【作者】 谭建庄；

【导师】 呙于明；

【作者基本信息】 中国农业大学 ， 动物营养与饲料科学， 2014， 博士

【摘要】 本论文研究了日粮精氨酸的添加对不同疾病模型肉鸡免疫功能的影响。首先评定了因传染性法氏囊病病毒攻毒引起的免疫抑制肉鸡的精氨酸需要量,以及精氨酸的添加对肉鸡免疫抑制的调节作用；继而采用球虫攻毒诱导肉鸡肠道损伤,研究了日粮精氨酸的添加对肉鸡肠道炎症及因肠道炎症引起的肠道损伤的调控作用；采用了大肠杆菌脂多糖注射诱导肉鸡系统性炎症反应,研究了日精氨酸添加对肉鸡系统性炎症反应的影响：最后采用沙门氏菌攻毒探讨日粮精氨酸的添加对因革兰氏阴性菌感染引起的肉鸡肠道粘膜屏障功能紊乱的调控作用。试验—探讨了因传染性法氏囊病病毒攻毒引起的免疫抑制肉鸡达到最佳生产性能和最佳免疫功能时的精氨酸需要量,以及日粮精氨酸的添加对因传染性法氏囊病病毒攻毒引起的肉鸡免疫抑制的调节作用。试验采用2×5因子安排的随机试验设计,包含5个精氨酸水平(前期：10.0、15.0、20.0、25.0、和30.0g/kg;后期：8.8、13.2、20.0、22.0和26.4g/kg),和2个攻毒处理。500只雌性罗氏308肉鸡被随机分配到10个处理组(每个处理5个重复,每个重复10只鸡)。在第14日龄,处理组肉鸡腿部肌肉注射传染性法氏囊病病毒(IBDV),对照组注射生理盐水。分别在第1、21和42天统计生产性能和器官指数。于第21d采集法氏囊和血液样本。本试验结果显示,IBDV注射显著抑制了(P<0·05)肉鸡的免疫功能,如血清溶菌酶活性、IgA浓度、外周血淋巴细胞转化率(Con A),以及外周血淋巴细胞体外培养液中NO和H202浓度。IBDV注射显著降低了(P<0·05)肉鸡血清抗新城疫抗体滴度。随着日粮精氨酸水平的升高,血清溶菌酶活性、IgA水平、和抗传染性法氏囊病抗体滴度显著升高(P<0·05)。攻毒组肉鸡达到最佳料重比时的精氨酸需要量为18.9g/kg,高于(P<0·05)对照组(16.0g/kg)。攻毒组肉鸡(IgA:16.5g/kg;淋巴细胞转化率：17.4g/kg)达到最佳免疫功能时的精氨酸需要量大于(P<0·05)对照组肉鸡(IgA：12.4g/kg;淋巴细胞转化率：13.1g/kg)。IBDV攻毒显著降低了(P<0.05)攻毒后第二天外周血CD3+、CD4+、和CD8+比例；降低了(P<0.05)攻毒后第2天法氏囊IL-1β的mRNA表达量;降低了(P<0.05)攻毒后第4天血清IL-6浓度。攻毒后第2天,随着日粮精氨酸水平的升高,外周血CD4+和CD8+比例显著升高(P<0.05),血清IL-6水平显著升高(P<0.05)。攻毒后第4天,随着日粮精氨酸水平的升高,IBDV攻毒组肉鸡CD3+淋巴细胞比例线性升高(P<0.05)。试验二探讨了日粮精氨酸添加对球虫攻毒肉鸡肠道结构和免疫功能的影响。试验采用2×3因子安排的试验设计,包括3个精氨酸水平(10.0、14.0和20.0g/kg)和2个攻毒处理。在14日龄时,肉鸡强饲球虫疫苗或等量生理盐水。21日龄时,屠宰肉鸡采集空肠组织和空肠黏膜样品,用于组织切片、基因表达和肠道免疫指标的测定。在球虫攻毒期间(14-21d),球虫攻毒显著降低了肉鸡的体增重、采食量和饲料效率(P<0.05)。球虫攻毒显著提高了(P<0.05)空肠黏膜IgA浓度和肠道炎症因子的mRNA表达量(iNOS、IL-1β、IL-8和MyD88)。显著降低了空肠Mucin-2、IgA和IL-1RI的mRNA表达量。随着日粮精氨酸水平的升高,空肠绒毛高度、空肠隐窝深度、黏膜麦芽糖酶活力显著提高(P<0.05)。日粮精氨酸的添加显著降低了球虫攻毒组肉鸡空肠黏膜IgG浓度和TLR4的mRNA表达量。随着日粮精氨酸水平的升高,mTORCl通路基因mTOR和RPS6KB1,以及抗凋亡基因BcI-2mRNA的表达量呈现出二次曲线变化趋势。试验三探讨了日粮精氨酸的添加对肉鸡全身性炎症反应的调控作用。本试验采用了2×3因子安排的随机试验设计,分别设置2个攻毒处理(LPS注射和生理盐水注射)和3个精氨酸水平(10.0、14.0和19.0g/kg),每个处理8个重复,每个重复6只鸡。LPS采用隔天连续注射,分别在第14、16、18和20天进行腹腔注射(1mg/kg体重),对照组注射生理盐水。21d采样,并统计14-21d生产性能数据。研究结果表明,LPS注射期间(14-21d),肉鸡体增重和采食量显著降低(P<0·05),饲料效率显著升高(P<0·05)。LPS注射降低了(P<0·05)脾脏CD11+和B淋巴细胞比例,以及脾脏噬异细胞和巨噬细胞吞噬指数。随着日粮精氨酸水平的升高,脾脏CD11+, CD14+和B淋巴细胞比例线性降低(P<0·05)。同时LPS注射提高了(P<0·05)脾脏和盲肠扁桃体IL-1β和IL-6的mRNA表达量,而日粮精氨酸的添加降低了(P<0·05)脾脏IL-1β、TLR4、NFκB和PPAR-y的mRNA表达量,以及降低了(P<0·05)盲肠扁桃体IL-1β、IL-10、TLR和NFκB的mRNA表达量。试验四探讨了日粮精氨酸的添加对沙门氏菌攻毒肉鸡肠粘膜屏障功能的影响。试验采用了2×3因子安排的随机试验设计,分别设置2个攻毒处理(强饲沙门氏菌或生理盐水)和3个精氨酸水平(10.0、14.5和19.0g/kg)。试验组肉鸡分别在第7、8和9天口腔强饲1ml含沙门氏菌的培养液,对照组强饲相同量的无菌LB肉汤培养基。分别于第10和16天采样,同时统计攻毒期间肉鸡的生产性能。研究结果表明,沙门氏菌攻毒降低了攻毒组肉鸡7-16d体增重和采食量,然而随着日粮精氨酸水平的升高,体增重和采食量下降的幅度也随之升高。攻毒后第一天,沙门氏菌攻毒显著提高了肉鸡血清D-木糖浓度(P<0.05),并呈现出显著降低肉鸡血清尿酸浓度的趋势(P<0.10)；随着日粮精氨酸水平的升高,肉鸡血清尿酸浓度呈现出二次曲线变化趋势(P<0.05),而血清D-木糖浓度呈现出线性降低的趋势(P<0.05)。攻毒后第七天,随着日粮精氨酸水平的升高,攻毒组肉鸡血清尿酸浓度呈现出二次曲线变化趋势(P<0.05),D-木糖浓度则呈现出线性降低的趋势(P<0.05)。攻毒后第七天,随着日粮精氨酸水平的升高,攻毒组肉鸡血清内毒素含量呈现出线性降低的趋势(P<0.10)。攻毒后第一天,饲喂含19.0g/kg精氨酸日粮的肉鸡肝脏沙门氏菌含量显著高于饲喂含10.0和14.5g/kg精氨酸日粮的肉鸡(P<0.05)。以上结果表明,在传染性法氏囊病引起的免疫抑制状态下肉鸡需要添加更多的精氨酸以维持其最佳生产性能和免疫功能;日粮精氨酸的添加提高了外周血T淋巴细胞比例,缓解了因IBDV攻毒引起的免疫抑制；日粮精氨酸的添加能够通过抑制TLR4通路以及降低脾脏CD14+淋巴细胞比例来缓解因LPS注射引起的全身性炎症反应,同时本试验还表明过量精氨酸的添加能够降低脾脏淋巴细胞比例；日粮精氨酸的添加通过抑制TLR4通路缓解了肉鸡因球虫攻毒引起的肠道炎症,并通过激活mTORCl通路促进了肉鸡肠道的损伤修复；日粮精氨酸的添加未能缓解肉鸡因沙门氏菌攻毒引起的肠粘膜屏障功能紊乱和体增重下降。更多还原

【Abstract】 This study was performed to determine the effects of dietary L-arginine (Arg) supplementation on growth performance, immunosuppression, inflammation, and intestinal barrier dysfunction in broiler chickens.Expt.1was conducted to determine the Arg requirement of broiler chickens immunized with infectious bursal disease vaccine (IBDV) based on growth performance and immunological responses and the effects of dietary Arg on immunosuppression of chickens following infectious bursal disease virus (IBDV) inoculation. A total of500one-day-old female Ross broilers were randomly assigned into10treatments (5replicates per group,10birds per replicate). The design of this study was a5x2factorial arrangement (n=5) with5Arg concentrations (starter:10.0,15.0,20.0,25.0, and30.0g/kg; grower-finisher:8.8,13.2,20.0,22.0and26.4g/kg) with or without IBDV inoculation (IBDV or saline inoculation at14days). Chickens were sampled at21,42days of age and2,4and6days post-inoculation (DPI). The IBDV inoculation significantly (P<0.05) suppressed immune functions of broilers, i.e. the serum lysozyme and IgA content, mitogen-stimulated peripheral blood mononuclear cells (PBMC) proliferation (Concanavalin A, Con A), PBMC in vitro NO and H2O2production. The IBDV inoculation caused a suboptimal response to vaccination which was evidenced by the suppression of anti-newcastle disease virus (NDV) antibody titers. Dietary Arg supplementation quadratically increased (P<0.05) the serum lysozyme and IgA concentrations, PBMC proliferation (LPS), and in vitro PBMC H2O2yield in the IBDV inoculated treatments. Serum anti-IBDV antibody titer increased quadratically with increasing dietary Arg concentration (P=0.06). The Arg requirement of vaccinated broilers (1.89%) for minimum FCR in a quadratic model was higher (P<0.05) than that of control broilers (1.60%). The Arg requirements of vaccinated broilers (IgA:1.65%; PBMC proliferation (LPS):1.74%) for optimal immune status in a broken-line model was higher (P<0.05) than control broilers (IgA:1.24%; PBMC proliferation (LPS):1.31%). The IBDV inoculation:decreased (P<0.05) CD3+, CD4+, and CD8+T cell counts at2days post-inoculation (DPI) and monocyte counts at6DPI; reduced (P<0.05) bursal IL-1/? mRNA expression at2DPI and serum IL-6concentration at4DPI. Increasing Arg concentration:increased (P<0.05) CD4+and CD8+T cell counts at2DPI; linearly increased (P<0.05) CD3+T cell counts in IBDV inoculated groups and monocyte counts in control groups at4DPI; increased (P<0.05) serum IL-6concentration in IBDV inoculated groups at2DPI; and increased (P<0.05) serum anti-IBDV antibody titers at42days of age.Expt.2was performed to investigate the effects of dietary Arg supplementation on intestinal structure and functionality in broiler chickens undergoing coccidial challenge. The design of this study was a randomized complete block employing a3×2factorial arrangement (n=8) with3dietary concentrations of Arg (10.0,14.0, and20.0g/kg) with or without coccidial vaccine challenge (unchallenged and coccidial challenge). On day14, birds were gavaged with coccidial vaccine or saline. Birds were killed on day21to obtain jejunal tissue and mucosal samples for histological, gene expression and mucosal immunity measurements. Within7days of challenge, the coccidial challenge decreased BW gain and feed intake, and increased feed-to-gain ratio (P<0.05). The jejunal inflammation was evidenced by villus damage, crypt dilation, and goblet cell depletion. Coccidial challenge increased (P<0.05) mucosal IgA concentrations and inflammatory gene (iNOS, IL-1β, IL-8and MyD88) mRNA expression as well as reduced (P<0.05) jejunal Mucin-2, IgA, and IL-1RI mR HA expression. Increasing Arg concentration:(a) increased (P<0.05) jejunal villus height and linearly increased (P<0.05) jejunal crypt depth;(b) quadratically increased (P<0.05) mucosal maltase activity and linearly decreased (P<0.05) mucosal IgG concentration within coccidiosis-challenged groups;(c) linearly decreased (P<0.05) jejunal TLR4mRNA expression within coccidiosis-challenged groups. The mTOR complex1pathway genes(mTOR and RPS6KB1) and anti-apoptosis gene Bcl-2mRNA expression quadratically (P <0.05) responded to increasing Arg supplementation.Expt.3was conducted to investigate the effect of dietary Arg supplementation on inflammatory response and innate immunity of broiler chickens. Two experiments were conducted in this part. Experiment1was designed as a2×3factorial arrangement (n=8cages/treatment;6birds/cage) with3dietary Arg concentrations (1.05,1.42, and1.90%) and2immune treatments (injection of lipopolysaccharide (LPS) or saline) which were given in48h interval between14and21d of age. Correlation between dietary Arg concentration (0.99,1.39,1.76,2.13, or2.53%) and circulating B cell percentage (%of circulating lymphocytes) was determined in Experiment2. In Experiment1, LPS injection decreased BW gain and feed intake, and increased feed-to-gain of challenged broilers (14to21d; P<0.05). LPS injection suppressed (P<0.05) splenic CD11+and B cell percentages (%of splenic lymphocytes) and phagocytosis by splenic heterophils and macrophages; Arg supplementation linearly decreased the percentage of CD11+, CD14+and B cells in the spleen (P<.10). LPS injection increased (P<0.05) IL-1β and IL-6mRNA expression in the spleen and cecal tonsils. Arginine supplementation decreased (P<0.05) the mRNA expression of IL-1β, TLR4, and PPAR-γ in the spleen, and IL-1β, IL-10, TLR4, and NFκB in the cecal tonsils. In Experiment2, increasing dietary Arg concentration linearly and quadratically reduced circulating B cells percentage (P<0.01).Expt.4was conducted to investigate the effects of dietary Arg supplementation on intestinal barrier functions of broiler chickens undergoing Salmonella enterica serovar Typhimurium challenge. The design of this study was a randomized complete block employing a3×2factorial arrangement with3dietary concentrations of Arg (10.0,14.5, and19.0g/kg) with or without Salmonella challenge (unchallenged and coccidial challenge). On day7,8and9, birds were gavaged with Salmonella or saline. Birds were killed on day10and16to obtain ileum tissue and liver samples. Within9days of challenge, the Salmonella challenge decreased BW gain and feed intake. With the increase of dietary Arg concentration, the reduction percentage of growth performance increased. At1DPC, Salmonella challenge significantely increased (P<0.05) serum D-xylose concentration and tended to decrease (P<0.10) serum Uric acid concentration; increasing dietary Arg concentration quadratically affected (P<0.05) srume Uric acid concentration and linearly decreased (P<0.05) serum D-xylose concentration; the chickens fed diet containing19.0g/kg Arg had the highest (P<0.05) liver viable Salmonella colonies (CFU). At7DPC, increasing dietary Arg concentration quadratically effected (P<0.05) serum Uric acid concentration in Salmonella challenged groups, linearly decreased (P<0.05) srum D-xylose concentration, and tended to decease (P<0.10) serum endotoxin concentration.Collectively, these results indicate that additional dietary Arg supplementation is required to get the optimal growth performance and immune function for immunosuppressive broilers, and Arg supplementation attenuated IBDV inoculation induced immunosuppression via modulating circulating T cell subpopulations. Dietary Arg supplementation attenuated intestinal mucosal disruption of coccidiosis-challenged chickens probably through suppressing TLR4and activating mTOR complex1pathways, and attenuated the overexpression of pro-inflammatory cytokines probably through the suppression of the TLR4pathway and CD14+cells percentage, while, it failed to ameliorate Salmollena challenge induced intestinal barrier dysfunction and growth performance reduction.更多还原