Ansari et al., J. Anim. Plant Sci. 24(4):2014 1256 THIOGLYCOL IN EXTENDER IMPROVES THE POST-THAW QUALITY OF BUFFALO (BUBALUS BUBALIS) BULL SPERMATOZOA M. S. Ansari, B. A. Rakha * , S. M. H. Andrabi ** , N. Ullah ** , R. Iqbal, W. V. Holt *** and S. Akhter ,** Department of Zoology, University of Gujrat – Gujrat, Pakistan * Department of Wildlife Management, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, 46300, Pakistan ** Animal Physiology Laboratory, Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, 46300, Pakistan, *** Institute of Zoology, Zoological Society of London, Regent’s Park, London NW1 4RY, UK Corresponding Author: Email: sashraf1993@gmail.com ABSTRACT This study was designed to evaluate the effect of thioglycol in extender on post-thaw quality of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa. Semen from three buffalo bulls was collected at weekly intervals (three replicates; two ejaculates per bull per replicate) and qualifying ejaculates (n=18) were cryopreserved in extenders containing thioglycol at 0.0 or 0.5 or 1.0mM. Sperm motility (%; visually), plasma membrane integrity, (%;supravital hypo-osmotic swelling test), viability (%;dual staining technique using Trypan blue and Giemsa stains) and DNA integrity (%;acridine orange test) was assessed at 0, 2 and 4 hour post-thaw. Sperm motility, plasma membrane integrity and viability of buffalo bull spermatozoa were improved in a dose dependent manner with the supplementation of thioglycol at 0.5 mM and 1.0mM compared control. Sperm DNA integrity was similar (P>0.05) in extenders containing 0.5 mM and 1 mM thioglycol that remained higher (P < 0.05) than the values of DNA integrity in control. It is concluded that 1.0mM thioglycol in extender improved the motility, plasma membrane integrity, viability and DNA integrity of buffalo bull spermatozoa. Keywords: Buffalo bull spermatozoa; thioglycol; cryopreservation INTRODUCTION Excessive generation of reactive oxygen species (ROS) molecules are evident during cryopreservation of mammalian semen that reduces the viability and fertilization capacity of the spermatozoa (Bilodeau et al., 2000). ROS molecules can cause damage to sperm motility, plasma membrane, acrosomal and DNA integrity (Aitken et al., 1998; Bilodeau et al., 2001; Lenzi et al., 2002; Kumar et al., 2011). Buffalo sperm plasma membrane have high content of polyunsaturated fatty acids than cattle bull spermatozoa that makes it highly susceptible to the oxidative stress during freeze/thawing process due to the presence of double bonds (Parks et al. 1987; Cheshmedjieva and Dimov 1994; Lenzi et al., 2002; Andrabi, 2009). Buffalo semen is equipped with endogenous antioxidant system consisted of enzymatic and non-enzymatic antioxidative agents but this is not sufficient for sperm protection during cryopreservation (Kumar et al., 2011). It suggests that antioxidant supplementation is necessary for protection against ROS- mediated damage/stress in buffalo semen. Thioglycol, a low molecular weight thiol compound with a reducing power can interact directly with oxidized radicals. It protects cysteine, a precursor of glutathione, from oxidation into cystine and increases its entry into the sperm cells, which are known to trigger glutathione synthesis. Increased concentration of glutathione decreases the occurrence of reactive oxygen species and increases sperm motility and viability (de Matos et al., 2002; Feugang et al., 2004). It was hypothesized that thioglycol addition in semen extender may improve the post-thaw quality of buffalo bull semen. Therefore, this study was designed to evaluate the effect of thioglycol in extender on quality (motility, plasma membrane integrity, viability and DNA integrity) of buffalo bull spermatozoa at 0, 2 and 4 h post thaw. MATERIALS AND METHODS Preparation of extenders: The stock extender was consisted of 1.56 g citric acid, 3.0 g tris– (hydroxymethyl)-aminomethane, 0.2 g fructose, 7.0 mL glycerol and 20 mL egg yolk in 73 mL distilled water. Three experimental extenders were prepared by adding thioglycol 0.0, 0.5 or 1.0mM in stock extender. Collection and initial evaluation of semen: Semen was collected from three Nili-Ravi buffalo bulls (Bubalus bubalis) of known fertility and similar age (7–8 years) at weekly intervals (three replicates; two ejaculates per bull per replicate) with artificial vagina (42°C) during summer 2010. Sperm progressive motility (%) was assessed (200X) with phase contrast microscope. Sperm Short Communication The Journal of Animal & Plant Sciences, 24(4): 2014, Page: 1256-1259 ISSN: 1018-7081