| Pigs were superorganisms made up of hosts and microbes.Gut microbes more than one thousand species,for a total of 10~144 cells,was host's 5 to 10 times of own cell numbers.Therefore,it couldn't be studied without intestinal microbiota,especially in the early life of pigs.Microbial colonization was closely related to intestinal development,which would determin the later growth performance and health of piglets.Studies on germ-free mice had found that microbiota colonization at different time stages would have various effects on host.On this basis,this study was focused on two issues,experiment one was to study the effect of microbial intervention at different time stages on the development of piglets.Experiment two was to verif the effect of microbial intervention on intestinal development of piglets by germ free pig model.Experiment one focused on the influence of early fecal microbiota transplation(eFMT)on growth of newborn common piglets at different time periods.Adult sow microbiota was performed at 3 days age(group A)and 7 days age(group B)of piglets,and control group(group C)without treatment.Influence of eFMT on intestinal morphology development,intestinal microbiota communities and their metabolic products were researched.Growth performance results showed that transplation at 3 days age and 7 days age could promote the growth.After birth,the birth weight of group A and group B were significant lower than group C,and group A were significant lower than group B;Slaughtering at 28 days age,the average weight of group A and group B were significant higher than group C,and group B were significant higher than group A;during the experiment,compared with group C,the average daily gain of group A and group B were significantly increased,and group B were significant higher than group A.In addition,results showed that FMT could reduce the diarrhea incidence of piglets.The diarrhea rate of group A and group B were significant lower than group C,and group A were significant lower than group B.Analysis of duodenal and jejunal morphology by HE staining suggested that the influence of FMT to the duodenal villi development was not significant,but that of jejunum villi development was significant.Compared with group C,villi height of group A and group B were increased,and group A significantly higher than group B;the crypt depth of group A(P<0.01)and group B(P<0.05)were significantly decreased;and compared with group C,the ratio of villi to crypt(V/C)in group A was significantly increased significantly(P<0.01),and that of group B was decreased significantly.16S rRNA gene sequencing was used to determine the microflora difference of each group before and after colonization.Results showed that FMT was beneficial to increase the bacterial diversity,induced the probiotics colonization and reduce the proportion of opportunistic pathogens.Compared to the pre-transplantation,the proportion of Firmicutes of group A was increased significantly(P<0.01),and that of Proteobacteria was reduced significantly(P<0.05);that of group A and group B no significant change.At the genus level,the relative abundance of Lactobacillus and Ruminococcaceae_UCG-002 was significantly increased in group A(P<0.05),and Dialister,Peptostreptococcus and Acidaminococcus were significantly increased(P<0.01),while Escherichia-Shigella significantly decreased(P<0.01).GC-MS machine was used to analyze the content of short chain fatty acids(SCFAs)in pigs colon,testing data showed that FMT contributed to the formation of SCFAs.Compared to group C,FMT at 3 days age and 7 days age could incduce the SCFAs;the content of propionic acid and isobutyric acid in group A and group B were increased significantly;the isobutyric acid content of group B was significantly decreased,others had no significant change.The above results indicate that eFMT was conducive to improve animal production performance,disease prevention and promote the intestinal morphology development.Moreover,FMT at 3 days age of pig increased the proportion of beneficial microorganism such as Firmicutes and Bacteroidetes phylum in the colon contents,reduce the counts of opportunistic pathogens and promoted the generation of SCFAs.Comprehensive analysis showed that FMT at 3 days age had more significant effect on intestinal development of piglets.The next experiment in order to verify the effect of FMT on intestinal development by the germ free pigs,we used the germ-free pig model as control group and experiment group transplated the above microbita,studied the effect of FMT on intestinal physical barrier,microbial barrier,chemical barrier and immune barrier of piglets.16S rRNA sequencing was used to determine the reproduce of transplated microbiota on germ free models,and data showed that it reconstructed well.At the phylum,the intestinal microflora structure and proportion of grem-free piglets were similar to the composition of fecal bacterial biopreparate(FBB)and Donor sows(D_SOW),and the abundance of Firmicutes and Bacteroidetes here was no significant difference between the FMT group and control.Furthermore,there was also no significant difference of the top two bacteria between groups at the genus level.GC-MS method was used to analyze the content of short chain fatty acids(SCFAs)in pigs colon,testing data showed that the transplated community were ontributed to the metabolic product SCFAs production.The acetic acid,butyric acid,isobutyric acid,isopentric acid and total SCFAs acid content of group FMT were higher than group GF.HE staining was used to measure the changes of jejunal intestinal morphology of piglets.Measurement results indicated that FMT contributed to the jejunal villi development.Compared to GF group,the jejunal villi height were longer(P<0.05),the crypt depth was lower(P<0.01)and the ratio of V/C was greater(P<0.05).In addition,goblet cells number of jejunum epithelium cells of GF group was significantly higher than that of FMT group(P<0.01).To further explored whether the intestinal barrier function changes by Western blot,results showed that FMT promoted the expression of intestinal tight junction proteins.Compared to GF group,the expression of ZO-1 and Occludin protein was increased in FMT group.Quantitative the related intestinal homeostasis genes expression by reverse transcription quantitative real-time PCR,we found that the receptor genes expression of TLR2 and TLR4 in the jejunal tissues were increased after FMT(P>0.05).Gene expression of mucosal protection factor TFF3 in group F almost unanimous in that of group G(P>0.05),intestinal antimicrobial peptide Reg3 gamma gene expression was 153 times than that of group G(P<0.05),and the expression of inflammatory genes IL-22 increased about 15 times in group F(P>0.05).The results showed that the transplanted bacteria could reproduce well in the germ-free pigs and formated the intestinal microbial barrier.FMT increased the expression of tight junction proteins ZO-1 and Occludin of intestinal epithelial cells,and altered the intestinal villi morphology and then promoted the intestinal physical barriers.Moreover,FMT could increase the SCFAs content in the colon and promote the formation of intestinal chemical barrier and increased the expression of disease-resistant cytokines and constructed intestinal immune barrier.In conclusion,eFMT improved the growth and development of piglets,and bacteria intervention at 3 days age better than 7 days age.Study on germ free pig model,proved that FMT could promote the establishment of intestinal microbial barrier,physical barrier,chemical barrier and immune barrier function. |
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