Suprazero Cooling Rate Rather Than Freezing Rate Determines Post Thaw Quality Of Rhesus Macaque Sperm

摘要

Sperm become most sensitive to cold shock when cooled from 37ºC to 5ºC at rates that are too fast or too slow; cold shock increases the susceptibility to oxidative damage due to its influence on reactive oxygen species (ROS) production ([]. ROS are significant stress factors that are generated during cooling and low temperature storage, and may be a main cause of decreased motility and fertility upon warming. ROS have been shown to change cellular function through the disruption of the sperm plasma membrane and through damage to proteins and DNA. The objective of this study was to determine which cryopreservation rates result in the lowest degree of oxidative damage and greatest sperm quality. In the rhesus model it has not been determined whether suprazero cooling or subzero freezing rates causes a significant amount of ROS damage to sperm. Semen samples were collected from male rhesus macaques, washed, and resuspended in TEST-yolk cryopreservation buffer to 100 x 10⁶ sperm/mL. Sperm were frozen in 0.5mL straws at four different combinations of suprazero and subzero rates. Three different suprazero rates were used between 22ºC and 0ºC: 0.5ºC/min (Slow), 45ºC/min (Medium), and 93ºC/min (Fast). These suprazero rates were used in combination with two different subzero rates for temperatures 0ºC to −110ºC: 42ºC/min (Medium) and 87ºC/min (Fast). The different freezing groups were as follows: Slow-Med (SM), Slow-Fast (SF), Med-Med (MM), and Fast-Fast (FF). Flow cytometry was used to detect lipid peroxidation (LPO), a result of ROS generation. Motility was evaluated using a computer assisted sperm motion analyzer. The MM and FF treated sperm had less viable (P < 0.0001) and motile sperm (P < 0.001) than the SM, SF, or fresh sperm. Sperm exposed to MM and FF treatments demonstrated significantly higher oxidative damage than SM, SF, or fresh sperm (P < 0.05). The SM and SF treated sperm showed decreased motility, membrane integrity, and LPO compared to fresh semen (P<0.001). Slow cooling from room temperature promotes higher membrane integrity and motility post thaw, compared to medium or fast cooling rates. Cells exposed to similar cooling rates with differing freezing rates were not different in motility and membrane integrity, whereas comparison of cells exposed to differing cooling rates with similar freezing rates indicated significant differences in motility, membrane integrity, and LPO. These data suggest that sperm quality appears to be more sensitive to the cooling, rather than freezing rate and highlight the role of the suprazero cooling rate in post thaw sperm quality.