Evaluating the quantitative accuracy and application of DNA metabarcoding for dietary reconstruction in ruminants

DNA metabarcoding offers a powerful, non-invasive tool to identify dietary composition with high taxonomic resolution, yet its quantitative accuracy and bias remain a well-recognised limitation across taxa and sample types. This universal challenge is particularly evident in herbivores, where plant material introduces additional amplification constraints. This study evaluates the accuracy of DNA metabarcoding in reconstructing the diets of sheep under controlled feeding trials involving high and low digestibility forage, using two widely used plant DNA barcodes (ITS2 and trnL). A secondary trial tested the detectability and proportional representation of a target species, Medicago sativa, when added to the diet in varying amounts (1%, 5%, 10%). ITS2 provided greater species-level resolution, while trnL showed broader taxonomic coverage but reduced precision. Both markers distinguished diet treatments effectively; however, faecal DNA showed proportional discrepancies from vegetation input, particularly under low-digestibility conditions. M. sativa was reliably detected even at 1% inclusion but was consistently overrepresented in sequence reads. Our findings highlight the strengths and limitations of DNA metabarcoding for herbivore diet studies and underscore the importance of marker choice and the effects of differential digestion biases. These findings demonstrate the need for multi-marker approaches and calibration controls in dietary studies, especially when quantitative interpretation is required. Despite limitations in quantitative accuracy, faecal DNA metabarcoding provides valuable insights into herbivore diet composition and preferences, with future refinements expected to improve its resolution and reliability for ecological monitoring and grazing management.