Global amphibian loss is considered a major factor in the contemporary "sixth mass extinction" according to Wake and Vreedenberg . The potential detrimental ecological impacts have been discussed and debated extensively [1–5]. Perhaps 30% of known amphibians are endangered. Significantly, it was Nace  more than 40 years ago, who recognized that some populations of Lithobates pipiens (formerly Rana pipiens) in the U.S.A. were already declining in the mid-1960s. Today, Western North American populations, including those in Alberta, British Columbia, Colorado, and Nevada have dramatically declined [7–10]. Northeastern Ontario populations of L. pipiens have also been declining over the last 40 years .
Nace and colleagues [6, 12] were probably amongst the earliest to suggest the need for captive breeding and eventual domestication of frogs. Timed breeding in captivity is therefore a critical step for the propagation of any threatened or endangered species. To address this challenge, Nace et al. established the University of Michigan Amphibian Facility in the late 1960s [6, 12]. They reported [6, 12] that spawning in captivity was possible but involved injections of pituitary extracts, which necessitates sacrifice of adult leopard frogs, thus defeating the main purpose of a captive colony of a species in decline. The costs, risks, inefficiencies and inappropriateness of injection of pituitary extracts have been discussed by Clulow et al. . Unfortunately, the early attempts [6, 12] at induced breeding did not lead to establishment of a spawning method for captive leopard frogs. This was at a time before the discovery of the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH), and little was known about the neuronendocrine control of reproduction in frogs.
While it is now known that GnRH and GnRH agonist treatments can stimulate pituitary luteinizing hormone (LH) release in frogs [14, 15], they may not effectively induce spawning without co-treatments with other hormones or neuroactive agents [16–18]. This is indicative of the existence of an inhibitory neuroendocrine mechanism controlling the surge release of LH required for ovulation and spawning. Until recently, this possibility had not been considered important in frog reproduction despite clear but limited evidence to the contrary. Electrolytic lesions in the hypothalamus and infundibular regions of the hibernating frog Rana temporaria increased GnRH and LH release and advanced spawning times, thereby establishing the existence of an LH-inhibitory system in the frog neuroendocrine brain. It is known that these brain areas contain the catecholamine dopamine (DA)  and DA type 2 receptors have been found in the frog pituitary [21, 22]. Immunocytochemical visualization of DA neuronal fibres in the hypothalamus and median eminence of Rana ridibunda indicates that DA can be delivered to the pituitary . The DA agonist bromocriptine can inhibit LH release and ovulation in R. temporaria in some situations . Moreover, long-term implantation of silastic pellets containing the DA antagonist metoclopromide (MET) induced ovulation in hibernating R. temporaria. These data indicate that DA is an important inhibitor of LH release in frogs as it is in numerous fish species, birds and some mammals, including sheep and humans . Browne et al.  explored the effects of combinations of hormones on spawning in Bufo fowlerii. In that study, they used the DA antagonist pimozide and concluded that pimozide may increase spawning in some situations and hormone combinations. However, pimozide is not specific to DA receptors, and acts on adrenergic and serotoninergic receptors in addition to DA receptors , so it’s use should be avoided. Nevertheless, together these studies led us to test several dopamine antagonists in L. pipiens.
In our first report on hormonal induction of spawning in L. pipiens, it was clear that the combination of des-Gly10, D-Ala6, Pro-NHEt9-GnRH (GnRH-A) and the specfic DA D2-receptor antagonist MET gave the best results. We named the approach the AMPHIPLEX method, a term that derives from the combination of the words amphibian and amplexus. Amplexus refers to the specific reproductive behaviour of frogs where the male grasps the female, helping to stimulate ovulation and after some delay fertilizes the eggs as they are laid. In one year in the breeding season, we obtained fertilized egg masses from 100% of females, while in the second year this was only 60% . We considered these in-season spawning results for L. pipiens a good first step . On the other hand, out-of-season breeding success was very low in L. pipiens, indicating that significant improvements are needed.
Here we report on the efficient, large-scale spawning induction during the reproductive season of L. pipiens following co-injection of GnRH-A and either of the DA antagonists MET or domperidone. Given the efficacy and ease of use of the GnRH-A+MET combination, we also successfully induced breeding out-of season and obtained thousands of viable tadpoles. In our case, the main reason for this planned breeding is to obtain healthy tadpoles in a timed manner for physiological, ecotoxicological and epidemiological studies [28, 29]. In the long-term our goal is to establish captive colonies so that harvesting of wild eggs can be stopped because the iconic North American frog L. pipiens is unfortunately in decline in several regions of the traditional range [7–12].