Modulation of Rumen Microbial Communities in Tropical Holstein Dairy Cows through Lemon Peel Essential Oil Supplementation: A Metagenomic Analysis

Lemon peel essential oil (LPEO) is a promising natural additive for modulating the rumen microbiota in dairy cattle. This study evaluated the effects of LPEO supplementation on fecal microbial diversity and composition in tropical Holstein cows, with focus on methanogenic archaea and fiber-degrading bacteria. Cows received 0, 2.5, or 5.0 mL/head/day of LPEO. Overall microbial diversity remained stable across treatments (Shannon index: P = 0.4838; Simpson index: P = 0.497), and species richness increased (Control: 28.0, High LPEO: 31.0; P = 0.0572), indicating preserved ecosystem stability. The Firmicutes/Bacteroidetes (F/B) ratio remained optimal (>3.0) and increased with LPEO dose (Control: 3.57; High LPEO: 3.94), suggesting enhanced fiber degradation. Despite non-significant p-values, strong dose-dependent trends were observed: Bacteroidota declined (slope = –0.819, R² = 0.702), Firmicutes increased (slope = 0.557, R² = 0.467), and the Archaea/Bacteria ratio decreased by 8.9% (0.00981→0.00891), indicating reduced methanogenic potential. Inter-phylum ecological relationships were preserved, including a perfect negative correlation between Bacteroidota and Firmicutes (ρ = –1.000, P < 0.0001), and network connectivity indices remained stable (0.980–0.982). These results highlight LPEO’s selective antimicrobial activity—modulating microbial structure without broad-spectrum disruption. Limonene (40.60% of LPEO) likely mediated the selective suppression of archaea and enrichment of fiber degraders. The strong predictive capacity of dose-response models supports the biological relevance of observed trends. LPEO supplementation at 5 mL/head/day offers potential for improved rumen function and environmental sustainability in tropical dairy systems, warranting larger-scale validation.