Effects of the Freezing Diluent Tonicity and Prefreezing Cooling Rates on Post-Thaw Sperm Quality Markers in the Pure-Bred Arabian Stallion.

Stallion semen cryopreservation remains limited by inconsistent post-thaw sperm quality and fertility. This study evaluated the effects of freezing extender effective tonicity and prefreezing cooling rate on post-thaw sperm characteristics in pure-bred Arabian stallions. In a two-phase design, ejaculates (n = 40) from 10 stallions were processed using extenders with different osmotic conditions (330, 390, and 450 mOsm/kg) and subjected to cooling durations of 45 min, 1.5 h, or 3 h. Sperm quality was assessed using CASA, functional assays, flow cytometry, transcriptomic analysis, and a pilot fertility trial. Moderate hypertonicity (∼356 mOsm/kg effective tonicity) significantly improved sperm function, including higher proportions of live/nonapoptotic cells, enhanced mitochondrial activity, improved acrosome integrity, and reduced DNA fragmentation (p < 0.05). In contrast, excessive hypertonicity (∼383 mOsm/kg) increased necrosis and acrosome damage and tended to elevate the Bax/Bcl2 ratio although this effect was not statistically significant. Sperm kinematic parameters were not affected by osmolarity. Extending prefreezing cooling from 45 min to 1.5-3 h significantly improved motility, viability, mitochondrial function, acrosome stability, and DNA integrity, while reducing apoptosis and total cell death (p < 0.05), with no consistent additional benefit beyond 1.5 h. Oxidative stress (MDA) was not significantly affected. The optimized protocol resulted in pregnancy rates of 75.0% at Day 21 and 62.5% at Day 60. In conclusion, moderate hypertonicity combined with controlled slow cooling enhances stallion sperm cryosurvival and provides a practical strategy to improve fertility outcomes.