Background: Many recent studies have gravitated towards manipulating the gastrointestinal (GI) microbiome of livestock to improve host nutrition and health using dietary interventions. Few studies, however, have evaluated if inoculation with rumen fluid could effectively reprogram the development of GI microbiota. We hypothesized that inoculation with rumen fluid at an early age could modulate the development of GI microbiota because of its low colonization resistance.Results: In this study, we tested the above hypothesis using young lambs as a model. Young lambs were orally inoculated repeatedly (four times before or twice during gradual weaning) with the rumen fluid collected from adult sheep. The oral inoculation did not significantly affect starter intake, growth performance, or ruminal fermentation. Based on sequencing analysis of 16S rRNA gene amplicons, however, the inoculation (both before and during weaning) affected the assemblage of the rumen microbiota, increasing or enabling some bacterial taxa to colonize the rumen. These included operational taxonomic units (OTUs) belonging toMoryella,Acetitomaculum,Tyzzerella4,Succiniclasticum,Prevotella1,Lachnospiraceae,Christensenellaceae R-7group,Family XIIIAD3011, andBacteroidales S24–7corresponding to inoculation before weaning; and OTUs belonging toSucciniclasticum,PrevotellaceaeUCG-003,ErysipelotrichaceaeUCG-004,Prevotella1,Bacteroidales S24–7gut group uncultured bacterium, and candidateFamily XIIIAD3011 corresponding to inoculation during weaning. Compared to the inoculation during weaning, the inoculation before weaning resulted in more co-occurrences of OTUs that were exclusively predominant in the inoculum. However, inoculation during weaning appeared to have more impacts on the colonic microbiota than the inoculation before weaning. Considerable successions in the microbial colonization of the GI tracts accompanied the transition from liquid feed to solid feed during weaning.Conclusions: Repeated rumen fluid inoculation during early life can modulate the establishment of the microbiota in both the rumen and the colon and co-occurrence of some bacteria. Oral inoculation with rumen microbiota may be a useful approach to redirect the development of the microbiota in both the rumen and colon.

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