Reduced glutathione in the freezing extender improves the in vitro fertility of ram epididymal spermatozoa

Abstract

Post-mortem recovery and freezing of epididymal spermatozoa is an option (in some instances the only option available) for preserving genetic material in wild species at risk of extinction and in farm animals when a genetically valuable male dies accidentally. Evidences exist that oxidative stress may involve in the cell death and reduced functionality of spermatozoa following cryopreservation. This study was aimed to improve the freezability and post-thaw fertility of ram epididymal spermatozoa by the addition of antioxidants into the freezing extender. Ram epididymal spermatozoa were frozen either at the presence of l-glutamine (2.5 or 5 mM), sericin (0.25 or 0.5%), reduced glutathione (GSH, 2.5 or 5 mM), or in freezing medium without supplement (Control). Immediately after thawing, the motility, functional membrane integrity and DNA damage of the thawed spermatozoa were not significantly different between groups. After 3 h post-thaw incubation at 39 °C, however, the total motility, progressive motility, and some kinematic parameters of GSH5 group were higher than other groups (P < 0.05). Oocytes inseminated with frozen-thawed spermatozoa of GSH5 and Control group had lower cleavage and blastocyst (blastocyst/oocyte ratio) rates compared to those inseminated with fresh epididymal sperm (P < 0.05). However, the blastocyst rate (both blastocyst/oocyte and blastocyst/cleavage) in GSH5 group was higher than control (P < 0.05). In conclusion, the addition of GSH to the freezing extender could improve the in vitro fertility of frozen/thawed ram epididymal spermatozoa.