基于体外产气法探究不同假单胞菌添加量对人工瘤胃内环境的影响

doi:10.16431/j.cnki.1671-7236.2025.06.011

摘要/Abstract

摘要： 【目的】探究不同假单胞菌添加量对人工瘤胃内环境的影响，筛选出适宜的假单胞菌添加量。【方法】选用4头高产奶牛作为瘤胃液的供体，以供体牛的全混合饲粮(TMR)为发酵底物。采用单因素四水平试验设计，对照组(CON)发酵底物中添加0.5 g TMR，试验组(A～D)在此基础上分别添加1×10⁹、2×10⁹、4×10⁹、8×10⁹ CFU/mL假单胞菌，每个水平设置3个重复，发酵2、4、6、8、10、12、24 h时记录pH及产气量，发酵结束后测定氨态氮(NH₃-N)、微生物蛋白(microbial protein，MCP)和挥发性脂肪酸(volatile fatty acid，VFA)含量；基于16S rRNA、内转录间隔区(internal transcribed spacer，ITS)进行宏基因组测序，分析瘤胃发酵液中微生物区系的变化。【结果】①发酵10 h时，C、D组瘤胃发酵液中pH显著低于CON组(P＜0.05)；各时间点所有处理组间的产气量、NH₃-N、MCP含量均无显著变化(P＞0.05)。②发酵10 h时，B组瘤胃发酵液中乙酸、丁酸、总VFA含量显著或极显著高于CON组(P＜0.05；P＜0.01)；发酵12 h时，A、B组瘤胃发酵液中异丁酸含量显著高于D组(P＜0.05)；发酵24 h时，各试验组瘤胃发酵液中丙酸、丁酸、异戊酸、乙丙比和总VFA含量显著或极显著高于CON组(P＜0.05；P＜0.01)。③C、D组瘤胃发酵液中16S rRNA和ITS的Chao1、Ace指数显著或极显著低于CON组(P＜0.05；P＜0.01)。④在16S rRNA门和属水平上，与CON组相比，D组瘤胃发酵液中变形杆菌门和克雷伯菌属相对丰度均显著升高(P＜0.05)，C和D组琥珀菌属_UCG-001、理研菌属_RC9相对丰度显著或极显著降低(P＜0.05；P＜0.01)；B组瘤胃发酵液中解琥珀酸菌属相对丰度显著高于C、D组(P＜0.05)，norank_F082菌属相对丰度显著高于D组(P＜0.05)。⑤在ITS门和属水平上，与CON组相比，B、C、D组瘤胃发酵液中柯达酵母属相对丰度显著升高(P＜0.05)；B组放线菌属相对丰度显著升高(P＜0.05)。【结论】本试验条件下，2×10⁹ CFU/mL假单胞菌对奶牛瘤胃发酵参数无副作用，增加了VFA的产生，同时促进了纤维降解菌的增殖，因此，假单胞菌适宜添加量为2×10⁹ CFU/mL。

关键词: 体外产气法; 假单胞菌; 瘤胃发酵液; 微生物区系

Abstract: 【Objective】 This study was aimed to investigate the effect of different Pseudomonas additions on the artificial rumen internal environment,and screen the appropriate Pseudomonas additions.【Method】 Four high-yielding dairy cows were selected as the donors of rumen fluid,and the total mixed ration (TMR) of donor cows was used as the fermentation substrate.A one-way four-level experimental design was used,the fermentation substrate in control group (CON) was supplemented with 0.5 g TMR,and fermentation substrate in experimental groups (A-D) were supplemented with 1×10⁹,2×10⁹,4×10⁹ and 8×10⁹ CFU/mL Pseudomonas,respectively,three replicates were set up for each level.pH and gas production were recorded at 2,4,6,8,10,12 and 24 h of fermentation,and the contents of ammoniacal nitrogen (NH₃-N),microbial protein (MCP) and volatile fatty acid (VFA) were measured at the end of fermentation.16S rRNA and internal transcribed spacer (ITS) were used for macro-genome sequencing to analyze the changes in the microbiota of rumen fluid.【Result】 ①At 10 h of fermentation,the pH of rumen fermentation broth in groups C and D was significantly lower than that in CON group (P＜0.05),and there were no significant changes in gas production,NH₃-N and MCP contents among all treatment groups at each time point (P＞0.05).②At 10 h of fermentation,the contents of butyric acid and total VFA in rumen fermentation broth of group B were significantly or extremely significantly higher than those of CON group (P＜0.05 or P＜0.01).At 12 h of fermentation,the isobutyric acid content of rumen fermentation broth in groups A and B were significantly higher than that in group D (P＜0.05).At 24 h of fermentation,the contents of propionic acid,butyric acid,isovaleric acid,acetic acid/propionic acid and total VFA in rumen fermentation broth of each experimental group were significantly or extremely significantly higher than those of CON group (P＜0.05 or P＜0.01).③The Chao1 index and Ace index of 16S rRNA and ITS in rumen fermentation broth of groups C and D were significantly or extremely significantly lower than those of CON group (P＜0.05 or P＜0.01).④At the level of phylum and genus of 16S rRNA,compared with CON group,the relative abundance of Proteobacteria and Klebsiella in rumen fermentation broth of group D was significantly increased (P＜0.05),and the relative abundance of Succinivibrionaceae_UCG-001 and Rikenellaceae_RC9_gut_group in groups C and D were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).The relative abundance of Succiniclasticum in rumen fermentation broth in group B was significantly higher than that in groups C and D (P＜0.05),the relative abundance of norank_F082 was significantly higher than that in group D (P＜0.05).⑤At the level of phylum and genus of ITS,compared with CON group,the relative abundance of Kodamaea in rumen fermentation broth of groups B,C and D was significantly increased (P＜0.05),and the relative abundance of Anaeromyces in group B was significantly increased (P＜0.05).【Conclusion】 Under the condition of this experiment,2×10⁹ CFU/mL Pseudomonas had no side effects on rumen fermentation parameters in dairy cows,which increased the production of VFA,and also promoted the proliferation of fiber-degrading bacteria.Therefore,the appropriate amount of Pseudomonas to be added was 2×10⁹ CFU/mL.

Key words: in vitro gas production method; Pseudomonas; rumen fermentation broth; microbiota

中图分类号:

S831.1

韩一铭, 张照杰, 高宇凤, 杨文华, 任晓丽, 宋超, 石冬梅, 章越, 马梦辉, 皇甫和平, 王金明. 基于体外产气法探究不同假单胞菌添加量对人工瘤胃内环境的影响[J]. 中国畜牧兽医, 2025, 52(6): 2569-2581.

HAN Yiming, ZHANG Zhaojie, GAO Yufeng, YANG Wenhua, REN Xiaoli, SONG Chao, SHI Dongmei, ZHANG Yue, MA Menghui, HUANGFU Heping, WANG Jinming. Exploring the Effect of Different Pseudomonas Additions on the Artificial Rumen Internal Environment Based on in vitro Gas Production Method[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2569-2581.

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