Supplementary Value of Methionine, Cystine and Choline in a Practical Soybean Oil Meal Starter Ration

D. R. CLANDININ, W. W. CRAVENS, J. G. HALPIN AND E. B. HART University of Wisconsin, Madison, Wisconsin (Received for publication April 26, 1946)

I

N A series of papers; Rose and co- workers (1936); Rose (1937); Womack, Kemmerer and Rose (1937); Womack and Rose (1941), presented evidence proving the dispensability of cystine and the indispensability of methionine in the diet of the rat. These workers also showed that cystine is capable of stimulating growth in rats only if the diet is suboptimal in methionine. Their findings indicate that on a cystine deficient diet, body cystine arises from dietary methionine and that on a suboptimal methionine diet, cystine included in the diet has methionine sparing action. That the sulphur of cystine synthesized in the body actually originates from methionine was established by Tarver and Schmidt (1939) and confirmed by Rose and Wood (1941). The relationship between choline, homocystine and methionine was worked out by du Vigneaud et al. (1939) who showed that in the presence of choline, homocystine is capable of replacing methionine in the diet of the rat. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from The Borden Company, New York, N. Y. We are indebted to Merck and Co., Rahway, N. J. and Merck and Co. Ltd., Montreal, Que. for substantial amounts of the dl methionine and vitamins used in this work.

That the relationship between cystine and methionine and between choline, homocystine and methionine as prevails in the rat, is applicable to the chick has been ably proven by Almquist and coworkers: Klose and Almquist (1941); Grau and Almquist (1943); Almquist and Grau (1944, 1945). Mitchell and Smuts (1932) were the first workers to attempt supplementation of soybean meal with sulphur containing amino acids. They showed that growth of rats on a ration containing raw soybean meal could be improved by the addition of 1(—) cystine. This observation was confirmed by Shrewsbury and Bratzler (1933). Three years later Hayward, Steenbock and Bohstedt (1936), also working with rats, demonstrated that a diet containing heated soybean meal could be likewise improved by the inclusion of 1( —) cystine. It was, however, not until about 1941 that sulphur containing amino acids were used to supplement soybean meal in the diet of the chick. In this connection Hayward and Hafner (1941) investigated cystine and methionine as supplements to raw and cooked soybean meal for rats and chicks and reported that the protein of raw soybeans was greatly improved by 0.3% 1( —) cystine and even more so by

509

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Supplementary Value of Methionine, Cystine and Choline in a Practical Soybean Oil Meal Starter Ration

510

D. R. CLANDININ, W. W. CRAVENS, J. G. HALPIN AND E. B. HART

The latter paper was followed by a series of reports by Carrick and coworkers: Berry, Carrick, Roberts and Hauge (1943a), (1943b); Marvel, Carrick, Roberts and Hauge (1944), (1945a), (1945b), (1945c) in which evidence is presented to support their contentions that choline and methionine may be used interchangeably as supplements to a practical soybean oil meal starter ration, that soybean oil meals differ in their content of available choline and hence differ in their ability to be supplemented by choline additions, and that their practical soybean oil meal starter may be made adequate by supplementation with choline and certain B-vitamins. On the other hand, Bird and Mattingly (1945) present data which indicate that choline and methionine cannot be considered interchangeable as supplements to a practical soybean oil meal ration. These

workers also indicate that their diet probably requires no supplement other than methionine for optimum growth. In a later paper Bird and Rubin (1946) suggest on the basis of certain feeding trials that the growth responses which Carrick and co-workers obtained with choline were due to the high levels of certain B-vitamins fed in conjunction with choline rather than to the choline alone. In this paper we are reporting on results which we obtained on supplementing different samples of soybean oil meal with methionine, cystine, choline and certain B-vitamins as well as with a number of other supplements. EXPERIMENTAL

Single Comb White Leghorn chicks (cockerels) were used in experiments I, II, and III while New Hampshire chicks (mixed sexes) were employed in experiment IV. In experiments I and I I I lots of thirtyone and forty-three chicks respectively were started on each of the rations shown in Tables 1 and 3. In experiments II and IV duplicate lots of twenty-one were started on the rations given in Tables 2 and 4. At the end of the first week the number of chicks in the lots of experiment I was reduced to twenty-eight; in experiment II to forty; in experiment III to thirty-three and in experiment IV to forty. The practice of removing the poorest chicks from each lot at the end of the first week was adopted in an effort to decrease variability. Each lot of chicks occupied a separate compartment of a battery brooder. All chicks were serially wing banded at hatching time. Individual weighings were made at weekly intervals until the end of the fourth week. Experiments I and I I I were conducted during the summer while experiments II and IV were undertaken

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0.3% dl methionine and that the protein •of cooked soybeans was equally improved by 0.3% l ( - ) cystine or 0.3% dl methionine. A year later, Almquist et al. (1942) reported that the addition of choline to a synthetic type raw soybean meal diet produced little or no increase in the rate of gain but that the addition of dl methionine resulted in a rate of gain which was comparable to that obtained on practical diets of the same total crude protein level. In the same study these workers found that the addition of choline to a synthetic type heated soybean meal diet resulted in a small but definite increase in rate of gain and that the addition of methionine produced a distinct increase in the growth rate. They concluded that the principal growth limiting factor in raw soybean protein is that of methionine and that heated soybean protein is slightly deficient in methionine for the chick at the 20% protein level, but is complete in . all other amino acids required by the chick.

METHIONINE, CYSTINE, CHOLINE SUPPLEMENTS TO SOYBEAN MEAL RATION

511

TABLE 1.—Experiment I.—Rations and growth Lot number Ingredients 1 42.5 10 10 5 28 2 1.5 .5 .5 3.3

35 10 10 5 28 2 1.5 .5 .5 3.3. 7.5

3 42.4 10 10 5 28 2 1.5 .5 .5 . 3.3

4

5

42.2 10 10 5 28 2 1.5 .5 .5 3.3

42.4 10 10 5 28 2 1.5 .5 .5 3.3 .1

.1 .3 1 4 2 15

Growth (Leghorn cockerels) Number chicks at 4 weeks Av. wt. in gms. at 4 weeks

27 200

28 213

28 202

28 227

27 197

*..Manganese sulphate molohydrate added at rate of 24 grams per pound of salt.

during the spring of the year. Formulas of the rations fed the various lots are given in Tables 1, 2, 3, and 4. It should be noted that all supplementations were made at the expense of ground corn. No attempt was made to adjust the protein content of the supplemented rations since the soybean oil meal control ration contained approximately 20% protein which was considered adequate for rapid growth. Attention is called to the fact that the soybean oil meal used in each experiment was a different sample and that the alfalfa and grain ingredients used were those on hand at the time the trials were conducted. RESULTS AND DISCUSSION

Number of chicks and average weights at four weeks are given in the tables. Experiment I The results of experiment I (Table 1)

indicate that the addition of choline chloride (lot 3) or choline chloride plus thiamine hydrochloride, calcium pantothenate, pyridoxine hydrochloride and nicotinic acid (lot 5) to the soybean oil meal ration was not beneficial. .Sup* plementation with dl mclliiimiine (lot 4), on the other hand, resulted in nn tip preciable increase in growth over that of the soybean oil meal control Clot 1). These results would seem to indicate that the soybean oil meal control ration used in the experiment was suboptimal in .methionine hut./iot low in choline. Since Berry, Carrick, Roberts and Hauge (1943a) had reported that a soybean oil meal ration in which the grain portion of the ration consisted entirely of corn was improved by the inclusion of choline plus certain B-vitamins it was thought that possibly the reason we had not obtained similar results was because our ration contained ten percent bran and ten percent middlings. To determine

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Ground corn Wheat bran Wheat middlings Alfalfa meal Soybean oil meal #1 Limestone grit Steamed bone meal Iodized salt* Fish oil (1500A 200D) Riboflavin, mg./kg. Labco casein Choline chloride dl methionine Thiamine hydrochloride, mg./kg. Calcium pantothenate, mg./kg. Pyridoxine hydrochloride, mg./kg. Nicotinic acid, mg./kg.

2

512

D. R. CXANDININ, W. W. CRAVENS, J. G. HALPIN AND E. B. HART

whether or not this was actually the case and also to investigate choline and methionine supplementation further, experiment II was undertaken. Experiment II

TABLE 2.—Experiment II.—Rations and growth Lot number Ingredients 6

7

8

9

10

11

12

13

14

15

so

42.5 10 10 5 28

42.5 10 10 5 28

42.5 10 10 5 28

42.5 10 10 5 28

62.5

62.5

62.5

62.5

62.5

5 28

5 28

5 28

5 28

5 28

2 1.5 5 5 3.3

2 1.5 5 .5 3.3 3

2 1.5 5 .5 3.3

Ground corn 10 Wheat bran 10 Wheat middlings 5 Alfalfa meal 6 Soybean oil meal §2 6 Fish meal (67% protein) 6 Meat meal (50% protein) Buttermilk powder 5 1 Limestone grit Steamed bone meal .5 Iodized salt* .5 Fish oil (1500A 200D) Riboflavin, mg./kg. dl methionine Choline chloride Thiamine hydrochloride, mg./kg. Calcium pantothenate, mg./kg. Pyridoxine hydroch oride, mg./kg. Nicotinic acid, mg./kg.

.15

2 1.5 .5 .5 3.3 3 .15

2 1.5 5 5 3.3

2 1.5 .5 .5 3.3 .3

2 1.5 .5 .5 3.3 .15

2 1.5 .5 .5 3.3 .3 .15

2 1.5 .5 5 3.3 .15 2 15 3 20

Growth (Leghorn cockerels) Number chicks at 4 weeks Av. wt. in gms. a t 4 weeks

40 331

40 226

39 279

39 267

39 291

39 198

39 265

* Manganese sulphate monohydrate added at rate of 24 grams per pound of salt.

39 243

40 265

40 238

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From the results shown in Table 2 it is apparent that both the soybean oil meal ration containing bran and middlings (lot 7) and the one in which the bran and middlings were replaced by ground corn (lot 11) were improved by either choline chloride (lots 9 and 13) or dl methionine (lots 8 and 12). It should, however, be noted that choline chloride did not give as good growth as dl methionine and hence cannot be considered as being equal in supplementary value. Choline chloride plus dl methionine (lots 10 and 14) gave no better results than dl methionine alone (lots 8 and 12). Further, it should be noted that supplementation of the corn

soybean oil meal ration with choline chloride plus thiamine hydrochloride, calcium pantothenate, pyridoxine hydrochloride and nicotinic acid (lot 15) at levels fairly comparable to those used by Berry, Carrick, Roberts and Hauge (1943a) and Marvel, Carrick, Roberts and Hauge (1945c) gave no better results than choline chloride alone. None of the various treatments produced as good growth as that obtained on the positive control diet (lot 6). The results of this experiment seem explainable on the basis that the soybean oil meal rations concerned we rA g nh^pt :r "nl in both choline and methionine. The fact that 0.3% dl methionine gave as good growth as 0.3% dl methionine plus 0.15% choline chloride indicates that 0.3%_dJa methionine adequately rmm>r-t»A ^c p ^ _ tial Hiolineand methionine defjcienrips However"since dl methionine supple-

METHIONINE, CYSTINE, CHOLINE SUPPLEMENTS TO SOYBEAN MEAL RATION

mentation did not give as good growth as the positive control diet, it is apparent that it is not the only deficiency in these rations. That thiamine, pantothenic acid, pyridoxine and nicotinic acid are not limiting factors is suggested by the results obtained in lots 13 and 15.

From the data presented in Table 3 it will be seen that choline chloride (lot 21)

K~) cystine gave an appreciable growth response. Thus it would appear that the soybean oil meal ration used in this experiment, like the ration used in experiment I, is not deficient in choline but is suboptimal in methionine. Methionine, however,, is not the only limiting factor as 0.5% dl methionine did not give as good growth as the positive control diet (lot 16). The fact that 7.5% Labco casein (lot 18) did not give as good growth as the

TABLE 3.—Experiment III.—Rations and growth Lot number Ingredients Ground corn Wheat bran Wheat middlings Alfalfa meal Soybean oil meal #3 Fish meal (67% protein) Meat meal (50% protein) Buttermilk powder Limestone grit Steamed bone meal Iodized salt* Fish oil (1500A 200D) Riboflavin mg./kg. Labco casein dl methionine 1 (-) cystine Choline chloride

16

17

18

19

20

21

22

23

50 10 10 5 6 6 6 5 1

42.5 10 10 5 28

42.5 10 10 5 28

42.5 10 10 5 28

42 5 10 10 5 28

42.5 10 10 5 28

42 5 10 10 5 28

42.5 10 10 5 28

2 1.5 .5 .5 3.3

2 1.5 .5 5 3.3 7.5

2 1.5 .5 .5 3.3

2 1 5 .5 .5 3.3

2 1.5 .5 .5 3.3

2 1.5 .5 .5 3.3

2 1 5 .5 5 3.3

.5

.3 .3

5 .5

.3 .15

.3 .15

Growth (Leghorn cockerels) Number chicks at 4 weeks Av. wt. in gms. at 4 weeks

33 291

33 212

33 243

33 258

32 254

31 215

32 237

32 247

* Manganese sulphate monohydrate added at rate of 24 grams per pound of sait.

did not improve the soybean oil meal ration (lot 17) used in experiment III. A comparison of the group receiving 0.3% 1( —) cystine and 0.15% choline chloride (lot 23) with the group receiving 0.3% K ~ ) cystine (lot 22) indicates that choline is not a limiting factor even at this higher plane of nutrition. It will be noted, however, that 0.5% dl methionine (lot 19) and 0.3% dl methionine plus 0.3% l ( - ) cystine (lot 20) gave a significant and equal growth response and that 0.3%

positive control diet (lot 16) was surprising indeed, since 5 | % , 6 | % , and 7 | % commercial casein in another experiment (unpublished data) and involving a different soybean oil meal gave a growth equal to the same positive control diet. The results of experiment IV suggest a good reason why commercial casein was able to complete the latter soybean oil meal ration, namely because the ration concerned was probably only deficient in methionine.

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Experiment III •

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514

D. R. CLANDININ, W. W. CRAVENS, J. G. HALPIN AND E. B. HART

TABLE 4.—Experiment IV.—Rations and growth Lot number

Ingredients Ground corn Wheat bran W h e a t middlings Alfalfa meal Soybean oil meal #4 Fish meal ( 6 7 % protein) M e a t meal ( 5 0 % protein) Buttermilk powder Limestone grit Steamed bone meal Iodized salt* Fishoil(1500A200D) Riboflavin, m g . / k g . Labco casein Condensed fish press water dl methionine 1 (-) cystine Choline chloride Thiamine hydrochloride nig. / k g . Calcium pantothenate mg./kg. Pyridoxine hydrochloride m g . / k g . Nicotinic acid m g . / k g .

24

25

50 10 10 5 6 6 6 5 1

42.4 10 10 5 28

.5 .5

2 1.5 .5 .5 3.3

26

27

42.35 42.35 10 10 10 10 5 5 28 28

2 1.5 .5 .5 3.3

2 1.5 .5 .5 3.3

28

29

30

31

32

33

34

35

38.5 10 10 5 28

38.35 10 10 5 28

42.05 10 10 5 28

38.05 10 10 5 28

42.2 10 10 5 28

38.2 10 10 5 28

36.5 10 10 5 28

32. 32.5 10 10 5 28

2 1.5 .5 .5 3.3

2 1.5 .5 .5 3.3

2 1.5 .5 .5 3.3

2 1.5 .5 .5 3.3

2 1.5 .5 .5 3.3

2 1.5 .5 .5 3.3

2 1.5 1. .5 .5 3! 3.3 6 4

.3

4 .3

2 1.5 .5 .5 3.3 6

4 .15

.15 2 20 4 20

4

4 .15

.3 .15

.3 1.5

)

Growth (New Hampshire chicks) Number chicks at 4 weeks Av. wt.ingms. at4weeks Number chicks at 4 weeks Av. wt. in gms. at 4 weeks

o"'s cf's 9 's 9 's

26 359 14 317

22 317 18 285

19 298 21 302

18 319 22 294

25 343 15 316

21 355 19 322

17 356 23 310

28 375 21 332

20 328 20 316

27 363 13 318

19 344 19 310

24 365 15 323

* Manganese sulphate added at rate of 24 grams per pound of salt.

to the positive control diet (lot 24). Apparently this soybean oil meal ration is only slightly suboptimal in methionine and not low in any other factor required for optimum growth. From the experiments reported it would appear that soybean oil meals purchased on the open market when incorporated in what might be called a practical starter ration may or may not be improved by choline supplementation. These experiments also indicate that choline and methionine cannot be considered interchangeable supplements to the soybean

.1this work, may be only slightly improved Iby methionine supplementation others may be made entirely adequate for growth Iby such supplementation. SUMMARY AND CONCLUSIONS Experiments involving supplementation of practical soybean oil meal starter rations with methionine, cystine, choline and certain B-vitamihs are reported. The results obtained indicate: 1. That soybean oil meals purchased on the open market when incorporated in what might be called a practical starter

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oil meal practical starter ration used in this study. Further the results seem to indicate that calcium pantothenate, pyridoxine hydrochloride and nicotinic acid, in addition to choline chloride, cannot be considered effective supplements to our soybean oil meal starter. Finally it would appear from these experiments that while certain soybean oil meals, incorporated in a starter ration such as the one used in

Experiment IV The results of this experiment shown in Table 4 indicate that the soybean oil meal ration used (lot 25) was not improved by choline chloride supplementation (lot 26). Likewise supplementation with choline chloride plus B-vitamins (lot 27) proved of no value. It will be noted, however, that all the other supplementations tried (lots 28 to 35) gave growth comparable

METHIONINE, CYSTINE, CHOLINE SUPPLEMENTS TO SOYBEAN MEAL RATION

REFERENCES

Almquist, H. J., and C. R. Grau, 1944. Interrelation of methionine, choline, betaine and arsenocholine in the chick. J. Nutrition. 27: 263-269. , 1945. Further studies in cystine, methionine and choline in chick diets. J. Nutrition. 29: 219222. Almquist, H. J., E. Meccbi, F. H. Kratzer and C. R. Grau, 1942. Soybean protein as a source of amino acids for the chick. J. Nutrition. 24: 385-392. Berry, E. P., C. W. Carrick, R. E. Roberts and S. M. Hauge, 1943a. Whey solubles as a source of growth factors in chick rations. Poultry Sci. 22: 252-263.

, 1943b. A deficiency of available choline in soybean oil and soybean oil meal. Poultry Sci. 22:442-445. ^ Bird, H. R., and J. P. Mattingly, 1945. Addition of dl. methionine to starting and growing rations. Poultry Sci. 24: 29-33. , and M. Rubin, 1946. The value of high levels of calcium pantothenate and pyrodixine hydrochloride in chick diets free of animal protein. Poultry Sci. 25:87-89. Grau, C. R., and H. J. Almquist, 1943. The utilization of sulphur amino acids by the chick. J. Nutrition. 26:631-640. Hayward, J. W., and F. H. Hafner, 1941. The supplementary effect of cystine and methionine upon the protein of raw and cooked soybeans as determined by chicks and rats. Poultry Sci. 20: 139150. , H. Steenbock and G. Bohstedt, 1936. The . effect of cystine and casein supplements upon the nutritive value of the protein of raw and heated soybeans. J. Nutrition. 12: 275-283. Klose, A. A., and H. J. Almquist, 1941. Methionine in the diet of the chick. J. Biol. Chem. 138: 467469. Marvel, J. A., C. W. Carrick, R. E. Roberts and S. M. Hauge, 1944. The supplementary value of choline and methionine in a corn and soybean oil meal chick ration. Poultry Sci. 23:294-297. , 1945a. A comparison of soybean oils and soybean oil meals in chick rations containing distiller's dried solubles. Poultry Sci. 24:46-52. , 1945b. The value of choline additions to a corn soybean oil meal chick ration containing distillers' dried solubles. Poultry Sci. 24: 181; 186. , 1945c. Distillers' dried solubles in chick rations containing corn and vegetable protein supplements. Poultry Sci. 24:252-258. Mitchell, H. H., and D. B. Smuts, 1932.The amino acid deficiencies of beef, wheat, corn, oats and soybeans for growth in the white rat. J. Biol. Chem. 95:263-281. Rose, W. C , 1937. The nutritional significance of the amino acids and certain related compounds. Science 86: 298-300. , K. S. Kemmerer, M. Womack, E. T. Mertz, J. K. Gunther, R. H. McCoy and C. E. Meyer, • 1936. The present status of the amino acids in nutrition. J. Biol. Chem. 114:LXX i XV. , and T. R. Wood, 1941. The synthesis of cystine in vivo. J. Biol. Chem. 141: 381-389. Shrewsbury, C. L., and J. W. Bratzler, 1933. Cystine deficiency of soybean protein at various levels, in a purified ration and as a supplement to corn. J. Agr. Res. 47: 889-895.

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ration may or may not be improved by choline supplementation. None of the meals tested were made adequate for optimum growth by choline supplementation. 2. That soybgan-Qil mcalo are-nnproved by methiomnejupplefaentdLioii. All of the mrnTrfHtrriTiTrrhrnnfitrrl h)'mrthinninr addition and_one-wao made adequate, for "pHrnriTnjngwth by g n r h fliippjernentation. 3. That choline and methionine cannot be considered as interchangeable supplements to practical soybean oil meal starter rations. Three out of four meals tested were not improved by choline addition whereas all four meals were improved by methionine supplementation. 4. That calcium pantothenate, pyridoxine hydrochloride, nicotinic acid and choline as a supplement to practical spybean oil meal rations cannot be relied upon to produce growth increases superior to choline alone. In the trials reported this combination of vitamins was in no instance found to be superior to choline. 5. That it is not possible to indicate what pure supplements should be added to a chick starter composed of soybean oil meal, alfalfa meal, grain, grain byproducts and mineral feeds in order to ensure optimum growth.

515

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BOOK REVIEW

Tarver, H., and C. L. A. Schmidt, 1939. The conversion of methionine to cystine: Experiments with radio active sulphur. J. Biol. Chem. 130:6780. du Vigneaud, V., J. P. Chandler, A. W. Moyer and D. M. Keppel, 1939. The ability of homocystine plus choline to support growth of the white rat on a methionine free diet. J. Biol. Chem. 128: CVIII.

Womack, M., K. S. Kemmererand W. C. Rose, 1937. The relation of cystine and methionine to growth. J. Biol. Chem. 121:403-110. , and W. C. Rose, 1941. The partial replacement of dietary methionine by cystine for purpose of growth. J. Biol. Chem. 141: 375-379.

G. T. Starting Right with Turkeys. The Macmillan Company, New York, 1946.129 pages. Price $2.50.

KLEIN,

This book is an excellent guide for the small lot beginner in turkey raising. It begins with a discussion of the practicability of raising a few turkeys for home or freezer locker use and points out the advantage for boys' and girls' club work. Brooding, growth and turkey troubles are discussed in a very plain and readable form. Methods of killing, dressing, preserving and cooking turkeys are given in the various chapters. Also included are details on the construction of small houses and sun porches.

Whether "Starting Right with Turkeys" outlines the correct method for a beginner who might develop turkeys into a commercial unit of his farm enterprise is open to question. It emphasizes backyard turkey raising and clearly presents a method of raising a few turkeys in a small space as has been done with rabbits, pigeons and chickens. The plan of rearing is the confinement and the sun porch method which may not be desirable in all geographic regions. Any person who contemplates developing a turkey project into a business enterprise might find bulletins or books which emphasize larger scale production more useful sources of information.

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Book Review