21 Saving salamanders with sonograms: tracking follicular development with ultrasonography in a variety of caudate species

Captive breeding colonies are crucial hedges against the current amphibian extinction crisis because of their role as genetic refuges for a plethora of threatened species. In caudates, there has been extensive research on assisted reproductive technology (ART) in males, but there is still a need for advancing ART in females. Previous research in female salamander ART has typically administered hormones to encourage spawning, without knowledge of the natural follicular state, which has resulted in unsuccessful breeding events. A solution to these limitations is ultrasonography, which provides a noninvasive method to assess follicular development. This tool is cost effective and transportable, allowing for the assessment of both in situ and ex situ populations. The focus of this study was to use ultrasonography in female caudates to determine the effectiveness of hormone therapy in advancing oocyte development. We hypothesised that follicular maturation would occur as a result of hormone administration and would be detectable via ultrasonography. The target species used in this study were the spotted salamander (Ambystoma maculatum, Least Concern) and the Iranian newt (Neurergus kaiseri, Critically Endangered). Prior to hormone administration, transabdominal ultrasound images were obtained from A. maculatum (N = 5) and N. kaiseri (N = 6) to determine their follicular development state. Each female was graded on an ordinal scale of 1 to 5, looking at the presence or absence of matured oocytes (i.e. 1 signifying pre-vitellogenesis and 5 signifying complete oocyte maturation) and was then intramuscularly administered a prime of 2 IU g⁻¹ of bodyweight of human chorionic gonadotrophin to induce follicular advancement. Follicular development was assessed 24 h later via ultrasound to determine whether hormone administration promoted follicular maturation. Paired t-tests were used to analyse the average female ultrasound grades before and after hormone treatment. For A. maculatum, the average ultrasound grade pre- and post-treatment was 3.0 ± 1.4 and 4.4 ± 0.5, respectively. This analysis found no significant difference between ultrasound grades pre- and post-hormone administration (P = 0.052, t = −2.7); however, post-hormone grades were numerically higher for each individual, indicating that there may have been a positive trending effect of hormones on follicular maturation. For N. kaiseri, the mean ultrasound grades pre- and post-hormone administration were 2.3 ± 1.3 and 3.7 ± 0.8, respectively. The analysis showed a significant increase in ultrasound grade after hormone administration (P = 0.01, t = −4.0), indicating the positive effect that hormones had in aiding development. Ultrasonography is a key tool within ART because it allows for immediate assessment of follicular development in female amphibians. Having the ability to synchronise female oviposition is vital for efficient genetic crossing in threatened and endangered species and it should be utilised in conjunction with other ART to increase the success of captive breeding in amphibian species.