Genome wide profiling of buffalo (Bubalus bubalis) sperm DNA methylation in relation to bull fertility

Sperm carries information to the presumptive embryo upon fertilization in terms of epigenetic codes and transcripts along with the haploid genome. The epigenetic code includes DNA methylation and histone modifications. During spermatogenesis, the DNA of sperm undergoes overall methylation changes and this could have some role to play in fertilizing ability of the sperm. Many of the studies have shown that the altered methylation can cause sub fertility. In the present study we report the development of first comprehensive 4X180K buffalo (Bubalus bubalis) CpG island/promoter microarray for studying the global DNA methylation profile of buffalo sperm. The array has been developed by employing microarray based comparative genomic hybridization (aCGH) technique with bovine and buffalo DNA using bovine genome sequence as reference. The array represents 157084 features assembled from CDS, Promotor and CpG regions covering 2,967 unique genes. We also report the comparison of genome wide methylation differences in buffalo sperm from high fertile and sub fertile bulls which indicated profound discrepancies in their methylation status. A total of 96 individual genes along with another 55 genes covered under CpG islands were found differentially methylated and and were associated with different cellular functions and biological processes affecting germ cell development, spermatogenesis, capacitation and embryonic development. Agilent two-color Methylation experiment, Organism: Bos taurus , 4X180K buffalo (Bubalus bubalis) CpG island/promoter microarray designed by Genotypic Technology Private Limited (AMADID-037258). To see the differential DNA methylation pattern between high and sub fertile buffalo bull sperm, the cryopreserved semen samples from the identified bulls were procured. The DNA was isolated from all the samples, sheared, immunoprecipitated for methylated DNA (meDIP) and hybdidized on array to get information for all the probes printed on the array. The data was analyzed and the differentially methylated genes between the two group were identified.