College of Veterinary Science Animal Husbandry

Behera,M.
2016‘Age-specific Gross Morphometrical and Histomorphological Changes in Long Leg Bones of Post-Hatch Broiler Chickens with
Age-specific Gross Morphometrical and

Histomorphological Changes in Long Leg Bones
of Post-hatch Broiler Chickens with Special
Reference to Growth Cartilage
MINATI BEHERA
Adm. No. 02 VAN / 14
Special Reference to Growth Cartilage
DEPARTMENT OF VETERINARY
ANATOMY AND HISTOLOGY
COLLEGE OF VETERINARY
SCIENCE & ANIMAL HUSBANDRY
ORISSA UNIVERSITY OF AGRICULTURE
AND TECHNOLOGY, BHUBANESWAR
2016
Age-specific Gross Morphometrical and
Histomorphological Changes in Long Leg Bones
of Post-hatch Broiler Chickens with Special
Reference to Growth Cartilage
A THESIS SUBMITTED TO
ORISSA UNIVERSITY OF AGRICULTURE & TECHNOLOGY,
BHUBANESWAR
IN PARTIAL FULFILLMENT OF THE REQUIREMENT
FOR THE DEGREE OF
MASTER OF VETERINARY SCIENCE

IN
VETERINARY ANATOMY AND HISTOLOGY
by
Minati Behera
Adm.No.-02VAN/14
DEPARTMENT OF VETERINARY ANATOMY

AND HISTOLOGY
COLLEGE OF VETERINARY SCIENCE
AND ANIMAL HUSBANDRY
ORISSA UNIVERSITY OF AGRICULTURE AND
TECHNOLOGY, BHUBANESWAR
2016
ORISSA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY
Department of Veterinary Anatomy and Histology
College of Veterinary Science and Animal Husbandry
Dr. Arun Kumar Mandal, Ph.D. Bhubaneswar

Associate Professor Date:
CERTIFICATE - I
This is to certify that the thesis entitled “Age-Specific Gross
Morphometrical and Histomorphological Changes in Long Leg Bones of
Post-Hatch Broiler Chickens with Special Reference to Growth Cartilage’’
submitted in partial fulfillment of the requirements for the award of degree of
Master of Veterinary Science in the subject of Veterinary Anatomy and
Histology, to the Orissa University of Agriculture and Technology,
Bhubaneswar is a faithful record of bona fide and original research work
carried out by Minati Behera under my guidance and supervision. No part of
this thesis has been submitted for any other degree or diploma.
It is further certified that the assistance and help received by her from
various sources during the course of investigation has been duly acknowledged.
CHAIRMAN
ADVISORY COMMITTEE
CERTIFICATE – II
This is to certify that the thesis entitled ‘‘Age-specific Gross
Morphometrical and Histomorphological Changes in Long Leg Bones of
Post-hatch Broiler Chickens with Special Reference to Growth Cartilage’’
submitted by Minati Behera to the Orissa University of Agriculture and
Technology, Bhubaneswar in partial fulfillment of the requirements for the
degree of Master of Veterinary Science (Anatomy and Histology) has been
approved/disapproved by the student’s advisory committee and the external
examiner.
Advisory Committee
Chairman:
Dr. Arun Kumar Mandal
___________________
Associate Professor,
Dept. of Veterinary Anatomy & Histology
Members:
1. Dr. U.K. Mishra ___________________
Associate Professor and Head,
Dept. of Veterinary Anatomy & Histology
2. Dr. A.K. Kundu ___________________

Professor and Head,
Dept. of Veterinary Physiology
3. Dr. A. Maity ___________________

Assistant Professor
Dept. of Veterinary Biochemistry
4. Dr. S. Sathapathy ___________________

Assistant Professor
Dept. of Veterinary Anatomy and Histology
External Examiner
(Name & Designation)
ACKNOWLEDGEMENT
Blessed are those who can give without remembering and take without forgetting.
Elizabeth Asquith Bibesco.
The perspicuous piece of acknowledgement provides me the opportunity to express

my heartfelt regards and gratitude towards all those who always stand by me through the ups
and downs of this itinerary and whose help is indispensable for the completion of this
manuscript.
I would like to avail this golden opportunity in life to express my heartfelt regards
and deepest sense of gratitude and indebtedness from the core of my heart to the Honourable
Chairman of my Advisory Committee Dr. Arun Kumar Mandal, Associate Professor.
Department of Anatomy and Histology, College of Veterinary Science and Animal
Husbandry, Orissa University of Agriculture and Technology, Bhubaneswar for his adroit
supervision, pertinent suggestions, intellectual guidance, consistent inspiration &
encouragement, constructive criticism & discussion, valuable suggestions, affectionate
attitude and advice that helped me at every step of my research work. His innovative ideas,
meticulous nature and close monitoring of the research work, the manuscript with perfection.
My sense of obligation is negligible and unquantifiable in respect of his energy and time spent
on this endeavour. It was a life time opportunity to work with him, to know about an
experimental man in work and to gather valuable knowledge and experience, suggestions
during the course of my investigation. It is my golden opportunity to have him as the
Chairman of my Advisory committee.
I am greatly beholden beyond words to express my deep sense of obligation and

gratefulness to my respected Dr. Uma Kanta Mishra, Associate Professor & Head cum
Member of Advisory Committee, Department of Anatomy and Histology for his skillful
guidance enlightened views, inspiring attitude and valuable suggestions and making timely
arrangements for supply of necessary chemicals, instruments and above all keeping full faith
on me during my entire period of research work and keen interest during the entire course of
my present study to bring this manuscript into its present existence. It is the matter of great
pride to work under the dynamic guidance of an able and affectionate academician of his
calibre.
I gratefully articulate my sincere gratification and obligation to the member of the

Advisory Committee Dr. A.K. Kundu. Professor and Head. Department of Veterinary
Physiology for his valuable advice, constant encouragement and requisite help during the
entire course of this research work.
I take privilege at expressing my gratefulness to Dr. A. Maity, Assistant Professor.
Department of Biochemistry for providing co-operation, encouragement during the course of
research work. I duly acknowledge the Laboratory facilities extended by him. His guidance
and critical analysis of my Laboratory work have helped me a lot and it is my golden chance
to have him as the member of my Advisory committee.
I wish to acknowledge the invaluable help and co-operation extended by Dr. Srinivas
Sathapathy, Assistant Professor, Department of Anatomy and Histology. I thank him for his
kind words, immense patronage and encouragement and untired help in the study period and
it is my golden chance of my life to have him as the member of Advisory committee.
It is my pleasure to express my deepest sense of regards to Dr. R K Das Professor,

Department of Anatomy and Histology cum Director, Agro Polytechnic, OUAT,
Bhubaneswar, for his valuable suggestions, moral encouragement and inspiration in each and
every step of my research work.
I feel previleged to express my heartiest gratefulness to Dr.(Mrs) Ritun Patra and

Dr.(Miss) Sagarika Dehury, Assistant Professor. Department of Anatomy and Histology for
their kind support at each and every step of my need.
It is a unique opportunity to express my sincere and dear sense of indebtedness thanks

for Dr. Niranjan Sahoo, Professor and Head. Department of Preventive Medicine for his
kind words, necessary guidance and providing timely co-operation as and when required. His
help and advice is always invaluable.
I take privilege of expressing gratitude to Dr. Sushant Kumar Dash, Associate

Professor, for his concern and magnanimity in providing timely co-operation in all my
statistical formulation.
I owe my heartiest gratefulness to Dr. Kamadev Sethi, Assistant Professor,

Department of Animal Nuitrition for providing co-operation and encouragement during the
course of my research work.
I also pay my deep regards to Dr. R.C. Patra, The Dean, College of Veterinary
Science and Animal Husbandry for his generous attitude in providing necessary facilities for
carrying out the research work.
I am greatly beholden beyond words to express my sincere and deepest sense of

gratitude and indebtedness from the core of my heart to my Honourable Commissioner
cum Secretary, Government of Orissa, Shri Bishnupada Sethi Sir, IAS for allowing me to
prosecute higher studies in the only institute of our beloved state as a study leave candidate.
I gratefully articulate my sincere gratification and obligation to my Respected

Director, Animal Husbandry and Veterinary Services, Odisha, Cuttack for allowing me to
prosecute M V Sc study in OUAT , Bhubaneswar.
It is my great pleasure as well as a golden opportunity to express my deepest sense of

heartfelt regards and gratitude to Dr. R. K. Sahoo, CDVO, Dhenkanal, Dr. S.K.Sahoo,
ADVO, Dhenkanal, Dr. N.C. Rath, SDVO, Hindol and Dr. S.K.Nanda, VAS for allowing
me to prosecute MVSc study in this noble institute. Their kind co-operation, moral
encouragement, inspiring attitude, skillful guidance, valuable suggestion and requisite help as
and when required are really admirable. Their help and advice is always invaluable.
I express my obligation to Sri A. K. Punja, General Manager, Eastern Hatchery,

Bhubaneswar for providing the Vencob broiler chicks for my research work. I am very much
thankful to him.
Anything said will be less to Mrs. Sabita Sahu, Laboratory Assistant who provided
me the whole hearted support throughout my PG course and will be remembered forever. I
am very much thankful to Sri Sanjeeb Kumar Das, Laboratory Technician for his timely
suggestions and co-operation as and when required.
I duly acknowledge to the help and cooperation provided by the non-teaching staffs of
the department Ajaya bhaina, Kashi bhaina and Golekh bhaina who helped me in handling
the huge research work. They cooperated me for rearing of chicks upto the last part of the
period of my investigation.
I express my sincere appreciation and deep love to my lovely friends Sasmita, Sarita,
Narmada, Vineela. I also acknowledge the lovely cheerful cooperation from my younger
brothers Kuldeep, Chinmaya, Santosh.
I solicit the benediction of my parents Sri. Bipin Behari Behera and Mrs. Ramarani
Behera for my progress and prosperity. My parents prayer and pious advice kept all
negativity and failures in bay. I respect the abundant love and shower of blessings of my
sisters and relatives. I find acknowledgement inadequate to quantify the sacrifices, love
affection and constant confidence instilled in me by them and my gratitude are beyond words.
The author is very much thankful to Sri Hemant Kumar Sahu, Auro X ray
Diagnostic,Forest Park for taking X-Ray film during the course of my research work.
The author is also thankful to Ali Bhai, The Creations, Siripur for their co-operation for
preparing this manuscript in time.
It’s not possible to take everybody name if someone is forgotten, I am highly thankful
to them either they help me directly or indirectly.
Last but not the least I bow down before ALMIGHTY LORD JAGANNATH whose
eternal blessing could help me to pass over all the obstacles and enable me to complete this
investigation at Bhubaneswar.
Bhubaneswar
Date: (Minati Behera)

ABSTRACT
The present study was conducted on forty five (45) ‘Vencob’ day old broiler chicks
which were reared up to day 42 (market age). The gross morphometrical, radiographical
and histomorphological studies were carried out on the long leg bones (femur, tibiotarsus
and tarsometatarsus) and associated growth cartilages in broiler chickens at different ages
(on days 1,7, 14, 21, 28, 35 and 42). Simultaneous measurement of body weight and
biochemical evaluation of serum and bone Ca and P level was also done at weekly
interval. The average weight of the bones in day old male and female chicks gradually
increased on day 7, followed by a marked increase from day 14 up to day 42. There was
simultaneous increase in length of the bones in both male and female birds throughout the
experimental period. The thickness of marrow cavity and cortical bone gradually increased
with age of the broiler birds. The diameter (width) of all the bones in broiler birds showed
a steep increase from day 7 up to day 21 and a steady increase thereafter till day 42. The
proximal and distal end widened more than the mid shaft and the distal end is the widest.
The growth of the gross dimensions of the bones slowed down a bit towards the end of the
experiment (days 35 and 42). Though initially mid shaft cortical bone was thinner than
epiphyseal ends, it gradually became thicker from day 21 (in femur and tarsometatarsus) or
from day 28 (in tibiotarsus) till day 42. The gross anatomical observation did not reveal the
presence of growth plates in all day old chicks. But radiographical and histomorphological
study confirmed the presence of both proximal as well as distal growth plates in all these
three bones from day 1 till day 42, the latter one being more authentic. In femur presence
of growth plates are reported for the first time. Histology of growth plates revealed five
histological zones, i.e. resting or reserve zone, zone of proliferation, zone of
prehypertrophy, zone of hypertrophy and degeneration, and zone of ossification (bone
formation) from epiphyseal towards diaphyseal or metaphyseal end. Secondary centre of
ossification in broiler chicken appeared in the epiphyseal ends of tibiotarsus and
tarsometatarsus (but not in femur) on day 21. There was sparse amount of matrix and
connective tissue elements (collagen) in growth cartilage. There was variable PAS and
alcian blue reactivity of matrix (with predominance of alcianophilia) in different zones of
growth cartilage in all the bones. Different histological zones of growth cartilage exhibited
age and sex related variation in thickness as well as chondrocyte diameter (size). The
compact bone tissue of the bones in the broiler birds of both sexes revealed a common
histoarchitecture in a particular age. Each osteon or Haversian system consisted of 1- 4
concentric lamellae or layers of bone matrix around the central osteonal or Haversian
canal. The shape and size of these canals and osteons varied with age of the birds. The
cortical bone appears relatively porous and is made up of interlacing woven bone
resembling a network like pattern in younger birds, which is gradually transformed into a
compact mass subsequently. The average serum Ca and P levels in plasma and bone (%) in
broiler birds gradually increased with age. The ratio between serum Ca and P level (Ca: P)
and bone Ca and P percentage remains almost constant during the growing period of the
broiler chickens to maintain a proper balance for adequate mineralization of bones.
CONTENTS
Chapter Particulars Page No
No
1 Introduction 1-5
2 Review of Literature 6-34
2.1 Skeletal or Leg problems (deformities) and 6
animal welfare in broiler chickens
2.2 Gross morphometrical study 10
2. 3Radiographical study 14
2.4 Histomorphological study 16
2.4.1 Normal Growth plate (cartilage) structure 16
2.4.2 Growth plate (cartilage) abnormality 23
2.4.3 Bone structure and its alteration 27
2.5 Biochemical study 29
2.5.1 Blood calcium and phosphorus level 29
2.5.2 Bone calcium and phosphorus content 32
(Bone ash)
3 Materials and Methods 35 -39
3.1 Preamble 35
3.2 Experimental Design 35
3.3 Collection of Specimen 35
3.4 Gross Morphometrical Study 36
3.5 Radiographical Study 36
3.6 Histomorphological Study 37
3.7 Biochemical Study 38
3.8 Statistical Analysis 39
4 Results 40-60
4.1 Gross Morphometrical Observations 40

4.2 Gross Morphometrical Study of Leg Bones 40
a) Femur 40
b) Tibiotarsus 42
c) Tarsometatarsus 45
4.3Radiographical Study of Leg Bones and 47
Associated Growth Cartilages
4.4 Histomorphological Study of Growth 48
Cartilages and Bones
4.4.1 Histomorphological Study of Growth 48
Cartilages
a) Femur 48
b) Tibiotarsus 51
4.4.2 Histomorphological Study of Bones 57
4.5.1 Estimation of Blood calcium (Ca) and 60
Phosphorus (P) level
4.5.2 Estimation of Bone calcium (Ca) and 60
Phosphorus (P) Content
5 Discussion 111- 129
5.1 Gross Morphometrical Observations 111

5.1.1 Body weight 111
5.2 Gross Morphometrical Study of Leg Bones 112
and Associated Growth Cartilages
a) Femur 111
b) Tibiotarsus 114
5.3 Radiographical Study of Leg Bones and 117
Associated Growth Cartilages
5.4Histomorphological Study of Growth 119
Cartilages and bones
5.4.1 Histomorphological Study of Growth 119
Cartilages
a) Femur 119
b) Tibiotarsus 120
5.4.2 Histomorphological Study of Bones 125
6 Summary and Conclusion 130-139
6.1 Summary 130
6.2 Conclusion 137
Bibliography i-xiv
LIST OF FIGURES
Fig Particulars Page
No. No
Change in weight and length of right and left femur in male broiler chicken at
1 75
different ages
2 Change in width of right and left femur in male broiler chicken at different ages 75
Change in cortical bone thickness of right and left femur in male broiler chicken at
3 75
different ages
Change in thickness of growth cartilage and its different zones in femur of male broiler
4 76
chicken at different ages
5 Change in weight of right and left tibiotarsus in male broiler chicken at different ages 76
6 Change in length of right and left tibiotarsus in male broiler chicken at different ages 76
7 Change in width of right and left tibiotarsus in male broiler chicken at different ages 77
Change in cortical bone thickness of right and left tibiotarsus in male broiler chicken
8 77
at different ages
Change in thickness of growth cartilage and its different zones in tibiotarsus of male
9 77
broiler chicken at different ages
Change in weight of right and left tarsometatarsus in male broiler chicken at different
10 78
ages
Change in length of right and left tarsometatarsus in male broiler chicken at different
11 78
ages
Change in width of right and left tarsometatarsus in male broiler chicken at different
12 78
ages
Change in cortical bone thickness of right and left tarsometatarsus in male broiler
13 79
Change in thickness of growth cartilage and its different zones in tarsometatarsus of
14 79
male broiler chicken at different ages
15 Change in weight of right and left femur in female broiler chicken at different ages 79
16 Change in length of right and left femur in female broiler chicken at different ages 80
17 Change in width of right and left femur in female broiler chicken at different ages 80
Change in cortical bone thickness of right and left femur in female broiler chicken at
18 80
different ages
Change in thickness of growth cartilage and its different zones in femur of female
19 81
20 Change in weight of right and left tibiotarsus in female broiler chicken at different ages 81
21 Change in length of right and left tibiotarsus in female broiler chicken at different 81
22 Change in width of right and left tibiotarsus in female broiler chicken at different ages 82
Change in cortical bone thickness of right and left tibiotarsus in female broiler chicken
23 82
at different ages
Change in thickness of growth cartilage and its different zones in tibiotarsus of female
24 82
Change in weight of right and left tarsometatarsus in female broiler chicken at different
25 83
ages
Change in length of right and left tarsometatarsus in female broiler chicken at different
26 83
ages
Change in width of right and left tarsometatarsus in female broiler chicken at different
27 83
ages
28 Change in cortical bone thickness of right and left tarsometatarsus in female broiler 84
Change in thickness of growth cartilage and its different zones in tarsometatarsus of
29 84
female broiler chicken at different ages
30 Photograph of right and left leg bones (F, TT and TM) of day old broiler chick 85
31 Photograph of left leg bones (F, TT and TM) of 7 days old broiler chick 85
32 Photograph of right leg bones (F, TT and TM) of 7 days old broiler chick 85
33 Photograph of right leg bones (F, TT and TM) of 14 days old broiler chick 85
Photograph of right and left leg bones (F, TT and TM) of 21 days old male broiler
34 85
chicken
35 Photograph of right and left femur bone of 28 days old male broiler chicken 86
36 Photograph of right and left TT bone of 28 days old male broiler chicken 86
37 Photograph of right and left TM bone of 28 days old male broiler chicken 86
38 Photograph of right and left femur bone of 35 days male old broiler chicken 86
39 Photograph of right and left TT bone of 35 days male old broiler chicken . 86
40 Photograph of right and left TM bone of 35 days male old broiler chicken. 86
41 : Photograph of right and left femur bone of 42 days male old broiler chicken 87
42 Photograph of right and left TT bone of 42 days male old broiler chicken 87
43 Photograph of right and left TM bone of 42 days male old broiler chicken 87
44 Photograph of right and left femur bone of 42 days female old broiler chicken. 87
45 Photograph of right and left TT bone of 42 days female old broiler chicken 87
46 Photograph of right and left TM bone of 42 days female old broiler chicken 87
Fig. 47a: Sectional view of Femur bone of day old broiler chick showing growth cartilages
47a 88
(arrows)
Fig. 47b: Sectional view of Tibiotarsus bone of day old broiler chick showing growth cartilages
47b
(arrows)
Fig. 47c: Sectional view of Tarsometatarsus bone of day old broiler chick showing growth
47c
cartilages (arrows)
Sectional view of F, TT & TM bones o f 7 days old broiler chick showing growth
48 88
cartilages
Sectional view of F, TT & TM bones of 14 days old broiler chick showing growth
49 89
cartilages
Sectional view of right and left F, TT & TM bones of 21 days old broiler chicken
50 89
(male) showing growth cartilages (arrows
Sectional view of right and left F, TT & TM bones of 21 days old broiler chicken
51 90
(female) showing growth cartilages (arrows)
Sectional view of left F, TT & TM bones of 28 days old broiler chicken (male)
52 90
showing growth cartilages (arrows)
Sectional view of left F, TT & TM bones of 28 days old broiler chicken (female)
53 91
showing growth cartilages (arrows
Sectional view of left and right F, TT & TM bones of 35 days old broiler chicken
54 91
(male) showing growth cartilages (arrows)
Sectional view of left and right F, TT & TM bones of 35 days old broiler chicken
55 92
Sectional view of left and right femur (F) bones of 42 days old broiler chicken (male)
56 92
Sectional view of left and right TT bones of 42 days old broiler chicken (male)
57 92
Sectional view of left and right TM bones of 42 days old broiler chicken (male)
58 93
59 Sectional view of left and right Femur (F) bones of 42 days old broiler chicken 93
Sectional view of left and right TT bones of 42 days old broiler chicken (female)
60 93
Sectional view of left and right TM bones of 42 days old broiler chicken (female)
61 93
Sectional view of femur (F) bone of 42 days old broiler chicken showing proximal
62 94
growth cartilage with associated zones (arrows)
Sectional view of femur (F) bone of 42 days old broiler chicken showing distal growth
63 94
cartilage with associated zones (arrows
Sectional view of Tibiotarsus (TT) bone of 42 days old broiler chicken showing
64 94
proximal growth cartilage with associated zones (arrows)
Sectional view of Tibiotarsus (TT) bone of 42 days old broiler chicken showing distal
65 94
growth cartilage with associated zones (arrows
Sectional view of Tarsometatarsus (TM) bone of 42 days old broiler chicken showing
66 95
proximal growth cartilage with associated zones (arrows)
Sectional view of Tarsometatarsus (TM) bone of 42 days old broiler chicken showing
67 95
proximal growth cartilage with associated zones (arrows
Radiograph of left and right leg bones of day old broiler chick showing growth
68 95
cartilages (arrow)
Radiograph of left and right leg bones of 7 days old broiler chick showing growth
69 96
cartilages (arrow)
Radiograph of left and right leg bones of 14 days old broiler chick showing growth
70(a) 96
cartilages (arrow)
Radiograph of left and right leg bones of 21 days old male and female broiler chickens
70(b) 97
showing growth cartilages (arrow)
71 98
72 99
73 100
showing growth cartilages (arrow
Photomicrograph of growth cartilage in femur of day old broiler chick showing
74 101
different zones –a) RZ, b)PZ, c) PHZ, d) HZ, e) OZ. H & E, 10X
Photomicrograph of growth cartilage in femur of day old broiler chick showing
75 different zones a) RZ, b)PZ, c) PHZ, d) HZ, e) OZ and invading blood vessel (BV) in 101
OZ. H & E, 40X
Photomicrograph of compact bone in femur of day old broiler chick showing – a)
76 Periosteum, b) Endosteum, c) Bone marrow, d) Woven compact bone. 101
H & E, 10X
Photomicrograph of distal growth cartilage in femur of day old male broiler chick
77 101
showing Alcianophilia of matrix in different zones. AB - PAS, 10X
Photomicrograph of compact bone in femur of day old broiler chick showing – a)
78 Periosteum, b) Woven / trabecular compact bone with developing Haversian system. H 101
& E, 40X
Photomicrograph of proximal growth cartilage in femur of day old male broiler chick
79 showing collagen fibers (Green) around lacunae of different zones and in wall of the 101
blood vessel (BV) Masson’s trichrome, 40X
Photomicrograph of compact bone in femur of 7 days old broiler chicken showing – a)
80 Periosteum, b) Endosteum, c) Woven/trabecular bone with developing osteon H & E, 102
10X
Photomicrograph of proximal growth cartilage in femur of 7 days old broiler chick
81 102
showing different zones – HZ and OZ with trabecular bone. H & E, 40X
Photomicrograph of compact bone in femur of 14 days old broiler chick showing
82 collagen fibers (green) in matrix of developing osteons & lining of osteonal canals. 102
Masson’s trichrome, 40X
Photomicrograph of proximal growth cartilage in femur of 14 days old female broiler
83 102
chick showing collagen fibers (Green) in different zones Masson’s trichrome, 10X
Photomicrograph of compact bone in femur of 14 days old broiler chick showing
84 102
alcianophilia& weak PAS reaction in matrix . AB -PAS 10X
Photomicrograph of compact bone in femur of 14 days old broiler chickn showing – a)
85 Periosteum, b) Endosteum, c) Bone marrow, d) compact bone with developing osteons 102
H & E, 10X
Photomicrograph of compact bone in femur of 21 days old broiler chicken showing
86 103
compact bone with developing osteons and peripheral porous part. H & E, 10X
87 compact bone with developing osteons containing lacunae (L) around osteonal canals 103
(OC). H & E, 40X
88 103
collagen fibers (green) in matrix of developing osteons.. Masson’s trichrome, 10X
89 103
collagen fibers (green) in matrix of developing osteons. Masson’s trichrome, 40X
Photomicrograph of compact bone in femur of 28 days old male broiler chicken
90 showing compact bone with developing osteons with outer peripheral porous part. 103
H & E, 10X
Photomicrograph of distal growth cartilage in femur of day old broiler chick showing
91 103
different zones – a) RZ, b)PZ, c) PHZ, d) HZ, e) OZ. H & E, 10X
92 104
showing AB and PAS reaction of matrix. AB - PAS, 10X
93 104
: Photomicrograph of distal growth cartilage in femur of 28 days old broiler chicken
94 104
showing AB and PAS reaction of matrix of different zones H & E, 10X
95 showing normal definitive osteons, osteonal canals (OC) and perforating canals (PC). 104
H & E, 10X
96 showing normal definitive osteons, osteonal canals (OC), lacunae (L) and perforating 104
canals (PC). H & E, 40X
Photomicrograph of compact bone in femur of 35 days old female broiler chicken
97 104
Photomicrograph of distal growth cartilage in femur of 42 days old female broiler
98 105
chicken showing different zones – a) RZ, b)PZ, c) PHZ, d) HZ, e) OZ. H & E, 10X
Photomicrograph of growth cartilage in tibiotarsus of day old broiler chick showing
99 different zones –a) RZ, b)PZ, c) PHZ, d) HZ, e) OZ and blood vessels (BV). H & 105
E, 10X
Photomicrograph of compact bone in tibiotarsusof day old male broiler chick showing
100 porous compact bone with developing, immature osteons. 105
H & E, 10X
Photomicrograph of compact bone in tibiotarsus of 7 days old male broiler chick
101 showing porous compact bone with developing, immature osteons and larger osteonal 105
canals (OC). H & E, 40X
Photomicrograph of proximal growth cartilage in tibiotarsus of 7 days old broiler chick
102 showing distribution of sparse collagen fibers (green) in different zones. Masson’s 105
trichrome 10X
Photomicrograph of proximal growth cartilage in tibiotarsus of 7 days old broiler chick
103 105
Photomicrograph of distal growth cartilage in tibiotarsus of 14days old broiler chick
104 showing different zones –a) RZ, b)PZ, c) PHZ, d) HZ, e) OZ and blood vessels (BV). 106
H & E, 10X
Photomicrograph of compact bone in tibiotarsus of 14days old male broiler chick
105 showing compact bone with developing osteons with outer peripheral porous part, 106
marrow cavity (MC). H & E, 10X
Photomicrograph of compact bone in tibiotarsus of 14days old male broiler chick
106 showing compact bone with developing osteons containing lacunae (L) around 106
osteonal canals (OC). H & E, , 40X
Photomicrograph of compact bone in tibiotarsus of 14 days old broiler chick showing
107 106
Photomicrograph of compact bone in tibiotarsus of 14days old broiler chick showing
108 106
Photomicrograph of distal growth cartilage in tibiotarsus of 7 days old broiler chick
109 106
showing AB reactivity of matrix. AB - PAS, 10X
Photomicrograph of distal growth cartilage in tibiotarsus of 21 days old broiler chicken
110 showing distribution of sparse collagen fibers (green) in different zones. Masson’s 107
trichrome 10X
Photomicrograph of proximal growth cartilage in tibiotarsus of 21 day old broiler
111 107
chicken showing different zones and 2dary ossification centre(SOC) H & E, 10X
Photomicrograph of compact bone in tibiotarsus of 28 days old male broiler chicken
112 showing maturing, definitive osteons containing lacunae (L) around osteonal canals 107
(OC). H & E, 40X
113 107
showing compact bone with maturing, definitive osteons. H & E, 10X
114 107
showing normal definitive osteons, osteonal canals (OC), lacunae (L). H & E, 40X
Photomicrograph of distal growth cartilage in tibiotarsus of 42 days old female broiler
115 107
chicken showing different zones. H & E, 10X
Photomicrograph of compact bone in tarsometatarsus of day old male broiler chick
116 108
showing porous compact bone with developing, immature osteons. H & E, 10X
Photomicrograph of compact bone in tarsometatarsus of day old male broiler chick
117 showing collagen fibers (green) in matrix of developing osteons. Masson’s trichrome, 108
40X
Photomicrograph of compact bone in tarsometatarsus of 35 days old female broiler
118 108
chicken showing AB and PAS reaction of matrix. AB - PAS, 10X
Photomicrograph of proximal growth cartilage in tarso-metatarsus of 7 days old male
119 108
broiler chick showing different zones. H & E, 10X
Photomicrograph of proximal growth cartilage in tarso-metatarsus of 14days old male
120 broiler chick showing different zones - a) RZ, b)PZ, c) PHZ, d) HZ, e) OZ 108
H & E, 10X
121 108
broiler chick showing lower zones - a) HZ, b) OZH & E, 40X
Photomicrograph of distal growth cartilage in tarso-metatarsus of 21 days old male
122 109
broiler chicken showing different zones. H & E, 10X
123 Photomicrograph of proximal growth cartilage in tarso-metatarsus of 21 days old male 109
broiler chicken showing trabeculae of cartilage cells in HZ & OZ with Blood vessels
(BV) H & E, 40X
Photomicrograph of compact bone in tarsometatarsus of 21 days old female broiler
124 109
chicken showing AB and PAS reaction of matrix. AB - PAS, 10X
Photomicrograph of proximal growth cartilage in tarsometatarsus of 21 days old broiler
125 109
chicken showing AB reactivity of matrix. AB - PAS, 10X
Photomicrograph of compact bone in tarsometatarsus of 21 days old male broiler
126 chicken showing collagen fibers (green) in matrix of developing osteons. 109
127 chicken showing collagen fibers (green) in matrix of developing osteons. 109
128 110
broiler chicken showing different zones. H & E, 10X
129 broiler chicken showing different zones –a) RZ, b)PZ, c) PHZ, d) HZ, e) OZ. 110
H & E, 40X
130 110
chicken showing compact bone with mature, definitive osteons. H & E, 10X
131 chicken showing compact bone with mature, definitive osteons with Osteonal Canals 110
(OC) & lacunae (L). H & E, 40X
132 110
broiler chicken showing different zones (indistinct). H & E, 10X
133 chicken showing compact bone with mature, definitive osteons with Osteonal Canals 110
(OC), lacunae (L) & Cement Lines (CL). H & E, 40X
LIST OF TABLES
Table No Particulars Page No
1 Age related changes in gross biometrical parameters of femur in post- 61
hatch male broiler chicken
2 Age related changes in gross biometrical parameters of tibiotarsus in 62
post-hatch male broiler chicken
3 Age related changes in gross biometrical parameters of tarsometatarsus 63
in post-hatch male broiler chicken
4 Age related changes in gross biometrical parameters of femur in 64
post-hatch female broiler chicken
5 Age related changes in gross biometrical parameters of tibiotarsus in 65
post-hatch female broiler chicken
6 Age related changes in gross biometrical parameters of tarsometatarsus 66
in post-hatch female broiler chicken
7 Age related changes in histomorphometrical parameters of growth 67
cartilage of femur in post hatch male broiler chicken
cartilage of tibiotarsus in post hatch broiler chicken
cartilage of tarsometatarsus in post hatch male broiler chicken
cartilage of femur in post hatch female broiler chicken
cartilage of tibiotarsus in post hatch female broiler chicken
cartilage of tarsometatarsus in post hatch female broiler chicken
13 Age related changes in histomorphometrical parameters of compact 73
bone of femur, tibiotarsus and tarsometatarsus in post hatch male and
female broiler chicken
14 Age related changes in Serum Calcium and Phosphorus level (mg/ dl ) 74
in post hatch broiler chicken at different ages
15 Age related changes in Bone Calcium and Phosphorus level (%) in post 74
hatch male broiler chicken at different ages
ABBREVIATIONS USED
µm Micrometer
AB PAS Alcian Blue Periodic Acid Schiff
BFD Bone Formation Distal
BFP Bone Formation Proximal
BW Body weight
Ca Calcium
cm Centimetre
D Day
dl decilitre
et al. And others
F Femur
F Female
FFD Film Focus Distanca
Fig Figure
gm gram
H& E Haematoxylin &Eosin
HCl Hydrochloric Acid
HDD Hypertrophic and Degeneration Distal
HDP Hypertrophic and Degeneration Proximal
HZ Hypertrophic zone
i.e Id est (That is, that says )
KV KiloVolt
M Male
mA Mili Ampere
mg milligram
mm millimeter
n number
NS Non-Significant
OCPC O-Cresolpthalene
OZ Ossification Zone
P Phosphorus
PHD Prehypertrophic zone Distal
PHP Prehypertrophic zone Proximal
PHZ Prehypertrophic zone
ppm Parts per meter
PZ Proliferation Zone
PZD Proliferation Zone Distal
PZP Proliferation Zone Proximal
RZ Reserve Zone
RZD Reserve Zone Distal
RZP Reserve Zone Proximal
TD TibialDyschondroplasia
TM Tarsometatarsus
TPI Teeth per inch
TT Tibiotarsus
TTD Total thickness Distal
TTP Total thickness Proximal
TVCC Teaching Veterinary Clinical Complex
INTRODUCTION
A broiler is a type of domestic chicken specifically raised for meat production.

Modern commercial broilers grow much faster than other breeds of chicken. During
the last three decades or so, there has been remarkable growth in poultry farming in
India. Backyard poultry has now been transformed into poultry industry. It has
important contribution to the economy of rural and semi-urban India. Broiler farming
has become very popular among the small and marginal farmers as a means of their
livelihood. There is tremendous scope of growth in poultry sector in India. In recent
decades, there has been much genetic selection for faster growth and tender meat
production in broilers (Kirkwood et al., 1989; Williams et al., 2000; Manohar et al.,
2015). This has resulted in an imbalance between the developments of various body
systems, including increased demands being placed on skeletal integrity. The desired
genetic manipulation produces early muscle growth without a simultaneous increase
in the skeletal development, thus resulting in leg weakness and disorders (Manohar et
al., 2015). Moreover, there has been frequent incidence of skeletal problems in
general and leg problems in particular (Williams et al., 2000; Applegate and Lilburn,
2002).
It is well recognized that the long leg bones (femur, tibiotarsus and
tarsometatarsus) are principal weight bearing bones in poultry birds. Upper leg bones
(femur and tibiotarsus) are heavily muscled and play very important role in
locomotion and support. Legs play an important role in determining the degree of
mobility of the limbs in birds (Sreeranjini et al., 2013). Biometrical parameters of
these bones like length, width or diameter etc. are considered as good indicators of
musculoskeletal growth in broiler chicken. Over 98% of the variability in tibia and
femur length was correlated with the change in body weight and age in broilers
(Applegate and Lilburn, 2002). In adult poultry the length of the tarsometatarsal bone
was correlated to the body weight (Naldo et al., 2000, Williams et al., 2000). The
length and diameter of the leg bones were reported to be larger in the fast growing
broilers than in layer chickens (Breugelmans et al., 2007).
1
A typical long bone has two (proximal and distal) ends called epiphyses with
spongy bone, a compact shaft or diaphysis with medullary cavity within and in
growing stage, epiphyseal or growth plate cartilage intervening between respective
epiphysis and diaphysis. The transitional area of newly formed spongy bone joining
the growth plate cartilage and adjacent part of diaphysis is termed metaphysis. The
other features characterizing the appendicular long bones are presence of outer fibro-
cellular periosteum, inner lining of endosteum, nutrient foramen with blood vessels
and accompanying nerves, articular cartilage at the ends ( Goff, 2004; Akers and
Denbow, 2008; Dyce et al., 2009).
The solid compact bone varies in thickness along the shaft. It is composed of
mineralized interstitial substance (matrix) deposited in 3-20 concentric layers
(lamellae) around the central canals or Haversian canals. These canals are parallel to
the long axis of the bone and carry small blood vessels. Transversely oriented
perforating or Volkmann’s canals connect the blood vessels of periosteum with those
of Haversian canals. There are a number of uniformly spaced small cavities (lacunae)
containing osteocyte (s) within the matrix. The lacunae are interconnected by small
canaliculi to form a continuous network, thereby providing communicating channels
between the entrapped osteocytes within the lacunae. However, immature, growing
bone called woven bone is non lamellar, which is replaced subsequently in mature
stage by lamellar bone (Samuelson, 2007; Akers and Denbow, 2008; Dyce et al.,
2009).
The growth plate is a highly organized cartilage structure, i.e. a connective

tissue composed of cells (chondrocytes) embedded in a highly hydrated extracellular
matrix. It is entrapped between the epiphyseal and metaphyseal bone at the ends of
the long bones (Getty, 1975; Orth and Cook, 1994; Van der Eerden et al., 2003;
Valteau et al., 2011).
Growth plate cartilage is the key to the longitudinal growth (elongation) of

long bones. This specialized hyaline cartilage is present at physis (metaphysis) region,
i.e. junction of respective epiphysis and adjacent diaphysis (shaft) (Farquharson and
Jefferies, 2000; Villemure and Stokes, 2009). The growth plate consists of
chondrocytes (cartilage cells) embedded in abundant extracellular matrix.
Microscopically, five distinct zones are identified in the growth plate. There is highly
2
ordered differentiation of the chondrocytes into three zones: the reserve, proliferative
and hypertrophic zones, followed by zone of provisional calcification and resorption,
where chondrocytes are replaced by bone through the action of osteoblasts and
osteoclasts (Farquharson and Jefferies, 2000; Akers and Denbow, 2008; Villemure
and Stokes, 2009).
The resting chondrocytes initially proliferate slowly and then accelerate their
rate of proliferation along the longitudinal axis of the bone and form columns of cells.
Ultimately, the most distal cells of the columnar layer stop proliferating, exit the cell
cycle, and differentiate into hypertrophic chondrocytes. Proliferation and hypertrophy
of chondrocytes, along with extracellular matrix (ECM) synthesis, are the main
drivers of endochondral bone growth (Provot and Schipani, 2005; Mackie et al.,
2008). The hypertrophic chondrocytes mineralize their ECM and undergo apoptosis
or autophagy, and the region of hypertrophic cartilage is invaded by blood vessels
(Burdan et al., 2009; Srinivas et al., 2009). The capillary invasion mediated by
vascular endothelial growth factor (VEGF) is a key mechanism for the precise
coupling of chondrogenesis and osteogenesis and any changes in this balance might
induce pathological conditions (Ortega et al., 2010).
Eventually, the new cartilage production ceases and the growth plate is replaced
by spongy bone at maturity. This is called epiphyseal closure leaving behind only a
scar mark termed epiphyseal line. Age of epiphyseal closure varies with species,
breed, bone and even proximal or distal end (epiphysis) of a bone (Getty, 1975; Akers
and Denbow, 2008; Dyce et al., 2009). However, epiphyseal growth plates are not
reported in all limb bones except tibiotarsus and tarsometatarsus in birds (Naldo et al.,
2000 and Breugelmans et al., 2007). Even slight histoarchitectural difference of avian
growth plates is also reported (Howlett, 1979; Farquharson and Jefferies, 2000).
Any sort of disruption in normal development and growth of the epiphyseal

growth plate cartilage is sure to affect the normal bone growth, specifically elongation
of long bones. This may lead to deformity of the bones. Some of the common health
problems observed in broilers are skeletal disorders namely tibial dyschondroplasia
(TD), which is due to growth plate cartilage anomaly (Williams et al., 2000,
Farquharson and Jefferies, 2000, Applegate and Lilburn, 2002). Tibial
3
dyschondroplasia is a disease of rapid growth rate that occurs in many avian species
(Leach and Ornan, 2007).
The skeletal problems compromise the welfare of the birds, by reducing normal
growth, increasing mortality and downgrading carcass quality, thus affecting overall
performance of the broilers (Williams et al., 2000). There is high prevalence of such
musculoskeletal disorders at the peak of long bone growth (Naldo et al., 2000).
Abnormalities of broiler extremities cause important economical losses due to
decreased weight gain and increased mortality (Rowland, 1988). Tibial
dyschondroplasia (TD) is a prevalent skeletal abnormality associated with rapid
growth rate in many avian species that causes enormous economic losses and is an
animal welfare problem (Simsa et al., 2007; Imik et al., 2012; Genin et al., 2012).
An important factor in the diagnosis of skeletal abnormality is an understanding

of the normal anatomy and development of the long bones. Radiography is an
important tool used to document bone development and abnormality (Naldo et al.,
2000). Moreover, epiphyseal closure of long leg bones by radiography along with
other gross morphometrical parameters is still useful for age determination. Besides,
this non-invasive imaging technique may prove very useful for fast and reliable age
determination of frozen samples used for broiler meat export (Breugelmans et al.,
2007).
Calcium (Ca) is the most abundant mineral in the body of animals (McDonald
et al., 1995). About 90% of Ca is found in the bones (skeleton), where it combines
with phosphorus (P), the second most abundant mineral in bone, to form calcium
phosphate crystals or hydroxyapatite to provide structural strength and hardness to the
bones (Scott et al., 1982; Goff, 2004). Rest 2% Ca is primarily found in extracellular
fluids. About 50% blood plasma Ca is bound to plasma proteins and organic as well
as inorganic elements of blood (Goff, 2004).
Phosphorus, the other major constituent of bone, is an essential component of

almost all metabolic processes (McDonald et al., 1995). The phosphorus content of
the body is somewhat less than that of the calcium content and 80 to 85% of the total
body phosphorus is found in the bones. Skeletal strength and hardness are dependent
on normal blood calcium and phosphorus level. Bone mineralization occurs when
4
plasma Ca and P concentrations are normal. Under stable conditions the rate of bone
resorption is equal to the rate of bone formation so that the mineral content of the
skeleton remains unchanged (Goff, 2004).
Generation of the base line data on morphometry of leg bones and associated
growth cartilage can help address different skeletal deformities (specifically of long
leg bones) like TD, ricket, fracture etc. Besides, determination of age in broiler from
radiographic analysis of the growth plates and its closure along with morphometrical
data on the leg bones could be very useful and authentic. Detailed gross
morphometrical, radiographical, histomorphological and biochemical data on the long
leg bones in broiler chickens are scarce. Literature on these data in Vencob broilers is
lacking. Changes in conformation and ever - increasing body weight on a physically
less mature skeleton poses the greatest challenge to prevent skeletal anomalies in
modern broilers and in turn, to minimize the economic loss due to leg bone deformity
in commercial broiler farms. In the context of the aforesaid facts, the present study
has been planned with the following objectives.
Objectives:-
i) To record the age-specific gross morphometrical data on long leg bones in

broiler chickens,
ii) To analyze radiographically the age - related changes in long leg bones and
associated epiphyseal or growth plate cartilages in broiler birds,
iii) To elucidate the histoarchitectural changes with age in these bones and
associated growth plate cartilages in broilers, and
iv) To record age specific changes in serum calcium and phosphorus level
(concentration) in broilers and to correlate with these mineral contents in the
bones.
5
REVIEW OF LITERATURE
2.1 Skeletal or Leg Problems (Deformities) and Animal Welfare in Broiler

Chickens
Wise (1975) reviewed the skeletal abnormalities in table poultry. The rapid
growth rates achieved by modern table poultry appear to have resulted in an increase
in conditions which are often grouped under the term "leg weakness". Only skeletal
abnormalities of economic consequence in table poultry were discussed and the
literature pertaining to them was reviewed. The disorders include osteomyelitis,
rickets, chondrodystrophy, tibial dyschondroplasia, spondylolisthesis and twisted leg.
Some of the most common health problems observed in broilers are skeletal
problems (Kestin et al., 1992). It is evident from the high incidence of leg problems
observed among modern high-yield broiler chickens. The modern high-yield broiler
chicken has been selected very successfully to reduce the time taken to reach target
body weight and feed efficiency. However, there have been several indirect
consequences of the selection programs with evidence of adverse effects on the
skeletal systems in broilers by the fast growth at a given age (Hester, 1994; Lilburn,
1994).
Skeletal deformities can be caused in a variety of ways. Nutritional

deficiencies can result in skeletal disease in all birds. Rapidly growing birds have a
higher requirement for specific nutrients, and many skeletal defects in broiler and
roaster chickens are rare or absent in slower growing strains (Havenstein et al., 1994).
Mechanically induced or trauma-associated problems are also much more frequent in
fast-growing broilers. These problems may have more to do with immaturity and
weight than rapid growth because tissue becomes stronger and more resilient with
age. This age-related effect is particularly true of bone, tendon, and ligament.
6
Bone abnormalities, causing major leg problems in meat-type chickens, are
thought to be related to a chicken’s growth potential (Thorp and Waddington, 1997).
They opined that cortical bones of fast-growing (FG) broilers are highly porous,
which may easily lead to bone deformities.
Julian (1998) gave a detailed overview on rapid growth problems: ascites and
skeletal deformities in broilers. Over the last 4 decades, genetic selection for rapid
growth and improved feed efficiency has been very effective in meat-type poultry.
Combined with changes in the feed that have increased both the nutritional and
physical density to encourage a high nutrient intake, growth rate has more than
doubled. The effect of genetic selection for high muscle to bone ratio and high calorie
intake of a ration that supplies all nutritional requirements causes significant mortality
from cardiovascular disease. Rapid growth induced by high nutrient intake alone can
cause severe lameness, bone defects, and deformity, as these problems are seen in
animals that have not been selected for rapid growth: dogs, horses, pigs, ratites and
wild birds kept in zoological gardens. In meat-type poultry, growth-related disease
can be reduced or eliminated by reducing feed intake without affecting final body
weight.
The advances made in the areas of genetics and nutrition during this century
have resulted in improved growth rates for livestock. However, one drawback has
been the increased prevalence of long bone growth problems, such as rickets, avian
tibial dyschondroplasia, and osteochondrosis (Orth, 1999).
Acute and chronic pain, and mortality resulting from osteoporotic fractures
increase economic loss (Cook, 2000) and pose serious animal welfare concerns
(Webster, 2004) by increased mortality resulting from skeletal disease.
Leg weakness is one of the major welfare problems in rapid growing meat-
type chickens (Europe Commission, 2000). Leg problems are one of the most
common causes of culling, early and late mortality in broiler flocks, and are
considered to be one of the more serious welfare problems facing the broiler industry
(Mench, 2004).
7
Simsa et al. (2007) reported induction of tibial dyschondroplasia in turkeys by
tetramethylthiuram disulfide (thiram). Tibial dyschondroplasia (TD) is a prevalent
skeletal abnormality associated with rapid growth rate in many avian species; it
causes enormous economic losses and is an animal welfare problem.
Selection in development of commercial chicken meat hybrids focused on

increase in growth rate, feed efficiency and meatiness. Increasing growth rate and
slaughter yield resulted in increased incidence of abnormalities of extremities
particularly tibia and other loaded components of the skeleton. The most frequent
abnormalities of legs observed in rapidly growing birds included perosis, rachitis,
osteoporosis and tibial dyschondroplasia. Changes in mechanical properties of bones
developing at their disorders can further worselt their condition and contribute to
lameness (Baranova et al., 2008). Decreased elasticity of bones may result in damage
to growth plate and subsequent morphological changes and locomotion disorder.
Shim (2010) reported leg problems of modern broilers as affected by
incubation temperature, fluoride and fast growth. Fast growing broiler chickens are
especially susceptible to bone abnormalities, causing major problems for broiler
producers. No main effects or interactions between incubation temperature or time
and bone abnormalities were detected. Fluoride (F) had varying degrees of beneficial
effects on bone mineralization and strength, despite its toxic effects on growth and leg
disorders. Even low levels of F had the potential to create measurable effects.
Leg angulations, described in the twisted legs syndrome as valgus and bilateral
or unilateral varus, were investigated in 2 subpopulations of mixed-sex Arkansas
random bred broilers (Shim et al., 2012). Mean lesion scores of left and right valgus
and tibial dyschondroplasia (0.40, 0.38, and 0.06) of fast-growing broilers were higher
than those (0.26, 0.28, and 0.02) of slow-growing broilers (P = 0.0002, 0.0037, and
0.0269), respectively. Growth rate was negatively associated with the twisted legs
syndrome and a bone abnormality (TD) in this random bred population.
Many of the skeletal disorders in chickens are common and easy to observe,
but very little is known about tendons, cartilage, synovial fluid, neural innervations,
and biomechanics of movement in broilers and other avian species. This is one of the
reasons that determining definite solutions to leg problems in poultry has been so
8
difficult in the past 30 years. Breeder management and nutrition, and adequate
incubation are important to diminish the prevalence of locomotion issues and leg
disorders in broiler flocks (Oveido – Rondon and Wineland, 2012).
Comparative clinical and morphological studies on the incidence of tibial

dyschondroplasia were conducted by Dinev et al. (2012) in commercial broiler
chickens. Tibial dyschondroplasia (TD) was the most common skeletal anomaly
associated with fast growth in broiler and turkey lines and often resulted in the
occurrence of bone deformation and lameness. It was a fairly common cartilage defect
associated with the prehypertrophic cartilage of the growth plate being retained in the
metaphysis. The number of observed macroscopic TD lesions was usually higher in
males than in females. A comparatively high prevalence of TD lesion-associated
deformities was found in the proximal tibial metaphysis.
Genin et al. (2012) experimented on thiram-induced tibial dyschondroplasia.

Tibial dyschondroplasia (TD) was a skeletal abnormality that can cause economic
losses and animal welfare concerns. Thiram-induced TD was characterized by
enlarged, unvascularized growth plates, low levels of the vascular endothelial growth
factor receptor Flk-1, abnormal chondrocyte differentiation, and lameness.
The selection for growth and carcass traits in poultry meat species has
contributed to increased interest in understanding and characterizing skeletal growth
as the birds struggle to balance skeletal development with increased BW and muscle
mass. A comparative study was carried out on the physical characteristics and
mineralization of the tibia and femur from commercial Pekin ducks (Van Wyhe et al.,
2012).
Robison et al. (2015) reported duck gait and its relationship to hip angle, bone
ash, bone density, and morphology. The rapid growth meat birds, including ducks,
undergo requires skeletal integrity; however, fast growth may not be conducive to
adequate bone structure. A relationship likely exists between skeletal changes and
duck mobility. Reduced mobility in meat ducks may have impacts on welfare and
production.
9
Manohar et al. (2015) gave an overview on leg weakness in commercial
broiler chicken. According to them, success in commercial broiler chicken enterprise
depends to a greater extent on minimizing the incidence of disease occurrence and
elimination of conditions producing stress to the birds which may result in economic
losses. Diseases of the locomotor system are gaining economic significance in poultry
industry. Broiler Chicken has a capacity to grow very fast and produce tender meat.
They are being developed for high growth rate through genetic selection. This
produces early muscle growth without a simultaneous increase in the skeletal
development resulting in leg weakness and disorders. A normal gait is an integrated
function of the nervous, muscular and skeletal systems. A failure in any of the above
component clinically results in leg weakness or lameness and this, in turn, causes
increased culling rate or mortality in broilers.
2.2 Gross morphometrical study
Females usually have a lighter skeleton than males, even in avian domestic
species (Rose et al., 1996). Male and female broilers were compared for the growth of
cortical bone. Morphology, histomorphometry, composition and biomechanical
properties of the tibiotarsi were analysed in both sexes at 1, 12, 26 and 42 days of age.
The quantity of bone tissue of the tibiotarsus (weight, volume, diameter of the
diaphysis, area of the cortex) was smaller in females. The tibia became significantly
lighter in females from 26 d of age. Differences in tibia length and volume became
significant at 42 d of age only, while cross-sections of the diaphysis were smaller in
females from the hatching, leading to thinner bones in females. They opined that in
female broilers, the thinness of bone diaphysis is counter-balanced by modifications
in the composition of the matrix and in the porosity of the cortex, leading to equal
biomechanical characteristics of tibiotarsi in both sexes.
Male chickens from lines divergently selected for fast (FGL) and slow (SGL)
growth were compared for the growth of cortical bone (Leterrier and Constantin,
1999). Morphology, histomorphometric, compositional and biomechanical properties
of the tibiotarsi were analysed in both lines at 1, 8, 15 and 22 d of age. Tibial
morphology (length, volume, cross-section and diameters) was similar in FGL and
10
SGL chickens when compared at an equal body weight. Cortical area was also similar
in both lines at an equal body weight but cortex porosity was higher in FGL. Tibial
mineral density (ash:volume) was higher in FGL than in SGL at hatching and at 8 d of
age. The very slow growth rate in SGL did not modify bone size when chickens were
compared at equal body weight. Bone quality was modified in various ways: in SGL
bone structure was strengthened by a lower porosity of the cortex while bone tissue
was less mineralized up to 22 d of age. In both lines, cortical growth was slower than
in commercial cross-breeds and bone quality (structure and composition) was
improved compared to broilers.
Naldo et al. (2000) determined the growth rate of long bones in different types
of bustard chicks. The growth rates of the tarsometatarsus and tibiotarsus in the
bustard species investigated were similar to those in domestic fowl (Gallus
domesticus) and some long-legged avian species.
Williams et al. (2000) studied skeletal development in the meat-type chicken

up to day 39. A tibiotarsus from each bird was X-rayed and its dimensions and
estimated resistance to bending were determined. The selected strain grew faster and
heavier than the control strain.The length (mm) and width(mm) of tibiotarsus in
control birds on day 4,11,18,25,32 and 39 were (34, 48, 63, 76, 89, 94) and (1.6, 2.6,
3.8, 4.7, 5.1, 5.3) respectively.
Rutten et al. (2002) studied bone development and activity in meat type
Hubbard chickens in response to reduced weight-load on legs. Different bone
morphometrical parameters were compared. According to them, body weight was
113.8gm on day 5 and 642.3 gm on day 19 in control birds. Other data on control
birds on day 19 were as follows. The weight of femur bones was 3.8 gm and that of
tibia was 5.3–5.4 gm. Length of these two bones were 48 mm and 67.1mm
respectively. The diameter of tibia varied from 5.1 -5.8 mm and the width of the tibial
growth plate was 4.92mm. Bone stiffness, composition and histological parameters
were not significantly different in the 3 groups. They concluded that a reduction in
load-bearing on the legs of young chicks enhanced locomotor activity and
longitudinal growth of leg bones. Bone quality was not affected, probably due to the
contradictory effects of increased exercise and reduced weight.
11
Reich et al. (2005) studied the effect of weight loading on young fast growing
chicks. The mechanical stimuli resulting from weight loading play an important role
in mature bone remodelling. The increased load reduced the length and diameter of
the long bones.
Hassanabadi et al. (2007) evaluated the effect of microbial phytase and dietary
calcium and phosphorus on productive (growth) traits, as well as some blood and
bone mineral parameters in male broiler chickens. Phytase addition increased body
weight (on days 21 and 42) in treated birds as compared to control birds (560gm and
2114 gm respectively).
The age-related degree of ossification of the sternum and the long leg bones
(femur, tibiotarsus and tarsometatarsus) of chickens was determined both
macroscopically and radiographically by Breugelmans et al. (2007) in ten broilers
ranging from 7 to 14 weeks in age, and in four laying hens of different ages and
breeds. The dimensions (length and width) of femur, tbiotarsus and tarsometatarsus in
broilers aged 42 days were (8.75±0.19, 1.05±0.06), (11.5±0.84, 0.85) and (8.85±0.07,
1.15±0.07) cm respectively. Similar data for laying hens aged 8 weeks were
(8.7±0.34, 0.8±0.08), (12.1±0.35, 0.7±0.16) and (8.1±0.36, 0.8±0.09) cm respectively.
The length of the long bones of the shank varied with age but was also breed
dependent. In contrast, the diaphyseal diameter of the long leg bones and the
thickness of the articular cartilage covering the femoral head, femoral condyles and
proximal tibiotarsal surface varied little in 7 to 14-week-old broilers.
Simsa et al. (2007) compared body growth in tibial dyschondroplasia (TD)

affected broilers with control birds. The average BW (body weight) at 10th day was
241.1 ± 1.97 g for the control group and 180.3 ± 3.42 g for the 50-mg/kg of TD
group.
Coto et al. (2008) reported effects of dietary levels of calcium and nonphytate
phosphorus in broiler starter diets on live performance, bone development and growth
plate conditions in male chicks fed a corn-based diet. Ca: NPP ratios and Ca levels
similar or higher than NRC recommendations appeared necessary for adequate bird
12
performance. Phytase supplementation improved FCR, whereas the addition of 25-
OH to diets already containing 5500 IU kg-1 of cholecalciferol had a negative effect
on FCR due to a possible hypercalcemia condition. Bone development was improved
by increasing NPP and Ca levels. Body weight was significantly affected only by the
dietary calcium level. There was no significant effect of the dietary phosphorus level
and phytase supplementation on body weight.
Shim (2010) conducted an experiment to test the hypothesis that fast growing
(FG) broiler chicks were susceptible to bone abnormalities, causing major leg
problems. Tibia weights (15.3550±2.28) of FG (fast growing) were also greater than
those of SG (slow growing) broilers (11.2318±1.81; P < 0.0001). Shank length
(81.5003±4.71) and tibia length (104.3360±4.45) of FG broilers were longer than
those of SG broilers (71.8797±4.66 and 95.9749±4.85; P < 0.0001). Shank diameter
(11.5933±1.60) and tibia diameter (8.1987±0.62) of FG broilers were wider than
those of SG broilers (9.4526±1.74, 6.8151±0.58; P < 0.0001).
For the first time computed tomography was used by Charuta et al. (2013) to
analyze densitometric and geometric parameters in proximal metaphyses and the mid-
diaphyses of tibiotarsal bones in Ross 308 broiler chickens in post-hatching
development as influenced by age and sex. The body weight (gm) in male and female
birds at 2wks, 4 wks and 6 wks were (395, 339), ( 1580, 1341) and ( 2550, 2263)
respectively. The weight (gm) of tibiotarsus bones in male and female birds of the
said age groups recorded as (4, 3.4), (13.3,10) and ( 20.9, 203) respectively. The
decreasing relative bone weight from 1.03% to 0.79%, led to deformities and breaks
of the tibiae, which may have a negative effect on the productivity of the broiler
chickens flock.
Paxton et al. (2014) studied anatomical and biomechanical traits of broiler

chickens across ontogeny. In broiler chickens, genetic success for desired production
traits is often shadowed by welfare concerns related to musculoskeletal health. Whole
limb morphology was not uniform relative to body size, with broilers obtaining large
thighs and feet between four and six weeks of age. This implied that the energetic cost
of swinging the limbs was markedly increased across this growth period, perhaps
contributing to reduced activity levels. Hind limb bone length did not change during
13
this period, which may be advantageous for increased stability despite the increased
energetic costs.
Tibial bone morphometry, bone and serum mineral profile and carcass
characters in broiler chicken were studied in Cobb 400 broiler chicken from day 1 to
28 (Vijayakumar and Balakrishnan, 2014). There was no significant variation in the
parameters studied. The tibial morphometrical parameters (weight, length and width)
were recorded as percentage (0.23,7.24 and 1.04 respectively) of body weight
(968.00gm) at 4 weeks of age.
ash, bone density, and morphology. This study examined the relationships among gait
score, bone parameters, and hip angle. Body weight increased with age, but within an
age, body weight decreased as walking ability became worse (P < 0.01). There was
linear increase in tibia and femur bone width (tibia- 4.64 to 7.16mm, femur – 4.89 to
6.90 mm) and length (tibia- 7.83 to 11.24 cm, femur – 4.88 to 6.90 cm) the ducks
aged (P < 0.01).
2.3 Radiographical study
Hogg (1980) re-investigated the centres of ossification in the avian skeleton at

and after hatching up to 182 days. He employed radiography (X-rays) as well as
alizarin S staining. The numerous centres already present at hatching were identified.
Those first appearing around the time of hatching were for the uncinate processes and
the phalanx of digit IV. The centres appearing after hatching were those for
metacarpal II, four carpal centres, the orbitosphenoid, rostal and caudal
basibranchials, epibranchials, entoglossal, proximal tibial centre, patella and tarsal
sesamoid. Some evidence of slight variation in sequence of appearance was detected.
The proximal tibial centre was the only true secondary centre in the long bones and
corresponded to the traction epiphysis of this region in the mammal. The only
sesamoidean centre found, other than the patella and the tarsal sesamoid, was in the
carpal region and was termed the dorsal carpal sesamoid. Some of the many
controversies existing in the previous literature have been assessed in the light of the
findings of this study.
14
Sadler (1991) studied the fusion of proximal epiphysis in tibiotarsus and
development of spur core and its fusion with tarsometatarsus in domestic fowl with
the help of radiography. The male tarsometatarsus stopped increasing in length at an
average of 142 days (about 5 months). The same bone in females stopped increasing
in length at an average of 110 days.
A serial radiographic study was conducted by Naldo et al. (2000) on four

types of bustard chicks to determine the growth rate of long bones and to establish
radiographic standards for assessing skeletal maturity. Maturation of long bones
occurred earlier in houbara bustards compared with rufous-crested, white-bellied and
kori bustards.Ossification at proximal tarsometatarsus was completed earlier in
females.
Williams et al. (2000) investigated skeletal development in meat –type

chicken. They used X-rays to measure different dimensions (length and width of the
bone, width of marrow cavity etc.) of tibiotarsus bones in two lines of Ross broiler
birds and compared the data.
Breugelmans et al. (2007) determined the age-related degree of ossification of

the sternum and the long leg bones (femur, tibiotarsus and tarsometatarsus) of
chickens both macroscopically and radiographically. The birds included ten broilers
ranging from 7 to 14 weeks in age, and four laying hens of different ages and breeds.
The relatively slow ossification rate of the sternum interfered with accurate age
determination. Morphometric analysis of tibiotarsus was performed on radiographs
and the data were compared with the gross morphometric measurements.The length of
the long bones of the shank varied with age but was also breed dependent. A femoral
and tarsometatarsal length of ≥ 10 cm and a tibiotarsal length of > 14 cm indicate that
the animals are at least 10 weeks old. The anatomical and radiographic disappearance
of the tibiotarsal and tarsometatarsal growth plates suggested that the broilers were
slaughtered at the age of 14 weeks or older. The closure of the growth plates in the
tibiotarsus and the tarsometatarsus was the most reliable criterion for age
determination in broilers.
15
Charuta et al. (2011) evaluated computer-generated radiological imagery of
the structure of the spongious substance in the postnatal development of the tibiotarsal
bones of the Peking domestic duck (Anas platyrhynchos var. domestica) aged 4 to 8
wks. The Trabecula program used in the study identified a map of radiological
trabeculae and calculated the number, average volume, density, and width of
trabeculae. The number of trabeculae differed significantly (P ≤ 0.05) variant on age,
sex, and a unique fragment of the studied bone. Six-week-old hens whose TT bones
were most often exposed to deformities and fractures possessed attenuated bone mass.
Results showed a unique linear relationship between BW and the volume of
trabeculae. Indeed, the larger the BW, the more numerous the trabeculae observed. No
significant correlation was determined between the BW and the number of recognized
trabeculae nor their density and width. A small number of trabeculae and the lowered
density may be the cause of fractures and deformities of the TT bones of the domestic
duck.
2.4 Histomorphological study

2.4.1 Normal Growth plate (cartilage) structure
Similar to the situation in fetal mammals, primary ossification centers arise

prenatally in the middle part of the diaphyseal cartilage of the avian long bones (Fell,
1925). However, unlike in mammals, no secondary ossification centers are formed in
the epiphyses of the appendicular skeleton of birds. Wise (1975) opined that the fowl
typically lacks the presence of bony epiphyseal plates, except in few long bones.
According to Church and Johnson (1964), Hogg (1980), and Naldo et al. (1998),
epiphyseal growth plates were lacking in avian bones, except at the distal end of the
tibiotarsus and the proximal end of the tarsometatarsal bone, both of which had a
growth plate formed by the fusion lines between tarsal bones and the tibia or the
metatarsal bones, respectively. Additionally, a secondary ossification centre was
reported in the craniodorsal part of the tibia corresponding to the tibial crest center of
mammals.
16
Longitudinal growth occurs in the growth plate that links the epiphyseal and
the metaphyseal regions within bone (Orth and Cook, 1994). Longitudinal growth
takes place by a process called endochondral ossification, in which a cartilaginous
scaffold is replaced by bone in a coordinated fashion (Van der Eerden et al., 2003).
Elongation of bones results from a synchronized spatio-temporal process involving
chondrocyte differentiation within the three histological zones of the growth plate: the
reserve, proliferative and hypertrophic zones (Valteau et al., 2011). The growth plate
cartilage is divided into horizontal zones of chondrocytes at different stages of
differentiation (Van der Eerden et al., 2003).There are three principal zones: the
reserve, proliferative, and hypertrophic zones (Howlett, 1979; Orth and Cook, 1994).
The main physiological events of endochondral ossification are chondrocyte

proliferation, extracellular matrix production, chondrocyte hypertrophy, matrix
calcification, vascular invasion, matrix degradation, and primary bone formation. The
fibrous peripheral structure is composed of the ossification groove, which provides
chondrocytes for latitudinal expansion of the growth plate, and the perichondrial ring,
which provides mechanical support for the tenuous chondro-osseous junction
(Valteau et al.,2011).
Each zone differs in chondrocyte size and arrangement as well as extracellular

matrix composition and plays a specific role in the bone growth. Bone growth is a
complex interplay between rapid cell division in the proliferative zone (where
chondrocytes are flat and arranged into longitudinal columns), substantial cell
enlargement in the hypertrophic zone (where chondrocytes begin their terminal
differentiation) and extracellular matrix synthesis (Hunziker and Schenk, 1989; Breur
et al., 1997). Wilsman et al. (1996) ranked the contribution of these parameters to
bone growth as follows: (1) the expansion of chondrocytes in the hypertrophic zone,
(2) the synthesis of extracellular matrix in both proliferative and hypertrophic zones
and (3) the cellular division in the proliferative zone.
At the epiphyseal end of the growth plate, the reserve zone lies either adjacent
to the secondary ossification center in the epiphysis or, as in the case of some avian
bones with no secondary ossification center (i.e., the proximal end of the femur),
directly underneath the articular cartilage. This reserve zone, also called germinal or
stem cell zone, contains the resting chondrocytes. These cells have been shown to be
17
crucial for orientation of the underlying columns of chondrocytes and therefore
unidirectional bone growth, probably by secreting a growth plate-orienting factor
(Abad et al., 2002). The chondrocytes in this zone are approximately the same size as
the proliferating chondrocytes but have no real organization in the surrounding matrix
(Brighton et al., 1973). The cells proliferate sporadically at best (Kember,1960). The
vascularization of this zone is poor. Apparently the small arterial branches from the
epiphyseal artery pass through this zone in cartilage canals. This zone contains the
lowest amount of ash and proteoglycan. However, it has the highest concentration of
collagen fibrils, composed primarily of type II collagen. The fibrils are randomly
distributed and oriented. In the avian reserve zone, the intercellular matrix is
composed of finely beaded strands that are lightly packed and often swirl around the
chondrocytes (Howlett, 1979). The exact function of this zone is not known. The cells
contain more lipid bodies and vacuoles than do chondrocytes found in other zones,
which suggests that these cells store materials such as lipids for later nutritional
requirement.
Moving distally from the reserve to the proliferative zone, spherical resting
chondrocytes give way to flattened chondrocytes. Upon some unknown trigger, the
stem cells enter into the proliferating zone. In this matrix-rich zone, the flattened
chondrocytes undergo cell divisions in a longitudinal direction and organize in a
typical column wise orientation. However, in faster growing species of domestic
animals, such as pigs, turkeys, and broiler chickens, columnar organization is poorer
than in their slowly growing counterparts. For the most part, only these cells
proliferate in the growth plate (Kember, 1960). The germinal cell layers appear to be
at the top of the proliferative zone rather than in the reserve zone. The proliferating
cells synthesize and have the highest rate of sulfate incorporation into proteins, such
as proteoglycans and hexosamine. It has the highest concentrations of sodium
chloride, potassium, and inorganic pyrophosphate, an inhibitor of calcification. The
proliferative zone has the highest percentage of extracellular matrix volume, which is
essential for the structure of the growth plate (Abad et al., 2002).The major
proteoglycan in the matrix is a large aggregating proteoglycan (aggrecan). They are
also hydrophilic molecules that help maintain structural integrity during mechanical
loading and also regulate cation concentration in the rnatrix. These macromolecules
are found between the columns of proliferating chondrocytes.
18
At a given moment, either by a finite number of cell divisions or by changes in
exposure to a local growth factor (for example PTHrP) (Gafni et al., 2001, Minina et
al., 2002), flattened proliferating chondrocytes lose their capacity to divide and start
to differentiate and become prehypertrophic, coinciding with an increase in size. Their
location is called the transition zone. Collagen II synthesis occurs in the proliferative
zone, whereas synthesis of type X collagen, a marker for chondrocyte hypertrophy, is
initiated in the prehypertrophic zone (Chen et al., 1993).
Then these cells further progress in the differentiation pathway to become

hypertrophic chondrocytes, which have a round (spherical) appearance with increased
volume and secrete large amounts of matrix proteins. The proliferative zone thus
becomes finally the hypertrophic zone. The zone begins where the chondrocytes begin
to change shape and ends at cartilage-bone junction. The average cells have increased
to over five times their original size at the bottom of the zone. The environment
surrounding the cells becomes increasingly anaerobic and void of a nutrient supply.
Increased collagenase activity in the hypertrophic zone enables the cells to expand by
degrading the pericellular matrix and to facilitate vascular penetration by breaking
down collagen in the extracellular matrix. This stage is characterized by an increase
in intracellular calcium concentration. This is essential for the production of matrix
vesicles, which are small membrane-enclosed particles released from chondrocytes
(Wang and Kirsch,2002, Anderson, 2003).
The matrix vesicles contain large amounts of annexins, which mediate calcium
uptake into the matrix vesicles (Kirsch et al., 2000, Wang and Kirsch, 2003). The
vesicles secrete calcium-phosphates, hydroxyapatite, and matrix metalloproteinases
(MMPs), resulting in mineralization of the vesicles and their surrounding matrix.
Chondroitin sulfate has been reported to be an important molecule which, as an ion-
exchanger, controls calcification of the cartilage matrix (Hunter, 1991).The
mineralization process, in combination with low oxygen tension, attracts blood
vessels from the underlying primary spongiosum (Schipani et al., 2001). Calcification
(mineralization) of the extracellular matrix occurs in the lower hypertrophic zone,
approximately 100 pm from the tip of the most advanced metaphyseal blood vessels
(Howlett,1979). In the chick growth plate cartilage, mineralization is apparently
controlled by chondrocytes closest to the metaphyseal vessels, because they have a
19
relatively high level of oxidative metabolism as compared with the majority of
hypertrophied cells.
Subsequently, the mineralized chondrocytes at the bottom of the zone,

undergo programmed cell death (apoptosis), leaving a scaffold for new bone
formation. The apoptotic process is, among other factors, regulated by elevated
intracellular calcium levels (leading to activation of proteases, lipases, and nucleases),
retinoic acids, and vitamin D (Waring and Beaver, 1996, Escargueil-Blanc et al.,
1997, Srinivasan et al., 1998, Boyan et al., 2001). In either case, vessels penetrate
through these cells, led by phagocytic (i.e., chondroclasts or macrophage) and
endothelial cells, from the metaphyseal region into the cartilage (Farquharson et al.,
1992). The growth plate receives its vascular supply from 2 sources of blood vessels:
proximal and metaphyseal (Hunt et al., 1979; Howlett et al., 1984). Because the 2
blood supplies do not meet, this creates an avascular zone referred to as the transition
zone, located between the proliferative and prehypertrophic zones. The fact that all
cartilages are poorly vascularized leads to some interesting challenges in terms of
nutrient and oxygen supply.
Longitudinal and transverse septae, which keep the chondrocytes in a

columnwise orientation, are resorbed by chondroclasts or osteoclasts from the
underlying primary spongiosum (Lewinson and Silbermann, 1992, Vu et al., 1998).
Following behind the endothelial cells, osteoblasts enter the area to lay down new
metaphyseal trabecular bone on top of the calcified cartilage (Lewinson and
Silbermann, 1992).The matrix thus eventually is resorbed and replaced by trabecular
bone. The combination of chondrocyte proliferation, the enlargement of maturing
chondrocytes in the hypertrophic zone, and the production of ECM are the major
contributors to longitudinal bone growth.
Barreto and Wilsman (1994) demonstrated the relationship between the

hypertrophic chondrocyte volume and growth rates in avian growth plates. A positive
linear relationship between the mean hypertrophic chondrocyte volume and the rate of
bone elongation in birds was tested like that in growing mammals. The control of
chondrocytic volume in the growth plate was a major determinant in controlling bone
elongation in mammals. A scheme of fluorochrome labelling was devised to enable
direct measurement of bone elongation per unit time. Growth plate cartilage was fixed
20
in the presence of ruthenium hexamine trichloride and embedded in Epon araldite.
Regression analysis revealed a positive linear relationship between the two
parameters, rate of bone elongation and mean hypertrophic cell volume in both
species.
Nakano and Sim (1995) studied growth dependent changes in the chemical
composition of proximal tibial articular cartilage and growth plate of broiler chickens
on days 1, 8, 14, 22, 29, 36 and 43. In the articular cartilage, the contents of dry
matter, collagen measured as hydroxyproline and keratan sulfate increased (P < 0'05),
and the content of sialic acid decreased (P < 0.05) as age advanced. The content of
chondroitin sulfate measured as uronic acid and the proportion of hyaluronic acid in
total glycosaminoglycan (GAG) were relatively constant (P > 0.05) among the age
groups. In the growth plate, the contents of chondroitin sulfate and keratan sulfate
increased (P < 0.05) with age, while the contents of dry matter, sialic acid, and
collagen were similar among the age groups. The proportion of hyaluronic acid in
total GAG was greater in l-d-old chickens than in the remaining age groups. It
decreased at the age of 8 d, and remained relatively constant thereafter.
Van der Eerden et al. (2003) reviewed systemic and local regulation of the
growth plate. The growth plate is the final target organ for longitudinal growth and
results from chondrocyte proliferation and differentiation. At the end of puberty,
growth plates fuse, thereby ceasing longitudinal growth. It has been recognized that
receptors for many hormones such as estrogen, GH, and glucocorticoids are present in
or on growth plate chondrocytes, suggesting that these hormones may influence
processes in the growth plate directly. Moreover, many growth factors, i.e., IGF-I,
Indian hedgehog, PTHrP, fibroblast growth factors, bone morphogenetic proteins, and
vascular endothelial growth factor, are now considered as crucial regulators of
chondrocyte proliferation and differentiation.
Reich et al. (2005) studied the effect of weight loading on young fast growing
chicks. The mechanical stimuli resulting from weight loading play an important role
in mature bone remodelling. The average width of the bag-loaded group's growth
plates was 75 ± 4% that of the controls, and the plates showed increased
mineralization. Moreover, weight loading enhanced the penetration of blood vessels
into the growth plates. The present study addressed the effect of mild mechanical
21
loading on bone elongation in fast-growing young chicks to evaluate the effect of
weight loading on differentiation, mineralization, and ossification. The significance of
mechanical stress studies on bones in their rapid elongation phase could be relevant to
intense sports activities during adolescence, the act of bearing heavy loads, and
possibly childhood obesity.
Samples of growth plates from birds on control and calcium-deficient diets

were visually assessed for enlargement and stained with hematoxylin and eosin
(H&E) by Rutt (2008). Both control and rachitic growth plates showed changes in
chondrocyte morphology throughout the plate. A region of flattened cells oriented in
columns was closest to the articular cartilage, followed by a gradual increase in cell
size, a region of round chondrocytes with a large cytoplasm, and mineralization with
the introduction of blood vessels. Enlarged growth plates from broilers on the
calcium-deficient diet had a considerably larger proliferative region compared to the
smaller growth plates from birds on the control diet In addition, the length of the
perforating epiphyseal blood vessels seemed to be longer in the deficient growth
plate, the concentration of blood cells in the marrow spaces was lower, and the vessels
show portions of thin, non-functional remains.
Burdan et al. (2009) reported morphology and physiology of the epiphyseal

growth plate. The epiphyseal growth plate develops from the cartilaginous-orientated
mesenchymal cells. This multilayer structure is formed by the proliferation and
hypertrophy of cells that synthesize the extracellular matrix composed of collagen
(mainly type II, IX, X, XI) and proteoglycans (aggrecan, decorin, annexin II, V and
VI). The resting zone is responsible for protein synthesis and maintaining a germinal
structure. In the proliferative zone, cells rapidly duplicate. The subsequent
morphological changes take place in the transformation zone, divided into the upper
and lower hypertrophic layers. In the degenerative zone, the mineralization process
becomes intensive due to increased release of alkaline phosphate, calcium and matrix
vesicles by terminally differentiated chondrocytes and some other factors e.g.,
metaphyseal ingrowth vessels. At this level, as well as in the primary and secondary
spongiosa zones, chondrocytes undergo apoptosis and are physiologically eliminated.
Chondrocyte proliferation and differentiation are regulated by various endocrine,
paracrine, and autocrine agents such as growth, thyroid and sex hormones, beta-
22
catenin, bone morphogenetic proteins, insulin-like growth factor, iodothyronine
deiodinase, leptin, nitric oxide, transforming growth factor beta and vitamin D
metabolites. Any disturbances of the epiphyseal development and its physiology
result in various skeletal abnormalities known as dysplasia.
Villemure and Stokes (2009) gave an overview on growth plate mechanics and
mechanobiology. The longitudinal growth of long bones occurs in growth plates
where chondrocytes synthesize cartilage that is subsequently ossified. Altered growth
and subsequent deformity resulting from abnormal mechanical loading is often
referred to as mechanical modulation of bone growth. The longitudinal growth of
bones is apparently controlled by modifying the numbers of growth plate
chondrocytes in the proliferative zone, their rate of proliferation, the amount of
chondrocytic hypertrophy and the controlled synthesis and degradation of matrix
throughout the growth plate. These variables may be modulated to produce a change
in growth rate in the presence of sustained or cyclic mechanical load.
According to Serrat (2014), environmental temperature can have a surprising

impact on extremity growth in homeotherms. Limbs of animals raised at warm
ambient temperature are significantly and permanently longer than those of littermates
housed at cooler temperature. Environmental modulation of tissue temperature can
have direct and immediate consequences on cell proliferation, metabolism, matrix
production, and mineralization in cartilage. Temperature can also indirectly influence
cartilage growth by modulating circulating levels and delivery routes of essential
hormones and paracrine regulators. Recent advances in imaging modalities enabling
the dynamic study of cartilage growth plates in vivo could be the key to elucidating
fundamental physiological mechanisms of long bone growth regulation.
2.4.2 Growth plate (cartilage) abnormality
Orth and Cook (1994) detailed avian tibia1 dyschondroplasia, the growth plate
lesion and its causes. Avian tibial dyschondroplasia is a disease found in fast growing
strains of chickens, ducks, and turkeys worldwide in which growth plate cartilage
accumulates in the metaphyseal region of the tibiotarsus. The growth plate
chondrocytes did not reach their expected size in the hypertrophic zone and necrosed
prematurely. The chondrocytes also produced decreased amounts of extracellular
23
proteins. This immature cartilage became highly crosslinked in the collagen
molecules and apparently resistant to resorption and vascularization by the
metaphyseal vessels. The dyschondroplastic cartilage remained in the metaphysis for
several weeks.
Collagen expression in growth plate cartilage derived from broiler chickens

with tibial dyschondroplasia was studied and compared with samples from unaffected
birds (Wardale and Duance,1996). Normal growth plate contained 12% collagen (dry
weight) and dyschondroplastic growth plate 19% collagen compared. Normal and
dyschondroplastic growth plate cartilages contained similar amounts of type I
collagen (5% of the total collagen) but dyschondroplastic growth plate cartilage
contains slightly less type II and type XI collagens, and significantly more type X
collagen (25% as compared to 11%) than in normal growth plate. Normal growth
plate chondrocytes retained their polygonal morphology whereas chondrocytes
derived from dyschondroplastic cartilage initially exhibited both fibroblastic and
polygonal phenotypes but gradually changed to totally fibroblastic. The results
suggested that the chondrocytes in dyschondroplastic growth plate cartilage were at a
different stage of maturity than normal resulting in a cartilage that was failing to turn
over at a normal rate.
Growth plate cartilage, which regulates long bone development, must maintain
a tightly controlled balance between cartilage synthesis and degradation as well as
chondrocyte proliferation and apoptosis. Some of the growth plate diseases were
discussed with an emphasis on how a breakdown in growth plate metabolism is
related to the observed problems (Orth, 1999).
Vitamin D deficiency in particular, resulting in rickets is associated with

failure of growth plate calcification, vascularization, and subsequent decrease in bone
formation, phenomena that have also been observed in rachitic mice (Malloy et al.,
1999; Lin et al., 2002).
Tibial dyschondroplasia (TD) is characterized by an avascular lesion in which

the life span of the growth plate chondrocyte is essentially doubled. A characteristic
pattern of gene expression and gene product localization has emerged that mimics the
pattern observed with endoplasmic reticulum (ER) stress in growth plate
24
chondrocytes. This activates a cell-survival mechanism called autophagy. The initial
phases of this mechanism appear to originate in the avascular transition zone of the
growth plate. Because specific genes and gene products were associated with
autophagy and ER stress, it could be possible to identify the mechanisms involved in
the development of this cartilage abnormality (Leach and Ornan, 2007).
Tibial dyschondroplasia was characterized by the presence of a non-

vascularized, non-mineralized lesion extending from the epiphyseal growth plate into
the metaphysis of the proximal tibiotarsal bones. They found that dramatically longer
exposures to much higher concentrations of thiram were required to induce TD in
turkeys as compared to broilers. In contrast to broilers, in which 50 mg/kg of thiram
induced a high incidence of severe TD within 10 days, in turkeys, an exposure to 400
mg/kg of thiram for 11 wks was necessary for the development of severe TD lesions
(Simsa et al., 2007).
Almeida et al. (2005) compared techniques for tibial dyschondroplasia

assessment in Cobb broiler chickens. At 39 days, forty birds were selected and tibial
dyschondroplasia status was assessed by four different techniques: evaluation using
the lixiscope, macroscopic examination, histological examination and bone mineral
density assessment using optical radiographic densitometry. The efficacy of each
technique to assess dyschondroplasia lesions in the tibial growth plate was determined
in comparison to histology, which was considered to be 100% efficient. Correlation
analysis showed that macroscopic analysis and bone mineral density assessment by
optical densitometry using radiographs are able to characterize the status of the
growth plate in the proximal epiphysis of the tibia in broiler chickens.
Clinical, serological, gross anatomy and histological investigations were

carried out in broiler chickens with signs of rickets (Dinev, 2011). Gross lesions in
chickens from two of farms were indicative of hypophosphataemic rickets and the
other two- of hypocalcaemic rickets types. Microscopic lesions correlated to
macroscopic ones. The results showed that lesions resulting from calcium deficiency
were very different from those occurring after phosphorus deficiency or calcium
excess. It was suggested that macroscopic lesions in the proximal tibiotarsus could be
used to distinguish field cases of Ca-deficiency and P-deficiency rickets in broiler
chickens.
25
Valteau et al. (2011) stated that longitudinal bone growth, which occurs in
growth plates, has important implications in pediatric orthopedics. Mechanical loads
are essential to normal bone growth, but excessive loads can lead to progressive
deformities. Loading effects on growth plate histomorphometry were greater in the
static than dynamic groups with significant reductions (pb0.001) observed for growth
plate thickness, proliferative chondrocyte number per column and hypertrophic
chondrocyte height in the static group when compared to the sham group. However,
dynamic loading causes less detrimental effects on growth plate histomorphometry
compared to static loading.
The effect of inhibition of heat-shock proteins on thiram-induced tibial

dyschondroplasia was studied by Genin et al. (2012). Thiram-induced TD was
characterized by enlarged, unvascularized growth plates, low levels of the vascular
endothelial growth factor receptor Flk-1, abnormal chondrocyte differentiation, and
lameness. The results demonstrated the specificity and the major role of Hsp90 in
chondrocyte differentiation and growth-plate vascularization. In contrast to the
antiangiogenic effect of 17-DMAG observed in mammals, inhibition of Hsp90
activity in the unvascularized TD affected growth plates resulted in activation of the
angiogenic switch and restored normal growth-plate morphology.
The histopathological examination of the tissues of the tibiotarsal joint of the

birds diagnosed with TD revealed degenerative alterations characterized by the
dominance of hypertrophy and the lack of urate crystals and calcification in
chondrocytes (Imik et al., 2012).
Zhang et al. (2013) studied expression and identification of recombinant

chicken vascular endothelial growth factor in pichia pastoris and its role in the
pathogenesis of tibial dyschondroplasia. Vascular endothelial growth factor (VEGF)
was an essential mediator of angiogenesis and endochondral ossification. To explore
the role of VEGF in avian diseases such as tibial dyschondroplasia (TD), a typical
disorder of endochondral ossification, they expressed and identified recombinant
chicken VEGF (ch- VEGF) protein in Pichia pastoris and evaluated its effects on
thiram-induced TD in broiler chickens.
26
2.4.3 Bone structure and its alteration
In three groups of broilers (Ross 1) consisting of 50 birds each and fed rations
supplemented with growth promoters, cross- sections of femoral epiphyses and
diaphyses were observed on days 11, 28, 42 and 56 after hatching for
histopathological changes and compared with findings in control birds (Neumann,
1986). There were no substantial differences observed as to the effects of the three
growth promoters on the bone. Tissue of broilers. Incipient changes in femurs of the
treated birds occurred on day 11, and they became more evident in the course of
fattening culminating in 56-d-old birds as against controls. Treatment with growth
promoters under study resulted in increased numbers of mesenchymal cells in
periosteum and enhanced periosteal apposition. Simultaneously, compact bone
medullarized and endosteal resorption occurred. Thus, remodeling of long bones in
broilers generally ascribed to one-sided selection for meat production was enhanced
appreciably in response to treatment with growth promoters.
Leterrier and Nys (1992) compared tibial growth in slowly (X) and rapidly (F)
growing chicks weekly until the birds weighed 500 g. A peripheral active zone of
greater porosity was predominantly characterised by radial fibrolamellar tissue in fast
growing birds and covered a larger area in crossbreed F than in crossbreed X. They
concluded that an increased rate of growth was associated with a lower mineral
density and higher porosity of the tibial cortices resulting in reduced bending
resistance.
Bone tissue is continuously produced by osteoblasts, modified with the help of

osteocytes and destroyed by osteoclasts. The process of mineralization occurs in two
tightly linked steps. The first step consists of the laminar secretion of the bony matrix,
by osteoblasts. The second stage involves the actual osteoid mineralisation. Bone
hardness and rigidity are conferred by the presence of mineral salts in the osteoid
matrix, and especially of calcium salts and of phosphate hydroxide precipitated as
thermodynamically stable hydroxyapatite (HAP) crystals. These fix
Themselves between and on the collagen fibers, thus ensuring osteoid

mineralisation. The osteoblasts generate matrix vesicles rich in alkaline phosphatase,
which causes accumulations of Ca2+ and PO4 3-, and in pyrophosphatases which
27
cleave PO4 3- ions from larger molecules. The matrix vesicles bud from the
osteoblast apical pole and are stored in the matrix. They provide the control element
for mineral deposition in osteoid. After initial precipitation of HAP crystals, they
grow rapidly through accretion and intermesh to generate larger crystaline entities. In
this way, mineralization is propagated in the newly formed osteoid. Alkaline
phosphatase (ALP) facilitates osteoid matrix mineralisation, and can be used as a
phenotypicaly marker indicator for osteoblasts. On the matrix produced mineral
deposits (calcium phosphates and carbonates) then form, creating crystals of
hydroxyapatite – Ca10(PO4)6(OH)2 – and thus forming a core mineral deposit,
surrounding the osteoblasts, that remain included in the gaps of the newly formed
tissue. From this point, the osteoblast reduces its synthetic activity and is transformed
into an osteocyte adult cell, which persists in the lacunae of the formed tissue (Banks,
1993).
Microstructure and mineral content of femora in male turkeys with and

without fractures was evaluated by Crespo et al. (2002). In general, the femoral cortex
of the turkeys at the mid diaphysis from all groups was similar to lamellar bone of
mammals. Most collagen fibers were arranged in circumferences, parallel to the bone
cross-section surface. The outer (periosteum) and inner (endosteum) margins of the
cortex contained densely packed, circumferential lamellar bone. Between these two
layers, the femoral cortex had an underlying pattern of network bone, which is made
up of intercalating woven bone tissue containing osteons and layers of lamellar bone
arranged in circumferential rows.
Influence of the enzyme phytase on the clinical status, some plasma

macroelements and the histostructure of femur and tibia in commercial chickens (3
experimental groups and 1 control group) was carried out by Tsokova (2006 ).The
first analysis of femur and tibia specimens from broiler and commercial chickens (on
the 30th day of the experiment) did not exhibit any histostructural changes in the
control groups, and only insignificant changes in the experimental groups were
observed. In several samples from the 3rd group of broilers, the bone trabeculae in the
epiphyseal spongiosa were thinned at places, and the spaces between them were filled
with traces of bone marrow. On the 60th day, the histological study on chicken femur
and tibia revealed intact general histological structure in control and experimental
28
commercial chickens and control broiler chickens, while for the experimental broilers,
there were changes, such as thinned compact bone at places, appearance of resorption
surfaces (on the account of spongiosa of the diaphyses and the belonging part of the
compacta), increases in the central (Haversian) canals and loss of bone trabeculae
within the epiphyseal spongiosa. The histological study of femur and tibia showed a
thinned compacta and enlargement of the central (Haversian) canals in the broiler
chickens at the end of the trials.
Dumitrescu et al. (2012) studied the effect of boron supplementation in the

feed of broiler chickens on the histological structure of the tibia. Histomorphometric
study of transverse sections of the tibia in treatment group 1 showed that the body of
the tibia was formed of cortical bone consisting of osteones of circular profiles which
were in the process of formation. Median cortical bone thickness was 1081.3 μ and
the overall diameter of the tibia was 0.75 cm. The cortical bone of the tibial shaft was
formed of circular osteones. Median thickness of the cortical bone was 1365.3 μ in
treatment group 2. In treatment group 3, transverse sections of tibial shaft showed an
intense process of ossification, with involvement of the periosteum, in particular the
osteoprogenitor cells in cellular layers of the periosteum. These cells established
themselves in the previously established cavities and, following their osteoblasts in
the cavities had the strongly basophilic cytoplasm associated with active protein
synthesis. Cortical bone thickness was 1514 μ. After the transformation into
osteoblasts, they began to produce the young connective tissue.
2.5 Biochemical Study
2.5.1 Blood calcium and phosphorus level
Heller et al. (1934) reported changes in the blood calcium and phosphorus
partition during the life cycle of the chicken. The inorganic calcium and phosphorus
content of the blood serum has been of interest in mineral metabolism studies for
many years. With an increased knowledge of the importance of these ions in certain
deficiency diseases, numerous investigations have been made of the calcium-
phosphorus ratios existing in these conditions. In a previous article it was reported
that fowl is peculiar in that it is much greater than that of mammals in respect to the
inorganic calcium and phosphorus content of the serum, as usually determined,
29
composes only a small part of the whole. In mammals this ion changes very little
throughout the life cycle but in fowls the calcium increases over 100 per cent at the
time of egg production. Serum Ca and P levels (mg/100ml) in non-laying birds were
estimated as 15.1 and 5.9 respectively.
Relationship between inorganic phosphorus and inorganic calcium in chicken

blood plasma was evaluated by Gardiner (1969). Male crossbred broiler-type chickens
were fed graded levels of dietary phosphorus from hatching to 4 weeks of age. Plasma
inorganic phosphorus and plasma inorganic calcium were determined on aliquots of
blood plasma samples taken when the chickens were 4 weeks of age. The level of
plasma inorganic phosphorus decreased when the chickens were fed low levels of
dietary phosphorus. The level of plasma inorganic calcium was inversely related to
the plasma inorganic phosphorus level. He recorded plasma inorganic P and Ca levels
(mg %) as (3.10, 4.94, 5.87, 5.541) and (14.92, 12.73, 12.05, 12.33) at 0.1, 0.2, 0.3
and 0.4 % added dietary P respectively. Regression equations were different for each
of the four dietary phosphorus levels fed. The sum of plasma inorganic and plasma
inorganic calcium was very similar for the four treatments.
Deo et al. (2003) studied growth performance and blood biochemicals of

starting broiler chicks as influenced by different levels of dietary phosphorus. Three
test diets were formulated with 0.30, 0.40 and 0.50% available phosphorus by
manipulating the inclusion level of dicalcium phosphate in a corn-soya based basal
diet. The feeding of diets varying in available phosphorus contents did not influence
the growth performance of broiler chicks during starting phase (0–3 wks). Similarly,
the activity of serum alkaline phosphotase and serum calcium and phosphorus levels
also remained statistically (P>0.05) similar among different dietary treatments. It was
inferred that the lowest dietary inclusion level of dicalcium phosphate at 1.2%
(equivalent to 0.30% available phosphorus) in a corn-soya based basal diet was
adequate for optimum performance of broiler chicks during starting phase in tropical
climate.
Hassanabadi et al. (2007) conducted a study to evaluate the effect of microbial

phytase and dietary calcium and phosphorus on productive traits, tibia ash percentage,
apparent retention of Ca, P, N also on number of blood factors in male broiler
chickens. Phytase supplementation increased serum P concentration, tibia ash
30
percentage, and Ca, P and N retention. Serum ca and P level (mg/dL) in control
broilers were 6.69 and 7.21 respectively on day 28. Its tibia ash percentage was
recorded as 39.25. Low dietary ca and nPP levels, regardless of phytase levels,
improved body weight, average daily gain and FCR.
In 18 -21 days old broiler chickens, plasma Ca concentration (mg/dL) varied

from 10.9 to 12.7 in birds with control diet and 5.5 to 8.6 in birds on Ca deficient diet.
No significant differences in blood phosphorus levels were found. The blood calcium:
phosphorus ratio was greater in birds fed the control diet compared to the deficient
diet (p=0.005, n=10; Fig. 3.3B). Control birds had ratios ranging between 1.4 and 2.1
(mean=1.69 ± 0.14), while calcium-deficient birds had ratios between 0.6 and 1.3
(mean=0.96 ± 0.13) (Rutt, 2008).
Rani et al. (2011) studied hematological and biochemical changes of stunting

syndrome in broiler chicken from day old ( group I) and 3 weeks (group III) of age to
8 weeks of age in two phases along with group II and group IV as control. Serum Ca
and P levels (g %) were 10.92 and 5.41 respectively in control birds. The
corresponding values in experimental birds were recorded (9.43 -10.58) and (4.79 –
5.17). The decreased hematological and biochemical parameters in the present study
indicates a decrease in the absorption and digestion of protein and damage to liver and
intestines in stunting syndrome of broiler chicken.
Bogusawska -Tryk et al. (2012) determined the concentration of total protein

and its fractions as well as the concentration of selected mineral components in the
blood serum of male broiler chickens Cobb 500 fed diets with different cellulose
content. Cellulose preparation had no influence on the content of total protein and its
fractions, inorganic phosphorus, sodium, potassium, chloride and iron concentrations
in blood serum. The highest calcium concentration (P<0.05) was detected in the blood
serum of males fed a diet with the highest cellulose content (0.75-0.95%). They
recorded normal serum calcium and phosphorus level as 2.00 ±0.25 mmol/l and
2.07±0.02 mmol/l in control birds on day 42. Simultaneously, a tendency of
increased calcium content was observed along with an increased amount of pure
cellulose in diets. They suggested that introduction of a limited amount of pure
cellulose into the diet of broiler chickens does not affect total protein concentration
and protein fractions but can influence the mineral content in the blood serum.
31
Imik et al. (2012) reported the effect of tibial dyschondroplasia on metabolic
parameters in Ross 308 broiler chickens. For this purpose, blood serum mineral, lipid,
protein, glucose, uric acid and enzyme levels, and histopathological alterations in the
tissues of the tibiotarsal joint were determined. Analyses showed that calcium,
phosphorus and free fatty acid levels in blood serum were lower, whilst the lipid
profile and amount of uric acid were high in birds suffering from tibial
dyschondroplasia, compared to healthy ones (P<0.05). On day 42, they recorded
serum Ca and P level (mg/dL) as 11.53± 0.64 and 7.30± 0.68 respectively in control
(healthy) birds.
Vijayakumar and Balakrishnan (2014) evaluated the bioavailability of calcium

phosphate nanoparticles as mineral supplement in Cobb 400 broiler chicken. Tibial
bone morphometry, bone and serum mineral profile and carcass characters in broiler
chicken were studied from day 1 to 28. There was no significant variation in the
parameters studied. The tibial bone ash, calcium and phosphorus content were unison
for both the control and 50% calcium phosphate nanoparticles supplemented groups.
Serum Ca and P level (mg/dL) were recorded as 10.56 and 7.15 respectively in these
control birds. The results indicated that the bioavailability of phosphorus in calcium
phosphate nanoparticles was 200 per cent when compared to dicalcium phosphate.
This finding may help the poultry managers to minimize the feed transport cost and
also minimize the mineral wastages.
2.5.2 Bone Calcium and Phosphorus Content (Bone Ash)
Williams et al. (2000) studied skeletal development as regards to mineral

contents and porosity assessment in the meat-type chicken up to day 39. Cortical bone
samples were ashed to measure total mineral, calcium and phosphorus content as well
as for porosity assessment. The cortical bone of the selected strain was less well
mineralised and more porous than that of the control strain and showed a significant
increase in the molar Ca:P ratios above the expected range of values during the first 2
to 3 weeks of life. The cortical bone Ca and P content (mg/g) in control birds on day
4, 11, 18, 25, 32 and 39 were (120, 156, 173, 199, 139, 269) and (71, 75, 80, 95, 73,
114) respectively. Despite production of bones with the correct dimensions for load
support, the relatively poor density and mineral content of bone in the selected strain
is likely to reduce effective breaking strength of the tibiotarsus. Possible reasons may
32
be either inadequate dietary supply of Ca and P or impaired utilisation of the minerals
due to a rapid growth rate or genetic factors.
Hall et al. (2003) advocated and compared two methods for the estimation of
bone ash in broilers. An experiment was conducted to compare two common methods
of estimating bone ash from growing broiler chicks (A = autoclaving; B =
boiling/extracting). Tibial bone ash% varied (day 7- 33.5, day14 – 34.9, day 21 -39.6
in autoclaving method and 25.8, 28.7 and 33.3 in boiling method) with age.
According to the report of Shim (2010), tibia ash content (39.7556±2.81) of

fast growing (FG) broiler chicken was lower than those of slow growing (SG) broiler
chicken (39.9920±2.67; P = 0.1725). Fluoride (F) had varying degrees of beneficial
effects on bone mineralization and strength, despite its toxic effects on growth and leg
disorders.
A comparative study was carried out on the physical characteristics and

mineralization of the tibia and femur from commercial Pekin ducks representing circa
1993 and 2010 commercial strains (Van Wyhe et al., 2012). The percentage ash in the
diaphyseal region of the tibia was 3% greater (P < 0.05) in the 2010 versus 1993
ducks. The percentage epiphyseal ash in the femur was 10% lower (P < 0.01) at 10 d
and 14 d in the 2010 ducks but there were no significant differences by 18 d of age.
The lower epiphyseal ash values at both younger ages and smaller BW in the 2010
contemporary ducks suggested that it was critical to monitor those factors that
influence bone mineralization in contemporary ducklings that could achieve market
BW at earlier chronological ages.
Vijayakumar and Balakrishnan (2014) evaluated the bioavailability of calcium

phosphate nanoparticles as mineral supplement in Cobb 400 broiler chicken. Tibial
bone morphometry, bone and serum mineral profile and carcass characters in broiler
chicken were studied from day 1 to 28. Estimation of bone ash (per cent tibia ash) of
the dried and defatted bone was carried out as per the method of
There was no significant (p<0.05) difference among the various treatments in

tibial bone mineral contents. The tibial bone ash, calcium and phosphorus content
were unison for both the control and 50% calcium phosphate nanoparticles
33
supplemented groups. In control birds of 4 weeks age, total ash percentage was 51.03
and that of Ca and P were 17.56 and 7.05 respectively.
ash, bone density, and morphology. This study examined the relationships among gait
score, bone parameters, and hip angle. Femur ash content (%) was lowest in 32-day-
old ducks and ducks with GS1 and GS2 (P < 0.0001). It varied from 47.52 on day 14,
followed by 48.3 on day 21 and 46.36 on day 32. Tibia ash content increased with
age (49.66 on day 14, 50.93 on day 21 and 51.63 on day 32), but decreased as gait
score increased (P < 0.001).
34
MATERIALS AND METHODS
3.1 Preamble
In order to achieve the said objectives, the present study was conducted on
forty five (45) ‘Vencob’ broiler birds. The day old chicks were reared up to day 42
(market age) in the experimental pen of the Department of Anatomy & Histology,
College of Veterinary Science & A.H., O.U.A.T., Bhubaneswar. The gross
morphometrical, radiographical and histomorphological studies were carried out on
the long leg bones and associated growth cartilages along with biochemical
observations in broiler chickens at different ages (on days 1,7, 14, 21, 28, 35 and 42).
3.2 Experimental Design
For the present investigation, forty five (45) day old ‘Vencob’ broiler chicks
were procured from Eastern Hatcheries Pvt. Ltd., Bhubaneswar, Odisha (A subsidiary
of Venkateswara Hatcheries Group, Pune). The sexing of chicks was done by the
conventional vent method, followed by wing banding. Accordingly, the male and
females were separately reared up to 6 weeks (i.e. market age) under deep litter
system in the experimental pen of the Department of Anatomy & Histology, College
of Veterinary Science & A.H., O.U.A.T., Bhubaneswar. The routine management
practice was followed that included housing, vaccination, medication and feeding.
Proper ventilation and lighting arrangement was done. The feeding regimen offered
ad libitum feed and water to all the birds throughout the experimental period. The
chicks were fed commercially available starter feed (up to 3 weeks) and then the
grower feed (up to day 42).
3.3 Collection of Specimen
Birds of either sex were sacrificed by cervical dislocation after their body
weight measurement and blood collection. The specimen samples (Femur, tibiotarsus
and tarsometatarsus bones, and associated growth plate cartilages) were collected
after careful dissection to record the gross morphometrical, radiographical,
35
histomorphological and biochemical data at weekly interval (on days 1,7, 14, 21, 28,
35 and 42).
3.4 Gross Morphometrical Study
The selected birds were sacrificed, followed by careful dissection of both the
limbs (right and left) to collect the intact long leg bones, i.e. Femur, tibiotarsus and
tarsometatarsus. Gross morphometrical parameters of the said bones under study
included weight, length, width or diameter, width (thickness) of marrow cavity,
thickness of cortical bone and growth plates (proximal and distal), and distance
between proximal and distal growth plates.
The bones were longitudinally sectioned very carefully by using a small hand
saw (30 TPI or teeth per inch) to record some of the gross morphometrical parameters
viz. width or diameter, width of bone marrow, thickness of cortical bone (proximal,
distal and mid shaft), thickness of growth plates (proximal and distal) and distance
between proximal and distal growth plates. The gross morphometrical measurement
was taken in mm and cm (as required) with the help of a digital slide calliper, a non-
distensible thread and a measuring tape (scale ruler). Six number (n =06) of samples
were used for each measurement.
Gross photography of the intact as well as sectioned bones from each of the
respective bird was done before radiographical and histological study.
3.5 Radiographical Study
Digital radiography of the said bones (right and left side) from both the male
and female birds along with associated growth or epiphyseal plates (cartilages) were
performed in a local radiography centre (Auro X-Ray Diagnostic Centre, Forest Park,
Bhubaneswar) before and after taking the longitudinal section of these bones. The
radiographic exposure factors were taken into account, i.e.50 kV, 0.1 second,15 mA,
100cm FFD and 12 x10 film size.
36
Some of the gross morphometrical parameters such as width of bone and bone
marrow, and thickness of cortical bone and growth plates were also recorded and
analyzed on radiography.
3.6 Histomorphological Study
After recording all the gross morphometrical data and radiography, a small
piece of tissue from proximal as well as distal epiphysis (end) from each of the said
bones from either sex was cut carefully with the help of a scalpel blade and a small
hand saw for histomorphological study. These tissue pieces included the articular
cartilage, growth plate (cartilage) and adjacent part of the bone. Small representative
bone tissue samples of 3 to 5 mm thickness were also collected from the mid shaft
region of the bones. The collected tissue pieces were fixed properly in 10% buffered
neutral formalin (BNF) for further tissue processing.
Decalcification: All the tissue pieces were subjected to the standard

decalcification process (Luna, 1968 and Bancroft and Stevens, 1996). The
decalcification fluid contained 5% nitric acid (HNO3) and 95% BNF. The tissues were
immersed in the decalcification fluid with the help of a string for 6 to 7 days. Care
was taken to ensure that the lowest tissue of the string never touched the bottom of the
fluid container. The tissues were periodically agitated or stirred, and the fluid was also
changed 2-3 times to facilitate better decalcification. The completion of the
decalcification process was assessed, rather confirmed by the calcium oxalate test
(Luna, 1968) with the help of 5% ammonia and ammonium oxalate. It was performed
regularly. The resultant colour (whether turbid or clear) of the test decalcification
fluid was the indicator of the success of the test.
After proper decalcification, the tissue pieces were thoroughly washed under
the tap water for 2 days before routine tissue processing. The tissues were dehydrated
through the ascending grades of ethyl alcohol (70%, 80%, 90%, 95% and absolute
alcohol), cleared in xylene, followed by paraffin impregnation inside a
thermostatically controlled hot air oven to prepare the paraffin tissue blocks. After
trimming, the paraffin blocks were cut with the help of a semi motorized rotary
microtome (Leica RM 2245, Germany) to obtain the serial tissue sections of 5 to 7
37
micron (µm) thickness. Good sections were selected, flattened in hot water bath and
finally mounted on clean, grease – free, albumenized glass slides before air drying.
The tissue sections were subjected to the following histological staining techniques.
o Routine histological staining - Haematoxylin and Eosin ( H & E) staining for

routine histomorphology ( Bancroft and Stevens, 1996),
o Special histological staining - Masson’s Trichrome staining with light green
for connective tissue elements and PAS – Alcian blue ( at pH 2.5) combined
staining for mucopolysaccharide content ( Bancroft and Stevens, 1996),
The stained tissue sections (slides) were observed under light microscope
(Leica DM 2500, Germany) under lower (10X) as well as higher (40X) magnification.
The photomicrography was done from the selected microscopic fields with the help of
Leica DFC 290 camera in Department of Anatomy & Histology, College of
Veterinary Science & A.H., O.U.A.T., Bhubaneswar.
Histomorphometry:
Histomorphometric measurement was done in micrometer (µm) by the

conventional ocular micrometry after due calibration of ocular micrometer with a
stage micrometer as well as by the Leica microscope software (LAS) of Leica DM
2500 microscope (Germany). The histomorphometric parameters included diameter of
Haversian systems (osteons) and Haversian canals, thickness of periosteum, thickness
of growth plate and its zonation pattern as well as diameter (size) of chondrocytes in
different zones.

Estimation of Serum Calcium and Phosphorus level:
5-7 ml of blood samples were collected from each of the birds sacrificed
through the wing vein venipuncture. Care was taken to avoid formation of haematoma
during the blood collection. The specimen or collection tubes with blood samples
were left undisturbed in a slanting position for 4-5 hours at room temperature so as to
facilitate the serum formation. The serum was collected (harvested) carefully in sterile
38
Eppendorf tubes (Sigma) with the help of a syringe with needle (22 gauge). Then
these serum samples were kept immediately in a deep freezer at - 20⁰ C temperature
before serum calcium (Ca) and phosphorus (P) estimation in TVCC and Dept. of
Preventive Veterinary Medicine, College of Veterinary Sc. & A.H., O.U.A.T.,
Bhubaneswar. The estimation of serum calcium (Ca) and phosphorus (P) level (in mg/
dl) was done by a semi auto analyzer (Merck Minilab 300) using commercially
available calcium kit (Coral Clinical Systems, Goa) as per OCPC method (Bagainski,
1973) and phosphorus kit (Coral Clinical Systems, Goa ) as per Modified Gomorri’s
method (Gomorri, 1942).
Estimation of bone Calcium and Phosphorus content (Bone Ash):
Representative samples from diaphysis (shaft) of all the leg bones (right)
under study were used for determination bone ash, calcium and phosphorus on dry, fat
– free bones in the Department of Nutrition, College of Veterinary Sc. & A.H.,
O.U.A.T., Bhubaneswar. The total ash content was analyzed and assayed for calcium
and phosphorus content as per the methods of Talapatra et al. (1940) and AOAC
(1995). The calcium and phosphorus content of the bone was expressed in % per gram
(DM basis) of the respective bone.
3.8 Statistical Analysis

All the gross morphometrical, histomorphometrical and biochemical data
generated were subjected to the standard statistical analysis (Snedecor and Cochran,
1989).
39
RESULTS
4.1 Gross Morphometrical Observations
The present study was carried out in post – hatch “Vencob” broiler birds. The
day old chicks were reared up to day 42. All the gross morphometrical parameters
were recorded at weekly interval, i.e. days 1, 7, 14, 21, 28, 35 and 42 in both the sexes
(male and female). Each parameter was replicated for six (n=06) birds.
Body weight: The average body weight of day old male chicks were recorded
to be 49.025± 0.591 gm, whereas the female chicks weighed 49.333± 0.648 gm. On
day 7, the body weight of male chicks were 159.408± 3.711 gm and those of female
ones were 154.291± 3.563 gm. On day 14, the body weight measured in male and
females were 555.083± 3.363 gm and 540.05± 6.385 gm respectively. The values for
the parameter in male birds on days 21, 28, 35 and 42 were found to be 868.216±
5.117 gm, 1317.667± 15.247 gm, 1658.65± 3.847 gm and 1798.2± 7.583 gm
resepectively. While these values in female birds of respective age were recorded to
be 847.783± 3.1780 gm, 1103.267± 17.377 gm, 1502.933± 6.370 gm and 1665.783±
9.271 gm respectively.
4.2 Gross Morphometrical Study of Leg Bones
a) Femur
The general gross morphological features of femur (thigh bone) in male as

well as female broiler chickens did not vary much except the size of the bone at
different ages. The femur consisted of a proximal end (epiphysis), a distal end
(epiphysis) and a cylindrical, slightly bent shaft. Its proximal end had a small, almost
spherical head (medially), a constricted neck below the head and a single greater
trochanter (laterally) with a small articular facet (antitrochanter) for pelvic bone. The
distal end consisted of two widely apart trochlear ridges and two condyles (medial
and lateral) behind with a small intervening fossa. The lateral condyle was marked by
a sagittal groove for articulation with the fibula. The sectional view of the bones
showed the presence of marrow cavity of different size enclosed within the cortical
(compact) bones of the shaft and a narrow transversely oriented, almost translucent
40
strip of growth (epiphyseal) cartilage at the respective end (Figs.30, 31,32, 33, 34, 35,
38, 41,44,47a,48,49,50,51,52,53,54,55,56,59,62 & 63).
In day old male chicks, the average weight of the femoral bones (right leg)
was 0.311 gm. It gradually increased on day 7 (0.711 gm), followed by a marked
increase from day 14(1.648 gm) up to day 42 (9.688 gm). The bones of female day
old chicks weighed 0.305 gm that gradually increased from day 7 (0.615 gm) till day
42 (9.14 gm). The average weight of left femur in male and female broiler birds were
measured to be (0.313 gm and 0.301 gm ) on day 1 and the values showed gradually
increasing trend from day 7 ( 0.627 gm and 0.610 gm) till the end of the experimental
period ( 9.637 gm and 9.05 gm) respectively (Table 1 and 4, Fig. 1).
In male birds, the average length of femur varied from 2.9 cm (on day 1) to 7.9
cm (on day 42). The values in female birds ranged from 2.88 cm (on day 1) to 7.7 cm
(on day 42). There was simultaneous increase in length of left femoral bones in both
male and female birds under study like that of right leg. The average values in male
birds varied from 2.85 cm (on day 1) to 7.75 cm (on day 42), while in females these
ranged from 2.80 cm to 7.55 cm respectively (Table 1 and 4, Fig.1, 16).
In male birds, the diameter at proximal end, mid shaft and distal end of right
femur were 2.35 mm, 2.20 mm and 2.98 mm on day 1, followed by a steep increase
from day 7 (3.94 mm, 3.38 mm and 4.14 mm) up to day 21(8.64 mm, 5.15 mm and
9.10 mm) and a steady increase thereafter till the end of the study period, i.e. day
42(10.88 mm, 7.09 mm and 11.15 mm) respectively. The bones of the left side
showed the values ranging from (2.32 mm, 1.88 mm and 2.89 mm) in day old chicks
so as to reach the highest values (10.82 mm, 7.75 mm and 11.05 mm) in 42 days old
birds respectively. In females, the values ranged from (2.31 mm, 2.15 mm and 2.65
mm) for right side and (2.30 mm, 1.77 mm and 2.52 mm) for left side of day old
chicks and increased up to (10.15 mm, 6.75 mm and 10.34 mm) and (10.20 mm, 7.25
mm and 10.32 mm) on day 42 respectively (Table 1 and 4, Fig. 2, 17).
In male birds, the values for size (thickness) of marrow cavity of right and left
bones recorded were 0.7mm and 0.83 mm respectively on day 1. The values became
almost double (1.59 mm and 1.74 mm) on day 7. Then there was a progressive
increase in the bone marrow size till day 42 (4.28 mm and 4.54 mm). However, the
41
growth slowed down a bit towards the end of the experiment (days 35 and 42). In
females, the lowest values for right and left bones were 0.71 mm and 0.75 mm
respectively on day 1. The highest values (4.01 mm and 4.31 mm respectively) were
recorded at the end of the experimental period (Table 1 and 4).
In males, average cortical bone (proximal, mid shaft and distal) thickness were
(0.49mm, 0.71mm and 0.51mm) and (0.66 mm, 0.53mm and 0.6 mm) on day 1 and
increased from day 7 onward till it became the thickest one (1.25 mm, 1.42 mm and
1.25 mm, and 1.30 mm, 1.59 mm and 1.30 mm) respectively for right and left side on
day 42. Its values in females ranged from (0.45mm, 0.70 mm and 0.48 mm) and (0.52
mm, 0.50 mm and 0.58 mm) in day old chicks to (1.22 mm, 1.36 mm and 1.25 mm)
and (1.15 mm, 1.45 mm and 1.22 mm) respectively in the eldest birds (Table 1 and 4,
Fig. 3, 18).
The growth plate (cartilage) could not be traced (identified) grossly in the
femur of both sides and sexes on day 1. However, from day 7 till day 42 the same
could be easily traced and studied for gross parameters on sectional view of the bones.
The distance between proximal and distal growth cartilage was measured to be (2.45
cm and 2.60 cm) and (2.37 cm and 2.53 cm) in male and female birds respectively on
day 7 and reached the highest respective values, i.e. (7.1 cm and 6.95 cm) and (6.6 cm
and 6.52 cm) on day 42. The values for the growth plates (proximal and distal) in
femur of either side of male birds were (0.62 mm and 0.58 mm) and (0.41 mm and
0.38 mm) respectively on day 7. The values gradually increased up to (0.95 mm and
0.85 mm) and (0.98 mm and 0.91 mm) respectively on day 28, followed by a slight
decline in the values thereafter. In females, the respective values on day 7 were (0.60
mm and 0.56 mm) and (0.35 mm and 0.31 mm) and (0.81 mm and 0.73 mm) and
(0.80 mm and 0.75 mm) on day 42 (Table 1 and 4).
b) Tibiotarsus
The tibiotarsus bones in male as well as female broiler chickens did not vary
much gross anatomically, except the size of the bone at different ages. It was the
longest among all the leg bones. Its shaft was nearly straight, slightly prismatic
proximally and more or less cylindrical distally. Tibial crest was very prominent. The
lateral side of the upper shaft showed an elongated ridge for attachment of a narrow,
42
thin fibula with pointed distal end. Its distal end had two condyles (larger medial and
smaller lateral) for articulation with tarsometatarsus. Proximal facets (condyles) were
made for distal condyles of femur. A cnemial crest also characterized its proximal
end. The sectional view of the tibiotarsus showed the presence of marrow cavity of
different size enclosed within the cortical (compact) bones of the shaft and a narrow
transversely oriented, almost translucent strip (distinctly visible) of growth
(epiphyseal) cartilage at the respective end (Figs.30,31, 32,33, 34, 36, 39, 42, 45, 47b,
48, 49, 50, 51, 52, 53, 54, 55, 57, 60, 64 & 65).
In day old male chicks, the average weight of the tibiotarsal bones (right leg)
were 0.55 gm. It sharply increased on day 7 (1.514 gm) and followed this increasing
trend from day 14(2.38 gm) up to day 42 (14.180 gm). The bones of female day old
chicks weighed 0.51 gm that markedly increased from day 7 (1.350 gm) till day 42
(13.520 gm). The average weight of left femur in male and female broiler birds were
measured to be (0.491 gm and 0.411 gm ) on day 1 and the values showed sharp
increasing trend from day 7 ( 1.6567 gm and 1.350 gm) till the end of the
experimental period ( 14.105 gm and 13.630 gm) respectively (Table 2 and 5, Fig. 5,
19).
In male birds, the average length of right tibiotarsus varied from 3.8 cm (on
day 1) to 11.4 cm (on day 42). The values in case of female birds varied from 3.52 cm
(on day 1) to 10.88 cm (on day 42). The average values for left side bones in male
birds ranged from 3.50 cm (on day 1) to 11.30 cm (on day 42), while in females these
ranged from 3.42 cm to 10.75 cm respectively (Table 2 and 5, Fig. 6, 21).
tibiotarsus were 2.66 mm, 2.28 mm and 3.22 mm on day 1, followed by a marked
increase from day 7 (4.68 mm, 3.73 mm and 5.25 mm) up to day 21(9.77 mm, 5.93
mm and 10.30 mm) and a steady increase thereafter till the end of the study period,
i.e. day 42(14.850 mm, 8.85 mm and 15.32 mm) respectively. The bones of the left
side showed almost similar trend with values ranging from (2.41 mm, 2.16 mm and
3.13 mm) in day old chicks so as to reach the highest values (14.35 mm, 8.90 mm and
14.85 mm) in 42 days old birds respectively. In females, the values for right as well as
left bones were slightly lesser than those recorded in case of male birds. These ranged
from (2.59 mm, 2.15 mm and 3.19 mm) for right side and (2.32 mm, 1.75 mm and
43
2.92 mm) for left side bones of day old chicks and increased up to (14.105 mm, 7.35
mm and 14.75 mm) and (13.75 mm, 7.38 mm and 13.95 mm) ) on day 42 respectively
(Table 2 and 5, Fig. 7, 22).
In male birds, the values for size (thickness) of marrow cavity in right and left
tibiotarsal bones recorded were 0.74mm and 0.85 mm respectively on day 1. The
values became almost double (1.65 mm and 1.54 mm) on day 7. Then there was a
progressive increase in the bone marrow size till day 42 (5.45 mm and 5.10 mm). In
females, the size of bone marrow followed a similar pattern with advancement of age
of the birds. The lowest values for right and left bones were 0.70 mm and 0.88 mm
respectively on day 1. At the end of the experimental period, the highest values (4.12
mm and 4.05 mm respectively) were recorded (Table 2 and 5).
In males, the cortical bone thickness were (0.98mm, 0.75mm and 0.88mm)
and (1.02 mm, 0.48mm and 0.77 mm) on day 1 and increased from day 7 onward till
it became the thickest one ( 1.55 mm, 1.68 mm and 1.53 mm, and 1.78 mm, 1.85 mm
and 1.65 mm) respectively for right and left side bones on day 42. In female birds its
values varied from (0.91mm, 0.70 mm and 0.82 mm) and (0.94 mm, 0.41 mm and
0.74 mm) in day old chicks to ( 1.48 mm, 1.60 mm and 1.45 mm) and (1.55 mm, 1.65
mm and 1.57 mm) respectively in the eldest birds (Table 2 and 5, Fig. 8, 23).
The growth plate (cartilage) was identified on gross observation in the

tibiotarsus of all the day old chicks under study. However, from day 7 till day 42 the
same was easily identified and studied grossly on sectional (longitudinal) view of the
bones. The distance between proximal and distal growth cartilage was measured to be
(2.22 cm and 2.80 cm) and (2.11 cm and 2.32 cm) in male and female birds
respectively on day 1. The distance between proximal and distal growth cartilage
showed a gradual increase from day 7 onward and reached the highest respective
values, i.e. (9.88 cm and 9.75 cm) and (8.95 cm and 8.88 cm) on day 42. The values
for the growth plates (proximal and distal) in tibiotarsus of either side of male birds
were (0.65 mm and 0.41 mm) and (0.53 mm and 0.46 mm) respectively on day 1. The
values for this parameter gradually increased up to (1.74 mm and 1.61 mm) and (1.52
mm and 1.45 mm) respectively on day 28, followed by a little decline in the values
thereafter. In females, the respective values on day 1 were (0.61 mm and 0.38 mm)
44
and (0.48 mm and 0.42 mm) and (1.26 mm and 1.19 mm) and (1.16 mm and 1.10
mm) on day 42 (Table 2, 5).
c) Tarsometatarsus
The general gross morphological features of tarsometatarsus in male as well as

female broiler chickens did not vary much except the size of the bone at different
ages. It was characterized proximally by two (internal and external) condyles like
structures for articulation with distal condyles of tibiotarsus. Proximally it also
showed calcaneal ridges forming tendinal canals on planter side (This part is often
known as hypotarsus) and several foramina dorsally Distally it had three condyles for
articulation with 2nd , 3rd and 4th digits. A large distal foramen was located between
3rd and 4th condyles (area of fusion between these two). It also presented planterly a
small, non-fused first metatarsal at the distal end (serving the articulation for 1st digit
(Figs.30,31,32,33,34,37,40,43,46,47c,48,49,50,51,52,53,54,55,58,61,66 & 67).
The average weight of the tarsometatarsus bones of right legs were 0.318 gm
in day old male chicks. It gradually increased on day 7 (0.409 gm), followed by a
marked increase from day 14 (1.612 gm) up to day 42 (9.593 gm). The bone of female
day old chicks weighed 0.310 gm that gradually increased from day 7 (0.390 gm) till
day 42 (9.263 gm). The average weight of left side bones in male and female broiler
birds were measured to be (0.376 gm and 0.351 gm ) on day 1 and the values showed
gradually increasing trend from day 7 ( 0.387 gm and 0.366 gm) till the end of the
experimental period ( 9.823 gm and 9.350 gm) respectively (Table 3 and 6, Fig. 10,
25).
In male birds, the average length of tarsometatarsus (right side) varied from
2.7 cm (on day 1) to 9.1 cm (on day 42). The values in female birds ranged from 2.50
cm (on day 1) to 8.80 cm (on day 42). The average values for left side bones in male
birds varied from 2.50 cm (on day 1) to 8.90 cm (on day 42), while in females these
ranged from 2.40 cm to 8.25 cm respectively (Table 3 and 6, Fig. 11, 26).
tarsometatarsus were 4.22 mm, 2.81 mm and 4.06 mm on day 1, followed by a steep
increase from day 7 (5.7 mm, 4.43 mm and 5.59 mm) up to day 21(10.71 mm, 7.40
45
mm and 10.32 mm) and a steady increase thereafter till the end of the study period,
i.e. day 42(14.88 mm, 9.85 mm and 14.25 mm) respectively. The bones of the left
side showed almost similar trend with values ranging from (4.09 mm, 3.51 mm and
4.10 mm) in day old chicks so as to reach the highest values (14.61 mm, 9.74 mm and
14.12 mm) in 42 days old birds respectively. In female birds, the values for right as
well as left bones were slightly lesser than those recorded in case of males. These
ranged from (4.10 mm, 2.60 mm and 3.95 mm) for right side and (3.88 mm, 3.28 mm
and 3.92 mm) for left side of day old chicks and increased up to (14.12 mm, 9.15 mm
and 13.78 mm) and (13.90 mm, 9.20 mm and 13.65 mm) ) on day 42 respectively
(Table 3 and 6, Fig. 12, 27).
The size (thickness) of marrow cavity in the tarsometatarsal bones of both sex
and side revealed a rapid increase from day 1 to day 7, followed by a gradual and
steady rise thereafter till day 42. In male birds, the values for right and left bones
recorded were 1.30mm and 1.91 mm respectively on day 1. The values became nearly
double (2.40 mm) for right side bones and little less than twice the size of previous
age (2.6 mm) for left side bones on day 7. Then there was a progressive increase in
the bone marrow size till day 42 (6.75 mm and 6.548mm). However, the growth
slowed down a bit towards the end of the experiment (days 35 and 42). In females, the
size of bone marrow followed a similar pattern with age of the birds. The lowest
values for right and left bones were 1.20 mm and 1.84 mm respectively on day 1. The
highest values (6.22 mm and 6.15 mm respectively) were recorded at the end of the
experimental period (day 42) (Table 3 and 6).
In male birds, the average cortical bone (proximal, mid shaft and distal)
thickness were (0.94mm, 0.73mm and 0.93mm) and (0.91 mm, 0.77mm and 1.01
mm) on day 1 and increased from day 7 onward till the cortical bone became the
thickest one (1.48 mm, 1.53 mm and 1.46 mm, and 1.52 mm, 1.58 mm and 1.44 mm)
respectively for right and left side bones on day 42. Its values in female birds ranged
from (0.85mm, 0.69 mm and 0.88 mm) and (0.81 mm, 0.70 mm and 0.92 mm) in day
old chicks to (1.38 mm, 1.45 mm and 1.41 mm) and (1.43 mm, 1.52 mm and 1.38
mm) respectively in the oldest birds. The values were slightly lesser (lower) than
those of male birds of respective age. (Table 3 and 6, Fig.13, 28).
46
Though the proximal growth plate (cartilage) was identified, the distal one
could not be traced (identified) grossly in the tarsometatarsus of both sides and sexes
on day 1. However, from day 7 till day 42 the same were easily studied for gross
parameters on sectional view of the bones. The distance between proximal and distal
growth cartilage was measured to be (2.6 cm and 2.40 cm) and (2.35 cm and 2.28 cm)
in male and female birds respectively on day 7. Consistent with the increase in length
of the bone, the distance between proximal and distal growth cartilage also showed
gradual increase from day 14 onward and reached the highest respective values, i.e.
(7.90 cm and 7.70 cm) and (6.98 cm and 7.22 cm) on day 42. The thickness of
proximal growth plate were (0.57 mm and 0.54 mm) and (0.48 mm and 0.51 mm) for
both side bones of males and females respectively. The values for the growth plates
(proximal and distal) in tarsometatarsus of either side of male birds were (0.85 mm
and 0.25 mm) and (0.89 mm and 0.62 mm) respectively on day 7. The values for this
parameter gradually increased up to (1.66 mm and 0.59 mm) and (1.43 mm and 1.32
mm) respectively on day 28, followed by a slight decline in the values thereafter. In
females, the respective values on day 7 were (0.69 mm and 0.25 mm) and (0.78 mm
and 0.60 mm), and on day 42 these were (1.25 mm and 0.32 mm) and (1.15 mm and
1.05 mm) respectively (Table 3 and 6).
4.3 Radiographical Study of Leg Bones and Associated Growth Cartilages
Some of the gross morphometrical parameters like width of the bones and
bone marrow, and cortical bone thickness were measured and analyzed on
radiography of the long leg bones (Femur, tibiotarsus and tarsometatarsus) of broiler
chickens at different ages (days 1, 7, 14, 21, 28, 35 and 42) and the data (not shown
separately) were compared with the gross morphometrical observations of the bones
and compiled for inclusion in the Tables of gross morphometrical data (Table1, 2, 3,
4, 5 and 6 ). Besides, the presence or absence of growth plates or cartilages (proximal
and /or distal) in each of these bones at different ages was distinctly evidenced on
radiography even in day old chicks. The thickness of the growth plates was also
measured directly on radiographs by using image analysis software and the data (not
shown separately) obtained were compared and included with those of gross
observations.
47
femur of both sides and sexes on day 1. But on radiograph the same was marked
easily. The growth plate (cartilage) could even be identified on gross observation in
the tibiotarsus of all the day old chicks under study. The same was also evident on
radiograph of the bones.
However, from day 7 till day 42 the growth plates were easily identified
grossly and radiographically for gross as well as radiographic observations of
different morphometric parameters of the bones. Though the proximal growth plate
(cartilage) was identified, the distal one could not be traced (identified) grossly in the
tarsometatarsus of both sides and sexes on day 1. But the growth cartilages of both
ends were clearly visible on radiograph of the bones from day 1 till day 42 and
radiographic analysis of the morphometrical parameters was done. The growth
cartilages in female birds appeared slightly thinner than those of male birds of
respective ages. And there was minor variation in thickness between proximal and
distal growth cartilage at a given age. The thickness of the growth cartilages in all the
bones became a little bit thinner after day 28 (Tables1, 2, 3, 4, 5 and 6). However,
epiphyseal closure was not seen in any of the bones during the experimental period.
Radiographs of different bones of male and female birds at different ages are shown at
Figs. 68, 69, 70a, 70b, 71,72 & 73.
4.4 Histomorphological Study of Growth Cartilages and Bones
4.4.1 Histomorphological Study of Growth Cartilages
a) Femur
Both the proximal and distal ends (epiphyses) showed the presence of growth
plates (cartilages) even in all day old chicks (males and females) despite the fact that
the same could not be identified on gross observation. However, these growth plates
were better recognized from day 7 onward till day 42. Histomorphology of the growth
plates (proximal) revealed horizontal zones of chondrocytes at different stages of
differentiation. These characteristic histological zones were arranged chronologically
as resting or reserve zone, zone of proliferation, zone of prehypertrophy, zone of
hypertrophy and degeneration, and zone of ossification (bone formation) from
48
epiphyseal towards diaphyseal/metaphyseal end of the growth cartilage. The zonation
pattern was better elucidated thereafter till day 28. But the typical arrangement of
zones of chondrocytes was not clearly depicted in the birds of older age (Days 35 and
42). Each zone showed difference in chondrocyte size and arrangement as well as
extracellular matrix (ECM). The reserve or resting zone was located just underneath
the articular cartilage. The chondrocytes were flattened and comparatively smaller
than those of other zones. These chondrocytes were scattered (without any specific
arrangement) or clustered within the lacunae. The lacunae within the matrix contained
usually one or even two chondrocytes (cartilage cells). Vascularization was poor in
this zone. The rapid proliferation of these flattened chondrocyes and their polarized
arrangement with columnar orientation was the distinguishing feature of the zone of
proliferation. However, a typical row–wise or columnar organization of the
proliferating chondrocytes was lacking (poor) in the true sense. More or less flattened
proliferating chondrocytes gradually increased in size to form the zone of
prehypertrophy (a thin zone of transition) and subsequently enlarged to the maximum
extent so as to form the zone of hypertrophy. The largest chondrocytes (among all
zones) of the hypertrophic zone were more or less spherical or rounded in appearance
in consonance with the shape of the lacunae. Scanty matrix of this zone (in lower part)
revealed provisional calcification resulting in normal death and degeneration
(apoptosis) of the hypertrophied chondrocytes. This zone continued into the zone of
true bone formation (ossification) that revealed the laying down of bony matrix
surrounded by seam of cartilage cells. It was accompanied by formation of trabecular
bone (primary spongiosa) and developing marrow. Very rich vascularization was
evident in this zone. The amount of extracellular matrix was comparatively more in
reserve and proliferation zones, whereas a scanty amount of matrix surrounded the
closely arranged lacunae containing chondrocytes in rest of the zones. Masson’s
trichrome stain demonstrated the presence of connective tissue elements, namely
collagen fibers in the matrix, walls of the invading blood vessels, lining of endosteum
of developing marrow spaces and newly formed bony matrix of zone of ossification.
Trichrome reaction was very intense in the zone of ossification as compared to other
zones. But the cartilage matrix did not reveal any collagen fibers as such, except those
forming an anastomosing, thread – like network around the lacunae of lower zones as
well as in the newly formed bony matrix in the ossification zone.
49
The matrix of the growth plate showed different staining reactivity for PAS –
alcian blue stain. The matrix in different zones was scanty. There was moderate PAS
reaction in the cytoplasm of enlarged chondrocytes of hypertrophic zone. The matrix
of newly formed bony trabeculae revealed moderate to weak reaction for PAS with
alcianophilia in some parts. However, the alcianophilic reaction was more intense
than the PAS reactivity in the matrix of different cartilage zones.
Practically there was no variation as such in the connective tissue content and
matrix reactivity for PAS – alcian blue stain in male and female birds. There was age
and sex related variation in the histomorphometrical parameters, such as thickness of
different zones and diameter of residing chondrocytes. Even there was individual
variation between the proximal and distal growth plates of a particular bone. The
histomorphological features of growth cartilages of femur bones in male as well as
female birds are presented in Figs.74, 75, 77, 78, 79, 81, 83, 91, 94 & 98.
The proximal and distal growth plates of male day old chicks measured
401.234µm and 392.230 µm respectively. The thickness of respective cartilage
gradually increased with age of the birds from day 7 (620.212 µm and 582.368 µm)
up to day 28 (957.241 µm and 859.632 µm). Thereafter there was a slight decline in
these values from day 35 (859.783 µm and 818.991 µm) till day 42 (818.652 µm and
797.321 µm) respectively. In females, the corresponding values for proximal as well
as distal growth cartilage were (400.984 µm and 391.984 µm) on day 1, (619.962 µm
and 582.117 µm) on day 7, (956.991 µm and 859.382 µm) on day 28, followed a
decrease thereafter till day 42 (818.402 µm and 797.074 µm) respectively (Table 7
and 10).
Different zones also exhibited variation in thickness as well as chondrocyte

diameter (size). Reserve zone of both the proximal as well as distal growth cartilages
(plates) of femur in males were measured to be ( 35.013 µm and 33.21 µm) on day1,
followed by a gradual increase up to day 28 ( 131.572 µm and 125.217 µm) and
became slightly thinner from day 35 till day 42 (87.358 µm and 87.542 µm)
respectively. The thickness of zone of proliferation varied from (120.659 µm and
119.321 µm) on day 1 to (219.32 µm and 234.354 µm) on day 28 respectively. It was
followed by a little decline till day 42 (145.781 µm and 193.321 µm). The
prehypertrophy zone was comparatively thinner than other zones. This zone was
50
(18.564 µm and 18.021 µm) thick on day1, that increased to day 7, 14 and 21 and
became maximum (127.153 µm and 99.279 µm) on day 28 that decreased a little
(85.365 µm and 83.321 µm) respectively on day 42.
The zone of hypertrophy and ossification measured (118.414 µm and 104.670

µm, and 115.256 µm and 103.254 µm) on day 1, (218.872 µm and 258.872 µm, and
152.62 µm and 248.034 µm) on day 28 and (134.069 µm and 372.321 µm, and
133.589 µm and 281.321 µm) on day 42 respectively.
The corresponding values for different zones in females followed almost a

similar trend with age like those of males. However, all the values were a little bit
lower (lesser) than those observed in respective male birds at different ages (Table 7
and 10).
Chondrocytes in different zones of proximal and distal growth cartilages in

male and female birds showed variation in respect to their size (diameter) at different
ages. The chondrocytes of reserve zone were the smallest ones, followed by those of
proliferative and prehypertrophic zones. In hypertrophic zone the cells were the
largest ones in the broiler chickens of different ages. The cell size of these zones in
both growth cartilages in day old male chicks were (8.269 µm and 8.102 µm), (8.834
µm and 8.701 µm), (8.924 µm and 8.825 µm) and (9.201 µm and 9.121 µm)
respectively. The cells grew a little larger from day 7 and became the largest on day
28, i.e. (11.063 µm and 11.02 µm), (12.042 µm and 12.064 µm), (12.421 µm and
12.121 µm) and (13.369 µm and 13.223 µm). Then the size of these cells became
slightly smaller from day 35 till day 42 (Table 7 and 10).
In females, the cell size in different zones varied accordingly like that of
males. However, there was very little variation (change) in the chondrocyte diameter
at different ages as compared to respective male birds (Table 7 and 10).
b) Tibiotarsus
Both the proximal and distal growth plates in males and female birds were
better recognized from day 1 onward till day 42. These growth plates revealed all the
histological zones distinctly like those observed in case of femur. The zonation
51
pattern was better elucidated from day 7 till day 28. But the typical arrangement of
zones was not clearly seen in the older age groups towards the end of experimental
period (Days 35 and 42). The reserve or resting zone contained one or two flattened or
oval chondrocytes (cartilage cells) within the lacunae scattered throughout the matrix
of this zone. There was poor vascularization in this zone as observed also in case of
femoral bones. The zone of proliferation did not show a typical row – wise or
columnar organization of the proliferating chondrocytes. The proliferating
chondrocytes gradually became larger in size in the zone of prehypertrophy (a thin
transitional zone) and subsequently enlarged the maximum to form the zone of
hypertrophy. The chondrocytes of the hypertrophic zone were usually spherical or
rounded in appearance in consistent with the shape of the lacunae. Scanty matrix of
this zone (in lower part) revealed provisional calcification resulting in entrapment of
the degenerating chondrocytes. This zone abutted on the zone of ossification that
revealed the presence of newly formed spongy or trabecular bone with intervening
marrow spaces and many invading blood vessels. Appearance of 2ndary centre of
ossification in both the epiphyseal ends on day 21 was a characteristic feature of this
bone. The amount of extracellular matrix was comparatively more in reserve and
proliferation zones, whereas a scanty amount of matrix surrounded the closely
arranged lacunae containing chondrocytes in rest of the zones. Masson’s trichrome
reaction was variable in different zones of the growth cartilage. The cartilage matrix
did not demonstrate the presence of collagen fibers as such, except those forming an
anastomosing, thread – like network around the lacunae of lower zones as well as in
the newly formed bony matrix in the ossification zone. Rest of the zones had very less
connective tissue elements. The connective tissue elements, namely collagen fibers
were seen in the walls of the penetrating blood vessels, lining of endosteum of
developing marrow spaces and newly formed trabecular bone matrix in zone of
ossification. Distal cartilage showed comparatively more collagen than the proximal
one, specifically on day 21.
The matrix of proliferative and hypertrophic zone was strongly alcianophilic.

Hypertrophic zone also revealed the presence of PAS – positive material. Sparse
amount of cartilage matrix was predominantly reactive to alcian blue than PAS. The
matrix of newly formed bony trabeculae in zone of ossification revealed moderate to
weak reaction for PAS with alcianophilia in some parts. No such difference in matrix
52
reactivity to PAS – alcian blue was observed between proximal and distal cartilages
as well as male and female birds. The histomorphological features of growth
cartilages of tibiotarsus bones in male as well as female birds are presented in Figs.99,
102, 103, 104, 109, 110, 111 & 115.
There was age and sex related variation in the histomorphometrical parameters
of the growth cartilages. The proximal and distal growth plates of male day old chicks
measured 652.112 µm and 413.356 µm in thickness respectively. The respective
cartilage gradually thickend with age of the birds from day 7 (993.213 µm and
896.965 µm) up to day 28 (1745.352 µm and 1614.890µm). Thereafter there was a
slight fall in these values from day 35 (1388.526 µm and 1493.568 µm) till day 42
(1302.785 µm and 1358.256 µm) respectively (Table 8). In females, the
corresponding values for proximal as well as distal growth cartilage were (651.863µm
and 412.856 µm) on day 1, (992.963 µm and 896.715 µm) on day 7, (1745.022 µm
and 1614.552µm) on day 28, followed a decrease thereafter till day 42 (1302.534 µm
and 1358.007 µm) respectively (Table 11).
There was variation in thickness as well as residing chondrocyte diameter

(size) in different zones. Reserve zone of both the proximal as well as distal growth
cartilages (plates) of tibiotarsus in males were measured to be ( 69.361 µm and 68.351
µm) on day1, followed by a gradual increase up to day 28 ( 203.041 µm and 179.256
µm) and became slightly thinner from day 35 till day 42 (84.198 µm and112.526 µm)
respectively. The thickness of zone of proliferation varied from (95.305 µm and
92.523 µm) on day 1 to (219.021 µm and 209.257 µm) on day 28 respectively. It was
followed by a slight decline till day 42 (89.895 µm and 114.325 µm). The
prehypertrophy zone appeared relatively thinner than other zones. This zone was
(61.698 µm and 58.652 µm) thick on day1, that increased gradually and became the
maximum (203.703 µm and 122.982 µm) on day 28 that decreased a little thereafter
to reach (74.527 µm and 74.658 µm) respectively on day 42(Table 8).
The zone of hypertrophy and ossification measured (93.102 µm and 91.356

µm, and 330.173 µm and 102.123 µm) on day 1, (215.237 µm and 197.359 µm, and
904.361 µm and 906.026 µm) on day 28 and (93.578 µm and 98.758 µm, and 999.672
µm and 948.567 µm) on day 42 respectively (Table 8).
53
The corresponding values for different zones in female birds followed almost a
similar trend like those of males throughout the experimental period. However, all the
values were a little bit lesser than those observed in respective male birds at different
ages (Table11).
The size (diameter) of chondrocytes in different zones of proximal and distal

growth plates in male and female birds showed variation at different ages. The
chondrocytes of reserve zone were the smallest ones among all the zones studied. The
cells of hypertrophic zone were larger than those of other zones in the broiler chickens
of different ages. The cell size of these zones in both of the growth cartilages in day
old male chicks were (11.982 µm and 11.003 µm), (12.022 µm and 12.050 µm),
(12.215 µm and 12.117 µm) and (13.235 µm and 13.009 µm) respectively. The cells
enlarged a little from day 7 and became the largest on day 28, i.e. (13.023 µm and
13.102 µm), (13.609 µm and 13.520 µm), (13.468 µm and 13.321 µm) and (14.418
µm and 14.224 µm). Then the cells became slightly smaller on day 35 and day 42
(Table 8).
In females, the cell size in different zones varied accordingly like that of
males. However, there was very little variation (change) in the size of chondrocytes at
different ages as compared to respective male birds (Table 11).
c) Tarsometatarsus
plates (cartilages) even in day old chicks (males and females) though the distal one
could not be identified grossly. Both the growth plates in males and female birds were
better recognized from day 7 onward till the end of the experimental period. These
growth plates elucidated all the histological zones distinctly like those observed in
case of femur as well as tibiotarsus. The pattern of different zones was better visible
from day 7 till day 28. However, the typical arrangement of zones was not clear cut in
the older age groups towards the end of experimental period (Days 35 and 42). The
reserve or resting zone had comparatively few, scattered, flattened or oval
chondrocytes (cartilage cells) usually one per each lacuna surrounded by relatively
more amount of matrix. Poor vascularization characterized this zone like that
observed in case of femoral and tibiotarsal bones. Proliferating chondrocytes (the
54
zone of proliferation) were not uniformly arranged in typical rows or columns. The
proliferating chondrocytes gradually became larger in the zone of prehypertrophy (a
thin transitional zone) and these subsequently attained the largest size in the zone of
hypertrophy. The enlarged, spherical or rounded chondrocytes of the hypertrophic
zone were usually housed inside the lacunae of similar appearance. Scanty matrix of
this zone (in lower part) with provisional calcification resulted in trapping of the
dying and degenerating chondrocytes. This zone finally continued into the zone of
ossification that revealed the presence of newly formed spongy or trabecular bone
with intervening marrow spaces and a number of invading blood vessels. Zone of
ossification of the distal cartilage appeared incomplete with less amount of bony
matrix surrounded by cartilage cells in the developing trabecular bone. Secondary
centre of ossification appeared in the proximal epiphyseal end on day 21 like that of
tibiotarsus bone. However, no secondary ossification centre appeared in the distal end
of the bone. The amount of extracellular matrix was comparatively more in reserve
and proliferation zones, whereas a scanty amount of matrix surrounded the closely
arranged lacunae containing chondrocytes in rest of the zones as also observed in
other two bones. Masson’s trichrome reaction was variable in different zones of the
growth cartilage. Collagen fibers as such were not discernible in the cartage matrix,
except those forming an anastomosing, thread – like network around the lacunae of
lower zones as well as in the newly formed bony matrix in the zone of ossification.
Other zones showed very sparse connective tissue elements. The connective
tissue elements, namely collagen fibers were seen in the walls of the invaded blood
vessels, lining of endosteum of developing marrow spaces and newly formed
trabecular or spongy bone matrix in the zone of ossification.
There was strong alcianophilia of the matrix of proliferative and hypertrophic

zone. Presence of PAS – positive substance was also revealed in the hypertrophic
zone. Sparse amount of cartilage matrix was predominantly reactive to alcian blue
than PAS. The matrix of newly formed bony trabeculae in zone of ossification was
moderate to weakly reactive for PAS along with alcianophilia. Matrix did not show
any conspicuous difference in PAS – alcian blue reactivity between proximal and
distal cartilages as well as between the male and female birds.The histomorphological
55
features of growth cartilages of tarsometatarsus bones in male as well as female birds
are presented in Figs. 119, 120, 121, 122, 123, 125, 128, 129 & 132.
Histomorphometrical parameters of the growth cartilages varied with the age

and sex of the broiler chickens. The distal cartilage was much thinner than the
proximal one in all the birds under study. The thickness of proximal and distal growth
plates of male day old chicks was 578.254 µm and 178.856 µm respectively. The
respective cartilage gradually thickened with age of the birds from day 7 (858.862 µm
and 285.354 µm) up to day 28 (1662.393 µm and 595.325µm). Thereafter there was a
slight decrease in these values on day 35 (1586.325 µm and 443.526 µm) and day 42
(1538.652 µm and 406.235 µm) respectively (Table 9). In females, the corresponding
values for proximal as well as distal growth cartilage were (651.863 µm and 413.106
µm) on day 1, (992.963 µm and 896.715 µm) on day 7 (1745.102 µm and 1614.641
µm) on day 28, followed a decrease thereafter till day 42 (1302.535 µm and 1358.007
µm) respectively (Table12).
The thickness of different zones as well as residing chondrocyte diameter

(size) varied. Reserve zone of both the proximal as well as distal growth cartilages
(plates) of tarsometatarsus in males were measured to be ( 65.93 µm and 62.356 µm)
on day1, followed by a gradual increase up to day 28 ( 136.407 µm and 101.211 µm)
and became slightly thinner from day 35 till day 42 (111.523 µm and 69.356 µm)
respectively. The thickness of zone of proliferation varied from 101.475 µm and
79.262 µm) on day 1 to (204.021443 µm and 97.568 µm) on day 28 respectively. It
was followed by a slight decrease till day 42 (168.023 µm and 58.658 µm). The
prehypertrophy zone appeared comparatively thinner than other zones. This zone was
(59.774 µm and 34.256 µm) thick on day1, that gradually increased and became
maximum (98.839 µm and 73.352 µm) on day 28 that declined a little thereafter to
reach (89.250 µm and 38.325 µm) respectively on day 42.The zone of hypertrophy
and ossification measured (85.788 µm and 78.324 µm, and 265.325 µm and 13.985
µm) on day 1, (250.509 µm and 82.658 µm, and 971.416 µm and 240.558 µm) on day
28 and 111.356 µm and 79.658 µm, and 1058.254 µm and 277.252 µm) on day 42
respectively (Table 9).
The corresponding values for different zones in female birds followed almost a
similar trend like those of males throughout the experimental period. However, all the
56
values were a little bit lesser than those observed in respective male birds at different
ages (Table12).
The size (diameter) of chondrocytes in different zones of proximal and distal

growth plates in birds of both sex showed variation at different ages. The
chondrocytes of reserve zone were the smallest ones among all the histological zones
in the present study. The cartilage cells of hypertrophic zone were the largest ones.
The cell size of these zones in both of the growth cartilages in day old male chicks
were (8.325 µm and 8.124 µm), (9.235 µm and 9.003 µm), (9.586 µm and 9.875 µm)
and (10.254 µm and 10.895 µm) respectively. The cells enlarged a little from day 7
and became the largest on day 28, i.e. (11.857 µm and 11.005 µm), (11.986 µm and
11.064 µm), (12.065 µm and 12.658 µm) and (12.245 µm and 12.654 µm). Then the
cells became slightly smaller on days 35 and day 42 (Table9).
In female birds, the chondrocyte size in different zones varied accordingly like
that of males. However, there was very little variation (change) in the values of this
parameter at different ages as compared to respective male birds (Table12).
4.4.2 Histomorphological Study of Bones
Histomorphological study of cortical (compact) bone from mid shaft region of

all the bones under study ( Femur, tibiotarsus and tarsometatarsus) in male as well as
fmale broiler chickens during post – hatch period ( from day 1 to day 42) was carried
out to assess the skeletal maturity and age – related changes in compact bone.
Presence of outer (peripheral) fibrous periosteum covering and inner, thin lining of
endosteum was a constant feature of the compact bone in the birds of both the sexes at
different ages. Although the compact bone appeared solid on gross observation, it
showed considerable detail microscopically. The characteristic feature of the compact
bone was the presence of several structural (histological) units called the osteons or
Haversian systems. Each osteon ( on cross section) appeared as a cylindrical (circular)
profile consisting of 1-4 concentric lamellae or layers of bone matrix around the
central osteonal or Haversian canal running parallel to the long axis of the bone. The
osteonal canals contained small blood vessels. The shape and size of these canals
varied with age of the birds. In younger stage, these canals were usually oval
elongated with few circular and even quadrangular profiles. The appearance of the
57
osteons also varied with the age of the birds. The compact bone appeared more porous
and was made up of interlacing woven bone resembling a network like pattern
containing ill – defined, primitive osteons. The periosteal and endosteal margins of
the cortical bone contained densely packed circumferential lamellar bone.This porous
bone gradually transformed into a compact mass later on and consisted of usual
(typical) osteons of circular profiles. Relatively porous woven bone was observed
from day 1 up to day 21 followed by a true compact bone thereafter. The outer
peripheral part of the compact bone appeared still porous and was characterized by
radial fibro-lamellar tissue. This peculiar histological feature was observed in all the
bones up to day 28. Then it was completely replaced by more typical osteons. A
second variety of canals carrying blood vessels was also seen to be connected with the
adjacent Haversian canals. These were more transversely oriented and endosteum
lined elongated profiles called perforating or Volkmann’s canals. The connective
tissue elements, namely bundles of collagen fibers were demonstrated (by Masson’s
trichrome stain) in the bone matrix as well as lining of endosteum of Haversian and
Volkmann’s canals. Collagen fibers were also present in the outer part of periosteum.
In younger birds, collagen fibers were randomly oriented around the developing
central (Haversian) canals. But in later stage, these fibers had orderly arrangement in
definitive osteons. Scanty matrix showed strong reactivity to alcian blue stain with
variable shades of PAS reaction. The histomorphology of different compact bones
under study are shown in Figs.76, 78, 80, 82, 84, 85, 86, 87, 88, 89,90,92, 93,95,96,
97, 100, 101, 105,106,107,108,112,113,114,116,117,118,124,126,127,130,131 &133.
Though the compact bone of femur, tibiotarsus and tarsometatarsus in the

birds of both sexes shared a common histoarchitecture, it showed individual variations
in respect to some histometrical parameters like thickness of periosteum and compact
bone, size of Haversian canal and system, and number of lamella with lacuna
containing osteocytes at different ages.
In femur of male birds, the cortical bone thickness varied from 713.958µm on
day 1 to 1425.256µm on day 42, whereas in females it ranged from 713.707µm to
1425.003µm. The diameter of Haversian canals and systems were (62.712 µm and
69.81 µm) on day 1 that gradually changed to (50.993 µm and 69.557 µm) on day
21and finally reached (26.102 µm and 125.256 µm) on day 42 respectively. In
58
females, the respective values were (62.462 µm and 69.57µm), (50.742µm and
91.057µm) and (25.851 µm and 125.007 µm).
The thickness of periosteum was measured as (26.662 µm and 26.302 µm) on

day 1 that became increasingly thicker with age and subsequently was the thickest one
on day 42(76.235 µm and 75.985 µm) respectively in male and females (Table13).
In tibiotarsus of male birds, the cortical bone thickness varied from

758.522µm on day 1 to 1690.214µm on day 42, whereas in females it ranged from
713.707µm to 1425.003µm. The diameter of Haversian canals and systems were
(74.283 µm and 84.593 µm) on day 1 that gradually changed to (39.760 µm and
95.325 µm) on day 21and finally reached (25.325 µm and 103.889 µm) on day 42
respectively. In females, the respective values were (74.003µm and 84.332 µm),
(39.51µm and 95.007µm) and (25.005 µm and 103.639 µm).
The thickness of periosteum was measured as (35.477µm and 335.227 µm) on

day 1 that became increasingly thicker with age and subsequently was the thickest one
on day 42(72.021 µm and 71.771 µm) respectively in male and females (Table13).
In tarsometatarsus of male birds, the cortical bone thickness varied from

737.350 µm on day 1 to 1541.740 µm on day 42, whereas in females it ranged from
737.101 µm to 1514.109 µm. The diameter of Haversian canals and systems were
(56.350 µm and 72.673 µm) on day 1 that gradually changed to (40.126 µm and
86.206 µm) on day 21and finally reached (23.726 µm and 118.325 µm) on day 42
respectively. In females, the respective values were (56.647µm and 72.421 µm),
(39.876µm and 85.906 µm) and (23.475 µm and 117.575 µm).
The thickness of periosteum was measured as (26.320µm and 26.001 µm) on

day 1, that became gradually thicker with age and subsequently became the thickest
one on day 42(58.321 µm and 57.328 µm) respectively in male and females
(Table13).
59
4.5.1 Estimation of Blood calcium (Ca) and Phosphorus (P) level
Serum Ca and P level (concentration) was estimated in male and female

broiler chickens on days 1, 7, 14, 21, 28, 35 and 42. The average serum Ca levels in
male and female birds were recorded to be (5.78 mg/dl and 5.6 mg/ dl) in day old
chicks, followed by a gradual increase (7.215 mg/dl and 6.85 mg/ dl) from day 7
onward. The values were the maximum (11.78 mg/dl and 10.50 mg/ dl) on day 42
(Table14).
Serum P level in male as well as female birds showed a similar increasing

trend with age. The respective values were (2.82 mg/dl and 2.61 mg/ dl) on day 1,
(3.06 mg/dl and 2.93 mg/ dl) on day 7 and (5.73 mg/dl and 5.145 mg/ dl) in the birds
of 42 days age (Table14).
The ratio between serum Ca and P level (Ca : P) varied accordingly with age
and sex of the birds. The ratios ranged from 2.05:1 to 2.35:1 in males, whereas it
varied from 2.04:1 to 2.32:1 in female birds (Table14).
4.5.2 Estimation of Bone calcium (Ca) and Phosphorus (P) Content
Bone Ca content (percentage) was estimated in male broiler chickens on days

1, 7, 14, 21, 28, 35 and 42. The average bone Ca percentage for femur, tibiotarsus and
tarsometatarsus in male birds were recorded to be (14.82, 14.5 and 14.2) in day old
chicks, followed by a gradual increase (16.11, 15.9 and 14.62) respectively from day
7 onwards. The corresponding values were the maximum (29.12, 26.52 and 24.73) on
day 42 (Table 15).
The respective values of bone P content (percentage) was estimated in male

broiler chickens on days 1, 7, 14, 21, 28, 35 and 42. The average bone Ca percentage
for femur, tibio-tarsus and tarso-metatarsus in male birds were recorded to be (7.15,
6.90 and 7.02) in day old chicks, followed by a gradual increase (7.75, 7.83 and 7.25)
from day 7 onwards. The values were the maximum (14.25, 13.0 and 12.3) on day 42
(Table 15).
The ratio between bone Ca and P percentage (Ca : P) varied accordingly with
age of the birds. The ratios ranged from 2.02:1 to 2.13:1 in femur, 2.03:1 to 2.1:1 in
tibio-tarsus and 2.01:1 to 2.06:1 in tarso-metatarsus in male birds (Table 15).
60
Table 1: Age related changes in gross biometrical parameters of femur in post-hatch male broiler chicken
Parameters Day 1 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42
Weight (gm) 0.311±1.34 NS 0.711 ±1.42NS 1.648±1.44* 3.542±1.36* 5.34 ±1.46* 7.362±1.52* 9.688±1.56
Length (cm) 2.9±0.71 NS 3.2 ±0.73 NS 4.6 ±0.75* 5.8 ±0.74* 6.6 ±0.76* 7.6±0.79 NS 7.9±0.81
Proximal 2.35±1..21* 3.94 ±1.23 * 4.95 ±1.29* 8.64 ±1.30* 9.82 ±1.31 NS 10.64±1.33 NS 10.88±1.38
Width/Diameter(mm) Mid shaft 2.20±0.59* 3.38 ±0.61* 3.95 ±0.61 NS 5.15 ±0.63* 6.32 ±0.65 NS 6.59±0.69 NS 7.09±0.71
Distal 2.98±1.07* 4.14 ±1.11* 5.19 ±1.21* 9.10 ±1.22* 10.11±1.24 11.07±1.28 NS 11.15±1.31
Marrow cavity thickness(mm) 0.7±0.48* 1.59 ±0.49 NS 1.95 ±0.51* 2.79 ±0.50* 3.67±0.55 NS 3.85±0.59 NS 4.28±0.63
Right Cortical bone Proximal 0.49±0.11 NS 0.60 ±0.12 NS 0.72 ±0.12 NS 0.80 ±0.15 NS 1.17 ±0.19 NS 1.22±0.21 NS 1.25±0.29
thickness(mm) Mid shaft 0.71±0.10 NS 0.87 ±0.11 NS 0.96 ±0.17 NS 1.15 ±0.19 NS 1.32 ±0.21 NS 1.38±0.22 NS 1.42 ±0.25
Distal 0.51±0.11 NS 0.59 ±0.15 NS 0.63 ±0.17 NS 0.76 ±0.19 NS 1.19 ±0.21 NS 1.22±0.22 NS 1.25 ±0.29
Distance between proximal & distal growth -- 2.4 ±0.96* 3.6 ±0.97* 5.3 ±0.99 NS 5.8 ±1.01* 6.8±1.07 NS 7.1 ±1.09
cartilage(cm)
Growth cartilage Proximal -- 0.62 ±0.10 NS 0.76 ±0.11 NS 0.94 ±0.13 NS 0.95 ±0.17 NS 0.85±0.21 NS 0.81±0.25
thickness(mm) Distal -- 0.58 ±0.10 NS 0.72 ±0.11 NS 0.74 ±0.15 NS 0.85± 0.17 NS 0.81±0.19 NS 0.79±0.21
Weight (gm) 0.313 ±1.33 NS 0.627 ±1.41* 1.892±1.42 * 3.374 ±1.39* 5.309 ±1.45* 7.871±1.49* 9.637±1.55*
Length (cm) 2.8 ±0.72 NS 3.1 ±0.71* 4.6 ±0.73* 5.6 ±0.74* 6.8 ±0.75 NS 7.6 ±0.78 NS 7.7±0.79
Width/Diameter (mm) Proximal 2.32 ±1.31* 3.66 ±1.33* 5.61 ±1.34* 9.05 ±1.36* 10.40 ±1.37 NS 10.78±1.39 NS 10.82±1.40
Mid shaft 1.88 ±0.29* 3.57 ±0.31 * 4.77 ±0.32* 5.85 ±0.33* 6.93 ±0.33 NS 7.38 ±0.36 NS 7.75±0.39
Distal 2.89 ±1.27 NS 3.96 ±1.29 * 6.88 ±1.22* 9.21 ±1.30* 10.59±1.33 NS 10.96±1.38 NS 11.05±1.41
Marrow cavity thickness (mm) 0.83 ±0.46* 1.74 ±0.49 NS 2.24 ±0.51 NS 3.14 ±53 NS 4.00 ±0.56 NS 4.25±0.58 NS 4.54±0.61
Left Cortical bone thickness Proximal 0.66 ±0.10 NS 0.76 ±0.11 NS 0.83 ±0.12 NS 0.95 ±0.13 NS 1.25±0.19 NS 1.28±0.21 NS 1.30±0.25
(mm) Mid shaft 0.53 ±0.10 NS 0.84 ±0.13 NS 1.22 ±0.15 NS 1.35 ±0.17 NS 1.50 ±0.19 NS 1.55±0.21 NS 1.59±0.26
Distal 0.6 ±0.11 NS 0.68 ±0.13 NS 0.76 ±0.15 NS 0.84 ±0.17 NS 1.22±0.19 NS 1.25±0.22 NS 1.30±0.23
Distance between proximal & distal growth -- 2.6 ±0.89* 3.7 ±0.91* 5.2 ±0.93* 5.8 ±0.95 NS 6.6±0.96 N S 6.9±0.99
cartilage (cm)
Growth cartilage thickness Proximal -- 0.41 ± 0.11 NS 0.6 ±0.10 NS 0.88 ±0.12 NS 0.98±0.15 NS 0.88±0.19 NS 0.75±0.23
(mm) Distal -- 0.38 ±0.11 NS 0.56 ±0.12 NS 0.79 ±0.13 NS 0.91±0.16 NS 0.85±0.17 NS 0.78±0.19
*Values in a row vary non-significantly ( p ≥ 0.05) and values for the same parameter in a column ( between right and left) show non-significant variation ( p ≥ 0.05).
61
Table 2: Age related changes in gross biometrical parameters of tibiotarsus in post-hatch male broiler chicken
Parameters Day 1 Day 7 Day14 Day21 Day28 Day35 Day42
Weight (gm) 0.55±1.96* 1.514±1.97* 2.38 ±1.99* 5.443 ±2.01* 7.893 ±2.12* 11.343 ±2.31* 14.180 ±2.33
Length (cm) 3.8±1.00 NS 4.3±1.01* 6.3 ±1.08* 7.9±1.11* 8.5 ± 1.15* 10.2±1.16* 11.4±1.19
Proximal 2.66±1.26* 4.68 ±1.37* 8.52±1.60* 9.77 ±1.87 NS 9.98 ±1.95* 12.67 ±1.99* 14.850±2.01
Width/Diameter(mm) Mid shaft 2.28±0.79* 3.73 ±0.83 NS 4.15±0.86* 5.93 ±0.87 NS 6.55 ±0.89 NS 7.35 ±0.90* 8.85 ±0.98
Distal 3.22 ±1.49* 5.25 ±1.57* 8.67±1.59* 10.30 ±2.0 NS 10.83 ±2.01* 12.84 ±2.11* 15.320 ±2.13
Marrow cavity thickness(mm) 0.74±0.57* 1.65 ±0.59 NS 2.02±0.61* 3.20 ±0.65 NS 3.33 ±0.67 NS 4.04 ±0.69* 5.45 ±0.71
Right Cortical bone Proximal 0.98±0.03 NS 1.09 ±0.07 NS 1.25±0.09 NS 1.45 0.11 NS 1.49±0.13 NS 1.52 ±0.14 NS 1.55 ±0.17
thickness(mm) Mid shaft 0.75±0.09 NS 0.96±0.11 NS 0.99±0.14 NS 1.35±0.15 NS 1.59 ±0.16 NS 1.64 ±±0.18 NS 1.68 ±0.19
Distal 0.88±0.05 NS 0.97 ±0.08 NS 1.19±0.11 NS 1.35±0.13 NS 1.45 ±0.16 NS 1.50 ± 0.18 NS 1.53 ±0.19
Distance between proximal & distal growth 2.22±1.01* 3.6 ±1.03* 5.4±1.08* 6.9±1.11* 7.8 ±1.13* 9.3 ±1.16* 9.88 ±1.19
cartilage(cm)
Growth cartilage Proximal 0.65±0.10 NS 0.99±0.11 NS 1.40±0.13 NS 1.52±0.14 NS 1.74±0.17 NS 1.38±0.19 NS 1.30±0.21
thickness(mm)
Distal 0.41 ±0.11 NS 0.89±0.13 NS 1.23±0.16 NS 1.40±0.17 NS 1.61±0.18 NS 1.49±0.19 NS 1.35±0.21
Weight (gm) 0.491 ±1.98* 1.567±1.99* 2.945±2.01* 6.027±2.22 * 8.424±2.26* 12.490±2.27* 14.105±2.31
Length (cm) 3.5±1.01 NS 4.2 ±1.01* 6.3±1.08* 8.2±1.11 NS 8.6±1.17* 10.3±1.19* 11.3±1.2
Width/Diameter (mm) Proximal 2.41 ±1.49* 4.47 ±1.51* 8.57±1.56* 10.06 ±1.57 NS 10.50±1.59 NS 11.45±1.61* 14.35±1.63
Mid shaft 2.16 ±0.82 NS 3.05± 0.87* 4.74±0.91* 6.10 ±0.96 NS 6.35±0.98* 7.4±1.01* 8.9±1.03
Distal 3.13 ±1.46* 4.74±1.49* 9.13±1.53* 10.18 ±1.54 NS 10.85±1.57 NS 11.79±1.59* 14.85±1.62
Marrow cavity thickness (mm) 0.85 ±0.49 NS 1.54±0.52 NS 1.97±0.54 NS 2.76±0.56 NS 2.80±0.59 NS 3.78±0.61* 5.10±0.63
Left Cortical bone thickness Proximal 1.02 ±0.09 NS 1.08 ±0.11 NS 1.19±0.12 NS 1.54±0.14 NS 1.70±0.15 NS 1.75±0.17 NS 1.78±0.19
(mm) Mid shaft 0.48 ±0.13 NS 0.83 ±0.16 NS 1.34±0.20 NS 1.66±0.21 NS 1.75±0.24 NS 1.8±0.27 NS 1.85±0.30
Distal 0.77 ±0.12 NS 0.86 ±0.13 NS 0.93±0.15 NS 1.38±0.17 NS 1.59±0.18 NS 1.63±0.19 NS 1.65±0.22
Distance between proximal & distal growth 2.8 ±0.97 NS 3.2 ±1.01* 5.6±1.03* 7.1±1.06 NS 7.7±1.09* 9.2±1.13 NS 9.75±1.16
cartilage (cm)
Growth cartilage thickness Proximal 0.53 ±0.10 NS 0.69 ±0.11 NS 0.86±0.12 NS 0.96±0.14 NS 1.52±0.17 NS 1.42±0.19 NS 1.36±0.20
(mm) Distal 0.46 ±0.09 NS 0.58 ±0.11 NS 0.91±0.15 NS 1.30±0.13 NS 1.45±0.16 NS 1.40±0.17 NS 1.28±0.19
62
Table 3: Age related changes in gross biometrical parameters of tarsometatarsus in post-hatch male broiler chicken
Weight (gm) 0.318±1.23NS 0.409±1.29* 1.612±1.31* 3.309±1.35* 5.067±1.38* 7.474±1.39* 9.593±1.41
Length (cm) 2.7±0.86 NS 3.2±0.89* 4.7±0.91* 6.2±0.92 NS 6.67±0.97* 8.3±0.99 NS 9.1±1.01
Proximal 4.22±1.39* 5.77±1.42* 9.93±1.45* 10.71±1.49 NS 11.15±1.51* 14.11±1.53 NS 14.88±1.56
Width/Diameter(mm) Mid shaft 2.81±0.92* 4.43±0.96 NS 5.32±0.99* 7.40±1.01 NS 8.1 0 ±1.05* 9.3±1.08 NS 9.85±1.11
Distal 4.06±1.39* 5.59±1.41* 9.44±1.43 NS 10.32±1.45 NS 10.90±1.47* 13.86±1.49 NS 14.25±1.52
Marrow cavity thickness(mm) 1.30±0.69* 2.4±0.72 NS 2.96±0.73* 4.07±0.77* 5.18±0.79* 6.29±0.83 NS 6.75±0.86
Right Cortical bone Proximal 0.94±0.03 NS 1.09±0.05 NS 1.15±0.06 NS 1.33±0.07 NS 1.40±0.11 NS 1.45±0.12 NS 1.48±0.14
thickness(mm) Mid shaft 0.73±0.04 NS 1.06±0.06 NS 1.18±0.09 NS 1.35±0.11 NS 1.45±0.12 NS 1.5±0.12 NS 1.53±0.13
Distal 0.93±0.03 NS 1.15±0.04 NS 1.20±0.05 NS 1.27±0.07 NS 1.42±0.09 NS 1.45±0.11 NS 1.46±0.15
Distance between proximal & distal growth -- 2.6±1.01 NS 3.2±1.03* 4.8±1.04* 5.8±1.09* 7.0±1.13 NS 7.9±1.17
cartilage(cm)
Growth cartilage Proximal 0.57±0.08 NS 0.85±0.09 NS 1.14±0.12 NS 1.32±0.15 NS 1.66±0.17 NS 1.58±0.18 NS 1.53±0.21
thickness(mm)
Distal -- 0.28±0.03 NS 0.42±0.05 NS 0.55±0.07 NS 0.59±0.09 NS 0.44±0.11 NS 0.40±0.17 NS
Weight (gm) 0.376±1.22 NS 0.387±1.27* 1.812 ±1.30* 5.183±1.48* 6.887±1.46* 8.548±1.38* 9.823±1.39
Length (cm) 2.5±0.84 NS 2.9±0.88* 4.8±091* 6.3±0.93 NS 6.8±0.95* 8.1±0.98 NS 8.9±0.99
Width/Diameter (mm) Proximal 4.09±1.38* 5.10±1.41* 8.83±1.47* 10.53±1.57* 11.72±1.51* 14.33±1.52 NS 14.61±1.56
Mid shaft 3.51±0.85 NS 4.41±0.87* 5.51±0.89* 7.41±0.91 NS 8.16±0.98* 9.24±1.07 NS 9.74±1.10
Distal 4.10±1.37 NS 4.93±1.38* 8.31±1.42* 9.90±1.49 NS 10.85±1.47* 13.81±1.48 NS 14.12±1.49
Marrow cavity thickness (mm) 1.91±0.66 NS 2.60±0.67 NS 3.2±0.68* 4.73±0.68 NS 5.20±0.71 NS 6.14±0.73 NS 6.58±0.78
Left Cortical bone thickness Proximal 0.91±0.03 NS 1.03±0.05 NS 1.12±0.07 NS 1.25±0.09 NS 1.44±0.10 NS 1.48±0.11 NS 1.52±0.13
(mm) Mid shaft 0.77±0.02 NS 0.85±0.05 NS 1.15±0.11 NS 1.31±0.12 NS 1.49±0.11 NS 1.55±0.13 NS 1.58±0.13
Distal 1.01±0.01 NS 1.13±0.03 NS 1.18±0.05 NS 1.27±0.06 NS 1.33±0.08 NS 1.4±0.11 NS 1.44±0.14
Distance between proximal & distal growth -- 2.4±0.99* 3.8±1.01* 5.7±1.06 NS 6.1±1.07* 7.3±1.11 NS 7.7±1.15
cartilage (cm)
Growth cartilage thickness Proximal 0.54±0.06 NS 0.89±0.08 NS 1.06±0.11 NS 1.23±0.12 NS 1.43±0.15 NS 1.36±0.18 NS 1.30±0.20
(mm) Distal -- 0.62±0.08 NS 0.74±0.09 NS 0.95±0.17 NS 1.32±0.16 NS 1.23±0.18 NS 1.19±0.21
63
Table 4: Age related changes in gross biometrical parameters of femur in post-hatch female broiler chicken
Parameters Day 1 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42
Weight (gm) 0.305±1.22NS 0.615±1.24NS 1.520±1.27* 3.155±1.30* 4.85±1.32* 7.475±1.34* 9.14±1.39
Length (cm) 2.8±0.58NS 3.0±0.59NS 4.5±0.66* 5.6±0.72* 6.3±0.75* 6.9±0.79NS 7.7±0.81
Proximal 2.31±1.20* 3.89±1.22* 4.77±1.23* 8.23±1.24* 9.75±1.27NS 10.07±1.29NS 10.15 ±1.33
Width/Diameter(mm) Mid shaft 2.15±0.58* 3.21±0.59* 3.85±0.61NS 5.01±0.64* 5.86±0.67NS 6.20±0.69NS 6.75 ±0.72
Distal 2.65±1.19* 4.00±1.20* 5.01±1.21* 8.37±1.23* 9.81±1.24 10.33±1.25NS 10.34 ±1.27
Marrow cavity thickness(mm) 0.71±0.33* 1.52±0.36NS 1.90±0.41* 2.65±0.45* 3.37±0.47NS 3.52±0.49NS 4.01 ±0.51
Right Cortical bone Proximal 0.45±0.08NS 0.55±0.09NS 0.72±0.10NS 0.79±0.11NS 0.87±0.13NS 1.18±0.17NS 1.22 ±0.21
thickness(mm) Mid shaft 0.70±0.07NS 0.82±0.08NS 0.90±0.09NS 1.08±0.10NS 1.22±0.14NS 1.32±0.15NS 1.36 ±0.19
Distal 0.48±0.04NS 0.55±0.05NS 0.58±0.08NS 0.71±0.12NS 1.03±0.15NS 1.19±0.19NS 1.25 ±0.21
Distance between proximal & distal growth -- 2.3±0.61* 3.4±0.79* 5.1±0.90NS 5.5±0.91* 6.1±0.93NS 6.6 ±0.97
cartilage(cm)
Growth cartilage Proximal -- 0.60 ±0.09NS 0.73 ±0.11NS 0.90 ±0.12NS 0.92 ±0.14NS 0.85 ±0.17NS 0.81 ±0.19
thickness(mm)
Distal -- 0.56 ±0.06NS 0.70 ±0.07NS 0.68±0.10NS 0.81 ±0.13NS 0.78 ±0.15NS 0.73 ± 0.19
Weight (gm) 0.301 ±1.13NS 0.610 ±1.18* 1.724 ±1.26* 3.105 ±1.29* 4.901± 1.31* 7.501 ±1.35* 9.05 ± 1.38
Length (cm) 2.8 ±0.59NS 3.0 ±0.62* 4.5±0.66* 5.1 ±0.70* 6.4 ±0.72NS 6.8 ±0.75NS 7.5 ±0.79
Width/Diameter (mm) Proximal 2.30 ±1.19* 3.61 ±1.21* 5.32 ±1.25* 8.75 ±1.27* 9.86 ±1.29NS 10.18 ±1.31NS 10.20± 1.33
Mid shaft 1.77 ±0.69* 2.66 ±0.72* 4.35 ±0.77* 5.73 ±0.81* 6.52 ±0.83NS 6.93 ±0.85NS 7.25 ±0.88
Distal 2.52 ±1.01NS 3.87 ±1.12* 6.81± 1.19* 9.11 ±1.22* 9.92 ±1.25NS 10.21 ±1.27NS 10.32 ±1.29
Marrow cavity thickness (mm) 0.75 ±0.46* 1.15 ±0.51NS 2.00 ±0.53NS 3.05 ±0.55NS 3.75 ±0.57NS 4.12 ±0.59NS 4.31 ±0.61
Left Cortical bone thickness Proximal 0.52 ±0.03NS 0.60 ±0.05NS 0.80± 0.07NS 0.88 ±0.09NS 1.02 ±0.10NS 1.11 ±0.13NS 1.15 ±0.17
(mm) Mid shaft 0.50 ±0.09NS 0.70 ±0.10NS 1.15 ±0.11NS 1.25 ±0.14NS 1.38 ±0.15NS 1.41 ±0.17NS 1.45 ±0.19
Distal 0.58 ±0.06NS 0.62 ±0.08NS 0.66 ±0.09NS 0.85 ±0.10NS 1.05 ±0.13NS 1.15 ±0.15NS 1.22 ±0.19
Distance between proximal & distal growth -- 2.5 ±0.73* 3.6 ±0.88* 5.6 ±0.91* 5.7 ±0.98NS 6.1 ±1.02NS 6.5 ±1.07
cartilage (cm)
Growth cartilage thickness Proximal -- 0.35 ±0.06NS 0.62 ±0.08NS 0.82 ±0.13NS 0.95 ±0.15NS 0.81 ±0.17NS 0.80 ±0.19
(mm) Distal -- 0.31 ±0.05NS 0.51 ±0.07NS 0.76 ±0.12NS 0.88 ±0.14NS 0.79 ±0.17NS 0.75 ±0.19
64
Table 5 : Age related changes in gross biometrical parameters of tibiotarsus in post-hatch female broiler chicken
Weight (gm) 0.51 ±1.77* 1.350±1.79* 2.18±1.83* 5.122±1.87* 7.135±1.89* 10.750±1.93* 13.520±1.96
Length (cm) 3.5 ±0.97NS 4.1±0.99* 5.9±1.00* 7.5±1.02* 8.1±1.07* 9.5±1.10* 10.8±1.13
Proximal 2.59 ±1.41* 4.32±1.46* 8.15±1.51* 9.50±1.53NS 9.73±1.57* 11.98±1.59* 14.105±1.61
Width/Diameter(mm) Mid shaft 2.15± 0.56* 3.20±0.59NS 3.71±0.64* 5.30±0.73NS 5.65±0.75NS 6.79±0.76* 7.35±0.79
Distal 3.19±1.21* 5.10±1.26* 8.22±1.38* 9.85±1.50NS 10.21±1.53* 12.12±1.54* 14.75±1.58
Marrow cavity thickness(mm) 0.70±0.35* 1.52±0.39NS 1.91±0.41* 2.86±0.46NS 3.01±0.48NS 3.75±0.49* 4.120±0.51
Right Cortical bone Proximal 0.91±0.03NS 1.01±0.05NS 1.18±0.07NS 1.32±0.08NS 1.39±0.11NS 1.45±0.15NS 1.48±0.19
thickness(mm) Mid shaft 0.70±0.09NS 0.82±0.10NS 0.85±0.11NS 1.20±0.13NS 1.31±0.16NS 1.52±0.18NS 1.60±0.21
Distal 0.82±0.04NS 0.93±0.07NS 1.10±0.08NS 1.26±0.09NS 1.35±0.13NS 1.40±0.15NS 1.45±0.18
Distance between proximal & distal growth 2.1±0.88* 3.0±0.91* 5.1±0.94* 6.1±0.99* 7.0±1.02* 8.6±1.08* 8.9±1.11
cartilage(cm)
Growth cartilage Proximal 0.61±0.06NS 0.89±0.08NS 1.36±0.11NS 1.43±0.12NS 1.52±0.15NS 1.35±0.17NS 1.26±0.21
thickness(mm)
Weight (gm) 0.411±1.12* 1.35±1.39* 2.30±1.63* 5.256±1.91* 7.55±1.92* 11.105±1.97* 13.63±2.01
Length (cm) 3.4±0.99NS 4.0±1.00* 5.9±1.02* 7.8±1.03NS 7.9±1.08* 9.6±1.11* 10.7±1.18
Width/Diameter (mm) Proximal 2.32±1.38* 4.10±1.42* 8.20±1.47* 9.48±1.50NS 9.84±1.53NS 11.12±1.55* 13.75±1.58
Mid shaft 1.75±0.63NS 2.80±0.68* 3.95±0.73* 5.30±0.78NS 5.85±0.82* 6.73±0.85* 7.38±0.89
Distal 2.92±1.38* 4.28±1.41* 8.68±1.44* 9.75±1.47NS 10.10±1.49NS 11.38±1.51* 13.95±1.57
Marrow cavity thickness (mm) 0.88±0.38NS 1.33±0.39NS 1.72±0.41NS 2.38±0.43NS 2.69±0.45NS 3.49±0.48* 4.05±0.51
Left Cortical bone thickness Proximal 0.94±0.02NS 0.99±0.06NS 1.05±0.07NS 1.25±0.11NS 1.46±0.12NS 1.50±0.13NS 1.55±0.16
(mm) Mid shaft 0.41±0.06NS 0.72±0.07NS 1.10±0.13NS 1.45±0.18NS 1.57±1.18NS 1.62±0.20NS 1.65±0.21
Distance between proximal & distal growth 2.3±0.86NS 2.7±0.88* 5.0±0.93* 6.2±0.99NS 7.1±1.01* 8.7±1.06NS 8.8±1.09
cartilage (cm)
Growth cartilage thickness Proximal 0.48±0.05NS 0.55±0.06NS 0.77±0.08NS 0.83±0.12NS 1.25±0.13NS 1.21±0.15NS 1.16±0.17
(mm) Distal 0.42±0.05NS 0.49±0.07NS 0.80±0.09NS 0.88±0.11NS 1.18±0.13NS 1.11±0.16NS 1.10±0.19
65
Table 6: Age related changes in gross biometrical parameters of tarsometatarsus in post-hatch female broiler chicken
Weight (gm) 0.310±1.11NS 0.390±1.17* 1.510 ±1.22* 3.210±1.30* 4.850± 1.33* 7.105±1.37* 9.253±1.39
Length (cm) 2.5 ±0.81NS 2.9 ±0.83* 4.4 ±0.86* 5.8 ±0.89NS 6.2±0.91* 7.7±0.93NS 8.8±0.98
Proximal 4.10±1.21* 5.35 ±1.28* 9.55±1.35 * 10.15 ±1.41NS 10.65 ±1.44* 13.25±1.47NS 14.12±1.51
Width/Diameter(mm) Mid shaft 2.60 ±0.77* 4.15 ±0.81NS 4.75 ±0.88* 6.25 ±0.91NS 7.50±0.93* 8.55±0.95NS 9.15±0.98
Distal 3.95 ±1.39* 4.45 ±1.41* 9.15 ±1.43NS 9.85 ±1.45NS 10.28±1.47* 13.12±1.49NS 13.78±1.53
Marrow cavity thickness(mm) 1.2 ±0.62* 2.25 ±0.65NS 2.55 ± 0.69* 3.75 ±0.72* 4.82±0.75* 5.78±0.76NS 6.22±0.79
Right Cortical bone Proximal 0.85 ±0.68NS 0.98 ±0.69NS 1.05 ±0.71NS 1.20± 0.73NS 1.26±0.76NS 1.32±0.79NS 1.38±0.81
thickness(mm) Mid shaft 0.69 ±0.06NS 0.94 ±0.07NS 1.09 ± 0.08NS 1.23± 0.10NS 1.31±0.13NS 1.38±0.17NS 1.45±0.21
Distal 0.88 ±0.02NS 1.05 ±0.04NS 1.10 ±0.05NS 1.18 ±0.07NS 1.31±0.11NS 1.35±0.15NS 1.41±0.17
Distance between proximal & distal growth -- 2.3 ±0.82NS 2.9 ±0.89* 4.1 ±0.91* 4.9±0.94* 6.1±0.96NS 6.9±0.99
cartilage(cm)
Growth cartilage Proximal 0.48 ±0.03NS 0.69 ±0.07NS 0.95 ±0.10NS 1.18 ±0.13NS 1.42±0.15NS 1.32±0.17NS 1.25±0.21
thickness(mm)
Distal -- 0.25±0.02NS 0.38±0.03NS 0.49 ±0.06NS 0.52 ±0.09NS 0.35 ±0.11NS 0.32±0.17NS
Weight (gm) 0.35 ±1.10 NS 0.366 ±1.22* 1.422 ±1.39* 4.85 ±1.41* 6.15 ±1.45* 7.95 ±1.48* 9.350±1.51
Length (cm) 2.4 ±0.77NS 2.7 ±0.81* 4.5 ±0.84* 6.1 ±0.86NS 6.4 ±0.89* 7.6 ± 0.91NS 8.2± 0.95
Width/Diameter (mm) Proximal 3.88 ± 1.11* 4.92 ±1.29* 8.52± 1.37* 10.05± 1.48* 10.85 ±1.49* 13.65 ±1.50NS 13.90±51
Mid shaft 3.28 ±0.66NS 4.10± 0.72* 4.95±0.79* 6.81± 0.88NS 7.82 ± 0.91* 8.80 ±0.93NS 9.20±0.96
Distal 3.92 ±1.33NS 4.85±1.38* 8.18 ±0.40* 9.66 ±1.41NS 10.15 ±1.42* 13.15 ±1.44NS 13.65±1.47
Marrow cavity thickness (mm) 1.84 ±0.55NS 2.48±0.61NS 2.93 ±0.62* 4.35 ±0.64NS 5.01 ±0.71NS 5.88 ±0.77NS 6.15±0.78
Left Cortical bone thickness Proximal 0.81 ±0.05NS 0.93±0.06NS 1.02 ±0.07NS 1.18±0.09NS 1.30 ±0.11NS 1.38±0.14NS 1.43±0.19
(mm) Mid shaft 0.70 ±0.05NS 0.78±0.06NS 0.96 ±0.09NS 1.18 ±0.12NS 1.39 ±0.13NS 1.45±0.14NS 1.52±0.19
Distal 0.92 ±0.01NS 0.99±0.02NS 1.10 ±0.04NS 1.18 ±0.06NS 1.22 ±0.08NS 1.31±0.11NS 1.38±0.18
Distance between proximal & distal growth -- 2.2 ±0.77* 3.1 ±0.91* 4.8 ±1.00NS 5.6 ±0.98* 7.0±1.01NS 7.2±1.03
cartilage (cm)
Growth cartilage thickness Proximal 0.51 ±0.02NS 0.78 ±0.06NS 0.95 ±0.07NS 1.15 ±0.10NS 1.22 ±0.11NS 1.18±0.15NS 1.15±0.16
(mm) Distal -- 0.60 ±0.04NS 0.68 ±0.09NS 0.85 ±0.15NS 1.12 ±0.17NS 1.10±0.16NS 1.05±0.17
66
Table 7 : Age related changes in histomorphometrical parameters of growth cartilage of femur in post hatch male broiler chicken
Parameters Day 1 Day7 Day 14 Day 21 Day 28 Day 35 Day 42
Total thickness( micro metre) 401.234±12.22* 620.212±12.49* 762.561±13.46* 942.324±14.57* 957.241±14.63* 859.783±14.75* 818.652±14.88
Reserve zone 35.013±11.62* 73.224±11.98* 111.227±12.32* 124.369±12.53 NS 131.572±12.64* 89.758±12.78 NS 87.358±12.93
Proliferation 203.654±15.22 148.368±15.66
120.659±15.07* 135.057±15.16* 209.614±15.30* 219.32±15.53* 145.781±15.72
NS NS
Prehypertrophy 18.564±12.96* 66.481±13.11* 93.379±13.19* 112.365±13.23* 127.153±13.27* 87.259±13.35 NS 85.365±13.41
Proximal Hypertrophy & Degeneration 118.414±16.22* 125.986±16.39* 208.321±16.55* 218.738±16.63* 136.064±16.75 134.069±16.81
200.79±16.43 NS
NS
Bone formation/Ossification zone 217.257±16.01 219.002±16.09 376.668±16.21
104.67±15.96* 225.021±16.11* 258.872±16.18* 372.321±16.33
NS NS NS
Diameter of cells of Reserve zone 8.269±0.48 NS 8.522±0.51* 9.517±0.55 NS 9.826±0.58* 11.063±0.61* 12.325±0.67 NS 11.586±0.71
Diameter of cells of Proliferation
8.834±0.52 NS 9.775±0.55* 10.882±0.58 NS 11.369±0.62 NS 12.042±0.67* 13.258±0.71 NS 13.104±0.77
zone
Diameter of cells of prehypertrophy
8.924±0.49* 9.948±0.53* 10.989±0.58 NS 11.698±0.61 NS 12.421±0.65 NS 13.261±0.69 NS 13.023±0.72
zone
Diameter of cells of hypertrophy zone
9.201±0.53* 10.332±0.65* 11.939±0.71 NS 12.334±0.79 NS 13.369±0.81* 14.987±0.83 NS 14.356±0.88
Reserve zone 122.321±12.52
33.21±12.13* 72.014±12.38* 123.254±12.80 NS 125.217±12.90* 99.985±13.13* 87.542±13.25
NS
Proliferation 201.121±16.95
119.321±16.58 NS 122.563±16.79* 207.365±17.01* 234.354±17.15* 204.325±17.28* 193.321±17.38
NS
Prehypertrophy
Distal 18.021±9.56* 64.231±9.77* 78.352±9.95* 87.325±10.10* 99.279±10.18* 85.321±10.25 NS 83.321±10.37
Hypertrophy & Degeneration 115.256±4.22* 124.321±4.35 135.981±4.63* 152.262±4.73* 144.025±4.88* 133.589±4.97
129.635±4.47 NS
NS
Bone formation 199.124±13.35 201.012±13.55 287.254±13.93
103.254±13.10* 203.654±13.76* 248.034±13.84* 281.321±13.99
NS NS NS
Diameter of cells of Reserve zone 8.102±0.21 NS 8.112±0.34* 9.351±0.48 NS 9.321±0.60* 11.02±0.72* 12.254±0.88* 11.032±0.97
8.701±0.33 NS 9.521±0.41* 10.742±0.53 NS 11.324±0.65 NS 12.064±0.72* 13.254±0.76 NS 13.022±0.87
zone
8.825±0.33* 9.823±0.45 NS 10.321±0.59* 11.625±0.62 NS 12.121±0.73* 13.123±0.81 NS 13.024±0.89
zone
Diameter of cells of hypertrophy zone 9.121±0.34* 10.321±0.45* 11.651±0.59* 12.522±0.61* 13.266±0.69 NS 13.223±0.76 NS 13.025±0.81
*Two successive values in a row vary significantly (P ≤ 0.05) and NS indicates non-significant (P ≥ 0.05) changes between two successive values in a row.
67
Table 8: Age related changes in histomorphometrical parameters of growth cartilage of tibiotarsus in post hatch male broiler chicken
Reserve zone 69.361±16.11* 102.369±16.25* 120.893±16.31 NS 124.623±16.35* 203.041±16.41* 132.256±16.53* 84.198±16.61
Proliferation 95.305±19.53* 111.921±19.73* 154.971±19.81* 211.365±19.92* 219.021±19.97* 135.325±20.04* 89.895±20.13
Prehypertrophy 61.698±17.08* 98.954±17.11* 109.38±17.21 NS 113.466±17.26* 203.703±17.32* 107.523±17.44* 74.527±17.53
Hypertrophy & Degeneration 93.102±18.14* 109.365±18.33* 147.122±18.67* 201.695±18.97* 215.237±19.01 122.356±19.17*
Proximal 93.578±19.31
NS
Bone formation 330.173±13.31* 570.054±13.42* 856.321±13.68* 871.853±13.98* 904.361±14.01* 989.689±14.28* 999.672±14.33
Diameter of cells of Reserve zone 11.982±0.09 NS 12.321±0.18 NS 12.389±0.25 NS 12.718±0.43 NS 13.023±0.51* 11.235±0.62 NS 9.662±0.71
Diameter of cells of Proliferation zone 12.022±0.22* 13.101±0.31 NS 13.285±0.45 NS 13.316±0.51 NS 13.609±0.63* 11.289±0.68* 9.935±0.72
Diameter of cells of Prehypertrophy zone 12.215±0.21* 13.221±0.29 NS 13.357±0.36 NS 13.473±0.45 NS 13.486±0.54* 11.235±0.57* 10.523±0.61
Diameter of cells of hypertrophy zone 13.398±0.24 NS
13.235±0.11 NS 14.033±0.31 NS 14.269±0.37 NS 14.418±0.41* 13.273±0.49* 11.528±0.53
68.351±15.42* 99.214±15.53* 158.321±15.76* 179.256±15.92* 151.523±16.01* 112.526±16.20
NS
Proliferation 92.523±15.89* 149.523±15.93* 174.223±16.02* 198.324±16.05* 209.257±16.11* 148.235±16.24* 114.325±16.37
Prehypertrophy 117.325±8.98
Distal 58.652±8.24* 87.481±8.46* 108.354±8.66* 122.982±9.01* 107.354±9.21* 74.658±9.33
NS
Hypertrophy & Degeneration 91.356±16.01* 141.869±16.05* 168.225±16.09* 195.321±16.11* 197.359±16.19 135.452±16.22* 98.758±16.37
NS
Bone formation 102.123±27.89* 418.278±27.97* 621.902±28.01* 716.488±28.08* 906.026±28.19* 919.952±28.27* 948.567±28.39
Diameter of cells of Reserve zone
11.003±0.01* 12.281±0.22 NS 12.285±0.36 NS 12.703±0.50 NS 13.102±0.68* 11..123±0.77* 9.234±0.91
Diameter of cells of Proliferation zone
12.050±0.22* 13.034±0.29 NS 13.258±0.31 NS 13.297±0.58 NS 13.52±0.62* 11.112±0.68* 9.324±0.75
Diameter of cells of prehypertrophy zone
12.117±0.21* 13.134±0.31 NS 13.286±0.42 NS 13.362±0.47 NS 13.321±0.53* 11.146±0.67* 10.237±0.77
Diameter of cells of hypertrophy zone
13.009±0.28 NS 13.357±0.29 NS 14.105±0.33 NS 14.182±0.37 NS 14.224±0.48* 12.982±0.56* 11.485±0.63
68
Table 9: Age related changes in histomorphometrical parameters of growth cartilage of tarsometatarsus in post hatch male broiler
chicken
Reserve zone 65.93±10.42* 98.623±10.53* 155.961±10.65* 106.954±10.88* 136.407±10.91* 124.365±11.04* 111.523±11.17
Proliferation 101.475±13.22* 133.401±13.39* 163.187±13.67* 198.652±13.73 NS 204.443±13.82* 178.658±13.91* 168.023±14.01
Prehypertrophy 59.774±5.01* 90.538±5.14* 81.268±5.27* 94.237±5.35 NS 98.839±5.41 NS 92.35±5.53 NS 89.25±5.67
Hypertrophy & Degeneration 85.788±22.10* 113.115±22.31* 184.325±22.53* 205.746±22.95* 250.509±23.01* 121.256±23.11* 111.356±23.34
Proximal
Bone formation 265.325±1.09* 421.368±1.13* 572.569±1.15* 718.155±1.18* 971.416±1.21* 1018.256±1.34* 1058.254±1.43

Diameter of cells of Reserve zone 10.785±0.63
8.325±0.33 NS 9.214±0.38* 10.583±0.41 NS 11.244±0.46 NS 11.857±0.51* 10.056±0.71
NS
9.235±0.28 NS 9.567±0.31* 10.608±0.35 NS 11.405±0.39 NS 11.986±0.42* 10.264±0.47* 11.528±0.53
zone
Diameter of cells of 12.005±0.53
9.586. ±0.29* 10.652±0.31 NS 10.911±0.33 NS 11.801±0.34 NS 12.065±0.41 NS 11.256±0.67
prehypertrophy zone NS
Diameter of cells of hypertrophy
10.254±00.21 NS 10.966±0.28 NS 11.277±0.31 NS 11.449±0.37 NS 12.245±0.43 NS 12.046±0.51* 13.258±0.63
zone
* * *
Total thickness( micro metre) 178.856±4.11 NS 285.354±4.32 429.856±4.53 555.782±4.77 595.325±4.81* 443.526±4.92* 406.235±5.02
* *
Reserve zone 62.356±5.11 NS 66.235±5.23 NS 69.325±5.31 89.201±5.37 101.211±5.41* 82.235±5.52* 69.356±5.67
Proliferation 79.262±5.79* 88.657±5.86* 99.256±5.91* 109.657±6.60* 97.568±6.71* 73.256±6.79* 58.658±6.88
Prehypertrophy 34.256±5.98* 58.685±6.01 NS 62.223±6.08* 77.253±6.17 NS 73.352±6.22* 62.985±6.34* 38.325±6.48
Distal Hypertrophy & Degeneration 78.324±3.21 * 86.258±3.34 * 79.658±3.55 * 101.736±3.76 * 82.658±3.82* 69.256±3.89* 79.658±3.91
* * * *
Bone formation 13.985±20.21 28.785±20.32 117.852±20.55 177.464±20.64 240.558±20.75* 252.325±20.81* 277.252±20.91
Diameter of cells of Reserve zone 8.124±0.28* 10.009±0.31 NS 10.008±0.33 NS 10.272±0.35 NS 11.005±0.41 NS 10.235±0.52* 9.256±0.61
9.003±0.11* 10.523±0.17 NS 10.759±0.21 NS 10.889±0.28 NS 11.064±0.32 NS 10.985±0.41* 9.985±0.58
zone
Diameter of cells of
9.875±0.18* 11.085±0.27 NS 11.984±0.31 NS 12.246±0.39 NS 12.658±0.42* 11.523±0.47* 10.253±0.56
prehypertrophy zone
Diameter of cells of hypertrophy
10.895±0.11 NS 11.256±0.17 NS 11.307±0.21* 12.333±0.27 NS 12.654±0.32* 11.216±0.41* 10.856±0.53
zone
69
Table 10 : Age related changes in histomorphometrical parameters of growth cartilage of femur in post hatch female broiler chicken
Total thickness( micro metre) 400.984±14.11* 619.962±14.24* 762.311±14.38* 942.074±14.57* 956.991±14.63˟ 859.432±14.75˟ 818.402±14.86
Reserve zone 124.117±12.50 89.508±12.67
34.763±12.10* 72.973±12.22* 110.977±12.38* 131.326±12.58˟ 87.108±12.72
NS NS
Proliferation 203.403±15.28 148.218±15.48
120.409±15.11* 134.807±15.23* 209.363±15.30* 219.073±15.39˟ 145.531±15.67
NS NS
18.562±13.04* 66.231±13.11* 93.129±13.18* 112.114±13.20* 126.902±13.31˟ 85.114±13.53
NS
Proximal
Hypertrophy & Degeneration 118.164±16.21* 125.716±16.32* 200.541±16.44 208.077±16.55* 218.488±16.63˟ 135.814±16.71 133.819±16.85
NS NS
Bone formation 218.752±16.01 376.418±16.31
104.67±15.86* 217.007±15.92 NS 224.778±16.09* 258.632±16.17˟ 372.077±16.36
NS NS
Diameter of cells of Reserve zone 8.521±0.13 NS 8.723±00.23* 8.810±0.39 NS 9.007±0.42* 9.214±0.51˟ 11.426±0.62 NS 10.658±0.73
Diameter of cells of Proliferation zone
8.562±0.21 NS 8.776±0.28* 8.893±0.32 NS 9.125±0.38 NS 9.362±0.43˟ 11.214±0.53 NS 10.559±0.64
8.882±0.24* 8.985±0.36* 9.006±0.53 NS 9.243±0.62 NS 9.882±0.71 NS 12.887±0.82 NS 11.987±0.97
zone
Diameter of cells of hypertrophy zone 8.987±0.16* 9.012±0.34* 9.112±0.44 NS 9.426±0.63 NS 10.468±0.74˟ 13.002±0.88 NS 12.223±0.98
Total thickness( micro metre) 391.984±11.34* 582.117±11.58* 726.432±11.73* 749.738±11.91 859.382±12.02˟ 818.741±12.11˟ 797.074±12.34
Reserve zone 122.073±12.65 123.001±12.81
32.89±12.21* 71.764±12.43* 124.967±12.94˟ 99.735±13.02˟ 87.291±13.21
NS NS
Proliferation 200.872±10.99
119.072±10.86 NS 122.313±10.93* 207.106±11.01 234.104±11.12˟ 204.075±11.27˟ 193.071±11.31
NS
17.771±9.84* 63.982±9.92* 78.102±10.02* 87.076±10.09 99.019±10.16˟ 83.071±10.38
NS
* *
Hypertrophy & Degeneration 115.007±4.13 124.072±4.33 129.385±4.51
Distal 135.732±4.60 152.012±4.71˟ 143.025±4.83˟ 133.349±4.91
NS
Bone formation 200.762±23.63 287.254±23.94
103.006±23.22* 198.874±23.47 NS 203.404±23.72 247.784±23.81˟ 281.082±23.99
NS NS
Diameter of cells of Reserve zone 8.321±0.22 NS 8.654±0.37* 8.789±0.44 NS 8.985±0.53 9.032±0.61˟ 12.004±0.73˟ 11.121±0.82
Diameter of cells of Proliferation zone 8.456±0.21 NS 8.557±0.38* 8.859±0.57 NS 8.965±0.63 NS 9.231±0.71˟ 13.004±0.83 NS 11.036±0.91
Diameter of cells of prehypertrophy 8.654±0.22* 8.839±0.39 NS 8.952±0.52* 9.025±0.63 NS 9.726±0.68˟ 11.771±0.90
zone 12.873±0.71 NS
Diameter of cells of hypertrophy zone 8.965±0.19* 9.001±0.41* 9.021±0.53* 9.235±±0.62* 10.321±0.69 NS 12.089±0.88
12.973±0.73 NS
70
Table11: Age related changes in histomorphometrical parameters of growth cartilage of tibiotarsus in post hatch female broiler chicken
Total thickness( micro metre) 651.863±1.11˟ 992.963±1.23˟ 1408.073±1.29˟ 1522.753±1.36˟ 1745.022±1.42˟ 1388.026±1.51˟ 1302.534±1.58
Reserve zone 120.643±16.27 83.947±16.67
69.112±16.08˟ 102.117±16.13˟ NS 124.373±16.34˟ 202.792±16.42˟ 131.756±16.53˟
Proliferation 95.056±19.31˟ 111.673±19.45˟ 154.721±19.73˟ 211.116±19.92˟ 218.773±20.01˟ 134.825±20.13˟ 89.645±20.22
Prehypertrophy 109.113±17.21 74.227±17.53
61.447±17.08˟ 98.703±17.16˟ NS 113.217±17.26˟ 203.453±17.33˟ 107.003±17.46˟
Hypertrophy & Degeneration 214.987±19.03 93.329±19.29
Proximal 109.105±18.39˟ 146.872±18.67˟ 201.408±18.96˟ NS
93.102±18.24˟ 121.856±19.17˟
Bone formation 330.173±23.22˟ 569.804±23.37˟ 856.063±23.49˟ 871.623±23.53˟ 904.112±23.64˟ 989.189±23.76˟ 999.328±23.84
Diameter of cells of Reserve zone 11.072±0.17 11.139±0.21 11.468±0.26 9.662±0.47
10.733±0.08˟ NS NS NS 11.776±0.32˟ 10.735±0.39˟
Diameter of cells of Proliferation zone 11.602±0.19 12.036±0.26 12.067±0.33 9.935±0.59
10.773±0.08˟ NS NS NS 12.359±0.41˟ 10.789±0.52˟
Diameter of cells of prehypertrophy zone 10.966±0.08 11.978±0.17 12.108±0.22 12.224±0.30 10.735±0.43 10.523±0.48
NS NS NS NS 12.465±0.38˟ NS
Diameter of cells of hypertrophy zone 12.148±0.13 12.782±0.21 13.026±0.23 11.528±0.46
11.985±0.08˟ NS NS NS 13.168±0.34˟ 12.773±0.39˟
Reserve zone 67.852±15.33˟ 174.726±15.91 179.006±16.02˟ 151.273±16.18˟ 112.277±16.23
98.964±15.52˟ 158.072±15.73˟ NS
Proliferation 92.023±15.86˟ 149.273±15.93˟ 173.974±16.01˟ 198.075±16.05˟ 209.009±16.09˟ 147.984±16.21˟ 114.077±16.31
Prehypertrophy 58.156±8.35˟ 117.077±9.00 122.732±9.16˟ 107.104±9.21˟ 74.408±9.28
87.232±8.67˟ 108.106±8.91˟ NS
Distal Hypertrophy & Degeneration 91.107±15.88˟ 141.619±16.01˟ 167.976±16.08˟ 195.071±16.11˟ 197.107±16.24 135.202±16.30˟ 98.508±16.38
NS
Bone formation 102.123±27.87˟ 418.278±27.93˟ 621.902±28.01˟ 716.488±28.08˟ 906.026±28.11˟ 919.952±28.19˟ 948.567±28.22
Diameter of cells of Reserve zone 9.803±0.03˟ 10.034±0.11 10.046±0.16 10.453±0.17 10.778±0.21˟ 11.123±0.32˟ 10.234±0.41
NS NS NS
Diameter of cells of Proliferation zone 10.771±0.05˟ 10.789±0.09 11.008±0.11 11.048±0.13 11.221±0.18˟ 11.412±0.22˟ 10.324±0.26
NS NS NS
Diameter of cells of prehypertrophy zone 10.017±0.06˟ 10.885±0.13 11.036±0.16 11.113±0.20 11.321±0.23˟ 11.446±0.28˟ 10.237±0.35
NS NS NS
Diameter of cells of hypertrophy zone 10.759±0.08 11.107±0.12 11.855±0.17 11.933±0.26 12.224±0.29˟ 12.782±0.35˟ 11.485±0.46
NS NS NS NS
71
Table 12: Age related changes in histomorphometrical parameters of growth cartilage of tarsometatarsus in post hatch female broiler
chicken
Reserve zone 69.111±16.01˟ 102.119±16.22˟ 120.643±16.28˟ 124.373±16.34˟ 202.792±16.47˟ 132.006±16.55˟ 83.948±16.68
Proliferation 211.116±±19.92
95.056±19.21˟ 111.671±19.35˟ 154.721±19.75˟ 218.771±19.99˟ 135.076±20.04˟ 89.644±20.18
NS
Prehypertrophy 113.216±17.26 203.453±17.32 107.273±17.41
61.448±17.02˟ 98.704±17.11˟ 109.133±17.20˟ 74.277±17.53
NS NS NS
Proximal Hypertrophy & Degeneration 92.852±18.23˟ 109.117±18.47˟ 146.872±18.68˟ 201.445±18.97˟ 214.987±19.01˟ 122.107±19.11˟
93.328±19.31
Bone formation 329.923±23.07˟ 569.805±23.24˟ 856.071±23.49˟ 871.603±23.58˟ 904.112±23.61˟ 989.439±23.66˟ 999.422±23.71
11.732±0.08 NS 12.072±0.16˟ 12.133±0.33 NS 12.468±0.43 NS 12.773±0.49˟ 9.412±0.63
NS
Diameter of cells of Proliferation zone 11.772±0.25 NS 12.851±0.33˟ 13.035±0.42 NS 13.066±0.51 NS 13.359±0.55˟ 11.039±0.67˟ 9.685±0.71
Diameter of cells of prehypertrophy 13.236±0.51 10.985±0.53
11.965±0.11˟ 12.971±0.25 NS 13.107±0.39 NS 13.226±0.46 NS 10.273±0.63
zone NS NS
Diameter of cells of hypertrophy zone 14.168±0.41
12.985±0.08 NS 13.144±0.17 NS 13.784±0.22 NS 14.009±0.37 NS 13.023±0.48˟ 11.278±0.62
NS
Total thickness( micro metre) 413.106±1.24 NS 896.715±1.35˟ 1230.775±1.47˟ 1402.118±1.53˟ 1614.641±1.61˟ 1403.318±1.72˟ 1358.007±1.81
68.101±15.27 NS 158.071±15.71˟ 174.735±15.89˟ 179.007±15.93˟ 151.273±16.05˟ 112.276±16.22
NS
Proliferation 92.273±15.89˟ 149.273±15.97˟ 173.973±16.03˟ 198.074±16.05˟ 209.009±16.18˟ 147.889±16.27˟ 114.076±16.38
Prehypertrophy 58.402±8.31˟ 87.232±8.46 NS 108.104±8.76˟ 117.075±8.97 NS 122.732±9.01˟ 107.104±9.17˟ 74.406±9.31
Hypertrophy & Degeneration 91.106±15.86˟ 141.619±15. 167.975±16.01˟ 195.073±16.11˟ 197.109±16.13˟ 135.202±16.27˟ 98.508±16.38
Distal 94˟
Bone formation 101.873±27.37˟ 418.278±27.89˟ 621.652±28.01˟ 716.238±28.07˟ 905.776±28.11˟ 919.702±28.24˟ 948.317±28.30
10.753±0.22˟ 12.281±0.31 NS 12.035±0.38 NS 12.453±0.47 NS 10.873±0.62˟ 8.984±0.73
NS
Diameter of cells of Proliferation zone 11.852±0.61
11.771±0.27˟ 13.034±0.34 NS 13.008±0.48 NS 13.047±0.55 NS 10.862±0.68˟ 9.124±0.76
NS
11.866±0.14˟ 13.134±0.27 NS 13.233±0.31 NS 13.112±0.44 NS 12.071±0.48˟ 10.896±0.53˟ 10.237±0.61
zone
Diameter of cells of hypertrophy zone 12.759±0.01 NS 13.357±0.17 NS 13.856±0.28˟ 13.833±0.33 NS 13.977±0.39˟ 12.733±0.43˟ 11.535±0.52
72
Table 13: Age related changes in histomorphometrical parameters of compact bone of femur, tibiotarsus and tarsometatarsus in post hatch male and female broiler
chicken
Male Female
Cortical bone Diameter of Periosteum Diameter of Cortical bone Diameter of Periosteum Diameter of
Parameters thickness(Mid Haversian canal thickness (µm) Haversian system thickness(Mid Haversian canal thickness (µm) Haversian
shaft) (µm) (µm) (µm) shaft) (µm (µm) system (µm)
Day 1 713.958 ±8.42* 62.712±4.98* 26.662±5.98* 69.81±7.87 NS 713.707±8.01* 62.462±4.89* 26.302±5.82* 69.57±7.79 NS
Day 7 877.129 ±8.51* 56.923±5.02 NS 36.138±5.99* 72.323±7.89* 876.879±8.13* 56.673±4.91 NS 35.887±5.87* 72.072±7.81*
Day 14 968.853 ±8.69* 54.295±5.03 NS 46.483±6.22 NS 83.592±8.12* 968.701±8.17* 54.032±4.96 NS 46.233±5.97 NS 83.342±7.93*
Femur Day 21 1157.068±8.91* 50.993±5.08* 49.557±6.31 NS 91.306±8.22* 1156.817±8.21* 50.742±5.07* 49.307±6.11 NS 91.057±8.01*
Day 28 1135.264 ±8.95* 41.350±5.09* 52.456±6.35* 110.890±8.24* 1135.004±8.24* 41.102±5.09* 52.207±6.16* 110.502±8.04*
Day 35 1391.066±8.97* 32.690±5.19 NS 65.253±6.37* 118.305±8.25* 1390.816±8.27* 32.711±5.11* 65.003±6.19* 118.057±8.08*
Day 42 1425.256±8.99 26.102±5.22 76.235±6.39 125.256±8.26 1425.003±8.29 25.851±5.17 75.985±6.22 125.007±8.11
Day 1 758.522±1.08* 74.283±5.58* 35.477±4.08* 84.593±1.85 NS 758.271±1.08* 74.003±5.96* 35.227±4.12* 84.332±1.95 NS
Day 7 966.92±1.12* 53.512±5.72* 43.751±4.22* 88.515±1.92 NS 966.07±1.10* 53.226±6.01* 43.502±4.27* 88.237±2.06 NS
Day 14 1002.45±1.37* 46.31±5.98* 55.214±4.42 NS 92.742±2.11 NS 1001.231±1.27* 46.006±6.09* 54.963±4.38 NS 92.493±2.10 NS
TibioTarsus Day 21 1361.213±1.43* 39.76±6.12 NS 58.321±4.63 NS 95.325±2.21 NS 1360.963±1.33* 39.51±6.11 NS 58.073±4.52 NS 95.007±2.12 NS
Day 28 1599.723±1.47* 38.31±6.14* 63.851±4.68 NS 96.53±2.27 NS 1599.373±1.37* 38.001±6.15* 63.501±4.61 NS 96.11±2.17 NS
Day 35 1647.64±1.49* 27.812±6.22 NS 67.45±4.73 NS 98.355±2.31* 1647.33±1.38* 27.551±6.19 NS 67.112±4.65 NS 98.102±2.21*
Day 42 1690.214±1.52 25.325±6.31 72.021±4.77 103.889±2.36 1689.963±1.42 25.005±6.27 71.771±4.71 103.639±2.26
Day 1 737.350±9.91* 56.896±4.01 NS 26.32±3.88 NS 72.673±5.92 NS 737.101±9.08* 56.647±4.07 NS 26.001±3.89 NS 72.421±4.89 NS
Day 7 1070.325±9.97* 53.256±4.12 NS 28.324±3.92 NS 76.321±5.99* 1070.025±9.18* 53.006±4.09 NS 28.024±3.94 NS 75.821±4.93*
Day 14 1188.351±9.99* 51.325±4.19* 32.251±4.05* 82.321±6.06 NS 1187.851±9.26* 50.825±4.11* 32.001±3.99 NS 82.071±5.01*
TarsoMetatarsus Day 21 1359.706±10.2* 40.126±4.23* 38.743±4.11* 86.206±6.10* 1359.026±9.32* 39.876±4.13 NS 38.339±4.03* 85.906±5.09*
Day 28 1456.321±10.5* 35.327±4.27* 45.321±4.17* 95.325±6.18* 1455.821±9.34* 34.827±4.19* 44.820±4.08* 94.805±5.13*
Day 35 1508.250±10.7* 26.720±4.31 NS 51.230±4.21* 106.728±6.26* 1507.625±9.39* 26.470±4.21 NS 50.980±4.13* 106.321±5.17*
Day 42 1541.740±10.9 23.726±4.37 58.321±4.32 118.325±6.29 1541.109±9.42 23.475±4.28 57.328±4.21 117.575±5.19
73
Table 14: Age related changes in Serum Calcium and Phosphorus level (mg/ dl ) in post hatch broiler chicken at different ages
Calcium Phosphorus Calcium phosphorus ratio
Days
Male Female Male Female Male Female
Day 1 5.78 ±0.69˟ 5.6 ±0.55˟ 2.82 ± 0.33 NS 2.61 ±0.25 NS 2.05:1 2.14:1
Day 7 7.215 ±0.71 NS 6.815 ±0.57 NS 3.06± 0.34 NS 2.93 ±0.27 NS 2.35:1 2.32:1
Day 14 7.675 ±0.73 NS 6.91 ±0.59 NS 3.49±0.36 NS 3.285 ±0.29 NS 2.20:1 2.10:1
Day 21 8.270 ±0.76 NS 7.26 ±0.61 NS 3.705±0.37 NS 3.435±0.32 NS 2.23:1 2.11:1
Day 28 8.885 ± 0.79˟ 7.635 ±0.63˟ 3.865± 0.39 NS 3.730 ±0.35 NS 2.30:1 2.04:1
Day35 10.50 ±0.81˟ 9.03 ±0.65˟ 4.585±0.41 NS 4.250±0.37 NS 2.29:1 2.12:1
Day 42 11.78 ±0.85 10.500 ±0.68 5.730 ± 0.43 5.145 ±0.41 2.05:1 2.04:1
Table 15: Age related changes in Bone Calcium and Phosphorus level (%) in post hatch male broiler chicken at different ages
Days Femur Tibiotarsus Tarsometatarsus
Ca(%) P (%) Ratio Ca (%) P (%) Ratio Ca (%) P (%) Ratio
Day 1 14.82 ±0.69* 7.15 ±0.28 NS 2.06:1 14.50±0.69* 6.90±0.29 NS 2.1:1 14.20±0.69 NS 7.02±0.32 NS 2.02:1
Day 7 16.11 ±0.72* 7.75±0.31 NS 2.08:1 15.90±0.71* 7.83±0.31 NS 2.03:1 14.62±0.71 NS 7.25±0.37 NS 2.01:1
Day 14 17.30 ± 0.74* 8.50±0.35 NS 2.03:1 16.83±0.75 NS 8.20±0.33 NS 2.05:1 15.23±0.75 NS 7.45±0.38 NS 2.04:1
Day 21 18.62 ±0.76 * 9.15±0.37 NS 2.03:1 17.61±0.77 NS 8.60±0.39 NS 2.05:1 16.11±0.78 NS 7.95±0.40 NS 2.03:1
Day 28 20.90 ±0.78* 9.80±0.38 NS 2.13:1 18.23±0.79* 8.90±0.41* 2.05:1 16.50±0.81* 8.10±0.43* 2.04:1
Day 35 24.22 ±0.79* 11.95±0.41* 2.02:1 22.40±0.81* 11.00±0.43* 2.04:1 21.80±0.84* 10.6±0.45* 2.06:1
Day 42 29.12 ±0.81 14.25±0.44 2.04:1 26.52±0.83 13.0±0.47 2.04:1 24.73±0.89 12.3±0.49 2.01:1
74
Fig. 1: Change in weight and length of right and left femur in male broiler chicken at different
ages
Fig. 2: Change in width of right and left femur in male broiler chicken at different ages
Fig. 3: Change in cortical bone thickness of right and left femur in male broiler chicken at
different ages
75
Fig. 4: Change in thickness of growth cartilage and its different zones in femur of
Fig. 5: Change in weight of right and left tibiotarsus in male broiler chicken at
different ages
76
Fig. 6: Change in length of right and left tibiotarsus in male broiler chicken at
different ages
Fig. 7: Change in width of right and left tibiotarsus in male broiler
Fig. 8: Change in cortical bone thickness of right and left

tibiotarsus in male broiler chicken at different ages
77 of growth cartilage and its different

Fig. 9: Change in thickness
zones in tibiotarsus of male broiler chicken at different ages
Fig. 10: Change in weight of right and left tarsometatarsus in male broiler chicken
at different ages
Fig. 11: Change in length of right and left tarsometatarsus in male broiler
Fig. 12: Change in width of right and left tarsometatarsus in male broiler chicken
at different ages
78
Fig. 13: Change in cortical bone thickness of right and left tarsometatarsus in
Fig. 14: Change in thickness of growth cartilage and its different zones in
tarsometatarsus of male broiler chicken at different ages
Fig. 15: Change in weight of right and left femur in female broiler chicken at
different ages
79
Fig. 16: Change in length of right and left femur in female broiler
Fig. 17: Change in width of right and left femur in female broiler
Fig. 18: Change in cortical bone thickness of right and left femur in
female broiler chicken at different ages
80
Fig. 19: Change in thickness of growth cartilage and its different
zones in femur of female broiler chicken at different ages
Fig. 20: Change in weight of right and left tibiotarsus in female

Fig. 21: Change in length of right and left tibiotarsus in female

81
Fig. 22: Change in width of right and left tibiotarsus in female broiler
Fig. 23: Change in cortical bone thickness of right and left tibiotarsus
in female broiler chicken at different ages
Fig. 24: Change in thickness of growth cartilage and its different

82 chicken at different ages
zones in tibiotarsus of female broiler
Fig. 25: Change in weight of right and left tarsometatarsus in female broiler
Fig. 26: Change in length of right and left tarsometatarsus in female broiler
Fig. 27: Change in width of right and left tarsometatarsus in female broiler
83
Fig. 28: Change in cortical bone thickness of right and left tarsometatarsus
in female broiler chicken at different ages
Fig. 29: Change in thickness of growth cartilage and its different zones
in tarsometatarsus of female broiler chicken at different ages
84
F
TT
F TT
TMM
TM
LEFT
Fig. 30: Photograph of right and left leg bones (F, TT Fig. 31: Photograph of left leg bones (F, TT and TM) of
and TM) of day old broiler chick 7 days old broiler chick
F TT
TM
RIGHT
LEFT
Fig. 32: Photograph of right leg bones (F, TT and TM) of Fig. 33: Photograph of right leg bones (F, TT and TM) of
7 days old broiler chick 14 days old broiler chick
F TT TM
Fig. 34: Photograph of right and left leg bones (F, TT and TM) of 21 days old male broiler
chicken
85
TR
H PE
FI FI
S S
CONO
N CONO
N
Fig. 36: Photograph of right and left TT bone of 28 days

Fig. 35: Photograph of right and left femur bone of 28
old male broiler chicken
days old male broiler chicken
F
MALE
F
MALE
Fig. 37: Photograph of right and left TM bone of 28 days Fig. 38: Photograph of right and left femur bone of 35
old male broiler chicken days male old broiler chicken
F
MALE MALE F
TT TM
Fig. 39: Photograph of right and left TT bone of 35 days Fig. 40: Photograph of right and left TM bone of 35 days
male old broiler chicken male old broiler chicken
86
TT
Fig. 41: Photograph of right and left femur bone of 42 Fig. 42: Photograph of right and left TT bone of 42 days
days male old broiler chicken male old broiler chicken
TM
PE
CON
Fig. 43: Photograph of right and left TM bone of 42

Fig. 44: Photograph of right and left femur bone of 42
days male old broiler chicken
days female old broiler chicken
TT TM
Fig. 45: Photograph of right and left TT bone of 42 days Fig. 46: Photograph of right and left TM bone of 42 days
female old broiler chicken female old broiler chicken
87
F TT
Fig. 47a: Sectional view of Femur bone of day old Fig. 47b: Sectional view of Tibiotarsus bone of day old
broiler chick showing growth cartilages (arrows) broiler chick showing growth cartilages (arrows)
TM
Fig. 47c: Sectional view of Tarsometatarsus bone of day old broiler chick
TT
F TM
Fig. 48 : Sectional view of F, TT & TM bones o f 7 days old broiler chick

showing growth cartilages
88
TM
F
TT
Fig. 49: : Sectional view of F, TT & TM bones of 14 days old broiler chick
showing growth cartilages
F TM
TT
Fig. 50: : Sectional view of right and left F, TT & TM bones of 21 days old
broiler chicken (male) showing growth cartilages (arrows)
89
F TT TM
Fig. 51: : Sectional view of right and left F, TT & TM bones of 21

days old broiler chicken (female) showing growth cartilages (arrows)
Fig. 52: Sectional view of left F, TT & TM bones of 28 days old

broiler chicken (male) showing growth cartilages (arrows)
90
Fig. 53: Sectional view of left F, TT & TM bones of 28 days old broiler
chicken (female) showing growth cartilages (arrows)
F TT TM
Fig. 54: : Sectional view of left and right F, TT & TM bones of 35 days old broiler
chicken (male) showing growth cartilages (arrows)
91
TT TM
F
Fig. 55: Sectional view of left and right F, TT & TM bones of 35 days old
broiler chicken (female) showing growth cartilages (arrows)
F
TT
Fig. 56: Sectional view of left and right femur (F) bones Fig. 57: Sectional view of left and right TT bones of
of 42 days old broiler chicken (male) showing growth 42 days old broiler chicken (male) showing growth
cartilages (arrows) cartilages (arrows)
92
TM F
s
k
s
k
k
s
k
Fig. 58: Sectional view of left and right TM bones Fig. 59: : Sectional view of left and right Femur
of 42 days old broiler chicken (male) showing (F) bones of 42 days old broiler chicken (female)
growth cartilages (arrows) showing growth cartilages (arrows)
TT
TM
Fig. 60: Sectional view of left and right TT bones of 42 Fig. 61: Sectional view of left and right TM bones of 42
days old broiler chicken (female) showing growth days old broiler chicken (female) showing growth
cartilages (arrows) cartilages (arrows)
93
F
F
OZ OZ
OZ
Fig. 62: Sectional view of femur (F) bone of 42 days old Fig. 63: Sectional view of femur (F) bone of 42 days old
broiler chicken showing proximal growth cartilage with broiler chicken showing distal growth cartilage with
associated OZ zone (arrows) associated OZ zone (arrows)
F F
TT TT
OZ
OZ
Fig. 64: Sectional view of Tibiotarsus (TT) bone of 42 Fig. 65: Sectional view of Tibiotarsus (TT) bone of 42
days old broiler chicken showing proximal growth days old broiler chicken showing distal growth cartilage
cartilage with associated zones (arrows) with associated zones (arrows)
94
F
TM TM F
skskksk
skskksk
OZ
OZ
Fig. 66: Sectional view of Tarsometatarsus (TM) bone of Fig. 67: Sectional view of Tarsometatarsus (TM) bone of
42 days old broiler chicken showing proximal growth 42 days old broiler chicken showing proximal growth
cartilage with associated zones (arrows) cartilage with associated zones (arrows)
skskk
sk
Fig. 68: Radiograph of left and right leg bones of day old broiler chick showing growth
cartilages (arrow)
95
skskks
sk
k sk
ks
k
skskksk
Fig. 69: Radiograph of left and right leg bones of 7 days old broiler
chick showing growth cartilages (arrow)
s
k
Fig. 70a: Radiograph of left and right leg bones of s
14 days old
broiler chick showing growth cartilages (arrow) k
k
96 s
k
ss
kk
ss
kk
kk
ss
kk
s
skskksk
k
s
k
k
s
k
Fig. 70b: Radiograph of left and right leg bones of 21 days

old male and female broiler chickens showing growth
cartilages (arrow)
97
skskk
sk
skskk
skskks
skskksk
skk
Fig. 71: Radiograph of left and right leg bones of 28 days old male and female
broiler chickens showing growth cartilages (arrow)
98
skskksk skskksk
skskksk skskksk
skskksk
Fig. 72: Radiograph of left and right leg bones of 35 days

old male and female broiler chickens showing growth
cartilages (arrow)
99
skskksk skskksk
skskksk skskk skskks

sk k
Fig. 73: Radiograph of left and right leg bones of 42 days old male and female broiler chickens
100
RZ
RZ
PZ
PZ
PHZ
PHZ
HZ
HZ
OZ BV
OZ
Fig. 74: Photomicrograph of growth cartilage in femur of Fig. 75: Photomicrograph of growth cartilage in femur of
day old broiler chick showing different zones –a) RZ, day old broiler chick showing different zones a) RZ,
b)PZ, c) PHZ, d) HZ, e) OZ. H & E, 10X b)PZ, c) PHZ, d) HZ, e) OZ and invading blood vessel
(BV) in OZ. H & E, 40X
CB
BM
Fig. 76: Photomicrograph of compact bone in femur of Fig. 77: Photomicrograph of distal growth cartilage in
day old broiler chick showing- Periosteum(P),) femur of day old male broiler chick showing
Endosteum (E), Bone marrow(BM), Woven compact Alcianophilia of matrix in different zones.
bone(CB). H & E, 10X AB - PAS, 10X
HC P
BV
CB
Fig. 78: Photomicrograph of compact bone in femur of Fig. 79: Photomicrograph of proximal growth cartilage in
day old broiler chick showing – Periosteum (P), Woven / femur of day old male broiler chick showing collagen
trabecular compact bone (CB) with developing Haversian fibers (Green) around lacunae of different zones and in
canal (HC). H & E, 40X wall of the blood vessel (BV) Masson’s trichrome, 40X
101
HZ
P
CB
OZ
Fig. 80: Photomicrograph of compact bone in femur of 7 Fig. 81: Photomicrograph of proximal growth cartilage in
days old broiler chick showing – a) Periosteum, b) femur of 7 days old broiler chick showing different zones
Endosteum, c) Woven/trabecular bone with developing – HZ and OZ with trabecular bone.
osteon H & E, 10X H & E, 40X
Fig. 82: Photomicrograph of compact bone in femur of Fig. 83: Photomicrograph of proximal growth cartilage in
14 days old broiler chick showing collagen fibers (green) femur of 14 days old female broiler chick showing
in matrix of developing osteons & lining of osteonal collagen fibers (Green) in different zones
canals. Masson’s trichrome, 40X Masson’s trichrome, 10X
CB
BM E
Fig. 84: Photomicrograph of compact bone in femur of Fig. 85: Photomicrograph of compact bone in femur of
14 days old broiler chick showing alcianophilia & weak 14 days old broiler chick showing – a) Periosteum, b)
PAS reaction in matrix . AB -PAS 10X Endosteum, c) Bone marrow, d) compact bone with
developing osteons H & E, 10X
102
L
OC
CB
OC L
Fig. 86: Photomicrograph of compact bone in femur of Fig. 87: Photomicrograph of compact bone in femur of
21 days old broiler chicken showing compact bone with 21 days old broiler chicken showing compact bone with
developing osteons and peripheral porous part. developing osteons containing lacunae (L) around
H & E, 10X osteonal canals (OC). H & E, 40X
Fig. 89: Photomicrograph of compact bone in femur of

21 days old broiler chicken showing collagen fibers
21 days old broiler chicken showing collagen fibers
(green) in matrix of developing osteons.
(green) in matrix of developing osteons..
RZ
PZ
PHZ
CB HZ
OZ
Fig. 90: Photomicrograph of compact bone in femur of Fig. 91: Photomicrograph of distal growth cartilage in
28 days old male broiler chicken showing compact bone femur of day old broiler chick showing different zones –
with developing osteons with outer peripheral porous a) RZ, b)PZ, c) PHZ, d) HZ, e) OZ. H & E, 10X
part. H & E, 10X
103
28 days old male broiler chicken showing AB and PAS 28 days old male broiler chicken showing AB and PAS
reaction of matrix. AB - PAS, 10X reaction of matrix. AB - PAS, 40X
PC OC
OC
Fig. 94: Photomicrograph of distal growth cartilage in Fig. 95: Photomicrograph of compact bone in femur of
femur of 28 days old broiler chicken showing AB and PAS 35 days old male broiler chicken showing normal
reaction of matrix of different zones H & E, 10X definitive osteons, osteonal canals (OC) and perforating
canals (PC). H & E, 10X
PC
OC

35 days old male broiler chicken showing normal Fig. 97: Photomicrograph of compact bone in femur of
definitive osteons, osteonal canals (OC), lacunae (L) and 35 days old female broiler chicken showing AB and PAS
perforating canals (PC). H & E, 40X reaction of matrix. AB - PAS, 10X
104
RZ
PZ
RZ
PHZ PZ BV
PHZ
BV
HZ
HZ OZ
OZ
Fig. 98: Photomicrograph of distal growth cartilage in Fig. 99: Photomicrograph of growth cartilage in
femur of 42 days old female broiler chicken showing tibiotarsus of day old broiler chick showing different
different zones – a) RZ, b)PZ, c) PHZ, d) HZ, e) OZ. H & zones –a) RZ, b)PZ, c) PHZ, d) HZ, e) OZ and blood
E, 10X vessels (BV). H & E, 10X
CB
CB OC
OC
Fig. 100: Photomicrograph of compact bone in Fig. 101: Photomicrograph of compact bone in
tibiotarsus of day old male broiler chick showing porous tibiotarsus of 7 days old male broiler chick showing
compact bone(cb) with developing, immature osteons. porous compact bone with developing, immature
H & E, 10X osteons and larger osteonal canals (OC). H & E, 40X
Fig. 102: Photomicrograph of proximal growth cartilage Fig. 103: Photomicrograph of proximal growth cartilage
in tibiotarsus of 7 days old broiler chick showing in tibiotarsus of 7 days old broiler chick showing AB and
distribution of sparse collagen fibers (green) in different PAS reaction of matrix. AB - PAS, 10X
zones. Masson’s trichrome 10X
105
CB
MC
OZ
PZ
PHZ
HZ
RZ
Fig. 104: Photomicrograph of distal growth cartilage in Fig. 105: Photomicrograph of compact bone in
tibiotarsus of 14 day old broiler chick showing different tibiotarsus of 14 days old male broiler chick showing
zones –a) RZ, b)PZ, c) PHZ, d) HZ, e) OZ and blood compact bone(CB) with developing osteons with outer
vessels (BV). H & E, 10X peripheral porous part, marrow cavity (MC).
H & E, 10X
OC L
OC
L
OC
tibiotarsus of 14days old male broiler chick showing tibiotarsus of 14 days old broiler chick showing collagen
compact bone with developing osteons containing fibers (green) in matrix of developing osteons..
lacunae (L) around osteonal canals (OC). H & E, 40X Masson’s trichrome, 10X
Fig. 108: Photomicrograph of compact bone in Fig. 109: Photomicrograph of distal growth cartilage in
tibiotarsus of 14 days old broiler chick showing collagen tibiotarsus of 7 days old broiler chick showing AB
fibers (green) in matrix of developing osteons. reactivity of matrix. AB - PAS, 10X
Masson’s trichrome,
106
SOC
Fig. 110: Photomicrograph of distal growth cartilage in Fig. 111: Photomicrograph of proximal growth cartilage
tibiotarsus of 21 days old broiler chicken showing in tibiotarsus of 21 day old broiler chicken showing
distribution of sparse collagen fibers (green) in different different zones and 2dary ossification centre(SOC) H &
zones. Masson’s trichrome 10X E, 10X
OC
L
L
OC OC CB
tibiotarsus of 28 days old male broiler chicken showing tibiotarsus of 28 days old male broiler chicken showing
maturing, definitive osteons containing lacunae (L) compact bone(CB) with maturing, definitive osteons.
around osteonal canals (OC). H & E, 40X H & E, 10X
L
L
L
OC
OC
L
Fig. 114: Photomicrograph of compact bone in Fig. 115: Photomicrograph of distal growth cartilage in
tibiotarsus of 35 days old male broiler chicken showing tibiotarsus of 42 days old female broiler chicken showing
normal definitive osteons, osteonal canals (OC), lacunae different indistinct zones. H & E, 10X
(L). H & E, 40X
107
tarsometatarsus of day old male broiler chick showing tarsometatarsus of day old male broiler chick showing
porous compact bone with developing, immature collagen fibers (green) in matrix of developing osteons.
osteons. H & E, 10X Masson’s trichrome, 40X
Fig. 118: Photomicrograph of compact bone in Fig. 119: Photomicrograph of proximal growth cartilage
tarsometatarsus of 35 days old female broiler chicken in tarso-metatarsus of 7 days old male broiler chick
showing AB and PAS reaction of matrix. AB - PAS, 10X showing different zones. H & E, 10X
RZ PHZ
PZ
PHZ
HZ
HZ
OZ
OZ
Fig. 120: Photomicrograph of proximal growth cartilage in Fig. 121: Photomicrograph of proximal growth cartilage
tarso-metatarsus of 14 days old male broiler chick showing in tarso-metatarsus of 14 days old male broiler chick
different zones - a) RZ, b)PZ, c) PHZ, d) HZ, e) OZ showing lower zones - a) PHZ, b) HZ & c )OZ
H & E, 40X
H & E, 10X 108
HZ
BV
BV
OZ
Fig. 122: Photomicrograph of distal growth cartilage in Fig. 123: Photomicrograph of proximal growth cartilage
tarso-metatarsus of 21 days old male broiler chicken in tarso-metatarsus of 21 days old male broiler chicken
showing different zones. H & E, 10X showing trabeculae of cartilage cells in HZ & OZ with
Blood vessels (BV) H & E, 40X
Fig. 124: Photomicrograph of compact bone in Fig. 125: Photomicrograph of proximal growth cartilage
tarsometatarsus of 21 days old female broiler chicken in tarsometatarsus of 21 days old broiler chicken
showing AB and PAS reaction of matrix. AB - PAS, 10X showing AB reactivity of matrix. AB - PAS, 10X
tarsometatarsus of 21 days old male broiler chicken tarsometatarsus of 21 days old male broiler chicken
showing collagen fibers (green) in matrix of developing showing collagen fibers (green) in matrix of developing
osteons. Masson’s trichrome, 10X osteons. Masson’s trichrome, 40X
109
RZ
PZ
PHZ
HZ
OZ
Fig. 128: Photomicrograph of proximal growth cartilage Fig. 129: Photomicrograph of proximal growth cartilage
in tarso-metatarsus of 28 days old male broiler chicken in tarso-metatarsus of 28 days old male broiler chicken
showing different zones. H & E, 10X showing different zones –a) RZ, b)PZ, c) PHZ, d) HZ, e)
OZ. H & E, 40X
L
L
L OC
L
OC
L OC
tarsometatarsus of 35 days old male broiler chicken tarsometatarsus of 35 days old male broiler chicken
showing compact bone with mature, definitive osteons. showing compact bone with mature, definitive osteons
H & E, 10X with Osteonal Canals (OC) & lacunae (L). H & E, 40X
CL
OC
OC L
CL L
OC CL
Fig. 132: Photomicrograph of proximal growth cartilage Fig. 133: Photomicrograph of compact bone in
in tarso-metatarsus of 42 days old male broiler chicken tarsometatarsus of 42 days old male broiler chicken
showing different zones (indistinct). H & E, 10X showing compact bone with mature, definitive osteons
with Osteonal Canals (OC), lacunae (L) & Cement Lines
110 (CL). H & E, 40X
DISCUSSION
5.1 Gross Morphometrical Observations
5.1.1 Body weight
From the present observations, it is evident that the average body weight of
male (49.335 gm) day old chicks did not vary significantly (p ≥ 0.05) from those of
female chicks (49.025 gm). Thereafter, the body weight of male chicks significantly
increased (p ≤ 0.05) with age of the birds till the end of experimental period (day 42).
The female chicks followed almost a similar growth pattern like those of males. There
was faster growth in body weight in all the birds under study till day 28, followed by
relatively slower body weight gain towards the end of study period (between day 35
and day 42). However, the female birds were comparatively lighter than the male
birds of respective age. The data on body weight in the present study are comparable
with those observed by Rutten et al. (2002) in meat type Hubbard chickens (113.8 gm
on day 5 and 642.3 gm on day 19). Hassanbadi et al. (2007) reported almost similar
data (560 gm on day 21 and 2114 gm on day 42) on body weight in male broiler
chickens. A similar observation was made by Charuta et al. (2013) in male and female
Ross 308 broiler chickens, Simsa et al. (2007), and Vijayakumar and Balakrishnan
(2014) in different broiler birds. However, a little variation in the present values for
body weight may be attributed to the related strain difference of broiler chicken as
well as variable feeding and management practice. According to Coto et al. (2008),
the body weight in broiler chicks is significantly affected by the dietary Ca level, but
not by the P level and phytase supplementation. Early nutritional supplementation
immediately after hatching helps development of digestive system that evokes better
utilization of nutrients and overall body growth leading to increasing body weight
(Noy and Sklan, 1999; Handerson et al., 2008).
111
5.2 Gross Morphometrical Study of Leg Bones and Associated Growth
Cartilages
a) Femur
The average weight of the femoral bones (right leg) in male day old chicks
gradually increased on day 7, followed by a marked increase from day 14 up to day
42. The femur of day old female chicks gradually increased from day 7 till day 42.
The average weight of left femur in male and female broiler birds showed gradually
increasing trend from day 7 ill the end of the experimental period. There was
insignificant difference (p ≥ 0.05) in weight of femoral bones when compared
between right and left side and male and female birds. However, the bones in female
birds were slightly lighter than those of respective male birds.
There was concomitant increase in length of the right and left femoral bones in
both male and female birds during the post – hatch experimental period. However, the
bones of male birds were slightly taller than those of respective female birds. There
was insignificant difference (p ≥ 0.05) in length of femoral bones when compared
between right and left side and male and female birds.
The diameter (width) of femur bones of both sides and sexes, in general,
showed a gradual increase during the experimental period. In male birds, the diameter
at proximal end, mid shaft and distal end of right femur showed a steep increase (p ≤
0.05) from day 7 up to day 21 and a steady increase thereafter till the end of the study
period, i.e. day 42. The bones of the left side showed almost similar trend. In females,
the values for right as well as left bones were slightly lesser than those observed in
case of males. In both sexes and sides, the proximal and distal diameter increased
more than the mid shaft diameter. And the diameter of distal end was invariably
greater than those of other two throughout the experimental period.
The size (thickness) of marrow cavity in the femoral bones of the either side in
male and female birds revealed a marked increase from day 1 to day 7, followed by a
gradual and steady rise thereafter till day 42. In male birds, the values for right and
left bones became almost double on day 7 as compared to that of day 1. Then there
was a progressive increase in the bone marrow size till day 42. However, the growth
slowed down a bit birds, the diameter at proximal end, mid shaft and distal end of
112
right femur showed a steep increase (p ≤ 0.05) towards the end of the experiment
(days 35 and 42). In females, the size of bone marrow followed a similar pattern with
age of the birds, but was slightly smaller than those of male birds of respective age.
The average thickness of cortical bone (proximal, mid shaft and distal) of the
either side in both sexes revealed a general trend of gradual increase birds, the
diameter at proximal end, mid shaft and distal end of right femur showed a steep
increase in post – hatch broiler chickens. However, its growth slightly slowed down
towards the end of the present study, i.e. days 35 and 42. The thickest cortical bone
was recorded in the eldest birds (on day 42). The values in females were slightly
lesser (lower) than those of male birds of respective age. Though initially mid shaft
cortical bone was thinner than epiphyseal ends, it gradually overtook to become
thicker from day 21 till the end of experimental period. Age –wise variation and
variation between male and female as well as between right and left side bones were
insignificant (p≥ 0.05).
femur of both sides and sexes on day 1. However, from day 7 till day 42 the same
could be easily traced and studied for gross parameters on sectional view of the bones.
In consistent with the increase in length of the bone, the distance between proximal
and distal growth cartilage also showed a gradual increase (far away from each other)
from day 7 onward and reached the highest respective values on day 42. The
thickness of growth plate (cartilage) gradually and slowly increased with age (from
day 7 onward up to day 28) in bones of both sides and sexes. Thereafter, the growth
plates became slightly thinner. The growth cartilages in male birds appeared slightly
thicker than those of female birds of respective ages. And there was minor variation
(p≥ 0.05 in thickness between proximal and distal growth cartilages at a given age.
The present findings on different gross morphometrical parameters in broiler

chickens corroborate well with the observations of earlier workers. According to Rose
et al. (1996), female birds have lighter skeleton than males. The weight, length and
diameter of femur as recorded earlier by Rutten et al. (2002) in meat type Hubbard
chickens on day 19 and Breugelmans et al. (2007) in 42 days old broiler chickens are
more or less similar to the present finding. Robinson et al. (2015) reported linear
increase in length and width of femur with age in case of ducks, who recorded almost
113
similar data as observed in the present study. The minor difference in these values
may be due to strain or breed variation of the broiler birds studied as well as variable
management and feeding practice offered. However, the detailed data on different
gross dimensions of the bones (right and left) at different ages during post – hatch
period in both male and female broiler birds are not available for comparison.
b) Tibiotarsus
The average weight of the tibiotarsal bones (right and left side) in day old male
chicks sharply increased (p ≤ 0.05) on day 7 and followed this increasing trend up to
day 42. The female birds showed similar increasing trend (as also seen in case of
femoral bones) from day 7 till the end of the experimental period.
There was simultaneous increase in length of the bones like that of femur in
both male and female birds throughout the experimental period. It showed significant
variation increased (p ≤ 0.05) except the first and last week.
The diameter (width) of tibiotarsal bones of both sides and sexes, in general,
showed a gradual increase during the experimental period. The diameter at proximal
end, mid shaft and distal end of right and left tibiotarsus in male as well as female
birds showed almost similar trend like that of femur. The mid shaft diameter followed
a gradual and steady increase from day 1 up to day 42.
The size (thickness) of marrow cavity in the tibiotarsal bones of both sides in
male as well as female birds revealed a steep increase from day 1 to day 7, followed
by a gradual and steady rise thereafter till day 42. However, its growth was slower (p
≥ 0.05) between days 21 and 28. In male and females, the size of bone marrow
followed a similar pattern like that of femur with advancement of age of the birds.
The average thickness of cortical bone (proximal, mid shaft and distal) of
either side in both sexes revealed a general trend of gradual increase (p ≥ 0.05) in post
– hatch broiler chickens, with slight slowdown towards the end of the study period,
i.e. days 35 and 42. Its sex variation revealed a similar pattern like femur. Though
initially mid shaft cortical bone was thinner than epiphyseal ends, it gradually became
thicker from day 28 till the end of experimental period.
114
bones. The distance between proximal and distal growth cartilage also showed a
gradual increase from day 7 onward like that of femur bones. The trend in change or
variation (p ≥ 0.05) in thickness of growth plate (cartilage) in bones of both sides and
sexes at different ages appeared similar to that observed in case of femur bones.
The present observations on different gross dimensions of tibiotarsus bones

(namely weight, length and diameter or width) in broiler chickens are well
comparable with those reported by Rose et al. (1996), Leterrier and Constantin
(1999), Williams et al. (2000), Rutten et al. (2002), Breugelmans et al. (2007), Shim
et al. (2012), Paxton et al. ( 2014), Vijayakumar and Balakrishnan (2014) and
Robinson et al. (2015) in different meat type domestic birds ( chicken and duck).
These biometrical values on tibiotarsus of male and female Ross 308 broiler chicken
as observed by Charuta et al. (2013) are in agreement with the present findings. The
slight variation in these values might be attributed to the strain or breed variation of
the chicken as well as due to difference in feeding and management practice, as also
opined earlier by Noy and Sklan (1999), Handerson et al. (2008) and Coto et
al.(2008). However, the literature on the detailed side, age and sex – wise variation in
different gross morphometrical parameters is lacking to be compared with and
discussed on.
c) Tarsometatarsus
The average weight of the tarsometatarsus bones of right and left legs in day
old male and female chicks gradually increased on day 7, followed by a marked
increase from day 14 up to day 42, i.e. till the end of the experimental period (as also
observed in case of femoral and tibiotarsus bones). There was insignificant difference
(p ≥ 0.05) in weight of tarsometarsal bones when compared between right and left
side and male and female birds. However, the bones in female birds were slightly
lighter than those of respective male birds.
The right as well as left side bones in both male and female birds elongated
concomitantly like that of femur during the post – hatch experimental period. There
was simultaneous lengthening of left tarsometatarsal bones in both male and female
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birds under study like that of right ones. The sex and side – wise variation was similar
to that of femur and tibiotarsus bones.
The tarsometatarsus bones of both sides and sexes, in general, gradually

increased (p≤ 0.05) in diameter throughout the experimental period. The diameter at
proximal end, mid shaft and distal end of right and left side bones in male as well as
female birds showed almost similar trend like that of femur and tibiotarsus.
The size (thickness) of marrow cavity in the tarsometatarsal bones of both

sides in male as well as female birds revealed a steep increase (p ≤ 0.05) from day 1
to day 7, followed by a gradual and steady rise thereafter till the end of experimental
period. However, its growth slowed down (p ≥ 0.05) between days 35 and 42. In male
and females, the size of bone marrow followed a similar pattern like that of femur and
tibiotarsus with age of the birds.
either side in both sexes revealed a general trend of gradual increase in post – hatch
broiler chickens. It slightly slowed down towards the end of the study period, i.e. days
35 and 42. Its sex variation revealed a similar pattern like femur and tibiotarsus.
Though initially mid shaft cortical bone was thinner than epiphyseal ends, it gradually
became thicker from day 21 till the end of experimental period.
on day 1. However, from day 7 till day 42 the same were easily observed and studied
for gross parameters on sectional view of the bones. The distance between proximal
and distal growth cartilage showed a gradual increase (p ≤ 0.05) from day 7 onward
like that of femur and tibiotarsal bones. The trend in change or variation in thickness
of growth plate (cartilage) in bones of both sides and sexes at different ages appeared
similar to that observed in case of femur and tibiotarsus.
Some of the gross morphometrical values (namely length and width) on

tarsometatarsus in the present study agree with those observed by Breugelmans et al.
(2007) in broiler chickens of 7 weeks of age.
From the present observations, it is evident that the age – wise variation in
different gross morphometrical dimesions (weight, length, diameter or width of the
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bones and thickness of marrow cavity, cortical bone and growth plate cartilage etc.
followed almost a similar trend in all the bones under study (femur, tibiotarsus and
tarsometatarsus). The results of the concurrent radiographic and histomorphological
studies of the bones confirm rather authenticate well different gross morphometrical
observations on the same.
Elongation of all these appendicular long bones continued with advancement

of age of the birds till day 42, i.e. as long as the growth plates were present. This is
due to the fact that lengthening of long bones occurs at the expense of growth plates
and increase in length of the bone caeses after closure of the growth plate. Similar
opinion was put forth by Getty (1975), Farquharson and Jefferies (2000), Nilsson et
al. (2005), Akers and Denbow (2008), Dyce et al. (2009), and Villemure and Stokes
(2009) in domestic animals as well as birds.
The presence of growth plates was doubly confirmed by simultaneous

radiographic as well as histomorphological observations along with gross anatomical
finding in the present study.
Increase in diameter of the long bones is the result of intramembranous

ossification effected by deposition of bony matrix by appositional method on the
periosoteal surface of the bone. At the same time bone resorption on endosteal surface
results in enlargement of marrow cavity. This process called bone modeling continues
till the bone attains its definite shape (Banks, 1993; Eurell and Sickle, 1998 and Dyce
et al., 2009). Different morphometrical parameters of the leg bones increased
simultaneously with age as well as with increase in body weight of the broiler birds.
Moprphometrical dimensions of the long leg bones like length, width etc. are
considered as good and reliable indicators of musculoskeletal growth in broiler
chicken (Naldo et al., 2000; Williams et al., 2000 and Appplegate and Lilburn, 2002).
The general gross morphological features of these three leg bones in the
present study are similar to the text description of Getty (1975), Dyce et al. (2009)
and Ghosh (2015).
5.3 Radiographical Study of Leg Bones and Associated Growth Cartilages
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chickens at different ages during post – hatch period and the data (not shown
separately) were compared with the gross morphometrical observations of the bones.
Besides, the presence or absence of growth plates or cartilages (proximal and /or
distal) in each of these bones at different ages was distinctly evident on radiography
even in day old chicks. The growth plate (cartilage) could not be traced (identified)
grossly in the femur of both sides and sexes on day 1. But on radiograph the same was
marked easily.The growth plate (cartilage) was identified on gross observation in the
tibiotarsus of all the day old chicks under study. The same was also confirmed on
different morphometric parameters of the bones. Though the proximal growth plate
ends were clearly visible on radiograph of the bones from day 1 till day 42. The
growth cartilages in female birds appeared slightly thinner than those of male birds of
respective ages. And there was minor variation in thickness between proximal and
distal growth cartilage at a given age. The growth cartilages in all the bones became a
little bit thinner after day 28.
The radiographical data on some bone dimensions of the said bones under
study corroborate exactly with those of gross morphometrical values. Radiographical
analysis is the mere confirmation of the gross morphometrical observations in the
present study. Moreover, radiography authenticates and confirms the presence of
growth plates in these bones from day 1 till day 42. Hogg (1980) investigated the
centre of ossification in avian skeleton at and after hatching. He opined that proximal
tibial centre was the only true secondary cntre in the long bones. However, both
tibiotarsus and tarsometatarsus bones showed the presence of secondary ossification
centre as revealed by the simultaneous histological observation of these bones in the
present study. Sadler (1991) studied fusion of proximal epiphysis in tibiotarsus and
development of spur core and its fusion with tarsometatarsus in domestic fowl. On the
contrary, none of the tarsometatarsus bones under study showed the spur core and its
fusion as also reported earlier by Sadler (1991) who observed much late appearance
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of spur core and its fusion with tarsometatarsus. Different dimensions of the leg bones
were radiographically analyzed by Naldo et al. (2000), Williams et al. (2000) and
Breugelmans et al. (2007) in domestic chickens and Charuta et al. (2013) in domestic
ducks to assess and evaluate the skeletal maturity and development.
5.4 Histomorphological Study of Growth Cartilages and Bones
5.4.1 Histomorphological Study of Growth Cartilages
a) Femur
plates (cartilages) even in all day old chicks (males and females). However, these
growth plates were better recognized from day 7 onward till day 42. Histomorphology
of the growth plates (proximal) revealed horizontal zones of chondrocytes at different
stages of differentiation. These histological zones were arranged as resting or reserve
zone, zone of proliferation, zone of prehypertrophy, zone of hypertrophy and
degeneration, and zone of ossification (bone formation) from epiphyseal towards
diaphyseal or metaphyseal end of the growth cartilage. The zonation pattern was
better elucidated thereafter till day 28. But the typical arrangement of zones of
chondrocytes was not clearly depicted in the birds of older age (Days 35 and 42).
Each zone showed difference in chondrocyte size and arrangement as well as
extracellular matrix (ECM). The scattered or clustered chondrocytes of reserve or
resting zone were flattened and comparatively smaller than those of other zones. The
lacunae within the matrix contained usually one or even two chondrocytes (cartilage
cells). Vascularization was poor in this zone. The rapid proliferation of these flattened
chondrocyes and their characteristic arrangement was the distinguishing feature of the
zone of proliferation. However, a typical row – wise or columnar organization of the
proliferating chondrocytes was lacking (poor) except that of Femur and tibiotrsus on
day 1, where such characteristic arrangement was to some extent evident. The
proliferating chondrocytes gradually increased in size to form the zone of
prehypertrophy (a thin zone of transition) and subsequently enlarged (more or less
spherical or rounded in appearance) much to form the zone of hypertrophy. Lower
part of this zone elucidated provisional calcification resulting in normal death and
degeneration (apoptosis) of the hypertrophied chondrocytes. This zone continued into
the zone of true bone formation (ossification) that revealed the laying down of bony
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matrix accompanied by formation of trabecular bone (primary spongiosa) and
developing marrow. This zone had very rich vascularization. The amount of
extracellular matrix was comparatively more in reserve and proliferation zones,
whereas a scanty amount of matrix surrounded the closely arranged lacunae
containing chondrocytes in rest of the zones. The connective tissue elements, namely
collagen fibers were demonstrated in the matrix, walls of the invading blood vessels,
lining of endosteum of developing marrow spaces and newly formed bony matrix of
zone of ossification. The cartilage matrix did not reveal any collage fibers as such,
except those forming an anastomosing, thread – like network around the lacunae
containing chondrocytes as well as in the newly formed bony matrix in the
ossification zone.
alcian blue stain. There was moderate PAS reaction in the cytoplasm of enlarged
chondrocytes of hypertrophic zone. The matrix of newly formed bony trabeculae
revealed moderate to weak reaction for PAS along with alcianophilia in some parts.
However, the alcianophilic reaction was more intense than the PAS reactivity in the
matrix of different cartilage zones. The intensity of alcianophilia in the cartilage
matrix remained almost unchanged throughout the experimental period. The collagen
content was also nearly constant in all age groups.
b) Tibiotarsus
Both the proximal and distal growth plates in males and female birds were
better recognized from day 1 onward till day 42. These growth plates revealed all the
histological zones distinctly like those observed in case of femur. The zonation
pattern was better elucidated from day 7 till day 28.Appearance of 2ndary centre of
ossification in both the epiphyseal ends on day 21 was a characteristic feature of this
bone. The amount and distribution of extracellular matrix and the presence of
collagen fibers and its arrangement were almost similar to those observed in case of
femur bones.
PAS and alcian blue reactivity of matrix in different zones followed much
similarity with that revealed in case of femur bones. No variation in matrix reactivity
to PAS – alcian blue was observed between proximal and distal cartilages as well as
male and female birds like those of femoral bones.
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c) Tarsometatarsus
plates (cartilages) even in day old chicks (males and females) though the distal one
could not be identified grossly. These growth plates elucidated all the histological
zones distinctly like those observed in case of femur as well as tibiotarsus. The pattern
of different zones was better visible from day 7 till day 28. Normal histological
features including vascularization of different zones didn’t vary from those of femur
as well as tibiotarsus. Secondary centre of ossification appeared in the proximal
epiphyseal end on day 21 like that of tibiotarsus bone. However, no secondary
ossification centre appeared in the distal end of the bone.
The amount and distribution of extracellular matrix and the presence of

femur and tibiotarsus bones.
similarity with that seen in case of femur bones. No variation in matrix reactivity to
PAS – alcian blue was observed between proximal and distal cartilages as well as
male and female birds like those of other two bones.
From the above observations it is evident that histomorphological study

confirms the presence of both proximal as well as distal growth plates in all these
three bones from the very beginning, i.e. day 1 till day 42.
Wise (1975) opined that the fowl typically lacks the presence of bony
epiphyseal plates, except in few long bones. According to Church and Johnson
(1964), Hogg (1980), and Naldo et al. (1998), epiphyseal growth plates were lacking
in avian bones, except at the distal end of the tibiotarsus and the proximal end of the
tarsometatarsal bone, both of which had a growth plate formed by the fusion lines
between tarsal bones and the tibia or the metatarsal bones, respectively. None of the
previous workers reported the presence of both proximal as well as distal growth
plates in all the leg bones, specifically in femur. Presence of both proximal as well as
distal growth plates in all these three bones is in accordance with the fact that the
longitudinal growth of the long bones occurs in the growth plate (Orth and Cook,
1994; Eurell and Sickle, 1998) by a process called endochondral ossification, in which
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a cartilaginous scaffold is replaced by bone in a coordinated fashion (Van der Eerden
et al., 2003). According to Villemure and Stokes (2009), the longitudinal growth of
long bones occurs in growth plates where chondrocytes synthesize cartilage that is
subsequently ossified.
Distinct histological zonation pattern of the growth plates was earlier reported
by several workers. Three principal zones: the reserve, proliferative, and hypertrophic
zones were observed by Howlett (1979) and Orth and Cook (1994). Burdan et al.
(2009) reported resting zone, proliferative zone, transformation zone (divided into the
upper and lower hypertrophic layers), degenerative zone, and the primary and
secondary spongiosa zones. An extra prehypertrophic zone (a zone of transition) was
mentioned by Chen et al. (1993), Gafni et al. (2001) and Minina et al. (2002).
Frandson and Spurgeon (1992), Eurell and Sickle (1998), Samuelson (2007), and
Akers and Denbow (2008) stated about five zones: the reserve, proliferative, and
hypertrophic zones, followed by zone of provisional calcification and resorption, and
zone of ossification. The presence of zone of resorption between hypertrophic and
ossification zone describing the invading capillaries is well included in the zones of
hypertrophy and degeneration as well as ossification in the present study.
The common histological features of resting and proliferative zones observed

in the present study are similar to the findings of Kember (1960), Brighton et al.
(1973), Howlett (1979), Abad et al. (2002) in avian species. Eurell and Sickle (1998),
and Akers and Denbow (2008) gave almost similar account on these zones in
domestic animals. The unique columnar or row –wise arrangement of proliferating
chondrocytes as seen in domestic animals was usually lacking in the present study as
reported previously by Howlett (1979) and Farquharson and Jefferies (2000).The
chondrocytes of reserve zone act as stem cells that give rise to proliferative
chondrocytes. These cells have been shown to be crucial for orientation of the
underlying columns of chondrocytes and therefore unidirectional bone growth,
probably by secreting a growth plate-orienting factor (Abad et al., 2002). Brighton
(1978) and Howlett (1979) suggested that these cells store materials such as lipids for
later nutritional requirement. The proliferating chondrocytes were related with each
other as a result of mitotic cell division (Goff, 2004). Either by a finite number of cell
divisions or by changes in exposure to a local growth factor (Gafni et al., 2001 and
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Minina et al., 2002), flattened proliferating chondrocytes lose their capacity to divide
and start to differentiate and become prehypertrophic, coinciding with an increase in
size.
The hypertrophy of the chondrocytes is due to accumulation of inclusion

bodies like glycogen, calcium and vacuoles (Brighton et al., 1973; Eurell and Sickle,
1998 and Akers and Denbow, 2008). This stage is characterized by an increase in
intracellular calcium concentration. This is essential for the production of matrix
vesicles, which are small membrane-enclosed particles released from chondrocytes
(Wang and Kirsch, 2002; Anderson, 2003). The matrix vesicles contain large amounts
of annexins, which mediate calcium uptake into the matrix vesicles (Kirsch et al.,
2000; Wang and Kirsch, 2003). The vesicles secrete calcium-phosphates,
hydroxyapatite, and matrix metalloproteinases (MMPs), resulting in mineralization of
the vesicles and their surrounding matrix. This leads to the degeneration of these cells
due to void of nutritional supply (Brighton et al., 1973; Eurell and Sickle, 1998 and
Akers and Denbow, 2008). Chondroitin sulfate has been reported to be an important
molecule which, as an ion-exchanger, controls calcification of the cartilage matrix
(Hunter, 1991). The calcified zone merged with the zone of ossification. The calcified
matrix provides the scaffolding for spongy or trabecular bone formation (Eurell and
Sickle, 1998; Goff, 2004).
The mineralization process, in combination with low oxygen tension, attracts

blood vessels from the underlying primary spongiosum (Schipani et al., 2001; Rutt,
2008; Burdan et al., 2009 and Srinivas et al., 2009). The vessels penetrate through
these cells, led by phagocytic (i.e., chondroclasts or macrophage) and endothelial
cells, from the metaphyseal region into the cartilage (Farquharson et al., 1992). These
blood vessels provide medium for entry of osteoblasts that lay down bony matrix on
top of the calcified cartilage (Lewinson and Silbermann, 1992).
The most part of the growth cartilage matrix did not show collagen fibers as
such in the present study. The different zones usually contain type II, IX, X and type
XI collagen fibers (Howlett, 1979; Chen et al., 1993; Burdan et al., 2009). These are
very thin fibril forming collagen, which could not be elucidated in the present study.
However, the bony matrix in zone of ossification contains mostly thicker type I
collagen. These are comparatively thicker collagen fibers and were readily
123
demonstrated by Masson’s trichrome stain (Bancroft and Stevens, 1996; Eurell and
Sickle, 1998).
Variable reactivity of the growth cartilage matrix to PAS – alcian blue stain
indicates the presence of variable (different) concentration of proteoglycans (GAGs).
The predominance of alcian blue stained matrix is suggestive of presence of mainly
acidic mucopolysaccharides in the matrix (Bancroft and Stevens, 1996). Active
secretion of proteins in the adjacent matrix by metabolically very active chondrocytes
of proliferative, pre hypertrophic and hypertrophic zones may be attributed for the
variation in mucopolysaccharide content (concentration). Moderate to weak PAS
reactivity of the matrix, specifically in the hypertrophic zone is indicative of presence
of neutral mucopolysaccharides (usually glycogen) as earlier suggested by Brighton et
al. (1973), Eurell and Sickle (1998). Presence of mainly acidic (hyaluronic acid) as
well as sulfated (chondroitin sulfate and keratan sulfate) proteoglycans characterized
the growth cartilage (Nakano and Sim, 1995; Abad et al., 2002). So was the trend of
alcianophilia of the matrix. The proteoglycan rich matrix plays a vital role in water
and electrolytes transportation necessary for the growing epiphyseal plate (cartilage).
There was age and sex related variation in the histomorphometrical

parameters, such as thickness of different zones and diameter of residing
chondrocytes. Even there was individual minor (p ≥ 0.05) variation in this regard
between the proximal and distal growth plates of a particular bone.
From the present observations (Table7, 8, 9, 10, 11 and 12) it is quite obvious
that the proximal and distal growth plates in all the bones (right) of male and female
chicks gradually thickened (p≤ 0.05) with age of the birds from day 7 up to day 28.
Thereafter there was a slight decline in these values from day 35 till day 42. This is in
conformity with the gross observation of the respective growth plate.
Different zones also exhibited variation in thickness (p≤ 0.05) as well as

chondrocyte diameter (size). The values for different zones in male and females
followed almost a similar trend with age. The reserve zone was relatively thinner and
the zone of proliferation was by far the thickest one. The zone of prehypetrophy was
invariably the thinnest one among all the zones. Similar observations were made by
Chen et al. (1993) and Rutt (2008).These zones became gradually thicker with age up
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to day 28, followed by a little lag period till day 42. But zone of ossification showed
gradual and continuous increase throughout the experimental period. However, all the
values in females were a little lesser than those observed in respective male birds at
different ages. The detailed data on different zones in growth pales of male and
female birds at different ages (in the present investigation) could not be compared
with due to paucity of related literature.
Chondrocytes in different zones of proximal and distal growth cartilages in

male and female birds showed variation in respect to their size (diameter) at different
ages. The chondrocytes of reserve zone were the smallest ones, followed by those of
proliferative and prehypertrophic zones. The cells of hypertrophic zone were the
largest ones in the broiler chickens of different ages. These observations are in
agreement with those reported by Brighton et al. (1973), Chen et al. (1993), Breur et
al. (1997), Wang and Kirsch (2002), Anderson, (2003) and Rutt (2008).There was a
gradual and steady increase in chondrocyte diameter up to day 28, followed by a
slight decline thereafter till day 42. All the values in females were comparatively
lesser than those observed in respective male birds at different ages. The detailed data
on chondrocyte size (diameter) different zones in growth pales of male and female
birds at different ages (in the present investigation) could not be compared with and
discussed on due to paucity of related literature.
5.4.2 Histomorphological Study of Bones

all the bones under study (Femur, tibiotarsus and tarsometatarsus) was carried out to
assess the skeletal maturity and age – related variation in compact bone structure. The
compact bone tissue of these bones in the birds of both sexes revealed a common
histoarchitecture in a particular age.
Presence of outer (peripheral) fibrous periosteum covering and inner, thin

lining of endosteum was the usual feature of the compact bone in the birds of both the
sexes at different ages. The characteristic feature of the compact bone was the
presence of several structural (histological) units called the osteons or Haversian
systems, each consisting of 1- 4 concentric lamellae or layers of bone matrix around
the central osteonal or Haversian canal. The shape and size of these canals varied with
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age of the birds. The appearance of the osteons also varied with the age of the birds.
The cortical bone appeared relatively porous and was made up of interlacing woven
bone resembling a network like pattern containing atypical, ill - developed osteons in
younger birds. This porous bone gradually transformed into a compact mass later on
and consisted of usual definitive (typical) osteons of circular profiles. Even the outer
peripheral part of the compact bone in elder birds was still porous and was
characterized by radial fibro-lamellar tissue and it finally became a true (typical)
compact bone. This observation is similar to the finding of Leterrier and Nys (1992),
who concluded that an increased rate of growth was associated with a lower mineral
density and higher porosity of the tibial cortices in rapidly growing chicks. Crespo et
al. (2002) reported that the femoral cortex had an underlying pattern of network bone
made up of intercalating woven bone tissue containing osteons in male turkeys. The
general histoarchitecture of the avian compact bone, specifically the osteons was
similar to those described by Neumann (1986), Banks (1993), Crespo et al. (2002),
Tsokova (2006) and Dumitrescu et al. (2012). Similar description was given by
Fawcett (1994), Eurell and Sickle (1998), Samuelson (2007), and Akers and Denbow
(2008). However, number of concentric lamellae and associated lacunae varied from
1-4 in the present study in contrary to 3-20 lamellae per osteon in domestic animals.
This variation may be due to the species difference and lesser compact bone thickness
in avians. The collagen fibers were stained by Masson’s trichrome. The collagen
fibers in the bone matrix are usually thick Type I collagen. So, these fibers were
readily demonstrated by trichrome reaction. The presence of sulfated proteoglycans
(GAGs) namely keratan sulfate and small amount of chondroitin sulfate in the scanty
bone matrix may be attributed for predominant alcianophilia over moderate to weak
PAS reactivity. Similar opinion was given by Fawcett (1994), Eurell and Sickle
(1998), and Samuelson (2007).
Though the compact bone of femur, tibiotarsus and tarsometatarsus in the

birds of both sexes shared a common histoarchitecture, it showed individual variations
in respect to some histometrical parameters like thickness of periosteum and compact
bone, size of Haversian canal and system, and number of lamella with lacuna
containing osteocytes at different ages.
126
In all these bones of male and female birds, the cortical bone thickness
gradually increased from day 7 to up to day 42. This is in agreement with the
respective gross observations in the present study. The micrometric dimension of
compact bone (tibiotarsus) was comparable with those observed by Dumitrescu et al.
(2012) in broiler chickens. The diameter of Haversian canals progressively enlarged
with age, whereas the size of Haversian system followed a reverse trend throughout
the experimental period. This is due to the fact that immature, growing woven bone of
younger stage is subsequently replaced in mature stage by typical, lamellar compact
bone tissue. The laying down of more amount of calcified bone matrix with age leads
to gradual narrowing of the Haversian or osteonal canals.
The periosteum became increasingly thicker (p≤ 0.05) with age and
subsequently was the thickest one on day 42 in all the birds. This might be due to the
fact that a growing bone needs more protection to prevent more possible insults as
well as the fact that increasing diameter of the bone with age is the result of continued
intra membranous ossification on periosteal surface of the bone (Fawcett, 1994; Eurell
and Sickle, 1998). In females, all these parameters showed slightly lower values as
compared to those of male birds. The literature on the detailed data on these
histometrical parameters in male and female birds at different ages (in the present
investigation) is not available and accordingly these could not be compared with.
Serum Ca and P level (concentration) was estimated in male and female

broiler chickens during post – hatch period to assess its effect on the skeletal
morphology and integrity as well as to correlate it with the simultaneous changes in
bone mineral content. The average serum Ca levels in male and female birds
gradually increased from day 7 onward. The values were the maximum on day 42.
Serum P level in male as well as female birds showed a similar increasing

trend with age like that of serum Ca. The ratio between serum Ca and P level (Ca : P)
varied accordingly with age and sex of the birds.
Heller et al. (1934) estimated serum Ca and P levels (mg/100ml) as 15.1 and
5.9 respectively in non-laying birds. According to Hassanabadi et al. (2007), serum
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Ca and P level (mg/dL) in control broilers were 6.69 and 7.21 respectively on day 28.
As per the observation of Rutt (2008), plasma Ca concentration (mg/dL) varied from
10.9 to 12.7 in 18 -21 days old broiler chickens with control diet. Rani et al. (2011)
recorded serum Ca and P levels (g %) as 10.92 and 5.41 respectively in control broiler
chickens. Imik et al. (2012) reported serum Ca and P level (mg/dL) to be 11.53± 0.64
and 7.30± 0.68 respectively in control (healthy) Ross 308 broiler chickens on day 42.
Serum Ca and P level (mg/dL) were recorded as 10.56 and 7.15 respectively in the
control Cobb 400 broiler chickens (Vijayakumar and Balakrishnan, 2014). The
present observation on serum Ca and P levels (mg/dl) in broiler chicken is well
comparable with those of the said workers. A little variation in these values may be
attributed to strain or breed difference of the chicken studied and variation in
management and feeding practice offered (Deo et al.,2003; Bogusawska -Tryk et al.,
2012), and variable bioavailability as well as absorption of these minerals (Rani et al.,
2011 and Vijayakumar and Balakrishnan, 2014). Comparatively much higher level of
serum Ca and P levels (as observed in the present study) in chicken (avians) than
mammals were reported earlier by Heller et al. (1934) and Brar et al. (2014).
Serum Ca levels observed in the present study were inversely proportional to

serum P levels throughout the experimental period. It is in accordance with the
finding of Gardiner (1969), who also advocated similar inverse relationship between
serum Ca and P levels in broiler type chickens. The ratio between serum Ca and P
levels in the present investigation varied from 2.05:1 to 2.35:1 in males and from
2.04:1 to 2.32:1 in female birds respectively. Rutt (2008) reported this ratio range to
be between 1.4 and 2.1. Similar observations in this regard were made by Rani et al.
(2011). The Ca : P ratio was more or less constant throughout the experimental period
in the present study. Skeletal strength and hardness are dependent on normal blood
calcium and phosphorus level. Bone mineralization occurs when plasma Ca and P
concentrations are normal. Under stable conditions the rate of bone resorption is equal
to the rate of bone formation so that the mineral content of the skeleton remains
unchanged (Goff, 2004).
The bone mineral content, i.e. percentage of Ca and P in all the representative
bones of male birds gradually increased with age of the broiler birds in the present
study. Ca% varied from 14.8 to 29.1 in femur, 14.5 to 26.5 in tibiotarsus and from
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14.2 to 24.7 in tarsometatarsus repectively. The respective values for bone P (%) were
7.15 to 14.25, 6.90 to 13.0 and 7.04 to 12.3. The present observation compares well
with those of Williams et al. (2000), Salamatdoustnobar et al. (2011) Vijayakumar
and Balakrishnan (2014), and Robinson et al. (2015) in different broiler birds. The
ratio between bone Ca and P percentage appeared more or less constant for all the
bones at different ages. This is in consonance with the simultaneous observation on
plasma Ca and P level in the present study. Lower bone mineral content (%) in
younger birds indicates the peresnce of comparatively more porous and less
mineralized cortical bone that gradually became more compact and highly
mineralized in later stages. Williams et al. (2000) opined alike. Plasma Ca and P
profile is complementary to bone mineral (Ca and P) content. Bone mineralization
takes place only when plasma Ca and P concentrations are normal (Goff,
2004).Continued modeling and remodeling of mature skeleton provides a pool for
maturing osteons and plays a vital role for maintaining a proper balance between
mineral content of plasma and bone, thus effecting proper mineralization and
structural integrity of the bones (Fawcett, 1994).
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SUMMARY AND CONCLUSION
6.1 Summary
The present study was conducted on forty five (45) ‘Vencob’ broiler birds.
The day old chicks were reared up to day 42 (market age). The gross morphometrical,
radiographical and histomorphological studies were carried out on the long leg bones
(femur, tibiotarsus and tarsometatarsus) and associated growth cartilages in broiler
chickens at different ages (on days 1,7, 14, 21, 28, 35 and 42). Simultaneous
measurement of body weight and biochemical evaluation of serum and bone Ca and P
level was also done at weekly interval.
The average body weight of day old male and female chicks was recorded to
be 49.025 gm and 49.333gm respectively. Then the body weight gradually increased
up to day 42 (1798.2 gm and 1665.783gm respectively).
In day old male and female chicks, the average weight of the femoral bones
(right leg) were 0.311 gm and 0.305 gm respectively. It gradually increased on day 7
(0.711 gm and 0.615 gm), followed by a marked increase from day 14 up to day 42
(9.688 gm and 9.14 gm). The average weight of left femur in male and female broiler
birds varied from (0.313 gm and 0.301 gm) on day 1 to (9.637 gm and 9.05 gm) on
day 42 respectively.
In male birds, the average length of femur varied from 2.9 cm (on day 1) to 7.9
cm (on day 42). The values in female birds ranged from 2.88 cm (on day 1) to 7.7 cm
(on day 42). There was simultaneous increase in length of left femoral bones in both
male and female birds under study like that of right leg.
The diameter (width) of femur bones of both sides and sexes, in general,
showed a gradual increase during the experimental period. In male birds, the diameter
at proximal end, mid shaft and distal end of right femur were 2.35 mm, 2.20 mm and
2.98 mm on day 1, followed by a steep increase from day 7 (3.94 mm, 3.38 mm and
4.14 mm) up to day 21(8.64 mm, 5.15 mm and 9.10 mm) and a steady increase
thereafter till the end of the study period, i.e. day 42(10.88 mm, 7.09 mm and 11.15
130
mm) respectively. The bones of the left side showed almost similar trend. In females,
the values for right as well as left bones were slightly lesser than those observed in
case of males.
The size (thickness) of marrow cavity in the femoral bones of either sex and
side revealed a marked increase from day 1 to day 7, followed by a gradual and steady
rise thereafter till day 42. In male birds, the values for right and left bones recorded
were 0.7mm and 0.83 mm respectively on day 1. The values became almost double
(1.59 mm and 1.74 mm) on day 7. Then there was a progressive increase in the bone
marrow size till day 42 (4.28 mm and 4.54 mm). However, the growth slowed down a
bit towards the end of the experiment (days 35 and 42). In females, the size of bone
marrow followed a similar pattern with age of the birds. The size of the marrow cavity
in the female birds was slightly smaller than those of male birds of respective age.
In males, the cortical bone thickness were (0.49mm, 0.71mm and 0.51mm)
and (0.66 mm, 0.53mm and 0.6 mm) on day 1 and increased from day 7 onward till it
became the thickest one ( 1.25 mm, 1.42 mm and 1.25 mm, and 1.30 mm, 1.59 mm
and 1.30 mm) respectively for right and left side on day 42. The female birds showed
almost a similar growth pattern of cortical bone with slightly lesser (lower) values
than those of male birds of respective age.
Consistent with the increase in length of the bone, the distance between
proximal and distal growth cartilage also showed gradual increase from day 14
onward and reached the highest respective values, i.e. (7.1 cm and 6.95 cm) and (6.6
cm and 6.52 cm) on day 42. The thickness of growth plate (cartilage) gradually and
slowly increased with age (from day 14 onward up to day 28) in bones of both sides
and sexes. Thereafter, the growth plates became a little thinner. The growth cartilages
in females appeared slightly thinner than those of male birds of respective ages. And
there was minor variation in thickness between proximal and distal growth cartilage at
a given age.
The average weight of the tibiotarsal bones (right and left side) in day old male
chicks sharply increased on day 7 and followed this increasing trend up to day 42. The
female birds showed similar increasing trend (as also seen in case of femoral bones)
from day 7 till the end of the experimental period.
131
There was simultaneous increase in length of the bones like that of femur in
both male and female birds throughout the experimental period.
The diameter (width) of tibiotarsal bones of both sides and sexes, in general,
showed a gradual increase during the experimental period. The diameter at proximal
end, mid shaft and distal end of right and left tibiotarsus in male as well as female
birds showed almost similar trend like that of femur. The mid shaft diameter followed
a gradual and steady increase from day 1 up to day 42.
The size (thickness) of marrow cavity in the tibiotarsal bones of both sides in
male as well as female birds revealed a steep increase from day 1 to day 7, followed
by a gradual and steady rise thereafter till day 42. However, its growth was slower
between days 21 and 28. In male and females, the size of bone marrow followed a
similar pattern like that of femur with advancement of age of the birds.
broiler chickens, with slight slowdown towards the end of the study period, i.e. days
35 and 42. Its sex variation revealed a similar pattern like femur. Though initially mid
shaft cortical bone was thinner than epiphyseal ends, it gradually became thicker from
day 28 till the end of experimental period.

bones. The distance between proximal and distal growth cartilage also showed a
gradual increase from day 7 onward like that of femur bones.
The average weight of the tarsometatarsus bones of right and left legs in day
old male and female chicks gradually increased on day 7, followed by a marked
increase from day 14 up to day 42, i.e. till the end of the experimental period (as also
observed in case of femoral and tibiotarsus bones).
The right as well as left side bones in both male and female birds elongated
concomitantly like that of femur during the post – hatch experimental period. There
132
was simultaneous lengthening of left tarsometatarsal bones in both male and female
birds under study like that of right ones.
The tarsometatarsus bones of both sides and sexes, in general, gradually

increased in diameter throughout the experimental period. The diameter at proximal
end, mid shaft and distal end of right and left side bones in male as well as female
birds showed almost similar trend like that of femur and tibiotarsus.
The size (thickness) of marrow cavity in the tarsometatarsal bones of both

sides in male as well as female birds revealed a steep increase from day 1 to day 7,
followed by a gradual and steady rise thereafter till the end of experimental period.
However, its growth slowed down between days 35 and 42.
broiler chickens. It slightly slowed down towards the end of the study period, i.e. days
35 and 42. Its sex variation revealed a similar pattern like femur and tibiotarsus.
Though initially mid shaft cortical bone was thinner than epiphyseal ends, it gradually
became thicker from day 21 till the end of experimental period.
on day 1. However, from day 7 till day 42 the same were easily observed and studied
for gross parameters on sectional view of the bones. The distance between proximal
and distal growth cartilage showed a gradual increase from day 7 onward like that of
femur and tibiotarsal bones.
chickens at different ages (days 1, 7, 14, 21, 28, 35 and 42) and the data (not shown as
separately) were compared with the gross morphometrical observations of the bones
and compiled for inclusion in the Tables of gross morphometrical data. Besides, the
presence or absence of growth plates or cartilages (proximal and /or distal) in each of
these bones at different ages was distinctly evidenced on radiography even in day old
133
chicks. The thickness of the growth plates was also measured directly on radiographs
by using image analysis software and the data (not shown separately) obtained were
compared and included with those of gross observations.
femur of both sides and sexes on day 1. But on radiograph the same was marked
easily. The growth plate (cartilage) was identified on gross observation in the
tibiotarsus of all the day old chicks under study. The same was also evident on
different morphometric parameters of the bones.Though the proximal growth plate
ends were clearly visible on radiograph of the bones from day 1 till day 42 and
radiographic analysis of the morphometrical parameters was done.
Histomorphology of the growth plates (proximal) in femur revealed distinct

histological zones - resting or reserve zone, zone of proliferation, zone of
prehypertrophy, zone of hypertrophy and degeneration, and zone of ossification
(bone formation) from epiphyseal towards diaphyseal or metaphyseal end of the
growth cartilage. The zonation pattern was better elucidated thereafter till day 28. But
the typical arrangement of zones of chondrocytes was not clearly depicted in the birds
of older age (Days 35 and 42). The scattered or clustered chondrocytes of reserve or
resting zone were flattened and comparatively smaller than those of other zones. The
lacunae within the matrix contained usually one or even two chondrocytes (cartilage
cells). Vascularization was poor in this zone. The rapid proliferation of these flattened
chondrocyes and their characteristic arrangement was the distinguishing feature of the
zone of proliferation. However, a typical row – wise or columnar organization of the
proliferating chondrocytes was lacking (poor) except that of femur and tibiotarsus of
day old chicks. The proliferating chondrocytes gradually increased in size to form the
zone of prehypertrophy (a thin zone of transition) and subsequently enlarged the
maximum to form the zone of hypertrophy. Lower part of this zone elucidated
provisional calcification resulting in normal death and degeneration (apoptosis) of the
134
hypertrophied chondrocytes. This zone continued into the zone of true bone formation
(ossification) that revealed the laying down of bony matrix accompanied by formation
of trabecular bone (primary spongiosa) and developing marrow. This zone had very
rich vascularization. The amount of extracellular matrix was comparatively more in
reserve and proliferation zones, whereas a scanty amount of matrix surrounded the
closely arranged lacunae containing chondrocytes in rest of the zones. The connective
tissue elements, namely collagen fibers were demonstrated in the matrix, walls of the
invading blood vessels, lining of endosteum of developing marrow spaces and newly
formed bony matrix of zone of ossification. The cartilage matrix did not reveal any
collage fibers as such, except those forming an anastomosing, thread – like network
around the lacunae containing chondrocytes as well as in the newly formed bony
matrix in the ossification zone.
alcian blue stain. The matrix of the reserve zone was comparatively pale than
proliferative, prehypertrophic and hypertrophic zones.There was moderate PAS
reaction in the cytoplasm of enlarged chondrocytes of hypertrophic zone. The matrix
of newly formed bony trabeculae revealed moderate to weak reaction for PAS along
with alcianophilia in some parts. However, the alcianophilic reaction was more
intense than the PAS reactivity in the matrix of different cartilage zones. The intensity
of alcianophilia in the cartilage matrix slightly increased with age up to day 21 and
remained almost unchanged thereafter throughout the experimental period. The
collagen content was also nearly constant in all age groups.
Both the proximal and distal growth plates of tibiotarsus bones in males and
female birds were better recognized from day 1 onward till day 42. These growth
plates revealed all the histological zones distinctly like those observed in case of
femur. The zonation pattern was better elucidated from day 7 till day 28. Appearance
of seconndary centre of ossification in both the epiphyseal ends on day 21 was a
characteristic feature of this bone. The amount and distribution of extracellular matrix
and the presence of collagen fibers and its arrangement were almost similar to those
observed in case of femur bones.
similarity with that revealed in case of femur bones. No variation in matrix reactivity
135
to PAS – alcian blue was observed between proximal and distal cartilages as well as
male and female birds like those of femoral bones.
Both the proximal and distal ends (epiphyses) of tarsometatarsus showed the
presence of growth plates (cartilages) even in day old chicks (males and females)
though the distal one could not be identified grossly. These growth plates elucidated
all the histological zones distinctly like those observed in case of femur as well as
tibiotarsus. The pattern of different zones was better visible from day 7 till day 28.
Normal histological features including vascularization of different zones didn’t vary
from those of femur as well as tibiotarsus. Secondary centre of ossification appeared
in the proximal epiphyseal end on day 21 like that of tibiotarsus bone. However, no
secondary ossification centre appeared in the distal end of the bone.
The amount and distribution of extracellular matrix and the presence of

femur and tibiotarsus bones.
PAS and alcian blue staining affinity of matrix in different zones was much
similar to that seen in case of femur bones. No variation in matrix reactivity to PAS –
alcian blue was observed between proximal and distal cartilages as well as male and
female birds like those of other two bones.

all the bones under study (Femur, tibiotarsus and tarsometatarsus) was carried out to
assess the skeletal maturity and age – related variation in compact bone structure. The
compact bone tissue of these bones in the birds of both sexes revealed a common
histoarchitecture in a particular age.
In the birds of both the sexes at different ages, the characteristic feature of the
compact bone between outer periosteum covering and inner lining of endosteum was
the presence of several structural (histological) units called the osteons or Haversian
systems, each consisting of 1-4 concentric lamellae or layers of bone matrix around
the central osteonal or Haversian canal. The shape and size of these canals varied with
age of the birds. The appearance of the osteons also varied with the age of the birds.
The cortical bone appeared relatively porous and was made up of interlacing woven
136
bone resembling a network like pattern containing atypical osteons in younger birds.
This porous bone gradually transformed into a compact mass later on and consisted of
usual (typical) osteons of circular profiles. Even the outer peripheral part of the
compact bone in elder birds was still porous and was characterized by radial fibro-
lamellar tissue and it finally became a true (typical) compact bone.
The average serum Ca levels in male and female birds gradually increased
from day 7 onward. The values were the maximum on day 42. Serum P level in male
as well as female birds showed a similar increasing trend with age like that of serum
Ca. The ratio between serum Ca and P level (Ca : P) varied accordingly with age and
sex of the birds. The ratios ranged from 2.05:1 to 2.35:1 in males, whereas it varied
from 2.04:1 to 2.32:1 in female birds.
The bone mineral content, i.e. percentage of Ca and P in all the representative
bones of male birds gradually increased with age of the broiler birds in the present
study. Ca% varied from 14.8 to 29.1 in femur, 14.5 to 26.5 in tibiotarsus and from
14.2 to 24.7 in tarsometatarsus repectively. The respective values for bone P (%) were
7.15 to 14.25, 6.90 to 13.0 and 7.04 to 12.3.
6.2 Conclusion
From the results of the present study after due comparison with the available
literature, the following conclusions can be inferred.
1. There is faster growth in broiler chickens till day 28, followed by relatively
slower body weight gain towards the end of study period (between day 35 and
day 42). However, the female birds are comparatively lighter than the male
birds of respective age.
2. The average weight, length, size (thickness) of marrow cavity and cortical bone
thickness in all the bones gradually increase with age of the broiler birds.
3. The diameter (width) of all the bones in broiler birds shows a steep increase
from day 7 up to day 21 and a steady increase thereafter till day 42. The
proximal and distal end widens more than the mid shaft and the distal end is the
widest.
137
4. The growth of the gross dimensions of the bones slows down a bit towards the
end of the experiment (days 35 and 42).
5. Though initially mid shaft cortical bone is thinner than epiphyseal ends, it
gradually becomes thicker from day 21 (in femur and tarsometatarsus) or from
day 28 (in tibiotarsus) till day 42.
6. The gross anatomical observation may not reveal the presence of growth plates
in all day old chicks. But radiographic and histomorphological study confirms
the presence of both proximal as well as distal growth plates in all these three
bones from day 1 till day 42, the latter one being more authentic. In femur
presence of growth plates are reported for the first time.
7. There are horizontal histological zones, i.e. resting or reserve zone, zone of
proliferation, zone of prehypertrophy, zone of hypertrophy and degeneration,
and zone of ossification (bone formation) from epiphyseal towards diaphyseal
or metaphyseal end of the growth cartilage. The histological zonation pattern is
better elucidated from day 7 till day 28 in post –hatch broiler chickens.
8. Unlike the mammals, characteristic columnar or row- wise arrangement of
proliferating chondrocytes is usually lacking in broiler chickens.
9. Secondary centre of ossification in broiler chicken appears in the epiphyseal
ends of tibiotarsus and tarsometatarsus (but not in femur) on day 21.
10. There is sparse amount of matrix and connective tissue elements (collagen) in
growth cartilage. Arrangement of collagen fibers in growth cartilage of different
bones is almost similar.
11. There is variable PAS and alcian blue reactivity of matrix (with predominance
of alcianophilia) in different zones of growth cartilage in all the bones. There is
no variation in matrix reactivity to PAS – alcian blue between proximal and
distal cartilages as well as male and female birds.
12. Different histological zones of growth cartilage exhibit age and sex related
variation in thickness as well as chondrocyte diameter (size).
13. The compact bone tissue of the bones in the broiler birds of both sexes reveals a
common histoarchitecture in a particular age. Each osteon or Haversian system
consists of 1- 4 concentric lamellae or layers of bone matrix around the central
osteonal or Haversian canal. The shape and size of these canals and osteons
varies with age of the birds.
138
14. The cortical bone appears relatively porous and is made up of interlacing woven
bone resembling a network like pattern in younger birds, which is gradually
transformed into a compact mass subsequently.
15. There is insignificant (very little) variation in different gross morphometrical
and histometrical parameters of different bones when compared between right
and left side and male and female birds at a particular age.
16. The average serum Ca and P levels in plasma and bone (%) in broiler birds
gradually increase with age.
17. The ratio between serum Ca and P level (Ca: P) and bone Ca and P percentage
remains almost constant during the growing period of the broiler chickens to
maintain a proper balance for adequate mineralization of bones.
139
REFERENCES
Abad V, Meyers J L, Weise M, Gafni R I, Barnes K M, Nilsson O, Bacher J D and

Baron J. 2002. The role of the resting zone in growth platechondrogenesis.
Endocrinology, 143:1851–1857.
Akers R M and Denbow D M. 2008. Anatomy and Physiology of Domestic Animals.

1stEdn., pp.133 – 168, Blackwell Publishing, USA & UK.
Almeida P I, Mendes A A, Takita T S, Vulcano L C, Guerra P C, Wechsler F S,

Garcia R G, Takahashi S E, Moreira J, Pelícia K, Komiyama C M and
Quinteiro R R. 2005. Comparison of techniques for
tibialdyschondroplasiaassessment in broilerchickens.Brazilian Journal of
Poultry Science,7(1): 27 – 31.
Anderson H C. 2003. Matrix vesicles and calcification. Curr.Rheumatol. Rep., 5:222–

226.
AOAC. 1995. Official Methods of Analysis of the Association of Analytical

Chemists. 16th Ed., Association of Official Analytical Chemists,
Washington DC.
Applegate T J and Lilburn S. 2002.Growth of the Femur and Tibia of a Commercial

Broiler Line. Poultry Science,81:1289–1294.
Bagainski E S1973. Calcium estimation by OCPC method.Anal. Biochem.,18:521.
Bancroft J D and Stevens A. 1996. Theory and Practice of Histological Techniques.

4thEdn., Churchill Livingstone, New York, London and Tokyo.
Banks W J.1993. Supportive tissues – bone. In: Applied Veterinary Histology, 3

rdEdn., pp. 107 – 126, Mosby Year Book Inc: Baltimore.
Baranova D, Pesek L, and Saly J. 2008. Mechanical properties of bones. Folia

Veterinaria, 52 (3 -4): 168-173.
i
Barreto C and Wilsman N J. 1994. Hypertrophic chondrocyte volume and growth
rates inavian growth plates. Research in Veterinary Science, 56 (1): 53- 61.
Bogusawska -Tryk M, Szymeczko R, and Piotrowska A. 2012.The level of major

proteins and minerals in the blood serum of chickensfed diets with pure
cellulose.Folia Biologica (Krakow),60: 65-70.
Boyan B D, Sylvia V L, Dean D D and Schwartz Z. 2001. 24,25- (OH)(2)D(3)

Regulates cartilage and bone via autocrine and endocrine mechanisms.
Steroids, 66:363–374.
Brar R S, Sandhu S and Singh A. 2014. Veterinary Clinical Diagnosis by Laboratory

Methods. Kalyani Publishers, pp.148 – 149, New Delhi, Chennai, Kolkata.
Breugelmans S, Muylle S, Cornillie P, Saunders J and Simoens P. 2007. Age

determination of poultry: a challenge for customs. Vlaams
Diergeneeskundig Tijdschrift, 76: 423-430.
Breur G J, Lapierre M D, Kazmierczak K, Stechuchak K M and McCabe G P 1997.

The domain of hypertrophic chondrocytes in growth plates growing at
different rates. Calcif. Tissue Int., 61:418–25.
Brighton C T, Sugioka Y and Hunt R.1973. Cytoplasmic structures of epiphyseal-

plate chondrocytes: quantitative evaluation using electron micrographs of
real costochondral junctions with special reference to the fate of
hypertrophic cells. J. Bone Jt. Surg. Am., 55:771-784.
Brighton C T. 1978. Structure and function of growth .Clin. Orthopaed., 136 : 22-32.
Burdan F, Szumilo J, Korobowicz A, Farooquee R, Patel S, Patel A, Dave A,

Szumi M, Solecki M, Klepacz R and Dudka J. 2009. Morphology and
physiology of the epiphyseal growth plate. Folia Histochemica
EtCytobiologica, 47(1): 5-16.
ii
Charuta A, Dzierzęcka M, Majchrzak T,Czerwiński E and Cooper R G. 2011.
Computer-generated radiological imagery of the structure of the
spongioussubstance in the postnatal development of the tibiotarsal bones of
the Pekingdomestic duck (Anasplatyrhynchosvar. domestica).Poultry
Science,90:830–835.
Charuta A, Dzierzecka M, Komosa M, Kalinowski L and Pierzcha M. 2013. Age- and

sex-related differences of morphometric, densitometric andgeometric
parameters of tibiotarsal bone in ross broiler chickens. Folia Biologica
(Krakow),61: 211-220.
Chen Q, Gibney E P, Leach R M and Linsenmayer T F. 1993. Chicken

tibialdyschondroplasia: A limb mutant with two growth plates and possible
defects of collagen crosslinking. Dev. Dyn.196:54–63.
Church L E and Johnson L C. 1964. Growth of long bones in the chicken – Rates of
growth in length and diameter of the humerus, tibia, and metatarsus.
American Journalof Anatomy, 114:521-538.
CookM E. 2000. Skeletal deformities and their causes: introduction. Poult. Sci.,
79:982-984.
Coto C, Yan F, Cerrate S, Wang Z,Sacakli P, Halley Z T, WiernuszC J, Martinez A

and Waldroup PW 2008. Effects of dietary levels of calcium and
nonphytate phosphorus inbroiler starter diets on live performance, bone
development andgrowth plate conditions in male chicks fed a corn-based
diet.International Journal of Poultry Science,7 (7): 638-645.
Crespo R, Stover S M, Shivaprasad H L and Chin R P. 2002. Microstructure and

mineral content of femora in male turkeys with and without fractures.
Poultry Science, 81:1184–1190.
Deo C, Shrivastava H P, Singh N B and Tyagi P K. 2003. Growth performance and

blood biochemicals of starting broiler chicks as influenced by different
levels of dietary phosphorus. Indian Journal of Poultry Science, 38(2):
45-49.
iii
Dinev I. 2011.Comparative pathomorphological study of rickets typesin broiler
chickens. Iranian Journal of Veterinary Science and Technology(IJVST),
3(1):1-10.
Dinev I, Denev S A and Edens F W. 2012. Comparative clinical and morphological

studies on the incidence of tibialdyschondroplasia as a cause of lameness in
three commercial lines of broiler chickens. J. Appl. Poult. Res., 21: 637–
644.
Dumitrescu G, Drinceanu D, Ştef L, Petculescu Ciochina L, Julean C and Boca L.

2012. Study of the effect of boron supplementation in the feed of broiler
chickens on the histological structure of the tibia. Animal Science and
Biotechnologies, 45 (2):149 – 156.
Dyce K M, Sack W O and Wensing C J G. 2009. Textbook of Veterinary Anatomy.

3rdEdn. (Indian), pp.12 -74, Saunders Elsevier, Noida, India.
Escargueil-Blanc I, Meilhac O, Pieraggi M T, Arnal J F, Salvayre R and Negre-

Salvayre A. 1997. Oxidized LDLs induce massive apoptosisof cultured
human endothelial cells through a calcium-dependentpathway. Prevention
by aurintricarboxylic acid. Arterioscler Thromb Vasc Biol.,17:331–339.
Eurell J A C and Sickle D C V.1998. Connective and supportive tissues. In: Text book
of Veterinary Histology, (Eds.) Dellman H D and Eurell J A., 5thedn. Pp 32-
61, Williams and Wilkins, Philadelphia, London.
Europe Commission. 2000.The welfare of chickens kept for meat production

(broilers), Scientific committee on Animal Health and Animal Welfare, pp.
149.
FarquharsonC and Jefferies D. 2000. Chondrocytes and longitudinal bone growth:

The development of tibialdyschondroplasia. Poultry Sc.,79: 994-1004.
iv
Farquharson C, Whitehead C, Rennie S, Thorp B and Loveridge N. 1992. Cell
proliferation and enzyme activities associated with the development of
avian tibialdyschondroplasia: An in situ biochemical study. Bone,13:59–67.
Fawcett D W .1994. A Text Book of Histology. 12thEdn., Chapman and Hall, pp. 182
– 229.
Fell H B. 1925. The histiogenesis of cartilage and bone in the long bones of the
embryonic fowl. Journal of Morphology and Physiology, 4:417-459.
Frandson R D and Spurgeon T L. 1992. Endocrinology. In: Anatomy and Physiology

of Farm Animal, 5th Edition. Lea and Febiger, Philadelphia, Pp.503-505.
Gafni R I , Weise M , Robrecht D T , Meyers J L , Barnes K M , De Levi S and

Baron J. 2001. Catch-up growth is associated with delayed senescenceof
the growth plate in rabbits. Pediatr Res., 50:618–623.
Gardiner E E. 1969. Relatioship between inorganic phosphorus and inorganic calcium

in chicken blood plasma. Canadian Journal of Animal Science, 49: 193-
195.
Genin O, Hasda A, Shinder D and Pines M. 2012. The effect of inhibition of heat-
shock proteins on thiram-induced tibialdyschondroplasia. Poultry
Science,91:1619–1626.
Getty R. 1975. General osteology. In: Sisson and Grossman’s The Anatomy of the
Domestic Animals, vol.I, 5thEdn.,W.B.SaundersCompany, Philadelphia.
Ghosh R K.2015. Primary Veterinary Anatomy, 6thEdn., Current Books Internatinal,

Kolkata, Chennai, Mumbai.
Goff P J. 2004. Cartilage, Bones and Joints,In: Dukes Physiology of Domestic

Animals, (Ed.) Reece, W.O., 12thEdn., Panima Publishing Co.,New Delhi,
Bangalore,pp. 600-618.
Gomorri G. 1942. A modification of the colorimetric phosphorus determination for

use with the photoelectric colorimeter. J. Lab. Clinic. Med., 27: 955.
v
Hall L E , Shirley R B , Bakalli R I , Aggrey S E , Pesti G M and Edwards H M Jr.
2003. Power of two methods for the estimation of bone ash of broilers.
Poultry Science,82:414–418.
Handerson S N, Vincente J L, Pixiey C M, Hargis B M and Tellez G. 2008. Effect of

an early nutritional supplement of broiler performance. Int.J. Poult.Sci.,
7:211-214.
Hassanabadi A, Alizadeh- Ghamsari A and Leslie M A. 2007. Effects of dietary

phytase, calcium and phosphorus on performance, nutrient utilization and
blood parameters of male broiler chickens. Journal of Animal and
Veterinary Advances, 6(12): 1434-1442.
Havenstein G B, Ferket P R, Scheideler S E and Larson B T. 1994. Growth, livability,

and feed conversion of 1957 vs 1991 broilers when fed “typical” 1957 and
1991 broiler diets. Poultry Sci., 73:1785–1794.
Heller V G, Paul H and Thompson R B. 1934. Changes in the blood calcium and
phosphorus partition during the life cycle of the chicken. J. Biol. Chem.,
106:357-364.
Hester P Y. 1994. The role of environment and management on leg abnormalities in

meat-type fowl. Poult. Sci. 73:904-915.
Hogg D A. 1980. A re-investigation of the centres of ossification in the avian skeleton

at and after hatching. Journal of Anatomy, 130:725-743.
Howlett C R. 1979. The fine structure of the proximal growth plate of the avian tibia.
J. Anat.,128:377-399.
Howlett C R, Dickson M and Sheridan A K. 1984. The fine structure of the proximal
growth plate of the avian tibia: Vascular supply. J. Anat., 139:115–132.
Hunt C D, Ollerich D A and Nielsen F H. 1979. Morphology of the perforating

cartilage canals in the proximal tibial growth plate of the chick. Anat.
Rec,.194:143–158.
vi
Hunter G K. 1991. Role of proteoglycan in the provisional calcification of cartilage. A
review and reinterpretation. Clin. Orthop.,262: 256-280.
Hunziker E B and Schenk R K. 1989. Physiological mechanisms adopted by

chondrocytes in regulating longitudinal bone growth in rats. J. Physiol.,
414:55–71.
Imik H, KapakinK A T, Gumus R, Kapakin S, Kurt A. 2012. The effect of

tibialdyschondroplasia on metabolic parameters in broiler chickens. Ankara
üniv vet fakderg, 59, 271-277.
Julian R J. 1998. Rapid growth problems: ascites and skeletal deformities in

broilers.Poultry Science,77:1773–1780.
Kember N F. I960.Cell division in endochondral ossification: a study of cell

proliferation in rat bones by the method of tritiated thymidine
autoradiography. J Bone JtSurg Br., 42:824-839.
Kestin S C, Knowles T G, Inch A E and Gregory N G. 1992. Prevalence of leg

weakness in broiler chickens and its relationship with genotype. Vet.
Rec.,131:190-194.
Kirkwood J K, Duignan P J, Kember N F, Bennet P M and Price D J. 1989.The

growth rate of the tarsometatarsus bone in birds. Journal of
Zoology,London,217: 403–416.
Kirsch T, Harrison G, Golub E E and Nah H D. 2000. The roles of annexinsand types
II and X collagen in matrix vesicle-mediated mineralization of growth plate
cartilage. J Biol Chem.,275:35577– 35583.
Leach R M jr and Ornan E M. 2007. Tibialdyschondroplasia 40 years later.Poultry

Science,86:2053–2058.
Leterrier Cand ConstantinP. 1999. Early bone growth in chickens genetically selected
for a high and low growth rate.Growth Dev Aging, 63(3):75-84.
vii
Leterrier C and Nys Y. 1992. Composition, cortical structure and mechanical
properties of chicken tibiotarsi: effect of growth rate.Br. Poult. Sci., 33(5):
925-39.
Lewinson D and Silbermann M. 1992. Chondroclasts and endothelial cells collaborate

in the process of cartilage resorption. Anat Rec., 233:504–514.
Lilburn M S. 1994. Skeletal growth of commercial poultry species. Poult. Sci. 73:897-
903.
Lin R, Amizuka N, Sasaki T, Aarts M M , Ozawa H, Goltzman D, Henderson J E

and White J H. 2002. 1,25-Dihydroxyvitamin D3 promotesvascularization
of the chondro-osseous junction by stimulatingexpression of vascular
endothelial growth factor and matrixmetalloproteinase 9. J Bone Mineral
Res., 17:1604–1612.
Luna L G. 1968. Manual of Histologic Staining Methods of the Armed Forces

Institute of Pathology, 3rdEdn., McGraw Hill Book Company, New York.
Mackie E J, Ahmed Y A, Tatarczuch L, Chen K S and Mirams M. 2008.

Endochondral ossification: How cartilage is converted into bone in the
developing skeleton. Int. J. Biochem. Cell Biol., 40:46–62.
Malloy P J, Pike J W and Feldman D. 1999. The vitamin D receptor and the syndrome
of hereditary 1,25-Dihydroxyvitamin D-resistant rickets. Endocr Rev.,
20:156–188.
Manohar G R, Omprakash A V and Kanagaraju P. 2015. Leg weakness in commercial

broiler chicken an overview. International Journal of Science,
Environmentand Technology, 4(2): 482 – 487.
McDonald P, Edwards R A, Greenhalgh J F D and Morgan C A. 1995. Minerals.In:

Animal Nutrition, 5th Edition. Longman Singapore Publishers Ltd.
Singapore, pp. 101-105.
viii
Mench J. 2004. Lameness, In: Weeks, C. andButterworth, A., (Eds) Measuring and
AuditingBroiler Welfare, CAB International.
Minina E , Kreschel C, Naski M C, Ornitz D M and Vortkamp A. 2002. Interaction

of FGF, Ihh/Pthlh, and BMP signaling integrates chondrocyteproliferation
and hypertrophic differentiation. Dev Cell,3:439–449.
Nakano T and Sim J S. 1995. Growth dependent changes in the chemical composition
of proximal tibial articular cartilage and growth plate of broiler chickens.
Canadian Journal of Animal Science,75: 433 - 437.
NaldoL, Bailey T A and Samour J H. 2000. Radiographic analysis of the growth rate
of long bones in bustards. Research in Veterinary Science, 69: 233–240.
Naldo J L, Samour J H, Bailey T A. 1998. Radiographic monitoring of the ossification

of long bones in kori (Ardeotiskori) and white-bellied
(Eupodotissenegalensis)bustards. Research in Veterinary Science, 65:161-
163.
Neumann J. 1986. Changes in bone tissue .of broilers treated with growth promoters.
Acta Vet. Brno, 55: 285-291.
Nilsson O , Milchum R D Jr, Schrier L , Ferns S P, Barnes K M, Troendle J F and

Baron J. 2005. Growth plate senescence is associated with loss of DNA
methylation. Journal ofEndocrinology, 186: 241 -249.
Noy Y and Sklan D.1999. Different types of early feeding and performance in chicks
and poults. J. Appl. Poult.Res., 8:16-24.
Oveido – Rondon E O and Wineland M J. 2012. Effect of breeder nutrition and

management, and incubation on broiler leg health. XXIV World’s Poultry
Congress, 5-9 August, Salvador – Bahia, Brazil.
Ortega N, Wang K, Ferrara N, Werb Z and Vu T H. 2010. Complementary interplay

between matrix metalloproteinase-9, vascular endothelial growth factor and
ix
osteoclast function drives endochondral bone formation. Dis. Model Mech.,
3:224–235.
Orth M W. 1999. The regulation of growth plate cartilage turnover. J. Anim. Sc.,
77(2):183-189.
Orth M W and Cook M E. 1994. Avian tibia1 dyschondroplasia: a morphological and

biochemical review of the growth plate lesion and its causes. Vet.
Pathol.,31:403-414.
Paxton H, Tickle P G, Rankin J W, Codd J R and Hutchinson J R.2014. Anatomical

and biomechanical traits ofbroiler chickens across ontogeny. Body segment
inertial properties andmuscle architecture of the pelvic limb. Peer J.,
e473:1-24.
Provot S and SchipaniE. 2005. Molecular mechanisms of endochondral bone

development. Biochem. Biophys. Res. Commun.328:658–665.
Rani M P, Ahmad N N, Prasad, P E. and Latha C S. 2011. Hematological and

biochemical changes of stuntingsyndrome in broiler chicken.Veterinary
World,4(3):124-125.
Reich A, Jaffe N A, Tong A, Lavelin I , Genina O, Pines M, Sklan D, Nussinovitch

A, Ornan E M. 2005. Weight loading young chicks inhibits bone elongation
and promotes growth plate ossification and vascularisation. Journal of
Applied Physiology,98(6): 2381-2389.
RobisonC I, RiceM, MakagonM Mand Karcher D M. 2015. Duck gait: relationship to

hip angle, bone ash, bone density, and morphology. Poultry
Science,94:1060–1067.
Rose N, Constantin P, LeterrierC. 1996. Sex differences in bone growth of broiler

chickens.Growth Dev Aging, 60(2):49-59.
Rowland G N. 1988. Leg problems in broilers and broiler breeders (Skeletal system).
Indian River Int. Breeder Update,iv (1).
x
Rutt J E. 2008. Molecular analysis of the epiphyseal growth plate in rachitic broilers:
evidence for the etiology of the condition. Ph. D. Thesis submitted tothe
Graduate School of The Ohio State University.
Rutten M, Leterrier C, Constantin P, Reiter K and Bessei W. 2002. Bone development

and activity in chickens in response to reduced weight-load on legs.Anim.
Res. 51: 327–336.
Sadler P. 1991. The use of tarsometatarsi in sexing and ageing domestic fowl (Gallus
domesticus L.) and recognizing five – toed breeds in archaeological
material. Circaea, 8(1): 41 -48.
SalamatdoustnobarR , Ghorbani A, Nazerad K , ShahriyarH A, Fani A,GhiyasiJand

KiyaniNahandM. 2011. Influence of Dietary Levels of canola oil on the
broiler chicks tibiamain minerals contents.Annals of Biological Research, 2
(2):346-350.
Samuelson D A. 2007. Textbook of Veterinary Histology. 1stEdn., Saunders Elsevier,

Missouri, pp. 100 – 129.
Schipani E , Ryan H E , Didrickson S, Kobayashi T, Knight M and Johnson R S.

2001. Hypoxia in cartilage: HIF-1_ is essential forchondrocyte growth
arrest and survival. Genes Dev., 15:2865–2876.
Scott M L, Nesheim M C and Young R J. 1982. Essential inorganic nutrients. In:

Nutrition of the Chicken, 3rd Edition. M.L. Scott and Associates,Ithaca,
New York, Pp. 288-304.
Serrat M A. 2014. Environmental temperature impact on bone and cartilage growth.

American Physiological Society. Compr. Physiol.,4:621‐655.
Shim M Y. 2010. Leg problems of modern broilers as affected by incubation

temperature, fluoride and fast growth. Ph.D. Dissertation submitted tothe
Graduate School, The University of Georgia, Athens, Georgia.
xi
Shim M Y, KarnuahA B, AnthonyN B, PestiG M and AggreyS E. 2012.The effects
of broiler chicken growth rate on valgus, varus, and
tibialdyschondroplasia.Poultry Science, 91:62-65.
Simsa S, Hasdai A, Dan H and Ornan E M. 2007. Induction of tibialdyschondroplasia

in turkeys by tetramethylthiuram disulfide (thiram).Poultry
Science,86:1766–1771.
Snedecor G W and Cochran W G. 1989. Statistical Methods. 9thEdn., The Iowa State
University Press, Ames, Iowa.
Sokolova VV, RadtkeI, Heumann R., EppleM. 2006. Effective transfection of cells
with multi-shell calcium phosphate-DNA nanoparticles.
Biomaterials,27:3147–53.
Sreeranjini A R , Ashok N, Indu V R, Lucy K M, Maya S and Syam K V.2013.

Morphological studies on the femur, tibiotarsus and fibula of pea hen
(Pavocristatus).Tamilnadu J. Vet. Anim.Sc.,9(4) :248 – 252.
Srinivas V, Bohensky J and Shapiro I M. 2009. Autophagy: A new phase in the

maturation of growth plate chondrocytes is regulated by HIF, mTOR and
AMP kinase. Cells Tissues Organs,189:88–92.
Srinivasan S, Stevens M J, Sheng H, HallK E and Wiley J W. 1998. Serum from

patients with type 2 diabetes with neuropathy induces complement-
independent, calcium-dependent apoptosis in cultured neuronal cells. J Clin
Invest., 102:1454–1462.
Talapatra S K, Ray S C and Sen K C.1940. Estimation of phosphorus, chlorine,

calcium, magnesium, sodium and potassium in foodstuffs. Indian J. Vet.
Sc. Anim. Husb., 10: 243 – 246.
Thorp B H, Waddington D. 1997. Relationships between the bone pathologies, ash,

and mineral content of long bones in 35-day-old broiler chickens. Res. Vet.
Sci.,62:67–73.
xii
Tsokova L T. 2006. Influence of the enzyme phytase on the clinical status, some
plasma macroelements and the histostructure of femur and tibia in chickens.
Bulg. J. Vet. Med., 9(3): 201−209.
Valteau B, Grimard G, Londono I, Moldovan F and Villemure I. 2011. In vivo

dynamic bone growth modulation is less detrimental but as effective as
static growth modulation. Bone, 49: 996–1004.
Van der Eerden B C J, Karperien M and Wit J M. 2003. Systemic and local regulation
of the growth plate. Endocrine Reviews, 24: 782–801.
VanWyhe R C, Applegate T J, Lilburn M S and Karcher D M. 2012. A comparison of

long bone development in historicaland contemporary ducks. Poultry
Science,91: 2858–2865.
Vijayakumar M Pand Balakrishnan V. 2014. Evaluating the bioavailability of calcium

phosphate nanoparticles as mineral supplement in broiler chicken.Indian
Journal of Scienceand Technology, 7(10), 1475–1480.
Villemure I and Stokes I A F. 2009. Growth plate mechanics and mechanobiology. A

survey of present understanding. Journal of Biomechanics,42: 1793–1803.
Vu T H, Shipley J M, Bergers G, Berger J E , Helms J A, Hanahan D , Shapiro S D,

Senior R M and Werb Z. 1998. MMP-9/gelatinase B is a keyregulator of
growth plate angiogenesis and apoptosis of hypertrophicchondrocytes. Cell,
93:411–422.
Wang W and Kirsch T. 2002. Retinoic acid stimulates annexin-mediated growth plate
chondrocyte mineralization. J Cell Biol., 157:1061–1069.
Wang W, Xu J and Kirsch T. 2003. Annexin-mediated Ca2_ influx regulates growth

plate chondrocyte maturation and apoptosis. J Biol Chem.,278:3762–3769.
Wardale R J and Duance V C. 1996.Collagen expression in chicken

tibialdyschondroplasia. Journal of Cell Science, 109: 1119-1131.
xiii
Waring P and Beaver J. 1996. CyclosporinA rescues thymocytes from apoptosis
induced by very low concentrations of thapsigargin: effects on
mitochondrial function. Exp Cell Res., 227:264–276.
Webster A B. 2004. Welfare implications of avian osteoporosis. Poult. Sci., 83:184-

192.
Williams B, Solomon S, Waddington D, Thorp B and Farquharson C. 2000. Skeletal

development in the meat-type chicken.British Poultry Science,41: 141–149.
Wilsman N J, Farnum C E , Leiferman E M , Fry M and Barreto C. 1996.

Differential growth by growth plates as a function of multiple parameters of
chondrocytic kinetics. J. Orthop. Res., 14:927–36.
Wise D R. 1975. Skeletal abnormalities in table poultry - a review. Avian Pathology,

4: 1-10.
Zhang Z P, Deng Y F, Zhou Z L and Hou Z F. 2013. Expression and identification of

recombinant chicken vascular endothelial growth factor in
pichiapastorisand its role in the pathogenesis of tibialdyschondroplasia.
Poultry Science, 92:3214–3227.
xiv

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Behera,M.

Age-specific Gross Morphometrical and

MASTER OF VETERINARY SCIENCE

DEPARTMENT OF VETERINARY ANATOMY

Dr. Arun Kumar Mandal, Ph.D. Bhubaneswar

2. Dr. A.K. Kundu ___________________

3. Dr. A. Maity ___________________

4. Dr. S. Sathapathy ___________________

Elizabeth Asquith Bibesco.

The perspicuous piece of acknowledgement provides me the opportunity to express

I am greatly beholden beyond words to express my deep sense of obligation and

I gratefully articulate my sincere gratification and obligation to the member of the

It is my pleasure to express my deepest sense of regards to Dr. R K Das Professor,

I feel previleged to express my heartiest gratefulness to Dr.(Mrs) Ritun Patra and

It is a unique opportunity to express my sincere and dear sense of indebtedness thanks

I take privilege of expressing gratitude to Dr. Sushant Kumar Dash, Associate

I owe my heartiest gratefulness to Dr. Kamadev Sethi, Assistant Professor,

I am greatly beholden beyond words to express my sincere and deepest sense of

I gratefully articulate my sincere gratification and obligation to my Respected

It is my great pleasure as well as a golden opportunity to express my deepest sense of

I express my obligation to Sri A. K. Punja, General Manager, Eastern Hatchery,

Date: (Minati Behera)

4.1 Gross Morphometrical Observations 40

5.1 Gross Morphometrical Observations 111

6.1 Summary 130

6.2 Conclusion 137

A broiler is a type of domestic chicken specifically raised for meat production.

The growth plate is a highly organized cartilage structure, i.e. a connective

Growth plate cartilage is the key to the longitudinal growth (elongation) of

Any sort of disruption in normal development and growth of the epiphyseal

An important factor in the diagnosis of skeletal abnormality is an understanding

Phosphorus, the other major constituent of bone, is an essential component of

i) To record the age-specific gross morphometrical data on long leg bones in

2.1 Skeletal or Leg Problems (Deformities) and Animal Welfare in Broiler

Skeletal deformities can be caused in a variety of ways. Nutritional

Selection in development of commercial chicken meat hybrids focused on

Comparative clinical and morphological studies on the incidence of tibial

Genin et al. (2012) experimented on thiram-induced tibial dyschondroplasia.

2.2 Gross morphometrical study

Williams et al. (2000) studied skeletal development in the meat-type chicken

Simsa et al. (2007) compared body growth in tibial dyschondroplasia (TD)

Paxton et al. (2014) studied anatomical and biomechanical traits of broiler

2.3 Radiographical study

Hogg (1980) re-investigated the centres of ossification in the avian skeleton at

A serial radiographic study was conducted by Naldo et al. (2000) on four

Williams et al. (2000) investigated skeletal development in meat –type

Breugelmans et al. (2007) determined the age-related degree of ossification of

2.4 Histomorphological study

Similar to the situation in fetal mammals, primary ossification centers arise

The main physiological events of endochondral ossification are chondrocyte

Each zone differs in chondrocyte size and arrangement as well as extracellular

Then these cells further progress in the differentiation pathway to become

Subsequently, the mineralized chondrocytes at the bottom of the zone,

Longitudinal and transverse septae, which keep the chondrocytes in a

Barreto and Wilsman (1994) demonstrated the relationship between the

Samples of growth plates from birds on control and calcium-deficient diets

Burdan et al. (2009) reported morphology and physiology of the epiphyseal

According to Serrat (2014), environmental temperature can have a surprising

2.4.2 Growth plate (cartilage) abnormality

Collagen expression in growth plate cartilage derived from broiler chickens

Vitamin D deficiency in particular, resulting in rickets is associated with

Tibial dyschondroplasia (TD) is characterized by an avascular lesion in which