AAPH-treated blastocysts vs CTL-treated blastocysts

In order to understand how in vitro culture affects embryonic quality, we analyzed survival and global gene expression in bovine blastocysts after exposure to increased oxidative stress conditions of IVC. A pro-oxidant agents that act extra-cellularly by promoting ROS production (0.01 mM 2,2'-azobis (2-amidinopropane) dihydrochloride [AAPH]) was added from days 3 to 7, and transcriptomic analysis was then performed in resulting blastocysts. Precisely, after in vitro maturation and fertilization, bovine zygotes were culture in vitro in normal condition, then at day 3, embryos were allocated into culture in control or supplemented with AAPH (0.01 mM) until day 7. At this time, blastocysts were harvested and analyzed. Our hypothesis was that AAPH treatment will affect blastocyst survival and gene expression associated with low embryo quality 4 replicates of 10 blast/rep was produced for AAPH and control treatment. According to a dye swap design with 2 colors, 4 arrays were used to compare AAPH (green) against CTL (red) and 4 arrays were used to compared AAPH (red) againt CTL (green).

为探究体外培养（in vitro culture, IVC）如何影响胚胎质量，本研究分析了牛囊胚在暴露于增强氧化应激的体外培养条件后的存活情况与全基因表达谱。本研究使用一种可通过促进活性氧（reactive oxygen species, ROS）生成发挥胞外促氧化作用的试剂——0.01 mM 2,2'-偶氮二(2-脒基丙烷)二盐酸盐（2,2'-azobis (2-amidinopropane) dihydrochloride, AAPH），在胚胎培养第3天至第7天添加至培养基中，随后对所得囊胚开展转录组分析。具体而言，在体外成熟与体外受精后，牛受精卵先在正常条件下进行体外培养；至第3天时，将胚胎分配至对照组或添加0.01 mM AAPH的实验组，持续培养至第7天。此时收集囊胚并进行分析。本研究的核心假设为：AAPH处理会影响囊胚存活情况，以及与低胚胎质量相关的基因表达。实验组与对照组各设置4次生物学重复，每次重复包含10枚囊胚。根据双色染料互换实验设计，本研究使用4张芯片以绿色荧光标记AAPH组、红色荧光标记对照组进行对比，另使用4张芯片以红色荧光标记AAPH组、绿色荧光标记对照组开展反向对照实验。