Cholesterol-loaded cyclodextrin pretreatment of ram spermatozoa protects structural integrity of plasma membrane during osmotic challenge and reduces their ability to undergo acrosome reaction in vitro

The effect of cholesterol-loaded cyclodextrin (CLC) pretreatment on the structural integrity of ram sperm membranes during osmotic challenges and the ability of those spermatozoa, in the presence of cholesterol, to undergo acrosome reaction in vitro were examined in two successive experiments. In experiment 1, ejaculates (n = 9) were extended with Tris-citric acid-glucose (TCG) solution to a concentration of 400 × 106 spermatozoa/mL and then divided into two equal aliquots. The first aliquot was treated with CLC (3 mg/120 × 106 spermatozoa), and the second aliquot remained as control. The sperm samples were then incubated at 35 °C for 15 min in one of the five fructose solutions, adjusted to 20, 80, 290, 500, or 1500 mOsm/L. After osmotic challenges, the percent live sperm and the sperm plasma membrane integrity were estimated in both groups by a modified hypo-osmotic swelling test associated with supravital eosin staining. CLC pretreatment significantly increased the percentage of live, intact, and live-intact spermatozoa (P < 0.05) following exposure to different osmotic challenges. In experiment 2, ejaculates (n = 8) were first centrifuged twice to remove seminal plasma and extended with TCG to concentration of 200 × 106 spermatozoa/mL. After CLC supplementation, the ability of spermatozoa to undergo acrosome reaction was evaluated in the presence of an acrosome reaction inducing substance, lysophosphatidylcholine. The percentage of sperm that underwent acrosome reaction in CLC-supplemented group was lower (P < 0.05) compared to control. In conclusion, CLC pretreatment enhanced the structural integrity of ram sperm membranes in wide range of osmotic pressures, between 20 and 1500 mOsm/L. Furthermore, CLC pretreatment reduced the ability of ram spermatozoa to undergo acrosome reaction in vitro.