191 Effect of different concentrations of eugenol in maturation medium on the maturation, oxidative status, and developmental competence of porcine oocytes

Eugenol (EU) is a bioactive compound with defined attributes, including a role in reducing oxidative stress. Therefore, EU is an ideal candidate for improving in vitro embryo production (IVEP) in an environment that favors the formation of reactive oxygen species (ROS). Our aim was to assess the effect of different concentrations of EU on the oxidative status and embryonic development of porcine oocytes during IVM. Oocytes were cultured in IVM medium consisting of TCM-199 with 10% (vol/vol) porcine follicular fluid, 0.6 mM cysteine, 50 µg mL⁻¹ gentamicin, 50 µM sodium pyruvate, 2 mg mL⁻¹d-sorbitol, 10 IU mL⁻¹ eCG, and 10 IU mL⁻¹ hCG, covered with mineral oil at 39°C and 5% CO₂ for 22 h, and then incubated for 22 h in the absence of hormone. Oocytes were allocated into five groups and treated with EU at 0, 10, 40, 80, and 120 µM to assess their antioxidant effects. As a control, oocytes were cultured in IVM medium without EU and ethanol. In Experiment 1, oocytes were assessed for meiotic competence and DNA integrity by TUNEL assay. In Experiment 2, oxidative status was measured by intracellular ROS and glutathione (GSH) levels. Fluorescence signals were detected under fluorescence microscopy. Images were captured as TIFF files, and fluorescence intensity was analyzed using NIH ImageJ software. In Experiment 3, matured oocytes were co-incubated with frozen-thawed sperm (1 × 10⁶ sperm mL⁻¹) for 5 h and then cultured in vitro for 7 days at 39°C, 5% CO₂, 5% O₂, and 90% N₂ to assess subsequent fertilization, development, and embryo quality. Four to six replicates (30–40 oocytes per replicate) were conducted. Data were analyzed by variance analysis (ANOVA) followed by Fisher’s PLSD test using STATVIEW. All concentrations of EU increased the MII rate compared with the untreated groups (73.7%–80.9% vs. 56.0%–60.1%; P < 0.05); however, there was no difference in the DNA integrity of oocyte nucleus between the groups. ROS levels decreased in the 10, 40, and 80 µM EU groups compared with the untreated groups (8.8–9.3 pixels/oocyte vs. 10.6–11.2 pixels/oocyte; P < 0.05); GSH levels increased in the 10, 40, and 80 µM EU groups compared with the control group (69.5–73.0 pixels/oocyte vs. 64.4 pixels/oocyte; P < 0.05). The monospermic fertilization rates of EU-treated oocytes were significantly higher than those of the untreated groups (83.9%–94.8% vs. 60.0%–63.4%; P < 0.05). Only 80 µM EU significantly improved blastocyst development compared with the untreated group (13.0% vs. 3.6%–7.0%; P < 0.05). Moreover, the addition of EU reduced the proportion of DNA fragments in blastocysts compared with controls (2.1%–2.9% vs. 8.3%; P < 0.05) but had no effect on the total cell number of blastocysts. Overall, 80 µM EU is sufficient to generate a significant effect to mitigate the oxidative status and optimize IVM and embryo development. This study provides information on EU, a novel natural antioxidant that improves the efficiency of IVEP in pigs.