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Background:The establishment of stable microbiota in early life is beneficial to the individual.Changes in the intestinal environment during early life play a crucial role in modulating the gut microbiota.Therefore,early intervention to change the intestinal environment can be regarded as a new regulation strategy for the growth and health of poultry.However,the effects of intestinal environmental changes on host physiology and metabolism are rarely reported.This study was conducted to investigate the effects of early inoculation with caecal fermentation broth on small intestine morphology,gene expression of tight junction proteins in the ileum,and cecum microbial metabolism of broilers.Results:Our data showed that early inoculation with caecal fermentation broth could improve intestine morphology.The small intestine villus height was significantly increased (P ＜ 0.05) in the intervened broilers compared to the control group,especially on day 28.A similar result was observed in the ratio of villus height to crypt depth (P ＜ 0.05).Meanwhile,we found early inoculation significantly increased (P ＜ 0.05) the expression levels of zonula occludens-1 (ZO1) on days 14 and 28,claudin-1 (CLDN1) on day 28,whereas the gene expression of claudin-2 (CLDN2) was significantly decreased (P ＜ 0.05) on days 14 and 28.Gas chromatography time-of-flight/mass spectrometry (GC-TOF/MS) technology was further implemented to systematically evaluate the microbial metabolite profiles.Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) displayed a distinct trend towards separation between the fermentation broth group (F group) and the control group (C group).The differentially expressed metabolites were identified,and they were mainly functionally enriched in beta-alanine metabolism and biosynthesis of unsaturated fatty acids.In addition,1,3-diaminopropane was selected as a key biomarker that responded to early inoculation with caecal fermentation broth.Conclusions:These results provide insight into intestinal metabolomics and confirm that early inoculation with caecal fermentation broth can be used as a potential strategy to improve intestinal health of broilers.