88 Co-culture with bovine oviduct epithelial cells during in vitro culture affects the bioenergetic profile of bovine expanded blastocysts

The embryo in vitro production (IVP) results in altered embryo quality, which limits the efficient usage. A detailed understanding of the embryo bioenergetic profile as a consequence of culture conditions could be helpful. In vitro bovine oviduct epithelial cell (BOEC) co-culture is related with improved embryo vitality. However, insights into the detailed mechanisms are rare and a deeper understanding of BOEC co-culture effects on the bioenergetic profile would contribute to fill knowledge gaps. For this purpose, slaughterhouse oviducts were prepared and BOECs were stripped out with a tweezer. The BOECs were cultured in suspension in SOFaa-HA (5% HA, 5 µL/mL insulin-tranferrin-selen (100×) and 5 µL/mL EGF (10 ng/mL), 38.5°C, 5% CO₂ and O₂). Simultaneously, IVP was conducted and embryos were randomly separated into two groups and cultured in SOFaa-HA from zygote to blastocyst stages either in the presence or absence of BOECs. For metabolic measurements expanded blastocysts of Day 7 (expanded blastocysts [EX] were removed from BOEC co-culture as well as control and analysed in pools of 10 EX (10 replicates) using an extracellular FLUX analyser (Agilent). In addition, as a model of altered embryo quality, measurements were also carried out for Day 8 EX (11 replicates). The bioenergetic profile was investigated in terms of mitochondrial oxygen consumption (OCR) and extracellular acidification rate (ECAR) related to glycolysis. In addition, Cell Mito-Stress Test (Agilent) consisting of three serial injections (oligomycin 1.5 µM; FCCP 4.0 µM and rotenone/AA 0.25 µM) was used to determine reserve capacity, ATP-linked respiration and compensatory glycolytic activity. Two-way ANOVA (P < 0.05) was performed for comparisons between treatment (control vs BOEC) and morphokinetics (Day 7 vs Day 8). The results of our study showed no effect of BOECs on blastocyst rates compared to control for Day 7 (18.01 ± 2.18% vs 17.68 ± 2.54%) and Day 8 EX (26.65 ± 2.62% vs 27.96 ± 3.03%). In contrast, a significant effect on the bioenergetic profile was observed (P < 0.05) as indicated by reduced mitochondrial activity of BOEC co-culture, in particularly in Day 8 EX (0.46 ± 0.024 vs 0.78 ± 0.039 pmol/min per embryo). Consistently, ECAR was reduced in BOEC co-culture, irrespectively of day (Day 7: 0.12 ± 0.012 vs 0.23 ± 0.021 mpH/min per embryo; Day 8: 0.11 ± 0.009 vs 0.24 ± 0.033 mpH/min per embryo). Furthermore, a similar reserve capacity is received between BOEC and control (Day 7: 299.2% vs 297.7%; Day 8: 244.0% vs 249.8%). Finally, BOEC co-culture resulted in lower levels of ATP-linked respiration in Day 8 EX (0.24 ± 0.016 vs 0.46 ± 0.017 pmol/min per embryo) and caused a significantly higher compensatory glycolytic activity, independent of morphokinetic (Day 7: 207.8% vs 127.6%; Day 8: 207.1% vs 129.8%), Finally, BOECs co-culture resulted in higher resting mitochondrial and glycolytic activities, which were more pronounced in embryos of lower developmental capacity. While BOECs did not affect reserve capacity, but may enable a better compensation for mitochondrial limitations via glycolysis.