Temporal dynamics of ruminal metabolome and fermentation in response to molasses-treated Guinea grass-cowpea silage

Molasses is widely used to improve silage quality, but little is known about how its inclusion during the ensiling of tropical forages affects ruminal degradability kinetics and metabolomic responses upon subsequent microbial fermentation. This study evaluated the temporal dynamics of ruminal fermentation, nutrient degradability, and metabolomic responses to molasses-treated Guinea grass-cowpea silage using in vitro assays. Two treatments were compared: untreated silage (GC) and silage treated with 8% molasses (GCM), each prepared in quadruplicate. Ruminal fermentation parameters and metabolomic profiles were assessed over 24 h, whereas nutrient degradability kinetics and cumulative gas production were determined over 72 h. GCM increased short-chain fatty acids production and estimated CO₂ and CH₄ concentrations during the first 4 h of incubation, while improving dry matter (DM) and organic matter (OM) effective degradabilities (51.66 vs. 48.52% and 49.48 vs. 45.72%, respectively). In contrast, crude protein effective degradability was reduced in GCM (47.57 vs. 53.72%), accompanied by transiently lower branched-chain fatty acids production. Metabolomics revealed increased abundance of furanone derivatives together with selected antioxidant and plant-derived metabolites such as biochanin A-β-d-glucoside (log₂FC = 2.54) under GCM. Meanwhile, several amino acid deamination products and lipid oxidation-related metabolites, including indole-3-acetate (log₂FC = -9.06) and 9-HODE (log₂FC = -1.38), were decreased. These metabolic shifts suggest that molasses supplementation altered substrate utilization patterns during ruminal fermentation by favoring carbohydrate- and antioxidant-associated metabolism while moderating protein and lipid catabolism. Overall, molasses supplementation during the ensiling of Guinea grass-cowpea improved DM and OM ruminal fermentability and modified subsequent metabolic responses.