Melatonin Added to Cryopreservation Extenders Improves the Mitochondrial Membrane Potential of Postthawed Equine Sperm

Reactive oxygen species (ROS) can induce sperm damage, especially after cryopreservation. Melatonin (MEL) is a potent amphiphilic antioxidant, allowing free penetration to any compartment of cell. Ferulic acid (FA) is a phenolic compound exhibiting a wide range of beneficial effects against several pathologies due to its antioxidant property. This study aimed to evaluate the effect of MEL and FA on sperm quality of cryopreserved equine semen. Five ejaculates from four stallions were collected. For each ejaculate, the control group (conventional freezing extender BotuCrio; Botupharma, Botucatu, SP, Brazil) and two different concentrations of each antioxidant (MEL 2 mM, MEL 1 μM, FA 0.5 mM, and FA 1.2 mM) were tested. Sperm kinetics were assessed by CASA system (SCA). Integrity of plasma and acrosomal membranes and mitochondrial potential were assessed using fluorescent probes PI, Hoechst 33342, FITC-PSA, and JC-1. Sperm ROS production was evaluated by CellRox Deep Red. Comparisons among treatments were analyzed by generalized linear model (PROC GLM in SAS, version 9.3), and differences were compared with Duncan's test (a value of P ≤ .05 was considered significant). MEL 1 μM treatment demonstrated higher percentage of sperm cells presenting intact plasma membrane, intact acrosome, and high mitochondrial membrane potential, which was due to higher percentage of sperm cells with high mitochondrial membrane potential. No differences among treatments were observed on ROS evaluation. It was concluded that semen treatment with 1 μM MEL improves mitochondrial function of equine sperm cells submitted to cryopreservation process.