اثر عصاره نسترن بر پارامترهای میکروسکوپی و بیوشیمیایی و آنزیمهای پلاسمای منی پس از فرآیند انجماد-یخگشایی

Abstract

Introduction Semen cryopreservation has detrimental effects on sperm cell organelles, including cell membranes, mitochondria, and DNA due to the increased level of reactive oxygen species (ROS). Oxidation of polyunsaturated fatty acids in the sperm cell membranes is caused by high levels of ROS produced during the freezing-thawing process. This problematic condition causes decreased sperm motility, membrane integrity, increased metabolic changes and, ultimately, decreased fertility of the sperm. The addition of antioxidant compounds to the semen extender before semen cryopreservation can decrease ROS levels and their deleterious effects on spermatozoa. This study was conducted to assess the antioxidant effect of different levels of Rosa canina extract on microscopic and biochemical parameters and antioxidant enzyme activities of Ghezel ram semen after freezing-thawing process. Materials and Methods Semen samples were collected from five Ghezel rams. Ejaculates were collected twice a week. To eliminate individual effects, ejaculates containing sperm with >80% progressive motility, volume of 0.75-2 mL, sperm concentrations greater than 3×109 sperm/mL and sperm abnormalities of less than 10% were pooled. Different concentrations of Rosa canina extract (0, 100, 150, 200 μL/mL) were added into the tris-egg yolk based diluent. After processing and freezing, the samples were stored in liquid nitrogen until the time of evaluation. Sperm motility characteristics were analyzed using computer-assisted sperm analysis (CASA). Sperm motility parameters including total motility, progressive motility, average path velocity, straight-line velocity, curvilinear velocity, linearity, straightness, amplitude of lateral head displacements and beat/cross frequency of sperm, viability, membrane integrity, sperm abnormalities, lipid peroxidation, antioxidant enzymes of glutathione peroxidase, superoxide dismutase and total antioxidant capacity were evaluated after thawing. Statistical analyses were performed using SAS software. The data were analyzed using the GLM procedure. Tukey–Kramer test were used to determine the significance differences between the experimental treatments. Significant differences were reported at the level of 5 percent. Results and Discussion The results of lipid peroxidation (LPO) measurements show that adding 100 μl / ml Rosa canina extract to the diluent medium reduced the amount of malondialdehyde, which was not significant in comparison to the control group. This could be indicative of the beneficial effect of Rosa canina extract to reduce the process of lipid peroxidation of the sperm membrane. The results showed no significant difference in malondialdehyde and morphology of sperm in treatments containing Rosa canina extract compared to the control group. The addition of Rosa canina extract was not significantly effect in morphology and structure of sperms compared to the control group. However, the percentage of abnormal sperms decreased at levels of 100 and 150 μL/mL compared to the control group. The addition of 150 μL/mL of Rosa canina extract significantly improved the viability and plasma membrane integrity of sperms after freezing-thawing compared to the control and other treatment groups (P <0.05). This is probably due to inhibition of free radicals, especially ROS, by the Rosa canina extract, which improves the viability of sperms. Previous studies have suggested that phenolic compounds, especially flavonoids, can alter and stop lipid peroxidation by coating lipids. Also through reducing fluidity of cell membranes, preventing excessive production of reactive oxygen species and limiting the peroxidation of phospholipids and unsaturated fatty acids protect cell membranes. (6, 9). Addition of 150 μL/mL Rosa canina extract to diluent significantly improved total motility compared to the control and other treatment groups (P <0.05). Progressive motility was significantly increased due to addition of 150 μL/mL of the Rosa canina extract compared to the control group (P<0.05). Curvilinear velocity and lateral head displacement parameters were significantly higher in the group receiving 100 and 150 μL/mL of Rosa canina extract compared to the control group (P <0.05). The total antioxidant capacity was significantly lower in group receiving 100 and 200 μL/mL compared to the control group (P<0.05). Reducing the total antioxidant capacity of semen may have a significant role in sperm morphology. Because there are various inhibitors of SOD, GPX and CAT in semen, which prevent sperm from oxidative damage by eliminating aloxcyle and proxyl radicals. The amount of the superoxide dismutase increased significantly at groups receiving 150 and 200 μL/mL of Rosa canina extract as compared to control (P <0.05). The activity of glutathione peroxidase at level of 150 μL/mL tended to increase. Total antioxidant activity decreased significantly at concentrations of 100 and 200 μL/mL (P <0.05). Conclusion In conclusion, the findings of this study showed after freezing-thawing process, the diluent containing 150 μL/mL Rosa canina extract improved sperm parameters significantly when compared to other levels.