| Summary: | Development of alternative forage resources is of great importance to provide necessary nutrients and minimize greenhouse gas emissions in ruminant production. The aim of this study was to examine the effects of dietary supplementation of <i>Moringa oleifera</i> on the production performance and fecal methanogenic community in dairy cows using methyl-coenzyme M reductase α-subunit gene. Sixty-four cows were allocated to one of four treatments: basal diet without <i>M. oleifera</i> (control) or low (3% <i>w/w</i>, M3), medium (6%, M6), or high (9%, M9) supplementation with <i>M. oleifera</i>. This study demonstrated that different supplementation levels of <i>Moringa oleifera</i> in the diet achieved similar feed intake and milk production, but adding 6% of <i>Moringa oleifera</i> improved milk fat content. Two families, two phyla, three genera, and three species in total were identified among the four treatments. The fecal archaeal community in the control treatment was predominated by <i>Methanobrevibacter</i> (39.1% of the total sequence reads) followed by <i>Methanosphaera</i> and <i>Methanocorpusculum</i> at the genus level. The increased abundance of the <i>Methanosphaera</i> genus and <i>Methanosphaera</i> sp. ISO3-F5 species was induced by secondary metabolites of <i>Moringa oleifera</i> in the diet. Results indicated that <i>Moringa oleifera</i> supplementation not only improved dairy product quality but could also potentially reduce methane emissions.
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