Large Intestinal Microbes And Host Metabolism Response To Low Protein Amino Acid Supplementation Diets And Ileum Terminal Antibiotic Intervention In Pigs

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.