52 Extracellular vesicles from oviduct and uterus in sequential invitro culture affects mitochondrial activity and lipid metabolism transcripts in bovine embryos

Oviducal fluid (OF) and uterine fluid (UF) improve the quality of embryos during invitro culture, and their extracellular vesicles (EV) may be involved in such an effect. We aimed to evaluate the effect of EV from OF and UF in sequential invitro culture on the development and quality of bovine embryos. Zygotes were cultured in synthetic oviduct fluid supplemented either with 3mg mL−1 BSA (n=1584) or 5% EV-depleted fetal calf serum (dFCS, n=1594) in absence or presence (BSAEV, n=1853 and dFCSEV, n=1473) of 3×105 EV mL−1 from OF (Day 1 to Day 4) and UF (Day 5 to Day 8), mimicking invivo conditions. EV from oviducts (early luteal phase) and uterine horns (mid luteal phase) from slaughtered heifers were isolated by size exclusion chromatography; size and concentration were assessed by nanotracking analysis (NTA) and morphology by transmission electron microscopy (TEM). Blastocyst rate was recorded on Days 7–8 and their quality was assessed for mitochondrial activity by staining with Mitotracker Deep Red (ThermoFisher Scientific), survival rate after vitrification/warming by invitro culture for up to 72h, and relative mRNA abundance of lipid metabolism-related transcripts by quantitative PCR. Housekeeping genes were H2AFZ and ACTB. Data were analysed by one-way ANOVA and Tukey test. TEM confirmed the presence and morphology of EVs, and NTA indicated mode size and concentration of particles (137.2 and 151.2 nm; 2.97×1010 and 7.98×1010 particles mL−1, for OF and UF, respectively). Blastocyst yield was lower (P