Delivering cholesterol or cholestanol to bull sperm membranes improves cryosurvival

This study compared the effect of adding other cholesterol conjugates, which should incorporate into and increase sperm membrane fluidity at low temperatures thereby increasing cryosurvival. Ejaculates from each of four bulls were diluted to 120 million cells/ml in a Tris diluent and used in two experiments. Each experiment contained four treatments: No additive (control); 1.5 mg CLC/120 million sperm (positive control); and 1.5 mg cyclodextrin pre-loaded with cholestanol or desmosterol/120 million sperm. In the first experiment, fresh sperm were treated with cyclodextrins that were pre-loaded with cholesteryl conjugates and incubated for 15 min at 22 °C to allow incorporation of the conjugates. The percentages of motile sperm after exposure to solutions ranging from 0 to 1200 mOsm were used to evaluate the osmotic tolerance of the cells. The ability of treated sperm to bind to the bovine zona pellucida (ZP) and chicken egg perivitelline membrane (CEPM) was evaluated to determine if altering the sperm membrane affected the ability of sperm to bind to oocytes. In the final experiment, the cryosurvival rates of control and treated sperm were determined. Control and treated sperm were cryopreserved in a Tris diluent containing 10% egg yolk (EY) and 8% glycerol (final concentrations). Samples were thawed to determine the motility and ability of sperm to bind to the ZP and to CEPM using a CASA and epifluorescence microscope, respectively. Fresh sperm treated with CLC resulted in more binding to the ZP compared to all other treatments (P < 0.05). No differences were observed between ZP and CEPM binding (P > 0.05). The percentages of motile sperm were greater for fresh samples treated with cholesterol, cholestanol or desmosterol loaded cyclodextrin than control cells (P < 0.05) when the sperm were exposed to anisosmotic conditions, and then returned to isosmolality. After cryopreservation the percentages of motile sperm and number of sperm binding to each CEPM were similar for sperm treated with CLC and cholestanol compared to sperm treated with desmosterol (P > 0.05). All treatments provided greater motility and binding efficiency than control sperm (P < 0.05). Therefore, adding cholesterol or cholestanol to bull sperm membranes improved cell cryosurvival.