柴毛毛1, 郭玉光1, 李阳源1, 彭宇1,2
, 王永华2 1. 广东溢多利生物科技股份有限公司, 广东 珠海 519060;
2. 华南理工大学食品科学与工程学院, 广东 广州 510640
收稿日期：2019-11-13；修回日期：2020-01-16；网络出版日期：2020-06-04
基金项目：珠海市引进创新创业团队项目(ZH01110405170028PWC)
*通信作者：彭宇, E-mail:pengyu@vtrbio.com.
摘要：[目的] 本研究旨在探讨博落回提取物(Macleaya cordata extract，MCE)替代促生长抗生素(Antibiotic growth promoters，AGPs)对黄羽肉鸡生长性能、盲肠微生物及紧密连接mRNA表达的影响。[方法] 试验选取体重相近、体型均匀、健康状况良好的1日龄温氏新黄鸡二号公鸡300只，随机分为5组，每组6个重复，每个重复10羽。分别饲喂基础日粮(NC)、抗生素日粮(ANT，基础日粮添加50 mg/kg那西肽和50 mg/kg金霉素)和试验日粮(基础日粮中添加200、400、800 mg/kg MCE)，试验期60 d。[结果] 日粮添加400 mg/kg MCE替代AGPs显著降低了料肉比(P < 0.05)，并显著增加了盲肠长度(P < 0.05)。日粮添加MCE显著提高了肉鸡盲肠食糜中Firmicutes细菌数量和Clostridium cluster XIVa数量(P < 0.05)；MCE替代AGPs显著降低了盲肠Escherichia coli数量(P < 0.05)。400 mg/kg和800 mg/kg MCE替代日粮中AGPs显著增加了肉鸡盲肠食糜中总短链脂肪酸、乙酸和丁酸含量(P < 0.05)；400 mg/kg MCE替代AGPs显著提高了盲肠中支链脂肪酸异丁酸和异戊酸的浓度(P < 0.05)。日粮添加MCE显著上调了肉鸡盲肠Claudin-1、JAM2、ZO-1的mRNA表达量(P < 0.05)，并降低了黏蛋白MUC2、MUC5ac和MUC13的表达量(P < 0.05)。[结论] MCE替代AGPs可通过提高盲肠有益菌数量和短链脂肪酸浓度，促进肠道发育，提升肠道屏障功能等途径，改善黄羽肉鸡生长性能，本研究中其最适添加量为400 mg/kg。
关键词：博落回提取物黄羽肉鸡盲肠微生物紧密连接
Effects of Macleaya cordata extracts instead of antibiotics on growth performance, caecum microbes and tight junction gene expression of yellow-feathered broilers
Maomao Chai1, Yuguang Guo1, Yangyuan Li1, Yu Peng1,2
, Yonghua Wang2 1. Guangdong VTR BIO-Tech Co., Ltd, Zhuhai 519060, Guangdong Province, China;
2. School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong Province, China
Received: 13 November 2019; Revised: 16 January 2020; Published online: 4 June 2020
*Corresponding author: Yu Peng, E-mail:pengyu@vtrbio.com.
Foundation item: Supported by the Project of Zhuhai Innovation and Entrepreneurship Team (ZH01110405170028PWC)
Abstract: [Objective] The aim of this study was to evaluate the effects of Macleaya cordata extracts (MCE) instead of antibiotics on growth performance, caecum microbes, short chain fatty acids (SCFAs) and tight junction mRNA expression in yellow-feathered broilers. [Methods] A total of 300 one-day-old Wenshi new yellow broilers No. 2 with similar body weight were randomly allotted to 5 groups, with 6 replicates and 10 broilers per replicate. Broilers in control group were fed a basal diet (NC) and broilers in antibiotic group (ANT), fed a basal diet with 50 mg/kg Nosiheptide and 50 mg/kg chlortetracycline, while those in the treatment groups were fed a basal diet supplemented with 200, 400 and 800 mg/kg MCE. The experiment was lasted for 60 days. [Results] The addition of 400 mg/kg MCE to replace antibiotic growth promoter in yellow-feathered broilers diet significantly reduced (P < 0.05) the feed conversion ratio, and significantly increased (P < 0.05) the length of the cecum. Dietary supplementation with MCE significantly increased (P < 0.05) the cecal Firmicutes and Clostridium cluster XIVa counts and significantly decreased (P < 0.05) the Escherichia coli counts. Supplemented with 400 and 800 mg/kg MCE to replace AGPs significantly increased (P < 0.05) the cecal total short-chain fatty acids, acetic acid and butyric acid, and 400 mg/kg MCE significantly increased the branched-chain fatty acids, including isobutyrate and isovalerate (P < 0.05). Supplementation with MCE in no AGPs diet significantly increased the expression of Claudin-1, JAM2 and ZO-1 (P < 0.05). And MCE instead of AGPs significantly increased the expression of JAM2 (P < 0.05). However, the addition of MCE to the diet significantly reduced the gene expression of MUC2, MUC5ac and MUC13 (P < 0.05). [Conclusion] The MCE replace dietary AGPs of yellow-feathered broilers improves the growth performance promotes caecum growth, and help to establish a stable and healthy intestinal barrier which through increasing the number of beneficial bacteria, SCFAs concentration and regulate the expression of tight junction proteins expression. The optimal addition amount of MCE under this experiment is 400 mg/kg.
Keywords: Macleaya cordata extractsyellow-feathered broilerscaecum microbestight junction
自2006年欧盟全面禁止在饲料中添加促生长类抗生素以来，世界各国纷纷出台了相应措施。我国农业农村部发布的第194号公告显示，将于2020年7月1日起全面停止生产除中药外的促生长药物添加剂。幼龄动物由于抗病机制不完善，日粮中常添加饲用抗生素来预防感染、促进生长，然而长期添加抗生素会造成动物免疫力下降、畜产品药物残留超标等不良后果。因此，寻找天然绿色的抗生素替代品，促进动物肠道健康成为动物营养的研究热点。
博落回是我国特有草本植物，其提取物(Macleaya cordata extract，MCE)的主要生物活性成分为异喹啉类生物碱，包括血根碱、白屈菜红碱、别隐品碱等[1]，具有抗菌、消炎、抗病毒、增强免疫力等功能。研究显示，MCE在畜禽及水生动物养殖中具有促进动物生长、降低腹泻率、增强机体免疫力等作用[2-3]。胡贵丽等[4]将MCE单独或与茶籽多糖混合替代AGPs可显著提高肉鸡日平均采食量。将MCE添加于肉鸡日粮中和饮水中可显著促进肉鸡生长后期增重[5]，显著降低坏死性肠炎的发生率，改善不使用抗生素饲养条件下肉鸡的生长性能[6]。也有研究表明，MCE具有调节仔猪空肠紧密连接蛋白mRNA表达，增强生长仔猪肠道屏障功能[7]。综上所述，MCE替代AGPs具有促进动物生长、维持动物肠道健康的功效，但其是否通过调控肠道微生物和肠道紧密连接mRNA表达的途径来发挥作用，目前尚不清楚。因此，本试验通过在日粮中添加MCE替代AGPs，研究其对黄羽肉鸡生长性能、盲肠微生物、短链脂肪酸和盲肠紧密连接mRNA表达的影响，旨在探讨MCE应用于肉鸡生产中替代AGPs的可能机制。
1 材料和方法 1.1 试验材料 本试验中所用博落回提取物由湖南美可达生物资源股份有限公司提供，商品名为美佑壮(血根碱含量1.5%)。那西肽(硫多肽含量4%)和金霉素(10%)均为市售产品。
1.2 试验设计与动物饲养管理 试验选取生长状况良好的1日龄温氏新黄鸡二号公鸡300只，随机分为5组，每组6个重复，每个重复10只。试验分为两个阶段：肉仔鸡阶段(1-21 d)和中大鸡阶段(22-60 d)，试验期60 d。分别饲喂不含抗生素的基础日粮(NC)、基础日粮添加50 mg/kg金霉素和50 mg/kg那西肽(ANT)，以及基础日粮中分别添加200、400、800 mg/kg的MCE。试验于2019年2-3月在广东溢多利生物科技股份有限公司试验农场进行。所有试验鸡采用4层立体网上饲养，自由采食和饮水。试验期间采取自然通风和光照，其他按照常规饲养和免疫流程进行。
基础日粮参照NRC (1994)并结合我国农业行业标准《黄羽肉鸡营养需求》(NY/T33-2004)进行配制，基础日粮组成及营养水平如表 1。
表 1. 基础日粮组成及营养水平(风干基础) Table 1. Composition and nutrient levels of basal diets (air-dry basis)
| Ingredients | Day 1-21 | Day 22-60 | Nutritional levels2 | Day 1-21 | Day 22-60 |
| Corn | 60.10 | 64.38 | Crude protein | 21.02 | 19.03 |
| Soy bean meal (CP 43%) | 30.00 | 27.00 | Metabolic energy (kcal/kg) | 2900 | 3000 |
| Corn gluten meal | 3.00 | 3.00 | Crude fat | 3.60 | 3.78 |
| Fish meal | 1.50 | Total Ca | 0.90 | 0.80 | |
| DL-Met | 0.14 | 0.14 | Total P | 0.65 | 0.60 |
| L-Lys | 0.11 | 0.15 | Available P | 0.42 | 0.37 |
| Soy-bean oil | 1.00 | 1.50 | Lys | 1.10 | 1.00 |
| Salt | 0.25 | 0.23 | Met | 0.48 | 0.45 |
| Mountain | 1.30 | 1.00 | Met+Cys | 0.83 | 0.77 |
| Calcium bicarbonate | 1.50 | 1.50 | |||
| Choline chloride | 0.10 | 0.10 | |||
| Premix1 | 1.00 | 1.00 | |||
| Total | 100 | 100 | |||
| 1: The premix provided the following per kg of diets: D-pantothenic acid 10.9 mg, nicotinic acid 30 mg, folic acid 0.95 mg, biotin 0.16 mg, VA 8000 IU, VD 2800 IU, VE 19 mg, VK 33.32 mg, VB1 1.7 mg, VB2 8.2 mg, VB6 2.78 mg, VB12 0.015 mg, Mn (as manganese sulfate) 82 mg, Zn (as zinc sulfate) 68 mg, Fe (as ferrous sulfate) 81 mg, Cu (as copper sulfate) 9 mg, I (as potassium iodide) 0.50 mg, Se (as sodium selenite) 0.27 mg. 2: Crude protein was a measured value, while the others were calculated values. | |||||
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1.3 生长性能与样品采集 试验第0和60天，对试验鸡进行称重、计算平均日增重(Average daily gain，ADG)、平均日采食量(Average daily feed intake，ADFI)、耗料增重比(Feed conversion ratio，FCR)。每组随机选取6只接近平均体重的试验鸡，通过前腔动脉放血处死、解剖、分离盲肠组织并分别称重和测量长度。于超净台采集盲肠食糜并保存于-80 ℃冰箱，用于提取盲肠食糜总DNA和短链脂肪酸测定(Short-chain fatty acids，SCFAs)。采集盲肠组织保存于-80 ℃冰箱，用于提取RNA。
1.4 检测指标
1.4.1 盲肠食糜总DNA提取及实时荧光定量PCR: 采用天根粪便基因组DNA试剂盒(天根生化科技北京有限公司)，提取盲肠食糜总DNA。采用特异性引物(表 2)，对Total bacteria、Firmicutes、Bacterodietes、Clostridium cluster IV group、Clostridium cluster XIVa group、Escherichia coli和Lactobacillus进行菌群定量分析。采用Chowdhury的方法[8]，挑取代表细菌单克隆的16S rRNA基因制备质粒，梯度稀释后建立标准曲线。采用SYBR (TaKaRa Biotechnology，中国)试剂盒，使用(Bio-Rad，CFX96 PCR System，美国)系统进行实时荧光定量PCR (Quantitative Real-time PCR，RT-qPCR)。
表 2. 细菌基因荧光定量PCR引物及其序列 Table 2. Primers used for bacterial counts quantitative real-time PCR
| Target organisms | Sequences (5′→3′) | References |
| Total bacteria | GTGSTGCAYGGYYGTCGTCA | [9] |
| ACGTCRTCCMCNCCTTCCTC | ||
| Firmicutes | GGAGYATGTGGTTTAATTCGAAGCA | [10] |
| AGCTGACGACAACCATGCAC | ||
| Clostridium cluster IV | GCACAAGCAGTGGAGT | [11] |
| CTTCCTCCGTTTTGTCAA | ||
| Clostridium cluster XIVa | CGGTACCTGACTAAGAAGC | [11] |
| GACGACAACCATGCACCACCTG | ||
| Lactobacillus | AGCAGTAGGGAATCTTCCA | [12] |
| CGTCCCGATTAACAGAGCTT | ||
| Bacterodietes | GGARCATGTGGTTTAATTCGATGAT | [10] |
| CTCTACGAGACTCAAGCTTGC | ||
| Escherichia coli | CATGCCGCGTGTATGAAGAA | [13] |
| GGTGTTCTTCCCGATATCTACA |
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1.4.2 盲肠食糜短链脂肪酸浓度测定: 取0.3 g肠道食糜于2 mL灭菌后的离心管中，加入1.2 mL双蒸水，混匀后3000×g离心10 min，取1 mL上清液，按5:1的比例加入25% (W/V)偏磷酸，-20 ℃保存。使用气相色谱(岛津，日本)检测分析各样品中短链脂肪酸浓度，气相色谱配备氢火焰离子检测器和毛细管柱(30 m×0.32 mm×0.25 μm，Agilent Technologies Inc.美国)。
1.4.3 盲肠组织RNA提取、逆转录及实时荧光定量PCR: 取约0.1 g空肠或盲肠组织，采用液氮研磨法对肠道组织进行匀浆，使用EASYspin Plus组织/细胞RNA提取试剂盒(北京艾德莱生物科技有限公司)。采用NanoDrop1000微量分光光度计测定RNA浓度和纯度，OD260/OD280值为2.0-2.2的样品于-20 ℃保存用于反转录反应，反转录采用试剂盒(TaKaRa，RR047A，日本)合成cDNA。实时荧光定量PCR以cDNA为模板，引物序列见表 3。反应体系为20 μL，SYBR? Premix Ex Taq 10 μL，cDNA模板2 μL，上下游引物各0.3 μL，7.4 μL灭菌水。使用CFX96 PCR System (Bio-Rad，美国)进行实时荧光定量PCR，具体程序如下：95 ℃ 30 s，95 ℃ 5 s，60 ℃ 30 s，40个循环。以GAPDH作为内参基因，每个样品重复3次。
表 3. 基因荧光定量PCR引物及其序列 Table 3. Primers used for gene expression Quantitative Real-time PCR
| Target organisms | Sequences (5′→3′) | References |
| Catenin | CGACAACTGCTCCCTCTTTGA | [14] |
| GCGTTGTGTCCACATCTTCCT | ||
| Claudin-1 | TGGCCACGTCATGGTATGG | [14] |
| AACGGGTGTGAAAGGGTCATAG | ||
| Occludin | GCTTGATGTGGAAGAGCTTGTTG | [14] |
| ACCTCTGCCATCTCTCCACA | ||
| JAM2 | AGCCTCAAATGGGATTGGATT | [14] |
| CATCAACTTGCATTCGCTTCA | ||
| JAM3 | CCGACGGCTGTTTGTGTTT | [14] |
| GGCGGTGCAAAGTTTTGG | ||
| ZO-1 | CCGCAGTCGTTCACGATCT | [14] |
| GGAGAATGTCTGGAATGGTCTGA | ||
| MUC2 | GCCTGCCCAGGAAATCAAG | [14] |
| CGACAAGTTTGCTGGCACAT | ||
| MUC5ac | TGTGGTTGCTATGAGAATGGA | [14] |
| TTGCCATGGTTTGTGCAT | ||
| MUC13 | GCATTCCTCAAGCAGAGGTG | [14] |
| CTCAGGCTGCCGTGATATTT | ||
| GAPDH | GGCACGCCATCACTATC | [15] |
| CCTGCATCTGCCCATTT |
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1.5 数据处理与分析 试验数据采用Excel 2010进行整理，SPSS 21.0进行单因素方差(one-way-ANOVA)分析，Graphpad Prism 8.0制图，以P < 0.05为差异显著性标准。结果最终以“平均值±标准误”呈现。
2 结果和分析 2.1 博落回提取物替代抗生素对黄羽肉鸡生长性能的影响 由表 4可知，与ANT相比，日粮添加400 mg/kg和800 mg/kg MCE提高了黄羽肉鸡末重，但差异不显著(P > 0.05)。与NC相比，日粮添加400 mg/kg MCE显著降低了(P < 0.05)黄羽肉鸡的ADFI和FCR。
表 4. 博落回提取物替代抗生素对黄羽肉鸡生长性能的影响 Table 4. Effects of Macleaya cordata extracts instead of antibiotics on growth performance of yellow-feathered broilers
| Items | NC | ANT | 200 mg/kg MCE | 400 mg/kg MCE | 800 mg/kg MCE |
| Initial weight/g | 34.05±1.01 | 34.26±0.91 | 34.21±1.00 | 34.14±1.05 | 34.22±0.80 |
| Final weight/g | 2196.80±63.55 | 2202.71±24.15 | 2180.00±46.36 | 2226.74±35.11 | 2240.31±72.55 |
| ADG/g | 36.04±0.55 | 36.14±0.21 | 35.76±1.09 | 36.54±0.54 | 36.77±0.85 |
| ADFI/g | 78.54±1.58b | 77.63±1.74ab | 75.29±2.85ab | 75.04±1.33a | 78.14±2.19b |
| FCR | 2.18±0.03c | 2.15±0.01bc | 2.11±0.02ab | 2.05±0.02a | 2.13±0.03bc |
| Values in the same row with different letters superscripts mean significant difference (P < 0.05). The same as below. | |||||
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2.2 博落回提取物替代抗生素对黄羽肉鸡盲肠微生物数量的影响 图 1结果显示，与NC和ANT相比，日粮添加MCE显著提高了盲肠食糜中Firmicutes细菌数量(P < 0.05)。与NC相比，日粮添加MCE均显著增加了盲肠Clostridium cluster XIVa细菌数量(P < 0.05)，有显著增加Lactobacillus数量的趋势(P=0.069)。与ANT相比，日粮中添加MCE均显著降低了(P < 0.05)盲肠Escherichia coli数量。
 |
| 图 1 博落回提取物替代抗生素对黄羽肉鸡盲肠微生物的影响 Figure 1 Effects of Macleaya cordata extracts instead of antibiotics on cecal microbes counts of yellow-feathered broilers. Value columns with different small letters mean significant difference (P < 0.05). The same as below. |
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2.3 博落回提取物替代抗生素对黄羽肉鸡盲肠食糜短链脂肪酸的影响 由表 5可知，与NC组相比，日粮中添加400 mg/kg和800 mg/kg MCE显著提高了盲肠食糜中总短链脂肪酸、乙酸、丁酸和戊酸浓度(P < 0.05)；而使用400 mg/kg和800 mg/kg MCE替代AGPs显著增加了(P < 0.05)黄鸡盲肠食糜中总短链脂肪酸、乙酸和丁酸浓度。此外，400 mg/kg MCE替代AGPs显著增加了支链脂肪酸浓度，包括异丁酸和异戊酸(P < 0.05)。
表 5. 博落回提取物替代抗生素对黄羽肉鸡盲肠短链脂肪酸的影响 Table 5. Effects of Macleaya cordata extracts instead of antibiotics on cecal SCFAs of yellow-feathered broilers
| Items/mmol | NC | ANT | 200 mg/kg MCE | 400 mg/kg MCE | 800 mg/kg MCE |
| Total SCFA | 116.58±6.55a | 120.84±17.28a | 139.74±15.91ab | 174.68±11.79bc | 188.40±9.79c |
| Acetic acid | 86.50±5.51a | 86.80±11.92a | 97.05±10.86ab | 121.89±8.68bc | 129.91±8.19c |
| Propionic acid | 8.19±1.58 | 9.08±1.40 | 9.88±0.94 | 11.27±1.27 | 12.52±0.80 |
| Butyric acid | 15.69±1.50a | 18.15±3.95a | 23.04±3.90ab | 28.73±3.37bc | 37.26±2.66c |
| Valeric acid | 2.41±0.31a | 3.46±0.44ab | 4.53±0.56bc | 5.57±0.62c | 4.66±0.30bc |
| BCFA | 3.79±0.94a | 3.35±0.25a | 5.23±0.60ab | 7.22±1.02b | 4.04±0.77a |
| Isobutyric acid | 1.66±0.40ab | 1.31±0.12a | 1.93±0.18ab | 2.59±0.41b | 1.56±0.33ab |
| Isovaleric acid | 2.13±0.57a | 2.04±0.16a | 3.30±0.44ab | 4.63±0.63b | 2.48±0.45a |
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2.4 博落回提取物替代抗生素对黄羽肉鸡盲肠长度和重量的影响 由表 6可知，与NC和ANT相比，日粮添加400 mg/kg MCE显著增加黄羽肉鸡盲肠的长度(P < 0.05)，但对盲肠重量无显著影响(P > 0.05)。
表 6. 博落回提取物替代抗生素对黄羽肉鸡盲肠长度和重量的影响 Table 6. Effects of Macleaya cordata extracts instead of antibiotics on caecum length and weight of yellow-feathered broilers
| Items | NC | ANT | 200 mg/kg MCE | 400 mg/kg MCE | 800 mg/kg MCE |
| Cecum length/cm | 14.92±0.55a | 14.60±0.53a | 16.25±0.48ab | 18.25±1.20b | 16.33±0.40ab |
| Cecum weight/g | 9.89±0.75 | 9.50±1.60 | 12.77±0.92 | 11.84±3.08 | 9.81±0.64 |
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2.5 博落回提取物替代抗生素对黄羽肉鸡盲肠组织紧密连接mRNA表达的影响 由图 2可知，与NC相比，不同添加量MCE均显著上调了(P < 0.05)盲肠紧密连接蛋白Claudin-1、JAM2和ZO-1的mRNA表达量(P < 0.05)；添加800 mg/kg的MCE显著上调了(P < 0.05)JAM3的mRNA表达量。800 mg/kg的MCE替代AGPs显著提高了(P < 0.05)盲肠组织JAM2和ZO-1的mRNA表达量。
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| 图 2 博落回提取物替代抗生素对黄羽肉鸡盲肠组织紧密连接mRNA表达的影响 Figure 2 Effects of Macleaya cordata extracts instead of antibiotics on caecum tight junction mRNA expression of yellow-feathered broilers. |
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2.6 博落回提取物替代抗生素对黄羽肉鸡盲肠组织黏蛋白mRNA表达的影响 由图 3可知，与NC组相比，不同添加量的MCE均显著降低了(P < 0.05)盲肠黏蛋白MUC2、MUC5ac和MUC13的mRNA表达量。
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| 图 3 博落回提取物替代抗生素对黄羽肉鸡盲肠组织黏蛋白mRNA表达的影响 Figure 3 Effects of Macleaya cordata extracts instead of antibiotics on caecum mucoprotein mRNA expression of yellow-feathered broilers. |
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3 讨论 3.1 博落回提取物替代抗生素对黄羽肉鸡生长性能的影响 肉鸡日粮中AGPs的停用会导致其发病率上升，生长性能下降，因此饲用抗生素的禁用将对肉鸡产业产生巨大影响。博落回提取物是一种具有抗炎杀菌、抗肿瘤功效的生物活性提取物，可用于替代AGPs促进动物生长和维护肠道健康，但其促进动物生长的作用受提取方法和添加剂量等因素的影响有所差异。胡贵丽等研究发现，日粮添加20 mg/kg MCE (含60%血根碱和20%白屈菜红碱)显著提高了黄羽肉鸡试验全阶段的ADFI[4]。黄海等研究也显示，添加40 mg/kg (含35%血根碱)的MCE于麻花肉鸡日粮中，显著增加了麻花鸡的ADG，并显著降低其FCR[5]。本研究中，400 mg/kg的MCE替代AGPs显著降低了黄羽肉鸡FCR。由此可知，MCE替代AGPs能维持黄羽肉鸡的生产性能，且MCE添加量不宜过高或过低，本试验结果建议添加量为400 mg/kg (血根碱含量1.5%)。
3.2 博落回提取物替代抗生素对黄羽肉鸡盲肠微生物和短链脂肪酸的影响 肠道微生物对动物生长和健康具有重要作用，长期低剂量AGPs在促进动物生长的同时也会抑制肠道有益菌生长，增加大肠杆菌等有害菌的数量[6]。与AGPs的广谱抗菌特性不同，MCE的主要成分血根碱可通过诱导细菌细胞壁溶解，干扰菌体蛋白合成，抑制细菌繁殖[7]。本研究中，MCE替代AGPs显著降低了盲肠食糜中Escherichia coli数量，这与早期研究的结果一致。另一方面，采用MCE替代AGPs增加了盲肠食糜中的Lactobacillus数量，此结果与博落回提取物显著增加黄羽肉鸡空肠食糜中大肠杆菌数量的结果相似[16]。乳酸杆菌是肉鸡肠道内的主要有益菌，对维持肠道低pH值、竞争性抑制病原菌增殖等具有重要作用[17]。由此可知采用MCE替代AGPs可通过促进肉鸡肠道内Lactobacillus生长、抑制肠道有害菌增殖维护其肠道健康。Clostridium cluster XIVa是动物肠道中最主要的产丁酸菌，日粮中长期添加抗生素会抑制肠道Clostridium cluster XIVa增殖，减少肠道内丁酸生成[18]。本研究中，基础日粮添加400 mg/kg和800 mg/kg MCE显著增加了盲肠Clostridium cluster XIVa的数量，并增加了盲肠食糜中的总短链脂肪酸、乙酸和丁酸浓度。盲肠是禽类消化道生成SCFAs的主要场所，其中乙酸和丁酸占其总量的70%以上。乙酸作为脂肪生成和糖异生的基质，参与上皮细胞能量代谢[19]，而丁酸则为肠上皮细胞提供能量，参与肠细胞的分化和增殖[20]。肠道中SCFAs浓度的增加将促进肠道上皮细胞代谢，调节肠细胞更新，增强肠道粘膜屏障功能[21]。本研究在肉鸡日粮中添加400 mg/kg MCE增加盲肠长度的结果也表明梭菌XIVa和丁酸浓度的增加促进了肉鸡盲肠的生长和发育。因此，肉鸡日粮中添加MCE替代AGPs可通过促进肠道中有益菌增殖，增加食糜中SCFAs，促进肉鸡肠道生长和发育等方式维护动物健康，提高生产性能。
3.3 博落回提取物替代抗生素对黄羽肉鸡盲肠组织紧密连接和黏蛋白mRNA表达的影响 完整的肠道结构对于维持肠道健康具有重要作用，紧密连接蛋白是肠道屏障的重要组成部分，主要介导肠道内物质交换、能量代谢过程。研究表明，肠道组织中Claudin-1、Occludin和ZO-1表达量降低会导致肠道通透性增加，肠屏障功能失效[22-23]，因此肠道炎症通常伴随紧密连接蛋白的减少和损伤。博落回提取物具有温和的抗炎和免疫调节特性，能提升动物肠道免疫机能，维持肠道屏障功能[24]。Liu等在仔猪日粮中添加MCE可显著上调其空肠组织Claudin-1、Occludin和ZO-1的表达量[25]。本研究也显示，MCE可显著上调盲肠Catenin、Claudin-1、ZO-1的表达量，增强黄羽肉鸡肠道屏障功能。JAM2属于粘附分子家族，可调节蛋白的形成和胞间分布[26]。研究指出，JAM2通过特殊的蛋白间互作结构域(PDZ结构域)与ZO-1等TJ蛋白结合，增加TJ蛋白的数量，促进肠道屏障生成[27]。本研究发现，MCE在上调JAM2 mRNA表达的同时也促进了Claudin-1、Occludin和ZO-1等紧密连接蛋白的mRNA表达，这暗示着MCE可能通过上调JAM2的表达来促进紧密连接蛋白生成，进而增强肠道屏障功能。
杯状细胞分泌的黏蛋白是一种覆盖在肠上皮细胞表面的大分子糖蛋白，能防止外源致病菌入侵，保护肠上皮细胞。在鸡肠道组织中的黏蛋白中MUC2和MUC5ac属于分泌型黏蛋白，MUC13为跨膜黏蛋白。MUC2是肠道黏液屏障的重要组成，可作为判断肠道黏液屏障完整性的标记基因[28]。Forder等利用艾美耳球虫和产气荚膜梭菌双重感染显著降低了肉鸡空肠MUC2 mRNA水平表达量[29]，而van Klinken等利用产气荚膜梭菌感染肉鸡则导致回肠组织MUC2 mRNA水平表达显著升高[30]。本研究中，基础日粮添加抗生素和MCE均显著降低了黄羽肉鸡盲肠MUC2 mRNA表达量的结果暗示，肉鸡饲喂无抗日粮时可能引起肠道感染，导致MUC2 mRNA表达量上升，但此结果还需进一步研究验证。先前的研究显示，MUC5ac和MUC13在健康完整的肠道中表达量较低，但当肠道黏液屏障受损或发生炎症时，肠组织中MUC5ac和MUC13 mRNA水平表达量显著升高[31-32]。本研究中添加MCE下调盲肠组织MUC5ac和MUC13 mRNA表达量的结果表明MCE可减少肠道损伤或炎症。由此可知，日粮中添加MCE可通过上调盲肠组织紧密连接蛋白的mRNA表达，减少肠受损黏蛋白mRNA表达，增强肉鸡肠道屏障功能，改善肉鸡生长性能。
4 结论 黄羽肉鸡日粮中添加MCE替代抗生素显著改善了肉鸡FCR，增加了盲肠长度，促进有益菌增殖，抑制有害菌生长，增加短链脂肪酸生成，上调紧密连接蛋白mRNA表达，改善肠道屏障功能。本试验条件下MCE的最适添加量为400 mg/kg。
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