Marta Cittoni, Patricio Razquin, J.K. Graham
Membrane cholesterol affects sperm membrane fluidity and stability at low temperatures. Sperm from species with high cholesterol:phospholipid ratios (human and rabbit) are resistant to cooling damage whereas equine sperm with low cholesterol:phospholipid ratios (~ 0.3) are sensitive to cooling and freezing damage. Cholesterol-loaded cyclodextrins (CLC) improve sperm cryosurvival; however, cholesterol-treated sperm have always been diluted with egg yolk or milk-based cryopreservation media that also protect sperm from cooling damage. Therefore, cholesterol’s effect on mitigating cooling or freezing damage remains unclear. We examined how CLC affect stallion sperm membrane cholesterol composition and whether cholesterol alone can mitigate cold shock and cryopreservation damage. Stallion sperm treated with CLC (0-1.8 mg) were subjected to cold shock (0°C) or cryopreservation, with or without egg yolk, using 1 or 2 step cryopreservation protocols; CLC increased sperm cholesterol:phospholipid ratios 2.7 fold. After cold shock, sperm motility was lower (p < 0.05) in control (8%) than CLC-treated sperm (39-65%), and CLC maintained high motility in cold shocked sperm, similar to fresh sperm. Sperm motility was similar for sperm cryopreserved in medium with egg yolk in either 1 or 2 step cryopreservation protocols. Although CLC alone provided modest cryopreservation protection, the combination of CLC and egg yolk resulted in greater motility and viability (p < 0.05) than either CLC or egg yolk alone. Furthermore, the cholesterol:phospholipid ratio that protected sperm from cold shock damage varied widely among stallions (0.33-0.79). In conclusion, adding cholesterol to sperm membrane eliminated cold shock damage but egg yolk provided additional cryoprotection, particularly at low CLC concentrations.