Water-induced hypo-osmotic test for the evaluation of canine sperm membrane integrity

The purpose of this study was to investigate the efficiency of the water test to evaluate the functional membrane integrity of ejaculated canine sperm after collecting, cooling, and freezing-thawing. Twenty-five ejaculates were obtained from 12 stud dogs by digital manipulation. Sperm-rich fractions were evaluated, diluted, cooled at 5oC and frozen-thawed. A conventional hypo-osmotic swelling test (HOST) using a fructose solution (60 mOsm/l) was compared with a HOST using distilled water (0 mOsm/l), for evaluation of the functional integrity of the plasma membrane in fresh, cooled, and frozen-thawed semen. Distilled water detected a higher percentage of reacted sperm in fresh semen (91.2 ± 0.1%) than the conventional HOST (90.6 ± 0.1%; P < 0.05). For cooled and frozen-thawed semen, the water test was as efficient as the conventional HOST (81.2 ± 0.3% and 80.3 ± 0.3%, respectively), for detecting functional membrane integrity (P > 0.05). Regardless of the test used, there was a decrease in the mean percentage of reacted sperm after equilibrium and freezing-thawing (P < 0.05). In conclusion, this study demonstrated that water can efficiently replace conventional fructose-based hypo-osmotic media for evaluation of the functional integrity of the plasma membrane of ejaculated fresh, cooled, and frozen– thawed canine sperm.