Strontium Metabolism in Chicks1

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J. G. WILSON AND C. C. BEUNSON

of handling. C0 2 immobilization of broilers following gentle handling by the wings produced the lowest amount of hemorrhaging. The incidence of hemorrhaging was higher in the right thigh of birds handled by the right leg in comparison with hemorrhaging in the left thigh. There was no apparent relationship between blood loss and incidence of hemorrhaging. The results indicated that hemorrhaging in thighs of broilers used in this study was caused primarily by handling of the birds during loading and processing and by struggling of birds after they were placed on the killing line. With few exceptions, gentle handling of birds by the wings followed by C0 2 immobilization resulted in a complete absence of hemorrhaging. REFERENCES Crothers, J. L., and N. V. Helbacka, 1960. Evaluation of broiler grade defects by quality control

methods. Misc. Ext. Publ. 226, University of Maryland. Hamdy, M. K., K. N. May and J. J. Powers, 1961. Some physical and physiological factors affecting poultry bruises. Poultry Sci. 40: 790-795. Jewett, L. J., and R. F. Saunders, 1960. Handling and processing broilers in Maine. Part 2. Maine Agr. Exp. Sta. Bull. 593. Kaiser, W. K., and R. C. Smith, 1958. Factors affecting the bruising of broilers. Delaware Agr. Exp. Sta. (Tech.) Bull. 323. Kotula, A. W., and N. V. Helbacka, 1966. Blood volume of live chickens and influence of slaughter technique on blood loss. Poultry Sci. 45: 684-688. May, K. N., and M. K. Hamdy, 1966. Bruising of poultry—a review. World's Poultry Sci. J. 22: 316-322. Ringrose, A. T., 1953. Bruised poultry challenges processor's profits. Am. Egg Poultry Rev. 3 : 36-39. Saunders, R. F., and R. K. Lanson, 1954. Handling with care and profit. Maine Farm (Res.) Bull., pp. 11-13.

Strontium Metabolism in Chicks 1 C. W. WEBER, A. R. DOBERENZ,2 R. W. G. WYCKOFF AND B. L. REID Departments of Physics and Poultry Science, University of Arizona, Tucson, Arizona 85721 (Received for publication January 17, 1968)

T

HIS study of the metabolic uptake of stable strontium in chicks had its origins in the need for more knowledge concerning the amounts of this element that can be absorbed by living animals. There were two reasons for the investigation. On 'This work was supported by National Institutes of Health Grant No. 5R01-DE-01919 and National Science Foundation Grant No. GB-4117. 2 Research Career Development Awardee 5K3-DE-32, 542, National Institute of Dental Research. Arizona Agricultural Experiment Station Journal Article #1283

the one hand it is of practical importance for a better understanding of the relation of strontium to dietary calcium levels in geographic areas where the uptake of strontium may be high. It is also important for its bearing on the interpretation that can be given to the high strontium contents of many fossil bones and teeth (Wyckoff and Doberenz, 1967). In this work we have sought, using chicks, (a) to evaluate the utilization of large amounts of strontium in relation to the dietary calcium level, (b) to determine the maximum amounts of strontium that can be incorporated into the

1319

STRONTIUM METABOLISM

growing chicks without seriously impairing their health and (c) to see how this strontium is distributed within the developing bones. There have been many studies involving the ingestion of strontium but over the last decade emphasis has been on radioactive strontium and problems concerned with its absorption and retention by animals and plants. This subject has been well documented by numerous articles and by several reviews. In the course of this earlier work a strontium-calcium relationship has been demonstrated in which calcium but not strontium was under homeostatic control. Regulation of strontium absorption has been reported in one study to be determined by the dietary calcium level or the level of total alkaline earths (Comar and Bronner, 1964). In this regulation the gastrointestinal membrane has been considered to act as a barrier which discriminates between strontium and calcium since it has been shown that calcium, but not strontium, is transported against a concentration gradient. Using radiostrontium and radiocalcium as indicators, other investigators have stated that though there are differences in the strontium deposition in new and mature bones, the skeletal discrimination between these elements is small compared to other processes and seems to favor an overall retention of calcium (Robertson, 1960). In rats, too, little skeletal discrimination has been found between calcium and strontium (Bauer et al., 1955; Comar et al., 1956). In earlier dietary experiments high levels of strontium added at the expense of calcium were found to result in reduced body weights and in bone abnormalities (Bartley and Reber, 1961; Colvin and Creger, 1967); it has also been stated (Jones, 1938) that such strontium in the diet gives rise to insoluble phosphates during digestion and to a phosphorus deficiency.

TABLE 1.—Composition of experimental diets Ingredient

Ground yellow corn Soybean meal Fish meal Dehydrated alfalfa meal Dried whey Corn dist. dried solubles Animal fat DL-methionine Vitamin premix 1 Dicalcium phosphate Calcium carbonate Salt Magnesium sulfate Cr 2 0 3 Bentonite Total

0.72% Ca diet

%

47.50 31.33 S.00 2.00 1.00 1.00 5.00 0.10 2.50 1.40

0.1% Ca diet

%

0.20 0.02 0.20 2.75

47.50 31.33 5.00 2.00 1.00 1.00 5.00 0.10 2.50 1.40 0.75 0.20 0.02 0.20 2.00

100.00

100.00

—

1 Supplied the following per kg. of diet: 9,900 I.U. vitamin A, 1,540 I.C.U. vitamin D3, 4.4 mg. riboflavin, 27.5 mg. niacin, 11.0 mg. D-calcium pantothenate, 440 mg. choline chloride, 13.2 meg. vitamin B12. 5.5 I.U. alpha tocopheryl acetate, 2.2 mg. menadione sodium bisulfite, 4.4 mg. procaine penicillin, and 22.0 mg. chlortetracycline.

METHODS AND PROCEDURES

Feeding experiments were begun with day-old female Hubbard chicks and continued for a four-week period. During this time the chicks were housed in electrically heated batteries with raised wire floors, feed and water being supplied ad libitum. The two diets (Table 1) supplied all known requirements at calcium levels of 0.72 and 1.0%; both provided a level of 0.5% available phosphorus. Two groups of chicks fed one or the other of these diets served as controls; five groups were given these diets supplemented by added strontium as carbonate; the 0.72% calcium diet was fed at strontium levels of 3,000 and 6,000 p.p.m. while to the calcium diet was added 3,000, 6,000 and 12,000 p.p.m. of strontium. A total of 18 chicks were used per dietary treatment in three replications of six chicks for each diet. After the fourth week, the chicks were weighed and feed conversions calculated. Feed and feces were collected during the

1320

C. W. WEBER, A. R. DOBERENZ, R. W. G. WYCKOFF AND B. L. REID

fourth week and analyzed for Cr 2 0 3 , calcium, phosphorus (Weber and Reid, 1967) and strontium. Strontium was determined by x-ray spectroscopy described below. At the termination of the experiment, the birds were bled and analyzed for plasma calcium, potassium, sodium and total blood phosphorus (Coleman, 19S8; Oser, 1965). In addition, six birds per dietary treatment were sacrificed and tibia samples taken. The tibias were cleaned, dried and weighed. Experimental data were analyzed statistically by Duncan's multiple range test (Duncan, 1955). All measurements of strontium were made by the conventional techniques of xray spectroscopy. Feed and feces samples for this were dried and powdered to 100 mesh. The dried tibial bones were ashed and powdered. Rate meter readings of the heights of the Ka peaks of strontium from these various samples were obtained using a standard Philips fluorescent vacuum spectrometer equipped with a tungsten xray tube (operated at 40 KV, 10 Ma), an LiF analyzing crystal and an Nal scintillator as detecting device. Heights of the strontium peaks above background were taken as measurements of the strontium contents of the samples. These were standardized by reference to peaks obtained from powdered synthetic calcium phosphate (as hydroxy apatite) to which known amounts of strontium had been added; correction was made for the amount of strontium always present in the commercially available phosphate. Strontium contents of many of the bones were so high that the powdered samples were diluted with known amounts of the synthetic apatite to bring these contents down into the range where the standardization curve was linear. Bone samples were examined under the electron probe to ascertain the relative distribution of their strontium and calcium. Samples for this were prepared by cutting

millimeter thick transverse sections through the tibia embedded in bioplastic. The upper surfaces of these sections mounted on one inch disc of glass or aluminum were ground, polished and vacuum coated with a thin, electrically conducting layer of carbon. The probe was the prototype of the Philips Model MRC instrument operated to scan a square area 160 micra on a side for first its calcium and then its strontium content. X-ray diffraction patterns were also obtained from the powdered bones to furnish information about the chemical state of the strontium they contained. RESULTS AND DISCUSSION Four week old female chicks fed different levels and combinations of calcium and strontium exhibited reduced body weights at the higher strontium levels (Table 2). The basal and 3,000 p.p.m. strontium fed birds with either 0.73 and 1.0% Ca were not significantly different in body weights. These data suggest that a change in the Ca:Sr ratio did not affect the growth rate at the lower treatment. Previous studies (Bartley and Reber, 1961; Colvin and Creger, 1967) had indicated a growth reduction at moderate levels of strontium but in these earlier experiments calcium was removed when the strontium was added. The possibility exists that the reduced body weights noted were a reflection of calcium insufficiency. In the present study, the reduction in weight at the 6,000 p.p.m. strontium level was less on the higher calcium diet. This suggests an antagonistic effect of the increased calcium, but such a conclusion is debatable in view of the strontiumretentions measured. As Table 2 indicates, the measured feed conversions were not greatly different for the several dietary treatments. Analysis of the blood samples showed no effect of dietary strontium or calcium on the plasma sodium, potassium or calcium

1321

STRONTIUM METABOLISM

TABLE 2.—Effect of strontium on body weights, feed conversion, calcium and phosphorus retention Diet No.

Dietary treatment

0.72% Ca 0 p.p.m. Sr 0.72% Ca 3000 p.p.m. Sr 0.72% Ca 6,000 p.p.m. Sr 1.0% Ca 0 p.p.m. Sr 1.0% Ca 3,000 p.p.m. Sr 1.0% Ca 6,000 p.p.m. Sr 1.0% Ca 12,000 p.p.m. Sr

1 2 3 4 5 6 7

Retention

4th Week body weight (gm.)

Feed conversion

562a1

%

%

Calcium

Phosphorus

1.58

44.55

39.20

592a

1.54

44.56

39.10

384c

1.53

39.38

34.43

598a

1.62

42.91

39.22

596a

1.60

31.62

38.89

482b

1.44

39.04

42.23

255d

1.84

30.82

39.53

1 Means having different superscripts are significantly different at the 0.05 level of probability (Duncan, 1955).

levels (Table 3). Total blood phosphorus was essentially the same for all dietary treatments. These data conflict with the earlier conclusions (Jones, 1938) that high dietary strontium leads to a phosphorus deficiency; instead our blood phosphorus levels and phosphorus retention values indicate no alteration in phosphorus metabolism as the result of strontium feeding. The determined levels of strontium retention are given in Table 4. These data indicate that for a stated level of dietary strontium the amount retained per bird was substantially the same at the two levels of calcium fed. Tibia weights, as percent of body weight, increased significantly with increasing dietary levels of strontium with the exception of treatment 6. Evidently, the greater retention of strontium with inTABLE 3.—Effect

of strontium on blood components Plasma

Diet No.

Na meq./l.

K meq./l.

Ca mg./lOO ml.

Total blood phosphorus mg./lOO ml.

1 2 3 4 5 6 7

159 160 160 160 162 160 160

7.1 6.5 7.3 6.5 6.6 6.3 7.0

10.9 10.6 9.2 11.1 10.5 10.1 10.3

100.7 107.3 94.7 99.8 102.6 101.6 111.0

creased dietary levels was paralleled by an increase in bone weight. The x-ray spectroscopic data show, as might be expected, that the strontium content of the tibia increased with dietary intake. They also seem to demonstrate that an increase in the dietary calcium level decreased the strontium uptake. For example, bones from birds fed a diet containing 0.72% calcium and 0.6% strontium contained 9.4% strontium in the bone ash; whereas bones from birds fed 1.0% calcium and 0.6% strontium had 7.7% strontium in their bone ash. The presence of such large amounts of strontium in the chick bones led to attempts to determine how this strontium TABLE 4.—Strontium analysis of feed, bone ash Diet No.

Sr (p.p.m.) added t o feed

Analyzed level of feed Sr (p.p.m.)

0 3,000 6,000 0 3,000 6,000 12,000

205 ,598 ,526 215 ,867 ,596 12,363

% Sr in Tibia as %of tibia bone bodyash weight 0.1 5.7 9.4 0.1 4.1 7.7 10.0

0.719c' 0.7971 0.853b 0.718c 0.812b O.609d 1.090a

Sr2 (p.p.m.) retained per bird 19 869 2,072 118 951 2,238 5,629

1 Means having different superscripts are significantly different a t the 0.05 level of probability (Duncan, 1955). 2 Calculated from percent retention and feed intake d a t a .

1322

C. W. WEBER, A. R. DOBERENZ, R. W. G. WYCKOFF AND B. L. REID

"* i*%&fyffiiv'

FIG. 1. X-ray electron probe scan of the distribution of calcium (top) and strontium (bottom) from the same cross sectional of a tibia from treatment seven.

was distributed. When the tibial sections already described were subjected to electron probe examination by photographing the distribution, first of the calcium and then of the strontium characteristic x-rays emitted from a cross section of bone, patterns such as that of P'igure 1 were obtained. In such a pattern each dot represents a detected quantum of the characteristic radiation, and it is apparent that except for the large voids which are the osteons there is no conspicuous localization of either element; they were deposited more or less simultaneously as the bone hardened. Electron microscopic examination has repeatedly shown that the apatite crystals of bone are exceptionally small and this accounts for the poor x-ray diffraction patterns which bone furnishes. Nevertheless it is not inconceivable that the crystallites

might be larger in strontium-rich bone or that the strontium might be present in a non-apatitic phase. It has been reported that rat bone rich in strontium gives the pattern of apatite with altered cell dimensions indicative of strontium in the apatite structure (MacDonald et al., 1951). The enamel of rat teeth rich in strontium has been found to contain hexa-strontiumtrihydrogen pentaorthophosphate (Johnson and Singer, 1967; Johnson, 1967). In view of these observations powder patterns were made of chick bone containing the highest percentage of strontium, before and after ashing. As usual, ashing somewhat sharpened the lines in the apatite pattern as a consequence of an increase in the mean size of the crystallites; but this sharpening was not sufficient to demonstrate the small increase in cell dimensions that would result from the presence of strontium in the structure. No lines due to a non-apatitic phase were seen. Two of the present authors have recently shown that many fossil bones, especially those from certain localities, contain relatively large amounts of strontium, and it has seemed important for a variety of reasons to try to decide if this strontium was part of the original animal bone (Wyckoff and Doberenz, 1967). The experiments described here demonstrate that apparently healthy chicks can, in response to a strontium-rich diet, have as much of this element in their bones as has been found in the fossils. Such data about minor constituents in bones presumably can furnish helpful information about the environment which supported earlier forms of life. SUMMARY

The utilization of large amounts of strontium in relation to dietary calcium level has been investigated with chicks. Dietary calcium levels of 0.72 and 1.0% were each fed with 3,000 and 6,000 p.p.m. stron-

STRONTIUM METABOLISM

tium as strontium carbonate. Feeding 6,000 p.p.m. strontium reduced growth rate more severely at the lower calcium level. Higher levels of strontium appeared to reduce calcium retentions while phosphorus utilization was apparently unaffected. Tibia weights were significantly increased with increased levels of strontium in the diet. Higher strontium levels in bone ash were found at the lower calcium level than when 1.0% calcium was fed. Electron probe examination of tibia sections exhibited no conspicuous localization of either calcium or strontium. X-ray diffraction studies failed to exhibit a non-apatitic phase as a result of strontium feeding. REFERENCES Bartley, J. C , and E. F. Reber, 1961. Toxic effects of stable strontium in young pigs. J. Nutri. 75: 21-28. Bauer, G. C. H., A. Carlsson and B. Lindquist, 1955. A comparative study on the metabolism of Sr90 and Ca". Acta Physiol. Scand. 35: 56-66. Colvin, L. B., and C. R. Creger, 1967. Stable Sr and experimental bone anomalies. Fed. Proc. 26: 416. Comar, C. L., and F. Bronner, 1964. Mineral metabolism. Academic Press, New York, Vol. 2, p. 523-572. Comar, C. L., R. H. Wasserman and M. M.

1323

Nold, 1956. Strontium-calcium discrimination factors in the rat. Proc. Soc. Exptl. Biol. Med. 92: 859-863. Duncan, D. B., 1955. The new multiple range and F test. Biometrics, 1 1 : 1-42. Johnson, A. R., 1967. X-ray diffraction patterns of rat incisor tooth enamel with low or high strontium content. J. Dent. Res. 46: 79-81. Johnson, A. R., and L. Singer, 1967. An electron microprobe study of rat incisor teeth with low or high concentrations of strontium. Archs. Oral Biol. 12: 389-399. Jones, J. A., 1938. Metabolism of calcium and phosphorus as influenced by addition to the diet of salts of metals which form insoluble phosphates. Am. J. Physiol. 124: 230-237. MacDonald, N. S., F. Ezmirlian, P. Spain and C. MacAuthur, 1951. The ultimate site of skeletal deposition of strontium and lead. J. Biol. Chem. 189: 387-399. Operating Directions for the Coleman Model 21 Flame Photometer, 1958. Coleman Instruments, Inc., p. 19. Oser, B. L., 1965. Hawk's Physiological Chemistry, McGraw-Hill Book Co., New York, p. 1116. Robertson, J. S., 1960. Radioisotopes in the Biosphere. ed. R. S. Caldecott and L. A. Snyder. U. of Minnesota Center for Continuation Study, Minneapolis, Minn., p. 423. Weber, C. W., and B. L. Reid, 1967. Protein requirements of Coturnix quail to five weeks of age. Poultry Sci. 46: 1190-1194. Wyckoff, R. W. G., and A. R. Doberenz, 1967. The strontium content of fossil teeth and bones. Geochim. Cosmochim. Acta, in press.

Goitrogenic Effects of (—)-5-Vinyl-2-Oxazohdinethione, a Goitrogen in Rapeseed, in Growing Chicks T. MATSUMOTO, H. ITOH AND Y. AKIBA Department of Animal Husbandry, Faculty of Agriculture, Tohoku University, N-6, Sendai, Japan (Received for publication January 24, 1968)

I

T HAS been reported that when rapeseed meal is fed to an animal as the main source of protein, the thyroid gland is enlarged (Turner, 1946; Blakely and Anderson, 1948a, b; Witz et al., 1950; and Klain et al., 1956), and the growth rate is

decreased (Kratzer et al., 1954; Clandinin et al., 1959; and Holmes and Roberts, 1963). (—) -5-Vinyl-2-oxazolidinethione (goitrin) in rapeseed (Astwood et al., 1949; Carroll, 1949) has been known as the goitrogen which enlarges the thyroid