Reading, Berkshire. Greenfield, 1952) and sheep (Hyd6n, 1961) have all suggested small intestine

Transcription

1 J. Physiol. (1964), 172, pp With 4 text-figures Printed in Great Britain PASSAGE OF DIGESTA THROUGH THE CALF ABOMASUM AND SMALL INTESTINE BY R. H. SMITH From the National Institute for Research in Dairying, Shinfield, Reading, Berkshire (Received 18 February 1964) A considerable amount of data exists on the time taken for food residues to pass through the bovine reticulo-rumen and the whole alimentary tract (Balch, 1961; Campling & Freer, 1960) but little is known of the time taken to pass through the small intestine. Experiments with humans (Mattson, Perman & Lagerl6f, 1960), rats (Goodman, Lewis, Schuck & Greenfield, 1952) and sheep (Hyd6n, 1961) have all suggested small intestine transit times of about 1-2 hr. With milk-fed calves the value was found to be rather greater, about 2-5 hr (Smith, 1963). Although the absorption of some dietary constituents, for which the main site of exchange is the small intestine, may not be greatly influenced by transit time variations [it seems probable, for example, that glucose absorption is so rapid that it would be virtually complete at any normal transit rate (Reynell & Spray, 1956)], the absorption of other, more slowly absorbed, constituents may be limited by the time that they spend in the small intestine. It has in fact been shown for milk-fed calves that net magnesium absorption efficiency is positively correlated with transit time through the small intestine (Smith, 1963). In the present work variations in small intestine transit times have been examined after feeding different diets to ruminating and non-ruminating calves with the possible effects on absorption, particularly magnesium absorption, in mind. Polyethylene glycol (mol. wt. 4000) and phenol red were used as markers. Some information on abomasal emptying has also been obtained. METHODS Animals Friesian bull calves obtained within four days of birth were used. For experiments involving liquid diets they were efficiently muzzled from about 1 week of age and, except during experiments, given 4-40 l./day of milk in two feeds. Milk intake was increased to /day from 10 weeks of age. A daily mineral supplement containing 0-08 g iron, 0-01 g copper and 0-01 g manganese was added from 3 weeks of age. Calves used for experiments with solid diets were weaned before 7 weeks of age and then fed on hay (up to 0-9 kg/day) and a normal calf rearing mixture (1.6 kg/day) consisting of 47-5 % flaked maize, 24 % bruised

2 306 R. H. SMITH oats, 9-5 % extracted, decorticated ground-nut meal, 6 % dried skim milk, 3 % fish meal and 10 % molasses/peat mixture with supplementary vitamins A and D and calcium phosphate, sodium chloride and trace elements. All the calves were equipped with re-entrant cannulae in the most distal part of the ileum and some were also equipped with simple cannulae in the most proximal part of the duodenum. The cannulae were made of Perspex and were similar to those described previously (Smith, 1962) except that they were only about 3-5 cm long. Analyses Polyethylene glycol. This was determined in the ileal effluent as described previously (Smith, 1958, 1962). When phenol red was present it was necessary to remove it during the determination. This was done by adding about 0-25 g 'Amberlite' IRA-400 ion-exchange resin (chloride form) to the filtrate from the barium hydroxide, zinc sulphate, lead acetate treatment and shaking vigorously for about 15 min, with addition of more resin if necessary, until the solution was colourless. This solution was filtered and treated with barium hydroxide and mercuric chloride as before. Polyethylene glycol values obtained with this treatment for six ileal effluent samples containing added phenol red were not significantly different from values obtained by the usual method without added phenol red. Phenol red. Samples of ileal effluent were homogenized in an 'Ato-Mix' blender (Measuring and Scientific Equipment Ltd.), sometimes with the addition of an equal weight of water, and centrifuged until the supernatant liquid was reasonably clear. A sample (2 ml.) of this supematant was pipetted into a 25 ml. conical flask, 4 ml. 0 4 N-NaOH added and 4 ml. ZnSO4 solution (5 %, wt./vol.) then added drop by drop with shaking. The mixture was shaken vigorously, filtered (Whatman 541 paper) and the filtrate poured back through the same paper until it was clear. This filtrate was diluted with 0-05 N-NaOH to contain less than 0-8 and, if possible, more than 0 4 mg phenol red/100 ml. Phenol red concentration was determined by comparison with suitable standard phenol red solutions in 0-05 N-NaOH using a Spekker absorptiometer (Hilger and Watts Ltd.) with a green filter (Ilford No. 5). These standard solutions lost colour rapidly on storage and were prepared as required from a stock 1 % (wt./vol.) solution which was stable for at least 2 months when kept in the refrigerator. Ileal effluent samples, when not analysed immediately, were stored in the deep freeze (- 140 C) where no colour loss occurred. Samples from calves fed on liquid diets did not contain any coloured materials interfering in the determination and recovery of known amounts of phenol red added to six ileal effluent samples was %. A blank correction, using samples obtained just before the appearance of the marker, was necessary with ileal effluent from ruminating calves. Mea8urement of Transit Time In many experiments with calves fed on liquid diets, a complete collection of ileal effluent was made, in successive fractions, for about 7 hr after giving a feed containing polyethylene glycol (about 1 g/l.) or phenol red (about 0-05 g/l.) as marker. The marker was determined in these fractions, its cumulative elimination plotted against the time after feeding, and the time at which it first appeared estimated by back extrapolation of the curve (Fig. 1 a). This time will be referred to as transit time (mouth-ileum). In ruminating calves receiving solid diets and sometimes in liquid-fed calves a sterile solution (usually 5 ml.) of polyethylene glycol (usually 20 %, wt./vol.) or phenol red (usually 1 %, wt./vol.) in 0-9 % NaCl was injected into a cannula in the proximal duodenum. Subsequent collection from the distal ileum was carried out as before and the time between injection and first emergence of marker estimated (Fig. 1 b). This time, will be referred to as transit time (duodenum-ileum). It will be shown later that there were wide calf to calf differences in transit time values and that, even in an individual liquid-fed calf, there was sometimes a slow, unpredictable,

3 SMALL INTESTINE TRANSIT 307 change with age. However, successive similar experiments with any one calf over fairly short time intervals (say 2-3 weeks) usually gave consistent values. Consequently, in comparing different treatments for liquid-fed calves, the following procedure was adopted. Each treatment to be compared was carried out successively on one calf with an interval of 2-3 days between one and another to avoid carry-over effects and overstraining of the calf. The group of treatments was then repeated one or more times. Thus any changes in the intrinsic properties of the calf during the experiments were made apparent. Comparisons were made only between treatments carried out within a maximum of 2 weeks of each other and when no appreciable intrinsic changes were indicated. Relative values for one treatment in terms of another were obtained, usually for more than one calf, and expressed as an over-all mean + S.E. of the mean. RESULTS Suitability of markers Polyethylene glycol is recovered almost completely after passing through the calf alimentary tract as far as the ileum (Smith, 1962). The possibility of its affecting gut function in the fairly high concentrations usually employed was examined in some experiments by also administering it in still larger amounts, and in amounts so small as to be only just detectable. For calves aged 9-11 weeks receiving milk the transit time (mouth-ileum) with 0x25 g polyethylene glycol in the diet was % (mean for 4 experiments with 2 calves + S.E. of mean) of the transit time (mouth-ileum) with 2 5 g polyethylene glycol. The transit time (duodenumileum) with injections of 0.1 g and 2-0 g polyethylene glycol into the proximal duodenum were % (4 experiments, 2 calves) and % (4 experiments, 2 calves) respectively of the transit time (duodenum-ileum) with an injection of 05 g polyethylene glycol. The widely differing concentrations had no significant effect on transit time. Phenol red has been used as a marker within the alimentary tract of rats (Reynell & Spray, 1956) and humans (Hunt & Spurrell, 1951). Its suitability for use with calves was examined by administering it simultaneously with polyethylene glycol. With calves aged 8-12 weeks receiving milk the recovery in ileal effluent of phenol red as a percentage of polyethylene glycol recovery was % when 0-05 g was injected into the proximal duodenum (6 experiments, 2 calves) but only % when 0-1 g was added to a feed (6 experiments, 2 calves). Despite the poor recovery, the pattern of emergence of phenol red was always identical to that of polyethylene glycol (Fig. la, b) at least during the first few hours. Thus, although phenol red appears to be of doubtful value as a reference substance in the gut, it is apparently suitable for determining transit time. In view of its chemical similarity to substances having a pharmacological effect on gut function (e.g. phenolphthalein) a series of experiments was 20 Physiol. 172

4 308 R. H. SMITH carried out with calves aged 8-12 weeks receiving milk using polyethylene glycol as marker with and without the addition of phenol red. The transit time (mouth-ileum) with 0 1 g phenol red in the diet and the transit time (duodenum-ileum) with injection of 0 05 g phenol red into the proximal duodenum were % (5 experiments, 2 calves) and 97 ± 3 %/ (6 experiments, 2 calves) respectively of the corresponding transit times without phenol red addition. Phenol red, in the concentrations used, had no significant effect on transit time. ~~~100 ~ ~1- Inj. either given with the milk (2+5 g polyethylene glycol or 0@1 g phenol red) inj. or co ~~~~Fed En. 0 E 4) E s5 _ F~~~~~ed a b c Time after feeding or injection of marker (hr) Fed Fig. 1. Cumulative emergence of polyethylene glycol (0) and phenol red (e) from the distal ileuim of calves aged 7-9 weeks and fed on milk. The markers were either given with the milk (2-5 g polyethylene glycol or 0.1 g phenol red) or injected into the proximnal duodenum about 1 hr after feeding (1-0 g polyethylene glycol or 0-05 g phenol red). Flow from the abomasum in calve fed on liquid diets Previous results have suggested that although the majority of a milk feed usually goes directly to the abomasum of a non-ruminating calf (Smith, 1961), substantial amounts of material do not begin to leave the abomasum until min later (Smith, 1962). A similar effect was shown radiographically in goats by Benzie & Phillipson (1957). This was further studied in the present experiments with calves aged 8-12 weeks by feeding liquid diets ( ) containing 2-5 g polyethylene glycol and, about 1 hr later, injecting 0 05 g phenol red in sterile, isotonic solution into the proximal duodenum. A typical result for the emergence of the two markers from the distal ileum is shown in Fig. 1 c. Transit times (mouth-ileum) were consistently longer than transit times (duodenum-ileum) presumably owing to the effect of abomasal hold-up on the former. Mean differences were

5 SMALL INTESTINE TRANSIT hr (5 experiments, 2 calves) for a milk feed, hr (2 experiments, 1 calf) for a 5 % glucose solution feed and hr (4 experiments, 2 calves) for a 5 % glucose, M magnesium chloride solution feed. All these diets were apparently held in the abomasum for about the same time after feeding before appreciable flow into the duodenum began. For calves aged 5-15 weeks and fed on unsupplemented milk ( ) the proportion of dietary polyethylene glycol leaving the ileum in the first 2 hr after its first appearance ranged between 27 and 67 % (25 experiments, 7 calves) but with more values at the upper end of the range (the mean was %). There were no consistent changes with age. It has been shown previously (Smith, 1961) that although in most experiments with milk-fed calves almost the whole of the feed goes directly to the abomasum, occasionally a substantial amount enters the rumen instead. It seems probable, therefore, that the lower values usually represented occasions when this happened, and that a closer approximation to the marker flow rate without leakage into the rumen can be estimated by taking into account only the upper 60 % of the values. A mean value of % was obtained in this way. If it is assumed that the small intestine transit time remained approximately constant during a collection this mean value can also be regarded as a measure of the extent to which the abomasum was emptied of marker in the first 2 hr after flow began. With a 5 % glucose solution feed (7 experiments, 3 calves) the values were little different from those for milk (range 33-66%, over-all mean %, mean for upper 60 % of values %), but with a 5 % glucose, M magnesium chloride solution feed (28 experiments, 9 calves) they were markedly higher (range %, over-all mean %, mean for upper 60 % of values 73±2 %). Only three results (two calves) were obtained for milk supplemented to a similar magnesium concentration (about 35 mg/100 ml.) with magnesium chloride but two values were higher than any for unsupplemented milk (containing about 12 mg magnesium/ 100 ml.) and the mean was %. It appears probable that a higher magnesium concentration led to a higher rate of abomasal emptying on both milk and glucose solution diets. The addition of 3-1 % casein (in solution as sodium caseinate) to a 5 % glucose, M magnesium chloride feed (6 experiments, 2 calves) had no apparent effect on the percentage of marker appearing in the first 2 hr after its flow began (range %, over-all mean %) but the additon of 3*1 % finely dispersed wheat gluten to a similar feed (6 experiments, 2 calves) caused a marked reduction in this value (range 40-52%, over-all mean ).

6 310 R. H. SMITH Variations in small intestine transit time within and between calves fed on a milk diet Transit times (mouth-ileum) were determined in sixteen calves aged between 1 and 20 weeks after they had been given (up to 10 weeks) or unsupplemented milk. A total of 54 experiments were made with between two and seven determinations on each animal. There was relatively little variation within each calf, the standard deviation for individual determinations being hr. There was, however, considerable variation between calves and the mean values derived for each calf ranged from 2-25 to 5-25 hr. The over-all mean value of the means for each of the sixteen calves (± S.E. of the mean) was hr. Using a figure of 0-8 hr for abomasal hold-up this corresponds to a value of 3-2 hr for small intestine transit time. Transit times (duodenum-ileum) determined in four calves within this age range, with the marker injected into the duodenum about 1 hr after feeding, showed a mean value of the means for each calf of hr. Variations within each calfwere usually less than is indicated above when replicate determinations were carried out within 2 or 3 weeks of each other but were sometimes greater over longer periods of time. Thus, for example, one calf gave transit time (mouth-ileum) values of 4.75, 4.75, 4-25, 4 25, 3-50 and 3-50 hr at 11, 12, 15, 17, 22 and 23 weeks of age, respectively. Such variations, however, differed, both in extent and direction, from calf to calf and there were no consistent changes during the experiments due either to change in age (and size) or to temporary disturbance of gut function as a result of the operation to insert the cannulae (at least after the first week). Thus two groups of calves, one aged 2-4 weeks (four calves) and the other aged 8-12 weeks (five calves), both examined 1-2 weeks after the operation, gave mean transit times (mouth-ileum) of and hr, respectively. Another group aged 8-12 weeks (seven calves) but examined 4-8 weeks after the operation gave a mean transit time (mouth-ileum) of 4* hr while yet another group also examined 4-8 weeks after the operation but at weeks of age (four calves) gave a mean transit time (mouth-ileum) of hr. No significant differences existed between any of these groups. The above results and those given elsewhere in this paper were obtained on calves in which the volume rate of flow and appearance of the ileal effluent on any one diet remained little changed from that observed shortly after the operation. This flow rate was about ml./hr after the first appearance of food residues from a milk feed. Frequently, after 2 or 3 months on a liquid diet, there was a marked increase in the rate of flow of ileal effluent and its appearance and odour sometimes changed.

7 SMALL INTESTINE TRANSIT 311 Apparently normal conditions were restored and usually maintained when aureomycin (0 1 g/day) was added to the diet suggesting that the development of an unsuitable bacterial flora was responsible. The small intestine transit time usually remained little changed under these conditions and in four calves, when the ileal effluent rate of flow had more than doubled, the transit time (mouth-ileum) was % of that with normal flow. In another calf, however, the transit time (mouth-ileum) fell from about 3 5 hr when the ileal effluent flow rate was normal, to 2 0 hr, one week later, when this flow rate had increased to about 4 times normal. Aureomycin was then added to the diet and 1 week later the flow rate was again normal and the transit time (mouth-ileum) was 3-75 hr. 100 Elu 14(6) 15(8), Magnesium in diet (mg/100 ml.) Fig. 2. Relative transit times (mouth-ileum) after milk (S) and 5 % glucose solution (0) feeds with different magnesium contents. Transit times obtained after feeding unsupplemented milk (about 12 mg magnesium/l00 ml.) were used as a basis for comparing the other diets and always assigned a value of 100. Each individual result for one of the other diets was obtained by comparing, in one calf, a transit time after feeding this diet with a transit time after feeding the same volume ( ) of unsupplemented milk, the one determined within 2 weeks (almost always within 1 week) of the other. The numbers of individual determinations used to derive each mean are shown on the figure with the numbers of calves examined in parentheses. Standard errors of the means are represented by vertical bars. Effect of diet on small intestine transit time Calves fed on liquid diets. The results given in Fig. 2 show that transit times (mouth-ileum) were shorter after feeds of 5 % glucose, M magnesium chloride or of milk supplemented to about this magnesium concentration than after feeds of unsupplemented milk. These differences were significant (P < and P < 0.01, respectively). Direct comparison of 5 % glucose solution feeds with and without the addition of magnesium

8 312 B. H. SMITH chloride (0-014 M) also showed a significant (P < 0-01) decrease in transit time (mouth-ileum) with magnesium addition (Table 1). The results in Fig. 2 suggest that at comparable magnesium concentrations transit times were shorter with 5 % glucose solution feeds than with milk feeds. This is confirmed for one magnesium intake level by a result given in Table 1 where a direct comparison showed the transit time (mouthileum) on a milk diet containing about 35 mg magnesium/100 ml. to be significantly greater (P < 0.05) than on a 5 % glucose solution diet with a similar magnesium content. That this difference was due to the casein, fat or sodium and potassium contents of the milk was not supported by further experiments (Table 1) in which these constituents were separately added to 5 % glucose, 0 014M magnesium chloride solutionfeeds without significant effect. The omission of glucose from an M magnesium chloride solution feed was also without apparent effect, as was the addition of wheat gluten in another experiment. It also appeared from the results given in Table 1 that the decrease in transit time produced by magnesium chloride addition to the diet was, in fact, due to the magnesium ion since magnesium acetate and nitrate gave similar effects. The addition to a 5 % glucose solution feed of another cathartic, sodium sulphate [in a concentration which gave a similar increase (3-4 times) in ileal effluent volume to that given by M magnesium chloride addition], did not cause any apparent decrease in transit time in one calf. Determinations of transit time (duodenum-ileum) for liquid-fed calves reported elsewhere in this paper were made when the marker was injected into the duodenum about 1 hr after feeding, i.e. during the early flow of food constituents from the abomasum. In 4 experiments with two calves, transit time (duodenum-ileum) determined in this way, was compared with transit time (duodenum-ileum) determined after 18 hr fasting. Values for the latter, expressed as a percentage of the former, ranged between 122 and 200 with a mean value of Statistically the difference was of low significance (P < 0.1) because of the great variation. Calves fed on solid diets. Series of transit time (duodenum-ileum) determinations were carried out in a number of ruminating calves and are shown in Fig. 3. From about 9-16 weeks of age the calves were given (and usually completely consumed) 1-6 kg/day of the normal calf-rearing concentrate mixture described earlier p. 305). This was given in two equal feeds at about 9 00 a.m. and 5.00 p.m. The calves were also given 0 9 kg/day of hay and average consumption increased from 0 4 kg/day at 10 weeks to 0-7 kg/day at 16 weeks. Occasionally the concentrate mixture was temporarily varied and transit times determined after at least 6 days on the new diet. Results obtained at these times have not been distinguished in Fig. 3 as they were not apparently different from those obtained on the

9 SMALL INTESTINE TRANSIT 313 d z0 V V X = Cw~~~~~C~v v CQ ++.- r l > O e0~ ~~~~~~~~~~ 0 9 E.3 t I II + 0 =~~~~~~~~Co; t : f CO A i~ ~ ~ 0" b0o -' D 1+O co=rs +"0 +. o s o0 s 0 0 ~ ~ 00~~ ~~ 0 0 E X -F 0 0 * -> 0 Ca z O'

10 314 B. H. SMITH normal stall diet. They were those shown at 124 and 211 weeks for calf 8G and 11 weeks for calf 9G, when a high-protein mixture containing 40 % flaked maize, 50 % extracted de-corticated ground-nut meal and 10 % molasses/peat was given, and those shown at 11 weeks and 204 weeks for calf 8G, when a low-protein mixture containing 90 % flaked maize and 10 % molasses/peat was given. At about 16 weeks of age some of the calves were abruptly put out to graze a new growth of spring grass. They were tethered to graze an area of about 500 square feet and were moved to a new site usually every 2 days o Approximate age (weeks) Fig. 3. Transit times (duodenum-ileum) at different ages in ruminating calves 7F(O), 8F(E), 9F(A), 8G(E), 9G(+) and 10G(v). Age intervals for each calf are accurately represented but absolute ages varied by ± 1 weeks from those shown. Where duplicate determinations were carried out within 5 days of each other a mean value (with standard error shown as a vertical bar) is given and shown at the mean age. Details of the diets are given in the text. No measurements of intake were made but inspection of the grazed areas showed considerable amounts to have been eaten. Calves 7 F and 9F grazed a ley of H. 1. ryegrass previously fertilized with nitrochalk (32 kg N/acre) and potassium chloride (39 kg K/acre). Calves 8G and log grazed a ley of timothy and meadow fescue previously fertilized with 31 kg N/acre and 20 kg K/acre. At about 18 weeks of age one of the calves (8G) was returned to stall feeding with 1-8 kg/day concentrate mixture and 1-1 kg/day hay. The determinations shown in Fig. 3 were made with marker (polyethylene glycol for calves 7F, 8F, 9F and phenol red for calves 8G, 9G, log) injections into the duodenum at about a.m. This was about 1 hr after the morning feed was given during stall feeding and about 1 hr

11 SMALL INTESTINE TRANSIT 315 after removing from pasture during grazing. Excluding the high and low protein stall diets the over-all means of the mean values for each calf were hr for stall feeding (six calves) and 2' hr for grazing (four calves). Within 2-4 days of some of the stall feeding determinations reported above the calves were fasted (but allowed water) for 24 hr and transit time re-determined. In each case the transit time after fasting was compared with a corresponding transit time after feeding. The results, expressed as a mean value (8 experiments with 3 calves), showed no significant difference between them; the former were % of the latter. Effect of certain drugs on transit of liquid diets from mouth to ileum Intramuscular injection of propantheline bromide ('Probanthine', G. D. Searle and Co., Ltd.), an anticholinergic drug found to inhibit gastrointestinal motor activity in humans (Roback & Beal, 1953), had a variable effect on transit from the mouth to the ileum in calves fed on 5 % glucose, 0 la 100 c~~~~~~i E 501.0PBPBN b 7 o Co / E. I uj Time after feeding (hr) Fig. 4. Cumulative emergence of polyethylene glycol marker from the distal ileum of calves aged 7-10 weeks and fed on % glucose, Mmagnesium chloride solution containing the marker. Comparative experiments, within one week of each other, were carried out with (@-@) and without (O---O) injection (I.M.) of 0 5 mg/kg body weight propantheline bromide (PB) or 0-04 mg/kg body weight neostigmine methyl sulphate (NS) at times indicated by arrows M magnesium chloride solution. In some experiments an injection of 0*5 mg/kg body weight caused an almost complete inhibition of the flow of ileal effluent within 1 hr and maintained this condition for about 4-5 hr. In other experiments, with an injection of up to 0-9 mg/kg body weight, there was little or no interference with the ileal flow or transit time (mouthileum) during the first 3-4 hr but then there was a partial or complete

12 316 R. H. SMITH inhibition of flow for about 2-5 hr. These extreme effects are illustrated in Fig. 4a, b. Oral administration of the drug (about 1-7 mg/kg body weight) produced a delayed response similar to that shown in Fig. 4b. Intramuscular injection of neostigmine methyl sulphate ('Prostigmin', Roche Products Ltd.) (0.04 mg/kg body weight), which stimulates small intestine motility in the human (Youmans, Haney, Rush & Zavin, 1941), given within 15 min of feeding 5 % glucose, M magnesium chloride solution, consistently caused a very big increase in the volume flowing from the distal ileum. In 5 experiments with 2 calves the mean volume flow for the first hour after feeding was ml. without neostigmine injection and ml. with neostigmine injection. This increased volume continued for several hours. Nevertheless, the pattern of marker emergence was not grossly affected. Neostigmine injection caused the appearance ofvery small amounts ofmarker before the main front appeared and a lower rate of marker emergence after the front appeared (these effects are exemplified in Fig. 4c), but the transit times (mouth-ileum) with neostigmine injection were not significantly different from those estimated without neostigmine injection; the former were % of the latter. DISCUSSION Transit time through the small intestine can be defined in different ways and can differ for different constituents of the digesta. In this discussion it is defined as the time for a front of water-soluble non-absorbed material to pass from the proximal duodenum to the distal ileum. In many experiments with liquid diets this was not measured directly but the results on abomasal hold-up show that the more easily determined time between giving a marker by mouth and its first arrival at the distal ileum gives, for comparative purposes, a good measure (although a small, fairly constant overestimate) of this value. Transit time defined in this way may be subject to diurnal variation since the volume and composition of abomasal effluent varies greatly as the time after feeding increases (Smith, 1962). Comparative measurements therefore were always made during the early flow of digesta following a feed. Nevertheless, it is unlikely that moderate differences in abomasal flow had much effect on transit time since even after 18 hr without food it was not drastically changed (it increased by an average of 45 % possibly due in part to a decreased magnesium concentration). Standardization of conditions was not possible in examining ruminating calves which, particularly when on pasture, eat and ruminate at indeterminate times and in which food materials pass steadily from the reticulo-rumen to the abomasum throughout the day. However, small intestine transit times in such calves were not appreciably influenced by a period of 24 hr without food so that it appears probable that small intestine

13 SMALL INTESTINE TRANSIT 317 transit times in ruminating calves are largely independent of the immediately preceding pattern of food intake. Any variations in small intestine transit time may be associated with variations in the efficiency with which slowly absorbed dietary constituents, such as magnesium, are absorbed (Smith, 1963). Two dietary factors have been clearly shown to influence this transit time in liquid-fed calves; the use of milk rather than water as the basic food component and the magnesium content of the diet. Attempts to characterize the milk component responsible for the former effect were unsuccessful. Under some conditions dietary fat (Annegers & Ivy, 1947) and pepsin/trypsin digested casein (Schneider, Bishop & Shaw, 1960) have been shown to inhibit gastro-intestinal motility but in the present experiments neither butterfat nor casein, added to a water-based diet in concentrations similar to those in milk, appeared to affect transit time. Dietary wheat gluten, examined because of its inhibitory action on intestinal motility under some conditions (Schneider et al., 1960), was also without apparent effect on transit time. Variations in the total flow rates of fluid through the small intestine are not necessarily accompanied by similar changes in the transit times of non-absorbed markers. This was demonstrated in experiments in which the flow of fluid from the distal ileum increased considerably with bacterial infection, the addition of sodium sulphate to the diet or injection of neostigmine while the transit time was usually unaffected. A further experiment showed that the addition of 144% L-lysine to a magnesium chloride solution diet caused a large increase (to about 8-10 times normal) in the volume flow rate of ileal effluent but no appreciable change in transit time (mouthileum) (unpublished observation). Thus the effect of magnesium on small intestine transit time which, despite the long history of magnesium salts as cathartics, does not appear to have been previously demonstrated, is not apparently merely a secondary result of increased water retention in the intestine. Increasing the osmotic pressure of the diet leads to slower gastric emptying in the human (Hunt, MacDonald & Spurrell, 1951). Making the reasonable assumption (see above) that small intestine transit time did not vary much during the first few hours after a feed the results on the rate of marker flow after its first appearance at the distal ileum suggest that addition of magnesium chloride to liquid diets had the opposite effect. This may have been a secondary consequence of the more rapid rate of passage of digesta through the small intestine. Similar evidence suggests that dietary wheat gluten, but not casein, caused a slowing of abomasal emptying and although another possible interpretation might be a greater leakage of feeds containing wheat gluten into the rumen, this seems unlikely.

14 318 R. H. SMITH The present results confirm preliminary observations (Smith, 1963) that, on one liquid diet, variations in small intestine transit time between calves are considerably greater than within an individual calf. It appears (Fig. 3) that individual ruminating calves also tend to have characteristic small intestine transit times which may vary considerably from one calf to another. There are no data on the effect of magnesium intake on small intestine transit time in ruminating calves. In the six calves examined however the magnesium content of the abomasal effluent was almost always between 6-12 mg/100 ml. during both stall feeding and grazing. This was not grossly different from the magnesium content of the abomasal effluent from milk-fed calves one hour after feeding (about 7-9 mg/100 ml. Smith, 1962). It seems probable therefore that magnesium concentration was not an important source of variation in small intestine transit times for experiments comparing differently fed ruminating calves and milk-fed calves. In fact, the mean small intestine transit time for stall fed ruminating calves (2.8 hr) was only slightly lower than that for milk-fed calves (3.2 hr) and was similar to what might be expected for a water-based liquid diet of similar magnesium content (Fig. 2). This gives a further example of the independence of volume of fluid flow and small intestine transit time since the volume of ileal effluent from the ruminating calves was about 5-10 times greater than that from milk-fed calves. The period when cattle are turned out to graze early spring grass after having been fed in stalls is associated with a clinical disorder characterized by hypomagnesaemia ('grass tetany'). This is believed by many authorities to be due to relatively poor utilization of dietary magnesium (Rook & Storry, 1962) although the reason for this is still unknown. The possibility that one causative factor is a decreased small intestine transit time is not supported by the present experiments. In no case was there any marked immediate change in small intestine tiansit time when stall fed calves were put out to graze spring grass. One calf showed a fall in transit time but this developed slowly over several weeks while 'grass tetany' is associated with the first change in diet. Nevertheless, differences in small intestine transit time between animals, if shown also by adults, were sufficient partly to explain the wide animal to animal variations in susceptibility to clinical hypomagnesaemia which are characteristic of this disorder (Rook & Storry, 1962). SUMMARY 1. Small intestine transit time, defined as the time for a water-soluble marker front to travel from the proximal duodenum to the distal ileum, was estimated in fistulated calves. 2. For non-ruminating calves the mean transit time shortly after a

15 SMALL INTESTINE TRANSIT 319 milk feed was 3-2 hr. Withholding food increased this time. There were considerable variations between animals but no consistent changes with age between 1 and 20 weeks. 3. Transit time decreased with increasing dietary magnesium but was longer after a milk feed than after a water feed ofsimilar magnesium content. With a water-based diet it was not appreciably affected by adding butterfat, casein, wheat gluten, sodium and potassium chlorides, sodium sulphate or glucose to the diet. Propantheline bromide (I.M.) and neostigmine methyl sulphate (I.M.) respectively inhibited and stimulated ileal effluent flow. The latter did not however appreciably affect transit time. 4. Liquid diets were held up in the abomasum for about 45 min after feeding, before appreciable flow into the duodenum began. Once begun, emptying was apparently more rapid after higher magnesium intakes and slower with wheat gluten in the diet. 5. For ruminating calves fed on a stall ration of hay and cereals etc. the mean small intestine transit time (2.8 hr) was similar to that for nonruminating calves. Transit times varied considerably from calf to calf but were not apparently affected by temporarily withholding food or, usually, by transferring the calves to spring pasture. 6. Phenol red, used as a marker in some experiments, was poorly recovered after passing through the alimentary tract to the ileum. I should like to thank Dr A. T. Cowie for carrying out all surgical operations, and Mr H. S. Hallett, B.E.M.,Mr B. D. Lawrence, Miss P. Lewis andmiss S. Castlefortechnicalassistance. REFERENCES ANNEGERS, J. H. & Ivy, A. C. (1947). The effect of dietary fat upon gastric evacuation in normal subjects. Amer. J. Physiol. 150, BALCH, C. C. (1961). Movement of digesta through the digestive tract. In: Digestive Physiology and Nutrition of the Ruminant, ed. LEWIS, D. London: Butterworths. BENZIE, D. & PHILIIPSON, A. T. (1957). The Alimentary Tract of the Ruminant, p. 17. Edinburgh: Oliver and Boyd. CAMPLING, R. C. & FREER, M. (1960). Rate of passage of inert particles through the alimentary tract of the cow. Nature, Lond., 188, GOODMAN, R. D., LEWIS, A. E., SCHUCK, E. A. & GREENFIELD, M. A. (1952). Gastrointestinal transit. Amer. J. Physiol. 169, HUNT, J. N. & SPURRELL, W. R. (1951). The pattern of emptying of the human stomach. J. Physiol. 113, HuNT, J. N., MAcDONALD, I. & SPURRELL, W. R. (1951). The gastric response to pectin meals of high osmotic pressure. J. Physiol. 115, HYDEN, S. (1961). The use of reference substances and the measurement of flow in the alimentary tract. In: Digestive Physiology and Nutrition of the Ruminant, ed. LEWIs, D. London: Butterworths. MATTSON, O., PERMAN, G. & LAGERLOF, H. (1960). The small intestine transit time with a physiologic contrast medium. Acta radiol., Stockh., 54, REYNELL, P. C. & SPRAY, G. H. (1956). The simultaneous measurement of absorption and transit in the gastro-intestinal tract of the rat. J. Physiol. 131, ROBACK, R. A. & BEAL, J. M. (1953). Effect of a new quatemary ammonium compound on gastric secretion and gastro-intestinal motility. Gastroenterology, 25,

16 320 R. H. SMITH ROOK, J. A. F. & STORRY, J. E. (1962). Magnesium in the nutrition of farm aninials. Nutr. Abstr. Rev. 32, SCHNEIDER, R., BISHOP, H. & SHAW, B. (1960). The inhibition of the peristaltic reflex by substances from protein sources. Brit. J. Pharmacol. 15, SMITH, R. H. (1958). Substances in the calf alimentary tract interfering in the determination of polyethylene glycol. Nature, Lond., 182, SMITH, R. H. (1961). The development and function of the rumen in milk-fed calves. J. agric. Sci. 56, SMITH, R. H. (1962). Net exchange of certain inorganic ions and water in the alimentary tract of the milk-fed calf. Biochem. J. 83, SMITH, R. H. (1963). Small intestine transit time and magnesium absorption in the calf. Nature, Lond., 198, YOUMANS, W. B., HANEY, H. F., RUSH, H. P. & ZAVIN, W. (1941). Observations and moving picture studies of the motility of the human small intestine. Amer. J. dig. Di8. 8,