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. 2022 Dec 21;102(3):102434. doi: 10.1016/j.psj.2022.102434

Compound mycotoxin detoxifier alleviating aflatoxin B1 toxic effects on broiler growth performance, organ damage and gut microbiota

Hongwei Guo *,†, Ping Wang ‡, Chaoqi Liu ‡, Juan Chang ‡, Qingqiang Yin ‡,1, Lijun Wang ‡, Sanjun Jin ‡, Qun Zhu §, Fushan Lu #
PMCID: PMC9811249  PMID: 36586389

Abstract

The aim of this study was to evaluate the effects of compound mycotoxin detoxifier (CMD) on alleviating the toxic effect of aflatoxin B1 (AFB1) for broiler growth performance. One-kilogram CMD consists of 667 g aflatoxin B1-degrading enzyme (ADE, 1,467 U/g), 200 g montmorillonite and 133 g compound probiotics (CP). The feeding experiment was divided into 2 stages (1–21 d and 22–42 d). In the early stage, a total of 300 one-day-old Ross broilers were randomly divided into 6 groups, 5 replications for each group, 10 broilers (half male and half female) in each replication. In the later feeding stage, about 240 twenty-two-day-old Ross broilers were randomly divided into 6 groups, 8 replications for each group, 5 broilers in each replication. Group A: basal diet; group B: basal diet with 40 μg/kg AFB1; group C: basal diet with 1 g/kg CMD; groups D, E, and F: basal diet with 40 μg/kg AFB1 plus 0.5, 1.0 and 1.5 g/kg CMD, respectively. The results indicated that AFB1 significantly decreased average daily gain (ADG), protein metabolic rate, organ index of thymus, bursa of Fabricius (BF), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and catalase activities in serum, and increased AFB1 residues in serum and liver (P < 0.05). Hematoxylin-Eosin (HE) staining analysis of jejunum, liver and kidney showed that AFB1 caused the main pathological changes with different degrees of inflammatory cell infiltration. However, CMD additions could alleviate the negative effects of AFB1 on the above parameters. The gut microbiota analysis indicated that AFB1 could significantly increase the abundances of Staphylococcus-xylosu, Esherichia-coli-g-Escherichia-Shigella, and decrease Lactobacillus-aviarius abundance (P < 0.05), but which were adjusted to almost the same levels as the control group by CMD addition. The correlative analysis showed that Lactobacillus-aviarius abundance was positively correlated with ADG, SOD and BF (P < 0.05), whereas Staphylococcus-xylosus abundance was positively correlated with AFB1 residues in serum and liver (P < 0.05). In conclusion, CMD could keep gut microbiota stable, alleviate histological lesions, increase growth performance, and reduce mycotoxin toxicity. The optimal CMD addition should be 1 g/kg in AFB1-contaminated broilers diet.

Key words: detoxifier, aflatoxin B1, broiler, organ damage, gut microbiota

INTRODUCTION

Aflatoxins (AF) B1, B2, G1, and G2 are produced by Aspergillus flavus and Aspergillus parasiticus, among which aflatoxin B1 (AFB1) holds the highest toxicity (Liu et al., 2020). AFB1 is well known for its teratogenic, carcinogenic, and immunosuppressive effects on human and animals, consumption of AFB1-contaminated feeds by poultry may decrease feed intake and growth performance as well as increase intestinal damage and mortality (Fan et al., 2021). Liver is the target organ for AFB1 to induce liver injury involving inflammation, oxidative stress and DNA damage caused by metabolic enzymes (Murcia and Diaz, 2021). The previous study has shown that in the state of inflammation and stress, muscle protein synthesis will be decreased, and energy will be mobilized to support immunity, resulting in poor growth and immunosuppression (Zheng et al., 2021).

The damage of AFB1 to broilers is multifaceted and comprehensive. The damage degree of AFB1 to the organism has a linear relationship with AFB1 content in chicken diet. When dietary AFB1 concentration was less than 100 μg/kg, it was mainly manifested as growth retardation, low nutrient digestibility and disturbed intestinal microbiota (Ortatatli et al., 2005); when dietary AFB1 concentration was above 100 μg/kg, microscopic anatomical defect of internal organization began to occur except for the low growth performance (Hernández-Ramírez et al., 2020). Visceral organs (liver and kidney) presented the substantial damage when dietary AFB1 concentration was higher than 1,000 μg/kg (Śliżewska et al., 2019). However, it is difficult to determine the quantization relationship because it is affected by many factors such as broiler breeds, AFB1 resources and feeding managements (Bryden, 2001). The investigation report of animal feed pollution in China from 2018 to 2020 showed that the detective rate of AFB1 was 99.6%, and the maximal concentration was 54 μg/kg (Zhao et al., 2021a). According to AFB1 threshold in broiler diets (10–20 μg/kg) in China and Europe, growth retardation will be induced by more than 10 μg/kg AFB1, which will restrict the healthy development in broiler industry (Zhao et al., 2021a).

Physical adsorbent such as montmorillonite has been reported to absorb AFB1 to alleviate its toxic effects (Mao et al., 2022). Biological methods such as microorganisms and enzymes have good applications in the degradation of mycotoxin. Microorganisms including Lactobacillus, Yeast and Bacillus can degrade mycotoxin by adsorption and fermentation degradation (Liu et al., 2020). Some enzymes such as laccase and aflatoxin-degrading enzyme can degrade mycotoxin effectively (Liu et al., 2021). In addition, the combination of different antidotes (bentonite, yeast cell wall and glucose biopolysaccharides) has been proven to have synergistic effect for attenuating the negative effects of AFB1 on production performance, immunological function and intestinal health in broilers and laying hens (Zhao et al., 2021b; Lai et al., 2022). A published report has confirmed AFB1-degrading efficacy in broilers by combining probiotics and aflatoxin B1-degrading enzyme (ADE) (Chang et al., 2020). However, few data have been reported about the compound mycotoxin detoxifier (CMD) which contained ADE, montmorillonite, and compound probiotics (CP) application in alleviating AFB1 toxic effects on broilers performance, histopathology, and gut microbiota.

Our preliminary results showed that a triple-action compound mycotoxin detoxifier (CMD) could effectively degrade AFB1 in vitro, which contained AFB1-degrading enzyme, montmorillonite and CP consisting of Lactobacillus casein, Bacillus subtilis, Candida utilis and Enterococcus faecalis (Guo et al., 2021a); the further research indicated that CMD without montmorillonite could alleviate cell cytotoxicity and inflammation induced by AFB1 exposure through suppressing the activations of nuclear factor kappa B p65 (NF‑κB), inducible nitric oxide synthase (iNOS), nucleotide-binding oligomerization domain containing 1 (NOD1) and toll like receptor 2 (TLR2) pathways in chicken embryo primary intestinal epithelium, liver and kidney cells (Guo et al., 2021b). The aim of this research was to investigate the effects of CMD on growth performance, gut microbiota, and tissue injury of broilers in high-AFB1-level (40 μg/kg) diet.

MATERIALS AND METHODS

Materials Preparation

The moldy corn containing 72.14 μg/kg AFB1, 85.71 μg/kg zearalenone (ZEA) and 147.22 μg/kg deoxynivalenol (DON) was purchased from the market in Henan Province, China. The mycotoxin contents were determined by ELISA method (R-Biopharm, Darmstadt, Germany) according to manufacturer's standard instructions. According to the calculation of dietary formulation for broilers, all the normal corn meals in the basal diet were replaced by the moldy corn meals to make the diets with high-level AFB1, in which the dietary AFB1 concentration was determined as 40 μg/kg. Montmorillonite was provided by Henan Delin Biological Product Co., Ltd. Xinxiang, China.

Aspergillus oryzae, Lactobacillus casein, Bacillus subtilis, Candida utilis, and Enterococcus faecalis were purchased from China General Microbiological Culture Collection Center (CGMCC). Bacillus subtilis was inoculated in LB medium (g/L): peptone 10 g, yeast extract 5 g, NaCl 10 g, pH 7.0, and cultured in a rotary shaker with 180 rounds per min (rpm) at 37°C for 24 h. Lactobacillus casein and Enterococcus faecalis were inoculated in MRS medium (g/L): peptone 10 g, yeast extract 10 g, glucose 20 g, Tween 80 1 mL, K2HPO4 2 g, sodium acetate 5 g, sodium citrate 2 g, MgSO4 0.2 g, MnSO4 0.05 g, pH 6.20 to 6.60, cultured statically at 37°C for 24 h. Candida utilis was inoculated in YPD medium (g/L): yeast extract 10 g, peptone 20 g, glucose 20 g, cultured at 30°C for 24 h in 180 rpm shaker. After incubation, 4 species of microbes were placed statically for 2 h, and then the supernatant was removed. Skimmed milk powder, trehalose dihydrate, sodium glutamate and silica were added and mixed for freeze-drying. The microbial counts were expressed as colony forming units per gram (CFU)/g.

AFB1-degrading enzyme (ADE) was prepared from Aspergillus oryzae. The A. oryzae incubation was prepared as follows: A. oryzae spores were scraped off from the incubating plate with sterilized normal saline, and its concentration was adjusted to 1 × 108 spores/mL. The solid-state medium formula was as the following(w/w): the ratio of wheat bran, corn meal and soybean meal were 7:1:2, 15 g sample was taken, mixed with 9 mL distilled water, put in a 250 mL triangle bottle, autoclaved at 121°C for 30 min, and then cooled to room temperature. The medium was inoculated with 2 mL of the above spore fluid, incubated at 30°C for 5 d, and then dried. The activity of AFB1-degrading enzyme was 1,467 U/g. Enzyme activity was defined as the following: the amount of enzyme that could degrade 1 ng AFB1 per min at pH 8.0 and 37°C was defined as one unit (Guo et al., 2021a). One kilogram of CMD consisted of 667 g ADE, 200 g montmorillonite and 133 g CP in which the visible counts of Bacillus subtilis, Lactobacillus casein, Enterococcus faecalis and Candida utilis were 1.0 × 108, 1.0 × 108, 1.0 × 1010, and 1.0 × 108 CFU/g, respectively.

Animals and Managements

The feeding experiment was divided into 2 stages (1–21 d and 22–42 d). In the early stage, a total of 300 one-day-old Ross broilers were randomly divided into 6 groups, 5 replications for each group, 10 broilers (half male and half female) in each replication. In the later feeding stage, about 240 twenty-two-day-old Ross broilers were randomly divided into 6 groups, 8 replications for each group, 5 broilers in each replication. All animals used in this experiment were managed according to the guidelines of Animal Care and Use Ethics Committee in Henan Agricultural University (SKLAB-B-2010-003-01). All husbandry practices and euthanasia were performed with full consideration of animal welfare. The broilers were fed in the multi-layer cages with 24-h light and natural ventilation. The mesh diets and water were given to the birds ad libitum. The immune process was conducted with bivalent vaccine at the age of 7 d and Newcastle vaccine at the age of 21 d. The feed compositions and nutrient levels in the earlier and later stage of broilers are presented in Tables 1 and 2, respectively. The feeding experiment was designed as follows:

  • Group A: Basal diet containing 4.31 μg/kg AFB1

  • Group B: Basal diet with 42.15 μg/kg AFB1

  • Group C: Group A added with 1 g/kg CMD

  • Group D: Group B added with 0.5 g/kg CMD

  • Group E: Group B added with 1.0 g/kg CMD

  • Group F: Group B added with 1.5 g/kg CMD

Table 1.

Feed compositions and nutrient levels in the earlier stage of broilers (%).

Groups A B C D E F
Corn meal 58.50 1.50 58.50 1.50 1.50 1.50
Soybean meal 33.00 33.00 33.00 33.00 33.00 33.00
Moldy corn meal 0.00 57.00 0.00 57.00 57.00 57.00
Fish meal 2.00 2.00 2.00 2.00 2.00 2.00
Soybean oil 3.00 3.00 3.00 3.00 3.00 3.00
CaCO3 1.40 1.40 1.40 1.40 1.40 1.40
Dicalcium phosphate 1.40 1.40 1.40 1.40 1.40 1.40
Methionine 0.10 0.10 0.10 0.10 0.10 0.10
Salt 0.30 0.30 0.30 0.30 0.30 0.30
Premix 0.20 0.20 0.20 0.20 0.20 0.20
Choline chloride 0.10 0.10 0.10 0.10 0.10 0.10
CMD 0 0 0.10 0.05 0.10 0.15
Total 100 100 100 100 100 100
ME(MJ/kg) 12.43 12.43 12.4 12.43 12.43 12.43
CP 20.51 20.48 20.50 20.51 20.49 20.48
Fat 3.61 3.62 3.65 3.62 3.61 3.62
Ca 0.98 0.97 0.99 0.99 0.97 0.98
TP 0.64 0.65 0.66 0.64 0.65 0.66
AP 0.46 0.46 0.46 0.46 0.46 0.46
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Note: Premix of vitamins and minerals provided per kilogram of diet: VA 12 000 IU; VD3 3,000 IU; VE 20 IU; VK3 1.0 mg; VB1 2.0 mg; VB6 3.5 mg; VB12 0.01 mg; Cu (as copper sulfate) 8 mg; Fe (as ferrous sulfate) 100 mg; Mn (as manganese sulfate) 80 mg; Zn (as zinc oxide) 60 mg; I (as calcium iodate) 0.45 mg; Se (as sodium selenite) 0.35 mg; Biotin 0.15 mg; Folic acid 1.25 mg; VB2 (riboflavin) 6 mg; Nicotinic acid 35 mg; Calcium pantothenate 10 mg. The contents of crude protein (CP), fat, calcium, (Ca), and total phosphorus (TP) were determined, and the others were calculated. One kilogram of CMD consisted of 667 g aflatoxin B1-degrading enzyme (ADE), 200 g montmorillonite and 133 g compound probiotics (CP) in which the visible counts of Bacillus subtilis, Lactobacillus casein, Enterococcus faecalis, and Candida utilis were 1.0 × 108, 1.0 × 108, 1.0 × 1010 and 1.0 × 108 CFU/g, respectively.

Table 2.

Feed compositions and nutrient levels in the later stage of broilers (%).

Groups A B C D E F
Corn meal 65.50 8.50 65.50 8.50 8.50 8.50
Soybean meal 27.00 27.00 27.00 27.00 27.00 27.00
Moldy corn meal 0.00 57.00 0.00 57.00 57.00 57.00
Fish meal 1.00 1.00 1.00 1.00 1.00 1.00
Soybean oil 3.00 3.00 3.00 3.00 3.00 3.00
CaCO3 1.30 1.30 1.30 1.30 1.30 1.30
Dicalcium phosphate 1.40 1.40 1.40 1.40 1.40 1.40
Methionine 0.10 0.10 0.10 0.10 0.10 0.10
Salt 0.30 0.30 0.30 0.30 0.30 0.30
Premix 0.20 0.20 0.20 0.20 0.20 0.20
Choline chloride 0.10 0.10 0.10 0.10 0.10 0.10
CMD 0.00 0.00 0.10 0.05 0.10 0.15
Total 100 100 100 100 100 100
ME, MJ/kg 12.66 12.66 12.66 12.66 12.66 12.66
CP 17.90 17.91 17.92 17.90 17.92 17.91
Fat 5.93 5.92 5.91 5.91 5.93 5.91
Ca 0.89 0.88 0.89 0.89 0.88 0.90
TP 0.60 0.59 0.61 0.59 0.60 0.61
AP 0.42 0.42 0.42 0.42 0.42 0.42
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Note: Premix of vitamins and minerals provided per kilogram of diet: VA 12 000 IU; VD3 3,000 IU; VE 20 IU; VK3 1.0 mg; VB1 2.0 mg; VB6 3.5 mg; VB12 0.01 mg; Cu (as copper sulfate) 8 mg; Fe (as ferrous sulfate) 100 mg; Mn (as manganese sulfate) 80 mg; Zn (as zinc oxide) 60 mg; I (as calcium iodate) 0.45 mg; Se (as sodium selenite) 0.35 mg; Biotin 0.15 mg; Folic acid 1.25 mg; VB2 (riboflavin) 6 mg; Nicotinic acid 35 mg; Calcium pantothenate 10 mg. The contents of crude protein (CP), fat, calcium, (Ca), and total phosphorus (TP) were determined, and the others were calculated. One kilogram of CMD consisted of 667 g aflatoxin B1-degrading enzyme (ADE), 200 g montmorillonite and 133 g compound probiotics (CP) in which the visible counts of Bacillus subtilis, Lactobacillus casein, Enterococcus faecalis, and Candida utilis were 1.0 × 108, 1.0 × 108, 1.0 × 1010, and 1.0 × 108 CFU/g, respectively.

(The proportion of moldy corn meal substituting for normal corn meal in diets was 57% in groups B, D, E, and F).

Determinations of Nutrient Metabolic Rates

At the 17 to 19th d and 37 to 39th d during feeding experiments, metabolic experiment was performed using total excreta collection method. The plastic sheet was placed under the cages in 5 replications in each group to collect the excreta in both stages, which was collected for 3 d by changing the plastic sheets every day. Fresh excreta was collected without containing dander, feather, and feed, then mixed and added with 10% sulfuric acid to fix nitrogen, frozen at -20°C in refrigerator every day. The excreta samples of each replication from 3-d collections were mixed, dried and ground. Crude protein, crude fat, calcium and phosphorus contents in diet and excreta were determined with Kjeldahl, ether extract, potassium permanganate (KMnO4) and ammonium molybdate (NH4)6Mo7O24), respectively (Caldas et al., 2018). The calculation of apparent nutrient metabolic rate was made as follows: Nutrient apparent metabolic rate (%) = 100 × (nutrient content in diet - nutrient content in excreta)/nutrient content in diet.

Determination of Antioxidant Indexes in Serum

At the end of feeding experiment, 3 male broilers with average body weight from groups A, B, C, and F were selected to be sacrificed, respectively. The blood samples, organs and jejunal contents were collected for further analysis.

The superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) contents in serum were detected as antioxidant indexes. SOD activity was analyzed by monitoring inhibition rate of reducing nitroblue tetrazolium (NBT). GSH-PX, CAT, and MDA were determined according to the former method (Shi et al., 2006). T-AOC determination was based on measuring the fluorescence decay of R-Phycoerythrin induced by the peroxyl radicals obtained with thermal decomposition of 2,2’-azobis[2-methylpropionamidine] dihydrochloride (Zuo et al., 2013).

Organ Index and AFB1 Residues Analyses and Hematoxylin–Eosin (HE) Staining

Three samples of liver, kidney, thymus, spleen and bursa of Fabricius in groups A, B, C, and F were selected for the assays of organ indexes. The calculation of organ index was made as follows: Organ index = organ weight / live weight; intestinal length index = intestinal length / live weight.

The tissue samples of small intestine, liver, kidney, and muscle were prepared for AFB1 residue detection as the following: 5 g tissues were added with 25 mL 70% methanol (v/v), homogenized by an Ultra Turrax (T10, IKA Instrument Company, Staufen, Germany) at 10,000 rpm for 2 min. The following procedure was conducted (Ghali et al., 2008).

The same parts of jejunum, liver and kidney were taken, cut at 2 cm3, cleaned with physiological saline, and dried with filter paper. The samples were fixed in 10% formalin, dehydrated in ethanol, embedded in paraffin, cut at thickness of 0.6 μm, stained with Hematoxylin and Eosin, trans-parented with xylene, encased in neutral resin and then observed with a microscope.

Intestinal Microbiota Analysis

Three samples of jejunal contents were selected for microbiota analysis in groups A, B, C and F. The total genomic DNA of each sample was extracted by DNA Kit (Omega Biotek, Norcross, GA) according to manufacturer's instructions. The V3 and V4 variable regions of the bacterial 16S rRNA genes were amplified by the universal primer pairs: 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Reaction conditions consisted of an initial 95°C for 3 min followed by 27 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, and a final extension of 72°C for 10 min. PCR reactions were conducted in 20 μL reaction mixtures containing 10 ng template DNA, 0.8 μL each primer (5 μM), 2 μL 2.5 mM dNTPs, 0.4 μL FastPfu polymerase, and 4 μL 5 × FastPfu buffer. The PCR products were extracted with 2% agarose gels and purified using the AxyPrep DNA Extraction Kit (Axygen Biosciences, CA), and then quantified using QuantiFluor-ST (Promega Corporation, Madison, WI). The purified amplifications were pooled in equimolar and paired-end sequenced on an Illumina platform (Biomarker Technology Co., Ltd., Beijing, China).

Bioinformatics Analysis

The high-quality tags were clustered into the operational taxonomic units (OTUs) using UPARSE (version 7.1, http://drive5.com/uparse/) with a similarity threshold of 0.97. The phylogenetic affiliation of each 16S rRNA gene sequence was analyzed using the RDP Classifier (https://rdp.cme.msu.edu/) at 0.80 confidence level. The coverage percentage was calculated by Good's method (1953).

Statistical Analyses

Experimental data were sorted out by Excel and the experimental data expressed as means and standard errors (means ± SE). The data were analyzed using the ANOVA procedures and Duncan's multiple comparisons of the SPSS software (SPSS17.0). Differences were considered statistically significance at P < 0.05.

RESULTS

Effects of CMD on Alleviating AFB1 Damage for Production Performance of Broilers

Table 3 indicated that ADG in group B was significantly lower than that in groups A and C in the first feeding stage (P < 0.05). The F/G in group D was the highest and that in group B was the lowest among the 6 groups (P < 0.05). The mortality was 2% and 4% in groups B and D, respectively.

Table 3.

Effects of AFB1 and CMD on production performance of broilers.

Stages Items Group A Group B Group C Group D Group E Group F
1–21 d
IBW, g 50.31 ± 0.57 50.12 ± 0.61 50.02 ± 0.48 50.14 ± 0.61 50.09 ± 0.53 50.13 ± 0.48
FBW, g 922.02 ± 30.98 842.45 ± 68.76 980.17 ± 66.05 825.18 ± 68.15 847.17 ± 59.33 890.25 ± 43.91
ADFI, g 61.56 ± 5.42a 51.89 ± 2.35b 62.15 ± 2.51a 59.31 ± 2.63a 58.83 ± 4.87 a 59.21 ± 3.57a
ADG, g 41.51 ± 1.26ab 37.73 ± 3.03c 44.43 ± 1.18a 36.77 ± 1.89bc 37.94 ± 2.38bc 39.97 ± 0.8bc
F/G 1.48 ± 0.10b 1.37 ± 0.22 c 1.39 ± 0.04c 1.61 ± 0.09a 1.55 ± 0.12 b 1.48 ± 0.09b
Mortality, % 0 2 0 4 0 0
22–42 d
IBW, g 910.37 ± 13.15 909.18 ± 34.37 911.18 ± 29.65 908.35 ± 38.73 909.18 ± 34.37 909.18 ± 34.37
FBW, g 2,207.11 ± 46.29 ab 2,081.26 ± 59.68 b 2,263.12 ± 63.57 a 2,131.71 ± 47.65 ab 2,167.35 ± 57.56 ab 2,186.27 ± 47.16 ab
ADFI, g 118.29 ± 11.14 122.63 ± 6.76 115.14 ± 7.99 112.24 ± 11.6 107.12 ± 11.2 108.94 ± 10.72
ADG, g 60.71 ± 6.61ab 55.37 ± 5.68b 64.61 ± 6.7ab 58.33 ± 2.85ab 60.03 ± 7.14ab 60.77 ± 2.41a
F/G 1.95 ± 0.12ab 2.31 ± 0.27a 1.78 ± 0.18b 1.92 ± 0.21ab 1.78 ± 0.15b 1.79 ± 0.16b
Mortality, % 0 5 0 2.5 0 0
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Abbreviations: ADFI, average daily feed intake; ADG, average daily gain; FBW, final body weight; F/G, ADFI/ADG; IBW, initial body weight.

Group A: Basal diet; Group B: Basal diet with 42.15 μg/kg AFB1; Group C: Basal diet + 1 g/kg CMD; Group D: Group B + 0.5 g/kg CMD; Group E: Group B + 1.0 g/kg CMD; Group F: Group B + 1.5 g/kg CMD. In the same row, significant differences at P < 0.05 levels are indicated by the different lowercase letters (a, b, and c), whereas insignificant differences at P > 0.05 levels are indicated by the same lowercase letters or without letters.

In the second feeding stage, ADG in group B was significantly lower than that in group F (P < 0.05); with the addition of CMD increasing, ADG was gradually increased but without significant difference (P > 0.05). The mortality was 5% and 2.5% in group B and D, respectively. Based on the above comprehensive analysis, more than 1.0 g/kg CMD addition in broiler diet with 40 μg/kg AFBl could remove AFBl hazards completely.

Effects of AFB1 and CMD on Nutrient Metabolic Rate of Broilers

Table 4 indicated that crude protein metabolic rate in group B was significantly lower than that in the other groups in both feeding stages except for group D in the first stage (P < 0.05); indicating that addition of CMD could improve nutrient metabolic rates of broilers challenged with AFB1.

Table 4.

Effects of AFB1 and CMD on nutrient metabolic rate of broilers (%).

Stages Items Group A Group B Group C Group D Group E Group F
1–21 d Crude protein 58.79 ± 1.06ab 50.71 ± 1.89c 60.31 ± 1.27a 53.23 ± 1.08c 57.81 ± 0.67ab 57.04 ± 1.79b
Crude fat 78.87 ± 1.41 78.99 ± 1.51 78.49 ± 1.33 79.37 ± 0.84 79.93 ± 0.87 80.33 ± 1.71
Calcium 35.63 ± 1.39 34.88 ± 1.73 36.37 ± 1.53 34.28 ± 1.18 36.03 ± 0.74 36.29 ± 0.98
Phosphorus 42.49 ± 0.60 41.78 ± 0.59 43.11 ± 1.40 42.20 ± 0.73 41.96 ± 0.68 42.36 ± 0.52
22–42 d Crude protein 55.07 ± 1.56a 51.06 ± 1.86b 54.61 ± 3.04a 55.61 ± 1.99a 56.13 ± 1.28a 57.10 ± 1.57a
Crude fat 74.06 ± 1.47 73.14 ± 1.78 74.80 ± 2.96 72.78 ± 1.83 73.11 ± 1.28 74.12 ± 1.60
Calcium 32.46 ± 1.00 31.05 ± 0.74 32.40 ± 0.64 32.96 ± 1.46 31.09 ± 1.02 31.13 ± 1.13
Phosphorus 39.71 ± 1.13 38.63 ± 0.7 39.66 ± 1.29 38.47 ± 0.75 38.91 ± 1.07 40.28 ± 1.44
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Note: Group A: Basal diet; Group B: Basal diet with 42.15 μg/kg AFB1; Group C: Basal diet + 1.0 g/kg CMD; Group D: Group B + 0.5 g/kg CMD; Group E: Group B + 1.0 g/kg CMD; Group F: Group B + 1.5 g/kg CMD. In the same row, significant differences at P < 0.05 levels are indicated by the different lowercase letters (a, b, and c), whereas insignificant differences at P > 0.05 levels are indicated by the same lowercase letters or without letters.

Effects of AFB1 and CMD on Relative Organ Indexes of Broilers

Table 5 showed that organ indexes such as jejunum, thymus, and bursa of Fabricius in group B were significantly lower than that in other groups (P < 0.05).

Table 5.

Effects of AFB1 and CMD on relative organ weights of broilers (%).

Items Group A Group B Group C Group F
Jejunum 3.03 ± 0.19b 2.80 ± 0.28b 3.54 ± 029a 3.02 ± 0.19b
Liver 2.36 ± 0.37 2.31 ± 0.30 2.27 ± 0.14 2.52 ± 0.26
Kindy 0.36 ± 0.02 0.30 ± 0.08 0.34 ± 0.03 0.38 ± 0.05
Spleen 0.23 ± 0.05 0.20 ± 0.02 0.24 ± 0.05 0.22 ± 0.02
Bursa of Fabricius 0.20 ± 0.03a 0.14 ± 0.03b 0.20 ± 0.03a 0.19 ± 0.01a
Thymus 0.22 ± 0.06a 0.16 ± 0.01b 0.21 ± 0.03a 0.20 ± 0.02a
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Note: Group A: Basal diet; Group B: Basal diet with 42.15 μg/kg AFB1; Group C: Basal diet + 1.0 g/kg CMD; Group F: Group B + 1.5 g/kg CMD. In the same row, significant differences at P < 0.05 levels are indicated by the different lowercase letters (a and b), whereas insignificant differences at P > 0.05 levels are indicated by the same lowercase letters or without letters.

Effects of AFB1 and CMD on AFB1 Residues in 42 d Broilers

Table 6 showed that AFB1 residues in serum, jejunum, liver, and kidney tissues in group B were significantly higher than that in group A and C (P < 0.05), indicating that AFB1 residues were increased by adding AFB1 in broiler diets. AFB1 residues in serum, jejunum, liver, and kidney tissues in group F were significantly lower than that in group B (P < 0.05), indicating that CMD could effectively reduce AFB1 residues in tissues.

Table 6.

AFB1 residues in serum, jejunum, liver, and kidney (μg/kg).

Group Diet Serum Jejunum Liver Kidney
A 4.36 ± 0.10B,a ND 1.05 ± 0.07C,b 0.92 ± 0.04C,b 0.52 ± 0.03C,c
B 42.3 ± 1.24A,a 0.72 ± 0.05A,d 7.31 ± 0.22A,b 5.75 ± 0.14A,c 5.16 ± 0.28A,c
C 4.35 ± 0.01B ND ND ND ND
F 42.18 ± 0.89A,a 0.21 ± 0.07B,e 5.35 ± 0.31B,b 3.28 ± 0.33B,c 1.12 ± 0.31B,d
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Note: Group A: Basal diet; Group B: Basal diet with 42.15 μg/kg AFB1; Group C: Basal diet + 1.0 g/kg CMD; Group F: Group B + 1.5 g/kg CMD. In the same column, significant differences at P < 0.05 levels are indicated by the different capital letters (A, B, and C), whereas insignificant differences at P > 0.05 levels are indicated by the same capital letters. In the same row, significant differences at P < 0.05 levels are indicated by the different lowercase letters (a, b, and c), whereas insignificant differences at P > 0.05 levels are indicated by the same lowercase letters. ND: no detected.

Effects of AFB1 and CMD on Antioxidant Index of Broilers

Table 7 showed that the catalase, SOD and GSH-PX activities in group B were significantly lower (P < 0.05), and the MDA was significantly higher than that in other groups (P < 0.05). However, these indexes in group F were significantly improved (P < 0.05). Meanwhile, SOD and GSH-PX activities in group C were significantly higher than that in group A, inferring that CMD could improve the antioxidant level.

Table 7.

Effects of AFB1 and CMD on serum antioxidant indexes of broilers.

Items Group A Group B Group C Group F
Total antioxidant, mmol/mL 0.48 ± 0.04 0.41 ± 0.02 0.47 ± 0.04 0.45 ± 0.08
Catalase, U/mL 8.89 ± 0.29a 4.39 ± 0.23b 8.87 ± 0.69a 8.76 ± 0.57a
SOD, U/mL 42.08 ± 4.95b 10.98 ± 0.38c 51.49 ± 0.27a 55.86 ± 6.15a
MDA, nmol/mL 3.41 ± 0.13c 4.92 ± 0.05a 3.44 ± 0.02c 3.72 ± 0.08b
GSH-PX, μmol/mL 510.43 ± 6.85b 496.96 ± 19.57b 579.57 ± 33.91a 511.3 ± 2.61b
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Abbreviations: CAT, catalase; GSH-PX, glutathione peroxidase; MDA, malondialdehyde; SOD, superoxide dismutase.

Note: Group A: Basal diet; Group B: Basal diet with 42.15 μg/kg AFB1; Group C: Basal diet + 1.0 g/kg CMD; Group F: Group B + 1.5 g/kg CMD. In the same row, significant differences at P < 0.05 levels are indicated by the different lowercase letters (a, b, and c), whereas insignificant differences at P > 0.05 levels are indicated by the same lowercase letters or without letters.

Effects of AFB1 and CMD on Microstructure of Jejunum, Liver, and Kidney in Broilers

Effects of AFB1 and CMD on microstructure of jejunum.Figure 1 showed that villi in group A were normal in shape with clear boundary, but typical villi rupture and lymphocyte infiltration were observed in group B. The structure of villi in group C was normal with clear boundary and villi rupture was still observed in group F.

Figure 1.

Figure 1

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The micrograph of jejunum microstructure (HE 200×). Group A: Basal diet; Group B: Basal diet with 42.15 μg/kg AFB1; Group C: Basal diet + 1.0 g/kg CMD; Group F: Group B + 1.5 g/kg CMD. (A) Rupture of intestinal villi; (B) the increased eosinophile granulocyte and monocytes; (C) the increased mucosal hyperplasia and crypt depth.

Table 8 showed that villus height in group B was significantly lower than that in group A and C (P < 0.05), but that there was no significant difference between group B and F (P > 0.05), suggesting that the AFB1-induced reduction in villus height was irreversible. The crypt depth in group B was significantly higher than that in other groups, which lead to V/C value in group B to be significantly lower than that in other groups (P < 0.05).

Table 8.

Effects of AFB1 and CMD on villus height and crypt depth of jejunum in broilers.

Groups Villus height, µm Crypt depth, µm Villus height / Crypt depth
A 0.89 ± 0.11a 0.17 ± 0.01b 5.05 ± 0.67b
B 0.47 ± 0.02b 0.19 ± 0.01a 2.47 ± 0.10c
C 0.98 ± 0.06a 0.14 ± 0.01c 7.22 ± 0.56a
F 0.55 ± 0.02b 0.10 ± 0.01c 5.59 ± 0.41b
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Note: Group A: Basal diet; Group B: Basal diet with 42.15 μg/kg AFB1; Group C: Basal diet + 1.0 g/kg CMD; Group F: Group B + 1.5 g/kg CMD. In the same column, significant differences at P < 0.05 levels are indicated by the different lowercase letters (a, b, and c), whereas insignificant differences at P > 0.05 levels are indicated by the same lowercase letters.

Effects of AFB1 and CMD on microstructure of liver in broilers.Figure 2 showed that hepatocytes were normal without necrosis, arranged in order, and the hepatic cellular nuclei were big and round, even cytoplasm in group A and C. However, hepatocytes were yellowish brown with enlarged gaps, and macrophagocytes and mechanocytes were found in group B, indicating that steatosis and inflammatory response caused by AFB1 were increased in liver. The enlarged gaps of liver cells and reduced inflammatory cells were observed in group F, inferring that the CMD was able to protect liver from damage caused by AFB1.

Figure 2.

Figure 2

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The micrograph of liver microstructure (HE 200×). Group A: Basal diet; Group B: Basal diet with 40 μg/kg AFB1; Group C: Basal diet + 1.0 g/kg CMD; Group F: Group B + 1.5 g/kg CMD. (A) The increased eosinophile granulocyte and monocytes; (B) lipid vacuoles.

Effects of AFB1 and CMD on microstructure of kidney in broilers.Figure 3 showed the renal tissue micrographs of 4 groups. The glomerular morphology was complete with clear boundary, and the diameter of glomerular was about 50 microns in group A and C. The inflammatory cell infiltration and freed out nuclear cells with a part of glomerular structure destroyed were obviously found in group B. The phenomenon of inflammatory cell infiltration and glomerular structure destruction in group F were significantly decreased, compared to group B, inferring that the CMD was effective in protecting the kidney from damage caused by AFB1.

Figure 3.

Figure 3

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The micrograph of kidney microstructure (HE 400×). Group A: Basal diet; Group B: Basal diet with 40 μg/kg AFB1; Group C: Basal diet + 1.0 g/kg CMD; Group F: Group B + 1.5 g/kg CMD. (A) Diffuse infiltration of inflammatory cells; (B) glomerular basement membrane thickening and stromal cells increasing; (C) renal tubule edema.

Effects of AFB1 and CMD on Jejunal Microflora in Broilers

Effects of AFB1 and CMD on the microbial community in broiler jejunum at species level.Figure 4 showed that the relative abundances of the top 5 species from high to low were Lactobacillus-aviarius, Staphylococcus-xylosus, Lactobacillus-agilis, Esherichia-coli-g-Escherichia-Shigella, and Lactobacillus-salivarius. The relative abundance of Staphylococcus-xylosus in group B was higher than that in other groups (P < 0.05), whereas the relative abundances of Staphylococcus-xylosu and Esherichia-coli-g-Escherichia-Shigella in group F were significantly decreased compared to group B (P < 0.05). The relative abundance of Lactobacillus-aviarius in group C was higher than that in other 3 groups (P < 0.05), whereas the group B was the lowest (P < 0.05). The results showed that CMD was effective in maintaining the balance of a healthy microflora.

Figure 4.

Figure 4

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Composition of microbiota at species level in jejunal contents of broilers. Group A: Basal diet; Group B: Basal diet with 42.15 μg/kg AFB1; Group C: Basal diet + 1.0 g/kg CMD; Group F: Group B + 1.5 g/kg CMD.

Correlation analysis between intestinal microbiota at species level and other indexes. In order to evaluate the correlation between jejunal microbiota (top 5 at the species level) and other indexes such as mycotoxin residues, serum oxidation indexes and BF, a Pearson's correlation matrix was performed by calculating the Pearson's correlation coefficients (Figure 5). This research showed that jejunal Lactobacillus aviarius abundance was positively correlated with ADG, BF and SOD (P < 0.05), and negatively correlated with AFB1 residue in serum or AFB1 and MDA in liver (P < 0.05). However, the Staphylococcus-xylosus and Esherichia-coli-g-Escherichia-Shigella abundances showed the opposite correlation with the above indexes, illustrating that Lactobacillus aviarius played an important role in promoting broiler growth and decreasing AFB1 residues, but Staphylococcus-xylosus and Esherichia-coli-g-Escherichia-Shigella had negative effects on broilers health.

Figure 5.

Figure 5

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Correlation analysis of jejunal microflora with growth performance and AFB1 residues for broilers. Abbreviations: ADG, average daily gain; BF, bursa of Fabricius; MDA, malondialdehyde; Residue 1, AFB1 residue in serum; Residue 2, AFB1 residue in liver; SOD, superoxide dismutase. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

DISCUSSION

Compared with 10 to 20, 500, and 3,000 μg/kg for AFB1, ZEA, and DON thresholds in broiler diets in China, only AFB1 content (40 μg/kg) exceeds the threshold in this study. Therefore, the harm caused by moldy corn to broilers will be attributed to AFB1. Poultry is more sensitive to AFB1 than the other kinds of animals, and AFB1 can cause liver and gastrointestinal damages, increase feed conversion rate as well as reduce production performance (Gao et al., 2022). The mechanism of AFB1-induced injury in broilers is due to liver phase I metabolic enzyme (cytochromes P450, CYP450) in the mitochondrial to form AFB1-exo-8,9-epoxide (AFBO), which can bind to DNA to inhibiting DNA replication and protein expression, resulting in the damage and cancelation of hepatocytes (Fan et al., 2021). It was reported that genetic factors, environmental factors and physiological factors determined the toxicity degrees of mycotoxins to animals by affecting the absorption, metabolism, biotransformation and excretion of mycotoxins in animals; and the degrees of toxicity included functional and microscopic defects as well as death (Bryden, 2001).

The high residual level of AFB1 in organs will irritate the cells to cause tissue damage continuously. It was reported that AFB1 was absorbed from the gastrointestinal tract and extensively penetrated various tissues, AFB1 residue in animal edible issues and products was also a potential threat to human health (Caceres et al., 2020). In addition, small intestine is the physical barrier which usually first contacts with AFB1 (Pinton and Oswald, 2014), this is the reason why AFB1 residues in intestinal tissues were significantly higher than that in liver and kidney tissues. However, kidney as reabsorption and excretory organ is usually neglected, about 20% to 25% of the total amount of circulating toxins can reach the kidney; therefore, high levels of AFB1 residues also exist in renal tissues (Yu et al., 2015). Probiotics in CMD were composed of Lactobacillus casein, Bacillus subtilis, Candida utilis and Enterococcus faecalis in this experiment. It was reported that Lactobacillus could degrade AFB1 by the adsorption of peptidoglycan in cell wall, and this adsorption was reversible (Zhang et al., 2021). Bacillus subtilis can degrade AFB1 by fermentation, and its degradation essentially is an enzymatic reaction caused by oxidoreductase biosynthesized by bacteriocin to destroy the structure of AFB1 (Mao et al., 2020; Wan et al., 2021). Lactobacillus casein incubating components (teichoic acids on the peptidoglycan layer and β-D-glucan) have been shown to exhibit AFB1 removal ability and neutralize the adverse effects of toxins on rat body weight and gut (Liew et al., 2018). Montmorillonite can alleviate the toxic harm by inhibiting the absorption of aflatoxin. The interlayer of montmorillonite could absorb AFB1 by its medicinal system and the planarity of AFB1 ring. It has been proven that the main mechanism in adsorption process is the chemisorptive mechanism with high enthalpy (Phillips et al., 2019). It was reported that the broiler contaminated diets with 1 mg/kg AFB1 was supplemented with compound probiotics of Lactobacillus and yeast for 35 d, AFB1 residue levels were decreased from 8.9 to 3.7µg/kg in liver, from 7.9 to 2.5 µg/kg in kidney, respectively (Śliżewska et al., 2019).

Blood antioxidant indexes induced by AFB1 belong to the range of biochemical defects in this study. Liver injury induced by AFB1 is characterized by abnormal metabolism of nutrients, which was directly expressed as the decreases of serum total protein, triglyceride and glucose (Ates and Ortatatli, 2021). A published report showed that adding 1,000 μg/kg AFB1 to broiler diet significantly decreased the concentrations of serum total protein and triglyceride, and increased the activity of alkaline phosphatase and high-density lipoprotein content (Rashidi et al., 2020). AFB1 can also reduce the filtration capacity of glomerulus, which leads to the decreases of serum calcium, phosphorus and glucose concentrations, and the increases of serum urea, creatinine, and ammonia (Yu et al., 2015). However, only the levels of serum total protein and aspartate aminotransferase were decreased significantly in this study, indicating that protein metabolism seems to be more easily affected by AFB1 than sugar and lipid metabolism.

Meanwhile, AFB1-induced lipid oxidation begins by disrupting cell membrane integrity through stimulating phospholipid A2, and then oxidation of polyunsaturated fatty acids induced formation of reactive oxygen species to reduce antioxidant activity, thus further aggravating liver damage (Wan et al., 2021). The previous research showed that many hepatic enzyme gene expressions including CYP450 A1, 1A2, 2A6 and 3A4 involved in conversion of AFB1 to the highly reactive epoxide intermediate AFB1-8,9-epoxide (AFBO) were improved to decrease serum SOD, catalase and GSH-PX activity as well as increase serum MDA content (Sun et al., 2015). Probiotics supplementation can significantly improve the antioxidant indexes, mainly because it can improve the oxidation level by down-regulating CYP1A1 and CYP2H1 genes expressions, up-regulating GPX gene expression and increasing SOD enzyme activity (Ramadan et al., 2018; Salem et al., 2018) The other research showed that SOD was significantly decreased, and MDA was significantly increased by feeding 100 μg/kg AFB1 in boiler diet for 42 d, whereas the negative effects were significantly decreased by adding 3 g/kg montmorillonite in broiler diet (Shi et al., 2006). Because low-level addition of montmorillonite has little effectiveness for AFB1 adsorption, and high-level addition of montmorillonite has some side effects including adsorbing nutrients and decreasing nutrient concentrations, the study on the optimal-level addition of montmorillonite becomes important. In this experiment, the addition amount of montmorillonite was only 0.3 g/kg to have the good AFB1–removal effect, which may be related to the synergistic effect of montmorillonite, compound probiotics and AFB1-degrading enzyme.

The damages of AFB1 to intestinal tract mainly present barrier function loss and inflammatory reaction, lymphocyte, or monocyte infiltration as well as mucosal hyperplasia and vacuolar degeneration, and even decrease intestinal villus height and increase intestinal crypt depth (Hernández-Ramírez et al., 2020). Liver is the primary organ attacked by AFB1 which can cause many microscopic damages including high-level eosinophile granulocyte and monocytes (Rashidi et al., 2020), lipid vacuoles (Magnoli et al., 2017), inflammatory cell proliferation and infiltration, the edema and hepatocytes degeneration (Perali et al., 2020). Kidney is also the main organ attacked by AFB1. The renal toxicity of dietary AFB1 in broilers presented the increased glomerular basement membrane thickening and stromal cells, glomerular enlargement, tubular epithelial cell cytoplasmic vacoulation, renal glomerulus collapse and structural damage (Ortatatli, et al., 2005). This research showed that there was no change of glomerular structure in kidney by HE staining; however, mainly inflammatory cell infiltration and significant microscopic anatomical defects were observed in intestinal and liver tissues. The degree of tissue damage may be due to the sensitivity of the different tissues to AFB1 or the different contents of AFB1 residues in the different tissues.

Probiotics have been proved to reduce AFB1-induced microscopic damage to intestinal, liver and kidney tissues. It was reported that supplementation of Lactobacillus and Saccharomyces cerevisiae could produce significant protective effect against liver and kidney microscopic damages in broilers fed with 5 mg/kg AFB1 (Śliżewska et al., 2019). According to the results of this experiment, the damage of AFB1 to intestinal tissue appearance of villus rupture was the greatest, and CMD had the good protective effect on AFB1-induced liver injury. In the previous experiment, the mechanism of CMD on regulating AFB1-induced inflammatory response in chicken embryo primary intestinal epithelium, liver and kidney cells had been studied (Guo et al., 2021b), in agreement with this study.

This result showed that the relative weights of thymus and bursa of Fabricius were decreased by AFB1 addition; however, CMD addition could restore the above results. When AFB1 enters blood circulation through intestinal epithelial cells, it will enter the lymphatic circulation to cause damages of immune organs such as thymus (Guan et al., 2019), spleen (Li et al., 2019), bursa of Fabricius (Guo et al., 2021c), further cause atrophy and weight loss of these immune organs. AFB1 damaged the spleen tissue of poultry by activating the NF-κB signaling pathway, which directly presented atrophy and immunosuppression of spleen tissue (Wan et al., 2022). Similar, AFB1 could cause apoptosis of bursa of Fabricius cells by up-regulating Bax, Caspase-3 and p53 protein expressions, down-regulating Bcl-2 protein expression, and improving genes expressions involved in catalase and glutathione peroxidase, which finally lead to bursa of Fabricius atrophy (Rajput et al., 2019).

The previous studies have shown the following reasons for the reduction of nutrient metabolic rates of broilers induced by AFB1: 1) mycotoxin-contaminated corn had low nutrient contents (Dänicke et al., 2007); 2) decreased digestive enzyme activity such as trypsin and amylase in the intestinal lumen (Magnoli et al., 2017); 3) liver damage led to low nutrient metabolic rate (Chang et al., 2020). AFB1-contaminated feed may cause broilers refuse diet which may be related to neurochemical and physical damage of salivary glands and oral cavity (Brake et al., 2000). The previous report showed that AFB1 could significantly reduce the utilization of protein, decrease feed intake and growth performance (Chen et al., 2016), which is consistent with this study. Several beneficial microorganisms and bentonite have been used for reducing toxic effects on animal production. Holanda and Kim evaluated the effect of a mycotoxin-detoxifying additive compositing of clay, yeast cell wall, plant extracts and antioxidants on alleviating deoxynivalenol-induced toxicity in newly weaned pigs, which showed that the compound agent significantly improved piglet production performance and reduced oxidative stress (Holanda and Kim, 2020). Therefore, the combined materials may be the effective method to alleviate AFB1 toxicity.

The results showed that the abundance of Lactobacillus-aviarius was positively correlated with ADG and SOD. It was reported that Lactobacillus-aviarius and Lactobacillus-salivarius were the dominant bacteria in the small intestine of broilers (Gong et al., 2007). The previous report showed that AFB1 increased potentially pathogenic bacteria and reduced beneficial bacteria abundance in rat feces, and the microbiota composition was normalized by oral Lactobacillus casein (Huang et al., 2021). Another study showed that Lactobacillus had the ability to produce bacteriocin and organic acids, which could inhibit pathogens and inflammatory reaction, thus increasing body weight and improving immunity (Yan et al., 2016). Gut microbial metabolite could also affect health through gut-liver interaction (Saeedi et al., 2020) The high abundance of Lactobacillus was used as an indicator of healthy intestine and positive correlated with growth of broilers (Torok et al., 2011), in agreement with this study. In addition, there is a negative correlation between the abundance of Lactobacillus-aviarius and AFB1 residue in serum and liver, maybe due to the ability of Lactobacillus to degrade AFB1.

Esherichia-coli-g-Escherichia-Shigella as a kind of pathogenic bacterium could cause diarrhea and intestinal inflammation in human and broilers (Gong et al., 2019; Liu et al., 2020). In general, the optimal microbiota influences gastrointestinal development, prevents colonization of pathogens, and modulates the gut-associated systemic immunity. It can be inferred that CMD addition is able to decrease AFB1 residues and increase broiler growth by increasing the proliferations of beneficial bacteria such as Lactobacillus species and decreasing the proliferations of detrimental bacteria such as E. coli and Staphylococcus.

The results showed that the growth performance and antioxidant capacity in boilers were significantly reduced, and AFB1 residues and micro-pathological changes of intestines, liver and kidney were increased when the broilers were fed with corn contaminated by AFB1 for 42 d. However, the CMD composed of compound probiotics, ADE and montmorillonite could effectively alleviate mycotoxin negative effects on the above parameters. The combination of physical and biological detoxification provides a new strategy for alleviating AFB1 toxicity through AFB1 degradation and absorption to reduce inflammation and improve the antioxidant level of broilers.

DISCLOSURES

The authors declare no competing financial interest.

ACKNOWLEDGMENTS

This research was funded by the Natural Science Foundation of Henan Province, China (222300420238).

REFERENCES

  1. Ates M.B., Ortatatli M. Phase-1 bioactivation mechanisms of aflatoxin through AhR, CAR and PXR nuclear receptors and the interactions with Nigella sativa seeds and thymoquinone in broilers. Ecotox. Environ. Saf. 2021;208 doi: 10.1016/j.ecoenv.2020.111774. [DOI] [PubMed] [Google Scholar]
  2. Brake J., Hamilton P.B., Kittrell R.S. Effects of the trichothecene mycotoxin diacetoxyscirpenol on feed consumption, body weight, and oral lesions of broiler breeders. Poult. Sci. 2000;79:856–863. doi: 10.1093/ps/79.6.856. [DOI] [PubMed] [Google Scholar]
  3. Bryden W.L. Mycotoxin contamination of the feed supply chain: Implications for animal productivity and feed security. Anim. Feed Sci. Tech. 2001;83:134–158. [Google Scholar]
  4. Caceres I., Khoury A.A., Khoury R.E., Lorber S., Oswald I.P., Khoury A.E., toui A.A., Puel O., Bailly J.D. Aflatoxin biosynthesis and genetic regulation: a review. Toxins. 2020;12:150. doi: 10.3390/toxins12030150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Caldas J.V., ignale K.V., Boonsinchai N., Wang J., Putsakum M., England J.A., Coon C.N. The effect of β-mannanase on nutrient utilization and blood parameters in chicks fed diets containing soybean meal and guar gum. Poult. Sci. 2018;97:2807–2817. doi: 10.3382/ps/pey099. [DOI] [PubMed] [Google Scholar]
  6. Chang J., Wang T., Wang P., Yin Q., Liu C., Zhu Q., Lu F., Gao T. Compound probiotics alleviating aflatoxin B1 and zearalenone toxic effects on broiler production performance and gut microbiota. Ecotox. Environ. Saf. 2020;194 doi: 10.1016/j.ecoenv.2020.110420. [DOI] [PubMed] [Google Scholar]
  7. Chen J., Chen K., Yuan S., Peng X., Fang J., Wang F., Cui H., Chen Z., Yuan J., Geng Y. Effects of aflatoxin B1 on oxidative stress markers and apoptosis of spleens in broilers. Toxicol. Ind. Health. 2016;32:278–284. doi: 10.1177/0748233713500819. [DOI] [PubMed] [Google Scholar]
  8. Dänicke S., Valenta H., Matthes S. On the interactions between Fusarium toxin-contaminated wheat and nonstarch polysaccharide hydrolyzing enzymes in diets of broilers on performance, intestinal viscosity, and carryover of deoxynivalenol. Poult. Sci. 2007;86:291–298. doi: 10.1093/ps/86.2.291. [DOI] [PubMed] [Google Scholar]
  9. Fan T., Xie Y., Ma W. Research progress on the protection and detoxification of phytochemicals against aflatoxin B1-Induced liver toxicity. Toxicon. 2021;195:58–68. doi: 10.1016/j.toxicon.2021.03.007. [DOI] [PubMed] [Google Scholar]
  10. Gao S., Zhang L., Zhu D., Huang J.Y., Yang J.M., Jiang J.J., Wu H., Lv G. Effects of glucose oxidase and bacillus subtilis on growth performance and serum biochemical indices of broilers exposed to aflatoxin B1 and endotoxin. Anim. Feed Sci. Tech. 2022;286 [Google Scholar]
  11. Ghali R., Hmaissia-khlifa K., Ghorbel H., Maaroufi K., Hedili A. Incidence of aflatoxins, ochratoxin A and zearalenone in Tunisian foods. Food Control. 2008;19:921–924. [Google Scholar]
  12. Gong J., Si W., Forster R.J., Huang R., Yu H., Yin Y., Yang C., Han Y. 16S rRNA gene-based analysis of mucosa-associated bacterial community and phylogeny in the chicken gastrointestinal tracts: from crops to ceca. Fems Microbiol. Ecol. 2007;59:147–157. doi: 10.1111/j.1574-6941.2006.00193.x. [DOI] [PubMed] [Google Scholar]
  13. Gong Y., Yang H., Wang X., Xia W., Lv W., Xiao Y., Zou X. Early intervention with cecal fermentation broth regulates the colonization and development of gut microbiota in broiler chickens. Front. Microbiol. 2019;10:1422. doi: 10.3389/fmicb.2019.01422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guan K., Li H., Zuo Z., Wang F., Hu P., Peng X., Fang J., Cui H., Shu G., Ouyang P. The molecular mechanisms of protective role of Se on the G0/G1 phase arrest caused by AFB1 in broiler's thymocytes. Biol. Trace Elem. Res. 2019;189:556–566. doi: 10.1007/s12011-018-1491-y. [DOI] [PubMed] [Google Scholar]
  15. Guo Y., Balasubramanian B., Zhao Z.H., Liu W.C. Marine algal polysaccharides alleviate aflatoxin B1-induced bursa of Fabricius injury by regulating redox and apoptotic signaling pathway in broilers. Poult. Sci. 2021;100:844–857. doi: 10.1016/j.psj.2020.10.050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Guo H.W., Chang J., Wang P., Yin Q.Q., Liu C.Q., Li S., Zhu Q., Yang M., Hu X.F. Detoxification of aflatoxin B1 in broiler chickens by a triple-action feed additive. Food Addit. Contam. B. Part A. 2021;38:1583–1593. doi: 10.1080/19440049.2021.1957159. [DOI] [PubMed] [Google Scholar]
  17. Guo H.W., Chang J., Wang P., Yin Q.Q., Liu C.Q., Xu X.X., Dang X.W., Hu X.F., Wang Q.L. Effects of compound probiotics and aflatoxin-degradation enzyme on alleviating aflatoxin-induced cytotoxicity in chicken embryo primary intestinal epithelium, liver and kidney cells. AMB Express. 2021;11:35. doi: 10.1186/s13568-021-01196-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hernández-Ramírez J.O., Nava-Ramírez M.J., Merino-Guzmán R., Téllez-Isaías G., Vázquez-Durán A., Méndez-Albores A. The effect of moderate-dose aflatoxin B1 and Salmonella Enteritidis infection on intestinal permeability in broiler chickens. Mycotoxin Res. 2020;36:31–39. doi: 10.1007/s12550-019-00367-7. [DOI] [PubMed] [Google Scholar]
  19. Holanda D.M., Kim S.W. Efficacy of mycotoxin detoxifiers on health and growth of newly-weaned pigs under chronic dietary challenge of deoxynivalenol. Toxins. 2020;12:311. doi: 10.3390/toxins12050311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Huang Y., Lv H., Song Y., Sun C., Zhang Z., Chen S. Community composition of cecal microbiota in commercial yellow broilers with high and low feed efficiencies. Poult. Sci. 2021;100 doi: 10.1016/j.psj.2021.01.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lai Y., Sun M., He Y., Lei J., Han Y., Wu Y., Bai D., Guo Y., Zhang B. Mycotoxins binder supplementation alleviates aflatoxin B1 toxic effects on the immune response and intestinal barrier function in broilers. Poult. Sci. 2022;101 doi: 10.1016/j.psj.2021.101683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li H., Guan K., Zuo Z., Wang F., Peng X., Fang J., Cui H., Zhou Y., Ouyang P., Su G., Chen Z. Effects of aflatoxin B1 on the cell cycle distribution of splenocytes in chickens. J. Environ. Pathol. Tox. 2019;32:27–36. doi: 10.1293/tox.2018-0015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liew W.P., Nurul-Adilah Z., Than L., Mohd-Redzwan S. The binding efficiency and interaction of Lactobacillus casein shirota toward aflatoxin B1. Front. Microbiol. 2018;9:1503. doi: 10.3389/fmicb.2018.01503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Liu Y., Mao H., Woldemariam Y.K., Wan Z., Cao Y., Tron T., Lin J., Jiang Y., Li H., Wang J. Degradation of aflatoxin B1 by a recombinant laccase from Trametes sp. C30 expressed in Saccharomyces cerevisiae: a mechanism assessment study in vitro and in vivo. Food Res. Int. 2021;145 doi: 10.1016/j.foodres.2021.110418. [DOI] [PubMed] [Google Scholar]
  25. Liu Y., Yuan X., Li L., Lin L., Zuo X., Cong Y., Li Y. Increased ileal immunoglobulin a production and immunoglobulin a-coated bacteria in diarrhea-predominant irritable bowel syndrome. Clin. Transl. Gastroen. 2020;11:e00146. doi: 10.14309/ctg.0000000000000146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Magnoli A.P., Rodriguez M.C., González Pereyra M.L., Poloni V.L., FPeraltam M., Nilsonm A.J., Miazzo R.D., Bagnis G., Chiacchiera S.M., Cavaglieri L.R. Use of yeast (Pichia kudriavzevii) as a novel feed additive to ameliorate the effects of aflatoxin B1 on broiler chicken performance. Mycotoxin Res. 2017;33:273–283. doi: 10.1007/s12550-017-0285-y. [DOI] [PubMed] [Google Scholar]
  27. Mao M., Wang P., Shi K., Lu Z., Lv F. Effect of solid-state fermentation by Enterococcus faecalis M2 on antioxidant and nutritional properties of wheat bran. J. Cereal Sci. 2020;94 [Google Scholar]
  28. Mao J., Zhou Y., Lv G., Zhou R. Simultaneous detoxification of aflatoxin B1, zearalenone and deoxynivalenol by modified montmorillonites. Molecules. 2022;27:315. doi: 10.3390/molecules27010315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Murcia H.W., Diaz G.J. Protective effect of glutathione S-transferase enzyme activity against aflatoxin B1 in poultry species: relationship between glutathione S-transferase enzyme kinetic parameters, and resistance to aflatoxin B1. Poult. Sci. 2021;100 doi: 10.1016/j.psj.2021.101235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ortatatli M., Oğuz H., Hatipoğlu F., Karaman M. Evaluation of pathological changes in broilers during chronic aflatoxin (50 and 100 ppb) and clinoptilolite exposure. Res. Vet. Sci. 2005;78:61–68. doi: 10.1016/j.rvsc.2004.06.006. [DOI] [PubMed] [Google Scholar]
  31. Perali C., Magnoli A.P., Aronovich M., Rosa C., Cavaglieri L.R. Lithothamnium calcareum (Pallas) Areschoug seaweed adsorbs aflatoxin B1 in vitro and improves broiler chicken's performance. Mycotoxin Res. 2020;36:371–379. doi: 10.1007/s12550-020-00402-y. [DOI] [PubMed] [Google Scholar]
  32. Phillips T.D., Wang M., Elmore S.E., Hearon S., Wang J.S. NovaSil clay for the protection of humans and animals from aflatoxins and other contaminants. Clay Miner. 2019;67:99–110. doi: 10.1007/s42860-019-0008-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pinton P., Oswald I.P. Effect of deoxynivalenol and other Type B trichothecenes on the intestine: a review. Toxins. 2014;6:1615–1643. doi: 10.3390/toxins6051615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rajput S.A., Zhang C., Feng Y., Wei X.T., Khalil M.M., Rajput I.R., Baloch D.M., Shaukat A., Rajput N., Qamar H., Hassan M., Qi D. Proanthocyanidins alleviates aflatoxin B₁-induced oxidative stress and apoptosis through mitochondrial pathway in the bursa of fabricius of broilers. Toxins. 2019;11:157. doi: 10.3390/toxins11030157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ramadan S., Nagwan E.H., Fadl S.E., Sakr O.A., Elbialy Z.I. Effect of probiotic supplement on aflatoxicosis and gene expression in the liver of broiler chicken. Environ. Toxicol. Phar. 2018;60:118–127. doi: 10.1016/j.etap.2018.04.015. [DOI] [PubMed] [Google Scholar]
  36. Rashidi N., Khatibjoo A., Taherpour K., Akbari-Gharaei M., Shirzadi H. Effects of licorice extract, probiotic, toxin binder and poultry litter biochar on performance, immune function, blood indices and liver histopathology of broilers exposed to aflatoxin-B1. Poult. Sci. 2020;99:5896–5906. doi: 10.1016/j.psj.2020.08.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Saeedi B.J., Liu K.H., Owens J.A., Hunter-Chang S., Camacho M.C., Eboka R.U., Chandrasekharan B., Baker N.F., Darby T.M., Robinson B.S., Jones R.M., Jones D.P., Neish A.S. Gut-resident lactobacilli activate hepatic Nrf2 and protect against oxidative liver injury. Cell Metab. 2020;31:956–968. doi: 10.1016/j.cmet.2020.03.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Salem R., El-Habashi N., Fadl S.E., Sakr O.A., Elbialy Z.I. Effect of probiotic supplement on aflatoxicosis and gene expression in the liver of broiler chicken. Environ. Toxicol. Pharm. 2018;60:118–127. doi: 10.1016/j.etap.2018.04.015. [DOI] [PubMed] [Google Scholar]
  39. Shi Y.H., Xu Z.R., Feng J.L., Wang C.Z. Efficacy of modified montmorillonite nanocomposite to reduce the toxicity of aflatoxin in broiler chicks. J. Anim. Feed Sci. Technol. 2006;129:138–148. [Google Scholar]
  40. Śliżewska K., Cukrowska B., Smulikowska S., Cielecka-Kuszyk J. The effect of probiotic supplementation on performance and the histopathological changes in liver and kidneys in broiler chickens fed diets with aflatoxin B₁. Toxins. 2019;11:112. doi: 10.3390/toxins11020112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sun L.H., Zhang N.Y., Zhu M.K., Zhao L., Zhou J.C., Qi D.S. Prevention of aflatoxin B1 hepatoxicity by dietary selenium is associated with inhibition of cytochrome P450 isozymes and up-regulation of 6 selenoprotein genes in chick liver. J. Nutr. 2015;146:655–661. doi: 10.3945/jn.115.224626. [DOI] [PubMed] [Google Scholar]
  42. Torok V.A., Hughes R.J., Mikkelsen L.L., Perez-Maldonado R., Balding K., MacAlpine R., Percy N.J., Ophel-Keller K. Identification and characterization of potential performance-related gut microbiotas in broiler chickens across various feeding trials. Appl. Environ. Microbiol. 2011;77:5868–5878. doi: 10.1128/AEM.00165-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wan X.L., Li N., Chen Y.J., Chen X.S., Yang Z., Xu L., Yang H.M., Wang Z.Y. Protective effects of lycopene on mitochondrial oxidative injury and dysfunction in the liver of aflatoxin B1-exposed broilers. Poult. Sci. 2021;100 doi: 10.1016/j.psj.2021.101441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wan F., Tang L., Rao G., Zhong G., Jiang X., Wu S., Huang R., Tang Z., Ruan Z., Chen Z., Hu L. Curcumin activates the Nrf2 Pathway to alleviate AFB1-induced immunosuppression in the spleen of ducklings. Toxicon. 2022;209:18–27. doi: 10.1016/j.toxicon.2022.01.010. [DOI] [PubMed] [Google Scholar]
  45. Yan Y.S., Zheng Y., Huang Z., Min W.X., Yang H. Lactococcus nasutitermitis sp. nov. isolated from a termite gut. Int. J. Syst. Evol. Microbiol. 2016;66:518–522. doi: 10.1099/ijsem.0.000743. [DOI] [PubMed] [Google Scholar]
  46. Yu Z., Wang F., Liang N., Wang C., Peng X., Fang J., Cui H., Mughal M., Lai W. Effect of selenium supplementation on apoptosis and cell cycle blockage of renal cells in broilers fed a diet containing aflatoxin B1. Biol. Trace Rlem. Res. 2015;168:242–251. doi: 10.1007/s12011-015-0344-1. [DOI] [PubMed] [Google Scholar]
  47. Zhang Y., Wang P., Kong Q., Cotty P.J. Biotransformation of aflatoxin B1 by Lactobacillus helviticus FAM22155 in wheat bran by solid-state fermentation. Food Chem. 2021;341(Pt 1) doi: 10.1016/j.foodchem.2020.128180. [DOI] [PubMed] [Google Scholar]
  48. Zhao L., Feng Y., Wei J.T., Zhu M.X., Zhang L., Zhang J.C., Karrow N.A., Han Y.M., Wu Y.Y., Guo Y.M., Sun L.H. Mitigation effects of bentonite and yeast cell wall binders on AFB1, DON, and OTA induced changes in laying hen performance, egg quality, and health. Toxins. 2021;13:156. doi: 10.3390/toxins13020156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zhao L., Zhang L., Xu Z., Liu X., Chen L., Dai J., Karrow N.A., Sun L. Occurrence of aflatoxin B1, deoxynivalenol and zearalenone in feeds in China during 2018-2020. J. Anim. Sci. Biotechnol. 2021;12:74. doi: 10.1186/s40104-021-00603-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zheng A., Zhang A., Chen Z., Pirzado S.A., Chang W., Cai H., Bryden W.L., Liu G. Molecular mechanisms of growth depression in broiler chickens (Gallus Gallus domesticus) mediated by immune stress: a hepatic proteome study. J. Anim. Sci. Biotechnol. 2021;12:90. doi: 10.1186/s40104-021-00591-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zuo R.Y., Chang J., Yin Q.Q., Wang P., Yang Y.R., Wang X., Wang G.Q., Zheng Q.H. Effect of the combined probiotics with aflatoxin B₁-degrading enzyme on aflatoxin detoxification, broiler production performance and hepatic enzyme gene expression. Food Chem. Toxicol. 2013;59:470–475. doi: 10.1016/j.fct.2013.06.044. [DOI] [PubMed] [Google Scholar]

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