Avaliação in vitro de espermatozóides caprinos criopreservados em diluente à base de leite desnatado acrescido de glutationa reduzida e trolox em diferentes concentrações

Abstract

The objetive of this study was to verify the effect of different concentrations of Trolox and Glutathione (GSH) it was used sperm from five bucks, collected by artificial vagina with six replicates. After subjective analysis(wave moving, motility, vigor and sperm concentration) of each ejaculate, a pool of samples was performed and diluted (240 x 106 sperm/mL) in skimmed-milk plus glycerol 7% with antioxidants, according to the experiment: Exp. 1) G1= Control, G2= Trolox 30μM, G3= Trolox 60μM and G4=Trolox 120μM; Exp. 2) G1= Control, G2= GSH 2mM, G3= GSH 5mM and G4= GSH 7mM. Samples were packed in straws (0.25 mL), frozen and stored in Liquid Nitrogen (-196 oC). After thawing (37 oC/30 seconds), semen aliquots of each group were submitted to iPM, iAc, MMP, kinematic by CASA and ultrastructure analysis. In the Exp. 1 and 2, it was observed that iMP, iAc, PMM and kinematics parameters showed no significant difference (P>0.05) among groups. Exp. 1, in ultrastructural analysis, acrosome, plasma membrane on the tail region and axoneme integrity did not differ (P>0.05) among groups, but they were observed in lower (P<0.05) percentage than those of fresh samples. However, Trolox 60μM/mL and Trolox 120μM/mL groups had higher (P<0.05) percentage of sperms with intact plasma membrane on the head region than control and Trolox 30μM/mL groups, but they did not differ (P>0.05) of the fresh samples. Higher percentage (P<0.05) of sperms with intact mitochondrias wereobserved on fresh, control and Trolox 60μM/mL samples than those of Trolox 30μM/mL and 120μM/mL. Exp. 2, in ultrastructural analysis, it was observed that percentages of acrosome and plasma membrane (tail and head region) intacts did not differ (P>0.05)between Control and GSH (2, 5 and 7 mM/mL) groups, but they were lower (P<0.05) than in nature semen. However, semen samples of in nature and Control groups had higher percentage (P<0.05) of gametes with intact axonemes than those of GSH (2, 5 and 7 mM/mL) groups. Higher percentage (P<0.05) of sperms with intact mitochondrias were observed on Control group than those of GSH (5 and 7 mM/mL) groups, but it did not differed of in nature semen and GSH 2mM/mL. Based on ultrastructural analysis, it can be concluded that Trolox 60μM/mL can be used in skimmed-milk plus glycerol 7% diluent to preserve the integrity of sperm plasma membrane on the head region. However, the GSH (2, 5 and 7mM/mL) addition in skimmed-milk and glycerol 7% diluent to freezing goat semen did not preserve sperm integrity.