Significant variability among bulls in the sperm membrane permeability for water and glycerol: Possible implications for semen freezing protocols for individual males

The aim of this study was to test the hypothesis that bulls have significant intra-individual differences in the hydraulic conductivity (Lp) and permeability coefficient for glycerol (Ps) of the sperm cell membrane. The permeability parameters were determined at 22, 10, and 0 °C of sperm from 7 Holstein Frisian artificial insemination (AI) bulls, using four ejaculates per bull. A stopped-flow approach was applied to provide temporal resolution sufficient to measure rapid cell volume changes under anisosmotic conditions in the absence or presence of glycerol. This technique utilizes a concentration-dependent self-quenching entrapped fluorophore. The resulting cell volume changes were used in three-parameter fitting calculations to compute Lp in the absence glycerol, and Lp in the presence of glycerol (Lpgly) and Ps. Averaged over all bulls, Lp in the absence of glycerol was 0.28 ± 0.01, 0.15 ± 0.01 and 0.10 ± 0.01 μm min−1 atm−1 (mean ± SD) at 22, 10 and 0 °C, respectively, yielding an Arrhenius activation energy (Ea) of 7.39 kcal/mol. The average Lpgly value at 22 °C, was 3.8 times lower than Lp in the absence of glycerol (P < 0.05). Lpgly, Ps, and the reflection coefficient (σ) at 22 °C were 0.073 ± 0.015 μm min−1 atm−1, 0.80 ± 0.33 × 10−3 cm min−1, and 0.92 ± 0.10 (mean ± SD), respectively. Subsequent experiments were performed at 10 and 0 °C. Activation energies for Lpgly and Ps were 10.08 and 8.77 kcal/mol, respectively. The significant differences between individual bulls in Lp and Ps indicate that individual males may require individual adjustments of the cooling protocol. Application of these data in a theoretical model to simulate the osmotic events during freezing resulted in predicted optimal cooling rates in the range of published empirical values.