CN113273650A - 一种改善肉鸡肠道健康的植物添加剂及应用效果评估 - Google Patents
一种改善肉鸡肠道健康的植物添加剂及应用效果评估 Download PDFInfo
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Abstract
本发明公开了一种可改善肉鸡肠道健康的植物饲料添加剂的配置及应用效果。为改善鸡的肠道健康,本饲料以五加科植物A、毛茛科植物B、黄杨科植物C以一定比例混合均匀,以2%的比例添加于肉鸡饲料中。结果表明,日粮添加2%中药后,湘黄鸡空肠绒毛高度和基线长度高于对照组(P<0.05),回肠绒毛高度及绒毛高度与隐窝深度的比值于对照组(P<0.05)。回肠粘膜白介素IL1β的mRNA表达量以中药组低于对照组(P<0.05),B淋巴细胞瘤‑2基因(抑癌基因BCL2)的mRNA表达量中药组高于对照组(P<0.01)。中药组空肠粘膜肿瘤坏死因子TNFα的mRNA表达量低于对照组(P<0.05)。采食含有中药复方的饲料后,湘黄鸡脾脏的TNFα的mRNA表达量低于对照组(P<0.05),而BCL2的mRNA表达量高于对照组(P<0.05)。湘黄鸡肠道健康因该植物添加剂的添加获得明显改。
Description
技术领域
本发明涉及一种可改善鸡肠道健康的植物饲料添加剂生产领域,特别是通过中药组合改善鸡肠道健康的生物饲料生产,属于饲料添加剂技术领域。
背景技术
近年来,随着人民生活水平的提高,对绿色肉品的需求量也随之增大,大量化学添加剂及抗生素的使用极大地影响了家禽业的健康发展。因此,用健康无污染的传统中药来防治鸡群疾病,以及改善鸡群肠道健康和增加免疫力就显得尤为重要。肠道中大量微生物对其营养、免疫、疾病防治以及生理功能发挥具有重要作用,影响鸡的生长和健康。肠道是鸡最大的免疫器官,鸡的肠道黏膜与皮肤、呼吸道黏膜等共同组成了机体抵抗外界异物(微生物、灰尘等)入侵的第一道防线,更是有60—70%的免疫细胞都位于肠道内,所以肠道对机的免疫力与抗病力都是举足轻重的,有人说:禽病防控,要从“肠”计议。另外,鸡体80%以上的毒素、代谢物要靠肠道排出体外。肠道不仅是机体消化、吸收营养物质的主要场所,也是机体重要的防御屏障,是宿主防御的关键部位,但鸡的消化道较其他哺乳动物的短,仅为体长的6倍,通过增加肠道长度来增加肠道表面积,是促进肠道消化吸收的有效途径。又因为肠道黏膜含有大量的免疫细胞和腺体,所以肠道长度的增加使得免疫细胞含量增多,更有利于机体免疫功能的提升。鸡拥有健康的肠道不仅可以提高饲料营养的消化吸收率,而且能够抵抗肠道致病菌的侵害,减少因疾病死亡和并发症造成的经济损失,是鸡群发挥良好生产性能的关键因素。
中药可影响畜禽肠道微生物菌群多样性,增加肠道有益菌群。中药是我国的“天然医药宝库”,具有多成分、多途径、多靶点、不易产生耐药性的优势。中药中含有的活性成分有很多,主要包括有生物碱、多糖、苷类、有机酸、类黄酮和生物类黄酮等等,这些活性物质都可以提高机体的免疫细胞活性,增强机体免疫细胞的生长和抗体的合成能力,从而调节机体的免疫能力,维持内环境的稳态,最终进一步提高机体的抵抗力,具有显著的免疫增强作用。中草药还可以避免西药类免疫抑制剂对动物机体有交叉反应的副作用等弊端,同时还可以促进以及提高机体的细胞免疫和非特异性免疫功能。
中药饲料添加剂是以提高畜禽生产性能、改善产品品质、以及增强畜禽机体的免疫性能和预防疾病发生为目的的一种加入到饲料当中的添加物质。优质的饲料原料由于含有抗原较少,有助于维护肠道健康。
基于上述研究进展,我们拟以日粮中添加复方中药研究鸡免疫性能的变化,通过系统的研究不同的中药对鸡肠道健康的影响,寻找最合适的中药复方为添加剂。期望通过中药在鸡生产上的应用,提高畜禽对疾病的免疫力,促进畜禽肠道健康,提高畜禽生长性能,达到中药的高效利用,为鸡养殖行业服务。
发明内容
本发明的目的是提供一种植物饲料添加剂的配置方法及其应用效果,以解决上述现有技术存在的问题,使中药营养价值得以改善,鸡肠道健康获得提升。
为实现上述目的,本发明提供了如下方案:本发明提供一种植物饲料添加剂的配置方法,该植物饲料添加剂和饲料按一定比例混合,投喂给湘黄鸡:
五加科植物A、毛茛科植物B、黄杨科植物C以一定比例混合均匀,按照一定比例添加于肉鸡饲料中。
本发明所述的植物饲料添加剂具有改善鸡肠道健康,促进湘黄鸡肠道发育以及肠道黏膜屏障完整的作用。
本发明公开了以下技术效果:与对照组相比,日粮添加2%植物饲料添加剂后,湘黄鸡空肠绒毛高度和基线长度显著高于对照组 (P<0.05),回肠绒毛高度以及绒毛高度与隐窝深度的比值显著高于对照组(P<0.05)。回肠粘膜白介素IL1β的mRNA表达量以中药组显著低于对照组(P<0.05),B淋巴细胞瘤-2基因(抑癌基因BCL2)的 mRNA表达量中药组显著高于对照组(P<0.01)。中药组空肠粘膜肿瘤坏死因子TNFα的mRNA表达量显著低于对照组(P<0.05)。采食含有中药复方的饲料后,湘黄鸡脾脏的TNFα的mRNA表达量显著低于对照组(P<0.05),而BCL2的mRNA表达量显著高于对照组(P<0.05)。湘黄鸡的肠道健康因植物饲料添加剂的添加而得到明显改善。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1是肠道绒毛测定位置图。
具体实施方式
下面将结合本发明实施例中的附表,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合附表和具体实施方式对本发明作进一步详细的说明。
实施例(一)
1试验动物的选择及饲养管理
选择1日龄湘黄鸡100只作为试验动物,试验鸡舍位于衡阳师范学院动物房,自然通风,光照充足。试验湘黄鸡采用立体式单笼饲养,每日早晚各投料一次.在第1到第14日龄时,雏鸡采取地面平养,地上铺纸板和锯木屑,同时每日饲喂添加应激多维的水。每日按时进行卫生清理和消毒。每天记录观察鸡群的健康状况。
2试验设计
2.1试验设计
本实验分为对照组和实验组.每组12个重复,每重复4只鸡。A 组饲喂玉米-豆粕基础日粮(表1),B组在基础日粮上添加2%的植物饲料添加剂。
表1基础日粮营养成分表
每千克饲粮提供以下物质:VA 12500IU,VD3 3500 IU,VE 25mg, VB1 3.5 mg,VB28.5 mg,VB6 5.0 mg,VB12 0.03 mg,VK3 2.5 mg,酸烟酸30mg,D-泛酸15mg,叶酸1.0mg,生物素0.1mg,锌(硫酸锌)110mg,锰(硫酸锰)110mg,Fe(硫酸亚铁)80mg,Cu(硫酸铜)8mg,I(碘化钾)0.35mg,Se(亚硒酸钠)0.15mg。
2.2样品采集与处理
每周对鸡进行称重,对饲料摄入量进行统计。第45日龄时,每个重复随机选取2只鸡,电击致晕后屠宰。采集脾脏、法氏囊、肠道等组织器官进行称重。用生理盐水轻轻冲洗食糜后,取十二指肠、空肠、回肠肠段进行4%多聚甲醛固定。用载玻片轻轻刮取空肠、回肠粘膜,锡箔纸包裹,液氮速冻,-80℃保存。采集脾脏,锡箔纸包裹,液氮速冻,-80℃保存。
2.3指标测定与方法
2.3.1生产性能测定
计算平均日增重、平均日采食量、料肉比。
2.3.2器官指数的计算
按照公式:器官指数(%)=免疫器官鲜重(g)*100/活体重(g) 计算器官指数。
2.3.3肠道组织形态学的测定
(1)冲洗:
将取出的空肠组织放到烧杯中,用纱布将烧杯口遮住,然后用流水进行冲洗12h。
(2)石蜡切片包埋:
脱水:用乙醇进行脱水,将空肠组织依次放入不同浓度的乙醇中进行脱水,浓度和时间依次是85%乙醇浸泡120min、95%乙醇浸泡120min、100%乙醇Ⅰ浸泡60min、100%乙醇Ⅱ40min。
透明:将空肠组织从100%乙醇Ⅱ中拿出后放入二甲苯Ⅰ中浸泡 30min,然后取出组织浸泡到二甲苯Ⅱ中,15min观察一次,直至完全透明后取出。
浸蜡:将组织放入蜡Ⅰ(低熔点54-56℃)中60min,脱去二甲苯,接着放入蜡Ⅱ(高熔点60-62℃)中60min,浸石蜡和脱去二甲苯,然后放到蜡Ⅲ(高熔点60-62℃)中50min,浸石蜡以及彻底脱去二甲苯。
包埋:将融好的蜡Ⅳ(高熔点60-62℃)用来进行包埋,将已经浸透石蜡的空肠组织放入已经预热过的容器中,倒入已经融化好的蜡Ⅳ。包埋后放置于室温环境,待完全冷却后取出用保鲜膜包好,进行编号。
切片与贴片:将包埋好的蜡块固定于徕卡切片机上,进行切片,切出4μm的薄片,用毛笔和镊子将切片放置于水浴锅(恒温43℃) 中展平,选取肠组织完整的切片从左至右放置于涂有蛋白甘油的载玻片上,编号,等水分沥干,放到40℃烘箱中30min烘干。
(3)石蜡切片HE染色
脱蜡:将切片依次放入二甲苯Ⅰ和二甲苯Ⅱ中各自浸泡10min;
复水:将切片依次放入不同浓度的乙醇溶液中,浓度和时间依次是100乙醇Ⅰ1min、100%乙醇Ⅱ1min、95%乙醇Ⅰ1min、85%乙醇Ⅰ2min、75%乙醇Ⅰ2min;
去乙醇:用双蒸水冲洗切片3min;
染色:将切片浸泡至苏木精染液中7min;
去染色液:自来水冲洗5min;
褪色:将切片放置于1%盐酸酒精分化液30s;
返蓝;自来水冲洗10min;
脱水:依次放入75%乙醇Ⅱ、85%乙醇Ⅱ中各1min;
染色:将切片放入伊红染液中4-6s;
去染色液:将切片依次放置于85%乙醇Ⅲ溶液中30s;
脱水:将切片放置于不同浓度的乙醇溶液中,浓度和时间分别是 95%乙醇Ⅱ1min、100%乙醇Ⅲ1min、100%乙醇Ⅳ1min;
透明:将切片依次放置于二甲苯Ⅰ中5min,二甲苯Ⅱ中10min。
封固:将已透明的切片滴上中性树胶,然后盖上盖玻片封固。
(4)观察
待树胶略干后可以将切片放置于徕卡三目生物显微镜下观察,然后用LAS EZ软件拍照记录。最后用ipp软件(Media Cybernetics,美国)测量肠绒毛高度、隐窝深度、基线、绒毛宽度等,见说明书附图。
2.3.4基因表达量的测定
(1)磨样
从-80℃冰箱取出样品,剪切0.5g左右样品放入预冷的研钵中,加入液氮迅速研磨。
(2)RNA的提取及鉴定
使用TRIZOL(宝生物,山东,中国)按照试剂盒方法进行RAN 提取。
取1ul溶液放置于核酸蛋白质分析仪上,RioSpec-nano软件测量 RNA浓度。
(3)cDNA反转录
采用Evo M-MLV反转录试剂盒Ⅱ(艾科瑞,湖南,中国)进行反转录,严格按照说明书进行,得到cDNA,-80℃保存。
(4)荧光定量PCR
以β-Actin为内参基因,对目的基因TNF-α、IL1β、BCL-2、IL8L2、 TLR4、CLDN1的mRNA表达量进行检测分析,各基因及其引物序列见表2,引物由上海生工合成。采用Green Premix Pro Taq HS预混型qPCR试剂盒(含Rox)(艾科瑞,湖南,中国),反应总体系为10μL,具体配制见表3。PCR扩增采用QuantStudio3(Theme Fisher Scientific),将装有反应体系的96孔板放入QuantStudio3中,设置反应参数和条件,经历三个步骤具体荧光定量程序见表4:待反应完成后,对溶解和扩增曲线进行分析,将Ct值导出,以2-△△Ct公式计算目的基因的相对表达量。
表2引物序列
表3荧光定量PCR反应体系组分
反应体系组成 | 体积(μL) |
SYBR Green | 5 |
引物上 | 0.4 |
引物下 | 0.4 |
DNA | 2 |
水 | 2.2 |
总计 | 10 |
表4荧光定量PCR程序
2.4数据处理与分析
实验数据Excel 2010进行初步的处理,然后试验数据是采用SAS 8.0软件进行GLM单因素方差分析,组间差异采用Ttest检验,结果以平均值±标准误(mean±SE)来表示,显著水平为P<0.05。
3结果与分析
3.1中药的添加对湘黄鸡生产性能的影响
中药的添加对湘黄鸡正式试验期的平均日增重、平均日采食量、料肉比没有显著影响(P>0.05)(表5)。
表5中药的添加对湘黄鸡生产性能的影响
备注:ADFI:平均日采食量;ADG:平均日增重;F:G:料肉比
3.2中药的添加对湘黄鸡器官指数的影响
中药的添加对肝脏、脾脏、肠道、肾脏、心脏、法氏囊的重量及器官指数没有显著影响,对肠道长度没有显著影响(P>0.05)(表6)。
表6中药的添加对湘黄鸡器官指数的影响
3.3中药的添加对湘黄鸡肠道形态结构的影响
中药的添加对湘黄鸡十二指肠肠绒毛基线高度、绒毛长度、绒毛宽度、隐窝深度及绒毛高度与隐窝深度的比值没有显著影响(P>0.05) (表7)。添加中药后,鸡的空肠绒毛基线高度、绒毛款苏显著高于对照组(P<0.05),绒毛长度(P=0.068)及绒毛高度与隐窝深度的比值(P=0.061)有显著高于对照组的趋势,隐窝深度与对照组无显著差异(P>0.05)。回肠绒毛基线、绒毛宽带、隐窝深度与对照组无显著差异(P>0.05),绒毛高度以及绒毛高度与隐窝深度的比值以中药组显著高于对照组(P<0.05)。
表7中药的添加对湘黄鸡肠道形态的影响
项目 | 对照组 | 中药组 | SEM | P值 |
十二指肠 | ||||
基线,um | 108.59 | 144.19 | 16.18 | 0.264 |
绒毛高度,um | 431.43 | 553.34 | 37.85 | 0.108 |
绒毛宽度,um | 71.03 | 103.07 | 11.01 | 0.144 |
隐窝深度,um | 58.34 | 75.51 | 6.40 | 0.177 |
绒毛高度/隐窝深度 | 9.23 | 7.77 | 0.56 | 0.188 |
空肠 | ||||
基线,um | 174.96 | 220.74 | 9.11 | 0.025 |
绒毛高度,um | 960.30 | 1089.47 | 32.69 | 0.068 |
绒毛宽度,um | 111.04 | 137.62 | 4.56 | 0.011 |
隐窝深度,um | 147.92 | 128.84 | 6.67 | 0.175 |
绒毛高度/隐窝深度 | 7.04 | 8.75 | 0.42 | 0.061 |
回肠 | ||||
基线,um | 215.22 | 207.10 | 9.84 | 0.686 |
绒毛高度,um | 512.87 | 665.79 | 29.03 | 0.020 |
绒毛宽度,um | 183.33 | 173.50 | 8.91 | 0.589 |
隐窝深度,um | 129.90 | 110.78 | 9.15 | 0.314 |
绒毛高度/隐窝深度 | 4.41 | 6.04 | 0.32 | 0.022 |
3.4中药的添加对湘黄鸡肠粘膜及脾脏基因表达量的影响
与对照组相比,添加中药后,鸡回肠粘膜IL1β基因的mRNA表达量显著下降(P<0.05),BCL2和IL8L2基因的mRNA表达量显著增加(P<0.05),CLDN1、TNFα、TLR4基因的mRNA表达量没有显著变化(P>0.05)(表8)。空肠粘膜基因TNFα以及TLR4的mRNA表达量因中药的添加而显著下降(P<0.05),其他基因如IL1β, CLDN1,BCL2,IL8L2的mRNA表达量没有显著影响(P>0.05)。采食含有中药的饲料后,湘黄鸡脾脏的IL1β和CLDN1基因的mRNA表达量没有显著变化(P>0.05),但TNFα的mRNA表达量显著下降(P<0.05),而BCL2,IL8L2,TLR4的mRNA表达量显著增加(P<0.05)。
表8中药的添加对湘黄鸡肠粘膜及脾脏基因表达量的影响
备注:IL1β:白介素1β;CLDN1:紧密连接蛋白;TNFα:肿瘤坏死因子α;BCL2:B细胞淋巴瘤-2;IL8L2:白介素8;TLR4:Toll样受体4。
4结论
该植物饲料添加剂可通过增加肠绒毛高度,降低肠粘膜促炎因子的表达量,增加肠粘膜及脾脏抗凋亡因子的基因表达量而改善肉鸡肠道健康,增强免疫力。
本发明所述的植物饲料添加剂具有增加肠绒毛高度,降低肠粘膜炎症因子的mRNA表达量,维护肠道形态结构完整性的显著效果。
以上所述仅是本发明的一种实施方式,应当指出,对于本领域普通技术人员来说,在不脱离本发明创造构思的前提下,还可以做出若干相似的变形和改进,这些也应视为本发明的保护范围之内。
Claims (3)
1.一种可改善肉鸡肠道健康的植物饲料添加剂的组成,其特征是,以五加科植物A、毛茛科植物B、黄杨科植物C以一定比例混合均匀。
2.一种制备权利要求1所述植物饲料添加剂的生产方法,其特征是,包括以下步骤:
(1)分别将植物A、B、C晒干,逐级粉碎,过40目筛;
(2)按照一定比例对A、B、C进行混合,以2%的比例添加于肉鸡饲料中。
3.根据权利要求1和2所制作的植物饲料添加剂的应用效果,其特征是:
(1)日粮添加2%的中药后,湘黄鸡空肠绒毛高度和基线长度显著高于对照组(P<0.05),回肠绒毛高度以及绒毛高度与隐窝深度的比值显著高于对照组(P<0.05);
回肠粘膜白介素IL1β的mRNA表达量以中药组显著低于对照组(P<0.05),B淋巴细胞瘤-2基因(抑癌基因BCL2)的mRNA表达量中药组显著高于对照组(P<0.01),中药组空肠粘膜肿瘤坏死因子TNFα的mRNA表达量显著低于对照组(P<0.05),采食含有中药复方的饲料后,湘黄鸡脾脏的TNFα的mRNA表达量显著低于对照组(P<0.05),而BCL2的mRNA表达量显著高于对照组(P<0.05),湘黄鸡肠道健康因该植物饲料添加剂的添加获得明显改善。
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