IT HAS become accepted that feed protein(s)

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1 METABOLIZABLE ENERGY 333 on the utilization of dietary energy. Poultry Sci. 42: Sibbald, I. R., and S. J. Slinger, 1963b. A biological assay for metabolizable energy in poultry feed ingredients together with findings which demonstrate some of the problems associated with the evaluation of fats. Poultry Sci. 42: Sibbald, I. R., and S. J. Slinger, 1963c. Factors affecting the metabolizable energy content of poultry feeds. 12. Protein quality. Poultry Sci. 42: Sibbald, I. R., S. J. Slinger and G. C. Ashton, Factors affecting the metabolizable energy content of poultry feeds. 5. The level of protein and IT HAS become accepted that feed protein(s) must be balanced in essential amino acids to insure a maximal growth rate in the chick. In most instances, diets formulated for the chick are deficient or limiting in one or more of the essential amino acids. Three basic approaches have been taken to formulate diets so that each essential amino acid is supplied at an adequate level: (1) Increasing total protein in the diet, (2) Providing several protein sources, including both vegetable and animal proteins, (3) Supplementing the diet with crystalline amino acids. The first approach has one serious drawback protein wastage. The use of several protein sources results in an increased total cost of the feed. Supplementation with crystalline amino acids, with the 1 Journal Paper No. J-4686 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Project No Present address: Chas. Pfizer and Company, Inc., Terre Haute, Indiana. of test material in the diet. 6. A note on the relationship between digestible and metabolizable energy values. Poultry Sci. 41: Slinger, S. J., W. F. Pepper, I. Motzok and I. R. Sibbald, 1962a. Studies on the calcium requirements of turkeys. 3. Influence of chlortetracycline and reserpine during the starting and growing periods. Poultry Sci. 41: Slinger, S. J., W. F. Pepper and I. R. Sibbald, 1962b. Interrelationships between methionine, choline, sodium chloride and reserpine in growing turkeys. Poultry Sci. 41: Snedecor, G. W., Statistical Methods, 5th Edition, Iowa State College Press, Ames, Iowa. Amino Acid Supplementation of a Corn-Soybean Meal Chick Ration 1 C. E. ASKELSON 2 AND S. L. BALLOUN Department of Poultry Science, Iowa State University, Ames, Iowa (Received for publication August 6, 1963) exception of lysine and methionine, has not been feasible nor practical because of their cost and availability. A major problem in developing a diet that supports maximal growth is the lack of valid information concerning the chick's requirement for many of the essential amino acids. The development of purified diets, in which crystalline amino acids serve as the sole source of amino nitrogen, has provided additional information concerning dietary amino acid requirements and their interrelationships (Klain et al, 1960; Adkins et al, 1962). However, Fox et al. (1958) and Adkins et al. (1962) reported that the growth of chicks fed an amino acid diet was considerably below that of chicks fed a purified protein. It would be desirable, therefore, to develop a practical diet in which the majority of the essential amino acids were supplied from intact proteins and, at the same time, to utilize small quantities of

2 334 C. E. ASKELSON AND S. L. BALLOUN TABLE 1. Composition of diets used in experiment 1 Ingredients & ^ +a.a. Ground yellow corn Soybean oil meal, 50% protein Soybean oil Cellulose, Alphacel Dicalcium phosphate Ground oyster shell Vitamin and antibiotic mix" Salt and trace mineral mix b Monosodium glutamate L-lysine HC1 DL-methionine Glycine L-arginine HC1 DL-threonine DL-valine Di.-histidine HC1 DL-tryptophan ra.-isoleucine Percentage composition a Supplied in the following amounts per lb.: vitamin A, 3,000 I.U.; vitamin D 3, 500 I.C.U.; vitamin E, 5 I.U.; riboflavin, 2 mg.; niacin, 24mg.; calcium pantothenate, 4 mg.; choline, 304 mg.; menadione, 1 mg.; vitamin B meg.; and penicillin, 5.0 mg. b Supplied in the following amounts per lb.: NaCl, 2.3 gm.; Mn, 34.2 mg.; Zn, 13.8 mg.; Fe, 13.8 mg.; and Cu, 1.4 mg. crystalline amino acids in order to satisfy the chick's essential amino acid requirements, without supplying an excess of the other essential amino acids. The primary objective of the studies reported here was to investigate the feasibility of developing an amino acid-supplemented 18 percent protein chick diet, in which corn and soybean meal are the only sources of intact protein, capable of supporting a rapid and efficient production performance. PROCEDURE General. Vent-sexed Ames In-Cross male chicks hatched at the Iowa State University Poultry Farm were used to conduct the four-week feeding experiments reported herein. Day-old chicks were individually weighed, grouped into lots of ten chicks per pen and immediately placed on experimental diets. Chicks had accessto feed and water ad libitum. Individual chick weights and pen feed consumption were recorded at 2 and of age. Experiment 1. The experiment consisted of 3 dietary treatments, each triplicated, giving a total of 9 experimental pens. Chicks were assigned to a battery brooder in accordance with a completely randomized design. The composition of experimental diets is given in Table 1. The 18 percent protein basal diet was formulated so that crystalline amino acids would be supplemented in place of monosodium glutamate on an equal weight basis. It was assumed that all crystalline amino acids, including monosodium glutamate, contributed 100 percent crude protein. Thus, the two lower protein diets used in this experiment were considered isoproteinous, even though they were not exactly isonitrogenous. Amino acid levels reported by Dean and Scott (1962) were used as the criteria for supplementation of crystalline amino acids to the 18 percent protein basal diet. The essential amino acid levels reported by these workers and the calculated amino acid composition of the 18 percent protein basal diet used in this experiment are shown in Table 2. For comparative purposes, the essential amino acid requirements recommended by the National Research Council (1960) for starting chicks receiving a 20 percent protein diet are also shown in Table 2. With the exception of phenylalanine and leucine, it was necessary to supplement all essential amino acids in order to achieve the individual amino acid levels reported by Dean and Scott (1962). Since

3 AMINO ACID SUPPLEMENTATION 335 crystalline cystine was not used as a supplemental ingredient, DL-methionine was added to result in a combined methionine and cystine level of 0.8 percent of the diet. Experiment Z. This experiment was divided into 3 separate feeding trials and, with the exception of experimental diets, the procedures followed were similar. Each feeding trial consisted of 8 dietary treatments, each duplicated, giving a total of 16 experimental pens. Chicks were assigned a battery brooder in accordance with a 2X2X2 completely randomized factorial design. The composition of the experimental diets in experiment 2 was the same as the 18 percent protein, amino acid-supplemented diet given in Table 1, with the following modifications: 1. In trial 1, the basal diet included all supplemental amino acids with the exception of lysine, methionine and glycine, which were replaced by monosodium glutamate on an equal weight basis. The remaining 7 experimental treatments were obtained by adding lysine, methionine and glycine individually and in all possible combinations. As in experiment 1, amino acid supplementation was accomplished at the expense of monosodium glutamate. 2. A similar procedure as outlined above was used in the formulation of diets used in the remaining 2 feeding trials of this experiment. The basal diet in trial 2 included all supplemental amino acids except arginine, threonine and valine; whereas, in trial 3, all amino acids were added to the basal diet except histidine, tryptophan and isoleucine. In each of the respective trials, these amino acids were then supplemented individually and in all possible combinations to fulfill the requirements for a 2 X 2 X 2 factorial design. Experiment 3. The experiment consisted of 3 dietary treatments, each triplicated, TABLE 2. Comparison of amino acids in basal diet with levels reported by Dean and Scott {1962) and the National Research Council" Amino Acid Lysine Methionine Cystine Glycine Arginine Threonine Valine Histidine - Tryptophan Isoleucine Leucine Phenylalanine 18% 0.81 b = Dean and Scott 1.12 b NRC 0.90 b a National Research Council, Nutrient requirements of domestic animals. No. 1 Nutrient requirements for poultry. b Expressed as percent of total diet. giving a total of 9 experimental pens. Chicks were assigned a battery brooder in accordance with a randomized block design. The experimental treatments were as follows: (1) 18 percent protein basal, (2) 18 percent protein basal plus three amino acids, (3) 18 percent protein basal plus nine amino acids. The composition of diets 1 and 3 is given in Table 1. Diet 2 was a modification of the basal diet in that only lysine, methionine and glycine were supplemented in place of monosodium glutamate on an equal weight basis. RESULTS Experiment 1. Chick growth and feed efficiency data for this experiment are given in Table 3. At the end of two weeks, chicks receiving the amino acid-supplemented diet were 8.1 percent heavier and 11.1 percent more efficient in feed conversion than chicks receiving the unsupplemented 18 percent protein diet. Analyses of variance indicated that the improved growth was statistically significant at P<.05, while the improved feed efficiency was statistically significant at

4 336 C. E. ASKELSON AND S. L. BALLOTJN TABLE 3.- -Chick weight and feed efficiency data for experiment 1 Dietary treatment 2 weeks 18% 18% +a.a. 22% Control 18% 18% +a.a. 22%, Control Chick weight 3 (gms.) '- Feed efficiency a Average per chick of triplicate experimental b Grams of feed per gram of grain. P<.01. The magnitude of response to supplemental amino acids (8.9 percent improved growth and 12.7 percent improved feed efficiency) was not appreciably altered at the end of. Both of these differences were statistically significant at P<.01. The 22 percent protein control diet was included as an experimental treatment to observe whether or not chick growth obtained with the amino acid-supplemented 18 percent protein diet was comparable to that of chicks fed a practical, high-energy 22 percent protein diet. Chicks fed the higher protein diet were 7.1 and 9.4 percent heavier at 2 and of age, respectively, than were chicks fed the amino acid-supplemented 18 percent protein diet. In contrast to the differences in body weight, feed conversions for chicks fed these two diets were similar, especially at the end of. The failure of the 18 percent protein diet to produce maximum weight gains, even when amino acids were added at the levels indicated, may have been the result of several factors. These results suggest that, even though the essential amino acid requirements were fulfilled, total nitrogen intake may have been a limiting factor for maximal growth. However, it is possible that amino acid supplementation still did not provide adequate amounts of one or more of the essential amino acids. Although growth rate and feed efficiency were improved by amino acid supplementation, consideration must also be given to the possibility that an amino acid imbalance and/or toxicity was induced by adding one or more of the crystalline amino acids, thereby offsetting or masking the full effect of the other amino acids added. Experiment 2, Having observed a significant improvement in weight gain and feed efficiency by supplementation of nine essential amino acids to the 18 percent protein diet in experiment 1, this study was designed to observe which of the added amino acids were responsible for the improvement in chick performance. Chick weight and feed efficiency data for trial one of experiment 2 are shown in Table 4. At two weeks, the only statistically significant (P <.05) response in chick weight was observed when the basal diet was SUP- TABLE 4. Chick weight and feed efficiency data for trial 1 of experiment 2 Dietary treatment 2 weeks +lysine (L) +methionine (M) +glycine (G) +L+M +L+G +M+G +L+M+G +L +M +G +L+M +L+G +M+G +L+M+G Chick Fee(] 7 ^ (gms.) efficiency" * a Average per chick of duplicate experimental b Grams of feed per gram of gain.

5 AMINO ACID SUPPLEMENTATION 337 plemented with lysine, methionine and glycine in combination. Chicks receiving this diet were 11.3 percent heavier than were chicks receiving the basal diet. No significant response in body weight gains was observed when these three amino acids were supplemented singly and in two-way combinations. In addition to statistically significant (P <.05) main effects due to supplemental lysine and methionine, analysis of the 4 week weight data also revealed significant (P<.05) lysinexmethionine (LXM) and lysinexglycine (LXG) interactions. Supplementing the diet with lysine alone resulted in an improved growth rate of less than 1 percent. When methionine was supplemented alone, an improved growth rate of 2.9 percent was observed. In contrast, chicks fed diets supplemented with lysine plus methionine, and with lysine, methionine and glycine, were 4.0 and 10.1 percent heavier, respectively, than were chicks fed the basal diet. Adding glycine in the absence of supplemental lysine did not result in an improvement in chick growth. However, when glycine was added in the presence of supplemental lysine, chick growth was improved. The nature of the LXM and LXG interactions would lead one to suspect that a significant LXMXG interaction, as was observed at 2 weeks, would be present. However, statistical analysis failed to verify that this three-way interaction was significant at the probability of.05, although it did approach significance at this level. At 2 weeks of chick age, adding lysine, methionine and glycine, either individually or in combination, had no significant effect on feed efficiency. At, however, significant differences in feed efficiency were observed among the various dietary treatments. Similar to body weight data, statistically significant (P TABLE 5. Chick weight and feed efficiency data for trial 2 of experiment Z Dietary treatment Z weeks +arginine (A) +threonine (T) -j-valine (V) +A+T +A+V +T+V +A+T+V +A +T +V +A+T +A+V +T+V +A+T+V Chick weight* (gms.) Feed efficiency a Average per chick of duplicate experimental b Grams of feed per gram of gain. <.05) lysine and LXM interaction effects upon feed efficiency were revealed. In addition, the LXMXG interaction was also found to be statistically significant at the same level of probability. These results suggest that a maximal response in feed efficiency was obtained by supplementation of all three amino acids in combination. The response in feed efficiency to supplemental glycine in the presence of supplemental methionine was found to be statistically different (P<.05) from that observed when glycine was added in the absence of supplemental methionine. When glycine was added in the absence of supplemental methionine, feed efficiency was improved. However, when these two amino acids were supplemented in combination, there was a tendency to increase the feed required per unit of gain. The results of trial 2 of experiment 2 (Table 5) indicate that no significant improvements in chick weight and/or

6 338 C. E. ASKELSON AND S. L. BALLOUN TABLE 6. Chick weight and feed efficiency data for trial 3 of experiment 2 Dietary treatment 2 weeks +histidine (H) +tryptophan (T) +isoleucine (I) +H+T +H+I +T+I +H+T+I +H +T +1 +H+T +H+I +T+I +H+T+I Chick weight* (gms.) Feed efficiency 1, Average per chick of duplicate experimental b Grams of feed per gram of gain. feed efficiency were obtained at 2 or 4 weeks of age when arginine, threonine and valine were supplemented, either singly or in various combinations, to the basal diet. In fact, body weights of chicks receiving diets supplemented with these three amino acids were lower, although not significantly lower, than that observed for chicks receiving the basal diet. There was, however, a statistically significant (P<.05) threonine X valine interaction in the ' weight data. Addition of threonine or valine individually resulted in weight gains of only 92.0 and 96.5 percent, respectively, of that observed for chicks receiving the basal diet. When these two amino acids were supplemented in combination, chick growth was increased up to 97.2 percent of that observed for the basal diet. It would appear that, when threonine was added alone, an amino acid imbalance occurred, which resulted in a tendency to depress chick growth. Adding histidine, tryptophan and isoleucine, (Table 6) individually or in various combinations to the basal diet did not improve weight gains. The only statistically significant (P<.05) effect due to amino acid supplementation in trial 3 of experiment 2 was the increased feed requirement per unit of gain of chicks that received supplemental tryptophan. Histidine and isoleucine supplementation had no significant effect on feed utilization. The results of these three feeding trials indicate that the basal diet was deficient in lysine, methionine and glycine in its ability to support maximal chick growth. Of these three amino acids, methionine was the first limiting amino acid, followed by lysine, then glycine. A maximal response in weight gain and feed efficiency was not obtained unless the basal diet was supplemented with these three amino acids in combination. Further supplementation of the basal diet with arginine, threonine, valine, histidine, tryptophan and isoleucine did not improve growth or feed efficiency. Experiment 3. This experiment was designed to verify that the basal diet used in experiment 1 required only the supplementation of lysine, methionine and glycine to result in maximal chick growth and feed efficiency. Chick weight and feed efficiency data for experiment 3 are given in Table 7. At 2 weeks of age, chicks fed diets supplemented with either three or nine amino acids were 14.0 and 11.2 percent heavier, respectively, than chicks fed the basal diet. These differences were statistically significant at P<.01. As would be expected, chicks receiving the amino acidsupplemented diets demonstrated a significantly (P<.01) improved feed efficiency when compared with chicks receiving the basal diet.

7 AMINO ACID SUPPLEMENTATION 339 The magnitude of response in weight gains and feed efficiency to amino acid supplementation was not altered at the end of. Again, chicks fed the amino acid-supplemented diets showed significantly (P<.01) improved weight and feed efficiency over that of chicks fed the basal diet. At both 2 and of age, there were no significant differences in weight and feed efficiency between chicks receiving diets supplemented with lysine, methionine and glycine, and chicks receiving diets supplemented with all nine amino acids. The results of this experiment support the observations made in experiment 2, i.e., that a maximal response in chick growth can be obtained when the basal diet is supplemented with lysine, methionine and glycine in combination, and that further supplementation of this diet with arginine, threonine, valine, histidine, tryptophan and isoleucine does not significantly improve chick weight and feed efficiency. DISCUSSION Compared with the 18 percent protein standard diet reported by Dean and Scott (1962), the basal diet formulated for the presently reported experiments was calculated to be deficient in nine essential amino acids. However, it was found that the inclusion of only lysine, methionine and glycine was necessary to result in a maximal growth response. It is reasonable to expect that observations concerning the chicks' requirement for a given essential amino acid would differ, depending upon the dietary source of that amino acid. In the standard diet reported by Dean and Scott (1962), all the amino acids were supplied in the free, crystalline form, whereas, the majority of the amino acids supplied in the presently reported experiments were from TABLE 7. Chick weight and feed efficiency data for experiment 3 Dietary Chick weight" Feed treatment (gms.) efficiency b 2 weeks a.a a.a a.a a.a Average per chick of triplicate experimental h Grams of feed per gram of gain. intact protein sources. Furthermore, the isomeric form(s) of the crystalline amino acids supplemented, either D, L or DL, used in any particular experiment would also be expected to influence the apparent requirement for any given amino acid. Dean and Scott (1962) used only the L isomer of the amino acids, with the exception of methionine, in developing their standard diet. In the present experiments, the DL racemic form of crystalline amino acids, with the exception of lysine and arginine, was used for supplementation. Because of differences in utilization of the isomeric form(s) of amino acids, it would be logical to assume that the dietary requirement for a given amino acid supplied only as the L isomer would be less than that observed when part of the total amino acid requirement was supplied as the racemic mixture. It appears, however, from the experiments reported here, that the chicks' amino acid requirement for arginine, valine, histidine, tryptophan and isoleucine is lower than that reported by Dean and Scott (1962). There are two factors which must be considered as possible explanations why Dean and Scott (1962) observed a higher requirement for these amino acids than was observed in these experiments. First,

8 340 C. E. ASKELSON AND S. L. BALLOTJN the amino acid composition of intact proteins was calculated from values determined by microbiological assay a number of years ago. In view of newer and more reliable assay methods, the values used to obtain the amino acid composition of corn and soybean oil meal in the basal diet may or may not reflect the true amino acid composition of these protein sources. Secondly, differences in availability of amino acids for absorption from the gastrointestinal tract, between a purified diet and a diet which contains intact proteins, may also affect the apparent requirement for a given amino acid. In the former diet, the amino acids are readily available for absorption without aid of certain enzymatic digestive processes. In contrast, a diet containing intact proteins must be subjected to a selective degradative process, which results in the release of individual amino acids in a relatively slow and orderly fashion. For a period of time immediately following ingestion of a purified diet, it would be anticipated that there would be an excess of amino acids present in the gastrointestinal tract. This excess of amino acids would probably result in a lower efficiency of absorption than that observed when an intact protein is fed. If there was an excessive quantity of amino acids in the gastrointestinal tract, a high blood plasma amino acid concentration would also be expected. Thus, when a purified diet is fed, the concentration of a given amino acid in the blood plasma may be greater than the body tissues' ability to efficiently utilize that amino acid. This would result in a further loss of the amino acid through deamination and/or excretion in the urine. The concept that tissue protein synthesis, assuming that weight gain is a valid index of this synthesis, can proceed only up to a point where one amino acid becomes the most limiting, regardless of the adequacy or inadequacy of any other essential amino acid, was illustrated in experiment 2. In this experiment, the supplementation of lysine and glycine individually did not result in any apparent improvement in chick growth and feed efficiency, whereas, the supplementation of methionine individually resulted in a slight improvement in growth rate. However, when lysine was added in the presence of methionine, a further improvement in growth rate was observed. Furthermore, a response from glycine supplementation was not observed unless both lysine and methionine had been added to the basal diet. Even though the basal diet was deficient in all three amino acids, the supplementation of any one amino acid did not result in a growth response unless the needs for the most limiting amino acid in the diet had been first met. SUMMARY Experiments were designed to determine the effects of crystalline amino acid supplementation of an 18 percent crude protein diet, which contained corn and soybean oil meal as the only sources of intact proteins, on chick growth and feed efficiency. The amino acid standard diet reported by Dean and Scott (1962) was used as the criterion for essential amino acid supplementation. Amino acids were added to the basal diet in lieu of monosodium glutamate on an equal weight basis. The results of these experiments indicate that the basal diet was deficient in lysine, methionine and glycine as measured by its ability to support maximal chick growth and feed utilization. Of these three amino acids, methionine was the first limiting amino acid, followed by lysine, then glycine. Maximal chick growth and feed efficiency was not obtained unless all three of these amino

9 AMINO ACID SUPPLEMENTATION 341 acids were supplemented in combination. Further supplementation with arginine, threonine, valine, histidine, tryptophan and isoleucine did not improve chick growth or feed efficiency. REFERENCES Adkins, J. S., M. L. Sunde and A. E. Harper, The development of a free amino acid diet for the growing chick. Poultry Sci. 41: Dean, W. F., and H. M. Scott, The develop- WIDESPREAD occurrence among turkey flocks in the United States and Canada of a fatal condition characterized by massive internal hemorrhage was first reported by Durrell, Pomeroy, Carr and Jerstad (1952). A detailed description of the disorder by McSherry et al. (1954) reported that the large blood clots most commonly originated from a rupture of the posterior aorta between the origins of the external iliac and sciatic arteries. Examination of such aortas indicated that rupture occurred subsequent to a dissecting aneurysm. An apparently similar disorder was reported in Great Britain (Carnaghan, 1955; and Gibson and de Gruchy, 1955). Several workers (Carnaghan, 1955; Gibson and de Gruchy, 1955; and Gresham and Howard, 1961) 1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by the Research Committee of the Graduate School from funds supplied by Wisconsin Alumni Research Foundation, Madison. 2 Present address, Department of Animal Science, University of Illinois, Urbana, Illinois. ment of an amino acid standard for the early growth of chicks. Poultry Sci. 41: Fox, S. M. R., G. M. Briggs and L. 0. Ortis, Studies of amino acid diets for the chick. J. Nutrition, 64: Klain, G. J., H. M. Scott and B. C. Johnson, The amino acid requirement of the growing chick fed a crystalline amino acid diet. Poultry Sci. 39: National Research Council, Nutrient requirements of domestic animals. No. 1. Nutrient requirements for poultry. The Incidence of Spontaneous Pathologic Changes in Aortas of Growing Turkeys 1 B. E. MCDONALD, 2 H. R. BIRD AND J. J. LALICH Departments of Poultry Science and Pathology, University of Wisconsin, Madison, Wisconsin (Received for publication August 6, 1963) have reported the presence of atherosclerotic lesions adjacent to the dissecting aneurysms in birds dying with massive abdominal hemorrhage. In fact the presence of accompanying atheroma led Carnaghan (1955) to suggest that these plaques may predispose the aorta to rupture. Other workers (Pritchard et al., 1958), however, believe that degenerative changes in the elastic fibers in the media represent the primary lesion in aortic rupture in turkeys. Surveys on spontaneous aortic rupture in turkeys show males to be most commonly affected with losses usually occurring between 10 and 2 of age. Studies of aortas from apparently healthy birds have been inadequate to explain the age and sex incidence of spontaneous aortic rupture although aortic degeneration has been found at autopsy in apparently healthy survivors (Carnaghan,1955; Pritchard et al., 1958; and Lalich et al., 1957). The marked susceptibility of young male turkeys to aortic rupture may be partly due to the transient rise in systolic