65 Effect of oviductal fluid extracellular vesicle supplementation during invitro culture on development and quality of bovine embryos

Recently, it has been postulated that oviductal extracellular vesicles (oEV) might act as natural nanoshuttles bringing key components (small noncoding RNAs and proteins) of the oviduct into gametes and embryos. Furthermore, co-incubation of frozen-thawed oEV with invitro-produced bovine embryos was reported to increase blastocyst rate and quality (Almiñana et al. 2017 Reproduction 154, 153-168). The objective of this study was to determine the dose-dependent effect of oEV supplementation of embryo culture medium on the invitro development and cryotolerance of embryos. Briefly, oEV were isolated by ultracentrifugation from a pool of oviductal fluids (8 cows/sample) collected at the slaughterhouse at the post-ovulatory stage and ipsilateral to ovulation and stored at −80°C until used. Slaughterhouse-derived bovine oocytes were invitro matured and fertilised with frozen-thawed semen from one bull (4 replicates; 194 presumptive zygotes per group), according to our standard procedures. After IVF, groups of presumptive zygotes (n=20/drop) were cultured under humidified air with 5% CO2, 5% O2 at 38.8°C for 7 days in 30µL of synthetic oviductal fluid-bovine serum albumin supplemented with oEV at different protein concentrations: 0.5, 0.05, or 0.005mgmL−1 and without (control). Cleavage rates were evaluated on Day 2 and blastocyst rates were assessed on Days 6 and 7 (IVF as Day 0). At Day 7, expanded grade 1 blastocysts were evaluated (International Embryo Technology Society classification) and embryos at the expanded grade 1 blastocyst stage were slow frozen in 1.5M ethylene glycol + 0.1M sucrose and stored in liquid nitrogen. For cryotolerance evaluation, embryos were thawed and cultured for 48h in synthetic oviductal fluid-bovine serum albumin + 1% estrous cow serum. Hatching rates were assessed at 48h post-thawing. Data were analysed by a logistic regression mixed model (SAS, SAS Institute Inc.; Glimmix procedure) followed by post-hoc Tukey for multiple comparisons. Differences were considered significant at P<0.05. No differences were observed among the different oEV concentrations tested for cleavage and Day 6 blastocysts. A tendency (P=0.0535) was observed for Day 7 blastocyst rates (19.1±2.8, 29.4±3.3, 16.0±2.6, and 20.6±2.9 for 0.5, 0.05, 0.005mgmL−1, and control, respectively) in favour of the 0.05mgmL−1 group. However, a significant difference (P<0.0288) for Day 7 grade 1 expanded blastocyst rates in favour of the 0.05mgmL−1 group was observed (5.2±1.6, 12.9±2.4, 3.1±1.2, and 9.8±2.2 for 0.5, 0.05, 0.005mgmL−1, and control, respectively). For cryopreserved embryos, hatching rates of frozen-thawed embryos were not significant among experimental groups (81.6±10.2 (n=19), 89.6±6.6 (n=27), 77.2±12.2 (n=10), and 60.2±13.6 (n=23) for 0.5, 0.05, 0.005mgmL−1, and control, respectively). In conclusion, under our experimental conditions, the supplementation of the embryo culture medium with frozen-thawed post-ovulatory oEV at the protein concentration of 0.05mgmL−1 increased the Day 7 grade 1 expanded blastocyst rate. Moreover, we showed a tendency to improve Day 7 blastocyst rates but with no apparent effects on the cryotolerance of embryos. This work was supported by APIS GENE.