We evaluated the effects of supplementing bacterial direct-fed microbial (DFM) on performance, apparent total-tract digestibility, rumen fermentation, and immune parameters of lactating dairy cows. One-hundred and 14 multiparous Holstein cows (41 ± 7 DIM) were used in a randomized complete block design with an experiment comprising 14-d covariate (pre-experimental sample and data collection) and 91-d of experimental period. Cows were blocked based on energy-corrected milk yield during the covariate period and the following treatments were randomly assigned within each block: 1) Control (CON): Corn silage-based total mixed ration (TMR) without DFM; 2) PRO-A: Basal diet top-dressed with a mixture of Lactobacillus animalis and Propionibacterium freudenreichii at 3 × 10⁹ cfu/day; and 3) PRO-B: Basal diet top-dressed with a mixture of L. animalis, P. freudenreichii, Bacillus subtilis and B. licheniformis at 11.8 × 10⁹ cfu/day. Milk yield, DMI, and BW were measured daily while milk samples for component analysis were taken on 2 consecutive days of each week of data collection. Feces, urine, rumen, and blood samples were taken during the covariate period, wk 4, 7, 10, and 13 for estimation of digestibility, N-partitioning, rumen fermentation, plasma nutrient status and immune parameters. Treatments had no effect on DMI and milk yield. Fat-corrected milk (3.5% FCM) and milk fat yield were improved with PRO-B, while milk fat percent and feed efficiency (ECM/DMI) tended to increase with PRO-B compared with PRO-A and CON. Crude fat digestibility was greater with PRO-B compared with CON. Feeding CON and PRO-A resulted in higher total VFA concentration relative to PRO-B. Percentage of neutrophils tended to be reduced with PRO-A compared with CON and PRO-B. The mean fluorescence intensity (MFI) of anti-CD44 antibody on granulocytes tended to be higher in PRO-B compared with CON. The MFI of anti-CD62L antibody on CD8+ T cells was lower in PRO-A than PRO-B, with PRO-A also showing a tendency to be lower than CON. This study indicates the potential of DFM to improve fat digestibility with consequential improvement in fat corrected milk yield, feed efficiency and milk fat yield by lactating dairy cows. The study findings also indicate that dietary supplementation with DFM may augment immune parameters or activation of immune cells, including granulocytes and T cells; however, the overall effects on immune parameters are inconclusive.

We examined the effects of 2 multispecies 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 and were a subset from a lactation performance study, which used 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 a mixture of Lactobacillus animalis and Propionibacterium freudenreichii at 3 × 10⁹ cfu/d (PRO-A), or (3) basal diet top-dressed with a mixture of L. animalis, P. freudenreichii, Bacillus subtilis, and Bacillus licheniformis at 11.8 × 10⁹ cfu/d (PRO-B). The basal diet was fed ad libitum daily as a TMR at 0600 and 1200 h for a 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. An in-depth, untargeted metabolome profile of the composite rumen and plasma samples and the d -3 samples was developed by using a chemical isotope labeling/liquid chromatography-mass spectrometry (LC-MS)-based technique. Differentially abundant metabolites (taking into account fold change [FC] values and false discovery rates [FDR]) were identified with a volcano plot. In the rumen, compared with the CON diet, 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, and 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 the CON diet, relative concentrations of 13 plasma metabolites, including myxochelin A and glyceraldehyde, were increased (FC ≥1.2; FDR ≤0.05) by PRO-A supplementation, 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, and reduced 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 are involved in AA and fatty acid metabolism and have antimicrobial and immune-stimulating properties. The results of this study demonstrated that dietary supplementation with either PRO-A or PRO-B altered the plasma and ruminal metabolome. Notably, ruminal and plasma metabolites involved in the metabolism of AA and fatty acids and those with immunomodulatory properties were altered by either or both of the 2 microbial additives.

We examined the effects of two direct-fed microbial (DFM) products containing multiple microbial species and their fermentation products on ruminal metatranscriptome and carboxyl-metabolome of beef steers. Nine ruminally-cannulated Holstein steers were assigned to 3 treatments arranged in a 3 × 3 Latin square design with three 21-d periods. Dietary treatments were (1) Control (CON; basal diet without additive), (2) Commence (PROB; basal diet plus 19 g/d of Commence), and (3) RX3 (SYNB; basal diet plus 28 g/d of RX3). Commence and RX3 are both S. cerevisiae-based DFM products containing several microbial species and their fermentation products. Mixed ruminal contents collected multiple times after feeding on day 21 were used for metatranscriptome and carboxyl-metabolome analysis. Partial least squares discriminant analysis revealed a distinct transcriptionally active taxonomy profiles between CON and each of the PROB and SYNB samples. Compared to CON, the steers fed supplemental PROB had 3 differential (LDA ≥ 2.0; p ≤ 0.05) transcriptionally active taxa, none of which were at the species level, and those fed SYNB had eight differential (LDA > 2.0, p ≤ 0.05) transcriptionally active taxa, but there was no difference (p > 0.05) between PROB and SYNB. No functional microbial genes were differentially expressed among the treatments. Compared with CON, 3 metabolites (hydroxylpropionic acid and 2 isomers of propionic acid) were increased (FC ≥ 1.2, FDR ≤ 0.05), whereas 15 metabolites, including succinic acid and fatty acid peroxidation and amino acid degradation products were reduced (FC ≤ 0.83, FDR ≤ 0.05) by supplemental PROB. Compared with CON, 2 metabolites (2 isomers of propionic acid) were increased (FC ≥ 1.2, FDR ≤ 0.05), whereas 2 metabolites (succinic acid and pimelate) were reduced (FC ≤ 0.83, FDR ≤ 0.05) by supplemental SYNB. Compared to SYNB, supplemental PROB reduced (FC ≤ 0.83, FDR ≤ 0.05) the relative abundance of four fatty acid peroxidation products in the rumen. This study demonstrated that dietary supplementation with either PROB or SYNB altered the ruminal fermentation pattern. In addition, supplemental PROB reduced concentrations of metabolic products of fatty acid peroxidation and amino acid degradation. Future studies are needed to evaluate the significance of these alterations to ruminal fatty acid and amino acid metabolisms, and their influence on beef cattle performance.

The study evaluated the effects of supplementing a multi-species direct-fed microbial (DFM) on the milk lipidome of lactating dairy cows. Twenty-four multiparous Holstein cows (41 ± 7 d in milk) were used in a randomized complete block design with experimental duration of 91 d. Cows were blocked based on energy-corrected milk yield from a 14-d pretreatment period, and were assigned randomly within each block to the following treatments: (1) control (CON): corn silage-based total mixed ration without DFM; or (2) BOV+: basal diet top-dressed with a DFM containing a mixture of Lactobacillus animalis (LA-51), Propionibacterium freudenreichii (PF-24), Bacillus subtilis (CH201), and Bacillus licheniformis (CH200) at 11.8 × 10⁹ cfu/d. Milk samples were taken from morning and evening milkings on 2 consecutive days of each week of the pretreatment and treatment periods. Separate composites of pretreatment period and treatment period samples were prepared for individual cows and used for lipidome analysis. Lipidome analysis of the milk samples was performed using an ultra-high-performance liquid chromatograph linked to a quadrupole time-of-flight mass spectrometer in both positive and negative ionizations. The relative concentrations of 14 lipid species, including long-chain polyunsaturated fatty acids (LC-PUFA) such as FA 20:8 and FA 28:7 and triacylglycerides (TG) such as TG 40:3 and TG 54:2, were increased [false discovery rate (FDR) ≤0.05], whereas 13 lipid species, including saturated FA 24:0 and TG 40:0 were decreased (FDR ≤0.05) by supplemental BOV+. The relative concentration of de novo FA in milk was greater, whereas that of preformed FA was lower in dairy cows supplemented with BOV+. Results from this study demonstrate the potential of a DFM containing L. animalis, P. freudenreichii, Bacillus subtilis, and B. licheniformis to alter the milk lipidome in lactating dairy cows toward increased relative concentration of LC-PUFA, which might offer a healthier profile of FA to consumers with its associated health benefits.

We applied an untargeted metabolomics technique to analyze the plasma carboxyl-metabolome of beef steers with divergent average daily gain (ADG). Forty-eight newly weaned Angus crossbred beef steers were fed the same total mixed ration ad libitum for 42 days. On day 42, the steers were divided into two groups of lowest (LF: n = 8) and highest ADG (HF: n = 8), and blood samples were obtained from the two groups for plasma preparation. Relative quantification of carboxylic-acid-containing metabolites in the plasma samples was determined using a metabolomics technique based on chemical isotope labeling liquid chromatography mass spectrometry. Metabolites that differed (fold change (FC) ≥ 1.2 or ≤ 0.83 and FDR ≤ 0.05) between LF and HF were identified using a volcano plot. Metabolite set enrichment analysis (MSEA) of the differential metabolites was done to determine the metabolic pathways or enzymes that were potentially altered. In total, 328 metabolites were identified. Volcano plot analysis revealed 43 differentially abundant metabolites; several short chain fatty acids and ketone bodies had greater abundance in HF steers. Conversely, several long chain fatty acids were greater in LF steers. Five enzymatic pathways, such as fatty acyl CoA elongation and fatty-acid CoA ligase were altered based on MSEA. This study demonstrated that beef steers with divergent ADG had altered plasma carboxyl-metabolome, which is possibly caused by altered abundances and/or activities of enzymes involved in fatty acid oxidation and biosynthesis in the liver.