Identification and Validation of Operational Pain Indicators in Anurans

Amphibian welfare, particularly pain assessment in anurans, remains understudied despite their ecological and biomedical significance. This study aimed to identify and validate operational pain indicators for adult anurans under professional care. A four-phase approach was used: a systematic literature review, expert validation with risk analysis, field validation in a zoological facility, and development of a preliminary pain index. From 158 publications, 16 potential indicators were identified, encompassing behavioural, clinical, and physiological signs. Expert evaluation by 28 professionals from 12 institutions refined this to seven indicators, achieving over 60% consensus: feeding behaviour changes, abnormal behaviour, impaired locomotion, oedema, reduced movement, retained skin post-moulting, and altered respiration. Field validation in 53 anurans confirmed high observability and ease of measurement, with feeding behaviour changes and oedema scoring highest for practicality (93.5% and 93.0%, respectively). These validated indicators provide a science-based foundation for routine welfare monitoring, enabling timely interventions. Their integration into husbandry protocols can enhance ethical standards, improve conservation outcomes, and increase public confidence in amphibian care, paving the way for a standardised anuran pain index.