animals
Article
Evaluation of Clinical and Biochemical Traits in Egyptian Barki
Sheep with Different Growth Performances
Ragab M. Fereig 1, * , Rawia M. Ibrahim 2 , Atef M. Khalil 3 , Caroline F. Frey 4
Citation: Fereig, R.M.; Ibrahim, R.M.;
Khalil, A.M.; Frey, C.F.; Khalifa, F.A.
Evaluation of Clinical and
Biochemical Traits in Egyptian Barki
Sheep with Different Growth
Performances. Animals 2023, 13, 962.
https://doi.org/10.3390/
ani13060962
Academic Editors: Wei Liu,
Liping Jiang and Yisong Liu
Received: 14 February 2023
Revised: 26 February 2023
Accepted: 28 February 2023
Published: 7 March 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Animals 2023, 13, 962. https://doi.org/10.3390/ani13060962
and Fatma A. Khalifa 5
1 Division of Internal Medicine, Department of Animal Medicine, Faculty of Veterinary Medicine,
South Valley University, Qena 83523, Egypt
2 Division of Clinical and Laboratory Diagnosis, Department of Animal Medicine, Faculty of Veterinary
Medicine, South Valley University, Qena 83523, Egypt
3 Division of Clinical Pathology, Department of Pathology and Clinical Pathology, Faculty of Veterinary
Medicine, South Valley University, Qena 83523, Egypt
4 Institute of Parasitology, Department of Infectious Diseases and Pathobiology, Vetsuisse-Faculty,
University of Bern, Länggassstrasse 122, CH-3012 Bern, Switzerland
5 Division of Infectious Diseases, Department of Animal Medicine, Faculty of Veterinary Medicine,
South Valley University, Qena 83523, Egypt
* Correspondence: ragab.feraeg2@vet.svu.edu.eg
Simple Summary: Our randomized controlled clinical study revealed novel and valuable information
on detecting biomarkers for growth performance in Barki sheep. Our study revealed that in most
cases, the stunted Barki sheep exhibited a normal clinical picture, including appetite, and normal
biochemical profiles for minerals and liver function markers. However, levels of nutritionally relevant
biomarkers, such as total protein, albumin, cholesterol, and triglycerides, were markedly decreased
in stunted sheep compared with normal sheep. These findings were also confirmed by lower levels
of growth and thyroid hormones in stunted sheep than those in the control sheep. This approach is
useful from economic and financial perspectives as it can be applied to exclude animals with poor
growth and development from the livestock industry.
Abstract: The Barki sheep industry is becoming increasingly important in Egypt because of the high
quality of their meat and wool. This sheep breed is also commonly known for its resistance to arid
and harsh environmental conditions. Such characteristics can be exploited in solving the problematic
situation of inadequate animal protein for human consumption, particularly under climatic changes.
However, very few studies have investigated aspects of breeding, nutrition, and susceptibility to
infectious or non-infectious diseases in Barki sheep. Herein, we propose to unravel the differences
in the clinical and biochemical profiles among Barki sheep of different growth rates. We measured
clinical and biochemical parameters in stunted (n = 10; test group) and in good body condition (n =
9; control group) Barki sheep. Animals subjected to this experiment were of the same sex (female),
age (12 months old), and housed in the same farm with similar conditions of feeding, management
practice, and vaccination and deworming regimens. Regarding clinical examination, stunted/tested
sheep showed a significantly higher pulse and respiratory rate compared to sheep with a good body
condition/control group. The appetite, body temperature, and digestion processes were the same in
both groups. In biochemical investigations, nutritional biomarkers were reduced markedly in stunted
sheep compared with the control sheep, including total protein (p = 0.0445), albumin (p = 0.0087),
cholesterol (p = 0.0007), and triglycerides (p = 0.0059). In addition, the Barki sheep test group suffered
from higher levels of urea and blood urea nitrogen than the control group. Consistently, growth and
thyroid hormone levels were lower in stunted sheep than the control sheep, although the differences
were not statistically significant (p > 0.05). No significant differences were detected in both groups for
serum levels of calcium, phosphorus, magnesium, iron, and zinc (p > 0.05). To detect the reasons for
emaciation, certain debilitating infections were tested. All tested sheep showed negative coprological
tests for gastrointestinal parasites, and had no obvious seropositivity to brucellosis, toxoplasmosis,
neosporosis, or Q fever. This study demonstrates the useful biochemical markers for monitoring
https://www.mdpi.com/journal/animals
Animals 2023, 13, 962 2 of 13

growth performance in Egyptian Barki sheep and unravels the usefulness of this breed in nationwide
breeding and farming.

Keywords: Barki sheep; parasite; mineral; biochemical; clinical; protein

1. Introduction
Sheep are one of the primary sources of animal protein in human diets in Egypt and
worldwide. Sheep can even endure in the desert by grazing on poor-quality fodder [1].
With 470,000 heads (11% of the total Egyptian sheep population), Barki sheep are raised in
the Northwestern Coastal Desert of Egypt using a transhumant animal farming technique.
Additionally, it is well known that this breed can survive in the arid conditions of Egypt [2].
Individual sheep of the same breed vary in terms of their final body weight and rate of
growth. Thus, choosing lambs with rapid growth and a high final body weight is essential
for enhancing meat production, which is the primary goal of sheep breeding projects [3].
Farm animal growth performance is a crucial economic feature that is influenced by
both genetic and non-genetic variables, such as type of birth, sex, breed, season, age, and
pre-mating weight of the dam [4,5].
The estimation of normal values of blood biochemical parameters is a highly significant
tool for the laboratory interpretation of the clinical condition of tested sheep. Numerous
factors, including age, sex, stress, weather, season, physical exercise, and pregnancy, can
affect biochemical parameters. The degree of soundness and sickness of animals can be
evaluated using biochemical parameters [6].
Growth hormone administration led to early puberty in Rahmani ewe lambs. This may
be due to increased provision of trophic signals represented by increased gonadotropin-
releasing hormone (GnRH) and luteinizing hormone (LH) secretions, which may arise from
the early activation of the GnRH pulse generator. Furthermore, increased nutrient avail-
ability is crucial for ovarian function and/or local enhancement of ovarian activity. These
effects may result from direct and/or indirect influences of somatotropin on the previously
mentioned levels. Serum IGF-1 and/or blood-borne metabolites (mainly glucose) may be
the potential signals by which somatotropin exerted its indirect effect [7].
The thyroid hormones, triiodothyronine (T3) and thyroxine (T4), relevant metabolic
indices of animals’ nutritional state, maintain nutrient homeostasis, thermoregulation,
growth, and productivity [8]. They are essential for normal growth and development of
the fetus. T3 and T4 are required for the general accretion of fetal mass, elicit discrete
developmental events in the fetal brain and somatic tissues from early gestation, and
promote terminal differentiation of fetal tissues closer to term [9].
Toxoplasma gondii and Neospora caninum are intracellular protozoan parasites affecting
sheep and can induce serious illness in lambs and severe reproductive disorders in pregnant
ewes. In addition, Brucella spp. and Coxiella burnetii are bacterial agents with similar clinical
forms of the above-mentioned protozoa. Previous reports document the high prevalence of
the four pathogens among sheep in different regions of Egypt [10–12].
Despite the increasing significance of Barki sheep in different regions in Egypt, few
studies on various aspects of biology and infection have been conducted on this breed.
Herein, we measure clinical and biochemical parameters in stunted and good body con-
dition Barki sheep. Animals used for this experiment were of the same sex (female), age
(12 months old), and housed in the same farm with similar conditions of feeding, man-
agement practice, and vaccination and deworming regimens. A previous report had
investigated some endocrine and biochemical parameters among fast- and slow-growing
Muzaffarnagari male and female lambs during the postweaning period [13]. However, in
the current study, we investigated sheep of a different breed (Barki), sex (female), location
(Egypt), and demonstrated novel data on various examined clinical aspects, biochemical
parameters, and metabolites. Furthermore, we provided more evidence for the health status
Animals 2023, 13, 962 3 of 13

of tested sheep using serological screening of a group of highly debilitating infectious dis-
eases (brucellosis, Q fever, toxoplasmosis, and neosporosis). Additionally, we established
a correlation profile of various clinical, biochemical, and hormonal parameters against
the recorded body weight of each animal. Our study revealed differences in numerous
variables of the clinical and biochemical profile among the two tested groups.

2. Materials and Methods

2.1. Ethical Statement
This study was performed according to standard procedures identified by the Research
Board of the Faculty of Veterinary Medicine, South Valley University, Qena, Egypt. The
study was approved by the Research Bioethics Committee at South Valley University
VM/SVU/23(1)-05. This study was conducted according to ARRIVE guidelines for animal
handling and experimentation (https://arriveguidelines.org/arrive-guidelines accessed
on 22 December 2022).

2.2. Animals of the Study

The current study was conducted on a private Barki sheep flock at Qena governorate,
southern Egypt. Barki sheep have been bred from sheep that were raised in the Barca
region in eastern Libya, from which the name was derived. These sheep were transported
to western Egypt to the Matrouh governorate by the free movement of Bedouins. High
numbers of Barki sheep are usually transported from Matrouh to the Beheira governorate
because of plentiful agricultural land, animal farms, and also its proximity to Cairo and
other big Egyptian cities (Figure S1). Our tested animal flock of Barki sheep was purchased
from a private farm in the Beheira governorate in September 2021 at the age of 4 months
old with an approximate body weight of 11–13 kg for the purpose of commercial breeding.
These animals were female and were kept and fed together from the first day of arrival until
12 months of age. The animals in this study consumed drinking water and a diet composed
of dry berseem (Trifolium alexandrinum L.) ad libitum and concentrates based on wheat bran,
crushed soybean and yellow corn, vitamin premix, and common salt, formulated to meet
the proper rations for the growing animals. Qena is located in the southern region of Egypt,
which is a hotter and drier place than the Beheira region [14]. During that time, all sheep
were subjected to the same conditions of housing, feeding, vaccination, and deworming
regimens. All sheep in this experiment received a primary (at 4 months old) and a booster
dose (at 5.5 months old) of Covexin® 8 vaccine according to the manufacturer’s protocols
(Merck, Rahway, NJ, USA), which is protective against infection with Clostridium chauvoei,
C. septicum, C. novyi type B, C. haemolyticum (known also as C. novyi type D), C. tetani, or
C. perfringens types C and D. In addition, the sheep flock was treated with Trimec according
to the manufacturer’s instructions (Pharma SWEDE, 10th of Ramadan city, Sharkia, Egypt),
which is a broad spectrum anti-parasitic oral suspension (1 mL contains triclabendazole
50 mg and ivermectin 1 mg), at 7 and 10 months old at two drenching times, each 21 days
apart to combat the prepatent stages. Sanitary measures were applied to the farm for
routine cleanliness and disinfection by glutaraldehyde. At the age of 12 months, despite
exposure to similar conditions, a remarkable difference in body weight was observed in
some animals (herein referred as stunted/test animals, n = 10) compared to others (referred
as good body condition/control animals, n = 9) (Figure 1). All the investigated sheep
showed no obvious differences in appetite and digestive behaviors.
Animals 2023, 13, 962 4 of 13
Animals 2023, 13, x FOR PEER REVIEW 4 of 14

Figure 1. Illustration diagram showing the observational and experimental approaches in this study.
Figure 1. Illustration diagram showing the observational and experimental approaches in this study.
Our sheep were purchased from the Beheira governorate in September 2021 at the age of 4 months
Our sheep were purchased from the Beheira
with an approximate governorate
body weight in They
of 11–13 kg. September 2021 at
were subjected the age of 4procedures
to experimental months at
12 months old. During that time, all sheep were subjected to
with an approximate body weight of 11–13 kg. They were subjected to experimental procedures the same conditions of housing, feed-
ing, vaccination, and deworming regimens. All sheep in this experiment received a primary (at 4
at 12 months old. Duringmonthsthat
old) time, all sheep
and a booster dose were
(at 5.5 subjected
months old),to the
and same
were conditions
treated with a broadofspectrum
housing, anti-
feeding, vaccination, and deworming
parasitic regimens.
oral suspension at 7 and 10 All sheep
months old.inAtthis experiment
the age of 12 months,received a primary
despite expose to similar
(at 4 months old) and conditions,
a boosteradoseremarkable difference in body weight was observed in some animals (herein referred
(at 5.5 months old), and were treated with a broad spectrum
as stunted/test animals, n = 10) compared to others (referred as good condition/control animals, n =
anti-parasitic oral suspension
9). The 2 arrows above10
at 7 and months
refer old. At of
to the drenching the age of
animals 12anthelmintic
with months, despite expose
drugs 2 times to
at 3-week
intervals, according to the manufacturer’s instructions.
similar conditions, a remarkable difference in body weight was observed in some animals (herein
referred as stunted/test animals, n = 10) compared to others (referred as good condition/control
2.3. Case History and Clinical Examination
animals, n = 9). The 2 arrows above refer to the drenching of animals with anthelmintic drugs 2 times
A specified questionnaire was designed to record the information and complaints of
at 3-week intervals, according
the animal toowner,
the manufacturer’s instructions.
the exhibited clinical signs, and management practices. The collected
data were organized with our recorded signs and observations on each animal and breed-
2.3. Case History anding
Clinical Examination
site. Careful routine clinical examination of these studied cases was carried out ac-
cording to thewas
A specified questionnaire methods described
designed topreviously
record the [15].
information and complaints of
the animal owner, the exhibited clinical signs, and management
2.4. Fecal Collection and Parasitological Investigations
practices. The collected
data were organized with our recorded signs and observations on each animal and breeding
Fecal samples were collected directly from the rectum of stunted and good body con-
site. Careful routinedition
clinical examination
animals in a clean, of
drythese
plasticstudied cases was carried
bag for examination out
of specific according
eggs using direct
to the methods described
smear andpreviously [15]. sedimentation-saturated salt-based flotation technique [16].
a concentration

2.5. Blood
2.4. Fecal Collection and Collection Investigations
Parasitological
At the end of study, 5 mL blood samples were collected via jugular venipuncture
Fecal samples from
wereeach
collected directly from the rectum of stunted and good body
animal at 12 months old after disinfecting the puncture area with ethyl alcohol
condition animals in70%.
a clean, dry plastic bag for
The samples were placed intoexamination of specific
a vacutainer tube eggs using for
without anticoagulant direct
serum
smear and a concentration sedimentation-saturated
separation salt-based
from stunted sheep (n = 10) and flotation
those of good body technique
condition (n [16].
= 9). Serum
was separated by leaving the sample in an upright position for 2 h in a cooling box fol-
2.5. Blood Collection lowed by centrifugation at 4000 rpm/20 min. Next, the separated serum samples were
stored at −20 °C until biochemical analyses.
At the end of study, 5 mL blood samples were collected via jugular venipuncture
from each animal at2.6.
12 Biochemical
months old after disinfecting the puncture area with ethyl alcohol
Investigation
70%. The samples wereSerum placed intowere
samples a vacutainer tube
used to estimate without
total protein, anticoagulant for
albumin, urea, urea serumcre-
nitrogen,
atinine,sheep
separation from stunted cholesterol, triglycerides,
(n = 10) and those total
ofbilirubin,
good body directcondition
bilirubin, alanine transaminase
(n = 9). Serum
was separated by leaving the sample in an upright position for 2 h in a cooling box followed
by centrifugation at 4000 rpm/20 min. Next, the separated serum samples were stored at
−20 ◦ C until biochemical analyses.

2.6. Biochemical Investigation

Serum samples were used to estimate total protein, albumin, urea, urea nitrogen, crea-
tinine, cholesterol, triglycerides, total bilirubin, direct bilirubin, alanine transaminase (ALT),
aspartate transaminase (AST), and glucose using colorimetric methods and commercial
kits (Spectrum Diagnostics, Cairo, Egypt) according to the manufacturer’s instructions.
Calcium, phosphorus, magnesium, iron, and zinc were measured using colorimetric meth-
Animals 2023, 13, 962 5 of 13

ods and commercial kits (Bio-Diagnostic, Giza, Egypt) according to the manufacturer’s
instructions. Values were read using a UV spectrophotometer (SEAC, Slim, Florence, Italy).
Globulin concentration was calculated by subtracting albumin from the corresponding
total protein value.

2.7. Hormonal Analysis

Quantitative determinations of sheep thyroid stimulating hormone (TSH) and sheep
growth hormone (GH) were estimated in undiluted serum samples using an ELISA Kit (My-
Biosource, Mumbai, Maharashatra, India). The detection assay procedures and calculations
were undertaken according to the manufacturer’s guidelines and protocols.

2.8. Serological Investigations and ELISAs

To detect antibodies of T. gondii, the serum samples were analyzed with an indirect multi-
species ELISA for toxoplasmosis (ID.vet, Grabels, France) according to the manufacturer’s
instructions. Serum samples and controls were diluted 1:10. The optical density (OD) obtained
was used to calculate the percentage of sample (S) to positive (P) ratio (S/P%) for each of
the test samples according to the following formula: S/P (%) = (OD sample − OD negative
control)/(OD positive control − OD negative control) × 100. Samples with an S/P% of less
than 40% were considered negative; if the S/P% was between 40% and 50%, the result was
considered doubtful, and considered positive if the S/P% was greater than 50%.
For antibodies of N. caninum, serum samples were analyzed with a competitive multi-
species ELISA for neosporosis (ID.vet, Grabels, France). Serum samples and controls were
diluted 1:2. The ODs obtained were used to calculate the percentage of sample (S) to
negative (N) ratio (S/N%) for each of the test samples according to the following formula:
S/N (%) = OD sample/OD negative control × 100. Samples with an S/N% greater than
60% were considered negative; if the S/N% was between 50% and 60% the result was
considered doubtful and was considered positive if the S/N% was less than 50%.
Concerning Brucella spp., serum samples were analyzed with an indirect multi-species
ELISA for brucellosis (ID.vet, Grabels, France). Serum samples and controls were di-
luted 1:20. The ODs obtained were used to calculate the percentage of sample (S) to
positive (P) ratio (S/P%) for each of the test samples according to the following formula:
S/P (%) = (OD sample − OD negative control)/(OD positive control − OD negative control)
× 100. Samples with an S/P% of less than 110% were considered negative; if the S/P% was
between 110% and 120% the result was considered doubtful and was considered positive if
the S/P% was greater than 120%.
Similarly, in Coxiella burnetii (Q fever) antibody detection, serum samples were ana-
lyzed with an indirect multi-species ELISA for Q fever (ID.vet, Grabels, France). Serum
samples and controls were diluted 1:50. The ODs obtained were used to calculate the per-
centage of sample (S) to positive (P) ratio (S/P%) for each of the test samples according to
the following formula: S/P (%) = (OD sample − OD negative control)/(OD positive control
− OD negative control) × 100. Samples with an S/P% of less than 40% were considered
negative; if the S/P% was between 40% and 50% the result was considered doubtful, from
50% to 80% were regarded as positive, and it was considered strong positive if the S/P%
was greater than 80%.
The ODs of all ELISA results were read at 450 nm and measured with an Infinite®
F50/Robotic ELISA reader (Tecan Group Ltd., Männedorf, Switzerland). A summary of
commercial kit ELISAs details is described in Table 1.
Animals 2023, 13, 962 6 of 13

Table 1. Commercially available ELISA test kits used for detecting antibodies to T. gondii, N. caninum,
Brucella spp., and Q fever.

Infectious Agent ELISA Test Kit Antigen Serum Dilution Conjugate Interpretation
ID Screen® Negative < 40
Anti-multi-species
Toxoplasma gondii Toxoplasmosis P30 antigen 1:10 Doubtful 40–50
IgG-HRP
Indirect Multi-species Positive > 50
ID Screen® Neospora Anti-N. Negative > 60
Purified extract of
Neospora caninum caninum competition 1:2 caninum-HRP(detect Doubtful 50–60
Neospora caninum
Multispecies IgG or IgM) Positive < 50
ID Screen® Negative < 110
LPS of Brucella Anti-multi-species-
Brucella species Brucellosis Serum 1:20 Doubtful 110–120
species IgG-HRP
Indirect Multispecies Positive > 120
Negative < 40
ID Screen® Q fever Phase I and II Anti-multi-species
Coxiella burnetii (Q fever) 1:50 Doubtful 40–50
Indirect Multispecies proteins of C. burnetii IgG-HRP conjugate
Positive > 50
All kits were purchased from ID. Vet., Innovative Diagnostics, Grabels, France.

2.9. Statistical Analyses

Statistical analyses of clinical and biochemical variables between lean and good condi-
tion groups were measured by unpaired Student’s t-test using GraphPad Prism version 5
(GraphPad Software Inc., La Jolla, CA, USA). The 95% confidence intervals of a proportion,
including continuity correction, were calculated using www.vassarstats.net (accessed on 22
December 2022). The results were considered significant when the p-value was <0.05 or
highly significant when the p-value was <0.0001.

2.10. Pearson’s Correlation Coefficient

The correlation between the values of body weight and those against body condition
score, and a number of biochemical parameters that showed significant differences and growth
hormone between the 2 groups, were analyzed using Pearson’s correlation coefficient test.
Correlation coefficients were calculated using Pearson’s correlation coefficient: |r| = 0.70,
strong correlation; 0.5 < |r| < 0.7, moderately strong correlation; and |r| = 0.3–0.5 weak-to-
moderate correlation [17].

3. Results
In this study, a BCS scale consisting of four grades was evaluated (in the poor condition
or stunted sheep group, 1 is emaciated and 2 is thin; in the good body condition or
control sheep group, 3 is moderate and 4 is good body condition). There is a highly
significant (p ≤ 0.0001) difference between the poor body condition test group and the
group with good body conditions in terms of body weight and BCS (Table 2) (Figure 2).
Regarding clinical systemic parameters, there was a highly significant increase (p ≤ 0.0001)
in pulse rate (beat/min) in poor body condition animals (87.1 ± 8.9) compared with the
control group (68.6 ± 5.3). Similarly, but with a lower degree of significance, there was
a significant increase (p ≤ 0.0134) in respiratory rate (breath/min) in stunted animals
(30.1 ± 4.3) compared with the control group (24.6 ± 4.5). However, the body temperature
in both groups did not change markedly (p = 0.203). Concerning other items of clinical
investigation, no obvious changes were observed between the test and control groups,
including characteristics of digestive, respiratory, nervous, or urogenital systems, or skin,
mucous membrane, or lymph nodes (Table 2) (Figure 2). These results suggest the minimal
effect of stunted growth on clinical pictures of tested sheep.
 Animals 2023, 13, x FOR PEER REVIEW 7 of 14
Animals 2023, 13, 962 7 of 13

Table 2. Clinical findings among Barki sheep of good and poor condition.

Parameter (Unit) Stunted/Test (n = 10) Good Condition/Control (n = 9) p Value

Table 2. Clinical findings among Barki sheep of good and poor condition.
Body weight (Kg) 20.5 ± 2.4 27.8 ± 2.5 <0.0001
Body condition score (grade 1–4) 1.5 ± 0.52 3.3 ± 0.51 <0.0001
Parameter (Unit) Stunted/Test (n = 10) Good Condition/Control (n = 9) p Value
Body temperature (Celsius degree) 39.6 ± 0.23 39.5 ± 0.25 0.203
Body weight (Kg)
Respiratory rate (breath/min) 20.5 ± 2.4 30.1 ± 4.3 27.8 ± 24.6
2.5 ± 4.5 <0.0001
0.0134
Body condition score (grade 1–4) 1.5 ± 0.52 3.3 ± 0.51 <0.0001
Pulse rate (beat/min) 87.1 ± 8.9 68.6 ± 5.3 <0.0001
Body temperature (Celsius degree) 39.6 ± 0.23 39.5 ± 0.25 0.203
Ruminal
Respiratory rate movement (contraction/2 min)
(breath/min) 30.1 ± 4.3 3.8 ± 1.3 24.6 ± 4.2
4.5 ± 0.97 0.442
0.0134
Pulse rate (beat/min) Appetite 87.1Normal
± 8.9 to slightly reduced Normal
68.6 ± 5.3 -
<0.0001
Ruminal movement (contraction/2 min)Feces 3.8 ± 1.3 Normal Normal
4.2 ± 0.97 -
0.442
Appetite Nasal discharges Normal to slightlyScanty
reduced to absent Normal
Scanty to absent - -
Feces Coat NormalRough, rusty, alopecia Normal
Shiny, smooth - -
Nasal discharges Mucous membrane Scanty to absent
Pale to rosy-red Scanty to Rosy-red
absent - -
Coat Rough, rusty, alopecia Shiny, smooth -
Lymph node abnormalities None None -
Mucous membrane Pale to rosy-red Rosy-red -
Lymph node abnormalities Eyes lesions None None None None - -
Eyes lesions Urogenital abnormalities None None None None - -
Nervous disorders
Urogenital abnormalities None None None None - -
Nervous disordersLocomotor disorders None None None None - -
Locomotor disorders Noneat <0.05 p value and highly significant
Significance values Noneat <0.0001 p values measured by -t-test
(unpaired).
Significance values at <0.05 p value and highly significant at <0.0001 p values measured by t-test (unpaired).

Figure 2. Clinical findings of examined Barki sheep. Body weight, body condition, and systemic
Figure 2. Clinical findings of examined Barki sheep. Body weight, body condition, and systemic
parameters were investigated
parameters in stunted
were investigated andand
in stunted good
goodbody condition
body condition sheep.
sheep. Each
Each bar bar represents
represents the
the mean ± mean
SD with
± SD with p-value
the the p-valuecomparing
comparing thethe
twotwo groups
groups using
using an an Student’s
unpaired unpaired Signifi- t-test.
Student’s
t-test.
cance at p = <0.05.
Significance at p ≤ 0.05.
The results (Figure 3) (Table S1) show the serum biochemical variables between the
The results (Figure
stunted 3) (Table
and control groups.S1) show
These therevealed
results serumthe biochemical variables
significant decrease between
(p = <0.05) in the
stunted andcholesterol
control (35.7
groups. These
± 5.8 vs. results
49.8 ± 8.8), revealed
triglycerides (36 ±the
3.3 significant
vs. 46.9 ± 10.4),decrease (p ≤ 0.05)
creatinine (0.58
in cholesterol (35.7 ± 5.8 vs. 49.8 ± 8.8), triglycerides (36 ± 3.3 vs. 46.9 ± 10.4), creati-
nine (0.58 ± 0.09 vs. 0.81 ± 0.1), total protein (6.7 ± 0.44 vs. 7.2 ± 0.48), and albumin
(3.6 ± 0.27 vs. 4.1 ± 0.4) for stunted vs. good body condition groups, respectively. Op-
positely, a marked increase (p ≤ 0.05) was observed in stunted sheep compared to the
good body condition group in the serum urea level (29.6 ± 3.2 vs. 26.9 ± 2.9) and BUN
(13.8 ± 1.5 vs. 12.3 ± 1.4), respectively. However, non-statistically significant variations
(p ≥ 0.05) were obtained liver function tests (total bilirubin, direct and indirect bilirubin,
ALT, AST), globulin, and glucose levels. These results indicate the suffering of stunted
sheep from nutritional deficiency and a degree of renal insufficiency.
Similarly, non-statistically significant variations (p ≥ 0.05) were observed when a
number of macro- and micro-elements were tested (calcium, phosphorus, magnesium, zinc,
and iron) (Figure 4) (Table S1).
 ± 0.09 vs. 0.81 ± 0.1), total protein (6.7 ± 0.44 vs. 7.2 ± 0.48), and albumin (3.6 ± 0.27 vs. 4.1
± 0.4) for stunted vs. good body condition groups, respectively. Oppositely, a marked in-
crease (p = <0.05) was observed in stunted sheep compared to the good body condition
group in the serum urea level (29.6 ± 3.2 vs. 26.9 ± 2.9) and BUN (13.8 ± 1.5 vs. 12.3 ± 1.4),
respectively. However, non-statistically significant variations (p = >0.05) were obtained
Animals 2023, 13, 962 liver function tests (total bilirubin, direct and indirect bilirubin, ALT, AST), globulin, and
8 of 13
glucose levels. These results indicate the suffering of stunted sheep from nutritional defi-
ciency and a degree of renal insufficiency.

Cholesterol Triglycerides Creatinine Blood urea BUN

p = <0.0001 40 p = 0.0381 20 p = 0.0381
80 1.0
80
p = 0.0007 p = 0.0059
0.8 30 15
60 60

mg/dl

mg/dl
0.6

mg/dl
mg/dl
mg/dl
40 20 10
40
0.4

20
20 10 5
0.2

0 0.0 0 0
0
d ed on te
d on
on nt iti d on

d
iti

on
te iti te
un

te
un tu nd nd un iti

iti
St

un
St nd S
co nd

nd
co co St

St
d co

co
d oo
d
oo oo d

d
G
oo

oo
G G

G
G

Total biliruben Direct bilirubin Indirect bilirubin ALT AST

p = 0.738 p = 0.473 p = 0.858 p = 0.823

1.0 0.8 60 p = 0.370 150
0.25
0.8
0.20 0.6
40 100
0.6
mg/dl

mg/dl
0.15

mg/dl

U/l

U/l
0.4
0.4
0.10
20 50
0.2 0.2
0.05

0.0 0.00 0.0 0 0

d on d
te iti te on

on
iti

d
un

d
on

on
te
un

te
nd

te
St nd

iti
un
St

iti

iti
un

un
co

nd
co

nd

nd
St
St

St
co
d

co
d

co
oo oo

d
d

d
G

oo
G

oo

oo
G
G

G
Albumin Globulin A/G ratio Glucose
Total protein
p = 0.0087 p=1 p = 0.193
10 4 2.0 p = 0.956
p = 0.0445 5 60
8 4 1.5
3
6 40

Ratio
3
g/dl

g/dl

mg/dl
g/dl

2 1.0
4 2
20
1 0.5
2 1

0 0 0 0.0 0
d d
on on

d
d

on
on

d
te te

on
te
te

iti iti

te
un
iti

iti
un

un
un

iti
un
nd nd
nd

nd
St St

nd
St

St

St
co co
co

co

co
d d
d

d
oo oo
oo

oo

oo
G

G
G
G

Figure 3. Biochemical
Animals 2023, 13, x FOR PEER REVIEW analysis
Figure of Barki
3. Biochemical sheepofserum.
analysis Values
Barki sheep ofValues
serum. various serumserum
of various biochemical variables
biochemical 9variables
of 14
in Barkiand
in Barki sheep of stunted sheep of stunted
good bodyand good bodyEach
condition. condition. Each bar represents
bar represents the mean ± SD
the mean ± SD
withwithp-value
p-value
comparing the two groups using an unpaired Student’s t-test. Significance at p = <0.05.
comparing the two groups using an unpaired Student’s t-test. Significance at p ≤ 0.05.

Calcium
Similarly, non-statistically
Phosphorus
significant variations (p = >0.05) were observed when a
Magnesium
number of macro-
8 and
p = micro-elements
0.432 were
3 tested
p = (calcium,
0.0962 phosphorus, magnesium,
15
p = 0.700
zinc, and iron) (Figure 4) (Table S1).
6
2
10
mg/dl
mg/dl
mg/dl

4
1
5
2

0 0 0
d

ed
n
d

on
n

te

tio
te

tio

nt iti
un
un

tu
i

nd
nd
nd

St
St

S
co
co
co

d
d
d

oo
oo
oo

G
G
G

Iron Zinc
200 80
p = 0.499
p = 0.124
150 60
ug/dl
ug/dl

100 40

50 20

0 0
d

n
te

tio

te

tio
un

un
i
nd

i
nd
St

St
co

co
d

d
oo

oo
G

Figure 4. Mineral analysis of Barki sheep serum. Values of various serum variables in Barki sheep of
Figure 4. Mineral analysis of Barki sheep serum. Values of various serum variables in Barki sheep
stunted
of and
stunted andgood
goodbody
bodycondition.
condition.Each
Eachbar
barrepresents
representsthe mean±
themean SD with
± SD p-valuecomparing
with p-value comparingthe
the
twogroups
two groupsusing
usingan
anunpaired
unpairedStudent’s t-test. Significance
Student’s t-test. atpp =≤<0.05.
Significanceat 0.05.

Although we
Although we observed
observed differences
differencesbetween
betweenstunted
stuntedandandcontrol
control groups
groups concerning
concerning the
thyroid
the and and
thyroid growth hormones,
growth they were
hormones, theynot statistically
were significantsignificant
not statistically (p > 0.05) (Figure 5) (Table
(p > 0.05) S1).
(Figure
These results demonstrate the balanced ratio of minerals between the two sheep groups.
5) (Table S1). These results demonstrate the balanced ratio of minerals between the two
Pearson’s correlation coefficient was used to investigate the association between body
sheep groups.
weights with values of BCS and biochemical parameters and showed significant differences
between the two groups. A better correlation was identified for BCS (strong; Pearson
rTSH
= 0.917) and albumin (moderately strong; Pearson Growth r =hormone
0.683), followed by triglycerides
(moderately strong; Pearson r = 0.537), cholesterol (weak-to-moderate;
p = 0.250 Pearson r = 0.489),
0.006 p = 0.343 0.15
ng/ml)

0.004 0.10
(uIU)
 0 0

n
te

tio

te

tio
un

un
i
nd

i
nd
St

St
co

co
d

d
oo

oo
G

G
Figure 4. Mineral analysis of Barki sheep serum. Values of various serum variables in Barki sheep
of stunted and good body condition. Each bar represents the mean ± SD with p-value comparing the
Animals 2023, 13, 962 two groups using an unpaired Student’s t-test. Significance at p = <0.05. 9 of 13

Although we observed differences between stunted and control groups concerning

the thyroid and growth hormones, they were not statistically significant (p > 0.05) (Figure
growth hormone (weak-to-moderate;
5) (Table Pearson rthe
S1). These results demonstrate = balanced
0.4005),ratio
andofeventually totalthe
minerals between protein
two
sheep groups.
(weak-to-moderate; Pearson r = 0.393) (Figure 6).

TSH Growth hormone

0.006 p = 0.343 p = 0.250

0.15

GH (ng/ml)
0.004 0.10
TSH (uIU)

0.002 0.05

0.000 0.00

on
d

on

te
te

iti
un
iti
un

nd
nd

St
St

co
co

d
d

oo
oo

G
G

Figure 5. Serum levels of thyroid-stimulating hormone and growth hormone in stunted and good
Animals 2023, 13, x FOR PEER REVIEW 10 of 14
Figure 5. Serum levels of thyroid-stimulating hormone and growth hormone in stunted and good
body condition Barki sheep. Serum level of TSH and growth hormone between stunted Barki sheep
body condition Barki sheep. Serum level of TSH and growth hormone between stunted Barki sheep
(n = 10) and good(nbody condition
= 10) and sheep
good body or control
condition sheep orgroup
control(n = 9).(nEach
group bar bar
= 9). Each represents
representsthe mean ± ±
the mean SDSD
with p-value
with p-value comparing comparing
thegrowth
0.489), the two
two groups
hormone groups
using an using an unpaired
unpaired
(weak-to-moderate; Student’s
Student’s
Pearson t-test.
rt-test. Significance
Significance
= 0.4005), atatp p
and eventually = <0.05.
≤ 0.05.
total pro-
tein (weak-to-moderate; Pearson r = 0.393) (Figure 6).
Pearson’s correlation coefficient was used to investigate the association between
body weights with values of BCS and biochemical parameters and showed significant dif-
ferences between the two groups. A better correlation was identified for BCS (strong; Pear-
son r = 0.917) and albumin (moderately strong; Pearson r = 0.683), followed by triglycer-
ides (moderately strong; Pearson r = 0.537), cholesterol (weak-to-moderate; Pearson r =

Figure 6. Correlation
Figure 6. Correlation between different between
clinicaldifferent clinical and parameters
and biochemical biochemical parameters
against theagainst
bodytheweight
body weight
of
of all examined sheep. Scatter graphs show the correlation between body weight and different pa-
all examined sheep. Scatter graphs show the correlation between body weight and different parameters
rameters including body condition score (A), cholesterol (B), triglycerides (C), total proteins (D),
albumin
including body condition (E), and
score (A),growth hormone
cholesterol (F)triglycerides
(B), from stunted (n(C),
= 10)total
and good body condition
proteins sheep (n(E),
(D), albumin = 9).
The equation represents the approximation formula. The break line represents the calculated line of
and growth hormonebest (F) from stunted (n = 10) and good body condition sheep (n = 9). The equation
fit. Correlation coefficients were calculated using Pearson’s correlation coefficient: |r| = 0.70
represents the approximation formula.0.5The
strong correlation; < |r|break line represents
< 0.7 moderately the calculated
strong correlation; line
and |r| of best
= 0.3–0.5 fit. Correla-
weak-to-moderate
tion coefficients were correlation.
calculated using Pearson’s correlation coefficient: |r| = 0.70 strong correlation;
0.5 < |r| < 0.7 moderatelyAdditionally,
strong correlation; and |r| = 0.3–0.5 weak-to-moderate correlation.
we estimated the possibility of growth retardation in the studied ani-
mals because of debilitating infections. We tested for internal parasites using a direct fecal
Additionally, we estimated
smear the possibility
and a concentration of growth retardation
sedimentation/flotation technique.inDifferent
the studied
kindsanimals
of ELISAs
because of debilitating
wereinfections.
used to reveal We tested for
debilitating internal(T.parasites
protozoan gondii and using a direct
N. caninum) fecal smear
or bacterial (Brucella
and a concentrationspp., and C. burnetii) infections that
sedimentation/flotation are endemicDifferent
technique. in our locality.
kindsThere
ofwas no evidence
ELISAs wereof
infection of tested pathogens, except for T. gondii, in which only one and two cases were
used to reveal debilitating protozoan (T. gondii and N. caninum) or bacterial (Brucella spp.,
identified as seropositive in test and control groups, respectively (Table 3) (Figure 7).
and C. burnetii) infections that are endemic in our locality. There was no evidence of
infection of tested pathogens, except
Table 3. Serological for
testing T. gondii,
of Barki sheep inin which
good only
and poor one and two cases were
condition.
identified as seropositive in
Parameter testMethod
Used and control groups, respectively
Stunted (n = 10) (Table 3) (Figure
Good Condition7).(n = 9)
Brucella spp. antibodies Indirect ELISA 0 0
Coxiella burnetii antibodies Indirect ELISA 0 0
Toxoplasma gondii antibodies Indirect ELISA 1 (10%) 2 (22.2%)
Neospora caninum antibodies Competitive ELISA 0 0
Animals 2023, 13, 962 10 of 13

Table 3. Serological testing of Barki sheep in good and poor condition.

Parameter Used Method Stunted (n = 10) Good Condition (n = 9)

Brucella spp. antibodies Indirect ELISA 0 0
Coxiella burnetii antibodies Indirect ELISA 0 0
Animals 2023,
Toxoplasma 13, antibodies
gondii x FOR PEER REVIEW Indirect ELISA 1 (10%) 11 of 14
2 (22.2%)
Neospora caninum antibodies Competitive ELISA 0 0

Brucella species Coxiella burnetii

b
0.8 2.0 b

0.6 ac 1.5
OD 450

OD 450
0.4 1.0 a a
ad
0.2 a 0.5
a
0.0 0.0
C d d
N PC te oo

PC

d
d

oo
un

te
N
G

un
St

G
St
Type of samples Type of samples

Toxoplasma gondii Neospora caninum

2.5 2.5 a
a a
2.0 2.0
a a

1.5
OD 450

1.5 a
OD 450

1.0 1.0

0.5 0.5 b
a
0.0 0.0
C

PC

PC

d
d

d
oo

oo
te

te
N

N
un

un
G

G
St

St

Type of samples Type of samples

Figure 7.7.Different
Figure DifferentELISAs for examined
ELISAs Barki sheep.
for examined Serasheep.
Barki from stunted
Sera (n = 10)stunted
from and good(nbody
= 10) and good body
condition (n = 9) Barki sheep were tested using a commercial ELISA for detection of specific anti-
condition (n = 9) Barki sheep were tested using a commercial ELISA for detection
bodies against Brucella species, Coxiella burnetii, Toxoplasma gondii, and Neospora caninum and for of specific antibodies
against Brucella
significant species,
differences Coxiella
in different seraburnetii, Toxoplasma
against the same testedgondii, and
antigen. EachNeospora caninum
bar represents the and for significant
mean ± standard deviation. The different letters above the bars in the graphs indicate statistically
differences in different sera against the same tested antigen. Each bar represents the mean ± standard
significant differences of other groups (one-way ANOVA with Tukey–Kramer post hoc analysis, p
deviation.
< 0.05). RedThe
dots different letterssamples
refer to positive above either
the bars
frominthe
thecontrol
graphsor indicate statistically
test samples. significant differences
NC; negative
control,
of otherPC; positive
groups control. ANOVA with Tukey–Kramer post hoc analysis, p < 0.05). Red dots refer to
(one-way
positive samples either from the control or test samples. NC; negative control, PC; positive control.
4. Discussion
The most common sheep breeds in Egypt are Ossimi, Rahmani, Saidi, and Barki
4. Discussion
sheep. The latter has a superior quality of meat and is well adapted to the challenging
desertThe most common
environment, sheepbybreeds
characterized in Egypt
food shortage highare Ossimi, Rahmani,
temperature fluctuations. Saidi, and Barki
sheep.
Growth The latter has
performance a superior
of Barki sheep hasquality of meat
an important andvalue
economic is well adapted
in terms to the challenging
of mini-
mizing environment,
desert the shortage of mutton meat in Egypt.
characterized Furthermore,
by food shortagemilk production
high temperatureis of great
fluctuations. Growth
importance for feeding newborn lambs [18].
performance of Barki sheep has an important economic value in terms of minimizing the
In our comparative study to evaluate health hazards, systemic clinical examinations
shortage
were appliedof mutton meatdistinct
and revealed in Egypt. Furthermore,
clinical symptoms among milksheep
production is ofgood
of poor and great importance for
feeding newborn
growth. Our systemiclambs
clinical[18].
examination revealed higher respiratory and pulse rates in
stunted
In sheep than in control study
our comparative sheep. However,
to evaluatethis variation might be attributable
health hazards, systemic toclinical
the examinations
smaller
were size of the
applied andstunted sheep, distinct
revealed as can be observed
clinical from body andamong
symptoms BCS findings
sheep [15].
of poor and good
Most of the other examined clinical parameters were within the normal limits. However,
growth. Our systemic clinical examination revealed higher respiratory and pulse rates in
the same stunted sheep also showed rough and rusty body coats, revealing the growth
stunted sheep than of
disorders in this group insheep.
control sheep. However, this variation might be attributable to the
smaller
Serumsize of the stunted
cholesterol sheep,represent
and triglycerides as can thebe most
observed from
important body and
parameters whenBCS findings [15].
a lipid profile is studied; both were decreased in stunted
Most of the other examined clinical parameters were within the normal sheep, which showed poor limits. However,
growth rates compared with the control group in a significant manner. This was consistent
the same stunted sheep also showed rough and rusty body coats, revealing the growth
with Singh et al. (2018) [13], who also reported a lower level of such lipid metabolites in
disorders in this group of sheep.
Serum cholesterol and triglycerides represent the most important parameters when a
lipid profile is studied; both were decreased in stunted sheep, which showed poor growth
rates compared with the control group in a significant manner. This was consistent with
Animals 2023, 13, 962 11 of 13

Singh et al. (2018) [13], who also reported a lower level of such lipid metabolites in slow
growth sheep compared with rapid growth sheep, although it was significant only in the
case of triglycerides. In addition, the triglycerides contribute in female puberty to the
preparatory stage of conception and gestation [19].
The serum urea and urea nitrogen concentrations were increased significantly in the
stunted group compared to the good body condition group. Previous studies also demon-
strated that urea levels can be increased during stages of increased nutritional demands.
This increase is due to the ability of ruminants to recycle endogenous urea partly into the
forestomach to synthesize microbial protein [20]. This compensatory mechanism took place in
cases of decreased protein intake or imbalanced endogenous protein homeostasis. This can be
achieved via a reduction in urea excretion by the kidneys to maintain a high entry of urea to
the rumen. The conservation of urea by the kidney occurs through a decrease in renal plasma
flow and glomerular filtration rate, causing a reduction in the filtration of urea by glomeruli
and an increase in the reabsorption of urea from tubules to blood [21]. On the contrary, the
serum creatinine level was reduced significantly in sheep with a poor growth rate compared
with control sheep. This result agrees with the study of Piccione et al. (2009) [22], which
determined that the amount of formed creatinine depends on the content of creatinine in the
body, which is related to muscle mass, food intake, and synthesis rate of creatinine.
Our results demonstrated that the serum total protein and albumin were decreased
significantly in sheep with poor growth rather than good growth sheep. These observations
agree with the results of Piccione et al. (2009) [22], who attributed the results to low intake
of protein and dehydration.
Unexpectedly, our results revealed that all tested parameters of liver function and
glucose level remained within the physiological levels in the investigated groups. Similarly,
our tested macro-elements, such as calcium, phosphorus, and magnesium, and micro-
elements, such as iron and zinc levels, did show statistical differences between stunted and
good growth sheep, although lower levels were recorded in the former group in the case of
magnesium and iron. Minerals are required by animals to grow and reproduce. They play
important roles as structural components of enzymes and regulate metabolic reactions in
the animal body [23]. Normal mineral levels in the serum of all tested sheep are indicative
of the normal food supply and food intake, which is also supported by our findings of
normal appetite [6].
Lower levels of growth and thyroid hormones were observed in stunted sheep than
in good growth sheep. This difference was apparent but not statistically recorded; other
observations and previous literatures confirmed the hormonal role in growth disorders in
stunted groups. In a previous study, somatotropin administration enhanced puberty in
Rahmani ewe lambs. This is due to increased provision of trophic signals (represented by
increased Serum IGF-1 secretions) and/or blood-borne metabolites (glucose, cholesterol,
and lipids) [7]. Another study reported that melatonin implantation improved the sheep
growth and development via increasing the growth hormone release [24]. In the same
context, appropriate thyroid gland function and activity of thyroid hormones (TH) are
considered crucial to sustain productive performance in domestic animals (growth, milk,
hair fiber production), and circulating TH can be considered as an indicator of the metabolic
and nutritional status of the animals [25,26].
In an attempt to recognize the reasons behind the difference in growth rate between
the test and control groups, we also estimated the subclinical infections of a group of
endemic infectious diseases affecting sheep. There was no strong evidence of involvement
of infections of T. gondii, N. caninum, Brucella spp., or C. burnetii, internal or external
parasitic infections. As a result of close observations, when we noticed a difference in the
body weight in sheep, infection with endoparasites was the first suspicion. Thus, our first
procedure for investigation of the reason for weakness in some sheep was fecal analysis for
the detection of different parasitic stages. However, none of the tested sheep were positive
for any type of parasites, including coccidian oocysts. This finding might be attributable to
the parasitological methods used (screening methods) and the sanitary measures applied
Animals 2023, 13, 962 12 of 13

to animals (deworming by anthelmintics) and farms (routine cleanliness and disinfection

by glutaraldehyde and using clean food).
One limitation for this study is the lack of genetic line/pedigree information for the
sheep and data on the relationship/degree of relationship between these sheep. Further
studies are required to confirm our hypothesis and the obtained results, particularly using
animals with a well-known genetic background and close pedigree.

5. Conclusions
Our randomized controlled clinical trial revealed valuable information on detecting
biomarkers for growth performance in Barki sheep. This information is crucial to address
economic and financial concerns as it is useful in excluding animals with poor growth
and development from the livestock industry. Our study revealed that in most cases, the
stunted Barki sheep exhibited a normal clinical picture, including appetite, and a normal
biochemical profile for minerals and liver function markers. Additionally, the serological
testing negated the suffering of such animals from brucellosis, Q fever, toxoplasmosis,
and neosporosis. However, levels of nutritionally relevant biomarkers, such as total
protein, albumin, cholesterol, and triglycerides, were markedly decreased in stunted sheep
compared with normal sheep. These findings were also confirmed by lower levels of
growth and thyroid hormones in stunted sheep than in the control sheep. Additional
studies are required to investigate the productive and reproductive capacities of Barki
sheep to validate the usefulness of this breed in nationwide breeding and farming.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/ani13060962/s1, Figure S1: Map of Egypt showing the location and
movement of Barki sheep of this study; Figure S2: Representative image for Barki sheep used in the
current study.; Table S1: Biochemical variables among stunted and good body condition Barki sheep.
Author Contributions: Conceptualization, R.M.F.; formal analysis, R.M.F. and C.F.F.; methodology,
R.M.F., R.M.I., A.M.K., and F.A.K.; project administration, R.M.F.; resources, R.M.F., R.M.I., A.M.K.,
F.A.K., and C.F.F.; writing—original draft, R.M.F.; writing—review and editing, R.M.F., R.M.I., A.M.K.,
F.A.K., and C.F.F. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The protocols were approved by the Research Bioethics
Committee at South Valley University VM/SVU/23(1)-05.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated and analyzed during this study are included in this
published article. Raw data supporting the findings of this study are available from the corresponding
author upon request.
Acknowledgments: We would like to appreciate the great help of our colleagues at the Department of
Animal Medicine, Faculty of Veterinary Medicine, South Valley University, Qena, for their cooperation
and technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.

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