Adult and Juvenile Sex Identification in Threatened Monomorphic Geocrinia Frogs Using Fecal Steroid Analysis
A reliable method for the confirmation of sex in Geocrinia frogs is currently unavailable. A captive colony of Geocrinia frogs was used to determine whether fecal steroid analysis could be used as a noninvasive method of sex diagnosis in mature frogs. Linear discriminate analysis, using measured concentrations of fecal hormones, also was applied to establish whether a reliable model could be developed to verify the sex of juvenile frogs. Fecal samples were collected over a 3-month period from all frogs in 2010, and again from 12 frogs in 2011. Samples were pooled for each individual and tested for testosterone metabolites (TMs) and estrone conjugate metabolites (EMs) using enzyme immunoassays. Mature male frogs had higher (P < 0.01) mean TM : EM ratios than those of mature females, with mature male and female ratio ranges being 100% distinct. Mature female frogs had higher (P < 0.01) mean EM concentrations than those of mature males, but EM concentrations overlapped between the sexes in 18.7% of cases. There was no statistical difference (P = 0.28) between mature males and females in mean or range of TM concentration, with overlap between the sexes in 75% of cases. Juveniles had hormone values similar to those recorded for mature male and female frogs. Generated discriminate rules were able to distinguish (100%) between mature males and females and correctly classified the sex of 75% of the juvenile frogs. It was concluded that fecal steroid analysis offers a promising, noninvasive approach to sex identification in Geocrinia frogs.Abstract
Parallelism between a dilution of standard (A) testosterone (closed circles) and (B) estrone-conjugate (closed circles) and serial dilutions of mature male (open circles), mature female (closed triangles), and juvenile (open triangles) fecal extracts.
Box and whisker plot of raw concentrations of fecal steroids from Geocrinia frogs by using antibodies for testosterone (TM) and estrone conjugate (EM). Center bars represent medians, boxes represent the inter-quartile range and whiskers represent the total range in hormone concentrations (ng/g of dried feces) for each group (M = mature males; F = mature females; J = juveniles).
Projection of individual (N = 64) fecal hormone measurements of Geocrinia frogs, based on two variables (logeEM, logeTM) from a linear discriminate function analysis. Key: univariate discriminate model rule (solid line); bivariate discriminate model rule (dashed line); known males (closed squares), known females (closed circles), juveniles of unknown sex (open triangle and asterisks), juveniles retained by Perth Zoo (asterisks), TM = testosterone metabolites; EM = estrone conjugate metabolites.
Shifts in hormone measurements and sex classifications for eight Geocrinia frogs tested as both juveniles (2010) and as mature frogs (2011). Key: univariate discriminate model rule (solid line); bivariate discriminate model rule (dashed line); initial (2010) juvenile values (closed circles); final (2011) mature values (open circles).
