Magnesium Deficiency, Requirement and Toxicity in the Young Japanese Quail1

Magnesium Deficiency, Requirement and Toxicity in the Young Japanese Quaill B . F . HARLAND, M .

R. SPIVEY F O X AND B . E .

FRY, JR.

Division of Nutrition, Food and Drug Administration, Department of Health, Education, and Welfare, Washington, D.C. 20204 (Received for publication May 20, 1975)

POULTRY SCIENCE 55: 359-364,

INTRODUCTION

of factors that may influence ItheN studies utilization of an element, it is important to know the animal's requirement and the responses to deficiency and toxicity. In the case of Japanese quail (Coturnix coturnix japonica), most workers have utilized salt mixtures adequate for the chick. Although they were also adequate for the quail, they may have supplied unknown excesses of individual elements. Vohra (1972) recently reported that 150 p.p.m. magnesium was adequate for growth of the young quail and that 500 p.p.m. magnesium was toxic. Both of these levels were considerably lower than corresponding values for the chick (N.R.C., 1971). The characteristic signs of magnesium deficiency vary with species, as reviewed by Scott et al. (1969). When newly hatched 1. Part of this work was presented at the Annual Meeting of the Poultry Science Association, Morgantown, West Virginia, August 5-9, 1974 (Poultry Sci. 53: 1932, 1974).

1976

chicks received a magnesium deficient diet, they grew slowly, were lethargic and often panted and gasped (Almquist, 1942). They sometimes had convulsions, went into a comatose state and died. Mortality was usually high. A level of 6,000 p.p.m. magnesium in the diet caused reduced growth in young chicks (Chicco et al., 1967) while 6,400p.p.m. caused reduced growth and high mortality (Nugara and Edwards, 1963). Gardiner et al. (1960) obtained no adverse effect with 4,058 p.p.m. magnesium in the diet. The purpose of the present study was to extend the observations of Vohra to additional responses that might be useful for development of a bioassay of magnesium in foodstuffs. EXPERIMENTAL PROCEDURE Day-old quail of both sexes from our stock colony were maintained in continuously lighted, heated batteries under conditions to minimize environmental mineral contamination (Jacobs et al., 1969). The birds were

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ABSTRACT Studies of magnesium deficiency, requirement and toxicity in the young Japanese quail (Coturnix coturnix japonica) were conducted. Day-old birds were fed an adequate purified diet containing 35% soy protein to 2 weeks of age. Residual magnesium in the diet without any added magnesium salts was 21 p.p.m. Magnesium was supplied by graded amounts of MgS0 4 to a total of 11 levels ranging from 125 to 2,000 p.p.m. Deficiency signs included poor growth, and occasionally excitability, gasping and convulsions. Most mortality occurred during the first 7 days. The maximum dietary magnesium concentrations that produced the minimal significant deviation from normal values for mortality, body weight, hemoglobin and tibia ash were 225, 200, 250 and 250 p.p.m., respectively. Based on these measurements, 300 p.p.m. magnesium is considered adequate to meet the young quail's requirement under the conditions of these experiments. With 2,000 p.p.m. magnesium, the only adverse effect was an increase in mortality. Between 200 and 1,000 p.p.m. magnesium there was a linear relationship between concentration of magnesium in the tibia and the log of the concentration of dietary magnesium. This suggests that tibia magnesium concentration might be useful for bioassay of magnesium in foodstuffs.

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B. F. HARLAND, M. R. S. FOX AND B. E. FRY, JR.

Zinc, iron, copper, manganese and magnesium were determined in dietary components. Zinc, iron, manganese and magnesium were determined in tibias that had been wet ashed. To suppress anion interference, the final sample dilutions contained (v./v.) 10% glycerine and 0.7% perchloric acid. By these procedures, values for these elements in the National Bureau of Standards Liver Reference Material fell within the sampling error certified for the liver. The data were statistically evaluated by Student's ttest (Steel and Torrie, 1960).

TABLE 1.—Composition of the magnesium deficient diet1 Amount Ingredient g/kgSoybean protein2 350.0 Glycine, N.R.C.3 5.0 3 DL-methionine 6.0 Salts (no Mg salt)4 57.0 Choline chloride3 2.0 Corn oil5 40.0 Ethoxyquin6 0.1 Glucose monohydrate7 539.9 'The B vitamins were mixed with 10 g. glucose/kg. diet and the fat-soluble vitamins were mixed with 5 g. corn oil/kg. diet. Vitamins were present (mg./kg. diet) as follows: thiamine• HO 8, riboflavin 8, D-calcium pantothenate 40, nicotinic acid 100, pyridoxine • HC1 8, folic acid 3, cyanocobalamin 0.02, biotin 0.6, retinyl acetate 6, menaquinone 1, cholecalciferol 0.02, DL-a-tocopheryl acetate 25, and DL-a-tocopherol 25. 2 One lot of Purina Assay Protein, RP-100, Ralston Purina Co., St. Louis, Mo. 3 General Biochemicals, Inc., Chagrin Falls, Ohio. 4 Except for Mg, the macroelements and I were supplied according to Fox-Briggs Salts (1960). For other elements, the amount added and the dietary total (p.p.m.), respectively, were: Zn 19 and 30; Fe 44 and 100, Cu 0 and 5, Mn 10 and 12 and Se 0.2 and unknown. The Mg-deficient diet contained 21 p.p.m. Mg. Individual powders were prepared from glucose monohydrate and zinc carbonate, ferric citrate (<200 mesh), M n S 0 4 H 2 0 , Na 2 Se0 3 or MgS04, when present. Except for Se, each was assayed by atomic absorption spectrophotometry. All chemicals were reagent grade and were obtained from J. T. Baker Chemical Co., Phillipsburg, N.J., except ferric citrate, which was U.S.P., obtained from Merck & Co., Inc., Rahway, N.J., and Na 2 Se0 3 from Alpha Inorganics, Inc., Beverly, Mass. 5 Mazola, Best Foods, Englewood Cliffs, N.J. 6 Monsanto Chemical Co., St. Louis, Mo. The ethoxyquin was dissolved in 5 ml. of the corn oil. 7 Cerelose, Corn Products Refining Co., New York, N.Y.

RESULTS Signs typical of magnesium deficient animals, such as excitability, gasping and convulsions, were observed in only a few birds.

The kidneys and ureters of deficient birds appeared grossly normal. With the two highest levels of dietary magnesium, 1,500 and 2,000 p.p.m., there were watery feces.

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fed an adequate purified diet containing 35% soybean protein. Composition of the magnesium deficient diet, details of dietary mineral content and preparation are described in Table 1. There were negligible quantities of the inorganic elements present in any dietary component except the specific element source and the soybean protein. Eleven magnesium levels, ranging from 125 to 2,000 p.p.m., were fed. The groups within experiments consisted of ten to twelve birds, which were wing-banded at seven days of age. A record of mortality was kept daily as well as an examination for signs of gross physical and behavioral abnormalities. Birds were weighed at day seven and day fourteen, the final day of the experiment. Microhematocrit and hemoglobin (Drabkin and Austin, 1932) were measured in two experiments on blood collected from the vein at 14 days of age. At the end of the experimental period the birds were killed by decapitation and the tibias were removed and treated as previously described (Fox et al., 1971) for determination of fat-free dry weight. One bone from each bird was ashed at 600° C. for 24 hours.

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QUAIL MAGNESIUM REQUIREMENT TABLE 2 . --Effect

of magnesium intake on mortality and body weight Body weight 1

Mortality No. of experiments

125 200 225 250 275 300 500 750 1000 1500 2000

5 4 3 7 3 4 5 4 4 3 3

No. of birds 57 44 32 77 32 42 55 43 47 34 34

0-7 days

8-14 days

%

%

98.2 50.0 21.9 10.3 6.2 7.1 0 9.3 8.5 14.7 26.4

0 13.6 12.5 1.4 3.2 7.2 1.8 0 2.1 0 3.0

7 days g11.6 17.0 ± 0.53 ab 19.5 ± 0.57 ab 20.3 ± 0.33 20.7 ± 0.41 20.8 ± 0.38 19.2 ± 0.35 ab 18.0 ± 0.46 ab 19.6 ± 0.42 ab 20.7 ± 0.42 19.7 ± 0.61

14 days g25.1 36.5 ± 1.19ab 41.8 ± 1.08" 41.9 ± 0.62a 41.1 ± 0 . 8 6 a 41.9 ± 0.74 42.2 ± 0.72 41.0 ± 0.76 40.9 ± 0.91 45.2 ± 0.83 ab 42.8 ± 1.06

'Pooled means ± S.E. aWithin experiments, mean was significantly different from that of group receiving 300 or 500 mg. Mg/kg. in at least one experiment (P < 0.05). bPooled mean value was significantly different from pooled mean of birds fed 300 mg. Mg/kg. diet (P < 0.05).

Table 2 summarizes data from seven experiments for mortality and body weight at the various dietary magnesium levels. Not all levels were represented in each experiment. With either deficient (125, 200 or 225 p.p.m.) or the highest (2,000 p.p.m.) levels of magnesium, most of the mortality occurred during the first week. From 250-1,500 p.p.m. magnesium, there was little mortality. It was only at the lowest levels that body weight was severely depressed by magnesium deficiency. In the early experiments, 500 p.p.m. magnesium was used as a reference point and in the later experiments, 300 p.p.m. was used. At seven days of age mean body weights were similar from 225-2,000 p.p.m. in the diet. At 14 days of age growth was normal with 225 p.p.m. or higher levels of magnesium. Small differences in means within some experiments were significantly different from the reference groups. In the three experiments with 1,500 p.p.m. magnesium, the pooled body weight mean was significantly higher than that of the groups receiving 300 or 500 p.p.m. magnesium; however, this difference was significant in only one of the three experiments.

At 14 days of age there were no significant differences in hematocrit values as dietary magnesium levels increased from 225 to 1,000 p.p.m. (Table 3). There were, however, significantly lower hemoglobin values with 225 p.p.m. magnesium and with 250 p.p.m. in one of two experiments. There was a significant decrease in tibia ash at dietary magnesium levels of 200 and 225 p.p.m. and in one of four experiments with 250 p.p.m. (Table 4). From 275 through 2,000 p.p.m. the tibia ash values were similar; however, the values for 1,500 p.p.m. and 2,000 p.p.m. were significantly higher than the values for 300 or 500 p.p.m. A plot of tibia magnesium concentration vs. the log of dietary magnesium concentration is presented in Figure 1. Between 200 and 1,000 p.p.m. dietary magnesium, there was a linear response in tibia magnesium concentration. At the very low and very high levels of intake, there were plateaus in tibia magnesium concentration. DISCUSSION The young quail, like the chick, responded quickly to magnesium deficiency. The physi-

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Dietary Mg mg./kg.

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B. F. HARLAND, M. R. S. FOX AND B. E. FRY, JR.

TABLE 3.—Effect Dietary Mg mg. /kg.

of magnesium intake on hematocrit and hemoglobin at 14 days of age in two experiments No. of birds

Hemoglobin'

%

g./lOOml.

±1.36 ±1.12 ± 0.77 ± 0.98

10.9 12.1 11.1 12.0

± 0.33" ±0.39 ±0.27 ± 0.45

± ± ± ±

10.5 11.7 11.8 12.2

±0.61a ± 0.68 ± 0.20 ± 0.36

250 500 750 1000

7 11 10 8

Experiment 1 46.6 45.9 44.0 45.6

225 250 275 300

5 9 9 7

Experiment 2 48.0 47.7 45.3 46.2

2.10 1.15 1.43 1.11

'Mean ± S.E. "Significant difference from mean values of birds fed 500 mg. Mg/kg. in experiment 1 and 300 mg. Mg/kg. in experiment 2 (P < 0.05). TABLE 4.—Effect Dietary Mg

of magnesium intake on tibia ash

mg. /kg.

No. of experiments

No. of birds

125 200 225 250 275 300 500 750 1000 1500 2000

1 1 1 4 1 2 4 4 3 1 1

1 7 8 37 11 20 41 39 30 9 9

Tibia ash 1

% 48.6 45.8 ± 0.90 a 47.3 ± 0.88 a 50.4 ± 0.50 a 50.4 ± 0.24 50.2 ± 0.34 51.0 ± 0.42 51.2 ± 0.68 51.4 ± 0.53 52.8 ± 0.66" 51.7 ± 0.56 a

'Pooled means ± S.E. Ash values are expressed as % of fat-free dry weight. "Within experiments, mean was significantly different from that of group receiving 300 mg. Mg/kg. in at least one experiment (P < 0.05). ological measurements of survival, growth, hematopoiesis and mineralization of the bone behaved in an almost all-or-none manner. Without more extended dose-response effects, it was not possible to use the intercept of maximal response with the dose-response curves, as has been done for other elements (Davis et al., 1968). None of these responses appeared suitable for developing a bioassay. Growth by two weeks was essentially normal with 225 p.p.m. magnesium. Although

some of the small differences between groups were statistically significant, it would appear that this was related to normal variations within and between experiments rather than to magnesium level, per se. Mortality was a sensitive indicator of both deficiency and toxicity. Some mortality occurred in most groups. It is not always possible to identify weak birds among newlyhatched quail, so some mortality is to be expected even with adequate diets during the

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Hematocrit'

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QUAIL MAGNESIUM REQUIREMENT

O)

E

Z3

<

1 00

200 DIETARY

3 00

5 00

M A G N E S I UM,

1000

2000

mg./ kg.

FIG. 1. Effects of dietary magnesium on tibia magnesium concentration (mg./g. fat-free dry weight). The slope and regression coefficient (0.899) were calculated from individual values for birds fed 200-1,000 mg. Mg./kg. (Steel and Torrie, 1960). first week. Also, mortality may occur later in normal birds if they are startled and fly into the top of the cage. Therefore, the mortality of significance is that observed with the lowest and the highest levels of magnesium. Although anemia was minimal in deficient birds at two weeks of age, it is possible that anemia may have occurred at an earlier time period. This was not investigated. It is not clear why Vohra obtained such low values for magnesium requirement, 150 p.p.m., and toxicity, 500 p.p.m. Since his diet contained higher levels of zinc (4x), manganese (9x) and copper (20 x) than were present in the diet used in this study, it may be that these would account for some of the differences.

Vohra did not report the magnesium content of his basal diet, which may have been a source of some magnesium. Even if it were, this still would not account for the difference in toxicity between his study and this. The magnesium content of a stock diet 2 for young quail was found to be 2,500-2,800 p.p.m. We have obtained no evidence of toxicity with this diet. Differences in strains of quail may be a factor; however, we have recently compared responses to the same diets in different strains at different locations and closely similar results were obtained (Harland et al., 1975).

2. Game-bird Startena, Ralston-Purina Co.. St. Louis, Mo.

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Z

o <

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B. F. HARLAND, M. R. S. FOX AND B. E. FRY, JR.

In other species it has been shown that the magnesium requirement a n d / o r metabolism may be affected by phytate (McWard, 1969), potassium (Grace and O'Dell, 1970), high dietary fluoride (Chan et al, 1973) an J calcium and phosphorus (Kruse et al., 1932). It is reasonably certain that significant dietary alterations with respect to one or more of these components might affect magnesium requirements in the young quail. The concentration of magnesium in the tibia increased in response to dietary level before was constant with higher levels of magnesium to 1,000 p.p.m. It appears that tibia magnesium may be a useful index of

biological

availability of magnesium in foodstuffs.

ACKNOWLEDGMENTS The authors wish to thank Mildred Johnson, James Etherith and Sylvia Small for their fine technical assistance.

REFERENCES Almquist, H. J., 1942. Magnesium requirement of the chick. Proc. Soc. Exp. Biol. Med. 49: 544-545. Chan, M. M., R. B. Rucker, F. Zeman and R. S. Riggins, 1973. Effect of fluoride on bone formation and strength in Japanese quail. J. Nutr. 103: 14311440. Chicco, C. F.,C. B. Ammerman, P. A. van Wallehgem, P. W. Waldroup and R. H. Harms, 1967. Effects of varying dietary ratios of magnesium, calcium and phosphorus in growing chicks. Poultry Sci. 46: 368-373. Davis, P. N., L. C. Norris and F. H. Kratzer, 1968. Iron utilization and metabolism in the chick. J. Nutr. 94: 407-417. Drabkin, D. L., and J. H. Austin, 1932. A method

MARCH 31,1976. THIRTY-FIRST ANNUAL DISTILLERS FEED CONFERENCE, NETHERLAND HILTON HOTEL, CINCINNATI, OHIO AUGUST 8-11, 1976. JOINT MEETING OF THE AMERICAN INSTITUTE OF NUTRITION, THE AMERICAN SOCIETY OF CLINICAL NUTRITION, AND THE NUTRITION SOCIETY OF CANADA, MICHIGAN STATE UNIVERSITY, EAST LANSING, MICHIGAN

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the requirement was met. The rate of increase

for hemoglobin determination. J. Biol. Chem. 98: 719-733. Fox, M. R. S., and G. M. Briggs, 1960. Salt mixtures f or purif ied-type diets. III. An improved salt mixture for chicks. J. Nutr. 72: 243-250. Fox, M. R. S., B. E. Fry, Jr., M. E. Schertel and B. F. Harland, 1971. Effect of ascorbic acid on cadmium toxicity in the young coturnix. J. Nutr. 101: 1295-1305. Gardiner, E. E., J. C. Rogler and H. E. Parker, 1960. Magnesium requirement of the chick. Poultry Sci. 39: 1111-1115. Grace, N. D., and B. L. O'Dell, 1970. Interrelationship of dietary magnesium and potassium in the guinea pig. J. Nutr. 100: 37-44. Harland, B. F., J. H. Soares, Jr., M. R. S. Fox, B. E. Fry, Jr. and J. W. Boehne, 1975. Tibia mineral responses of two colonies of young Japanese quail fed identical diets. Fed. Proc. 34: 923. Jacobs, R. M., M. R. S. Fox and M. H. Aldridge, 1969. Changes in plasma proteins associated with the anemia produced by dietary cadmium in Japanese quail. J. Nutr. 99: 119-128. Kruse, H. D., E. R. Orent and E. V. McCollum, 1932. Studies on magnesium deficiency in animals. I. Symptomatology resulting from magnesium deprivation. J. Biol. Chem. 96: 519-539. McWard, G. W., 1969. Effects of phytic acid and ethylene-diaminetetraacetic acid (EDTA) on the chick's requirement for magnesium. Poultry Sci. 48: 791-792. National Research Council, 1971. Nutrient requirements of poultry. Sixth rev., National Academy of Sciences, Washington, D.C. Nugara, D., and H. M. Edwards, Jr., 1963. Influence of dietary Ca and P on the Mg requirement of the chick. J. Nutr. 80: 181-184. Scott, M. L., M. C. Nesheim and R. J. Young, 1969. Nutrition of the Chicken. M. L. Scott and Associates. Ithaca, New York. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc., New York, N.Y. Vohra, P., 1972. Magnesium requirements for survival and growth of Japanese quail (Coturnix coturnix japonica). Poultry Sci. 51: 2103-2105.