Effect of cooling rates and equilibration times on post-thaw sperm quality of Kail rams

Abstract

Context The conflicting findings regarding the impact of equilibration time on post-thawed sperm quality underscore the need for further research to evaluate the impact of equilibration time and cooling rate on post-thaw sperm quality of ram semen. Aims The current study aimed to assess the combined impact of cooling rates and pre-freezing equilibration times on post-thaw sperm quality in Kail ram semen (n = 5). Methods Semen collection was performed using an artificial vagina at 42°C. The pooled semen was divided into equal aliquots and subjected to either slow cooling (SC, −0.27°C/min) or moderate cooling (MC, −0.36°C/min) rates, transitioning from 37°C to 4°C. Equilibration times of 0, 4, 8, and 12 h were employed before freezing. Key results Semen samples undergoing the SC rate and equilibrated for 4 h exhibited higher (P < 0.05) percentages of progressive motile (PM), rapid progressive (RP), and medium progressive (MP) sperm compared with the MC rate. However, total motility remained unaffected by the cooling rate (P < 0.05). Semen equilibrated for 4 h demonstrated higher (P < 0.05) percentages of PM and RP sperm, as well as improved kinematics (curvilinear velocity, average path velocity, and straight-line velocity) compared with other equilibration times. Nevertheless, equilibration time had no (P > 0.05) impact on the amplitude of the lateral head displacement for semen samples subjected to the MC rate. Notably, the cooling rate did not affect post-thaw sperm kinematics, plasma membrane integrity, or live-sperm percentage (P > 0.05). Semen samples equilibrated for 4, 8, and 12 h exhibited a higher (P < 0.05) percentage of sperm with intact plasma membrane and viability than did those equilibrated for 0 h. Conclusions In conclusion, slow cooling rate and a 4 h equilibration period were shown to be optimal for preserving post-thaw sperm quality in Kail rams. Implications The findings highlighted the combined effect of cooling rate and equilibration time on post-thaw sperm quality for optimising sperm cryopreservation protocols in the context of ram semen.