Divergence Analyses of Sperm DNA Methylomes between Monozygotic Twin AI Bulls.

Semen quality is critical for fertility. However, it is easily influenced by environmental factors and can induce subfertility in the next generations. Here, we aimed to assess the impacts of differentially methylated regions and genes on semen quality and offspring fertility. A specific pair of monozygotic (MZ) twin artificial insemination (AI) Holstein bulls with moderately different sperm qualities (Bull1 > Bull2) was used in the study, and each twin bull had produced ~6000 recorded daughters nationwide in China. Using whole genome bisulfite sequencing, we profiled the landscape of the twin bulls' sperm methylomes, and we observed markedly higher sperm methylation levels in Bull1 than in Bull2. Furthermore, we found 528 differentially methylated regions (DMR) between the MZ twin bulls, which spanned or overlapped with 309 differentially methylated genes (DMG). These DMG were particularly associated with embryo development, organ development, reproduction, and the nervous system. Several DMG were also shown to be differentially expressed in the sperm cells. Moreover, the significant differences in DNA methylation on gene INSL3 between the MZ twin bulls were confirmed at three different age points. Our results provided new insights into the impacts of AI bull sperm methylomes on offspring fertility.