Validation of enzyme immunoassays for quantifying sex steroid hormones in tropical screech owls (Megascops choliba).

ABSTRACT

Androgens and estrogens are steroid hormones that regulate reproductive processes in both males and females. Monitoring plasma levels of these steroids or their metabolites present in feces, offers diagnostic support for assessing the reproductive status of animals. Immunoassays are commonly used methods for quantifying these hormones, but their protocols require species-specific validation to ensure reliability. The objective of this study was to perform analytically and biologically validation of enzyme immunoassay (EIA) kits for measuring testosterone (T), estradiol (E2), faecal androgen metabolites (fAM), and faecal estrogenic metabolites (fEM) in the tropical screech owl (Megascops choliba). Serum and fecal samples were collected from six adult females and six males both before and during breeding season, with males' gonadal activity assessed using electroejaculation (EE). The parallelism test confirmed the immunogenic similarity of the antigens in the estradiol and testosterone standards and the antigens in the serum samples and fecal extracts of M. choliba. Additionally, the EIA kits displayed nearly 100% recovery rates, and showed coefficients of variation ranging from 8% to 14% at the intra-assay level and from 10% to 16% at the inter-assay level, underscoring result reliability and consistency. In males, the highest serum T and fAM levels were recorded concurrently with the presence of spermatozoa in samples collected via EE. Although females did not exhibit oviposition events, significantly higher E2 and fEM levels were observed in August compared to May, suggesting potential seasonal variations in estrogenic hormone production. Fecal androgen and estrogen levels were significantly different between sexes in August, with males having higher fAM and females having higher fEM levels. Overall, the immunoassays validated in this study were found to be efficient in diagnosing reproductive activity in owls.