Cholestanol-loaded-cyclodextrin improves the quality of stallion spermatozoa after cryopreservation

• We tested adding cholestanol and cholesterol to stallion sperm to improve sperm cryopreservation.
• Different levels of cholestanol were compared to the optimal concentration of cholesterol.
• Cryosurvival rates for cholestanol-treated sperm were similar to cholesterol-treated sperm.
• Cryosurvival was optimal when used 0.75 mg of cholestanol-loaded cyclodextrin in 120 million sperm.

This study was to compare the effect of adding cholesterol or cholestanol loaded cyclodextrins in stallion sperm prior to cryopreservation to optimize sperm cryosurvival. Ejaculates from each of eight stallions were diluted to 120 million cells in a S-MEDIUM diluent. The diluted sperm were sub-divided into three treatments: no additive (control); 0.75 mg of cyclodextrin pre-loaded with cholesterol (CLC)/120 million sperm (positive control); 1.5 mg CLC/120 million sperm; 0.75 mg of cyclodextrin pre-loaded with cholestanol (CnLC)/120 million sperm; and 1.5 mg CnLC/120 million sperm. To set the experiments, the treated sperm were incubated for 15 min at 22 °C to allow for the incorporation of cholesterol or cholestanol. In each experiment, treated sperm incubated for 15 min at 22 °C to allow for incorporation of cholesterol or cholestanol. The samples were then diluted 1:5 (v/v) with Lactose-Egg Yolk diluent and cooled to 5 °C over a 2 h period. Loaded into 0.25 ml polyvinylchloride straws, frozen in liquid nitrogen vapor for 10 min, and then plunged into liquid nitrogen until further use. Higher percentages of motile sperm and viable cells were achieved after thawing for stallion sperm treated with CLC and CnLC compared to control (P < 0.05). Addition of CnLC also resulted in more number sperm binding to chicken egg perivitelline membrane (CEPM) after cryopreservation than cholestanol and control sperm (P < 0.05). In conclusion, CnLC and CLC improved the percentage of post-thaw motility of equine sperm and CnLC provided greater binding efficiency.