Blood samples were collected from 69 Sahiwal cows maintained at Instructional Livestock Farm Complex (ILFC), College of Veterinary Science and Animal Husbandry, Mathura. The genomic DNA was extracted from WBCs of collected blood samples by phenolchloroform method (Sambrook and Russell, 2001) and further optical density of extracted DNA was determined at wavelength 260 and 280 nm using UV-Visual spectrophotometer for estimation of quality and quantity of DNA.

A fragment of LEPR gene was amplified using 5’-ACTACAGATGCTCTACTTTGG-3’ as forward and 5’-TGCTCCTCCTCAGTTT-3’ (Almeida et al., 2008) as reverse primers which resulted in amplification of 197 bp amplicon. The amplification was done in a total volume of 25 μl PCR mix in thermal cycler (Bio-Rad, USA). The PCR mix was prepared containing 2.5 μl PCR dream buffer 10X, 2.5 μl of 2 mM dNTPs, 10 pM (0.5 μl) from each primer, 1 U Taq DNA polymerase and 100-150 ng of template DNA. The PCR program was 94°C for 3 min, followed by 35 cycles of 94 °C for 30s, 51.6 °C for 50s and 72 °C for 50 sec. The final step prolonged for 7 min at 72°C.

LEPR/BseGI polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was executed for determining the genetic polymorphism in LEPR gene. Each digestion reaction carried out at 50°C for 4.5 hours in 10 μl reaction mixture containing 5 μl PCR products, 2 μl buffer 10X, 5 U (0.5 μl) BseGI restriction enzyme (Fermentas) and 7.5 μl deionized water. The restriction fragments were visualized on 2% agarose gels in 1X TBE stained with ethidium bromide (0.5 μg/ ml) (Fermentas).

The allelic and genotypic frequencies of LEPR/BseGI polymorphism were determined for estimating the deviation from Hardy-Weinberg equilibrium using χ² test. The statistical significance was determined by ANOVA followed by Tukey’s post-hoc multiple comparison test using SPSS software (version 16.0). The data are presented as mean ± SE and a p value < 0.05 was considered to be statistically significant.

The amplified fragments of the LEPR gene revealed amplicon of 197 bp (Fig. 1) which on digestion with BseGI restriction enzyme revealed three types of band patterns (genotypes); CC, CT and TT genotypes. The amplicon of CC genotypes were having single restriction site thus revealed two fragments of 130 and 67 bp. The amplicon of CT genotype was containing 3 restriction sites thus revealed 4 fragments of 130, 93, 67 and 37 bp. The third TT genotype containing 2 restriction sites revealed 3 fragments of 93, 67 and 37 bp (Fig. 2). This result revealed that the studied population of Sahiwal cattle were polymorphic in nature with two types of alleles C and T with three types of genotypes CC, TT and CT genotypes in the population.

The results disclosed that in studied population of Sahiwal cattle, the most frequent genotype was CT (47.83%) followed by the homozygote CC (36.23%), and TT genotype (15.94%) and the allele frequency of C and T alleles were 0.60 and 0.40, respectively. The allelic and genotypic frequencies of LEPR/BseGI in Sahiwal cattle were calculated and presented in Table 1.

The χ² calculated value for LEPR/BseGI genes in Sahiwal cattle were 10.78, while χ² table values were 9.210 at 5% and 1% level of significance, respectively for degree of freedom 2. These results revealed that χ²_(cal) >χ²_(tab) at 1% level of significance i.e. selected population of Sahiwal cattle was not found in Hardy-Weinberg equilibrium.

The association studies of LEPR/BseGI assay on reproduction traits of Sahiwal cows has been depicted in Table 2. The LEPR/BseGI PCR-RFLP assay did not reveal significant influence of CC, CT and TT genotypes on reproduction traits in Sahiwal cattle. The association of LEPR/BseGI on milk production traits of Sahiwal cows have been presented in Table 3. The LEPR/BseGI assay revealed significant association of genetic polymorphism of LEPR/BseGI on LP, TMY, MY300, and PY in third lactation in which TT genotype showed longest LP and highest TMY, MY300 and PY compared to CT and CC genotypes in Sahiwal cows.

The present study revealed genetic polymorphism in LEPR/ BseGI with two types of alleles C and T and showed three types of genotypes; CC, CT and TT genotypes. Similarly, Komisarek and Dorynek (2006), Komisarek (2010), Silva et al. (2012) and Ghanbari Baghenoey et al. (2014) also reported all the three genotypes in cattle population, corroborated the findings of present study. The frequency pattern of C and T alleles in LEPR/BseGI assay was 0.60 and 0.40, respectively. This finding was in agreement with the observations of various authors in different breeds of cattle who reported higher allelic frequency of C than T (Komisarek and Dorynek, 2006; Komisarek, 2010; Silva et al., 2012; Trakovicka et al., 2011, 2013a, 2013b, 2015; Ghanbari-Baghenoey et al., 2014). This difference in allele frequency may be due to different history of the breeds, long-term geographical isolation, and selection towards high milk yield.

However, Trakovicka et al. (2011, 2013a, 2013b, 2015) did not observed TT genotype in the studied population. In present investigation the genotypic frequency of CT genotype was highest which was similar to the findings of other scientists (Komisarek, 2010; Silva et al., 2012; Ghanbari-Baghenoey et al., 2014). The high frequency of heterozygous animals indicated effective selection for polymorphism in the population of animals. However, Komisarek and Dorynek (2006) and Trakovicka et al. (2011, 2013, 2013a, 2015) reported highest frequency of CC genotype compared to CT and TT genotypes in cattle population. This variation in frequency of genotypes might be due to population size and geographical distribution.

The association studies of LEPR gene by BseGI/PCRRFLP assay revealed significant influence of genetic polymorphism on LP, TMY, MY300 and PY in studied population of Sahiwal cows. The association study showed that T may be selected as a desirable allele because TT genotype exhibited longer LP, higher TMY, MY300 and PY compared to CT and CC genotypes. However, the LEPR/BseGI genetic polymorphism did not show significant association with AFC, DP, CI, and DRPY in Sahiwal cattle breeds. Almeida et al. (2008), and Giblin et al. (2010) also did not observe significant effect of T945M on milk production and reproduction performance in Holstein cattle, but significant associations was observed between other polymorphisms in LEPR gene and energetically expensive process of lactogenesis, energy storage and fertility performance, therefore they considered LEPR gene as a potential candidate gene for selection of improved fertility in dairy cows. Additionally, Komisarek (2010) and Trakovicka et al. (2013a) did not observed significant influence of T945M of LEPR gene on production or reproduction traits except for calving interval and age at first insemination, respectively.

However, in contrast to the findings of present study, Trakovicka et al. (2015) reported significant higher milk, protein and fat yield in CC genotype of LEPR gene in Pinzgau cows and Komisarek and Dorynek (2006) reported negative significant association between fat and protein content in milk and T allele of T945M polymorphism in Jersey cows. Besides, Chen et al. (2004), Mackowski et al. (2005) and Schenkel et al. (2006) also found genetic variations of the bovine LEPR gene which were associated with milk production traits and fatness traits.

Carvajal et al. (2010) reported significant associations of T945M for milk production, fat and protein percentages but not with milk yield in Chilean dairy cattle. Clempson et al. (2011) reported only a weak association of this SNP with milk yield and days to first service in Holstein cows. De Matteis et al. (2012) reported four novel SNPs in LEPR gene (LEPR01, LEPR03, LEPR09 and LEPR16) which significantly influenced milk production traits (ME fat%, ME protein%, test day fat% and test day protein %) in Holstein cows. For SNP LEPR/T945M effect on milk production (Glantz et al., 2012), reproduction (Clempson et al., 2011; Glantz et al., 2011; Komisarek, 2010) and growth traits (Almeida et al., 2008, Silva et al., 2012) were confirmed.