Assessing the effects of bovine embryo-derived extracellular vesicles on the development of individually cultured bovine embryos

In vitro embryo production requires an enriched microenvironment with various vital cell-secreted factors. In vitro cultured single bovine embryos have demonstrated lower blastocyst rate compared to grouped cultured embryos. We assumed that extracellular vesicles (EVs) within an embryo culture system may affect normal in vitro development. This study aimed to assess the supplementation effects of bovine embryo-derived EVs on the development of individually cultured bovine embryos. Bovine oocytes were in vitro maturated (IVM) for 24 h and then in vitro fertilized (IVF). In preliminary experiments, we established that group cultured embryos in EV depleted Bovine Serum Albumin (BSA) media successfully completed their development; while single cultured embryos were only able to reach the morula stage and then degenerated. Hence, we tested EVs supplementation effects in droplets of EV depleted BSA media covered by mineral oil. EVs used for supplementation were produced from single embryos cultured for 8 days in droplets of BSA culture media under mineral oil. Conditioned medium was collected on day 5. EVs were purified, using Izon columns, from embryos which reached the blastocyst stage and embryos which cleaved on day 2 then degenerated. Non-EV supplemented single embryos cultured in BSA media were considered as control. Purified EVs were characterized by nanoparticle tracking analysis and transmission electron microscope (TEM). A total of 8.8 ×106 particles/ml, which we assumed to be the approximate amount of EVs that a single embryo may release during in vitro culture, was supplemented to each droplet on day 4 post-fertilization. Cleavage rates were 70 and 80% for the supplemented groups and 86% for the control. Morula rates were 40%, 47%, and 47% respectively. No blastocyst was observed within the supplemented groups while the control group counted 33% of blastocysts. Our study suggests that BSA EVs support single cultured embryos to complete their development and that a single embryo needs a significant amount of EVs to reach the blastocyst stage. More researches are needed to understand the role of culture media EVs in supporting single embryo development.