The World Conservation Union (IUCN) currently recognises Eleutherodactylus locustus from Puerto Rico and Plectrohyla acanthodes from Mexico and Guatemala as critically endangered, and they are recommended for ex situ breeding by several amphibian conservation organizations (e.g. AZA, ATAG, RCP, and AARK). Because of their very small and delicate bodies, conventional injectable methods developed for hormonally stimulating spawning in larger frogs and toads are undesirable, because this can result in trauma and injury. Recently, a protocol was developed for inducing spawning in frogs by co-injection of a gonadotropin-releasing hormone agonist (GnRHa) with metoclopramide-HCl (MET), a dopamine antagonist (Amphiplex; Trudeau et al. 2010 Rep. Biol. Endo. 8, 36). The goal of this study, therefore, was to determine the effectiveness of Amphiplex using 3 routes of exogenous treatments: (1) direct IP injection (0.4 µg of GnRHa + 10 µg of MET per gram of body weight), (2) injection into prey (1 µg of GnRHa + 25 µg of MET per insect) before feeding, and (3) a 60-min immersion in 10 mL of pond water containing 0.17 µg of GnRHa + 0.42 µg of MET per microliter. The northern cricket frog (Acris crepitans), a very common local species, was used as a model because it is also small, delicate, and difficult to handle. A total of 110 cricket frogs were caught locally either late or after their normal breeding season, sexed as males or females, and segregated into a control group that was housed similarly ex situ but not exposed to Amphiplex and the 3 Amphiplex experimental groups. The frogs were monitored daily for egg production and behavioural amplexus (potential spermiation). The results are summarised as follows (Table 1): of the frogs exposed to the Amphiplex-supplemented pond water, 84% of 38 females deposited eggs, and 6 pairs were observed in amplexus (indicating spermiation). Approximately 27% of more than 2242 eggs were found to develop cleavage divisions, and tadpoles were first observed in vitro after 4 days. In conclusion, to our knowledge this is the first report indicating that transdermal absorption of a reproductive hormone cocktail (e.g. Amphiplex) may serve in the application of assisted reproductive technology to increase the populations of critically endangered amphibians such as frogs and toads that may be too small or delicate for conventional methods of exogenous hormone treatments that require handling and injections.
Table 1.Treated northern cricket frogs
Ex situ seed baiting to isolate germination-enhancing fungi for assisted colonization in Paphiopedilum spicerianum, a critically endangered orchid in China
