THE USE OF THE CHICK IN VITAMIN B1 AND Bz STUDIES*

BY 0. L. KLINE, J. A. KEENAN, C. A. ELVEHJEM, AND E. B. HART

(From the Department of Agricultural Chemistry, University of blisconsin, Madison)

(Received for publication, October 21, 1932)

The chicken was used as an experimental animal very early in the history of vitamin research. Eijkman (l), using this animal, demonstrated in 1897 the existence of an antineuritic substance obtainable from rice polishings, and indispensable to the growth and health of the fowl. Grijns, Vedder and Clark, and Fraser and Stanton also used the chicken for beriberi studies. This early work is reviewed by Funk (2).

Since the food requirements of pigeons were found to be much simpler than those of chicks, and because beriberi was readily produced in this animal, the pigeon replaced the chick to a large extent in vitamin B studies. The early work with pigeons is also reviewed by Funk (2).

After McCollum and Kennedy (3) demonstrated that those natural foods which cured polyneuritis in chickens and pigeons also promoted growth in rats fed a vitamin B-deficient diet, the rat was used extensively as the experimental animal. This animal has been used almost exclusively in the investigations concerning the dual nature of vitamin B.

Emmett and Luros (4) and Levene and Muhlfeld (5) used both rats and pigeons in their attempts to prove that the antineuritic factor for pigeons and the growth-promoting factor for rats are not identical. In 1926 Hauge and Carrick (6) used the chicken exclusively for the differentiation between the water-soluble growth-promoting factor and the water-soluble antineuritic sub- stance. Hauge and Carrick used the rate of growth of the chickens for evaluating the second factor of the vitamin B complex. In

* Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station.

295

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Vitamins B1 and B,

1930 Norris and Ringrose (7) described a pellagrous-like syndrome in chicks which could be prevented by the addition of autoclaved yeast which is rich in the growth-promoting factor.

Williams and Waterman (8) used the pigeon for demonstrating that the vitamin B complex contains a third, highly thermolabile factor necessary for maintenance of weight and general condition of adult pigeons. Further evidence was presented by Eddy, Gurin, and Keresztesy (9) substantiating the existence of a bird growth and weight restoration factor designated by Williams and Waterman as Bs and distinct from vitamins B, and Bz. Eddy and coworkers showed that this factor is also necessary for the chick.

Carter, Kinnersley, and Peters (10) accepted the Ba factor studied by Williams and Waterman and by Eddy, Gurin, and Keresztesy, and concluded that the pigeon requires for maintenance nutrition an additional factor, designated as vitamin B,, which is distinct from vitamins B,, B,, BS, and Bq.

From the work of Williams and Waterman, Eddy, Gurin, and Keresztesy, and Carter, Kinnersley, and Peters it is necessary to conclude that of the five vitamin B factors which have been studied, the pigeon needs vitamins B,, B,, and Bs and the rat needs vitamins B,, B,, and Bq. Only one component, the anti- neuritic vitamin B1, is required in common by the two species. The results which we have obtained with chickens indicate that the heat-labile vitamin BS factor is not necessary for this species, but that vitamin Ba is required in relatively large amounts. While carrying out these experiments, methods for rendering a ration of natural foodstuffs deficient in vitamins B1 and B2 were developed. A description of the preparation and use of these rations will be given in this paper.

EXPERIMENTAL

For these experiments day-old white Leghorn chicks with an Initial weight of 30 to 35 gm. were procured from the University Poultry Department or from commercial hatcheries. They were placed in individual cages (14 X 16 X 14 inches) in order to allow the recording of individual food consumption. The cages were individually heated and equipped with raised wire screen (three meshes to the inch) floors.

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Kline, Keenan, Elvehjem, and Hart 297

During the first 2 weeks of the experiment the ration was placed in small, round Daton feeders (No. 1015). Five of the six holes were stoppered with corks and an additional hole made in the center, which left two openings through which the chicks could consume the ration. This type of feeding served to minimize the loss of food due to scratching and spilling.

At 2 weeks of age, the small feeders were replaced by 2 X 3 X 10 inch feed pans equipped with 3 inch flanges to prevent the scatter- ing of feed. Each chick was separated from the feeding pan by means of two wire grids, superimposed, and sliding one upon the other so that the openings could be made just large enough for the chick to place its head through the grid. The feed pan was then placed flush with the opening of the cage and the grids imme- diately behind to allow the chick as much space as possible. The water cup was placed a short distance from the feed pan to avoid the splashing of water into the food.

The basal ration formulated for use in these experiments con- sisted of the following ingredients.

Yellow corn.. . . 58 Salt (common). . . . . . . . . . 1 Middlings.. . . . 25 Cod liver oil.. . . . . 2 Casein.. . . 12 CaC03.. . . . . . . 2

The yellow corn used was obtained directly from the field and dried at room temperature (20”). The middlings were standard wheat middlings and the casein was a commercial preparation. This ration will hereafter be designated as Ration 240.

The ability of this ration to promote normal growth of the chick was demonstrated in preliminary experiments. To render this ration deficient in vitamins B, and Bt, it was subjected to various heat treatments. In this procedure only the yellow corn, middlings, casein, and salt were treated thermally; the CaC03 and cod liver oil were added to the heated portion as the rations were prepared for feeding each week. The feed was weighed into the pans at the beginning of each week. At the end of the week the pans were reweighed, the ration not consumed discarded, and the pans refilled. In some cases it was necessary to make further weighed additions during the week. Weekly consumption records have been kept throughout all our experiments.

Since forty-eight individual cages were available for the work,

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Vitamins B, and B,

it was possible to study at one time twelve different rations with four chicks on each ration. However, two groups were always used for positive and negative controls in each series. In t.his paper no attempt will be made to present a complete record of ail the experimental work; only those results will be given which are needed to demonstrate that vitamins B, and B, can be destroyed independently in a natural ration. In all cases the growth curves for four individual animals on any specific ration will be given. In most cases many more animals have been reared on the same ration but the four curves are typical of all the animals.

When chicks are placed on the basal unheated ration (Ration 240) good growth is obtained and the animals appear normal in every way. The growth curves are given in Chart I. The growth is comparable to that observed for white Leghorn chicks grown on any complete ration. Chicks placed on a ration identical in all respects except that it had been autoclaved at 120’ and 15 pounds pressure for 5 hours failed to grow normally and developed polyneuritis at about 8 days of age. Growth curves for these chickens are also given in Chart I. The development of poly- neuritis in all chicks receiving this ration was exceedingly uniform, usually not before 7 days and not later than 10 days of age. The animals became extremely emaciated before death and exhibited a general lack of muscular coordination and sense of balance. This condition usually lasted about 1 day before the typical opistho- tonos of the neck muscles was observed. Death followed shortly after these characteristic symptoms developed. Fig. 1 illustrates a bird exhibiting typical vitamin B1 deficiency. Ration 240 which has been autoclaved at 120” for 5 hours and which produces this typical vitamin B, deficiency will be designated as Ration 240-A.

This condition was cured by the administration of yeast or was prevented by incorporating 4 per cent of air-dried yeast in the ration. The dried yeast was prepared from fresh bakers’ yeast which was obtained from the Red Star Yeast Company, Mil- waukee, and dried immediately at 65”. The results in Chart I show that 2 per cent of this yeast was insufficient to prevent poly- neuritis but that 4 per cent was ample for complete protection. The chicks receiving 8 per cent showed no improvement over those getting 4 per cent yeast. These results demonstrate that vitamin B1 is destroyed in a ration of natural foodstuffs by autoclaving

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Kline, Keenan, Elvehjem, and Hart

WeIghtI I I I I IA I

GrouP 6 i ) mhea'ted Ration .

4 6 0 2 4 6 age In Weeks

CHART 1. Typical growth records of chicks reared on the unheated ration (Ration 240) and on the autoclaved ration (Ration 240-A) with and without vitamin 13, supplements. The chicks receiving Ration 240-A and Ration 240-A plus 2 per rent of air-dried yeast, exhibited typical poly- neuritis before death.

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300 Vitamins B, and B,

and that the deficiency can be prevented by the addition of 4 per cent of air-dried yeast. They do not eliminate the possibility that other factors are also destroyed by this treatment and in turn supplied by the yeast. However the administration of a vitamin B1 concentrate prevented polyneuritis and allowed growth.

The vitamin B, preparation was made as follows: 2000 gm. of fresh bakers’ yeast were suspended in 1000 cc. of boiling water acidified with 1 cc. of glacial acetic acid, and boiled 5 minutes with constant stirring. The yeast was filtered off and reextracted under the same conditions. The filtrates were combined and adjusted

FIG. 1 FIG. 2

FIQ. 1. Typical opisthotonos of the neck ml&es exhihited hy chicks suffering from vitamin B, deficiency.

FIG. 2. Scabby incrustations of the corner of the mouth and lower mandible exhibited by pellagric chick.

to pH 4.5 with HCl, and 80 gm. of fullers’ earth were added. The suspension was stirred frequently and allowed to stand overnight. The fullers’ earth was filtered off and the filtrate treated with an additional 40 gm. of fullers’ earth. After standing 6 hours the suspension was filtered and the two samples of fullers’ earth were combined and dried at room temperature. 1 gm. of activated fullers’ earth was equivalent to 16 gm. of fresh yeast.

The results obtained when four chicks were fed the autoclaved ration (Ration 240-A) plus the B1 preparation equivalent to 20 per cent of air-dry yeast are given in Chart I. The good growth indicates that vitamin Bi is the only factor destroyed during autoclaving.

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Kline, Keenan, Elvehjem, and Hart 301

Since a ration compounded from grains and grain products may be low in vitamin B,, the effect of additional amounts of this factor in the form of autoclaved yeast was studied. The growth curves for four chicks raised on the autoclaved ration plus 4 per cent autoclaved yeast and 4 per cent air-dried yeast are given in Chart II. The growth of these chicks is no different from that of

Weight gmS* Group 59

Autoclaved Ratibn + 'I$ A+ocl$ved jeast

CHART II. Growth records of chicks raised on autoclaved ration plus additional vitamin Bz in the form of autoclaved yeast with and without vitamin B1 supplements.

those receiving 4 per cent of air-dried yeast alone. It is readily seen from the growth records of t,he chicks fed 8 per cent of auto- claved yeast that autoclaved yeast furnishes no vitamin B1. The autoclaved ration can therefore be used for testing the vitamin B1 potency of materials containing large amounts of vitamin B,.

The results obtained when Ration 240-A was used for testing the vitamin B, potency of the original unheated ration are given in

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302 Vitamins B1 and B,

Chart II. The chickens receiving 16 per cent of the ration were protected to some extent, while those receiving 32 per cent were protected completely and grew normally. These results show that the natural ration contains about one-eighth as much vitamin B1 as the yeast used. In much of our work we have used Ration 240-A plus 4 per cent autoclaved yeast as the basal ration in order to insure a plentiful supply of vitamin Bz. The ration containing autoclaved yeast has been designated as Ration 241-A. Either of these rations is well suited for testing the vitamin B1 potency of any food material, for two reasons; first, because they are easily prepared and, secondly, because the vitamin B, activity is determined under conditions which are comparable to those under which the vitamin is generally consumed.

While testing the stability of the vitamin B factors at tem- peratures lower than 120”, it was found that chicks reared on the basal diet heated dry at 100” for 144 hours developed definite symptoms of pellagra. For the heat treatment, the ration was placed in suitable pans and allowed to remain in an oven kept at 95-100” for 6 days. To insure uniform treatment in the different batches, a 2 inch layer of feed was always placed in each pan. Ration 240 which has been heated dry at 100” for 144 hours is designated as Ration 240-H.

All chickens grown on this ration invariably developed pellagra at about 3 weeks of age. The symptoms are very similar to those described by Norris and Ringrose (7). As these workers state, the external manifestations appear chiefly at the eyes, the corners of the mouth, and upon the legs and feet. The feathering is retarded, and the feathers are very ruffled because the birds peck themselves continually. The crusty scabs at the corners of the mouth gradually enlarge and often involve the margins of t.he skin around the nostrils and underneath the lower mandible. The condition develops gradually and the animals often survive 2 weeks or more after the first appearance of the disease. The appetite does not decrease as is the case with chicks suffering from vitamin B, deficiency. The average weekly food consumption for a number of normal chicks, vitamin B1-deficient chicks, and vitamin Bz-deficient chicks is given in Table I.

The growth records for four chicks on the unheated ration (Ration 240) and four chicks on the ration heated at 100” for 144 hours (Ration 240-H) are given in Chart III. It is readily seen

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Wk Ration 240-A;. basal,

Ration 240-H; basel, Rati,oo;;~O&aml, autoclaved; mtamin heated at 100” for

Bl-deficient 144 hrs.: vitamin Bpdefieient

Kline, Keenan, Elvehjem, and Hart 303

TABLE I

Average Weekly Consumption Records (in Gm.) of Normal, Vitamin Bl-, and Vitamin Bs-Deficient Chicks

32 30 45 20 90

115 204 240

30 46 62 65 85

weigt gme.

It cmxlp 31 , llnheated Easal

200

100

0

300

200

100

0

CHART III. Gir0wt.h records of chicks fed Ration 240 and Ration 240 heated at 100” for 144 hours (Ration 240-H) with and without vitamins B1 and BZ supplements. P denotes the approximate time at which the symp- toms of pellagra were first noted.

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304 Vitamins B, and B,

that this heat treatment develops in the ration a deficiency characterized by retarded growth and a pellagrous condition in the chick. Fig. 2 illustrates this deficiency in the chick.

When 6 per cent of autoclaved yeast is added to the ration heated at 100” for 144 hours (Ration 240-H), the development of pellagra is prevented and normal growth is obtained. The addition of a

Weight- gms.

/ -+--p"A Ra CLir

200. crouo bs I

0

300

200

100

0 6

CHART IV. Growth records of chicks fed Ration 240-H supplemented with skim milk powder and liver extract (Lilly, No. 343). P denotes the approximate time at which the symptoms of pellagra were first observed.

vitamin B1 preparation did not prevent pellagra and had practi- cally no effect on growth. These results demonstrate conclu- sively that chicks require vitamin Bz for normal development. Williams and Waterman (8) concluded, as did Seidell (ll), that pigeons do not need this vitamin. However, Eddy and coworkers (9) showed that, unlike the adult pigeon, growing chicks showed

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Kline, Keenan, Elvehjem, and Hart 305

some need for vitamin B,. Our results are in complete accord with those of Norris and Ringrose who showed the very great require- ment of this species for vitamin Bz.

We have used Ration 240-H for testing the vitamin Bz potency of a number of foodstuffs. The results obtained for skim milk powder and liver extract (Lilly, No. 343) are given in Chart IV. 8.0 per cent of skim milk powder and 0.4 per cent of liver extract supplied sufficient vitamin B.J to meet the requirements of the chick.

It is possible, therefore, to destroy the vitamin Bz in a ration of natural foodstuffs by heating dry at 100” for 144 hours and at the same time allow sufficient amounts of vitamin B, to remain to supply the requirement of the chick. Ration 240-H is especially valuable, because it eliminates the necessity of preparing and adding a vitamin B1 concentrate to a ration deficient in both vitamins B, and Bz, which is the usual procedure in vitamin Bt assay.

DISCUSSION

In the past the majority of the workers have used either a rice diet for vitamin B1 studies with pigeons or synthetic diets for vitamins B1 and B, studies with rats. In the case of the rice diet no serious difficulties have been encountered because pigeons develop polyneuritis so rapidly that no other factors complicate the results; nevertheless, the rice is far from a complete diet. The use of synthetic diets has introduced many complicated problems in vitamin assay. This is especially true in the case of chickens as has been pointed out in several papers by Hogan and coworkers (12). When a synthetic ration is compounded it is difficult to determine whether the ration is deficient only in the factor to be studied or if it is lacking in other nutrients as well. Generally the ration is so constructed that one or more factors in addition to the one being investigated must be added in con- centrated form. In this case it is difficult to prepare these concentrates so that they are exceedingly low in or devoid of the factor in question. The existence or non-existence of some of the recently reported factors rests upon the purity of the preparations used.

Whether workers are justified in concluding that the requirements

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306 Vitamins B, and B,

of an animal for a certain vitamin are the same when it is added to a synthetic diet as when added to a diet of natural food materials is a question which may be raised also. In this paper we have described methods for rendering a diet of natural foodstuffs de- ficient in vitamin B1 or Bz respectively. The original ration is compounded from materials which are ordinarily consumed by humans or farm animals and is complete in all known nutrients before it is subjecOed to either of the treatments for the destruction of the vitamins.

SUMMARY

1. A ration of natural foodstuffs (Ration 240) has been devised which can be rendered deficient in vitamin B1 or Bz respectively by different heat treatments.

2. When Ration 240 was autoclaved at 120’ and 15 pounds pressure for 5 hours (Ration 240-A) and fed to chicks, the chicks developed severe polyneuritis at about 8 days of age. The incorporation of air-dried yeast or a vitamin B1 concentrate inthe ration corrected this deficiency. Ration 240-A was usually supple- mented with additional vitamin Bz in the form of 4 per cent of autoclaved yeast (Ration 241-A) when it was used as a basal ration for vitamin B1 studies.

3. When Ration 240 was heated dry at 95-100’ for 144 hours (Ration 240-H) and fed to chicks, the chicks developed pellagra at about 3 weeks of age. This ration was rendered complete by the addition of autoclaved yeast.

4. A few results obtained when Rations 241-A and 240-H were used in assaying the vitamin B1 and vitamin B2 content of various foods are given.

BIBLIOGRAPHY

1. Eijkman, C., Firchows Arch. path. And., 149, 187 (1397) 2. Funk, C., The vitamins, Baltimore, 2nd edition (1922). 3. McCollum, E. V., and Kennedy, C., J. Biol. Chem., 24, 491 (1916). 4. Emmett, A. D., and Luros, G. O., J. Biol. Chem., 43, 265 (1920). 5. Levene, P. A., and Muhlfeld, M., J. Biol. Chem., 67, 341 (1923). 6. Hauge, S. M., and Carrick, C. W., J. Biol. C&m., 69,403 (1926). 7. Norris, L. C., and Ringrose, A. T., Science, 71,643 (1930). 8. Williams, R. R., and Waterman, R. E., J. Biol. Chem., 78, 311 (1928). 9. Eddy, W. H., Gurin, S., and Keresztesy, J., J. Biol. Chem., 87, 729

(1930).

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Kline, Keenan, Elvehjem, and Hart 307

10. Carter, C. W., Kinnersley, H. W., and Peters, R. A., &o&m. J., 24, 1832 (1930).

11. Seidell, A., Bull. Sot. chim. biol., 8, 746 (1926). 12. Hogan, A. G., Guerrant, N. B., and Kempster, H. L., J. Biol. Chem.,

64, 113 (1925); Missouri Agric. Exp. Station, Research Bull. 81 (1925). Hogan, A. G., and Shrewsbury, C. L., J. Nutr., 3,39 (1930).

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E. B. HartO. L. Kline, J. A. Keenan, C. A. Elvehjem and

STUDIES2 AND B1BTHE USE OF THE CHICK IN VITAMIN

1932, 99:295-307.J. Biol. Chem. 

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