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Calcium-Phosphorus Metabolism in the Chicken
Calcium-Phosphorus Metabolism in the Chicken IV. ERGOSTEROL REQUIREMENT OF GROWING CHICKS EARL J. KING, HARRY HULL AND G. EDWARD HALL Department of Medical Research, University of Toronto, Canada (Received for Publication June 29, 1932)
T
fectiveness by including dried carrots as a source of vitamin A. That the difference between the antirachitic potency for the chicken of irradiated ergosterol and cod liver oil may not be so great as that stated by Massengale and Nussmeier (1930) was indicated by Hall and King (1931a), who found that normal chickens could be obtained by administering one drop daily of an irradiated ergosterol solution containing 100 times the potency of cod liver oil by rat assay, i.e., 100X. One drop of this solution weighed about 20 mg., and was equivalent, therefore, to 2 grams of a IX solution. By this treatment King and Hall (1931) were able to obtain an average bone ash of 46.8 percent, which is almost the same as that obtained by Massengale and Nussmeier for 10 weeks old chickens when 2 percent of cod liver oil was added to the rickets-producing ration. If it be assumed that the chickens consumed on. the average about 100 grams of feed daily, then the birds receiving 1 drop of the ergosterol solution were getting the same amount of vitamin D (by rat assay) as were those receiving 2 percent of cod liver oil. The apparent discrepancy between the two experiments may perhaps be explainable in two ways: first, King and Hall gave the ergosterol by dropper per os, while Massengale and Nussmeier mixed the oily solution with the feed. In a control experi-
[129]
Downloaded from http://ps.oxfordjournals.org/ at ULB Bonn on May 27, 2015
HE action of various irradiated ergosterol preparations in preventing rickets in chickens has been reported on by several investigators. Kreitmair and Moll (1928), in a study of the results of overdosage with irradiated ergosterol, reported that the chicken required larger amounts to produce toxic effects than any other of the several species of animals tried, and also that the minimum antirachitic dose for the chicken (0.01 mg.) was considerably higher than that of other animals. Irradiated ergosterol preparations are standardized by reference to their capacity for curing rickets in the rat, and it has been customary to state their strength in terms of their antirachitic potency relative to cod liver oil. When irradiated ergosterol solutions have been given to chickens receiving a rickets-producing diet, it has been found that much more irradiated erogsterol, relative to cod liver oil, is necessary to prevent rickets than is the case with the rat. Thus Massengale and Nussmeier (1930) have found that it is necessary to give 200 times as much irradiated ergosterol as cod liver oil (by rat unitage) to produce a normal ash content of the femurs of chickens. Hess and Supplee (1930) and Mussehl and Ackerson (1930) have also reported on the inferiority of irradiated ergosterol solutions relative to cod liver oil for the prevention of rickets in chickens, and Russell and Klein (1931) have failed to increase its ef-
130
POULTRY
ment which was run concurrently with that to be described, we have had some evidence that ergosterol is not as effective when mixed with the feed and left thus in the hoppers as when administered by mouth. Secondly, the calcium phosphorus ratio of the ration may have had a definite influence on the results. King and Hall used a ration containing calcium and phosphorus in the ratio of 1.8 to 1, which ratio falls in the normal range in which rickets in the chicken is easily prevented by the addition of a small amount of vitamin D or by irra-
SCIENCE
chicks* were placed on a basal ration, the composition of which is given in Table 1. The analysis of the different constituents of this ration and the amounts of calcium and phosphorus contributed by each, together with the total amounts of these elements present, are shown in the table. This basal ration was supplemented with vitamin D in the form of two percent of cod liver oil or two percent of sesame oil containing various amounts of irradiated ergosterol so mixed as to give solutions of several strengths which are referred to in
Ca
P percent
Yellow corn Wheat Meat meal Buttermilk powder Calcium carbonate Sodium chloride Oil (cod liver oil or sesame oil+irradiated ergosterol)
Number of parts of constituent in ration
0.01 0.05 8.61 1.26 40.00
0.31 0.37 3.98 0.88 0.00
—
—
62 15 10 9 1 1
—
—
2 100
diation (see Wilgus, 1931, for a discussion of the Ca:P ratio). It is well known that to produce rickets in the rat it is necessary to use a ration containing a very abnormal calcium-phosphorus ratio; and it was with a ration similar to the Steenbock ricketsproducing diet for rats and containing a Ca:P ratio of about 3:1 that Massengale and Nussmeier found so marked a difference between the rickets-preventing power of cod liver oil and that of irradiated ergosterol. The present experiment was devised in order to determine what was the basal requirement of irradiated ergosterol for the prevention of rickets in chickens fed a ration containing a normal Ca: P ratio. Seven groups of fifteen Barred Plymouth Rock
Parts Ca and P contributed by constituent to ration Ca P 0.006 0.007 0.861 0.114 0.400
0.192 0.055 0.398 0.079 0.000
—
.—
—
—
1.388 0.724 Ratio Ca: P 1.91:1
terms of their potency relative to cod liver oil by rat assay. Thus "10 percent of irradiated ergosterol solution" means that 2 percent of a sesame oil solution was used containing an amount of irradiated ergosterol equivalent in rat units to 10 percent of cod liver oil. These solutions were prepared fresh each week by appropriate dilutions of Mead's Viosterol** with sesame oil; two parts of the oil solution were intimately mixed with ninety-eight parts of the basal
•Kindly supplied to us by Professor W. R. Graham, of the Ontario Agricultural College, Guelph. ** The Viosterol (irradiated ergosterol in corn oil) was kindly supplied to us by the Mead Johnson Company, Evansville, Indiana.
Downloaded from http://ps.oxfordjournals.org/ at ULB Bonn on May 27, 2015
TABLE 1. Calcium and phosphorus content of ration
MARCH,
1933.
VOL.
XII,
No.
131
2
The percentages of ash for the bones of the chickens in the several groups, together with the averages, are shown in Table 2. Two conclusions can be drawn from the figures, namely, that with the amounts of ergosterol used the results were better than those obtained by Massengale and Nussmeier and poorer than those obtained by King and Hall. Although 10 and 20 percent of irradiated ergosterol solution (of the same rat unitage strength as cod liver oil) were insufficient to give quite as high
TABLE 2. Percentage ash of long bones of chickens receiving basal ration su by various amounts of vitamin D Basal
Irradiated Ergosterol Solution
Cod Liver Oil 20%
0.5%
2%
4%
10%
37.0 39.3 37.0 39.6 42.7 35.5 37.8 36.2 35.5 42.8
37.9 45.0 38.0 24.6 38.8 41.1 41.8 39.3 42.6 46.9
38.3 40.3 40.8 41.9 43.3 51.2 37.4 40.7 43.8 38.3 44.1
36.3 41.3 37.8 40.0 39.8 38.7 46.0 41.9 47.8 48.4 44.4 47.2
42.1 43.6 47.5 46.9 45.2 42.3 47.0 40.6 46.4 43.5 51.0 50.3 52.3
51.4 50.1 42.2 48.9 45.0 45.2 44.6
44.6 48.5 48.5 46.1 45.1 49.4 50.6 51.0 48.2 47.4 46.5 46.2
Average 38.3
39.6
41.8
42.6
46.1
46.8
47.7
for ash analysis. After removing the adhering tissue as completely as possible, the bones were split lengthwise and the marrow scraped out with a blunt knife. The bones were dried in an oven at 110°C. and then fat extracted with ether in a Soxhlet apparatus for six hours. The dried fat-free bones thus obtained were passed through a grinding mill and reduced to a powder. Weighed samples of the powder were placed in porcelain crucibles and ashed in an electric muffle furnace at red heat. The weight of the residue times 100 over the weight of the sample was taken as the percentage ash of the bone.
2%
an ash content of the bones, yet the results were only 1 percent lower than those got with 2 percent cod liver oil, and the general condition of the birds appeared to be quite as good. As mentioned previously, it is felt that these better results with ergosterol are due to the use of a ration with a normal calcium-phosphorus ratio; and further, that they more truly represent the relative antirachitic values of cod liver oil and irradiated ergosterol when mixed with the feed than do results obtained with diets containing the mineral elements calcium and phosphorus in abnormal proportions. Diets similar to the Steenbock rickets-producing diet for
Downloaded from http://ps.oxfordjournals.org/ at ULB Bonn on May 27, 2015
ration, and the mixtures kept before the birds in metal hoppers. The chickens were kept on the rations for eleven weeks behind ordinary window glass in a room with western exposure. Birds which died during the course of the experiment were discarded. Unfortunately, the mortality was somewhat higher in some groups than in others, which has rendered the interpretation of the results less clear than might have been desired. During the eleventh and twelfth weeks the birds were killed and the tibiae taken
132
POULTRY
SUMMARY
Irradiated ergosterol is not as effective in preventing rickets in chickens as is cod liver oil. The difference in the antirachitic power of these two sources of vitamin D is not as
marked with a ration containing a normal calcium-phosphorus ratio as when these elements are present in abnormal ratio. There is some indication that irradiated ergosterol is more effective when given by mouth than when mixed with the feed. REFERENCES
Hess, A. F. and G. C. Supplee, 1930. The action of irradiated ergosterol on rats and chickens. Proc. Soc. Exp. Biol. Med., 27:609-610. Hall, G. E. and E. J. King, 1931a. Calcium phosphorus metabolism in the chicken. I. Poul. Sci., 10:132-152. , 1931b. Calcium phosphorus metabolism in the chicken. II. Poul. Sci., 10:259-268. King, E. J. and G. E. Hall, 1931. Calcium phosphorous metabolism in the chicken. III. Poul. Sci., 10:332-352. Kreitmair, H. and T. Moll, 1928. Hypervitaminosis through large doses of vitamin D. Munch. Med. Woch., 75:637-639. Massengale, O. N. and M. Nussmeier, 1930. The action of activated ergosterol in the chicken. II. J. Biol. Chem., 87:423-426. Mussehl, F. E. and C. W. Ackerson, 1930. Irradiated ergosterol as an antirachitic for chicks. Poul. Sci., 9 :334-338. Russell, W. C. and D. Klein, 1931. The vitamin A content of a ration and the antirachitic potency of irradiated ergosterol. Poul. Sci., 10: 269-274. Wilgus, H. S., 1931. The quantitative requirement of the growing chick for calcium and phosphorus. Poul. Sci., 10:107-117.
Downloaded from http://ps.oxfordjournals.org/ at ULB Bonn on May 27, 2015
the rat when used for chickens represent an unnatural dietary condition and results thus obtained are of experimental rather than of practical significance. That irradiated ergosterol may not be as effective when mixed with the feed as when given by mouth would seem to be indicated by the very much lower ash content in the birds receiving 2 percent of irradiated ergosterol solution in the present experiment than that of birds receiving an approximately equal amount (1 drop daily of Viosterol) in the former investigation, both series of birds having received almost identical basal diets. A control group of forty chickens receiving 1 drop of Viosterol daily which were maintained concurrently with the present experiment but which were kept for another purpose until five months of age, were in excellent condition and had an average bone ash of 52.3 percent, which is quite as good as that of normal range birds of the same age which have been raised on a ration supplemented by cod liver oil (Hall and King, 1931b).
SCIENCE
T
fectiveness by including dried carrots as a source of vitamin A. That the difference between the antirachitic potency for the chicken of irradiated ergosterol and cod liver oil may not be so great as that stated by Massengale and Nussmeier (1930) was indicated by Hall and King (1931a), who found that normal chickens could be obtained by administering one drop daily of an irradiated ergosterol solution containing 100 times the potency of cod liver oil by rat assay, i.e., 100X. One drop of this solution weighed about 20 mg., and was equivalent, therefore, to 2 grams of a IX solution. By this treatment King and Hall (1931) were able to obtain an average bone ash of 46.8 percent, which is almost the same as that obtained by Massengale and Nussmeier for 10 weeks old chickens when 2 percent of cod liver oil was added to the rickets-producing ration. If it be assumed that the chickens consumed on. the average about 100 grams of feed daily, then the birds receiving 1 drop of the ergosterol solution were getting the same amount of vitamin D (by rat assay) as were those receiving 2 percent of cod liver oil. The apparent discrepancy between the two experiments may perhaps be explainable in two ways: first, King and Hall gave the ergosterol by dropper per os, while Massengale and Nussmeier mixed the oily solution with the feed. In a control experi-
[129]
Downloaded from http://ps.oxfordjournals.org/ at ULB Bonn on May 27, 2015
HE action of various irradiated ergosterol preparations in preventing rickets in chickens has been reported on by several investigators. Kreitmair and Moll (1928), in a study of the results of overdosage with irradiated ergosterol, reported that the chicken required larger amounts to produce toxic effects than any other of the several species of animals tried, and also that the minimum antirachitic dose for the chicken (0.01 mg.) was considerably higher than that of other animals. Irradiated ergosterol preparations are standardized by reference to their capacity for curing rickets in the rat, and it has been customary to state their strength in terms of their antirachitic potency relative to cod liver oil. When irradiated ergosterol solutions have been given to chickens receiving a rickets-producing diet, it has been found that much more irradiated erogsterol, relative to cod liver oil, is necessary to prevent rickets than is the case with the rat. Thus Massengale and Nussmeier (1930) have found that it is necessary to give 200 times as much irradiated ergosterol as cod liver oil (by rat unitage) to produce a normal ash content of the femurs of chickens. Hess and Supplee (1930) and Mussehl and Ackerson (1930) have also reported on the inferiority of irradiated ergosterol solutions relative to cod liver oil for the prevention of rickets in chickens, and Russell and Klein (1931) have failed to increase its ef-
130
POULTRY
ment which was run concurrently with that to be described, we have had some evidence that ergosterol is not as effective when mixed with the feed and left thus in the hoppers as when administered by mouth. Secondly, the calcium phosphorus ratio of the ration may have had a definite influence on the results. King and Hall used a ration containing calcium and phosphorus in the ratio of 1.8 to 1, which ratio falls in the normal range in which rickets in the chicken is easily prevented by the addition of a small amount of vitamin D or by irra-
SCIENCE
chicks* were placed on a basal ration, the composition of which is given in Table 1. The analysis of the different constituents of this ration and the amounts of calcium and phosphorus contributed by each, together with the total amounts of these elements present, are shown in the table. This basal ration was supplemented with vitamin D in the form of two percent of cod liver oil or two percent of sesame oil containing various amounts of irradiated ergosterol so mixed as to give solutions of several strengths which are referred to in
Ca
P percent
Yellow corn Wheat Meat meal Buttermilk powder Calcium carbonate Sodium chloride Oil (cod liver oil or sesame oil+irradiated ergosterol)
Number of parts of constituent in ration
0.01 0.05 8.61 1.26 40.00
0.31 0.37 3.98 0.88 0.00
—
—
62 15 10 9 1 1
—
—
2 100
diation (see Wilgus, 1931, for a discussion of the Ca:P ratio). It is well known that to produce rickets in the rat it is necessary to use a ration containing a very abnormal calcium-phosphorus ratio; and it was with a ration similar to the Steenbock ricketsproducing diet for rats and containing a Ca:P ratio of about 3:1 that Massengale and Nussmeier found so marked a difference between the rickets-preventing power of cod liver oil and that of irradiated ergosterol. The present experiment was devised in order to determine what was the basal requirement of irradiated ergosterol for the prevention of rickets in chickens fed a ration containing a normal Ca: P ratio. Seven groups of fifteen Barred Plymouth Rock
Parts Ca and P contributed by constituent to ration Ca P 0.006 0.007 0.861 0.114 0.400
0.192 0.055 0.398 0.079 0.000
—
.—
—
—
1.388 0.724 Ratio Ca: P 1.91:1
terms of their potency relative to cod liver oil by rat assay. Thus "10 percent of irradiated ergosterol solution" means that 2 percent of a sesame oil solution was used containing an amount of irradiated ergosterol equivalent in rat units to 10 percent of cod liver oil. These solutions were prepared fresh each week by appropriate dilutions of Mead's Viosterol** with sesame oil; two parts of the oil solution were intimately mixed with ninety-eight parts of the basal
•Kindly supplied to us by Professor W. R. Graham, of the Ontario Agricultural College, Guelph. ** The Viosterol (irradiated ergosterol in corn oil) was kindly supplied to us by the Mead Johnson Company, Evansville, Indiana.
Downloaded from http://ps.oxfordjournals.org/ at ULB Bonn on May 27, 2015
TABLE 1. Calcium and phosphorus content of ration
MARCH,
1933.
VOL.
XII,
No.
131
2
The percentages of ash for the bones of the chickens in the several groups, together with the averages, are shown in Table 2. Two conclusions can be drawn from the figures, namely, that with the amounts of ergosterol used the results were better than those obtained by Massengale and Nussmeier and poorer than those obtained by King and Hall. Although 10 and 20 percent of irradiated ergosterol solution (of the same rat unitage strength as cod liver oil) were insufficient to give quite as high
TABLE 2. Percentage ash of long bones of chickens receiving basal ration su by various amounts of vitamin D Basal
Irradiated Ergosterol Solution
Cod Liver Oil 20%
0.5%
2%
4%
10%
37.0 39.3 37.0 39.6 42.7 35.5 37.8 36.2 35.5 42.8
37.9 45.0 38.0 24.6 38.8 41.1 41.8 39.3 42.6 46.9
38.3 40.3 40.8 41.9 43.3 51.2 37.4 40.7 43.8 38.3 44.1
36.3 41.3 37.8 40.0 39.8 38.7 46.0 41.9 47.8 48.4 44.4 47.2
42.1 43.6 47.5 46.9 45.2 42.3 47.0 40.6 46.4 43.5 51.0 50.3 52.3
51.4 50.1 42.2 48.9 45.0 45.2 44.6
44.6 48.5 48.5 46.1 45.1 49.4 50.6 51.0 48.2 47.4 46.5 46.2
Average 38.3
39.6
41.8
42.6
46.1
46.8
47.7
for ash analysis. After removing the adhering tissue as completely as possible, the bones were split lengthwise and the marrow scraped out with a blunt knife. The bones were dried in an oven at 110°C. and then fat extracted with ether in a Soxhlet apparatus for six hours. The dried fat-free bones thus obtained were passed through a grinding mill and reduced to a powder. Weighed samples of the powder were placed in porcelain crucibles and ashed in an electric muffle furnace at red heat. The weight of the residue times 100 over the weight of the sample was taken as the percentage ash of the bone.
2%
an ash content of the bones, yet the results were only 1 percent lower than those got with 2 percent cod liver oil, and the general condition of the birds appeared to be quite as good. As mentioned previously, it is felt that these better results with ergosterol are due to the use of a ration with a normal calcium-phosphorus ratio; and further, that they more truly represent the relative antirachitic values of cod liver oil and irradiated ergosterol when mixed with the feed than do results obtained with diets containing the mineral elements calcium and phosphorus in abnormal proportions. Diets similar to the Steenbock rickets-producing diet for
Downloaded from http://ps.oxfordjournals.org/ at ULB Bonn on May 27, 2015
ration, and the mixtures kept before the birds in metal hoppers. The chickens were kept on the rations for eleven weeks behind ordinary window glass in a room with western exposure. Birds which died during the course of the experiment were discarded. Unfortunately, the mortality was somewhat higher in some groups than in others, which has rendered the interpretation of the results less clear than might have been desired. During the eleventh and twelfth weeks the birds were killed and the tibiae taken
132
POULTRY
SUMMARY
Irradiated ergosterol is not as effective in preventing rickets in chickens as is cod liver oil. The difference in the antirachitic power of these two sources of vitamin D is not as
marked with a ration containing a normal calcium-phosphorus ratio as when these elements are present in abnormal ratio. There is some indication that irradiated ergosterol is more effective when given by mouth than when mixed with the feed. REFERENCES
Hess, A. F. and G. C. Supplee, 1930. The action of irradiated ergosterol on rats and chickens. Proc. Soc. Exp. Biol. Med., 27:609-610. Hall, G. E. and E. J. King, 1931a. Calcium phosphorus metabolism in the chicken. I. Poul. Sci., 10:132-152. , 1931b. Calcium phosphorus metabolism in the chicken. II. Poul. Sci., 10:259-268. King, E. J. and G. E. Hall, 1931. Calcium phosphorous metabolism in the chicken. III. Poul. Sci., 10:332-352. Kreitmair, H. and T. Moll, 1928. Hypervitaminosis through large doses of vitamin D. Munch. Med. Woch., 75:637-639. Massengale, O. N. and M. Nussmeier, 1930. The action of activated ergosterol in the chicken. II. J. Biol. Chem., 87:423-426. Mussehl, F. E. and C. W. Ackerson, 1930. Irradiated ergosterol as an antirachitic for chicks. Poul. Sci., 9 :334-338. Russell, W. C. and D. Klein, 1931. The vitamin A content of a ration and the antirachitic potency of irradiated ergosterol. Poul. Sci., 10: 269-274. Wilgus, H. S., 1931. The quantitative requirement of the growing chick for calcium and phosphorus. Poul. Sci., 10:107-117.
Downloaded from http://ps.oxfordjournals.org/ at ULB Bonn on May 27, 2015
the rat when used for chickens represent an unnatural dietary condition and results thus obtained are of experimental rather than of practical significance. That irradiated ergosterol may not be as effective when mixed with the feed as when given by mouth would seem to be indicated by the very much lower ash content in the birds receiving 2 percent of irradiated ergosterol solution in the present experiment than that of birds receiving an approximately equal amount (1 drop daily of Viosterol) in the former investigation, both series of birds having received almost identical basal diets. A control group of forty chickens receiving 1 drop of Viosterol daily which were maintained concurrently with the present experiment but which were kept for another purpose until five months of age, were in excellent condition and had an average bone ash of 52.3 percent, which is quite as good as that of normal range birds of the same age which have been raised on a ration supplemented by cod liver oil (Hall and King, 1931b).
SCIENCE