| Mycotoxins are a class of toxic and harmful secondary metabolites produced by fungi.More than 500 mycotoxins have been identified,among which deoxynivalenol（DON）,aflatoxin B1（AFB1）and zearalenone（ZEN）are the main mycotoxins that contaminate feed.Under the current trend of global warming,the risk of crops being contaminated with mycotoxins is also increasing,seriously endangering livestock and poultry health.Therefore,monitoring the contamination of common mycotoxins in feed and carrying out research on the toxicological mechanism of related mycotoxins is of great significance for the prevention and control of the harm of mycotoxins to livestock and poultry production and human food safety.In addition,our survey results show that the excess rate of DON in pig compound feed in our country is as high as 8.9%.Ingestion of DON-contaminated feed will lead to anorexia and vomiting of livestock and poultry,endangering the health and production of livestock and poultry.Pigs are most sensitive to DON.Ingestion of high concentrations of DON can lead to intestinal inflammation and cell necrosis in pigs,and damage to the mucosal epithelial cell barrier,thereby affecting the absorption of nutrients in the intestines,thereby endangering the healthy production of livestock and poultry.This is also one of the important reasons why DON contamination in feed aggravates the diarrhea of piglets after weaning.However,the mechanism of DON-induced porcine intestinal damage has not been fully elucidated.Therefore,further analysis of the toxicological mechanism of DON-induced intestinal injury in pigs can provide a new theoretical basis for the prevention and treatment of DON-induced damage to pigs.This study is divided into the following three parts.Part 1:Occurrence of Aflatoxin B1,deoxynivalenol and zearalenone in feeds in China during 2018–2020From 2018 to 2020,a total of 3,507 test samples were collected from feed companies and farms in different regions of my country,including 2,090 feed ingredients and 1,417compound feeds.The common mycotoxins AFB1,DON and ZEN in these feed samples were analyzed.The results showed:1)The positive rates of AFB1,DON and ZEN in the feed samples were 81.9%-100%,96.4%-100%and 96.9%-100%,respectively;the average contamination concentrations of AFB1,DON and ZEN in the feed were 1.2-27.4μg/kg,458.0-1,925.4μg/kg and 48.1-326.8μg/kg.2)It is worth noting that the exceeding rate of AFB1,DON and ZEN in feed ingredients were 0.9%,0.5%and 0.1%,respectively,while the exceeding rate of AFB1,DON and ZEN in pig,poultry and ruminant compound feeds were 1.2%-12.8%,0.9%-2.9%and 0%-8.9%,respectively.3)In addition,more than 81.5%of feed ingredients and 95.7%of compound feeds were simultaneously contaminated with the above two or three mycotoxins.In conclusion,the results of this study showed that during 2018-2020,there were widespread individual and combined contamination of AFB1,DON and ZEN in feed ingredients and compound feeds in China.Therefore,in feed processing and animal husbandry,it is very important to strictly monitor the occurrence of mycotoxins in feed and implement effective prevention and control measures.Part Ⅱ:Effects of DON on growth performance and intestinal health of weaned pigletsIn this experiment,21 21-day-old male castrated and grown-up ternary crossbred piglets were randomly divided into 3 treatment groups,with 7 replicates in each group,and1 pig in each replicate.Piglets were fed basal diet,basal diet+1.0 mg/kg DON and basal diet+3.0 mg/kg DON,respectively.After 4 weeks of feeding,piglets were slaughtered and blood,intestines and their contents were collected for experimental analysis.The main experimental results showed:1)Compared with the control group,the dietary supplementation of 1.0 and/or 3.0 mg/kg of DON reduced（P<0.05）the body weight gain of piglets throughout the experiment（16.0%-60.8%）and feed intake（18.1%-38.7%）,at the same time,increased（P<0.05）feed-to-weight ratio（12.8%-33.8%）on days 1-28.In addition,compared with the 1.0 mg/kg DON group,the 3.0 mg/kg DON group could further reduce（P<0.05）the body weight gain and feed intake of the piglets.2)Compared with the control group,the dietary supplementation 1.0 and 3.0 mg/kg of DON could cause different degrees of damage to the duodenum,jejunum and ileum of piglets,mainly including villus breakage and loss,lamina propria edema,increased inflammatory cells and other pathological symptoms.In addition,dietary supplementation of DON at both 1.0 and 3.0mg/kg significantly increased（P<0.05）the concentration of LPS（21.7%-24.3%）in piglet serum.The CARD-FISH results showed that DON could increase（P<0.05）the content of microorganisms in the intestinal tissue of piglets.3)Dietary supplementation of DON at1.0 and 3.0 mg/kg significantly increased MDA（17.5%-86.4%）and protein carbonyl content（58.1%-170.1%）in duodenum,jejunum and ileum compared with the control)（P<0.05）.4)16S r RNA sequencing showed that DON significantly changed（P<0.05）the flora structure of duodenum,jejunum and ileum,mainly including the ecological imbalance of Actinomycetes,Cyanobacteria,Firmicutes and Proteus.5)Compared with the control group,dietary supplementation of DON at 1.0 and/or 3.0 mg/kg significantly up-regulated（P<0.05）the key genes Dmt1,Fth1 and Ftl in the ferroptosis signaling pathway and down-regulated（P<0.05）the key genes Steap3,Lpcat3,Cisd1、Fsp1 in the ferroptosis signaling pathway;WB results showed that 1.0 mg/kg DON and 3.0 mg/kg DON did not change the expression of STEAP3 and ACSL4 proteins in the duodenum（P>0.05）,but significantly increased DMT1 in the duodenum（19.0%-28.5%）,FTH1（98.6%-99.7%）and FTL（28.7%-53.1%）protein expression（P<0.05）and decreased FPN（27.9%-31.6%）and FSP1（41.0%-46.3%）protein expression（P<0.05）.Part Ⅲ:Mechanism of ferroptosis signaling pathway mediating DON-induced cytotoxicity of piglet intestinal epithelial cellsThe results of the second part of the experiment indicated that the ferroptosis signaling pathway may play an important role in mediating DON-induced intestinal injury in piglets.Therefore,we selected porcine intestinal epithelial cells IPEC-J2 as the research object,iron chelator DFP and gene knockdown expression technology to explore the role and mechanism of ferroptosis signaling pathway in DON-induced piglet intestinal injury.The main findings of the trial:1)Compared with the control group,the addition of 500μg/L DON in the medium can reduce（P<0.05）the cell viability of IPEC-J2,while the addition of 20μM iron chelator DFP in the medium can alleviate（P<0.05）DON cytotoxicity to IPEC-J2.In addition,DON significantly upregulated（P<0.05）the expression of DMT1,STEAP3,ACSL4 and FTL proteins in IPEC-J2 cells compared with the control group,while DFP addition antagonized（P<0.05）DON-induced ACSL4 and FTL Changes in protein expression in IPEC-J2 cells.2)After inhibiting the expression of Ftl gene in IPEC-J2 cells by siRNA technology,DON-induced IPEC-J2 cytotoxicity can also be alleviated.Conclusion:1)During the period from 2018 to 2020,the individual and combined contamination of AFB1,ZEN and DON in feed in China is very serious;2)The dietary supplementation of DON at 1.0 and/or 3.0 mg/kg can significantly damage the growth performance of piglets,which is related to the damage to the intestinal health of piglets;3)The ecological imbalance of intestinal flora induced by DON and the ferroptosis of intestinal cells are the important reasons for the intestinal damage of piglets. |
