198 Seasonal effect of in vitro prematuration with follicular fluid and follicular fluid small extracellular vesicles on bovine oocyte developmental competence

Elevated environmental temperature and humidity during summer can disrupt follicular function and compromise oocyte competence. Both parameters are accounted for by the temperature-humidity index (THI). In vitro prematuration of oocytes in the presence of follicular fluid (FF) or follicular-fluid small extracellular vesicles (FF-sEV), could prevent spontaneous oocyte maturation, allowing the oocyte time to complete the required nuclear and cytoplasmic processes for developmental competence. The aim of this study was to assess the effect of a 6-h pre-IVM with FF or FF-sEV, collected during summer or during winter, on oocyte meiotic resumption and embryo development. Independent FF samples (n = 3 per season) were obtained from 2–8 mm follicles from abattoir ovaries, during the austral winter (July–August 2021, average THI: 54 ± 7) and summer (January–March 2022, THI: 73 ± 3). The FF was centrifuged at 3000g for 30 minutes at 4°C and the supernatant was filtered using a 0.2-µm filter. The FF-sEV were extracted from 1 mL of FF using ExoQuick^TM, resuspended in PBS, and stored at −80°C until analysis. A volume of 10 µL from purified FF-sEV was used to determine the concentration and size distribution through microfluidic resistive pulse sensing. The small extracellular vesicles were prepared by adding 5 µL from FF-sEV to 500 µL of maturation medium. Immature cumulus–oocyte complexes (COCs) were collected during winter (June–August 2022, THI: 54 ± 6) and distributed into five treatments: IVM for 24 h with 10% fetal bovine serum (control group), pre-IVM for 6 h with 10% winter or summer FF and pre-IVM for 6 h with winter or summer FF-sEV (~7.0 × 10⁹ particles mL⁻¹). The COCs from both prematuration groups followed a 22-h IVM. The COCs were collected after pre-IVM (40 per group) and after IVM (52–55 per group) for meiotic progression assessment. The remaining COCs were fertilized, and presumptive zygotes (106–116 per group) were cultured for 8 days. Expanded blastocysts (6–7 per group) were used for cell counting and apoptotic index evaluation. Three replicates were conducted, and data were analysed by one-way analysis of variance with Tukey’s test. The concentration and size of FF-sEV from winter and summer were not different (P > 0.05, data not shown). Prematuration with winter FF tended to decrease the rate of germinal vesicle breakdown and increase embryo quality by decreasing apoptosis (Table 1). These findings suggest that, during summer, the capacity of FF to delay oocyte spontaneous maturation is decreased. Additionally, summer reduces the ability of FF and FF-sEV to increase oocyte developmental competence.