Original Research ArticleGlutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa
Introduction
The freeze–thawing of mammalian semen is partially associated with the oxidative stress that causes overproduction of reactive oxygen species (ROS) resulting in lipid peroxidation (LPO) of the plasma membrane [1]. Buffalo sperm has a higher plasma membrane content of polyunsaturated fatty acids compared to cattle bull sperm [2], [3]. This makes the buffalo sperm more susceptible to oxidative stress during cryopreservation [4]. Consequently, there is a reduction in the motility as well as in the integrity of the plasma membrane, acrosome and chromatin of buffalo spermatozoa [5], [6], [7]. Likewise, there is also a decrease in the fertility of buffalo spermatozoa compared to cattle bull spermatozoa [8]. Buffalo semen is equipped with an anti oxidative-stress system consisting of enzymatic (superoxide dismutase, glutathione peroxidase, glutathione reductase, catalase) and non-enzymatic (vitamin C, vitamin E, glutathione) antioxidants [8], [9]. However, the endogenous defense system of buffalo semen is believed to be insufficient against the oxidative stress during the process of freezing and thawing. This weakness is mainly due to low concentrations of naturally occurring antioxidants in buffalo semen, which further decreases during the extension and freezing process [6], [8], [10].
Glutathione is a tripeptide (gamma-glutamylcysteinyl-glycine; GSH), naturally present in buffalo semen and has been recognized as an essential intracellular antioxidant [8]. Cryopreservation resulted in a decreased GSH level in bovine semen. Moreover, extender supplementation with GSH resulted in a better post-thaw semen quality [11], [12]. It was demonstrated that GSH added to bovine semen extender resulted in a decreased LPO level during freezing, and ultimately improved fertility rates [13]. In our previous studies, GSH at a concentration of 0.5 mM improved the quality of chilled buffalo bull semen [14], while the use of 2 mM GSH was useful in the improvement of the post-thaw quality (motility, normal apical ridge and plasma membrane integrity) of buffalo bull spermatozoa [15]. Since a delicate balance of antioxidants and ROS is important for normal sperm functions, it is necessary to more precisely refine the concentrations of GSH in extender for improved post-thaw quality and in vivo fertility of buffalo bull spermatozoa. The present study was therefore planned to evaluate the effects of semen extender supplementation with GSH (0, 0.5, 1.0, 1.5 or 2.0 mM) on motility, functional and structural plasma membrane integrity, viability (live sperm with intact acrosome), DNA integrity and in vivo fertility rate of buffalo bull spermatozoa.
Section snippets
Experimental extender
The stock extender consisted of 74.4 mM citric acid (Fisher Scientific, Loughborough, Leicestershire, UK), 274.6 mM tris–(hydroxymethyl)-aminomethane (Research Organics, Cleveland, OH, USA), 11.1 mM fructose (Scharlau, Barcelona, Spain), 7% (v/v) glycerol (Merck, Darmstadt, Germany) and 20% (v/v) hen egg yolk in distilled water. Finally, five experimental extenders were prepared by adding 0, 0.5, 1, 1.5 or 2 mM GSH (Merck, Darmstadt, Germany) to the stock extender [14], [15].
Semen collection and initial evaluation
Semen ejaculates were
Results
In the present study, the effects of post-thaw incubation time and different concentrations of GSH on post-thaw semen quality parameters were significant (p < 0.05), but the interaction between these two factors was not significant (p > 0.05). Extender supplementation with 0.5, 1.0, 1.5 and 2.0 mM GSH increased sperm motility (Fig. 1), plasma membrane integrity (Fig. 2) and viability (Fig. 3) of buffalo bull spermatozoa (p < 0.05) in a dose dependent manner when compared to the respective controls.
Discussion
Extender supplementation with GSH (0.5, 1.0, 1.5 and 2.0 mM) increased sperm motility, plasma membrane integrity and viability of buffalo bull spermatozoa in a dose dependent manner. A dose dependent response of semen quality parameters in response to GSH extender supplementation was reported for cryopreserved [14] but not for chilled [15] buffalo bull semen. Progressive motility is a basic criterion for assessment of semen. It is pertinent to mention that subjective motility assessed post-thaw
Acknowledgment
The authors thank to the Higher Education Commission of Pakistan for financial assistance under the scheme “Indigenous 5000 PhD Fellowship Program”.
References (28)
- et al.
A comparative study on lipid peroxidation, activities of antioxidant enzymes and viability of cattle and buffalo bull spermatozoa during storage at refrigeration temperature
Animal Reproduction Science
(2006) - et al.
Storage of buffalo (Bubalus bubalis) semen
Animal Reproduction Science
(2000) - et al.
Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen
Theriogenology
(2001) - et al.
The effect of cysteine and glutathione on sperm and oxidative stress parameters of post-thawed bull semen
Cryobiology
(2010) - et al.
Prognostic value of spermatological parameters as predictors of in vitro fertility of frozen-thawed bull semen
Theriogenology
(2004) - et al.
Effects of freeze–drying on cytology, ultrastructure, DNA fragmentation, and fertilizing ability of bovine sperm
Theriogenology
(2007) - et al.
A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence
Fertility and Sterility
(1984) - et al.
Assessment of in vitro sperm characteristics in relation to fertility in dairy bulls
Animal Reproduction Science
(2008) - et al.
Lipid peroxidation, mitochondrial membrane potential and DNA integrity of spermatozoa in relation to intracellular reactive oxygen species in liquid and frozen-thawed buffalo semen
Animal Reproduction Science
(2009) - et al.
Motility and fertility of bull sperm in whole milk extender containing antioxidants
Animal Reproduction Science
(2002)
The effect of glutathione on the motility, enzyme leakage and fertility of frozen goat semen
Theriogenology
Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa
Biology of Reproduction
Effect of freezing on phospholipid distribution of buffalo spermatozoa plasma membranes
Lipids of plasma membrane and outer acrosomal membrane from bovine spermatozoa
Biology of Reproduction
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