Effect of slow-cooling, cooled storage, and centrifugation prior to cryopreservation on post-thaw spermatological parameters in stallions classified as good or poor freezers

The use of cryopreserved semen has become a standard technique in equine reproduction and is significantly affected by the stallion's individual aptitude for the technique. The aim of the study was to compare the effect of different semen handling protocols before cryopreservation on post-thaw semen quality of stallions (n = 9) either classified as good (n = 5) or poor freezers (n = 4). Ejaculates (n = 6) were collected, splitted and subjected to five different protocols: Group 1 - ejaculates frozen immediately after collection and centrifugation at room temperature (25 degrees C); Group 2 - ejaculates frozen after cooling at 5 degrees C for 6 hours with centrifugation before storage; Group 3 - ejaculates frozen after cooling at 5 degrees C for 6 hours (slow cooling: 0.1 degrees C/min) without centrifugation before storage; Group 4 - ejaculates frozen after cooling at 5 degrees C for 24 hours with centrifugation before storage; Group 5 - ejaculates frozen after cooling at 5 degrees C for 24 hours (slow cooling: 0.1 degrees C/min) without centrifugation before storage. Spermatological examination (directly before freezing (T1) and after thawing (T2) included assessment of motility, membrane integrity, acrosomal status and DNA fragmentation index (DFI). Differences between good and bad freezers were observed in all groups. While good freezers had the highest post-thaw progressive motility (T2PM) in Group 1 and 2, poor freezers showed the best results for T2PM in Group 3. Cooled storage for 24 hours resulted in lower T2PM for all groups. Post-thaw DFI and membrane integrity were not affected by treatment group but freezability of the individual stallion. More acrosomal defects were observed in poor freezers as compared to good freezers and after cooled storage for 24 h in poor freezers in comparison to cooled storage for 6 hours. In conclusion, good freezers showed the best post-thaw semen quality after immediate centrifugation and cryopreservation or after centrifugation, followed by cooled storage for 6 hours. Post-thaw semen quality of poor freezers benefitted from slow cooling and cooled storage (6 hours) without previous centrifugation