Mouse spermatozoa in high concentrations of glycerol: chemical toxicity vs osmotic shock at normal and reduced oxygen concentrations.

The cryobiological preservation of mouse spermatozoa has presented difficulties in the form of poor motilities or irreproducibility. We have identified several likely underlying problems. One is that published studies have used concentrations of the cryoprotectant glycerol that are substantially lower (0.3 M) than the approximately 1 M concentrations that are optimal for most cells. Another may arise from the known high susceptibility of mouse sperm to free radical damage. We have identified two contributors to damage from higher concentrations of glycerol, namely, chemical toxicity proportional to concentration and exposure time and osmotic damage arising from too rapid an addition and removal of the glycerol. When toxicity is minimized by restricting the exposure time to 1 or 5 min and osmotic shock is minimized by adding and removing the glycerol stepwise, relatively high percentages of the sperm survive contact with 0.8 M glycerol. Free-radical damage in mouse sperm is known to be proportional to the oxygen concentration. We have determined the consequences of reducing the oxygen to <3% of atmospheric by the use of a bacterial membrane preparation, Oxyrase. Oxyrase reduced damage from centrifugation and substantially reduced damage from osmotic shock; however, it did not significantly reduce glycerol toxicity.