The effect of ellagic acid on rabbit sperm in vitro parameters after cryopreservation

Abstract

Cryopreservation reduces the antioxidant activity of spermatozoa and makes them more sensitive to damage caused by reactive oxygen species (ROS). The addition of antioxidants to the freezing medium could prevent cryo-damage by mitigating the harmful effects of ROS and, thus, protecting the spermatozoa. This study aimed to evaluate the effect of ellagic acid (EA) on the rabbit sperm traits after freezing-thawing. Semen samples collected from New Zealand White rabbit males were cryopreserved in a BotuCrio freezing medium (Nidacon, Sweden) supplemented with different concentrations of ellagic acid (EA at 0, 0.5, 1.5 and 2.5 mM) using the manual slow freezing procedure. After thawing, sperm motility parameters were evaluated by CASA. The parameters of viability (DRAQ7), apoptosis (Yo-Pro-1), acrosome integrity (peanut agglutinin; PNA), intracellular ROS (Cell-ROX) and mitochondrial activity (MitoTracker) were evaluated by flow cytometry. EA added to the freezing medium at all concentrations led to a significant reduction (P < 0.05) in intracellular ROS in frozen-thawed sperm cells. However, this effect was not reflected in motility parameters. Semen supplemented with 1.5 mM EA also yielded a lower proportion of apoptotic cells compared to the control group. In conclusion, EA supplementation of semen extender demonstrated its antioxidative properties protecting spermatozoa against oxidative damage during cryopreservation. Nevertheless, to draw a definitive conclusion regarding the effect of EA on spermatozoa functionality, additional research is necessary.