In embryos subjected to assisted reproductive techniques, epigenetic modifications may occur that can influence embryonic development and the establishment of pregnancy. In horses, the storage temperature during transport of fresh embryos before transfer is a major concern. The aim of this study was, therefore, to determine the effects of two storage temperatures (5 °C and 20 °C) on equine embryos, collected at day seven after ovulation and stored for 24 h, on: (i) morphological development; (ii) expression of candidate genes associated with embryo growth and development, maternal recognition of pregnancy, methylation and apoptosis, and (iii) gene-specific and global DNA methylation. Embryos (n = 80) were collected on day seven or day eight after ovulation and assigned to four groups: day seven control (E7F, fresh); day seven, stored for 24 h at 5 °C (E5C); day seven, stored for 24 h at 20 °C (E20C) and day eight control (E8F, fresh 24h time control). The embryos and the storage medium (EquiHold, holding medium, Minitube, Tiefenbach, Germany) from all treatment groups were analyzed for (i) medium temperature, pH, and lipid peroxidation (malondialdehyde; MDA) and (ii) embryo morphology, mRNA expression and DNA methylation (immunohistochemistry and gene-specific DNA methylation). The size of embryos stored at 5 °C was larger (p < 0.01), whereas embryos stored at 20 °C were smaller (p < 0.05) after 24 h. There were no changes in pH and MDA accumulation irrespective of the group. The mRNA expression of specific genes related to growth and development (POU5F1, SOX2, NANOG), maternal recognition of pregnancy (CYP19A1, PTGES2), DNA methylation (DNMT1, DNMT3A, DNMT3B) and apoptosis (BAX) in the E5C and E20C were either up or downregulated (p < 0.05) when compared to controls (E7F and E8F). The immune expression of 5mC and 5hmC was similar among treatment groups. Percentage of methylation in the CpG islands was lower in the specific genes ESR1, NANOG and DNMT1 (p < 0.001) in E20C embryos when compared to E8F (advanced embryo stage). Therefore, our study demonstrates for the first time the gene-specific and global DNA methylation status of fresh equine embryos collected on days seven and eight after ovulation. Although our results suggest some beneficial effects of storage at 20 °C in comparison to 5 °C, the short-term storage, regardless of temperature, modified gene expression and methylation of genes involved in embryo development and may compromise embryo viability and development after transfer.

中文翻译：

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在不同温度下短期储存后马胚的表观遗传变化

在接受辅助生殖技术的胚胎中，可能会发生表观遗传修饰，从而影响胚胎发育和怀孕。在马中，转移前新鲜胚胎在运输过程中的储存温度是一个主要问题。因此，本研究的目的是确定两个排卵温度（5°C和20°C）对在排卵后第7天收集并储存24 h的马胚胎的影响：（i）形态发育；（ii）表达与胚胎生长发育相关的候选基因，母体对妊娠，甲基化和凋亡的识别，以及（iii）基因特异性和整体DNA甲基化。胚胎（ñ= 80）在排卵后的第七天或第八天收集，并分为四组：第七天对照（E7F，新鲜）；第七天，在5°C（E5C）下保存24小时；第七天，在20°C（E20C）下保存24小时，第八天进行对照（E8F，新鲜的24小时对照）。分析所有治疗组的胚胎和存储培养基（EquiHold，保存培养基，Minitube，蒂芬巴赫，德国）的（i）培养基温度，pH和脂质过氧化作用（丙二醛； MDA）和（ii）胚胎形态，mRNA表达和DNA甲基化（免疫组织化学和基因特异性DNA甲基化）。储存在5°C的胚的大小较大（p <0.01），而储存在20°C的胚的体积较小（p<0.05）24小时后。不论哪个组，pH和MDA积累均无变化。E5C和E20C中与生长发育相关的特定基因（POU5F1，SOX2，NANOG），母体对妊娠的识别（CYP19A1，PTGES2），DNA甲基化（DNMT1，DNMT3A，DNMT3B）和细胞凋亡（BAX）的mRNA表达分别为与对照组（E7F和E8F）相比，上调或下调（p <0.05）。在治疗组之间，5mC和5hmC的免疫表达相似。在特定基因ESR1，NANOG和DNMT1中，CpG岛的甲基化百分比较低（p与E8F（高级胚胎阶段）相比，E20C胚胎中的<0.001）。因此，我们的研究首次证明了在排卵后第7天和第8天收集的新鲜马胚胎的基因特异性和整体DNA甲基化状态。尽管我们的研究结果表明与20°C相比5°C贮藏有一些有益的影响，但短期贮藏，无论温度如何，与胚胎发育有关的基因的修饰基因表达和甲基化程度都可能降低，并且可能损害胚胎活力和转移后的发育。