52 Vitrification of equine germinal vesicle oocytes: an ongoing challenge

Equine oocyte vitrification still represents a challenge for researchers. Overnight holding (H) of equine immature oocytes may induce a preselection of the most competent oocytes (Merlo et al. 2022 Anim. Reprod. Sci. 245, 107071). Naloxone (Nx), an opioid receptor antagonist, was shown to exert a positive effect on bovine oocyte meiotic progression (Dell’Aquila et al. 2002 Mol. Reprod. Dev. 63, 210–222), and its antioxidant activity has been investigated (Migheli et al. 2023 Biochem. Biophys. Rep. 34, 101441). The aim of this study was to evaluate the effect of H and Nx on vitrified equine immature cumulus–oocyte complexes (COCs). The COCs were collected from abattoir ovaries from February to July, 2023. Combining the addition of Nx (8–10 mmol) and H, COCs were split into six groups: fresh control (F), holding control (H), vitrified (VIT), vitrified + Nx (VIT-Nx), holding + vitrified (H-VIT), holding + vitrified + Nx (H-VIT-Nx). COCs were vitrified using cryotops after three-step cryoprotectant exposure and warmed using three decreasing sucrose-containing solutions. Two hours after warming, COCs were denuded, evaluated, and only intact oocytes were stained. Annexin V/propidium iodide (PI) was used to assess oocyte viability and apoptosis (viable: no/low signal in the cytoplasmic membrane; apoptotic: a clear green signal in the membrane; necrotic: a discontinuous green signal in the membrane and red nucleus). H2DCFDA, Celltraker Blue CMF2HC, and JC1 were used to evaluate the concentrations of reactive oxygen species (ROS), glutathione (GSH), and the mitochondrial potential (MP), respectively. ImageJ was used to assess staining intensity. Data are expressed as mean ± standard deviation. Chi-square test was used to compare COCs integrity and AnnexinV/PI results; GLM and Tukey’s honestly significant difference were used to compare the intensity of the other stainings; significance was assessed for P < 0.05 (SPSS 25). Overall, 482 COCs were analysed. The rate of intact oocytes was greater (P < 0.05) for F (88.4%) than VIT-Nx (76.5%) but not different from H (81.1%), VIT (82.1%), H-VIT (82.9%) and H-VIT-Nx (84.9%). AnnexinV/PI viable oocytes were greater in F (13/17) and H (16/17) than in all vitrification groups (VIT 7/17; VIT-Nx 4/16; H-VIT 2/17; H-VIT-Nx 3/13). Apoptotic oocytes were greater (P < 0.05) in H-VIT (7/17) than F and H (1/17, for both). Necrotic oocytes were lower in H (0/17) than VIT-Nx (9/16), H-VIT (8/17) and H-VIT-Nx (8/13), while no significant difference was found with F (3/17) and VIT (5/17). ROS and GSH concentrations were not different among groups, while MP was greater (P < 0.05) for H-VIT-Nx (107.7 ± 64.1) than the others (F 34.6 ± 36.9, H 46.5 ± 49.5, VIT 49.9 ± 36.2, VIT-Nx 63.0 ± 45.1, H-VIT 36.8 ± 35.3). Although morphological evaluation after warming and staining for ROS, GSH, and MP were promising, Annexin/PI results showed that neither holding nor Nx addition exerted a positive effect on the success of vitrification of equine oocytes at germinal vescicle (GV) state. Vitrification induces oocyte apoptosis, a major cause that prevent GV oocytes from reaching maturity. Further research is needed to clearly understand which vitrification protocol is less harmful and to better explain the mechanisms behind these findings.