Effects Of Antibiotic Intervention On Small Intestinal Microbiota And Amino Acid Metabolism In Pigs

The small intestinal bacteria of pigs can metabolism essential amino acids in vitro.However,it is still unclear that the effects of small intestinal bacteria on dietary amino acids metabolism in vivo.Antibiotics have been widely used in animal husbandry for growth promotion,but its impact on small intestinal microbiota and amino acid metabolism of pigs at different growth stage is large unknown.Early-life environment variation has long-lasting effects on host metabolism and gut microbiota.However,it is still unclear whether the early life administration of antibiotics has long-lasting effects on the different growth stages of pig’s small intestinal bacteria and amino acids metabolism.Furthermore,whether ileum infusion antibiotic intervene the large intestinal bacteria has a feedback regulation the composition of small microbiota and amino acid absorption still not clear.Thus,this study used pigs as subjects,and using continuous feeding experiment and ileum infusion experiment,aimed to investigate the effects of antibiotic intervention on the small bacteria and amino acids metabolism of the pigs.1.Effects of Antibiotics Intervention on Small Intestinal Mmicrobiota and Amino Acids Metabolism of Suckling PigletsSixteen litters of suckling piglets were randomly allocated to 2 groups and were fed a creep feed diet with or without antibiotics(olaquindox,oxytetracycline calcium,and kitasamycin,50 mg/kg)at postnatal day 7.The experiment lasted for 16 days.The results showed that antibiotic treatment did not affect the growth performance(P>0.05),but significantly increased the relative pancreas weight(P<0.05)when compared with control group.Antibiotic treatment increased serum urea and blood ammonia concentrations(P<0.05),but decreased serum glucose concentration(P<0.05).Antibiotic treatment significantly decreased the number of total bacterial,Firmicutes,and Lactobacillus in the digesta of jejunum(P<0.05),but increased the number of Escherichia coli in ileal digesta(P<0.05).High throughput analysis results showed that antibiotic significantly altered the bacterial community composition in the ileum.At genus level,antibiotic treatment significantly increased the abundance of Streptococcus,Rothia,Actinomyces,and Corynebacterium in the ileum(P<0.05),while decreased the abundance of Lactobacilli(P<0.05).Amino acids analysis showed that antibiotic significantly decreased amino acids concentrations in jejunal and ileal digesta(P<0.05),while increased serum,jejunal and ileal tissue amino acids concentrations(P<0.05).Antibiotic treatment upregulated the mRNA expression of jejunal amino acid transporters(y+LAT1 and b0.+ AT),peptide transporters(PepT1),and Na+/K+-ATPase(ATP1A1),and ileal AA transporters(EAACI,ASCT2,y+LAT1,and ATB0.+)(P<0.05).Antibiotics also upregulated the mRNA expression of jejunal and ileal AAs receptors T1R3(P<0.05).These results indicated that antibiotic treatment significantly decreased the quantity of bacteria and altered bacteria composition in the small intestine,reduced amino acids concentrations in jejunal and ileum lumen,but increased them in serum and jejunal and ileum tissue,and upresulated the expression of amino acid transporters and receptors in the jejunum and ileum.2.Effects of Antibiotics Intervention on Small Intestinal Microbiota and Amino Acids Metabolism of Weaned PigletsBased on the second chapter of animal experiment,the control and antibiotic group piglets were weaned on d 23 of age and fed the same diets,respectively.The experiment lasted for 18 days and slaughtered at d 42 of age.The results indicated that antibiotic did not affect the ADG and ADFI(P>0.05),but increased the weight gain to feed ratio and the relative spleen weight(P<0.05),and increased the concentration of serum urea(P<0.05).Antibiotics decreased ileal total bacteria and Firmicutes counts(P<0.05),and tended to decrease jejunal total bacteria counts(P=0.069).At phyla level,antibiotic significantly decreased the abundance of Firmicutes in ileal digesta(P<0.05),while increased Proteobacteria and Bacteriodetes(P<0.05).At genus level,antibiotic treatment increased the abundance of Escherichia-Shigella and Actinomyces in the ileum(P<0.05).At the phylotype level,antibiotic treatment significantly decreased the abundance of Lactobacillus mucosae and Bacillus methylotrophicus(P<0.05),but increased the abundance of Escherichia coli,Actinobacillus minor,and Streptococcus gallolyticus(P<0.05).Amino acids analysis showed that antibiotic treatment increased concentrations of most AAs in serum,jejunal and ileual tissue(P<0.05),and decreased the concentrations of most AAs in jejunal and ileal digesta.In jejunum,antibiotics upregulated(P<0.05)the mRNA expression levels of jejunal AAs transporters(CAT1,EAACI,ASCT2,and y+LAT1)and ATP1A1,and also upregulated the mRNA and protein expression of PepTl.In ileum,antibiotic treatment upregulated the mRNA expression level of ASCT2,y+LAT1,b0,+AT,and B0AT1,and ATP1A1(P<0.05).The antibiotics also upregulated the mRNA expression of jejunal AAs receptors T1R3 and CaSR,and ileal T1R3(P<0.05).Our results showed that antibiotic treatment significantly increased the feed conversion ratio,decreased the quantity of bacteria,and altered bacteria composition in the small intestine of weaned piglets,upregulated mRNA expression levels for key AA transporters,and then improved the absorption of AAs into blood.3.Effects of Early Antibiotic Intervention on Subsequent Small Intestinal Microbiota and Amino Acids Metabolism in Growing-pigsBased on the third chapter of animal experiment,pigs within control and antibiotic groups were mixed,respectively,fed the same and had no any antibiotics diets during d 42 to 77 and 77 to 120 of age.The pigs were slaughtered at d 77 and 120 of age.Compared with control group,at d 77,early antibiotic intervention(EAI)significantly reduced the number of Bacteroidetes(P<0.05).Meanwhile,EAI decreased the diversity and richness index of ileal microbiota(P<0.05).At the genus level,EAI significantly decreased the abundance of Clostridium sensu stricto 1,Blautia,and Butyrivibrio(P<0.05),while increased the abundance of Streptococcus(P<0.05).EAI increased the concentrations of some amino acids in serum,jejunal and ileal tissue(P<0.05),but had no effects on jejunal and ileal digesta amino acids concentrations(P>0.05).EAI upregulated the mRNA expression of EAAC1、b0,+AT,and B0AT1 in jejunal mucosa(P<0.05),but had no effect on ileal amino acid transporters expression(P>0.05),and did no effect on the expression of amino acid receptors in jejunal and ileal mucosa(P>0.05).At d 120,EAI significantly decreased the number of total bacteria and Bacteroidetes in jejunum(P<0.05).EAI also decreased the abundance of Streptococcus and Escherichia-Shigella(P<0.05),but increased Bifidobacterium,Blautia,unclassified Ruminococcaceae,and Mogibacterium(P<0.05).EAI increased some amino acids concentrations in serum,jejunal and ileal tissue and digesta(P<0.05).EAI upregulated the mRNA expression of CAT1,ASCT2,ATB0,+,B0AT1,and ATP1A1 in jejunal mucosa(P<0.05),and ileal CAT1,ASCT2,B0AT1,and ATP1A1(P<0.05),while downregulated the mRNA expression of PepTl(P<0.05).EAI also upregulated the mRNA expression of jejunal AAs receptors T1R3 and CaSR(P<0.05).These results indicated that EAI had long-lasting effects on the different growth stages of pig’s intestinal bacteria and amino acids metabolism,and varied between age.4.In vitro Metabolism of Amino Acids by Pigs Ileal Bacteria Intervened with AntibioticThere are two source of ileal digesta:one was collected from the piglet infused with antibiotics;the other was collected from the piglet infused with saline.The digesta was inoculated into the bottles containing anaerobic media with 10 mmol/L single amino acid as substrate,respectively.The bottle was incubated for 24 h.Samples were collected to analyze the concentrations of free amino acid,ammonia,MCP and biogenic amine at 0 h and 24 h.The results showed that in antibiotic intervention group,the utilization of lysine,threonine or arginine by microbiota was significantly decreased compared to the control group(P<0.05).The concentration of ammonia nitrogen in antibiotic intervention group with lysine,threonine,arginine,and valine as substrate was significantly decreased compared with that in control group(P<0.05).In antibiotic intervention group,the putrescine,cadaverine and spermine in bottles with lysine as substrate were significantly lower than those in control group(P<0.05).The putrescine in antibiotic intervention group with arginine as substrate was significantly decreased compared with that in control group(P<0.05).However,there was no significant difference between the groups with arginine as substrate.DGGE and real-time PCR showed that though the antibiotics decreased the diversity of the microbiota,no significant difference in the number of total bacteria was observed between the antibiotic intervention group and the control group.The diversity in treatment group with lysine,threonine or arginine as substrate was significantly decreased compared to that in control group(P<0.05).The number of Lactobacillus in treatment group with lysine or threonine as substrate was significantly lower than those in control group(P<0.05).The number of Escherichia coli in treatment group with threonine or arginine as substrate was significantly lower than those in control group(P<0.05).The number of Escherichia coli in antibiotic intervention group with lysine as substrate tended to be lower than those in control group(P=0.057).Overall,our results indicated that antibiotics infused to piglet significantly altered the microbiota in ileal digesta.The microbiota of ileal digestain treatment group was able to metabolize amino acids,but there was difference in metabolism of different amino acids by the microbiota.The antibiotics decreased the utilization of lysine,threonine and arginine,However,the utilization of glutamic acid and branched chain amino acids was not influenced.5.Effects of Terminal Ileum Infusion Antibiotics on Amino Acids Concentrations and Gene Expression of Amino Acids Transporters in the Small Intestine in PigletsThis study aimed to investigate the effects of ileal infusion antibiotics on amino acids composition and amino acid transporters and receptors expression in intestinal epithelial cells.Based on the chapter five,the pigs were fed 25 d and slaughtered at d 26.These results indicated that ileum infusion antibiotic had no effect on the number of main bacteria(P>0.05).However,ileal infusion antibiotic altered the amino acids profiles,significantly decreased the concentrations of some amino acids in serum and jejunal tissue(P<0.05),while increased their concentrations in jejunal and ileal digesta(P<0.05).Compared with control group,ileal infusion antibiotics downregulated the mRNA expression levels of jejunal amino acids transporters(EAAC1,CAT1,b0,+AT,ASCT2,and ATB0,+)in jejunal mucosa(P<0.05),and decreased ASCT2 expression in ileal mucosa(P<0.05),while tended to increase the mRNA expression level of PepTl(P=0.064).Furthermore,infusion antibiotic also significantly increased the mRNA expression of ileal amino acids receptors CaSR(P<0.05),and tended to increase T1R3 expression(P=0.054),while had no significantly effects on jejunal mucosa amino acids receptors(P>0.05).These results indicated that ileal infusion antibiotics altered the amino acids profiles,decreased serum and jejunal and ileal tissue level of most amino acids while increased most amino acids in the small intestine,and this change was correlated with the downregulations of mRNA expression levels for key amino acids transporters and receptors in the small intestine,but had no effect on the main bacterial population.Employing continuous feeding of pigs,the current experiment found that antibiotic decreased the number of small intestinal microbiota and changed the composition,upregulated the expression of amino acid transporter,and then make more amino acids absorbed into blood.Meanwhile,antibiotic intervention had long-lasting effect on small intestinal microbiota and amino acid metabolism.In vitro,inoculation of ileal bacteria intervened with antibiotic reduced the utilization of lysine,threonine,and arginine.Ileum infusion antibiotic downregulated the expression amino acid transporters and inhibited the absorption of amino acid in jejunum.