Determination of CAST/MspI Gene Polymorphism and its Effects on 180-Day Weight in Meat Production Line of Kazakh Saryarka Lambs

The study was conducted at the Zhenis breeding facility in the Zhanaarka district of the Ulytau (former Karaganda) region of Khazakistan. The test material consisted of a total of 60 male lambs of the Zhanaarka type Saryarka sheep breed.

Lambs raised by their mothers until weaned. The lambs were weighed on day 180 after birth, and blood samples were taken under veterinary supervision in 15 ml tubes containing an anticoagulant (EDTA). DNA was obtained from whole blooed using DNA-sorb-B extraction kit (REF K1-2-100-CE, AMPLISENS, Moscow, Russia).

A 622 bp fragment of the CAST gene was amplified with the primer pairs; 5-TGGGGCCCAATGACGCCATCGATG-3 and reverse 5-GTGGAGCAGCACTTCTGATCACC-3 using a SimpliAmp thermal cycler (Applied Biosystem). The PCR cycle was performed: an incubation step at 95 °C for 12 mins, followed by 35 cycle of 95 °C for 1 min, annealing at 64 °C for 1 min and an extraction step at 72 °C for 1 min, and 10 mins at 72 °C as final extraction.

PCR products (622 bp) were digested using the restriction endonuclease enzyme MspI (EURX) to genotype the animals for the CAST gene. The PCR products were incubated for 1 h at 37°C in a final volume of 50 µl, the 10 µl PCR product, 5 µl 10x One buffer, 0.5 µl BSA [100x], 0.3 µl MspI enzyme and finally ultrapure contained water brought to final volume and after digestion heated to 65oC for 20 min for inactivation. A 50 bp DNA ladder was used to determine fragment sizes of PCR-RFLP results.

Allele and genotype frequencies for the CAST gene were calculated using PopGene 32 software (Yeh et al., 1999). Chi-sequere analyzes were performed to determine whether the studied population was in Hardy-Weinberg equilibrium. The general linear model (GLM) was used to demonstrate the effects of CAST gene genotypes on live weight at day 180 using SPSS 22 (IBM SPSS Inc., Chicago, IL, USA). The effect of farm, feeding and age factors etc. was not used in the GLM analysis as all lambs were reared on the same farm.

The 622 bp fragment of the CAST gene was successfully amplified by PCR and all individuals were genotyped by digestion of the MspI enzyme. In the population of Saryarka sheep from Khazakistan, PCR-RFLP results showed two genotypes, heterozygous MN and homozygous MM In the study, NN genotypes were not observed in the examined samples. Animals with MN genotypes showed three fragments of 622 bp, 336 bp and 286 bp, while homozygous animals MM showed two fragments of 336 bp and 286 bp on 3 % agarose gel. The most of the studied animals were of heterozygous genotype. In the study, the calculated allele and genotype frequencies were given in Table 1.

NS, not significant; Ho, observed heterozygosity; He, expected heterozygosity; x2, Chi−sequare.

The M and N allele frequencies for the CAST gene in Saryarka sheep were 0.80 and 0.20, respectively, while the genotype frequencies were 0.59 for MM and 0.41 for MN. The result of the Chi-square test showed that the examined Saryarka sheep was in HWE (P>0.05).

Lamb live weight at day 180 in the study was calculated as 48.01±3.03 kg for MM genotypes and 52.49±2.96 kg for MN genotypes, indicating that MN genotype lambs gained an average of 4.48 kg more weight than lambs with MM genotype. As a result, live weight at day 180 was statistically (P<0.01) influenced by the genotype. The values of observed (Ho) and expected (He) heterozygosity were estimated as 0.4107 and 0.3293, respectively. The chi-square analysis showed that the studied population was in Hardy-Weinberg equilibrium.

The study showed that the Saryarka breed has CAST/MspI gene polymorphism, and the gene has a significant effect on live weight at day 180. This polymolymorphism has also been reported in many sheep breeds from different part of the world such as Bulgarian (Bozhilova-Sakova et al., 2020), Brazil (Santos et al., 2016), New Zeland (Byun et al., 2008), Russian (Kolosov et al., 2021), Turkey (Kırıkçı et al., 2021; Kırıkçı, 2022) and Iranian (Eftekhari Shahroudi et al., 2006). The M allele frequency in the Saryarka sheep was higher than the N allele frequency. Heterozygous MN genotype frequency value for the Saryarka breed were higher than the some previous studies reported for Awassi-0.35 (Jawasreh and Ismail, 2019), Prydniprovska -0.13 (Pomitun et al., 2019), Colombian Creole hair sheep-15.5 (Vergara et al., 2019) and West Siberian mutton breed − 0.23 (Afanasyeva et al., 2019). The present study could not detected the NN genotype in the lambs examined. This result was consistent with the study by Suleman et al. (2012) who examined three Pakistani sheep breeds– Thali, Lohi and Kajli. Similar results were also observed for the Turkish Akkaraman and Karayaka sheep breeds (Kırıkçı et al., 2021; Kırıkçı, 2022).

No study has been found in the literature that demonstrates the genetic structure for the CAST gene in the Saryarka breed and its effects on body weight. To the best of our knowledge, the current study was the first to examine the CAST gene and its impact on day 180 live weight in Saryarka lambs. Therefore, the results obtained were limited to comparison with other Kazakh breeds. In the study, the frequency percentage (0.41) of the heterozygous genotype was quite high. It can be said that this is mainly caused by the selection of the studied animals in terms of 180th day live weight for many years. Afanasyeva et al. (2019) investigated the CAST gene polymorphism and its impact on live weight in the West Siberian sheep breed at various ages. According to the findings, heterozygous lambs produced 3.2 kg more live weight than homozygous animals. This finding was comparable to the current study, which showed that heterozygous Saryarka lambs produced 4.48 kg more live weight than homozygous lambs, which was a desired result. On the 180th day, heterozygous Saryarka lambs averaged 52,49±2.96 whereas homozygous lambs averaged 48,01±3.03. There were clearly significant differences (P<0.01) between genotypes for live weight at day 180. Likewise, heterozygous Volgograd sheep have been reported to have 3.7 kg more live weight before slaughter than homozygous ones (Kolosov et al., 2021).

Bayraktar and Shoshin (2022) found an association between CAST genotypes and body weight at one year of age in Awassi sheep. According to their findings, the body weight difference between MN and MM genotypes was 0.54 kg. Another study found no association between the CAST gene and some live weight elements (Bayram et al., 2019). Therefore, it is worth noting that the live weight differences obtained for Saryarka lambs between MN and MM genotypes were quite higher than in most of the studies mentioned above. The only limitation of the current study may be the sample size. Despite this limitation, the results of this study are important as they provide the polymorphism of the related gene and an association with live weight.

This study was the first to show the genetic variation of Saryarka lambs for the CAST gene and provided evidence for the relationship between genotype and phenotype. The CAST gene MN genotype significantly affected the live weight of lambs at 180 days. Consequently, this gene may be used as a potential molecular marker to increase meat production and integrated into the current selection program in the Saryarka breed meat production line. However, given the lack of molecular studies and sample size in the present study, further investigations should be done in the Saryarka and other Kazakh sheep breeds.

The study was not financially supported by any organization.

The authors have declared no conflict of interest.