Diet starch and fiber contents influence the rumen microbial profile and its fermentation products, yet no information exists about the effects of these dietary carbohydrate fractions on the metabolic activity of these microbes. The objective of this experiment was to evaluate the effects of dietary carbohydrate profile changes on the rumen meta-proteome profile. Eight cannulated Holstein cows were assigned to the study as part of a 4 × 4 Latin square design with a 2 × 2 factorial treatment arrangement including four 28-d periods. Cows received 1 of 4 dietary treatments on a dry matter (DM) basis. Diets included different concentrations of rumen fermentable starch (RFS) and physically effective undigested NDF (peuNDF240) content in the diet: (1) low peuNDF240, low RFS (LNLS); (2) high peuNDF240, low RFS (HNLS); (3) low peuNDF240, high RFS (LNHS); and (4) high peuNDF240, high RFS (HNHS). Rumen fluid samples were collected from each cow on the last 2 d of each period at 3 time points (0600, 1000, and 1400 h). The microbial protein fraction was isolated, isobarically labeled, and analyzed using liquid chromatography combined with tandem mass spectrometry techniques. Product ion spectra were searched using the SEQUEST search on Proteome Discoverer 2.4 (Thermo Scientific) against 71 curated microbe-specific databases. Data were analyzed using PROC MIXED procedure in SAS 9.4 (SAS Institute Inc.). A total of 138 proteins were characterized across 26 of the searched microbial species. In total, 46 proteins were affected by treatments across 17 of the searched microbial species. Of these 46 proteins, 28 were affected by RFS content across 13 microbial species, with 20 proteins having higher abundance with higher dietary RFS and 8 proteins having higher abundance with lower dietary RFS. The majority of these proteins have roles in energetics, carbon metabolism, and protein synthesis. Examples include pyruvate, phosphate dikinase (Ruminococcus albus SY3), 30S ribosomal protein S11 (Clostridium aminophilum), and methyl-coenzyme M reductase subunit α (Methanobrevibacter ruminantium strain 35063), which had higher abundances with higher dietary RFS. Conversely, glutamate dehydrogenase (Butyrivibrio fibrisolvens) and 50S ribosomal protein L5 (Pseudobutyrivibrio ruminis) and L15 (Ruminococcus bromii) had lower abundances with higher dietary RFS content. Among the remaining 18 proteins unaffected by RFS content alone, 5 proteins were affected by peuNDF240 content, and 13 were affected by peuNDF240 × RFS interactions. Our results suggest that the RFS content of the diet may have a greater influence on rumen microbial protein abundances than dietary peuNDF240 content or peu-NDF240 × RFS interactions. This research highlights that dietary carbohydrate profile changes can influence rumen microbial protein abundances. Further research is needed to fully characterize the effects of diet on the rumen meta-proteome and manipulate the various roles of rumen microbes. This will aid in designing the strategies to maximize the efficiency of ...

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Identifying milk biomarkers impacted by dietary starch and fiber profiles can support optimization of feeding and concurrent promotion of animal health. The objective of this study was to characterize the rumen meta-proteome and milk whey proteome in Holstein cows fed different levels of physically effective undigestible NDF (peuNDF240) and rumen fermentable starch (RFS). Eight ruminally cannulated Holstein cows were included in a 4 x 4 Latin square design with four 28-day periods. From this study, samples were collected from cows in each period receiving one of two dietary treatments: 1) low peuNDF240, high RFS (LNHR) or 2) high peuNDF240, low RFS (HNLR). Rumen fluid and milk samples were collected at the end of each period at three timepoints, snap-frozen, and stored at -800C. Rumen microbial proteins and milk whey proteins were isolated, quantified, isobarically labeled, and analyzed by LC-MS/MS. The acquired product ion spectra from rumen fluid and the milk samples were searched against 71 composite databases and a cattle database, respectively. Data were analyzed using the PROC MIXED procedure in SAS 9.4. Of the 130 rumen-associated proteins identified across 22 searched microbial species, the abundances of 13 proteins were impacted by diet across eight microbial species. Six of these proteins were associated with core metabolism. Among the 159 quantified milk proteins, the abundances of 17 proteins were impacted by diet. Many of these proteins were associated with host defense, nutrient synthesis, and transportation. The abundance of CD14 and lactadherin was less with the LNHR diet (P < 0.05), whereas Prostaglandin-H2 D-isomerase and lipoprotein lipase abundances were greater with LNHR diet (P < 0.05). Our results indicate feeding a higher concentration of RFS has a greater influence on milk whey proteome than the rumen meta-proteome profile and highlight the potential use of milk whey proteins as biomarkers indicative of nutrition.

Locust bean gum (LBG) is a galactomannan extracted from the seeds for use as a food thickener and stabilizer and is claimed to have applications in the biopharmaceutical field. The possible immunomodulatory effects of LBG have been studied in cell lines. The objective of this study was to evaluate the effect of LBG on the expression of genes associated with innate and adaptive immune responses in cow blood. Blood was collected from (n=3) clinically healthy Holstein-Friesian cows from the North Carolina A&T State University Dairy Unit. Whole blood (1 mL) from three cows was treated individually with 10 ng/mL of LBG (Sigma-Aldrich St. Louis, MO) or maintained in PBS, incubated at 37ºC for 30 minutes. Total RNA was extracted, reverse transcribed, and real-time PCR was carried out using the RT2 Profiler human Innate & Adaptive Immune Responses PCR Array containing 84 genes, as recommended by the manufacturer (Qiagen). The Livak method was used to calculate fold change (FC >2 considered significant). The analysis showed that 40 genes out of 84 genes were affected by treatment with LBG. Among 40 genes, 4 were upregulated, and 17 genes were downregulated. The highest up-regulated and down-regulated genes following exposure to LBG were Metaxin 1(MX1) and Recombination activation gene 1 (RAG1) respectively. The MX1 gene, which encodes a protein located on the outer membrane of mitochondria, is a component of the mitochondrial protein translocation apparatus. The RAG-1 gene is involved in the activation of immunoglobulin V-D-J recombination and immune homeostasis. Thus, gums may have bioactive roles in cow blood. The observed differential effects seen have implications for the function of the molecules examined possible epigenetic regulation of V(D)J recombination and potential for dietary modulation of animal health using gums and dietary fiber and warrant further study.

Dual-flow continuous culture fermenters were used to evaluate the impacts of forage mixtures on ruminal fermentation. Diets (DM basis) contained 40% red clover combined with 1) 60% orchardgrass (OG); 2) 30% orchardgrass + 30% meadow fescue (MF); 3) 20% orchardgrass + 20% meadow fescue + 20% Kentucky bluegrass (KYBG); or 4) 15% orchardgrass + 15% meadow fescue + 15% Kentucky bluegrass + 15% perennial ryegrass (PRG). Treatments were randomly assigned to fermenters in a 4 x 4 Latin square design. Each 10-d period included a 7-d adaptation and 3-d sampling period. Diets (110 g DM/d) were added across 4 feedings/d (33% each, 0700 h and 1600 h; 17% each, 0820h and 1720 h). Fermenter pH was recorded continuously for 10-d. Methane was measured on d 7-10 at 0630 and 1530 h. Effluent samples were collected on d 8-10. Results were analyzed using PROC GLIMMIX of SAS with significance at P ≤ 0.05. Apparent digestibility of DM, OM, NDF, and ADF was greatest (P ≤ 0.05) for fermenters receiving KYBG, followed by PRG. Apparent DM and OM digestibility for OG and MF were less than KYBG by 18.0% and 19.6%, respectively. Fermenters receiving KYBG spent the greatest amount of time under pH 5.8 (avg. 15.5 h/d; P = 0.0004). Acetate:propionate and acetate + butyrate:propionate ratios were greater for fermenters receiving KYBG (4.09 and 4.71) and PRG (4.03 and 4.65; P < 0.0001). Water soluble carbohydrate concentration of effluent was least for KYBG (0.80 mg/ mL; P ≤ 0.05). Methane output for KYBG (116.8 mg/dL; P < 0.0001) was greater than that of OG (13.7 mg/dL), MF (31.7 mg/dL), and PRG (11.9 mg/dL) which did not differ from one another. These results suggest that diets containing KYBG and PRG were more digestible with PRG having the added benefit of decreased methane output.