77 ADDITION OF CHOLESTEROL IN FRESH GOAT SPERM IMPROVES CRYOSURVIVAL RATES

Abstract

Cryopreservation causes irreversible damage to goat sperm membranes, measured by a loss of motile and functional normal cells, compared with fresh sperm. The objective of this study was to determine if the addition of cholesterol-loaded cyclodextrin (CLC) to goat semen improved sperm cryosurvival. The CLC was prepared as described by Purdy and Graham (2004 Cryobiology 48, 36–45) with some modifications: 200 mg of cholesterol were dissolved in 1 mL of chloroform and 1 g of methyl-beta-cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of the cholesterol solution was added to the cyclodextrin solution, after which the mixture was poured into a glass Petri dish and the solvents allowed to evaporate on a warm plate for 24 h. The resulting crystals were removed from the dish and stored at 22°C. A working solution of the CLC was prepared by adding 50 mg of CLC to 1 mL TALP at 37°C. Thirty ejaculates from 5 bucks were collected, diluted 1 : 1 in Tris diluent, divided into 7 equal aliquots, and centrifuged at 800g for 10 min. The sperm pellets were resuspended in Tris diluent, to which 0, 0.75, 1.5, 3.0, 4.5, 6.0, or 7.5 mg of CLC/120 million sperm were added. All treatments were incubated for 15 min at room temperature and then cooled to 4°C over 2 h. The samples were then diluted with Tris-egg-yolk diluent containing 2% glycerol, and the sperm were packaged into 0.5-mL straws, frozen in static liquid-nitrogen vapour for 20 min, and plunged into liquid nitrogen. Straws were thawed in 37°C water for 30 s, extended in Tris, and analyzed using optic microscopy. To test thermal resistance, after thawing, 0.5 mL of semen from each treatment were placed in 1.5-mL Eppendorf tubes in a water bath at 37°C for 3 h. At 0, 60, 120, and 180 min, subsamples were evaluated for sperm progressive motility. A hyposmotic test was also conducted by adding 10 µL of sperm to 2 mL of each solution and incubating them for 1 h/37°C. Sequentially, 20 µL of sperm was diluted in hypoosmotic solution (150 mOsm), and the samples were evaluated using phase-contrast microscopy. A total of 100 spermatozoa were counted in at least 5 different fields, and sperm tails were classified as either noncoiled or coiled. Data were analyzed using ANOVA, and treatment means were separated, using the SNK test at 5% probability. The sperm motility (50.4, 33.8, and 22.5%) was significantly higher for sperm treated with 0.75 mg of cholesterol after 0, 60, and 120 min of incubation after thawing, when compared with other treatments. No treatment differences in the hypoosmotic swelling test were observed. The addition of 0.75 mg of cholesterol to fresh goat semen improved sperm motility after cryopreservation for up to 3 h.

Supported by FACEPE and CAPES.