IN THE experiments of the preceding

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1 408 R. E. SMITH AND H. M. SCOTT and W. W. Cravens, Liberation of essential amino acids from raw, properly heated, and overheated soybean oil meal. J. Biol. Chem. 167: Schweigert, B. S., Availability of tryptophan from various products for growth of chicks. Arch. Biochem. 19: Schweigert, B. S., and B. T. Guthneck, Utilization of amino acids from foods by the rat. I. Methods of testing for lysine. J. Nutrition, 49: Smith, R. E., and H. M. Scott, Biological evaluation of fish meal proteins as sources of amino acids for the growing chick. Poultry Sci. 44: Spackman, D. H., W. H. Stein and S. Moore, Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30: Measurement of the Amino Acid Content of Fish Meal Proteins by Chick Growth Assay 1 2. THE EFFECTS OF AMINO ACID IMBALANCES UPON ESTIMATIONS OF AMINO ACID AVAILABILITY BY CHICK GROWTH ASSAY R. E. SMITH 2 AND H. M. SCOTT Department of Animal Science, University of Illinois, Urbana IN THE experiments of the preceding paper (Smith and Scott, 1965), the levels of 9 of 10 essential amino acids in fish meal determined by chick growth assay proved to be in excess of those determined by chemical means. It is possible that the amino acid content of the meal was either underestimated by the chemical method or overestimated by the chick bio-assay. The analysis of the amino acid content of the fish meal has since been confirmed by another laboratory 3 and it appears that an overestimation has been made by the bio-assay. Overestimation of the amino acid content by the chick growth method could have been due to (1) increased growth re- 1 Part of a doctoral dissertation submitted by the senior author in partial fulfillment of the requirements for the Ph.D. degree while on leave of absence from the Canada Department of Agriculture. 2 Present address: Division of Animal and Poultry Science, Canada Department of Agriculture, Research Branch, Nappan, Nova Scotia. s Courtesy of O. G. Rasmussen, American Meat Institute Foundation, Chicago 37, Illinois. (Received for publication August 4, 1964) sponses of the birds on the test diets brought about by so-called unidentified growth factors present in the fish meals but not in the amino acid mixture which was to simulate them, and/or (2) supplementing the 6 standard diets with an amino acid mixture simulating fish meal created an amino acid imbalance capable of depressing chick growth. This report is concerned with the latter point. In order to gain additional information about the effect of the addition of an amino acid mixture to simulate the test protein in these biological assays, three experiments were conducted to measure the effect of adding various mixtures and various levels of amino acids to the standard reference diet when the diet was fully balanced and when the diet was deficient in one amino acid. EXPERIMENTAL PROCEDURE Male chicks originating from the mating of New Hampshire males to Columbian females were used in this study. The pre-experimental feeding and handling of

2 AMINO ACIDS IN FISH MEAL 409 the stock and the subsequent analysis of the data were carried out in a manner similar to that described by Smith and Scott (1965). The object of Experiment 1 was to determine the effect of adding various levels of amino acids, either in the form of intact protein or free crystalline amino acids, to the standard reference diet (Smith and Scott, 1965) which was already balanced and complete in every known respect. Three levels, 2.0, 4.0 and 6.0% of Fish Meal L, heated Fish Meal L (autoclaved 2 hr. at 20 pounds/in. 2 ) and an amino acid mixture to simulate these levels of Fish Meal L (Table 1 amino acid mixture No. 2) were incorporated into the standard reference diet at the expense of corn starch (Table 2). Experiment 2 was designed to determine if the effect of the addition of the fish meals or the amino acid mixture which simulated them could be influenced by increasing the protein equivalent of the standard reference diet. In order to determine this, the standard reference diet was fed at 100% and 110% of its amino acid TABLE 1. Composition of amino acid mixtures Amino acid L-arginine HC1 L-histidine HC1 H2O L-lysine HC1 L- tyrosine L- tryptophan L-phenylalanine DL-methionine L-cystine L-threonine L-leucine L-isoleucine L-valine glycine L-glutamic acid L-proline L-aspartic acid DL-alanine DL-senne Total No. 11 No. 22 No. 33 No. 4< % contribution to the diet (l) (l) C Amino acid mixture of Dean and Scott (1962). 2 Amino acid mixture first calculated to simulate 5% Fish Meal L (C.P.=70.3%) based on literature values. 3 Final amino acid mixture to simulate 5% Fish Meal L (Amino acid mixture No. 2 revised by results of the biological assays of Smith and Scott, 1965). 4 Amino acid mixture to simulate 5% Fish Meal L based upon the chemical analysis of the amino acid content of Fish Meal L (C.P.=72.0%). TABLE 2. The effect of the addition of Fish Meal L and a mixture of amino acids to simulate Fish Meal L upon the growth of chicks fed a standard reference diet Diet No X 9 10 Supplement to standard reference diet 1 None 2% Fish Meal L 4% Fish Meal L 6% Fish Meal L 2% heated Fish Meal L 2 4% heated Fish Meal L 6% heated Fish Meal L Amino acid mix No. 2 to simulate 2% Fish Meal L 3 Amino acid mix No. 2 to simulate 4% Fish Meal L Amino acid mix No. 2 to simulate 6% Fish Meal L Gain/chick/ day (gm.) Gain Feed i Smith and Scott, Autoclaved 2 hours at 20 pounds/in. 2 3 Amino acid mixture No. 2 of Table 1 minus aspartic, alanine and serine. This mixture as shown in Table 1 simulates 5% Fish Meal L, consequently only the required proportions of the mixture were fed in treatments 8, 9 and 10 above. content. To both these diets was also added 5% Fish Meal L, 5% heated Fish Meal L and a mixture of amino acids (Table 1 amino acid mixture No. 2) to stimulate 5% Fish Meal L (Table 3). Experiment 3 was designed to determine the effect of the addition of amino acid mixtures to the standard reference diet when the diet was deficient in one amino acid. This experiment consisted of 10 treatments (Table 4). Treatments 1 to 3 involved the feeding of the standard reference diet devoid of isoleucine as a basal diet with supplemental additions of 0.44, 0.52 and 0.60% L-isoleucine shown TABLE 3. The effect of the addition of Fish Meal L and a mixture of amino acids to simulate Fish Meal L upon growth of chicks fed two levels of the amino acid mixture in the standard reference diet Diet No. Supplement to standard reference diet 1 Gain/chick/ day Cgm.) Gain 1 None % standard amino acid mix % Fish Meal L As % heated Fish Meal L' As Amino acid mix =5% Fish Meal L As See Smith and Scott, (The amino acid mixture is also presented in Table 1 of this paper as amino acid mixture No. 1.) 2 Table 1 amino acid mixture No Autoclaved 2 hours at 20 pounds/in. 2 4 Table 1 amino acid mixture No. 2.

3 410 R. E. SMITH AND H. M. SCOTT TABLE 4. The eject of amino acid imbalances upon estimations of amino acid availability by chick growth assay Diet No Supplement to standard reference diet ( isoleucine) 0.44% L-isoleucine As % L-isoleucine As % L-isoleucine Amino acid mix No. 3'+0.44% L-isoleucine As % L-isoleucine As % L-isoleucine Amino acid mix No % L-isoleucine As % L-isoleucine As % L-isoleucine 5% Fish Meal L+0.40% L-isoleucine (Total =0.52% L-isoleucine) uain/cnick/ day (gm.) oain Feed Amino acid mix No. 3 of Table 1. Mixture simulates 5% Fish Meal L based on revised book values. 2 Amino acid mix No. 4 of Table 1. Mixture simulates 5% Fish Meal L based on chemical analysis. to support suboptimal growth ranging between 6 and 12 grams/chick/day (Smith and Scott, 1965). To a similar series of diets was added a mixture of amino acids to simulate 5% fish meal based on literature values (Table 1 amino acid mixture No. 3) yielding treatments 4 to 6. To a second series of 3 diets similar to those of treatments 1 to 3 was added a mixture of amino acids patterned after the chemical analysis of the fish meal (Table 1 amino acid mixture No. 4) yielding treatments 7 to 9. In treatment 10, 5% Fish Meal L was added to the standard reference diet devoid of isoleucine. Since the contribution of isoleucine by the fish meal was not sufficient the total isoleucine content was brought up to the 0.52% level by supplementation with the crystalline form of the amino acid. RESULTS AND DISCUSSION The addition of Fish Meal L to the standard reference diet in Experiment 1 improved growth and feed utilization (Table 2). Heating the meal prior to its incorporation did not alter this response. The addition of the simulating mixture of amino acids, on the other hand, did not improve growth or feed efficiency over the.60 standard reference diet at any of the levels tested. Differences were not observed among the levels of incorporation of any of the 3 amino acid sources studied. The significant growth response upon the addition of fish meal to the standard reference diet was observed by Dean (1963) when dried egg white was added to this same diet. However, this response was not attained with the simulated amino acid mixture. The question arose as to whether this growth response to the intact protein might be indicative of an overall deficiency of nitrogen in the standard diet, and if it might be overcome by providing a higher level of protein in the standard reference diet. Experiment 2 was designed to test this possibility. Individual degree of freedom comparisons show that increasing the amino acid mixture of the standard reference diet by 10% did not alter the growth promoting ability of the diet (Table 3). Once again it was demonstrated that the amino acid mixture simulating Fish Meal L had no effect upon growth when added to the standard reference diet, while the addition of either of the intact fish meals increased growth to a significant degree. If the standard reference diet supplies all of the amino acids at their required level, then the addition of any other amino acid mixture to this diet may or may not result in an adverse effect upon growth depending upon the extent to which the diet is imbalanced by this addition. If, however, one amino acid is made severely limiting in the standard diet, and then the additions are made, as in the biological assay experiments described previously (Smith and Scott, 1965), a detrimental effect upon growth could be anticipated (Fisher et al., 1960), especially when the added mixture is itself imbalanced. Moreover, if amino acid mixtures of different patterns are added, then their influence

4 AMINO ACIDS IN FISH MEAL 411 upon growth might be expected to differ. In Experiment 4 two mixtures of amino acids were added to the standard reference diet which had been made deficient to varying degrees in isoleucine. There was a highly significant growth depression among the chicks fed the diets containing the added amino acid mixtures compared with those not receiving them (Table 4). Growth response on the diets containing the two simulated mixes of amino acids were not statistically different, indicating no significant error was encountered in the experiments of Smith and Scott (1965) by the use of the amino acid mixture based on literature values as opposed to those arrived at by chemical analysis. There was a highly significant linear effect due to the addition of the limiting amino acid isoleucine. The interactions or measurement of parallelism between the diets containing the additional amino acid mixtures and those not receiving them, as well as between those diets receiving the amino acid mixtures were not significant. This can be interpreted to mean that even though the simulated mixtures depressed growth in this experiment, the reaction to these mixes throughout the growth curve is similar but of different magnitude. Thus potency measured by these curves could be expected to be the same over the entire segment of the curve studied. Figure 1 shows the plot isoleucine response (A) in the presence of no additional imbalancing mixture (B) in the presence of the amino acid mixture to simulate 5% Fish Meal L as per literature values, and (C) in the presence of the amino acid mixture based on the chemically determined amino acid content of the fish meal. If these curves are assumed to represent standard growth curves and were used to estimate the potency of a protein source resulting in an arbitrary growth of _ i (A) No addition -(B) Old simulation (C) New simulation x(p) Flshmeal addition * I - iaoleucino (total) FIG. 1. The effect of amino acid imbalances upon estimations of amino acid availability by chick growth assay. grams/chick/day, it would be concluded from plot A, that the isoleucine content of the unknown was 0.494%. If the amino acid mixture patterned after literature values was added to the standard, plot B, it would be concluded from an equivalent growth response, that the isoleucine content of the unknown was 0.544%, and similarily, if the simulation based on the chemical analysis were used, plot C, the value of 0.588% would be accepted. The reason for this variation can be explained on the basis of amino acid imbalance. Fisher el al. (1960) pointed out that an imbalance may be created through the addition of an essential amino acid mixture deficient in one amino acid which is also limiting in the dietary protein. Apparently the addition of the simulating amino acid mixtures to the standard diet did bring about an imbalance and furthermore, this imbalance inhibited

5 412 R. E. SMITH AND H. M. SCOTT growth more with the simulated amino acid additions than with the fish meal protein. As a consequence of this growth retardation, the calculated isoleucine content taken from Figure 1 appears greater in the diets containing the simulating mixtures than in those without them. It may be inferred therefore, that in the biological assay previously described (Smith and Scott, 1965), the addition of the simulating mixture to the six diets constituting the standard curve created an imbalance which depressed growth to a greater extent than did the intact proteins in the seventh and eighth treatments. As a consequence an "inflated" amino acid content was calculated. It is therefore clear that by merely adding a mixture of amino acids to the standard diet, the assayed value of one amino acid may be altered considerably. This observation illustrates the danger of adding various proteins to a basal diet with no attempt to equate the additional amino acids or other components in the protein. It is also clear that the growth depression caused by adding the simulating amino acid mix to the diets constituting the standard assay curve could bring about higher biological potency values in the unknown than if no simulation mixture had been added. In addition, reference to Figure 1, point D, indicates that the constituents of 5% Fish Meal L, as expressed at the one isoleucine level (0.52%), promote growth closer to that where no amino acids were added (plot A) than to those where the simulation mixtures were incorporated (plots B and C). It is thus apparent that the components of the fish meal were not truly simulated by the amino acid mixtures used. However, it still may be inferred that had the mixture approached more nearly the true overall addition resulting from the feeding of Fish Meal L, the potency evaluations of the amino acids would have been closer to the theoretical value of 100% of that determined chemically or even the more practical value beneath this figure. It is also conceivable that the detrimental effect caused by the simulated amino acid additions would vary under conditions of differing limiting amino acid composition. This could account for the range of % in availability observed in the experiments of the original investigation (Smith and Scott, 1965). SUMMARY Three experiments were carried out to measure the effect of adding various mixtures of crystalline amino acids or intact proteins at several levels to a standard purified diet when the diet was balanced and when the diet was deficient in one amino acid. The addition of an intact protein (fish meal) to the balanced diet improved chick growth and feed utilization but the addition of a mixture of amino acids simulating those of the fish meal had no effect. Increasing the amino acid mixture of the standard diet by 10% did not alter the growth response of either the intact protein or the simulating amino acid mixture. The addition of two mixtures of amino acids (one patterned after the chemical and one after the biological analysis of fish meal) to a diet deficient in one amino acid had an adverse effect upon growth. The extent of this growth inhibition varied depending upon the amino acid pattern of the mixture added. The consequence of this relationship upon biological assays of amino acid availability in intact proteins is discussed. REFERENCES Dean, W. F., The development of a crystalline amino acid reference diet for chicks with special reference to its use in studying the effect of suboptimal and superoptimal dietary concentrations of amino acids on the free-amino acid content of

6 AMINO ACIDS IN FISH MEAL 413 blood plasma. Doctoral Thesis, University of Illinois, Urbana. Dean, W. F., and H. M. Scott, The development of an amino acid standard for the early growth of chicks. Poultry Sci. 41: Fisher, H., P. Griminger, G. A. Leveille and R. Shapiro, Quantitative aspects of lysine de- Piperazine was the second outstanding poultry anthelmintic to be developed. Riedel (1950) first reported the use of piperazine for the removal of ascarids from poultry. In 1951 Riedel used piperaficiency and amino acid imbalance. J. Nutrition, 71: Smith, R. E., and H. M. Scott, Measurement of the amino acid content of fish meal protein by chick growth assay. I. Biological assay of amino acid availability in fish meal protein before and after heat treatment. Poultry Sci. 44: The Effects of Piperazine, Phenothiazine and Di-N-Butyltin Dilaurate Combinations on Egg Production and Egg Quality in Chickens D. L. PEAEDON, W. O. HABERMAN, J. E. MARR, F. W. GARLAND, JR. AND H. L. WILCKE Veterinary and Poultry Research Departments, Ralston Purina Company, St. Louis, Missouri THREE outstanding poultry anthelmintics have emerged during the past two and one half decades. The first of these was phenothiazine which McCulloch and Nicholson (1940) employed for the removal of Heterakis gallinae from chickens. They reported an average effectiveness of from 95 to 100% when dosages of from 0.05 to 0.5 gm. per chicken were administered. They also reported that enormous doses up to 500 times the smallest effective therapeutic dose caused no apparent harm to chickens and a 0.5 gm. dosage level was used without any appreciable effect on the egg production of a test flock. Since then the safe and effective use of phenothiazine for removal of H. gallinae from chickens and turkeys has been confirmed by Roberts (1940), Nicholson and McCulloch (1942), Olivier el al. (1943), Harwood and Guthrie (1944), Wehr (1959) and others. (Received for publication August 6, 1964) zine at 0.25, 0.5, 1.0 and 2.0% continuously in the regular poultry ration for 1 week. Although this was reportedly nontoxic, the efficacy in eliminating ascarids left something to be desired. The best results he obtained, using 1.0% piperazine in the feed for 2 weeks continuously, were less than 90% effective. Since then, many workers have used a variety of piperazine compounds in different dosages and other methods of administration with more effective results, and a wide margin of safety in poultry has been established. The reports of Edgar el al. (1957), Shumard (1957), and Ross and Alicata (1959) indicated that piperazine could be used effectively against Ascaridia galli in laying hens without reducing their production. Due to the efficacy, ease and safety of its use, piperazine soon became the poultry ascaricide of choice. [A comprehensive review of piperazine compounds as poultry ascaricides was published by Tudor (1962).] Combinations of chemotherapeutics for the removal of H. gallinae and A. galli in a single treatment was first conceived by