Mild hydrostatic pressure preconditioning improves post-thaw motility of cryopreserved stallion semen

The success of stallion semen cryopreservation is limited by substantial variation among stallions and by temporal variation within individual donors. However, the equine breeding industry requires predictable, stable, and efficient cryopreservation protocols to support the increasing use and dissemination of valuable genetics. One possible strategy for improving semen freezing is to introduce a preconditioning step before cryopreservation to prepare spermatozoa for the unavoidable stresses associated with freezing and thawing. This study evaluated the effects of mild hydrostatic pressure preconditioning on semen samples from Arabian and Shagya-Arabian stallions. Sixty-three semen samples from ten stallions were included in the study, spanning between July and April, in 31 collection days. Before routine cryopreservation, each sample was split into one control and six treatment aliquots, subjected to one of six hydrostatic pressure preconditioning treatments or maintained as control. After freezing and thawing, total motility (TM) and progressive motility (PM) were assessed using computer-assisted sperm analyser. Five of the six preconditioning treatments significantly improved both TM and PM compared with the control (p < 0.05). TM increased from 51.87% in control samples to 56.39-59.17% in mild-hydrostatic-pressure-treated samples, while PM increased from 39.56% to 43.81-47.00%. In our study, mild hydrostatic pressure preconditioning was readily integrated into the routine semen cryopreservation workflow and improved post-thaw motility. These findings indicate improved motility-based cryosurvival of stallion spermatozoa; however, the study did not assess pregnancy rate, foaling rate, or other in vivo fertility outcomes. Artificial insemination trials are therefore required before conclusions can be drawn regarding reproductive performance.