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