Some studies have shown that the excessive metabolic heat production is the primary cause for dead chicken embryos during late embryonic development. Increasing heat shock protein (HSP) expression and adjusting metabolism are important ways to maintain body homeostasis under heat stress. This study was conducted to investigate the effects of in ovo injection (IOI) of vitamin C (VC) at embryonic age 11th day (E11) on HSP and metabolic genes expression. A total of 320 breeder eggs were randomly divided into normal saline and VC injection groups. We detected plasma VC content and rectal temperature at chick's age 1st day, and the mRNA levels of HSP and metabolic genes in embryonic livers at E14, 16 and 18, analysed the promoter methylation levels of differentially expressed genes and predicted transcription factors at the promoter regions. The results showed that IOI of VC significantly increased plasma VC content and decreased rectal temperature (P < 0.05). In ovo injection of VC significantly increased heat shock protein 60 (HSP60) and pyruvate dehydrogenase kinase 4 (PDK4) genes expression at E16 and PDK4 and secreted frizzled related protein 1 (SFRP1) at E18 (P < 0.05). At E16, IOI of VC significantly decreased the methylation levels of total CpG sites and -336 CpG site in HSP60 promoter and -1137 CpG site in PDK4 promoter (P < 0.05). Potential binding sites for nuclear factor-1 were found around -389 and -336 CpG sites in HSP60 promoter and potential binding site for specificity protein 1 was found around -1137 CpG site in PDK4 promoter. Our results suggested that IOI of VC increased HSP60, PDK4 and SFRP1 genes expression at E16 and 18, which may be associated with the demethylation in gene promoters. Whether IOI of VC could improve hatchability needs to be further verified by setting uninjection group.

中文翻译：

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卵内注射维生素C对热休克蛋白和代谢基因表达的影响。

一些研究表明，过多的代谢热产生是胚胎后期发育过程中死鸡胚胎的主要原因。增加热休克蛋白（HSP）的表达和调节新陈代谢是在热应激下维持人体稳态的重要途径。进行了这项研究以调查在卵胎龄11天（E11）时经卵内注射（IOI）维生素C（VC）对HSP和代谢基因表达的影响。将总共​​320个种蛋随机分为生理盐水和VC注射组。我们检测了雏鸡第一天的血浆VC含量和直肠温度，以及E14、16和18日胚胎肝脏中HSP和代谢基因的mRNA水平，分析了差异表达基因的启动子甲基化水平，并预测了启动子区域的转录因子。结果表明，VC的IOI显着增加血浆VC含量并降低直肠温度（P <0.05）。在卵内注射VC时，E16和PDK4处的热休克蛋白60（HSP60）和丙酮酸脱氢酶激酶4（PDK4）基因表达显着增加，E18处分泌了卷曲的相关蛋白1（SFRP1）（P <0.05）。在E16，VC的IOI显着降低了HSP60启动子中总CpG位点和-336 CpG位点的甲基化水平，以及PDK4启动子中的-1137 CpG位点的甲基化水平（P <0.05）。在HSP60启动子的-389和-336 CpG位点附近发现了核因子-1的潜在结合位点，在PDK4启动子的-1137 CpG位点附近发现了特异性蛋白1的潜在结合位点。我们的研究结果表明，VC的IOI增加了E16和18时HSP60，PDK4和SFRP1基因的表达，这可能与基因启动子中的去甲基化有关。VC的IOI是否可以提高孵化率需要通过设置未注入组来进一步验证。