Moringa oleifera leaf extract enhances rumen degradability and modifies methanogen communities in vitro

The development of innovative feed resources for livestock is crucial for ensuring nutrient adequacy while reducing greenhouse gas emissions. We aimed to evaluate the effects of Moringa oleifera leaf extract (ML) supplementation on in vitro nutrient degradability, net gas production (GP), ruminal fermentation, methane (CH₄) emissions, and methanogen community structure using a semi-automated in vitro gas production system. Methanogen-specific 16S rRNA genes were amplified through nested PCR and then sequenced with Sanger sequencing. Microbial analyses were conducted using 16S rRNA sequencing. A basal diet (50% concentrate and 50% forage) was incubated in vitro for 24 h as a control (no additives) and compared to diets supplemented with ML at 1.0, 2.0, and 3.0 mL/100 g dry matter (DM), designated ML1, ML2, and ML3, respectively. GC-MS profiling of ML revealed that glycerin (82.08%), unsaturated fatty acid derivatives such as linoleic acid, and minor bioactive sulfur- and nitrogen-containing compounds (e.g., L-cystathionine, homocysteine derivatives) were the major constituents. These compounds exert antimicrobial, membrane-disrupting, and redox-modulating effects, which provide the basis for the proposed mechanisms by which ML influences rumen fermentation and methanogenesis. Supplementation with ML significantly reduced net GP (linear, P < 0.001; quadratic, P = 0.002) and CH₄ production (linear, P = 0.033) across all levels. Similarly, truly degradable dry matter (TDDM; linear, P = 0.038) and truly degradable organic matter (TDOM; linear, P = 0.016) decreased, whereas the partitioning factor increased with ML1 and ML2 supplementation (quadratic, P = 0.002). Ruminal pH and ammonia nitrogen (NH₃-N) concentrations remained unaffected. However, ML treatments reduced total volatile fatty acids (linear, P = 0.009; quadratic, P = 0.003) and butyrate concentrations (linear, P < 0.001). Acetate and propionate concentrations were reduced by ML1 and ML2 (quadratic, P = 0.005). In contrast, ML3 increased isobutyrate (linear, P = 0.004; quadratic, P = 0.012) and isovalerate (linear, P = 0.023; quadratic, P = 0.012) levels. Protozoal enumeration showed that Diplodinium spp. counts decreased with ML (linear, P = 0.008), while Epidinium spp. counts were reduced by ML1 (quadratic, P = 0.048). Phylogenetic analysis of 16S rRNA gene sequences indicated that ML supplementation altered the rumen methanogen community, with distinct shifts toward Methanobrevibacter smithii and M. woesei in ML2 and ML3, respectively. These findings suggest that ML selectively inhibits methanogenic archaea, potentially contributing to reduced CH₄ emissions and altered fermentation profiles.