Antioxidant Effect of Melatonin on Arabic Ram Semen Parameters after Freeze - Thawing Process

Semen collection evaluation and addition of preservatives to increase storage period of sperm are essential for successful artificial insemination. Cryopreservation of spermatozoa is becoming more important because of new clinical requirements and current clinical practice. Although frozen-thawed semen has great practical benefits for reproduction, it is widely reported that the cryopreservation process involving cooling, freezing, and thawing induces serious detrimental changes in sperm functions. The viability, motility and membrane integrity of mammalian spermatozoa decrease during the cryopreservation process. Many studies exist as regards the effects of antioxidants on the cryopreservation aimed at improving the quality of post-thaw semen. Antioxidant molecules could decrease the impact of oxidative stress and therefore improve Sperm quality following the freeze–thawing process. Melatonin (N-acetyl-5-methoxytryptamine), a derivative of tryptophan, is mainly synthesized and secreted by the pineal gland during the night in reaction to changes in light levels. Melatonin can stimulate the activity of antioxidant enzymes such as SOD and GSH-Px. melatonin scavenges a variety of reactive oxygen and nitrogen species in vivo and in vitro, signifying it has a powerful non-enzymatic antioxidant property. It has been shown that the spermatozoa undergo a freeze–thawing process produced high concentrations of reactive oxygen species. The aim of this study was to investigate the effects different levels of melatonin supplementation (0, 0.5, 1, 2 and 4 mM/ml) in extender on semen characteristics the Arabic ram after freezing-thawing. This research was performed at Ramin Agriculture and Natural Resources University of Khuzestan in the fall in 2015. Semen samples were collected from 6 Arabic ram with an average weight 73 ± 3 kg by electro ejaculator twice a week. Sperm samples motility were assessed by Computer Aided
Sperm Analysis (CASA) after freezing and thawing. The pH, viability, abnormal sperm percentage, membrane integrity and total antioxidant capacity were also assessed after freezing-thawing process. Pearson correlation test was used to assess correlation of melatonin with routine sperm parameters (pH, viability, abnormal sperm percentage, membrane integrity and total antioxidant apacity of plasma). Data analysis was performed using SAS software. P<0.05 was considered significant. Results and Discussion the Results of this experiment showed that the diluent of ram semen containing 0.5 mM/ml of melatonin improved the motility, membrane integrity and viability of Arabic ram spermatozoa compared to the control. The total antioxidant capacity was highest in diluent contained whit 1 mM/ml of melatonin. The effect of melatonin on the pH of semen at all levels was not significant. Cryopreservation causes an irreversible damage to enzymatic activity and sperm organelles leading to a reduction in the sperm kinetic parameters. Composition of extender may also affect the freeze ability of spermatozoa and their fertilizing ability. Many studies reported that melatonin has beneficial effects on preservation of mammalian sperm function and improves the microscopic parameters of spermatozoa. Melatonin supplementation to ram semen freezing extender protected spermatozoa from the cryopreservation injuries, as proved by post-thaw viability, motility, intracellular ATP concentrations, DNA integrity, and fertilizing ability. It is suggested that melatonin stimulates the activities of antioxidant enzymes. In consequence, melatonin reduces the number of free radicals, ROS, and also may increases the production of molecules protecting sperm cells against oxidative stress. As a conclusion, supplementation of melatonin in the freezing medium can counteract the adverse effects of the freeze–thawing process on the motility, viability, normal morphology and plasma membrane integrity in ram spermatozoa. The results were suggested that the protective effects of melatonin on spermatozoa were associated with a reduction in LPO as a consequence of increasing the TAC and antioxidant enzymes activity. Cryopreservation of sperm is an applicable technique in infertility management but it may influence the post-thaw qualities of sperm, including morphology, motility, viability and DNA integrity. In this research, we show that supplementation of cryopreservation extenders with melatonin provide a cryoprotective effect on pH, viability, abnormal sperm percentage, membrane integrity and total antioxidant capacity. Overall addition of 0.5 mM/ml of melatonin to the extender improved the most of spermatozoa quality characteristics as well as total antioxidant capacity of semen after freezing-thawing process in Arabic ram. In the current study, we observed no correlation between melatonin and pH, viability, abnormal sperm percentage, membrane integrity and total antioxidant capacity of plasma.