Evaluation of the effect of adding cysteamine on Ros 308 sperm quality parameters and reduction of lipid peroxidation rate during freezing-thawing process

Long-term storage of semen is essential for achieving the benefits of artificial insemination (Tuncer et al., 2010). This is carried out by sperm cryopreservation, which stops the sperm metabolic activities, allowing long storage (Bailey et al., 2000). This process affects the sperm quality (Wang et al., 1991) by introducing mechanical and chemical damage, production of reactive oxygen species, oxidative stress, and reducing antioxidant activity (cysteamine is an aminothiol antioxidant as an effective scavenger). Cysteamine is known to have been reported in some studies to improve the freezing of ram sperm (Bucak et al., 2007). The aim of the present study was to determine the antioxidant effects of cysteamine on the functional parameters of cystemine in Lake Extender based on soybean lecithin.

This study was carried out in University of Tabriz research station. For this purpose, 15 adult roosters of 25 weeks were used. Sperm collection was done by dorsal-abdominal massage. The roosters were habituation for one month and sperm collection was performed twice a week. First, sperm were examined for volume, concentration and color, and only samples with volume of 0.2 to 0.7 ml and motility greater than 80% were used. To eliminate the individual effects, the confirmed samples were pooled. Four levels containing control, 15, 30, and 45 μM cysteamine were then added to Lake Extender containing 1/4 glycerol and sperm added to the extender containing different levels of antioxidant. The cooling process was carried out in two steps. The samples were adjusted to 4°C and after two hours the samples were cooled to 4°C. Then, samples were transferred to the refrigerator for one hour more, then they were drawn into 0.25 ml straws, placed 4 cm above nitrogen vapor for 7 min, and then immersed in liquid nitrogen. They were stored in liquid nitrogen until carrying out the tests. For thawing, the straw containing the samples was removed from the tank and immersed in water for 30 seconds at 37°C in water bath. The motility parameters were evaluated using CASA, viability by Eosin-Nigrosin staining, membrane integrity by Host tests, sperm abnormality by Hancock test and lipid peroxidation by MDA.

Addition of cysteamine at all levels significantly improved motility parameters. At the 30 and 45 μM levels total motility significantly increased and 30 μM level improved progressive motility, VAP, VSL and VCL parameters (P <0.05). The results show that the addition of cysteamine amino acid improves sperm quality. Addition of 30 μM level significantly increased viability and plasma membrane integrity of rooster sperm (P <0.05); Also significantly decreased malondialdehyde compared to the control group (P <0.05). The addition of cysteamine reduced sperm abnormality which was not significant. Discussion Freezing causes oxidative stress and induces eversible damage such as reduced viability and fertility (Najafi et al., 2014). This study was conducted to evaluate the addition of cysteamine as a supplement to counter oxidative damage during freezing and thawing (Blesbois, 2011). Normally, the antioxidant system in the semen eliminates the free radicals in the semen which are disturbed by cryopreservation process (Safa et al., 2017). Adding antioxidants can control ROS production (Amini et al., 2015). Antioxidants in different species and at different doses have shown different results. Addition of cysteamine to rooster spermatozoa during freezing and thawing significantly increased motility and survival parameters, which was in agreement with the results of Bucak et al., (2007) reported that the addition of cysteamine to ram sperm improved sperm motility parameters (Najafi et al., (2014) also reported an increase in motility and viability of ram sperm with addition of 2, 4 and 6 mM cysteamine levels, but at 8 mM level these parameters were significantly reduced. This was probably due to the high dose of diluent used. But it was in contrast to the study of Thananurak et al., (2019) who reported that adding levels of 0.001, 0.002, 0.004 and 0.01 decreased motility and viability. Low levels of cysteamine induce cysteine to enter the cell, producing glutathione as an intracellular antioxidant. Cysteamine at high doses produces large amounts of hydrogen peroxide, causing oxidative stress and reducing glutathione peroxidase activity (Besouw et al., 2013). According to reports (Partyka et al., 2013) cysteine increases motility and viability of rooster sperm. Glutathione peroxidase is one of the enzymes with peroxidative activity that plays an important role in sweeping high oxidations and protecting the cell from oxidative stress. Glutathione can also restore the oxidized vitamins E and C and restore them to the original antioxidant structure (Almasi et al., 2014). Cysteamine as a glutathione synthase can play a major role in reducing free radicals. In the present study, the amino acid cysteamine decreased malondialdehyde concentration and increased plasma membrane integrity and normal sperm during the freezing-thawing process, which is in agreement with the report of Najafi et al., (2014) which reported that addition of 6 mM reduced the concentration of malondialdehyde in ram semen compared with the control group. Not only does cysteamine improve sperm quality after freeze-thawing, but also it increases sperm resistance during artificial insemination in the reproductive female tract (Najafi et al., 2014). In the present study, cysteamine did not significantly decrease the percentage of abnormal sperm compared to control group.

Antioxidants have different results depending on the doses which are used and the treated species, and by understanding the antioxidant mechanism and achieving the optimal dose, the best results can be obtained. In the present study, the use of cysteamine amino acid as an antioxidant supplement improved sperm functional parameters and significantly reduced lipid peroxidation at level 45 μM.