The objective of the present study was to determine the concentration of cholesterol or cholestanol-loaded-cyclodextrin that needs to be added to goat sperm before cryopreservation to optimize its survival. The cholesterol or cholestanol loaded methyl-β-cyclodextrin was prepared as described by Moraes et al. (2010 Anim. Reprod. Sci. 118, 148–154). A working solution of the cholesterol or cholestanol-loaded cyclodextrin was prepared by adding 50 mg of each one to 1 mL of TALP at 37°C and mixing the solution briefly using a vortex mixer. Ejaculates (n = 24) from 5 bucks were used for this experiment. Sperm from each ejaculate were diluted 1 : 1 (vol : vol) in Tris diluent (200 mM Tris, 65 mM citric acid, and 55 mM glucose) and centrifuged at 800 × g for 10 min. The pellets were resuspended to a concentration of 120 × 106 sperm mL–1 in Tris and subdivided into 7 aliquots of 5 mL each (600 × 106 total sperm). Sperm were treated in 7 treatment groups that received no additive (0 mg; control) or different levels of cholesterol or cholestanol (0.75, 1.5, or 3.0 mg/120 × 106 sperm). All treatments were incubated for 15 min at room temperature and then cooled to 4°C over 2 h. The samples were diluted with Tris-egg yolk diluent containing 2% glycerol. The sperm were packaged into 0.5-cc straws and frozen in static liquid nitrogen vapor for 20 min and then straws were plunged into liquid nitrogen and stored until analysed for motility and thermal resistance test using a computer-assisted semen analysis system (CASA). Two straws from each treatment were thawed in a 37°C water bath for 30 s and extended in Tris. For the thermal resistance test, after thawing, 0.5 mL of semen from each treatment was placed in 1.5-mL tubes in a water bath at 37°C for 3 h. At 0, 60, 120, and 180 min, subsamples were evaluated for sperm total and progressive motility using a computer-assisted sperm motion analyzer. A total of 200 spermatozoa were counted in at least 5 different fields. Data were analysed using ANOVA and treatment means were separated, using the SNK test at 5% probability. Cholesterol (0.75 mg; 46.7%) and cholestanol (1.5 mg; 40.5%) produced an increase in progressive motility compared with other treatments after 1 h of incubation (P < 0.05). However, cholestanol (0.75 mg; 39.5 and 31%) was higher for total and progressive motility after 3 h of sperm incubation compared with the control (27 and 17.8%; P < 0.05), respectively. The addition of 0.75 mg of cholestanol in fresh sperm before cryopreservation improved the motility of freeze-thawed goat sperm compared with cholesterol. Therefore, adding cholestanol to goat sperm membranes improved cell cryosurvival.
Supported by Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
Thermal-wet model of knitted double jersey based on backpropagation algorithm of neural network
