Ultrasound imaging improves hormone therapy strategies for induction of ovulation and in vitro fertilization in the endangered dusky gopher frog (Lithobates sevosa)

Establishing captive breeding populations of amphibians is an important conservation strategy to safeguard against ongoing declines of wild populations and provide broodstock for reintroduction programs. The endangered dusky gopher frog (DGF) has never naturally reproduced in captivity and requires breeding intervention to sustain the population. Methods for inducing ovulation in female DGFs using hormone therapies have not been evaluated. To address this need, we tested four exogenous hormone treatments to induce ovulation in DGFs (n = 11/treatment), including: treatment (A) gonadotropin-releasing hormone agonist (GnRHa); (B) GnRHa with dopamine antagonist metoclopramide hydrochloride; (C) GnRHa and human chorionic gonadotropin (hCG) and (D) GnRHa with hCG following two low hCG priming doses. Treatments B, C and D resulted in a significantly greater (P < 0.0125) number of ovulating females compared to the control (no hormone); Treatment A was not different from control. For ovulating females, the number of eggs, relative fecundity and cleavage rates of eggs were compared between the four hormone treatments and initial ultrasound grade. Between treatments, there was no difference in number of eggs or relative fecundity; however, Treatments A and D resulted in higher (P < 0.05) cleavage rates than Treatment C, but were not different from Treatment B. Ultrasound imaging was used to assess the ovarian state of DGF females prior to and following hormone therapy. A grading scale (Grades 1-5) was developed to characterize ovarian states. Ultrasound grade was found to be a significant (P = 0.002) predictor for ovulation following hormone treatment, with only high-grade females (Grades 3-4) ovulating in response to hormones. Ultrasound grade did not influence egg numbers or cleavage rate (P > 0.05). Results demonstrate multiple hormone therapies are available for stimulating ovulation in female DGFs and ultrasonography is a valuable tool to inform hormone therapy. Ultimately, these reproductive technologies are critical to enhance breeding and reintroduction efforts for the DGF.