change. The diet was changed from grass cubes to grass, then to a dry CHANGES IN SODIUM REQUIREMENT OF THE SHEEP ASSOCIATED

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1 Quart. J. Exper. Physiol. (1966) 51, CHANGES N SODUM REQUREMENT OF THE SHEEP ASSOCATED WTH CHANGES OF DET. By A. DOBSON,* D. SCOTT and J. B. BRuCE. From the Physiology Department, Rowett Research nstitute, Bucksburn, Aberdeen. (Received for publication 23rd May 1966) Observations were made on 3 sheep while their rations were changed from dried grass cubes to fresh frozen grass, then to a dry diet of hay and meals and finally back to fresh frozen grass. n spite of the low sodium intake on the fresh grass, the sheep were neither losing nor gaining sodium up to the time the change to hay and meals was made. When hay and meals were given, the excretion of sodium in the urine fell to very low levels, and the composition of the saliva changed as if aldosterone secretion were increased. Since the hay and meals ration had the highest sodium content, a net retention of sodium was observed. This retention, accompanied by an increase in liveweight, appeared to be due to a sequestration of sodium and water within the rumen. On returning to the grass ration the sodium retained during the period when hay and meals were given was rapidly lost in the urine with a corresponding fall in the liveweight. The transient changes in sodium requirement associated with certain dietary changes thus appeared to reflect changes in the amount of sodium in the rumen. Less potassium tended to be retained during the period when hay and meals were given, but more was retained when the final ration of grass was begun; these changes were smaller than the changes in sodium balance. A transient mild hypomagnesaemia accompanied an initial period of magnesium loss both times that grass feeding began, with recovery of the concentration of magnesium in the plasma during the diet of hay and meals. WHEN sheep accustomed to a diet of hay and meals were fed grass cut from a heavily fertilized pasture, a loss of sodium from the rumen was observed [Dobson and McDonald, 1963]. This loss was so large that it was presumed that sodium must have been excreted and this response would be remarkable since the grass contained little sodium. The object of the experiments reported here was to measure the sodium retention over this period of dietary change. The diet was changed from grass cubes to grass, then to a dry ration of hay and meals and then back to grass. Samples of saliva, rumen contents and plasma were taken, and potassium and magnesium retentions were observed. The loss of sodium from the sheep on changing from hay and meals to grass was confirmed but, more important, an intense retention of sodium was observed when the hay and meals diet was given. t is suggested that dietary change can induce a change in sodium requirement which is related to changes in the amount of sodium in the rumen. A brief summary of these results was given at the Second nternational Symposium on the Physiology of the Ruminant [Dobson, 1965]. * Present address: Department of Physiology, University, thaca, N.Y , U.S.A. N.Y.S. Veterinary College, Cornell 311

2 312 Dobson, Scott and Bruce METHODS Anirnmls. - Four 2-year-old Blackface ewes were fitted with permanent in. bore ebonite cannulae into the rumen several months before the observations. Since leakage around a cannula, which is exacerbated by a diet of grass, would vitiate the observations made, fibrous formation around the cannula was encouraged, in order to ensure a tight fit, by passing each cannula through a polythene disc stitched to the serosal surface of the rumen [Ash, 1957]. Diets. - A ration containing about 700 g./day dry matter was offered before and during the experiment in two equal portions at 9 a.m. and 4 p.m. The grass cubes were consumed for at least a month before observation began and this ration continued through the first 6 days of the experiment. After this the sheep were offered fresh frozen grass, then hay and meals and finally the fresh frozen grass once more. Each of these rations was consumed for 9 days. The meals consisted of two parts linseed cake and one part of crushed oats with 1% NaCl. One hundred grams (dry) of this was offered with 250 g. hay at each meal. TABLE. COMPOSTON OF RATONS (g./kg. DRY MATTER). Cubes Grass Hay Meats Na * *39 K Ca *6 Mg * Cl 7* N Crude fibre Ash Crude fat Water 140 4, The grass was cut from the first growth of a mixture of talian rye grasses sown in the spring of 1962 and dressed with 7j cwt./acre of ammonium sulphate and 7 cwt./acre of potassium chloride in two applications in March On May 28th, after the dew had evaporated, about a ton was cut using a mower set high to ensure a preponderance of leaf. The cut grass was picked up with a forage harvester, dumped in the shade, mixed and weighed into 10 kg. lots in hessian sacks. This procedure ensured a well mixed ration with minimum damage. These sacks were transported to a commercial blast freezer, frozen at F and stored at F. The time from beginning of cutting until freezing started was 4 hr., and the rapid processing ensured no spoilage. Observations on grazing the second growth of this pasture have been reported [Dobson, Scott and McDonald, 1966]. Each diet was mixed and weighed out in smaller batches suitable for feeding before the experiment began. The composition of each ration is given in Table, and the dry matter and minerals consumed are given in Table. Three sheep ate the rations provided, but the fourth refused a part of the hay offered. This sheep was also unsatisfactory since its rumen fistula leaked slightly when it was given fresh grass. Observations with this sheep have therefore been excluded even though the general pattern of changes was similar to that in the other sheep. Procedure. - The sheep were put into metabolism cages [Duthie, 1959] 3 days before observations were begun. Since separators were used for faeces and urine, a small cross contamination can be expected, but the balances are unaffected. Excess tap water was provided for drinking. The daily water consumption and the volume of

3 Sodium Requirement and Dietary Change urine were recorded and the faeces removed and weighed at 9.30 a.m. Faeces or urine samples were often bulked over 3-day periods. The food and faeces were ashed and extracted as described previously [Scott and Dobson, 1965]. Mixed saliva [Dobson and McDonald, 1963], blood and rumen contents were sampled at a.m. The volume of blood and rumen contents taken was less than 10 ml. per sample to avoid depleting the sheep of sodium. Each day at a.m. each sheep was weighed. Analytical Methods. - Sodium and potassium in saliva, plasma, food, urine, faeces and rumen contents were estimated by flame photometry with corrections for mutual interference. Magnesium in food, faeces, urine and plasma was estimated by atomic absorption spectroscopy [Willis, 1960]. Calcium in the food and plasma was determined by titration with E.D.T.A. as described previously [Scott and TABLE. MEAN DALY CONSUMPTON OF MNERALS AND DRY MATTER. Na K Mg D.M. Ration (m-mole) (m-mole) (g.) (kg.) Cubes '70 Grass 9* *82 0*73 Hay and Meals Dobson, 1965]. Plasma protein was calculated from the specific gravity of the plasma by the method of Phillips et al. [1950]. Chloride in food was estimated by the method of Sanderson [1952], following extraction by 01N HNO3 for 24 hr. at room temperature. n calculating the retention of sodium the intake of sodium in the drinking water was included. The amounts of potassium and magnesium supplied in drinking water were small enough to be neglected. The effects of treatments were generally either so large as to render statistical analysis superfluous, or so small that the biological significance of differences was doubtful. Hence no statistical analysis was undertaken, but the observations on plasma protein concentration were subjected to an analysis of variance. RESULTS Sodium Retention. - When given grass cubes and fresh grass during the first two periods, the sheep were approximately in sodium balance in spite of the marked drop in the amount of sodium consumed (fig. 1, Table ). Two sheep lost sodium following this dietary change, but the mean excursion from balance was no greater than observed with cubes. The sheep appeared not to be fully adapted to the cages before the observations began, but these departures from balance were small compared to those encountered subsequently. When the ration of hay and meals was given, very little sodium was excreted at all. During the whole nine-day period mole sodium was retained, which represented 96 per cent of the intake. The mean urinary excretion was 0.6 m-mole/day (fig. 4) and the mean faecal excretion was 1.5 m-mole/day. Both were lower than when the other diets were given. mmediately the sheep were given fresh grass again, the output of sodium in the urine increased about a hundred fold. Eighty-eight per cent (81-95%) of the sodium retained du'ring the preceeding period when the dry VOL. L, No. NO

4 314 Dobson, Scott and Bruce diet was given had been lost before balance was attained. During the last three days of both the periods when fresh grass was consumed, the retention of sodium was close to zero. The cumulative sodium retention over the 33 days of observation was 6 per cent of the intake. Cubes (mole/ 3 day) Grass Hay a Meals Grass Na ntake x+0x-x4 Ho x+x 0 ScvE ; x ~ oxj 1 Na Balance + + (mole/3day) 2 K ntake----- x0 x (mole/3 day) 0-x O : 0 F ) + + F 0 V -01C x x XOl l x - K Balance + x Time (day) FG. 1. The intake and balance of sodium and potassium over 3-day periods during 3 changes of diet. ndividual balances are given for 3 sheep and the solid line represents the mean for each 3-day period. The same symbol for each sheep is retained throughout the figures. Potassium Retention. - Over the whole 33 days of observation 5 per cent of the total potassium intake was retained. There was no marked change in potassium retention during the last 3 days of feeding the grass cubes and the following 9 days when fresh grass was given (fig. 1). However, when the diet of hay and meals was given, the potassium retention was progressively reduced with each successive 3-day period. On returning to the grass diet, a greater retention of potassium took place over the first 3-day period, but during the following six days the retention was similar to that noted when the first two diets were given. The changes in potassium retention tended to be of the opposite sign but somewhat smaller in magnitude than

5 Sodium Requirement and Dietary Change 31-5 the changes in retention of sodium. Expressed on a molar basis the results for potassium retention were more erratic than those for sodium due, no doubt, to the much higher intake. Magnesium Retention. - The cumulative retention of magnesium during 33 days of observation was 6 per cent of the intake. On both occasions when grass feeding was begun the magnesium retention became negative (fig. 2). Maximal retention was observed during the first 3 days of feeding the diet of (g./ 3 day) 4 - O - (g./ 3d Cubes -----, :Mg ntake r L. Grass Hay a Meals Gross 2 18 Time (day) FG. 2. The intake and balance of magnesium over 3-day periods during 3 changes of diet, together with concentration of magnesium in the plasma of 3 sheep. hay and meals, but this gain in magnesium was partly compensated in the following collection period, when one sheep lost most of this retained magnesium. Estimates of Changes in Body Water. - The apparent water retention was calculated by subtracting the sum of the urine volume and faecal water from the sum of the moisture in the feed and the water drunk (fig. 3). While consuming the diet of hay and meals each sheep retained more water than when grass cubes were given. This excess retention occurred mainly during the first six days when hay and meals were given. By a similar comparison between the two grass periods, an excess loss of /sheep during the second period of grass feeding was observed, mainly during the first 3-day period.

6 316 Dobson, Scott and Bruce Changes in liveweight also reflect in the main cumulative changes in total body water. Although somewhat erratic, the weight increase during the period when hay and meals were given and the decrease in the following period on grass were conspicuous. The mean weights for the three sheep over the last three days of cubes, the first fresh grass, hay and meals, and the second fresh grass diets were 41.0, 42.0, 43.4 and 41-7 kg. respectively (fig. 3). Cubes Grass Hay a Meals Grass 2 l (./3day) Apparent H20 Balance + 0 o (Kg.) Live Weight , Time (day) FG. 3. The apparent water balance over 3-day periods and liveweight of 3 sheep during 3 changes of diet. Salivary Composition. - The concentration of potassium in the samples of mixed saliva remained low throughout the experiment except when hay and meals were ingested (fig. 4). The potassium concentration rose promptly after starting this diet and for the following four or five days was two or three times the level that was observed with the other diets. After the fifth day the concentration of potassium fell, and, in two of the three sheep, had reached the baseline established during the rest of the experiment by the time the diet was changed again. During the two periods when fresh grass was given, the potassium concentration was lowest over the first three days, when sodium excretion in the urine was maximal. The sodium concentration in the saliva tended to vary in the opposite

7 Sodium Requirement and Dietary Change 317 direction to the concentration of potassium so that the sum of the concentrations of these two ions remained approximately constant. Composition of Rumen Fluid. - The concentration of potassium in the rumen contents was high with the fresh grass diets, intermediate with the grass cubes and low on hay and meals ration (fig. 5). t thus reflected the potassium intake. The sodium concentration was high with the two dry (MM) Cubes Grass Hay a Meals Grass 2 Salivary K Concn. j (m-mole/day) loo Time (day) FG. 4. The concentration of potassium in the saliva, and amount of sodium excreted in the urine of 3 sheep during 3 changes of diet. Note the logarithmic scale for output of sodium in urine. rations and low on the fresh grass. After the first few days on each new ration, the changes in concentration of sodium and potassium were almost complete. The mean concentrations in the last samples taken during each experimental period were as follows; cubes, Na 81 mm, K 63 mm: Grass 1, Na 48 mm, K 77 mm: hay and meals, Na 89 mm, K 32 mm : Grass 2, Na 53 mm, K 85 mm. Plasma Composition. - n both periods when fresh grass was eaten a mild hypomagnesaemia was observed (fig. 2). The minimum concentration of magnesium was reached on the second or third day when grass was given. Thereafter, the concentration of magnesium tended to rise. The concentration of magnesium in the plasma on the diet of hay and meals rose after the sixth day to a similar level to that on cubes.

8 318 Dobson. Scott and Bruce (mm) 100 r. Cubes z9l.. Gross Hay and Meals Grass 2 Rumen Na Concentriation 50 F 80k 60 0-SK Rumen K Concentration L Time (day) FG. 5. The concentration of sodiuim and potassium irn rumen fluid of 3 sheep during 3 changes of diet. - (mm) [ Na 140- (mm) 6 - Cubes Grass Hay s Meals Grass 2 O,A 4 L-o ' Time (day) FG. 6. The concentration of sodium, potassium and protein in the plasma of 3 sheep during 3 changes of diet.

9 Sodium Requirement and Dietary Change 319 The changes in concentration of the other plasma constituents were not particularly marked (fig. 6). The concentration of sodium was somewhat lower initially on two of the rations, the first period when fresh grass was given, and also when hay and meals was given. However, by the end of each period, the differences between diets had largely disappeared; the mean concentration of sodium in the plasma for the three sheep was 144, 145, 143 and 145 mm on the last day of each diet in the order in which they were fed. The potassium concentrations showed little consistent trend apart from a peak on the second day of the first period of eating grass. This was not observed the second time grass was fed. The mean of the concentrations at the end of each three-day period were 4.5, 4.5, 4.5 and 4.8 mm for the four diets in order. There was no suggestion that changes in potassium intake were reflected in the plasma levels, except for the peak already noted. The concentration of protein in the plasma was, on an average, about 5 per cent (P < 0.05) lower when fresh grass was given than on the two dry rations. There was a suggestion that the fresh grass gave a lower plasma protein concentration the second time it was consumed than it did the first time. There was no evidence of any sequential change between days with any of the rations. Faecal Changes. - The output of dry matter in the faeces was very similar in the last two collection periods on the two dry rations, but the grass was much more digestible. Using the mean apparent digestibility coefficient, namely 100 x (intake-output)/intake, the results during the last two collection periods for each ration were 62 and 66 per cent for cubes, 82 and 85 per cent for Grass 1, 63 and 63 per cent for hay and meals, 84 and 82 per cent for Grass 2. DSCUSSON The change from a dry diet of hay and meals to one of fresh grass was accompanied by a rapid and consistent loss of 0.4 mole of sodium in the urine (fig. 1, fig. 4). This was expected from the results of the previous experiments in which the fate of sodium in the rumen following a similar dietary change was studied [Dobson and McDonald, 1963]. This loss took place at a time when the animal was passing from a ration with a moderate amount of sodium to one with a very low sodium content, and thus appeared to be an inappropriate response. n the dietary change from fresh grass to hay and meals we expected a transitory retention of sodium, if only to replenish the quantity of sodium in the rumen, but the intensity of the retention of sodium following the dietary change came as a surprise. n spite of the higher sodium content of this dry ration, sodium virtually disappeared from the urine even on the first day that the hay and meals were given, and showed no sign of increasing until after six days. The increase in potassium concentration in the mixed saliva was closely related to the change in diet, and can be taken as evidence of increased

10 320 Dobson, Scott and Bruce levels of aldosterone in the plasma. This response was much more consistent among these animals than has been noted previously either in sheep kept indoors or when grazing [Dobson and McDonald, 1963]. Although the saliva sample was representive of only a short part of the feeding cycle, the low excretion of sodium in the urine indicated that an effective stimulus to sodium retention lasted throughout each day. Sheep fed for some time on a similar diet of hay and meals showed no tendency to have raised levels of potassium in the saliva and so the change here must have been transitory. ndeed, the potassium concentration in the saliva appeared to fall five or six days after the change from grass to hay and meals, and this was reflected in a slight rise in excretion of sodium in the urine over the last days in which this dry ration was given. The sheep apparently adapted rapidly to the low dietary intake of sodium the first time they were fed grass. Although two of the sheep lost a little sodium when they first ate the grass, during the rest of the period they were in sodium balance. There was no evidence of a loss during the first period of grass similar to the gain when eating hay and meals nor was there any evidence of sodium deficiency when the diet of grass cubes was given in the preceeding period. n addition, on changing the diet back to grass the sodium retained from the ration of hay and meals was almost quantitatively excreted. This indicated that the sheep had no large deficit of sodium at the end of the first grass feeding period. Since this dry ration itself was not deficient in sodium the avid retention of sodium associated with the dietary change from fresh grass to hay and meals could therefore be attributed to a temporary increase in sodium requirement associated with the dietary change. n order to explain the changes in sodium requirement over the final two dietary changes it would be desirable to partition the transitory retention of sodium between the three main body compartments involved, the gut and the extracellular and intracellular fluids. Our experiments do not, however, permit this to be done with certainty because there were no direct observations of changes in any of these compartments. Some of the changes, however, may be inferred. The apparent water retention suggested that water were gained during the period on hay and meals. The mean weight increase from the end of the period on cubes until the end of the period on hay and meals was 2.3 kg. This occurred almost entirely during the period on hay and meals, since the increase from the end of the period on grass cubes to the end of the first period when grass was given, namely 1.0 kg., could be attributed to the greater weight eaten on the grass diet. Similarly both the weight loss during the second period when grass was given and apparent water balance change suggested that most of the water retained during hay and meals was subsequently lost. The plasma protein concentration was slightly higher during the period on hay and meals than on either grass ration. This was compatible with a decrease of about of extracellular fluid during the hay and meals period. The increase in body water over this period was thus due to an

11 Sodium Requirement and Dietary Change 321 expansion of gut or intracellular water to the extent of This can tentatively be ascribed to changes in rumen volume. n an analogous situation, the transfer of cattle to pasture, the loss in body weight was almost entirely due to loss of rumen fill [Balch and Line, 1957]. f an expansion of is taken in conjunction with the higher sodium concentration in the rumen, more than 0.3 mole of the sodium retention on hay and meals, which amounted to 0.45 mole during the nine-day period, would be accounted for by an increase in sodium in the rumen. The sudden excretion of sodium in the urine on the following diet would then largely reflect a displacement of sodium from the rumen. While the amount of sodium in the rumen was increasing on the ration of hay and meals, there was a suggestion that the extracellular sodium was minimal. Compared to the periods on grass, the plasma protein was high, and the sodium concentration was more consistently low. The evidence of increase in aldosterone secretion at this time, namely the raised concentration of potassium in the saliva and the reduced output of sodium in the urine, confirms that the tissues of the animal were depleted of sodium. Perhaps the simplest hypothesis is to regard the depletion of the extracellular space as a consequence of the accumulation of sodium in the rumen. Each day the sheep can secrete an amount of sodium in the saliva approximately equal to that in the extracellular space [Kay, 1960]. The prompt absorption of this sodium is therefore necessary in order not to deplete the sodium in the extracellular fluid. A time lag between secretion and absorption appears to give rise to changes in volume and composition of the urine [Stacy and Brook, 1964]. A greater amount of sodium in the rumen on hay and meals would reflect an increased lag between secretion and absorption of sodium compared with the grass. The amount of sodium in the rumen is determined by an interaction between the rate of inflow of sodium in saliva and food, the rate of absorption through the rumen wall and its rate of passage on down the gut. t is possible that the high intake of salts of the grass ration would give a faster volume outflow since potassium is not as readily absorbed as sodium, and osmosis tends to keep the osmotic pressure near the plasma level. n addition the coarseness of the diet may be important, since in calves a 30 per cent increase in rumen fluid has been associated with a high proportion of poor quality hay, in spite of a lower intake of dry matter and ash [Nicholson, Loosli and Warner, 1960]. The dry matter intake, and its overall digestibility are not the sole factors, since these were similar with the rations of hay and meals and dried grass cubes. However, it is apparent that much more must be known about factors controlling the volume, composition and outflow from the rumen before further analysis of these changes becomes profitable. An undersanding of these factors is likely to be of more than academic interest. since it appears that a low sodium intake alone does not account for changes in salivary composition of the grazing ruminant [Dobson, 1965] indicative of increased aldosterone secretion. When- discussing ruminants under arid conditions, Blair-West et al. [1963] state, without details, that the change

12 322 Dobson, Scott and Bruce from a lush to a dry diet under laboratory conditions is accompanied by a decrease in Na/K ratio of the saliva. An increase in ruminal sodium at the expense of extracellular sodium presupposes that the sodium in the rumen is outside the extracellular space for the purposes of regulation of extracellular sodium. This concept was introduced [Beilharz and Kay, 1963] to explain certain aspects of salt appetite in the sheep. t has been suggested that the sodium in the rumen forms a store which can be drawn upon at a time of reduced dietary intake [Denton, 1957: Kay and Hobson, 1963]. Although in other circumstances this function of the rumen may be important, during the dietary changes reported here, the sodium accumulated within the rumen on one diet was not available for the use of the animal on another, since most of it was jettisoned at the stage when the dietary intake was low. The potassium retention.tended to be positive throughout the experiment This may have been due in part to the high content of potassium in suint, the secretion retained in the wool [Blaxter and Rook, 1957]. t was possible that the declining potassium retention during hay and meals was due to the displacement of potassium from the rumen by sodium. f the rumen volume was expanding at this time, less potassium would be lost than the gain in sodium. The opposite changes in the rumen could account for the increased retention following the change back to grass. Hypomagnesaemia was produced by feeding this grass, as indeed it was produced by grazing grass receiving similar fertilization [Dobson, Scott and McDonald, 1966]. However, the effect indoors was not associated with a rise in the concentration of potassi-um in the saliva, as was found outdoors. The three largest excursions in magnesium balance were associated with periods of dietary change, with the two negative balances corresponding with a drop in plasma magnesium, and the positive balance corresponding with a rise in the plasma magnesium. A change of 1 mg./100 ml. plasma would involve about 60 mg. of magnesium in the extracellular space. Thus the changes in retention were sufficient to account for the decrease in concentration observed in the plasma providing no other compartments were involved. The effect on the balance and plasma level appeared to be transient since the balance rapidly returned to normal and the plasma concentrations rose after passing through a minimum on the second or third day. Once again the dietary change itself appeared to be important, rather than the change in mineral intake itself. ACKNOWLEDGMENTS We wish to thank Professor A. T. Phillipson and Dr. K. B. Blaxter for advice and criticism of the manuscript. Appreciation is also offered to Mr. J. ngram and Mr. W. Fettes for skilled technical assistance during the course of these experiments. We also wish to thank Dr. F. Raymond, Grassland Research nstitute, Hurley for advice on the cutting and storing of the frozen herbage.

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