196 Sustainable effect of heat stress during maturation on the bioenergetics profile of bovine blastocysts

Heat stress is one of the most challenging factors affecting fertility in high-yielding dairy cows. Elevated temperature within in the period of oocyte maturation and early embryonic development is associated with decreased developmental potential and increased mitochondrial dysfunction, implicating the loss of membrane potential and lowering of production capacity during oxidative phosphorylation. The aim of this study was to understand the detrimental effect of heat stress during the maturation on subsequent developmental characteristics. Therefore, slaughterhouse-derived ovaries were used to collect cumulus–oocyte complexes (COCs). A total of 688 immature oocytes were subjected to maturation under elevated temperature (41°C) for 22 h, whereas a total of 646 oocytes matured under routine procedures (TCM + 5% serum, 5% CO₂, 38.8°C, humidified air). Subsequently, COCs were fertilized and were cultured in Syntetic oviducal fluid with 0.6% BSA fatty acid free (5% CO₂, 5% O₂, 38.8°C, humidified air) for 8 days to analyse the bioenergetic profile of the resulting blastocysts using extracellular flux analysis. In addition to the basal oxygen consumption rate, a mitochondrial stress test was applied to get deeper insights into mitochondrial metabolism and health. Likewise, gene expression analysis of candidate genes related to oxidative stress response and apoptosis was conducted using quantitative real-time PCR. In addition to lower cleavage rates of heat-stressed oocytes (77.1 vs 89.2; P < 0.05), a significant impact on blastocyst rates on Day 7 (25.6 vs 40.3; P < 0.05) and Day 8 (30.3 vs 50.3; P < 0.05) was observed. It was also noteworthy that extracellular flux analysis discovered a tendency toward a lower mitochondrial oxygen consumption rate (OCR, 0.79 ± 0.08 vs 0.99 ± 0.04 pmol/minute/embryo) and higher non-mitochondrial OCR (1.25 ± 0.15 vs 0.99 ± 0.09 pmol/minute/embryo), indicating a sustainable effect of heat stress during maturation on the efficiency of mitochondrial metabolism at the blastocyst stage. Furthermore, our results revealed a significantly lower spare capacity of embryos resulting from heat-stressed oocytes (0.66 ± 0.09 vs 1.01 ± 0.08 pmol/minute/embryo), implicating a lower ability of the mitochondria to meet the energy demand in the event of a deficiency. Quantitative real-time PCR revealed a significant higher abundance of genes regulating oxidative stress response like GPX1, SOD, NRF2, and PRDX1 in embryos suffering from heat stress, indicating the necessity of a higher defence mechanism against oxidative stress. Furthermore, genes like BAX and CASP3, which are related to apoptosis, were significantly up-regulated in heat-stressed embryos, whereas the apoptosis inhibitor BLC2 was almost 2-fold down-regulated in this group. Taken together, heat stress during maturation does not inhibit the developmental competence completely, but affects the embryo to such an extent that the metabolism is under heavy strain, resulting in lower mitochondrial spare capacities. Moreover, blastocysts derived from oocytes subjected to heat stress during maturation appear to be under greater oxidative stress, resulting in higher levels of programed cell death.