| چکیده انگلیسی مقاله |
Extended abstract
Introduction and Objective: Artificial insemination technique is widely used as a basic technique in livestock and poultry breeding programs. The requirement to use this technique is sperm freezing. Sperm freezing is a process during which superior genes are widely expanded and more progeny can be obtained from a male animal with high genetic characteristics. However, during the cooling and freezing process, due to the drop in temperature and cold shock, and the subsequent excessive production of free radicals, many damages are caused to the sperm. Lipid peroxidation of the sperm plasma membrane (LPO) occurs as a result of the production of reactive oxygen species (ROS), which damages the structure of the sperm membrane. The attack of free radicals on intracellular organelles, especially mitochondria and sperm DNA, can often disrupt sperm functions and have a very negative effect on sperm fertility. Therefore, adding antioxidant compounds to sperm thinner is considered essential for freezing and maintaining the quality of sperms. Curcumin, as a natural antioxidant compound obtained from turmeric root, with the characteristic of having a phenolic ring and a beta-di-ketone part in its structure, is capable of neutralizing free radicals and thus maintaining the quality of sperm cells. This compound has been introduced as a very strong antioxidant compound in most pharmaceutical, medical and food industry studies, and even in some studies, it has been reported to be the co-base of known antioxidant compounds such as vitamins E and C and superoxide dismutase enzyme. The purpose of this experiment was to investigate the effects of adding different levels of curcumin to diluents on the physical, structural and biochemical parameters (oxidative stress) of ram epididymal sperm after freezing-thawing process.
Materials and methods: In this experiment, testicular tissue was collected from the slaughterhouse and transferred to the laboratory, and sperm cells were collected by cutting the tail of the epididymis. Sperm samples were selected after initial evaluation and diluted in Tris-egg yolk based diluent with a concentration of 50 million sperm per milliliter. Different concentrations of curcumin, including 0, 10, 25, and 50 μM, were added to the diluent containing sperm at 37°C. Samples containing different concentrations of curcumin in different experimental groups were poured into a 15 ml Falcon and transferred to 5°C in isothermal water for cooling. After about two hours, the samples reached equilibrium at this temperature. Then the experimental groups were filled and sealed at the same temperature in one-quarter straws at the same temperature. The peyotes were frozen at a distance of four centimeters from the surface of liquid nitrogen by nitrogen vapor for seven minutes and then immersed in liquid nitrogen. The frozen peyote were kept in a nitrogen tank until the evaluation (about one month). After thawing at 37°C for 30 seconds, physical parameters, survival and apoptosis status, plasma membrane health, abnormality percentage, biochemical parameters including measurement of malondialdehyde (MDA) concentration, activity of superoxide dismutase enzymes (SOD), glutathione peroxidase (GPX), catalase (CAT) and H202 concentration assay were evaluated. The data obtained from the evaluations were analyzed by SAS software and GLM procedure at a significance level of 0.05.
Findings: The results of this experiment showed that in the case of kinematic parameters such as the total kinematics (TM) of the diluent containing 10 and 25 μM curcumin, the progressive kinematics (PM) of the diluent containing all three concentrations of curcumin (10, 25 and 50 μM) and the speed in the mean path (VCL) diluent containing 25 μM curcumin performed significantly better than the control group (P<0.05). However, no significant difference was observed between the experimental groups for other kinematic parameters such as velocity in curved path (VAP), velocity in straight path (VSL), transverse head movement (ALH), linearity of kinematics (LIN) and percentage of straight movement (STR) (P>0.05). In the analysis of structural parameters, it was found that the diluent containing all three levels of curcumin (10, 25 and 50 µM) had significantly increased the percentage of sperm plasma membrane health after thawing, and regarding the viability percentage, the diluent containing 25 µM curcumin significantly increased compared to Other treatment groups had better performance and the addition of the same level significantly reduced the percentage of early apoptosis compared to other groups (P<0.05). No significant difference was observed among the treatments regarding the percentage of secondary apoptosis, necrosis and abnormalities (P>0.05). The results of the evaluation of biochemical parameters (oxidative stress) showed that MDA concentration and CAT enzyme activity did not differ significantly among the experimental groups (P>0.05), but in the case of SOD enzyme activity, dilutions containing 10 and 25 μM curcumin and for GPX dilutions containing 25 Micromolar curcumin significantly increased the activity of these antioxidant enzymes (P<0.05). Also, the addition of all three concentrations of curcumin (10, 25 and 50 μM) significantly reduced H202 concentration compared to the control group (P<0.05).
Conclusion: The results of this experiment showed that the use of curcumin in freezing diluent can improve the quality of ram epididymal sperm after freezing-thawing process. Therefore, it is recommended to use a concentration of 25 μM curcumin in the freezing diluent.
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