221 Supplementation of bovine oocyte maturation media with umbilical cord-derived exosomes

The supplementation of maturation medium with mesenchymal stem cell (MSC)-conditioned media (CM) has been shown to increase blastocyst rates in bovine, porcine, and murine models. MSCs are commonly utilised in therapies due to their wide range of action and paracrine effects; the paracrine capacity of MSC is partly due to its secretion of exosomes (EX). This study aims to compare the effects of MSC-conditioned media and EX as supplements for oocyte maturation on cleavage and blastocyst rates as well as assess the incorporation of MSC EX. MSC samples were isolated from the Wharton jelly of equine foals post-parturition and cultured at 37°C in 5% CO₂ and air; CM was collected from MSC cultured in a basal media containing either EX-depleted fetal bovine serum ([FBS] ED-CM) or regular FBS (R-CM). Exosomes were isolated from CM via the ExoQuick-TC Ultra kit (System Biosciences) and diluted to reach initial sample volume. In five replicates, in vitro-produced (IVP) embryos were produced by aspirating cumulus-oocyte complexes from 2 mm to 8 mm follicles of abattoir bovine ovaries. COCs were randomly divided into four treatments (10% R-CM, ED-CM, R-EX, ED-EX) and one control. COCs (n = 1,725) were matured for 23 h at 38.5°C in 5% CO₂ and air, fertilised with frozen-thawed semen from one of two bulls, and cultured in SCSF1 medium at 38.7°C, 6.2% CO₂, 7% O₂, and 86.8% N₂. On Days 7.5 and 8.5 post-fertilisation, cleavage and blastocyst rates were evaluated. Cleavage and blastocyst rates were analysed utilising ANOVA and l.s.d. with JMP. To assess EX incorporation, EX samples were fluorescently tagged utilising Exo-Glow (System Biosciences) and diluted to initial conditioned media sample volumes or remained concentrated. Additional COCs (n = 21) were matured in media supplemented with 10% fluorescently labelled EX samples, under diluted or concentrated conditions, and after 12 h were imaged using confocal microscopy. Fluorescent intensity was measured by Image J software (National Institutes of Health) and assessed in JMP utilising the Shapiro-Wilk test. Results indicated similar (P > 0.05) cleavage and blastocyst rates between all groups (Control 82.77 ± 2.13 and 25.63 ± 2.37%, R-CM 84.67 ± 1.50 and 21.70 ± 3.98%, ED-CM 84.59 ± 2.25 and 24.03 ± 5.60%, R-EX 81.33 ± 4.32 and 16.39 ± 7.62%, and ED-EX 85.54 ± 1.69 and 16.39 ± 5.68%, respectively). The undiluted R-EX incorporated into the COC during the maturation period (P < 0.0125), however, concentrated ED-EX and diluted R-EX and ED-EX did not show evidence of incorporation (P > 0.0125). These results suggest that MSC-conditioned media and EX do not increase developmental competence of in vitro-matured bovine oocytes and displayed that EX uptake can occur at high concentrations. EX uptake in R-EX group was visually limited to the cumulus cells. Further studies must be done to analyse abundance of EX produced by MSC in R and ED medium and quantify localisation of EX uptake.