Abstract
BACKGROUND AND AIM: Indigofera zollingeriana is a high-protein tropical legume with potential as a sustainable ruminant feed; however, its ensiling is challenged by rapid proteolysis and ammonia accumulation. Incorporating lactic acid bacteria (LAB) inoculants and natural tannin sources may enhance fermentation quality and nitrogen preservation. This study evaluated the effects of Lactiplantibacillus plantarum inoculant and Acacia mangium leaf tannin extract, individually and in combination, on the chemical composition, ensiling characteristics, in vitro rumen fermentation, digestibility, and metabolomic profiles of Indigofera silage. MATERIALS AND METHODS: A completely randomized design was used with four treatments: (R0) control, (R1) L. plantarum (10(6) colony forming units/g FM), (R2) 1% A. mangium extract, and (R3) combination of both additives, each with five replicates. Silages were fermented anaerobically for 30 days. Analyses included proximate composition, pH, lactic acid, ammonia nitrogen (NH(3)-N), in vitro gas and methane (CH(4)) production, volatile fatty acids (VFA), digestibility, and untargeted metabolomics of both silage and rumen fluid using gas chromatography-mass spectrometry. Data were evaluated using a one-way analysis of variance, Duncan's test, principal component analysis, and partial least squares discriminant analysis. RESULTS: L. plantarum lowered (p < 0.05) silage pH and fiber fractions but increased NH(3)-N due to enhanced deamination. A. mangium tannins effectively suppressed proteolysis, reducing NH(3)-N by 11.85%. Their combination improved (p < 0.05) dry- and organic-matter digestibility (↑ ≈ 9%), increased the propionate proportion by 6.82%, and lowered the acetate-to-propionate ratio, indicating a shift toward more energy-efficient rumen fermentation without significant methane (CH(4)) inhibition. Metabolomic profiling identified 23 key metabolites in silage and 11 in rumen fluid; acacia tannins increased fatty acyl compounds (+14.3%) while LAB enhanced prenol lipids, reflecting modified lipid and oxygen-derived pathways that improve nutrient utilization. CONCLUSION: The combination of L. plantarum and A. mangium leaf extract synergistically improved Indigofera silage quality, nutrient preservation, and digestibility while modulating beneficial metabolites associated with rumen fermentation efficiency. This integrated additive strategy represents a sustainable and locally adaptable approach for tropical ruminant feed production.