Calotropis gigantea flavonoid blends reduce wasteful ammoniagenesis and methanogenesis by inhibiting rumen protozoa

Ammonia (NH3-N) and enteric methane (CH4) contribute to nutritional inefficiencies and environmental impacts. This study evaluated the effects of Calotropis gigantea flavonoid-rich extract (CGFE), individual flavonoids (kaempferol [KAE], hesperidin [HES], naringin [NAR]), and binary (KAE + NAR, KAE + HES, NAR + HES) or ternary combinations (KAE + HES + NAR), each at a dose of 4.5% dry matter (DM), on rumen fermentation parameters and rumen protozoa in an in-vitro batch fermentation system incubated for 24 h. Rumen protozoa were counted microscopically, while bacteria and methanogens were quantified by qPCR targeting 16S rRNA and mcrA genes. Data were analysed using one-way ANOVA followed by Tukey's test and Spearman correlation in R v4.4.0. CGFE has a total flavonoid content (TFC) of 48,552 ± 510 µg/g, with HES (37.6%) and NAR (26.8%) as the major components, followed by KAE (14.6%). CGFE and KAE + HES + NAR showed the most pronounced effects. Both increased total volatile fatty acids (VFA) and microbial crude protein (MCP), reduced the NH3-N, acetate-to-propionate ratio, and methanogenesis (p < 0.001). Entodinium was suppressed by CGFE (5.32), KAE + HES + NAR (5.34), KAE + HES (5.44), and KAE (5.45), as compared to the control (CON, 5.5) (p < 0.001), while other genera remained unaffected (p > 0.05). In conclusion, CGFE and ternary flavonoid combinations reduce ammonia and methane, likely through protozoa inhibition and acetate reduction, suggesting the potential of CGFE as a sustainable feed additive for ruminants.