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

Abstract

The aim of this study was to test the hypothesis that bulls have significant intra-individual differences in the hydraulic conductivity ( L p) and permeability coefficient for glycerol ( P s) 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 L p in the absence glycerol, and L p in the presence of glycerol ( L p gly ) and P s. Averaged over all bulls, L p 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 ( E a) of 7.39 kcal/mol. The average L p gly value at 22 °C, was 3.8 times lower than L p in the absence of glycerol ( P < 0.05). L p gly , P s, 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 L p gly and P s were 10.08 and 8.77 kcal/mol, respectively. The significant differences between individual bulls in L p and P s 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.