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An Unidentified Factor Required by Chicks Fed Practical Rations
An Unidentified Factor Required by Chicks Fed Practical Rations M. L. SUNDE, W. W. CRAVENS, C. A. ELVEHJEM AND J. G. HALPIN Departments of Poultry Husbandry and Biochemistry, University of Wisconsin, Madison (Received for publication August 27, 1949)
T
EXPERIMENTAL METHODS Straight run chicks (New Hampshire male X Single Comb White Leghorn female) were the progeny of hens fed, managed and cared for as described previously (Robblee, et al., 1948). After wingbanding, the chicks were kept in standard electrically heated batteries. They were weighed weekly. Mortality was negligible in all lots. Each trial was for a four week period except for one broiler trial. New Hampshire male XBarred Plymouth Rock female chicks were used in this ten week trial. These chicks were from hens fed a Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from the Western Condensing Company, San Francisco, California. We are indebted to Merck & Company, Rahway, New Jersey, for the crystalline vitamin Bi 2 and the B12 concentrate used in these studies.
RESULTS AND DISCUSSION
The results are shown in Table 1. The data show that the vitamin Bi2 concentrate did not promote as rapid growth as did the 3 percent fish solubles, either alone or when additional vitamins or choline were added to the basal diet. I t might be suggested that the B i2 requirement of the chick is higher than the amounts of the vitamin fed. One percent * Supplied through the courtesy of Merck & Co., Rahway, N. J. Standardized to contain 2.0 mg. vitamin Bu per lb.
204
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 5, 2015
standard breeder ration and maintained in floor pens. The basal ration was composed of the following in percent: ground yellow corn 46, wheat bran 5, wheat middlings 5, alfalfa leaf meal 5, soybean oil meal 22, corn gluten meal 10, granite grit 2, ground oyster shell 2, bone meal 2, iodized salt 0.5, vitamin A and D feeding oil (3000A400D) 0.2, MnS(VH 2 0 and riboflavin were fed at levels of .22 gm./kg. and 2.8 mg./kg., respectively. Vitamin Bi2 concentrate* and choline were added at the expense of the entire ration. The fish solubles was added at the expense of the corn. When the known vitamins were added, they were added at the following levels in mg. per kg. of ration: thiamine HC1 3, riboflavin 6, Ca pantothenate 20, niacin 50, pyridoxine HC1 4, biotin .2, menadione .5, inositol 1000, folic acid .5, choline chloride 1500, tocopherol 3, para-aminobenzoic acid 1.
INTRODUCTION HE availability of vitamin B12 concentrates makes it desirable to determine the value and limitations of such products in practical chick starting rations. The activity of vitamin B12 for growing chicks has been demonstrated by Ott, et al, (1948), Lillie, et ah, (1948) and Nichol, et al., (1948). However, none of the diets used were of a practical nature. Therefore, studies were initiated to compare the effect of a vitamin Bi2 concentrate with fish solubles in rations such as might be used in practice.
UNIDENTIFIED FACTOR REQUIRED BY CHICKS
205
TABLE 1.—The comparative effect of vitamin Bu, a Bn concentrate,fishsolubles and liver on chick growth 1
2
4
3
7t
6
5
Number of chicks started Addition to basal
30
25
19
m
25
32
25
25*
Percent gain
Grams None
204
214
250
1,201
223
. 2 % choline CI
221
276
1,278
84
266
1% B12 concentrate #1
240
274
303
1,356
Fish solubles
248
292
324
1,454
1% B12 concentrate #1 Known vitamins
205
290
287
209
228
240
108
255
116
2 % B12 concentrate #1 3 % fish solubles Known vitamins
244
113
309
1% B12 concentrate #1 . 2 % choline CI
277
312
1,417
261
287
3 % fish solubles . 2 % choline CI.
290
353
1,474
312
320
3 % fish solubles 1% B12 concentrate #1
303
1% B12 concentrate #1 . 5 % liver extract (1-20)
312
5% 40° whole liver powder
213 317
256 256
121
291
118
Crystalline B12 1 7/day/chick
104
1% B12 concentrate 7% casein
114
* .04% iodinated casein added, t 0.25% L cystine added. % 10 week weight.
of the B12 concentrate, according to the assay values reported, would add approximately 44 micrograms of the vitamin per kg. of ration. In one experiment, it was found that 0.25 percent of vitamin B12 concentrate in the ration caused a growth response equal to that obtained with .7 percent. After the first trial, one percent
of the concentrate was used in order to be sure that there was plenty of the vitamin in the ration. The addition of fish solubles or 1-20 liver powder to rations containing one percent of the B12 concentrate increased the rate of growth of the chicks. In trials not reported here three other types of rations were tried and in
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 5, 2015
Known vitamins
10
206
M. L. SUNDE, W. W. CRAVENS, C. A. ELVEHJEM AND J. G. HALPIN
out that the vitamin B i2 concentrate plus fish solubles group was as good as the 5 percent whole liver powder group. It might be suggested that the response obtained is due to an amino acid since the addition of casein, together with the concentrate, gave a growth response above that obtained with the concentrate. This seems unlikely since, as previously stated, added fish meal failed to nullify the difference in growth observed with the vitamin Bj2 concentrate and fish solubles. Further, the quantity of protein added by 3 percent fish solubles is negligible (1 percent). Hill (1948) has postulated additional factors required by the chicks, but in our studies the addition of dried whey to our ration containing the Bi2 concentrates has not resulted in additional stimulation of growth. Menge, Combs and Shorb (1949) reported the existence of an unidentified chick growth factor in dried whey. Since this manuscript was submitted for publication, Stokstad, et al., (1949) and Carlson et al., (1949) have presented data suggesting the existence of additional unidentified chick growth factors. The addition of known vitamins in earlier experiments resulted in increased growth when either fish solubles or a vitamin Bi2 concentrate were used. The data indicate that the major portion of the increased growth is due to the choline. This is not in agreement with data presented by Schaeffer et al (1949) and Gillis et al (1949). No explanation can be given at this time. SUMMARY Eleven experiments have been conducted comparing vitamin B12 with fish solubles and liver products and in each case the chicks fed the fish solubles or liver have been larger at the end of the test period than those in the groups re-
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 5, 2015
each case the response was less when vitamin Bi2 was used in place of fish solubles. A ration containing 4 percent fish meal was also tried. Responses of the same trend, but of a lower magnitude were observed. This is to be expected since fish meal is a good source of the animal protein factor (s). Several additional natural materials have been tested for activity and data will be presented in further publications. In order to further study these additional growth promoting properties of fish solubles and liver, iodinated casein was tested in experiment 6 to see if the differences could be magnified. From this data it appears that rations containing iodinated casein will be useful in testing various materials for activity. Whole liver powder and fish solubles apparently are very effective in overcoming the depressing action of the iodinated casein. When sufficient amounts of crystalline B12 became available, it was deemed advisable to test the crystalline material against the concentrate, whole liver powder and fish solubles. The chicks were fed the basal ration and distributed according to the method described by Clandinin, et al., (1947) except that the heavy and light chicks were removed at two weeks instead of one week. The test period was for 10 days. The crystalline B12 was injected into the pectoral muscle each day. It was decided that one microgram per day per chick would exceed the daily requirements of the chick. The data of experiment 7 indicate that crystalline Bi2 did not promote growth equal to that obtained with whole liver powder, fish solubles, fish solubles and B12 concentrate #1, 7 percent casein plus B12 concentrate §\, or 2 percent Bi2 concentrate #1. One percent of the concentrate was slightly more effective than the crystalline Bi2. It should be pointed
UNIDENTIFIED FACTOR REQUIRED BY CHICKS
ceiving crystalline Bi2 or the B i2 concentrate. I t is suggested, therefore, that chicks fed all vegetable rations require a factor or factors in addition to vitamin B12 and other known vitamins for maximum growth. The relation between the factor indicated from these data and other postulated unidentified factors cannot be determined at this time. REFERENCES
Lillie, R. J., C. A. Denton and H. R. Bird, 1948. Relation of vitamin B12 to the growth factor present in cow manure. J. Biol. Chem. 176: 1477-78. Menge, H., G. F. Combs a n d M . S. Shorb. 1949. Evidence for an unidentified chick growth factor in the presence of Vitamin B12. Poultry Sci. 28: 775. Nichol, C. A., L. S. Dietrich, W. W. Cravens, and C. A. Elvehjem, 1949. Activity of vitamin B i 2 in the growth of chicks. Proc. Soc. Exp. Biol, and Med. 70: 40-42. Ott, W. H., E. L. Rickes and T. R. Wood, 1948. Activity of crystalline Bi 2 for chick growth. J. Biol. Chem 174: 1047-1048. Robblee, A. R., C. A. Nichol, W. W. Cravens, C. A. Elvehjem and J. G. Halpin, 1948. The effect of hen diet and chick diet on the need of chicks for an unidentified growth factor. Poultry Sci. 27: 442447. Schaefer, A. E., W. D. Salmon and D. R. Strength, 1949. Interrelationship of vitamin B12 and choline. I. Effect on growth of the chick. Proc. Soc. Exp. Biol. Med. 71:202-204. Stokstad, E. L. R., T. H. Jukes, J. Pierce, A. C. Page, Jr., and A. L. Franklin, 1949. The multiple nature of the animal protein factor. J. Biol. Chem. 180: 647-654.
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 5, 2015
Carlson, C. W., R. F. Miller, H. T. Peeler, L. C. Norris, G. F. Heuser, 1949. The complex nature of the animal protein factor. Poultry Sci. 28: 750752. Clandinin, D. R., W. W. Cravens, C. A. Elvehjem and J. G. Halpin. 1947. Deficiencies in overheated soybean oil meal. Poultry Sci. 26:150-156. Gillis, M. B. and L. C. Norris, 1949. Effect of the animal protein factor on the requirement for methylating compounds. J. Biol. Chem. 179: 487-488. Hill, F. W., 1948. The multiple nature of the deficiency of unidentified nutrients in crude allvegetable protein chick starter rations. Poultry Sci. 27: 536-541.
207
T
EXPERIMENTAL METHODS Straight run chicks (New Hampshire male X Single Comb White Leghorn female) were the progeny of hens fed, managed and cared for as described previously (Robblee, et al., 1948). After wingbanding, the chicks were kept in standard electrically heated batteries. They were weighed weekly. Mortality was negligible in all lots. Each trial was for a four week period except for one broiler trial. New Hampshire male XBarred Plymouth Rock female chicks were used in this ten week trial. These chicks were from hens fed a Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from the Western Condensing Company, San Francisco, California. We are indebted to Merck & Company, Rahway, New Jersey, for the crystalline vitamin Bi 2 and the B12 concentrate used in these studies.
RESULTS AND DISCUSSION
The results are shown in Table 1. The data show that the vitamin Bi2 concentrate did not promote as rapid growth as did the 3 percent fish solubles, either alone or when additional vitamins or choline were added to the basal diet. I t might be suggested that the B i2 requirement of the chick is higher than the amounts of the vitamin fed. One percent * Supplied through the courtesy of Merck & Co., Rahway, N. J. Standardized to contain 2.0 mg. vitamin Bu per lb.
204
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 5, 2015
standard breeder ration and maintained in floor pens. The basal ration was composed of the following in percent: ground yellow corn 46, wheat bran 5, wheat middlings 5, alfalfa leaf meal 5, soybean oil meal 22, corn gluten meal 10, granite grit 2, ground oyster shell 2, bone meal 2, iodized salt 0.5, vitamin A and D feeding oil (3000A400D) 0.2, MnS(VH 2 0 and riboflavin were fed at levels of .22 gm./kg. and 2.8 mg./kg., respectively. Vitamin Bi2 concentrate* and choline were added at the expense of the entire ration. The fish solubles was added at the expense of the corn. When the known vitamins were added, they were added at the following levels in mg. per kg. of ration: thiamine HC1 3, riboflavin 6, Ca pantothenate 20, niacin 50, pyridoxine HC1 4, biotin .2, menadione .5, inositol 1000, folic acid .5, choline chloride 1500, tocopherol 3, para-aminobenzoic acid 1.
INTRODUCTION HE availability of vitamin B12 concentrates makes it desirable to determine the value and limitations of such products in practical chick starting rations. The activity of vitamin B12 for growing chicks has been demonstrated by Ott, et al, (1948), Lillie, et ah, (1948) and Nichol, et al., (1948). However, none of the diets used were of a practical nature. Therefore, studies were initiated to compare the effect of a vitamin Bi2 concentrate with fish solubles in rations such as might be used in practice.
UNIDENTIFIED FACTOR REQUIRED BY CHICKS
205
TABLE 1.—The comparative effect of vitamin Bu, a Bn concentrate,fishsolubles and liver on chick growth 1
2
4
3
7t
6
5
Number of chicks started Addition to basal
30
25
19
m
25
32
25
25*
Percent gain
Grams None
204
214
250
1,201
223
. 2 % choline CI
221
276
1,278
84
266
1% B12 concentrate #1
240
274
303
1,356
Fish solubles
248
292
324
1,454
1% B12 concentrate #1 Known vitamins
205
290
287
209
228
240
108
255
116
2 % B12 concentrate #1 3 % fish solubles Known vitamins
244
113
309
1% B12 concentrate #1 . 2 % choline CI
277
312
1,417
261
287
3 % fish solubles . 2 % choline CI.
290
353
1,474
312
320
3 % fish solubles 1% B12 concentrate #1
303
1% B12 concentrate #1 . 5 % liver extract (1-20)
312
5% 40° whole liver powder
213 317
256 256
121
291
118
Crystalline B12 1 7/day/chick
104
1% B12 concentrate 7% casein
114
* .04% iodinated casein added, t 0.25% L cystine added. % 10 week weight.
of the B12 concentrate, according to the assay values reported, would add approximately 44 micrograms of the vitamin per kg. of ration. In one experiment, it was found that 0.25 percent of vitamin B12 concentrate in the ration caused a growth response equal to that obtained with .7 percent. After the first trial, one percent
of the concentrate was used in order to be sure that there was plenty of the vitamin in the ration. The addition of fish solubles or 1-20 liver powder to rations containing one percent of the B12 concentrate increased the rate of growth of the chicks. In trials not reported here three other types of rations were tried and in
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 5, 2015
Known vitamins
10
206
M. L. SUNDE, W. W. CRAVENS, C. A. ELVEHJEM AND J. G. HALPIN
out that the vitamin B i2 concentrate plus fish solubles group was as good as the 5 percent whole liver powder group. It might be suggested that the response obtained is due to an amino acid since the addition of casein, together with the concentrate, gave a growth response above that obtained with the concentrate. This seems unlikely since, as previously stated, added fish meal failed to nullify the difference in growth observed with the vitamin Bj2 concentrate and fish solubles. Further, the quantity of protein added by 3 percent fish solubles is negligible (1 percent). Hill (1948) has postulated additional factors required by the chicks, but in our studies the addition of dried whey to our ration containing the Bi2 concentrates has not resulted in additional stimulation of growth. Menge, Combs and Shorb (1949) reported the existence of an unidentified chick growth factor in dried whey. Since this manuscript was submitted for publication, Stokstad, et al., (1949) and Carlson et al., (1949) have presented data suggesting the existence of additional unidentified chick growth factors. The addition of known vitamins in earlier experiments resulted in increased growth when either fish solubles or a vitamin Bi2 concentrate were used. The data indicate that the major portion of the increased growth is due to the choline. This is not in agreement with data presented by Schaeffer et al (1949) and Gillis et al (1949). No explanation can be given at this time. SUMMARY Eleven experiments have been conducted comparing vitamin B12 with fish solubles and liver products and in each case the chicks fed the fish solubles or liver have been larger at the end of the test period than those in the groups re-
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 5, 2015
each case the response was less when vitamin Bi2 was used in place of fish solubles. A ration containing 4 percent fish meal was also tried. Responses of the same trend, but of a lower magnitude were observed. This is to be expected since fish meal is a good source of the animal protein factor (s). Several additional natural materials have been tested for activity and data will be presented in further publications. In order to further study these additional growth promoting properties of fish solubles and liver, iodinated casein was tested in experiment 6 to see if the differences could be magnified. From this data it appears that rations containing iodinated casein will be useful in testing various materials for activity. Whole liver powder and fish solubles apparently are very effective in overcoming the depressing action of the iodinated casein. When sufficient amounts of crystalline B12 became available, it was deemed advisable to test the crystalline material against the concentrate, whole liver powder and fish solubles. The chicks were fed the basal ration and distributed according to the method described by Clandinin, et al., (1947) except that the heavy and light chicks were removed at two weeks instead of one week. The test period was for 10 days. The crystalline B12 was injected into the pectoral muscle each day. It was decided that one microgram per day per chick would exceed the daily requirements of the chick. The data of experiment 7 indicate that crystalline Bi2 did not promote growth equal to that obtained with whole liver powder, fish solubles, fish solubles and B12 concentrate #1, 7 percent casein plus B12 concentrate §\, or 2 percent Bi2 concentrate #1. One percent of the concentrate was slightly more effective than the crystalline Bi2. It should be pointed
UNIDENTIFIED FACTOR REQUIRED BY CHICKS
ceiving crystalline Bi2 or the B i2 concentrate. I t is suggested, therefore, that chicks fed all vegetable rations require a factor or factors in addition to vitamin B12 and other known vitamins for maximum growth. The relation between the factor indicated from these data and other postulated unidentified factors cannot be determined at this time. REFERENCES
Lillie, R. J., C. A. Denton and H. R. Bird, 1948. Relation of vitamin B12 to the growth factor present in cow manure. J. Biol. Chem. 176: 1477-78. Menge, H., G. F. Combs a n d M . S. Shorb. 1949. Evidence for an unidentified chick growth factor in the presence of Vitamin B12. Poultry Sci. 28: 775. Nichol, C. A., L. S. Dietrich, W. W. Cravens, and C. A. Elvehjem, 1949. Activity of vitamin B i 2 in the growth of chicks. Proc. Soc. Exp. Biol, and Med. 70: 40-42. Ott, W. H., E. L. Rickes and T. R. Wood, 1948. Activity of crystalline Bi 2 for chick growth. J. Biol. Chem 174: 1047-1048. Robblee, A. R., C. A. Nichol, W. W. Cravens, C. A. Elvehjem and J. G. Halpin, 1948. The effect of hen diet and chick diet on the need of chicks for an unidentified growth factor. Poultry Sci. 27: 442447. Schaefer, A. E., W. D. Salmon and D. R. Strength, 1949. Interrelationship of vitamin B12 and choline. I. Effect on growth of the chick. Proc. Soc. Exp. Biol. Med. 71:202-204. Stokstad, E. L. R., T. H. Jukes, J. Pierce, A. C. Page, Jr., and A. L. Franklin, 1949. The multiple nature of the animal protein factor. J. Biol. Chem. 180: 647-654.
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 5, 2015
Carlson, C. W., R. F. Miller, H. T. Peeler, L. C. Norris, G. F. Heuser, 1949. The complex nature of the animal protein factor. Poultry Sci. 28: 750752. Clandinin, D. R., W. W. Cravens, C. A. Elvehjem and J. G. Halpin. 1947. Deficiencies in overheated soybean oil meal. Poultry Sci. 26:150-156. Gillis, M. B. and L. C. Norris, 1949. Effect of the animal protein factor on the requirement for methylating compounds. J. Biol. Chem. 179: 487-488. Hill, F. W., 1948. The multiple nature of the deficiency of unidentified nutrients in crude allvegetable protein chick starter rations. Poultry Sci. 27: 536-541.
207