In vitro Gas Production and Rumen Microbiome Structural Shifts in Response to Graded Inclusion of Indigofera zollingeriana Pellet Concentrate

This study aimed to evaluate the dose-dependent effects of graded inclusion levels of Indigofera zollingeriana pellet concentrate on in vitro gas production, fermentation kinetics, and microbial community structure. A completely randomized design was applied with three treatments: T0 (0% Indigofera), T1 (13.57% Indigofera), and T2 (27.13% Indigofera), each with four replicates. In vitro gas production was measured over a 48 h incubation period, digestibility was determined post-incubation, and rumen microbiome composition was characterized using 16S rRNA gene sequencing targeting the V3–V4 region. Cumulative gas production decreased significantly with increasing Indigofera inclusion (P < 0.05), with the highest value observed in T0 (184.6 mL), followed by T1 (171.3 mL) and T2 (158.4 mL), indicating reduced fermentability at higher inclusion levels. At the genus level, the relative abundance of key fibrolytic taxa such as Ruminococcus and Xylanibacter decreased with increasing Indigofera inclusion, consistent with reduced gas production and altered fermentation kinetics. In addition, Succiniclasticum, involved in succinate conversion, declined at higher inclusion levels, suggesting shifts in downstream fermentation pathways. The increased representation of fiber-adaptive groups such as the Christensenellaceae R-7 group indicates microbial restructuring under elevated fiber and tannin pressure rather than enhanced fermentative efficiency. Beta diversity analyses (PCoA and UPGMA) demonstrated clear separation of microbial communities in response to graded Indigofera inclusion. Overall, increasing levels of Indigofera zollingeriana in pellet concentrates were associated with reduced in vitro gas production and altered gas fermentation kinetics, accompanied by dose-dependent restructuring of the rumen microbiome, whereas low-level inclusion (T1) maintained relatively balanced fermentation characteristics and microbial stability.