Effects of direct-fed microbial supplement on ruminal and plasma metabolome of early-lactation dairy cows: Untargeted metabolomics approach

We examined the effects of 2 multi-species direct-fed microbial (DFM) supplements on ruminal and plasma metabolome of early-lactation dairy cows using a high-coverage untargeted metabolomics approach. A total of 45 multiparous Holstein cows (41 ± 7 DIM) were enrolled for the 14-d pre-experimental and 91-d experimental period study, and were subset from a lactation performance study, which utilized a total of 114 cows. Cows were blocked using pre-experimental energy-corrected milk yield and randomly assigned within each block to 1 of 3 treatments: 1. Corn silage-based diet with no DFM supplement (Control; CON), 2. Basal diet top-dressed with mixture of Lactobacillus animalis and Propionibacterium freudenreichii at 3 × 109 cfu/day (PRO-A), and 3. Basal diet top-dressed with a mixture of L. animalis, P. freudenreichii, Bacillus subtilis, and B. licheniformis at 11.8 × 109 cfu/day (PRO-B). The basal diet was fed ad libitum daily as a total mixed ration at 0600 and 1200 h for a total duration of 91 d. Rumen fluid and blood samples were taken on d −3, 28, 49, 70, and 91, and immediately stored at −80°C. Before analysis, ruminal and plasma samples from d 28, 49, 70, and 91 were composited. In-depth untargeted metabolome profile of the composite rumen and plasma samples and the d −3 samples was done using a chemical isotope labeling (CIL)/liquid chromatography–mass spectrometry (LC-MS)-based technique. Differentially abundant metabolites (fold change (FC) values ≥1.2 or ≤0.83 and false discovery rate (FDR)-adjusted P-value ≤0.05) were identified with a Volcano plot. In the rumen, compared with CON, supplemental PRO-A increased (FC ≥1.2; FDR ≤0.05) the relative concentrations of 9 metabolites including 2-hydroxy-2,4-pentadienoic acid, glutaric acid, quinolinic acid, and shikimic acid, while PRO-B increased relative concentrations of 16 metabolites including 2-hydroxy-2,4-pentadienoic acid, glutaric acid, 16-hydroxypalmitic acid, and 2 propionate precursors (succinic and methylsuccinic acids). Relative to PRO-A, supplemental PRO-B increased (FC ≥1.2; FDR ≤0.05) relative rumen concentrations of 3 metabolites: 16-hydroxypalmitic acid, indole-3-carboxylic acid, and 5-aminopentanoic acid, but reduced relative rumen concentrations of 13 metabolites including carnitine, threonic acid, and shikimic acid. Compared with CON, relative concentrations of 13 plasma metabolites including myxochelin A and glyceraldehyde were increased (FC ≥1.2; FDR ≤0.05) by PRO-A, whereas those of 9 plasma metabolites including 4–2-aminophenyl-2,4-dioxobutanoic acid, N-acetylornithine, and S-norlaudanosolin were reduced (FC ≤0.83; FDR ≤0.05). Supplemental PRO-B increased (FC ≥1.2; FDR ≤0.05) relative concentrations of 9 plasma metabolites including trans-o-hydroxybenzylidenepyruvic acid and 3-methylsalicylaldehyde, while reducing relative concentrations of 4 plasma metabolites including β-ethynylserine and kynurenine. Pathway analysis of the differentially abundant metabolites in both rumen and plasma revealed that these metabolites were involved in amino acid and fatty acid metabolisms and have antimicrobial and immune-stimulating properties. The results of this study demonstrated that dietary supplementation of either PRO-A or PRO-B altered plasma and ruminal metabolome. Notably, ruminal and plasma metabolites involved in the metabolism of amino acids and fatty acids and those with immunomodulatory properties were altered by either or both of the 2 microbial additives.