Subacute ruminal acidosis is induced by high concentrations of short-chain fatty acids (SCFA, mainly acetate, propionate, and butyrate) that release protons to decrease the pH of the ruminal digesta. This low pH, in turn, is thought to damage epithelial barrier function. The present study applied a model of simulated ruminal acidosis ex vivo to investigate if SCFA directly contribute to epithelial barrier failure beyond their role as proton donors. Epithelial tissues from the rumen of slaughtered sheep were mounted in Ussing chambers and incubated under 3 different conditions. Two groups were incubated in the absence of SCFA at mucosal pH 6.1 (control) and pH 5.1, respectively, for 7 h. A third group was first incubated in a mucosal solution containing 100 mM SCFA at pH 5.1 for 2 h and, thereafter, in a mucosal solution without SCFA at pH 6.1 for the remaining 5 h. Transepithelial conductance (G), short-circuit current (I), and fluorescein fluxes were determined. After 7 h of incubation, the expression levels of claudin-1, claudin-4, claudin-7, and occludin were measured by quantitative reverse-transcription PCR and Western blot. Furthermore, the local distribution of these tight junction (TJ) proteins was examined by confocal laser scanning microscopy. A 7-h incubation at pH 5.1 in the absence of SCFA did not influence either G or fluorescein flux rates of ruminal tissues ex vivo compared with the control. In contrast, incubation at pH 5.1 with SCFA for only 2 h induced increases in G and fluorescein flux rates that continued even after tissues were returned back to pH 6.1. Expression analysis showed that pH 5.1 without SCFA for 7 h induced no changes in mRNA expression of claudin-1, claudin-4, claudin-7, and occludin and a selective decrease in protein expression of only claudin-4 compared with the control. However, a 2-h incubation at pH 5.1 in the presence of SCFA decreased the mRNA-expression of claudin-7, as well as the protein expression of claudin-4, claudin-7, and occludin. The decreased expression of these TJ proteins in the group incubated with SCFA was also evident in immunohistochemistry. Immunohistochemistry additionally evidenced a considerable retraction of all tested TJ proteins out of the TJ in that group. We conclude that a low mucosal pH of 5.1 is tolerated well by ruminal epithelia for several hours. However, a low pH in combination with SCFA induces damage to the TJ and disturbs barrier function, which is not immediately reversible upon the removal of the acidotic insult.
The aim of the study was to investigate the influence of oral rehydration solutions (ORS) on milk clotting, abomasal pH, electrolyte concentrations, and osmolality, as well as on the acid-base status in blood of suckling calves, as treatment with ORS is the most common therapy of diarrhea in calves to correct dehydration and metabolic acidosis. Oral rehydration solutions are suspected to inhibit abomasal clotting of milk; however, it is recommended to continue feeding cow's milk or milk replacer (MR) to diarrheic calves to prevent body weight losses. Three calves with abomasal cannulas were fed MR, MR-ORS mixtures, or water-ORS mixtures, respectively. Samples of abomasal fluid were taken before and after feeding at various time points, and pH, electrolyte concentrations, and osmolality were measured. The interference of ORS with milk clotting was examined in vivo and in vitro. To evaluate the effects of ORS on systemic acid-base status, the Stewart variables strong ion difference ([SID]), acid total ([A(tot)]), and partial pressure of CO2 (pCO2) were quantified in venous blood samples drawn before and after feeding. Calves reached higher abomasal pH values when fed with MR-ORS mixtures than when fed MR. Preprandial pH values were re-established after 4 to 6 h. Oral rehydration solutions prepared in water increased the abomasal fluid pH only for 1 to 2 h. Oral rehydration solutions with high [SID(3)] ([Na(+)] + [K(+)] - [Cl(-)]) values produced significantly higher abomasal pH values and area under the curve data of the pH time course. Caseinomacropeptide, an indicator of successful enzymatic milk clotting, could be identified in every sample of abomasal fluid after feeding MR-ORS mixtures. The MR-ORS mixtures with [SID(3)] values > or =92 mmol/L increased serum [SID(3)] but did not change venous blood pH. Oral rehydration solutions do not interfere with milk clotting in the abomasum and can, therefore, be administered with milk. In this study, MR-ORS mixtures with high [SID(3)] values caused an increase of serum [SID(3)] in healthy suckling calves and may be an effective treatment for metabolic acidosis in calves suffering from diarrhea.
Ruminal fermentation products such as short-chain fatty acids (SCFA) and CO2 acutely stimulate urea transport across the ruminal epithelium in vivo, whereas ammonia has inhibitory effects. Uptake and signaling pathways remain obscure. The ruminal expression of SLC14a1 (UT-B) was studied using polymerase chain reaction (PCR). The functional short-term effects of ammonia on cytosolic pH (pHi) and ruminal urea transport across native epithelia were investigated using pH-sensitive microelectrodes and via flux measurements in Ussing chambers. Two variants (UT-B1 and UT-B2) could be fully sequenced from ovine ruminal cDNA. Functionally, transport was passive and modulated by luminal pH in the presence of SCFA and CO2, rising in response to luminal acidification to a peak value at pH 5.8 and dropping with further acidification, resulting in a bell-shaped curve. Presence of ammonia reduced the amplitude, but not the shape of the relationship between urea flux and pH, so that urea flux remained maximal at pH 5.8. Effects of ammonia were concentration dependent, with saturation at 5 mmol/l. Clamping the transepithelial potential altered the inhibitory potential of ammonia on urea flux. Ammonia depolarized the apical membrane and acidified pHi, suggesting that, at physiological pH (< 7), uptake of NH4 (+) into the cytosol may be a key signaling event regulating ruminal urea transport. We conclude that transport of urea across the ruminal epithelium involves proteins subject to rapid modulation by manipulations that alter pHi and the cytosolic concentration of NH4 (+). Implications for epithelial and ruminal homeostasis are discussed.
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