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The effect of increasing concentrations of short-chain fatty acids [SCFA; mixture of the Na+ salts of acetic acid (62.5%), propionic acid (25.0%) and of butyric acid (12.5%)] on Na+ transport of sheep rumen epithelium was studied in vitro. The conventional Ussing chamber method was used for measuring Na+ transport rates (22Na+), short-circuit current (Isc) and tissue conductance (GT) of isolated rumen epithelium. SCFA in the buffer solution on the mucosal side caused a linear increase of Jnet Na+ from 1.14, to 1.22, 1.78 and 2.50 microeq/cm2/h in hay-fed sheep at 0, 15, 40 and 80 mmol/l SCFA, respectively. In a second study, the effect of higher SCFA concentrations [0 (control), 80, 100 and 120 mmol/l] was investigated with epithelia from two groups of sheep. One group was subjected to hay ad libitum, whereas the other received concentrate feed (800 g/day in equal portions at 7.00 am and 3.00 pm) and hay ad libitum. Epithelia from concentrate-fed sheep again showed a significant (p < 0.05) and linear increase in Jnet Na+ at 80, 100 and 120 mmol/l. However, in hay-fed sheep, the difference in increase among 80, 100 and 120 mmol/l SCFA was not significant, indicating that, above 80 mmol/l SCFA Jms and Jnet exhibit saturation. Moreover, Na+ fluxes (Jms and Jnet) were generally higher in concentrate-fed than in hay-fed sheep at all SCFA concentrations and significant differences were observed at 100 and 120 mmol/l SCFA. The obtained results confirm the effect of SCFA on Na+ transport and are in agreement with studies regarding feeding regimes and electrolyte transport in the rumen. The important new observation is the increase of Na+ transport in concentrate-fed sheep even at high concentrations of SCFA (100 and 120 mmol/l). The enhanced activity of the Na+/H+ exchanger at these SCFA concentrations supports the assumption that the capacity for regulating the intracellular pH by extrusion of protons is increased, suggesting an adaptation in concentrate-fed sheep. This adaptation could prevent possible disturbances of epithelial functions (transport and barrier) under conditions of increased SCFA absorption.