75 Influence of paternal exposure to high temperature-humidity index on the oxygen consumption rate of in vitro-produced Bos taurus embryos

Bulls exposed to high temperature-humidity index (THI) experience adverse effects on sperm functionality and subsequent in vitro embryo development. Recently, extracellular flux analysis (EFA) has been established to measure real-time oxygen consumption rate (OCR) in mammalian embryos as an indicator of mitochondrial function. As embryo oxygen consumption and implantation potential are interrelated, the aim of this study was to evaluate the influence of bull exposure to high THI on subsequent OCR of frozen-thawed in vitro-produced embryos by means of EFA. Cumulus-oocyte complexes retrieved from slaughterhouse-derived ovaries were in vitro matured for 22 h and underwent IVF with sperm collected from four Simmental bulls who were sequentially exposed to high (≥60) and low (≤55) THI during the epididymal maturation phase (seven days before sperm collection). 24 h after IVF, presumptive zygotes were denuded from their cumulus cells and cultured in vitro for nine days. Expanded, hatching, and hatched blastocysts were cryopreserved by slow freezing at Days 7, 8, and 9 of in vitro culture (IVC) and stored in liquid nitrogen until EFA. The cryopreserved embryos were thawed and cultured for 24 h. Blastocysts that re-expanded or advanced in their developmental stage after post-thawing IVC were classified as viable. A total of 85 frozen-thawed viable blastocysts (high-THI = 45, low-THI = 40) were evaluated using the Seahorse XFp Analyser (Agilent Technologies). The selected blastocysts were washed out from the culture media in 100 µL of synthetic oviducal fluid (assay media), placed into a pre-warmed plate, and loaded into the Seahorse XFp machine. Embryos were analysed with a protocol consisting of 12 min of equilibration followed by six cycles (M1–M6) of 3 min OCR measurement and 3 min re-equilibration via lifting of sensor cartridge and mixing. For each treatment well, five blastocysts (one expanded, one hatching, and three hatched blastocysts) were used. In addition, two wells filled only with assay media (blank) served as background wells to account for changes in oxygen concentration in the media independent of the presence of embryos and thus, OCR is given as a function of these blank wells. To calculate the mean OCR, most stable measurements (M3&4) were used. Data of OCR was expressed as ppm/min and differences in mean OCR were explored using the least squares means procedure and Tukey’s HSD pairwise comparisons. Embryos from the high-THI group showed lower (P < 0.05) OCR than their low-THI counterparts (9 ± 2 ppm/min vs 15 ± 2 ppm/min, respectively; mean ± s.e.M). This suggests a link between bull high-THI exposure and the mitochondrial function of the subsequent in vitro-produced embryo, which might impact on its implantation potential.