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Background Targeted modification of the dairy calf ruminal microbiome has been attempted through rumen fluid inoculation to alter productive phenotypes later in life. However, sustainable effects of the early life interventions have not been well studied, particularly on the metabolically active rumen microbiota and its functions. This study investigated the sustained effects of adult-derived rumen fluid inoculations in pre-weaning dairy calves on the active ruminal microbiome of post-weaned dairy calves analyzed via RNA-sequencing. Results Two different adult-derived microbial inocula (bacterial- or protozoal-enriched rumen fluid; BE or PE, respectively) were administered in pre-weaned calves (3–6 weeks) followed by analyzing active rumen microbiome of post-weaned calves (9 weeks). The shared bacterial community at the genus level of 16S amplicon-seq and RNA-seq datasets was significantly different ( P  = 0.024), 21 out of 31 shared major bacterial genera differed in their relative abundance between the two analytic pipelines. No significant differences were found in any of the prokaryotic alpha- and beta-diversity measurements ( P  &gt; 0.05), except the archaeota that differed for BE based on the Bray–Curtis dissimilarity matrix ( P  = 0.009). Even though the relative abundances of potentially transferred microbial and functional features from the inocula were minor, differentially abundant prokaryotic genera significantly correlated to various fermentation and animal measurements including butyrate proportion, body weight, and papillae length and counts. The overall microbial functions were affected quantitatively by BE and qualitatively by PE ( P  &lt; 0.05), and this might be supported by the individual KEGG module and CAZymes profile differences. Exclusive networks between major active microbial (bacterial and archaeal genera) and functional features (KEGG modules) were determined which were differed by microbial inoculations. Conclusions This study demonstrated that actively transcribed microbial and functional features showed reliable connections with different fermentations and animal development responses through adult rumen fluid inoculations compared to our previous 16S amplicon sequencing results. Exclusive microbial and functional networks of the active rumen microbiome of dairy calves created by BE and PE might also be responsible for the different ruminal and animal characteristics. Further understanding of the other parts of the gastrointestinal tract (e.g., abomasum, omasum, and small intestine) using metatranscriptomics will be necessary to elucidate undetermined biological factors affected by microbial inoculations.

animal microbiome

The rumen liquid metatranscriptome of post-weaned dairy calves differed by pre-weaning ruminal administration of differentially-enriched, rumen-derived inocula