Melatonin supplementation to the freezing medium enhances post-thaw sperm quality and fertility of giant grouper (Epinephelus lanceolatus)

Abstract

Giant grouper (Epinephelus lanceolatus) is not only favored in the international live reef-fish trade but also a promising species for mariculture in coastal communities throughout Asia. Sperm cryopreservation contributes to the extensive utilization of artificial insemination in grouper aquaculture industry. Inevitable damage during the process of freezing can negatively affect post-thaw sperm quality and fertility, thus hindering its routine application in practice. Melatonin is an antioxidant that can protect sperm from the adverse effects of cryopreservation in a variety of species. This study was aimed to assess the effect of melatonin addition on post-thaw sperm function of giant grouper. Semen samples were cryopreserved with freezing medium containing different final concentrations of melatonin (1, 2 and 4 μM) and the control group (without melatonin). After thawing, multiple sperm functional parameters were evaluated. Generally, a positive effect on cryopreservation was observed for all doses of melatonin. Melatonin treatment at 1 and 2 μM seemed to be efficient in preventing sperm cryodamage, which yielded significantly higher total motility, progressive motility, mitochondrial membrane potential (P < 0.05) and improved velocity (VCL, VAP, VSL) compared to controls. Lower apoptotic-like changes were detected at 2 μM treatment. Samples frozen with melatonin also contributed to greater sperm viability and lesser DNA damage. Although not statistically different from control, a reduction in intracellular ROS was found under the melatonin treatments. Moreover, melatonin supplementation improved the fertility of frozen-thawed sperm, which increased significantly the hatching rate under a limited sperm/egg ratio (10,000:1). Hence, our study suggests inclusion of melatonin to semen extender can improve the post-thaw quality of cryopreserved giant grouper spermatozoa, the recommended concentration of melatonin is 1–2 μM in the cryomedium.