动物营养学报    2021, Vol. 33 Issue (7): 3790-3798    PDF    
大蒜粉对肉鸡生长性能、抗氧化能力、免疫力和肠道形态的影响
余洋1 , 沈媛媛1 *, 杨彩梅2 , 刘金松2 , 曾新福2 , 冯杰3     
1. 浙江农林大学动物科技学院·动物医学院, 浙江省畜禽绿色生态健康养殖应用技术研究重点实验室, 动物健康互联网检测技术浙江省工程实验室, 临安 311300;
2. 浙江万方生物科技有限公司, 安吉 313300;
3. 浙江大学动物科学学院, 杭州 310058
摘要: 本试验旨在研究大蒜粉对肉鸡生长性能、抗氧化能力、免疫力和肠道形态的影响。选取480羽体重相近、健康状况良好的1日龄白羽肉鸡,随机分成4组,每组8个重复,每个重复15羽,进行为期42 d的饲养试验。对照组饲喂基础饲粮,抗生素组饲喂基础饲粮+75 mg/kg金霉素,2个大蒜粉组分别饲喂基础饲粮+1 000 mg/kg大蒜粉(1 000 mg/kg大蒜粉组)、基础饲粮+2 000 mg/kg大蒜粉(2 000 mg/kg大蒜粉组)。结果表明:1)与对照组相比,饲粮中添加1 000 mg/kg大蒜粉可显著提高1~42日龄肉鸡的平均日增重(P < 0.05),添加1 000和2 000 mg/kg大蒜粉可显著降低1~21日龄肉鸡的料重比(P < 0.05);2)与对照组和抗生素组比,饲粮中添加1 000和2 000 mg/kg大蒜粉均可显著提高肉鸡血清免疫球蛋白A(IgA)、免疫球蛋白Y(IgY)、免疫球蛋白M(IgM)含量(P < 0.05),显著降低血清肿瘤坏死因子-α(TNF-α)含量(P < 0.05),且大蒜粉组血清白细胞介素-1β(IL-1β)含量显著低于抗生素组(P < 0.05);3)2 000 mg/kg大蒜粉组血清谷胱甘肽过氧化物酶(GSH-Px)活性显著高于对照组(P < 0.05);4)与对照组相比,饲粮中添加1 000 mg/kg大蒜粉可显著提高肉鸡21日龄绒毛高度,添加1 000和2 000 mg/kg大蒜粉均可显著降低42日龄肉鸡隐窝深度(P < 0.05),并可显著提高绒毛高度/隐窝深度(V/C)(P < 0.05);5)与对照组相比,饲粮中添加1 000和2 000 mg/kg大蒜粉均可显著降低回肠黏膜Toll样受体2(TLR2)的相对表达量(P < 0.05)。综上可知,饲粮中添加大蒜粉能促进肉鸡生长,调节机体免疫力,促进肠道发育,减少炎症反应。本试验中,肉鸡饲粮中大蒜粉的最佳添加量为1 000 mg/kg。
关键词: 大蒜    肉鸡    免疫力    抗氧化能力    肠道形态    基因表达    
Effects of Garlic Powder on Growth Performance, Antioxidant Ability, Immunity and Intestinal Morphology of Broilers
YU Yang1 , SHEN Yuanyuan1 *, YANG Caimei2 , LIU Jinsong2 , ZENG Xinfu2 , FENG Jie3     
1. Key Laboratory of Applied Technology on Green-Eco Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health and Internet Technology, College of Animal Sci-Technology and Animal Medicine, Zhejiang Agriculture and Forestry University, Lin'an 311300, China;
2. Zhejiang Wanfang Biotechnology Co., Ltd., Anji 313300, China;
3. College of Animal Science and Technology, Zhejiang University, Hangzhou 310058, China
Abstract: This experiment was conducted to study the effects of garlic powder on the growth performance, antioxidant ability, immunity and intestinal morphology of broilers. A total of 480 healthy 1-day-old white-feathered broilers with the similar body weight were randomly divided into 4 groups with 8 replicates per group and 15 birds per replicate, and a 42-day feeding trial was conducted. Broilers in control group were fed a basal diet, those in antibiotic group were fed the basal diet+75 mg/kg chlortetracycline, those in 1 000 mg/kg garlic powder group were fed the basal diet+1 000 mg/kg garlic powder, and those in 2 000 mg/kg garlic powder group were fed the basal diet+2 000 mg/kg garlic powder. The results showed as follows: 1) compared with the control group, the average daily gain of broilers at 1 to 42 days of age was significantly increased by adding 1 000 mg/kg garlic powder (P < 0.05), and the feed to gain ratio of broilers at 1 to 21 days of age was significantly decreased by adding 1 000 and 2 000 mg/kg garlic powder (P < 0.05). 2) Compared with the control group and the antibiotic group, adding 1 000 and 2 000 mg/kg garlic powder could significantly increase the contents of immunoglobulin A (IgA), immunoglobulin Y (IgY) and immunoglobulin M (IgM) in serum (P < 0.05), and significantly decrease serum tumor necrosis factor-α (TNF-α) content (P < 0.05). The serum interleukin-1β (IL-1β) content in the two garlic powder groups was significantly decreased compared with the antibiotic group (P < 0.05). 3) The serum glutathione peroxidase (GSH-Px) activity in the 2 000 mg/kg garlic powder group was significantly higher than that in the control group (P < 0.05). 4) Compared with the control group, adding 1 000 mg/kg garlic powder could significantly increase the villus height of 21-day-old broilers, and adding 1 000 and 2 000 mg/kg garlic powder could significantly decrease the crypt depth and significantly increase the ratio of villus height to crypt depth (V/C) of 42-day-old broilers (P < 0.05). 5) Compared with the control group, adding 1 000 and 2 000 mg/kg garlic powder could significantly decrease the relative expression level of Toll-like receptor 2 (TLR2) in ileum mucosa (P < 0.05). These results indicate that adding garlic powder in the diet can promote the growth, regulate the immunity, promote the intestinal development, and reduce the inflammatory reaction of broilers. The best addition of garlic powder in the broilers' diet is 1 000 mg/kg in this experiment.
Key words: garlic    broilers    immunity    antioxidant ability    intestinal morphology    gene expression    

大蒜为百合科葱属植物,含有糖类、蛋白质、脂类、酶类,同时还富含维生素和微量矿物元素[1]。研究表明大蒜素有“天然抗生素”之称,其具有广泛的抗菌作用,可通过竞争性抑制菌体巯基酶、损伤菌体膜系统、抑制菌体生物被膜、产生多靶点抑制效应、影响核酸及蛋白质合成等方式达到抑菌效果[2]。大蒜还可间接通过抑制病毒合成而发挥抗病毒作用,其所抗病毒包括腺病毒、冠状病毒、甲型流感病毒等[3]。此外,大蒜还具有抗糖化、抗氧化及抗癌作用[4-5]。随着大蒜药理、生化、临床等方面研究的深入,其生物功效(抗菌、消炎、杀虫、抗氧化等作用)也逐步引入畜牧研究,大蒜作为一种天然的食、药两用原材料,有望发展为绿色、健康、高效的饲料添加剂。研究表明,饲粮中添加大蒜可提高断奶仔猪的生长性能,改善健康状况[6]。Shojai等[7]发现,大蒜对鸡胚传染性支气管炎病毒有抑制作用,添加大蒜可提高肉鸡先天免疫力,抑制肠道致病菌增殖[8-9]。大蒜应用于蛋鸡可提高其产蛋性能、蛋品质[10]。目前大蒜在肉鸡上的应用研究较少,本研究旨在探究大蒜粉对肉鸡生长性能、抗氧化能力、免疫力和肠道形态的影响,以期为大蒜在畜禽养殖中的应用提供理论基础。

1 材料与方法 1.1 试验材料

试验用金霉素和大蒜粉均为市购,大蒜粉中大蒜总糖含量≥40%、大蒜蛋白含量≥16%。

1.2 试验设计

试验采用单因子完全随机化设计,选取体重相近、健康状况良好的1日龄白羽肉鸡480羽(公母各占1/2),随机分为4组,每组8个重复,每个重复15羽,进行为期42 d的饲养试验。对照组饲喂基础饲粮,抗生素组饲喂基础饲粮+75 mg/kg金霉素,2个大蒜粉组分别饲喂基础饲粮+1 000 mg/kg大蒜粉(1 000 mg/kg大蒜粉组)、基础饲粮+2 000 mg/kg大蒜粉(2 000 mg/kg大蒜粉组)。基础饲粮参照NRC(1994)肉鸡营养标准配制,其组成及营养水平见表 1

表 1 基础饲粮组成及营养水平(风干基础) Table 1 Composition and nutrient levels of basal diets (air-dry basis) 
1.3 饲养管理

饲养试验期间480羽肉鸡饲养于同一鸡舍,每个重复1笼,采用普通平养模式,按常规方法定期进行免疫接种及疾病预防、消毒。

1.4 样品采集

42日龄时每组取8只鸡,颈静脉采血5~6 mL,室温静置凝血后放入高速冷冻离心机(Heal Force-Neofuge 13R,上海智城分析仪器制造有限公司),以3 000 r/min离心15 min,取血清于500 μL离心管中,-80 ℃保存待测。采血后将试验鸡屠宰,用无菌载玻片刮取回肠黏膜,迅速放入2 mL无RNA酶冻存管中,于-80 ℃保存待测;另用无菌组织剪剪取一段0.5 cm左右的回肠中段,用0.9%生理盐水冲洗干净后放入4%甲醛溶液中固定24 h以上。

1.5 指标测定及方法 1.5.1 生长性能

空腹称量1、21、42日龄体重,试验期间每天记录每组每个重复的采食量、死淘率,计算1~21日龄、22~42日龄以及1~42日龄的平均日采食量(ADFI)、平均日增重(ADG)和料重比(F/G)。

1.5.2 肠道形态

取出固定好的回肠组织,采用由低到高梯度浓度的酒精作为脱水剂,将组织脱水后置于二甲苯(透明剂)中透明,然后经石蜡包埋处理后切成4 μm厚的薄片,置于载玻片上制作切片,常规苏木精-伊红(HE)染色,最后中性树脂封片。采用光学显微镜(ae-2000,Nikon,日本)观察切片,记录绒毛高度、隐窝深度,并计算绒毛高度/隐窝深度(V/C)。

1.5.3 血清和回肠黏膜抗氧化指标

取血清及回肠黏膜,按试剂盒说明测定丙二醛(MDA)含量以及谷胱甘肽过氧化物酶(GSH-Px)、总超氧化物歧化酶(T-SOD)活性,测定所用仪器主要有恒温水浴锅(HH-2,方科仪器有限公司)、多功能酶标仪(Multiskan FC,Thermo Fisher,美国),所用试剂盒均购于南京建成生物工程研究所。

1.5.4 血清免疫指标

按照酶联免疫吸附测定(ELISA)试剂盒说明测定血清中免疫球蛋白A(IgA)、免疫球蛋白Y(IgY)、免疫球蛋白M(IgM)及炎症相关因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)含量,测定所用仪器主要有生化培养箱(SPX-105,宁波海曙赛福光照培养箱)、多功能酶标仪(Multiskan FC,Thermo Fisher,美国),所用ELISA试剂盒均购于南京建成生物工程研究所。

1.5.5 肠道炎症相关基因表达

取回肠黏膜,匀浆后提取RNA,再利用普通PCR仪(TC-96,BioRad,美国)反转录成cDNA,以β-肌动蛋白(β-actin)为内参基因,采用实时定量PCR仪(CFX96,BioRad,美国)测定Toll样受体2(TLR2)、髓样分化因子88(MYD88)、TNF-αIL-1β的mRNA相对表达量。参照文献设计TLR2[11]MYD88[12]TNF-α[13]IL-1β[14]引物,并委托擎科生物科技有限公司合成,引物序列见表 2。目的基因mRNA相对表达量的计算采用2-ΔΔCt法,反转录和实时定量PCR所用试剂盒均购于TaKaRa。

表 2 实时定量PCR引物 Table 2 RT-qPCR primers
1.6 数据统计与分析

试验数据经Excel 2013整理后采用SPSS 26.0软件进行单因素方差分析(one-way ANOVA),并采用Duncan氏法进行多重比较。P<0.05作为差异显著性判断标准,结果以平均值±标准差(mean±SD)表示。

2 结果 2.1 大蒜粉对肉鸡生长性能的影响

表 3可以看出,与对照组相比,添加大蒜粉显著降低了1~21日龄肉鸡的料重比(P < 0.05),且2 000 mg/kg大蒜粉组1~21日龄肉鸡的料重比显著低于抗生素组(P < 0.05);与对照组相比,添加1 000 mg/kg大蒜粉可显著提高1~42日龄肉鸡的平均日增重(P < 0.05)。

表 3 大蒜粉对肉鸡生长性能的影响 Table 3 Effects of garlic powder on growth performance of broilers
2.2 大蒜粉对肉鸡肠道形态的影响

表 4可以看出,21日龄时,大蒜粉组与抗生素组肉鸡绒毛高度较对照组显著提高(P < 0.05),且抗生素组绒毛高度显著高于1 000 mg/kg大蒜粉组(P < 0.05);2 000 mg/kg大蒜粉组隐窝深度显著高于对照组和抗生素组(P < 0.05),而1 000 mg/kg大蒜粉组隐窝深度较对照组和抗生素组显著降低(P < 0.05),且1 000 mg/kg大蒜粉组V/C较对照组和抗生素组显著升高(P < 0.05)。42日龄时,大蒜粉组隐窝深度较对照组和抗生素组显著降低(P < 0.05),V/C较对照组显著提高(P < 0.05),但与抗生素组无显著差异(P>0.05)。

表 4 大蒜粉对肉鸡肠道形态的影响 Table 4 Effects of garlic powder on intestinal morphology of broilers
2.3 大蒜粉对肉鸡血清和回肠黏膜抗氧化指标的影响

表 5可以看出,2 000 mg/kg大蒜粉组血清GSH-Px活性显著高于对照组(P < 0.05)。各组间血清MDA含量、T-SOD活性差异不显著(P>0.05)。回肠黏膜中,各组间MDA含量及GSH-Px、T-SOD活性差异均不显著(P>0.05)。

表 5 大蒜粉对肉鸡血清和回肠黏膜抗氧化指标的影响 Table 5 Effects of garlic powder on antioxidant indexes in serum and ileum mucosa of broilers
2.4 大蒜粉对肉鸡血清免疫指标的影响

表 6可以看出,大蒜粉组血清IgA、IgY、IgM含量较对照组和抗生素组显著提高(P < 0.05),血清TNF-α含量则较对照组和抗生素组显著降低(P < 0.05)。1 000 mg/kg大蒜粉组血清IL-1β含量显著低于抗生素组(P < 0.05),2 000 mg/kg大蒜粉组血清IL-1β含量显著低于对照组和抗生素组(P < 0.05)。大蒜粉组血清IL-6含量显著低于抗生素组(P < 0.05)。

表 6 大蒜粉对肉鸡血清免疫指标的影响 Table 6 Effects of garlic powder on serum immune indexes of broilers
2.5 大蒜粉对肉鸡肠道炎症相关基因表达的影响

表 7可以看出,大蒜粉组回肠黏膜TLR2相对表达量较对照组显著降低(P < 0.05),但与抗生素相比则无显著变化(P>0.05);抗生素组回肠黏膜MYD88相对表达量显著低于对照组和大蒜粉组(P < 0.05);回肠黏膜IL-1β相对表达量各组间无显著差异(P>0.05)。

表 7 大蒜粉对肉鸡肠道炎症相关基因表达的影响 Table 7 Effects of garlic powder on expression of intestinal inflammation-related genes of broilers
3 讨论 3.1 大蒜粉对肉鸡生长性能的影响

研究显示,饲粮中添加大蒜多糖可提高断奶仔猪的平均日增重[15],添加大蒜叶粉可改善肉鸡生长性能[16]。但目前关于大蒜粉对肉鸡生长性能影响的研究很少,本试验结果表明添加1 000 mg/kg大蒜粉可显著提高1~42日龄肉鸡的平均日增重,降低1~21日龄肉鸡的料重比,说明大蒜作为饲料添加剂可提高肉鸡的生长性能。

3.2 大蒜粉对肉鸡肠道形态的影响

肠道是营养物质消化吸收的最终场所,绒毛高度及隐窝深度影响机体对营养物质的吸收,绒毛高度增加,肠道吸收面积及对营养物质的吸收率也增加,隐窝深度越浅,分泌酶的细胞成熟度越高,分泌功能越强,V/C增加说明肠道消化吸收能力增强[17-18]。本试验中,大蒜粉组肉鸡21日龄回肠绒毛高度显著高于对照组,42日龄时V/C优于对照组,这与Peinado等[9]在肉鸡饲粮中添加大蒜衍生物所得试验结果基本一致,说明添加大蒜粉有利于肉鸡肠道组织形态发育和成熟,有利于营养物质的消化吸收,促进肉鸡生长。

3.3 大蒜粉对肉鸡抗氧化能力的影响

肠道作为机体防御体系的第1道屏障,其健康是保障动物生长的重要基础。氧化应激极易触发炎症反应,诱发肠道健康问题,且氧化应激问题在畜牧养殖中非常普遍,相关经济损失巨大[19]。大蒜中的黄酮类物质、硒元素、多酚及大蒜素等可通过清除活性氧,增强细胞中超氧化物歧化酶、过氧化氢酶和GSH-Px活性,并增加细胞中谷胱甘肽的含量,进而发挥抗氧化作用[20];此外,大蒜中的烯丙基半胱氨酸可通过防止细胞中谷胱甘肽的消耗、抑制过氧化物的表达来抵抗氧化应激损伤[21]。本试验结果表明,饲粮中添加2 000 mg/kg大蒜粉可显著提高肉鸡血清中GSH-Px活性,说明大蒜粉可提高肉鸡血清抗氧化能力,虽然添加大蒜粉对肉鸡回肠黏膜中抗氧化酶活性无显著影响(可能与肉鸡处于非应激状态有关),但结合血清抗氧化指标和肠道形态结果,可知大蒜粉对解决氧化应激引起的肠道健康问题具有一定潜力,但具体作用机理仍需进一步探究。

3.4 大蒜粉对肉鸡免疫力的影响

血清免疫球蛋白含量可衡量动物免疫机能状况,IgA在病原感染时对维持机体功能性免疫应答至关重要,IgM、IgY是机体受抗原诱导产生的抗体,可杀菌、抗病毒,有益于机体健康。血清白细胞介素含量可反映机体炎症状况,同时体现机体免疫力[22]。大蒜中的大蒜素是主要的免疫功效成分。研究显示,大蒜油对大鼠的辅助型T细胞(Th1/Th2)有双重影响,高剂量时,可触发Th2型反应;低剂量时,Th1型反应增强[23]。Keiss等[24]的研究显示,大蒜可抑制脂多糖(LPS)诱导的炎症反应,降低血清中细胞因子IL-1β及TNF-α含量,抑制核因子-κB(NF-κB)的活性。本试验结果表明,饲粮中添加大蒜粉可显著提高肉鸡血清IgA、IgY、IgM含量,显著降低血清促炎因子TNF-α的含量,这与Keiss等[24]的结果研究一致,说明大蒜可调节机体免疫力,减轻炎症反应。

TLR2可识别各种微生物成分(各种病原体的脂蛋白、脂肽,来自革兰氏阳性菌的肽聚糖和脂蛋白酸),引发信号转导并导致炎症介质的释放[25]TLR2表达下调表明炎症反应降低,本试验中,相比对照组和抗生素组,饲粮中添加大蒜粉显著降低了肉鸡回肠黏膜中TLR2的相对表达量,与Simon等[26]的研究结果基本一致。这在血清免疫指标的基础上进一步说明大蒜粉可提高机体免疫力,有效缓解机体炎症反应,并可能通过提高免疫力对肉鸡肠道、血清抗氧化能力和平均日增重产生积极影响。

4 结论

饲粮中添加1 000 mg/kg大蒜粉可提高肉鸡的生长性能,提升免疫力,缓解炎症反应与氧化应激,并有助于改善肠道形态。

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