Sperm cryopreservation may lead to adverse effects on sperm structure and function. Cyanocobalamin (vitamin B12) has antioxidant potential and can protect DNA from free radical‐induced damages. Recent studies have shown that vitamin B12 preserves glutathione that leads to modulate oxidative stress responses. Also, vitamin B12 might act directly as a scavenger of ROS. The aim of this study was to investigate the effects of vitamin B12 supplementation on human sperm parameters during the cryopreservation process. Thirty semen samples were obtained from normozoospermic men. Using cryopreservation medium supplemented with different concentrations of vitamin B12 (0, 0.5, 1, 2, 2.5 mg/ml), the semen samples were cryopreserved. After thawing, all samples were evaluated for motility and viability. Based on results, 2 mg/ml was considered as the optimal concentration of vitamin B12 for evaluating sperm DNA fragmentation. The results showed that 1 and 2 mg/ml vitamin B12 significantly increased post‐thawing motility and viability compared with the 0 mg/ml vitamin B12 (p < .05). Also, by supplementing with 2 mg/ml vitamin B12, DNA fragmentation decreased when compared to the control. The present study showed that cryopreservation medium supplemented with vitamin B12 at 2 mg/ml could improve sperm quality after freeze–thaw process.

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