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Taurine is considered one of the main scavengers that improves the defence of sperm against ROS and reduce lipid peroxidation (Sharma and Agarwal, 1996; Saleh and Agarwal, 2002). Taurine has a variety of physiological roles including viability, cell propagation, osmoregulation, inhibition of oxidative stress in the cell (Buff et al., 2001); (Chatiza et al., 2018), transport and binding of calcium (Lazarewicz et al., 1985), and the biological stabilisation of membranes (Balkan et al., 2002). It has been stated that taurine has an important role as an antioxidant in cell defence mechanisms (Green et al., 1991). This study was aimed to determine the optimal timing and concentration of taurine supplementation of the semen extender that can decrease oxidative stress and enhance the integrity of cryopreserved ram spermatozoa. The percentage of motility, acrosome integrity, viability, penetrability, and ROS production were assessed as parameters to rectify of oxidative damage to the sperm membrane and the resulting effect on sperm function during cryopreservation. The addition of different concentrations of taurine to ram semen extender before freezing and after thawing was thought to reduce this damage by promoting the stabilization of the sperm membrane. The results obtained were in agreement with previous studies which showed that supplementation with antioxidants such as taurine improved and maintained the motility and viability of cryopreserved spermatozoa of various species such as the bull (Foote et al., 2002; Chhillar et al., 2012), buffalo (Shiva Shankar Reddy et al., 2010), goat (Salvador et al., 2006), turkey (Donoghue and Donoghue, 1997; Slanina et al., 2018), rabbit (Alvarez, and Storey, 1983), rat (Yang et al., 2010) donkey (Dorado et al., 2014; Bottrel et al., 2018), and horse semen (Ijaz and Ducharme, 1995). Chatiza et al. (2018) stated that 75 mM taurine supplementation during liquid storage of boar semen have the greatest effect at 17ºC for 4h, compared with other types of antioxidants such as cysteine and vitamin E. Taurine has the ability as an antioxidant to protect and maintain the integrity of the acrosome (Sariözkan et al., 2009a), stabilizes and protects cytoplasmic membrane and improves the motility of mammalian spermatozoa (Slanina et al., 2018).

Moreover, some studies have been conducted on ram sperm using different concentration of taurine Previous studies (Bucak et al., 2007; Rather et al., 2016; Banday et al., 2017) have demonstrated the effect of taurine on spermatozoa using concentrations of25-50 mM taurine compared to 8 mM in the present study during pre-freeze supplementation on the semen of different species of animals including rams. These high concentrations of taurine improved ram sperm integrity and reduced the level of ROS and oxidative stress in most species. It is possible that at these high concentrations used pre-freeze, there is a reduced

supplementation of 1.0 mg/ml of taurine (8 mM) PF and PT improved sperm integrity and reduced the level of ROS. Therefore, this method of using taurine pre-freezing and post- thawing (PF + PT) seemed to be more effective in protecting sperm function as well as being more economical through detecting the optimal time and concentration of taurine in ram semen extender compared with previous studies.

The significant effect of taurine on sperm parameters on the present study could be due to the fact that taurine acts as a sulfonated amino acid which is an essential defender for cells against the production of ROS when exposed during aerobic circumstances (Alvarez and Story, 1983; Holmes et al., 1992). Furthermore, taurine has the ability to penetrate cryoprotectants and reacts as an antioxidant to induce the rearrangement of proteins and lipids within the sperm membrane, which improves membrane fluidity (Yang et al., 2010), and regulates the energy metabolism in the cell (Agarwal and Allamaneni et al., 2004). This effect could minimize the effect of low temperatures to which the sperm are exposed during cryopreservation and subsequently improve the capability of spermatozoa to resist the harmful effects of freezing and thawing ( Sanchez-Partida et al., 1993; Maxwell and Watson, 1996). In addition, taurine supplementation of 0.5 and 1.0 mg/ml (PF + PT) improved sperm motility post cryopreservation, and displayed as antioxidative role. This could be due to the ability of taurine to improve the level of antioxidant enzymes in sperm cell through reducing the damaging effect of superoxide anion, which has high activity and cannot distribute easily through the cell membrane, and dismute to generate hydrogen peroxide (H2O2) either naturally or by superoxide dismutase (SOD) enzyme (Slimen et al., 2014).

Taurine has the ability to protect and enhance the function of other antioxidant enzymes including catalase (Perumal et al., 2013), superoxide dismutase (Higuchi et al., 2012), glutathione peroxidase (Nonaka et al., 2001), and thioredoxin reductase (Yildirim et al.,

2007). The protective feature of taurine on the antioxidant system of spermatozoa improved sperm ability to resist the harmful effect of ROS (Holmes et al., 1992; Bucak et al., 2007; Paal et al., 2018). Additionally, this synergistic effect protects sperm integrity (Slanina et al.,

2018). This system can assist to inhibit cholesterol efflux from the cell membrane, reduce production of malondialdehyde, and decrease the damage of cholesterol from cell membranes which is essential for acrosome integrity (Perumal et al., 2013). Moreover, it has been reported that taurine has the ability to transport across the mitochondrial plasma membrane (Suzuki et al., 2002), and supports stabilisation of the chain of electron transportation (Jong et al., 2012). This could explain the role of taurine in the improvement of sperm integrity such as motility, acrosome integrity, viability and penetrability in the present study.

In studies that have been conducted to assess the ability of taurine to protect sperm integrity at 5°C such as in turkey (2.5-7.5 mg/ml, Slanina et al., 2018), and in bull (50 mM, Perumal

et al., 2013), high concentrations of taurine were beneficial to sperm function. However, liquid storage/colled semen is not suitable for long term storage of semen and also the sperm experienced high stress as in cryopreservation (Salamon and Maxwell, 2000). Conversely, 75 mM taurine supplementation did not improve boar sperm integrity in liquid storage at 17 °C (Chatiza et al., 2018), highlighting the beneficial technique in the present study for the optimized time and concentrations of taurine supplementation in cryopreserved semen.

Contrariwise, Partyka et al. (2017) found that pre-freeze supplementation of chicken sperm by 10 mM of taurine improved sperm parameters such as motility, mitochondrial activity and viability, and decreased apoptosis and DNA fragmentation of sperm, however they did not test the post-thaw treatment. In the present study, the penetrability of frozen-thawed ram sperm through the artificial mucus was greatest in samples treated with 1.0 mg/ml taurine PF + PT 1.0 mg/ml This effect could be related to the beneficial effect of taurine on endogenous antioxidant capability to improve catalase level in the presence of ROS (Bucak

et al., 2007; Sariözkan et al., 2009). Additionally, taurine can maintain sperm integrity at post thaw through stabilization of the mitochondrial membrane and electron passage chain inhibiting it from high production of superoxide anion (Jong et al., 2012). Lopes et al. (2018) reported that taurine supplementation of tambaqui sperm extender improved the regulation and transport of Ca2+_{, which is required to generate sperm activity. This positive effect of} taurine on the sperm membrane could also explain their ability to maintain the integrity and improve sperm penetrability.

The direct effect of taurine as an antioxidant could be through its ability to detoxify and reduce some volatile intermediates such as hypochlorous acid generated by myeloperoxidase, and the indirect effect through taurine penetrates into the cell membrane and stabilizing it (Schaalan et al., 2018). In the current study, taurine reduced ROS thus inhibiting the harmful effect of ROS and providing stability to the sperm membrane specifically in the post-thaw phase. Moreover, the presence of amine group in taurine plays an important role through its relationship with nucleic acids and can consequently reduce the ROS production and the damage to DNA (Sokól et al., 2009).

Taurine has the ability to improve either the primary post-thaw motility of sperm or the longevity of motility of frozen–thawed ram sperm (Sanchez-Partida et al., 1997). Consequently in the present study, the use of taurine both pre-freeze and post-thaw could be particularly beneficial.The pre-freeze supplementation allowed taurine to function as an osmoregulater and protect the sperm membrane from the effect of cold shock damage

support the systemic enzymes of the sperm to reduce the effect of ROS and oxidative stress (Das et al., 2009; Yang et al., 2010). In addition, the sudden variation in temperature through thawing of frozen spermatozoa from (-196 ºC to 37 ºC), that leads to a fast phase change from solid to liquid and severe ROS production by switching on the O2 _{creation or a release} of free radicals produced along the stages of cryopreservation (Chatterjee and Gagnon, 2001). Therefore, these findings in the current study could determine the effect of optimal timing and /or concentration of taurine supplementation on the frozen-thawed ram semen.