The potential and short-circuit current across isolated rumen epithelium of the sheep.

1. A technique is described for working with isolated sheets of rumen epithelium. Epithelium was obtained at operations for rumen fistulation or in acute experiments and was rapidly mounted between two cone-shaped chambers each of which held 40 ml. of bathing fluid. The fluids were gassed with 95% O2/5% CO2 and 20 cm2 of epithelium was exposed to the fluids. 2. Electrical measurements were made of (a) the potential generated across the epithelium, (b) the short-circuit current which flowed and (c) the resistance of the epithelium. 3. The results are grouped according to the composition of the sheep Ringer solution used to bathe the epithelium. The most stable preparation was obtained when the solutions contained 5 mM each of glucose, acetate, propionate and butyrate as well as bicarbonate and phosphate buffers. 4. In Cl- media, the average initial p.d., short-circuit current (s.-c.c.) and resistance were 9.3 mV, 11.6 microA/cm2 and 1156 omega x cm2 respectively. Higher values of 17.6 mV, 17.3 microA/cm2 and 1501omega x cm2 were recorded in SO4(2-) media. In both media, the s.-c.c. declined at approximately 1.5 microA/cm2. hr and preparations were suitable for study over periods from 4 to 6 hr. 5. When the concentration of potassium was varied on either side of the epithelium at constant [Na+], the potential showed a linear relation with log [K+] for both sides of the epithelium. 6. Changing the sodium concentrations at constant [K+] on either side of the epithelium caused negligible alterations in the potential. 7. Exclusion of sodium from the media bathing both sides of the epithelium abolished the potential and s.-c.c. 8. The addition of ouabain to the blood side of the preparation abolished the potential and s.-c.c. whereas on the rumen side these parameters were unaffected.     

The effect of acetzolamide on Ion transport across isolated sheep rumen epithelium.

1. The net fluxes of sodium and chloride from the lumen to the blood side of isolated sheep rumen epithelium were reduced by treatment of both sides of the epithelium with acetazolamide. 2. The changes in the net fluxes of sodium and chloride were significantly correlated and showed recovery after removal of acetazolamide. 3. The net flux of potassium from blood to lumen side of the epithelim was not affected by treatment with acetazolamide. 4. It is suggested that acetazolamide blocks coupled sodium and chloride transport which may be mediated through 'low-activity' carbonic anhydrase enzymes.     

The effect of ouabain on ion transport across isolated sheep rumen epithelium.

1. The net flux of Na from the lumen to the blood side of isolated sheep rumen epithelium was reduced to zero in the third 30 min period after treatment with ouabain. 2. The net flux of K from blood to lumen side of the epithelium was reduced from 0-34 to 0-13 mumole/cm2. hr in the third 30 min period after ouabain treatment. 3. The net flux of Cl from lumen to blood side of the epithelium was reduced from 1-4 to 0-72 mumole/cm2. hr in the third 30 min period after ouabain treatment. 4. The results support the existence of a Cl pump independent of Na transport and also a K pump in sheep rumen epithelium.     

Influence of short-chain fatty acids on ammonia absorption across the rumen wall in sheep.

The effects of short-chain fatty acids (SCFA) on ammonia net absorption from the sheep rumen in vivo and on ammonia transport across rumen wall mucosa in vitro were studied. Ammonia net absorption was directly, though in a non-linear manner, correlated with the SCFA concentration in the artificial rumen fluid. Almost 70% of total ammonia absorption was dependent upon the presence of SCFA when 12 mmol l-1 ammonia and 67.5 mmol l-1 SCFA were present. Lactic acid was ineffective. Incubation experiments showed that mucosal disappearance and serosal appearance of ammonia were reduced by 38% and 32%, respectively, when SCFA (63 mmol l-1) were replaced by lactic acid. The SCFA effect was independent of the type of SCFA used. In part of the experiments up to 54% of the ammonia taken up by the tissue was not recovered in the serosal incubation solution and must have been metabolized in the mucosa.     

Ion transport across an isolated preparation of sheep rumen epithelium

1. The fluxes of isotopically labelled sodium, potassium and chloride passing in each direction across isolated sheets of rumen epithelium from the sheep have been measured under short-circuit conditions.2. With both sides of the epithelium bathed in chloride Ringer the mean sodium fluxes were 2.85 mumole/cm(2).hr from rumen to blood and 1.28 mumole/cm(2).hr in the reverse direction. In sulphate Ringer the sodium fluxes were 1.64 mumole/cm(2).hr from rumen to blood and 0.54 mumole/cm(2).hr from blood to rumen.3. In chloride Ringer the mean potassium fluxes were 0.18 mumole/cm(2).hr from rumen to blood and 0.54 mumole/cm(2).hr from blood to rumen. In sulphate Ringer the potassium fluxes were 0.07 mumole/cm(2).hr from rumen to blood and 0.35 mumole/cm(2).hr from blood to rumen.4. In chloride Ringer the mean chloride fluxes were 4.89 mumole/cm(2).hr from rumen to blood and 3.78 mumole/cm(2).hr from blood to rumen.5. In chloride Ringer the mean value of the short-circuit current was 13 muA/cm(2), corresponding to a flux of 0.49 muequiv/cm(2).hr. When sulphate was substituted for chloride, the short-circuit current was increased by about 40%, and the net flux of sodium from rumen to blood fell by 30%.6. Neither the sodium nor the chloride fluxes changed significantly when the epithelium was temporarily open-circuited.     

Mechanism of electrically silent Na and Cl transport across the rumen epithelium of sheep

This study was designed to study the mechanism of electroneutral Na and Cl transport across the isolated rumen epithelium of sheep. Net sodium transport (5.75 +/- 0.35 microequiv cm-2 h-1) was significantly higher than the short-circuit current (0.95 +/- 0.08 microequiv cm-2 h-1). Both, net sodium and net chloride transport were markedly reduced by replacement of chloride, bicarbonate and sodium, respectively, but were not changed by the absence of mucosal potassium. Net sodium and net chloride absorption was significantly decreased by 1.0 mM-amiloride. Mucosal addition of bumetanide, furosemide, hydrochlorothiazide or low concentrations of amiloride (less than 0.1 mM) did not change sodium fluxes. These results provide compelling evidence consistent with the presence of Na-H exchange as the predominant electroneutral mechanism for transepithelial sodium movement. The ion replacement studies and data from literature suggest that the Na-H exchange is working in parallel with a Cl-HCO3 exchange although luminal addition of DIDS (4,4'diisothiocyanatostilbene-2,2'-disulphonate, 1 mM) did not significantly influence Cl transport.     

Na(+) transport across rumen epithelium of hay-fed sheep is acutely stimulated by the peptide IGF-1 in vitro

An energy-rich diet leads to enhanced ruminal Na(+) absorption, which is associated with elevated plasma insulin-like growth factor 1 (IGF-1) levels and an increased number of IGF-1 receptors in rumen papillae. This study examined the in vitro effect of IGF-1 on Na(+) transport across the rumen epithelium of hay-fed sheep, in which the IGF-1 concentration in plasma is lower than in concentrate-fed animals. At concentrations ranging from 20 to 100 μg l(-1), serosal LR3-IGF-1, a recombinant analogue of IGF-1, rapidly (within 30 min) stimulated the mucosal-to-serosal Na(+) flux (J(ms)Na) and consequently the net Na(+) flux (J(net)Na). Compared with controls, J(net)Na increased by about 60% (P < 0.05) following the serosal application of LR3-IGF-1 (20 μg l(-1)). The IGF-1-induced increment of J(ms)Na and J(net)Na was inhibited by mucosal amiloride (1 mmol l(-1)). Neither IGF-1 nor amiloride altered tissue conductance or the short-circuit current of the isolated rumen epithelium. These data support the assumption that the stimulating effect of serosally applied IGF-1 on Na(+) transport across the rumen epithelium is mediated by Na(+)-H(+) exchange (NHE). A further study was performed with cultured rumen epithelial cells and a fluorescent probe (BCECF) to estimate the rate of pH(i) recovery after acid loading. The pH(i) of isolated rumen epithelial cells was 6.43 ± 0.15 after butyrate loading and recovered by 0.26 ± 0.02 pH units (15 min)(-1). Application of LR3-IGF-1 (20 μg l(-1)) significantly increased the rate of pH(i) recovery to 0.33 ± 0.02 pH units (15 min)(-1). Amiloride administration reduced the recovery rate in both control and IGF-1-stimulated cells. These results show, for the first time, that an acute effect of IGF-1 on Na(+) absorption across rumen epithelium results from increased NHE activity. Insulin-like growth factor 1 is thus important for the fast functional adaptation of ruminal Na(+) transport via NHE.     

Fluid absorption from the rumen during rehydration in sheep

Water movements across the rumen wall were studied during rehydration in four fistulated sheep. The animals were dehydrated for 48 h which increased total plasma protein, plasma osmolality, plasma Na+, arginine vasopressin and plasma renin activity. Two series of experiments were performed: Expt I with no food available during rehydration, and Expt II where the animals were fed hay. On the rehydration day, a fluid marker (cobalt-EDTA) was administered into the rumen. To avoid water outflow from the rumen a stopper was inserted into the reticulo-omasal orifice. When the animals were provided with water they immediately drank 9 l. The water offered contained the same marker concentration (Co2+) as in the rumen liquid. In Expt I, Co2+ concentration increased after drinking, and remained elevated until the stopper was removed. The highest value was obtained after 20 min, and this corresponded to at least a 11 water absorption in individual animals. In Expt II, the sheep immediately started to eat following drinking and the inflow of saliva caused a dilution of the marker. Plasma osmolality and Na+ concentration decreased in both experiments indicating that water absorption occurred in both experiments. Removal of the stopper did not cause any significant changes in the parameters measured. Vasopressin concentration fell immediately on the sight of water, and then continued to decrease. It is concluded that in the sheep, voluntary drinking is followed by an immediate and substantial absorption of water from the rumen.     

Transfer of R factors from Escherichia coli to salmonellas in the rumen of sheep.

Adult sheep were given into the rumen c. 10(8) cells each of donor strains of E. coli containing an R factor and prospective salmonella-recipient organisms and were maintained on a diet of lucerne chaff; the animals excreted the organisms, remained healthy, and no transfer of the R factor was detected. When the animals were starved for 48 h before inoculation, the ruminal environment was altered so that, on resumption of feeding, small numbers (c. 10(2)-10(4) cells--less than one cell per ml of rumen fluid) of the introduced organisms were able to multiply and reach sufficient numbers for the transfer of R factors to occur within the rumen. One animal, given 7-8 X 10(3) cells of recipient S. lomita after starvation for 48 h, became a carrier of this organism. A second animal, given 4-4 X 10(2) cells of S. typhimurium after starvation for 48 h, developed acute, fatal salmonellosis 5 days later; at the time of death, large numbers of salmonella organisms (c. 10(9) cells per g) were present in the faeces; these included many (c. 10(6) cells per g) that had received the R factor by transfer in vivo. These results indicate that short periods of starvation may enhance the transfer of R factors and possibly other plasmids between suitable micro-organisms in vivo, and may increase the susceptibility of animals to pathogenic micro-organisms.     

Observations on the pathogenesis of Chlamydia psittaci infection of pregnant sheep

Susceptible pregnant sheep were infected with Chlamydia psittaci at different stages of early and mid-gestation. Placentas and foetuses were examined by microbiological, serological, histopathological and immunochemical methods at various intervals thereafter. While ewes were susceptible to infection from early gestation onwards and infections of placentas and foetuses occurred from about 60 days gestation (dg), pathological changes were not detected until after 90 dg. These changes consisted of initial rapid replication of C. psittaci in the trophoblast at the limbus of the placentomal hilus, leading to local necrosis and contiguous spread of infection to involve the cotyledonary and intercotyledonary placenta and apposing endometrium. Foetal pathology consisted of focal necrosis in liver and other organs suggestive of embolic dissemination of infection from the placenta. An early foetal immune response was detected including the demonstration of IgM and IgG antibody to C. psittaci and quantitative evidence of a B cell response to infection. Pathogenetic mechanisms are discussed.     

Involvement of carbonic anhydrase in ammonia flux across rumen mucosa in vitro.

Carbonic anhydrase activity in mucosa of the sheep rumen wall was completely inhibited by acetazolamide and ethoxyzolamide. This inhibition significantly reduced ammonia flux across mucosal discs in vitro but was ineffective if short-chain fatty acids were present in the bathing solution. The findings are discussed in relation to the mechanism of ruminal ammonia absorption.     

The sequential contractions of the rumen associated with eructation in sheep

1. The orderly sequential movements of the reticulum and the rumen were studied in conscious sheep by electromyography using enamelled stainless-steel wires implanted in various regions of the stomach wall and by recording mechanical changes within the various parts of the organ. Electrical activity of the rumen and/or pressure changes were related to eructation when the animals were at rest, feeding or ruminating.2. Secondary contractions of the rumen were found to originate in the ventral blind sac immediately following a primary contraction or independently. The wave of contraction originating in the ventral blind sac was seen to pass in a circular manner to the dorsal blind sac, the dorsal sac, the ventral sac and finally once more to the ventral blind sac. Eructation occurs at the end of the contraction of the dorsal sac. In each case, the time required to initiate the secondary cycle depended on the strength of contraction of the ventral blind sac.3. Sustained gaseous distension elicited numerous secondary contractions of the rumen concurrent with a lower frequency of reticular contractions. Although some secondary contractions were incomplete, all began with contraction of the ventral blind sac and were associated with eructation.4. Chemical stimulation of the rumen by fatty acids at pH 5.5-5.9 increased the ratio of secondary to primary contractions of the rumen to a varying extent depending on their initial rate.5. It was concluded that the seemingly random occurrence of a secondary cycle of the rumen was dependent on the activity of the ventral blind sac and its pattern could be altered by both mechanical and chemical stimulation.     

Adrenergic influences on the electrical potential across the colonic mucosa of the rabbit.

1. An investigation was undertaken of the role of adrenergic influences on the transient change in colonic potential difference (p.d.) induced by handling in the rabbit. 2. P.d. was increased from 9-5 +/- 0-5 to 27-05 +/- 1-94 mV during a 2 hr period of handling and wrapping. Atropine and alpha-adrenergic block during this 2 hr period did not alter the magnitude of the response and the response in adrenalectomized animals was not significantly different from that of normal animals. 3. 'Total' adrenergic block and selective beta-adrenergic block abolished the change in p.d. with handling and infusion of an alpha-receptor agonist during the 2 hr period significantly reduced the response. 4. The changes in colonic p.d. with handling were greatly increased by the infusion of a beta-receptor agonist during the 2 hr test period. 5. The results indicate that the change in colonic p.d. with handling is a direct effect of the autonomic nervous system mediated via beta-receptors and that the changes were not related to the effects of circulating catecholamines or to changes in aldosterone production.