Examining the interplay of gut microbiome and diet-induced ruminal acidosis in dairy cattle

Polson, Shawn W.Dairy cattle support an important industry worldwide and produce about 378 billion liters of milk per year. During industrial milk production, cows are switched from a diet rich in fiber to a diet rich in grain to support their increased energy output. Some cows respond to that diet in a healthy manner, while approximately 26 % develop metabolic diseases including ruminal acidosis, obesity, diabetes type 2, and laminitis, costing the dairy cattle industry $ 1 billion annually through decreased milk production and the culling of diseased animals from production. There are similarities at the mechanistic and characteristic levels between cattle metabolic disease and type 2 diabetes in humans. The current study investigates the rumen and intestinal microbiome of the dairy cattle to better understand the interaction between metabolic disease and gut microbiome due to a shift to a high starch diet. We propose that the rumen and intestinal microbiome distinctly shift among dairy cattle fed a high starch diet and exhibit ruminal acidosis. A total of 6 rumen-cannulated dairy cattle were randomly assigned to a control diet (high fiber) group or treatment diet (high starch) group (N=3 for each). The current study conducted a multiphase experimental trial which was divided into two phases based on the switch to a high starch diet and the subsequent addition of periodic corn starch infusions. During the diet phase, cows were switched from a standard non-lactation (dry) diet to an experimental diet. Cows in the control group were fed a total mixed ration (TMR) consisting of corn silage, alfalfa silage, by product feeds high in neutral detergent soluble fiber and soybean meal, whereas cows in the treatment group were fed ground barley (20 % on a dry matter basis) which was top dressed in TMR. During the infusion phase, cows continued to receive their corresponding diets and experienced three infusion cycles, each consisting of a control (water) or treatment (corn starch) infusion into the abomasum twice daily for one week followed by three weeks of recovery (no infusions). The infusion phase cycles were followed by a final 14 -day infusion prior to euthanasia. Rumen fluid and feces were collected throughout the study, and intestinal contents (digesta and tissue) samples from two small intestine (jejunum and ileum) and large intestine (cecum and colon) locations were collected at euthanasia to evaluate the intestinal bacterial community using a high-throughput 16S rRNA bacterial gene amplification and barcoding pipeline for Illumina MiSeq sequencing. In a companion study, rumen fluid and fecal samples were collected to determine pH and the concentrations of volatile fatty acid (VFA) and lactate. Gene expression analysis of pro-inflammatory cytokines was determined using real time PCR. Immune cell phenotype was determined using flow cytometer. ☐ Rumen fluid microbial analysis showed that the treatment diet impacted rumen fluid-associated microbial communities during the infusion phase. A high starch diet reduced (p < 0.01) the diversity (richness and evenness) and shifted the composition of rumen-fluid associated microbial communities as indicated by the Kruskal Wallis results of alpha diversity (Shannon index and Pielou’s Evenness) and the PERMANOVA results of beta-diversity (Unweighted-Unifrac and Weighted-Unifrac) metrics. At the family level, a high starch diet and periodic corn starch infusion increased the relative abundance of some taxa including Bifidobacteriaceae and Erysipelotrichaceae and decreased the proportion of Lactobacillaceae. There was no treatment effect based on rumen pH and VFA concentrations. ☐ Fecal microbial analysis results showed that corn starch infusion significantly reduced (p < 0.01) the richness and evenness of fecal associated microbial community, and shifted their composition as indicated by the Kruskal Wallis results of alpha diversity (Shannon index, OTU numbers, and Pielou’s Evenness) and beta-diversity (Unweighted-Unifrac and Weighted-Unifrac) metrics. The principal coordinate analysis (PCoA) results showed that the fecal-associated bacterial communities of the treatment group significantly shifted in each infusion week then drifted back toward the control community during the subsequent three recovery weeks. At the family level, corn starch infusion increased the relative abundance of some taxa including Succinivibrionaceae, Erysipelotrichaceae, Lachnospiraceae, Clostridiaceae 1, Prevotellaceae, Bacteroidales S24 - 7 group, and Bifidobacteriaceae and decreased the proportion of Spirochaetaceae, p – 2534 - 18B5 gut group, and unknown Bacteroidetes. Fecal pH level decreased (p < 0.01) in treatment cows compared to the control during infusion and recovery week 1. The concentration of acetic acid increased (p < 0.01) in treatment recovery week 1 group compared to control group whereas the concentration of butyric acid increased (p < 0.01) in treatment infusion week group compared to control group. The concentrations of propionic acid and lactic acid were not affected by starch infusion. ☐ Intestinal microbial analysis at the conclusion of the experiment showed that the digesta-associated bacterial community distinctly clustered by location (small intestine and large intestine) whereas no diet influence on tissue-associated bacterial community. Cytokine expression levels and immune cell populations were not affected by treatment (barley diet and periodic corn starch infusion). ☐ Collectively, our study demonstrates that high starch diet (barley diet and periodic corn starch infusion) did not result in rumen acidosis. However, high starch diet can lower the diversity and change the ruminal bacterial community composition over time. Also, corn starch infusion can temporarily lower fecal pH, increase acetic and butyric acids, reduce the diversity and change the structure of fecal-associated bacterial community. That suggests that corn starch infusion is a selective force that might open the door for diseases. Also, the study demonstrated that high starch diet had no effect on the bacterial diversity among digesta and tissue, or on pro-inflammatory cytokine expression and immune cell population. That suggests that the homeostasis in the intestinal luminal and mucosal immune system resists microbiome disturbances associated with starch challenge. Thus, the distinct variation in how different part of the gastrointestinal tract respond to the dietary challenge may provide the basis to further explore the ways in which the gut microbiome contributes to animal health or disease.University of Delaware, Department of Biological SciencesM.S