Quality, oxidative markers and DNA damage (DNA) fragmentation of red deer thawed spermatozoa after incubation at 37 °C in presence of several antioxidants

Antioxidants may be useful for supplementing sperm extenders. We have tested the effects of dehydroascorbic acid (DHA), TEMPOL (TPL), N-acetyl-cysteine (NAC) and rutin (RUT) on epididymal spermatozoa from red deer, during a long incubation at 37 C. Cryopreserved spermatozoa were thawed, washed and incubated with 1 mM or 0.1 mM of each antioxidant, including oxidative stress (Fe2+/ascorbate). Motility (CASA), viability, mitochondrial membrane potential, and acrosomal status were assessed at 2 and 4 h, and lipoperoxidation, intracellular ROS and DNA status (TUNEL) were checked at 4 h. Oxidative stress increased ROS, lipoperoxidation and DNA damage. In general, antioxidants had a negative effect on motility and physiological parameters. Only DHA 1 mM protected motility, improving some kinematic variables. However, it had a significantly detrimental effect on acrosomal status and DNA integrity, in absence of oxidative stress. TPL and RUT efficiently reduced lipoperoxidation, ROS, and DNA damage in presence of oxidative stress. NAC was not as efficient as TPL or RUT reducing lipoperoxidation or protecting DNA, and did not reduce ROS, but overall its negative effects were lower than the other antioxidants when used at 1 mM. Our results show that these antioxidants have mixed effects when spermatozoa are incubated at physiological temperatures. DHA may not be suitable due to pro-oxidant effects, but TPL, NAC and RUT may be considered for cryopreservation trials. NAC might be tried at higher concentrations. In general, exposure of red deer spermatozoa to these antioxidants should be limited to low temperatures, when only protective effects may develop