Common goals, different stages: the state of the ARTs for reptile and amphibian conservation

Keywords: assisted reproduction, biobanking, biodiversity, cryopreservation, gametes, genome resource banks, herpetofauna, IVF.

This foreword accompanies the publication in Reproduction, Fertility and Development (RFD) of a two-part special issue on amphibian and reptile reproductive biology and assisted reproductive technologies (ARTs). This special issue is intended to focus attention on and promote the expanding contribution of the reproductive sciences to conservation of those taxa. The need is pressing. The biodiversity extinction crisis that marked the 20th century continues to accelerate in the 21st. Amphibians and reptiles, traditionally grouped as the herpetological taxa, are amongst the most threatened of vertebrate groups. Grouping amphibians and reptiles under the banner of the herpetological sciences is more a matter of convenience and shared discipline histories rather than reflecting real phylogenetic, ecological and reproductive homologies. However, considering the number of reproductive biologists who work on both taxa, it is useful to compare and contrast the progress of ARTs and biobanking in these groups, despite the differences in reproductive anatomy and physiology. What meaningful contribution can a better understanding of basic reproductive biology, and the development of reproductive technologies make to the conservation of these taxa? The use of assisted reproductive technologies (ARTs) in concert with genome resource banks (GRBs) are now recognised and increasingly promoted as linked tools in the fight against genetic and biodiversity loss of vertebrates (Holt et al. 2003; Pukazhenthi and Wildt 2004; Pukazhenthi et al. 2006; Ryder and Onuma 2018; Holt and Comizzoli 2021), and amphibians and reptiles are no exception. GRBs and ARTs are not a new concept in conservation per se (Wildt et al. 1986; Holt et al. 2003), but the investment of research resources and development of technologies has heavily favoured avian and mammalian models. Even within those taxa, the focus has been on sperm cryopreservation paired with artificial insemination (Rodger and Clulow 2021) rather than more advanced stem and cell biology approaches, with some notable exceptions involving advanced technologies such as stem cell generation and nuclear transfer (Folch et al. 2009; Hildebrandt et al. 2021; Holt and Comizzoli 2021; Sandler et al. 2021). For reptiles and amphibians, the development of technologies has lagged the other vertebrate taxa both in resources and time by a couple of decades (Browne et al. 2011; Clulow et al. 2012; Clulow and Clulow 2016; Della Togna et al. 2020). This two-part special issue of RFD (which should be read in conjunction with an upcoming book on amphibian reproductive technologies; Silla et al. 2022) indicates how that situation is rapidly changing, but also highlights how much more needs to be done.

ARTs and biobanking (typically cryobanks) are complementary and interdependent processes (Holt et al. 2003; Clulow and Clulow 2016; Della Togna et al. 2020; Holt and Comizzoli 2021; Silla and Kouba 2022). First, retrievable genetic material such as gametes, embryos and somatic cells are stored in long-term repositories (i.e. GRBs, typically cryobanks (Clulow and Clulow 2016) to insure against the complete loss of genes and species, or to provide the genomic material for managing the gene pools of captive and wild populations. Second, ARTs allow the stored material to be revived as viable, reproductively competent individual organisms, potentially restoring genetic diversity stored prior to declines and even extinctions many decades previously (Howard et al. 2016; Holt and Comizzoli 2021). The potential conservation implications of these tools are enormous. ARTs and GRBs provide conservation practitioners the tools to reverse inbreeding and restore lost genetic diversity by introducing or re-introducing critical genes to wild populations (genetic rescue; Howard et al. 2016; Madsen et al. 2020; Holt and Comizzoli 2021), manage or reverse inbreeding depression and limit selection for domesticity in captive breeding programs (Howard et al. 2016; Howell et al. 2021a), and even revive lost species (Jørgensen 2013), while optimising the allocation and use of conservation resources (Howell et al. 2021a).

This two-part special issue of Reproduction, Fertility and Development comprises 10 papers on amphibian and 12 papers on reptile ARTs. The authors form a broad international cohort, which is a hopeful sign for the future use and uptake of these conservation tools. However, while the goals and ultimate applications of ARTs in conservation for amphibians and reptiles are the same, the challenges and progress are not. The reproductive systems differ greatly between the groups, as does progress in developing ARTs. A push for the development of ARTs for amphibians gained momentum in the decade prior to the turn of the century in response to the emerging amphibian extinction crisis that became increasingly obvious from as early as the 1980s (Browne et al. 1998; Clulow et al. 1999). In large part, the acuteness of the crisis was due to the rapid spread of the emerging amphibian chytrid fungal pandemic (Berger et al. 1998; Bower et al. 2017; Scheele et al. 2019). The decline or extinction of more than 500 species of amphibians around the world in just a few decades has been attributed to this disease alone, with few options available to prevent or reverse ongoing severe declines in the wild (Clulow et al. 2018b; Scheele et al. 2019). Combined with other threats such as climate change and habitat loss, there are now estimates that more than half the world’s amphibians are at risk of extinction (Stuart et al. 2004; González-del-Pliego et al. 2019).

Meanwhile, a lower profile but similarly devastating crisis of decline has unfolded among the reptiles, driven by sometimes similar and sometimes different threatening processes. Threats from exploitation due to over-harvesting, habitat loss, invasive species and emerging disease have led to recent estimates of more than 20% of reptiles now threatened with extinction (Whitfield Gibbons et al. 2000; Böhm et al. 2013; Doody et al. 2017). These estimates jump to as high as 50% in some groups such as the chelonians (turtles and tortoises) and crocodilians (Grigg and Kirshner 2015; Rhodin et al. 2018). Like amphibians, recognition of threats to the reptiles has led to more recent, but equally persuasive, calls for the development of reptile ARTs (Clulow and Clulow 2016); nevertheless, the lower profile of reptile threats and fewer resources committed in reproductive and biobanking research compared to amphibians has inevitably resulted in this group being left behind.

Here, we briefly review and summarise the recent developments in the state of reproductive technologies for amphibians and reptiles with particular emphasis on the progress captured through this two-part special issue of Reproduction, Fertility and Development. Comparisons to a comprehensive review on the state of the ARTs for reptiles and amphibians in 2016 (Clulow and Clulow 2016) and more recent reviews on individual technology areas and taxa (Clulow et al. 2018a, 2019; Browne et al. 2019; Silla and Byrne 2019; Della Togna et al. 2020) are illuminating. It is clear that ARTs for amphibians have come a long way over the past two decades, particularly in technology and protocol development for the storage of sperm cells and the recovery of male germlines from biobanked material. Further breakthroughs may be on the horizon for storing and retrieving maternal lines and embryos (Clulow et al. 2019, 2022; Holt and Comizzoli 2021). Research and application to on-ground programs translating technology to significant, applied conservation outcomes is the next stage required for amphibians (Clulow et al. 2019; Burger et al. 2021; Johnson and Mendelson 2022; Kouba 2022; Silla and Kouba 2022).

Reptiles, on the other hand, are missing many of the advances and milestones at the most basic levels of technology development (e.g. production of live young from artificial insemination in many groups, or from cryopreserved sperm in any group). Arguably, the field for reptiles is at a stage similar to the development of ARTs in amphibians in the decades before the turn of the century. The most pressing need for reptiles in the immediate future is technology development supported by studies of basic reproductive physiology and endocrinology to fill critical knowledge gaps that take into account the extreme variation in reproductive anatomy and physiology of the reptilian orders. The most appropriate targets, with the highest probability for success in the short-term, lies in the storage and retrieval of the male germline through sperm cryopreservation and artificial insemination.

Reptiles have been neglected for a long time compared to other vertebrates when it comes to the development of ARTs (Clulow and Clulow 2016). This may be due to indifference or negative public perceptions towards reptiles compared to other vertebrates, and to the widespread lack of awareness of the conservation issues and level of threat to reptiles. This is unfortunate, as reptiles are not only complex and social animals (Doody et al. 2013, 2021a; Gardner et al. 2016), a role previously considered relatively exclusive to mammals and birds, but also play critical ecosystem roles ranging from apex-predators to ecosystem engineers (Doody et al. 2015, 2021b).

The development of ARTs in reptiles is further hampered by their diverse and complex reproductive systems. These differences are a product of ancient evolutionary divergence within the Reptilia. Extant orders include Testudines (turtles and tortoises), Rhynchocephalia (tuatara), Squamata (snakes, lizards and amphisbaenians) and Crocodylia (crocodiles, alligators, caimans, gharials), which separated from one another between 150 and 250 Mya (Chiari et al. 2012). Highlighting just how divergent these groups are, modern crocodiles are more closely related to birds than to the other orders of Reptilia (Walker 1972; Whetstone and Martin 1979; Chiari et al. 2012). Thus, non-avian reptiles have evolved perhaps the most challenging variety of reproductive systems and functions of all vertebrates when it comes to the development of ARTs. These include: multiple paternity, sperm competition and post-copulatory sexual selection; significant genital variation; large, yolky amniotic eggs; development ranging from oviparity to viviparity; complex sex-determination systems including genetic and temperature-dependent sex determination; sex reversal; long-term sperm storage in the female tract; environmentally determined control over timing of birth; and parthenogenesis (reviewed in detail in Van Dyke et al. 2021). Despite the challenges of working with such complexity and variability, and historical neglect, the number of articles submitted on reptiles for the special issue of RFD marginally surpassed those submitted on amphibians. This is promising for the field, and reflects a recognition by reproductive scientists, especially those involved in conservation, of the urgent and growing need for the development of ARTs for this taxon.

Nevertheless, the number of published studies in the field remains low despite the valuable contributions from the impressive number of studies contributed to this two-part special issue, which represents a substantial increase in the published pool of research to date. Cryopreservation studies of reproductive cells and tissues in the reptile literature exclusively deal with the cryopreservation of sperm (Clulow and Clulow 2016), but the total number of cryopreservation studies remains low. For example, two new protocols for sperm cryopreservation in lizards (Campbell et al. 2021b; Hobbs et al. 2022) almost doubles the number of studies of sperm cryopreservation published for lizards worldwide, but only raises the number from three to five (Young et al. 2017; Campbell et al. 2020, 2021a). This compares to at least 26 reported studies on sperm cryopreservation for more than 35 species of amphibians prior to the RFD special issue (Clulow and Clulow 2016; Browne et al. 2019; Clulow et al. 2019). The small number of published lizard protocols stands in stark contrast to the more than 6000 species of lizard worldwide that constitute half of all extant reptile species (Böhm et al. 2013). The study by Hobbs et al. (2022) extends the range of cryoprotectant and extender formulations available for lizards while recognising interactions between cooling rates and cryoprotectant type. The study by Campbell et al. (2021b) demonstrates it is possible to cryopreserve sperm of the yellow-spotted monitor lizard Varanus panoptes after 4 days of cold-storage and still recover acceptable levels of motility (∼30%) and plasma membrane integrity (∼50%). This represents the first report of short-term cold-storage of lizard sperm prior to cryopreservation and retrieval and is another small step forward in reptile ARTs since the handful of reports of successful cold storage in reptile sperm without cryopreservation reviewed in Clulow and Clulow (2016).

The other two new reports of sperm cryopreservation in reptiles in this issue of RFD advanced our understanding of snake sperm cryopreservation (Young et al. 2021, 2022), adding to one prior published study reviewed in Mengden et al. (1980) and another also recently published outside of this special issue (Sandfoss et al. 2021). Taken together, this literature, with all the other reptile cryopreservation literature, confirms the focus of cryopreservation technology development in reptiles has been restricted to sperm, fulfilling the prediction by Clulow and Clulow (2016) that ‘sperm cryopreservation will be the ART that yields the most progress in the near future’ for reptiles. However, the prediction that this would be soon followed by the production of live young from cryopreserved sperm when combined with improvement and optimisation of AI protocols (Clulow and Clulow 2016) has still yet to come to fruition. The production of live young from cryopreserved sperm remains an urgent area of research and the next logical step in the continued development of reptile ARTs.

It is also worth noting that all the new cryopreservation studies in the RFD special issue, along with others recently published elsewhere (Campbell et al. 2021a; Sandfoss et al. 2021) were in squamates. This might not be surprising considering that squamates make up >95% of reptile species globally. However, considering that there is a large overrepresentation of testudines (turtles and tortoises) and crocodilians among threatened reptiles (Böhm et al. 2013; Rhodin et al. 2018), these other taxa should not be neglected. To date, the only published attempts for sperm cryopreservation in non-squamate reptiles include a few testudines (Platz et al. 1980; Perry and Mitchell 2022) and two crocodilians (Larsen et al. 1984; Johnston et al. 2014; Johnston et al. 2017).

Interestingly, the reptile species most represented in the special issue of RFD was the saltwater crocodile (Crocodylus porosus), one of the few non-squamate reptiles in which sperm cryopreservation has been previously attempted (Johnston et al. 2014, 2017). Four papers were submitted on the saltwater crocodile, making it the most studied reptile for ARTs in the world in recent times, and cementing it as a model species. A comprehensive review of the progress of ARTs in saltwater crocodiles reported that non-lethal semen collection through manual stimulation has been highly successful, although sperm cryopreservation attempts to date have resulted in low rates of recovery (∼10–15% motility at best) (Johnston et al. 2021). The authors conclude that future studies are needed on fundamental anatomy, physiology and behaviour of females in particular, a point applicable to all the reptile groups. Nixon et al. (2021a, 2021b) and Miller et al. (2021) contributed three studies on fundamental reproductive physiology for the saltwater crocodile, ranging from post-testicular sperm maturation and gross and micro-anatomy of the male reproductive duct (Nixon et al. 2021a, 2021b) to characterising the chemical lipid composition of the sperm plasma and acrosomal membranes (Miller et al. 2021). Sandmaier et al. (2022) also contributed a study characterising sperm production in a male Philippine crocodile, further filling knowledge gaps in crocodilians.

It is worth noting that a total of six studies contributed to the special issue of RFD characterised basic reproductive physiology in reptiles, a critical step to filling the substantial knowledge gaps on fundamental reproductive physiology to enable development of ARTs in this group. These included one on the role of extracellular vesicles in the male reproductive tract of the Chinese softshell turtle Pelodiscus sinensis (Chen and Holt 2021) and another on predicting optimal sperm collection times in iguanas (Perry et al. 2022), in addition to the four on crocodiles.

Despite the significant proportional increase in studies on reptile ARTs in the RFD special issue, reptile ARTs are still in their infancy and behind our understanding and technology development for other vertebrate classes. As predicted more than 5 years ago in an earlier review (Clulow and Clulow 2016) most of the development of ARTs in reptiles has focussed on the male germ line through collection, storage and retrieval of sperm. There have been few other reports (e.g. Oliveri et al. 2018) of AI in reptiles since the 2016 review and successful AI with cryopreserved sperm remains elusive.

Encouragingly, while the majority of ARTs studies to date have been focussed on squamates and the saltwater crocodile, a recent study reported the first collection, characterisation, and storage of tuatara (Sphenodon punctatus) sperm (Lamar et al. 2021). Although preliminary, the authors reported low levels of live sperm recovery post-thaw using the commercial cryoprotectant Synth-a-Freeze™ (containing 10% DMSO buffered with HEPES), making it the first ART study for the ancient reptile order Rhynchocephalia (Lamar et al. 2021). That means there has now been at least one published study on ARTs in all four of the extant reptile orders, a small but important step forward in reptile conservation. Despite this milestone, there have still been no reports of ARTs leading to successful storage or retrieval of maternal germ lines or embryos. This is perhaps not surprising considering the substantial challenges preventing storage and retrieval of eggs or embryos in reptiles and highlights the huge task still confronting ARTs development in this historically neglected taxon.

Looking to the future, we suggest three key areas of research moving forward in the short to medium terms for reptile ARTs. These include: (1) continued development and refinement of sperm cryopreservation protocols, testing their ubiquity across a broader phylogenetic range of species; (2) development of non-lethal methods of sperm collection that lead to good quality and quantities of sperm for successful cryopreservation; and (3) development of AI with cryopreserved sperm. We note many sperm cryopreservation protocols developed to date have used sperm collected post-mortem, due to the large quantities and quality of sperm obtained and the ease of protocol development this confers (Young et al. 2017, 2021, 2022; Campbell et al. 2021a, 2021b; Sandfoss et al. 2021). However, encouragingly, two recent studies have looked at sperm collected non-lethally, including the first study on tuatara (Lamar et al. 2021; Hobbs et al. 2022). We also believe that further studies on fundamental reptile reproductive ecology and physiology, along with testing fundamental hypotheses around cryopreserving reptile reproductive cells and tissues (Campbell et al. 2021a) will aid with these conservation and research goals.

ARTs for amphibians are much closer to a suite of utilisable technologies for conservation programmes than are reptiles, a conclusion supported by a comparison of the amphibian and reptile papers in this special issue. For example, in amphibians there are now many examples of stored sperm (cryopreserved, but also sperm held for periods without cryopreservation) resulting in the production of live offspring. In this special issue there are five papers reporting the production of live progeny in anurans (Arregui et al. 2022a; Kaurova et al. 2021; Silla and Byrne 2021; Upton et al. 2021) and caudates (McGinnity et al. 2022). The advent of an increasing number of caudate species from which progeny have been produced with ARTS (see also Kouba 2022) is of particular note, including with frozen sperm as reported by McGinnity et al. (2022) in this issue. There are also another four research papers that refine our understanding of the underpinning reproductive processes supporting these reproductive technologies including endocrinology and ovarian senescence (Jacobs et al. 2021), DNA integrity of induced urinic sperm (Arregui et al. 2022b), effect of over-wintering history on spermiation (Kouba et al. 2022), the challenges of inducing sperm release in the unique leiopelmatids of New Zealand (Germano et al. 2022) and finally one that modelled the econonomic and genetic benefits of utilising biobanking in amphibian captive breeding and conservation programmes (Howell et al. 2021b).

This two-part special issue of RFD highlights the ongoing refinements and extension of protocols for amphibian biobanking, gamete induction, and artificial fertilisation, whilst recognising some applications remain challenging and indicate the need to persist with research. The generation of live progeny for externally fertilising caudates using cryopreserved sperm (McGinnity et al. 2022) is one of those important achievements, an important step forward since the Clulow and Clulow (2016) review. Improving the knowledge of the reproductive biology of target species (such as the mountain yellow-legged frog, (Jacobs et al. 2021) with expanded use of technologies, including ultrasound for ovarian assessments and facilitated application of analysis of steroids for rapid assessment of reproductive condition, will facilitate improved protocols. As well, the use of tools to assess DNA integrity of sperm collected non-invasively (Arregui et al. 2022a) will further inform protocol development involvement utilising spermiation.

Nevertheless, despite the advances, there is still much left to do for the development of ARTs, even for the amphibians. Not all studies result in outcomes that are hoped for. Such is the case with the archaic New Zealand leiopelmatid lineage, Hamilton’s frog (Leiopelma hamiltoni), a threatened and unique EDGE (evolutionarily distinct, globally endangered) species. Attempts to hormonally induce mating and the release of sperm met with limited success, and no matings were achieved with hormonal induction in this species (Germano et al. 2022). Further, despite recent successes in ARTs development in caudates, as in our 2016 review (Clulow and Clulow 2016), the third order of amphibian – the caecilians (order: Gymnophiona) – are still completely absent. This mirrors information on their ecology and status which remains elusive for the group due to their cryptic nature (Clulow and Clulow 2016).

The achievements in collection and cryopreservation of sperm, and their use in artificial fertilisation are the outstanding milestones in ARTs for amphibian species, with obvious implications for applications in conservation. In some ways, the development of amphibian ARTs for conservation based on sperm technology and artificial fertilisation parallel the development of sperm cryopreservation and AI technologies for conservation in mammal and bird species (Rodger and Clulow 2021). Nevertheless, as with mammals and birds, there are gaps in the amphibian technologies that are highlighted by the topics and papers that are not in the current issue. In particular, there are no reports of progress towards the storage and recovery of either the maternal haploid genome or the somatic diploid embryonic genome. These objectives really need to be the target of concerted research efforts by the amphibian ARTs community, but there is cause for optimism for advances in this area if the resources are available to pursue these goals (Lawson et al. 2013; Clulow et al. 2019; Clulow et al. 2022).

Such optimism comes from recent advances in the field of fish and aquaculture ARTs (Clulow et al. 2019; Holt and Comizzoli 2021; Clulow et al. 2022). Fish are challenging to cryopreserve because of the size and yolk content of the oocytes and embryos, and permeability barriers to cryoprotectants which together result in lethal intracellular ice formation events during thawing (Hagedorn and Kleinhans 2000; Hagedorn et al. 2004). For fish, there are now ways around these blocks to cryopreservation that have recently emerged such as laser warming of vitrified embyros (Khosla et al. 2017; Khosla et al. 2019; Khosla et al. 2020); the generation of viable gametes generated from cryopreserved primordial germ cells (Lin et al. 1992; Higaki et al. 2010; Higaki et al. 2013); or spermatogonial stem cell transfer into host larvae from the same or different species (Marinović et al. 2018; Marinović et al. 2019). These approaches were recently reviewed outside this special issue by Clulow et al. (2022). Other technologies, known to be effective in amphibians and/or fish, such as ICSI, nuclear transfer, the generation of chimeras and the xenotransplantation of ovarian tissues offer many possibilities for the future expansion of amphibian ARTs (Clulow et al. 2014; Clulow et al. 2019). Overall, there should be great optimism for technology transfer between these vertebrate classes with aquatic life history phases.

While the status quo for reptile ARTs outlined in our review half a decade ago (Clulow and Clulow 2016) has been more or less maintained, amphibian ARTs have undergone considerable refinement and generated further proofs of concept for their effectiveness in the intervening period. This can be seen both across this special issue and more broadly in the literature (Upton et al. 2018; Browne et al. 2019; Clulow et al. 2019; Della Togna et al. 2020; Upton et al. 2021; Silla et al. 2022). For amphibians, ARTs are now available and are in fact being utilised in conservation programs (for a global perspective see chapters in (Silla et al. 2022). In Australia, sperm of threatened amphibians are being collected from wildfire impacted ecosystems and biobanked by University of Newcastle and Taronga Conservation Society with Australian Federal Government support (Clulow et al. unpubl. data). Encouragingly, the broad availability of amphibian ARTs targeting storage and retrieval of sperm has resulted in a nascent focus on demonstrating not only the genetic, but also the economic value of adopting these tools and approaches in amphibian conservation programs (Howell et al. 2021a, 2021b). This is sure to help to increase uptake. Looking to the future, we suggest two main goals for amphibian ARTs in the short to medium terms: (1) translating the successful technology development for storing and retrieving male germ lines into tangible conservation outcomes. ‘End product’ proof of concept is needed to gain social licence for wildlife ARTs and encourage further funding. (2) Focus technology development on the emerging possibilities for storing and retrieving maternal haploid and embryonic diploid cell lines.

While the goals of developing ARTs for amphibian and reptile conservation are similar, the progress for each taxon are at different stages. There has been strong progress in understanding and developing reproductive technologies for amphibians with the goal of utilising these as conservation tools. In the case of amphibians, ARTs have developed to the point where they can be utilised now. The availability of protocols that allow the generation of live progeny from cryopreserved and biobanked sperm mean that ARTs can be used extensively in amphibian conservation programs, captive breeding and for extinction insurance, despite the lack of maternal line/embryo banking technologies. Technologies to break this block in amphibian ARTs will likely come soon for at least some species. Meanwhile, the field should be, and is in some cases, focussed on shifting technology development to application in on-ground conservation management programs for ultimate end product proof of concept and tangible conservation outcomes.

For reptiles, progress in the development of ARTs has perhaps two decades of catch up to match the range of technological applications available for amphibians, but there is the high probability that advances for reptiles will accelerate greatly if more resources are invested into the field. Technologies for sperm storage for at least some reptile species now exist and should continue to be optimised and ready for use in the near future if research resources are available. There should be no fundamental block to the application of artificial insemination with cryopreserved sperm, or even the application of advanced stem cell technologies to reptiles eventually. The storage of maternal and embryonic lines are likely still some way off, but may be accelerated by the development of these technologies in amphibians. The papers published in this two-part special issue of RFD on ARTs for reptiles and amphibians highlight the progress for both threatened taxa.

The authors declare no conflict of interest.