The assessment of embryo quality aims to enhance subsequent pregnancy and live birth outcomes. Metabolic analysis of embryos has immense potential in this regard. As a step towards this goal, here we assess the metabolism of bovine embryos using label-free optical imaging. We compared embryos defined as either on-time or fast-developing, as fast dividing embryos are more likely to develop to the blastocyst stage. Specifically, bovine embryos at 48 (Day 2) and 96 (Day 4) hours post fertilization were fixed and separated based on morphological assessment: on-time (Day 2: 2 cell; Day 4: 5-7 cell) or fast-developing (Day 2: 3-7 cell; Day 4: 8-16 cell). Embryos with different developmental rates on Day 2 and Day 4 were correlated with metabolic activity and DNA damage. Confocal microscopy was used to assess metabolic activity by quantification of cellular autofluorescence specific for the endogenous fluorophores NAD(P)H and FAD with a subsequent calculation of the optical redox ratio. Separately, hyperspectral microscopy was employed to assess a broader range of endogenous fluorophores. DNA damage was determined using γH2AX immunohistochemistry. Hyperspectral imaging showed significantly lower abundance of endogenous fluorophores in fast-developing compared to on-time embryos on Day 2, indicating a lower metabolic activity. On Day 4 of development there was no difference in the abundance of FAD between on-time and fast-developing embryos. There was, however, significantly higher levels of NAD(P)H in fast-developing embryos leading to a significantly lower optical redox ratio when compared to on-time embryos. Collectively, these results demonstrate that fast-developing embryos present a 'quiet' metabolic pattern on Day 2 and Day 4 of development, compared to on-time embryos. There was no difference in the level of DNA damage between on-time and fast-developing embryos on either day of development. To our knowledge, this is the first collective use of confocal and hyperspectral imaging in cleavage-stage bovine embryos in the absence of fluorescent tags.

中文翻译：

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使用高光谱和共聚焦方法对卵裂期牛胚胎进行光学成像，揭示准时和快速发育胚胎之间的代谢差异

胚胎质量评估旨在提高随后的妊娠和活产结果。胚胎的代谢分析在这方面具有巨大的潜力。作为实现这一目标的一步，我们在这里使用无标记光学成像评估牛胚胎的代谢。我们比较了定义为准时或快速发育的胚胎，因为快速分裂的胚胎更有可能发育到囊胚阶段。具体而言，受精后 48（第 2 天）和 96（第 4 天）小时的牛胚胎根据形态学评估进行固定和分离：准时（第 2 天：2 个细胞；第 4 天：5-7 个细胞）或快速发育（第 2 天：3-7 细胞；第 4 天：8-16 细胞）。在第 2 天和第 4 天具有不同发育速率的胚胎与代谢活动和 DNA 损伤相关。共聚焦显微镜用于通过量化内源性荧光团 NAD(P)H 和 FAD 特异性的细胞自发荧光以及随后计算光学氧化还原比来评估代谢活性。另外，高光谱显微镜被用来评估更广泛的内源性荧光团。使用 γH2AX 免疫组织化学确定 DNA 损伤。高光谱成像显示，与第 2 天准时胚胎相比，快速发育中的内源性荧光团丰度显着降低，表明代谢活动较低。在发育的第 4 天，准时和快速发育的胚胎之间的 FAD 丰度没有差异。然而，与准时胚胎相比，快速发育的胚胎中 NAD(P)H 水平显着更高，导致光学氧化还原比显着降低。总的来说，这些结果表明，与准时胚胎相比，快速发育的胚胎在发育的第 2 天和第 4 天呈现出“安静”的代谢模式。在发育的任何一天，准时和快速发育的胚胎之间的 DNA 损伤水平没有差异。据我们所知，这是在没有荧光标签的情况下，在卵裂期牛胚胎中首次集体使用共聚焦和高光谱成像。