18 Epigenetic makeup of cloned buffalo (Bubalus bubalis) bull spermatozoa

Somatic cell nuclear transfer (SCNT) can be used for faster multiplication of superior buffalo bulls, which could be helpful in faster dissemination of superior germplasm and breed improvement. However, very few studies have been conducted on the genetics and epigenetics of cloned bull sperm. This study was conducted to compare the DNA methylation status and sperm apoptosis level in cloned bulls with their respective donor bulls. Frozen-thawed semen from two cloned bulls and their donor bulls were used for the study. Whole genome bisulfite sequencing revealed similar methylation percentages at cytosine residues in spermatozoa of cloned bulls compared with their donor bulls. Differential methylation analysis suggested 413 hypermethylated and 354 hypomethylated differentially methylated cytosines (DMCs) were observed, with a methylation cutoff of 25% (q < 0.05). Gene annotation of the DMCs identified hypermethylation in the promoter-TSS region of nine genes and hypomethylation in six genes involved in different regulatory functions. In addition, the relative mRNA abundance of pro-apoptotic genes (MCL1, BCL2, BCL2L1) and anti-apoptotic genes (BAX, BID, BAD) were found to be similar between the cloned and donor bulls spermatozoa (P < 0.05). This finding suggested that the apoptosis level in the sperm of cloned bulls is normal and comparable to that of their donor bulls (normal population bulls). In conclusion, this study provides preliminary insight into the epigenetic status of cloned bull’s spermatozoa. It suggests that not only are the genetics comparable, but the epigenetic makeup of gametes of cloned bulls is also comparable to that of their donor bulls.