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Influence of a Low Chloride Practical Diet on Acid-Base Balance and Other Factors of Blood in Young Dairy Calves1
I n f l u e n c e of a L o w C h l o r i d e Practical D i e t o n Acid-Base Balance and O t h e r Factors o f B l o o d in Y o u n g D a i r y Calves t D. L. BURKHALTER, M. W. NEATHERY, W. J. MILLER, R. H . W H I T L O C K 2,
J. C. ALLEN, and R. P. GENTRY Departments of Animal and Dairy Science and Large Animal Medicine University of Georgia Athens 30602
ABSTRACT
INTRODUCTION
Control (.5% chloride) or low-chloride (.038% chloride) practical diets and low-chloride (.00038% chloride) well water were fed to male Holstein calves for 7 wk. Substantial alterations in characteristics of blood associated with acid-base balance developed in those fed the low-chloride diet. Along with reduced potassium in plasma, concentrations of chloride in synovial fluid, saliva, and plasma also were reduced. Solids in plasma, blood pH, packed cell volume, carbon dioxide pressure (PCO2) in blood, and bicarbonate were increased. Most of the changes in plasma occurred within 1 wk with little change thereafter, indicating an adaptation by the calves to the lowchloride diet. The adaptation by the low-chloride calves was primarily due to a tremendous reduction in urinary chloride excretion. Change in blood factors was insufficient to cause severe alkalosis in calves. The low-chloride diet had no significant effect on sodium metabolism as measured by sodium absorption, retention, or distribution in body fluids. Potassium in plasma and aqueous humor were reduced in calves fed the low-chloride diet, but potassium concentrations in other body fluid were not affected significantly.
Next to sodium (Na), the chloride (C1) ion is the most common ion and is b y far the most common anion in the extracellular body fluids of animals. In vertebrates, the CI concentration [CI-] in body fluids normally is regulated within narrow limits. However, a severe derangement of the acid-base system and metabolic alkalosis could occur under conditions of dietary insufficiency of C1 or by excessive C1 losses from the body due to profuse sweating and by some pathological defects (kidney damage) affecting C1 metabolism (8). The influence of feeding a tow-el practical diet on the various factors affecting acid-base balance have not been investigated thoroughly in cattle. Likewise, there is little information on the effects of low-C1 intake on Na and potassium (K) metabolism in cattle. However, the interaction of Na and C1 in their influence on various acid-base characteristics has been studied in several other species (10 to 12). The objective of this research was to study the effects of a low-el diet on the acid-base balance and other blood factors in young dairy calves. The effects of low C1 intake on Na and K metabolism also were investigated.
Received August 27, 1979. 1Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations. ZSchool of Veterinary Medicine, New Bolton Center, Kennett Square, PA 19348. 1980 J Dairy Sci 63:269--276
EXPERIMENTAL PROCEDURES A control, practical-type diet containing .5% C1 was fed ad libitum for a 1-wk standardization period to eight male Holstein calves which averaged 78 kg and 2 mo of age and which were in wooden metabolism crates. The calves were divided into two groups balanced for feed intake, body weight, and plasma CI. The groups were assigned at random to the control diet or a tow-C1 diet (.038% Ct) for a 7-wk experimental period. The basal diet was 269
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59% cracked yellow corn, 22% soybean meal, and 15% cottonseed hulls with adequate minerals and vitamins. Sodium bicarbonate replaced sodium chloride of the low-C1 diet in amounts to equalize the Na content of the diets. In dry matter, the diets contained (by analyses) .35% Na and .98% K. Both groups received well water ad libitum containing .00038% CI, .00041% Na, and .00021% K. Feed and water intake were measured daily, and body weight was measured once each week. Special precautions were taken to avoid contamination between animal groups with extraneous C1. The two groups were separated in the metabolism barn by about 9 m. Calves always were handled (feeding, weighing, sampiing, etc.) in order of low CI calves first. Fecal and urine collection containers were washed with low-C1 water. Workers wore disposable plastic gloves for handling calves. Total fecal and urinary excretions were measured during the first 4 days of standardization and the first 4 days of each week during the 7-wk experimental period. Feces were mixed thoroughly, and about 100 g of each collection were composited daily. Feed samples were dried, ground in a Wiley mill, and analyzed for [C1-], [Na+], and [K +] concentrations. Urine samples also were composited and analyzed for [CI'], [Na+], and [K+]. Saliva was collected weekly by procedures described by Whitlock et al. (18) with modifications. A clean synthetic sponge gripped with long forceps was placed in the submaxillary area of the jaw. The sponge absorbed mixed saliva that then was squeezed into test tubes. Care was taken to obtain relatively contamination-free samples. The mouth was cleared of feed prior to sampling, and any saliva appearing contaminated was discarded. Samples were centrifuged, and the supernatant was analyzed for [C1-], [Na+], and [K+I. Jugular venous blood was collected three times during standardization and the first 2 wk of the experimental period, twice weekly
3Models 213 and 329, manufactured by International Laboratory, Inc., Lexington, MA 02173. 4 Model CR, manufactured by International Equipment Co., Needham Heights, MA 02194 s Model 10400, manufactured by American Optical, Keene, NH 03431. Journal of Dairy Science Vol. 63, No. 2, 1980
during the 3rd wk of the experimental period, and once weekly thereafter. Twenty-five milliliters of blood were drawn from the jugular vein into heparinized test tubes which were put on ice along with the remaining 15-ml sample left in the syringe. Care was taken to expel all air from the syringe. The syringe was made airtight by sticking the needle into a rubber stopper. Samples then were transported immediately to the laboratory where a 1-ml sample o f blood was injected from the syringe into a blood gas analyzer 3 for pH and PCO2 measurements. The [HCO3-] was calculated from these data by the Henderson-Hasselbalch equation (17). Packed cell volume (PCV) was determined with a microhematocrit 4 . Plasma was harvested following centrifugation of the heparinized blood and analyzed for [CI-], [Na+], and [K+]. Plasma total solids were determined by refractometry s . Total b o d y chloride was estimated from data collected during the experiment and from published data. Blood volumes of the body were obtained by multiplying the b o d y weight by 7% (7). Plasma, as a percentage of whole blood, was calculated by subtracting the determined percentage packed cell volume from 100%. Total plasma chloride was estimated by multiplying the percent chloride in plasma by total plasma volume. Before necropsy each calf was sedated with 100 mg Xylazine ( R o m p u m obtained from Haver-Lockhart Laboratories) at approximately 5 to 10 min prior to receiving 5 to 7 ml of euthanasia solution containing approximately 25% pentobarbital. Synovial fluid samples were taken from the right carpus and specifically the carpal-metacarpal joint space. Cerebrospinal fluid (CSF) was obtained by an 18-gauge 8.75 cm spinal needle inserted into the alantoocipital space with the head in the flexed position. A sample of aqueous humor was taken from the anterior chamber of the eye by aspiration with a syringe and 18-gauge needle, tn all cases, fluid samples were taken prior to death. Calves were bled by cannulation o f the carotid artery. These samples were centrifuged and the supernatant fluids analyzed for [C1-], [Na+], and [K +] . Samples of feed, water, urine, feces, saliva, plasma, and other b o d y fluids were extracted with nitric acid and shaken. Feed and feces samples in solution also were filtered. Chloride in the solution of these samples was determined
METABOLISM OF CHLORIDE IN CALVES by the specific ion electrode manual method, by a Corning 125 pH meter 6 equipped with Ct and double function reference electrodes. Millivolt readings then were plotted on a standard curve to determine chloride. Urine, water, plasma, and other body fluids were centrifuged, and the Na and K content of the supernatant was measured directly by aspiration of the liquids into an emission flame photometer equipped with a lithium (Li+) internal standard 7. Feed and fecal samples were wet ashed with nitric-perchloric acid and filtered before analysis (1). Variance was analyzed as described by Steel and Torrie (14). RESULTS AND DISCUSSION
Lowering dietary CI from .5% to .038% caused a reduction (P<.01) in plasma [CI-] concentration (Figure 1A) and in total plasma C1 (Figure 2A). The increase in total plasma chloride in both groups of calves with respect to time on the diets (Figure 2A) was due to an increase in body weight gain. Concentration decreased rapidly within the 1st wk, then remained constant for the remainder of the experiment. The reduced steady state of concentration of plasma CI from wk 2 through wk 7 in the low-C1 fed calves, following the early rapid decrease, indicated that some adaptation had occurred and that concentrations were in equilibrium with CI intake. The major adaptation was a rapid decrease in urinary concentration of C1. By the 3rd wk there were only barely detectable amounts in the urine (Figure 2C). Total urinary and fecal C1 losses were less (P<.01) in the low CI fed calves (Table 1). Earlier studies have indicated that the main homeostatic control mechanism in C1 metabolism in most species is endogenous excretion via urine (4, 5, 17). Other excretion routes for C1 of minor importance include feces and sweat (4, 17). In spite of the drastic reduction in dietary C1 intake, the low-C1 fed calves maintained a positive C1 balance as calculated from total plasma C1 (Figure 2A). Since body C1 is mostly
6Model 125, manufactured by Corning Glass Works, New York, NY 10001. Model 143, manufactured by International Laboratory, Inc., Lexington, MA 02173.
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Figure 1. Plasma [CI-] (A), [Na+] (B), and [K+] (C) concentrations in dairy calves fed a control, practical diet (.5% C1) or a practical, low-chloride diet (.038% C1) for 7 wk. Both groups received the control diet for a 1-wk standardization (STD) period. SE = standard error of treatment means calculated from error mean square with 4 calves per treatment.
in extracellular fluid (about 84%), of which plasma may be a major part, total plasma C1 gives a reasonable estimate of total body C1. However, extracellular fluids other than plasma, e. g., cerebrospinal, aqueous humor, synovial, Journal of Dairy Science Vol. 63, No. 2, 1980
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and saliva, contain some C1 (6, 17)• In the calves, calculated total plasma C1 increased 32% over the 7-wk feeding period compared to 64% for controls (Figure 2A). Since growth rate of the two groups was similar (1.13 kg vs. 1.23 kg/day), the major difference
low-Cl
Journal of Dairy Science Vol. 63, No. 2, 1980
in body C1 was due to the Cl concentration in plasma. The 64% increase in plasma CI was comparable to the 77% increase in body weight of the control group• However, the 32% increase in plasma C1 of the low CI calves was less than half of the 71% increase in their weight. One may wonder whether a continuation of the low C1 diet would cause serious consequences. The data indicate that the total plasma CI differences were not becomming wider at the end of the 7-wk period (Figure 2A). This suggests that the calves probably had begun to adapt to the low C1 intake. Lowering the dietary C1 to .038% caused significant alterations in the measures associated with acid-base balance. Blood pH increased (P<.01) rapidly within the 1st wk in the low-C1 calves; then higher pH remained parallel to those of controls until the end of the experiment (Figure 3A). Over the 7 wk, blood pH in the low-C1 calves increased from 7.41 to 7.50 as compared to 7.42 to 7.43 for controls. The blood pH of 7.50 in the low-Cl calves during the 7th wk slightly exceeded the normal pH range of 7.27 to 7.49 quoted for cattle (2). Although the differences between the two groups were statistically significant (P< .01 ), the increase in blood pH of the low-C1 calves did not produce severe alkalosis. With a steady intake of .038% C1 (which was absorbed almost completely), extremely little C1 was in urine by the 2nd wk of treatment; by only minor losses in feces and sweat, CI homeostasis in plasma and blood pH were maintained effectively. Because of the effective C1 homeostasis, growth rate of the calves was not affected adversely for the remaining 5 wk. It is u n k n o w n whether the calves could have remained on the low CI diet indefinitely without blood pH increasing sufficiently to produce severe alkalosis. In the calves fed low CI, the rise in blood pH was associated with a corresponding rise in PCO2 and [HCO3"] (Figures 3B and 3C). The increase (P<.01) in blood [HCO3-], as a result of reduced plasma [CI'], may represent an adaptation by the animal to offset the increased pH. The bicarbonate disassociates into carbonate and free [H+], thus lowering the pH to the original level. In our study, the amount of [HCO3-] produced was not sufficient to restore fully blood pH in the low C1 calves to control pH. Phillips (13) reported that the normal mean bicarbonate concentration of adult cattle
METABOLISM OF CHLORIDE IN CALVES
273
TABLE 1. Salivary, urinary, and fecal chloride and sodium and potassium in calves fed control or low-chloride diets for 7 wk. Diets a
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SEb
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23.56 62.70 13.22
.98 3.70 1.40
18.21 * 75.97 18..45
1.45 4.48 2.41
Feces Chloride, g/day Sodium, g/day Potassium, g/day
1.38 1.76 3.57
.208 .425 .666
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.086 .180 .564
Urine Chloride, g/day Sodium, g/day Potassium, g/day
17.53 11.88 50.76
1.13 .332 2.90
.93 * * 11.98 51.18
.144 1.50 4.46
acontrol diet contained .5% CI; low-chloride diet, .038% C1 (dry basis). bFour calves per treatment. CThe mineral values for feces and urine represent an average of 7 balance trials of 4 days each with 4 calves per treatment: mixed saliva is an average of 7 weekly samples. *P<.05. **P<.01.
blood was 23.4 mEq/liter and that 95% of the individual observations fell between 20.2 and 26.6 mEq/liter. The mean [HCO3-] in steers (about 336 kg body weight) was 28.0 -+ 2.12 mEq/liter. Our 31.3 mEq/liter for control calves agrees reasonably well with the literature, but the 39.7 mEq/liter of the low-C1 calves is considerably higher. Increased lactic acid in plasma due to physical stress causes a decrease in plasma bicarbonate (13). Since these blood samples were taken under minimum stress, this probably did not affect blood [HCO3-] of the calves. Water balance was not maintained in the low-C1 calves. Likewise, symptoms of polydipsia and polyuria became more pronounced with time in the calves fed low CI. Thus, a dehydrating effect occurred as indicated by increased total solids in plasma and percentage PCV in blood (Figures 4A and 4B). This effect of dietary C1 has been reported in other species (16). The concentration of Ct in mixed saliva of the low-C1 calves was substantially less (P<.05) over an average 7-wk experimental period than
in controls (Table 1). The concentration was reduced significantly (P<.05) beginning with the 3rd wk (Figure 2B). The C1 concentration of mixed saliva in control calves was comparable to 22.9 mEq/liter in parotid saliva of saltsupplemented lactating cows as reported by Whitlock et al. (18), but it was higher than reported by Coppock et al. (5) in lactating cows receiving diets containing .4% C1. Coppock et al. (5) reported that chloride in parotid saliva was not reduced significantly in lactating cows fed a low C1 diet (.18% C1) for 11 wk as compared to cows fed the control diet (.4% C1). Their data indicated that saliva Cl concentration did not reflect dietary C1 intake truly. Chloride, Na, and K content of the cerebrospinal, aqueous humor and synovial fluid o f the bovine have not been reported. In some other species, plasma is hypotonic to CSF with ratios of C1 content in CSF to that in plasma ranging from 1.07:1 to 1.15:1 (6, 15). In our study, Cl contents of synovial and ruminal fluids were considerably less (P<.O1) in the low-Cl fed calves (Table 2). Cerebrospinal, aqueous humor, bile, and abomasal fluid C1 was Journal of Dairy Science Vol. 63, No. 2, 1980
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not (P<.05) affected by dietary C1. Maintaining an adequate C1 concentration is important for synthesis of hydrochloric acid in the abomasum. Hydrochloric acid is essential for normal Journal of Dairy Science Vol. 63, No. 2, 1980
p r o t e i n digestion in t h e a b o m a s u m . P r o t e i n m e t a b o l i s m was a f f e c t e d severely in t h e c h l o r i d e d e f i c i e n t rat (16). T h e C S F C1 in t h e calves was c o n s i d e r a b l y l o w e r t h a n t h a t in t h e cat (3), dogfish (9), r a b b i t , dog, goat, a n d h u m a n (6). T h e ratios o f C S F to p l a s m a C1 at t h e e n d o f t h e 7-wk e x p e r i m e n t were .59:1 a n d .66:1 for c o n t r o l and low-C1 calves. T h e r e a s o n f o r t h e large d i f f e r e n c e s b e t w e e n calves a n d o t h e r species is n o t k n o w n . L o w e r i n g t h e d i e t a r y C1 t o .038% h a d little e f f e c t o n Na m e t a b o l i s m in calves. S o d i u m c o n c e n t r a t i o n s in u r i n e , feces, plasma, saliva, a n d in all o t h e r b o d y fluids s a m p l e d were n o t s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s (Tables 1 a n d 2). In t h e r a b b i t , cat, dog, goat, a n d h u m a n , t h e Na c o n t e n t o f C S F was slightly less t h a n t h a t o f p l a s m a w i t h C S F to p l a s m a Na r a t i o s
METABOLISM OF CHLORIDE IN CALVES
275
TABLE 2. Chloride, sodium, and potassium content of body fuids in calves fed control and low-chloride d i e t s for 7 wk. Chloride Body fluids c
Control a
Low chloridea
Sodium SE b
Controla
Low chloridea
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Low chloridea
SE b
.49 3.9 2.3 ...d 4.0 2.8 .23
4.4 .97 5.1 .... d 2.6 35.8 72.0
3.5** .98 3.8" .... d 1.9 35.8 66.0
.07 .10 .32 ...d 21" 3.0 3.2
(mEq/liter) PlasmaC Cerebrospinal Aqueous humor Synovial Bile Abomasal Rumen a
.
104.7 62.1 103.3 100.1 21.9 124.1 14.5
92.0** 61.2 95.9
88.9" * 16.5 128.3 8.2**
.54 7.7 2.2 1.6 1.6 4.4 1.2
143.5 45.3 139.3 .... d 59.8 25.9 73.8
142.6 47.4 141.0 .... d 51.7 35.3 77.2
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Control dmt contained .50% chloride; low chloride diets, .038% chloride. bFour calves per treatment. Cplasma chloride, sodium, and potassium concentrations determined during the 7th wk of experiments. Cerebrospinal, aqueous humor, and synovial fluid values were obtained while animals were under anesthesia just prior to death; other during necropsy. dsynovial fluid sodium and potassium were not determined due to the fluid being too viscous to be aspirated properly into the flame photometer. *P<.05. * *P<.01.
ranging f r o m .95:1 to 1.00:1 (6). The ratios o f CSF Na to plasma Na at the end o f the 7-wk e x p e r i m e n t were .97:1 and .99:1 for control and low-C1 calves. Thus, calves are similar to o t h e r species. In man, ratios of Na in synovial fluid and a q u e o u s h u m o r to that in plasma were 1.21:1 and 1.30:1 (16). T h e r e is little i n f o r m a t i o n on effects o f feeding a low-C1 diet on Na m e t a b o l i s m in cattle. In swine, feeding a low-C1 diet significantly reduced C1 and o n l y slightly reduced Na in plasma. However, feeding a low Na and normal C1 diet caused a significant decrease in plasma CI and a small decrease in Na. Evidently b o d y Na, at least in swine, is regulated m o r e critically in the b o d y than C1. Beginning with the 2nd wk, plasma K was reduced (P<.05) in the low-Cl calves (Figure 1C) and by the 7th wk, plasma K was a b o u t 80% o f control (Table 2). Potassium is n o t a major cation in plasma, and the explanation for this effect o f dietary C1 on plasma K is not evident. However, these results agree with reduced plasma K in lactating cows fed a .18% C1 diet (5). The low CI diet had no significant effect on fecal or urinary K excretion, on K c o n c e n t r a t i o n
in saliva, or in most b o d y fluids sampled (Tables 1 and 2). In a d d i t i o n to plasma, K c o n t e n t of s o m e o t h e r b o d y fluids was reduced slightly in the low C1 calves with differences f r o m controls in aqueous h u m o r being statistically significant (P<.05) (Table 2). The ratios of K c o n t e n t of CSF to those of plasma in rabbit, cat, dog, goat, dogfish, and h u m a n range f r o m .62:1 in humans to 1.12:1 in the dogfish (6). The ratios of CSF K to plasma K were .22:1 and .28:1 for control and low-Cl calves. Like Na, K was slightly higher in saliva o f the low-C1 calves (Table 1). The increase of these t w o cations in saliva w h e n C1 is lowered agreed with (18). Results of this research with cattle agree with those f r o m o t h e r species indicating that the acid-base system is a highly regulated system. In spite of a drastic r e d u c t i o n in dietary CI, b o d y CI was maintained f u n c t i o n a l for 7 wk. A l t h o u g h various factors associated with acid-base balance were altered significantly, these changes were insufficient to cause severe alkalosis in the calves. The t r e m e n d o u s r e d u c t i o n in urinary C1 e x c r e t i o n in the low C1 calves was the primary h o m e o s t a t i c m e c h a n i s m by which b o d y CI was maintained. More research is Journal of Dairy Science Vol. 63, No. 2, 1980
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BURKHALTER ET AL.
n e e d e d t o d e t e r m i n e t h e d i e t a r y C1 r e q u i r e d b y cattle a n d / o r if feeding t h e .038% C1 longer w o u l d p r o d u c e severe m e t a b o l i c alkalosis in calves.
ACKNOWLEDGMENTS
The a u t h o r s gratefully a c k n o w l e d g e R o b e r t A. Isaac and William C. J o h n s o n , Jr., o f t h e Georgia C o o p e r a t i v e E x t e n s i o n Service Soil Test L a b o r a t o r y for their t e c h n i c a l advice and assistance c o n c e r n i n g t h e c h l o r i d e analysis o f samples. Special t h a n k s are e x t e n d e d to Sheila Heinmiller for h e r overall supervision o f e x p e r i m e n t a l animal care.
REFERENCES
1 Allan, J. E. 1961. The determination of zinc in agricultural materials by atomic absorption spectro photometry. The Analyst 86(1025) : 530. 2 Altman, P. L., and D. S. Dittmer. 1964. Page 259 in Biology data book. Fed. Am. Soc. Exp. Biol., Washington, DC. 3 Bourke, R. S., H. L. Gabelnick, and O. Young. 1970. Mediated transport of chloride from blood into cerebrospinal fluid. 4 Church, D. C. 1971. Digestive physiology and nutrition of ruminants. Vol. 2. Oregon State Book Store, Inc., Corvallis. 5 Coppock, C. E., R. A. Aguirfe, L. E. Chase, G B. Lake, E. A. Oltenacu, R. E. McDowell, M. J. Fettman, and M. E. Woods. 1979 Effect of a low chloride diet on lactating Holstein cows. J. Dairy Sci. 62:723. 6 Davson, H. 1970. Physiology of the cerebrospinal fluid. J. and A. Churchill, London.
Journal of Dairy Science Vol. 63, No. 2, 1980
7 Hansard, S. L., W. O. Butler, C. L. Comar, and C. S. Hobbs. 1953. Blood volume of farm animals. J. Anita. Sci. 12:402. 8 Hiatt, E. P. 1940. Extreme hypochlorernia in dogs induced by nitrate administration. Am. J. Physiol. 129:597. 9 Hobgen, C.A.M., P. Wistrand, and T. H. Maren. 1960. Role of active transport of chloride in formation of dogfish cerebrospinal fluid. Am. J. Physiol. 199:124. 10 Hurwitz, S., I. Cohen, A. Bar, and S. Bornstein. 1973. Sodium and chloride requirements of the chick: relationship to acid-base balance. Poultry Sci. 52:903. 11 Luke, R. G., V. Warren, M. Kashgarian, and H. Levitin. 1970. Effects of chloride restriction and depletion on acid-base balance and chloride conservation in the rat. Clin. Sci. 38:385. 12 Meyer, J. H., R. H. Grummer, R. H. Phillips, and G. Bohstedt. 1950. Sodium, chloride, and potassium requirements of growing pigs. J. Anim. Sci. 9: 300. 13 Phillips, G. D. 1970. Plasma standard bicarbonate and chloride concentations in cattle and sheep. Br. Vet. J. 126:409. 14 Steel, R.G.D., and J. H. Torrie. 1960. Principles and procedures of statistics. McGraw-Hill, New ¥ork. 15 Tschirgi, R. D., and J. L. Taylor. 1954. A steady bioelectric potential between blood and the cerebrospinal fluid (CSF). Fed. Proc. 13:154. (Abstr.) 16 Voris, L., and E. J. Thacker. 1941. The effects of the substitution of bicarbonate for chloride in the diets of rats on growth, energy and protein metabolism. J. Nutr. 23:365. 17 White, A., P. Handler, and E. L. Smith. 1964. Principles of biochemistry. McGraw-Hill Book Co., New York. 18 Whitlock, R. It., M. J. Kessler, and J. B. Tasker. 1975. Salt (sodium) deficiency in dairy cattle: polyuria and polydipsea as prominent clinical features. Cornell Vet. 65:512.
J. C. ALLEN, and R. P. GENTRY Departments of Animal and Dairy Science and Large Animal Medicine University of Georgia Athens 30602
ABSTRACT
INTRODUCTION
Control (.5% chloride) or low-chloride (.038% chloride) practical diets and low-chloride (.00038% chloride) well water were fed to male Holstein calves for 7 wk. Substantial alterations in characteristics of blood associated with acid-base balance developed in those fed the low-chloride diet. Along with reduced potassium in plasma, concentrations of chloride in synovial fluid, saliva, and plasma also were reduced. Solids in plasma, blood pH, packed cell volume, carbon dioxide pressure (PCO2) in blood, and bicarbonate were increased. Most of the changes in plasma occurred within 1 wk with little change thereafter, indicating an adaptation by the calves to the lowchloride diet. The adaptation by the low-chloride calves was primarily due to a tremendous reduction in urinary chloride excretion. Change in blood factors was insufficient to cause severe alkalosis in calves. The low-chloride diet had no significant effect on sodium metabolism as measured by sodium absorption, retention, or distribution in body fluids. Potassium in plasma and aqueous humor were reduced in calves fed the low-chloride diet, but potassium concentrations in other body fluid were not affected significantly.
Next to sodium (Na), the chloride (C1) ion is the most common ion and is b y far the most common anion in the extracellular body fluids of animals. In vertebrates, the CI concentration [CI-] in body fluids normally is regulated within narrow limits. However, a severe derangement of the acid-base system and metabolic alkalosis could occur under conditions of dietary insufficiency of C1 or by excessive C1 losses from the body due to profuse sweating and by some pathological defects (kidney damage) affecting C1 metabolism (8). The influence of feeding a tow-el practical diet on the various factors affecting acid-base balance have not been investigated thoroughly in cattle. Likewise, there is little information on the effects of low-C1 intake on Na and potassium (K) metabolism in cattle. However, the interaction of Na and C1 in their influence on various acid-base characteristics has been studied in several other species (10 to 12). The objective of this research was to study the effects of a low-el diet on the acid-base balance and other blood factors in young dairy calves. The effects of low C1 intake on Na and K metabolism also were investigated.
Received August 27, 1979. 1Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations. ZSchool of Veterinary Medicine, New Bolton Center, Kennett Square, PA 19348. 1980 J Dairy Sci 63:269--276
EXPERIMENTAL PROCEDURES A control, practical-type diet containing .5% C1 was fed ad libitum for a 1-wk standardization period to eight male Holstein calves which averaged 78 kg and 2 mo of age and which were in wooden metabolism crates. The calves were divided into two groups balanced for feed intake, body weight, and plasma CI. The groups were assigned at random to the control diet or a tow-C1 diet (.038% Ct) for a 7-wk experimental period. The basal diet was 269
270
BURKHALTER ET AL.
59% cracked yellow corn, 22% soybean meal, and 15% cottonseed hulls with adequate minerals and vitamins. Sodium bicarbonate replaced sodium chloride of the low-C1 diet in amounts to equalize the Na content of the diets. In dry matter, the diets contained (by analyses) .35% Na and .98% K. Both groups received well water ad libitum containing .00038% CI, .00041% Na, and .00021% K. Feed and water intake were measured daily, and body weight was measured once each week. Special precautions were taken to avoid contamination between animal groups with extraneous C1. The two groups were separated in the metabolism barn by about 9 m. Calves always were handled (feeding, weighing, sampiing, etc.) in order of low CI calves first. Fecal and urine collection containers were washed with low-C1 water. Workers wore disposable plastic gloves for handling calves. Total fecal and urinary excretions were measured during the first 4 days of standardization and the first 4 days of each week during the 7-wk experimental period. Feces were mixed thoroughly, and about 100 g of each collection were composited daily. Feed samples were dried, ground in a Wiley mill, and analyzed for [C1-], [Na+], and [K +] concentrations. Urine samples also were composited and analyzed for [CI'], [Na+], and [K+]. Saliva was collected weekly by procedures described by Whitlock et al. (18) with modifications. A clean synthetic sponge gripped with long forceps was placed in the submaxillary area of the jaw. The sponge absorbed mixed saliva that then was squeezed into test tubes. Care was taken to obtain relatively contamination-free samples. The mouth was cleared of feed prior to sampling, and any saliva appearing contaminated was discarded. Samples were centrifuged, and the supernatant was analyzed for [C1-], [Na+], and [K+I. Jugular venous blood was collected three times during standardization and the first 2 wk of the experimental period, twice weekly
3Models 213 and 329, manufactured by International Laboratory, Inc., Lexington, MA 02173. 4 Model CR, manufactured by International Equipment Co., Needham Heights, MA 02194 s Model 10400, manufactured by American Optical, Keene, NH 03431. Journal of Dairy Science Vol. 63, No. 2, 1980
during the 3rd wk of the experimental period, and once weekly thereafter. Twenty-five milliliters of blood were drawn from the jugular vein into heparinized test tubes which were put on ice along with the remaining 15-ml sample left in the syringe. Care was taken to expel all air from the syringe. The syringe was made airtight by sticking the needle into a rubber stopper. Samples then were transported immediately to the laboratory where a 1-ml sample o f blood was injected from the syringe into a blood gas analyzer 3 for pH and PCO2 measurements. The [HCO3-] was calculated from these data by the Henderson-Hasselbalch equation (17). Packed cell volume (PCV) was determined with a microhematocrit 4 . Plasma was harvested following centrifugation of the heparinized blood and analyzed for [CI-], [Na+], and [K+]. Plasma total solids were determined by refractometry s . Total b o d y chloride was estimated from data collected during the experiment and from published data. Blood volumes of the body were obtained by multiplying the b o d y weight by 7% (7). Plasma, as a percentage of whole blood, was calculated by subtracting the determined percentage packed cell volume from 100%. Total plasma chloride was estimated by multiplying the percent chloride in plasma by total plasma volume. Before necropsy each calf was sedated with 100 mg Xylazine ( R o m p u m obtained from Haver-Lockhart Laboratories) at approximately 5 to 10 min prior to receiving 5 to 7 ml of euthanasia solution containing approximately 25% pentobarbital. Synovial fluid samples were taken from the right carpus and specifically the carpal-metacarpal joint space. Cerebrospinal fluid (CSF) was obtained by an 18-gauge 8.75 cm spinal needle inserted into the alantoocipital space with the head in the flexed position. A sample of aqueous humor was taken from the anterior chamber of the eye by aspiration with a syringe and 18-gauge needle, tn all cases, fluid samples were taken prior to death. Calves were bled by cannulation o f the carotid artery. These samples were centrifuged and the supernatant fluids analyzed for [C1-], [Na+], and [K +] . Samples of feed, water, urine, feces, saliva, plasma, and other b o d y fluids were extracted with nitric acid and shaken. Feed and feces samples in solution also were filtered. Chloride in the solution of these samples was determined
METABOLISM OF CHLORIDE IN CALVES by the specific ion electrode manual method, by a Corning 125 pH meter 6 equipped with Ct and double function reference electrodes. Millivolt readings then were plotted on a standard curve to determine chloride. Urine, water, plasma, and other body fluids were centrifuged, and the Na and K content of the supernatant was measured directly by aspiration of the liquids into an emission flame photometer equipped with a lithium (Li+) internal standard 7. Feed and fecal samples were wet ashed with nitric-perchloric acid and filtered before analysis (1). Variance was analyzed as described by Steel and Torrie (14). RESULTS AND DISCUSSION
Lowering dietary CI from .5% to .038% caused a reduction (P<.01) in plasma [CI-] concentration (Figure 1A) and in total plasma C1 (Figure 2A). The increase in total plasma chloride in both groups of calves with respect to time on the diets (Figure 2A) was due to an increase in body weight gain. Concentration decreased rapidly within the 1st wk, then remained constant for the remainder of the experiment. The reduced steady state of concentration of plasma CI from wk 2 through wk 7 in the low-C1 fed calves, following the early rapid decrease, indicated that some adaptation had occurred and that concentrations were in equilibrium with CI intake. The major adaptation was a rapid decrease in urinary concentration of C1. By the 3rd wk there were only barely detectable amounts in the urine (Figure 2C). Total urinary and fecal C1 losses were less (P<.01) in the low CI fed calves (Table 1). Earlier studies have indicated that the main homeostatic control mechanism in C1 metabolism in most species is endogenous excretion via urine (4, 5, 17). Other excretion routes for C1 of minor importance include feces and sweat (4, 17). In spite of the drastic reduction in dietary C1 intake, the low-C1 fed calves maintained a positive C1 balance as calculated from total plasma C1 (Figure 2A). Since body C1 is mostly
6Model 125, manufactured by Corning Glass Works, New York, NY 10001. Model 143, manufactured by International Laboratory, Inc., Lexington, MA 02173.
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in extracellular fluid (about 84%), of which plasma may be a major part, total plasma C1 gives a reasonable estimate of total body C1. However, extracellular fluids other than plasma, e. g., cerebrospinal, aqueous humor, synovial, Journal of Dairy Science Vol. 63, No. 2, 1980
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and saliva, contain some C1 (6, 17)• In the calves, calculated total plasma C1 increased 32% over the 7-wk feeding period compared to 64% for controls (Figure 2A). Since growth rate of the two groups was similar (1.13 kg vs. 1.23 kg/day), the major difference
low-Cl
Journal of Dairy Science Vol. 63, No. 2, 1980
in body C1 was due to the Cl concentration in plasma. The 64% increase in plasma CI was comparable to the 77% increase in body weight of the control group• However, the 32% increase in plasma C1 of the low CI calves was less than half of the 71% increase in their weight. One may wonder whether a continuation of the low C1 diet would cause serious consequences. The data indicate that the total plasma CI differences were not becomming wider at the end of the 7-wk period (Figure 2A). This suggests that the calves probably had begun to adapt to the low C1 intake. Lowering the dietary C1 to .038% caused significant alterations in the measures associated with acid-base balance. Blood pH increased (P<.01) rapidly within the 1st wk in the low-C1 calves; then higher pH remained parallel to those of controls until the end of the experiment (Figure 3A). Over the 7 wk, blood pH in the low-C1 calves increased from 7.41 to 7.50 as compared to 7.42 to 7.43 for controls. The blood pH of 7.50 in the low-Cl calves during the 7th wk slightly exceeded the normal pH range of 7.27 to 7.49 quoted for cattle (2). Although the differences between the two groups were statistically significant (P< .01 ), the increase in blood pH of the low-C1 calves did not produce severe alkalosis. With a steady intake of .038% C1 (which was absorbed almost completely), extremely little C1 was in urine by the 2nd wk of treatment; by only minor losses in feces and sweat, CI homeostasis in plasma and blood pH were maintained effectively. Because of the effective C1 homeostasis, growth rate of the calves was not affected adversely for the remaining 5 wk. It is u n k n o w n whether the calves could have remained on the low CI diet indefinitely without blood pH increasing sufficiently to produce severe alkalosis. In the calves fed low CI, the rise in blood pH was associated with a corresponding rise in PCO2 and [HCO3"] (Figures 3B and 3C). The increase (P<.01) in blood [HCO3-], as a result of reduced plasma [CI'], may represent an adaptation by the animal to offset the increased pH. The bicarbonate disassociates into carbonate and free [H+], thus lowering the pH to the original level. In our study, the amount of [HCO3-] produced was not sufficient to restore fully blood pH in the low C1 calves to control pH. Phillips (13) reported that the normal mean bicarbonate concentration of adult cattle
METABOLISM OF CHLORIDE IN CALVES
273
TABLE 1. Salivary, urinary, and fecal chloride and sodium and potassium in calves fed control or low-chloride diets for 7 wk. Diets a
Low
Itemsc
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SEb
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23.56 62.70 13.22
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1.45 4.48 2.41
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1.38 1.76 3.57
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17.53 11.88 50.76
1.13 .332 2.90
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acontrol diet contained .5% CI; low-chloride diet, .038% C1 (dry basis). bFour calves per treatment. CThe mineral values for feces and urine represent an average of 7 balance trials of 4 days each with 4 calves per treatment: mixed saliva is an average of 7 weekly samples. *P<.05. **P<.01.
blood was 23.4 mEq/liter and that 95% of the individual observations fell between 20.2 and 26.6 mEq/liter. The mean [HCO3-] in steers (about 336 kg body weight) was 28.0 -+ 2.12 mEq/liter. Our 31.3 mEq/liter for control calves agrees reasonably well with the literature, but the 39.7 mEq/liter of the low-C1 calves is considerably higher. Increased lactic acid in plasma due to physical stress causes a decrease in plasma bicarbonate (13). Since these blood samples were taken under minimum stress, this probably did not affect blood [HCO3-] of the calves. Water balance was not maintained in the low-C1 calves. Likewise, symptoms of polydipsia and polyuria became more pronounced with time in the calves fed low CI. Thus, a dehydrating effect occurred as indicated by increased total solids in plasma and percentage PCV in blood (Figures 4A and 4B). This effect of dietary C1 has been reported in other species (16). The concentration of Ct in mixed saliva of the low-C1 calves was substantially less (P<.05) over an average 7-wk experimental period than
in controls (Table 1). The concentration was reduced significantly (P<.05) beginning with the 3rd wk (Figure 2B). The C1 concentration of mixed saliva in control calves was comparable to 22.9 mEq/liter in parotid saliva of saltsupplemented lactating cows as reported by Whitlock et al. (18), but it was higher than reported by Coppock et al. (5) in lactating cows receiving diets containing .4% C1. Coppock et al. (5) reported that chloride in parotid saliva was not reduced significantly in lactating cows fed a low C1 diet (.18% C1) for 11 wk as compared to cows fed the control diet (.4% C1). Their data indicated that saliva Cl concentration did not reflect dietary C1 intake truly. Chloride, Na, and K content of the cerebrospinal, aqueous humor and synovial fluid o f the bovine have not been reported. In some other species, plasma is hypotonic to CSF with ratios of C1 content in CSF to that in plasma ranging from 1.07:1 to 1.15:1 (6, 15). In our study, Cl contents of synovial and ruminal fluids were considerably less (P<.O1) in the low-Cl fed calves (Table 2). Cerebrospinal, aqueous humor, bile, and abomasal fluid C1 was Journal of Dairy Science Vol. 63, No. 2, 1980
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not (P<.05) affected by dietary C1. Maintaining an adequate C1 concentration is important for synthesis of hydrochloric acid in the abomasum. Hydrochloric acid is essential for normal Journal of Dairy Science Vol. 63, No. 2, 1980
p r o t e i n digestion in t h e a b o m a s u m . P r o t e i n m e t a b o l i s m was a f f e c t e d severely in t h e c h l o r i d e d e f i c i e n t rat (16). T h e C S F C1 in t h e calves was c o n s i d e r a b l y l o w e r t h a n t h a t in t h e cat (3), dogfish (9), r a b b i t , dog, goat, a n d h u m a n (6). T h e ratios o f C S F to p l a s m a C1 at t h e e n d o f t h e 7-wk e x p e r i m e n t were .59:1 a n d .66:1 for c o n t r o l and low-C1 calves. T h e r e a s o n f o r t h e large d i f f e r e n c e s b e t w e e n calves a n d o t h e r species is n o t k n o w n . L o w e r i n g t h e d i e t a r y C1 t o .038% h a d little e f f e c t o n Na m e t a b o l i s m in calves. S o d i u m c o n c e n t r a t i o n s in u r i n e , feces, plasma, saliva, a n d in all o t h e r b o d y fluids s a m p l e d were n o t s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s (Tables 1 a n d 2). In t h e r a b b i t , cat, dog, goat, a n d h u m a n , t h e Na c o n t e n t o f C S F was slightly less t h a n t h a t o f p l a s m a w i t h C S F to p l a s m a Na r a t i o s
METABOLISM OF CHLORIDE IN CALVES
275
TABLE 2. Chloride, sodium, and potassium content of body fuids in calves fed control and low-chloride d i e t s for 7 wk. Chloride Body fluids c
Control a
Low chloridea
Sodium SE b
Controla
Low chloridea
Potassium sEb
Controla
Low chloridea
SE b
.49 3.9 2.3 ...d 4.0 2.8 .23
4.4 .97 5.1 .... d 2.6 35.8 72.0
3.5** .98 3.8" .... d 1.9 35.8 66.0
.07 .10 .32 ...d 21" 3.0 3.2
(mEq/liter) PlasmaC Cerebrospinal Aqueous humor Synovial Bile Abomasal Rumen a
.
104.7 62.1 103.3 100.1 21.9 124.1 14.5
92.0** 61.2 95.9
88.9" * 16.5 128.3 8.2**
.54 7.7 2.2 1.6 1.6 4.4 1.2
143.5 45.3 139.3 .... d 59.8 25.9 73.8
142.6 47.4 141.0 .... d 51.7 35.3 77.2
.
Control dmt contained .50% chloride; low chloride diets, .038% chloride. bFour calves per treatment. Cplasma chloride, sodium, and potassium concentrations determined during the 7th wk of experiments. Cerebrospinal, aqueous humor, and synovial fluid values were obtained while animals were under anesthesia just prior to death; other during necropsy. dsynovial fluid sodium and potassium were not determined due to the fluid being too viscous to be aspirated properly into the flame photometer. *P<.05. * *P<.01.
ranging f r o m .95:1 to 1.00:1 (6). The ratios o f CSF Na to plasma Na at the end o f the 7-wk e x p e r i m e n t were .97:1 and .99:1 for control and low-C1 calves. Thus, calves are similar to o t h e r species. In man, ratios of Na in synovial fluid and a q u e o u s h u m o r to that in plasma were 1.21:1 and 1.30:1 (16). T h e r e is little i n f o r m a t i o n on effects o f feeding a low-C1 diet on Na m e t a b o l i s m in cattle. In swine, feeding a low-C1 diet significantly reduced C1 and o n l y slightly reduced Na in plasma. However, feeding a low Na and normal C1 diet caused a significant decrease in plasma CI and a small decrease in Na. Evidently b o d y Na, at least in swine, is regulated m o r e critically in the b o d y than C1. Beginning with the 2nd wk, plasma K was reduced (P<.05) in the low-Cl calves (Figure 1C) and by the 7th wk, plasma K was a b o u t 80% o f control (Table 2). Potassium is n o t a major cation in plasma, and the explanation for this effect o f dietary C1 on plasma K is not evident. However, these results agree with reduced plasma K in lactating cows fed a .18% C1 diet (5). The low CI diet had no significant effect on fecal or urinary K excretion, on K c o n c e n t r a t i o n
in saliva, or in most b o d y fluids sampled (Tables 1 and 2). In a d d i t i o n to plasma, K c o n t e n t of s o m e o t h e r b o d y fluids was reduced slightly in the low C1 calves with differences f r o m controls in aqueous h u m o r being statistically significant (P<.05) (Table 2). The ratios of K c o n t e n t of CSF to those of plasma in rabbit, cat, dog, goat, dogfish, and h u m a n range f r o m .62:1 in humans to 1.12:1 in the dogfish (6). The ratios of CSF K to plasma K were .22:1 and .28:1 for control and low-Cl calves. Like Na, K was slightly higher in saliva o f the low-C1 calves (Table 1). The increase of these t w o cations in saliva w h e n C1 is lowered agreed with (18). Results of this research with cattle agree with those f r o m o t h e r species indicating that the acid-base system is a highly regulated system. In spite of a drastic r e d u c t i o n in dietary CI, b o d y CI was maintained f u n c t i o n a l for 7 wk. A l t h o u g h various factors associated with acid-base balance were altered significantly, these changes were insufficient to cause severe alkalosis in the calves. The t r e m e n d o u s r e d u c t i o n in urinary C1 e x c r e t i o n in the low C1 calves was the primary h o m e o s t a t i c m e c h a n i s m by which b o d y CI was maintained. More research is Journal of Dairy Science Vol. 63, No. 2, 1980
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n e e d e d t o d e t e r m i n e t h e d i e t a r y C1 r e q u i r e d b y cattle a n d / o r if feeding t h e .038% C1 longer w o u l d p r o d u c e severe m e t a b o l i c alkalosis in calves.
ACKNOWLEDGMENTS
The a u t h o r s gratefully a c k n o w l e d g e R o b e r t A. Isaac and William C. J o h n s o n , Jr., o f t h e Georgia C o o p e r a t i v e E x t e n s i o n Service Soil Test L a b o r a t o r y for their t e c h n i c a l advice and assistance c o n c e r n i n g t h e c h l o r i d e analysis o f samples. Special t h a n k s are e x t e n d e d to Sheila Heinmiller for h e r overall supervision o f e x p e r i m e n t a l animal care.
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