CN111011598A - 一种提高安徽白山羊消化能力的组合物及其制备方法、使用方法 - Google Patents
一种提高安徽白山羊消化能力的组合物及其制备方法、使用方法 Download PDFInfo
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Abstract
本发明属于羊养殖领域,具体涉及一种提高安徽白山羊消化能力的组合物,按照重量百分比计包括90%‑95%的茶皂素以及5%‑10%辅料;所述辅料按照重量份数计包括3‑5份陈皮、2‑3份五味子、6‑8份山楂、2‑3份枸杞、3‑5份三七和2‑3份麦冬。本发明的有益效果是:通过喂食本发明制备的组合物可以提高安徽白山羊羔羊的胰腺消化酶活性和瘤胃挥发性脂肪酸含量,还能够改变小肠的上皮黏膜形态,有效促进机体的消化功能。
Description
技术领域
本发明属于羊养殖领域,具体涉及一种提高安徽白山羊消化能力的组合物及其制备方法、使用方法。
背景技术
对于反刍动物,瘤胃是它们十分重要的,也是特有的消化器官,是食物的处理必经之地,瘤胃的消化能力能够反应机体的消化能力,当动物体的消化能力得到改善时,也反映了动物的生产性能的提高,对生产的成本和养殖的经济效益也有很大的作用。许多试验针对反刍动物的瘤胃研究其机体的消化力,而瘤胃中有大量的瘤胃液,其中有丰富的消化酶,它可以帮助动物体分解食物,其发酵参数可以测量瘤胃液中的挥发性脂肪酸。瘤胃中的微生物通过发酵产生大量的VFA,主要成分是乙酸、丙酸与丁酸,会降低机体的消化能力。
发明内容
为了解决上述问题,本发明的提供一种一种提高安徽白山羊消化能力的组合物,制备出的组合物可以显著的提高山羊的消化能力,提高山羊的经济产出。
本发明提供了如下的技术方案:
一种提高安徽白山羊消化能力的组合物,其特征在于,按照重量百分比计包括90%-95%的茶皂素以及5%-10%辅料;
所述辅料按照重量份数计包括3-5份陈皮、2-3份五味子、6-8份山楂、2-3份枸杞、3-5份三七和2-3份麦冬。
优选的,按照重量百分比计包括93%的茶皂素以及7%辅料。
本发明还提供了一种上述组合物的制备方法,分别将陈皮、五味子、山楂、枸杞、三七和麦冬在30-50℃下干燥并粉碎,过80-100目筛子,按照上述配比均匀混合得到辅料,将茶皂素与辅料按比例均匀混合,即得所述组合物。
本发明还提供了一种上述组合物的使用方法,将组合物与山羊精料均匀搅拌后进行喂食,其中所述组合物的添加量通过如下方式确定,组合物中茶皂素的重量:单只山羊体重=(0.625-2.5g):1kg
本发明的有益效果是:
通过喂食本发明制备的组合物可以提高安徽白山羊羔羊的胰腺消化酶活性和瘤胃挥发性脂肪酸含量,还能够改变小肠的上皮黏膜形态,有效促进机体的消化功能。
附图说明
图1是小肠空肠组织切片(×10),其中A为对照组空肠、B为低浓度茶皂素组空肠、C为高浓度茶皂素组空肠;
图2是小肠回肠组织切片(×10),其中A为对照组回肠、B为低浓度茶皂素组回肠、C为高浓度茶皂素组回肠;
图3是小肠十二指肠组织切片(×10),其中A为对照组十二指肠、B为低浓度茶皂素组十二指肠、C为高浓度茶皂素组十二指肠;
图4是组间阿尔法多样性盒图;
图5是韦恩分析图;
图6是基于OTU丰度的PCA分析图;
图7是物种累计曲线图;
图8是OTU Rank曲线图;
图9是门分类水平中物种柱状图;
图10是纲分类水平中物种柱状图;
图11是目分类水平中物种柱状图;
图12是科分类水平中物种柱状图。
具体实施方式
下面结合具体实施例对本发明做具体说明。
实施例1
分别将陈皮、五味子、山楂、枸杞、三七和麦冬在40℃下干燥并粉碎,过100目筛子,按照3份陈皮、3份五味子、6份山楂、2份枸杞、3份三七和2份麦冬均匀混合,得到辅料,在将茶皂素与辅料按重量百分比90%:10%混合,即得所述组合物。
实施例2
本实施例与实施例1的区别之处在于“在将茶皂素与辅料按重量百分比93%:7%混合,即得所述组合物。”
实施例3
本实施例与实施例1的区别之处在于“在将茶皂素与辅料按重量百分比95%:5%混合,即得所述组合物。”
关于本发明的组合物在对山羊消化能力的影响试验
1、试验方法
1.1试验时间与地点
试验于2018年4月至6月在安徽寿县临淮畜牧养殖有限公司。
1.2试验设备和试验动物
本发明实施例2制备的组合物。
安徽白山羊由安徽淮南安徽寿县临淮畜牧养殖有限公司提供。
BK-500全自动生化分析仪:。
1.3试验设计与饲养管理方法
将21只山羊随机分为3组,每组7只,公母随机,分为三圈饲养,栅栏隔开,为对照组、低茶皂素组和高茶皂素组。预饲养一周后,在同一水平下,每日将组合物手动搅拌在山羊精料中,草料与饮水不限量,记录每天精料采食量,正常检疫和驱虫护理,以确保通风和清洁。如表1,是对各组山羊的称重和所添加组合物中的茶皂素量。
表1每组山羊的初始体重和试验处理(kg)
Tab1 Initial weight of each group of goats and experimental treatment(kg)
正式试验前经过7天预试验,预试验结束后开始正式试验,正试期63天,整个试验期共70天。
试验山羊饲养模式为自由采食、饮水,自由运动,散放式管理,
对照组喂食山羊精料;
低茶皂素组按照如下方式喂食,将实施例2制备的组合物与山羊精料均匀搅拌后进行喂食,其中所述组合物的添加量通过如下方式确定,组合物中茶皂素的重量:单只山羊体重=0.625g:1kg;
高茶皂素组按照如下方式喂食,将实施例2制备的组合物与山羊精料均匀搅拌后进行喂食,其中所述组合物的添加量通过如下方式确定,组合物中茶皂素的重量:单只山羊体重=2.5g:1kg;
各组基础日粮相同。饲喂时间及方式为每天早晨上料时将组合物一次性添加在精料中逐头饲喂,每天草料无限供应。
2、样品采集
2.1胰腺消化酶活力测定
在试验结束后,每组选取一只按标准解剖山羊,分离出胰腺。选取不同的部位修整好放入离心管,测定时,称重,按其重量加入相应体积的生理盐水(1:9),并用高速匀浆器2500r/min,温度4℃,离心10min,呈10%的组织匀浆,待静置后吸取其上清液,用试剂盒说明书的方法测定Trypsin、PAMY和Lipase的活性指标。
2.1肠粘膜形态学测定
2.1.1组织切片制作
(1)水洗与组织处理:将组织从固定液中取出,选取合适的部位,切成1mm左右的平整小块,并除去多余脂肪,按组放入包埋框内,用流水冲洗过夜。
(2)脱水与透明:将洗过的组织置于滤纸上吸收水分,然后通过70%→80%→90%→95%→100%的酒精梯度脱水,再于70%的酒精中放置7h,其余的酒精放置1h,再按放入苯醇(二甲苯与无水乙醇)中15min,接着放入二甲苯中10min,至组织透明为止。
(3)浸蜡:取出组织并置于预熔的二蜡(二甲苯与石蜡)中1h,再先后放入熔点在50℃-60℃和58℃-60℃在的石蜡液中,全过程在60℃烘箱中操作。
(4)包埋:点燃酒精灯,烧红镊子,将充分浸好蜡的组织用镊子取出后放在大小合适的容器正中,摆正,小心倒入融化好的石蜡液,用镊子轻轻搅动两下,带出容器中的气泡,常温下静放至石蜡冷却凝固。
(5)修块:将包埋好的蜡块取出,用小刀修整蜡块为合适的形状(主要看组织的大小),适合载玻片为宜。
(6)切片:将修好的蜡块放在切片机上,放置切片机上,先调整厚度为30μm,切至组织位置后调整厚度为5μm,切至出完整组织为止(切前对蜡块哈气更易切出完整组织)。
(7)展片与烤片:将切出的完整片子小心取下,放入53℃-56℃的温水中漂浮,待完全展开后用粘性载玻片取出,沥干水分后放入37℃烘箱过夜。
2.1.2HE染色
(1)复水:取出烤完的片子并先后放入二甲苯Ⅰ和二甲苯Ⅱ各10min后,再苯醇(二甲苯和无水乙醇)中放5min,取出并以100%→95%→80%→70%的无水乙醇梯度分别放置5min,取出后放入蒸馏水中冲洗2min。
(2)苏木素染色:将冲洗干净的片子取出发放平,在有组织的位置滴上少量苏木素染液(覆盖组织为宜),保留4min,再用蒸馏水冲洗1min,冲干净为止。置于分化溶液(盐酸1ml和95%的酒精99ml)中,约15s,再次冲洗1min。
(3)伊红染色;将片子平放,滴上伊红染色液,染色操作如上,保留30s,用蒸馏水冲洗两次,干净为止。再按100%的无水乙醇→苯醇(二甲苯和无水乙醇各半)→二甲苯Ⅰ→二甲苯Ⅱ的顺序放入片子,先后各停留5min。
(4)封片:将片子取出后平放在滤纸上,将树脂轻滴两至三滴在片子有组织的位置,盖上载玻片,轻轻按压封片,常温通风情况下待树脂干即可放在显微镜下观察小肠的组织形态变化。
瘤胃液的发酵参数
先提取瘤胃液的VFA样本,放于仪器检测。
仪器:气相色谱仪(GC6890N),自动进样器,检测器
色谱柱:DB-FFAP,(30m×0.2323mm×1μm)
柱压:0.05MPa
柱温:程序升温,初始温度60℃,并以4℃/min将温度升至100℃,持续5min。
分流比;1:30
检测器温度:250℃
进样器温度:200℃
进样量:1μL,分流进样
载气:氮气
流量:30mL/min
高纯氢发生器流速:30mL/min
3.1小肠菌群测序
3.1.1样品检测方法
样品的测试包含其浓度,完整性与纯化。样品浓度通过分光光度计或酶标仪(如Qubit分光光度计,表达载体)检测。琼脂糖凝胶电泳(1%琼脂糖凝胶,150V恒压电泳40分钟)测定其纯度与完整性。
3.1.2总体工作流程概述
DNA样品被接收后,对样品进行检测;检测合格的样品构建文库:回收目的Amplicon片段,用T4 DNA Polymerase、Klenow DNA Polymerase和T4 PNK将打断形成的粘性末端修复成平末端,此外,通过碱基“A”添加到3'末端,DNA片段可以连接3'末端具有“T”碱基的特殊接头;或者,设计并合成含有测序衔接头的双指数融合引物,并使用基因组DNA作为模板进行融合引物PCR,筛选磁珠用于扩增子片段,最后,使用合格的文库进行簇制备和测序。由机器获得的数据用于执行相应的生物信息分析。
3.1.3信息分析流程
通过过滤机器的数据,能滤掉低质量的读数,剩余高质量的Clean数据可用以后的分析;通过reads之间的Overlap关系将reads拼接成Tags;将给定的相似的标签聚合到聚成OTU(Operational Taxonomic Unit),然后将OTU与数据库进行比较,以在OTU执行物种注释;基于OTU和物种注释结果物种间的样本复杂性分析和物种差异分析。
3.1.4基于Illumina HiSeq平台的Meta 16S rDNA的V6区域测序
检测合格的样本用于上机建库。该库使用双Index双端测序方法。设计融合引物包括P5和P7 Illumina接头序列,8-nt Index序列和基因特异性序列。创建一个双索引排序的方法,可以让我们产生大量高质量的序列数据,同时最大限度地减少文库建设的成本。提取合格样本总DNA,加入准备好的4μV6引物、30ng DNA、25μPCR Master Mix PCR和ddH2O构建扩增体系,体外变性后循环33次,变性(98℃,45min),退火(55℃,45min),延伸(72℃,45min),最终在72℃下取出扩增样品,用AmpureXPbeads(AGENCOURT)纯化以去除非特异性PCR产物,然后与End Repair Mix混合,在20℃下孵育30min。用QIAquick PCR纯化试剂盒(Qiagen)修复末端DNA,然后与A-Tailing混合,37℃孵育30min。将纯化的腺苷酸3'末端DNA与载体链接,在16℃孵育连接反应12-16h。选择适合连接的DNA通过运行2.5%琼脂糖凝胶约2.5-3h以回收目标片段。使用QIAquick凝胶提取试剂盒验证净化结果和数据库结果。最终的文库将以两种方式定量:使用Agilent 2100生物分析仪(Agilent DNA 1000 Reagents)确定平均分子长度,并通过实时荧光定量PCR(QPCR)进行量化(EvaGreenTM)。在MiSeq系统上对合格的文库进行测序策略PE250(PE251+8+8+251)或PE300(PE301+8+8+301)(MiSeq试剂盒)。
3.1.5生物信息学和统计分析
宏基因组研究通常涉及原核16S rDNA基因(16S rDNA)的分析,包括9个可变区和10个恒定区。16S rDNA可变区经常被用于系统发育分类,例如在原核生物和线粒体,18SrRNA是16S rRNA真核细胞核同系物。所谓的16S指的是16S rDNA(或16S rRNA),16S rRNA基因是编码原核核糖体的小亚基的基因,长度约1542bp,包括9个可变区和10个保守区,可变区序列9可以反映物种之间的差异。16S测序是指选择16S rDNA一个或几个变异区域,以及选择用于PCR环境样品(肠道、土壤、水体等)的通用引物,然后对其产物高通量测序,并将测序数据与现有的16S rDNA数据库进行比较,因此,研究环境群落的多样性,核心是物种分析,包括微生物的种类,不同种类间的相对丰度,不同分组间的物种差异以及系统发育演化等。
3.1.6OTU统计分析
根据97%的序列相似将这些序列聚类成为OTUs,并获得每个聚类的序列及其代表序列(即为OTU),可下游分析计算每个OTU序列与丰度。
得到OTU代表序列后,通过RDP classifer(v2.2)软件将OTU代表序列与数据库Greengene_2013_5_99比对,对于物种注释,置信度阈值设置为0.5。
比对数据库:
16S(包括细菌与古菌):Greengene(默认):V201305:RDP Release 11_5,20160930
18S真菌:Silva(默认):SILVA_V132,20180410
ITS真菌:UNITE(默认):Version 7.2,20171201
对注释结果进行如下过滤:
1.删除没有注释结果的OTU;
2.删除不属于分析项目的注释结果。例如,样品为细菌16S,如果OTU注释上古菌则去除。
剩余的OTU方可用于后期分析。
3.1.7数据分析及图片制作
本试验的所有数据值以±SD的形式表示,数据采用Excel进行统计,再用SPSS20.0对数据进行one-way ANOVA显著性分析,组间进行Duncan多重比较。上标是小写字母,不相同的表示差异显著(P<0.05),上标是大写字母,不同则表示为差异极显著(P<0.01)。使用ImageJ对小肠的切片图像处理,测量绒毛的长度和隐窝的深度。
4.胰腺淀粉酶的影响
如表2所示,低浓度茶皂素组和高浓度茶皂素组两组的胰蛋白酶、PAMY和脂肪酶含量均极显著高于对照组(P<0.01)。与高浓度茶皂素组相比,低浓度茶皂素的胰蛋白酶的含量有显著升高(P<0.05),PAMY和脂肪酶的含量无显著变化(P>0.05),但有上升趋势。
表2不同剂量的茶皂素对血清中胰腺淀粉酶的影响(IU·mg-1)
Table 2 Effects of different doses of tea saponin on pancreaticamylase in serum
5.瘤胃液的发酵参数影响
如表3所示,与对照组相比,低浓度茶皂素组和高浓度茶皂素组的丙酸、正丁酸、正戊酸均有显著增加(P<0.05)。
表3不同剂量的茶皂素对瘤胃液的发酵参数影响(μg/g)
Tab 3 Effects of different doses of tea saponin on the fermentationparameters of gastric juice
6.小肠黏膜形态的影响
由图1可以看到,相比对照组,低浓度茶皂素组和高浓度茶皂素组的空肠肠绒毛都有增长,且隐窝深度变浅。
由表4可以看出,与对照组相比,低浓度茶皂素组和高浓度茶皂素组的空肠绒毛高极显著增加(P<0.01),V/C值也极显著上升(P<0.01):而隐窝深度,与对照组相比,高浓度茶皂素组极显著降低(P<0.01),而低浓度茶皂素组相对于其他两组无显著变化(P>0.05)。
表4不同剂量的茶皂素对空肠黏膜形态的影响
表4 Effects of different doses of tea saponin on jejunal mucosamorpHology
由图2可以看出,与对照组相比,低浓度茶皂素组和高浓度茶皂素组的回肠肠绒毛都有增长,且隐窝深度变浅。
通过表5以看出,低浓度茶皂素组和高浓度茶皂素组的回肠绒毛高度极显著增加(P<0.01),隐窝深度显著下降(P<0.05),V/C值极显著增加(P<0.01)。
表5不同剂量的茶皂素对回肠黏膜形态的影响
Table 5 effects of different doses of tea saponin on ileal mucosamorpHology
由图3可以看出,与对照组相比,低浓度茶皂素组和高浓度茶皂素组的十二指肠肠绒毛都有增长,且隐窝深度变浅。
由表6能看出,低浓度茶皂素组和高浓度茶皂素组的十二指肠绒毛高度极显著高于对照组(P<0.01),隐窝深度显著降低(P<0.05),V/C值极显著增加(P<0.01)。
表6不同剂量的茶皂素对十二指肠黏膜形态的影响
Tab 6 effects of different doses of tea saponin on duodenal mucosamorpHology
7.不同剂量茶皂素对山羊小肠菌群影响
7.1 OTU统计结果
OTU用于系统发育学研究或群体遗传学研究以促进分析。由人为分类单元(品系,种,属,分组等)设定的相同标志。根据97%的相似性阈值,序列通常被分为不同的OTU,并且每个OTU通常被认为是个微生物物种。如果相似度小于97%,可以认为它属于不同的物种,相似性小于95%,可以认为属于不同的属。
Paired End Reads通过reads之间的overlap关系拼接成Tags,所有样品一共得到1143532条Tags,平均每个样品103957条,SD值为675;Tag平均长度为252bp,SD值为1bp。
由表7可知,试验采取的11样本分析共得到1143532标记,而且各组样本标记数的有效率都达到了99%以上,直接说明了测试数据的可靠性。11个样品一共有9593个OTU,对照组有2692个,低浓度茶皂素组有3282个,高浓度茶皂素组3619个,说明了三组之间存在差异性,且高浓度茶皂素组的丰富度最高。
表7读取拼接成标签统计数据及OTU统计结果
Tab 7 Reads the tags spliced into the statistical results of markingand OTU statistical results
7.2基于OTU的组间阿尔法多样性分析
阿尔法多样性是衡量群落生态中物种丰富度的常见指标,也是反应物种丰富度与均匀度的综合指标。一般在文献中多次出现的多样性指数为observed_species、shannon、chao和simpson。
observed species指数:表示该样品中含有的物种数目,数值越高表明样品物种丰富度越高。
chao指数:估算样品中所含OTU数目的指数,数值越大代表样本中所含物种越多。
shannon指数:对样品中物种组成的丰富度与均匀性的评估。其数值高表明该物种在环境中丰富度更高,每个物种分布越均匀。
simpson指数:随机选取一个样本数据的两个OTU,它们是不同物种的概率。概率越大,样品的物种多样性越高,反之亦然。优势种在群落中的地位与作用也可用该指数评估。
由图4和表8可以看出,低浓度茶皂素组和高浓度茶皂素组的样本Simpson指数大多数低于对照组,表明它们的多样性更高,覆盖值近似于1,表明样品检测度高。
表8阿尔法多样性分析结果
Tab 8 AlpHa diversity analysis results
7.3基于OTU的样本韦恩图和PCA分析
韦恩图(Venn Diagram),用于显示元素集的重叠区域的图示。每个圈代表一个(组),两个圈重叠部分内的数字就是两个样品或者两组之间的共同拥有的OTU数,没有重叠部分上面的数字就是两个样本或者两组各自特有的OTU数。我们可以通过韦恩图的分析结果看到样本或者组间的共有或者特有的OTU,从而能够比较出在不同情况下的差异性。
对于已经重叠混合的三组样本数据,可以通过图5看出,三个组的样本一共有2581种细菌,其中不重叠的细菌有940种,三个组共有的细菌有1023种,其中低浓度茶皂素组和高浓度茶皂素组共2078个细菌种,两个组共有1375种细菌重叠,低浓度茶皂素组与对照组共2352个细菌种,两组共有1227个细菌种相重叠,对照组与高浓度茶皂素组共2311细菌种,两组共有1145种细菌相重叠。
7.4 OTU PCA分析
主成分分析(Principal Component Analysis),它可以用于分析与简化数据集。它可降低数据集的维数,从而可以对低阶的主成分保留,而对高阶的忽略,从而保留最大化数据集中方差贡献的特征。假如在图中的两个样品距离越接近,就表示这两个样品的组成是相似越高的。对于不同条件下的样品,是可能出现分散或者聚集的情况的,从而可以用于判断相同在相同条件下的样品的相似度如何。
根据每个样品的OTU从而计算每个样品OTU的相对丰度,并将该丰度信息用于OTU的PCA分析。通过R(v3.1.1)语言中ade4包进行统计与作图。
图中的横坐标是第一主成分,括号中的百分比则为第一主成分对样本差异的贡献数;纵坐标是第二主成分,括号中的百分比则为第二主成分对样品差异的贡献数。图中点分别表示各个样品。不同颜色代表属于不同组的样品。
由图6可看出,样本的三组存在分散,有差异性,而圆心之间的距离不等,则表示三组的差异度也不等,表示了茶皂素对样本有着不同的影响
7.5物种累积曲线和稀释曲线图
物种累积曲线(species accumulation curves)表示当样品增加时,物种数量的表达,并且是对样品的物种组成的研究和对样品的物种丰度预测的重要工具。它常被用于判断样本量是否足够以及物种丰富度的估计,因此,该曲线不但对样品量的充分程度可以进行判断,还能够在其前提下,对物种的丰富度判断。
经过Rank-abundance曲线的绘制,可对OTU的丰富度进行测量,并与不同的样品稀释曲线相比较,可以更为直观的显示样品间物种多样性的差异。可以用来展示多样性的两个方面:物种丰度和物种均匀度。该曲线水平方向的宽度是对物种的丰度的反应,物种丰度随着横轴的跨度范围增大而升高;此外,曲线平缓程度越高,其分布均匀度越高,反之亦然。该曲线对测序数据结果与数量的合理性可以直接体现,数据的丰富度可以间接体现。当测序数据逐渐合理时,数据量增多但新物种产生少,曲线也会逐渐平缓。
由图7能够看到,横坐标是样本量,纵坐标是抽样后OTU数目,物种的数目证明其丰富度和多样化,能够形成一条趋向平缓的曲线,物种的数目不会随着样本增加而剧烈上升,证明抽样充分,符合样本抽取原理,可以进行分析。
由图8所示,可以看到样本的丰富度与均匀度,横坐标是样品数目,纵坐标是检测到的物种数。在该曲线中,曲线在横轴上的跨度接近1200,较长,表示样本的物种含量丰富;且曲线相对平坦,表示样品的物种组成较均匀。
7.6物种注释分析
所获得的结果比较数据库,在不同物种分类等级作物种柱状图。可以展示了各样品在不同分类等级上的物种柱状图。每个样品中不同物种所占的比例从图中可以直观看出。从门水平开始画所有物种的柱状图。从纲水平开始,所有样品丰度均低于0.5%的物种被合并成到其他物种中。在每个样品中,大多数比例的是优势细菌,少数比例的是劣势细菌。
由图9和表9可以看出,与对照组相比,高茶皂素的Euryarchaeota(广古菌门)含量显著增加(P<0.05),Bacteroidetes(拟杆菌门)的含量显著下降,Verrucomicrobia(疣微菌门)含量相较其他两组显著增加(P<0.05),Actinobacteria(放线菌门)无显著变化但有上升趋势(P>0.05),Cyanobacteria(蓝藻菌门)无显著变化但有下降趋势(P>0.05)。
由图10和表10可以看出,与对照组相比,高浓度茶皂素组的Verrucomicrobia(疣微菌纲)和Actinobacteria(放线菌纲)显著下降(P<0.05),而高浓度茶皂素组的Coriobacteriia(红椿杆菌纲)和Erysipelotrichi(产芽胞菌纲)含量相对其他两组都显著上升(P<0.05),低浓度茶皂素组与高浓度茶皂素组的Bacteroidia(拟杆菌纲)变化不显著(P>0.05),但有下降趋势。
由图11和表11可以看出,我们可以看出与对照组相比,高浓度茶皂素组的Bifidobacteriale(双歧杆菌目)和Methanobacteriales(甲烷杆菌目)含量显著上升(P<0.05),高浓度茶皂素组的Coriobacteriales(红蝽菌目)含量相较其他两组都显著上升(P<0.05),其他两组相较对照组Bacteroides(拟杆菌目)含量菌目无显著变化但有下降趋势,Clostridiales(梭菌目)无显著变化。
由图2和表12可以看出,与对照组相比,高浓度茶皂素组的Bifidobacteriaceae(双歧杆菌科)和Methanobacteriaceae(甲烷杆菌科)含量显著上升(P<0.05),高浓度茶皂素组的Coriobacteriaceae(红蝽菌科)和Erysipelotrichaceae(丹毒丝菌科)含量相较其他两组都显著上升(P<0.05),其他两组相较对照组Bacteroidaceae(拟杆菌科)含量菌目无显著变化但有下降趋势。
表9肠道微生物在门水平的物种丰度显著性分析
Table 9 pHylum level significance analysis of intestinal microbes
表10肠道微生物在纲水平的物种丰度显著性分析
Table 10 Class level significance analysis of intestinal microbes
表1肠道微生物在目水平的物种丰度显著性分析
Table 1 Order level significance analysis of intestinal microbe
表12肠道微生物在科水平的物种丰度显著性分析
Table 12 Family level significance analysis of intestinal microbe
胰腺是机体的重要器官,它有外分泌的功能,其胰液中分泌的消化酶可以消化食物,特别是脂肪的消化中起着重要作用,它分泌物主要是胰液,它可以通过胰液进入小肠中,并对其的消化功能起着重要影。Trypsin是胰腺分泌的一种蛋白酶,在动物机体内起消化酶的作用,对呼吸道疾病等有重要作用。PAMY是胰腺分泌的一种水解酶,属于淀粉酶,可以促进消化。Lipase是胰腺分泌的脂酶,对食物脂质的消化、处理和运输过程中不可或缺的部分。人体内胰脂肪酶是消化系统中的重要酶分子。因此,胰脂酶检测也常被用作诊断阶段性回肠炎等病的一个佐证。胰腺的损伤会导致血脂酶水平在短时间上升,这三项都是判断胰腺的分泌和消化作用的重要指标。本试验结果中,与对照组相比,低浓度茶皂素组和高浓度茶皂素组的Trypsin、PAMY和Lipase的值有极显著升高,证明茶皂素可以提高胰腺的消化酶活性,对机体消化功能有着积极作用。
低浓度茶皂素组与高浓度茶皂素组的值相比,PAMY和Lipase的值无显著区别,但有升高趋势,而Trypsin的值有显著升高,证明低浓度茶皂素组的剂量0.625g/kg对促进胰腺消化酶活性更有效。
瘤胃作为反刍动物重要消化器官,它能够对食物蛋白质吸收过程中产生大量的会发向脂肪酸,为机体提供能量,并维持其环境的内稳态。有试验证明,挥发性脂肪酸含量的升高可以促进瘤胃发酵,在瘤胃的发酵参数上会升高。乙酸、丙酸、异丁酸、正丁酸、正戊酸和异戊酸都是瘤胃中的游离脂肪酸,参与发酵,加快食物消化吸收。试验结果表明,低浓度茶皂素组和高浓度茶皂素组在丙酸、正丁酸、正戊酸上均有显著提高,而乙酸、异丁酸和异戊酸的含量没有显著变化,但与对照组相比,其他两组的含量均有所增加,说明了茶皂素能够提高山羊瘤胃发酵,从而对消化有促进作用。
8.不同剂量的茶皂素对山羊羔羊小肠黏膜组织的影响
小肠里面含有消化液,如肠液、胰液和胆汁。小肠内壁上的环形皱襞上有小肠绒毛,还有其中毛细血管和毛细血管淋巴管,都是由一层上皮细胞构成的,有利于营养物质的吸收.可以消化和吸收,小肠绒毛主要作用是吸收,消化功能改善后绒毛结构会发生改变,形状会变得更规则,绒毛内细胞数也会升高,会提高小肠的消化吸收能力。隐窝深度是对肠细胞的生成率的响应,如果变浅则说明了细胞成熟率的升高,分泌能力也相应上升。小肠的V/C值可以更直观和综合的反应了小肠的主要的消化功能变化。有试验证实,油茶粕对肉鸡养殖的中对其肠道形态的影响,并提高了其消化功能。因此,对小肠的十二指肠、空肠和回肠的这三个指标能够反应机体的消化能力。
本试验结果中所示,低浓度茶皂素组和高浓度茶皂素组的绒毛高度相极显著升高,隐窝深度显著降低,V/C值极显著升高,表示茶皂素对山羊羔羊的肠道消化功能的促进作用。其中低浓度茶皂素组相对高浓度茶皂素组无显著区别,但是有升高趋势,说明低浓度茶皂素组的剂量0.625g/kg对肠道消化功能相对更好。
9.不同剂量的茶皂素对山羊羔羊肠道菌群的影响
肠道微生态中有100万亿量真菌、细菌等的微生物,细菌成分含量最高,这些菌群不仅具有消化免疫等功能,对维持肠道内稳态,促进新陈代谢与发育。茶皂素中的低聚糖可以被双歧杆菌等肠道有益菌利用,促进生长和繁殖,从而对有害菌有抑制作用,对机体和肠道微生态都有益处。谭婷等发现茶皂素可以调节肠道菌群,从而对机体的免疫、消化和肠道功能的作用。
上述试验证明了茶皂素对肠道菌群的调节作用,添加了茶皂素的组在Euryarchaeota和Verrucomicrobia的含量显著上升,Bacteroidetes的含量显著下降,Coriobacteriia和Erysipelotrichi的含量显著上升,Verrucomicrobia和Actinobacteria的含量显著下降,Bifidobacteriale、Methanobacteriales和Coriobacteriales的含量显著上升,Bifidobacteriaceae、Methanobacteriaceae、Coriobacteriaceae和Erysipelotrichaceae的含量都显著上升。放线菌因为菌落形状是线性的,所以它被称为放线菌,它是原核生物,能够产生多样的酶制剂和维生素等,当其含量过高会导致机体感染,形成肉芽肿性疾病。广古菌门包括了许多古菌种,例如常见于动物肠道中的产甲烷菌,以及盐杆菌和一些需氧和厌氧细菌,这种菌种经常被发现在一些极端的条件下。疣微菌门是被刚划分出的菌门,包括的种类较少,可存在于粪便中。双歧杆菌目能产生有机酸,从而对肠道的腐生菌有抑制作用,参与机体的多种生理活动,还能影响肠黏膜免疫,它早就被发现能治疗肠道感染,后来又发现该菌株可被用作制作益生菌用于饲料行业,它的抗癌、助代谢和提高免疫等作用都是不可忽视的。红蝽菌目存在于肠道,当机体免疫力提升的时候,此种菌群含量会有所上升,梭菌则是可以产生毒素的肠道致病菌,对机体有一定危害。
结合上述结果,当有益菌群含量提高时,机体的免疫力和消化能力等相应提高,有害菌减少时,能减少机体的损伤。因此,本试验结果可以表明茶皂素对山羊羔羊的小肠肠道菌群有着良好的影响,能够提高优势菌群的含量,抑制有害菌种含量,改善肠道内稳态,增加机体的免疫力和消化能力4结论。
茶皂素可以提高安徽白山羊羔羊的胰腺消化酶活性和瘤胃挥发性脂肪酸含量,还能够改变小肠的上皮黏膜形态变小肠的上皮黏膜形态,促进机体的消化功能,优选0.625g/kg。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.一种提高安徽白山羊消化能力的组合物,其特征在于,按照重量百分比计包括90%-95%的茶皂素以及5%-10%辅料;
所述辅料按照重量份数计包括3-5份陈皮、2-3份五味子、6-8份山楂、2-3份枸杞、3-5份三七和2-3份麦冬。
2.根据权利要求1所述的一种提高安徽白山羊消化能力的组合物,其特征在于,按照重量百分比计包括93%的茶皂素以及7%辅料。
3.一种如权利要求1或2所述组合物的制备方法,其特征在于,分别将陈皮、五味子、山楂、枸杞、三七和麦冬在30-50℃下干燥并粉碎,过80-100目筛子,按照上述配比均匀混合得到辅料,将茶皂素与辅料按比例均匀混合,即得所述组合物。
4.一种如权利要求1或2所述组合物的使用方法,其特征在于,将组合物与山羊精料均匀搅拌后进行喂食,其中所述组合物的添加量通过如下方式确定,组合物中茶皂素的重量:单只山羊体重=(0.625-2.5g):1kg。
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