Gotthold Gäbel

RUMINANT NUTRITION SYMPOSIUM: Role of fermentation acid absorption in the regulation of ruminal pH12

Highly fermentable diets are rapidly converted to organic acids [i.e., short-chain fatty acids (SCFA) and lactic acid] within the rumen. The resulting release of protons can constitute a challenge to the ruminal ecosystem and animal health. Health disturbances, resulting from acidogenic diets, are classified as subacute and acute acidosis based on the degree of ruminal pH depression. Although increased acid production is a nutritionally desired effect of increased concentrate feeding, the accumulation of protons in the rumen is not. Consequently, mechanisms of proton removal and their quantitative importance are of major interest. Saliva buffers (i.e., bicarbonate, phosphate) have long been identified as important mechanisms for ruminal proton removal. An even larger proportion of protons appears to be removed from the rumen by SCFA absorption across the ruminal epithelium, making efficiency of SCFA absorption a key determinant for the individual susceptibility to subacute ruminal acidosis. Proceeding initially from a model of exclusively diffusional absorption of fermentation acids, several protein-dependent mechanisms have been discovered over the last 2 decades. Although the molecular identity of these proteins is mostly uncertain, apical acetate absorption is mediated, to a major degree, via acetate-bicarbonate exchange in addition to another nitrate-sensitive, bicarbonate-independent transport mechanism and lipophilic diffusion. Propionate and butyrate also show partially bicarbonate-dependent transport modes. Basolateral efflux of SCFA and their metabolites has to be mediated primarily by proteins and probably involves the monocarboxylate transporter (MCT1) and anion channels. Although the ruminal epithelium removes a large fraction of protons from the rumen, it also recycles protons to the rumen via apical sodium-proton exchanger, NHE. The latter is stimulated by ruminal SCFA absorption and salivary Na(+) secretion and protects epithelial integrity. Finally, SCFA absorption also accelerates urea transport into the rumen, which via ammonium recycling, may remove protons from rumen to the blood. Ammonium absorption into the blood is also stimulated by luminal SCFA. It is suggested that the interacting transport processes for SCFA, urea, and ammonia represent evolutionary adaptations of ruminants to actively coordinate energy fermentation, protein assimilation, and pH regulation in the rumen.

Microbial butyrate and its role for barrier function in the gastrointestinal tract

Butyrate production in the large intestine and ruminant forestomach depends on bacterial butyryl‐CoA/acetate‐CoA transferase activity and is highest when fermentable fiber and nonstructural carbohydrates are balanced. Gastrointestinal epithelia seem to use butyrate and butyrate‐induced endocrine signals to adapt proliferation, apoptosis, and differentiation to the growth of the bacterial community. Butyrate has a potential clinical application in the treatment of inflammatory bowel disease (IBD; ulcerative colitis). Via inhibited release of tumor necrosis factor α and interleukin 13 and inhibition of histone deacetylase, butyrate may contribute to the restoration of the tight junction barrier in IBD by affecting the expression of claudin‐2, occludin, cingulin, and zonula occludens poteins (ZO‐1, ZO‐2). Further evaluation of the molecular events that link butyrate to an improved tight junction structure will allow for the elucidation of the cofactors affecting the reliability of butyrate as a clinical treatment tool.

Transfer of energy substrates across the ruminal epithelium: implications and limitations

Abstract The ruminal epithelium has an enormous capacity for the absorption of short-chain fatty acids (SCFAs). This not only delivers metabolic energy to the animal but is also an essential regulatory mechanism that stabilizes the intraruminal milieu. The epithelium itself, however, is endangered by the influx of SCFAs because the intracellular pH (pHi) may drop to a lethal level. To prevent severe cytosolic acidosis, the ruminal epithelium is able to extrude (or buffer) protons by various mechanisms: (i) a Na+/H+ exchanger, (ii) a bicarbonate importing system and (iii) an H+/monocarboxylate cotransporter (MCT). Besides pHi regulation, the MCT also provides the animal with ketone bodies derived from the intraepithelial breakdown of SCFAs. Ketone bodies, in turn, can serve as an energy source for extrahepatic tissues. In addition to SCFA uptake, glucose absorption has recently been identified as a potential way of eliminating acidogenic substrates from the rumen. At least with respect to SCFAs, absorption rates can be elevated when adapting animals to energy-rich diets. Although they are very effective under physiological conditions, the absorptive and regulatory mechanisms of the ruminal epithelium also have their limits. An increased number of protons during the state of ruminal acidosis can be eliminated neither from the lumen nor the cytosol, thus worsening dysfermentation and finally leading to functional and morphological alterations of the epithelial lining.

Epithelial Capacity for Apical Uptake of Short Chain Fatty Acids Is a Key Determinant for Intraruminal pH and the Susceptibility to Subacute Ruminal Acidosis in Sheep

Subacute ruminal acidosis (SARA) is a common digestive disorder occurring in ruminants, with considerable variation in the severity of SARA observed among animals fed the same diet. Our aim in this study was to determine whether differences in the capacity of the ruminal epithelium for the apical uptake of acetate and butyrate (determined in Ussing chambers after slaughter) explains differences observed for the severity of a preceding episode of SARA in vivo. Adult sheep with an indwelling small ruminant ruminal pH measurement system (SRS) were randomly assigned to either a SARA induction treatment (oral drench containing 5 g glucose/kg body weight; n = 17) or a sham treatment (SHAM; n = 7; 12 mL water/kg body weight). Sheep receiving the glucose drench were further classified as nonresponders (NR; n = 7) or responders (RES; n = 7) according to their ruminal pH profile for the 3 h following the oral drench. Mean ruminal pH for the 3 h following the drench differed among groups (P < 0.001), with it being highest for SHAM (6.67 +/- 0.08), intermediate for NR (5.97 +/- 0.05), and lowest for RES (5.57 +/- 0.08) sheep. The apical uptake of acetate and butyrate did not differ between SHAM and RES sheep. However, NR sheep had greater in vitro apical uptake of acetate and butyrate and a higher plasma beta-hydroxybutyrate concentration than RES sheep, suggesting greater absorptive capacity for NR. Differences between NR and RES were attributed to greater bicarbonate-independent, nitrate-sensitive uptake of acetate (P = 0.007), a tendency for greater bicarbonate-dependent uptake of acetate (P = 0.071), and greater bicarbonate-independent uptake of butyrate (P = 0.022). These data indicate that differences in the rates and pathways for the uptake of acetate and butyrate explain a large proportion of the individual variation observed for the severity of SARA.

Bicarbonate-dependent and bicarbonate-independent mechanisms contribute to nondiffusive uptake of acetate in the ruminal epithelium of sheep

The present study investigated the significance of apical transport proteins for ruminal acetate absorption and their interaction with different anions. In anion competition experiments in the washed reticulorumen, chloride disappearance rate (initial concentration, 28 mM) was inhibited by the presence of a short-chain fatty acid mixture (15 or 30 mM of each acetate, propionate, and butyrate). Disappearance rates of acetate and propionate, but not butyrate (initial concentration, 25 mM each) were diminished by 40 or 80 mM chloride. In isolated ovine ruminal epithelia mounted in Ussing chambers, an increase in chloride concentration from 4.5 to 90 mM led to a decrease of apical acetate uptake at a concentration of 0.5 mM. Mucosal nitrate inhibited acetate uptake most potently whereas sulfate had no effect. Decreasing mucosal pH from 7.4 to 6.1 approximately doubled uptake of acetate both at 0.5 and 10 mM, but this doubling was almost abolished when HCO(3)(-) was absent. The stimulated uptake at mucosal pH 6.1 consisted of a bicarbonate-dependent, nitrate-inhibitable part (K(m) = 54 mM) and a bicarbonate-independent component (K(m) = 12 mM) that was also sensitive to nitrate inhibition. Maximal uptake was three times larger for bicarbonate-dependent vs. bicarbonate-independent uptake. Mucosal addition of 200 microM DIDS, 400 microM p-chloromercuribenzene sulfonic acid, 800 microM p-hydroxymercuribenzoic acid, or 100 microM phloretin had no effects on acetate uptake although the latter two inhibited l-lactate uptake. Our data conclusively show a dominant involvement of proteins in apical acetate uptake. Previously described pH effects on acetate absorption originate mainly from modulation of acetate/bicarbonate exchange. Additionally, there is bicarbonate-independent uptake of acetate anions that is protein coupled but not via monocarboxylate cotransporter.

A single mild episode of subacute ruminal acidosis does not affect ruminal barrier function in the short term.

Twenty-four German Merino sheep (72.3±10.1 kg of body weight) were fed an all-hay diet and assigned to either the subacute ruminal acidosis (SARA) treatment (n=17) or sham treatment (n=7). The SARA sheep were orally dosed with a 2.2 M glucose solution to supply 5 g of glucose/kg of body weight, whereas sham sheep received an equal volume of water. Ruminal pH was measured for 48 h before and 3 h after the oral dose. Sheep were then killed and ruminal epithelia from the ventral sac were mounted in Ussing chambers. The serosal-to-mucosal flux rate of partially (3)H-labeled mannitol (J(mannitol-SM)), an indicator of barrier function, was measured while epithelia were exposed to 3 sequential in vitro measurement periods lasting 1 h each. The measurement periods consisted of baseline, challenge, and recovery periods and were interspersed by 30-min periods for treatment equilibration. Baseline conditions were pH 6.1 (mucosal solution) and pH 7.4 (serosal solution) with a bilateral osmolarity of 293 mOsm/L. During the challenge period, the mucosal side of the epithelia was exposed to either an acidotic challenge (pH 5.2, osmolarity 293 mOsm/L) or an osmotic challenge (pH 6.1, osmolarity 450 mOsm/L); a third group served as control (pH 6.1, osmolarity 293 mOsm/L). The mucosal buffer solution was replaced for the recovery period. In vivo, sheep on the SARA treatment had lower mean (5.77 vs. 6.67) and nadir (5.48 vs. 6.47) ruminal pH for the 3h following the oral drench compared with sham sheep, indicating the successful induction of SARA with the oral glucose dose. Despite the marked reduction in pH in vivo, induction of SARA had no detectable effects on the baseline measurements of J(mannitol-SM), tissue conductance (G(t)), and short-circuit current (I(sc)) in vitro. However, reducing mucosal pH to 5.2 in vitro had negative effects on epithelial barrier function in the recovery period, including increased J(mannitol-SM), increased G(t), and decreased I(sc). The osmotic challenge increased J(mannitol-SM) and G(t) and decreased I(sc) during the challenge period, which was reversible in the recovery period except for slight reduction in I(sc). Interactions between the in vitro treatment and measurement period were detected for J(mannitol-SM), G(t), and I(sc). These data indicate that a mild episode of SARA (nadir pH, 5.48; duration ruminal pH <5.8, 111 min relative to the 180-min measurement period) does not affect ruminal epithelial barrier function immediately after the episode but that a rapid and more severe acidification (pH 5.2) in vitro increases epithelial permeability following the insult.

Increased papillae growth and enhanced short-chain fatty acid absorption in the rumen of goats are associated with transient increases in cyclin D1 expression after ruminal butyrate infusion

We tested the hypothesis that the proliferative effects of intraruminal butyrate infusions on the ruminal epithelium are linked to upregulation in cyclin D1 (CCND1), the cyclin-dependent kinase 4 (CDK4), and their possible association with enhanced absorption of short-chain fatty acids (SCFA). Goats (n=23) in 2 experiments (Exp.) were fed 200 g/d concentrate and hay ad libitum. In Exp. 1, goats received an intraruminal infusion of sodium butyrate at 0.3 (group B, n=8) or 0 (group C, n=7) g/kg of body weight (BW) per day before morning feeding for 28 d and were slaughtered 8 h after the butyrate infusion. In Exp. 2, goats (n=8) received butyrate infusion and feeding as in Exp. 1. On d 28, epithelial samples were biopsied from the antrium ruminis at 0, 3, and 7 h after the last butyrate infusion. In Exp. 1, the ruminal molar proportional concentration of butyrate increased in group B by about 110% after butyrate infusion and remained elevated for 1.5 h; thereafter, it gradually returned to the baseline (preinfusion) level. In group C, the molar proportional concentration of butyrate was unchanged over the time points. The length and width of papillae increased in B compared with C; this was associated with increased numbers of cells and cell layers in the epithelial strata and an increase in the surface area of 82%. The mRNA expression of CCND1 increased transiently at 3 h but returned to the preinfusion level at 7 h following butyrate infusion in Exp. 2. However, it did not differ between B and C in Exp. 1, in which the ruminal epithelium was sampled at 8 h after butyrate infusion. The mRNA expression of the monocarboxylate transporter MCT4, but not MCT1, was stably upregulated in B compared with C. The estimated absorption rate of total SCFA (%/h) increased in B compared with C. We conclude that transient increases in cyclin D1 transcription contribute to butyrate-induced papillae growth and subsequently to the increased absorption of SCFA in the ruminal epithelium of goats.

Role of Na+/ H+ exchange and HCO3− transport in pHi recovery from intracellular acid load in cultured epithelial cells of sheep rumen

Abstract This study sought to investigate effects of short-chain fatty acids and CO2 on intracellular pH (pHi) and mechanisms that mediate pHi recovery from intracellular acidification in cultured ruminal epithelial cells of sheep. pHi was studied by spectrofluorometry using the pH-sensitive fluorescent indicator 2′,7′-bis (carboxyethyl)-5(6′)-carboxyfluorescein acetoxymethyl ester (BCECF/AM). The resting pHi in N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-buffered solution was 7.37 ± 0.03. In HEPES-buffered solution, a NH4+/NH3-prepulse (20 mM) or addition of butyrate (20 mM) led to a rapid intracellular acidification (P < 0.05). Addition of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 μM) or HOE-694 (200 μM) inhibited pHi recovery from an NH4+/NH3-induced acid load by 58% and 70%, respectively. pHi recovery from acidification by butyrate was reduced by 62% and 69% in the presence of EIPA (10 μM) and HOE-694 (200 μM), respectively. Changing from HEPES- (20 mM) to CO2/HCO3−-buffered (5%/20 mM) solution caused a rapid decrease of pHi (P < 0.01), followed by an effective counter-regulation. 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 100 μM) blocked the pHi recovery by 88%. The results indicate that intracellular acidification by butyrate and CO2 is effectively counter-regulated by an Na+/H+ exchanger and by DIDS-sensitive, HCO3−-dependent mechanism(s). Considering the large amount of intraruminal weak acids in vivo, both mechanisms are of major importance for maintaining the pHi homeostasis of ruminal epithelial cells.

SCFA Transport in the Forestomach of Ruminants

Short-chain fatty acids are the main end-products of microbial metabolism in the forestomach of ruminants. SCFA produced by the microorganisms are rapidly absorbed across forestomach epithelia and can cover up to 80% of the energy requirement of the animal. Although there is a great concentration gradient for SCFA between the forestomach content and the blood favoring passive transport, (secondary) active transport mechanisms are likely involved in SCFA permeation across the epithelia. (Secondary) active SCFA transport seems to be mediated by an anionic exchange system. The system interacts with other anions like chloride and bicarbonate. Similar to the large intestine of various species, SCFA can stimulate sodium transport probably by activating a Na+/H+ exchange located in the apical membrane. However, in contrast to the large intestine, SCFA transport itself seems to be independent from sodium. Part of the absorbed SCFA does not reach the blood side in the original form because it is metabolized in the epithelial cell. Metabolism, in turn, influences SCFA transport.

Portal recovery of short-chain fatty acids infused into the temporarily- isolated and washed reticulo-rumen of sheep

The present study was undertaken to study the metabolism of short-chain fatty acids (SCFA) by the reticulo-ruminal epithelium and the portal-drained viscera (PDV) under in vivo conditions with no interference from the metabolism of the rumen microbes. The technique of temporary isolation of the reticulo-rumen was applied to wethers implanted with catheters in a mesenteric artery, the hepatic portal vein and the right ruminal vein. Portal blood flow was measured by downstream dilution of p-aminohippuric acid; the PDV uptake of arterial acetate, as well as the whole-body irreversible loss rate (ILR) of acetate, was estimated by [2-(13)C]acetate infusion into the right ruminal vein. The sheep were maintained with a bicarbonate-buffered solution of SCFA in the reticulo-rumen along with continuous intraruminal infusion of SCFA for 4 h. The portal appearance of SCFA of non-reticulo-ruminal origin was estimated before and after the infusion protocol. Of the acetate absorbed by the sheep, 89 (SE 5), 109 (SE 7) and 101 (SE 7)% was recovered as portal net appearance of acetate, portal net appearance of acetate corrected for PDV uptake of arterial acetate and increase in the ILR of acetate respectively. Of the propionate, isobutyrate, butyrate, isovalerate and valerate absorbed by the sheep, 95 (SE 7), 102 (SE 9), 23 (SE 3), 48 (SE 5) and 32 (SE 4)% respectively was recovered as portal net appearance. In contrast to current concepts, the present study showed that the reticulo-ruminal epithelium metabolizes none (or only a small proportion) of the acetate and propionate absorbed from the rumen. This observation could lead to the more efficient use of results obtained with multi-catheterized animals to quantify the net metabolite output of the rumen microbes.