茶皂素调控肉牛瘤胃微生物菌群结构及甲烷生成的研究

发布时间：2018-04-30 21:18

本文选题：茶皂素 + 瘤胃菌群结构　；参考：《四川农业大学》2016年博士论文

【摘要】：瘤胃是反刍动物的重要消化器官,也是甲烷生成的重要场所,瘤胃内微生物的菌群结构变化会影响瘤胃内环境的稳定、饲料利用效率和甲烷排放,由于瘤胃纤毛原虫、细菌和甲烷菌间存在紧密的种间氢转移、共生和相互制约的关系,因此纤毛原虫的菌群结构变化可能进一步影响瘤胃细菌和甲烷菌的菌群结构变化。近年来发现茶皂素(TSS)因其特殊的生物学活性被作为一种瘤胃发酵调控剂,本研究以TSS为试验材料,利用qPCR和高通量测序技术,从微生物分类学上研究TSS对肉牛瘤胃纤毛原虫、细菌和甲烷菌的丰度和区系组成的影响,探明纤毛原虫、细菌和甲烷菌的菌群结构组成与肉牛瘤胃发酵和甲烷生成间的关系,以及TSS调控肉牛瘤胃发酵和甲烷生成的微生物学机制。试验一茶皂素对肉牛瘤胃纤毛原虫区系组成和甲烷生成的调控作用本试验选用6头安有瘤胃瘘管的贝尔蒙特红杂牛(体重为363±8.5 kg)作为研究对象,考察添加茶皂素前、添加期间和停止添加茶皂素后这三个阶段肉牛瘤胃中纤毛原虫区系组成和甲烷产量的变化。首先经过56d的驯饲期让肉牛从放牧饲喂到适应舍内饲喂高精料基础日粮(精粗比为77：23,DM基础)后进行正式试验,前2周继续饲喂基础日粮作为添加TSS前的对照阶段(BD),然后在晨饲基础日粮中添加TSS(30 g/d)饲喂20d(TSS添加阶段,TSS),最后2周饲喂不添加TSS的基础日粮(停止添加TSS后的对照,BDP)。分别在三个阶段的最后2d检测肉牛甲烷排放量并采集瘤胃液,利用qPCR和Illumina Miseq高通量测序技术分析纤毛原虫的丰度和区系组成变化。结果发现：1)内毛属、后毛属、真双毛属、多甲属和等毛虫属原虫是三个阶段肉牛瘤胃中的优势原虫属,添加TSS降低了利用淀粉的内毛属原虫的相对丰度(P0.05),增加了利用纤维物质的多甲属和真双毛属原虫的相对丰度(P0.05);2)在31个核心原虫OTUs中,添加TSS降低了内毛属和等毛虫属OTUs的比例,增加了多甲属和真双毛属OTUs的比例,但停止添加TSS后,内毛虫属OTUs的比例增加；3)添加TSS增加了纤毛原虫的总体丰度(P0.05),但BDP阶段又降低恢复到BD阶段85%的水平；4)BDP阶段肉牛瘤胃中利用可溶性糖类的等毛虫属原虫的丰度低于BD和TSS阶段(P0.05),而降解利用淀粉的后毛属原虫和降解纤维的多甲属原虫的丰度高于BD阶段(P0.05)；5)添加TSS对肉牛的甲烷排放量无显著影响(P0.05),但BDP阶段的甲烷排放量显著低于BD和TSS阶段(P0.05)；6)等毛虫属原虫的丰度与肉牛甲烷排放量呈显著正相关(P0.05),后毛属原虫的丰度与肉牛甲烷排放量呈显著负相关(P0.05)。以上结果表明,在肉牛的高精料日粮中添加30 g/d TSS后,TSS通过抑制利用淀粉的内毛属原虫的增殖和促进利用纤维物质的多甲属和真双毛属原虫的增殖来改变瘤胃纤毛原虫的区系组成；另外,可通过降低与肉牛甲烷排放量呈显著负相关的等毛虫属的丰度或增加与肉牛甲烷排放量呈显著正相关的后毛属原虫的丰度来降低反刍动物瘤胃的甲烷排放量。试验二茶皂素对瘤胃细菌菌群组成和瘤胃发酵模式的调控作用瘤胃纤毛原虫与瘤胃细菌间存在捕食与被捕食、共生或寄生,以及竞争和协同关系,在了解茶皂素改变了瘤胃纤毛原虫区系组成的基础上,利用qPCR和Illumina Miseq高通量测序技术进一步探讨TSS对瘤胃细菌的丰度和菌群组成,以及瘤胃发酵的调控作用。结果发现：1)厚壁菌门(46.1%)和拟杆菌门(44.4%)是三个阶段肉牛瘤胃中的优势细菌菌门,普雷沃氏菌科(32.1%)、氨基酸球菌科(19.9%)和瘤胃菌科(11.3%)为优势菌科,普雷沃氏菌属(24.3%)和琥珀酸菌属(18.7%)为优势菌属；2)添加TSS降低了厚壁菌门的丰度,增加了变形菌门和纤维杆菌门的丰度(P0.05),在属水平上,降低了丙酸生成菌(琥珀酸菌属)的丰度,增加了纤维降解菌(瘤胃球菌属和丁酸弧菌属)的丰度(P0.05)；3)添加TSS降低厚壁菌门、氨基酸球菌科和琥珀酸菌属OTUs的比例,而增加了拟杆菌门、变形菌门和瘤胃球菌科OTUs的比例；4)添加TSS降低了黄色瘤胃球菌的丰度(P0.05),增加了白色瘤胃球菌和产琥珀酸丝状杆菌的丰度(P0.05)；5)添加TSS降低了丙酸的摩尔浓度,增加了异丁酸的摩尔浓度(P0.05),也提高了乙酸：丙酸的比例(P0.05),使瘤胃发酵模式由丙酸型转为乙酸型。以上结果表明,TSS通过对瘤胃细菌在门、属和种水平上的差异性调控来改变其区系组成,即抑制丙酸生成菌(琥珀酸菌属)和黄色瘤胃球菌的增殖,促进了纤维降解菌中白色瘤胃球菌、产琥珀酸丝状杆菌和丁酸弧菌属的增殖,使瘤胃发酵模式由丙酸型转为乙酸型。试验三茶皂素对肉牛瘤胃甲烷茵丰度和菌群组成的调控作用瘤胃纤毛原虫和细菌能够为甲烷菌生成甲烷提供所需的底物(代谢氢和一碳化合物),纤毛原虫还为一些甲烷菌提供生长繁殖生境。因此在了解TSS改变了瘤胃纤毛原虫和细菌区系组成的基础上,利用qPCR和Illumina Miseq高通量测序技术进一步探讨TSS对甲烷菌丰度和菌群组成的调控作用。结果发现：1)甲烷短杆菌属是肉牛瘤胃中的绝对优势甲烷菌属(95%),另外系统进化树分析发现：在15个优势甲烷菌OTUs中,有86%以上(占13个OTUs)位于SGMT簇内,即SGMT簇甲烷菌是主要的优势甲烷菌簇；2)添加TSS增加了RCC甲烷菌的绝对丰度(P0.05),但对总甲烷菌和甲烷短杆菌属甲烷菌的丰度无显著影响(P0.05)。3)在种簇水平上,添加TSS降低了丰度高的SGMT簇甲烷菌的相对丰度,增加了丰度低的RO簇甲烷菌的相对丰度：4)系统进化树分析发现与等毛虫属原虫选择性共生的甲烷菌主要是SGMT簇甲烷菌,与多甲属原虫选择性共生的甲烷菌主要是RO簇甲烷菌。以上结果表明,TSS通过对瘤胃甲烷菌在种簇水平上的差异性调控改变了肉牛瘤胃甲烷菌的区系组成,即抑制与等毛虫属原虫选择性共生的丰度高的SGMT簇甲烷菌增殖和促进与多甲属原虫选择性共生的丰度低的RO簇甲烷菌的增殖。试验四不同甲烷菌产甲烷活性差异的比较研究本试验旨在比较优势甲烷短杆属甲烷菌中低丰度的RO簇的主要代表菌Mbr. ruminantium和高丰度的SGMT簇的主要代表菌Mbr. gottschalkii的生长指数和产甲烷活性差异。对2种甲烷菌进行时程(Time-course)培养试验,分别在13个时间点(0、8、12、16、20、24、30、36、48、60、72、84、96h)测定甲烷菌的生长指数和甲烷产量,并在DNA和RNA水平上研究甲烷菌的甲烷产量与甲烷菌丰度和产甲烷能力间的相关关系。结果发现：1)Mbr. ruminantium的对数增长期(40h)和稳定持续期(24h)均比Mbr. gottschalkii的对数增长期(36h)和稳定持续期(12h)长；2) Mbr. ruminantium的最大菌体量和最大比生长速率均显著高于Mbr. gottschalkii (P0.05); 3)两种甲烷菌的累积甲烷产量与其丰度和mcrA基因表达水平均呈显著正相关(P0.05),而且甲烷菌的丰度与其mcrA基因表达水平也呈显著正相关(P0.05)；4)Mbr. ruminantium和Mbr. gottschalkii所消耗H2量差异不显著(P0.05),但Mbr. ruminantium生成的CH4量显著高于Mbr. gottschalkii (P0.05); 5) Mbr. ruminantium可在低氢浓度下继续生长产生少量甲烷,而Mbr. gottschalkii则不能利用低浓度的氢生成甲烷。以上结果表明,甲烷短杆菌属中低丰度的RO簇代表菌Mbr. ruminantium的产甲烷活性和对低浓度环境的适应能力要高于丰度高的SGMT簇代表菌Mbr. gottschalkii;另外甲烷菌的丰度和mcrA基因表达水平均可用于评定甲烷菌的产甲烷活性。综上所述,不同种属的纤毛原虫、细菌和甲烷菌对TSS的调控作用存在种属敏感差异性,在肉牛的高精料日粮中添加30 g/d TSS后,TSS改变了瘤胃微生物的菌群结构组成。对于瘤胃纤毛原虫,添加TSS选择性抑制了利用淀粉的原虫(内毛属)的增殖,促进了利用纤维物质的原虫(多甲属和真双毛属)的增殖；对于细菌,添加茶皂素选择性抑制了丙酸生成菌(琥珀酸菌属)和黄色瘤胃球菌的增殖,而促进了纤维降解菌中白色瘤胃球菌、产琥珀酸丝状杆菌和丁酸弧菌属的增殖,使肉牛瘤胃发酵模式由丙酸型转为乙酸型；对于甲烷菌,添加茶皂素选择性抑制了甲烷短杆菌属中丰度高的SGMT簇甲烷菌的增殖,而促进了丰度低的RO簇甲烷菌的增殖。低丰度的RO簇甲烷菌代表菌Mbr. ruminantium产甲烷活性和对低浓度环境的适应能力均高于丰度高的SGMT簇甲烷菌代表菌Mbr. gottschalkii。茶皂素主要通过抑制与等毛虫属原虫选择性共生的丰度高产甲烷活性较低的SGMT簇甲烷菌的增殖和促进与多甲属原虫选择性共生的丰度低但产甲烷活性高的RO簇甲烷菌增殖来影响肉牛瘤胃的甲烷生成。
[Abstract]:Rumen is an important digestive organ of ruminants and an important place for methane production. The change of microbial flora in the rumen will affect the stability of the rumen environment, feed efficiency and methane emission, and the close interspecies hydrogen transfer, symbiotic and mutual restriction among the rumen ciliated protozoa, bacteria and methanogens. The changes in the structure of protozoa may further affect the changes in the structure of the bacteria in the rumen and methanogens. In recent years, it is found that the tea saponin (TSS) has been used as a rumen fermentation regulator because of its special biological activity. In this study, TSS was used as a test material. QPCR and high throughput sequencing technology were used to study the TSS pairs from microbiological taxonomy. The effects of the abundance and flora of the ciliated protozoa, bacteria and methanogens, the relationship between the structure of protozoa, bacteria and methanogens and the relationship between the rumen fermentation and methane production of beef cattle, as well as the microbiological mechanism of TSS regulation of rumen fermentation and methane production in beef cattle. The regulation of floristic composition and methane production in this experiment selected 6 Baer Mont red cattle (363 + 8.5 kg) with rumen fistula (weight 363 + 8.5) as the research object. The changes in the composition of the faunal flora and the methane production in the rumen of beef cattle were investigated before the addition of tea saponin and the addition of tea saponin, and the changes of methane production in the rumen of beef cattle. In the period of taming of 56d, beef cattle were fed from grazing to the adaptation house to feed the high concentrate base diet (the ratio of 77:23, DM). The first 2 weeks continued to feed the basal diet as the control stage (BD) before adding TSS, and then added TSS (30 g/d) to the basal diet to feed 20d (TSS addition stage, TSS), and the last 2 weeks were not fed. Add the basal diet of TSS (stop adding TSS after the control, BDP). At the final 2D of the three stages, the methane emission of beef cattle was detected and the tumor gastric juice was collected. The abundance and floristic composition of cilium protozoa were analyzed by qPCR and Illumina Miseq high throughput sequencing technology. The results were found: 1) internal hair, posterior hair, true double hair, multi a, and so on. The genus protozoa of the genus caterpillar is the dominant protozoa in the rumen of three stages of beef cattle. Adding TSS reduces the relative abundance of protozoa (P0.05) with starch and increases the relative abundance (P0.05) of the use of fibrous material and true bis protozoa (P0.05); 2) in the 31 core protozoan OTUs, the addition of TSS reduces the genus and the OTUs of the genus caterpillar. In proportion, the proportion of the genus OTUs was increased, but after the addition of TSS, the proportion of OTUs of the genus caterpillar increased; 3) adding TSS increased the total abundance of cilium protozoa (P0.05), but the BDP phase decreased to the level of the BD stage 85%; 4) the abundance of the soluble sugar like parasite protozoa was lower in the BDP stage of the bovine tumor. In the BD and TSS stages (P0.05), the abundance of protozoa protozoa and degrading fibers degraded using starch was higher than that in BD phase (P0.05); 5) the addition of TSS had no significant effect on the methane emission of beef cattle (P0.05), but the methane emission at BDP stage was significantly lower than that of BD and TSS stages (P0.05); 6) the abundance of protozoa and beef beetle. The number of alkanes showed significant positive correlation (P0.05), and the abundance of protozoan protozoa was negatively correlated with the amount of methane emission from beef cattle (P0.05). The above results showed that after adding 30 g/d TSS to the high concentrate diet of beef cattle, TSS inhibited the proliferation of the protozoa protozoa using starch and promoted the use of fibrous material and the protozoa. Proliferation to change the floristic composition of the rumen ciliated protozoa; in addition, the methane emission of the rumen of ruminants can be reduced by reducing the abundance of the genus caterpillar and increasing the abundance of the genus protozoa with significant positive correlation with the methane emission of beef cattle to reduce the methane emission of the rumen of the ruminant. Test two tea saponin to the rumen bacteria The regulation of group composition and rumen fermentation mode in rumen protozoa and rumen bacteria exists between predatory and predatory, symbiotic or parasitic, and competition and synergy. On the basis of understanding the changes in the composition of the rumen ciliated protozoa, qPCR and Illumina Miseq high throughput sequencing technology are used to further explore the rumen fines of the rumen. The abundance and composition of bacteria and the regulation of rumen fermentation. Results: 1) 1) the phylum (46.1%) and the bacteriobacteriaceae (44.4%) were the dominant bacteria in the rumen of the three stages of beef cattle, the Poulet Was bacteria family (32.1%), the amino acid bacteria (19.9%) and the tumor stomach bacteria (11.3%) as the dominant bacteria, the genus Poulet Was (24.3%) and succinic acid. The genus (18.7%) was the dominant genus; 2) the addition of TSS reduced the abundances of the thick walled bacteria and increased the abundance of the Proteus and fibrobacteria (P0.05). At the level of the genus, the abundances of the propionic acid producing bacteria (succinic bacteria) were reduced and the abundance of the fibrous degrading bacteria (rumen and Vibrio butyrate) was increased (P0.05); and 3) TSS was added to the thicker wall. The proportion of the bacteria gate, the amino acid family and the succinic bacteria OTUs increased the proportion of the bacteriobacteria, the deformable bacteria and the rumen family OTUs; 4) the addition of TSS decreased the abundance of the Yellow rumen coccus (P0.05), increased the abundance of the white rumen cocci and the production of the filamentous succinate (P0.05), and 5) added TSS to reduce the mole of propionic acid. The molar concentration of isobutyric acid (P0.05) was increased, and the proportion of acetic acid, propionic acid (P0.05) was increased, and the rumen fermentation mode was transformed from propionic acid to acetic acid. The above results showed that TSS changed its floristic composition by differential regulation of the rumen bacteria at the gate, genus and species level, that is, the inhibition of propionic acid producing bacteria (succinic bacteria) and yellowish tumor. The proliferation of gastro cocci promoted the proliferation of white rumen coccus, producing succinic succinic and Vibrio butyrate in the degrading bacteria, and transforming the rumen fermentation mode from propionic acid to acetic acid. The regulation of three tea saponin to the rumen abundance and group composition of rumen of beef cattle was capable of producing methanogens. Alkanes provide the required substrates (metabolic hydrogen and carbon compounds), and ciliated protozoa also provides a growth and reproduction habitat for some methanogens. Therefore, on the basis of understanding the changes in the composition of the rumen ciliated protozoa and bacterial flora by TSS, qPCR and Illumina Miseq sequencing techniques are used to explore the regulation of TSS on the abundance of methanogens and the composition of the bacteria. The results were as follows: 1) 1) the genus Methanobacterium was the absolute dominant Methanobacterium in the rumen of beef cattle (95%). In addition, phylogenetic tree analysis found that in 15 dominant methane bacteria OTUs, more than 86% (13 OTUs) were located in the SGMT cluster, that is, SGMT methanogens were the main high potential methanogens; 2) added TSS to increase the absolute abundance of methanogens. Degree (P0.05), but there was no significant effect on the abundance of methanobacteria and methanobacteria (P0.05).3). At the cluster level, adding TSS decreased the relative abundance of the high abundance of SGMT methanogens and increased the relative abundance of RO methanogens with low abundance: 4) phylogenetic tree analysis found a selective symbiosis with the parasite and other protozoa. The main species of alkanes are SGMT methanogens, and the methanogens which symbiotic with the protozoa are mainly RO methanogens. The above results show that TSS changes the floristic composition of methanogens in the rumen of beef cattle by regulating the diversity of the rumen methanogens at the cluster level, that is, the inhibition of the high abundance of SGMT cluster a with the selective paragenesis of the protozoan protozoa. Proliferation and promoting the proliferation of RO methanogens with low abundance of selective paragenesis with protozoa. Test four comparison of methane production differences between different methanogens in different methanogens, this experiment aims to compare the main representative bacteria Mbr. ruminantium of the low abundance RO cluster in the dominant methanum genus methanogens and the main representative bacteria M of high abundance SGMT clusters The growth index and methane production activity of br. gottschalkii were different. The growth index and methane production of methanogens were measured at 13 time points (0,8,12,16,20,24,30,36,48,60,72,84,96h) at the time history (Time-course) culture of 2 methanogens, and methane production and methanogens abundance and armour production were studied at DNA and RNA levels. The results were as follows: 1) the logarithmic growth period (40H) and stable duration (24h) of Mbr. ruminantium were both higher than that of Mbr. gottschalkii (36h) and stable duration (12h); 2) the maximum biomass and maximum growth rate of Mbr. ruminantium were significantly higher than that of Mbr. gottschalkii (3) two methane. The cumulative methane yield of the bacteria was significantly positively correlated with the abundance and mcrA gene expression level (P0.05), and the abundance of methanogens and the expression level of mcrA genes also showed significant positive correlation (P0.05). 4) the difference of H2 consumed by Mbr. ruminantium and Mbr. gottschalkii was not significant (P0.05), but the quantity of Mbr. ruminantium generated was significantly higher than that of those. Gottschalkii (P0.05); 5) Mbr. ruminantium can continue to grow a small amount of methane at low hydrogen concentration, while Mbr. gottschalkii can not use low concentration of hydrogen to produce methane. The above results show that the low abundance RO cluster of the genus brevis represents the methanogenic activity of Mbr. ruminantium and the high adaptability to low concentration environment. The SGMT cluster of high abundance represents Mbr. gottschalkii, and the abundance of methanogens and the level of mcrA gene expression can be used to evaluate methanogenic activity of methanogens. In summary, the regulatory effect of cilium protozoa, bacteria and methanogens on TSS is different, and 30 g/d TSS is added to the high concentrate diet of beef cattle. After that, TSS changed the structure of the microbial flora of the rumen. For the rumen ciliated protozoa, the addition of TSS selectively inhibited the proliferation of protozoa (internal hair) using starch and promoted the proliferation of protozoa using fibrous material (multi and true double hairy genus); for bacteria, adding tea saponin selectively inhibited propionic acid producing bacteria (succinic bacteria). And the proliferation of yellowish rumen cocci promoted the proliferation of white rumen cocci, filamentous succinic acid and Vibrio butyrate in the degrading bacteria, and the rumen fermentation mode of beef cattle was converted from propionic acid to acetic acid; for methanogens, the addition of tea saponin selectively inhibited the proliferation of SGMT methanogens of high abundance in the genus brevibacilli. The proliferation of RO methanogens with low abundance was promoted. The low abundance of RO methanogens representing the bacteria Mbr. ruminantium and the ability to adapt to the low concentration environment were higher than the high abundance of SGMT methanogens, representing the Mbr. gottschalkii. tea saponin mainly by inhibiting the selective symbiotic abundance of methanogens with the parasite protozoa. The proliferation of low active SGMT methanogens and the promotion of the proliferation of RO methanogens with high methane producing activity with low abundance but high methane producing activity, which affect the methane production in the rumen of beef cattle.

【学位授予单位】：四川农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S823

【参考文献】