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Alfalfa (Medicago sativa L.) silage (AS) is an important feedstuff in ruminant nutrition. However, the high content of non-protein nitrogen in AS often leads to a poor ruminal nitrogen fixation. Different pre-ensiling treatments, i.e. varying dry matter concentrations, wilting intensities and sucrose addition, improve the quality and true protein preservation in AS and show substantial effects on the in vitro ruminal fermentation pattern. Hitherto, it is unknown how these pre-ensiling treatments affect the ruminal microbiota composition and whether alterations at the microbial level can explain the observed differences in ruminal fermentation. Therefore, differently treated AS were incubated in a rumen simulation system and samples from the liquid and solid phase were collected two and seven days after first incubating AS, i.e. representing an early (EAT) and late (LAT) adaption time point, respectively. Subsequently, microbial DNA was extracted and both qPCR and 16S rRNA gene amplicon sequencing analyses were performed. At the EAT, high dry matter concentration and sucrose addition increased archaea in the liquid (P = 0.001) and anaerobic fungi in the solid phase (P < 0.001). At the LAT, sucrose addition increased anaerobic fungal concentrations in the solid phase (P < 0.001). In contrast, archaea in the solid phase were decreased by sucrose treatment (P = 0.050), but increased in the liquid phase (P = 0.014). Bacterial loads were not affected by pre-ensiling treatments at both adaption time points. From EAT to LAT, prokaryotic alpha diversity was not affected in the solid phase (P = 0.060), but decreased in the liquid phase (P = 0.034), which may indicate an adaption towards the altered conditions that were provoked by AS incubation. Likewise, redundancy analysis of the sequencing data at genus level revealed that sucrose addition (P = 0.001) and adaption time point (P = 0.001) as well as its interaction (P = 0.001) significantly contributed to explaining the variation in the microbial community composition. In summary, sucrose addition was the main influencing factor among the pre-ensiling treatments and together with the adaption time point predominantly affected the microbial community composition in both phases of the in vitro rumen model. |