Abstract
This study aimed to investigate the effects of different storage times of the mixed inoculum on in vitro rumen fermentation characteristics, microbial diversity, and community composition. The experiment was divided into five groups, with mixed inoculum composed of fresh rumen fluid and culture medium being stored at 39 °C for 0 h (H0), 12 h (H12), 24 h (H24), 36 h (H36), and 48 h (H48). After 48 h of in vitro fermentation, the fermentation fluid was collected to assess rumen fermentation characteristics and microbial community composition. The H24 group showed higher total gas production, ammoniacal nitrogen levels, and total volatile fatty acids, as well as higher concentrations of individual volatile fatty acids except propionate, compared to the H0 and H48 groups (p < 0.05). The Shannon and Simpson evenness indices were significantly higher in the H0, H12, and H24 groups than in the H48 group (p < 0.05). A total of nine phyla and sixteen genera involved in starch and fiber degradation were found to be more abundant in the H24 or H48 groups (p < 0.05). Moreover, nine predicted metabolic pathways were observed to be significantly enriched in either the H24 or H48 group (p < 0.05). Both principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) analysis revealed distinct clustering patterns among the H0, H12, H24, H36, and H48 groups, and analysis of similarities (ANOSIM) confirmed these significant differences (R = 1.00, p < 0.05). This study demonstrates that the storage time of mixed inoculum influences rumen fermentation characteristics and microbial community composition in a time-dependent manner. It is recommended to use a mixed inoculum that has been stored within 24 h in an anaerobic environment at 39 °C for in vitro rumen fermentation tests. This study offers valuable microbial insights into the storage strategies for mixed inoculum, thereby improving the methodologies for variable control in in vitro rumen fermentation techniques.