Effect of Different Levels of Glycerol and Cholesterol‐Loaded Cyclodextrin on Cryosurvival of Ram Spermatozoa

Abstract

This study was conducted to determine the optimum level of glycerol and cholesterol-loaded cyclodextrin (CLC) in a Tris-based diluent for cryopreservation of ram spermatozoa. Ram semen was treated with 0, 1.5, 3 or 4.5mg CLC/120×10(6) cells in Tris-based diluents containing 3, 5 or 7% glycerol in a factorial arrangement 3×4 and frozen in liquid nitrogen vapour. Sperm motility, viability (eosin-nigrosin staining) and functional membrane integrity (hypo-osmotic swelling test) were assessed immediately after thawing (0h) and subsequently after 3 and 6h at 37°C. There was an interaction between CLC and glycerol on the functional membrane integrity (p<0.05). In the presence of 3% glycerol, the highest functional membrane integrity (32.2%) was found in the spermatozoa treated with 1.5mg CLC/120×10(6) sperm. Post-thaw sperm motility was highest in 1.5mg CLC immediately after thawing (40.5%) and after 3-h (30.6%) incubation at 37°C (p<0.05). Viability of spermatozoa was higher in all CLC treatments than in the untreated samples, and it was highest (33.9%) in the spermatozoa treated with 1.5mg CLC (p<0.05). These data indicate that the addition of cholesterol to sperm membranes by 1.5mg CLC/120×10(6) cells may allow the use of a lower concentration of glycerol (3%), which is sufficient to mitigate the detrimental effects of freezing and thawing.