INTRODUCTION. Y. Pang and T. J. Applegate 1. Department of Animal Sciences, Purdue University, West Lafayette, IN 47906

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1 Effects of Dietary Copper Supplementation and Copper Source on Digesta ph, Calcium, Zinc, and Copper Complex Size in the Gastrointestinal Tract of the Broiler Chicken Y. Pang and T. J. Applegate 1 Department of Animal Sciences, Purdue University, West Lafayette, IN ABSTRACT An experiment was conducted to study the 100,000 to 30,000, 30,000 to 5,000, and <5,000 molecular effects of high dietary and source on the ph of digesta from the gizzard, duodenum + jejunum, ileum, and complex size of Ca, Zn, and in the duodenum + jejunum digesta of broiler chickens. Ross 308 male broiler chicks were randomly assigned to 32 cages and fed 1 of 4 treatments: control, 250 ppm from sulfate, 250 ppm from lysinate, and 250 ppm tribasic from chloride from 15 to 21 d of age. Copper supplementation and source had no effects on ph of gizzard or duodenum + jejunum contents. Copper supplementation, however, increased the ph of the ileal contents (P < 0.05) but was not affected by source. Neither supplementation nor source had significant effects on the solubility of Ca in the duodenum + jejunum contents, and the portions of Ca existing in different soluble complex sizes: >100,000, weight (MW) in the duodenum + jejunum digesta. About 80% of soluble Ca,, and Zn was associated with either large complexes (>100,000 MW) or small complexes (<5,000 MW). The solubility of supplemental in digesta was from 59 to 61% (P < 0.05), but solubility was not affected by source. No effects on portions of existing in different sizes of complexes in the supernatant were noted. Copper lysinate decreased the Zn solubility in the digesta (P < 0.05), but sulfate and tribasic chloride supplementation did not. Copper supplementation increased (P < 0.05) the percentage of Zn associated with large complexes (>100,000 MW) and decreased (P < 0.05) the percentage of Zn associated with small complexes (<5,000 MW; P < 0.05), thereby suggesting an antagonism between and Zn. Key words: chicken, copper, intestinal ph, solubility, zinc 2007 Poultry Science 86: INTRODUCTION Copper is an essential trace mineral for poultry. The requirement for broilers is 8 ppm (NRC, 1994). In the poultry industry, 125 to 250 ppm from sulfate pentahydrate ( SUL) is normally added in the United States as a growth promoter (Pesti and Bakalli, 1996). However, little information is known about the growth stimulation mechanisms. One of the possible mechanisms could be attributed to the bactericidal, bacteriostatic, or both, effects of on the gastrointestinal tract (GIT) microbiota (Hawbaker et al., 1961; Bunch et al., 1965). The bactericidal action of is dependent on the concentration of free ionic in solution (Zevenhuizen et al., 1979; Menkissoglu and Lindow, 1991), whereas the free ionic concentration is affected by ph and solubility. The ph in the GIT is an important factor to determine solubility of as well as other minerals in the digesta. The extent of mineral absorption is influenced by solubil Poultry Science Association Inc. Received September 7, Accepted November 27, Corresponding author: applegt@purdue.edu ity of the compound in the small intestine, because insoluble compounds are not available for the birds to absorb (Maenz et al., 1999). Therefore, intestinal ph and solubility of in the small intestine may eventually affect intestinal microbiota, mineral absorption, and bioavailability in birds. Absorption and bioavailability of minerals by birds may be affected by their distribution within different sizes of intestinal complexes (Shafey et al., 1991). Shafey et al. (1991) observed that high dietary Ca (2.18 and 2.26%) reduced the proportion of soluble Zn associated with small complexes (<5,000 MW) and increased the proportion of soluble Zn associated with large complexes (>100,000 MW). Further, high dietary Ca (2.26%) and high available P (0.83%) also reduced the proportion of soluble Mg associated with small complexes (<5,000 MW). Because the smaller complexes have relatively larger surface areas from which minerals can be exposed to enzymes or proteins to binding and transporting, the minerals associated with the smaller complexes may have a better chance for absorption. Therefore, the reduced proportion of soluble Mg and Zn associated with small complexes explains the mechanism of the reduced availability of Mg and Zn in high Ca and high available P diets (Shafey et al., 1991). 531

2 532 PANG AND APPLEGATE Similarly, sources may have different effects within the digestive tract and therefore different bioavailability. In addition, different sources of have different effects on birds. For example, tribasic chloride (TBCC) is more bioavailable to broilers than SUL and chemically less active than SUL in promoting the oxidation of vitamin E in feed (Luo et al., 2005). Pang and Applegate (2006) found that high concentrations of up to 500 ppm inhibited phytate-p hydrolysis in vitro at intestinal ph 5.5 and 6.5, with the inhibition dependent on source. Moreover, Banks et al. (2004b) observed that supplementation with 250 ppm from SUL or citrate decreased apparent P retention by 8.11 and 14.49%, respectively; however, supplementation with 250 ppm from lysinate ( LYS) or chloride did not affect apparent P retention. The different effects of these sources of on growth performance, vitamin E oxidation, and phytate-p utilization in birds may result from their different solubility. The most commonly used source of as a dietary supplement for poultry is SUL. It is normally used as a reference point for comparing bioavailability of various sources and is very soluble in both water and acidic solvents (Guo et al., 2001; Pang and Applegate, 2006). Other sources are being used and considered for use by the poultry producers. For example, LYS is a chelated compound with high solubility (99.4%) at ph 2.5 and low solubility (47.1%) at ph 6.5 (Pang and Applegate, 2006). Tribasic chloride is another relatively new product that is not soluble in water but soluble in acidic solutions (Cromwell et al., 1998; Pang and Applegate, 2006). These 3 sources show different in vitro solubility. Therefore, this experiment was conducted to determine how SUL, LYS, and TBCC influence digesta ph,, Ca, and Zn solubility and soluble complex size in the small intestine and P retention in broiler chickens. MATERIALS AND METHODS The experiment described herein was approved by the Purdue University Animal Care and Use Committee. Two hundred twenty-four male newly hatched Ross 308 broiler chicks were randomly placed in thermostatically controlled battery cages at d 0. They had ad libitum access to water via nipple and jug waterers and mash starter feed that met or exceeded the NRC (1994) requirements, with the exception of lower Ca (0.81%) and total P (tp; 0.62%) level until 14 d of age. At 5 d of age, all jug waterers were removed. The chicks were on a lighting schedule of 24 h light from hatch to d 2 and 22L:2D per day from d 3 to 21. All the chicks were then individually weighed, and 192 birds were randomly assigned to 32 cages, such that BW differences were minimized among cages. Each diet had 8 replicate cages, and each cage had 6 birds. The birds had ad libitum access to water and experimental diets from d 15 to 21. A 6-d experimental period was chosen to reduce the experimental time to limit tissue accumulation and minimize biliary concentration, Table 1. Starter and basal diet formulation and nutrient composition (%, as-fed basis) for broiler chickens Ingredient (%) Starter and basal diet Corn Soybean meal Soy oil 3.42 Salt 0.42 DL-Met 0.19 L-Lys 0.01 Limestone 1.44 Monocalcium phosphate 1.36 Vitamin and mineral premix Formulated analysis ME n, kcal/kg 3,100 CP, % Lys, % 1.20 Ca, % 0.90 Total P, % 0.71 Nonphytate P, % 0.45 Determined analysis CP, % Ca, % 0.81 Total P, % 0.62, ppm Vitamin and mineral premix supplied the following per kilogram of diet: vitamin A, 13,233 IU; vitamin D 3, 6,636 IU; vitamin E, 44.1 IU; vitamin K, 4.5 mg; thiamine, 2.21 mg; riboflavin, 6.6 mg; pantothenic acid, 24.3 mg; niacin, 88.2 mg; pyridoxine, 3.31 mg; folic acid, 1.10 mg; biotin, 0.33 mg; vitamin B 12, 24.8 g; choline, mg; Fe from ferrous sulfate, 50.1 mg; from sulfate, 7.7 mg; Mn from MnO 2, mg; Zn from ZnO 2, mg; I from ethylene diamine dihydroidide, 2.10 mg; and Se from Na selenite, 0.30 mg. thereby limiting confounding effects of biliary on dietary. The experimental diets were made from a large basal diet to minimize any mixing error. The formulation and nutrient analysis of the starter diet and the basal diet from which the experimental diets were derived are detailed in Table 1. The experimental diets were made by adding each source on top of the basal diet and remixing. The weight contribution of each source and subsequent diet nutrient was considered to be negligible. Experimental diets consisted of a control corn and soybean meal-based diet (basal diet), basal diet plus 250 ppm from SUL, basal diet plus 250 ppm from LYS (Monarch Nutritional Laboratories Inc., Park City, UT), and basal diet plus 250 ppm from TBCC [ 2 (OH) 3 Cl; Micronutrients, Indianapolis, IN]. Experimental design and analyzed dietary concentrations are presented in Table 2. Each experimental diet was analyzed for DM, tp, CP,, and Ca. Excreta samples were collected and pooled within each cage from 18 to 20 d. Excreta samples were freeze-dried and ground using a mill grinder (ZM100, F. Kurt Retsch Table 2. Experimental design and analyzed concentration Formulated Analyzed source (ppm) (ppm) sulfate lysinate Tribasic chloride

3 COPPER SOURCE AND DIGESTA MINERAL SIZE AND SOLUBILITY 533 GmbH., Haan, Germany) through a 0.5-mm screen. Samples were then analyzed for DM by being dried at 105 C overnight and tp according to the procedures of Sands et al. (2001). Birds and feed were weighed on 20 d of age by cage. On d 21, birds were euthanized by CO 2 asphyxiation. The gizzard digesta, duodenum + jejunum digesta, and ileum digesta of 1 bird per cage were immediately collected and placed into clean and preweighed beakers. Nine-fold of distilled deionized water of the digesta weight (wt/vol) was added to the beaker and stirred for 5 min. The ph of the solution was then measured by a ph meter (Corning Glass Works, Medfield, MA) and assumed as the ph of the intestinal contents. The digesta from the duodenum + jejunum and from the ileum of the remaining birds per cage were immediately excised into clean preweighed 50-mL centrifuge tubes and pooled by cage. Fractionation of Ca,, and Zn in the digesta samples was done using the combined techniques of centrifugation (to measure the solubility of these minerals) and ultrafiltration of the supernatant (to define the solubility of these minerals more accurately) modified from that of Wien and Schwartz (1985). The weights of the wet samples were determined. The samples were then centrifuged at 10,000 g for 30 min at 4 C. The precipitate samples were dried at 70 C for 24 h and then ashed at 600 C overnight for determination of the insoluble fraction. The ashed samples were digested according to Hurwitz (1980). Calcium,, and Zn were determined by atomic absorption spectrophotometry (AAS; FS240 AA, Varian Inc., Palo Alto, CA). The supernatant was poured into clean, 50-mL centrifuge tubes, and the volumes were recorded. The supernatant (7.5 ml) was added to the ultrafiltration cell with 100,000 molecular weight (MW) cut-off membrane (30625, Vivascience AG, Hannover, Germany) and centrifuged at 6,000 g until no liquid remained in the filter chamber. The supernatant was collected and the volume determined. Then, 4.5 ml of the supernatant was added to the ultrafiltration cell with 30,000 MW cut-off membrane and centrifuged at 10,000 g until no liquid remained in the filter chamber. The supernatant was collected, and the volume was determined. Lastly, 2.5 ml of the supernatant was added to the ultrafiltration cell with 5,000 MW cutoff membrane and centrifuged at 10,000 g until no liquid remained in the filter chamber. The supernatant was collected, and the volume was determined. The Ca,, and Zn concentrations in all the supernatants were measured using AAS. Feed samples were ground using a centrifugal mill grinder (ZM100, F. Kurt Retsch GmbH) through a 0.5- mm screen and dried at 105 C overnight for DM determination. Crude protein for the feed samples was measured with a LECO model FP 2000 N combustion analyzer (LECO Corp.; St. Joseph, MI). Total P was determined colorimetrically according to the procedures of Sands et al. (2001). Calcium,, and Zn were analyzed by AAS. Apparent P retention was determined by accounting for the differences between tp consumption in the feed and excretion in the excreta corrected to DM basis. In Vitro Solubility of Sources Solubility of 3 sources of ( SUL, LYS, and TBCC) was measured at concentrations of 83.4 mg of / L in 0.2 mm Gly-HCl (ph 2.5) and 0.2 mm Na acetate buffers (ph 5.5 and 6.5). The 83.4 mg of /L is an assumed concentration to be representative of digesta content based on 250 ppm dietary at a feed-to-water ratio of 1:2. Each source was mixed with 40 ml of buffer in triplicate, incubated at 41 C in a shaking water bath for 1 h, and filtered through 42- m Whatman filter paper for analysis (Brown and Zeringue, 1994) by AAS. Solubility was calculated as follows: solubility (%) = (soluble /total ) 100. Statistical Analysis Statistical analysis was completed by ANOVA, using the GLM procedures of SAS (SAS Institute Inc., Cary, NC). Differences between means were determined by Duncan s multiple comparison test when significance of the model was Table 3. Effects of concentration and source on the ph of gastrointestinal tract (GIT) contents in broiler chickens 1 GIT region (ph) concentration 2 Duodenum source (ppm) Gizzard + jejunum Ileum b sulfate a lysinate a TBCC a SD Probability of diet effects a,b Means within a column with no common superscripts differ significantly (P 0.05) as a result of a Duncan s means comparison. 1 Means represent 8 replicate cages of 1 bird per cage for tribasic chloride (TBCC) treatment and 7 replicates for the other 3 treatments. 2 Formulated dietary concentration.

4 534 PANG AND APPLEGATE Table 4. Effects of supplementation at 250 ppm and source on solubility and soluble mineral complex size in duodenum + jejunum contents in chickens 1 Mineral complex size 2 concentration 3 solubility 4 >100, ,000 to 30, ,000 to 5,000 5 <5,000 5 source (ppm) (%) (%) (%) (%) (%) b sulfate a lysinate a TBCC a SD Probability of diet effect a,b Means within a column with no common superscripts differ significantly (P 0.05) as a result of a Duncan s means comparison. 1 Means represent 8 replicate cages of 5 birds per cage for tribasic chloride (TBCC) treatment and 7 replicates for the other 3 treatments in mineral complex size. Means represent 7 replicate cages for TBCC and lysinate treatments and 6 replicates for control and sulfate treatments in solubility. 2 Fractionation of soluble using molecular weight cut-off membranes. 3 Formulated dietary concentration. 4 Percentage of in the duodenum + jejunum supernatant after centrifugation at 10,000 g at 4 C for 30 min of the total in the duodenum + jejunum digesta. 5 Percentage of in a range of molecular weight size of in the duodenum + jejunum supernatant after centrifugation of the duodenum + jejunum digesta at 10,000 g at 4 C for 30 min. RESULTS The effects of supplementation at 250 ppm and source in the diet on the ph of the GIT contents are shown in Table 3. Neither concentration nor source had significant effects on the ph of gizzard contents or duodenum + jejunum contents. Copper addition increased the ph of the ileal contents, but source had no effect. Not enough supernatant was derived after centrifugation from most of the ileal samples for determining mineral concentration and serial ultracentrifugation; therefore, the ileal samples were not analyzed. The analysis results of duodenum + jejunum samples are reported below. The effects of supplementation at 250 ppm and source on complex size in duodenum + jejunum contents in broiler chickens are shown in Table 4. Copper supplementation increased the solubility in the duodenum + jejunum contents. Copper source, however, had no effect on the solubility of. In the supernatant, 37 to 44% was bound to large complexes (>100,000 MW), 29 to 40% was bound to small complexes (<5,000 MW), and little (1 to 3%) existed in the complex sizes from 100,000 to 30,000 MW. Neither supplementation at 250 ppm nor source had significant effects on the portions of among the different sizes of complexes. The effects of supplementation at 250 ppm and source on the Ca complex size in duodenum + jejunum contents in broiler chickens are shown in Table 5. Neither supplementation at 250 ppm nor source had significant effects on the solubility of Ca in the duodenum + jejunum contents. In the supernatant, 58 to 59% Ca was bound to small complexes (<5,000 MW), 26 to 31% was bound to large complexes (>100,000 MW), and little Ca (1 to 4%) existed in the complex sizes from 100,000 to 30,000 MW. Neither supplementation at 250 ppm nor source had significant effects on the portions of Ca among the different size complexes. The effects of supplementation at 250 ppm and source on the Zn complex size in duodenum + jejunum contents in broiler chickens are shown in Table 6. Copper lysinate decreased the solubility of Zn in the duodenum + jejunum contents, but SUL and TBCC did not. In the supernatant, 50 to 60% Zn was bound to small complexes (<5,000 MW), 20 to 35% Zn was bound to large complexes (>100,000 MW), and little Zn (3 to 4%) existed in complex sizes from 100,000 to 30,000 MW. Copper addition increased the percentage of Zn associated with large complexes (>100,000 MW) and decreased the percentage of Zn associated with small complexes (<5,000 MW). Copper source had no effect on the portions of Zn among the different size complexes. At ph 2.5, 5.5, and 6.5, the order of the solubility of the 3 sources was SUL > LYS > TBCC (Table 7). As ph increased toward neutrality, solubility decreased. Copper sulfate pentahydrate decreased by the lowest rate, and LYS decreased by the moderate rate. Tribasic chloride decreased the most, with a solubility of 81% at ph 2.5 and with a solubility of 9% at ph 6.5. Copper supplementation at 250 ppm and source had no effect on BW gain, feed intake, ratio of gain to feed, or the apparent P retention (Table 8). DISCUSSION Maintenance of proper ph in the GIT is imperative for proper digestive enzyme functionality. Changes in ph outside normal ranges can result in decreased digestion and absorption and eventually reduced growth performance (Bristol, 2003). In addition, intestinal ph is also important to keep animals healthy by maintaining the balance between pathogenic and nonpathogenic microorganisms. For example, Dinsmore et al. (1997) reported that gastric acidity is protective against intestinal colonization and translocation of potentially pathogenic bacteria in a neonatal rabbit model.

5 COPPER SOURCE AND DIGESTA MINERAL SIZE AND SOLUBILITY 535 Table 5. Effects of supplementation at 250 ppm and source on Ca solubility and soluble mineral complex size in duodenum + jejunum contents in chickens 1 Mineral complex size 2 Ca concentration 3 solubility 4 >100, ,000 to 30, ,000 to 5,000 5 <5,000 5 source (ppm) (%) (%) (%) (%) (%) sulfate lysinate TBCC SD Probability of diet effect Means represent 8 replicate cages of 5 birds per cage for tribasic chloride (TBCC) treatment and 7 replicates for the other 3 treatments. 2 Fractionation of soluble Ca using molecular weight cut-off membranes. 3 Formulated dietary concentration. 4 Percentage of Ca in the duodenum + jejunum supernatant after centrifugation at 10,000 g at 4 C for 30 min of the total in the duodenum + jejunum digesta. 5 Percentage of Ca in a range of molecular weight size of Ca in the duodenum + jejunum supernatant after centrifugation of the duodenum + jejunum digesta at 10,000 g at 4 C for 30 min. In the current study, results showed that neither concentration nor source had significant effects on the ph of gizzard contents and duodenum + jejunum contents. This is important for birds to digest and absorb nutrients and remain healthy. However, addition increased the ph of the ileal contents. Surprisingly, feeding high dietary doses of did not promote one of the conditions often associated with an optimal gut ecosystem, namely reduced ph in the proventriculus, gizzard, and ileum (Jensen, 1998). Changes in HCl secretion by peptic cells in the proventriculus that regulate the ph of proventricular contents may explain why high intake did not affect the ph of the gizzard contents. Any tendency toward a change in intestinal ph in response to high intake may be balanced by changes in secretion of alkaline components such as bicarbonate ions secreted by the pancreas and glycocholate, taurocholate (Lindsay and March, 1967), and acidic components such as bile acids (Elkin et al., 1990), or all three. Because pancreatic and bile secretions enter the GIT near the anterior jejunum to digest nutrients, this may decrease buffering action of the ileum, which results from the increased ileum ph by higher dietary. This conclusion, however, is not in agreement with the report by Hurwitz and Bar (1968) that the ph of the ileum returned to normal more quickly than the jejunum when they were perfused in vivo with solutions of different ph. If microorganism profile was affected by high dietary, decreasing the concentrations of acids such as short chain fatty acids, lactate, and succinate, produced by microorganism fermentation, may have caused the increase in ileal ph. Derivations and ramifications of this ph shift may warrant further investigation and cannot be fully explained through measurements made in this experiment. Supplementation with 250 ppm greatly increased the portion of soluble in the duodenum + jejunum contents. However, source had no effect between different soluble complex sizes or the solubility, Table 6. Effects of supplementation at 250 ppm and source on Zn solubility and soluble mineral complex size in duodenum + jejunum contents in chickens 1 Mineral complex size 2 Zn concentration 3 solubility 4 >100, ,000 to 30, ,000 to 5,000 5 <5,000 5 source (ppm) (%) (%) (%) (%) (%) a b a sulfate a a b lysinate b a b TBCC ab a b SD Probability of diet effect a,b Means within a column with no common superscripts differ significantly (P 0.05) as a result of a Duncan s means comparison. 1 Means represent 7 replicate cages of 5 birds per cage for control and 6 replicates for the other 3 treatments in mineral complex size. Means represent 7 replicate cages for control and tribasic chloride (TBCC) treatments and 6 replicates for sulfate and lysinate treatments in Zn solubility. 2 Fractionation of soluble Zn using molecular weight cut-off membranes. 3 Formulated dietary concentration. 4 Percentage of Zn in the duodenum + jejunum supernatant after centrifugation at 10,000 g at 4 C for 30 min of the total Zn in the duodenum + jejunum digesta. 5 Percentage of Zn in a range of molecular weight size of Ca in the duodenum + jejunum supernatant after centrifugation of the duodenum + jejunum digesta at 10,000 g at 4 C for 30 min.

6 536 PANG AND APPLEGATE Table 7. The in vitro solubility of from different sources 1 Solubility concentration source (ppm) ph 2.5 (%) ph 5.5 (%) ph 6.5 (%) sulfate a a a lysinate b b b TBCC c c 9.11 c SEM Probability of solubility < < a c Means within a column with no common superscripts differ significantly (P 0.05) as a result of a Duncan s means comparison. 1 Means represent 3 replicates. 2 Tribasic chloride. thereby contradicting the in vitro results. In the in vitro study, these 3 sources of showed different solubility. Therefore, the in vitro solubility values may not provide good prediction of bioavailability. Part of the inconsistencies between in vitro and in vivo results may be due to either disassociation or chemical shifts of each respective source as it traverses the intestinal tract. Centrifugation of duodenum + jejunum contents elucidated that most (88 to 94%) of the Ca and Zn was in insoluble forms. High dietary had no effect on the solubility of Ca and the proportions of soluble Ca associated with different sizes of complexes. Copper lysinate supplementation decreased the soluble Zn percentage in the duodenum + jejunum contents, but SUL and TBCC did not. However, high diets reduced the proportion of soluble Zn associated with small complexes (<5,000 MW) and increased the proportion of soluble Zn associated with large complexes (>100,000 MW). Minerals associated with smaller complexes may have a better chance for absorption, because the smaller complexes have relatively larger surface areas from which minerals can be exposed to enzymes or proteins for binding and transporting. Therefore, high dietary may inhibit Zn from being digested and absorbed. And the change of portions of soluble Zn in different sizes of complexes may explain the antagonism between Zn and observed by Southern and Baker (1983a,b). Shafey et al. (1991) reported that high dietary Ca (2.18 and 2.26%) reduced the proportion of soluble Zn associated with small complexes (<5,000 MW) and increased the proportion of soluble Zn associated with large complexes (>100,000 MW). Therefore, the high dietary Ca (2.18 vs. 1.53%) affected the Zn in a similar way as the high dietary. Supplementation with 250 ppm from 3 different sources did not significantly affect apparent P retention, which contradicts the results reported by Banks et al. (2004a). In that report, supplementation with 250 ppm SUL from 9 to 21 d of age reduced apparent P retention by percentage units of the diet as compared with the control diet, whereas LYS did not affect P retention. This disparity may result from the different experiment period or different dietary tp concentration (0.62 vs. 0.49%). In addition, the effects of source on apparent P retention observed by Banks et al. (2004a,b) cannot be explained by in vivo solubility values. In conclusion, supplementation with 250 ppm did not affect ph of the contents of gizzard or duodenum + jejunum but increased that of the ileal contents. However, ramifications of the effect this ph has on the intestinal tract or luminal environment is uncertain. Neither supplementation at 250 ppm nor source had significant effects on the portions of Ca nor associated with different sizes of complexes. High dietary increased the solubility in the duodenum + jejunum contents. Table 8. Effects of supplementation at 250 ppm and source on the growth performance and apparent P retention in broiler chickens from 15 to 21 d of age 1 concentration 2 Feed intake BW gain Gain:feed Apparent P source (ppm) (g/d per bird) (g/d per bird) (g:g) retention (%) ab a sulfate ab ab lysinate b b TBCC a ab SD Probability of diet effect a,b Means within a column with no common superscripts differ significantly (P 0.05) as a result of a Duncan s means comparison. 1 Means represent 8 replicate cages of 6 birds per cage for tribasic chloride (TBCC) treatment, 7 replicates for sulfate and lysinate treatments, and 6 replicates for control in BW gain, feed intake, and gain:feed. Means represent 8 replicates for lysinate treatment and 7 replicates for the other 3 treatments in apparent P retention. 2 Formulated dietary concentration.

7 COPPER SOURCE AND DIGESTA MINERAL SIZE AND SOLUBILITY 537 Copper addition significantly increased the percentage of Zn associated with the large complexes (>100,000 MW) and decreased the percentage of Zn associated with the small complexes (<5,000 MW), therefore partly explaining the antagonism between dietary and Zn. REFERENCES Banks, K. M., K. L. Thompson, P. Jaynes, and T. J. Applegate. 2004a. The effects of copper on the efficacy of phytase, growth, and phosphorus retention in broiler chicks. Poult. Sci. 83: Banks, K. M., K. L. Thompson, J. K. Rush, and T. J. Applegate. 2004b. Effects of copper source on phosphorus retention in broiler chicks and laying hens. Poult. Sci. 83: Bristol, R. H Phytate update. Pages 1 4 in Mineral Writes. Iowa Limestone Co., Des Moines. Brown, T. F., and L. K. Zeringue Laboratory evaluations of solubility and structural integrity of complexed and chelated trace mineral supplements. J. Dairy Sci. 77: Bunch, R. J., J. T. McCall, V. C. Speer, and V. W. Hays Copper supplementation for weanling pigs. J. Anim. Sci. 24: Cromwell, G. L., M. D. Lindemann, H. J. Monegue, D. D. Hall, and D. E. Orr Jr Tribasic copper chloride and copper sulfate as copper sources for weanling pigs. J. Anim. Sci. 76: Dinsmore, J. E., R. J. Jackson, and S. D. Smith The protective role of gastric acidity in neonatal bacterial translocation. J. Pediatr. Surg. 32: Elkin, R. G., K. V. Wood, and L. R. Hagey Biliary bile acid profiles of domestic fowl as determined by high performance liquid chromatography and fast atom bombardent mass spectrometry. Comp. Biochem. Physiol. B 96: 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: Hawbaker, J. A., V. C. Speer, V. W. Hayes, and D. V. Catron Effects of copper sulfate and other chemoprophylactics in growing swine rations. J. Anim. Sci. 20: Hurwitz, W Official Methods of Analysis. 13th ed. Assoc. Off. Anal.Chem. Int., Washington, DC. Hurwitz, S., and A. Bar Activity, concentration, and lumen-blood electrochemical potential difference of calcium in the intestine of the laying hen. J. Nutr. 95: Jensen, B. B The impact of feed additives on the microbial ecology of the gut in young pigs. J. Anim. Feed Sci. 7: Lindsay, O. B., and B. E. March Intestinal absorption of bile salts in the cockerel. Poult. Sci. 46: Luo, X. G., F. Ji, Y. X. Lin, F. A. Steward, L. Lu, B. Liu, and S. X. Yu Effects of dietary supplementation with copper sulfate or tribasic copper chloride on broiler performance, relative copper bioavailability, and oxidation stability of vitamin E in feed. Poult. Sci. 84: Maenz, D. D., C. M. Engele-Schan, R. W. Newkirk, and H. L. Classen The effect of minerals and mineral chelators on the formation of phytase-resistant and phytase-susceptible forms of phytic acid in solution of canola meal. Anim. Feed Sci. Technol. 81: Menkissoglu, O., and S. E. Lindow Chemical form of copper on leaves in relation to the bactericidal activity of cupric hydroxide deposits on leaves. Phytopathology 81: NRC Nutritional Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Pang, Y., and T. J. Applegate Effects of copper source and concentration on in vitro phytate phosphorus hydrolysis by phytase. J. Agric. Food Chem. 54: Pesti, G. M., and R. I. Bakalli Studies on the feeding of cupric sulfate pentahydrate and cupric citrate to broiler chickens. Poult. Sci. 75: Sands, J. S., D. Ragland, C. Baxter, B. C. Joern, T. E. Sauber, and O. Adeola Phosphorus bioavability, growth performance, and nutrient balance in pigs fed high available phosphorus corn and phytase. J. Anim. Sci. 79: Shafey, T. M., M. W. McDonald, and J. G. Dingle Effects of dietary Ca and available phosphorus on digesta ph and on the availabilities of Ca, iron, magnesium and zinc from the intestinal contents of meat chicken. Br. Poult. Sci. 32: Southern, L. L., and D. H. Baker. 1983a. Eimeria acervulina infection and the zinc-copper interrelationship in the chick. Poult. Sci. 62: Southern, L. L., and D. H. Baker. 1983b. Zinc toxicity, zinc deficiency and zinc-copper interrelationship in Eimeria acervulina-infected chicks. J. Nutr. 113: Wien, E. M., and R. Schwartz Dietary calcium exchangeability and bioavailability in. Pages 1 16 in Nutritional Bioavailability of Calcium. C. Kies, ed. Am. Chem. Soc., Washington, DC. Zevenhuizen, L. P. T. M., J. Dolfing, E. J. Eshuis, and I. J. Scholten-Koerselman Inhibitory effects of copper on bacteria related to the free ion concentration. Microb. Ecol. 5: