Muhammad Fadhlirrahman Latief, Osfar Sjofjan, Jasmal Ahmari Syamsu, Suyadi Suyadi
This study evaluated rumen fermentability and volatile fatty acid (VFA) profiles in response to graded inclusion levels of Indigofera zollingeriana pellets, interpreted through predicted rumen microbial metabolic functions. An in vitro experiment was conducted using a completely randomized design with three dietary treatments: T0 (control), T1 (low Indigofera inclusion), and T2 (moderate Indigofera inclusion), all supplemented with 0.25% arginine–tannin complex on a dry matter basis. After 48 h of incubation, in vitro dry matter degradation (IVDMD), in vitro organic matter degradation (IVOMD), rumen pH, ammonia nitrogen (NH3–N) microbial protein synthesis (MPS), and VFA profiles and ratios were determined. Microbial metabolic functions were predicted using 16S rRNA gene sequencing data, followed by PICRUSt2 analysis to infer shifts in microbial functional potential. In vitro dry matter degradation (57.19–62.99%) and organic matter degradation (57.29–66.71%) did not differ among the treatments (P > 0.05), indicating that fermentation responses were not driven by differences in substrate degradation. The concentrations of individual VFAs, total VFA production, ammonia nitrogen (NH3-N) concentration, and rumen pH were not significantly affected by the dietary treatments (p > 0.05). However, significant shifts in VFA ratios were observed. T1 showed a lower acetate-to-propionate ratio and a higher proportion of propionate relative to the total VFA compared with T0 (p < 0.05), indicating a more glucogenic fermentation pattern. At higher inclusion levels (T2), similar ratio shifts were accompanied by numerical reductions in fermentative outputs, suggesting a state of metabolic constrained ruminal response rather than enhanced fermentation patterns. Principal component analysis (PCA) and PICRUSt2-based heatmap visualization revealed that T1 was associated with enrichment of carbohydrate degradation and propionate-related fermentation pathways, whereas T2 showed increased adaptive metabolic pathways related to respiration, sulfur metabolism, and purine degradation. In conclusion, a low inclusion level of Indigofera zollingeriana pellets shifted rumen fermentation toward a more glucogenic metabolic profile, whereas moderate inclusion induced adaptive microbial responses associated with altered fermentative routing rather than reduced substrate degradation. These findings reflect the predicted microbial functional potential inferred from 16S rRNA–based analysis, not direct measurements of metabolic activity. Copyright: 2026 by the authors.
Doctoral Program in Animal Science, Faculty of Animal Science, Universitas Brawijaya, Malang, Ketawanggede, 65145, Indonesia; Faculty of Animal Science, Universitas Brawijaya, Malang, Ketawanggede, 65145, Indonesia; Faculty of Animal Science, Universitas Hasanuddin, Makassar, Tamalanrea, 90245, Indonesia
