【作者】 魏金涛；

【导师】 齐德生；

【作者基本信息】 华中农业大学 ， 动物营养与饲料科学， 2022， 博士

【摘要】 T-2毒素在玉米、小麦及其加工副产物中的检出率较高。雏鸡对T-2毒素较为敏感,采食被T-2毒素污染的日粮后会诱导肠道出现局灶性溃疡和发炎,进而危害雏鸡的健康。玉米、小麦及其加工副产品是肉鸡主要饲料原料,肉鸡配合饲料中T-2毒素污染现象十分常见。因此,有必要综合评价T-2毒素对雏鸡的毒性效应;采用转录组学、代谢组学和微生物组学技术等多组学技术系统解析T-2毒素致雏鸡肠道损伤的分子机制;并根据多组学研究结果筛选外源营养素,研究营养素缓解T-2毒素致雏鸡危害的效果,为T-2毒素危害的防控提供科学依据和新的思路。本研究通过3个试验开展研究工作,主要研究内容及试验结果如下:试验1,选取96只1日龄体重相近的快大型肉鸡(科宝500),随机分为4个组,每组4个重复,每个重复6只鸡,分别饲喂基础日粮(对照组)和在基础日粮中分别添加1.0 mg/kg T-2毒素(试验1组)、3.0 mg/kg T-2毒素(试验2组)、6.0mg/kg T-2毒素(试验3组)的日粮。雏鸡于14日龄屠宰采样,测定并分析T-2毒素对雏鸡生长性能、血液生化指标、营养物质表观消化率及肠道组织病理学变化的影响。本试验的主要结果如下:1.和对照组相比,试验第一周,试验2组和试验3组雏鸡平均日增重分别显著的(P<0.05)降低了7.58%和15.15%,平均日采食量分别显著的降低了(P<0.05)6.28%和12.13%。试验第二周,三个试验组雏鸡平均日增重分别显著的(P<0.05)降低了11.75%、16.25%和31.75%,平均日采食量分别显著的(P<0.05)降低了9.64%、16.18%和20.55%,同时,试验3组雏鸡料重比显著的(P<0.05)提高了15.94%。整个试验周期,三个试验组雏鸡平均日增重分别显著的降低了(P<0.05)8.36%、13.38%和26.09%,平均日采食量分别显著的(P<0.05)降低了7.09%、13.42%和18.23%。说明T-2毒素对雏鸡生产性能影响较大,而且影响的幅度随着日粮中T-2毒素剂量的增加而增加。2.和对照组相比,3个试验组雏鸡血清中天冬氨酸转氨酶(AST)的活性分别显著的(P<0.05)提高了14.49%、11.18%和17.69%,试验3组雏鸡血清中甘油三酯(TG)的含量显著的(P<0.05)提高了28.57%。说明T-2毒素对雏鸡的肝脏功能以及脂类代谢有一定的影响。3.和对照组相比,试验3组雏鸡粗蛋白质、钙和总磷的表观消化率分别显著的(P<0.05)降低了14.93%、18.02%和16.13%。说明日粮中含有6.0 mg/kg T-2毒素可以影响雏鸡对日粮主要营养物质的消化能力。4.病理切片结果显示,T-2毒素可引起雏鸡十二指肠、空肠和回肠的绒毛上皮细胞严重变性、坏死、脱落,肠固有层充血等病变。说明T-2毒素可以造成雏鸡肠道的损伤。试验2,在试验1的基础上,选取对照组和试验3组雏鸡十二指肠为试验材料,通过转录组学、代谢组学和微生物组学技术解析T-2毒素致雏鸡肠道损伤的分子机制。主要结果如下:1.转录组学研究结果表明,T-2毒素处理后雏鸡十二指肠基因共有148个表达下调和2388个表达上调,这些差异表达基因主要富集在免疫反应途径、细胞生长和死亡、细胞凋亡及细胞色素P 450(CYP 450)等信号通路及细胞进程中。2.对雏鸡十二指肠内容物进行差异代谢物筛选,共获得了41个差异显著的代谢物,其中有机酸和核苷酸类差异代谢物均多为下调,而氨基酸和脂质类代谢物多为上调。对这些差异显著的代谢物进行功能预测,结果表明,T-2毒素能够影响核酸代谢、氨基酸代谢、果糖和甘露糖代谢、碳水化合物消化和吸收、AMPK信号途径、磷酸转移酶系统等多种信号通路。3.对十二指肠内容物进行微生物组学分析,结果表明,与对照组相比,T-2毒素显著降低了(P<0.05)十二指肠厚壁菌门、杆菌纲及乳酸杆菌属微生物丰度,显著提高了(P<0.05)布劳特氏菌属、短状杆菌属、瘤胃球菌属、慢生根瘤菌和鞘氨醇单胞菌属等微生物丰度。通过功能预测发现氨基酸等主要营养物质的代谢以及膜转运等功能受到T-2毒素的影响。试验3,根据试验2结果,筛选出核苷酸和乳酸菌,评价其缓解T-2毒素对雏鸡损伤的效果,并与常见市售脱毒剂及无T-2毒素污染日粮做比较。选取196只1日龄体重相近的快大型肉鸡(青脚麻),随机分为6个组,每个组4个重复,每个重复8只鸡,分别饲喂:1)基础日粮+6.0 mg/kg T-2毒素(T-2毒素组);2)基础日粮+6.0 mg/kg T-2毒素+0.5%核苷酸(核苷酸组,NT组);3)基础日粮+6.0 mg/kg T-2毒素+5×10~6cfu/g乳酸菌(乳酸菌组,LA组);4)核苷酸+乳酸菌组,基础日粮+6.0mg/kg T-2毒素+0.5%核苷酸+5×10~6cfu/g乳酸菌(NT+LA组)。5)基础日粮+6.0mg/kg T-2毒素+500 g/T脱毒剂(DA组);6)基础日粮(CK组)。T-2毒素组、NT组、LA组和NT+LA组采用两因素两水平析因设计,DA组和CK组为对照。雏鸡于14日龄屠宰采样,测定并分析T-2毒素及不同营养素对雏鸡生长性能、血液生化指标、十二指肠组织病理学变化的影响及机理。本研究的主要结果如下:1.和T-2组相比,试验第一周,NT组和CK组雏鸡平均日增重分别显著的提高了9.86%和23.94%(P<0.05),NT组、LA组、NT+LA组和DA组雏鸡料重比没有显著性差异(P>0.05),但是均显著低于CK组(P<0.05)。试验第二周,CK组雏鸡平均日增重和平均日采食量分别显著的提高了15.87%和3.81%(P<0.05)。整个试验周期,NT+LA组和CK组雏鸡平均日增重分别显著提高了8.08%和18.18%(P<0.05),CK组雏鸡平均日采食量显著提高了3.81%(P<0.05)。在试验的不同阶段,核苷酸和乳酸菌对雏鸡平均日增重、平均日采食量及料肉比的影响均不存在显著的交互作用(P>0.05)。2.各试验组雏鸡血清中AST、ALP、TP、ALB、TBIL、CHOL、TG、BUN和CREA的活性(含量)均没有显著性差异,且没有显著的交互作用(P>0.05),但是,NT组、LA组、NT+LA组、DA组、CK组雏鸡血清中ALT和T-2毒素组相比显著降低(P<0.05),且NT和LA对雏鸡血清中ALT的影响具有显著的交互作用(P<0.05)。3.从雏鸡十二指肠病理变化来看,T-2毒素组雏鸡肠绒毛顶端上皮细胞重度变性、坏死和脱落、固有层充血。LA组和LA+NT组雏鸡十二指肠绒毛末端仍有上皮细胞脱落,固有层局部充血。但是,整体而言,在含有T-2毒素的日粮中补饲NT、LA及DA对雏鸡十二指肠的病理变化均有一定的缓解作用。4.q-PCR结果显示,T-2毒素可引起雏鸡十二指肠中IL-1β、IL-6、TNF-α、NF-κB等炎症相关细胞因子表达的显著上调(P<0.05),核苷酸、乳酸菌和脱毒剂均可以显著下调IL-1β、TNF-α和NF-κB的表达(P<0.05)。除此之外,T-2毒素还可引起雏鸡十二指肠中RIPK3、P53、BCL2和Caspase3等凋亡相关基因表达上调,NT、LA和DA均可以显著下调RIPK3、P53、BCL2和Caspase3的表达水平(P<0.05)。免疫印迹检测结果显示,T-2毒素处理显著降低雏鸡十二指肠中紧密连接蛋白Occludin的表达,而NT、LA添加可上调Occludin,并且NT与LA组合添加可显著上调Occludin的表达(P<0.05)。综上,本研究得出的结论如下:1.日粮中T-2毒素可影响雏鸡对粗蛋白质、钙和总磷的表观消化率,降低雏鸡生长性能,并可危害雏鸡的健康,造成肝脏功能受损、脂类代谢异常及十二指肠、空肠和回肠损伤。2.T-2毒素造成十二指肠损伤的机制是其通过改变免疫反应途径等信号通路及细胞进程中的基因表达、影响十二指肠代谢及改变肠道菌群结构。3.日粮中添加核苷酸和乳酸菌可以在一定程度上缓解T-2毒素对雏鸡生产性能、及肠道健康造成的不良影响,其机理可能是核苷酸和乳酸菌的抗炎作用及通过干扰细胞凋亡和坏死性凋亡程序抑制T-2毒素对雏鸡的损伤作用。更多还原

【Abstract】 T-2 toxin is commonly found in corn,wheat,and subsidiary agricultural products.Broiler chickens are very sensitive to T-2 toxin,and focal ulcers and inflammation in the gastrointestinal tract could be induced through diets contaminated with T-2 toxin,which impairs the health of broilers.Corn,wheat,and subsidiary agricultural products were main energy feedstuffs for broilers,and T-2 toxin pollution in broiler feed was very common.Therefore,it’s necessary to study the toxic effects of T-2 toxin,clarify the mechanism of T-2 toxin on intestinal damage in broilers,and develop nutrients to provided theoretical basis and new ideas for the prevention and treatment of the T-2toxin-induced detriment.The research was carried out through three trails,the main results are as follows:Trial 1,96 one-day-old male broilers with similar body weight were randomly allocated to 4 dietary treatment groups:control diet based on corn and soybean-meal(Control),Control+1.0 mg/kg of T-2 toxin(Group 1),Control+3.0 mg/kg of T-2 toxin(Group 2)and Control+6.0 mg/kg of T-2 toxin(Group 3).Each group contained 4replicate cages with 6 birds per cage.On d 14,the broilers were sampled to analyze the effects of T-2 toxin on the growth performance,blood biochemical indicators,apparent digestibility rates of nutrients,and intestinal histopathology.The main results obtained in this trial are as follows:1.Compared with Control,the body weight gain of Group 2 and Group 3 was decreased by 7.58%and 15.15%(P<0.05)respectively,and their feed intake was decreased by 6.28%and 12.13%(P<0.05)respectively during the first week.In the second week,the body weight gain of Group 1,2 and 3 was reduced by 11.75%,16.25%and 31.75%(P<0.05)respectively,and their feed intake was reduced by 9.64%,16.18%,and 20.55%(P<0.05)respectively.In addition,the feed/gain ratio was increased by15.94%in Group 3(P<0.05)during the second week.During the whole feeding period,the body weight gain of Group 1,2 and 3 was inhibited by 8.36%,13.38%and 26.09%(P<0.05)respectively,and their feed intake was inhibited by 7.09%,13.42%,18.23%(P<0.05)respectively.The results showed that T-2 toxin had influence on the performance of chicks,and the extent of the influence increased with the increase of T-2 toxin dosage in the diet.2.Compared with control group,the dietary treatments with T-2 toxin had no influence on the following serum indexes including alanine aminotransferase(ALT),total protein(TP),albumin(ALB),total bilirubin(TBIL),total cholesterol(CHOL),urea nitrogen(BUN),alkaline phosphatase(ALP),creatinine(CREA),blood glucose(GLU),andγ-glutamyl transpeptidase(GGT)(P>0.05).However,the aspartate aminotransferase(AST)activity in the serum was significantly increased by 14.49%,11.18%,and 17.69%(P<0.05)respectively in Group 1,2 and 3.Besides,the triglyceride(TG)in the serum was increased by 28.57%(P<0.05).The results indicated that T-2 toxins have certain effects on liver function and lipid metabolism in broilers.3.In comparison with Control,the apparent digestibility rates of crude protein,calcium,and total phosphorus in Group 3 were reduced by 14.93%,18.02%,and 16.13%(P<0.05),respectively.The results showed that 6.0 mg/kg T-2 toxin in the diet could affect the digestibility of main nutrients in the diet of broilers.4.According to the histopathological analysis,dietary addition T-2 toxin resulted in severe degeneration,necrosis,and shedding of villous epithelial cells in duodenum,jejunum,and ileum.It indicated that T-2 toxin could cause intestinal damage in broilers.Trial 2,based on the results obtained in the trial 1,the duodenum samples of broilers in Control and Group 3(addition with 6.0 mg/kg of T-2 toxin)were selected for transcriptomics,metabolomics and microbiome study to reveal the mechanism of T-2toxin induced intestinal injury in broilers.The main results in this trial are as follows:1.Transcriptomics results shows,a total of 148 genes were down-regulated while2388 genes were up-regulated in the duodenal tissue treated with T-2 toxin.These differentially expressed genes are mainly enriched in immune response pathways,cell growth and death,apoptosis,the metabolism by CYP450.2.The differential metabolites in the duodenal contents showed that a total of 41significantly different metabolites were obtained,among which organic acid and nucleotide metabolites were mostly down-regulated,while amino acids and lipid metabolites were mostly up-regulated.Functional prediction of these significantly different metabolites showed that T-2 toxin affected the nucleic acid metabolism,amino acid metabolism,fructose and mannose metabolism,carbohydrate digestion and absorption,AMPK signaling pathway,phosphotransferase system and other signaling pathways.3.According to the microbiome analysis of the duodenal contents,T-2 toxin was found to reduce the abundance of Pachybacteria,Bacteroides and lactobacillus(P<0.05),and increase the abundance of Breutella,Brevibacterium,Rumenococcus,Bradyrhizobium and Sphingomonas(P<0.05).Functional prediction showed that the metabolism of major nutrients such as amino acids metabolism and the membrane transport were influenced by T-2 toxins.Trial 3,According to the results of trial 2,nucleotide and lactobacillus were screened.The effects of nucleotide and lactobacillus on alleviating the damage of T-2toxin to broilers were evaluated,and compared with common commercial detoxification agents and diets without T-2 toxin pollution.196 one-day-old cyan-shank partridge chicken with similar body weight were randomly allocated to 6 dietary treatment groups,including T-2 Group(basal diet+6.0 mg/kg T-2 toxin),NT Group(basal diet+6.0mg/kg T-2 toxin+0.5%nucleotide),LA Group(basal diet+6.0 mg/kg T-2 toxin+5×10~6cfu/g lactobacillus),NT+LA Group(basal diet+6.0 mg/kg T-2 toxin+0.5%nucleotide+5×10~6cfu/g lactobacillus),DA Group(basal diet+500 g/T detoxification agent)and CK Group(basal diet).In a 2×2 factorial design arrangement for T-2 Group,NT Group,LA Group and NT+LA Group,DA group and CK group were used as controls.Each group contained 4 replicate cages with 8 birds per cage.On d 14,the broilers were sampled to analyze the effects of T-2 toxin and detoxication agent on the growth performance,blood biochemical indicators,apparent digestibility rates of nutrients,and intestinal histopathology of broilers.The main results obtained in this trial are as follows:1.Compared with T-2 Group,the body weight gain of NT Group and CK Group was increased by 9.86%and 23.94%(P<0.05)respectively,the feed/gain ratio of NT Group,LA Group,NT+LA Group and DA Group have no different significant difference(P>0.05),but significantly lower than the CK Group(P<0.05)during the first week.In the second week,the body weight gain and daily feed intake of CK Group was increased by15.87%and 3.81%(P<0.05)respectively,NT Group,LA Group,NT+LA Group and DA Group have no different significant difference(P>0.05).During the whole feeding period(d 1-14),the body weight gain of NT+LA Group and CK Group was increased by 8.08%and 18.18%(P<0.05)respectively,and daily feed intake of CK Group was increased by 3.81%(P<0.05).At different stages of the trial,there was no significant interaction between nucleotides and lactobacillus on average daily gain,average daily feed intake and feed-meat ratio of chicks(P<0.05).2.In all Trial,there were no influence and interaction on serum AST,ALP,TP,ALB,TBIL,CHOL,TG,CREA,BUN or GLU(P>0.05),but in comparison with T-2 Group,activity of ALT was significant decreased in NT Group,LA group,NT+LA Group,DA Group and CK Group(P<0.05).Moreover,NT Group and LA Group have significant interaction on the influence of serum ALT in broilers(P<0.05).3.According to the duodenum pathological of broilers,T-2 Group broilers epithelial cell severe degeneration,necrosis and shedding,congestion of lamina propria.LA Group and LA+NT Group broilers still had epithelial cells shedding at the end of duodenum villi,and local congestion in lamina propria.However,on the whole,supplementation of nucleotides,lactobacillus and detoxification agent in the diet containing T-2 toxin can alleviate the pathological changes of chick duodenum to some extent.4.The results of q-PCR showed that T-2 toxin could significantly up-regulation the expression of inflammation-related cytokines and such as IL-1β,IL-6,TNF-αand NF-κB in broilers duodenum(P<0.05),nucleotides,lactobacillus and detoxification agent can significantly down-regulate the expression of IL-1β,TNF-αand NF-κB(P<0.05).In addition,T-2 toxin can also up-regulation the expression of apoptosis-related genes such as RIPK3,P53,BCL2-2 and Caspase3 in broilers duodenum(P<0.05),nucleotides,lactobacillus and detoxification agent could significantly down-regulate the expression of RIPK3,P53,BCL2-2 and Caspase3(P<0.05).Western blot analysis showed that T-2toxin can significantly reduce the expression of tight junction protein Occludin in broilers duodenum,nucleotide and lactobacillus were added simultaneously can significantly down-regulate the expression of Occludin(P<0.05).In summary,this study draws the following conclusions:1.Diet addition of T-2 toxin can inhibit apparent digestibility rates of crude protein,calcium and total phosphorus,reduce the growth performance of broilers,and endanger the health of broilers,including hepatic function,abnormal lipid metabolism,duodenum,jejunum and ileum injury,and so on.2.The mechanism of duodenum injury caused by T-2 toxin was changed the signal pathways such as duodenal immune response pathway and gene expression in cell process,affects duodenal metabolism and changes the structure of intestinal flora.3.Dietary addition of nucleotides and lactobacillus can effectively alleviate the T-2toxin induced impairment in the growth performance and intestinal histopathology in broiler chickens.The mechanism may be the anti-inflammatory effects of nucleotides and lactobacillus and the inhibition of T-2 toxin on chicken damage by interfering with apoptosis and necrotic apoptosis procedures.更多还原