In the cave salamander, Proteus anguinus, sex identification is difficult due to the absence of sexual dimorphism and homomorphic sex chromosomes. The aim of this work was to develop a non-destructive and reliable approach for sex identification based on analysis of (i) sex-specific genetic markers with polymerase chain reaction, (ii) the protein vitellogenin (Vtg) by sodium dodecyl sulfate polyacrylamide gel electrophoresis and (iii) the testosterone (T) and 17-β-estradiol (E2) and their metabolites by enzyme-linked immunosorbent assays. The results were partially evaluated by histological analysis of the gonads. We found that females are identified when the concentration of E2 in the blood exceeds 450 pg/ml. In addition, Vtg allows the recognition of vitellogenic stage of oogenesis and is detected after a gradual increase in E2 over several months, followed by the visible oocytes. Multiple measurements of E2 and Vtg in female confirmed the maturation of the oocytes, while an unexpected decrease in E2 concentration and Vtg during oogenesis coicides with their degeneration, which was also confirmed by a decrease in the size and number of visible oocytes. Males are identified when the blood T concentration exceeds 450 pg/ml. The increase in T conc. coincides with a swollen cloaca, but sex identification by a swollen cloaca is not reliable. With the approaches used, the analysis of T and E2 in mucus and their metabolites in faeces was not successful. Vtg was not detected in mucus, liver and ovarian homogenates. Genetic sex-specific markers known in some amphibians could not be confirmed in P. anguinus. The developed approaches allow sex identification during the active gametogenesis and will be an important tool for the purpose of captive breeding of P. anguinus.
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