Impact of extended equilibration periods on in vitro post-thaw sperm quality in rams

Equilibration, a critical phase in cryopreservation, allows sperm cells to adapt to cryoprotective agents (CPAs) before freezing. This study investigated the impact of extended equilibration durations on the quality of frozen-thawed ram spermatozoa. Semen samples from five healthy Merino rams were pooled, extended with a Tris-based extender, and equilibrated for 3 h (control), 24, 48, or 72 h. post-equilibration and post-thaw sperm quality parameters, including motility, plasma membrane and acrosome integrity (PMAI), high mitochondrial membrane potential (HMMP), mitochondrial reactive oxygen species (MROS) levels, lipid peroxidation (LPO), and intracellular calcium levels (ICL) were analysed. Short equilibration durations (3-24 h) preserved motility, PMAI, and HMMP while minimizing MROS production, lipid peroxidation, and calcium influx. Conversely, prolonged equilibration (48-72 h) leads to CPA toxicity, oxidative stress, and premature capacitation-like changes, impairing sperm viability. Notably, 3-hour equilibration maintained post-thawed sperm with the highest motility (83.96 ± 1.87%) and lowest lipid peroxidation levels (11.46 ± 2.12%). These findings emphasize that shorter equilibration times optimize CPA penetration and minimize cryodamage, thereby enhancing post-thaw sperm function. These results provide valuable insights into refining cryopreservation protocols to improve freezing -thawing outcomes in ram sperm freezing centres.