Effects Of Intestinal Microbiota On Metabolism And Toxicity Mitigation Of Zearalenone In Broilers

Zearalenone(ZEN)is an estrogenic mycotoxin widely found in grain and feed materials.ZEN and its metabolites compete with estradiol to bind estrogen receptors(ERs)in animals,causing hyperestrogenism and liver and kidney damage in animals.Sensitivity to ZEN varies among species.Chickens are not very sensitive to ZEN and only at high doses can serious reproductive toxicity occur.Finding out the reason why chickens are insensitive to ZEN has important reference value for the targeted implementation of ZEN hazard control.There are many reasons for chicken’s insensitivity to ZEN,among which intestinal microbe may be an important reason.This study was conducted in vitro and in vivo to preliminarily explore the effects of intestinal microorganisms on ZEN metabolism and the mitigation of ZEN toxicity in broilers.The results can provide reference for ZEN hazard prevention and control in feed and rational utilization of feed materials.The main experimental studies are as follows:Experiment 1.The metabolic effect of intestinal microbe on ZEN of broilers in vitro6 healthy 42-day-old broilers were selected.After slaughter,about 1 g of duodenum,jejunum,ileum,cecum and colonic digme were taken and diluted 10 times with DPBS solution to prepare test solution.Part of the test solution was added into ZEN standard solution and prepared with a content of 3 μg/m L Zen-intestinal chyme solution.The solution was anaerobic incubated at 39℃ for 6 h.Another part of the test solution was added with cadmium chloride to inactivate microorganisms,and the same solution was prepared into 3μg/m L Zen-intestinal chyme solution,and the solution was anaerobic incubated at 39℃ for6 h as the control group.HPLC was used to detect the content of ZEN and its metabolites in the reaction system,and ZEN degradation rate was calculated based on the control group.The results showed that jejunum had no significant degradation effect on ZEN,while duodenum,ileum,cecum and colon had degradation rates of 56%,12%,15% and 17%,respectively.Zearalenol can be metabolized by intestinal microorganisms in duodenum,cecum and colon,and the proportion of β-ZOL metabolized by ZEN is higher than that of α-ZOL in duodenum and colon.These results indicate that intestinal microorganisms of broilers have the ability to degrade and biotransform ZEN,and duodenum microorganisms have the highest activity to degrade ZEN.Experiment 2.The effect of intestinal microbe on ZEN metabolism and toxicity mitigation in broilersIn vitro experiments showed that intestinal microbiota of broilers could degrade ZEN and biotransform ZEN,but whether intestinal microbiota in vivo had the same effect is uncertain.In this study,antibiotic treatment was used to inhibit microbial activity in order to investigate the effects of intestinal microorganisms on ZEN metabolism and toxicity mitigation in broilers.A total of 120 one-day-old AA+ broilers were random Ly divided into 4 groups with 6replicates.The treatment groups were: 1)control group: basal diet;2)ZEN group: 2.5 mg/kg ZEN diet;3)Microbial inhibition group: streptomycin(1 g/kg)+ ampicillin(1 g/kg)+neomycin(1 g/kg);4)"Microbial inhibition +ZEN" group: streptomycin(1 g/kg)+ampicillin(1 g/kg)+ neomycin(1 g/kg)+ 2.5 mg/kg ZEN diet.Fresh feces were collected on day 10 and slaughtered for sampling on day 21.The test results are as follows:1.Effects of ZEN on intestinal microbe of broilersDietary exposure of 2.5 mg/kg ZEN significantly increased the relative abundance of Firmicutes and significantly decreased the relative abundance of Bacteroidetes in cecum of broilers at phylum classification level(P<0.05).At the level of genus classification,ZEN significantly increased the relative abundance of Roboutisa and Enterococcus in duodenum and cecum,and significantly decreased the relative abundance of Streptococcus in ileum(P<0.05)and cecum(P<0.01).These results indicate that 2.5 mg/kg ZEN can change the intestinal microbial composition of broilers.2.Effects of ZEN on broiler health2.5 mg/kg ZEN significantly increased the average daily gain and reduced the feed/gain ratio(P<0.05),and there was no significant loss in intestinal tissue of broilers.However,2.5mg/kg ZEN exposure significantly reduced the contents of BUN and ALP in serum(P<0.05),and the serum T-AOC content had a decreasing trend(P<0.1).Meanwhile,liver GSH enzyme activity was significantly decreased(P<0.05),and T-SOD content had a downward trend(0.05<P<0.1).The activity of GSH in serum was significantly increased(P<0.05).In addition,the ZEN group showed inflammatory cell infiltration,vascular congestion,peripheral vascular edema and cell vacuolization,a little congestion in ovary,and enhanced cytoplasm eosinophilic in testicular tissue.These results indicate that exposure to 2.5 mg/kg ZEN has no effect on growth performance and intestinal health of broilers,but can cause damage to antioxidant capacity,liver and reproductive organs of broilers.3.Effects of intestinal microorganisms on ZEN metabolism in broilersCompared with the ZEN group,the fecal ZEN and β-Zol contents in the "microbial inhibition +ZEN" group were significantly increased(P<0.05)by about 55%,and the ZEN excretion rate was increased by 9%.The relative abundances of Firmicutes and proteobacteria in ileum and bacteroidetes and actinobacteria in cecum in "microbial inhibition +ZEN" group were significantly different from those in ZEN group(P<0.05).At the genus level,there were significant differences in the relative abundance of many bacteria.It was noteworthy that the relative abundance of Enterococcus in duodenum and cecum was significantly decreased,that of Lactococcus in ileum was significantly decreased,and that of Ruminococcus was significantly different(P<0.05).These results indicate that intestinal microorganisms of broilers also have adsorption,degradation and biotransformation effects on ZEN in vivo,and the effects may be enterococcus,Lactococcus and ruminococcus.4.Alleviating effects of intestinal microorganisms on ZEN toxicity in broilersThe serum contents of TG,BUN and ALP in "microbial inhibition + ZEN" group were significantly decreased(P<0.05),the serum antioxidant index T-AOC content was significantly decreased(P<0.05),the liver GSH activity and T-SOD content were significantly decreased(P<0.05).Meanwhile,serum GSH content and GSH-Px activity were significantly increased(P<0.05).And compared with other groups,"microbial inhibition +ZEN" set of liver tissue and blood vessels within a large number of protein mucus,a serious phenomenon of inflammatory cell infiltration and vascular peripheral edema,liver cells appear circular cavity,ovarian tissue and a small amount of serious situation,cell infiltration,testicular tissue capsule partial thick,eosinophilic cytoplasm enhancement and small vacuoles.There was a significant correlation between the unclassifed tests and the relative abundance of Streptococcus,norank＿f＿Muribaculaceae,Alistipes,Bilophila,and unclassified＿o＿lactobacillales.The contents of ZEN and its metabolites in feces were significantly negatively correlated with the relative abundance of norank＿f＿Muribaculaceae,Streptococcus,Bacteroides,Alistipes and Bilophila.It has significant positive correlation with Faecalibacterium and partial Ruminococcaccae.These results indicate that intestinal microbe of broilers can alleviate the damage of antioxidant capacity,liver and reproductive organs caused by ZEN to broilers through metabolism.In conclusion,this study concluded that:(1)there were differences in the metabolic ability of microorganisms in different intestinal segments to ZEN,jejunum microbes had no significant degradation of ZEN,while duodenum,ileum,cecum and colon microbes had degradation and biotransformation ability of ZEN,and duodenum microbes had the strongest degradation activity.(2)2.5 mg/kg ZEN can cause intestinal microbiome disorder,decrease antioxidant capacity,and histomorphological damage of liver and reproductive organs of broilers.(3)Intestinal microorganisms affected the metabolism of ZEN and alleviated the damage of ZEN to serum and liver antioxidant capacity,liver and reproductive organs of broilers.(4)Beneficial bacteria such as Lactobacillus,Streptococcus,Enterococcus and Ruminococcus may play an important role in the metabolism of ZEN in the intestinal microorganisms of broilers.