67 DELIVERING CHOLESTANOL OR DESMOSTEROL TO BULL SPERM MEMBRANES IMPROVES CRYOSURVIVAL

Abstract

Altering the lipid composition of sperm plasma membranes affects sperm cryosurvival. Cryopreservation induces many stresses on the spermatozoa, including destabilization of the plasma membrane, which results in the loss of sperm motility and function. Treating bull spermatozoa with cholesterolloaded cyclodextrin (CLC) prior to cryopreservation increases sperm cryosurvival rates. This study compared the effect of adding other sterols, which should incorporate into the membrane and increase membrane fluidity at low temperatures, thereby increasing cryosurvival. Ejaculates from four bulls were divided into two experiments (E). In E1, ejaculates were extended with Tris, and then subdivided into four treatments: No additive (control), 1.5 mg CLC/120 million sperm (positive control), and 1.5 mg/120 million sperm in cyclodextrin pre-loaded with either cholestanol or desmosterol. Spermatozoa were incubated for 15 min at 22°C after which both the ability of fresh spermatozoa to bind to the zona pellucida (ZP) and chicken egg perivitelline membrane (EPM) and their osmotic tolerance were evaluated. In E2, sperm were diluted to 120 million cells mL^–1 in a Tris diluent and treated as described for E1. Then, samples were diluted 1:1 (v:v) in Tris with 20% Egg Yolk (EY) and cooled to 5°C. After dilution 1:1 (v:v) with Tris containing 10% EY and 16% glycerol, samples were allowed to equilibrate for 15 min, and then were packaged into 0.5-mL straws, frozen in static liquid nitrogen vapor for 20 min, and plunged into liquid nitrogen for storage. Straws were thawed and the motility and zona-binding ability were determined using a Hamilton Thorne Motility Analyzer (Hamilton Thorne Biosciences, Beverly, MA, USA) and epifluorescence microscopy, respectively. Treatment differences for sperm motility, osmotic tolerance, and zona binding were determined using analysis of variance. Treating spermatozoa with CLC resulted in more fresh bull spermatozoa binding to the EPM and ZP compared to cholestanolor desmosterol-loaded cyclodextrin-treated spermatozoa or control cells (P < 0.05). No differences were observed between EPM and ZP binding (P > 0.05). The percentages of total and progressively motile spermatozoa were higher for fresh samples treated with cholesterol-, cholestanol-, or desmosterol-loaded cyclodextrin than for control cells (P < 0.05) when spermatozoa were exposed to anismotic conditions, and then returned to isosmolality. After cryopreservation, the percentages of motile spermatozoa and number of spermatozoa binding to ZP were similar for spermatozoa treated with CLC (56% and 115 sperm/ZP) and cholestanol (53% and 108 sperm/ZP) compared to spermatozoa treated with desmosterol (42% and 86 sperm/ZP; P < 0.05). All treatments provided higher motility and binding efficiency than control spermatozoa (32% and 62 sperm/ZP; P < 0.05). Therefore, adding cholesterol or cholestanol to bull sperm membranes improved cell cryosurvival. Studies to determine if cholestanol affects sperm capacitation need to be conducted.