Metatranscriptome Analysis of Sheep Rumen Reveals Methane Production Changes Induced by <i>Moringa oleifera</i> as a Dietary Supplement

Global warming has become a significant public health concern, with intensive livestock farming as a major contributor. To mitigate greenhouse gas emissions, strategies such as manipulating the ruminal environment with dietary additives are essential. This study evaluated <i>Moringa oleifera</i>, a globally widespread tree with antioxidant, multivitamin, protein-rich, and anti-inflammatory properties, as a feed additive. Rumen fluid was collected from three Pelibuey sheep, homogenized, and subjected to an in vitro fermentation study for 48 h with three alfalfa/moringa ratio treatments: T0 Control (100:0), T1 Low (85:15), and T2 High (70:30). Total RNA was extracted, followed by high-definition sequencing of the metatranscriptome. The sequencing yielded approximately 456 million sequences. A total of 117 phyla were identified and approximately 1300 genera were mapped. Predominant phylum differed by treatment: T0, <i>Firmicutes</i>; T1, <i>Proteobacteria</i>; and T2 with <i>Synergistetes,</i> at least one sample per treatment. Archaea were nearly absent in T1, which explains a statistically significant decrease in methane production. In the Gene Set Enrichment Analysis (GSEA), it was observed that one of the metabolic pathways with a statistically significant difference (<i>p</i>-value < 0.05) was that of methane, specifically in the low moringa treatment (T1) compared to the control (T0). From the functional analysis, differentially expressed enzymes were identified, some of which are involved in the methane metabolic pathway, such as formate dehydrogenase (EC 1.17.1.9) and glycine hydroxymethyltransferase (EC 2.1.2.1), which are intermediates in methane formation. These results suggest that 15% <i>Moringa oleifera</i> supplementation alters ruminal microbiota, reduces archaeal activity, and suppresses methane-related pathways. These findings provide molecular evidence supporting the potential of <i>M. oleifera</i> as a methane mitigation strategy in ruminant nutrition.