Melatonin Alleviates Vitrification Damage in Sheep Oocytes by Enhanced Mitochondrial Function

Abstract

Vitrification plays an important role in protecting germplasm resources, but the vitrification method can have adverse effects on oocytes. In order to investigate the role of melatonin (MLT) in vitrification of oocytes and improve the vitrification freezing efficiency of oocytes, this study first examined the effects of adding different concentrations of MLT in vitrification/thawing solution on the survival and in vitro maturation rate of oocytes. Adding 10-9 M MLT to vitrification/thawing solution significantly enhanced the survival rate of oocytes (76.04% compared to 65.65%) and in vitro maturation rate (72.83% compared to 66.19%); consequently, 10⁻⁹M was determined as the optimal concentration and employed for subsequent experiments. Secondly, in contrast to the cryopreserved control group, MLT significantly reduced the early apoptotic rate of ovine cumulus-oocyte complexes (COCs) and enhanced the expansion ability of cumulus cells. Meanwhile, MLT significantly enhanced the expression of oocyte cytoskeletal protein F-actin and the spindle assembly ability. In addition, subsequent to the addition of MLT, the concentrations of reactive oxygen species (ROS) and Ca²⁺ within oocytes were significantly reduced, whereas the levels of glutathione (GSH), ATP content, mitochondrial activity, and membrane potential were significantly increased. In conclusion, our results demonstrate that the addition of 10-9 M MLT in the vitrification/thawing solution can alleviate the freeze-induced oxidative stress, enhance the antioxidant capacity of ovine oocytes, and restore the normal function of oocyte mitochondria as well as improve the quality of in vitro-matured ovine oocytes.

Keywords: ovine, oocytes, vitrification, melatonin, mitochondria