Oxidative Studies in Cryopreserved Mammalian Spermatozoa

I. Oxidative Studies in Cryopreserved Mammalian Spermatozoa.;Preservation of the male gamete has and will continue to have significant impacts on selective breeding, conservation of valuable genetic material, protection of endangered species, maintenance of biodiversity, and clinical research. Cryopreservation is still one of the most effective cell preservation methods and involves storage of sperm at extremely low temperatures. Successful cryopreservation requires optimal interactions between multiple factors: cooling/freezing rates, sperm membrane composition, freezing medium, cryoprotectant type and concentration, cellular oxidative balance, osmotic balance, and sperm cell handling technique. The varying morphology of mammalian sperm from different species, as well as varying species requirements in the aforementioned factors limit progress in standardization of cryopreservation techniques for all species. Therefore a deeper understanding of sperm cryobiology is a particularly integral part of successful assisted reproduction for the majority of animal species, whose gametes have not been genetically selected for cryo-survivability.;II. Cooling rate, rather than freezing rate, determines post thaw quality of rhesus macaque spermatozoa.;Spermatozoa become most sensitive to cold shock when cooled from 37°C to 5°C at rates that are too fast or too slow. Cold shock increases membrane permeability and loss of important intracellular components, and in combination with exposure to atmospheric oxygen, increases susceptibility to oxidative damage due to reactive oxygen species (ROS) production (Bucak et al., 2008). 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 change cellular function through the disruption of the sperm plasma membrane and through damage to proteins and DNA. 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. The objective of this study was to determine which cryopreservation rates result in the lowest degree of oxidative damage and greatest sperm quality. Nine semen samples were collected from three electro--ejaculated male rhesus macaques, washed, and resuspended in TEST--yolk cryopreservation buffer to 100 x 106 sperm/mL. Sperm were frozen in 0.5 mL straws at four different combinations of suprazero and subzero rates. Three different suprazero rates were used between 22°C and 0°C: 0.5°C/min (slow), 45°C/min (medium), and 93°C/min (fast). These suprazero rates were used in combination with two different subzero rates for temperatures 0°C to -110°C: 42°C/min (medium) and 87°C/min (fast). The different freezing groups were as follows: slow--medium (SM), slow--fast (SF), medium--medium (MM), and fast--fast (FF). Flow cytometry was used to detect cell viability and lipid peroxidation. Sperm motility was evaluated using a computer assisted sperm analyzer. 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 and viability, and increased lipid peroxidation compared to fresh semen (P < 0.001). Slow cooling from room temperature to 0°C promotes better viability and motility post thaw, compared with medium or fast cooling rates. 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.