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Introduction

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.

Until now, Ascorbic acid has not been used in semen extenders to examine its influence on the cryopreservation of Sahiwal bull spermatozoa. The objective of present study is to evaluate the effect of Ascorbic acid supplementation in diluent on post thaw quality of Sahiwal bull spermatozoa.

Review of Literature

In various studies, it has been observed that addition of antioxidants in the extender to preserve unfrozen semen increases sperm quality by controlling oxidation. Uysal et al. (2007) demonstrated that during semen cryopreservation attempts, addition of various antioxidants in different concentrations in extender showed beneficial effects on the quality of bull semen after freezing-thawing proess. Inclusion of natural antioxidants such as a-tocopherol and ascorbate had a protective effect on metabolic activity and cellular viability of cryopreserved bovine sperm (Beconi et al., 1991; Beconi et al., 1993). In vitro studies strongly suggest that the antioxidant effect of ascorbate is related to direct vitamin E regeneration by reducing the tocopheroxyl radical in the one-electron redox cycle (Dalvit et al., 1998). Similarly, Raina et al. (2002) found that incorporation of vitamin C or E in TCA based extender improved the motility of liquid buffalo bull semen.

Vitamin C (Ascorbic acid) may act as an oxidant at low concentrations and as an antioxidant at high concentrations (Breininger et al., 2005). Ascorbic acid, at a concentration of 5 mM in the freezing diluent acts as an antioxidant during freezing and thawing of bovine spermatozoa (Beconi et al., 1993). Singh et al. (1996) studied effect of vitamin C addition in the diluent on the quality of deep frozen Murrah buffalo bull (Bubalus bubalis) semen. They concluded that insertion of ascorbic acid (2.5mM) in the semen diluent produced significantly higher post-thawing sperm motility (37.5 vs. 46.25%) and percentage of live spermatozoa (58.12 vs. 67.58%) compared with untreated controls.

Aurich et al., 1997 J.E. Aurich, U. Schonherr, H. Hoppe and C. Aurich, Effects of antioxidants on motility and membrane integrity of chilled-stored stallion semen, Theriogenology 48 (1997), pp. 185–192. Article | PDF (558 K) | View Record in Scopus | Cited By in Scopus (41)Aurich et al. (1997) observed a positive effect of addition of ascorbic acid on preservation of membrane integrity of cooled equine sperm. Verma and Kanwar (1998) stated that Ascorbic acid when added in the semen is known to improve the post-thaw motility and feasibility of bull and buffalo sperm.

Salem et al. (2001) studied the protective role of ascorbic acid to enhance semen quality of rabbits treated with sublethal doses of aflatoxin B1. Treatment with ascorbic acid increased (P<0.05) live body weight (LBW), dry matter intake (DMI), relative testes weight (RTW), serum testosterone concentration, improved semen characteristics. Results showed the useful effects of ascorbic acid in decreasing the negative effects of aflatoxin B1 on production and reproduction of male rabbits.

Andrabi et al. (2008) examined the effect of non-enzymatic antioxidants (vitamins C or E) in tris-citric acid (TCA) extenders on post-thaw motility, membrane integrity, and morphology of buffalo bull spermatozoa. In their study, the inclusion of nonenzymatic antioxidants (vitamin C or E) in the cryodiluent improved the motility of buffalo spermatozoa at 0 and 6 h after thawing and incubation (37°C).

Paudel et al. (2008) assessed the usefulness of ascorbic acid, catalase, chlorpromazine and their mixtures in reducing the cryodamages to crossbred bull (Bos taurus × Bos indicus) spermatozoa. It was inferred that addition of ascorbic acid, catalase and ascorbic acid + chlorpromazine in semen extender enhanced the post-thaw semen quality in crossbred bulls.

Michael (2008) studied the effect of different concentrations of vitamin C in semen extenders on post thaw quality of dog spermatozoa. He concluded that addition of vitamin C to semen extenders does not improve the quality of extended canine semen preserved at 4 °C.

Yoshimoto, T. (2008) evaluated that the post-thaw qualities of fragile Agu sperm can be improved by the addition of ascorbic acid 2-O-α-glucoside (AA-2G), a stable ascorbate derivative to the freezing extender. Among the concentrations tested treatment with 200 μM AA-2G has the most valuable effect on the sperm motility and the plasmalemma integrity after cryopreservation

Materials and Methods

Experimental extenders

Tris-citric acid (TCA) containing 1.56 g citric acid (Merck, Germany) and 3.0 g tris(hydroxymethyl)- aminomethane (Sigma, USA) in 74 ml distilled water was used as a buffer for the experimental extenders. The pH of buffer was 7.00 and the osmotic pressure was 320 mOsmol/Kg. Egg yolk (20% vol/vol), fructose (0.2%; wt/vol; Riedel-DeHaen, Switzerland), glycerol (7%; vol/vol; Merck, Germany), benzyl penicillin (1000 I.U/ml; Hebei, China) and streptomycin sulphate (1000 μg/ml; Sigma, USA) were added to each of the three experimental extenders (Andrabi et al., 2008). The first extender contained vitamin C (TCAC) as sodium ascorbate (Sigma, USA), which was added at the rate of 5 mM (Beconi et al., 1993; Raina et al., 2002). The second extender contained vitamin E (TCAE) available as α-tocopherol acetate (Sigma, USA), added at the rate of 1 mg/ml (Beconi et al., 1993; Raina et al., 2002). The third extender did not contain any antioxidant and served as control (TCAN). Aliquots of each extender were stored frozen at -20°C and thawed before use.

Semen collection

Ejaculates were collected by artificial vagina 42°C from three adult Nili-Ravi buffalo bulls (Bubalus bubalis) of known fertility. The bulls were kept under uniform feeding and handling conditions during the entire study. Ejaculates were collected at weekly intervals for a period of 5 weeks (replicates; n = 5). The frequency of collection from each bull was two ejaculates on one day each week. Visual motility of each ejaculate was assessed at 37°C using a phase contrast microscope (X 400; Leica, Leitz Wetzlar, Germany) observed on closed circuit television by two operators.

Progressive motility of spermatozoa was assessed to the nearest 5%. Sperm concentration was assessed by digital photometry (Dr. Lange LP 300 SDM, Minitub, Germany) at 546 nm. Ejaculates containing more than 70% progressively motile spermatozoa and 0.5×109 spermatozoa/ml were pooled in order to have sufficient semen for a replicate (Rasul et al., 2000, 2001; Andrabi et al., 2008). At least one ejaculate on every collection from each bull did qualify for freezing.

Semen cryopreservation

Buffalo bull semen was cryopreserved according to Rasul et al. (2000). After a holding time of 15 min at 37°C, three aliquots of semen were diluted (37°C) in a single step with one of the three experimental extenders to a concentration of 50×106 motile spermatozoa/ml. After dilution, the semen was cooled to 4°C in 2 hours and equilibrated for 4 h at 4°C. Precooled 0.5 ml straws were then filled with the cooled semen at 4°C in the cold cabinet unit (Minitub, Germany) and frozen in a programmable cell freezer (KRYO 10 series III, UK) from 4°C to -15°C at the rate of 3°C/minute and from -15°C to -80°C at the rate of 0°C/minute. Straws were then plunged into liquid nitrogen (-196°C) for storage. After 24 h storage, semen straws were thawed at 37°C for 30 seconds.

Post-thaw spermatozoal evaluation

Visual motility

The motility of spermatozoa was assessed at 0 and 6 h post-thaw. Thawed semen sample was placed on a pre-warmed glass slide and cover-slipped. Visual motility of spermatozoa was assessed at 37°C using phase contrast microscope observed on closed circuit television by two operators.

Statistical analysis

Results are presented as means ± SD. Effect of non-enzymatic antioxidants for different variables was analyzed by the analysis of variance (ANOVA). When the F–ratio was significant (P<0.05), Tukey’s Honestly significant difference was used to compare the treatment means (SYSTAT, 1996).

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