234 Effect of sericin supplementation in synthetic oviductal fluid medium for in vitro maturation of canine oocytes

Assisted reproductive technologies in the canine species are limited due to the low efficiency of IVM. Unlike other mammals, female dogs (bitches) ovulate oocytes in the germinal vesicle stage and complete the Metaphase II (MII) after 48–72 h in the oviducal environment, becoming fertilisable. The aim of this study was to evaluate the effect of sericin (a protein source from the Bombyx mori silkworm, SER) supplementation on the IVM medium of canine oocytes. Previous study in our laboratory demonstrated the efficiency of sericin supplementation in the IVM medium and embryo culture in bovine (Velasquez Vasquez et al. 2023 Reprod. Fertil. Dev. 35, 181, https://doi.org/10.1071/RDv35n2Ab108). Other studies in our laboratory demonstrated that synthetic oviducal fluid (SOF) with insulin-transferrin-selenium (ITS) and low O₂ tension improves maturation rates up to 36.5% (Duque Rodriguez et al. 2021 Front. Cell Dev. Biol. 9, 694889, https://doi.org/10.3389/fcell.2021.694889). Additionally, bovine serum albumin (BSA) and fetal bovine serum (FBS) were evaluated as alternative protein sources. Synthetic oviducal fluid without protein sources (SOF-WPS) was used as a negative control. Ovaries were obtained from ovariohysterectomy at veterinary clinics and transported to the laboratory within 2 hours. Cumulus–oocyte complexes (COCs) were matured in vitro for 48 h in SOF medium supplemented with 1 μL mL⁻¹ ITS, 1% v/v ATB and divided into four groups: (1) SOF + 8% BSA + 2.5% FBS, (2) SOF + 1% SER, (3) SOF + 8% BSA, and (4) SOF-WPS. Subsequently, COCs were incubated in a modified atmosphere with 5% CO₂, 5% O₂, and 90% N₂ at 38.5°C with humidified air. Maturation was evaluated through the extrusion of the first polar body, and oocyte degeneration and viability were also assessed. Mature oocytes were separated from immature oocytes and treated with CellTracker™ Blue (CTB) and fluorescein diacetate (FDA). Infinity Analyzer software was used to measure glutathione (GSH) and reactive oxygen species (ROS) levels. Data were analysed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc.), and differences were considered significant at P < 0.05. The experiment was repeated six times. The results suggest that SOF + 8% BSA + 2.5% FBS (88/192, 45.8%) reached a higher maturation rate than SOF + 8% BSA (67/191, 35.08%) but showed a similar maturation rate to the other groups (SOF + 1% SER, 73/178, 41%; SOF-WPS, 65/169, 38.4%). Significant difference was found in GSH levels of mature oocytes in SOF + 1% SER group compared with the other groups, indicating a possible antioxidant property of sericin. In conclusion, the implementation of optimized supplementation in oocyte maturation medium is crucial for achieving better maturation rates and open up new possibilities to improve the efficiency of assisted reproductive technologies in canines.