The use of reduced glutathione (GSH) as antioxidant for cryopreserved sperm in dogs

Abstract

The aim of this study was to evaluate the effect of supplementation with different concentrations of reduced glutathione GSH (0; 5; 7.5; 10mM) in the extender for cryopreservation in dogs with evaluations performed after glycerolization (chilled) and thawing (thawed). For this purpose, we used 8 dogs and two semen collections were performed in a weekly interval, totaling 16 semen samples. The sperm were analyzed by automatic sperm motility (CASA) and flow cytometry analysis of mitochondrial potential (JC1 dye) and membrane/acrosome integrity (FITC-PI dyes). We evaluated subjectively the membrane and acrosome integrity, mitochondrial activity and DNA integrity. Seminal plasma was evaluated for lipid peroxidation (TBARS concentration). Chilled and thawed samples supplemented with 7.5 and 10mM of GSH had lower percentage of sperm with high (DAB -Class I) and medium (DAB -Class II) mitochondrial activity. And 10mM of GSH had higher percentage of low mitochondrial activity (DABClass III). Moreover, thawed samples of 10mM of GSH had high DNA fragmentation rates. Probably by a reductive stress effect on mitochondria which lead to an increase in reactive oxygen species, and a mitochondrial malfunction.Keywords: reactive oxygen species, cryopreservation, antioxidant, canine RESUMOO objetivo deste estudo foi avaliar o efeito da suplementação com diferentes concentrações de glutationa reduzida 5; 7,5; 10mM)

Intermediate-Term Storage of Spotted Halibut (Verasper variegatus) Sperm: Effects of Storage Methods, Extenders Supplemented with Antibiotics and Antioxidants on Sperm Quality

“…The application of 2 mM RG enhanced boar sperm cryopreservation [63], and 2 and 5 mM RG promoted cryopreservation of ram spermatozoa [64]. RG at 10 mM prevented DNA damage in cryopreserved dog sperm [65].…”

Effects of Inositol Supplementation in Sperm Extender on the Quality of Cryopreserved Mesopotamian Catfish (Silurus triostegus, H. 1843) Sperm

“…Recently, clear evidence has been presented that this potent antioxidant specifically acts by enhancing OXPHOS activity both at baseline and, to a greater extent, at capacitation in vitro [ 54 ]. In a study applying different concentrations of GSH (5, 7.5 and 10 mM), it was possible to observe high mitochondrial activity in refrigerated or thawed semen samples at a dose of 5 mM GSH [ 73 ]. The reducing stress effect of high doses of inositol on mitochondria causes an increase in reactive oxygen species and mitochondrial dysfunction, resulting in dysfunctions in ATP synthesis [ 74 ].…”

“…The reducing stress effect of high doses of inositol on mitochondria causes an increase in reactive oxygen species and mitochondrial dysfunction, resulting in dysfunctions in ATP synthesis [ 74 ]. Thus, it can be stated that high doses of inositol do not cause a stress effect [ 73 ]. Low-intensity oxidative stress can be helpful to cells, whereas high levels can bring about degradation of nucleic acids, proteins, lipids and carbohydrates, ultimately leading to cell death [ 38 , 43 , 44 ].…”