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Study of Genetic Polymorphism in Leptin Exon 3 Region and its Association with Milk Production and Reproduction Traits in Indian Sahiwal Cattle

Sumit Kumar Deepak Sharma Satyendra Pal Singh Madhu Tiwari Rakesh Goel
Vol 8(8), 84-91

This study investigated the status of leptin (LEP) exon 3 polymorphism in Indian Sahiwal cattle breed (n = 124) using hphI/PCR-RFLP assay and its association with milk production and reproduction traits. The 454 bp amplified fragment upon restriction digestion revealed two types of genotypes; namely; CT genotype (454, 309 and 145 bp fragments) and TT genotype (309 and 145 bp fragments) with frequencies 29.03% and 70.97%, respectively. We could not find any CC genotype (454 bp; uncut fragments) in screened population. Allelic frequency of C and T alleles were 0.15 and 0.85, respectively. Chi square analysis revealed that screened population was not found in Hardy-Weinberg equilibrium. Association studies reveled that TT genotype was associated with shorter age at first calving (AFC) and days to reach peak yield (DRPY) in the first lactation, while TT has shorter dry period (DP) than CT genotype in all the five lactation. TT genotype was also associated with shorter calving interval (CI) in all the studied lactation except lactation I. Therefore, the present study indicating that T allele of this SNP in LEP exon 3 regions could be used as a potential genetic marker for improvement in reproduction and production performance in cattle.

Keywords : Cattle hphI Leptin Milk Production Traits PCR- RFLP Sahiwal

Leptin is a 16-kDa polypeptide hormone product of the leptin (LEP) gene. It is synthesized and secreted predominantly by adipose tissue and regulating body weight, food Intake, energy expenditure, reproduction and immune system. The milk production traits are quantitative traits and these are directly related to feed intake and energy balance (Block et al., 2001). It was observed that the feed intake-induced leptin increase was eliminated during lactation and they speculated that the hypo-leptinemia may be an important factor promoting the hyperphagia of lactation. This data demonstrated that the onset of the negative energy balance is largely responsible for the declining leptin concentrations towards parturition and the low leptin level during lactation probably induces the hyperphagia of lactation. Therefore, LEP may be important in regulating metabolic adaptation of nutrient partitioning during the energy-consuming processes of pregnancy and lactation (Moschos et al., 2002). The gene encoding leptin was mapped to bovine chromosome 4 and it consists of 3 exons and 2 introns, of which only two exons are translated into protein. The coding region of the LEP gene (501 nucleotides length) is contained in exon 2 and 3, which are separated by intron of approximately 2 kb (Stone et al., 1996) and it is considered as a strong candidate gene for milk performance related traits in cattle.

Several single nucleotide polymorphisms (SNPs) in the LEP gene have been identified by previous authors (Liefers et al., 2002; Madeja et al., 2004, Yoon et al., 2005; Oztabak et al., 2010). However, research on associations between LEP gene polymorphism and milk production traits in dairy cattle is rather scanty (Buchanan et al., 2003; Sadeghi et al., 2008; Dandapat et al., 2009; Singh et al., 2014). A SNP resulting from a C>T substitution, causes amino acid change from alanine to valine (A59V) has been observed in LEP exon 3 in various cattle breeds (Haegeman et al., 2000; Madeja et al., 2004; Kulig and Kmiec, 2009; Oztabak et al., 2010; Silva et al., 2014), but a few reports about this LEP exon 3 polymorphism and its association with milk production and reproduction traits have been reported in Sahiwal cattle breed (Dandapat et al., 2009; Dandapat et al., 2010; Singh et al., 2014). Hence, the present study was undertaken with the objective of investigation of this SNP (A59V) within LEP exon 3 region and its association with milk production and reproduction traits in Indian Sahiwal cattle.

Materials and Methods

Animals and DNA Isolation

A total of 124 animals (only female) of Sahiwal breed of cattle were utilized in the present investigation, out of them 92 maintained at Instructional Dairy Farm, National Dairy Research Institute (NDRI), Karnal (Haryana) and remaining 32 maintained at Instructional Livestock Farm Complex (ILFC), DUVASU, Mathura (Uttar Pradesh). Genomic DNA was isolated using the standard phenol–chloroform extraction method (Sambrook and Russel, 1991).

Amplification of LEP Exon 3 Gene Fragment and HphI/PCR-RFLP Assay

C>T SNP containing region of LEP gene fragment has been amplified from isolated DNA as per Oztabak et al., 2010 (Table 1). The restriction digestion was carried out at 37oC for 14-16 hr in a total volume of 15μl containing 10μl PCR products, 0.5 μl HphI (Fermentas, USA; 10U/ μl) and 1.5μl 10X RE buffer. For restriction fragment analysis, digested products were checked on 1.5% agarose gel in 1X TAE buffer for 4-5 hrs at 5 V/cm.

SNP Region Amino Acid Change Primer Sequence Product






454 bp 57°c



Table 1: SNPs, Primers, annealing temperature and restriction enzyme details of amplified regions of LEP gene

Statistical Analysis

The data will be generated by estimating the frequency of different amplified products. The allelic frequency and genotypic frequencies of LEP gene was estimated by standard procedure (Falconer and Mackay, 1996) using following formulae:

Gene Frequency = (2D + H)/ 2N

Where, D = No. of homozygote of particular gene, H = No. of heterozygote having that gene, N = Total No. of individuals

The chi square (χ2) test (P ≤ 0.05) was also performed to test whether the distribution of the genotype frequencies was in the Hardy-Weinberg equilibrium (Snedecor and Cochran, 1989).

Association Study

The association study of LEP/hphI genotypes with the following milk production and reproduction traits: age at first calving (AFC), dry period (DP), lactation period (LP), total milk yield (TMY), milk yield in 300 days (MY300), calving interval (CI), peak yield (PY), days to reach peak yield (DRPY). Analysis of associations between the LEP genotypes, and production traits, were carried out with the General Linear Model (GLM) using SPSS software (ver. 16). The following linear model was applied:

Yij =  μ + Gi + eij

where: Yij– observed trait value of ith genotype in animal; μ – mean trait value; Gi – effect of ith genotype; eij– random error. Significant differences among least square means of different genotypes were calculated using Duncan’s multiple-range test, and P values of 0.05 were considered statistically significant.

Results and Discussion

The amplified fragments of the LEP gene revealed 454 bp product (Fig. 1). The LEP/HphI PCR-RFLP assay revealed two types of banding pattern (genotypes); one of them was of 309 and 145 bp (TT genotype); second of 454, 309 and 145 bp (CT genotype) and wild type homozygous pattern (CC genotype) was completely absent in the sample population (Fig. 1). This revealed that the Sahiwal cattle used in the present study were polymorphic in nature with two types of alleles C and T.        

Fig. 1: LEP/HphI PCR-RFLP assay showing genotype pattern in 1.5% agarose gel; Lane 1: M1: 50bp ladder, 2: TT genotype (309 & 145 bp), 3: CT genotype (454, 309 & 145 bp) 4: M2 – 100 bp ladder, 5: Undigested PCR product (454 bp).

LEP/HphI PCR-RFLP assay revealed that the TT genotype was the most frequent (70.97%) in all the screened samples, followed by the heterozygote CT (29.03%), whereas the CC genotype was not found in these samples. The frequency of LEP/HphI C and T alleles was 0.15 and 0.85, respectively. Chi square  test revealed that χ2(cal)2(tab) at 1% level of significance for degree of freedom 1 indicating that screened population of Sahiwal cattle was not found in Hardy- Weinberg equilibrium. The means with standard errors of mean (Mean ± S.E.M.) for each trait related to each genotype in all the five lactations are presented in Table 2. In the present study, the CC genotype was not found (0.0%), which was similar to study in Nellore bulls (Silva et al., 2014). This report was dissimilar with the findings in Sahiwal cattle; monomorphic; 100% (Dandapat et al., 2009; 2010), 96.0% (Singh et al., 2014) and in other breeds of cattle; Polish Black and White bull (42.7%; Madeza et al., 2004), Jersey X HF X Sahiwal crossbred (57.0%; Dandpat et al., 2010), Iranian Holstein (58.8%; Yazdani et al., 2010), Frieswal (58.0%; Singh et al., 2014). However, its frequency was 100% in Mehsana buffalo (Deshpande et al., 2014). It might be possible due to small population size and selective breeding towards T allele.

The genotypic frequency of CT genotype in the present investigation was 29.03%, which was in the range (30 – 47%) with the observations in different breeds of cattle; Polish Black and White bull (47.0%, Madeza et al., 2004), Jersey X HF X Sahiwal crossbred (36.0%; Dandpat et al., 2010), Iranian Holstein (38.8%; Yazdani et al., 2010), Frieswal (38.0%; Singh et al., 2014) and Nellore bulls (30.0%; Silva et al., 2014).

Table 2: Association studies of LEP/Hph1 genotypes with milk production and reproduction traits in Sahiwal cattle

Lactation Genotype n AFC (days) DP (days) LP (days) TMY (liters) MY300 (liters) CI (days) PY (liters) DRPY (days)
I TT 88 1103.6±10.69** 133.78±4.41* 347.85±5.98 1617.3±42.9 1424.52±37.36 481.64±6.87 6.34±0.16 54.38±0.81*
(N=124) CT 36 1550.8±42.3** 176.11±16.7* 348.53±12.5 1550.1±90.58 1409.44±80.72 524.64±21.9 5.8±0.34 58.06±1.59*
II TT 70 —– 133.81±5.13** 319.99±5.24 1613±51.60 1513.09±44.95 452.51±6.53** 6.91±0.2 53.05±1.06*
(N=88) CT 18 —– 228.17±22.83** 350.56±20.9 1480.2±161.97 1355.44±127.79 584.28±25.56** 5.64±0.66 59.22±2.0*
III (N=62) TT 51 —– 129.04±7.87* 328.33±6.24 1648.6±54.12 1518.24±48.45 456.39±9.09* 6.79±0.24 52.53±1.27
CT 11 —– 221.27±34.04* 323.27±19.15 1495.8±181.59 1409.18±171.16 544.55±31.79* 5.59±0.90 59.73±4.08
IV TT 32 —– 130.29±7.93* 328.26±6.45 1664.6±71.23 1550.60±64.13 455.92±10.63* 7.18±0.28 47.29±1.31
(N=50) CT 18 —– 227.67±34.29* 317.75±17.21 1419.6±172.74 1328.42±137.21 545.42±31.04* 7.33±0.56 50.83±2.69
V TT 32 —– 125.22±6.82* 324.25±7.66 1688.2±73.59 1587.97±72.45 449.78±10.47* 7.31±0.33* 50.19±1.81
(N=41) CT 9 —– 219.01±31.46* 318.22±17.19 1344.0±169.92 1266.33±140.91 537.22±26.17* 5.22±0.78* 60.0±4.45

Superscript (*) and (**) of a given row  indicates significant difference between genotypes at P<0.05 and  at P<0.01, respectively, n — number of individuals in particular genotype,  N — total number of individual in particular lactation, n= number of animals; GP= Gestation Period; LP= Lactation Period; TMY= Total Milk Yield; MY300= Milk Yield in 300 days; DP= Dry Period; CI= Calving Interval; PY= Peak Yield; DRPY= Days to Reach Peak Yield

But it was much higher than the previous findings in Sahiwal; 4.0% (Singh et al., 2014). In contrast, Dandapat et al. (2009) and (2010) could not observe any CT and TT genotypes in studied Sahiwal cattle. The value of TT genotype obtained in present study was 70.97%, similar to result in Nellore bulls (70.0%; Silva et al., 2014). This study was not consistent with the results of previous reports. It was much higher than the other findings in Sahiwal; 0.0% (Dandpat et al., 2009; 2010; Singh et al., 2014) and other cattle breeds; Polish Black and White bull (10.25%, Madeza et al., 2004), Jersey X HF X Sahiwal crossbred (7.0%; Dandpat et al., 2010), Iranian Holstein (2.4%; Yazdani et al., 2010), Frieswal (4.0%; Singh et al., 2014). However, its frequency was 100% in Murrah buffalo (Datta et al., 2013). Since in India, Sahiwal cattle are reared for milch purpose, this mutant allele might have undergone selection.

In the present study, allelic frequency of LEP/HphI for C and T allele were 0.15 and 0.85, respectively. In contrast, higher frequency of LEP/HphI C allele was reported in Sahiwal; 1.0 (Dandpat et al., 2009; 2010), 0.98 (Singh et al., 2014) and all other breeds of cattle; Polish Black and White bull (0.66, Madeza et al., 2004), Jersey X HF X Sahiwal crossbred (0.75; Dandpat et al., 2010), Iranian Holstein (0.782; Yazdani et al., 2010), Nellore (0.99; da Silva et al., 2012), Frieswal (0.77; Singh et al., 2014). It might be possible due to small sample population and selective breeding towards T allele.

Association analysis revealed that TT genotype had significantly (P = 0.000) smaller AFC in comparison to heterozygote CT. These results of AFC were dissimilar to the findings in Jersey X HF X Sahiwal (Dandpat et al., 2009) and frieswal (Singh et al., 2014) crossbred cattle. It might be due to difference in crossbred and indigenous cattle population. GP were not affected by genotypes in all the five lactations, which was also reported in crossbred cattle (Dandpat et al., 2009), while CC genotype had significant higher GP than other genotypes in Iranian Holstein cattle (Yazdani et al., 2010). LP was not affected by genotypes in all the lactations but CT genotype had higher LP in crossbred cattle (Dandpat et al., 2009). However, DP of TT genotype was significantly (P<0.05) lower than CT genotype in all the lactations. It can be explained by the selective breeding towards T allele, while in Iranian Holstein cattle there was no significant difference for DP among genotypes (Yazdani et al., 2010). CI of TT genotype (except Lactation I) was also significantly (P<0.05) lower than CT genotype in all the lactations, while in Iranian Holstein (Yazdani et al., 2010) and crossbred (Dandpat et al., 2009) cattle, CI was not affected by the genotypes. In the present study, no significant difference was found for TMY and MY300 between CT and TT genotypes in all the lactations, which was similar to the findings in Frieswal cattle (Singh et al., 2014) but CT genotype had higher milk yield in first and second lactation in crossbred cattle (Dandpat et al., 2009).

In the current study, T allele was found to be responsible for higher milk production without negatively affecting energy balance and fertility, which was similar to previous findings (Liefers et al., 2002, Madeja et al., 2004). Finally, allele T of LEP gene can be considered as a good indicator for milk production in the Sahiwal cattle breed.


Only two genotypes (CT and TT) in LEP exon 3 was identified in studied Sahiwal cattle population using hphI/PCR-RFLP assay. Association studies confirmed that C>T/hphI genotypes had significant effect on milk related traits. TT genotype had shorter AFC and DRPY in first lactation, while TT genotype has shorter DP than CT genotype in all the five lactation. TT genotype was also associated with shorter CI in all the lactation except lactation I. LEP/hphI genotypes had no effect on LP, TMY, MY300, GP and PY. Sahiwal cows with TT genotype tended to have a better reproduction performance than the heterozygote cows. Therefore, this SNP could be useful in marker assisted selection to improve the reproduction and milk production performance in dairy cattle.


The Instructional Dairy Farm, NDRI, Karnal (Haryana) and Instructional Livestock Farm Complex, DUVASU, Mathura (U.P.) is gratefully acknowledged for providing help during sample and data collection. The authors are thankful to Vice Chancellor, DUVASU, Mathura, (U.P) for providing necessary facilities and fund during entire research work at this esteemed university.



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