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To understand how the capacity for fat metabolism (uptake, synthesis, modification) changes in rumen epithelia immediately before and after onset of lactation in dairy cows, rumen fluid Short Chain Fatty Acid (SCFA) concentrations and mRNA expression profiles of rumen epithelia was determined in twelve Holstein dairy cows at three weeks prior to calving (wk -3, n = 12), one week post calving (week +1, n = 12) and six weeks (week +6, n = 12) after calving. The diet was modified from a dry cow formulation to a lactating cow formulation immediately following parturition and raised the non-fiber carbohydrate level from 34 to 43%. All data was analyzed using the mixed procedure of SAS, with cows blocked by anticipated calving date and week of sampling as the repeated measure. Propionate, butyrate, isovalerate and valerate levels rose significantly following the diet change (p≤0.001), although acetate and isobutyrate levels were unchanged (p>0.05). Mean rumen pH also changed during the transition period (6.38 Vs 5.81 and 5.85±0.08; -3 Vs +1 and +6; p

Energy availability in epithelial cells is a crucia l link for maintaining epithelial barrier integrity ; energy depletion is linked to impaired barrier function in several epithelia. This study aimed to elucidate t he effects of exogenous butyrate on mRNA abundance of genes indirectly involved in rumen epithelial barrier integrit y. Sixteen mid-lactation Holstein cows fed a total mix ed ration received a concentrate mix to induce Suba cute Ruminal Acidosis (SARA). For 7 days, while being fed the concentrate mix, cows were assigned either a control treatment or a butyrate treatment, in which cows were fed butyrate at 2.5% daily dry matter in take in the form of a calcium salt. On days 6 and 7, rumen pH was measured continuously and on day 7, rumen biopsies took place. Rumen pH fell below 5.6 for more than 3 hours per day in both treatments, con-fir ming the occurrence of SARA. Microarray and pathway analysis, confirmed by real time PCR, showed that exogenous butyrate significantly increased the mRNA abundance of hexokinase 2 (fold change: 2.07), pyruvate kinase (1.19), cytochrome B-complex 3 (1.18) and ATP Synthase, F0 subunit (1.66), which en-code important glycolytic enzymes. Meanwhile, butyrate decreased mRNA abundance of pyruvate dehydrogenase kinase 2(-2.38), ATP citrate lyase (-2.00) and mito chondrial CoA transporter (-2.27), which en-code enzymes involved in lipogenesis. These data suggest exogeno us butyrate induces a shift towards energy mobiliza tion in the rumen epithelium, which may aid barrier functio n in the rumen epithelium during SARA.

The objective of this study was to elucidate the ef fects of butyrate on the Short Chain Fatty Acids (S CFA) membrane transport proteins and proton membrane transport proteins in the rumen epithelium. Sixteen mi dlactation cows were fed a 44% Non-Fibre Carbohydrate (NFC) diet and divided into a control treatment a nd a butyrate treatment. For 7 days, the cows on the c ontrol treatment received a carrier treatment and t he cows on the butyrate treatment received a ruminal butyra te dose at the rate of 2.5% of Dry Matter Intake (D MI). Rumen pH was measured on days 6 and 7 and rumen biopsies were taken on days 1 and 7. Rumen pH measurements confirmed the occurrence of ruminal acidosis in both treatment groups, defined as a rumen pH of 5.6 for at least 3 h per day. Between the con trol and butyrate treatment, there was no differenc e in rumen pH profile. Immunofluorescence analysis performed on longitudinal ruminal papillae cross-section s showed that for the duration of the study, protein abundance in the stratum basale increased for Monocarboxylate Cotransporter Isoform 1 (MCT1), sodium/proton exchanger isoform 3 (NHE3) and sodium/Bicarbonate Cotransporter Isoform 1 (NBC1). There was a time*treatment interaction for MCT1 and NBC1, with the butyrate treatment group showing a higher abundance of MCT1 and a lower abundance of NBC1 at day 7. Luminal butyrate appears to increase SCFA uptake capacity by increasing the abundance of MCT1 transport proteins on the basolateral membrane and decreasing basolateral bicarbonate uptake capacity through decreased NBC1 protein expression. These effects decrease bicarbonate uptake capacity through NBC1 and help to offset the increased MCT1, since MCT also creates alkalotic pressure by expelling protons from the cytosol.

Subacute Ruminal Acidosis (SARA) is a disorder in cattle which can lead to chronic inflammation in the rumen epithelium, known as rumenitis. Butyrate has been shown to attenuate some of the detrimental effects of inflammatory gastroenteral disorders but the molecular mechanisms mediated by butyrate have not been defined. The objective of this study was to define the inflammatory-related genomic changes responsible for the beneficial effects of butyrate. Experimentally, 16 fistulated dairy cows at mid-lactation were fed a SARA-inducing (45% non-fiber carbohydrate) diet beginning 2 days before the beginning of treatment and continuing throughout the experiment. Cows were then evenly divided into treatment groups where a carrier with (n = 8) or without (n = 8) supplemental butyrate (2.5% initial DM intake) was deposited into the rumen daily for 7 days. The minimum rumen pH was higher in cows with supplemental butyrate (4.96±0.09 to 5.20±0.05, p = 0.040), but mean pH, maximum pH and the duration for which rumen pH was below 5.6 was unaffected. Free plasma Lipopolysaccharide (LPS) concentration was unaffected by treatment as was the concentration of Serum Amyloid A (SAA), although the LPS Binding Protein (LBP) concentration was increased by the addition of butyrate to the rumen (6.91±0.29 to 7.93±0.29 µg mL-1, p = 0.024). Of the rumen Short Chain Fatty Acids (SCFA) tested, only butyrate showed a pronounced treatment effect, rising from 8.60±0.94 to 21.60±0.94 mM (p≤0.0001). Plasma Beta-Hydroxybutyrate (BHBA) concentration also increased (799.50±265.24 to 3261.63±265.24 µM, p≤0.001). Butyrate infusion did not affect milk parameters (total fat, lactose, total protein and LOS); however, when related to dry matter intake, milk production efficiency was increased (p = 0.035). Microarray and qRT-PCR analyses of rumen papillae biopsies collected on day 7 found that butyrate administration affected (p≤0.05) the expression of genes involved in Non-Specific Host Defense (NSHD), Remodeling or adaptation (RM) and Immune Response (IR). Of the 49 genes tested by qRT-PCR, 9 (LCN2, MMP1, MUC16, GPX2, CSTA, FUT1, SERPINE2, BCAM, RAC3) were upregulated, 20 (MTOR, AKIRIN2, NFKBIZ, NFKB2, ACVR2A, LAMB1, FRS2, PPARD, LBP, NEDD4L, SGK1, DEDD2, MAP3K8, PARD6B, PLIN2, ADA, HPGD, FMO5, BMP6, TCHH) were downregulated and 20 were unchanged due to butyrate administration in the proximal gastrointestinal tract. These results demonstrate the potential protective effect and molecular mechanisms involved in a novel butyrate treatment for inflammatory gastrointestinal conditions.

The objective of this study was to characterize the mRNA and protein expression of inflammatory and structural genes in the rumen epithelium during gra in-induced ruminal acidosis in lactating dairy catt le. A total of 16 rumen-fistulated, lactating Holstein da iry cattle (618±35 kg of body weight, 221±32 days i n milk) were used in a randomized complete block design study. All cattle were initially fed a high-forage die t (HF; 88.9% of dry matter) and after a baseline (wk 0) me asurement, half of the cattle were randomly assigne d and transitioned to a high-concentrate diet (HC; 62 .2% of dry matter) which was fed for 3 weeks (weeks 1, 2 and 3). Continuous ruminal pH, ruminal LPS and plasma LPS-binding protein were measured each week followed by a rumen papillae biopsy used for mRNA and protein quantification. After the baseline perio d, ruminal LPS was higher in HC compared to HF cattle (28851±6905 vs. 5771±3042 EU mL -1 ). There was no difference in mRNA expression of inflammatory and structure genes in rumen papillae between HF and HC cattle during all weeks. With regard to protein exp ression, there was an up regulation (p = 0.02) of n uclear factor of activated T-cells cytoplasmic 2 expressio n during weeks 1, 2 and 3; however, all other infla mmatory markers within the rumen epithelium were unchanged by treatment. These results suggest that although g raininduced ruminal acidosis leads to characteristic wh ole-animal inflammatory response, only marginal changes in inflammatory and structural gene and protein expres sion in the rumen epithelium were detected.

Problem statement: Subacute Ruminal Acidosis (SARA) is a metabolic disorder affecting approximately 20% of all dairy cattle in North America. Although the presence of SARA has been described for some time, the etiology of the disorder remains uncertain. For example, many animals diagnosed with SARA seem to remodel and adapt their epithelium to accommodate the stresses imposed by SARA, but not before exacting a significant health and economic toll. Specifically, a search is on in which a desire to identify the system and associated pathways that are causative agents in the progression and development of SARA is evident. We hypothesize that adaptation to SARA is facilitated by the immune system. Approach: In order to answer of this question, 4 mature, non-lactating dairy cattle were transitioned from a High Fiber (HF; 0% grain) diet to High Grain (HG; 65% grain) diet. Having fed the HG diet for three weeks, the cattle were then transitioned back to the HF diet for an additional three weeks to facilitate adaptation. SARA was diagnosed by pH data only during the first week and not during the remaining weeks, indicating that adaptation to the HG diet took place within one week. Results: In this study, significant (p