Commercial turkey breeding relies almost entirely on artificial insemination, yet avian sperm are unusually vulnerable to cooling and freezing injury. As a result, extender chemistry and processing steps, especially low-temperature equilibration, are pivotal for post-thaw performance. We evaluated how extender choice, paired with equilibration time, shapes the post-thaw quality of turkey semen. Ejaculates were diluted in Beltsville, Sperm Motility Medium (SMM), Botucrio, or Kobidil⁺, then equilibrated for 20 or 40 min before freezing; samples were cryostored for one month and assessed immediately after thawing. The outcomes included motility/kinematics, membrane integrity, mitochondrial activity and membrane potential, apoptosis/necrosis, reactive oxygen species (ROS), DNA fragmentation, and bacteriological load. Overall, 20 min equilibration improved post-thaw motility and membrane integrity, and reduced DNA fragmentation and ROS. Among extenders, Beltsville delivered the best overall sperm quality. Considering the extender × time interaction, Beltsville, Botucrio, and Kobidil⁺ performed best at 20 min, whereas SMM performed best at 40 min. Thus, Beltsville and SMM provide strong, time-specific options for turkey semen cryopreservation—Beltsville at 20 min and SMM at 40 min.

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.