Citation: Silla, A.J.; Hobbs, R.J.; Gilbert, D.J.; Goodall, D.; Parrott, M.L.; Lee, A.; O’Brien, J.K.; Byrne, P.G. Application of Reproductive Technologies to the Critically Endangered Baw Baw Frog, Philoria frosti. Animals 2023, 13, 2232. https://doi.org/10.3390/ ani13132232 Academic Editor: Jean-Marie Exbrayat Received: 8 June 2023 Revised: 1 July 2023 Accepted: 3 July 2023 Published: 7 July 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). animals Article Application of Reproductive Technologies to the Critically Endangered Baw Baw Frog, Philoria frosti Aimee J. Silla 1, * , Rebecca J. Hobbs 2 , Deon J. Gilbert 1,3 , Damian Goodall 3 , Marissa L. Parrott 3 , Adam Lee 3 , Justine K. O’Brien 2 and Phillip G. Byrne 1 1 School of Earth, Atmospheric and Life Sciences, University of Wollongong, Wollongong, NSW 2522, Australia 2 Taronga Institute of Science and Learning, Taronga Conservation Society Australia, Mosman, NSW 2088, Australia 3 Wildlife Conservation and Science, Zoos Victoria, Elliott Avenue, Parkville, VIC 3052, Australia * Correspondence: asilla@uow.edu.au Simple Summary: The integration of reproductive technologies into conservation breeding programs can contribute to the propagation and genetic management of threatened amphibian species. Here, we report on the first application of reproductive technologies to the critically endangered Baw Baw frog, Philoria frosti. We present the results of the successful use of protocols for hormone-induced spawning in male–female pairs, hormone-induced sperm-release in isolated males, and sperm cryopreservation. Abstract: Reproductive technologies (RTs) can assist integrated conservation breeding programs to attain propagation targets and manage genetic diversity more effectively. While the application of RTs to enhance the conservation management of threatened amphibians has lagged behind that of other taxonomic groups, a recent surge in research is narrowing the divide. The present study reports on the first application of RTs (hormone-induced spawning, hormone-induced sperm-release, and sperm cryopreservation) to the critically endangered Baw Baw frog, Philoria frosti. To determine the effect of hormone therapy on spawning success, male–female pairs were administered either 0 μg/g gonadotropin-releasing hormone agonist (GnRHa), 0.5 μg/g GnRHa, or 0.5 μg/g GnRHa + 10 μg/g metoclopramide (MET) (n = 6–7 pairs/treatment), and the number of pairs ovipositing, total eggs, and percent fertilisation success were quantified. To determine the effect of hormone therapy on sperm-release and to establish the peak time to collect sperm post-hormone administration, males were administered 0 IU/g (n = 4), or 20 IU/g hCG (n = 16). Total sperm, sperm concentration, and percent viability were quantified at 0, 2, 4, 6, 8, 10, and 12 h post-hormone administration. Overall, the percentage of pairs ovipositing was highest in the GnRHa + MET treatment, with 71% of pairs ovipositing, compared to 57% and 33% of pairs in the GnRHa and control treatments, respectively. The quantity of sperm released from males in response to hCG peaked at 4 h post-hormone administration, though it remained high up to 12 h. The percent sperm viability also peaked at 4 h post-administration (94.5%), exhibiting a steady decline thereafter, though viability remained above 77% throughout the 12 h collection period. The remaining sperm samples (n = 22) were cryopreserved using established protocols and biobanked for long-term storage and future conservation applications. The mean post-thaw sperm viability was 59%, and the percent total motility was 17%. The results from this preliminary study will direct further applications of RTs to the critically endangered Baw Baw frog to assist with species recovery. Keywords: amphibian; captive breeding; reproductive technologies; ART; gamete-release; oviposition; spawning; sperm; spermiation; cryopreservation; biobanking 1. Introduction The primary objective of conservation breeding programs (CBPs) is to aid threat- ened species recovery by maintaining genetically representative captive assurance colonies Animals 2023, 13, 2232. https://doi.org/10.3390/ani13132232 https://www.mdpi.com/journal/animals