The aim of this project was to examine the significance of oxidative stress in the etiology of sperm cryostorage injury and determine whether antioxidant supplementation might improve the efficacy of this process.
Human semen samples were initially tested (i) unprocessed, (ii) following addition of Quinn's Advantage™ Sperm Freeze medium, (iii) immediately after a freeze-thaw cycle and (iv) 3 h after being resuspended in culture medium. Tests included: basic sperm parameters, reactive oxygen species production (ROS), lipid peroxidation, DNA fragmentation, and antioxidant activity. To determine the value of antioxidant supplementation samples (n=15) were cryopreserved with resveratrol (0.625 - 320µM), vitamin C (0.1-1.6 mM) melatonin (2.2 mM) or N-acetylene cysteine (NAC; 0.1 mM).
Cryoprotectant addition significantly lowered motility and vitality as well as seminal antioxidant capacity (P < 0.001). Cryopreservation and subsequent incubation led to further deterioration in sperm motility, vitality, antioxidant activity, ROS production, lipid peroxidation and fragmented DNA (P < 0.01).
A significant improvement in vitality was found with 1.25 – 5 µM resveratrol addition (P < 0.01). Moreover, supplementation with ≥80 µM resveratrol significantly improved antioxidant status but proved cytotoxic to spermatozoa at this dose.
Both pre- and post-cryopreservation, vitamin C, but neither melatonin nor NAC, significantly improved motility and vitality. (P < 0.05). A subsequent dose-dependent study with vitamin C established an optimal dose of 0.4 mM, this antioxidant level significantly improved motility, vitality and DNA fragmentation levels post-cryopreservation (P < 0.05).
Cryopreservation is harmful to human spermatozoa because this process induces oxidative stress. Utilising vitamin C to enhance the level of antioxidant protection during cryopreservation, led to significant improvements in total and progressive motility (P < 0.05) and a significant decrease in DNA fragmentation levels (P < 0.05) post freeze. This information has the potential to inform the design of improved cryoprotectant media.