Effect of different protein supplements on motility and plasma membrane integrity of frozen-thawed stallion spermatozoa.

Three experiments were conducted to evaluate the effect of different macromolecule components (egg yolk, skim milk, and BSA) in a widely employed extender for cryopreservation of horse semen. Spermatozoal motility (MOT) and the percentage of spermatozoa with an intact plasma membrane (IPM) were evaluated in frozen-thawed samples. In the first experiment (four Draft Horse stallions, four ejaculates each) a standard freezing extender containing 20% whole egg yolk was modified by replacing extender components (glucose-EDTA solution, 11% lactose solution) with an increasing volume of a skim milk diluent (0, 25, 50, and 75%, v/v). The best results were obtained with an extender containing 75% (v/v) skim milk solution (MOT 24.7%; IPM 46.6%). This extender was further tested in Experiment 2 (three Draft Horse stallions, four ejaculates each) by varying the amount of egg yolk (10 or 20%) and by addition of 5% BSA. Neither the amount of egg yolk nor the addition of BSA significantly influenced postthaw results. Selected extenders from the previous experiments were tested in Experiment 3 (three stallions, four ejaculates each). Again, a mixture of 20% whole egg yolk and a skim milk solution provided the best results (MOT 41.7%; IPM 54.2%). The results of this study indicate that the inclusion of different sources of macromolecules (e.g., egg yolk and skim milk) can improve motility and plasma membrane integrity of frozen-thawed horse spermatozoa.