在肉鸡饲养过程中，由病原体、非病原体等应激源引起的免疫应激普遍存在且不可避免。由于肉雏鸡肠黏膜屏障发育不健全，免疫应激易引起肠黏膜屏障受损、炎性细胞浸润，导致生长性能和健康状况下降。因此，使用营养调控方法缓解肉鸡生产中出现的免疫应激现象对提高生产水平具有重要意义。绿原酸（Chlorogenic acid，CGA）是一种天然缩酚酸，具有抗炎、抗氧化和抗菌等生物学功能，应用于饲料中可以缓解动物免疫应激，改善肠道健康。然而，对于CGA缓解免疫应激造成肉鸡肠黏膜损伤的研究仍相对缺乏。为了探讨CGA对于缓解肉鸡免疫应激和改善肠道健康状况的作用，本论文首先进行正常肉鸡饲养试验，研究不同添加水平CGA在肉鸡日粮中的应用效果，以确定CGA适宜的添加水平；在此基础上，利用腹腔注射脂多糖（Lipopolysaccharide，LPS）诱导免疫应激，以进一步探讨CGA对免疫应激肉鸡肠道健康的保护作用。全文共包括以下三个试验：

试验一旨在研究不同添加水平CGA对肉鸡生长性能、血清生化指标、肠道完整性、抗氧化能力和免疫功能的影响。选取1日龄雄性爱拔益加（AA）肉雏鸡240只，随机分为5组，每组6个重复，每个重复8只肉鸡。在玉米-豆粕型基础日粮中添加不同水平CGA（0、125、250、500和1000 mg/kg），试验为期14 d。结果表明，与对照组相比，500 mg/kg CGA添加水平提高了肉鸡平均体重（ABW）和平均日增重（ADG）（P<0.05），降低了料重比（F/G）（P<0.05）。与对照组相比，不同添加水平CGA均提高了血清葡萄糖（GLU）浓度（P<0.05），125 mg/kg CGA降低了血清总胆固醇（TC）水平（P<0.05），250 mg/kg CGA降低了血清甘油三酯（TG）水平（P<0.05）。与对照组相比，500 mg/kg CGA降低了血清二胺氧化酶（DAO）活性和D-乳酸（D-LA）水平（P<0.05），提高了十二指肠和空肠绒毛高度与隐窝深度比（VH/CD）（P<0.05）；提高了血清和十二指肠、空肠及回肠黏膜总抗氧化能力（T-AOC）（P<0.05），降低了血清和空肠黏膜丙二醛（MDA）含量（P<0.05）。CGA对GLU浓度、十二指肠VH/CD、血清还原型谷胱甘肽（GSH）水平的影响呈线性效应（P<0.05）。CGA对F/G、血清TC及TG水平、空肠黏膜谷胱甘肽过氧化物酶活性（GSH-Px）和过氧化氢酶活性（CAT）回肠黏膜GSH水平和血清免疫球蛋白M（IgM）含量的影响呈二次效应（P<0.05）。CGA对ABW、ADG、血清DAO活性和D-LA水平、血清T-AOC和GSH-Px活性、十二指肠黏膜超氧化物歧化酶（SOD）活性和血清免疫球蛋白A（IgA）含量的影响呈线性效应和二次效应（P<0.05）。

试验二旨在研究日粮添加CGA对免疫应激肉鸡免疫功能的保护作用。试验选取1日龄雄性AA肉雏鸡192只，随机分为4组，每组6个重复，每个重复8只肉鸡。试验为期21 d。试验处理如下：（1）对照组（CON）：正常肉鸡饲喂基础日粮；（2）免疫应激组（LPS）：LPS应激肉鸡饲喂基础日粮；（3）绿原酸组（CON-CGA）：正常肉鸡饲喂基础日粮添加500 mg/kg CGA的试验日粮；（4）免疫应激-绿原酸组（LPS-CGA）：免疫应激肉鸡饲喂基础日粮添加500 mg/kg CGA的试验日粮。于试验第16、18和20 d对肉鸡腹腔注射LPS溶液（LPS注射剂量为1 mg/kg体重）。结果表明，日粮添加CGA提高了免疫应激肉鸡十二指肠相对重量（P<0.05）；提高了免疫应激肉鸡血清白细胞介素-10（IL-10）水平（P<0.05），降低了免疫应激肉鸡空肠黏膜白细胞介素-6（IL-6）水平（P<0.05）、回肠黏膜肿瘤坏死因子-α（TNF-α）水平（P<0.05）以及空肠黏膜核因子κB p65（NF-κB p65）蛋白表达水平（P<0.05）。

试验三旨在研究日粮添加CGA对免疫应激肉鸡肠道屏障功能的保护作用。试验动物和试验设计同试验二。结果表明，日粮添加CGA有提高免疫应激肉鸡十二指肠VH和降低十二指肠CD的趋势（P>0.05）。日粮添加CGA降低了免疫应激肉鸡血清内毒素含量（P<0.05），并有降低免疫应激肉鸡血清D-LA含量的趋势（P>0.05）。日粮添加CGA降低了免疫应激肉鸡回肠黏膜闭合蛋白-3（CLDN-3）的mRNA相对表达量（P<0.05），并有提高空肠和回肠黏膜闭锁小带蛋白-1（ZO-1）mRNA相对表达量的趋势（P>0.05）。日粮添加CGA降低了免疫应激肉鸡回肠黏膜半胱天冬酶-9（Casp-9）活性（P<0.05） 以及空肠黏膜Casp-9活性的mRNA相对表达量（P<0.05），并有降低空肠和回肠黏膜半胱天冬酶-3（Casp-3）活性的趋势（P>0.05）。日粮添加CGA提高了免疫应激肉鸡空肠黏膜B淋巴细胞瘤2与B淋巴细胞瘤2相关X蛋白（BCL-2/BAX）的mRNA相对表达量比值（P<0.05），降低了免疫应激肉鸡回肠上皮细胞凋亡率（P<0.05）。

综上所述，可得出以下结论：

1.日粮添加CGA可提高肉鸡生长性能和肠道完整性，改善血清和肠黏膜抗氧化状态，增强机体免疫功能，且CGA在肉鸡饲料中的添加量以500 mg/kg为宜。

2.日粮添加CGA可通过抑制炎症因子的分泌和炎症信号通路的活化来缓解免疫应激肉鸡肠道炎症反应、提高免疫功能。

3.日粮添加CGA可降低免疫应激肉鸡肠黏膜通透性，提高肠道完整性和改善紧密连接功能，抑制小肠上皮细胞凋亡水平，缓解肠黏膜屏障损伤，从而提高肉鸡肠道健康水平。

Immulogical stress caused by pathogens, non-pathogens and other stressors is common and unavoidable during broiler production. Due to the immature intestinal mucosal barrier, immulogical stress can easily damage the intestinal mucosal barrier and induce inflammatory cell infiltration in broilers, resulting in compromised performance and inferior health status. Therefore, it is important to develop nutritional regulation measures to alleviate immulogical stress in broilers production in order to improve production efficiency. Chlorogenic acid (CGA), a natural phenolic acid, possesses a variety of biological functions, such as anti-inflammatory, antioxidant and antibacterial effects, which can be supplemented to animal feed to alleviate inflammatory response and improve intestinal health in animals. However, studies regarding the protective effects of CGA on immulogical stress induced intestinal mucosal damage in broilers are still lacking. In order to investigate the effects of CGA on alleviating immulogical stress and improving intestinal health of broiler chickens, a feeding trial was firstly conducted on normal broilers to investigate the application effects of different levels of CGA in order to select the optimum level of CGA in broiler diet. After that, an intraperitoneal injection of lipopolysaccharide (LPS) was used to induce immulogical stress and to further investigate the protective effect of CGA on intestinal health of immulogical stressed broilers. This paper includes the following three experiments:

Experiment 1 was conducted to investigate the effects of different supplemental levels of CGA on growth performance, serum biochemical indices, intestinal integrity, antioxidant capacity and immune function of broilers. A total of 240 one-day-old male Arbor Acres (AA) broiler chicks were randomly divided into 5 groups with 6 replicates of 8 chicks each. Different levels of CGA (0, 125, 250, 500 and 1000 mg/kg) were added to a corn-soybean meal basal diet. The experiment lasted for 14 days.The results showed that compared with the control group, 500 mg/kg CGA increased the average body weight (ABW) and average daily gain (ADG) (P<0.05), and decreased feed to gain ratio (F/G) (P<0.05). Compared with the control group, CGA supplementation, irrespective of its dosage, increased serum glucose (GLU) concentration (P<0.05), 125 mg/kg CGA decreased serum total cholesterol (TC) level, 250 mg/kg CGA decreased serum triglyceride (TG) level (P<0.05). Compared with the control group, 500 mg/kg CGA decreased serum diamine oxidase (DAO) activity and D-lactic acid (D-LA) level (P<0.05), increased the ratio of duodenal and jejunal villus height to crypt depth (VH/CD) (P<0.05), increased serum and total antioxidant capacity (T-AOC) in duodenal, jejunal and ileal mucosa (P<0.05) and decreased serum and jejunal mucosal malondialdehyde (MDA) level (P<0.05). The effect of CGA on serum GLU concentration, duodenal VH/CD, and serum reduced glutathione (GSH) level showed a linear effect (P<0.05). The effects of CGA on F/G, serum TC and TG level, jejunal mucosal glutathione peroxidase activity (GSH-Px) and catalase activity (CAT), ileal mucosal GSH level and serum immunoglobulin M (IgM) level showed a quadratic effect  (P<0.05). The effects of CGA on ABW, ADG, DAO activity, serum D-LA level, serum T-AOC and GSH-Px activities, duodenal mucosal superoxide dismutase (SOD) activity and serum immunoglobulin A (IgA) level showed a linear and quadratic effect (P<0.05).

Experiment 2 was conducted to study the protective effect of dietary CGA on immune function in immulogical stressed broilers. A total of 192 one-day-old male AA broilers were randomly divided into 4 groups with 6 replicates of 8 chicks each. The experiment lasted for 21 days. The experimental treatments were as follows: (1) Control group (CON): normal broilers were fed a basal diet; (2) Immulogical stress group (LPS): LPS-challenged broilers were fed a basal diet; (3) Chlorogenic acid group (CON-CGA): normal broilers were fed a basal diet supplemented with 500 mg/kg CGA; (4) Immulogical stress-chlorogenic acid group (LPS-CGA): LPS-challenged broilers were fed a basal diet supplemented with 500 mg/kg CGA. On the day 16th, 18th and 20th of the experiment, broilers were intraperitoneally injected with LPS solution (LPS injection dose was 1 mg/kg body weight). The results showed that the addition of CGA to the diet increased the relative weight of duodenum in immulogical stressed broilers (P<0.05). It also increased the level of serum interleukin-10 (IL-10) (P<0.05) and decreased the level of jejunal mucosal interleukin-6 (IL-6) (P<0.05), ileal mucosal tumor necrosis factor-α (TNF-α) (P<0.05), and nuclear factor-κB p65 (NF-κB p65) protein expression in immulogical stressed broilers (P<0.05).

Experiment 3 was designed to investigate the protective effects of dietary supplementation with CGA on intestinal barrier function in immulogical stressed broilers. The test animals and experimental design were the same as that of experiment 2. The results showed that dietary supplementation with CGA tended to increase duodenal VH and decrease duodenal CD in immulogical stressed broilers (P>0.05). The addition of CGA to the diet reduced the serum endotoxin level (P<0.05) and tended to reduce D-LA level (P>0.05) in immulogical stressed broilers. The addition of CGA to the diet reduced the relative expression level of ileal mucosal claudin-3 (CLDN-3) mRNA in immulogical stressed broilers (P<0.05) and tended to increase the relative expression level of jejunal and ileal mucosal zonula occluden-1 (ZO-1) mRNA (P>0.05). Dietary supplementation with CGA reduced the activity of ileal mucosal Caspase-9 (Casp-9) (P<0.05) and jejunal mucosal Casp-9 mRNA relative expression level (P<0.05) in immulogical stressed broiler chickens, and tended to reduce the activity of jejunal and ileal mucosal Caspase-3 (Casp-3) (P>0.05). The addition of CGA to the diet increased the ratio between mRNA expression level of B cell lymphocytoma-2 (BCL-2) (P<0.05) and B cell lymphocytoma-2 associated X protein (BCL-2/BAX) (P<0.05) in the jejunal mucosa of immulogical stressed broiler chickens, and decreased the ratio of apoptotic cells positive in the ileum of immulogical stressed broiler chickens (P<0.05).

It can be concluded as follows:

1.Dietary supplementation with different levels of CGA can improve growth performance and intestinal integrity, improve the antioxidant status of serum and intestinal mucosal, and improve immune function of broilers, with its optimal dosage being 500 mg/kg.

2.Dietary supplementation with CGA can alleviate the intestinal inflammatory response and improve immune function of immulogical stressed broilers by inhibiting the secretion of inflammatory factors and inactivating inflammatory signaling-related pathway.

3.Dietary supplementation with CGA can reduce intestinal mucosal permeability, improve intestinal integrity and tight junction function of immulogical stressed broilers, inhibit the level of small intestinal epithelial cell apoptosis, and alleviate intestinal mucosal barrier damage, resulting an improved intestinal health of broiler chickens.