Captive breeding in the endangered alpine tree frog, Litoria verreauxii alpina

Introduction

Amphibians are experiencing some of the most devastating population declines on the planet (Houlahan et al., 2000; Stuart et al., 2004; Collins, 2010). Many species around the world require intensive conservation measures to protect against extinction. However, captive breeding of amphibians can be a challenging endeavor because species often rely on specific and particular environmental stimuli to induce breeding, which can be difficult to simulate under captive conditions (Kouba, Vance & Willis, 2009; Silla & Byrne, 2019). Assisted reproduction technologies like exogenous hormones can be effective at reducing the need for particular abiotic or biotic stimuli and induce breeding behaviours in captivity (Silla & Byrne, 2019). There are two commonly used exogenous hormones that induce breeding in amphibians, gonadotropin-releasing hormone agonist (GnRH-a) and human chorionic gonadotropin (hCG) (Silla & Roberts, 2012; Brannelly et al., 2015; Silla, McFadden & Byrne, 2019). Both of these exogenous hormones are used to elicit spermiation in males and spawning in females (Mansour, Lahnsteiner & Patzner, 2009; Vu, Weiler & Trudeau, 2017; Silla & Byrne, 2019). In some species, hormone induction will result in spontaneous egg laying in females, such that sperm and eggs can be collected separately and fertilized in the laboratory (Mansour, Lahnsteiner & Patzner, 2009; Wlizla et al., 2017). In other species, spawning is enhanced by hormone induction, but normal mating behaviours between males and females are still required (e.g., amplexus) (Trudeau et al., 2013; Vu, Weiler & Trudeau, 2017; Brannelly, Ohmer & Richards-Zawacki, 2019).

Finding appropriate captive breeding protocols for amphibian species can be challenging because species often respond differently to different methods. Protocols have been refined and there are reliable methods for collecting sperm and eggs in model species like Xenopus laevis and X. tropicalis (Mansour, Lahnsteiner & Patzner, 2009; Wlizla et al., 2017). Whereas, protocols for non-model species are less reliable and require species-specific troubleshooting (Silla & Roberts, 2012; Clulow et al., 2018; Pham & Brannelly, 2022). In other amphibian species, researchers have been unable to identify exogenous hormones that are successful at inducing gamete release and spawning, or there are protocols for males but not for females (Clulow et al., 2018). In cases where exogenous hormones are ineffective, amphibian captive breeding colonies might require specific environmental stimuli to induce breeding. In these cases, outdoor enclosures might offer a sustainable alternative where animals can court and spawn under semi-natural conditions, and then fertilized egg masses can be retrieved.

The alpine tree frog, Litoria verreauxii alpina, is endemic to the alpine regions of New South Wales and Victoria, Australia and is considered endangered. The species has declined dramatically since the 1980s, and has been extirpated from greater than 80% of its former distribution (Gillespie et al., 2015). The primary cause of decline in this species is the deadly disease chytridiomycosis, caused by the fungal pathogen, Batrachochytrium dendrobatidis (Brannelly, Scheele & Grogan, 2020). The eight to 10 populations that remain are physically isolated from each other with virtually no possibility of geneflow among the populations (Banks et al., 2020). However, these remaining populations are seemingly stable, where adults breed and recruitment is successful each year. The dramatic declines throughout its range and restricted current distribution makes this species an ideal candidate to test captive breeding protocols. There are no current captive assurance colonies of L. v. alpina, yet it is well understood that there is a risk of further declines due to climate change and other factors (Brannelly et al., 2016b; Scheele et al., 2016). Wildfire frequency and intensity as well as drought are a high risk in the Australian Alps (Whetton, Haylock & Galloway, 1996) and could decimate the remaining populations. Identifying reliable methods for captive breeding are critical to the longevity of captive assurance colonies as well as research captive colonies (Clulow, Trudeau & Kouba, 2014; Clulow et al., 2019). Having a healthy and sustainable captive colony allows for improved management capacity and high quality research on endangered species without threatening current population stability.

The aim of this study was to test methods for captive breeding in the alpine tree frog. Breeding under laboratory conditions can be challenging in Litoria species, therefore we trialed two different methods: indoor laboratory controlled conditions using exogenous hormones following brumation, and an outdoor breeding mesocosm following brumation. In the first year we tested if a captive environment alone could produce breeding behaviours (no-hormone control), and also tested the efficacy of two exogeneous hormone types (hCG [Chorulon]; GnRH-a [Lucrin]) on breeding behaviours and reproductive success under stable laboratory conditions. In the second year, we tested the efficacy of using outdoor enclosures without exogeneous hormone assistance on reproductive behaviours and reproductive success. Identifying an effective and reliable method for captive breeding in L. v. alpina is critical for the sustainability of this species under captive conditions. Furthermore, understanding captive breeding in this species can help direct captive breeding in other similar species.

Methods

Results

Induction with exogeneous hormones

While all females were visibly gravid following brumation, no eggs were released, either through the attempted stripping procedures or through amplexus with male frogs. Although no spawning took place, we found signs of breeding activity in all hormone-treated groups. No-hormone control animals did not display signs of breeding activity. GnRH-a-treated animals displayed significantly more reproductive displays (amplexus, calling males) than the hCG-treated animals (GLM: χ² = 11.550, p = 0.003). Over the four-week breeding trial we recorded 13 instances of amplexus and 3 instances of calling in GnRH-a-treated animals. Breeding activity appeared to be concentrated on or one day after the hormone was injected (Fig. 1); however, days after injection was not a significant effect on the probability of a mating display (GLM: χ² = 1.114, p = 0.291). While GnRH-a-treated animals displayed breeding activity, no spawning events occurred, even after extended amplexus. We recorded one instance of amplexus (one day after the first injection) and one instance of male calling (one day after the second injection) in the hCG-treated animals. Breeding activity did not occur in any treatment after the third injection.

Outdoor breeding

In the 169 days that animals inhabited their outdoor breeding mesocosm enclosures, 23 distinct egg masses were laid by the 15 females placed in the enclosures. Of those 23 laid masses, 15 masses were viable (65%; at least one tadpole successfully hatched from the mass). The first egg mass was laid on day 23, and the last viable egg mass was laid on day 163 (Fig. 2). No egg masses contained 100% viable eggs, but 20% of the viable egg masses (three masses) hatched over 75% of eggs laid. 13.33% of the viable egg masses (two masses) had less than 25% of eggs hatch. The majority of viable egg masses laid had between 25% and 75% hatching success and were laid in the middle of the experimental time frame (day 65–90; Fig. 2).

On day 100, nine females that were no longer visually gravid were moved to a female only mesocosm. After that shift in sex ratio (from 6F:3M in each tank to 3F:5M and 3F:4M per tank) nine egg masses were laid (Fig. 2). Four of those egg masses came from the tank with only females.

There was no effect of day since animals were placed in outdoor breeding enclosures on the viability of the egg masses (GLM: χ² = 0.348, p = 0.556), or the size of the egg mass laid (MR: χ² = 04.463, p = 0.107). There was no effect of day or size of the egg mass laid on the proportion of eggs that were viable within the egg mass laid (LM: day, F_1,19 = 0.113, p = 0.740; egg mass size, F_2,19 = 1.767, p = 0.198).

Neither males nor females lost a significant amount of body mass during their brumation period, but both males and females lost significant body mass between brumation at the end of the experiment (Fig. 3). Female frogs lost 43.4% of their body mass (6.9 ± 1.2 g before brumation, 3.9 ± 0.8 g after breeding; d = 2.88) from the time they entered brumation to the end of the experiment, and males lost 26.7% of their body mass (4.5 ± 0.9 g before brumation, 3.3 ± 0.7 g after breeding; d = 1.49) (LM: Females; F_2,43 = 30.786, p <0.001: Males; F_2,23 = 4.929, p = 0.017).

Discussion

The hormone induction methods trialed here using Lucrin (GnRH-a) and Chorulon (hCG), as well as the no-hormone control, were unsuccessful at inducing spawning. Litoria species are notoriously difficult to induce spawning in a captive indoor setting (Clulow et al., 2018); therefore, this result was not surprising. Since this trial we have begun optimizing an effective hormonal induction dose for male L. v. alpina in captivity, where an effective dose to induce spermiation using GnRH-a was lower (1.5 µg GnRH-a per animal) than trialed here (8 µg per animal), and hCG dose was higher (120 IU per animal) than trialed here (54 IU per animal; Pham & Brannelly, 2022). The dose of GnRH-a used in this study did not produce sperm in a later study (Pham & Brannelly, 2022), yet it did result in male breeding behaviour like amplexus and calling. These behaviours without sperm release might indicate that the physiological mechanism for breeding behaviour and sperm production is different; therefore, an optimum dose of hormonal induction would need to induce both breeding behaviour and spermiation. There is more work to be done improving the use and dose of exogeneous hormones in L. v. alpina, particularly for inducing ovulation in females (Clulow et al., 2018; Pham & Brannelly, 2022).

We successfully bred L. v. alpina in outdoor mesocosms following an artificial brumation period at 4 °C for 8–13 weeks. The temperature in Melbourne, Victoria, Australia during winter is approximate to the temperature in the Australian Alps during spring when breeding would naturally occur (average of 5–17 °C over a 24 hr period; Brannelly et al., 2015); therefore, it is possible to captively breed these animals following the protocol used in Victoria, Australia. However, because egg masses were laid over a period of four months, adapting this protocol to a warmer climate might be less successful because this species is cool-adapted and would require a shorter breeding window. Because we were able to breed animals in outdoor enclosures but not in the lab with or without hormone induction there is likely one or several environmental parameters (such as temperature, humidity, rain events, barometric pressure) that encourage the animals to breed. In this study we did not collect detailed environmental data during the breeding experiment, but this would be important to know if which weather events are important to encourage breeding within their natural environment and outside their natural environment (e.g., our facility in Melbourne), and this could then be adapted for indoor breeding facilities. Although, sometimes environmental parameters are not enough to encourage breeding: in other species, like Rana pipiens, brumation and then placing the animals in outdoor breeding mesocosms within their natural habitat range was not enough to induce breeding: hormonal induction was required (Trudeau et al., 2013; Vu, Weiler & Trudeau, 2017). Captive breeding requirements are species specific, and troubleshooting protocols are necessary for captive breeding to be successful.

Because the animals were housed in communal mesocosms it is unclear which females laid egg masses, which did not lay, and which ones laid multiple egg masses. Egg masses were laid over 140 days; it is possible that females who spawned early in the experiment developed new eggs by the end of the experiment. Female frogs with seasonal breeding cycles often regenerate eggs once per year, but some frog species do breed at multiple times throughout the year (Rastogi et al., 2005). The sympatric species, Crinia signifera, is known to occasionally lay multiple egg clutches within a single breeding season when ecological conditions will allow (Lemckert & Shine, 1993), indicating that multiple clutches within a single female is possible in species that share the habitat.

In amphibian species with long or continuous breeding cycles the ovaries often have eggs at multiple stages of development at any one time, while in species that have an annual duration ovarian cycle, the eggs within the ovary mature at approximately the same time (Rastogi et al., 2005). For L. v. alpina, the previous work on oogenesis indicates asynchronous follicular development (Brannelly et al., 2016b; Brannelly et al., 2021), such that it might be physiologically possible for one animal to lay multiple egg clutches within the duration of this experiment. Some species are known to lay two egg clutches within a short timeframe (30 days) (Rastogi et al., 1983). A previous experiment on L. v. alpina noticed increased oogenesis after exposure to the pathogen Batrachochytrium dendrobatids after just 3–12 weeks (Brannelly et al., 2016b), suggesting that oogenesis can occur more quickly than on an annual cycle.

The short breeding season and annual ovulatory cycle in L. v. alpina is likely due in part to the limiting environmental conditions of the Australian Alps and infectious disease causing high adult mortality before the breeding season would historically end (Brannelly et al., 2015; Brannelly, Scheele & Grogan, 2020; Scheele et al., 2015). Litoria. v. alpina is part of species complex with species L. ewingii, L. paraewingii, and L. v. verreauxii, which all have an extended breeding season and can breed throughout much of the year (Hero, Littlejohn & Marantelli, 1991; Lauck, Swain & Barmuta, 2005; Smith et al., 2013; Scheele et al., 2014). Retaining an asynchronous breeding capacity might be due to their phylogenetic history rather than their seasonal breeding ecology.

It is also possible that the distinct egg masses represent partial ovulations for the females. Partial ovulation of mature eggs seems to be relatively rare in natural amphibian populations, but some captive breeders have experienced partial ovulation in female frogs particularly following hormone inductions (Lehman, 1977; Jorgensen, 1984; Sive, Grainger & Harland, 2000). Egg clutches resulting from partial ovulation can result in poor fertilization success, reduced egg viability, or few tadpoles hatching (Silla & Byrne, 2021). For example, the species Heleioporus eyrei only partially ovulated following hormonal inductions and had much lower early embryo survival compared to other species that ovulated completely following hormonal induction (Silla & Byrne, 2021). The egg masses collected in this study where overall much smaller than expected for this species (a range of 50–400 eggs per clutch with many clutches being approximately 100–200 eggs per clutch), which typically lays 300–400 eggs per clutch in the wild (unpublished data; Gillespie, Osborne & McElhinney, 1995). Furthermore, egg mass viability was low, with 43% of the masses producing no or less than 25% viable tadpoles. However, the low hatching success rate of the majority of the egg masses produced in this experiment is consistent with the low viability of other studies that explored methods of captive breeding (McFadden et al., 2008; Trudeau et al., 2013; Vu, Weiler & Trudeau, 2017; Brannelly, Ohmer & Richards-Zawacki, 2019). It is understood that eggs and egg masses in captive breeding colonies are less viable than they would be in the wild, although this is not universally the case (see Reichling et al., 2022).

When we moved the females that were no longer visibly gravid into a female-only mesocosm, four unviable egg masses were laid in that tank. We are confident that those four non-viable clutches came from females that already laid, but we are unsure whether these spawning events represent new ovulation or a partial ovulation. Gravid females were determined visually, if eggs could be seen though a clear patch in their groin skin, but this is not a fool-proof method. Female frogs injected with exogenous hormones will spontaneously spawn (Mansour, Lahnsteiner & Patzner, 2009; Wlizla et al., 2017), but it is unclear why females would spontaneously spawn under the conditions in this trial. Male mating calls can elicit a spawning position in some female frog species (Knorr, 1976), such that calling and other breeding stimuli might elicit spontaneous spawning. Spontaneous spawning without amplexus can occur in frogs (Jungfer & Weygoldt, 1990), and we have seen it in our captive facility in L. v. alpina and L. ewingii (LA Brannelly, pers. obs. 2022). Spontaneous spawning might be a mechanism for the animals to reduce their risk of becoming egg-bound when there are no prospective breeding opportunities and they cannot reabsorb the eggs. Or it could occure following stimulation from calling males. If L. v. alpina are spontaneously spawning due to breeding stimuli, it might indicate that they are a good species for exogenous hormone trials for female spawning. It is clear that the hormones and doses used in this study were unsuccessful, but more research could be done to try to identify a more successful dose—possibly using hCG at a higher dose because it was successful in producing spermic urine in males (Pham & Brannelly, 2022).

Notably, the animals used in the outdoor breeding experiment were the same animals that underwent the failed hormone induction trial the year before. All females remained visibly gravid over that year of recovery from the hormonal induction trial, but it is possible that the mature eggs present in the ovaries atrophied by the time the outdoor mesocosm experiment began. Poor quality of eggs due to atrophy or follicular atresia (Rastogi et al., 2005) might explain the low viability observed in many of the egg masses laid. However, captive breeding frequently produces egg masses that are less than 100% viable (Trudeau et al., 2013; Vu, Weiler & Trudeau, 2017; Brannelly, Ohmer & Richards-Zawacki, 2019); therefore, the low viability might be due to captive breeding methodology and/or suboptimal eggs. To determine if the egg quality was poor due to the prior hormonal treatment, a follow up study should follow the same methods but use animals that had not been used in a breeding experiment before.

Both females and males lost a substantial amount of body mass during the outdoor breeding experiment. There is no data from wild L. v. alpina comparing pre- and post-breeding mass to assess if this loss in mass is typical. Female frogs of a variety of species tend to lose 20–30% of their body mass following spawning (Ryser, 1989; Lemckert & Shine, 1993; Rastogi et al., 2005), yet we observed a loss in mass of over 40% between brumation and the end of the experiment. Animals were fed ad libitum on feeding days; therefore, starvation is unlikely (and all survived well following this experiment). It is possible that during this prolonged breeding season, females in good body condition had the capacity to invest their energy reserves into oogenesis to produce a second clutch, and thus leading to a more profound loss of mass. Male frogs also lost mass during the experiment, with over 20% loss of mass observed. This loss of mass in males could be due to the production of calling displays, which are energetically costly. Frogs in the breeding season will often forgo food and self-maintenance in order to maximize their breeding success (Robertson, 1986; Ryser, 1989), with some males losing over 30% of their body mass over the breeding season (Robertson, 1986).

Conclusion

Captive breeding of understudied species can be challenging but is critically important to help protect declining or vulnerable species. In this study, we tested two methods of captive breeding on an endangered amphibian species that had never been bred in captivity: exogeneous hormone induction and the use of outdoor mesocosm enclosures. We found that outdoor breeding mesocosms with brumation were successful in producing viable egg clutches in Litoria verreauxii alpina, while exogenous hormones at the doses trialed here were not successful in producing any egg masses, viable or not. If outdoor conditions can loosely mimic the temperatures of the alpine region during L. v. alpina’s natural breeding season, then it could be an effective captive breeding strategy. Egg mass viability was fairly low, with 35% of clutches laid being unviable. Therefore, more work needs to be done to optimize breeding success.

Supplemental Information