miR-31-5p Is Associated with Apoptotic and Mitochondrial Alterations in Fresh and Frozen Bull Spermatozoa

This study investigated whether miR-31-5p abundance in fresh Holstein bull semen is associated with post-thaw sperm resilience. The initial cohort comprised 36 Holstein bulls, from which 16 ejaculates representing the highest- and lowest-ranking post-thaw freezers were selected for epigenetic and molecular analyses. Fresh semen was analyzed for miR-31-5p, while BAX and BCL2 were evaluated at transcript and protein levels before and after cryopreservation. Post-thaw sperm quality was assessed using total motility, viability, JC-1 mitochondrial membrane potential, Annexin-negative spermatozoa, reactive oxygen species (ROS) and sperm DNA fragmentation. Cryopreservation significantly impaired sperm quality, increased ROS and DNA fragmentation, and shifted the molecular profile toward increased BAX and reduced BCL2. Ejaculates with superior post-thaw motility showed higher miR-31-5p levels, accompanied by better preservation of viability, mitochondrial function, non-apoptotic membrane status, lower ROS burden and DNA fragmentation after thawing. Fresh miR-31-5p was positively associated with post-thaw motility, viability, mitochondrial function, and Annexin-negative spermatozoa and negatively associated with post-thaw ROS. Our data support miR-31-5p as a potential candidate biomarker of sperm freezability in Holstein bulls.