Sperm cryopreservation is widely used in large-scale artificial insemination programs, sperm selection advancements, genetic resource conservation in various animals, and fertility preservation for individuals with fertility-threatening conditions. Despite significant progress in semen freezing technology, the process still involves physical and chemical stress, which remains a major drawback. These stresses reduce the fertility potential of sperm stored under laboratory conditions. High concentrations of superoxide radicals contribute to oxidative damage after sperm freezing. Superoxide dismutase (SOD) is an antioxidant enzyme that can neutralize oxygen free radicals. Another harmful factor during cell freezing is hydrogen peroxide, which catalase mitigates by converting it into water. In this study, recombinant forms of animal catalase and plant-based SOD enzymes were used to investigate their effects. To produce SOD, an engineered strain of E. coli bacteria, encoding the CuZnSOD gene from the mangrove plant (AmSOD1), was cultured. By inducing the bacteria with IPTG, the recombinant protein, tagged with His at the amino end, was produced. This particular SOD was chosen due to its high enzyme activity and stability under varying temperatures and pH levels, as demonstrated in previous research. Protein production was confirmed through SDS-PAGE, and the enzyme was purified using two stages: affinity chromatography and dialysis. Enzyme activity was measured at each stage of production. To determine the optimal enzyme concentrations, the effects of different levels of each enzyme on sperm motility and viability were eva‎luated. The impact of AmSOD1, catalase, and a mixture of both enzymes on sperm motility, viability, lipid peroxidation, and ROS levels were analyzed before and after freezing and thawing. Sperm treated with AmSOD1 and the enzyme mixture showed significantly higher motility and viability (P ≤ 0.01) compared to the control and catalase-treated groups. Sperm membrane integrity also improved significantly (P ≤ 0.01) in all treated groups compared to the control. Additionally, all treatments significantly reduced ROS levels (P ≤ 0.01) compared to the control. However, there was no significant difference in lipid peroxidation levels between the groups. Sperm freezing was performed using each of the two enzymes, separately or in combination, and samples were thawed after three months. The same factors were re-eva‎luated. Sperm motility, viability, and membrane integrity were significantly higher in the AmSOD1 and combined enzyme treatments compared to catalase alone and the control group (P ≤ 0.01). DNA damage, ROS levels, and lipid peroxidation were also lower in all treated groups compared to the control (P ≤ 0.01). Based on these results, AmSOD1 appears to play a crucial role in improving sperm cryopreservation. Since the combination of the two enzymes showed a similar effect to AmSOD1 alone, using AmSOD1 is likely more cost-effective. However, final IVF outcomes should also be considered for a comprehensive eva‎luation.

آذر شاه پيري , مجيد طالبي , فرنوش جعفرپور

ابوذر سورني , اميرحسين مهدوي