were sent to reference laboratories for reconfirmation of the
identification. The species identification carried out on adult parasites was further validated by
measurement of eggs for species differentiation using stage micrometer of the compound
microscope.

Prevalence of fasciolosis and evaluation of forage source for Fasciola metacercarias
The prevalence studies carried out in different parts of the country showed that fasciolosis in
buffaloes was more than 50 percent of the population in most parts of the country (Table 1) even at
high altitude regions of the hills. However, in some high hills, where the forage resource was not
obtained from the rice field, the prevalence rate was only about 10 percent, in the animals purchased
from the lower valleys. This finding further supports that under the existing management system,
fasciolosis in Nepal is very closely dependent upon rice cultivation and forages obtained from these
fields. This fact was further supported by the finding that buffaloes managed under the stall fed
system on rice straw showed higher prevalence than the grazed animals (Table 2). Two
1083
experimental studies (Joshi, 1986, 1989) carried out to evaluate the contamination of rice straw
with Fasciola metacercaria further supported the fact that rice straw feeding was one of the major
cause of Fasciola transmission in stall-fed buffaloes (Table 3). This study was further supported by
the findings of Mahato and Harrison, (2005) that the water submerged parts of the straw was only
contaminated with metacercaria and the upper parts of the straw was free from contamination.
No specific studies have been carried out to determine the prevalence of paramphistomiasis
in buffaloes, however, under the field conditions; the common observation has been the mixed
infection of both parasites in almost all cases. Prevalence of ascariasis in new-borne buffalo calves
was recorded to be 57 percent in hill environment (Joshi and Ghimire, 1993), however, no studies
have been conducted so far for the tropical Terai environment.
Intermediate snail hosts and their distribution
Four Lymnaea spp were identified in the hills and terai regions of eastern Nepal, while other
parts of the country have not been surveyed so far. The intermediate snail hosts for Fasciola
identified were Lymnaea auricularia race rufescens, L auricularia sensu stricto, L. viridis, and for
Paramphistomes were L. luteola Planorbis and Indoplanorbis exaustus. Among the habitats studied,
rice fields and ditches along the road side, permanent spring fed rice fields with slow flowing water
were the major habitats for the snails, while the river banks, stagnant marshy ditches with red
flocculent precipitates on the bottom, big water hole were not found to be suitable habitats for the
snails. Altitude wise, the fields around 1500-1600 meter above sea level (masl) with permanent
spring irrigation were found to be with high concentration of snail population.
Fasciola burdens in the slaughtered buffaloes
Mahato (1993) examined the livers of slaughtered buffaloes for seasonal variation of fluke
burdens (Table 4) and its relation with altitude, age and sex of buffaloes (Table 5). These studies
showed that 80 percent of the slaughtered buffaloes were infected with Fasciola infection and the
mean fluke burden was around 200-350 flukes with the recorded highest burden of 2071 flukes in
one buffalo. The flukes recovered were of adult stage except during the month of January, which
indicates that infection was acquired during October-November months, which is the rice
harvesting season and animals are fed on forages collected from rice fields and/or grazed on the
crop harvested paddy fields.
Species of Fasciola and Paramphistomes infecting the animals
Mahato (1993) studied the Fasciola recovered from slaughtered animals by morphometric
measurement and identified the fluke species as Fasciola gigantica, F. hepatica and intermediate
form. He further measured the egg size of these parasites and recorded considerable variation in the
size of the eggs recovered from these three species (Table 6). It was also recorded that in most of
the slaughtered animals from the hills, only F. gigantica was the predominant species recovered
with a low proportion of F.gigantica and intermediate form mixture (Table 7). Singh et al (1973)
reported four species of Paramphistome infecting the animals in Nepal. These are Cotylophoron
cotylophorum, Gastrothylax crumenifer, Fischoedirus crumenifer and Gigantocotyle explanatum,
however, it could be considered that they might not have included the representative parasite
samples from all over the country.
Implication of climate change on Fasciola and Paramphistome infection in Nepal
Global circulation model (GCM) projections indicate that the temperature over Nepal will
increase between 0.5ºC and 2.0ºC with a multi-model mean of 1.4ºC and a wide range of
precipitation changes especially during the monsoon from a decrease of 14% to an increase of 40%
by the 2030s. On the ground, perceptions of farmers suggest that precipitation is growing more
erratic, days are becoming hotter, the pattern of winds, fog and hailstorms have been altered and
that farmers are becoming more vulnerable (Dixit, 2011). The implication of these climatic changes
would likely be on expansion of rice cultivation areas to the higher elevations of the hills, and thus
expansion of snail habitats in new areas for the transmission of Fasciola and Paramphistome
parasites even at the higher altitude regions of the country and the increased prevalence of these
parasitic diseases in the wider population of farm livestock.
Management of gastrointestinal parasitic diseases
1084
In Nepal, management of fasciolosis, paramphistomosis in farm animals is mostly related to

feeding of forage resources obtained from the rice fields and grazing on these fields after the rice
harvest. This has become even more crucial because, the open pasture area is very limited and the
rain fall is mostly concentrated during monsoon months (June-September) with dry environment
during rest of the year. Thus the management of these diseases could be possible with strategic
anthelmintic treatment taking in to account the feeding of forage resources obtained from these rice
fields. For the management of ascariasis, the strategic treatment of anthelmintics during early age
could provide adequate protection throughout the life.
REFERENCES
Dixit, Ajaya. 2011. Climate Change in Nepal: Impacts and adaptive strategies. World resources
Report. Institution for Social and environmental transformation-Nepal.
Joshi, B.R. 1989. A study on rice-straw feeding to evaluate its role in Fasciola (liverfluke) infection
in Nepalese farm animals. In: Livestock Production in the Tropics (eds. H. Kuil, R.W.
Paling and J.E. Huhn). Proceedings of the sixth International Conference of Institutes for
Tropical Veterinary Medicines, 28 Aug.-1 Sept, 1989, Wageningen, The Netherlands. Pp-
354-357.
Joshi, B.R and S.C. Ghimire. 1993. Strategic piperazine drenching against Toxocara vitulorum
infection of buffalo calves. Proceedings of the 4th National Veterinary Conference of Nepal
Veterinary Association, 17-19 Nov, 1992. 21-23, Pp 91-96.
Joshi, B.R. 1986. Evaluation of rice-straw as a potential source for Fasciola infection of ruminants
in Nepal. Lumle Agriculture Centre, Technical paper no 14/86.
Joshi, B.R. 1988. Prevalence of Fascioliasis (Liverfluke) in cattle and buffaloes in the mid-western
hills of Nepal. Journal of Institute of Agriculture and Animal Science 9: 111-114.
Joshi, B.R. and S.C. Ghimire. 1993-95. Strategic piperazine drenching against Toxocara vitulorum
infection in buffalo calves. Proceedings of the 4th National Veterinary Conference of Nepal
Veterinary Association, 17-19 November, 1992, 21-23, pp 91-96.
MAFF. 1984. Manual of Parasitological Laboratory Techniques, Ministry of Agriculture, Fisheries
and Food, United Kingdom.
Mahato, S.N. and L.J.S. Harrison 2005. Control of Fasciolosis in stall fed buffaloes by managing
the feeding of rice-straw. Tropical animal Health and Production 37: 285-291.
Mahato, S.N. 199). Epidemiology and Pathogenesis of fasciolosis in Eastern Nepal. Ph.D. Thesis,
University of Edinburgh.
MOAD. 2012. Livestock statistics, Ministry of Agriculture Development, Government of Nepal.
Singh, N.B., B.M. Basnyat, G.Eichenberger,and W.Bommmeli 1973. Report on Preparatory Phase
of Parasite Control Project, HMG/SATA, Kathmandu, Nepal.
Singh, S.B. and D.P. Chapagain. 1998. Livestock sector in the Agriculture Perspective Plan. In:
Proceedings of the first national workshop on animal genetic resources conservation and
genetic improvement of domestic animals in Nepal- April 11-13, 1994 (Ed. J.N.B.
Shrestha). Nepal Agricultural Research Council, Khumaltar, Nepal. pp. 117-128.
1085
Table 1. Prevalence of fasciolosis in buffaloes at different altitudes of the hills.
Altitude Prevalence Percent- Western Prevalence Percent-

hills Eastern hills
(Joshi,1988) (Mahato,1993)
Low hill (< 1000 masl) 79-92 64

Mid hill (1000-1500 masl) 78-85 70
High hill (at 2200 masl) 10-70 42
Table 2. Prevalence of fasciolosis in buffaloes under stall fed/grazing managements in the eastern
hills.
Management system Prevalence percent

Stall-fed 69.4
Grazing 47.5
Table 3. Number of Fasciola recovered from rice-straw fed buffalo calves in in-housed study.
Groups Mean number of Fasciola recovered Range

First study Second study First study Second study
Feeding period 2 months 5 months
Rice straw fed 13.5 ± 4.87 19.71 ± 4.8 7-28 8-46
Control 0.0 0.0 0.0 0.0
Table 4. Mean fluke burden of slaughtered buffaloes from hills during different months.
Months Number of livers Mean burden Mature Flukes (%)

examined
August 9 179.4± 44.4 100
September 14 223.9± 57.6 100
October 6 91.2 ± 29.6 100
November 7 234.8± 57.1 100
December 8 148.8±46.1 100
January 12 326.0±155.8 45.7
February 11 221.4±33.1 100
March 10 218.1±39.9 93.5
April 17 147.5±37.2 80.9
May 18 175.4±28.0 99
June 11 120.9±37.7 99
July 8 99.9±36.4 97
1086
Table 5. Mean burdens of Fasciola species at different altitudes, age and sex of buffaloes.
Variable Group Number of Fluke Burdens Statistics

livers Range Mean SE
examined
Area High hill 6 9-550 247.0 88.1 P=0.05
Mid hill 65 1-2071 218.5 38.4
Low hill 62 2-771 167.1 19.1
Terai 30 22-500 235.6 23.0
Age 1-2 26 1-597 77.1 24.1 P=0.0001
(Years) 3-4 18 13-550 144.3 28.5
5-6 16 30-771 260.1 53.2
7-8 30 6-760 185.6 30.1
9-10 39 48-787 242.7 23.3
11& over 34 3-2071 274.1 63.8
Sex Male 63 1-597 138.0 16.9 P=0.005
Female 100 3-2071 244.1 26.3
Overall 163 1-2071 203.2 17.9
Table 6. Mean body size and egg size of F.gigantica, F.hepatica and intermediate form.
Fasciola spp Body size (mm) Egg length (µm) Egg width (µm)
Length Width Range Mean SE Range Mean SE
F. hepatica 22.9-35.5 9.8-13.5 107.1-166.6 139.9 1.4 59.5-106.0 82.9 1.7
Intermediate 28.5-39.8 9.2-11.3 121.2-175.7 153.1 1.1 71.4-136.4 93.9 1.4
form
F.gigantica 28.8-63.6 7.2-11.1 139.4-224.2 170.5 1.4 78.8-145.4 115.4 1.5
Table 7. Species of Fasciola recovered in slaughtered buffaloes.
Number of liver F.gigantica only F. hepatica only F.gigantica F.gigantica and

examined and F. hepatica intermediate form
111 102 (92%) 0 0 9 (8%)
1087
Detection of bovine viral diarrhea virus prevalent in dairy herds of Punjab,

Pakistan
Humayun GOHARa, Masood RABBANIa*, Arfan AHMADa, Nasim AHMADb, Ali Ahmad
SHEIKHa and Khushi MUHAMMADc
a
University Diagnostic Laboratory (UDL). University of Veterinary & Animal Sciences, Lahore, 54000,
Pakistan.
b
Department of Theriogenology, University of Veterinary & Animal Sciences, Lahore, 54000, Pakistan.
c
Department of Microbiology, University of Veterinary & Animal Sciences, Lahore, 54000, Pakistan
*Corresponding email: mrabbani@uvas.edu.pk
ABSTRACT
Bovine viral diarrhea (BVD) virus is a positive sense RNA virus having genome of
approximately 12.3 kb. in length and is one of the most insidious and ubiquitous virus of bovines
throughout the world. Isolation and identification of BVD virus on cell culture is labour intensive.
Other diagnostic techniques used are not cost effectiveness. Therefore in this study an antigen-capture
ELISA was used to detect BVDV in dairy animals at selected areas of Punjab and to evaluate
comparative prevalence of the virus in cattle and buffaloes. Out of the test blood samples (n=184)
originated from public (n=4) and private sector (n=3) dairy farms, 16.85% cattle and 6.31% buffalo
were positive for BVD virus. Prevalence of the virus on public farms was significantly higher than that
of private farms (p<0.05). The virus was undetectable in all samples of Friesian breed (0%) whereas
prevalence was the highest (50%) was in cattle of Sahiwal breed. Overall prevalence of BVD in buffalo
was lower than that of cattle population. It is concluded that BVD virus is prevalent in dairy herds of
Punjab, Pakistan therefore, a comprehensive screening program along with biosecurity measures may
be initiated to minimize its losses and further spread to other herds.
Keywords: antigen capture ELISA, BVDV, cattle, buffalo
INTRODUCTION
In Pakistan, livestock contributes approximately 45 million tons of milk and is the 3rd largest
milk producing country in the world. The major problem for small dairy farms is the dismal milk
productivity of Pakistani cattle and buffalos. One of the reasons of this low production potential of
Pakistan animals is involvement of infectious agents including bacteria, viruses and protozoans, etc.
Among the viruses, Bovine viral diarrhea (BVD) virus is believed to be a silent killer of dairy animals
in the world. It is a positive-strand RNA virus having genome of approximately 12.3 kb in length and is
one of the most insidious and ubiquitous pathogens of dairy animals throughout the world. Multiple
clinical manifestations including abortion, repeat breeding problems, congenital defects and fetal
mummification have been associated with the virus (Barlow et al., 1986; Baker, 1987; Bolin et al.,
1987). Nowadays, trend of modern dairy farming is common in Pakistan. Many livestock producers
are importing exotic dairy animals without testing their BVDV status which could lead to spread of this
malady to other herd mates. BVD virus infection is common in exporting countries; therefore, they are
recommending vaccination against BVDV; however main hindrance is cost effectiveness of vaccines
and unknown BVDV status in animals due to unavailability of diagnostic facilities. Unfortunately little
attention has been paid until now to investigate the involvement of BVD virus in productive and
reproductive losses in dairy animals of Pakistan. Present study, therefore, was undertaken to investigate
the status BVDV in dairy animals of Punjab, Pakistan.

1088

A total of 184 bovine blood samples originated from Sahiwal, crossbred cattle and Nili Ravi
buffalo were collected from 7 dairy farms in Punjab. The dairy farms include four public (MF, BF, LP,
and RG) and three private sector farms (RDF, SDF, and LDPF). Complete history of animals like milk
production, age, sex, breed, abortion and BVDV vaccine, etc. was also noted. After collection of blood,
sera were separated and transported to University Diagnostic Laboratory (UDL), University of
Veterinary and Animal Sciences, Lahore in cold chain for further processing.
Confirmation of BVDV in collected samples
For confirmation of BVD virus, all the collected samples were subjected to commercial antigen-
capture ELISA kit (IDEXX) as per manufacturer’s instructions. The suspected samples were assayed
twice, first with standard working detector reagent and then with a modified working detector reagent
as per manufacturer’s recommendations.

BVD virus is one of the major economic threats for dairy industry all over the World (Baker,
1987; Barlow et al., 1986; Bolin et al.,1987). In this study prevalence of Bovine viral diarrhea (BVD)
virus was determined using antigen-capture ELISA on government and private dairy farms located in
Punjab, Pakistan. Out of 184 test animals, twenty one (11.41%) was found positive for BVD virus
(Table1).The prevalence observed in this study did not match with already reported prevalence of
BVD virus in Europian countries (0.75% to 1.4% )(O'Neill et al., 2009). The possible reason of low
prevalence in other countries might be due to use of good management practices, availability of quality
fodder, implementation of routine vaccination and BVD virus control programs by elimination of
persistently infected animals with virus. Regarding farm wise prevalence of BVD virus, higher
positivity was observed in public sector dairy farms (50% at LP farm, farm followed by 15% at MF) as
compared to farms of private sector (SDF and 10 % at LDPF) (Table 1). The high positivity at Govt.
farms might be due to lack of routine herd screening and vaccination practices. Furthermore one of the
other reasons may be due to availability of insufficient funds for diagnosis of diseases which lead to
their spread to herd mates. In this study prevalence of the disease in cattle was also compared with
buffaloes. Higher positivity percentage was recorded in cattle (16.85%) as compared to buffaloes
(Table 1). This finding is line with the previously reported observations of few researchers that BVD
infection is most common in cattle than in buffaloes (Akhtar and Asif, 1996; Zaghawa, 1998). The
prevalence observed in cattle is almost similar with already reported prevalence of BVD virus in Greek
cattle herds (14%) (Billinis et al., 2005). Regarding correlation of BVD virus with history of abortion,
out of twenty one positive animals, three (n=3/4, 75%) Friesian X Local crossbred cattle and two Nili
Ravi buffaloes (2/6, 33.3%) were found with history of abortion (Table-2). Such findings have already
been reported by many researchers that BVD may cause abortion in infected dams depending upon
their gestation age and immune status (Bielefeldt-Ohmann, 1995). This study also depicted more
positivity of BVD virus in Sahiwal cattle breed (6/12, 50%) as compared to Friesian X Local (4/30,
13.3%) and Friesian X Sahiwal crossbred cattle (5/43, 12.8%) (Table-2). High positivity in Sahiwal
cattle as compared to crossbred animals may be due to the reasons that BVDV vaccines are not being
applied in Pakistan whereas exotic animals are mostly vaccinated and have with good immune status.
In this study no pure breed Friesian cattle were found positive with BVDV (Table-2). In order to
investigate the reason about freeing of Friesian cattle from BVDV, there is a need to establish detailed
study to rule out its actual reason. Based on this study it is concluded that BVD virus is prevalent in
dairy animals of Punjab, Pakistan and it needs to be addressed at the earliest to control its spread to
other healthy animals.
1089
REFERENCES
Akhtar, S. and M. Asif. 1996. Epidemiologic association between antibody titres against bovine virus
diarrhoea virus, rinderpest disease virus and infectious bovine rhinotracheitis virus in a buffalo
herd. Tropical Animal Health and Production 28: 207-212.
Baker, J.C. 1987. Bovine viral diarrhea virus: a review. J. Am. Vet. Assoc. 190: 1449-1458.
Barlow, R.M, P.F. Nettleton, A.C. Gardiner, A. Greig, J.R. Campbell, and J.M. Bonn. 1986. Persistent
bovine virus diarrhea virus infection in a bull. Vet. Rec. 118, 321-324.
Bielefeldt-Ohmann, H. 1995. The pathologies of bovine viral diarrhea virus infection. A window on the
pathogenesis. Veterinary Clinics of North America. Food Animal Practice11: 447-76
Billinis, C., L. Leontides, G.S. Amiridis, V. Spyrou, P. Kostoulas and M. Sofia. 2005. Prevalence of
BVDV infection in Greek dairy herds. Prev. Vet. Med. 72:75-79
Bolin, S.R., J.A. Roth, E.K. Uhlenhopp and J.F. Pohlenz. 1987. Immunologic and virologic findings in
a bull chronically infected with noncytopathic bovine viral diarrhoea virus. J. Am. Vet. Assoc.
190: 1015-1017.
O'Neill R., B. Wilson, C. Regan, E. Connaghan and J. Mooney. 2009. Patterns of BVD virus in
laboratory submissions. Ir Vet J. 62:679-683
Zaghawa, A. 1998. Prevalence of antibodies to bovine viral diarrhea virus and/or border disease virus
in domestic rumiants. Zentralblatt fu¨r Veterina¨rmedizin 45: 345-351
Table1. Prevalence of BVDV in cattle and Buffaloes
Farms ID Positive Animals(Cattle) Positive Animals(Buffaloes)

LP 6/12 (50%) 1/14 (7.1%)
MF 3/20 (15%) 0/11 (0%)
LDPF 1/10 (10%) -
RDF 0/4 (0%) -
SDF 5/43 (11.6%) 4/48 (8.3%)
RG - 1/5 (20%)
BF - 0/17 (0%)
Total 15/89 (16.85%) 6/95 (6.3%)
Table 2. Breed wise prevalence of BVDV and correlation with history of abortion
Breed of animal BVDV Positivity Animals with history of abortion

FRIESIAN 0/4 (0%) 0/4 (0%)
FRIESIAN X SAHIWAL 5/43 (12.8%) 0/5 (0%)
FRIESIAN X LOCAL 4/30 (13.3%) 3/4 (75%)
SAHIWAL 6/12 (50%) 0/6 (0%)
NILI RAVI 6/95 (6.3%) 2/6 (33.3%)
Total 21/184 (11.4%) 21(23.8%)
1090
Clinical Evaluation of Hypertonic Saline Solution for Treatment of Lactic

Acidosis in Water Buffaloes
Frederico Augusto Mazzocca Lopes RODRIGUESa*, Antonio Humberto Hamad
MINERVINOb, Raimundo Alves BARRÊTO-JÚNIORc, Carolina Akiko Sato Cabral de
ARAÚJOa, Francisco Leonardo Costa de OLIVEIRAa, Rodrigo Nogueira Fernandes
FERREIRAa, Thales dos Anjos Faria VECHIATOa, Clara Satsuki MORIa, Enrico Lippi
ORTOLANIa
a
School of Veterinary Science, University of São Paulo, 05508-270, São Paulo-SP, Brazil
b
Institute of Biodiversity and Forest, Federal University of Western Pará, 68035-110, Santarém-
PA, Brazil
c
Institute of Animal Science, Federal Rural University of the Semiarid, 52695-900, Mossoró-RN,
Brazil.
*Corresponding e-mail: fmazzocca@usp.br
ABSTRACT
Hypertonic saline solution (HSS) is known as an important treatment for hypovolemic
shock. Buffaloes with acute ruminal lactic acidosis (ARLA) usually present different degrees of
dehydration. This study evaluated the efficiency of HSS for treatment of ARLA in water buffaloes.
Twelve yearling, male Murrah buffalos were used. After an adaptation period, when a rumen
cannula was implanted, the animals were subjected to induction of ARLA through administration of
sucrose into the rumen in accordance with the classical model for cattle, but with a 15% reduction
in the indicated amount of sucrose. Twenty hours later, the animals were randomly divided into two
groups. The first group received 5 mL/kg of body weight (BW) of HSS (7.5% NaCl solution)
within 15 min, followed by 20 mL/kg BW of isotonic saline solution (ISS) over the next 165 min
(the authors should state the route (s) of treatment). The second group was treated only with ISS (5
mL/kg over 15 min and 20 mL/kg over 165 min). Five liters of ruminal contents were removed and
replaced with five liters of water in both groups. Clinical examinations were performed and blood
samples were taken at the baseline time (M0), 20 hours after induction (M20h) and throughout the
treatment, after 30, 60, 120 and 180 minutes (M30', M60', M120' and M180', respectively). Blood
samples were used for blood gas analysis (ABL-5, Radiometer, Copenhagen, Denmark) and
globular volume (GV) determination. Statistical analysis was done using repeated-measure two-way
ANOVA with Dunnett's multiple comparisons test. HSS caused mild acidemia followed by slight
hypercapnia (at which time point?). No side effects were observed in buffaloes treated with HSS, as
has previously been seen among cattle. Mean GV in both groups rose from 33% at M0 to 38% at
M20h (p < 0.05), without any difference between the groups, thus indicating that dehydration due to
lactic acidosis occurred similarly in both groups. The blood pH decreased from 7.357 at M0 to
7.288 at M20h (P <0.05) for the ISS group and from 7.372 to 7.301 for the HSS group at same
times, without any difference between the experimental groups. The GV values for the HSS group
at M0 and M20h were 34 and 39% and for ISS group, 32 and 36%, respectively. At M30', the HSS
group present a significant decrease in GV, reaching values of 26% and differing (P < 0.05) from
the ISS group (33%). After the treatment, at M180', the pH was 7.295 for the ISS group and 7.323
for HSS, also without any difference (P < 0.05). Infusion of HSS decreased the globular volume,
thus indicating that fluids were passing from the rumen into the bloodstream and correcting the
dehydration. Use of HSS is a possible additional treatment for correction of dehydration caused by
ARLA in water buffaloes, with plasma volume expansion due to water passage from the rumen to
the blood stream.
Keyword: globular volume, blood pH, dehydration, HSS.


1091
Influence of Storage Temperature on Blood Gas Analyses on Buffalo Venous

Blood
Antonio Humberto Hamad MINERVINOa*, Frederico Augusto Mazzocca Lopes
RODRIGUESb, Carolina Akiko Sato Cabral de ARAÚJOb, Rejane Santos SOUSAb, Clara
Satsuki MORIb, Raimundo Alves BARRÊTO-JÚNIORc, Adriana Caroprezo MORINIa,
Felipe Nascimento STELMACHTCHUKa and Enrico Lippi ORTOLANI b
a
PA, Brazil.
b
School of Veterinary Science, University of São Paulo, 05508-270, São Paulo-SP, Brazil.
c
Brazil.
*corresponding e-mail: ah.minervino@gmail.com
ABSTRACT
With the objective of determining the effects of storage temperature over time on blood gas
analyses on water buffalo, six 16-month-old male Murrah buffalos were used. From each animal,
duplicated samples were collected from the jugular vein, using disposable needles and 5 mL plastic
syringes containing around 1,000 IU of sodium heparin. Immediately after venipuncture, the tip of
the needle was sealed with a rubber stopper in order to prevent gas from moving into or out of it.
Unpreserved samples were kept at room temperature (between 22 and 26 ºC), and those to be kept
at refrigeration temperatures were placed in a cooler containing four liters of cold water and four
kilograms of ice, in order to keep the temperatures between 1 and 4 ºC. Blood gas analyses were
carried out immediately after collection and after 1, 2, 3, 4, 5, 6, 8, 10, 12 and 24 hours. The
samples were analyzed in an automatic blood gas apparatus (ABL-5, Radiometer, Copenhagen,
Denmark). This measured pH and the partial pressures of oxygen (PO2) and carbon dioxide (PCO2)
by means of an electrode system, and automatically calculated plasma bicarbonate concentration
(HCO3), base excess (BE) and oxygen saturation in blood (SO2). After the blood aliquot had been
placed in the blood gas analyzer, the rectal temperature values were corrected for each animal
evaluated. The results were analyzed by means of repeated-measure two-way ANOVA followed by
the Bonferroni test, taking into consideration the effects of the storage temperature and time until
analysis. Regarding pH, the time until analysis caused a decrease among the samples stored at room
temperature. We found a difference between the refrigerated and room temperature results, 10 and
12 hours (P < 0.01) and 24 hours (P < 0.0001) after blood collection. Unrefrigerated samples were
different from the baseline at 8, 10, 12 and 24 hours (P < 0.01). The pH remained stable, even 24
hours after the baseline, among the refrigerated samples, with a numerical increase of 0.2% (7.367
at baseline and 7.351 at 24th hour). Regarding PO2 and PCO2, it was observed that, besides a
continuous increase over time, the refrigerated samples did not present any difference from the
baseline. However, the room temperature samples showed a significant difference starting at the
10th hour for PO2 (P < 0.05) and at the 8th hour for PCO2 (P < 0.01). The refrigerated and
unrefrigerated samples were significant different (P < 0.05) at 12 and 24 hours for both PO2 and
PCO2. HCO3 remained stable throughout the study period and did not present any difference in
relation to either the time or the storage temperature. The BE in the unrefrigerated samples
decreased (P < 0.05) from the baseline values from the 6th until the 24th hour. SO2 also remained
stable (P > 0.05), but with a numerical increase of 15% even in the refrigerated group. The variables
SO2 and BE exhibited greater discrepancy in relation to the baseline, while pH and HCO3 were
more stable. It was concluded, therefore, that blood gas analysis on buffalo venous blood may be
safely carried out up to 24 hours after blood collection, if the samples are kept under adequate
refrigeration.
Keywords: blood pH, PCO2, PO2, temperature, venous blood
1092
Experimental Induction of Lactic Acidosis in Buffaloes with Sucrose

Raimundo Alves BARRÊTO-JÚNIORa*, Antonio Humberto Hamad MINERVINOb,
Frederico Augusto Mazzocca Lopes RODRIGUESc, Carolina Akiko Sato Cabral de
ARAÚJOc, Francisco Leonardo Costa de OLIVEIRAc, Rejane Santos SOUSAc, Leonardo
Frasson dos REISc; Clara Satsuki MORIc, Enrico Lippi ORTOLANIc
a
Brazil. bInstitute of Biodiversity and Forest, Federal University of Western Pará, 68035-110,
Santarém-PA, Brazil. cSchool of Veterinary Science, University of São Paulo, 05508-270, São
Paulo-SP, Brazil.
*corresponding e-mail: barreto@ufersa.edu.br
ABSTRACT
Experimental induction of lactic acidosis in non-adapted ruminants is the best model for
studying intrinsic mechanisms of pathogenesis or for evaluating treatments and prophylactic
measures. Different types and amounts of grains and carbohydrates have been used to induce this
condition, but the sucrose model has been used safely with satisfactory results in cattle. The
objective of this study was to validate the use of sucrose to induce mild lactic acidosis in buffaloes
to obtain a standard outcome based on ruminal fluid pH. Twelve yearling, male Murrah buffaloes
were used. After an adaptation period, a rumen cannula was implanted. The animals were subjected
to induction of lactic acidosis through administration of sucrose into the rumen in accordance with
the classical model for cattle, using the corrected metabolic weight (MW). Thus, the formula used
was: amount of sucrose = 1057 + 43.1*MW; but with a reduction of 15% in the amount of sucrose,
in order to avoid extreme acute acidosis and risk of death to the animals. Clinical examinations
were performed at the baseline (M0) and 8, 15 and 20 hours after induction (M8, M15 and M20,
respectively). At the same times, blood and ruminal content samples were collected. Blood samples
were used for blood gas analysis (ABL-5, Radiometer, Copenhagen, Denmark) and ruminal
samples for pH determination (PG 1800, Gehaka, São Paulo, Brazil). Statistical analysis was done
using the paired T test. In all the buffaloes, rumen lactic acidosis was successfully induced with the
rumen fluid pH ranging from 4.20 to 4.72 at M20. We used ruminal pH lower than 5.0 as the
threshold to consider an animal with ruminal lactic acidosis. In general, buffaloes presented
anorexia, dehydration, apathy and sternal recumbency. Rumen pH differed (P < 0.05) from M0
(6.9) at M8 (4.7), M15 (4.6) and M20 (4.5). Rumen movements decreased from M0 to every other
time and were absent in all buffaloes at M20. In most cases (9/12), the buffaloes presented watery
diarrhea. Heart and respiratory rates did not change over the experiment and the mean remained
within the normal reference range for the species. The rectal temperature increased from M0 (38.0)
to M20 (39.3) (P < 0.05). Fluid rumen content was observed in every buffalo. The mean blood pH
decreased from 7.365 at M0 to 7.295 at M20 (P < 0.05). The partial pressure of O2 rose from 34 to
44 mmHg and the partial pressure of CO2 decreased from 51 to 43 mmHg at the same times (P >
0.05). After M20, all the animals were properly treated and returned to normal within 24 hours. The
experimental model successfully produced rumen lactic acidosis in all buffaloes and can be used
safely in this species.
Keywords: Murrah, acidosis, rumen pH, model


1093
Mineral Status of Buffaloes Raised in the Wetlands of the Lower Amazon Basin
Ecosystem
Antonio Humberto Hamad MINERVINOa, Elyzabeth da Cruz CARDOSOb, Wellington
Cunha MORENOb, Ermino BRAGAb, Enrico Lippi ORTOLANIc*
a
PA, Brazil
b
Federal Rural University of the Amazon (UFRA), 66077-901, Belém-PA, Brazil
c
School of Veterinary Science, University of São Paulo, 05508-270, São Paulo-SP, Brazil
*corresponding e-mail: ortolani@usp.br
ABSTRACT
Floodplain ecosystems have great economic importance for the livestock industry in the
lower Amazon basin, since they are used for cattle and buffalo production. In this, the large areas of
native pastures on the margins of the Amazon River are used as soon as they have dried out after
the last flood, taking advantage of the nutrient-rich soil left behind by the river. In such ecosystems,
the animals are raised extensively without additional mineral supplementation. Thus, the present
study aimed to evaluate the performance and mineral status of buffaloes in the floodplain
ecosystems of the Amazon basin. A total of 50 buffalo calves of the Mediterranean breed, with an
average age of 9 months and weighing 202 kg, were used. At the beginning of the experiment, these
animals were weaned and transported to a typical lowland area of the Amazon region in the dry
season (i.e. after the rivers started to decrease in volume), with native pastures. The animals
remained without receiving mineral supplementation until the end of the study (duration of 168
days). They were weighed at baseline and every 28 days. From 10 randomly selected buffaloes,
blood and bone samples were taken at the beginning and end of the study for mineral analysis. The
bone samples were obtained throughout rib biopsy. Calcium (Ca), phosphorus (P), magnesium,
copper, zinc, cobalt, iron and manganese were determined from blood and ash density. Ca and P
were evaluated on bone samples. The average daily weight gain (ADWG) calculated during the
study was 0.422 kg/day, and there was significant evolution of the ADWG over the first two
months (-0.167 and 0.235 kg/day) and the last months of the study (reaching 0.735 kg/day). The
mean bone mineral analyses obtained in the beginning and end of study were 13.81% Ca, 8.77% P,
44.22% ash and bone density 1.26 g/ml; and 18.49% Ca, 9.20% P, 44.22% ash and bone density
1.51 g/ml, respectively, thus showing that the average Ca and P concentrations and bone density
were higher at the end of the period in these lowland areas. Regarding the blood analysis, we
observed reductions in serum P and Cu and increases in Co, Zn, Mn and Mg levels. Therefore, the
results indicated that buffaloes raised in wetland systems in the Amazon basin should receive
mineral supplementation, especially of P and Cu, despite the excellent pastures and nutrient-rich
soil. Further studies must be conducted to evaluate the mineral requirements and the productivity
increase that can be expected from this additional supplementation in the wetland system.
Keywords: mineral nutrition, buffaloes, bone, macrominerals


1094
Serological Profile of Buffalo (Bubalus bubalis) Female Calves Vaccinated with

Standard Brucella abortus Strain 19 Vaccine using Rose Bengal,
2-Mercaptoethanol and Complement Fixation Tests
Geraldo de NARDI JÚNIORa*, André Mendes JORGEb and Márcio Garcia RIBEIROc
a
Professor Ass. Doctor Discipline of Animal Production-Course of Technology in Agribusiness,
Faculty of Technology Botucatu - Fatec-Bt, state of Sao Paulo, Brazil.
b
Animal Production Department, School of Veterinary Medicine and Animal Science,Univ Estadual
Paulista UNESP, 18618-000, Botucatu, Sao Paulo, Brazil. CNPq Researcher.
c
School of Veterinary Medicine and Animal Science, UNESP – Univ Estadual Paulista, Department
of Veterinary Hygiene and Public Health, Infectious Diseases of Domestic Animals, Botucatu, Sao
Paulo, Brazil.
*Corresponding email: gjunior@fatecbt.edu.br
ABSTRACT
The serological profiles of 21 female buffaloes vaccinated between 3 to 8 months of age
using Brucella abortus strain 19 (S19) were evaluated by rose bengal (RBT), 2-mercaptoethanol
(2ME) and complement fixation (CFT) tests. The serum strains were collected in day zero (day of
vaccination), 15, 30, 45, 60th days and subsequently to each 30 months, until 720th day after
vaccination. No animal showed reaction in day zero. In 15th day, above 95% of animals revealed
reaction in all tests. The maximum levels of antibodies detected by the 2ME were found between 15
and 45th days after vaccination. Among 120 and 150 days after vaccination was observed decreased
of reactions in all the tests. All the animals presented absence of reactions in CFT, RBT and 2ME
tests at 270, 300 and 360 days after vaccination, respectively. Our finding highlighted early
response in CFT compared than other conventional agglutination tests. None of animals presented
oscillation of titers or reactions in any test after 360 day of study, which enables the use of CFT,
RBT and 2ME after this period without interference of antibodies from vaccine origin using S19
between 3 to 8 months in buffalo heifers.
Keywords: brucellosis, buffalo, vaccination, strain 19, serodiagnosis
INTRODUCTION
Brucellosis remains one of the most common zoonotic diseases worldwide (Acha and
Szyfres, 2003). Beside the public health concern, Brucella infections in livestock species also
represent a great economic impact particularly in developing countries due to reproductive
problems, reduced milk production (Seleem et al., 2010), and restrictions on animal movements and
trade, imposed by international regulatory norms (Godfroid et al., 2010).
Brucellosis in buffalo is caused by Brucella abortus (B. abortus), characterized by abortion
predominantly in third trimester of gestation, non viable calves, infertility and reduction of milk
production (Nielsen and Duncan, 1990).
In countries and regions with high prevalence of disease in livestock species, the most
effective measures to controlling and eradicating the disease are based on vaccination of all
susceptible host, serologic tests and elimination of positive animals (Gall and Nielsen, 2004; Olsen
and Tatum, 2010; Seleem et al., 2010). Vaccination is a critical tool to control or eradicate bovine
and buffalo brucellosis, because prevents abortion and consequent pasture contamination,
recognized as major form to transmission of B. abortus to these species (Olsen and Tatum, 2010).
The use of S19 vaccine in bovine leads to protection in cattle approximately up to nine years
of age, with 65-75 % of protection in all vaccinated animals throughout their productive life.
However, production of immunoglobulins (Ig) and persistence of titers induced by strain S19
depend particularly of age of animals at the time of vaccination (Seleem et al., 2010).
1095
Conventional serologic test for bovine and buffalo brucellosis detect antibody against the
LPS antigens induced by vaccination with S19 or exposure to virulent field Brucella. Indeed, no
single serological test can differentiate without any doubt animals vaccinated with S19 and animals
infected with virulent field strains (Godfroid et al., 2010). However, little information is available
about serological profile and persistance of Ig in buffalo calves vaccinated with standard S19
(Ribeiro et al., 2001; Paulin and Ferreira Neto, 2008). The persistence of Ig in buffalo calves
vaccinated with standard S19 can cause difficulties in serodiagnosis of disease using conventional
methods (Nielsen and Duncan, 1990; Nielsen, 2002). Thus, the purpose of this study was
investigate the serologic profile anti-Brucella abortus in buffalo calves vaccinated between 3 to 8
months of age with the S19, using rose bengal, 2-mercaptoethanol and complement fixation tests.

Twenty-one female buffalo calves (Bubalus bubalis), between 3 to 8 months old received
B. abortus vaccine S19. The vaccine used consisted of a commercial product, prepared with the
standard strain S19 (B. abortus). Prior to vaccination, the commercial vaccine was evaluated for
purity, dissociation and number of viable cells (Nielsen and Ewalt, 2004). The animals were
vaccinated with a single 2 mL dose, by subcutaneous route, containing 5,0–8,0 x1010 viable cells
(Organizacion Mundial de La Salud-OMS, 1986; Office International des Epizooties-OIE, 2010),
according to the manufacturer's recommendations.
Before brucellosis vaccination, blood samples of 21 animals were collected constituted day
zero of study. Subsequently, all the animals were sampled every 15 days (15 th, 30th, 45th and 60th
days post-vaccination) and after in intervals of 30 days (90th, 120th, 150th, 180th, 210th, 240th, 270th,
300th, 330th, 360th, 390th, 420th, 450th, 480th, 510th, 540th, 570th, 600th, 630th, 660th, 690th and 720th
day post-vaccination), resulting in the monitoring of animals for more than two years old. Serum
samples were centrifuged, separated in aliquots and stored at −20 °C.
The serological diagnosis was performed using rose bengal (RBT), 2-mercaptoethanol
(2ME) and complement fixation (CFT) tests, were performed using antigens and procedures as
described previously (FAO/OMS, 1986; Alton, 1988; MacMillan, 1990).

Figure 1 show the serological profile of heifers vaccinated between 3 to 8 months of age
using S19, during 720 days after vaccination. In day zero (day of vaccination) the 21 animals were
negative in all tests. On the 15th day post vaccination, over 95% of the animals revealed reactions in
all tests. Higher titers on 2ME were obtained between 15th to 45th days after vaccination. Among
120 and 150 days post vaccination was finding the decline of the reactions in animals in all tests.
None of the animals were reactors on days 270, 300 and 360 after vaccination in CFT, RBT and
2ME, respectively. None animals showed any oscillation of reactions in the three tests after 360
days (approximately 12 months).
The RBT is a well-know buffered Brucella acidified antigen test introduced in many
countries as the standard screening test because it is simple, quickly and present sensitive results
(Godfroid et al., 2010). Acidification of the antigen in RBT limits reactions with IgM, which
persists in vaccinated animals, and prioritizes reactions with IgG, which predominates in infected
animals (MacMillan, 1990; Nielsen, 2002).
Most of the animals were reactive to RBT at 15th day post-vaccination in present study,
revealed decrease at 150th day and the absence of reagent animals in the 300th day of monitoring. In
Brazil, (Ribeiro et al., 2001) also observed early reaction of RBT in the first two weeks post-
vaccination in buffalo calves vaccinated with standard S19, and no reaction at 300 days of
monitoring.
The 2ME is a conventional serologic test characterized by high specificity and moderate
sensitivity, used mainly as a confirmatory test in diagnosis of animal brucellosis in some countries
(Stemshorn et al., 1985; Megid et al., 2000; Gall and Nielsen, 2004). In the current study, were
observed maximum titers on 2ME between 15th and 45th days post-vaccination of buffalo. This
1096
finding is in agreement with similar studies in buffalo (Ribeiro et al., 2001) that founded maximum
titers in 2ME between the second and sixth week after vaccination, in animals vaccinated with
standard S19 vaccine. This result is justified by the ability of 2ME in prioritizing agglutination with
IgG, which show a peak of titers in heifers vaccinated with the standard S19 between the 28th and
42 days post-vaccination (Butler et al., 1981; MacMillan, 1990). The buffalo calves studied
presented gradual decline of titers in 2ME between 90th and 180th days after vaccination, and at 360
days no reaction was observed in these test. Similar study in Brazil showed the serological profile
of buffalo heifers vaccinated with S19 from 3 to 8 months old and the absence of reaction in 2ME
after 360 days of monitoring (Ribeiro et al., 2001).
The CFT enables the detection of anti-Brucella antibodies in bovine and buffalo that are
able to activate complement fixation factors, represented mainly by IgG and IgM classes
(MacMillan, 1990; Godfroid et al., 2010). The CFT has been used as a confirmatory test in some
countries that have achieved the status of eradication of brucellosis (Stemshorn et al., 1985;
MacMillan, 1990). Office International des Epizooties (OIE, 2010) consider CFT as a prescribed
test for international trade, despite the complexity of method, and recommends the harmonization
of laboratories in order to improve diagnostic performance of test (Gall and Nielsen, 2004). CFT is
characterized by detect mainly IgG, particularly IgG1 subclass (Chappel, 1989). In our 21 buffalo
calves, 95% were reactive in CFT at 15 days post-vaccination, with subsequent decline in number
of reagents at 120th day post-vaccination, and absence of reagent animals with 270 day of study.
(Godfroid et al., 2010) described that CFT identify early IgG1 around the 14th day. This result is in
agreement with the high number of CF-positive animals in the 15th day post-vaccination observed in
the present study.
The majority of animals studied presented reactions in three tests 15th and 60th days post-
vaccination, which coincide approximately with the peaks of IgM and IgG classes, respectively, in
calves vaccinated with S19 (Godfroid et al., 2002). The animals revealed also an early lack of
response in CFT compared than other conventional agglutination tests used, probably due to
prioritization of detection of IgG class in CFT, which tends to decline earlier in these test in heifers
vaccinated with standard S19 (MacMillan, 1990).
Our finding highlighted early response in CFT (270 days post-vaccination) compared than
other conventional agglutination tests. Routine and confirmatory tests used in current study showed
no reactions in any animals vaccinated after approximately 360 days of monitoring, which enables
the safe use of these tests after this period without any interference of Ig induced by standard S19
vaccine, in buffalo heifers vaccinated between 3 to 8 months of age.
REFERENCES
Acha, P.,N. and Szyfres, B. 2003. Zoonosis y enfermidades transmisibles comunes al hombre y a
los animales. 3.ed. Washington: Organización Panamericana de la Salud, p.28-56.
Alton, G.,G. 1988. Techniques for the Brucellosis Laboratory. Paris: Intitut National de la
Recherche Agronomique, 545p.
Butler, J.,E. Seawright, G.,I. Mcgivern, P.,L. and Gilsdrof, M. 1981. Class and subclass antibody
response of B. abortus strain 19-vacinated and field-strain-challenged cattle: evidence for
a predominant IgG1 response in infected animals. Adv. Exp. Med. Biol., v.137, p.790-
791.
Chappel, R.,J. 1989. Diagnosis of bovine brucellosis: Principles, practice and problems.
Surveillance, v.16, n.2, p.3-5.
Godfroid, J. Nielsen, K. and Saegerman, C. 2010. Diagnosis of Brucellosis in livestock and
wildlife. Croat Med J, v.51, n.4, p.296-305.
Godfroid, J. Saegerman, C. Wellemans, V. Walravens, K. Letesson, J.,J. Tibor, A. Mc, Millan., A.
Spencer, S. Sanna, M. Bakker, D. Pouillot, R. and Garin-Bastuji, B. 2002. How to
substantiate eradication of bovine brucellosis when specific serological reactions occur in
the course of brucellosis testing. Veterinary Microbiology, v.90, p.461-477.
1097
Gall, D. and Nielsen, K. 2004. Serological diagnosis of bovine brucellosis: a review of test
performance and coast comparison. Rev. Sci. Off. int. Epiz., v.23, n.3, p.989-1002.
MacMillan, A. 1990. Conventional serologic tests. In: Nielsen, K; Duncan, Jr. Animal brucellosis.
USA: CRC Press, p.155-300.
Megid, J. Ribeiro, M.,G. Marcos, Júnior., G. And Crocci, A.,J. 2000. Avaliação das provas de
soroaglutinação rápida, soroaglutinação lenta, antígeno acidificado e 2-mercaptoetanol no
diagnóstico da brucelose bovina. Brazilian Journal of Veterinary Research and Animal
Science, v. 37, n. 5, p. 1-13.
Nielsen, K. and Duncan, J.,R. 1990. Animal brucellosis. Boca Raton: CRC Press, 453p.
Nielsen, K. 2002. Diagnosis of brucellosis by serology. Veterinary Microbiology, v.90, p.447-459.
Olsen, S. and Tatum, F. 2010. Bovine brucellosis. Vet Clin Food Anim, v.26, p.15-27.
Organizacion Mundial De La Salud-OMS. Comité Mixto FAO/OMS de expertos en brucelosis.
1986. Genebra, 149p. (Série de informes técnicos, 740).
Office International des Epizooties-OIE. Bovine brucellosis. In: Manual of diagnosis test and
vaccines for terrestrial animals. 2010.
Paulin, L.M.S. and Ferreira Neto, J.S. 2008. Brucelose em búfalos. Arquivos do Instituto Biológico,
São Paulo. v.75, n.3, p.389-401.
Ribeiro, M.,G. Megid, J. Nardi Junior, G. Kuroda, B.,S. and Jorge, A.,M. 2001. Perfil de aglutininas
anti-Brucella abortus em provas de triagem e confirmatórias, em bezerras búfalas
vacinadas com a B19. In: CONGRESSO BRASILEIRO DE MEDICINA
VETERINÁRIA, 28., 2001. Salvador. Anais.Salvador, p.160.
Seleem, M.,N. Boyle, S.,M. And Sriranganathan, N. 2010. Brucellosis: A re-emerging zoonosis.
Veterinary Microbiology, v.140, p.392-398.
Stemshorn, B.,W. Forbes, L.,B. Eaglesome, M.,D. Nielsen, K.,H. Robertson, F.,J. and Samagh,
B.,S. 1985. A comparison of standard serological tests for the diagnosis of bovine
brucellosis in Canada. Can J Comp Med, v.9, n.4, p. 391–394.
2ME
RBT
21
20
CFT
19
18
17
16
15
14
13
number of animals
12
11
10
9
8
7
6
5
4
3
2
1
0
0 15 30 45 60 90 120 150 180 210 240 270 300 330 360 390 420 720
Days post v accination
Figure 1. Serological profile of buffalo heifers vaccinated with standard Brucella abortus S19
strain, between 3 to 8 months of age, using rose bengal (RBT), 2-mercaptoethanol (2ME) and
complement fixation (CFT) test, during 720 days of monitoring after vaccination.
1098
Interference of Vaccinal Antibodies on Serological Diagnostic of Leptospirosis in

Vaccinated Buffalo using Two Types of Commercial Vaccines
Geraldo de NARDI JÚNIORa*, André Mendes JORGEb and Márcio Garcia RIBEIROc
a
Professor Ass. Doctor Discipline of Animal Production-Technology Course in Agribusiness,
Faculty of Technology Botucatu - Fatec-Botucatu, SP, Brazil.
b
School of Veterinary Medicine and Animal Science, Animal Production Department, UNESP –
Univ Estadual Paulista, Botucatu, SP, Brazil. CNPq Researcher.
c
School of Veterinary Medicine and Animal Science, UNESP – Univ Estadual Paulista, Veterinary
Hygiene and Public Health Department, Infectious Diseases of Domestic Animals, Botucatu, SP,
Brazil
*Corresponding Email: gjunior@fatecbt.edu.br
ABSTRACT
The vaccinal antibodies interference represents one of the problems in the leptospirosis
diagnostic on serum. The present study aimed to determine the pattern of serum agglutinins anti-
Leptospirae spp in vaccinated female buffaloes against leptospirosis using two types of commercial
vaccines: bacterin and extern membrane. The temporal interference of vaccinal titers on serum
diagnostic was evaluated. Three groups of 11 adult female buffaloes were established as follows:
G1 control, non-vaccinated; G2: vaccinated with bacterin containing six serovars and G3 with
extern membrane vaccine containing five serovars. A booster was administered at 30 days from the
first vaccination (dfv) and two re- vaccinations were performed in each semester (days 210 and
390). Serum samples were collected on days 0, 15, 30, 45 and 60 and every 30 days until 540 dfv,
being submitted to Serum Agglutination Microscopy (SAM) against the serovars present in the
vaccine. G1 remained always negative. Both vaccines induced serologic responses when assessed
by SAM at 150 days post first vaccination against all serovars and they revealed maximum titers
around days 45 and 60 after first vaccination. At the re-vaccination there was an increase on
agglutinin levels, but of less intensity than the levels previously observed. After six months from
the second revaccination (540 dfv), they were almost zero, which demonstrates the short duration of
diagnostic interference. The serologic monitoring of the vaccinated herds can be an efficient
method to evaluate the status of protection provided by the vaccine.
Keywords: leptospirosis, buffalo, vaccination, serodiagnosis
INTRODUCTION
The extensive system of raising buffaloes provides access to a diversity of ecosystems
(rivers, creeks and water reservoirs) added to a close contact with bovines, which are risk factors
regarding leptospirosis for this species. The disease can be manifested by abortion, infertility,
sterility, week newborns, and it can finally compromises the reproductive and productive indexes
for cattle raising (Radostits et al., 2007). The leptospirosis diagnostic is performed by the detection
of agglutinins anti-Leptospirae on serum, suffering interference by the antibodies from vaccination,
compromising the decisions made about treatment and control, upon the presence of serologic
reaction in field animals (Rao and Keshavamurthy, 1985; Faine et al., 1999).
Although, Buffaloes have been considered a viable alternative of protein production from
animal source in Brazil, there are very few studies approaching the humoral response provided by
the vaccination (Nardi Junior, 2005). The present work aimed to determine the patterns of
agglutinins anti-Leptospirae spp on serum of buffalo cows previously vaccinated against
leptospirosis, using two types of commercial vaccines: bacterin and extern membrane. In addition,
the study evaluated the temporal interference of vaccine titers in serum-diagnostic.

1099

Three groups (G1, G2 and G3) of 11 adult buffalo females (Bubalus bubalis) mixed-bred
from Murrah (2 to 10 calving), clinically healthy and non-vaccinated against leptospirae.
Serum samples were collected before vaccination (day 0) and analyzed by Serum
Agglutination Microscopy (SAM) as described by Faine et al., (1999) against 6 serovars: Canicola,
Icterohaemorrhagiae, Grippotyphosa, Pomona, Hardjo and Wolffi. Then, the samples were
assessed at 15, 30, 45, 60 and every 30 days until 540 days post first vaccination (dfv) by SAM
against the serovars present in the vaccines. It was used a serial dilution with ratio 2 and
considering as reacting, the serum with >1:25 dilution presenting 100% of agglutination and a final
titer as the highest dilution showing 50% or more of agglutination.
G1- control, received 2.0 mL of sterile saline solution IM; G2 received 3.0 mL IM of
bacterin vaccine, containing the following serovars: sorovares Canicola, Icterohaemorrhagiae,
Grippotyphosa, Pomona, Hardjo and Wolffi. G3 received 5.0 mL IM of extern membrane vaccine,
containing the same serovars cited above, except Wolffi. The groups received a booster after 30
(dfv) and re-vaccinations within a six months interval (days 210 and 390).

The animals previously selected before vaccination had no reaction by SAM (<25). The
figures 1 to 6 represent the geometric means of titers from agglutinins in each group of animals, for
the serovars present in the vaccine.
The control group G1 were maintained in the same conditions of groups G2 and G3, even
though, they had non-static serologic titers when compared with day 0, remaining always inferior to
100 (all animals negative), which is represented by the geometric curve of agglutinin titers (always
below 2) supporting the efficacy of the sanitary control.
Once being confirmed negatives before vaccinations, all vaccinated buffaloes either with
bacterin or extern membrane presented serologic responses, which began at the first 15 days after
first-vaccination for all serovars from the respective vaccines. The same fact was also observed by
Rao and Keshavamurthy (1985), reporting that all buffaloes presented seroconversion to the vaccine
serovars (Pomona, Canicola, Hebdomadis, Tarassovi and Shermani) at 15 dfv.
The serologic monitoring of animals throughout 540 days after first vaccination revealed
maximum titers around 45 and 60 dfv, for both vaccines and all serovars. The same responses were
described by Nardi Júnior et al., (2006), observing maximum titers by SAM between 45 and 60
days after vaccination in 17 buffalo heifers vaccinated with pentavalent bacterian
(Icterohaemorrhagiae, Canicola, Grippotyphosa, Pomona, Hardjo), showing the similarity of
responses regarding the antibodies production upon the use of different commercial vaccines.
The agglutinin curves for both vaccines presented a similar pattern, with close serological
responses, but different intensity, as represented by the superiority of G3 to G2 in the majority of
times and for the most part of serovars. G3 presented precocity in their seroconversion, showing
elevated titers when compared to G2. In general, six months after the second dose (210 dfv), G2
and G3, had no positive animals (titers >100), with exception of the serovar Pomona (G2 presenting
one animal with a titer of 100 and G3 presenting three animals with a titer of 100), and Hardjo (G2
and G3, with one animal per group presenting a titer of 100). Rao and Keshavamurthy (1985)
described the persistency of agglutinins from the vaccines during three months in buffalo calves
previously vaccinated with a pentavalent bacterin. After this period, the geometric means of the
titers rarely exceeded the cut of point 2 (titer 100) but this fact was not observed for Pomona
serovar, which showed elevated means at 240 and 420 dfv in G3 (Fig. 1 to 6). After the first re-
vaccination (210 dfv) elevations of lower intensity were observed, and they presented a tendency to
rapidly become negative when compared with the animals receiving a booster (30 dfv), in which the
elevations occurred after the second re- vaccination. In the present study, at 540 days after first
vaccination (6 months after the second re-vaccination), the two treated groups did not presented
positive animals for the two serovars, showing the non-interference for vaccination upon the
diagnostic after this period. Thus, in average, at 6 months after each re-vaccination, positive
1100
animals were no more observed by the SAM. However this test does not permit any inferences
regarding the bacterin protection against leptospirosis.
Oliveira (2000) described the interference of vaccine antibodies, while performing SAM in
three herds of buffaloes submitted to the control measures for leptospirosis (chemotherapy and
systematic vaccination), comparing it to a control herd, which was non-vaccinated and non-treated.
They observed that the vaccinated herd presented a percentage of reacting animals significantly
superior than the numbers observed for the control herd, suggesting that even respecting the interval
of four to six months between the last vaccination and the blood collection, there was an
interference of vaccination in the results of serologic monitoring. One explanation for this
disagreement regarding the results would be supported by the inclusion of non-reacting animals,
which had a seroconversion upon vaccination, but probably would have a tendency to become
negative throughout the re-vaccinations. Other hypothesis would be related to the strains used in the
preparation of commercial vaccines, specially under seeds maintenance and growth conditions, and
furthermore due to the particularities of each manufacture, including number of serovars and
adjuvant used.
In conclusion, the serologic monitoring of vaccinated herds can be an interesting tool to
evaluate the status of protection provided by the vaccine used in the field by farmers, but always
having in mind that a significant increase of vaccine serovar titers, or even an altered herd serologic
pattern can reflect a failure in the capability of protection from the vaccine and thus it demands a
review on prophylaxis measures. In this case, the attempt in isolating a strain from the circulation
could be of great help for the knowledge of its virulence and capability of dissemination, possibly
including it in a new immunization protocol. This study is an important tool for the health of the
buffalo herd and increment the agribusiness of buffalo.
REFERENCES
Radostits, O., M. Gay, C., C. and Hinchcliff, K.W. 2007. Veterinary medicine: A textbook of the
diseases of cattle, horses, sheep, pigs, and goats. 10.ed. Philadelphia: W.B. Saunders,
2156p.
Rao, A., S. and Keshavamurthy, B., S. 1985. Study of the immune response of buffalo calves to
heat-killed pentavalent leptospiral vaccine. Indian Veterinary Journal, v .62, p. 357-361.
Faine, S. Adler, B. Bolin, C. and Perolat, P. 1999. Leptospira and Leptospirosis. 2. ed. Melboune:
MediSci, 272 p.
Nardi Junior, G. 2005. Perfil de anticorpos anti-Leptospira spp em búfalas (Bubalus bubalis)
vacinadas com dois tipos de vacinas comerciais anti-leptospirose (Bacterina e Membrana
externa)[Dissertação]. São Paulo: Faculdade de Medicina Veterinária e Zootecnia,
Universidade de São Paulo.
Nardi Junior, G. Ribeiro, M., G. Vasconcellos, S., A. Megid, J. Jorge, A., M. Geronuti, L. And
Morais, Z., M. 2006. Perfil de aglutininas anti-Leptospira em bezerras búfalas vacinadas
com bacterina pentavalente contra leptospirose. Arquivo Brasileiro de Medicina
Veterináriae Zootecnia, Belo Horizonte, v. 58, p. 299-304.
Oliveira, J.,C.,F. 2000. Isolamento de Leptospira santarosai sorovar Guaicurus em búfalos
(Bubalus
bubalis) do Vale do Ribeira/SP, Brasil: Efeito de dois esquemas de controle da leptospirose
sobreo desempenho reprodutivo das búfalas. 63f. Tese (Doutorado em Reprodução animal)
– Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo
1101
Group 1 Group 1
Group 2
Group 2
2,1 Group 3
2,55 Group 3
1,95
2,4
1,8
2,25
2,1 1,65
1,95 1,5
1,8 1,35
Geometric means
1,65
Geometric means
1,2
1,5
1,05
1,35
0,9
1,2
1,05 0,75
0,9 0,6
0,75 0,45
0,6
0,3
0,45
0,15
0,3
0
0,15 0* 15 30** 45 60 90 120 150 180 210*** 240 270 300 330 360 390**** 420 450 480 510 540
0
Days post vaccination
(* = first vaccination, ** = booster, *** = 1ª re-vaccination, **** = 2ª re-vaccination)
0
0
**
15
45
60
90
**
0*
***
12
15
18
24
27
30
33
36
42
45
48
51
54
30
0*
0*
21
39
Figure 1. Figure 2.
Group 1
Group 1 Group 2
2,4
Group 2 Group 3
2,25
Group 3 2,25
2,1 2,1
1,95 1,95
1,8 1,8
1,65 1,65
1,5 1,5
Geometric means
Geometric means
1,35 1,35
1,2 1,2
1,05 1,05
0,9 0,9
0,75 0,75
0,6 0,6
0,45 0,45
0,3 0,3
0,15 0,15
0 0
0
**
15
45
60
90
**
0*
*
0
0
**
15
45
60
90
**
0*
**
12
15
18
24
27
30
33
36
42
45
48
51
54
**
12
15
18
24
27
30
33
36
42
45
48
51
54
30
0*
30
0*
0*
0*
21
21
39
39
Days post vaccination Days post vaccination

(* = first vaccination, ** = booster, *** = 1ª re-vaccination, **** = 2ª re-vaccination) (* = first vaccination, ** = booster, *** = 1ª re-vaccination, **** = 2ª re-vaccination)
Figure 3. Geometric means of agglutinin titers Figure 4. Geometric means of agglutinin titers
anti-Leptospirae spp. serovar Grippotyphosa in anti-Leptospirae spp. serovar Pomona in serum
serum of vaccinated buffaloes. of vaccinated buffaloes.
Group 1
Group 1 Group 2
2,4
Group 2 Group 3
3 2,25
Group 3
2,85
2,1
2,7
2,55 1,95
2,4 1,8
2,25
1,65
2,1
1,5
Geometric means
1,95
Geometric means
1,8 1,35
1,65 1,2
1,5
1,05
1,35
1,2 0,9
1,05 0,75
0,9
0,6
0,75
0,6 0,45
0,45 0,3
0,3
0,15
0,15
0
0
0
0
**
15
45
60
90
**
0*
*
**
12
15
18
24
27
30
33
36
42
45
48
51
54
0
0
**
15
45
60
90
**
0*
30
0*
**
12
15
18
24
27
30
33
36
42
45
48
51
54
0*
30
0*
21
0*
39
21
39

Figure 5. Geometric means of agglutinin titers Figure 6. Geometric means of agglutinin titers
anti-Leptospirae spp. serovar Hardjo in serum anti-Leptospirae spp. serovar Wolffi in serum
of vaccinated buffaloes. of vaccinated buffaloes.
1102
Vaccine Trial of Recombinant Schistosoma japonicum Paramyosin in Water

Buffaloes
Mario Antonio L. JIZ IIa, Claro N. MINGALAb*, Ivy Fe M. LOPEZc, Mike CHUAd,
Francisco G. GABONADA, Jr.c, Luz P. ACOSTAa, Haiwei WUd and Jonathan D. KURTISe
a
Research Institute for Tropical Medicine, Alabang, Muntinlupa City, Metro Manila, Philippines
b
Animal Health Unit, Philippine Carabao Center National Headquarters and Gene Pool, Science City
of Munoz 3120, Nueva Ecija, Philippines
c
Philippine Carabao Center at Visayas State University, Visca, Baybay, Leyte, Philippines
d
Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, People's Republic of
China
e
Department of Pathology and Laboratory Medicine, Rhode Island Hospital, Rhode Island, USA
*Corresponding email: cnmingala@hotmail.com
ABSTRACT
The overall aims of this project were to assess the safety and immunogenicity of the
Schistosoma japonicum vaccine Paramyosin among water buffalos residing in endemic areas. The
study was conducted in four villages in Leyte, Philippines, an area highly endemic for
Schistosomaaponicum. One hundred fifteen (N=115) animals provided baseline stool samples for
coprologic examination, with preliminary results using FLOTAC showing a 10% prevalence of
schistosomiasis. Forty-nine (N=49) animals consented to treatment with 25 mg/kg Praziquantel, and
40, 36 and 32 animals consented to the first, second and third dose of the paramyosin vaccine,
respectively. The safety trial involved the first 20 animals and included skin testing, vaccination,
anaphylaxis monitoring, and hematology and serum chemistry analysis. Skin tests revealed that only 3
out of 20 animals exhibited redness at the injection site, with none greater than 1 cm. None of the
animals exhibited anaphylaxis. All hematology and serum chemistry markers were within normal
range or were similar to pre-vaccination levels. None of the 40 animals administered with the first
dose exhibited anaphylaxis, or any of the subsequent vaccine doses. Immunogenicity assessment of
sera collected prior to every vaccination and one month after the last dose showed that the paramyosin
vaccine induced robust antibody responses to all animals, as assessed by ELISA. Analysis of cytokine
levels of whole blood culture supernates are forthcoming. Overall, this project has demonstrated that
the Schistosoma japonicum paramyosin vaccine is safe, well-tolerated, and immunogenic among water
buffalos residing in endemic areas.
Keywords: Schistosoma japonicum, paramyosin, vaccine, immunogenicity, water buffaloes


1103
Seroepidemiological Investigation, Risk Factors Analysis of Brucellosis in

Ruminants and Their Owners in District Buner of Pakistan
Mansur-ud-Din AHMAD,a Muhammad IDREES,a Shakera SADIQ,a Abdul REHMAN a and

Muhammad Hassan MUSHTAQ *a
a
Epidemiology and Public Health, University of Veterinary and Animal Sciences, Lahore, Pakistan.
Corresponding email: hassan.mushtaq@uvas.edu.pk
ABSTRACT
Brucellosis is important from two main aspects as economic loss to livestock industry and
impacts on public health. Brucellosis is worldwide infectious zoonotic disease of ruminants. In
Pakistan, brucellosis is endemic in cattle and buffalo, sheep and goat population, so keeping in view
the significance of this disease in the ruminants and livestock owners population the study was
designed to detect prevalence of brucellosis and to analyze various risk factors of brucellosis in
ruminants and livestock farmers in ruminants and livestock farmers in Buner district of Pakistan. A
two stage sampling technique was used in which small and large ruminants and their owners were
included. In the first stage sampling, two villages were selected by systemic way and in second
stage sampling; five households having livestock holdings were selected. Information about risk
factors in man and animals was gathered by separate structured questionnaires. Blood samples were
collected and serum was isolated for screening with Rose Bengal Plate Test (RBPT). The
prevalence of Brucellosis in animals was 6.25%, 5.55%, 5.59%, 6.14%, and 3.27% in buffalo,
cattle, sheep, goat and livestock owners respectively. Herd level prevalence for Brucellosis in
buffalo, cattle, sheep, goat, and mix herds was 10.33%, 15.55%, 35%, 7.89%, and 19.51%
respectively. Individual herd level prevalence was from was 4.76%, 25% and 13.38% minimum,
maximum and average respectively. Among the risk factors associated with Brucellosis in
ruminants; type of farm operation (p-value=0.000), type of flooring system (p-value=0.095 &
OR=0.36), ventilation. i.e. (p-value=0.252 & OR=0.55), housing condition (p-value=0.157 &
OR=0.692), animal health status (p-value=0.000). Interestingly the results showed a significant
relationship between natural breeding of the animals and positive cases of Brucellosis. i.e. (p-
value=0.033 & OR=9.98). No animal suffered from Brucellosis for whom Artificial Insemination
was used for breeding. Artificial Insemination was significantly associated with negative cases of
Brucellosis. i.e. (p-value=0.033 * OR=0.10). Among the risk factors in humans, significant
association was found between the occupation of the person and positive test results for Brucellosis.
These results highlight the importance of zoonotic diseases and indicate that there is need of
creating awareness among farmer for vaccination of livestock and adopting control/preventive
measures like use of artificial insemination.
INTRODUCTION
Brucellosis is one of the chronic infections of the animals and man caused by a bacteria
belonging to Brucella species. Commonly Brucella abortus is found in bovine, Brucella melitenses
in small ruminants and Brucella suis in pigs. The causative agent of Brucellosis targets the
reproductive system of the sexually matured animals (Radostits et al., 2007). Brucellosis is usually
transmitted to humans by direct contact through abraded skin or mucosal surfaces, consumption of

1104
contaminated milk and milk products, or by inhalation (Fosgate et al., 2002). Person to person
transmission of Brucella is extremely rare (Seric-Haracic et al., 2008). In animals, brucellosis
causes abortion and breeding-associated problems such as repeat breeding, retained placenta and
metritis in female animals while in male animals epididymitis and sterility are common (Plummet et
al. 1998, Quinn et al., 2002,Chakrabarati, A., 2006). In human, it causes undulant fever (Al-Eissa et
al., 1999).The highest infection rate (72.9%) has been reported in the Palestinian Authority
(Shuaibi, 1999). In cattle and buffalo it has been reported that the incidence of brucellosis occur
3.25% and 4.4% in different areas of Pakistan (Masoumi et al., 1992). In Pakistan, brucellosis is
endemic in cattle, buffalo, sheep and goat population (Ahmed and Munir, 1995). Khyber
Pukhtunkhwa province owns a developing livestock sector where 90% farmers are landless and
dependent on agriculture and livestock for their livelihood. So the present study was designed to
detect prevalence and to analyze various risk factors of brucellosis in ruminants and livestock
farmers in Buner District.
The present study was carried out in District Buner of Khyber Pukhtunkhwa Province. A
two stage sampling technique was applied. In first stage sampling two villages were selected from
each Tehsil by systemic sampling technique including twelve villages. In second stage sampling
five households with livestock farming background were selected from each selected village and
thus 60 households in total were selected. For seroprevalence of Brucellosis in ruminants and
humans, total 534 samples were collected including humans (61), sheep (91), goat (146), cattle
(128) and buffalo (108) and then screening was done through Rose Bengal Plate Test. (Berhe et al.,
2007). Statistical analysis was done through SPSS version 13.
RESULTS AND DISCUSSION:

Survey for risk factors in livestock:
The results of the survey of risk factors in livestock revealed that maximum number of the
herds was consisted of multiple species including sheep, goat cattle and buffalo. Among the total
farmers 93.4% practiced natural breeding while remaining was practicing artificial insemination. A
total of 67.2% farmers were raising their own animals while other purchased animals from local
markets. All the animals were kept indoor. Animal’s health status at herd level was scored by their
physical appearance and was recorded 32.8%, 52.5% and 14.8% good, medium and poor as shown
in Table: 1
1105
Table 1. Risk factors survey for farmers

Variables Frequency Percentage
Small (up to 4 animals) 6 10
Herd size Medium (up to 11 animals) 43 71.7
Large (up to 31 animals) 11 18.3
Single specie herd 5 8.3
Type of herd
Multi specie herd 55 91.7
Introduction of new animals to Yes 20 32.8
herd No 40 67.2
Natural breeding 56 93.4
Breeding practices
Artificial insemination 4 6.6
Intensive 25 41.0
Farm operation Backyard 20 34.0
Mix 15 25.0
Open 0 0.0
Animal Housing design
Closed 61 100
Sufficient 42 70.0
Space per animal
Insufficient 18 30.0
Satisfactory 28 46.6
Over all housing condition
Unsatisfactory 32 53.4
Good 32 53.4
Animals waste management
Poor 28 46.6
Tap water 40 66.6
Drinking water supply
Pond water 20 33.4
Yes 14 23.3
Veterinary services
No 46 76.7
Respiratory 1 1.6
Reproductive 4 6.6
Parasitic 12 19.7
Prevalent diseases at herd level
Infectious diseases 9 14.8
Multiple diseases 12 19.7
No diseases 22 36.1
Animal health status at herd level Good 20 33.3
Seroprevalence
The results of seroprevalence among the animals and humans showed that the prevalence of
Brucellosis in animals was 6.25%, 5.55%, 5.59%, 6.14%, and 3.27% in buffalo, cattle, sheep, and goat
and livestock owners respectively. Herd level prevalence for Brucellosis in buffalo, cattle, sheep, goat,
and mix herds was 10.33%, 15.55%, 35%, 7.89%, and 19.51% respectively. Individual herd level
prevalence was from was 4.76%, 25% and 13.38% minimum, maximum and average respectively. Herd
level prevalence of Brucellosis in buffalo population was 10.34% by RBPT. (Khan et al. 2010)
detected seroprevalence of Brucellosis in buffalo population in Swat valley NWFP as 4.85% and
5.62% by screening the serum with RBPT and ELISA respectively. A significant difference is found
with the present study. This may be due to inclusion of animals of slaughter houses. In this study
they also detected seroprevalence of Brucellosis at herd level and were 9.2% and6.8% in private
herds and 9.5% and 7% in animals of slaughter house by RBPT and ELISA respectively. It can be
1106
concluded from their study that seroprevalence of Brucellosis is higher in slaughtering animals. In
the present study seroprevalence of Brucellosis in sheep was 20%, 5.81% and 5.59% male, female
and overall respectively. Herd prevalence and out of positive herd’s prevalence was 35% by RBPT.
Seroprevalence of Brucellosis in goat population was 0%, 6.57% and 6.14% male, female and
overall respectively. Herd level prevalence was 7.89% by RBPT. (Koud et al. 2010) detected
seroprevalence of Brucellosis in sheep and goat 26.66% and 18.88% by RBPT and ELISA
respectively. The difference may due to that they have screened commercial herds at farm level and
secondly they have used ELISA for screening as in the present study only RBPT is used as
screening test. (Zafer et al. 2007) detected seroprevalence of Brucellosis in sheep and goat in
Turkey 6.47% collectively. (Abeer at al. 2003) stated in his study the seroprevalence of Brucellosis
in sheep in Spain that 14.3% and 7.2% by RBPT and ELISA respectively while herd level
prevalence was 56% and 45% by RBPT and ELISA respectively. The difference may be due to
they had monitored the sheep flock for three months and also included in the study with history of
abortion. Among the risk factors associated with Brucellosis in ruminants; type of farm operation (p-
value=0.000), type of flooring system (p-value=0.095 & OR=0.36), ventilation. i.e. (p-value=0.252 &
OR=0.55), housing condition (p-value=0.157 & OR=0.692), animal health status (p-value=0.000).
Interestingly the results showed a significant relationship between natural breeding of the animals and
positive cases of Brucellosis. i.e. (p-value=0.033 & OR=9.98). No animal suffered from Brucellosis for
whom Artificial Insemination was used for breeding. Artificial Insemination was significantly associated
with negative cases of Brucellosis. i.e. (p-value=0.033, OR=0.10). Results of the present study do not
agree with (Ahmad et al. 2009) as they have found a significant association between herd size, use
of disinfectants, and lack of veterinary services. It may be due to seasonal difference or
geographical location. But mix farming in both of the studies has a significant association. Results
about risk factors in the present studies in ruminants are agreed with the ( Sofian et al. 2008; and
Aguir et al. 2007).The results of seroprevalence among owners revealed that among total 3.27 were
infected with brucella. Among the risk factors in humans, significant association was found between the
occupation of the person and positive test results for Brucellosis. Out of the positive one sample was
positive with positive herd while one was positive with negative herd. The risk factors association of
the present study agreed with (Kaud et al. 2010) in terms of waste management and sanitary
measures at farm.
1107
Table 2. Test Results for Brucellosis for (Animals) with different Risk Factors
Variables Test result for
p- Odds CI
Brucellosis X2
value Ratio
+ve -ve
Type of Intensive 5 20
Operation Backyard Farm 13 8 15.57 0.000* - -
Mixed 12 3
Waste Good 11 22 0.080-
7.224 0.007* 0.23
Management Poor 19 9 0.693
Drinking Tap Water 21 19 0.508-
0.512 0.474 1.47
Water Pond Water 9 12 4.27
Veterinary Yes 5 9 0.142-
1.318 0.251 0.48
Services No 25 22 1.68
Good 2 18
Animal
Medium 22 10 18.28 0.000* - -
Health Status
Poor 6 3
Feeding Grazing 16 11 0.744-
1.969 0.161 2.07
system Stall feeding 14 20 5.806
Natural Yes 30 27 0.513-
3.34 0.033* *9.98
Breeding No 0 4 193.9
Artificial Yes 0 4 0.005-
3.34 0.033* 0.10*
Insemination 1.946
Purchase of Yes 11 9 0.403 0.525 1.41 0.484-
Animal No 19 22 4.141
from Out
Side
Current Respiratory 0 1
Disease Reproductive 3 1
Problems in Parasitosis 6 6
the Herd Infectious 7 15 6.294 0.279 - -
Multiple 5 4
Diseases
No Disease 8 4
REFERENCES
Abeer, H., A. Talafhah, S.Q. Lafi, and Y.A. Tarazi (2003). Epidemiology of ovine brucellosis in
Awassi sheep in Northern Jordan. Preventive Vet. Med. (60): 297–306.
Aguiar, D. M., G. T. Cavalcante, M. B. Labruna, S. A. Vasconcellos, A. A. R. Rodrigues, Z. M.

Morais, L. M. A. Camargo and S. M. Gennari (2007). Risk factors and seroprevalence of
brucella spp. In cattle from western amazon, brazil. Arq. Inst. Biol., Sao Paulo. 74(4): 301-
305.
Ahmad, M., A. Majali, Q. Abdelsalam, M. Mustafa, M. Ababneh and M. Ababneh (2009).

Seroprevalence and risk factors for bovine brucellosis in Jordan. J. Vet. Sci. 10(1): 61-65.
1108
Al-Eissa, Y,A (1999) Brucellosis in Saudi Arabia: past, present and future. Ann Saudi Med. 19:403-
5.
Berhe, G., K. Belihu and Y. Asfaw (2007). Seroepidemiological investigation of bovine brucellosis
in the extensive cattle production system of tigray region of ethiopia: Intern. J. Appl. Res.
Vet. Med. (2): 65-71.
Chakrabarati, A. (2006). T. B of animal husbandry sciences 1st edition Kalyani publishers.
Fosgate, G.T., T.E. Carpenter; B.B. Chomel; J.T. Case; E.E. Debess; and K.F. Reilly; (2002). Time
space clustering of human brucellosis, California, 1973 1992. Emerg. Infect. Dis., 8: 672-
678.
Kaoud, H.A., M. M. Zaki, A.R. El-Dahshan and S, A. Nasr (2010). Epidemiology of Brucellosis
Among Farm Animals: Nature and Science. 8(5):190-197.
Khan, K., M. Rabbani, K. Muhammad, A. Maqbool and M. Z. Shabbir ( 2010). Seroprevalence of

Brucellosis in Buffalo and Human in Swat Valley, NWFP, Pakistan: Pakistan J. Zool. Suppl.
Ser. (9) 111-114.
Masoumi, J. P., M. A. Sheikh, R. Ahmad, M. Naeem, M. Ahmad and I. Hussain, (1992)

Seroprevalence of brucellosis in sheep, goats and man in Lahore area. Indian J. Dairy Sci.,
45: 298-299.
Plummet, M., R. Diaz and J. M. Verger (1998). Zoonosis: biology, clinical practice and public
health control: Oxford Univ. Press, New York, USA, pp: 23-35.
Quinn, P. J., B. K. Markey, M. E. Carter, W. J. Donnelly and F. C. Leonard (2002). Veterinary

Microbiology and microbial diseases: Blackwell Ltd. Pp 162-167.
Radostits, O. M., C. C. Gay, K. W Hinchcliff and P. D. Constable, (2007). A text book of the
diseases of cattle, horses, sheep, pigs and goats 10th Ed. Elsevier Ltd. Pp. 966-996.
Seric-Haracic, S., M. Salman, N. Fejzic and S. Cavaljuga (2008). Brucellosis of ruminants in

Bosnia and Herzegovina: disease status, past experiences and initiation of a new surveillance
strategy. Bosn. J. Basic Med. Sci.(8):27–33.
Shuaibi, A (1999). Palestinian brucellosis contro programme. Country reports. Brucellosi

Information Workshop, Ramallah, Palestine.
Sofian, M., A. Aghakhani, A. A. Velayati, M. Banifazl, A. Eslamifar and A. Ramezani (2008). Risk
factors for human brucellosis in Iran: a case-control study: Int. J. of Inf. Dis. (12) 157—161.
Zafer, T., M. Yildirim and E. Ustanbulluoulu (2007). Seroprevalence of Brucellosis in Human,

Sheep, and Cattle Populations in Kyrykkale Turkey: Turk. J. Vet. Anim. Sci. 31(1): 75-78.
1109
Disease susceptibility of Buffalo in Pakistan

Sidra MANZOOR, Asif NADEEM, Maryam JAVED and Masroor Ellahi BABAR1
54000, Pakistan.
1
*Corresponding email: sidzm@yahoo.com
ABSTRACT
Buffalo is the leading dairy animal of Pakistan and provides beef, traction and tilling power.
In Pakistan, there are 32.7 million buffaloes which are responsible for 76.41% of the total gross
milk production. Some diseases such as mastitis, hemorrhagic septicemia, foot and mouth disease
and brucellosis threaten animal health and enormously cause economic losses. Mastitis is a familiar
disease with incidence of clinical (40.35%) and sub-clinical (59.64%) form in buffaloes of Pakistan.
Haemorrhagic septicaemia is ranked as the most important infectious disease with its prevalence up
to 49.0%. While Foot and mouth disease is considered the most prevalent disease as high morbidity
61.7% and mortality rates 20.8% were reported in buffaloes and young stock of District Sahiwal,
Pakistan. Buffalo also suffers from a large number of viral, bacterial, parasitic, fungal and
reproductive disorders which cause major hindrance in the growth and physiology of animal.
Besides other, commonly prevalent diseases in buffalo are also reported.
Keywords: Disease susceptibility, Buffalo, Pakistan
INTRODUCTION
Livestock is the most important sub sector of agriculture in Pakistan that accounts for 55.1
percent to the agricultural value added and 11.6 percent to national GDP (Economic Survey of
Pakistan, 2011-12). Gross value added of the livestock sector at constant factor cost has increased
from Rs. 672 billion (2010-11) to Rs. 700 billion (2011-12); showing an increase of 4.0 percent as
compared to previous year. The major products of livestock are milk and meat. Buffalo is the main
dairy and meat producing animal in the country. Pakistan owns about 32.7 million heads of buffalo.
About 76.41 % (29.5 million tons) of the total milk (38.69 million tons) produced in the country is
supplied by this specie (Economic Survey of Pakistan, 2011-12). Pakistan is regarded as the 4th
largest milk producing country in the world (FAO, 2010). Overall, the contribution of dairy industry
to the national economy is of the order of Rs. 540 billion and is expected to grow at 4 percent/year
under current scenario.
Previous studies have revealed that animal diseases caused huge economic losses with a
silent continuation over time. Buffalo is considered to be a valuable being as compared to other
domesticated animal species, which suffers a large number of bacterial, viral, parasitic, fungal,
protozoan, nutritional and metabolic diseases.
Mastitis: Mastitis has been recognized as one of the most economically important disease
throughout the world including Pakistan (Mustafa et al., 2011). It adversely affects animal health,
quality and quantity of milk (Raza et al., 2000; Sharma et al., 2007). Mastitis is responsible for the
production losses in the form of reduced milk yield (up to 70%), milk discard after treatment (9%),
veterinary services cost (7%) and earlier culling of animals (14%) (Bhikane and Kawitkar, 2000).
The estimated annual losses due to mastitis are about $ 184 per animal. Apart from its economic
importance it also poses the threat for the transmission of zoonotic diseases like tuberculosis,
brucellosis, leptospirosis and streptococcal sore throat to human beings (Radostits et al., 2000;

1110
Bachaya et al., 2011). The incidence of clinical and sub-clinical mastitis was reported as 40.35%
and 59.64% in buffaloes from Lahore district (Mustafa et al., 2011).
Haemorrhagic septicaemia: Haemorrhagic septicaemia (HS) has been ranked as the most
communicable disease caused by the gram-negative bacterium Pasteurella multocida (Tabatabaei et
al., 2007). Serotypes B:2, B:2,5 of Pasteurella multocida are strongly associated with disease in
animals of Asia (Farooq et al., 2007). The disease has a major impact on the livestock industry in
countries of South Asia including Pakistan, where it causes huge economic losses. A survey was
conducted on the importance of hemorrhagic septicaemia to buffalo rearing in Pakistan indicated its
prevalence up to 49% (Farooq et al., 2007). In another recent outbreak, 100% morbidity and
31.48% mortality was recorded in buffalo calves in Pakistan (Khan et al., 2011). The mortality rate
extensively peaked on 8th day (37%), is attributed to its acute clinical nature which does not allow
to treat the animals (Khan et al., 2011).
Foot and mouth disease Foot and mouth disease (FMD) is endemic, regarding 153 point
outbreaks are reported during 2002-2007 in Pakistan. The most prevalent serotypes are O (70%),
Asia-I (25%) and A (4.67%) causing a loss of Rs. 6.00 billions/year to farmers (Zulfiqar, 2003). But
there is no national control plan being executed currently. Foot and mouth disease is considered the
most prevalent disease as high morbidity 61.7 % and mortality rates 20.8 % were reported in
buffaloes and young stock of District Sahiwal, Pakistan (Afzal, 2008).
Tuberculosis: Bovine tuberculosis is caused by a group of bacilli referred as the
Mycobacterium tuberculosis complex. Mycobacterium bovis is most important causative agent and
has great tendency to infect human and other animals due to its wide host range (Khan and Khan,
2007). It is considered to be of socio-economic importance and is of high impact to the public
health. It is a problem of serious concern in developing countries like Pakistan, where milk is not
pasteurized on large scale and insufficient systems for disease management. A recent survey
conducted in the Punjab Pakistan signified the prevalence of tuberculosis in Nili Ravi buffaloes as
5.48% (Javed et al., 2006), 10.06% (Imtiaz et al., 2008) to 12.72% (Khan and Khan, 2007).
Brucellosis: Brucellosis is another important contagious and zoonotic disease of animals
worldwide. It causes large economic losses together with abortion, low fertility rates, reduced milk
production, and cost of replacement of animals (McDermott and Arimi, 2002). The overall
prevalence of brucellosis was found to be 3% and 8.5% in cattle and buffaloes using milk ring test
(MRT) and i-ELISA, respectively (Shafee et al., 2011).
Leptospira: Different serotypes of a bacterial genus Leptospira are responsible for another
zoonotic disease, Leptospirosis in dairy animals. In Nili Ravi buffaloes of Pakistan, the overall
incidence of leptospirosis was 11.88% (Bhatti, 2008).
Rinderpest: Due to the success of the national rinderpest eradication plan pursued by the
Government of Pakistan with assistance from the European Union and FAO, Asia is now free from
rinderpest for the first time in millennia. Roeder (2002) reported that Indus River buffalo tract of
Sindh Province (Pakistan) was the last reservoir in Asia and there is no recent outbreak of
rinderpest in Punjab.
Reproductive disorders: The occurrence of certain reproductive disorders in buffaloes is
also responsible for the huge economic losses. The overall incidence of reproductive disorder in
buffaloes of District Faisalabad, Pakistan was recorded as 46.18%. Repeat breeding was illustrating
the highest prevalence (15.69%) among all reproductive disorders. The subsequently important
disorders were anestrous (9.74%), genital prolapse (7.73%) and abortion (5.99%), retained placenta
(2.58%), uterine torsion (2.39%). The least occurrence of dystocia in buffaloes was found as 2.06%.
The overall economic losses due to genital prolapse in buffaloes in eight villages of the studied area
were estimated to be Rs. 4,59,500/- The maximum losses were due to dam mortality (39.17%),
followed by loss in milk (25.14%), service charges (21.33%) and cost medicine (14.36%) were
reported (Rabbani et al., 2010). Thus, it was concluded that genital prolapsed, repeat breeding and
anoestrus were the major reproductive problems in buffaloes of District Faisalabad, Pakistan.
1111
REFERENCES
Afzal, M. 2008. Pakistan: FMD current scenario. Livestock and Dairy Development Board
Islamabad.
Bachaya, H., A. M. A. Raza, S. Murtaza and I. U. R. Akbar. 2011. Sub-clinical bovine mastitis in
Muzaffar Garh District of Punjab (Pakistan). J. Anim. Plant Sci. 21(1):16-19.
Bhatti, S. U. 2008. Prevalence and epidemiology survey of leptospirosis in buffalo, cattle and hu
man beings in rural and peri urban areas of Punjab. PhD Thesis, University of Agriculture
Faisalabad, Pakistan.
Bhikane, A.V. and S.B. Kawitkar. 2000. Hand book for Veterinary Clincian.Venkatesh Books.
Udgir, India.
Farooq, U., M. Hussain, H. Irshad, N. Badar, R. Munir and Q. Ali. 2007. Status of haemorrhagic
septicaemia based on epidemiology in Pakistan. Pak. Vet. J. 27:67-72.
Javed, M. T., M. Usman1, M. Irfan and M. Cagiola. 2006. A study on tuberculosis in buffaloes:
some epidemiological aspects, along with haematological and serum protein changes. Vet.
Arhiv. 76 (3): 193-206
Khan, A., M. K. Saleemi, M. Z. Khan, S. T. Gul, M. Irfan and M. S. Qamar. 2011. Hemorrhagic
Septicemia in buffalo (Bubalus bubalis) calves under subtropical conditions in Pakistan.
Pak. J. Zool. 43(2): 295-302.
Khan, I. A. and A. Khan. 2007. Prevalence and risk factors of bovine tuberculosis in Nili –Ravi
buffaloes in the Punjab, Pakistan. Italian J. Anim. Sci. 6: 817-820.
Khan, I. A., A. Khan, A. Mubarak and S. Ali. 2008. Factors affecting prevalence of bovine
tuberculosis in Nili Ravi buffaloes. Pak. Vet. J. 28(4): 155-158.
McDermott, J. J. and S. M. Arimi. 2002. Brucellosis insubSaharan Africa: epidemiology, control
and impact. Vet. Microbio. 90(4): 111–134.
Mustafa, Y. S., F. N. Awan, T. Zaman, S. R. Chaudhry and V. Zoyfro. 2011. Prevalence and
antibacterial susceptibility in mastitis in buffalo and cow in and around the district Lahore,
Pakistan. Pak. J. Pharm. 24(2): 29-33.
Pakistan Economic Survey, 2011-12. Government of Pakistan, Finance Division, Economic
Advisor Wing, Islamabad.
Rabbani, R. A., I. Ahmad, L. A. Lodhi, N. Ahmad and G. Muhammad. 2010. Prevalence of various
reproductive disorders and economic losses caused by genital prolapse in buffaloes. Pak.
Vet. J. 30(1): 44-48.
Radostitis, O.M., C.C. Gay, D.C. Blood and K.W. Hichiff, 2000. Veterinary Medicine, 9th edition.
W.B. Saunders Company, London, UK
Raza, S.H., A. Ullah, S. Khan and N. Teufel. 2000. Buffalo milk production by small, medium and
large farmers and their response to different innovations in the Punjab (Pakistan). Proc.of
the Third Asian Buffalo Cong. Kandy, Sri Lanka. Pp. 191-196.
Roeder, P. 2000. A preliminary appraisal of current rinderpest epidemiology in Pakistan and
implications for rinderpest control, at National workshop on Rinderpest epidemiology,
surveillance and control, held in Islamabad, Pakistan. TCP/ Pak/8923 A.
Shafee, M., M. Rabbani, A. A. Sheikh, M. Ahmad and A. Razzaq. 2011. Prevalence of bovine
brucellosis in organized dairy farms, using milk ELISA, in Quetta city, Balochistan,
Pakistan. Vet. Med. Intern.
Sharma, Neelesh, S.K. Gupta, U. Sharma and K. Hussain. 2007. Treatment of clinical mastitis in
buffalo-A case report. Buffalo Bulletin. 26(2):56-58.
Tabatabaei, M., Moazzeni Jula, G. R. Jabbari and A. M. Esmail. 2007. Vaccine efficacy in cattle
against hemorrhagic septicemia with live attenuated aroA mutant of Pasteurella multocida
B:2 strain. J. Cell Anim. Biol. 1:62-65.
1112
Zulfiqar, M. 2003. Draft report for development of National disease control policy for foot and
mouth disease in Pakistan under the FAO project “Support for emergency prevention and
control of main trans-boundary animal diseases in Pakistan rinderpest, FMD, PPR”.
Table 1. Comparative disease susceptibility percentage in Pakistani buffalo
Condition Prevalence Percentage Referances
Foot and mouth

61.7 Afzal, 2008
disease
Clinical=40.35
Mastitis Mustafa et al., 2011
sub-clinical=59.64
Haemorrhagic septicaemia 49 Farooq et al., 2007
46.18
Reproductive disorders Rabbani et al., 2010
Tuberculosis 12.72 Khan and Khan, 2007
Brucellosis 3 Shafee et al., 2011
leptospirosis 11.88% Bhatti, 2008
1113
Epidemiological studies on mastitis in pakistani buffalo

Sidra MANZOOR, Asif NADEEM, Masroor Ellahi BABAR1, Tanveer HUSSAIN and
Maryam JAVED
54000, Pakistan.
1
*
Corresponding email: sidzm@yahoo.com
ABSTRACT
One of the most prevalent diseases of the dairy industry throughout the world that affects
both quality and quantity of milk in dairy animals is mastitis. It is the inflammation of the
parenchyma cells of the mammary glands, characterized by microbial and physiological changes.
Epidemiological studies showed that the risk factors of mastitis are the age, lactation number, stage
of pregnancy and lactation, dry period length, hard milking and calf suckling. Highest prevalence is
usually examined in 3-4 lactation and between the ages of 7-9 years of buffaloes. The occurrence of
disease is also high in non-pregnant but lactating animal and increased as the stage of lactation
proceeded. Mastitis was found to be inversely proportional to the dry period length. Staphylococcus
aureus and Streptococcus agalactiae are considered as most etiological agents of mastitis in
buffaloes. The current study is intended to determine the frequency, distribution and risk factors of
mastitis in buffalo population. Another aspect of the study is to determine different types of
microorganisms associated with mastitis and to see the effectiveness of commercially available
antibiotics against these agents.
Keywords: Mastitis, Epidemiology, Buffalo, Pakistan
INTRODUCTION
Mastitis (Greek Mastos, breast + it is, inflammation) is complex disease due to the
inflammation of parenchyma cells of mammary glands in mammals. It is usually characterized by
bacteriological, physical and chemical changes (Baloch et al., 2011; Akhtar et al., 2012) in milk and
pathological changes in glandular tissues (Radostits et al., 2000; Sudhan and Sharma 2010).
Mastitis has been recognized as one of the most economically important disease throughout the
world (Chishty et al., 2007; Ali et al., 2011; Mustafa et al., 2011) as it adversely affects animal
health, composition, quality and quantity of milk (Raza et al., 2000; Sharma et al., 2007). It causes
production losses in the form of reduction in milk production (up to 70%), milk discard after
treatment (9%), veterinary services cost (7%) and earlier culling of animals (14%) (Bhikane and
Kawitkar, 2000). About $ 184 per animal annual losses were estimated due to mastitis. Apart from
its economic importance it also enhances the chances for the transmission of major zoonotic
diseases like tuberculosis, leptospirosis, brucellosis and streptococcal sore throat to humans
(Radostits et al., 2000; Bachaya et al., 2011).
Mastitis has two forms, clinical and subclinical mastitis. Clinical mastitis exhibit all five
basic signs of udder inflammation including redness, swelling, heat, pain and loss in milk yield.
(Bachaya et al., 2011; Akhtar et al., 2012). The sub clinical form of mastitis is characterized by
having no evident symptoms either in the udder or in the milk. However, a negative relationship
exists between somatic cell count (SCC) and the milk yield (Khan and Khan 2006). It is reported
that sub-clinical form is 15-40 times more prevalent as compared to the clinical mastitis (Shearer
and Harris, 2003), responsible for two third losses of the total milk production due to affected
quarters of animal (Radostits et al., 2007). The sub-clinical mastitis infections in United States are
responsible for 60-70% of total economic losses (Merill and Galton, 1989). These losses may even

1114
be higher in Pakistan due to lack of mastitis prevention practices like teat dipping and dry period
antibiotic therapy. (Shakoor, 2004; Sharif and Ahmad 2007).
Etiology
Mastitis is considered to be a multifactorial disease, caused by a group of pathogenic
bacteria (Bezek and Hull, 1995), viruses (Wallenberg et al., 2003), fungi and algae (Radostit et al.,
1996). The bacteria responsible for udder inflammation are grouped as contagious or
environmental, based on their primary reservoir and transmission mode. The Staphylococcus
aureus, Streptococcus agalactiae and Corynebacterium bovis are referred as the contagious
pathogens of mastitis in buffaloes (Singh et al., 2005; Sharma et al., 2007) usually transmitted from
infected to clean udder, contact with infected milk and through flies. Staphylococcus aureus is
considered as most etiological agent, has reduced an average of 34.5% of udder’s potential milk
production while the total loss of milk yield is estimated as 6.8% per animal (Gebreyohannes et al.,
2010). Among the environmental pathogens the most common are the Coliforms (E. coli,
Enterobacter, Klebsiella), Strepcococcus uberis, Streptococcus bovis and Streptococcus
dysgalactiae, usually transmitted between and during milking (Sudhan and Sharma 2010). In
Pakistan, Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli are the most
prevailing pathogens which accounts for about 78% cases of mastitis in country (Ahmed, 2001;
Akhtar et al., 2012). In Sindh province of Pakistan; Staphylococcus aureus, Bacillus cereus,
Escherichia coli, Micrococcus luteus, Proteus vulgaris, Pseudomonas aeruginosa, Streptococcus
dysgalactiae, Streptococcus uberis and Citrobacter species and their incidence in milk samples was
48.57, 2.85, 10.0, 15.71, 4.28, 1.42, 11.42, 4.28 and 1.42% respectively. (Baloch etal., 2011).
Approximately 70-80% of Coliform infections is manifested by abnormal milk, udder swelling and
systemic disturbances including high fever, swollen quarters, diluted milk and reduced appetite, is
often responsible for the clinical cases.
Prevalence studies from Pakistan
Among various reports on prevalence studies of mastitis in buffaloes include Bilal et al.,
2004 who studied peri-urban and rural areas of Faisalabad and reported that the clinical mastitis
prevalence of was (25.12%) in peri-urban while (19.74%) in rural areas, however subclinical
mastitis prevalence was found 51% (A. Sharif and T. Ahmad 2007). Staphylococcus aureus was
found major while Streptococcus agalactiae was the second most isolated mastitis-pathogen found
in buffalo milk in Faisalabad. (Ashfaq and Muhammad, 2008). The study from tehsil Burewala,
district Vehari, Pakistan indicated highest relative prevalence of Staphylococcus aureus (53.85%)
followed by Streptococcus agalactiae (23.07%), Escherichia coli (15.38%), Streptococcus
dysagalactiae (3.85%), and Corynebacterium bovis (3.85%) in buffaloes as cause of mastitis.
(Hameed et al., 2008). Prevalence of subclinical mastitis in four districts (Lahore, Sialkot, Narowal
and Okara) of Punjab, Pakistan was found 44% in lactating dairy buffaloes (Ali et al., 2011). The
prevalence of clinical and subclincal mastitis was 40.35% and 59.64% in buffaloes from Lahore
district. (Mustafa et al., 2011). In Attock district 77.98% prevalence of sub-clinical mastitis was
found (Bachaya et.al., 2005). The overall 53% prevalence of sub-clinical mastitis was found in
buffaloes from district Dera Ismail Khan of Khyber Pakhtunkhwa, Pakistan. (Akhtar et al., 2012)
Other Risk Factors
Certain physiological (stage of lactation and lactation number) and managemental (source of
milk let down, method of milking and floor condition) factors are also attributed to the prevalence
of clinical mastitis in buffaloes (Bilal et al., 2004). As the stage of lactation proceeds, the udder
tissues are more prone to mastitis infection. This might be due to retention of milk in the teat canal
to develop udder infection during this period (Schalm et al., 1971). An association exists between
the number of lactations and incidence of mastitis. Highest prevalence is usually examined in 3-4
lactation and between the ages of 7-9 years of buffaloes. Management also acts as predisposing
factor when better practices reasonably reduce the occurrence of mastitis (Faull et al., 1985). The
milking practice is one of the significant risk factors for clinical mastitis as well as high SCC.
1115
Proper milking procedure is important to reduce the incidence of mastitis (Nickerson 1990). Data
revealed that high frequency is present when folded thumb method is used. The teat is might be
injured by full handed approach leads to the development of adverse effects on teat canal (Bilal et
al., 2004).
Effect of mastitis on milk composition
The milk quality is affected in mastitis in terms of decrease in protein, fat, milk, sugar
contents and increase in SSC. The processing of such milk results in decrease in the quality of milk
products like yogurt and cheese (Sharif and Ahmad 2007) and reduction in shelf life of processed
milk (Urech et al., 1999). Due to increased vascular permeability, Serum albumin,
immunoglobulins, transferrin and other serum proteins pass into milk. The milk composition of
normal with that of mastitis milk contain high SCC as described by Jones (2006) is illustrated in
(Table 1).
The quality of milk and udder health is usually monitored by the screening of SCC in milk.
The elevated level of SCC consists primarily of leucocytes including macrophages, lymphocytes
and neutrophils. During inflammation, there is a rapid influx of neutrophils into milk. Jones (2006)
reported that elevated SCC poses the higher risk of milk contamination with pathogens while lower
level results in high milk production and better milk quality.
Antibiotic therapy
Mastitis is among the primary factors responsible for the shortfall of milk supply in
Pakistan. The prevalence of the pathogens causing mastitis can be controlled by using antibiotic
susceptibility of the pathogens for different antibiotics. The effectiveness of different antibiotics
against the pathogenic isolates is in descending order to Enrofloxacin, Chloramphenicol,
Gentamycin, Oxytetracycline and Amoxicillin (Hameed et al., 2008). Enrofloxacin is reported to be
the most effective and Amoxicillin has a slightest effect against various mastitis pathogens. Dhakal
et al. (2007) found that Enrofloxacin had the highest sensitivity (91 %) for all types of bacteria and
the effectiveness of Gentamycin and Chloramphenicol was 87% and 82% respectively. Less
effectiveness of Amoxicillin to all the isolates may be due to the resistance produced in the bacteria
due to extensive use of this antibiotic in buffaloes.
REFERENCES
Ahmad, R. 2001. Studies on mastitis among dairy buffaloes. Pak. Vet. J. 21:220-221.
Akhtar, A., Habibullah, M. Ameer, Hidayatullah and M. Arshad. 2012. Prevalence of sub clinical
mastitis in buffaloes in district D. I. Khan Pakistan. Pak. J. Sci. 64(2).
Ali, M. A., M. D. Ahmad, K. Muhammad and A. A. Anjum. 2011. Prevalence of sub clinical
mastitis in dairy buffaloes of Punjab. J. Ani. Plant Sci. 21(3):477-480.
Ashfaq, K. and G. Muhammad. 2008. Pathogens Associated with Bovine and Bubaline Mastitis in
Peri-Urban Areas of Faisalabad, Pakistan. Pak. J. Life Soc. Sci. 6(2): 86-88.
Bachaya, H. A., M. A. Raza, S. Murtaza and I. U. R. Akbar. 2011. Sub-clinical bovine mastitis in
Muzaffar Garh District of Punjab (Pakistan). J. Anim. Plant Sci. 21(1):16-19.
Bachaya, H., A. Z. Iqbal, G. Muhammad, A. Yousaf and H. M. Ali. 2005. Sub-clinical mastitis in
buffaloes in Attock District of Punjab (Pakistan). Pak. Vet. J. 25(3):134-136.
Baloch, H., R. Rind, D. H. Kalhoro and A. B.Kalhoro. 2011. Study on the incidence of clinical mastitis
in buffaloes caused by bacterial species. Pak. J. Agri., Agril. Engg., Vet. Sci. 27 (1):83-93.
Bezek, D., M. and B. L. Hull. 1995. Peracute gangrenous mastitis and chelitis associated with
enterotoxin secreting Staphylococci.Canad. Vet. J. 36:106-107.
Bhikane, A.V. and Kawitkar, S.B. 2000. Hand book for Veterinary Clincian.Venkatesh Books.
Udgir, India.
Bilal, M. Q., M. U. Iqbal, G. Mohammad, M. Avais and M. S. Sajid. 2004. Factors affecting the
prevalence of clinical mastitis in buffaloes around Faisalabad district (Pakistan). Int. J. Agri.
Biol. 6:185-187.
1116
Chishty, M., A. M. Arshad, M. Avais and M. Ijaz. 2007. Cross-sectional epidemiological studies on
mastitis in cattle and buffaloes of tehsil Gojra Pakistan, Buff. Bull. 26:50-55.
Dhakal, I.P., P. Dhakal, T. Koshihara and H. Nagahata. 2007. Epidemiological and bacteriological
survey of buffalo mastitis in Nepal. J. Vet. Med. Sci. 69:1241-1245.
Faull, W.B., J.W. Hughes, M.J. Clearkson and G.S. Walton. 1985. Mastitis notes for the Dairy
Practitioner. Liverpool Univ. Press, Liverpool L69 3BX, UK.
Gebreyohannes Y., Tesfaye, F. G. Regassa and B. Kelay. 2010. Milk yield and associated economic
losses in quarters with subclinical mastitis due to Staphylococcus aureusin Ethiopian
crossbred dairy cows. Trop. Anim. Health Prod. 42:925–931.
Hameed, S., M. Arshad, M. Ashraf, M. Avis and M. A. Shahid. 2008. Prevalence of common
mastitogens and their antibiotic susceptibility in tehsil Bhurewala, Pakistan. Pak. J. Agri.
Sci. 45(2).
Jones, G. M. 2006. Understanding the basics of mastitis. Virginia Cooperative Extension. Publica-
tion No. 404-233. Virginia State University, USA. pp:1-7.
Khan, M. Z. and A. Khan. 2006. Basic facts of mastitis in dairy animals: A review Pakistan Vet. J.
26(4): 204-208.
Merill, W.G. and D.M Galton, 1989. Mastitis and its control. In: Milk Quality.A pro-dairy
management focus workshop for farm managers, Cornell University. New York, USA
Mustafa, Y. S., F. N. Awan, T. Zaman, S. R. Chaudhry and V. Zoyfro. 2011. Prevalence and
antibacterial susceptibility in mastitis in buffalo and cow in and around the district Lahore,
Pakistan. Pak. J. Pharm. 24(2): 29-33.
Nickerson, S. C. 1990. Production of quality milk and control of mastitis in Mexico. Diary
Research Report. Hill Farm Research Station Route-1,Box 10. Homer, LA 71040, USA.
Radostit. O. M., D. C. Blood and C. C. Gray. 1996. Veterinary Medicine. Text Book of the diseases
of cattle, sheep,goats and horses. 8th Ed. London Baillere Tindal.
Radostitis, O.M., C.C. Gay, D.C. Blood and K.W. Hichiff, 2000. Veterinary Medicine, 9th edition.
W.B. Saunders Company, London, UK
Radostits, O. R., D. C. Blood and C. C. Gay. 2007. Mastitis. Veterinary Medicine: A textbook of
the diseases of cattle, horses, sheep, pigs and goats, 9th Edn., Bailer tindall, London.
pp:563-614.
Raza, S.H., A. Ullah, S. Khan and N. Teufel. 2000. Buffalo milk production by small, medium and
large farmers and their response to different innovations in the Punjab (Pakistan). Proc. Of
the Third Asian Buffalo Cong. Kandy, Sri Lanka. Pp. 191-196.
Schalm, O.W., E.J. Carroll and N.C. Jain, 1971. Bovine Mastitis.Lea and Febiger, Philadelphia.
Shakoor, A. 2004. Preparation and evaluation of staphylococcus aureusvaccines for the control of
mastitis in dairy buffaloes (Bubalis bublais). PhD Thesis, University of Agriculture
Sharif, A. and T. Ahmad. 2007. Prevalence of severity of mastitis in buffaloes in district
Faisalabad (Pakistan). J. Agric. Soc. Sci. 3:34–36.
Sharma, Neelesh, Gupta, S.K., Sharma, U. and Hussain, K. 2007. Treatment of clinical mastitis in
buffalo-A case report. Buffalo Bulletin. 26(2):56-58.
Shearer, J. K. and B. Harris. 2003. Mastitis in dairy goats. Anim. Sci. Dept. Florida Coop. Ext.
Serv. Inst. Food Agri. Sci; Univ. Fl. Gainesville, USA. pp:1-6.
Singh, R., Sharma, Neelesh, J.S. Soodan and N.A. Sudhan. 2005. Etiology and sensitivity of
bacterial isolates from sub clinical mastitis in cattle from Jammu region. J. Res. SKUAST-J.
4(2):223-224.
Sudhan, N. A. and N. Sharma. 2010. Farm management and Diseases. Mastitis- An Important
Production Disease of Dairy Animals. SMVS-181102. Jammu, India.
Ullah, S., 2004. Effect of mastitis on milk composition in buffaloes under field conditions. MSc
(Hons.) Thesis. University of Agriculture Faisalabad, Pakistan.
Urech, E., Z. Puhan and M. Schallibaum, 1999. Changes in milk protein fraction as affected by sub-
clinical mastitis. J. Dairy Sci. 82:2402–2411.
1117
Wallenberg G. J., V. D. Poal, W. H. M. Vana and J. T. Oirschot. 2003. Viral infections and bovine
mastitis. A review. Vet. Microbiol.
Yousaf, M., G. Muhammad, M. Q. Bilal and S. Firyal. 2012. Evaluation of non-antibiotics alone
and in combination with cephradine in the cure rates in clinical bubaline mastitis. J. Anim.
Plant Sci. 22(3):207-211.
Table 1. Comparison of percentage of normal milk with that of mastitis milk.
Constituent Normal milk Mastitis milk with high SCC

Fat 3.5 3.2
Lactose 4.9 4.4
Total protein 3.61 3.56
Total casein 2.8 2.3
Whey protein 0.8 1.3 ↑
Serum albumin 0.02 0.07 ↑
Lactoferrin 0.02 0.1 ↑
Immunoglobulin 0.1 0.60 ↑
Sodium 0.057 0.105 ↑
Chloride 0.091 0.147 ↑
Source: Jones (2006)
1118
Excretion of Aflatoxin M1 in milk of Mediterranean Italian buffalo cow

fed diet naturally contaminated of Aflatoxin B1
Gilberto GIANGOLINI, Carlo BOSELLI, Francesco FILIPPETTI, Remo ROSATI, and
Simonetta AMATISTE
Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, - Via Appia Nuova, 1411 –
00178, Roma – Italy
*Corresponding email: gilberto.giangolini@izslt.it
ABSTRACT
Aflatoxins (AFs) are a group of mycotoxins produced by Aspergillus genus. Dairy animals
fed with diet containing aflatoxin B1 (AFB1) excrete aflatoxin M1 (AFM1) in milk. The buffalo also
excretes small amounts of AFB1. In European Union (EU) the law limit is 0.05 μg/kg (50 ng/kg) of
AFM1 in the milk. During the experimental period, eight Mediterranean Italian buffalo cow in
lactating were used to study the conversion of dietary AFB1 into AFM1. The buffaloes were fed
individually for six days with naturally contaminated corn meal (217 µg AFB1/buffalo/day) and for
additional three days with a diet free of AFB1. The milk yield by each animal during the test
(morning and evening milking) was collected and analyzed. The concentration of AFM 1 and AFB1
in milk was determined by HPLC method with fluorimetric detection. AFB1 was determined after
derivatization of the extract purified with trifluoroacetic acid. The carry over of AFM1, determined
during the steady-state phase was 0.190% (Range 0.123% - 0.280%). The rate of transfer of AFB1
from diet to milk was 0.019% (Range 0.012% - 0.020%). After 48 hours from the last
administration the concentration of AFM1 in milk drops below the law limit (29.86 ± 9.77 ng/kg)
and after 24 hours falls to 6.65 ± 2.39 ng/kg.
Keywords: aflatoxins, buffalo milk, carry over
INTRODUCTION
Aflatoxins, a class of mycotoxins, are produced by filamentous fungi particularly by certain
strains of Aspergillus flavus and Aspergillus parasiticus. Aflatoxin B1 (AFB1) is considered one of
the most potent known natural hepatic-carcinogen for mammals. Aflatoxin M1 (AFM1) is the
principal hydroxylated metabolite of AFB1 (Yiannikouris et al., 2002) and is excreted on small
amounts of the metabolite in milk (Wood 1991). The concentration of AFM1 in animal milk is
related to the dose of AFB1 ingested with the diet and is affected by milk yield (Masoero et al.,
2007), stage of lactation (Veldman et al., 1992), the duration of the period of ingestion and other
factors such as breed, health status and mammary alveolar cell membrane health. Studies in
different experimental conditions showed Carry Over (CO) values between 1% and 3% for the cow,
and lower values (<1%) for the buffalo (Pietri et al., 2003), sheep (Battacone et al., 2005) and goat
(Mazzette et al., 2009; Giangolini et al., 2011).
In Italy, buffalo milk is used for the production of mozzarella cheese and other typical dairy
products. Several studies have signalled the occurrence of AFB1 in buffalo milk (Pietri et al., 2003,
Fedele et al., 2007). The aim of this study was to investigate the transfer of AFM1 in milk of
Mediterranean Italian buffalo cow fed diets naturally contaminated with AFB1 and observe the time
that elapses from the last administration of AFB1 up to the AFM1 get below the European law limit
in milk (50 ng/kg).

Eight Mediterranean Italian buffalo cow (parity of 3 ± 1, in mid-lactation), were fed for 6
days with naturally contaminated corn meal (217 µg AFB1/buffalo/day) and monitored for 3 days
with a diet free of AFB1. The buffalo were milked twice a day, at 07:00 a.m. and 18:00 p.m., in a
Herringbone milking parlour, set at 44 kPa, 60 cycles/minute and pulsation rate 50:50.
1119
Each buffalo cow was milked and the milk was collected in a buklet and measured by graduated
cylinder. For each buffalo at each milking were collected one sample (200 mL) representative of
total milk yield and stored at -20°C before analysis.
AFM1 and AFB1 were determined in each sample of milk by HPLC with fluorimetric
detection (HPLC chromatograph connected to a reverse-phase C18 column). The extraction of AF
was done using an immunaffinity tecnique (Vicam Aflatest WB). The determination of AFB1 was
performed after derivatization of the purified extract with trifluoroacetic acid.
Fat, protein, lactose, casein, urea were determined in milk by MilkoScan FT 6000 (Foss
Electric, Hillerød, Denmark) and Somatic Cell Count (SCC) by Fossomatic 5000 (Foss Electric,
Hillerød, Denmark).
The transfer rate of Aflatoxin from feeds to milk during the experimental period was
calculated by AFM1/AFB1 ratio (AFM1 excreta in milk / AFB1 ingesta with feed x 100). The Carry
Over in milk of AFM1 was determined during the steady-state phase by ratio between the AFM1
excreted in milk and the intake of AFB1.
AFM1 concentrations during the experimental period (9 days) were analysed by ANOVA
using SPSS software (ver.13.0). The differences of means were tested with Bonferroni adjustment.
Results are presented as means ± standard deviation.

AFM1 in milk was <6 ng/kg before the start of trial. The daily average concentration of
AFM1 in milk after the first day of administration was 24.08 ± 8.94 ng/Kg.
During the experimental period the average concentration of AFM1 was 40.51 ± 27.92 ng/kg, while
the average concentration of AFB1was 3.39 ± 2.17 ng/kg. Excretion of AFM1 increased rapidly,
reaching the maximum value at the sixth day of trial (70.01 ± 16.19 ng/kg), then decreases linearly
until it reaches 6.65 ± 2.39 ng/kg in the last day of study (Tab.1).
The maximum value of AFB1 was observed at the third day of trial (5.89 ± 1.39 ng/kg)
(Tab.1).The rate of transfer of AFB1 from feeds to milk AFM1 (AFM1/AFB1 ratio) during the
experimental period was 0.240%. The Carry Over of AFM1, determined during the steady-state
occurred from the third to sixth day, was 0.190% (range: 0.123% - 0.280%).
The rate of transfer of AFB1 in milk was 0.019% (AFB1 escreta/AFB1 ingesta) (range: 0.012% -
0.020%).
The individual daily milk production did not show significant variation during the
experimental period, the average was 6.88 ± 1.39 kg / buffalo / day.
The average content of fat, protein, lactose, casein and urea was 9.67 ± 1.17%, 4.89 ±
0.23%, 4.61 ± 0.18%, 4.02 ± 0.23%, 33.2 ± 11.4 mg/dL respectively, while the SCC was 5.86 ±
0.14 log10 cell/mL. We observed significant differences (P<0.001) for the content of urea that
increase until the seventh day (47.1 ± 9.4 mg/dL) and then decrease progressively at the end of the
trial (32.8 ± 3.4 mg/dL).
After the last AFB1 feed administration, the AFM1 decreased and went below the law limit
after 48 hours (29.86 ± 9.77 ng/kg) and the clearance time was 72 hours. In other studies clearance
times were similar and comprised between 24 and 48 hours (Pietri et al., 2003; Fedele et al., 2007).
The low Carry Over observed in this study may be due to several factors: the low level of
milk yield, similarly as observed in dairy cows where AFM1 excretion depends on the level of
production (Britzi et al., 2013) and the capacity of the ruminal microbial ecosystem to degrade or
convert AFB1 into other metabolites (Fedele et al., 2010). In addition we have to consider the
individual variability of the hepatic bio-transformation capacity.
In Europe the regulation establishes the maximum limits authorized only for AFM1 in milk, while
no indication exists for AFB1.
The Carry Over of AFM1 obtained in our study (0.190%) for Mediterranean Italian buffalo
is similar to those obtained by other authors.
In the experimental period were not observed changes in milk yield and main chemical physical
parameter except for urea.
1120
In our experiment the contamination of milk with AFM1 leads to clearance times 72 hours after the
last administration of AFB1.
The presence of AFB1 in buffalo milk showed that not all of AFB1 ingested was bio-transformed in
AFM1. In consideration of the carcinogenic properties of AFB1 more studies are needed to
determine the Carry Over in milk of AFB1.
ACKNOWLEDGEMENTS
This study was supported by the Ministry of Health (Current Research Grant IZS LT 12/05
RC
REFERENCES
Battacone, G., A. Nudda, M. Palomba, M. Pascale, P. Nicolussi and G. Pulina. 2005. Transfer of
aflatoxin B1 from feed to milk and from milk to curd and whey in dairy sheep fed
artificially contaminated concentrates. J. Dairy Sci. 88: 3063-3069.
Britzi, M., S. Friedman, J. Miron, R. Solomon, O. Cuneah, J.A. Shimshoni, S. Soback, A. Rina,
A and Sima, A. Shlosberg. 2013. Carry-Over of Aflatoxin B1 to Aflatoxin M1 in High
Yielding Israeli Cows in Mid- and Late-Lactation. Toxins 5:173-183.
Fedele, V., L. Sepe, G.F. Cifuni, S. Claps and M.A. Di Napoli. 2010 in vitro degradability of
aflatoxin in rumen liquid of buffalo. Revista Veterinaria 21: Suppl. 1 672-67.
Fedele, V., G.F. Cifuni, L. Sepe and M.A. Di Napoli. 2007. Effect of two aflatoxin treatments on
contamination of mozzarella di bufala cheese in vitro degradability of aflatoxin in rumen
liquid of buffalo. Ital. J. Anim. Sci. 6: 2 1120-1122.
Galvano, F., A. Galofaro, M. De Angelis, M. Galvano, M. Bognanno and G. Galvano. 1998. Survey
of the occurrences of aflatoxin M1 in dairy products marketed in Italy. J. Food Prot. 61:
738-741.
Giangolini, G., F. Filippetti, C. Boselli, A. Gulli, A. Proietti, S. Amatiste and R. Rosati. 2011.
Excretion of Aflatoxin M1 in milk of Maltese goats fed diet naturally contaminated of
aflatoxins. IDF (Poster session) 16-18 May 2011 Athens, Greece.
Masoero, F., A. Gallo, M. Moschini, G. Piva and D. Díaz. 2007. Carry over of aflatoxin from feed
to milk in dairy cows with low or high somatic cell counts. Animal, 1:1344–1350.
Mazzette, A., M. Decandia, M. Acciaro, A. Fenu, A. Francesconi and G. Battacone. 2009.
Excretion of Aflatoxin M1 in milk of goats fed diet contaminated by Aflatoxin B1.
Italian Journal of Animal Science, Vol. 8 (Suppl. 2), p. 631-633.
Pietri, A., T. Bertuzzi and A. Fortunati Gualla. 2003. Excretion pattern of aflatoxins in buffalo milk
and carry-over in mozzarella cheese. Ital. J. Anim. Sci. 2: 302-304.
Veldman, A., J.A.C. Meijs, G.J. Borggreve and J.J. Heeres-van der Tol. 1992. Carry-over of
aflatoxin from cows’ food to milk. Journal of Animal Production, 55:163-168.
Wood., G.E. 1991. Aflatoxin M1. In: Sharma, R.P., Salunkhe, D.k. (Eds.), Mycotoxins and
Phytoalexins. CRC, London, pp. 145–163.
Yiannikouris, A. and J.P. Jouany. 2002. Mycotoxins in feeds and their fate in animals: a review.
Anim. Res. 51:81-99.
1121
Table 1. Excretion of Aflatoxins (AFM1 - AFB1) in milk during the experiment.
Days of trial AFM1 (ng/Kg) AFB1 (ng/Kg)

1 (diet with AFB1) 8.01±1.93c 0.24±0.05b
2 (diet with AFB1) 24.08± 8.94bc 4.04±1.73ac
3 (diet with AFB1) 42.54± 21.91bdefg 5.89±1.39a
4 (diet with AFB1) 59.37± 10.88ae 5.73±1.64a
5 (diet with AFB1) 62.72± 22.06ad 4.19±0.61a
6 (diet with AFB1) 70.01± 16.19a 4.41±1.23a
7 (diet without AFB1) 61.32± 30.97af 3.22±1.33bc
8 (diet without AFB1) 29.85± 9.77cg 1.31±0.35b
9 (diet without AFB1) 6.65± 2.39c 1.47±0.14b
Different superscripts within a column indicate significant differences (p<0.05).
1122
Preliminary Results on the Utilization of Phytotherapy in Organic Italian

Mediterranean Buffaloes
Gianluca NEGLIA, Roberta CIMMINO, Domenico VECCHIO, Valentina LONGOBARDI,

Giuseppe ALBERO, Francesco SALZILLO, Luca DE LUISE, Rossella DI PALO.
Department of Veterinary Medicine and Animal Production, Federico II University, Via F. delpino
1, 80137 Napoli, Italy
*Corresponding email: neglia@unina.it
ABSTRACT
It is known that the use of allopathic drugs is forbidden in organic agriculture. For this
reason a growing interest has been recently focused on homeopathy and phytotherapy. The aim of
this study was to evaluate the efficacy of some phytotherapic treatments in organic buffalo
breeding. The trial was performed between November and July on 127 pluriparous Italian
Mediterranean buffalo cows bred in an organic farm located in the South of Italy. Before the
calving period, all buffaloes were divided into four groups according to days of gestation, number
of lactations and milk production recorded in the previous year. After calving subjects in each
Group underwent the following treatments: 1) Group A (n=36): no treatments during the first 30
days post-calving; 2) Group B (n=32): Intrauterine treatment by Aloe Arborescens, Daucus Carota,
Calendula, Propoli, Tea tree oil (Aloe Lesionex gel, Nutrizoo, Caserta, Italia), between 20 and 25
days post-calving; 3) Group C (n=32): treatment by 4 vaginal pessaries constituted by Aloe
Arborescens, Tea tree oil, Calendula Officinalis, Propolis (Aloe Lesionex Ovuli, Nutrizoo,
Caserta, Italia), within 4 days post-calving and 4)Group D (n=28): treatment with both pessaries
constituted by Aloe Arborescens, Tea tree oil, Calendula Officinalis, Propolis (Aloe Lesionex
Ovuli, Nutrizoo, Caserta, Italia), within 4 days post-calving and the intrauterine treatment by Aloe
Arborescens, Daucus Carota, Calendula, Propoli, Tea tree oil (Aloe Lesionex gel , Nutrizoo,
Caserta, Italia), between 20 and 25 days post-calving. Buffalo bulls of proven fertility were present
in the herd throughout the experimental period. Clinical examinations were performed 15 Days
apart on all the subjects until pregnancy was assessed (30-35 days) and confirmed (90 days). The
calving-conception interval (CCI) and the fertility rate (FR) was assessed for each Group.
Differences among groups in FR were analyzed by Chi-square test and differences in CCI by
ANOVA. No differences were observed in terms of pregnancy rate among groups. Fertility rate
ranged from 62.5% in Group C to 52.8% in Group A and intermediate value were recorded in
Group B (59.4%) and D (57.1%). However, a significantly lower (P<0.05) calving-conception
interval was observed in buffaloes treated by vaginal pessari within 4 days post-calving (Group C)
compared to Group A and Group B (54.1±18.5, 68.0±22.4 and 71.8±16.3 in Group C, A and B,
respectively). According to these results, in organic buffalo farming a phytoterapic treatment by
Aloe Arborescens, Tea tree oil, Calendula Officinalis and Propolis within 4 days post-partum
reduces the calving-conception interval.
Keywords: Organic, phytotherapy, fertility, Calving-conception interval.


1123
Buffalo Physiology
Bone Marrow´S Harvest in The Coxal Tuberosity for Isolation and Culture of
Mesenchymal Stem Cells of Buffaloes (Bubalus Bubalis)
Eunice OBAa, Leandro MAIAa, Carolina Nogueira de MORAESa*, Amanda Jerônimo
LISTONIa, Carla Martins de QUEIROZa, Flávia Caroline DESTROa and Fernanda da Cruz
LANDIM- ALVARENGAa
a
Department of Animal Reproduction, São Paulo State University – Botucatu, Brazil. Rubião
Júnior, s/n CEP: 18618-970, Botucatu, Brazil
*Corresponding email: carolnmoraes@fmvz.unesp.br
ABSTRACT
Several studies with mesenchymal stem cells (MSCs) have been developed in many species
because of its ability to differentiate into other mesoderm lineages, capacity of self-regeneration,
low immunogenicity, paracrine, anti-inflamatory, immunomodulatory and antiapoptotic effects
which make then a promissory source to be used in therapeutic strategies. The aim of this study is to
report the technique of harvest of bone marrow (BM) in the coxal tuberosity (CT) of buffaloes. For
this, the animals were selected, identified and contained in a stock. Then trichotomy was performed
in the region corresponding to the CT. After identifying the anatomic site it was performed
antisepsis, local anesthetic block and introduction of a myelogram´s needle (Lang ®) for BM
aspiration. Once the needle was firmly fixed in the CT, the mandril was removed and proceeded to
BM aspiration with a syringe (20 mL) containing 1 ml of heparin at 1000 IU / mL and 1 mL of
PBS. After the collection, each sample collected was manually homogenized, identified and
referred to the LRACT - FMVZ / UNESP-BRAZIL for the correct processing. The anatomical site
tested showed to be an alternative site of harvest of BM once provided the appropriate isolation and
culture of the mononuclear fraction. Moreover, the procedure was performed without difficulty and
with great security. Based on this, it can be conclude that CT is an excellent anatomical site for
isolation and culture of MSCs and the proposed technique is viable and feasible to be held in
buffaloes.
Keywords: aspiration, buffaloes, mscs, anatomical site, coxal tuberosity, therapeutic
INTRODUCTION
The ability of mesenchymal stem cells (MSCs) to differentiate into other mesoderm
lineages such as bone and cartilage opened a variety of experimental strategies to investigate the
possibility of these cells to be used in tissue engineering since MSCs derived from bone marrow
(BM ) have been applied in the treatment of musculoskeletal disease in several species (Hepsibha et
al. 2011). Besides the ability to differentiate into other mesoderm lineages, those cells have the
capacity of self-regeneration, low immunogenicity, paracrine, anti-inflamatory, immunomodulatory
and antiapoptotic effects which make these cells a great alternative to be used in regenerative
therapies.
Bone marrow-derived mesenchymal stem cells (BM-MSCs) are the most studied source of
MSCs and because of this have received special attention and are best characterized (Fortier &
Travis 2011). Bone marrow´s harvest on CT has already been described in other animal species
such as horses (Delling et al. 2012; Maia et al. 2011), dog and cat (Townsend 2008), sheep
(Amorim et al. 2012) and pig (Abukawa et al. 2009).
In buffaloes, almost all works in this area focus on studies with embryonic stem cells or
MSCs derived from adipose tissue. Based on this, this work aims to evaluate an alternative site of
harvest of bone marrow in the coxal tuberosity (CT) to verify if this anatomical site was viable,
feasible and provides material properly for isolation and culture of the mononuclear fraction.

1125

Harvest of Bone Marrow
Six healthy adult females, derived from the Buffaloe´s Department of FMVZ-UNESP-
BOTUCATU were selected and used in this experiment. For bone marrow´s harvest the animals
were contained in a stock and the anatomical site of right CT was properly identified prior
trichotomy of the region. After this, it was performed antisepsis with chlorhexidine and alcohol
(Riohex, Rioquímica - Brazil) followed by local anesthetic block with 2% lidocaine (Cristália,
Brazil). Past approximately five minutes, a myelogram´s needle (Lang) was introduced for bone
marrow´s harvest at an angle of approximately 45 degrees relative to the animal in dorsoventral and
craniocaudal direction (Figure 1).
Figure 1. Myelogram´s needle position for bone marrow harvest on CT in buffaloes.
Once the needle was firmly fixed in CT, the mandril was removed and proceeded to BM
aspiration with a syringe of 20 mL properly identified containing 1 ml of heparin at 1000 IU / mL
and 1 mL of PBS (Figure 2). After BM´s harvest, each sample collected was manually
homogenized, properly identified, cooled and referred to the Laboratory of Reproduction and
Advanced Cell Therapy (LRACT), Department of Animal Reproduction and Veterinary Radiology
of FMVZ / UNESP-BRAZIL for the correct isolation, cultivation and characterization of
mononuclear fraction obtained from Ficoll-hipaque (1077, Sigma) in a laminar flux environment.
Figure 2. Bone marrow´s harvest on CT with the use of a syringe of 20 mL.

From each animal it was collected on average 3 mL of BM and this volume collected was
sufficient for the processing of the sample from the animals, promoting correct isolation, culture
1126
and characterization of the cells. Only in one animal it was noted that the volume collected was
lower when compared with the other animals probably because of its age, because this animal was
older than the others. Beside this bone marrow was yellowish and the culture of this sample did not
work probably because of the substitution of red bone marrow for yellow bone marrow.
Additionally comparing bone marrow´s harvest from adipose tissue (Hepsibha et al. 2011)
with bone marrow´s harvest on CT there is the advantage that the second can be done with a live
animal, there is no need to make a surgical wound, and the cells can be used in the same animal.
With this protocol proposed it was evident that the antisepsis, local block and site of
harvest were efficient on buffaloes and can be used in this specie for bone marrow´s harvest from
CT. The anatomical site tested showed to be to be an alternative site of harvest of BM once
provided appropriate isolation, culture and characterization of mononuclear fraction. Beside, the
procedure was performed without difficulty and with great security. Based on the above, it can be
conclude that CT is an excellent anatomical site for isolation and culture of MSCs and the proposed
technique is viable and feasible to be held in buffaloes and can be used in futures researches.
REFERENCES
Abukawa, H., M. Phelps, P. Jackson, R.M. Smith, J.P. Vacanti, L.B. Kaban and M.J. Troulis. 2009.
Effect of ibuprofen on osteoblast differentiation of porcine bone marrow-derived progenitor
cells. J Oral Maxillofac Surg. 67:2412–2417.
Amorim M.R., G.D. Nascimento, L. Maia L., A.J. Listoni, B.S. Paola, D.L.O. Ferreira, R.M.
Cavalcanti and F.C. Landim-Alvarenga. 2012. Bone marrow mesenchymal stem cells from
sheep. In: Procedings of the XXVII World Buiatrics Congress., Lisbon, Portugal. pp.323.
Delling, U., K. Lindner, I. Ribitsch, H. Jülke and W. Brehm. 2012.
Comparison of bone marrow aspiration at the sternum and the tuber coxae in middle-
agedhorses. Can J Vet Res. 76:52-56.
Fortier, L.A. and A.J. Travis. 2011. Stem cells in veterinary medicine. Stem cell Research &
Therapy 2:1-6.
Hepsibha, P., T.V. Meenambigai, A. Mangalagowri, A. Palanisamy, A. Stalin, S. Nithya and K.
Humanan. 2011. Multipotent differentiation potential of buffalo adipose tissue derived
mesenchymal stem cells. Asian J Anim Vet Adv. 6: 772-788.
Maia, L., R.M. Venturini, M.O. Taffarel, N.P.P. Freitas, G.A. Monteiro, B.de Vita, B.A Monteiro,
F.C.Landim-Alvarenga and R.M. Amorim. 2011. Técnica de colheita da medula óssea na
tuberosidade coxal de eqüinos para isolamento e cultivo de células tronco mesenquimais.
Procedings of XII Conferência Anual da ABRAVEQ, Campinas, Brasil. pp 117.
Townsend, F.I. 2008. Bone marrow aspiration in dogs and cats Lab Anim. 37:497–498.
1127
Isolation, Culture and Differentiation of Buffaloes Bone Marrow Mesenchymal

Stem Cells Obtained from the Coxal Tuberosity
Eunice OBA, Amanda Jeronimo LISTONI, Leandro MAIA, Carolina Nogueira de Moraes e
Fernanda da Cruz Landim e Alvarenga
Department of Animal Reproduction, São Paulo State University – Botucatu, Brazil. Rubião
Júnior, s/n CEP: 18618-970, Botucatu, Brazil
ABSTRACT
Currently, much attention has been devoted to the renewal of knowledge about Stem Cells
and Cell Therapy in domestic species. In this sense, the present work aimed to develop a
methodology for collecting, processing and cultivation of mesenchymal stem cells obtained from
bone marrow of coxal tuberosity in buffaloes. The collection was performed using a Komiyashiki
needle, which was introduced in the coxal tuberosity and the bone marrow aspirated into a
heparinized syringe with the aid of negative pressure. Directly after collection samples were
processed at the laboratory at FMVZ - UNESP. The samples took approximately 32 days to reach
80% confluence, when the first passage and differentiation was performed. To confirm the
mesenchymal origin, cells were induced to differentiate into adipogenic and osteogenic lineages.
Samples showed morphological changes during differentiation protocol, but not all presented
production of extracellular deposits of calcium or intracellular fat droplets, observed after staining
with Alizarin Red and Oil Red respectively. Compared with the material obtained from other
species and processed in the same laboratory, the primary culture was longer. Therefore, more
studies are needed to standardize the age of animals used and to test other inducers of cell
differentiation.
Keywords: bone marrow, buffalo, coxal tuberosity, cell differentiation stem cells
INTRODUCTION
The stem cells biology have been widely studied in several species due to their high
therapeutical potential. This unlimited potential is partly because they are immature stromal cells
with ability to self-renewal and differentiation into multiple lineages (LOTTEMBERG e
MOREIRA-FILHO, 2004).
The mesenchymal stem cells are maintained as a reserve tissue, being called multipotent
since they present the capacity to give rise to various cell types originated from the same embryonic
layer. The main sources of mesenchymal cells are bone marrow and adipose tissue. However, this
cell type is also found in several other adult tissues and in the embryonic annexes as in amniotic
fluid, amniotic membrane and in the blood and wall of the umbilical cord. One of the problems
encountered in the therapeutic application of MSCs is a lack of studies comparing the therapeutic
potential of cells derived from different tissues. (ZAGO e COVAS, 2006).
In this sense, the aim of the present study was to develop a methodology for collecting,
processing and cultivation of buffaloes mesenchymal stem cells obtained from bone marrow of the
coxal tuberosity. For this study material collected from six adult animals of varying ages were
collected and cells were differentiated into adipogenic and osteogenic lineage.

For this experiment we used six adult female Buffaloes of varying ages. The collection was
performed by introducing a Komiyashiki needle into the coxal tuberosity after local anesthesia. The
contents of the bone marrow were aspirated into heparinized syringe with the aid of negative
pressure. A total volume of 10 ml blood was aspirated. (KATHELEEN, 2000).

1128
The media used for processing the samples was Low Glucose DMEM ® (Invitrogen /
Gibco) + Glutamax ® F12 (Invitrogen / Gibco) supplemented with 10% Fetal Bovine Serum
(Invitrogen / Gibco) and antibiotics and antimycotics.
After collection, the samples were filtered and centrifuged at 1500 rpm for ten minutes to
separate the plasma. The supernatant was removed and culture medium previously prepared was
added. The separation was performed in a Histopaque ® (1077 – Sigma) gradient, trough 40
minutes centrifugation at 1500 rpm. Cell separation was followed by two washes with fresh culture
medium, and cells were cultured in 25 cm2 bottles in a humidified incubator with temperature of
37°C and 5% CO2 in air atmosphere. The adhesion time for these cells was four days, allowing the
first medium exchange. When the cells reached 80% confluence first passage was performed by
ressuspending the cells with TrypLE Express ® - (Invitrogen / Gibco), and transfer to 6 wells
culture dishes.
The cells were differentiated into osteogenic and adipogenic lineage. The differentiation
protocol took about 10 days using media and supplements prepared from the stem-pro-Invitrogen /
Gibco. At the end of differentiation period the morphological changes of cells were analyzed and
samples were stained with Alizarin Red and Oil Red, for the observation of the deposit of calcium
in the extracellular matrix and the presence of fat droplets into the cells, respectively.

The period between the adhesion and 80% confluence was approximately 32 days (Figure
1), depending on the sample. Compared with samples from other species processed in the same
laboratory, a longer primary culture time was observed. For example, in horses and dogs time to
reach the confluence is on average 20 days.
Morphological changes in cells in culture were observed during the differentiations period.
Of the six samples, four were able to differentiate in osteogenic lineage (Figure 2A and 2B) and two
were able to differentiate in the adipogenic lineage (Figure 2C and 2D). All samples showed
morphological changes, but some presented no production of extracellular deposits of calcium or
intracellular fat droplets, observed by staining with Alizarin Red and Oil Red, respectively.
Furthermore, we noted heterogeneity in response to differentiation after induction. Perhaps this
response is linked to an inadequacy of the differentiation media used. However, is important to
remember that intrinsic difference between the samples cannot be ruled out, since the population of
mesenchymal stem cells contained in bone marrow environment are heterogeneous and often
already committed to diverse differentiation processes.
CONCLUSION
Bone marrow mesenchymal stem cells culture, was successfully established in Buballinos
buballis species using a standardized technique. However, although the osteogenic potential was
evident the adipogenic differentiation needs to be improved. More studies should be made to
standardize the age of the animals and use of appropriate media for other mesenchymal tissues
differentiation.
REFERENCES
Katheleen, P.F. Bone marrow evaluation. 2000. In. FELDMAN, B. F.; ZINKL, J.G.; JAIN, N.C.
Shalm’s Veterinary Hematology. Philadelphia: Lippencott Williams and Wilkins, cap.
5, p. 29-32,.
Lottemberg, C. L. and C. A. Moreira-filho. 2004. Aplicações terapêuticas das células-tronco:
perspectivas e desafios, Com Ciência.
Zago, M. A. and D.T. Covas. 2006. Células-Tronco, a Nova Fronteira da Medicina, Editora
Atheneu, São Paulo.
1129
Figure 1: Aspect of Buffalo mesenchymal stem cells during culture

A. Begeaning of cell addesio (4 days in culture); B. cells after 10 days in culture; C. Cell after
25 days in culture. D. Aspect of the ceels after 50 days in culture.
Figure 2: Aspect of samples after differentiation

A. Begeaning of osteogenic differentiation; B. The re dods represented the extracellular depoist of
calcium stained by the Alizarin Red; C. Begeaning of adipogenic differentiation; D. Cells stained
redish presented lipid droplets inside their cytoplasm.
1130
Haematochemical and Hormonal Parameters Related to Buffalo Calves Welfare
Antonio FAGIOLOa, Olga LAIa*, Tiziana ZOTTOLAa, Cristina RONCORONIa, Vittoria

Lucia BARILEb and Lavinia ALFIERIa
a
Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana -via Appia Nuova 1411-00178
Roma-Italy.
b
Consiglio per la Ricerca e la Sperimentazione in Agricoltura - CRA PCM- via Salaria 31-00014
Monterotondo- Roma- Italy
*Corresponding Email: olga.lai@izslt.it
ABSTRACT
The aim of this preliminary study was to verify the application of veal calves welfare rules
to buffalo rearing and as a result, the related metabolic and hormonal parameters response. Data
regarding housing and management of two buffalo farms (A and B) were collected by means of a
specific farm record card in order to verify the compliance to European legislation.
In both farms, blood samples were taken from 14 newborn buffalo calves and followed up
monthly from one week to 5 months aged. The haemogram was carried out by automatic counter
Cell-Dyn 3700 (12 parameters) on blood samples with ethylenediaminetetraacetic acid (EDTA)
within 4 hours. Furthermore, iron and γ-Glutamyltransferase (γGT) serum levels were measured by
an automatic biochemical analyzer (Olympus 4000). Finally, cortisol serum concentration was
detected by radioimmunoassay (RIA) method. In comparison with farm B, farm A showed lesser
attention to the compliance of some management parameters and weaning procedures. Farm A
showed a significant lower average value for RBC and haemoglobin (P< 0.05). Iron level was
lightly higher in farm B whereas a significant increase of cortisol was observed in farm A (P< 0.05).
Nevertheless, in both farms, an increase of γGT values as a result of a correct colostrum ingestion
was observed. In conclusion, farm B could be considered as a control for what concerns the veal
calves rules application to buffalo rearing and the results of welfare related haematochemical
parameters.
Keywords: buffalo calves, welfare, legislation, haematochemical, cortisol
INTRODUCTION
The compliance with animal welfare is considered a topical matter, since it has been
assessed that stress interferes on physiological metabolism, endocrine and immune system. The
European Animal Welfare directive 98/58/CE concerns the protection of animals kept for farming
purposes. Regarding cattle, there are specific rules applying to veal calves breeding (91/629/CEE),
whereas there is a need to establish minimum standards for the buffalo species. Many studies about
welfare assessment in buffalo calves have focused on feeding, housing, space allowance and social
behaviour (Grasso et al. 1999; Napolitano et al. 2004). The aim of this preliminary study was to
verify the application of veal calves welfare rules to buffalo rearing and as a result, the related
metabolic parameters response.

The study was carried out in two buffalo intensive farms located in Latium region, in areas
recognised for Denomination of Protected Origin (D.O.P.) Mozzarella cheese production. In every
farm, buffalo calves breeding, housing and management were considered and data were collected
by means of a specific farm record card in order to verify the compliance to European legislation. In
the cards, several housing features, such as box dimension, light, temperature, and ventilation of the
building were included. In addition, dietary and nutritional aspects together with social ones were
studied.

1131
In every farm, blood samples were taken from 14 newborn buffalo calves and followed up
monthly from one week to 5 months aged. From each subject blood samples were taken from
jugular vein with and without EDTA.
Serum samples were frozen at – 20° C and stored under these conditions until analysed,
whereas samples with EDTA were refrigerated and analysed within 4 hours. The haemogram was
carried out using an automatic counter Cell-Dyn 3700 on blood samples with EDTA. Furthermore
iron and γ-Glutamyltransferase (γGT) serum levels were measured by using an automatic
biochemical analyzer (Olympus 4000). Finally, cortisol serum concentration (Diasorin kit) was
determined by radioimmunoassay (RIA) method. To compare overall parameters, repeated
measures Analysis of Variance (ANOVA) was used with significance set at P< 0.05. Data were
analyzed using SPSS/PC statistics package.

In our study we have mainly taken into account some of the parameters values mentioned by
the present legislation that provided useful indications to evaluate welfare conditions.
In farm A, 350 heads, buffalo calves were located, two by two, in 2.7 m2 boxes indoors. Ventilation
was guaranteed by a large window, but the light was scarce due to the bushiness. Buffalo calves
were fed with colostrum just for the first day, they were then fed buffalo milk and gradually passed
to milk replacer from the sixth to the tenth day. In the second week, buffalo calves were nursed by
an automatic calf milk feeder but, many of them needed to return to the nipple bottle since they
were frightened to the machine. Solid diet was introduced at 2 months of age. In the farm B, 400
heads, buffalo calves were separated from their dams after birth and housed in multiple pens (25
m2) indoors where they were fed colostrum for a week. Afterwards, they were transferred in
individual crates (1.45 m2) where they were given milk replacer from nipple bottle or bucket. At 2
weeks, buffalo calves were moved two by two in multiple pens (5 m2) indoors and they were fed
with hay and cereal flour ad libitum.
The haematic parameters assessment performed during the study, showed, in both farms, the
mean red blood cells (RBC) count included within 8 to 10 106/μl.
The RBC tended to be almost constant throughout the study in farm B while farm A was
characterised by a significant (P< 0.05) increase at 4 months of age (Table 1). Moreover, farm A
showed significant lower RBC mean value (P< 0.0001) throughout the experimental period.
Regarding haemoglobin (Hb) level, usually physiological values are higher than those considered in
the legislation limits for the veal calves (7.25 g/dl according to 2008/119/CE). In our study, as
previously described (Thangaraj et al., 1979; Karram et al., 1981), we observed, in both farms, a
significant (P< 0.05) decreasing trend in haemoglobin values as the age advanced (one week to 5
months) in all the calves, however the level was within physiological range indicating a proper
management (Table 2).
Furthermore, it must be highlighted that farm A did not comply with the European rules
about weaning procedures as calves were fed roughage only after 2 months of age, in spite of the 2
weeks prescribed.
physiological limits according to Radostis et al. (1994) cattle reference values (57-162 μg/dl).
About metabolic response, in both farms, serum iron mean value resulted within
Nevertheless, farm A showed the lowest mean iron level (66 μg/dl) in the 2 months old buffalo
calves as until this age a solid diet has not been administered. Furthermore, in the selected farms,
the γGT mean values trend indicate a proper colostrum administration, as these progressively
decreased up to the last sample (5 month of life) (Table 3). According to previous studies, the
correct colostrum ingestion both in cattle and buffalos leads to an increase of γGT values in the first
days of life whereas with age, as mature milk is assumed, the γGT values decrease (Lombardi et al.,
1996; Hadorn et al., 1997). Finally, the higher cortisol concentration has been observed in the
youngest calves of both farms with significant differences, probably due to some stressful events
such as calving, dam separation and environment changes (Table 4). In conclusion, since farm A
administers roughage beyond the prescribed age, has lack of space allowance and scarce light
1132
levels, farm B could be considered as a control for what concerns the veal calves rules application
and the results of welfare related haematochemical parameters.
REFERENCES
Grasso F., F. Napolitano, G. De Rosa, T. Quarantelli, L. Serpe and A. Bordi. 1999. Effect of pen
size on behavioral, endocrine, and immune responses of water buffalo (Bubalus bubalis)
calves. J. Anim. Sci.77:2039-2046.
Hadorn U. and J.W. Blum. 1997. Effects of feeding colostrum, glucose or water on the first day of
life on plasma immunoglobulin G concentrations and γ-glutamyltransferase activities in
calves. J.Vet.Med. 44:531-537.
Karram M.H., S.A. El Amrousi, M.F. Raghib, A.A. Amer. 1981. Studies on the red and white blood
cells of buffalo calves from birth up to 6 months age. J. Egipt Vet Med Assoc. 39:133-141.
Lombardi, P., L. Avallone, A. D’Angelo and E. Bogin. 1996. Gamma-glutamyltransferase and
serum proteins in buffalo calves following colostral ingestion. Eur. J. Clin. Chem. Clin.
Biochem. 34:965-968.
Napolitano F., G. De Rosa, F. Grasso, C. Pacelli, A. Bordi. 2004. Influence of space allowance on
the welfare of weaned buffalo (Bubalus bubalis) calves. Livest. Prod. Sci. 86:117-124.
Thangaraj T.M, V.N. Seshagiri, A.R. Krishnan and V. Venkataswami. 1979. Haematological
changes in the neonate of Bubalus bubalis. Indian J. Dairy Sci. 32:240-242.
Table 1. Mean absolute number of RBC (x106/μl) in the farms, according to the different ages (9 to
148 days)*.
RBC (x106/μl)
Age (days) Farm A Farm B
9 8.67 b 9.96
34 8.45 b 9.34
70 8.32 b 9.56
104 8.79 b 9.66
128 9.7 a 9.59
148 8.78 b 9.27
*Different superscripts within a column indicate significant differences (P<0.05)
1133
Table 2. Mean values of haemoglobin (g/dl) in the farms, according to the different ages (9 to 148
days)*.
Haemoglobin (g/dl)
9 13.3 a 15.7 a
34 12.6 ab 13.8 b
70 11.7 b 13.7 bc
104 11.8 b 13.5 bc
128 13 a 13.5 bc
148 12.3 ab 12.7 c
Table 3. Mean values of γGT (U/L) in the farms, according to the different ages (9 to 148 days)*.
γGT (U/L)
9 90 a 104 a
34 29 b 20 b
70 14 b 11 b
104 11 b 13 b
128 9b 15 b
148 19 b 16 b
*Different superscripts within a column indicate significant differences (P<0.05).
1134
Table 4. Mean values of cortisol in the farms, according to the different ages (9 to 148 days)*.
Cortisol (μg/dl)
9 2,65a 2,37a
34 1,73c 1,89ab
70 1,85b 2,14a
104 2,59ab 1,6b
128 2,53ab 1,34b
148 1.9ab 1,27b
1135
A Preliminary Study about Lymphocyte Subset of Water Buffalo Calves
Lavinia ALFIERIa, Antonio FAGIOLOa, Cristina RONCORONIa, Tiziana ZOTTOLAa,

Roberta CAVALLINAa and Olga LAIa*
a
Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, via Appia Nuova 1411, 00178
Rome, Italy.
*Corresponding Email: olga.lai@izslt.it
ABSTRACT
The aim of this study was to monitor the age related variation of the T-lymphocyte subsets
in peripheral blood of water buffalo calves. Additionally, lymphocytes and neutrophils percentage
was measured.
Monoclonal antibodies (mAbs) against bovine lymphocyte cell surface antigens CD4, CD8
and WC1 (γ/δ) were tested in sequential peripheral blood samples prepared from 36 different aged
water buffalo calves. Results showed the percentage of positive stained cells expressing CD4, CD8
and WC1 (γ/δ), as determined by flow cytometry. The subsets proportions varied significantly (P<
0.05) from the youngest age groups to the older ones as CD4+ increased while CD8+ and WC1+
decreased.
The present study shows that the T-cell subpopulations are present in peripheral blood of
buffalo calves as well as veal calves. Furthermore, there is a half reduction of neutrophil :
lymphocytes ratio at the age of 5 months.
Keywords: buffalo, t-lymphocyte, flow cytometry
INTRODUCTION
In Italy, the Mediterranean water buffalo (Bubalus bubalis) breeding represents an important
component of national livestock. Despite many studies describe immune response features in cattle
and calves, however there are very scarce data about immune response mechanisms in buffalo
(Davis et al., 2001).
The complex and sophisticated system providing the acquired immune response develops
slowly and takes several days to become effective relying partly upon the interaction and balance
between several different cell types, particularly lymphocyte subsets (Tizard, 2004).
In cattle, the importance of the different subpopulations of lymphocytes and the differences between
calves and adult animals have been described (Wyatt et al., 1994; Ayoub et al., 1996; Kampen et
al., 2006).
On the contrary, studies investigating lymphocyte subpopulations in buffaloes in
physiological conditions are scarce, although a previous research successfully found a large set of
bovine cross-reactive monoclonal antibodies (Davis et al., 2001). Additionally, reports of changes
in buffalo lymphocyte subsets refer to observations in diseased animals (Raj et al., 2006) and a
description of lymphocyte subpopulations changes in buffalo calves, followed over time, is lacking.
The aim of the present study was to provide preliminary data on the major T-lymphocyte subsets:
CD4+, CD8+ and WC1 (γ/δ), in buffalo calves, kept under conventional conditions, by sequential
measurements from the first week to the fifth month of age.

36 healthy water buffalo calves were randomly chosen from three different intensive farms.
12 animals per herd received, in every farm, the dam colostrum. Peripheral blood samples, 4 ml
each, were collected through jugular venipuncture, using vacutainer tubes with
ethylenediaminetetraacetic acid (EDTA). Sampling was performed at 9, 22, 35 days of life and
thereafter every month until the age of approximately 5 months. A complete blood cell count
1136
(haemogram) was performed using an automated counter Cell-Dyn 3700 (ABBOTT - 12

parameters) in order to assess leukocytes, neutrophils and lymphocytes count. Finally, flow
cytometric immunophenotyping of leukocytes was performed by a whole blood method according
to Shapiro (2003). The T lymphocytes subset concentration, CD4+, CD8+ and WC1 (γ/δ)
percentage, was characterised using monoclonal antibodies (mAbs) against the bovine antigen CD4
(CACT138A), CD8 (CACT80C) and WC1 (IL-A29) (VMRD, Pullmann, WA, USA).
The samples were analysed on a FACSCalibur flow cytometer (Becton Dickinson
Biosciences) and Cell quest Pro software. The results are reported as the percentage of gated cells
positive for each cell surface marker.
Data were statistically analysed using SPSS/PC statistics package (ANOVA Test) to
compare analytical results and relate them to farm and time of sampling with significance set at P
value < 0.05.

In this study, the absolute number of leucocytes (x103/μl) throughout the investigated period
significantly increased in an age dependent manner (P< 0.05). Particularly, it has been observed a
significant (P<0.05) increasing trend of mean lymphocytes percentage from the first to the last
sampling as well as a significant decreasing one of neutrophils percentage throughout the whole
study period (P< 0.05). In agreement with previous results in veal calves (Kramer, 2000), the
neutrophil to lymphocyte ratio (N/L ratio) at the beginning of the study was 1.3 (8 days) and at the
end was about 0.6 (5 months) with a progressive significant (P< 0.05) decrease (Table 1).
Moreover, on comparing the investigated farms, significant differences were found both in
leukocytes count, between farm 2 and 3, and lymphocytes count, between farm 3 and the others
(P<0.05) (Table 2). In veal calves, after birth, the neutrophils are the dominant white blood cells
(WBC) whereas by 3 months of age, lymphocytes concentration doubled and comprehending 70 to
80% of the total WBC (Kramer, 2000; Taylor, 2000). In buffalo species, calves begin theirs life
with fewer lymphocytes than granulocytes as well as cattle. The water buffalo haematological
changes with age have been studied from birth to one month of life, showing that the differential
leukocytes count consisted of 60% neutrophils and 39.7% lymphocytes at birth and becoming
32.2% and 66.4%, respectively, by day 28 (Lal Vegad, 2000).
The flow – cytometric phenotyping of lymphocytes showed that the mean percentage of
CD4+ cells increased significantly (P<0.05) during the first month of life (from 14.16% at 8 days of
age to 16.6% at 22 days of age) and as they grow up, from third to fifth month of age (17.8% at 96
days of age to 21% at 145 days of age (Table 3).
According to the results of a previous research of Kampen et al. (2006) in veal calves,
CD4+ levels in buffalo calves displayed ranging from 7% to 36%. Moreover, our studies
demonstrated that CD4+ cell reached 20% at the age of four months of life, as already reported in
veal calves by Kampen et al. (2006). Since the same value was observed in the 5 months aged
buffalo calves, this trend could suggest a first step towards the immune cells maturity in the buffalo
species and further investigations should involve longer periods of sampling. As a matter of fact, in
the bovine species, T cells CD4+,CD8+ and WC1+(γ/δ ) reach stable levels at 8 months of age
(Hein, 1994).
On the contrary, as for the mean proportion of CD8+, it was observed a fluctuating trend
throughout the whole study and finally, however, at the end of the study period there was a slight
decreasing trend and significant difference was observed (P<0.05) (Table 3). Besides, CD8+ levels
ranged from 5% to 75% which were different from the narrower range obtained in veal calves
(Kampen et al., 2006).
As Kampen et al. (2006) observed in veal calves, in the first week of life, in buffalo calves,
the WC1+ (γ/δ) levels ranged from 13% to 79% throughout the whole study, there was a
progressively reducing to the half from the first week of life to the oldest age group (5 month of
life) which demonstrated significant differences (P<0.05). Furthermore, buffalo calves WC1 (γ/δ)
cells percentage in the forth and fifth month of life reported the lowest percentages as well as CD8.
1137
The present study represents a preliminary research about immune cells maturation stages in buffalo
species. Further investigations on older calves and adult buffaloes could provide more complete
data of age related changes in immune cells maturation.
REFERENCES
Ayoub, I.A. and T.J. Yang. 1996. Age dependent changes in peripheral blood lymphocyte
subpopulations in cattle: a longitudinal study. Dev. Comp. Immunol. 20:353-363.
Davis, W.C., A.M. Khalid, M.J. Hamilton, J.S. Ahn, Y.H. Park and G.H. Cantor. 2001. The use of
crossreactive monoclonal antibodies to characterize the immune system of the water buffalo
(Bubalus bubalis). J. Vet. Sci. 2:103-109.
Hein, W.R. 1994. Ontogeny of T cells. In: Cells Mediated immunity in Ruminants (Ed. B.M.L.
Goddeeris and W.I. Morrison). CRC Press, Taylor &Francis Group, London, UK. pp. 19-36.
Kampen, A.H., I. Olsen, T. Tollersrud, A.K. Storset and A. Lund. 2006. Lymphocyte
subpopulations and neutrophil function in calves during the first 6 month of life. Vet.
Immun. Immunonopat. 113:53-63.
Kramer, J.W. 2000. Normal haematology of cattle, sheep, and goats. In: Schalm’s Veterinary
Hematology, 5th Ed. (Ed. B.F. Feldman, J.G. Zinkl and N.C. Jain). Blackwell Publishing,
Philadelphia, PA, USA. pp. 1075-1084.
Lal Vegad, J. 2000. Normal blood values of the Water Buffalo (Bubalus bubalis) In: Schalm’s
Veterinary Hematology, 5th Ed. (Ed. B.F. Feldman, J.G. Zinkl and N.C. Jain). Blackwell
Publishing, Philadelphia, PA,USA. pp. 1085-1088.
Raj, D.G., B. Mathivanan, K. Matheswaran, K. Nachimuthu and W.C. Davis. 2007. Lymphocyte
subset distribution in apparently normal and single intradermal test positive water buffalos
analysed by flow cytometry. Res. in Vet. Sci. 82:34-38.
Shapiro, H. M. 2003. Practical flow cytometry. 4th Ed. Wiley & Sons Hoboken, NJ, USA.
Taylor, J.A. 2000. Leukocyte responses in ruminants. In: Schalm’s Veterinary Hematology, 5th Ed.
(Ed. B.F. Feldman, J.G. Zinkl and N.C. Jain). Blackwell Publishing, Philadelphia, PA, USA.
pp. 391-404.
Tizard, I. R. 2004. Veterinary Immunology. 7th Ed. Saunders, Philadephia, PA,.USA.
Wyatt, C.R., C. Madruga, C. Cluff, S. Parish, M.J. Hamilton, W. Goff and W.C. Davis. 1994.
Differential distribution of γδ T-cell receptor lymphocyte subpopulations in blood and
spleen of young and adult cattle. Vet. Immun. Immunonopat. 40:187-199.
1138
Table 1. Mean (± S.D.) absolute number of leucocytes, mean percentage of neutrophyls and
lymphocytes and neutrophyls : lymphocytes (N/L) ratio, according to the different ages (8 to 145
days)*.
Mean (± S.D.)
Age Leukocytes
Neutrophils Lymphocytes
(days) (x 103cells/ul) N/L ratio
(%) (%)
8 10.18 b ± 2.17 52.27 a ± 9.8 41.88 d ± 7.92 1.33 a ± 0.49
22 10 b ± 2.4 48.27 a ± 9.74 48.21 c ± 10.2 1.08 b ± 0.45
35 10.02 b ± 2.75 49.96 a ± 9.54 45.09 cd ± 8.6 1.18 ab ± 0.51
65 10.29 b ± 1.79 41.08 b ± 9.22 54.32 b ± 9.11 0.81 c ± 0.36
96 11.73 a ± 2.48 36.16 c ± 13.45 59.92 a ± 12.25 0.68 c ± 0.4
123 12.66 a ± 3.53 37.13 bc ± 13.87 57.38 ab ± 12.34 0.76 c ± 0.57
145 12.63 a ± 2.64 33.84 c ± 9.14 59.09 ab ± 8.48 0.59 c ± 0.26
Table 2. Mean (± S.D.) absolute number of leucocytes (x103/μl), neutrophyls and lymphocytes
mean percentage and N/L ratio according to the selected farms*.
Mean (± S.D.)
Farm
Leukocytes Neutrophils Lymphocytes N/L ratio
(x103cells/ul) (%) (%)
1 11.2 ab ± 2.67 43.14 ± 12.67 49.88 b ± 11.03 1 ± 0.49
2 11.97 a ± 2.13 42.91 ± 9.52 50.04 b ± 9.57 0.93 ± 0.38
3 10.05 b ± 2.65 41.97 ± 14.06 56.89 a ± 13.48 0.82 ± 0.58
1139
Table 3. Mean (± S.D.) percentage of CD4+, CD8+ and WC1+ cells trend according to the
different ages (8 to 145 days)*.
Mean (± S.D.)
Age (days)
CD4 (%) CD8 (%) WC1 (%)
8 14.16 c ± 4.5 21.94 ab ± 11.48 61.78 a ± 8.46
22 16.6 b ± 5.42 24.83 a ± 10.34 51.8 b ± 10.26
35 17.58 b ± 3.57 18.18 bc ± 9.87 44.75 c ± 7.89
65 17.79 b ± 3.7 20.66 b ± 10.3 41.82 cd ± 8.38
96 17.87 b ± 5.25 22.9 ab ± 6.72 39.73 d ± 9.29
123 20.88 ab ± 6.23 12.13 c ± 2.88 33.17 e ± 5.95
145 21.08 a ± 5 16.44 bc ± 4.53 27.85 e ± 6.33
1140
Impact of Thermal Stress on Rectal, Skin Surface Temperatures, Respiration

Rate, Heat Load Index and Heat Storage in Lactating Murrah Buffaloes (Bubalus
Bubalis)
Vijay KUMARa*and Parveen KUMARb
a
Equine Production Campus, National Research Centre on Equines, Jorbeer, Bikaner-334001 India.
b
Division of Dairy Cattle Physiology, National Dairy Research Institute, Karnal-132001 (Haryana)
India
*Corresponding email: jaysutra@gmail.com
ABSTRACT
Impact of seasonal thermal stress on lactating Murrah buffaloes (n=9) was studied in the hot
dry and hot humid seasons as compared with spring season. The thermal stress was measured in terms
of the temperature humidity index (THI). The THI was significantly higher (p<0.01) in the hot dry
and hot humid seasons when compared to that in spring. The heat stress created a significant rise
(p<0.01) in the daily rectal and skin surface temperatures as compared to the spring season. During
the hot humid season, the baseline morning rectal (38.37±0.05⁰C) and skin temperature (31.04
±0.27⁰C) itself were higher (P<0.01) than in the other seasons (38.20±0.03⁰C & 26.36±0.17⁰C in hot
dry season; 37.90±0.03⁰C & 19.99±0.15⁰C during spring respectively) leading to a much higher heat
storage in the animals and high heat load as indicated by the increased Benezra’s heat load index
(near/above 2). By regression equation, the normal rectal temperature of 38.33 ⁰C corresponded to a
skin surface temperature of 27.42⁰C. Heat storage (KJ) in the animals in the hot dry and hot humid
seasons was more than 4 times (p<0.01) as in the spring season. The milk yield of the animals varied
significantly (p<0.01) between the seasons and was maximum during spring season. The climatic heat
stress caused significant changes in the physiological reactions, heat storage and heat load index in
lactating buffaloes and also negatively impacted milk yield which necessitates adequate mitigation
strategies to improve their productive potential in the tropics and subtropics.
Keywords: Heat stress, lactating Murrah buffaloes, rectal temperature, skin surface temperature,
respiration rate, heat storage and heat load index
INTRODUCTION
The most basic and simplest form of indicating thermal stress is the “Temperature-Humidity
Index” wherein the combined effect of high environmental temperature and relative humidity is
considered as the prominent contributor to physiological strain in the animal system. Upon exposure
to a hot environment, cattle and buffaloes respond initially with an acceleration of certain
physiological processes to increase the rate of heat loss (Upadhyay and Aggarwal, 1997, Aggarwal
and Upadhyay, 1998). Thermal stress from solar radiation, high ambient temperatures and high
relative humidity during summer and hot humid seasons not only causes significant changes in the
physiological responses such as rectal temperature, respiration rate with high respiratory rates
followed by panting and sweating being the prime responses of heat dissipation (Upadhyay and
Aggarwal, 1997; Silanikove, 2000). The impact of heat stress in the lactating Murrah buffaloes has
been reported only in a few studies (Das et al., 1999, Aggarwal and Singh, 2008). Moreover, the
relationship between skin surface temperature and rectal temperature in order to derive heat storage is
not well documented in lactating buffaloes. This study was therefore conducted to elucidate the

1141
impact of climatic thermal stress on various physiological responses, heat storage and heat load index
in the lactating Murrah buffaloes.

The data is from apparently healthy lactating Murrah buffaloes (n=9), studied at National
Dairy Research Institute (NDRI), Karnal in each of the three seasons spring (March 2008), hot dry
(April-May 2008) and hot humid (June 2008). The body weights in respective seasons were
560.22±09.12, 595.00±12.01 and 608.63±14.29 kg. The animals were selected to have milk yield
near and above herd average and fed as per standard practices at institute on a concentrate mixture
having CP 20% and TDN 70% and roughages (ad lib berseem, maize or oats fodder as per the
availability in the farm). The Temperature-Humidity Index THI=0.72 X (Tdb +Twb) + 40.6 {Where,
Tdb & Twb are dry and wet bulb temperatures in Celsius) NRC, 1971}, rectal (RT) and skin surface
temperatures (ST) and respiration rates (RR) and dry matter intakes were recorded on three
consecutive days in each of the above stated seasons at 0630-0730 hrs and 1430 hrs to 1530 hrs. The
skin temperature was recorded by an infra-red sensor thermometer (Surrey Scientific, Surrey
and udder. Heat storage (HS, Finch, 1984) was calculated as HS (KJ) = (0.8 x ΔRT+0.2 x ΔST) x
UK) at five different locations on the animal body i.e., forehead, rump, right flank, left flank
Specific heat of tissues x Body weight. The Coefficient of adaptability (Benezra, 1954); i.e,
(RT/38.33) + (RR/23) was considered as Heat load Index (HLI). Statistical analysis (ANOVA and
pair wise comparison of means was carried out by LSD) were carried out using SYSTAT 7.0
statistical software.

All results have been presented in the Table 1. It is evident that thermal stress due to high THI
had a significant effect on rise in rectal temperature, respiration rate and skin surface temperature as
visible signs of thermal stress. The heat storage and HLI provided the estimates of the extent of heat
load on the animals. The increased rectal temperature (p<0.01) at high THI in our experiment is of the
similar extent as observed by other authors (Das et al., 1999 in buffaloes; Koubkova et al., 2002 in
cows). The ST increased significantly (p<0.01) during exposure to seasonal thermal stress and was
perhaps the first response along with the significant rise (p<0.01) in RR to maintain homeothermy.
An increase in ST consequent to rising ambient temperature excites the thermosensory impulses from
receptors located in the skin (Joshi et al., 1968). The increase in respiration rate per degree rise in
temperature was of the measure 13.76, breaths/min with the increase in THI from 68-78. The
significant rise indicates that buffaloes are not very efficient in heat dissipation as they have fewer
sweat glands and black pigmented skin which absorbs more radiant heat and thereby more stress
(Benzamin, 1982, Das et al, 1999; Aggarwal and Upadhyay, 1998). High THI in hot-humid climate
caused thermal stress on the animals upon which they resorted to different compensatory mechanisms
to lose heat (Hahn, 1999; Das et al., 1999; Beatty et al., 2006).
To the best of our knowledge this is the first report that provides skin temperature (27.47°C )
corresponding to the normal rectal temperature of 38.33°C as derived by the linear regression
equation ST=9.343 x RT-330.7 (R2= 0.79). Therefore, with reference to 27.47°C as normal skin
temperature and 38.33°C as the normal rectal temperature, the novel estimates of heat storage were
calculated. These estimates were far more realistic than the conventional estimate of heat storage
based on the differences in the morning and afternoon rectal and skin temperatures. The primary
reason for the contrasting estimates is that during spring season (thermoneutral), the increase in the
RT & ST at afternoons from the morning time points is of a higher measure than the increase in hot
humid season. The morning RT and ST were comparatively higher during the Hot humid season than
in spring. The increases in RT & RR were responsible for high Heat load index which was near or
1142
above 2 in heat stress periods indicating that increased THI was above the adaptive capacity of these
buffaloes and impacted it adversely. Increased heat storage and high heat load are suggested to be
responsible for the reduced dry matter intake/100 kg BW (2.52 ±0.05vs 2.06±0.05 vs 1.98±0.06 kg
respectively in spring, hot dry and hot humid seasons) in buffaloes. This is supposed to have reduced
milk yield in heat stress (10.39±0.47, 8.22±0.43 and 6.57±0.28 kg/d respectively in spring, hot dry
and hot humid seasons, p<0.01). The Mean THI was negatively correlated with milk yield (r=-0.618,
p<0.01) and dry matter intake (r=-0.65 p<0.01). In such cases, animals do not have enough energy
and nutrients for regular milk synthesis in mammary glands. The finding is in line with the earlier
reports (Johnston et al., 1966; McDowell et al., 1976; De Rosa et al., 2009). It is concluded that heat
stress adversely impacts physiological responses such as temperature, respiration rate, skin surface
temperature and increases heat load index and heat storage in lactating buffaloes. These changes
might be responsible for reduced milk production suggesting the need for adequate heat stress
mitigation strategies for improving the productive potential of lactating buffaloes.
REFERENCES
Aggarwal, A and M. Singh. 2008. Changes in skin and rectal temperature in lactating buffaloes
provided with showers and wallowing. Trop Anim Health Prod. 40(3): 223-228.
Aggarwal, A. and Upadhyay, R.C. 1998. Studies on evaporative heat losses from skin and pulmonary
surfaces in male buffaloes exposed to solar radiations. Buffalo J. 2: 179-187.
Beatty, D.T, A. Baknes, E. Taylor, D. Pethick, M McCarthy and S. K. Maloney. 2006. Physiological
responses of Bos Taurus and Bos indicus cattle to prolonged continuous heat and humidity. J.
Anim. Sci. 84: 972-985.
Benezra, M.V. 1954. A new index for measuring the adaptability of cattle to tropical conditions. Proc.
J. Anim. Sci. 13: 1915
Benzamin, B.R. 1982. The thermoregulatory mechanism and its regulation in exocrine glands and
skin structures (histological) in buffaloes. Buffalo Seminar on Reproduction and Meat
Production, Tanaku, Andhra Pradesh, India, pp. 15–17.
Das, S.K, R. C. Upadhyay, and M. L. Madan. 1999. Heat stress in Murrah buffalo calves. Livest.
Prod. Sci. 61: 71-78.
De Rosa, G, F. Grasso, A. Braghieri, A. Bilancione, A. Di Francia and F. Napolitano. 2009. Behavior
and milk production of buffalo cows as affected by housing system. J. Dairy Sci. 92:907–912.
Finch, V.A. 1986. Body temperature in beef cattle: its control and relevance to production in the
tropics. J. Anim. Sci. 62: 531-542.
Hahn, G. L. 1999. Dynamic Responses of Cattle to Thermal Heat Loads. J Anim Sci. 1999 77: 10-20
Johnston, J. E, E. J. Stone, and J. B. Jr. Frye. 1966. Effects of hot weather on the productive function
of dairy cattle. Louisiaua Agr. Exp. Sta., Bull. 608.
Joshi, B. C, R. E. McDowell and D. P. Sadhu. 1968. Body surface evaporation rates at low and high
temperatures for Gir and Haryana Cattle. J. Dairy Sci., 51: 1693-1697.
Koubkova, M, I. Knizkova, P. Kunc, H. Hartlova, J. Flusser and O. Dolezal. 2002. Influence of high
environmental temperatures and evaporative cooling on some physiological, hematological
and biochemical parameters in high yielding dairy cows. Czech. J. Anim.Sci. 8: 309.
McDowell, R. E, N. W. Hooven and T. K. Camoens. 1976. Effects of climate on performance of
Holstein cows in first lactation. J. Dairy Sci. 59: 966–973.
National Research Council. 1971. A guide to environmental research on animals. Natl. Acad. Sci.,
Washington, DC.
Silanikove, N. 2000. Effects of heat stress on the welfare of extensively managed domestic
ruminants. Livest. Prod. Sci. 67: 1-18.
1143
Upadhyay, R. C and A. Aggarwal. 1997. Pulmonary and skin evaporative heat loss during exercise in
hot dry conditions in crossbreds. J. Anim. Sci. 67(1): 51-53
Table 1: Physiological and heat stress responses in the Lactating Murrah buffaloes
Physiological and heat Spring Season Hot Dry Season Hot Humid Season
stress responses
Morning
Micro En THI 61.96±0.29a 69.21±0.54b 78.76±0.12c
RT (⁰C) 37.90±0.03a 38.20±0.03b 38.37±0.05c
ST (⁰C) 19.99±0.15a 26.36±0.17b 31.04±0.27c
a
RR 13.00±0.25 20.30±0.40b 23.16±0.54c
B HLI 1.55±0.01a 1.88±0.02b 2.01±0.02c
a
HS (KJ)* -3552.84±78.65 -633.47±100.90b 1609.75±173.05c
a
HS (KJ/kg BW)* -6.36±0.12 -1.10±0.18b 2.62±0.28c
Afternoon
Micro En THI 70.84±0.25a 82.57±0.36b 83.68±0.35b
a
RT (⁰C) 38.50±0.04 39.31±0.03b 39.17±0.04b
ST (⁰C) 28.68±0.28a 35.82±0.32b 35.85±0.29b
a
RR 18.26±0.59 39.82±0.93b 48.15±1.74c
B HLI 1.80±0.03a 2.76±0.04b 3.12±0.08c
a
HS (KJ)* 752.18±143.57 5109.77±223.80b 4999.33±221.72b
a
HS (KJ/kg BW)* 1.35±0.26 8.54±0.30b 8.19±0.28b
Differences (Morning to Afternoon)
Micro En THI 8.88±0.38a 13.36±0.61b 4.92±0.43c
a
(14.40±0.68%) (19.47±0.98%)b (6.26±0.55%)c
a
RT (⁰C) 0.60±0.05 1.11±0.04b 0.80±0.05c
(1.59±0.12%)a (2.90±0.11%)b (2.10±0.13%)c
a
ST (⁰C) 8.67±0.32 9.47±0.32a 4.81±0.37b
a
(43.71±1.73%) (36.01±1.31%)b (15.69±1.31%)c
a
RR 5.26±0.68 19.52±1.08b 24.96±1.88c
a
(42.03±5.44%) (98.54±6.59%)b (111.10±9.91%)b
HS (KJ)† 4305.01±171.20a 5743.23±208.96b 3389.57±222.20c
a
HS (KJ/kg BW)* 7.71±0.30 9.64±0.29b 5.56±0.35c
Micro EN THI: Microenvironment THI, RT: Rectal Temperature, RR: Respiration rate, ST : Skin Surface
Temperature, B HLI: Benezra’s Heat Load Index (Benezra, 1953), HS= Heat Storage, *= Heat Storage
calculated by difference of RT and ST from the Normal RT 38.33⁰C and ST 27.47⁰C, †= Heat Storage
calculated by difference of RT and ST in the morning from the afternoon RT and ST. a,b,c: values within a
row with different superscripts vary significantly (p<0.01)
1144
Scrotal Thermography and Doppler Ultrasonography of the Testicular Artery

of Buffaloes Subjected to Environmental Heat Stress
Carlos Ramires Netoa*, Hélène Lacerda de Resendea, Gabriel A. Monteiroa, Mariana F.

Zorzettoa, Yamê F. R. Sancler da Silvaa, Eunice Obaa
a
and Animal Science, Sao Paulo State University, Botucatu, Brazil.
* Corresponding email: carlosramiresneto@hotmail.com
ABSTRACT
The process of spermatic division and differentiation (spermatogenesis) occurs with
intratesticular temperature lower that the corporal temperature and for that is essential that the
testicular thermoregulation mechanism occurs properly. For evaluation of the scrotal surface
temperature can be used the infrared thermography or testicular sensors, besides that, can be
evaluated the blood flux in the spermatic cord through the Doppler ultrasonography. Therefore the
objective of this study was the evaluation of the scrotal thermography and Doppler flowmetry of the
testicular artery of buffaloes subjected to environmental heat stress. For that were used seven
healthy buffaloes, with age of 3 and 4 years, of the Murrah breed. For the surface scrotal
temperature measurement (SST, oC) and superficial neck temperature (SNT, oC) was used the
infrared termography (Infra CamTM of the brand FLIR Systems Inc.), then Doppler flowmetry of the
testicular artery in the region of the spermatic cord through the ultrasonography (Mylab 5, Esaote®)
and measurement of the rectal temperature (RT, oC). The evaluations were done in two moments:
moment 1 (M1) with all the animals in the shade (Temperature=32,2oC) and moment 2 (M2) after 3
hours of exposure of animals to the sun (Temperature=38,7oC To calculate the resistivity index (RI)
and pulsatility index (PI), spectra were obtained from pulsed Doppler in three random regions of the
testicular artery in the spermatic cord. Data were subjected to analysis of variance (ANOVA)
followed by T test, using a significance level of 5%. There was an increase (p<0,05) of RT
(37,4±0,4a vs 39,0±0,3b; M1 and M2 respectively), SST (30,6±1,4a vs 35,2,0±1,0b; M1 and M2
respectively) and SNT (33,1±2,5a vs 38,5,0±0,3b; M1 e M2 respectively) e RI (0,67±0,1a vs
0,74±0,1b; M1 e M2 respectively) in M2. Increasing trend was observed (0,05>p>0,01) in PI
(1,10±0,4a vs 1,23±0,2b; M1 and M2 respectively) in M2. The results of the present study allow us
to conclude the healthy buffaloes have the scrotal average surface temperature 3oC lower that the
body temperature and that the exposure of 3 hours to sun in healthy buffaloes causes thermal stress
to the animals and changes in its surface scrotal temperature, and the Doppler flowmetry of the
testicular artery demonstrating the importance of thermal management for breeding buffaloes.
Besides that, the thermography and the Doppler ultrasonography presented great potential to detect
changes of testicular perfusion, being a promising additional test in the buffalo andrological
evaluation.
Keywords: buffalo, testicular thermoregulation, thermography, Doppler ultrasonography, thermal

stress.

1145
Blood Biochemical Profiles of Mehsana Riverine and Thai Swamp Buffaloes

under Tropical Conditions in the Northeast Thailand
Tossaporn SRISAKDI,a Panya CHAROENPOJANA,b Pichit CHUCAROEN,a Chalongchai

CHUMCHEEN,a Nikorn SANGHUAPHRAI,a Prapawan SAWASDEEa and Suvit
BOONPRONGa**
a
Department of Livestock Development, Ministry of Agriculture and Cooperatives,
Bangkok 10401, Thailand
b
Animal Science Program, Faculty of Agricultural Technology, Buriram Rajabhat University,
Buriram 31000, Thailand
*Corresponding email: suvit_dld@hotmail.com
ABSTRACT
Objectives of this study were to estimate and compare some blood parameters between
Mehsana riverine and Thai swamp buffaloes under tropical conditions of the Northeast Thailand.
Plasma biochemical profiles were studied in 50 mature (4 to 6-year-old) healthy cyclic buffaloes
comprised of 25 Mehsana riverine which were given to His Majesty the King of Thailand by the
Government of India in 2002 as the Golden Jubilee gifts and 25 Thai swamp buffaloes. These
animals were raised and managed under Buriram (northeast Thailand) Livestock Research and
Testing Station, Thailand conditions at 14°26′18″N, 102°43′30″E and 150 m above sea level with
an average temperature 27°C, average relative humidity of 75% and average of 1,096.60 mm annual
rainfall. The data were recorded from February to April, 2012 which was summer season in
Thailand. The results found that the plasma the glucose, creatinine, albumin, blood urea nitrogen
levels and albumin : globulin ratio in Thai swamp were significantly (P<0.05) higher than in
Mehsana riverine buffaloes. In the other hand, total protein, globulin in Mehsana riverine were
significantly (P<0.05) higher than in Thai swamp buffaloes. However, aspartate amino transferase
(AST) alanine amino trasferase (ALT), alkaline phosphatase (ALP) and gamma-glutamyl
transferase (GGT) did not significantly differ between Mehsana riverine and Thai swamp buffaloes.
In conclusion, the data presented in this communication suggest that Mehsana riverine buffaloes
could be well adapted, and are a dual purpose (meat and milk) buffaloes under tropical conditions
of Thailand.
Keywords: Mehsana riverine buffalo, Thai swamp buffalo, blood biochemical profiles
INTRODUCTION
Environments of high temperature and humidity are detrimental to the productivity of non-
adapted farm animals (Morrison, 1983). Thermoneutral zones (TNZ) for the animals are primarily
dependent on the species, the physiological status of the animal, the relative humidity, velocity of
ambient air and the degree of solar radiation (Yousef, 1985). Considerable work has been
undertaken to identify the physiological effects of heat stress and the mechanisms by which animal
productivity is reduced. In growing animals, heat stress may reduce dry matter intake (West, 1994),
the rate of weight gain (Mader, 2003), the fertility in males (Meyerhoeffer et al., 1985) and females
(Wilson et al., 1998). Quantification of these effects is complicated because of acclimation of
animals (Robinson et al., 1986) and breed differences in susceptibility to heat stress (Hammond et
al., 1998; Gaughan et al., 1999).
In the last decades, the animal production was mainly focused on the maximal production of
egg, milk, meat, etc. In recent years, the consumer has expressed his concern towards animal
welfare and food safety. The intensive animal production is forced to produce high quality products
with special attention to animal health and food safety. Exogenous factors such as management,
diseases and stress have a major influence on growth, product quality and animal welfare.
1146
Estimation of blood biochemicals such as enzymes, metabolites and proteins are helpful
complementary diagnostic tools and support the common veterinary diagnostic techniques for a
differentiated diagnosis of health and welfare status of the animal and completion of more specific
treatment regimen (for review see Bogin, 1994; Jain, 1996; Kaneko et al., 1997; Stockham and
Scott, 2002). On the other hand, blood parameters can be influenced by several factors including
breed, age, nutritional status, diseases and stress. In 2002, the Government of India presented 50
Meshana riverine buffalo (5 males and 45 females) to His Majesty the King of Thailand in the
occasion of Golden Jubilee gifts. The King had an initiative for Department of Livestock
Development (DLD) to study and research with efficiency in growth performance and milk yield
for promotion of these animals to Thai farmers (DLD, 2012). The aim of this research was to
determine and compare some blood parameters in Mehsana riverine and Thai swamp buffaloes
under tropical conditions of the Northeast Thailand.

Fifty mature (4 to 6-year-old) healthy buffaloes including 25 (18 non-pregnant cows and 7
bulls) Mehsana riverine and 25 (16 non-pregnant cows and 9 bulls) Thai swamp buffaloes from
different breeders were utilized. Animals were maintained as a buffaloes nucleus herd at Buriram
Livestock Research and Testing Station, DLD in Pakum District, Buriram Province, Thailand
conditions which were at 14°26′18″N, 102°43′30″E and 150 m above sea level with an average
temperature 27°C, average relative humidity (RH) of 75% and average of 1,096.60 mm annual
rainfall (Thai Meteorology Department, 2012).
They were provided with mixed pasture containing Para grass (Brachiaria mutica), Ruzi
grass (Brachiaria ruziziensis), Guinea grass (Panicum maximun) and Hamata (Stylosanthes
hamata). At night, all animals were housed indoors and fed soilage crop or silage or hay and 0.5-1.0
bodyweight/kg/day of 14-18% crude protein concentrate modified from the National Research
Council (NRC, 1987). Mineral supplements and clean water were also freely accessed. The animals
had a body condition score (BCS; Long et al., 1979; Herd and Sprott, 1986) at the time of blood
sampling as assessed on a scale from 1 (severe emaciation) to 9 (obese). The health of animals was
monitored by veterinarians regularly. The animals were vaccinated against foot and mouth disease
and were free from brucellosis and tuberculosis. They were treated for control of endoparasites
twice a year. All buffaloes were accustomed to humans and procedure of blood sampling. The
Mehsana dairy buffaloes were also kept likewise dairy cows.
Sample collections and measurements: blood samples (5 ml each) were collected via the
puncture of a jugular vein from each animal during February 2012 to April 2012 which was summer
season in Thailand between 08:00 and 10:00 AM. These animals were collected blood samples once
(10 μl/1 ml blood). Samples were immediately centrifuged (1000 × g) for 20 min at 4°C. Plasma
a month. The blood samples were withdrawn in heparinized syringes containing 10% Na-heparin
was separated and stored at -20°C until analyzed. All analyses were conducted within 7 days.
Plasma concentrations of the enzymes aspartate amino transferase (AST), alanine amino transferase
(ALT), alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) as well as glucose
(GLU), blood urea nitrogen (BUN), globulin and creatinine (CRT) were determined by standard
enzymatic methods. Total protein (TP) and albumin (ALB) levels were determined by Biuret
method (Scoffone and Fontana, 1975) and Bromocressol-green method (Drupt et al., 1974),
respectively.
Quantitative data were analyzed using GLM procedures of SAS (SAS, 1998). Differences
were determined using Least Significant Differences (LSD) procedure. The effects of sires, dams,
age of dams, year, season of birth and sex were also included in the model for unbiased adjustment.
(Harvey, 1975) was used for differences between groups. All statistical analyses were performed,
differences (LSD) using SAS Program Package (SAS, 1998). Values are presented as mean ± SEM.
and comparisons of mean were tested for differences among groups by the least significant
1147
RESUILTS AND DISCUSSION

There were no significant differences in age and BCS between the two breeds (Tables 1).
BCS of the animals ranged between 4 and 7 with an average of 5.62±0.11 in the Mehsana riverine
and 5.56±0.23 in the Thai swamp buffaloes. The levels of plasma GLU, CRT, ALB, TB and A/G
ratio and GGT in the two breeds were significantly (P<0.05) different (Table 1). Whereas, the AST,
ALT, ALP and GGT levels were not significantly differ between Mehsana riverine and Thai swamp
buffaloes.
The plasma levels of GLU in Thai swamp buffalo was significantly (P<0.05) higher than in
Mehsana riverine. Nevertheless, the levels were found to be within the range of reported values for
buffalo (1.38 – 5.88 mmol/l; Ellah, 2010). Plasma levels of GLU in Thai swamp buffalo was
significantly (P<0.05) higher than in Mehsana riverine. It is, however, to consider that the role of
glucose as a superior indicator for energy supply in the mature cattle is a matter of dispute, because
it is regulated by an array of different factors (Mudron et al., 2005). For BUN is a nutritional
indicator related to protein intake (Kaneko et al., 1997; Coppo, 2004) and useful in evaluating
kidney function in conjunction with creatinine which originates from the non-enzymatic conversion
of creatinine in muscle and is filtered by the kidney (Jain, 1996; Stockham and Scott, 2002). In the
present study, mean levels of plasma and creatinine were significantly higher in Thai swamp
buffalo than in Mehsana riverine animals. BUN values in all animals were within the range given
for the buffaloes (2.49-10.29 mmol/L; Ellah, 2010) but the mean concentration of creatinine
appeared to be higher than the range given for the buffaloes. However, all animals used in this study
showed on clinical signs or pathological symptoms.
Albumin levels in Mehsana riverine were lower than in Thai swamp buffalo (P<0.05). The
levels in all animals were within the range given for the buffaloes (Ellah, 2010). This biochemical
parameter has been shown to be significantly correlated with the health in animals and good
nutritional condition (Kaneko et al., 1997). Albumin concentration would significantly decrease
during malnutrition (Bogin, 1994; Singh et al., 2002); alimentary restriction alters quickly the
albumin levels in plasma (Jain, 1996).
Total protein and globulin levels of plasma are associated with the evaluation of hydration
status or possible haemorrhage and are a useful marker for acute and chronic active inflammation
(Stockham and Scott, 2002). In this study, total protein and globulin in Mehsana riverine were
significantly (P<0.05) higher than in Thai swamp buffalo. Furthermore, the mean of total protein
levels in Mehsana riverine were slightly higher than the range of 53-79 g/L which is estimated for
buffaloes in Egypt (Ellah, 2010). Several investigators have mentioned that the plasma protein
profile of a particular individual is relatively constant over a considerable length of time (Rowlands
et al., 1973; Anderson et al., 1987; Knowles et al., 2000; Coppo, 2004). This constancy is
apparently controlled genetically. In fact, Kaneko et al. (1997) suggested that the plasma protein
patterns were so characteristic that it can be used to differentiate species and in some cases the
strain and the sex of an animal. Furthermore, heritability of plasma protein in the cattle has been
reported by Smithies and Hickman (1958). For globulin which is produced by the liver, while others
are made by the immune system. Globulin carries essential metals through the bloodstream and
carries them to the various parts of the body and helps the body to fight infections. Globulin is the
'antibody' protein important for fighting disease (Jain, 1996; Stockham and Scott, 2002). In this
study, globulin levels in all animals were within the range given for the buffaloes (Ellah, 2010).
The enzymes AST, ALT, ALP and GGT are considered as indicators of the health of the
animal. They reveal the damage to tissue or cells in the muscle, liver, skeleton and heart (Canfield
et al., 1985; Kaneko et al., 1997; Stockham and Scott, 2002). Our results showed that AST, ALT,
ALP and GGT levels were not significantly differ between Mehsana riverine and Thai swamp
buffaloes. Furthermore, the enzymes AST, ALT, ALP and GGT levels were within the range given
for the buffaloes (Ellah, 2010).
In conclusion, the data presented in this communication can serve as reference values for
Mehsana riverine and Thai swamp buffaloes grown in Thailand and the other Asian countries
having similar climatic and nutritional conditions. Moreover, the Mehsana riverine buffaloes which
1148
were given to His Majesty the King of Thailand by the Government of India could be well adapted
under tropical conditions of Thailand.
REFERENCES
Anderson, K. L., T.G. Nagaraja and J. L. Morrill. 1987. Ruminal metabolic development in calves
weaned conventionally or early. J. Dairy Sci. 70. 1000–1005.
Bogin, E. 1994. Handbook for Veterinary Clinical Chemistry. Kodak Publishing, Rochester, NY.
Canfield, P. J., F. G. Best, A. J. Fairburn, J. Purdie and M. Gilham. 1985. Normal haematological
and biochemical values for the swamp buffalo (Bubalus bubalis). Aust. Vet. J. 61: 89-93.
Coppo, J. A. 2004. Biochemistry demonstration of malnutrition state in early weaned half-bred
Zebu calves. Rev. de Invest. Agropecuarias 33: 81–100.
Department of Livestock Development (DLD). 2012. Buffaloes Production and Management
Systems in Livestock Research and Breeding Center of DLD. Department of Livestock
Development, Minister of Agriculture and Cooperative, Bangkok. (in Thai)
Drupt, F., M. Paris, A. Frydman and M. Leclerec. 1974. Serum albumin assay by bromocresol
green method: application to different automatic apparatus. Ann. Pharm. Fr. 32: 249–256.
Ellan, M. R. A. 2010. Serum biochemical reference values for female buffaloes in Egypt. Buffalo
Bulletin 29: 141-147.
Gaughan, J. B., T. L. Mader, S. M. Holt, M. J. Josey and K. J. Rowan. 1999. Heat tolerance of
Boran and Tuli crossbred steers. J. Anim. Sci. 77: 2398–2405.
Hammond, A. C., C. C. Chase Jr., E. J. Bowers, T.A. Olson and R. D. Randel. 1998. Heat tolerance
in Tuli-, Senepol-, and Brahman-sired F1 Angus heifers in Florida. J. Anim. Sci. 76: 1568–
1577.
Harvey, R. W. 1975. Least Square Analysis of Data with Unequal Subclass Number. ARS H-4,
USDA Report, Washington, DC.
Herd, D. B. and L. R. Sprott. 1986. Body condition, nutrition and reproductive of beef cows. Texas
Agric. Ext. Serv. B. 1526.
Jain, N. C. 1996. Schalm’s Veterinary Hematology, 5th rev ed. Lea & Febiger, Philadelphia, PA.
Kaneko, J. J., J. W. Harvey and M. L. Bruss. 1997. Clinical Biochemistry of Domestic Animals, 5th
rev. ed. Academic Press, Inc., NY.
Kaneko, J. J., J. W. Harvey and M. L. Bruss. 1997. Clinical Biochemistry of Domestic Animals, 5th
revised edn. Academic Press, Inc., New York.
Knowles, T. G., G. E. Edwards, K. J. Bazeley, S .N. Brown, A. Butterworth and P. D. Warriss.
2000. Changes in blood biochemical and haematological profile of neonatal calves with age.
Vet. Rec. 147: 593–598.
Long, C. R., T. S. Stewart, T. C. Cartwright and T. E. Jenkins. 1979. Characterization of cattle of a
five breed diallel: I. measures of size, condition and growth in bulls. J. Anim. Sci. 49: 418–
431.
Mader, T. L. 2003. Environmental stress in confined beef cattle. J. Anim. Sci. 81(E. Suppl. 2):
E110–E119.
Meyerhoeffer, D. C., R. P. Wettemann, S. W. Coleman and M. E. Wells. 1985. Reproductive
criteria of beef cattle bulls during and after exposure to increased ambient temperature. J.
Anim. Sci. 60: 352–357.
Morrison, S. R. 1983. Ruminant heat stress: effect on production and means of alleviation. J. Anim.
Sci. 57: 1594–1600.
Mudron, P., J. Rehage, H. P. Sallmann, M. Holtershinken and H. Scholz. 2005. Stress response in
dairy cows related to blood glucose. Acta Vet. Brno 74: 37–42.
National Research Council (NRC). 1987. Predicting Feed Intake of Food Producing Animals. Natl.
Acad. Press, Natl. Res. Counc., Washington, DC.
Robinson, J. B., D. R. Ames and G. A. Milliken. 1986. Heat production of cattle acclimated to cold,
thermoneutrality and heat when exposed to thermoneutrality and heat stress. J. Anim. Sci.
62: 1434–1440.
1149
Rowlands, G. J., J. M. Payne and S. M. Dew. 1973. A potential use of metabolic profiles in the
selection of superior cattle. Vet. Rec. 93, 48–49.
Scoffone, E. and A. Fontana. 1975. Proteins analysis. In: Needleman, S. B. (ed.), Protein Sequence
Determination: A Source Book of Methods and Techniques, pp. 162–203. Springer-Verlag,
New York.
Singh, A. S., D. T. Pal, B. C. Mandal, P. Singh and N. N. Pathak. 2002. Studies on changes in some
of blood constituents of adult cross-bred cattle fed different levels of extracted rice bran.
Paksitan J. Nutr. 1: 95–98.
Smithies, O. and C. G. Hickman. 1958. Inherited variations in the serum protein of cattle. Genetics
43, 374–385.
Statistical Analysis Systems (SAS). 1998. SAS Users Guide: Statistics. Version 8, SAS Institute,
Cary, NC.
Stockham, S. L. and M. A. Scott. 2002. Fundamentals of Veterinary Clinical Pathology. Iowa State
University Press, Ames, IA.
Thai Meteorology Department. 2012. Agroclimatological Data for Thailand. Ministry of
Transport and Communication, Bangkok. (in Thai)
West, J. W. 1994. Interactions of energy and bovine somatotropin with heat stress. J. Dairy Sci. 77:
2091–2102.
Wilson, S. J., C. J. Kirby, A. T. Koenigsfeld, D. H. Keisler and M. C. Lucy. 1998. Effects of
controlled heat stress on ovarian function of dairy cattle. 2. Heifers. J. Dairy Sci. 81:2132–
2138.
Yousef, M. K. 1985. Stress Physiology in Livestock, Vol. I, Basic Principles. CRC Press, Florida
Table 1. Comparison on Means±SEM values of plasma levels of metabolites and proteins between
Mehsana riverine and Thai swamp buffaloes.
∼5
Parameter Mehsana riverine (n = 25) Thai swamp (n = 25) Reference range*
Age (year) 4.40±0.23 4.51±0.31
BCS 5.62±0.11 5.56±0.23 -
GLU (mmol/L) 3.58±0.22b 4.16±0.16a 1.38-5.88
BUN (mmol/L) 7.86±0.48b 9.37±0.74a 2.49-10.29
b a
CRT (μmol/L) 128.10±3.14 136.12±2.16 32-114
b a
ALB (g/L) 25.58±1.86 29.78±0.96 22-44
GLB (g/L) 55.45±1.77a 49.90±2.27b 21-54
TP (g/L) 81.02±0.27a 79.68±0.87b 53-79
A/G ratio 0.46±0.13b 0.60±0.14a 0.44-1.69
AST (U/L) 118.97±4.15 115.64±7.08 44-125
ALT (U/L) 35.25±0.85 37.74±1.44 7-38
ALP (U/L) 150.91±8.91 146.65±5.05 23-237
GGT (U/L) 17.22±1.42 15.44±2.02 8-52
Means±SEM in the same row with different superscripts were significantly different (P<0.05).
BCS, Body Condition Score; GLU, Glucose; BUN, Blood Urea Nitrogen; CRT, Creatinine; ALB,
Albumin; GLB, Globulin; TP, Total protein; A/G ratio, Albumin : globulin ratio; AST, Aspartate
amino transferase; ALT, Alanine amino transferase; ALP, Alkaline phosphatase; GGT, Gamma-
glutamyl transferase.
* Ellah (2011)
1150
Changes in Insulin-Like Growth Factor-Binding Proteins in Swamp Buffalo

Uterus during Estrous Cycle
Sirima THONGRUAY,a* Kitiya SRISAKWATTANA,a Wanvipa SUTHIKRAI,a Mariem

YUSUKSAWADb and Prakong TANGPRAPRUTIGULc
a
Science, Chulalongkorn University, Henri Dunant Street, Phathumwan, Bangkok 10330, Thailand
b
Department of Physiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330,
Thailand
c
Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
*Corresponding email: sirima.t@chula.ac.th
ABSTRACT
Insulin-like growth factor-binding proteins (IGFBPs) play a role in regulating insulin-like
growth factor (IGFs) action in a wide variety of cell types. The present study characterized the
changes in concentration of IGF binding protein-2 (IGFBP-2) and IGF binding protein-3 (IGFBP-3)
in uterine obtained from swamp buffalo during the estrous cycle. The uteri of 36 slaughtered swamp
buffaloes were collected, the stages of estrous cycle were considered by macroscopic examination
of the ovaries and were classified to be early, mid, late luteal and follicular phase. Western blot
analysis of endometrial tissue showed that IGFBP-2 was affected by the stage of estrous cycle. The
concentration of endometrial IGFBP-2 protein was increased at early and peak at mid luteal phase
of the cycle. However, endometrial IGFBP-3 protein was faintly detectable and did not change
significantly. Plasma progesterone by radioimmunoassay was also harmonized with endometrial
IGFBP-2 protein. Results suggest that increased in endometrial IGFBP-2 at the early and mid luteal
phase of the estrous cycle may regulate the activity of the IGFs which may be required to promote
the development of the uterus at this stage.
Keywords: insulin-like growth factor binding protein, uterus, swamp buffalo, estrous cycle
INTRODUCTION
The Insulin-like growth factors (IGFs) system has been reported in the uterus during the
oestrus cycle and play important role in regulating the development, differentiation and function in
the uterus and placenta of many species. The actions of IGF-I and IGF-II are mediated through the
IGF receptor and modulated by specific proteins called insulin-like growth factor-binding proteins
(IGFBPs) which there are at least six different proteins (IGFBP-1,-2,-3,-4,-5 and -6). IGFBPs
prolong the half-life of the IGFs and have been shown to either inhibit or stimulate the growth
promoting effects of the IGFs. The expression of mRNA and protein for IGFBPs has been
demonstrated in uterine tissue from several species. The synthesis of IGFBPs is regulated by steroid
hormones (Giudice et al., 1991). The changes in IGFBPs in uterine tissues according to the stages
of estrous cycle have been reported in rat (Davenport et al., 1992, Ghahary et al., 1993), ovine
(Osgerby et al., 1999), ewe (Gadds et al., 2000) and bovine (Robinson et al., 2000). However, there
is no information concerning IGFBPs in swamp buffalo uterus, therefore the objective of this study
was to determined the changes in IGFBP-2 and IGFBP-3 in swamp buffalo uterus during the
estrous cycle.

Sample Collection
Blood samples were collected from the jugular vein for progesterone analysis. Immediately
after slaughtered, the healthy uteri of 36 slaughtered swamp buffaloes were removed and placed on
iced, the endometrium tissues were dissected and frozen immediately in liquid nitrogen and stored
at -80°C until processed for western blot analysis. The stages of estrous cycle were considered by
1151
macroscopic examination of the ovaries and were classified to be early, mid, late luteal and
follicular phase.
RIA
Plasma progesterone concentrations were measured by non-extracted method as previously
described by Kamonpatana et al. (1983).
IGFBPs were extracted from frozen endometrium, after homogenization 30 μg of protein

Western blot analysis
extract was subjected to 12% SDS polyacrylamide gels followed by electrotransfer onto
nitrocellulose membranes. The membrane was incubated with primary antibody, a specific rabbit
anti-bovine IGFBP-2 (1:1000, US Biological), goat polyclonal antibody to IGFBP-3 (1:600,
Abcam, Cambridge, UK), mouse anti-β-actin monoclonal antibody (1:4000, Sigma Chemical) and
then incubated in the secondary antibody conjugated to horseradish peroxidase. Bands were
visualized by chemiluminescence and exposed to X-Omat film (Kodak ). For data analysis of each
band corresponding to IGFBP-2, IGFBP-3 and β-actin was analysed by measuring changes in
optical density using image analysis.
Results were expressed as means + standard deviation (SD). Statistical analysis was made
by analysis of variance (ANOVA) using the least significant difference (LSD). A probability (P)
less than 0.05 was considered to be significantly different.

In the present study, plasma concentrations of progesterone were determined to ranged
between 0.34+0.19 to 3.15+0.76 ng/ml during the estrous cycle in swamp buffalo. Plasma
progesterone concentrations were increased during early luteal phase and then further increased to a
maximum concentration during mid luteal phase and then declined during late luteal phase and were
lowest during the follicular phase (Figure 1). The pattern of progesterone was similar to those
reported by other investigations (Kamonpatana et al., 1976, Arora and Pandey, 1982).
Western Blot analysis of endometrial IGFBP-2 and IGFBP-3 protein in the swamp buffalo
throughout the estrous cycle was shown in Figure 2. There was a different pattern of endometrial
IGFBP-2 and IGFBP-3. The concentration of IGFBP-2 increased at early and peak at mid luteal
phase of the cycle and declined at the late luteal and follicular phase while IGFBP-3 was low
throughout the estrous cycle and there was no difference in concentration of IGFBP-3 during the
different stages of cycle. The timing of increased endometrial IGFBP-2 coincided with plasma
progesterone concentrations. These results supported the previous study that IGFBP-2 was
regulated by progesterone since it increased similarly during the luteal phase of estrous cycle in cow
(Geisert et al., 1991) and pig (Simmen et al., 1990). Moreover, the expression of IGFBP-2 was
increased by elevated progesterone (McCarthy et al., 2012).
In the present study, endometrial IGFBP-2 was greater than IGFBP-3 throughout the estrous
cycle. IGFBP-2 was the most abundant in cow endometrium (Kirby et al., 1996) thus IGFBP-2 may
play a role in modulate the action of IGFs in the endometrium. The important role of IGFBP-2 was
to modulate endometrial epithelial cell mitogenesis (Badinga et al., 1999).
Endometrial IGFBP-3 protein was low and there was no significant difference in
concentration of IGFBP-3 throughout the estrous cycle. The present result was similar to the study
in cow which there was no significant change in the expression of IGFBP-3 throughout the oestrous
cycle (Robinson et al., 2000). This result suggested that endometrial IGFBP-3 was not regulated by
progesterone.
Although the expression of IGFBP-3 protein in myometrium was not shown in this study,
the previous study reported that IGFBP-3 was more prevalent in myometrium than in endometrium
and myometrial IGFBP-3 production had a role in regulating transport of circulating IGFs into the
myometrium and subsequent distribution of IGFs to the surround tissues (Keller et al., 1998).
In summary, the present study demonstrated that endometrial IGFBP-2 protein may be
regulated by progesterone and an increase in endometrial IGFBP-2 in the luteal phase may regulate
1152
the activity of the IGFs which may be required to promote the development of the uterus at this
stage.
REFERENCES
Arora, R.C. and R.S. Pandey. 1982. Pattern of plasma progesterone, oestradiol-17β, luteinizing
hormone and androgen in non-pregnant buffalo (Bubalus bubalis). Acta. Endocrinol.
100:279-284.
Badinga, L., S. Song, R.C. Simmen, J.B. Clarke, D.R. Clemmons and F.A. Simmen. 1999. Complex
mediation of uterine endometrial epithelial cell growth by insulin-like growth factor-II (IGF-
II) and IGF-binding protein-2. J. Mol. Endocrinol. 23:277-285.
Davenport, M.L., J. Pucilowska, D.R. Clemmons, R. Lundblad, J.A. Spencer and L.E. Underwood.
1992. Tissue-specific expression of insulin-like growth factor binding protein-3 protease
activity during rat pregnancy. Endocrinology 130:2505-2512.
Gadd, T.S., J.C. Osgerby and D.C. Wathes. 2000. Regulation and localization of insulin-like growth
factor binding protein-5 gene expression in the uterus and placenta of the cyclic and early
pregnant ewe. Biol. Reprod. 62:1415-1421.
Geisert, R.D., C.Y. Lee, F.A. Simmen, M.T. Zavy, A.E. Fliss, F.W. Bazer and R.C. Simmen. 1991.
Expression of messenger RNAs encoding insulin-like growth factor-I, -II and insulin-like
growth factor binding protein-2 in bovine endometrium during the estrous cycle and early
pregnancy. Biol. Reprod. 45:975–983.
Ghahary, A., J. Luo and L.J. Murphy. 1993. Expression and regulation of insulin-like growth factor
binding protein-1 in the rat uterus throughout estrous cycle. Mol. Cell. Biochem. 124:43-49.
Giudice, L.C., D.A. Milkowaski, G. Lamson, R.G. Rosenfled and C. Irwin. 1991. Insulin-like
growth factor binding proteins in human endometrium: steroid-dependent messenger
ribonucleic acid expression and protein synthesis. J. Clin. Endocrinol.& Metab. 72:779-787.
Kamonpatana,M., R. Parnpai, C. Ngramsuriyaroj and K. Srisakwattana. 1983. Plasma progesterone,
oestrone and oestrone sulphate levels during the first half of gestation in swamp buffaloes.
Br. Vet. J. 139:256-261.
17-hydroxyprogesterone and β-oestradiol during oestrous cycle in the swamp buffalo in

Kamonpatana, M., Y. Luvira, P. Bodhipaksha and A. Kunawongkrit. 1976. Serum progesterone,
Thailand. In Nuclear Techniques in Animal Production and Health pp. 569-578. IAEA,
Vienna.
Keller, M.L., A.J. Robert and G.E. Seidel. Jr. 1998. Characterization of insulin-like growth factor-
binding proteins in the uterus and conceptus during early conceptus elongation in cattle. Biol
Reprod. 59:632-642.
Kirby, C.J., W.W. Thatcher, R.J. Collier, F.A. Simmen and M.C. Lucy. 1996. Effects of growth
hormone and pregnancy on expression of growth hormone receptor, insulin-like growth
factor-1, and insulin-like growth factor binding protein-2 and -3 genes in bovine uterus,
ovary and oviduct. Biol. Reprod. 55: 996–1002.
McCarthy, S.D., J.F. Roche and N. Forde. 2012. Temporal changes in endometrial gene expression
and protein localization of members of the IGF family in cattle: effects of progesterone and
pregnancy. Physiol. Genomics. 44:130-140.
Osgerby, J.C., T.S. Gadd and D.C. Wathes. 1999. Expression of insulin-like growth factor binding
protein-1 (IGFBP-1) mRNA in the ovine uterus throughout the oestrous cycle and early
pregnancy. J. Endocrinol. 162:279-287.
Robinson, R.S., G.E. Mann, T.S. Gadd, G.E. Lamming and D.C. Wathes. 2000. The expression of
the IGF system in the bovine uterus throughout the oestrous cycle and early pregnancy.
J. Endocrinol. 165:231–243.
Simmen, R.C., F.A. Simmen, A. Hofig, S.J. Farmer and F.W. Bazer. 1990. Hormonal regulation of
insulin-like growth factor gene expression in pig uterus. Endocrinology 127: 2166–2174.
1153
Figure 1 Plasma progesterone concentrations in swamp buffalo during the different

stages of the estrous cycle; early luteal(EL), mid luteal(ML), late luteal
(LL) and follicular phase (F). Values are mean+SD (n=9 in each stage).
Different letters above each column indicate significant difference at
p<0.05.
Figure 2 Endometrial IGFBP-2 and IGFBP-3 concentrations in swamp buffalo
endometrial IGFBP-2, IGFBP-3 and β-actin. (B) The ratios of

during the different stages of the estrous cycle. (A) Western blots for
endometrial IGFBP-2 and IGFBP-3 to β-actin. Representative data are

mean+SD which obtained from at least 6 experiments in each stage.
Different letters above each column indicate significant difference at
p<0.05.
1154
Use of Oxytocin and Milking Management of Buffaloes in

(Urban) Peri-Urban Area of Faisalabad
Muhammad TARIQ1 and Muhammad YOUNAS2*
1
University of Kassel/ University of Göttingen, Animal Husbandry in the Tropics and Subtropics,
Germany
2
Department of Livestock Management, University of Agriculture, Faisalabad, Pakistan
*Corresponding email: tariq7337@gmail.com
ABSTRACT
A study was conducted to evaluate the efficiency of (urban)peri-urban production system in
Faisalabad, third-largest city of Pakistan. Interviews with 145 milk-producing (urban)peri-urban
households (HH) were carried out. Based on cluster analysis, four types of dairy farmers were
identified, (i) semi-commercial smallholder mixed dairy-crop farmers (n=43), (ii) semi-commercial
smallholder dairy farmers (n=30), (iii) smallholder commercial (n=53) and (iv) large-holder
commercial dairy farmers (n=14). Of each type five HH were randomly selected for qualitative and
quantitative on-farm monitoring of management of buffalo and cattle. Milking was done twice
daily, exclusively by hand. Before milking intra-muscular injection of 2 ml oxytocin was practiced
by 90% of the (urban)peri-urban dairy farmers to stimulate milk letdown in 61% of the monitored
lactating buffaloes and 19% of the cattle whereas 36% buffalo and 48% cattle calves suckle their
dams. There was significant difference (P<0.05) in use of oxytocin for buffalo and cattle among the
four production systems. Oxytocin can be easily purchased for as little as 18–20 rupees/50 ml from
local shops, even at village level. In general oxytocin was used for those animals whose calves had
died, sold or were not accepted by dams. Some were injected once daily while most received
oxytocin at both milkings. This study suggests that regular use of oxytocin for milk let down should
be checked, should be prescribed on actual need and awareness should be created among farmers.
Prolonged use of oxytocin interferes and inhibits the normal milk ejection reflex and causes fertility
disorders such as poor estrus signs, low conception rate, high embryonic mortality, shortened
lactation period, increased abortion, calf death and incidences of mastitis. These problems are
currently being addressed in progeny-history interviews targeting each milking animal in the 20
monitored herds.
Keywords: Cattle, estrus, milking, milk let down, milk ejection reflex
INTRODUCTION
Urban and peri-urban dairy production has been growing constantly during the past decades
and is continuously getting importance with time; about 5 % of Pakistan's milk comes from urban
and 15 % from peri-urban producers (Jonas et al., 2010). Buffaloes and tropical cattle breeds are
known to be difficult to milk because of their thicker and longer teats, slow milk ejection reflex and
their hard teat sphincter muscle; there are several studies from different parts of the world that
reported disturbed milk ejection and quick ending of lactation in buffaloes when the calves died or
the normal milker was replaced as cited by Thomas (2004). Same phenomenon is also true for
tropical cattle, because in many breeds calves are used to stimulate and induce milk let down
(Pegram et al., 1991; Syrstad, 1993). As described by Thomas (2004) other ways beside calf
suckling or calf presence to stimulate milk flow are feeding of concentrates, manual pre-stimulation
and injection of oxytocin before milking.
Oxytocin plays an important role in milk ejection in buffaloes. Oxytocin influences milk
production by removal and evacuation of the gland by the help of the oxytocin-mediated milk
ejection reflex and decreasing intra-alveolar pressure and re-establishing normal mammary blood
flow (Belo & Bruckmaier, 2010; Lollivier et al., 2002). A survey in India revealed that 65 % of the

1155
surveyed farms used concentrate feeding to improve milk let down and 13 % used oxytocin
injections (Varma & Sastry, 1994 cited in Thomas, 2004). In Pakistan, calf suckling, oxytocin
injections and feeding of concentrate are frequently used for stimulus in dairy buffaloes on farms
(Bilal et al., 2008; Khan et al., 2007; Qureshi & Ahmad, 2008).
Dairy farmers in Pakistan, use oxytocin without any prescription and is readily available even
in general stores at village level on minimal prices; its use for stimulus of milk flow and increase of
milk production and thus income is motivated by both quacks and medical store owners (Mustafa et
al., 2008), as well as researchers and scientists (Ijaz & Aleem, 2006). The magnitude of the increase
in milk production from oxytocin injections is quite variable, ranging from 10 to 12% and some
reported up to 15.5% of milk production in some studies, but showing no significant effects on milk
production in others. In general, the factors that control milk production vary and are dependent on
dosage and time of oxytocin injection (Borghese et al., 2007).
The objectives of this study was to describe the effects of different management practices and
milking routines and use of oxytocin on the variation observed in milk production and its
physiological effects on dairy buffalo and cattle kept in urban and per-urban area.

Study Location
This study was conducted in the District Faisalabad, third-largest city of Pakistan which is
located on 31,4190° N and 73,0792° E (city center, own measurement) at an altitude of about 184 m
a.s.l. (Cheema et al., 2006). 145 households (HH) keeping dairy buffaloes and dairy cattle in the
urban and peri-urban zone of Faisalabad within a radius of about 4 to 9.4 km from the city center
were interviewed face to face using a structured questionnaire from 27.08. - 21.10.2009. Figure
(map) 1 here
Data Collection
A snowball sampling procedure (Babbie, 2009) was used to randomly select and interview
the 145 HH. Based on cluster analysis, four types of dairy farmers were identified, namely semi-
commercial smallholder mixed dairy-crop farmers (n=43), semi-commercial smallholder dairy
farmers (n=30), commercial smallholder dairy farmers (n=53) and commercial large-scale dairy
farmers (n=14). From each type, five HH were randomly selected for qualitative and quantitative
on-farm monitoring of management of dairy buffalo and cattle during 06/2010–12/2011.
All steps of data analysis were performed with SPSS 17.0 (SPSS Inc, Chicago, Illinois).
Frequencies were calculated and Chi-Square test was used for differences between production
systems, significance was declared at P<0.05.
RESULTS
On studied farms dairy animals were milked twice daily, exclusively by hand. During the
period of high milk flow during (06/2010–12/2011), intra-muscular injection of 2 ml oxytocin
before milking was practiced by overall 90 % of the farmers to stimulate milk letdown and increase
yield in 61 % of the monitored lactating buffaloes and 19 % of the lactating cattle (Fig. 2) whereas
36% buffalo and 48% cattle calves suckle their dams to stimulate milk let down. There was
significant difference (P<0.05) in use of oxytocin for buffalo and cattle among the four production
systems (Table. 1).
The use of oxytocin in small scale (SCD= 66%) and large scale (LCD=65%) commercial
dairy farmers for milk letdown in dairy buffalo was significantly higher than semi-commercial mix
(SSM=57%) and semi-commercial small (SSD= 47%) dairy farmers (Table.1). It was observed that
oxytocin could easily be purchased for as little as 18–20 rupees/50 ml from local shops, even at
village level.
1156
DISCUSSIONS
The profitability of a dairy farm has direct link with the wellbeing of the dairy animals
which results from good management practices. In this regard proper milking technique is of utmost
importance among other management practices. Amongst the major activities performed on a dairy
farm any of the duty does not carry as much responsibility as does the actual milking management
of dairy animals. The proper milking management results in high yields and better quality milk,
least udder health problems and longer productive life of a dairy animal. All these aspects finally
contribute to the better dairy farm profitability per dairy animal.
Buffaloes and also tropical cattle breeds are known to be difficult to milk because of their
thicker and longer teats, slow milk ejection reflex and their hard teat sphincter muscle. In Pakistan,
calf suckling, oxytocin injections and feeding of concentrate are frequently used as stimulus in dairy
buffaloes on farms (Bilal et al., 2008; Khan et al., 2007; Qureshi & Ahmad, 2008).
The magnitude of the increase in milk production from oxytocin injections is quite variable,
ranging from 10 to 12% and some reported up to 15.5% of milk production in some studies, but
showing no significant effects on milk production in others. In general, the factors that control milk
production vary and are dependent on dosage and time of oxytocin injection (Borghese et al., 2007).
Whereas two studies on Murrah buffaloes in India did not show an effect of oxytocin injections on
milk yield (Bidarimath & Aggarwal, 2007; Maurya & Ludri, 1992). A survey in India revealed that
65% of the surveyed farms used concentrate feeding to improve milk let down and 13% used
oxytocin injections (Varma & Sastry, 1994 cited in Thomas, 2004).
In general the calves of the injected animals had died, had been separated for sale or were not
accepted by the dams. The high frequencies of oxytocin use in peri-urban Faisalabad might be due
to the high turnover rates of many farms, leading to unfamiliar milkers for the animals, since small
changes in milking routines adversely affect milk ejection in buffaloes (Thomas, 2004). Also the
high calf mortality rates might make another form of stimulus necessary to replace calf suckling.
Animals regularly treated with oxytocin become drug habitual, as repeated injections
interfere with normal milk secretion and inhibit the normal ejection reflex. Prolonged use of
oxytocin also causes fertility disorders such as poor estrus signs, low conception rate, high
embryonic mortality, shortened lactation period, increased abortion rate, calf death and incidences
of mastitis. With respect to animal health, oxytocin use did not affect somatic cell counts in
buffaloes (Bidarimath & Aggarwal, 2007) nor cows (Nostrand et al., 1991). One study on buffaloes,
however, found that the frequent use of oxytocin for milk let down may delay postpartum ovulation
(Qureshi & Ahmad, 2008). On the other hand, there is evidence from the literature that short term
treatment of buffaloes with oxytocin to ease birth or placenta removal increased the reproductive
performance of buffaloes by decreasing involution time and service period (Mavi et al., 2004).
Cameron and Fosgate (1964) reported no effect on diestrus interval and conception rates of
oxytocin use on cows, whereas Armstrong and Hansel (1959) concluded that oxytocin may affect
estrous cycle length of cows. As reported by Afzal (2003) the effects of long-term use of oxytocin
in buffaloes are not known. These problems are currently being addressed by author in progeny-
history interviews targeting each milking animal in the 20 monitored herds.
CONCLUSIONS
The present study suggests that that the effects of oxytocin on milk production are
numerous, complex, and difficult to quantify presently and require more future studies. It has
significant effect on milking management of dairy buffaloes however the regular use of oxytocin for
milk let down should be checked properly, should be prescribed on actual need basis and awareness
should be created among farmers about the detrimental effects of oxytocin under the urban and peri-
urban dairy farming system.
1157
REFERENCES
Afzal, M. 2003. Livestock production-A tool for poverty alleviation in Pakistan. In: Poverty
Alleviation Through Sustainable Agriculture Development. F Ullah and F Ullah (eds).
NWFP Agriculture University, Peshawar, Pakistan. pp: 59-67.
Babbie, E. R. 2009. The practice of social research (12th Ed). Bedmont, California, USA:
Wadsworth Publishing. pp. 193–195.
Belo, C. J. and R. M. Bruckmaier. 2010. Suitability of low-dosage oxytocin treatment to induce
milk ejection in dairy cows. J. Dairy Sci. 93: 63-69.
Bidarimath, M. and A. Aggarwal. 2007. ‘Studies on cisternal and alveolar fractions and its
composition and mammary health of Murrah buffaloes administered oxytocin’. Trop. Anim.
Health Pro. 39(6): 433-438.
Bilal, M., Hameed, A. and B. B. Khan. 2008. ‘Assessment Of Dairy Farm Management Practices
Under Field Conditions Of Toba Tek Singh’. Pak. J. Agri. Sci. 45(2): 237-241.
Borghese, A., Rasmussen, M. and C. Thomas. 2007. Milking management of dairy buffalo. Ita. J.
Anim. Sci. 6(2): 39-50.
Cameron, N. and O. Fosgate. 1964, ‘Effects of Oxytocin upon the Reproductive Tract of the
Lactating Bovine’. J. Dairy Sci. 47: 79-81.
Cheema, M. A., Farooq M., Ahmad R. and H. Munir. 2006. Climatic Trends in Faisalabad, Pakistan
over the Last 60 Years (1945 - 2004). J. Agri. Soc. Sci. 2(1): 42-45.
Ijaz, A. and M. Aleem. 2006. ‘Exogenous Administration of Oxytocin and Its Residual Effects’.
Pak. Vet. J. 26(2): 99-100.
Jonas, H., M. Tariq and E. Schelcht. 2010. Factors Influencing Producer Milk Prices in Peri-urban
Faisalabad Punjab Province, Pakistan. Tropentag, September 14-16, 2010, Zurich “World
Food System (Abstract).
Khan, Z. U., Khan, S., Ahmad, N. and A. Raziq. 2007. ‘Investigation of Mortality Incidence and
Managemental Practices In Buffalo Calves At Commercial Dairy Farms In Peshawar City’. J.
Agric. & Biol. Sci. 2(3): 16-22.
Lollivier, V., J. G. Flament, M. O. Bousquet and P. G. Marnet. 2002. Oxytocin an milk removal:
two important sources of variation in milk production and milk quality during and between
milkings. Reprod. Nutr. Dev. 4: 173–186.
Maurya, V. and S. Ludri. 1992, ‘Effect of oxytocin administration on milk letdown time, milking
rate and composition of milk in buffaloes’. Ind. J. Anim. Sci. 62: 210-214.
Mavi, P. S., Pangaonkar, G. R., Sharma, R. K. and V. K. Gandotra. 2004. ‘Effect of certain
hormones on the reproductive performance of post-parturient buffaloes’. Buffalo J. 20(2):
193-201
Mustafa, M. Y., Saleem, K., Munir, R. and T. M. Butt. 2008. ‘Effect of oxytocin on the productive
and reproductive performance of buffalo and cattle in Sheikhupura-Pakistan (A field study)’.
Livest. Res. Rural Dev. 20(12), Article 193. URL:
http://www.lrrd.org/lrrd20/12/must20193.htm, accessed on January, 2013.
Nostrand, S. D., Galton, D. M., Erb, H. N. and D. E. Bauman. 1991. ‘Effects of Daily Exogenous
Oxytocin on Lactation Milk Yield and Composition’. J. Dairy Sci. 74(7): 2119-2127.
Pegram, R., Smith, R., Franklin, R., Gallagher, T., Oosterwijk, G. and A.Wilsmore. 1991.
‘Comparison of the calf suckling technique and milk oxytocin test for estimation of milk
yields’. Trop. Anim. Health Prod. 23(2): 99-102.
Qureshi, M. S. and N. Ahmad. 2008. ‘Interaction of calf suckling, use of oxytocin and milk yield
with reproductive performance of dairy buffaloes’. Anim. Reprod. Sci. 106(3-4): 380-392.
SPSS, 17.0 for Windows, Version 17.0.1 SPSS Inc., Chicago III.
Syrstad, O. 1993. ‘Milk yield and lactation length in tropical cattle’, World Anim. Rev. 1-2.
Thomas, C. S. 2004. ‘Milking management of dairy buffaloes’, PhD thesis. Department of Animal
Nutrition and Management, Swedish University of Agricultural Sciences, Uppsala.
1158
Figure 1. GIS-based map of Faisalabad city with the approximate expansion of the dense
housing area (within red border) and the 145 interviewed households.
Figure 2. Percentage of dairy buffalo and cattle being administered oxytocin for milk letdown
among the interviewed households in urban and peri-urban area of Faisalabad.
1159
Figure 3. Percentage of dairy buffalo and cattle with calf suckling and their distribution among four
production Systems in urban and peri-urban area of Faisalabad.
SSM=small-scale semi-commercial mixed farmers, SSD= small-scale semi-commercial dairy farmers and SCD= small-
scale commercial dairy farmers LCD= Large-scale commercial dairy farmers
Table 1. Percentage of dairy buffalo and cattle being administered oxytocin and those using calf
suckling and their distribution among four production systems in urban and peri-urban area of
Faisalabad.
Production System*
SSM SSD SCD LCD
Variable P< **
(n=43) (n=30) (n=52) (n=14)
Buffalo Oxytocin
Yes 54 47 66 65 0.004
No 46 53 34 35 n.s.
Cattle Oxytocin
Yes 15 38 18 20 n.s
No 85 62 82 80 0.001.
Buffalo Suckling
Yes 46 44 32 33 n.s.
No 54 56 68 67 0.009
Cattle Suckling
Yes 57 38 43 50 n.s.
No 43 62 57 50 n.s
Chi-Square test for differences between production systems, significance at P<0.05;
n.s. not significant.
*SSM=small-scale semi-commercial mixed farmers, SSD= small-scale semi-commercial dairy farmers and SCD=
small-scale commercial dairy farmers LCD= Large-scale commercial dairy farmers
1160
Environmental Factors Affecting Live Weight and Morphological Traits in Nili

Ravi Buffaloes of Pakistan
*Khalid JAVED, Riaz Hussain MIRZA, Muhammad ABDULLAH and Maqsood AKHTAR
Department of Livestock Production, University of Veterinary and Animal Sciences, Lahore Pakistan
*Corresponding email: khalidjaved@uvas.edu.pk
ABSTRACT
A study was conducted to assess the influence of some environmental factors in body weight
and some morphological traits in Nili Ravi buffaloes of Pakistan. The data were recorded on 498 adult
buffaloes in different parities maintained at 5 institutional herds and some private farms located in
different agro ecological zones in The Punjab province of Pakistan. The means±SD for different
morphological traits; neck length (63.299±6.046 cm), body length (139.55 ±6.29 cm), diagnal body
length (154.01±7.60 cm), height at withers (132.04±4.56 cm), heart girth (194.45±10.31cm) paunch
girth (238.51±13.96 cm), and body weight (523.13±81.63 kg) were recorded. Herd and parity were
significant sources of variation for all body measurements and body weight. Effect of stage of lactation
on neck length, height at withers, heart girth and paunch girth was non-significant but it influenced
body length and weight. Season of scoring did not affect the neck length, diagonal body length, height
at withers and heart girth, however, body length, paunch girth and body weight varied in different
seasons. The present study indicates that the body weight and some body measurements in Nili Ravi
buffaloes fall near the intermediate value when compared with other buffalo breeds. A large number of
environmental factors affect body size and body measurements which may mask the actual genetic
worth for these traits. Their accurate and precise assessment can be helpful in formulating selection
strategies for improved milk production under field conditions because these traits have been reported
to be positively correlated with milk yield in many studies.
Keywords: morphological traits, Nili Ravi Buffaloes
INTRODUCTION
Buffalo is the main dairy animal in Pakistan. Nili-Ravi breed constitutes about 38% the total
buffalo population (Khan et al., 2007). Efforts for its improvement through selection have been
initiated at institutional as well as field level. The physical appearance of the animal is more important
than any other quality including milk yield. Type and conformation in dairy cattle has received very
much attention both in the show ring and from breeders in selecting animals. Type and conformation
are valuable because superiority in these traits may help the animal to maintain a long and highly
productive life. Type traits are also very important because of their relationship with performance of
dairy animals however environmental factors often affect the performance of animals in terms of
growth, production and reproduction. The most important factors include herd, parity, season, stage of
lactation and age of the cow or buffalo at the time of recording. The purpose of this study was to
examine the variation in body weight and some conformation traits due to these factors in Nili Ravi
buffaloes of Pakistan.

1161

Nili Ravi buffalo herds maintained at Buffalo Research Institute, Pattoki, Distt. Kasur,
Livestock Experiment Station Haroonabad Distt. Bahawalnagar, Livestock Experiment Station, Chack
Katora Distt. Bahawalpur, Livestock Experiment Station Khushab, distt. Khushab, Livestock
Experiment Station Rakh Ghulaman distt. Bhakkar and few private breeders were utilized in the
present study. A total of 1180 records from 498 adult buffaloes in several parities on different type
traits and body measurements were generated over a scoring period of 2 years. Sources of variation
due to environmental factors like herd, stage of lactation, parity and season of scoring were included in
the model along with genetic and residual effects body weight and body measurements. Several
Statistical analysis:
Data recorded were subjected to analysis of variance technique under PROC MIXED using SAS
(2010).

The mean±SD, co-efficient of variation and range for different type traits and body
measurements are presented in Table 1. Average score for neck length was found greater than average
score for Azi Khale buffalo (Khan et al., 2009), Chilika buffaloes (Patro et al., 2003) and the difference
might be due to breed differences. Mean score for body length was similar to the findings of Khan et
al. (2009) in Azi Khale buffalo. Swamp and Banni (of Western India) buffaloes had longer body as
compared to Nili Ravi buffaloes (Kalita et al.,2010; Mishra et al., 2009) whereas Iranian and Chilika
buffaloes are smaller in body length (Dezfuli et al., 2010; and Patro et al., 2003). The differences in
body length are mainly due to breed differences and environmental factors.
Table 1. Least squares means for body measurements in Nili Ravi buffaloes.
Trait N Mean±Std Dev C V (%) Range
Neck length (along the surface(cm)) 1129 63.29±6.04 9.55 42-85
Body length (cm) 1176 139.55±6.29 4.50 118-160
Diagnal body length (cm) 1178 154.01±7.60 4.94 127-185
Height at withers (cm) 1179 132.04±4.56 3.45 117-146
Heart girth (cm) 1179 194.45±10.31 5.30 121-226
Paunch girth (cm) 1179 238.51±13.96 5.85 135-288
Body weight (Kg) 839 523.13±81.63 15.60 285-770
Table 2. F value and level of significance of environmental factors on body weight and some measurements.
Trait Herd Stage of lactation Parity Season of scoring
Neck length (along the surface(cm)) 41.96*** 1.06NS 4.23** 2.37NS
Body length (cm) 16.97*** 7.23*** 20.82*** 13.91***
Diagnal body length (cm) 13.5*** 13.20*** 24.13*** 0.96NS
Height at withers (cm) 12.31*** 1.29NS 3.18* 0.76NS
Heart girth (cm) 31.55*** 1.19NS 5.36** 0.63 NS
Paunch girth (cm) 21.79*** 0.81NS 17.73*** 6.76***
Body weight 97.45*** 4.22** 11.27*** 6.37***
*=P<.05, ** =P<.01, ***=P <.0001, NS=non significant
Average score for body diagonal length in the current study 154.013±7.608 cm was comparable
to already reported 151.6±8.7 cm and 147.8±0.71cm in Nili Ravi (Mehboob, 1996 & Khalid, 2011),
149.76 cm in Nili and 151.04 cm in Ravi buffaloes (Wahid, 1976). Average score for height at withers
has been recorded as 132.041±4.566 cm which is compareable to Azi Khale (Khan et al., 2009)
1162
whereas higher than Vietnamese Swamp (Berthouly et al., 2010), Chilika buffaloes (Patro et al., 2003)
and Iranian (Dezfuli et al., 2010). The variation might be due to management, breed and within breed
variation among the members of the same breed. Average value for heart girth has been estimated as
194.45±10.31cm in the current study. Various workers have reported different measurements for heart
girth (208.3 to 251.46 cm) in Nili Ravi buffaloes (Wahid, 1976; Mehboob,1996); 170.0 ± 0.3 cm in
Chilika buffaloes ( Patro et al. 2003). Estimated paunch girth has been observed as 238.51±13.96 cm.
Some workers studied paunch girth (barrel girth) in different breeds of buffaloes and average values
are given as 198.20± 0.96 cm in Swamp buffaloes (Kalita et al. 2010), 217.7± 2.00 cm in Azi Khale
buffalo (Khan et al.,2009), 235.3±15.2 cm in Nili Ravi buffaloes (Mehboob,1996). The findings of
current study regarding average body weight as 523.133±81.631 kg when compared to other reports
differ considerably. Cockrill, (1974) and Khan et al., (2009) has reported 450 and 454 kg body weight
in Nili Ravi buffaloes.
Effect of Environmental factors on body weight and body measurements
Herd and parity were found to be significant sources of variation for all body measurements
and body weight (Table 2). Various workers (Patro et al., 2003; Kayastha, et al., 2011) have reported a
similar finding in different buffalo breeds and indigenous cattle. Season of scoring was also found to
be non significant source of variation for neck length, diagonal body length, height at withers and heart
girth, however body length, paunch girth and body weight were found to be highly different in
different seasons. Body weight has been found significantly different in different herds, parities,
stages of lactation and seasons. Effect of parity has been reported to be significant on body weight in
grade Murrah, Diara and non descript buffaloes by Shankar and Mandal (2010). A large number of
environmental factors affect body size and body measurements which may mask the actual genetic
worth for these traits. Their accurate and precise assessment can be helpful in formulating selection
strategies for improved milk production under field conditions because these traits have been reported
to be positively correlated with milk yield in many studies
CONCLUSIONS
The results of the present study indicate that the body weight and some body measurements in
Nili Ravi buffaloes fall near the intermediate value when compared with other buffalo breeds.
However a large number of environmental factors affect body size and body measurements including
the herd, parity, stage of lactation and season.
REFERENCES
Berthouly, C., X. Rognon, T. Nhu Van, A. Berthouly, H. Thanh Hoang, B. BedHom, D. Laloe, C. Vu
Chi, E. Verrier and J. C. Maillard. 2010. Genetic and morphometric characterization of a local
Vietnamese swamp buffalo population. J. Anim. Breed. & Genet. 127 (1): 74-84.
Cockrill, W. R. 1974.The husbandry and health of the domestic buffalo, FAO, Rome, Italy.
Dezfuli, B. T., M. Babaee, M. R. Mashayekhi and S. S. Sofia, 2010. Prediction of live weight from
linear body measuremets in Iranian buffaloes. Advances in Anim. Bio. Sci. 1 (1): 277
Kalita, R., A. Dandapat, B. D. C. Kamal, G. C. Das and R. N. Goswami, 2010. Conformation traits of
Swamp buffalo of Assam at different age groups.Indian J. anim. Res. 44(4): 300-302
Kayastha, R. B., G. Zaman, R. N. Goswami and A. Haque, 2011. Physical and morphometric
characterization of indigenous cattle of Assam. Open Vet. J.1:7-9
1163
Khalid, M. S. 2011. Correlation response of udder and body measurements as affected by age and
parity on milk contents and yield in Nili Ravi buffaloes in peri urban areas of Lahore. M Phil
thesis University of Veterinary and Animal Sciences Lahore, Pakistan
Khan, M. S., N. Ahmad and M. A. Khan, 2007. Genetic resources and diversity in dairy buffaloes of
Pakistan. Pakistan Vet. J. 27(4): 201-207.
Khan, S. A., M. S. Khan, Y. Muhammad, S. Sultan, F. Muhammad , S .A. Khan and G. Jabbar,
2009. On-Farm performance of Azi Kheli buffalo. Pakistan J. Zool. Suppl. Series. No 9:209-
212
Mehboob-ur-Rehman. 1996. Studies on phenotypic characteristics and body conformation of Nili Ravi
buffaloes and their relationship with milk production. M.Sc. thesis, University of Agriculture,
Mishra, B. P., K. P. Singh, D. B. Chavan, D. K. Sadana, R. S. Kataria, P. Kathiravan and S. P. S.
Ahlawat. 2009. Characterization of Banni buffalo of Western India. AGRI 44:77-86
Patro, B. N., P. K. Mishra and P. K. Rao. 2003. Chilika buffaloes in Orissa: a unique germplasm. AGRI
33: 73-79.
SAS Instt. Inc.2010. SAS/STAT user’s guide, Release 9.1.Carry, North Carolina, USA
Shankar, S. K. and G Mandal, 2010. Genetic and non genetic factors affecting body weight of
buffaloes. Vet. World 3(5):227-229
Wahid, A. 1976. Livestock resources of Pakistan, Monograpg-7‘‘Pakistani Buffaloes’’. University of
Karachi, Karachi (Pakistan).
1164
BUN and Total Protein levels of Buffalo Population

in Udonthani Province of Thailand
Chanachai BOONPERM a, Cholawit YUWACHITb , Tanom TATHONGc,

Somkiat TOOJINDAd,
a
Program in Animal Production Technology, Faculty of Technology, Udonthani Rajabhat
University, Udonthani 4100,0 Thailand
b
Program in Veterinary Technology, Faculty of Technology, Udonthani Rajabhat University,
Udonthani 41000 Thailand
c
Program in Agro-Industry, Faculty of Agriculture and Technology, NakhonPhanom University
48000, Thailand
d
Khumpawapi Livestock Office, Khumpawapi, Udontthani 41110, Thailand
*Corresponding Email : cboonperm@gmail.com
ABSTRACT
The aim of this research was to study on BBP value (Biochemical Blood Parameters) and
healthy management model of Thai buffalo (Bubalus bubalis) in Udonthani Province, Thailand. The
animal used buffalo female at more than two years old and had at least one baby. The experimental
was sampling BUN value and total protein, TP at 150 head in Kumpawapee Districts, Udonthani
Province. The result showed that the healthy management model follow up by advice of
Development of Livestock with health management of management program for the year and
showed that BBP value of buffalo had 6.74 mg/dl and 7.10 g/dl respectively, However; all data was
stay in standard value.
Keywords: Blood urea nitrogen, total protein, Thai buffalo
INTRODUCTION
A buffalo population of Thailand had trend reduce, because; economics system and social
was rapid expansion, city social expansion, farmer had debt burden by agriculture system. All of
these factors were effect on buffalo population to reduce and less land tenure. The present had many
machine in all farmer for compensated labor of buffalo. Udonthani province is located in the north
east, NE of Thailand and all area had buffaloes population is second level of NE. The household
population about 13,137, them had buffalos 46,833 head consist with 15,299 male, 21,643 calf and
heifer 9,891. The first parity on purpose of buffalos production in Udonthani was for sale in the
local market in traditional production most of holder were fed the buffalo by rice straw, Si-jae
village is located in Kumphawapi district. Udonthani province had a pilot project in Behind Bhudda
project under cover by royal project under the program on extension in the development of buffalo
farming. In terms of promoting the breeding of bull by the best semen with good quality health
services by DLD staff. A distribution of mineral block and promote knowledge about the buffalo
correctly technical farmers respectively (DLD, 2013).
However; buffalo production will success by consider appropriateness of the roughage,
concentrate, management and household. The buffalo can use low quality roughage as well better
than other ruminant. The healthy management programs was requirement and important for
assessing on nutritional state and base on buffalo health. The advantages of the modern animal were
the results indicate that the nutritional integrity. which fed by good quality and reasonable health
and can indicate the overall health of the animals. Such as the Liver, Kidney and the others as well
as to indicate the exposure conditions that affect the health of buffalo (Junlun, 2013).
The model of healthy management and hematology in buffalo in certain had the purpose of
this study for used as a basis for identifying buffalo production. The health of the buffalo population
in Si-jae village, Khumphawapi District, Udonthani Province, Thailand.

1165
MATTERIALS AND METHODS

Animal: Hundred and fifty swamp buffalo (Bubalus bubalis ) at two year old and first parity
were sampling and calculated by G-power programing (Suk-oun, 2011). The buffalo raised in
public grass land and ad libitum with rice straw and water.
Data collection: The experimental design and data analysis were collected type of roughage
and stocking health program, vaccinations and deworming. Blood collection 10 ml form jugular
vein by MONO vet tube according to Junlun (2013) and then centrifuge at 1200 rpm for separation
serum. The serum analyzed total protein, Blood Urea Nitrogen, BUN (mg / dl) and total protein
determination (mg / dl) with BS-400 (Mindray).
Data analysis: data analyzed by SPSS program version 17 (KKU license, 2008).

The result showed that BUN value and total protein value had 0.74 mg/dl (4-14 ±2.33
mg/dl) , TP 7.10 g/dl (5.9-8.0±0.56 g/dl) according to Sil (1992) reported in cattle clave on rice
straw based ration. BUN value found on dietary treatment are with in normal physiological value of
10.30 mg/100 ml for buffalo, Total protein value 5.9-8.0 g/dl observed in present study are higher
than the value reported by Sil (1992) in buffalo and slightly lower than the value 7.75 g. reported by
Singh (1991) in buffalo fed rice straw –based ration, However, value are close to the normal range
of 6.75 -7.82 g/100ml as reported by Gupta et al (1988) Significantly higher values for serum
protein in animals fed on ammoniated rice straw than untreated straw fed group has been observed
(Sato et al., 1988;Mishra, 1991). Significantly (P < 0:05) higher serum protein in buffalo
supplemented with Fish meal might be due to differences in quality and quantity of protein
available forabsorption at the intestinal level, differences in the rate of uptake of amino acids which
are due to the changes in hormonal levels to favour anabolic processes (Walli, 1993; Kaufmann and
Luping, 1982), Pattanaik et al. (1999) observed no significant effect on BUN and TP in buffalo fed
green oat supplemented this might be due to the absence urea in buffalo feed. The height of BUN
value may be attribute to season in Thailand.
ACKNOWLEDGEMENT
The authors gratefully thank Faculty of Technology UDRU for financial supported.
Khumpawapi Livestock Office for all supported in this experiment
REFERENCES
Chantalukana, C. and P. Skunmaun. 2002. Sustainable small holder animal system. Kasetsart
University press, Bangkok. 302p.
Jun-lun, A. 2010. Buffalo healh management Faculty of veterinary science, Khon Khean
University.
Suk-Oun, P. 2010. Experimental and study design in veterinary science. Faculty of vete –
rinary science, Khon Khean University.
Sil, B. 1992. Comparative studies on feeding of different conventional proteins in urea

containing diet on performance of crossbred calves. M.V.Sc. Thesis, Deemed
University, IVRI, Izatnagar, India.
Singh, P. 1991. Utilisation of nutrients from straw as affected by mode of NPN
supplementation. Ph.D. Thesis, Deemed University, IVRI, Izatnagar, India.
.
Wannapat, M. and R. Pilajun. 2000. Effect of energy source and urea levels in concentrate for
swamp buffalo.. Department of Animal Science, Faculty of Agriculture, Khon khean
University. In Proc. the 3th Animal science congress.January 23, 2000. 215-226p.
Wanapat, M., F. Sundstol and J.M.R. Hall. 1986. A comparison of alkali treatment methods to
1166
improve the nutritive value of straw. II. In sacco and in vitro degradation relative to in vivo
digestibility. Anim. Feed Sci. Technol., 14 (1986), 125 p.
Wora-anu, S., M., Wannapat, C.Wachirapakorn and N. Notaso. 2000. Effect of Roughage to
concentration ratio on ruminal ecology and voluntary feed intake in cattle and swamp
buffaloes fed on urea treated rice straw. In Proc.the 19th Congress of the Asian-
Australasian of Animal Production Societies and 23th Biemial Conference of the
Australian Society of Animal Production, University of New South Wales, Sydney
Australia, July 3-7,2000, Vol 13:236.
Walli, T.K. 1993. Critical assessment of rumen escape protein feeding systems and its
importance in practical rations. In: Compedium I (Review papers) at the Proceedings of the
6th Animal Nutrition Research Workers Conference, 13ฑ16 September, 1993. OUAT,
Bhubaneshwar, India.
Table 1. Blood parameter in Thai native buffalo 150 on traditional production

Item Unit value SD
Blood urea nitrogen, BUN mg/dl 6.74 2.33
Total protein g/dl 7.10 0.56
Figure1. Frequency of BUN and TP
1167
Preliminary Study on Energy Metabolism and Energy Requirement of Early

Lactation Water Buffalo
Caixia ZOU, Bingzhuang YANG *, Shengju WEI, Xianwei LIANG, Shulu LI, Guangsheng
QIN, Chenjian YANG
*Corresponding author, professor, E-mail: Yangbri@126.com
ABSTRACT
The objective of this study was to investigate the energy requirement of early lactation water
buffalo. Thirty healthy early lactation water buffalo, similar milk yield in the last lactation, 2 or 3
parities, were divided into 3 groups by randomized complete block design to carry out an energy
balance trial. The animals were divided according to their milk yield, parity and received one of 3
diets containing the same forage but different concentrate mixtures of varying levels of net energy
for lactation: 6.0, 6.6 and 7.2 MJ/kg, respectively. The elephant grass was the main ingredient of
forage. The whole experiment was lasted 45 days with the first 15 days for adaptation. Seven-day
digestion and 3 d respiratory gas metabolism tests were conducted on the 24th and 28th day of feeding
trial, respectively. From the relationship between energy intake and heat production, the results
showed that NEm=263.40KJ/(W0.75·d)×120%=316.10 KJ/(W0.75·d) for early lactation water buffalo,
the average milk energy was 4.4 MJ/kg, according to the formula: standard buffalo milk (kg) =milk
yield (kg)×[(milk fat g 40)+(milk protein g 31)]×0.01155+1.0) Di Palo`s equation (1992) ,
based on the average water buffalo milk protein, fat were 4.7% and 6.8% respectively, 1 kg water
buffalo amount to 1.51 kg standard water buffalo milk, thus, produce 1 kg standard water buffalo
milk require 2.915 MJ for net energy. The requirement for total net energy of early lactation water
buffalo could be estimated by the following equations: NE (MJ/d) =0.316W0.75·d
0.2907×ΔW×W0.75 2.915 FCM, where ΔW was average daily body weight change (kg/d).
Keywords: energy requirement, water buffalo, early lactation

1168
Buffalo Production and
Management
Inter-relationship of Milk Constituents with Body and Udder Measurements in

Nili-Ravi Buffaloes Raised at Commercial Farms of Pakistan
Khalid JAVEDa*, Muhammad ABDULLAHa, Muhammad Sulman KHALIDa, Nisar AHMADa,

Jalees Ahmad BHATTIa, Umair YOUNASa
a
Department of Livestock Production, University of Veterinary and Animal Sciences, Lahore, Pakistan.
*Corresponding author e-mail: javeddrkhalid@yahoo.com
ABSTRACT
The current study was aimed at evaluating the inter-relationship of milk constituents with
various body as well as udder measurements of Nili-Ravi buffalo so as to estimate its dairy potential
for making buffalo selection policies on the basis of these measurements in future. The parameters
studied for the current research were body measurements including average Heart Girth (HG), Body
Length (BL), Body Height (BH), Pin to Pin distance (PP), Hook to Hook (HH) and Body Depth (BD).
Similarly, udder measurements included Udder Depth (UD), Udder Width (UW), Udder Length (UL),
Teat Length (TL), Teat Diameter (TD) and Milk Vein (MV) size. Different Milk constituents studied
were Fat%, SNF%, Lactose %, Protein % and Ash %. Nili-ravi buffaloes (n= 200) were selected
randomly from commercial dairy herds in peri-urban vicinity of Lahore city. The selected animals were
belonged to different parities. The findings of this study showed negative correlation (-0.007) between
Milk Production (MP) and Fat % in animals of different parities. However, positive correlation was
observed between MP and SNF% (0.177), Lactose% (0.149), Protein% (0.144) and Ash% (0.152).
Similarly, negative correlation was observed between milk constituents and body measurements except
for Fat % with HG and BD (0.206 and 0.010, respectively).Milk Vein (MV) size did correlate positively and
significantly (P<0.05) with SNF% (0.086), Lactose% (0.044), Ash% (0.031) and Protein% (0.064).
Significant (P<0.05) but negative correlation (-0.116) was noticed between MV size and Fat%. The
current study will be supportive for making selection policies in Nili Ravi buffaloes to enhance and
evaluate the production potential of Nili-Ravi buffalo.
Keywords: Nili-Ravi buffalo, Milk constituents, Udder measurements, Body measurements.
INTRODUCTION
Various studies reported that milk constituents may show strong and significant association
with mammary measurements like udder depth, udder width, udder length, teat length, teat diameter
and milk vein size. The findings of Heins et al. (2008) and Iniguez et al. (2009) has shown significant
affect of mammary measurements on milk composition. Rao et al. (2007) investigated the relationship
of udder with milk fat and protein contents and found significant influence of mammary dimensions on
milk contents. Similarly Ghosh and Parasad (1998) found significant changes in milk composition
associated with udder measurements. The shape of udder also reported to influence the milk production
in dairy animals (Bhuiyan et. al., 2004, Pawlina et. al., 2000). There is immense need to develop
certain standards for such traits in the prime dairy animals of Pakistan as there no work has been done
in respect of this important selection criterion.

1170

Nili-Ravi buffaloes (n=200) were selected for this study randomly. Animals were grouped
according to the stage of lactation. Various body measurements were recorded like body length (BL)
which was taken as distance from point of shoulder to the pin bone. Body height (BH) was measured as
distance from the withers of the animal to the surface of the platform. Heart girth (HG) was measured
by measuring the circumference of the chest. Pin to pin (PP) distance was measured as distance straight
between two pin bones of each side of animal’s body. Hook to hook (HH) distance was measured
between two hook bones of each side of animal’s body. Body depth (BD) of the animal was measured
by subtracting the two different measurements i.e. height at top line and height at bottom line. Whereas,
height at top line was measured by placing the side hand of the measuring scale/rod on the ventral
region of the animal at withers. And height at bottom line was taken by placing the side hand of the
measuring scale/rod on the dorsal region of the animal perpendicular to the place from where height at
top line was measured before. The udder measurements like udder depth (UD), udder width (UW) and
udder length (UL) were taken as described by Bhuiyan et al. (2004). Similarly, teat length (TL) and teat
diameter (TD), milk vein (MV) size was determined using measuring tape and vernier caliper,
respectively. Milk samples (30ml) were taken from each animal after thorough mixing in the Falcons
tube. The milk was analyzed through the Lacto-scan machine for various constituents like fat, protein,
solid-not-fat, lactose and ash.
The mean and standard errors values of different udder, teat and body measurements were
worked out. The Pearson’s Correlation between the different measurements, lactation stage and milk
contents was determined (Steel et al., 1997).
The animals in the study were observed in three different lactation stages. 23.5% buffaloes were
found in first post partum stage (≤100 days). Similarly, number of buffaloes in second (100> to≤200
days) and third post partum stage (200≥ days) were found as 38% and 38.5%, respectively. The
average measurements (mean±SD) for various body measurements like HG, BL, BH, PP, HH and BD
were found as 203.2±11.0, 147.3 ±7.2, 140.2±7.2, 30.2±3.7, 56.9±4.5 and 85.8±5.8cm. Similarly, average
value (mean±SD) for udder measurements like UD, UW, UL and teat measurements including TL, TD
were found as 10.8±1.6, 29.1±4.1, 64.2±7.3cm and 9.6±1.2, 4.08±0.6cm, respectively. The average UL in
lactating Nili-Ravi buffaloes of different parities was found to be 64.2±7.3cm. The average UD found
in lactating Nili-Ravi buffalo animals was 10.8±1.6cm. The UL, UD and UW manifest the pattern of
increasing in size as the lactation number increases. These findings are in parallel with the findings of
Bhuiyan et al. (2004) and also with agreement of findings of Prasad et al. (2010), worked on Murrah
buffaloes. The mean±SD value for UW was found 29.1±4.1cm with CV as 14.2%. The UW was found
to have high positive and significant (P<0.01) relation with the average TL, TD and distance between
the fore teats. These findings do not coincide with the findings of Prasad et al. (2010). The average TL
(mean±SD) in lactating Nili-Ravi buffaloes was found 9.6±1.2 cm. Similarly, mean±SD value for TD
was found 4.08±0.66 cm. The mean diameter of MV measured in two hundred buffaloes was 7.4±1.3cm,
ranged from 2.4 cm to 9.6 cm. Milk vein was found highly positive and significantly (P<0.01)
correlated with the average TL and TD. Among these dimension, UD and distance between the rear
teats shows positive and significant relation with each other only, it is also not in agreement of findings
of Prasad et al. (2010), worked on Murrah buffalo which may imply the importance of basis about
genetic difference among these breeds. The buffaloes were randomly selected for milk samples and
1171
milk composition was analyzed through the Lacto-scan. The average fat%, protein%, SNF%, lactose%
and ash % were 5.98±1.5, 3.3±0.2, 8.67±0.54, 58±0.3 and 0.83±0.04 respectively (table-2). The correlation
among the five milk components was studied (table-3). Most of observed elements of milk were highly
positive and significantly correlated with each other. Fat percentage was found to be negatively
correlated (P<0.01) with MP (Table-6). Correlations between the milk constituents and different
mammary dimensions were studied too (table-4). All milk constituents showed positive association
with MV size except for Fat%. Non-significant association was found between milk constituents and
mammary measurements. These results are in agreement with Kominakis et al. (2009) and were against
the findings of Heins et al. (2008) and Iniguez et al. (2009). Similarly, correlations between the milk
constituents and various body measurements were studied (table-5). Non-significant relationship was
found between five different milk constituents with age, body weight, lactation and milk production (table-
6) which don’t comply with findings of Sieber et al. (1988) However, fat% showed positive association
with age, BW and LN (table-6).
REFERENCES
Bhuiyan, M. M., M. R. Islam, M. L. Ali. M. K. Hossain, M. A. Kadir, N. S. Lucky and B. R. Das.
2004. Importance of mammary system conformation traits in selecting dairy cows on milk yield
in Bangladesh. J. Biolog. Sci. 4(2): 100-102.
Ghosh, B. and J. Prasad. 1998. Milk yield and composition as influenced by udder measurements in
Jersey x Red Sindhi crosses. Ind. J. of Anim. Prod. Mngmnt. 14: 23 - 25.
Heins, B. J., L. B. Hansen, A. J. Seykora, D. G. Johnson, J. G. Linn, J. E. Romano and A. R. Hazel.
2008. Crossbreds of Jersey X Holstein compared with pure Holsteins for production, fertility
and body and udder measurements during first lactation. J. Dairy Sci. 91: 1270–1278.
Iniguez. L., M. Hiliali., D. L. Thomas and G. Jesry. 2009). Udder measurements and milk production in
two Awassi sheep genotypes and their crosses. J. Dariy. Sci. 92: 4613-4620.
Kominakisa, A.P., D. Papavasilioub and E. Rogdakis. 2009. Relationships among udder characteristics,
milk yield and non-yield traits in Frizarta dairy sheep. Sml. Rumin. Res. 84: 82-88.
Prasad, R. M. V., E. R. Rao, K. Sudhakar, B. R. Gupta and M. Mahender. 2010. Studies on udder and
teat measurements as affected by parity and their relationship with milk yield in Murrah
buffaloes. Buf. Bullet. 29(3): 194-198.
Pawlina, E., W. Kruszyński and M. Kuczaj. 2000. An investigation of changes in udder size of Red and
White cows in first and third lactation. Med. Weter. 56: 672-674 [in Polish].
Rao, T. K. S., A. K. Dang and C. Singh. 2007. Effect of udder and teat characteristics on milk
composition and yield of Karan fries cows. Ind. J. Dairy Sci. 60(5).
Sieber. M., A. E. Freeman and D. H. Kelley. 1988. Relationships between body measurements, body
weight, and productivity in Holstein dairy cows. J. Dairy. Sci. 71: 3437-3445.
Steel, R. G. D., J. H. Torrie and D. A. Dickey. 1997. Principles and Procedures of Statistics 3rd Ed.
Table-1: Mean with S.D, C.V and range of milk components

Variables Mean ± S.D C.V (%) Range
FAT (%) 5.98±1.5 25.9 2.9-8.2
SNF (%) 8.67±0.5 6.2 8.01-10.6
LACTOSE (%) 4.58±0.3 6.2 4.2-5.6
SOLIDS/ASH (%) 0.83±0.04 5.7 0.78-0.98
1172
PROTEIN (%) 3.3±0.2 5.9 3.10-3.94

S.D=Standard Deviation C.V=Coefficient of Variations
Table-2: Correlations among milk components

FAT SNF LACTOSE ASH PROTEIN
Fat 1 -0.193 -0.235 0.164 0.084
SNF 1 0.977** 0.836** 0.902**
Lactose 1 0.839** 0.878**
Ash 1 0.964**
Protein 1
UL UD UW MV TL TD
FAT -0.111 0.004 -0.084 -0.116 -0.088 -0.241
SNF -0.162 0.136 -0.225 0.086 0.166 -0.061
LACTOSE -0.228 0.086 -0.250 0.044 0.189 0.019
ASH -0.160 0.069 -0.208 0.031 0.194 -0.191
PROTEIN -0.123 0.100 -0.180 0.064 0.159 -0.194
Table-3: Correlations between milk constituents and mammary measurements

UL= Udder Length, UD= Udder Depth, UW=Udder Width, MV=Milk Vein, TL= Avg. Teat Length & TD=Teat
Diameter
Table-4: Correlations between milk constituents and body measurements

HG BL BH PP HH BD
FAT 0.206 -0.262 -0.167 -0.178 -0.167 0.010
SNF -0.187 -0.177 -0.085 -0.083 -0.033 -0.095
LACTOSE -0.213 -0.195 -0.089 -0.077 -0.045 -0.094
ASH -0.134 -0.253 -0.206 -0.193 -0.056 -0.164
PROTEIN -0.212 -0.278 -0.239 -0.157 -0.060 -0.226
HG= Heart Girth, BL= Body Length, BH= Body Height, PP= Pin to Pin distance, HH= Hook to Hook distance,
BD= Body Depth.
Table-5: Correlations between milk components, age, body weight, parity and milk production
Age BW LN MP
FAT 0.113 0.108 0.112 -0.007
SNF -0.070 -0.073 -0.135 0.177
LACTOSE -0.146 -0.132 -0.182 0.149
ASH -0.117 -0.178 -0.200 0.144
PROTEIN -0.129 -0.173 -0.225 0.152
BW=Body Weight, LN= Lactation Number, MP= Milk Production
1173
An integrated study on milk and beef production conducted at Macún

Buffalo Enterprise in Cuba. Some results and recommendations.
Luis Mateo FRAGA BENITEZ 1*, Delia Maria CINO NODARSE1, Gladys GUZMÁN
MARTINEZ1, Yenny GARCÍA ORTA1, Marcelo DULZAIDE2, Diosnel FERNÁNDEZ
GARCÍA2 and Alain RODRÍGUEZ LEÓN2
1
Institute of Animal Science. Carr. Ceentral Km 47 ½. San José de las Lajas. La Habana.
Cuba.
2
Macún Buffalo Enterprise. Sagua la Grande. Villa Clara. Cuba
*
Corresponding email: lfraga@ica.co.cu
ABSTRACT
At the “Macún” Buffalo Enterprise in the Villa Clara county in Cuba were conducted
several technologic (2003 – 2009 period) and socio-economics (2008 – 2009 period) integrated
activities between technicians, managers and workers of this center in order to analyse and
propose solutions for a better efficient milk and beef buffalo production, which are their
primary objective productions. There were employed modern methods such as Mixed Models
and SWOT crossed techiques in processing data (SAS 2007) which were used with an integral
analysis in the enterprise. Productive results were goods and the reproductive ones higher than
the expected for this species in Cuba. Lactation curves were obtained for the enterprise and
their 7 herds indicating the atipic ones. Male daily gains in performance tests were the best in
comparison with other Cuban enterprise stations (0.800 – 0.500 kg/day). The strategic analysis
expressed the higher “Strengths – Opportunities” evaluations indicating the possibility to adopt
an offensive action to face the existing threats and also a favourable enterprise economic
situation. Some recommendations are given to reach a more efficient activity in the enterprise.
It was recommended to conduct a similar study in other enterprises considering university
professors, researchers, managers and workers of the region into consideration.
Keywords: búffaloes, perfomance, SWOT, milk, beef


1174
Productivity of Mehsana Riverine Buffalo under

Tropical Conditions of Thailand
Pramorn MUANGPROMa, Prapawan SAWASDEE a, T. Srisakdia, Pichit CHUMCHAROEN
a
, Charlongchai CHUMCHEEN a, C. Pinyotheppratana and Suvit BOONPRONGa*
a
ABSTRACT
The objectives of this research were to study production performance of Mehsana riverine
buffaloes which imported parent stock breeds from India since 2002. These animals were kept as a
herd in Buriram Livestock Research and Testing Station conditions which were at 142618N,
1024330E and 150 m above sea level. All animals were kept unrestrained in an open animal
house and had free access to mixed pasture containing Para grass (Brachiaria mutica), Ruzi grass
(Brachiaria ruziziensis), Guinea grass (Panicum maximun) and Hamata (Stylosanthes hamata). At
night, they were also fed soilage crop or silage or hay and 0.5-1.0% bodyweight /kg /day of 14-18%
(of dry matter) crude protein concentrate. The animals had free access to mineral supplements and
clean water. These data consisted of 107 records of growth performance, and 27 records of fertility,
milk yields and milk compositions were used in this survey from 2005 to 2012. Least Square
Analysis was used for statistical analysis. The effects of sires, dams, age of dams, season and year
at birth of calves were also included in the model for unbiased adjustment. The research revealed
that bodyweight at birth as well as at 240, 400 and 600 days of age was 29.61, 205.90, 237.50 and
348.76 kg, respectively. The fertility such as: age at first calving and calving interval were 39.57
months and 476.00 days, respectively. The average milk yields per lactation, length of lactation and
milk yields per head per day were 1,062.39 kg, 273.53 days and 3.95 kg/head/day, respectively. The
milk compositions found that milk fat, protein, lactose, solid not fat and total solid were 7.28%,
4.69%, 4.75%, 10.14% and 16.13%, respectively.
Keywords: Mehsana riverine buffalo, growth rate, fertility, milk, Thailand
INTRODUCTION
The organized dairy sector in India is largely dependent on buffalo milk because of their
contribution to total milk production, rich fat and total solid content. However, the majority of
buffaloes in Thailand are classified as swamp buffalo, traditionally kept by small farm holders for
multi-purpose roles complementary to crop production. Buffalo farming essentially had three
functions: farm integration; insurance, capital formation and income; and food production
(Indramangala, 2002). There used to be raising Murrah dairy buffalo in Thailand in the last one or
two decades. However, those raising dairy buffalo was not popular among farmers at that time. As a
result, Department of Livestock Development (DLD) stopped producing Murrah riverine buffalo
production. There have been subsequences generations of Murrah crossbred buffaloes in the
villages, which are of high growth rate suitable for fattening.
Mehsana buffaloes are one of the best milk breeds of buffalo in India and are spread in the
northern part of the Gujarat State (Gupta, 1997; Trivedi, 1997; Pundir et al., 2000). The name of
Mehsana buffalo was derived from the town “Mehsana” in the North Gujarat State. In 2002, the
Government of India presented 50 Meshana riverine buffalo (5 males and 45 females) to His
Majesty the King of Thailand in the occasion of Golden Jubilee gifts. The King had an initiative for
DLD to study and research with efficiency in growth performance and milk yield for promotion of
these animals to Thai farmers (DLD, 2012).
Thus, the objective of this study was to examine, productive and reproductive performance
of the Mehsana riverine buffalo under tropical conditions of Thailand.
1175

Animals were maintained as a herd at Buriram Livestock Research and Testing Station,
Department of Livestock Development (DLD) in Pakum District, Buriram Province, Thailand
conditions which were at 142618N, 1024330E and 150 m above sea level with an average
temperature 27.00C (22.00-32.40C), average relative humidity (RH) of 75% (43-81%) and
average of 1,096.60 mm annual rainfall (Thai Meteorology Department, 2012).
They were provided as one group with mixed pasture containing Para grass (Brachiaria
mutica), Ruzi grass (Brachiaria ruziziensis), Guinea grass (Panicum maximun) and Hamata
(Stylosanthes hamata). At night, all animals were housed and fed soilage crop or silage or hay and
0.5-1.0 bodyweight/kg/day of 14-18% crude protein meal concentrate modified from the National
Research Council (NRC, 1987). Mineral supplements and clean water were also freely accessed.
They were vaccinated against foot and mouth disease and were free from brucellosis and
tuberculosis. Treatment for control of endoparasites was performed twice a year. The Mehsana
dairy buffaloes were also kept likewise dairy cows. Milk yields from 17 dairy cows were recorded
every day, and milk compositions were also analyzed ones every three months.
Historical records for the period 2005 to 2012 were used. These data consisted of 107
records from animals. Following data from each animal were analyzed: weight at birth, 240-, 400-
and at 600-day of age. The 27 records of fertility such as: age at first calving and calving interval,
and milk production as follow: milk yields and milk compositions were also determined. Those
Meshana riverine buffalo were offspring of 3 sires and 30 dams which were imported parent stock
breeds from India. Least Square Analysis was used for statistical analysis. The effects of sires,
dams, age of dams, season and year at birth of calves were also included in the model for unbiased
and female group by least significant difference (LSD). Values are presented as means±SEM. 
adjustment. Comparison on means of growth performance was tested for difference between male
RESUILTS AND DISCUSSIONS

Table 1 shows the growth performances of Mehsana riverine buffalo under tropical
conditions of Thailand. A sex difference was found in weight at 600 days of age with these male
animals having significantly higher bodyweight (P<0.01) than female groups. However,
bodyweight at birth, 240- and at 400-day of age did not significantly differ between male and
female animals. This table did not details of bodyweight of mature male and female, and had no
details of nutrient composition and digestibility of the diets fed during the period of this study.
Trivedi (2000) and Sethi (2003) reported the existence of the Meshana breed in north
Gujarat, India, was referred to in 1940. This breed was the result of selection of Indian breeds of
buffalo. Characteristics are intermediate between Surti and Murrah. Jet black skin and hair are
preferred. Horns are sickle-shaped but with more curve than the Surti. The udder is well developed
and well set. Milk veins are prominent. Bodyweight of mature animals with 3 to 5-year-old were
570 kg in males and 430 kg in females, respectively. However, this survey could not seek the
bodyweight at birth, 240-, 400- and at 600-day of age in Mehsana riverine buffalo under the other
countries having similar climatic and nutritional conditions.
The results revealed the fertility and milk production of Mehsana dairy buffalo under
tropical conditions of Thailand are shown in Table 2. The age at first calving and calving interval in
this survey showed that the levels were found to be within the range of reported values for Mehsana
dairy buffalo in India (McDowell et al., 1995; Rao and Nagarcenkar, 1997). However, milk
yields/lactation, length of lactation and milk yield/head/days were slightly lower than the means of
these milk productions (McDowell et al., 1995; Rao and Nagarcenkar, 1997) given for Mehsana
dairy buffalo in India (Table 2). According to Sethi (2003), the average milk yield/head/day in
Mehsana buffaloes ranges from 4.37 to 4.81 kg.
The milk compositions such as: colostrum and milk of Mehsana dairy buffalo under tropical
conditions of Thailand, and raised in India are shown in Table 3.
The results from this survey showed that milk compositions of Mehsana dairy buffalo under
tropical monsoon climate conditions of Thailand are the range of reported values for Mehsana dairy
1176
buffalo in India (McDowell et al., 1995; Trivedi, 2000; Sethi, 2003). Furthermore, these animals
kept in Thailand which had higher percentage protein than the animals raised in India (Table 3), and
the milk production of these animals had lower somatic cell count (cell/ml) than the range of GPM
which limit of DLD are somatic cell count (cell/ml) in milk should be lower 500,000 cell/ml.
In conclusion, it is assumed that the Mehsana to be a dual purpose (meat and milk) buffalo
in Thailand. Moreover, the demand for high quality buffalo has been increasing due to the change in
the socio-economic pattern of population in Thailand. The market system in the country demands
animals that produce heavy carcasses with less fat trims. Animals with high fertility, efficiency in
growth performance and high milk compositions are preferred. This survey confirms that Mehsana
riverine buffalo can keep in Thailand with high production under tropical climate.
REFERENCES
Department of Livestock Development (DLD). 2012. Buffaloes Production and Management
Systems in Livestock Research and Breeding Center of DLD. Department of Livestock
Development, Minister of Agriculture and Cooperative, Bangkok. (in Thai)
Gupta, P. R., 1997. Dairy India. 4th ed., Statistics, 153-195.
Indramangala, J. 2002. Buffalo development, pp. 117-123. In J. Allen and A. Na-Chiangmai, eds.
Development Strategies for Genetic Evaluation for Beef Production in Developing
Countries. ACIAR Proceeding, no. 108, Watson Ferguson & Co., Brisbane, Australia.
McDowall, R. E., J. C. Wilk, S. K. Shah, D. S. Balain and G. H. Melry. 1995. Potential of
Commercial Dairying with Buffalo. North Carolina State University, USA.
National Research Council (NRC). 1987. Predicting Feed Intake of Food Producing Animals. Natl.
Acad. Press, Natl. Res. Counc., Washington, DC.
Pundir, R. K., G. Sahana, N. K. Navani, P. K. Jain, D. V. Singh, S. Kumar and A. S. Dave. 2000.
Characterization of Mehsana Buffaloes in India. Anim. Gent. Res. Infrom. 28: 53-62
Rao, M. K. and R. Nagarcenkar. 1997. Potentialities of the buffalo. World Rev. Anim. Prod. 13:
53-62.
Sethi, R. K. 2003. Buffalo Breeds of India. Proc. of Fourth Asian Buffalo Congress, New Delhi,
India, 25 to 28 Feb.
Trivedi, K. R. 1997. Dairy herd improvement program actions. Buffalo Newsletter 7: 4-6.
Trivedi, K. R. 2000. Buffalo recording systems in India. Proc. Workshop on Animal Recording and
Management Strategies for Buffalo. ICAR Technical Series 4: 5-12.
Table 1 Means±SEM values of growth performance of Mehsana riverine buffalo under tropical
conditions of Thailand
Parameter Sex Average (n=107)
Male (n=64) Female (n=43)
Weight at birth (kg) 30.82±1.01 29.84±1.10 30.33±1.08
Weight at 240 days (kg) 214.42±8.52 209.08±7.68 211.75±8.10
Weight at 400 days (kg) 258.32±13.58 241.88±11.46 250.10±12.52
a b
Weight at 600 days (kg) 382.97±17.11 348.77±12.90 365.87±15.00
Means±SEM of sex within the same row with different superscripts were significantly different
(P<0.01).
1177
Table 2 Means±SEM values of fertility and milk production of Mehsana dairy buffalo under
tropical conditions of Thailand and under India conditions reference (Figures in the parenthesis
were range of values reported.)
Parameters No. Means±SEM In India1/
Age at first calving (AFC; months) 27 39.571.97 46.80

(32.47 – 46.67) (44.2-54.5)
Average calving interval (CI; days) 27 476.0021.38 492
(360 – 707) (457-526)
- CI from 1 to 2 (days) 24 465.0431.79
(333 – 707)
- CI from 2 to 3 (days) 20 475.1433.63 -
(315 – 718)
- CI from 3 to 4 (days) 10 375.2034.43 -
(328 – 512)
- CI from 4 to 5 (days) 9 433.1359.87 -
(342 – 679)
Milk yields/lactation (kg) 17 1,062.39129.56 1,610
(155.80 – 2,292.80) (1,308-1,838)
Length of lactation (days) 17 273.53±26.93 294
(150 – 450) (267-314)
Milk yield/head/days (kg) 17 3.950.28 5.48
(1.67 – 6.15)
1/
Sources: McDowell et al. (1995); Rao and Nagarcenkar (1997)
Table 3 Means±SEM values of milk compositions of Mehsana dairy buffalo under tropical
conditions of Thailand (Figures in the parenthesis are range of values reported.)
Milk compositions In Thailand (n = 17) In India 1/
Colostrum Milk Milk
Fat (%) 3.380.32 7.280.32 7.38 (6.1-8.3)
Protein (%) 9.710.72 4.690.22 3.60 (3.4-4.1)
Lactose (%) 3.510.17 4.750.13 5.48 (4.5-9.9)
Solid not fat (%) 14.090.64 10.140.24 9.8 (9.5-10.9)
Total solid (%) 17.140.51 16.130.65 17.24 (16.2-18.9)
Somatic cell count (cell/ml) 156,071.36±25,206.05 77,619.05±8,320.76 -
1/
Sources: McDowell et al. (1995); Trivedi (2000); Sethi (2003)
1178
Status and Perspectives of Buffalo in Bangladesh
Quazi M EMDADUL HUQUE and Antonio BORGHESE
Bangladesh Buffalo Center, Lal Teer Livestock, Anchor Tower, 108 Bir Uttam C R Dutta Road,
Dhaka 1205, Bangladesh and International Bufallo Federation, Rome, Italy.
*Corresponding Email:qmehuque@gmail.com
ABSTRACT
Buffalo has never been addressed in Bangladesh and it is a neglected species despite
their important role in the national economy. Most of the population are reverine type with the
exception of some swamp type found in Bangladesh. Different crossbreds population with
Murrah, Nili-Ravi, Surti and Jaffrabadi blood lebel are also available in scanty sorrounding
Indian border of Bangladesh. The buffaloes are 1.39 million heads and they are managed in
household subsistance farming in the villages as a draught animal that can give milk to the
family in low quantity, as producing about 800 kg per lactation and the most part is consumed
by calf and a little is sold out. Many buffaloes rear in saline coastal region under extensive
farming as Bathan farming, they are partially milked and are mostly used for meat production.
Recently the Lal Teer Livestock established Bangladesh Buffalo Centre where prime animals
of Bangladeshi breed were introduced, with the good condition of housing, management and
nutrition. Semen of high genetic merit of Mediterranean Italian breed was used to apply
artificial insemination after sinchronyzation with ovsynch method which will be discussed.
The goal of the centre is to obtain milk purpose crossbreds to utilize the females in the milk
line buffalo improvement, the males as breeding bulls in Bangladesh and in the household
subsistance farming and in the saline coastal region as extensive farming locally called Bathan
farming for Buffalo improvement in Bangladesh. Cheese and meat industries will be also
developed.
Keywords: Buffallo, Farming Systems, Households, Synchronization, Improvement
INTRODUCTION
All countries in the south of Asia, especially Bangladesh, should give more emphasis
policies to ensure food safety as very high densely populated country in the world. In the world,
many people are in poverty and or malnourished, and they mainly rely on local livestock and plant
for food. Livestock production critically needs to address these circumstances. Increase livestock
production requires large scale improvement and a high intensity of individual animal production.
In Bangladesh, recent studies and reports also reveal the rapid growth of human population
(approximately 1.60% per annum) with urbanization (10.00%, 1990-2010) (PPRC, 2011).
Simultaneously growing population, poverty reduction, increase in middle class, and their increased
income have changed their food preferences. These recent developments have major impacts on
demand for animal derived products such as milk, meat, butter, cheese, ice-cream, baby foods,
locally made sweets which are heavily dependent on milk plus sugar. While the consumption per
capita of livestock products is much higher in developed countries, substantial growths have also
occurred in developing countries of Asia except Bangladesh (FAO, 2009). The global buffalo
population is 180.70 million, Asian buffalo dominate the world population, representing 96.40% of
the total buffalo population as of 2008 (FAO, 2010). Within the Asian region, about 74.80% of
buffaloes in the South Asia, 12.80% in East Asia, and only 8.40% found in South-East Asia. The
total buffalo population is about 1.39 million (BER, 2012) in Bangladesh of which coastal regions
posses about 40% (Faruque et al., 1990).

1179

Buffalo breeds and genetic diversity
Dometic indigenous buffaloes of Bangladesh belongs to the Bubalus bubalis with most of
the population are reverine type with exception of some swamp type found in eastern part of
Bangladesh. Its crosses population (Faruque et. al,1990) with Murrah, Nili-Ravi, Surti and
Jaffrabadi blood lebel are scantly available sorrounding Indian border of Bangladesh due to border
migration from India (Huque and Borghese, 2012). Bangladesh imports a small number of Nili-
Ravi from Pakistan during 1960 without any scientific improvement program. Department of
Livestock Service (DLS) again imported 100 live Nili-Rav pregnant heifer and 1st lactating cows
from Pakistan during 1990 that were reared in newly established buffalo farm at Bagherhat district,
in south-west part of Bangladesh. But the breed was not maintain properly and mixed up with local
buffalo through male distribution program of the DLS. At present, the buffaloes in the farm were
also mixed up and no record keeping was maitained. Phenotypically Nili-Ravi charater is also
scantly found in southern-west part population of buffalo in Bangladesh. Most of the buffaloes are
non-descriptive in type and variable composition being either non-descriptive or crosses among
various breeds and can not be catagorized in any well-established breed.This diversity of intercross
breed resource narrows down the scope to selective breeding. Though most part of Bangladesh raise
riverine breeds, the quality of breeds are reducing everyday due to lack of any scientific
improvement program.
Production system
Buffaloes are raised in Bangladesh through out the countrty with some specific distribution
of concentration in coastal saline region, plain land, marshy land and hilly area which are fully
depends on feed resources availability. On the basis of land areas and type, major buffaloes are
raised under three production system: household subsistance, semi-intensive and extensive system
in coastal saline region which covers about 23.00% of total land areas. The houshold subsistance
farming (HF) buffaloes are reared under stall feeding with 6 to 7 h grazing in and around backyard
or public land with very little feed supply. The average herd size in HF is about 1 to 3 with highest
number 10. The semi-intensive farming (SIF) buffaloes are raised in combination of seasonal based
household during rice cultivation and free range system during common land free which is mostly
upper part of coastal areas. Seasonal rice cultivation is the main occupation in the areas. The
average herd size is 4 to 15 animals in highest. Buffaloes in the lower part of the coastal area are
raised under an extensive farming system (EF) locally called Bathan farming. The extensive
farming system in Bathan coastal region includes offshore islands, mudflats, chars (accreted land)
and new accretions. The important occupations include fisheries and salt production and buffalo
rearing. Many of the coastal areas have extensive areas of grasslands. These are used as grazing
lands for the buffalo. In the coastal area, buffalo are used for milk purpose along with live animal
for meat. The herd size is about 51 to 200 with highest number 600 animals and are reared
completely under natural grazing system with almost no extra feed supply.
Milk production and value chain development
Buffalo milk production in Bangladesh remained more or less stagnant due to absence of
any milk improvement program. According to WHO per capita requirement of milk per day is 250
ml. By taking this into account the country’s requirement of milk is about 13 MT (million tons).
However, Bangladesh produce only about 2.95 MT, showing a shortage of 10.05 MT. As the
poverty is decreasing and income is increasing, the demand for not only milk but also milk
products; like butter cheese, ghee, yogurt, ice cream is also increasing. Therefore, it is becoming
very important to increase the supply of milk and milk products to meet the increasing demand of
milk. Though total milk production of Bangladesh is about 2.95 MT in 2011 out of which about 3 to
4% is produced by the buffalo in spite of the number buffalo growth rate are increasing (Huque and
Borghese,2012).The average milk yield of water buffalo in Bangladesh is approximately 620 kg to
1161kg in 270 days to 330 days expansion (Islam et al., 2004). Faruque et al., (1990) reported that
average lactation yield was 730 kg for 328 days lactation period where fat in milk was found to
range 6.80 to 13.20 percent. Daily milk yield per buffalo was lower (2.00 to 3.50 liter) than the
1180
crossbred cattle (3.50 to 7.00 liter) but it is higher than indigenous cattle (Huque et al., 2010). As
the buffaloes have high milk production than local cattle in the same climate which shows higher
potentiality of buffalo for diminishing gaps of milk production in Bangladesh.The milk value chain
development of buffalo milk is required to improve the situation to keep the higher trend of income
of the farmers from buffalo milk.
Meat production
Buffalo meat is not popular in Bangladesh, but its low cholesterol level and higher quality
than cattle may attract by the consumers, if quality tender meat is available like cattle. Hasnath
(1985) reported that the average live weight of buffalo slaughter age was about 320 kg where the
dressing percentage was 44% only. It was reported that the average live wight of adult buffalo was
about 427 kg (BBC, 2012). From long passed, all most all buffalo in Bangladesh are slaughtered at
older age after completing their whole life in works and animals are usually very emaciated. The
meat fiber is very sticky and hard to chewing. However, a big number of buffaloes are slaughtered
every day in the city market and it has been sold in disguise of beef with lower price than beef. In
general, the quality of meat is one of the main reason not to well accepted by the consumers. The
buffalo meat price is about taka 250/kg against cattle meat price taka 285/kg which is always lower
in between taka 25 to 35 per kilogram (One US$= Taka 81) as tender aged quality buffalo meat is
not at all available in common market. This taboos of low quality buffalo meat can be changed by
introducing tender aged buffalo meat marketing through buffalo improvement program in the
country.
Buffalo breeding policy
The broad framework of buffalo breeding policy was formulated for crossing or grading up
of non-descriptive and low producing buffalo with high yielding genetic merit milk buffalo like
Nili-Ravi, Murrah or Mediterreanean Buffalo. However, buffalo breeding policy is not well defined.
Framers use to keep buffaloes for milk, draught and meat as surplus after work. The indigenous
buffaloes are considered to have higher disease resistance, better tolerance to high hot and humidity
conditions prevalent in Bangladesh.These animals are also more efficient in feed conversion
efficiency of crop residues and naturally available low quality roughages.
Feeding practices
The crop residues are mainly constitute the feed mterials for the Buffalo. Farmers are
generally follow the traditional feeding practices and are fully dependent on their own farm
produces dry roughages rice straw, grazing in common land and some concentrates ingredients like
wheat bran, rice bran, pulse bran. Small green grasses are available from rice field, road side grass
and Char land grazing. Grazing on coastal salt rich herbage in submerged Char land areas are also
practiced in coastal region. Migratory grazing on river basin areas are practiced.Variation of buffalo
feedings are depending on production systems. Only domestic salt are used as mineral
supply in upland areas.
Breeding and reproduction
Buffalo is still considered as difficult breeders due to silent heat for reproduction. Buffaloes
reared under household subsistance farming are subjected to controlled natural mating wheres
animals in SIF and extensive farming are mated rendomly with natural mating. Stud bull are one of
the main problems for buffalo reproduction in all production systems. Castration is done early in
life for male, by open method for producing work buffalo. Artificial insemination and time fixed
synchrinization are introduced by Lal Teer Livestock Limited first time in Bangladesh.
Health care practice
Most of the diseases that occurs in cattle also inflict harmful effects on the buffaloes. Foot
and mouth disease is the most problematic disease in Bangladesh as quality vaccine is not freely
available. Diseases such as Haemorrhagic septicaemia (HS), fasciolosis, ascariosis, anthrax,
brucellosis, tuberculosis and black quarter are also important diseases causing more economic
losses. Health care practice is not available in most cases of buffaloes as it reared in most remote
areas. Now a days very few farmers use Hemorrhagic Septicemia and antrax vaccines that are low
cost produced in country. Most of the farmers do not use any anthelmintic for deworming in buffalo
1181
except a few for only fasciolosis.

Ovsynch method of synchrinization in Bangladesh
Bangladesh Buffalo Centre of Lal Teer Livestock inroduced first time ovsynch treatment
protocol for buffaloes in Bangladesh in which follicular and luteal development was monitored after
treating with GnRH-PGF2α-GnRH for ovulation synchronization, which is commonly known as
‘ovsynch protocol’. Ovulation was synchronized by the administration of a GnRH analogue at day 0
(Overelin®, Gonadorelin 100mcg/ml, BOMAC, New Zealand), followed by PGF2α treatment
(Cloprostenol 250mcg/ml , BOMAC, New Zealand) on day 7 and a second GnRH treatment at
24:00 h after PGF2α (on day 9). The animals subjected to receive ovsynch treatment regimen
regardless of stages of their oestrus cycle. Ovsynch treated buffaloes were inseminated irrespective
of the onset of prominent oestrus signs. Each animal received a single artificial insemination with
frozen semen from Mediterranean buffalo bulls at 16:00 to 20:00 h (timed AI) following second
GnRH injection. Detection of ovulation was confirmed by ultrasound machine. Pregnancy diagnosis
was confirmed by the application of real-time transrectal ultrasonography at day 35 to 45 post
breeding. In using fixed time AI with ovsynch protocol, twenty buffaloes out of thirty five cyclic
buffaloes (20/35) were confirmed as pregnant giving a conception rate of 57.14%. In our ovsynch
method it has been observed that the protocol is effective to resume ovarian activities as early as 30
days postpartum, and appears to be a useful estrus synchronization method as well in
improving the reproductive performance of water buffalo-cows in Bangladesh.
Buffalo improvement program of lalteer livestock

Bangladesh Buffalo Center (BBC) has been established in 2010 by Lal Teer livestock Limited, a
private company of the country for improvement of buffaloes. As all buffaloes in Bangladesh have
constraints of very low milk yield, high age at first calving, longer dry period with many other low
merits (Table 1) cross breeding or grading up is taken
Table 1. Production Characteristics of Buffalo at
by Lal Teer Livestock to combine the desirable genes Bangladesh Buffalo Centre
in parental breeds in order to improve the milk
production potential. The main objectives of program Traits Number Results
are to improve the production potential of buffalo of
through assessment of genetic merits of sires and to observa-
tion
increase the production by breeding, feeding and
Adult average live weight 66 426.75
management. Buffalo improvement through bull in kg
selection on the basis of progeny performance is Average birth weight, 14 29.80
taken up. The purpose of the project is to increase local calves in kg
quality seed (semen) production from locally Average birth weight, F1 14 36.50
produced environmentally supported proven bull at in kg
Average growth rate local 14 352.65
desired blood levels for higher milk and meat yield calves up to 6 month
and its distribution/marketing for artificial (g/day)
insemination for increasing milk and meat through Average growth of rate F1 14 803.25
out the country. Improvement of knowledge and skill calves up to 6 month
of buffalo farmers will be upgraded. At present, (g/day)
Average lactation period 14 289.60
project have nucleus farm, bull station for semen in days
production, contract growing farm for quality bull Average lactation yield in 14 541.93
production. The semen processing lab and disease litre
diagnostic lab will be shared with the dairy semen Average gestation period 14 304.25
production project that has been taken by Lal Teer in days
F1= Crosses between Mediterranean and local
Livestock. The results will be the creation of dairy
Source: BBC,2012
purpose buffalo which will contribute very much to
the human needs in Bangladesh of animal protein as
milk and meat. Moreover, the creation of a market of milk and meat products as mozzarella and
other cheese, butter, ghee, ricotta, salami, sausages could be useful for Bangladesh economy. The
BBC was established in 2010 with selected elite cows for crossing with the imported Mediterranean
1182
frozen semen from Mediterranean buffalo for producing F1 bull for semen production and
distribution. A program for buffalo improvement is undertaken in household farming, semi-
intensive and extensive farming in slected regions. Very recently, DLS has also started buffalo
improvement program through cross breeding with Mediterrean breed in selected district of
Bangladesh.
CONCLUSION
Buffalo could be a major source of milk to reduce the milk demand gap in Bangladesh.
Buffalo improvement program through scientific crossbreeding with high yielding quality breed
with feed and management improvement and technical knowledge along with buffalo milk value
chain in different farming systems will increase the production of buffalo in Bangladesh.
REFERENCES
BBC (Bangladesh Baffalo Centre). 2012. Lal Teer Livestock Report (personal communication).
BER (Bangladesh Economic Review). 2012. Finance division, Ministry of Finance, Government of
Bangladesh.
FAO. 2009. Food and Agricultural Organization. The State of Food and Agriculture 2009.
FAO. 2010. Food and Agricultural Organization. Production Yearbook 2008.
Faruque, M. O., M. A. Hasnath and N. U. Siddique.1990. Present status of buffaloes and their
productivity. Asia-australian J. Anim Sci. 3:287-292.
Hasnath, M. A. 1985. Breeding, feeding and management of water buffalo in Bangladesh. In
proceedings of 3rd AAAP Anim. Sci. Congress. Tokyo, Japan. 1: 70-79.
Huque, Q. M. E., S. K. Fouzder and R. Islam. 2010. Buffalo production system in Bangladesh and
its economic return based on input-output. Rev.Vet. 21(Sup. 1): 1009-1012.
Huque, Q. M. E. and A. Borghese. 2012. Production potentiality and perspective of buffalo in
Bangladesh. In:Procedings of the 15th AAAP Animal Science Congress, 26-30 November,
2012, Thailand. pp.244 (Abstr.)
Islam, M. A., M. A . Mazed., Islam, M. S. and M. K. Uddin. 2004. Some productive performance
of Nili-Ravi and crossbred (Nili-ravi X Local) buffaloes at Government buffalo farm,
Bagerhat, Bangladesh. J.Anim. and Vet. Adv. 3(12): 895-897.
PPRC. 2011. Power and Participation Research Centre. 2011. URBAN Bangladesh.
1183
Buffalo Farming in Bhutan: Challenges and Opportunities
Dr. Nar Bahadur TAMANGa, Dr. Dhan Bahadur RAIb and Dr. Min Prasad TIMSINAc
a
Livestock Specialist, Department of Livestock HQ, Thimphu, Bhutan,
b
Program Director, National Dairy Development Centre, Yusipang, Bhutan,
c
Livestock Specialist, National Dairy Development Centre, Yusipang, Bhutan,
Corresponding email: timsinampdr2003@gmail.com
ABSTRACT
This study was undertaken to improve understanding on traditional buffalo husbandry
practices, its role to improve rural livelihood, investigate reasons for fast population decline and
identify main challenges and opportunities to strengthen buffalo farming in Bhutan. Data was
collected from 80 buffalo farming households in four districts in the sub-tropical belts of Bhutan.
Results indicated that average herd size is around two buffaloes per household and are managed
with simple housing and feeding. Buffaloes are found to support rural livelihood providing milk,
meat and manure. Ability to survive on coarser feed, better productive and reproductive efficiency
than local Siri cattle (Bos indicus) are found to be advantages of buffalo farming. Main challenge to
sustain buffalo farming are: inability to withhold population decline because of frequent culling,
inbreeding and controlled breeding in the herd due to unavailability of quality breeding bull/semen
and shrinking of grazing areas. However opportunities exist to improve buffalo farming as many
farmers continued to show interest in rearing buffaloes because of its multiple utilities and steps are
being taken by government recently to support farmers. Study concluded that though buffalo rearing
is more profitable than keeping equal number of local cattle, no due attention have been given to
improve them. Adequate policy and technical support to revamp buffalo farming could broaden
farmer’s income-base and save the buffalo population from further decline. Support in terms of
buffalo breed improvement and capacity building of farmers is crucial to sustain buffalo farming.
Keywords: Bhutan, Sub-tropical belt, Buffalo farming, Population decline
INTRODUCTION
Buffalo (Bubalus bubalis) farming is popular in south-east Asia and south Asia. In contrast
to cattle, with their ubiquitous distribution all over the globe, buffaloes are found only in certain
regions. Similarly, buffaloes in Bhutan are found only in warmer sub-tropical belt viz. Chukha,
Samtse, Sarpang, Tsirang and Dagana dzongkhag (district). The buffalo population in Bhutan was
28,000 heads in 1984 (FAO, 1984). But over the last two decades, the number has gone down
drastically. The present population is only about 851 heads (DoL, 2011). Joshi (2007) pointed out
that there has been a significant decline in buffalo population in Bhutan, which calls for an
investigation.
Buffaloes are multipurpose animal and contribute to a large portion of milk for dairy
industry in India (Banerjee, 1991). In Bhutan, buffaloes are used for milk, meat and manure. But
there is limited government intervention in promotion of buffalo farming for the last two decades.
The last effort to improve buffalo farming in Bhutan was made during fifth five year plan (1982-
87), wherein some Surti buffalo bulls were supplied to farmers (Planning Commission, 1986).

1184
Realizing the potential of buffalo farming in the sub-tropical belt of Bhutan some Murrah buffalo
breeding bulls and heifers were procured and distributed to farmers (DoL, 2010).
Despite several benefits farmers derive from buffaloes, in the recent years, farmers are
giving up buffalo farming for reasons not very clear. Moreover, buffalo husbandry practices in
Bhutan and its importance are not well documented. Therefore, field study on buffalo husbandry
practices, its roles to the farming communities and reason for buffalo population decline was carried
out with the following objectives;
 To understand traditional buffalo husbandry practices and its role in Bhutan
 To get insight of challenges and opportunities for buffalo farming in Bhutan and
 To suggest necessary policy support needed to revamp buffalo farming in the country

Study area, sampling techniques and data collection
The study was carried out in two geogs (block) each of Sarpang, Tsirang, Dagana, and
Samtse Dzongkhas. The geogs and villages were selected based on buffalo population density and
farmers involvement in buffalo farming. Twenty buffalo farming households each from two
selected geogs were purposively sampled (Table 1). Semi-structured questionnaires were used to
collect information on buffalo types, feeding and management, breeding and production/
reproduction parameters. Informal discussions were held with some of the resourceful farmers to
gather information on challenges and opportunities in buffalo farming tradition in Bhutan.
Table 1. Sample collection areas and sample size.
Dzongkhag Geog selected Household sampled (no)
Tsirang Kikhorthang, Cholingkhor 20
Sarpang Bhur, Umling 20
Dagana Goshi, Tshandeygang 20
Samtse Dorokha, Ugyentse 20
Quantitative variables such as production and reproduction parameters were entered in
spread sheet. Mean and Standard Deviation (SD) were calculated. Qualitative data acquired through
informal discussion, answers in many cases that fall into patterns with the same answer appearing
frequently were coded and entered in spreadsheet. The frequency of each answer was sorted,
counted manually and, when appropriate, converted into percentage.

Buffalo breed types and herd sizes
Buffaloes in Bhutan are non-descript type. Farmers in some areas categorize these buffaloes
into three types: Kagay with entire black body, Hyakulae with white or light grey stripes below the
neck region and Dobla; the cross between the local and Surti buffalo (Tamang, et. al., 2009). The
average herd size in sampled households is around two buffaloes (2.46, n =45).
Feeding and housing
The suckling calves are stall-fed with tender green fodder. Rice bran, maize grit and hulls
are cooked and fed in a form of a porridge/gruel. When the calves are about three weeks old, they
are provided with some green fodder and this practice is continued till they are weaned. The adults
are let loose to graze with other livestock. Lactating buffaloes are provided with little amount of
gruel and some green grass/fodder as supplementary feed to the day’s grazing. Milking buffaloes
1185
are housed in homestead and calves are kept inside the shed all the time till they are weaned (12- 15
months of age). After weaning they are tethered along with other buffaloes either inside or outside
depending on weather condition and availability of shed.
Buffalo health management
Buffaloes are found to be susceptible to Foot and Mouth Disease, Haemorrhagic
Septicaemia, bloat and parasitic infestation. Mortality of calves due to worm infestation is also
reported. Payne (1990) supported the view that round worm (Neoascaries vitulorum) is transferable
from dam to foetus during pregnancy.
Breeding and reproduction parameters
Farmers breed their buffaloes mostly in alternate year. Intentional delayed calving allows
farmers to harvest more milk per lactation but at the cost of a calf crop. Average age at first calving
is 42 months and inter-calving period ranges from one to two years with an average of 540 days
(Table 2).
Table 2. Reproductive parameters of buffaloes.
Reproductive parameters Respondents (no) Mean ±SD
Age of first calving(months) 18 42±5.00
Gestation period(days) 10 313±4.59
Calving interval (days) 9 540±97.46
Lactation length (days) 9 315±28.28
Productive life (years) 12 14.3±2.46
Life span(years) 14 21.0±3.36
No of calving in lifetime(Nos) 9 10.4±1.30
* Figure are based on interview method and are farmer's best estimate
The above table indicates that reproductive efficiency of buffaloes is slightly better than Siri cattle
in Bhutan. Siri cows bear their first calves at about five years and have short lactation length of
about eight months (Tamang & Perkins, 2005). Payne and Hodges (1997) are also of the view that
average age at first calving for Bos indicus was about 60-63 months.
Milk production and other products
Buffaloes are reared primarily for milk and average production is about 5kg per cow/ day.
This yield is higher than local Siri cattle (1.5 litres/ day) (Arbenz and Tshering, 2000). Milk is
processed into butter and cheese, and sold for cash income. By-product-whey is consumed at home.
Main challenges in buffalo farming
Major challenges to buffalo farming and reasons for decline in buffalo population include:
 Inadequate Government support and interventions on buffalo farming
 Unavailability of quality breeding bull/semen for crossbreeding at the village level
 Frequent culling of males and infertile/sterile females for meat purpose
 Drying-up of wallowing pond (water pools) due to change in land use system
 Fodder scarcity due to shrinking of grazing areas and labour shortage at farm household
 Farmers preferences and Govt. subsidy support for Jersey cattle
Opportunities in buffalo farming
Lots of potential for revamping of Buffalo faming in the southern districts:
 Farmers interested to expand buffalo farming because of its higher economic return
 Farmers are taking initiatives to procure high yielding buffalo breeds from India
 Interventions are being made to procure quality breeding bulls and frozen semen to encourage
buffalo farming
1186
CONCLUSIONS
Higher milk yield than local Siri cattle, easy management and market value suggest that
rearing buffalo could supplement dairy development efforts of Royal Government of Bhutan.
However, for the last two decades, limited attention is given to encourage buffalo farming in
Bhutan. Looking at quality and rapid decline in buffaloes, there is every chances for their extinction
and loss of domestic animal bio-diversity in Bhutan. To boost farmer’s income and conserve the
scarce buffalo population it is imperative to revive buffalo farming in potential areas. This could be
achieved through enabling Government policy support in place.
Key recommendations of the field study are:
 Encourage Buffalo farming with special stimulus package through supply of quality breeding
bulls and progeny tested semen to improve buffalo production
 Diversify utility of buffaloes to bring greater benefits to the rural communities such as draft,
dairy products and dried carabeef
 Provide training on improved buffalo husbandry practices including fodder development
periodically to build capacity of farmers on buffalo farming
REFERENCES
Arbenz, M. and G. Tshering. 2000. Bos indicus and Bos taurus cattle in Bhutan, special publication
no. 4, RNR-RC, Jakar, Bhutan.
Banerjee, G. C. 1991. Textbook of Animal Husbandry, 7th Edn, Oxford & PBH Publications Pvt.
Ltd.
DoL. 2011. Statistics bulletin, Department of Livestock, Bhutan.
DoL. 2010. Mid-term Review Report of 10th Five Year Plan (2008-2013), Department of
Livestock, Thimphu.
FAO. 1984. FAO Production Year Book, 38. Rome.
Joshi, B. K. 2007. Consultancy Report, Village herd recording scheme and selective breeding of
native domestic animals in Bhutan. RNR-RC Jakar, Bhutan.
Payne, W. J. A. 1990. Introduction to Animal Husbandry in Tropics, Longman Scientific and
Technical, New York.
Planing Commision. 1986. 5th Five Year Plan, an interim review, p.37.
Tamang, N. B. and J. M. Perkins. 2005. Milk Yield of Mithun cross and Siri cattle Managed in
village herds in Bhutan, SAARC Journal of Agriculture, 5: 91-100.
Tamang, N. B., D. L. Sherpa, B. N. Sharma, G. Tshering, G. Thinley and D. B. Rai. 2009. Buffalo
farming in Bhutan, vanishing before its full potential is explored. Journal of Renewable
Natural Resources, Bhutan: 5: 82-93.
1187
Transition of Milk Production and Reproduction of Dairy Buffaloes in Nepal
Yoshiaki HAYASHIa*, Manoj Kumar SHAHb and Hajime KUMAGAIc
a
Experimental Farm, Faculty of Agriculture, Meijo University, Takaki-cho, Kasugai 486-0804,
Japan
.bRegional Agricultural Research Station, Nepal Agricultural Research Council, Lumle, Kaski,
Nepal
c
Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
*
Corresponding email: yoshiha@meijo-u.ac.jp
ABSTRACT
Consumption of buffalo milk has been increasing in Nepal. Thus, further development of
dairy buffalo is required for efficient production. However, the transition of milk production and
reproduction of buffaloes in the country remains obscure. Hence, the study is conducted for
identifying the changes of dairy production and reproduction of the animals. The records of daily
milk yield from July 1997 to July 2012 were collected at Livestock Development Farm, Nepal.
Total milk production per lactation, days in milk, daily milk yield, calving interval days and days in
dry were calculated. The results were summarized every 5 years (Period 1: July 1997-July 2002,
Period 2: July 2002-July 2007, Period 3: July 2007-July 2012). The total milk production per
lactation was lowest in Period 1. The days in milk had no significant difference among the periods.
The daily milk yield was lowest in Period 1. The milk production was assumed to improve from
Period 1 to Period 2. The calving interval days were longer in Period 3 than in Period 2. The
variance of the calving interval days tended to be longer in Period 3 than in Period 1. The days in
dry were longest in Period 3. The variance of drying days tended to be longer in Period 3 than in the
other periods. The efficiency of buffalo reproduction during the last five years assumed to be
declined. The deterioration of reproduction possibly has been caused by changes in nutritional and
environmental condition during the surveyed period.
Keywords: Buffalo, Milk, Nepal, Production, Reproduction, Transition
INTRODUCTION
Buffaloes are essential animals which have been raised for draught, meat and dairy in
Nepal for a long time. The milk yield of buffaloes was 1.1 million metric tons and contributed to
67.4% of the national milk production in 2011 (FAO, 2013). The amount of consumption and
supply of milk products has been growing in Nepal for the last decade (FAO, 2005; FAO, 2013).
The major local milk products available in the markets are cheese, paneer, butter, ghee, ice cream
and yoghurt (FAO, 2010). The other various dairy products are imported for fulfilling the national
dairy demand. However, there are several constraints relating to dairy buffalo development in the
country, such as low milk productivity, inadequate breeding support services and feeding
1188
management. Therefore, the comprehensive study on improvement of dairy buffaloes is required for
the efficient production. Some studies have been conducted for identifying the trait of milk
production in the country (Hayashi et al., 2005). The daily milk yield of buffaloes in small-scale
farms was from 6.4 to 7.7 L. The periods divided by the environments of pasture and fodder caused
the variance in feed constituents. The variance induced different milk yield of buffaloes. The other
research has also been done for improving the milk production of buffaloes in the country. The
additional feeding of field pea hay tended to rise the milk production of buffaloes (Hayashi et al.,
2008). In addition, dairy buffaloes fed corn silage increased daily milk production and fat content in
milk (Hayashi et al., 2009). For further improvement of dairy productivity, the past and present
status of milk production and reproduction in dairy buffaloes should be understood. However, the
transition of milk production and reproduction of dairy buffaloes in the country remains obscure.
Hence, the present study is conducted for identifying the changes of milk production and
reproduction of dairy buffaloes raised in a national dairy farm during the last 15 years.
MATERIALD AND METHODS

Data collection site and animals
The records of daily milk yield from July 15, 1997 to July 15, 2012 were collected in the
total 513 head of dairy Murrah buffaloes raised by Livestock Development Farm in Lampatan,
Pokhara, Kaski, Nepal. The farm was located approximately 140 km northwest of Kathmandu, and
at an altitude of around 830 m above mean sea level in the Hill region of Nepal. The region was in a
temperate climate. The average monthly temperature ranged from 15.2 to 26.2 degree Celsius with
3952 mm total precipitation from 1971 to 2011 (Normal Maximum, Minimum Temperature and
Rainfall through 2000, Meteorological Forecasting Division, Government of Nepal). The buffaloes,
had the first to fourth lactations, were fed Napier grass and commercial concentrate feed twice a day,
and grazed in pasture during day time. The animals were milked manually twice daily in early
morning and afternoon, and each milk yield was determined using a weighing scale and recorded.
In this survey, the data of milk contents and cost of milk yield was not collected due to the shortage
of data accumulation during the survey period.
Data analysis
Total milk production per lactation, days in milk, daily milk yield, calving interval days
and days in dry were calculated. The results of the production and reproduction were summarized
every five years (Period 1: from July 1997 to July 2002, Period 2: from July 2002 to July 2007,
Period 3: from July 2007 to July 2012) and compared the means among the three periods. The
means of each period were adjusted for multiple comparisons and were separated statistically using
Tukey’s multiple range test. Significance was defined as p<0.01 or p<0.05.

Transition of milk production in buffaloes
The total milk in a lactation period was lower in Period 1 than in Period 2 and 3 (p<0.01)
(Table 1). The days in milk had no significant difference among the periods. Although the days in
1189
milk were similar among the three periods, the milk production was assumed to improve from
Period 1 to Period 2. The daily milk yield was also lower in Period 1 than in Period 2 and 3
(p<0.01). The research on dairy production of lactating buffaloes in small-scale farms in Tarai,
Nepal was conducted from 2002 to 2004 when these years belong to Period 2 in the present survey
(Hayashi et al., 2005; Hayashi et al., 2006). The average milk yield of 305-day lactation of the
buffaloes was from 1619 L to 2196 L in different parities. The days in milk were from 118 days to
173 days in the different survey periods. Although the lactation days were longer in the present
survey than in the past reports, the milk yield was considered to be similar. On the other hand, FAO
(2013) showed the population and total milk yield of lactating buffaloes in Nepal every year.
According to these data, the average annual milk production of a buffalo was calculated as 829 kg
from 1997 to 2001, 848 kg from 2002 to 2006 and 853 kg from 2007 to 2011. This average milk
production of the first five years from 1997 was significantly lower than that of the other years
(p<0.01). This transition coincides with the results of present survey. Hayashi et al. (2005) reported
the daily milk yield of a buffalo in small-scale farms was from 6.4 to 7.7 L. The buffaloes at the
present research site had a longer lactation days and lower daily milk yield than those of the
buffaloes in small-scale farms. The dissimilarity might have been attributed to the different feeding
management.
Transition of reproduction in buffaloes
The calving interval days were longer in Period 3 than in Period 2 (p<0.01) (Table 2).
Although the interval days showed no significant difference between Period 3 and Period 1, the
variance of the days tended to be longer in Period 3 than in Period 1. The days in dry were longer in
Period 3 than in Period 1 and 2 (p<0.05). The variance of drying days tended to be longer in Period
3 than in the other periods. The efficiency of the buffalo reproduction during the last five years
assumed to be declined. In general, the productivity of buffaloes in Nepal is reported to be low. One
of the reasons for the low condition is degradation of reproduction in buffaloes. Anestrus due to
ovarian dysfunction and silent ovulation, and repeat breeding are major reproductive disorders. Sah
and Nakao (2006) reported the main clinical features of repeat breeding buffaloes were longer
interval between calving and treatment, a relatively high incidence of cervicities. The deterioration
of reproduction possibly has been caused by changes in nutritional and environmental condition
during the surveyed period. These issues should receive further attention.
In conclusion, the dairy production of the buffaloes was improved and maintained during
the last 15 years. However, the reproduction of the buffaloes tended to have constraints for efficient
milk production. More attention for improving breeding and nutritional management should be paid.
The further reformed management of raising environment for keeping health in buffaloes is also
necessary for efficient dairy production.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge Dr. N. P. Sharma and the staffs of Livestock
Development Farm, in Lampatan, Pokhara, Kaski, Nepal for providing the data and technical
assistance in this study.
1190
REFERENCES
FAO. 2005. Livestock Sector Brief, Nepal. Livestock Information, Sector Analysis and Policy
Branch. Food and Agriculture Organization of the United Nations, Rome, Italy.
FAO. 2010. Dairy Sector Study of Nepal. Food and Agriculture Organization of the United Nations,
Kathmandu, Nepal.
FAO, 2013. Livestock Primary, Production, FAOSTAT. Food and Agriculture Organization of the
United Nations, Rome, Italy.
Hayashi, Y., S. Shah, S.K. Shah and H. Kumagai. 2005. Dairy production and nutritional status of
lactating buffalo and cattle in small-scale farms in Terai, Nepal. Livest. Res. Rural Dev. (In
press).
Hayashi, Y., K.L. Maharjan and H. Kumagai. 2006. Feeding traits, nutritional status and milk
production of dairy cattle and buffalo in small-scale farms in Tarai, Nepal. Asian-Aust. J.
Anim. Sci. 19:189-197.
Hayashi, Y., N.R. Devkota and H. Kumagai. 2007. Effects of field pea (Pisum sativum L.) hay
feeding on dry matter intake and milk production of Murrah buffaloes (Bubalus bubalis)
fed rice straw ad libitum. J. Anim. Sci. 78:151-158.
Hayashi, Y., B.B. Thapa, M.P. Sharma, M. Sapkota and H. Kumagai. 2009. Effects of maize (Zea
mays L.) silage feeding on dry matter intake and milk production of dairy buffalo and
cattle in Tarai, Nepal. J. Anim. Sci. 80:418-427.
Sah, S.K. and T. Nakao. 2006. Characteristics of repeat breeding buffaloes in Nepal. J. Reprod. Dev.
52:335-341.
Table 1. Milk productivity of the buffaloes.

Period 1 Period 2 Period 3
n 174 191 148
Total milk in a lactation/buffalo 1263±453b 1776±610a 1652±567a
Days in milk 351±114 355±96 335±97
Daily milk yield/buffalo 3.6±0.9b 4.9±1.0a 4.9±1.0a
Period 1: from July 1997 to July 2002, Period 2: from July 2002 to July 2007,
Period 3: from July 2007 to July 2012.
ab
Means within the same row with superscripts significantly differ (p<0.01).
Table 2. Reproductivity of the buffaloes.

Period 1 Period 2 Period 3
Calving interval days 661±175ab (n=92) 601±149b (n=75) 699±258a (n=63)
Days in dry 306±135d (n=103) 255±117d (n=87) 371±279c (n=82)
Period 1: from July 1997 to July 2002, Period 2: from July 2002 to July 2007,
Period 3: from July 2007 to July 2012.
abc
Means within the same row with superscripts significantly differ (abp<0.01, cdp<0.05)
1191
Buffalo Socio-economic and
Sustainable Production
Community Management for Buffalo Eco-tourism in Udon Thani
Krisdakorn WONGWAIa, Atthasat WISIANSATa and Chakrit WANGKAORMb

a
Program in Agribusiness, Faculty of Technology, Udon Thani Rajabhat University, Udon Thani
41000 Thailand
b
Program in Environmental Science, Faculty of Science, Udon Thani Rajabhat University, Udon
Thani 41000 Thailand
*Corresponding e-mail: W_krisdakorn@yahoo.com
ABSTRACT
The purpose of this research is to study on the potential of Ban Si Jae, Thai buffalo village,
as a eco-tourism area in Udon Thani. The tools used in this research are two questionnaires; Sample
groups of this research are 12 government officials, 12 private officers, 12 local population, 30
tourists and 40 copies of buffalo keepers. The tools used to gather information are questionnaire and
interviews. The result of the research found that, for the general information Ban Si Jae has 3
clusters village, 456 families and 3,080 people. The majority occupation is a farmer and the famous
product is “KAO-LARM”. In the second part of questionnaire is about buffalo information by
surveying found that; the number of buffalo in the target area were 19 masculine gender and 255
feminine gender and 2 categories to divided with 57 to become pregnant and 37 still young and
less than 2 year amount 131. In term of a travel location that Ban Si Jae far from Udon Thani about
40 kms and a time to go by car half an hour. This area is close to Ban Chiang, the Red Lotus Lake,
Wat Pa Ban Tad and Phu Foi Lom Park. The management of buffalo eco-tourism, the researchers
offered the Thai buffalo eco-tourism village, Ban Si Jae, Udon Thani, into three directions to
develop; travel resources, travel market and travel preparedness which lead to the development of
sustainable tourism.
Keywords: eco-tourism, Thai buffalo village
INTRODUCTION
Eco-tourism is the form of tourism involving visiting fragile, pristine, and relatively
undisturbed natural area, intended as a low-impact and often small scale alternative to standard
commercial tourism. Its purpose may be to educate the traveler, to provide funds for ecological
conservation, to directly benefit the economic development and political empowerment of local
community, or to foster respect for different culture and for human rights. Udon Thani is a province
in the northeastern region of Thailand. Its civilization has been settled for thousands of years. Udon
Thani is strategically located as a center of economics, development, education and tourism in the
region. Currently, Udon Thani has emerged a new attraction known “The Lake of Red Lotus” such
as; it is a great opportunity to develop eco-tourism in Kumphawapi district. Ban si jae is one of
village in Kumphawapi district which a lot of Thai buffaloes. The objectives of this research were
study buffalo villages as a tourist attraction in Udon Thani to study SWOT Analysis: strengths,
weaknesses, opportunities and threats (Boontong, 2007) to development of buffalo villages as a
tourist attraction in Udon Thani and to study strategies in order to support area-based tourism
development and management to sustainable tourism.

The study population was a purposive sampling. The research instruments were three types
of questionnaires. The tools used in this research are tourism resource inspection form, tourism
infrastructure inspection form, and tourism potential evaluation form. Sample groups in this
research are 40 of buffalo keepers, 12 government officials, 12 private officers, 12 local population
and 30 tourists. The tools used to gather information are questionnaire and interviews. The process
and statistic used to analyze the information are content analysis, internal and external environment
1193
analysis, average, percentage and variance. The researcher uses an instrument to observe and collect
data in five places to bring in analyze and combine by descriptive analysis. The general information
of responder and potentiality to estimate of a travel location can explain by descriptive analysis and
descriptive statistic by percentage (%), average and standard error (SD) and the score to assign for
analyze are 1,2,3,4 and 5. The interviews data researcher would explain by descriptive analysis.

Research question 1: General information
Site of a community: A study in community found that a buffalo keepers in Ban Si Jae by
the interviews and area survey that Ban Si Jae had a 3 clusters village to consist of cluster 3, cluster
5 and cluster 10 (to be present of 3 clusters in a table No.1) was a random sample specific
(Purposive Sampling) to considered a people who had a buffalo and a questionnaires to obtain a
detail of basic foundation in a scope of a travel site of researcher.
Administration: Ban Si Jae was settles by a people who moved from Nakornrachasima for a
200 years. Nowadays, its to be under the control of Tambon Pasuk municipal, Kumphawapi district.
The name Si Jae is E-sarn language it means a four corners (a junction) and 456 families in here and
population is 3,080 people.
Surrounding: Ban Si Jae had a plateau and plain and area about 66.5 ha. Soil textural
classification is sandyloam. There are composed of temple, school and public health centre and all
around cover by a forest and swamp for planting a vegetable and consume.
Economics: In this place has a cash crop such as cassava, sugar cane, rubber but not much
because the people are a small scale farmers holding small area of cash crop. But the famous
product of this place is Kao-Larm (Kao-larm:Sticky rice cooked in fresh bamboo tube) it is the best
product of One Tambon One Product of Kumphawapi district. For an animal husbandry just for
consume but not for trade and buffalo still use in agriculture activities over 60 families.
Tradition: A tradition is slightly similar to other place in Udon Thani. It is E-sarn culture
and the people are conservative their tradition such as: to make merit, Boon Bunk Fai (A festival
that people make a kind of fireworks with a long tail) and Song Kran festival.
Obstacles: The major problem of this area is drought and high temperature in summer so a
glass is not enough for buffalo. The buffalo just only consume a straw.
Research question 2: Buffalo information and a travel location development

Buffalo information: A survey of insights about the number of buffalo in the target area
were 19 masculine gender and 255 feminine gender and 2 categories to divided with 57 to become
pregnant and 37 still young and less than 2 year amount 131.
Travel location: A study in community found that buffalo keepers in Ban Si Jae by the
interviews and area survey in buffalo villages as a tourist attraction to study strengths, weaknesses,
opportunities and threats (SWOT Analysis) all of area. The questionnaires to ask about the attitude
for travel location and a feedback from travel location development and a study found that:
Travel resource: A travel resource in Kumphawapi district to be composed of nature,
history, tradition and festival.
Travel location: A travel location to be composed of infrastructure, service.
Travel information: A travel information to be composed of information, comfortable and
transport.
Strengths: A research area is proximate by other location. A tourist can travel by car not
over 50 km and a time to travel less than 1 hour.
Weakness: A people who are the buffalo keeper nonsensical in management.
Opportunities: The local administrations prepare to support and they can put a capital to
make in the project and we are going to AEC (ASEAN Economic Community) in 2015. A travel
business will be an important project to run a local economics in Udon Thani.
1194
Threats: Researcher needs a time to explain a purpose of the project for a people and get them to
accept a method to make an eco-tourism.
The eco-tourism line (including buffalo village)

The evaluation of tourist spots in Kumphawapi, Udon Thani 1) Historical and
archaeological tourism at Ban Chiang: Ban Chiang is considered the most important prehistoric
settlement so far discovered in South-East Asia. It marks an important stage in human cultural,
social and technological evolution. The site presents the earliest evidence of farming in the region
and of the manufacture and use of metals. It far from Udon Thani 30 minute by car. 2) Natural
tourism includes study area in The Red Lotus Lake: There is a lake in the north part of
Kumphawapi district which is wortha visit. From December untill March this large lake is filled
with lotusses. It is amazing to see this, the whole lake looks like a large lotus field. It is called
"Talee Bua Daeng" (actually the lotusses are pink and not red). The tourist can take a boat to
looking a red lotus around the lake and fishing, take a picture, but no red lots pinch off and get an
adventure activities and camping at Phu Foi Lom Park. 3) Agricultural tourism includes agricultural
tourism in Buffalo village: visit the buffalo conservation village with activities like plowing
demonstrations and peasant life and then into mode of peaceful at Wat Pa Ban Tad: visit pilgrimage
route to pay obeisance to the bone of the elements of sacred L.D.S. theology (Luang Ta Ma Ha
Bua). From the study, it can be found that the overall tourism has intermediate potential.
Considering in each aspect, the opinion in art and cultural aspect and natural aspect are in high
level, which has the average score of 3.60 and 3.55, respectively. Considering each tourist spot, it
can be found that "Talee Bua Daeng" is in high level condition.
Research question 3: The direction to management for buffalo eco-tourism

The researcher would like to present the direction to management for buffalo eco-tourism
that:
3.1 The direction to develop travel resources
-Part of nature: A study found that the forests are decreasing and glass not for buffalo consume. So
the people have to supplement planting a forest and conserves water to keep food for buffalo.
-Part of historical and tradition: Buffalo village nearly to Ban Chiang World heritage. The tourist
can travel by car about 1 hour to visit the buffalo conservation village with activities like plowing
demonstrations and peasant life to raise awareness and recognize the importance of the buffalo and
setting up to be a source of knowledge. For a sample of the local population and traveling officers
found a valuable source of travel as a low level and the lack of a tour guide and tourist not impulse.
Community life has not changed much that it is strength of them.
3.2 The direction to develop travel market
The people should be settling a target group and the number of visitors or make a tourist map and
travel schedule and strengthen link to other nearly location with buffalo village. Only the buffalo
village cannot be developed to a sustainable tourism. It does not have the ability and factor to
persuade to be permanently tourists. In the form of eco-tourism including nature tourism,
Archaeology, Culture, Historical and the life of people and buffalo have to make information to
advertise.
3.3 The direction to develop travel preparedness
Part of location and environment: The location and environment have to be in agreement with art
and culture because it will illustrate the identical of area.
Part of product: In part of product the people have to be specific in a local product such as KAO-
LARM is the best product in Kumphawapi district and very famous or the product which the tourist
cannot found in other place to get them to come back again. It will lead to the development of
sustainable tourism.
1195
CONCLUSIONS
The results from this research can be conclude, Ban Si Jae has many supporting resources to
establish Thai buffalo eco-tourism village because there are consist of natural, human and travel
resources including a comfortable transportations and close to a famous travel places, so Ban Si Jae,
Thai buffalo eco-tourism village is the one of the important travel place in Udon Thani. It will be
lead to develop of sustainable eco-tourism.
ACKNOWLEDGEMENTS
The authors would like to thank the Faculty of Technology, Udon Thani Rajabhat
University for supporting by grant fund and thanks to the Office of Domestic Animals Province of
Udon Thani and all the people in Ban Si Jae for giving the useful data for this research.
REFERENCES
Auntonio. 1971. Environmental Menagement Planning for Traffic. UK: Mcgraw Hill Book
Boontong, A. 2007. strengths, weaknesses, opportunities and threats and the way to manage eco-
tourism elephant village.
Ceballos-Lascurain. 1988. The Future of Ecotourism. Mexico Journal. 17(1): 13-14.
Inskeep, E. 1999. Tourism Planning: An Integrated and Sustainable Development Approach. New
York: Van Nostrand Reinhold.
Godfrey, K. and J. Clarke. 2000. The Tourism Development Handbook: A Practical Approach to
Planning and Marketing. New York: Continuum.
Harvey, M. R. 1992. Pedestrain Mall, Street Scap and Urban Scapes. USA: John Wille & Son Ins.
Richardson, J. 1993. Ecotourism & Nature based Holidays. Sydney: Australiam Print Group.
Lafferty, G. and A. Van Fossen. 2001. Integrating the tourism industry: problems and strategies.
Tourism Management 22(1), pp. 11-19.
Mcfarland E. D. (1979). Management foundations and Practices. New York : Macmillan.
McIntosh R.W. and C. Goeldner. (1986). Tourism Principles, Practices, Philosophiers. New York :
John Willey & Son.
Piboonrungroj, P. and S.M. Disney. 2009. Tourism supply chains: a conceptual framework.
Proceeding on PhD Networking Conference, 1-2 July. Nottingham, UK.
Richard K. U. 1984. Accommodation The Pedestrian. NY: Vannostrand reinhold. The Ecotourism
Society. 1991.
Sinclair, M. T. and M. Stabler. 1997. The economics of tourism. London: Routledge. The
Ecotourism Society’s definitation. The Ecotourism Society Newsletter.
1196
Economical and Social Importance of Buffalo in a Small Milk Production

System
Eduardo BASTIANETTOa, Mayara BRITOa, Denise OLIVEIRAa, Rômulo LEITEa.
a
Brazil
ABSTRACT
In tropical areas buffaloes are used to improve a milk and meat production in small farmers
and have the social impact in keep the population in the rural areas. This paper describes the
economical results of a milk production system composed by 10 lactation buffaloes and one sire,
calves and weaned animals breed in Braquiaria sp. pasture at Minas Gerais State, Brazil. Using the
production index and products prices the value of the work and economical results were calculated.
In a year this production system controlled by a single man allows an income of USD 16527,03.
The natural capacity of buffaloes to produce more milk and meat in tropical areas reduces the
spending with modern corrals and medications, improving the economical results and
environmental welfare.
Keywords: Small production system, economy, social, environmental welfare.
INTRODUCTION
Buffaloes have a huge capacity to survive and produce in tropical conditions in function of
the physiological characteristics (Bastianetto, 2009). In Brazil this potential are been explored to
produce milk, meat and power draft particularly in small production system, familiar agriculture
type (Quinzeiro Neto et al., 2009). The aim of this work was evaluate the productive and financial
results of a milk and meat production system recently created at a school farm. The principal
porpoise of this production system is progress technologies and human technical skill.

During the period 27/03/2012 at 15/12/2012 were evaluated the milk production of 10
buffaloes (Murrah, Jafarabadi and crossbreed), growth calves and weight gain of others 20 weaned
male. One man was responsible for milking buffaloes once a day, care calves and organize all
others necessary functions correlated to animals production (vaccination, parasitic control, etc...).
The animals was created in Brachiaria sp. pasture with additional supplementation with 01 kg of
ration (24% Proteion and 87% de TDN/DM = USD 0,38). The milk production was measured
monthly and animals weight gain were often checked. Using the production index, the local price of
milk (USD 0,67), calves prices (USD 428,57) and meat price (USD 1,04/kg) the value of the work
and economical results were calculated.

The rate of milk production per day was 6,5 liters milk, with composition of 6,40% FAT and
4,43% PROTEIN, being this index according to the mean buffalo production capacity considering
the animals nutrition and number of daily milking (Campanile et al., 2007). Relative to the
development of weaned animals the daily weight gain was 0,73±0,11 kg (table 01). The animal ID 14
and 17 had an inferior development in function of a unexplained illness. According to necropsy study the
animal ID 16 and 18 died after a acute helminthic infection. The total production of milk and calves value
make a income equal USD 15.985,00 plus a income of other a income of USD 542,00 relative to a
weight gain totalizing a final of USD 16527,03 in the evaluated period.

1197
Is important to note the possibility to increase the production values observed with the
adoption of ideal practices of sanity, nutrition and management, as a twice milking and parasites
control. These numbers are substantially superior when compared to other livestock production
systems, in particular with sheep and bovine, and buffalos breeding can be an important tool for
increase food production and human population in rural areas of Brazilian territory.
Considering the lower production costs and the higher milk prices, the social importance of
create a buffalo in small areas in Brazil is related to the maintenance of population in their original
regions with a respectable quality of life.
IMPLICATIONS
Using corrects production practices in familiar production system buffalos in Brazil are
enable to produce more food and increase income in many situations and provide a better life for
the quality for small farmers.
REFERENCES
Bastianetto, E. and J.D. Barbosa. 2009. Diferenças Fisiológicas Entre Bubalinos e Bovinos:
Interferência na Produção. Proceedings of VIIICongresso Brasileiro de Buiatria, Belo
Horizonte, Brazil.
Campanile, G., O. Bernardes, E. Bastianetto, P. S. Baruselli, L. Zicarelli and D. Vecchio. Manejo de
Búfalas Leiteiras Bufalotec – ABCB (www.bufalo.com.br), 2007.
Quinzeiro, N., T.A.R. Garcia, J.C. Santos, M.T. Garcia, A.K.O. Homma and J.B. Lourenço Júnior,
Socio economical Importance of Buffalo Breeding to Small Farm Agriculture at Xingu
River Floodplains, Pará, Brazil. Proccedings of X Word Buffalo Congress, Buenos Aires,
Argentina 2009.
1198
Table 1. Mean weight gain of weaned animals created at Brachiaria sp. pasture at Minas Gerais
State, Brasil, 2012.
ID Age (days) Mean weigth
Weigth data (kg)
ANIMALS 15/12/12 gain/day
Birth 27/03/2012 26/07/2012 13/09/2012 30/10/2012 15/12/2012
1 03/2011 145 235 245,00 301 350 205 0,78
2 03/2011 197 305 300,00 357 410 213 0,81
3 03/2011 166 280 305,00 330 375 209 0,79
4 03/2011 158 240 303,25 291 335 177 0,67
5 03/2011 175 280 289,00 363 405 230 0,87
6 03/2011 156 255 266,00 307 360 204 0,78
7 04/2011 185 270 267,00 330 365 180 0,68
8 04/2011 164 260 250,00 310 360 196 0,75
9 04/2011 146 230 240,00 290 335 189 0,72
10 04/2011 175 300 266,00 362 415 240 0,91
11 04/2011 158 240 255,00 292 340 182 0,69
12 04/2011 152 250 278,00 295 345 193 0,73
13 04/2011 125 210 220,00 260 305 180 0,68
14 05/2011 112 160 165,00 - 220 108 0,41
15 05/2011 117 215 215,00 255 315 198 0,75
16 05/2011 112 DIED
17 05/2011 124 170 190 230 275 151 0,57
18 05/2011 113 DIED
19 06/2011 207 310 305 355 415 208 0,79
20 09/2011 175 260 250 310 370 195 0,74
Average daily mean weigth and standard deviation 0,73±0,11
1199
Coordination of the Chain of Buffalo Milk in São Paulo State (Brazil)
Fabrício Pini ROSALESa* and Mário Otávio Batalhaa

a
Agribusiness and Agri-food Research Group (GEPAI), Department of Industrial Engineering,
Federal University of Sao Carlos, Highway Washington Luís, km 235, Sao Carlos (SP) Brazil.
*Corresponding email: fprosales@gmail.com
ABSTRACT
The goal of this paper is to analyze the relationship between producers of buffalo milk and
dairy industries in order to characterize the chain of buffalo milk in Sao Paulo State, the major
producer of buffalo milk in Brazil. As a result of the increasing demand for cheeses and other
products derived from the buffalo milk, we can observe the emergence of belts of buffalo milk
producers nearby dairy industries specialized in processing this raw material. However, the
production of buffalo milk is unable to supply this demand and most dairy industries work in idle
capacity. In this context, it is fundamental that milk producers and dairy industries choose
coordination mechanisms capable of transmitting information, stimuli, and control, and providing a
win-win situation. This research is qualitative, descriptive, and exploratory, characterized as a multi
case study. The data were collected through a semi-structured questionnaire applied to 63 buffalo
milk producers and 14 dairy industries working exclusively with buffalo milk. The study was based
on the Theory of Transaction Costs, in order to identify the characteristics of transactions and the
existing type of governance. It was observed that the transaction between buffalo milk producers
and dairy industries is often based on informal agreement, although there are few transaction based
on formal agreement. Despite the predominance of informal agreement, there is an established long
term relationship between these players.
Keywords: Transaction cost, buffalo, milk production, chain coordination, agribusiness
INTRODUCTION
The increased demand for buffalo milk and its derivatives has driven the buffalo breeding in
Brazil. Consumer demand for cheeses made with buffalo milk stimulated the increase in the number
of dairy industries that work exclusively with buffalo milk and the appearance of belts that produce
buffalo milk. The State of São Paulo is responsible for 24.58% of Brazilian industrialization of
buffalo milk. However, milk production in this State has been unable to meet the demand and most
dairy industries that work with this raw material have idle production capacity and do not even meet
the State market demands (Rosales et al., 2011).
The different ways that firms are established in the market and transact with each other
characterize the coordination of a given chain. The ability of the actors to maintain a good
relationship and adapt to the market reality improve the chain coordination and lead to structural
changes that increase its efficiency (Mikkola, 2008). An efficient coordination must transmit stimuli
and information that facilitate physical, financial and information flows within the chain, allowing
the alignment of goals and performance improvements of the chain as a whole. The research showed
important information gaps that raise difficulties to the coordination in the entire chain of buffalo
milk in São Paulo State, Brazil. The lack of knowledge of the transactional mechanisms between
producers of buffalo milk and dairy industries in São Paulo State is among these major shortages
and will be the goal of this article.
This paper is divided into three sections. After this brief introduction, the aspects linked to
the research method will be covered. In the third and last section, results will be presented and
discussed.

1200

This study is a qualitative, exploratory and descriptive one. A descriptive and exploratory
research provides an overview about little-known phenomena and describes their features. As for
the qualitative research, the researcher seeks to raise opinions or meanings under the participants'
perspectives through the interaction with people (Gil, 1,999; Vieira, 2009).
The most appropriate research method in this situation is the case study. This method can be
seen as an approach empirical character that investigates a given phenomenon within a real
contemporary context and provides deep insight into the phenomenon studied (Miguel, 2010).
The major tool for collecting research data depicted on this article was a semi-structured
questionnaire applied personally to 63 milk producers and 14 dairy industries that work with buffalo
milk in São Paulo State from July 2012 to November 2012. The questionnaire was discussed with
researchers and key agents (a milk producer and an owner of a dairy industry) of the chain studied
prior to data collection. After the interviews, telephone contacts with the interviewees to clarify
possible doubts were made.
The option to select milk producers and dairy industries is due to their importance to the
dynamics and performance of the chain as a whole. As the chain is formed by small producers and
small dairy industries, the preference was given to interview directly the owners and, in cases where
this was not possible, managers or responsible for the trade department were interviewed.

It can be affirmed that the chain of buffalo milk in São Paulo State is formed mainly by
small dairy industries and small milk producers. The daily milk reception capacity of these
industries ranges from 80 L to 30,000 L with an average of 5,429 L/day. However, only three of
these companies work with 100% of the production capacity, and, on average, the dairy industries
work with only 68.50% of production capacity. During the inter-harvest period, with the fall in milk
production, the dairy industries use 42.61% of production capacity.
It was also noticed that dairy producers produce on average 250.73 L/day during the harvest and
during the inter-harvest period this production falls to 97.80 L/day. One can see that the production
of buffalo milk in that State is very seasonal and that some producers stop producing during the
inter-harvest period.
The research has indicated that the pricing system of buffalo milk in the State is quite
precarious. The price of this raw material is determined by the dairy industries with no periodical
pattern for adjustments and just nine producers said they received a raise during the inter-harvest
period. Another aggravating factor in this situation is the lack of information or quotation practiced
in several regions or States, which prevents the efficient monitoring of the market.
The relationship between milk producers and dairy industries will be analyzed in view of this the
theories of Transaction Cost Economics (TCE). TCE has been used successfully to explain how
companies shape and are affected by the coordination of productive chains in which they
participate. Transaction costs can be defined as the costs of conducting a transaction and are
inducers of alternative ways of organization (coordination) of the chain (Williamson, 1985;
Peterson et al., 2001; Ménard, 1995). According to Williamson (1991), once a particular
governance structure (coordination of the chain) is chosen, organizations seek to minimize
transaction costs. The various governance structures proposed by this author can be seen as a
continuous which starts by the spot market (transaction resolves itself in a single moment of time),
through hybrid structures (use of agreements formal or not) and ending at vertical integration (two
or more operations are performed by only one company).
Concerning to the vertical integration upstream the chain, it was noticed that only one of the
interviewee does not have his own production of buffalo milk and four others produce all the milk
that they industrialize. On average, each dairy industry produces 38% and 51% of the milk that is
industrialized during the harvest and the inter-harvest period, respectively. The verticalization of the
milk production can be effective to avoid very steep falls on the uptake of milk during the inter-
harvest period, period of increased product demand. One out of the four dairy industries that
produce 100% of milk has an organic certification and finds it difficult to acquire raw materials
1201
with the same certification, the other three have low production capacity, and two of them sell
surplus production to other agricultural industries.
The decision to adopt vertical integration has important trade-offs. On one hand the largest
chain control allows the adoption of actions focused on quality improvements, cost reduction,
strategies against rivals or the exploration of the consumer market of the final product. On the other
hand, it diverts managerial efforts of its main focus, raises the bureaucratic costs and prevents the
gains that have come from market characteristic incentives (Peterson et al., 2001; Azevedo, 2011).
An agreement may be defined as an deal between the parties of a transaction to reduce the
uncertainties and risks in terms of trade. The agreement is not always formal or written and can be
very different in relation to the purpose. Oral business agreements between the parties, even if they
hamper the adoption of penalties for the breach of the agreement, are characterized as informal and
are often used in many agro-industrial chains (Zhang and Aramyan, 2009, Azevedo, 2011).
The chain of buffalo milk in São Paulo State is marked by extensive use of informal
agreements, being rare the use of formal agreements between the parties. Despite the low degree of
formalization, one can see that the relationship producer/dairy industry is based on fidelity by both
of the dairy industry and the producer. For most producers interviewed, the most important item in
this relationship is the price paid for the milk and the dairy industry's accomplishment of the
commitments established - something that has been done.
Although less frequent, the occurrence of some formal agreements between producers
interviewed and dairy industries was also noticed. These agreements are annually negotiated and are
based on two aspects: production forecast of the harvest and the inter-harvest period and quality.
The interviewees’ opinion, however, diverges about the use of this mechanism of
commercialization. Some of them say that it is unnecessary while others claim that the agreement
provides greater security for the transaction. However, it was noticed that the agreements are not
always followed faithfully and, in these cases, the understanding between the parties prevails.
An intermediary, in this context, can be defined as a player (company or person) who acts by
purchasing the raw material of the rural producers and reselling it to the agro-industry, in this case,
the dairy industries. The presence of intermediaries is positive when it allows the reduction of trade
costs, optimizes the productive agents’ efforts and regulates the flow of product demand, providing,
thus, gains in productivity to the system. However, the intermediary will have a negative effect for
the chain if he works opportunistically; practicing very high margins without aggregating any value
to the product, or if he compromises the approach and exchange of information between producer
and processor (Sproesser and Lima Filho, 2011).
The research found that in the chain of buffalo milk in the State of São, milk production
usually follows the rural producer directly to agribusiness, without the presence of the intermediary.
However, it was noticed the intermediary took part only in three cases and in all of them this agent
was seen as beneficial to the chain, since the agent collects the milk from small producers and
resells it to the dairy industries. Without this intermediary, the cost for companies to collect milk
from small producers would be very high, which would make the access of these producers to dairy
industries not viable.
The seasonality in buffalo milk production appeared to be one of the main difficulties faced
by the sector. However, the pricing system practiced does not provide sufficient stimuli for milk
producers to change this situation. A bonus that during the inter-harvest period could give producers
conditions to employ existing biotechnology techniques to change the calendar of raising animals.
Vertical integration upstream seems to be an interesting strategy. Many dairy industries use their
own milk production to compensate the lack of milk on the market.
Informal agreements proved to be an efficient tool to coordinate transactions. Despite the low
adoption of formal agreements, it was noticed that there are, in most cases, long-term relationships
between milk suppliers and dairy industries.
Still considering the results, it is worthy highlighting that the option for the qualitative
methodology and for the small number of evaluated cases makes the effort of result generalization
unfeasible.
1202
ACKNOWLEDGMENTS
The authors are thankful to FAPESP (São Paulo Research Fundation) and to CNPq for the
financial support received.
REFERENCES
Azevedo, P.F. 2011. Comercialização de produtos agroindustriais. In: Gestão Agroindustrial (Ed.
M. O. Batalha). Ed. Atlas. São Paulo. pp. 63 – 112
Gil, A.C. 1999. Método e Técnicas de Pesquisa Social. São Paulo: Editora Atlas. IBGE,Censo
agropecuário 2006. Disponível em <
http://www.ibge.gov.br/home/estatistica/economia/agropecuaria/censoagro/default.shtm>
Acesso em 10/05/2012.
Ménard, C. 1995. Market as institutions versus organizations as markets? Disentangling some
fundamental concepts. Journal of Economic Behavior and Organization. 28: 161 – 182.
Miguel, Paulo A.C. 2010 Adoção do estudo de caso na engenharia de produção. In: Metodologia de
pesquisa em engenharia de produção e gestão de operações (Ed. MIGUEL, Paulo A.M). Rio
de Janeiro: Elsevier. pp.129-143.
Mikkola, M. 2008. Coordinative structures and development of food supply chains. British Food
Journal. 110: 189 – 205.
Peterson, H.C., A. Wysocki, and S.B. Harsh. 2001. Strategic choice along the vertical coordination
continuum. International Food and Agribusiness Management Review, 4: 149-66.
Rosales, F.P., M.O. Batalha and R.N. Tomas. 2011 Produção de leite de búfala no Estado de São
Paulo: um estudo de caso sobre a colaboração na cadeia de suprimentos. In: Proceedings of
thje XVIII Simpósio de Engenharia de Produção.
Sproesser, R.L and D.O. Lima Filho. 2011. Varejo de alimentos: estratégia e marketing. In: Gestão
Agroindustrial (Ed. M. O. Batalha). Ed. Atlas. São Paulo. pp. 257 – 331.
Vieira, S. 2009. Como elaborar questionários. São Paulo: Atlas.
Zhang, X. and L.H. Aramyan. 2009. A conceptual framework for supply chain governance: an
application to agri-food chains in china. China Agricultural Economic Review, 1: 136 -154.
Williamson, O. 1985 The economic institutions of capitalism: Firms, Markets, Relational
Contracting. The Free Press, New York.
Williamson, O. 1991. Comparative economic organization: the analyses of discrete structural
alternatives. Administrative Science Quarterly 36: 269 – 296.
1203
Swamp Buffalo Production System and Needs for Extension on Local Scale
Farmers in the Lower Northeast of Thailand
Sornnarong SUPHACHAVALITa*, Pinij SRICHAROENa, Thoranong LUESOPHAa,

Tossaporn SRISAKDIa, Anchalee Na-CHIANGMAIa and Suvit BOONPRONGa
a
*Corresponding email: sornnarong_s@hotmail.co.th
ABSTRACT
Objectives of this study were to estimate on the raising swamp buffaloes and the needs for
extension on swamp buffalo production of small scale farmers. The data were recorded from 405
swamp-buffalo-raising farmers located in 6 provinces of the lower northeast of Thailand. The
survey was conducted during 2 February 2009 and 24 September 2009. The records were also
analyzed by using the Statistical Packages for the Social Science (SPSS). The basic records showed
that the farmers who raised buffaloes were male with 52 years of age. On the average, their
household size was 5 persons. Within the household, there were about 3 persons working on both
rice and animal farming. Each household owned about 19.39 rai of agricultural land of which was
allocated for 16.77 rai of rice farming and the rest went for raising 6 swamp buffaloes. Household
income was derived from 2 major sources: rice farming (28,298.27 Baht) and animal farming
(9,673.58 Baht), respectively.
The results of this study found that the main objectives of the farmers were to utilize
buffalo’s manure (43.8%), sell their animals (40.8%) and receive from their inheritances (12.4%)
and from the DLD (3.0%), respectively. They had raised swamp buffaloes approximately for 17
years. The majority of farmers (93.7%) bred their buffaloes by sires although they did not mainly
know sires’ pedigree. They slightly know respective breed (6.3%), and they had to pay about
100.86 Baht each. Moreover, majority of the farmers did not plan for selection and breeding
improvement of sires or dams of swamp buffalo in the future. For raising buffalo, they would
mainly restrain with grassing in public lands during the day and provide rice straw supplementary
during the night. Slightly, someone may be chop natural grass for raising their buffaloes at night.
Their buffaloes were restrained separately from their homes, and the farmers did not have area for
forage production. Eighty point three percentages of farmers kept their buffaloes by rice straw in
summer. They fed slightly concentrate (23.0%) and mineral (26.4%) for buffaloes. Their 90.6% of
animals were vaccinated against diseases by the authorities of DLD, and 68.5% of them were
treated for endoparasites. When buffaloes had health problems, they would be cured by authorities
of the DLD (70.3%), by volunteers of the DLD (12.1%) and by the owner (17.6%), respectively.
In addition, needful for extension on raising buffaloes can be showed as follows: breeding,
farm management, buffalo production interval, artificial insemination, ruminant nutrition, disease
control, animals’ treatment and promoting for buffaloes of farmer grouping.
Keywords: raising buffalo, production, need for extension, northeast Thailand
INTRODUCTION
The most of Thailand’s buffalo production could be referred to as backyard, subsistence
production, involving smallholders in remote areas. There is a great variety in forms of husbandry
and management techniques from region and even from farm to farm. These variations in
husbandry and management are a result of the natural conditions of the grazing area, the crop
production system, and lifestyle and economic framework of framers. Knowledge about assessing
the suitability of animals’ species in different environments has been passed down through
generations of farmers. Swamp buffaloes are indigenous to Thailand and are well-adapted to the
poor conditions, especially in the northeast of the country which keep the most buffalo. They are
1204
capable of utilizing agricultural waste products or low quality to convert into meat while providing
manure for as a fertilizer. Excess animals are also sold when necessary for extra income
(Chantalakhana, 1994, 1999; Na-Chiangmai, 2002).
Environment factors determine the status of buffalo production in farming systems and
influence the type of farming carried out. It is evident that human influence and socio-economic
factors relate to the location of the production also has a decisive impact on the goals of livestock
production. Population growth, local economic development and increasing urbanization have
created a demand for buffalo productions. As a result, the number of buffalo in Thailand is sharply
decreasing (Indramangala, 2002).
Thus, the aims of this study were to estimate on the raising swamp buffaloes and the needs
for extension on swamp buffalo production of small scale farmers.

The samples of population were 405 farmers which raised buffaloes on local villages in
Amnatcharoen, Ubon Ratchathani, Srisaket, Surin, Buriram and Nakhon Ratchasima provinces. The
samples were selected with purposive sampling (Yamane, 1976 Cited by Prasitrathasindhu, 2003).
Data were collected using interview schedule which consisted of closed-ended questions and open-
closed questions via interview from each farmer. The general data of farmers, raising swamp
buffaloes and needs for extension on swamp buffalo production of small scale farmers during 2
February 2009 to 24 September 2009 were recorded. As, the needs for extension consisted of 3
levels as follow: 1 = slight need, 2 = moderate need and 3 = high need. For the means of needful
levels such as: 1.00 to 1.66 = slight need, 1.67 to 2.33 = moderate need and 2.34 to 3.00 high need,
respectively (Prasitrathasindhu, 2003). Data were analyzed using the Statistical Packages for the
Social Science (SPSS). Values are presented as percentage and meansSE.

The basic records showed that the farmers who raised buffaloes were male with 52 years of
age. On the average, their household size was 5 persons. Within the household, there were about 3
persons working on both rice and animal farming. Each household owned about 19.39 rai of
agricultural land of which was allocated for 16.77 rai of rice farming and the rest went for raising 2
cattle and 4 swamp buffaloes. Household income was derived from 2 major sources: rice farming
(28,298.27 Baht) and animal farming (9,673.58 Baht), respectively.
The results of this study found that the main objectives of the farmers were to utilize
buffalo’s manure (43.8%), sell their animals (40.8%) and receive from their inheritances (12.4%)
and from the DLD (3.0%), respectively. They had raised swamp buffaloes approximately for 17
years (15-20 years). The majority of female buffaloes (93.7%) were mated with the sires by natural
mating although they did not mainly know sires’ pedigree. They know slightly respective breed
(6.3%), and they had to pay about 100.86 Baht each.
The small scale farmers raised buffaloes about 1-5 head/family (74.2%). Moreover, 65.5%
of farmers did not plan for selection and breeding improvement of sires or dams of swamp buffalo
in the future. For raising buffalo, they would mainly restrain with grassing in public lands during
the day and provide rice straw supplementary during the night. Slightly, someone maybe chop
natural grass for raising their buffaloes at night. Their buffaloes were restrained separately from
their homes (65.5%), and 85.2% of farmers did not have area for forage production. Eighty point
three percentages of farmers kept their buffaloes by rice straw in summer. The animals were fed
slightly concentrate (23.0%) and mineral (26.4%) for buffaloes. Their 90.6% of animals were
vaccinated against diseases by the authorities of DLD, and 68.5% of them were treated for
endoparasites. When buffaloes had health problems, they would be cured by authorities of the DLD
(70.3%), by volunteers of the DLD (12.1%) and by the owner (17.6%), respectively.
Table 1 shows the case of need for extension on raising buffaloes, and it was found that they
need high levels of extension for buffaloes breeding, buffaloes production interval, artificial
insemination, ruminant nutrition, disease control, treatment of control for endoparasites and
1205
promoting for buffaloes of grouping. However, promoting for buffaloes fattening, domestic
marketing and standard slaughterhouse set up were found to be moderately needed.
The problems and obstacles on raising buffaloes in lower Northeast of Thailand were lacks
of public land for raising buffaloes. Now Thai farmers have a shorter time on raising their animals
than in the past. Because it due to lack of plantation land, unpredictable weather and potential
damage to crop. Furthermore, the industrial revolution has had a strong impact on the farmers’
lives. Young village people, who traditionally supervised the animals during the day, can also
change their careers to non-farm enterprise that is quite often city-base. These factors influence the
reduction in buffalo’s population (Na-Chianhmai, 2002). Likewise, Indramangala (2002) reported
that traditional farming practices could not use extensively and generally include daytime grazing
such as: edge of fields, roadsides marginal land, communal grazing areas in the present. Note that
animals are usually supervised while grazing with confinement and protection reasons. This has
been traditionally the role of children. In the other hand, this now conflict with parents desires to
send their children to school and improve their education. Thus, buffaloes’ production in the future
cannot be sustained under traditional practices. However, rather fewer animals are raised without
planning for long-term sustained production (Na-Chianhmai, 2002).
For in the future development of buffalo, the Ministry of Agriculture and Cooperatives has
approached the objective strategies on buffaloes’ development in the lower northeast of Thailand
(Indramangala, 2002; Na-Chianhmai and Allen, 2000; Na-Chianhmai, 2002) as follow:
(1) To increase the efficiency in the Government official for breeding development and
relay it to the farmers systematically.
(2) Extend the results of basic animal health programs in collaboration with the promotion
of the use of good sires, with participation of the farmers in the development by setting up group
support organizations.
(3) As swamp buffalo farming is mostly practiced in the lower northeast region, there is a
constraint on research to build up the technology in this direction. It is therefore necessary to
support researchers to be able to work in this area and employ high technology towards swamp
buffalo production.
(4) Expand the ability of the existing national database to record adaptation information
along with pedigree and performance information. The initial focus of this work will utilize the
historical pedigree data combined with the adaptive traits, body temperature and health data as these
measurements become available.
(5) Help and sustain the in situ conservation of buffalo genetics by helping to establish
sustainable buffalo production systems at the village level while providing improved genetics using
the government breeding program.
In conclusion, the demand for high quality meat has been increasing due to the change in the
socio-economic pattern of the population in the lower northeast of Thailand such as an increasing
standard of living and education. The marketing system in the country demands animals that
produce heavy carcasses with less fat trims. Animals with high fertility and efficiency in growth are
preferred. A selection program which incorporates these two characteristics is suggested when
determining which animal to keep for mating to produce progeny in the next generation.
REFERENCES
Chantalakhana, C. 1994. Swamp buffalo development in past three decades and sustainable
production beyond 2000. In: Proceedings 1st Asian Buffalo Association Congress, Khon
Kaen, Thailand.
Chantalakhana, C. 1999. Buffalo breeding program in Thailand. Proceedings of the Workshop on
Developing Breeding Strategies for Lower input Animal Production Environments. Bella,
Italy, 2225 September, 1999.
Indramangala, J. 2002. Buffalo development in Thailand, pp. 117-123. In J. Allen and A. Na-
Chiangmai, eds. Development Strategies for Genetic Evaluation for Beef Production in
1206
Developing Countries. ACIAR Proceeding, no. 108, Watson Ferguson & Co., Brisbane,
Australia.
Na-Chiangmai, A. 2002. Current situation and development trends of beef production in Thailand,
pp. 93-97. In J. Allen and A. Na-Chiangmai, eds. Development Strategies for Genetic
Evaluation for Beef Production in Developing Countries. ACIAR Proceeding, no. 108,
Watson Ferguson & Co., Brisbane, Australia.
Na-Chiangmai, A. and J. Allen. 2000. Genetic improvement of swamp buffalo in Thailand.
Proceedings of the 9th Congress of the AAAP Societies and 23rd Biennial Conference of
ASAP. July 37, 2000. University of New South Wales, Sydney, Australia.
Prasitrathasindhu, S. 2003. The System of Social Science Research Method. 12th ed., Fuangfa
Printing Ltd., Bangkok, Thailand. (in Thai)
Table 1. Needs for extension on raising buffaloes of farmers in the lower Northeast of Thailand.
n = 405
Values (meansSE)
2.600.65
Needs for extension on raising buffaloes Means of needful levels
2.400.65
Buffalo breeding high
2.850.40
Farm management high
2.630.60
Buffaloes production interval high
2.750.55
Artificial insemination high
2.800.50
Ruminant nutrition high
2.800.50
Disease control high
2.430.69
Treatment of control for endoparasites high
2.130.60
Promoting for buffaloes of grouping high
1.800.70
Promoting for buffaloes fattening moderate
1.700.80
Domestic marketing moderate
Standard slaughterhouse set up moderate
1207
The Problems and Obstacles on Raising Buffaloes of Local Farmers in Central

Thailand: A Case Study of Saraburi Province
Sirisukh SAPANAN, a K. RIMKEEREE,a P. CHUSEN,a C. DONGPALEETAN,a G.
PHOTIKANITa Anchalee Naa and Suvit BOONPRONG a*
a
ABSTRACT
Swamp buffaloes are indigenous to Thailand and are well-adapted to the poor conditions.
However, the industrial revolution has affected a strong impact on the farmers’ lives. Young village
people who traditionally supervised the animals during the days can also change their careers to
non-farm enterprises that are often city-based. Thus, the objectives of this study were to investigate
problems and obstacles on raising buffaloes. The samples were derived from 330 buffalo-raising-
farmers in Saraburi province (143143.59N, 1005435.58E and 70 m above sea level with an
average temperature 26.90C, average relative humidity (RH) of 75% and average of 1,250 mm
annual rainfall) located in central Thailand. The data were analyzed in terms of percentage, mean
and standard deviation. The results showed that the main objectives of the farmers were to utilize
buffalo’s manure (38.0%), sell their animals (35.2%) and receive from their inheritances (26.8%),
respectively. 45.6% of the farmers had raised buffaloes approximately for 11 years. The labours for
raising buffalo were 1 to 2 persons each farm (80.3%) and 82.4% of them raised buffaloes by
themselves. Each household had about 1 to 5 heads of buffaloes (74.2%). Ninety-three point seven
percentages of the farmers bred their buffaloes by sires with random mating. However, 15.5% of
famers knew to animal selection. Moreover, 97.5% of the farmers restrained their buffaloes
separately from their homes, and 85.0% of them did not have enough space for forage production.
The farmers (88.6%) stored rice straw for their buffaloes in summer. The minority famers kept
concentrate (34.3%) and mineral (38.5%) for their buffaloes. However, 96.5% of their animals were
vaccinated against diseases, and 78.5% were treated for endoparasites by authorities of the
Department of Livestock Development (DLD). When the animals were sick, they were treated by
authorities of the DLD (78.5 %), by volunteers of the DLD (12.2 %) and by owner (10.3 %),
respectively. The problems and obstacles on raising buffaloes were: lacks of space for raising
buffaloes, forage and knowledge of farm management. Moreover, the farmers in the study area
suggested that the DLD should provide financial support in terms of: funding for buffalo raising
development in the villages in each district; training and promoting sire selection; improving public
land for raising buffaloes; forage production; using animal for agriculture; and using buffalo’s
manure efficiently.
Keywords: raising buffalo, problems, obstacles, production, need for extension, Saraburi province
INTRODUCTION
In Thailand, the swamp buffalo is an indigenous animal and is well-adapted to the poor
conditions. This animal is capable of utilising agricultural waste products or low quality feed to
convert into meat while also providing manure for use as a fertilizer. Manure does not only provide
a direct supply of fertilizer but also improves soil and pH value of the soil especially in areas where
are used for higher quantities of chemical fertilizer, as in today’s rice paddy fields. Moreover,
excess animals are also sold when they are necessary for extra income (Indramangala, 2002; Na-
Chiangmai, 2002). The swamp buffalo is a sturdy draught animal. Its body structure, especially the
distribution of body over the feet and legs, is an important advantage. Its large boxy hooves allow it
to move in the soft mud of rice fields. This animal has a high capacity for work hard in marshy and

1208
flood-prone environments where tractors cannot work as well (Chantalakhana, 1994; Indramangala,
2002).
Moreover, Thai swamp buffalo is also considered a friend of the family who is a loveable
family member and has many legends about them. It is recreated in a part of many festivals such as:
“Buffalo Racing Festival” at Chonburi province, “Fighting Buffalo” at Samui Island, Suratthani
province and agro-tourism as “Ban Kwai Thai” at Chiang Mai province. Furthermore, foreign
tourists also would like to see swamp buffalo at work in Thailand (Indramangala, 2002).
Saraburi province is in central Thailand. It is located at 143143.59N, 1005435.58E and
70 m above sea level with an average temperature 26.90C, average relative humidity (RH) of 75%
and average of 1,150 mm annual rainfall (Thai Meteorology Department, 2012). The areas in this
province are generally alternation between flat and low hill with good soil fertility. There is a higher
population density and a high level of mechanization in the irrigated paddy fields. The buffaloes in
Saraburi province graze on low hill and on tiny public land. In general Thailand, buffaloes had a
sharp decline in numbers (Indramangala, 2002). However, these animals had a slight decreasing in
numbers. Department of Livestock Development (DLD) (2012) reported that buffaloes in Saraburi
province from 2004 to 2011, numbers decreased from 15,729 to 13,028 head. As, environment
factors determine the status of buffalo production in farming systems and influence the type of
farming carried out.
Thus, the objectives of this study were to investigate problems and obstacles on raising
buffaloes in Saraburi province.

The samples of population were 330 from 1,886 buffalo-raising-farmers in Saraburi
province, Thailand. The samples were selected with purposive sampling (Cohen and Manion,
1989). Data were collected using interview schedule which consisted of closed-ended questions and
open-closed questions via interview from each farmer. The general data of farmers, raising swamp
buffaloes and problems and obstacles on raising buffaloes were recorded in the period of October
2008 to September 2010. As, the problems and obstacles consisted of 3 levels as follow: 1 = slight
problem, 2 = moderate problem and 3 = high problem. For the means of problem on raising
buffaloes levels such as: 1.00 to 1.66 = slight problem, 1.67 to 2.33 = moderate problem and 2.34 to
3.00 high problem, respectively (Prasitrathasindhu, 2003). Data were analyzed using the Statistical
Packages for the Social Science (SPSS). Values were presented as percentage and meansSE.

The results showed that the main objectives of the farmers were to utilize buffalo’s manure
(38.0%), sell their animals (35.2%) and receive from their inheritances (26.8%), respectively.
45.6% of the farmers had raised buffaloes approximately for 11 years. The labours for raising
buffalo were 1 to 2 persons each farm (80.3%) and 82.4% of them raised buffaloes by themselves.
Each household had about 1 to 5 heads of buffaloes (74.2%). Ninety-three point seven percentages
of the farmers bred their buffaloes by sires with random mating. However, 15.5% of famers knew to
animal selection. Moreover, 97.5% of the farmers restrained their buffaloes separately from their
homes, and 85.0% of them did not have enough space for forage production. The farmers (88.6%)
stored rice straw for their buffaloes in summer. The minority famers kept concentrate (34.3%) and
mineral (38.5%) for their buffaloes. However, 96.5% of their animals were vaccinated against
diseases, and 78.5% were treated for endoparasites by authorities of the DLD. When the animals
were sick, they were treated by authorities of the DLD (78.5 %), by volunteers of the DLD (12.2 %)
and by owner (10.3 %), respectively.
Table 1 is shown that the problems and obstacles on raising buffaloes were high levels in
buffalo selection and improvement, in knowledge of farm management (such as: raising buffalo
interval, weaning, fattening, conserving forage), in the price of concentrate which were expensive,
in forage production and lacks of space for raising buffaloes. Their problem in buffaloes’
production interval management, in artificial insemination and funding for raising buffalo
1209
development were moderate levels. However, the farmers were slight problems in disease control,
treatment of control for endoparasites and domestic marketing. Moreover, the farmers in the study
area suggested that the DLD should provide financial support in terms of: funding for buffalo
raising development in the villages in each district; training and promoting sire selection; improving
public land for raising buffaloes; forage production; using animal for agriculture; and using
buffalo’s manure efficiently.
In fact, the central Thailand gets almost all the annual rainfall during the monsoon season
between July and October. The dry season stretches for 6-8 months and can be generally divided
into two phases; cool and dry (November to February) and hot and dry (March to June) (Thai
Meteorology Department, 2012). The first part of the dry season is characterized by food shortage
with low quality of the food. The later part of the dry season is the most critical with limited
quantity and quality of food (Crowder and Cheda, 1982). Rainfall is the most important factor
controlling the composition and productivity of a pasture (Butterworth, 1985). This study revealed
that swamp buffaloes in Saraburi province graze on low hill or on tiny public land and do not graze
on specific pastures. Furthermore, the farmers lacked of forage species with high production, and
concentrates were expensive. As these results, the animals were low fertility and low efficiency in
growth performance.
Thus, promoting for farmers grow forages which are quantity and quality of food for
buffaloes under limited farmers’ areas should consider. Moreover, DLD officials need to study how
to the method which provide financial support in terms of: funding for buffalo raising development
in the villages in each district, training and promoting sire selection, improving public land for
raising buffaloes, forage production, using animal for agriculture as drought power, and using
buffalo’s manure instead of chemical fertiliser and promoting with sustentation and conservation
on raising buffaloes of local farmers which breed by sire with high fertility and efficiency in
growth performance.
Furthermore, the roles of buffalo in contributing to human needs were discussed by Peters
(1999); Chantalakhana (1994); Indramangala (2002) and can be grouped as follows:
1.) Food security, it can be defined by a combination of balance between availability and
need, avoidance of temporary food shortage and nutritional deficiencies and adequate food quality.
Buffalo are living manufacturers who transform non-edible human food (such as crop residue, rice
straw, and weeds) into high protein food for human consumption. Thus, increased buffalo
production may add to food security in several ways. Firstly, many poor farmers will have direct
access to more food with lower cost. Secondly, increased domestic production will reduce imports
and save foreign currency.
2.) Insurance, capital formation and income, this function is most relevant to securing the
socioeconomic status of the farmers. It can serve as a long-term capital reserve by using local
natural resource as long as fodder resources are freely accessible at no charge. Rearing buffalo as a
means of a ‘savings bank’ gives some financial security for the household. In addition, sales of
progeny or unproductive buffalo and their dung provide direct cash income to the farmers.
Therefore, buffalo represent buffer assets (with some security against inflation) which can be
realised at any time, adding further stability to the self-sufficiency crop-livestock system.
3.) Farm integration, the integration of cropping and livestock production makes efficient
use of farm waste and crop by-products such as rice straw and weeds, and enables a transfer of such
nutrients to cultivated soils using manure. Manure not only provides a direct supply of fertiliser but
also improves soil structure and pH value of the soil especially in areas which use higher quantities
of chemical fertilizer.
In conclusion, this study could be enlisted for farming system relating to buffalo production
in Saraburi province and in the other locations of Thailand. It needs to be a multi-discipline research
program to identify, diagnose and design programs which make the greatest impact on farmers’ net
income. This needs to be supported by an extension program conducted in a suitable manner to
implement the strategies at the village level.
1210
REFERENCES
Butterworth, M.H. 1985. Beef Cattle Nutrition and Tropical Pastures. Longman, London, New
York, NY.
Chantalakhana, C. 1994. Swamp buffalo development in past three decades and sustainable
production beyond 2000. In: Proceedings 1st Asian Buffalo Association Congress, Khon
Kaen, Thailand.
Cohen, L. and L. Manion. 1989. Research Method in Education. 3rd ed., Routledge, London.
Crowder, L.V. and H.R. Cheda. 1982. Tropical Grassland Husbandry. Longman, London, New
York, NY.
Department of Livestock Development (DLD). 2012. Annual Livestock Reported during 2004 to
2012. Information Technology Center, Department of Livestock Development, Bangkok. (in
Thai)
Indramangala, J. 2002. Buffalo development in Thailand, pp. 117-123. In J. Allen and A. Na-
Chiangmai, eds. Development Strategies for Genetic Evaluation for Beef Production in
Developing Countries. ACIAR Proceeding, no. 108, Watson Ferguson & Co., Brisbane,
Australia.
Na-Chiangmai, A. 2002. Current situation and development trends of beef production in Thailand,
pp. 93-97. In J. Allen and A. Na-Chiangmai, eds. Development Strategies for Genetic
Evaluation for Beef Production in Developing Countries. ACIAR Proceeding, no. 108,
Watson Ferguson & Co., Brisbane, Australia.
Peters, J.K. 1999. Livestock production and food security consequences for the environment?
Agriculture and Rural Development. Vol. 6, No. 1, Frankfurt.
Prasitrathasindhu, S. 2003. The System of Social Science Research Method. 12th ed., Fuangfa
Printing Ltd., Bangkok, Thailand. (in Thai)
Table 1. Problems and obstacles on raising buffaloes of farmers in Saraburi province, Thailand.
Values (meansSE)
n = 330
2.450.56
Problems and obstacles Means of problem levels
2.760.48
Buffalo selection and improvement high
2.250.40
Farm management high
2.130.46
Buffaloes production interval moderate
2.880.25
Artificial insemination moderate
2.900.23
Prices of concentrate high
2.850.40
Forage production high
1.460.44
Land for raising buffaloes high
1.600.45
Disease control slight
2.300.45
Treatment of control for endoparasites slight
1.560.57
Funding for raising buffalo development moderate
Domestic marketing slight
1211
Assessment of Village-based Artificial Insemination Technician (VBAIT)

Scheme as a Strategy towards Privatization of Artificial Insemination (AI)
Services in Nueva Ecija
Sonia D. POLa, F. L. PORCIUNCULAb and Felomino V. MAMUADa
a
Philippine Carabao Center, National Headquarters and Gene Pool, Maharlika Hi-way,
Science City of Munoz, 3120, Nueva Ecija, Philippines. bDepartment of Rural Development
and Development Communication, Institute of Graduate Studies, Central Luzon State
University,
Science City of Munoz, 3120, Nueva Ecija, Philippines.
*Corresponding email: soniadillapol@yahoo.com
ABSTRACT
The study aimed to assess the Village-based Artificial Insemination Technician
(VBAIT) scheme as a strategy towards privatization of AI services in the villages of Nueva
Ecija. A total of 28 VBAITs and three Local Government Unit (LGU) AI technicians were
identified from 8 municipalities of Nueva Ecija through a purposive sampling method and
additional 182 farmers and 20 key informants served as respondents of the study. Sets of
questionnaires were developed for each and personal interview was done to generate data
from the respondents. Secondary data covering the period of 2005 to 2010 and the data
obtained from the key informants were analysed using averages and percentages. The study
revealed that the technical training for VBAITs at PCC at CLSU was “very satisfactory” in
acquiring knowledge, skills and positive attitude deemed necessary to carry out AI services in
the field. The provision of post training AI support from PCC contributed positively to
VBAITs’ desire to carry out AI services in their communities. The cost of VBAIT training
amounted to PhP32,627.00. However, the cost to produce a calf through VBAIT was
PhP3,252, lower by 32.0 percent than LGU-AIT (PhP3,932). There was no difference in the
cost of post-training support between VBAIT and LGU-AIT. The VBAIT’s additional
income from artificial insemination service amount to PhP19,550/year on purely carabao
services as recorded from the database. However, this additional income was estimated to be
PhP 52,350/year if based on survey result as recalled by VBAIT respondents covering their
carabao and cattle AI services. These represents 13.2 percent and 35.2 percent increment
respectively in the VBAITs’ average annual income of PhP148,517. About 64.3 percent of
the VBAITs have already established loyal clients in a span of five years of their services in
the field. This ensures them of sustainable clients, hence, sustainable income thereby creating
a pool of VBAITs with high sense of self-esteem and accomplishment willing to do AI
services on a sustainable basis.
Keywords: VBAIT, LGU-AIT, PhP, AI
INTRODUCTION
The government through Philippine Council for Agricultural Resources Research and
Development – Department of Science and Technology (PCARRD-DOST) launched a 10-
year United Nations Development Program (UNDP)-FAO-assisted research project (1982-
1992) and demonstrated that the crossbreds between swamp buffaloes and dairy buffaloes are
fertile, grow 2x more than the native parents, produce 3 to 4x more milk and are acceptable to
farmers. It was on the basis of this above-cited UNDP-FAO-assisted project that the
government institutionalized the Carabao Development Program (CDP) thru the creation of
the Philippine Carabao Center (PCC) in 1993 (Cruz, 2009). The CDP relies on artificial

1212
insemination (AI) as the approach to massive crossbreeding in view of the absence of

sufficient number of good genetics of dairy breed in the country. During the early years,
government-led AI services in carabaos were done by limited technicians of PCC and the
devolved LGU technicians of the Department of Agriculture (DA). Over the years, the
number of AI services rendered by LGU technicians has not significantly increased in
magnitude due to their limited number, added with the inherent inability of many LGUs to
provide basic requirements for a truly responsive AI service due to budgetary constraints.
In view of these facts, the PCC introduced Village-based Artificial Insemination
Technicians (VBAITs) in 2006 to act as private AI technician and render AI services for a fee
in villages. The logic behind VBAIT approach is to increase AI service coverage as a
component of the Expanded AI Program without much cost on the national government. By
privatization thru VBAIT, farmers can be gradually weaned from relying on dole-out services
from the government and will learn to invest on animal breeding as part of their dairying
enterprise (BAI, 2004). Moreover, the privatization of AI services thru VBAIT approach
capacitates and empowers selected willing individuals to work in their villages as AI
technicians and earn additional income for their families and in the process develop a higher
sense of self-worth. In Nueva Ecija, the number of VBAIT has considerably increased in
number (Mamuad et al., 2010) and is now perceived to contribute significantly to the
upgrading efforts of carabaos towards dairying.

The theoretical framework of this study was based on the Context, Input, Process, and
Product evaluation (CIPP) Model developed by Stufflebeam et al., (1971). The CIPP Model
is a simple systems model applied to program evaluation and adopted the basic open system
that includes input, process, and output.
The scheme under evaluation focused on the envisaged privatization of AI services
towards the much sought responsive, expanded and sustainable genetic upgrading of carabaos
towards dairying in the province of Nueva Ecija. The assessment was anchored on basic
understanding that the inputs are transformed through a process into outputs that could
ultimately gauge the outcomes of the scheme as shown in Figure 1.
Sampling and respondents of the study
Following purposive sampling method, two municipalities with the highest number of
trained VBAITs in every district were identified for the survey. In these eight municipalities,
respondents VBAIT, respondents LGU AI technicians, respondent farmer-clients and key
informants were identified. Only 19 (50.0%) VBAITs out of the 28 interviewed were found
active. For each active VBAIT, seven farmer respondents were interviewed thus a total of
182 farmers, representing 30% of the total population of the dairy farmers (840) served by
VBAITs in the selected municipalities were covered. For nine (23.7%) in-active VBAITs,
only some of their farmer clients were interviewed. It was very difficult to find their clients
due to their long time of in-activity. On the other hand, only 50% or three LGU-AIT trained
by PCC at CLSU were interviewed from the covered municipalities since three of them
cannot be accessed for interview at the time of the study despite several attempts. It was
learned from their colleagues that they were no longer doing AI services for several years.
Instrumentation
Three sets of questionnaires were developed and pre-tested. The first set of
questionnaire was for the trained VBAITs, the second set of questionnaire for LGU AI
technicians and the third set for randomly selected dairy farmer beneficiaries. Also, key
informant interviews, using a guided questionnaire, were done to gather the additional
information.
1213
Data Gathering Procedure

Primary data were gathered from the VBAIT, LGU AI technicians and from farmer
beneficiaries of the AI services through face to face interview guided by the designed
questionnaires. Secondary data on the performance of VBAIT were gathered from the
available reports and documents of the training center, PCC at CLSU.
Methods of Data Analysis
Frequencies and percentages were used to summarize the descriptive data particularly
on the inputs used and outputs of the scheme. Secondary data from available reports and
documents on AI activities carried out in Nueva Ecija for the last five (5) years were
reviewed and analyzed. Cost analysis was used to compare the cost of AI service, cost of
producing a crossbred carabao, cost of training, and the cost of post-training support between
VBAIT versus LGU-AIT. Four-point criterion-referenced scales were used to interpret the
knowledge, skills and attitude of VBAIT in doing AI service in the field.

Resources and stakeholders
It was found that PCC at CLSU as a training center has adequate training facilities,
training animals, well trained trainers, and an approved module for training AI technicians. It
can handle 10-15 trainees per batch and was funded with MOOE of Php23,000/trainee to
cover food and lodging, supplies and materials and other miscellaneous expenses during the
training. Other stakeholders are the Nueva Ecija Provincial Government (NEPG), municipal
government of the Local Government Units (LGUs) and Municipal/City Veterinarian. Their
roles were assistance in the selection of would-be VBAITs and in nurturing and monitoring
the trained VBAITs in their respective areas.
Socio-demographic profile of VBAITs and farmer respondents
The average age of VBAIT respondents is 39.3 years (n=28), that of LGU-AIT is 46.6
years (n=3) and were of quite similar age group as noted by Acoba (1990). In an earlier study
it was reported that age is not affecting AI services (Macalandag, 1989). All of the LGU-AI
technicians and some 82.1 percent of the VBAITs are married. All of LGU-AIT (100%) are
college graduates whereas only 32.1 percent of the VBAIT had college education.
Incomewise, VBAIT respondents have a mean of Php148,517/year (Php10,000-420,000)
whereas LGU-AITs have a mean of Php307,806 (Php266,000-348,000/year). On the other
hand, all farmer recipients of AI services (n=182) owned dairy buffaloes. Of these farmers,
85.5 percent are males; 94.0 percent are married and only 11.0 percent had college education.
Average age is 47.7 years old with average family size of 4.62. The average income is
Php117,098.00/annum with rice farming as the primary source of income of 72.5 percent
whereas vegetable farming is the source of income of the 35.7 percent of the respondents.
Selection, training and post-training interventions
The qualified candidates for AI training were recommended by barangay captain
(10.7%), endorsed by LGU through the MAO to PCC (42.9%), and by Chairman of the
Cooperatives (25.0%). Final screening was done by PCC and from 2006 to 2010, all the final
recommendees were accepted for training. Selected candidates underwent one month live-in
training on AI and pregnancy diagnosis (PD). The program consisted of 30 percent
theoretical and 70 percent practical and was designed to provide VBAIT adequate knowledge
and skills. Towards the final phase of the training, VBAITs were given written and oral
exams as well as actual exam on AI and PD. Only those trainees that passed the exams were
given certificate. After graduation, VBAITs were given free of charge LN2 field tank, AI
gun, and thermometer. The liquid nitrogen and frozen semen were given also free of charge
during the initial year of field service by VBAIT. PCC assisted the VBAITs in the field
during the initial period of their service and they were gathered once a month to assess
1214
problems and concerns related to their services. Outstanding VBAITs were recognized and
were given special awards. They were also given cash incentive of Php200/calf born. This
was on top of the fee they charged the clients for their services. Among VBAIT respondents,
71.4 percent (10/14) of the AI related knowledge items used in the survey were learned “very
satisfactorily” and the remaining 28.6 percent (4/14) were learned at “satisfactory level” only.
VBAIT respondents obtained “very satisfactory” ratings in all nine skills used in the survey
except on the examination on the normality of female reproductive organ, and on
examination of ovarian status. In these two skills, LGU-AIT respondents only registered
“satisfactory” rating. All of the five references positive attitudes used in the survey were
learned “very satisfactory” by active VBAITs and LGU-AITs. However, the ratings noted
among in-active VBAITs were comparatively lower than those among the active VBAITs.
Cost of producing a VBAIT
Based on the records of PCC at CLSU, the training cost for an AI technician is
pegged at Php23,000/trainee. Based on the PCC at CLSU database, of the 61 VBAITs trained
for Nueva Ecija, only 43 are now active, thus training efficiency is 70.5 percent and 71.42
percent for LGU-AIT. It appears that the cost to produce an active VBAIT is Php32, 627.90
and Php427.00 more than the cost for producing an active LGU-AI technician. This
difference is not at a striking magnitude, however.
Cost of AI service by VBAIT
The total cost of AI service to produce a calf via LGU-AIT is Php3,932.00 and
Php3,252 by VBAIT service. While the total cost through LGU-AIT is 100 percent
subsidized by the government, through VBAIT is only 68.1 percent (Table 1). The rest of the
costs are shared by VBAIT and client farmer, representing 16.5 and 15.4 percent,
respectively.
Effectiveness of the VBAIT scheme
When VBAIT was introduced in Nueva Ecija, the contribution of LGU and PCC
significantly went down to only 20.0 and 44.9 percent, respectively, whereas that of the
VBAITs has consistently grown from 34.9 percent in 2004 to 74.1 percent in 2011. This
increase represents a growth of 16.0 percent in average annual AI output from 2005 to 2010
despite the decline in AI services in Nueva Ecija of the PCC and LGU-AITs. Effectiveness of
VBAIT scheme is very visible in terms of improvement on the number of AI services done in
Nueva Ecija.
Respondents’ satisfaction rating for VBAIT services in their communities and in their
animals are summarized in Table 2. Based on the criteria in the survey, farmer-respondents
are generally satisfied with the VBAIT services in their respective areas. Repeat calls for AI
service in this study were interpreted as customer loyalty. Those farmers that kept on calling
the same VBAIT for AI service for many years can be described as loyal customer. The term
customer loyalty is used to describe the behaviour of repeat customers, as well as those that
offer good ratings or testimonials (Wynn, 2012). Loyal customers do a great favour to
VBAIT as they tell friends about positive attributes of VBAIT and their services. About 64.3
percent of VBAIT respondents indicated that they have served the same client every year for
the period 2005 to 2010 (Table 3). It is clear that majority of active VBAITs have already
established loyal clients their service area.
Generating additional income. VBAITs are interested to do AI service for additional
income on top of what they normally derive from their main income generating activities.
Based on the data gathered from the field surveys among active VBAIT respondents, they
would have an estimated average income of Php52,350/year from their services on carabao
and cattle combined. Given the mean income of VBAITs of Php148,517/year, the estimated
average annual income of VBAIT of Php52,350/year from AI services is a 35.2 percent
increment in their annual income.
1215
The feeling of worthiness in serving the community is expressed by 92.8 percent

(26/28) of the VBAIT respondents. This feeling of self-esteem out of being able to serve their
respective communities is the single indicator of the positive attitude and outlook derived
from the training, nurturing and through the course of their service. More so, effectiveness
has also been revealed in the customer satisfaction and customer loyalty survey wherein 64.3
percent of VBAITs have already established loyal clients in a span of five years of service in
the field. This ensures VBAITs of sustainable clients, hence, sustainable income thereby
creating a pool of VBAITs with high sense of self-esteem and accomplishment willing to do
AI services on a sustainable basis.
REFERENCES
Acoba, T. 1990. Role Performance of Central Luzon State University-Philippine Carabao
Research and Development Center Trainees on Artificial Insemination and Pregnancy
Diagnosis of Water Buffaloes. Unpublished Ph.D. Dissertation, CLSU, Nueva Ecija.
Bureau of Animal Industry (BAI). 2004 and 2011. Farm Integrated Animal Health and
Production Project (FIAHPP) Status and Annual Report.
Cruz, L.C. 2009. Transforming Swamp Buffaloes to milk producers thru backcrossing.
Proceeding: Asian Buffalo Congress, Lahore, Pakistan.
Cruz, L.C. 2012. Philippine Carabao Center 49th Program Management Committee
Meeting. Proceeding: 49th PMC. Tuguegarao.
Macalandag, E.C. 1989. Assessment of social and technical factors affecting effective AI of
buffaloes in regions III and VIII. Unpublished MS Thesis, CLSU, Nueva Ecija,
Philippines
Mamuad, F.V., H.V. Venturina, E.C. Atabay, R.S. Hibionada, E.C. Encarnacion, M.M. Jr.
Roguel, R.T. Morcoso and C.M. Adriano. 2010. Increasing efficiency of artificial
insemination (AI) program in Nueva Ecija. Proceeding: In-House R&D Review PCC,
May 26-28, 2010.
Stufflebeam, D.L; W.J. Foley, W.J. Gephart, E.G. Guba, R.L. Hammond, H.O. Merriman and
M. M. Provus. 1971. Educational Evaluation and Decision Making. Itasca, Ill.: F.E.
Peacock. www.southalabama.edu/coe/bset/Johnson
Wynn, L.S. 2012. What is Customer Loyalty? Conjecture Corporation. Retrieved on
October 2, 2011 from http://www.wisegeek.com/what-is-customer-loyalty.htm
Table 1. Breakdown of sources of cost to produce a calf for VBAIT and LGU-AIT,
Nueva Ecija, 2011.
LGU-AIT VBAIT
SOURCE
Php % Php %
Government 3,932 100 2,216 68.1
Clients/Farmers - - 500 15.4
VBAIT - - 536 16.5
Total 3,932 100 3,252
1216
Table 2. Respondents’ satisfaction ratings for VBAIT services in Nueva Ecija.

AVERAGE DESCRITIVE
CRITERIA SD
RATING RATING
Dealing with clients 3.73 0.52 Very satisfactory
Respond to calls promptly 3.76 0.48 Very satisfactory
Friendly and approachable 3.85 0.36 Very satisfactory
Explain clearly VBAIT policy 3.75 0.47 Very satisfactory
Pay attention to AI service 3.75 0.53 Very satisfactory
details
Mean Rating Descriptive Rating Mean Rating Descriptive Rating
1.00– 1.74 Unsatisfactory 2.50-3.24 Satisfactory
1.75-2.49 Fairly satisfactory 3.25-4.00 Very satisfactory
Table 3. Total number of farmers and frequency of calls to VBAIT, 2005 – 2010.
REPEAT CALLS FOR AI VBAIT
Percentage
SERVICE Frequency(n=28)
Once in five years 4 14.3
Twice in five years 2 7.1
Thrice in five years 4 14.3
Five times in five years 18 64.3
* Multiple responses
Figure 1. Conceptual framework in assessing VBAIT scheme as a strategy towards

privatization of AI services in Nueva Ecija.
1217
Condition and Specifics of Buffalo Breeding in Republic of Macedonia
Bone PALASHEVSKI,1* Zoran NALETOSKI,1 Natasa MATEVA1 and Ana

PALASHEVSKA1
1
University, Ss. Cyril and Methodius”, Institute of Animal Science, 1000 Skopje, Republic of
Macedonia
*Corresponding email: palasevskib@yahoo.com
ABSTRACT
Buffalos are almost extinct in Europe, with the exception of Italy and the Balkan Peninsula,
where the majority is located in Bulgaria, Albania, Romania, and, in minor numbers, in Macedonia.
By using contemporary selection methods in some of the countries, during the course of lactation
within 230 – 270 days, produce over 4000 kg of milk with high content of milk fats (6,7%).
Buffalos which are not part of the selection programme usually produce low quantities of milk. In
the conditions in Macedonia, the average milk production capacity is around 700 kg, whereas in
conditions of improved nutrition the milk quantity can increase up to 1300 kg. The percentage of
milk fats is 9%. At present the number of buffalos in Republic of Macedonia is reduced to almost
insignificant – around 400 populations (an estimation of which 100 are determined). Our research
included 36 buffalos, bread in an extensive system with very low production inputs. For the purpose
of intensive husbandry, the buffalo does not have larger significance, except as an indigenous
genetic resource.
Keywords: buffalo, indigenous genetic resource
INTRODUCTION
Initial data on buffalo spread on the territory of Republic of Macedonia date from the XII
century (Ulmanski, 1926) and have been entered in the so called “eastern spread” from Bulgaria and
Greece.
Frangesh,1926 based on analysis carried out in 1920, presents the following data on the
number of buffalos on the Balkan Peninsula (Table 1).
According to Belic, 1995, 82 – 84% of the total number of buffalos, were located on the
present terriory of Republic of Macedonia (the area around the river Vardar).
The largest problem in Republic of Macedonia is the close kinship, which results from the
lack of male reproductive buffalos i.e. the lack of fertilization sperm.

Currently, in the Republic of Mcedonia several herds of buffalos have been determined,
mostly deployed in several villages. In the village of Debreshta, in the Prilep region, the largest
number of buffalo breeders have been noted, in total four breeders with eleven (11) populations of
buffalos. In the village of Mojanci, in the Kochani region, one buffalo breeder has been noted with a
total number of eight (8) populations of buffalos. In the village of Poeshevo, in the Bitola region,
one buffalo breeder has been noted with a total number of ninteen (19) populations of buffalos, of
which 5 are male and 14 are female. These populations had been taken over from a buffalo breeder
in Mariovo and are in an exceptionally poor condition. Since 2008 a project has been set up for
preservation of the buffalos as an indigenous genetic resource (Table 2).
Reduction in the number of buffalos occurs predominantly due to the lack of demand for
buffalo milk and diary products, as well as the high cost for their breeding, and the absence of long-
term strategy for preservation of buffalos as an indigenous genetic resource. There is demand for
the milk fat as a product used in alternative and traditinal medicine to eliminate the fat in the milk,
whereas the milk is being used for cheese production, however largely for private rather than for
commercial purposes.
1218
With the amendments to the Law for Animal Husbandry, 2008, in which buffalos have been
inserted as endangered species in the Republic of Mcedonia, the present condition of the bufalos is
expected to improve.
RESULTS AND DISCUSION

During the course of the realisation of the project, samples of milk and white salty cheese,
produced from boiled milk and, by the use of an acid coagulant, curded with vinegar acid, were
obtained and their chemical composition was determined. The alterations which took place during
the course of maturing (fermentation) of the cheese samples were sent for analysis after the 30th
day of the fermentation process (Table 3).
The results of the chemical composition of the cheese produced from buffalo milk are in
accordance with the results of Peeva, 1993 i.e. there are minor differences in terms of the content of
the total amount of dry matter.
 Preservation (conservation) of buffalos “in situ”, as one of the best methods for preservation
IMPLICATIONS
 Examinining the productional, reproductive and morphological properties in buffalos in

of domestic animals;
 Proposed measures for genetic enhancement of the buffalos in Republic of Macedonia;

Republic of Macedonia;
 Defining future prospects for research of buffalos in Macedonia and the potential for
 Introduction of buffalos in the Registry of Indigenous breeds of domestic animals at MZSV

international colaboration;
including the water buffalo, as addition to the indigenous breeds of the Law for Animal
Husbandry, and thus including the buffalo in the biodiversity programme of Republic of
 Need for further finalising of the project post the amendments of the law and bylaws.
Macedonia.
REFERENCES
Belic, J. Saric, M. 1995. Animal husbandry in Yugoslavia from his establishment until
disintegration 1991. Serbian Academy of science and arts, ISBN 8670252147, Belgrade,
Serbia.
Borghese, A. 2009. Situation and perspectives of buffalo in the World, Europe and Macedonia
Maced. J. Anim. Sci. 1 (2) 281–296 (2011) SSN 1857 – 7709.
Čobić Timotej M. 2000. Origin, domestication and expansion of domestic buffalo
(Bubalus bubalis) in Yugoslavia. Zbornik Matice srpske za prirodne nauke, 2000, iss. 99,
pp. 87-96. University of Novi Sad, Faculty of Agriculture, Novi Sad, Serbia.
Peeva, C. 1993, Everything about buffalo. Agriculture university, Agriculture faculty, Plovdiv,
Bulgaria.
Palasevski, et al. 2008. Macedonian buffalo as an indigenous genetic resource. Final Report
Ministry of Agriculture, Forestry and Water Economy of the Republic of Macedonia.
Ulmanski, S. 1926. Buffalo. Serbian-Croatian-Slovenian National encyclopedia.
1219
Table 1. Number of determined buffalo grla in the Republic of Macedonia.
Adult Calves and

Location Total
population heifers
Debreshte (Prilep) 13 7 20
Presil (Krushevo) 3 1 4
Berovci (Prilep) 2 2
Mojanci (Kochani) 7 1 8
Vinica 2 2
Poeshevo (Bitola) 12 5 17
Total 35 18 53
Table 2. Number of buffalos in Republic of Macedonia during the period 1957-2006*.
populations populations populations populations of

Year Year Year Year
of buffalos of buffalos of buffalos buffalos
1957 20247 1974 9825 1985 4725 1996 1013
1960 18919 1975 10316 1986 2316 1997 908
1961 15530 1976 9768 1987 1780 1998 899
1962 15530 1977 11100 1988 1709 1999 725
1963 17470 1978 9426 1989 1647 2000 632
1964 15663 1979 9343 1990 1602 2001 605
1965 17366 1980 9006 1991 1488 2002 599
1966 12645 1981 9200 1992 1179 2003 566
1967 13631 1982 9020 1993 1125 2004 565
1969 10653 1983 7800 1994 1021 2005 474
1973 11255 1984 6750 1995 1017 2006 409
*
(Source: State Statistics Office of RM)
1220
Table 3. Chemical and microbiological composition of buffalo milk and cheese sampled from
individual populations.
Total count
Dry matter Fat Protein Lactose
Trial рН of somatic
% % % %
cells
1 26,98 10,95 5,26 5,37 6.76 45.000

2 24,48 9,35 5,07 5,36 6.88 35.000
3 23,73 9,05 4,45 5,23 6,68 27.000
4 24,89 9,56 5,10 5,25 6,63 56.000
5 25,37 10,28 4,96 5,17 6,71 48.000
Dry
Fat Protein Lactose Ash Salt
Sample matter рН °ЅН
% % % % %
%
1 54,96 22,72 25,105 1,933 2,43 2,99 5.69 9,8
2 57,09 30,13 24,374 1,77 5,66 3,78 5.28 12,0
3 56,42 32,5 22,0 0,3 4,24 3,22 4.65 82,6
4 55,94 34,2 20,18 0,25 4,75 3,56 4.47 90,5
1221
Assessing the Performance of Village-Based Artificial Insemination

Technicians for Water Buffaloes in Nueva Ecija Province, Philippines
Eric PALACPACa, Moses Gil HONORIOa and Erwin VALIENTEa*
a
Operations Research Task Force, Philippine Carabao Center National Headquarters and
Gene Pool, Science City of Muñoz, Nueva Ecija Province, Philippines
*Corresponding email: ericclap@gmail.com
ABSTRACT
A case study was conducted to assess the performance of village-based artificial
insemination technicians (VBAITs) for water buffaloes in Nueva Ecija Province, Philippines.
Secondary data on the number of breedable animals in the service areas, number of artificial
insemination (AI) services, and calving percentage were gathered and analyzed. Using the
“number of AI services” in the villages as an indicator, a sample size of 36 VBAITs was
categorized into “high”, “medium”, and “low” performers. Based on these categorizations,
the VBAITs were characterized as to their age, education, and income derived from AI
services. Their stock knowledge was also assessed by administering a 30-item quiz on animal
reproduction and AI while their practical skills as AI technicians were gauged from actual
field evaluation by an external expert. Results showed an increasing trend in the number of
AI services of VBAITs from years 2004 to 2011 with an estimated calving percentage of
23% in 2011. In terms of performance categories, there was more or less an equal distribution
of VBAITs with the “high” performers deriving more income and covering more breedable
animals compared to the “medium” and “low” performers. Mean percentage quiz and
practical skills scores did not vary among all VBAIT categories. The “number of breedable
buffaloes in the service areas” and “income derived from AI services” were found to be
significant positive regressors for the “number of AI services” performed by the VBAITs
indicating that the predictor variables in the current study are important considerations for
increasing AI service delivery.
Keywords: artificial insemination, water buffalo, village-based technicians
INTRODUCTION
The application of artificial insemination (AI) in improving the genetics of native
(swamp) buffaloes has been the banner program of the Philippine Carabao Center (PCC)
since its establishment in 1992. Over the years, the AI technicians operating in Nueva Ecija
province have consistently provided the biggest share to the PCC’s outputs in terms of
number of AI services and number of calves monitored post AI. In 2011, total number of AI
services by all types of technicians in Nueva Ecija is 7,370, which is equivalent to
approximately 3,685 animals serviced (at an average of two services per animal). However,
given an estimated 16,778 breedable female buffaloes in the province (PCC at CLSU, 2011)
and at a rejection rate of approximately 30% at the time of AI, the total AI accomplishment in
2011 would only represent a diffusion rate of around 31%. Likewise, in terms of calving
percentage, the cumulative success rate in 2011 following AI in 2010 is estimated at only
23%. Thus, there is a lot of room for improvement. Also, field data from the PCC at Central
Luzon State University (PCC at CLSU), who oversees the AI activities in Nueva Ecija, would
indicate a wide variation in the individual outputs of technicians. There has not been any
previous research that was focused on assessing the performance or proficiency of AI
technicians for water buffaloes in Nueva Ecija. Likewise, previous evaluation studies about
AI in water buffaloes elsewhere in the country have been very minimal and usually gave
emphasis on its implication on genetics and economics (Bondoc, 1996 and 1999; Sarabia,
1998), on factors influencing its adoption or acceptability among farmers (Porciuncula,

1222
Paderes, and Battad, 2000; Undong, 2004), or on farmers’ perceptions about the PCC’s
crossbreeding program and its field technicians (Antalan, Paderes, Porciuncula, and Trimor,
2006). The current study aims to fill this literature gap with emphasis on analyzing the
possible influencing factors related to the performance of professional village-based AI
technicians (VBAITs) in Nueva Ecija province that PCC has helped develop.

Face-to-face individual interviews with a sample of 36 VBAITs were conducted using
a pre-tested and semi-structured questionnaire. To measure their stock knowledge, a 30-item
quiz on the topics of reproduction and AI were administered individually. Likewise, an
external expert was commisioned to evaluate the practical skills of the VBAITs during their
actual AI activities in the villages using an evaluation instrument. Secondary data were also
collected from the PCC’s Program Monitoring and Evaluation Division and PCC at CLSU to
categorize the VBAITs based on the number of AI services, to calculate the calf drop
percentage, and to estimate the number of breedable animals in the service areas. The
gathered data were consolidated and analyzed using MS Excel and SPSS Version 17.
Descriptive statistics and tabular presentations were generated. Linear correlation and
regression were analyzed as regards the number of AI services by VBAITs in 2011, which
was assumed to be related to selected independent variables that were gathered from the
research instruments, namely (1) quiz scores; (2) 2010 income of VBAITs derived from AI
services; and (3) number of breedable buffaloes in the service areas.

AI performance, socio-demographics, income, service areas, and breedable buffaloes
The number of AI services in water buffaloes by VBAITs in Nueva Ecija has steadily
increased from 1,122 in 2004 (performed by 18 VBAITs) to 5,232 in 2011 (performed by 45
VBAITs). Likewise, the 3,552 AI services conducted by VBAITs in 2010 resulted in 830
calves (or 23% normal preterm) in 2011. The 36 sample VBAITs are more or less equally
distributed over the three performance categories, i.e., “high”, “medium”, and “low” (Table
1). A majority of the VBAITs is middle-aged (average of 38 years) across performance
categories. Likewise, some 45% of the VBAITs reached high school, 42% reached college,
and 10% earned a vocational education. High performing VBAITs earned from 1.75 to 3.15
times more in 2010 and 2.3 times more in 2011 than the medium and low performers (Table
2). Also, the high performers had more service barangays or villages covered in 2011 and
were naturally exposed to more breedable buffaloes compared to the medium and low
performers (Table 3).
Stock Knowledge and Practical Skills
The mean scores of VBAITs for the quiz administered by the research team and for
the practical skills evaluation by an external expert did not vary regardless of performance
categories (Table 4) and were found to be below the passing percentage of 75%. These results
indicate a need for PCC to revisit its training program and re-tool the VBAITs through
appropriate refresher trainings.
Correlation and Regression Analysis
Of the three independent variables that were considered, only the “2010 income
derived from AI services” and the “number of breedable carabaos in the service areas” were
found to have a significant positive linear relationship with the “number of AI services by
VBAITs in 2011” (Table 5). Using standard multiple regression, a significant model emerged
(Table 6a) with both predictors (“2010 income” and “breedable buffaloes”) being significant
(Table 6b). Likewise, “2010 income” has a larger Beta value, i.e., has the largest impact on
the “number of AI services in 2011”. In summary, efforts to increase the AI output in Nueva
Ecija province should focus on how to enhance the income of the VBAITs and how to locate
or cover more breedable animals.
1223
REFERENCES
Antalan Jr, R.V., A.S. Paderes, F.L. Porciuncula and B.P. Trimor. 2006. Towards
Strengthening the Philippine Carabao Center’s (PCC) Crossbreeding Program: An
Evaluation of Outcomes. Central Luzon State University.
Bondoc, O.L. 1996. An Assessment of the Genetic and Economic Impacts of Artificial
Insemination (AI) in Cattle and Carabao Breeding in the Philippines. PCARRD-
DOST Project No. 89-540-21.
Bondoc, O.L. 1999. Genetics and Economics of Artificial Insemination of Cattle and
Carabaos in the Philippines. The Philippine Agricultural Scientist, 82 (2): 213-229.
Philippine Carabao Center at Central Luzon State University. 2011. Summary Report of
Carabao Inventory in Nueva Ecija.
Porciuncula, F.L., A.S. Paderes and L.G. Battad. 2000. Evaluation of the Philippine Carabao
Center Crossbreeding Program. PCARRD Highlights ‘99.
Sarabia, A.S. 1998. Recent developments in the use of artificial insemination in the genetic
improvement of water buffaloes in the Philippines. A country report presented at the
SEM-32-97: Seminar on Livestock Artificial Insemination Program, Mirah Hotel,
Bogor, Jawa Barat, Indonesia, 16-20 February 1998. 17p.
Undong, M.E. 2004. Factors Affecting the Artificial Insemination Program of Carabao in the
Province of Maguindanao. Master of Science Thesis. Central Luzon State University.
Unpublished.
Table 1. Categories of VBAITs based on number of AI services in 2011.
Performance Categories Number %

High Performers (>120 AI services per year) 12 32
Medium Performers (61-119 AI services per year) 13 36
Low Performers (<60 AI services per year) 11 32
Total 36 100
Table 2. Estimated median annual income from AI services by VBAITs.
Median Annual Income from AI services per VBAIT (Php)

Performance Categories
N CY 2010 N CY 2011
High Performers 12 63,000 12 85,000
Medium Performers 11** 36,000 12* 36,500
Low Performers 9** 20,000 9** 37,000
Total 32 33
*One has no data; **Two have no data
Table 3. Number of service barangays, breedable carabaos, and AI services in 2011.
No. of
No. of Service No. of AI
Performance Categories N Breedable
Barangays Covered Services in 2011
Carabaos
High Performers 12 251 5,965 3,266
Medium Performers 13 216 3,554 1,072
Low Performers 11 138 2,736 398
Total 36 4,736
1224
Table 4. Mean quiz scores and practical skills scores by VBAITs.
Mean Quiz Score Overall Mean Score

Performance Categories N N
(%) for Practical Skills (%)
High Performers 12 57 10 67
Medium Performers 13 55 5 68
Low Performers 11 55 3 65
TOTAL 36 56 18* 67
*Only 18 VBAITs were evaluated by the external expert due to scheduling difficulties
Table 5. Correlation analysis.
Quiz 2010 Income from No. of Breedable

Scores AI services Buffaloes
Number of Pearson Correlation, r 0.138 0.616** 0.492**

AI services Sig. (one-tailed), p 0.230 0.000 0.002
in 2011 N 31 31 31
**Correlation is significant at the 0.01 level (one-tailed)
Table 6a. Regression ANOVA (b).
Model Sum of Df Mean F Sig.

Squares Square
1 Regression 12.935 2 6.468 13.348 0.000 (a)

Residual 13.567 28 0.485
Total 26.502 30
a Predictors: (Constant), Number of Breedable Carabaos, 2010 Income from AI
b Dependent Variable: Number of AI services in 2011
Table 6b. Regression coefficients*.
Unstandardized Standardized
Coefficients Coefficients
Variables t Sig.
B Std. Beta
Error
Constant -3.319 1.561 -2.127 0.042
2010 income from AI services 0.538 0.147 0.518 3.669 0.001
Number of breedable buffaloes 0.398 0.163 0.344 2.442 0.021
*Dependent variable: Number of AI services in 2011
1225
Sustainability of Philippine Carabao Center and Primary Cooperative

Partnership in Carabao-Based Enterprise
Wilma T. Del ROSARIOa and Danilo S. VARGASb
Philippine Carabao Center, National Headquarters and Gene Pool, Maharlika Hi-way, Science City
a
of Munoz, 3120, Nueva Ecija, Philippines. bDepartment of Rural Development and Development
Communication, Institute of Graduate Studies, Central Luzon State University, Science City of
Munoz, 3120, Nueva Ecija, Philippines.
*Corresponding email: delrosariowilma@yahoo.com
ABSTRACT
The partnership between the Philippine Carabao Center (PCC) and farmer-cooperatives
in Nueva Ecija is an initiative in the enhancement and sustainability of carabao-based enterprise.
Three farmer-cooperatives (FCs) were studied to describe the condition of partnerships through
experiences, lessons learned including gaps and issues of development. Results reveal that the
respondents invested an amount of PhP 46,897.00 for every head of dairy animal for the
construction of animal shed, establishment of potable drinking water, forage plantation. PCC
provided the 25 head dairy modules including necessary support services such as training,
marketing assistance, milk collection and delivery system, credit assistance, provision of
agricultural inputs, milk processing, animal production management, and strengthening
cooperative development and management including the provision of credit assistance
particularly provident fund, emergency loans and educational loan during cooperative activities.
Systems and policies in all aspects of cooperative were developed and implemented by partner-
cooperatives in support of the Carabao-Based Enterprise (CBE). Officers and hired employees
handled operation and management of dairy enterprises resulting to viable carabao-based
enterprises for economic gains. FCs generated an accumulated 618,697.96 liters supply of milk
from 2000 to 2011 with an average milk production of 4.77 liters in single milking session per
one lactating buffalo and increasing number of dairy buffalo stocks from 100 heads to 580 heads
by dairy cooperatives. All loaned animals were paid, heifer replacement stocks were produced
and farmers owned an average of five to twelve dairy buffaloes by the farmers. Village-based
milk collection centers (VBMCCs) were established serving as collecting hub for milk
production of carabao-dairy farmers. The VBMCC is operated by identified primary
cooperatives enabling it to provide additional employment in the community for the milk
collectors, delivery man, milk processors, managers, bookkeepers involved in daily operations.
Keywords: 25 Dairy Buffalo Module, Partnership, Carabao-Based Enterprise
INTRODUCTION
Local partnership in agricultural development is a vital factor in community
development. This is common in areas where scarce resources and support institutions prevail.
One of the institutions supportive to local partnership is the Philippine Carabao Center (PCC).
The PCC was established and created to support the local carabao industry. This is done through
the Carabao-Based Enterprise (CBE) development program. The CBE was conceived to provide
better nutrition, higher level of income and improved general well-being of rural farming
families. Furthermore, the CBE as a development model embarked in developing Nueva Ecija as

1226
the National Impact Zone for dairy buffalo development project between the PCC and Nueva
Ecija Provincial Government (NEPG).
Through this effort, CBE development envisions the establishment of additional source of
income for dairy farmers while transforming small-scale dairying activities and potentials of
small farmers into cooperative-led viable village-based dairy enterprises; build collaborative
relationships among public and private community organizations to address vital community
needs and improve quality of life of residents of communities and to empower cooperative
members to actively develop processes within their own communities to counter social
challenges, and ultimately enhance quality of their life through sustainable local partnerships.
MATEAILS AND METHODS

The study used three farmer cooperatives with total membership of 59 dairy farmers (15
officers and 44 members) to describe the sustainability of local partnerships through historical
and descriptive research design. The study was guided by the structural-functional theory
approach (Keel, 2012). Structured questionnaires were used to gather pertinent data which were
described and presented through frequency counts, mean, and percentage, analysed and
interpreted to define the sustainability of partnership based on experiences, lessons learned, ideas
and perception of subject cooperatives. Also, these were further supported by primary and
secondary data from PCC and farmer cooperatives’ records.
Roles, functions and contributions of the farmer cooperatives were assessed including
gaps and issues, cooperative resources and institutional support services were identified to post
for conclusive results of PCC and people’s organization partnership such as best practices,
experiences and sustainability indicators towards attainment of workable and viable CBE.

The PCC and Its Partner-Cooperatives
Philippine Carabao Center (PCC) operates as an attached agency to the Department of
Agriculture mandated by Republic Act No. 7307 to conserve, propagate and promote carabaos as
source of draft animal power, meat, milk, and hide to benefit rural farmers. Its mission is to
improve the general well-being of rural farming communities through genetic improvement,
technology development and dissemination, and establishment of carabao-based enterprises,
thus, ensuring higher income and better nutrition. In 1999, PCC identified Nueva Ecija as its
National Impact Zone for Dairy Buffalo Development and integrated carabao as component of
rice-based farming system. The carabao-based enterprise (CBE) partnership used one- module of
25-dairy buffaloes with appropriate production technologies and loaned to qualified cooperative-
partners in propelling local dairy industry in Nueva Ecija (NIZ Annual Report, 2011).
The partner-cooperatives conformed to the requirements as stipulated in Republic Act
9520 like leadership and management, policy development and decision-making of the farmer-
cooperatives. Also, the cooperatives adapted the accounting system and standard chart of
accounts developed by CDA including proper bookkeeping and record keeping of financial
transactions. The cooperatives’ records show that they have annual audited financial statements
from 2002 to 2011 in relation to their financial performances.
Through the efforts of PCC and its partners needs, all support mechanisms and strategies
were provided to sustain partnerships like necessary trainings and services to enable the farmer-
cooperatives to function accordingly. Counterpart contributions necessary in the 25 dairy module
were assessed and highly satisfied by the farmers with an investment of Php 46,897.00 per dairy
1227
animal including animal shed (4 x 4 m2 with concrete floor), supply of drinking water and for
cleaning animals during milking session, Napier plantation measuring 16 m2 , and payment of
mortuary guaranty fund as insurance of loaned animals in case of death or negligence.
Moreover, PCC and its partner-cooperatives developed programs, services, systems and
policies in support to CBE particularly acceptance of new memberships, marketing assistance,
collection and delivery services, credit assistance, provision of agricultural inputs and
processing of milk collected from members, provision of loan services like provident fund,
emergency loans and educational loan, and policy support like prevention of selling dairy
buffaloes, guidelines on milk delivery and collection system to prevent dairy farmers from
selling milk production to other affiliated cooperatives.
Based on these, it was noted that there was an improvement of capital build-up formation,
implementing transparent type of financial management, sufficiency and development of short
and medium term plans for the cooperative and moderately satisfactory on the provision of
appropriate educational training programs for their members; satisfactory ratings on supervision
and management of CBE; regular provision of support services to members, and regular
monitoring and evaluation of the cooperative on CBE activities.
Sustainability Indicators
The sustainability factors are classified into social and economic aspects supported by
environmental dimensions. For social factors, partnerships in CBE are related to enhanced skills,
development of business sense, better sense of belongingness among members, and intensified
self-confidence in managing the enterprise. Also, it created more job opportunities for other
members like milk feeding program and improved internal and external communication within
cooperatives and among members and PCC. Furthermore, it established and attracted stronger
partnerships in CBE in Nueva Ecija from local, national and international organizations like DA-
BAR, PCARRD, and the Korean International Cooperation Agency (KOICA).
On the other hand, the economic factors provided additional source of income especially the sale
of milk produced by the animals. Specifically, the dairy farmers obtained from two dairy animals
(1 lactating and 1 female calf) an additional net income of PhP 51,491.20 for one year or an
average monthly net income of PhP 4,290.93; with four dairy buffaloes (2 lactating, 1 male and 1
female calves) with additional net income of PhP 89,132.40 in one year or an average monthly
net income of PhP 7,427.70; with six heads (3 lactating, 2 female and 1 male calves) with
additional annual net income of PhP 131,623.60 or an average monthly net income of PhP
10,968.63; and farmers with 10 heads (4 lactating, 2 heifers, 2 female and 2 male calves) earned
annual net income of PhP 194,264.80 or an average monthly net income of PhP 16,188.73 and
dairy farms with 12 buffaloes earned additional annual net income of Php220, 955.80 with
average monthly income of PhP 18,412.98.
Other indicators include increase productivity on the volume of milk collected from the
cooperatives, better access to resources, improved and contributed better living conditions and
most especially increased cooperative financial performance like better capitalization, assets and
income generation that led them to acquire other home appliances and even the purchase of
female buffaloes to increase their herd.
In the case of environmental dimensions, there was a positive response on the CBE
particularly on community livelihood opportunities and improve community relationships. It also
changed the agricultural landscape of communities from traditionally non-dairying communities
to more defined and intensified producers and sources of fresh buffalo milk. Also, the
partnerships provided other source of organic fertilizer to the cooperatives for rice and vegetable
production and even the Napier plantation.
1228
Best Practices In The Sustainability Of Partnership

After 12 years of experience, best practices were created and noted in the implementation
of CBE in the rural areas of Nueva Ecija; namely: complementation of resources – PCC and
partner cooperatives share and enhance the use and intensification of technologies and local
resources; cooperation and participation – these are manifested by years of membership
and continuous support of dairy farmers to their respective cooperatives during cooperatives
meetings, payment of annual mortuary funds and capital shares and the alliance of all primary
cooperatives in Nueva Ecija through the Nueva Ecija Federation of Dairy Carabao Cooperatives
(NEFEDCCO) and other government agencies, non-government agencies and international
organization that provided financial and technical assistance to primary cooperatives for
improvement and enhancement of the dairy industry in Nueva Ecija was also attained as a result
of partnership; creation of social organizational patterns within the partner-cooperatives -
the cooperatives had the legal accreditation as primary cooperative duly recognized allowing
them to enter into other partnership with other institutions to avail the programs and services of
cooperatives; and continuous flow of benefits - PCC and partner-cooperatives benefited
from CBE partnership created tangible benefits to members with additional and regular source of
income through sales of daily milk collection from dairy buffaloes and created 155 jobs and
employments for dairy farmers, service providers and residents of the community and resulted to
positively contributed better living conditions, better income, improved nutrition and
development of empowered cooperatives in the rural farming communities of Nueva Ecija and
the dairy farmers..
REFERENCES
Basics Of The Cooperative Model, Module No. 1, Government of New Foundland and Labrador.
New Foundland. Retrieved on July 13, 2012 from
http://www.ibrd.gov.nl.ca/regionaldev/Basics_of__Co_operative.pdf. pp 6.
European Commission. 2004. Directorate-General Regional Policy. Resource Book on PPP Case
Studies. Brussels. Retrieved on July 26, 2012 from
http://ec.europa.eu/regional_policy/sources/docgener/guides/ pppresourcebook.pdf.
Guatam, J. C. 2007. The impact study on farmer's group approach. Adopted in Agricultural
Extension System of Nepal, Retrieved on June 6, 2012 from abtraco@wlink.com.np.
Institutional Support To Agriculture And Rural Institutions, Nigeria Federal Ministry of
Agriculture. Retrieved on July 26, 2012 from
www.academicjournals.org/.../Ugwu%20and%.
Keel, R. O. 2012. Structural Functionalism, URL: http://www.umsl.edu/~keelr/url.html.
Khan, M. A. 2000. Planning for and monitoring of project sustainability: A Guideline on
Concepts, Issues and Tools. Retrieved on June 25, 2012 from
http://www.mande.co.uk/docs/khan.htm.
National Impact Zone, 2011. Annual Report, Philippine Carabao Center, Science City of Muñoz,
Nueva Ecija.
Philippine Carabao Center, 2009. Dairy Production Handbook, Science City of Muñoz, Nueva
Ecija.
Republic Act 7307. Philippine Carabao Act Of 1992, Philippines.
Republic Act 9520. Cooperative Code Of The Philippines, 2008, Article 3,p.1.
1229
A Model of Sustainable Herd Buffalo Farming in Songkhram Wet Land,

Nakhon Phanom
Tanapat SURANARAKUL a *, Mattaneeya SARAKULa and Sornchai KONGSOOKb
a
Department of Animal Science, Faculty of Agriculture and Technology,
Nakhon Phanom University, Nakhon Phanom 48000, Thailand
b
Nakhon Phanom Livestock Research Testing Station, Nakhon Phanom 48000, Thailand
*
Corresponding E-mail : suraball@yahoo.com
ABSTRACT
The researchers conducted a qualitative and descriptive analysis research on the lesson
learned visualizing on herd buffalo farming of farmers in Song Khram Basin at Nawa District, Sri
Songkhram District and Tha Utane District, Nakhon Phanom. It was found that each household
raised 7.68 buffaloes by average and income from buffalo and buffalo dunk selling was 21,650 baht
per year per household. Their strategy for buffalo conservation and increasing were: 1) setting
buffalo farmer club in each Sub-district, 2) the club making an agreement to sell male buffalo or
sick buffalo or spent female buffalo only, 3) the availability of waters and community forest for
buffalo raising, and 4) joining of a member from each family to raise the club’s buffalo under the
policy of interdependence, Sufficiency Economy Philosophy, conservation-oriented management
and sustainability of the club.
Keywords: buffalo, Songkhram wet land, sustainable, Nakhon Phanom
INTRODUCTION
Most of buffalo farmers are small farmers because it is easier to raise a small flock of
buffalo which grazed some grass and weeds available in common areas of villages. The buffalo can
live on less feed quality than cattle’s feed quality. Raising buffalo need not use techniques and
complicated technology. In Thailand, the population of buffalo in 2010 was 1,234,179 heads while
in 2011 it increased was 43,293 heads (+3.64%) due to the implementation of a royal project,
“Buffalo Bank for Farmers” to helped poor farmers. The largest number of buffaloes (901,630) or
73.05% of them was raised in the northeast of Thailand. The buffalo number was increased from the
number in 2010 (23,280 buffaloes). The number of buffaloes raised by farmers in the country in
descending order were: the northeast, the central, the north and the south. Nakhon Phanom had the
eight largest numbers of buffalo in Thailand, 62,802 buffaloes. The proportion of buffalo per
household was 4.77. The buffaloes raised by farmers in districts of the province in descending order
were: Muang, Nawa, Sri Songkhram, and Tha Utane (Department of Livestock Development,
2010).

According to cluster sampling, this field study was conducted in Nawa District, Sri
Songkhram District and Tha Utane District of Nakhon Phanom which represented buffalo farmers
of Songkhram Wetland.
The researchers employed participation observation and interview schedule with closed-
ended and open-ended questions to collect data. It was a face-to-face interview. The obtained
quantitative data descriptive statistics and qualitative data were analyzed by percentage, arithmetic
means.

It was found that farmers in Songkhram wetland set a buffalo raising group in each
community. There were 18 buffalo raising groups and the president of each group acted as the
group representative to participate government-held workshops. The president of each group then
1230
brought his obtained knowledge from workshop to transfer to group’s members. The presidents
were trained how to prepare buffalo’s vaccine, artificial insemination, primary treatment for sick
buffaloes etc. This was to create cooperation for buffalo conservation and buffalo number
development.
There were 26,558 heads raised by 4,652 households or 5.71 heads per household ranging
from 2- 43 heads. Farmers bred their buffaloes by sires; the proportion of adult male buffalo per
adult female buffalo was 1 : 10.51 while the proportion of male buffalo calves to female buffalo
calves was 1 : 1.64. The farmers herded their buffaloes to buffalo’s grazing lands. There were 20-30
heads per herd. Farmers helped each other to herd the buffaloes, to discuss some topics and to have
lunch. This made the raising more convenient and easier.
The buffaloes were allowed to graze grass from 9.00-11.00 A.M. then they drank water from
water sources such as Songkhram stream, reservoirs, water ways, ponds etc. The buffaloes laid in
muddy place from 11.00 A.M. to 12.30 P.M. The buffaloes began to graze grass again from 12.30-
15.00 P.M. then they drunk some water. The farmers herded buffaloes home and the buffaloes
might graze some grass on their way home.
The buffaloes were raised in paddy fields after rice was harvested or during December to
July of each year. Some farmers used their field to do out- of- seasoned rice planting during
November to
March of each year so 52.46% of farmers raised their buffaloes at common areas, around
reservoirs, near roads where natural grass was grown. Forty-seven point five four percent of the
farmers raised their buffaloes in the areas and plant Brachiaria ruziziensis and violet Guinea grass
for their buffalo feed while 68.85% of the farmers supplemented the buffaloes’ feed with straw and
mineral bars.
For buffalo breeding, 98.36% of the farmers let 3.5 to 4 year-old adult male buffalo to
breed adult female buffaloes in the herd with the ratio of adult male buffalo to adult female buffalo
was 1: 20-25. Some farmers did not have their adult male buffalo so they used neighbor’s adult
male buffalo to breed their adult female buffaloes. Sixty-five point five seven percent of adult
female buffaloes were ready for breeding during October to December after they had born their
offspring about 60-90 days. For adult female buffaloes at estrus interval with 82% of conception
rate, they bore their offspring during September to December. The carving rate of the adult female
buffaloes was 92%. Most of the farmers (95.08%) vaccinated their buffaloes against foot and mouth
disease, 70.49% of the farmers vaccinated the buffalo twice a year and 80.33% of the farmers gave
their buffaloes some vermifuges once a year. The cost of buffalo rising was 137 baht per head per
year. Thirty one point one five percent of the farmers used buffaloes to plough their fields. Farmers
used buffalo dunk for their paddy field, vegetable gardens, rubber farms, making organic compose,
1231
and watermelon planting (54.10%) while 45.90% of the farmers sold the dunk or swapped the dunk
for rice straw.
Table 1. Areas using for buffalo raising in Song Khram wet land.
Month
September
November
December
February
January
October
Activities
August
March
April
June
May
July
1) Public spaces
2) Near river areas
3) Community forest
4) Fields after in-
season rice planting
5) Fields after out-
of-season rice
planting
CONCLUSIONS
To conserve buffalo in Songkhram wetland, to sustain the buffalo population and to increase
the buffalo population, they needed: 1) waterbody, 2) public areas with grass for buffalo raising and
3) buffalo raising club intended to conserve buffalo.
ACKNOWLEDGEMENTS
The researcher’s worlds like to thank National Research Council of Thailand (NRCT) for
financial support in 2010.
REFERENCES
Department of Livestock Development. 2010. Buffalo Classified by Number and Farmer.
Information and Statistics Group, Information Center, Department of Livestock
Development. (March 10, 2010). Available from: URL:http://www.dld.go.th/ict/
Department of Livestock Development. 2010. Buffalo for Ploughing Bank Recovery Project under
Her Majesty’s Project. Livestock Development and Technology Transfer Office.
Department of Livestock Development.
Information Center, Department of Livestock Development. The Information of Buffalo and
Buffalo Farmer Statistics in 2011. Information and Statistics Group, Information Center,
Department of Livestock Development.
Inthamongkol, J. 2005. Thai Buffalo for Sustainable Development. National
Agriculture Cooperative Club Printing. Co. Organic Livestock Development Project,
Department of Livestock Development, Bangkok.
Janthalakkhana, J. 1991. Research on Buffalo from the Past to the Present. Buffalo Magazine, 14th
Year, Vol. 1 January-February. Aksorn Siam Printing, Bangkok.
Jungsisipornpakorn, P.S., P.C. Choosep and C. Chumchuen. 2004. Buffalo’s Manure Passing
Study. Surat Thani Livestock Breeding and Research Center. Punpin District, Surat Thani.
12 Pages
Sajjaphan, B., T. Sukyoi and P. Wuthipranee. 2004. Native Buffalo Conservation and Breeding
Development for Sustainable Uses. Department of Livestock Development, Livestock
Breeding and Research Division, Phayathai Road, Rajadevi District, Bangkok.
1232
Contextualizing the GatasngKalabaw Festival in Support to the

Carabao-based Enterprise in Nueva Ecija, Philippines
Marilou A. SANTOSa and Marlowe U. AQUINOb
a
Community Development Officer, Philippine Carabao Center, National Headquarters and Gene Pool,
Science City of Munoz, Nueva Ecija, Philippines
Corresponding e-mail address: loulimry@yahoo.com
b
NIZ Coordinator, Philippine Carabao Center, National Headquarters and Gene Pool, Science City of
Munoz, Nueva Ecija, Philippines
*Corresponding e-mail address: marloweaquino@yahoo.com
ABSTRACT
Festivals are part of people and community culture. They define the existence and development
of communities especially on the physical landscape, activities and events. The carabao dairy, locally
termed as GatasangKalabaw Festival (GKF) conducted annually in the province of Nueva Ecija is an
interesting dimension of dynamism and agricultural development especially the carabao-based
enterprise. Its evolution is an interesting social scientific inquiry that combines culture, community
development and economic activity. The study described the process of evolution and development of
GKF; identified and assessed the dairy stakeholders’ involvement; and extent of contribution to the
Carabao-based Dairy Enterprise to the overall community development through quantitative and
qualitative methodologies using purposive sampling procedure supported by key informant interview,
document analysis using historical interpretation and participants observation. The research participants
are composed of the NEFEDCCO and 11 primary cooperatives from Rizal, Llanera, Science City of
Munoz, San Jose City, and Talavera including the primary stakeholders and representatives from PCC,
DTI, CDA, NEPG, and LGU’s. The GKF was evaluated over a period of five-year implementation
from 2007 to 2011 as a mechanism to improve the process of implementing local development
programs anchored on people and community capacity particularly in planning, implementation,
monitoring and evaluation activities. It also provides an avenue to capture the interactions and
relationships of government organizations and non-government organizations, the dairy convergence
system aiming to sustain the proliferation of dairy enterprises as a way of improving farmers lives
better including generation of additional employment and income. Furthermore, it provided an avenue
for partnership that intensify and enhance the carabao-based enterprise in the province as the Dairy
Capital of the Philippines and strengthening of partnerships of the different stakeholders through
knowledge sharing on knowledge on cooperative management, promotion of the dairy product,
enterprise and business development, community and cooperative camaraderie and employment
generation.
Keywords: Gatasang Kalabaw Festival, community development, carabao-based enterprise
INTRODUCTION
The Philippine Carabao Center (PCC), an attached agency of the Department of Agriculture is
mandated to promote the development of the carabao not just for draft but as source of meat, milk and
hide (Republic Act 7307, 1992). Through the Carabao Development Program (CDP), PCC is
continuously organizing its efforts to increase the genetic potential of the native carabao results to
development of the buffalo and buffalo-based and related enterprises aimed to increase income and
improve the nutritional status of the farming communities.PCC identified Nueva Ecija as its National

1233
Impact Zones (NIZ) which serves as the frontline in implementing national carabao development
program, making carabao-based dairy enterprise productive, profitable and sustainable.
Inspired by the One-Town-One-Product (OTOP) development strategy, local chief executives
of the province took the lead and transformed the carabao-dairy enterprise in Nueva Ecija as its OTOP.
The carabao dairy became the commodity associated with the province creating a ripple effect and
drumbeat the efforts of PCC and the local government to make Nueva Ecija the hub of carabao
dairying. Nueva Ecija Dairy Convergence System collectively worked together and prioritize the dairy
industry development as one of the major projects; conceptualize promotional activities for the industry
which resulted to the evolution of the First Gatas ng Kalabaw Festival (GKF) in 2007. GKF expressed
several developments, showed improvements and innovations which triggered significant and relevant
aspect in the lives of the carabao-based farmers, the efforts of the stakeholders and to the Carabao-
based Dairy Enterprise.

Research participants were purposively selected based on their direct involvement in the
implementation of GKF; as active recipients of the program; and their active contribution and
collaborative effort in the dairy convergence system. Pertinent data were collected using interview
schedule to the member of the Dairy Convergence System, the primary cooperatives and the different
government agencies; document analysis through historical interpretation and secondary report analysis
on GKF reports and accomplishments from different stakeholders. In support to this, participant
observations and documentation from various GKF and related activities were further analyzed and
presented in order to have a more comprehensive presentation and discussions. Also, the researcher
observed phenomenon of interests in the study area to draw information which was not obtainable from
other methods. What had been observed in the whole environment was related to the physical setting
and landscape within which the event took place and gained close and intimate familiarity.

Gatas ng Kalabaw Festival was a collaborative effort from the different cooperating agencies
supporting and promoting the dairy industry in the province such as Philippine Carabao Center (PCC),
Department of Trade and Industry (DTI), Department of Agrarian Reform (DAR), Nueva Ecija
Provincial Government (NEPG), Local Government Units of Llanera, San Jose City, Science City of
Muñoz and Talavera, Cooperative Development Authority (CDA), Nueva Ecija Federation of Dairy
Carabao Cooperative (NEFEDCCO) and its primary cooperative members, Nueva Ecija Small and
Medium Enterprise Development Council (NESMEDC) and Likhang Novo Ecijano Marketing
Association (LINEMA) and private sectors. This festival started in 2007, initiative of the Department
of Trade and Industry(DTI) under the Comprehensive Agrarian Reform Program (CARP) support
services and the One Town One Product marketing program.GKF strongly supports the direction of the
provincial government to promote and support dairy development in the province because of the
success of dairy farmers in dairying enterprise who attribute improvements not only in carabao
production but also to augment dairy farmers status of economics condition. The objective of the
festival is to promote the province as the dairy capital and first of its kind recognizing the role of the
dairy industry in the economy of the province (MO Manzano 2009) and served as promotional
activities to highlight the best dairy processed products in the province and the launching of the milk
feeding program, celebrated in consonance with Nutrition Month Celebration.
Another highlight of GKF was the first Dairy Stakeholder Forum entitled “Onwards Stronger
Convergence for Nueva Ecija’s Dairy Buffalo Entrepreneurship Program” participated with all the
member of the convergence system, that harmonize efforts of various stakeholders collaboratively,
intensify commitment support and identify the needs of the industry in the value chain and make
strategies to address such needs. Dairy Development Plan 2009-2010 was realized for better programs
1234
implementations were work and resource complementation strategy is best used and proven to be
effective ways of implementing programs and projects, incorporated in the Medium Term
Development Plan of the Dairy Industry in Nueva Ecija.
The fourth and fifth year (2010, 2011) festival create remarkable to the dairy industry because it
was the first localization of the Gatas ng Kalabaw Festival, a member of the second level stakeholder,
LGU Llanera and LGU Talavera sponsored the GKF and symbolized the start of a more broaden
celebration and localization. Local government take the lead, recognized that they have to take an
active role in securing the economic well-being of their constituents and provide an environment that is
conducive to growth and to fulfill their alternative function as an economic entity that play a crucial
role in the national economic strategy through the dairy industry as their municipal OTOP. The hosts
provide their full manpower and resources in tandem with cooperating agencies as they also poured
their financial and manpower to make this festival a big success, making it extravagant most expensive
GKF having the most numbered of guests and participants.
Activities in the five years celebration of Gatas ng Kalabaw Festival increases on its fourth and
fifth year implementation due to increase of budget, dairy farmers and local government unit
participated in the festival tremendously improved. This signifies full cooperation and support to the
dairy industry because they strongly agree that GKF support to the Carabao Based Dairy Enterprise,
increase of farmers’ income, nutrition of the community, employment generation, creation of market
outlet, promotion of the product, community camaraderie, promotion of Nueva Ecija as dairy capital of
the Philippines, promote tourism in the province and promote dairy being the one town one product.
RECOMMENDATION
1.Continue the localization of the festival and active involvement of the dairy cooperative, to implies
the attitude of assertiveness, self-reliance, and confidence that the local government and the local
community of the dairy farmers knows better where its interest lie and how to best pursue them. This
will allow them to administer their own affairs freely and pursue the development of their selected
industry.
2. Ensure the participation and empowerment all primary and second level stakeholders of the dairy
industry in development decision-making and processes. There should be overall management and
coordination among stakeholders to harmonize dairy development. Exercise institutional viability,
recognizing that sustainable development of GKF festival is shared; collective responsibility calls
institutional structures that build the spirit of solidarity, partnership and convergence among the
different stakeholders.
3. Additional funds are also in advantage, to suffice adequate funds needed for effective and efficient
promotion of the event and the dairy industry. Provincial and local government must see to it that they
are the one patronizing first their local industry. If the whole province supported this industry, it is
possible to gain support from the national level, through policy formulation that will not only support
and development the dairy industry but also protect the local economy of the province
REFERENCES
Aquino, M.U. 2008. Carabao Dairy. Mainstreaming the carabao dairy products for
community dairy enterprise and nutrition program retrieved from
http://www.bar.gov.ph/bardigest/2008/octdec08_carabao_dairy.asp on December 2, 2011.
Battad, L.G. 2010. Nueva Ecija Dairy Convergence System.
Banu, S.A. 1996. Impact of the Philrice Training Program on Rice Production and other
Agricultural Support Services for Farmer-Borrowers of ATFI in Cabiao, Nueva Ecija.
Philippines, Institute of Graduate Studies, CLSU, Munoz, Nueva Ecija.
Carbonel, R. and A. M. Lyndon. 2011. 7th Nuang Fest in San Agustin brings out
novel amusement, Philippine Carabao Center Newsletter.
1235
Cooperative Development Authority Mandate, Vision, Mission. 2011. Retrieved from

http://125.5.51.147/website/html/mission.html#. October 11, 2011.
Dar, Carlito, March 1, 2012. Article entitled PANAGBENGA shows community convergence at
work, Ugnayan.
Department of Agrarian Reform Vision, Mission, Mandate, Programs and Services. 2011.
Retrieved from http://www.dar.gov.ph/misvis.html. October 10, 2011
Department of Trade and Industry One Town One Product (OTOP Philippines). 2011. Retrieved
from http://www.dti.gov.ph/dti/index.php?p=442. November 3, 2011.
Department of Trade and Industry Small and Medium Enterprise Development Plan 2004-2010
Department of Trade and Industry Vision, Mission, Programs and Services. 2011. Retrieved
from http://www.dti.gov.ph/dti/index.php?p=134. October 6, 2011.
Family Income and Expenditure Survey (FIES). 2000. Integrated Survey of Households Bulletin.
National Statistics Office. Manila. 1 (98)
Fleicher, D. and Aliza. May 2003. Local Festivals and Tourism Promotion: The role of public
assistance and visitor expenditures.
Florencio, C.A. 1997. Dietary Guideline in Asia Pacific-Philippines. ASEAN. New Zealand
IILP project 5.
Goyagoy, J. G. 2011. Kneeling of Carabaos Regale, Cajole Throng in Bulacan. PCC
Newsletter.
IIRR, LGSP, SANREM CRSP/Southeast Asia. 2001. Enhancing Participation in Local
Governance: Experience from the Philippines, International Institute of
Rural Reconstruction, Philippines-Canada Local Government Support Program and
SANREM CRSP/Southeast Asia. 197p.
Kotler et. all. 2008. Principles of Marketing
National Impact Zone Milestone: The Dawning of Carabao Industry, March 2007
Nigro, F. and Lloyd. 1992. Modern Public Administration, seventh edition
Nueva Ecija Development Plan 2010-2015, Onwards Stronger Convergence for Nueva Ecija’s
Dairy Buffalo Entrepreneurship Program
Nueva Ecija Medium Term Development Plan 2011-2013
Philippine Carabao Center Vision, Mission, Programs and Services.2011. Retrieved from
http://www.pcc.gov.ph/. October 14, 2011.
Salud, J. 2010, Article on Carabao’s Milk
Selden, P.H. 1997. Sales Process Engineering
Tim, U. October 2005. Partnership in Development Practice: Evidence from multi-
stakeholder ICT4D partnership practice in Africa. UNESCO Publications for the World
Summit on the Information Society.
Vargas D. S. and M.A. Santos. 2010. NEFEDCCO Organizational Assessment. Unpublished PCC
Document. Science City of Munoz, Nueva Ecija
Wikipedia Encyclopedia. 1997. Household Size of Filipino Families
1236
Economics of Raising Calves on Milk and/or Milk Replacer in Nili-Ravi Buffaloes

Ahsan-ul-HAQUEb, Muhammad ABDULLAHa*, Muhammad SAADULLAHa , Zeeshan
Muhammad IQBALa, Jalees Ahmad BHATTIa, Nisar AHMADa, Dilshad RASHEEDa and Imran
MOHSINa
a
Department of Livestock Production, University of Veterinary and Animal Sciences, Lahore 54000,
Pakistan
b
Buffalo Research Institute, Pattoki, Distt. Kasur, Pakistan
*Corresponding Email: mabdullah@uvas.edu.pk
ABSTRACT
Water buffalo (Bubalus bubalis) is the main stay of Pakistan dairy industry, but buffalo calves
are mostly deprived of feeding whole milk in the areas where the animal milk is sold in the market to
fetch higher price. The objective of this experiment was to compare the economics of buffalo calves
reared on whole milk and/or milk replacer. Thirty six Nili Ravi buffalo calves were randomly assigned
to treatments A(whole milk), B(50% whole milk and 50% milk replacer) and C(milk replacer) upto
120 days. The data regarding average daily dry matter intake, average daily gain, feed cost and cost per
kg weight gain were recorded. Average daily dry matter intake of treatment A, B and C was
443.68±73.15, 419.64±60.28 and 386.14±48.29 g, respectively. The average daily gain of treatment A,
B and C was 457.38±110.13, 426.67±78.70 and 362.22±107.83 g, respectively. There was a significant
(P<0.05) difference in the dry matter intake and weight gain of treatment A and C while there was a
non-significant (P>0.05) difference in the weight gain of treatment A&B and B&C. The daily cost for
feeding dry matter (from milk and/or milk replacer) in treatment A, B and C was Rs. 186.35±30.72,
155.06±22.27 and 117.78±14.73, respectively. There was a significant difference in the cost per kg
gain of treatment A and C while there was non-significant difference between treatments A&B and
B&C, respectively. It can be concluded from the present study that it is economical to raise Nili Ravi
calves on blend of milk and milk replacer (50:50) rather than to rise on whole milk or milk replacer
only.
Keywords: whole milk, milk replacer, feed cost, buffalo calves, weight gain
INTRODUCTION
Buffalo calves are conventionally reared, due to the increasing price of whole milk day by day,
calf rearing on whole milk is becoming very costly. Dairy farmer prefer to sell milk in commercial
market instead of feeding to calves (Azim et al. 2011). Calf rearing on milk replacer or on whole milk
alone is uneconomical because of narrow margin of this system. Furthermore the method and amount
of milk feeding to dairy calves can affect behavioral, immunological, physiological, and economic
traits (Khan et al., 2007). The amount of milk feeding to dairy calves during pre-weaning period not
only influences dry feed intake, health and growth but also effect gut maturity, mammary development
and capability of milk production. Dairy farmers are in search of new strategies to improve calf
performance by decreasing labor cost and improved health. In modern feeding system, calves are
separated from their dam and fed a restricted amount of colostrums. These calves are then offered milk
though buckets or nipples (Khan et al., 2007). Milk feeding through nipples allows calves to show
natural suckling behavior. The daily recommended amount of milk for dairy calves is 10% of their
body weight (Khan et al., 2007). It is essential to meet the nutritional requirements of dairy breed
calves during the first week of life for their normal biological development. This will ensure more
1237
weight gain and health in later life (Khan et al., 2011). Previous studies shows that increasing the
energy and protein intake of calves during preweaning life, increases rapid body size, body weight,
early mammary growth and improves milk producing ability. Information on calf growth and rearing
cost are very rear. Several studies predict that milk replacer is beneficial for calf raiser and dairy
producer, including ease and flexibility of storage, good calf performance, disease control and
economic (wagenaar and Langhout, 2007). The price of whole milk is increasing day by day and
farmers are offering small quantity of milk to their calves and major portion of milk is sold in the
market for human consumption. The rearing of calves on milk replacer saves more milk for human
consumption and sells to protect economical considerations (BAMN, 2002).
The study was conducted with the object of evaluating the effect of whole milk, 50% whole milk+50%
milk replacer and milk replacer on calf growth, performance and its economic feasibility.

Thirty six Nili Ravi buffalo calves born during October-November 2011 are selected from
Livestock Experiment Station Bhunikey, Pattoki, were separated from their mothers within 3 hours
after their birth, weighed, and moved into individual pens (1 X 2 m) bedded with wheat straw. In these
pens these animals were offered colostrums (10% of body weight) through bucket feeder for the first
three days. Calf starter ration and water offered in bowels fixed in each cage. The above selected thirty
six calves were randomly divided into three experimental treatments (A, B and C) under completely
randomized design. There were twelve replicates in each treatment. The animals of treatment A were
fed whole milk @ 10% of their body weight while treatment B were given 50% whole milk : 50% milk
replacer @ 10% of their body weight the animals belonging treatment C were offered milk replacer @
10% of their body weight, all treatments were offered calf starter ad lib, green fodder ad lib and
antibiotic in milk.
All the calves received liquid diet for the first 8 weeks at 10% of their body weight and then 1
% decline every week for the other eight weeks. The duration of experiment was 120 days. During the
whole trial feeding frequency was twice daily. At the age of one month all the calves were vaccinated
against FMD and HS by using UVAS, FMD & HS vaccine. The deworming of calves was done on 25
day of age. Feed intake and refusal was recorded on daily basis to calculate the individually intake of
calf through the trial period.
Sampling parameters: The weighing of all the animals was done at the start of the experiment and then
weighted weekly to calculate the weekly weight gain of calves. Dry matter of milk, milk + milk
replacer and milk replacer, calf starter and green fodder was calculated on fortnightly by using official
methods of AOAC (2000). The cost of milk, milk + milk replacer, milk replacer, calf starter and green
fodder was calculated on dry matter basis in Pak Rs./Kg. The cost for production of per Kg live weight
was also calculated in Pak Rupees.
Statistical analysis: Data on milk intake (dry matter basis) and average daily weight gain are expressed
as mean ± S.D. Data was collected and analyzed using ANOVA technique under Completely
Randomized Design by using SAS 9.1. Differences among treatment means was tested through LSD
test (Steel et al, 1997).

Table 1 shows that the fresh milk is costly, Least square mean of initial body weight on the
start of experiment was non-significant (P>0.05) but there is significant difference (P<0.05) in the
body weight gain (105 days) of calves raised on whole milk (47.94a±11.52) and milk replacer
1238
(38.03b±4.38). There was non-significant (P>0.05) difference in weight gain of calves raised on whole
milk & Whole milk + milk replacer. There was also non-significant (P>0.05) difference in the weight
gain of calves raised on Whole milk + milk replacer and milk replacer.
Least square mean of liquid feed intake is non-significant (P>0.05) contradicted form previous
56 days research (Lee et al., 2008) which represents 4.01 Liter of milk replacer/day per calf. This
contradiction may be due to duration of trial period. Least square mean for average daily dry matter
intake is significantly (P<0.05) different in whole milk group and milk replacer group. This difference
may be due to the difference in the presence of dry matter contents per liter of whole milk and milk
replacer. Least square mean for average daily dry matter intake cost was significantly (P<0.05) higher
in whole milk (186.35a±30.72) group then 50% whole milk+50% milk replacer (155.06b±22.27) and
milk replacer group (117.78c±14.73). This difference is due to the higher cost for fresh milk then milk
replacer. Least square mean for average daily weight gain was significantly (P<0.05) higher in whole
milk group (457.38a±110.12) then replacer group (362.22b±107.82). These finding are in line with
Shamay et al., 2005. Who reported that calves gain higher if given free access to whole milk then milk
replacer. There was non-significant (P>0.05) difference in the average daily gain of calves raised on
Treatment A (whole milk) & B (mixed milk group) and B (mixed milk group) & C (milk replacer
group). Least square mean for cost of production for per Kg weight was significantly (P<0.05) higher
in whole milk group then milk replacer group (Table 2). The pre-weaning cost of whole milk and milk
replacer for rearing of calves under same managerial conditions was in agree with the study of BAMN
(2002) and El-Jack and Ahmed, 2012, who reported that the cost for rearing of calves on milk replacer
is less than the cost for rearing of calve on whole milk There was non-significant difference in the least
square mean of cost per kg weight gain in whole milk and mix milk group. There was also non-
significant difference in the least square mean of cost per kg weight gain in mix milk group and milk
replacer group. This cost for obtaining per kg weight was higher than the cost calculated by Azim et
al., 2011, who reported that feed cost/Kg of weight gain was 211.2 PKR. The contradiction in feed
cost/kg weight gain may be due to that Azim et al., 2011 has offered milk replacer @ 8% of body
weight for the period of 90 days.
IMPLICATIONS
Rearing of dairy calves with whole milk gave better calf performance then with milk replacer
in terms of calf growth. The cost of milk replacer is cheaper but results in less weight gain. The growth
rate of calves on whole milk and combination of 50% whole milk and 50% milk replacer was non-
significant regarding growth performance. So, combination of whole milk and milk replacer can replac
whole milk without effecting performance of calves during pre-weaning period and it is economically
feasible then whole milk.
REFERENCES
Association of Official Analytical Chemists (AOAC) (2000). Official Methods of Analysis of
Association of Analytical Chemists international, 17th ed. Horwitz, W. (Ed). Vol I and
II. AOAC International Publs, Maryland USA. Ch. 45: 12 - 20.
Azim, A., A. G. Khan, M. I. Anjum and M. A. Nadeem. 2011. Effect of milk replacer and early
weaning diets on growth performance of buffalo calves during weaning period. Pak Vet J,
31(1): 23-26.
BAMN Bovine Alliance on Management and Nutrition. 2002. A guide to modern calf milk replacer.
Contact information: AFIA, Jim Rydell, and 1501 Wilson Blvd., Suite 1100. Arlington,
1239
El-Jack, A. A. and K. E. E. Ahmed. 2012. The effects of using milk replacer on body growth and its
economic feasibility in feeding dairy calves. Agri.. Sci. Res. J. 2(4): 183-188
Khan, M. A., D. M. Weary, M. A. G. Von Keyserlingk. 2011. Invited review: effects of milk ration on
solid feed intake, weaning and performance in dairy heifers. J. Dairy Sci., 94: 1071–1081.
Khan, M. A., H. J. Lee, W. S. Lee, H. S. Kim, S. B. Kim, K. S. Ki, J. K. Ha, H. G. Lee and Y. J. Choi.
2007. Pre- and post-weaning performance of Holstein female calves fed milk through step-
down and conventional methods. J. Dairy Sci. 90: 876-885
Lee, H. J., M. A. Khan, W. S. Lee, H. S. Kim, K. S. Ki, S. J. Kang, T. Y. Hur, M. S. Khan and Y. J.
Choi.2008. Growth, blood metabolites, and health of Holstein calves fed milk replacer
containing different amounts of energy and protein. Asian-Aust. J. Anim. Sci. 21(2): 198-203.
Shamay, A., D. Werner, U. Moallem, H. Barash and I. Bruckental. 2005. Effect of nursing
management and skeletal size at weaning on puberty, skeletal growth rate, and milk production
during first lactation of dairy heifers. J. Dairy Sci. 88:1460–1469.
Steel, R. G. D., J. H. Torrie, and D. A. Dickey. 1997. Principles and procedures of statistics: A
Biometrical Approach. 3rd Ed. McGraw Hill Book Co. Inc., New York, USA. pp. 481.
Wagenaar, J. P. and J. Langhout. 2007. Practical implications of increasing ‘natural living’ through
suckling systems in organic dairy calf rearing. Nordic J. Afri. Stud. 54(4): 375-386
The data regarding cost of feed stuff and weight is shown in table 1.
Table: 1 Least square mean for Cost of feed stuff and animal weight:
Treatments
Parameters A C
B
DM in milk/lit (gm) 159.5 152.4 149.5
Milk cost on DM basis (PKR/Kg) 420 362 290
Concentrate cost on DM basis (PKR./Kg) 30.5 30.5 30.5
GF cost on DM basis (PKR./Kg) 12.5 12.5 12.5
Initial B. WT (Kg) day 0 33.62a±5.065 32.81a±5.16 33.84a±4.38
B. WT gain(Kg) day 105 47.94a±11.52 44.68ab±8.35 38.03b±4.38
DM= dry matter, GF= green fodder, B. WT= Body weight, Treatment A= whole milk, B=(50% WM+ 50%MR), C= MR
Table: 2 Least square mean of dry matter based feed intake and cost of production
Parameters Treatment A Treatment B Treatment C
a a
AVG. daily Liquid Feed (L) 2.95 ±0.48 2.92 ±0.42 2.74a±0.34
AVG. daily DM (gm) 443.68a± 73.15 419.64ab±60.28 386.15b±48.29
AVG. daily milk DM cost PKR. 186.35a±30.72 155.06b±22.27 117.78c±14.73
AVG. daily gain (gm) 457.38a±110.12 426.67ab±78.70 362.22b±107.82
Cost per Kg gain PKR 422.72a±70.66 369.73ab±65.70 352.97b±97.49
AVG= Average, L= liter, DM= dry matter, Kg= Kilogram, Treatment A= whole milk, B=(50% WM+ 50%MR), C= MR
1240
Biohydrogen Production from Buffalo Manure Codigested with Agroindustrial

By-products in an Anaerobic Reactor
Serafino CONCETTIa, Antonella CHIARIOTTIa, Chiara PATRIARCAa, Antonella

MARONEa, Cristiano VARRONEb, Giacomo CONTÒa, Massimo CALÌa and Antonella
SIGNORINIb
a
Animal Production Research Centre (PCM), Agriculture Research Council (CRA) 00015,
Monterotondo, Roma, Italy
b
ENEA-UTRINN-BIO, via Anguillarese 301, 00123 Roma (RM) Italy.
Corresponding email: antonella.chiariotti@entecra.it
ABSTRACT
Most on-farm biogas plants in Europe use animal waste and co-substrates for biogas
production (CH4 and CO2 mixture). However combined hydrogen and methane production in a two-
stage process is a concept which has been developed in recent years and it is more promising from
an energy perspective. The aim of this research was to explore hydrogen production from buffalo
manure alone and in co-digestion with suitable feedstocks (low protein cheese whey and crude
glycerol from biodiesel manufacturing). Experiments were carried out in batch reactor at 37 °C
using a microbial consortia as inoculum. In a first set of batch trials the sterilization effect on
hydrogen production from each individual substrate was explored. Results showed that sterilization
increased hydrogen production yield (mL H2/g VS) in all substrates, even reaching a three times
higher yield in the case of buffalo manure. In a second set of experiments hydrogen production
using different mixing ratios of sterilized substrates were investigated. Results showed that the
hydrogen production yield from manure codigested with 10% glycerol or 10% of low protein
cheese whey (LPCW), was increased from 37.7 mL H2/g VS to 47.2 and 65.4, respectively.
Moreover the co-digestion decreased H2 production time from 114 hrs to 24 hrs. The yields further
increased up to 116 mL H2/g VS when a combination of 70% manure, 20% LPCW and 10%
glycerol was used. During the experiments CH4 was not detected. Buffalo manure, when codigested
with LPCW and glycerol gave interesting yield together with an optimum buffering capacity
avoiding the use of any external buffer/pH control system.
Keywords: biohydrogen, anaerobic digestion, buffalo manure, cheese whey, glycerol
INTRODUCTION
Combined hydrogen and methane production in a two-stage process is a concept which has
been developed in recent years and it is more promising from an energy perspective (Kyazze et al.,
2007; Liu et al., 2006; Ueno et al., 2007). It is similar to the traditional two-phase process that
separates hydrolysis/acidogenesis and methanogenesis, and optimizes each process separately,
leading to a larger overall reaction rate and biogas yield (Fox and Pohland, 1994). The main
difference is that hydrogen is retrieved in the first stage of the two-stage process for hydrogen and
methane production. Moreover hydrogen is considered one of the most promising candidates as a
substitute for fossil fuel, because it is clean, renewable and has a high energy yield. Biological H2
production using fermentative bacteria is an environmental and energy saving process. The major
advantages of this process are low cost, high energy efficiency, and process simplicity compared to
other waste treatments. Buffalo manure contains complex organics, such as polysaccharides,
proteins, amino acids and fatty acid (Hawkes et al., 2002) and a high chemical oxygen demand
(COD). H2 production from manure is influenced by various factors such as microbial inocula,
pretreatment, pH, temperature (Fang and Liu, 2002; Hussy et al., 2003; Kim et al., 2006). Several
pretreatments have been employed, but little information is available on pH and T° influence on H 2
production from cattle manure and none on buffalo manure. The aim of the present study is to
investigate the feasibility of biohydrogen production by dark fermentation using buffalo manure
1241
alone and in co-digestion with agricultural and food processing waste, namely: de-proteinized
cheese whey (LPWC) and crude glycerol.

Bio-hydrogen production was studied in batch reactors (125 mL serum bottles with a
working volume of 50 mL) at 37°C with a volatile solids (VS) concentration of 8 g/L. The first
experiment on single substrate was carried out to test the effects of sterilization (121 °C for 15 min)
on hydrogen production. The inoculum was collected from Fogliano coastal lake near Latina (Italy),
acclimated on glucose after 210 hrs in a continuous-type bioreactor and stored at -20°C with 30%
glycerol. Before being seeded into the bioreactors the mixed microorganisms were activated in a
medium culture (BFM) and (10g/L glucose), buffer pH 6.7. After inoculating 20% v/v the bottles
were flushed with nitrogen gas to establish anaerobic condition and maintained in orbital shaking
(120 rpm/min). The total biogas volume was measured using a water displacement system (Kalia et
al., 1994). Biogas in reactors headspace was analyzed using a gas chromatograph (Focus GC, by
Thermo) equipped with a thermal conductivity detector (TCD) and a 3 m Stainless Steel column
packed whit Hayesep Q (800/100 mesh).
The volume of produced H2 was calculated by the mass balance equation (1) (Logan et al., 2002)
(1)
where VH,i and VH,i–1 are cumulative H2 gas volumes at the current (i) and previous (i–1)
time intervals, respectively; VG,i and VG,i–1 are the total biogas volumes at the current (i) and
previous (i–1) time intervals; CH,i and CH,i–1 are the fraction of H2 gas in the headspace at the current
(i) and previous (i–1) time intervals, and VH is the total volume of headspace in the reactor.
The metabolic products of fermentation (volatile fatty acids, lactate, and ethanol) and sugars were
analyzed by a HPLC (Thermo Spectrasystem P4000) equipped with both an UV detector (λ= 210
nm) and a refractive index detector. The column (300 mm × 7.8 mm Rezex ROA-Organic Acid H+
(8%) (Phenomenex)) with a 4 × 30 mm security guard cartridge Carbo-H (Phenomenex), was
operated at 65 °C, using a solution of 5 mN H2SO4 as mobile phase (flow rate, 0.6 mL/min). The
liquid samples were diluted 1:20 in H2SO4 5 mN and filtered with 0.22 µm membrane before
injection into the HPLC.

Results reported in table 2 show the all feedstocks gave different hydrogen yield (mL H2/g
VS): 10.4 (manure), 52.4 (glycerol), and 142.7 (LPCW). As reported in table 2 sterilization
increased hydrogen production (mL H2/g VS): 37.7 (manure), 130 (glycerol), and 164 (LPCW).
This is probably due to the elimination of autochthonous H2-consuming bacteria (Guo et al., 2008;
Sung et al., 2002). This experiment revealed that manure, which is usually considered unsuitable for
hydrogen production, gave higher yield when codigested both with LPWC and glycerol or alone
(from 10.4 up to 170 mL H2/g VS) because of the presence of highly fermentative compounds
which increased hydrogen-producing bacteria activity. Nevertheless buffalo manure buffering
action allowed to maintain the pH at an optimum range throughout the fermentation process
(Weiland, 2010; Tenca et al., 2011). VFA analysis at the end of fermentative process (table 3)
showed that the main metabolic pathway was towards butyric acid (concentration between 36 and
77%), the same trend observed by Kim et al. (2009) using vegetable wastes
CONCLUSIONS
The feedstocks tested (buffalo manure, LPCW and glycerol) alone are suitable for hydrogen
production via anaerobic dark fermentation, however the codigestion as well as sterilization
procedures allowed a yield increase. Buffalo manure when codigested with 20% LPCW and 10%
glycerol gave a yield of 116 mL H2/g VS, showing an optimum buffering capacity and avoiding the
use of any external buffer/pH control system. Bio-hydrogen production from buffalo manure, via
1242
anaerobic digestion could, thus, offer a good solution either in terms of energy production/savings,
or waste management or pollution control.
REFERENCES
Fang, H.H.P. and H. Liu. 2002. Effect of pH on hydrogen production from glucose by mixed culture.
Bioresource Technol. 82:87-93.
Fox, P. and F.G. Pohland. 1994. Anaerobic treatment applications and fundamentals substrate-specificity
during phase-separation. Water Environ. Res. 66:716–724.
Guo L., X. M. Li, X. Bo, Q. Yang, G. M. Zeng, D. X. Liao and J. J. Liu. 2008. Impacts of sterilization,
microwave and ultrasonication pretreatment on hydrogen producing using waste sludge. Bioresource
Technol. 99:3651-3658.
Hawkes, F. R., R. Dinsdale, D. L. Hawkes and L. Hussy. 2002. Sustainable fermentative hydrogen
production: challenges for process optimization. Int. J. Hydrogen Energy. 27:1339-1347.
Hussy, I., F. R. Hawkes, R. Dinsdale and D. L. Hawkes. 2003. Continuous fermentative hydrogen
production from wheat starch co-product by mixed microflora. Biotechnol. Bioeng. 84:619-626.
Kalia, V.C., S. R. Jain, A. Kumar and A. P. Joshi. 1994. Fermentation of biowaste to H2 by Bacillus
licheniformis. World J. Microb. Biotechnol. 10:224-227.
Kim S. H., S. H. Han and H. S. Shin. 2006. Effect of substrate concentration on hydrogen production and
16S rDNA-based analysis of the microbial community in a continuous fermenter. Process Biochem.
41:199-207.
Kim D. H., S. H. Kim and H. S. Shin. 2009. Hydrogen fermentation of food waste without inoculum
addition. Enzyme Microb. Technol. 45:181.
Kyazze, G., R. Dinsdale, A. I. Guwy, F. R. Hawkes, G. C. Premier and D. L. Hawkes. 2007. Performance
characteristics of a two-stage dark fermentative system producing hydrogen and methane continuously.
Biotechnol. Bioeng. 97:759–770.
Liu, D.W., D. P. Liu, R. J. Zeng and I. Angelidaki. 2006. Hydrogen and methane production from
household solid waste in the two-stage fermentation process. Water Res. 40:2230–2236.
Logan, B.E., S. E. Oh, I. S. Kim and S. Van Ginkel. 2002. Biological Hydrogen Production Measured in
Batch Anaerobic Respirometers. Environ. Sci. Technol. 36:2530 -2535.
Ren N. Q., H. Chua, S. Y. Chan, Y. F. Tsang, Y. J. Wang and N. Sin. 2007. Assessing optimal fermentation
type for bio-hydrogen production in continuous-flow acidogenic reactors. Bioresource Technol.
98:1774-1780.
Sung S., L. Raskin, T. Duangmanee, S. Padmasiri and J. J. Simmons. 2002. Hydrogen production by
anaerobic microbial communities exposed to repeated heat treatments. U. S. DOE Hydrogen Program
Review. NREL/CP-610-32405.
Tenca, A., A. Schievano, F. Perazzolo, F. Adani and R. Oberti. 2011. Biohydrogen from thermophilic co-
fermentation of swine manure with fruit and vegetable waste: Maximizing stable production without pH
control. Bioresource Technol. 102:8582-8588.
Ueno, Y., H. Fukui and M. Goto. 2007. Operation of a two-stage fermentation process producing
hydrogen and methane from organic waste. Environ. Sci. Technol. 41:1413–1419.
Weiland P. 2010. Biogas production: current state and perspective. Appl. Microbiol. Biotechnol. 85:849-
860.
Table 1. Chemico-physical analysis of feedstocks.
COD TS VS Energy Density pH

gO2/g DM % % KJ/g DM g/mL
manure 6.28 13.51 11.08 18.10 1.01 6.08
glycerol 1.05 91.78 86.61 14.90 1.28 6.04
LPCW 0.59 6.22 5.46 8.36 1.02 6.05
TS: total solids
1243
Table 2. H2 production parameters from different feedstocks both alone and codigested.
Feedstock Composition Pretreatment Fermentation H2 (% ) H2 (ml/l) ml H2 / g VS pH at start pH at the end

(% ) time (h)
LPCW 100 - 144 42,4 ± 1,29 1028 ± 5,62 143 ± 0,78 6,5 4,5
manure 100 - 24 4,26 ± 0,49 83,3 ± 11,3 10,4 ± 1,41 6,8 6,2
glycerol 100 - 120 20,9 ± 0,63 420 ± 8,20 52,4 ± 1,02 6,4 5,0
LPCW 100 sterilization 72 39,2 ± 0,58 1447 ± 0,69 164 ± 0,08 6,5 4,7
manure 100 sterilization 114 17,8 ± 1,10 301 ± 14,5 37,7 ± 1,81 6,8 6,1
glycerol 100 sterilization 138 32,4 ± 0,59 1037 ± 142 130 ± 17,8 8,0 5,6
manure, glycerol 90:10 sterilization 24 17,15 ±1,9 377 ± 10,8 47,16 ± 5,2 6,8 5,8
manure, LPCW 90:10 sterilization 24 20,8 ±2,12 516 ± 52,5 65,4 ± 6,57 6,5 6,1
manure, LPCW 50:50 sterilization 72 33,6 ± 0,44 1028 ± 75,8 128 ± 9,48 6,5 5,0
LPCW, manure, glycerol 33:33:33 sterilization 72 34,7 ± 0,19 818 ± 4,24 102 ± 0,25 6,3 5,5
LPCW, manure, glycerol 70:20:10 sterilization 72 40,7 ±0,58 1499 ± 24 170 ± 2,73 5,6 4,7
LPCW, manure, glycerol 20:70:10 sterilization 72 36,7 ± 0,53 924 ± 59,4 116 ± 7,42 6,5 5,5
Table 3. Metabolic products (VFAs, lactate, 1-3 propanediol and ethanol) at the end of fermentation
(mg/L).
Feedstock Composition (% ) Lactate Formate Acetate Propionate Butyrate 1,3 propanediol Ethanol
manure 100 13,5 ± 10,5 82,3 ± 19,3 330 ± 24,8 31,6 ± 0,9 960 ± 95,9 126 ± 24,3 0
glycerol 100 36,1 53,9 106 42,6 1087 2588 278
LPCW 100 22,6 ± 0,9 133 ± 5,5 324 ± 9,1 347 ± 12,6 2062 ± 38,3 530 ± 3,8 193 ± 91,2
manure, glycerol 90:10 186 ± 0,77 50,9 ± 1,12 94,7 ± 1,69 14,4 ± 0,72 957 ± 137 720 ± 8,00 635 ± 240
manure, LPCW 90:10 44,1 ± 16,9 20,4 ± 11,2 108 ± 3,49 49,9 ± 3,82 1189 ± 119 157 ± 110 587 ± 135
manure, LPCW 50:50 18,9 ± 13,1 33,7 ± 0,37 331 ± 84,1 26,9 ± 0,65 1958 ± 13,5 159 ± 81,6 778 ± 47,6
LPCW, manure, glycerol 33:33:33 58,7 ± 2,68 145 ± 0,26 498 ± 12,7 29,2 ± 1,84 1524 ± 74,7 128 ± 63,7 118 ± 61,6
LPCW, manure, glycerol 20:70:10 56,8 ± 5,18 120 ± 2,04 302 ± 8,10 54,4 ± 3,07 1881 ± 27,3 191 ± 48,1 92,4 ± 40,5
LPCW, manure, glycerol 70:20:10 21,2 ± 0,72 55,3 ± 4,56 318 ± 4,77 283,5 ± 7,45 1688 ± 51,3 540 ± 22,4 116±0,0
1244
The Herd Size and Production Performances of Buffalo in Romania
Livia VIDUa* and Adrian BOTAb

a
University of Agricultural Sciences and Veterinary Medicine Bucharest, Faculty of Animal
Science,Marasti Blvd. 59, Bucharest, Romania,
b
Institute of Research and Development for Buffalo Breeding Sercaia Brasov, Str. Campului 2,
Sercaia, Romania
*Corresponding email: vidulivia@yahoo.com
ABSTRACT
In Romania, buffalo have been known since the fifth century after Christ, and were brought
by migratory peoples who come from Asia. The number of animals increased or decreased,
depending on social and economic conditions the country has passed. The highest number of
buffalos in Romania was registered in 1987, 228 000 heads. At present the total number of
buffaloes is 22 375 heads, of which 91.5% in the counties of central and northwestern Transylvania.
Buffalos reproductive indicators of small farms studied in the year 2012 were as follows: age at first
mating 33.71 months, the first calving 43.92 months, calving interval 423.83 days, the service
period 169.54 days, birth rate 90.8% and fecundity rate 80.10%. Data collected and analyzed from
all over Romania indicate that milk production recorded following indicators: the average quantity
of milk from 1329 to1420 kg milk, lactation period 272-275 days, fat content from 7.65 to 7.9%,
protein content 4.52% -4.96% and 4.85% lactose. The experiments organized in recent years have
demonstrated that the production of buffalo meat were good. Young buffalos for meat had good
capacity intensive fattening and the slaughter indicators are economic efficiently. At the age of
18.35 months live weight was 470, 83 kg, 240.75 kg carcass weight. Carcass meat percentage was
53.16%, 15.25% bone percentage and fat in carcass 26.64%.
Keywords: buffalo, dairy buffalo, meat buffalo, reproduction
INTRODUCTION
The world crosses a major food crisis because water and oil reserves have dropped
dramatically. Increasing global demand for food has three essential causes: increasing world
population, increasing welfare by the consumption of foods of animal origin and use of cereals for
fuel production. At present the world's population increases with 80 million people annually. In this
social and economic context must rethink the use of genetic resources. The statistical data
demonstrates that buffalo herd is focused on 29 countries, but milk production is recorded only in
19 countries. Buffalo provides meat, milk, hides, jobs, fuel for many rural marginal areas in the
world. Buffalo milk is used by direct consumption or as raw material for the dairy industry. In
Romania, buffalo has been raising since 5 th century and came from Asia to the South of the country
and from North Africa in the west of the country.

We have performed research in all areas of the country where there are buffalos; we
analyzed databases of Romanian livestock. The data were processed and interpreted statistically and
compared with the ones obtained by other researchers. The indicators of reproduction were studied
in 2012 years, in the small farms on a population of 231 buffaloes in 3 localities in Brasov County
(Rupea, Fagaras, Avrig). The indicators of milk production were obtained from the study of 960
lactations completed over a period of 3 years. To determine the chemical composition of milk
commonly classical methods have been used. For meat production we processed the data obtained
from the slaughter of 53 Romanian breed young male buffalos.

1245

Buffalo herds dynamics in Romania between 1920-2012.
The evolution of buffalo herds within 9.2 decades revealed the following (Table 1): in
1920, in Romania 145,856 buffaloes were raised, if we look at the size of herds in the first quarter
of the twentieth century the stock is large; in the period 1920-1980 the growth had a sinuous curve
that registered two peaks, in 1946, when growth was 37% and in 1980 the number of buffaloes
increased by 56.3% from 1920, the higher growth rate was in the 8th decade of the last century; the
largest decline has occurred in the last 25 years when the number has dropped from 228,000 to
22,400 heads today, the decrease was of 9.84 times. This decrease in the number of buffaloes in
Romania was caused by the unfavorable agricultural policy for buffalo breeders, increased exports
of live animals to countries in North-West Europe and slaughter buffalo youth at an uneconomic
age.
The reproduction indicators.
Regarding milk buffaloes in Transylvania (the center of Romania) we have observed that
the age of the first conception was 33.71 months, with variations between 32.55 and 34.73 months
(Table 2). These values are close to those found by Zicarelli et al. (1977) to milk buffaloes in Italy,
respectively 26-25 months. Considering the age of the first calving value obtained by us is on
average 43.92 months. The previous researches made in Romania showed values between 41.82 and
48 months. (Georgescu & Vidu, 2008). For this indicator the values are different depending on the
area and breed of the buffalo. Thus, Phathodiya (1999) determined the age to first calving to Surti
breed of 56.1 months, Zicarelli et al. (1977) to milk buffaloes in Italy 44.7 months. The calving
interval ranged from 397.47 days and 463.42 days. This value is smaller by 23.35 days in
comparison with to the value found by Velea (2006) of 447.18 days. The birth rate indicates the
number of viable product obtained. The average value found by us is 90.80% with variations
between 88.52 and 92.45%. Pucheanu (2000) determined the average birth rate for milk buffaloes in
Romania at the value of 89.82%. Regarding fecundity we obtained an average value of 80.1%,
varying between 77.45% and 82.32%. These values for the reproduction indicators are within the
limits found by other authors for European or Mediterranean buffalo.
Milk production.
Dairy buffaloes in Romania have the average of lactation period of 272.93 days (Table 3),
with variations depending on the area of raising from 275 days in southern Romania to 272 days in
the north-west area. These values are close to those found by Borghese (2005) for the
Mediterranean breed (270 days). The quantity of milk buffaloes in Romania is on average 1356.33
kg with 1320 kg in the south, in the valley of the Danube and 1420 kg in the North-West of
Romania. Romanian buffalo breed has a level of milk production by 1.4 to 2 times lower compared
to other breeds worldwide. Thus, Pucheanu (2000) determined an average of 1109.27 kg milk per
normal lactation in buffaloes in the Fagaras area with limits between 1047 kg at buffalo’s first
lactation and 1130 kg milk for multiparous. Buffaloes kept in Germany, coming mostly from
Romania have 2028 kg milk in first lactation, in 279.6 days of lactation, and the secundipare have
made 1793 kg milk in 208 days of lactation. These values demonstrate good genetic potential of
buffalos in Romania, and the need to improve raising conditions. Buffalo milk quality is a
distinctive factor, especially for milk processing industry. The chemical composition varies
depending on the region, season and feeding level. The average dairy buffaloes in Romania have a
fat content of 7.8%. This value is higher than those found in previous research by Pucheanu (2000)
in dairy buffaloes from Şercaia area where fat percentage was 6.94%, with low variability (5.12%).
Comparing with buffaloes kept on farms in Germany fat percentage is 11% lower, but higher than
the European average (6.8%). Bulgarian Murrah breed fat percentage is 7.04% at a quantity of 1800
kg milk, and in Italy it increased from 7.09% in the period 1977-1981 to 8.31% in 2001year
(Borghese, 2005). The analysis of data from control milk production in buffaloes was found that the
percentage of protein varies so: in counties in southern Romania there has been registered the
highest percentage of protein, respectively 4.96% and 4.73% for the entire population studied.
Comparing with the specialty literature data, these values are superior to those found by Pucheanu
1246
(2000) in dairy buffaloes from Şercaia area (4.69%). The average value found by us is comparable
to that found by Borghese (2005) in dairy buffaloes in Italy (4.72%). Kundi breed of Pakistan
obtained higher values of protein percentage (6%), while the remaining breeds the value varies
between 4.2 and 4.6%. Regarding the lactose content of milk he had obtained an average of 4.85%.
Meat production.
In Romania the research on meat skills of buffalos were have been developed since the 70's
and continued to the present day. For the young buffalos intensively grown and slaughtered at since
the age of18.3 months, the body weight was 470.83 kg and the carcass weight of 240, 75 kg. The
carcass meat content was 53.16%, fat 26.64% and 15.25% bones. Comparison with the young males
slaughtered in Italy (Borghese et al. 2005) have found the following differences: the animals were
slaughtered in Italy with 2.3 months earlier than in Romania and the carcass weight was 234.6 kg,
the weight of carcass meat by 8.8% higher than in young buffalos in Romania. This experiment
demonstrates that young male buffalos are an important source of meat for the communities in the
area.
ACKNOWLEDGEMENTS
This scientific work was funded by the Sectoral Programme ADER 713/2011, project "The
elaboration selection criteria in Buffalo Carpathian Indigenous populations in order to Improve the
breed"
REFERENCES
Borghese A. 2005. Buffalo production and research, FAO, Rome.
Georgescu G.H., L.Vidu. 2008. The monograph buffalos in Romania and worldwide, Ceres
Publishing House, Bucharest.
Phathodiya O.P. 1999. Age at first calving and its correlation with economic traits in Surti buffalo.
Indian Vet. J. 76: 902-905.
Pucheanu C. 2000. Research on knowledge of morphological, physiological and productive
parameters at buffaloes, depending on various factors of influence, PhD thesis, University
of Agricultural Sciences and Veterinary Medicine Bucharest.
Velea C. and G. Marginean. 2006. Actuality and perspective in raising buffalos, Agrotehnica
Zicarelli L. 1997. Reproductive seasonality in buffalo, Bubalus bubalis, Suppl 4: 29-52.
Table 1. Buffalo herds dynamics in Romania 1920-2012.
The reference year Number of animals Differences compared to

reference year % (+ / -)
1920 145.856 -
1946 200.000 +37,12
1966 154.113 +5,66
1970 175.240 +20,14
1980 228.000 +56,31
2005 64.028 -56,10
2012 22.400 -84,64
1247
Table 2. The reproduction indicators of milk buffaloes in Transylvania.
Area
The indicator Rupea Făgăraş Avrig Average
( n=120) (n=56) (n=55)
Age at first conception 32,55 34,73 33,85 33,71
(months)
Age at first calving (months) 42,87 45,06 43,83 43,92
Calving-intervalul (days) 463,42 397,47 410,82 423,83
Service-periode (days) 194,93 165,14 148,55 169,54
Birth rate (%) 88,52 91,41 92,45 90,80
Fecundity rate (%) 77,45 82,32 80,45 80,10
Table 3. Indicators of milk production in buffaloes in Romania.
The indicator Number of animals Average

Southern Romania
Lactation duration (days) 167 275
Milk yield (kg/lactation) 167 1320
Milk fat (%) 167 7,65
Milk protein (%) 167 4,96
Milk lactose (%) 167 4,22
Central of Romania
Milk fat (%) 388 7,9
Northwest of Romania
Milk fat (%) 405 7,72
Total population of buffalo in Romania
Lactation duration (days) 960 272,93
Milk yield (kg/lactation) 960 1356,33
Milk fat (%) 960 7,8
1248
Buffalo Meat and Meat Products
Fatty Acids in the Muscle and Fat Layer of Buffaloes Supplemented

with Fish Oil
Exequiel Maria PATIÑOa, Jose Feliciano CEDRESa, Maria Alicia JUDISb, Marcial
SANCHEZ NEGRETTEa, Ana Maria ROMEROb, Mirtha Marina DOVALb , Gladys Isabel
REBAKa, Gustavo Angel CRUDELIa, Eduardo Luis FAISALa and Gonzalo TUÑONc
a
Facultad de Ciencias Veterinarias, Universidad Nacional del Nordeste (UNNE), Sargento Cabral
2139, Corrientes (3400), Argentina. b Departamento de Ciencias Basicas y Aplicadas. Universidad
Nacional del Chaco Austral (UNCAus). Presidencia Roque Sáenz Peña, Chaco, Argentina. c New
Zealand Farming Systems Uruguay (NZFSU)
*Corresponding email: exepa@vet.unne.edu.ar
ABSTRACT
The aim of this work was to investigate the influence of fish oil supplementation on the con-
centration of conjugated linoleic acid (CLA) and Ω 6 and 3 in muscle and in the fat layer samples of
Mediterranean buffaloes. A total of 20 animals were randomly divided into two groups, fed daily
for 60 days on Brachiaria brizantha, supplemented with 3 kg rice flour, 500 g corn and 500 g
sunflower pellet (group I) and also supplemented daily with 100 ml fish oil per animal (group II). In
both samples the results showed a significant decrease in the palmitic fatty acid, in group II (232.67
mg/g fat) compared to group I (254.73 mg/g fat) in the muscle and in the group II (259.72 mg/g fat)
compared to group I (280.84 mg/g of fat) in fat layer. In the muscle Ω 6 (linoleic 18:02 n6) content
in group II (28.85 mg/g fat) decreased significantly compared to group I (47.00 mg/g fat) and fat
layer in the group II (13.21 mg/g fat) increased slightly compared to group I (12.71 mg/g fat). In
muscle the Ω 3 (20: 1 c 11 + alpha eicosenoic (18:3) n3) content for groups I and II was very
similar (10.31 and 10.70 mg/g fat, respectively) and in fat layer was of 4.70 and 6.05 mg/g fat,
respectively. In muscle the Ω 6/3 relationship in group II was narrower (2.69:1) than in group I
(4.55:1). The proportion of UFA / SFA in group II was 0.72 in the muscle and 0.53 in the fat layer.
The diet including fish oil, both in muscle and fat layer, increased the CLA content, improved the
ratio Ω 6/3 and decreased the palmitic fatty acid content, in addition to decreased the Ω 6 content in
muscle and increased in fat layer of Ω 3 content. Fish oil supplementation may effectively increase
the CLA content and lower values of palmitic fatty acid in both muscle and fat layer, and improve
the relationship Ω 6/3.
Keywords: buffalo, muscle, fat layer, CLA, omega 6 and 3, fish oil
INTRODUCTION
The generic term ‘functional feed’ is used to identify feeds and/or components of feeds that
have additional properties for consumers that exceed the usual nutritional benefit of a feed (Milner,
1999). The fat content of bovine products such as milk and meat, in many cases, is considered to be
unhealthy due to the relatively high saturated fatty acid content. In recent years it has been discov-
ered that there is a component in the fat, the conjugated linoleic acid (CLA), which has anticarcino-
genic properties (NRC, 1996), lipolitic activities and can prevent ateroesclerosis and diabetes.
Therefore, feeds with high CLA content should be considered functional feeds (McGuire and
McGuire, 2000).
Feed products coming from ruminants make up a major source of CLA for humans and have
a potential role in the production of functional feeds (Bauman et.al 2000; Gagliostro, 2004). The
CLA is a natural component of adipose tissue of ruminants. It is created in the rumen as an interme-
diate derived from the biohydrogenation of linoleic acid by the enzyme linoleic isomerase, pro-
duced by anaerobic bacteria of Butyrivibrio genera, which transforms this fatty acid into an isomer
cis-9 y trans-11 (Parodi, 1977). The main source of CLA in the human diet is ruminant milk and
meat, which contain mainly cis-9, trans-11 C18:2 (rumenic acid) and trans-9, cis-11 C18:2, the first
1250
contributing with 60% of the total CLA in the muscle (Monson, 2004). Research determining CLA
content in buffalo meat in Italy (Romano et al., 2007), Brasil (Rodrigues et al. 2004; Lira et al.
2005; Oliveira et al., 2008), Venezuela (Giuffrida-Mendonza et al., 2008) and Argentina (Cedres,
2004) obtained contrasting values due to the differences in the diets used. It has been shown that
eicopentanoic (EPA) y docosahexanoic (DHA) Ω 3 acids have hypocolesterolemic, antitrombic and
antiinflamatory properties (Williams 2000). Several authors consider that for human health it is
convenient to use the relationship Ω 6 /3 between 5 and 2 or similar, and this would be optimum for
human nutrition (Gagliostro 2004). Diets with this relationship allow us to infer that these feeds
could be considered functional feeds.
The population of buffaloes in Argentina is estimated to be 100,000, concentrated mainly in
the wet subtropic northeastern region, in Corrientes, Chaco, Misiones, Formosa and north of Santa
Fe, Corrientes having the highest number of animals, approximately 45,000. There are also buffa-
loes in other provinces such as Buenos Aires, Entre Ríos, Tucumán, Mendoza and San Luis. Meat
production continues to be the main purpose of breeding of this species in Argentina. The Northeast
has the highest potential for the production of buffalo meat.
The objective of this work was to investigate if buffaloes grazing Brachiaria brizantha and
supplemented with concentrates based on rice bran, corn, sunflower and fish oils, would have a dif-
ferent CLA concentration, Ω 3 and 6 concentrations and the close relationship Ω 6/3 in the meat and
fat. It is crucial for this research to find out if the objective is achieved due to the habit in Argentina
of consuming meat with fat layer.

The animals used came from La Florencia farm, located in General Paz department, Corri-
entes province, Argentina. Twenty castrated 11-month old buffaloes of Mediterranean breed were
divided into two groups of 10 and identified with alphanumeric tags. All animals were fed with
Brachiaria brizantha grass and a daily ration per animal of 3 kg rice bran, 500 g corn and 500 g
sunflower pellets during the experimental period (60 days). The animals on group I (control) only
received pasture and ration; animals on group II received 100 ml fish oil daily included in the ra-
tion. The fish oil came from Argentinian hake (85%, Merluccis hubbsi) and anchovy (15%, Anchoa
marinii). The buffaloes received the daily ration in individual feeders to follow the independence
criteria.
The samples (n=20) of the Longissimus dorsi muscle and the fat covering the striploin be-
tween the 10th and 11th rips were obtained at day 60 of the experiment, which was carried out on the
months of February and March 2011. Meat and fat samples were taken from each animal, which
were conditioned in disposable containers, freezed to -20ºC and maintained in polyurethane boxes
unitil they arrived to the lab. Each sample was processed twice to obtain the lipidic profile. To ex-
tract the total lipids a mix of chloroform and methanol was used according to the method in Bligh
and Dyer (1959) maintaining the nitrogen atmosphere. Conversion of the fatty acids to methyl es-
ters was carried out with NaOH y methanolic BF3 at 14% boiling for eight minutes. Standard methyl
esters of fatty acids with 99% purity were used (Lipid Standard 189-19 Sigma-Aldrich). The fatty
acid composition was obtained with a gas chromatographer from Agilent firm with a capilar column
of 60 m long and 0.25 mm internal diameter (Supelco 2340) and a flame ionisation detector. The
gas chromatographic method used GC-FID) was adapted to the norm ISO 15304 (2010).
Descriptive statistics were applied to evaluate the sample estimated for each the treatments
(average, standard deviation, variation coefficient and minimum and maximum ranges). Prior to the
analyses the descriptive behaviour of the sample was evaluated using confidence intervals and box
and whisker graphs. The basic assumptions for the analysis of variance, homogeneity and normality
model ( X ij = μ + τ j + eij ). The analyses were carried out using the software Infostat (2009), which
were tested. To estimate diet effects a completely randomised deign was used, with an aditive linear
belongs to the Faculty of Veterinary Science of the National University of the Northeast.
1251

Palmytic fatty acid in muscle was lower in group II (232.72 mg/g fat) than in group I
(254.73 mg/g fat); in the fat layer was also lower in group II (259.72 mg/g fat) than in group I
(280.84 mg/g fat). Unsaturated fatty acids (UFA) in muscle and in fat layer showed a higher rate of
increase in group II than in group I. In muscle, the CLA content (18:2) 9 c, 11 t of group II showed
an increase of 21.23 mg/g fat, while the increase in group I was 15.80 mg/g fat. In the fat layer the
CLA content increased by 16.80 mg/g fat for group II, while the increase was 11.75 mg/g fat for
group I.
In muscle the content of Ω 6 (linoleic 18:02 n6) of group II (28.85 mg/g fat) decreased sig-
nificantly compared to group I (47.00 mg/g fat). In fat layer, on the other hand, group II (13.21
mg/g fat) had a slight increase compared to group I (12.71 mg/g fat). In muscle the content of Ω 3
(20: 1 c 11 + eicosenoic alpha (18:3) n3) for groups I and II were 10.31 and 10.70 mg/g fat, respec-
tively, with no statistical difference between both groups, while the values of fat layer were 4.70
and 6.05 mg/g fat, respectively. In muscle the relationship Ω 6/3 of group II was narrower (2.69:1)
than in group I (4.55:1). The proportion UFA/SFA in group II was 0.72. In fat layer the relationship
Ω 6/3 of group II was narrower (2.18:1) than in group I (2.70:1). The proportion UFA/SFA of group
II was 0.53.
The average values for CLA in the fat layer obtained in this experiment with the two diets
were higher than the values reported in Brazil by Oliveira et al. (2008) in Murrah buffaloes, with
diets based on corn silage, corn grain, soy flower, soy grain, soy oil and urea (1.6 – 3.4 mg/g fat)
but smaller than the diets with soy oil (10.80 mg/g fat). In terms of the ratio Ω 6/3 it was observed
that this relationship was narrower (1.37:1) in group II, which contained fish oil.
CONCLUSIONS
This experiment shows that supplementation with fish oil leads to increased CLA content,
decreased values for palmitic acid in muscle and fat layer, and an improved Ω 6/3 relationship.This
study has shown the beneficial effect of fatty acid composition on the meat and fat of buffaloes.
The clear understanding of the effects of the relative toxicity of fatty acids on the physiology
of the biohydrogenic bacteria in the rumen will allow us to suggest further modifications and doses
in the strategic rations that are given to these animals to obtain functional feeds that have and ho-
mogeneous CLA and Ω 3 content. This would result in a benefit for human health.
ACKNOLEDGMENTS
“Florencia” farm in General Paz, Corrientes province, Argentina because they provided the
animals and the infrastructure to carry out the experiment. Thanks to Omega Sur from Batan, Mar
del Plata, Buenos Aires province, Argentina, who gave us the fish oil.
REFERENCES
Bauman, D. E., L.H. Baumgard, B.A. Corl and J.M. Griinari. (2000). Biosynthesis of Conjugated
Linoleic Acid in Rumiants. Proc. Am.Soc. Anim. Sci.
http://www.asas.org./jas/symposia/procedings.
Bligh, E.G., W .J. Dyer. 1959. A rapid method for total lipid extraction and purification. Can. J.
Biochem. Physiol. 37: 911-917.
Cedres, J.F. (2004). Rendimiento carnicero del búfalo. En el libro Búfalos en Argentina pags. 81 a
113. Editado por Moglia S:R:L: Corrientes, Argentina ISBN Nº 987-43-7388-1 230 p.
Gagliostro G .A. (2004). Manejo Nutricional para la Producción de Leches de Vaca y de Cabra con
Alto Impacto sobre la Salud Humana. Area de Investigación en Producción Animal. INTA.
Unidad Integrada Balcarce. Estación Experimental Agropecuaria. Fac.de Ciencias Agrarias /
UNMdP. 84 p.
1252
Giuffrida, M., L. Arenas de Moreno, M.J. Beriain, N.O. Huerta-Leidenz, G. C. Smith. (2005). Oc-
currence of Conjugated Linoleic Acid in Longissimus Muscle of Water Buffalo (Bubalus bu-
balis) and Zebu-Type Cattle Raised Under Savannah Conditions. Meat Science 69: 93-100.
ISO 15304. 2002. Animal and vegetable fats and oils- Determination of the content of trans fatty
acid isomers of vegetable fats and oils – Gas chromatographic method.
McGuire, M.A. and M.K. McGuire. (2000). Conjugate Linoleic Acid (CLA): A Ruminant Fatty
Acid with Benefical Efects on Human Health. Proc. Am. Soc. Anim. Sci. 1999.
http://www.asas.org./jas/symposia/procedings (Consulta 28/05/09).
Monson, F. D. (2004). Suplementación con Lípidos en Bovinos de Carne: Metabolismo, Efectos
sobre la Calidad de la Canal, de la Carne y sobre la Salud Humana. Tesis Doctoral. Instituto
Mediterráneo de Zaragoza. España. 110 p. 2004.
Milner, J.A. (1999). Funcional Foods and Health Promotion. J.Nutr. 129: 1395S –1397S.
National Research Council (NRC). (1996). Carcinogenesis and Anticarcinogenesis in the Human
Diet. National Academy Press. 242.p. Washington D.C.
Lira, G.M., J. Mancini Filho, R.P. Torres, A.C. Oliveira, A.M.A. Vasconcelos, C.M.B. Omena and
M.C. S. Almeida. (2005). Composição centesimal, valor calórico, teor de colesterol e perfil de
ácidos graxos da carne de búfalos (Bubalus bubalis) da cidade de Sao Luiz do Quintunde- AL.
Rev. Ins. Adolfo Lutz 64: 31-38.
Oliveira, R.L., M.M. Ladeira, M.A.A.F. Barbosa, D.M.P. Assunção, M. Matsushita, G.T. Santos,
and R.L. Oliveira. (2008). Acido linoleico conjugado e perfil de ácidos graxos no musculo
e na capa de gordura de novilhos bubalinos alimentados com diferentes fontes de lipídos.
Arq. Bras. Med.Zootec. 60: 169-178.
Parodi, P.W. (1977). Conjugated Octadecaienoic Acids of Milk Fat. J. Dairy Sci. 60: 1550-1553
Rodríguez, V.C., M.C. Bressan,; , M.das G. Cardoso and R.T. Fonseca de Freitas. (2004). Acidos
Graxos na Carne de Búfalos e Bovinos Castrados e Inteiros. R.Bras. Zootec. 33: 434-443.
Romano, R., I. Borriello, L. Chianese and R. Sachi. (2007). Efect od dietary vitamin E content on
the CLA,cholesterol and triglycerides composition of Italian Mediterranean buffalo meta. Ital.
J.Anim. Sci. 6: 1202-1206.
Williams, C.M. (2000) Dietary fatty acid and human health. Ann. Zootech. 165-180.
1253
Using Slaughter Weight to Predict Weight and Yield

of Primal Cuts of Carcass from Buffaloes
André Mendes JORGEa*, Rafael Silvio Bonilha PINHEIROb, Caroline de Lima

FRANCISCOc, André Michel de CASTILHOSc, José Nicolau Próspero PUOLI FILHOc,
Maurícia BRANDÃO DA SILVAc, Dayane Cristina RIVAROLIc, Carolina Cesarino
COUTINHOc, Alexandre COMINOTTEc, Isabela de Mello PADOVANc
a
Estadual Paulista UNESP, 18618-000, Botucatu, Sao Paulo, Brazil. CNPq Researcher.
b
Biology and Animal Science Department, Faculty of Engineering of Ilha Solteira, Univ
Estadual Paulista UNESP,15385-000, Ilha Solteira, Sao Paulo, Brazil.
c
Estadual Paulista UNESP, 18618-000, Botucatu, Sao Paulo, Brazil.
* Corresponding email: andrejorge@fmvz.unesp.br
ABSTRACT
The knowledge of the meat production from different buffalo breeds and their crossings
in different feeding systems becomes necessary for the supply of subsidies to whole productive
meat chain. Some quantitative carcass traits of Mediterranean buffaloes bulls, finished in feedlot,
with initial age of fourteen months and 330 kg live weight, slaughtered with 450, 480, 510 and
540 kg, were evaluated. The diet contained 13% crude protein, 2.68 Mcal digestible energy/kg
DM and a roughage : concentrate ratio of 25:75. Regression equations for prediction weight and
yield of primal cuts of carcass as a function of slaughter weight were obtained. Carcass dressing
percent increased as the slaughter weight increased (49.2; 49.5; 49.7; and 49.9%). The Pistola
Style cut weight although increasing linearly in weight (108.2; 117.6; 124.0 and 130.7 kg) as the
slaughter weight increased, declined linearly when expressed in relation to cold carcass weight
(49.5; 49.0; 48.6 and 48.2%). In this experimental conditions Mediterranean young bulls
slaughtered between 450 to 540 kg of live weight showed increasing yields of cold carcass,
forequarter and thin flank.
Keywords: Cold carcass, forequarter, pistol style cut, thin flank, carcass dressing
INTRODUCTION
Knowledge of meat potential production from different buffalo breeds and their
crossbreds, as well performance rearing and feeding regimes is necessary to provide subsidies in
the meat production chain, ranging from the breeder cattle until the consumer (Jorge and
Andrighetto 2005). Carcass and commercial cuts yield as well as carcass weight are measures
of interest from slaughterhouses to assess the value of the purchased product and operational
costs (Jorge et al., 1997, Jorge, 2004; Jorge and Andrighetto, 2005 ).
The slaughter weight (SW) has great importance in feedlot by changing costs and quality
of the final product (Jorge et al., 2002; Jorge, 2004). The production of young animals, called
early and "superprecoce", has raised interest of producers.
In Brazilian conditions, there are few works with buffaloes, particularly about evaluation the
effect of different slaughter weights on carcass traits of young animals. Therefore this experiment
aimed to study the effect of different slaughter weights on quantitative carcass traits from non-
castrated Mediterranean buffaloes feedlot from fourteen months old.

This research was carried out at Universidade Estadual Paulista, UNESP, Botucatu
campus. Twenty-eight non-castrated Mediterranean buffaloes, taken at random from the same
herd, with average initial 330 kg ± 1.52 body weight (BW) and 14 months old were used.
1254
Carcass traits from animals slaughtered at different slaughter weights (SW): 450, 480,
510 and 540kg BW. The animals were fed ad libitum in feedlot with a diet containing, on
average, 13% crude protein and 2.68 Mcal/kg dry matter (DM) of digestible energy.
Diet was composed of 7.8% of corn whole plant silage, 20.6% of coast-cross hay, 8.2%
cottonseed, 46% corn silage wet grain and 17.4 % concentrate (42.2% citrus pulp, 29.2% of
cassava meal, 13.4% soybean meal, 11.9% of gluten, 2.6% mineral mixture, 0.7% urea and
0.02% Rumensin). Composition of the mineral mixture (per kg) was 180g calcium, 130g
phosphorus, 1.250mg copper, 5.270mg zinc, 2000mg manganese, 100mg cobalt, 90mg iodine,
15 mg selenium, 2.200mg iron and 1.300mg fluorine.
Weighing were taken each 28 days and the animals were monitored their growth during
the period of feedlot according Perkins et al. (1992) procedure using ultrasound vet "PIE
MEDICAL - Scanner 200" with probe "Sector Curved Array Scanner" model 51B04UM02 to
check the development of back fat thickness between the 12th and 13th ribs (BFUS), back fat
thickness in P8site Biceps femoris (BFP8US) and rib eye area (REAUS) from Longissimus dorsi
muscle.
This procedure was performed after the first month of feedlot and on boarding of animals
to the slaughterhouse. Once they reached the predetermined SW the animals were fasting by 16
hours and sent for slaughterhouse 280 km distant from feedlot, which were held carcasses
evaluation after cooling 24 hours at 0º Celsius. The measurement of back fat thickness was
performed at 12th rib in the Longissimus dorsi (BFC) surface of left carcass and in the Biceps
femoris (BFP8C).
Also at the 12th rib was taken rib eye area (REAC). The right half carcass was divided
into the primary or basic cuts: forequarter, composed of the neck, shoulder, arm and five ribs; the
Pistola Style or "Serrote", comprising the posterior region of carcass, separated from the
forequarter between the fifth and sixth rib to a distance of approximately 20 cm of the spine, and
spare ribs or "Costilhar" comprising the region from the sixth rib plus the abdominal muscles.
The experimental design was completely randomized with four treatments and seven
repetitions. The data were subjected to polynomial regression analysis (SAS Inst. Inc., Cary,
NC).

Increasing in slaughter weight (SW) promoted linear increase in cold carcass weight
(CCW) (Table 1), and the correlation between SW and CCW was 0.74. This same trend was
observed by researchers when working with cattle and buffaloes slaughtered at different stages
of physiological maturity (Jorge et al., 1997). Carcass weight is an important feature because it is
directly associated with the commercial value of the animal (Jorge, 2004; Jorge and Andrighetto,
2005 ). Currently, the most widely form of marketing for slaughterhouse is payment per " arroba
(@)" (15 kg of chilled carcass) (Jorge and Andrighetto, 2005).
The carcass yield showed an increase as a function of SW, these results corroborate those
of Jorge et al. (2002) who worked with Mediterranean buffalo bulls and steers slaughtered at 450
and 500 kg. Back fat thickness on the carcass (BFC) and in Biceps femoris muscle (BFP8C),
increased by 0.036 mm and 0.057 kg more per kg of SW. In this experiment, we observed values
back fat thickness higher than those reported by Jorge et al. (1997) who worked with diets with
forage: concentrate ratio of 50:50, whereas in the present experiment this ratio was 25:75.
Regarding the percentage of primal cuts due to increased slaughter weight, it was found that the
variation of Pistola Style Cut or "Serrote", which is the best reward for slaughterhouses, showed
a decreased linearly.
According to Berg and Walters (1983), higher slaughter weights improve carcass
conformation and fat cover, but cause decrease in the hindquarter percentage. Likewise,
increased fat carcass deposition influenced positively in meat tenderness. The forequarter and
spares ribs percentages showed small linear increments, i.e., 0.009 and 0.004% more per kg of
SW.
1255
The same trend was observed by Jorge et al. (2004) working with Mediterranean
buffaloes fed diets containing two protein levels (13 and 16%) and slaughtered at 450 and 500 kg
body weight. When using the regression equations to estimate the weight of the primal cuts as a
function of slaughter weight, it was found out in this study that for each 30 kg heavier SW
weight gain was similar between the forequarter (6.43 kg) and Pistola Style cut (6.72 kg), and the
spare ribs has the smallest absolute increments in weight (2.66 kg).
Increases in the percentage of spare ribs (Costilhar) with greater weight and degree of
finishing can be attributed to increased fat deposition in this area. In this study the correlation
between BFC and BFP8C with Costilhar weight was 0.63 and 0.67 and with Costilhar
percentage was 0.48 and 0.52, respectively.
Rib eye area (REAC) increased 0.054 cm ² more per kg of slaughter weight (SW). When
REAC was adjusted to 100 kg carcass weight, a linear decrease, in agreement with the data
reported by Jorge et al. (1997) when they worked with cattle and buffaloes slaughtered at
different stages of physiological maturity. REAC values of this study, in all weight ranges were
well above the average of 40.85 cm ² reported by Jorge (2004) with Mediterranean buffaloes.
In this experimental conditions early non-castrated Mediterranean buffaloes slaughtered
at weights ranging from 450 to 540 kg showed increasing yields of cold carcass, forequarter and
spare ribs and decreasing values in Pistola Style cut yield. Back fat thickness and rib eye area
increased with increasing slaughter weight.
REFERENCES
Berg, R.T. and L.E. Walters. 1983. The meat animal: changes and challenges. Journal of Animal
Science, v.57, Supplement 2, p.133-146.
Jorge, A.M., C.A.A. Fontes and J.E. Soares. 1997. Características quantitativas da carcaça de
bovinos e bubalinos abatidos em diferentes estágios de maturidade. Revista Brasileira de
Zootecnia, v.26, n.5, p.1039-1047, 1997.
Jorge, A.M., M.G. Calixto and C. Andrighetto. 2002. Effect of sexual condition and slaughter
weight on carcass traits from buffaloes finished in feedlot. In: 1ST BUFFALO
SYMPOSIUM OF AMERICAS, Belém, 2002 Proceedings... Belém:International Buffalo
Federation, 2002. v.1, p.506-509.
Jorge, A.M., C. Andrighetto and D.D. Millen. 2004. Características de carcaça de bubalinos
Mediterrâneo não castrados alimentados em dois níveis de proteína bruta e abatidos em
dois pesos de abate. In: VI CONGRESSO INTERNACIONAL DE ZOOTECNIA, XIV
CONGRESSO NACIONAL DE ZOOTECNIA, X REUNIÃO NACIONAL DE ENSINO
DE ZOOTECNIA, XVII FÓRUM DE ENTIDADES DE ZOOTECNISTA, Brasília. A
Zootecnia e o Agronegócio. Brasília:ABZ, AZOO-DF, Faculdades UPIS, v.1, p.1-6
Jorge, A.M. Produção de carne bubalina. 2004. In: VI CONGRESSO INTERNACIONAL DE
ZOOTECNIA, XIV CONGRESSO NACIONAL DE ZOOTECNIA, X REUNIÃO
NACIONAL DE ENSINO DE ZOOTECNIA, XVII FÓRUM DE ENTIDADES DE
ZOOTECNISTA, 2004, Brasília. Anais Palestras. A Zootecnia e o Agronegócio.
Brasília:ABZ, AZOO-DF, Faculdades UPIS, 2004. v.1, p.1-16
Jorge, A.M. and C. Andrighetto. 2005. Características de carcaça de bubalinos. In: VII
CONGRESSO INTERNACIONAL DE ZOOTECNIA, XV CONGRESSO NACIONAL
DE ZOOTECNIA, XI REUNIÃO NACIONAL DE ENSINO DE ZOOTECNIA, XVIII
FÓRUM DE ENTIDADES DE ZOOTECNISTA, Campo Grande. Anais Palestras.
Produção Animal e Responsabilidade. Campo Grande: ABZ, AZOO-MS, UFMS-UEMS,
v.1, p.1-29.
Perkins, T.L., R.D. Green and K.E. Hamlin. 1992. Evaluation of ultrasonic estimates of carcass
fat thickness and longissimus muscle area in beef cattle. Journal of Animal Science, v.70,
p.1002-1010.
1256
Table 1. Carcass traits from non-castrated Mediterranean young buffaloes finished in feedlot and
slaughtered at different weights
Slaughter Weight (SW) (kg)

Parameters 450 480 510 540 Regression equations
Actual Slaughter Weight (kg) 443.1 484.9 513.3 543.4 -
Cold carcass weight (kg) 218.2 240.2 255.2 271.0 Ŷ = -15.280 + 0.5269*SW
Dressing (%) 49.2 49.5 49.7 49.9 Ŷ = +46.762 + 0.0057*SW
REAUS (cm2) 62.3 65.9 68.1 70.4 Ŷ = +28.962 + 0.0763*SW
REAC (cm2) 66.6 68.9 70.4 72.1 Ŷ = +42.447 + 0.0545*SW
REAC/100 kg carcass (cm2) 30.5 28.8 27.7 26.4 Ŷ = +48.451 - 0.0405*SW
BFUS (mm) 8.1 9.5 10.5 11.6 Ŷ = -7.128 + 0.0344*SW
BFC (mm) 8.5 9.9 11.0 12.0 Ŷ = -7.309 + 0.0356*SW
BFC/15 kg (mm) 0.57 0.61 0.64 0.67 Ŷ = +0.1253 + 0.0010*SW
BFP8US (mm) 9.0 11.3 12.9 14.5 Ŷ = -15.563 + 0.0554*SW
BFP8C (mm) 10.2 12.6 14.2 16.0 Ŷ = -15.004 + 0.0570*SW
BFP8C/15 kg (mm) 0.70 0.77 0.82 0.88 Ŷ = -0.0981 + 0.0018*SW
Forequarter (%) 36.0 36.4 36.6 36.9 Ŷ = +31.984 + 0.0091*SW
Pistola Style cut (%) 49.5 49.0 48.6 48.2 Ŷ = +55.53 - 0.01350*SW
Spare ribs (%) 14.5 14.6 14.8 14.9 Ŷ = +12.471 + 0.0044*SW
Forequarter (kg) 78.6 87.6 93.7 100.1 Ŷ = -16.275 + 0.2142*SW
Pistola Style cut (kg) 108.2 117.6 124.0 130.7 Ŷ = +8.9841 + 0.2204*SW
Spare ribs (kg) 31.3 35.0 37.6 40.2 Ŷ = -8.0204 + 0.0888*SW
1257
Carcass Composition and Meat Quality of Buffalo by Raised Alongside

Mekong River: Nakhon Phanom Province
Tanom TATHONGa, Teerawat KWEANGMAUNGb, Chanachai BOONPERMc, Sutipong

URIYAPONGSONd and Sornchai KONGSOOKe
a
Program in Food Technology, Faculty of Agriculture and Technology, Nakhon Phanom
University, Thailand,
b
Renu-Nakhon Local Government, Renu-Nakhon District, Nakhon Phanom, Thailand,
c
Program in Animal Production Technology, Faculty of Technology, Udonthani Rajabaht
University, Udonthani, Thailand,
d
Department of Animal Science, Faculty of Agriculture, KhonKaen University, Thaialnd,
e
NakhonPhanom Livestock Research Testing Station, Tha-u-ten District, Nakhon Phanom,
Thailand.
*Corresponding email: bangson_tim@hotmail.com
ABSTRACT
The objective of this research was to study on carcass composition of buffalo and meat
quality of male and female buffalo between West and North Distric in NakhonPhanom Province.
Twenty buffaloes were randomized from 4 Districts of NakhonPhanom Province involve in an area
of Renu-Nakhon and Plaplak (West District), Srisongkram and Banpeang (North District). The
buffaloes were slaughtered and used Thai style of cutting in the slaughter house of Renu-Nakhon
and Banpeang Districts.
The result of carcass compositions showed that hind shank, lean from fore leg, pin strips
from hind leg, pin strips leg, breast meat, rib meat, loin, neck, flat and tongue + head meat of
buffaloes from west districts were higher than those from the north districts (P<0.05) while lean
from hind leg, tender loin and skin of buffaloes from north districts were higher than those from
west districts (P<0.05). The difference of guts composition showed that kidney, kidney fat, lung,
reticulum and omasum of buffaloes from the west districts were higher than those from the north
districts (P<0.05), however; buffaloes from the north districts had speel and back fat higher than
those from the west districts (P<0.05). For meat quality showed that cooking loss at 24 h of
Loingissimus dorsi (LD) from buffaloes in the west districts were higher than those from the north
districts (P<0.05) while cooking loss of Semi membranosus (SM) and LD of buffaloes from the
north districts were higher than those from the west districts (P<0.05). The pH1 and pH2 of SM and
LD of buffaloes from the north districts were also higher than those from the west districts
(P<0.05).
Keywords: North and west districts, Nakhon Phanom, carcass composition, meat quality
INTRODUCTION
Buffalo is an animal that important to the farmers throughout Thailand as part of the
agricultural production system, with a growing income. Long ago, the buffalo were used as a source
of labor in agriculture. The manure was used as a fertilizer and when necessary, it can be sold as an
additional way. At the same product can be used in fields which have been widely used as a feed to
the high price of meat (Kanjak, 2007).In Nakhon Phanom Province has a population of buffaloes
5.05% of the country, 6.85% in the Northeast and 17.96% of livestock fourth zone (Nakhon
1258
Phanom Livestock office, 2011) involving an area of Ta-u-ten District, Nawa District, Nathom
District, Wangyang District, Srisongkram District and Banpeang District. The buffalo raising in the
west and north districts will be allowed to graze at difference area all seasons. The data of carcass
of buffalo from difference area of Nakhon Phanom are rare. Therefore, a study on comparative of
carcass composition and meat quality of west and north districts will be used as basic data for
buffalo development of Nakhon Phanom province in the future.
MATERIAL AND METHOD

The experiment was conducted by using buffaloes (five years old) that raised in 4 Districts
of Nakhon Phanom Province involve in an area of Renu-Nakhon and Plaplak (West District)
Srisongkram and Banpeang (North District). Twenty buffaloes from west and north district 10
males and 10 females with an average weight of 550kg and 450 kg respectively. The buffaloes were
randomly selected and slaughtered in the slaughter house of Renu-Nakhon and Banpeang District.
Buffalo carcasses were cut using theThai style of cutting. The carcass compositions were separated
into 21 details of cut and 11 details of gut compositions. All compositions were weighted and
means of all composition were determined for analysis of variance and compared between sex using
group t-test and the SPSS version 16.0 program. (Steel and Torrie, 1980).
RESULT AND DISCUSSION

Carcass composition of buffalo
The carcass composition of buffalos from west and north District of Nakhon Phanom
Province were showed in Table 1. It was found that hind shank, lean fore leg, pin strips hind leg,
pin strips leg, breast meat, rib meat, LD, flat and tongue+head meat of buffalo in west were higher
than north (P<0.05) while lean hind leg, tender loin and skin of buffalo in north were higher than
west (P<0.05) and another composition showed that fore shank, bone fore leg, rib bone, breast bone
and head were no difference between two places (P>0.05) respectively.
Gut compositions of buffalo
The guts compositions of buffaloesin west and north district showed in Table 2. It was found
that kidney, kidney fat, lung, reticulum and omasum of west were higher than north (P<0.05) while
spleen and BF were lower (P<0.05). The other gut composition (heart, liver, abomasum and total
fat) were similar between west and north (P>0.05) distracts.
Meat quality of buffalo

The LD quality of buffaloes meat in west and north districts were showed in Table 3. It was
found that cooking loss at 1 h of west were higher than north district (P<0.05) and cooking loss at
24 h, pH1 and pH2were lower than north district (P<0.05) while drip loss were similar between two
place (P>0.05).
The SM quality of buffaloes meat in west and north districts showed in Table 4. The
cooking loss at 24 h, pH1 and pH2 of north district were higher than west district (P<0.05) while
cooking loss at 24 h and drip loss were similar between two group (P>0.05).
CONCLUSION
Buffaloes from west district had higher hind shank, lean fore leg, pin strips hind leg, pin
strips leg, breast meat, rib meat, LD, flat and tongue+head meat while lean, hind leg, tender loin and
skin were lower. Most gut composition of west district (kidney, kidney fat, lung, reticulum and
omasum) were higher than north while spleen and BF were lower. Meat quality of LD was cooking
loss at 1 h of west were higher than north district and cooking loss at 24 h, pH1 and pH2were lower.
The SM on the other hands, cooking loss at 24 h, pH1 and pH2 of north were higher than west
district.
1259
ACKNOWLEDGEMENTS
The authors would like to express their most sincere thanks Nakhon Phanom University and
Faculty of Agriculture and Technology, Nakhon Phanom University for their kind financial support
and use of the research facilities.
REFERENCE
Chantalakana, C. 1984. Buffalo in Thai farmer system. Bangkok; Wattanapanich. 150 p.
Kanjak, K. 2007. Book Elactronics “Teacher of Wisdom”. Faculty of Technology, Rajabhat Maha
Sarakham University, Thailand. 243 p.
Nakhon Phanom Livestock office. 2011. Livestock Statistics. Nakhon Phanom, Thailand.
(http://www.dld.go.th/pvlo_kop/index.html).
Steel, R.G.D. and J.H. Torrie. 1980. Principle and Procedures of Statistics: A Biometrical
Approach. 2ndedn. McGraw-Hill Book Company, New York, New York. 631 p.
Table 1. Carcass compositions of buffaloes (% of hot carcass) from difference districts.
Carcass Composition West North Means P-value

Fore shank 2.05 1.98 2.02 0.59
Hind shank 2.03a 1.64b 1.84 0.00
Lean fore leg 17.61a 15.28b 16.45 0.04
Bone fore leg 3.78 3.95 3.87 0.24
Lean hind leg 20.28b 23.42a 21.85 0.00
Bone hind leg 4.10 3.76 3.93 0.15
Pin strips hind leg 3.71a 1.74b 2.73 0.00
Pin strips leg 1.99a 1.55b 1.77 0.03
Breast meat 5.47a 4.31b 4.89 0.01
Rib meat 8.41a 4.34b 6.38 0.00
Rib bone 8.51 8.04 8.28 0.10
Longissimus dorsi (LD) 8.51a 6.19b 7.35 0.00
Neck 4.35a 9.29b 6.82 0.00
Breast bone 1.89 2.02 1.96 0.14
Flat 6.39a 4.48b 5.44 0.00
Tender loin 2.82b 4.26a 3.54 0.00
Head 6.32 6.94 6.63 0.19
Tongue + Head meat 3.49a 1.93b 2.71 0.00
Skin 22.84b 27.00a 24.92 0.01
ab
In difference row was significant at P<0.05 (95%)
1260
Table 2. Guts compositions of buffaloes (% of hot carcass) from difference districts.
Guts composition West North Means P-value

Heart 1.09 1.03 1.06 0.40
Spleen 0.52b 0.67a 0.60 0.01
Kidney 0.64a 0.50b 0.57 0.01
Kidneyfat 0.41a 0.29b 0.35 0.04
Lung 3.38a 2.20b 2.79 0.00
Liver 3.32 2.47 2.90 0.07
Rumen 5.62 5.07 5.35 0.29
Reticulum 1.08a 0.75b 0.92 0.00
Omasum 2.32a 1.42b 1.87 0.01
Abomasum 0.92 0.97 0.95 0.77
Total fat 2.22 3.17 2.70 0.09
BF (back fat thickness) 0.50b 1.12a 0.81 0.00
ab
Table 3. Quality of the Longissimus dorsi (LD) of buffalo from difference districts.
Meat quality West North Means P-value

Cooking loss at 1 h (%) 15.42a 8.15 11.79 0.00
Cooking loss at 24 h (%) 8.23b 9.44a 8.84 0.05
Drip loss at 24 h (%) 7.10 6.89 7.00 0.59
pH1 5.39b 5.83a 5.61 0.01
pH2 4.71b 4.88a 4.80 0.01
ab
Table 4.Quality of the Semimembranosus (SM) of buffalo from difference districts.
Meat quality West North Means P-value

Cooking loss at 1 h (%) 8.38 7.68 8.03 0.07
Cooking loss at 24 h (%) 8.48b 9.65a 9.07 0.03
Drip loss at 24 h (%) 8.81 6.87 7.84 0.55
pH1 5.20b 5.59a 5.40 0.01
pH2 4.70b 4.82a 4.76 0.03
ab
1261
Effect of Gender on Carcass Composition and Meat Quality of Buffalo

in wet-land in Nakhorn Phanom
Tanom TATHONGa, Chanachai BOONPERMb, Suthipong URIYAPONGSONc, Sorat

PRAWEENWONGWUTd
a
Program in Food Technology, Faculty of Agriculture and Technology, Nakhon Phanom University,
Thailand,
b
Program in Animal Production Technology, Faculty of Technology, Udonthani Rajabaht University,
Udonthani, Thailand,
c
Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Thaialnd,
d
Program in Plant Science, Faculty of Agriculture and Technology, Nakhon Phanom University,
Thailand
*Corresponding email: bangson_tim@hotmail.com
ABSTRACT
The objective of this research was aim to study on effect of gender on carcass composition and
meat quality of buffalo in Nakhon Phanom Province. The method of this research used group t-test
comparison at significantly 95% by Steel and Torrie (1980). Twenty buffalos were randomized from 3
Districts of Nakhon Phanom Province involving in an area of Ta-u-ten District, Srisongkram District
and Banpeang District. The buffalos were slaughtered and used Thai style cutting in the slaughter
house of Banpeang district. The data of this study composed of 21 carcass compositions and 11 gut
compositions. Meat samples form Longissimus dosi (LD) and Semimembranosus (SM) were collected
from each carcass and determined for meat quality (cooking loss and pH at 24 hour after slaughtered).
The result of carcass compositions showed that pin strips leg and flat in female were higher than male
(P<0.05) while the skin were higher in male (P<0.05). However, guts composition demonstrated that
reticulum of female were higher than male (P<0.05) while the others were not difference between
gender. Meat quality showed that cooking loss at 24 h of LD in female were higher than those from
male (P<0.05). More over the pH2 of SM and LD from male buffaloes were higher than those from
female buffaloes (P<0.05).
Keywords: carcass composition, gut compositions, meat quality, buffalo, Nakhon Phanom
INTRODUCTION
Buffalo is an animal that important to the farmers throughout Thailand as part of the
agricultural production system, with a growing income. Long ago, the buffalo were used as a source of
labor in agriculture. The manure were used as a fertilizer and when necessary, it can be sold as an
additional way. At the same product can be used in fields which have been widely used as a feed to the
high price of meat (Kanjak, 2007). In wet-land of Nakhon Phanom Province has a population of
buffaloes 5.05% of the country, 6.85% in the Northeast and 17.96% of livestock fourth zone. In 2011
with Buffalos 60,188 heads are mostly in wetlands 61.51% (Nakhon Phanom Livestock office, 2011)
involve an area of Ta-u-ten District, Nawa District, Nathom District, Wangyang District, Srisongkram
District and Banpeang District. The buffalo raising in the wetlands will be allowed to graze in the
underwater and water front that is rich in grass all seasons. The data of carcass of buffalo from wetland
area are rare. Therefore, a study on comparative of carcass composition and meat quality of male and
female will be the basic data for buffalo development of Nakhon Phanom province in the future.
1262
MATERIAL AND METHOD

The experiment was conducted by using buffaloes (five years old) that raised in wet-land of
Nakhon Phanom Province (Ta-u-ten District, Srisongkram Districts and Banpeang Districts). Twenty
buffaloes (10 females with an average weight of 450 kg and 10 males with an average body weight of
550 kg) were randomly selected from selected districts and slaughtered in the slaughter house of
Banpeang District. Buffalo carcasses were cut using the Thai style of cutting. The carcass compositions
were separated into 21 details of cut and 11 details of gut compositions, respectively. All compositions
were weighted and means of all composition were determined for analysis of variance and compared
between sex using group t-test and the SPSS version 16.0 program. (Steel and Torrie, 1980).

Carcass composition
The carcass compositions of buffalos from wet-land were showed in Table 1. It was found that
pin strip leg and flat of female were higher than male (P<0.05) while skin were lower (P<0.05).
However; fore shank, hind shank, lean fore leg, bone fore leg, lean hind leg, bone hind leg, pin strips
hind leg, breast meat, rib meat, rib bone, longissimus dorsi (LD), neck, breast bone, tender loin, head
and tongue + head meat were similar between male and female (P>0.05).
Gut composition
The gut compositions of buffaloes in wetland showed were showed in (Table 2). It was found
that reticulum of female was higher than female (P<0.05) while the other gut composition (heart, speel,
kidney, kidney fat, lung, liver, rumen, Omasum, abomasum, total fat) were similar (P>0.05)
Meat quality of buffalo
The LD quality of buffaloes meat in wet-land were showed in Table 3. It was found that
cooking loss at 24 h from female meat was higher than male (P<0.05). However, pH2 (pH after 24
hours) of meat from female buffalo was lower than male (P<0.05) while the other quality (cooking loss
at 1 h, drip loss and pH2) were similar (P>0.05).
The SM quality of buffaloes meat in wet-land showed in Table 4. It was pH2 from female was higher
than male (P<0.05) while other quality (cooking loss at 1 h, cooking loss at 24 h, drip loss and pH1)
were similar between male and female (P>0.05).
CONCLUSION
Female buffaloes from wet-land had higher pin strip and flat cut but had lower skin part
compared to male while other compositions were similar. Most gut composition of female buffaloes
was similar except reticulum was higher. Meat quality of LD was similar between gender except
cooking loss was higher and pH2 was lower in female. SM on the other hands, the pH2 was higher
while other quality were similar compared to male buffaloes.
ACKNOWLEDGEMENTS
The authors would like to express their most sincere thanks Nakhon Phanom University and
Faculty of Agriculture and Technology, Nakhon Phanom University for their kind financial support and
use of the research facilities.
REFERENCES
Kanjak, K. 2007. Book Elactronics “Teacher of Wisdom”. Faculty of Technology, Rajabhat Maha
Sarakham University, Thailand.
Nakhon Phanom Livestock office. 2011. Livestock Statistics. Nakhon Phanom, Thailand.
1263
Steel, R.G.D. and J.H. Torrie. 1980. Principle and Procedures of Statistics: A Biometrical Approach.
2nd edn. McGraw-Hill Book Company, New York, New York.
Table 1. Carcass composition of buffalo (% of hot carcass)

Carcass Composition Male Female Means P-value
Fore shank 1.98 2.08 2.03 0.61
Hind shank 1.64 1.63 1.64 0.92
Lean fore leg 15.28 15.15 15.22 0.68
Bone fore leg 3.95 3.54 3.75 0.09
Lean hind leg 23.42 22.13 22.77 0.07
Bone hind leg 3.76 3.27 3.52 0.15
Pin strips hind leg 1.74 1.79 1.77 0.82
Pine strips leg 1.55b 1.90a 1.73 0.05
Breast meat 4.31 4.35 4.33 0.89
Rib meat 4.34 4.45 4.40 0.78
Rib bone 8.04 8.31 8.17 0.48
Longissimus dorsi (LD) 6.19 6.33 6.26 0.65
Neck 9.29 8.99 9.14 0.66
Breast bone 2.02 1.85 1.94 0.18
b
Flat 4.48 5.47a 4.97 0.03
Tender loin 4.26 4.49 4.38 0.66
Head 6.94 7.06 7.00 0.79
Tongue + Head meat 1.93 2.00 1.96 0.57
a
Skin 27.00 23.82b 25.41 0.00
ab
Table 2. Guts composition of buffalo in wet land (% of hot carcass)

Guts composition Male Female Means P-value
Heart 1.02 1.04 1.03 0.81
Spleen 0.67 0.69 0.68 0.79
Kidney 0.50 0.54 0.52 0.38
Kidney fat 0.29 0.33 0.31 0.18
Lung 2.20 2.15 2.18 0.70
Liver 2.47 2.61 2.54 0.53
Rumen 5.07 4.61 4.84 0.36
b a
Reticulum 0.75 0.90 0.82 0.02
Omasum 1.42 1.64 1.53 0.42
Abomasum 0.95 0.95 0.95 0.92
Total fat 3.17 3.90 3.53 0.19
BF (back fat thickness) 1.12 1.12 1.12 1.00
ab
1264
Table 3. Longissimus dorsi (LD) quality of buffalo (%)

Meat quality Male Female Means P-value
Cooking loss at 1 h 8.88 8.15 8.52 0.22
b
Cooking loss at 24 h 9.00 9.44a 9.22 0.01
Drip loss at 24 h 5.91 6.89 6.40 0.21
pH1 5.98 5.83 5.91 0.13
a
pH2 4.94 4.88b 4.91 0.02
ab
Table 4 Semimembranosus (SM) quality of buffalo (%)

Meat quality Male Female Means P-value
Cooking loss at 1 h 7.42 7.68 7.55 0.35
Cooking loss at 24 h 9.33 9.65 9.49 0.35
Drip loss at 24 h 6.57 6.87 6.72 0.55
pH1 5.65 5.59 5.62 0.32
a
pH2 4.98 4.82b 4.90 0.02
ab
In difference low was significant at P<0.05 (95%)
1265
Tenderization Effect on the Physical Characteristics of Commercial Cuts of Second

and Third Buffalo´s Meat (Bubalus Bubalis) During Ripening Process
Luisa Fernanda GARCÍA DELVASTOa*, Laura Marcela MENDOZA ARAGÓNa* and Javier
Francisco REY RODRÍGUEZa*.
a
Food Engineer, Universidad de La Salle, Bogotá, Colombia. bTeacher, research and Food Engineer,
Universidad de La Salle, Bogotá, Colombia.
*Corresponding email: felho717@hotmail.com, lauramarcela_28@hotmail.com,
jrey@unisalle.edu.co.
ABSTRACT
Buffalo’s meat production in Colombia, is becoming an alternative for, consumption of a lean,
low cholesterol and tasty product in accordance with market regarding new trends in meat products;
however, sub-primal cuts have some marketing issues mainly due to its increased toughness. The
purpose of this research is testing the effect of tenderization in the ripening process to decrease the
toughness indexes sub-primal cuts, in order to standardize that way the most effective treatment.
Selected sections were arm loin, paletero and arm murillo. Every section was submitted to the
following processes: ripening (M), tenderization- ripening (TM), ripening-tenderization (MT),
ripening-tenderization-ripening (MTM) and tenderization-ripening vacuum packaging (TV) by testing
both water retention capacity (WRC) under cooking and texture, using the Warner Bratzler method in
three weeks. The process of TM for paletero, arm murillo, and MT for arm loin showed decreased
toughness without the WRC effect, based on these results, were selected for a more demanding test of
these processes about the properties of the cuts. (pH, color, hardness, WRC by dripping and WRC by
cooking). Further than demonstrating that day 14th is the most appropriate for the ripening of these
products at 2°C, the results didn’t show any pH alteration of tested samples; in addition, sections under
both of these methods showed to have higher WRC, decreased toughness and stronger color, when
compared with the samples that weren’t submitted to tenderization. From this study, it was concluded
that ripening process can be improvised the sub-primal cuts of buffalo’s meat.
Keywords: Tenderization, ripening, buffalo meat, hardness
INTRODUCTION
Buffalo’s meat production in Colombia is becoming an alternative for compsuntion of a lean,
and good tasty product, in accordance with marketing and regard news trends (Martínez, 2003), so in
comparison with the bovine meat is more nutritive, in addition it has contain 40%t less cholesterol,
55% less calories, 11% more of protein and 10% more minerals (ABCB, sf.). Cuts of second and third
buffalo’s meat show difficult to marketing, for this reason it used as raw material in the manufacturing
of some subproducts, with the consequent decrease of incomes for the producer. Bos Indicus Ltda.
(Personal communication. November 6 th /2011). The purpose of this research was decrease the
hardness of these cuts with the application of tenderization and ripening process. Based on the above
were selections cuts of second and third to work, evaluating the physical characteristics of the cuts
during the ripening process.

Selection of buffalo’s meat cuts and definition of the repetition in the tenderization.
By means of the test of texture Warner Bratzler , following the protocol of Herrera, Bolaños and
Lutz (2003) modifying the dimensions of the pieces of meat (4x5x0 5cms) for the cogote, arm ball, arm
1266
murillo, paletero and arm loin at a height of shearing of 28 mm. From each cut, it took a sample of
approximately of 300g, this at a time was divided into 3 pieces, passing the first fillet once for the
tenderization , the second twice and the third three times. These fillets were visually examined for a
sensory panel not trained, which determined the quantity of recommended times to submit the cuts of
tenderization.
Tenderization and ripening buffalo’s meat
Buffalo’s meats cuts were submitted to tenderization and ripening to 2°C during 21 days,
according to the protocol proposed in the research “Evaluación comparativa de las propiedades,
fisicoquímicas, funcionales y sensoriales de la carne de búfalo (Bubalus Bubalis) y vacuno (bos
indicus), maduradas a bajas temperaturas” developed by Guatava and Trujillo (2011). These were
placed in polystyrene trays, these are wrapped with PVC retractable material for the procedure from 1
to 4, and for the procedure 5 were packaged in vacuum. [Table 1].
Monitoring physical characteristics of the meat.
Was monitored physical characteristics of the meat, such as color, through Minolta coordinates
L, A, B, pH, WCR drip and cooking (steam) and texture by Warner Bratzler shear and Volodkevich
test. This monitoring was performed on days 1, 7, 14 and 21.

Selections of buffalo’s meat cuts and definitions of repetitions in the tenderization.
The selected cuts proved to be the arm loin , arm murillo and paletero, as they presented an
index of increased hardness into of the 5 cuts studied in this section. The arm loin is tenderization once
and the other cuts twice.
Effect of the tenderization in the physical characteristics of the meat
In the present study, the effect of the tenderization over the color was not significant, for as the
days, pass the tone of the meat became darker, normal procedure to the oxidation reaction in
myoglobin. This behavior responds to pseudoequilibrio (oxidation-reduction) and the final oxidation
that occurs in the metamyoglobin as indicated Lopez and Vanaclocha (2004), this was product of the
breakdown of muscle fibers during the tenderization and contact with oxygen.
It can be said that the implementation of the tenderization does not affect then pH , when
performed prior and subsequent to the ripening, because the values are within the normal range of
ripening, according to the values set by the academy of the area of pilots plants for food (2004), that
expose that the pH to be found in values between 5,8 and 6,4 show the optimum phase of normal
ripening (meat slightly acid). Happening the contrary in the MTM process, when the values increased
in accordance with spend the week (Table 2).
The behavior of the WRC by drip is optimal, because there are not significant differences
between the cuts, nor between the days of the monitoring. With regard to the implementation of the
tenderization, this does not affect significantly the WRC, because the water that is lost during the
procedure is superficial, as they say Moron and Zambrano (2004), the free water is located in low
proportions while in the course of the ripening the bound water is stable. With regard to the WRC by
cooking, the arm murillo was the cut had a better retention of water during all procedures, with a
percentage of 78,94± 9,14% registered during the day 21 and the arm loin cut that less water retained in
it’s structure with a percentage of 58,,41±3,92% during the day 14 when performing the ripening to the
tenderization later.
In our study, the decrease of the hardness by mean of the shear Warner Bratzler with the
implantation of the tenderization was efficient especially for the cut of arm loin down played
considerably that feature, starting with 3,30±1,25Kgf for the day 1 and ending with 0,77±0,08Kgf for
the day 21.
1267
With the Volodkevich test (Table 3) it became apparent that the decrease of the hardness was
effective corresponded to the day 7, in the MT method with a force of 0,71±0,13Kgf for the arm loin.
REFERENCE
ABCB. (Associação Brasileira de criadores de Búfalos). (s.f). Extraído El 28 de Octubre de 2011 desde
http://www.bufalo.com.br/carne.html
Academia del Área de Plantas Piloto de Alimentos. (2004). (2ed). Introducción a la tecnología de
alimentos. México: Limusa
Guatava C. y Trujillo L. (2011). Evaluación comparativa de las propiedades, fisicoquímicas,
funcionales y sensoriales de la carne de búfalo (BubalusBubalis) y vacuno (bosindicus),
maduradas a bajas temperaturas. Trabajo de grado. Universidad de la Salle. Bogotá-Colombia.
Herrera, C., Bolaños, N. y Lutz, G.(2003).Química de Alimentos: Manual de Laboratorio. San Jose:
Universidad de Costa Rica.
López, y Vanaclocha, A. (2004). Tecnología de mataderos. Tecnología de alimentos. España: Mundi-
Prensa
Martínez, P. (2003). Que la carne de búfalo no le saque cuerno. Catering. Año 2 No.6.
Morón O. y Zamorano L. (2004). Perdida por goteo en carne cruda de diferentes tipos de animales.
Revista Científica. Vol XIV - No. 001. Recuperado el 28 de julio de 2012 desde
http://www.saber.ula.ve/bitstream/123456789/28013/2/art5.pdf
http://www3.sympatico.ca/howard.swatland/Brazil.htm
Table 1. Ripening and tenderization procedure.
Procedures
M
1. Ripening at a temperature of 2 degrees during 21n days.
Patron
1. Tenderization
TM 2. Ripening during 21 days at a temperature of 2 degrees
1. Ripening during 21 days a t 2 degrees

MT 2. Tenderization
1. Ripening during 10 days at a temperature of 2 degrees

MTM 2. Tenderization
3. Ripening during 11 days at 2 degrees.
1. Tenderization
TV
2. Based of the vacumm packing and leave at a temperature of 2 degrees.
1268
Tabla 2. Effect of the tenderization in the pH of the buffalo´s meat.
Cut Day 1 Day 7 Day 14 Day 21

Ripening
Arm murillo 5,83±0,09 6,33±0,43 6,23±0,17 7,07±0,76
Arm loin 5,64±0,04 5,77±0,10 5,79±0,02 6,42±0,11
Paletero 5,89±0,16 5,54±0,12 5,71±0,26 6,50±0,24
Tenderization-ripening
Arm murillo 6,21±0,11 6,31±0,31 6,88±0,33 7,23±0,33
Arm loin 5,59±0,02 5,52±0,03 5,75±0,04 5,96±0,05
Paletero 5,74±0,13 5,64±0,11 6,11±0,19 6,76±0,63
Ripening-tenderization
Arm murillo 6,23±0,34 6,38±0,37 6,48±0,54 7,01±0,19
Arm loin 5,47±0,11 5,55±0,15 5,86±0,10 6,08±0,19
Paletero 5,72±0,05 5,47±0,10 5,63±0,15 6,50±0,20
Ripening-tenderization-ripening
Arm murillo 6,11±0,17 6,30±0,32 6,58±0,45 6,90±0,19
Arm loin 5,58±0,04 5,93±0,47 6,30±0,51 6,12±0,25
Paletero 5,82±0,03 5,49±0,09 6,86±0,45 7,47±0,36
Tenderization-vacum ripening
Arm murillo 6,41±0,03 6,08±0,17 5,77±0,19 5,92±0,11
Arm loin 5,71±0,06 5,38±0,01 5,58±0,05 5,48±0,06
Paletero 5,76±0,05 5,40±0,13 5,77±0,19 5,56±0,15
Tabla 3. Effect of the tenderization in the texture with Volodkevich test in the buffalo´s meat.
Cut Day 1 Day 7 Day 14 Day 21
Ripening
Arm murillo 1,78±0,67 1,68±1,00 0,43±0,40 0,81±0,59
Arm loin 0,54±0,41 1,56±0,67 1,04±0,18 0,66±0,07
Paletero 1,25±0,97 1,13±0,74 0,63±0,38 0,44±0,24
Tenderization-ripening
Arm murillo 1,27±0,68 1,10±0,59 0,87±0,06 1,24±0,76
Arm loin 0,45±0,09 2,00±0,59 1,78±0,43 1,12±0,32
Paletero 0,12±0,05 0,97±0,64 0,79±0,16 1,68±1,09
Ripening-tenderization
Arm murillo 2,01±0,67 1,70±0,93 0,78±0,05 1,13±0,23
Arm loin 1,12±0,45 0,71±0,13 0,87±0,21 1,05±0,31
Paletero 0,91±0,03 0,87±0,28 0,39±0,12 0,38±0,23
Ripening-tenderization-ripening
Arm murillo 1,40±0,35 1,44±0,47 0,74±0,16 1,71±0,89
Arm loin 1,64±0,35 1,33±0,44 1,81±0,42 1,35±0,35
Paletero 1,45±0,67 1,50±0,48 0,35±0,12 1,40±0,79
Tenderization-vacum ripening
Arm murillo 1,89±0,20 1,39±0,83 1,09±0,81 1,76±1,02
Arm loin 1,28±0,89 1,96±0,24 1,69±0,28 1,12±0,56
Paletero 1,21±0,09 1,50±0,48 0,86±0,25 1,40±0,79
1269
Conjugated Linoleic Acid and Fatty Acids Profile in Buffalo Meat
Monica I. CUTRIGNELLIa* Serena CALABRÒa, Raffaella TUDISCOa, Biagina

CHIOFALOb, Nadia MUSCOa, Oswaldo J. GONZALEZ, Micaela GROSSIa, Giovanni
MONASTRAa, Federico INFASCELLIa
a
Department of Veterinary Medicine and Animal Production, University of Napoli Federico II, Via
F. Delpino 1 80137, Napoli, Italy
b
Department of Veterinary Sciences, University of Messina Polo Universitario Annunziata, 98168
Messina Italy
*Corresponding email: cutrigne@unina.it
ABSTRACT
Aim of the present study was to evaluate the fatty acids profile of buffalo meat. The trial
was carried out on 16 buffalo calves fed a total mixed ratio (2.7% body weight) composed by a
commercial concentrate, mixed hay and corn silage (CP: 14.9 % DM; 0.91 VFU/kg DM). All the
animals were slaughtered when the BW of 350 kg was reached (about 420 d of age). Samples of
Longissimus thoracis (LT), Semitendinosus (ST), Iliopsoas plus Psoas minor (IP) were analysed for
fatty acids profile by gas chromatography (GC). To this purpose total fat was extracted and
subsequently turned into methyl esters (FAMEs) by direct methylation. The Atherogenic Index (AI)
and the Thrombogenic Index (TI) were also calculated. The muscle type highly affected the results:
ST showed the most favourable fatty acids profile and consequently, lower values for both index
(AI: 0.60, 0.42, 0.57 and TI: 1.50, 1.02, 1.51, for IP, ST and LT, respectively) as well as CLAs
contents (0.20, 0.25, 0.21 g/100g; for IP, ST and LT, respectively). In each case, the results of
present trial should confirm the favourable assessment of the nutritional characteristics of the meat
from buffalo young bulls.
Keywords: Meat, Italian Mediterranean Buffalo, fatty acid profile, CLA.
INTRODUCTION
Ruminant meat has been longer criticized for its high levels of mutagens and carcinogens
(Belury, 1995) and considered one of the factors that may lead to the development of human
cardiovascular diseases, obesity, hypertension, and cancer, especially due to the high levels of
saturated fatty acids (SFA) and cholesterol. However, mainly in the case of grass-fed ruminants,
meat and milk are the best dietary sources of CLAs, which has been shown to have health
promoting effects, as well as immunomodulating and anticarcinogenic activity (Pastuschenko et al.,
2000; Whigham et al., 2000). Additionally, CLAs has been reported to reduce atherosclerosis and
total serum cholesterol (Lee et al., 1994). The CLAs are a group of positional and geometric fatty
acid isomers derived from linoleic acid (Parodi, 1999) of which the main representative isoform is
the cis-9, trans-11 CLA, known as rumenic acid (Kramer et al., 1998). Comparing bovine and
buffalo meat, Infascelli et al. (2004) reported several favourable nutritional characteristics for
buffalo meat: lower SFA and higher monounsaturated fatty acids (MUFA) and polyunsaturated
fatty acids (PUFA) percentages. Recently, Giordano et al. (2010) found that consumption of buffalo
meat is associated with several beneficial effects on cardiovascular risk profile, including lower
carotid atherosclerotic burden and susceptibility to oxidative stress. Aim of the present study was to
evaluate the fatty acids profile and, in particular, CLA content of the meat in the Italian
Mediterranean Buffalo bred in an intensive system.

The trial was carried out on 16 Italian Mediterranean buffalo calves (age 30 d, BW 55 kg)
that received 6 l/head/d of acidified milk replacer (180 g/l of water) until 56 d. Subsequently, the
replacer amount was gradually decreased, whilst administering the same volume. Roughly chopped
1270
alfalfa hay and weaning concentrate were available from the fifth week of life and corn silage was
administered from 70 d of age. After weaning until the trial beginning, the animals were fed ad
libitum hay and corn silage; the concentrate was administered in the amount of 2 kg/d. At the age of
84 d (average 30 kg BW), each animal was placed in individual box up to the slaughtering weigh
and was fed (2.7% body weight) a total mixed ratio (CP: 14.9 % DM; 0.91 VFU/kg DM) composed
by a commercial concentrate, mixed hay and corn silage. All the animals were slaughtered in an
authorized slaughterhouse according to EU legislation (EU Regulation EC No 882/2004) when the
BW of 350 kg was reached (about 420 ± 59.4 d of age). Samples of Longissimus thoracis (LT),
Semitendinosus (ST), Iliopsoas plus Psoas minor (IP) muscles were collected seven days (at 3-5°C)
after slaughter, to simulate what usually is done in Italy to improve meat tenderness and water
holding capacity (Cutrignelli et al., 2008a). Fatty acids profile was analysed by gas chromatography
(Chiofalo et al., 2011). To this purpose total fat was previously extracted (Folch et al., 1957) and
subsequently turned into methyl esters (FAMEs) by direct transesterification (Christie, 1993). The
FAMEs were analyzed by GC-FID (Agilent Technologies 6890N, Palo Alto, CA, U.S.A.) with a
split/splitless injector, a flame ionization detector and fused silica capillary column Omegawax 250
(Supelco, Bellefonte, PA, U.S.A.), 30m x 0.25mm I.D., 0.25 μm film thickness. Column
temperature was programmed: initial isotherm of 160°C (6 min.), increment of 3°C/min and final
isotherm of 250°C (30 min.). Temperature of the injector and detector: 250°C. Injection volume:
1.0 μL. Carrier gas: helium (1 mL/min). Split ratio: 1:50. Identification of fatty acids was made by
comparing the relative retention times of FAME peaks from samples with standards from Supelco
(Bellefonte, PA, U.S.A.). Chromatogram peak areas were acquired and calculated by Chemstation
software (Agilent, Palo Alto, CA, U.S.A.). The concentration of each fatty acid was expressed as
g/100 g, considering 100 g the summation of the areas of all fatty acid methyl esters identified. For
each sample the chromatographic analysis was replicated three times.. Identification of fatty acids
was made by comparing the relative retention times of FAME peaks from samples with an external
standards (Supelco). The Atherogenic Index (AI) and the Thrombogenic Index (TI) were also
C12 : 0  (4 x C14 : 0)  C16 : 0

calculated as follows:
n - 6 PUFA  n - 3 PUFA  MUFA

AI =
C14 : 0  C16 : 0  C18 : 0

(0.5 x MUFA)  (0.5 x n - 6 PUFA)  (3 x n - 3 PUFA)  (n - 3 PUFA/n - 6 PUFA)
TI =
were MUFA and PUFA were expressed as g/100 g. Data set were statistically processed using the
SAS (2000) GLM procedure.
most favourable fatty acids profile: lower SFA, higher PUFA, MUFA, -3, -6, and consequently,
The muscle type highly affected the results (Table 1). The semitendinosus (ST) showed the
lower values for both index (AI: 0.60, 0.42, 0.57 and TI: 1.50, 1.02, 1.51, for IP, ST and LT,
respectively). In each case, these last values are particularly interesting; indeed the AI was lower
than those reported by Ulbricht and Southgate (1991) for raw minced beef, Cutrignelli et al (2008b)
for Marchigiana meat, as well as TI was lower than that reported by Cutrignelli et al (2008b). As
concerns the CLAs, also in this case the ST had the more favourable results. Indeed, the contents of
either the single isomers or of the total CLA were significantly higher in this muscle compared to
the LT and IP. In each case, the average value found in this trials were very interesting (0.23
g/100g), taking into account that meat was from animals bred in intensive system, without use of
pasture or linseed in the diet, and close to the data reported by other authors for Italian
Mediterranean buffaloes (Infascelli et al., 2004). It is well known that the presence of CLA in the
intramuscular lipids of large ruminants depends, in part, on the ruminal biohydrogenation of the
diets LA (Bauman et al., 1999). As reported by de Mendoza et al. (2005), many authors recorded
differences in rumen microbial metabolism, rumen ciliate protozoa population, forage-eating
behaviour, consumption and rumination, development and size of the rumen-reticulum between
1271
domestic buffalo as compared to cattle. These data suggest that the rumen supply of CLA readily
available for intestine absorption and further deposition in the intramuscular lipids of these
herbivorous would also differ. In conclusion, the results of present trial should confirm the
favourable assessment of the nutritional characteristics of the meat from buffalo young bulls.
REFERENCES
Bauman, D.E., L.H. Baumgard, B.A. Corl and J.M. Griinari. 1999. Biosynthesis of conjugated
linoleic acid in ruminants. Proc. American Society of Animal Science (pp. 1–15), Ithaca,
NY.
Belury, M.A. 1995. Conjugated dienoic linoleate: A polyunsaturated fatty acid with unique
chemoprotective properties. Nutrition Reviews, 53, 83–89.
Chiofalo B., V. Lo Presti, G. Savoini, E. D'Alessandro, V. Chiofalo and L. Liotta. 2011.
Nucleotides administration in broiler chichen diet: Effect on breast muscles quality. Czech.
J. Food Sci., 29 (4): 308-317.
Christie W.W. 1993. Preparation of ester derivatives of fatty acids for chromatographic analysis.
69–111. In W.W. Christie (ed.) Advances in lipid methodology, 2nd ed. Oily Press, Dundee,
UK.
Cutrignelli M.I., G. Piccolo, F. Bovera, S. Calabrò, S. D’Urso, R. Tudisco and F. Infascelli. 2008a.
Effects of protein source and energy value of diet on the performance of young Marchigiana
bulls: 1 Infra vitam performance and carcass quality. Ita. J. of Anim. Sci., 7: 259-270.
Cutrignelli M.I., S. Calabrò, F. Bovera, R. Tudisco, S. D’Urso, M. Marchiello, V. Piccolo and F.
Infascelli. 2008b. Effects of protein source and energy value of diet on the performance of
young Marchigiana bulls: 2. meat quality. Ita. J. of Anim. Sci., 7: 271-285.
de Mendoza M.G., L.A. de Moreno, N. Huerta-Leidenz, S. Uzcàtegui-Bracho, M.J. Beriain and
G.C. Smith. 2005. Occurrence of conjugated linoleic acid in longissimus dorsi muscle of
water buffalo (Bubalus bubalis) and zebu-type cattle raised under savannah conditions. Meat
Science 69: 93–100.
Folch J., M. Lees, and Sloane Stanley. 1957. A simple method for the isolation and purification of
total lipides from animal tissues. J Biol Chem., 226(1): 497-509.
Giordano G., P. Guarini, P. Ferrari, G. Biondi-Zoccai, B. Schiavone and Giordano A. 2010.
Beneficial impact on cardiovascular risk profile of water buffalo meat consumption. Europ.
J Clin. Nutr., 64: 1000–1006.
Infascelli, F., S. Gigli and G. Campanile. 2004. Buffalo meat production: Performance infra vitam
and quality of meat. Veterinary Research Communications. 28 (suppl. 1): 143-148.
Kramer, J.K.G., P.W. Parodi, R.G. Jensen, M.M. Mossoba, M.P. Yurawecz and R.O. Adlof. 1998.
Rumenic acid: a proposed common name for the major conjugated linoleic acid isomer
found in natural products. Lipids, 33: 835.
Lee, K.N., D. Kritchevsky and M.W. Pariza. 1994. Conjugated linoleic acid and atherosclerosis in
rabbits. Atherosclerosis, 108: 19–25.
Parodi P.W. 1999. Conjugated linoleic acid and other anticarcinogenic agents of bovine milk fat. J.
Dairy Sci., 82: 1339–1349.
Pastuschenko V., H.D. Matthes, T. Hein and Z. Holzer. 2000. Impact of cattle grazing on meat fatty
acid composition in relation to human nutrition. In: Alföldi, T., Lockeretz, W., Niggli, U.
(Eds.), Proc. 13th Int. IFOAM Scientific Conf., Basel, Switzerland, 293–296.
SAS. 2000 SAS/STAT User’s Guide, Version 8.2, Vol. 2, 4th Edition. SAS Institute Inc., Cary, NC.
Whigham L.D., M.E. Cook and R.L. Atkinson. 2000. Conjugated linoleic acid: implications for
human health. Pharmacol. Res., 42: 503–510.
1272
Table 1. Fatty acid profile (g/100 g) in the three muscles.
IP ST LT MSE
A B A
C14:0 1.42 0.92 1.31 0.026
C16:0 21.6A 20.3B 21.0AB 0.955
A B A
C18:0 27.5 20.0 27.2 2.50
C18:1n-9 27.8B 33.1Aa 31.2Ab 5.11
B A B
C18:1-cis11 1.17 1.48 1.16 0.012
C18:2n-6 8.12Ab 9.12Aa 6.47B 1.04
B A B
C18:3n-6 0.09 0.14 0.09 0.0069
C18:3n-3 0.56b 0.66a 0.51b 0.0073
b a b
cis9-trans11 CLA 0.05 0.06 0.04 0.00017
trans10-trans12 CLA 0.15b 0.19a 0.17ab 0.0017
SFA 50.0A 42.2B 49.9A 1.12
B A A
MUFA 28.4 34.2 32.0 4.9
9.52 A 10.50 A 8.56 B
 CLA
PUFA 0.87
0.20b 0.25a 0.21b 0.0001
A B A
AI 0.60 0.42 0.57
TI 1.50 A 1.02 B 1.51 A
IP: Iliopsoas plus Psoas minor; ST: Semitendinosus; LT: Longissimus thoracis.
MSE: Mean square error. Along the row: A,B,C: P<0.01 and a,b,c: P<0.05.
1273
Determination of Muscularity and Correlation with Body Weight in Buffalo in

the Northeast of Argentina
Gladis REBAK a*, Gustavo JAUREGUIb, Adriana CAPELLARIa and Natalia PRESTER b
a
Food Technology and Bovine Production courses. b Fellowship of the Science and Technology
Secretary. Veterinary School. University of the Northeast. Sargento Cabral 2139 (3400)
Corrientes. Argentina. Phone: 54- 379-4425753 int 145.
*Corresponding email: grebak@vet.unne.edu.ar
ABSTRACT
Muscularity index corresponds to an equation used to determine in living animal and
instantaneous real increased musculature and therefore higher yield performance, comparing the
ribeye area by live weight of the animal. This is a very important tool to evaluate carcass
characteristics necessary to identify the animal extensively raised for slaughter and at the age in
which meat tenderness have conditions demanded by consumers. In the last years the number of
buffalos in the northeast region of Argentina has been increased, especially in Corrientes province.
The aim of this study was to perform the calculation of the index of muscularity and determine its
relationship with the body weight. We worked with buffaloes of 250-710 kg in farms in the
provinces of Corrientes and Formosa, identifying them individually recording each animal, plus:
live weight, approximate age by teeth, high to the scapula, high at the rump, chest circumference
and long body, then proceeded to collect information by ultrasound ribeye area (REA), rib eye
thickness (AL), backfat thickness (EGD) and rump fat thickness (EGC or GC). Collected data was
performed to calculate the index of muscularity dividing the ribeye area per body weight of each
individual to give an index average of 0.11, then, use statistical software to perform a calculation
InfoStat correlation between this index and the weight of individuals determined that there is a
negative correlation between them. Rate of muscularity in buffalos (0.11) is lower than in cattle
(0.15) and that it varies with the weight of animals.
Keywords: Muscularity, Index, Buffalo, Carcass, Meat, Quality
INTRODUCTION
In the Argentina Republic beef cattle have been the traditional source of red meat (Irurueta
et. al 2008). National trade in meat systems are made: pay per kilogram of live animals, pay for
performance or yield (kilogram of meat and bone obtained after the slaughter). A current concept is
the industrial performance pay, is per kilogram of cuttings obtained from the slaughtering to be
boned carcasses, making the commercial cuts (retail cuts). In the trial of live animals is not always
possible to predict the performance of the covers will sacrifice no performance or industrial
(commercial cuts). For that industry is looking up animals with better conformation (muscle
building) and finishing (degree of fatness), the industry does not always get the best yields of
sacrifice and boned. The sale of buffalo meat is currently side-lined by differentiated be sold as
beef, the industry paid a lower price than beef steer for the misconception of the lower yield in
slaughterhouse (caused by the high weight of leather, head, feet and gastrointestinal tract) (Rebak et
al. 2004).
Regardless of the species, breed and sex, it should be noted that the composition of the muscles
vary with advancing age of the animal, there being a general raising of most parameters, other than
water, although the increase in speed so some are identical in all muscles. Even adults components
reach values at different times (Lawrie, 1998).

1274
Ultrasound has been used to calculate the measurements of fat and muscle in cattle for more than 30
years. However, the great advances in terms of portability and image resolution of the new
generation of ultrasound, in addition to the use of computerized image analysis finally made
possible the practical application of ultrasound. The study of the composition and quality in animal
production is one of the trends that is used as a tool in marketing systems with high added value,
and consists in measuring the ultrasound or ultrasound fat thickness or subcutaneous loin, loin eye
area or steak (REA), rump fat or P8 Australian and intramuscular fat or "marbling". In selecting
programs bulls and their progeny, evaluated a number of production parameters (conformation,
fertility, maternal ability, gain kg/d, etc.), but are capital importance, those other parameters that are
directly related to meat production, fattening and quality of carcasses and their products (Wilson,
1992).
Good correlations were observed between the ultrasound measurements of loin eye area of
fat cover, with actual measurements post-mortem. Muscularity index corresponding to an equation
used to determine in live animal instantly which has the largest muscles and therefore greater
performance postmortem. The result is obtained after dividing the ribeye area by the weight of the
animal. The index of muscularity is an important tool of choice to evaluate carcass characteristics.
Usually in cattle with ribeye areas of 6.87 cm2 achieve a live weight of approximately 45.3 kg. In
animals heavier than 453 kg, index of muscularity is less. The ribeye area is the measure of eye
muscle area (Longuisimus dorsi) in cm. It is a true indicator of the quality of the beef and carcass
and has negative correlation with fatness: with increasing muscle decreases fatness and vice versa,
which requires a good balance. At the same time, the greater muscular development increased yield.
The rib eye area is a measure of heritability half and has a high positive correlation with the genetic
percentage of retail (Ferrario and Fernandez, 2007).
The aim of this study was to perform the calculation of the index of muscularity in buffaloes from
the northeast of Argentina and determine its relationship with the body weight of the animals.

The work was conducted in the provinces of Formosa and Corrientes, Argentina, located
between parallels 22 and 30 degrees south latitude in the subtropical region. For the study analyzed
30 buffaloes from each provinces with 250-710 kg live weight, identifying them individually
recording each animal, plus: live weight with electronic scale, approximate age by teeth, high to the
scapula, high at the rump, chest circumference and long body with extensible tape measure, then
proceeded to collect information by ultrasound using Aquila Vet AS equipment sound THE 3.5-
MHz 30C 180 mm coupled with stand-off guide of ribeye area (REA) taken between the 12th and
13th rib, with the placement of the transducer perpendicular to the animal's position. In the
laboratory of Food Technology, Faculty of Veterinary Science, National University of the Northeast
collected data was performed to calculate the index of muscularity dividing the ribeye area per body
weight of each animal. Then we use InfoStat program for proceeded to perform the calculation of
correlation between this index and the live weight of animals.

The results obtained by relating parameters ribeye area and weight of buffaloes studied
determined that the rate of average muscularity of buffaloes tested is 0.11 and has a negative
correlation with the weight of 0.35. Figure I show the dispersion achieved by relating index of
muscularity and body weight. This negative correlation is because as increase the weight of the
animals increases the percentage of skin, bones and viscera in turn decreasing the muscle mass in
relation to the total weight.
Rate of muscularity in buffalos is lower than in cattle (0.15) and that it varies with the
weight of animals (Ferrario and Fernandez, 2007).
Garriz and Vranic (2008) found muscularity index (0.18 ±0.01) in crossbreeding carcasses from
Hereford, Shorthorn and Aberdeen Angus x Criollo Argentino cattle.
1275
This information is remarkable, knowing conformation traits have low to media heritability values
(Tonhati et al. 2008).
The routine use of this technology, which we call business, specifically applied to the
determination of the degree of fatness of animals for slaughter. This is to optimize feed resources so
as not to feed more animals that already have the degree of completion just for marketing.
REFERENCES
Ferrario J. D. and M. A. Fernández. 2007. Rev. Braford, Buenos Aires, 23(58):72-75.
Irurueta M., A. Cadoppi, L. Langman, G. Grigione and F. Carduza. 2008. “Effect of aging on the
characteristics of meat from water buffalo grown in the Delta del Paraná region of
Argentina”. J. Meat Science 79:529-533.
Garriz C.A. and L. Vranic. 2008. Genotype effects on muscularity index in meat steers. Rev.
Argentina de Producción Animal 28. Sup I: 177-233.
Lawrie R.A. 1998. Ciencia de la carne. 3ra. Ed Acribia. Zaragoza.367 p.
Rébak, G.Iand J.E. Cedrés. 2004.Características de la Carne de Búfalos como alternativa de
diversificación productiva. Comunicación de la Dirección de Estudios Económicos de la
Bolsa de Cereales. p 25- 26. Buenos Aires.
Rébak, G.I. 2006. La carne bubalina en el NEA. Caracteres de faena de novillos vacunos y bubillos.
Revista del Consejo Profesional de Médicos Veterinarios de Chaco y Formosa Nº 2. p 30-
32.
Tonhati H., G. Mendoza Sanchez. R.C. Sesana and P.R. Aspilcueta Borquis. Galvao de
Albuquerque l. 2008. Programa de mejoramiento de búfalos. Rev Argentina de Producción
Animal 28: 53-67.
Wilson, D.E. 1992. Application of ultrasound for genetic improvement. J. Anim. Sci., 70: 973- 983.
Figure 1. Shows dispersion of the index of muscularity and body weight of the buffaloes tested.
1276
Relation of the Approximate Age and Ultrasound Data in Buffalo in the Northeast
of Argentina
Gladis REBAK a*, Gustavo JAUREGUI b, Adriana CAPELLARI a and Macarena NAVARRO
KRILICH b
a
Food Technology and Bovine Production courses. b Fellowship of the Science and Technology
Secretary. Veterinary School. University of the Northeast. Sargento Cabral 2139 (3400) Corrientes.
Argentina. Phone: 54- 379-4425753 int 145.
*Corresponding email: grebak@vet.unne.edu.ar
ABSTRACT
The buffaloes in Argentina are distributed in the north eastern provinces of the country. Ultrasound is
used in breeding as a tool for assessing the meat composition in live animals, enabling determine
objectively and in real time the ability to produce meat. The four characteristics stand that ultrasound
can identify are: Rib eye area (REA) in Longissimus dorsi muscle, thickness or height of the steak
(AL), marbling, back fat thickness (EGD) and fat hip or rump (EGC or GC). The present work
described the effect of the ultrasound data in relation of the approximate age in buffaloes steers from
two provinces (states) of Argentina. For the experience were analysed 30 animals of each province,
recording live weight, approximate age by teeth and ultrasound data. We conclude that the values
associated with the meat production characteristics change with the age of animals in both provinces.
Keywords: Ultrasound, Meat, Age, Quality, Argentina, Buffaloes
INTRODUCTION
The stock buffalo in Argentina is distributed approximately 100.000 head in the northeastern
provinces of the country, focusing on meat and milk production (Rébak et al.2004). The production of
buffalo (Bubalus bubalis) in the last three decades is presented as an alternative to large agricultural
growing areas of Argentina on farms with poor quality forage supply, fitted with low digestibility
grass, high parasite loads, floors inadequate drainage, which means that they are waterlogged most or
all of the year and with a climate that allows to express the maximum traditional production systems. In
these circumstances, in the last years the population of buffalos has been increased in the northeast
area, where the stock is increasing every year (Arias Mañoti, 2007). Scientific studies evidence that
young buffalo meat is tender will be highly valuable in improving the image and price of buffalo meat
in developing countries, where the common perception has been that buffalo meat is of lower quality
than beef, a result from years of consuming buffalo meat from old retired draught animals. (Neath et. al
2007) There is currently sufficient stock to meet potential demand after a good promotion of the
species, so it is necessary to characterize the buffalo meat reared extensively and destined for slaughter
at an age when the meat has the tenderness requirements demanded by consumers. Previous studies
have shown that the use of ultrasound in real time provides predictive data on carcass quality and meat
cuts in live animals. Change of buffalo teeth at an early age (14-18 months) and always has tartar on their
incisors, even in young animals (Rebak, 2011). The aim of this study was correlating carcass quality
characteristics by ultrasound and approximate age of buffaloes steers reared in extensive systems from
the provinces of Corrientes and Formosa, Argentina.

1277

We worked with 30 buffaloes steers from Corrientes y Formosa provinces reared on natural
pastures identifying them individually recording each animal, live weight through electronic scale,
approximate age by teeth, then proceeded to collect information by ultrasound using Aquila Vet AS
equipment sound THE 3.5-MHz 30C 180 mm coupled with stand-off guide of: ribeye area (REA), rib
eye thickness (AL), backfat thickness (EGD) and rump fat thickness (EGC).
In the laboratory of Food Technology, Faculty of Veterinary Science, National University of the
Northeast with the collected data we proceed to perform a classification, grouping them according the
recording teeth (estimated age) and ultrasound data the following results.

The results of recording estimate age and ultrasound data of buffaloes steers from two
Argentina provinces are presented in Table 1. In this study we found that buffaloes baby tooth have
higher values of ribeye area, backfat and rump fat and buffaloes with four permanent teeth have higher
values of rib eye thickness, while animals with two permanent teeth shown intermediate values,
without statistically significant differences.
These values are similar to those reported by Zava (2011) and the same work group (Rébak
2010- 2011) in Argentina. Jorge et al (2010) achieved parameters higher than ours in Brazil in
buffaloes of 14 month of age, fed by 120 days with 70 % of concentrate diet.
We conclude that it is necessary to continue studies on these correlations with a greater number
of animals to provide utility to the productive sector, given that the dentition of buffalo is different to of
bovine.
REFERENCES
Arias Mañotti A. 2007. Panorama de la ganadería en diferentes ambientes del NEA. 1er Seminario de
Ganaderia del NEA. Amanecer Rural. Resistencia. Chaco. Argentina. p. 5-11.
Jorge A.M; Andrighetto C.; Francisco C.L; Pinheiro R.S.; Tavares S.A; Santos Silva T.L; Andrade
C.R.M.; Surge C.A; Pouli Filho J.N.P. 2010. Real Time Ultrasound to Predict Beef Carcass
Retail Products of Buffaloes. Proccedings 9th World Buffalo Congress. Rev. Vet. 21. Sup. 1:
527-529.
Neath K.E.; Del Barrio A.N.; Lapitan R.M.; Herrera J.R.V.; Cruz L.C.; Fujihara T.; Muroya S.;
Chchuni K.; Hirabayashi M.; Kanai Y. 2007. J. Meat Science 77: 389-396.
Rebak, G.I; Cedres, J.E.2004.Características de la Carne de Búfalos como alternativa de diversificación
productiva. Comunicación de la Dirección de Estudios Económicos de la Bolsa de Cereales. p
25- 26. Buenos Aires. Argentina.
Rébak G.; Capellari A.; Ynsaurralde Rivolta A.; Alarcón A. 2010.Exploratory study of ultrasound on
properties of meat in buffaloes in the Northeast of Argentina. Proceedings 9th World Buffalo
Congress. Rev. Vet. 21. Sup. 1: 512-414.
Rébak G. 2011. Bubalinocultura de las Americas. (Ed Moglia. Argentina). Cap 13. p 231-237.
1278
Zava M. 2011. El Búfalo Doméstico. (Ed. Orientación Gráfica Editora. INTA. Argentina). p 283-286.
Table 1. Please write down the title of this table precisely Results presented in Table 1 shown values
found for rib eye area (REA), rib eye thickness (AL), back fat thickness (EGD) and rump fat thickness
(EGC) according the approximate age by teeth: 0 (baby tooth), 2 (two permanent teeth) and 4 (four
permanent teeth).
TEETH REA AL EGD EGC

0 50,87±4,32 5,62±0,26 0,91±0,23 0,89±0,15
2 53,20±12,19 5,54±0,82 0,83±0,23 0,80±0,17
4 58,86±13,46 5,67±0,82 0,90±0,24 0,83±0,24
1279
Research Regarding the Dynamics of Body Development in Young Buffaloes,

Depending on Various Factors
Livia VIDUa*, Alina UDROIUa, Razvan POPAa, Vasile BACILAa, Adrian BOTAb
a
University of Agricultural Sciences and Veterinary Medicine Bucharest, Faculty of Animal
Science, Marasti Blvd. 59, Bucharest, Romania
b
Institute of Research and Development for Buffalo Breeding Sercaia Brasov, Str. Campului 2,
Sercaia, Romania
Corresponding email: vidulivia@yahoo.com
ABSTRACT
At present the world market is facing a deficit of beef. In terms of the total world
production of meat, buffalo meat contributes 1.3 percent. The quality and quantity of buffalo
meat depends on various factors. The most important are breed, age, feeding, natural and
artificial environmental conditions. The research was conducted on a total of 30 young male
buffaloes, of the Romanian breed, for 10 months. This study analyzes the evolution of body
development in buffaloes which can be used for meat production, depending on age, level of
feeding, and season. The experiment started when the buffaloes were 8 months old, and had a
body weight of 176 kg. At 17 months of age, the animals weighed 454.3 kg, with a total weight
gain of 278.3 kg. Daily average weight gain ranged from 750 g at the age of 8 months to 1135 g
at 17 months. The highest daily growth in weight was achieved at the age of 12 months in the
winter season - 1207 g. During the feeding, young buffalo meat, alfalfa hay or tare hay, green
grass or maize silage, and concentrates were used. Every month we made body measurements
(body weight, size at withers, the thoracic perimetre, the spiral perimetre of the pulp, oblique
length of the body), and at the end of the experiment we calculated synthetic indicators. Thus the
size at withers increased from 111cm (10 months) to 127.4 cm (17 months). The spiral perimetre
of the pulp increased to 165 cm at the age of slaughter (17 months).
Keywords: youth buffalos, weight, feed, size at withers, weight increase
INTRODUCTION
The growth and development of cattle represent the changes that occur when the single
celled -zygote transforms into an adult animal. Pradal (1989) explained these two phenomena by
the following: cell multiplication (hyperplasia), increased cell size (hypertrophy) and cell
specialization, empower their role in tissues and organs. During growth, the buffalo body passes
through two major changes: increasing live weight and reaching adult status by developing
tissues, organs and functions. The body weight growth of buffaloes is achieved by: accelerated
growth in the youth period, adulthood reduced growth, and decline of old growth stage.
Regarding the growth and development, Soltner (1987) cited by Georgescu and Vidu
(2008) presented the following succession for tissues and organs: the tissue-nervous, bone,
muscle and fat, the body regions-head, neck, trunk, members region - metacarpus, metatarsus,
radius, tibias, humerus, femur, shoulder girdle and pelvic, the fat - internal fat, intramuscular and
intramuscular and coverage fat.

1280
The growth of the young buffalo is continuous and high for the young animals for
intensive increased meat. The growth is discontinuous with a period of slowdown or more,
during the winter season.
The chemical composition of meat is different, depending on the age of the buffalo: meat
content in terms of water, proteins, and minerals, decreases with age. The dry matter content of
meat and fat increases with age.

The study was made on a herd of 30 male young animals of the Romanian buffalo breed.
The animals were 8 months old, and had a body weight of 176 kg at the beginning of the
experiment. The experiment lasted 10 months. The animals were carefully monitored regarding
the feeding technology, sheltering, food ingestion behavior, and social behavior.
Accommodation for the young buffaloes was made in collective boxes (three boxes), with a
capacity of 10 animals in each box, and providing a space of 3.5 square meters per animal. The
feeding was seasonally differentiated.
Every month the animals were individually weighed, the daily average gain was
calculated, and body measurements were performed.
Principal body measurements that were made are: body weight, size at withers, the
thoracic perimetre, the spiral perimetre of the pulp, oblique length of the body, theperimeter of
the shinebone, the chest width and croup width. On the basis of monthly measurements
performed, we calculated some body indices that provided information on levels of growth and
fattening of young buffalos. These indices are: index of lateral body shape (oblique length of the
body x 100 / size at withers) index of development (perimeter of the chest x 100 / size at
withers), index of bone (the perimeter of the shinebone x 100 / size at withers). The resulting
data were statistically processed and interpreted.

The experiment began in July and ended in April of the next year (2011), when the
animals were slaughtered. The animals in the experiment caught two seasons. The longest of
them were summer and winter. The animals which were studied in the experiment were fed with
rations that were seasonally differentiated. This system for growth is called the fattening in
intensive system. In the summer period youth buffalo which were meant for fattening were fed
with alfalfa hay or tare hay, green grass, and concentrates (Table 1). The volume of feed had a
rate ranging from 78% to 81.5%. In the winter, from the age of 12 months food rations consisted
of alfalfa hay, maize silage, and concentrates. The maize silage had a percentage of 79.36% and
reached 84.4% in the last month of fattening. The maize silage is the forage with high energy
value commonly used in intensive fattening of cattle.
Regarding body weight at the beginning of the experiment, from the age of 8 months the
weight was 176 kilograms (kg) and from 17 months of age to slaughter it was 454.3 kilograms
(Table 2). Romita et al. (1982) cited by Borghese (2005) presents for the Italian buffalo breed,
the following evolution of body weight: from 151.8 kilograms at the age of 5 months, to 466.4
kilograms at 18 months.
Regarding the average daily weight gain, the rate that was observed in Romania, in the
male buffalo, ranged from 750 grams , at the beginning of the period of intensive fattening, to
1207 grams at the age of 12 months. The Achievement of this daily growth gain at 12 months of
1281
age was due to the introduction into the feeding ration of maize silage in the quantity of 20 kg (a
percentage increase of 150% from the previous month). After this age the average daily gain
decreased slightly. Average daily gain in the last month was 1135 grams. Concerning the Italian
buffalo breed, the average daily gain ranged between 795 grams at 5 months old, and 930 grams
at 12 months old.
The size at withers is a morphological trait of animals that provides information on the
development of the animal body. Depending on the size at withers the buffalos are grouped in
populations and breed, with other large size (above 135 cm), medium-size (130-135 cm) and
small size (122-130 cm) (Velea, 2006). The size of the animals we studied at withers increased
from 111 cm at the age of 10 months, to 127.4 cm at 17 months (Table 3).
The oblique length of the body increased by 21% from the start of the experiment, and
the chest width by 48%, indicating good aptitude for meat production. The shinebone perimeter
indicated a well developed bone structure, with variations between 19.7 cm and 24.2 cm. The
dimensions of the hindquarters are positively correlated with meat production in the quantitative
and qualitative report (the spiral perimeter of the pulp ranged between 133.2 cm and 165.0 cm,
and the croup width between32.5 and 53.3 cm).
The index of lateral body shape has values between 102.4% and 108.9%. With regard to
the adult dairy buffaloes, Georgescu and Vidu (2008) we found an average value of 103%. The
lateral body shape is variable depending on age, sex, breed, and the level of breeding. The lateral
body shape has values over 110% in populations of buffalo which showed signs of improvement,
and 107-109% for the intermediary and mixed meat-milk populations. The index of development
has values ranging from 131.37% for animals that are 10 months old and 141.6% for those that
are 16 months old. The index of bone ranges between 17.74% and 18.99%.
The index of bone has an average value of 16.54% in terms of the Murrah buffalo breed,
16.13% in terms of the Caucasian breed and 16.18% in terms of the Italian breed. For males the
report is higher than for that of females by 1-3%.
The ratio of shinebone perimeter and thoracic perimeter emphasizes values which decline
between birth (16.5%) and adulthood (10.65%). The ratio of shinebone perimeter and body
weight is between 4 and 4.5% with regard to selected breeds of buffalos, and around 5% for the
unimproved. The forehand body has a rate of 20-25%, the middle a rate of 40-45%, and the
posterior a rate of of 30-37%.
ACKNOWLEDGEMENT
This scientific work was Funded by the Sectoral Programme ADER 713/2011, project
"The elaboration selection criteria in Buffalo Carpathian Indigenous populations in order to
Improve the breed".
REFERENCES
Borghese, A. (2005). Buffalo production and research, FAO, Rome.
Georgescu, Gh., L. Vidu. (2008). The monograph buffalos in Romania and worldwide, Ceres
Pradal, M. (1989). Productia de carne de bovina astăzi, Ed.Tec-Doc, Lavoisier, Paris.
Romita et al. (1982).Water buffaloes and Frisian bovine males performances at different ages Io-
In vivo and at slaughtering characteristics. Atti IIo Convegno Internazionale su ll
Allevamento Bufalino nel Mondo, Caserta Italy, Sett.29-Ott.2: 573-592.
Solner, D. (1987).Producera carnii de bovina, Ed. Collection Ses, Tehn. Agric, Angers.
1282
Velea, C. et al. (2006). Actuality and perspective in raising buffalos, Agrotehnica Publishing
House, Bucharest.
Table 1. The structure of forage rations depending on age and season (kg per capita per day)
Forage Age (months)

8 9 10 11 12 13 14 15 16
Tare hay 2.6 2.8 3.0 3.8 - - - - -

Alfalfa hay - - - - 1.2 2.0 1.7 1.1 1
Green grass 4.0 4.4 - - - - - - -
Maize silage - - 5 8 20 25 25 26 27
Concentrates 1.5 2.0 2.3 3 4 3 4.2 4.1 4
Table 2. Dynamic of body weight and daily gain average
Parameter Average-Age (months)

8 9 10 11 12 13 14 15 16-17
The initial weight 176 198.5 225.1 256.1 287.9 350.7 376.9 419.1 454.3
(kg)
The total weight - 22.5 26.6 31 31.83 36.21 26.2 29.2 34.0
gain(kg)
The daily average - 750 858 1000 1061 1207 1167 942 1135
gain (g)
Table 3. Evolution of body development at young male buffalo
Parameter (cm) Average-Age (months)

10 11 12 13 14 15 16 17
Size at withers 111 113.5 118.4 121.6 122.8 124.2 126 127.4
Oblique length of 114.6 116 118.1 123 125.6 127.9 133.2 138.7
the body
Thoracic 145.8 151 160.2 167 171.3 173.8 176.9 180.4
perimetre
Chest width 33.2 35.3 39.9 43.1 44.4 46.9 47.3 49.1
Shinebone 19.7 19.8 21.6 22 22.7 23 23.8 24.2
perimeter
Spiral perimeter of 133.2 144.2 146.1 155.3 157.7 159.8 163.7 165
the pulp
Croup width 32.5 36.6 40.1 45.7 47.9 48.9 49.7 53.3
1283
Estimation of Fine Cuts of Meat Production Performance in Mediterranean

Italian Buffalo Young Bulls
Roberta VITTORIAa, Antonella DE GREGORIOb, Concettina FEZZAb, Chiara CASOa,

Giovanni DI PAOLAc and Angelo COLETTAa*
a
ANASB,Associazione Nazionale Allevatori della Specie Bufalina, Via Petrarca 42-44 Centurano,
81100 Caserta – Italy – Tel. +390823356743 – Fax. 0823320964.
b
Dottore in Scienze delle Produzioni Animali.
c
Laureando in Scienze e Tecnologie delle Produzioni Animali – Università degli studi di Napoli -
Federico II.
*
Corresponding email: progettiesviluppo@anasb.it
ABSTRACT
Although it is known worldwide that the main purpose of Mediterranean Italian Buffalo
breeding, in Italy, is the production of Mozzarella cheese. It’s necessary to bear in mind that the
selection has not deleted the ancestral features of this race so these are still strongly present. The
aim of this study was to evaluate the performance, in vivo and post mortem, of the first cuts of meat
production in Mediterranean Italian Buffalo young bulls through the survey of the daily weight gain
(DWG) and the yield at slaughter (YS) of the subjects under observation. In vivo some biometric
measurements were made on a significant number of animals, randomly chosen. Post mortem the
sudden yield at slaughter (hot yield) was calculated and the comparison between the last one and the
latter yield at slaughter (cold yield) was evaluated. The authors have enclosed the first cuts of MIB
meats in a trade set of Campania Region called “Tunno and Coscia” (TC) which includes, in
addition to the shank, the following fine cuts of meat: loin, silverside, rump, haunch and knob. The
TC was considered the large part of the most fine cuts of MIB meats. All the weight of individual
cuts for each animal slaughtered were collected. Has been established behind this work the
existence of a positive relationship between TC and dead weight (DW) in the subjects of the trial. In
other words has been highlighted that the dead weight influenced positively the rate of “tunno and
coscia” in the carcass. This study is useful to help in make decisions to all operators in the field.
Keywords: Buffalo, meat, growth, yield, slaughter, carcass
INTRODUCTION
The meat market has undergone a radical transformation in recent years, in Italy in relation
to changes in eating habits and lifestyle of consumers. Consumer demand consist of a genuine
product and safe from a health point of view. In addition, the move toward value based marketing
has underlined the importance for beef producers to be concerned about the final products they
produce (Greiner et al., 2003). Italian buffalo breeders have the opportunity to remain on the market
only through a quality product also avoiding waste due to poor information on the performance of
animals (yields, growths) and enhancing the best cuts. The objective of this study was to determine
the efficacy of the in vivo prediction of the future performance of young buffalo bulls in high
quality meat production.

1284

The trial was carried out on 139 male buffaloes coming from different farms. Calves were
fed with a steady diet in energy and protein levels, composed by corn silage and oat hay as based
forage; soy, cornmeal and bran as concentrated feed and a supplement of vitamins and mineral salts
until the animals gained nearly 320 kg of body weight. Beyond all subjects had touched this value,
the hay took silage place and with concentrated feed and supplements to give a good diet for
animals finishing. Animals were slaughtered at the average life weight (LW) of 395.97 Kg (37.70
std. dev.) and mean daily weight gain (DWG) of 0.82 (820 g; std. dev. 0.11). During the trial
animals very different in age were considered (days of life DL mean 486.61 std. dev. 64.31) to
verify the existence of a positive relation between life weight and yield at slaughter, in addition to
the final carcass weight and daily weight gain. The life weight of subjects was detected before of
the slaughter phases, buffaloes were slaughtered at a common facility of a private. The carcass
weight was detected two times, at the end of the slaughter phases firstly, and at 24 hours
postmortem, at the arrival at a commercial sectioning facility. The weight of the single cuts was
taken at the sectioning facility too, at 24 hours postmortem, a number of 139 cuts of meat (of the
high quality cuts of meat: loin, silverside, rump, haunch and knob) were taken for both carcass
halves for a total of 1390 weightings.

Table 1 list the means, standard deviations, and minimum and maximum values for live animal and
carcass traits.
Table 1. Means, standard deviations, minimum and maximum values for live animal,
carcass and fine cuts weightings.
TRAIT MEAN STD. DEV. MINIMUM MAXIMUM
n 139
TC µ 38,75 4,31 25,88 46,53
DL µ 486,61 64,31 399 778
LW µ 395,97 37,70 284 474
DWG µ 0,82 0,11 0,54 1,05
CW µ 192,95 19,15 128,4 228,5
YIELD µ 49% 0,03 0,39 0,59
HY
n 278
LOIN 4,86 0,59 3 6,07
SILVERSIDE 2,00 0,36 1 4,5
KNOB 3,92 0,52 2,5 5,6
RUMP 5,00 0,68 3 6,8
HAUNCH 3,63 0,61 1,9 4,8
n = number of observations DL = days of life LW = live weight CW = carcass weight
DWG = daily weight gain HY = hot yield at slaughter (few minutes after the
slaughter phases) YIELD = cold yield at slaughter (at 24 hours postmortem)
The environmental impact was investigated first of all and no association rate there was
between either DWG or CW and farms. The subjects were arranged evenly albeit coming from
different companies, so it possible to exclude the environmental effect.
1285
The impact of single fine cuts is describes in Table 2. and it is more clear in graph. 1.
Table 2. Impacts of single fine cuts on Tunno e Coscia TC,
Carcass Weight CW, Live Weight LW and Yield.
TC CW LW YIELD
LOIN 13% 3% 1% 10%
SILVERSIDE 5% 1% 0,5% 4%
KNOB 10% 2% 1% 8%
RUMP 13% 3% 1,3% 10%
HAUNCH 9% 2% 1% 8%
Graph. 1. Impact of single fine cuts on Tunno e Coscia TC value
10% LOIN
13%
5% SILVERSIDE
9% KNOB
13%
RUMP
HAUNCH
For a number of subjects randomly chosen, the hot yield at slaughter was calculated. The
value of the hot yield at slaughter was taken few minutes after the end of the slaughter phases. The
Table 3 describes the differences between hot and cold yield at slaughter, mean, standard deviation,
and minimum and maximum values.
Table 3. Differences between hot and cold yield at slaughter, mean,

standard deviation, and minimum and maximum values.
CW0 HY CW24 CY D
MEAN 190,0 49,1 187,3 48,3 0,7
STD. DEV. 21,4 2,5 21,0 2,4 0,8
MINIMUM 130 45,8 128,4 44,6 0
MAXIMUM 223 59,4 220,8 58,7 4,8
Carcass Weight at 0 time (CW0), Hot Yield (HY), Carcass Weight at 24
hours postmortem (CW24), Cold Yield (CY), Differences between the
two yields (D).
1286
The study showed that in reference to a significant number of observations, the substantial
difference between the hot and the cold yield (at 24 hours postmortem) was in the order of seven
percentage points.
As you can see from Figure 2, there is a positive relationship between TC and the cold yield
at slaughter (R2= 0,22). The trial also showed that there is a positive relationship among TC, LW
and CW values as appears from the Figure 3. Below. These values allow us to affirm that it is
possible in vivo to predict the production performance of fine meat cuts in buffalo males through
the knowledge of the best time to slaughter the animals, related to LW, CW, DWG, DL, Yield and
TC.
Figure 3. Relationship among TC, LW and CW
1287
For this reason the authors have constructed 7 classes grouping the subjects by age as you
can see from Table 4.
Table 4. Subjects grouped in classes related to the age (days of life DL), with all the
features.
a b c d e f g
DL 400/450 451/500 501/550 551/600 601/650 651/700 >700
LW 389,0 392,2 406,7 404,6 419,7 402,0 424,5
DWG 0,89 0,84 0,78 0,70 0,66 0,61 0,58
CW 196,0162 190,0269 192,7333 198 196,0222 198,2 198,1
YIELD 0,504348 0,484724 0,473848 0,489968 0,467201 49,30% 0,466673
TC 38,58868 37,89215 40,722 40,46286 40,55222 39,7 39,56
n 37 68 15 7 9 1 2
% 27% 49% 11% 5% 6% 0,7% 1,4%
Taking also into account that the evidence has shown a positive association (R2= 0,46)
between DWG and days of life DL, and that the yield has a constant trend in relation to DWG
values. But although what, this not mean that those who have increased more had TC value higher,
in fact the relation between TC and DG is not significant (R2= 0,05). In conclusion, looking at the
Table 4.with the aim to help in make decisions to all operators in the field, we can say that the best
class is the “a” in the Table, that consist of the animals between 400 and 450 days of life i.e. from
13 to 15 months which express the best percentage in yield in addition to being more cost-effective.
The TC value increase with a pair of percentage points in the class “c”, but just this value does not
justify the fact that raising animals for 3 months more.
REFERENCES
Faulkner, D.B., D.F Parret and M.C. Keith. 1990. Prediction of fat cover and carcass
composition from live and carcass measurements. J. Anim. Sci. 68:604-610.
Greiner, S.P., G.H. Rouse and D.E. Wilson. 2003. Predicting beef carcass retail products using real
time ultrasound and live animal measures.www.extension.iastate.edu/Pages/ansci/beefreports/asl-
1327.pdf
Rouse, G.H. and D.E. Wilson. 2003. Predicting beef carcass retail products using Real-time
ultrasound and live animal measures. www.extension.iastate.edu/Pages/ansci/beefreports/asl-
1327.pdf
Hamlin, K.E., D. Green, and L.V. Cundiff. 1995. Real-time ultrasonic measurement of fat thickness
and longissimus muscle area: II. Relationship between realtime ultrasound measures and
carcass retail yield. J. Anim. Sci. 73:1725-1734.
Herring, W.O., S.E. Williams and J.K. Bertrand. 1994. Comparison of live and carcass equations
predicting percentage of cutability, retail product weight, and trimmable fat in beef cattle. J.
Anim. Sci. 72:1107-1118.
May, S.G., W.L. Mies and J.W. Edwards. 2000. Using live estimates and ultrasound measurements
to predict beef carcass cutability. J. Anim. Sci. 78:1255-1261.
Mendes, A.J., C. Andrighetto, C.F. De Lima, A.P. Neto and M.R. De Castro. 2007. Predicting
beef carcass retail products of Mediterranean buffaloes by real-time ultrasound measures. J.
Anim. Sci. 6:1157-1159.
Mendes, A.J., C. Andrighetto, C.F. De Lima and R.A. De Amorim. 2007. Correlations among
1288
carcass traits taken by ultrasound and after slaughter in Mediterranean (Bubalus bubalis) young
bulls. J. Anim. Sci. 6:1160-1162.
1289
Body Condition Score (BCS) system in Murrah buffaloes
Sarjan Rao KAPA1 and Anitha ALAPATI2

1
Department of Livestock Production and Management, College of VeterinaryScience,Hyderabad
500030, A.P.,India
2
Department of Livestock Production and Management,College of Veterinary Science, Tirupati – 517
502, A.P.,India.
*Corresponding email: kapasarjanreddy@gmail.com
ABSTRACT
A new body condition score (BCS) system was developed for Murrah buffaloes. The skeletal
check points were identified based on the anatomical features and carcass fat reserves. A new BCS
chart in a 1-5 scale using 0.5 increments examining eight skeletal check points was developed. The
ultrasonography assessment of the precision of BCS system in 10 buffaloes for each point of the 1-5
scale indicated that BCS adequately reflected in the actual fat reserves. The influence of body
condition score at calving (BCS) on the reproductive and productive performance studied in 24 (4 x 6
Completely Randomized Design) and 40 (4 x 10 Completely Randomized Design) buffaloes
respectively revealed that buffaloes of BCSc group 3.5 – 3.99 showed the best performance among the
four BCSc groups with earlier (P < 0.05) resumption of ovarian activity (29.33 days), less (P < 0.01)
postpartum estrus period (46.66 days), less (P < 0.05) service period (58.83 days), less number of
services per conception (1.50), higher rate of 1st service conception (66.66%) with higher (P < 0.01)
breeding efficiency (90.64 per cent). The milk production traits like total milk yield upto 18 weeks of
lactation (1658.67 kg), 305 day predicted lactation yield (3187.3 kg), peak milk yield (16.5 kg), milk
protein and solids not fat were also higher in BCSc of 3.5 – 3.99 followed by the BCSc groups of 4.0 –
4.49, 3.0 – 3.49 and 2.5 – 2.99.
Keywords: Body condition score, ultrasonography, reproduction, production, buffaloes.
INTRODUCTION
Body Condition Score (BCS) system is based on evaluation of the outer appearance of the
animal that interacts with its body fat reserves and therefore is directly influenced by energy balance. It
gives an immediate appraisal of the body state of the animal and is readily incorporated in operational
decision making (Gransworthy, 1988). The BCS systems were developed earlier by many scientists
like Jefferies (1961) using 0 to 5 scale in ewes, Lowman et al. (1976) using a 0 to 5 scale in beef cattle
and Earle (1976) using a 8 grade system in dairy cows. Edmonson et al. (1989) developed a chart for
body condition scoring of Holstein dairy cows in a 1 to 5 scale using 0.25 increments. Sarjan Rao et al.
(2002) and Anitha et al. (2005) have utilized this chart for scoring the crossbred dairy cows in India.

The skeletal check points were identified taking into consideration the anatomical features and
amount of fat reserves in 50 slaughtered buffaloes. The amount of fat reserves were measured at six
skeletal check points which include 1. points between 12th and 13th ribs, 2. spinous and transverse
processes of lumbar vertebrae, 3. sacral crest and tuber sacrale, 4. sacral crest and hooks, 5. hooks and
pins, 6. tail head and 7. pins. Based on the amount of fat reserves the scores were prioritized in a 1 to 5
scale of the new BCS proposed. A score of 1 indicates emaciated, 2 indicates thin, 3 indicates average,
4 indicates fat and 5 indicates obese condition (Chart 1). The eight locations observed were : Tail head
1290
to pin bones, spinous processes of the lumbar vertebrae, depression between the spinous and transverse
processes, transverse processes of lumbar vertebrae, point between 12th and 13th ribs, sacral crest,
depression between sacral crest and hooks and depression between hooks and pins. After each check
point was observed by vision and palpation the scores were recorded and average BCS was assigned to
a herd of 200 buffaloes. Murrah buffalo showing the skeletal check points for BCS as presented in
Fig.1.
3, 4, 5) of the scale by ultrasonography measurements of body fat reserves An LOGIQ  100

The BCS system developed was subjected for its precision in 10 buffaloes for each point (1, 2,
ultrasound machine (GE Medical Systems) with a 5.5 MHz convex transducer was used to determine
the amount of subcutaneous fat at five body locations through a coupling gel on each buffalo
(Bruckmaier and Blum, 1992). Body locations were selected based on the skeletal check points used
for body condition scoring and ease of obtaining and reading ultrasonography measurements.
Twenty four Murrah and Graded Murrah buffaloes from Buffalo Research Station,
Vekataramannagudem, Sri Venkateswara Veterinary University, Tirupati were selected to study the
influence of BCS at calving (BCSc) on the reproductive performance. The buffaloes selected were in
first to third lactation. The buffaloes selected were grouped in 4 groups, 6 buffaloes each in group, in a
4 x 6 completely randomized design to study the relationship between BCSc and reproductive traits.
To study the postpartum resumption of ovarian activity, the serum progesterone (P4) concentration was
estimated as per the procedures of Hubl et al. (1982). The serum P4 concentration was estimated by
using Enzyme Linked Immuno Sorbent Assay (ELISA) technique with the help of P 4 kits (Biotron
Diagnostics Inc. Hemet California, USA) and was expressed as ng/ml. Breeding Efficiency (BE):The
Breeding Efficiency (BE) of experimental buffalo herd in relation to BCSc was calculated by using the
n365  1040100 where n is the number of calving intervals; AC is the age at first calving and
formula:
AC  C i
Ci is the calving interval in days (Jagdish Prasad and Neeraj, 2007).The data regarding the calving
intervals and age at first calving were obtained from the data records of the farm. The influence of
body condition on milk production traits was studied in a herd of 40 buffaloes from calving upto 18
weeks postpartum in a 4 x 10 CRD (4 groups divided based on BCSc).The production data including
the daily milk yield (kg) upto 18 weeks of lactation was measured everyday both morning and evening
after separating the milk for pail feeding the calves. The peak milk yield (kg) pertaining to the test herd
was obtained from the computed data of the farm. The 305 day predicted lactation yields were
calculated by using the ratio estimates of partial lactations of Murrah buffaloes (Thomas and Sastry,
1991). The lactation yield up to 18 weeks was multiplied by the corresponding ratio estimate of 1.9216
to obtain estimate of lactation yield.The milk components include fat, protein and solids not fat (SNF)
were studied in relation to BCS. (IS : 1224, Part-I, 1977);(IS 1479, Part II, 1961) ;(IS : 1224-
1958).Analysis of variance was used to study the variation in carcass fat thickness at various skeletal
check points, the variation in ultrasonography fat reserves within BCS and among different BCS
groups, the relationship of BCSc with the parity and postpartum estrus, parity and service period,
parity and number of services per conception, 1st service conception rate, resumption of ovarian
activity, breeding efficiency, total milk yield up to 18 weeks after calving, predicted lactation, yield,
peak milk yield, persistency index, milk fat, protein and SNF and for comparison of scores assigned
with carcass fat reserves. Correlation coefficients were used to study the relationship among BCS,
carcass, fat and ultrasonographic fat reserves (Snedecor and Cochran, 1994).
RESULTS
The carcass fat reserves measured at six skeletal check points showed that the fat thickness
(mm) at tail head to pin bones (6.28 ± 0.37) was significantly (P < 0.01) higher than the fat other
1291
skeletal check points, followed by the fat thickness at lumbar area (4.43 ± 0.28), between 12th and 13th
ribs (4.19 ± 0.27), sacral crest to tuber sacrale and sacral crest to hooks (3.56 ± 0.23), hooks to pins
(3.22 ± 0.19). Based on the amount of fat reserves the scores were assigned in a 1 to 5 scale. The mean
values of carcass fat thickness for the scores 1 to 5 at individual skeletal check points and the mean of
all the six check pints were presented in Table I. Significant (P < 0.01) difference was observed in the
carcass fat thickness among the five scores at all the individual check points as well as the mean of the
six check points indicating that the scale was internally consistent. The BCS chart in a 1 to 5 scale
using 0.5 increments, examining 8 skeletal check points was developed (Chart 1) and BCS was
assigned to a herd of 200 buffaloes using the chart. Thus, the new BCS system was developed to score
the buffaloes.
Ultrasonic assessment of the precision of the BCS system
The ultrasonography fat measurements at the five skeletal check points within each BCS
showed that the fat thickness was highest at the tail (P < 0.01), followed by the lumbar area, ribs,
sacral crest to hooks and hook to pins. Fig. 2, 3, 4, 5 and 6 show the ultrasonography fat measurements
in a Murrah buffaloes of BCS 1, 2, 3, 4 and 5 respectively. The ultrasonography measurements of
mean body fat thickness for buffaloes of different body condition scores were presented in Table I.
Significant (P < 0.01) difference was observed in the fat thickness for buffaloes of various BCS
groups. As the BCS increased the amount of fat reserves also increased indicating that BCS were
adequately reflected in the amount of actual fat reserves. BCS was significantly (P < 0.01) correlated
with the carcass fat reserves (0.86) as well as ultrasonography fat reserves (0.85).
The reproductive performance of buffaloes of various BCSc groups in the test herd was
presented in Table II. The buffaloes of BCSc group 3.5 – 3.99 had earlier (P < 0.05) resumption of
ovarian activity, less postpartum estrus period (P < 0.01), service period (P < 0.05), number of services
per conception, higher 1st service conception rate and higher breeding efficiency (P < 0.01) of 29.33
days, 46.66 days, 58.83 days, 1.5, 66.66 per cent and 90.64 per cent respectively, followed by
buffaloes of BCSc group 4.0 – 4.49 with 39.33 days, 55.16 days, 77.16 days, 1.83, 50 per cent and
87.48 per cent respectively, followed by buffaloes of BCSc group. 3.0 – 3.49 with 42 days, 65.66 days,
85.66 days 2.0, 33.33 per cent and 80.58 respectively, followed by buffaloes of BCSc group 2.5 – 2.99
with 47.25 days, 77.16 days, 125.16 days, 2.66, 16.66 per cent and 70.49 per cent respectively. It was
observed that the reproductive performance improved as the BCSc increased upto 3.99, but beyond
that decline was noticed.
Buffaloes of BCSc group 3.5 – 3.99 had higher (P < 0.01) milk yield upto 18 weeks of
lactation (kg). 305 day predicted lactation yield (kg) and peak milk yield (kg) of 1658.67, 3187.31 and
16.5 respectively, followed by buffaloes of BCSc group 4.0 – 4.49 with 1359.92, 2613.23 and 13.75
respectively, followed by buffaloes of BCSc group, 3.0 – 3.49 with 1197.12, 2300.39 and 11.60
respectively, followed by buffaloes of BCSc group. 2.5 – 2.99 with 1030.93, 1981.05 and 9.50
respectively.(Table III). As the BCSc increased beyond 3.99, the milk yield showed a decline trend.
Table IV showed the relationship between BCSc and milk components. Buffaloes of BCSc group 4.0 –
4.49 had higher (P < 0.01) milk fat per cent followed by BCSc groups of 3.5 – 3.99, 3.0 – 3.49 and 2.5
– 2.99 at 6-8 weeks after calving as well as the 16-18 weeks after calving whereas buffaloes of BCSc
group. 3.5 – 3.99 had higher (P < 0.01) milk protein and SNF per cent followed by BCSc groups of 4.0
– 4.49, 3.0 – 3.49 and 2.5 – 2.99 at 6-8 weeks and at 16-18 weeks after calving.
DISCUSSION:
The mean ± SE (mm) values of carcass fat thickness for the scores 1 to 5 ranged from 1.67 ±
0 .0.7 to 7.82 ± 0.21 at the point between 12th and 13th ribs whereas the values recorded by Apple et al.
(1999) by assigning scores to beef cows on a 9 point scale ranged from 0.5 ± 1.5 to 27.3 ± 1.5 at the
12th / 13th rib interface. The difference in the fat thickness measurements with that of the present study
might be attributed to the species difference. The new BCS chart in a 1 to 5 scale using 0.5 increments
examining 8 skeletal check points was developed to score the Murrah buffaloes. For beef cattle, a 9
1292
point scale is commonly used (Wagner et al., 1988). Concerning dairy cows, 8 and 10 points scales are
used in Australia and New Zealand (Roche et al., 2004). Prevailing scoring systems in the United
States and Ireland use a 5-point scale. The BCS is determined by vision and palpation of the skeletal
check points in the present study which was in tune with Wildman et al. (1982) and Ferguson et al.
(1994) whereas Edmonson et al. (1989) evaluated the body locations only visually. BCS was assigned
using the chart developed and the new BCS system was found to be precise and consistent. Thus, the
present study suggested that anatomical studies, amount of fat reserves and the assessment of scores
helped in the development of a valid BCS system.
The ultrasonography fat thickness measurements were significantly (P < 0.01) higher at the
check point between tail head to pin bones which was in accordance to the findings of Gentry et al.
(2004) who observed that tail head area accounted for the majority of the variation in BCS in mares.
As the BCS increased the amount of fat reserves increased significantly (P < 0.01) indicating that BCS
adequately reflected in the amount of actual fat reserves. Significant (P < 0.01) correlation was
observed between BCS and ultrasonography fat reserves (0.85) which was in accordance to the
findings of Lubis and Fletcher (1985) who reported significant correlation (0.87) between subjectively
determined BCS and ultrasonically determined back fat thickness in swamp buffalo cows and Zulu et
al. (2001) who reported significant correlations between BCS and ultrasonography measurements of
subcutaneous fat in dairy cows.
The results of the present study highlighted the importance of body condition at calving in
achieving good reproductive performance. Studies on the interaction of BCSc with parity showed no
significant effect on the reproductive performance. The resumption of ovarian activity was observed at
a mean values of 47.25±2.39, 42.0±2.91, 29.33±3.33 and 39.33±4.21 days for the BCSc groups of 2.5-
2.99, 3.0-3.49, 3.5-3.99 and 4.0-4.49 respectively whereas Nosier and Hussein (1988) reported that
postpartum ovarian activity resumed in the 4th week after calving in Egyptian buffaloes was earlier
than the values of the present study.
The results showed that body condition at calving was the critical factor related to
reestablishment of ovarian function. Buffaloes of BCSc range 3.5-3.99 have shown early resumption
of ovarian activity which is an indicator of good reproduction performance whereas buffaloes of BCSc
range 2.5-2.99 have taken longer period for resumption of ovarian activity showing poor reproductive
performance which was in tune with the findings of Beam and Butler (1997) and Reksen et al. (2002)
who reported the delayed resumption of luteal function in thinner cows. Similarly, Markusfeld et al.
(1997) reported that thinner cows at calving were more likely to have inactive ovaries. For every one
unit increase in BCS a decrease of 20.5 days in postpartum estrus period, 37.41 days in service period,
0.66 in the number of services per conception and an increase of 33.33 per cent in the conception rate
at first service was observed. The reproductive performance improved as the BCSc increased up to
3.99 but beyond, that a decline was noticed
Studies on the influence of BCSc on productive performance showed that for every one unit
increase in BCSc an increase of 395.27 kg, 795.55 kg and 4.57 kg was observed in the 18 weeks
lactation yield, 305 day predicted lactation yield and peak milk yield respectively. These findings were
in tune with the reports of Mohammed et al. (1988) that cows with BCSc of 2.5 produced less milk
than those with 3 to 3.5. For every one unit increase in BCSc an increase of milk fat per cent of 1.8 and
2.0 and milk protein / SNF per cent of 0.55 and 0.54 was observed at 6-8 weeks after calving and 16-
18 weeks after calving respectively. Holter et al. (1990) observed that cows considered as under
conditioned at calving had reduced milk fat concentration which was in accordance to the present
trendsThe results of the study revealed that the reproductive performance and milk production
increased with BCSc up to a score of 3.99 but beyond this there was a decline. The present study
1293
suggested that a BCSc of 3.5 – 3.99 was ideal for better reproductive and productive performance of
Murrah buffaloes and hence the feeding management should be monitored such that the buffaloes
maintain a BCS of 3.5 – 3.99 at the time of calving.
REFERENCES
Anitha, A., K. Sarjan Rao, J.V. Ramana and P.V.V. Satyanarayana Reddy. 2005. Body condition score
and its relation to age and physical parameters in crossbred cows. Indian Veterinary Journal 82
: 305-308.
Apple, J.K., J.C. Davis, J. Stephenson, J.E. Hankins, J.R. Davis and S.L. Beaty. 1999. Influence of
body condition score on carcass characteristics and sub-primal yield from cull beef cows.
Journal of Animal Science 77: 2660-2669.
Beam, S.W. and W.R. Butler. 1997. Energy balance and ovarian follicle development prior to the first
ovulation postpartum in dairy cows receiving three levels of dietary fat. Biology of
Reproduction 56: 133-142.
Bruckmaier, R.M. and J.W. Blum. 1992. B-mode ultrasonoography of mammary glands of cows, goats
and sheep during a and p-adrenergic agonist and oxytocin administration. Journal of Dairy
Research 59: 151-159.
Earle, D.F. (1976). A guide to scoring dairy cow condition : Australian Department of Agriculture
Journal, Victoria 74: 228.
Edmonson, A.J., I.J. Lean, L.D. Weaver, T. Farver and G. Webster. 1989. Body condition scoring
chart for Holstein Dairy Cows. Journal of Dairy Science 72: 68-78.
Ferguson, J.D., T.D. Galligan and N. Thomsen. 1994. Principal descriptors of body condition score in
Holstein cows. Journal of Dairy Science 77: 2695-2703.
Gentry, L.R., D.L. Thompson, Jr. G.T. Gentry, G. R.P. del-Vecchio, K.A. Davis, and P.M. Del-
Vecchio. 2004. The relationship between body condition score and ultrasonic fat measurements
in mares of high versus low body condition. Journal of Equine Veterinary Science 24: 198-203.
Gransworthy. 1988. The effect of energy reserves at calving on performance of dairy cows. In nutrition
and lactation in the dairy cow. 1st Edition, Butterworths, London, pp. 157-170.
Holter, J.B., M.J., Slotnick, H.H. Hayes and C.K. Bozak. 1990. Effect of prepartum dietary energy on
condition score, postpartum energy, nitrogen partitions and lactation production responses.
Journal of Dairy Science 73: 3502-3511.
Hubl, W., T. Fehert, W. Ronde, G. Dormer, H.H. Tanbert and E. Freymann. 1982. Relationships
among estradiol, progesterone and postpartum ovarian activity in Holstein cows.
Endokrinologie, 79: 165.
IS: 1224-1958. IS: 1479 (Part-II) 1961; IS: 1224 (Part-I), 1977 Methods of test for dairy industry.
Indian Standards Institute, New Delhi.
Prasad, J. and Neeraj. 2007. Principles and practices of dairy farm management. Fifth revised and
enlarged edition. Kalyani publishers, New Delhi, pp. 260-261.
Jefferies, B.C. 1961. Body condition scoring and its use in management. Tasmanian Journal of
Agricultural Ministry of Agriculture 32: 19.
1294
Langley, D.H. and J. Sherington. 1983. Effect of body condition scoring at calving on subsequent
reproductive performance. Animal Production Research Report. Dunsinea Mcorepark and
Western Research Centre, Dublin, p. 59.
Lowman, B.G., N.A. Scott and S.H. Somerville. 1976. Condition scoring of cattle Bull No. 6, East
Scotland College of Agricultural Animal Production Advisory Development Department.
Lubis, A. and I.C. Fletcher 1985. Body condition score in swamp buffalo cows. Research Report,
Research Institute for Animal Production, Indonesia, 31.
Markusfeld, O., N. Gallon and E. Ezra. 1997. Body condition score, health, yield and fertility in dairy
cows. Veterinary Record 141: 67-72.
Mohammed, H.O., G.A. Donovan and R.K. Braun. 1988. The importance of body condition scoring as
a predictor for the productivity and reproductivity of dairy cattle. Proceedings VI World
Conference on Animal Production, 598.
Nosier, M.B. and F.M. Hussein. 1988. Progesterone variations in buffalo’s milk and blood during the
postpartum period. 11th International Congress on Animal Reproduction and Artificial
Insemination – University College – Dublin – Ireland. June 26-30, Volume 2, Brief
Communications (Paper 54): 3.
Reksen, O., O. Havrevoll, Y.T. Grohn, T. Bolstad, A. Waldmann and E. Ropstad. 2002. Relationships
among body condition core, milk constituents and postpartum luteal function in Norwegian
dairy cows. Journal of Dairy Science 85: 1406-1415.
Roche, J.R., P.G. Dillon, C.R. Stockdale, L.H. Baumgard and M.J. Van Baale. 2004. Relationships
among international body condition scoring systems. Journal of Dairy Science 87: 3076-3079.
Sarjan Rao, K., G. Dilip Kumar and M.M. Kailash, M.M. 2002. Influence on body condition score.
Indian Journal of Animal Sciences 72: 882-886.
Snedecor, G.W. and W.G. Cochran. 1994. Statistical methods Eighth Edition, Iowa State University
Press, Ames, Iowa.
Stevenson, J.S. and J.H. Britt. 1979. Relationship among luteinizing harmone, estradiol, progesterone,
glucocorticoides, milk yield, body weight and postpartum ovarian activity in Holstein cows.
Journal of Animal Science 48: 570-577.
Wagner, J.J., K.S. Lusby, J.W. Oltjen, J. Rakestrow, R.P. Wettemann and L.E. Walters. 1988. Carcass
composition in mature Hereford cows. Estimation and effect on daily metabolizable energy
requirement during winter. Journal of Animal Science 66: 603-612.
Wildman, E.E., G.M. Jones, P.E. Wagner, R.L. Boman, H.F. Troutt and T.N. Lesch 1982. A dairy
cow body condition scoring system and its relationship to select production characteristics.
Journal of Dairy Science 65: 495-501.
Yaylak, E. 2003. Effects of body condition score on reproductive traits of Holstein cows. Hayvansal-
Uretim. Journal of Animal Production 44: 44-51.
Zulu, V.C., T. Nakao, M. Moriyoshi, K. Nakada, Y. Sawamukai, Y. Tanoka and Zhang-Wenchang
2001. Relationship between body condition score and ultrasonographic measurement of
subcutaneous fat in dairy cows. Asian-Australian Journal of Animal Sciences 14: 816-820.
1295
TABLE III.: RELATIONSHIP BETWEEN BCSc AND MILK YIELD IN THE TEST HERD
At 6-8 weeks after calving At 16-18 weeks after calving
BCSc
Fat % Protein % SNF % Fat % Protein % SNF %
2.5 – 2.99 5.82d 3.12d 8.73d 6.44d 3.39d 8.99d
3.0 – 3.49 6.80c 3.47c 9.07c 7.54c 3.74c 9.34c
3.5 – 3.99 7.76b 3.96a 9.56a 8.62b 4.24a 9.84a
4.0 – 4.49 8.46a 3.74b 9.34b 9.37a 3.97b 9.57b
a, b, c, d : values with different superscripts vary significantly (P < 0.01)
TABLE IV. Relationship between Bcsc and Milk Components in the Test Herd
Milk yield
upto 18 Predicted
Peak milk
BCSc weeks of ‘F’ Value lactation ‘F’ Value ‘F’ Value
yield (kg)
lactation yield (kg)
(kg)
2.5–2.99 1030.93d 1981.05d 9.50d
3.0–3.49 1197.12c 2300.39c 11.60c

150.33** 150.33** 78.73**
3.5–3.99 1658.67a 3187.31a 16.50a
4.0–4.49 1359.92b 2613.23b 13.75b
7 7
8 6 3 5
1
4
Figure1. Skeletal Check Points
1296
Table I. The Mean Carcass and Ultrasonographic Fat Thickness (Mm) For the Five Scores Of Body Condition
Between
Between Between
Between 12th sacral crest Between Tail Mean of all
Lumbar area sacral crest hooks and
and 13th ribs and tuber head and pins check points
and hooks pins
BCS sacrale
Ultra- Ultra- Ultra- Ultra- Ultra- Ultra- Ultra-
Car- Car- Car- Car- Car- Car- Car-
sono- sono- sono- sono- sono- sono- sono-
cass cass cass cass cass cass cass
graph graph graph graph graph graph graph
1. 1.67 1.6 2.21 1.8 1.73 --- 1.73 1.3 1.64 1.2 2.70 2.7 1.95 1.72
2. 2.68 3.2 3.08 3.3 2.37 --- 2.37 2.8 2.50 2.6 4.23 4.6 2.97 3.3
3. 4.16 4.3 4.27 4.6 3.48 --- 3.48 3.8 3.33 4.0 6.50 6.4 4.35 4.62
4. 6.06 6.2 6.24 6.6 5.20 --- 5.20 5.6 4.45 5.0 8.69 8.5 6.15 6.38
5. 7.82 8.0 8.22 8.0 6.68 --- 6.68 6.8 5.34 5.9 11.02 12.5 7.83 8.06
Table II. Reproductive Performance of Buffaloes of Various Bcsc Groups in the Test Herd
1297
Reproduction BCSc
Parameters 2.5 – 2.99 3.0 – 3.49 3.50 – 3.99 4.00 – 4.49
Post-partum resumption 47.25 ± 2.39 42.00 ± 2.91 29.33 ± 3.33 39.33 ± 4.21
of ovarian activity
Post-partum estrus (days) 77.16 ± 5.33 65.66 ± 5.46 46.66 ± 4.26 55.16 ± 4.19
Service period (days) 125.16 ± 17.42 85.66 ± 5.83 58.83 ± 9.01 77.16 ± 14.76
No. of services per 2.66 ± 0.61 2.00 ± 0.40 1.50 ± 0.37 1.83 ± 0.52
conception
1st service conception 16.66 33.33 66.66 50
rate (%)
Breeding efficiency 70.49 ± 2.35 80.58 ± 2.01 90.64 ± 1.98 87.48 ± 1.10
CHART 1. Body condition scoring chart for Murrah and Graded Murrah buffaloes in a 1 to 5 scale using 0.5 increments
1298
Buffalo Milk and Milk Products
Flavored Probiotic (Acidophilus) Buffalo Milk: Development and Quality

Assessment
Muhammad JUNAIDa*, Imran JAVEDb, Muhammad GULZARa, Muhammad AYAZa,

Muhammad ABDULLAHb, Umair YOUNASb and Jamal NASIRc
a
Department of Dairy Technology, University of Veterinary & Animal Sciences, Lahore, Pakistan
b
Department of Livestock Management, University of Veterinary & Animal Sciences, Lahore, Pakistan
c
Department of Meat Technology, University of Veterinary & Animal Sciences, Lahore, Pakistan
*
Corresponding e-mail: mjunaid@uvas.edu.pk
ABSTRACT
Functional foods containing probiotic bacteria (lactic acid bacteria and bifidobacteria) are
getting popularity in the world, due to tremendous health benefits conferred by these bacteria.
However, the total viable count of bacteria in the final product and the sensory attributes of the product
are of higher importance for its consumer acceptability. In this study, flavored (strawberry, pineapple,
and mango) probiotic acidophilus milk (from buffalo) using probiotic starter culture (Lactobacillus
acidophilus) was prepared and its microbiological, physicochemical and sensory quality studies were
carried out up to 6 days of storage. A slight increase in acidity of the milk was observed after 6 days of
storage resulting in a decrease of pH (from pH 4.5 to 4.3). Total viable count of L. acidophilus bacteria
was decreased after 6 days of storage due to increase in acidity but it was still within acceptable range
(>106). Sensory evaluation data shows that the quality of sensory attributes (color, taste, aroma,
appearance and overall acceptability) was slightly decreased after 6 days of storage but still had
considerable acceptability.
Keywords: Functional foods, Probiotics, Lactobacillus Acidophilus, Acidophilus milk, Sensory

evaluation
INTRODUCTION
In recent years, there has been an increasing trend about incorporation of the health promoting
bacterial species; Lactobacillus acidophilus and Bifidobacterium longum, into fermented milk products.
In fact, these bacteria having prescribed population of viable cells, when administered orally as
supplement or through food products impart a variety of beneficial health effects (Gilliland, 1990;
Azlin et al., 1997; Anderson and Gilliland 1999; Andrade and Borges 2009).
A number of Lactobacillus acidophilus strains are used in the processing of various dairy
products such as acidophilus yoghurt and sweet acidophilus milk. The nutritional and therapeutic
benefits derived through the consumption of dairy products containing viable Lactobacillus acidophilus
as a food or supplement have been the focus of studies for the last two decades (Walker and Gilliland,
1993; Salminen et al., 1996; Azlin et al., 1997; Anderson and Gilliland, 1999; Andrade and Borges
2009). However, the production of high-quality fermented milk products containing these probiotic
bacteria is a major challenge (Yeung et al., 2002). This challenge in part is due to particular attributes
of the bacteria such as rapid acid production from lactose and development of suitable quantities of the
volatile compounds such as diacetyl and acetaldehyde etc. These compounds must not be over or under
produced nor should they be accompanied by off-flavoring compounds. A lot of information has been
accumulated relating to starter culture for milk fermentation (Marshall, 1987) and now even the efforts
are going on to use the mixed cultures of bacterial species to produce specific flavor.
Although significant attention has been placed towards the organoleptic characteristics of the
product, enriched with probiotic bacteria but still most of the publications concerning probiotic bacteria
1300
have not paid much attention on the consumer acceptability and preference of the finished product. For
enhancing the consumption of functional probiotic products, the consumer satisfaction must be
achieved keeping in view the cost effectiveness and health perspectives (Hilde et al., 2003).
Keeping in mind the beneficial and therapeutic values of Lactobacillus acidophilus culture the
present study was designed to prepare the probiotic acidophilus milk using Lactobacillus acidophilus
as probiotic starter culture. The microbiological, physicochemical and sensory quality assessment of
the product up to 6 days of storage time was carried out. In order to appeal the consumer acceptance,
flavoring with different food grade flavors like strawberry, pineapple and mango was also carried out.

Probiotic Lactobacillus acidophilus culture used in this study was procured from starter culture
collection center (Danisco, Denmark). All the reagents and glass ware sterilized either by autoclaving
or hot air oven for each set of experiment.
Probiotic Acidophilus Milk Development
The fresh whole Buffalo milk was standardized at 3.5% fat and 8.5% SNF befor pasteurization
(500 ml quantities) at 72 °C for 15 minutes in a water bath and then cooled to 4 ± 1 °C in an ice bath.
Then samples (200 ml) were equilibrated for one hour at fermentation temperature (40ºC) in a water
bath before inoculation with the starter cultures. The freeze-dried culture used for fermentation of milk
was activated according to the recommendations of suppliers and was grown in milk at 40 ± 1ºC and
then maintained at 4 ± 1ºC.
Preliminary studies were carried out to optimize the conditions of culture concentration used for
fermentation of milk. For this purpose, milk samples (200ml) were inoculated with overnight active
probiotic culture of Lactobacillus acidophilus at different concentrations ranging from 1-5%. It was
filled into clean plastic containers (250ml) and incubated in a shaker water bath at different
temperatures; 30 °C, 35 °C and 40 °C for different time; 04 and 08 hours. The aim of preliminary
studies was to find out the best combination of culture concentration with temperature and incubation
time for product development. It was observed that by increasing temperature, time for incubation and
concentration of culture, the rate of fermentation was increased. With 1% culture incubated at 40°C for
40 minutes a good gel with pH 4.4 was observed. This combination was then used for further study.
Mango, strawberry and pineapple flavors were incorporated to produce flavored probiotic acidophilus
milk. The flavored probiotic acidophilus milk obtained was cooled and stored at 4±1°C for six days.
The microbiological, physicochemical and sensory evaluation of the product at day 1 and day 6 was
carried to study the acceptability of the product.
Sample Analysis
The samples were then analyzed for their sensory attributes (appearance, taste, color, flavor and
overall acceptability) (Peryam et al., 1952), shelf life (titratable acidity, pH up to six days of storage)
and Microbiological quality (Total viable count). The results of data were analyzed through analysis of
variance technique (ANOVA) (Steel et al., 1997). The significant difference comparisons were done
using Duncan’s Multiple Range (DMR) test with a probability P ≤ 0.05 (Steel and Torrie, 1980).

Optimization and Standardization of Conditions for Acidophilus Milk development
For determining the better conditions for the development of the product a preliminary study
was conducted, where culture concentration (1 – 5%), temperature (30 – 40°C) and incubation time (04
– 8h) was varied. All the three parameters accelerated the rate of fermentation, which was observed in
the form of increased acidity and pH of the final product. These parameters affected the consistency of
the gel, as well as physicochemical and microbiological quality. Overall, keeping in mind the curd
consistency, pH, acidity and its sensorial attributes like aroma, taste, texture and overall acceptability,
1% culture concentration incubated in milk at 40 °C for 04hrs was found to be the best possible
1301
combination of variables and thus used for further studies. It resulted in a good gel with final pH of 4.4.
In fact the pH 4.4 is around the iso-electric pH of the milk proteins and help in efficient aggregation of
these proteins molecules.
Determination of keeping quality of Acidophilus milk
Table 1 show that the titratable acidity in all the flavoured milk samples on 1st day was
around 0.81, which is acceptable for a fermented dairy product. During the course of storage, the
acidity was continuously increased and after 6 days, the acidity was found to be almost 0.91 in all the
samples. No effect on acidity as a result of different flavouring was observed. The increase of acidity
may be attributed to the production of lactic acid as a result of microbial fermentation of lactose
(Tamine and Robinson, 2004). These results were also correlates with findings for pH which also
decreased with storage period. Increased production of lactic acid decreases the pH of the product is in
agreement with the results of Salwa et al. (2000). The pH of all the flavoured milk samples at 1st day
was around 4.5 but was continuously decreased during storage. After 6 days of storage the pH in all the
samples was around 4.32. This decrease in pH is related to the production of lactic acid. The overall
acidity and pH results after 6 days of storage are under the acceptable range.
Total Viable Bacterial Count
In order to claim a product to be probiotic the viability of the organism is of primary
importance. The acidophilus milk was evaluated at day-1 and day-6 of storage (Table 2) for total viable
count of Lactobacillus acidophilus per ml. It is important to maintain the viability of probiotic
microorganisms until the products are consumed in order to ensure delivery of sufficient number of live
microorganisms. According to international standards, the total viable count in a probiotic product must
be at least 105 /g (Robinson, 1987) at the time of consumption. In fact, the beneficial effects of
Lactobacillus acidophilus can be expected only when ingesting significant count of viable cells which
then can colonize the human gut (Ishibashi and Shimamura, 1993). Average values of total viable count
for Lactobacillus acidophilus observed in this work were 2.50x106, 3.60x106 and 2.87x106 for mango,
pineapple and strawberry flavor milk respectively at Day-1 of the development of flavored probiotic
acidophilus milk (Table 2). However a slight decrease in total viable count of product from day-1 to
day-6 was observed. Average values of total viable count for mango, pineapple and strawberry flavored
probiotic acidophilus milk at day 6 were 1.50x106, 3x106 and 2.50x106, respectively (Table 2). These
results are in agreement with the results of Shah et al. (1995) who also observed a decrease of total
viable count in samples of commercial yoghurt. In fact increase in acidity has lethal effect on bacterial
population. However, the total viable count was under acceptable range even after 6 days of storage
indicating good keeping quality up to 6 days.
Sensory evaluation of Acidophilus Milk
Color serves as a preliminary parameter for the acceptance of food and indicates the fitness of
milk products for consumption. Table 3 shows that the Acidophilus milk with different flavors was
rated about 7.8 by the panelists, which is quite reasonable for a product. This rating was decreased to
about 6.6 after 6 days of storage. At both days, the difference of color among three different milk
samples was very slight.
Flavor means an overall integrated perception of taste and aroma associated with the product
(Meilgaard et al., 1987). The flavor for mango, pineapple and strawberry flavored milk was rated as 7.6
± 1.1, 7.4 ± 1 and 8.0 ± 0.7 respectively. After 6 days of storage the rating was decreased to 6.6 ± 0.6,
5.8 ± 0.9 and 6.3 ± 1 respectively (Table 3).
Overall acceptability is based on multiple organoleptic quality parameters i.e. color, flavor,
texture etc. and shows the accumulative perception and acceptance by the panelists. Table 3 shows that
the overall acceptability was around 7.7 in mango and pineapple flavored milk; however it was slightly
higher (8.2) in strawberry flavored milk. The overall acceptability was decreased after 6 days of storage
and was around 6.3. The overall acceptability after 6 days of storage is considered still reasonable for a
fermented dairy product.
1302
In conclusion, the probiotic Acidophilus milk produced with different flavors in this study has
been seen to have higher overall acceptability. The overall acceptability was slightly decreased after 6
days of storage. The total viable count was within the acceptable range up to 6 days of storage without
affecting deleteriously other physicochemical parameters. Keeping in view all above results, the
production of flavored Acidophilus milk at commercial level is highly recommended.
ACKNOWLEDGMENTS
We are highly grateful to Danisco for provision of probiotic culture along with Dairy Animal
Training and Research Center (DAT&RC), UVAS, Ravi campus, Pattoki for the provision of Buffalo
milk.
REFERENCES
Azlin, M., T. Jiang and D. A. Savaiano. 1997. Improvement of lactose digestion by humans following
ingestion of unfermented acidophilus milk: influence of bile sensitivity, lactose transport, and
acid tolerance of Lactobacillus acidophilus. J. Dairy. Sci. 80: 1535-1547.
Anderson, J.W. and S.E. Gilliland. 1999. Effect of fermented milk (yogurt) containing Lactobacillus
acidophilus L1 on serum cholesterol in hypercholesterolemic humans. Amer. J. C. Nutr. 18(1):
43–50.
Andrade, S. and N. Borges. 2009. Effect of fermented milk containing Lactobacillus acidophilus and
Bifidobacterium longum on plasma lipids of women with normal or moderately elevated
cholesterol. J. Dairy Res. 76: 469-474.
Gilliland, S. 1990. Health and nutritional benefits from lactic acid bacteria. FEMS Micro. Review. 87:
175–188.
Hilde, M.O., M.H. Helland and J.A. Narvhus. 2003. Growth and metabolism of selected strains of
probiotic bacteria in milk. Int J. Food Microbiol. 87: 17-27.
Kerr, T.J. and B.B. McHale. 2001. Applications in General Microbiology, A Laboratory Manual,
Winston-Salem.
Marshall, V. M. 1987. Lactic acid bacteria: starters for flavor. J. Elsevier Sci. B. V. 46: 327-336.
Peryam, D.R. and N.F. Girardot. 1952. Advanced taste test method. Food Eng. 24: 58-61.
Ritcher. R.L. and R. Vadamuthu. 2001. Microbiological Examination of Food. 4th ed. American Public
Health Association. pp: 483-485.
Salminen, S., E. Isolauri and E. Salminen. 1996. Clinical uses of probiotics for stabilising the gut
mucosal barrier: successful strains and future challenges. Antonie van Leeuwenhoek 70: 251-
262.
alwa, A.A. and H. Diekmann. 2000. Behavior of aflatoxin during manufacture and storage of yoghurt.
Alex. J. Vet. Sci. 16: 1-7.
Steel, R.G.D. and J.H. Torrie. 1980. Principles and procedures of statistics: A biometrical Approach.
2nd Ed. McGraw- Hill Book Company, New York, New York.
Steel, R.G.D. and J.H. Torrie and D.A. Dickey. 1997. Principles and procedures of statistics: A
biometrical approach. 3rd edn. Mc-Graw Hill book Company, New York, New York.
Tamine, A.Y. and K. Robinson. 2004. Yoghurt Science and Technology, Institute of Applied Science.
Walker, D.K. and S.E. Gilliland. 1993. Relationship among bile tolerance, bile salt deconjugation and
assimilation of cholesterol by Lactobacillus acidophilus. J. Dairy Sci. 76: 956-961.
Yeung, P. S. M. and M.E. Sanders, C.L. Kitts, R. Cano and P.S. Tong. 2002. Species specific
identification on commercial probiotic strains. J. Dairy Sci. 85: 1039-1051.
1303
Table 1. Determination of titratable acidity and pH of Flavored Probiotic Acidophilus Milk at Day-1
and Day-6.
Flavor
Mango Pineapple Strawberry
Interval Day-1 Day-6 Day-1 Day-6 Day-1 Day-6
Titratable Acidity 0.81±0.01 0.91±0.01 0.81±0.01 0.92±0.01 0.80±0.01 0.91±0.01
pH 4.50±0.01 4.32±0.01 4.52±0.01 4.33±0.01 4.50±0.01 4.30±0.01
Table 2. Total Viable Count of Lactobacillus Acidophilus in Flavored Probiotic Acidophilus Milk.
Flavor
Interval Day-1 Day-6 Day-1 Day-6 Day-1 Day-6
Total viable count 2.50x106 1.50x106 3.60x106 3x106 2.87x106 2.50x106
Table 3. Summary of Sensory Evaluation Scores of Flavored Probiotic Acidophilus Milk Conducted at
Day-1 (a) and Day-6 (b).
a)
Attributes Flavor
Color 7.8 ±0.92 8.0 ±1.05 7.5 ±1.08
Flavor 7.6 ±1.074 7.4 ±0.96 8.0 ±0.66
Over Acceptability 7.6 ±1.26 7.8 ±0.78 8.2 ±0.78
b)
Attributes Flavor
Color 6.4 ± 0.84 6.7 ±0.95 6.8 ± 1.13
Flavor 6.6 ±0.69 5.8 ±1.032 6.3 ±1.05
Over Acceptability 6.9 ±0.57 5.9 ±0.88 6.1 ±0.74
A 9-point hedonic rating scale (9 = excellent; 1 = extremely poor) was used for sensory evaluation.
1304
Effective Environmental Factors on Milk Composition, Rennet Coagulation

Time and Urea Content of in Anatolian Buffaloes Milk of Ilıkpınar Village
Hatay Province
Özel ŞEKERDENa*, Yahya Kemal AVŞARb
a
Mustafa Kemal Univ. Fac. of Agric. Dept. of Anim. Sci., Antakya, Turkey
b
Mustafa Kemal Univ. Fac. of Agric. Dept. of Food Engineering, Antakya, Turkey
Corresponding e-mail: sekerden@mku.edu.tr*
ABSTRACT
The objectives of this study were to investigate determining environmental factors on
composition, renneting time, urea concentration, acidity, density and pH of Anatolian Buffaloes
milk. As a total of 115 milk samples from 53 cows that were calved in 2004 and 2005 in 8 units of
Ilıkpınar Village were collected in morning milkings in June, September, December and March.
The cows were on their lactation days 30±15, 60±15, 90±15, 120±15, 150±15, 180±15, 210±15,
240±15 and 270±15. The milk samples were analysed for total dry matter, fat, protein, ash, density,
pH, acidity, renneting time and urea content. Rennet coagulation time, urea, protein and fat contents
were determined using Berridge, photometric, formol titration and Gerber methods, respectively.
Data were classified as follows; lactation stages: 1 (30±15, 60±15, 90±15 days): 2 (120±15,
150±15, 180±15): 3 (210±15, 240±15, 270±15); calving year: 1 (2004), 2 (2005); calving season: 1
(January-May), 2 (September and October); month of samples collection: 1 (June), 2 (September), 3
(December), 4 (March); lactation order: 1 and 2 : 1, 3 and 4: 2, 5 and 6: 3. Effects of environmental
factors on each variable were investigated separately and analysed using analysis of variance.
Production mount on all the characteristics; calving year and lactation stage on most of the
characteristics; lactation order on fat and protein contents; unit and calving season on some of the
characteristics were found to be effective significantly.
Keywords: Buffalo, milk properties, variation sources
INTRODUCTION
It is a well established fact that there are several factors affecting milk composition. It varies
from one genotype to another (1, 2). Feeding (3), lactation stage (4, 5, 6) production season (7, 8)
and calving season (7, 9, 10, 11) have significant effects on fat, casein, protein and total dry matter
(TDM). Lactation order also has important influence on milk yield and its constituent contents (4,
10) although Şekerden et al (5), reported that lactation order did not have an important effect on any
milk component percentage in Anatolian buffaloes.
Milk coagulation properties [rennet coagulation time, firming time and firmness of clot] are
very important to cheese production and can be affected by genotype (2, 12), season, lactation
order, lactation stage and feeding (13). These properties change throughout lactation as milk yield,
protein and fat concentrations change and are detected best at the beginning and the end of
lactation. Lactation number does not have a significant effect on milk coagulation ability (12),
whereas season has such an effect owing to the reduction in urea content of milk (14). Foltys et al.
(15) determined that urea content of milk rises from 29.2 mg/100 g in winter to 36.07 mg/100 g in
May; protein, fat and lactose contents decreased in the same period from 3.06% to 2.77%, 4.27% to
3.92%, 4.80% to 4.60%, respectively. Feeding level is effective on urea content of milk (16).
Milk coagulation properties differ significantly from one unit to another. The differences are
due most likely to feeding and management factors (12). Povinelli et al. (2) found that breed, herd
and lactation stage had a significant effect on milk coagulation ability on five different dairy cattle
breeds unlike the urea content. pH has a negative effect on milk coagulation ability (17).
1305
Milk urea concentration can be used as a tool to monitor crude protein and energy intake
(18) and is related to the rate of protein-energy in ration and crude protein intake (19, 20). In order
to use milk urea concentration as a tool to identify any imbalances related to feeding, in addition to
feeding related factors such as food intake and ration composition and other factors and their levels
of effect have to be determined and should be taken into consideration to interprete urea
concentration (21). These factors can be ordered as sample collection season, analysis method, live
weight of animal, parity and milk yield of cow (22). Roy et al. (23) reported that a significant
reduction occurred in milk urea concentration as the lactation number increased. However, lactation
stage did not have significant effects on urea and protein concentrations of milk.
Hojman et al. (24) showed that milk urea level was higher in summer and increased with
lactation number for adult cows. Relationships with milk urea content and crude protein, ruminal
digestive protein and fibre content of ration were positive, but the relationship between urea content
and ration energy was negative.
The objectives of this study were to investigate effective environmental factors on milk
composition, rennet coagulation time, urea concentration, titratable acidity, density and pH of
Anatolian Buffaloes’ milk

The material of the study consisted of 115 milk samples from 53 Anatolian buffalo cows of
Ilıkpınar Village of Kırıkhan District of Hatay Province in 8 units that they were calved in 2004 and
2005. Milk samples were collected from the morning milkings in June, September, December and
March from the cows on lactaidohn dayıs 30±15, 60±15, 90±15, 120±15, 150±15, 180±15, 210±15,
240±15 and 270±15.
From the beginning of June 2004, milk samples were collected from all the buffalo cows in
morning milkings monthly on milk control days of June, September, December and March. The
samples were analysed for total dry matter, fat, protein, ash contents, pH, density, rennetting time
and milk urea content. Protein and fat contents were determined by formol titration (25) and Gerber
methods (26), respectively. Rennet coagulation time was determined by recording time from the
addition of enzyme to milk to the appeareance of first clot using Berridge method (27). Milk urea
content determined with diacetyl monoxime using photometric method, as described in Merck
handbook (28). Data were classified as follows; 30±15., 60±15, 90±15 days: 1st.; 120±15, 150±15,
180±15: 2nd.; 210±15, 240±15, 270±15: 3rd lactation stages. 2004: 1st, 2005: 2nd calving years;
January-May period: 1st, September and October Months: 2nd calving seasons; June: 1st, September:
2nd, December: 3rd, March: 4th production months (samples collection months); 1st and 2nd : 1st, 3rd
and 4th: 2nd, 5th and 6th: 3rd lactation order groups.
The effect of environmental factors on each characteristic were analysed separately using
variance analysing technique. The means and correlation coefficients of each character were
calculated. SPSS programme (standard version, SPSS Inc.) were used in the statistical analysis.
RESULTS
Variance analysis are given in Table 1, 2 and 3.
1306
Table 1. Variance analysis for morning and daily milk yields, rennet coagulation time and pH
Variation source f.d. F
Morning milk Daily milk Rennet coagulation pH
yield yield time
Unit 7 11.400*** 12.149*** 1.193 2.841*
Production month 3 7.275*** 8.531*** 12.931*** 3.246*
Calving season 1 6.516* 0.474 4.563* 0.066
Lactation stage 2 0.067 5.424** 10.049*** 7.076**
Calving year 1 1.371 5.295* 13.169*** 2.918*
Lactation order 2 1.915 1.360 0.972 1.699
Total N 115 115 115 115
*P < 0.05, **P < 0.01, ***P < 0.001
Table 2. Variance analysis for TDM, fat, ash contents and density
Variation f.d. F
source TDM Fat Ash Density
Unit 7 0.997 0.644 0.781 1.508
Production month 3 6.017** 3.025* 19.797*** 22.553***
Calving season 1 0.002 0.842 0.003 0.085
Lactation stage 2 3.611* 10.758*** 4.610* 3.534*
Calving year 1 38.739*** 46.880*** 14.403*** 35.519***
Lactation order 2 0.356 3.377* 0.805 0.740
Total N 109 109 107 107
*P < 0.05, **P < 0.01, ***P < 0.001
1307
Table 3. Variance analysis for titratable acidity, protein and urea contents
Variation f.d F
source Titratable acidity Protein Urea
Unit 7 5.497*** 1.225 1.831*
Production month 3 4.898** 9.191*** 6.081**
Calving season 1 1.758 5.425* 1.293
Lactation stage 2 9.687*** 3.869* 0.689
Calving year 1 12.733** 110.153*** 1.110
Lactation order 2 1.185 3.538* 1.223
Total N 115 109 100
*P < 0.05, **P < 0.01, ***P < 0.001
DISCUSSION
As can be seen in Table 1, morning milk yield was affected by unit (P < 0.001), production
month (PM) (P < 0.001) and calving season (CS) (P<0.05); daily milk yield was affected by unit (P
< 0.001), PM (P < 0.001), lactation stage (LS)(P < 0.01) and calving year (CY) (P < 0.05)
significantly. Differences in daily milk yield between CY can be explained by differences in
feeding level during the year, year to year and unit to unit. As opposed to the literature (4, 10), the
effects of lactation order (LO) on morning and daily milk yields were found not significant in this
study (Table 1).
As is clear from Table 2, PM (P < 0.01), LS (P < 0.05) and CY (P < 0.001 were influential
on TDM content. The effects of PM and CY can be explained by feeding conditions since a pasture-
based feeding in the Village was commonly employed. The literature also supported that PM (4, 7,
8) and LS (5, 6) effects on TDS were significant. However, the effect of lactation order effect on
TDM content was found insignificant on Anatolian buffaloes in an earlier study (7).
PM (P < 0.05), LS (P < 0.001), CY (P < 0.001) and LO (P < 0.05) were influential on fat
content significantly. The effects of PM and CY on fat can be explained by feeding level. The
literature supported the significant effects of PM (7, 15) and LS (4, 5, 6) on fat content, except for
one study (5) where the effect of LO on fat content was reported insignificant (4, 10) (Table 2).
Ash content was also affected by PM (P < 0.001), LS (P < 0.05) and CY (P < 0.001) (Table
2). PM (P < 0.001), CS (P < 0.05), LS (P < 0.05), CY (P < 0.001) and LO (P < 0.05) were found to
be effective on protein content significantly. Alteration in milk fat and protein contents are related
to feeding level and climatic conditions. Literature also confirms that PM (7, 8, 15), and CS (7, 9)
are influential on protein content of milk. The significant effect of LO on milk protein content was
also reported (4, 10), as opposed to the findings of Şekerden et al (5). The milk yield varies due to
LS and there are negative relationship between milk yield with fat and protein contents of milk.
Protein and fat contents were highest at the beginning and end of lactation, and lowest during peak
lactation associated with milk milk yield (1, 4, 11) (Table 3). Roy et al (23) reported that LS did not
have a significant effect on milk protein concentration in Murrah buffaloes.
The pH of milk samples were affected by unit (P < 0.05), PM (P < 0.05), LS (P < 0.01) and
CY (P < 0.05); the density was similarly affected by PM (P < 0.001), LS (P<0.05) and CY (P<
0.001) significantly. PM (P < 0.001), CS (P < 0.05), LO (P < 0.001) and CY (P < 0.001) were
effective significantly on RCT (Table 1). Literature reports that milk coagulation properties can be
1308
affected by production season and feeding level (12, 13, 14), LS (2, 12, 13); coagulation properties
are well related to alteration in fat and protein contents at the beginning and end of lactation.
However, the significant effect of LO on coagulation properties are reported by some researchers
(13) whereas findings supporting our results were reported by the others (12).
In spite of literature indicating that milk coagulation properties vary from one unit to another
significantly, this was found insignificant in our study since feeding was based mainly on village
pasture, and supplement fodders were almost the same in every unit (Table 1). Titratable acidity
was affected by unit (P < 0.001), PM (P < 0.01), LS (P < 0.001), CY (P < 0.01) at significant levels
(Table 3). Similarly, urea content of milk was affected by unit (P < 0.05) and PM (P < 0.01)
significantly (Table 3). It can be suggested that urea concentration was affected by only feeding
level since both sample collection months and unit factors are related to feeding levels. It is reported
that production season (14) and feeding level (15, 16, 24) are effective on milk urea concentration.
It was also reported that milk urea concentration is affected by LO significantly (23, 24), but LS
does not have an important effect on milk urea concentration (23) as was found in our study (Table
3).
REFERENCES
Agabriel, C., J.B. Coulon, G. Marty and B. Bonaiti. Changes in fat and protein concentrations in
farm with high milk production. Anim. Bred., 1993; 61: 532.
Anonymous, Urea in Milk. http://photometry. Merck.de/servlet/PB/menu/ 1169740_ePRJ-MERCK-
EN-pcontent_12/content.html, 15.09.2005.
Baker, L.D., J.D. Ferguson and W. Chalupa. Responses in urea and true protein of milk to different
protein feeding schemes for dairy cows. J. Dairy Sci., 1995; 78: 2424-2434.
Broderick, G.A. and M.C. Clayton, A statistical evaluation of animal and nutritional factors
influencing concentrations of milk nitrogen. J. Dairy Sci., 1997; 80: 2964-2971.
Erbersdobler, H.F. and H. Zucker. Harnstoffgehalt der Milch-ein Indicator der Protein versorgung
von Milchkühen Kraftfutter, 1990; 1: 11-12.
Foltys, V., J. Pazmova, E. Chobotova and V. Zatopkova, V.: Influence of season on composition of
bulk milk in relation to its technological processing. Cattle Commission, Free
Communications. EAAP 46th Meeting European Association for Animal Production,
Prague. 1995; 210.
Hanus, O., F. Malina, J. Kopecky, R. Fedelska and A. Beranova, A.: Sezonni kolisani slozeni
bazenoveho mleka. Mliekarstvo, 1994; 25: 36-37.
Hojman, D., O. Kroll, G. Adin, M. Gips, B. Hanochi and E. Ezra, E.: Relationships between milk
urea and production, nutrition, and fertility traits in Israel Hers. J. Dairy Sci., 2004; 87:
1001-1011.
Hojman, D., G. Adin, M. Gips and E. Ezra. Association between live body weight and milk urea
concentration in Holstein cows. J. Dairy Sci., 2005; 88: 580-584.
Ikoneen, T. Possibilities of Genetic Improvement of milk coagulation properties of dairy cows.
Academic Dissetation, Univ. of Helsinki, Dept. Anim. Sci., Publications, 2000; No: 49.
James, C.S. Analytical Chemistry of Foods. Elsevier Publisher, 1998; New York.
Koçak, C. and H. Devrim. Effect of heat procedure on coagulation ability of goat milks. Gıda, 1994;
19: 125-129.
Kreuzer, M., J.P. Schulz, C. Fry and H. Abel. Rennet coagulation properties of milk from cows at
three stages of lactation supplied with graded levels of an antimicrobial feed supplement.
Milchwissenchaft, 1996; 51: 243-247.
1309
Kurt, A. Guide of Analysis Methods of Milk and Milk’s Products. A.Ü.Publ. No: 18. Lecture book
No: 252, Erzurum. 1984.
Patel, K.S., S.V. Majmudar, H.B. Patel and L.H. Saiyed, L.H.: Lactation curve for milk fat content
in Surti buffaloes. Gujarat Agricultural Univ. Research, 1991; 16: 82-83.
Piironen, T., M. Ojala, T. Niini, E.L. Syvaoja and J. Setala, J.: Effects of milk protein genetic
variants and lactation stage on renneting properties of bovine milk. Commission on Cattle
Production, Session II. 43rd EAAP Meeting, Madrid. 1992; 1-12.
Polanski, S., H. Czaka and M. Latocha. The effect of some factors on milk fat and protein
percentage of Simmental cows at the Brzozow pedigree farm. Roczniki Naukowe
Zootechniki., 1992; 19: 55-65.
Povinelli, M., D. Marcomini, , R.D. Zotto, G. Gaiarin, L. Gallo, P. Carnier, M. Casandro. Sources
of variation of milk rennet-coagulation ability of five dairy cattle breeds reared in Trento
Province. IX. World Animal Prod. Cong., Porto Alegre. 2003.
Şekerden, Ö., H. Erdem, B. Kankurdan, and B. Özlü. Factors affecting milk composition and
changes in milk composition with lactation stage in Anatolian bufaloes. Turk. J. Vet. Anim.
Sci., 1999a; 23: 505-509.
Şekerden, Ö., M. Tapkı, and M. Şahin. Changing of milk composition in first ten days and along of
the lactation in Black Pied cattle, J. Atatürk Univ., Fac. of Agric., 1999b; 30: 37-40.
Şekerden, Ö. Effects of calving season and lactation order on milk yield and milk components in
simmental cows. Turk. J. Vet. Anim. Sci., 1999; 23: 79-86.
Şekerden, Ö., İ.Tapkı, and Ş. Kaya. Changing of milk yield and composition with lactation stage
and production season at village conditions of Hatay Province in Anatolian buffaloes. J.
Atatürk Univ. Fac. of Agric., 1999c; 30: 161-168.
Sethi, R.K., M.S. Khatkar, S.N. Kala and V.N. Tripathi. Effect of pregnancy on milk constituents
during later stages of lactation in Murrah Buffaloes. Proc. 4th World Buffalo Cong.. San
Paolo. 1994.
Rajala-Schultz P.J. and W.J.A. Saville. Sources of variation in milk urea nitrogen in Ohio Dairy
Herds. J. Dairy Sci., 2003; 86: 1653-1661.
Roseler, D.K., J.D. Ferguson, C.J. Sniffen and J. Herrema. Dietary protein degradability effects on
plasma and milk urea nitrogen and milk nonprotein nitrogen in Holstein cows. J. Dairy Sci.,
1993; 76: 525-534.
Roy, B., R.K. Mehla and S.K. Sirohi. In fluence of milk yield, parity, stage of lactation and body
weight on urea and protein concentration in milk Murrah
buffaloes.(http://www.ajas.info/contents/abr/03-9-9 htm), 2004.
Waldner, D.N., S.R. Stokes, E.R. Jordan, and M.L. Looper. 2003. Managing milk composition:
Normal sources of variation. http://www.ansi.okstate.edu/exten/ dairy/wf-4016.html,
06.06.2003.
Yadav, S.B.C., A.S. Yadav and M.S. Yadav, Seasonal fluctations in milk yield composition at
various stages of lactation in crossbred dairy cattle. Indian J. Dairy Sci., 1991; 44: 33-36.
1310
A Study on the Composition and Microbiology of Raw Milk

from Three Breeds of Buffalo in Thailand
Krittin CHUAYCHOOa, Onrampa THEMPACHANAa, Puriya NGAMWONGSATITa and

Jitkamol THANASAKa*
a
Department of Clinical Medicine and Public Health Section, Faculty of veterinary science,
Mahidol University, 999, Phutthamonthon Sai 4 Road, Salaya, Nakornpathom, 73170, Thailand
*Corresponding email: jitkamol.tha@mahidol.ac.th
ABSTRACT
The objective of this study is to investigate the milk quality parameters and microbiological
status of raw buffalo milk. Seventy nine samples of raw buffalo milk were collected from three
types of buffalo including swamp buffalo (n=10), river buffalo (Murrah) (n=55) and crossbreed
buffalo (Murrah x swamp buffalo; F1) (n=14). Average milk compositions were; fat 6.35%, protein
4.13%, lactose 4.79%, solid not fat (SNF) 9.8% and total solid (TS) 16.22% with freezing point
(FP) at -0.61 oC and pH 6.72. Instead of lactose that was higher in Murrah milk rather than in
swamp buffalo milk, most of other parameters; fat, protein, SNF, TS, FP and pH of milk from
swamp buffalo were higher than those of Murrah milk. Moreover, those parameters of the
crossbreeds were presented in between those of milk from swamp buffalos and Murrah. Average of
electrical resistance (ER) and somatic cell counts (SCC) were 659 units and 147,494 cells/ml
respectively. Microbiological analysis of an average total plate count (TPC) was 4.43 x 105 cfu/ml,
which was not shown any significantly different among three types of samples. Some pathogenic
microorganisms were isolated such as Staphylococcus aureus, Pseudomonas spp., Enterobacter
spp. and Escherichia coli which most presented in milk samples of Murrah. All milk quality and
microbiological values reveal the need of upgrade breeding to improve the quality and hygienic
status of buffalo milk production in Thailand.
Keywords: Milk, Milk composition, Microbiology, Murrah, Swamp, Buffalo
INTRODUCTION
Thailand is one of the countries that breed buffaloes. Most of them are Swamp type. A study
on buffalo rising situation and attitude of farmers raising buffalos in the northeastern part of
Thailand indicated that the primary objective of buffalo used is to produce the fertilizer made from
bufffaloes’ feces for 80.70 % instead of considering for work. However, the farmer sales buffaloes
for their beneficial income. Moreover another study reveals that primary purpose of buffaloes rising
of farmers is to be used as a heritage which accounted for 75.8%. Recently, Thai people start to pay
attention in buffalo milks by more consumption. Occurrence of buffalo dairy farm will make
buffalo become an economic livestock in the future. River type buffaloes are breed. In addition,
crossbreeding the native swamp buffalo with river buffalo breeds such as Murrah and Thai local
swamp buffalos are thought to be an improvement of production and feeding management. To
awareness of food safety and quality, the microbial and chemical composition of buffalo milks need
to be investigated. Even though those general data and knowledge from the studies in others
countries are available (Han and Ding., 1994; Fundora et al., 2001; Supino et al., 2004; Han et al.,
2007), the information reported in Thailand will be great significance for further development of
higher quality buffalo milk production.

Animal and milk sampling
Samples of buffalo milk were obtained from a Murrah farm in Chachoengsao province of
Thailand. A total of 79 samples of buffalo milk, tested with California Mastitis test (CMT) did not
exceed +1 score, were collected from three types of buffalo. These included 10 samples from
1311
swamp buffalo, 55 samples from pure river buffalo (Murrah) and 14 samples from crossbreed
(murrah buffalo x swamp buffalo; F1).
milk pH, Electrical resistance (ER) and California Mastitis Test (CMT)
A part of each sample was used to be measured for pH, electrical resistance (ER) and
California Mastitis Test (CMT) parameters immediately. A digital pH meter and a mastitis detector
(Draminski®) were utilized to detect the pH and the ER respectively. CMT was conducted
according to Gomes et al. (2006).
Milk composition analysis and SCC
The composition of fat , protein, lactose, solid not fat (SNF), total solids (TS) and freezing
point (FP) were measured using automated infrared analysis (MilkoScanTM , IDF and AOAC
approved), while SCC was analyzed using Fossomatic milk cell counter (FossomaticTM, AOAC
approved).
Microbiological analysis
A tenfold serial dilution of each milk sample was prepare in sterile NSS 0.9% up to 10-3
dilution.Subsequently,1 ml of each dilution was spread onto Plate Count agar and 1 ml of milk was
spread onto sheep blood-agar plate and MacConkey.The plates were incubate aerobically at 37°C
and examined at 24 h post-seeding.The colonies were provisionally identified based on
morphology, haemolysis pattern and Gram stain. The number of each distinct colony type were
recorded. Representative colonies were subculture on sheep blood agar plate and incubated
aerobically at 37°C, 24 h to obtain pure colony. Gram positive cocci were tested for catalase and
coagulase production.
All data were subjected to one way ANOVA according to a completely randomized design.
The multiple comparisons were used for significant differences among each parameter from three
types of buffalos.

Milk composition analysis
Fat, protein, Solid not fat (SNF), Total solid (TS), and pH of swamp buffaloes were
significantly higher (P<0.05) than those of river buffalo Murrah. The lactose content of Murrah was
significantly higher (P<0.05) than swamp buffalo. Interestingly that protein, SNF and TS of F1
were higher than the value of Murrah but lower than those of swamp buffalo. Even though the fat
content of Murrah and F1 milk were not difference but F1 also show the lower value than swamp
buffalo (Table 1). These results are in agreement with the previous study that reported by Han et al.
(2007). Their results show the content of multi-crossbreed-buffalo milk were higher than those of
river buffalo Murrah but lower than crossbreed F1 and F2 buffaloes. This ranking was to be
expected that the swamp buffalo milk has more nutritive value than river buffalo milk (Amarjit and
Toshihiko, 2003). Considering this investigation with the others, it can be reported that most of the
nutrients in swamp buffalo milk has higher levels than the river buffalo milk which in between of
those milk composition values is expected to be Crossbreed F1. Concerning of breeding Murrah in
Thailand, Murrah milk in this study had lower fat, protein, lactose and total solid contents than
other reported of other countries such as China, Cuba, Bulgaria and Pakistan (Petrova and
Tzankova, 2000; Han et al., 2007; Mahmood and Usman, 2010). According to fat (%), protein (%),
lactose (%), total solid (%) of F1 buffalo in this study indicated that the lower nutrient of Murrah
milk that breed in Thailand can be improved by cross breeding between River buffalo Murrah and
Thai local swamp buffalo.
Electrical Resistance (ER) and Somatic cell count (SCC)
There is no significant statistical difference of ER and SCC‘s values among three types of
buffalo milk were observed. Normally, the mastitis detector that equipped for cows was assigned
the range of 250 to 300 units for milk quality interpretation, however, all ER values in this study
were exceed 600 units. This result indicated that the standard range used with cows was not
1312
appropriate for buffalo milk determination, thus the further study can be conducted to adjust the
new range for buffalo milk.
Microbiology analysis
There is no significant statistical difference of total plate count (TPC) values among three
types of buffalo milk were observed. Buffalo milk contained an average TPC of 4.43±1.45
×105cfu/ml which is lower than the maximum level of the EU specification (EU Directive
92/46/EEC), 5×105 cfu/ml. There are 13 types of bacteria were isolated from 79 buffalo milk
samples such as Acinetobacter spp., Acinetobacter baumanni, Group D Streptococcus,
Streptococcus not Group.A,B,D, Viridans streptococcus, Pseudomonas spp., Pseudomonas
aeruginosa, Enterococcus spp., Enterobacter spp., Moraxella spp., S.aureus, Coagulase-negative
staphylococcus and E.coli. The majority of bacterial isolates were Coagulase-negative
Staphylococcus (83.54 %), Group D streptococcus (25.32 %) and Staphylococcus aureus (18.99 %)
with most encountered in Murrah milk. In addition, 3 majorities of bacterial isolated from Murrah
milk were Coaulase-negative Staphylococcus (85.45 %), Group D streptococcus (30.91 %) and
Enterobacter spp. (20.00 %). Concerning pathogenic microorganism, S.aureus, Enterobacter spp,
Pseudomonas spp. and E.coli founded in this study are in accordance with those of previous finding
(Gupta, 1987; Han et al., 2007; Maniruzzaman et al., 2010). E.coli is microorganism indicates
contamination of fecal. The presence of pathogenic and indicator bacteria, such as E. coli,
Enterobacter spp., Pseudomonas spp. and S. aureus indicate a public health problem. As most of
the microorganisms were isolated from Murrah milk, this evidence indicated that pure breed murrah
breeding in Thailand was susceptible to infection rather than swamp buffalo and those crossbreed.
IMPLICATIONS
Upgraded crossbreed (Murrah×Swamp) should be recommended to improve immunity and
milk quality of milking buffalo in Thailand. This investigation also indicated the safety of raw
buffalo milk production in Thailand.
ACKNOWLEDGEMENTS
The author gratefully acknowledges Mrs. Runjuan Hengtragoolsin for cooperation of
sampling at the Murrah farm and Miss. Sukanya Apiratworasakul and Miss Suwanna Saenyuttitham
for laboratory procedure suggestion.
REFERENCES
Fundora, O., M.E. González, O. Lezcano, A. Montejo, N. Pompa, and A.V. Enriquez. 2001. A
comparative study of milk composition and stability of Murrah river buffaloes and Holstein
cows grazing star grass. Cuban Journal of Agriculture Science 35: 219-222.
Gomes, V., A.M.M.P.D. Libera, M. Paiva, K.M. Madureira and W.P. Araujo. 2006. Effect of the
stage of lactation on somatic cell counts in healthy goat (Caprae hircus) breed in brazil.
Small Rumin. Res. 64: 30-34
Gupta, R.S. 1987. Bacteriological analysis of buffalo milk. Indian Veterinary Journal 63: 254-255
Han, G. and Q.B. Ding. 1994. A physiochemical study on buffalo milk in China. Journal of South
China Agricultural University 15: 92–97.
Han, B.Z., Y. Meng, M. Li, Y.X. Yang, F.Z. Ren, Q.K. Zeng and M.J.R. Nout. 2007. A survey on
the microbiological and chemical composition of buffalo milk in China. Food Control. 18:
742–746.
Mahmooh, A. and S. Usman. 2010. A comparative study on the physicochemical parameters of
milk samples collected from buffalo, cow , goat and sheep of Gujrat, Pakistan. Pakistan
Journal of Nutrition 9: 1192-1197.
Maniruzzaman, M., M.F.R. Khan, M.M. Amin, A.K. Paul and M. Islam. 2010. Isolation and
identification of bacterial flora from milk of apparently healthy buffalo-cows. Int. J. BioRes.
1: 13-16
1313
Petrova, H. and M. Tzankova. 2000. Production and composition of milk from three breeds of
buffaloes in Shoumen region. Bulgarian journal of agriculture science 103-106
Supino, M.T., M. Gallo, G. Capo, C. Morena, G. Durante and G. Galiero. 2004. Buffalo milk
produced in the province of Salerno:Evaluation of sanitary and product parameters. Bubalus
Bubalis 10: 22–26.
1314
Table 1. Milk compositions analysis of buffalo milk.
Breed Fat (%) Protein (%) Lactose (%) SNF (%) TS (%) FP pH
S (n=10) 8.30± 0.46*,a 5.41± 0.18a 4.61± 0.05b 10.75± 0.14a 19.30± 0.55a -0.61±0.00 6.83± 0.02a
M (n=55) 5.90 ± 0.18b 3.79± 0.08b 4.83± 0.03a 9.51± 0.07b 15.44± 0.20b -0.62±0.00 6.69± 0.02b
6.75 ±
F1 (n=14) 6.74 ± 0.54b 4.60± 0.25c 4.75 ± 0.10a,b 10.19± 0.16c 17.10± 0.66c -0.62±0.01
0.04a,b
Average 6.35±0.19 4.13±0.09 4.79±0.03 9.80±0.08 16.22±0.24 -0.61±0.00 6.72±0.01
S (Swamp buffalo), M (Murrah), F1 (Murrah×Swamp buffalo) abc different letters in the same column show significant statistical difference
(P<0.05) *Mean±SEM
1315
Quality Evaluation of Olive Oil Coated Labneh Cheese Mixed with Culinary Herbs
Sarfraz AHMAD a*, Hafsa UMARa, Faqir Muhammad ANJUM a and Mian Anjum MURTAZA b
a
National Institute of Food Science and Technology, University of Agriculture, Faisalabad, 38000,
Pakistan; bInstitute of Food Science and Nutrition, University of Sargodha, 40100, Pakistan
*Corresponding email: sarfraz.ahmad@uaf.edu.pk
ABSTRACT
Labneh cheese is a concentrated yoghurt based cheese with 40% solids, generally stored in
olive oil at room temperature for extended shelf life. It is mostly served in Middle Eastern countries
with shredded herbs on consumer demand which are excellent source of vitamins, minerals and
biologically active compounds. The objective of this study was to analyze the effects of four
culinary herbs (mint, poppy seeds, cinnamon and basil) on the properties and shelf life of labneh
cheese prepared from whole buffalo milk. Five samples were prepared (one with each herb as four
treatments and one without herb as a control) and were coated with sufficient quantity of olive oil
for preservation. The samples were analyzed on weekly basis for physicochemical (pH, acidity),
compositional (moisture, ash, fat, protein fractions (total protein, non-protein nitrogen and non-
casein nitrogen), total solids, lactose, mineral contents), rheological (texture-firmness),
microbiological (total viable count, coliforms) and organoleptic properties during storage period of
21 days at 4±1°C. The least pH decrease was observed in basil added cheese i.e. 1.46 whereas the
highest was in cinnamon added cheese i.e. 2.15 after 21 days. The highest increase in acidity was
observed in control i.e. 0.64% as compared to culinary added cheese samples which showed
increase in the range of 0.3-0.4%. The moisture, fat, total protein, non-casein nitrogen, non-protein
nitrogen, total solids and lactose contents showed slight differences during storage. A slight
decrease in ash contents was observed throughout the storage period in all samples, the least in
poppy seeds added cheese i.e. 0.02% whereas the highest in control and cinnamon added cheese i.e.
0.14%. The calcium contents of labneh cheese samples decreased with the addition of herbs;
whereas the phosphorous contents increased. Firmness of labneh cheese samples decreased
significantly during storage may be due to penetration of oil into the product. Total viable counts
started to decrease from second week of storage. Culinary herbs and storage time influenced the
organoleptic scores as well. Results indicated that culinary herbs significantly affected the overall
quality of labneh cheese during storage and covering with olive oil after further concentration upto
40% total solids improved its keeping quality by preserving the nutrients.
Keywords: Buffalo milk, labneh cheese, culinary herbs, olive oil, storage time
INTRODUCTION
Fermented milk products are popular worldwide among which yoghurt is the most important
one. Yoghurt has limited shelf life which can be extended by concentrating it and the product
named “Labneh” (Tamime, 1978). Labneh can be defined as a semisolid dairy product obtained by
draining out water and water soluble compounds from yoghurt (Lebanese Standards, 1965). It is
perceived to be slightly acidic whereas its titratable acidity ranges from 1.8-2.0% on the basis of
lactic acid (Rasic, 1987). Bacterial growth is expected to be limited at this level, but mould, yeast
and some lactic acid producing bacteria can silently harm the quality of product. For this motive,
labneh can additionally be concentrated upto 40% total solids or above and placed into cups or
polyester bags with a covering of olive oil, concentrated product known as “Labneh anbaris”.
Labneh anbaris also known as labneh cheese, produced and sold unpacked in some rural areas of
Middle East (Rosenthal et al., 1980). Traditional and modern sophisticated methods are used for
labneh cheese preparation. Labneh cheese can be prepared from milk of different species. Malek et
al., (2001) found labneh cheese prepared from buffalo’s whole milk brighter, whiter and more
acceptable than that prepared from cow and reconstituted skim milk powder. Labneh cheese is
1316
considered appropriate for lactose intolerant individuals than simple yogurt because of low lactose
content which are lost in drained water whereas the fat content can be diversified according to
consumer requirement (Salji, 1991).
The organoleptic quality of labneh can be enhanced by herbs addition. Herbs are plants with
leaves, seeds and flowers and are categorized on the basis of use as culinary and medicinal herbs.
Herbs used in this research along with their benefits are: mint has good taste and relieves
suppressed, painful or scalding urine; poppy seed used against pain, insomnia, nervousness and
chronic cough (Pelder and Ros, 1996); cinnamon used as a blood purifier, infection preventer and
provides digestion aid (Keith, 2008) and basil is used against stomach cramps, vomiting,
constipation, headaches and anxiety. Olive oil preservation is known to affect the microbiological
quality of labneh cheese (Rao et al., 1987). Yeast and mold are counted as the main cause of
bacterial spoilage in Labneh even when stored at ambient temperature 25-30°C or at 7°C
(Suriyarachchi and Fleet, 1981). Olive oil forms a coating on the surface of labneh balls creating
anaerobic conditions and preventing the growth of microorganisms (Keceli et al., 1999). In Pakistan
cow or buffalo milk is used to prepare regular unstrained yoghurt by the name of dahi (Younus et
al., 2002). When dahi is kept for several hours in clay pot some of the water evaporates through the
clay pores leaving behind true strained yoghurt ‘Chakka’. Previously very limited researches have
been conducted on labneh cheese. The aim of this study was to use buffalo milk for preparation of
labneh cheese and to analyze the effect of four culinary herbs (mint, poppy seeds, cinnamon and
basil) on physico-chemical/quality attributes of labneh cheese.

Material: Fresh raw whole bulk buffalo milk was obtained from Dairy farm, Department of
Livestock Management, University of Agriculture, Faisalabad. Commercially available freeze dried
cultures (blend of Streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgaricus) was
provided by commercial suppliers (Rhodia, France). The herbs (mint, poppy seeds, cinnamon and
basil) used in this study were bought from local market. The herbs were dried and then properly
ground before use. Olive oil with the brand name “Sasso” was purchased from the local market.
Commercially available salt was added in Labneh cheese.
Preparation of Labneh Cheese: Raw whole buffalo milk samples were pasteurized at 95oC for 10
min then homogenized at 4000 bar pressure using FT9 homogenizer (Arm field). The milk samples
were then cooled at 45oC and inoculated with 2.5% yoghurt mixed culture and incubated at 42°C
until the pH reached 4.6. Incubation was terminated by cooling the yoghurt at 4oC. Yoghurt was
hanged in cloth bag under refrigerated conditions for whey removal overnight to obtain labneh
cheese. Labneh samples were divided into 5 equal portions of 1 kg. The first batch was used as a
control (To) and the other batches were supplemented with different culinary herbs @3.0% as
treatments: mint (T1), poppy seeds (T2), cinnamon (T3) and basil (T4). All these samples were screw
processed and coated with olive oil. The labneh cheese samples were then analyzed in triplicates.
Physicochemical analysis: Heated milk and labneh cheese samples were analyzed for pH through
electronic digital pH meter (Inolab WTW Series 720) and acidity by simple titration method
(AOAC, 2000).
Compositional analysis: Heated milk and labneh cheese samples were analyzed for moisture, ash,
fat, protein fractions (total protein, non protein nitrogen and non casein nitrogen), total solids, and
lactose and mineral contents using standard methods (AOAC, 2000) whereas protein fractions were
analyzed by International dairy federation methods (IDF, 1993).
Microbial analysis: Cappuccino and Sherman (1996) method was used to conduct total viable and
Coliform count.
Rheological analysis: TA-XT plus texture analyzer was used to assess the firmness of the labneh
cheese samples according to Kumar and Mishra (2008).
Organoleptic properties:The organoleptic properties of labneh cheese samples were evaluated by 7
panelists after every week. In terms of intensity, the evaluation was done on the basis of nine point
hedonic scale by Meilgarrd et al. (1999). The parameters judged were: surface appearance,
1317
color/brightness, water on surface, firmness on cutting, stickiness with cutting, taste, flavor,
mouthfeel and acidity. The overall acceptability was calculated as the sum of scores of judged
parameters.
Statistical analysis: Data obtained was subjected to statistical analysis using analysis of variance
technique (ANOVA) under two factor factorial completely randomized designs (CRD). The mean
of all treatments were also compared by using LSD test adopting the method as described by Steel
et al (1997).

Physico-Chemical analysis: The pH decreased whereas acidity increased during storage
(Table 1). The interaction between storage time and samples were significant (P<0.01). The
difference in pH values and their decreasing trend among samples was probably due to the
herbs used and their buffering action. The least pH decrease was observed in basil added cheese
i.e. 1.46 followed by control (1.78), poppy seed added cheese (1.86), mint added cheese (1.91)
and cinnamon added cheese (2.15) after 21 days. Similar observation has been reported by
Tarakci et al. (2011). Acidity of samples ranged from 1.32-1.49%. The highest increase in acidity
was observed in control i.e. 0.64% as compared to culinary added cheese samples which showed
increase in the range of 0.3-0.4%. Similar examination was conducted by Bilal (1995), Shin et al.
(1991), Cho-Ah-Ying (1990), Peterson (1989) and Rehman (1987) who concluded that
microbial activity causes conversion of lactose into lactic acid causing an increase in acidity
during storage.
Table 1. Changes in pH and acidity of labneh cheese samples during storage.

Storage days
Mean
Treatments 0 7 14 21
pH Acidity pH Acidity pH Acidity pH Acidity pH Acidity
T0 5.56±0.03 1.06±0.04 5.44±0.04 1.15±0.02 4.68±0.02 1.35±0.02 3.78±0.01 1.70±0.01 4.86C 1.32E
T1 5.87±0.03 1.30±0.01 5.59±0.01 1.37±0.01 4.58±0.02 1.54±0.04 3.96±0.01 1.58±0.01 5.00A 1.45B
T2 5.78±0.02 1.15±0.02 5.72±0.02 1.46±0.02 4.64±0.04 1.44±0.02 3.92±0.03 1.52±0.01 5.01A 1.39D
T3 5.79±0.00 1.19±0.02 5.54±0.01 1.38±0.01 4.59±0.02 1.47±0.03 3.64±0.01 1.61±0.01 4.89B 1.41C
T4 5.49±0.01 1.32±0.02 5.31±001 1.39±0.03 4.76±0.02 1.61±0.04 4.03±0.06 1.64±0.01 4.90B 1.49A
A D B C C B D A
Mean 5.70 1.20 5.52 1.35 4.65 1.48 3.87 1.61
T0, control; T1, mint; T2, poppy seeds; T3, cinnamon and T4, basil added Labneh cheese; ABCDE Letters indicate significant difference among storage
time and labneh cheese samples, P<0.01.
Compositional Analysis: Table 2 shows mean values for compositional parameters. The moisture
content of labneh cheese samples was determined as between 47.96-57.54%.The difference in
moisture among samples is probably due to the herbs used. In accordance with these results
Dublin-Green and Ibe (2005) evaluated the quality of commercially produced yoghurt and found an
increase in moisture content with storage time. The mean values for lactose and fat contents were
3.86-4.05% and 24.88-34.81%. The ash of samples decreased significantly during storage
(P<0.01). Results concerning fat content of labneh cheese show that herbs and storage
conditions have significant affect on fat. The mean fat content of the labneh samples was similar
to that found by Kucukoner et al. (2006), but lower than that reported by Nergiz and Seckin (1998).
Total protein, non-protein nitrogen and non-casein nitrogen ranged from 10.96-16.36%, 2.35-
3.36% and 0.45-0.66%, respectively. Results show a significant decrease in nitrogenous
fractions of samples during storage. Mean values of ash and total solids, ranging from 0.97-
1.32%, 42.46-52.04%, respectively which were decreased significantly during storage.
1318
Table 2. The compositional changes in labneh cheese samples during storage.

Storage days
Treatments Mean
0 7 14 21
T0 53.45±0.94 53.00±1.01 52.44±0.07 53.20±0.52 53.03C
T1 55.08±0.45 55.21±0.29 56.25±0.06 56.57±0.37 55.78B
T2 47.56±0.42 47.41±0.37 48.19±0.56 48.68±0.36 47.96D
Moisture
T3 55.32±0.38 55.53±0.39 56.42±0.46 57.24±0.25 56.13B
T4 57.18±0.54 57.19±0.49 57.40±0.41 58.378±0.47 57.54A
Mean 53.72C 53.67D 54.14B 54.81A
T0 4.21±0.01 4.13±0.02 4.07±0.02 3.79±0.01 4.05A
T1 4.26±0.02 4.23±0.02 3.86±0.02 3.38±0.03 3.93B
T2 4.40±0.03 4.28±0.03 3.69±0.02 3.41±0.01 3.94B
Lactose
T3 4.31±0.01 4.27±0.02 3.59±0.02 3.26±0.03 3.86D
T4 4.16±0.01 4.06±0.02 3.96±0.02 3.37±0.03 3.89C
A B C D
Mean 4.27 4.20 3.83 3.44
T0 24.23±0.40 24.60±0.51 25.30±0.51 25.03±0.05 24.79C
T1 26.03±0.05 26.00±0.00 25.83±0.05 25.90±0.17 25.94B
T2 34.57±0.05 34.90±0.17 34.87±0.11 34.90±0.17 34.81A
Fat
T3 24.93±0.11 24.83±0.11 24.87±0.11 24.90±0.17 24.88D
T4 25.73±0.23 25.87±0.11 25.97±0.05 25.83±0.05 25.85BC
Mean 27.10B 27.24AB 27.37A 27.31A
T0 16.45±0.08 16.45±0.10 16.37±0.09 16.17±0.07 16.36A
T1 12.76±0.04 12.67±0.02 12.51±0.02 12.36±0.02 12.58C
T2 11.30±0.08 11.24±0.07 11.09±0.06 10.94±0.08 11.14D
Total protein
T3 13.40±0.06 13.34±0.01 13.16±0.02 12.96±0.09 13.21B
T4 11.21±0.06 11.02±0.04 10.87±0.02 10.72±0.02 10.96E
Mean 13.03A 12.94B 12.80C 12.63D
T0 3.39±0.01 3.38±0.02 3.36±0.02 3.32±0.02 3.36A
T1 2.62±0.08 2.61±0.03 2.57±0.03 2.54±0.03 2.58C
Non-casein T2 2.33±0.02 2.31±0.01 2.28±0.01 2.25±0.01 2.29D
nitrogen T3 2.75±0.02 2.74±0.05 2.70±0.01 2.66±0.03 2.72B
T4 2.31±0.02 2.27±0.01 2.24±0.01 2.19±0.01 2.25E
A B C D
Mean 2.68 2.66 2.63 2.59
T0 0.66±0.04 0.66±0.03 0.66±0.03 0.65±0.03 0.66A
T1 0.51±0.02 0.51±0.01 0.51±0.01 0.50±0.01 0.51C
Non-protein T2 0.46±0.01 0.45±0.03 0.45±0.01 0.44±0.03 0.45D
nitrogen T3 0.54±0.03 0.54±0.01 0.53±0.01 0.52±0.03 0.53B
T4 0.45±0.02 0.45±0.01 0.44±0.01 0.43±0.01 0.45E
A B C D
Mean 0.53 0.52 0.52 0.51
T0 1.10±0.13 0.98±0.02 0.97±0.01 0.96±0.01 1.00C
T1 1.00±0.02 0.96±0.03 0.95±0.02 0.94±0.03 0.97D
T2 1.32±0.01 1.32±0.01 1.32±0.04 1.30±0.07 1.32A
Ash
T3 1.19±0.03 1.18±0.02 1.12±0.04 1.05±0.01 1.13B
T4 1.03±0.01 1.01±0.02 0.95±0.02 0.95±0.01 0.98CD
Mean 1.13A 1.09B 1.06BC 1.04C
T0 46.55±0.03 47.01±0.04 47.56±0.04 46.80±0.02 46.98B
T1 44.92±0.15 44.79±0.15 43.75±0.06 43.43±0.04 44.23C
T2 52.44±0.02 52.59±0.01 51.81±0.01 51.32±0.01 52.04A
Total solids
T3 44.68±0.02 44.48±0.02 43.58±0.02 42.76±0.01 43.87C
T4 42.82±0.02 42.81±0.02 42.60±0.01 41.63±0.01 42.46D
Mean 46.28A 46.34A 45.86B 45.19C
Mineral Analysis: There was significant difference in calcium and phosphorous content of the
samples. The Ca contents were found to be high in the T0 as 1345 ppm, but the lowest Ca contents
were determined in the cinnamon added labneh samples to be 739 ppm at the end of storage
duration (Table. 3). The P contents were found to be high in the T1 as 1300 ppm, but the lowest P
contents were determined in the T3 as 920 ppm. The phosphorous contents show significant
decrease on day 0, 7, 14 and 21 among various groups (Table. 3). According to similar
observation by Tarakci et al. (2011) calcium content of the labneh decreased with the addition of
herbs, but phosphorus contents increased.
1319
Table 3. Calcium (Ca) and Phosphorous (P) contents (ppm) of labneh cheese samples.
Storage days
Mean
Treatments 0 7 14 21
Ca P Ca P Ca P Ca P Ca P
T0 1423±11 986±6 1329±9 1007±5 1351±9 1031±7 1277±11 1063±11 1345A 1021.6D
T1 1065±9 1235±10 1019±7 1277±7 1033±12 1315±7 976±8 1355±12 1023D 1300.1A
T2 1272±5 1044±11 1209±71 1093±9 1232±10 1130±8 1171±7 1164±10 1221C 1107.7C
E
T3 1014±7 873±7 716±10 899±10 775±6 938±4 448±14 971±6 739 920.3E
T4 1378±11 1168±9 1213±6 1201±8 1247±6 1253±7 1142±3 1278±3 1245B 1224.9B
Mean 1230A 1065D 1128B 1095C 1097C 1133B 1003D 1166A
Microbial Analysis: Total viable count of all cheese samples decrease during storage interval.
Maximum total viable count was observed in plain Labneh (control) whereas minimum total viable
count was observed in cinnamon added Labneh cheese. The data shows a significant increase in
viable count from 1st day to 7th day which gradually decline till 21st day (Table. 4). Results were in
the agreement with Keceli et al. (1999) who consider olive oil to be the cause of decrease in
bacterial count. Coliform was absent in Labneh cheese samples and it remained absent throughout
the storage intervals. Results indicated that proper packaging, olive oil covering and good quality of
raw material results in no coliform till 21st day of storage. Al-Hadethi et al. (1992) and Dave et al.
(1992) work is in accordance with the experimental results explaining that raw milk quality, proper
storage and packaging conditions can prevent the occurrence of coliform in final product.
Rheological and sensorial Analysis: Maximum firmness was observed in control whereas
cinnamon added labneh cheese had poor firmness as compared to control, mint, poppy seeds and
basil. The firmness showed significant decrease on 0, 7, 14 and 21 days among various groups
(Table. 4). Mahdian and Tehrani (2007) work supported these results. According to his research
it was found that samples with higher total solids had better textural properties. The results of
sensory evaluation showed that significant difference was observed among samples for surface
appearance, color/brightness, water on surface, taste, flavor and overall acceptability (P<0.01). The
color and appearance value of mint were the lowest among all treatments while mint gave best
results with respect to firmness and stickiness. Basil got the lowest points assessing water on
surface of samples. Control show best scores in assessing taste and flavor. The overall
acceptability scores decreased during storage time (P< 0.01). The lowest overall acceptability
scores were detected for mint while highest acceptability score were detected for control.
1320
Table 4. Microbiological, rheological and organoleptic parameters of labneh cheese samples.

Storage days
Treatments 0 7 14 21 Mean
T0 2.86±0.04 4.12±0.02 3.33±0.02 2.44±0.05 3.19A
T1 2.41±0.03 3.65±0.01 3.31±0.04 2.39±0.03 2.94B
T2 2.21±0.02 3.10±0.10 2.62±0.01 1.94±0.06 2.47D
TVC (cfu.mL-1)
T3 2.27±0.14 3.31±0.05 2.27±0.07 1.78±1.00 2.41E
T4 2.55±0.08 3.67±0.02 2.24±0.07 1.91±0.02 2.60C
Mean 2.46C 3.57A 2.76B 2.09D
T0 5.48±0.06 5.48±0.06 5.48±0.11 5.43±0.40 4.05A
T1 5.40±0.06 5.40±0.06 5.40±0.06 5.35±0.01 3.93B
T2 5.38±0.06 5.38±0.05 5.38±0.05 5.32±0.01 3.94B
Firmness (N)
T3 5.19±0.05 5.19±0.05 5.18±0.05 5.13±0.01 3.86D
T4 5.37±0.05 5.37±0.01 5.36±0.05 5.30±0.01 3.89C
Mean 4.27A 4.20B 3.83C 3.44D
T0 7.57±0.05 7.20±0.05 6.43±0.01 6.07±0.04 6.82A
T1 6.47±0.05 6.13±0.05 6.03±0.06 5.13±0.01 5.94D
Surface T2 7.23±0.05 6.93±0.05 6.40±0.05 5.20±0.01 6.44B
appearance T3 6.63±0.06 6.27±0.05 5.97±0.05 5.60±0.01 6.12C
T4 6.81±0.05 6.73±0.07 6.30±0.05 5.93±0.01 6.44B
Mean 6.94A 6.65B 6.23C 5.59D
T0 7.15±0.01 7.17±0.20 7.07±0.05 6.57±0.15 6.99A
T1 6.77±0.11 6.53±0.05 6.07±0.11 5.77±0.05 6.28C
T2 7.03±0.05 6.93±0.11 6.43±0.11 5.57±0.11 6.49B
Color/Brightness
T3 6.70±0.20 6.47±0.15 6.10±0.10 5.97±0.05 6.31C
T4 6.94±0.09 6.73±0.05 6.47±0.11 6.00±0.00 6.55B
Mean 6.92A 6.77B 6.43C 5.97D
T0 7.63±0.11 7.33±0.05 6.90±0.17 6.53±0.05 7.10A
T1 6.97±0.05 6.53±0.05 6.23±0.05 5.53±0.11 6.32D
T2 7.53±0.05 7.13±0.05 6.83±0.11 6.37±0.05 6.97B
Surface water
T3 7.27±0.05 7.20±0.17 7.00±0.17 6.33±0.11 6.95B
T4 7.08±0.20 6.93±0.11 6.47±0.11 5.80±0.11 6.57C
Mean 7.30A 7.03B 6.69C 6.11D
T0 7.54±0.06 6.97±0.01 6.48±0.02 5.33±0.11 6.58A
T1 6.27±0.05 5.80±0.17 5.23±0.05 4.37±0.11 5.42D
T2 7.13±0.05 6.57±0.20 5.70±0.17 4.90±0.17 6.07B
Taste
T3 6.90±0.17 6.43±0.11 5.80±0.17 5.27±0.23 6.10B
T4 6.59±0.13 6.19±0.06 5.68±0.03 4.59±0.01 5.76C
Mean 6.89A 6.39B 5.78C 4.89D
T0 7.53±0.08 6.94±0.07 6.34±0.09 5.37±0.04 6.55A
T1 6.41±0.02 5.89±0.01 5.23±0.11 4.27±0.05 5.45E
T2 7.06±0.03 6.23±0.11 5.73±0.11 4.77±0.23 5.95C
Flavor
T3 6.72±0.05 6.50±0.005 5.93±0.05 5.38±0.04 6.13B
T4 6.40±0.17 6.22±0.02 5.83±0.11 4.73±0.05 5.80D
Mean 6.82A 6.36B 5.82C 4.90D
T0 7.38±0.01 7.03±0.02 6.60±0.17 6.043±0.02 6.764A
T1 6.60±0.05 6.27±0.04 6.06±0.05 5.450±0.05 6.096D
T2 7.14±0.06 6.86±0.01 6.257±0.02 5.400±0.05 6.414B
Overall acceptability
T3 6.91±0.62 6.69±0.02 6.393±0.04 5.730±0.10 6.431B
T4 6.77±0.10 6.69±0.02 6.417±0.02 5.463±0.01 6.334C
Mean 6.96A 6.71B 6.345C 5.617D
CONCLUSION
It is evident from this research that traditional cloth bag method employed for the
production of labneh cause reduction of handling equipments resulting in safe and good quality end
product. Labneh pH decreased during storage; despite of low pH it constituted a selective
environment for the growth of yeasts and or moulds, even under refrigeration. Olive oil coating
helped to improve the keeping quality of labneh and shelf life as well. Culinary herbs significantly
affected quality parameters of labneh cheese during storage but in sensory evaluation, plain labneh
cheese samples were preferred. Conclusively, culinary herbs can be replaced with dry fruits, chilies
and cumin seeds etc.
1321
REFERENCES
Al-Hadethi, H. A., D. A. Hammed and E. R., Al-Kannay. 1992. Incidence of coliform bacteria in
milk products in Mosul city. Iraqi, Journal of Veterinary Science. 5: 151-158.
AOAC. 2000. Official Methods of Analysis. The Association of Analytical Chemists. 17th Ed.
Arlington, USA.
Bilal, A. 1995. Effect of some hydrocolloids on the physio-chemical characteristics of yogurt.
M.Sc. (Hons.) Thesis, Department of Food Science and Technology, University of
Agriculture, Faisalabad.
Cappuccino, J. G. and N. Sherman. 1996. Microbiology A Laboratory Manual. The
Benjamin/Cummings Pub. Co. Inc. New York. 59:137-149.
Cho-Ah-Ying. 1990. Population of CD4 city that produce transforming growth factor β and
interleukin-10. Laboratory of Immunology. 8:695-701.
Dave, R. L., J. M. Dave and S. S. Sannabhadti. 1992. Micorbiological quality of some market and
household dahi samples. Asian Journal of Dairy Research. 10:111-114.
Dublin-Green, M. and S. N. Ibe. 2005. Quality evaluation of yoghurts produced commercially in
Lagos. Nigeria. African Journal of applied zoology and environmental biology. 7:78-82.
IDF. International Dairy Federation. 1993. Milk Determination of Nitrogen Content (Kjeldhal
Method). Brussels Provisional Standard. IDF-20B.
Keceli, T., R. Robinson and M. H. Gordon. 1999. The role of olive oil in the preservation of yogurt
cheese (Labneh Anbaris). International Journal of Dairy Technology. 52:68-72.
Keith, S. 2008. Cinnamon: Overview of Health Benefits. Nutrition Today. 43:263-266.
Kucukoner. E., Z. Tarakci, and O. Sagdic. 2006. Physicochemical and microbiological
characteristics and mineral content of herby cacik: a traditional Turkish dairy product.
Journal of the Science of Food and Agriculture. 86:333–338.
Kumar, S. Y. and H. N. Mishra. 2008. Modelling of acidification kinetics and textural properties in
dahi (Indian yoghurt) made from buffalo using response surface methodology.
International Journal of Dairy Technology. 61:284-289.
Lebanese Standards. LS-24. 1965. Lebanese standards institution. PO Box 2806. Beirut. Lebanon
Mahdian, E. and M. M. Tehrani. 2007. Evaluation the Effect of Milk Total Solids on the
Relationship between Growth and Activity of Starter Cultures and Quality of Concentrated
Yoghurt. Journal of Agriculture and Environmental Sciences. 2:587-592.
Malek, A., S. Shadarevian and I. Toufeili. 2001. Sensory Properties and Consumer Acceptance of
Concentrated Yoghurt Made from Cow’s, Goat’s And Sheep’s Milk. Milchwissenschaft.
56:687-690.
Meilgarrd, M. M., G. V. Civille and T. Carr. 1999. Descriptive analysis techniques. In: Sensory
evaluation techniques. 3rd Ed.
Nergiz, C. and A. K. Seckin. 1998. The losses of nutrients during the production of strained (torba)
yoghurt. Food Chemistry. 61:13–16.
Pelder, M. G. and J. J. Ros. 1996. Poppy seeds: difference in morphine and codeine content and
variation in inter- and intra-individual excretion. Journal of forensic science. 41: 209-
212.
Peterson, K. 1989. Lactobacillus plantarum 299: beneficial in vitro immunomodulation in cells
extracted from inflamed human colon. Journal of Gastroenterol Hepatol. 19:166-173.
Rao, D. R., A. Alphajali and C. B. Chawan. 1987. Nutritional, sensory and microbiological qualities
of Labneh made from goat and cow’s milk. Journal of Food Science. 52: 1228-1230.
Rehman, A. 1987. Some microbiological aspects in relation to quality control of yogurt. M.Sc.
(Hons.) Thesis, Department of Food Science and Technology, University of Agriculture,
Faisalabad.
Rosenthal I., B. J. Juven and S. Gordin. 1980. The characteristics of the concentrated yoghurt
(Labneh) produced in Israel. Journal of Dairy Science. 63:1826-1828.
Salji, J. 1991. Concentrated yoghurt: A challenge to our food industry. Food Science and
Technology Today. 5:18-19.
1322
Shin, J. G., J. T. Weid and C. Bulliard. 1991. Induction by a lactic acid bacterium of a population of
CD4(+) T cells with low proliferative capacity that produce transforming growth factor β
and interleukin-10. Clinical and Diagnostic Laboratory Immunology. 8:695–701.
Biometrical Approach 3rd Ed. Mcgraw Hill Book Co., Inc., Singapore: 172-177.
Suriyarachchi, V. R. and G. H. Fleet. 1981. Occurrence and growth of yeasts in yoghurts. Applied
and Environmental Microbiology. 42:574-519.
Tamime, A. Y. 1978. Concentrated yoghurt labneh. A potential new dairy spread. Milk Industry.
80:4-7.
Tarakci, Z., H. Temiz and A. Ugur. 2011. The effect of adding herbs to labneh on physicochemical
and organoleptic quality during storage. International Journal of Dairy Technology.
64:108-115.
Younus, S., T. Masud and T. Aziz. 2002. Quality evaluation of market yoghurt/dahi. Pakistan
Journal of Nutrition. 1:226-230.
1323
Seasonal Variations in Chemical Composition of Buffalo Milk

Sarfraz AHMADa, Tiehua ZHANGb, Frank LEEe, Yanyan LIUc, Xiaodong LId, Mingruo GUOe*
a
National Institute of Food Science and Technology, University of Agriculture, Faisalabad, 38040,
Pakistan,
b
Food College, Heilongjiang Bayi, Agricultural University, Daqing, 163319, P.R. China cCollege of
Quartermaster and Technology Jilin University, Changchun, 130062, P.R. China dNortheast
Agricultural University, Harbin, 150000, P.R. China
e
351 Marsh Life Science Building, University of Vermont, Burlington, VT 05405 USA
*Corresponding email: mguo@uvm.edu
ABSTRACT
Buffaloes are the second most widely available milk source in the world after cow milk with
~13.0% (more than 93 out of 727 billion liters) annual production (FAOSTAT, 2011). The overall
volume of buffalo milk produced can be considered twice due to higher total solids contents as
compared to cow milk. Typical average milk compositions are readily available for major and minor
components but information on the seasonal variations in chemical composition of buffalo milk is
limited rather unavailable. Data presented in this study can be useful for the manufactures of diverse
dairy products with respect to compositional variations and desired characteristics based on major
components in different seasons during the whole year. Physicochemical, compositional and mineral
contents of buffalo milk as a function of a one-year’s cycle, were analyzed. Average contents of total
solids, fat, lactose, crude protein, ash and conjugated linoleic acid in the milk were 17.2%, 7.3%, 4.6%,
5.0%, 0.91%, and 6.0 mg.g-1 fat, respectively which varied throughout the year. The average mineral
contents of calcium, phosphorous, potassium, magnesium, sodium and zinc in the milk were 1799,
1217, 844, 337 and 7 mg.kg-1, respectively, and remained about steady throughout the year. The results
indicated that the buffalo milk is a rich source of nutrients and are nutritionally preferable to cows’
milk for developing children, pregnant and lactating women as little quantity as compared to cow, goat,
camel and human milk can provide recommended daily allowance of nutrients to them.
Keywords: Buffalo milk, chemical composition, conjugated linoleic acid, mineral composition,
physicochemical properties
INTRODUCTION
Globally buffalo milk production (93.02 billion liters; 12.8%) ranks second after cow milk
(606.66 billion liters, 84.4%) (FAOSTAT, 2011). In buffalo milk-producing countries (India, Pakistan
and China), Buffalo milk is more preferred owing to its rich nutrition, by consumers. It is drunk as
liquid or transformed into valuable products such as cheese curd locally known as dahi, yogurt and ice
cream (Moio et al., 1993; Jayamanne and Adams, 2004). Buffalo milk mozzarella cheese is the most
highly valued pasta filata cheese in Italy (Romano et al., 2001) and USA. Furthermore, during last few
years, buffaloes as exotic animals are becoming a hope to build a market for special dairy products like
cheeses due to higher yield higher nutritive values higher sensory quality and desirable textural
parameters than that of cow milk (Ligda, 1996; Walstra et al., 1999). Buffalo milk contains two times
higher fat contents 40 g.kg-1, 10 g.kg-1 higher caseins and ultimately higher total solids (TS) contents
than that of cow milk. These attributes make it highly suitable for processing of various types of dairy
products and result into whiter color, creamy textures, rich flavor and firmer texture profiles. For
humans, buffalo milk is richer source of protein, fat, lactose, minerals and conjugated linoleic acid
(CLA) (Khanal and Olson, 2004; Ahmad et al., 2008; Menard et al., 2010). CLA contents of animal

1324
products like milk, meat and egg can be enhanced through animal diet (Khanal and Olson, 2004).
Buffalo milk composition varies due to geographical location, breed, management practices and
feeding. These compositional variations govern the changes in manufacturing conditions, nutritional
properties and acceptability of products. The information on chemical composition of buffalo milk
including CLA contents is available but variations due to one of major factors i.e. seasons are very
limited rather unavailable. The aim of this study was to evaluate the seasonal variations over a period
of one year in milk composition with particular emphasis on CLA contents and physico-chemical
characteristics and mineral contents.

Milk samples
Bulk buffalo milk samples of three breeds (Murrah, Nili-Ravi and Jafrabadi) were collected
during 2nd week of every month from a commercial water buffalo dairy farm (Bufala di Vermont,
USA) for whole year. They were fed on a mixture of baylage, corn silage and palletized supplements
The samples were transported to the Analytical Foods Laboratory at the University of Vermont at 4°C.
Buffalo milk samples were stored at -70°C prior to analysis of fatty acids contents.
Physico-chemical, compositional and minerals analysis
Chemical composition and physicochemical characteristics of milk samples was measured by
using standard AOAC methods (Bradley et al., 1992). Minerals were measured according to Christie
(1995).
Lipid extraction, methylation, FAME and CLA analysis
Lipid extraction was done according to the Mojonnier procedure in duplicate then methylation
was done according to Christie (1995). A simple procedure for rapid transmethylation of glycerolipids
and cholesteryl esters was followed as described by Christie (1982) and modified by Shahin et al.
(2003). All Analytical grade chemicals and reagents were obtained from well-known industries like
Tridecanoate and CLA isomers mixture from Matreya Pleasant Gap, PA; 0.5 N sodium methoxide in
methanol and methyl acetate from SigmaAldrich Co St Louis, MO; nitrogen evaporator from
Organomation Associates Inc Berlin, MA.

During whole year, the minimum and maximum values found for pH: 6.76 and 6.98; fat: 6.57
and 7.97%; crude protein (CP): 4.59 and 5.37%; lactose: 4.49 and 4.73%; ash: 0.91 and 0.92% and TS:
16.39 and 18.47%, respectively (see Table 1). All the readings were within normal range with data
reported earlier (Ligda, 1996; Ahmad et al., 2008; Barlowska et al., 2011). However, all the values
varied during the whole year showing significant impact of seasons. The highest pH value was
observed in April, whereas the lowest was found in February. The lowest content of fat was observed
in July during summer, whereas the highest was found in November during winter. Fat contents were
higher during September to January as compared to February to August. The results of fat contents are
comparable to results from a study on 7770 Nili-Ravi buffaloes in herds at the Pakistan Research
Institute showing an average fat content of 6.4% (value based on 10 tests over 10 months). Regardless
of season, fat content is shown to be at least 5.0% and average found higher (6.8%) than that of cow
milk (3.5%) (Christie, 1982; Barlowska et al., 2011). In another study, Ligda (1996) found 77%
buffaloes ranged between 5 and 8% fat and 12% buffaloes were below the 5% fat content. CP content
was the highest in June during the peak of summer season and the highest was found in January during
the peak of winter season. It is evident that fat and protein contents are directly proportional to each
other as both were found the lowest in summer and the highest in winter. The lactose contents were the
lowest in the month of April when the highest pH was observed, whereas the highest lactose contents
were found in the month of February when the lowest pH was observed. So it is evident that both are
inversely proportional to each other as the similar trend of increase vs. decrease was observed for other
1325
months for both of these parameters. Ash remained unchanged throughout the year. TS contents were
the higher from November to March as compared to April to October. This study showed that buffalo
milk contains 16-19% of TS as compared to12-14% for cow milk which is an important indicator for
better adaptability of this milk for already existing cow milk based dairy products having better
rheological characteristics.
During whole year, the minimum and maximum values (mg.kg-1) found for Ca: 1022 and 2059;
P: 707 and 1513; K: 468 and 984; Na: 235 and 462; Mg: 90 and 202 and Zn: 4 and 16, respectively (see
Table 2). All these values were within the normal global averages for buffalo milk (Barlowska et al.,
2011). Mineral contents of buffalo milk are higher as compared to that of cow milk except for K which
found lower. Ca and P in buffalo milk are found twice the amount in buffalo milk than cow milk.
Major fatty acid esters including the cis9 trans11CLA isomer (the principal CLA in milk fats)
are shown in Table 3. During the whole year, these fatty acids contents varied. The major saturated
fatty acids were palmitic followed by stearic and myristic acids. Similar findings for buffalo milk were
reported by Bergamo et al. (2003) and Menard et al. (2010). CLA have numerous benefits for human
health so much interest has been given while measuring buffalo milk fat to exploit its maximum
benefits for dairy product development during different seasons. During whole year, the minimum and
maximum values (mg.g-1 fat) for cis9 trans11CLA: 4.4 and 7.6, respectively with an average value of
5.9. Bergamo et al. (2003) reported 7.3 mg.g-1 fat of cis9trans11CLA in conventional buffalo milk
which is near to the maximum value found in this study, whereas Lal (1984) found 8.0 mg.g-1 fat in
Asian water buffalo milk. If we observe cis9 trans11CLA concentration on year round, it was found to
be lower with an average value of 4.6 mg.g-1 fat during the month of July, August and September.
Parodi (1977) gave reasons for the variation in CLA concentration of milk among which the season is
an important one.
CONCLUSIONS
Buffalo breeding and milk production must be promoted due to its higher nutritional value and
greater potential for processing into value added dairy products such as yoghurt, cheese, butter with
desirable quality attributes. It is evident from this study that buffalo milk’s physico-chemical
characteristics and composition particularly CLA contents (the focused content of the study) varied as a
function of seasons. Cheeses from the said milk can give better yield in winter season as compared to
summer on the basis of higher TS contents. Further studies are needed to transform buffalo milk into
dairy products already made by cow milk to exploit it maximally and beneficially particularly
economic benefits to the consumers.
REFERENCES
Ahmad, S., I. Gaucher, F. Rousseau, E. Beaucher, M. Piot, J.F. Grongnet and F. Gaucheron. 2008.
Effects of acidification on physicochemical characteristics of buffalo milk a comparison with
cow milk. Food Chem. 106: 11-17.
Barlowska, J., M. Szwajkowska, Z. Litwińczuk and J. Król. 2011. Nutritional value and technological
suitability of milk from various animal species used for dairy production. Compr. Rev. Food. Sci.
F. 10: 291-302.
Bergamo, P., E. Fedele, L. Iannibelli and G. Marzillo. 2003. Fat soluble vitamin contents and fatty acid
composition in organic and conventional Italian dairy products. Food Chem. 82:625-631.
Bradley, R.L., E. Arnold, D.M. Barbano, R.G. Semerad, D.E. Smith and B.K. Vines. 1992. Chemical
and physical methods. In: Standard methods for the examination of dairy products, 16 ed.
(Marshall R. T. editor). Washington D. C. American Public Health Association. 433-531.
Christie, W. 1982. A simple procedure for rapid transmethylation of glycerolipids and cholesteryl
esters. J. Lipid Res. 23: 1072-1075.
1326
Christie, W. 1995. Composition and structure of milk lipids. In: Advanced dairy chemistry, 2nd ed.
(Fox PF editor). London Chapman Hall 136.
FAOSTAT. 2011 Food and Agriculture Organization of the United Nations Available at
http://faostat.fao.org/site/569/DesktopDefault.aspx?PageID=569#ancor. Accessed at March 4,
2013.
Jayamanne, V.S. and M.R. Adams. 2004. Survival of probiotic bifidobacteria in buffalo curd and their
effect on sensory properties. Int. J. Food Sci. Tech. 39: 719-725.
Khanal, R.C. and K.C. Olson. 2004. Factors affecting conjugated linoleic acid (CLA) content in milk,
meat and egg: A Review. Pak. J. Nutr. 3(2): 82-98.
Lal, D.M. 1984. Effect of lactation number on the polyunsaturated fatty acids and oxidation stability of
milk fats. India J. Dairy Sci. 225-229.
Ligda, D. 1996. The water buffalo management. Accessed at http://www./users/djligda/waterbuf.htm.
Menard, O., S. Ahmad, F. Rousseau, V. BriardBion, F. Gaucheron and C. Lopez. 2010. Buffalo vs cow
milk fat globules: size distribution, zeta-potential, compositions in total fatty acids and in polar
lipids from the milk fat globule membrane. Food Chem. 120: 544-551.
Moio, L., J. Dekimpe, P.X. Etievant and F. Addeo. 1993. The neutral volatile compounds of water
buffalo milk. Italian J. Food Sci. 5(1): 43-56.
Parodi, P.W. 1977. Conjugated octadecadienoic acids of milk fat. J. Dairy Sci. 60: 1550-1553.
Romano, P., A. Ricciardi, G. Salzano and G. Suzzi. 2001. Yeasts from water buffalo mozzarella a
traditional cheese of the Mediterranean area. Int. J. Food Microbiol. 69: 45-51.
Shahin, A.M., M.K. McGuire, M.A. McGuire, K.L. Ritzenthaler and T.D. Shultz. 2003. Determination
of c9t11CLA in major human plasma lipid classes using a combination of methylating
methodologies. Lipids 38(7): 793-800.
Walstra, P., T.J. Geurts, A. Noomen, A. Jellema and M.A.J.S. Van Boekel. 1999. Dairy technology
principles of milk properties and processes. In: Dairy Science and Technology. (Gustavo V.
Barbosa-Cánovas et al., eds.) (2nd ed). CRC Group, Taylor & Francis, New York.
Table 1. Physicochemical characteristics and composition of buffalo milk (Mean±sd; n= 3).
Months pH TS (%) Fat (%) CP (%) Lactose (%) Ash (%)

Jan 6.79±0.01 18.45±0.04 7.63±0.03 5.37±0.11 4.50±0.08 0.92±0.01
Feb 6.76±0.01 18.20±0.09 6.58±0.03 5.23±0.03 4.73±0.04 0.92±0.03
Mar 6.82±0.01 18.48±0.03 7.07±0.06 5.25±0.02 4.59±0.10 0.92±0.01
Apr 6.98±0.01 16.39±0.01 6.68±0.03 4.65±0.04 4.49±0.06 0.91±0.05
May 6.88±0.01 17.51±0.03 6.90±0.01 5.12±0.06 4.56±0.03 0.91±0.02
Jun 6.85±0.01 16.61±0.02 6.80±0.01 4.59±0.37 4.50±0.07 0.91±0.03
Jul 6.82±0.01 17.29±0.08 6.57±0.06 4.70±0.09 4.60±0.08 0.92±0.01
Aug 6.89±0.01 17.78±0.01 7.02±0.03 5.14±0.10 4.49±0.02 0.92±0.04
Sep 6.91±0.01 17.79±0.01 7.40±0.01 5.11±0.13 4.55±0.04 0.92±0.03
Oct 6.91±0.01 17.98±0.02 7.60±0.01 5.10±0.07 4.66±0.06 0.92±0.01
Nov 6.78±0.03 18.47±0.01 7.97±0.06 4.98±0.26 4.69±0.01 0.92±0.02
Dec 6.83±0.03 18.40±0.03 7.37±0.06 4.94±0.02 4.70±0.03 0.92±0.05
1327
Table 2. Mineral contents of buffalo milk (mg.kg-1) (Mean±sd; n= 3).
Month Ca P K Na Mg Zn
Jan 1694±9 1217±5 835±8 419±4 150±1 7±1
Feb 1835±17 1307±14 890±13 418±6 183±2 10±1
Mar 1505±5 1034±9 721±7 350±1 128±2 8±1
Apr 1022±14 707±17 468±10 245±4 90±1 4±1
May 2010±35 1393±33 984±18 384±6 172±3 9±1
June 1671±11 1215±14 759±5 267±1 135±1 16±4
Jul 2034±8 1317±4 947±7 296±1 146±1 6±1
Aug 2059±9 1243±3 920±6 235±1 137±1 5±1
Sep 1984±20 1242±29 851±34 261±13 143±3 7±2
Oct 1974±21 1136±8 854±18 295±12 146±1 5±1
Nov 1892±19 1115±25 785±23 301±17 155±5 6±1
Dec 1747±28 1513±131 954±31 462±15 202±7 8±1
1328
Table 3: Fatty Acid contents of buffalo milk mg.g-1 fat (Mean±sd; n= 2)
Fatty Acida
Months 10:0 12:0 14:0 14:1 15:0 16:0 16:1 17:0 18:0 18:1 18:2 CLAb 18:3
Jan 7.5±0.0 16.9±0.2 80.1±0.1 5.6±0.6 10.8± 0.6 210.0±1.6 10.4±0.0 4.6±0.0 108.6±1.3 190.5± 1.4 12.2± 0.1 6.2± 0.1 3.1± 0.0
Feb 13.0±0.2 20.5±0.3 88.2±0.4 4.6±0.0 11.2±0.0 228.5±2.6 10.3±0.0 5.5±0.1 122.4±1.2 198.4±1.6 11.4±0.1 6.2±0.1 4.0±0.0
Mar 12.6±0.5 19.8±0.6 84.4±0.5 4.6±0.1 9.8±0.0 255.2±3.0 10.5±0.8 4.7±0.0 127.0±2.7 198.2±2.5 12.8±0.5 6.9±0.4 3.3±0.3
Apr 7.3±0.3 15.6±0.0 69.5±0.6 4.0±0.1 8.7±0.2 190.2±5.3 8.8±0.2 4.1±0.0 110.4±3.4 174.3±4.3 11.2±0.5 6.2±0.1 2.8±0.1
May 5.4±0.2 16.0±0.2 75.0±0.2 4.4±0.1 8.8±0.0 207.4±1.9 9.9±0.1 3.9±0.2 111.3±1.8 187.6±4.0 13.3±0.2 6.6±0.1 3.6±0.4
June 6.3±0.0 15.1±0.4 79.0±0.6 4.0±0.1 8.9±0.1 215.5±0.9 8.3±0.0 4.5±0.1 109.7±1.5 173.5±2.5 9.5±0.0 7.6±0.1 3.3±0.1
July 5.5±0.2 17.6±0.0 91.5±0.1 3.2±0.1 9.2±0.1 256.4±1.7 12.7±0.3 5.08±0.1 111.5±0.8 165.4±1.2 9.3±0.0 4.7±0.1 3.7±0.0
Aug 6.0±0.0 20.4±0.3 106.0±1.1 4.6±0.0 10.2±0.3 316.0±11.0 11.2±0.1 5.8±1.5 111.3±3.7 162.5±5.8 10.7±0.3 4.8±0.2 3.3±0.1
Sept 4.6±0.1 17.0±0.1 85.0±0.0 4.2±0.0 9.7±0.0 234.8±0.7 9.8±0.0 4.4±0.1 91.3±0.1 142.2±0.1 11.3±0.8 4.4±0.0 3.1±0.0
Oct 12.5±0.1 23.9±0.1 94.0±0.2 5.1±0.1 9.1±0.1 243.1±0.8 10.4±0.1 4.1±0.1 99.2±0.5 152.0±1.0 12.3±0.1 5.9±0.0 4.3±0.1
Nov 19.2±0.1 30.3±0.2 114.4±0.4 6.9±0.0 11.7±0.0 287.4±0.8 14.0±0.1 4.9±0.1 105.7±0.3 183.3±3.8 11.7±0.1 5.9±0.1 3.0±0.0
Dec 11.0±0.1 22.8±0.1 90.0±0.2 5.6±0.0 9.9±0.0 228.3±1.5 11.9±0.7 4.3±0.1 93.3±1.0 158.4±1.8 10.3±0.1 5.5±0.1 2.9±0.0
Average 9.24 19.66 88.09 4.73 9.83 239.40 10.68 4.66 108.48 173.86 11.33 5.91 3.37
1329
Capric acid (10:0); lauric acid (12:0); myristic acid (14:0); myristoleic acid (14:1); pentadecanoic acid (15:0); palmitic acid (16:0);
palmitoleic acid (16:1); margaric acid (17:0); stearic acid (18:0); oleic acid (18:1); linoleic acid (18:2); CLA (18:2) and linolenic acid (18:3)
b
Conjugated linoleic acid cis9 trans11CLA isomer
Milk CLA Content and Δ9 Desaturase Activity in Buffalo Cows along the
Lactation
Raffaella TUDISCO*, Monica I. CUTRIGNELLI, Serena CALABRÒ, Micaela GROSSI,
Nadia MUSCO, Giovanni MONASTRA and Federico INFASCELLI
Department of Veterinary Medicine and Animal Production, University of Napoli Federico II, via
F. Delpino 1, 80137 Napoli, Italy
*Corresponding email: tudisco@unina.it
ABSTRACT
Significant influence of the stage of lactation was found on milk CLA in grazing cow, sheep
and goat. However, differences in pasture composition and plant vegetative stage can affect the
results; when animals were fed the same diet, conflicting data are reported. CLA is formed as an
intermediate during the biohydrogenation of linoleic acid to stearic acid in the rumen or from the
endogenous conversion of t-11 C18:1 by Δ9-desaturase (SCD) in the mammary gland. Aims of
present trial were to study the influence of lactation stage on milk CLA content and SCD activity in
forty lactating Italian Mediterranean buffalo cows fed TMR (0.90 UFL/kg DM; 15.4 CP/DM).
From 30th to 120th d of lactation, individual milk yield was daily registered and representative
samples were monthly analyzed for protein, fat and fatty acids profile. SCD activity was calculated
as Desaturation index (14:1n-9/14:0). Average milk yield, fat and protein were kg/d 9.8, 8.56% and
4.70% respectively. Milk fat was significantly (P<0.01) lower at the 1st compared to the following
months of sampling. cis 9-trans 11 CLA was affected by lactation stage: the highest value was
registered at the 3rd sampling while the lowest at the 1st and 4th sampling (0.75% vs. 0.57 and 0.58%
of total fatty acids; P<0.05). No correlation was found between milk CLA content and SCD
different at the 4th compared to the 1st sampling (0.125 ± 0.02 vs 0.096 ± 0.01; P<0.05).
activity. Indeed, the Desaturation index increased with the lactation stage and was significantly
Keywords: buffalo milk, CLA, Δ9-desaturase
INTRODUCTION
Conjugated linoleic acid (CLA), suggested to have immunomodulating, anticarcinogenic
and antiartheriosclerosis properties (Pastuschenko et al., 2000, Whigham et al., 2000), is a group of
positional and geometric fatty acid isomers derived from octadecadienoic acid of which the major
isomer is cis-9, trans-11 or rumenic acid (up to 80% of total CLA in the food). The presence of
CLA in ruminants milk results from the isomerization and biohydrogenation of unsaturated fatty
acids [octadecatrienoic (C18:3) and ocatdecadienoic (C18:2) acids] by rumen bacteria as well as the
activity of Stearoyl-CoA desaturase (SCD) in the mammary gland on trans-11 C18:1 (TVA,
transvaccenic acid), intermediate product of several polyunsaturated fatty acids (PUFA)
biohydrogenation (Griinari and Baumann, 1999). SCD is also the rate-limiting enzyme in the
biosynthesis of mono-unsaturated fatty acids (MUFAs) by the introduction of a cis double bond
between carbons 9 and 10 in a spectrum of saturated fatty acids, with preference for C14:0, C16:0
and C18:0. SCD activity can be measured by comparing the product:substrate ratios of certain fatty
acids. According to Lock and Garnsworthy (2003), the best indicator of SCD activity is the c9
C14:1/C14:0 ratio because all of the C14:0 in milk fat is produced via de novo synthesis in the
mammary gland; consequently, desaturation is the only source of C14. Several dietary and
environmental factors are known to affect milk CLA content (Tudisco et al., 2010) as well as stage
of lactation (D’Urso et al. 2008; Tudisco et al., 2012). However these last results were from animals
grazing pastures throughout the experimental period; when animals were fed the same diet, no
effect of lactation stage on milk CLA content were found in dairy cows (Stanton et al., 1997) and in
sheep (Tsiplakou et al., 2006) while Kelsey et al. (2003) reported that stage of lactation had little
effect on CLA content of cow milk. Aim of present trial was to study the influence of lactation
1330
stage on milk CLA content and SCD activity in buffalo cows raised in open yards fed the same diet
along the experimental period.

The trial was carried out on forty dairy buffalo cows (3 number of calving; MW: kg 128.7 ±
0.6), raised in open yards that allowed 15 m2 and a feeding trough space of around 1.0 m for each
buffalo, fed 14.7 kg DM/head of total mixed ration composed (% on DM basis) by 23.3 alfalfa hay,
42.2 concentrate and 34.5 corn. Individual milk yield was daily registered (from 30 days until 120
days in milk) while monthly samples of milk, representative of the two daily milking, were
collected and analyzed for fat and protein (Milko Scan 133B, Foss Matic, Hillerod, Denmark,
calibrated with an appropriate standard for buffalo milk). Moreover, milk fat was extracted using a
hexane/isopropanol mixture (3/2 v/v) according to Hara and Radin (1978). The extracted fatty acids
were trans methylated by using the base-catalyzed procedure described by Christie (1982) and
modified by Chouinard et al. (1999). The methyl esters fatty acids were quantified using a gas
chromatograph series FOCUS, equipped with a detector flame-ionization (ThermoElectron
Corporation, Rodano -Milano- Italia) through a capillary column (CP-SIL 88 fused silica capillary
column, 100 m x 0.25 mm diameter with film thickness 0.2-µm; Varian, Inc. Walnut Creek, CA).
The peaks of fatty acids were identified in comparison with a standard mix of methyl esters fatty
acids; the identification of CLA isomers was effected comparing the sample chromatograms with
that of each purified isomer (cis-9, trans-11 CLA; trans-10, cis-12 CLA; cis-9, trans-11 CLA; trans-
9, trans-11 CLA) (Larodan Fine Chemicals, AB, Limhamnsgårdens Malmö, Sweden). From data of
fatty acid profile, the SCD activity (Δ9 Desaturase index) was calculated by the ratio C14:1/C14:0
(Lock and Garnsworthy, 2003). Data of milk yield and quality were analysed using GLM (General
Linear Model) procedure of SAS (2000).
RESULTS
Average milk yield, fat and protein were 9.8 kg/d, 8.56% and 4.39%, respectively. Milk
yield was significantly (P<0.01) affected by month of sampling; the highest milk yield was recorded
at 60 days of lactation, the lowest at 120 days after calving (Table 1). The concentrations of milk fat
and protein along the trial showed a physiological trend for buffalo species; significant (P<0.01)
differences were detected only for the fat, in particular among the first and the following months.
Table 2 shows, as percent of total fatty acids, the main fatty acids, the values of total CLA and of
addition the Δ9 Desaturase index is reported. Month of sampling significantly (P<0.01) affected
the four detected isomers (cis-9, trans-11; trans-10, cis-12; cis-9, cis-11; trans-9, trans-11). In
octadecatrienoic acid (C18:3n6) percentage, which was higher at the third sampling. Total CLA
content was significantly higher at the 3rd sampling compared to the first and fourth one (0.862 vs.
9 trans-11 CLA (0.75% vs. 0.57 and 0.58% of total fatty acids; P<0.05). Δ9 Desaturase index
0.689 and 0.679 % of total fatty acids; P<0.05). The result was mainly affected by the values of cis-
increased with the lactation stage and was significantly different at the 4th compared to the 1st
sampling (0.125 ± 0.02 vs 0.096 ± 0.01; P<0.05).
DISCUSSIONS
Lactation stage is one of the major factor influencing milk yield and chemical composition
in buffaloes; milk yield decrease during the lactation, but there is increase in milk fat (Bovera et al.,
2007). Milk fatty acids profile is affected by several factors, such as lactation stage, breed, genetic
variation, age, health and feed composition (Qureshi et al, 2012). Auldist et al. (1998) stated that
there is an increase in saturated fatty acids with the advancement of lactation from early to late. In
present trial the peak of milk yield was registered at 60 days of lactation when percentage of fat was
significantly higher than that of previous sampling. In addition, the percentage of the main saturated
fatty acids (C14:0, myristic and C16:0, palmitic) were the lowest registered along the trial. A
significant influence of lactation stage on milk CLA has been reported by several authors (D’Urso
et al. 2008; Tudisco et al., 2012). However these last results were from animals grazing pastures
1331
throughout the experimental period, thus such feeding regime does not guarantee comparable data,
because differences in pasture botanical composition and plant vegetative stage can affect the CLA
content of milk fat (Cabiddu et al., 2004). In present trial, buffalo cows were fed the same diet along
3rd sampling, cis-9 trans-11 CLA showed the highest value but the Δ9 Desaturase activity. It
the experimental period, thus milk CLA variation could not be attributed to dietary factors. At the
probably means that in this period milk CLA resulted mainly from the isomerization and
biohydrogenation of unsaturated fatty acids [octadecatrienoic (C18:3) and ocatdecadienoic (C18:2)
acids] by rumen bacteria. According to Kelsey et al (2003) days in milk should account for about
2.0% of total variation of milk CLA in cows fed the same diet along the experimental period. SCD
activity can be measured by comparing the product:substrate ratios of certain fatty acids. There are
four main products of SCD activity in the mammary gland of ruminants: c9 C14:1 , c9 C16:1 , c9
C18:1 and CLA, which are produced from C14:0 , C16:0 , C18:0 and trans11 C18:1, respectively.
According to Lock and Garnsworthy (2003), the best indicator of SCD activity is the c9
C14:1/C14:0 ratio because all of the C14:0 in milk fat is produced via de novo synthesis in the
mammary gland; consequently, desaturation is the only source of C14. Increasing c9 C14:1/C14:0
ratio values would indicate an increase of SCD activity. In present trial, the ratio was higher at the
4th sapling; in each case our findings of the desaturase activity were closely related to Qureshi et al.
(2012) where a ratio of 0.11 was reported for buffalo cows with a lactation number between 1 and 4.
REFERENCES
Auldist, M.J., B.J. Walsh and N.A. Thomson. 1998. Seasonal and lactation influences on bovine
milk composition in New Zealand. J. Dairy Res. 65:401-411.
Bovera, F., M.I. Cutrignelli, S. Calabrò, G. Piccolo, R. Tudisco, S. D’Urso and F. Infascelli. 2007.
Use of two different dietary energy and protein contents to define nutritive requirements of
lactating buffalo cows. J. Anim. Physiol. Anim. Nutr. 91:181-186.
Cabiddu, A., M. Addis, S. Spada, M. Sitzia, G. Molle and G. Piredda. 2004. The effect of different
legumes-based pastures on the fatty acid composition of sheep milk with focus on CLA. In:
Proc. 20th Meeting of European Grassland Federation, vol. 9. Luzern, Switzerland.
Blackwell Publishing Ltd., Oxford, UK, pp. 1133–1135.
Chouinard, P.Y., L. Corneau, D.M. Barbano, L.E. Metzger and D.E. Bauman. 1999. Conjugated
linoleic acids alter milk fatty acid composition and inhibit milk fat secretion in dairy cows.
J. Nutr. 129: 1579-1584.
Christie, W.W. 1982. A simple procedure of rapid transmethylation of glycerolipids and cholesteryl
esters. J. Lipid Res. 23:1072-1075.
D’Urso, S., M.I. Cutrignelli, S. Calabrò, F. Bovera, R. Tudisco, V. Piccolo and F. Infascelli. 2008.
Influence of pasture on fatty acid profile of goat milk. J. Anim. Physiol. Anim. Nutr. 92:
405–410.
Griinari, J.M. and D.E. Baumann. 1999. Biosynthesis of conjugated linoleic acid and its
incorporation into meat and milk in ruminant. In: Advances in Conjugated Linoleic Acid
Research (Ed. M.P. Yurawecz, M.M. Mossoba, J.K.G. Kramer, M.W. Pariza and G.J.
Nelson). American Oil Chemistry Society, Champaign, IL, pp. 180–200.
Hara, A. and N.S. Radin. 1978. Lipid extraction of tissues with a low-toxicity solvent. Anal.
Biochem. 90: 420-426.
Kelsey, J.A., B.A. Corl, R.J. Collier and D.E. Bauman. 2003. The effect of breed, parity, and stage
of lactation on conjugated linoleic acid (CLA) in milk fat from dairy cows. J. Dairy Sci. 86:
2588-2597.
Lock, A.L. and P.C. Garnsworthy. 2003. Seasonal variation in milk conjugated linoleic acid and
D9-desaturase activity in dairy cows. Livest. Prod. Sci. 79: 47–59.
Pastuschenko, V., H.D. Matthes, T. Hein and Z. Holzer. 2000. Impact of cattle grazing on meat
fatty acid composition in relation to human nutrition. In: Proc. 13th Int. IFOAM Scientific
Conf. 28–31 September 2000, Basel, Switzerland, pp. 293–296.
1332
Qureshi, M.S.S., A. Jan, I.U. Mushtaq, Rahman, M. Jan and Ikramullah. 2012. Effect of age on
milk fatty acids in dairy buffaloes. The J. of Anim. and Plant Sci. 22: 108-112.
SAS Institute Inc. 2000. SAS/STAT® Software: Changes and Enancements through Relase 8.1.
Stanton, C., F. Lawless, G. Kjellmer, D. Harrington, R. Devery, J.F. Connolly and J. Murphy. 1997.
Dietary influences on bovine milk cis-9, trans 11-conjugated linoleic acid content. J. Food
Sci. 62: 1083-1086.
Tsiplakou, E., K.C. Mountzouris, and G. Zervas. 2006. Concentration of conjugated linoleic acid in
grazing sheep and goat milk fat. Livest. Sci. 103: 74–84.
Tudisco, R., M.I. Cutrignelli, S. Calabrò, G. Piccolo, F. Bovera, A. Guglielmelli, G. Moniello and F.
Infascelli. 2010. Influence of organic systems on milk fatty acid profile and CLA in goats.
Small Rum. Res. 88: 151-155.
Tudisco, R., S. Calabrò, M.I. Cutrignelli, G. Monello, M. Grossi, O.J. Gonzalez, V. Piccolo and F.
Infascelli. 2012. Influence of organic systems on Stearoyl-CoA desaturase gene expression
in goat milk. Small Rum. Res. 106S: S37-S42.
Whigham, L.D., M.E. Cook and R.L. Atkinson. 2000. Conjugated linoleic acid: implications for
human health. Pharmacol. Res. 42: 503–510.
Table 1. Milk yield (kg/d) and chemical composition (%).
Yield Protein Fat

Average 9.8 4.39 8.56
1st month 9.3B 4.45 7.59B
2nd month 11.1A 4.31 8.54A
3rd month 9.3B 4.31 8.46A
th C
4 month 7.7 4.50 8.90A
SEM 1.47 0.14 0.19
Different capital letters among columns mean P < 0.01; SEM: standard error of mean
Table 2. Milk fatty acid profile and CLA (% of total fatty acids).
Average Sampling month

1 2 3 4 SEM
C10:0 1.92 1.94 1.88 1.81 2.04 0.50
C12:0 2.59 2.65 2.50 2.47 2.72 0.58
C14:0 11.25 11.5 10.9 11.4 11.3 1.62
C14:1 1.26 1.11 1.23 1.29 1.42 0.30
C16:0 32.55 32.6 31.1 34.2 32.0 6.16
C16:1 1.41 1.25 1.28 1.52 1.54 0.31
C18:0 13.20 13.2 13.3 12.9 13.4 1.56
C18:1 23.95 22.3 22.7 26.1 25.0 4.06
C18:2 2.45 2.50 2.44 2.63 2.25 0.40
C18:3n6 0.53 0.46B 0.59AB 0.65A 0.44B 0.071
C18:3n3 0.08 0.063 0.090 0.088 0.085 0.023
CLA c9t11 0.644 0.585b 0.666ab 0.750a 0.575b 0.115
CLA t10c12 0.056 0.055 0.054 0.059 0.056 0.009
CLA c9c11 0.039 0.036 0.050 0.041 0.035 0.010
CLA t9t11 0.012 0.012 0.013 0.012 0.012 0.003
0.689b 0.783ab 0.862a 0.679b
Δ9
tot CLA 0.751 0.117
Desaturase
0.112 0.0965b 0.1128ab 0.1133ab 0.1255a 0.04
index
Different letters among rows mean P < 0.01 (capital) and P < 0.05 (lower); SEM: standard error of
mean.
1333
Milk Yield And Milking Characteristics in Murrah Buffaloes Submitted to

Machine Milked With or Without Calf
Alberto de GUSMÃO COUTOa*, Alcides de AMORIM RAMOSb, Elizabeth SAMPAIOa, Janira

Lúcia ASSUMPÇÃO COUTOa, João ALBERTO NEGRÃOc, Jayasooriya APPUHAMYd,
Patrícia MENDES and Guimarães BEELENa
a
Centro de Ciencias Agrárias, Universidade Federal de Alagoas, Maceió, Alagoas,Brazil
b
Universidade Estadual Paulista, Botucatu, São Paulo, Brazil
c
Laboratorio de Fisiologia, Universidade de Sao Paulo, Pirassununga, São Paulo, Brazil
d
University of California Davis, CA, USA
*Corresponding email: couto.a@uol.com.br
ABSTRACT
This work was carried out on a Murrah breed buffalo farm located in São Luiz do Quitude,
Alagoas, Brazil. The objective was to compare the milk yield and milking characteristics between
buffaloes machine milked with or without calf. Eighteen buffaloes used in the experiment were in their
second to fourth lactation, having body weights of 650 ± 100 kg and a total milk yields of 2,127.47 ±
462.99 in the previously lactation. The animals were randomized in two treatments: system 1) machine
milking without calf and system 2) machine milking with calf. Each animal attended the experiment in
the day of parturition and data were collected every five days, from day 20 to day 45 of experiment.
The data consisted in milk yield, milk letdown time, milking time, milk yield per milking, milk flow
rate and cortisol. The milking system did not affect milk yield (P> 0.05), which showed a a day mean
of 9.259 ± 1.228 kg, with 5.248 ± 1.228 kg ± 4.200 in the morning and 0.950 kg in the afternoon
milking. The milk letdown time varied (p <0.05) between 1.55 min, in the group without suckling
calves to 3.42 minutes in the system without suckling calves. However The milking system did not
affect the total time of milking or the amount of residual milk, that was, on average, 9.81 ± 2.35
minutes and 165 g (3.88%), respectively. The cortisol level in the milk, although low, was higher in
buffaloes milked with calf at foot (0.22 mg/dl vs 0.14 mg/dl). The management of buffaloes allowed
the milking machine without suckling calves with no differences in daily milk production.
Keywords: Buffaloes, Milking time


1334
Relationship of Udder and Teat Morphology with Milk Production in Nili-Ravi

Buffaloes of Pakistan
Muhammad ABDULLAHa*, Khalid JAVEDa, Muhammad Salman KHALIDa, Nisar AHMADa,
Jalees Ahmad BHATTIa and Umair YOUNASa
a
Department of Livestock Production, University of Veterinary and Animal Sciences, Lahore, Pakistan
*Corresponding email: mabdullah@uvas.edu.pk
ABSTRACT
The study was planned to investigate the milk production (MP) efficiency of Nili-Ravi
buffaloes belonging to different parities, by assessing the MP relationship with the various
morphometric measurements of udder. Nili-Ravi buffaloes (n= 200) were selected randomly from
commercial dairy herds at peri-urban areas of Lahore city. The selected animals were in different
parities. The various morphometric parameters for udder measurements used in this study were udder
depth (UD), udder width (UW), udder length (UL), teat length (TL) and teat diameter (TD). Body
Condition Score (BCS), udder and teat shape also gave better understanding of milk production
enhancement. 78% of Nili-Ravi animals were found to be having bowl shape udder followed by 19.5 %
round and 2.5% goaty shape udder. Similarly, 89% Nili-Ravi buffalo were observed for cylindrical
shape teat followed by 7% funnel and 4% bottle shape teats. Results showed significant (P<0.01) and
positive correlation of UW with MP (0.573) and lactation number (0.341). Milk vein (MV) size also did
correlate significantly (P<0.01) and positively with various parameters like body weight (BW),
lactation number (LN) and milk production (MP) illustrating values as 0.633, 0.390 and 0.799,
respectively. Whereas, negative correlation of MV size with lactation stage (-0.183) was observed. TL and TD
showed positive and significant (P<0.01) relationship with MP (0.315 and 0.494, respectively). The present
study will be helpful for selection and evaluate the production potential of Nili-Ravi buffaloes.
Keywords: Nili-Ravi buffalo, Milk production, Udder measurements, Teat measurements
INTRODUCTION
Selection is crucial tool to enhance and promote the productive potential and good genetic for
the dairy production of buffalo on the basis of phenotypic characteristics. In this regards body
appearance and udder conformation may play vital role for propagation of high conformation traits in
subsequent generations. Reports on teat length and udder height had shown significant effect on milk
production in Brown Swiss animals (Tilk et al., 2005). Similarly, positive correlations were determined
by Lin et al. (1987) for udder measurements with high milk yield in Holstein cattle. Bardakcioglu et al.,
(2011) reported that Holstein cows with increase teat diameter had more milk yield and positive
correlation was found between udder measurements and milk yield (Petersen et al., 1985). The udder
shape also reported to influence the milk production (Pawlina et al., 2000; Bhuiyan et al., 2004).

Study site
The study was conducted on Nili Ravi (n=200) buffaloes in the immediate vicinity of Lahore,
the capital of Punjab province in Pakistan. The animals belonging to different parities were selected for
this study. These animals were grouped further according to the stage of lactation (Table 1).
Morphometric measurements of udder and teat
The udder measurements were taken as described by Bhuiyan et al. (2004). Udder depth (UD)
was measured by subtracting the distance of platform to the udder floor from the distance of platform
1335
to the base of udder. Udder width (UW) was measured as a distance between widest point of udder near
the stifle joint of animal and passing it in between the fore and rear teats, to the widest point of udder
near the stifle joint of the other side. Udder length (UL) was measured as a distance from rear
attachment of udder below the vagina and moving along udder body up to the fore attachment, where
fore udder blends smoothly with the body of the animal. Two different teat measurements were taken
including teat length (TL) as a distance of teat attaches with the udder body moving along the teat
down to the tip of the teat and teat diameter (TD) was measured in the middle of the teat with the help
of vernier caliper. Milk vein (MV) was also determined using vernier caliper. The buffaloes were
selected randomly during the study and the milk production was calculated after hand milking of
buffaloes with steel bucket underneath.
The mean and standard errors values of various udder and teat measurements were worked out.
The Pearson’s Correlation between the different measurements, lactation stage and parity was
determined (Steel et al., 1997).

The animals belonged to three different stages of lactation (early, mid and late) were observed
in this study. The mean±S.D value for BW (kg), lactation number (LN), MP (liter) and age were found
as 554.5±84.2, 3.6±1.2, 12.3±3.4, 10.5±2.3 among commercially reared Nili Ravi buffaloes (n=200).
The UD, UW and UL showed the pattern of increasing size as lactation number increases. These
findings are parallel to the findings of Bhuiyan et al. (2004) and also in agreement with the findings of
Prasad et al. (2010) which worked on dairy cows of Bangladesh and Murrah buffaloes, respectively
(Table-1). The highest values for UD, UW, UL and MV size were found as 15.4cm, 40cm, 82cm and
9.6cm as compared to the lowest values as 7cm, 18.5cm, 46cm and 2.4cm, respectively.The average
mean±S.D measurements for TL and TD in lactating Nili-Ravi buffaloes were found as 9.6±1.2 cm and
4.08±0.66 cm (table-2). The UW was found to have high positive and significant (P<0.01) relation with
the average TL, TD and distance between the fore teats (FF), (Table-3). However, these findings do not
coincide with the findings of Prasad et al. (2010). Milk vein size was also found to be significantly
(P<0.01) correlated with the average TL and TD. The UW and MV size of these dairy animals were
found to be significantly (P<0.01) correlated with the BW, parity and MP (table-4). The UW and MV
size of these dairy animals were found positive and significantly (P<0.01) correlated with the BW,
parity and MP. However, negative and significant (P<0.01) association was recorded between the MV
size and lactation stage. Positive relationship was found among the udder measurements and between
udder measurements and MP. This is in accordance with the findings of Kominakis et al. (2009).
Average TL and TD also found to have positive and significant (P<0.01) relation with the BW,
lactation number (LN) and MP of lactating Nili-Ravi buffaloes. Tilki et al. (2005) also found similar
results on significant relationship of teat measurements with the MP in Brown Swiss cows. These
results also coincide with the findings of Bardaakcioglu et al. (2011) who observed significant
correlation of TD of right side of animal with the MP. Prasad et al. (2010) also reported that teat
diameter is significantly and positively correlated with milk production. Whereas, Roa et al. (2007)
reported that TL was positively but TD was negatively correlated with MP in Karan fries cows. The
positive and significant (P<0.05) association of FF with the milk production was noted, which is in
agreement with findings of Tilki et al. (2005). Sabuncuoglu and Coban (2007) found moderate
correlations daily MP and FF (P<0.01), RR (P <0.05) and front to rear (P<0.01) teat distances. Rests of
the associations were found weak and non-significant. It was found that average teat length and teat
diameter had high positive and significant relationship with the BW, parity and MP of lactating Nili-
Ravi buffaloes at level P<0.01.
1336
REFERENCES
Bardakcioglu, H.E., S. Sekkin and H.D.O. Toplu. 2011. Relationship between some teat and body
measurements of Holstein cows and sub-clinical mastitis and milk yield. J. Anim. Vet. Adv.,
10(13): 1735-1737.
Bhuiyan, M.M., M.R. Islam, M.L. Ali, M.K. Hossain, M.A. Kadir, N.S. Lucky and B.R. Das. 2004.
Importance of mammary system conformation traits in selecting dairy cows on milk yield in
Bangladesh. J. Biolog. Sci. 4(2): 100-102.
Kominakisa, A.P., D. Papavasilioub and E. Rogdakis. 2009. Relationships among udder characteristics,
milk yield and non-yield traits in Frizarta sheep. Sml. Rumin. Res. 84: 82-88.
Lin, C.Y., A.J. Lee, A.J. Mcallister, T.R. Batra, G.L. Roy, J.A. Vesely, J.M. Wauthy and K.A. Winter.
1987. Intercorrelations among milk production traits and body and udder measurements in
Holstein heifers. J. Dairy. Sci. 70: 2385-2393.
Pawlina, E., W. Kruszyński and M. Kuczaj. 2000. An investigation of changes in udder size of Red and
White cows in first and third lactation. Med. Weter. 56: 672-674.
Petersen, M.L., L.B. Hansen, C.W. Young and K.P. Miller. 1985. Correlated response of udder
dimensions to selection for milk yield in Holsteins. J. Dairy Sci., 68: 99-113.
Prasad, R.M.V., E.R. Rao, K. Sudhakar, B.R. Gupta and M. Mahender. 2010. Studies on udder and teat
measurements as affected by parity and their relationship with milk yield in Murrah buffaloes.
Buf. Bullet. 29(3): 194-198.
Rao, T.K.S., A.K. Dang and C. Singh. 2007. Effect of udder and teat characteristics on milk
composition and yield of Karan fries cows. Ind. J. Dairy Sci. 60(5).
Sabuncuoglu, N. and O. Coban. 2007. Relationship between udder and teat conformation and milk
yield performance in dairy cows pre- and post-milking. Can. J. Anim. Sci. 87: 285-289.
Steel, R.G.D., J.H. Torrie and D A. Dickey. 1997. Principles and Procedures of Statistics. 3rd Ed.
Tilki, M., M. Colak, S. Inal and T. Caglayan. 2005. Effects of teat shape on milk yield and milking
traits in Brown Swiss cow. Turk J. Vet. Anim. Sci. 29: 275-278.
Table 1. Average (mean±S.D) values of various udder measurements in specified lactations.
Order of No. of UL UD UW MV Diameter

Lactation Animals (cm) (cm) (cm) (cm)
Lactation No. 1 4 57.0±6.1 13.3±6.1 23.9±0.6 5.6±0.7
Lactation No. 2 32 62.4±6.1 10.7±2.1 28.0±3.2 6.6±1.1
Lactation No. 3 63 63.6±7.8 10.8±1.6 28.3±4.2 7.3±1.2
Lactation No. 4 53 65.9±6.9 14.7±19.2 29.2±4.6 7.4±1.6
Lactation No. 5 34 66.1±6.8 11.1±1.3 31.6± 3.2 8.1±0.8
Lactation No. 6 14 62.7±9.0 10.5±1.5 30.7±1.3 7.9±0.77
UL= udder length, UD= udder depth, UW=udder width, MV=milk vein
1337
Table 2. Mean with S.D, coefficient of variance and range of various udder measurement.
Variables No. of Mean±S.D C.V (%) Range (cm)

Animals (cm)
Udder Measurements
Udder Depth (UD) 200 10.8±1.6 15.05 7-15.4
Udder Width (UW) 200 29.1±4.1 14.02 18.5-40
Udder Length (UL) 200 64.2±7.3 11.4 46-82
Teat Measurements
Avg. Teat Length (TL) 200 9.6±1.2 12.6 6.5-13.5
Teat Diameter (TD) 200 4.08±0.66 16.2 2.27-5.3
Fore to Fore Distance (FF) 200 14.4±3.2 22.1 8-24
Rear to Rear Distance (RR) 200 6.3±3.2 33.2 2-13
Right Fore to Right Rear (RFRR) 200 6.4±2.1 28.4 3-12
Left Fore to Left Rear (LFLR) 200 6.4±1.8 28.7 3-11.5
Milk Vein (MV) 200 7.4±1.3 17.9 2.4-9.6
S.D= standard deviation C.V= coefficient of variations
Table 3. Phenotypic correlations between udder and teat measurements.
UL UD UW MV
Avg. Teat Length (TL) 0.092 -0.088 0.215** 0.333**
Teat Diameter (TD) 0.092 -0.079 0.333** 0.501**
Fore to Fore Distance (FF) 0.106 0.081 0.191** 0.085
Rear to Rear Distance (RR) -0.074 0.187** -0.002 -0.104
Right Fore to Right Rear (RFRR) -0.071 0.057 0.118 -0.009
Left Fore to Left Rear (LFLR) -0.032 0.074 0.071 0.011
UL= udder length, UD= udder depth, UW=udder width, MV=milk vein
Table 4. Phenotypic correlations between udder and teat measurements with Age, BW, LN & MP.
AGE BW LN MP
Udder length (UL) -0.028 0.062 -0.007 0.128
Udder depth (UD) -0.002 0.024 0.031 0.031
Udder width (UW) 0.129 0.447** 0.341** 0.573**
Avg. Teat Length (TL) 0.077 0.328** 0.217** 0.315**
Teat Diameter (TD) 0.001 0.399** 0.318** 0.494**
Fore to Fore Distance (FF) -0.001 0.111 0.018 0.152*
Rear to Rear Distance (RR) -0.005 -0.047 -0.056 -0.108
Right Fore to Right Rear (RFRR) 0.046 0.069 0.047 0.069
Left Fore to Left Rear (LFLR) 0.051 0.051 0.087 0.088
Milk vein (MV) size 0.173* 0.633** 0.390** 0.799**
BW= body weight, LN= lactation number and MP= milk production
1338
Differences in Lactation Curves and Peak Production in Mediterranean

Italian Buffaloes Bred According to Three Levels of Production
Angelo COLETTAa* and Luigi ZICARELLIb

a
ANASB,Associazione Nazionale Allevatori della Specie Bufalina, Via Petrarca, 42-44 - Centurano
- 81100 Caserta – Italy–Tel. +390823356743 – Fax. +39 0823320964.
b
DISCIZIA, sez. B. Ferrara, Università degli Studi di Napoli Federico II - Via F.Delpino,
1 - 80137 - Napoli – Italy - Tel. +39 081 2536188 – Fax. +39 081 2536186.
*
Corresponding email: progettiesviluppo@anasb.it
ABSTRACT
The aim of this study was to make a comparison among the lactations of the buffalo cows that
were both members of the Herd Book and under Milk Recordings in order to identify some
parameters useful in the planning of milk production and then to improve the deseasonalization of
the quantity of produced milk. The milk production primarily depends by the trend of the lactation
curve, and the factors which composed it are: peak efficiency and persistence. The previous
parameters are the key in the description of the model of the release of milk, particularly: the peak
efficiency is the maximum production during the lactation, and the persistence describes the
magnitude of the decline in production. The Wood model is the most widely used function for the
description of lactation curves because it provide to extrapolate a lot of information relatives to the
curve when the data are not available. The Wood model also helps the breeders in the management
of the herd, in fact a rational management would implies to estimate the amount of milk that will be
produced. The work presented below has availed of the Wood mathematical model (1967) and has
established that it is possible to develop a forward estimate of the lactation curve with a good
approximation of what will be the future lactations.
Keywords: Buffalo, lactation, curve, milk, production, peak
INTRODUCTION
The breeding of buffaloes intended for the production of milk appears to be strongly
influenced by both fluctuations in the demand and the price of milk itself. This is due to the well-
known seasonal demand for mozzarella market. Know in advance the future production is an
essential tool for the buffalo breeder who is paid by transformation companies according to the
synchrony between demand and supply.
Of considerable importance is the knowledge of the productive potential of primiparae
through objective parameters which integrate and confirm the information that are already through
the known production pedigree of the parents. Anticipate the knowledge of the productive
capability of a primipara is essential for the estimation of the bulls in Progeny test. As soon the
assessment takes place sooner the bulls will be chosen to use with huge economic benefits of the
company. In addiction a previous estimation of individual production enables the distribution of
both the primiparae and the female in groups featuring by diets with different nutritional
characteristics. The aim of this work was to identify some parameters that allow to predict the milk
production, according to their production level, in buffaloes both registered in the Herd Book and
under Milk Recordings.

Subjects
All the buffalo female enclosed in the Herd Book of ANASB (National Association of
Buffalo Species Breeders – Italy), that were under Milk Recordings and that they had finished at
least one lactation between January 2005 and August 2010 were considered. All lactations, for each
1339
subject, that had at least nine Milk Recordings were considered. This study regarded the production
level of buffaloes that were divided into three groups thanks to the milk recording currently used in
buffaloes reared in Italy and that are the same in bovine(AIA): high, medium and low production
capability (x) (x < 2.500 Kg; 2.501 < x > 3.500 Kg e x > 3.500 Kg, respectively). The Milk
Recordings are done in accordance to the methods established by ICAR (International Committee
for Animal Recording). The method used is called A4 and the average interval between two
subsequent controls is 30 days. The animals under observation, belonged to 279 stables located in
35 Italian provinces. 6274 subjects and 6750 lactations (Table. 2) (with at least 9 Milk Recordings),
for a total of 64.049 Milk Recordings, were considered. The average age of 6274 subjects
considered was 5 years and 3 months (Table. 3).
Production:
y = an b e (− cn )
The lactation curves were estimated through the followed Wood function:
In which:
y is the milk quantity produced in the n day;
a is a constant that describes the initial production level of buffalo female considered;
b describes the speed with which the milk production increase until the lactation peak;
c represents the speed of milk production reduction after the lactation peak;
n is the time (days/weeks).
After estimating the 3 parameters (a, b and c) that describe the lactation curve of the observed
subjects, both the production peak (ymax) and the day scheduled for the peak (tmax) were
calculated.
- the day scheduled for the peak (tmax) = b/c
- production peak (ymax) = a(b/c)b e(-cn) (n is the day of the peak).
6750 lactations were used (tab. 4). The production level of the lactations was divided into three
ranges according to the milk quantity produced in a standard lactation (270 days), in: “low
production” lower than 2500 Kg (LP); “medium production” ranging between 2501 and 3500 Kg
(MP) and “high production” over than 3500 Kg (HP).

The considering lactations refers to all the buffalo female that were under Milk Recordings
and that they had finished at least one lactation between January 2005 and August 2010. The
average duration of conventional lactations used was of 269.43 days. Compared to the total
observations (full sample used), the three ranges were 57,5% LP; 39,0% MP and 3,5% HP. The
average lactation of the full sample (64049 Milk Recordings) was 2421,47 Kg (Table. 1), tended to
be higher than that produced in 2009 by ANASB (2182 Kg), which shows the progression of
genetic trend of recent years. In other words while the ANASB data referred to lactations closed
each year of publication of the bulletin (that of 2009 in this case), in this study were considered all
lactations of the subjects alive until August 2010. In the processed series the lactations of the
primiparae and pluriparae affecting more (36,56% vs. 27,13%) and less (40,17% vs. 52,77%) than
the ANASB data respectively. It should be emphasized that the oldest buffalo in ANASB series are
generally at the end of their career and for this reason they do record the lowest production before
of their elimination. While those of the present study are not necessarily at the end of their career.
From the explanations set it follows that if the ANASB data were adjusted for those of buffaloes
both oldest and less productive, i.e. those that were likely be eliminated in the following year would
give a more real picture of the potential production of the Mediterranean Italian Buffalo.
Below are percentage distribution of the three production levels (LP, MP and HP) for each of
the six groups of subjects ranked by number of calving, i.e. from the first to the sixth lactation
(Table. 5, 6, 7, 8, 9 and 10). Is noted that in the table no. 5 (first lactation), the percentage of LP
subjects is much higher than the tables 6 and 7 (second and third lactation) 68,7%, 52,7% and
47,8% respectively. On the contrary, if it see the percentage of the HP subjects, the values takes an
opposite trend: 0.8% in the first lactation (Table.5) vs. 5.7% in the third lactation (Table. 7). This
1340
study confirm the fact that the second buffalo lactation is better than the first and that the third
appear better than the second, as what has already been published in (Coletta et al., 2007).
In the table 11 it is noted that the 50% of the average production of the whole lactation is
realized in the early 112 days of lactation and this value is almost constant in all different levels of
production. The table 12 contains the estimation of the parameters of Wood model for the three
production levels (LP, MP and HP). The a parameter (initial production level) was higher in HP
group (8,91) than LP (6,13). The b parameter (speed with which the milk production increase until
the lactation peak) was higher in HP (0.26) than MP (0.21) and LP (0.19). the c parameter (speed of
milk production reduction after the lactation peak) is similar in the groups (0,006=HP; 0,005=MP;
0,005=LP). The tmax (day scheduled for the peak) and the ymax (maximum quantity of milk
produced in Kg, in the peak day) are represented in the same table 12 where comparing LP and HP
groups, appear that tmax is reached at 37th day in LP, at 46th day in HP and that ymax is
considerably higher in HP (18,52 Kg) compared to LP (9,99 Kg).
The graphic 1 shows that the peak is even more belated as higher will be the production. The
graphic 2 shows the percentage production of buffalo population (all) in 270 days and compare this,
with the production in HP (> 3500 kg), MP (between 2500 and 3500 kg) and LP (< 2500 kg). If one
observes the percentage differences between the three levels of production and the average
production of whole sample (all) it can be noted the different trend of the three production levels.
Taking in consideration the LP group is clear a trend almost decreasing depending on the distance
from calving: at 30 days of lactation it is produced less of 16,00%; at 120 days it is produced less of
18,50% and at 240 days less of 19,00% than the average production of the whole sample.
Taking in consideration the MP group is clear a trend almost increasing depending on the
distance from calving: at 30 days of lactation it is produced more of 13,50%; at 120 days it is
produced more of 15,50% and at 240 days more of 16,50% than the average production of the
whole sample.
Lastly the HP group takes a trend almost constant but increase depending on the distance from
calving: at 30 days of lactation it is produced more of 36,50%; at 120 days it is produced more of
37.50% and at 240 days more of 36,00% than the average production of the whole sample.
CONCLUSIONS
The lactation curve of the LP group reached the maximum peak of production at 37th day
(tmax) and the milk quantity produced in that day was 9,99 Kg (ymax). In LP, these curve
anticipates the tmax compared with MP and HP, had lower b values (speed with which the milk
production increase until the lactation peak); the c parameter, instead, (speed of milk production
reduction after the lactation peak) was almost similar compared with MP and HP.
The lactation curve of the MP group reached the maximum peak of production at 43th day
(tmax) and the milk quantity produced in that day was 13,57 Kg (ymax).
The lactation curve of the HP group reached the maximum peak of production at 46th day
(tmax) and the milk quantity produced in that day was 18,52 Kg (ymax). This curve belated the
tmax through an higher b value, while the c value was similar to the same in MP and LP groups.
The study reveals also that the best production (HP) was verified in subjects with a and b
parameters higher. The c parameter was influenced by the previous two but it is constant in the
three production levels. So higher is the production so will be both belated and higher the lactation
peak.
Ultimately it is possible to confirm that further investigations are necessary to deepen the
results obtained in this study since in the literature are traceable only few references in this regard
(Aziz et al., 2006). The processing of forward estimate of the lactation curves, allows to estimate
with good approximation the productions that will make by buffaloes in the current lactation.
REFERENCES
Aziz, M.A., N.A. Shalaby, O.M. El-Shalifie, A.T. Mahdy and A. Nishida. 2006. Comparison
between the shapes of lactation curve of Egyptian buffalo milk yield estimated by the
1341
incomplete gamma function and a new model. Livestock Research for Rural Development 18
(5).
Bollettino, AIA dei Controlli Funzionali. 2010.
Bufalo è – il valore dei migliori tori n° 1 Giugno. 2010.
Circolare del, M., D.P. Agricole and A. e Forestali. 2009. Programma dei controlli funzionali svolti
dalle associazioni provinciali degli allevatori per ogni specie, razza o tipo genetico.
Coletta, A. and C. Caso. 2008. Bulletin of the International Dairy Federation Milking Management
of Dairy Buffaloes Cap. Milk recording 11: 101 – 104.
Coletta, A., C. Caso, M. Castrillo, M. Parlato, A. Zullo and L. Zicarelli. 2007. “Fit of the wood
function to milk yield datacollected by different recording systems in Mediterranean Italian
Buffalo” (Misurazione della Funzione di Wood con i dati produttivi raccolti con due differenti
sistemi di registarzione nella Bufala Mediterranea Italiana), XVII Congresso Nazionale
A.S.P.A. (Associazione Scientifica di Produzione Animale), Alghero. pp. 29.
Dimauro, C., G. Catillo, N. Bacciu, N.P.P. Macciotta, 2005. Fit of different linear models to the
lactation curve of Italian water buffalo. Ital. J. Anim. Sci. 4: 22-24.
Disciplinare del Libro Genealogico dell’ANASB .
Groenewald, P., A. Ferreira, H. Van Der Merwe and S. Slippers. 1995. J. Anim. Sci. 61: 95-101.
Macciotta, N.P., C. Dimauro, G. Cavillo and A. Cappio-Borlino. 2005. Lactation curve
characteristics of Italian River Buffalo. Proc III Cong. Naz. All. Buf. Capaccio (SA), Roma.
pp.67-68.
Macciotta, N.P., P.D. Vicario, and A. Cappio-Borlino. 2005. Detection of different shapes of
lactation curve for milk yield in dairy cattle by empirical mathematical models. J. Dairy Sci.
88:1178-1191.
Norme tecniche di selezione del Libro Genealogico dell’ANASB.
Relazione annuale Assemblea dell’ANASB del 29 giugno 2010.
Wood, P.D.P. 1969. Factors affecting the shape of the lactation curve in cattle. Animal Production
11: 307-316.
Wood, P. D. P. 1967. Algebraic model of the lactation curve in cattle. Nature 216: 164-165.
Zullo, A., C. M. A. Barone, P. Colatruglio, M. Occidente, M. Troisi, D. Matasssino. 2001. Primo
Contributo alla conoscenza dell’effetto di alcuni fattori ambientali sulla galattopoiesi
individuale di bufale allevate in Italia e sottoposte ai controlli funzionali. Proc. I Cong. Naz.
All. Buf. Capaccio (SA). pp. 322-326.
Table 1. Comparison of data (number and % of total lactations) ANASB 2009 and those of the
present study.
Order of lactation Data of the present study Data of ANASB 2009

n. % n. %
1° 2.468 36,56 8.900 27,13
2° 1.570 23,26 6.594 20,09
> 2° 2.712 40,17 17.315 52,77
Produced Kg 2.421 2.182
Table 2. Lactation order, number of lactations and percentage distribution in the whole sample.
Lactation order number of lactations percentage distribution (%)

1° 2.468 36,56
2° 1.570 23,26
3° 1.066 15,79
4° 730 10,81
5° 514 7,61
6° 402 5,96
TOTAL 6.750 100,00
1342
Table 3. Lactation order, number of lactation and average age / lactation order in months.
Lactation order Number Age in months (years, months)

1° 2.468 40,64 (3 years 5 months)
2° 1.434 56,26 (4 years, 8 months)
3° 910 70,81 (5 years, 11 months)
4° 657 86,63 (7 years, 3 months)
5° 454 101,17 (8 years, 5 months)
6° 351 116,67 (9 years, 8 months)
AVERAGE 5 years 3 months
Table 4. Number of observation, average, standard deviation and percentage distribution of

lactations in buffaloes having different production level.
Production level Kg N. observation Average Std. Dev. (%) distribution
Low LP < 2500 36.917 2.049,50 327,13 57,5

Medium MP 2501-3500 25.072 2.851,60 253,60 39,0
High HP > 3500 2.060 3.804,24 334,12 3,5

lactations in buffaloes at first calving having different production level.
Production level N. observation Average Std. Dev. (%) distribution
LP < 2500 kg 1.695 2.024,11 324,88 68,7

MP 2501-3500 kg 754 2.785,52 230,24 30,6
HP > 3500 kg 19 3.735,21 328,23 0,8

lactations in buffaloes at second calving having different production level.
LP < 2500 kg 827 2.073,25 313,98 52,7

MP 2501-3500 kg 677 2.876,01 258,65 43,1
HP > 3500 kg 66 3.772,07 272,17 0,4

lactations in buffaloes at third calving having different production level.
LP < 2500 kg 510 2.082,96 321,14 47,8

MP 2501-3500 kg 495 2.894,56 253,61 46,4
HP > 3500 kg 61 3.827,30 337,43 5,7
1343

lactations in buffaloes at fourth calving having different production level.
LP < 2500 kg 365 2.087,58 320,53 50,0

MP 2501-3500 kg 328 2.874,84 255,29 44,9
HP > 3500 kg 37 3.778,00 305,75 5,1

lactations in buffaloes at fifth calving having different production level.
LP < 2500 kg 264 2.032,05 385,94 51,4

MP 2501-3500 kg 225 2.863,23 260,25 43,8
HP > 3500 kg 25 3.885,10 469,00 4,9
lactations in buffaloes at sixth calving having different production level.
LP < 2500 kg 218 2.035,43 321,03 54,2

MP 2501-3500 kg 171 2.862,31 264,19 42,5
HP > 3500 kg 13 3.882,73 334,22 3,2
Table 11. Day in which is reached the 50% of the production of the whole lactation.
Production level Day

Basso < 2500 kg 111
Medio 2501-3500 kg 113
Alto > 3500 kg 112
Media 112
Table 12. Estimation of Wood parameters and number of observation (N) in buffaloes that were
calving in 2005 and beyond with high, medium and low production and in whole population.
Produzione N. osservazioni a b c tmax ymax

Alta 2.060 8,91 0,26 0,006 46,1 18,52
Media 25.072 7,48 0,21 0,005 43,09 13,57
Bassa 36.917 6,13 0,19 0,005 37,39 9,99
tutto il campione 64,049 6,7 0,2 0,005 40,62 11,66
1344
20
18
16
14 Tutte
All
Milk,kg
Kg
12
< 2.500 kg
Latte,
10
8 2.500-3.500 kg
6 > 3.500 kg
4
2
0
4 18 32 46 60 74 88 102116130144158172186200214228242256270
giorno del day

Milk Recording controllo
Graph 1. Daily milk production estimated through gamma function (Wood, 1967) of buffaloes
with high (> 3.500 kg), medium (between 2.500 and 3.500 kg), low (< 2.500 kg) production and
in whole population (all).
50,00
40,00
30,00
< 2.500 kg
20,00
2.500-3.500 kg
10,00
%
> 3.500 kg
0,00
4 18 32 46 60 74 88 102116130144158172186200214228242256270 Tutte
All
-10,00
-20,00
-30,00
giorno del controllo
Milk Recording day
Graph 2. Percentage difference between the production of the whole buffaloes of population
(All) and high (> 3.500 kg), medium ( 2.500 and 3.500 kg), low (< 2.500 kg).
1345
Microstructure, Rheological and Textural Characteristics of Low Fat Buffalo

Milk Cheddar Cheese
Mian Anjum MURTAZAa,b,*, Alistair S. GRANDISONb, Nuzhat HUMAb,c, Sarfraz AHMADc

and Mian Shamas MURTAZAc
a
Institute of Food Science and Nutrition, University of Sargodha, Sargodha-40100, Pakistan
b
Department of Food and Nutritional Sciences, University of Reading, Whiteknights, Reading, UK
c
National Institute of Food Science and Technology, University of Agriculture, Faisalabad-38040,
Pakistan
*Corresponding email: anjum_ft@yahoo.com/mian.anjum@uos.edu.pk
ABSTRACT
Buffalo milk is at the top in Indo-Pak milk production; however, not characterized and
studied in depth. Cheddar cheese is produced in the world from cow milk and low fat product is
preferred on compositional basis but fells short in quality. The study was designed to manufacture
the low fat Cheddar cheese from buffalo milk and assess that how fat content affects its structure
and rheology. Cheddar cheese was manufactured from buffalo milk standardized at 2% and 4% fat
levels and was investigated for chemical composition, microstructure, hardness and rheological
characteristics. Reduction in milk fat from 4% to 2% enhanced the moisture and protein levels and
reduced the fat contents in cheese. Confocal scanning laser microscopy showed that the size and the
number of non-spherical fat globules reduced on lowering the fat levels in cheese. Reducing the fat
contents of buffalo cheese increased the elastic and viscous modulus at 20°C but no perceptible
variations were observed at 40°C. The discrepancy in cheese rigidity (elastic and viscous modulus)
might be owing to the modifications in gel network between buffalo milk proteins and fats that was
consequence of variations in fat content and their distribution. Total hardness in low fat cheese
found by compression and texture profile as 783.33g and 8194.67g respectively was almost two
times than of full fat cheese (340.00g and 4947.33g, respectively). The established results have
momentous variations as compared to those reported in cow milk cheese. Hence, it can be
concluded that reduced fat buffalo milk produced cheese with acceptable textural and structural
quality.
Keywords: Buffalo milk, Cheddar cheese, fat content, microstructure, texture, rheology
INTRODUCTION
The ambition for caloric reduction in food, distinctively targeting the calories from fat,
persuades the development of low-fat alternatives to traditional products (Rogers et al., 2010).
Cheese is rich in fat food and some consumers hesitate to include it within diet, although it is an
excellent source of dietary calcium (Johnson et al., 2009). Reduced and fat free cheeses are
preferred due to their compositional profile but mostly their overall quality fell diminutive (Rogers
et al., 2010).
Fat in cheese is not only of nutritional importance but also confers the sensory and
functional characteristics by playing an influential role in texture, flavor and aroma development in
cheese (Mistry, 2001). However, reduction in fat content poses a negative impact on sensory
characteristics and develops an unwanted rubbery and firm texture (Rogers et al., 2009). Studies are
still underway to develop the techniques and alternatives for lucratively producing the reduced fat
cheese.
The consumers consider the rheological characteristics commonly referred as cheese body
and texture as the imperative quality traits. In addition to flavor and appearance, the texture is a key
feature of cheese quality (Messens et al., 2000). Being a solid food, Cheese has viscoelastic
character, so it shows both viscous and elastic behaviors. This feature demands the performance of
rheological and textural measurements of cheese (Pinho et al., 2004).
1346
Buffalo’s milk is rich in fat, lactose, caseins, calcium, magnesium and phosphate as
compared to cow (Ahmad et al., 2008) and on account of the composition, it proffers exceptional
prospects for the manufacturing of various milk products (Murtaza et al., 2008). Cheddar cheese is
manufactured from cow’s milk worldwide however; buffalo milk is at top in milk production of
Pakistan and is more appropriate for the manufacturing of cheese (Murtaza et al., 2012).
Many studies have conducted to produce and evaluate the low fat cheese from cow milk.
However; investigations to manufacture reduced and low fat cheeses from buffalo milk are still
deficient. The present research was designed to manufacture the low fat Cheddar cheese from
buffalo milk and assess the impact of fat content on its microstructure, rheological and textural
characteristics.

Cheddar Cheese Manufacturing and Ripening
Buffalo milk procured from Farm House, University of Agriculture, Faisalabad, Pakistan
was standardized at 2.0% and 4.0% fat levels. Cheddar cheese samples were manufactured using
direct in vat (DVS) Mesophilic starter cultures of CHR-Hansen, Denmark, following the standard
protocol, with some modifications, as described by Rehman et al. (2000). Cheese was stored for
ripening at 6-8°C for a period of 60 days. After ripening, samples were evaluated for chemical
composition, microstructure, hardness and rheological characteristics.
Moisture, fat and protein contents were determined following the protocols of AOAC
(1990).
Cheese Hardness
Hardness of cheese samples was evaluated by performing the compression and texture
profile analysis (TPA) on texture analyzer (Texture Pro CT V1.2 Build 9, Brookfield Engineering
Labs, Inc. USA) as described by O’Mahony et al. (2005).
Rheological characteristics
Rheological analysis were performed on a Stress Tech controlled rheometer (ATS
Rheosystems, Bordentown, NJ) fitted with a 20-mm smooth parallel plate geometry, according to
the method of Rogers et al. (2009). Cheese samples were tested for temperature and time sweeps
and their elastic and viscous modulus at 20°C and 40°C at 0, 5 and 10 minutes each.
Microstructure
Cheese microstructure was imaged using confocal scanning laser microscopy (CSLM) as
done by Rogers et al. (2010). Cheese samples were sliced into sections approximately 5 mm × 5
mm × 1 mm thick using a razor blade. Fluorescent dyes Nile Red and phosphate Green (Invitrogen
Molecular Probes, Eugene, OR) were used to image the fat and protein phases of the cheese,
respectively. Samples were imaged using an inverted Leica TCS SP1 CSLM using a PL Fluotar
40.0× 1.0 oil UV objective (Leica Microsystems GmbH, Wetzlar, Germany).
All the cheese samples were prepared and analyzed in triplicates and results drawn were
statistically analysed using Analysis of Variance (Steel et al., 1997).

The composition of cheese showed that lowering the milk fat reduced the fat content in
cheese (Table 1). As anticipated, moisture (38.29±0.21%) and protein (28.86±0.37%) contents were
higher in low fat cheese as compared to full fat cheese (35.98±1.10% and 24.38±0.18%
respectively). These variations contribute to discrete texture, microstructure and rheological
characteristics (Rogers et al., 2009).
Cheese Hardness
Cheese hardness was the force required to compress the cheese sample to 30% of its original
height during the first compression cycle. The hardness calculated by compression and TPA showed
1347
that half reduction in fat level increased the hardness up to double (Table 2). The increase in
hardness was owing to the fact that low fat cheese has more dense protein network and firm hard
texture (Rogers et al., 2009). However, the comparatively less hardness in buffalo cheese as
compared to cow might be due to differences in fat globules size, distribution and composition
(Menard et al., 2010).
Rheological characteristics
Regarding rheological characteristics, storage (elastic) modulus and loss (viscous) modulus
increased on reducing the fat levels and similarly the tan delta values (ratio) were affected.
However, the time sweep showed inverse effects on both modulus (Tables 3, 4 and 5). At start, the
elasticity in low fat cheese was almost two times than full fat however, with time, the differences
were reduced. At higher experimental temperature (40°C), the deviations among the full fat and low
fat cheeses were not of that extent as at low temperature (20°C). It might be due to melting of fat at
higher temperature. The variations in rheological parameters because of fat contents and
temperatures are similar as reported by Rogers et al. (2010). The temperature behavior can be
endorsed to the changes of fat from solid to fluid phase in cheese and modifications in protein
network (Vithanage et al., 2009).
Microstructure
The CSLM images of cheese microstructure (Figure 1 and 2; having red and green colors
representing fat and protein, respectively) showed that the size of fat globules decreased on
reducing the cheese fat content and also showed variation in the shape of fat globules. Moreover,
low fat cheese had more spherical fat globules dispersed all through the matrix, however; fat
globules in full fat cheese emerged clustered and coalesced into non-spherical shapes. The globules’
dispersion in low fat cheese also supports the increase in elastic and viscous modulus. This imaging
is comparable to the observations of low fat Cheddar cheeses by Guinee et al. (2000) and Rogers et
al. (2010).
CONCLUSION
The present study characterized the low and full fat buffalo milk cheese and it was
concluded that low fat cheese with acceptable texture and structure can be produced from buffalo
milk.
REFERENCES
Ahmad, S., I. Gaucher, F. Rousseau, E. Beaucher, M. Piot, J. F. Grongnet and F. Gaucheron. 2008.
Effects of acidification on physico-chemical characteristics of buffalo milk: a comparison
with cow’s milk. Food Chem. 106:11-17.
AOAC. 1990. Official Methods of Analysis. 15th Edn. The Association of Official Analytical
Chemists, Arlington, Virginia.
Guinee, T.P., M.A.E. Auty and M.A. Fenelon. 2000. The effect of fat content on the rheology,
microstructure and heat-induced functional characteristics of Cheddar cheese. Int. Dairy J.
10:277-288.
Johnson, M.E., R. Kapoor, D.J. McMahon, D.R. McCoy and R.G. Narsimmon. 2009. Reduction of
sodium and fat levels in natural and processed cheese: Scientific and technological aspects.
Comp. Rev. Food Sci. Food Safety 8:252-268.
Menard, O., S. Ahmad, F. Rousseau, V. Briard-Bion, F. Gaucheron and C. Lopez. 2010. Buffalo vs.
cow milk fat globules: Size distribution, zeta-potential, compositions in total fatty acids and
in polar lipids from the milk fat globule membrane. Food Chem. 120:544-551.
Messens, W., D. Van de Walle, J. Arevalo, K. Dewettinck and A. Huyghebaert, 2000. Rheological
properties of high-pressuretreated Gouda cheese. Int. Dairy J. 10:359-367.
Mistry, V. 2001. Low fat cheese technology. Int. Dairy J. 11:413-422.
Murtaza, M.A., S.U. Rehman, F.M. Anjum and H. Nawaz. 2008. Nutritional comparison of cow and
buffalo milk Cheddar cheese. Pak. J. Nutr. 7:509-512.
1348
Murtaza, M.A., S.U. Rehman, F.M. Anjum, N. Huma, O.M. Tarar and G. Mueen-ud-Din. 2012.
Organic acids contents of buffalo milk Cheddar cheese as influenced by accelerated ripening
and sodium salt. J. Food Biochem. 36:99-106.
O’Mahony, J.A., J.A. Lucey and P.L.H. McSweeney. 2005. Chymosin-mediated proteolysis,
calcium solubilisation, and texture development during the ripening of Cheddar cheese. J.
Dairy Sci. 88:3101-3114.
Pinho, O., E. Mendes, M.M. Alves and I.M.P.L.V.O. Ferreira. 2004. Chemical, physical, and
sensorial characteristics of ‘‘Terrincho” ewe cheese: change during ripening and
intravarietal comparison. J. Dairy Sci. 87:249-257.
Rehman, S.U., J.M. Banks, E.Y. Brechany, D.D. Muir, P.L.H. McSweeney and P.F. Fox. 2000.
Influence of ripening temperature on the volatiles profile and flavour of Cheddar cheese
made from raw or pasteurized milk. Int. Dairy J. 10:55-65.
Rogers, N.R., D.J. McMahon, C.R. Daubert, T.K. Berry, E.A. Foegeding. 2010. Rheological
properties and microstructure of Cheddar cheese made with different fat contents. J. Dairy
Sci. 93:4565-4576.
Rogers, N.R., M.A. Drake, C.R. Daubert, D.J. McMahon, T.K. Bletsch and E.A. Foegeding. 2009.
The effect of aging on low-fat, reduced-fat, and full-fat Cheddar cheese texture. J. Dairy Sci.
92:4756-4772.
Steel, R.G.D., J.H. Torrie and D.A. Dickey. 1997. Principles and Procedures of Statistics. 3rd Ed. A
Bio-Metrical Approach, McGraw Hill Book Company, New York, USA.
Vithanage C.R., M.J. Grimson and B.G. Smith. 2009. The effect of temperature on the rheology of
butter, a spreadable blend and spreads. J. Texture Stud. 40:346-369.
Table 1. Chemical composition of Cheddar cheese.
Parameters Full fat (4%) Low Fat (2%)

Mositure (%) 35.98±1.10b 38.29±0.21a
Fat (%) 38.28±0.11a 20.36±0.29b
Protein (%) 24.38±0.18b 28.86±0.37a
Table 2. Total hardness (g) of Cheddar cheese.
Method Full fat (4%) Low Fat (2%)

Compression 340.00±27.06b 783.33±16.43a
TPA 4947.33±267.36b 8194.67±611.26a
1349
Table 3. Loss Modulus Values (Mean±SD) of Full and Low Fat Cheddar cheese (in KPa).
°C Time Full Fat Low Fat
0 min 1.06E+05 ± 6578.249 1.97E+05 ± 23373.52

20°C
5 min 9.07E+04 ± 4758.858 1.09E+05 ± 7105.689
10 min 8.50E+04 ± 7824.809 8.37E+04 ± 6123.06
0 min 4.31E+04 ± 7101.017 3.69E+04 ± 554.0708

40°C
5 min 1.19E+04 ± 169.8141 3.31E+04 ± 257.3018
10 min 8049.1 ± 188.2702 2.66E+04 ± 5918.252
Table 4. Storage Modulus Values (Mean±SD) of Full and Low Fat Cheddar cheese (in KPa).
0 min 3.75E+05 ± 8230.078 6.39E+05 ± 20585.55

20°C
5 min 3.08E+05 ± 3510.046 3.24E+05 ± 6729.972
10 min 2.85E+05 ± 9052.479 2.07E+05 ± 12906.80
0 min 1.04E+05 ± 19575.83 8.75E+04 ± 1465.727
40°C 5 min 1.09E+04 ± 872.6333 7.28E+04 ± 1781.059
10 min 5282.4 ± 722.5632 5.65E+04 ± 9851.161
Table 5. Tan Delta Values (Mean±SD) of Full and Low Fat Cheddar cheese.
0 min 0.28303 ± 0.018768 0.33549 ± 0.031529

20°C
5 min 0.29421 ± 0.016181 0.33139 ± 0.036123
10 min 0.297955 ± 0.027041 0.33111 ± 0.110339
0 min 0.41757 ± 0.016265 1.2166 ± 0.031225

40°C
5 min 1.0996 ± 0.084358 1.41305 ± 0.057437
10 min 1.5433 ± 0.151081 1.3703 ± 0.0427
1350
Figure 1. Microstructure of full fat cheese samples.
Figure 2. Microstructure of half fat cheese samples.
1351
BUFFALO BULLETIN
IBIC, KASETSART UNIVERSITY, P.O. BOX 1084
BANGKOK 10903, THAILAND
URL : http://ibic.lib.ku.ac.th
E-mail : libibic@ku.ac.th
Tel : 66-2-9428616 ext. 344
Fax : 66-2-9406688
View publication stats

BuffaloBulletinVol 32 SP2

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

BuffaloBulletinVol 32 SP2

Uploaded by

Copyright:

Available Formats

See discussions, stats, and author profiles for this publication at: https://www.researchgate.

Article in Buffalo Bulletin · January 2013

Sarfraz Ahmad Faqir Muhammad Anjum

SEE PROFILE SEE PROFILE

Mian Anjum Murtaza

Research work of my Msc(hons.) thesis View project

The user has requested enhancement of the downloaded file.

Buffalo Reproduction Symposium

1. Prof. Yindee Kitiyanant

3. Prof. Dr. Dheer Singh

4. Dr. Sunpetch Sophon

5. Assoc. Prof. Petai Pongpiachan

6. Assoc. Prof. Dr. Yupaporn Chaiseha

7. Assoc. Prof. Dr. Bunlue Kornmatitsuk

8. Assoc. Prof. Dr. Suneerat Aiumlamai

9. Assoc. Prof. Dr. Siriwat Suadsong

10. Asst. Prof. Pongthorn Suwannatada

11. Asst. Prof. Dr. Theerawat Tharasanit

12. Dr. Takashi Nagai

13. Dr. Kei Imai

14. Dr. Tamas Somfai

15. Dr. Giorgio Antonio Presicce

16. Dr. Vibuntita Chankitisakul

17. Dr. Nutthee Amin

18. Dr. Anucha Sathanawongs

19. Dr. Krittiya Lertchunhakiat

20. Dr. Thuchadaporn Chaikhun

21. Mr. Anawat Sangmalee

22. Ms. Jakkhaphan Chasombat

23. Mr. Ashit Pual

1. Prof. Dr. Kanchana Markvichitr

2. Prof. Masroor Ellahi Babar

3. Prof. Leopoldo Iannuzzi

4. Assoc. Prof. Dr. Monchai Duangjinda

5. Assoc. Prof. Dr. Marina Ketudat-Cairns

6. Assoc. Prof. Dr. Neramit Sukmanee

7. Asst. Prof. Dr. Sirinporn Sindhuvanich

8. Asst. Prof. Dr. Skorn Koonawootrittririon

9. Asst. Prof. Dr. China Supakorn

10. Asst. Prof. Dr. Amornrat Molee

11. Asst. Prof. Dr. Wuttigrai Boonkum

12. Dr. Nalinee Imboonta

13. Dr. Supawadee Manatrinon

14. Dr. Warangkana Kitpipit

15. Dr. Yuanyuan Liang

16. MS. Kanokwan Srirattana

17. Mr. Phakphume Saowaphak

Buffalo Nutrition and Feeding Symposium

1. Assoc. Prof. Dr. Wisitiporn Suksombat

Buffalo Health Symposium

1. Prof. Dr. Chaleow Salakij

2. Assoc. Prof. Dr. Anudep Rungsipipat

3. Assoc. Prof. Dr. Wijit Banlunara

4. Asst. Prof. Dr. Somporn Techangamsuwan

5. Asst. Prof. Dr. Burin Nimsuphan

6. Asst. Prof. Khamphee Pattanatanang

8. Asst. Prof. Dr. Nareerat Viseshakul

9. Asst. Prof. Dr. Worakij Cherdchutham

10. Asst. Prof. Dr. Sirikachorn Tangkawattana

11. Asst. Prof. Dr. Piyanan Taweethavonsawat

12. Dr. Kidsadagon Pringproa

Buffalo Physiology Symposium

1. Assoc. Prof. Dr. Apassara Choothesa