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

D.S.R Angrimani,M Nichi,B.R Rui,N.M.G Vieira,D.S.G Cruz,N Queiroz-Hazarbassanov,C.O Massoco,G.K.V Kawai,J.D.A Losano,C.I Vannucchi,M.M Brito,M.C.P Francischini

doi:10.1590/1678-4162-9684

D.S.R Angrimani, M Nichi + Show 10 more

Open Access

https://doi.org/10.1590/1678-4162-9684

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Abstract

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 (DAB - Class 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.

Highlights

The advances of assisted reproduction techniques in dogs are extremely offset when compared to farm animals (Goodrowe et al, 2000; Luvoni et al, 2005)
Recebido em 11 de janeiro de 2017 Aceito em 8 de junho de 2017 *Autor para correspondência E-mail: cacavann@usp.br. These efforts occur because the semen cryopreservation allows the preservation and propagation of genetic material, through long distances, even post mortem (Thomassen and Farstad, 2009)
No statistical difference was observed between the different concentrations of GSH in refrigerated semen for all Computer Assisted Sperm Analysis (CASA) variables (Table 1), lipid peroxidation, DNA integrity (Table 2) and all flow cytometry variables (Table 3)

Summary

Introduction

The advances of assisted reproduction techniques in dogs are extremely offset when compared to farm animals (Goodrowe et al, 2000; Luvoni et al, 2005). Some reproductive biotechnologies are being highly studied, such as the artificial insemination and semen cryopreservation in canines (Lucio et al, 2016b). These efforts occur because the semen cryopreservation allows the preservation and propagation of genetic material, through long distances, even post mortem (Thomassen and Farstad, 2009). The process of seminal cryopreservation promotes loss of sperm motility and decrease of survival post-thaw (Belala et al, 2016). Such effects occur mainly through the reactive oxygen species (ROS) generated by the spermatozoa (Goes et al, 2011). OS promotes injury in proteins, carbohydrates, plasma membrane, sperm motility, and in spermatic DNA integrity (Birben et al, 2012)

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