Primer-Dependent Insights into Rumen Microbiota and Methanogen Shifts Induced by Orange Peel Secondary Feed in Dairy Sheep

Ruminant livestock production faces rising challenges related to feed costs, sustainability, and methane (CH₄) emissions, with the rumen microbiome playing a central role. This study evaluated the effects of processed and unprocessed orange peel waste, valorized as secondary feedstuff, on rumen microbial composition and methanogen abundance in dairy sheep while assessing primer-dependent biases in microbial detection. Eighteen mid-lactation Chios ewes were assigned to three isonitrogenous and isoenergetic diets: control, 11% processed orange peel, and 11% unprocessed orange peel, over an 84-day trial. Rumen samples collected on days 0 and 84 were analyzed using Oxford Nanopore sequencing with full-length 16S (V1-V9) and prokaryotic (V3-V4) primers. Firmicutes (39.5-58.0%) and Bacteroidota (20.0-37.4%) predominated across diets, while Methanobacteria (6.9-8.8%) were detected exclusively with the prokaryotic primer. Orange peel inclusion attenuated the rise of Proteobacteria in controls and stabilized Prevotella populations. Notably, the processed orange peel diet reduced Methanobacteria abundance by 19.3% (<i>p</i> < 0.05) after 84 days, suggesting enhanced antimethanogenic effects. These results highlight both the methodological relevance of primer selection and the potential of citrus by-products as sustainable feed ingredients that promote rumen microbial stability and contribute to methane mitigation in dairy sheep production.