Effects of melamine in young broiler chicks L. M. Brand,* R. A. Murarolli,* R. E. Gelven,* D. R. Ledoux,*1 B. R. Landers,* A. J. Bermudez,† M. Lin,‡ and G. E. Rottinghaus§ *Department of Animal Sciences, †Department of Veterinary Pathobiology, ‡Food Science Program, and §Department of Biomedical Sciences, University of Missouri, Columbia 65211 ABSTRACT A study was conducted to determine the toxicity of melamine in young broilers fed graded levels of melamine. An additional objective was to determine melamine residual levels in selected tissues. One hundred and seventy-five 1-d-old male Ross broiler chicks were sorted to a randomized block design in stainless steel battery pens. Chicks were assigned to 7 dietary treatments containing 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0% melamine. Each dietary treatment was fed to 5 replicate pens of 5 chicks for 21 d. Mortality increased quadratically (P < 0.001) with increasing dietary concentrations of melamine. However, compared with controls, mortality was only higher (P < 0.001) in birds fed ≥2.5% melamine. Feed intake decreased linearly (P < 0.001), whereas BW gain decreased quadratically (P < 0.02) with increasing dietary concentrations of melamine. Compared with controls, both feed intake and BW gain were lower (P < 0.001) only in birds fed ≥1.0% melamine. Relative kidney weights increased linearly (P < 0.001), whereas relative liver weights increased quadratically (P < 0.05) with increasing di-
etary concentrations of melamine. Melamine residues in breast muscle and liver tissue increased linearly (P < 0.001) with increasing dietary concentrations of melamine, whereas melamine residues in kidney and bile increased quadratically (P < 0.02) with increasing dietary concentrations of melamine. Compared with controls, melamine concentrations in liver and kidney were higher (P < 0.001) in birds fed all levels of melamine, whereas melamine concentrations in breast muscle and bile were only higher (P < 0.001) in birds fed ≥1.0% melamine. Serum albumin, total protein, globulin, and calcium increased quadratically (P < 0.02) in birds as dietary melamine increased, whereas serum aspartate transaminase and gamma gluatamyltransferase increased linearly (P < 0.01) with increasing levels of melamine in the diet. Renal histopathology revealed nonpolarizable melamine crystals in the collecting tubules and ducts of birds fed ≥1.5% melamine. In summary, dietary melamine was toxic to broilers at concentrations ≥1.0%.
Key words: broiler, crystal, kidney, melamine, toxicity 2012 Poultry Science 91:2022–2029 http://dx.doi.org/10.3382/ps.2011-02044
INTRODUCTION There have been recent concerns regarding intentional and illegal adulteration of protein ingredients, used in pet and human foods, with melamine. Melamine (1,3,5-triazine-2,4,6-triamine) is a white, crystalline compound with a molecular formula of C3H6N6 (NIOSH, 2006; Safety Data, 2008; OSHA, 2009; Figure 1). Because melamine contains 66% nitrogen by mass, analysis of feed or food containing melamine by Kjeldahl or Dumas analytical methods would suggest that these products are rich in protein. Recently, it was discovered that melamine was intentionally and illegally added to animal feed ingredients in an attempt to in©2012 Poultry Science Association Inc. Received November 23, 2011. Accepted April 4, 2012. 1 Corresponding author:
[email protected]
crease the protein content and thus the value of the ingredients (Ingelfinger, 2008). On March 16, 2007, Menu Foods placed a voluntary recall on wheat gluten and rice protein products that were imported from China (FDA, 2007b). From April 5 to June 6, 2007, 235 cats (61% mortality) and 112 dogs (74% mortality) were diagnosed with “pet foodinduced nephrotoxicity” (Burns, 2007b). The formation of “spoke-like” crystals in the kidneys of affected animals was suspected of compromising kidney function, leading to acute renal failure and death of cats and dogs (He et al., 2008). Furthermore, pigs and chickens consuming melaminecontaminated pet food by-products were traced to various states across the nation (Burns, 2007a; FDA, 2007a). The FDA (2007b) determined that melamine residues in the tissue of the affected animals were not high enough to pose a threat to human health. Upon further investigation, it was determined that melamine
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MELAMINE TOXICITY IN BROILERS
Table 1. Ingredient and nutrient composition of basal ration Composition (%)
Item
Figure 1. Melamine structure.
and its related triazine compounds are generally excreted via the kidneys and are not metabolized in mammals (Filigenzi et al., 2007; Dobson et al., 2008). Lü et al. (2009) found that melamine residue levels in tissues of broiler chickens were lower on d 42 compared with d 28, suggesting that animals may clear melamine from the body better with advancing age or that there is tissue dilution due to the larger BW with increasing age. Yang et al. (2011) reported that birds fed 200 and 1,000 mg of melamine/kg of diet had increasing residue levels of melamine in the egg from day one to 18 and day one to 19, respectively. However, Yang et al. (2011) found that residue levels of melamine in the egg decreased slightly after d 18 and 19. It was also reported that birds fed 200 mg of melamine/kg of diet did not require a withdrawal period, although birds fed 1,000 mg of melamine/kg required a 4-d withdrawal period for eggs to reach an acceptable residue level of less than 2.5 mg/ kg (Yang et al., 2011). The objective of this study was to determine if melamine causes toxic effects in young chicks, and if so, to determine the minimum concentration of melamine that is toxic.
MATERIALS AND METHODS The animal care and use protocol was reviewed and approved by the University of Missouri-Columbia Animal Care and Use Committee.
Diet Preparation Seven dietary treatments were prepared using graded levels of melamine purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). Melamine was included at 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0% to a basal corn-soybean meal diet (Table 1) that was formulated to meet or exceed requirements of broiler chicks as rec-
Ingredient Corn Soybean meal (48% CP) Pork meal Corn oil Fish meal Dicalcium phosphate Limestone Salt Methionine Trace mineral1 Vitamin mix2 Selenium mix3 Lysine Copper sulfate Sand Total Nutrient composition (calculated) CP (%) ME (kcal/kg) Lysine (%) Methionine (%) Methionine + cysteine (%) Threonine (%) Calcium (%) Nonphytate phosphorus (%)
55.45 30.25 4.74 4.21 3.68 0.36 0.47 0.35 0.19 0.11 0.08 0.06 0.04 0.00 3.00 100 23.00 3,200 1.30 0.55 0.90 0.85 1.00 0.45
1Trace mineral mix provided (mg/kg of diet): manganese, 110 mg from MnSO4; iron, 60 mg from FeSO4∙7H2O; zinc, 110 mg from ZnSO4; iodine, 2 mg from ethylenediamine dihydroidodide. 2Vitamin mix supplied (per kg of feed): vitamin A (retinyl acetate), 8,800 IU; cholecalciferol, 3,855 ICU; vitamin E (dl-α-tocopheryl acetate), 14 IU; niacin, 55 mg; calcium pantothenate, 17 mg; riboflavin, 6.6 mg; pyridoxine, 2.2 mg; menadione sodium bisulfate, 1.7 mg; folic acid, 1.4 mg; thiamin mononitrate, 1.1 mg; biotin, 0.2 mg; cyanocobalamin, 11 μg. 3Selenium premix provided 0.2 mg of Se/kg of diet from NaSeO . 3
ommended by the National Research Council (NRC, 1994). Melamine was substituted for sand to obtain the desired dietary melamine concentrations.
Birds, Management, and Response Variables One hundred and seventy-five 1-d-old male Ross broiler chicks were purchased from a commercial hatchery. Chicks were weighed, wing-banded, and sorted to a randomized block design in stainless steel battery pens. Housing was in an environmentally controlled room with a 24 h constant light schedule. Feed and water were supplied for ad libitum consumption. One-dayold chicks were divided among 7 dietary treatments (5 birds per pen and 5 replicate pens per treatment) and fed dietary treatments for 21 d. On d 21, chicks were euthanized using carbon dioxide, weighed, and serum was collected from 3 birds per pen. Chicks that died before termination were weighed and necropsied. Residual feed was measured and recorded. At termination, serum, kidney, liver, bile, and breast muscle samples were collected from 3 birds per pen and frozen for further analysis. Response variables included feed intake, BW gain, feed conversion (feed:gain), mortality, serum chemistry, relative kidney
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and liver weights, gross and histopathology of kidneys, and melamine concentrations in breast muscle, kidney, liver, and bile. Five sections of spleen, thymus, bursa of Fabricius, liver, kidney, trachea, proventriculus, heart, and jejunum were collected per treatment and examined for pathological effects.
Melamine Analysis Extraction of melamine from samples was based upon a standard FDA method for melamine detection with some modifications (FDA, 2007c). Briefly, 50% (vol/ vol) acetonitrile in water was used to extract melamine from all samples. For feed samples, melamine was extracted at a ratio of 0.1 g of sample to 10 mL of extraction solvent. The samples were then sonicated using an ultrasonic processor equipped with a 6.5-mm tapered microtip (Sonics & Materials Inc., Newtown, CT) for 2 min with 30 s working and 30 s interval at an amplitude of 36%. For bile samples, melamine was extracted at a ratio of 0.5 mL of sample to 0.5 mL of extraction solvent. For breast muscle, liver, and kidney samples, melamine was extracted at a ratio of 2.0 g of sample to 15 mL of extraction solvent and homogenized for 1 min. All extracts were centrifuged at 2,266 × g for 5 to 20 min, and the supernatant from each sample was collected. Extracts of breast muscle, liver, and kidney samples, were defatted using methylene chloride (extract: methylene chloride = 1:1, vol/vol). All extracts were then diluted with a stock solution of 0.1 N HCl and filtered through a 0.45-μm nylon syringe filter. A melamine standard stock solution (1.0 mg/mL) was prepared with acetonitrile:water (60:40, vol/vol). A series of concentrations of standard melamine solutions were prepared by diluting the stock solution with 0.1 N HCl to obtain melamine concentrations of 1, 5, 10, 25, 50, 75, 100, 200, 300, and 400 μg/mL, respectively. For the HPLC analysis, an Agilent 1100 series HPLC system with a 1200 series automatic injector was used. The system consisted of a quaternary pump, a degasser, a column oven, and a diode array detector. The mobile phase was an 85:15 (vol/vol) buffer containing 10 mM citric acid and 10 mM sodium octanesulfonate (pH 3.0):acetonitrile. Test conditions included: Zorbax SB-C8 (4.6 mm × 75 mm, 3.5 μm particle, Agilent) column; column temperature of 40°C; flow rate of 1.0 mL/ min; DAD spectra, 190 to 400 nm, detected at 240 nm. For HPLC data analysis, a melamine standard curve was obtained by establishing a plot correlating the concentrations of standards to the peak areas. Melamine in samples was confirmed by retention time and a specific absorbance spectrum with a λmax at 236 nm. Melamine contents in samples were quantified by plotting peak areas into the standard curve. The detection limit of the assay for melamine was 1 mg/kg.
Statistical Analyses Data for all response variables were subjected to regression analysis using the GLM procedure of SAS (SAS
Institute, 2006). Regression analysis best fit the means relative to a linear (yi = a + bxi + Ei) or quadratic (yi = a + bxi + cx2i + Ei) response. Dunnett’s test was run to calculate means among treatments and determine at which level of inclusion the specific parameter was significantly different from the control. Statistical significance was accepted at a P-value of
