【作者】 佘德勇；

【导师】 陈耀星； 董玉兰；

【作者基本信息】 中国农业大学 ， 预防兽医， 2016， 博士

【摘要】 禽类产品是我国人民肉类消费的重要来源。近30年来,我国居民对禽产品的消费需求不断增加。但是受国内养殖业技术水平的限制,家禽养殖常常遭受疾病、营养失衡、应激等影响,使得禽产品供应经常发生较大波动。因此,如何提高家禽健康水平,提升家禽生产成绩,保障禽产品稳定供应以满足居民需求是我国农业产业的重要课题。转移因子(Transfer factor, TF)是一组由T淋巴细胞释放的没有抗原性的小分子多肽、核苷酸、氨基酸类物质组成的混合液,近年来作为增强免疫制剂在家禽生产领域的应用引起了越来越多人的关注。本试验对出壳后5日龄雏鸡灌服0.1mg、0.25mg及1.0mg剂量的鸡脾转移因子,对照组灌服生理盐水,至40日龄时取十二指肠、空肠、回肠及直肠组织,从肠道黏膜组织学结构、细胞因子、MUC2(黏蛋白基因2)及TLR2/4 (Toll样受体2/4)表达及肠道抗氧化功能等途径研究了鸡脾转移因子的作用机制。试验同时研究了鸡脾转移因子在生产中的应用,分析了其对鸡群增重、免疫器官发育和新城疫治疗效果的影响。结果如下：1.鸡脾转移因子对鸡肠黏膜组织学结构影响1.0mg剂量鸡脾转移因子可以显著增加40日龄试验鸡小肠的绒毛高度、肠绒毛高度／隐窝深度比值(V/C)(P<0.05)和杯状细胞密度(P<0.05)；但各剂量鸡脾转移因子对直肠杯状细胞数目的影响与对照组相比较差异不显著。结果表明鸡脾转移因子可改善鸡小肠的黏膜屏障功能。2.鸡脾转移因子对鸡肠道细胞因子的影响1.0mg剂量的鸡脾转移因子显著提高40日龄鸡十二指肠、空肠、回肠和直肠的IL-10含量(P<0.05)。0.25mmg剂量鸡脾转移因子显著提高试验组空肠、回肠、直肠IL-13含量(P<0.05)。O.1mg和0.25mg剂量的鸡脾转移因子显著降低40日龄鸡十二指肠的IL-1水平(P<0.05)。0.25mg和1.0mg剂量水平的鸡脾转移因子显著降低试验组十二指肠内TNF-α表达水平(P<0.05)。结果表明鸡脾转移因子可提高鸡肠道抗炎因子表达和抑制促炎因子分泌,改善肠道黏膜免疫屏障功能。3.鸡脾转移因子对鸡肠道MUC2、TLR2及TLR4 mRNA表达的影响灌服1.0mmg剂量的鸡脾转移因子可显著增加40日龄试验鸡十二指肠、回肠、直肠MUC2 mRNA水平(P<0.05)。灌服0.25mg和1.0mg剂量鸡脾转移因子均可以显著降低TLR2、TLR4基因在十二指肠(P<0.05)、空肠(P<0.05)、回肠(P<0.01；P<0.05)和直肠(P<0.01；P<0.05)的表达水平,且这种作用在一定程度上表现出剂量依赖性。4.鸡脾转移因子对鸡肠道抗氧化功能的影响灌服0.25mg鸡脾转移因子显著提高40日龄鸡十二指肠、空肠、直肠CAT活性(P<0.05)。0.25mg剂量组的十二指肠、空肠GSH-Px活力与对照组相比显著升高(P<0.05)。0.25mg和1.0mg剂量的鸡脾转移因子可显著降低十二指肠、空肠及回肠内MDA含量(P<0.05)。0.25mg剂量的鸡脾转移因子可显著性提高十二指肠、回肠、直肠的T-AOC(P<0.05)。结果表明鸡脾转移因子可提高鸡肠道组织的抗氧化功能。5.鸡脾转移因子对免疫器官发育及疾病抵抗能力的影响对5日龄雏鸡隔周灌服1.0mg剂量的鸡脾转移因子,40日龄试验组鸡体重显著高于对照组(P<0.05)。雏鸡5日龄起每日灌服0.25mg剂量的鸡脾转移因子可以显著提高14日龄和22日龄脾脏与胸腺重量,极显著提高新城疫免疫后的抗体效价(P<0.001)。6.鸡脾转移因子在养鸡生产中的应用大群试验结果显示鸡脾转移因子可有效提高规模化生产鸡群的成活率、个体体重,降低料肉比,提高养殖户的综合效益。对感染新城疫的生产鸡群应急处理显示,鸡脾转移因子可以显著提高鸡群的抵抗力,降低感染新城疫鸡群的死亡率,大大降低因疫病传播带来的损失。结论：鸡脾转移因子可以显著提高鸡小肠绒毛高度,增加鸡小肠V/C比值,诱导肠道干细胞增殖分化发育,增加杯状细胞密度,改善肠道黏膜机械屏障功能；改变肠道细胞因子的动态分布,促进肠道抗炎因子IL-10及IL-13的表达,降低促炎因子IL-1和TNF-α水平,改善肠道黏膜免疫屏障功能,进而提高鸡群抗病能力和生长性能。其作用机制可能与鸡脾转移因子提高肠道MUC2基因的表达水平、下调各个肠段TLR2及TLR4 mRNA水平和提高肠道抗氧化功能有关。更多还原

【Abstract】 Poultry product is an important source of Chinese meat consumption. Over the past 30 years, China consumer’s demand for poultry products was increased rapidly. Being restricted by domestic breeding and feeding technology level, poultry farming suffered from disease, nutritional imbalance, stress and so on, which results the poultry product market volatility sometimes. It became an important subject in our agriculture that how to prove poultry health and meet the demand of residents. Chicken spleen transfer factor (TF) has attracted the attention of more and more people in poultry production. It is a set of small molecular peptides, nucleotides and amino acid composition with no antigenicity, released by T lymphocytes. TF was widely used to enhance the immune preparations in poultry production. Therefore, we divided the 5th-day-old chicken to four groups fed with different dose of TF (0mg、0.1mg、0.25mg and 1mg) in this experiment. When chicken grew to the 40th day old, duodenum, jejunum, ileum and rectum were taken out and gut mucosal histology structure, anti-oxidation ability, immune cytokines, MUC2 and TLR2/4 (Toll-like receptor 2/4, TLR2/4) mRNA expression were studied to verify the mechanism of chicken spleen transfer factor. At the same time, the application of chicken spleen transfer factor in production was studied too. Chicken’s body weight and growth rate of immune organs were observed to explore their growth and intestine mucosal barrier, which provides a certain theoretical support for the application of TF to livestock and poultry production. The results of those studies are as follows:1. The role of TF on the chicken intestine mucosa histological structureThe villus height and the ratio of V/C (villus height to crypt depth, V/C) were significantly enhanced in the small intestine of 1.0 mg-TF-fed chicken (P<0.05). Goblet cell density was significantly increased in 1.0 mg-TF-fed 40th-day-old chicken’s duodenum, jejunum and ileum (P<0.05). Three doses of TF have no effect on goblet cell density in the rectum. These results indicate that the TF improved the mucosal mechanical barrier in the small intestine of chicken.2. The rolet of TF on the secretion of cytokines intestineThe IL-10 level in the duodenum, jejunum, ileum and rectum of 1.0 mg-TF-fed 40-day-old chicken were significantly enhanced (P<0.05), the IL-13 level in 0.25 mg-TF-fed 40-day-old chicken’s jejunum, ileum and rectum were also enhanced significantly (P<0.05).After the treatments with 0.1 mg and 0.25 mg TF, the IL-1 (interleukin-1) content of chicken duodenum was significantly declined at 40-day-old as compared to control group (P<0.05). Treatments with 0.25 mg and 1.0 mg TF declined significantly the content of TNF-α (Tumor necrosis factor-α) in duodenum compared to control group (P<0.05). These results indicated that the TF improved the mucosal immunologic barrier in the chicken intestine by increasing antiinflammatory cytokines and decreasing proinflammatory cytokines.3. The role of TF on the expression of MUC2、TLR2 and TLR4 mRNA in intestineThe intestine’s MUC2 mRNA expression level in 1.0 mg-TF-fed 40-day-old chicken’s duodenum, ileum and rectum were significantly enhanced (P<0.05). Treatments with 0.25 mg and 1.0 mg TF can significantly reduce TLR2 and TLR4 mRNA expressions of duodenum (P<0.05), jejunum (P<0.05), ileum (P<0.01, P<0.05) and rectum (P<0.01) in the 40-day-old chicken. This role showed dose-dependent feature.4. TF’s influence on chicken intestines antioxidant functionTreatment with 0.25 mg TF significantly improved the duodenum, jejunum and rectum CAT (catalase, CAT) activity (P<0.05). The intestinal GSH-Px (glutathione peroxidase, GSH-Px) activity in the 0.25mg-TF-fed 40-day-old chicken’s duodenum, jejunum was significantly increased (P<0.05) which compared with control group. Treatment with 0.25 mg and 1 mg TF can significantly reduce the MDA (Malondialdehyde, MDA) content of duodenum, jejunum and ileum (P<0.05). Treatment with 0.25 mg TF can significantly enhance T-AOC (the total antioxidant capacity, T-AOC) in duodenum, ileum and rectum (P<0.05).5. The effect of TF on the body weight, immune organs and disease resistanceThe body weight of 40-day-old group treated with 1.0 mg TF was significantly higher than control group (P<0.05). Treatment with 0.25 mg TF-fed can significantly increase the weights of spleen and thymus in the 14th day old and 22th day old chickens. Meanwhile, the antibody titer of Newcastle disease vaccine was greatly increased (P<0.001).6. TF application in poultry productionLarge group of test results showed that TF could effectively improve the survival rate and body weight of the chicken population in large-scale production, reduce feed to meat ratio and improve the efficiency of farmers. TF could also significantly improve the disease resistance of chickens; reduce the mortality of Newcastle disease chicken group. As a result, the loss due to the spread of the disease would be greatly reduced.Conclusion:The villus height, the ratio of V/C, the proliferation of intestinal stem cells and the goblet cell density could be significantly increased by TF to improve the mucosal mechanical barrier in the chicken intestine. TF could also significantly promote the expression of anti-inflammatory cytokines and inhibit the level of proinflammatory factor to improve the mucosal immunologic barrier in the chicken intestine, and resulted in to promote the growth, performance and disease resistance of chickens. Its effect mechanism was involved in up-regulation of MUC2 mRNA transcription level, down regulation of TLR2 and TLR4 mRNA transcription and increase of antioxidant function in the chicken intestines by TF.更多还原