Development of a Methodology for Measuring Oxytocin in Feces: Insights from a Preliminary Study in Captive Lions ().

Oxytocin (OT) has emerged as a central focus in stress regulation and animal welfare research. While commonly measured in blood, saliva, and urine, its presence in feces remains unexplored. Fecal OT (fOT) could be highly valuable for wildlife studies, where direct handling is impractical. This study explored OT detection in feces using lions () as a model species. The objectives included developing a reliable fOT extraction protocol, describing baseline fOT patterns in captive lions, and evaluating its relationship with fecal glucocorticoid metabolites (fGM) under non-stressful conditions. Fecal samples from 16 lions in stable social groups across five Spanish zoos were collected over two to six weeks. A methanol-based extraction and commercial enzyme immunoassay were successfully validated for fOT quantification. Concentrations ranged from 3.00 to 296.64 ng OT/g of feces, with notable intra- and inter-individual variability. Age, sex, and contraceptive status had no significant influence on fOT ( > 0.05), but concentrations differed significantly between zoos ( < 0.001). No consistent relationship was found between fOT and fGM levels under baseline conditions. This study is the first to demonstrate OT detection in feces. The validated methodology provides a foundation for investigating fOT as a biomarker of stress and welfare in wild and domestic species.