Inclusão de colesterol na membrana plasmática de espermatozoides caprinos

• Autor(a) principal: Silveira, Camila Oliveira
• Data de Publicação: 2013
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/5132
• topic: Colesterol; Ciclodextrina; Cromatografia de camada delgada; Cholesterol; Ciclodextrines; Thin-layer chromatography; CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::REPRODUCAO ANIMAL

The aim of this study was to evaluate the fertilizing capacity, acrosomal integrity, and qualify and quantify by chromatographic techniques the incorporation of cholesterol to the sperm membrane by cyclodextrin in different diluents on cryopreservation of goat sperm. Four males, Saanen (2) and Parda Alpine (2) breeds were used. It was performed a completely randomized design, with each semen sample divided into the following treatments: TG - Tris-glycerol diluent; TGCCC - Cyclodextrin-cholesterol complex (CCC) + Tris-glycerol diluent; TG15CCC - CCC diluted in isosmotic solution (saline) to semen with 15 minutes of incubation before addition of the Tris-glycerol diluent; EE - egg yolk + ethylene glycol diluent; EECCC - CCC + egg yolk + ethylene glycol diluent; EE15CCC - CCC diluted in isosmotic solution (saline) to semen with 15 minutes of incubation before addition of the egg yolk + ethylene glycol diluent. Fresh semen, after its examination, was submitted to the cryopreservation process and stored in a cryogenic cylinder for 10 days. After thawing, the following analysis were performed: acrosomal integrity, sperm fertilizing capacity through perivitelline membrane of hen egg yolk binding test (MPEY) and analysis of sperm motility and vigor. Besides, techniques of gas and thin layer chromatography were conducted to evaluate the incorporation of cholesterol to the sperm membrane. Data were submitted to Lilliefors and Cochran and Bartlett tests to verify normality and homogeneity of variances, respectively. The characteristics that met the assumptions of these tests were submitted to ANOVA and means were compared by Duncan s test at 5% of probability. When data did not meet the assumptions of normality and homogeneity of variances, the means were compared by Kruskal-Wallis test. Pearson s correlation coefficient was calculated in all features.Addition of cyclodextrin-cholesterol complex did not increase binding of sperm to MPEY (P > 0.05). The EE15CCC treatment was superior to EECCC treatment in maintaining the acrosomal integrity but did not differ from control (EE; P > 0.05). In Tris-glycerol diluent, the values observed in the control treatment were higher (P < 0.05) than values of the other treatments (TGCCC and TG15CCC) in maintaining the acrosomal integrity. There was a negative correlation between the binding assay and acrosomal integrity (r = -0.25) and positive correlation between the binding assay and sperm motility (r = 0.20). The sperm motility and acrosomal integrity were negatively correlated (r = -0.26). In quantitative and qualitative evaluation of cholesterol by chromatographic techniques (gas and thin layer) there was no difference between the samples of semen in different treatments (P > 0.05). Both techniques showed no incorporation of cholesterol to spermatozoa. It was concluding that addition of cholesterol-cyclodextrin complex to the medium did not improve the physical aspects of goat semen, or the sperm binding capacity. Pre-incubation of semen with CCC for 15 minutes before addition of ethylene + egg yolk diluent (EE15CCC) did not enhance the integrity of the acrosome in relation to control (EE). The gas and thin layer chromatography showed, respectively, an efficient method for quantitatively and qualitatively determining the cholesterol present in the cryopreserved goat sperm. The concentration of 1 mg of cholesterol-cyclodextrin complex added to the goat semen was not effective for increasing the concentration of cholesterol in sperm.