210 Effect of carvacrol and sericin supplementation on in vitro maturation medium of porcine oocytes

The ability to produce porcine embryos in vitro has improved over the years, allowing genetic manipulation and selection of healthy, high-quality embryos for use in xenotransplantation. However, IVM of porcine oocytes generates high levels of reactive oxygen species (ROS). The use of antioxidant substances in the IVM medium decreases the levels of ROS, increases the viability and quality of the oocytes and embryo development. The objective of this study was to analyse the effects of carvacrol (phenol from oregano that has demonstrated high efficiency in porcine cryopreserved semen) and sericin (silk protein derived from silkworms) in porcine IVM medium. Cumulus–oocyte complexes (COCs) were obtained by follicular aspiration from slaughterhouse ovaries. Immature COCs with up to three layers of cumulus cells and compact, homogeneous cytoplasm were in vitro matured for 44 h in tissue culture medium 199, supplemented with 0.3 mM Na pyruvate, 5 µg mL⁻¹ myo-inositol, 1 µL mL⁻¹ ITS, 1% v/v antibiotic-antimycotic, 10 μg mL⁻¹ FSH and 10% v/v porcine follicular fluid and divided into three groups: (1) 20 µM carvacrol (CARV), (2) 100 µM cysteamine (CYS) and (3) 1% v/v of sericin + 100 µM cysteamine (SER + CYS). The maturation process was carried out at 38.5°C in humidified air with 6.5% CO₂. Oocyte maturation was evaluated by extrusion of the first polar body, and oocyte degeneration and viability were also evaluated. Mature oocytes were separated from immature oocytes and treated with CellTracker^TM Blue (CTB) and fluorescein diacetate (FDA). Infinity Analyzer software was used to measure glutathion (GSH) and 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 seven times. Significant differences in maturation were found between the SER + CYS (160/321, 49.8 ± 13%) and CYS (128/321, 39.8 ± 13%) groups. No differences were found in the CARV group compared with the others. Additionally, the SER + CYS group (246/321, 76.6 ± 15%) showed higher viability compared with CYS (210/321, 65.4 ± 20%). Regarding ROS level, mature oocytes from SER + CYS group showed significantly lower levels compared with the CYS group. On the other hand, no significant difference was found in GSH levels of mature oocytes between all groups. Remarkably, GSH levels were higher in SER + CYS compared with the CYS group. Based on the results obtained in this study, SER + CYS supplementation in the IVM medium decreases intracellular levels of ROS, which contributes to an increase in oocyte viability and maturation rates. Further studies should be conducted to evaluate the effect of sericine in porcine IVF and embryo development.