Study Of Bacillus Amyloliquefaciens Repairs The Intestinal Damage Of Intrauterine Growth Retardation Weanling Piglets And Underlying Mechanism

Intrauterine growth retardation(IUGR)is characterized by impaired growth and development of embryo/fetus during pregnancy.Approximately 15%-20%of newborn piglets suffer from IUGR in commercial swine industry,resulting in a huge economic loss to the producer in China.Many reports indicated that IUGR would impair the intestinal morphological structure and physiological function of piglets,directly affecting their feed efficiency(FE)and whole health status.Recent evidence demonstrated that Bacillus amyloliquefaciens(BA)has some beneficial roles in improving animal's FE,maintaining intestinal flora balance,regulating immune function,and decreasing diarrhea rate.Therefore,this study used IUGR piglets as the research subjects.Firstly,the present study was to investigate the effects of IUGR on the intestinal development and tissue damage of piglets at 21 d of age,so as to confirm the importance of nutritional intervention to improve their intestinal function.After that,this study would systematically evaluate the protective effects of BA on the intestinal function of IUGR weanling piglets from the perspective of growth performance,digestive function,mucosal barrier,goblet cell differentiation and the activity of signaling pathways.The aim of trial 1 was to investigate the effect of IUGR on growth performance and digestive function of suckling piglets at 21 d of age.Ten normal birth weight(NBW)neonatal piglets and ten IUGR littermates were selected at birth.The newborn piglets with a birth weight near the mean birth weight[within 0.5 standard deviation(SD)]were defined as NBW,whereas those 2 SD lower birth weight were identified as IUGR.Piglets were distributed into groups of two treatments(NBW and IUGR groups),ten replicate per group.All piglets were allowed to suckle the dam naturally up to 21 d of age.Before the piglets were given solid feed,their plasma and intestinal samples were collected at 21 d of age,to determine the biochemical parameters.The results obtained from the present study indicated that(1)compared with NBW,IUGR significantly decreased the body weight of piglets at 21 d of age and reduced their average daily gain(ADG)from 1 to 21 d of age(P<0.05).(2)IUGR obviously increased plasma diamine oxidase(DAO)activity and decreased jejunal villus height(VH)and crypt depth(CD)and ileal VH and VH:CD ratio(VCR)(P<0.05).(3)Compared with the NBW piglets,the IUGR piglets exhibited lower reduced glutathione(GSH)content and the ratio of GSH to oxidized glutathione(GSSG)whereas higher MDA concentration in both jejunum and ileum,as well as a greater protein carbonyl content in jejunum(P<0.05).(4)At 21 d of age,the IUGR piglets had decreased contents of secretory immunoglobulin A(slgA)and interleukin-10(IL-10)in jejunum and reduced level of interleukin-1?(IL-1?)in ileum,when compared with the NBW piglets(P<0.05).(5)IUGR resulted in a greater number of Escherichia coli but a lower number of Bifidobacterium in the ileal digesta of piglets(P<0.05).In conclusion,IUGR impairs the intestinal integrity and villus morphology,resulting in mucosal oxidative damage,compromised immune function,and altered microbial population,which may be the important mechanism underlying the decreased ADG of IUGR suckling piglets.Trial 2 was conducted to study the effect of BA on growth performance and digestive function of IUGR weanling piglets.Eighteen NBW neonatal piglets and thirty-six IUGR littermates were selected at birth according to the same selection as mentioned above.Piglets were allowed to suckle the dam naturally up to 21 d of age and then allocated to receive the basal diet supplemented with 0(NBW-CON),0(IUGR-CON),and 2.0 g/kg BA ES-2(IUGR-BA)from 21 to 49 d of age.Each group consisted of six replicates with thr2ee piglets per replicate.Fresh fecal sample were collected from 46 to 48 d of age.Plasma and intestinal samples were collected from one piglet per replicate at the end of this trial and analyzed for the biochemical parameters.The results indicated that(1)IUGR substantially decreased the ADG and average daily feed intake from 21 to 49 d of age in the IUGR-CON group than those in the NBW-CON group;dietary supplementation of BA obviously increased the ADG and FE of IUGR weanling piglets during the first 4 weeks postweaning(/P<0.05).(2)Compared with the NBW-CON group,IUGR decreased the apparent digestibility of dry matter and crude protein of the IUGR-CON group;BA supplementation effectively elevate the apparent digestibility of-dry matter of IUGR weanling piglets(P<0.05).(3)The activities of jejunal amylase and trypsin were significantly decreased in the IUGR-CON group than those in the NBW-CON group;after BA feeding,the activities of jejunal and ileal amylase and ileal trypsin were dramatically increased in the IUGR-BA group.than those in the IUGR-CON group(P<0.05).(4)Compared with the NBW-CON group,the IUGR-CON group had decreases in the jejunal and ileal VH and VCR;dietary supplementation of BA strongly increased the jejunal and ileal VCR and ileal VH of IUGR piglets(P<0.05).In conclusion,dietary supplementation of BA improves the intestinal morphology and amylase activity of IUGR piglets,as well as increases their apparent digestibility of dry matter and growth performance during the early period after weaning.Trial 3 was designed to investigate the effects of BA on intestinal mucosal barrier function of IUGR weanling piglets.The present study shared the same details of the experiment design as that of Trial 2.The present observations showed that(1)IUGR-CON piglets showed higher levels of plasma DAO,immunoglobulin M,and endotoxin than those in the NBW-CON piglets;however,these increases were obviously prevented by BA supplementation(P<0.05).(2)Compared with NBW,IUGR dramatically increased the activity of jejunal glutathione peroxidase and the contents of ileal GSSG and MDA,whereas decreased the jejunal and ileal GSH/GSSG ratio;dietary supplementation of BA significantly increased the jejunal and ileal GSH contents and GSH/GSSG ratio of the IUGR-BA group,whereas decreased their jejunal and ileal GSSG and MDA concentrations(P<0.05).(3)Compared with the NBW-CON group,the IUGR-CON group exhibited higher monocyte chemoattractant protein contents and MPO activities in both jejunum and ileum,greater concentrations of jejunal tumor necrosis factor alpha(TNF-a)and IL-1?,and a lower level of ileal IL-10;compared with the IUGR-CON group,the IUGR-BA group had increased IL-10 but decreased TNF-? contents in both jejunum and ileum,as well as reduced ileal MPO activity(P<0.05).(4)The expression level of occluding(OCLN)was down-regulated in the ileum of IUGR-CON piglets in comparison with the NBW-CON piglets;feeding the IUGR weanling piglets with BA significantly elevated their jejunal and ileal OCLN expression at transcription level(P<0.05).(5)Compared with the NBW-CON piglets,IUGR-CON piglets showed increased Escherichia coli and decreased Bifidobacterium abundance in the ileal digesta.The decreased numbers of jejunal Escherichia coli and increased numbers of ileal Lactobacillu and Bifidobacterium were observed in the IUGR-BA piglets than those of the IUGR-CON piglets(P<0.05).(6)The relative abundance of Other phylum was significantly increased in the cecal contents of IUGR-BA group than that in the IUGR-CON group.Among the dominant bacteria whose relative expression levels were more than 1%,the abundance of Faecalibacterium was remarkedly decreased in the cecal digesta of IUGR-CON piglets than that of the NBW-CON piglets.Dietary supplementation with BA obviously increased the relative abundance of cecal Lactobaeillu,Faecalibacterium,CF231,and Parabacteroides of IUGR weanling piglets.Also,the IUGR piglets fed a BA diet exhibited less Escherichia and more Bacillus in cecal contents when compared with those fed a basal diet(P<0.05).In conclusion,dietary supplementation with BA effectively alleviates the increases in intestinal permeability,mucosal oxidative damage,down-regulation of the key protein of tight junction of IUGR piglets.Also,BA improves the bacterial community structural of IUGR piglets.These observations may provide potential mechanisms for the improvement of intestinal damage of IUGR weanling piglets after BA supplementation.Trial 4 was aimed to study the effects of BA on intestinal cell proliferation and apoptosis and goblet cell differentiation of IUGR weanling piglets.The present study shared the same details of the experiment design as that of Trial 2.The results demonstrated that(1)IUGR decreased the jejunal and ileal trefoil factor 3(TFF3)contents,ileal goblet cell density and mucosal mucin 2(MUC2)level;supplementation with BA obviously alleviated the decreases in the ileal goblet cell density,mucosal MUC2 and TFF3 contents of IUGR weanling piglets(P<0.05).(2)Compared with the NBW-CON group,the IUGR-CON group had the increases in ileal crypt cell proliferation rate,jejunal and ileal villus cell apoptotic rates;BA supplementation prevented the above increases effectively(P<0.05).(3)IUGR-CON piglets showed decreased mRNA abundance of B-cell lymphoma 2 in both jejunum and ileum than those in the NBW-CON piglets(P<0.05).(4)Compared with NBW,IUGR decreased the protein expression of activated Notchl in the cell nucleus of ileal mucosa of weanling piglets;feeding the IUGR weanling piglets with a BA diet exhibited a lower activated Notch1 protein content in the cell nucleus of ileal mucosa,when compared with their counterparts fed a basal diet(P<0.05).(5)The transcription expression of growth factor independence lb(Giflb)in the jejunal mucosa and Mathl in the ileal mucosa was significantly decreased of IUGR-CON piglets,meanwhile,the transcription expression of Hesl was significantly increased when compared with the NBW-CON piglets;BA obviously increased the transcriptional activities of ileal Giflb,SAM pointed domain-containing Ets transcription factor and T cell transcription factor 4 in the IUGR-BA group than those in the IUGR-CON group(P<0.05).In conclusion,dietary supplementation with BA decreases the ileal crypt cell proliferation and villus cell apoptotic rates,improves the activities of Notch pathways,regulates the transcription expression of genes related to goblet cell differentiation,and then increases the ileal goblet cell numbers and MUC2 and TFF3 contents in the IUGR weanling piglets.In conclusion,IUGR not only decreases the ADG of piglets during the suckling period,but also impairs their intestinal mucosal morphology,function and intestinal microflora,resulting in a compromised adaptive capacity to weaning stress.Supplementation with BA effectively improves intestinal morphology,amylase activity and the apparent digestibility of dry matter of IUGR weanling piglets.Also,BA has some beneficial roles in improving the intestinal mucosal barrier function,microflora,cell proliferation and apoptotic balance,and the differentiation and secretion capacity of goblet cells,which may be the important factors contributing to the better growth performance of IUGR weanling piglets after BA feeding.