This study aimed to explore the dynamic variations of rumen fermentation characteristics and bacterial community composition during a 24 h in vitro fermentation. A total of twenty-three samples were collected from original rumen fluid (ORF, n = 3), fermentation at 12 h (R12, n = 10), and fermentation at 24 h (R24, n = 10). Results showed that gas production, concentrations of microbial crude protein, ammonia nitrogen, and individual volatile fatty acids (VFA), as well as total VFA and branched-chain VFA concentrations, were higher in R24 when compared with R12 (p < 0.05). However, no significant differences were observed in acetate to propionate ratio and fermentation efficiency between R12 and R24 (p > 0.05). Bacterial diversity analysis found that Shannon index and Simpson index were higher in R24 (p < 0.05), and obvious clusters were observed in rumen bacterial community between R12 and R24. Taxonomic analysis at the phylum level showed that the abundances of Proteobacteria and Fibrobacteres were higher in R12 than that in R24, and inverse results were observed in Bacteroidetes, Firmicutes, Cyanobacteria, Verrucomicrobia, Lentisphaerae, and Synergistetes abundances. Taxonomic analysis at the genus level revealed that the abundances of Rikenellaceae RC9 gut group, Succiniclasticum, Prevotellaceae UCG-003, Christensenellaceae R-7 group, Ruminococcaceae UCG-002, Veillonellaceae UCG-001, and Ruminococcaceae NK4A214 group were higher in R24, whereas higher abundances of Succinivibrionaceae UCG-002, Ruminobacter, and Fibrobacter, were found in R12. Correlation analysis revealed the negative associations between gas production and abundances of Proteobacteria, Succinivibrionaceae UCG-002, and Ruminobacter. Moreover, the abundances of Firmicutes, Rikenellaceae RC9 gut group, Christensenellaceae R-7 group, and Ruminococcaceae UCG-002 positively correlated with VFA production. These results indicate that both rumen fermentation characteristics and bacterial community composition were dynamic during in vitro fermentation, whereas the fermentation pattern, efficiency, and bacterial richness remained similar. This study provide insight into the dynamics of rumen fermentation characteristics and bacterial composition during in vitro fermentation. This study may also provide a reference for decision-making for the sampling time point when conducting an in vitro fermentation for bacterial community investigation.

Recent studies have revealed that LuxS/AI-2 quorum sensing (QS) is the most universal cell-to-cell communication in rumen bacteria; however, it remains unknown how they respond to nutritional stress from a diet shift. This study aimed to explore whether a diet shift could trigger rumen bacterial LuxS/AI-2 QS and its influences on rumen fermentation characteristics and bacterial community diversity and composition. A total of fifteen Hu sheep were selected to undergo a pre-shift diet (Pre, concentrate to forage ratio 75:25) for one month and then abruptly switch to a post-shift diet (Post, concentrate to forage ratio 49:51). Results showed that the serum cortisol and immunoglobulin G concentrations were higher in Post than in Pre (p < 0.05). The microbial density, AI-2 concentration, biofilm formation, and gene expression of ftsH were higher in Post when compared with Pre (p < 0.05), whilst the expression of luxS tended to be lower in Post (p = 0.054). The molar concentration of valerate and fermentation efficiency decreased after the diet shift, while the acetate to propionate ratio and the molar proportion of butyrate were higher in Post compared to Pre (p < 0.05). Moreover, the diet shift increased the richness of ruminal bacteria and the relative abundance of Roseburia, and decreased the relative abundances of Prevotella, Megasphaera, Dialister, Prevotellaceae UCG-001, and Lachnospira (p < 0.05). A difference in trends was also observed in an analysis of similarity (R = 0.1208 and p = 0.064). This study suggests that a diet shift could trigger rumen bacterial LuxS/AI-2 QS by altering microbial density, AI-2 concentration, biofilm formation, and related gene expression, as well as affect the rumen fermentation pattern and bacterial community diversity and composition. This study may provide insight into a potential strategy for relieving nutritional stress via regulating bacterial communication.

This study aimed to explore the dynamic variations in fermentation characteristics, bacterial diversity and community composition at two preservation temperatures as preservation time extended. Six rumen fluid samples collected from high-grain feeding sheep were stored at −20 °C or −80 °C for 0 day, 7 days, 14 days, 30 days, 60 days, 120 days, and 240 days. The results showed that the current preservation temperature did not alter the fermentation characteristics, bacterial diversity and community composition (p > 0.05). The concentrations of ammonia, microbial crude protein, acetate, propionate, butyrate, valerate, and total volatile fatty acids were higher when stored at 60 days (p < 0.05). Preservation time had no influence on bacterial richness and evenness (p > 0.05), whilst the relative abundances of Bacteroidota and Prevotella were numerically higher when stored at 30 days, and the opposite results were observed regarding Firmicutes. Both principal co-ordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) showed clusters among treatments in terms of preservation time and preservation temperature. Analysis of similarities (ANOSIM) also revealed similarities between treatments (p > 0.05). This study indicates that most fermentation characteristics in rumen fluid were altered after a 60-day preservation, whilst the preservation time for rumen bacterial community profile alteration was 30 days. It is recommended to finish the sample determination of rumen fluid within 30 days. This study may assist decision-making regarding the practicable time for rumen fluid determination, as well as viable preservation conditions for inoculum used for in vitro fermentation testing.