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Calcium and Phosphorus Requirements for Growing Chicks*
CALCIUM AND PHOSPHORUS REQUIREMENTS FOR GROWING CHICKS* I.
STUDIES W I T H SALT MIXTURES UNDER. RACHITIC CONDITIONS
W. C. TULLY, S. M. HAUGE, C. W. CARRICK, AND ROY E. ROBERTS Agricultural Experiment Station, Purdue University
* Published with the approval of the Director of the Agricultural Experiment Station, Purdue University.
299
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(Received for Publication 4-4-31) I t is well known that vitamin D or its equivalent, ultraviolet rays, has a corrective effect on the assimilation of calcium and phosphorus from rations with unfavorable mineral combinations. In other words, vitamin D masks the true biological action of the minerals as such. Therefore, to study the true mineral value of the diet, it is necessary to conduct experiments in the absence of this influencing factor. Shipley and co-workers1 demonstrated that rickets can be produced by either low calcium diets or low phosphorus diets. Sherman and Pappenheimer 2 showed that rickets could be produced in rats by a diet low in phosphorus and that it could be prevented simply by increasing the phosphorus and decreasing the calcium content of the diet. In 1924 Goldblatt 3 showed that when the calcium-phosphorus ratio was 1:0.8, rickets was only very slightly evident in rats fed a purified ration containing no vitamin D. However, in rats fed the same ration, but with the calcium-phosphorus ratio altered either way, rickets was very severe. The ratio of calcium and phosphorus in the ration is the determining factor for their assimilation, providing the availability of the compounds of these elements is the same. Investigations by Steenbock, Hart, Sell, and Jones 4 , Buckner and Martin 5 , Buckner, Martin, and Insko0, and Bethke, Kennard, and Kick 7 have shown that there is no difference in the availability of calcium in calcium carbonate, calcium sulphate, calcium lactate, tricalcium phosphate, dicalcium phosphate, steamed bone, dolomitic limestone, and oyster shells. Forbes 8 states: "the utilization of calcium and phosphorus compounds appears to be determined largely by the relative
300
POULTRY
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proportions in which they and magnesium are available in the ration . . . . " Haag and Palmer 9 showed the importance of a more or less balanced condition of calcium, phosphorus, and magnesium salts in the ration to secure good growth and mineral retention. Wheeler10 found that rations deficient in calcium but carrying an abundance of magnesium "produced a notable shortage of calcium and of mineral matter in the bones of fowls and ducks." Hart, Steenbock, Halpin, and Johnson 11 reported very good growth in chicks up to eight weeks of age on a ration of 80 parts of yellow corn, 20 parts of wheat middlings, five parts of raw bone, five parts of pearl grits, one part of common salt, and skimmed milk ad libitum. They stressed the fact that the ration was low in the antirachitic factor and stated '' If the antirachitic factor is low in a ration, then a more liberal supply of calcium and phosphorus will aid in delaying the extreme effects of rickets. On the other hand, if the antirachitic factor is liberally supplied in a ration, then the calcium and phosphorus supply may be lowered without causing rickets." Hart, Steenbock, Kletzien, Halpin, and Johnson 12 have demonstrated that the calcium-phosphorus ratio can be varied within certain wide limits, at least as much as 7 to 1, without affecting growth or the development of the skeleton, provided that there is a generous supply of the antirachitic factor in the diet. They state that while in winter, with a limited amount of the antirachitic factor, the calcium-phosphorus ,ratio is probably of very great importance, in summer the mineral ration may be greatly increased in its lime content, as compared to the plfbsphorus, or vice versa, with no ill effects. Mussehl, Blish, and Ackerson13 have reported that excess calcium and phosphorus added to a complete all-mash ration resulted in inhibition in growth and, in some cases, rickets. Some recent investigations by Bethke, Kennard, Kick, and Zinzalian14 have shown that "the optimum, or near optimum, ratio of calcium to phosphorus for the growing chick lies between 3:1 and 4:1. The requirements for the antirachitic factor are at a minimum at this relationship. Within certain limits of concentration, the ratio of calcium to phosphorus is of greater
SALT MIXTURES
UNDER RACHITIC CONDITIONS
301
EXPERIMENTAL
The general plan of these experiments as regards the varieties of chicks used and the methods of selection, feeding, and management were the same as have been previously described15. The indoor brooder tables16 were equipped with one-half inch mesh wire bottoms to prevent access to litter and excreta. Precautions were taken to prevent exposure of chicks to direct sunshine, or even direct skyshine. The health and growth conditions of the chicks throughout the experiments were carefully watched. The first signs of rickets as well as the severity of the attack, in each case were carefully recorded. Dead or diseased chicks were removed from their pens for post-mortem examination. In addition at the end of each experiment a number of chicks were killed for postmortem examination. Since the calcium and phosphorus content of the ingredients of the ration contribute to the total mineral diet, analyses were made for these elements. In addition, similar analyses were made of all calcium and phosphorus containing substances used as minerals, when not chemically pure. The mineral mixtures used in the various lots of these experiments were either McCollum's Mineral No. 185 or modifications of it. The composition of these mixtures is given in Table I. By substituting an equal weight of calcium carbonate for the calcium lactate of McCollum 's No. 185, the Ca :P ratio was changed from 1:1.449 to 1:0.608 in Mineral No. 1. In Mineral No. 2, the calcium lactate of No. 185 was replaced by an equal weight of calcium oxide, giving a Ca :P ratio of 1:0.363. Mineral No. 3
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significance in calcification and growth than the amounts of these elements in the ration." The purpose of our investigation was to study the calciumphosphorus requirements of growing chicks. Since vitamin D aids in the assimilation of calcium and phosphorus, correcting the deleterious effects of disproportions of these elements, it seemed advisable to conduct these studies chiefly in the absence of this influencing factor. Various ratios and levels of calcium and phosphorus were tested as well as different sources of these elements.
POULTRY
302
SCIENCE
was based on the ash analysis of the all-mash ration, corrected to furnish magnesium, sodium, and potassium in amounts equivalent to those supplied by McCollum's No. 185. The Ca:P ratio of Mineral No. 3 was 1:0.74 which was the same as that of the all-mash ration. COMPOSITION OP M I N E R A L M I X T U R E S U S E D
Mg So4 Naj H P 0 4 K, H P 0 4 CaH. ( P 0 4 ) 2 H 2 0 F e (C 3 H 3 0 3 ) 2 .5H 2 0
.266 .347 .954
-., x, gm. .173 .266 .347 .954
.540 .118
.540 .118
Ca(C 3 H 5 O s ) 2 .511,0
1.300
Ingredients Na CI
Ca C0 3 CaO
KCL, C a : P Ratio
McCollum's 1 No. 185 | gm. .173
No
No. 2 gm. .173
No. 3 gm. 0.70
.266 .347 .954
0.44
.540 .118
6.94 0.20
— —
-—
—
— — —
1.300
0.22
— —
1.300
—
1.30
| 1:1.449
1:0.608
1:0.363
| 1:0.74
—
The composition of the rations and the number and kind of chicks used in each trial are given in Table II. The results obtained in all of the trials are given in Table I I and Chart I. Trial I—This was a preliminary trial to determine the effect of varying the calcium-phosphorus ratio in the absence of vitamin D. Lot I received Mineral No. 185; Lot 2, Mineral No. 1; Lot 3, Mineral No. 2; and Lot 4, Mineral No. 185 plus irradiation for 20 minutes daily at a distance of 30 inches with an ultra violet lamp. This was the only pen receiving vitamin D in any form. The results from the three pens which received no vitamin D indicated that the ration containing the higher level of calcium gave the best results as measured by mortality and rate of growth. Rickets was severe in all three lots. The first symptoms of rickets appeared as early as the eleventh day and at 24 days of age all chicks were very stiff, walking awkwardly and with difficulty. At six weeks of age, the surviving chicks in Lots 1, 2, and
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T A B L E I.
Basal Basal
5
6 7 8
2 Barred Plymouth
3
3
3
Basal (5 casein replaced by 5 liver meal)
Basal (5 casein replaced by 5 liver meal; agar omitted)
14
15
13
12 3
9.8
9.8
9.8
9.8
1.998
1.998
1.998
1.998
1.998
9.8
Basal (agar omitted) Basal
-
3
1.998
1.31
.655 .655 .983
1.09 .275
-
9.8
8
4 4 6
4 4
.275 .655
Cain Ration Per Cent 1.93J
Standard! Basal
10 11
3
9
Per Cent 4 4
Ca:P Ratio
P in Ration Per Cent 1.47
1.47
1.47
1.47
1.47
1:0.735 16
1:0.735 16
1:0.735 20 1:0.735 16
1:0.735 20
1.47 1:0.735 10 1.466$ l:0.76i 20
.398 11:0.608 16 .398 |1:0.608 !6 .597 1:0.608 16 .796 1:0.608 16
.398 |1:0.363| 20 .398 |1:1.449| 20
.398 |1:1.449 20 | .398 |1:0.608| 20
99
79
212
211
14
16
11
20
215
227
201
234
10 | 135
17
9
5 ! 158
15 | 323 5 | 152
2 |
17 | 152 19 | 291
6 | 104
4 |
No. Chicks Started No. Chicks Alive
Eight Weeks
14
16
19 9
14
—
302
315
333 275
505
—
+
+
+ +
+
+
—
1 | 155 | — 15 179 | — 19 449 | +
" 1" 1 -
Irradiation
0)
.. --
35.7
35.4
--
30.3
40.2
—
28.7 32.2
•Basal Ration: Casein 20; Dried Brewer's Yeast 5; Corn Germs 5; Lard 2; Agar 2; Degerminated Yellow Corn to make 100 (after addition of minerals as indicated). t Standard Ration: Ground Yellow Corn 53; Wheat Bran 15; Wheat Middlings 15; Meat Scraps 12; Dried Buttermilk 4; Salt 1. t In entire ration.
4 S. C. White Leghorns
3 S. C. White Leghorns
1
1 1
Basal (0.6 casein replaced by 0.6 cystine)
Basal
Basal
4
Breds
Rocks
Basal Basal
3
Cross-
|
2 185
Basal Basal
1 2
1
1
185 1
6
Trial No.
=5
Ration
Av. Weights
Six-Weeks
No. Chicks Alive Av. Weights
Mineral Additions
Ash in Fi Free Tib Per Cent
T A B L E I I . — S H O W I N G T H E P L A N S OF T H E E X P E R I M E N T S W I T H SUMMARY OF T H E R E S U L T S
m http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on May 2, 2015 OS
o
CO
Os
O
g
C5 ©
SB
ft
4
OS
304
POULTRY
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3 averaged respectively 99, 104, a n d 152 grams. Since the usual weight for this variety a n d age of chicks-grown u n d e r laboratory conditions approximates 350 grams, it is readily seen t h a t the
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CHART 1
Growth curves of the chicks that lived through the experiments. The figures under each lot indicate the number of chicks started and the number which lived through (20-4 under Lot 1, means that 20 chicks were started and 4 lived through). Lots which were irradiated are indicated by an asterisk (*).
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UNDER RACHITIC CONDITIONS
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growth in all lots was decidedly unsatisfactory. In the irradiated group, however, 18 of the 20 chicks grew almost normally to ten weeks of age. One died in the sixth week and one in the ninth. At eight weeks the survivors average 449 grams. The good growth obtained in this lot indicated that our basal semipurified ration was practically complete in nutritional factors. While it proved nothing as to the optimum calcium-phosphorus ratio, it did suggest that, with adequate or perhaps excess vitamin D supplied, good growth would result when a mineral mixture having a Ca :P ratio of 1:1.449 was used. Trial II—In this trial Lots 5, 6, 7, and 8 received 4, 4, 6, and 8 per cent of Mineral No. 1. Lot 6 which was the only lot receiving vitamin D in any form, was irradiated. At six weeks of age the surviving chicks in Lots 5, 7, and 8 average respectively 79, 152, and 158 grams. The mortality in all three lots was very high, only two chicks surviving in Lot 5 and five surviving in each of Lots 7 and 8. All chicks in these three lots had severe rickets. These results suggest that possibly six or eight per cent of mineral was closer to the optimum level than was four per cent. In the irradiated lot, 14 of the 16 chicks lived and grew well to eight weeks of age and showed no indications of rickets. These chicks averaged 323 grams at six weeks of age and 505 grams at eight weeks. Trial HI—In the two previous trials, the results were disappointing. We had noticed in other work conducted at this station, that chicks fed!-a standard all-mash ration (ground yellow corn 53, wheat bran 15, wheat middlings 15, 50 per cent protein meat scraps 12, dried buttermilk 4, salt 1)„ without any vitamin D, did not show signs of rickets until they were four weeks of age or older. Most of the rachitic chicks in the previous trials showed unmistakable sighs of the disease in three weeks, and in some cases as early as the eleventh day. Since it seemed probable that the superior ability of the all-mash ration to offset rickets is due to its more favorable mineral balance, the calcium and phosphorus content of this ration was duplicated with the semipurified diet in this trial. Analyses of the standard all-mash ration showed that it contained 1.93 per cent elemental calcium and 1.466 per cent of ele-
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mental phosphorus. The calcium-phosphorus ratio was therefore approximately 1:0.76. Allowing for the calcium and phosphorus present in the nonmineral ingredients of the semipurified ration, Mineral No. 3 was formulated to furnish the same Ca :P ratio as the all-mash ration. By using 9.8 per cent of this mineral mixture in the basal semipurified ration, practically the same amounts of these elements were furnished as in the all-mash ration. Pour lots of S. C. White Leghorn chicks, 10 in Lot 9 and 20 each in Lots 10, 11, and 12 were used. All lots received 9.8 per cent of Mineral No. 3, except Lot 10 which received the all-mash ration. Lot 9 was designed to determine whether or not the addition of cystine (0.6 per cent) would correct the ruffling of feathers which was common in all chicks fed the semipurified diets. The chicks in this lot were irradiated. Cystine failed to show any improvement as to this condition. In Lot 10, receiving the all-mash ration in the absence of vitamin D, rickets appeared in several chicks at 25 days of age while a week later only three or four showed decided rickets. Lot 11 was fed the regular semipurified ration with mineral. No. 3. In this lot rickets was noticed as early as the nineteenth day, and at four weeks the disease was very severe in all chicks. Lot 12 was the same as Lot 11 except that agar was omitted and the chicks were irradiated. The chicks in this lot did not make satisfactory growth, and feather ruffling was prevalent. The lack of agar seemed to have no ill effect, though no pen was an actual check on this one factor. At six weeks of age surviving chicks in these consecutive lots averaged respectively 211, 212, 135, and 234 grams, and the mortality was 10, 15, 50, and 5 per cent. From the results of this experiment, it was evident that Mineral No. 3 was not satisfactory as a mineral supplement. Lot 10, fed the standard all-mash ration without any vitamin D, grew as well to six weeks as Lot 9 on the semipurified ration plus Mineral No. 3 and with irradiation. Trial IV—In this trial three lots of 16 chicks each were used. Mineral No. 3 was fed at the same level as in the previous trial.
SALT MIXTURES
UNDER RACHITIC CONDITIONS
307
DISCUSSION
Although much work has been conducted to establish favorable calcium-phosphorus ratios for the rat, very little work has been reported on the calcium-phosphorus requirements for growing chicks. In a similar manner as rickets has been prevented and cured in rats in the absence of vitamin D, by altering the calcium-phosphorus ratio, it might be possible by proper mineral balance to prevent or at least delay the onset of rickets in chicks in the absence of vitamin D. The practicability of growing chicks to six or eight weeks of age without vitamin D may be questionable, but if with proper mineral balance, the quantitative requirement for vitamin D could be reduced to such an extent that chicks would not suffer from the lack of insufficient sunshine during unfavorable weather conditions, this would be of practical importance. When a chick is hatched it probably carries with it, from the egg yolk, a supply of vitamin D sufficient to prevent the visible external appearance of rickets over a period of three or four weeks, if it is fed a ration complete except for the antirachitic factor. Miller, Dutcher, and Ktiandel 17 have recently demonstrated that chicks fed a rachitogenic ration show a decided drop in the ash of their tibiae from 36.3 per cent at one week of age to ap-
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Lot 13 received the semipurified ration with 9.8 per cent of Mineral No. 3. Lot 14 was the same, except that five per cent of dried liver meal replaced an equal amount of casein. This change was made to see if such an addition of protein would improve growth. Since the liver meal contained less than six per cent of ash, it was not thought that the use of five per cent of it would measurably affect the mineral content of the ration. Lot 15 was the same as Lot 14 except that agar was omitted. All three lots were irradiated. At six weeks of age Lots 13, 14, and 15 averaged respectively 201, 227, and 215 grams, and the mortality was 5, 0, 2 chicks. Growth in all lots was below the average expected. While the use of liver meal appeared to increase the rate of growth to some extent, the omission of agar seemed to have little, if any, effect. All lots had severe rickets after five weeks despite irradiation.
308
POULTRY
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SUMMARY
1. MeCollum's Mineral No. 185, and substitutions of calcium
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proximately 28 per cent at six weeks. The chicks did not show severe rickets till the fifth week, although the time at which rickets was first observed is not mentioned. This suggests that a chick may be visibly non-rachitic for the first three or four weeks of its life and yet have the ash content of its bones continually decreasing. Little work has been done to show whether a variation in the calcium-phosphorus ratio of the ration would prevent this bone deterioration where no vitamin D was supplied. The results of these four trials indicate that even though the amounts of mineral in the ration, as well as the calcium-phosphorus ratio, were varied widely, the chicks did not grow satisfactorily without some form of vitamin D in the diet. In Trial I, the calcium-phosphorus ratio was varied from 1:1.449 to 1:0.363 without augmenting growth to anywhere near a usual or so-called normal rate. That this failure in growth cannot be attributed to inadequacy of the ration other than minerals and vitamin D was established when good growth was secured with some of the same rations simply by irradiation of the chicks. This indicated that the basal semipurified ration was practically complete in nutritional factors. The unsatisfactory growth of the chicks when no vitamin D was supplied can be attributed only to a lack of the vitamin and improper mineral balance. Attempts to duplicate the amounts as well as the ratio of calcium and phosphorus in the all-mash ration by adding diabasic calcium phosphate and calcium carbonate to the semipurified ration were not successful, since chicks thus fed did not make satisfactory growth even with irradiation. Therefore, it appears that Mineral No. 3 did*not meet the mineral requirements of the chicks. Although these trials using various salt mixtures in the absence of vitamin D did not contribute materially to our knowledge of the calcium-phosphorus ratio necessary for growing chicks, they did suggest that the problem might be approached more readily with a limited supply of vitamin D. Such studies were made and will be reported later.
SALT
MIXTURES
UNDER
RACHITIC
CONDITIONS
309
BIBLIOGRAPHY i S H I P L E Y , P . G., P A B K , E . A., MCCOLLUM, E , V., AND SIMMONDS,
N.,
Amer. Jour. Dis. Child., 1922, X X I I I , 91. 2 SHERMAN, H. C , AND PAPPENHEIMER, A. M., Proc. Soe. Exper. Biol, and Med., 1921, XVIII, 193. s GOLDBLATT, H., Biochem. Jour., 1924, X V I I I , 414. * STEENBOCK, H., H A B T , E . B., S E L L , M. E., A N D J O N E S , J. H., J. Biol.
Chem., 1923, LVI, 375. s BUCKNEB,
G.
DAVIS
AND M A R T I N ,
J.
HOLMES, POULTRY
SCIENCE,
1929, X V I I I , 284. s BUCKNEB,
G.
DAVIS, MABTIN,
J.
HOLMES,
A N D I N S K O , W.
M.
Jr.,
P O U L T R Y SCIENCE, 1929, IX, 1. ? B E T H K E , R. M., K E N N A R D , D. C , A N D K I C K , C. H., P O U L T R Y SCIENCE,
1929, I X , 45. s FORBES, E. B., R e p r i n t a n d Circular Series of t h e National Research Council, 1924, No. 60. s HAAG, J. R., AND PALMER, L. S., Jour. Biol. Chem., 1928, LXXVI, 367. io W H E E L E R , W. P., New York Agr. E x p . Sta. Bui. No. 468, 1919. i i H A R T , E . B., STEENBOCK, H.,
H A L P I N , J. G., AND JOHNSON, 0 .
N.,
Wisconsin Agr. E x p . Sta. Bui. No. 371, 1927. 12 Wisconsin Agr. E x p . Sta. Bui. No. 386, 1927. is M U S S E H L ,
F.
E.,
SCIENCE, 1927, VI,
BUSH,
M.
J.,
A N D ACKEBSON,
C.
W.,
POULTEY
239.
1* B E T H K E , R. M., K E N N A R D , D. C , K I C K , C. H., AND Z I N Z A N L I A N , P O U L T R Y SCIENCE, 1928, VII, is P R A N G E ,
R.
W.,
S C I E N C E , 1928, V I I ,
G.,
95.
CARRICK,
C.
W.,
AND HAUGE,
S.
M.,
POULTRY
C,
POULTRY
95.
ie CARRICK, C. W., P O U L T R Y SCIENCE, 1925, V, 98. "
MILLER,
R.
J.,
DUTCHER,
SCIENCE, 1929, V I I I , 113.
R.
A.,
AND KNANDEL,
H.
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on May 2, 2015
salts in it to produce a wide variation in the calcium-phosphorus ratio, when used with the semipurified ration in the absence of vitamn D, did not result in satisfactory growth or proper bone formation. 2. With the addition of vitamin D, the chicks approached optimal growth. 3. In the absence of vitamin D, better results were obtained with six or eight per cent of Mineral No. 1, than with four per cent. 4. Good growth was not obtained in young chicks fed a mineral mixture composed chiefly of dibasic calcium phosphate, at the level at which it was used in the semipurified ration, either with or without an adequate supply of vitamin D in the form of irradiation.
STUDIES W I T H SALT MIXTURES UNDER. RACHITIC CONDITIONS
W. C. TULLY, S. M. HAUGE, C. W. CARRICK, AND ROY E. ROBERTS Agricultural Experiment Station, Purdue University
* Published with the approval of the Director of the Agricultural Experiment Station, Purdue University.
299
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on May 2, 2015
(Received for Publication 4-4-31) I t is well known that vitamin D or its equivalent, ultraviolet rays, has a corrective effect on the assimilation of calcium and phosphorus from rations with unfavorable mineral combinations. In other words, vitamin D masks the true biological action of the minerals as such. Therefore, to study the true mineral value of the diet, it is necessary to conduct experiments in the absence of this influencing factor. Shipley and co-workers1 demonstrated that rickets can be produced by either low calcium diets or low phosphorus diets. Sherman and Pappenheimer 2 showed that rickets could be produced in rats by a diet low in phosphorus and that it could be prevented simply by increasing the phosphorus and decreasing the calcium content of the diet. In 1924 Goldblatt 3 showed that when the calcium-phosphorus ratio was 1:0.8, rickets was only very slightly evident in rats fed a purified ration containing no vitamin D. However, in rats fed the same ration, but with the calcium-phosphorus ratio altered either way, rickets was very severe. The ratio of calcium and phosphorus in the ration is the determining factor for their assimilation, providing the availability of the compounds of these elements is the same. Investigations by Steenbock, Hart, Sell, and Jones 4 , Buckner and Martin 5 , Buckner, Martin, and Insko0, and Bethke, Kennard, and Kick 7 have shown that there is no difference in the availability of calcium in calcium carbonate, calcium sulphate, calcium lactate, tricalcium phosphate, dicalcium phosphate, steamed bone, dolomitic limestone, and oyster shells. Forbes 8 states: "the utilization of calcium and phosphorus compounds appears to be determined largely by the relative
300
POULTRY
SCIENCE
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proportions in which they and magnesium are available in the ration . . . . " Haag and Palmer 9 showed the importance of a more or less balanced condition of calcium, phosphorus, and magnesium salts in the ration to secure good growth and mineral retention. Wheeler10 found that rations deficient in calcium but carrying an abundance of magnesium "produced a notable shortage of calcium and of mineral matter in the bones of fowls and ducks." Hart, Steenbock, Halpin, and Johnson 11 reported very good growth in chicks up to eight weeks of age on a ration of 80 parts of yellow corn, 20 parts of wheat middlings, five parts of raw bone, five parts of pearl grits, one part of common salt, and skimmed milk ad libitum. They stressed the fact that the ration was low in the antirachitic factor and stated '' If the antirachitic factor is low in a ration, then a more liberal supply of calcium and phosphorus will aid in delaying the extreme effects of rickets. On the other hand, if the antirachitic factor is liberally supplied in a ration, then the calcium and phosphorus supply may be lowered without causing rickets." Hart, Steenbock, Kletzien, Halpin, and Johnson 12 have demonstrated that the calcium-phosphorus ratio can be varied within certain wide limits, at least as much as 7 to 1, without affecting growth or the development of the skeleton, provided that there is a generous supply of the antirachitic factor in the diet. They state that while in winter, with a limited amount of the antirachitic factor, the calcium-phosphorus ,ratio is probably of very great importance, in summer the mineral ration may be greatly increased in its lime content, as compared to the plfbsphorus, or vice versa, with no ill effects. Mussehl, Blish, and Ackerson13 have reported that excess calcium and phosphorus added to a complete all-mash ration resulted in inhibition in growth and, in some cases, rickets. Some recent investigations by Bethke, Kennard, Kick, and Zinzalian14 have shown that "the optimum, or near optimum, ratio of calcium to phosphorus for the growing chick lies between 3:1 and 4:1. The requirements for the antirachitic factor are at a minimum at this relationship. Within certain limits of concentration, the ratio of calcium to phosphorus is of greater
SALT MIXTURES
UNDER RACHITIC CONDITIONS
301
EXPERIMENTAL
The general plan of these experiments as regards the varieties of chicks used and the methods of selection, feeding, and management were the same as have been previously described15. The indoor brooder tables16 were equipped with one-half inch mesh wire bottoms to prevent access to litter and excreta. Precautions were taken to prevent exposure of chicks to direct sunshine, or even direct skyshine. The health and growth conditions of the chicks throughout the experiments were carefully watched. The first signs of rickets as well as the severity of the attack, in each case were carefully recorded. Dead or diseased chicks were removed from their pens for post-mortem examination. In addition at the end of each experiment a number of chicks were killed for postmortem examination. Since the calcium and phosphorus content of the ingredients of the ration contribute to the total mineral diet, analyses were made for these elements. In addition, similar analyses were made of all calcium and phosphorus containing substances used as minerals, when not chemically pure. The mineral mixtures used in the various lots of these experiments were either McCollum's Mineral No. 185 or modifications of it. The composition of these mixtures is given in Table I. By substituting an equal weight of calcium carbonate for the calcium lactate of McCollum 's No. 185, the Ca :P ratio was changed from 1:1.449 to 1:0.608 in Mineral No. 1. In Mineral No. 2, the calcium lactate of No. 185 was replaced by an equal weight of calcium oxide, giving a Ca :P ratio of 1:0.363. Mineral No. 3
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on May 2, 2015
significance in calcification and growth than the amounts of these elements in the ration." The purpose of our investigation was to study the calciumphosphorus requirements of growing chicks. Since vitamin D aids in the assimilation of calcium and phosphorus, correcting the deleterious effects of disproportions of these elements, it seemed advisable to conduct these studies chiefly in the absence of this influencing factor. Various ratios and levels of calcium and phosphorus were tested as well as different sources of these elements.
POULTRY
302
SCIENCE
was based on the ash analysis of the all-mash ration, corrected to furnish magnesium, sodium, and potassium in amounts equivalent to those supplied by McCollum's No. 185. The Ca:P ratio of Mineral No. 3 was 1:0.74 which was the same as that of the all-mash ration. COMPOSITION OP M I N E R A L M I X T U R E S U S E D
Mg So4 Naj H P 0 4 K, H P 0 4 CaH. ( P 0 4 ) 2 H 2 0 F e (C 3 H 3 0 3 ) 2 .5H 2 0
.266 .347 .954
-., x, gm. .173 .266 .347 .954
.540 .118
.540 .118
Ca(C 3 H 5 O s ) 2 .511,0
1.300
Ingredients Na CI
Ca C0 3 CaO
KCL, C a : P Ratio
McCollum's 1 No. 185 | gm. .173
No
No. 2 gm. .173
No. 3 gm. 0.70
.266 .347 .954
0.44
.540 .118
6.94 0.20
— —
-—
—
— — —
1.300
0.22
— —
1.300
—
1.30
| 1:1.449
1:0.608
1:0.363
| 1:0.74
—
The composition of the rations and the number and kind of chicks used in each trial are given in Table II. The results obtained in all of the trials are given in Table I I and Chart I. Trial I—This was a preliminary trial to determine the effect of varying the calcium-phosphorus ratio in the absence of vitamin D. Lot I received Mineral No. 185; Lot 2, Mineral No. 1; Lot 3, Mineral No. 2; and Lot 4, Mineral No. 185 plus irradiation for 20 minutes daily at a distance of 30 inches with an ultra violet lamp. This was the only pen receiving vitamin D in any form. The results from the three pens which received no vitamin D indicated that the ration containing the higher level of calcium gave the best results as measured by mortality and rate of growth. Rickets was severe in all three lots. The first symptoms of rickets appeared as early as the eleventh day and at 24 days of age all chicks were very stiff, walking awkwardly and with difficulty. At six weeks of age, the surviving chicks in Lots 1, 2, and
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T A B L E I.
Basal Basal
5
6 7 8
2 Barred Plymouth
3
3
3
Basal (5 casein replaced by 5 liver meal)
Basal (5 casein replaced by 5 liver meal; agar omitted)
14
15
13
12 3
9.8
9.8
9.8
9.8
1.998
1.998
1.998
1.998
1.998
9.8
Basal (agar omitted) Basal
-
3
1.998
1.31
.655 .655 .983
1.09 .275
-
9.8
8
4 4 6
4 4
.275 .655
Cain Ration Per Cent 1.93J
Standard! Basal
10 11
3
9
Per Cent 4 4
Ca:P Ratio
P in Ration Per Cent 1.47
1.47
1.47
1.47
1.47
1:0.735 16
1:0.735 16
1:0.735 20 1:0.735 16
1:0.735 20
1.47 1:0.735 10 1.466$ l:0.76i 20
.398 11:0.608 16 .398 |1:0.608 !6 .597 1:0.608 16 .796 1:0.608 16
.398 |1:0.363| 20 .398 |1:1.449| 20
.398 |1:1.449 20 | .398 |1:0.608| 20
99
79
212
211
14
16
11
20
215
227
201
234
10 | 135
17
9
5 ! 158
15 | 323 5 | 152
2 |
17 | 152 19 | 291
6 | 104
4 |
No. Chicks Started No. Chicks Alive
Eight Weeks
14
16
19 9
14
—
302
315
333 275
505
—
+
+
+ +
+
+
—
1 | 155 | — 15 179 | — 19 449 | +
" 1" 1 -
Irradiation
0)
.. --
35.7
35.4
--
30.3
40.2
—
28.7 32.2
•Basal Ration: Casein 20; Dried Brewer's Yeast 5; Corn Germs 5; Lard 2; Agar 2; Degerminated Yellow Corn to make 100 (after addition of minerals as indicated). t Standard Ration: Ground Yellow Corn 53; Wheat Bran 15; Wheat Middlings 15; Meat Scraps 12; Dried Buttermilk 4; Salt 1. t In entire ration.
4 S. C. White Leghorns
3 S. C. White Leghorns
1
1 1
Basal (0.6 casein replaced by 0.6 cystine)
Basal
Basal
4
Breds
Rocks
Basal Basal
3
Cross-
|
2 185
Basal Basal
1 2
1
1
185 1
6
Trial No.
=5
Ration
Av. Weights
Six-Weeks
No. Chicks Alive Av. Weights
Mineral Additions
Ash in Fi Free Tib Per Cent
T A B L E I I . — S H O W I N G T H E P L A N S OF T H E E X P E R I M E N T S W I T H SUMMARY OF T H E R E S U L T S
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o
CO
Os
O
g
C5 ©
SB
ft
4
OS
304
POULTRY
SCIENCE
3 averaged respectively 99, 104, a n d 152 grams. Since the usual weight for this variety a n d age of chicks-grown u n d e r laboratory conditions approximates 350 grams, it is readily seen t h a t the
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CHART 1
Growth curves of the chicks that lived through the experiments. The figures under each lot indicate the number of chicks started and the number which lived through (20-4 under Lot 1, means that 20 chicks were started and 4 lived through). Lots which were irradiated are indicated by an asterisk (*).
SALT MIXTURES
UNDER RACHITIC CONDITIONS
305
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growth in all lots was decidedly unsatisfactory. In the irradiated group, however, 18 of the 20 chicks grew almost normally to ten weeks of age. One died in the sixth week and one in the ninth. At eight weeks the survivors average 449 grams. The good growth obtained in this lot indicated that our basal semipurified ration was practically complete in nutritional factors. While it proved nothing as to the optimum calcium-phosphorus ratio, it did suggest that, with adequate or perhaps excess vitamin D supplied, good growth would result when a mineral mixture having a Ca :P ratio of 1:1.449 was used. Trial II—In this trial Lots 5, 6, 7, and 8 received 4, 4, 6, and 8 per cent of Mineral No. 1. Lot 6 which was the only lot receiving vitamin D in any form, was irradiated. At six weeks of age the surviving chicks in Lots 5, 7, and 8 average respectively 79, 152, and 158 grams. The mortality in all three lots was very high, only two chicks surviving in Lot 5 and five surviving in each of Lots 7 and 8. All chicks in these three lots had severe rickets. These results suggest that possibly six or eight per cent of mineral was closer to the optimum level than was four per cent. In the irradiated lot, 14 of the 16 chicks lived and grew well to eight weeks of age and showed no indications of rickets. These chicks averaged 323 grams at six weeks of age and 505 grams at eight weeks. Trial HI—In the two previous trials, the results were disappointing. We had noticed in other work conducted at this station, that chicks fed!-a standard all-mash ration (ground yellow corn 53, wheat bran 15, wheat middlings 15, 50 per cent protein meat scraps 12, dried buttermilk 4, salt 1)„ without any vitamin D, did not show signs of rickets until they were four weeks of age or older. Most of the rachitic chicks in the previous trials showed unmistakable sighs of the disease in three weeks, and in some cases as early as the eleventh day. Since it seemed probable that the superior ability of the all-mash ration to offset rickets is due to its more favorable mineral balance, the calcium and phosphorus content of this ration was duplicated with the semipurified diet in this trial. Analyses of the standard all-mash ration showed that it contained 1.93 per cent elemental calcium and 1.466 per cent of ele-
306
POULTRY
SCIENCE
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mental phosphorus. The calcium-phosphorus ratio was therefore approximately 1:0.76. Allowing for the calcium and phosphorus present in the nonmineral ingredients of the semipurified ration, Mineral No. 3 was formulated to furnish the same Ca :P ratio as the all-mash ration. By using 9.8 per cent of this mineral mixture in the basal semipurified ration, practically the same amounts of these elements were furnished as in the all-mash ration. Pour lots of S. C. White Leghorn chicks, 10 in Lot 9 and 20 each in Lots 10, 11, and 12 were used. All lots received 9.8 per cent of Mineral No. 3, except Lot 10 which received the all-mash ration. Lot 9 was designed to determine whether or not the addition of cystine (0.6 per cent) would correct the ruffling of feathers which was common in all chicks fed the semipurified diets. The chicks in this lot were irradiated. Cystine failed to show any improvement as to this condition. In Lot 10, receiving the all-mash ration in the absence of vitamin D, rickets appeared in several chicks at 25 days of age while a week later only three or four showed decided rickets. Lot 11 was fed the regular semipurified ration with mineral. No. 3. In this lot rickets was noticed as early as the nineteenth day, and at four weeks the disease was very severe in all chicks. Lot 12 was the same as Lot 11 except that agar was omitted and the chicks were irradiated. The chicks in this lot did not make satisfactory growth, and feather ruffling was prevalent. The lack of agar seemed to have no ill effect, though no pen was an actual check on this one factor. At six weeks of age surviving chicks in these consecutive lots averaged respectively 211, 212, 135, and 234 grams, and the mortality was 10, 15, 50, and 5 per cent. From the results of this experiment, it was evident that Mineral No. 3 was not satisfactory as a mineral supplement. Lot 10, fed the standard all-mash ration without any vitamin D, grew as well to six weeks as Lot 9 on the semipurified ration plus Mineral No. 3 and with irradiation. Trial IV—In this trial three lots of 16 chicks each were used. Mineral No. 3 was fed at the same level as in the previous trial.
SALT MIXTURES
UNDER RACHITIC CONDITIONS
307
DISCUSSION
Although much work has been conducted to establish favorable calcium-phosphorus ratios for the rat, very little work has been reported on the calcium-phosphorus requirements for growing chicks. In a similar manner as rickets has been prevented and cured in rats in the absence of vitamin D, by altering the calcium-phosphorus ratio, it might be possible by proper mineral balance to prevent or at least delay the onset of rickets in chicks in the absence of vitamin D. The practicability of growing chicks to six or eight weeks of age without vitamin D may be questionable, but if with proper mineral balance, the quantitative requirement for vitamin D could be reduced to such an extent that chicks would not suffer from the lack of insufficient sunshine during unfavorable weather conditions, this would be of practical importance. When a chick is hatched it probably carries with it, from the egg yolk, a supply of vitamin D sufficient to prevent the visible external appearance of rickets over a period of three or four weeks, if it is fed a ration complete except for the antirachitic factor. Miller, Dutcher, and Ktiandel 17 have recently demonstrated that chicks fed a rachitogenic ration show a decided drop in the ash of their tibiae from 36.3 per cent at one week of age to ap-
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Lot 13 received the semipurified ration with 9.8 per cent of Mineral No. 3. Lot 14 was the same, except that five per cent of dried liver meal replaced an equal amount of casein. This change was made to see if such an addition of protein would improve growth. Since the liver meal contained less than six per cent of ash, it was not thought that the use of five per cent of it would measurably affect the mineral content of the ration. Lot 15 was the same as Lot 14 except that agar was omitted. All three lots were irradiated. At six weeks of age Lots 13, 14, and 15 averaged respectively 201, 227, and 215 grams, and the mortality was 5, 0, 2 chicks. Growth in all lots was below the average expected. While the use of liver meal appeared to increase the rate of growth to some extent, the omission of agar seemed to have little, if any, effect. All lots had severe rickets after five weeks despite irradiation.
308
POULTRY
SCIENCE
SUMMARY
1. MeCollum's Mineral No. 185, and substitutions of calcium
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proximately 28 per cent at six weeks. The chicks did not show severe rickets till the fifth week, although the time at which rickets was first observed is not mentioned. This suggests that a chick may be visibly non-rachitic for the first three or four weeks of its life and yet have the ash content of its bones continually decreasing. Little work has been done to show whether a variation in the calcium-phosphorus ratio of the ration would prevent this bone deterioration where no vitamin D was supplied. The results of these four trials indicate that even though the amounts of mineral in the ration, as well as the calcium-phosphorus ratio, were varied widely, the chicks did not grow satisfactorily without some form of vitamin D in the diet. In Trial I, the calcium-phosphorus ratio was varied from 1:1.449 to 1:0.363 without augmenting growth to anywhere near a usual or so-called normal rate. That this failure in growth cannot be attributed to inadequacy of the ration other than minerals and vitamin D was established when good growth was secured with some of the same rations simply by irradiation of the chicks. This indicated that the basal semipurified ration was practically complete in nutritional factors. The unsatisfactory growth of the chicks when no vitamin D was supplied can be attributed only to a lack of the vitamin and improper mineral balance. Attempts to duplicate the amounts as well as the ratio of calcium and phosphorus in the all-mash ration by adding diabasic calcium phosphate and calcium carbonate to the semipurified ration were not successful, since chicks thus fed did not make satisfactory growth even with irradiation. Therefore, it appears that Mineral No. 3 did*not meet the mineral requirements of the chicks. Although these trials using various salt mixtures in the absence of vitamin D did not contribute materially to our knowledge of the calcium-phosphorus ratio necessary for growing chicks, they did suggest that the problem might be approached more readily with a limited supply of vitamin D. Such studies were made and will be reported later.
SALT
MIXTURES
UNDER
RACHITIC
CONDITIONS
309
BIBLIOGRAPHY i S H I P L E Y , P . G., P A B K , E . A., MCCOLLUM, E , V., AND SIMMONDS,
N.,
Amer. Jour. Dis. Child., 1922, X X I I I , 91. 2 SHERMAN, H. C , AND PAPPENHEIMER, A. M., Proc. Soe. Exper. Biol, and Med., 1921, XVIII, 193. s GOLDBLATT, H., Biochem. Jour., 1924, X V I I I , 414. * STEENBOCK, H., H A B T , E . B., S E L L , M. E., A N D J O N E S , J. H., J. Biol.
Chem., 1923, LVI, 375. s BUCKNEB,
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AND M A R T I N ,
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HOLMES,
A N D I N S K O , W.
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ie CARRICK, C. W., P O U L T R Y SCIENCE, 1925, V, 98. "
MILLER,
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R.
A.,
AND KNANDEL,
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salts in it to produce a wide variation in the calcium-phosphorus ratio, when used with the semipurified ration in the absence of vitamn D, did not result in satisfactory growth or proper bone formation. 2. With the addition of vitamin D, the chicks approached optimal growth. 3. In the absence of vitamin D, better results were obtained with six or eight per cent of Mineral No. 1, than with four per cent. 4. Good growth was not obtained in young chicks fed a mineral mixture composed chiefly of dibasic calcium phosphate, at the level at which it was used in the semipurified ration, either with or without an adequate supply of vitamin D in the form of irradiation.