The Protective effect of Vitamin E on DNA integrity of frozen-thawed Beluga (Huso huso) sperm Using Alkaline Comet assay

One of the hypotheses of cell damage after cryopreservation is the deleterious effects of oxygen free radicals generated during freezing and thawing. This study evaluated the protective effect of Vitamin E supplementation as antioxidant on post-thaw DNA integrity of beluga (Huso huso) sperm. Collected sperm was divided into three parts and using DMSO alone as control group or in combination with vitamin E supplementation in 600 and 1200 μM has been diluted and was maintained for 30 days in liquid nitrogen storage tank. We used the Comet assay, a microelectrophoretic method to detect DNA damage in response to cryopreservation process in beluga spermatozoa. This technique was used to assess DNA integrity in the cells by measuring damages reflected as strand break. According to the results DNA damage rate in control freeze-thawed sperm was significantly higher than levels measured in fresh sperm. Adding the vitamin E had a dose dependent effect on the amount of DNA damage, as in 600 μM concentration, non-significant reduction in the level of damage than the control group was observed, but in 1200 μM, probably due to the pro-oxidant properties of the high-dose antioxidants significant increase in damage was assessed. In addition, the results indicated that the extent of damage to sperm motility caused by freeze-thawing was correlated with DNA breakage.