Using the Ratio of Urine Testosterone to Estrone-3-Glucuronide to Identify the Sex of Chinese Giant Salamanders (Andrias davidianus).

Simple Summary: Due to environmental pollution, habitat loss, and overutilization, the wild population of Chinese giant salamanders (Andrias davidianus) is decreasing continuously. Therefore, artificial proliferation and release are effective ways to restore the wild population of this species. However, the sex of Chinese giant salamanders (CGSs) needs to be confirmed before reintroduction or rejuvenation into the wild. The sex identification of amphibians is difficult and is usually conducted through blood hormone testing, ultrasound, or even the observation of gonads and other invasive sampling methods, and the operation is cumbersome and expensive. As one of the main aspects of animal welfare, non-invasive or minimally invasive sampling is increasingly important in wildlife research. As one of the raw materials of non-invasive sampling, urine has been considered by more and more researchers engaged in wildlife physiology and biochemistry. Our objective was to identify the sex of CGSs by detecting the ratio of hormone testosterone (T) to estrone-3-glucuronide (E1G) in urine collected by minimally invasive sampling. Perhaps this method can be used for the determination of sex in other animals, especially amphibians. Minimally invasive sampling was used to determine the sex of Chinese giant salamanders (Andrias davidianus). Urine samples (n = 25) were collected from 6 adults in the breeding season and from 19 individuals (7 adults and 12 juveniles) in the non-breeding season. The hormone testosterone (T) and estrone-3-glucuronide (E1G) in urine were collected from Chinese giant salamanders (CGSs), and the hormone extracts were analyzed by enzyme immunoassays (EIA). The data demonstrated that the urine T concentration of the male CGSs was significantly higher than that of the females during the breeding season (p < 0.05) and even more pronounced during the non-breeding season (p < 0.01). The urine E1G concentration of the males was less pronounced than that of the females during the breeding season (p < 0.01) and significantly lower during the non-breeding season (p < 0.05). The urine T/E1G values of all the male salamanders were significantly higher than those of the females (p < 0.01) during both the breeding season and the non-breeding season. An interesting pattern was found in this study: the value of urine log10(T/E1G) of the male CGSs was higher than 1, whereas the value for the females was lower than 1, during both the breeding and non-breeding seasons, and in the adult and sub-adult age groups of CGSs. There were 25 salamanders in this study and the accuracy rate reached 100% by using a log10(T/E1G) value of 1. The results of the log10(T/E1G) value provide new insight into the future development of the sex identification of CGSs and also lay the foundation for accurate sex identification in the preparation for artificial release. This is the first study to show that the T/E1G ratio in urinary hormones is reliable for the sex identification of CGSs. Additionally, urinary hormone T/E1G measures are promising sex identification tools for amphibian or monomorphic species and for those whose secondary sex characteristics are visible only during the breeding season. [ABSTRACT FROM AUTHOR]