Abstract
BackgroundAmphibian declines are now recognized globally. It is also well known that many anurans do not reproduce easily in captivity, especially when held over long periods, or if they require hibernation before breeding. A simple method to induce spawning and subsequent development of large numbers of healthy tadpoles is therefore required to meet research and conservation goals.MethodsThe method is based on simultaneous injection of both female and male leopard frogs, Lithobates pipiens (formerly called Rana pipiens) with a cocktail of a gonadotropin-releasing hormone agonist (GnRH-A) and a dopamine antagonist. We call this the AMPHIPLEX method, which is derived from the combination of the words amphibian and amplexus. Following injection, the animals are thereby induced, and perform amplexus and natural fertilization under captive conditions.ResultsWe tested combinations of a GnRH agonist with 2 different dopamine antagonists in L. pipiens in the breeding season. The combination of des-Gly10, D-Ala6, Pro-NHEt9-GnRH (0.4 micrograms/g body weight; GnRH-A) with metoclopramide hydrochloride (10 micrograms/g body weight; MET) or domperidone (DOM) were equally effective, producing 89% and 88% successful spawning, respectively. This yielded more than 44,000 eggs for the 16/18 females that ovulated in the GnRH-A+MET group, and more than 39,000 eggs for the 15/17 females that ovulated in the GnRH-A+DOM group. We further tested the GnRH-A+MET in frogs collected in the wild in late autumn and hibernated for a short period under laboratory conditions, and report a low spawning success (43%). However, GnRH-A priming 24 hours prior to injections of the GnRH-A+MET cocktail in animals hibernated for 5–6 weeks produced out-of-season spawning (89%) and fertilization (85%) comparable to those we observed for in-season spawning. Assessment of age and weight at metamorphosis indicated that L. pipiens tadpoles resulting from out-of-season spawning grew normally and metamorphosed successfully.ConclusionWe provide evidence for successful captive breeding of the leopard frog, L. pipiens. This simple protocol can be used to obtain large numbers of eggs in a predictable, timed manner.
Highlights
Experiment 1: Large-scale induction of spawning in the spring breeding season of L. pipiens One pair of saline-injected control animals was observed in amplexus on Day 3 This couple remained in amplexus for 2 days but did not spawn
On Day 3, 5/18 pairs were in amplexus and 2 egg masses were laid in the gonadotropin-releasing hormone agonist (GnRH-A)+MET group
On Day 3, 9/17 pairs were in amplexus and one egg mass was laid in the gonadotropin-releasing hormone (GnRH)-A+DOM group
Summary
It is well known that many anurans do not reproduce in captivity, especially when held over long periods, or if they require hibernation before breeding. Global amphibian loss is considered a major factor in the contemporary "sixth mass extinction" according to Wake and Vreedenberg [1]. Perhaps 30% of known amphibians are endangered. It was Nace [6] 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. Northeastern Ontario populations of L. pipiens have been declining over the last 40 years [11]
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