Validation of two enzyme immunoassays for non-invasive glucocorticoid measurement in a lacertid lizard (Podarcis muralis): Effects of pharmacological and biological stimuli on faecal corticosterone metabolites and behaviour

The assessment of stress-related hormone levels using non-invasive methods has gained popularity in mammal and bird welfare, yet its application in reptiles remains limited. Particularly, the exploration of physiological measures such as faecal corticosterone metabolites (FCMs) for reptilian welfare has scarcely been explored. This study aims to validate two enzyme immunoassays (5α-pregnane-3ß,11ß,21-triol-20-one and 11-oxoaetiocholanolone EIA) for monitoring FCM levels in the European common wall lizard (Podarcis muralis). We collected daily faecal samples before (baseline) and after (post-treatment phase) inducing elevated corticosterone levels using transdermal administration of corticosterone (pharmacological treatment) and handling/confinement (biological treatment). We also conducted daily behavioural observations to explore the relationship between stress-related corticosterone changes and behaviour. Although treatments induced significant increases in FCM levels, the effect was much larger in the pharmacological one. Transdermal corticosterone induced a cumulative increase in FCMs over the treatment period, with a higher response observed in females. In contrast, the biological treatment yielded smaller FCM peaks, with no significant sex differences. Overall, 5α-pregnane-3ß,11ß,21-triol-20-one EIA appeared to be more sensitive in detecting these effects. Regarding lizard behaviour, both treatments led to increased hiding and decreased basking compared to baseline. The effects were more pronounced in animals subjected to handling/confinement, despite smaller FCM increases. Our results confirm the suitability of an EIA for monitoring FCMs in both male and female common wall lizards and provide insights into the complexities of using integrated approaches to assess stress, highlighting the need for further research on direct measures to evaluate reptile welfare.