Adaptive responses in short-chain fatty acid absorption,gene expression,and bacterial community of the bovine rumen epithelium recovered from a continuous or transient high-grain feeding

The feeding of high-grain diets to dairy cows commonly results in lowered pH and ruminal dysbiosis, characterized by changes in absorption dynamics of short-chain fatty acids (SCFA) across the reticuloruminal wall, epithelial function, and the epithelial bacteria community structure. Therefore, the present study evaluated the effect of high-grain feeding persistence on the absorption kinetics of reticuloruminal SCFA, gene expression in the rumen epithelium, and the associated shifts in the epithelial bacteria in cows recovering from either a long-term continuous high-grain feeding model or a long-term transient high-grain feeding model. In a crossover study design, 8 nonlactating Holstein cows were fed 60% concentrate either continuously for 4 wk (continuous) or with a 1-wk break in the second week of the high-grain feeding (transient). After the high-grain feeding, all animals were fed a diet of 100% forage (recovery) for an additional 8 wk. Rumen papilla biopsies and SCFA absorption measurements were taken at the start of the trial (baseline), after the 4-wk high-grain feeding (49 d), after 2-wk recovery forage feeding (63 d), and after 8-wk recovery forage (105 d). Absorption of SCFA was determined in vivo using the washed and isolated reticulorumen technique. Rumen papillae biopsies were used for adherent bacterial DNA and host RNA extraction. The epithelial bacteria were determined using Illumina MiSeq (Microsynth AG, Balgach, Switzerland) sequencing of the 16S rRNA gene. No significant effects of the high-grain feeding model were seen for bacterial diversity. However, bacterial diversity increased with time spent in the recovery forage feeding period regardless of feeding model. The relative abundance of Acidobacteria phyla and Acetivibrio spp. increased when animals were fed a transient high-grain feeding model. A trend toward increased CLDN4 expression was observed in the continuous model. Furthermore, there were interactions between feeding model and sampling day for gene targets CD14, DRA, NHE2, NHE3, and MCT2. When comparing length of recovery, in the continuous model increased relative absorption of SCFA was sustained at 63 d but dropped to baseline measurements at 105 d. A similar pattern was found with the transient model but it did not reach significance. The only gene target that was found to significantly correlate to relative absorption of SCFA was DRA (correlation coefficient ≤ −0.41). Whereas, genera Alkalibaculum, Anaerorhabdus, Coprococcus, and Dethiobacter all showed positive correlations to gene targets for pH regulation (NHE2 and NHE3) and SCFA uptake (MCT1) but negative correlations to SCFA absorption. We conclude that while the rumen absorption and epithelial bacteria were able to recover to baseline levels after 8 wk of forage feeding, the time needed for re-establishment of homeostasis in host gene expression is longer, especially when high-grain feeding is interrupted.     

Chloride, gluconate, sulfate, and short-chain fatty acids affect calcium flux rates across the sheep forestomach epithelium

In ruminants, more than 50% of overall gastrointestinal Ca absorption can occur preintestinally, and the anions of orally applied Ca salts are thought to play an important role in stimulating ruminal Ca absorption. This assumption is based mainly on ion-exchange studies that have used gluconate as the control anion, which may bind Ca²⁺ ions and interfere with treatment effects. In the present study, we investigated the distinct effects of different anions on Ca absorption across the sheep rumen and on the concentration of free Ca²⁺ ions ([Ca²⁺]_ion). We showed that gluconate, sulfate, and short-chain fatty acids (SCFA) remarkably reduced [Ca²⁺]_ion in buffer solutions. Nevertheless, increasing the Cl or SCFA concentration by 60 mM stimulated net ruminal Ca absorption 5- to 7-fold, but these effects could be antagonized by gluconate. Therefore, ion-exchange experiments must be (re)evaluated very carefully, because changes in [Ca²⁺]_ion in the presence of gluconate, sulfate, or SCFA not only might entail an underestimation of Ca flux rates, but also might have effects on other cellular pathways that are Ca²⁺ dependent. Concerning the optimal Ca supply for dairy cows, the present study suggests that CaCl₂ formulations and Ca salts of the SCFA stimulate Ca absorption across the rumen wall and are beneficial in preventing or correcting a Ca deficiency.     

Effect of supplemental concentrate during the dry period or early lactation on rumen epithelium gene and protein expression in dairy cattle during the transition period

We previously reported 2 experiments with rumen-cannulated Holstein-Friesian dairy cows showing that during the transition period, rumen papillae surface area, and fractional absorption rate of volatile fatty acids (VFA) increase after calving. However, supplemental concentrate during the dry period and rate of increase of concentrate allowance during lactation affected papillae surface area, but not VFA absorption. Here we report the changes in gene and protein expression in rumen papillae related to tissue growth and VFA utilization. The lactation experiment treatment consisted of a rapid [RAP; 1.0 kg of dry matter (DM)/d; n = 6] or gradual (GRAD; 0.25 kg of DM/d; n = 6) increase of concentrate allowance (up to 10.9 kg of DM/d), starting at 4 d postpartum (pp). The dry period experiment treatment consisted of 3.0 kg of DM/d of concentrate (n = 4) or no concentrate (n = 5) during the last 28 d of the dry period. Real-time quantitative PCR analysis of rumen papillae showed that the expression of apoptosis-related genes was neither affected by day nor its interaction with treatment for both experiments. Expression of epithelial transporter genes was not affected by day or treatment in the lactation experiment, except for NBC1. In the dry period experiment, expression of MCT1, NBC1, DRA, NHE2, NHE3, and UT-B generally decreased after calving. A day and treatment interaction was observed for ATP1A1 in the dry period experiment, with greater expression at 18 and 8 d antepartum for concentrate than no concentrate. Generally, expression of VFA metabolism-related genes was not affected by day or its interaction with treatment. In the lactation experiment, immunoblotting of 5 selected genes showed that protein expression of DRA and PCCA was greater at 16 d pp compared with 3 and 44 d pp. Expression of NHE2 was greater, and that of ATP1A1 lower, at 16 and 44 d pp compared with 3 d pp, suggesting alterations in intracellular pH regulation and sodium homeostasis. Both MCT1 and PCCA protein were upregulated by RAP from 3 to 16 d pp, indicating modulations in VFA metabolism. Our data suggests that VFA absorption and metabolic capacity changed little per unit of surface area during the transition period, and suggests that a change in mitosis rate rather than apoptosis rate is associated with the increased ruminal VFA production, resulting in tissue growth. A significant but weak correlation between the examined gene and protein expression levels was observed only for PCCA, indicating that care must be taken when interpreting results obtained at either level.     

Rumen volatile fatty acid molar proportions,rumen epithelial gene expression,and blood metabolite concentration responses to ruminally degradable starch and fiber supplies

The objective of this work was to characterize rumen volatile fatty acid (VFA) concentrations, rumen epithelial gene expression, and blood metabolite responses to diets with different starch and fiber sources. Six ruminally cannulated yearling Holstein heifers (body weight = 330 ± 11.3 kg) were arranged in a partially replicated Latin square experiment with 4 treatments consisting of different starch [barley (BAR) or corn (CRN)] and fiber [timothy hay (TH) or beet pulp (BP)] sources. Treatments were arranged as a 2 × 2 factorial. Beet pulp and TH were used to create relative changes in apparent ruminal fiber disappearance, whereas CRN and BAR were used to create relative changes in apparent ruminal starch disappearance. Each period consisted of 3 d of diet adaptation and 15 d of dietary treatment. In situ disappearance of fiber and starch were estimated from bags incubated in the rumen from d 10 to 14. From d 15 to 17, rumen fluid was collected every hour from 0500 to 2300 h. Rumen fluid samples were pooled by animal/period and analyzed for pH and VFA concentrations. On d 18, 60 to 80 papillae were biopsied from the epithelium and preserved for gene expression analysis. On d 18, one blood sample per heifer was collected from the coccygeal vessel. In situ ruminal starch disappearance rate (7.30 to 8.72%/h for BAR vs. 7.61 to 10.5%/h for CRN) and the extent of fiber disappearance (22.2 to 33.4% of DM for TH vs. 34.4 to 38.7% of DM for BP) were affected by starch and fiber source, respectively. Analysis of VFA molar proportions showed a shift from propionate to acetate, and valerate to isovalerate on TH diets compared with BP. Corn diets favored propionate over butyrate in comparison to BAR diets. Corn diets also had higher molar proportions of valerate. Expression of 1 gene (SLC9A3) were increased in BP diets and 2 genes (BDH1 and SLC16A4) tended to be increased in TH diets. Plasma acetate demonstrated a tendency for a starch by fiber interaction with BAR-BP diets having the highest plasma acetate, but other metabolites measured were not significant. These results suggest that TH has the greatest effect on shifts in VFA molar proportions and epithelial transporters, but does not demonstrate shifts in blood metabolite concentrations.     

Milk odd- and branched-chain fatty acids in relation to the rumen fermentation pattern

The objectives of this study were 1) to determine whether a relationship exists between molar proportions of volatile fatty acids in the rumen and milk odd-and branched-chain fatty acid concentrations (i.e., iso C13:0, anteiso C13:0, iso C14:0, C15:0, iso C15:0, anteiso C15:0, iso C16:0, C17:0, iso C17:0, anteiso C17:0, and cis-9 C17:1); and 2) to evaluate the accuracy of prediction of the latter equations using an independent data set. For development of the regression equations, individual cow data from 10 feeding experiments with rumen-fistulated dairy cows were used, resulting in a data set of 148 observations. Milk odd- and branched-chain fatty acids were closely related to the molar proportions of acetate (SE = 15.3 mmol/mol), propionate (SE = 14.7 mmol/mol), and butyrate (SE = 9.2 mmol/mol). These regression equations were further validated using data from the literature (n = 14). Evaluation of these prediction equations using the independent data set resulted in a root mean square prediction error of 3.0, 9.0, and 8.9% of the observed mean for acetate, propionate, and butyrate, respectively. In addition, less then 5% of the mean square prediction error was due to line bias. This suggests that the currently developed prediction equations based on milk odd- and branched-chain fatty acids show potential to predict molar proportions of individual volatile fatty acids in the rumen.     

The effect of supplemental concentrate fed during the dry period on morphological and functional aspects of rumen adaptation in dairy cattle during the dry period and early lactation

Ten rumen-cannulated Holstein-Friesian cows were used to examine the effect of feeding supplemental concentrate during the dry period on rumen papillae morphology and fractional absorption rate (k_a) of volatile fatty acids (VFA) during the dry period and subsequent lactation. Treatment consisted of supplemental concentrate [3.0 kg of dry matter (DM)/d] from 28 d antepartum (ap) until the day of calving, whereas control did not receive supplemental concentrate. Cows were fed for ad libitum intake and had free access to the dry period ration (27% grass silage, 28% corn silage, 35% wheat straw, and 11% soybean meal on a DM basis) and, from calving onward, to a basal lactation ration (42% grass silage, 42% corn silage, and 16% soybean meal on a DM basis). From 1 to 3 d postpartum (pp), all cows were fed 0.9 kg DM/d of concentrate, which increased linearly thereafter to 8.9 kg of DM/d on d 11 pp. At 28, 18, and 8 d ap, and 3, 17, 31, and 45 d pp, rumen papillae were collected and k_aVFA was measured in all cows. On average, 13.8 (standard deviation: 3.8) papillae were collected each from the ventral, caudodorsal, and caudoventral rumen sacs per cow per day. The k_aVFA was measured by incubating a standardized buffer fluid (45 L), containing 120 mM VFA (60% acetic, 25% propionic, and 15% butyric acid) and Co-EDTA as fluid passage marker, in the evacuated and washed rumen. Treatment did not affect ap or pp DM and energy intakes or milk yield and composition. Treatment increased papillae surface area, which was 19 and 29% larger at 18 and 8 d ap compared with 28 d ap, respectively. Surface area increased, mainly due to an increase in papillae width. However, treatment did not increase k_aVFA at 18 and 8 d ap compared with 28 d ap. In the control group, no changes in papillae surface area or k_aVFA were observed during the dry period. In the treatment group, papillae surface area decreased between 8 d ap and 3 d pp, whereas no decrease was observed for control. From 3 to 45 d pp, papillae surface area and k_aVFA increased for all cows by approximately 50%, but the ap concentrate treatment did not affect k_aVFA pp. In conclusion, the efficacy of supplemental concentrate during the dry period to increase papillae surface area and k_aVFA in preparation for subsequent lactation is not supported by the present study. Current observations underline the importance of functional measurements in lieu of morphological measurements to assess changes in the adapting rumen wall.     

Effect of forage:concentrate ratio on fatty acid composition of rumen bacteria isolated from ruminal and duodenal digesta

Four dairy cows were used to examine the effect of the dietary forage:concentrate ratio [35:65, 50:50, 65:35, and 80:20 on a dry matter (DM) basis] on the fatty acid composition of rumen bacteria isolated from the liquid (LAB) and solid (SAB) phase of the rumen and duodenal digesta. Rumen contents were sampled 4 h after the morning feeding. Solid and liquid phases were separated from rumen contents and duodenal bacteria from a composite duodenal sample by differential centrifugation. Total fatty acid content in bacterial DM was 1.6 to 2.8 times higher in SAB compared with LAB, and increased with dietary concentrate. In combination with published reports, the data show that bacterial fatty acid content and composition is closely related to dietary fatty acids except for C18:2n-6 and C18:3n-3. A decrease in forage:concentrate ratio increased bacterial concentration of trans-10 C18:1, and this increase was 3.4 times higher in LAB compared with SAB. Analysis of odd- and branched-chain fatty acids showed large differences between SAB and LAB, which probably reflected a difference in species composition. The variation in odd- and branched-chain fatty acids between SAB and LAB was used to estimate their relative proportions in duodenal bacteria by means of linear programming, and showed an increased proportion of SAB from 64.7 to 74.8% with increasing forage:concentrate ratio. In addition, increasing the proportion of dietary forage was closely related to the proportion of anteiso C15:0 in total odd- and branched-chain fatty acids (r_pearson = −0.771). The bacterial concentration of iso C17:0 closely reflected the bacterial growth rate as shown by the relation with cytosine:N (r_pearson = −0.729). These strong relationships suggest that odd- and branched-chain fatty acids might be used as tool to evaluate nutrient supply to rumen bacteria.     

Technical note: Use of laser capture microdissection for the localization of tissue-specific global gene expression in rumen papillae

Gene expression profiling of bovine rumen tissue has provided insight into dietary regulation of rumen epithelial function. However, most studies have relied on a heterogeneous sample with multiple tissue and cell types. The objective of this study was to use laser capture microdissection to characterize RNA expression profiling of epithelial and connective tissues of rumen papillae. Papillae were biopsied from 3 lactating dairy cows, frozen in cryomolds, cut into sections, stained, and dehydrated, and epithelial and connective cells were collected using laser capture microdissection. Total RNA was isolated from epithelial and connective tissue and global gene expression was assessed using the Affymetrix GeneChip Bovine Gene 1.0 ST array (Affymetrix, Santa Clara, CA). Data preprocessing was conducted using the robust multi-array average method, and detection of differentially expressed genes (DEG) was determined using ANOVA. The model included the fixed effect of tissue, and a Benjamini-Hochberg false discovery rate of 0.1 was applied to DEG. We found 382 DEG between epithelial and connective tissues. Analysis of these DEG using Ingenuity Pathway Analysis (Redwood City, CA) found that epithelial and connective tissues in rumen papillae expressed distinct RNA profiles (signatures). The epithelial signature was enriched with RNA encoding tight junction and metabolic genes, whereas connective signatures were enriched with RNA encoding proteins involved in cell structure and extracellular matrix composition. The molecular functions enriched within the top networks between the 2 tissues from the Ingenuity Pathway Analysis included connective tissue disorders, dermatological diseases and conditions, gastrointestinal disease, tissue morphology, and tissue development. In summary, it is possible to use laser capture microdissection for the localization of tissue-specific global gene expression in rumen papillae. This approach may be useful to improve the accuracy and interpretation of molecular measurements in future studies.     

Form of calf diet and the rumen. II: Impact on volatile fatty acid absorption

Diet is known to affect rumen growth and development. Calves fed an all-liquid diet have smaller and less developed rumens and a decreased ability to absorb volatile fatty acids (VFA) compared to calves fed both liquid and dry feed. However, it is unknown how rumens respond when challenged with a defined concentration of VFA. The objective of this study was to assess the effects of 2 different feeding programs on VFA absorption in preweaned calves. Neonatal Holstein bull calves were individually housed and randomly assigned to 1 of 2 diets. The diets were milk replacer only (MRO; n = 5) or milk replacer with starter (MRS; n = 6). Diets were isoenergetic (3.87 ± 0.06 Mcal of metabolizable energy per day) and isonitrogenous (0.17 ± 0.003 kg/d of apparent digestible protein). Milk replacer was 22% crude protein, 21.5% fat (dry matter basis). The textured calf starter was 21.5% crude protein (dry matter basis). Feed and ad libitum water intakes were recorded daily. Calves were exposed to a defined concentration of VFA buffer (acetate 143 mM, propionate 100 mM, butyrate 40.5 mM) 6 h before euthanasia on d 43 ± 1. Rumen fluid samples were obtained every 15 to 30 min for 6 h to measure the rate of VFA absorption. Rumen tissues were obtained from the ventral sac region and processed for morphological and immunohistochemical analyses of the VFA transporters monocarboxylate transporter 1 (MCT1) and 4 (MCT4). Body growth did not differ between diets, but empty reticulorumens were heavier in MRS than MRO calves (0.67 vs. 0.39 ± 0.04 kg) and MRS calves had larger papillae areas (0.76 vs. 15 ± 0.08 mm²). We observed no differences between diets in terms of the abundance of MCT1 and MCT4 per unit area. These results indicate that the extrapolated increase in total abundance of MCT1 or MCT4 in MRS calves was not due to increased transporter density per unit area. Modeled VFA absorption metrics (flux, mmol/h, or 6 h absorbed VFA in mmol) were not different across diets. These results demonstrate that the form of calfhood diet, whether solely MR or MR and starter, does not alter VFA absorption capacity when the rumen is exposed to a defined concentration of VFA at 6 wk of age.     

Comparison of techniques to determine the clearance of ruminal volatile fatty acids

The objective of this experiment was to compare measurements of fractional clearance rates obtained by using an unlabeled valerate-CoEDTA technique with measurements obtained by using a ¹³C-labeled volatile fatty acids (VFA) technique. The exponential decay rate of the ¹³C/¹²C ratio after pulse-dosing ¹³C-acetate, ¹³C-propionate, or ¹³C-butyrate into the rumen was compared with the decay rate of rumen valerate concentration following a simultaneous pulse dose. The unlabeled valerate, CoEDTA, and each labeled VFA, one at a time, were concurrently mixed with the evacuated ruminal content of 6 lactating cows in two 3 × 3 Latin squares. The clearance of VFA by passage to the omasum was assumed to be equivalent to the decay in ruminal Co concentration and was around 50% of the total clearance. Acetate, propionate, and butyrate had similar fractional clearance rates (31.2, 33.4, 30.4%/h, respectively), but propionate had a higher absorption rate (19.2%/h) than butyrate (14.2%/h). Linear regression determination coefficients using the valerate clearance rate as an estimator for acetate, propionate, and butyrate rumen clearance were 0.51, 0.56, and 0.99, respectively. In a second experiment, the ¹³C-valerate fractional clearance rate estimate (33.7%/h) was similar to the estimate obtained with unlabeled valerate (35.0%/h) by the valerate-Co technique. No ¹³C enrichment of rumen microbes was noted 4 h after the intraruminal infusion of ¹³C-valerate. Fractional VFA absorption rate estimates obtained in both techniques were similar, although both were lower than estimates reported in the literature by other methods.     

Short-chain fatty acid content and pH in caecum of rats given various sources of carbohydrates

The caecal content of short-chain fatty acids (SCFA; acetic, propionic and butyric acid), caecal pH, fermentability and dry matter digestibility (DMD) were examined through balance experiments in rats fed 11 various indigestible carbohydrates. The following carbohydrate sources were incorporated into test diets: cellulose, oat husk, wheat bran, oat bran, pea fibre, linseed fibre, low methoxylated (LM)-pectin, guargum, β-glucans, neosugar and raffinose. The indigestible carbohydrates, except for those in wheat bran, oat husk and cellulose, were highly fermented, ie > 90%. Caecal pH varied between 5·6 and 7·8, with neosugar and raffinose causing the lowest pH and the fibre-free diet and the diet with oat husk the highest. The caecal pool sizes of SCFA were highest with raffinose, β-glucans, LM-pectin, guargum and linseed fibre (335-400 μmol) while pea fibre, wheat bran, oat bran and neosugar gave intermediate levels (137–227 μmol). The pool size with oat husk and cellulose was similar as with the basal diet (45–64 μmol). A high proportion of propionic acid was obtained with guargum and linseed fibre, whereas acetic acid was the predominant product in case of LM-pectin. On the other hand, linseed fibre gave a remarkably low proportion of butyric acid. The quantity fermented and caecal pH correlated well to the amount of SCFA with most materials (r = 0·96 and r = ?0·87, respectively), an exception was neosugar and in case of fermentability also oat bran. DMD values with most of the easily fermented carbohydrates were high (>96%). Exceptions were diets with β-glucans and oat bran which caused low DMD values, about 93%. It is concluded that indigestible carbohydrates may differ in ability to lower caecal pH and to form SCFA during fermentation.     

Using artificial neural networks to predict pH,ammonia, and volatile fatty acid concentrations in the rumen

The objectives of this study were (1) to predict ruminal pH and ruminal ammonia and volatile fatty acid (VFA) concentrations by developing artificial neural networks (ANN) using dietary nutrient compositions, dry matter intake, and body weight as input variables; and (2) to compare accuracy and precision of ANN model predictions with that of a multiple linear regression model (MLR). Data were collected from 229 published papers with 938 treatment means. The data set was randomly split into a training data set containing 70% of the observations and a test data set with the remaining observations. A series of ANN with a range of 1 to 9 artificial neurons in 1 hidden layer were examined, and the best one was selected to compare with the best-fitted MLR model. The performance of model predictions was evaluated by root mean square errors (RMSE) and concordance correlation coefficients (CCC) using cross-evaluations with 100 iterations. When using the ANN to predict ruminal pH and concentrations of ammonia, total VFA, acetate, propionate, and butyrate, the RMSE were 4.2, 41.4, 20.9, 22.3, 32.9, and 29.7% of observed means, respectively. The RMSE for the MLR were 4.2, 37.8, 18.3, 19.9, 29.8, and 26.6% of the observed means. The CCC for ruminal pH, ruminal concentrations of ammonia, total VFA, acetate, propionate, and butyrate were 0.57, 0.49, 0.45, 0.40, 0.52, and 0.40, using the ANN, and 0.37, 0.48, 0.40, 0.29, 0.43, and 0.35, using the MLR. Evaluations of the MLR and the ANN indicated that these 2 model forms exhibited similar prediction errors, with 4.2, 39.6, 19.6, 21.1, 31.3, and 28.1% of observed means for pH, ammonia, total VFA, acetate, propionate, and butyrate. Although the ANN increased the precision of predictions related to ruminal metabolism, it failed to improve the accuracy compared with the linear regression model.     

Effect of a monensin controlled-release capsule on rumen and blood metabolites in Florida Holstein transition cows

The objective of this study was to determine the effect of a monensin controlled-release capsule administered intraruminally at dry-off on rumen volatile fatty acids, NH3, lactate, pH, and energy blood metabolites in transition dairy cows fed Florida typical diets. In March 2003, 24 cows (10 primiparous and 14 multiparous) dried-off 50 to 70 d before expected parturition were randomly assigned to a treatment (n=12, oral capsule of monensin) or a control group (n=12, no capsule). Both groups received the same diet and were exposed to the same environment and management conditions. At assignment, at 21 d before expected parturition, at calving, and at 7, 14, and 21 d postpartum, blood samples were taken and body condition scores were determined. At 10 d postpartum, rumen and blood samples were obtained in the morning before the first feeding and at 2, 4, and 6 h after feeding. Serum nonesterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA), and glucose were measured. Rumen samples were analyzed for concentrations of acetic, propionic, butyric, L- and D-lactic acids, and NH3. Data for rumen and blood metabolites were analyzed by ANOVA, mixed models for repeated measures. Volatile fatty acids were not different between groups. Multiparous treated cows had a significant reduction in rumen NH3 at 6 h after feeding. Treatment with monensin significantly increased body condition score at calving in multiparous cows. During the postpartum period, NEFA and BHBA were noticeably lower in treated than in control primiparous cows. This difference was not observed in multiparous cows.