β-Carotene addition into a Tris-based diluent improves Hu ram sperm parameters after cryopreservation

Abstract

The effect of β-Carotene on ram semen cryopreservation has not been previously evaluated. The present research aimed to investigate the impact of different concentrations of β-Carotene in the diluent on the quality of ram semen and to analyze its antioxidant effects by the addition of tert-butyl hydroperoxide (TBHP) during cryopreservation. Semen samples were diluted with a Tris-based extender containing the β-Carotene (0, 10, 20, 30 and 40 mg/L). The motility and biokinetic characteristics, membrane and acrosome integrity, related parameters of oxidative stress, mitochondrial membrane potential (MMP) and apoptosis rate were measured after the cryo-preservation. The results indicated that the 20 mg/L β-Carotene group significantly improved total motility (TM), progressive motility (PM), average motion degree (MAD), membrane and acrosome integrity, total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and catalase (CAT) compared to the other groups (P < 0.05). Additionally, this group significantly reduced reactive oxygen species (ROS) and malondialdehyde (MDA) content compared to the control group (P < 0.05). Furthermore, the TM, PM, wobble (WOB), MAD, membrane and acrosome integrity, CAT, SOD, T-AOC and MMP in the 100 μM TBHP+20 mg/ml β-Carotene group were higher than those in the 100 μM TBHP group (P < 0.05). The combined supplementation group also significantly decreased the ROS, MDA, the protein level of Cytochrome C and sperm apoptosis rates (P < 0.05). Therefore, 20 mg/L is identified as the optimal concentration for the cryopreservation of ram semen, and β-Carotene can improve the oxidative stress damage during the frozen-thawed process by enhancing the antioxidant capacity of sperm and maintaining mitochondrial function.