The shift in animal welfare standards towards free-range housing for laying hens in the EU has raised questions about changes in dietary composition. Accurate assessment of outdoor plant material intake is crucial for effective feeding strategies. This study introduces an approach using n-alkanes as markers to determine plant intake in laying hens, involving n-alkane recovery rate assessment, discriminant analysis and linear equation-solving for both qualitative and quantitative assessment, respectively, considering systematic n-alkane combinations. Two diets: a standard commercial diet and a diet incorporating 1% alfalfa were tested. Chemical analyses showed an altered n-alkane profile due to alfalfa inclusion, resulting a recovery rates ranging from 30-44% depending on the n-alkane type and diet. Statistical analysis revealed significant differences in recovery rates among the different alkanes for the same diets and between the diets for the same alkane, together with an interaction between n-alkane carbon chain length and initial concentration in the diet. The method accurately predicted plant inclusion, with a slight overestimation (2.80%) using the combination C25-C29-C33. Accurate qualitative classification of the animals based on fecal n-alkanes profiles was observed. The study successfully demonstrated the utility of n-alkanes for estimating dietary composition, providing a non-invasive approach for future free-range studies.
Keywords: alfalfa; free-range; laying hens; n-alkanes; outdoor consumption; recovery rate.