This study evaluated degradable nutrient supply effects on VFA concentrations, fluid flux and pool sizes, rumen epithelial metabolic and absorptive genes, and post-absorptive muscle and blood responses. Six ruminally cannulated Holstein heifers (BW=330 ± 11.3 kg) were used in a partially replicated Latin Square experiment with four treatments consisting of beet pulp or timothy hay and barley or corn grain. Periods were18 d with 3 d diet adaptation and 15 d of treatment. During each period, d 10 to 14 was used for in situ nutrient degradation assessment, d 16 to 18 was used for rumen fluid sampling, and d 18 was used for rumen papillae and skeletal muscle biopsies and blood sampling. In situ ruminal starch disappearance rate (barley 7.61 to 10.5 %/h vs corn 7.30 to 8.72%/h; P = 0.05) and extent of fiber disappearance (timothy hay 22.2 to 33.4 % DM vs beet pulp 34.4 to 38.7 % DM P=0.0007) differed significantly among diets. Acetate (P = 0.02) and isovalerate (P = 0.008) molar percentages (% mol) were increased by timothy hay, but propionate (P = 0.06) and valerate (P = 0.10) molar percentages were decreased. Corn increased propionate (P = 0.02) and valerate (P = 0.049) molar percentage, but decreased butyrate (P = 0.04) molar proportion. Fluid volume and fluid passage rate, and individual VFA pool sizes were not influenced by diet (P > 0.05). Four epithelial genes, two metabolic and two absorptive, had increased expression on timothy hay diets (P < 0.15). Blood acetate concentration was influenced by treatment (P = 0.067) but no other blood metabolites were. Skeletal muscle metabolic rate was significantly increased on corn diets (P = 0.023). The results of this study provide a whole-system snapshot of how the rumen environment changes on diets differing in nutrient degradability and how the post-absorptive system adapts in response. / Master of Science / Over the last 50 years, dairy cattle have been bred to optimize milk production to meet growing population demands for milk and dairy products. The world population continues to grow and is projected to reach 9.7 billion people by 2050. Because of this growing population, there is an overwhelming need for dairy nutritionists to optimize the conversion of human inedible fibers into human edible food. The ruminant animal accomplishes this conversion through microbial fermentation of feedstuffs into volatile fatty acids (VFA), which account for approximately 70% of total energy available for meat, milk, and fiber production. Because rumen fermentation is a complex biochemical system, it is influenced by myriad factors including the substrate provided, the pH of the environment, and the absorptive and metabolic capacity of the rumen wall, among others. Although we understand how diet influences individual aspects of rumen fermentation, few studies have concurrently evaluated how diet influences the rumen chemical environment, the epithelium, and the resulting shifts in postabsorptive metabolism. Our study sought to understand the impacts of feedstuffs with different expected ruminally available starch and fiber supplies on these aspects of ruminant physiology. Six ruminally cannulated Holstein heifers were fed four different diets which used either beet pulp (low fiber ingredient) or timothy hay (high fiber ingredient), and ground corn (low starch ingredient) or ground barley (high starch ingredient). Heifers were fed each diet for a period of 18 days. From day 10 to day 14 of the period, nutrient degradability was assessed by incubating bags of feed in the rumen and conducting feed analysis after removed from the rumen. During the last four days of each period, rumen fluid samples, blood samples, muscle biopsies, and rumen papillae biopsies were collected. Feed analysis indicated that the starch sources differed in degradation rates (i.e. the speed of degradation) and fiber sources different in extent of rumen degradation (i.e. the percentage of feed degraded). Timothy hay caused greater concentrations of Total VFA, Total branched-chain VFA, acetate isobutyrate, and isovalerate. Timothy hay caused greater molar proportions of acetate and isovalerate. Corn caused greater molar proportions of propionate and valerate when barley caused greater molar proportions of butyrate. Rumen papillae biopsies were used to evaluate gene expression. Out of 14 genes, four were impacted by diet. Two rumen transporters responsible for the absorption of VFA had greater expression when animals were fed timothy hay diets versus beet pulp diets. Two metabolic genes also had greater expression due to timothy hay. The changes of both absorptive genes and metabolic genes is likely connected to the increased presence of VFA in the rumen. Lastly, blood acetate was increased, but there was not a specific ingredient or combination that caused the change. These results provide an overall snapshot of rumen fermentation characteristics and how changes in the rumen affect other biology.