Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 12: 7852 - 7864

DOI: https://doi.org/10.12418/CJAN2023.713

RESEARCH PAPER

Study on Changes of Rumen Fermentation Parameters and Microflora in Dairy Cows before and after Delivery

  • MA Liqin , 1, 2 ,
  • WEN Wan 2 ,
  • TIAN Jia 2 ,
  • LI Long 2 ,
  • WANG Kun 2 ,
  • ZHOU Jiamin 2 ,
  • LI Weiqi 2 ,
  • LU Tingting 2 ,
  • XU Han 2 ,
  • WANG Ling , 1, *
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  • 1 College of Agriculturel, Ningxia University, Yinchuan 750021, China
  • 2 Ningxia Hui Autonomous Region Animal Husbandry Workstation, Yinchuan 750002, China
* professor, E-mail:

Received date: 2023-04-20

  Online published: 2023-12-11

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Abstract

This experiment was conducted to study the changes of rumen fermentation parameters and microflora in dairy cows before and after delivery. Twenty Holstein cows (similar in age, body condition, number of lactation, due date and milk yield of last parity) were selected and six cows of them were randomly selected, rumen fluid was collected before morning feeding at 7 d prenatal (-7 d) and 7 d postnatal (+7 d) to detect rumen fermentation parameters and microbial composition. The results showed as follows: 1) the pH in postpartum period was extremely significantly lower than that in prenatal period (P<0.01), the concentrations of total volatile fatty acids (TVFA) and propionate in postpartum period were extremely significantly higher than those in prenatal period (P<0.01), the concentration of acetate in postpartum period was significantly higher than that in prenatal period (P<0.05), the difference between rumen ammoniacal nitrogen (NH3-N) before and after delivery was not significant (P>0.05), and rumen microprotein (MCP) concentration tended to decrease after parturition (0.05≤P<0.10). 2) The results of 16S rRNA high-throughput sequencing showed that the alpha diversity indexes of rumen bacteria in dairy cows was significantly lower than that in prenatal period (P<0.01). At the phylum level, the relative abundance of rumen Firmicutes, Patescibacteria, Kiritimatiellaeota, Spirochaetes, Fibrobacteres, Verrucomicrobia, Lentisphaerae and WPS-2 was extremely significantly lower than that in prenatal period (P<0.01), the relative abundance of Proteobacteria and Cyanobacteria was extremely significantly higher than that in prenatal period (P<0.01), and the relative abundance of Actinomycetes was significantly higher than that in prenatal period (P<0.05). At the genus level, the relative abundance of Prevotella_7 and Shuttleworthia was significantly higher than that in prenatal period (P<0.05), and the relative abundance of Ruminococcus, Anaerovorax, Rikenellaceae_RC9_gut_group, Christensenellaceae_R-7_group, [Eubacterium]_coprostanoligenes_group, Candidatus_Saccharimonas, Papillibacter, Saccharofermentans was extremely significantly lower than that in prenatal period (P<0.01). The relative abundance of Olsenella and Stenotrophomonas was significantly higher than that in prenatal period (P<0.01). 3) The results of ITS high-throughput sequencing showed that the Chao1 index and ACE index of rumen fungi were significantly higher than those in prenatal period (P<0.05), the Shannon index and Simpson index were extremely significantly lower than those in prenatal period (P<0.01). At the phylum level, the relative abundance of Anthophyta was extremely significantly higher than that in prenatal period (P<0.01), the relative abundance of Ascomycota and Basidiomycota was extremely significantly lower than that in prenatal period (P<0.01), the relative abundance of Chlorophyta was significantly lower than that in prenatal period (P<0.05). At the genus level, the relative abundance of Gossypium was extremely significantly higher than that in prenatal period (P<0.01). the relative abundance of Aspergillus, Chenopodium, Arocladium Acremonium was extremely significantly lower than that in prenatal period (P<0.01), the relative abundance of Glycine, Wallemia, Nigrospora and Penicillium was significantly lower than that in prenatal period (P≤0.05), and the relative abundance of Debaryomyces was decreased after parturition (0.05<P<0.10). It can be seen that there are significant differences in rumen fermentation before and after delivery, and the richness and diversity of rumen bacteria and fungi change significantly as cows enter the early lactation stage from the end of gestation.

Key words： Holstein dairy cows; before and after delivery; rumen index; rumen bacteria; rumen fungus

Cite this article

MA Liqin , WEN Wan , TIAN Jia , LI Long , WANG Kun , ZHOU Jiamin , LI Weiqi , LU Tingting , XU Han , WANG Ling . Study on Changes of Rumen Fermentation Parameters and Microflora in Dairy Cows before and after Delivery[J]. Chinese Journal of Animal Nutrition, 2023 , 35(12) : 7852 -7864 . DOI: 10.12418/CJAN2023.713

奶牛围产期又称过渡期,是指奶牛产犊前3周到产犊后3周这一阶段。此时,奶牛的生理、激素水平、瘤胃功能等方面都发生了巨大变化,是影响奶牛生产性能正常发挥和健康养殖的关键时期。有研究表明,瘤胃菌群结构和功能的稳态对机体的健康和营养等方面有着至关重要的作用[1]。奶牛在分娩后,其饲粮结构立即由高纤维干奶配方转变为高碳水化合物泌乳牛配方[2],此时,奶牛瘤胃微生物组成、瘤胃微生物活性及整体瘤胃环境等方面都发生了显著变化[3⇓-5]。Zhu等[6]通过16S rRNA高通量测序技术对10头初产荷斯坦奶牛过渡期瘤胃微生物区系进行分析,研究发现,在围产期期间,奶牛在应对饮食变化时,瘤胃细菌和古菌群落的组成发生显著变化。Sofyan等[7]研究发现,奶牛在产犊前14 d至产犊后21 d这一阶段,瘤胃微生物多样性呈先降低后升高的发展趋势,瘤胃微生物多样性在产后7 d最低,产后21 d恢复至产犊前水平。目前关于奶牛产犊前后瘤胃微生物变化规律相关报道较少,因此,本研究旨在通过16S rRNA、ITS高通量测序技术,探究奶牛产犊前后瘤胃发酵参数、瘤胃细菌、瘤胃真菌变化规律,以期为围产期奶牛饲养管理及营养调控提供理论依据。

1 材料与方法

1.1 试验设计

采用完全随机试验设计,选取20头年龄(3.58±1.34)、胎次[(1.55±0.69)次]、上一胎次产奶量[(9 116.05±1 839.79) kg]相近的健康荷斯坦围产期奶牛,随机选取6头奶牛分别于产前7 d(-7 d)和产后7 d(+7 d)晨饲前采集瘤胃液,测定奶牛产犊前后瘤胃发酵参数和微生物组成。试验期间,奶牛饲养管理安牧场标准化饲养管理流程执行(饲喂全混合日粮,每日2次;散栏式饲养,自由饮水,自由采食),基础饲粮组成及营养水平见表1。
表1 基础饲粮组成及营养水平(干物质基础)

Table 1 Composition and nutrient levels of basal diets (DM basis) %

项目
Items		 围产前期
Early perinatal period		 围产后期
Post perinatal period
原料Ingredients
玉米Corn 10.77 22.81
豆粕Soybean meal 2.27 8.31
棉籽粕Cottonseed meal 9.07 3.78
玉米干酒糟及其可溶物Corn DDGS 3.83 2.94
预混料Premix1) 2.27 2.82
其他Others2) 0.14 1.45
玉米青贮Corn silage 17.08 22.77
苜蓿干草Alfalfa hay 14.32
燕麦草Oat grass 31.89 2.40
稻草Straw 22.68
全棉籽Whole cottonseed 7.09
甜菜粕Beet pulp 4.77
发酵饲料Fermented feed 1.89
糖蜜Molasses 4.65
合计Total 100.00 100.00
营养水平Nutrient levels3)
泌乳净能NEL/(MJ/kg) 5.98 7.24
粗蛋白质CP 12.00 16.20
中性洗涤纤维NDF 52.30 31.30
钙Ca 0.77 0.98
磷P 0.34 0.52

1)围产前期每千克预混料含有 One kilogram premix contained the following in early perinatal period:VA 300 000 IU, VD 35 000 IU, VE 2 500 IU, I 80 mg, Se 25 mg, Co 60 mg, Cu 930 mg, Zn 6 000 mg, Mn 3 400 mg; 围产后期每千克预混料中含有 One kilogram premix contained the following in post perinatal period:VA 220 000 IU, VD 22 000 IU, VE 2 200 IU, I 70 mg,Se 20 mg,Co 45 mg,Cu 710 mg,Zn 4 100 mg,Mn 2 300 mg。

2)围产前期其他为:活性干酵母0.11%、脱霉剂0.03%;围产后期其他为:小苏打0.85%、氧化镁0.28%、过瘤胃蛋氨酸0.05%、活性干酵母0.22%、脱霉剂0.05%。Others in the early perinatal period were: active dry yeast 0.11%, mildew remover 0.03%; others in the post perinatal period were: baking soda 0.85%, magnesium oxide 0.28%, rumen protected methionine 0.05%, active dry fermentation 0.22%, mildew remover 0.05%.

3)泌乳净能为计算值,其他均为实测值。NEL was a calculated value, while the others were measured values.

1.2 试验材料

台式酸度计测定仪(PHS-3C,江苏盛蓝仪器制造有限公司)、气相色谱仪(CG-2014C,日本岛津)、琼脂糖凝胶电泳仪(DYY-6C,北京六一仪器厂)、凝胶成像系统(Tanon-2500,上海天能科技有限公司)、超纯水仪(明澈 TM-D,上海乐枫生物科技有限公司)、移液器(德国艾本德股份公司)、振荡器(米欧mix-28+,广州围古润仪器有限公司)、凯氏定氮仪(KDY-9820,山东海能科学仪器有限公司)、粗纤维测定仪(SLQ-200,上海纤检仪器有限公司)、脂肪测定仪(SOX-406,山东海能科学仪器有限公司)、16103马弗炉(SX-G,天津中环电炉股份有限公司)。

1.3 瘤胃液的采集

选取6头健康的荷斯坦奶牛于产前7 d(-7 d)和产后7 d(+7 d)晨饲前用胃管式牛瘤胃液采样器采集瘤胃液50 mL,经4层纱布过滤,用台式酸度计测定瘤胃液pH。将滤液分装于5和10 mL无菌冻存管中,随后置于液氮速冻后置于-80 ℃的冰箱内保存,用于测定奶牛产犊前后瘤胃发酵参数和微生物区系的变化。

1.4 指标测定

1.4.1 常规营养成分测定

泌乳净能(NEL)根据NRC(2001)提供的模型进行计算;饲粮干物质(DM)含量参照GB/T 6435—2006方法测定,粗蛋白质(CP)含量参照GB/T 6432—2018方法测定,粗纤维(CF)含量参照GB/T 6434—2006方法测定,粗脂肪(EE)含量参照GB/T 6433—2006方法测定,粗灰分(Ash)含量参照GB/T 6438—2007方法测定,中性洗涤纤维(NDF)含量参照GB/T 20806—2006方法进行测定,钙(Ca)含量参照GB/T 6436—2018方法测定,磷(P)含量参照GB/T 6437—2018方法测定。

1.4.2 挥发性脂肪酸(VFA)、氨态氮(NH3-N)、微生物蛋白(MCP)浓度测定

用气相色谱仪测定瘤胃液中的总挥发性脂肪酸(TVFA)、乙酸、丙酸、丁酸浓度[7],利用比色法测定瘤胃液中NH3-N浓度[8],MCP浓度采用嘌呤碱基法[7]测定。

1.4.3 瘤胃液微生物总DNA提取、扩增和测序

使用Hipure Soil DNA提取试剂盒抽提瘤胃液总DNA,DNA提取完成后,利用1%琼脂糖凝胶电泳和分光光度法对DNA的纯度及浓度进行检测,质检合格的样品-20 ℃保存。取适量检测合格的DNA样品于离心管中进行稀释,将稀释后的基因组DNA作为模板,瘤胃细菌DNA扩增目的片段为V3~V4区(测序引物为341F:5'-CCTACGGGNGGCWGCAG-3';806R:5'-GGACTACHVGGGTATCTAAT-3'),瘤胃真菌DNA扩增目的片段为ITS2区[引物序列为 ITS3_KYO2(F):5'-GATGAAGAACGYAGYRAA-3';ITS4(R):5'-TCCTCCGCTTATTGATATGC-3'],PCR扩增完成后,分别用2%琼脂糖凝胶电泳进行检测,使用AMPure XP Beads对第二轮扩增产物进行纯化,用ABI Steponeplus Real-Time PCR System(Life Technologies)进行定量,根据Illumina HiSeq2500的PE250模式Pooling上机测序。测序完成后,原始读数据上传在NCBI SRA(Sequence Read Archive)数据库。
使用UPARSE流程将clean tag按≥97%相似度聚类为操作分类单元(OTU)。选取丰度最高的tag序列作为每个OTU的代表序列。OTU代表序列比对Greengene数据库使用RDP注释软件(版本2.2)的朴素贝叶斯模型进行物种分类注释,置信阈值设为0.8~1.0。使用Krona(版本2.6)显示每个物种分类的丰度统计数据。

1.5 数据统计与分析

用Excel 2016对试验数据进行初步整理后,采用SPSS 21.0软件中t检验进行显著性分析。以P≤0.05为差异显著,0.05<P<0.10为有显著趋势,P<0.01为差异极显著。

2 结果与分析

2.1 奶牛产犊前后瘤胃发酵参数的变化

由表2可知,与产前比较,奶牛产后瘤胃液pH极显著降低(P<0.01),瘤胃液NH3-N浓度在奶牛产犊前后差异不显著(P>0.05),瘤胃液MCP浓度在产后有降低趋势(0.05<P<0.10)。奶牛产后瘤胃液TVFA、丙酸浓度较产前极显著增加(P<0.01),乙酸浓度较产前显著增加(P<0.05)。
表2 奶牛产犊前后瘤胃发酵参数变化

Table 2 Change of rumen fermentation parameters in dairy cows before and after delivery

项目
Items		 时间Time/d SEM P值
P-value		 差异倍数
Difference multiple1)
-7 +7
pH 7.18A 5.58B 0.26 <0.01 0.78↓2)
氨态氮NH3-N/(mg/dL) 4.70 3.92 0.24 0.11 0.83↓
微生物蛋白MCP/(mg/dL) 2.22 1.36 0.26 0.10 0.61↓
总挥发性脂肪酸TVFA/(mmol/L) 67.25B 130.20A 11.96 <0.01 1.94↑
乙酸Acetate/(mmol/L) 51.78b 79.09a 6.64 0.03 1.52↑
丙酸Propionate/(mmol/L) 9.23B 33.58A 4.40 <0.01 3.64↑
丁酸Butyrate/(mmol/L) 6.24B 17.97A 1.97 <0.01 2.88↑
乙酸/丙酸Acetate/propionate 5.64A 2.54B 0.52 <0.01 0.45↓

同行数据肩标不同小写字母,表示差异显著(P≤0.05),不同大写字母表示差异极显著(P<0.01)。

1)差异倍数值为2组间微生物相对丰度平均值的比值。下表同。

2)↑ 或 ↓表示奶牛产犊前后微生物表达差异趋势。下表同。

In the same row, vales with different small letter superscripts mean significant difference (P≤0.05), while with different capital letter superscripts mean extremely significant difference(P<0.01).

Difference multiple is the ratio of the mean relative abundance of microorganisms between the two groups. The same as below.

↑ or ↓ shows the expression difference trend of microorganism in dairy cows before and after delivery. The same as below.

2.2 奶牛产犊前后瘤胃细菌的变化

2.2.1 瘤胃细菌高通量测序质量控制与alpha多样性指数

测序数据经过质量控制后,奶牛产犊前后clean tags数目、OTU数目以及alpha多样性指数见表3。本试验从12个瘤胃液样本中共得到1 408 157条高质量的细菌16S rRNA基因序列,每个样本平均的clean tags数目为117 346,奶牛产犊前后无显著差异(P>0.05)。样本平均OTU数目为3 420.25,奶牛产后OTU数目较产前极显著降低(P<0.01)。本试验平均测序覆盖率为96.94%,满足了后续分析要求。与产前比较,奶牛产后Chao1指数、ACE指数、香农指数和辛普森指数较产前极显著降低(P<0.01)。
表3 瘤胃细菌高通量测序质量控制与alpha多样性指数的变化

Table 3 Change of high-throughput sequencing quality control and alpha diversity indexes of rumen bacteria

项目
Items		 时间Time/d SEM P值
P-value		 差异倍数
Difference multiple
-7 +7
Clean tags数目Clean tags number 116 538.00 118 154.83 1 115.70 0.50 1.01↑
操作分类单元数目OTU number 3 786.50A 2 656.67B 207.88 <0.01 0.70↓
覆盖率Goods_coverage/% 96.55b 97.33a 0.20 0.05 1.01↑
Chao1指数Chao1 index 5 103.21B 5 361.27A 254.06 <0.01 1.05↑
ACE指数ACE index 5 249.06A 4 065.79B 250.21 <0.01 0.77↓
香农指数Shannon index 10.03A 8.31B 0.32 <0.01 0.83↓
辛普森指数Simpson index 0.997A 0.984B <0.01 <0.01 0.99↓

2.2.2 奶牛产犊前后瘤胃细菌门水平变化

由表4可知,奶牛产后瘤胃厚壁菌门(Firmicutes)、Patescibacteria、Kiritimatiellaeota、螺旋体门(Spirochaetes)、纤维杆菌门(Fibrobacteres)、疣状菌门(Verrucomicrobia)、黏胶球形菌门(Lentisphaerae)和WPS-2的相对丰度较产前极显著降低(P<0.01),变形菌门(Proteobacteria)和蓝藻门(Cyanobacteria)相对丰度较产前极显著增加(P<0.01),放线菌门(Actinobacteria)相对丰度较产前显著增加(P<0.05)。
表4 奶牛产犊前后瘤胃细菌门水平变化(在至少1个样本中占总序列的0.05%且差异倍数≥1)

Table 4 Changes in rumen bacterial phylum levels in dairy cows before and after delivery (accounting for 0.05% of the total sequences in at least one of the samples and difference multiple≥1) %

项目
Items		 时间Time/d SEM P值
P-value		 差异倍数
Difference multiple
-7 +7
厚壁菌门Firmicutes 58.18A 46.86B 2.25 <0.01 0.81↓
拟杆菌门Bacteroidetes 32.01 41.11 2.08 0.20 1.28↑
变形菌门Proteobacteria 2.35B 7.05A 0.73 <0.01 3.00↑
放线菌门Actinobacteria 0.79b 2.65a 0.43 0.02 3.37↑
Patescibacteria 1.93A 0.16B 0.30 <0.01 0.08↓
Kiritimatiellaeota 1.38A 0.13B 0.68 <0.01 0.09↓
广古菌门Euryarchaeota 0.62 0.52 0.09 0.60 0.83↓
无壁菌门Tenericutes 0.40 0.24 0.10 0.46 0.60↓
蓝藻门Cyanobacteria 0.18B 0.53A 0.07 <0.01 2.92↑
浮霉菌门Planctomycetes 0.36 0.16 0.05 0.06 0.45↓
螺旋体门Spirochaetes 0.41A 0.11B 0.06 <0.01 0.26↓
纤维杆菌门Fibrobacteres 0.23A 0.03B 0.03 <0.01 0.14↓
疣状菌门Verrucomicrobia 0.19A 0.05B 0.03 <0.01 0.26↓
黏胶球形菌门Lentisphaerae 0.13A 0.01B 0.02 <0.01 0.07↓
WPS-2 0.17A 0.01B 0.03 <0.01 0.05↓

2.2.3 奶牛产犊前后瘤胃细菌属水平变化

由表5可知,奶牛产犊后瘤胃普雷沃氏菌属7(Prevotella_7)、Shuttleworthia相对丰度较产前显著增加(P<0.05),瘤胃球菌属1(Ruminococcus_1)、瘤胃球菌科_NK4A214群(Ruminococcaceae_NK4A214_group)、瘤胃球菌科_UCG-005(Ruminococcaceae_UCG-005)、瘤胃球菌科_UCG-010(Ruminococcaceae_UCG-010)、厌氧弧菌属(Anaerovorax)、理研菌科RC9肠道群(Rikenellaceae_RC9_gut_group)、克里斯滕菌科R-7群(Christensenellaceae_R-7_group)、真杆菌群属([Eubacterium]_coprostanoligenes_group)、Candidatus_Saccharimonas、Papillibacter、Saccharofermentans相对丰度较产前极显著降低(P<0.01),欧氏菌属(Olsenella)和嗜麦芽窄食单胞菌(Stenotrophomonas)相对丰度较产前极显著增加(P<0.01)。
表5 奶牛产犊前后瘤胃细菌属水平变化(在至少1个样本中占总序列的0.05%且差异倍数≥1)

Table 5 Changes in rumen bacterial genus levels in dairy cows before and after delivery(accounting for 0.05% of the total sequences in at least one of the samples and difference multiple≥1) %

项目
Items		 时间Time/d SEM P值
P-value		 差异倍数
Difference multiple
-7 +7
普雷沃氏菌属1 Prevotella_1 5.35 7.58 1.45 0.47 1.42↑
普雷沃氏菌属7 Prevotella_7 0.04b 15.99a 3.95 0.04 399.75↑
瘤胃球菌属1 Ruminococcus_1 2.79A 0.96B 0.38 <0.01 0.34↓
瘤胃球菌科_NK4A214群
Ruminococcaceae_NK4A214_group		 4.32A 1.74B 0.54 <0.01 0.40↓
瘤胃球菌科_UCG-005
Ruminococcaceae_UCG-005		 2.62A 0.25B 0.38 <0.01 0.10↓
瘤胃球菌科_UCG-014
Ruminococcaceae_UCG-014		 1.88 1.28 0.20 0.16 0.68↓
瘤胃球菌科_UCG-010
Ruminococcaceae_UCG-010		 2.12A 0.23B 0.30 <0.01 0.11↓
解琥珀酸弧菌属Succiniclasticum 1.03 1.68 0.28 0.27 1.63↑
厌氧弧菌属Anaerovorax 1.05A 0.15B 0.15 <0.01 1.42↓
欧氏菌属Olsenella 0.35B 1.30A 0.19 <0.01 3.71↑
嗜麦芽窄食单胞菌
Stenotrophomonas		 <0.01B 2.91A 0.44 <0.01 363.75↑
理研菌科RC9肠道群
Rikenellaceae_RC9_gut_group		 9.35A 1.93B 1.22 <0.01 0.21↓
克里斯滕菌科R-7群
Christensenellaceae_R-7_group		 9.57A 1.44B 1.37 <0.01 0.15↓
真杆菌群属
[Eubacterium]_coprostanoligenes_group		 2.12A 0.63B 0.25 <0.01 0.30↓
毛螺菌科NK3A20群
Lachnospiraceae_NK3A20_group		 0.95 2.42 0.51 0.16 2.54↑
Shuttleworthia 0.08b 2.87a 0.61 0.01 35.88↑
Candidatus_Saccharimonas 1.53A 0.15B 0.25 <0.01 0.10↓
Papillibacter 1.01A 0.05B 0.15 <0.01 0.05↓
Saccharofermentans 3.28A 0.62B 0.48 <0.01 0.19↓

2.3 奶牛产犊前后瘤胃真菌的变化

2.3.1 瘤胃真菌高通量测序质量控制与alpha多样性指数

基于HiSeq2500 PE250高通量测序平台对12个样本瘤胃液真菌区系的ITS2区域进行测序,测序数据经过质量控制后,共得到1 428 120条优质序列数,每个样本平均的clean tags数目为119 010,奶牛产犊前后clean tags数目无显著差异(P>0.05)。样本平均OTU数目为1 111.25,奶牛产后OTU数目显著高于产前(P<0.05)。本试验平均测序覆盖率为99.58%,满足了后续分析要求。奶牛产后Chao1指数、ACE指数显著高于产前(P<0.05),香农指数、辛普森指数较产前极显著降低(P<0.01)。
表6 瘤胃真菌高通量测序质量控制与alpha多样性指数的变化

Table 6 Change of high-throughput sequencing quality control and alpha diversity indexes of rumen fungi

项目
Items		 时间Time/d SEM P值
P-value		 差异倍数
Difference multiple
-7 +7
Clean tags数目Clean tags number 117 852.00 120 168.00 1 544.37 0.48 1.02↑
操作分类单元数目OTU number 1 054.67b 1 167.83a 24.20 0.01 1.12↑
覆盖率Goods_coverage/% 99.61A 99.55B 0.01 <0.01 0.99↓
Chao1指数Chao1 index 1 750.79b 1 977.15a 48.38 0.01 1.13↑
ACE指数ACE index 1 797.16b 2 005.70a 45.28 0.01 1.12↑
香农指数Shannon index 4.63A 2.07B 0.42 <0.01 0.45↓
辛普森指数Simpson index 0.84A 0.38B 0.07 <0.01 0.45↓

2.3.2 奶牛产犊前后瘤胃真菌门水平变化

由表7可知,奶牛产后被子植物门(Anthophyta)相对丰度极显著高于产前(P<0.01),子囊菌门(Ascomycota)和担子菌门(Basidiomycota)相对丰度极显著低于产前(P<0.01),绿藻门相对丰度显著低于产前(P<0.05),新丽鞭毛菌门(Neocallimastigomycota)、纤毛亚门(Ciliophora)和被孢菌门(Mortierellomycota)相对丰度在产犊前后差异不显著(P>0.05)。
表7 奶牛产犊前后瘤胃真菌门水平变化(在至少1个样本中占总序列的0.05%且差异倍数≥1)

Table 7 Changes in rumen fungal phylum levels in dairy cows before and after delivery(accounting for 0.05% of the total sequences in at least one of the samples and difference multiple≥1) %

项目
Items		 时间Time/d SEM P值
P-value		 差异倍数
Difference multiple
-7 +7
被子植物门Anthophyta 46.67B 89.52A 7.13 <0.01 1.92↑
子囊菌门Ascomycota 41.11A 10.17B 5.34 <0.01 0.25↓
新丽鞭毛菌门Neocallimastigomycota 8.11 0.03 2.67 0.14 <0.01↓
担子菌门Basidiomycota 3.02A 0.17B 0.51 <0.01 0.06↓
绿藻门Chlorophyta 0.37a 0.01b 0.09 0.03 0.03↓
纤毛亚门Ciliophora 0.16 0.02 0.05 0.20 0.13↑
被孢菌门Mortierellomycota 0.09 0.01 0.03 0.13 0.11↓

2.3.3 奶牛产犊前后瘤胃真菌属水平变化

由表8可知,奶牛产犊后瘤胃棉属(Gossypium)相对丰度极显著高于产前(P<0.01),曲霉属(Aspergillus)、藜属(Chenopodium)、枝顶孢属(Arocladium)和Acremonium相对丰度极显著低于产前(P<0.01),Glycine、Wallemia、Nigrospora、青霉菌属(Penicillium)相对丰度显著低于产前(P≤0.05),脱重霉属(Debaryomyces)相对丰度产后有降低趋势(0.05<P<0.10)。
表8 奶牛产犊前后瘤胃真菌属水平变化(在至少1个样本中占总序列的0.05%且差异倍数≥1)

Table 8 Changes in rumen fungal genus levels in dairy cows before and after delivery(accounting for 0.05% of the total sequences in at least one of the samples and difference multiple≥1) %

项目
Items		 时间Time/d SEM P值
P-value		 差异倍数
Difference multiple
-7 +7
棉属Gossypium 39.87B 83.29A 7.12 <0.01 2.09↑
酵母菌属Saccharomyces 6.79 4.73 0.30 0.45 0.70↓
曲霉属Aspergillus 10.65A 1.01B 1.53 <0.01 0.10↓
根囊鞭菌属Orpinomyces 7.53 0.03 2.44 0.13 <0.01↓
Glycine 0.81b 4.19a 0.88 0.05 5.17↑
Wallemia 2.41a 0.02b 0.45 0.02 0.01↓
Nigrospora 1.92a 0.02b 0.34 0.02 0.02↓
青霉菌属Penicillium 1.79a 0.41b 0.26 0.02 0.23↓
藜属Chenopodium 1.34A 0.09B 0.22 <0.01 0.07↓
枝顶孢属Sarocladium 1.33A 0.02B 0.22 <0.01 0.01↓
Medicago 0.19 0.70 0.05 0.17 3.68↑
念珠菌属Candida 0.06 0.29 0.10 0.28 4.83↑
鳞孢菌属Scedosporium 0.65 0.06 0.20 0.16 0.09↓
黄孢菌属Chrysosporium 0.56 0.12 0.13 0.10 0.21↓
脱重霉属Debaryomyces 0.60 0.15 0.13 0.07 0.25↓
Acremonium 0.53A 0.03B 0.10 <0.01 0.06↓

3 讨论

3.1 奶牛产犊前后瘤胃发酵参数的变化

与单胃动物相比,反刍动物拥有丰富多样的瘤胃微生物生态系统,饲粮在瘤胃发酵过程中产生大量的VFA是机体主要能量来源[9],其浓度和组成是衡量瘤胃内环境稳态和瘤胃发酵状况的重要指标之一。王富伟等[10]研究结果表明,奶牛干奶期随着饲粮能量水平的提高,瘤胃丙酸浓度显著增加,乙酸/丙酸显著降低,有增加NH3-N浓度的趋势。Minuti等[11]研究发现,奶牛产后饲粮结构的变化导致瘤胃丁酸浓度极显著增加,乙酸浓度显著降低,有增加丙酸浓度、降低乙酸/丙酸的趋势。本试验中,围产期奶牛饲粮结构由分娩前的28.35%精补料(中性洗涤纤维含量为52.3%,粗蛋白质含量为12.0%)转变为分娩后42.11%精补料(中性洗涤纤维含量为31.3%,粗蛋白质含量为16.2%),随着奶牛产后饲粮能量水平的提高,奶牛产后瘤胃TVFA、丙酸浓度较产前极显著增加,乙酸浓度较产前显著增加,乙酸/丙酸极显著降低。在本试验条件下,奶牛产后瘤胃TVFA、丙酸、乙酸浓度的增加可能是由于产后饲粮能量水平的提高,导致易被降解的非结构性碳水化合物比例增加代替了不易被降解的结构性碳水化合物,使乙酸/丙酸也随之改变。
瘤胃pH是评价瘤胃内环境稳态和反映瘤胃发酵效率及健康状况的重要指标之一。在生产实践过程中,奶牛场通常通过增加奶牛产后饲粮中精料比例来满足奶牛泌乳需求,由此导致大量有机酸在瘤胃内蓄积,通常会诱发亚急性瘤胃酸中毒(SARA)的发生[12]。在临床上,SARA的发生往往伴随着瘤胃微生物区系的失衡和瘤胃生理生化状态的改变[13],本研究中,奶牛产后瘤胃液pH由产前7.18降低至产后5.58,同比降低22.28%,存在SARA风险。在本试验条件下,奶牛产犊前后瘤胃液pH存在显著差异,一方面可能是因为奶牛由妊娠末期向泌乳初期过渡,奶牛干物质采食量下降及内分泌激素的剧烈变化,导致奶牛出现免疫抑制和氧化应激,易诱发SARA;另一方面,可能是由于奶牛从围产后期到泌乳初期这一阶段机体能量需求剧增,加之奶牛产后饲粮中精料比例的增加,使精料中非结构性碳水化合物(淀粉、单糖等)在瘤胃微生物作用下快速降解,产生大量VFA使瘤胃pH降低,进而引起机体酸碱失衡,导致奶牛在泌乳早期易发生SARA。本试验初步探讨了奶牛产犊前后瘤胃液pH的变化规律,但泌乳早期SARA对奶牛消化、生产性能等方面的影响还需进一步探究。
NH3-N是饲粮蛋白质在瘤胃中降解的产物,也是微生物合成MCP的重要氮源。通常情况下,瘤胃内NH3-N浓度可以反映瘤胃可代谢蛋白的降解和瘤胃微生物对NH3-N的吸收和利用情况,NH3-N浓度过高或过低都会影响微生物对NH3-N的利用率。相关研究结果表明,随着饲粮精粗比的提高,瘤胃NH3-N浓度呈显著或极显著增加趋势[10,14],与本试验结果不一致。Yang等[15]报道,当瘤胃NH3-N浓度低于5 mg/dL时饲粮在发酵过程中易产生“解偶联”反应,使MCP合成效率降低,而本试验中奶牛在产犊前后瘤胃液NH3-N浓度均低于此值,且MCP浓度在奶牛产后有降低趋势。本试验中,奶牛产后饲粮中粗蛋白质含量较产前提高了35%,但瘤胃NH3-N及MCP浓度较产前分别降低16.60%和38.74%,可能是围产期奶牛经历分娩应激、饲粮结构的剧烈变化等原因,使瘤胃菌群结构发生剧烈变化而造成的结果。

3.2 奶牛产犊前后瘤胃细菌菌群结构的变化

在生产过程中,为应对奶牛产犊后干物质采食量(DMI)低而营养需要量高的挑战,奶牛产犊后饲粮通常由产前高粗料饲粮结构转变为产后高精料饲粮结构,瘤胃菌群结构也随之发生剧烈变化[4]。本试验中,奶牛产后瘤胃细菌OTU数目、Sobs指数、Chao1指数、ACE指数和Shannon指数均极显著低于产前,表明奶牛在产后瘤胃细菌的丰富度和多样性发生剧烈变化,与其他学者报道的关于奶牛产犊前后瘤胃菌群变化趋势[9,16]相似。奶牛产犊前后瘤胃微生物的结构和组成存在显著差异,可能是奶牛所处的生理阶段的不同使奶牛产犊前后瘤胃微生物区系存在差异,或是处于不同生理阶段的奶牛瘤胃微生物所面临的内部和外部应激反应不同所导致的结果。在本试验条件下,奶牛产犊前后瘤胃优势菌门为厚壁菌门、拟杆菌门、变形菌门和放线菌门等,与其他学者报道结果[17-18]一致。Zhu等[19]和Pitta等[4]通过对过渡期奶牛瘤胃菌群结构分析发现,随着奶牛由妊娠末期进入泌乳期,瘤胃厚壁菌门、拟杆菌门和变形杆菌门相对丰度较产前有着显著变化,并且奶牛产后厚壁菌门与拟杆菌门比值较产前均有下降。有研究报道,拟杆菌门与厚壁菌门的比值可作为肠道菌群生态失调的标志,比值上升提示肥胖性疾病的发生,比值下降提示炎症性肠道疾病的发生[20-21]。也有报道指出,变形菌门是动物胃肠道菌群平衡的关键指示物,如果变形菌门的相对丰度明显增加,可以间接表明动物胃肠道菌群失调[22]。本试验中,厚壁菌门相对丰度较产前极显著降低,变形菌门相对丰度较产前极显著升高,且厚壁菌门与拟杆菌门相对丰度由产前1.82%下降至1.14%,表明奶牛从妊娠末期到泌乳初期瘤胃微生物菌群结构出现明显变化。
普雷沃氏菌属是拟杆菌门的优势菌属,与机体淀粉、纤维素和蛋白质的降解密切相关,并且对调节瘤胃内环境具有积极作用[23]。本试验中,奶牛产后普雷沃氏菌属7相对丰度较产前显著增加,与Tajima等[24]研究结果一致,可能是由于低纤维饲粮能加速普雷沃氏属的增殖,使其在瘤胃微生物区系中快速定植优势生态位[25],奶牛产犊前后其饲粮结构存在明显差异,与产犊前比较,奶牛产后饲粮纤维含量降低,淀粉含量提升,可发酵底物量提升,从而加速了普雷沃氏菌属的增殖并定植有利生态位。瘤胃球菌属在瘤胃中可以降解粗料中的纤维素、半纤维素和木质素,产生大量的纤维素酶和木聚糖酶,通过酶作用生成乙酸[24],本试验中,奶牛产后瘤胃球菌属1、瘤胃球菌科_NK4A214群、瘤胃球菌科_UCG-005和瘤胃球菌科_UCG-010相对丰度较产前极显著降低,推测瘤胃球菌属相对丰度在奶牛产犊前后变化剧烈,可能是由于奶牛产后饲粮中精料比例提升粗料比例降低,且奶牛DMI处于较低水平,使瘤胃中纤维降解优势菌-普雷沃氏菌属的相对丰度也随之发生显著变化。研究表明,理研菌科在降解纤维多糖方面发挥重要作用[26],并且与肠道健康调控密切相关[27]。本研究中,随着奶牛产后饲粮精料水平的增加,奶牛理研菌科RC9肠道群相对丰度较产前极显著降低,与徐晓锋等[28]报道的在高精料条件下,理研菌科RC9肠道群相对丰度增加的结果不一致,推测可能由于奶牛所处生理阶段及饲粮组成不同而使理研菌科RC9肠道群相对丰度变化趋势出现差异。综上所述,为了深入了解奶牛产犊前后瘤胃发酵特点,对瘤胃细菌的动态学变化规律有待进一步研究。

3.3 奶牛产犊前后瘤胃真菌菌群结构的变化

相关研究报道,饲粮结构对瘤胃厌氧真菌丰度和多样性有重要影响,当饲喂粗料比例较高的低能饲粮时瘤胃真菌丰度高于高精料饲粮[29]。本试验条件下,随着奶牛产后饲粮能量水平的提升,奶牛产后瘤胃真菌相对丰度显著高于产前,与其他学者报道的关于奶牛产犊前后瘤胃真菌变化趋势[16,30]不一致,可能由于奶牛产后DMI尚未恢复及奶牛产犊前、取样间隔时间过短等原因有关。徐晓锋等[31]研究发现,随着饲粮精料水平的提高,奶牛瘤胃真菌多样性也随之提高,与本试验研究结果不一致,可能由于围产期奶牛受分娩应激和激素水平变化等因素的影响,瘤胃内环境发生变化,使瘤胃机能减弱,尽管奶牛产后饲粮的泌乳净能已由产前的5.98 MJ/kg提升至7.24 MJ/kg,但由于奶牛产后DMI仍处于较低水平,导致瘤胃真菌的适应能力降低。
研究发现,瘤胃内厌氧真菌是具有穿透植物细胞壁组织,并分泌纤维素酶、半纤维素酶和酯酶等多种高活性的纤维素降解酶作用的一类功能菌,在纤维性物质的消化和提高反刍动物粗料利用价值方面具有重要意义[32-33]。大量研究证明,目前已分离到的瘤胃真菌几乎都具有降解饲料纤维的功能,在消除瘤胃细菌影响的条件下,瘤胃真菌能降解62%的饲料纤维[34]。高爱武[35]的试验发现,在高纤维饲粮条件下去除瘤胃厌氧真菌后,显著降低了绵羊瘤胃干物质、中性洗涤纤维和酸性洗涤纤维的降解率,再次引入瘤胃厌氧真菌后瘤胃纤维物质的消化率有提升的趋势。通过对奶牛产犊前后瘤胃菌群结构分析发现,奶牛产犊前后优势菌门为被子植物门和子囊菌门,瘤胃棉属和酵母菌属为相对丰度最高的菌属,与其他学者[31,36]报道的关于奶牛瘤胃真菌区系菌群分布结果差异较大,猜测可能受奶牛所处生理阶段及饲粮结构不同等原因的影响。本研究发现,被子植物门和棉属为奶牛产犊前后瘤胃真菌优势群体,目前被子植物门和棉属在农作物领域研究较多,其在反刍动物瘤胃内的分布情况及其功能注释有待进一步研究。子囊菌门是瘤胃真菌界中最大的一类群体,主要参与木质素和角蛋白等物质的降解,隶属子囊菌门的曲霉属是一类分布广泛的丝状真菌,在纤维素、半纤维素和木质素等物质的降解过程中起重要作用[37],本试验中,奶牛产后子囊菌门和曲霉属的相对丰度极显著低于产前,推测随着奶牛产后饲粮中中性洗涤纤维含量降低(由产前52.3%降至产后31.3%),瘤胃纤维降解优势菌对粗纤维的降解能力也随之降低。本试验初步探讨了奶牛产犊前后瘤胃真菌结构组成,其优势菌属和核心菌属及其功能注释还需要进一步探究。

4 结论

本研究中,健康荷斯坦奶牛产犊前后瘤胃内环境发生了显著变化,瘤胃细菌、真菌丰富度和多样性发生剧烈变化。这表明随着奶牛由妊娠末期进入泌乳初期,由于奶牛所处生理阶段的不同和产后饲粮结构的改变等因素,奶牛在产前7 d至产后7 d这一阶段,瘤胃内环境和微生物区系发生了显著性应答改变。

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