Testosterone identifies hatchling sex for Mojave desert tortoises (Gopherus agassizii)

Abstract

The threatened Mojave desert tortoise (Gopherus agassizii) exhibits temperature-dependent sex determination, and individuals appear externally sexually monomorphic until sexual maturity. A non-surgical sex identification method that is suitable for a single in situ encounter with hatchlings is essential for minimizing handling of wild animals. We tested (1) whether plasma testosterone quantified by enzyme-linked immunosorbent assay differentiated males from females in 0-3 month old captive hatchlings, and (2) whether an injection of follicle-stimulating hormone (FSH) differentially elevates testosterone in male hatchlings to aid in identifying sex. We validated sex by ceolioscopic (laparoscopic) surgery. We then fit the testosterone concentrations to lognormal distributions and identified the concentration below which individuals are more likely female, and above which individuals are more likely male. Using a parametric bootstrapping procedure, we estimated a 0.01-0.04% misidentification rate for naïve testosterone samples, and a 1.26-1.39% misidentification rate for challenged (post-FSH injection) testosterone samples. Quantification of plasma testosterone concentration from small volume (0.1 mL) blood samples appears to be a viable, highly accurate method to identify sex of 0-3 month old hatchlings and could be a valuable tool for conservation measures and investigation of trends and variation in sex ratios for in situ wild nests.

Figure 1

Mean change in blood plasma testosterone concentration from naïve sample to sample collected after follicle-stimulating hormone (FSH) challenge for captive hatchling Mojave desert tortoise (Gopherus agassizii; n = 23) in Henderson, Nevada, USA in 2019. Sex was validated by coelioscopy at ~ 1.3 year old. Regression lines from a repeated-measures analysis of variance (rANOVA) by sex and challenge status are overlaid on box plots of the log-transformed data. Data points have been horizontally jittered for clarity.

Figure 2

Plasma testosterone concentrations for hatchling (0–3 months old) Mojave desert tortoises (Gopherus agassizii) from Clark Co., Nevada, USA before and after follicle-stimulating hormone (FSH) challenge performed in 2019, with sex validated by coelioscopy at ~ 1.3 years old. Data are ordered by naïve concentrations, and naïve/challenged samples are paired by individual. Individuals with only one sample are indicated with a letter above the column (N = naïve only, C = FSH-challenge only). Asterisk (*) indicates column was truncated for visualization and the actual concentrations are written below.

Figure 3

Coelioscopic images of gonads from captive hatchling (0–3 months old) Mojave desert tortoise (Gopherus agassizii) performed in Clark Co., NV, USA in 2021. (a) Well-differentiated immature testis; (b) Well-differentiated immature ovary demonstrating several follicles; (c) Poorly differentiated gonad; and (d) Small retained yolk sac or Meckel’s diverticulum on the small intestine.

Figure 4

Four-parameter logistic regression curves for the standard dilution series and the pooled sample dilution series, including 95% confidence intervals, for Mojave desert tortoise (Gopherus agassizii) blood plasma testosterone concentration determined from enzyme-linked immunosorbent assay. Samples were collected in San Bernardino Co., CA, USA in 2015.

Figure 5

Fitted probability density functions for blood plasma testosterone concentrations of captive hatchling (0–3 months old) Mojave desert tortoise (Gopherus agassizii) collected in Clark Co., Nevada, USA in 2019. Sex was confirmed by coelioscopic surgery in 2021. Observed concentrations shown as colored bands beneath density plot. Observed concentrations from wild hatchlings (0 day old) of unknown sex collected in Clark Co., NV, USA 2017–2021 shown as black bands above density plot. Area in gray indicates unobserved concentrations between male and female samples. (a) Naïve samples; (b) Follicle-stimulating hormone (FSH) challenged samples.