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

肖润 ^1,2, ,
郑旺 ^1,2, ,
郑琳 ^3, ,
蔡明 ^4, ,
徐泽平 ^1,2, ,
黄先智 ^1,2, ,
刘建勇 ^4, ,
沈以红 ^{1,2,
,}

1.
xinandaxuejiacanjiyinzushengwuxueguojiazhongdianshiyanshi，zhongqing 400715
2.
nongyebucansanggongnengjiyinzuyushengwujishuzhongdianshiyanshi，zhongqing 400715
3.
zhongqingshicanyejishuguanlizhan，zhongqing 400020
4.
yunnanshengcaodidongwukexueyanjiuyuan，yunnan kunming 650212

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。

关键词:

English

Effects of mulberry branch and leaf powder on rumen microflora of Yunnan Yunling cattle

1.
state key laboratory of silkworm genome biology, southwest university, chongqing 400715, china
2.
key laboratory for sericulture functional genomics and biotechnology, agricultural of ministry, chongqing 400715, china
3.
重庆欢乐生肖 chongqing sericulture technical management station, chongqing 400020, china
4.
grassland animal science research institute of yunnan province, kunming 650212, yunnan, china

Corresponding author: Yihong SHEN,

Received Date: 2018-11-28
Available Online: 2019-10-15

Abstract: The rumen microbial community plays a crucial role in the degradation and utilization of dietary nutrients. As a new unconventional feed resource, the effect of mulberry branches and leaves on rumen microflora of ruminants is still unclear. In this experiment, six healthy 30-month-old Yunling cattle with an average weight of (400 ± 20) kg were selected. The control group (Group C) and mulberry branch and leaf powder treatment group (Group V) were separated by the self-control method. Group C was fed a basic diet and Group V, a diet supplemented with 7.31% mulberry branch and leaf powder (MBLP). The experimental period was 30 days, including 10 days of pre-feeding and 20 days of normal feeding. The composition of rumen fluid microbiota was analyzed by 16S rRNA sequencing and the bacterial functions predicted using PICRUSt. The results showed that 1 342 operational taxonomic units were obtained by 16S rRNA gene sequencing, which belonged to 182 genera of 19 phyla. The dominant bacteria at the phylum level in both Groups C and V were Bacteroidetes and Firmicutes, and the dominant bacteria at the genus level were Prevotella, Bacteroides BS11 and Rikenellaceae RC9. At the phylum and genus levels, the number of bacteria related to fiber degradation increased, and the number of Bacteroidetes and Prevotella related to non-fiber polysaccharide degradation decreased. The main functions of the rumen microbial community, as predicted by PICRUSt, were amino acid and carbohydrate metabolism. Therefore, adding mulberry branch and leaf powder to the diet appears to have a positive effect on the rumen microbial community diversity and metabolic function, especially by increasing of the proportion of fiber degradation bacteria, a change conducive to the degradation of plant cellulose.

Key words:

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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

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图 2 OTUs Venn图

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

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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

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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

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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

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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/%^①	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)^②	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.

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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]

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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

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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

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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

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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^① 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.

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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 number	KO通路 KO pathway	C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