METABOLISM AND NUTRITION. Comparison of hydrated sodium calcium aluminosilicate and yeast cell wall on counteracting aflatoxicosis in broiler chicks

METABOLISM AND NUTRITION Comparison of hydrated sodium calcium aluminosilicate and yeast cell wall on counteracting aflatoxicosis in broiler chicks J. Zhao, 1 R. B. Shirley, 2 J. D. Dibner, F. Uraizee, 3 M. Officer, M. Kitchell, M. Vazquez-Anon, and C. D. Knight Novus International Inc., St. Charles, MO 63304 ABSTRACT The objective of this research was to determine the efficacy of 2 types of adsorbents [hydrated sodium calcium aluminosilicates (HSCAS) vs. a combination of clay and yeast cell wall] in preventing aflatoxicosis in broilers. A total of 275 one-day-old birds were randomly divided into 11 treatments, with 5 replicate pens per treatment and 5 chicks per pen. The 11 treatments included 3 diets without any adsorbent containing either 0, 1, or 2 mg/kg of aflatoxin B 1 (AFB 1 ) plus 8 additional treatments employing 2 dietary levels of AFB 1 (1 or 2 mg/kg), 2 different adsorbents [Solis (SO) and MTB-100 (MTB)], and 2 different levels of each absorbent (0.1 and 0.2%) in a 2 2 2 factorial arrangement. Solis is a mixture of different HSCAS and MTB is a combination of clay and yeast cell wall. Feed and water were provided ad libitum throughout the 21-d study period. Body weight gain and feed intake were depressed and relative liver weight was increased in chicks fed AFB 1 compared with the positive control (P < 0.05). Severe liver damage was observed in chicks fed 2 mg/kg of AFB 1 with lesions consistent with aflatoxicosis, including fatty liver and vacuolar degeneration. Serum glucose, albumin, total protein, Ca, P, and alkaline phosphatase concentrations were reduced by AFB 1 (P < 0.05). The addition of either SO or MTB ameliorated the negative effects of 1 mg/kg of AFB 1 on growth performance and liver damage (P < 0.05). However, supplemental MTB failed to diminish the negative effects of 2 mg/kg of AFB 1, whereas SO was more effective compared with MTB at 2 mg/kg of AFB 1 (P < 0.05). These data indicate that the HSCAS product effectively ameliorated the negative effect of AFB 1 on growth performance and liver damage, whereas the yeast cell wall product was less effective especially at the higher AFB 1 concentration. Key words: adsorbent, aflatoxin, broiler, hydrated sodium calcium aluminosilicate INTRODUCTION Aflatoxins, a class of mycotoxins produced by fungal species of the genus Aspergillus (flavus and parasiticus), are sometimes found in feed ingredients used for poultry rations. Major forms of aflatoxins include B 1, B 2, G 1, and G 2, with aflatoxin B 1 (AFB 1 ) being the most common and biologically active component (Busby and Wogan, 1981). Economic losses associated with aflatoxin exposure in broilers include poor growth and feed conversion, increased mortality, leg problems, and carcass condemnations. Aflatoxin causes a wide range of metabolic changes in poultry and is associated with liver damage, reduced digestive enzyme activities, and 2010 Poultry Science Association Inc. Received December 13, 2009. Accepted June 16, 2010. 1 Corresponding author: Junmei.zhao@novusint.com 2 Current address: Ajinomoto Heartland LLC, Chicago, IL 60631. 3 Current address: Dow Agro-Sciences LLC, Indianapolis, IN 46268. 2010 Poultry Science 89 :2147 2156 doi: 10.3382/ps.2009-00608 immunosuppression (Edds and Bortell, 1983). Recent studies indicated that aflatoxin affects hepatic gene expression (Yarru et al., 2009) and impairs gut morphology and function (Applegate et al., 2009) With the global shortage of grains and increase in international agricultural trade, mycotoxin contamination has become more of a reality for animal producers worldwide. A survey conducted by Biomin suggested that more than one-third of grains are contaminated with mycotoxin in the Asia-Pacific region (Binder et al., 2007). Hydrated sodium calcium aluminosilicates (HSCAS) have been shown to be effective in preventing aflatoxicosis (Araba and Wyatt, 1991; Scheideler, 1993; Ledoux et al., 1999). The β-carbonyl portion of the aflatoxin molecule binds to the uncoordinated edge site of aluminum ions of the HSCAS, making the aflatoxin molecule unavailable for adsorption (Phillips et al., 1990ab). Modified yeast cell wall has also been used to sequester aflatoxin and its effectiveness has been variable in ruminants (Diaz et al., 2004; Kutz et al., 2009). Yiannikouris et al. (2003, 2004) proposed that 2147

2148 Table 1. Analyzed dietary aflatoxin B 1 concentration 1 Treatment Designed aflatoxin level, mg/kg Analyzed aflatoxin level, mg/kg 1 0 0 2 1 0.955 3 2 2.135 4 1 0.990 5 1 1.175 6 1 0.950 7 1 0.960 8 2 2.290 9 2 1.930 10 2 2.050 11 2 2.255 1 Diets were analyzed in triplicate for aflatoxin by HPLC following Gowda et al. (2009). the glucan portion of the yeast cell wall interacts with the mycotoxin molecule and is the active component. The authors hypothesized that different types of adsorbents would differ in their effectiveness to sequester AFB 1 in feed and consequently in reducing aflatoxicosis in chickens. The objective of this study was to determine the efficacy of 2 types of adsorbents, one containing a mixture of HSCAS [SO, Solis, Novus International Inc., St. Charles, MO] and the other a combination of clay and yeast cell wall [MTB, MTB-100, Alltech Inc., Nicholasville, KY] in preventing aflatoxicosis in young broilers. MATERIALS AND METHODS Birds, Diets, and Aflatoxin Quantification The study was conducted in an environmentally controlled battery room at the Animal Science Research Center, University of Missouri, Columbia. The animal care and use protocol was reviewed and approved by the University of Missouri, Columbia Animal Care and Use Committee. A total of 275 one-day-old-broilers were weighed, wing-banded, and randomly assigned to 11 dietary treatments, with 5 replicate pens per treatment and 5 birds per pen. Chicks were maintained on a 24-h constant-light schedule and were allowed access to feed and water ad libitum. The 11 treatments included 3 diets without any adsorbent containing either 0, 1, or 2 mg/kg of AFB 1 plus 8 additional treatments employing 2 dietary levels of AFB 1 (1 or 2 mg/kg), 2 different adsorbents (SO and MTB), and 2 different levels of each absorbent (0.1 and 0.2%) in a 2 2 2 factorial arrangement (Table 1). The adsorbent doses used in this trial are within the commercial recommendation for both SO and MTB. The SO product is a combination of different HSCAS (Solis, Novus International Inc.) and MTB is a commercial product containing modified yeast cell walls based on the Saccharomyces cerevisiae strain 1026 with HSCAS stated as one of the ingredients on the label (MTB-100, Alltech Inc.). Zhao et al. A common corn-soybean meal basal diet was used and was formulated to meet or exceed the nutritional requirements of growing chicks as recommended by the NRC (1994, Table 2). Each dietary treatment was then made by adding AFB 1 and adsorbents in the common basal. Aflatoxin was supplied by Aspergillus parasiticus (NRRL-2999) culture material (815 mg/kg of AFB 1, University of Missouri, Columbia). Dietary concentrations of aflatoxin were confirmed by analysis (Gowda et al., 2009). In brief, feed samples were extracted with acetonitrile and water (86:14) and an aliquot of the extract was passed through a puritox TC-M160 cleanup column (Trilogy Analytical Laboratory Inc., Washington, MO) and suitably diluted with water before analysis using HPLC with cobra cell postcolumn derivatization with fluorescence detection at 365 nm excitation and 440 nm emission. All diets were screened by the methods of Rottinghaus et al. (1992) for the presence of citrinin, T-2 toxin, vomitoxin, zearalenone, fumonisins, and ochratoxin A. Sample Collection On d 21, birds were weighed by pen and total feed consumption was recorded for each pen. Average feed intake and weight gain were corrected for mortality when calculating feed conversion for each pen. Fifteen Table 2. Basal diet composition and nutrient profile Item Amount Ingredient (%) Corn 54.80 Soybean meal 36.91 Calcium carbonate 1.14 Dicalcium phosphorus 2.03 Soybean oil 3.49 Sodium chloride 0.27 l-lysine, 78% 0.01 HMTBA 1 0.21 Antioxidant (ethoxyquin) 0.01 Sodium bicarbonate 0.28 Cellulose 0.30 Vitamin-mineral premix 2 0.35 Silica or adsorbents to 100 Nutrient 3 ME, kcal/kg 3,070 CP, % 22.73 Ca, % 1.00 Available P, % 0.50 Available amino acids Lysine, % 1.15 Methionine + cystine, % 0.82 Threonine, % 0.77 1 HMTBA = 2-hydroxy-4-methylthiobutanoic acid, provides 88% methionine activity; product of Novus International Inc. (St. Charles, MO). 2 Vitamin-mineral premix provided (per kg of feed): Mn, 110 mg/kg; Fe, 60 mg/kg; Zn, 110 mg/kg; I, 2 mg/kg; Se, 0.2 mg/kg; vitamin A, 8,800 IU/kg; cholecalciferol, 3,855 ICU/kg; vitamin E, 14 IU/kg; niacin, 55 mg/kg; calcium pantothenate, 17 mg/kg; riboflavin, 6.6 mg/kg; folic acid, 1.4 mg/kg; thiamin mononitrate, 1.1 mg/kg; biotin, 0.2 mg/kg; and cyanocobalamin, 11 μg/kg. 3 Calculated value based on NRC (1994).

chicks (5 pens per diet, 3 birds/pen) from each treatment were selected randomly, anesthetized with carbon dioxide, bled, and then killed for tissue collection. About 5 ml of blood samples was collected via cardiac puncture using a 5-mL syringe with a needle that was 0.91 mm in diameter and 3.81 cm long for serum chemistry analysis. The liver weight of each bird was recorded and liver tissue samples from each of the 15 birds from each treatment were fixed in 10% neutralbuffered formalin and saved for histopathologic evaluation. Mean average of the 3 birds within a pen was used for statistical analyses. Serum Chemistry Blood sat at room temperature at least 1 h before being centrifuged at 1,400 g at 8 C for 30 min (Sorvall, RC 3 B plus) and serum was separated and preserved at 20 C until submitted for biochemical analysis. Serum samples were analyzed for urea N, Na, K, Cl, Ca, P, total protein (TP), albumin, globulin, uric acid, gamma glutamyltransferase, aspartate aminotransferase, and creatinine phosphokinase (CPK) using an autoanalyzer (Kodak Ektachem Analyzer, Eastman Kodak Co., Rochester, NY). Table 3. Efficacy of adsorbents to ameliorate the toxin effects of aflatoxin on performance and mortality in broiler chicks Treatment EFFICACY OF AFLATOXIN ADSORBENTS IN BROILERS 2149 Aflatoxin, mg/kg level, % Feed intake, 1 g BW gain, 1 g Feed conversion Percentage of mortality 2 1 0 0 0 1,046 a 817 a 1.281 4 c 2 1 0 0 919 b 732 abcd 1.261 12 abc 3 2 0 0 753 d 642 ef 1.191 28 a 4 1 SO 0.1 973 ab 798 ab 1.221 12 abc 5 1 SO 0.2 993 ab 788 ab 1.266 4 c 6 1 MTB 0.1 962 ab 744 abc 1.293 4 c 7 1 MTB 0.2 933 b 726 bcde 1.284 4 c 8 2 SO 0.1 810 cd 656 def 1.234 12 abc 9 2 SO 0.2 945 ab 761 abc 1.243 8 bc 10 2 MTB 0.1 703 d 544 g 1.296 24 ab 11 2 MTB 0.2 739 d 572 fg 1.299 24 ab SEM 38 31 0.046 7 P-value <0.01 <0.01 0.72 0.09 Main effect means AFB, 3 mg/kg 1 965 a 764 a 1.266 2 799 b 633 b 1.268 s SO 4 930 a 751 a 1.241 MTB 5 834 b 646 b 1.293 level, % 0.1 866 686 1.261 0.2 902 712 1.273 2-way means AFB 1 mg/kg + MTB (0.1 + 0.2%) 947 ab 735 ab 1.299 AFB 1 mg/kg + SO (0.1 + 0.2%) 983 a 793 a 1.241 AFB 2 mg/kg + MTB (0.1 + 0.2%) 720 c 557 c 1.289 AFB 2 mg/kg + SO (0.1 + 0.2%) 877 b 708 b 1.235 AFB 1 mg/kg + 0.1% (MTB + SO) 968 a 771 a 1.255 AFB 1 mg/kg + 0.2% (MTB + SO) 963 a 760 a 1.272 AFB 2 mg/kg + 0.1% (MTB + SO) 756 c 600 c 1.259 AFB 2 mg/kg + 0.2% (MTB + SO) 842 b 666 b 1.263 AFB (1 + 2 mg/kg) + MTB at 0.1% 832 b 644 b 1.290 AFB (1 + 2 mg/kg) + MTB at 0.2% 834 b 649 b 1.287 AFB (1 + 2 mg/kg) + SO at 0.1% 892 a 727 b 1.225 AFB (1 + 2 mg/kg) + SO at 0.2% 969 a 775 a 1.248 Source Factorial analyses, 6 probability A (aflatoxin) 0.01 <0.01 0.12 <0.01 B (adsorbent) <0.01 <0.01 0.95 0.30 L (level) 0.14 0.24 0.71 0.53 AB 0.03 0.04 0.83 0.06 AL 0.18 0.33 0.65 0.83 BL 0.10 0.07 0.86 0.53 ABL 0.64 0.44 0.71 0.83 a g Values within columns, within each analyses (1-way ANOVA, main effect means, 2-way means), with no common superscripts differ (P < 0.05). 1 Data are means of 5 replicate pens of 5 chicks each. 2 Total of 25 birds per treatment. 3 AFB = aflatoxin B. 4 SO = a combination of different types of hydrated sodium calcium aluminosilicates (HSCAS). 5 MTB = a combination of HSCAS and yeast cell walls. 6 Factorial analyses excluded treatments 1 to 3. Similar analyses applied to the following tables.

2150 Zhao et al. Table 4. Efficacy of adsorbents to ameliorate the toxic effect of aflatoxin on liver weight and liver lesion score 1 AFB 1, 2 mg/kg level, % Liver weight, g/100 g of BW Liver lesion score 3 Compared with 0 mg/kg of AFB 1, 4 P-value Compared with 2 mg/kg of AFB 1, 4 P-value 0 0 0 2.71 h 0.2 <0.01 1 0 0 3.42 efg 0.7 0.04 <0.01 2 0 0 4.49 bc 1.8 <0.01 1 SO 0.1 2.91 gh 1.4 <0.01 0.13 1 SO 0.2 2.90 gh 0.6 0.14 <0.01 1 MTB 0.1 3.42 efg 1.2 <0.01 0.03 1 MTB 0.2 3.34 fg 1.3 <0.01 0.06 2 SO 0.1 4.26 cd 0.8 0.04 0.02 2 SO 0.2 3.47 ef 0.2 0.66 <0.01 2 MTB 0.1 5.07 a 0.8 0.02 <0.01 2 MTB 0.2 4.96 ab 1.3 <0.01 0.10 SEM 0.19 P-value <0.01 Main effect means AFB, 5 mg/kg 1 3.14 b 2 4.44 a s SO 6 3.35 b MTB 7 4.20 a level, % 0.1 3.92 a 0.2 3.67 b 2-way means AFB 1 mg/kg + MTB (0.1 + 0.2%) 3.38 c AFB 1 mg/kg + SO (0.1 + 0.2%) 2.90 c AFB 2 mg/kg + MTB (0.1 + 0.2%) 5.01 a AFB 2 mg/kg + SO (0.1 + 0.2%) 3.86 b AFB 1 mg/kg + 0.1% (MTB + SO) 3.16 c AFB 1 mg/kg + 0.2% (MTB + SO) 3.12 c AFB 2 mg/kg + 0.1% (MTB + SO) 4.66 a AFB 2 mg/kg + 0.2% (MTB + SO) 4.21 b AFB (1 + 2 mg/kg) + MTB at 0.1% 4.24 a AFB (1 + 2 mg/kg) + MTB at 0.2% 4.15 a AFB (1 + 2 mg/kg) + SO at 0.1% 3.58 b AFB (1 + 2 mg/kg) + SO at 0.2% 3.18 c Source Factorial analyses, probability A (aflatoxin) <0.01 B (adsorbent) <0.01 L (level) 0.07 AB 0.01 AL 0.25 BL 0.13 ABL 0.17 a h Values within columns, within each analyses (1-way ANOVA, main effect means, 2-way means), with no common superscripts differ (P < 0.05). 1 Data are means of 5 replicate pens of 3 chicks each. 2 AFB 1 = aflatoxin B 1. 3 A score system of 0 = liver unremarkable; 1 = mild aflatoxicosis lesions; 2 = moderate aflatoxicosis lesions; and 3 = severe aflatoxicosis lesions. 4 GENMODE was used to analyze the liver score data. These 2 columns represented P-value of the GENMODE test when each treatment was compared to either the 0 mg/kg of AFB 1 or 2 mg/kg of AFB 1 group. 5 AFB = aflatoxin B. 6 SO = a combination of different types of hydrated sodium calcium aluminosilicates (HSCAS). 7 MTB = a combination of HSCAS and yeast cell walls. Histopathology The fixed liver samples were trimmed, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin for microscopic examination. Histopathological changes were evaluated blindly in the liver of birds fed AFB 1 and were scored based on descriptions of aflatoxin-induced hepatic pathology (Hoerr, 2003). Changes scored included vacuolar degeneration and fatty change in hepatocytes, both scored on a 0 to 3 scale with 0 indicating no change (0 = no changes, liver unremarkable; 1 = mild aflatoxicosis lesions; 2 = moderate aflatoxicosis lesions; 3 = severe aflatoxicosis lesions). Average score mean of vacuolar degeneration and fatty change was used for statistical analyses. Statistics Data were first analyzed as a 1-way ANOVA using the GLM procedure of SAS (SAS Institute, 1985), then

EFFICACY OF AFLATOXIN ADSORBENTS IN BROILERS 2151 as a 3-way ANOVA (2 levels of aflatoxin; 2 adsorbent types at 2 supplementation rates) excluding the first 3 treatments without adsorbent supplementation. The model included main effects of aflatoxin level, adsorbent type, adsorbent supplementation rates, and all 2-way and 3-way interactions. Pen served as the experimental unit. Effects were considered significant at 95% probability (P 0.05). The means for treatments showing significant differences in the ANOVA were compared using Fisher s protected least significant difference procedure. For the liver lesion score, GENMOD was used and data were presented as pairwise comparisons of odds ratios. RESULTS Dietary Mycotoxin Analyses Analytical values for dietary AFB 1 indicated that target concentrations were achieved for each treatment (Table 1). Diets were also screened for the presence of citrinin, T-2 toxin, vomitoxin, zearalenone, fumonisins, and ochratoxin A and were found to be below detection limits for these mycotoxins with detection limits of 1,000 μg/kg for T-2; 500 μg/kg for citrinin, vomitoxin, zearalenone, and fumonisins; and 50 μg/kg for ochratoxin A (data not shown). Growth Performance During the 21-d study, feed intake averaged 1,046, 919, and 753 g, and weight gain averaged 817, 732, and 642 g for the positive control, 1 mg/kg, and 2 mg/kg of AFB 1 groups, respectively (Table 3, P < 0.05). Birds fed 2 mg/kg of AFB 1 only consumed 72% of the feed and gained 78% of the weight of the positive control birds. Mortality increased with increased AFB 1, with mortality observed for the control, 1 mg/kg, and 2 mg/ kg of AFB 1 groups at 4, 12, and 28%, respectively (P < 0.01). Feed conversion was not affected by dietary treatments (P > 0.05). For the factorial arrangement, no 3-way interactions were observed on performance (P > 0.10). Significant 2-way AFB 1 level adsorbent interaction was observed Figure 1. Effects of aflatoxin on gross (panels A, B, C, and D) and histological appearance (panels E, F, G, and H) of livers in broilers. Gross and histological appearances of livers exhibiting lesion scores of 0, 1, 2, and 3 are shown with 0 = no changes, liver unremarkable; 1 = mild aflatoxicosis lesions; 2 = moderate aflatoxicosis lesions; and 3 = severe aflatoxicosis lesions. Note that with increased score, the severity of vacuolar degeneration and fatty infiltration increases. Histological sections were stained with hematoxylin and eosin. Color version available in the online PDF.

2152 Zhao et al. Figure 1 (Continued). Effects of aflatoxin on gross (panels A, B, C, and D) and histological appearance (panels E, F, G, and H) of livers in broilers. Gross and histological appearances of livers exhibiting lesion scores of 0, 1, 2, and 3 are shown with 0 = no changes, liver unremarkable; 1 = mild aflatoxicosis lesions; 2 = moderate aflatoxicosis lesions; and 3 = severe aflatoxicosis lesions. Note that with increased score, the severity of vacuolar degeneration and fatty infiltration increases. Histological sections were stained with hematoxylin and eosin. Color version available in the online PDF. for feed intake (P = 0.03) and BW gain (P = 0.04). At 1 mg/kg of AFB 1, both MTB and SO recovered more than 90% of the feed intake and weight gain, but only SO was effective at 2 mg/kg of AFB 1 (adsorbent difference, P < 0.01). It was found that MTB at 0.1 or 0.2% did not ameliorate the decreased feed intake and weight gain at 2 mg/kg of AFB 1, whereas 0.2% SO partially recovered the impaired growth performance (interaction, P < 0.05). A comparison of the 2 adsorbents indicated that birds fed SO consumed more feed (930 vs. 834 g, P < 0.01) and gained more (751 vs. 646 g, P < 0.01) than those fed MTB. No performance difference was observed between the 0.1 and 0.2% supplementation levels (P > 0.05). Liver Weight and Histological Evaluation Relative liver weight (g/100 g of BW) was increased with AFB 1 contamination (Table 4). Birds fed 2 mg/ kg of AFB 1 had livers that were 1.6-fold heavier than the control (4.49 vs. 2.71 g/100 g of BW, P < 0.05). Similar to growth performance, SO was more effective in reducing the increased liver weight compared with MTB, especially at 2 mg/kg of AFB 1 (interaction, P = 0.01). In addition, birds fed MTB had heavier livers compared with birds fed SO (4.20 vs. 3.35 g/100 g of BW, P < 0.01). Birds fed 0.2% adsorbent tend to have smaller livers compared with those fed 0.1% absorbent (3.67 vs. 3.92 g/100 g of BW, P = 0.07). To analyze liver scores as category data, GENMODE was used. The last 2 columns in Table 4 list the pairwise comparison P-values by comparing each treatment to either the positive control or the 2 mg/kg of AFB 1 group. Birds fed 2 mg/kg of AFB 1 had severe liver damage with a mean score of 0.2 for the positive control and a mean score of 1.8 for the 2 mg/kg of AFB 1 group (Table 4, P < 0.01). Birds fed 0.2% SO at both 1 mg/kg and 2 mg/kg of AFB 1 levels had similar liver scores as birds fed the positive control (P > 0.05) and significantly better scores than birds fed 2 mg/kg of AFB 1 without adsorbent (P < 0.05). Interestingly, birds fed 0.1% MTB with 2 mg/kg of AFB 1 appeared to have normal liver but not the birds fed the same amount of MTB with the lower level of AFB 1. Representative gross and histological appearances of livers of various lesion scores are depicted in Figure 1. Vacuolar degeneration and fatty changes were more severe and extensive with increased liver score.

EFFICACY OF AFLATOXIN ADSORBENTS IN BROILERS 2153 Table 5. Efficacy of adsorbents to ameliorate the toxic effects of dietary aflatoxin on serum metabolites in chicks 1,2 AFB1, 3 mg/kg level, % Glucose, BUN, ALB, TP, Globulin, ALP, AST, GGT, CPK, Na, K, Cl, Ca, P, UA, 0 0 0 303 ab 2.00 1.500 a 3.00 a 1.61 a 8,820 ab 225 14.33 8,037 149 8.27 109 10.68 a 8.27 a 7.61 1 0 0 284 abc 2.20 0.947 b 2.27 bc 1.19 bc 7,203 abc 200 14.60 7,774 144 8.08 107 9.83 abc 7.46 abcd 8.61 2 0 0 236 cde 2.80 0.427 ef 1.39 ef 0.99 cd 3,016 d 250 12.93 11,772 144 7.35 106 8.09 e 6.65 d 7.52 1 SO 0.1 281 abcd 2.13 0.913 bc 2.32 bc 1.39 ab 9,840 a 212 14.20 6,264 151 7.75 110 10.08 ab 7.62 abcd 7.07 1 SO 0.2 314 a 2.13 0.987 b 2.60 ab 1.61 a 7,374 ab 197 13.40 6,233 149 9.07 109 10.53 a 8.00 ab 7.74 1 MTB 0.1 258 abcde 2.73 0.547 def 1.69 def 1.15 bc 6,877 abc 252 13.60 8,963 149 8.14 111 9.55 bcd 7.55 abcd 9.79 1 MTB 0.2 256 abcde 2.40 0.693 cd 2.07 cd 1.38 a 6,761 bc 287 15.47 13,088 151 7.23 111 9.64 bcd 7.21 bcd 8.30 2 SO 0.1 208 e 3.07 0.360 f 1.19 f 0.83 ab 2,769 d 224 13.27 9,469 150 8.57 113 9.34 bcd 7.78 abc 10.15 2 SO 0.2 245 bcde 2.20 0.600 de 1.73 de 1.13 ab 7,035 abc 232 13.47 7,617 151 6.66 110 9.40 bcd 7.25 bcd 6.93 2 MTB 0.1 221 de 2.60 0.400 ef 1.45 ef 1.05 b 4,294 cd 205 14.60 6,434 150 7.00 109 8.87 de 6.84 cd 7.96 2 MTB 0.2 204 e 2.47 0.413 ef 1.60 def 1.17 ab 3,719 d 254 14.93 9,269 151 6.73 112 9.06 cd 6.80 cd 7.03 SEM 21 0.24 0.082 0.18 0.11 1,048 26 1.38 1,721 2 0.50 2 0.30 0.33 0.98 P-value 0.01 0.07 <0.01 <0.01 <0.01 <0.01 0.21 0.97 0.12 0.24 0.06 0.16 <0.01 0.04 0.32 Main effect means AFB, 4 mg/kg 1 277 a 2.35 0.785 a 2.17 a 1.38 a 7,713 a 237 14.17 8,637 150 8.05 111 9.95 a 7.60 8.22 2 220 b 2.58 0.443 b 1.49 b 1.05 b 4,454 b 229 14.07 8,197 150 7.24 111 9.17 b 7.17 8.02 s SO 5 262 2.38 0.715 a 1.96 1.24 6,755 216 13.58 7,396 150 8.01 110 9.84 a 7.66 a 7.97 MTB 6 235 2.55 0.513 b 1.71 1.19 5,413 249 14.65 9,439 150 7.28 111 9.28 b 7.10 b 8.26 level, % 0.1 242 2.63 0.555 b 1.66 b 1.11 b 5,945 223 13.92 7,782 150 7.87 111 9.46 7.44 8.74 0.2 255 2.30 0.673 a 2.00 a 1.33 a 6,222 242 14.32 9,052 150 7.42 111 9.66 7.31 7.50 2-way means AFB 1 mg/kg + MTB (0.1 + 0.2%) AFB 1 mg/kg + SO (0.1 + 0.2%) AFB 2 mg/kg + MTB (0.1 + 0.2%) AFB 2 mg/kg + SO (0.1 + 0.2%) AFB 1 mg/kg + 0.1% (MTB + SO) AFB 1 mg/kg + 0.2% (MTB + SO) AFB 2 mg/kg + 0.1% (MTB + SO) AFB 2 mg/kg + 0.2% (MTB + SO) AFB (1 + 2 mg/kg) + MTB at 0.1% AFB (1 + 2mg/kg) + MTB at 0.2% AFB (1 + 2 mg/kg) + SO at 0.1% AFB (1 + 2 mg/kg) + SO at 0.2% 257 b 2.57 a 0.620 b 1.88 b 1.26 b 6,729 ab 269 14.53 11,007 a 150 7.68 ab 111 9.59 b 7.38 a 9.04 297 a 2.13 b 0.950 a 2.45 a 1.50 a 8,607 a 204 13.80 6,248 b 150 8.41 a 110 10.31 a 7.81 a 7.40 213 c 2.53 a 0.406 c 1.53 c 1.11 bc 4,069 c 229 14.77 7,851 ab 151 6.87 b 111 8.96 c 6.82 b 7.49 227 c 2.63 a 0.480 c 1.46 c 0.98 c 4,902 bc 228 13.37 8,542 ab 151 7.62 ab 111 9.37 b 7.51 a 8.54 269 a 2.43 b 0.730 a 2.00 b 1.27 b 8,270 a 232 14.90 7,595 150 7.95 a 111 9.81 a 7.59 a 8.43 ab 285 a 2.27 b 0.840 a 2.34 a 1.50 a 7,067 ab 242 14.43 9,660 150 8.15 a 110 10.09 a 7.60 a 8.02 ab 214 b 2.83 a 0.380 c 1.32 d 0.94 c 3,535 c 214 13.93 7,951 150 7.79 a 111 9.10 b 7.31 ab 9.05 a 225 b 2.33 b 0.507 b 1.67 c 1.15 b 5,436 bc 243 14.20 8,442 151 6.70 b 111 9.23 b 7.02 b 6.98 b 240 b 2.67 a 0.473 c 1.57 b 1.10 b 5,500 228 14.10 7,680 b 150 7.57 ab 110 9.21 c 7.20 bc 8.87 230 b 2.43 ab 0.553 bc 1.84 b 1.28 ab 5,299 270 15.20 11,178 a 151 6.98 b 112 9.35 bc 7.00 c 7.66 244 b 2.60 a 0.637 b 1.75 b 1.11 b 6,304 218 13.73 7,866 ab 151 8.16 a 111 9.71 ab 7.70 a 8.61 280 a 2.17 b 0.793 a 2.17 a 1.37 a 7,204 214 13.43 6,925 b 150 7.86 ab 110 9.97 a 7.62 ab 7.33 Continued

2154 Zhao et al. Table 5 (Continued). Efficacy of adsorbents to ameliorate the toxic effects of dietary aflatoxin on serum metabolites in chicks 1,2 UA, P, Ca, Cl, K, Na, CPK, GGT, AST, ALP, Globulin, TP, ALB, BUN, Glucose, level, % AFB1, 3 mg/kg Source Factorial analyses, probability A (aflatoxin) 0.08 0.33 0.01 0.05 0.49 0.08 0.08 0.28 0.10 0.98 0.06 0.66 0.01 0.02 0.76 B (adsorbent) <0.01 0.17 <0.01 <0.01 <0.01 <0.01 0.65 0.92 0.72 0.70 0.04 0.70 <0.01 0.07 0.66 L (level) 0.39 0.06 0.05 0.01 0.01 0.71 0.31 0.68 0.30 0.82 0.25 0.88 0.36 0.57 0.07 AB 0.39 0.12 0.03 0.01 0.02 0.55 0.09 0.74 0.02 0.96 0.98 0.26 0.48 0.58 0.05 AL 0.14 0.14 0.51 0.53 0.57 0.41 0.22 0.48 0.08 0.56 0.57 0.71 0.79 0.81 0.22 BL 0.86 0.86 0.89 0.97 0.93 0.04 0.60 0.89 0.53 0.33 0.77 0.10 0.74 0.52 0.96 ABL 0.75 0.12 0.21 0.33 0.51 0.02 0.91 0.52 0.91 0.66 0.02 0.25 0.57 0.20 0.10 a f Values within columns, within each analyses (1-way ANOVA, main effect means, 2-way means), with no common superscripts differ (P < 0.05). 1 Data are means of 5 replicate pens of 3 chicks each. 2 BUN = serum urea N; ALB = albumen; TP = total protein; ALP = alkaline phosphatase; AST = aspartate aminotransferase; GGT = gamma glutamyltransferase; CPK = creatine phosphokinase; UA = uric acid. 3 AFB 1 = aflatoxin B1. 4 AFB = aflatoxin B. 5 SO = a combination of different types of hydrated sodium calcium aluminosilicates (HSCAS). 6 MTB = a combination of HSCAS and yeast cell walls. Serum Metabolites Compared with the positive control, glucose, albumin, TP, globulin, alkaline phosphatase (ALP), Ca, and phosphate were significantly reduced with increased AFB 1 contamination (Table 5, P < 0.05). No treatment difference was observed for aspartate aminotransferase, gamma glutamyltransferase, CPK, serum urea N, Na, and Cl (P > 0.05). For the factorial arrangement, 3-way interactions were observed on ALP (P = 0.02) and K (P = 0.02) with no appropriate physiological explanation. Significant 2-way interactions of aflatoxin and adsorbent were observed on albumin (P = 0.03), TP (P = 0.01), globulin (P = 0.02), CPK (P = 0.02), and uric acid (P = 0.05). It was found that SO, but not MTB, eliminated the negative effect of 1 mg/kg of AFB 1 ; neither SO nor MTB restored albumin, TP, and globulin at 2 mg/kg of AFB 1 contamination. Compared with MTB, birds fed SO had higher albumen (ALB; P < 0.01), TP (P < 0.01), globulin (P < 0.01), ALP (P < 0.01), glucose (P < 0.01), K (P = 0.04), Ca (P < 0.01), and uric acid (P = 0.04). Main effect of adsorbent levels was observed on ALB, TP, and globulin, with a higher level (0.2%) being more effective (P < 0.05). DISCUSSION Mycotoxin contamination is very costly for the animal industry and is a food safety concern because of potential mycotoxin residue in meat, dairy, and eggs (Pandey and Chauhan, 2007; Denli et al., 2009). The most significant economic cost of mycotoxin in poultry is reduced growth rate and spiked mortality. In this study, AFB 1 depressed feed intake and weight gain and increased mortality. The failure of AFB 1 to affect feed conversion could be attributed to feed rejection by birds and is probably a protection mechanism. These results are consistent with previous studies reported by Miazzo et al. (2000) and Rauber et al. (2007). s have been proven to be the most promising and economical approach of preventing aflatoxicosis in poultry. Various products are available on the market, including single ingredients of clays, bentonites, zeolites, phyllosilicates, synthetic aluminosilicates, or a blend of adsorbents with enzyme or yeast cell wall, or both. However, the efficacy of these products is not always known and their model of action is not well understood. The efficacy of HSCAS depends on the geographical source and surface characteristics of the clay, with products having a larger surface area being more effective. Solis is a combination of HSCAS that has been shown to have strong affinity for AFB 1 using in vitro adsorption-desorption studies (data not shown). The efficacy of SO on preventing aflatoxicosis has been demonstrated in swine (Harper et al., 2010) and ruminants (Kutz et al., 2009) by sequestering the toxin and preventing systemic adsorption from the gastrointestinal tract. In this study, SO effectively ameliorated the negative effects of AFB 1 and recovered the depressed

feed intake to 90% of that of the positive control group, whereas MTB partially recovered feed intake at 1 mg/ kg but had no effect at 2 mg/kg of AFB 1. The lack of efficacy of MTB especially at the higher level of AFB 1 is perhaps due to saturation or limited binding capacity of yeast cell walls and the type and concentration of clay that was used to prepare the MTB product. Similar results were reported in dairy cows by Kutz et al. (2009), with MTB at 0.5 and 0.56% of diets containing 170 and 112 µg of AFB 1 /kg of feed, respectively, not effective in reducing milk AFM 1 concentrations, AFB 1 excretion, or AFB 1 transfer from feed to milk. The liver is both the main organ for AFB 1 detoxification and the principal target organ (Miazzo et al., 2000). Livers from birds fed AFB 1 were pale and enlarged with relative liver weight doubled at 2 mg/kg of AFB 1. Damaged liver cells can account for most of the changes in serum chemistries because less functional proteins are synthesized and secreted from damaged livers. Chronic mycotoxicosis could be diagnosed by determining serum biochemical alterations even before major clinical symptoms appear (Oğuz et al., 2000). Birds fed AFB 1 had decreased blood glucose. This was perhaps due to reduced feed intake or a reduction in the activity of enzymes involved in carbohydrate utilization, or both. Fernandez et al. (1994) reported a decrease of 36% in plasma alanine aminotransferase activity in laying hens treated with 2.5 mg/kg of AFB 1. Stanley et al. (1993) found an alanine aminotransferase activity decrease of 17 to 42% in AFB 1 -intoxicated chickens. The results of this study are in agreement with Kubena et al. (1990), in that reductions in serum TP, albumin, and globulin, as indicators of protein synthesis, were observed in chicks consuming diets containing AFB 1. Serum chemistry profiles were generally in agreement with the performance data in that adsorbents partially restored the alterations in serum chemistry associated with AFB 1 contamination. It was found that SO was more effective than MTB at restoring impaired serum chemistries at both levels of AFB 1 contamination. In addition, a significant main effect of adsorbent supplementation levels was observed, in that a higher level was more effective than a lower level at restoring ALB, TP, and globulin. This may suggest that serum chemistry profiles are more sensitive than performance as an indicator of aflatoxicosis and can potentially be used as a diagnostic tool. In summary, AFB 1 dramatically depressed feed intake, weight gain, and resulted in high mortality. Birds fed AFB 1 -contaminated feed had enlarged livers and impaired liver function, with decreased serum proteins, Ca, P, and ALP. 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