Effect of Organically Complexed Copper, Iron, Manganese, and Zinc on Broiler Performance, Mineral Excretion, and Accumulation in Tissues

2007 Poultry Science Association, Inc. Effect of Organically Complexed Copper, Iron, Manganese, and Zinc on Broiler Performance, Mineral Excretion, and Accumulation in Tissues Y. M. Bao,* M. Choct, P. A. Iji,* 1 and K. Bruerton *School of Environmental and Rural Science, and Australian Poultry Cooperative Research Centre, University of New England, Armidale, New South Wales 2351, Australia; and Protea Park Nutrition, Palm Beach, Sorrento, Queensland 4217, Australia Primary Audience: Nutritionists, Nutrition Researchers, Feeding Managers SUMMARY Supplementation of trace minerals with a large safety margin in broiler chickens has resulted in a high level of mineral excretion that ends up in the environment. Organically complexed trace minerals (organic minerals) may be able to replace the inorganic trace minerals, because the former appear to have a greater bioavailability. Therefore, a 29-d cage study that included diets with supplemental trace minerals from organic and inorganic sources based on a trace mineral deficient control diet was conducted to examine the possible response of broiler chickens to organic mineral supplements. The results showed that supplementation with 4 mg of Cu and 40 mg each of Fe, Mn, and Zn from organic sources may be sufficient for normal broiler growth to 29 d of age. It is possible to use these lower levels of organic trace minerals in broiler diets to avoid high levels of trace mineral excretion. Key words: broiler, organic copper, iron, manganese, zinc, mineral excretion 2007 J. Appl. Poult. Res. 16:448 455 DESCRIPTION OF PROBLEM Trace minerals, such as Cu, Fe, Mn, and Zn, are essential for broiler growth and are involved in many digestive, physiological, and biosynthetic processes within the body. They function primarily as catalysts in enzyme systems within cells or as parts of enzymes. They are also constituents of hundreds of proteins involved in intermediary metabolism, hormone secretion pathways, and immune defense systems [1]. Traditionally, these trace minerals are supplemented in the form of inorganic salts, such as sulfates, oxides, and carbonates, to provide levels of minerals that pre- vent clinical deficiencies, allow the bird to reach its genetic growth potential, or both. Despite enormous advances in poultry production and technology, research into trace mineral nutrition has lagged behind other areas of nutrition. In 2006, the BW of a meat chicken reached 2 kg in 35 d, down from 64 d in 1979. However, the trace mineral requirements for broilers have still been thought to be the same level as those recommended by the NRC in the early 1990s [2], some of which are based on data as far back as the 1950s. Although most of the increase in BW via genetic selection has been an indirect response to selection for appetite, in- 1 Corresponding author: piji@une.edu.au

BAO ET AL.: RESPONSE OF BROILERS TO ORGANIC MINERALS 449 creased body growth has resulted in skeletal problems, which may be related to poor mineral nutrition. It is thus reasonable to consider the current NRC recommendation as unsuitable for the needs of the modern bird. Actually, the industry is still using a large safety margin in feed formulation because of higher dietary mineral needs and cheaper cost of trace mineral sources. So, in commercial practice, these supplemental inorganic trace minerals result in a high level of mineral excretion. Obviously, this is not only wasteful but also harmful to the environment. For example, poultry manure applied on a N basis contains Zn and Cu, 660 and 560%, respectively, in excess of crop requirements [3]. Due to the concern for build-up of heavy metals when applying poultry litter to cropland, environmental protection agencies around the world have pressed for lower levels of mineral waste applied to land. Organically complexed trace minerals provide alternative pathways for absorption, thus leading to a reduction in the excretion of minerals [4, 5]. However, research into the use of organic trace mineral supplementations in broiler chicken diets is still at a nascent stage, and there are not enough data to determine optimal levels of supplementation and quantify differences in excretion rates between inorganic and organic sources. Most studies on organic minerals for broilers have used conventional diets, which makes it difficult to separate the effect of the supplemental minerals from that of native minerals in the ingredients [6, 7, 8, 9, 10, 11]. In addition, purified diets usually decrease feed intake of broilers and cannot support the bird to reach growth potential, leading to compromised growth of the chick due to deficiency of other nutrients [12]. The present study was conducted to evaluate a semiconventional control diet, based mainly on sorghum and isolated soy, to evaluate possible response of broilers to organically complexed Cu, Fe, Mn, and Zn in performance, trace mineral excretion, and tissue accumulation. MATERIALS AND METHODS All methods used in this experiment regarding animal care were approved by the University of New England Animal Ethics Committee (AEC 04/147). Animal Husbandry During the first 2 wk, 160 one-day-old Cobb broilers [13] (45.48 ± 1.61 g/bird) were randomly allocated to 40 multicompartment brooder units located in 2 temperature-controlled rooms, with 8 replicates (4 chicks in each cage) per dietary treatment. Each cage contained a water trough and a feeder. Room temperature was maintained at 34 C during the first 3 d and was gradually reduced to 28 C at the end of wk 2. Body weight and feed intake were recorded weekly. At 14 d of age, groups of 4 chicks were individually weighed and transferred to metabolism cages. After 4 d of adaptation period, all excreta were collected over 4 d and analyzed to evaluate excretion of trace minerals. At d 29, all birds were killed, and blood, liver, and right tibia were sampled to analyze for their mineral contents. Dietary Treatments The experimental design consisted of 5 treatments with 8 replicate cages per treatment. The dietary treatments were as follows: 1) control diet (Table 1) was formulated to either meet or exceed NRC [2] nutrient requirements, with the exception of Cu, Fe, Mn, and Zn, which were added to the experimental diets separately (Table 2); 2) organic 1 (LOW-ORG) was control diet supplemented with 2 mg of Cu/kg of diet and 20 mg/ kg of diet each of Fe, Mn, and Zn; 3) organic 2 (MID-ORG) was control diet supplemented with 4 mg of Cu/kg of diet and 40 mg/kg of diet each of Fe, Mn, and Zn; 4) organic 3 (HIGH-ORG) was control diet supplemented with 8 mg of Cu/ kg of diet and 80 mg/kg of diet each of Fe, Mn, and Zn; and 5) inorganic positive control (INORG) was supplemented with 5 mg of Cu, 70 mg of Fe, 80 mg of Mn, and 50 mg of Zn (in sulfate form) per kilogram of diet. The organically complexed Cu, Fe, Mn, and Zn were provided as Bioplex-Cu, Bioplex-Fe, Bioplex-Mn, and Bioplex-Zn [14]. The vitamin-mineral premix [15] was free of Cu, Fe, Mn, and Zn. Measurements Birds were weighed individually at the start, then weekly and at the end of the experiment. Feed intake in each cage was recorded to determine FCR, and both were corrected for mortality.

450 JAPR: Research Report Table 1. Composition of the control diet Ingredients Amount, g/kg Sorghum 771.0 Isolated soy 175.0 Vegetable oil 16.00 Calcium carbonate 12.46 Calcium phosphate 18.20 NaCl 2.50 Lys-HCl 1.00 DL-Met 2.34 Vitamin-mineral premix 1 1.00 Choline chloride 0.50 Total 1,000 Calculated nutrient analysis ME (kcal /kg) 3,125 CP, % 22.5 Ca, % 0.84 Available P, % 0.42 Lys, % 1.12 Cu, mg/kg (as fed, analyzed) 4.20 Fe, mg/kg (as fed, analyzed) 42.19 Mn, mg/kg (as fed, analyzed) 14.82 Zn, mg/kg (as fed, analyzed) 20.38 1 Vitamin-mineral premix supplied the following per kilogram of diet: 10,000 IU of vitamin A, 2,500 IU of vitamin D 3, 50 mg of vitamin E, 2 mg of thiamine, 10 mg of riboflavin, 50 mg of niacin, 7 mg of D-calcium pantothenate, 7 mg of pyridoxine, 25 g of cyanocobolamin, 250 g of biotin, 0.3 mg of Se, 1 mg of I, 0.5 mg of molybdenum, and 0.25 mg of Co. Excreta Collection At 14 d of age, all chicks were weighed and transferred to metabolism cages. From 19 to 22 d of age, total excreta from each cage were collected daily and dried at 80 C in a forced draft oven. Fresh and dry weights of feces were recorded. Tissue and Blood Sample Collection At termination of the experiment, all birds were killed, and blood samples were individually collected into heparinized tubes. The tubes were then centrifuged at 1,000 g for 15 min [16], and the supernatant was transferred to 5-mL tubes and frozen at 20 C. The right tibia from each bird was pooled per cage and then frozen for analysis. The liver from 1 bird in each cage was weighed and frozen for analysis. Chemical Analysis Feed samples were prepared for inductively coupled plasma emission spectroscopy (ICP) [17] by grinding them to pass through a 0.5-mm screen in a stainless blade grinder. After grinding, 0.5 g of samples was placed in a Teflon tetrafluroethylene vessel. Eight ml of nitric acid (70%) was added along with 2 ml of hydrogen peroxide (30%). The solution was made to 50 ml of total volume with deionized water and mixed well for ICP analysis [18]. Fecal samples were prepared according to methods described by AOAC [19] and Dozier et al. [20] for ICP analysis. Tibia samples were boiled for approximately 10 min in deionized water and cleaned of all soft tissue. Tibia and liver samples were then dried and ashed for ICP analysis [21]. For measurement of Cu, Fe, Mn, and Zn contents in the plasma, 4 ml of plasma sample was wet-ashed in a beaker by adding 10 ml of nitric acid and heated to minimal volume (the solution was never allowed to dry). When the solution was cooled, it was filtered into a 25-mL flask and diluted to 25 ml with deionized water for ICP analysis. Statistical Analysis Statistical analyses were performed using STATGRAPHICS software [22]. The data were analyzed using 1-way ANOVA with diet as the factor. The significance of difference between Table 2. Dietary treatments fed to broilers Added Cu Added Fe Added Mn Added Zn Diet 1 (mg/kg) (mg/kg) (mg/kg) (mg/kg) Control 0 0 0 0 Low organic 2 20 20 20 Mid organic 4 40 40 40 High organic 8 80 80 80 Inorganic 5 70 80 50 1 Low-, mid-, and high-organic diets and inorganic diets were based on the control diet.

BAO ET AL.: RESPONSE OF BROILERS TO ORGANIC MINERALS 451 Table 3. Effect of different diets on feed intake, body growth, and FCR of broilers (0 to 29 d) Intake, 0 to 7 d Weight gain Intake FCR Diet (g/bird) (g/bird) (g/bird) (intake/gain) Control 147.6 979.9 c 1,567.5 b 1.590 ± 0.093 a Low organic 146.9 1,380.7 b 2,077.1 a 1.510 ± 0.094 b Mid organic 145.6 1,499.5 a 2,100.3 a 1.403 ± 0.067 c High organic 151.0 1,494.5 a 2,137.4 a 1.432 ± 0.052 bc Inorganic 154.6 1,481.9 a 2,210.0 a 1.493 ± 0.069 b Pooled SEM 4.95 57.95 37.37 0.027 P-value 0.70 <0.001 <0.001 <0.001 a c Means within a column with unlike superscripts differ significantly (P < 0.05). means was determined by Duncan s multiplerange test. Regression analysis was carried out only with control diet and different levels of organic treatments. RESULTS AND DISCUSSION Broiler Performance During wk 1, there was no significant difference in feed intake between the control and experimental groups (Table 3). After 1 wk, the birds on the control diet started to show symptoms of mineral deficiencies, including reduced feed intake and consequently reduced body growth. Supplemental Cu, Fe, Mn, and Zn, regardless of their source, improved (P < 0.01) broiler performance. The organic supplements had positive effects on live weight gain and FCR, but there was no significant difference (P > 0.05) in BW gain between organic trace minerals and the positive (inorganic) control. The MID-ORG diet achieved a superior (P < 0.01) FCR than the inorganic positive control due to relatively less feed intake by the MID-ORG treatment. However, there was no additional response in growth and FCR for the HIGH-ORG diet. The control diet had Cu, Fn, Mn, and Zn below NRC requirements for broilers. Therefore, the birds on the control diet grew poorly but survived for the entire experimental period. The deficiency of Cu, Fe, Mn, and Zn in the control diet strongly affected feed intake, which led to depressed growth of broilers. This is similar to the symptom of Zn deficiency described by King et al. [23] that a marked reduction in dietary Zn is invariably followed quickly by a reduction in food intake and growth failure. The mechanisms involved in the effects of deficiency of Zn on growth are unknown, but a reduction in food intake may be a protective response to ensure survival and maintain relatively normal, albeit downregulated, metabolic levels of these minerals [24]. The birds on the MID-ORG diet reached optimal BW gain and were 53% heavier than the birds fed the control diet. However, HIGH-ORG treatment with the highest supplemental levels of the 4 minerals, which was close to the commercial recommendation of the minerals, did not show any further response in weight gain and FCR. So, it may not be necessary to supplement these organically complexed minerals at levels as high as those currently used by industry. Mineral Excretion The excretion of Cu, Mn, and Zn increased (P < 0.001) linearly with increasing intakes of these trace minerals (Table 4). Thus, the birds on the MID-ORG diet, which supported the best FCR, had a lower (P < 0.001) trace mineral excretion than those on the HIGH-ORG treatments. This clearly suggests that the highest levels of organically complexed Cu, Mn, and Zn tested in the current study do not contribute to bird growth but are excreted. Indeed, it is well known that changes in trace mineral absorption and excretion in the gastrointestinal tract are primary mechanisms for maintaining trace mineral homeostasis [23]. Due to this pattern of excretion, the apparent absorption of Cu, Fe, Mn, and Zn is not suitable to assess the bioavailability of trace minerals [25]. A pronounced reduction in Zn and Cu excretion could only be achieved by dietary manipulation [20, 26]. The current experiment demonstrated that the highest organic trace mineral supplementation had no additional effects on broiler performance, and it is possible to use lower levels of organic trace mineral supplements without com-

452 JAPR: Research Report Table 4. Trace mineral excretion in birds fed different diets (mg/bird per d; 18 to 21 d) Diet Cu Fe Mn Zn Control 0.28 d 17.66 b 1.34 d 2.30 d Low organic 0.56 c 17.05 b 3.85 c 4.44 c Mid organic 0.83 b 24.74 b 6.22 b 6.47 b High organic 1.22 a 30.27 ab 10.12 a 10.91 a Inorganic 0.86 b 46.66 a 8.92 a 11.05 a Pooled SEM 0.047 7.143 0.425 0.547 P-value <0.001 0.039 <0.001 <0.001 Regression to intake (R 2 ) 90.61 7.22 90.28 88.38 P-value <0.001 <0.140 <0.001 <0.001 a d Means within a column with unlike superscripts differ significantly (P < 0.05). promising bird growth or increasing the rate of excretion. Mineral Concentrations in Tibia Table 5 shows that only the concentrations of Zn in tibia increased (P < 0.001) linearly with Zn intake. The Cu and Mn concentrations were also increased (P < 0.001) with supplemental dietary Cu and Mn, but there was no significant difference (P > 0.05) among supplemental organic levels. There was no significant difference (P > 0.05) in tibia Fe concentration between treatments. It has been observed that when the dietary Zn content was greater than the requirement for growth, there was an increase in the plasma and tibia concentration until a dietary concentration of 48 mg of Zn/kg of diet was reached [26, 27]. In the current experiment, tibia Zn concentrations were also strongly related to the dietary organic Zn intake (R 2 = 70.28%), but the tibia Zn concentration reached a plateau as birds attained optimal BW on the diet in which the dietary Zn concentration was 60 mg/kg of diet. The bone is a complex heterogeneous tissue that supports the musculature, and, thus, its growth and development are intimately connected with overall body growth [28], making tibia Zn concentration a good predictor of whole-body growth. Mineral Concentrations in Liver and Plasma At 29 d of age, the BW of birds fed the control diet was only 70% that of birds on supplemental treatments, but there was no significant difference (P > 0.05) in plasma trace mineral concentrations (Figure 1). Trace mineral concentrations in the liver of birds on the control diet were higher (P < 0.05) than those on the supplemental treatments, but there was no significant difference (P > 0.05) among different supplemental treatments (Figure 2). That chickens give priorities to their mineral requirements for vital functions in compromise of body growth is indicated by the normal concentrations of the minerals in the plasma of the control birds. The concentrations of the trace minerals were higher in the liver of birds on the control diet than in those on the supplemented Table 5. Trace mineral concentration of tibia bone of broiler chickens at 29 d of age ( g/g of dry bone) Diet Cu Fe Mn Zn Control 2.96 b 54.65 2.64 c 61.91 d Low organic 4.81 a 69.38 3.61 b 97.72 c Mid organic 5.90 a 67.46 3.56 b 139.73 b High organic 5.94 a 65.68 4.16 ab 148.91 ab Inorganic 6.37 a 66.62 4.47 a 160.16 a Pooled SEM 0.57 4.55 0.22 5.56 P-value <0.001 0.183 <0.001 <0.001 Regression to intake (R 2 ) 17.30 5.21 39.02 70.28 Regression to weight gain (R 2 ) 60.35 5.60 24.22 62.08 a d Means within a column with unlike superscripts differ significantly (P < 0.001).

BAO ET AL.: RESPONSE OF BROILERS TO ORGANIC MINERALS 453 Figure 1. Concentrations of Cu, Fe, Mn, and Zn in the plasma of chickens on different diets (means ± SD). diets. Similar findings have been attributed to a diluting effect as a result of rapid growth rate on the adequate diets and poor growth rate on the control diet [29]. With Zn, the results are consistent with the model for laboratory animals [30], which shows that if dietary deficiency of Zn is mild, the animal usually reduces the rate of growth and excretion to maintain normal tissue concentrations. This response is because an animal at a stage of development at which sensitivity to Zn deficiency is high stops growing immediately when given a low-zn diet, but it maintains a normal concentration of Zn in its tissues [31]. It indicates that the assessment of trace mineral Figure 2. Concentrations ( g/g of dry tissue) of Cu, Fe, Mn, and Zn in the liver of chickens on different diets (means ± SD).

454 status is difficult and remains an important, tricky challenge. This result is also in agreement with human Zn research in which there appears to be an adaptation to low-zn intake associated with a JAPR: Research Report reduction in Zn excretion to conserve this micronutrient and maintain normal plasma Zn concentration [32]. CONCLUSIONS AND APPLICATIONS 1. The control diet based on natural ingredients produced marked trace mineral deficiencies. When the broiler diet is deficient in trace minerals, birds will decrease their feed intake, resulting in poor growth. It is necessary to supplement trace minerals in broiler diets to allow the modern broiler to reach its genetic potential. 2. At lower supplemental levels, the organically complexed trace minerals were adequate to support optimum broiler chicken performance at reasonable rates of excretion. REFERENCES AND NOTES 1. Dieck, H. T., F. Doring, H. P. Roth, and H. Daniel. 2003. 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