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 (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.

摘要 本研究旨在评估在犬精液冷冻保存稀释液中添加不同浓度还原型谷胱甘肽（reduced glutathione, GSH）（0、5、7.5、10mM）的效果，分别在甘油化处理后（冷藏样本）与解冻后（解冻样本）开展检测。本研究共纳入8只实验犬，每周采集1次精液，累计完成2次采集，获得16份精液样本。采用计算机辅助精子分析（CASA）检测精子活力，通过流式细胞术使用JC1染料检测线粒体膜电位、使用异硫氰酸荧光素-碘化丙啶（FITC-PI）染料检测细胞膜与顶体完整性；同时主观评估精子细胞膜与顶体完整性、线粒体活性及DNA完整性，并对精浆开展脂质过氧化水平检测（测定硫代巴比妥酸反应物TBARS浓度）。结果显示，添加7.5mM与10mM GSH的冷藏及解冻样本中，具备高线粒体活性（DAB-Ⅰ类）与中等线粒体活性（DAB-Ⅱ类）的精子占比更低；而10mM GSH组中呈现低线粒体活性（DAB-Ⅲ类）的精子占比更高。此外，10mM GSH组的解冻样本DNA碎片化率更高。该现象可能源于还原应激作用于线粒体，会引发活性氧水平升高，进而造成线粒体功能异常。