红三叶草提取物对奶牛体外瘤胃发酵中微生物多样性及尿素分解的影响

doi:10.11843/j.issn.0366-6964.2024.06.022

摘要/Abstract

摘要：

旨在通过体外发酵研究红三叶草提取物对瘤胃微生物组成和尿素代谢的影响。试验分为空白对照组(不添加红三叶草提取物)和添加红三叶草提取物处理组，采集3头健康奶牛瘤胃液混合均匀，进行体外发酵培养。分别在培养0、0.5、1、2、4、6、24 h收集发酵液样品，测定尿素、氨氮(NH₃-N)、微生物蛋白、总游离氨基酸的含量。利用16S rRNA基因测序揭示瘤胃微生物多样性、群落组成及功能的变化。本研究发现，红三叶草提取物处理组在发酵4 h内显著降低尿素分解速率，在发酵至2 h后显著降低氨氮含量，在发酵6 h后显著提高瘤胃微生物蛋白的含量，对总游离氨基酸含量无显著影响。16S rRNA基因测序结果显示，添加红三叶草提取物显著增加了体外发酵中微生物α多样性的Chao 1和Shannon指标，改变了体外发酵中瘤胃微生物群落结构。添加红三叶草提取物改变了瘤胃蛋白分解菌瘤胃杆菌属(Ruminobacter)、普雷沃氏菌属(Prevotella)中ASV 35和s_unidentified_rumen_bacterium_RF18的丰度，降低了致病菌嗜胆菌属(Bilophila)的丰度。综上所述，添加红三叶草提取物可降低瘤胃微生物分解尿素的速率，提高瘤胃微生物利用氨氮转化为自身蛋白的能力。

关键词: 红三叶草提取物, 瘤胃, 尿素, 菌群多样性

Abstract:

The purpose of this study was to investigate the effects of red clover extract on rumen microbial composition and urea metabolism in vitro. The experiment was divided into control group (no red clover extract added) and supplement with red clover extract treatment group. Rumen fluid collected from 3 healthy cows were mixed for in vitro fermentation. The fermentation fluid samples were collected at 0, 0.5, 1, 2, 4, 6 and 24 h of fermentation, respectively, and the contents of urea, ammonia nitrogen (NH₃-N), microbial crude protein and total amino acids were determined. 16S rRNA sequencing was used to reveal the changes of microbial diversity, community composition and function in rumen. The results showed that in the red clover extract treatment group, urea decomposition rate was significantly reduced within 4 h of fermentation, ammonia nitrogen content was significantly reduced after 2 h of fermentation, rumen microbial protein content was significantly increased after 6 h of fermentation, and total free amino acid content was not significantly affected. 16S rRNA sequencing results showed that supplemented with red clover extract significantly increased the Chao 1 and Shannon indexes of microbial α diversity and changed the rumen microbial community structure in the in vitro fermentation. The abundance of Prevotella (ASV 35 and s_unidentified_rumen_bacterium_RF18) and Ruminobacter were changed, Bilophil were decreased. In summary, the addition of red clover extract can reduce the rate of urea hydrolyzing by rumen bacteria, and improving the ability of rumen bacteria to convert ammonia nitrogen into microbial crude protein.

Key words: red clover extract, rumen, urea, bacterial diversity

中图分类号:

S823.911.5

刘思佳, 郑楠, 王加启, 赵圣国. 红三叶草提取物对奶牛体外瘤胃发酵中微生物多样性及尿素分解的影响[J]. 畜牧兽医学报, 2024, 55(6): 2510-2518.

Sijia LIU, Nan ZHENG, Jiaqi WANG, Shengguo ZHAO. Effects of Red Clover Extract on Microbial Diversity and Urea Decomposition in Rumen Fermentation of Dairy Cows in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2510-2518.

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参考文献 36

1	KERTZ A F . Review: urea feeding to dairy cattle: a historical perspective and review[J]. Prof Anim Sci, 2010, 26 (3): 257- 272. doi: 10.15232/S1080-7446(15)30593-3
2	HAILEMARIAM S , ZHAO S G , HE Y , et al. Urea transport and hydrolysis in the rumen: a review[J]. Anim Nutr, 2021, 7 (4): 989- 996. doi: 10.1016/j.aninu.2021.07.002
3	刘思佳. 奶牛瘤胃中活性尿素分解菌群多样性分析[D]. 北京: 中国农业科学院, 2020.
	LIU S J. Diversity analyze of active ureolytic bacterial community in the rumen of dairy cows[D]. Beijing: Chinese Academy of Agricultural Sciences, 2020. (in Chinese)
4	GETAHUN D , ALEMNEH T , AKEBEREGN D , et al. Urea metabolism and recycling in ruminants[J]. Biomed J Sci Tech Res, 2019, 20 (1): 14790- 14796.
5	SOUZA V C , AGUILAR M , VAN AMBURGH M , et al. Milk urea nitrogen variation explained by differences in urea transport into the gastrointestinal tract in lactating dairy cows[J]. J Dairy Sci, 2021, 104 (6): 6715- 6726. doi: 10.3168/jds.2020-19787
6	WAGNER J J , ENGLE T E , BRYANT T C . The effect of rumen degradable and rumen undegradable intake protein on feedlot performance and carcass merit in heavy yearling steers[J]. J Anim Sci, 2010, 88 (3): 1073- 1081. doi: 10.2527/jas.2009-2111
7	CECONI I , RUIZ-MORENO M J , DILORENZO N , et al. Effect of urea inclusion in diets containing corn dried distillers grains on feedlot cattle performance, carcass characteristics, ruminal fermentation, total tract digestibility, and purine derivatives-to-creatinine index[J]. J Anim Sci, 2015, 93 (1): 357- 369. doi: 10.2527/jas.2014-8214
8	ZHAO S G , WANG J Q , ZHENG N , et al. Reducing microbial ureolytic activity in the rumen by immunization against urease therein[J]. BMC Vet Res, 2015, 11, 94. doi: 10.1186/s12917-015-0409-6
9	ZHANG Z Y , LI M , ZHANG X Y , et al. A novel urease inhibitor of ruminal microbiota screened through molecular docking[J]. Int J Mol Sci, 2020, 21 (17): 6006. doi: 10.3390/ijms21176006
10	HE Y , ZHANG X Y , LI M , et al. Coptisine: a natural plant inhibitor of ruminal bacterial urease screened by molecular docking[J]. Sci Total Environ, 2022, 808, 151946. doi: 10.1016/j.scitotenv.2021.151946
11	GUTIERREZ-BAÑUELOS H , ANDERSON R C , CARSTENS G E , et al. Effects of nitroethane and monensin on ruminal fluid fermentation characteristics and nitrocompound-metabolizing bacterial populations[J]. J Agric Food Chem, 2008, 56 (12): 4650- 4658. doi: 10.1021/jf800756c
12	PATRA A K , SAXENA J . Dietary phytochemicals as rumen modifiers: a review of the effects on microbial populations[J]. Antonie van Leeuwenhoek, 2009, 96 (4): 363- 375. doi: 10.1007/s10482-009-9364-1
13	赵玉超, 余诗强, 蒋林树. 生物类黄酮调控瘤胃微生态系统的作用——聚焦甲烷减排[J]. 动物营养学报, 2022, 34 (9): 5452- 5465. doi: 10.3969/j.issn.1006-267x.2022.09.002
	ZHAO Y C , YU S Q , JIANG L S . Roles of bioflavonoids in regulating rumen microecosystems: focus on methane emission reduction[J]. Chinese Journal of Animal Nutrition, 2022, 34 (9): 5452- 5465. doi: 10.3969/j.issn.1006-267x.2022.09.002
14	MA J , ZHENG Y M , TANG W J , et al. Dietary polyphenols in lipid metabolism: a role of gut microbiome[J]. Anim Nutr, 2020, 6 (4): 404- 409. doi: 10.1016/j.aninu.2020.08.002
15	STEINSHAMN H . Effect of forage legumes on feed intake, milk production and milk quality-a review[J]. Anim Sci Pap Rep, 2010, 28 (3): 195- 206.
16	ZHAN J S , LIU M M , SU X S , et al. Effects of alfalfa flavonoids on the production performance, immune system, and ruminal fermentation of dairy cows[J]. Asian-Australas J Anim Sci, 2017, 30 (10): 1416- 1424. doi: 10.5713/ajas.16.0579
17	WEATHERBURN M W . Phenol-hypochlorite reaction for determination of ammonia[J]. Anal Chem, 1967, 39 (8): 971- 974. doi: 10.1021/ac60252a045
18	JIN D , ZHAO S G , ZHENG N , et al. Differences in ureolytic bacterial composition between the rumen digesta and rumen wall based on ureC gene classification[J]. Front Microbiol, 2017, 8, 385.
19	CALLAHAN B J , MCMURDIE P J , ROSEN M J , et al. DADA2:high-resolution sample inference from Illumina amplicon data[J]. Nat Methods, 2016, 13 (7): 581- 583. doi: 10.1038/nmeth.3869
20	WANG Q , GARRITY G M , TIEDJE J M , et al. Naïve bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[J]. Appl Environ Microbiol, 2007, 73 (16): 5261- 5267. doi: 10.1128/AEM.00062-07
21	JIN D , ZHAO S G , ZHENG N , et al. Urea metabolism and regulation by rumen bacterial urease in ruminants-a review[J]. Ann Anim Sci, 2018, 18 (2): 303- 318. doi: 10.1515/aoas-2017-0028
22	WANG R , WANG M , UNGERFELD E M , et al. Nitrate improves ammonia incorporation into rumen microbial protein in lactating dairy cows fed a low-protein diet[J]. J Dairy Sci, 2018, 101 (11): 9789- 9799. doi: 10.3168/jds.2018-14904
23	TROYER A F , STOEHR H , Willet M . Hays, great benefactor to plant breeding and the founder of our association[J]. J Hered, 2003, 94 (6): 435- 441. doi: 10.1093/jhered/esg099
24	FLYTHE M , KAGAN I . Antimicrobial effect of red clover (Trifolium pratense) phenolic extract on the ruminal hyper ammonia-producing bacterium, Clostridium sticklandii[J]. Curr Microbiol, 2010, 61 (2): 125- 131. doi: 10.1007/s00284-010-9586-5
25	LIU S J , ZHANG Z Y , HAILEMARIAM S , et al. Biochanin A inhibits ruminal nitrogen-metabolizing bacteria and alleviates the decomposition of amino acids and urea in vitro[J]. Animals, 2020, 10 (3): 368. doi: 10.3390/ani10030368
26	COLEMAN G S , SANDFORD D C . The uptake and utilization of bacteria, amino acids and nucleic acid components by the rumen ciliate eudiplodinium maggii[J]. J Appl Bacteriol, 1979, 47 (3): 409- 419. doi: 10.1111/j.1365-2672.1979.tb01201.x
27	VANHATALO A , KUOPPALA K , AHVENJÄRVI S , et al. Effects of feeding grass or red clover silage cut at two maturity stages in dairy cows.1.Nitrogen metabolism and supply of amino acids[J]. J Dairy Sci, 2009, 92 (11): 5620- 5633. doi: 10.3168/jds.2009-2249
28	CHOWDHURY M R , WILKINSON R G , SINCLAIR L A . Feeding lower-protein diets based on red clover and grass or alfalfa and corn silage does not affect milk production but improves nitrogen use efficiency in dairy cows[J]. J Dairy Sci, 2023, 106 (3): 1773- 1789. doi: 10.3168/jds.2022-22607
29	BRODERICK G A . Utilization of protein in red clover and alfalfa silages by lactating dairy cows and growing lambs[J]. J Dairy Sci, 2018, 101 (2): 1190- 1205. doi: 10.3168/jds.2017-13690
30	SALAMI S A , VALENTI B , LUCIANO G , et al. Dietary cardoon meal modulates rumen biohydrogenation and bacterial community in lambs[J]. Sci Rep, 2021, 11 (1): 16180. doi: 10.1038/s41598-021-95691-3
31	DAO T K , DO T H , LE N G , et al. Understanding the role of Prevotella genus in the digestion of lignocellulose and other substrates in vietnamese native goats' rumen by metagenomic deep sequencing[J]. Animals, 2021, 11 (11): 3257. doi: 10.3390/ani11113257
32	BETANCUR-MURILLO C L , AGUILAR-MARÍN S B , JOVEL J . Prevotella: a key player in ruminal metabolism[J]. Microorganisms, 2022, 11 (1): 1. doi: 10.3390/microorganisms11010001
33	ACCETTO T , AVGUŠTIN G . The diverse and extensive plant polysaccharide degradative apparatuses of the rumen and hindgut Prevotella species: a factor in their ubiquity?[J]. Syst Appl Microbiol, 2019, 42 (2): 107- 116. doi: 10.1016/j.syapm.2018.10.001
34	KOVATCHEVA-DATCHARY P , NILSSON A , AKRAMI R , et al. Dietary fiber-induced improvement in glucose metabolism is associated with increased abundance of Prevotella[J]. Cell Metab, 2015, 22 (6): 971- 982. doi: 10.1016/j.cmet.2015.10.001
35	NATIVIDAD J M , LAMAS B , PHAM H P , et al. Bilophila wadsworthia aggravates high fat diet induced metabolic dysfunctions in mice[J]. Nat Commun, 2018, 9 (1): 2802. doi: 10.1038/s41467-018-05249-7
36	OLSON C A , IÑIGUEZ A J , YANG G E , et al. Alterations in the gut microbiota contribute to cognitive impairment induced by the ketogenic diet and hypoxia[J]. Cell Host Microbe, 2021, 29 (9): 1378- 1392.e6. doi: 10.1016/j.chom.2021.07.004

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