Sperm cryopreservation with supplementation of α-tocopherol and ascorbic acid in freezing media increase sperm function and fertility rate in atlantic salmon (salmo salar)

Abstract

The use of alpha-tocopherol and ascorbic acid in freezing medium prevents lipoperoxidation and damage by ROS (reactive oxygen species); however, the effects of these antioxidants in salmonid spermatozoa have not been studied. In this work the protective effects of antioxidants on the enzymatic activity and fertilization capacity of frozen Atlantic salmon spermatozoa were studied. Spermatozoa were frozen in Cortland (R) medium supplemented with 1.3 M DMSO, 0.3 M glucose, 2% BSA and different concentrations of alpha-tocopherol and ascorbic acid; spermatozoa frozen without antioxidants were used as control and fresh sperm. Samples loaded in 0.5 ml plastic straws were frozen at 68 degrees C/min (Freeze Control (R)). Thawing was carried out at 35 degrees C and post-thawing lipid peroxidation (LPO) was evaluated along with Glutathione (GSH/GSSG) levels; and Glutathione peroxidase (GPx) and Catalase (CAT) activity. Additionally, percentage of motility, mitochondrial membrane potential (ATM), and fertilization rate were assessed. Results showed significantly lower LPO values (expressed as malondialdehyde concentration) in spermatozoa cryopreserved with alpha-tocopherol (0.1 mM and 0.5 mM) and with combinations of alpha-tocopherol/ascorbic acid (0.1 mM/1.0 mM and 0.5 mM/10 mM ratios) (p < .05). Moreover, spermatozoa frozen with alpha-tocopherol/ascorbic acid also exhibited significantly higher GPx and CAT activity as well as GSH/GSSG ratio compared to controls (p < .05). The production of O-2(i)- also decreased with the combined treatment, however, no statistically significant difference was found compared to controls. Consistently with the improvement in antioxidant defences, the percentage of motility and Delta Psi M were highest and significantly different in spermatozoa cryopreserved with alpha-tocopherol/ascorbic acid 0.1 mM/1.0 mM compared to controls, supporting the significantly higher fertilization rate exhibited by spermatozoa under the same treatment.