Apoptotic-like changes in epididymal spermatozoa of soft-shelled turtles, Pelodiscus sinensis, during long-term storage at 4 ºC.

Apoptosis is a physiological phenomenon that has been recognized as a cause of sperm death during cryopreservation in endothermic mammals. There is, however, no data on its role in sperm death during cooled storage in ectothermic animals. In this study, spermatozoa from the epididymis of soft-shelled turtle were investigated to identify the mechanism of spermatozoa apoptotic-like changes during storage at 4 °C. In this study, there was survival of spermatozoa for more than 40 Days when stored at 4 °C. During cooled storage, sperm kinematics was evaluated using CASA system. Values for all sperm motility variables decreased during the period of storage; while for velocity curvilinear (VCL) there was a further decrease after 20 Days of storage. Results from flow cytometry analysis indicated that there was a significant increase in the percentage of apoptotic spermatozoa, but there was no change in the percentage of necrosis. Furthermore, the concentration of cellular ROS increased after 20 Days of storage at 4 °C. The results using JC-1 staining indicated there was a decrease in MMP of spermatozoa as the duration of storage at 4 °C increased. Nuclear fragmentation of spermatozoa was observed using TEM on Day 30 of storage. There were large amounts of pro-apoptotic cytochrome c (Cytc) and cleaved caspase-9/3 proteins detected using western blot analysis after 30 days of spermatozoa storage at 4 °C. These findings indicate ROS generation induces mitochondria damage after 20 days of storage at 4 °C, which can induce spermatozoa apoptotic-like changes during storage of soft-shelled turtle spermatozoa.