We present a novel method to successfully induce spawning for 1 North American and 3 South American anurans having different reproductive strategies. It is well known that ovulation and sperm release in vertebrates are under the control of the LH . The surge release of LH is naturally induced by species-specific environmental conditions and controlled by a multitude of endogenous neuroendocrine factors and sex steroids [15, 35]. However, reproduction can be inhibited by multiple stresses, including handling and captivity [43, 44]. For example, confinement stress can inhibit reproduction in male rough-skinned newts (Taricha granulosa). The combination of the absence of key environmental cues and confinement stress are among the main causes for unsuccessful breeding of many anurans in captivity. The AMPHIPLEX method was designed to maximally stimulate endogenous LH release thereby inducing a coordinated release of gametes in both females and males, overriding the detrimental effects of stress in captivity.
The combination of a highly active GnRH agonist and a specific DA receptor antagonist offers significant advantage over other hormone-based anuran spawning induction methods. Results using hCG seem to be highly variable in amphibians. One possible explanation for this is that hCG is a human hormone and it does not stimulate the animal to release its own pituitary gonadotropic hormones. Rather, it only partially mimics the actions of anuran LH to activate gonadotropin receptors in the ovary or testis. This was indicated earlier by Licht (1995), and ranids in particular respond poorly if at all to hCG [18, 46]. The AMPHIPLEX method stimulates endogenous LH release, thus avoiding some of the problems seen with heterologous gonadotropins. Most previous methods used multiple injections of a variety of GnRH agonists, sometimes in combination with hCG. This is more labour-intensive, and requires multiple handling of animals, which in itself can be highly stressful . Furthermore, the administration of multiple injections of hCG may also make the animals refractory to the treatment as already demonstrated in fish . By treating frogs with MET, the inhibitory actions of the catecholamine DA on LH are blocked, and GnRH-A more effectively stimulates spawning.
The research groups of R.E. Peter in Canada and H.R. Lin in China pioneered the use of DA antagonists for induction of spawning in fish. However, it was J. Sotowska-Brochocka [30–32] who first made the suggestion that DA may be an important inhibitor of LH release and ovulation in hibernating R. temporaria. Until the current report, this idea had been largely ignored by those attempting hormonal induction of spawning in Anura. Browne et al.  tested a single dose of pimozide in Anaxyrus fowleri. The authors concluded that its effects to improve hormonal induction in anurans were uncertain. Therefore, we are the first to use the combination of a highly active GnRH agonist and a DA antagonist to induce successful spawning in several anurans.
In L. pipiens caught as mature animals in spring 2008 and 2009, we achieved spawning rates (60-100%) highly comparable to those reported for cultured cyprinid fish with a similar single treatment of the combination of GnRH agonists and DA antagonists [26–28]. In frogs, similar doses of LHRH-A have been shown to induce sperm release in Lepidobatrachus laevis, while much higher doses of GnRH-A can induce ovulation in Eleutherodactylus coqui. However in the Wyoming Toads (Anaxyrus baxteri), animals must first be primed with multiple injections of low levels of hCG and GnRH-A in combination to achieve 70-89% spawning success in response to relatively high ovulatory doses of hCG plus GnRH-A .
Our data show that both the type of GnRH agonist and DA antagonist influences reproductive outcome. The results from experiment 1 indicate that GnRH-A plus MET is better than GnRH-A plus PIM, indicating that MET is more effective. Comparison of T2 and T3 in experiment 1 suggests that GnRH-A is more effective than GnRH-B. The exact reason for this is unknown but it likely relates to a combination of higher affinity for the pituitary GnRH receptor and a lower degradation rate of GnRH-A compared to GnRH-B in L. pipiens. Our results on DA antagonists are in contrast to data in goldfish where PIM is more potent than MET in regards to the potentiation of LH release and ovulation induced by GnRH-A . In experiment 2 in spring 2009, we repeated treatments of L. pipiens with GnRH-A and MET. It was shown that 10 μg/g Bwt MET in combination with 0.4 μg/g GnRH-A was somewhat more effective than 5 μg/g Bwt MET in combination with 0.4 μg/g GnRH-A. Therefore, we proceeded to test 10 μg/g Bwt MET in combination with 0.4 μg/g GnRH-A in several scenarios of captive breeding. Additionally, spawning success in L. pipiens was lower in 2009 than 2008. There are 2 main reasons for differences between years. In 2009, we recorded a major failure in juvenile recruitment in the wild population at Bishop's Mill, Ontario where the animals were harvested. This suggests that animals did not overwinter well in 2008-2009. Moreover, actual temperatures in the outdoor tanks at the time of spawning induction were on average 5°C lower in 2009 than in 2008. It was also reported in goldfish that a similar difference (8°C) in water temperature can dramatically affect the LH response to the combined injection of GnRH-A and a DA antagonist . For example, peak induced LH levels can be 3-7 times lower at 12°C compared to 20°C for sexually mature female goldfish in the breeding season .
Presumably mature female and male L. pipiens were caught during the autumn migration when these frogs return to deeper bodies of water for hibernation. We developed an artificial hibernation protocol in which there were no deaths recorded. In experiment 3, we were able to induce out-of-season breeding in 2/8 females that resulted in thousands of viable tadpoles. The reason for this low success rate compared to the in-season breeding is currently unknown, but likely relates to the short hibernation period we induced. Perhaps a longer period of hibernation at 4°C would increase spawning success by promoting vitellogenesis in the females [50, 51]. Nevertheless, the tadpoles grew and metamorphosed normally, although they resulted from spawnings in captivity that were approximately 6 months earlier that the expected time of natural spawning of L. pipiens in Eastern Ontario. An example of one representative animal resulting from this out-of-season breeding is shown (see Additional File 1, Figure S1).
The AMPHIPLEX method was also effective in the 3 tested species from Argentina. The single pair of large C. ornata tested in experiment 4 had been in captivity for 5 years without spawning naturally. This couple produced viable eggs following a single treatment. The tadpoles grew and metamorphosed quickly as expected for this species. Horned-frogs of the genus Ceratophrys are explosive breeders and their macrophagic carnivorous larvae develop in highly ephemeral ponds and have extremely short larval periods with rapid morphological development . We also tested C. cranwelli, which also produced fertilized eggs. It is important to note that this was in the winter period of the Southern Hemisphere, which also represents a successful out-of-season breeding because C. cranwelli is a fossorial species that usually breeds during spring and summer time in the first heavy rains of the year in Formosa Province, Argentina. The method was also successful in O. americanus, which is another fossorial explosive breeder, but occurs in open grasslands and savannahs. During breeding time it is found at shallow, temporary ponds and flooded areas. On average, from the 2 trials with O. americanus, we were able to successfully induce spawning and the production of tadpoles from 6/8 (67%) females. In the three tested species from Argentina, AMPHIPLEX promoted reproduction. Males started to sing and display reproductive behaviour ending in amplexus and spawning in a similar manner that has been described for breeding in nature. We provide photographic evidence of successful spawning and development of individual representatives of 3 Argentinian species at various metamorphic stages (see Additional File 1, Figure S1).