Naloxone supplementation during vitrification of equine in vitro matured oocytes after overnight holding: insights from a comparative study with the bovine model

Cryopreservation of equine mature oocytes remains a major challenge in assisted reproduction, mainly due to the limited availability of material and logistical constraints requiring a holding phase. To address these issues, this study evaluated whether naloxone (NX), an opioid receptor antagonist with reported antioxidant properties, could improve vitrification outcomes and whether bovine oocytes could serve as a suitable preliminary and supportive model. Two experiments were conducted. In experiment 1, immature (bGV) and mature (bMII) bovine oocytes were vitrified without (VIT) or with NX (VIT-NX) to assess protocol efficiency and evaluate whether results obtained in bovine MII oocytes (without holding) could serve as a counterpart to equine oocytes subjected to overnight holding. In experiment 2, equine in vitro matured oocytes after overnight holding (eMII) were vitrified with or without NX, and analysed for viability, reactive oxygen species (ROS), glutathione (GSH), high mitochondrial membrane potential (HMMP), and developmental competence following intracytoplasmic sperm injection (ICSI). Expression of apoptosis-related genes (BCL2, BAX, p53, survivin) was assessed by qRT-PCR. In bovine oocytes, NX did not affect vitrification efficiency or maturation rates, whereas oocytes vitrified at the MII stage yielded a higher proportion of viable mature oocytes after warming (bGV 13.5 %, vs. bMII 46.1 %) (P < 0.05). In equine oocytes, NX negatively affected post-warming viability (eMII-VIT-NX 64.7 ± 17.3) compared with non-vitrified oocytes (eMII 91.1 ± 16.9), with intermediate results observed for vitrification without NX (eMII-VIT 74.9 ± 25.0). NX did not significantly affect GSH or HMMP (P > 0.05), but prevented the increase in intracellular ROS levels induced by vitrification. Overall, vitrification reduced cleavage rates and increased degeneration (P < 0.05), with no differences between vitrification protocols. qRT-PCR revealed stable BCL2 expression, inconsistent detection of pro-apoptotic genes, and no significant differences among groups in the BAX:BCL2 ratio, indicating limited transcriptional activation of apoptosis. In conclusion, naloxone supplementation did not improve equine MII oocyte survival or developmental competence, although demonstrated antioxidant activity. Bovine oocytes confirmed the value of a preliminary model to test oocyte vitrification protocol efficiency but could not reliably predict equine responses to naloxone.