The effect of calpastatin gene on meat quality traits in Turkish sheep breeds

Abstract

This research aimed to examine the effects of haplotype groups observed in three different loci on the Calpastatin gene on ultrasonographic MLD measurements and seasonal live weight in five different types of sheep (GBK, HM, K, KM, and R). In the CAST intron 1, intron 5, and intron 12 regions, 15 SNPs were found. The HWE p-value for SNP2, SNP3, SNP5, SNP6, SNP7, SNP8, and SNP14 is less than 0.05, and except SNP9 and SNP10, all SNPs have a MAF of more than 0.01. SNP1, SNP2, and SNP7 made up one haplotype block. The haploblock has 3 haplogroups. The most common haplotype group was H1 (-AGG-), which had a frequency of 0.52; H2 (-TGG-) and H3 (-TAA-) had rates of 0.35 and 0.13, respectively. Based on ultrasonographic MLD readings and live weights, there were no statistically significant differences between haplotype H1 and H3, but there were statistically significant differences between haplotype H2 lambs. The effect of the H2 haplotype on 90-day MLD depth revealed a statistically significant difference between the HM and KM and K and KM breeds. This distinction persisted until the 180th day of life before disappearing into adulthood. Similarly, the effect of H2 haplotype on the skin thickness at day 90 was significant between K and KM and between K and R, whereas the effect of H2 haplotype on fat thickness demonstrated a substantial difference between HM and KM at one year of age.

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Iraqi Journal of Veterinary Sciences, Vol. 38, No. 4, 2024 (761-769)
761
Iraqi Journal of Veterinary Sciences
www.vetmedmosul.com
The effect of calpastatin gene on meat quality traits in Turkish sheep
breeds
V.K. Esen
Department of Breeding and Genetics, Sheep Breeding Research Institute, Balikesir, Turkiye
Article information
Abstract
Article history:
Received 07 April, 2024
Accepted 07 June, 2024
Published online 18 September, 2024
This research aimed to examine the effects of haplotype groups observed in three
different loci on the Calpastatin gene on ultrasonographic MLD measurements and seasonal
live weight in five different types of sheep (GBK, HM, K, KM, and R). In the CAST intron
1, intron 5, and intron 12 regions, 15 SNPs were found. The HWE p-value for SNP2, SNP3,
SNP5, SNP6, SNP7, SNP8, and SNP14 is less than 0.05, and except SNP9 and SNP10, all
SNPs have a MAF of more than 0.01. SNP1, SNP2, and SNP7 made up one haplotype block.
The haploblock has 3 haplogroups. The most common haplotype group was H1 (-AGG-),
which had a frequency of 0.52; H2 (-TGG-) and H3 (-TAA-) had rates of 0.35 and 0.13,
respectively. Based on ultrasonographic MLD readings and live weights, there were no
statistically significant differences between haplotype H1 and H3, but there were
statistically significant differences between haplotype H2 lambs. The effect of the H2
haplotype on 90-day MLD depth revealed a statistically significant difference between the
HM and KM and K and KM breeds. This distinction persisted until the 180th day of life
before disappearing into adulthood. Similarly, the effect of H2 haplotype on the skin
thickness at day 90 was significant between K and KM and between K and R, whereas the
effect of H2 haplotype on fat thickness demonstrated a substantial difference between HM
and KM at one year of age.
Keywords:
Calpastatin
Ultrasound measurements
Live weight
Lamb
Correspondence:
V.K. Esen
vasfiye.esen@gmail.com
DOI: 10.33899/ijvs.2024.148563.3599, ©Authors, 2024, College of Veterinary Medicine, University of Mosul.
This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).
Introduction
Sheep farming assumes a pivotal role in the
socioeconomic landscape, particularly within developing
countries, by simultaneously addressing nutritional,
economic, and sociocultural dimensions. It is a reliable
source of high-quality sustenance, fosters income growth,
and promotes societal inclusivity (1,2). Within such
contexts, expeditiously optimizing these multifaceted
benefits emerges as an imperative. To expedite the
realization of these objectives, marker-assisted selection
(MAS) programs have emerged as a prominent and widely
implemented methodology. Leveraging MAS programs,
livestock breeders can effectively augment livestock metrics,
including live weight gain and meat quality (3-5). Notably,
contemporary selection programs emphasize the effects of
candidate genes employed in MAS programs regarding meat
tenderness and composition, reflecting a strategic focus
aligned with the heightened consumer demand for premium
meat products (6,7). This strategic orientation not only
underpins the economic prosperity of sheep farming
communities but also satisfies the discerning preferences of
consumers. On the other hand, Calpastatin merits particular
attention due to its vital contributions in determining the
quantity and quality of meat (8). Calpastatin, a cellular
protein inhibiting calpains (Ca2+-dependent cysteine
proteinases) involved in diverse cellular processes such as
cytoskeleton modulation, cell migration, cell cycle
progression, and apoptosis, plays a crucial role in the
calpain-calpastatin system governing protein turnover,

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growth, myoblast migration, myoblast fusion, and meat
tenderness; given its influence on these activities and its
potential impact on meat quality, the calpastatin (CAST)
gene emerges as a promising candidate gene for elucidating
variations in meat traits (9-11). It has been shown that
calpains are crucial to the breakdown of myofibrillar proteins
in living muscle tissues and play a major role in postmortem
proteolysis, a biochemical process responsible for meat
tenderization (12-14). Thus, Calpastatin acts as an
endogenous inhibitor of calpains, influencing both the rate
and extent of postmortem tenderization (11,15,16). More
precisely, the augmentation in skeletal muscle growth can be
attributed to reduced muscle protein degradation. This
decrease is linked to lower calpain activity, which elevated
calpastatin levels facilitate (17). Additionally, prior studies
have indicated that elevated calpastatin activity within living
cells impedes calpains' capacity to degrade myofibrillar
proteins during postmortem storage (18,19). The CAST
gene, localized at the 5q15 locus on chromosome 5 of the
sheep genome and comprising 29 exons, exhibits
polymorphism across numerous sheep breeds. Research on
livestock species such as pigs, cattle, sheep, and goats has
revealed the significant impact of various polymorphisms
within the CAST gene. These polymorphisms influence
weight gain, carcass quality, and meat quality, particularly
tenderness, highlighting their substantial role in animal
production and meat processing (20-24). These researches
have demonstrated the significant influence of the CAST
gene on growth, attributed to its capacity to promote muscle
fiber proliferation. Specifically, it has documented its impact
on the birth weight and growth rate of Romney sheep (10)
until weaning and its influence on post-weaning weight and
daily weight in Targhee sheep (25). These findings
underscore the critical importance of assessing the varied
impacts of the CAST locus and its polymorphisms on a range
of traits throughout different developmental stages.
Therefore, evaluating the effects of the CAST locus and its
polymorphisms on various traits at different stages is crucial,
and a comprehensive understanding of their potential effects
from birth to adulthood before integrating them into MAS
programs can significantly improve the efficiency of
selection processes. Nevertheless, ultrasound technology,
designed for evaluating the composition and quality of
animal carcasses intended for market, facilitates swift and
cost-effective assessment of carcass properties in live
animals without causing harm (26-28). Utilizing ultrasound
measurements in live animals holds practical significance,
enabling the selection of particular carcass traits based on
measurement criteria for breeding purposes and predicting
the optimal timing for slaughtering or marketing (29-31).
Previous studies have identified polymorphic variants in
intron 1 (32), intron 5 (14), and intron12 (24,33) of the CAST
gene in sheep; however, the relationship between haplotypic
diversity and live weight, as well as ultrasonographic muscle
measurements, has not been explored.
Therefore, the current study aims to fill this gap by
investigating single nucleotide polymorphisms (SNPs) in
these regions within selected meat-type sheep breeds in
Turkey and determining the associations between haplotypes
and live weight and ultrasonographic muscle measurements
collected at various time points.
Materials and methods
Ethical approve
The Ethics Committee of the Sheep Breeding Research
Institute in Türkiye (approval number: 13360037) granted
consent for all animal trials on April 11, 2018. This research
was conducted at the Bandirma Sheep Breeding Research
Institute, Balikesir, Türkiye. Lambs used in this study were
sourced from the institute's farm and constituted the primary
animal material for our research endeavors.
Animals and DNA isolation
The study specifically focused on lambs born within the
2018 lambing season and restricted the inclusion criteria to
those born within a 10-day window after the lambing season.
In total, the study encompassed 202 lambs, encompassing
diverse breeds such as German Black‐Head Mutton ×
Kivircik (GBK), Hampshire Down × Merino (HM), Kivircik
(K), Karacabey Merino (KM), and Ramlic (R). It is pertinent
to note that our prior investigations have extensively
documented these animals, offering comprehensive insights
into their care and feeding regimens. As elucidated by Kader
Esen (3) and Kader Esen (2), these details provided a
foundational understanding of the subjects under scrutiny.
The methodology involved the collection of blood samples
from the lamb's Vena jugularis, ensuring meticulous
preservation in 10 ml EDTA tubes to obtain high-quality
genomic DNA. These samples were then stored at -20 °C
until the subsequent DNA extraction process was executed
with precision and accuracy.
Genetic analyses and identification of SNPs
DNA extraction from the samples followed the protocols
outlined in the GeneAll® kit. Specific primers were designed
to target three distinct regions with lengths of 565, 254, and
448 base pairs to investigate the CAST gene. The
amplification process was conducted in a 20 μL reaction
mixture containing DNA and each primer at a concentration
of 100 ng using a commercially available kit. Primers for
amplifying Intron 1 were described by Khederzadeh (17),
while those for Intron 5 and Intron 12 were sourced from the
work of Byun (10). The detailed polymerase chain reaction
(PCR) conditions are provided in table 1.
Following the Sanger sequencing method, PCR products
underwent sequencing using the ABI3500 genetic analyzer
(Applied Biosystems, Foster City, CA, USA). Geospiza's
FinchTV software (Version 1.4) was employed to visualize
and scrutinize the sequence of chromatograms. The obtained

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sequences were meticulously deposited in the GenBank
database with the following accession numbers: OP620911,
OP620912, OP620913, OQ513936, and OQ513937. To
discern the genetic variations, the DNA sequences obtained
in this study were systematically compared with the
reference sheep genome (Oar_v3.1) sourced from the
Ensembl Genome Database, thereby facilitating the precise
identification of SNP positions.
Table 1: PCR conditions for CAST loci
Reaction phase
Intron 1
Intron 5
Intron 12
H
T
C
H
T
C
H
T
C
First denaturation
95
5
1
94
2
1
94
2
1
Denaturation
94
1
94
0,5
94
0,5
Annealing
51
1
33
55
0,5
35
55
0,5
35
Extension
72
2
72
0,5
72
0,5
Final extension
72
8
1
72
5
1
72
5
1
H: Heat (°C); T: Time (min); C: Cycle.
Live weight and ultrasonographic muscle measurements
In the study's initial stages, lambs' birth weights were
meticulously documented within the first 12 hours
postpartum. Subsequent assessments involved carefully
recording live weights (LW) and precise ultrasonographic
measurements on the research period's 90th, 180th, and 360th
days. To ensure accuracy and reliability, lambs were
weighed before their morning feeding, thus mitigating
potential inaccuracies arising from the presence of stomach
content. The ultrasonographic evaluations were conducted
by a skilled technician employing a real-time ultrasound
system (Mindray DP-20) integrated with a linear veterinary
ultrasound transducer (Mindray 75L50EAV) operating at a
frequency of 7.5 MHz, as detailed in the work of Esen (34).
The ultrasonographic analysis focused on monitoring the
Musculus longissimus dorsi depth (MLDD), fat thickness
(FT), and skin thickness (ST) located between the 12th and
13th ribs. These assessments were conducted after recording
live weights at predetermined intervals, as stipulated in the
research protocol elucidated by Kader Esen and Elmaci (1).
Statistical analysis
The Hardy-Weinberg equilibrium (HWE) was assessed
for each SNP by comparing observed (HetOb) and predicted
(HetPre) heterozygosities. A threshold of 5 percent was
defined for HWE. Haploview software (Version 4.2) was
utilized to ascertain haplotypes and assess the linkage
disequilibrium (LD) between SNPs. SNPs were considered
eligible for inclusion in the linkage disequilibrium analysis
if they exhibited a p-value greater than 0.05 in the HWE test
and possessed a minor allele frequency (MAF) of at least 1%
(27). The dataset underwent rigorous analysis to explore the
relationship between the response variables (LW, MLDD,
FT, and ST) and various explanatory factors, including
breed, gender, birth type, dam age, and haplotype. An
analysis of variance (ANOVA) was performed using a mixed
model approach. This method, executed in the R
programming language using the 'lmer' function from the
'lme4' package, was chosen to account for potential data
correlations arising from the hierarchical structure. Post hoc
analysis was conducted using Tukey's test on the mixed
model to discern differences among the variables (35).
Results
Four lambs were omitted from the study due to indistinct
genotyping results, ensuring the integrity of the dataset.
Fifteen distinct SNPs were identified within three specific
regions of the CAST gene, as depicted in figure 1. Notably,
SNPs 1 to 8 were located in intron 1, 9 to 11 were in intron
5, and SNPs 12 to 15 were positioned in intron 12. Seven
SNPs detected in our study had been previously documented
in the reference genome (Sheep_texel Oar_v3.1),
underlining their relevance and consistency with existing
genetic data.
Genetic parameters, including observed heterozygosity,
predicted heterozygosity, and the assessment of HWE, were
calculated for all SNPs, as detailed in table 2. Notably, HWE
was only observed for some SNPs under consideration.
Specifically, SNPs 1, 9, 10, 11, 12, 13, and 15 exhibited p-
values exceeding 0.05. Additionally, except for SNPs 9 and
10, the minimal allelic frequencies of these SNPs were more
significant than 0.01.
SNP3, SNP4, SNP5, SNP6, SNP8, and SNP14 displayed
subpar performance and failed in one or more tests, as
delineated in figure 2. Examination of linkage disequilibrium
(LD) coefficients, encompassing D’ and r2 values,
demonstrated strong genetic associations between SNP1 and
SNP2 (D’= 1.0, LOD=20.61, r2=0.17), SNP2 and SNP7 (D’=
1.0, LOD=57.03, r2=1.0), SNP9 and SNP10 (D’= 1.0,
LOD=4.95, r2=1.0), SNP12 and SNP13 (D’= 1.0,
LOD=22.73, r2=1.0), and SNP13 and SNP15 (D’= 1.0,
LOD=22.73, r2=1.0). Conversely, a weak linkage was
observed between SNP10, SNP11, and SNP12 (LOD<2). It
is important to note that, despite their high LOD scores
represented by red diamonds, not all of these SNPs formed
haplotype blocks due to their placement outside Gabriel’s
confidence interval, as depicted in figure 2. Specifically, a

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haplotype block was established by SNP1, SNP2, and SNP7.
Through haplotype analysis, three distinct haplotype groups
were identified within the population, each with frequencies
exceeding 1%. The H1 (-AGG-) haplotype group was
prevalent, with a frequency of 0.52, while the H2 (-TGG-)
and H3 (-TAA-) haplotype groups exhibited frequencies of
0.35 and 0.13, respectively.
Figure 1: Nucleotide variants in introns 1, 5, and 12 of the
CAST gene.
Figure 2: Linkage disequilibrium (LD) plot of calpastatin
SNPs. The D' coefficient is depicted in graph (a), whereas
graph (b) represents the r² coefficient. LD is represented
through standard color codes: red signifies strong LD with
LOD > 2 and D′ = 1), blue denotes intermediate LD with
LOD < 2 and D′ = 1, while white signifies no LD with LOD
< 2 and D′ <1.
Table 3 presents the findings on the effect of CAST
haplotypes on lambs' live weight and ultrasonographic
muscle features. Neither live weight nor ultrasound
measurements were statistically significant among the three
haplotype groups (P>0.05). Although there were no
statistically significant differences between haplotypes in
LW and MLDD, notable patterns were observed. Compared
to other haplotype groups, lambs of haplotype H2 had higher
birth weights and adult weights. Furthermore, lambs in the
H1 haplotype group exhibited higher MLDD at both
weaning (LW90) and 180 days, while no significant
differences were observed in ST and FT among the
haplotypes.
Table 2: Hardy-Weinberg equilibrium, minor allele frequency, and heterozygosity of CAST SNPs in meat-type sheep breeds
SNP #
Chromosome Location
rs ID
Alleles
HetOb
HetPre
HWE
MAF
SNP1
5:93448534
rs421197310
A: T
0.432
0.499
0.0569
0.480
SNP2
5:93448548
-
G: A
0.264
0.229
0.0261
0.132
SNP3
5:93448577
rs399966367
A: G
0.432
0.339
4.1339E-6
0.216
SNP4
5:93448621
rs407174907
G: A
0.432
0.339
4.1339E-6
0.216
SNP5
5:93448696
rs412475054
G: A
0.432
0.339
4.1339E-6
0.216
SNP6
5:93448734
rs398259427
G: T
0.432
0.339
4.1339E-6
0.216
SNP7
5:93448760
-
G: A
0.264
0.229
0.0261
0.132
SNP8
5:93448820
rs161885148
A: G
0.432
0.339
4.1339E-6
0.216
SNP9
5:102025584
-
G: C
0.009
0.009
1.0
0.005
SNP10
5:102025590
-
C: T
0.009
0.009
1.0
0.005
SNP11
5:102025594
-
G: A
0.027
0.027
1.0
0.014
SNP12
5:102036450
-
C: T
0.064
0.062
1.0
0.032
SNP13
5:102036487
-
T: C
0.064
0.062
1.0
0.032
SNP14
5:102036502
rs422618244
G: C
0.936
0.498
2.1698E-47
0.468
SNP15
5:102036646
-
C: G
0.064
0.062
1.0
0.032
HetOb: observed heterozygosity; HetPre: predicted heterozygosity; HWE: Hardy-Weinberg equilibrium p-value; MAF: minor
allele frequency.

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Table 3: Effects of CAST haplotypes on live weight and ultrasonographic muscle measurements
Trait
Day
N
H1 (-AGG-)
N
H2 (-TGG-)
N
H3 (-TAA-)
P value
Mean
SE
Mean
SE
Mean
SE
LW
0
66
4.27
0.25
83
4.51
0.14
49
4.29
0.19
NS
90
66
32.87
1.36
83
30.52
0.76
49
31.74
1,04
NS
180
58
41.16
2.12
66
40.93
1.08
24
40.90
1.88
NS
360
57
58.14
2.80
63
62.87
1.33
21
62.49
2.55
NS
MLDD
90
66
2.37
0.14
83
2.34
0.08
49
2.33
0.11
NS
180
58
2.63
0.14
66
2.39
0.06
24
2.24
0.12
NS
360
57
2.79
0.17
63
2.96
0.08
21
2.76
0.15
NS
FT
90
66
0.45
0.05
83
0.38
0.03
49
0.50
0.04
NS
180
58
0.43
0.06
66
0.39
0.03
24
0.36
0.05
NS
360
57
0.38
0.07
63
0.46
0.03
21
0.49
0.06
NS
ST
90
66
0.22
0.01
83
0.23
0.01
49
0.21
0.01
NS
180
58
0.18
0.02
66
0.18
0.01
24
0.16
0.01
NS
360
57
0.23
0.03
63
0.22
0.01
21
0.25
0.03
NS
LW: live weight; MLDD: Musculus longissimus dorsi depth; FT: fat thickness; ST: skin thickness; N: sample size; SE: standard
error of the mean.
Figure 3 illustrates the effect of CAST haplotypes on LW
at various time intervals in meat-type sheep breeds. Male
lambs were absent in the studied populations from both H1
and H2 groups. Furthermore, the absence of H1 and H3
haplotypes in HM and R breeds and the complete lack of H3
haplotypes in the R breed was notable. Similarly, GBK
breeds displayed a complete absence of H2 haplotypes.
While H1 and H3 haplotypes demonstrated no significant
influence on the breeds, the H2 haplotype emerged as a
pivotal factor. Significant birth weight differences were
observed between KM and R (P<0.05). During the weaning
period, substantial variations were identified among K and
KM (P<0.05), K and R (P<0.001), and KM and R (P<0.05)
H2 haplotype lambs. By the 180th day, noteworthy
differences in LW were evident between HM and R
(P<0.01), K and R (P<0.01), and KM and R (P<0.01) H2
haplotype lambs. Moreover, on the 360th day, notable
differences in the live weights of H2 haplotype lambs were
observed, with significant distinctions between HM and R
(P<0.05) and KM and R (P<0.01).
Figure 4 depicts the effects of CAST haplotypes on
ultrasonographic muscle measurements in meat-type sheep
breeds at different time intervals. Particularly, on the 90th
day, noteworthy distinctions in MLDD were evident among
lambs with H2 haplotypes, with significant differences
observed between HM and K (P<0.05) and HM and R
(P<0.05). Moreover, substantial differences in ST were
noted among H2 haplotype lambs, with significant
disparities between K and KM lambs (P<0.01) and K and R
lambs (P<0.05). Notably, K lambs exhibited greater ST than
their counterparts, while the influence of CAST haplotypes
on FT on the 90th day did not yield a statistically significant
difference. Concerning the impact of H2 haplotype in lambs
on MLDD on the 180th day, significant differences were
noted between HM and K lambs (P<0.05) and between KM
and K lambs (P<0.05). Likewise, the impact of the H2
haplotype on ST on the 180th day showed statistical
significance, specifically between K and R lambs (P<0.05).
In contrast, no statistically significant FT differences existed
among haplotype groups at 180 days (P>0.05). In adulthood
(on the 360th day), there were no statistically significant
differences in MLDD or ST among the haplotype groups
(P>0.05). However, in H2 haplotype lambs, a significant
difference was observed between HM and K (P<0.05) as
well as between K and R (P<0.05) in terms of FT.
Figure 3: Effects of CAST haplotypes on live weights over
time in meat-type sheep breeds. BW: birth weight; LW90:
live weight on the 90th day LW180: live weight on the 180th
day; LW360: live weight on the 360th day; F: female; M:
male; GBK: German Black‐Head Mutton × Kivircik; HM:
Hampshire Down × Merino; K: Kivircik; KM: Karacabey
Merino; R: Ramlic; ns: not significant; *: P<0.05; **:
P<0.01; ***: P<0.001.

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Figure 4: Effects of CAST haplotypes on ultrasonographic
muscle measurements over time in meat-type sheep breeds
MLDD: Musculus longissimus dorsi depth; ST: skin
thickness; FT: fat thickness; F: female; M: male; GBK:
German Black‐Head Mutton × Kivircik; HM: Hampshire
Down × Merino; K: Kivircik; KM: Karacabey Merino; R:
Ramlic; ns: not significant; *: P<0.05; **: P<0.01; ***:
P<0.001.
Discussion
The effectiveness of MAS lies in its ability to curtail the
generation interval, thereby expediting genetic progress and
facilitating targeted improvements (15,36,37). Notably,
previous research has underscored the pivotal role of the
CAST gene as a prime candidate in meat quality selection
initiatives. This importance is attributed to inhibiting the
calpain system, a vital mechanism governing muscle
development, growth, and postmortem meat tenderness
(10,38,39). Combining this gene with ultrasonographic
muscle measurements has proven to enhance the precision of
genetic parameters, indicating its potential in genetic
selection strategies. Various studies have elucidated genetic
variations within the CAST gene, spanning both coding and
non-coding regions across diverse sheep breeds (11,32,40).
Integrating this gene with ultrasonographic muscle
measurements significantly augments the accuracy of
genetic parameters, underscoring its substantial promise in
genetic selection tactics (26,28).
Prior research has established that introns can influence
mRNA stability and transcriptional efficiency, eliciting
distinct biological effects on genes (41). Recent research has
revealed that introns also significantly impact growth,
carcass, and meat quality traits in sheep and cattle, with
specific effects on genes (42,43). The current study focused
on the intron 1, 5, and 12 regions of the CAST gene, believed
to influence LW and ultrasonographic muscle
measurements, to identify SNPs within these critical regions.
A comprehensive analysis uncovered 15 SNPs in total: 8
within intron 1 (SNP 1 to 8), three within intron 5 (SNP 9 to
11), and four within intron 12 (SNP 12 to 15). Notably, seven
of these SNPs identified in the current study had been
previously documented in existing literature, highlighting
their significance in genetic research (rs421197310,
rs399966367, rs407174907, rs412475054, rs398259427,
rs161885148, and rs422618244). Numerous SNPs within the
ovine CAST gene have been extensively explored in prior
studies, aligning with the present investigation's outcomes.
Notably, Roberts (43) delineated nine SNPs within intron 12,
forming distinctive haplotypes, a discovery later
corroborated by Byun (33), who identified a novel haplotype
encompassing previously reported ones. The genetic
variations within intron 12, as highlighted by Greguła-Kania
(44), exhibited robust correlations with growth rates,
underscoring the genetic significance in the context of ovine
development. Furthermore, Palmer (40) revealed three
unique haplotypes within the intron region spanning exons
1C and 1D, marked by nine SNPs, and established their
significant relationships with lamb growth and meat
tenderness. In a parallel vein, Chung and Davis (25) made a
groundbreaking discovery of a novel SNP (A/G) within
intron 25, establishing significant associations with birth
weight and average daily gain, illuminating critical genetic
determinants of these traits. Moreover, Esteves (43)
identified specific SNPs (c.679A>G; c.383A>G) within the
CAST gene, leading to the substitution of glutamic acid with
glycine and threonine with alanine, profoundly impacting pH
values. However, in contrast, Zhou (45) found no significant
links between tenderness in un-aged lamb and CAST
haplotypes or genotypes within the region encompassing
exon six and partial introns 5 and 6, emphasizing the nuanced
nature of genetic associations in this specific genomic area.
The intricate process of muscle growth and development
is significantly influenced by the regulation of new protein
degradation and synthesis within the calpain-calpastatin
system. The suppression of CAST leads to increased μ-
calpain expression and controlling cell proliferation,
survival, and apoptotic pathways, as demonstrated by Van
Ba et al. (46), and it also results in reduced calpain activity.
This reduction in calpain activity, in turn, decreases muscle
fiber breakdown, thereby facilitating muscle mass
accumulation. In this context, LW, employed to evaluate
body growth and partial development, plays a crucial role, as
highlighted by Greguła-Kania (44). Furthermore, grazing
Texel ewes have observed an established association
between an SNP in the CAST gene and birth weight and
growth rate (6). Notably, the influence of the A allele on birth
weight and pre-weaning daily gain was particularly
discerned in animals of the simple lambing type among
Romney lambs (10).
Furthermore, Chung and Davis (25) highlighted the
influence of the CAST gene on average daily gain and post-
weaning weight in Targhee sheep. This study observed no
significant impact of CAST gene haplotypes on birth weight

Iraqi Journal of Veterinary Sciences, Vol. 38, No. 4, 2024 (761-769)
767
and LW values recorded at various intervals. This outcome
distinguishes the present study from previous research in this
particular aspect. Nevertheless, the obtained results align
with the findings reported by Nikmard (47), where no
significant relationship was observed between SNPs and
metrics such as birth weight, weaning weight, weight at 6
and 9 months, as well as pre-and post-weaning weight gain
characteristics in Afshari sheep.
In previous studies, ultrasonographic muscle
measurements have demonstrated optimal reliability when
assessing muscling and fatness in live animals (28,42). The
strong correlation between fat depth at the C-site of the
carcass and its corresponding ultrasonic measurement was
established in previous research (48). This study, however,
found no statistically significant impact of the CAST gene
haplotypes on ultrasound muscle measurements.
Correspondingly, Knight (47) identified specific SNPs,
CAPN2_28672486 (m-calpain) and CAPN3_38942291
(calpain 3), associated with fat depth at the C-site of the
carcass; yet, subsequent analysis using a Restricted
Maximum Likelihood model revealed their lack of statistical
significance. The present study's findings indicate a breed-
specific influence of CAST haplotypes on ultrasonographic
muscle measurements in meat-type sheep breeds, with
particular emphasis on the H2 haplotype. Notably, a previous
study conducted on Lori-Bakhtiari (fat-tailed) and Zel (thin-
tailed) sheep highlighted polymorphic variations within the
CAST gene specific to the breed and tail type (23).
Moreover, consistent evidence demonstrates the additive
effects of the CAST gene variants on both FT and carcass fat
scores. In their study, Machado (27) identified six CAST
variants (rs423099226, rs428213368, rs400315475,
rs415186098, rs430517308, and rs418818682) with
significant additive impacts on carcass fat scores in Santa
Ines sheep, showing differences ranging from 0.170
(rs415186098) to 0.246 (rs418818682) between
homozygotes. Additionally, the CAST variant rs403339381
exhibited a 0.038 cm difference between homozygotes in
ultrasound images of FT.
Conclusion
To conclude, this study examined the intricate genetic
landscape of the CAST gene within specific meat-type sheep
breeds, revealing how genetic variations influence critical
characteristics like live weight and ultrasonographic muscle
measurements by shedding light on the complexity of
genetic variation. This study identifies 15 distinct SNPs
within the CAST gene's introns 1, 5, and 12, some of which
have been previously reported. While extensive exploration
was conducted, no significant associations were found
between the CAST gene haplotype and live weight or
ultrasonographic muscle measurements during various
periods. In light of this nuanced result, it is evident that
genetic influences on complex traits are multifaceted, and
comprehensive investigations across different breeds and
environments are necessary.
Acknowledgment
This study received funding from the Republic of
Türkiye, the Ministry of Agriculture and Forestry, and the
General Directorate of Agricultural Research (Project No:
TAGEM/HAYSUD/B/18/A4/P2/308).
Conflict of interests
The author declares no conflicts of interest regarding this
manuscript's publication and/or funding.
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H2 (-TGG-)H3 (-TAA-)
         MLD H1H3         
H2H2MLD
HMKMKKM

H2
KKMKR
H2HMKM

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