Abstract
A comprehensive understanding of the molecular differences between X and Y sperm in Holstein bull semen is crucial for advancing sex control technologies. While previous studies have primarily focused on proteomic and transcriptomic differences, the genome-wide DNA methylation differences between these sperm types remains largely unexplored. In this study, we employed whole-genome bisulfite sequencing to systematically compare the autosomal methylation profiles of X and Y sperm. Although global methylation patterns showed remarkable consistency between the two sperm types, our localized comparative analysis revealed 12,175 differentially methylated regions mapping to 2,041 genes (differentially methylated genes, DMGs). Functional enrichment analysis of these DMGs revealed their involvement in essential biological processes, particularly in energy metabolism and membrane voltage regulation. Notably, SPA17 and CHCHD3, identified as hypermethylated genes in X sperm in this study, have also been reported to show lower protein expression levels in X sperm compared to Y sperm. Furthermore, we identified 28 DMGs functionally associated with spermatogenesis and 5 DMGs related to fertilization. Our findings lay the foundation for thorough understanding of molecular differences between X and Y sperm in bull, providing essential insights for the development of more advanced sex control technologies in the future.