Stimulation of Chick Growth by Proline*

Stimulation of Chick Growth by Proline*

192 C. F. SMITH made from fresh and frozen egg whites. Cereal Chem. 20: 528-534. Mueller, W. J., 1958. Shell porosity of chicken eggs 1. CO2 loss an...

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made from fresh and frozen egg whites. Cereal Chem. 20: 528-534. Mueller, W. J., 1958. Shell porosity of chicken eggs 1. CO2 loss and CO2 content of infertile eggs during storage. Poultry Sci. 37: 437-444. Pearce, J. A., and C. G. Lavers, 1949. Liquid and frozen eggs. V. Viscosity, baking quality, and other measurements on frozen egg products. Can. J. Research, F27: 231-240. Rahn, O., 1932. Why cream or egg white whips? Food Ind. 4: 300-301.

Stimulation of Chick Growth by Proline* D. N. ROY AND H. R. BIRD Department of Poultry Husbandry, University of Wisconsin, Madison, Wisconsin (Received for publication Tune 26, 1958)


HESE experiments began as an attempt to attenuate the toxic effect of beta aminopropionitrile fumarate (BAPN —fumarate) by supplementation of the diet with different amino acids and other related compounds at various levels. Barnett et al. (1957) first described the toxic effect upon turkey poults of feeding beta aminopropionitrile hydrochloride (BAPN-HC1). The compound caused paralysis, degeneration of anterior motor neurons and growth depression when fed at levels of 0.12 percent to 0.25 percent and pericardial and pulmonary hemorrhage, ruptured aortas, leg and toe deformities and growth depression at lower levels. In these experiments on chicks, it was noted that BAPN fumarate (0.036 percent) caused marked mal-symptoms from

Published with the approval of the Director of the Wisconsin Agricultural Experiment Station, College of Agriculture, Madison, Wisconsin. * Supported in part by the Research Committee of the Graduate School from funds supplied by the Wisconsin Alumni Research Foundation, Madison, Wisconsin.

the early part of the first week. There was no attenuation of the toxic effects, but growth was improved by some amino acid supplements. This improvement apparently was not dependent on the presence of BAPN fumarate in the diet. EXPERIMENTAL

Day old straight-run, crossbred (New Hampshire male X Single Comb White Leghorn female) chicks were used for all the experiments. Each group contained ten chicks. They were housed in electrically heated wire floored batteries. The experimental diet and water were given ad libitum from the first day after hatching. The basal diets are described in Table 1. The high protein diet, Basal 1, was used only in experiment 2. The birds were weighed at weekly intervals and the severity of symptoms was recorded at each weighing. Birds that died during these experiments were examined grossly for internal hemorrhage and aortic rupture. At the conclusion of each experiment all the surviving chicks were killed

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Slosberg, H. M., H. L. Hanson, G. F. Stewart and B. Lowe, 1948. Factors influencing the effects

of heat treatment on the leavening power of egg white. Poultry Sci. 27: 294-301. St. John, J. L., and I. H. Flor, 1931. A study of whipping and coagulations of eggs of varying quality. Poultry Sci. 10: 71-82. Smith, M., 1931. The relations between yolk and white in the hen's egg III. Gas exchange in infertile eggs. J. Exp. Biol. 8: 312-318. Smith, M., and J. Shepherd, 1931. The relations between yolk and white in the hen's egg I I . Osmotic equilibration. J. Exp. Biol. 8: 298-311. Yushok, W. D., and A. L. Romanoff, 1949. Studies on preservation of shell eggs with plastic. Food Res. 14: 113-122.


STIMULATION or CHICK GROWTH BY PROLINE TABLE 1.—Basal diets 2 (gms./kg.) (gms./kg.)


480 50 50 200 50 50 50 5 10 5 0.9 0.4 0.2 0.25 0.5 50

1 ,005.98 6 40 10

1,005.98 6 40 10

Riboflavin, mg./kg. Niacin, mg./kg. Calcium pantothenate mg./kg.

2 0.33 1.4

Basal 1 contains Protein:Ca:P=29.00:1.59:0.997 (calculated) Basal 2 contains Protein : Ca: P = 21.92:1. 24:0.78 (calculated)

and examined for gross internal changes. BAPN fumarate was used in experiments 2, 3 and 4 as 0.036 percent of the diet. In experiment 6, mono beta aminopropionitrile fumarate (mono—BAPN fumarate) was used; the level was 0.053 percent


The chicks receiving BAPN fumarate showed a high incidence of leg deformities, and some died with ruptured aortas. The effects of the compound were quite similar to those observed in turkeys. There were no significant differences in the skeletal changes, death rate and other abnormalities between the chicks in the groups on BAPN fumarate and the chicks on BAPN fumarate supplemented with amino acids and hence those results are not given here. One of the principal effects of BAPN in turkeys is growth inhibition, and Table 2 shows that this is

TABLE 2.—Average weights of chicks fed BAPN-fumarate

plus various amino acids

Ave. weights at the end of the exps.8 Diets Basal only1 Basal+BAPN-fumarate 2 Basal+BAPN—fumarate+ . 5 % L-arginine Basal+BAPN—fumarate+1% L-arginine Basal+BAPN—fumarate+2% L-arginine Basal+BAPN—fumarate+ . 5 % L-proline Basal+BAPN—fumarate+1% L-proline Basal+BAPN—fumarate+2% L-proline Basal+BAPN—fumarate+ . 8 % DL-threonine Basal+BAPN—fumarate+ . 3 % DL-tryptophan Basal+BAPN—fumarate+ . 5 % L-cystine Basal+BAPN—fumarate+ . 5 % L-tyrosine Basal+BAPN—fumarate-r-1% L-tyrosine Basal+BAPN—fumarate+2% L-tyrosine Basal+BAPN—fumarate+1% L-lysine Basal+BAPN—fumarate+2% DL-norvaline Basal+BAPN—fumarate+2% DL-pyroglutamic acid 1

Exp. 2 (gms.)

Exp. 3 (gms.)

Exp. 4 (gms.)

Exp. 6 (gms.)

589.9 278.8

494.3 386.3

391.5 261.2



573.2 359.8 394.4 392.4 387.9 448.1 433.0 505.3

302.2 461.9


238.0 282.6 284.4 398.4

437.7 415.7 365.5

388.6 213.0 242.5

Basal 1 was used in Experiment 2 and Basal 2 was used in Experiments 3, 4 and 6. Level of beta aminopropionitrile fumarate (BAPN—fumarate) in experiments 2, 3 and 4 was 0.036 percent. In experiment 6 mono beta aminopropionitrile fumarate was used (.053%) which is equivalent to .036% of beta aminopropionitrile fumarate. 3 Experiments 2, 3 and 4 were run for 6 weeks, experiment 6 for 5 weeks. 2

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Ground yellow corn 305 Oat groats 50 Alfalfa meal (17% protein) 50 Soybean oil meal (44% protein) 325 Fish meal (60% protein) 100 Meat and bone meal (50% protein) 50 Dried skim milk 50 Oyster shell (chick size) 5 Granite grit (chick size) 10 Salt 5 Vitamin D supplement (15001.C.U./gm.) 0.9 Vitamin A supplement (15,000 units/gm.) 0.4 DL methionine 0.2 Vitamin Bu supplement (6 mg./lb.) 0.25 Procaine Penicillin supplement (4 gm./lb.) 0.5 White grease 50 Solution A (alpha-tocopheryl acetate, 1% in soybean oil) 2 MnS04 • H2O 0.33 Choline chloride (70%) 1.4

which is equivalent to 0.036 percent BAPN fumarate. The crystalline BAPN salts were added directly to the diets. The plan of the experiments and the levels of amino acids fed are shown in Tables 2 and 3. Experiments 2, 3 and 4 were conducted for six weeks, while experiment 6 was terminated at the end of the fifth week.



TABLE 3.—Average weights of chicks fed basal diet 2 plus various amino acids Average weights a t the end of the experiments 2 Diets

Basal only 1 B a s a l + 1 % L-arginine B a s a l + 1 % L-proline B a s a l + 2 % L-proline B a s a l + 2 % DL-norvaline B a s a l + 2 % DL-pyroglutamic acid Basal4-2% L-glutamic acid 1

573.2 586.4 637.6

391.5 430.6 418.1 427.9 372.3 413.5

Basal 2 was used in experiments 4 and 6. Experiment 4 was run for 6 weeks, experiment 6 for 5 wee ks

also true of chickens. The effect of amino acid supplementation on growth is shown in Tables 2 and 3. Table 2 shows the average weights of chickens on BAPN fumarate and amino acid supplements. In this table the average weights of chicks on different supple-

Feed conversion =

Total weight of feed consumed in gms. per week ; ; Total gain in weight in gms. per week

ments of amino acids are compared with those on BAPN salts alone. In experiment 2, the groups on 1 percent L-arginine and 0.5 percent L-proline had much better average weights. In experiment 3, 1 percent L-proline gave best weight. Weights of chicks fed 1 percent L-arginine or 1 percent L-tyrosine were but little better than weights of chicks fed no amino acid supplement. In experiment 4, the average weights of chicks on L-proline showed the best results and here 2 percent gave maximum growth, while chicks on 0.5 percent and 1 percent L-proline came next. In this experiment a substantial growth response resulted from feeding 0.5 percent of either L-arginine or L-tyrosine. In experiment 6, 2 percent L-proline gave the best result. At 5 weeks of age, the difference between the mean weight of groups fed BAPN and that of groups fed BAPN plus proline was significant at the 1% level. In these experiments, threonine, trypto-

The proline supplements consistently improved feed conversion. Table 5 shows some of the levels of amino acids in the two basal diets used in the experiments. These figures are calculated ones. DISCUSSION Table 2 shows that the growth of chicks fed BAPN fumarate was consistently improved by proline. There were six groups fed proline at levels of 0.5,1 and 2 percent in 4 experiments and they all grew better TABLE 4.—Effect

of amino acid supplements on feed conversion


Basal 2 Basal 2 + 1 % L-arginine Basal 2 + 1% L-proline Basal 2 + 2 % L-proline Basal 2 + 2 % DL-norvaline Basal 2 + 2 % DL-pyroglutamic acid Basal 2 + 2 % L-glutamic acid

Experiment 4 Experiment 6 6 weeks 5 weeks (gms. feed/ (gms. feed/ gm. gain) gm. gain) 2.07 3.27 1.96

2.38 1.94 2.02 1.88 2.03 1.97

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Experiment 4 Experiment 6 (gms.) (gms.)

phan, cystine, lysine, norvaline, and pyroglutamic acid were ineffective. Table 3 shows the average weights of chicks fed supplements of some amino acids without any BAPN. In experiment 4, 1 percent L-proline gave a substantial growth response, but L-arginine did not. In experiment 6, 1 percent L-proline gave best growth although it was nearly equalled by 2 percent DL-norvaline. In experiment 4 the effect of 1% L-proline closely approached significance at the 5% level. The calculated value of / was 2.044, whereas the required value was 2.064. The differences in experiment 6 were not significant. Figures for feed conversion are given in Table 4. They were calculated on the basis of the following formula:

STIMULATION OF CHICK GROWTH BY PROLINE TABLE 5.—Calculated percentages of amino acids in the Basal 1 and Basal 2

Amino acids

Basal 1



1.740 0.652 .507 1.305 .290 1.015 1.305 1.45 0.21 0.87 2.03 1.16 29.000

Basal 2

% 1.232 0.486 0.304 1.161 0.234 0.815 1.035 2.0 0.48 0.91 1.77 1.14 21.900

Requirement: NRC figures 21.9 X 20


1.314 0.492 0.383 0.985 0.219 0.766 0.985 1.09 0.16 0.65 1.53 0.87 21.900

than their respective basal groups, the margins varying from 23 to 145 grams. At first it was thought that this was an indication that proline was counteracting the toxic effect of BAPN, even though the incidence of leg deformity and aortic rupture was not influenced by proline. However, when proline was fed to three lots of chicks receiving no BAPN, their growth exceeded that of their respective basal groups by margins of 26 to 64 grams. Although poultry nutritionists have unhesitatingly classified proline as a nonessential amino acid for years, examination of the literature reveals some uncertainty. Almquist and Grau (1944) fed a very small number of chicks a purified diet with and without proline. Those receiving proline showed an immediate gain in weight whereas those without showed a lag of several days before they began to grow at the same rate as the chicks fed proline. The authors classified proline as non-essential but stated that there might be conditions under which the chick did not synthesize it rapidly enough for maximum growth. In 1957, Almquist stated that "the chick apparently cannot synthesize glycine, glutamic acid, or proline sufficiently fast for the demands of early rapid growth" but again he classified proline as non-essential. Benton et al. (1955) observed that proline stimulated growth

of chicks slightly in one experiment when it was added to an amino acid diet. It was much less effective than gelatin. Gelatin was ineffective in our experiments. There is almost no information on proline content of feedstuffs. The level of this amino acid in connective tissue proteins is quite high, so the growing chick needs to acquire considerable quantities of it from some source. Table 5 gives calculated levels of some other amino acids in the two basal diets used and compares these figures with the requirement figures given by Bird et al. (1954) corrected for protein level. Values for amino acid content of feedstuffs were taken from the National Research Council (1956), Mitchell and Block (1946), and Sievert and Fairbanks (1957). Both diets were a little low in arginine, so it is not surprising that this amino acid improved growth in several instances. Methionine and cystine were also low according to calculation, but the cystine supplement tried in one experiment did not improve growth. Levels of lysine, tryptophan, phenylalanine and tyrosine should have been adequate and apparently were, except that 0.5 and 1.0 percent of L-tyrosine improved growth in experiment 4. The 2 percent level of tyrosine in this experiment and the 1 percent level in experiment 3 were not effective. SUMMARY Chicks were fed diets composed of grains, alfalfa meal, soybean oil meal, fish meal, meat and bone meal, dried skimmilk, white grease, DL-methionine, procaine penicillin, and mineral and vitamin supplements. Beta aminopropionitrile fumarate added to these diets at a level of 0.036 percent produced leg deformities and aortic rupture similar to the effects previously reported for BAPN-HC1 in turkey poults. L-proline at levels from 0.5 to 2.0 percent had no effect on the leg de-

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Arginine 1.702 Methionine 0.639 Cystine 0.386 Lysine 1.663 Tryptophan 0.323 Tyrosine 1.002 Phenylalanine 1.360 Glycine 3.08 Histidine 0.62 Isoleucine 1.24 Leucine 2.26 Valine 1.54 Protein 29.000

Requirement: NRC figures 29 X— 20




formities or aortic damage but did result in increased growth, either in the presence or absence of BAPN fumarate. Addition of L-arginine also improved growth. Effects of the other amino acids tried were negative or equivocal. REFERENCES

Benton, D. A., H. E. Spivey, A. E. Harper and C. A. Elvehjem, 1955. Factors affecting the growth of chicks on amino acid diets. Arch. Biochem. Biophys. 57: 262-263. Bird, H. R., H. J. Almquist, W. W. Cravens, F . W. Hill and J. McGinnis, 1954. Nutrient requirements for poultry. Publication 301. National Research Council. Mitchell, H. H., and R. J. Block, 1946. Some relationships between the amino acid contents of proteins and their nutritive values for the rat. J. Biol. Chem. 163: 599-620. National Research Council, 1956. Composition of concentrate by-product feedingstuffs. Publication 449. Sievert, C. W., and B. W. Fairbanks, 1957. Feed Bag Red Book, p. 188.

The Respiratory System in the Normal White Pekin Duck R. H.


Department of Pathology, The University of Texas Medical Branch, Galveston, Texas (Received for publication June 26, 1958)


HE observations in this paper have resulted from a study of neoplasms induced with a carcinogenic agent in the respiratory tract of the white Pekin duck (Rigdon, 1957). The anatomical findings in the duck's respiratory tract have made it necessary to establish the normal for this bird. The respiratory system in the white Pekin duck is similar to that in other birds; however, there are some interesting variations when compared with similar structures in the chicken (McLeod and Wagers, 1939) and in the turkey (Cover, 1953; Rigdon et al., 1958). Some of these differences no doubt reflect physiologic functions.

This investigation was supported by research grants C-1469 (C5) from the National Cancer Institute of the National Institutes of Health, Public Health Service and Tobacco Industry Research Committee.

The respiratory tract in the bird has been studied extensively by Sappey (1847), Chauveau (1890), Bradley (1915), Locy (1916), Kaupp (1918), Gilbert (1939), Zietzschmann (1943), and Hazelhoff (1951). Sappey in 1847 gave an excellent description of the respiratory apparatus and emphasized certain variations in different types of birds. A large part of Sappey's monograph is devoted to a review of the literature, beginning with the observations made by Aristotle about 400 B.C. and including those of John Hunter in 1774. Harvey in 1651 was the first to describe carefully the air sacs. Juillet in 1911 published a treatise on the anatomical, embryological, histological, and comparative study of the bird's lung. Few anatomical observations have been made on the respiratory tract in the duck (Chauveau, 1890; and McLeod and

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Almquist, H. J., 1957. Proteins and Amino Acids in Animal Nutrition. Fourth Edition. Almquist, H. J., and C. R. Grau, 1944. The amino acid requirement of the chick. J. Nutrition, 28: 325-331. Barnett, B. D., H. R. Bird, J. J. Lalich and F . M. Strong, 1957. Toxicity of beta aminoproprionitrile for turkey poults. Proc. Soc. Exp. Biol. Med. 94: 67-70.