棉酚旋光异构体在泌乳奶牛体内转移规律及其代谢组研究

Free gossypol in cottonseed accounts for about 90% of total gossypol. There are two enantiomers of gossypol: (+)isomer and (-)isomer. (-)isomer is of more biological toxicity. Gossypol may affect production and productivity of cows, and may even cause death when too much gossypol is included in the diet. It is not clear how gossypol is transferred from diet to milk and how gossypol affects the metabolome of the cow. In relationto these problems, a feeding trial will be done using lactating cows to measure gossypol enantiomers concentration in blood, faeces, urine and milk as. Transfer regularity of gossypol enantiomers to the cow in vivo will be studied. And by using the continuous dynamic artificial rumen method, gossypol enantiomers concentration of rumen fluid will be determined by LC-MS, and the metabolic pathway of gossypol enantiomers will be investigated. Perfusion of gossypol through duodenal fistula will be performed in cows to demonstrate distributions of two different gossypol enantiomers, and gossypol enantiomers concentration in` blood, faeces, urine and milk will be determined by LC-MS. Metabolites in the cow's blood and urine will be determined by NMR and LC-MS, to explore the mechanism of how gossypol affectes metabolome of the cow and to identify its potential biological marker. The theoretical basis for regularity of gossypol transferred into the cow in vivo and effect of gossypol toxicity on cows will be determineded.

全棉籽中游离棉酚约占总棉酚的90%，而棉酚有左旋和右旋棉酚两种旋光异构体，左旋棉酚有更强的生物毒性。当棉酚超过瘤胃解毒能力，可影响奶牛生产性能和繁殖能力甚至造成奶牛死亡。棉酚旋光异构体在奶牛体内转移规律、棉酚毒性影响泌乳牛代谢组变化规律等科学问题尚不清楚。因此，拟通过奶牛饲养试验，利用LC-MS法测定奶牛血浆、粪、尿和乳中棉酚旋光异构体含量，研究日粮中棉酚旋光异构体向奶牛体内转移规律；采用持续动态人工瘤胃法，利用LC-MS法测定瘤胃液中棉酚旋光异构体含量，研究棉酚旋光异构体在瘤胃内代谢规律；通过奶牛十二指肠瘘管灌注试验，利用LC-MS法测定奶牛血浆、粪、尿和乳中棉酚旋光异构体含量，研究过瘤胃棉酚旋光异构体向奶牛体内转移规律；采用NMR、LC-MS法研究奶牛代谢组，探索棉酚毒性影响奶牛代谢组变化的规律和潜在的生物标志物。研究结果将为棉酚在奶牛体内转移及其对奶牛毒性作用提供理论依据。

棉酚是限制棉籽类产品在奶牛养殖中应用最主要的因素。针对棉酚对泌乳奶牛毒性机制尚不完全清楚等问题，本项目开展了以下四方面相关研究。.调研了解我国奶牛日粮棉酚饲喂情况。设计动物试验，将9头奶牛按随机区组分为3组，分别灌喂0、500、1000 mg/kg DM棉酚28天，比较棉酚旋光异构体在奶牛血浆中代谢规律。结果发现：（1）调研的三个牛场中有两个牛场日粮棉酚饲喂量超过EFSA限量；（2）相比于右旋棉酚，左旋棉酚在奶牛体内的积蓄作用更强，消除更慢。.开展体外静态培养试验，研究瘤胃微生物对棉酚旋光异构体的降解规律。结果发现：发酵6 h，500、1000 μg/g DM棉酚添加组左旋棉酚降解率分别为67.78%和85.85%，右旋棉酚降解率分别为65.65%和86.44%；48 h，左旋棉酚降解率分别为81.73%和85.22%，右旋棉酚降解率分别为84.08%和87.18%。结果表明：瘤胃微生物对棉酚的降解速率快且不存在异构体选择性。.开展泌乳奶牛饲养试验，研究高剂量棉酚对泌乳奶牛机体代谢的影响。12头中期泌乳奶牛按随机区组分为2组，分别灌喂0、1000 mg/kg DM 棉酚28天。结果发现：与对照组相比，棉酚组乳糖和乳蛋白率显著下降，血浆谷丙转氨酶、碱性磷酸酶、谷胱甘肽转移酶和超氧化物歧化酶活性升高，代谢组分析发现，棉酚组血浆赖氨酸及糖异生前体物质含量显著降低。结果表明棉酚通过引发氧化应激，造成肝损伤，影响氨基酸利用与糖异生过程，降低乳蛋白和乳糖合成，降低奶牛生产性能。.针对棉酚增加奶牛溶血这一现象，联合代谢组学、蛋白质组学及酶活性靶向测定技术，研究棉酚溶血毒性机制。结果从代谢物、酶蛋白表达量及活性水平联合证明：棉酚处理后导致红细胞内糖酵解代谢流量下降；磷酸戊糖途径代谢流量增加，该途径被激活；谷胱甘肽降解代谢增加；该结果揭示了能量缺乏以及氧化应激是棉酚溶血毒性的机制。.整体来讲，本研究系统阐明了棉酚旋光异构体在泌乳奶牛体内代谢规律以及棉酚对泌乳奶牛毒性机制，研究结果为棉籽类产品在奶牛上合理应用以及棉酚安全性评价奠定基础。