99 Effects of unfertilized oocytes and blastocysts in the culture environment on cleavage and blastocyst developmental rates of bovine in vitro embryos

The increased production of in vitro-fertilized bovine embryos necessitates an optimized culture system that will maximize blastocyst and offspring production. Previous research suggests that culturing embryos in groups of 5 or more elicits a cooperative helper effect that increases blastocyst development compared to embryos cultured singly. Pooled embryos are not always feasible if single identities need to be maintained. The objectives of this experiment were to investigate whether unfertilized oocytes (UFOs) or frozen–thawed blastocysts elicit the same helper effect as presumptive zygotes. Follicles were aspirated from slaughterhouse ovaries. Immature oocytes with compact cumulous cells were selected, matured, fertilized, vortexed to remove cumulus, and placed into culture groups under an oil overlay at 38.5°C in a fully humidified environment. Culture groups for the UFO experiment consisted of a group of 10 presumptive zygotes as controls, 8 presumptive zygotes with 2 UFOs, 2 presumptive zygotes, and 1 presumptive zygote with 1 UFO. In a separate study, bovine embryos frozen in either ethylene glycol (EG) or glycerol (GLY) were thawed and placed in culture media drops under an oil overlay at 38.5°C in a fully humidified environment overnight. Frozen–thawed embryos that were high quality and developed into blastocysts were placed into new culture drops with presumptive zygotes. Culture groups for the blastocyst experiment consisted of a control group of 10 presumptive zygotes, 10 presumptive zygotes with 1 EG blastocyst, and 10 presumptive zygotes with 1 GLY blastocyst. On the seventh day of culture, the cleavage (≥2 cells) and development to blastocyst rates were evaluated and analysed via chi square. Results indicated that although cleavage rates (mean % ± standard error of the mean) were similar among culture groups in the UFO study (10; 62.8% ± 3.6, 8 + 2; 70.4% ± 3.7, 2; 64.5% ± 3.2, 1 + 1; 71.4% ± 4.3), the development to the blastocyst stage (mean % ± standard error of the mean) was significantly different (P < 0.05) between the control groups of 10 (24.4% ± 3.2) and 2 (10.5% ± 2.1). There was no statistical difference between the UFO groups of 8 + 2 (17.1% ± 3.1) and 1 + 1 (18.8% ± 3.7). There was no statistic difference between the control group of 10 and 8 + 2 UFOs, but the group of 1 + 1 UFO tended (P = 0.053) to have a higher blastocyst rate than the group of 2 embryos. In the blastocyst study, cleavage was significantly lower (P < 0.05) in the GLY group (50.0% ± 4.8) compared to EG (63.1% ± 3.8) and control (64.4% ± 3.9). Blastocyst development was also significantly lower in the GLY group (16.4% ± 3.5) compared to EG (30.6% ± 3.6) and controls (27.5% ± 3.5). The addition of a UFO resulted in a tendency to have a higher blastocyst development rate than two presumptive zygotes, suggesting that UFOs may provide a helper effect. The addition of a GLY blastocyst resulted in lower cleavage and blastocyst development, but that effect was not observed in embryos frozen in EG. While frozen embryos offer the advantages of increased availability and greater flexibility in timing for inclusion in in vitro culture, it is essential to recognise that further investigations are warranted to explore the potential effects of cryoprotectants in culture media. The role that these additional embryos and UFOs play in culture is still unknown. There needs to be further investigation into whether these cells secrete beneficial factors into the media, or they take on a sacrificial role that removes harmful components in the media.