Effects of holding and the addition of naloxone on vitrification of equine immature oocytes

Abstract

This study investigates the effects of overnight holding and naloxone (Nx) supplementation on the vitrification outcomes of equine immature oocytes. Oocytes were divided into six experimental groups based on treatment combinations: fresh (F) and held (H) control oocytes, oocytes vitrified with or without Nx (10⁻⁸ M) (VIT and VIT-Nx), oocytes vitrified after overnight holding with or without Nx (10⁻⁸ M) (H-VIT and H-VIT-Nx). They were assessed for survival, meiotic competence, intracellular oxidative stress, mitochondrial activity and distribution, apoptosis, and apoptotic gene expression. At survival rate determination, the degeneration rate was higher in VIT and VIT-Nx compared to F (P < 0.05). The highest maturation rate was observed in VIT-Nx. A significant reduction in ROS levels was observed in H compared to F (P < 0.05). ROS levels were similar between F and VIT, while the Nx supplementation tended to increase them (VIT-Nx vs F: P = 0.053; VIT-Nx vs VIT: P = 0.069). Conversely, in oocytes vitrified after overnight holding, vitrification induced an increase in ROS levels (H vs VIT: P < 0.05), which was not observed in H-VIT-Nx. GSH intracellular levels showed significant differences only in held oocytes, with higher GH levels in H compared to H-VIT and H-VIT-Nx (P < 0.05). All treatments induced an increase in HMMP levels compared to F (P < 0.05). In H oocytes, mitochondria were distributed throughout the entire oolemma (TOMM20) and active mitochondria (D-LAT) were detected in the outermost region. Incontrast, in H-VIT-Nx, potentially active mitochondria were spread throughout the cytoplasm. AnnexinV/PI staining revealed that the percentage of viable oocytes was higher (P < 0.05) in F and H than in all vitrified/warmed oocytes, and H-VIT-Nx had the highest degeneration rate (P < 0.05). RT-PCR analysis confirmed the detection for both reference genes, and target genes BCL2 and Survivin in all samples. In contrast, BAX and p53 transcripts were consistently undetectable. No significant differences were observed in the expression of BCL2 and Survivin between groups. In conclusion, overnight holding at uncontrolled room temperature can alter oocyte characteristics and lead to variable results after vitrification. Nx demonstrated contrasting antioxidant effects depending on the vitrification timing, but it appeared to improve IVM outcomes in oocytes vitrified immediately after collection.