Quality improvement of post-thawed stallion epididymal spermatozoa with single layer centrifugation

BACKGROUND: Epididymal sperm cryopreservation represents the ultimate option to preserve spermatozoa of valuable stallions. OBJECTIVE: The study aims to evalute whether single layer centrifugation (SLC) prior to cryopreservation or after post-thawing improves the quality of stallion epididymal sperm. MATERIALS AND METHODS: Epididymal sperms of stallions were harvested (N=20). Sperm samples were subjected to treatments: conventional centrifugation, SLC prior to cryopreservation (SLC-PC) or SLC post-thaw (SLC+). All samples were cryopreserved, thawed and evaluated. SLC+ were thawed, single layer centrifuged and resuspended in freezing extender (SLC+F) or cooling extender (SLC+C). Total motility, progressive motility, morphology, mitochondrial functionality, membrane integrity and DNA integrity were evaluated. RESULTS: SLC-PC and SLC+F yielded higher total motility, while SLC+F yielded the highest progressive motility. Mitochondrial functionality was significantly higher in all SLC groups. Membrane integrity was higher in SLC-PC. The percentage of morphologically normal spermatozoa was higher in SLC-PC and SLC+F. CONCLUSION: SLC prior to cryopreservation or post-thaw improves the quality of stallion epididymal spermatozoa. When SLC is performed post-thaw, freezing extender is the best medium to resuspend the pelleted semen.