Effect And Mechanism Of Intervention During Window Period On Gut Microbiota And Intestinal Innate Immune Development In Piglets

Diarrhea is one of most important factors restricting the benefits of pig production,in the past few decades,antibiotic growth promoters(AGPs)were widely used to treat and prevent diarrhea.As the AGPs are gradually being banned as feed additives for animals,how to effectively prevent diarrhea and alleviate weaning stress under AGPs free condition has become an important problem,which should be solved urgently to ensure the efficient and healthy development of pig production.A mature immune system is an important factor for the host to resist disease and stress.However,the immune system of newborn piglet is immature and has poor resistance to pathogenic infections.The rapid development of the intestinal immune system after piglet birth is closely related to the succession of gut microbiota.There has been increasing evidence suggesting that early intervention of the gut microbiota during the critical “window of opportunity” period may be a promising method to improve intestinal microbial colonization,which can promote the development of immune system.Our previous research found that transplanting fecal microbiota(FMT)from pregnant sows into newborn piglets can regulate the gut microbiota and enhance the intestinal barrier function of piglets,thereby reducing diarrhea and promoting piglet growth performance,but the mechanism has not been elucidated.In addition,previous studies indicated that colonization history,donor-recipient compatibility,and gut oxygen play important roles in microbial colonization and community of neonatal,hence we surmised that there is the possibility of further optimization to improve the method of regulating gut microbiota of newborn piglets using fecal bacteria transplantation,and we also suppose that the probiotics with specific functions may achieve optimization the effect.Therefore,in this study,a meta-analysis was used to analyze the influencing factors of restricting the effect of probiotics in piglet production.Then,the early gut microbiota intervention of FMT combined with probiotics was used to promote growth performance of piglets.On this basis,the characteristics of early intervention affecting the composition and succession of gut microbiota were explored,and the mechanism of early intervention to promote immune system development,reduce piglet diarrhea,and alleviate weaning stress injury were revealed.The main contents and results are as follows:Part ? A Meta-analysis for the efficacy of probiotics in prevention of diarrhea in piglets.We collected the literatures of SCI published from January 1960 to February 2020 on probiotics affecting piglet diarrhea.A Meta-analysis of the available data from 49 randomized,placebo-controlled trials to evaluate the evidence for the efficacy of probiotics in preventing diarrhea,and evaluated the efficacy of probiotics by strain,age groups,and assigned quality score of literatures.Our results indicated that the combination of Lactobacillus and other probiotics have the best effect in preventing piglets' diarrhea significantly reduced diarrhea by 5.73%(95% CI 3.26 – 8.19,P < 0.05).Dietary supplementation with Clostridium butyricum can effectively reduce the diarrhea index of piglets by 0.08(95% CI 0.05,0.11,P < 0.05).The combination of multiple strains of probiotics significantly reduced the diarrhea rate of piglets by 4.25%(95%CI 1.97 – 6.53,P < 0.00001),and single strain of probiotics can reduce the diarrhea rate of piglets by 4.14%(95% CI 3.64 – 4.64;P <0.00001).Probiotics had better preventive effects on diarrhea in weaned piglets(4.58%(95% CI 4.05 – 5.1;P <0.05))than suckling piglets(3.21%(95% CI 1.38 – 5.04;P <0.05)).The Cochrane system was used to evaluate the literature,and the effectiveness of probiotics in preventing piglet diarrhea(4.42%(95% CI 3.95 – 4.88;P <0.05))reported in the literature with a score of no more than 4 points was better than the score of 5 Literature(1.89%(95% CI 074 – 3.05;P <0.05)).In conclusion,the available data provides sufficient evidence for the role of probiotics in the prevention of diarrhea.Probiotics can effectively prevent the occurrence of diarrhea in piglets.Lactobacillus probiotics have the best prevention effect on piglet diarrhea.The effect of using a combination of multiple strains of probiotics is better than that of individual strains.In addition,the effects of probiotics in different growth stages of piglets are different.Part ? Establishment of the model of FMT combined with probiotics to promote growth performance of piglets.A total of 6 sows(LY)with 70 days of gestation and no antibiotic treatment for at least 3 months were selected as the fecal donors for this experiment,and the concentration of fecal bacteria is about 1.0 × 109 CFU / m L.A total of 121 litters of piglets(DLY)with an average birth weight of 1.55 ± 0.35 kg were randomly divided into 6 groups.Piglets in antibiotic treated group(AB)were given a single subcutaneous injection of 0.3 m L of cephalosporin solution at age of 2-3 days.Piglets in groups of FMT treated group(FMT, maternal fecal suspension),FMT combined with C.butyricum group(FMT-C,maternal fecal suspension added 1.0 × 109 CFU/m L C.butyricum),FMT combined with S.boulardii group(FMT-S,maternal fecal suspension added 1.0 × 109 CFU/m L S.boulardii),and FMT combined with C.butyricum and S.boulardii group(FMT-CS,maternal fecal suspension added 1.0 × 109 CFU/m L C.butyricum and 1.0 × 109 CFU/m L S.boulardii)were given a single oral dose of 2ml of bacterial solution every day during age of 1 to 3 days.Piglets in control group(CON)were treated with PBS at the same time.All the piglets were weaned at the age of 27 d.500 weaned piglets were randomly raised in eight similar pens,with four pens for each group.The experiment was ended at the age of 56 d.During the experiment period,the piglets were allowed free access to water and antibiotics-free diets.Piglets were individually weighted weekly to determine average daily gain(ADG).The diarrhea rate was recorded every day during experimental period.Blood samples and fresh fecal samples were collected at 3d,7d,14 d,21d,35 d,and 56 d for biochemical indicators detection.The main results are as follows:1.Compared with CON group,the weaning BW and finial BW of FMT piglets were significantly increased(P < 0.05),FMT tended to show higher ADG than CON(0.05 < P < 0.1),and FMT significantly reduced the diarrhea during suckling period.However,it has no significant effect on the growth performance of piglets during the post-weaning period.When compared with CON group,FMT-CS significantly increased the BW(P < 0.05)and finial BW(P < 0.05),and significantly reduced the diarrhea rate both in suckling(P < 0.05)and post-weaning period(P < 0.05).Moreover,FMT-CS tended to show higher weaning body weight and ADG during suckling period than FMT,and show a significantly increase of ADG and a decrease of diarrhea during post-weaning period than FMT.2.ELISA was used to determine the fecal s Ig A and MUC2,and plasma DAO and D-LA level.Compared with CON group,FMT-CS significantly increased the fecal MUC2(P < 0.05),and reduced the plasma DAO and D-LA and age of 14 d,21 d,35 d,and 56 d(P < 0.05),which indicated FMT-CS can enhance the intestinal barrier function of piglets.3.Using gas chromatography(GC)to determine concentration of fecal SCFAs,and using ELISA to determine concentration of fecal succinate.Compared with CON group,FMT-CS significantly increased the concentration of SCFAs at age of 14 d,and FMT-CS increased the concentration of succinate,which suggested FMT-CS can increased the production of beneficial metabolites in the intestine of piglets.4.Quantitative PCR(q PCR)was perform for determining the relative abundance of selected flora in fecal.Compared with CON group,FMT-CS decreased the relative abundances of Escherichia coli,and increased the relative abundances of Bifidobacterium genus and Lactobacillus spp,which may indicate FMT-CS could increase the relative abundance of beneficial bacteria and reduce the relative abundance of harmful bacteria.Part ? Early-life intervention with FMT-cs promotes gut microbiota maturation,regulates immune system development,and alleviates weaning stress in piglets.16S r RNA gene sequencing was used to analysis the composition of fecal microbiota at age of 7 d,27 d,35 d,and 56 d,and ELISA kits were used to determine the level of IL-17,IL-22,IL-23,IFN-?,Ig G,Ig M,Glutathione peroxidase(GSH-px),catalase(CAT),total antioxidant capacity(T-AOC),malondialdehyde(MDA),total superoxide dismutase(T-SOD),DAO,and D-LA level.The main results are as follows:1.16 S r RNA gene sequencing showed the FMT-CS increased the ?-diversity and reduced the unweighted Uni Frac distances of the OTU community,and enhanced the relative abundance of genes related to cofactors and vitamin,energy,and amino acid metabolisms during the early-life period,these results indicated that FMT-CS can accelerate gut microbiota maturation in newborn piglets.2.Bugbase analysis predicted that FMT-CS reduced the relative abundance of genes of oxygen utilizing(7d)and potential pathogenic microorganisms(35d)(P <0.05),and increased relative abundance of genes of stress tolerant(27d)and mobile element containing microorganisms(P <0.05).Lefse and Metastats analysis found that Firmicutes and Bacteroidetes were the most dominant phyla in both control and treated piglets.In treated piglets,the third most dominant phylum was Spirochaetes before weaning,and was Actinobacteria after weaning.Notably,a rapid increase in the relative abundance of Actinobacteria might be a signal of weaning stress.In control piglets,Fusobacteria and Proteobacteria were the third and fourth most dominant phyla before weaning(7 d and 27 d);while after weaning(35 d and 56 d),the relative abundance of Fusobacteria drastically decreased,and Actinobacteria became the third most dominant phylum.At the genus level,Prevotella and Lactobacillus were dominant in both control and treated piglets.In control piglets,the other two major genera were Fusobacterium and Bacteroides at the age of 7 d and 27 d, Collinsella and Catenibacterium at the age of 35 d,and Roseburia and Faecalibacterium at the age of 56 d;while in treated piglets,the other two major genera were Treponema and p-75-a5,Treponema and Phascolarctobacterium,Collinsella and Bulleidia,and Faecalibacterium and Roseburia at the age of 7 d,27 d,35 d,and 56 d,respectively.These results indicated that FMT-CS can increase the relative abundance of beneficial bacteria,while decreasing that of opportunistic pathogens.3.FMT-CS treated significantly increased the plasma concentrations of IL-23(P <0.05),IL-17(P <0.05),and IL-22(P <0.05)at age of 7d,and there is increasing tendency for plasma concentrations of IL-17(P <0.1)and IL-22(P <0.1)at age of 14 d and 21 d.The plasma concentrations of IL-17(P < 0.05),IL-22(P < 0.05),IFN-?(P < 0.05)at age of 27 d were significantly increased in FMT-CS piglets.The plasma Ig G,Ig M,and fecal s Ig A level were significantly increased during the whole experimental period.Moreover,the plasma levels of anti-M.hyo(P < 0.05)and anti-PCV2 antibodies(P < 0.05).These results indicated that FMT-CS can promote the development of immune systems in piglets.4.FMT-CS-treated piglets at age of 35 d showed decreases of TNF-?,IL-6,and cortisol levels.The level of plasma CAT(P <0.05),GSH-px(P = 0.0514),T-AOC(P <0.05),and T-SOD(P <0.05)were significantly increased,while the MDA(P <0.05)was decreased in FMT-CS treated piglets.FMT-CS also reduced the level of plasma DAO and D-LA.These results indicated FMT-CS can alleviate the damage of weaning stress.In summary,the main conclusions of this study are as follows:(1)Lactobacillus probiotics and the combination of Lactobacillus and other probiotics can obtain the best results in preventing piglet diarrhea.(2)In the window period of gut microbiota development,the early intervention of FMT combined with Clostridium butyricum and Saccharomyces boulardii can promote the colonization and maturation of gut microbiota and the development and function of immune system in piglets,as well as reduce diarrhea,promote the growth performance and alleviates weaning stress in piglets.