In a preliminary survey of the distribution and fate of phosphorus-containing compounds in the alimentary tract of the sheep it was decided to estimate the inorganic phosphate levels of the fluid of the various divisions of the stomach. Information on this topic is very scanty, the only figure for phosphorus in sheep rumen liquor which could be found in the literature being 87 mg. P/100ml. (Brünnich & Winks, 1931). The initial experiments showed that inorganic phosphate occurred in higher concentration in abomasal liquor than was anticipated, and this led to an examination of the levels of soluble calcium and magnesium. Calcium and magnesium were chosen because their fate in the alimentary tract is often closely linked with that of inorganic phosphate. Some observations on the composition of sheep gastric juice were also made as one of the animals available for this investigation had an abomasal pouch of the Hollander type.
(a) Fistulated sheep
The animals used were two Cheviot wethers (sheep nos. 1 and 2) and a Cheviot ewe (sheep no. 3). Animals nos. 1 and 2 were fitted with permanent ruminal and duodenal fistulae, the latter being immediately caudal to the pylorus. Sheep no. 3 had permanent fistulae in the rumen and abomasum, together with an abomasal pouch. The author wishes to thank Dr A. T. Phillipson who carried out the surgical operations on these animals and for allowing him to use sheep no. 3 in this investigation.
(b) Slaughtered sheep
All were adult animals ; nos. 4−7 were Cheviot ewes, nos. 8 and 9 (kindly placed at the author’s disposal by Dr J. Duckworth) were cross-Border Leicester ewes and no. 10 was a Suffolk cross-ewe.
(a) Fistulated sheep
Sheep nos. 1 and 2 received 450 g. chopped meadow hay twice a day (8 a.m. and 8 p.m.). For two 2 hr. periods each day the animals had access to water. They consumed all the food and drank about 1·75 1. of water per day. Sheep no. 3 received 450 g. chopped meadow hay twice a day (8 a.m. and 8 p.m.) and 225 g. of a 2 :1 mixture of linseed oil cake and oats at 11 a.m. each day. The animal had access to water all day. All three sheep were housed indoors in individual pens and had access to salt and mineral licks.
(b) Slaughtered sheep
The pasture-fed sheep which were slaughtered (sheep nos. 4−7) consumed a mixture of rye-grass and clover during the months of May to September. Sheep no. 8 had been receiving an almost calcium-free diet, consisting mainly of oats, chopped oat straw, ground maize and blood meal. Sheep no. 9 received the same diet with the addition of 2·5 g. CaCO3 per day. Sheep no. 10 consumed 1000 g. chopped meadow hay per day and had free access to water and a mineral lick.
(a) Fistulated sheep
Ruminal samples (usually about 50 ml.) were withdrawn by inserting a glass tube into the fistula and removing the contents by suction. Although the ruminal samples cannot be considered as representative of the total ruminal contents the method of sampling consistently gave material of a similar composition in respect of the percentage of dry matter. Duodenal and abomasal samples (usually about 50 ml.) were obtained by withdrawing the gauze plug in the fistula and allowing the contents to flow out; the duodenal material is representative of the material leaving the abomasum (Phillipson, 1950). Samples of gastric juice were collected from the abomasal pouch of sheep no. 3 by attaching a rubber tube to the glass cannula and allowing the secretion to drain into a suitable receiver. The rate of flow of the juice varied (see Phillipson, Green, Reid & Vowles, 1949), but was usually about 50 ml./hr.
(b) Slaughtered sheep
The animals were slaughtered at 10 a.m., which in the case of sheep nos. 8 and 9 was 12 hr. after the last feed and 2 hr. in the case of sheep no. 10. As soon as possible after slaughter the various divisions of the stomach were ligated. The contents of each division were then removed, weighed, and representative samples taken for analysis.
All the reagents used were of A.R. quality (British Drug Houses Ltd.). For the estimation of inorganic phosphate, soluble calcium and magnesium the samples of ruminal, abomasal and duodenal contents were strained through three thicknesses of surgical gauze to yield the corresponding liquors. Omasal samples were found to be too dry to be strained, and so a representative sample of the total contents was wrapped in surgical gauze and squeezed in a hand-press to obtain liquor. The brown-green liquor obtained by straining or squeezing was then treated as follows :
The liquor (5 ml.) was diluted with distilled water (5 ml.) and the diluate centrifuged in the superspeed head of an M.S.E. centrifuge (20,000 × g) for 20 min. The supernatant liquid was then pipetted off and re-centrifuged at the same speed for to min. The resulting supernatant liquid was clear and bacteria-free.
Inorganic phosphate determination
The clear liquid (0·5 ml.) was diluted to 25 ml. and suitable aliquots taken for the estimation of phosphorus according to Berenblum & Chain (1938). The original colour of the liquors was diluted to such an extent that it did not interfere with the determination.
Soluble calcium and magnesium determination
Duplicate analyses on all samples were carried out. The clear liquid (2 ml.) was pipetted into a 15 ml. conical centrifuge tube and saturated ammonium oxalate solution (1 ml.) added, followed by thymol blue indicator (5 drops). Dilute trichloroacetic acid (10%, w/v) was then added dropwise until the solution turned pink. Dilute ammonia (10%, v/v) was carefully added dropwise until the indicator changed to yellow, thus bringing the mixture to pH 3 at which calcium is quantitatively precipitated aş oxalate uncontaminated with phosphate (see Washbum & Shear, 1932; Holth, 1949). The mixture was allowed to stand overnight and then centrifuged at 2000 r.p.m. for 10 min. The supernatant liquid was carefully decanted into a 15 ml. centrifuge tube and reserved for the determination of magnesium (see below). The precipitated calcium oxalate was washed with 2x5 ml. of dilute ammonia (2%, v/v) in the centrifuge, dissolved in N-HgSOf (2 ml.) and the solution titrated against N/100 KMnO4 in the usual way.
Magnesium was determined by adding 2% (w/v) diammonium hydrogen phosphate (1 ml.), followed by 7 drops of ammonia (sp.gr. 0·88) and then scratching the sides of the tube with a glass rod to facilitate crystallization of the magnesium salt. The mixture was left overnight and centrifuged at 3000 r.p.m. for 10 min. The precipitate was washed twice in the centrifuge with 5 ml. of ethanolic ammonia (Briggs, 1922) and then dissolved in N-H2SO4 (2 ml.). The solution was diluted to 25 ml. and suitable aliquots taken for P determination by the method of Berenblum and Chain (1938)..
Inorganic phosphate, soluble calcium and magnesium in gastric juice were determined by similar methods after removal of the small amount of protein present by precipitation with an equal volume of 10% (w/v) trichloroacetic acid.
Representative samples of unstrained organ contents were dried to constant weight at 105°.
These determinations were made with a glass electrode on the liquors after straining or squeezing as soon as possible after removal of the sample from the animal.
The initial experiments showed that the inorganic phosphate concentration of the ruminal and duodenal contents of sheep nos. 1 and 2 was of the order of 30−40 mg. P %, expressed on the strained liquors. The inorganic phosphate level did not depend on the time after feeding that the samples were taken. Typical results are shown in Table 1.
Since the concentration of inorganic phosphate in the material leaving the abomasum was of the same order as that found in rumen liquor in spite of the diluting effect of gastric juice, it was decided to investigate the levels of soluble calcium and magnesium as well as inorganic phosphate in the two liquors to see if a similar ‘concentration effect’ was apparent. The results are shown in Table 2, in which is also recorded the pH and percentage dry matter of several samples. The ruminal and duodenal or abomasal samples were withdrawn at the same time.
Food material entering the abomasum is subjected to dilution with gastric juice and so samples from the abomasal pouch of sheep no. 3 were examined. The pH was found to be usually between 1·0 and 2·0, the inorganic phosphate content 0·1−0·5mg.%, magnesium 0·7−1·7 mg.% and calcium about 1 mg.%.
Samples of total omasal contents cannot be obtained from a living animal even if fistulation were possible, the contents between the laminae are too solid and inaccessible to allow the withdrawal of a representative sample. Consequently, analyses of omasal contents had to be performed on slaughtered animals. The analyses of the organ contents of these animals are given in Table 3. In all cases the term ‘ruminal contents ‘includes reticulum contents, since early experiments showed no significant difference in the concentration of inorganic phosphate in these loosely separated divisions of the stomach.
Effect of gastric juice on ruminal contents
Samples were obtained from sheep no. 2 of ruminal contents (500 ml.) and duodenal contents (50 ml.), 4 hr. after the last hay feed. Gastric juice was collected from sheep no. 3 and centrifuged to remove a little mucus. The ruminal sample was strained through open-mesh gauze to remove large hay particles and so facilitate the measuring of volumes. The strained material is subsequently referred to as ruminal contents, since the straining only reduced the percentage of dry matter from 3·4 to 2·9%. The pH of the ruminal contents, duodenal contents and gastric juice was determined and inorganic phosphate, soluble calcium and magnesium estimated as already described, with the results shown in Table 4.
To simulate the acidity of duodenal (abomasal) contents a mixture of the gastric juice (65 ml.) and the ruminal contents (25 ml.) was made. The mixture (pH 2·65) was sampled immediately and after 3 hr. at 39°. In Table 4 are recorded the results of the analyses of these samples for inorganic phosphate, soluble calcium and magnesium.
Effect of synthetic saliva and gastric juice on hay
Since the experiments already described show a considerable release of calcium by gastric juice from the ruminal contents of hay-fed sheep, the effect of synthetic saliva and gastric juice on hay was studied. The sample of hay used was obtained from the bulk store used for feeding sheep nos. 1−3.
A solution of salts resembling in composition that of sheep saliva was prepared essentially according to Elsden (1946), as modified by Oxford (1951) and gastric juice was collected from sheep no. 3. The synthetic saliva was allowed to stand overnight at room temperature, after which it had a pH of 6·85.
Finely chopped hay (10 g.) was placed in a 250ml. conical flask and synthetic saliva (100 ml.) then added. The flask was shaken and the pH of the mixture determined. It was then loosely stoppered and put into the incubator at 390 along with another flask containing only synthetic saliva (too ml.). The flasks were shaken every half hour and removed from the incubator after 5 hr. The pH of the contents of the flasks was determined and the mixture containing hay was filtered. Inorganic phosphate, soluble calcium and magnesium were determined on the filtrate and on the sample of synthetic saliva which had been similarly incubated.
The experiment using gastric juice was carried out in a similar manner using hay (10 g.) and a mixture of gastric juice (75 ml.) and distilled water (25 ml.) in order to simulate conditions in the abomasum. The second (control) flask in this case contained 100 ml. of the same diluted gastric juice. The results of these experiments are given in Table 5.
The inorganic phosphate concentration in the fluid of the rumen and abomasum of the sheep appears to be fairly constant on a given diet. The rumen of the sheep has no secretory glands of its own and the saliva provides much of the liquid medium of ruminal contents. McDougall (1948), in a study of sheep saliva, found that the secretion of parotid saliva (the major component of mixed saliva) ranged from 2 to 4 1. per day. Phosphorus in mixed saliva was found to be entirely in the inorganic form (37−72 mg. P%), calcium, 1·6−3·0mg.% (considered to be largely ionized) and magnesium 0·6−1·0 mg. %.
It is clear that ruminal fluid is a good buffer in which phosphate, along with bicarbonate, plays an important role in buffering acid production resulting from bacterial fermentation. Van der Wath (1942) showed that the bacterial population of the sheep’s rumen was increased by the addition of phosphate to a diet low in phosphorus. The phosphate in the rumen doubtless plays an important part in the fermentation reactions themselves, most of which probably involve phosphorylation mechanisms.
The inorganic phosphate content of the ruminal liquor of sheep fed exclusively on hay (nos. 1, 2 and 10) or on rye-grass and clover (nos. 4−7) is of the same order as that given by McDougall (1948) for saliva, and it would seem that very little soluble phosphate is derived from such feeds in the rumen. If, however, the diet contains linseed oil cake and oats (sheep no. 3) or oats and ground maize (sheep nos. 8 and 9), the inorganic phosphate content of the ruminal liquor is considerably raised above the level found in hay- or pasture-fed animals. It seems likely that this extra phosphate is derived from the phytates present in the diet, for Reid, Franklin & Hallsworth (1947) have shown that phytates are almost completely hydrolysed quite rapidly in the sheep’s rumen, probably under the influence of enzymes of micro-organisms.
On the other hand, the soluble calcium and magnesium values found for the ruminal liquor of hay-fed sheep are higher than those reported for saliva and can be accounted for by the simple solution of these ions from the food. This has been demonstrated in vitro (Table 5) which shows that 20·5 mg. of calcium and 13·5 mg. of magnesium pass into solution from 10 g. of hay in 5 hr. at 390 in 100 ml. synthetic saliva over a pH range of 6·85−7·7. An interesting point in connexion with the results presented in Table 5 is that the free inorganic phosphate found in the synthetic saliva extract of hay was less than that originally present in the synthetic saliva itself. The pH of the extract rose from 6·85 to 7·7, resulting in the precipitation of a little of the phosphate, probably as calcium phosphate. This might occur in vivo if rumen contents ever become alkaline.
The work of Phillipson et al. (1949) has shown that the dilution by gastric juice of food matter leaving the sheep’s omasum is quite considerable, being of the order of 1−2 parts of gastric juice to 1 part of omasal material. Since gastric juice has been shown to contain very little phosphate, calcium and magnesium, an explanation must be sought to account for the maintenance of the concentration of inorganic phosphate and soluble magnesium in abomasal contents, and also to account for the greatly increased amounts of soluble calcium found in the abomasum. It could therefore be that there is a liberation of hitherto insoluble phosphate, calcium and magnesium in the abomasum under the influence of gastric juice or that absorption of water occurs in the omasum, resulting in the passage of a concentrate of ruminal and reticulum contents into the abomasum. The results presented in Table 4 show that gastric juice does not effect any significant release of phosphate from the rumen contents of a hay-fed sheep, though magnesium is liberated to a slight extent. Calcium, on the other hand, undoubtedly passes into solution in considerable amounts as soon as gastric juice and ruminal contents are mixed, or when hay is incubated with diluted gastric juice (Table 5).
For many years the omasum, with its highly specialized structure has attracted the attention of physiologists, giving rise to much speculation regarding its function in the ruminant. Favilli (1937) reviewed much of the data on this subject and concluded that the omasum does not function merely to press and squeeze the food, but also to effect comminution and to absorb water. The work on omasal contraction has been summarized by Dukes (1947, p. 315), who considers that food material entering the omasum is subjected to considerable pressure and ‘some of the liquid so released probably undergoes absorption’.
The percentage of dry matter in omasal contents is considerably higher than that found in ruminal or abomasal contents (see also Elsden, Hitchcock, Marshall & Phillipson, 1946; Dukes, 1947, p. 330). This is shown in Table 3, together with the figures for inorganic phosphate, soluble calcium and magnesium in omasal liquor, which lend support to the idea that water absorption occurs during the passage of food through this division of the stomach. In all cases the concentration of these three ions is greater in omasal liquor than in the corresponding ruminal liquor. The figures for sheep nos. 6−9 are particularly striking in this respect; the percentage of dry matter in the various divisions of the stomach, taken together with the inorganic phosphate concentrations of the corresponding liquors (Table 3), can be correlated with the findings of Phillipson et al. (1949). If the omasal material of these animals were diluted with about one volume of gastric juice the percentage of dry matter and the phosphate concentration of the liquor would be lowered to give figures in agreement with those found in abomasal contents.
The figure of 6·2 in Table 3 for the pH of the abomasal contents of grass-fed sheep nos. 6 and 7 is rather high. No explanation of this finding can be offered, though it may be related in some way to the comparatively small total weights of the abomasal contents of these animals. Another apparently anomalous figure in Table 3 is the low value for phosphate in the omasal liquor of sheep no. 10; this is associated with a much greater weight of total omasal contents than was found in any of the other animals.
However, the main findings of the present work point to the absorption of water from the omasum of the sheep playing an important part in the water economy of the alimentary tract, water added to the food in the form of gastric juice more or less compensating for that which is absorbed as the food passes through the omasum. The concentrations of inorganic phosphate and magnesium in abomasal liquor are thus kept at about the same level as are found in the corresponding ruminal liquor.
The picture in respect of calcium is slightly different from that of inorganic phosphate and magnesium in view of the much higher percentages of soluble calcium found in abomasal liquor than were found in ruminal or omasal liquor (see Tables 2 and 3). Omasal absorption of water probably accounts for this in some measure, but, in addition, the acidity of abomasal liquor (due to the presence of gastric juice), results in an immediate liberation of calcium as is clearly shown in Table 4. The combined form of calcium from which it is released is not a phosphate or phosphates in sheep on a hay diet, since no parallel increase in inorganic phosphate occurred when ruminal contents and gastric juice were mixed. The nature of the calcium compound or compounds from which it is liberated remains to be investigated further.
The inorganic phosphate, soluble calcium and soluble magnesium levels in the various divisions of the stomach of the sheep on different diets have been studied.
The concentration of inorganic phosphate and soluble magnesium in ruminal and abomasal liquors was of the same order in these two divisions of the stomachs of three fistulated animals independent of the type of feed or time after feeding at which the samples were taken.
The concentration of inorganic phosphate, soluble calcium and magnesium in omasal liquor of slaughtered sheep was higher in all cases than the corresponding value for rumen liquor, lending support to the idea that one of the functions of the omasum is to absorb water.
Although the concentration of soluble calcium in omasal liquor was higher than that found in the corresponding ruminal liquor, much higher levels were found in abomasal liquor than in omasal or ruminal liquors.
The author is indebted to many members of the staff of this Institute for helpful discussion, particularly Dr A. E. Oxford, Dr A. T. Phillipson and Mr P. J. Heald, and to Mr W. R. H. Duncan for his technical assistance.