Improvement of ram sperm cryopreservation protocols assessed by sperm quality parameters and heterogeneity analysis

Semen cryopreservation processes generally decrease sperm quality and fertility rate, but this is highly dependent on the specific susceptibility of sperm cells to low temperatures. In order to study the effect of cooling, freezing, and thawing on ram spermatozoa, sperm viability (plasma membrane integrity), HOS test (hyposmotic swelling test), and individual motility were assessed in fresh, cooled, and frozen-thawed samples. The cryoprotective ability of four extenders [Triladyl-yolk (20% Triladyl, 20% egg yolk), Salamon (9.9% raffinose, 2% sodium citrate, 15% egg yolk, 5% glycerol), I.N. I.A. (6.31% Tes, 1.51% Tris, 6% egg yolk, 3% glycerol), and Fiser (F1: 3.25% Tris, 9.3% fructose, 1.7% citric acid, 25% egg yolk, 2% glycerol; F2: 0.68% sodium citrate, 0.15% TES, 0.36% glycine, 10.18% lactose, 1.18% raffinose, 0.5% fructose, 3.95% dextran (150,000-200, 000 MW); 12% of the obtained solution was replaced by the same volume of glycerol)] was tested, as well as the effect of adding various compounds (bovine lactalbumin and serum albumin, vitamin E, dithiothreitol, bull seminal plasma, glycine betaine, proline, phosphatidylcholine, and cholesterol) to Fiser's medium. Simultaneously, centrifugal counter-current distribution in an aqueous two-phase system was carried out to analyze the effect of the four extenders on sperm surface heterogeneity. The results showed that ram spermatozoa undergo a dramatic loss of heterogeneity, viability, motility, and positive response to the HOS test during freezing and thawing. These changes were partially prevented by diluting samples in Fiser's extender containing egg yolk and glycerol. A considerable increase in sperm quality with respect to that obtained in Fiser's control sample was achieved by the addition of bovine lactalbumin, vitamin E, bovine serum albumin, and bovine and ram seminal plasma.