Effect of several antioxidants on thawed ram spermatozoa submitted to 37°C up to four hours

Abstract

Thawed ram spermatozoa were incubated at 37°C in the presence of dehydroascorbic acid (DHA), TEMPOL (TPL), N-acetyl-cysteine (NAC) and rutin (RUT), at 0.1 and 1mm, in order to test their effects on sperm physiology. Cryopreserved spermatozoa from four rams were thawed, pooled, washed and incubated in TALP-Hepes with 1mm or 0.1mm of each antioxidant, performing a replicate with induced oxidative stress (Fe 2+/ascorbate). Motility (CASA), viability and mitochondrial membrane potential (flow cytometry) were analysed at 2 and 4h. Lipoperoxidation (MDA production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were analysed at 4h. Antioxidants, except DHA 0.1mm, decreased motility and kinematic parameters, but had little effect on viability or mitochondrial activity. Except 1mm DHA, the antioxidants reduced ROS at 4h. Moreover, NAC 1mm, rutin and TEMPOL reduced ROS and DNA damage in the presence of oxidative stress. N-acetyl-cysteine, rutin 1mm and TEMPOL reduced lipoperoxidation in the presence of oxidative stress. However, DHA did not affect lipoperoxidation. At 1mm, DHA increased DNA damage in the absence of oxidative stress. Dehydroascorbic acid effects could arise from spermatozoa having a low capacity for reducing it to ascorbic acid, and it may be tested in the presence of other antioxidants or reducing power. Future research should focus in testing whether the inhibition of motility observed for NAC, rutin and TEMPOL is reversible. These antioxidants might be useful at lower temperatures (refrigerated storage or cryopreservation) when their protective effects could be advantageous.