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The Relationship of Choline and Cystine to the Methionine Requirement of Young Chickens1,2
T h e Relationship of Choline and Cystine to the Methionine Requirement of Young Chickens12 J. W. WEST, C. W. CARRICK, S. M. HATJGE AND E. T. MERTZ
Departments of Poultry Husbandry and Agricultural Chemistry, Purdue University Agricultural Experiment Station, Lafayette, Indiana (Received for publication February 28, 1951)
HE essential nature of methionine in the diet of the chick was demonstrated by Klose and Almquist (1941). Homocystine and choline together effectively replaced methionine in the diet of the chick, but neither homocystine nor cystine completely replaced methionine. Direct proof of the homocystine-cholinemethionine interrelationship in the rat was presented by Simmonds et al. (1943). Transmethylation was observed both in the absence of homocystine and in the presence of ordinary methionine when the diet contained deuterio-choline. Du Vigneaud et al. (1944) reported results which demonstrated the role of methionine in cystine formation. Grau and Almquist (1943) investigated the relationship between the sulfur amino acids and choline in chick nutrition. Choline-depleted chicks were able to utilize homocystine effectively in the place of cystine but not as a substitute for methionine. Almquist and Grau (1945), using a diet deficient in methionine and cystine, fed increasing levels of cystine up to 0.9 percent and various levels of choline up to 0.6 percent. In all cases growth was sub-optimal. Replacement of part of 1 Journal Paper No. 487 of the Purdue University Agricultural Experiment Station. 2 This research was supported in part by a grant from The Borden Company.
the cystine with methionine so that the ration contained 0.55 percent methionine resulted in optimal growth. It was concluded that cystine could not compensate for a methionine deficiency, even in the presence of high choline levels. Almquist (1947) stated that the growing chicken requires 0.9 percent sulfur amino acids, 0.5 percent of which must be methionine and 0.4 percent of which may be provided by cystine or methionine. Grau and Kamei (1950) concluded that when the diet contains approximately 20 percent total protein the sulfur amino acid requirement appears to be met with approximately 0.5 percent methionine and 0.3 percent cystine or with 0.8 percent methionine alone, McKittrick (1947) observed that the methionine and choline requirements for chick growth consist of essential and replaceable parts. For optimal growth it was found that 0.5 percent methionine and 0.1 percent choline constitute the essential'parts, and that 0.25 percent methionine or 0.45 percent choline or any equivalent mixture may comprise the replaceable parts. Grau and Almquist (1943) reported that DL-methionine is equivalent to Lmethionine for growth, whether in the presence or absence of choline in the diet of the chick. Treadwell (1948) observed poor growth, fatty livers and hemorrhagic kidneys when young rats were fed a
880
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T
881
METHIONINE REQUIREMENTS OF CHICKS
choline-free diet containing 18.6 percent protein, 0.5 percent methionine and 0.1 percent cystine. Increased cystine levels aggravated the fatty liver condition, but 0.2 percent choline produced good growth and a normal fat content of the liver. So far as we are aware similar studies with chicks have not been reported. EXPERIMENTAL
After the two-week depletion period, the chicks were weighed individually and sorted into blocks on the bases of weight and sex and distributed at random into experimental pens. The chicks were all reared in battery brooders with wire floors. Purified rations and tap water were supplied ad libitum during the twoto-six week experimental period. Individ-
TABLE 1.—-Methionine and cystine content of the protein ingredients
2 S S
T ri al(s)
Casein 1, 2 and 3 Gelatin 1 and 2 Gelatin 3 Blood fibrin 1 and 2 Blood fibrin 3 Lederle APF 1, 2 and 3
£ | £ r
5 - a t
2.283 0.832 0.S31 1.863 1.600 0.300
0.382 0.113 0.095 2.430 1.910 0.280
* Expressed as percent of air-dried material. In each trial each protein ingredient was thoroughly mixed and used throughout the trial.
chick was ascertained by the use of published literature values, and appropriate corrections made where necessary. Methionine was assayed by the method of Stokes et al. (1945) using Streptococcus fecalis R, and cystine by Dunn's (1945) procedure using Leuconosloc mesenteriodes P-60. The methionine and cystine content of the protein ingredients used in each of the three trials is given in Table 1. The composition of the basal diets used in the various trials is shown in Table 2. Variable ingredients are listed in the individual tables for the various trials. All diets, with few exceptions, contained approximately 20 percent protein. Liver samples were taken for choline determinations when the trials were terminated at six weeks, to study the effect of dietary levels of choline oh liver storage of that vitamin. In Trials 1 and 3
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Barred Plymouth Rock chicks were used in each of the trials. All chicks were sexed at one day of age by the method of Gerry and Mishler (1949), wingbanded and placed on the same diet for the firsttwo weeks. In order to deplete the body stores of choline, the chicks were fed a diet deficient in choline for the first week. The percentage composition of the depletion diet was ground yellow corn 59.45, casein 12, corn gluten meal 10, peanut meal 5, dried skimmilk 5, alfalfa leaf meal 3, fish solubles (dry basis) 1.5, steamed bone meal 2, ground limestone 1, glycine 0.5, iodized salt 0.45, manganese sulfate 0.05, and irradiated animal sterol product 0.05. Three hundred micrograms of riboflavin were added per 100 grams of diet. The choline content of this diet was determined by a slight modification of Engel's (1942) method, and was found to be less than 0.05 percent choline. During the second week the chicks were all fed on the same diet, previously given, except that 0.05 percent choline chloride was included in order to prevent excessive perosis.
ual weights were taken at biweekly intervals, the weighing operation being done at about the same time of day. Feedgain ratios were reported only for the 4-6 week periods unless otherwise indicated. The 2-6 week gains were analyzed statistically by the analysis of variance. The methionine and cystine content of each protein ingredient for each trial was determined by microbiological assay. The composition of the diet with respect to the other amino acids essential for the
882
J. W. WEST, C. W. CARRICK, S. M. HATJGE AND E. T. MERTZ TABLE 2.-—Composition
of basal diets
The basal ration (see Table 2) contained 0.4 percent methionine and 0.11 percent Trial 3 Ingredients Trial 1 Trial 2 cystine; cystine was held constant at that Percent Percent Percent Casein 12.0 6.0 level in this trial. The variable ingredients Gelatin 10.0 10.0 14.0 Blood fibrin 2.0 2.0 4.0 in the diets and the results are shown in DL-tryptophan 0.05 0.05 Choline chloride 0.1 0.1 Table 3. This trial was a factorial experiLederle A P F 0.75 0.75 0.75 3,000-A, 400-D oil 0.5 0.5 0.5 ment, with six levels of methionine within Vitamin mixture* 0.1 0.1 0.1 Ruff ex 5.0 5.0 5.0 each of the two choline levels. Except at 6.0 6.0 6.0 Mineral mixturet Cerelose 62.4-63.5 51.55-63.5 6 2 . 9 - 6 3 . 2 the basal level of 0.4 percent methionine, * Vitamin mixture (mg. per 100 gm. diet): Biotin 0.02, the growth and feed efficiency data indicalcium pantothenate 2, folic acid 0.15, inositol 100, menadione 0.1, niacin 5, pyridoxine 0.6, riboflavin 0.8, thiamine 0.4 and cated no sparing action due to choline, tocopherol (alpha) 0.5. within the limits of choline provided. For t Mineral mixture (gm. per 100 gm. diet): CaCOs 1.5, Ca.(PO.)» 1.3, K J H P O . 0.85, NaCl-iodized 0.87. NaJHPO* 0.73, the higher levels of methionine, no sigMgSO - 7H O 0.5, Fe(CsH>C<7)r 6H 0 0.14, MnSOr 4H 0 0.105, ZnSOj 0.002, CuSO • 5HjO 0.002, HiBO. 0.0009, and CoCl • 6HsO nificant differences in growth rate were 0.0001. where the choline and cystine levels were observed between 0.1 and 0.2 percent chovaried, respectively, fat determinations line. Maximum growth was obtained, were made of the dried liver composite with both choline levels, at 0.91 percent for each lot to study the relationship of methionine plus cystine (Lots 5 and 6). choline and cystine to lipotropism in With choline levels less than 0.1 percent, chickens. Chemical determinations for fat evidence of its sparing action might have and choline were made on dried liver sam- been obtainable, except that lower choline ples from certain of the lots by Engel's levels would cause such a high incidence (1942) method and the results are re- of perosis that growth data would not be reliable. Seven cases of perosis were noted corded in Tables 3, 4 and 5. in this trial, four of which occurred with RESULTS AND DISCUSSION the 0.2 percent choline level. On the basis Trial 1, which began in January 1950, of these results, it was concluded that 0.1 was designed to study the relationship of percent choline may be adequate for maxicholine to the methionine requirement. mum early growth. (
s
5
8
t
Variable ingredients (percent)
Lot 1
Lot 2
Lot 3
Lot 4
Lot 5
DL-methionine Choline chloride
0.1
0.2
0.2 0.1
0.2 0.2
0.4 0.1
0.4 0.2
0.6 0.1
0.6 0.2
0.8 0.1
0.8 0.2
1.0 0.1
1.0 0.2
Total protein Total methionine Total cystine Meth.-fcyst.
19.5 0.40 0.11 0.51
19.5 0.40 0.11 0.51
19.7 0.60 0.11 0.71
19.7 0.60 0.11 0.71
19.9 0.80 0.11 0.91
19.9 0.80 0.11 0.91
20.1 1.00 0.11 1.11
20.1 1.00 0.11 1.11
20.3 1.20 0.11 1.31
20.3 1.20 0.11 1.31
20.5 1.40 0.11 1.51
20.5 1.40 0.11 1.51
Av. gram gains* (2-5 weeks)1—-22 males , 22 females per lot: ] 336 390 Males 327 372 374 337 279 Females 252 324 326 363 308 Sexes combined 290 348 350
405 349 372
390 344 367
395 331 363
397 350 373
396 327 361
371 327 349
375 330 352
2.19 18.1 8.6
2.18 19.2 8.2
2.29 20.7 8.9
2.11
2.18
2.15
2.38
Feed-gain ratio 2 Liver fat (%) Liver choline 3
2.64 17.5 9.1
2.45 15.9 8.6
2.29 20.6 8.8
2.37 18.0 8.6
2.17 19.4 7.6
Lot 6
Lot 7
* Two-week weights (gms.): Males 127, females 116, both sexes 122. Approximate least significant differences:
1
2-5 week gainsi Males Females Sexes combined 2 Sexes combined (4-5 weeks). 3 Mg. per gm. of dried liver.
24 grams 23 grams 16 grams
1% 1<™>
31 grams 30 grams 21 grams
Lot 8
Lot 9
Lot 10
Lot 11
Lot 12
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TABLE 3.—Relationship of choline to methionine requirement (Trial 1)
883
METHIONINK REQUIREMENTS OF CHICKS
TABLE 4.—Comparative utilization of D7 and L-methionine (Trial 2) Lot 13
Lot 14
Lot 15
Lot 16
Lot 17
Lot 18
Lot 19
Lot 20
Lot 21
Casein DL-methionine i^cystine DL-tryptophan Choline chloride
12.0
16.0
20.0
24.0
0.05 0.1
0.03 0.1
0.1
12.0 0.1 0.01 0.05 0.1
12.0 0.2 0.02 0.05 0.1
12.0 0.3 0.04 0.05 0.1
12.0 0.4 0.02 0.05 0.1
12.0 0.6 0.04 0.05 0.1
Protein (percent) Methionine (percent) Cystine (percent) M e t h . + c y s t . (percent)
20.4 0.40 0.11 0.51
23.8 0.49 0.12 0.61
27.1 0.58 0.14 0.72
30.5 0.67 0.15 0.82
20.5 0.50 0.13 0.63
20.6 .60 0.14 0.74
20.8 .70 0.15 0.85
20.9 .80 0.14 0.94
21.1 1.00 0.15 1.15
Av. gram gains* (2-6 wks.) L -22 males, 21 females per lot: Males 447 516 552 Females 360 416 435 Sexes combined 403 466 494
520 457 488
496 413 454
555 450 503
526 442 484
548 468 508
545 459 502
2.50 12.0
2.49 12.9
2.34 12.1
2.40 13.0
2.35 12.0
2.25 11.3
Variable ingredients (percent)
Feed-gain ratio 2 Liver choline 3
2.69 12.5
2.56 12.0
2.31 12.5
* Two-week weights (grams): Males 124, females 116, both sexes 120. Approximate least significant differences: 5 % level 1% level 2-6 week gains Males 34 grams 45 grams Females 26 grams 34 grams Sexes combined 21 grams 28 grams 2 Sexes combined (4-6 weeks). 8 M g . per gm. of dried liver. 1
when compared within a given level of methionine. Although the differences were small, they indicated a possible lipotropic function of choline. Trial 2 began in February, 1950. The same basal diet was used as in Trial 1. Casein was varied to supply additional quantities of L-methionine. The variable ingredients in the rations and results are shown in Table 4. To evaluate the results obtained in Trial 1, it was necessary to determine whether both isomers of methi-
T A B L E 5.—Relationship of cystine to methionine requirement (Trial 3) Variable ingredients (percent) Dl^methionine L-cystine Protein (%) Methionine (%) Cystine (%) Total S (%)
Lot 22
Lot
0.3 20.8 0.28 0.41 0.69
Lot 24
Lot 25
Lot 26
Lot 27
Lot 28
Lot 29
Lot 30
Lot 31
Lot 32
Lot 33
0.5
0.6
0.2 0.1
0.2 0.2
0.2 0.3
0.2 0.4
0.3
0.4
0.3 0.1
0.3 0.2
0.3 0.3
20.9 0.28 0.51 0.79
21.0 0.28 0.61 0.89
21.1 0.28 0.71 0.99
20.8 0.48 0.21 0.69
20.9 0.48 0.31 0.79
21.0 0.48 0.41 0.89
21.1 0.48 0.51 0.99
20.8 0.58 0.11 0.69
20.9 0.58 0.21 0.79
21.0 0.58 0.31 0.89
21.1 0.58 0.41 0.99
505 425 465
526 436 481
510 443 476
536 432 484
510 426 468
515 450 482
517 453 485
523 446 484
2.50 15.7 9.5
2.54 17.6 -9.2
2.53 17.3 9.6
2.55 17.4 11.3
2.44 19.6 9.5
2.43 16.7 9.5
2.49 17.9 10.2
2.51 18.1 10.4
23
Av. gram gains* (2-6 wks.) 1 —•18 Males 470 Females 383 Sexes combined 426 Feed-gain ratio 2 Liver fat (%) Liver choline 3
2.68 15.4 8.2
males, 22 females 527 • 488 428 430 458 479
2.55 16.6 8.1
2.48 14.8 9.0
per lot: 499 432 465
2.61 18.6 8.8
* Two-week weights (grams): Males 136, females 129, both sexes 132. Approximate least significant differences: 5% level 1% level 2-6 week gains Males 44 grams 34 grams Females 32 grams 24 grams Sexes combined 27 grams - 20 grams 2 Sexes combined (4-6 weeks). a M g . per gm. of dried liver. 1
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At six weeks of age livers were removed from two male and two female chicks of average weight from Lots 1-8. These were cooked in an autoclave, vacuum dried, ground and composited for choline and fat determinations. These results are also shown in Table 3. No consistent relationship was established between dietary level of choline and liver storage of choline. Percent liver fat (ether-extractable material) seemed to bear an inverse relationship to the dietary choline level,
884
J. W. WEST, C. W. CARRICK, S. M. HAUGE AND E. T. MERTZ
Disregarding Lots 14-16, the remainder of the experiment served as a check on the total sulfur amino acid requirement. The data indicated that the optimal level for sulfur amino acids was 0.74 percent of the diet. Liver samples were analyzed for choline but the data obtained were not conclusive (see Table 4). Trial 3, which began in March, 1950, was designed to study the relationship of cystine to the methionine requirement. The variables in the rations and results are shown in Table 5. The basal diet (see Table 2) contained 0.28 percent methionine and 0.11 percent cystine. Additions of synthetic DL-methionine and L-cystine were made, as shown in Table 5, to provide three methionine-cystine combinations at each of four levels of total sulfur amino acids: 0.69, 0.79, 0.89 and 0.99 percent. With this design it was possible to check again the total sulfur amino acid requirement and to determine the minimum requirement for methionine. Optimum growth and feed efficiency
resulted both at the 0.48 and 0.58 percent methionine levels when the total sulfur amino acid level was 0.79 percent of the diet (Lots 27 and 31). Comparable growth and feed efficiency also resulted at the 0.28 percent methionine level when the total sulfur amino acid level was 0.89 percent of the diet (Lot 24). When the gains and feed efficiency obtained with 0.28 percent methionine and 0.41 percent cystine (Lot 22) were compared with their counterparts (Lots 26 and 30) ^ it was apparent that cystine can replace some of the methionine, but not all of it. It was concluded from these data that the growth requirement of the chicken can be met with 0.28 percent methionine and 0.61 percent cystine or with approximately 0.5 percent methionine and 0.3 percent cystine when the ration contains approximately 20 percent total protein. The latter level is in agreement with the results of Grau and Kamei(1950). When the livers of these chickens were analyzed for fat (ether-extractable material), no relationship was established between dietary level of cystine and fat storage in the liver. Results of the choline content of the livers were inconclusive. The results of these analyses are also shown in Table 5. SUMMARY , 1. With the diets used, levels of 0.1 and 0.2 percent choline failed to show any relationship to the methionine requirement. 2. The choline requirement for rapid early growth was adequately met with a dietary level of 0.1 percent when ample methionine was present. 3. With a limited supply of cystine (0.3 percent), the total sulfur amino acid requirement could be met with 0.5 percent of methionine. 4. Cystine could supply approximately two-thirds of the total sulfur amino acid
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onine were utilizable, since DL-methionine was used to arrive at the methionine plus cystine requirement. Increments of DLmethionine were added to the basal diet (Lot 13) to correspond to increased levels of the natural (L) form, which were provided by increment increases of casein to the basal diet (see Lots 14-16). Growth responses in Lots 17, 18 and 19 were expected to be comparable to those in Lots 14,15 and 16, respectively, if both isomers were utilizable. Lots 18, 20 and 21 were expected to compare with Lots 14, 15 and 16, respectively, if only the L-form were available. The data clearly indicated that both isomeric forms are utilizable by the growing chick. Minor adjustments using L-cystine were made in Lots 17-21 to compensate for additional cystine contributed by casein to Lots 14-16.
ASSOCIATION NOTES
885
cedures for the determination of amino acids by microbiological methods as employed in the chemistry laboratory. University of California, Los Angeles. Mimeograph. du Vigneaud, V., G. W. Kilmer, J. R. Rachele and M. Cohn, 1944. On the mechanism of the conversion in vivo of methionine to cystine. J. Biol. Chem. 155: 645-651. Engel, R. W., 1942. Modified methods for the chemical and biological determination of choline. J. Biol. Chem. 144: 701-710. Gerry, R. W., and D. H. Mishler, 1949. Sexing Barred Rock chicks. Poultry Sci. 28:479-485. Grau, C. R., and H. J. Almquist, 1943. The utilization of the sulfur amino acids by the chick. J. Nutrition 26: 631-640. ACKNOWLEDGMENT Grau, C. R., and M. Kamei, 1950. Amino acid imWe are indebted to E . I. du P o n t de balance and the growth requirements for lysine Nemours a n d Company for DL-tryptoand methionine. J. Nutrition 41: 89-101. phan, to Dow Chemical Company for Klose, A. A., and H. J. Almquist, 1941. Methionine DL-tryptophan and DL-methionine, to in the diet of the chick. J. Biol. Chem. 138: 467469. U.S. Industrial Chemicals for DL-methionine, to Armour and Company and Wil- McKittrick, D. S., 1947. The interrelations of choline and methionine in growth and the action of son a n d Company for gelatin, to T h e betaine in replacing them. Arch. Biochem. 15: Borden Company for casein and gelatin, 133-155. a n d to Lederle Laboratories for the biotin, Simmonds, S., M. Cohn, J. P. Chandler and V. du Vigneaud, 1943. The utilization of the methyl folic acid a n d A P F supplements used in groups of choline in the biological synthesis of these and in preliminary experiments. methionine. J. Biol. Chem. 149: 519-525. REFERENCES Stokes, J. L., M. Gunness, I. M. Dwyer and M. C. Casewell, 1945. Microbiological methods for the Almquist, H. J., and D. R. Grau, 1945. Further determination of amino acids. II. A uniform studies on cystine, methionine and choline in assay for the ten essential amino acids. J. Biol. chick diets. J. Nutrition 29: 219-222. Chem. 160: 35-49. Almquist, H. J., 1947. Editorial review. Evaluation of amino acid requirements by observations on Treadwell, C. R., 1948. Growth and lipotropism. II. The effects of dietary methionine, cystine, and the chick. J. Nutrition 34: 543-563. choline in the young white rat. J. Biol. Chem. Dunn, M. S., with collaboration of M. W. Camien, 176: 1141-1147. S. Shankland and L. B. Rockland, 1945. Prorequirement, but 0.28 percent of the methionine in the diet was irreplaceable. 5. Both optical isomers of methionine were utilizable. 6. Fat analyses of liver samples sug^gested an inverse relationship of dietary choline level to the amount of fat storage in the liver. 7. Choline content of the liver samples indicated no relationship of dietary level of choline, cystine or methionine to liver storage of choline.
{Continued from page 879) team and won the Chicago contest. He has served as major advisor for students in poultry husbandry and has cooperated extensively with other departments and divisions in training graduate students.
Professor Kempster was elected a Fellow of the Poultry Science Association in 1938 and was President of the Association in 1941. He is a member of Alpha Zeta, Gamma Sigma Delta, and Sigma Xi.
{Continued on page 892)
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Association Notes
Departments of Poultry Husbandry and Agricultural Chemistry, Purdue University Agricultural Experiment Station, Lafayette, Indiana (Received for publication February 28, 1951)
HE essential nature of methionine in the diet of the chick was demonstrated by Klose and Almquist (1941). Homocystine and choline together effectively replaced methionine in the diet of the chick, but neither homocystine nor cystine completely replaced methionine. Direct proof of the homocystine-cholinemethionine interrelationship in the rat was presented by Simmonds et al. (1943). Transmethylation was observed both in the absence of homocystine and in the presence of ordinary methionine when the diet contained deuterio-choline. Du Vigneaud et al. (1944) reported results which demonstrated the role of methionine in cystine formation. Grau and Almquist (1943) investigated the relationship between the sulfur amino acids and choline in chick nutrition. Choline-depleted chicks were able to utilize homocystine effectively in the place of cystine but not as a substitute for methionine. Almquist and Grau (1945), using a diet deficient in methionine and cystine, fed increasing levels of cystine up to 0.9 percent and various levels of choline up to 0.6 percent. In all cases growth was sub-optimal. Replacement of part of 1 Journal Paper No. 487 of the Purdue University Agricultural Experiment Station. 2 This research was supported in part by a grant from The Borden Company.
the cystine with methionine so that the ration contained 0.55 percent methionine resulted in optimal growth. It was concluded that cystine could not compensate for a methionine deficiency, even in the presence of high choline levels. Almquist (1947) stated that the growing chicken requires 0.9 percent sulfur amino acids, 0.5 percent of which must be methionine and 0.4 percent of which may be provided by cystine or methionine. Grau and Kamei (1950) concluded that when the diet contains approximately 20 percent total protein the sulfur amino acid requirement appears to be met with approximately 0.5 percent methionine and 0.3 percent cystine or with 0.8 percent methionine alone, McKittrick (1947) observed that the methionine and choline requirements for chick growth consist of essential and replaceable parts. For optimal growth it was found that 0.5 percent methionine and 0.1 percent choline constitute the essential'parts, and that 0.25 percent methionine or 0.45 percent choline or any equivalent mixture may comprise the replaceable parts. Grau and Almquist (1943) reported that DL-methionine is equivalent to Lmethionine for growth, whether in the presence or absence of choline in the diet of the chick. Treadwell (1948) observed poor growth, fatty livers and hemorrhagic kidneys when young rats were fed a
880
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T
881
METHIONINE REQUIREMENTS OF CHICKS
choline-free diet containing 18.6 percent protein, 0.5 percent methionine and 0.1 percent cystine. Increased cystine levels aggravated the fatty liver condition, but 0.2 percent choline produced good growth and a normal fat content of the liver. So far as we are aware similar studies with chicks have not been reported. EXPERIMENTAL
After the two-week depletion period, the chicks were weighed individually and sorted into blocks on the bases of weight and sex and distributed at random into experimental pens. The chicks were all reared in battery brooders with wire floors. Purified rations and tap water were supplied ad libitum during the twoto-six week experimental period. Individ-
TABLE 1.—-Methionine and cystine content of the protein ingredients
2 S S
T ri al(s)
Casein 1, 2 and 3 Gelatin 1 and 2 Gelatin 3 Blood fibrin 1 and 2 Blood fibrin 3 Lederle APF 1, 2 and 3
£ | £ r
5 - a t
2.283 0.832 0.S31 1.863 1.600 0.300
0.382 0.113 0.095 2.430 1.910 0.280
* Expressed as percent of air-dried material. In each trial each protein ingredient was thoroughly mixed and used throughout the trial.
chick was ascertained by the use of published literature values, and appropriate corrections made where necessary. Methionine was assayed by the method of Stokes et al. (1945) using Streptococcus fecalis R, and cystine by Dunn's (1945) procedure using Leuconosloc mesenteriodes P-60. The methionine and cystine content of the protein ingredients used in each of the three trials is given in Table 1. The composition of the basal diets used in the various trials is shown in Table 2. Variable ingredients are listed in the individual tables for the various trials. All diets, with few exceptions, contained approximately 20 percent protein. Liver samples were taken for choline determinations when the trials were terminated at six weeks, to study the effect of dietary levels of choline oh liver storage of that vitamin. In Trials 1 and 3
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Barred Plymouth Rock chicks were used in each of the trials. All chicks were sexed at one day of age by the method of Gerry and Mishler (1949), wingbanded and placed on the same diet for the firsttwo weeks. In order to deplete the body stores of choline, the chicks were fed a diet deficient in choline for the first week. The percentage composition of the depletion diet was ground yellow corn 59.45, casein 12, corn gluten meal 10, peanut meal 5, dried skimmilk 5, alfalfa leaf meal 3, fish solubles (dry basis) 1.5, steamed bone meal 2, ground limestone 1, glycine 0.5, iodized salt 0.45, manganese sulfate 0.05, and irradiated animal sterol product 0.05. Three hundred micrograms of riboflavin were added per 100 grams of diet. The choline content of this diet was determined by a slight modification of Engel's (1942) method, and was found to be less than 0.05 percent choline. During the second week the chicks were all fed on the same diet, previously given, except that 0.05 percent choline chloride was included in order to prevent excessive perosis.
ual weights were taken at biweekly intervals, the weighing operation being done at about the same time of day. Feedgain ratios were reported only for the 4-6 week periods unless otherwise indicated. The 2-6 week gains were analyzed statistically by the analysis of variance. The methionine and cystine content of each protein ingredient for each trial was determined by microbiological assay. The composition of the diet with respect to the other amino acids essential for the
882
J. W. WEST, C. W. CARRICK, S. M. HATJGE AND E. T. MERTZ TABLE 2.-—Composition
of basal diets
The basal ration (see Table 2) contained 0.4 percent methionine and 0.11 percent Trial 3 Ingredients Trial 1 Trial 2 cystine; cystine was held constant at that Percent Percent Percent Casein 12.0 6.0 level in this trial. The variable ingredients Gelatin 10.0 10.0 14.0 Blood fibrin 2.0 2.0 4.0 in the diets and the results are shown in DL-tryptophan 0.05 0.05 Choline chloride 0.1 0.1 Table 3. This trial was a factorial experiLederle A P F 0.75 0.75 0.75 3,000-A, 400-D oil 0.5 0.5 0.5 ment, with six levels of methionine within Vitamin mixture* 0.1 0.1 0.1 Ruff ex 5.0 5.0 5.0 each of the two choline levels. Except at 6.0 6.0 6.0 Mineral mixturet Cerelose 62.4-63.5 51.55-63.5 6 2 . 9 - 6 3 . 2 the basal level of 0.4 percent methionine, * Vitamin mixture (mg. per 100 gm. diet): Biotin 0.02, the growth and feed efficiency data indicalcium pantothenate 2, folic acid 0.15, inositol 100, menadione 0.1, niacin 5, pyridoxine 0.6, riboflavin 0.8, thiamine 0.4 and cated no sparing action due to choline, tocopherol (alpha) 0.5. within the limits of choline provided. For t Mineral mixture (gm. per 100 gm. diet): CaCOs 1.5, Ca.(PO.)» 1.3, K J H P O . 0.85, NaCl-iodized 0.87. NaJHPO* 0.73, the higher levels of methionine, no sigMgSO - 7H O 0.5, Fe(CsH>C<7)r 6H 0 0.14, MnSOr 4H 0 0.105, ZnSOj 0.002, CuSO • 5HjO 0.002, HiBO. 0.0009, and CoCl • 6HsO nificant differences in growth rate were 0.0001. where the choline and cystine levels were observed between 0.1 and 0.2 percent chovaried, respectively, fat determinations line. Maximum growth was obtained, were made of the dried liver composite with both choline levels, at 0.91 percent for each lot to study the relationship of methionine plus cystine (Lots 5 and 6). choline and cystine to lipotropism in With choline levels less than 0.1 percent, chickens. Chemical determinations for fat evidence of its sparing action might have and choline were made on dried liver sam- been obtainable, except that lower choline ples from certain of the lots by Engel's levels would cause such a high incidence (1942) method and the results are re- of perosis that growth data would not be reliable. Seven cases of perosis were noted corded in Tables 3, 4 and 5. in this trial, four of which occurred with RESULTS AND DISCUSSION the 0.2 percent choline level. On the basis Trial 1, which began in January 1950, of these results, it was concluded that 0.1 was designed to study the relationship of percent choline may be adequate for maxicholine to the methionine requirement. mum early growth. (
s
5
8
t
Variable ingredients (percent)
Lot 1
Lot 2
Lot 3
Lot 4
Lot 5
DL-methionine Choline chloride
0.1
0.2
0.2 0.1
0.2 0.2
0.4 0.1
0.4 0.2
0.6 0.1
0.6 0.2
0.8 0.1
0.8 0.2
1.0 0.1
1.0 0.2
Total protein Total methionine Total cystine Meth.-fcyst.
19.5 0.40 0.11 0.51
19.5 0.40 0.11 0.51
19.7 0.60 0.11 0.71
19.7 0.60 0.11 0.71
19.9 0.80 0.11 0.91
19.9 0.80 0.11 0.91
20.1 1.00 0.11 1.11
20.1 1.00 0.11 1.11
20.3 1.20 0.11 1.31
20.3 1.20 0.11 1.31
20.5 1.40 0.11 1.51
20.5 1.40 0.11 1.51
Av. gram gains* (2-5 weeks)1—-22 males , 22 females per lot: ] 336 390 Males 327 372 374 337 279 Females 252 324 326 363 308 Sexes combined 290 348 350
405 349 372
390 344 367
395 331 363
397 350 373
396 327 361
371 327 349
375 330 352
2.19 18.1 8.6
2.18 19.2 8.2
2.29 20.7 8.9
2.11
2.18
2.15
2.38
Feed-gain ratio 2 Liver fat (%) Liver choline 3
2.64 17.5 9.1
2.45 15.9 8.6
2.29 20.6 8.8
2.37 18.0 8.6
2.17 19.4 7.6
Lot 6
Lot 7
* Two-week weights (gms.): Males 127, females 116, both sexes 122. Approximate least significant differences:
1
2-5 week gainsi Males Females Sexes combined 2 Sexes combined (4-5 weeks). 3 Mg. per gm. of dried liver.
24 grams 23 grams 16 grams
1% 1<™>
31 grams 30 grams 21 grams
Lot 8
Lot 9
Lot 10
Lot 11
Lot 12
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TABLE 3.—Relationship of choline to methionine requirement (Trial 1)
883
METHIONINK REQUIREMENTS OF CHICKS
TABLE 4.—Comparative utilization of D7 and L-methionine (Trial 2) Lot 13
Lot 14
Lot 15
Lot 16
Lot 17
Lot 18
Lot 19
Lot 20
Lot 21
Casein DL-methionine i^cystine DL-tryptophan Choline chloride
12.0
16.0
20.0
24.0
0.05 0.1
0.03 0.1
0.1
12.0 0.1 0.01 0.05 0.1
12.0 0.2 0.02 0.05 0.1
12.0 0.3 0.04 0.05 0.1
12.0 0.4 0.02 0.05 0.1
12.0 0.6 0.04 0.05 0.1
Protein (percent) Methionine (percent) Cystine (percent) M e t h . + c y s t . (percent)
20.4 0.40 0.11 0.51
23.8 0.49 0.12 0.61
27.1 0.58 0.14 0.72
30.5 0.67 0.15 0.82
20.5 0.50 0.13 0.63
20.6 .60 0.14 0.74
20.8 .70 0.15 0.85
20.9 .80 0.14 0.94
21.1 1.00 0.15 1.15
Av. gram gains* (2-6 wks.) L -22 males, 21 females per lot: Males 447 516 552 Females 360 416 435 Sexes combined 403 466 494
520 457 488
496 413 454
555 450 503
526 442 484
548 468 508
545 459 502
2.50 12.0
2.49 12.9
2.34 12.1
2.40 13.0
2.35 12.0
2.25 11.3
Variable ingredients (percent)
Feed-gain ratio 2 Liver choline 3
2.69 12.5
2.56 12.0
2.31 12.5
* Two-week weights (grams): Males 124, females 116, both sexes 120. Approximate least significant differences: 5 % level 1% level 2-6 week gains Males 34 grams 45 grams Females 26 grams 34 grams Sexes combined 21 grams 28 grams 2 Sexes combined (4-6 weeks). 8 M g . per gm. of dried liver. 1
when compared within a given level of methionine. Although the differences were small, they indicated a possible lipotropic function of choline. Trial 2 began in February, 1950. The same basal diet was used as in Trial 1. Casein was varied to supply additional quantities of L-methionine. The variable ingredients in the rations and results are shown in Table 4. To evaluate the results obtained in Trial 1, it was necessary to determine whether both isomers of methi-
T A B L E 5.—Relationship of cystine to methionine requirement (Trial 3) Variable ingredients (percent) Dl^methionine L-cystine Protein (%) Methionine (%) Cystine (%) Total S (%)
Lot 22
Lot
0.3 20.8 0.28 0.41 0.69
Lot 24
Lot 25
Lot 26
Lot 27
Lot 28
Lot 29
Lot 30
Lot 31
Lot 32
Lot 33
0.5
0.6
0.2 0.1
0.2 0.2
0.2 0.3
0.2 0.4
0.3
0.4
0.3 0.1
0.3 0.2
0.3 0.3
20.9 0.28 0.51 0.79
21.0 0.28 0.61 0.89
21.1 0.28 0.71 0.99
20.8 0.48 0.21 0.69
20.9 0.48 0.31 0.79
21.0 0.48 0.41 0.89
21.1 0.48 0.51 0.99
20.8 0.58 0.11 0.69
20.9 0.58 0.21 0.79
21.0 0.58 0.31 0.89
21.1 0.58 0.41 0.99
505 425 465
526 436 481
510 443 476
536 432 484
510 426 468
515 450 482
517 453 485
523 446 484
2.50 15.7 9.5
2.54 17.6 -9.2
2.53 17.3 9.6
2.55 17.4 11.3
2.44 19.6 9.5
2.43 16.7 9.5
2.49 17.9 10.2
2.51 18.1 10.4
23
Av. gram gains* (2-6 wks.) 1 —•18 Males 470 Females 383 Sexes combined 426 Feed-gain ratio 2 Liver fat (%) Liver choline 3
2.68 15.4 8.2
males, 22 females 527 • 488 428 430 458 479
2.55 16.6 8.1
2.48 14.8 9.0
per lot: 499 432 465
2.61 18.6 8.8
* Two-week weights (grams): Males 136, females 129, both sexes 132. Approximate least significant differences: 5% level 1% level 2-6 week gains Males 44 grams 34 grams Females 32 grams 24 grams Sexes combined 27 grams - 20 grams 2 Sexes combined (4-6 weeks). a M g . per gm. of dried liver. 1
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At six weeks of age livers were removed from two male and two female chicks of average weight from Lots 1-8. These were cooked in an autoclave, vacuum dried, ground and composited for choline and fat determinations. These results are also shown in Table 3. No consistent relationship was established between dietary level of choline and liver storage of choline. Percent liver fat (ether-extractable material) seemed to bear an inverse relationship to the dietary choline level,
884
J. W. WEST, C. W. CARRICK, S. M. HAUGE AND E. T. MERTZ
Disregarding Lots 14-16, the remainder of the experiment served as a check on the total sulfur amino acid requirement. The data indicated that the optimal level for sulfur amino acids was 0.74 percent of the diet. Liver samples were analyzed for choline but the data obtained were not conclusive (see Table 4). Trial 3, which began in March, 1950, was designed to study the relationship of cystine to the methionine requirement. The variables in the rations and results are shown in Table 5. The basal diet (see Table 2) contained 0.28 percent methionine and 0.11 percent cystine. Additions of synthetic DL-methionine and L-cystine were made, as shown in Table 5, to provide three methionine-cystine combinations at each of four levels of total sulfur amino acids: 0.69, 0.79, 0.89 and 0.99 percent. With this design it was possible to check again the total sulfur amino acid requirement and to determine the minimum requirement for methionine. Optimum growth and feed efficiency
resulted both at the 0.48 and 0.58 percent methionine levels when the total sulfur amino acid level was 0.79 percent of the diet (Lots 27 and 31). Comparable growth and feed efficiency also resulted at the 0.28 percent methionine level when the total sulfur amino acid level was 0.89 percent of the diet (Lot 24). When the gains and feed efficiency obtained with 0.28 percent methionine and 0.41 percent cystine (Lot 22) were compared with their counterparts (Lots 26 and 30) ^ it was apparent that cystine can replace some of the methionine, but not all of it. It was concluded from these data that the growth requirement of the chicken can be met with 0.28 percent methionine and 0.61 percent cystine or with approximately 0.5 percent methionine and 0.3 percent cystine when the ration contains approximately 20 percent total protein. The latter level is in agreement with the results of Grau and Kamei(1950). When the livers of these chickens were analyzed for fat (ether-extractable material), no relationship was established between dietary level of cystine and fat storage in the liver. Results of the choline content of the livers were inconclusive. The results of these analyses are also shown in Table 5. SUMMARY , 1. With the diets used, levels of 0.1 and 0.2 percent choline failed to show any relationship to the methionine requirement. 2. The choline requirement for rapid early growth was adequately met with a dietary level of 0.1 percent when ample methionine was present. 3. With a limited supply of cystine (0.3 percent), the total sulfur amino acid requirement could be met with 0.5 percent of methionine. 4. Cystine could supply approximately two-thirds of the total sulfur amino acid
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onine were utilizable, since DL-methionine was used to arrive at the methionine plus cystine requirement. Increments of DLmethionine were added to the basal diet (Lot 13) to correspond to increased levels of the natural (L) form, which were provided by increment increases of casein to the basal diet (see Lots 14-16). Growth responses in Lots 17, 18 and 19 were expected to be comparable to those in Lots 14,15 and 16, respectively, if both isomers were utilizable. Lots 18, 20 and 21 were expected to compare with Lots 14, 15 and 16, respectively, if only the L-form were available. The data clearly indicated that both isomeric forms are utilizable by the growing chick. Minor adjustments using L-cystine were made in Lots 17-21 to compensate for additional cystine contributed by casein to Lots 14-16.
ASSOCIATION NOTES
885
cedures for the determination of amino acids by microbiological methods as employed in the chemistry laboratory. University of California, Los Angeles. Mimeograph. du Vigneaud, V., G. W. Kilmer, J. R. Rachele and M. Cohn, 1944. On the mechanism of the conversion in vivo of methionine to cystine. J. Biol. Chem. 155: 645-651. Engel, R. W., 1942. Modified methods for the chemical and biological determination of choline. J. Biol. Chem. 144: 701-710. Gerry, R. W., and D. H. Mishler, 1949. Sexing Barred Rock chicks. Poultry Sci. 28:479-485. Grau, C. R., and H. J. Almquist, 1943. The utilization of the sulfur amino acids by the chick. J. Nutrition 26: 631-640. ACKNOWLEDGMENT Grau, C. R., and M. Kamei, 1950. Amino acid imWe are indebted to E . I. du P o n t de balance and the growth requirements for lysine Nemours a n d Company for DL-tryptoand methionine. J. Nutrition 41: 89-101. phan, to Dow Chemical Company for Klose, A. A., and H. J. Almquist, 1941. Methionine DL-tryptophan and DL-methionine, to in the diet of the chick. J. Biol. Chem. 138: 467469. U.S. Industrial Chemicals for DL-methionine, to Armour and Company and Wil- McKittrick, D. S., 1947. The interrelations of choline and methionine in growth and the action of son a n d Company for gelatin, to T h e betaine in replacing them. Arch. Biochem. 15: Borden Company for casein and gelatin, 133-155. a n d to Lederle Laboratories for the biotin, Simmonds, S., M. Cohn, J. P. Chandler and V. du Vigneaud, 1943. The utilization of the methyl folic acid a n d A P F supplements used in groups of choline in the biological synthesis of these and in preliminary experiments. methionine. J. Biol. Chem. 149: 519-525. REFERENCES Stokes, J. L., M. Gunness, I. M. Dwyer and M. C. Casewell, 1945. Microbiological methods for the Almquist, H. J., and D. R. Grau, 1945. Further determination of amino acids. II. A uniform studies on cystine, methionine and choline in assay for the ten essential amino acids. J. Biol. chick diets. J. Nutrition 29: 219-222. Chem. 160: 35-49. Almquist, H. J., 1947. Editorial review. Evaluation of amino acid requirements by observations on Treadwell, C. R., 1948. Growth and lipotropism. II. The effects of dietary methionine, cystine, and the chick. J. Nutrition 34: 543-563. choline in the young white rat. J. Biol. Chem. Dunn, M. S., with collaboration of M. W. Camien, 176: 1141-1147. S. Shankland and L. B. Rockland, 1945. Prorequirement, but 0.28 percent of the methionine in the diet was irreplaceable. 5. Both optical isomers of methionine were utilizable. 6. Fat analyses of liver samples sug^gested an inverse relationship of dietary choline level to the amount of fat storage in the liver. 7. Choline content of the liver samples indicated no relationship of dietary level of choline, cystine or methionine to liver storage of choline.
{Continued from page 879) team and won the Chicago contest. He has served as major advisor for students in poultry husbandry and has cooperated extensively with other departments and divisions in training graduate students.
Professor Kempster was elected a Fellow of the Poultry Science Association in 1938 and was President of the Association in 1941. He is a member of Alpha Zeta, Gamma Sigma Delta, and Sigma Xi.
{Continued on page 892)
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Association Notes