羊驼第一胃室纤维降解微生物的多样性及相关优势酶的研究

Alpaca, with three stomach compartments, is a kind of ruminant that no bloat was reported. It can be fed on stalks, and also depends on microorganisms in forestomach for the degradation of plant fibre. Our previous study show that the density of bacteria in the first stomach compartment of alpacas was approximately tenfold lower than that of sheep, but with similar or higher digestibility of plant fibre compared to sheep. The production of methane, a kind of greenhouse gas and loss of ingested energy, was only 17%～30% of that from sheep. However, the reason and the mechanism are not clear. In view of this, we will enrich the fiber degrading microorganisms on maize stalk in nylon bag that set in the first stomach compartment of alpaca. Then construct the metagenome library of the microorganisms. By aligning the sequences in the library with those in the reference databases, the species were annotated and the function of the sequences was analysed. The predomiant enzymes for fiber degradation were expressed to detect their activity. We expect to find the gene that belong to the predomiant and typical microorganisms or enzymes on fiber degradation from the first stomach compartment. Compared the results with that of the sheep, the reasons that the higher cellulolytic activity and energy utilization efficiency of microbes in the forestomach of alpaca than that in the rumen of sheep may be uncovered. Moreover, the information in this study could provide ideas for improving the utilization efficiency of roughage and decreasing the methane production of ruminants.

羊驼是具有三个胃室、不发生瘤胃鼓胀病的反刍动物。它能有效利用秸秆等粗饲料，并且对植物纤维的消化也主要靠前胃微生物。本课题组初步研究发现其第一胃室中细菌的浓度比绵羊瘤胃低近一个数量级，但对植物纤维的消化能力与绵羊相似或更高，而作为温室气体和动物能量损失的甲烷排放量仅为绵羊的17%～30%，但其原理未知。本项目拟利用尼龙袋富集羊驼第一胃室中纤维降解微生物，建立其宏基因组文库；将文库序列与参考数据库进行比对，进行物种注释和基因功能分析；表达优势纤维降解酶基因，并检测其产物酶的活性。预期发现羊驼第一胃室内优势和特有的纤维降解微生物和酶的基因，并与绵羊瘤胃微生物进行比较，探索和分析羊驼第一胃室微生物纤维降解效率高而甲烷产量低的可能原因，为进一步提高反刍动物对粗饲料的利用效率、调控甲烷产量提供理论依据。

为了探讨羊驼对劣质粗饲料消化率比绵羊高而甲烷排放量低的原因，本研究在以玉米秸秆为粗饲料的条件下，测定了羊驼与绵羊采食量，饲料的表观消化率，尿嘌呤衍生物，第1胃室固相与液相外流速率、内环境和酶活，第1胃室微生物菌群和细菌、真菌、产甲烷古菌、原虫等微生物类群的高通量测序、以及微生物转录组测序。试验选用1 周岁健康的公羊驼（体重29.5±7.1 kg）和晋中公绵羊（体重27.9±2.7 kg）各6 只，根据动物的采食量和NRC绵羊的饲养标准，按照满足动物维持需要的1.5 倍配制日粮，设置精粗比为3:7、5:5和7:3的三种日粮，各种动物分别采用有重复的3×3拉丁方设计。研究结果表明，羊驼的干物质采食量显著低于绵羊（P＜0.01），DM、OM、ADF、NDF的表观消化率与绵羊之间没有显著差异，而粗脂肪（P=0.009）和磷（P＜0.001）的表观消化率显著高于绵羊，钙的表观消化率显著低于绵羊（P=0.039）；羊驼尿酸、尿囊素及尿嘌呤衍生物的排泄量均显著低于绵羊；羊驼食糜平均滞留时间（53.273 h）极显著高于绵羊（35.651 h），第1胃室液相外流速率与滞留时间两种动物差异不显著，但羊驼第1胃室体积与液体外流速度小于绵羊；羊驼第1胃室中乙酸、丙酸、丁酸和总挥发性脂肪酸的含量与绵羊之间没有显著差异，戊酸、异丁酸和异戊酸的含量显著高于绵羊（P＜0.001），滤纸酶、β-葡糖糖苷酶、羧甲基纤维素酶等酶活显著低于绵羊瘤胃；羊驼第1胃室中产甲烷古菌、真菌和产琥珀酸丝状杆菌、溶纤维丁酸弧菌等占总细菌的比例与绵羊瘤胃没有显著差异，而白色瘤胃球菌（P=0.048）和黄色瘤胃球菌（P=0.007）占总细菌的比例以及原虫数量（P=0.017）显著低于绵羊瘤胃；羊驼与绵羊第1胃室中的细菌优势菌门和优势菌属、优势产甲烷古菌属均相同，但两者细菌菌群、真菌菌群存在很大差异。微生物菌群的这些差异能够解释两种动物代谢等方面的差异。另外，原虫高通量测序的结果正在进行分析，产甲烷菌高通量测序的结果不太理想，转录组测序数据需要进一步证明，羊驼第1胃室发现特殊微生物有待进一步研究。