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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

Department of Animal Sciences, Purdue University, West Lafayette, IN 47906

¹ Corresponding author: applegt{at}purdue.edu

	ABSTRACT

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An experiment was conducted to study the effects of high dietary Cu and Cu source on the pH of digesta from the gizzard, duodenum + jejunum, ileum, and complex size of Ca, Zn, and Cu 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 Cu from sulfate, 250 ppm Cu from lysinate, and 250 ppm tribasic Cu from chloride from 15 to 21 d of age. Copper supplementation and Cu 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 Cu source. Neither Cu supplementation nor Cu 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, 100,000 to 30,000, 30,000 to 5,000, and <5,000 molecular weight (MW) in the duodenum + jejunum digesta. About 80% of soluble Ca, Cu, and Zn was associated with either large complexes (>100,000 MW) or small complexes (<5,000 MW). The solubility of supplemental Cu in digesta was from 59 to 61% (P < 0.05), but solubility was not affected by Cu source. No effects on portions of Cu existing in different sizes of complexes in the supernatant were noted. Copper lysinate decreased the Zn solubility in the digesta (P < 0.05), but Cu sulfate and tribasic Cu 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 Cu and Zn.

Key Words: chicken • copper • intestinal pH • solubility • zinc

	INTRODUCTION

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Copper is an essential trace mineral for poultry. The Cu requirement for broilers is 8 ppm (NRC, 1994). In the poultry industry, 125 to 250 ppm Cu from Cu sulfate pentahydrate (Cu 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 Cu on the gastrointestinal tract (GIT) microbiota (Hawbaker et al., 1961; Bunch et al., 1965). The bactericidal action of Cu is dependent on the concentration of free ionic Cu in solution (Zevenhuizen et al., 1979; Menkissoglu and Lindow, 1991), whereas the free ionic Cu concentration is affected by pH and solubility.

The pH in the GIT is an important factor to determine solubility of Cu as well as other minerals in the digesta. The extent of mineral absorption is influenced by solubility 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 Cu 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).

Similarly, Cu sources may have different effects within the digestive tract and therefore different bioavailability. In addition, different sources of Cu have different effects on birds. For example, tribasic Cu chloride (TBCC) is more bioavailable to broilers than Cu SUL and chemically less active than Cu SUL in promoting the oxidation of vitamin E in feed (Luo et al., 2005). Pang and Applegate (2006) found that high concentrations of Cu up to 500 ppm inhibited phytate-P hydrolysis in vitro at intestinal pH 5.5 and 6.5, with the inhibition dependent on Cu source. Moreover, Banks et al. (2004b) observed that supplementation with 250 ppm Cu from Cu SUL or Cu citrate decreased apparent P retention by 8.11 and 14.49%, respectively; however, supplementation with 250 ppm Cu from Cu lysinate (Cu LYS) or Cu chloride did not affect apparent P retention. The different effects of these sources of Cu on growth performance, vitamin E oxidation, and phytate-P utilization in birds may result from their different solubility.

The most commonly used source of Cu as a dietary supplement for poultry is Cu SUL. It is normally used as a reference point for comparing bioavailability of various Cu sources and is very soluble in both water and acidic solvents (Guo et al., 2001; Pang and Applegate, 2006). Other Cu sources are being used and considered for use by the poultry producers. For example, Cu 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 Cu chloride is another relatively new Cu product that is not soluble in water but soluble in acidic solutions (Cromwell et al., 1998; Pang and Applegate, 2006). These 3 Cu sources show different in vitro solubility. Therefore, this experiment was conducted to determine how Cu SUL, Cu LYS, and TBCC influence digesta pH, Cu, Ca, and Zn solubility and soluble complex size in the small intestine and P retention in broiler chickens.

	MATERIALS AND METHODS

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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 Cu accumulation and minimize biliary Cu concentration, thereby limiting confounding effects of biliary Cu on dietary Cu.

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 Cu source on top of the basal diet and remixing. The weight contribution of each Cu 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 Cu from Cu SUL, basal diet plus 250 ppm from Cu LYS (Monarch Nutritional Laboratories Inc., Park City, UT), and basal diet plus 250 ppm from TBCC [Cu₂(OH)₃Cl; Micronutrients, Indianapolis, IN]. Experimental design and analyzed dietary Cu concentrations are presented in Table 2. Each experimental diet was analyzed for DM, tP, CP, Cu, and Ca.

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, Cu, 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 x 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, Cu, 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, Viva-science AG, Hannover, Germany) and centrifuged at 6,000 x 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 x 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 x g until no liquid remained in the filter chamber. The supernatant was collected, and the volume was determined. The Ca, Cu, 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, Cu, 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 Cu Sources
Solubility of 3 sources of Cu (Cu SUL, Cu LYS, and TBCC) was measured at concentrations of 83.4 mg of Cu/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 Cu/L is an assumed concentration to be representative of digesta content based on 250 ppm dietary Cu at a feed-to-water ratio of 1:2. Each Cu 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-µm Whatman filter paper for Cu analysis (Brown and Zeringue, 1994) by AAS. Solubility was calculated as follows:

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 0.05.

	RESULTS

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The effects of Cu supplementation at 250 ppm and Cu source in the diet on the pH of the GIT contents are shown in Table 3. Neither Cu concentration nor Cu source had significant effects on the pH of gizzard contents or duodenum + jejunum contents. Copper addition increased the pH of the ileal contents, but Cu source had no effect.

The effects of Cu supplementation at 250 ppm and Cu source on Cu complex size in duodenum + jejunum contents in broiler chickens are shown in Table 4. Copper supplementation increased the Cu solubility in the duodenum + jejunum contents. Copper source, however, had no effect on the solubility of Cu. In the supernatant, 37 to 44% Cu was bound to large complexes (>100,000 MW), 29 to 40% Cu was bound to small complexes (<5,000 MW), and little Cu (1 to 3%) existed in the complex sizes from 100,000 to 30,000 MW. Neither Cu supplementation at 250 ppm nor Cu source had significant effects on the portions of Cu among the different sizes of complexes.

	DISCUSSION

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In the current study, results showed that neither Cu concentration nor Cu 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, Cu addition increased the pH of the ileal contents. Surprisingly, feeding high dietary doses of Cu 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 Cu intake did not affect the pH of the gizzard contents. Any tendency toward a change in intestinal pH in response to high Cu 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 Cu. 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 Cu, 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 Cu greatly increased the portion of soluble Cu in the duodenum + jejunum contents. However, Cu source had no effect between different soluble Cu complex sizes or the Cu solubility, thereby contradicting the in vitro results. In the in vitro study, these 3 sources of Cu showed different solubility. Therefore, the in vitro solubility values may not provide good prediction of Cu bioavailability. Part of the inconsistencies between in vitro and in vivo results may be due to either disassociation or chemical shifts of each respective Cu 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 Cu 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 Cu SUL and TBCC did not. However, high Cu 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 Cu 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 Cu 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 Cu.

Supplementation with 250 ppm Cu from 3 different Cu 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 Cu SUL from 9 to 21 d of age reduced apparent P retention by 0.029 percentage units of the diet as compared with the control diet, whereas Cu 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 Cu 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 Cu 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 Cu supplementation at 250 ppm nor Cu source had significant effects on the portions of Ca nor Cu associated with different sizes of complexes. High dietary Cu increased the Cu solubility in the duodenum + jejunum contents. 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 Cu and Zn.

Received for publication September 7, 2006. Accepted for publication November 27, 2006.

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