泌乳反刍动物主要组织器官AA代谢调控途径与机制研究

It was well established that effects of essential amino acid supply on milk protein synthesis in lactating ruminants could not be described by the single limiting amino acid theory, which justified the development of a new theory to direct researches on the balancing technologies of dietary amino acid in order to promote milk protein synthesis and to elevated the conversion efficiency of absorbed amino acid into milk protein in lactating ruminants. For lactating ruminants, absorbed amino acids are mainly metabolized in the digestive tracts, the liver, and the mammary glands. Previous studies revealed that metabolic losses of individual essential amino acids in the digestive tract might relate only to its supply; the liver might had active cross-talk mechanisms in amino acid metabolism with the peripheral tissues; and instead of mass action, the changed intracellular signals in the mammary epithelial cells by the varied EC fluid environment might be the main pathway for a limiting amino acid to depress milk protein synthesis. These are three key questions related to the basis of the new regulating theory of essential amino acid on milk protein synthesis in lactation ruminants. Aims of the present study were to elucidate the regulating pathways, mechanisms, and the major possible pathways and proteins that could be used as practical targets to regulate amino acid metabolism in the three organs by comprehensive utilization of blood vessel catheterization, cell culture, proteomics, and transcriptomic technologies. Results of the present study will contribute the development of the new regulation theory of amino acid in lactating ruminants.

有确切的证据表明，必需氨基酸供给对泌乳反刍动物乳蛋白合成的影响不符合单一限制性氨基酸理论，研究提高泌乳反刍动物乳蛋白产量和吸收氨基酸转化效率的日粮氨基酸平衡技术，需要用新的理论来指导。消化道、肝脏和乳腺是泌乳反刍动物氨基酸代谢的主要器官，已有的研究表明，消化道的单一必需氨基酸代谢损失可能仅与其供给量有关，肝脏可能存在与外周组织间的氨基酸代谢主动对话机制，限制性氨基酸抑制乳蛋白合成的途径很可能不是底物效应，而是体液内环境变化引起的乳腺上皮细胞内信号途径变化。三个问题涉及到泌乳反刍动物乳蛋白合成必需氨基酸调控理论的基础，本研究结合运用血管插管、细胞培养、蛋白组学、转录组学等技术手段，力图阐明必需氨基酸供给调控三个组织器官氨基酸代谢的途径与机理，发现必需氨基酸调控乳蛋白合成的重点途径和靶标蛋白，推动泌乳反刍动物氨基酸代谢调控理论的发展。

用低蛋白日粮平衡氨基酸技术来提高泌乳反刍动物吸收氨基酸的泌乳转化效率是一个重大的产业技术问题。已有的研究表明，消化道的单一必需氨基酸代谢损失可能仅与其供给量有关，肝脏可能存在与外周组织间的氨基酸代谢主动对话机制，限制性氨基酸抑制乳蛋白合成的途径很可能不是底物效应。本项目以泌乳奶牛和奶山羊为试验对象，研究了EAA供给和构成对消化道、肝脏和乳腺氨基酸代谢的影响及低蛋白日粮平衡氨基酸技术提高乳蛋白转化效率的可行性。.以泌乳奶山羊为试验动物，在0~100%范围内梯度缺失单一EAA，Met在PDV、肝脏和乳腺中的代谢损失率都显著降低，His只有在肝脏中的代谢损失率降低，Lys和Leu在PDV和乳腺中的代谢损失率降低，Arg在PDV和肝脏中的代谢损失率降低，说明组织器官的AA代谢损失可以进行营养调控。随后通过不同灌注部位给奶山羊供给AA，发现与肠腔吸收AA相比，静脉供给AA可以显著降低EAA在消化道中的代谢损失；对于主要在肝脏中代谢的Met、His、Phe和Thr，肝后外周组织供给量和乳腺提取量都没有明显变化，表明肝脏能根据外周组织的需要移除多余的氨基酸。在泌乳奶牛上用低蛋白日粮同时补饲过瘤胃Met、Leu、Ile和Thr可以显著提高乳蛋白产量，证实了低蛋白日粮平衡氨基酸技术的可行性并确定了补饲氨基酸时适宜的低蛋白日粮水平为14%。随后在14%低蛋白日粮上单独补饲Met和Leu，发现添加Met能够提高奶牛的乳产量、乳蛋白率和血清Met水平，降低乳腺eEF2蛋白磷酸化水平；添加Leu却无明显响应，说明单一EAA缺乏能够引起血液氨基酸浓度和构成发生变化，通过影响mTOR相关信号蛋白影响乳蛋白的合成。.综上所述，本研究证实，消化道、肝脏和乳腺面对单一EAA缺失的响应模式存在差异；肝脏与外周组织间存在主动对话机制，EAA供给和构成影响体内氨基酸的代谢分配；低蛋白日粮平衡氨基酸技术能够显著提高乳蛋白转化效率。低蛋白平衡日粮技术研究的重点是确定AA构成和供给影响组织器官氨基酸代谢的途径和机制，从而营养调控乳蛋白合成效能。