In India, cross-breeding of indigenous cattle with exotic cattle such as Holstein Friesian and Jersey has been going on since last four decades to improve milk production. Although it has led to increased milk yield, the subfertility in male cross-bred progeny has remained a significant problem. Epigenetic modifications (DNA methylation, histone modifications and chromatin remodelling) are regarded as key players influencing gene expression. DAZL gene plays an important role in germline development and gametogenesis. The methylation and mRNA expression level of this gene have been significantly negatively correlated in the testes of cattle-yak hybrids and their parents. This study analysed the methylation profile of DAZL gene promoter in bull spermatozoa in an attempt to speculate its role in cross-bred cattle subfertility. Semen samples from Sahiwal, Holstein Friesian and Frieswal bulls (Sahiwal X Holstein Friesian) with varying semen motility parameters were collected, and DNA was isolated. Methylation-specific primers were used to amplify part of promoter and exon 1 of DAZL gene using bisulphite-converted DNA. The amplified products were sequenced after cloning in pTZ57R/T vector. Sequence analysis revealed significantly higher DNA methylation of DAZL gene in Frieswal bulls with poor motility (28.26%) as compared to medium (15.21%) and high motility phenotype (6.52%). In pure-bred counterparts, Sahiwal and Holstein Friesian, epigenetic marks were more in the former (15.21%) than the latter (4.34%), but in both cases, the values were lower as compared to the poor motility Frieswal bulls. This suggests that differential hypermethylation of the CpG islands could possibly influence reproductive parameters in bovines.

Major credit for India being the largest producer of milk in the world, goes to crossbred cows produced by inseminating low-producing indigenous cattle with semen from high producing exotic bulls. However, over the years, the policy of crossbreeding has been confronted with a major problem of subfertility in crossbred male progenies, culminating into disposal of a major fraction of mature bulls. Many studies have demonstrated relationship between epigenetic alterations and male fertility across different species. PIWIL1 is an important candidate gene for spermatogenesis and germ line development. Negative correlation between DNA methylation and expression of this gene has been highlighted in inter species hybrids of cattle and yaks. The present study envisaged elucidating promoter methylation status and expression profile of PIWIL1 gene in exotic Holstein Friesian cattle, indigenous Sahiwal cattle and their crossbreds with varying semen motility parameters. Semen samples were collected from bulls for isolation of DNA and RNA from spermatozoa. Bisulfite converted DNA was used to amplify promoter of PIWIL1 gene using methylation specific primers. The amplified products were sequenced after cloning in pTZ57R/ T vector. The degree of methylation of the PIWIL1 promoter region was significantly higher in poor motility crossbred bulls (7.17%) as compared to good motility crossbreds (1.02%), Sahiwal (1.02%) and Holstein Friesian bulls (0.77%). PIWIL1 expression was 1.75, 1.71 and 1.59 folds higher in HF, Sahiwal and good motility crossbreds, respectively as compared to poor motility crossbreds.