【作者】 彭宇；

【导师】 朱伟云；

【作者基本信息】 南京农业大学 ， 动物营养与饲料科学， 2017， 博士

【摘要】 肠道微生物对宿主营养物质的消化吸收、肠道健康及功能具有重要作用。日粮是影响肠道微生物最主要的因素之一,近年来营养素与肠道微生物之间的互作成为研究热点。为了缓解我国蛋白质资源紧缺,在猪养殖中采用低蛋白氨基酸平衡日粮能提高氮营养素利用率,减少氮污染物排放。但日粮蛋白水平和氮营养素形式对肠道微生物和微生物代谢的影响还有待进一步研究。大肠微生物由于数量巨大、种类繁多,是维持机体肠道微生物稳态最主要的组成部分,建立大肠微生物干预模型有利于深入研究其对宿主营养、代谢和肠道功能的作用。本研究以猪为研究对象,首先研究蛋白质水平和氮营养素形式对肠道微生物及代谢产物的影响,然后建立了抗生素干预大肠微生物模型,研究大肠微生物对宿主氮营养素消化、吸收和宿主代谢的影响,并探讨了肠道微生物及其代谢的改变对肠道发育和蠕动功能的影响。1低蛋白必需氨基酸平衡日粮对生长猪肠道微生物及其代谢产物的影响试验选取40头杜长大阉公猪(日龄45±2天,初始体重13.50±0.50 kg),随机分为4组,每组10个重复,每个重复1头猪。分别饲喂20.00%(NP)、17.16%(MP)、15.30%(LP)和13.90%(ELP)粗蛋白日粮,平衡必需氨基酸使其满足NRC(2012)营养需求,试验期28天。试验结束选取体重接近平均体重的仔猪8头屠宰,采集回肠、盲肠、结肠食糜和黏膜组织,分析菌群结构和代谢产物。结果显示:较NP和ELP组,适当降低日粮粗蛋白显著增加了(P<0.05)LP组结肠食糜,MP和LP组结肠黏膜的菌群多样性指数。回肠、盲肠和结肠食糜中Bifidobacterium数量随日粮水平降低呈显著线性下降(Linear,P<0.05),ELP组盲肠和结肠食糜中Bifidobacterium的数量显著低于(P<0.05)NP和MP组,但ELP组结肠食糜中E.coli数量则显著高于(P<0.05)LP组。随日粮蛋白水平下降,回肠食糜中总短链脂肪酸(T-SCFA)、乙酸和丁酸浓度线性增加(Linear,P<0.05),而盲肠中所有短链脂肪酸(除甲酸外),结肠食糜中T-SCFA和乙酸浓度线性降低(Linear,P<0.05)。降低蛋白水平导致回肠、盲肠和结肠食糜中微生物蛋白(MCP)和氨氮浓度均线性降低(Linear,P<0.05),但盲肠食糜中亚精胺,结肠食糜中总生物胺、腐胺、甲胺和亚精胺浓度则呈现出先升高后降低的二次项变化规律(Quadratic,P<0.05),且LP组浓度最高。结果表明,适度降低日粮粗蛋白水平(降至15.30%)能提高大肠内菌群多样性,但更大程度的降低蛋白水平则可能对微生物结构和发酵产生不利影响。2低蛋白日粮补充不同形式氮营养素对生长猪结肠微生物、微生物代谢产物及肠道发育基因表达的影响试验选用80头杜长大阉公猪(初重15.57±0.13 kg,日龄48±2天),随机分为4组。每组5个重复,每个重复4头猪。分别饲喂粗蛋白为17.00%(MP)、15.00%(LP)、13.00%+AA(ELP-AA)和 13.90%+酪蛋白(ELP-CAS)的日粮。MP、LP和ELP-AA组日粮平衡所有AA,ELP-CAS组日粮中添加3%酪蛋白,均使其满足NRC(2012)中15-25kg猪AA需求,试验期28天。试验结束后每栏挑选1头接近栏平均体重的仔猪屠宰,采集结肠食糜和组织,分析食糜中营养物质浓度、微生物数量和微生物代谢产物,肠道组织中发育相关基因表达。结果显示ELP-AA和ELP-CAS组结肠食糜中粗蛋白含量显著低于(P<0.05)MP和LP组,而葡萄糖含量显著高于(P<0.05)MP和LP组。ELP-CAS组总糖和支链淀粉含量显著低于(P<0.05)MP和 LP 组。ELP-AA 组 Firmicutes、Bacteroidetes、Bacteroides、Prevotella数量显著低于(P<0.05)MP 组,Lachnospiraceae、Roseburia和Bifidobacterium数量显著低于(P<0.05)其他三组,而ELP-CAS则与MP组无显著差异。ELP-CAS组较MP组显著降低了(P<0.05)Clostridium cluster ⅣV 群,Peptostreptococcus数量显著低于(P<0.05)其他三组。对于细菌代谢产物,ELP-AA和ELP-CAS组较MP和LP组显著降低了(P<0.05)结肠中总短链脂肪酸(T-SCFA)和乙酸浓度,较MP组显著降低丁酸浓度(P<0.05),但ELP-CAS组甲酸浓度显著高于其他三组(P<0.05),氨氮和微生物蛋白浓度(MCP)浓度显著高于(P<0.05)MP和LP组。ELP-AA和ELP-CAS组总生物胺浓度显著升高(P<0.05),其中尸胺浓度较MP和LP组显著增加(P<0.05),而亚精胺和组胺浓度较LP组显著降低(P<0.05)。对于肠道组织中发育相关基因的表达,ELP-CAS 组 GLP-2、EGF、βFGF、βFGF-rI基因表达均较 LP 和 ELP-AA 显著增加。研究结果表明,日粮粗蛋白从17.00%降至13.00%补充AA或酪蛋白均导致结肠中碳水化合物、碳水化合物发酵菌和SCFA的降低。极低蛋白日粮中补充酪蛋白能改善大肠中部分菌群数量并促进肠道发育基因表达,但增加了微生物蛋白合成、氨氮和生物胺浓度。3回肠末端灌注抗生素对猪全肠道微生物结构的影响试验选用14头杜长大阉公猪(初重12.08±0.28 kg,日龄45天),手术安装回肠末端T型瘘管,术后恢复10天,并随机分为2组,每组7头猪。试验组从瘘管灌注复合抗生素(ANT组,氨辛西林150mg/kg/d,庆大霉素4mg/kg/d,甲硝唑30mg/kg/d,溶于10ml无菌生理盐水),对照组每天灌注相同体积无菌生理盐水(CON组)。试验期25天,试验期间每组各1头因健康不佳被淘汰,饲养试验结束每组屠宰6头猪,采集胃、十二指肠、空肠、回肠、盲肠、结肠食糜和黏膜,利用高通量测序分析了全肠道微生物结构,利用Real-time PCR技术分析空肠和结肠食糜中微生物数量。结果显示:回肠末端灌注抗生素显著(P<0.05)改变了大肠食糜和黏膜中的微生物结构,盲肠、结肠食糜和结肠黏膜中OTU数量、Ace和Chao1指数显著降低(P<0.05),但显著增加了(P<0.05)胃黏膜的微生物丰富度和多样性指数。属水平上,抗生素干预对前肠微生物影响较少,主要增加了Streptococcus的相对丰度。回肠末端灌注抗生素显著减少了(P<0.05)大肠中SCFA产生菌的相对丰度,如Ruminococcaceae、Clostridiales、Coprococcus、Campylobacter、Clostridium、Prevotella和Bacteroides等;增加了(P<0.05)大肠中乳酸产生菌和条件致病菌的相对丰度,如Lactobacillus、Stuterella和Collinsella等。对空肠和结肠食糜中的主要微生物进行定量,结果也显示回肠末端灌注抗生素显著降低了(P<0.05)结肠中 SCFA产生菌数量,如 Clostridium cluster XⅣVa、Roseburia、Ruminococcus和 Lachnospiraceae 等,但增加了乳酸产生菌数量,如 Enterococcus和Lactobacillus。由此说明,回肠末端灌注抗生素主要干预了大肠食糜和黏膜微生物的组成,对前肠微生物影响较小,此模型是研究大肠微生物影响机体代谢的一种有效手段。大肠中短链脂肪酸产生菌的减少和潜在病原菌的增加可能不利于猪的肠道发育和健康。4抗生素干预大肠微生物对猪生长、消化和代谢的影响在前一章动物试验基础上研究抗生素干预大肠微生物对猪生长、消化和代谢的影响。试验期每5天称取体重,每天记录采食量和猪健康状况,计算猪生产性能。试验第26天屠宰,计算屠宰性能并称取器官重量,测量肠道重量和长度;采集血液样品测定血液生理生化和血清激素;采集回肠和结肠食糜用于营养物质消化率分析;采集胃食糜、空肠食糜和胰腺组织分析消化酶活性。结果显示:回肠末端灌注抗生素干预猪大肠微生物对猪体增重和器官重均无显著影响,但降低了小肠(P<0.05)和大肠(P<0.10)的相对重量,显著增加了(P<0.05)大肠食糜重量。对于血液生理生化指标,抗生素干预显著降低了(P<0.05)血清总蛋白、白蛋白、低密度脂蛋白和总胆汁酸浓度,并显著增加了(P<0.05)血清葡萄糖水平和谷草转氨酶活性。与对照组相比较,抗生素组血清中胰岛素、GLP-1、胃泌素和PYY浓度升高。抗生素干预显著增加了粪氮的排放,因此降低了氮的表观利用率。虽然抗生素干预显著增加了(P<0.05)空肠糜蛋白酶活力和胰淀粉酶活力,但对回肠营养物质消化率无显著影响。抗生素干预显著降低了(P<0.05)结肠有机物、粗蛋白、酸性洗涤纤维、中性洗涤纤维以及所有AA(除Arg和Tyr)的消化率,增加了(P<0.10)粗脂肪的消化率。结果表明,猪大肠微生物的改变对猪生长性能和器官发育无显著影响,但可能通过改变大肠营养物质消化率和肠道激素分泌影响机体代谢和肠道发育。5抗生素干预大肠微生物对猪肠道发育及蠕动功能的影响在前两章基础上深入研究大肠中微生物代谢产物变化及干预肠道微生物对肠道发育和蠕动功能的影响。结果显示:抗生素干预显著增加了(P<0.05)回肠末端食糜流量和食糜在盲肠中滞留时间,降低了结肠肠壁肌肉层厚度下降(P=0.087),显著下调了(P<0.05)结肠组织中IGF-1和IGF-1受体的基因表达。分析全肠道微生物代谢产物发现,前肠中微生物代谢产物受抗生素影响较少,抗生素干预显著降低了(P<0.05)盲肠和结肠中SCFA和粪臭素浓度及结肠中氨氮浓度,并增加了(P<0.05)结肠中甲酸、乳酸和生物胺(P胺、色胺和精胺)浓度。对血清和肠道组织调节肠道蠕动的神经递质和激素分析发现,抗生素显著提高了(P<0.05)血清中5-HT、GLP-1和PYY的浓度,其中采食后4h血清中5-HT浓度和采食后8h血清中GLP-1较对照组显著上升(P<0.05)。回肠组织中,抗生素干预对激素和调节激素的基因表达均无显著影响,但显著提高了(P<0.05)结肠组织中5-HT、GLP-1和PYY的浓度,上调了(P<0.05)结肠组织中Tph1、GLP-1和PYY的基因表达,同时5-HT受体基因5HT2A和5HT1B基因表达量也显著提高(P<0.05)。以上结果表明,抗生素抑制大肠中SCFA产生,阻碍了大肠肌肉层的发育,减缓了肠道蠕动。肠道组织和血清中5-HT和促肠道蠕动激素GLP-1、PYY的mRNA表达和浓度增加,引起回肠末端食糜流速加快,延长了大肠中食糜滞留时间。大肠中乳酸和生物胺浓度增加,可能不利于肠道的健康。综上所述,日粮蛋白水平主要影响了猪大肠中微生物结构,极低蛋白日粮造成大肠中微生物多样性下降,有益菌减少,有害菌增加,SCFA产生减少等不利影响。在极低蛋白日粮中补充酪蛋白能部分改善SCFA产生菌数量,增加乙酸产生,促进肠道发育基因表达,但微生物对蛋白质利用和发酵增加不利于宿主对蛋白质的吸收和肠道健康。通过大肠微生物干预模型研究显示大肠微生物通过调节营养物质消化和肠道激素分泌影响宿主的代谢和肠道发育。大肠中SCFA产生菌的减少,导致肠道蠕动减缓、乳酸堆积和蛋白质发酵产生的生物胺增加可能是影响肠道健康的原因之一。更多还原

【Abstract】 The gut microbiota exist along the gastrointestinal tract(GIT)which can have a significant impact upon host metabolism and health via modulation of immunity,intestinal development,protection against pathogens and fermentation of substracts largely provide by the diet.In recent years,the interaction between diet and host microbies was widely studied.Low-protein diet was use to reduce the high-cost protein source and cut down nitrogen excretion in pigs production.However,the response of gut microbiota and fermentation to protein level or nitrogen sources still vary with the reduction extent.Compared with small intestine,the fermentation in single-stomached animals mainly occurs in the large intestine.Thus,development of a direct model to perturb large intestinal bacteria may offer a new idea for further study the interaction between large intestinal bacteria and host metabolism.This study used pigs as subjects,aimed to investigate the effect of low-protein diet on intestinal bacteria and the role of large intestinal bacteria on host metabolism and health.1 Effects of Low-protein Diet on Bacterial Community and Fermentation in Growing PigsForty pigs(Initial BW 13.50±0.50 kg,45 ±2 day of age)were randomly assigned to 4 dietary treatments containing CP levels at 20.00%(Normal crude protein,NP),17.16%(Medium crude protein,MP),15.30%(Low crude protein,LP)and 13.90%(Extremely low crude protein,ELP),respectively.Crystalline amino acids(AA)were added to meet the essential AA(EAA)requirement of pigs.After 4-week feeding,8 pigs per treatment(n=8)were randomly selected and slaughtered for sampling of ileal,cecal and colonic digesta and mucosa.Pigs with moderately reduced CP level had increased bacterial diversity,with the Shannon diversity indices for the colon digesta in MP group and mucosa in MP and LP groups significantly(P<0.05)higher than those in NP and ELP groups.As the CP level reducing,the Bifidobacterium population were linear decreased(P<0.05)both in ileum,cecum and colon,and the ELP group had the lowest Bifidobacterium population in the cecum and colon,with its value significantly lower than NP and MP groups(P<0.05).However,the ELP group had the highest population of Escherichia coli in the colon,with its value significantly higher than LP group(P<0.05).For bacterial metabolites,as CP level decreased,total short-chain fatty acid(T-SCFA),acetate and butyrate were linearly increased(Linear,P<0.05)in the ileum,while all SCFAs except formate in the cecum and T-SCFA and acetate in the colon,were linearly decreased(P<0.05).Reducing CP level led to a linear decrease of microbial crude protein(MCP)in the ileum(P<0.05)and ammonia in all intestine segments(P<0.05).The spermidine in cecum and total amines,cadaverine,methylamine,spermidine in colon were shown a quadratic change(P<0.05)with dietary CP decrease,with the highest concentration in LP group.These findings suggest that moderate reduction of dietary CP level may benefit large intestinal bacterial community and its fermentation,which were negatively affected by extremely low CP diet.2 Effect of supplemention AA or Casein in Extrmely Low-protein Diet on Colonic Bacterial Community,Metabolites and Gut Development Genes Expression in Growing pigsEighty pigs(Initial BW 15,57±0.13 kg,48±2 day of age)were randomly assigned to 4 dietary treatments,17%CP plus essential AA,His and Cys(Medium crude protein,MP),15%CP plus essential AA,His,Cys,Phe,Leu and Tyr(Low crude protein,LP),13%CP plus all AA(Extremely low crude protein,ELP-AA)and 13%CP plus casein(Extremely low crude protein,ELP-CAS),respectively.Crystalline AA or casein were added to meet the AA requirement of pigs.Pigs were housed in 4 pigs per pen with 5 pens per treatment.After 4-weeks feeding,five pigs per treatment(n=5)were selected and slaughtered for sampling of colonic digesta and tissues.Pigs fed the ELP-AA diet or ELP-CAS diet had lower(P<0.05)crude protein(CP)and higher(P<0.05)glucose content than pigs fed MP or LP diets.Compared with MP or LP groups,pigs fed ELP-CAS diet decreased the concentrations of carbohydrate(P<0.05)and amylopectin contents(P<0.05),For bacteria counts,pigs in ELP-AA group had lower(P<0.05)Firmicutes,Bacteroidetes,Bacteroides and Prevot.ella counts than MP group,and had lowest(P<0.05)Lachnospiraceae,Roseburia and Bifidobacterium counts than other three groups.However,these bacteria were partly normalized by feeding the ELP-CAS diet.Pigs fed ELP-CAS diet had lower(P<0.05)Clostridium cluster IV than MP diet and had lowest(P<0.05)Peptostreptococcus in the four groups.For bacteria metabolites,pigs fed ELP-AA or ELP-CAS diets decreased(P<0.05)the total short chain fatty acids(T-SCFA)and acetate level relative to pigs fed MP or LP diets,and decreased(P<0.05)the butyrate level relative to pigs fed MP diet.However,the ELP-CAS group had the highest(P<0.05)formate level in four groups.The ammonia(P<0.05)and microbial crude protein(MCP)(P<0.05)levels were increased in pigs fed ELP-CAS diet relative to pigs fed MP or LP diets.The total amines level was increased(P<0.05)both in ELP-AA and ELP-CAS groups,with the markedly higher cadaverine(P<0.05)level than MP and LP groups,and lower spermidine(P<0.05)and histamine(P<0.05)levels than LP group.Furthermore,the mRNA expression of GLP-2,eEGF,PFGF and FGF receptor I were significantly increased(P<0.05)in pigs fed ELP-CAS diet relative to pigs fed LP and ELP-AA diets.In summary,reducing dietary CP level from 17%to 13%decreased the CP and carbohydrate contents and consequently led to reduction of SCFA producing bacterial counts and SCFA production.However,bacterial counts,nitrogen fermentation and gut development gene expression were partly improved using casein to replace free AA in extremely low-protein diet,which may led to the normalized gut development and growth performance of pigs.3 Effects of Hindgut Microbiota Perturbation by antibiotics Infusion on Gastrointestinal Bacterial Community and Fermentation of PigsGut bacteria,especially the large intestinal bacteria,was critical to the host metabolism and health.However,seldom research focused on the effect of hindgut microbial on host metabolism.Fourteen DurocxLandracexLarge barrows(45 days of age,12.08±0.28 kg)were fitted with a T-cannula at the terminal ileum and randomly assigned to two groups and infused either saline(Control,CON)or antibiotics(ampicillin at 150 mg/lkg/day,gentamicin at 4 mg/kg/day and metronidazole at 30 mg/kg/day)(Antibiotics,ANT)into hindgut.After 25 days\experiment,six pigs from each group were euthanized,digesta and mucosa samples were collected for analysis of total gastrointestinal(GIT)microbiota through high throughput sequencing and bacterial counts through Real-time PCR.Meanwhile,microbial metabolites were determined,aiming to investigate the change of microbial composition in the GIT of pigs after antibiotics infusion into terminal ileum and the influence of antibiotics on bacterial fermentation.The results showed that antibiotics infusion mainly affected the cecal and colonic bacterial community with the significantly decreased the number of OTUs,the indices of Ace and Chaol both in digesta and mucosa.However,the bacterial diversity of stomach mucosa were increased.Antibiotics infusion has less effects on bacterial community of small intestine,mainly increased the genus of Streptococcus,The short chain fatty acids(SCFA)producing bacterial were significantly deceased in hindgut,such as Ruminococcaceae,Clostridiales,Coprococcus,Campylobacter,Clostridium,Prevotella and Bacteroides,while the potential pathogenic bacteria were increased,such as Stuterella and Collinsella.Real-time PCR results also showed that the antibiotics significantly decreased(P<0.05)the colonic SCFA producing bacteria counts,such as Clostridium cluster ⅩⅣa,Roseburia,Ruminococcus and Lachnospiraceae,while the lactic acid producing bacteria counts were increased(P<0.05),such as Enterococcus and Lactobacillus.These results suggested ileum terimal antibiotic infusion mainly affected large intestinal bacterial community and improved that we have developed a hindgut microbiota perturbation model through infusion antibiotics into terminal ileum.The suppression of commercial SCFA producing bacteria and stimulation the potential pathogenic bacteria may affect the gut development and health of pigs.4 Perturbation of Porcine Hindgut Microbiota by Antibiotics Changed Host Metabolism through Altering Nutrient Digestion and Hormone SecretionBased on the animal trial in chapter 4,after 25 days’ experiment,six pigs from each group were euthanized,blood samples were collected for analysis of blood profile and serum hormone;ileum and colon digesta samples were collected for analysis of nutrient digestion;stomach,jejunum and pancreas samples were collected for analysis enzyme activity.Results showed that antibiotics infusion had no effect on the weight gain,carcass weight,muscle and viscera organs weight,but decreased the relative weight of small(P<0.05)and large intestine(P=0.054),while increased(P<0.05)the content in large intestine.For blood profile,antibiotics reduced the concentrations of total protein(TP)(P<0.05),albumin(ALB)(P<0.05),low density lipoprotein cholesterol(LDL-C)(P<0.05))and total bile acids(TBA)(P<0.05)in serum while increased the serum level of glucose(GLU)(P<0.05)and the activities of aspartate aminotransferase(AST)(P<0.05).For serum hormones,the levels of insulin(P<0.05),gastrin(P<0.05)and peptide YY(PYY)(P<0.05)were elevated by antibiotics.The increased fecal nitrogen by antibiotics led to the retained nitrogen decrased.The jejunal chymotrypsin activity(P<0.05)and pancreatic amylase activity(P<0.05)were increased by antibiotics treatment,but did not affect the ileal nutrient digestibility.While antibiotics infusion reduced the colonic organic matter(OM)(P<0.05),crude protein(CP)(P=0.060),acidic detergent fiber(ADF)(P<0.05),neutral detergent fiber(NDF)(P<0.05)and all AA(except Arg and Tyr)digestibility,whereas increased the ether extract(EE)digestibility(P=0.080)in colon.Our findings indicate that antibiotics infusion into terminal ileum did not affect the weight gain of the pigs,but had evident impact on the large intestine development and host metabolism,possibly due to the alteration of large intestinal nutrient digestion and gut hormone secretion.5 Perturbation Hindgut Microbiota by antibiotics infusion inhibited Colon development and MotilityBased on the animal trial in chapter 4 and chapter 5,we deeply analyzed the influence of antibiotics on jejunal and colonic bacteria counts,gut development and motility function.The organic matter flow and nitrogen flow of the ileum,the retention time of cecal digesta were increased(P<0.05)by antibiotics.Antibiotics decreased(P=0.087)the muscle layers and down-regulated(P<0.05)the mRNA expressions of IGF-1 and IGF-1 receptor of colon.The SCFA and skatale concentrations were decreased both in cecum and colon,while the formate,lactic acid and bogenic amins(cadaverine,tryptamine and sperimine)decreased by antibiotics infusion.Antibiotics infusion significantly affected(P<0.05)the serum 5-HT and hormones with increasing the serum 5-HT concentration 4 hours after feeding and the GLP-1 concentration 8 hours after feeding.There were no significantly changes(P>0.05)of hormone levels in jejunum tissue,while,the antibiotics increased(P<0.05)the GLP-1,PYY and 5-HT level and up-regulated the mRNA expressions of GLP-1,PYY,Tphl of colonic tissue.Besides,the 5-HT receptor of 5-HT2A and 5-HT1B were increased(P<0.05)in antibiotics group.These results suggested that inhibition of SCFA producing bacteria decreased the colon smooth muscle growth and motility.As the potential feedback regulation to decreasing motility,the hormone 5-HT,PYY and GLP-1 secretion increased.However,these hormone increased ileum digesta flow and enlarged the large intestinal capacity.These changes may enhance bacterial protein fermentation and consequently affect host gut health.Above all,dietary protein level mainly affected large intestinal bacterial community and its fermentation.Extremely decreasing protein level negatively affected bacterial counts and metabolities.Although some SCFA producing bacteria and SCFA production were partly improved using casein to replace free AA in extremely low-protein diet,the increasing MCP and nitrogen fermentation may limited the host nitrogen absorbtion.Large intestinal bacteria affects host gut development and metabolism,possibly through the alteration of large intestinal nutrient digestion and gut hormone secretion.As the SCFA production decreasing,the large intestinal motility and the utilization of lactic acid decrased,while the biogenic amines concentration was dramatic rised,which may negatively affect host gut health.更多还原