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

## TL;DR

Adding orange peel waste to dairy sheep diets can reduce methane emissions and improve rumen health, offering a sustainable solution for livestock farming.

## Contribution

The study demonstrates that processed orange peel waste can reduce methane-producing microbes in sheep rumen, with insights into primer-dependent microbial detection biases.

## Key findings

- Processed orange peel reduced Methanobacteria abundance by 19.3% in sheep rumen.
- Orange peel inclusion stabilized Prevotella populations and limited Proteobacteria increase.
- Prokaryotic primers detected Methanobacteria, highlighting the importance of primer choice in microbial analysis.

## Abstract

Dairy sheep farming is an integral part of the food production chain, but it faces challenges related to high feed costs and environmental impact, especially the release of methane, a strong greenhouse gas produced during digestion. In this study, we tested whether orange peel waste from the juice industry, either used as is or after processing, could be added to sheep diets to improve sustainability and reduce emissions. Over a period of 84 days, we found that including orange peel waste changed the community of microbes in the animals’ rumen in ways that may benefit both them and the environment. Animals receiving orange peel waste did not show an increase in bacteria linked to poor digestive health, as observed in the control group, and the processed orange peel reduced the presence of methane-producing microbes. These results suggest that orange peel waste, normally discarded, can be transformed into a valuable feed ingredient that reduces waste and could help limit greenhouse gas emissions.

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% (p < 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.