The Effect of Probiotic Supplementation on Performance and the Histopathological Changes in Liver and Kidneys in Broiler Chickens Fed Diets with Aflatoxin B1

The aim of the study was to investigate the toxic effects of aflatoxin B1 (AFB1) and efficacy of a probiotic preparation containing L. reuteri, L. plantarum, L. pentosus, L. rhamnosus and L. paracasei and Saccharomyces cerevisiae yeasts to ameliorate their effects in broiler chickens. A total of 168 one-day-old female Ross 308 broilers were randomly allocated to six groups. Three wheat and soybean meal-based diets were prepared: Control diet and diets contaminated with 1 or 5 mg/kg AFB1 supplied in moldy wheat. All diets were unsupplemented or supplemented with probiotic, cold pelleted and fed from 1 to 35 day of life. Feeding diet with 1 mg AFB1/kg did not affect performance, but a diet with 5 mg AFB1 resulted in a significant reduction of feed intake and BWG, both diets induced liver and kidneys enlargement. The probiotic supplementation of the diets partially ameliorated those negative effects and resulted in a significant increase of AFB1 excretion. It was accompanied by the reduced level of AFB1 residues in the liver from 8.9 to 3.7 and from 11.8 to 5.9 µg/kg, in kidneys from 7.9 to 2.5 and from 13.7 to 4.1 µg/kg in birds fed the less and more contaminated diets, respectively. AFB1 exposure caused many severe histopathological changes in the liver and kidneys of broilers, probiotic supplementation significantly reduced the changes of these organs. It may be concluded that the probiotic supplementation can be used to alleviate the negative effects of contamination of broiler feed with AFB1 on bird health and product security.


Introduction
There are many biological toxins present in the natural environment, which may be dangerous for human and animal health. Mycotoxins are currently considered to be among the most dangerous

Performance Indices
No mortality was recorded among control birds and birds that were fed diets with 1 mg AFB 1 /kg, but during the last week of the experiment 2 birds died in the group fed the diet with 5 mg AFB 1 /kg and 1 bird in the group fed the diet with 5 mg AFB 1 /kg supplemented with probiotic (Table 1). Feeding chickens for 35 days with diet contaminated with 1 mg AFB 1 /kg had no significant effect on body weight gain (BWG), feed intake and feed conversion ratio (FCR), compared to chickens fed the control diet ( Table 1). The addition of the probiotic to control diet and to the diet contaminated with 1 mg AFB 1 /kg did not affect performance indices. Only a high contamination level (5 mg AFB 1 /kg diet) resulted in a significant reduction of feed intake and BWG. The probiotic supplementation of the diet Toxins 2019, 11, 112 3 of 15 containing 5 mg AFB 1 improved feed intake and BWG significantly, but not to the level obtained in birds fed control and the less contaminated diets. Lost birds 0 0 2 1 0 0 1 each value represents mean ± SD, the mean initial body weight of chick was 40 g, BWG = the body weight of chick at the end of week − the body weight at the end of preceding week; a,b,c means in a row marked with various letters are significantly different at p < 0.05; 2 initial number of birds was 16 (n = 16), but in the group 5 AFB 1 two birds died at 5th week (n = 14), in the group 5AFB 1 + probiotic one bird died at 5th week (n = 15).
Feeding chickens with both AFB 1 contaminated diet, increased the weight of liver and kidneys significantly, compared to the control group ( Table 2). The addition of the probiotic to the control diet did not affect the weight of liver and kidneys, the probiotic added to the diet with 1 mg AFB 1 /kg contributed to the restoration of a weight of liver and kidneys to a similar as in control group. The probiotic added to the diet with a high level of AFB 1 reduced the weight of liver and kidneys significantly but still they remained enlarged in comparison with other groups.

The Concentration of Aflatoxin B 1 in Excreta
Aflatoxin B 1 was not detected in excreta of chickens fed control diet without or with probiotic. The concentration of AFB 1 in excreta in chickens fed contaminated diets depended on its dietary concentration but throughout the whole period of the experiment was higher in chickens consuming diets supplemented with probiotic ( Figure 1). At the end of the experiment the concentration of AFB 1 in excreta of chickens eating diets contaminated with AFB 1 at a level of 1 and 5 mg/kg was, on average, 0.29 and 2.74 µg/kg, respectively, but was 3-fold and by 46% higher (p < 0.05) in chickens provided respective diets supplemented with probiotic (Table 3). respective diets supplemented with probiotic (Table 3). Table 3. Concentration of aflatoxin B1 in wet excreta (n = 14-16, see Table 1), and in the liver and kidney (n = 10) at 35th day of age 1,2 . 1 Each value represents mean ± SD; a,b,c,d means in a row marked with different letters differ significantly at p < 0.05; 2 Number of birds n = 14-16 (see Table 1).

Concentration of Aflatoxin B1 in Liver and Kidneys
Aflatoxin B1 was not found in livers and kidneys of chickens fed control diet without or with probiotic. The mean concentration of AFB1 in the liver tissue of chickens consuming diet contaminated with AFB1 at a level of 1 and 5 mg/kg was 8.86 and 11.84 µ g/kg, respectively. In chickens eating respective diets supplemented with the probiotic the concentration of AFB1 in the liver was, on average, by 58% and 50% lower as compared to chickens fed the diets without probiotic (Table 3). Differences between unsupplemented and supplemented groups are statistically significant (p < 0.05).
In chickens receiving the diet with 1 and 5 mg/kg AFB1 the mean concentration of AFB1 in kidney tissue was 7.93 and 13.71, respectively, whereas in chickens obtaining those diets supplemented with the probiotic it was, on average, by 68% and 70% lower (Table 3). Differences between unsupplemented and supplemented groups were statistically significant (p < 0.05).  Table 1): (A) chickens fed diets with 1 mg AFB 1 /kg; (B) chickens fed diets with 5 mg AFB 1 /kg; respective diets supplemented with probiotic (Table 3). Table 3. Concentration of aflatoxin B1 in wet excreta (n = 14-16, see Table 1), and in the liver and kidney (n = 10) at 35th day of age 1,2 . 1 Each value represents mean ± SD; a,b,c,d means in a row marked with different letters differ significantly at p < 0.05; 2 Number of birds n = 14-16 (see Table 1).

Concentration of Aflatoxin B1 in Liver and Kidneys
Aflatoxin B1 was not found in livers and kidneys of chickens fed control diet without or with probiotic. The mean concentration of AFB1 in the liver tissue of chickens consuming diet contaminated with AFB1 at a level of 1 and 5 mg/kg was 8.86 and 11.84 µ g/kg, respectively. In chickens eating respective diets supplemented with the probiotic the concentration of AFB1 in the liver was, on average, by 58% and 50% lower as compared to chickens fed the diets without probiotic (Table 3). Differences between unsupplemented and supplemented groups are statistically significant (p < 0.05).
In chickens receiving the diet with 1 and 5 mg/kg AFB1 the mean concentration of AFB1 in kidney tissue was 7.93 and 13.71, respectively, whereas in chickens obtaining those diets supplemented with the probiotic it was, on average, by 68% and 70% lower (Table 3). Differences between unsupplemented and supplemented groups were statistically significant (p < 0.05).

Item
Experimental Group 1 Each value represents mean ± SD; a,b,c,d means in a row marked with different letters differ significantly at p < 0.05; 2 Number of birds n = 14-16 (see Table 1).

Concentration of Aflatoxin B 1 in Liver and Kidneys
Aflatoxin B 1 was not found in livers and kidneys of chickens fed control diet without or with probiotic. The mean concentration of AFB 1 in the liver tissue of chickens consuming diet contaminated with AFB 1 at a level of 1 and 5 mg/kg was 8.86 and 11.84 µg/kg, respectively. In chickens eating respective diets supplemented with the probiotic the concentration of AFB 1 in the liver was, on average, by 58% and 50% lower as compared to chickens fed the diets without probiotic (Table 3). Differences between unsupplemented and supplemented groups are statistically significant (p < 0.05).
In chickens receiving the diet with 1 and 5 mg/kg AFB 1 the mean concentration of AFB 1 in kidney tissue was 7.93 and 13.71, respectively, whereas in chickens obtaining those diets supplemented with the probiotic it was, on average, by 68% and 70% lower (Table 3). Differences between unsupplemented and supplemented groups were statistically significant (p < 0.05).

Histopathological Changes in Liver and Kidneys
Histological examination showed significant damage in the liver of chickens that were fed the diets contaminated with AFB 1 at both concentrations (Table 4), whereas lesions in kidneys were found only in chickens consuming the diet with 5 mg AFB 1 /kg. Livers of chickens that were fed the diet with 1 mg of AFB 1 presented a slight infiltration of perilobular eosinophilic cells, and a slight to moderate vascular degeneration and fatty changes of hepatocytes. In chickens that were fed a diet with 5 mg of AFB 1 the infiltration of eosinophilic cells was more intense and occurred both in peri-and interlobular areas. A division of the hepatic parenchyma into fine nodules was also observed as signs of micronodular cirrhosis ( Figure 2). In addition to predominantly moderate vascular degeneration and fatty changes of hepatocytes, moderate hyperplasia of the bile tract epithelium and intensive fibrosis were found in livers of those chickens ( Figure 3). In the kidneys, histological changes involved enlargement of the renal glomeruli and an increase of the mezangium matrix ( Figure 4). Wilder staining revealed also thickening of the basal membrane in the glomeruli ( Figure 5). Table 4. Effects of aflatoxin B 1 and probiotic supplementation on the histological scores of the liver tissue at 35th day of life 1 .

Bile Duct Hypertrophy Fibrosis
Control Results are expressed as mean of 20 liver sections (2 sections of 10 chickens in each group). Sections with no, mild, moderate, or severe lesions were given scores of 0, 1, 2, and 3, respectively; a,b,c,d means in a row marked with different letters differ significantly at p < 0.05.

Histopathological Changes in Liver and Kidneys
Histological examination showed significant damage in the liver of chickens that were fed the diets contaminated with AFB1 at both concentrations (Table 4), whereas lesions in kidneys were found only in chickens consuming the diet with 5 mg AFB1/kg. Livers of chickens that were fed the diet with 1 mg of AFB1 presented a slight infiltration of perilobular eosinophilic cells, and a slight to moderate vascular degeneration and fatty changes of hepatocytes. In chickens that were fed a diet with 5 mg of AFB1 the infiltration of eosinophilic cells was more intense and occurred both in peri-and interlobular areas. A division of the hepatic parenchyma into fine nodules was also observed as signs of micronodular cirrhosis ( Figure 2). In addition to predominantly moderate vascular degeneration and fatty changes of hepatocytes, moderate hyperplasia of the bile tract epithelium and intensive fibrosis were found in livers of those chickens ( Figure 3). In the kidneys, histological changes involved enlargement of the renal glomeruli and an increase of the mezangium matrix ( Figure 4). Wilder staining revealed also thickening of the basal membrane in the glomeruli ( Figure 5).
The probiotic supplementation of the diet contaminated with 5 mg AFB1/kg reduced histopathological changes in kidneys, whereas changes in the liver were fully reduced only at a lower concentration of AFB1 in the diet. At a higher dose of AFB1 the probiotic failed to prevent histological changes in the liver, but accounted for the reduction of their intensity.   The probiotic supplementation of the diet contaminated with 5 mg AFB 1 /kg reduced histopathological changes in kidneys, whereas changes in the liver were fully reduced only at a lower concentration of AFB 1 in the diet. At a higher dose of AFB 1 the probiotic failed to prevent histological changes in the liver, but accounted for the reduction of their intensity.

Discussion
Biological elimination of mycotoxins from food and feed and lowering their negative effects in a human and animal organisms is a novel and a very promising method. Among organisms used in research on elimination of mycotoxins, one should mention Acinetobacter calcoaceticus [15], Bifidobacterium sp. [16,17], Enterococcus faecium [18], Flavobacterium aurantiacum [19], Lactobacillus sp.

Discussion
Biological elimination of mycotoxins from food and feed and lowering their negative effects in a human and animal organisms is a novel and a very promising method. Among organisms used in research on elimination of mycotoxins, one should mention Acinetobacter calcoaceticus [15], Bifidobacterium sp. [16,17], Enterococcus faecium [18], Flavobacterium aurantiacum [19], Lactobacillus sp.

Discussion
Biological elimination of mycotoxins from food and feed and lowering their negative effects in a human and animal organisms is a novel and a very promising method. Among organisms used in research on elimination of mycotoxins, one should mention Acinetobacter calcoaceticus [15], Bifidobacterium sp. [16,17], Enterococcus faecium [18], Flavobacterium aurantiacum [19], Lactobacillus sp.
The mechanism of removing mycotoxins from the environment by microorganisms has not been thoroughly explained so far. It arises from the available literature that in the case of LAB and yeast, it is the result of cell wall adhesion, which is additionally confirmed by the fact that even dead cells preserve the said activity [27,28].
In the mechanism of mycotoxin adsorption to bacterial cells an important role may be played by peptidoglycans and exopolysaccharides in the cell, teichoic acids and the reactions related to cell hydrophobicity and electrostatic properties [29]. In the case of yeast, the most significant role is ascribed to β-D-glucans and mannan oligosaccharides of the cell wall [30]. Pizzolitto et al. [31] additionally stated that van der Waals forces, hydrogen bonds or hydrophobic interactions are also involved in the absorption process.
Probiotics composed with LAB should arouse the greatest interest, considering their beneficial, physiological properties in human and animal organism, among others in broiler chickens [13,32,33]. Moreover, the LAB are able to inhibit the growth of moulds, and the production of mycotoxins [34]. The present study evaluated the ability of probiotic preparation composed of LAB and S. cerevisiae yeasts to counteract the toxic effects of AFB 1 in broiler chickens.
Probiotics are subject to the rules contained in the general food law, according to which they should be safe for animal and human health. In the USA, microorganisms used for oral intake should be classified as GRAS (generally regarded as safe), and be regulated by Food and Drug Administration. In Europe, the European Food Safety Authority (EFSA) introduced the term QPS (Qualified Presumption of Safety). The QPS concept takes into account additional criteria of the safety rating of bacterial additives, such as the history of safe use and the lack of risk becoming antibiotic-resistant. In the study, Lactobacillus bacteria and Saccharomyces cerevisiae yeast were used, classified as both GRAS and QPS and therefore safe for animal and human health.
In the present study feeding chickens for 35 days with the diet contaminated with 1 mg/kg of AFB 1 had a minor effect on feed intake, BWG and FCR compared to birds fed with the control diet. The addition of the probiotic to that diet resulted in insignificant improvement of BWG by 4%, on average. Supplementation with probiotic of the control diet and the diet contaminated with 1 mg/kg AFB 1 had no beneficial effect on the growth performance of broilers.
Rodricks and Stoloff [35] observed a reduction of BWG by 5-10% as a result of feeding chickens with the diet containing 2.5 mg AFB 1 /kg. Using the same dose of AFB 1 , Hamilton et al. [36] reported a reduction of BWG by 81 g, and with the dose of 5 mg AFB 1 /kg the reduction of BWG by 164 g, on average. Smith et al. [37] demonstrated that the administration of a mixture of aflatoxins (79% AFB 1 , 16% AFG 1 , 4% AFB 2 and 1% AFG 2 ) to chickens in the amount of 3.5 mg/kg diet resulted in reduced BWG, and an enlargement of liver and kidneys. An enlargement of liver and kidneys of chickens that were fed a diet contaminated with 5 mg/kg AFB 1 was also reported by Pimpukdee et al. [38]. Quezada et al. [39] feeding chickens with the diet containing 2 mg/kg AFB 1 obtained the increase of mass of kidneys at 21st day of age and 20-30% BWG reduction in 4-week-old chickens.
In the current study we have observed a reduction of BWG by 20-50% in chickens that were fed a diet with 5 mg of AFB 1 /kg and a significant increase in the relative weight of liver and kidneys on both AFB 1 contamination levels compared to the control group. The relative weight of those organs in chicken that were fed a diet contaminated with 1 mg of AFB 1 /kg was 34 and 24% higher, though in chickens that were fed a diet with 5 mg of AFB 1 /kg it was 73% and 122% higher compared to control. Obtained results are therefore consistent with those reported by Smith et al. [37], Pimpukdee et al. [38], and Quezada et al. [39].
In the present study the probiotic added to the diet containing 1 mg AFB 1 /kg contributed to the restoration of the relative weight of liver and kidneys to similar as in the control group. The positive effect of probiotic supplementation was also observed in chickens fed the diet containing 5 mg AFB 1 /kg but both organs remain enlarged by 59% and 100% compared to the control birds.
The aflatoxins present in the feed might be absorbed, transformed by intestinal tract microbiota to less toxic metabolites, or excreted in droppings. In the present study the excreta of chickens fed unsupplemented diets contaminated with 1 and 5 mg AFB 1 /kg contained, on average, 0.29 and 2.74 mg AFB 1 /kg, respectively. The addition of the probiotic to the AFB 1 -contaminated feed contributed to a significant increase in the concentration of AFB 1 in droppings. There is substantial evidence that LAB and S. cerevisiae yeasts, present in the probiotic preparation used in the experiment are capable of effective binding of toxins [13,14,21,26]. Moreover, it was evidenced by our in vitro study [40] that a probiotic composed with LAB and S. cerevisiae has been active in metabolic transformation of AFB 1 , as the concentration of toxin in broiler diets contaminated with 1 and 5 mg AFB 1 /kg decreased by 55 and 39%, respectively, after 6 h incubation.
The main site of absorption of mycotoxins in animals is the small intestine (in birds mainly the proximal part of the jejunum). The absorption takes place as a result of both an active transport, and a passive diffusion and with blood in the portal vein absorbed mycotoxins reach the liver and other organs [41]. The greater part of absorbed mycotoxins is removed with urine (in birds excreted with droppings), but residues are accumulated in liver, kidneys and muscles and pose a threat for animal and human health [42].
In the present study the concentration of AFB 1 in the liver tissue was 8.9 and 11.8 µg/kg; in kidneys 7.9 and 13.7 µg/kg in chickens that were fed diets with 1 and 5 mg AFB 1 /kg, respectively. Tejada-Castañeda et al. [43] reported, that after 21 days of feeding chickens with diet contaminated with 2 µg AFB 1 /kg the concentration of the toxin in the liver tissue was 1-1.2 µg/ kg and in the kidneys tissue it was 0.8 µg/kg. We cannot find reports concerning the protective effect of LAB probiotics on the AFB 1 residues accumulation in broilers body tissues, but Fan et al. [21] reported, that Bacillus subtilis probiotic lowered AFB 1 residues in the liver of broilers by 42-97%, depending on dose. In the present study the AFB 1 concentration in the liver of broilers that were fed diets containing 1 and 5 mg AFB 1 /kg and supplemented with the probiotic was reduced by about 50% (to 3.7 and 5.9 µg/kg, respectively), in kidneys by about 70% (to 2.5 and 4 µg/kg, respectively) compared to the birds fed contaminated but non-supplemented diets. These results show that the LAB and S. cerevisiae probiotic used in the study AFB 1 can be used against the occasional contamination of broiler feed with aflatoxins.
In the study we assessed changes in livers and kidneys, organs the most exposed to aflatoxin B 1 . The histopathological examination demonstrated the presence of kidney injuries only in chickens fed the diet contaminated with 5 mg AFB 1 /kg. Changes involved increased diameter of the glomeruli, an increased amount of the matrix and mesangial cells and a thickening of basal membranes of the glomeruli. The addition of the probiotic to the diet caused a reduction of the diameter of most of the glomeruli, even back to the normal value. Pathological changes were found in livers of chickens fed with both contaminated diets. They were: Eosinophil infiltrations, vascular degeneration and fatty changes of hepatocytes. Additionally, signs of micronodular cirrhosis with highly intensified eosinophilic infiltration were found in livers of chickens fed with the diet containing 5 mg AFB 1 /kg. Histological changes were not observed in livers and kidneys of chickens fed with the probiotic-supplemented diets containing 1 mg AFB 1 /kg and were of lesser intensity in chickens fed the probiotic-supplemented diet with 5 mg AFB 1 /kg. It confirmed the other reports that LAB and yeast from the probiotic are able to effectively bind AFB 1 [33] and/or to transform it to a non-toxic compound [40]. Due to that less toxin has reach liver and in kidneys and a resulted histological changes were not as pronounced as it was in case of chickens receiving contaminated but non-supplemented diets.
According to literature, in chickens aflatoxins may cause degradation of proximal tubules, the formation of hyaline concrements in the free space of the Bowman capsule, atrophic glomeruli and cortical fibrosis [44]. Symptoms of acute liver damage include increased fragility of blood vessels, coagulopathy, hemorrhage and prolonged presence of blood clots. The color of livers turns from yellow to copper, the gallbladder becomes enlarged and bile gets thinner. Hypertrophy of the biliary tract ensues [45]. According to Solcan et al. [46] the size of mycotoxin-induced lesions in chicken liver and kidneys are dose-dependent. The authors noted minor degenerative changes in the liver and kidneys for the lowest dose, more pronounced ones for 5 and 9 mg OTA/kg, and for the dose of 20 mg/kg changes were defined as serious. Pizzolitto et al. [47] demonstrated that Saccharomyces cerevisiae CECT 1891 yeast, added to a diet (10 10 cells/kg) or to drinking water (10 9 cells/L) had a beneficial protective effect on histopathological changes of the liver of broiler chickens fed with the diet contaminated with 1.2 mg of AFB 1 /kg.
The aim of the study was to evaluate the aflatoxin B 1 detoxification potential of the probiotic preparation composed with LAB and S. cerevisiae in the gastrointestinal tract of chickens. The results indicate that the probiotic preparation may therefore be used for prevention of mycotoxicoses in broiler chickens.

Conclusions
Addition of the probiotic preparation composed with LAB and S. cerevisiae to AFB 1 -contaminated diet has a positive effect on performance, reduces AFB 1 residues concentration and prevents or reduces degenerative changes in the liver and kidneys. As a consequence the probiotic preparation may be used in the prevention of mycotoxicoses in chicken, contributing to the improvement of animal health, and the quality of meat.

Preparation of Diets
Wheat grain was inoculated with the strain of Aspergillus flavus K30 and incubated to obtain high concentration of aflatoxin B 1 in accordance with a procedure described by Xiao et al., [47]. Twenty Erlenmeyer flasks (500 mL), each containing 30 g of feed-grade wheat and 30 mL of distilled water, were autoclaved for 30 min at 120 • C and then inoculated with Aspergillus flavus K30. The culture was maintained at 30 • C in a dark room. The fermentation was terminated on day 12 of incubation, the contaminated wheat was dried for 72 h, ground, and the aflatoxin B 1 concentration was determined.
Experimental diets were prepared, each covered satisfactorily the requirements suggested by the NRC [49] for broiler chickens. The composition of control diets are shown in Table 5, experimental diets were prepared to meet the final concentration of 1 or 5 mg of aflatoxin B 1 per kg of diet by substituting respective amounts of contaminated wheat by feed-grade wheat. Diets were prepared without or with probiotic preparation. All ingredients were thoroughly mixed and cold pelleted with the use of CL-2 CPM laboratory pellet mill (California Pellet Mill Co., Crawfordsville, IN, USA).

Experimental Design, Birds and Sample Collection
A total of 168 one-day-old male Ross broiler chicks were used. They were randomly assigned to six groups and housed in electrically controlled breeder (4 cages × 7 chickens per group) for the first week of life. On the 8th day of life, after 4 h of feed deprivation chickens were weighed, 16 birds with a body weight close to the group average were selected in each group and housed in individual metabolic cages in environmentally controlled room. A 3 × 2 factorial arrangement was used, the factors being dietary AFB 1 concentration (0, 1 and 5 mg per kg) and without or with probiotic (Table 6). Chickens were fed with Starter-type diets for the first 14 days of experiment, from day 15 to 28 with Grower-type diet, and during the last seven days with Finisher-type diet ( Table 5). The experimental protocol was approved by the Local Animal Care and Use Committee in Warsaw, Warsaw University of Life Sciences, Poland (1/2016, date of approval: 12 January 2016). Table 6. Experimental scheme.

Experimental Group Dietary Treatment
Control control diet without additives Control + probiotic control diet + probiotic 1 AFB 1 diet contaminated with 1 mg/kg aflatoxin B 1 1 AFB 1 + probiotic diet contaminated with 1 mg/kg aflatoxin B 1 + probiotic 5 AFB 1 diet contaminated with 5 mg/kg aflatoxin B 1 5 AFB 1 + probiotic diet contaminated with 5 mg/kg aflatoxin B 1 + probiotic Probiotic preparation was added at 2 g/kg diet between 1st and 14th day of life and at 1 g/kg diet until the end of the experiment.
Bird weight and feed consumption was recorded weekly, mortality was registered as it happens. Excreta were collected during the last day of each week, homogenized and 10 g samples were frozen for AFB 1 determination. After the last weighing on day 35 of life, birds were killed by cervical dislocation. Liver, kidneys and the alimentary tract filled with content were excised and weighed. Feed consumption, body weight gain, feed conversion ratio as well as the relative weight of liver and kidneys were calculated. Liver and kidneys tissues were sampled from 10 chickens in each group for AFB 1 determination and histopathological assessment.

Aflatoxin B 1 Concentration
The AFB 1 concentration was determined in excreta (16 samples in a group at each week of the experiment) and in the liver and kidneys with an enzyme linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC).

Reagents
All chemicals were analytical reagent grade, excepting methanol and acetonitrile (HPLC grade). They were purchased from Merck (Darmstadt, Germany). All solutions were prepared with deionized water. Standard of AFB 1 was purchased from Sigma Chemical Co. (St. Louis, MO, USA). AFB 1 standard stock solution was prepared in benzene-acetonitrile (98:2, v/v).

ELISA Method
Commercial ELISA kit AgraQuant Aflatoxin (Romer Labs Diagnostic, Singapore) was used in the study. These ELISA assay kit is a direct competitive immunoassay with horseradish peroxidase conjugate. The assays were performed according to the procedure described in the AgraQuant Assay kit manual. The AFB 1 was extracted by mixing 10 g of sample (excreta, liver or kidneys) with 50 mL methanol:water (70:30, v/v), the mixture was filtered. The 100 µL of horseradish peroxidase conjugate solution and 50 µL of filtrate or standard AFB 1 solution was added to the well and mixed thoroughly. Then 50 µL of the mixture were transferred to well containing antibodies and incubated for 15 min at room temperature. After incubation, wells were rinsed five times with distilled water and dried. The 50 µL of substrate (urea peroxide) was added to each well, after 5 min at room temperature the reaction was stopped by adding 50 µL of stopping solution (1M sulfuric acid). The absorbance of the solution was read with a UVM 340 microplate reader (Asys, Eugendorf, Austria) at a wavelength λ = 450 nm, and calculated by extrapolating the OD with the respective AFB 1 calibration curve [50].

HPLC Method
The samples (excreta, liver or kidneys) were analyzed for the presence of AFB 1 using an immunoaffinity column for clean up and HPLC with fluorescence detection. Using 10 g of sample, aflatoxin extraction was done with 50 mL methanol:water (70:30, v/v). The samples were centrifuged at 1600× g for 10 min and the upper fat layer was discarded. Then, 50 mL was passed through an immunoaffinity column (AflaStar, Romer Labs Diagnostic, Singapore). The column was washed with 25 mL of PBS buffer (pH 7.4) to remove extraneous non-specific material. The AFB 1 bound to the antibody was released by the elution with 2.0 mL methanol. The eluate was evaporated to dryness using an N 2 stream. The residue was dissolved in 400 µL mobile phase and a 20 µL aliquot was injected into the HPLC equipment.

Histopathological Examination
Specimens collected from the liver and the kidney were fixed in 4% buffered formalin (Chempur, PiekaryŚląskie, Poland) for 24 h at room temperature. The material in histopathological cases (Leica, Wetzlar, Germany) was placed in a tissue processor TP 1020-1 (Leica, Wetzlar, Germany), where it was passed through a series of increasing concentrations of alcohol (70%, 90%, 96%, 99.8%), xylene and paraffin. Then the material was embedded in paraffin, using the corresponding equipment EG-1160 (Leica, Wetzlar, Germany). The embedded material was sliced into 4-µm specimens using a metal knife (Shandon MX35, ThermoFisher Scientific, Waltham, MA, USA) with a microtome RM 2265 (Leica, Wetzlar, Germany). Specimens were placed on syalinised slides (Surgipath, Leica Biosystem, Wetzlar, Germany). Slides with specimens were then placed in an incubator at 60 • C for 24 h, and then de-paraffined. The material was de-paraffined with xylene and decreasing concentrations of ethanol (99.8%, 96%, 50%). The material was placed in each of the reagents for 3 min. Specimens were hematoxylin and eosin stained (H&E staining) for 1-2 min and closed with glycerogel (Mounting Medium, DakoCytomation, Glostrup, Denmark). Sections obtained from each organ were examined under the Olympus light microscope at magnifications ×100 (H&E and Azan staining), and ×400 (Wilder staining). Specimens of the liver were examined for previously described lesions: vascular changes and fatty degeneration of hepatocytes, inter-and perilobular inflammation, bile duct hyperplasia/hypertrophy and fibrosis. Sections with no, mild, moderate, or severe lesions were given scores of 0, 1, 2, and 3, respectively.

Azan Staining of Specimens
After de-paraffination, specimens were incubated for 7 min with 1% aniline alcohol (a solution of aniline and 96% ethanol), and then washed with 96% ethanol and running water, for 3 min. After washing specimens were incubated at 56 • C for 20 min in azocarmine G solution (0.1 g of azocarmine G, 100 mL of distilled water, 1 mL of acetic acid). After incubation, specimens were washed in distilled water and washed with 1% aniline alcohol solution and with acidic alcohol (a solution containing 1 mL of acetic acid and 99 mL of ethanol). Then, specimens were incubated at room temperature for 10 min with 10% phosphotungstic acid. After washing specimens with distilled water, they were incubated for 3 min with the Mallory's mixture (0.5 g of aniline blue, 2 g of yellow orange G, 100 mL of distilled water, 8 mL of glacial acetic acid). Specimens closed with glycerogel (Mounting Medium, DakoCytomation, Glostrup, Denmark) were examined under the Olympus light microscope (Olympus, Tokyo, Japan).

Wilder Staining of Specimens
De-paraffined specimens were incubated in 5% periodic acid for 15 min, and then in 5% oxalic acid for 2 min. After washing with distilled water, specimens were incubated for 2 min with 3% H 2 O 2 solution. Specimens, washed with tap water (3 min) and distilled water, were impregnated with ammonia silver for 7 min. Ammonia silver was obtained by combining 5 mL of 10% silver nitrate AgNO 3 with 5 mL of 3% NaOH, and ammonia added dropwise until dissolution of the formed sediment. The prepared solution was diluted with equal volume of distilled water. After washing with 96% ethanol, specimens were incubated for 2 min with 10% formalin solution, washed with distilled water and immersed in 1% gold chloride solution for 2 min. After washing specimens with distilled water, they were incubated for 2 min with 5% sodium thiosulphate solution, and then washed with distilled water. Specimens closed with glycerogel were examined under the Olympus light microscope.

Statistical Analysis
The data were analyzed using the STATISTICA 8.0 software package (Statsoft Co., Krakow, Poland). One-way analysis of variance (ANOVA) was applied to assess the effects of diets on the concentration of aflatoxin B 1 in excreta, liver, kidneys and on performance indices. If the analysis revealed a significant influence (p ≤ 0.05), the differences among the groups were analyzed with Duncan's multiple range post hoc test (p ≤ 0.05).