Good quality day 7 embryos display disturbed cellular functions during post-hatching development when oocytes were matured under lipotoxic conditions: novel insights from a bovine embryo-transfer model. Good quality day 7 embryos display disturbed cellular functions during post-hatching development when oocytes were matured under lipotoxic conditions: novel insights from a bovine embryo-transfer model

Objective: To study post-hatching development after transfer of morphologically good quality embryos that survived in vitro maturation under lipolytic conditions using a well-established and recognized bovine model. Design: prospective study with a crossover design. Setting: research laboratory. Animals: 8 healthy multiparous non-lactating Holstein cows used in repeated embryo transfers. Intervention(s): Bovine cumulus oocyte complexes (COCs) were in vitro matured in the presence of either 1) a combination of elevated pathophysiological concentration of palmitic acid (PA) and physiological concentrations of stearic acid (SA) and oleic acid (OA) (HIGH PA), or 2) physiological concentrations of PA, SA and OA (BASAL). Matured oocytes were fertilized and cultured in FA-free conditions until day 7.7 p.i. Equal proportions of normal and expanded blastocysts were subsequently transferred to synchronized cows. Conceptuses were transcervically recovered 7 days after the transfer. Main Outcome Measure(s): Recovered embryos were morphologically assessed and dissected into embryonic disc (ED) and extraembryonic tissue (EXT). Samples of EXT were used for analysis of interferon-τ (IFN-τ) secretion and measurement of cellular metabolic activity. ED and EXT samples were subjected to RNA sequencing in order to evaluate the genome wide transcriptome patterns of recovered day 14 embryos. Results: Recovered embryos resulting from metabolically compromised oocytes were retarded in growth or less elongated than their control counterparts. Furthermore, they exhibited a decrease in glucose consumption and lactate production. Importantly, lipolytic conditions resulted in reduced capacity of the embryo to signal its presence to the mother through IFN-τ secretion. The transcriptome profile was altered in the recovered embryos with main effects in pathways related to redox-regulating mechanisms, apoptosis, cellular growth, interaction and differentiation, energy metabolism, and epigenetic mechanisms. Overall design: A total of 53 samples (extraembryonic tissue (EX) and embryonic discs (ED)) were used for RNA/DNA extraction, representative for the pool of recovered day 14 embryos, the embryo morphology, the recipient cows (embryos from one of the eight cows were excluded for RNA sequencing) and replicates.

研究目标：采用成熟公认的牛模型，探究在脂解条件下经体外成熟且形态优质的胚胎移植后的孵化后发育情况。试验设计：采用交叉设计的前瞻性研究。试验场所：科研实验室。实验动物：选用8头健康的经产非泌乳荷斯坦奶牛，用于重复胚胎移植操作。干预措施：将牛卵丘-卵母细胞复合物（cumulus oocyte complexes, COCs）分别在两种条件下进行体外成熟：1) 升高浓度的棕榈酸（palmitic acid, PA）结合生理浓度的硬脂酸（stearic acid, SA）与油酸（oleic acid, OA）组合组（高PA组）；2) 生理浓度的PA、SA、OA组合组（基础组）。成熟卵母细胞在无脂肪酸的条件下受精并培养至受精后（post insemination, p.i.）7.7天，随后将等比例的正常扩张囊胚移植到同步化的受体奶牛体内，移植后7天经子宫颈回收胚胎。主要结局指标：对回收的胚胎进行形态学评估，并将其分为胚胎盘（embryonic disc, ED）与胚外组织（extraembryonic tissue, EXT）两部分；采集EXT样本用于干扰素-τ（interferon-τ, IFN-τ）分泌检测与细胞代谢活性测定；对ED与EXT样本进行RNA测序，以分析回收的第14天胚胎的全基因组转录组特征。结果：由代谢受损卵母细胞发育而来的回收胚胎，其生长发育迟缓或伸长程度低于对照组胚胎；此外，此类胚胎的葡萄糖消耗与乳酸生成量均降低。值得注意的是，脂解条件会削弱胚胎通过分泌IFN-τ向母体传递其存在信号的能力。回收胚胎的转录组谱发生改变，主要影响通路涉及氧化还原调控机制、细胞凋亡、细胞生长、相互作用与分化、能量代谢以及表观遗传调控机制。整体试验设计：共计53份样本（胚外组织与胚胎盘）用于RNA/DNA提取，样本覆盖回收的第14天胚胎群、胚胎形态学样本、受体奶牛（8头供体奶牛中的1头的胚胎未纳入RNA测序）以及重复试验组。