【作者】 冯焱；

【导师】 姚军虎；

【作者基本信息】 西北农林科技大学 ， 动物营养与饲料科学， 2012， 博士

【摘要】 集约化肉鸡饲养环境蕴含的各种病原和非病原微生物都会随时袭击动物，诱发机体产生免疫应激，破坏机体内环境的平衡，改变营养物质的消化吸收与体内重分配，损坏新陈代谢、延长饲养周期，导致机体免疫力和抗病力的下降。目前有关免疫应激对肉鸡消化系统、免疫机能和肠道微生物区系影响的研究十分有限。本文通过对不同免疫状态下肉鸡生产性能、肠道消化酶活性、养分转运载体、免疫功能和肠道微生物区系变化规律的研究，旨在探讨免疫状态影响养分利用的机理，为优化肉鸡免疫程序提供理论依据。1.免疫应激对肉鸡生产性能和消化系统的影响选用540只1日龄Cobb500肉鸡随机分为5个处理，每处理9重复，每重复12只。试验分为无免疫组（NV）、简化免疫（SV）、常规免疫（CV）、免疫亢进（LPS）和免疫抑制（CYP）5个处理组。免疫应激组分别于16、18、20、37、39和41d肌肉注射相应剂量的LPS和CYP，NV组、SV和CV组于同日龄注射等体积生理盐水。NV、SV及CV组前期、后期及全期采食量、体增重无显著差异（P>0.05），显著或极显著高于LPS组和CYP组（P<0.05或P<0.01），LPS和CYP可降低肉鸡采食量和体增重。21d，SV组十二指肠、空肠指数高于LPS组（P<0.05）；42d，处理组间的小肠指数无显著差异（P>0.05）。LPS组和CYP组空肠和回肠的淀粉酶和脂肪酶高于NV组（P<0.05或P<0.01）。胰蛋白酶活性在全期无显著变化（P>0.05）。LPS组和CYP组的肠道蠕动率低于NV组（P<0.01）。LPS和CYP组3部分小肠SGLT-1，CaBP-D28k和L-FABP mRNA的相对表达显著高于CV和NV组（P<0.05）。本研究表明，在合理控制饲养环境的前提下，对肉鸡采取简化免疫或者不免疫可提高肠道蠕动速率，有利于肉仔鸡生长；LPS和CYP刺激可降低肉鸡采食量和体增重。2.免疫应激对肉鸡免疫功能的影响选用180只1日龄AA肉公鸡随机分为4个处理，每处理5重复，每重复9只。试验分为无免疫组（NV）、常规免疫组（CV）、免疫亢进组（LPS）和免疫抑制组（CYP）4个处理组。免疫应激组分别于18、19、20、32、33和34d肌肉注射相应剂量的LPS和CYP，NV组和CV组于同日龄注射等体积生理盐水。21d和35d，LPS组胸腺指数高于其它组（P<0.05），CYP组趋于降低胸腺指数（P>0.05）；21d和35d，LPS组脾脏指数高于NV组（P<0.05），21、28、35和42d法氏囊指数CYP组显著低于其它组（P<0.05），LPS组和CYP组均降低sIgA的水平（P<0.01）；LPS组血清IgG比NV和CV组显著升高在42d（P<0.01），随日龄增加，处理组间抗体效价出现递增趋势；CYP和LPS均抑制T、B淋巴细胞增殖，LPS组CD4+/CD8+T细胞百分比显著低于NV组（P<0.05）。本研究表明，不进免疫增强肉鸡黏膜免疫机能，LPS主要激活体液免疫应答，CYP抑制法氏囊的发育，同时抑制细胞免疫应答反应。3.免疫应激对肉鸡肠道微生物区系及常见菌数量的影响选用180只AA肉公鸡随机分4个处理，每处理5重复，每重复9只。试验分为NV、CV、LPS和CYP处理组。免疫应激组分别于18、19、20、32、33和34d肌肉注射相应剂量的LPS和CYP，NV组和CV组于同日龄注射等体积生理盐水。分别于21、28、35和42d，每处理组各取6只鸡，无菌操作，宰杀后取十二指肠、空肠、回肠和盲肠内容物。ERIC-PCR结果显示，处理组间十二指肠细菌种群结构的相似性最高（75%），盲肠（40%）、回肠（39%）和空肠（38%）的相似性较低。图谱的条带数目为十二指肠＞回肠＞盲肠＞空肠。28和35d，十二指肠和空肠LPS组条带均低于21d，而回肠和盲肠的未见显著变化。21d，回肠CYP组的条带低于其它处理组。RT-PCR对肠段4种菌定量结果表明，与NV相比，4种细菌的基因拷贝数发生显著变化。集中在回肠和盲肠，大肠杆菌属：5.03-7.88log copies/mg；乳酸杆菌属：5.48-10.34log copies/mg；双歧杆菌属：4.11-7.98log copies/mg；肠球菌属：5.61-8.79log copies/mg。本研究表明，双歧杆菌属含量为4种菌属中细菌含量最低，乳酸杆菌含量高，盲肠含菌最多，十二指肠含量最少，后期各菌群趋于稳定。综上所述，合理控制饲养环境，对肉鸡NV或SV可达到与CV相同的生产性能。免疫应激改变肠道菌群组成，降低肠道黏膜免疫机能，影响体液和细胞免疫应答，对微生物区系的影响主要集中空肠、回肠和盲肠，且LPS和CYP的特异性条带主要是不可养的硬壁门细菌（Uncultured Firmicutes bacterium）、不可培养的拟杆菌属细菌（Uncultured Bacteroidales bacterium），NV组主要是不可培养的梭菌目细菌（UnculturedClostridiales bacterium）。十二指肠各时期的变化不明显，故未对此肠段进行条带分析。更多还原

【Abstract】 It is apparent that many factors can influence the immune response of an animal understress. Stress can suppress, enhance or have no effect on the immune response of an animal.There are many interacting factors influencing the immunological response of an animalunder stress, some factors in the homeostasis of broilers directly or indirectly affect theirimmune status, and immunosuppression induced by stress is manifested by failures invaccination and increased morbidity and mortality of flocks. This article attempted to studythe effect on different immune stress in growth performance, digestive enzyme activities,nutrient transporters, immune function and intestinal microflora in broiler chickens, aiming toreveal the mechanism of immune stress’ effects on nutrient utilization and to provide atheoretical basis for optimizing the broiler immune program.1. Effects of immune stress on performance parameters and digestive function in broilerchickensImmune stress is the loss of immune homeostasis by external forces. The purpose of thisexperiment was to investigate the effects of immune stress on the growth performance, smallintestinal enzymes and peristalsis rate, and mRNA expression of nutrient transporters inbroiler chickens. Five hundred and fourty1-day old broilers (Cobb500) were randomlyseparated into5groups with9replicates per group and12birds each replicate; Group1=novaccine (NV); Group2=simplified vaccination (SV); group3=conventional vaccine (CV);group4=lipopolysaccharide (LPS)+conventional vaccine (LPS); group5=cyclophosphamide (CYP)+conventional vaccine (CYP). The results demonstrated thatimmune stress by LPS and CYP reduced body weight gain (BWG), feed intake (FI). However,feed conversion ratio (FCR) remained unchanged during the feeding period. Relativeindicators were measured on d21and42. Duodenum and jejunum relative weight index ofLPS group is lower than other groups on d21(P <0.05); and there was no significantdifference of indicators between different groups on d42(P>0.05). LPS and CYP increased intestinal enzyme activity, relative expression of SGLT-1, CaBP-D28k and L-FABP mRNAs(P <0.05). Intestinal motility of LPS group and the CYP group were lower than the controlgroup and the conventional group (P <0.01), and there was no significant difference ofintestinal motility between different groups. LPS and CYP injection had a negative effect onthe growth performance of healthy broiler chicken. Immune stress demanded a higherconsumption of energy resulting in the reduction of the synthesis of inflammatory factors, FIand BWG, which finally inhibited the growth of broilers. The present study demonstrated thatNV and CV could improve growth performance while enzyme activity in small intestine andrelative expression of nutrient transporter mRNA of NV and CV were decreased under theconditions of a controlled rational feeding environment.2. Effects of immune stress on immune function in broiler chickensThis experiment was conducted to determine the effects of different immune statuses onimmune function of broiler chicken.1801-day AA broilers were allocated to four groups;each treatment had5replicate cages of9broilers per replicate pen. Different immune stressmodels, including the control group=no vaccines (NV), conventional vaccines group (CV),immune hyperthyroidism group (LPS) and immune suppression group (CYP) were conductedin this study. The T lymphocytes and subgroups were isolated from periphery blood fromimmunized and unvaccinated birds and were detected by CD3/CD4/CD8triple labelling andflow cytometric analysis and MTT assays, respectively. The results showed that, group LPSthymus index was higher than other treatment groups on day21and35(P <0.05); CYP grouphad reduced thymus index trend (P>0.05); LPS group increased significantly spleen index onday21and35(P <0.05), bursal index in CYP group was significantly lower than the othergroups on day21,28,35and42(P <0.05). Compared with NV group, sIgA level of LPSgroup and CYP group decreased (P <0.01); the serum IgG of42days LPS group increasedsignificantly (P <0.01) comparing to the NV and CV groups. With the increasing of day agebetween treated groups antibody titer appear increasing trend, CPY and LPS inhibited the T, Blymphocyte proliferation, and CD4+/CD8+T cell percentage of LPS group was significantlylower than NV group (P <0.05). LPS stress increased the cellular immune response ofchicken. Tolerance of chicken to CYP increased with age.3. Effects of immune stress on intestinal microflora and common bacteria changes ofbroiler chickens1801-day AA broilers were allocated to4groups; each treatment had5replicate cagesof9broilers per replicate pen. Different immune stress models, including the controlgroup=no vaccines (NV), conventional vaccines group (CV), immune hyperthyroidism group(LPS) and immune suppression group (CYP) were set in this study. On d21,28,35and42, each treatment group from each of6chickens, after slaughter, sterile operation collectedjejunum, ileum and duodenum and cecum intestinal contents. The results showed that theDNA fingerprinting of ERIC-PCR had high stability and repetition. Different immune statusaffected gut microflora to different degrees, and as age varied, the dominant microfloracorrespondingly changed. The diversity of cecal microflora was mostly obvious in LPS+general vaccination treatment and CYP+general vaccination treatment. In the present study,based on cluster analysis, the similarity of gut microflora in negative control was relativelystable; and among all treatments, the similarity of duodenal microflora was the highest at75%,followed by cecum (40%), jejunum (38%) and ileum (39%). These data suggest that immunestress can affect gut microflora in broilers. More specifically, changes happened in deodenumwere relatively stable, and for other tracts, compared with the CYP+vaccination treatment, theLPS+general vaccination treatment had more significant influence on microflora. Theapplication of RT-PCR on immunological stress test in the digestive tract of broilersEscherichia, Lactobacillus, Bifidobacterium and Enterococcus were analyzed. The dynamicdistribution and changes of the four bacterial species in the broiler’s digestive tract were alsoanalyzed systematically. Among the4genera of bacteria, changes mainly concentrated in theileum and cecum, E. coli content:5.03-7.88; Lactobacillus content:5.48-10.34;Bifidobacterium content:4.11-7.98; Enterococcus content:5.61-8.79. The results showed thatimmune stress makes the four bacterial species in broiler’s digestive tract changeconspicuously in their genic copy when comparing with CV. In which, the changes of the fourbacterial species mainly represent in ileum and cecum, while the changes of days byduodenum species is not clear.In summary, the reasonable control of broiler rearing environment, no immune orsimplified immunization led to the same production performance as conventional immunity.Immunological stress changes in the gut flora composition, reduces intestinal mucosalimmune function, subsequently affects the humoral and cellular immune responses. Themechanism of intestinal microflora change needs further research.更多还原