Fig. 1
Experimental design. A EVs isolation and characterization. Oviducts (n = 5) and uterine horns (n = 5), ipsilateral to a Stage 1 (oviduct) or Stage 2 (uterus) corpus luteum, were flushed with PBS (1 and 2 mL, respectively), and after centrifugation to remove cells and cellular debris, samples were filtered (0.22 μm) and EVs isolated by size exclusion chromatography. EVs samples were concentrated by ultracentrifugation and pellets resuspended in 100 μL PBS. Thirty microliters from each of the five EVs suspensions from oviduct (150 μL) and uterus (150 μL) were pooled and from this, 5 μL were used for EVs characterization by nanoparticle tracking analysis (NTA) and 5 μL for transmission electron microscopy (TEM), and the rest was frozen at − 20 °C and used in IVC. The remaining volume of the original five EVs samples (70 μL) was used for miRNA analysis. To have enough protein amount (35 μg) for EV biomarkers to be detectable by western blot, a pool of 10 oviducts (Stage 1) and 10 uterine horns (Stage 2) flushings was obtained and prepared the same way. B Embryo culture in vitro. At approximately 20 h after insemination, presumptive zygotes were cultured in 4 treatments: BSA: SOF with 0.3% BSA (3 mg/mL, w/v); dFCS: SOF with 5% dFCS; BSA-EV: SOF with 0.3% BSA supplemented with 3 × 105 EVs/mL from OF (D1 to D4) and 3 × 105 EVs/mL from UF (D4 to D9); and dFCS-EV: SOF with 5% dFCS supplemented with 3 × 105 EVs/mL from OF (D1 to D4) and 3 × 105 EVs/mL from UF (D4 to D9). BSA and dFCS treatments underwent media renewal at 96 hpi (day 4). Blastocyst development was assessed on days 7, 8 and 9. A representative number of day 7–8 blastocysts from each group was assessed for quality either by vitrification/warming (survival rate at 24, 48 and 72 h) or fixed and stained for total cell number, mitochondrial activity and lipid content and analysis. In addition, day 7–8 blastocysts were frozen in liquid nitrogen in groups of 10 and stored at − 80 °C for gene expression or western blot for protein analyses. C miRNA contents in EVs. Total RNA in oviducts and uteri EVs samples (n = 3) was extracted with a miRNeasy mini kit, reversed transcribed with the miScript PCR System and relative miRNA levels determined by qRT-PCR