70. Changes in rat sperm function after cooling, cryopreservation and centrifugation processes

Abstract

Sperm cryopreservation is an effective method of archiving valuable strains for biomedical research and handling of rat sperm is very important for successful cryopreservation. The aim of this study was to evaluate changes in rat sperm function during cryopreservation and centrifugation. Epididymal rat sperm were subjected to cooling and freezing-thawing processes and then motility, plasma membrane integrity, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were compared before and after minimum centrifugation force (200 g ). Cryopreservation decreased sperm motility, plasma membrane integrity, and MMP ( P < 0.05). Basal and stimulated ROS were increased in viable cooled sperm ( P < 0.01). ROS of all sperm were increased by TBHP and viabilities of fresh and cooled sperm were decreased by TBHP ( P < 0.01), with equal susceptibility to TBHP among them. Centrifugation decreased motility and plasma membrane integrity of frozen-thawed sperm ( P < 0.05). Centrifugation decreased basal ROS of all sperm ( P < 0.01), while it led to higher susceptibility to TBHP in viable cooled sperm, showing higher increased fold in ROS and decreased rate in viability by TBHP in viable cooled sperm ( P < 0.05). Cooling process was the major step of ROS generation, with loss in sperm motility, plasma membrane integrity, and MMP. Centrifugation affected function of cryopreserved sperm. These data suggest that centrifugation makes rat sperm susceptible to external ROS source, in particular during cooling process. Thus, protection from ROS damage and minimizing centrifugation should be considered during cryopreservation and post-thaw use of cryopreserved epididymal rat sperm.