Poult. Sci.
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Poult Sci 2008. 87:1353-1357. doi:10.3382/ps.2008-00058
© 2008 Poultry Science Association
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IMMUNOLOGY, HEALTH AND DISEASE

High Dietary Calcium Causes Metabolic Alkalosis in Egg-Type Pullets

X. Guo*,{dagger}, K. Huang*,1, F. Chen*, J. Luo* and C. Pan*

* Institute of Nutritional and Metabolic Disorder in Domestic Animals and Fowls, Nanjing Agricultural University, Nanjing 210095, China; and {dagger} College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China

1 Corresponding author: khhuang{at}njau.edu.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One hundred sixty healthy egg-type pullets were randomly divided into 2 groups at 35 d of age. The groups were fed either with a normal-calcium (control, 8.5 g of Ca/kg) diet or with a high-calcium (HC, 36.3 g of Ca/kg) diet, respectively. The experiment lasted for 32 d. The healthy situations of the egg-type pullets were observed. Blood gas values and serum and urine electrolyte content were determined. The HC diet caused watery excreta in pullets. The pullets raised on the HC diet had significantly higher blood pH, bicarbonate, base excess, and significantly lower blood partial pressure of oxygen, partial pressure of carbon dioxide, and oxygen saturation than the pullets raised on the control diet. Phosphorus and potassium decreased; meanwhile, calcium increased in the HC group as compared with the control group. Sodium, magnesium, and chloride in serum had no significant difference between the HC group and the control. The pullets raised on the HC diet had significantly higher urine pH than the pullets raised on the control diet. For HC pullets, urinary concentration and 24-h urinary excretion of calcium and chloride was significantly higher, and magnesium, inorganic phosphorus, and sodium was significantly lower than that of the control pullets. It is concluded that some metabolic disorders associated with high dietary calcium in egg-type pullets may be related to a state of metabolic alkalosis.

Key Words: pullet • high dietary calcium • metabolic alkalosis • electrolyte


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium is a macronutrient with important biological functions, constituting by far the greatest part of mineral matter in the bird body and eggshell (Wideman and Buss, 1985; Williams et al., 2000). Calcium is one of the major constituents to be supplemented in commercial layer rations. However, the beneficial effects of calcium can only be attained when the recommended doses are administered. Many metabolic problems, such as diuresis, urolithiasis, visceral gout, and poor pullet performance have been reported to be associated with excessive dietary calcium (Siller, 1981; Wideman and Cowen, 1987; Leeson and Summers, 1987; Wideman et al., 1993; Guo et al., 2005; Julian, 2005). Case histories of affected bird flocks often revealed that excessive calcium had been fed. These problems often occur in egg-type pullets fed on a commercial layer ration in China owing to poor knowledge. In addition, some feed mills prefer to sell rations with lots of calcium, because limestone is less expensive than corn or soybean meal. However, to date, the etiology and pathogenesis of these problems caused by high dietary calcium have yet not to be firmly established. A great number of studies about the metabolic problems were focused on the role of simplex calcium instead of holistic calcium carbonate.

For many years, it was a common practice for feed mills to use limestone as the source of calcium in the commercial layer ration in China. The major constituent of limestone is calcium carbonate, which contains lots of fixed cation (Ca) and carbonate that is not a fixed anion. Mongin (1981) demonstrated that feed ingredients are alkalinizing for animals whenever the ratio of fixed cations to fixed anions is excessive: (Na + K + Ca + Mg):(Cl + PO4 + SO4). Kidney plays a central role in the regulation of acid-base balance and electrolyte in the body. Kidney lesions as a result of excess dietary calcium have been reported. However, little research has been conducted on the relationship among the aforementioned metabolic problems, calcium carbonate as the source of calcium and acid-base balance. Obviously, such study is helpful to explain why field occurrences of diuresis and poor pullet performance can be triggered by the inappropriate addition of limestone to the feed. The objective of the present study was to determine the effects of calcium carbonate as the source of calcium on the arterial blood pH, partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), oxygen saturation (SO2), bicarbonate (HCO3–), and base excess (BE) in ISA Brown layer pullets. In addition, we have also measured the healthy situations and change of electrolyte in serum and urine of the affected pullets.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Design

One hundred eighty 1-d-old ISA Brown female chickens were placed in brooders and were fed commercial chick starter ration. The starter diet (Table 1Go) was prepared according to NRC (1994) recommendations. At 35 d of age, 160 healthy and similar-weigh pullets were chosen for the experiment. The design consisted of 2 dietary treatments with 4 replicate pens (8 pens, 2 blocks) of 20 birds. Over the entire experimental period (35 d old to 67 d old), the pullets were allowed ad libitum consumption of feed and water. The normal-calcium diet contained 8.5 g of Ca/kg, meeting the NRC (1994) recommendations. The high-calcium (HC) diet contained 36.3 g of Ca/ kg (a commercial layer ration), and the calcium of the diet was achieved by the addition of limestone. Nutritive values of different dietary treatments are shown in Table 1Go. The protocol was approved by the Institutional Laboratory Animal Care and Use Committee of the Nanjing Agricultural University.


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  		Table 1. Composition of experimental diets (g/kg): normal calcium (NC) or high calcium (HC)
 
Healthy Situations

Healthy situations of the pullets were inspected daily. Necropsies were performed on all birds that died during the experiment.

Blood Gas Analysis

Arterial blood samples for blood gas measurements were collected as described by Wideman et al. (2003) from 10 birds in each dietary treatment group at 67 d of age. Briefly, the pullets were restrained unanesthetized on the right side, and the left wing was extended laterally to expose the ventral surface. Lidocaine (2%) was injected intracutaneously and intramuscularly into the regions surrounding the brachial artery, and then the vessel was isolated and cannulated with 30-cm of PE-50 polyethylene tubing filled with heparinized saline (200 U/mL in 0.9% NaCl). The arterial cannula was advanced approximately 1 cm from its point of insertion. Cannulae were tied to the blood vessels with suture thread, flushed with 1 mL of heparinized saline, and clamped with a hemostat at the distal end. Arterial blood samples were collected anaerobically (1 mL each) with minimal disturbance to the bird. The clamp at the end of the cannula was removed, arterial blood pressure was allowed to force the saline out of the cannula, and then the tip of a 23-ga needle attached to a 1-mL tuberculin syringe was inserted into the cannula to withdraw the blood. Dead spaces in the needle and syringe hub previously had been filled with heparinized saline. Whole blood (1 mL) was injected within 60 s after collection into an I-Stat SN-245085 blood gas analyzer (i-Stat Corporation, East Windsor, NJ). The primary blood values for pH, PO2, PCO2, SO2, HCO3–, and BE were generated by the blood gas analyzer operating at a sample chamber temperature of 37°C and were recalculated by the I-Stat SN-245085 for a temperature of 41°C to match the normal body temperature of domestic fowl (Fedde and Wideman, 1996).

Blood Collection and Analysis

Heparinized blood samples were centrifuged immediately after collection by wing venipuncture before feeding at 0830 h, and serum samples were obtained from 5 birds in each replicate treatment pen at 67 d of age. The serum was divided into aliquots and stored at –20°C for analysis. A Cobas Bio Spectrophotometric Auto-Analyzer (Roche, Basel, Switzerland) was used to measure the concentrations of calcium, inorganic phosphorus, magnesium, sodium, potassium, and chloride in the serum.

Urinary Collection and Analysis

At 66 d of age, to prevent the backflow of urine from the cloaca to the cecum and to collect urine, 2 birds from each replicate treatment pen were colostomized as described by Belay et al. (1993). After 1 d, a urine collection bottle was attached to the anal collar of each colostomized bird for urine collection, and 24-h urine volume was determined. Freshly collected urine was used to measure urine pH. Urine samples were divided into aliquots and stored at –20°C for analysis. The concentrations of calcium, inorganic phosphorus, magnesium, sodium, potassium, and chloride in the urine were subsequently determined as described above.

Statistics

Blood pH, PO2, PCO2, SO2, HCO3–, BE, and the concentrations of calcium, inorganic phosphorus, magnesium, sodium, potassium, and chloride in the serum and in the urine were analyzed on an individual bird basis. Body weight, weight gain, and the ratio of feed and gain were analyzed on a pen basis. All data were subjected to AN-OVA using the GLM procedure of SAS software (SAS Institute, 2001). All data are shown as means ± SEM, and values were considered to differ significantly at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Healthy Situations

Healthy situations of the control birds and their feed and water intake were normal. Twenty-five percent of pullets in the HC group began to show signs of depression, loss of appetite, gradual emaciation, watery excreta, and dehydration by d 25 of the experiment. They had a tendency to hide. Almost all pullets in the HC group showed typical clinical signs as described above by d 32 of the experiment (i.e., 67 d of age). Five birds died in the HC group during the experiment. Postmortem examination showed that the dead pullets were emaciated and the breast, leg, and thigh muscles were atrophied and dehydrated. Enlarged kidneys with microscopic lesions and inflammation were observed, but typical visceral gout was not found in pullets raised on the HC diet in the experiment.

Blood Gas

Blood gas values for pullets are in Table 2Go. The pullets raised on the HC diet had significantly higher blood pH, HCO3–, and BE than the pullets raised on the control diet. It was found that PO2, PCO2, and SO2 decreased in blood in the HC group as compared with the control group. Especially, BE in blood in the HC group was 5 times higher than that in the control group.


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  		Table 2. Blood gas values for pullets raised on normal-calcium (NC) or high-calcium (HC) diets
 
Blood Chemistry

Serum values for pullets are in Table 3Go. Inorganic phosphorus and potassium were significantly lower for HC pullets, compared with the control pullets. Calcium was significantly greater for HC pullets than for the control pullets. Magnesium, sodium, and chloride were not significantly different between HC and the control group.


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  		Table 3. Serum concentrations of inorganic elements for pullets raised on normal-calcium (NC) or high-calcium (HC) diets
 
Urinary Chemistry

Urinary Volumes and Urinary Values. Urine volumes in pullets raised on normal and HC diets were 36 ± 5.4 mL and 40 ± 4.1 mL (data not shown), respectively. Urine pH in pullets raised on normal and HC diets was 6.61 ± 0.05 and 7.46 ± 0.07 (data not shown), respectively. Urine pH in the pullets raised on HC diets was significantly higher than the pullets raised on the control diet. Urinary values of different dietary treatments are shown in Table 4Go. Inorganic phosphorus, sodium, and magnesium for HC pullets were significantly lower when compared with the control pullets. Calcium and chloride were significantly higher for HC pullets than for the control pullets. Potassium was not significantly different between the control and HC groups.


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  		Table 4. Urinary concentrations of inorganic elements for pullets raised on normal-calcium (NC) or high-calcium (HC) diets
 
Total Quantity of 24-h Urinary Excretion. Total quantity of urinary excretion is calculated as the urinary concentration per unit volume multiplied the volume of urine that was excreted in 24 h, as shown for the 2 groups in Table 5Go. Inorganic phosphorus, magnesium, and sodium were significantly lower for HC pullets compared with the control pullets. Calcium and chloride were higher significantly for HC pullets than for the control pullets. Potassium was not significantly different between the control and HC groups.


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  		Table 5. Total quantity of urinary excretion for pullets raised on normal-calcium (NC) or high-calcium (HC) diets
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Administration of limestone as a calcium source to the egg-type pullets at the dosage used in this study resulted in significant changes in blood gas values, compared with the control treatment. The pullets raised on the HC diet had significantly higher blood pH, HCO3–, BE and significantly lower PO2, PCO2, and SO2 than that of the control group. It is common knowledge that metabolic alkalosis, an acid-base disorder, has 2 hallmarks: an elevated [HCO3–] and a lower [H+] in the extracellular fluid (Halperin and Goldstein, 1999). Therefore, the results in the experiment indicated that high dietary calcium, by addition of calcium carbonate, caused metabolic alkalosis in egg-type pullets. The resultant metabolic alkalosis can be regarded as an uncompensated condition. This may indicate the failure of the body buffering systems to correct acid-base balance. Also, this may point to the failure of the respiratory and renal regulatory systems to readjust blood pH.

Although there are multiple causes of metabolic alkalosis, in general, it is the result of acid loss or base gain. Acid losses occur either from renal secretion of H+ ions or from gastrointestinal losses (e.g., vomiting). Calcium carbonate as calcium source in the present experiment contains lots of fixed cation (Ca) and carbonate that is not a fixed anion. Content of calcium carbonate in the HC group diet was 70 g/kg (36.3 g of Ca/kg). We believed that the excessive ratio of fixed cations to fixed anions was the main cause of metabolic alkalosis in egg-type pullets in the study. Many metabolic problems have been reported to be associated with excess dietary calcium. Many researchers pay attention to the role of independent calcium instead of calcium carbonate (Wideman et al., 1993). In fact, metabolic alkalosis caused by calcium carbonate may be associated with many nutritional and metabolic disorders. In the presence of metabolic alkalosis and electrolyte imbalances (including disturbed cation:anion ratio), the constitution, osmotic pressure, and pH of the extracellular fluid are probably altered (Halperin and Goldstein, 1999), which may cause many metabolic disorders. For example, it is reported that accelerated breakdown and turnover of nucleoprotein in the presence of a state of alkalosis was probably the cause of overproduction of uric acid (Mubarak and Sharkawy, 1999). In addition, it has been stated that hyperuricemia does not usually lead to visceral urate deposition, and disturbance of the intercellular fluid constitution is needed to initiate urate deposition (Riddell, 1987). Precipitation and crystallization of urates in the cases were probably favored by the electrolyte changes, disturbed tissue osmotic pressure, and the altered pH of the extracellular fluid (Mubarak and Sharkawy, 1999). Therefore, metabolic alkalosis found in the present study may partly explain why high dietary calcium in domestic birds could induce gout in our previous study (Guo et al., 2005).

In agreement with a similar study (Leeson and Summers, 1987), the present results indicated that calcium levels, as commonly found in layer diets, could lead to watery excreta in growing layer. In our previous study (Guo et al., 2005), urine volumes were significantly higher in pullets fed on layer diets than the control diet, which may be associated with renal failure. Therefore, we think that the urine volume increase is one cause of watery excreta. In addition, the administration of calcium carbonate is likely to have resulted in fluid moving into the gastrointestinal tract from other fluid compartments. This is considered to be the other cause of watery excreta.

During metabolic alkalosis, there is an influx of K+ intracellularly and an efflux of H+ extracellularly, maintaining intracellular electrical neutrality (Willatts, 1987). The H+ efflux into the blood then buffers the increased HCO3– concentration. This was reflected in the current study, in which a decrease in the serum K+ concentration in the HC group was found, whereas the blood pH was increased. In addition, because kidneys are an essential regulator of K+ and Na+ levels, disturbance of their serum levels was reasonable in our pullets, which showed appreciable renal damage.

Hypokalemia is a frequent and important clinical problem with many possible causes. The balance between intra- and extracellular potassium concentrations is disturbed, which may interfere with cell functions and neuromuscular transmission. Although the signs and symptoms of hypokalemia are various, for example, muscle weakness, renal dysfunction, hyperglycemia, and rhabdomyolysis, its real danger lies in the cardiac ar-rhythmias it may cause. The severity of symptoms and signs is related to the degree of hypokalemia, individual variability, serum Ca2+ concentration, extracellular pH, and the rapidity in which the hypokalemia has developed (Kirschbaum, 2004).

The HC diet caused hypercalcemia and hypophosphatemia and had significant high urinary calcium excretion in the present experiment, suggesting that pullets raised on the HC diet had higher rates of intestinal calcium absorption and lower rates of intestinal phosphorus absorption. As we know, Na+ is the major extracellular cation, Cl– is the major extracellular anion, and HCO3– is the second major extracellular anion. Currently, levels of serum Ca2+ and HCO3– were increased, whereas levels of serum K+ were decreased. Thus, the cation:anion balance in the presently exposed pullets was disturbed.


   ACKNOWLEDGMENTS
 
This project was supported by the National Natural Science Foundation of China awarded to Kehe Huang (grant number: 30070575, 30671547) and the Natural Science Foundation of Jiangxi Province awarded to Xiao-quan Guo (grant number: 2007GQN0235).

Received for publication February 1, 2008. Accepted for publication March 21, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Belay, T., K. E. Bartels, C. J. Wiernusz, and R. G. Teeter. 1993. A detailed colostomy procedure and its application to quantify water and nitrogen balance and urine to thermoneutral and heat-distressed environments. Poult. Sci. 72:106–115.[Web of Science][Medline]

Fedde, M. R., and R. F. Wideman. 1996. Blood viscosity in broilers: Influence on pulmonary hypertension sydrome. Poult. Sci. 75:1261–1267.[Web of Science][Medline]

Guo, X. Q., K. H. Huang, and J. X. Tang. 2005. Clinicopathology of gout in growing layers induced by high calcium and high protein diets. Br. Poult. Sci. 465:641–646.

Halperin, M. L., and M. B. Goldstein. 1999. Pages 11–25 in Fluid, Electrolyte, and Acid-Base Physiology. 3rd ed. Saunders, St. Louis, MO.

Julian, R. J. 2005. Production and growth related disorders and other metabolic diseases of poultry – A review. Vet. J. 169:350–369.[CrossRef][Web of Science][Medline]

Kirschbaum, K. 2004. Effect of high bicarbonate hemodialysis on ionized calcium and risk of metastatic calcification. Clin. Chim. Acta 343:231–236.[CrossRef][Web of Science][Medline]

Leeson, S., and D. J. Summers. 1987. Effect of dietary calcium levels near the time of sexual maturity on water intake and excreta moisture content. Poult. Sci. 66:1918–1923.[Web of Science][Medline]

Mongin, P. 1981. Recent advances in dietary anion-cation balance: Applications in poultry. Proc. Nutr. Soc. 40:285–294.[CrossRef][Web of Science][Medline]

Mubarak, M., and A. A. Sharkawy. 1999. Toxopathology of gout induced in laying pullets by sodium bicarbonate toxicity. Environ. Toxicol. Pharmacol. 7:227–236.[CrossRef]

NRC. 1994. Nutrient Requirements of Poultry. 10th ed. National Academy Press, Washington, DC.

Riddell, C. 1987. Urinary system. Pages 67–73 in Avian Histopathology. American Association of Avian Pathologists, Kennett Square, PA.

SAS Institute. 2001. Proprietary Software Release, Version 8.02. SAS Institute Inc., Cary, NC.

Siller, W. G. 1981. Renal pathology of the fowl-A review. Avian Pathol. 10:188–261.

Wideman, R. F., B. C. Ford, R. M. Leach, D. F. Wise, and W. W. Robey. 1993. Liquid methionine analog (free acid) DL-methionine attenuate calcium-induced kidney damage in domestic fowl. Poult. Sci. 72:1245–1258.[Web of Science][Medline]

Wideman R. F., and E. G. Buss. 1985. Aterial blood gas, pH, and bicarbonate values in laying hens selected for thick or thin eggshell production. Poult. Sci. 64:1015–1019.[Web of Science][Medline]

Wideman, R. F., and B. S. Cowen. 1987. Effect of dietary acidification on kidney damage induced in immature chickens by excess calcium and infectious bronchitis virus. Poult. Sci. 66:626–633.[Web of Science][Medline]

Wideman, R. F., D. M. Hooge, and K. R. Cumming. 2003. Dietary sodium bicarbonate, cool temperatures, and feed withdrawal: Impact on arterial and venous blood-gas values in broilers. Poult. Sci. 82:560–570.[Abstract/Free Full Text]

Willatts, S. M. 1987. Pages 3–65 in Lecture notes on fluid and electrolyte balance. 2nd ed. Blackwell Scientific Publications, Melbourne, Australia.

Williams, B., D. Waddington, and C. Solomin. 2000. Dietary effects on bone quality and turnover, and Ca and P metabolism in chickens. Res. Vet. Sci. 69:81–87.[CrossRef][Web of Science][Medline]





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