Effect of Different Equilibration Times in the Freezing Process on Post-Thaw Motility Parameters in Equine Spermatozoa.

Cryopreservation of stallion semen remains suboptimal, with approximately one-third of stallions producing semen that is poorly tolerant to freezing. As optimization of pre-freezing procedures may improve post-thaw sperm quality, this study evaluated the effect of different equilibration times before freezing on the post-thaw quality of stallion semen. For this purpose, 21 ejaculates were initially collected from 5 stallions and 15 were included in the final analysis after predefined exclusions. These ejaculates were separated from the seminal plasma via centrifugation in INRA 96 medium. After removal of the supernatant, each ejaculate was divided into three aliquots and equilibrated at 4°C for 2, 4, or 6 h before freezing. The pellets were mixed with INRA FREEZE solution at a concentration of 100 million spermatozoa per millilitre. Subsequently, the semen was frozen in 0.5 mL straws after being kept at the designated temperature at +4°C for either 2, 4 or 6 h post-cooling. Post-thaw sperm total motility, progressive motility and kinematic parameters were assessed by computer-assisted sperm analysis (CASA). Total motility increased with equilibration time, from 27% ± 7% after 2 h to 34% ± 10% after 4 h and 40% ± 7% after 6 h (p < 0.05). Progressive motility was significantly lower after 2 h (15% ± 6%) than after 4 h (20% ± 7%) and 6 h (22% ± 7%) of equilibration (p < 0.05), whereas no significant difference was observed between 4 and 6 h. In contrast, equilibration time did not significantly affect sperm kinematic parameters, including VAP, VSL, VCL, ALH and LIN. These findings indicate that extending the equilibration period to 6 h at 4°C before freezing improves post-thaw total and progressive sperm motility in stallions without altering the movement characteristics of surviving spermatozoa.