No abstract

RESUMO.-Este estudo descreveu as características seminais, da membrana plasmática e do acrossoma de espermatozoide congelado/descongelado de 19 ejaculados de garanhões da raça Nordestina. Os aspectos analisados incluíram os parâmetros físicos do sêmen fresco; a motilidade e a longevidade do sêmen diluído e descongelado; a morfologia espermática, integridade funcional e estrutural da membrana plasmática do espermatozoide e a habilidade de ligação do espermatozoide à membrana perivitelina da gema do ovo de galinha do sêmen descongelado. This paper describes the seminal characteristics of the plasma membrane of frozenthawed sperm. Nineteen ejaculates of Nordestino horse breed. The aspects analyzed in the physical parameters of fresh semen were total and progressive motility and your longevity after dilution or thawed; sperm morphology, functional and structural integrity of the plasma membrane of the sperm and the sperm-binding ability to the perivitelline membrane of the yolk (MPV) after thawed. The variables were assessed by ANOVA with post hoc test of Student Newman-Keuls test (P<0.05). The total and progressive motility were higher in diluted semen than thawed (P<0.05). The average percentage of the major, minor and total defects was lower than the limit recommended by the CBRA. The percentage of reactive to hypo-osmotic swelling test was 14.21±1.12%, the intact membrane detected by supravitally test was 62.22±9.06% and the SYBR-14 was 81.47±26.9. The ability of sperm to bind to the MPV after thawing semen was 230.39±57.09. The total and progressive motility at time 0 min of termo resistance test was higher than to 150 minutes (P<0.05), and no difference was observed in the times 10 and 30 minutes. The results demonstrate that the use of additional laboratory tests help in the process of evaluation of samples, making possible to obtain more reliable and accurate information. Although cryopreservation has caused decrease in sperm motility and was used diluents with amides to diluted and cryopreservation protocol and this minimized the osmotic damage to sperm cells and maintained the morphological, functional and structural integrity of the plasma membrane of the sperm. These results are a reference for future studies since there are no comparative data on this breed.

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.

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).