Integrated microbiota-host-metabolome approaches reveal adaptive ruminal changes to prolonged high-grain feeding and phytogenic supplementation in cattle.

Diets rich in readily fermentable carbohydrates primarily impact microbial composition and activity, but can also impair the ruminal epithelium barrier function. By combining microbiota, metabolome, and gene expression analysis, we evaluated the impact of feeding a 65% concentrate diet for 4 weeks, with or without a phytogenic feed additive (PFA), on the rumen ecosystem of cattle. The breaking point for rumen health seemed to be the second week of high grain (HG) diet, with a dysbiosis characterized by reduced alpha diversity. While we did not find changes in histological evaluations, genes related with epithelial proliferation (IGF-1, IGF-1R, EGFR, and TBP) and ZO-1 were affected by the HG feeding. Integrative analyses allowed us to define the main drivers of difference for the rumen ecosystem in response to a HG diet, identified as ZO-1, MyD88, and genus Prevotella 1. PFA supplementation reduced the concentration of potentially harmful compounds in the rumen (e.g. dopamine and 5-aminovaleric acid) and increased the tolerance of the epithelium toward the microbiota by altering the expression of TLR-2, IL-6, and IL-10. The particle-associated rumen liquid microbiota showed a quicker adaptation potential to prolonged HG feeding compared to the other microenvironments investigated, especially by the end of the experiment.

Diet and phytogenic supplementation substantially modulate the salivary proteome in dairy cows.

Phytogenic compounds may influence salivation or salivary properties. However, their effects on the bovine salivary proteome have not been evaluated. We investigated changes in the bovine salivary proteome due to transition from forage to high-concentrate diet, with and without supplementation with a phytogenic feed additive. Eight non-lactating cows were fed forage, then transitioned to a 65% concentrate diet (DM basis) over a week. Cows were control (n = 4, CON) or supplemented with a phytogenic feed additive (n = 4, PHY). Proteomic analysis was conducted using liquid chromatography coupled with mass spectrometry. We identified 1233 proteins; 878 were bovine proteins, 189 corresponded to bacteria, and 166 were plant proteins. Between forage and high-concentrate, 139 proteins were differentially abundant (P < 0.05), with 48 proteins having a log2FC difference > |2|. The salivary proteome reflected shifts in processes involving nutrient utilization, body tissue accretion, and immune response. Between PHY and CON, 195 proteins were differently abundant (P < 0.05), with 37 having a log2FC difference > |2|; 86 proteins were increased by PHY, including proteins involved in smell recognition. Many differentially abundant proteins correlated (r > |0.70|) with salivary bicarbonate, total mucins or pH. Results provide novel insights into the bovine salivary proteome using a non-invasive approach, and the association of specific proteins with major salivary properties influencing rumen homeostasis. SIGNIFICANCE: Phytogenic compounds may stimulate salivation due to their olfactory properties, but their effects on the salivary proteome have not been investigated. We investigated the effect of high-concentrate diets and supplementation with a phytogenic additive on the salivary proteome of cows. We show that analysis of cows' saliva can be a non-invasive approach to detect effects occurring not only in the gut, but also systemically including indications for gut health and immune response. Thus, results provide unique insights into the bovine salivary proteome, and will have a crucial contribution to further understand animal response in terms of nutrient utilization and immune activity due to the change from forage to a high-energy diet. Additionally, our findings reveal changes due to supplementation with a phytogenic feed additive with regard to health and olfactory stimulation. Furthermore, findings suggest an association between salivary proteins and other components like bicarbonate content.

Bovine rumen epithelial miRNA-mRNA dynamics reveals post-transcriptional regulation of gene expression upon transition to high-grain feeding and phytogenic supplementation.

The rumen epithelium has a pivotal role in nutrient uptake and host health. This study aimed to explore the role of microRNAs (miRNAs) in the epithelial transcriptome during diet transition from forage to high-grain feeding and the modulation through supplementation with a phytogenic feed additive. Rumen biopsies were collected from 9 ruminally-cannulated non-lactating Holstein cows fed a baseline forage diet (FD) and then transitioned to high-grain feeding (HG; 65% concentrate on a dry matter basis). Cows were randomly allocated into a control group (CON, n = 5) and a group supplemented with a phytogenic feed additive (PHY, n = 4). MiRNA and mRNA sequencing was performed in parallel and transcripts were analyzed for differential expression, pathway enrichment analysis, and miRNA-mRNA interaction networks. We identified 527 miRNAs shared by all samples of the rumen epithelium, from which, bta-miR-21-5p, bta-miR-143 and bta-miR-24-3p were the most expressed. Six miRNAs were differentially expressed between CON and PHY and 8 miRNAs between FD and HG feeding, which were mainly associated with fat metabolism. Transcriptome analysis identified 9481 differentially expressed genes (DEGs) between FD and HG, whereas PHY supplementation resulted in 5 DEGs. DEGs were mainly involved in epithelium development and morphogenesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with tricarboxylic acid and short chain fatty acid (SCFA) metabolism were enriched in DEGs between diets. MiRNA target prediction and anti-correlation analysis was used to construct networks and identify DEGs targeted by DE miRNAs responsive to diet or PHY. This study allowed the identification of potential miRNA regulation mechanisms of gene expression during transition from FD to HG feeding and phytogenic supplementation, evidencing a direct role of miRNAs in host responses to nutrition.