Synergistic effects of

Background and Aim: Enteric methane emissions from ruminants represent a major loss of dietary energy and contribute significantly to greenhouse gas accumulation. Phytogenic feed additives and lipid supplements have emerged as sustainable strategies to modulate rumen fermentation and mitigate methanogenesis. This study evaluated the synergistic effects of Piper ornatum (PO) leaf powder and lemuru fish oil (LFO) on in vitro rumen fermentation characteristics, nutrient digestibility, and methane reduction. Materials and Methods: A 4 × 4 factorial randomized block design was used, with four levels of PO (0%, 3%, 6%, and 9%) and LFO (0%, 1.5%, 3%, and 4.5%), and three replications. The basal diet was incubated with buffered rumen fluid for 48 h at 39°C. Parameters measured included microbial protein synthesis (MPS), protozoa population, partial and total volatile fatty acids (VFAs), ruminal pH, ammonia (NH Results: The combined supplementation significantly influenced rumen fermentation (p < 0.05). Methane production and total gas output decreased markedly with increasing additive levels, with the lowest methane value (7.46 mL/g DM) observed at 6% PO and 4.5% LFO. Protozoa populations declined, indicating antimethanogenic effects. In contrast, MPS (up to 405.47 mg/100 mL), total VFA concentration (up to 123.51 mM), and NH Conclusion: The combination of 6% PO and 4.5% LFO provided the most favorable balance between methane mitigation, fermentation efficiency, and nutrient utilization. This synergistic strategy integrates phytogenic bioactive compounds and polyunsaturated fatty acid-mediated hydrogen diversion, offering a promising approach for sustainable ruminant production. However, in vivo validation is required to confirm long-term effectiveness under practical feeding conditions.