Supplementation of the Thawing Media With Reduced Glutathione Improves Function and the In Vitro Fertilizing Ability of Boar Spermatozoa After Cryopreservation

Abstract

ABSTRACT:In this study, we evaluated the effects of glutathione (Lg-glutamyl-L-cysteinylglycine; GSH) supplementation of the thawing extender on semen parameters to compensate for the decrease in GSH content observed during sperm freezing. To fully address these questions, we used a set of functional sperm tests. These included tests of motility and motion parameters, changes in sulfhydryl group content in membrane proteins, capacitation status, measures of intracellular reactive oxygen species generation, sperm chromatin condensation, and in vitro penetration of immature oocytes. The main findings emerging from this study were that addition of GSH to the thawing media resulted in a lower number of capacitated viable spermatozoa, a decrease in the number of spermatozoa with changes in the sulfhydryl groups in membrane proteins, a reduction of the reactive oxygen species generation, a lower chromatin condensation, and a higher penetration ability of oocytes in vitro and a higher proportion of decondensated sperm heads. GSH appears to play an important role in sperm antioxidant defense strategy. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen boar spermatozoa.

Effect of genistein supplementation of thawing medium on characteristics of frozen human spermatozoa

“…It could be related to the pro-oxidative and anti-oxidative capacity that the antioxidant substances present at a different concentration, thereby producing paradoxical effects. In our previous experiments with respect to antioxidant addition of reduced glutathione (GSH) to freezing and thawing media [29][30][31]48], we detected more significant activities when the antioxidant was added in the thawing, than in the freezing media. A plausible explanation for this question would be that cryodamage of antioxidant enzymes during the freezing process results in an overall decrease of enzy matic antioxidant defences in the sperm.…”

“…Previous studies in human and domestic animal spermatozoa have shown that freezing and thawing induce important changes in the sperm chromatin, resulting in greater compactness [30,31,42,43]. Thus, chromatin condensation and stability may be critical factors to consider when using frozen semen [44].…”

“…Production of ROS was measured by incubating the spermatozoa (1 × 10 6 sperms mL −1 ) in PBS in the presence of 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Invitrogen) (0.5 µmol L −1 ) for 60 min at 37ºC [30]. This dye is a fluorogenic probe commonly used to detect cellular ROS production.…”

“…Sperm chromatin was stained with propidium iodide (PI) for the determination of sperm chromatin condensation [30]. Thawed samples, after 60 min of incubation in the experimental media, were centrifuged (1 200 × g, 3 min) and the pellet was resuspended in a solution of ethanol and PBS (70/30, v/v) for sperm membrane permeabilization.…”

Efeito da adição de trolox e pentoxifilina na motilidade, integridade do acrossoma e do DNA de espermatozoides equinos após descongelação

“…Por isso, Gadea et al (2005), visando aumentar a viabilidade e a subsequente capacidade fertilizante de espermatozoides suínos, sugeriram a adição de antioxidante ao meio diluidor após a descongelação, em virtude de reduzir a produção de ROS e aumentar a habilidade de penetração no oócito.…”