Abstract
BackgroundConservation Breeding Programs (CBP's) are playing an important role in the protection of critically endangered anuran amphibians, but for many species recruitment is not successful enough to maintain captive populations, or provide individuals for release. In response, there has been an increasing focus on the use of Assisted Reproductive Technologies (ART), including the administration of reproductive hormones to induce gamete release followed by in vitro fertilisation. The objective of this study was to test the efficacy of two exogenous hormones to induce gamete release, for the purpose of conducting in vitro fertilisation (IVF), in one of Australia's most critically endangered frog species, Pseudophryne corroboree.MethodsMale frogs were administered a single dose of either human chorionic gonadotropin (hCG) or luteinizing hormone-releasing hormone (LHRHa), while female frogs received both a priming and ovulatory dose of LHRHa. Spermiation responses were evaluated at 3, 7, 12, 24, 36, 48, 60 and 72 h post hormone administration (PA), and sperm number and viability were quantified using fluorescent microscopy. Ovulation responses were evaluated by stripping females every 12 h PA for 5 days. Once gametes were obtained, IVF was attempted by combining spermic urine with oocytes in a dilute solution of simplified amphibian ringer (SAR).ResultsAdministration of both hCG and LHRHa induced approximately 80% of males to release sperm over 72 h. Peak sperm release occurred at 12 h PA for hCG treated males and 36 h PA for LHRHa treated males. On average, LHRHa treated males released a significantly higher total number of live sperm, and a higher concentration of sperm, over a longer period. In female frogs, administration of LHRHa induced approximately 30% of individuals to release eggs. On average, eggs were released between 24 and 48 h PA, with a peak in egg release at 36 h PA. IVF resulted in a moderate percentage (54.72%) of eggs being fertilised, however all resultant embryos failed prior to gastrulation.ConclusionsHormone treatment successfully induced spermiation and ovulation in P. corroboree, but refinement of gamete induction and IVF techniques will be required before ART protocols can be used to routinely propagate this species.
Highlights
Conservation Breeding Programs (CBP’s) are playing an important role in the protection of critically endangered anuran amphibians, but for many species recruitment is not successful enough to maintain captive populations, or provide individuals for release
An alternative approach is the use of synthetic hormones, in particular, commercially available analogues of luteinizing-hormone releasing hormone (LHRH) and human chorionic gonadotropin [8,17,18,19]
The number of males releasing sperm was significantly higher in response to hormone treatment compared to the control treatment, but the number of responding males did not significantly differ between hormone treatments
Summary
Conservation Breeding Programs (CBP’s) are playing an important role in the protection of critically endangered anuran amphibians, but for many species recruitment is not successful enough to maintain captive populations, or provide individuals for release. The objective of this study was to test the efficacy of two exogenous hormones to induce gamete release, for the purpose of conducting in vitro fertilisation (IVF), in one of Australia’s most critically endangered frog species, Pseudophryne corroboree. In almost all cases, breeding attempts have failed due to the inherent difficulties associated with simulating the complex combination of social and environmental factors that trigger amphibians to breed [6,5]. In reaction to this captive breeding crisis, there has been a growing interest in determining whether threatened amphibians can be propagated, and genetically managed, using assisted reproductive technologies (ART)[5,7,8]. An alternative approach is the use of synthetic hormones, in particular, commercially available analogues of luteinizing-hormone releasing hormone (LHRH) and human chorionic gonadotropin (hCG) [8,17,18,19]
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