Abstract
Success rates for cryopreservation of equine sperm show a large variation among individual stallions. Factors that have been implicated with this inter-male variation in cryosurvival are the ability of sperm to tolerate osmotic stress, and temperature-induced changes in membrane phase state and organization. Cells are exposed to osmotic stress both upon the addition of cryoprotective agents as well as during freezing and thawing. Moreover membranes undergo phase changes during cooling and warming causing transport of molecules that normally are membrane-impermeable. The aim of this study was to evaluate intra-individual variability in osmotic properties of stallion sperm and its correlation with cryosurvival. Furthermore, the temperature dependency of hypo-osmotic tolerance was studied and correlated with membrane phase behavior. Pre- and post-freeze sperm motility and plasma membrane integrity were assessed using computer assisted sperm analysis and flow cytometry. The critical osmolality, at which half of the sperm population survived exposure to hypotonic saline solution, was taken as a measure for osmotic tolerance. Temperature-dependent changes in membrane conformational disorder were evaluated using Fourier transform infrared spectroscopy. Sperm membranes display a main phase transition in the 10–30 °C temperature range, with two distinct melting events. The melting event around 30 °C becomes more pronounced with removal of cholesterol. Stallion sperm membranes exhibited good resistance towards hypotonic stress in the 15–30 °C temperature range, whereas membrane stability was found to be decreased at 4 and 37 °C. The decreased tolerance towards exposure to hypotonic stress at 4 °C could be caused by the membrane phase transition which the membranes undergo during cooling. The decreased hypo-osmotic tolerance at 37 °C might be due to the fluidity of stallion sperm membranes at this temperature. The critical osmolality at 22 °C, was found to vary between 55 and 170 mOsm kg −1 among sperm from different stallions. Clear correlations were found for pre- versus post-freeze sperm motility as well as membrane integrity, assessed in either freezing extender or after dilution in isotonic medium. Pre-freeze percentages of membrane intact sperm after exposure to hypotonic stress showed a correlation with sperm motility after cryopreservation. This correlation, however, disappeared when data were corrected for initial numbers of membrane intact sperm in the sample. It is concluded that studies on pre-freeze tolerance towards hypotonic stress cannot be used to predict sperm cryosurvival rates for individual stallions. Furthermore, membrane phase behavior and the overall membrane fluidity at a given temperature are involved in osmotic stability. Source of funding: This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy). Conflict of interest: None declared. harriette.oldenhof@tiho-hannover.de
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