Absorption of Volatile Acids From the Alimentary Tract of the Sheep and Other Animals

Observations of the fate of the volatile acids have been few. Wilsing (1885) calculated, on the basis of in vitro experiments, that 157 g. of volatile acids would be produced by rumen fermentation from1·5 kg. of hay which he fed to a goat. Of this only 2–5 g. was excreted in the urine and faeces. Phillip-son & McAnally (1942) showed that while the concentration of volatile acids in the rumen ingesta is high, that in the abomasal ingesta is very low. They suggested that absorption of the acids in question from the rumen must occur. It has now been possible to show that volatile acids are in fact absorbed into the blood coming from the rumen, that this organ, together with the reticulum, omasum and caecum are the only parts of the alimentary tract from which any considerable absorption occurs and that the amount of volatile acid absorbed is sufficient to supply an appreciable part of the energy requirements of the animal.

In all experiments except that performed upon a pony, a solution of nembutal (60 mg. in 1 c.c.), given by slow intravenous injection, was used for an anaesthetic.

The animal was laid on its left side, the abdomen was opened on the right side by a longitudinal incision extending backwards for 8–12 m. from the posterior border of the last rib and 4–6 in. below the transverse process of the lumbar vertebrae. A second incision from the anterior extremity of the previous one was made, parallel to the last rib, for 4–6 in. towards the mid line.

The abdominal viscera were covered by warm saline pads when it was necessary to withdraw them from the abdominal cavity, as was the case with the caecum, small intestine and abomasum.

Blood from the rumen was drawn, unless otherwise stated, from the posterior longitudinal rumen vein ; blood from the reticulum was drawn from the two veins draining the right side of that organ, while a special procedure had to be adopted to obtain blood draining the omasum owing to the small size of the vessels. The epiploic vessels from the abomasum run over the greater curvature of the omasum and receive vessels draining that organ so that the blood of the epiploic vessel carries blood from both organs. In order to obtain omasal blood only, small bulldogs were placed on the epiploic vessels at either extremity of the omasum so that blood subsequently drawn from the isolated area came from the omasum only. No difficulty was experienced in obtaining blood from the abomasum, small intestine or caecum.

The vessels draining the alimentary viscera are small, and blood samples can be collected with ease only from the large vessels into which they drain at the root of the mesentery. Blood from the stomach was drawn from the left epiploic vein coursing along the greater curvature, but blood from the small intestine and colon was obtained from the large vessels at the base of the mesentery; with the colon it was necessary to separate gently the loops of the spiral in which it is arranged in order to expose the root of the mesentery.

The size of the animal limited the amount of blood that could be withdrawn without seriously upsetting the blood pressure. For this reason, in the first two experiments 5 c.c. samples only were taken, although 10 c.c. samples were taken in the second two experiments as in the experiments upon other animals. No difficulty was experienced in sampling.

We are indebted to Prof. E. D. Adnan for kindly permitting us to obtain from a pony peripheral blood, and blood draining from the small intestine and colon.

The use of a needle bent into a hook was found especially useful in obtaining blood from vessels that were not easily accessible or which were draining in a direction opposite to the only possible approach to them.

In all except the first few experiments the condition of the animal was gauged by measuring the blood pressure in the carotid artery.

The separation of the volatile acids from the blood was accomplished by means of the technique described by McAnally (1944).

In the sheep numerous results show that the excess of volatile acid in the blood draining the rumen over that in the arterial blood is always considerable. Blood draining the reticulum contained much the same concentration of volatile acids as blood from the rumen. Absorption from the omasum, though much less than from the rumen or reticulum, is quantitatively significant. As might be expected from Philhpson & McAnally’s (1942) previous results on the concentration of the acids in the abomasal ingesta, absorption from the abomasum appears to be nil ; this is also the case with the small intestine; however, there is again absorption from the caecum where bacteria are again active and therefore a renewed production of volatile acid takes place.

The chief sites of absorption in the pig are the caecum and colon ; however, there is apparently some absorption from the stomach and small intestine. The possibility of there being some fermented material in the food was not excluded except in Exp. 3, where the concentration of volatile acid in the food was found to be only one-tenth of that in the stomach contents. It seems probable that even though the breakdown of the more readily fermentable constituents of the diet starts in the less acid cardiac end of the stomach and in the small intestine, conditions are not optimal until the caecum and colon are reached.

The results in the third table indicate that there is absorption from the caecum of the rabbit. Absorption from the small intestine was not significant.

Samples taken from a single pony showed that there is absorption of volatile acids from the colon of this species also.

Samples of blood were taken from a number of different vessels which drain the rumen of the sheep. The position of the various vessels is shown in FIG.1 and the concentration of volatile acids in the blood taken from them is given in Table 5.

The ventral sac of the rumen lay uppermost in the experiment as the sheep was lying on its side ; here therefore the gas collected. Comparing the figure and table, clearly the vessels which drain the portion exposed to gas carry little volatile acid while those which drain the lower parts in which the ingesta are lying carry much. The anterior vein contains blood which carries the greatest concentration of volatile acids.

Having obtained evidence of the absorption of volatile acids from the alimentary canal, clearly the next step was to determine precisely what these acids were and in what proportion they occurred. For this purpose a comparison was made between the volatile acids in the blood leaving the rumen and those in the rumen content. Similar estimations were also made in respect of the contents of the caecums of two sheep and one pig. One pony was also studied but in this case the blood only was analysed, not the contents of the intestine.

The following procedure was adopted:

Fifty c.c. of blood was taken from the posterior vein of the rumen, or the appropriate vein draining the portion of the alimentary canal under observation, and the separation of the mixed acids was performed in exactly the same way as for the smaller samples (McAnally, 1943).

A sample of ingesta was taken from the rumen at the same time that the blood sample was obtained. It was filtered through muslin and to the filtrate an equal volume of acid magnesium sulphate precipitant (McAnally, 1943) was added. The precipitate was filtered off and the mixture of acids was separated from the filtrate by steam distillation as from the blood.

The procedure was as in the case of the rumen.

For the determination of the nature and quantities of the various acids in these mixtures we have used the second distillation of Friedemann’s method (1938). The exact details are given in the appendix. The method, however, depends upon the following principle. If the whole, in this case 200 c.c., of the solution containing the acids be distilled, the acids of higher molecular weight will come over more rapidly than those of lower molecular weight and therefore if the distillate be collected in successive fractions the earlier fractions will contain relatively more of the heavier acids.

Taking butyric, propionic and acetic acids; were pure butyric present, 37% of it would come over in the first 25 c.c., 27 % (64–37) in the second 25 c.c. and so on according to the curve given in Fig. 2. Were the acid pure propionic acid, only 20·5 % would come over in the first 25 c.c. of distillate, 20 % in the second and so on—in the case of pure acetic acid 10% comes over in the first, 10·5 in the second and so on.

Within the limits of experimental procedure these proportions are unaltered by the rate of distillation and in a mixture of the acids the rate at which each distils over is independent of the presence of the others. When, therefore, as in the case of the distillate from the blood from the rumen the rate of distillation of the total acids present is almost coincident with but a little more rapid than that of acetic acid the inferences may be drawn: (1) that the great preponderance of acid present is acetic, (2) that there is some acid of higher molecular weight than acetic.

Further, when from the rumen content the earlier samples of distillate are richer in volatile acid than in the case of those from the blood, it may be inferred that there is less acetic relatively to acids of higher molecular weight in the rumen content than in the blood.

Finally, the figures in Table 6 show that the products of fermentation in the caecum of the sheep and the pig and in the colon of the pony are similar to those in the rumen of the sheep.

The conclusion was drawn from the results obtained in the previous section that blood draining the rumen contains a higher proportion of acetic acid to acids of higher molecular weight than do the contents of the rumen. There seemed thus to be an indication that acetic is more rapidly absorbed than are the higher acids. The following experiments were designed to see whether this was so.

Two 5 months old ram lambs and one adult. wether were fitted each with two rumen canulae one being in the usual position, in the flank, as already described by Philhpson & Innes (1939), and the other inserted in the ventral sac and brought out 2 in. to the left of the mid-line m the umbilical region. It has been found possible to wash out completely the rumen contents of such an animal if it has previously been fed on a finely divided fodder such as bran and oats. The rumen was filled with 2 1. of water and the animal then anaesthetized, the abdomen opened, and blood taken from the posterior rumen vein. 25 g. of sodium acetate in 2 l. of water at body temperature were then introduced into the rumen through the canula, and a sample of rumen blood was again taken. The acetate was washed out and replaced by warm water and blood again taken. The same process was repeated giving a dose of 25 g. of sodium propionate in 2 l. of water, followed by water, then 25 g. of sodium butyrate in 2 l. of water. In Exps. 2 and 3 as a control measure in case any abnormal condition of the rumen epithelium might have been induced by the experimental procedure, after measurement with water in the rumen the sodium acetate dose was again given.

Examination of Table 7 will show that while blood draining from the empty rumen or the rumen filled with water contains no detectable volatile acid, no more than 5 min. after filling the rumen with sodium acetate solution the concentration of the acids in the blood has risen very appreciably. The absorption of propionate after the same time is not so great as with acetate, while butyrate appears to be little absorbed in this space of time. Samples of peripheral blood were also taken in Exp. 1 and there was some indication that, even after so short a time, the concentration of volatile acid in the peripheral blood was influenced by the contents of the rumen, but the amounts of acid titrated in these samples were so small that the differences were not outside the range of experimental error. Acetate was again absorbed when given after the other two salts had been tested but the concentration in the blood was less than that found previously. Thus, though comparison of the butyrate absorption with the second acetate absorption clearly indicates that the former is very much less rapidly absorbed, the relative rates of absorption of propionate and acetate were not established with such certainty.

In order, therefore, to test this latter point a further experiment was performed. An eighteen months old wether was anaesthetized and a single canula was inserted into the ventral sac of the rumen ; the wound was closed around the neck of the canula. It was found possible to empty the rumen and wash it clean with warm water through this single canula (the animal was fed upon bran and oats for about a week before the experiment and starved for the last 24 hr.). The rumen was filled with 2 l. of warm water. The oesophagus was tied in the neck and the abdomen then opened on the right side in the usual manner. A ligature was then passed round the abomasum excluding the epiploic vessels immediately below the omasum so that no escape from the rumen was possible.

The previous experiments were then repeated with the following differences: (1) the order of the doses was water, propionate, water, acetate ; butyrate being omitted ; (2) the acetate was allowed to remain in the rumen for 90 min., samples of blood being withdrawn at intervals ; (3) equimolecular solutions were used.

The results shown in Table 8 demonstrate clearly that the rate of absorption of propionate is lower than that of acetate. At the end of the experiment the remainder of the dose containing acetate was carefully collected in addition to the washings from the rumen, so that the amount of acetate absorbed during the 90 nun. interval could be estimated. On post-mortem examination the rumen and reticulum were clean and empty but some ingesta were present in the omasum. As it was not impossible that some of the acetate solution had passed into the latter organ the ingesta were washed out and collected, the quantity of volatile acid found here was, however, negligible.

The total loss of sodium acetate during the 90 min. interval was 10 g. If this rate of absorption is calculated on the basis of grams of acetic acid per hour, it establishes the fact that the rumen and reticulum together can absorb at least 5 g. of acetic acid in this time. This quantity is rather greater than the amounts which we have calculated to be absorbed from the rumen containing normal ingesta. It is clear therefore that the calculated absorption is not outside the range of absorption of which the rumen is capable. It is interesting that the reduction of volume of the liquid in the rumen during the 90 min. interval was only 150 c.c.

The fact that the rate of absorption of propionate is less than that of acetic both at the start of an experiment, when physiological conditions are optimal and at the end when the conditions are less good (as shown by a fall in blood pressure) indicates clearly that the acid of higher molecular weight is less rapidly absorbed.

In order to determine the quantity of volatile acid absorbed in a given time from the rumen it was necessary to measure the rate at which blood flows from it as well as the concentration of volatile acids earned in this blood. It was found to be impossible to gain access to the vessel into which the tnbutary veins from the rumen flowed, neither was it possible to expose any vessel draining the rumen, except the posterior longitudinal vein, sufficiently to measure the rate of blood flow through it. It was decided, therefore, to measure the blood flow in the posterior vessel and from this to calculate the amount of blood draining both the rumen and reticulum in a given time on the assumption that the blood flow is directly proportional to the area drained.

The preliminary operative procedures have already been described. The blood pressure was taken from the carotid artery before and after opening the abdomen. A short length of the posterior vein in the vicinity of A, Fig. 1, was dissected free from the visceral peritoneum and a ligature was passed beneath it. A short needle with a bore of 1 ·5 –2 mm., to which was attached a short length of rubber tubing of approximately the same bore, was thrust into the vessel against the blood flow and the ligature was immediately tied so that the shaft of the needle was held in the vessel. The knot of the ligature was given a single hitch only so that it could be pulled tight immediately on the withdrawal of the needle from the vein. The needle and rubber tube were held horizontally and the end of the tubing was turned downwards into a measuring cylinder. The cylinder was changed every half minute, the time being taken by stop watch, and the half minute output of the vessel was thus measured over a period of from 2 $212$ to 3 mm. At the end of this time the needle was withdrawn from the vein and the ligature pulled tight and the carotid blood pressure was again taken in order to see if any serious fall had occurred through loss of blood. In one experiment the blood pressure tracing was taken throughout the period of collection and is shown in Fig. 3. An average figure for the half-minute readings was taken and doubled to give the output of the vessel per minute.

An important precaution in performing these manipulations was to ensure that the flow of blood from the vein was unimpeded in any way. It was essential to hold the needle firmly so that there was no danger of the walls of the vessel forming a valve over the oblique opening of the needle.

The course of one experiment is given below to illustrate the procedures described:

Sheep 25.

Blood pressure at start of experiment =140 mm. Hg

Abdomen opened and posterior vein of the rumen exposed

Blood pressure =128 mm. Hg

Blood flow of posterior vein measured

Blood collected during successive 30 sec. intervals: 42 ·0 c.c., 44 ·9 c c., 40 ·5 c.c., 39 ·5 c.c., 41 ·0 c.c.

Blood pressure at end of experiment =116 mm. Hg

Total blood collected in $2212$ min. =207 c.c.

.. Blood flow per minute =83 c.c.

Area of rumen and reticulum drained ^* = 31 %

Concentration of volatile acid in blood calculated = 15 c.c. o 01 N %

Blood flow from whole of rumen and reticulum = 268 c.c. per min.

Volatile acid drained from these organs = 40 ·2 c.c. o 01 N per min.

Volatile acid drained per minute in g. acetic acid = 0 ·024 g.

Volatile acid in g. acetic acid drained per hour = 1 44 g.

In most of the experiments the orifice of the rubber tube from which the blood was collected was held at the level of the vein; in sheep 29, however, the actual venous pressure was measured by a method devised by one of us for another purpose, namely, the insertion of a hypodermic needle into the vein ; to the needle is attached a piece of translucent rubber (bicycle valve) tubing containing Ringer’s solution, the solution runs into the vein till it falls to the level of the venous pressure. The meniscus can be seen through the wall and its height measured with a rule. The puncture in the vein causes no subsequent trouble. The pressure as measured in this experiment was 16 ·4 mm. Hg (mean of 16 ·1 and 16 ·7). During the subsequent collection of the blood samples the orifice of the collecting tubule was held at the level to which the meniscus had fallen. The venous pressure was thus taken into account and the resulting picture of blood flow was not significantly different from that in other experiments. The consecutive flows were: (1) 46, (2) 46, (3) 46, (4) 44 c.c. per 30 sec.

The blood pressure is given in Fig. 3.

At the end of the experiment the oesophagus and duodenum were ligated and the whole stomach was removed from the carcass. The area of the rumen drained by the posterior vein to the point of puncture was marked off by a line made with a solution of hexamethyl violet. This area was separated from the remainder of the organ and cut into pieces so that it could be spread flat upon a table and its area measured. The surface area of the remainder of the rumen and of the reticulum were similarly measured and the proportion of the whole represented by the area drained by the posterior vein was calculated.

The area drained was found to vary between 31 and 43 % of the total surface area of the rumen and reticulum, the average figure from fourteen measurements being 37 %. In each calculation the individual measurement for the sheep concerned was used in estimating the rate of absorption per hour.

Only those results are recorded in which :

1. The figures for the output of the posterior vessel were reasonably constant and not steadily falling.

2. The blood pressure did not fall below 80 mm. Hg during the experiment.

3. The sheep was found to be normal on postmortem examination.

The measurements of blood pressure made during the ten experiments given in Tables 10 and 11 were as follows :

The results are shown in Tables 10 and 11. Those in the former table were obtained from sheep which were taken from pasture in October and early November, while those shown in the latter table were obtained from sheep taken from grass in April and May. Of the five experiments done in the spring three were done at midnight, Nos. 23, 24 and 27, and two were done at mid-day, Nos. 25 and 26. This was done as previous results (Phillipson, 1942) showed that the maximum concentration of volatile acid in the ingesta in the rumen occurred at midnight, and it was thought that for this reason absorption would be greater at night than during the day. The results, however, show no significant difference between the quantity of volatile acid absorbed per hour in the daytime and at night. A tendency for absorption to be greater in the autumn than m the spring is apparent m the tables although there appears to be no reason why this should be so and in view of the small numbers of sheep employed no significance can be attached to this result.

The variation in the output per minute of the posterior vein of different sheep is related to the size of vessel. Thus in sheep in which the flow was above 100 c.c./min. the vessel was large while the vessel was small in sheep 24 in which the lowest figure, namely, 38 c.c./min., was recorded. The size of vessel cannot be correlated with either the weight of the animal or the weight of the ingesta.

The variation in the concentration of the volatile acid in the blood draining the rumen does not exceed the range found in previous experiments.

It is of interest to see whether or not the concentration of volatile acid in the ingesta of the rumen has any relation to the quantity of volatile acid absorbed. The concentration of volatile acid is expressed as gm. acetic acid per 100 g. dry matter in column 2 of Tables 10 and 11. It can be seen that the concentration in the autumn sheep was remarkably regular and so cannot account for the variation in the rate of absorption. In the spring sheep the average concentration was higher, yet in spite of this the rate of absorption in three animals was low. The only conclusion that can be drawn is that the rate of absorption does not depend upon the concentration of volatile acid in the ingesta.

The results obtained here fully confirm the previous inference that absorption of volatile acids occurs from the rumen. The fact that absorption in the omasum is less although still significant suggests that this organ, in addition to giving the solid ingesta a final comminution before it enters the abomasum, also removes the remainder of the volatile acids from the ingesta so that the material entering the abomasum is practically free from these acids.

The view that the omasum acts as a sponge was put forward by Favilli (1937); it is well known that the ingesta in the omasum has a higher percentage of dry matter than that of the rumen and as in addition volatile acids are absorbed from this organ the view put forward by Favilli has some support.

The fact that volatile acids reappear in the blood draining the caecum is a clear indication that fermentation of carbohydrate again commences in the large gut. The recent work of Trautmann & Asher (1939, 1941), m which the fermentation of cellulose in the caecum of goats was investigated, does not support the view that fermentation of this substance is responsible for the acids absorbed from the caecum although their conclusions are open to criticism (McAnally & Phillipson, 1944). It is sufficient here to point out that the mixture of volatile acids present in the caecum is essentially the same as that present in the rumen, in the caecum of the pig and in the blood draining the colon of the horse in all of which organs fermentation of carbohydrate, of which cellulose is an important component, is known to occur. It must, however, be allowed that the products of fermentation of the individual carbohydrates in these organs are not known, consequently this mixture of volatile acids may be characteristic of fermentation of carbohydrates other than cellulose.

The rate of absorption of volatile acid from the rumen was previously given as varying from 2 to 4 g./hr. (McAnally & Phillipson, 1942); this range must now be extended to 0 ·9 –5 ·3 g./hr.

These values must be regarded as minimal for the total absorption of volatile acid for the following reasons:

1. The papillae of the rumen greatly increase the absorbing area and as those of the anterior region are larger than those of the posterior region (the area drained by the posterior vein) measurements of the surface area, such as have been made, must give a higher value for the portion of the rumen drained by the posterior vein than is actually the case.

2. The anterior vessel of the rumen was found to carry a higher concentration of volatile acid in the blood than the posterior vessel.

3. Volatile acids are absorbed from both the omasum and the large intestine and no account has been taker) of this fact.

4. Volatile acids may be absorbed by the lymphatic system.

It is important to bear these errors in mind, for if the total carbohydrate digested by a 50 kg. sheep is calculated in terms of total carbon digested, on the assumption that polyhexoses predominate, it is found that after a deduction of 15 % for carbon lost as methane and carbon dioxide, the animal absorbs 264 g. carbon a day at maintenance level. If the highest rate of absorption, namely, 5 g. of vblatile acid per hour calculated as acetic acid, is taken, then only 48 g. of carbon can be accounted for in this way, approximately one-sixth of the total requirements. If this figure, however, is accepted, it does represent a significant portion of the digestible carbohydrate and as such is of nutritional interest. This is especially so when it is recalled that the production of volatile acids is common to carbohydrate fermentation in the horse, pig and rabbit and presumably in all herbivorous animals and possibly in man; consequently the nutritional value of these acids warrants further investigation.

1. The concentration of volatile acids in the blood draining the rumen is considerably higher than that of peripheral blood in which little volatile acid, if any, is present.

2. Volatile acid in significant amount is present in blood draining the omasum and the caecum but is absent from blood draining the abomasum and small intestine.

3. Volatile acid is also found in significant concentration in blood draining the caecum or colon of the horse, pig and rabbit.

4. Distillation curves indicate that acetic is the predominant acid present in both the rumen contents and in the blood draining the rumen, but the proportion of acids of higher molecular weight than acetic is greater in the rumen contents than in the blood. The same type of distillation curve is found for material taken from the caecum of the sheep and the large intestine of the horse and pig.

5. The rate of absorption of the sodium salts of acetic, propionic and butyric acids, as judged by the concentrations found in the blood leaving the rumen, appears to be in the order of their molecular weights ; acetate is rapidly absorbed, propionate less so, while butyrate is slowly absorbed.

6. The quantity of volatile acid calculated as acetic acid absorbed in an hour from the reticulum and rumen together is estimated to vary from 1 to 5 g., but this value must be regarded as minimal.

We wish to thank Mr H. Bowman and Mr D. Chilvers for valuable technical assistance.

The distillate containing mixed volatile acids was made slightly alkaline and then evaporated down to somewhat less than 200 c.c. The solution was made up to exactly 200 c.c. and this, with 10 c.c. of acid mercuric sulphate solution and solid magnesium sulphate (the quantities are given in Friedemann’s paper), was distilled. Four consecutive 25 c.c. fractions were collected and titrated against N/100 KOH, then a 50 c.c. fraction was similarly treated and finally distillation was continued until crystallization occurred in the flask; this final fraction of distillate thus obtained was about 50 c.c. in volume.

The amount of acids in the whole distillate was summed and the percentage of the whole in each fraction calculated.