Testing minimally invasive imaging methods for phenotypic sex determination in immature frogs

Amphibians are regularly encountered in exotic veterinary practice, and knowledge of their sex can be extremely important for various programs for breeding and species conservation, as well as for veterinary care. However, sex identification remains challenging because many amphibians lack secondary sexual characteristics until reaching maturity, and genetic sex may not always correspond to phenotypic sex. Consequently, visual inspection of the gonads is often the most reliable method for identifying sex in amphibians. However, non-invasive imaging methods for gonad examination have not been formally validated in juvenile amphibians, where the small size of the gonads may limit visualization success. In our study, we attempted to determine the phenotypic sex of small-sized, immature and mature agile frogs ( Rana dalmatina ) using three non-invasive imaging methods: general veterinary ultrasound, native computed tomography (CT), and magnetic resonance imaging (MRI). Ovaries in immature females were not unambiguously recognizable with any of the tested methods, but became clearly visible during oocyte development, enabling identification in mature females with MRI and ultrasonography. Testes were identifiable in both immature and mature males by MRI but not by ultrasonography or CT. Weighing the costs and benefits of each method in light of our results, we make recommendations for further research into non-invasive sexing of juvenile amphibians.