Effects of sub-optimal glycerol concentration and cholesterol-loaded cyclodextrin in a Tris-based diluent on cryopreserved ram sperm longevity and acrosomal integrity

The maintenance of motility, viability and acrosomal integrity of ram spermatozoa for an extended period of time after cryopreservation is important for achieving high pregnancy rates, when using frozen-thawed semen. The most frequently used cryoprotectant is glycerol, which has to be used at low concentrations (under 4%), due to its potential toxicity. The primary objective of this study was to determine the combined effect of sub-optimal glycerol inclusion and cholesterol-loaded cyclodextrin (CLC) in a Tris-based diluent on ram spermatozoal longevity and acrosomal integrity during in vitro incubation, after cryopreservation. Ram semen was treated with 0 or 2 mg CLC/100 × 106 cells in Tris-based diluents containing either 6 or 3% glycerol and frozen in 0.5 ml straws. After thawing, the responses of all sperm parameters recorded were not significantly different between sub-optimal glycerol concentration (3%) and optimal glycerol concentration (6%) for untreated semen groups (0 mg CLC/100 × 106). Similarly, no significant differences were recorded between glycerol concentrations when semen samples were treated with 2 mg CLC/100 × 106 cells prior to cryopreservation. However, sperm treated with CLC prior to cryopreservation in diluents containing a sub-optimal concentration of glycerol (3%), recorded significantly higher (P < 0.05) percentages of total motile sperm, progressive motility, viability and acrosomal integrity, than for optimal glycerol concentrations (6%) for up to 4 h of in vitro incubation, at 38.5 °C after cryopreservation and thawing. Results suggest that in ram spermatozoa, a combination of 2 mg cholesterol-loaded cyclodextrins (CLC)/100 × 106 and a sub-optimal concentration of glycerol (3%) in a Tris-based diluent helps to reduce the acrosomal damage observed during cryopreservation, maintaining sperm viability and motility.