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Metabolic Diseases and Immunology Unit, National Animal Disease Center, USDA-Agricultural Research Service, Ames, IA 50010
Corresponding author: J. P. Goff; e-mail: jgoff{at}nadc.ars.usda.gov.
| ABSTRACT |
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Key Words: milk fever anionic salts chloride sulfate
Abbreviation key: DCAD = dietary cation-anion difference, SBE = standard base excess
| INTRODUCTION |
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| MATERIALS AND METHODS |
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Experiment 1: Relative Urine Acidifying Activity of Hydrochloric Acid, Calcium Chloride, Ammonium Chloride, Magnesium Sulfate, Calcium Sulfate, and Elemental Sulfur
Six mature nonpregnant, nonlactating Jersey cows were limit fed 5.8 kg DM/d of a corn silage-alfalfa based diet (Table 1
) for 5 d to establish baseline urine pH values. Diets were limit fed to attempt to insure complete ingestion of the anion doses.
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Experiment 2. Effect of Chloride vs. Sulfate Sources at Graded Doses
Each trial assessed the acidifying activity of a sulfate and chloride source that shared the same accompanying cation at 3 doses and utilizing one of 2 diets (alfalfa- or corn silage [with potassium carbonate]-based diets). Seven nonlactating, nonpregnant Jersey cows, housed in a free-stall barn bedded with sand, were used in each trial. Each trial consisted of 7 periods, allowing each cow to receive each of 7 dietary treatments during the course of a trial in random order. Cows were limit fed basal ration (5.3 kg DM/d of the alfalfa-based diet or 4.8 kg DM/d of the corn silage based diet) (Table 1
) in 2 divided feedings (0800 and 2000 h for 10 d behind Calan gates (American Calan Inc, Northwood, NH) to which the cows were accustomed before hand. Then the appropriate anion supplement was added (one half the daily dose of anion suspended in 250 mL of water and mixed by hand into the morning and evening ration of each cow within the Calan gate tub) to the basal ration for the next 5 d. Two anion sources (a sulfate source and a chloride source sharing a common cation) were compared in each trial. The 2 anion sources compared in each trial were added to the diet at a rate of 0.75, 1.5, or 2.25 Eq/d. One treatment in each trial consisted of water only, for a total of 7 treatments/trial. At 3 h after the last feeding on d 5 of each treatment period, jugular venous blood was collected anaerobically, and a midstream urine sample was obtained, eliciting micturition by manual stimulation of the vulva. Blood gases, hemoglobin, and pH were determined on whole heparinized samples of jugular venous blood maintained at 4°C until analyzed (within 1 h of obtaining the sample) (Nova Ultra-D Blood Gas Analyzer, Nova Biomedical, Waltham, MA). Standard base excess (SBE) was then calculated from measured blood pH and partial pressure of carbon dioxide, using an algorithm (Thomas, 1972). To use this algorithm, the default value for blood hemoglobin of 14.3 g/100 mL whole blood was used. Not every cow has a blood hemoglobin concentration of 14.3 g/100 mL, but the blood hemoglobin concentration is relatively constant in individual cows. The equation utilized was: SBE = 0.7998 (HCO3 24) + 28.149 (blood pH 7.4). The SBE, which is generally positive in alkalosis and negative in acidosis, is defined as the amount of acid or base needed to restore 1 L of blood to "normal" acid-base composition (pH = 7.40) at a partial pressure of carbon dioxide of 40 mm mercury in the blood. The algorithm used was developed using "normal" human blood parameters. The typical forage-based cow diet contains cations far in excess of anions. When these cations are absorbed from the intestinal tract, they increase plasma strong ion difference creating a metabolic alkalosis (Constable, 1999). Thus the bovine samples usually appear "alkaline" in relation to the "standard" human. Determinations of urine pH were made within 1 h of urine collection. In one trial (cows fed corn silage-based diet receiving graded doses of either HCl or H2SO4), urine titratable base content was assessed by placing 25 mL of urine into a beaker and slowly adding 0.10 N HCl to the sample while stirring. The titratable base content was defined as the milliliter of acid required to acidify the urine sample to a pH of 4.5.
Experimental treatment periods within each trial were separated by 5-d rest periods in which the diet consisted of the basal ration only. Cows were also maintained on the basal ration during a 5- to 6-wk rest time between trials.
The first 3 trials used the same 7 cows, and the basal ration was an alfalfa hay-based diet. In trials 4, 5, and 6, seven different nonlactating Jersey cows were used, and the basal ration was a corn silage-based diet with added potassium bicarbonate (Table 1
). In the first and fourth trials, the effects of CaCl2 and CaSO4 were assessed. In the second and fifth trials, the effects of HCl and H2SO4 were assessed. In the third and sixth trials, the effects of MgCl2 and MgSO4 were assessed.
Statistical Analyses
Urine pH, blood pH, and SBE data collected within each trial were analyzed by an initial analysis of variance using diet, anion source (including water), dose, and anion source x dose interaction as main effects (Statview 5.0, SAS Institute, Cary, NC). With diet included as a main effect in the ANOVA model, the effect of diet was not significant when comparing the effects of the chloride and sulfate salts of magnesium and calcium. Therefore, the data from trials 1 and 4, and data from trials 3 and 6 were combined to allow comparison of these anion sources across both diets. The effect of diet was significant in the comparison of HCl and H2SO4. Examination of these data suggested the diet difference was related to the higher urine and blood pH induced by the corn silage-based diet, which is apparent during the water treatment (zero anion) periods. Because the relative changes appeared to be similar, the data from trials 2 and 5 were also combined to allow comparison of these acids across both diets. Data from the combined trials were subjected to ANOVA with anion source (including water), dose, and anion source x dose interaction as main effects. Treatment means were compared to the mean obtained during water-only treatment, and also to the equal dose of the opposing anion by the method of least significant difference.
The data obtained from all 6 trials were also combined across all 3 anion sources and both diets for ANOVA where anion source (chloride, sulfate, or water), dose and anion source x dose interactions served as main effects. Treatment means were compared to the mean obtained during water-only treatment, and also to the equal dose of the opposing anion by the method of least significant difference.
In the final ANOVA, data were combined across all 3 doses, all 3 anion sources, and both diets were combined with anion (chloride, sulfate, or water) as the main effect. Treatment means were compared to the mean obtained during water-only treatment and to the opposing anion by the method of least significant difference.
Animal Care and Use
All procedures employed on the cows were approved by the Animal Care and Use Committee of the National Animal Disease Center.
| RESULTS |
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Experiment 2
Urine pH and blood pH and SBE from the individual trials are summarized in Tables 2
to 4
. Because diet (corn silage vs. alfalfa-based) had no significant effect on the response to the calcium and magnesium salts of chloride and sulfate, these data were combined, and the data from individual trials will not be discussed further. Diet was a significant factor affecting the response to HCl and H2SO4. However, these data were also combined as stated in the Materials and Methods. Urine-titratable base data were obtained only in trial 5 examining the effect of HCl vs. H2SO4 in cows fed the corn silage-based ration. The amount of added acid needed to bring urine pH to 4.5 (titratable base) was 60 ± 9 mL during water treatment. There was an incremental decline in the titratable base left in the urine as increasing doses of either HCl or H2SO4 were added to the diet (Table 5
). There were no significant differences in titratable base content of urine from cows fed equivalent amounts of HCl and H2SO4.
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Effect of Magnesium Chloride vs. Magnesium Sulfate Across Both Diets
Urine pH of cows fed diet with only water added was 7.84 ± 0.09. There was no significant urine acidifying effect of 0.75 Eq of either MgCl2 or MgSO4 when compared to water alone. Higher doses of either magnesium salt caused significant decreases in urine pH (Table 6
). Urine pH during treatment with 2.25 Eq of MgCl2 was lower than urine pH during treatment with 2.25 Eq of MgSO4 (P < 0.10). All 3 doses of MgCl2 were able to cause a decrease in urine pH from that observed during water treatment (7.44 ± 0.01) (Table 7
). Only the 1.5 and 2.25 Eq doses of MgSO4 were able to significantly decrease blood pH from that observed during water treatment. The blood pH during treatment with 1.5 and 2.25 Eq doses of MgCl2 was significantly lower than the blood pH during treatment with equivalent doses of MgSO4. All 3 doses of MgCl2 were able to significantly reduce blood SBE from that of water alone (6.00 ± 1.05 mEq/L). Only the 1.5 Eq dose of MgSO4 caused a significant change in blood pH from that observed during water treatment (Table 8
). Blood SBE during treatment with 2.25 Eq of MgCl2 was significantly lower than blood SBE during treatment with 2.25 Eq of MgSO4.
Combined Effect of Chloride vs. Sulfate Sources Across Both Diets
The data were also analyzed by combining the 3 chloride source treatments into one treatment and the 3 sulfate source treatments into one treatment. Urine pH, blood pH, and blood SBE values were incrementally reduced as increasing doses of chloride or sulfate were added to the diet (Figure 2
). There was no difference in urinary acidifying effect of the chloride and sulfate salts. However, blood pH (P < 0.002) and blood SBE (P < 0.0001) were significantly lower when chloride served as the source of anions.
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| DISCUSSION |
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The difference between the number of cation and anion particles absorbed from the diet determines the pH of the blood (Stewart, 1983). The cation-anion difference of a diet is commonly described in terms of mEq/kg of just sodium, potassium, chloride, and sulfate as follows:
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Whereas this equation has proved quite useful, it assigns equal value to chloride and sulfate salts as contributors to DCAD. In the case of the close-up dry cow, we might presume this means that dietary chloride and sulfate anions contribute equally to changes in acid-base status of the cow and, therefore, the parathyroid-hormone sensitivity of her tissues. However, the data from experiments 1 and 2 demonstrate that dietary sulfate anions are less potent acidifiers of the blood than are dietary chloride anions. In experiment 1, treating with the 2-Eq dose of each chloride source reduced urine pH, blood pH more than the 2-Eq dose of the sulfate source. These results were similar to the effects observed in experiment 2 at the 2.25-Eq doses of the various chloride and sulfate sources. However, at the lower anion doses utilized in experiment 2, the differences between the chloride and sulfate sources on measures of acid-base physiology were small to imperceptible (Figure 2
). We speculate that there is some blockade of sulfate absorption at higher doses, while chloride absorption continues unabated. Another possibility is that sulfate is cleared from the blood faster than chloride, especially at higher blood levels. If absorbed sulfate is quickly excreted into the urine or bile, it might not exert as great an effect on blood pH, while continuing to add to the anion content of the urine and therefore decreasing urine pH. The results of this study, demonstrating a difference in acid-base effects between dietary chloride and sulfate, are not unique. Oetzel et al. (1991) screened a number of anionic salts for effects on urine pH and close examination of their data also demonstrates that the sulfate salts are less able to cause urinary acidification than are chloride salts. Returning to the traditional DCAD equation it might therefore seem appropriate to discount dietary sulfate when comparing it to chloride as a means of affecting blood pH.
It is difficult to determine just how much more potent chloride salts are. Tucker et al. (1991) compared addition of the same number of equivalents of chloride or sulfate into lactating cow diets to achieve similar DCAD [according to the equation DCAD = (Na + K)(Cl + S)] and concluded sulfate was about 60% as acidifying as chloride. Our data suggest that the acidifying effect of CaSO4 compares favorably with CaCl2 at the same dose, while MgSO4 is a much weaker acidifying agent than MgCl2. However, if we ignore these source effects and look at the grand total effect of chloride vs. sulfate across all anion sources, doses, and diets it would seem that sulfate is between 55 and 60% as effective as chloride at changing blood pH and SBE (Figure 3
), confirming the work of Tucker et al. (1991). Therefore, the DCAD equation could be more accurately written as (Na+ + K+) (Cl + 0.6 S ).
This equation may also be considered incomplete as it ignores the contribution other major cations (calcium and magnesium) and anions (phosphorus), present in significant amounts in most diets, can have on DCAD and acid-base status. It is tempting, but wrong, to express DCAD as (Na+ + K+ + Ca ++ + Mg++) (Cl + 0.6 S + P ) because this equation assumes that Ca and Mg are as strongly alkalinizing as Na and K and that P is as strongly acidifying as Cl. A discount must be applied to these other dietary cations and anions to adjust for their lower absorption (or perhaps retention) and acidifying activity. Unfortunately, experimental data required to assign coefficients to these other variables are lacking. However, in experiment 1, where many different salts were fed in the same trial, feeding CaSO4 or MgSO4 did reduce urine pH. It can therefore be assumed that the urine alkalinizing activity of calcium and magnesium cations is less than the acidifying activity of sulfate anions. Fewer dietary cations than anions were absorbed into the blood. Therefore, the DCAD equation coefficients for calcium and magnesium must be less than 0.6. Because urine pH of cows fed CaCl2 or NH4Cl was higher than urine pH of cows fed HCl, it is clear that the calcium and ammonium cations are contributing some alkalinity to the blood. At least some of these cations are being absorbed into the blood, and therefore the coefficient applied to these cations (as well as magnesium) in the equation should be greater than zero. In the traditional DCAD equation, there is no allowance made for the effect the ammonium cation could have on DCAD. However, it is clear from experiment 1 that NH4Cl did not acidify urine as strongly as did HCl. As with the work of Oetzel et al. (1991), CaCl2 and NH4Cl were nearly equipotent as urinary acidifiers. Just as with calcium and magnesium, ammonium should be included in the DCAD equation.
When cows received the water only added to their diet, all were producing alkaline urine. Their blood SBE was highly positive, consistent with observations that cows are normally in a state of compensated metabolic alkalosis. When placed on the highest doses of anions, all cows exhibited a decrease in blood SBE and urine pH while blood pH dropped only slightly. Larger total anion loads or more negative DCAD diets could have placed these cows in a state of uncompensated metabolic acidosis. The cows in this study (with the exception of those fed magnesium sulfate) continued to consume their entire ration, suggesting they had not entered a state of uncompensated metabolic acidosis. The cows were able to achieve a state of compensated metabolic acidosis with the doses of anion used in these studies.
The diet the cows were fed had little effect on the response of the cows to the anion supplementation. Whether potassium was from endogenous plant sources or from potassium carbonate mineral source did not seem to have an effect on the relative response to anions. No great interactions between basal diet protein, diet calcium, or diet magnesium content and anion supplementation were observed. Our goal in setting up the 2 diets was to try to determine the effects of a high nitrogen diet (which might be expected to result in ammonium cation production) on acid-base balance. Unfortunately any effect of NPN in the alfalfa diet was overwhelmed by the addition of potassium to the corn silage (low nitrogen) diet.
In some cases, the DCAD equations have been interpreted to suggest that elemental sulfur is equivalent to the sulfate anion. Results of experiment 1 demonstrate this interpretation is clearly wrong, as elemental sulfur is not capable of acidifying the urine.
Sulfur and sulfate are potentially toxic because they can be reduced to hydrogen sulfide in the rumen, a potent neurotoxin (Gould et al., 1991). Therefore the amount of sulfate added to the diet must be limited. The current maximum tolerable limit for dietary sulfur in cattle is thought to be 0.4% of the diet DM (National Research Council, 2001). Because low doses of sulfate coming from CaSO4 and H2SO4 appear to be equipotent to low doses of chloride sources, adding small amounts of these salts would be usefulso long as inclusion does not bring total sulfur content above 0.4%. Reagent grade concentrated H2SO4 is between 95 and 98% pure, while reagent grade HCl is just 36.5 to 38% HCl by weight. Thus the volume of H2SO4 required to add 1 Eq anion to the diet would be very low; there is 1 Eq of anion/28 mL of concentrated H2 SO4, while reagent grade HCl supplies 1 Eq of anion/83 mL.
In experiment 1 of this study, MgSO4 was the only anion not totally consumed by the cows. In experiment 2, all anions added to the rations were consumed, though it was observed that the diet with magnesium sulfate was consumed more slowly. These observations raise questions about the commonly held belief that sulfate salts, particularly magnesium sulfate, are more palatable than chloride salts.
Several of the variables in the above formulas are somewhat fixed when balancing rations. If diet Ca is set at 1%, P at 0.35%, Mg at 0.4%, and sulfur at 0.35% (above 0.22% to ensure adequate substrate for rumen microbial amino acid synthesis, but below 0.4% to avoid possible neurological problems associated with sulfur toxicity [Gould et al., 1991]), the only real variables in the equation become Na, K, and Cl. The goal for milk fever prevention is to keep sodium and potassium as close to the requirement of the cow as possible (~0.12% for Na and ~1.0% for potassium). The key to reduction of hypocalcemia is to then add chloride to the ration to counteract the effects of even low levels of potassium on blood alkalinity.
| CONCLUSIONS |
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| ACKNOWLEDGEMENTS |
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| FOOTNOTES |
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Received for publication May 6, 2003. Accepted for publication October 6, 2003.
| REFERENCES |
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