Tolerance of spermatozoa to hypotonic stress: role of membrane fluidity and correlation with cryosurvival.

The aim of this study was to evaluate inter-individual variability in osmotic properties of stallion spermatozoa and its correlation with cryosurvival. In addition, temperature dependency of hypo-osmotic tolerance and membrane fluidity were studied. 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. Bull spermatozoa showed greater hypo-osmotic tolerance compared with stallion spermatozoa, especially at temperatures above 30 °C, which coincided with decreased membrane fluidity of bovine spermatozoa in this temperature range. The critical osmolality at 22 °C, at which half of the sperm population survived exposure to hypotonic saline solution, was found to vary between 55 and 170 mOsm kg(-1) among different stallions. Clear correlations were found for pre- versus post-freeze sperm motility and membrane integrity. Pre-freeze percentages of membrane-intact spermatozoa after exposure to hypotonic stress showed a weak correlation with sperm motility after cryopreservation. This correlation, however, was not found when data were corrected for initial numbers of membrane-intact spermatozoa in the sample. We thus conclude that studies on pre-freeze tolerance towards hypotonic stress cannot be used to predict sperm cryosurvival rates for individual stallions.

Osmotic properties of stallion sperm subpopulations determined by simultaneous assessment of cell volume and viability.

The aim of this study was to determine the osmotic tolerance limits of stallion sperm as well as the osmotic behavior of different sperm subpopulations, including viable and non-viable cells as well as viable cells of different average sizes. A flow cytometric approach was used for simultaneous assessment of cell volume and permeability of the plasma membrane for the fluorescent dye propidium iodide while exposing the cells to media with different solute concentrations. Equine spermatozoa have limited osmotic tolerance limits: exposure to hypotonic conditions below approximately 240 mOsm kg(-1) already results in an increase in plasma membrane damaged cells, increasing up to 50% at an osmolality of 136 mOsm kg(-1). Plasma membrane damaged stallion sperm do not show an osmotic response after 10 min incubation in hypotonic conditions, and their volume is smaller as compared to viable cells. It is shown that inclusion or exclusion of different subpopulations greatly affects Boyle van 't Hoff behavior and therewith determination of the osmotic inactive volume. Osmotic inactive volumes were determined to be 76% and 46% of the isotonic volume for the whole sperm population and the plasma membrane intact viable cells, respectively. In addition, viable subpopulations with different average cell volumes also show different osmotic behavior. The main subpopulation of viable cells increased up to 1.6 times its isotonic volume upon exposure to 150 mOsm kg(-1), and exhibited an osmotic inactive volume of 79%.