Hyper- and hypoosmotic water permeability of bovine sperm as a function of age, breed, and ejaculate number: Implications for cryopreservation

The tolerance to cryoprotectant equilibration and the necessary cellular dehydration during equilibrium or quasi-equilibrium cooling that facilitates avoidance of intracellular ice is fundamentally connected to the ability of cells to transport water across the plasma membrane. Because of this, the relationship between the rate of water transport and post-thaw motility in bovine sperm has long been hypothesized, but has not been definitively established. Previous studies suggested that variability in bull sperm water permeability might explain post-thaw motility. Here we used a stopped-flow technique in which sperm were abruptly exposed to hypoosmotic and hyperosmotic media (220 mOsm/kg and 445 mOsm/kg final osmolalities, respectively) to critically evaluate this hypothesis and determine whether there is a relationship between the water permeability of fresh semen and the post-thaw motility rates in bull semen from various ejaculates, breeds, and age groups. We also evaluated additional possible predictive metrics of kinetic volume responses to anisosmotic medium, including the time to achieve 10%, 50% and 90% equilibrium volume (T10, T50, T90) in both hypoosmotic and hyperosmotic media. The total motility in the first of two ejaculates (Ejaculate 1) collected from each bull within a 10–15-minute interval was approximately 6.3% higher, and its progressive motility was about 8.1% higher than that of Ejaculate 2. Holstein bulls showed significantly higher total motility than Jersey bulls. No significant differences in either total or progressive motility of post-thaw bull semen were detected across the age groups of 12–24 months, 25–38 months, 40–57 months, and 69–116 months. Direct association analyses identified only weak relationships between water permeability and post-thaw motility, and none remained statistically significant after adjustment for multiple comparisons. The available sample size provided 80% power to detect absolute correlations of approximately 0.30 or greater. However, significant volume rectification was observed, with water permeability under hyperosmotic conditions being approximately 2% to 42% higher than under hypoosmotic conditions, depending on ejaculate number, breed, and age group. Principal component analysis indicated that L p under hypoosmotic conditions, followed by L p under hyperosmotic conditions, contributes most strongly to the separation of samples. Overall, the present paper demonstrates that both log-logistic and linear models provide reliable estimators of L p . Although L p was not a robust predictor of post-thaw motility under the freezing conditions examined, these modeling approaches provide a quantitative framework for future studies investigating sperm osmotic behavior during cryopreservation.