Effects Of Galacto-oligosaccharide Intervention On Intestinal Microbial Colonization And Colonic Function In Piglets

The microbiota,colonized in gastrointestinal(GI)tracts,affect the growth performance of pigs by influencing intestinal epithelial differentiation,affecting the development of the immune system and even modulating the appetite.During the early life,once piglets were born by sow,the "cross talk" between the gut microbiota and host has begun.The gastrointestinal microbial ecology of suckling piglets is unstable and frequently susceptible to the surrounding environment.Therefore,it is advantageous modulate and change the developmental program of the bacterial community and improve host health in the early life of piglets.It has been reported that several trials including early-life prebiotics supplement,fecal microbiota transplantation,early exposure to agricultural soil or early food introduction can alter the intestinal microbiota composition and modulate the gut function on the piglets.he barrier function and immune activity to maintain the normal colonic function and health of suckling piglets.Here,we hypothesized that the early-life GOS modulation alters the colonic microbiota composition,and these changes can influence the barrier function and immune response of piglets.The study is divided into the following five parts:1.Effects of early life galactooligosaccharides intervention on colonic microbial composition and metabolism of suckling pigletsIt is advantageous to modulate a developmental program of the bacterial community and improve gut health at early life of piglets.In the present study,newborn piglets with sow-rearing from six litters(10 piglets per litter)were used to investigated the response of colonic morphology,colonic microbiota composition and metabolism to early-life galactooligosaccharides(GOS)intervention.The piglets were assigned to the control group(CON)and the GOS group in each litter.From the age of 1 to 7 days,GOS piglets were intervened with 10 mL GOS solution(1g/kg body weight per day),and CON piglets were treated with the same dose of physiological saline.Six piglets from each group were euthanized,and colonic digesta and mucosa samples were collected for microbial composition 16S rRNA analysis on the day 8 and day 21.The results showed that early life GOS intervention decreased the diarrhea rate of suckling piglets(P=0.051).The colonic morphology study showed that compared with the control group,the colon length of the piglets in the treated group increased on day 8(P=0.056).High-throughput sequencing of 16S rRNA revealed that early life GOS intervention increased the diversity of colonic chyme and mucosal flora and the abundance of SCFAs,such as Prevotella,Unclassified Porphyromonadaceae,Unclassified Ruminococcaceae,Unclassified Bacteroidales,and so on.In addition,early life GOS intervention can also increase the concentration of total SCFAs in the colonic chyme of the treated group.The results indicated that early life GOS intervention changed colon morphology of suckling piglets,and changed the production of SCFAs in the colon by regulating the composition of the flora in the colonic chyme of the piglets.2.Effect of early life galacto-oligosaccharides intervention on colonic mucosal immune homeostasis and barrier function in suckling pigletsThis chapter aims to investigate the effects of early life galactooligosaccharides(GOS)intervention on colonic mucosal immune homeostasis and barrier function in suckling piglets.The tests in this chapter are divided into two parts,in vivo and in vitro.The in vivo test design is the same as the second chapter.After the test,piglet plasma samples and colonic mucosal tissue samples were collected for analysis of gene level and protein level.The results showed that early life GOS intervention significantly affected the concentration of sIgA in the colonic mucosa,plasma IgG on day 21 and sIgA in the colonic mucosa on day 8 of the treatment group(P<0.05).Detection of the gene expression level of Toll-like receptors revealed that early life GOS intervention significantly affected the relative expression of TLR2 and TLR5 genes in colonic mucosa on day 21(P<0.05).This experiment also examined the phosphorylation level of key proteins in the NFκB signaling pathway in the colonic mucosa.It was found that early life GOS intervention reduced the phosphorylation of IκB protein in the colonic mucosa of the treated piglets on day 8(P=0.079),but significantly reduced the phosphorylation level of IκB protein in the colonic mucosa of the treated piglets on the 21st day(P<0.05).With the down-regulation of IκB phosphorylation,the concentration of anti-inflammatory factor IL-10 in colonic mucosa of the treated group was significantly up-regulated on day 8 and day 21(P<0.05),while the concentration of pro-inflammatory factor IL-8 was down-regulated.In the study of colonic mucosal barrier function,early life GOS intervention significantly up-regulated the relative protein expression of colonic mucosal barrier proteins Claudin-1 and ZO-1 in the treatment group on day 8 and day 21,and in the colonic mucosa.Mucin and barrier protein gene expression also had a significant effect(P<0.05).In addition,on days 8 and 21,this study found that phosphorylation levels of key proteins(AMPK and ACC)of the AMPK signaling pathway,which are closely related to mucosal epithelial barrier function,are also up-regulated.In order to initially identify the activation mechanism of the AMPK pathway,this chapter conducted in vitro experiments and found that 2 mM sodium butyrate can up-regulate the phosphorylation level of AMPK in IPEC-J2 cells and promote the expression of the barrier protein ZO-1,and this promotion The AMPK upstream protein CaMMKⅡ is closely related.3.Effects of early life galacto-oligosaccharides intervention on liver metabolism and mitochondrial antioxidant function in suckling pigletsThis chapter aims to investigate the effects of early life galactooligosaccharides(GOS)intervention on liver metabolism and mitochondrial antioxidant function in suckling piglets.The experimental design of this chapter is the same as the second chapter.Before the slaughter,the piglet plasma samples were taken from the anterior vena cava for biochemical indicators.After slaughter,the liver tissue samples were collected for metabolomics(GC-MS),lipid metabolism related genes and mitochondrial antioxidants.The results showed that the early life GOS intervention significantly down-regulated the plasma total cholesterol concentration in the piglets on the 8th day(P<0.05),and also significantly increased the plasma high-density lipoprotein concentration on the 21st day of the treated piglets(P<0.05).Hepatic metabolomics analysis found that early life GOS intervention changed the metabolite composition of the liver of the two groups on day 8 and day 21.On day 8,PCA and PLS-DA analysis indicated that the control and treatment groups were in different clusters.Compared with the control group,the contents of 11 metabolites(glutamine,histidine and lysine)in the liver of the treated group were significantly increased(P<0.05),while 47 metabolites(stearic acid,oleic acid and cholesterol,etc.)were significantly down-regulated in the GOS group(P<0.05).The enrichment analysis of differential metabolite pathways revealed that eight metabolic pathways(lysine degradation,linoleic acid metabolism,and arachidonic acid metabolism)were significantly enriched.On day 21,the PLS-DA analysis also indicated that the control and treatment were in different clusters.Compared with the control group,the contents of five metabolites(taurine,yeast sterol,vitamin E,diacetic acid and glycolic acid)in the liver of the treated group were significantly increased(P<0.05),while 21 metabolites(lysine,glutamine and histidine,etc.)were significantly down-regulated(P<0.05),and differential pathways were found to identify 7 pathways(taurine and hypotaurine metabolism,lysine degradation,and ammonia,Acyl t-RNA synthesis,etc.)is clearly enriched.This chapter also tested genes related to liver lipid metabolism.The results showed that early life GOS intervention significantly down-regulated the relative gene expression of carnitine palmitoyl transferase(CPT-1)in the liver of the treated pigs on days 8 and 21(P<0.05).In addition,early life GOS intervention significantly increased the levels of antioxidant enzymes in the liver mitochondria of the treated piglets and decreased the content of lipid peroxidation product malondialdehyde(MDA)(P<0.05).4.Effect of different galactooligosaccharides intervention stages on colonic microbial composition and its metabolites in weaned pigletsThis chapter aims to investigate the effects of galactooligosaccharides(GOS)intervention on colonic microbial composition and metabolites in weaned piglets.12 litters of Duroc×Landrace ×Large White newborn piglets were selected and and divided into C/C group,G/C group,C/G group and G/G group,30 piglets per group.From dayl to day 7,piglets in G/C and G/G groups were intervented with galactooligosaccharide solution twice per day,and piglets in group C/C and C/G were given the same dose of sterile saline.The piglets were weaned at 21 days of age,the C/C and G/C piglets were fed a basal diet,and the C/G and G/G groups were fed a basal diet+2%GOS.During the weaning period,the health status and diarrhea of all pigs were monitored daily.Six piglets were slaughtered for sampling.After the piglets were slaughtered,the middle colonic digesta and mucosal samples were collected for analysis.The results showed that the GOS supplementation in weaning significantly affected the α-diversity of colonic mucosal microbiota(P<0.05).Microbial composition analysis showed that early GOS intervention significantly not only affected the relative abundance of Faecalibacterium in digesta and mucosa but also significantly affected the relative abundance of Unclassified Ruminococcaceae in digesta(P<0.05).GOS supplementation in weaning had significantly effect on the abundance of Clostridium in the mucosa(P<0.05).In addition,early GOS intervention and GOS supplementation in weaning significantly effect on the abundance of Phascolarctobacterium in the mucosa(P<0.05).With the interaction,the relative abundance of Actinobacillus in the mucosa of C/G,and the relative abundance of Anaerrotruncus in the mucosa of C/G group was significantly higher than that in C/C group(P<0.05).The relative abundance of Anaerrotruncus in the mucosa of C/G group was significantly higher than that of C/G group.The abundance of Unclassified Lachnospiraceae in piglets of G/C group was significantly higher than that in G/G group,and the relative abundances of Dorea and Phascolarctobacterium in digsta and the abundance of Prevotella in the mucosa of G/G group were all than those in the other three groups(P<0.05).The results of SCFAs concentration showed that early GOS intervention had a significant effect on the concentration of isovalerate in the weaning piglets(P<0.05),and GOS supplementation in weaning had a significant effect acetate concentrations(P<0.05).Early GOS intervention or GOS supplementation in weaning had a significant effect on the total SCFAs concentrationin the colonic digesta,respectively(P<0.05).With the interaction,the concentrations of butyrate and valerate in the G/G group were significantly higher than those in the other three groups.5.Effect of different galactooligosaccharide intervention stages on performance and colonic function in weaning pigletsThis chapter aims to investigate the effects of GOS intervention on growth performance and colonic function in weaning piglets.The experimental design was the same as that in Chapter 5.The data of feed intake and diarrhea rate were recorded during weaning.Before slaughter,the plasma samples of piglets were collected for biochemical indicators.After slaughter,the samples of colonic mucosa were collected for analysis.It was found that the early GOS intervention and GOS supplementation in weaning significantly affected the diarrhea frequency of piglets,and the GOS supplementation in weaning significantly affected the body weight and F/G riato of weaned piglet(P<0.05).Colonic index showed that GOS supplementation in weaning had a significant effect on ratio of colon weight/colon length and colon weight/body weight(P<0.05).Colonic function results showed that early GOS intervention significantly affected the mRNA expression of Claudin-2,MUC4,TLR5 and NOD-1 in colonic mucosa of weaning piglets(P<0.05),and GOS supplementation in weaning had significant effect on the mRNA expression of ZO-1,ZO-2,MUC-1 and TLR4 in the colonic mucosa(P<0.05).Early GOS intervention or GOS supplementation in weaning both had a significant effect on gene expression of Occludin and TLR2 in the colonic mucosa(P<0.05).With the interaction,the mRNA expression of NFκB,MyD88 and the protein content of IL1β and TNFα in colonic mucosa of G/G group were significantly lower than those of the other three groups(P<0.05),and the concentration of IL-8 in C/G group,G/C group and G/G group was significantly lower than that of C/C group,while the concentration of IL-10 in C/G group,G/C group and G/G group was significantly higher than that of C/C group(P<0.05).