A preliminary assessment of the reproductive morphology and endocrinology of European blind cave salamander Proteus anguinus

The European blind cave salamander Proteus anguinus , or olm, is an endangered species endemic to the Dinaric Karst with specific ecology, biology and reproduction. Despite its importance in sustainable conservation, the reproductive biology of the olm is still poorly understood due to the absence of sexual dimorphism, sex chromosomes being homomorphic, gonadal anomalies, a prolonged reproductive cycle and drawbacks of the current methodology. To gain insight into the sex identification and reproductive cycles of the olm, we combined visual observations and histological examinations of the gonads with in vitro analyses of vitellogenin, sex hormones and their metabolites. The applied approach revealed the hormonal oscillations during the reproductive cycles and the variability of sex-related traits and allowed a comparison of the methods used. Detection of cloacal swelling and observable oocytes has proven to be straightforward, yet only moderately reliable, and is limited to specific periods of the reproductive cycle. In contrast, analysis of vitellogenin and sex steroids in blood plasma provided insights into the dynamics of gametogenesis, while the ratio of testosterone to 17-β-estradiol was shown to be a reliable marker for sex identification. Analysis of sex steroid metabolites in fecal samples proved to be a promising non-invasive approach for long-term monitoring of hormonal oscillations. The advantages, disadvantages and reliability of methods for identification of sex and phases of the reproductive cycle, which are essential for further conservation efforts of this endangered amphibian, are discussed.