Factors Affecting Efficacy of Methionine Hydroxy Analogue for Chicks Fed Amino Acid Diets 1

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1 Factors Affecting Efficacy of Hydroxy Analogue for Chicks Fed Amino Acid Diets 1 AEN C. CHRISTENSEN, 2 J. O. ANDERSON, and D. C. DOBSON Animal, Dairy, and Veterinary Science Department, Utah State University, ogan, Utah (Received for publication December 3, 1979) ABSTRACT Chicks were fed mixtures of methionine, cystine, and the calcium salt of the hydroxy analogue of methionine (MHA) in a diet based on a mixture of amino acids. Rate of gain with the basic amino acid diet containing a mixture of methionine and cystine was about 90% of the rate noted with a practical type diet. Efficacy of MHA depended on its level in the diet and on the levels of methionine and cystine fed with it. It was least effective when fed as the only sulfur amino acid or when fed with cystine. When fed with methionine it had intermediate value and was most efficacious when fed with a mixture of methionine and cystine. Essentially it was fully effective when it provided 25% of the sulfur amino acids with the remainder as equal parts of methionine and cystine. Replacing part of the cystine in a mixture of cystine and MHA with methionine resulted in a marked improvement in performance with -methionine being slightly more effective than D-methionine. (Key words: methionine, methionine hydroxy analogue, cystine efficacy, growth rate) INTRODUCTION As indicated in the review found in a companion paper (Christensen and Anderson, 1980), agreement is not complete as to the relative efficacy of methionine and the calcium salt of the hydroxy analogue of methionine (MHA) for the growing chick. In general, the reports indicate that the two are equivalent on a molar basis when used in diets based on corn and soybean meal. In contrast, MHA appears to be less effective with diets based on a crystalline amino acid mixture (Baker, 1977). This implies that the metabolism of MHA differs with the two types of diets for some unknown reason. That there should be a difference due to the purified nature of the diet was unreasonable, so the studies to be presented in this paper were conducted using an amino acid diet developed at the Utah station. MATERIAS AND METHODS Results from four series of feeding tests with animo acid diets will be reported in this paper. Several amino acid diets containing different mixtures of methionine, cystine, and MHA 1 Journal paper number 94, Utah Agricultural Experiment Station, ogan, UT Associate Dean of Agriculture, California State Polytechnic University, Pomona, CA Poultry Science 59:80-84 were fed in each series. The composition of each sulfur amino acid (SAA) mixture is given with the results. Most of these mixtures were calculated to supply 48 mmol/kg of SAA. The basic diet fed in the last three series contained mmol/kg each of -methionine and - cystine; its composition is given in Table 1. The basic diet fed in the first series contained a slightly higher proportion of cystine, as indicated with the results, and all amino acid diets of this series contained 4% vegetable oil with extra glucose monohydrate to make up the difference. The indispensable amino acids, except for the SAA, are in these diets at the levels Dobson et al. (1964) found to be well balanced for the chick fed an 18% protein diet with a total of 8.95% indispensable amino acids. These levels are about 10% higher than what might be termed the minimum requirement for each. One mole of cystine was considered to add two moles of SAA. The 48 mmol/kg of SAA is near the minimum requirement and should make differences in efficacy of the mixtures more evident than the balanced ration level of.73% SAA. Except when noted, only the -isomer of each amino acid was used in preparing the diets. Amino acids were made by Ajinomoto, Inc., Tokyo, Japan, unless otherwise noted. Each was described by the supplier as having only the one amino acid in a chromatographically detectable quantity. The -methionine was Downloaded from at Penn State University (Paterno ib) on September 18,

2 MHA EFFICACY 81 TABE 1. Composition of the basal diet Ingredients Amino acid mix Amino acid mix Dicalcium phosphate Calcium carbonate Magnesium sulfate Magnesium trisilicate Aluminium hydroxide Trace mineral mixture a Vitamin mixture 0 Glucose monohydrate c Starch d Cellulose e Vegetable oil (g/kg) Arginine HC1 Arginine HistidineHCl'H 2 0 ysine HC1 eucine Isoleucine Valine Phenylalanine Tyrosine Tryptophan Cystine Threonine Glycine Proline Serine Asparagine Na glutamate"h 2 0 K glutamate'hjo Glutamic acid "Provided the following (mg): Fe, 51; Mn, 81; Zn, 67; Cu, 6; I, 6; Mo, 2; Cr,.68; and Se,.075. (g/239.: Provided the following: thiamin HC1, 8 mg; riboflavin, 8 mg; pyridoxine HCl, 8 mg; niacin, 100 mg; folic acid, 2 mg; biotin,.3 mg; vitamin B 12,.01 mg; menadione sodium bisulfite, 5.4 mg; D-alphatocopherol acetate, 40 mg; vitamin A, 10,000 IU, vitamin D 3, 1,500 ICU. c Cerelose, Corn Products Company, New York, NY; or Staleydex, A. E. Staley Manufacturing Company, Decatur, I. Powdered corn starch, A. E. Staley Manufacturing Co. e Alphacel, ICN Pharmaceuticals, Inc., Cleveland, OH assumed to supply 6.70 mmol/kg of SAA and the -cystine 8.32 mmol/kg. Two lots of MHA were used in these studies. The first was purchased from ICN Pharmaceuticals, Inc., Cleveland, OH, and the second was provided by Monsanto Chemical Co., St. ouis, MO. Based on Monsanto's description of the second sample, it was assumed to provide 5.50 mmol of SAA/g or the same amount assumed for the first sample. Male broiler type chicks (Hubbard) were used in all tests. They were fed a 21% protein diet based largely on corn, soybean, and meat and bone meals for either 8 or 12 days prior to being started on the test rations. Three chicks were used in some groups and only two in others. Four groups of chicks were fed each diet in each feeding test. Chicks were housed in electrically heated batteries with wire floors. The rations were fed from 8 through 19 days of age or 12 through 20 days of age; the period will be noted with the results of each series of tests. Each test involved feeding several amino acid diets containing different mixtures of methionine, cystine, and MHA. The practical diet used in starting the chicks also was included as one treatment in each feeding test. Individual chick weight gains and group feed consumption were recorded. The group average gains and the gain/feed ratios were analyzed by Duncan's multiple range test (1955). Data from groups fed the practical diet were not included in the analyses because the variance was so much lower with this diet. RESUTS AND DISCUSSION Table 2 gives the results of three series of tests in which different combinations of methionine, cystine, and MHA were used in the amino acid diet. The growth rate noted with the basic amino acid diet was about 91% of the rate noted with the practical type diet. These results are typical of results noted in other tests in which the two dietary types were fed. Even larger differences were noted when the practical Downloaded from at Penn State University (Paterno ib) on September 18, 2016

3 82 CHRISTENSEN ET A. TABE 2. Performances noted when chicks were fed amino acid diets with several sulfur amino acid combinations SAA in diet Cystine MHA Daily Gain/feed 1 gain 1 ratio (mmol/kg) (g) 28.4 a 23.7 C 18.4 d 26.3 b 28.7* a 21.lbc 15.7 d 23.5 a b.6 a 19.5C 21.2bc 10.9 e 26.4.ia 23.9 a b 22.5b 15.0C 15.2 C a.533b.467 c.556 b.601 a ab.606 b.520 d.616 b.652*.557c,57ic.415« a.620 ab.604 b.496 c.496 c h f d p ' ' ' Within each series, gains and gain/feed ratios not followed by the same letter are significantly different (P<.05). 'Results given for the first five diets in the top section are from a series of three tests with a total of 12 groups/treatment and those given in the lower two sections are each from a series of two tests with a total of 8 groups/treatment. Results with the practical diet and those with purified diets fed in only one of the three tests of the first series were not included in the statistical analyses. Tests of the first series were with chicks from twelve through twenty days of age. Those of the second and thirds series were with chicks from eight through nineteen days of age. 2 Addition was cysteine HC1 with the arginine HC1 of the diet being replaced by arginine. diet fed was higher in protein and contained added fat. It is recognized that growth rate with this amino acid diet is not as high as with a well balanced, 18% protein, practical diet, but it is higher than in most reports in the literature with amino acid diets. Chicks seem to hesitate to eat the purified diets when they are first placed before them. Feed regurgitation is much more common among the chicks fed the amino acid diets. These factors probably contribute to the slower growth rates. Gain/feed ratios are given in the table for the two types of diets, but these are considered less meaningful because of the wide difference in energy level of the diets. Consider the results with the first four diets listed in the upper section or in the middle section of Table 2. The MHA from ICN Pharmaceuticals was used in the series summarized in the upper section and that from Monsanto Chemical was used in the series summarized in the middle section. If the chick can convert MHA to methionine effectively and utilize the sulfur of methionine in cysteine production as effectively as is usually assumed, the four diets of each series should have produced similar performances. However, there were striking differences noted. The MHA-cystine combinations were least effective and produced per- Downloaded from at Penn State University (Paterno ib) on September 18, 2016

4 MHA EFFICACY 83 TABE 3. Effect of methionine level and form on chick performance when fed amino acid diets with half of the SAA as MHA CYS Dietary MHA evel Form Daily gain 1 Gain/feed (mmol/kg) D D D 2 (g) 15.7a 17.9 ab 19.4 b c 22.icde 23.0 d e 2l.7bcd 23.5 d <= 2i.icde.6 e.5 20a.560ab,570 bc.607cd.618 d.605 bcd.616 cd.606 bcd.633 d 1 b c d c ' ' ' ' Within each column values not followed by the same letter are significantly different (P<.05). 1 Values given are for the period from 8 to 19 days of age. Based on two feeding tests with a total of eight groups/treatment. The D- and -methionine used in the second through the fifth treatments were from California Corporation for Biochemical Research, os Angeles, CA. 2 Addition was homocysteine with 1 g/kg extra choline chloride. The homocysteine was from ICN Pharmaceuticals, Inc., Cleveland, OH. formances slightly poorer than the diets with 36 mmol/kg of the methionine-cystine combination. The methionine-mha combinations produced better performances but still not as good as with the methionine-cystine combinations. The fourth diet in each series could be regarded as a mixture of equal parts of the second and third, and for some tests it was prepared by mixing equal parts of the second and third diets. In each series this fourth diet produced better performance than either the second or third. Duncan's multiple range test (1955) was used in the statistical analyses reported in Table 2. When the gain or gain/feed ratio with the fourth diet was compared with the mean noted with the second and third diets by calculating the F value associated with this single degree of freedom, it was found that the probability of each difference arising by chance was less than.001 in both series. Thus, there appears to be an incompletely understood interaction among the three substances. The results are interpreted as indicating that MHA efficacy is influenced by the amount of methionine and cystine fed with it. This conclusion does not depend on knowing the exact composition of the MHA, methionine, and cystine used. A question might be raised as to why it was concluded that MHA efficacy differs when combinations primarily were compared in the tests. The reasons for this conclusion were 1) MHA was assumed to require amination before it could be utilized as a SAA, 2) methioninecystine combinations were much more effective than the MHA-cystine combinations or the MHA-cysteine combination, and 3) MHA alone was less effective than methionine alone. Somewhat similar results have been reported in several papers. Featherston and Horn (1974) and Marrett and Sunde (1965) reported that MHA is less effective than methionine in amino acid diets. The latter group also found that MHA was less effective when fed with cystine than when fed with methionine. Scott et al. (1966), Smith (1966), and Katz and Baker (1975) also have noted the increased efficacy of MHA when fed with a combination. The latter paper reports that one mixture of all three produced a gain as high as with the methionine-cystine combination, but feed efficiency was not as high. Baker (1977), in summarizing the Illinois work, states that the data clearly establish less than 100% utilization of MHA in amino acid diets (page 27). Differences in performance with the first and fourth diets listed in the upper two sections of Table 2 were small and statistically significant only in one series. Reviewing the results noted with all diets having about 50% of the SAA as MHA might lead to a conclusion that there was not 100% utilization of MHA with any com- Downloaded from at Penn State University (Paterno ib) on September 18, 2016

5 84 CHRISTENSEN ET A. bination fed. All three sections of Table 2 also give results noted with diets containing 25% of the SAA as MHA. When the remainder of the SAA was a mixture of equal parts of methionine and cystine, the mixture, and thus the MHA, appeared to be fully effective. It was recognized that the mixture of all three might appear to be fully effective because the 48 mmol/kg added was in excess of the SAA requirement. The two mixtures were compared in the second series at a level of 36 mmol/kg. This level was clearly suboptional yet the mixture of 25% MHA, 37.5% methionine, and 37.5% cystine was as effective as the mixture of 50% methionine and 50% cystine. The data certainly would not justify rejecting a hypothesis that the two mixtures were of equal value. In the third series a mixture of 25% MHA, 25% methionine, and 50% cystine appeared to be less effective than the mixture of equal parts of methionine and cystine. The data of Table 2 indicate that when MHA replaced all of the methionine in the diet, it was not as effective as when it was added to replace the cystine. Supposedly, additional metabolic steps are required for the conversion of MHA to cysteine vs. methionine. One would assume that some inefficiency is associated with each additional transformation, and it is difficult to understand why MHA should replace cysteine more effectively than methionine. It seemed possible that the activity of the enzyme systems functioning in the conversion of MHA to methionine might be affected by methionine level of the diet. If there is an effect, the two isomers of methionine might be quite different in activity. The tests summarized in Table 3 were conducted to compare the value of the - and D-isomers at low levels in diets in which half of the SAA was from MHA. The value of homocysteine also was tested. As little as 2.4 mmol/kg of -methionine significantly improved performance. Replacing one-fifth (4.8 mmol/kg) of the cystine with -methionine resulted in still further improvement in performance, and the improvement was as great as with any higher level of replacement. While the differences between the responses with the two isomers of methionine were not statistically significant, the -isomer produced the greatest responses. The response to homocysteine was lower, but not statistically different, than the response to methionine. These tests confirm reports of others that MHA is less efficacious in certain amino acid diets. Efficacy is affected by its level in the diet and by the levels of methionine and cystine also included. Its value was lowest when fed as the only SAA or with cystine alone. The methionine level of the diet was especially critical. Reduction in methionine fraction from three-eights to one-quarter, when MHA provided one-quarter of the SAA, seemed to make the mixture less effective. There is little to indicate what mechanism is involved in the difference in efficacy. The highly purified character of the amino acid diet is not itself responsible, although the dietary conditions needed to make MHA least effective can be developed only with amino acid diets. It would be almost impossible to formulate an 18% protein practical diet with less than 4.8 mmol/kg of methionine. Unless there are other factors that influence MHA efficacy, MHA should be effective in most practical diets based primarily on corn and soybean meal. REFERENCES Baker, D. H., Sulfur in nonruminant nutrition. Nat. Feed Ingredients Ass., West Des Moines, IA. Christensen, A. C, and J. O. Anderson, Factors affecting efficacy of methionine hydroxy analogue for chicks fed practical diets. Poultry Sci. 59: Dobson, D. C, J. O. Anderson, and R. E. Warnick, A determination of the essential amino acid proportions needed to allow rapid growth in chicks. J. Nutr. 82: Duncan, D. B., Multiple range and multiple F tests. Biometrics 11:1 42. Featherston, W. R., and G. W. Horn, Studies on the utilization of die a-hydroxy acid of methionine by chicks fed crystalline amino acid diets. Poultry Sci. 53: Katz, R. S., and D. H. Baker, Factors associated with utilization of the calcium salt of methionine hydroxy analogue by the young chick. Poultry Sci. 54: Marrett,. E., and M.. Sunde, The effect of other D amino acids on the utilization of the isomers of methionine and its hydroxy analogue. Poultry Sci. 44: Scott, H. M., M. Kelly, and R.. Huston, methionine versus methionine hydroxy analogue in basal diets containing either isolated soybean protein or crystalline amino acids. Poultry Sci. 45:1123. Smith, R. E., The utilization of -methionine, D-methionine and methionine hydroxy analogue by the growing chick. Poultry Sci. 45: Downloaded from at Penn State University (Paterno ib) on September 18, 2016