Mitochondrial function, blastocyst development and live foals born after ICSI of immature vitrified/warmed equine oocytes matured with or without melatonin

Abstract

Oocyte vitrification is considered experimental in the horse with only three live foals reported. The oxidative conditions induced by vitrification could in part explain the poor results and melatonin, a powerful antioxidant, could stimulate ROS metabolization and restore mitochondrial function in these oocytes. Our objective was to determine the oxidative status of vitrified equine oocytes and to analyze the effect of melatonin on mitochondrial-specific ROS (mROS), oocyte maturation, ICSI embryo development and viability. Immature, abattoir-derived oocytes were held for 15 h and vitrified in a final concentration of 20% EG, 20% DMSO and 0.65 M trehalose. In Experiment 1, overall ROS was determined by DCHF-DA; vitrification increased ROS production compared to non-vitrified controls (1.29 ± 0.22 vs 0.74 ± 0.25 a. u.; P = 0.0156). In Experiment 2, mROS was analyzed by MitoSOX™ in vitrified/warmed oocytes matured with (+) or without (−) supplementation of 10−9 M melatonin; mROS decreased in vitrified and non-vitrified oocytes matured in presence of melatonin (P < 0.05). In Experiment 3, we assessed the effect of melatonin supplementation on oocyte maturation, embryo development after ICSI, and viability by pregnancy establishment. Melatonin did not improve oocyte maturation, cleavage or blastocyst rate of non-vitrified oocytes. However, vitrified melatonin (+) oocytes reached similar cleavage (61, 75 and 77%, respectively) and blastocyst rate (15, 29 and 26%, respectively) than non-vitrified, melatonin (+) and (−) oocytes. Vitrified, melatonin (−) oocytes had lower cleavage (46%) and blastocyst rate (9%) compared to non-vitrified groups (P < 0.05), but no significant differences were observed when compared to vitrified melatonin (+). Although the lack of available recipients precluded the transfer of every blastocyst produced in our study, transferred embryos from non-vitrified oocytes resulted in 50 and 83% pregnancy rates while embryos from vitrified oocytes resulted in 17 and 33% pregnancy rates, from melatonin (+) and (−) treatments respectively. Two healthy foals, one colt from melatonin (+) and one filly from melatonin (−) treatment, were born from vitrified/warmed oocytes. Gestation lengths (considering day 0 = day of ICSI) were 338 days for the colt and 329 days for the filly, respectively. Our work showed for the first time that in the horse, as in other species, intracellular reactive oxygen species are increased by the process of vitrification. Melatonin was useful in reducing mitochondrial-related ROS and improving ICSI embryo development, although the lower pregnancy rate in presence of melatonin should be further analyzed in future studies. To our knowledge this is the first report of melatonin supplementation to an in vitro embryo culture system and its use to improve embryo developmental competence of vitrified oocytes following ICSI.