Relative Bioavailability of Novel Amino Acid Chelates of Manganese and Copper for Chicks 1

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1 2003 Poultry Science Association, Inc. Relative Bioavailability of Novel Amino Acid Chelates of Manganese and Copper for Chicks 1 R. D. Miles, 2 P. R. Henry, V. C. Sampath, M. Shivazad, 3 and C. W. Comer Department of Animal Sciences, P.O. Box , University of Florida, Gainesville, Florida Primary Audience: Nutritionists, Researchers SUMMARY Two experiments were conducted to estimate the relative bioavailability of two new organic chelates of Mn or Cu compared with either reagent-grade Mn sulfate monohydrate or reagentgrade Cu sulfate pentahydrate as the standards in chicks fed for 21 d. Based on either bone Mn concentration, regressed on added dietary Mn concentration, or log 10 liver Cu concentration, regressed on non-zero added dietary Cu intake, and assuming a value of 100% for either reagentgrade standard, relative bioavailability values were 84 ± 3.5 and 96 ± 14%, for the Mn and Cu chelates, respectively. Key words: manganese, copper, bioavailability, chick, chelate 2003 J. Appl. Poult. Res. 12: DESCRIPTION OF PROBLEM Manganese and Cu are commonly added to diets for poultry. Supplemental forms include the inorganic sulfates or possibly oxides in more humid climates, as well as the organic chelates and complexes. In most experiments, the common supplemental form of organic chelates and complexes of the trace elements have been shown to be at least equal in bioavailability to the reagent-grade and feed-grade sulfates [1]. The two new amino acid chelates of Mn and Cu examined herein were produced by a method (U.S. Patent No. 6,323,354) resulting in organic trace minerals that are defined as metal amino acid chelates based on the Association of American Feed Control Officials [2] criteria. The prod- ucts are derived from strong base hydrolysis and neutralization of lipoproteins. The source of lipoproteins is fractured cell walls of microbes generated from biological syntheses. The resulting amino acids are mixed with transition metal salts to form the metal amino acid chelates. Fatty acids are resident in the final product, originating from the lipoprotein substrate. Accumulation of trace elements in specific target tissues during high-level dietary supplementation has been shown to be a suitable criterion for estimating relative bioavailability of Cu [3, 4] and Mn [5, 6] from inorganic and organic sources for poultry. Bioavailability estimates for Cu and Mn in chicks were similar when sources from the same lot number were supplemented at pharmacological levels [4, 5] or conventional 1 Florida Agricultural Experiment Station Journal Series No. R To whom correspondence should be addressed: miles@animal.ufl.edu. 3 Visiting Professor, University of Tehran, Tehran, Iran.

2 418 TABLE 1. Composition of basal diets A Ingredient Amount B (%) Ground yellow corn Soybean meal (48% CP) Corn oil 2.50 Iodized salt 0.40 DL-Methionine 0.25 Ground limestone 1.01 Dicalcium phosphate 1.72 Microingredients C 0.50 Cornstarch + Mn or Cu supplement D 1.00 A Dry matter was 88.7%, and Mn was 118 ppm DM basis in experiment 1. Dry matter was 88.8% and Cu was 18.4 ppm DM basis in experiment 2. B As-fed basis. C Ingredients supplied per kilogram of diet: vitamin A palmitate, 6,600 IU; cholecalciferol, 2,200 ICU; menadione dimethylpyrimidinol bisulfite, 2.2 mg; riboflavin, 4.4 mg; pantothenic acid, 13 mg; niacin, 40 mg; choline chloride, 500 mg; biotin, 1 mg; vitamin B 12,22µg; ethoxyquin, 125 mg; iron, 50 mg; copper, 6 mg; zinc, 40 mg; manganese, 60 mg; selenium, 0.2 mg. D Manganese and copper supplements added in place of equivalent weights of cornstarch. levels [7, 8]. The objective of this study was to estimate the relative bioavailabilities of two new amino acid chelates compared with either reagent-grade Mn sulfate monohydrate or reagentgrade Cu sulfate pentahydrate as the standards in chicks fed for 21 d. MATERIALS AND METHODS Bird Management and Experimental Treatments Two experiments were conducted to estimate the relative bioavailability of two new amino acid chelates of Mn or Cu compared to either reagent-grade Mn sulfate monohydrate (MnSO 4 H 2 O) or reagent-grade Cu sulfate pentahydrate (CuSO 4 5H 2 O) as the standards in chicks fed for 21 d. In experiment 1, the basal diet (Table 1) containing 118 ppm Mn (DM basis by analysis) was formulated to meet or exceed the nutrient requirements of starting chicks [9]. The reagent-grade Mn sulfate or Mn amino acid chelate (MnAA) were added to the basal diet at 0, 500, 1,000, or 1,500 ppm Mn. For experiment 2, the basal diet (Table 1) contained 18.4 ppm Cu (DM basis by analysis) and reagent-grade Cu sulfate or the Cu amino acid chelate (CuAA) were added to the basal diet at 0, 150, 300, or 450 ppm Cu. In both experiments JAPR: Research Report chicks were assigned randomly to treatments and fed experimental diets for 21 d. There were six pens of five male Ross Ross broiler chicks for each of seven dietary treatments for a total of 210 chicks in a completely randomized design in each experiment. Birds were housed in two Petersime brooder units with stainless steel feeders, waterers, and gates and maintained on a 24- h constant light schedule. Tap water (containing no detectable Mn or Cu by analysis) and feed were available for ad libitum consumption. Chicks were managed according to guidelines approved by the University of Florida Animal Care and Use Committee. At the end of both experiments, feed intake was recorded per pen and birds were weighed individually. The heaviest and lightest chicks in each pen were discarded and remaining chicks were then killed by cervical dislocation. In experiment 1, right tibias were removed and frozen individually in heat-sealed plastic bags for Mn analysis. In experiment 2, a 5- to 6-g sample of liver (fresh weight) uncontaminated by bile was collected and frozen for Cu analysis. Chemical Analysis Manganese and Cu concentrations in diets, water, supplemental sources, and tissues were determined by flame atomic absorption spectrophotometry with a Perkin-Elmer Model 5000 [10]. Samples of diets and supplemental sources were dried at 105 C for 12 h, then were dry ashed at 550 C for 12 h, solubilized in HCl, and filtered through 42 Whatman paper. Bones were boiled for approximately 10 min in deionized water and cleaned of all soft tissue. Tibias with associated cartilages were dried for 12 h at 105 C, extracted in a Soxhlet apparatus with petroleum ether for 48 h prior to ashing, and then solubilized as indicated above. Livers were washed in deionized H 2 O to remove all blood, then blotted with ashless filter paper, cut with stainless steel scissors into pieces (<4 mm) and dried at 105 C for 12 h. The samples were preashed in 50% (v:v) HNO 3 on a hot plate before dry-ashing in a muffle furnace, then solubilized and filtered as described for diets. Water was concentrated tenfold by evaporation on a hotplate. Standards were matched for macroelement and acid concentrations as needed for Mn or Cu analysis. Citrus leaves-1572 and bovine liver-

3 MILES ET AL.: MANGANESE AND COPPER BIOAVAILABILITY b standard reference materials [11] were used as internal standards. Solubility of 0.1 g of each of the Mn or Cu sources was determined in 100 ml of deionized H 2 O, 0.4% HCl, 2% citric acid, or neutral ammonium citrate after 1 h of constant stirring at 37 C [12]. Statistical Analysis Data were analyzed by two-way ANOVA by the General Linear Models (GLM) procedure with a model that included Mn source, dietary Mn concentration, and their interaction in experiment 1, and Cu source, dietary Cu concentration, and their interaction in experiment 2 [13]. Pen was the experimental unit. For experiment 1, multiple linear regression of bone Mn concentration on added dietary Mn concentration was calculated using GLM. Relative bioavailability estimates were calculated using slope ratios, with reagent-grade Mn sulfate given a relative value of 100% [14]. Standard errors of the slopes and slope ratios were estimated with the method of error propagation as described by Kempthorne and Allmaras [15]. In experiment 2, simple linear regression of log 10 liver Cu concentration on added dietary Cu intake from non-zero added levels was used to calculate slope ratios. The log 10 transformation was needed due to heterogeneous variances, and Cu intake rather than dietary concentration was used as the independent variable because feed intake differed among treatment groups. Otherwise, calculations were as described for experiment 1, using Cu sulfate as the standard source. RESULTS AND DISCUSSION Solubility The Mn and Cu amino acid chelates evaluated were both 10 to 20% less soluble in water than their respective sulfate form, but their solubilities were within 10% of the respective sulfate s solubility in 0.4% HCl, 2% citric acid, and neutral ammonium citrate (Table 2). Reagentgrade Cu sulfate and CuAA chelate were less soluble in all solvents tested than the reagentgrade Mn sulfate, despite the fact that Guo et al. [3] reported values in all four solvents for the reagent-grade Cu sulfate and five organic Cu chelates and complexes in excess of 90%. High solubility values for reagent-grade Mn sulfate were also reported by Henry et al. [6]. Experiment 1 There was no effect (P > 0.10) of Mn source or dietary Mn concentration on feed intake of chicks fed for 21 d (Table 3); however, BW decreased (P < 0.05) as intake of Mn increased from an average of 738 g in birds given the control diet to 698 g in those given 1,500 ppm Mn from either source. Black et al. [16] reported lower feed intake in chicks fed 3,000 ppm Mn as reagent-grade Mn sulfate for 1, 2, or 3 wk. Southern and Baker [17] reported depressed weight gain in chicks fed 3,000 ppm Mn as the sulfate from 8 to 22 days posthatching. Wong- Valle et al. [18] and Henry et al. [6] reported no effect of dietary Mn concentration on feed intake or BW of chicks fed Mn sulfate or several forms of feed-grade Mn oxide or a Mn methionine complex at up to 3,000 ppm added Mn for 3 wk. Percentage of bone ash also decreased (P < 0.01) in birds given the greatest Mn supplementation level compared with those given other diets, which was unexpected in light of results reported earlier [6, 16], in which similar dietary Mn concentrations had no effect on bone ash. Black et al. [5] reported differences in percentage of bone ash in chicks fed three Mn sources at up to 4,000 ppm added Mn; however, there was no apparent pattern to the changes as found in the present study. There were effects (P < ) of Mn source and added Mn concentration on bone Mn concentration, as well as an interaction (P < ) in which bone Mn concentration was similar in chicks fed up to 1,000 ppm Mn from either source, but was greater in chicks given the standard source at 1,500 ppm compared with those fed the Mn AA chelate. This phenomenon probably represents a change in storage location or increase in excretion, but without a compartmental model describing kinetics of a tracer dose of Mn in chicks it is impossible to be more specific. A Mn methionine complex fed at similar dietary concentrations did not react similarly in an earlier experiment [6]. Henry et al. [6] reported bone Mn concentrations that were greater for the organic Mn complex than the standard reagent-grade sulfate at all levels of supplementation. A relative bioavailability value of 108% for the Mn methio-

4 420 JAPR: Research Report TABLE 2. Solubility of supplemental mineral sources A Item MnSO 4 H 2 O (%) Mn chelate (%) CuSO 4 5H 2 O (%) Cu chelate (%) Solvent Water % citric acid % HCl Neutral ammonium citrate Total Mn or Cu in source A From1hofconstant stirring at 37 C; expressed as a percentage of total element in the compounds, by analysis. nine compared with the sulfate was reported in that study. Baker and Halpin [19] fed 1,000 ppm added Mn as either a Mn proteinate chelate or MnSO 4 H 2 O to chicks in a casein-dextrose diet from 8 to 22 d post-hatching and found a relative value of 95% for the organic Mn based on bone Mn concentrations. Relative bioavailability values estimated from multiple linear regression slope ratios of bone Mn concentration (ppm ash weight basis) on added dietary Mn concentration (ppm) are found in Table 4. The coefficient of determination indicated an excellent fit to the linear model (R 2 = 0.94). The response for the standard source, reagent-grade Mn sulfate was set at 100%. The estimated relative bioavailability value was 84 ± 3.5 for the Mn chelate and the slopes differed (P < 0.05). A regression in which the highest level of supplementation (1,500 ppm) from both sources was omitted, resulted in a bioavailability estimate of 100%. Experiment 2 There was a linear effect (P < 0.001) of added Cu on feed intake in which increasing dietary concentrations had an inverse relationship in chicks given either of the supplemental sources (Table 5). Feeding birds the CuAA or Cu sulfate decreased (P < 0.05) feed intake compared with birds given the control diet. Body weight also decreased (P < 0.01) as intake of Cu increased to a level of 450 ppm from either source. Guo et al. [3] reported no effect of Cu source or level on feed intake in an experiment with similar amounts of Cu supplementation and duration. In a second experiment, however, in- TABLE 3. Effect of source and concentration of Mn on performance and bone Mn of chicks fed for 21 days in experiment 1 Total feed Added Mn A intake B BW B Bone ash C Bone Mn C Source (ppm) (g/bird) (g) (%) (ppm, ash basis) Control MnSO 4 H 2 O ,000 1, , Mn chelate 500 1, , , Pooled SE Probability ANOVA Source (S) NS D NS NS Added Mn (M) NS S M NS NS NS A Basal diet contained 118 ppm Mn, dry-matter basis. B Each value represents the mean of six pens of five chicks. C Each value represents the mean of six pens of three chicks (heaviest and lightest chicks in each pen deleted). D P > 0.10.

5 MILES ET AL.: MANGANESE AND COPPER BIOAVAILABILITY 421 TABLE 4. Multiple linear regression of bone Mn concentration (ppm ash weight) on added dietary Mn concentration (ppm) in experiment 1 A Source Slope ± SE Relative value ± SE MnSO 4 H 2 O ± a 100% Mn chelate ± b 84 ± 3.5 a,b Slopes differ (P < 0.05). A Intercept = 2.64; R 2 = 0.94; standard deviation = take decreased as Cu supplementation increased. Miles et al. [20] also reported decreased feed intake as the result of Cu supplementation at levels up to 450 ppm. Ledoux et al. [4] noted decreased feed intake in chicks given up to 450 ppm Cu as either sulfate or acetate but not in those fed Cu carbonate or oxide at similar dietary concentrations. Liver Cu concentration increased linearly (P < ) with increasing dietary Cu supplementation (Table 5). Chicks given Cu sulfate accumulated more (P < ) Cu in liver than birds given CuAA. The concentration of Cu found in liver was generally less than that reported previously in chicks given similar levels of supplementation for the same length of time [3, 4, 20, 21]. Despite the fact that greater liver Cu concentrations were found with Cu sulfate compared to the CuAA, the relative values from linear regression equations (Table 6) in which Cu intake rather than dietary concentration was used as the independent variable, due to differences in feed intake, were similar for Cu sulfate (100%) and CuAA chelate (96.4 ± 14.3%). Guo et al. [3] reported bioavailability values for organic Cu sources relative to that from Cu sulfate, ranging from 105 to 124% in two experiments. Pott et al. [21] reported that a Cu lysine complex was 99 ± 4.8% as available for chicks as reagentgrade Cu sulfate. Aoyagi and Baker [22] found a value of 120% for Cu lysine relative to reagentgrade Cu sulfate for chicks. The bioavailability values found in the present experiments for these two new chelated mineral products were similar to estimates for other organic products tested in other studies, which generally range from 90 to 120% [1] of a highly TABLE 5. Effect of source and concentration of Cu on performance and log 10 liver Cu concentration of chicks fed for 21 d in experiment 2 Total feed Added Cu A intake B BW B Liver Cu C Source (ppm) (g/bird) (g/bird) (ppm, dry basis) Control 0 1, CuSO 4 5H 2 O 150 1, , Cu chelate 150 1, Pooled SE Probability ANOVA Source (S) 0.05 NS D Added Cu (C) S C NS NS NS A Basal diet contained 18.4 ppm Cu, DM basis. B Each value represents the mean of six pens of five chicks. C Each value represents the mean of six pens of three chicks (heaviest and lightest chicks in each pen deleted). Log (10) transformation. D P > 0.10.

6 422 JAPR: Research Report TABLE 6. Linear regression of log 10 liver Cu concentration (ppm dry weight) on added dietary Cu intake (mg) in experiment 2 Source Intercept Slope ± SE Relative value ± SE r 2 SD CuSO 4 5H 2 O ± % Cu chelate ± ± soluble reagent-grade inorganic form. These two chelates are possibly more available than feedgrade sulfates with less hydration than the reagent-grade materials or the less soluble feedgrade carbonates or oxides, which weren t tested in the present experiments but have been reported as less available than reagent-grade sulfates of numerous required trace elements [1]. Feed-grade zinc sulfate, which is a monohydrate form compared to the heptahydrate reagentgrade material, has been shown to be less available for chicks [23] and turkeys [24]. Soares [25] also reported that hydrated forms of dicalcium phosphate were more available than anhydrous forms. CONCLUSIONS AND APPLICATIONS 1. The relative bioavailability of Mn in the MnAA chelate was found to be approximately 15% lower than that of reagent-grade Mn sulfate for chicks when all data were included in the regression model, but equal when the greatest level of supplementation was eliminated. 2. The relative bioavailability of Cu in the CuAA chelate was found to be similar to that of reagentgrade Cu sulfate for chicks. 3. Due to their lower solubility in water, both of these new compounds should be acceptable supplemental sources of Mn or Cu for inclusion in diets for poultry and may be less prone than sulfates to problems with caking in humid climates. REFERENCES AND NOTES 1. Ammerman, C. B., D. H. Baker, and A. J. Lewis, ed Bioavailability of Nutrients for Animals: Amino Acids, Minerals, and Vitamins. Academic Press, San Diego, CA. 2. American Association of Feed Control Officials Official Publication. Am. Assoc. Feed Control Officials, Oxford, IN. 3. Guo, R., P. R. Henry, R. A. Holwerda, J. Cao, R. C. Littell, R. D. Miles, and C. B. Ammerman Chemical characteristics and relative bioavailability of supplemental organic copper sources for poultry. J. Anim. Sci. 79: Ledoux, D. R., P. R. Henry, C. B. Ammerman, P. V. Rao, and R. D. Miles Estimation of the relative bioavailability of inorganic copper sources for chicks using tissue uptake of copper. J. Anim. Sci. 69: Black, J. R., C. B. Ammerman, P. R. Henry, and R. D. Miles Biological availability of manganese sources and effects of high dietary manganese on tissue mineral composition of broilertype chicks. Poult. Sci. 63: Henry, P. R., C. B. Ammerman, and R. D. Miles Relative bioavailability of manganese in a manganese-methionine complex for broiler chicks. Poult. Sci. 68: Zanetti, M. A., P. R. Henry, C. B. Ammerman, and R. D. Miles Estimation of the relative bioavailability of copper sources in chicks fed on conventional dietary amounts. Brit. Poult. Sci. 32: Henry, P. R., C. B. Ammerman, and R. D. Miles Bioavailability of manganese sulfate and manganese monoxide in chicks as measured by tissue uptake of manganese from conventional dietary levels. Poult. Sci. 65: National Research Council Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. 10. Anonymous Analytical Methods for Atomic Absorption Spectrophotometry. Perkin-Elmer Corp., Norwalk, CT. 11. National Institute of Standards and Technology, Gaitherburg, MD. 12. Watson, L. T., C. B. Ammerman, S. M. Miller, and R. H. Harms Biological assay of inorganic manganese for chicks. Poult. Sci. 49: SAS Institute SAS/STAT User s Guide. Version 6.04 ed. SAS Institute Inc., Cary, NC. 14. Littell, R. C., A. J. Lewis, and P. R. Henry Statistical evaluation of bioavailability assays. Pages 5 33 in Bioavailability of Nutrients for Animals: Amino Acids, Minerals, and Vitamins. C. B. Ammerman, D. H. Baker, and A. J. Lewis, ed. Academic Press, San Diego, CA. 15. Kempthorne, O., and R. R. Allmaras Errors of observation. Part 1. Page 1 in Methods of Soil Analysis. C. A. Black, ed. Am. Soc. Agron., Madison, WI. 16. Black, J. R., C. B. Ammerman, P. R. Henry, and R. D. Miles Effect of dietary manganese and age on tissue trace mineral composition of broiler-type chicks as a bioassay of manganese sources. Poult. Sci. 64:

7 MILES ET AL.: MANGANESE AND COPPER BIOAVAILABILITY Southern, L. L., and D. H. Baker Eimeria acervulina infection in chicks fed deficient or excess levels of manganese. J. Nutr. 113: Wong-Valle, J., C. B. Ammerman, P. R. Henry, P. V. Rao, and R. D. Miles Bioavailability of manganese from feed grade manganese oxides for broiler chicks. Poult. Sci. 68: Baker, D. H., and K. M. Halpin Efficacy of a manganese-protein chelate compared with that of manganese sulfate for chicks. Poult. Sci. 66: Miles, R. D., S. F. O Keefe, P. R. Henry, C. B. Ammerman, and X. G. Luo The effect of dietary supplementation with copper sulfate or tribasic copper chloride on broiler performance, relative copper bioavailability and dietary prooxidant activity. Poult. Sci. 77: Pott, E. B., P. R. Henry, C. B. Ammerman, A. M. Merritt, J. B. Madison, and R. D. Miles Relative bioavailability of copper in a copper-lysine complex for chicks and lambs. Anim. Feed Sci. Technol. 45: Aoyagi, S., and D. H. Baker Nutritional evaluation of copper-lysine and zinc-lysine complexes for chicks. Poult. Sci. 72: Sandoval, M., P. R. Henry, C. B. Ammerman, R. D. Miles, and R. C. Littell Relative bioavailability of supplemental inorganic zinc sources for chicks. J. Anim. Sci. 75: Sullivan, T. W The availability of zinc in various compounds to Broad Breasted Bronze poults. Poult. Sci. 40: Soares, J. H., Jr Phosphorus. Pages in Bioavailability of Nutrients for Animals: Amino Acids, Minerals, and Vitamins. C. B. Ammerman, D. H. Baker, and A. J. Lewis, ed. Academic Press, San Diego, CA. Acknowledgments The authors wish to acknowledge IMC Feed Ingredients, Lake Forest, IL, for supplying the organic chelates and funds in support of this research.