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肖润 郑旺 郑琳 蔡明 徐泽平 黄先智 刘建勇 沈以红

引用本文: 肖润,郑旺,郑琳,蔡明,徐泽平,黄先智,刘建勇,沈以红. 桑枝叶粉对云南云岭牛瘤胃微生物区系的影响. 草业科学, 2020, 37(10): 2069-2078 doi: shu
Citation:  XIAO R, ZHENG W, ZHENG L, CAI M, XU Z P, HUANG X Z, LIU J Y, SHEN Y H. Effects of mulberry branch and leaf powder on rumen microflora of Yunnan Yunling cattle. Pratacultural Science, 2020, 37(10): 2069-2078 doi: shu

桑枝叶粉对云南云岭牛瘤胃微生物区系的影响

    作者简介: 肖润(1995-),男,四川宜宾人,在读硕士生,主要从事饲料资源开发应用。E-mail: ;
    通讯作者: 沈以红,
  • 基金项目:重庆欢乐生肖 国家现代农业产业技术体系建设专项(CARS-18);公益性行业(农业)科研专项(201403049)

摘要: 瘤胃微生物群落对饲粮中营养物质的降解和利用起着至关重要的作用。桑(Morus alba)枝叶作为一种新型非常规饲料资源,对反刍动物瘤胃液微生物区系的影响尚不明确。本研究选用30月龄健康、平均体重为(400 ± 20) kg的云南云岭牛6头,采用自身对照法,分为对照组(C组)和桑枝叶粉处理组(V组),C组饲喂基础饲粮,V组饲喂添加7.31%桑枝叶粉(mulberry branches and leaves powder, MBLP)的饲粮。试验期30 d,其中预饲期10 d,正饲期20 d。通过16S rRNA测序分析瘤胃液菌群组成并进行PICRUSt功能预测。结果显示: 16S rRNA基因测序共获得1 342个操作分类单元(operational taxonomic units, OTUs),属于19个门182个属。C组与V组在门水平上的优势菌群均为拟杆菌门和厚壁菌门,属水平上的优势菌群均为普雷沃氏菌属、拟杆菌目BS11和理研菌科RC9。在门和属两个水平上,与纤维降解有关的菌群有升高趋势,与非纤维多糖物质降解相关的拟杆菌门和普雷沃氏菌属数量下降。PICRUSt预测菌群功能发现,瘤胃菌群功能主要集中在氨基酸和碳水化合物代谢。因此,在日粮中添加桑枝叶粉对瘤胃微生物群落多样性和代谢功能有积极影响,特别是纤维降解菌群比例提高,有利于促进植物纤维素的降解。

English

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  • 重庆欢乐生肖

    图 1  各样本的稀释曲线

    Figure 1.  重庆欢乐生肖 Rarefaction curve of each sample

    图 2  OTUs Venn图

    Figure 2.  重庆欢乐生肖 Operational taxonomic units (OTUs) Venn diagram

    图 3  重庆欢乐生肖 肉牛瘤胃微生物UPGMA分析

    Figure 3.  重庆欢乐生肖 UPGMA analysis of rumen microbes in beef cattle

    图 4  重庆欢乐生肖 肉牛瘤胃KEGG第一水平不同功能的相对丰度

    Figure 4.  Relative abundance of different functions of bacteria at the first level of KEGG in the rumen of beef cattle

    图 5  肉牛瘤胃KEGG第二水平主要功能的相对丰度

    Figure 5.  Relative abundance of the main functions of bacteria at the second level of KEGG in the rumen of beef cattle

    表 1  试验日粮的组成及营养水平

    Table 1.  重庆欢乐生肖 Dietary composition and nutrient level

    项目
    Item
    C组
    Group C
    V组
    Group V
    原料 Ingerdients
    青贮玉米秸秆 Corn straw silage/% 87.96 81.85
    桑枝叶粉 Mulberry branch
    and leaf powder (MBLP)/%
    0.00 7.31
    米糠 Rice bran/% 1.14 1.03
    麦麸 Wheat bran/% 0.90 0.81
    玉米 Corn/% 5.54 4.99
    豆粕 Soybean meal/% 0.66 0.60
    菜粕 Rapeseed meal/% 1.45 1.30
    棉粕 Cottonseed meal/% 0.36 0.33
    干酒糟及其可溶物
    Distillers dried grains with solubles/%
    0.84 0.76
    大麦 Barley/% 0.60 0.54
    预混料 Gunk/% 0.24 0.22
    小苏打 NaHCO3/% 0.06 0.05
    食盐 Salt/% 0.18 0.16
    植物油 Oil/% 0.06 0.05
    合计 Total/% 100.00 100.00
    营养水平 Nutritional level
    代谢能
    Metabolic energy/(MJ·kg−1)
    16.37 16.38
    粗蛋白质 Crude protein/% 9.91 10.03
    钙 Ca/% 0.12 0.25
    磷 P/% 0.13 0.14
     C组: 对照组,V组: 桑枝叶粉组,下表同。①根据美国国家研究委员会营养要求(2000),由云南省草地动物科学研究院配制。②代谢能为计算值,方法参考何亭漪[21],其余指标为实测值。营养物质测定以干物质为基础。
     Group C: control group, and Group V: mulberry branch and leaf powder treatment group; this is applicable for the following tables as well. ①According to the National Research Council nutrient requirement (2000), prepared by the Grassland Animal Science Research Institute of Yunnan Province. ②The metabolic energy was calculated with reference to He Tingyi[21], and the other indexes were measured. Nutrients were deermined after air drying.
    下载: 导出CSV

    表 2  重庆欢乐生肖 细菌qPCR特异性引物

    Table 2.  Bacterial qPCR specific primers

    目标菌
    Target bacteria
    引物序列 (5’ – 3’)
    Primer sequence (5’ – 3’)
    产物大小
    Product size/bp
    退火温度
    Annealing temperature/℃
    白色瘤胃球菌
    Ruminococcus albus
    F:CCCTAAAAGCAGTCTTAGTTCG
    R:CCTCCTTGCGGTTAGAACA
    176 58[22]
    黄色瘤胃球菌
    Ruminococcus flavefaciens
    F:CGAACGGAGATAATTTGAGTTTACTTAGG
    R:CGGTCTCTGTATGTTATGAGGTATTACC
    132 58[23]
    产琥珀酸丝状杆菌
    Fibrobacter succinogenes
    F:GTTCGGAATTACTGGGCGTAAA
    R:CGCCTGCCCCTGAACTATC
    121 58[23]
    栖瘤胃普雷沃氏菌
    Prevotella ruminicola
    F:GCGAAAGTCGGATTAATGCTCTATG
    R:CCCATCCTATAGCGGTAAACCTTTG
    78 58[24]
    牛链球菌
    Streptococcus bovis
    F:TTCCTAGAGATAGGAAGTTTCTTCGG
    R:ATGATGGCAACTAACAATAGGGGT
    127 54[25]
    溶纤维丁酸弧菌
    Butyrivibrio fibrisolvens
    F:CAGCAGGGAAGATAATGAC
    R:TGCAATACCGGAGTTGAG
    171 58[26]
    下载: 导出CSV

    表 3  瘤胃液细菌OTU数目及α多样性指数

    Table 3.  重庆欢乐生肖 Rumen bacterial operational taxonomic units (OTUs) number and alpha diversity index

    项目
    Item
    C组
    Group C
    V组
    Group V
    P
    操作分类单元
    OTUs
    1 065.00 ± 23.64 1 138.00 ± 28.48 0.001
    覆盖率
    Coverage
    0.99 ± 0.00 0.99 ± 0.00 0.868
    Ace指数 Ace 1 162.00 ± 11.60 1 194 ± 22.77 0.012
    Chao指数 Chao 1 166.00 ± 17.74 1 203 ± 32.32 0.033
    香农指数
    Shannon index
    5.82 ± 0.11 5.88 ± 0.12 0.391
    辛普森指数
    Simpson index
    0.01 ± 0.00 0.01 ± 0.00 0.606
    下载: 导出CSV

    表 4  重庆欢乐生肖 桑枝叶粉对瘤胃液菌群相对丰度的影响(门水平)

    Table 4.  重庆欢乐生肖 Effects of MBLP on the relative abundance of rumen bacteria (phylum level)

    项目
    Item
    C组
    Group C/%
    V组
    Group V/%
    P
    拟杆菌门 Bacteroidetes 76.29 ± 5.17 69.91 ± 5.94 0.075
    厚壁菌门 Firmicutes 18.66 ± 5.11 19.00 ± 4.12 0.902
    变形菌门 Proteobacteria 1.06 ± 0.48 1.68 ± 0.50 0.055
    黏胶球形菌门 Lentisphaerae 0.92 ± 0.22 2.45 ± 1.00 0.005
    纤维杆菌门 Fibrobacteres 0.85 ± 0.73 1.45 ± 1.53 0.411
    螺旋体门 Spirochaetae 0.58 ± 0.27 1.19 ± 0.28 0.003
    SR1 0.30 ± 0.09 1.12 ± 0.40 0.001
    蓝细菌门 Cyanobacteria 0.22 ± 0.08 0.95 ± 0.83 0.060
    软壁菌门 Tenericutes 0.34 ± 0.13 0.58 ± 0.34 0.134
    疣微菌门 Verrucomicrobia 0.04 ± 0.04 0.10 ± 0.12 0.299
    放线菌门 Actinobacteria 0.04 ± 0.03 0.01 ± 0.01 0.026
    绿弯菌门 Chloroflexi 0.02 ± 0.02 0.00 ± 0.00 0.010
    绿菌门 Chlorobi 0.00 ± 0.00 0.02 ± 0.02 0.209
    Saccharibacteria 0.18 ± 0.07 0.72 ± 0.19 < 0.001
    迷踪菌门 Elusimicrobia 0.14 ± 0.05 0.30 ± 0.08 0.002
    浮霉菌门 Planctomycetes 0.05 ± 0.02 0.22 ± 0.09 0.001
    互养菌门 Synergistetes 0.14 ± 0.11 0.11 ± 0.07 0.686
    SHA-109 0.15 ± 0.15 0.10 ± 0.06 0.411
    未分类Unclassified Bacteria 0.00 ± 0.01 0.09 ± 0.06 0.004
    下载: 导出CSV

    表 5  重庆欢乐生肖 桑枝叶粉对瘤胃液菌群相对丰度的影响(属水平)

    Table 5.  Effects of MBLP on the relative abundance of rumen bacteria (genus level)

    项目
    Item
    C组
    Group C/%
    V组
    Group V/%
    P
    克里斯滕森菌科 R-7
    Christensenellaceae R-7
    1.90 ± 0.54 1.32 ± 0.10 0.026
    解琥珀酸菌属
    Succiniclasticum
    4.24 ± 3.33 1.25 ± 1.62 0.076
    拟杆菌目 UCG-001
    Bacteroidales UCG-001
    1.87 ± 0.75 0.72 ± 0.51 0.011
    拟杆菌目 RF16
    Bacteroidales RF16
    2.02 ± 0.71 0.85 ± 0.62 0.012
    拟杆菌目 BS11
    Bacteroidales BS11
    14.56 ± 3.54 11.52 ± 3.36 0.158
    SR1 0.30 ± 0.09 1.12 ± 0.40 0.001
    拟杆菌目 S24-7
    Bacteroidales S24-7
    3.30 ± 1.04 3.75 ± 1.04 0.473
    纤维杆菌属
    Fibrobacter
    0.84 ± 0.73 1.44 ± 1.53 0.406
    普雷沃氏菌科 UCG-001
    Prevotellaceae UCG-001
    2.85 ± 0.87 2.93 ± 0.27 0.821
    黏胶球形菌门 RFP12
    Lentisphaerae RFP12
    0.49 ± 0.18 1.23 ± 0.96 0.094
    拟杆菌属
    Bacteroides
    1.03 ± 0.31 0.63 ± 0.21 0.025
    普雷沃氏菌属
    Prevotella
    28.84 ± 9.49 25.89 ± 7.97 0.573
    瘤胃球菌科 NK4A214
    Ruminococcaceae
    NK4A214
    1.15 ± 0.46 1.39 ± 0.51 0.402
    普雷沃氏菌科 UCG-003
    Prevotellaceae UCG-003
    5.22 ± 1.59 3.64 ± 1.16 0.078
    瘤胃球菌科 UCG-010
    Ruminococcaceae
    UCG-010
    0.90 ± 0.32 1.04 ± 0.43 0.551
    Papillibacter 1.24 ± 0.48 2.43 ± 1.15 0.041
    理研菌科 RC9
    Rikenellaceae RC9
    11.06 ± 4.20 14.83 ± 3.43 0.119
    理研菌科未分类属
    Unclassified Rikenellaceae
    1.82 ± 0.58 1.65 ± 0.54 0.609
    普雷沃氏菌科未分类属
    Unclassified Prevotellaceae
    0.63 ± 0.12 0.92 ± 0.26 0.032
    丁酸弧菌属
    Butyrivibrio
    0.33 ± 0.10 0.83 ± 0.43 0.019
    下载: 导出CSV

    表 6  桑枝叶粉对瘤胃液菌群相对丰度的影响(种水平)

    Table 6.  Effects of MBLP on the relative abundance of rumen bacteria (species level)

    项目
    Item
    C组
    Group C/%
    V组
    Group V/%
    P
    白色瘤胃球菌
    R. albus
    4.16 ± 1.06 1.64 ± 1.80 0.002
    黄色瘤胃球菌
    R. flavefaciens
    8.32 ± 2.28 6.50 ± 1.05 0.046
    产琥珀酸丝状杆
    F. succinogenes
    4.57 ± 1.67 5.01 ± 2.56 0.673
    栖瘤胃普雷沃氏菌
    P. ruminicola
    2.95 ± 0.44 3.13 ± 0.46 0.391
    牛链球菌
    S. bovis
    1.27 ± 0.25 0.54 ± 0.28 < 0.001
    溶纤维丁酸弧菌
    B. fibrisolvens
    2.08 ± 0.94 1.37 ± 1.01 0.143
     ①: 细菌数以每10 ng DNA 基因拷贝数的对数表示。
     ①: The number of microbes was shown by the logarithm of the values for gene copies per 10 ng DNA.
    下载: 导出CSV

    表 7  肉牛瘤胃细菌中10条丰度较高的KEGG第三水平通路

    Table 7.  重庆欢乐生肖 The ten most abundant KEGG third horizontal pathways in rumen bacteria of beef cattle

    KO号 KO numberKO通路 KO pathwayC组 Group C/%V组 Group V/%P
    KO03010 核糖体 Ribosome 4.66 ± 0.04 4.59 ± 0.06 0.044
    KO00230 嘌呤代谢 Purine metabolism 3.94 ± 0.05 3.88 ± 0.04 0.041
    KO00240 嘧啶代谢 Pyrimidine metabolism 3.50 ± 0.03 3.42 ± 0.05 0.006
    KO02010 ABC转运体 ABC transporters 3.06 ± 0.25 3.32 ± 0.28 0.121
    KO00190 氧化磷酸化 Oxidative phosphorylation 2.31 ± 0.02 2.34 ± 0.04 0.167
    KO00520 氨糖和核糖代谢
    Amino sugar and nucleotide sugar metabolism
    2.32 ± 0.03 2.30 ± 0.02 0.299
    KO00330 精氨酸和脯氨酸代谢 Arginine and proline metabolism 2.14 ± 0.01 2.11 ± 0.02 0.013
    KO00970 氨酰-tRNA生物合成 Aminoacyl-tRNA biosynthesis 2.06 ± 0.02 2.07 ± 0.01 0.298
    KO00250 丙氨酸、天冬氨酸和谷氨酸代谢
    Alanine, aspartate and glutamate metabolism
    2.04 ± 0.05 2.00 ± 0.04 0.138
    KO00680 甲烷代谢 Methane metabolism 1.99 ± 0.08 2.02 ± 0.06 0.510
    下载: 导出CSV
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                          • 通讯作者:  沈以红,
                          • 收稿日期:  2018-11-28
                          • 网络出版日期:  2019-09-21
                          • 刊出日期:  2019-10-15
                          通讯作者: 陈斌,
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