| It is a major task for current pig industry to improve disease resistense.Host gut characteristics plays a critical role in determining pig's resistance to diseases.Gut microbiota,known as animal second genome,is closely related to host gut development.It is not clear whether gut microbiota mediated the gut differences between different pig breeds.As the bacterial fermentation product of dietary fiber,SCFAs play an essential role in maintaining intestinal barrier function.However,whether SCFAs mediated the effects of gut microbiota on host's gut development remains unclear.The objective of present study was to reveal the correlationships between gut microbiota and host gut development and underlying crosstalk mechanisms.Six experiments were conducted to investigate the effects of dietary fiber and infusing SCFAs on gut microbiota composition,SCFAs profiles and the expression of gut development and function-related genes by using 16S rRNA sequencing,GC and RT-PCR after examining the difference of gut microbiota of different pig breeds and its relations to gut structure and functions by fecal microbiota transplanting to germ-free mice and suckling piglets.The results obtained in this study will give new insights into interactions among nutrition,microbiota and host,and interpreting the mediation of SCFAs.Experiment 1 The differences of gut microbiota composition and gut-phenotypes among pig breeds,and the transitivity of these characteristics in germ-free mice via fecal microbiota transplantaitonThe present study was conducted to compare the differences in gut microbiota composition and gut phenotypes among pig breeds,and determine whether these differences would transmit to mice colonized with fecal microbiota of different pig breeds.A total of 24 1-day-old germ-free BALB/C mice were divided into 3 groups(TFM,YFM and RFM),which were transplanted with intact fecal microbiota of Tibetan pig(TP).Yorkshire pig(YP)and Rongchang pig(RP),respectively.The whole trial lasted for 28 d.The results are as follows:1)Different pig breeds exhibited distinct gut microbiota profile based on high-throughput pyrosequencing.YP exhibited lower Firmicutes count and higher Bacteroidetes count compared with RP and TP,and higher levels of bacteria from Spirochaetes were observed in TP compared with RP and YP(P<0.05).Transplanting porcine microbiota into GF mice replicated the phenotypes of pig donors.2)TP and RP had higher intestinal weight and relative mRNA expression of CDX2 in ileum than YP,and longer intestine,higher villus height of duodenum and jejunum were observed in TP compared with YP and RP(P<0.05).TP exhibited higher GLP-2 mRNA expression in duodenum and jejunum than RP(P<0.05).Similarly,YFM had lower intestinal weight and CDX2 mRNA expression in ileum than TFM and RFM(P<0.05).The intestinal length in TFM was longer than that in RFM,and TFM had higher villus height in duodenum and jejunum and GLP-2 mRNA expression in ileum than the other two groups(P<0.05).3)The digestive and absorptive ability was different among the three groups in donor pigs and mice recipients.YP had higher jejunal lactase and maltase activities than TP and RP,while TP had higher activities of jejunal ATPase,y-GT,and relative SGLT1 mRNA expression in duodenum and jejunum than YP and RP(P<0.05).The relative ZO-1 mRNA expression of ileum in TP was higher than that in YP(P<0.05).Likewise,YFM had higher jejunal sucrase and maltase activities than TFM and RFM,whereas higher jejunal y-GT activity and relative SGLT1 mRNA expression of duodenum and ileum were observed in TFM compared with YFM and RFM(P<0.05).The relative ZO-1 mRNA expression of duodenum and ileum in TFM were higher than that in YFM(P<0.05).In conclusion,there were large differences in gut microbiota composition and gut characteristics among pig breeds,and gut microbiota could partially convey host characteristics from pigs to mice.Experiment 2 Effects of early fecal microbiota transplantation on gut development in sucking pigletsThe present study was conducted to investigate the effects of early fecal microbiota transplantation on gut development in sucking piglets.A total of 24 3-day-old DLY sucking piglets(2.11 ± 0.15)kg were divided into 4 group(TMP,YMP,RMP amd control group(CON)),which were transplanted with intact fecal microbiota of Tibetan pig(TP),Yorkshire pig(YP).Rongchang pig(RP),and without transplantation,respectively.The whole trial lasted for 56 d.The results are as follows:1)When compared with YMP and RMP,TMP and CON had lower diarrhea index(P<0.05).2)When compared with YMP and RMP,TMP had higher jejunal villus height:crypt depth and relative mRNA expression of colonic GLP-2(P<0.05),TMP and CON had higher relative mRNA expressions of GLP-2 and ANG4 in ileum(P<0.05).3)When compared with YMP and RMP,TMP and CON had higher activities of lactase and the relative mRNA expression of DMT1 in jejunum(P<0.05),CON had higher y-GT activity in jejunum(P<0.05),TMP had higher relative mRNA expression of DMT1 in duodenum(P<0.05).The jejunal Ca2+,Mg2+-ATPase activity in TMP was higher than that in CON,and the jejunal Na+,K+-ATPase activity in TMP was higher than that in the other three treantments(P<0.05).4)When compared with YMP and RMP,TMP had lower content of MDA and higher relative mRNA expression of MUC1 in jejunum(P<0.05),CON had higher SOD activity in jejunum(P<0.05),TMP and CON had higher concentration of butyric acid and lower centent of LPS in serum(P<0.05).When compared with TMP,CON had lower count of Lactobacillus spp in cecal and colonic digesta(P<0.05),YMP and CON had higher count of Eschericha coli(P<0.05),RMP had lower concentration of total SCFAs(P<0.05),CON,YMP and RMP had lower relative mRNA expression of IL-10 in colon(P<0.05).These data indicated that early transplantation of the fecal microbiota from the Yorkshire pigs and Rongchang pigs to DLY suckling piglets would destroy the gut microbiota balance and thus damage intestinal health.Experiment 3 Effects of early transplantation of the fecal microbiota from the Tibetan pigs on gut development in DSS-challenged pigletsThe present study was conducted to investigate the effects of early transplantation of the fecal microbiota from the Tibetan pigs on gut development in DSS-challenged sucking piglets.A total of 24 3-day-old DLY sucking piglets(2.08 ± 0.12)kg were divided into 4 groups with 6 replicates per treatment,and 1 pig per replicate.The experiment was designed by 2 × 2 factorial arrangement based on the factors of fecal microbiota transplantation(received the fecal microbiota from the Tibetan pigs and unreceived)and challenge status(dextra sulfate sodium(DSS)challenged and unchallenged).The whole trial lasted for 56 d.The results are as follows:1)On day 3,4 and 5 following DSS induction,the group treated with DSS without fecal microbiota transplantation showed higher clinical symptom of the colon,which was characterized by DAI,compared with the group treated with DSS with fecal microbiota transplantation(P<0.05).2)DSS infusion increased the density of large intestine,villus height and crypt depth in jejunum and ileum(P<0.05),decreased villus height:crypt depth in jejunum and the relative mRNA expressions of EGF,GLP-2,ANG4 and IGF-1 in colon(P<0.05).Fecal microbiota transplantation(FTM)increased the relative length of intestine,the relative mRNA expressions of GLP-2 and ANG4 in jejunum and GLP-2 in colon,and the content of colonic GLP-2(P<0.05),wherease decreased the density of large intestine,villus height and crypt depth in jejunum and ileum(P<0.05).FTM could attenuate the effect of DSS challenge on the density of large intestine,villus height and crypt depth in jejunum and the content of colonic GLP-2 of suckling piglets(P<0.05).3)DSS infusion decreased the activities of Na+,K+-ATPase and CK in jejunum and the relative mRNA expressions of ZNT1 and DMT1 in duodenum(P<0.05).FTM decreased the pH values of jejunal digesta,increased the activities of Na+,K+-ATPase,Ca2+,Mg2+-ATPase and CK in jejunum and the relative mRNA expressions of jejunal SGLT-1 and ileal DMT1(P<0.05).FTM could attenuate the effect of DSS challenge on the activity of CK in jejunum of suckling piglets(P<0.05).4)DSS infusion increased the activity of DAO in serum,the content of MDA in jejunum and colon(P<0.05),enhanced the numbers of Eschericha coli and the concentrations of propionic acid and total SCFAs in colonic digesta(P<0.05),reduced the numbers of goblet cells in colon,the count of Lactobacillus spp and the content of butyric acid in colonic digesta(P<0.05),decreased the relative mRNA expressions of MUC1 and MUC2 in jejunum,Occludin,MUC2.Reg?? and IL-10 in colon(P<0.05),decreased the activities of T-AOC and SOD in colon(P<0.05).FTM increased the T-AOC capacity and SOD activity in jejunum(P<0.05),enhanced the relative mRNA expressions of Reg??and IL-10 in colon(P<0.05),increased the number of Lactobacillus spp in cecal and colonic digests(P<0.05),decreased the content of MDA in colon,the numbers of Eschericha coli in cecal and colonic digesta,the relative mRNA expression of IL-1? in colon,the concentrations of acetic acid,propionic acid,and total SCFAs in colon and the activity of DAO in serum(P<0.05).FTM could attenuate the effect of DSS challenge on the activities of DOA in serum and SOD in colon,the content of MDA in colon,the number of Eschericha coli and the concentrations of propionic acid and total SCFAs in colonic digesta of suckling piglets(P<0.05).These data indicated that DSS infusion could damage the gut health in suckling piglets.Piglets received the fecal microbiota from the Tibetan pigs could attenuate the negative effect of DSS challenge on the gut development.Experiment 4 Effects of high fiber diet on gut development,microbiota composition and SCFAs in growing pigsThe present study was conducted to investigate the effects of dietary fiber levels on gut health in growing pigs.A total of 30 growing pigs with an initial average body weight of(45.8 ± 2.78)kg were divided into 3 groups with 10 replicates per treatment,and 1 pig per replicate,with the respective treatments being corn-soybean meal-based diet(control group,CF = 1.5%),corn-soybean meal-beet pulp-based diet(beet pulp group,CF = 5.74%)and corn-soybean meal-based diet(feed intake-pairing group,CF = 1.5%).The whole trial lasted for 28 d.The results are as follows:1)When compared with control group,beet pulp group had higher relative weight of small intestine and total intestine,relative density of large intestine and villus height in jejunum and ileum(P<0.05).The relative mRNA expressions of doudenal CDX2,EGF,GLP-2 and GLP-2R,jejunal EGF and GLP-2,ileal EGF and colonic GLP-2 in beet pulp group were higher than those in control group(P<0.05).2)When compared with control group,beet pulp group had lower ATTD of DM,crude ash,CP and energy(P<0.05).However,the ATTD of cude fiber(CF),the activity of jejunal lactase activity and the relative mRNA expression of doudenal GLUT-2 in beet pulp group were higher than those in control group and pairing group(P<0.05).3)When compared with control group,beet pulp group had higher number of goblet cells in colon(P<0.05).The relative mRNA expressions of duodenal Reg??,jejunal MUC2 and ileal GPR43 in beet pulp group were higher than those in control group(P<0.05).Beet pulp group had higher number of Bifidobacterium spp in cecal digesta,higher concentrations of propionic acid in cecal and colonic digesta,and total SCFAs in colonic digesta compared with control group(P<0.05).4)No differences were observed in control group and pairing group for the above measurements(P>0.05).These data indicated that pigs fed with high fiber diet(CF = 5.74%)could modulate gut microbiota composition,increase the contents of SCFAs in hindgut and promote gut health,which is independent of the feed intake.Experiment 5 Effects of distal ileal infusion of SCFAs on gut health in growing pigsThe present study was conducted to investigate the effects of distal ileal infusion of different compositions of SCFAs on gut health in growing pigs.In this study,24 barrows(Duroc × Landrance × Yorkshire)with average initial body weight of(30.72 ± 1.48)kg fitted with a T-cannula in the terminal ileum were randomly allotted to 4 treatments,with 6 replicates per treatment,and 1 pig per replicate.In order to suppress interference brought by intestinal endogenous SCFAs.we attempted to inhibit pigs of their intestinal microbiota by providing high dose of antibiotics.The proportions and concentrations of SCFAs used in this trial were observed from experiment 4.The treatment groups were:1)Control.2)Antibiotics,Ab,3)Antibiotics+SCFAs1,AS1,4)Antibiotics+SCFA2,AS2.The experimental period lasted for 28 days.The results are as follows:1)High dose of antibiotics supplementation with/without SCFAs infusion decreased total viable bacteria in feces for more than10 folds on d 7 and d 14,and less than 10 folds on d 28(P<0.05).Compared with the control group,Ab group had lower contents of acetic acid,propionic acid and total SCFAs in the serum and ileum,and lower content of butyric acid in ileum,along with lower relative mRNA expressions of GPR43 in ileum and GPR41 in colon(P<0.05).While compared with Ab group.AS1 and AS2 groups had higher concentrations of acetic acid,propionic acid,butyric acid and total SCFAs in the serum and ileum,higher concentrations of acetic acid and total SCFAs in cecum,and higher content of butyric acid in colon,along with higher relative mRNA expressions of GPR41 in colon,and GPR43 in both of ileum and colon(P<0.05).Besides,the contents of butyric acid and total SCFAs in the serum and the relative mRNA expressions of colonic GPR43 in AS2 group were higher than those in AS1 group(P<0.05).2)SCFAs infusion enhanced the relative length of small intestine and total intestine,and the relative weight of large intestine(P<0.05),increased the villus height and villus height:crypt depth in duodenum and jejunum(P<0.05).Besides,the proportions of ileal and colonic apoptotic cells and G0G1 phase cells in SCFAs infusion groups were lower,whereas the proportions of S phase cells,G2M phase cells and proliferation index were higher than those in Ab group(P<0.05).Meanwhile,compared with Ab group,SCFAs infusion groups had lower relative mRNA expressions of Bax,Caspase-3 and p21/Cipl,and higher relative mRNA expressions of Bcl-2 and Cyclin D1 in ileum and colon(P<0.05).Moreover,both of SCFAs infusion groups had higher colonic protein concentration,as well as higher relative mRNA expressions of ileal IGF-1R and GLP-2,colonic EGF,GLP-2 and IGF-1 than those in Ab group(P<0.05).Finally,the proportions of ileal G2M phase cells and the relative mRNA expressions of ileal Bcl-2 and IGF-1 in AS2 group were higher than those in AS1 group,whereas the proportions of colonic G0G1 phase cells in AS2 group were lower than that in AS1 group(P<0.05).3)Compared with Ab group,SCFAs infusion decreased the pH values of digesta in jejunum,cecum and colon(P<0.05),increased the ATTD of EE,DM,CP and crude ash,and the relative mRNA expression of SLC7A,in ileum(P<0.05).The pH values of jejunal digesta in AS2 group was lower than that in AS1 group(P<0.05).4)SCFAs infusion enhanced the relative mRNA expressions of ZO-1,Occludin,Claudin-1,MUC-2 and IL-10 and decreased the relative mRNA expressions of IL-8,IL-1? and TNF-? in ileum and colon compared with Ab group,which were accompanied by greater numbers of ileal and cecal Bacillus spp,Lactobacillus spp and Bifidobacterium spp,and less count of Eschericha coli in SCFAs infusion group(P<0.05).Additionally,the count of Eschericha coli in AS2 group was lower than that in AS1 group(P<0.05).This study provides the first systematic evidence in a pig model that distal ileal infusion of SCFAs could stimulate intestinal growth and improve gut barrier function.Experiment 6 Effects of gastric infusion of SCFAs on gut health in weaned pigletsThe present study was conducted to investigate the effects of gastric infusion of different concentrations of SCFAs on gut health in weaned piglets.In this study,21 barrows(Duroc x Landrance x Yorkshire)with average initial body weight of(8.31 ±0.72)kg were randomly allotted to 3 treatments,with 7 replicates per treatment,and 1 pig per replicate.The treatment groups were:1)control,2)SCFAs(low concentration),S1,3)SCFAs(high concentration),S2.The experimental period lasted for 7 days.The results are as follows:1)When compared with control group,gastric infusion of SCFAs increased the concentrations of acetic acid,propionic acid,butyric acid and total SCFAs in ileal,cecal and colonic digesta and butyric acid in serum,which is accompanied by higher relative mRNA expression of GPR43 in ileum and colon(P<0.05).Besides,the contents of acetic acid,propionic acid,butyric acid and total SCFAs in the serum and acetic acid,propionic acid and total SCFAs in the ileal digesta,and the butyric acid and total SCFAs in the colonic digesta in S2 group were higher than those in S1 group(P<0.05).2)When compared with control group,gastric infusion of SCFAs increased the jejunal villus height,proliferation index in jejunum and colon,and DNA and protein concentrations in jejunal mucosa(P<0.05),enhanced the relative mRNA expressions of IGF-1 in duodenum,IGF-1R in jejunum,GLP-2R in ileum and colon(P<0.05),decreased the proportions of colonic apoptotic cells and G0G1 phase cells in jejunum and colon,and the relative mRNA expressions of Bax and Caspase-3 in duodenum(P<0.05).In addition,the proliferation index in jejunum,protein concentrations in ileal mucosa in S2 group were higher than those in S1 group,whereas the proportions of jejunal G0G1 phase cells in S2 group were lower than that in S1 group(P<0.05).3)When compared with control group,gastric infusion of SCFAs decreased the pH values of digesta in duodenum and colon(P<0.05),increased the relative mRNA expression of SLC7A1 in jejunum and DMT1 in ileum(P<0.05).4)When compared with control group,gastric infusion of SCFAs increased the relative mRNA expressions of Occludin and Claudin-1 in duodenum and ileum,Claudin-1 and MUC1 in jejunum(P<0.05),enhanced the count of Lactobacillus spp in ileal digesta(P<0.05),decreased the relative mRNA expressions of IL-10 in jejunum,IL-1? in ileum,IL-1? and IL-8 in colon(P<0.05),reduced the number of Eschericha coli in ileal digesta(P<0.05).These data indicated that gastric infusion of SCFAs increased SCFAs concentrations in serum and intestine,decreased apoptosis of epithelial cells,stimulated intestinal DNA and protein concentrations,improved gut morphology and maintained the intestinal barrier function.In conclusion,there were apparent differences in gut microbiota composition and gut characteristics between Chinese indigenous pig breeds and imported foreign pig breed,and these differences can be transferred from pigs to germ free mice by fecal microbiota transplantation.Early transplantation of the fecal microbiota from the Yorkshire pigs and Rongchang pigs damage the balance of gut microbiota and thus be detrimental to intestinal development in conventional raised suckling piglets.However,piglets received the fecal microbiota from the Tibetan pigs could ameliorate DSS-induced gut injury.The interaction between dietary fiber level and gut microbiota composition can significantly regulate gut health of pigs,and SCFAs profiles especially SCFAs concentration play a mediator role in this interation.Therefore,gut microbiota is closely related to host's intestinal development and function,and SCFAs,the microbial metabolite,play a mediator role in the interactions among nutrition,microbiota and host. |
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