116 DEVELOPMENT OF ASSISTED REPRODUCTION TECHNOLOGIES FOR THE ENDANGERED MISSISSIPPI GOPHER FROG (RANA SEVOSA) AND SPERM TRANSFER FOR IN VITRO FERTILIZATION
Authors: Kouba A. , Willis E. , Vance C. , Hasenstab S. , Reichling S. , Krebs J. , Linhoff L. , Snoza M. , Langhorne C. , Germano J. 
Published: December 2011

Species-specific differences in breeding strategies and physiology have limited the application of assisted reproductive technologies (ART) for critically endangered amphibians in captive assurance colonies. In 2006, the Memphis Zoo (MZ) initiated a program to develop ART for the critically endangered Mississippi gopher frog after natural breeding failed. Standard gamete collection and IVF developed by MZ for reproducing endangered toads such as the Wyoming or boreal toad were applied to the gopher frog with little success, especially hormonal therapy for sperm production. Using the leopard frog as a model species for Ranids, we tested the time and dose dependence of a luteinizing hormone releasing hormone analogue (LHRHa) and hCG on sperm quantity and quality. Initial findings from the leopard frog study were critical in designing the study on gopher frogs. Our objectives were to (1) compare 2 different hormones administered intraperitoneal (500 IU hCG vs 15 μg LHRHa) or their combination on spermiation in gopher frogs; (2) develop in vivo oocyte maturation and ovulation protocols using LHRHa (15 μg) and hCG (500 IU); and (3) transfer this technology to another institution as proof of principle. In gopher frogs, 100 and 83% of the males produced sperm in response to the LHRHa and the combination treatment, respectively, whereas only 16% responded to hCG alone. Sperm concentration peaked at 1 h post-administration for all treatments, with the LHRH/hCG cocktail treatment producing the highest concentration of sperm (mean = 4.6 × 106 ± 1.2 × 106 sperm mL–1, n = 6). No differences in motility were observed between treatments (P > 0.05). For females, a series of priming hormones of hCG and LHRHa were given several months before an ovulatory hormone regimen resulting in ovulation by 100% of the females (n = 6), whereas animals not primed failed to ovulate (n = 4). These 3 separate priming and IVF trials conducted between 2008 and 2010 resulted in each female laying ∼2000 eggs, with an average fertilization rate of 76% for inseminated eggs and hundreds of tadpoles produced. These IVF tadpoles represent the first captive reproduction of gopher frogs and highlight how ART can be applied to conservation and genetic management of threatened species. Subsequently, we tested our IVF protocols on gopher frogs at Omaha's Henry Doorly Zoo using fresh (collected on site) and chilled, shipped sperm from MZ. We collected 6169 eggs from 9 hormone-primed females with all animals ovulating. A portion of the total eggs ovulated were inseminated, resulting in 2401 fertilized eggs (38.9% of total eggs collected) across 18 different male–female pairings leading to viable tadpoles. In addition, sperm transferred overnight from the MZ produced 202/441 fertilized eggs (46%). The transfer of this technology and production of endangered amphibians using chilled, shipped sperm from live animals is a conservation milestone that can be applied to other captive breeding programs.