120 THE DEVELOPMENTAL COMPETENCE OF TADPOLES PRODUCED IN VITRO FROM THE ENDANGERED DUSKY GOPHER FROG (RANA SEVOSA) USING EXOGENOUS HORMONE TREATMENT

Dusky gopher frogs once existed throughout the states of Mississippi, Alabama and Louisiana. Presently, the USA Fish and Wildlife Service estimates that there are less than 100 frogs left in the wild, with almost all of these residing in a single pond in Mississippi, making the dusky gopher frog America's most endangered frog species. Their habitat has been threatened by residential and forestry development, as well as from fire suppression and the decline of gopher tortoises, whose burrows the frogs use for shelter. The USA Fish and Wildlife Service brought the first dusky gopher frogs into captivity in 2001 and they have been kept at the Memphis Zoo since 2003 and at Omaha's Henry Doorly Zoo since 2004. Natural breeding attempts in captivity have been unsuccessful despite efforts to artificially mimic what is known to be the appropriate seasonal environmental (e.g. light, humidity, temperature) and social cues (e.g. calls from wild frogs during the breeding season) that stimulate reproduction in situ. Also, there was a concern about the incidences of abnormalities (e.g. spindly legs) from inadequate husbandry conditions (e.g. nutritional deficiencies) or diseases in tadpoles and froglets produced ex situ (e.g. dermomycoides). The objectives of this study were (1) to examine the developmental competence of tadpoles produced by exogenous hormone treatment for IVF to complete normal metamorphosis into viable froglets ex situ and (2) to determine the incidence of developmental abnormalities or dermomycoides in the froglets produced ex situ. Males and females were first separated by the identification of black male nuptial pads used during amplexus. Males and females were stimulated to spermiate and ovulate, respectively, using a variation of a standard amphibian hormonal stimulation protocol (Kouba et al. 2012 Reprod. Fertil. Dev. 24). As a result, more than 1460 viable tadpoles (61% of fertilized eggs) were produced from 18 segregated genetic pairings. To date, 602 (41%) of the tadpoles have completed normal metamorphosis into viable froglets (over 6 times the known existing population in situ), 51% were either culled to examine for dermomycoides or died for a variety of known (accidental) and unknown reasons (abnormal appendage development accounting for less than 7%) and 8% have yet to metamorphose after 9 months. The incidence of dermomycoides in the culled individuals was 100%; however, a study being conducted concurrently in their native habitat has confirmed that the organism does exist in situ with the dusky gopher frogs, which act as natural carriers. Efforts are currently in place to establish reintroduction programs for the dusky gopher froglets produced ex situ to increase the dwindling wild population now that tadpoles produced by IVF have been shown to be developmentally competent. In conclusion, IVF can be used to produce tadpoles of the dusky gopher frog with a low incidence of abnormalities, but the tadpoles were found to be carriers of the organism dermomycoides similar to their counterparts in the wild.

196 NONINVASIVE HORMONE MEASUREMENTS FOR GENDER IDENTIFICATION IN MONOMORPHIC ANURANS

Noninvasive techniques have been successful in distinguishing gender in dimorphic amphibian models (Szymanski DC et al. 2006 Zoo Biol. 25, 35-46). The aim of this study was to assess gender-specific fecal hormone metabolite concentrations in 2 dimorphic and 2 monomorphic, critically endangered species of amphibians to assist in ex situ breeding efforts. Fecal samples from 2 dimorphic anurans, the Puerto Rican Crested Toad (Bufo lemur) and the Wyoming Toad (Bufo baxteri) and 2 monomorphic species, the Mountain Chicken Frog (Leptodactylus fallax) and the Dusky Gopher Frog (Lithobates sevosa) were measured by enzyme immunoassay for sex-specific hormone differences (Student’s t-test; P < 0.05) among 6 variables (concentrations of estradiol (E2), progesterone (P), and testosterone (T), and ratios of P:E2, T:E2, and P:T). Values are presented as means ± SEM. Mountain Chicken Frog samples were obtained from individuals of known sex. Dusky Gopher Frog samples were from confirmed females but males were presumed based on subtle physical characteristics. The E2 concentrations (ng g-1) were significantly higher in females than males (P < 0.05) in all species except for the Dusky Gopher Frog. However, males in all 4 species, including the presumptive Dusky Gopher Frog males, exhibited significantly higher P:E2 and T:E2 ratios than females. Additionally, Wyoming Toad and Mountain Chicken Frog females had significantly higher P:T ratios than males (0.19 ± 0.05 v. 0.06 ± 0.00 and 0.34 ± 0.03 v. 0.20 ± 0.02, respectively). Similar to the American Toad (Szymanski et al. 2006), presumptive Dusky Gopher Frog males measured significantly higher T concentrations compared with females (376.4 ± 118.5 ng g-1 v. 174.0 ± 23.1 ngg-1, respectively). Although species-specific differences are apparent, fecal hormone measurements can be used to identify sex in monomorphic species and offer a potential tool for the captive breeding of endangered anurans. Table 1.