Effects of fumonisin B1 and mycotoxin binders on growth performance, tibia characteristics, gut physiology, and stress indicators in broiler chickens raised in different stocking densities

Effects of fumonisin B1 and mycotoxin binders on growth performance, tibia characteristics, gut... Abstract The present study was carried out to investigate the effects of stocking density, fumonisin B1 (FB), and mycotoxin binder (TB) on growth performance, bone quality, physiological stress indicators, and gut health in broiler chickens. Day-old Ross 308 male broiler chicks (n = 1,440/experiment) were randomly allocated to 72 floor pens in a 3 × 2 × 2 factorial arrangement, using 3 stocking densities (12.5 birds/m2 [HSD], 10 birds/m2 [MSD], or 7.5 birds/m2 [LSD]), 2 levels of purified FB (0 or 10 ppm), and 2 levels of TB (0 or 0.2%). Each treatment had 6 replicates (n = 6/treatment) and experiments lasted 34 days. All data were analyzed using 3-way ANOVA with stocking density level, FB, and TB as main factors. Body weight gain and feed intake were lower (P < 0.05) in broilers kept at HSD and MSD compared to LSD-housed counterparts. Birds fed an FB-contaminated diet exhibited a higher feed-to-gain ratio compared with those fed an FB-free diet (P < 0.05). None of the treatments affected intestinal morphology or ileal secretory immunoglobulin A levels. Stocking density affected tibia breaking strength (P < 0.05), which was lower in chickens housed at HSD compared with LSD-housed chickens. The heterophil/lymphocyte ratio (H/L ratio) was elevated (P < 0.05) in HSD and MSD groups compared with the LSD group. Serum nitric oxide (NO) levels were elevated (P < 0.05) in chickens fed the FB-contaminated diet compared with the control diet-fed counterparts. Significant interaction (P < 0.05) between FB and TB on serum NO levels was noted. In summary, increasing stocking density lowered growth performance and bone quality, but increased the H/L ratio. Dietary TB did not affect FB-induced increases in the feed-to-gain ratio. No interaction was observed between stocking density and FB for the measured variables. INTRODUCTION Many stress factors such as stocking density and/or diet-associated contaminants (i.e., mycotoxins) are known to negatively affect the welfare, health, and productivity of commercial poultry (Henry et al., 2000; Mosca et al., 2015; Murugesan et al., 2015). Dozier et al. (2006) reported that increasing stocking density from 25 to 40 kg of BW/m² linearly lowered body weight and feed intake, but increased feed conversion ratio in broiler chickens raised up to 35 d of age. Contradictory results have been reported with regard to the physiological stress responses to stocking density in chickens. For example, Dozier et al. (2006) demonstrated that stocking density did not alter corticosterone, glucose, cholesterol, or the heterophil/lymphocyte (H/L) ratio, which are indicative of stress in broiler chickens. However, other studies reported that increasing stocking density lowered physiological indices, including corticosterone and H/L ratio in chickens (Thaxton et al., 2006; Skomorucha et al., 2009). In addition, higher stocking density has been shown to affect the bone quality of broilers, reducing shear strength of the tibia due to insufficient rearing space (Buijs et al., 2012). The maximum recommended and allowed stocking density for broiler chickens has been set at less than 33 kg of BW/m² in Europe (European Commission, 2007). Mycotoxin contamination in feeds is an often-underestimated stress factor for broiler chickens. Mycotoxins are known to reduce growth performance and feed efficiency, increase mortality, and compromise the immune system in chickens (Awad et al., 2006; Yegani et al., 2006; Smith et al., 2012). Fumonisins are secondary metabolites of Fusarium verticillioides, and fumonisin B1 (FB) is the predominant form among fumonisin analogues (designated as A1, A2, B1, B2, B3, and B4) (Norred, 1993; Voss et al., 2007). FB-contaminated feed was reported to decrease villus height, villus/crypt ratio (Rauber et al., 2013; Antonissen et al., 2015), and body weight gain (Ledoux et al., 1992), and increase several FB-associated clinical signs, including liver necrosis, diarrhea, and rickets (Brown et al., 1992) in poultry. As FB can affect the intestinal epithelium (Bouhet et al., 2004; Antonissen et al., 2014), it is known to be a predisposing factor in necrotic enteritis in broiler chickens (Antonissen et al., 2015). In a recent survey in Korea, it was reported that more than 70% of commercial broiler diets were contaminated with FB, although the levels ranged from as low as 92 μg/kg to as high as 12.8 mg/kg (Kim et al., 2014). However, these levels are considered negligible, as the maximum recommended FB concentration in chicken feed varies from 20 to 50 mg/kg of diet (Food and Drug Administration [FDA], 2001; European Commission, 2006). Dietary strategies (e.g., use of toxin binders, TB) to form stable complexes with mycotoxins or to transform mycotoxins into nontoxic metabolites are commonly used to lessen toxicity in chickens (Huwig et al., 2001; Boudergue et al., 2009; Pappas et al., 2014). As both stocking density and FB-contaminated feed are important factors that negatively affect animal welfare or health of chickens, it was thought that they might act in an additive or synergistic manner. However, to the best of our knowledge, no reports have examined this hypothesis. Therefore, the present study was conducted to determine whether a combination of stocking density and/or FB-contaminated feed can affect growth performance, intestinal morphology, bone characteristics, and physiological stress indicators in broiler chicks. In addition, dietary TB was added to determine whether it can counteract FB-induced reduction in performance or physiological responses in broiler chickens. MATERIALS AND METHODS All animal care procedures were approved by Institutional Animal Care and Use Committee of Konkuk University (KU16138). Experimental Design A 3 × 2 × 2 factorial design (Table 1) was used with stocking density, FB, and TB as the main factors. A corn-soybean meal basal diet (Table 2) was fed to chickens. Experimental diets were formulated to mix the basal diet with or without 10 mg/kg purified FB and 0.2% of TB, and were fed to birds kept at 7.5 birds/m² (low stocking density: LSD), 10 birds/m² (medium stocking density: MSD), or 12.5 birds/m² (high stocking density: HSD). Each treatment had 6 replicated pens. Purified FB concentration was confirmed with liquid chromatography-mass spectrometry and was provided by Biomin Korea Ltd., Seoul, Korea. TB used in this study was a mixture of FB-degrading enzyme, mineral-based adsorbing agent, and plant and algae extracts. Table 1. Experimental design. Treatments        Density  FB  TB  Replicates/treatment  Total chicks/pen (birds/m2)  Total chicks/treatment   Low  No  No  6  15 (7.5)  90  Medium  No  No  6  20 (10)  120   High  No  No  6  25 (12.5)  150   Low  No  Yes  6  15 (7.5)  90  Medium  No  Yes  6  20 (10)  120   High  No  Yes  6  25 (12.5)  150   Low  Yes  No  6  15 (7.5)  90  Medium  Yes  No  6  20 (10)  120   High  Yes  No  6  25 (12.5)  150   Low  Yes  Yes  6  15 (7.5)  90  Medium  Yes  Yes  6  20 (10)  120   High  Yes  Yes  6  25 (12.5)  150  Treatments        Density  FB  TB  Replicates/treatment  Total chicks/pen (birds/m2)  Total chicks/treatment   Low  No  No  6  15 (7.5)  90  Medium  No  No  6  20 (10)  120   High  No  No  6  25 (12.5)  150   Low  No  Yes  6  15 (7.5)  90  Medium  No  Yes  6  20 (10)  120   High  No  Yes  6  25 (12.5)  150   Low  Yes  No  6  15 (7.5)  90  Medium  Yes  No  6  20 (10)  120   High  Yes  No  6  25 (12.5)  150   Low  Yes  Yes  6  15 (7.5)  90  Medium  Yes  Yes  6  20 (10)  120   High  Yes  Yes  6  25 (12.5)  150  View Large Table 2. Ingredients and nutrient composition of the basal diet (%, as-fed basis). Ingredients  g/100g  Corn  51.31  Soybean meal, 44% CP  29.34  Wheat, 12% CP  7.00  Corn gluten meal, 60% CP  4.00  Lysine-HCl, 78%  0.13  DL-methionine, 98%  0.24  Dicalcium phosphate  2.05  Threonine  0.07  Choline, 50%  0.10  Salt  0.30  Limestone coarse  1.14  NaHCO3  0.05  Tallow  4.00  Vitamin premix1  0.12  Mineral premix2  0.15  Total  100.00  Calculated or analyzed nutrient composition, %    AMEn3, kcal/kg  3170  Crude protein4  20.56  Crude fat4  6.27  Ca4  1.04  Available phosphorus3  0.46  Total phosphorus4  0.87  Lysine3  1.15  Methionine3  0.59  Met+Cys3  0.95  Ingredients  g/100g  Corn  51.31  Soybean meal, 44% CP  29.34  Wheat, 12% CP  7.00  Corn gluten meal, 60% CP  4.00  Lysine-HCl, 78%  0.13  DL-methionine, 98%  0.24  Dicalcium phosphate  2.05  Threonine  0.07  Choline, 50%  0.10  Salt  0.30  Limestone coarse  1.14  NaHCO3  0.05  Tallow  4.00  Vitamin premix1  0.12  Mineral premix2  0.15  Total  100.00  Calculated or analyzed nutrient composition, %    AMEn3, kcal/kg  3170  Crude protein4  20.56  Crude fat4  6.27  Ca4  1.04  Available phosphorus3  0.46  Total phosphorus4  0.87  Lysine3  1.15  Methionine3  0.59  Met+Cys3  0.95  1Vitamin premix provided following nutrients per kg of diet: vitamin A, 18,000 IU; vitamin D3, 3000 IU; vitamin E, 80 IU; vitamin K3, 5 mg; vitamin B12, 0.06 mg; thiamin, 5 mg; riboflavin, 20 mg; pyridoxine, 8 mg; niacin, 90 mg; biotin, 0.2 mg; folic acid, 1.1 mg; pantothenic acid, 50 mg. 2Mineral premix provided following nutrients per kg of diet: Fe, 80 mg; Mn, 40 mg; Zn, 12.5 mg; I, 0.25 mg; Se, 0.2 mg; Cu, 30 mg. 3Calculated value as-fed basis. 4Analyzed value as-fed basis. View Large A total of 1,440 day-old male broiler chicks (Ross 308) was purchased from a local hatchery, weighed upon arrival, and randomly allocated to one of 72 floor pens. Each pen had a pan feeder with a 52-cm diameter and had rice husks as a bedding material with a floor area of 2 m². Temperature of the facility was initially set at 34°C during the first wk, then gradually decreased to reach 24°C at 21 d, and then was maintained. Feed and water were provided ad libitum, and light was provided 23 h/day. Birds and feed intake on a pen basis were monitored weekly. The incidence of mortality was recorded daily to calculate the mortality-adjusted feed intake and feed conversion ratio. Production index (PI) was calculated with the following formula: PI = (livability × average daily gain)/ (feed conversion ratio × 10) (Gonzales et al., 2003). Sampling On d 34, one bird per pen with a weight close to the mean was randomly selected for sampling and euthanized with carbon dioxide. Immediately after euthanasia, blood was taken via cardiac puncture and collected in both heparinized and plain tubes. Serum samples were obtained by gentle centrifugation at 1,700 × g for 15 min and stored at –20 °C until use. Immediately after blood sampling, left breast and left leg meats were sampled, and the intestinal tract was removed. Then, organs (i.e., liver, spleen, and bursa of Fabricius) and abdominal fat were sampled and weighed. Relative organ weights were calculated from the ratio of organ weight to body weight and were expressed as grams per 100 grams of body weight. A segment of ileum (from Meckel's diverticulum to the ileocecal valve) was excised and an ileal segment 2-cm posterior to Meckel's diverticulum was sampled and fixed in 10% neutral buffered formalin solution for more than 48 hours. In addition, a 10-cm mid-ileal segment was dissected and washed with ice-cold saline, and ileal mucosal samples were collected by scraping the mucosal layer. The mucosal layers were then homogenized with 5 volumes of ice-cold saline and centrifuged at 15,000 × g for 10 min, and the supernatants were stored at –20 °C until use. Intestinal Morphology Formalin-fixed ileal segments were dehydrated with an alcohol-xylene sequence, embedded in paraffin, and sectioned in 5-μm slides. The sections were then stained with standard hematoxylin and eosin solution, and observed for villus height and width, and crypt depth at 100 × magnification by light microscopy (CKX53, Olympus, Tokyo, Japan) using a calibrated ocular micrometer. Ten microscopic fields per bird were measured. Villus surface area (μm2 × 10−3) was calculated with the following formula: villus surface area = villus height × (villus diameter at 1/3 of its height + diameter at 2/3 of its height)/2 (Miles et al., 2006; Incharoen and Yamauchi, 2009). Secretory Immunoglobulin A in Mucosal Scraping Secretory immunoglobulin A (sIgA) concentrations in ileal mucosal scrapings were measured (Tan et al., 2014; Du et al., 2016) using quantitative chicken IgA enzyme-linked immunosorbent assay (ELISA) kits (Bethyl Co., Montgomery, TX) as per the manufacturer's recommendation. The results were normalized against total protein concentration in each sample. Total protein concentrations in ileal scrapings were quantified as described by Bradford (1976) using bovine serum albumin. Tibia Characteristics The left tibia was obtained by manually removing attached meats. Breaking bone strength was measured using a testing machine (Instron 3342; Instron Corp., Canton, MA). The sheared tibia pieces were then dried overnight at 105 °C, ashed in a muffle furnace (Isotemp; Fisher Scientific, Pittsburgh, PA) at 600 °C for 2 h, and manually ground in a mortar before mineral content determination. Mineral (Ca, P, and Mg) contents were analyzed with an inductively coupled plasma emission spectrophotometer (Perkin Elmer 3300, Shelton, CT). Physiological Stress Indicators Heterophils and lymphocytes from heparinized fresh blood samples were counted using a Hemavet® HV950FS (Drew Scientific, Oxford, CT). Serum samples were used to measure nitric oxide (NO), corticosterone (CORT), and α-1-acid glycoprotein (AGP) using commercially available ELISA kits (NO: R&D Systems; CORT: ENZO; AGP: MyBioSource). Statistical Analysis Each pen was considered an experimental unit. Data for all variables were analyzed using 3-way analysis of variance (ANOVA), with the model including stocking density, mycotoxin, and TB as the main factors; interactions were analyzed using a general linear model (SAS 9.4, SAS Institute). Duncan's multiple range test was employed to determine means and differences among treatments. Significant differences among treatments were determined for P < 0.05, and trends were noted when 0.05 < P < 0.10. RESULTS Growth Performance and Production Index Body weight gain was higher (P < 0.001) in chickens kept at LSD compared with those at MSD or HSD during d 1 to 34 (Table 3). Chickens kept at MSD or HSD ate less (P < 0.001) compared with LSD-kept counterparts during d 22 to 34 or 1 to 34. In contrast, feed-to-gain ratio tended to decrease (P = 0.068) with increasing stocking density during d 1 to 21, but this trend was not found (P > 0.05) thereafter. Feeding FB-contaminated diets did not affect weight gain or feed intake during the entire period of the experiment. However, chickens fed the FB-contaminated diet exhibited higher (P = 0.019) feed-to-gain ratio compared with the control diet-fed chickens during d 1 to 34. Dietary TB did not influence growth performance. Significant interaction (P = 0.046) between stocking density and TB with regard to body weight gain during d 1 to 21 was observed. Body weight gain increased as the stocking density increased in the absence of TB, but this trend was reversed by the presence of TB. PI was high (P < 0.001) in LSD vs. MSD- or HSD-raised chickens (Table 4). On the other hand, there was no difference (P > 0.05) in mortality among treatments. Table 3. Effect of mycotoxin and toxin binder on growth performance in broiler chickens raised at different stocking densities.       Body weight gain (g/day/bird)  Feed intake (g/day/bird)  Feed conversion ratio (g:g)  Density1  FB1  TB1  1 to 21 d  22 to 34 d  1 to 34 d  1 to 21 d  22 to 34 d  1 to 34 d  1 to 21 d  22 to 34 d  1 to 34 d   LSD  +  +  29.603  85.51  50.98  44.08  132.26  78.1  1.50  1.55  1.54    +  −  28.78  89.23  51.89  42.85  131.95  80.2  1.49  1.49  1.55    −  +  29.66  87.71  51.86  43.40  129.35  75.9  1.47  1.48  1.48    −  −  28.33  88.93  51.50  42.19  131.63  74.0  1.49  1.48  1.44   MSD  +  +  28.03  78.33  47.27  43.71  119.47  71.1  1.57  1.51  1.52    +  −  29.84  75.91  47.46  43.15  118.02  71.6  1.45  1.57  1.52    −  +  29.40  81.27  49.23  42.77  121.52  72.3  1.46  1.50  1.48    −  −  28.82  79.39  48.15  41.90  119.23  71.1  1.45  1.50  1.48   HSD  +  +  28.32  74.64  46.03  40.64  115.78  69.3  1.44  1.56  1.52    +  −  30.44  75.80  47.78  43.12  117.49  73.0  1.42  1.55  1.53    −  +  29.08  76.52  47.22  41.22  120.87  70.6  1.42  1.58  1.52    −  −  29.84  76.79  47.79  43.28  119.37  71.9  1.45  1.56  1.52  Pooled SEM2      0.75  2.32  1.08  0.89  2.23  1.86  0.037  0.040  0.032  Main factors                         LSD      29.09  87.85a  51.56a  43.13  131.30a  77.10a  1.49  1.50  1.50   MSD      29.02  78.73b  48.03b  42.88  119.56b  71.53b  1.48  1.52  1.50   HSD      29.42  75.94b  47.21b  42.06  118.49b  71.21b  1.43  1.50  1.52    +    29.17  79.91  48.57  42.92  122.78  73.9  1.48  1.54  1.53a    −    29.19  81.77  49.29  42.46  123.66  72.7  1.46  1.52  1.49b      +  29.02  80.67  48.76  42.64  123.54  72.9  1.47  1.53  1.51      −  29.34  81.01  49.10  42.75  122.95  73.7  1.46  1.52  1.51  P-value                        Density (D)      0.730  <0.001  <0.001  0.213  <0.001  <0.001  0.068  0.097  0.627  Fumonisin (F)      0.966  0.170  0.249  0.369  0.350  0.259  0.348  0.333  0.019  TB (B)      0.455  0.800  0.596  0.825  0.793  0.468  0.487  0.772  0.990  D × F      0.938  0.772  0.768  0.488  0.224  0.158  0.521  0.497  0.202  F × B      0.106  0.725  0.322  0.816  0.841  0.202  0.126  0.970  0.608  D × B      0.046  0.372  0.574  0.147  0.610  0.506  0.325  0.519  0.911  D × F × B      0.680  0.899  0.999  0.985  0.609  0.894  0.693  0.575  0.876        Body weight gain (g/day/bird)  Feed intake (g/day/bird)  Feed conversion ratio (g:g)  Density1  FB1  TB1  1 to 21 d  22 to 34 d  1 to 34 d  1 to 21 d  22 to 34 d  1 to 34 d  1 to 21 d  22 to 34 d  1 to 34 d   LSD  +  +  29.603  85.51  50.98  44.08  132.26  78.1  1.50  1.55  1.54    +  −  28.78  89.23  51.89  42.85  131.95  80.2  1.49  1.49  1.55    −  +  29.66  87.71  51.86  43.40  129.35  75.9  1.47  1.48  1.48    −  −  28.33  88.93  51.50  42.19  131.63  74.0  1.49  1.48  1.44   MSD  +  +  28.03  78.33  47.27  43.71  119.47  71.1  1.57  1.51  1.52    +  −  29.84  75.91  47.46  43.15  118.02  71.6  1.45  1.57  1.52    −  +  29.40  81.27  49.23  42.77  121.52  72.3  1.46  1.50  1.48    −  −  28.82  79.39  48.15  41.90  119.23  71.1  1.45  1.50  1.48   HSD  +  +  28.32  74.64  46.03  40.64  115.78  69.3  1.44  1.56  1.52    +  −  30.44  75.80  47.78  43.12  117.49  73.0  1.42  1.55  1.53    −  +  29.08  76.52  47.22  41.22  120.87  70.6  1.42  1.58  1.52    −  −  29.84  76.79  47.79  43.28  119.37  71.9  1.45  1.56  1.52  Pooled SEM2      0.75  2.32  1.08  0.89  2.23  1.86  0.037  0.040  0.032  Main factors                         LSD      29.09  87.85a  51.56a  43.13  131.30a  77.10a  1.49  1.50  1.50   MSD      29.02  78.73b  48.03b  42.88  119.56b  71.53b  1.48  1.52  1.50   HSD      29.42  75.94b  47.21b  42.06  118.49b  71.21b  1.43  1.50  1.52    +    29.17  79.91  48.57  42.92  122.78  73.9  1.48  1.54  1.53a    −    29.19  81.77  49.29  42.46  123.66  72.7  1.46  1.52  1.49b      +  29.02  80.67  48.76  42.64  123.54  72.9  1.47  1.53  1.51      −  29.34  81.01  49.10  42.75  122.95  73.7  1.46  1.52  1.51  P-value                        Density (D)      0.730  <0.001  <0.001  0.213  <0.001  <0.001  0.068  0.097  0.627  Fumonisin (F)      0.966  0.170  0.249  0.369  0.350  0.259  0.348  0.333  0.019  TB (B)      0.455  0.800  0.596  0.825  0.793  0.468  0.487  0.772  0.990  D × F      0.938  0.772  0.768  0.488  0.224  0.158  0.521  0.497  0.202  F × B      0.106  0.725  0.322  0.816  0.841  0.202  0.126  0.970  0.608  D × B      0.046  0.372  0.574  0.147  0.610  0.506  0.325  0.519  0.911  D × F × B      0.680  0.899  0.999  0.985  0.609  0.894  0.693  0.575  0.876  1FB = fumonisin; TB = toxin binder; LSD = low stocking density; MSD = medium stocking density; HSD = high stocking density. 2SEM = pooled standard error of the means. 3Values are LS means of 6 replicates per treatment. a,bValues (n = 6/treatment) having a different superscript within a column differ significantly (P < 0.05). View Large Table 4. Effects of mycotoxin and toxin binder on production index and mortality in broiler chickens raised at different stocking densities. Density1  FB1  TB1  Production index2  Mortality (%)   LSD  +  +  312.14  5.56    +  −  315.3  5.56    −  +  339.9  3.33    −  −  342.5  3.33   MSD  +  +  291.6  5.83    +  −  307.8  1.67    −  +  313.6  5.00    −  −  312.2  3.33   HSD  +  +  294.2  3.33    +  −  302.6  2.67    −  +  292.2  6.40    −  −  302.6  4.00  Pooled SEM3      3.72  0.037  Main factors           LSD      327.4a  4.44   MSD      306.2b  3.96   HSD      298.1b  4.10    +    303.9  4.10    −    317.9  4.23      +  307.7  4.91      −  313.8  3.43  P-value          Density (D)      <0.001  0.470  Fumonisin (F)      0.075  0.752  TB (B)      0.404  0.105  D × F      0.315  0.254  F × B      0.708  0.948  D × B      0.938  0.521  D × F × B      0.846  0.619  Density1  FB1  TB1  Production index2  Mortality (%)   LSD  +  +  312.14  5.56    +  −  315.3  5.56    −  +  339.9  3.33    −  −  342.5  3.33   MSD  +  +  291.6  5.83    +  −  307.8  1.67    −  +  313.6  5.00    −  −  312.2  3.33   HSD  +  +  294.2  3.33    +  −  302.6  2.67    −  +  292.2  6.40    −  −  302.6  4.00  Pooled SEM3      3.72  0.037  Main factors           LSD      327.4a  4.44   MSD      306.2b  3.96   HSD      298.1b  4.10    +    303.9  4.10    −    317.9  4.23      +  307.7  4.91      −  313.8  3.43  P-value          Density (D)      <0.001  0.470  Fumonisin (F)      0.075  0.752  TB (B)      0.404  0.105  D × F      0.315  0.254  F × B      0.708  0.948  D × B      0.938  0.521  D × F × B      0.846  0.619  1FB = fumonisin; TB = toxin binder; LSD = low stocking density; MSD = medium stocking density; HSD = high stocking density. 2Production index = European production index = [body weight gain, kg × livability, %)/(feed conversion ratio × age, days)] × 100. 3SEM = pooled standard error of the means. 4Values are LS means of 6 replicates per treatment. a,bValues (n = 6/treatment) having a different superscript within a column differ significantly (P < 0.05). View Large Relative Organ and Abdominal Fat Weights Relative liver, spleen, and abdominal fat weights were not affected (P > 0.05) by stocking density, FB, or TB (Table 5). However, relative weight of the bursa of Fabricius was higher (P = 0.037) in the HSD-raised birds compared with those raised at LSD. The MSD-raised birds exhibited intermediate bursal weight. No interaction among stocking density, FB, and TB was detected. Table 5. Effects of mycotoxin and toxin binder on relative organ weights (organ weight (g)/live body weight (g) × 100) in broiler chickens raised at different stocking densities. Density1  FB1  TB1  Liver  Spleen  Bursa of Fabricius  Abdo-minal fat  Live body weight   LSD  +  +  6.423  0.129  0.209  1.165  1685    +  −  6.54  0.121  0.191  1.108  1707    −  +  6.75  0.147  0.199  1.096  1695    −  −  6.79  0.147  0.169  1.005  1684   MSD  +  +  5.80  0.127  0.167  1.219  1573    +  −  6.78  0.122  0.217  1.105  1575    −  +  5.96  0.126  0.219  1.094  1640    −  −  6.52  0.138  0.215  1.129  1598   HSD  +  +  5.80  0.124  0.264  0.937  1543    +  −  6.78  0.125  0.251  0.981  1615    −  +  5.96  0.128  0.223  0.989  1603    −  −  6.52  0.111  0.244  1.212  1630  Pooled SEM2      0.081  0.0043  0.030  0.030  36.52  Main factors     LSD      6.62  0.136  0.192b  1.093  1693   MSD      6.49  0.128  0.205a,b  1.137  1597   HSD      6.26  0.122  0.245a  1.030  1598    +    6.42  0.125  0.216  1.086  1616    −    6.50  0.133  0.212  1.088  1642      +  6.34  0.130  0.213  1.083  1623      −  6.58  0.127  0.214  1.090  1635  P-value    Density (D)      0.194  0.420  0.037  0.340  <0.001  Fumonisin (F)      0.592  0.349  0.779  0.975  0.237  TB (B)      0.138  0.740  0.951  0.907  0.596  D × F      0.662  0.458  0.465  0.250  0.558  F × B      0.961  0.872  0.744  0.413  0.331  D × B      0.076  0.847  0.541  0.315  0.399  D × F × B      0.468  0.691  0.577  0.728  0.992  Density1  FB1  TB1  Liver  Spleen  Bursa of Fabricius  Abdo-minal fat  Live body weight   LSD  +  +  6.423  0.129  0.209  1.165  1685    +  −  6.54  0.121  0.191  1.108  1707    −  +  6.75  0.147  0.199  1.096  1695    −  −  6.79  0.147  0.169  1.005  1684   MSD  +  +  5.80  0.127  0.167  1.219  1573    +  −  6.78  0.122  0.217  1.105  1575    −  +  5.96  0.126  0.219  1.094  1640    −  −  6.52  0.138  0.215  1.129  1598   HSD  +  +  5.80  0.124  0.264  0.937  1543    +  −  6.78  0.125  0.251  0.981  1615    −  +  5.96  0.128  0.223  0.989  1603    −  −  6.52  0.111  0.244  1.212  1630  Pooled SEM2      0.081  0.0043  0.030  0.030  36.52  Main factors     LSD      6.62  0.136  0.192b  1.093  1693   MSD      6.49  0.128  0.205a,b  1.137  1597   HSD      6.26  0.122  0.245a  1.030  1598    +    6.42  0.125  0.216  1.086  1616    −    6.50  0.133  0.212  1.088  1642      +  6.34  0.130  0.213  1.083  1623      −  6.58  0.127  0.214  1.090  1635  P-value    Density (D)      0.194  0.420  0.037  0.340  <0.001  Fumonisin (F)      0.592  0.349  0.779  0.975  0.237  TB (B)      0.138  0.740  0.951  0.907  0.596  D × F      0.662  0.458  0.465  0.250  0.558  F × B      0.961  0.872  0.744  0.413  0.331  D × B      0.076  0.847  0.541  0.315  0.399  D × F × B      0.468  0.691  0.577  0.728  0.992  1FB = fumonisin; TB = toxin binder; LSD = low stocking density; MSD = medium stocking density; HSD = high stocking density. 2SEM = pooled standard error of the means. 3Values are LS means of 6 replicates per treatment. a,bValues (n = 6/treatment) having a different superscript within a column differ significantly (P < 0.05). View Large Intestinal Morphology Stocking density, FB, and TB did not (P > 0.05) affect any parameters of ileal morphology in the chicks (Table 6). Marginal interactions between stocking density and TB for crypt depth (P = 0.074) and villus surface area (P = 0.054), and between FB and TB for crypt depth (P = 0.056) were noted (Table 6). Table 6. Effect of mycotoxin and toxin binder on ileal morphometry and secretory immunoglobulin A in broiler chickens raised at different stocking densities. Density1  FB1  TB1  Villus height (μm)  Crypt depth (μm)  Villus surface area2 (μm2 × 10−3)  Villus height/crypt depth ratio  Ileal sIgA (μg/mg of protein)   LSD  +  +  688.54  140.94  0.090  4.90  0.2672    +  −  842.9  163.92  0.127  5.12  0.187    −  +  711.8  136.38  0.105  5.23  0.178    −  −  741.9  164.85  0.132  4.53  0.280   MSD  +  +  788.7  144.94  0.102  5.38  0.204    +  −  590.9  118.53  0.094  5.05  0.258    −  +  758.7  142.63  0.115  5.32  0.251    −  −  741.4  164.81  0.115  4.47  0.396   HSD  +  +  773.8  149.00  0.117  5.14  0.359    +  −  833.0  144.74  0.120  5.74  0.233    −  +  718.8  147.55  0.111  4.87  0.293    −  −  734.4  153.32  0.106  4.79  0.241  Pooled SEM3      72.5  9.27  0.011  0.378  0.015  Main factors                 LSD      746.5  150.95  0.113  4.96  0.234   MSD      719.9  142.73  0.106  5.06  0.284   HSD      765.0  148.65  0.113  5.13  0.286    +    753.0  143.68  0.108  5.22  0.260    −    734.3  151.21  0.113  4.88  0.275      +  740.1  143.58  0.106  5.14  0.264      −  747.6  151.32  0.115  4.96  0.273  P-value                Density (D)      0.679  0.443  0.629  0.818  0.286  Fumonisin (F)      0.661  0.162  0.436  0.119  0.622  TB (B)      0.862  0.145  0.181  0.407  0.813  D × F      0.390  0.159  0.238  0.657  0.179  F × B      0.967  0.056  0.785  0.117  0.054  D × B      0.139  0.074  0.054  0.284  0.042  D × F × B      0.319  0.208  0.837  0.931  0.726  Density1  FB1  TB1  Villus height (μm)  Crypt depth (μm)  Villus surface area2 (μm2 × 10−3)  Villus height/crypt depth ratio  Ileal sIgA (μg/mg of protein)   LSD  +  +  688.54  140.94  0.090  4.90  0.2672    +  −  842.9  163.92  0.127  5.12  0.187    −  +  711.8  136.38  0.105  5.23  0.178    −  −  741.9  164.85  0.132  4.53  0.280   MSD  +  +  788.7  144.94  0.102  5.38  0.204    +  −  590.9  118.53  0.094  5.05  0.258    −  +  758.7  142.63  0.115  5.32  0.251    −  −  741.4  164.81  0.115  4.47  0.396   HSD  +  +  773.8  149.00  0.117  5.14  0.359    +  −  833.0  144.74  0.120  5.74  0.233    −  +  718.8  147.55  0.111  4.87  0.293    −  −  734.4  153.32  0.106  4.79  0.241  Pooled SEM3      72.5  9.27  0.011  0.378  0.015  Main factors                 LSD      746.5  150.95  0.113  4.96  0.234   MSD      719.9  142.73  0.106  5.06  0.284   HSD      765.0  148.65  0.113  5.13  0.286    +    753.0  143.68  0.108  5.22  0.260    −    734.3  151.21  0.113  4.88  0.275      +  740.1  143.58  0.106  5.14  0.264      −  747.6  151.32  0.115  4.96  0.273  P-value                Density (D)      0.679  0.443  0.629  0.818  0.286  Fumonisin (F)      0.661  0.162  0.436  0.119  0.622  TB (B)      0.862  0.145  0.181  0.407  0.813  D × F      0.390  0.159  0.238  0.657  0.179  F × B      0.967  0.056  0.785  0.117  0.054  D × B      0.139  0.074  0.054  0.284  0.042  D × F × B      0.319  0.208  0.837  0.931  0.726  1FB = fumonisin; TB = toxin binder; LSD = low stocking density; MSD = medium stocking density; HSD = high stocking density; sIgA = secretory immunoglobulin A. 2Villus surface area = calculated by villus height × (diameter of villi's 1/3 height part + diameter of villi's 2/3 height part)/2. 3SEM = pooled standard error of the means. 4Values are LS means of 6 replicates per treatment. View Large Ileal sIgA sIgA concentration in ileal mucosa was not affected (P > 0.05) by stocking density, FB, or TB (Table 6). Significant (P = 0.042) or marginal (P = 0.054) interactions between stocking density and TB, or FB and TB for ileal sIgA were observed. Tibia Characteristics Tibia breaking strength was low (P = 0.045) in the HSD- vs. LSD-raised chickens (Table 7). The MSD-raised chickens showed intermediate tibia breaking strength. However, no interaction between main factors for tibia breaking strength was noted. Calcium content in the tibia was highest (P = 0.005) in the LSD- vs. MSD- or HSD-raised chickens. On the other hand, tibia ash, P, and Mg contents were not affected by the treatments, and no interaction among stocking density, FB, and TB was detected (Table 7). Table 7. Effects of mycotoxin and toxin binder on tibia breaking strength and mineral content in broiler chickens raised at different stocking densities. Density1  FB1  TB1  Breaking strength (kg)  Tibia ash (%)  Ca (%)  P (%)  Mg (%)   LSD  +  +  24.053  0.346  35.01  18.19  0.900    +  −  24.43  0.331  35.52  17.96  0.890    −  +  26.72  0.353  34.84  17.94  0.915    −  −  28.73  0.355  34.82  18.15  0.885   MSD  +  +  23.83  0.355  34.53  18.16  0.917    +  −  24.02  0.351  34.49  18.28  0.893    −  +  21.50  0.356  34.17  17.64  0.898    −  −  22.13  0.353  34.66  18.10  0.888   HSD  +  +  21.94  0.350  33.86  17.78  0.927    +  −  22.90  0.356  34.59  18.09  0.918    −  +  20.28  0.333  34.23  17.73  0.898    −  −  23.85  0.357  34.38  18.05  0.908  Pooled SEM2      0.64  0.0085  0.34  0.22  0.023  Main factors                 LSD      25.98a  0.346  35.04a  18.06  0.898   MSD      22.87a,b  0.354  34.46b  18.05  0.899   HSD      22.24b  0.349  34.26b  17.91  0.913    +    23.53  0.348  34.66  18.08  0.908    −    23.87  0.351  34.52  17.94  0.899      +  23.05  0.349  34.44  17.91  0.909      −  24.34  0.350  34.74  18.11  0.897  P-value                Density (D)      0.045  0.471  0.005  0.593  0.578  Fumonisin (F)      0.792  0.567  0.445  0.285  0.514  TB (B)      0.317  0.733  0.124  0.127  0.367  D × F      0.197  0.136  0.541  0.519  0.745  F × B      0.543  0.227  0.619  0.312  0.883  D × B      0.838  0.170  0.884  0.522  0.786  D × F × B      0.942  0.730  0.424  0.786  0.811  Density1  FB1  TB1  Breaking strength (kg)  Tibia ash (%)  Ca (%)  P (%)  Mg (%)   LSD  +  +  24.053  0.346  35.01  18.19  0.900    +  −  24.43  0.331  35.52  17.96  0.890    −  +  26.72  0.353  34.84  17.94  0.915    −  −  28.73  0.355  34.82  18.15  0.885   MSD  +  +  23.83  0.355  34.53  18.16  0.917    +  −  24.02  0.351  34.49  18.28  0.893    −  +  21.50  0.356  34.17  17.64  0.898    −  −  22.13  0.353  34.66  18.10  0.888   HSD  +  +  21.94  0.350  33.86  17.78  0.927    +  −  22.90  0.356  34.59  18.09  0.918    −  +  20.28  0.333  34.23  17.73  0.898    −  −  23.85  0.357  34.38  18.05  0.908  Pooled SEM2      0.64  0.0085  0.34  0.22  0.023  Main factors                 LSD      25.98a  0.346  35.04a  18.06  0.898   MSD      22.87a,b  0.354  34.46b  18.05  0.899   HSD      22.24b  0.349  34.26b  17.91  0.913    +    23.53  0.348  34.66  18.08  0.908    −    23.87  0.351  34.52  17.94  0.899      +  23.05  0.349  34.44  17.91  0.909      −  24.34  0.350  34.74  18.11  0.897  P-value                Density (D)      0.045  0.471  0.005  0.593  0.578  Fumonisin (F)      0.792  0.567  0.445  0.285  0.514  TB (B)      0.317  0.733  0.124  0.127  0.367  D × F      0.197  0.136  0.541  0.519  0.745  F × B      0.543  0.227  0.619  0.312  0.883  D × B      0.838  0.170  0.884  0.522  0.786  D × F × B      0.942  0.730  0.424  0.786  0.811  1FB = fumonisin; TB = toxin binder; LSD = low stocking density; MSD = medium stocking density; HSD = high stocking density. 2SEM = pooled standard error of the means. 3Values are LS means of 6 replicates per treatment. a,bValues (n = 6/treatment) having a different superscript within a column differ significantly (P < 0.05). View Large Physiological Stress Indicators Blood H/L ratio was significantly low (P = 0.002) at LSD compared with the ratio at MSD or HSD (Table 8). Dietary FB did not affect (P > 0.05) blood H/L ratio. On the other hand, serum NO levels were significantly elevated (P = 0.042) in chickens fed the FB-contaminated diet compared with the control diet-fed chickens, but stocking density did not affect the level. Of interest, significant interaction (P = 0.007) between FB and TB for serum NO levels was noted. Dietary TB increased or decreased NO levels, depending on the absence or presence of FB, causing an interaction between the 2 factors. However, serum AGP and CORT levels were not affected by stocking density, dietary FB, or TB (P > 0.05). Table 8. Effects of mycotoxin and toxin binder on physiological stress indicators in broiler chickens raised at different stocking densities1. Density1  FB1  TB1  H/L2 ratio  NO2 (μM)  AGP2 (mg/ml)  CORT2 (pg/ml)   LSD  +  +  0.1954  31.4  2.55  161.5    +  −  0.208  35.5  3.35  143.5    −  +  0.166  43.8  2.99  138.7    −  −  0.175  19.8  2.93  47.9   MSD  +  +  0.242  25.5  3.18  45.0    +  −  0.210  39.3  3.90  142.1    −  +  0.303  21.6  3.57  153.8    −  −  0.220  27.9  3.37  61.5   HSD  +  +  0.267  29.2  3.69  100.3    +  −  0.295  48.1  3.32  112.9    −  +  0.268  27.7  2.74  118.6    −  −  0.243  23.8  2.67  177.5  Pooled SEM3      0.024  6.08  0.47  12.0  Main factors               LSD      0.189b  32.7  2.96  112.6   MSD      0.236a  28.6  3.50  98.7   HSD      0.273a  32.6  3.11  128.5    +    0.240  34.9a  3.33  115.5    −    0.230  27.6b  3.05  113.4      +  0.246  29.8  3.12  119.6      −  0.229  32.7  3.26  109.6  P-value              Density (D)      0.002  0.560  0.243  0.582  Fumonisin (F)      0.626  0.042  0.304  0.942  TB (B)      0.424  0.468  0.614  0.714  D × F      0.281  0.474  0.411  0.232  F × B      0.237  0.007  0.370  0.149  D × B      0.292  0.490  0.638  0.302  D × F × B      0.842  0.473  0.589  0.136  Density1  FB1  TB1  H/L2 ratio  NO2 (μM)  AGP2 (mg/ml)  CORT2 (pg/ml)   LSD  +  +  0.1954  31.4  2.55  161.5    +  −  0.208  35.5  3.35  143.5    −  +  0.166  43.8  2.99  138.7    −  −  0.175  19.8  2.93  47.9   MSD  +  +  0.242  25.5  3.18  45.0    +  −  0.210  39.3  3.90  142.1    −  +  0.303  21.6  3.57  153.8    −  −  0.220  27.9  3.37  61.5   HSD  +  +  0.267  29.2  3.69  100.3    +  −  0.295  48.1  3.32  112.9    −  +  0.268  27.7  2.74  118.6    −  −  0.243  23.8  2.67  177.5  Pooled SEM3      0.024  6.08  0.47  12.0  Main factors               LSD      0.189b  32.7  2.96  112.6   MSD      0.236a  28.6  3.50  98.7   HSD      0.273a  32.6  3.11  128.5    +    0.240  34.9a  3.33  115.5    −    0.230  27.6b  3.05  113.4      +  0.246  29.8  3.12  119.6      −  0.229  32.7  3.26  109.6  P-value              Density (D)      0.002  0.560  0.243  0.582  Fumonisin (F)      0.626  0.042  0.304  0.942  TB (B)      0.424  0.468  0.614  0.714  D × F      0.281  0.474  0.411  0.232  F × B      0.237  0.007  0.370  0.149  D × B      0.292  0.490  0.638  0.302  D × F × B      0.842  0.473  0.589  0.136  1FB = fumonisin; TB = toxin binder; LSD = low stocking density; MSD = medium stocking density; HSD = high stocking density. 2H/L ratio = heterophil/lymphocyte ratio; NO = nitric oxide; AGP = alpha-1-acid glycoprotein; CORT = corticosterone. 3SEM = pooled standard error of the means. 4Values are LS means of 6 replicates per treatment. a,bValues (n = 6/treatment) having a different superscript within a column differ significantly (P < 0.05). View Large DISCUSSION No strong interactions between 2 factors (i.e., FB and stocking density) were detected with respect to growth performance, bone quality, gut health, and blood stress indicators. In line with our findings, increasing stocking density was found to lower weight gain and feed intake (Dozier et al., 2006; Estevez, 2007; Cengiz et al., 2015), tibia breaking strength with concomitant decrease in ash calcium content (Buijs et al., 2012), and serum H/L ratio (Thaxton et al., 2006; Shakeri et al., 2014) in broiler chickens, while dietary FB alone increased feed-to-gain ratio in broiler chickens (Brown et al., 1992) and serum NO levels in rodents (Dombrink-Kurtzman et al., 2000). Thus, the lack of interaction between FB and stocking density was not due to the absence of FB or stocking density-induced effects in broiler chickens. FB at a concentration of 10 mg/kg of diet was not sufficient to affect the stocking density-induced effects in chickens, leading to the lack of apparent interaction. Thus, it is not known whether increasing FB levels up to 100 or 300 mg/kg of diet (Brown et al., 1992; Javed et al., 1993; Rauber et al., 2013) would aggravate the stocking density-induced depression in performance, health, or welfare of broiler chickens. The stocking density and FB concentration used in this study are considered to be moderate. For example, HSD was set at 0.08 m2/bird in this study, while HSD in previous studies ranged from 0.030 to 0.067 m2/bird (Guardia et al., 2011; Abudabos et al., 2013; Hongchao et al., 2014; Shakeri et al., 2014). It has been reported that increasing stocking density decreased growth performance or villus height (Beloor et al., 2010; Hongchao et al., 2014; Shakeri et al., 2014), increased the incidence of foot pad dermatitis (Hongchao et al., 2014), altered behaviors (Hall, 2001), and increased stress-associated indicators (i.e., acute phase protein, CORT, H/L ratio; Shakeri et al., 2014) in broiler chickens. However, the absence of a clear effect with increased stocking density in chickens (Nogueira et al., 2013; Wang et al., 2014) also has been reported. In addition to the absence of effects due to stocking density, the effects of FB levels also were considered negligible. A recent survey (Seo et al., 2013) found the presence of approximately 4 mg FB/kg of diet in commercial poultry feeds. However, when the diet was contaminated with less than 100 mg FB/kg of diet, no effect on growth performance was seen in broiler chickens (Henry et al., 2000; Antonissen et al., 2015; Antonissen et al., 2017), although the sphinganine: sphingosine ratio, a biomarker of FB exposure in animals, was increased (Henry et al., 2000; Antonissen et al., 2015). On the other hand, FB levels greater than 100 mg/kg of diet clearly decreased growth and increased mortality rates (Brown et al., 1992; Javed et al., 1993; Rauber et al., 2013). Thus, 10 mg FB/kg of diet used in this study, despite being within the contaminant levels in the chicken feeds (Kim et al., 2014), is considered low with respect to its impact on chickens. An unexpected observation emerged from this study. In sharp contrast to previous findings (Heckert et al., 2002; Ravindran et al., 2006), increasing stocking density significantly increased relative bursal weight. It is generally thought that environmental stressors (i.e., stocking density) may be associated with a decrease in primary and secondary lymphoid organs (Heckert et al., 2002), although no apparent effect on lymphoid weight (Buijs et al., 2012) has been reported. Unfortunately, we did not monitor humoral immune responses in this study. Thus, in-depth evaluation of local and systemic immune responses in broiler chickens raised at different stocking densities is warranted. As high stocking density and mycotoxin contamination in feeds are considered stress factors in poultry, several parameters, including H/L ratio, NO, AGP, and CORT, were determined as the main adaptive stress response indices of poultry (Puvadolpirod and Thaxton, 2000). It is clear from the current study that increasing stocking density increased H/L ratio, and FB increased serum NO levels. However, neither stocking density nor FB affected serum concentrations of AGP and CORT. Although increasing stocking density is known to affect stress indicators such as AGP, CORT, and H/L ratio (Shakeri et al., 2014), no clear stress-induced effect (Thaxton et al., 2006; Türkyilmaz, 2008) was noted. In addition, an increased H/L ratio was found in broilers fed 10 mg/kg of deoxynivalenol, which is also a Fusarium mycotoxin (Ghareeb et al., 2012), and in turkeys fed 75 mg/kg of FB (Bermudez et al., 1996). On the other hand, purified FB did not affect CORT in broiler chickens (Antonissen et al., 2015). Thus, it seemed that there is no single physiological indicator for detecting stressed birds. Serum NO level, which is a direct indicator of oxidative stress (Lancaster Jr, 1996) and a measure of immune status (Lillehoj and Li, 2004), increased by 26% in FB-fed chickens compared with the control counterparts, indicating that the mycotoxin could induce NO production in macrophages and monocytes. Uncontrolled NO production is known to induce gut damage (Allen, 1997), albeit that the FB-induced NO increase in this study was not accompanied by altered gut morphology or ileal sIgA levels. The FB-induced increase in NO may account for FB as a predisposing factor in the pathogenesis of necrotic enteritis or coccidiosis (Antonissen et al., 2015). In addition, the mitigating effect of dietary TB on FB-induced NO levels was noted. This is expected, as TB used in this study contained an FB-degrading enzyme, a mineral-based adsorbing agent, and plant and algae extracts. Although we failed to see interactions between stocking density and FB, and FB and TB (except for NO), a significant interaction between stocking density and TB was found for body weight gain and ileal sIgA levels at 21 days. Increasing stocking density increased weight gain in chickens fed a TB-free diet, but decreased weight gain in chickens fed a TB-added diet. Ileal sIgA levels were elevated in birds raised in HSD with a TB-added diet, and in those raised in MSD with a TB-free diet. These results were unexpected, as TB is presumed to act on mycotoxins present in feeds and/or gut contents. Indeed, TB alone did not affect any of the many parameters measured in this study. At this stage, the underlying basis for the observed interaction between stocking density and TB is not clear. In conclusion, the present study demonstrated that increasing stocking density decreased growth performance and tibial bone quality, and increased relative bursa weight and H/L ratio in broiler chickens. In addition, dietary FB increased the feed-to-gain ratio and serum NO levels in broiler chickens. A significant interaction between FB and TB with respect to the NO levels was noted. However, there was no interaction between stocking density and FB for any of the measurements. Further studies are warranted to determine whether failure of FB to interact with stocking density in broiler chickens may be due to the low level of FB used in this study. Finally, whether the effect of increasing stocking density on the increase in relative bursa weight is related to altered acquired immune response should be addressed. ACKNOWLEDGEMENTS This work was supported by the Korean Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fishery (IPET) through the Agri-Bio Industry Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA)(316036–3). REFERENCES Abudabos A. M., Samara E. M., Hussein E. O., Al-Ghadi M. A. Q., Al-Atiyat R. M.. 2013. Impacts of stocking density on the performance and welfare of broiler chickens. Ital. J. Anim. Sci.  12: e11. Google Scholar CrossRef Search ADS   Allen P. C. 1997. Nitric oxide production during Eimeria tenella infections in chickens. Poult. Sci.  76: 810– 813. Google Scholar CrossRef Search ADS PubMed  Antonissen G., Martel A., Pasmans F., Ducatelle R., Verbrugghe E., Vandenbroucke V., Li S., Haesebrouck F., Van Immerseel F., Croubels S.. 2014. The impact of Fusarium mycotoxins on human and animal host susceptibility to infectious diseases. Toxins . 6: 430– 452. Google Scholar CrossRef Search ADS PubMed  Antonissen G., Croubels S., Pasmans F., Ducatelle R., Eeckhaut V., Devreese M., Verlinden M., Haesebrouck F., Eeckhout M., De Saeger S.. 2015. Fumonisins affect the intestinal microbial homeostasis in broiler chickens, predisposing to necrotic enteritis. Vet. Res.  46: 98. Google Scholar CrossRef Search ADS PubMed  Antonissen G., Devreese M., De Baere S., Martel A., Van Immerseel F., Croubels S.. 2017. Impact of Fusarium mycotoxins on hepatic and intestinal mRNA expression of cytochrome P450 enzymes and drug transporters, and on the pharmacokinetics of oral enrofloxacin in broiler chickens. Food Chem. Toxicol.  101: 75– 83. Google Scholar CrossRef Search ADS PubMed  Awad W. A., Böhm J., Razzazi-Fazeli E., Zentek J.. 2006. Effects of feeding deoxynivalenol contaminated wheat on growth performance, organ weights and histological parameters of the intestine of broiler chickens. J. Anim. Physiol. Anim. Nutr.  90: 32– 37. Google Scholar CrossRef Search ADS   Beloor J., Kang H. K., Kim Y. J., Subramani V. K., Jang I. S., Sohn S. H., Moon Y. S.. 2010. The effect of stocking density on stress related genes and telomeric length in broiler chickens. Asian-australas. J. Anim. Sci.  23: 437– 443. Google Scholar CrossRef Search ADS   Bermudez A. J., Ledoux D. R., Turk J. R., Rottinghaus G. E.. 1996. The chronic effects of Fusarium moniliforme culture material, containing known levels of fumonisin B1, in turkeys. Avian Dis . 40: 231– 235. Google Scholar CrossRef Search ADS PubMed  Boudergue C., Burel C., Dragacci S., Favrot M. C., Fremy J. M., Massimi C., Prigent P., Debongnie P., Pussemier L., Boudra H.. 2009. Review of mycotoxin-detoxifying agents used as feed additives: mode of action, efficacy and feed/food safety. EFSA Supporting Publications . 6: e22. Google Scholar CrossRef Search ADS   Bouhet S., Hourcade E., Loiseau N., Fikry A., Martinez S., Roselli M., Galtier P., Mengheri E., Oswald I. P.. 2004. The mycotoxin fumonisin B1 alters the proliferation and the barrier function of porcine intestinal epithelial cells. Toxicol. Sci.  77: 165– 171. Google Scholar CrossRef Search ADS PubMed  Bradford M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.  72: 248– 254. Google Scholar CrossRef Search ADS PubMed  Brown T. P., Rottinghaus G. E., Williams M. E.. 1992. Fumonisin mycotoxicosis in broilers: Performance and pathology. Avian Dis . 36: 450– 454. Google Scholar CrossRef Search ADS PubMed  Buijs S., Van Poucke E., Van Dongen S., Lens L., Baert J., Tuyttens F. A.. 2012. The influence of stocking density on broiler chicken bone quality and fluctuating asymmetry. Poult. Sci.  91: 1759– 1767. Google Scholar CrossRef Search ADS PubMed  Cengiz O., Koksal B. H., Tath O., Sevim O., Ahsan U., Uner A. G., Ulutas P. A., Beyaz D., Buyukyoruk S., Yakan A., Onol A. G.. 2015. Effect of dietary probiotic and high stocking density on the performance, carcass yield, gut microflora, and stress indicators of broilers. Poult. Sci.  94: 2395– 2403. Google Scholar CrossRef Search ADS PubMed  Dombrink-Kurtzman M. A., Gomez-Flores R., Weber R. J.. 2000. Activation of rat splenic macrophage and lymphocyte functions by fumonisin B1. Immunopharmacology . 49: 401– 409. Google Scholar CrossRef Search ADS PubMed  Dozier W. A., Thaxton J. P., Purswell J. L., Olanrewaju H. A., Branton S. L., Roush W. B.. 2006. Stocking density effects on male broilers grown to 1.8 kilograms of body weight. Poult. Sci.  85: 344– 351. Google Scholar CrossRef Search ADS PubMed  Du E., Wang W., Gan L., Li Z., Guo S., Guo Y.. 2016. Effects of thymol and carvacrol supplementation on intestinal integrity and immune responses of broiler chickens challenged with Clostridium perfringens. J. Anim. Sci. Biotechnol.  7: 19. Google Scholar CrossRef Search ADS PubMed  Estevez I. 2007. Density allowances for broilers: Where to set the limits? Poult. Sci.  86: 1265– 1272. Google Scholar CrossRef Search ADS PubMed  European Commission. 2006. Commission recommendation of 17 August 2006 on the presence of deoxynivalenol, zearalenone, ochratoxin A, T-2 and HT-2 and fumonisins in products intended for animal feeding. Off. J. Eur. Union.  229, 7– 9. European Commission. 2007. Council Directive 2007/43/EC of 28 June 2007 laying down minimum rules for the protection of chickens kept for meat production. Off. J. Eur. Union.  182, 19– 28. FDA. 2001. Guidance for Industry: Fumonisin Levels in Human Foods and Animal Feeds . U.S. FDA Center for Food Safety and Applied Nutrition and Center for Veterinary Medicine, Washington, DC. Ghareeb K., Awad W. A., Böhm J.. 2012. Ameliorative effect of a microbial feed additive on infectious bronchitis virus antibody titer and stress index in broiler chicks fed deoxynivalenol. Poult. Sci.  91: 800– 807. Google Scholar CrossRef Search ADS PubMed  Gonzales E., Kondo N., Saldanha E. S., Loddy M. M., Careghi C., Decuypere E.. 2003. Performance and physiological parameters of broiler chickens subjected to fasting on the neonatal period. Poult. Sci.  82: 1250– 1256. Google Scholar CrossRef Search ADS PubMed  Guardia S., Konsak B., Combes S., Levenez F., Cauquil L., Guillot J.-F., Moreau-Vauzelle C., Lessire M., Juin H., Gabriel I.. 2011. Effects of stocking density on the growth performance and digestive microbiota of broiler chickens. Poult. Sci.  90: 1878– 1889. Google Scholar CrossRef Search ADS PubMed  Hall A. L. 2001. The effect of stocking density on the welfare and behaviour of broiler chickens reared commercially. Anim. Welfare.  10: 23– 40. Heckert R. A., Estevez I., Russek-Cohen E., Pettit-Riley R.. 2002. Effects of density and perch availability on the immune status of broilers. Poult. Sci.  81: 451– 457. Google Scholar CrossRef Search ADS PubMed  Henry M. H., Wyatt R. D., Fletchert O. J.. 2000. The toxicity of purified fumonisin B1 in broiler chicks. Poult. Sci.  79: 1378– 1384. Google Scholar CrossRef Search ADS PubMed  Hongchao J., Jiang Y., Song Z., Zhao J., Wang X., Lin H.. 2014. Effect of perch type and stocking density on the behaviour and growth of broilers. Anim. Prod. Sci.  54: 930– 941. Huwig A., Freimund S., Käppeli O., Dutler H.. 2001. Mycotoxin detoxication of animal feed by different adsorbents. Toxicol. Lett.  122: 179– 188. Google Scholar CrossRef Search ADS PubMed  Incharoen T., Yamauchi K.. 2009. Production performance, egg quality and intestinal histology in laying hens fed dietary dried fermented ginger. Poult. Sci.  8: 1078– 1085. Google Scholar CrossRef Search ADS   Javed T., Bennett G. A., Richard J. L., Dombrink-Kurtzman M. A., Cote L. M., Buck W. B.. 1993. Mortality in broiler chicks on feed amended with Fusarium proliferatum culture material or with purified fumonisin B1 and moniliformin. Mycopathologia . 123: 171– 184. Google Scholar CrossRef Search ADS PubMed  Kim N. Y., Lee I., Ji G. E.. 2014. Reliable and simple detection of ochratoxin and fumonisin production in black Aspergillus. J. Food Prot.  77: 653– 658. Google Scholar CrossRef Search ADS PubMed  Lancaster J. Jr. 1996. Nitric Oxide: Principles and Actions . Academic Press. Ledoux D. R., Brown T. P., Weibking T. S., Rottinghaus G. E.. 1992. Fumonisin toxicity in broiler chicks. J. Vet. Diagn. Invest.  4: 330– 333. Google Scholar CrossRef Search ADS PubMed  Lillehoj H. S., Li G.. 2004. Nitric oxide production by macrophages stimulated with coccidia sporozoites, lipopolysaccharide, or interferon-γ, and its dynamic changes in SC and TK strains of chickens infected with Eimeria tenella. Avian Dis . 48: 244– 253. Google Scholar CrossRef Search ADS PubMed  Miles R. D., Butcher G. D., Henry P. R., Littell R. C.. 2006. Effect of antibiotic growth promoters on broiler performance, intestinal growth parameters, and quantitative morphology. Poult. Sci.  85: 476– 485. Google Scholar CrossRef Search ADS PubMed  Mosca F., Madeddu M., Mangiagalli M. G., Colombo E., Cozzi M. C., Zaniboni L., Cerolini S.. 2015. Bird density, stress markers and growth performance in the Italian chicken breed Milanino. J. Appl. Poult. Res.  24: 529– 535. Murugesan G. R., Ledoux D. R., Naehrer K., Berthiller F., Applegate T. J., Grenier B., Phillips T. D., Schatzmayr G.. 2015. Prevalence and effects of mycotoxins on poultry health and performance, and recent development in mycotoxin counteracting strategies 1. Poult. Sci.  94: 1298– 1315. Google Scholar CrossRef Search ADS PubMed  Nogueira W., Velásquez P., Furlan R. L., Macari M.. 2013. Effect of dietary energy and stocking density on the performance and sensible heat loss of broilers reared under tropical winter conditions. Rev. Bras. Cienc. Avic.  15: 53– 57. Google Scholar CrossRef Search ADS   Norred W. P. 1993. Fumonisins-mycotoxins produced by fusarium moniliforme. J. Toxicol. Environ. Health A.  38: 309– 328. Google Scholar CrossRef Search ADS   Pappas A., Tsiplakou E., Georgiadou M., Anagnostopoulos C., Markoglou A., Liapis K., Zervas G.. 2014. Bentonite binders in the presence of mycotoxins: Results of in vitro preliminary tests and an in vivo broiler trial. Appl. Clay Sci.  99: 48– 53. Google Scholar CrossRef Search ADS   Puvadolpirod S., Thaxton J. P.. 2000. Model of physiological stress in chickens 1. Response parameters. Poult. Sci . 79: 363– 369. Google Scholar CrossRef Search ADS PubMed  Rauber R. H., Oliveira M. S., Mallmann A. O., Dilkin P., Mallmann C. A., Giacomini L. Z., Nascimento V. P.. 2013. Effects of fumonisin B1 on selected biological responses and performance of broiler chickens. Pesqui. Vet. Bras.  33: 1081– 1086. Google Scholar CrossRef Search ADS   Ravindran V., Thomas D. V., Thomas D. G., Morel P. C.. 2006. Performance and welfare of broilers as affected by stocking density and zinc bacitracin supplementation. Anim. Sci. J.  77: 110– 116. Google Scholar CrossRef Search ADS   Seo D.-G., Phat C., Kim D.-H., Lee C.. 2013. Occurrence of Fusarium mycotoxin fumonisin B1 and B2 in animal feeds in Korea. Mycotoxin Res . 29: 159– 167. Google Scholar CrossRef Search ADS PubMed  Shakeri M., Zulkifli I., Soleimani A. F., O’Reilly E. L., Eckersall P. D., Anna A. A., Kumari S., Abdullah F. F. J.. 2014. Response to dietary supplementation of L-glutamine and L-glutamate in broiler chickens reared at different stocking densities under hot, humid tropical conditions. Poult. Sci.  93: 2700– 2708. Google Scholar CrossRef Search ADS PubMed  Skomorucha I., Muchacka R., Sosnówka-Czajka E., Herbut E.. 2009. Response of broiler chickens from three genetic groups to different stocking densities. Ann. Anim. Sci.  9: 175– 184. Smith L. E., Stoltzfus R. J., Prendergast A.. 2012. Food chain mycotoxin exposure, gut health, and impaired growth: A conceptual framework. Adv. Nutr.  3: 526– 531. Google Scholar CrossRef Search ADS PubMed  Tan J., Applegate T. J., Liu S., Guo. Y., Eicher D.. 2014. Supplemental dietary L-arginine attenuates intestinal mucosal disruption during a coccidial vaccine challenge in broiler chickens. Brit. J. Nutr.  112: 1098– 1109. Google Scholar CrossRef Search ADS   Thaxton J. P., Dozier W. A., Branton S. L., Morgan G. W., Miles D. W., Roush W. B., Lott B. D., Vizzier-Thaxton Y.. 2006. Stocking density and physiological adaptive responses of broilers. Poult. Sci.  85: 819– 824. Google Scholar CrossRef Search ADS PubMed  Türkyilmaz M. K. 2008. The effect of stocking density on stress reaction in broiler chickens during summer. Turk. J. Vet. Anim. Sci.  32: 31– 36. Voss K. A., Smith G. W., Haschek W. M.. 2007. Fumonisins: toxicokinetics, mechanism of action and toxicity. Anim. Feed Sci. Technol.  137: 299– 325. Google Scholar CrossRef Search ADS   Wang B., Min Z., Yuan J., Zhang B., Guo Y.. 2014. Effects of dietary tryptophan and stocking density on the performance, meat quality, and metabolic status of broilers. J. Anim. Sci. Biotechnol.  5: 44. Google Scholar CrossRef Search ADS PubMed  Yegani M., Smith T. K., Leeson S., Boermans H. J.. 2006. Effects of feeding grains naturally contaminated with Fusarium mycotoxins on performance and metabolism of broiler breeders. Poult. Sci.  85: 1541– 1549. Google Scholar CrossRef Search ADS PubMed  © 2017 Poultry Science Association Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Effects of fumonisin B1 and mycotoxin binders on growth performance, tibia characteristics, gut physiology, and stress indicators in broiler chickens raised in different stocking densities

Loading next page...
 
/lp/ou_press/effects-of-fumonisin-b1-and-mycotoxin-binders-on-growth-performance-2UFqLNroZ4
Publisher
Oxford University Press
Copyright
© 2017 Poultry Science Association Inc.
ISSN
0032-5791
eISSN
1525-3171
D.O.I.
10.3382/ps/pex382
Publisher site
See Article on Publisher Site

Abstract

Abstract The present study was carried out to investigate the effects of stocking density, fumonisin B1 (FB), and mycotoxin binder (TB) on growth performance, bone quality, physiological stress indicators, and gut health in broiler chickens. Day-old Ross 308 male broiler chicks (n = 1,440/experiment) were randomly allocated to 72 floor pens in a 3 × 2 × 2 factorial arrangement, using 3 stocking densities (12.5 birds/m2 [HSD], 10 birds/m2 [MSD], or 7.5 birds/m2 [LSD]), 2 levels of purified FB (0 or 10 ppm), and 2 levels of TB (0 or 0.2%). Each treatment had 6 replicates (n = 6/treatment) and experiments lasted 34 days. All data were analyzed using 3-way ANOVA with stocking density level, FB, and TB as main factors. Body weight gain and feed intake were lower (P < 0.05) in broilers kept at HSD and MSD compared to LSD-housed counterparts. Birds fed an FB-contaminated diet exhibited a higher feed-to-gain ratio compared with those fed an FB-free diet (P < 0.05). None of the treatments affected intestinal morphology or ileal secretory immunoglobulin A levels. Stocking density affected tibia breaking strength (P < 0.05), which was lower in chickens housed at HSD compared with LSD-housed chickens. The heterophil/lymphocyte ratio (H/L ratio) was elevated (P < 0.05) in HSD and MSD groups compared with the LSD group. Serum nitric oxide (NO) levels were elevated (P < 0.05) in chickens fed the FB-contaminated diet compared with the control diet-fed counterparts. Significant interaction (P < 0.05) between FB and TB on serum NO levels was noted. In summary, increasing stocking density lowered growth performance and bone quality, but increased the H/L ratio. Dietary TB did not affect FB-induced increases in the feed-to-gain ratio. No interaction was observed between stocking density and FB for the measured variables. INTRODUCTION Many stress factors such as stocking density and/or diet-associated contaminants (i.e., mycotoxins) are known to negatively affect the welfare, health, and productivity of commercial poultry (Henry et al., 2000; Mosca et al., 2015; Murugesan et al., 2015). Dozier et al. (2006) reported that increasing stocking density from 25 to 40 kg of BW/m² linearly lowered body weight and feed intake, but increased feed conversion ratio in broiler chickens raised up to 35 d of age. Contradictory results have been reported with regard to the physiological stress responses to stocking density in chickens. For example, Dozier et al. (2006) demonstrated that stocking density did not alter corticosterone, glucose, cholesterol, or the heterophil/lymphocyte (H/L) ratio, which are indicative of stress in broiler chickens. However, other studies reported that increasing stocking density lowered physiological indices, including corticosterone and H/L ratio in chickens (Thaxton et al., 2006; Skomorucha et al., 2009). In addition, higher stocking density has been shown to affect the bone quality of broilers, reducing shear strength of the tibia due to insufficient rearing space (Buijs et al., 2012). The maximum recommended and allowed stocking density for broiler chickens has been set at less than 33 kg of BW/m² in Europe (European Commission, 2007). Mycotoxin contamination in feeds is an often-underestimated stress factor for broiler chickens. Mycotoxins are known to reduce growth performance and feed efficiency, increase mortality, and compromise the immune system in chickens (Awad et al., 2006; Yegani et al., 2006; Smith et al., 2012). Fumonisins are secondary metabolites of Fusarium verticillioides, and fumonisin B1 (FB) is the predominant form among fumonisin analogues (designated as A1, A2, B1, B2, B3, and B4) (Norred, 1993; Voss et al., 2007). FB-contaminated feed was reported to decrease villus height, villus/crypt ratio (Rauber et al., 2013; Antonissen et al., 2015), and body weight gain (Ledoux et al., 1992), and increase several FB-associated clinical signs, including liver necrosis, diarrhea, and rickets (Brown et al., 1992) in poultry. As FB can affect the intestinal epithelium (Bouhet et al., 2004; Antonissen et al., 2014), it is known to be a predisposing factor in necrotic enteritis in broiler chickens (Antonissen et al., 2015). In a recent survey in Korea, it was reported that more than 70% of commercial broiler diets were contaminated with FB, although the levels ranged from as low as 92 μg/kg to as high as 12.8 mg/kg (Kim et al., 2014). However, these levels are considered negligible, as the maximum recommended FB concentration in chicken feed varies from 20 to 50 mg/kg of diet (Food and Drug Administration [FDA], 2001; European Commission, 2006). Dietary strategies (e.g., use of toxin binders, TB) to form stable complexes with mycotoxins or to transform mycotoxins into nontoxic metabolites are commonly used to lessen toxicity in chickens (Huwig et al., 2001; Boudergue et al., 2009; Pappas et al., 2014). As both stocking density and FB-contaminated feed are important factors that negatively affect animal welfare or health of chickens, it was thought that they might act in an additive or synergistic manner. However, to the best of our knowledge, no reports have examined this hypothesis. Therefore, the present study was conducted to determine whether a combination of stocking density and/or FB-contaminated feed can affect growth performance, intestinal morphology, bone characteristics, and physiological stress indicators in broiler chicks. In addition, dietary TB was added to determine whether it can counteract FB-induced reduction in performance or physiological responses in broiler chickens. MATERIALS AND METHODS All animal care procedures were approved by Institutional Animal Care and Use Committee of Konkuk University (KU16138). Experimental Design A 3 × 2 × 2 factorial design (Table 1) was used with stocking density, FB, and TB as the main factors. A corn-soybean meal basal diet (Table 2) was fed to chickens. Experimental diets were formulated to mix the basal diet with or without 10 mg/kg purified FB and 0.2% of TB, and were fed to birds kept at 7.5 birds/m² (low stocking density: LSD), 10 birds/m² (medium stocking density: MSD), or 12.5 birds/m² (high stocking density: HSD). Each treatment had 6 replicated pens. Purified FB concentration was confirmed with liquid chromatography-mass spectrometry and was provided by Biomin Korea Ltd., Seoul, Korea. TB used in this study was a mixture of FB-degrading enzyme, mineral-based adsorbing agent, and plant and algae extracts. Table 1. Experimental design. Treatments        Density  FB  TB  Replicates/treatment  Total chicks/pen (birds/m2)  Total chicks/treatment   Low  No  No  6  15 (7.5)  90  Medium  No  No  6  20 (10)  120   High  No  No  6  25 (12.5)  150   Low  No  Yes  6  15 (7.5)  90  Medium  No  Yes  6  20 (10)  120   High  No  Yes  6  25 (12.5)  150   Low  Yes  No  6  15 (7.5)  90  Medium  Yes  No  6  20 (10)  120   High  Yes  No  6  25 (12.5)  150   Low  Yes  Yes  6  15 (7.5)  90  Medium  Yes  Yes  6  20 (10)  120   High  Yes  Yes  6  25 (12.5)  150  Treatments        Density  FB  TB  Replicates/treatment  Total chicks/pen (birds/m2)  Total chicks/treatment   Low  No  No  6  15 (7.5)  90  Medium  No  No  6  20 (10)  120   High  No  No  6  25 (12.5)  150   Low  No  Yes  6  15 (7.5)  90  Medium  No  Yes  6  20 (10)  120   High  No  Yes  6  25 (12.5)  150   Low  Yes  No  6  15 (7.5)  90  Medium  Yes  No  6  20 (10)  120   High  Yes  No  6  25 (12.5)  150   Low  Yes  Yes  6  15 (7.5)  90  Medium  Yes  Yes  6  20 (10)  120   High  Yes  Yes  6  25 (12.5)  150  View Large Table 2. Ingredients and nutrient composition of the basal diet (%, as-fed basis). Ingredients  g/100g  Corn  51.31  Soybean meal, 44% CP  29.34  Wheat, 12% CP  7.00  Corn gluten meal, 60% CP  4.00  Lysine-HCl, 78%  0.13  DL-methionine, 98%  0.24  Dicalcium phosphate  2.05  Threonine  0.07  Choline, 50%  0.10  Salt  0.30  Limestone coarse  1.14  NaHCO3  0.05  Tallow  4.00  Vitamin premix1  0.12  Mineral premix2  0.15  Total  100.00  Calculated or analyzed nutrient composition, %    AMEn3, kcal/kg  3170  Crude protein4  20.56  Crude fat4  6.27  Ca4  1.04  Available phosphorus3  0.46  Total phosphorus4  0.87  Lysine3  1.15  Methionine3  0.59  Met+Cys3  0.95  Ingredients  g/100g  Corn  51.31  Soybean meal, 44% CP  29.34  Wheat, 12% CP  7.00  Corn gluten meal, 60% CP  4.00  Lysine-HCl, 78%  0.13  DL-methionine, 98%  0.24  Dicalcium phosphate  2.05  Threonine  0.07  Choline, 50%  0.10  Salt  0.30  Limestone coarse  1.14  NaHCO3  0.05  Tallow  4.00  Vitamin premix1  0.12  Mineral premix2  0.15  Total  100.00  Calculated or analyzed nutrient composition, %    AMEn3, kcal/kg  3170  Crude protein4  20.56  Crude fat4  6.27  Ca4  1.04  Available phosphorus3  0.46  Total phosphorus4  0.87  Lysine3  1.15  Methionine3  0.59  Met+Cys3  0.95  1Vitamin premix provided following nutrients per kg of diet: vitamin A, 18,000 IU; vitamin D3, 3000 IU; vitamin E, 80 IU; vitamin K3, 5 mg; vitamin B12, 0.06 mg; thiamin, 5 mg; riboflavin, 20 mg; pyridoxine, 8 mg; niacin, 90 mg; biotin, 0.2 mg; folic acid, 1.1 mg; pantothenic acid, 50 mg. 2Mineral premix provided following nutrients per kg of diet: Fe, 80 mg; Mn, 40 mg; Zn, 12.5 mg; I, 0.25 mg; Se, 0.2 mg; Cu, 30 mg. 3Calculated value as-fed basis. 4Analyzed value as-fed basis. View Large A total of 1,440 day-old male broiler chicks (Ross 308) was purchased from a local hatchery, weighed upon arrival, and randomly allocated to one of 72 floor pens. Each pen had a pan feeder with a 52-cm diameter and had rice husks as a bedding material with a floor area of 2 m². Temperature of the facility was initially set at 34°C during the first wk, then gradually decreased to reach 24°C at 21 d, and then was maintained. Feed and water were provided ad libitum, and light was provided 23 h/day. Birds and feed intake on a pen basis were monitored weekly. The incidence of mortality was recorded daily to calculate the mortality-adjusted feed intake and feed conversion ratio. Production index (PI) was calculated with the following formula: PI = (livability × average daily gain)/ (feed conversion ratio × 10) (Gonzales et al., 2003). Sampling On d 34, one bird per pen with a weight close to the mean was randomly selected for sampling and euthanized with carbon dioxide. Immediately after euthanasia, blood was taken via cardiac puncture and collected in both heparinized and plain tubes. Serum samples were obtained by gentle centrifugation at 1,700 × g for 15 min and stored at –20 °C until use. Immediately after blood sampling, left breast and left leg meats were sampled, and the intestinal tract was removed. Then, organs (i.e., liver, spleen, and bursa of Fabricius) and abdominal fat were sampled and weighed. Relative organ weights were calculated from the ratio of organ weight to body weight and were expressed as grams per 100 grams of body weight. A segment of ileum (from Meckel's diverticulum to the ileocecal valve) was excised and an ileal segment 2-cm posterior to Meckel's diverticulum was sampled and fixed in 10% neutral buffered formalin solution for more than 48 hours. In addition, a 10-cm mid-ileal segment was dissected and washed with ice-cold saline, and ileal mucosal samples were collected by scraping the mucosal layer. The mucosal layers were then homogenized with 5 volumes of ice-cold saline and centrifuged at 15,000 × g for 10 min, and the supernatants were stored at –20 °C until use. Intestinal Morphology Formalin-fixed ileal segments were dehydrated with an alcohol-xylene sequence, embedded in paraffin, and sectioned in 5-μm slides. The sections were then stained with standard hematoxylin and eosin solution, and observed for villus height and width, and crypt depth at 100 × magnification by light microscopy (CKX53, Olympus, Tokyo, Japan) using a calibrated ocular micrometer. Ten microscopic fields per bird were measured. Villus surface area (μm2 × 10−3) was calculated with the following formula: villus surface area = villus height × (villus diameter at 1/3 of its height + diameter at 2/3 of its height)/2 (Miles et al., 2006; Incharoen and Yamauchi, 2009). Secretory Immunoglobulin A in Mucosal Scraping Secretory immunoglobulin A (sIgA) concentrations in ileal mucosal scrapings were measured (Tan et al., 2014; Du et al., 2016) using quantitative chicken IgA enzyme-linked immunosorbent assay (ELISA) kits (Bethyl Co., Montgomery, TX) as per the manufacturer's recommendation. The results were normalized against total protein concentration in each sample. Total protein concentrations in ileal scrapings were quantified as described by Bradford (1976) using bovine serum albumin. Tibia Characteristics The left tibia was obtained by manually removing attached meats. Breaking bone strength was measured using a testing machine (Instron 3342; Instron Corp., Canton, MA). The sheared tibia pieces were then dried overnight at 105 °C, ashed in a muffle furnace (Isotemp; Fisher Scientific, Pittsburgh, PA) at 600 °C for 2 h, and manually ground in a mortar before mineral content determination. Mineral (Ca, P, and Mg) contents were analyzed with an inductively coupled plasma emission spectrophotometer (Perkin Elmer 3300, Shelton, CT). Physiological Stress Indicators Heterophils and lymphocytes from heparinized fresh blood samples were counted using a Hemavet® HV950FS (Drew Scientific, Oxford, CT). Serum samples were used to measure nitric oxide (NO), corticosterone (CORT), and α-1-acid glycoprotein (AGP) using commercially available ELISA kits (NO: R&D Systems; CORT: ENZO; AGP: MyBioSource). Statistical Analysis Each pen was considered an experimental unit. Data for all variables were analyzed using 3-way analysis of variance (ANOVA), with the model including stocking density, mycotoxin, and TB as the main factors; interactions were analyzed using a general linear model (SAS 9.4, SAS Institute). Duncan's multiple range test was employed to determine means and differences among treatments. Significant differences among treatments were determined for P < 0.05, and trends were noted when 0.05 < P < 0.10. RESULTS Growth Performance and Production Index Body weight gain was higher (P < 0.001) in chickens kept at LSD compared with those at MSD or HSD during d 1 to 34 (Table 3). Chickens kept at MSD or HSD ate less (P < 0.001) compared with LSD-kept counterparts during d 22 to 34 or 1 to 34. In contrast, feed-to-gain ratio tended to decrease (P = 0.068) with increasing stocking density during d 1 to 21, but this trend was not found (P > 0.05) thereafter. Feeding FB-contaminated diets did not affect weight gain or feed intake during the entire period of the experiment. However, chickens fed the FB-contaminated diet exhibited higher (P = 0.019) feed-to-gain ratio compared with the control diet-fed chickens during d 1 to 34. Dietary TB did not influence growth performance. Significant interaction (P = 0.046) between stocking density and TB with regard to body weight gain during d 1 to 21 was observed. Body weight gain increased as the stocking density increased in the absence of TB, but this trend was reversed by the presence of TB. PI was high (P < 0.001) in LSD vs. MSD- or HSD-raised chickens (Table 4). On the other hand, there was no difference (P > 0.05) in mortality among treatments. Table 3. Effect of mycotoxin and toxin binder on growth performance in broiler chickens raised at different stocking densities.       Body weight gain (g/day/bird)  Feed intake (g/day/bird)  Feed conversion ratio (g:g)  Density1  FB1  TB1  1 to 21 d  22 to 34 d  1 to 34 d  1 to 21 d  22 to 34 d  1 to 34 d  1 to 21 d  22 to 34 d  1 to 34 d   LSD  +  +  29.603  85.51  50.98  44.08  132.26  78.1  1.50  1.55  1.54    +  −  28.78  89.23  51.89  42.85  131.95  80.2  1.49  1.49  1.55    −  +  29.66  87.71  51.86  43.40  129.35  75.9  1.47  1.48  1.48    −  −  28.33  88.93  51.50  42.19  131.63  74.0  1.49  1.48  1.44   MSD  +  +  28.03  78.33  47.27  43.71  119.47  71.1  1.57  1.51  1.52    +  −  29.84  75.91  47.46  43.15  118.02  71.6  1.45  1.57  1.52    −  +  29.40  81.27  49.23  42.77  121.52  72.3  1.46  1.50  1.48    −  −  28.82  79.39  48.15  41.90  119.23  71.1  1.45  1.50  1.48   HSD  +  +  28.32  74.64  46.03  40.64  115.78  69.3  1.44  1.56  1.52    +  −  30.44  75.80  47.78  43.12  117.49  73.0  1.42  1.55  1.53    −  +  29.08  76.52  47.22  41.22  120.87  70.6  1.42  1.58  1.52    −  −  29.84  76.79  47.79  43.28  119.37  71.9  1.45  1.56  1.52  Pooled SEM2      0.75  2.32  1.08  0.89  2.23  1.86  0.037  0.040  0.032  Main factors                         LSD      29.09  87.85a  51.56a  43.13  131.30a  77.10a  1.49  1.50  1.50   MSD      29.02  78.73b  48.03b  42.88  119.56b  71.53b  1.48  1.52  1.50   HSD      29.42  75.94b  47.21b  42.06  118.49b  71.21b  1.43  1.50  1.52    +    29.17  79.91  48.57  42.92  122.78  73.9  1.48  1.54  1.53a    −    29.19  81.77  49.29  42.46  123.66  72.7  1.46  1.52  1.49b      +  29.02  80.67  48.76  42.64  123.54  72.9  1.47  1.53  1.51      −  29.34  81.01  49.10  42.75  122.95  73.7  1.46  1.52  1.51  P-value                        Density (D)      0.730  <0.001  <0.001  0.213  <0.001  <0.001  0.068  0.097  0.627  Fumonisin (F)      0.966  0.170  0.249  0.369  0.350  0.259  0.348  0.333  0.019  TB (B)      0.455  0.800  0.596  0.825  0.793  0.468  0.487  0.772  0.990  D × F      0.938  0.772  0.768  0.488  0.224  0.158  0.521  0.497  0.202  F × B      0.106  0.725  0.322  0.816  0.841  0.202  0.126  0.970  0.608  D × B      0.046  0.372  0.574  0.147  0.610  0.506  0.325  0.519  0.911  D × F × B      0.680  0.899  0.999  0.985  0.609  0.894  0.693  0.575  0.876        Body weight gain (g/day/bird)  Feed intake (g/day/bird)  Feed conversion ratio (g:g)  Density1  FB1  TB1  1 to 21 d  22 to 34 d  1 to 34 d  1 to 21 d  22 to 34 d  1 to 34 d  1 to 21 d  22 to 34 d  1 to 34 d   LSD  +  +  29.603  85.51  50.98  44.08  132.26  78.1  1.50  1.55  1.54    +  −  28.78  89.23  51.89  42.85  131.95  80.2  1.49  1.49  1.55    −  +  29.66  87.71  51.86  43.40  129.35  75.9  1.47  1.48  1.48    −  −  28.33  88.93  51.50  42.19  131.63  74.0  1.49  1.48  1.44   MSD  +  +  28.03  78.33  47.27  43.71  119.47  71.1  1.57  1.51  1.52    +  −  29.84  75.91  47.46  43.15  118.02  71.6  1.45  1.57  1.52    −  +  29.40  81.27  49.23  42.77  121.52  72.3  1.46  1.50  1.48    −  −  28.82  79.39  48.15  41.90  119.23  71.1  1.45  1.50  1.48   HSD  +  +  28.32  74.64  46.03  40.64  115.78  69.3  1.44  1.56  1.52    +  −  30.44  75.80  47.78  43.12  117.49  73.0  1.42  1.55  1.53    −  +  29.08  76.52  47.22  41.22  120.87  70.6  1.42  1.58  1.52    −  −  29.84  76.79  47.79  43.28  119.37  71.9  1.45  1.56  1.52  Pooled SEM2      0.75  2.32  1.08  0.89  2.23  1.86  0.037  0.040  0.032  Main factors                         LSD      29.09  87.85a  51.56a  43.13  131.30a  77.10a  1.49  1.50  1.50   MSD      29.02  78.73b  48.03b  42.88  119.56b  71.53b  1.48  1.52  1.50   HSD      29.42  75.94b  47.21b  42.06  118.49b  71.21b  1.43  1.50  1.52    +    29.17  79.91  48.57  42.92  122.78  73.9  1.48  1.54  1.53a    −    29.19  81.77  49.29  42.46  123.66  72.7  1.46  1.52  1.49b      +  29.02  80.67  48.76  42.64  123.54  72.9  1.47  1.53  1.51      −  29.34  81.01  49.10  42.75  122.95  73.7  1.46  1.52  1.51  P-value                        Density (D)      0.730  <0.001  <0.001  0.213  <0.001  <0.001  0.068  0.097  0.627  Fumonisin (F)      0.966  0.170  0.249  0.369  0.350  0.259  0.348  0.333  0.019  TB (B)      0.455  0.800  0.596  0.825  0.793  0.468  0.487  0.772  0.990  D × F      0.938  0.772  0.768  0.488  0.224  0.158  0.521  0.497  0.202  F × B      0.106  0.725  0.322  0.816  0.841  0.202  0.126  0.970  0.608  D × B      0.046  0.372  0.574  0.147  0.610  0.506  0.325  0.519  0.911  D × F × B      0.680  0.899  0.999  0.985  0.609  0.894  0.693  0.575  0.876  1FB = fumonisin; TB = toxin binder; LSD = low stocking density; MSD = medium stocking density; HSD = high stocking density. 2SEM = pooled standard error of the means. 3Values are LS means of 6 replicates per treatment. a,bValues (n = 6/treatment) having a different superscript within a column differ significantly (P < 0.05). View Large Table 4. Effects of mycotoxin and toxin binder on production index and mortality in broiler chickens raised at different stocking densities. Density1  FB1  TB1  Production index2  Mortality (%)   LSD  +  +  312.14  5.56    +  −  315.3  5.56    −  +  339.9  3.33    −  −  342.5  3.33   MSD  +  +  291.6  5.83    +  −  307.8  1.67    −  +  313.6  5.00    −  −  312.2  3.33   HSD  +  +  294.2  3.33    +  −  302.6  2.67    −  +  292.2  6.40    −  −  302.6  4.00  Pooled SEM3      3.72  0.037  Main factors           LSD      327.4a  4.44   MSD      306.2b  3.96   HSD      298.1b  4.10    +    303.9  4.10    −    317.9  4.23      +  307.7  4.91      −  313.8  3.43  P-value          Density (D)      <0.001  0.470  Fumonisin (F)      0.075  0.752  TB (B)      0.404  0.105  D × F      0.315  0.254  F × B      0.708  0.948  D × B      0.938  0.521  D × F × B      0.846  0.619  Density1  FB1  TB1  Production index2  Mortality (%)   LSD  +  +  312.14  5.56    +  −  315.3  5.56    −  +  339.9  3.33    −  −  342.5  3.33   MSD  +  +  291.6  5.83    +  −  307.8  1.67    −  +  313.6  5.00    −  −  312.2  3.33   HSD  +  +  294.2  3.33    +  −  302.6  2.67    −  +  292.2  6.40    −  −  302.6  4.00  Pooled SEM3      3.72  0.037  Main factors           LSD      327.4a  4.44   MSD      306.2b  3.96   HSD      298.1b  4.10    +    303.9  4.10    −    317.9  4.23      +  307.7  4.91      −  313.8  3.43  P-value          Density (D)      <0.001  0.470  Fumonisin (F)      0.075  0.752  TB (B)      0.404  0.105  D × F      0.315  0.254  F × B      0.708  0.948  D × B      0.938  0.521  D × F × B      0.846  0.619  1FB = fumonisin; TB = toxin binder; LSD = low stocking density; MSD = medium stocking density; HSD = high stocking density. 2Production index = European production index = [body weight gain, kg × livability, %)/(feed conversion ratio × age, days)] × 100. 3SEM = pooled standard error of the means. 4Values are LS means of 6 replicates per treatment. a,bValues (n = 6/treatment) having a different superscript within a column differ significantly (P < 0.05). View Large Relative Organ and Abdominal Fat Weights Relative liver, spleen, and abdominal fat weights were not affected (P > 0.05) by stocking density, FB, or TB (Table 5). However, relative weight of the bursa of Fabricius was higher (P = 0.037) in the HSD-raised birds compared with those raised at LSD. The MSD-raised birds exhibited intermediate bursal weight. No interaction among stocking density, FB, and TB was detected. Table 5. Effects of mycotoxin and toxin binder on relative organ weights (organ weight (g)/live body weight (g) × 100) in broiler chickens raised at different stocking densities. Density1  FB1  TB1  Liver  Spleen  Bursa of Fabricius  Abdo-minal fat  Live body weight   LSD  +  +  6.423  0.129  0.209  1.165  1685    +  −  6.54  0.121  0.191  1.108  1707    −  +  6.75  0.147  0.199  1.096  1695    −  −  6.79  0.147  0.169  1.005  1684   MSD  +  +  5.80  0.127  0.167  1.219  1573    +  −  6.78  0.122  0.217  1.105  1575    −  +  5.96  0.126  0.219  1.094  1640    −  −  6.52  0.138  0.215  1.129  1598   HSD  +  +  5.80  0.124  0.264  0.937  1543    +  −  6.78  0.125  0.251  0.981  1615    −  +  5.96  0.128  0.223  0.989  1603    −  −  6.52  0.111  0.244  1.212  1630  Pooled SEM2      0.081  0.0043  0.030  0.030  36.52  Main factors     LSD      6.62  0.136  0.192b  1.093  1693   MSD      6.49  0.128  0.205a,b  1.137  1597   HSD      6.26  0.122  0.245a  1.030  1598    +    6.42  0.125  0.216  1.086  1616    −    6.50  0.133  0.212  1.088  1642      +  6.34  0.130  0.213  1.083  1623      −  6.58  0.127  0.214  1.090  1635  P-value    Density (D)      0.194  0.420  0.037  0.340  <0.001  Fumonisin (F)      0.592  0.349  0.779  0.975  0.237  TB (B)      0.138  0.740  0.951  0.907  0.596  D × F      0.662  0.458  0.465  0.250  0.558  F × B      0.961  0.872  0.744  0.413  0.331  D × B      0.076  0.847  0.541  0.315  0.399  D × F × B      0.468  0.691  0.577  0.728  0.992  Density1  FB1  TB1  Liver  Spleen  Bursa of Fabricius  Abdo-minal fat  Live body weight   LSD  +  +  6.423  0.129  0.209  1.165  1685    +  −  6.54  0.121  0.191  1.108  1707    −  +  6.75  0.147  0.199  1.096  1695    −  −  6.79  0.147  0.169  1.005  1684   MSD  +  +  5.80  0.127  0.167  1.219  1573    +  −  6.78  0.122  0.217  1.105  1575    −  +  5.96  0.126  0.219  1.094  1640    −  −  6.52  0.138  0.215  1.129  1598   HSD  +  +  5.80  0.124  0.264  0.937  1543    +  −  6.78  0.125  0.251  0.981  1615    −  +  5.96  0.128  0.223  0.989  1603    −  −  6.52  0.111  0.244  1.212  1630  Pooled SEM2      0.081  0.0043  0.030  0.030  36.52  Main factors     LSD      6.62  0.136  0.192b  1.093  1693   MSD      6.49  0.128  0.205a,b  1.137  1597   HSD      6.26  0.122  0.245a  1.030  1598    +    6.42  0.125  0.216  1.086  1616    −    6.50  0.133  0.212  1.088  1642      +  6.34  0.130  0.213  1.083  1623      −  6.58  0.127  0.214  1.090  1635  P-value    Density (D)      0.194  0.420  0.037  0.340  <0.001  Fumonisin (F)      0.592  0.349  0.779  0.975  0.237  TB (B)      0.138  0.740  0.951  0.907  0.596  D × F      0.662  0.458  0.465  0.250  0.558  F × B      0.961  0.872  0.744  0.413  0.331  D × B      0.076  0.847  0.541  0.315  0.399  D × F × B      0.468  0.691  0.577  0.728  0.992  1FB = fumonisin; TB = toxin binder; LSD = low stocking density; MSD = medium stocking density; HSD = high stocking density. 2SEM = pooled standard error of the means. 3Values are LS means of 6 replicates per treatment. a,bValues (n = 6/treatment) having a different superscript within a column differ significantly (P < 0.05). View Large Intestinal Morphology Stocking density, FB, and TB did not (P > 0.05) affect any parameters of ileal morphology in the chicks (Table 6). Marginal interactions between stocking density and TB for crypt depth (P = 0.074) and villus surface area (P = 0.054), and between FB and TB for crypt depth (P = 0.056) were noted (Table 6). Table 6. Effect of mycotoxin and toxin binder on ileal morphometry and secretory immunoglobulin A in broiler chickens raised at different stocking densities. Density1  FB1  TB1  Villus height (μm)  Crypt depth (μm)  Villus surface area2 (μm2 × 10−3)  Villus height/crypt depth ratio  Ileal sIgA (μg/mg of protein)   LSD  +  +  688.54  140.94  0.090  4.90  0.2672    +  −  842.9  163.92  0.127  5.12  0.187    −  +  711.8  136.38  0.105  5.23  0.178    −  −  741.9  164.85  0.132  4.53  0.280   MSD  +  +  788.7  144.94  0.102  5.38  0.204    +  −  590.9  118.53  0.094  5.05  0.258    −  +  758.7  142.63  0.115  5.32  0.251    −  −  741.4  164.81  0.115  4.47  0.396   HSD  +  +  773.8  149.00  0.117  5.14  0.359    +  −  833.0  144.74  0.120  5.74  0.233    −  +  718.8  147.55  0.111  4.87  0.293    −  −  734.4  153.32  0.106  4.79  0.241  Pooled SEM3      72.5  9.27  0.011  0.378  0.015  Main factors                 LSD      746.5  150.95  0.113  4.96  0.234   MSD      719.9  142.73  0.106  5.06  0.284   HSD      765.0  148.65  0.113  5.13  0.286    +    753.0  143.68  0.108  5.22  0.260    −    734.3  151.21  0.113  4.88  0.275      +  740.1  143.58  0.106  5.14  0.264      −  747.6  151.32  0.115  4.96  0.273  P-value                Density (D)      0.679  0.443  0.629  0.818  0.286  Fumonisin (F)      0.661  0.162  0.436  0.119  0.622  TB (B)      0.862  0.145  0.181  0.407  0.813  D × F      0.390  0.159  0.238  0.657  0.179  F × B      0.967  0.056  0.785  0.117  0.054  D × B      0.139  0.074  0.054  0.284  0.042  D × F × B      0.319  0.208  0.837  0.931  0.726  Density1  FB1  TB1  Villus height (μm)  Crypt depth (μm)  Villus surface area2 (μm2 × 10−3)  Villus height/crypt depth ratio  Ileal sIgA (μg/mg of protein)   LSD  +  +  688.54  140.94  0.090  4.90  0.2672    +  −  842.9  163.92  0.127  5.12  0.187    −  +  711.8  136.38  0.105  5.23  0.178    −  −  741.9  164.85  0.132  4.53  0.280   MSD  +  +  788.7  144.94  0.102  5.38  0.204    +  −  590.9  118.53  0.094  5.05  0.258    −  +  758.7  142.63  0.115  5.32  0.251    −  −  741.4  164.81  0.115  4.47  0.396   HSD  +  +  773.8  149.00  0.117  5.14  0.359    +  −  833.0  144.74  0.120  5.74  0.233    −  +  718.8  147.55  0.111  4.87  0.293    −  −  734.4  153.32  0.106  4.79  0.241  Pooled SEM3      72.5  9.27  0.011  0.378  0.015  Main factors                 LSD      746.5  150.95  0.113  4.96  0.234   MSD      719.9  142.73  0.106  5.06  0.284   HSD      765.0  148.65  0.113  5.13  0.286    +    753.0  143.68  0.108  5.22  0.260    −    734.3  151.21  0.113  4.88  0.275      +  740.1  143.58  0.106  5.14  0.264      −  747.6  151.32  0.115  4.96  0.273  P-value                Density (D)      0.679  0.443  0.629  0.818  0.286  Fumonisin (F)      0.661  0.162  0.436  0.119  0.622  TB (B)      0.862  0.145  0.181  0.407  0.813  D × F      0.390  0.159  0.238  0.657  0.179  F × B      0.967  0.056  0.785  0.117  0.054  D × B      0.139  0.074  0.054  0.284  0.042  D × F × B      0.319  0.208  0.837  0.931  0.726  1FB = fumonisin; TB = toxin binder; LSD = low stocking density; MSD = medium stocking density; HSD = high stocking density; sIgA = secretory immunoglobulin A. 2Villus surface area = calculated by villus height × (diameter of villi's 1/3 height part + diameter of villi's 2/3 height part)/2. 3SEM = pooled standard error of the means. 4Values are LS means of 6 replicates per treatment. View Large Ileal sIgA sIgA concentration in ileal mucosa was not affected (P > 0.05) by stocking density, FB, or TB (Table 6). Significant (P = 0.042) or marginal (P = 0.054) interactions between stocking density and TB, or FB and TB for ileal sIgA were observed. Tibia Characteristics Tibia breaking strength was low (P = 0.045) in the HSD- vs. LSD-raised chickens (Table 7). The MSD-raised chickens showed intermediate tibia breaking strength. However, no interaction between main factors for tibia breaking strength was noted. Calcium content in the tibia was highest (P = 0.005) in the LSD- vs. MSD- or HSD-raised chickens. On the other hand, tibia ash, P, and Mg contents were not affected by the treatments, and no interaction among stocking density, FB, and TB was detected (Table 7). Table 7. Effects of mycotoxin and toxin binder on tibia breaking strength and mineral content in broiler chickens raised at different stocking densities. Density1  FB1  TB1  Breaking strength (kg)  Tibia ash (%)  Ca (%)  P (%)  Mg (%)   LSD  +  +  24.053  0.346  35.01  18.19  0.900    +  −  24.43  0.331  35.52  17.96  0.890    −  +  26.72  0.353  34.84  17.94  0.915    −  −  28.73  0.355  34.82  18.15  0.885   MSD  +  +  23.83  0.355  34.53  18.16  0.917    +  −  24.02  0.351  34.49  18.28  0.893    −  +  21.50  0.356  34.17  17.64  0.898    −  −  22.13  0.353  34.66  18.10  0.888   HSD  +  +  21.94  0.350  33.86  17.78  0.927    +  −  22.90  0.356  34.59  18.09  0.918    −  +  20.28  0.333  34.23  17.73  0.898    −  −  23.85  0.357  34.38  18.05  0.908  Pooled SEM2      0.64  0.0085  0.34  0.22  0.023  Main factors                 LSD      25.98a  0.346  35.04a  18.06  0.898   MSD      22.87a,b  0.354  34.46b  18.05  0.899   HSD      22.24b  0.349  34.26b  17.91  0.913    +    23.53  0.348  34.66  18.08  0.908    −    23.87  0.351  34.52  17.94  0.899      +  23.05  0.349  34.44  17.91  0.909      −  24.34  0.350  34.74  18.11  0.897  P-value                Density (D)      0.045  0.471  0.005  0.593  0.578  Fumonisin (F)      0.792  0.567  0.445  0.285  0.514  TB (B)      0.317  0.733  0.124  0.127  0.367  D × F      0.197  0.136  0.541  0.519  0.745  F × B      0.543  0.227  0.619  0.312  0.883  D × B      0.838  0.170  0.884  0.522  0.786  D × F × B      0.942  0.730  0.424  0.786  0.811  Density1  FB1  TB1  Breaking strength (kg)  Tibia ash (%)  Ca (%)  P (%)  Mg (%)   LSD  +  +  24.053  0.346  35.01  18.19  0.900    +  −  24.43  0.331  35.52  17.96  0.890    −  +  26.72  0.353  34.84  17.94  0.915    −  −  28.73  0.355  34.82  18.15  0.885   MSD  +  +  23.83  0.355  34.53  18.16  0.917    +  −  24.02  0.351  34.49  18.28  0.893    −  +  21.50  0.356  34.17  17.64  0.898    −  −  22.13  0.353  34.66  18.10  0.888   HSD  +  +  21.94  0.350  33.86  17.78  0.927    +  −  22.90  0.356  34.59  18.09  0.918    −  +  20.28  0.333  34.23  17.73  0.898    −  −  23.85  0.357  34.38  18.05  0.908  Pooled SEM2      0.64  0.0085  0.34  0.22  0.023  Main factors                 LSD      25.98a  0.346  35.04a  18.06  0.898   MSD      22.87a,b  0.354  34.46b  18.05  0.899   HSD      22.24b  0.349  34.26b  17.91  0.913    +    23.53  0.348  34.66  18.08  0.908    −    23.87  0.351  34.52  17.94  0.899      +  23.05  0.349  34.44  17.91  0.909      −  24.34  0.350  34.74  18.11  0.897  P-value                Density (D)      0.045  0.471  0.005  0.593  0.578  Fumonisin (F)      0.792  0.567  0.445  0.285  0.514  TB (B)      0.317  0.733  0.124  0.127  0.367  D × F      0.197  0.136  0.541  0.519  0.745  F × B      0.543  0.227  0.619  0.312  0.883  D × B      0.838  0.170  0.884  0.522  0.786  D × F × B      0.942  0.730  0.424  0.786  0.811  1FB = fumonisin; TB = toxin binder; LSD = low stocking density; MSD = medium stocking density; HSD = high stocking density. 2SEM = pooled standard error of the means. 3Values are LS means of 6 replicates per treatment. a,bValues (n = 6/treatment) having a different superscript within a column differ significantly (P < 0.05). View Large Physiological Stress Indicators Blood H/L ratio was significantly low (P = 0.002) at LSD compared with the ratio at MSD or HSD (Table 8). Dietary FB did not affect (P > 0.05) blood H/L ratio. On the other hand, serum NO levels were significantly elevated (P = 0.042) in chickens fed the FB-contaminated diet compared with the control diet-fed chickens, but stocking density did not affect the level. Of interest, significant interaction (P = 0.007) between FB and TB for serum NO levels was noted. Dietary TB increased or decreased NO levels, depending on the absence or presence of FB, causing an interaction between the 2 factors. However, serum AGP and CORT levels were not affected by stocking density, dietary FB, or TB (P > 0.05). Table 8. Effects of mycotoxin and toxin binder on physiological stress indicators in broiler chickens raised at different stocking densities1. Density1  FB1  TB1  H/L2 ratio  NO2 (μM)  AGP2 (mg/ml)  CORT2 (pg/ml)   LSD  +  +  0.1954  31.4  2.55  161.5    +  −  0.208  35.5  3.35  143.5    −  +  0.166  43.8  2.99  138.7    −  −  0.175  19.8  2.93  47.9   MSD  +  +  0.242  25.5  3.18  45.0    +  −  0.210  39.3  3.90  142.1    −  +  0.303  21.6  3.57  153.8    −  −  0.220  27.9  3.37  61.5   HSD  +  +  0.267  29.2  3.69  100.3    +  −  0.295  48.1  3.32  112.9    −  +  0.268  27.7  2.74  118.6    −  −  0.243  23.8  2.67  177.5  Pooled SEM3      0.024  6.08  0.47  12.0  Main factors               LSD      0.189b  32.7  2.96  112.6   MSD      0.236a  28.6  3.50  98.7   HSD      0.273a  32.6  3.11  128.5    +    0.240  34.9a  3.33  115.5    −    0.230  27.6b  3.05  113.4      +  0.246  29.8  3.12  119.6      −  0.229  32.7  3.26  109.6  P-value              Density (D)      0.002  0.560  0.243  0.582  Fumonisin (F)      0.626  0.042  0.304  0.942  TB (B)      0.424  0.468  0.614  0.714  D × F      0.281  0.474  0.411  0.232  F × B      0.237  0.007  0.370  0.149  D × B      0.292  0.490  0.638  0.302  D × F × B      0.842  0.473  0.589  0.136  Density1  FB1  TB1  H/L2 ratio  NO2 (μM)  AGP2 (mg/ml)  CORT2 (pg/ml)   LSD  +  +  0.1954  31.4  2.55  161.5    +  −  0.208  35.5  3.35  143.5    −  +  0.166  43.8  2.99  138.7    −  −  0.175  19.8  2.93  47.9   MSD  +  +  0.242  25.5  3.18  45.0    +  −  0.210  39.3  3.90  142.1    −  +  0.303  21.6  3.57  153.8    −  −  0.220  27.9  3.37  61.5   HSD  +  +  0.267  29.2  3.69  100.3    +  −  0.295  48.1  3.32  112.9    −  +  0.268  27.7  2.74  118.6    −  −  0.243  23.8  2.67  177.5  Pooled SEM3      0.024  6.08  0.47  12.0  Main factors               LSD      0.189b  32.7  2.96  112.6   MSD      0.236a  28.6  3.50  98.7   HSD      0.273a  32.6  3.11  128.5    +    0.240  34.9a  3.33  115.5    −    0.230  27.6b  3.05  113.4      +  0.246  29.8  3.12  119.6      −  0.229  32.7  3.26  109.6  P-value              Density (D)      0.002  0.560  0.243  0.582  Fumonisin (F)      0.626  0.042  0.304  0.942  TB (B)      0.424  0.468  0.614  0.714  D × F      0.281  0.474  0.411  0.232  F × B      0.237  0.007  0.370  0.149  D × B      0.292  0.490  0.638  0.302  D × F × B      0.842  0.473  0.589  0.136  1FB = fumonisin; TB = toxin binder; LSD = low stocking density; MSD = medium stocking density; HSD = high stocking density. 2H/L ratio = heterophil/lymphocyte ratio; NO = nitric oxide; AGP = alpha-1-acid glycoprotein; CORT = corticosterone. 3SEM = pooled standard error of the means. 4Values are LS means of 6 replicates per treatment. a,bValues (n = 6/treatment) having a different superscript within a column differ significantly (P < 0.05). View Large DISCUSSION No strong interactions between 2 factors (i.e., FB and stocking density) were detected with respect to growth performance, bone quality, gut health, and blood stress indicators. In line with our findings, increasing stocking density was found to lower weight gain and feed intake (Dozier et al., 2006; Estevez, 2007; Cengiz et al., 2015), tibia breaking strength with concomitant decrease in ash calcium content (Buijs et al., 2012), and serum H/L ratio (Thaxton et al., 2006; Shakeri et al., 2014) in broiler chickens, while dietary FB alone increased feed-to-gain ratio in broiler chickens (Brown et al., 1992) and serum NO levels in rodents (Dombrink-Kurtzman et al., 2000). Thus, the lack of interaction between FB and stocking density was not due to the absence of FB or stocking density-induced effects in broiler chickens. FB at a concentration of 10 mg/kg of diet was not sufficient to affect the stocking density-induced effects in chickens, leading to the lack of apparent interaction. Thus, it is not known whether increasing FB levels up to 100 or 300 mg/kg of diet (Brown et al., 1992; Javed et al., 1993; Rauber et al., 2013) would aggravate the stocking density-induced depression in performance, health, or welfare of broiler chickens. The stocking density and FB concentration used in this study are considered to be moderate. For example, HSD was set at 0.08 m2/bird in this study, while HSD in previous studies ranged from 0.030 to 0.067 m2/bird (Guardia et al., 2011; Abudabos et al., 2013; Hongchao et al., 2014; Shakeri et al., 2014). It has been reported that increasing stocking density decreased growth performance or villus height (Beloor et al., 2010; Hongchao et al., 2014; Shakeri et al., 2014), increased the incidence of foot pad dermatitis (Hongchao et al., 2014), altered behaviors (Hall, 2001), and increased stress-associated indicators (i.e., acute phase protein, CORT, H/L ratio; Shakeri et al., 2014) in broiler chickens. However, the absence of a clear effect with increased stocking density in chickens (Nogueira et al., 2013; Wang et al., 2014) also has been reported. In addition to the absence of effects due to stocking density, the effects of FB levels also were considered negligible. A recent survey (Seo et al., 2013) found the presence of approximately 4 mg FB/kg of diet in commercial poultry feeds. However, when the diet was contaminated with less than 100 mg FB/kg of diet, no effect on growth performance was seen in broiler chickens (Henry et al., 2000; Antonissen et al., 2015; Antonissen et al., 2017), although the sphinganine: sphingosine ratio, a biomarker of FB exposure in animals, was increased (Henry et al., 2000; Antonissen et al., 2015). On the other hand, FB levels greater than 100 mg/kg of diet clearly decreased growth and increased mortality rates (Brown et al., 1992; Javed et al., 1993; Rauber et al., 2013). Thus, 10 mg FB/kg of diet used in this study, despite being within the contaminant levels in the chicken feeds (Kim et al., 2014), is considered low with respect to its impact on chickens. An unexpected observation emerged from this study. In sharp contrast to previous findings (Heckert et al., 2002; Ravindran et al., 2006), increasing stocking density significantly increased relative bursal weight. It is generally thought that environmental stressors (i.e., stocking density) may be associated with a decrease in primary and secondary lymphoid organs (Heckert et al., 2002), although no apparent effect on lymphoid weight (Buijs et al., 2012) has been reported. Unfortunately, we did not monitor humoral immune responses in this study. Thus, in-depth evaluation of local and systemic immune responses in broiler chickens raised at different stocking densities is warranted. As high stocking density and mycotoxin contamination in feeds are considered stress factors in poultry, several parameters, including H/L ratio, NO, AGP, and CORT, were determined as the main adaptive stress response indices of poultry (Puvadolpirod and Thaxton, 2000). It is clear from the current study that increasing stocking density increased H/L ratio, and FB increased serum NO levels. However, neither stocking density nor FB affected serum concentrations of AGP and CORT. Although increasing stocking density is known to affect stress indicators such as AGP, CORT, and H/L ratio (Shakeri et al., 2014), no clear stress-induced effect (Thaxton et al., 2006; Türkyilmaz, 2008) was noted. In addition, an increased H/L ratio was found in broilers fed 10 mg/kg of deoxynivalenol, which is also a Fusarium mycotoxin (Ghareeb et al., 2012), and in turkeys fed 75 mg/kg of FB (Bermudez et al., 1996). On the other hand, purified FB did not affect CORT in broiler chickens (Antonissen et al., 2015). Thus, it seemed that there is no single physiological indicator for detecting stressed birds. Serum NO level, which is a direct indicator of oxidative stress (Lancaster Jr, 1996) and a measure of immune status (Lillehoj and Li, 2004), increased by 26% in FB-fed chickens compared with the control counterparts, indicating that the mycotoxin could induce NO production in macrophages and monocytes. Uncontrolled NO production is known to induce gut damage (Allen, 1997), albeit that the FB-induced NO increase in this study was not accompanied by altered gut morphology or ileal sIgA levels. The FB-induced increase in NO may account for FB as a predisposing factor in the pathogenesis of necrotic enteritis or coccidiosis (Antonissen et al., 2015). In addition, the mitigating effect of dietary TB on FB-induced NO levels was noted. This is expected, as TB used in this study contained an FB-degrading enzyme, a mineral-based adsorbing agent, and plant and algae extracts. Although we failed to see interactions between stocking density and FB, and FB and TB (except for NO), a significant interaction between stocking density and TB was found for body weight gain and ileal sIgA levels at 21 days. Increasing stocking density increased weight gain in chickens fed a TB-free diet, but decreased weight gain in chickens fed a TB-added diet. Ileal sIgA levels were elevated in birds raised in HSD with a TB-added diet, and in those raised in MSD with a TB-free diet. These results were unexpected, as TB is presumed to act on mycotoxins present in feeds and/or gut contents. Indeed, TB alone did not affect any of the many parameters measured in this study. At this stage, the underlying basis for the observed interaction between stocking density and TB is not clear. In conclusion, the present study demonstrated that increasing stocking density decreased growth performance and tibial bone quality, and increased relative bursa weight and H/L ratio in broiler chickens. In addition, dietary FB increased the feed-to-gain ratio and serum NO levels in broiler chickens. A significant interaction between FB and TB with respect to the NO levels was noted. However, there was no interaction between stocking density and FB for any of the measurements. Further studies are warranted to determine whether failure of FB to interact with stocking density in broiler chickens may be due to the low level of FB used in this study. Finally, whether the effect of increasing stocking density on the increase in relative bursa weight is related to altered acquired immune response should be addressed. ACKNOWLEDGEMENTS This work was supported by the Korean Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fishery (IPET) through the Agri-Bio Industry Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA)(316036–3). REFERENCES Abudabos A. M., Samara E. M., Hussein E. O., Al-Ghadi M. A. Q., Al-Atiyat R. M.. 2013. Impacts of stocking density on the performance and welfare of broiler chickens. Ital. J. Anim. Sci.  12: e11. Google Scholar CrossRef Search ADS   Allen P. C. 1997. Nitric oxide production during Eimeria tenella infections in chickens. Poult. Sci.  76: 810– 813. Google Scholar CrossRef Search ADS PubMed  Antonissen G., Martel A., Pasmans F., Ducatelle R., Verbrugghe E., Vandenbroucke V., Li S., Haesebrouck F., Van Immerseel F., Croubels S.. 2014. The impact of Fusarium mycotoxins on human and animal host susceptibility to infectious diseases. Toxins . 6: 430– 452. Google Scholar CrossRef Search ADS PubMed  Antonissen G., Croubels S., Pasmans F., Ducatelle R., Eeckhaut V., Devreese M., Verlinden M., Haesebrouck F., Eeckhout M., De Saeger S.. 2015. Fumonisins affect the intestinal microbial homeostasis in broiler chickens, predisposing to necrotic enteritis. Vet. Res.  46: 98. Google Scholar CrossRef Search ADS PubMed  Antonissen G., Devreese M., De Baere S., Martel A., Van Immerseel F., Croubels S.. 2017. Impact of Fusarium mycotoxins on hepatic and intestinal mRNA expression of cytochrome P450 enzymes and drug transporters, and on the pharmacokinetics of oral enrofloxacin in broiler chickens. Food Chem. Toxicol.  101: 75– 83. Google Scholar CrossRef Search ADS PubMed  Awad W. A., Böhm J., Razzazi-Fazeli E., Zentek J.. 2006. Effects of feeding deoxynivalenol contaminated wheat on growth performance, organ weights and histological parameters of the intestine of broiler chickens. J. Anim. Physiol. Anim. Nutr.  90: 32– 37. Google Scholar CrossRef Search ADS   Beloor J., Kang H. K., Kim Y. J., Subramani V. K., Jang I. S., Sohn S. H., Moon Y. S.. 2010. The effect of stocking density on stress related genes and telomeric length in broiler chickens. Asian-australas. J. Anim. Sci.  23: 437– 443. Google Scholar CrossRef Search ADS   Bermudez A. J., Ledoux D. R., Turk J. R., Rottinghaus G. E.. 1996. The chronic effects of Fusarium moniliforme culture material, containing known levels of fumonisin B1, in turkeys. Avian Dis . 40: 231– 235. Google Scholar CrossRef Search ADS PubMed  Boudergue C., Burel C., Dragacci S., Favrot M. C., Fremy J. M., Massimi C., Prigent P., Debongnie P., Pussemier L., Boudra H.. 2009. Review of mycotoxin-detoxifying agents used as feed additives: mode of action, efficacy and feed/food safety. EFSA Supporting Publications . 6: e22. Google Scholar CrossRef Search ADS   Bouhet S., Hourcade E., Loiseau N., Fikry A., Martinez S., Roselli M., Galtier P., Mengheri E., Oswald I. P.. 2004. The mycotoxin fumonisin B1 alters the proliferation and the barrier function of porcine intestinal epithelial cells. Toxicol. Sci.  77: 165– 171. Google Scholar CrossRef Search ADS PubMed  Bradford M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.  72: 248– 254. Google Scholar CrossRef Search ADS PubMed  Brown T. P., Rottinghaus G. E., Williams M. E.. 1992. Fumonisin mycotoxicosis in broilers: Performance and pathology. Avian Dis . 36: 450– 454. Google Scholar CrossRef Search ADS PubMed  Buijs S., Van Poucke E., Van Dongen S., Lens L., Baert J., Tuyttens F. A.. 2012. The influence of stocking density on broiler chicken bone quality and fluctuating asymmetry. Poult. Sci.  91: 1759– 1767. Google Scholar CrossRef Search ADS PubMed  Cengiz O., Koksal B. H., Tath O., Sevim O., Ahsan U., Uner A. G., Ulutas P. A., Beyaz D., Buyukyoruk S., Yakan A., Onol A. G.. 2015. Effect of dietary probiotic and high stocking density on the performance, carcass yield, gut microflora, and stress indicators of broilers. Poult. Sci.  94: 2395– 2403. Google Scholar CrossRef Search ADS PubMed  Dombrink-Kurtzman M. A., Gomez-Flores R., Weber R. J.. 2000. Activation of rat splenic macrophage and lymphocyte functions by fumonisin B1. Immunopharmacology . 49: 401– 409. Google Scholar CrossRef Search ADS PubMed  Dozier W. A., Thaxton J. P., Purswell J. L., Olanrewaju H. A., Branton S. L., Roush W. B.. 2006. Stocking density effects on male broilers grown to 1.8 kilograms of body weight. Poult. Sci.  85: 344– 351. Google Scholar CrossRef Search ADS PubMed  Du E., Wang W., Gan L., Li Z., Guo S., Guo Y.. 2016. Effects of thymol and carvacrol supplementation on intestinal integrity and immune responses of broiler chickens challenged with Clostridium perfringens. J. Anim. Sci. Biotechnol.  7: 19. Google Scholar CrossRef Search ADS PubMed  Estevez I. 2007. Density allowances for broilers: Where to set the limits? Poult. Sci.  86: 1265– 1272. Google Scholar CrossRef Search ADS PubMed  European Commission. 2006. Commission recommendation of 17 August 2006 on the presence of deoxynivalenol, zearalenone, ochratoxin A, T-2 and HT-2 and fumonisins in products intended for animal feeding. Off. J. Eur. Union.  229, 7– 9. European Commission. 2007. Council Directive 2007/43/EC of 28 June 2007 laying down minimum rules for the protection of chickens kept for meat production. Off. J. Eur. Union.  182, 19– 28. FDA. 2001. Guidance for Industry: Fumonisin Levels in Human Foods and Animal Feeds . U.S. FDA Center for Food Safety and Applied Nutrition and Center for Veterinary Medicine, Washington, DC. Ghareeb K., Awad W. A., Böhm J.. 2012. Ameliorative effect of a microbial feed additive on infectious bronchitis virus antibody titer and stress index in broiler chicks fed deoxynivalenol. Poult. Sci.  91: 800– 807. Google Scholar CrossRef Search ADS PubMed  Gonzales E., Kondo N., Saldanha E. S., Loddy M. M., Careghi C., Decuypere E.. 2003. Performance and physiological parameters of broiler chickens subjected to fasting on the neonatal period. Poult. Sci.  82: 1250– 1256. Google Scholar CrossRef Search ADS PubMed  Guardia S., Konsak B., Combes S., Levenez F., Cauquil L., Guillot J.-F., Moreau-Vauzelle C., Lessire M., Juin H., Gabriel I.. 2011. Effects of stocking density on the growth performance and digestive microbiota of broiler chickens. Poult. Sci.  90: 1878– 1889. Google Scholar CrossRef Search ADS PubMed  Hall A. L. 2001. The effect of stocking density on the welfare and behaviour of broiler chickens reared commercially. Anim. Welfare.  10: 23– 40. Heckert R. A., Estevez I., Russek-Cohen E., Pettit-Riley R.. 2002. Effects of density and perch availability on the immune status of broilers. Poult. Sci.  81: 451– 457. Google Scholar CrossRef Search ADS PubMed  Henry M. H., Wyatt R. D., Fletchert O. J.. 2000. The toxicity of purified fumonisin B1 in broiler chicks. Poult. Sci.  79: 1378– 1384. Google Scholar CrossRef Search ADS PubMed  Hongchao J., Jiang Y., Song Z., Zhao J., Wang X., Lin H.. 2014. Effect of perch type and stocking density on the behaviour and growth of broilers. Anim. Prod. Sci.  54: 930– 941. Huwig A., Freimund S., Käppeli O., Dutler H.. 2001. Mycotoxin detoxication of animal feed by different adsorbents. Toxicol. Lett.  122: 179– 188. Google Scholar CrossRef Search ADS PubMed  Incharoen T., Yamauchi K.. 2009. Production performance, egg quality and intestinal histology in laying hens fed dietary dried fermented ginger. Poult. Sci.  8: 1078– 1085. Google Scholar CrossRef Search ADS   Javed T., Bennett G. A., Richard J. L., Dombrink-Kurtzman M. A., Cote L. M., Buck W. B.. 1993. Mortality in broiler chicks on feed amended with Fusarium proliferatum culture material or with purified fumonisin B1 and moniliformin. Mycopathologia . 123: 171– 184. Google Scholar CrossRef Search ADS PubMed  Kim N. Y., Lee I., Ji G. E.. 2014. Reliable and simple detection of ochratoxin and fumonisin production in black Aspergillus. J. Food Prot.  77: 653– 658. Google Scholar CrossRef Search ADS PubMed  Lancaster J. Jr. 1996. Nitric Oxide: Principles and Actions . Academic Press. Ledoux D. R., Brown T. P., Weibking T. S., Rottinghaus G. E.. 1992. Fumonisin toxicity in broiler chicks. J. Vet. Diagn. Invest.  4: 330– 333. Google Scholar CrossRef Search ADS PubMed  Lillehoj H. S., Li G.. 2004. Nitric oxide production by macrophages stimulated with coccidia sporozoites, lipopolysaccharide, or interferon-γ, and its dynamic changes in SC and TK strains of chickens infected with Eimeria tenella. Avian Dis . 48: 244– 253. Google Scholar CrossRef Search ADS PubMed  Miles R. D., Butcher G. D., Henry P. R., Littell R. C.. 2006. Effect of antibiotic growth promoters on broiler performance, intestinal growth parameters, and quantitative morphology. Poult. Sci.  85: 476– 485. Google Scholar CrossRef Search ADS PubMed  Mosca F., Madeddu M., Mangiagalli M. G., Colombo E., Cozzi M. C., Zaniboni L., Cerolini S.. 2015. Bird density, stress markers and growth performance in the Italian chicken breed Milanino. J. Appl. Poult. Res.  24: 529– 535. Murugesan G. R., Ledoux D. R., Naehrer K., Berthiller F., Applegate T. J., Grenier B., Phillips T. D., Schatzmayr G.. 2015. Prevalence and effects of mycotoxins on poultry health and performance, and recent development in mycotoxin counteracting strategies 1. Poult. Sci.  94: 1298– 1315. Google Scholar CrossRef Search ADS PubMed  Nogueira W., Velásquez P., Furlan R. L., Macari M.. 2013. Effect of dietary energy and stocking density on the performance and sensible heat loss of broilers reared under tropical winter conditions. Rev. Bras. Cienc. Avic.  15: 53– 57. Google Scholar CrossRef Search ADS   Norred W. P. 1993. Fumonisins-mycotoxins produced by fusarium moniliforme. J. Toxicol. Environ. Health A.  38: 309– 328. Google Scholar CrossRef Search ADS   Pappas A., Tsiplakou E., Georgiadou M., Anagnostopoulos C., Markoglou A., Liapis K., Zervas G.. 2014. Bentonite binders in the presence of mycotoxins: Results of in vitro preliminary tests and an in vivo broiler trial. Appl. Clay Sci.  99: 48– 53. Google Scholar CrossRef Search ADS   Puvadolpirod S., Thaxton J. P.. 2000. Model of physiological stress in chickens 1. Response parameters. Poult. Sci . 79: 363– 369. Google Scholar CrossRef Search ADS PubMed  Rauber R. H., Oliveira M. S., Mallmann A. O., Dilkin P., Mallmann C. A., Giacomini L. Z., Nascimento V. P.. 2013. Effects of fumonisin B1 on selected biological responses and performance of broiler chickens. Pesqui. Vet. Bras.  33: 1081– 1086. Google Scholar CrossRef Search ADS   Ravindran V., Thomas D. V., Thomas D. G., Morel P. C.. 2006. Performance and welfare of broilers as affected by stocking density and zinc bacitracin supplementation. Anim. Sci. J.  77: 110– 116. Google Scholar CrossRef Search ADS   Seo D.-G., Phat C., Kim D.-H., Lee C.. 2013. Occurrence of Fusarium mycotoxin fumonisin B1 and B2 in animal feeds in Korea. Mycotoxin Res . 29: 159– 167. Google Scholar CrossRef Search ADS PubMed  Shakeri M., Zulkifli I., Soleimani A. F., O’Reilly E. L., Eckersall P. D., Anna A. A., Kumari S., Abdullah F. F. J.. 2014. Response to dietary supplementation of L-glutamine and L-glutamate in broiler chickens reared at different stocking densities under hot, humid tropical conditions. Poult. Sci.  93: 2700– 2708. Google Scholar CrossRef Search ADS PubMed  Skomorucha I., Muchacka R., Sosnówka-Czajka E., Herbut E.. 2009. Response of broiler chickens from three genetic groups to different stocking densities. Ann. Anim. Sci.  9: 175– 184. Smith L. E., Stoltzfus R. J., Prendergast A.. 2012. Food chain mycotoxin exposure, gut health, and impaired growth: A conceptual framework. Adv. Nutr.  3: 526– 531. Google Scholar CrossRef Search ADS PubMed  Tan J., Applegate T. J., Liu S., Guo. Y., Eicher D.. 2014. Supplemental dietary L-arginine attenuates intestinal mucosal disruption during a coccidial vaccine challenge in broiler chickens. Brit. J. Nutr.  112: 1098– 1109. Google Scholar CrossRef Search ADS   Thaxton J. P., Dozier W. A., Branton S. L., Morgan G. W., Miles D. W., Roush W. B., Lott B. D., Vizzier-Thaxton Y.. 2006. Stocking density and physiological adaptive responses of broilers. Poult. Sci.  85: 819– 824. Google Scholar CrossRef Search ADS PubMed  Türkyilmaz M. K. 2008. The effect of stocking density on stress reaction in broiler chickens during summer. Turk. J. Vet. Anim. Sci.  32: 31– 36. Voss K. A., Smith G. W., Haschek W. M.. 2007. Fumonisins: toxicokinetics, mechanism of action and toxicity. Anim. Feed Sci. Technol.  137: 299– 325. Google Scholar CrossRef Search ADS   Wang B., Min Z., Yuan J., Zhang B., Guo Y.. 2014. Effects of dietary tryptophan and stocking density on the performance, meat quality, and metabolic status of broilers. J. Anim. Sci. Biotechnol.  5: 44. Google Scholar CrossRef Search ADS PubMed  Yegani M., Smith T. K., Leeson S., Boermans H. J.. 2006. Effects of feeding grains naturally contaminated with Fusarium mycotoxins on performance and metabolism of broiler breeders. Poult. Sci.  85: 1541– 1549. Google Scholar CrossRef Search ADS PubMed  © 2017 Poultry Science Association Inc.

Journal

Poultry ScienceOxford University Press

Published: Mar 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 12 million articles from more than
10,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Unlimited reading

Read as many articles as you need. Full articles with original layout, charts and figures. Read online, from anywhere.

Stay up to date

Keep up with your field with Personalized Recommendations and Follow Journals to get automatic updates.

Organize your research

It’s easy to organize your research with our built-in tools.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

Monthly Plan

  • Read unlimited articles
  • Personalized recommendations
  • No expiration
  • Print 20 pages per month
  • 20% off on PDF purchases
  • Organize your research
  • Get updates on your journals and topic searches

$49/month

Start Free Trial

14-day Free Trial

Best Deal — 39% off

Annual Plan

  • All the features of the Professional Plan, but for 39% off!
  • Billed annually
  • No expiration
  • For the normal price of 10 articles elsewhere, you get one full year of unlimited access to articles.

$588

$360/year

billed annually
Start Free Trial

14-day Free Trial