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The Effect of Minerals and Vitamin D on the Percentage Bone Ash of Young Chicks*
T h e Effect of Minerals and Vitamin D on the Percentage Bone Ash of Young Chicks* J. W. COOK AND E. I. ROBERTSON Divisions of Chemistry and Poultry Husbandry, Washington Agricultural Experiment Pullman, Washington
Station,
(Received for publication February 28, 1940)
T HAS been observed at this experiment station that when the preventative type of technic is used, unequal carry-over of vitamin D from the hen to the chick interferes with the assay of vitamin D. The curative method for the assay of vitamin D, reported by Carver, Heiman, and Cook (1939),f was designed to standardize the chicks by feeding a diet that was low in
diet and also of a control group fed on a diet believed adequate in these essentials. EXPERIMENTAL
PROCEDURE
One hundrd fifty day-old White Leghorn cockerels were wing-banded, individually weighed, and divided into groups 1 and 2 of 50 and 100 chicks, respectively. They were placed in wire bottom electrically heated
TABLE 1.—Organization and outline of experiment Time on ration 1-10 days, inclusive
11-15 days, inclusive
Group no.
No. chicks
Diet no.
Days sampled for bone ash
1
50
1
1,2,3,5,7,9
2
100
2
1,2,3,4,5, 6, 7,8, 9,10
No. chicks
Diet no.
Days sampled for bone ash
1
20
1
11, 13, 15,
2-a
22*
1
11,12,13,14
2-b
25
2
11,12,13, 14, 15
Group no.
* Three chicks died; one on the second, fourth, and seventh day.
minerals and vitamin D for a period of 9 or 10 days preceding the beginning of the assay period. It seemed desirable to make a study of the bone calcification of chicks from 1 to IS days of age when they were fed on the diet that was low in these essentials. This experiment was set up to determine the daily bone ash content of the tibiae of young chicks when fed on such a * Published as Scientific paper No. 436, College of Agriculture and Experiment Station, Washington State College. t Poultry Science 18:288.
battery brooders and fed according to the outline in Table 1. The diets used are described in Table 2. The chicks in group 1 were fed diet 1 throughout the entire trial from 1 to IS days, whereas the chicks in group 2 were fed diet 2 until 10 days of age, after which the chicks remaining in group 2 were divided into groups 2-a and 2-b. Group 2-a was fed an adequate diet from 11 to 15 days and group 2-b was continued on the inadequate diet 2. Five chicks were chosen daily at random,
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Downloaded from http://ps.oxfordjournals.org/ at Monash University on June 19, 2015
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386
J. W. COOK AND E. I. ROBERTSON TABLE 2.—Constituents of diets Diet 2—depletion diet
Diet 1—adequate chick starter ration Percent
Ingredients Ground yellow corn Finely ground heavy oats Dehydrated alfalfa Meat scrap Fish meal Skimmilk powder Ground oyster shell Salt Cod liver oil (85 units U.S.P.)
40.00 10.00 12.00 5.00 15.00 5.00 5.00 5.00 2.00 1.00 1.00
TABLE 3.—,
Days
0 1 2 3 5 7 9 11 13 IS*
Coarse ground yellow corn Wheat flour middlings Wheat bran Crude casein Unirradiated yeast Salt Sugar
Percent 50.00 20.00 5.00 12.00 3.00 0.50 9.50
Analysis 0.15 0.45
Calcium Phosphorus
which the bones were dried at 50°C. They were ashed in a muffle furnace for eight hours at 600°C. All the chicks were weighed before being killed and gain in body weight determined from their initial weight. RESULTS AND DISCUSSION When chicks were fed a diet adequate in minerals and vitamin D there was a consistent increase in the percentage of tibia bone ash, as is shown in Table 3 and Figure 1. These chicks increased in bone ash percentage from 36.10 to 44.37, an increase of 8.27 percent during the nine days on this diet. After the ninth day, and to the fifteenth, there was no increase in the tibia bone ash percentage in this group.
of results of chicks fed the adeqtiate diet Group 1
Average original weight of chicks
Average final weight of chicks
Average gain in weight
Average bone ash
grams
grams
grams
percent
38.2 45.2 40.2 40.6 39.8 41.0 40.0 41.6 39.2 40.3
38.2 46.2 47.4 49.2 51.0 63.8 62.8 87.8 99.4 104.3
0.0 1.0 7.2 8.6 11.2 22.8 22.8 46.2 60.2 64.0
36.10 37.43 39.61 41.00 41.73 43.73 44.37 43.95 44.42 44.26
* Average of four analyses.
Downloaded from http://ps.oxfordjournals.org/ at Monash University on June 19, 2015
killed, and both tibiae removed and preserved in 95 percent alcohol prior to the determination of the bone ash percentage. Bone ash determinations were made on the tibiae as follows: the bones were broken into four or five pieces over clean paper and all pieces were placed into a 43 by 123 mm. Whatman fatless extraction thimble and covered with a 7 cm. filter paper which was pressed into place with the funnel end of a small funnel. Approximately 25 bones were placed in each thimble. Very little difficulty was encountered in splintering of the bones and loss of particles if they were crushed with pliers while still wet with alcohol and fat. The thimbles were placed in a Sohxlet extractor and extracted for 72 hours in 95 percent ethyl alcohol, after
Ingredients
EFFECT OF MINERALS AND VITAMIN D ON THE PERCENTAGE BONE ASH
DAYS
FIG. 1. Bone ash at different days.
diet the bone ash percentage was 22.69, a total decrease from the original of 14.23 percent. The gain in body weight of chicks fed the
TABLE 4.—Summary of results of chicks fed the inadequate diet Group 2 Aveage original weight of chicks
Days
0 1 2 3 4 5 6 7 8 9 10
Average final weight of chicks
Average gain in weight
Average bone ash
grams
grams
grams
percent
36.8 43.2 43.0 38.2 44.6 39.2 41.4 39.8 39.8 37.8 39.8
36.8 46.2 49.8 48.6 57.4 52.0 57.2 60.2 54.8 60.2 63.2
0.0 3.0 6.8 10.4 12.8 12.8 15.8 20.4 15.0 22.4 22.0
36.92 36.98 35.72 35.13 34.43 33.10 31.59 32.97 31.65 29.97 28.36
Group 2-a Chicks changed to adequate diet
Days
11 12 13 14
Group 2-b Chicks continued on inadequate diet
Av. orig. wt. of chicks
Av. final wt. of chicks
Av. gain in weight
Av. bone ash
grams
grams
grams
percent
41.0 41.3 41.8 40.3
72.8 86.5 79.5 81.0
31.8 45.3 37.8 40.7
29.78 31.15 31.74 32.92
* Average of four analyses.
Days
11 12 13 14* 15*
Av. orig. wt. of chicks
Av. final wt. of chicks
Av. gain in weight
Av. bone ash
grams
grams
grams
percent
43.0 38.6 38.6 37.5 38.3
69.8 67.0 69.2 64.8 60.8
26.8 28.4 34.6 27.3 22.5
26.67 26.31 25.71 25.54 22.69
Downloaded from http://ps.oxfordjournals.org/ at Monash University on June 19, 2015
The chicks fed the diet inadequate in minerals and vitamin D decreased in bone ash percentage from 1 to 10 days of age, as is shown in Table 4 and Figure 1. This decrease was from 36.92 to 28.36 percent, a decrease of 8.56 percent. At 10 days of age, part of the chicks were changed to an adequate diet and their bone ash percentage immediately began to increase, as is shown in the results of group 2-a. During the four days the chicks were on the adequate diet the percentage of bone ash rose from 28.36 to 32.92 percent, a difference of 4.56 percent. Conversely, during the same period the chicks that were continued on the inadequate diet decreased to 25.54 percent, or a further decrease in bone ash of 2.82 percent. After 15 days on the inadequate
387
388
J. W. COOK AND E. I. ROBERTSON
further mortality despite the low ash content of their skeletons. Although the data reported here are the result of a sample of five chicks daily, these sample birds were chosen at random and the slight variation from day to day showed that this sample resulted in fairly consistent results. SUMMARY /Random samples of five cockerel chicks daily showed that when maintained on a diet believed adequate in minerals and vitamin D, the bone ash percentage increased from 36.1 to 44.4 percent in nine days, while similar chicks on a diet deficient in vitamin D and minerals decreased to 28.4 percent of bone ash in 10 days. When these depleted chicks were fed an adequate diet at this stage, the bone ash percentage increased to 32.9 in four days while in chicks continued on the inadequate diet the bone ash percentage decreased to 22.7 percent at IS days of age.
Downloaded from http://ps.oxfordjournals.org/ at Monash University on June 19, 2015
inadequate diet was approximately the same as chicks on the adequate diet to 10 days of age. However, after this time very little growth was observed in the depleted chicks, whereas those that were fed the adequate diet, or changed to it at 10 days of age, grew rapidly. Very little mortality was observed during the experiment. The three birds that died were on the inadequate diet but these deaths occurred on the second, fourth, and seventh day of age, and it is unlikely that the lack of vitamin D and minerals was the cause of death. It has been commonly observed in other vitamin D assays that the chicks show pronounced symptoms of vitamin D deficiency at about 9 or 10 days of age if they are placed on the depletion diet when one day old. The chicks in this experiment showed marked symptoms of vitamin D deficiency on the tenth day, but survived the continued depletion to IS days of age with no
Station,
(Received for publication February 28, 1940)
T HAS been observed at this experiment station that when the preventative type of technic is used, unequal carry-over of vitamin D from the hen to the chick interferes with the assay of vitamin D. The curative method for the assay of vitamin D, reported by Carver, Heiman, and Cook (1939),f was designed to standardize the chicks by feeding a diet that was low in
diet and also of a control group fed on a diet believed adequate in these essentials. EXPERIMENTAL
PROCEDURE
One hundrd fifty day-old White Leghorn cockerels were wing-banded, individually weighed, and divided into groups 1 and 2 of 50 and 100 chicks, respectively. They were placed in wire bottom electrically heated
TABLE 1.—Organization and outline of experiment Time on ration 1-10 days, inclusive
11-15 days, inclusive
Group no.
No. chicks
Diet no.
Days sampled for bone ash
1
50
1
1,2,3,5,7,9
2
100
2
1,2,3,4,5, 6, 7,8, 9,10
No. chicks
Diet no.
Days sampled for bone ash
1
20
1
11, 13, 15,
2-a
22*
1
11,12,13,14
2-b
25
2
11,12,13, 14, 15
Group no.
* Three chicks died; one on the second, fourth, and seventh day.
minerals and vitamin D for a period of 9 or 10 days preceding the beginning of the assay period. It seemed desirable to make a study of the bone calcification of chicks from 1 to IS days of age when they were fed on the diet that was low in these essentials. This experiment was set up to determine the daily bone ash content of the tibiae of young chicks when fed on such a * Published as Scientific paper No. 436, College of Agriculture and Experiment Station, Washington State College. t Poultry Science 18:288.
battery brooders and fed according to the outline in Table 1. The diets used are described in Table 2. The chicks in group 1 were fed diet 1 throughout the entire trial from 1 to IS days, whereas the chicks in group 2 were fed diet 2 until 10 days of age, after which the chicks remaining in group 2 were divided into groups 2-a and 2-b. Group 2-a was fed an adequate diet from 11 to 15 days and group 2-b was continued on the inadequate diet 2. Five chicks were chosen daily at random,
[385]
Downloaded from http://ps.oxfordjournals.org/ at Monash University on June 19, 2015
I
386
J. W. COOK AND E. I. ROBERTSON TABLE 2.—Constituents of diets Diet 2—depletion diet
Diet 1—adequate chick starter ration Percent
Ingredients Ground yellow corn Finely ground heavy oats Dehydrated alfalfa Meat scrap Fish meal Skimmilk powder Ground oyster shell Salt Cod liver oil (85 units U.S.P.)
40.00 10.00 12.00 5.00 15.00 5.00 5.00 5.00 2.00 1.00 1.00
TABLE 3.—,
Days
0 1 2 3 5 7 9 11 13 IS*
Coarse ground yellow corn Wheat flour middlings Wheat bran Crude casein Unirradiated yeast Salt Sugar
Percent 50.00 20.00 5.00 12.00 3.00 0.50 9.50
Analysis 0.15 0.45
Calcium Phosphorus
which the bones were dried at 50°C. They were ashed in a muffle furnace for eight hours at 600°C. All the chicks were weighed before being killed and gain in body weight determined from their initial weight. RESULTS AND DISCUSSION When chicks were fed a diet adequate in minerals and vitamin D there was a consistent increase in the percentage of tibia bone ash, as is shown in Table 3 and Figure 1. These chicks increased in bone ash percentage from 36.10 to 44.37, an increase of 8.27 percent during the nine days on this diet. After the ninth day, and to the fifteenth, there was no increase in the tibia bone ash percentage in this group.
of results of chicks fed the adeqtiate diet Group 1
Average original weight of chicks
Average final weight of chicks
Average gain in weight
Average bone ash
grams
grams
grams
percent
38.2 45.2 40.2 40.6 39.8 41.0 40.0 41.6 39.2 40.3
38.2 46.2 47.4 49.2 51.0 63.8 62.8 87.8 99.4 104.3
0.0 1.0 7.2 8.6 11.2 22.8 22.8 46.2 60.2 64.0
36.10 37.43 39.61 41.00 41.73 43.73 44.37 43.95 44.42 44.26
* Average of four analyses.
Downloaded from http://ps.oxfordjournals.org/ at Monash University on June 19, 2015
killed, and both tibiae removed and preserved in 95 percent alcohol prior to the determination of the bone ash percentage. Bone ash determinations were made on the tibiae as follows: the bones were broken into four or five pieces over clean paper and all pieces were placed into a 43 by 123 mm. Whatman fatless extraction thimble and covered with a 7 cm. filter paper which was pressed into place with the funnel end of a small funnel. Approximately 25 bones were placed in each thimble. Very little difficulty was encountered in splintering of the bones and loss of particles if they were crushed with pliers while still wet with alcohol and fat. The thimbles were placed in a Sohxlet extractor and extracted for 72 hours in 95 percent ethyl alcohol, after
Ingredients
EFFECT OF MINERALS AND VITAMIN D ON THE PERCENTAGE BONE ASH
DAYS
FIG. 1. Bone ash at different days.
diet the bone ash percentage was 22.69, a total decrease from the original of 14.23 percent. The gain in body weight of chicks fed the
TABLE 4.—Summary of results of chicks fed the inadequate diet Group 2 Aveage original weight of chicks
Days
0 1 2 3 4 5 6 7 8 9 10
Average final weight of chicks
Average gain in weight
Average bone ash
grams
grams
grams
percent
36.8 43.2 43.0 38.2 44.6 39.2 41.4 39.8 39.8 37.8 39.8
36.8 46.2 49.8 48.6 57.4 52.0 57.2 60.2 54.8 60.2 63.2
0.0 3.0 6.8 10.4 12.8 12.8 15.8 20.4 15.0 22.4 22.0
36.92 36.98 35.72 35.13 34.43 33.10 31.59 32.97 31.65 29.97 28.36
Group 2-a Chicks changed to adequate diet
Days
11 12 13 14
Group 2-b Chicks continued on inadequate diet
Av. orig. wt. of chicks
Av. final wt. of chicks
Av. gain in weight
Av. bone ash
grams
grams
grams
percent
41.0 41.3 41.8 40.3
72.8 86.5 79.5 81.0
31.8 45.3 37.8 40.7
29.78 31.15 31.74 32.92
* Average of four analyses.
Days
11 12 13 14* 15*
Av. orig. wt. of chicks
Av. final wt. of chicks
Av. gain in weight
Av. bone ash
grams
grams
grams
percent
43.0 38.6 38.6 37.5 38.3
69.8 67.0 69.2 64.8 60.8
26.8 28.4 34.6 27.3 22.5
26.67 26.31 25.71 25.54 22.69
Downloaded from http://ps.oxfordjournals.org/ at Monash University on June 19, 2015
The chicks fed the diet inadequate in minerals and vitamin D decreased in bone ash percentage from 1 to 10 days of age, as is shown in Table 4 and Figure 1. This decrease was from 36.92 to 28.36 percent, a decrease of 8.56 percent. At 10 days of age, part of the chicks were changed to an adequate diet and their bone ash percentage immediately began to increase, as is shown in the results of group 2-a. During the four days the chicks were on the adequate diet the percentage of bone ash rose from 28.36 to 32.92 percent, a difference of 4.56 percent. Conversely, during the same period the chicks that were continued on the inadequate diet decreased to 25.54 percent, or a further decrease in bone ash of 2.82 percent. After 15 days on the inadequate
387
388
J. W. COOK AND E. I. ROBERTSON
further mortality despite the low ash content of their skeletons. Although the data reported here are the result of a sample of five chicks daily, these sample birds were chosen at random and the slight variation from day to day showed that this sample resulted in fairly consistent results. SUMMARY /Random samples of five cockerel chicks daily showed that when maintained on a diet believed adequate in minerals and vitamin D, the bone ash percentage increased from 36.1 to 44.4 percent in nine days, while similar chicks on a diet deficient in vitamin D and minerals decreased to 28.4 percent of bone ash in 10 days. When these depleted chicks were fed an adequate diet at this stage, the bone ash percentage increased to 32.9 in four days while in chicks continued on the inadequate diet the bone ash percentage decreased to 22.7 percent at IS days of age.
Downloaded from http://ps.oxfordjournals.org/ at Monash University on June 19, 2015
inadequate diet was approximately the same as chicks on the adequate diet to 10 days of age. However, after this time very little growth was observed in the depleted chicks, whereas those that were fed the adequate diet, or changed to it at 10 days of age, grew rapidly. Very little mortality was observed during the experiment. The three birds that died were on the inadequate diet but these deaths occurred on the second, fourth, and seventh day of age, and it is unlikely that the lack of vitamin D and minerals was the cause of death. It has been commonly observed in other vitamin D assays that the chicks show pronounced symptoms of vitamin D deficiency at about 9 or 10 days of age if they are placed on the depletion diet when one day old. The chicks in this experiment showed marked symptoms of vitamin D deficiency on the tenth day, but survived the continued depletion to IS days of age with no