A significant national genetic resource of Pakistan is the Sahiwal cattle breed. Originated and developed in Pakistan, this breed is now reported to be present in 29 countries (FAO, 2007). Population of this breed is diminishing because of intensive crossbreeding for dairying which remains a main threat to its survival (Payne and Hodges, 1997). It is necessary to improve fertility rates in our national breeds, by the use of insemination with frozen-thawed semen (Barbas and Mascarenhas, 2009). This tropical dairy breed can be preserved through the conservation of its germplasm. Germplasm that have applications in agriculture, aquaculture, biotechnology and conservation of threatened species can be preserved by cryopreservation (Andrabi and Maxwell, 2007).
The development of reproductive techniques, such as artificial insemination (AI) and in vitro fertilization can be achieved by sperm cryopreservation (Medeiros et al., 2002). The cryopreservation of semen is a renowned industry worldwide, for agriculturally important animals, mainly for dairy cattle (Bailey, J. et al., 2000). Cryopreservation of spermatozoa is correlated with an oxidative stress (Salvador et al., 2006) due to the production of Reactive Oxygen Species (ROS) by malfunctioning and dead spermatozoa (Bailey et al., 2000), which eventually leads to membrane lipid peroxidation. Sperm cells are extremely prone to lipid peroxidation because their membranes are rich in unsaturated fatty acids and they have fewer amounts of antioxidants in their cytoplasm (Sinha et al., 1996).
The most common ROS are superoxide (O2-). anion, hydrogen peroxide (H2O2), peroxyl (ROO-). radicals, and the very reactive hydroxyl (OH-). radicals, nitric oxide and peroxynitrite anion (Sikka, 1996). At physiological concentrations, ROS play vital roles during normal sperm function, together with hyperactivation, capacitation and the acrosome reaction, and zona binding (De Lamirande et al., 1997). On the other hand, during cryopreservation increased generation of ROS is associated with harm to chromatin, proteins and membranes of sperm (Ball, 2008), early capacitation of sperm (Neild et al., 2003 D.M. Neild, B.M. Gadella, M.G. Chaves, M.H. Miragaya, B. Colenbrander and A. Aguero, Membrane changes during different stages of a freeze–thaw protocol for equine semen cryopreservation, Theriogenology 59 (2003), pp. 1693–1705. Article | PDF (279 K) | View Record in Scopus | Cited By in Scopus (24)Neild et al., 2003).
Semen represents a complex redox system that combines the antioxidant potential of seminal plasma and spermatozoa with the pro-oxidant potential of sperm through the production of ROS. Enzymatic antioxidant defense mechanisms in seminal plasma and spermatozoa contain catalase, glutathione reductase, gluthathione peroxidase and superoxide dismutase. Among non-enzymatic antioxidants there are reduced glutathione (GSH), urate, ubiquinones, Vitamin E, taurine, hypotaurine, carotenoids, and ascorbic acid.
The interaction of antioxidant and prooxidant mechanisms in semen determines the in general rate of lipid peroxidation in sperm (Gadea, J. et al., 2004). In present years, antioxidants in extenders have been used to save spermatozoa from the harmful effects of cryopreservation and free radicals are reduced by antioxidant systems (Baumber et al., 2000). Ascorbic acid, a foremost water soluble antioxidant, acts as a scavenger for a extensive range of ROS.
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