Background: In vitro production of bovine embryos is a well-established technology, but the in vitro culture (IVC) system still warrants improvements, especially regarding embryo quality. This study aimed to evaluate the effect of extracellular vesicles (EVs) isolated from oviductal (OF) and uterine fluid (UF) in sequential IVC on the development and quality of bovine embryos. Zygotes were cultured in SOF supplemented with either BSA or EVs-depleted fetal calf serum (dFCS) in the presence (BSA-EV and dFCS-EV) or absence of EVs from OF (D1 to D4) and UF (D5 to D8), mimicking in vivo conditions. EVs from oviducts (early luteal phase) and uterine horns (mid-luteal phase) from slaughtered heifers were isolated by Size Exclusion Chromatography. Blastocyst rate was recorded on Days 7-8 and their quality was assessed based on lipid contents, mitochondrial activity and total cell numbers, as well as survival rate after vitrification. Relative mRNA abundance for lipid metabolism related transcripts and levels of active hormone-sensitive lipase (pHSL) proteins were also determined. Additionally, the expression levels of 383 miRNA in OF- and UF-EVs were assessed by pRT-PCR.
Results: Blastocyst yield was lower (p<0.05) in BSA groups compared to dFCS groups. Survival rates after vitrification/warming were improved in dFCS-EVs compared to dFCS (p<0.05). EVs increased (p<0.05) blastocysts total cell number in dFCS-EV and BSA-EV compared to dFCS and BSA groups, while lipid content was decreased and mitochondrial activity increased in dFCS-EV (p<0.05). Lipid metabolism transcripts were affected by EVs ( PPARGC1B and ACACA , p<0.05) and protein source in the medium ( CD36, PLIN2 and ATGL , p<0.05). Levels of pHSL were lower in dFCS (p<0.05). Twenty miRNA were differentially expressed between OF- and UF-EVs and only bta-miR-148b was increased in OF-EVs (p<0.05).
Conclusions: Mimicking physiological conditions using EVs from OF and UF in sequential IVC does not affect embryo development but improves blastocyst quality regarding survival rate after vitrification/warming, total cell number, lipid content, mitochondrial activity and relative changes in expression of lipid metabolism transcripts and lipase activation. Besides, EVs miRNA contents may contribute to the observed effects.