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Fig. 2. Morphology assessment of Strabomantis bufoniformis eggs. Freshly laid eggs were collected and fixed in 4% PFA for 24 h, sectioned (A,B), stained with Coomassie Blue for 90 s (C,D) and washed with ARS. Staining revealed three jelly layers (E,F,G) when observed under the microscope (200X) (Della Togna, unpublished). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) can retain at least 90% of the genetic variation present in the wild countries, and mitigation of threats in the wild”, under the population for at least 100 years [13,14]. Achieving this goal is a assumption that successful and continuous breeding events will complicated task. First, amphibians are often cryptic in nature, have occur. Despite the actions set by the ACAP as the global standard, high levels of mortality or are non reproductive when brought into there is poor emphasis on reproduction as a vital part of Captive captivity. Second, inbreeding depression due to poor management, Breeding Programs. Reproduction is only mentioned as one of the too few founders (the minimum recommended number of found priority research gaps and as necessary for the production of ing pairs required to initiate a CBP is twenty according to Carrillo research and reintroduction animals. Strangely, given that repro et al., 2015), husbandry accidents or low genetic diversity in the duction is the cornerstone of these programs, the current edition of wild population on which CBP’s are founded, can jeopardize the the ACAP outlines the topics related to genome resource banking genetic variability of the captive population [15e17]. Third, suc (GRB’s) and ART’s as merely an appendix linked to Action 7, instead cessful unassisted breeding of amphibians in captivity can be of including it as an essential part of the Captive Program action, or difficult because of issues related to unsuccessful or asynchronous even dedicating an entirely new conservation action incorporating spawning, absence of breeding behaviour, dystocia, low fertiliza all these subjects. Given the great progress that has been made in tion rates, low survival rates of embryos and juveniles, and disease this field to date, the aim of this paper is to reassess the original [18]. These difficulties can be mitigated in a species specific objectives on amphibian reproduction and biobanking for captive manner by increasing research on the natural history and repro breeding programs as a contribution to the ACAP review process. ductive biology, followed by the development and application of artificial reproductive technologies (ART’s) and the incorporation of genome resource banks (GRB’s) [6,19]. Since Moore and Church’s 2. Genome resource banking (2008) paper on the implementation of the ACAP, the number of described species has increased, the number of CBP’s has increased, In Appendix A of the Captive Program section of the ACAP, but declines have not decreased, and as the second decade of the Pukazhenthi et al., 2007, emphasized the need of reproductive century comes to a close, the re evaluation of this document in success along with the application of emergent technologies to 2020 is imminent [20]. secure genetically valuable populations in captive breeding pro Action 7 of the ACAP establishes that “the captive assurance grams [4]. Browne & Zippel (2007) describe reproductive success strategy has to be implemented as part of an integrated plan that for amphibians as a complex process integrated by spermiation, includes research on amphibian biology and disease, development ovulation, oviposition, fertilization, embryonic development and of improved husbandry, training and building capacity in range metamorphosis, where all phases need to be accomplished [21]. Nevertheless, there is very limited information available on the

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Table 2 Stakeholder needs fulfilled by a centralized, Amphibian Biorepository (adapted from Ginsburg et al., 2015).

Research 1. Clarify use of samples for research. participants 2. Optimize the use of donated samples (collected opportunistically by field biologists). 3. Ensure transparency and standardization of collection methodology.

Researchers 1 Biospecimens collected in strategic and delineated manner from a diversity of individuals and populations to capture maximal genetic diversity. 2 Continual review of research needs. 3 Timely and widespread access to biospecimens and associated data through a centralized peer-review process. 4 Clear guidelines for acceptable sample use. CBPs 1 Provide valuable resources and infrastructure available to external researchers. 2 Optimize use of research space and increase opportunity for inter-institutional collaborative research funding. 3 Enable inter-institutional training and capacity building. 4 Facilitate inter-institutional collaborative research. 5 Provide long-term, permanent funding for curatorial and bioinformatics staff. Funding agencies 1 Minimize inefficiency in biobanking financial spending. 2 Ensure development of functional and enduring resources. 3 Apply for multi-institutional, collaborative funding.

concentration, integrity, viability and functionality [18,25]. research associate at SDZG. Thanks to Dr. Candace Williams, Following these initial processes, the only amphibian gamete that Michelle Curtis, Nicole Gardner and Rose Upton for their contri has been successfully cryopreserved is sperm. Notably, freezing bution to all of Natalie’s scientific endeavours. Cecilia and Natalie hormonally stimulated SU has proven more difficult than testicular thank our collaborators at the Colorado Division of Parks and sperm [35]. Hence, although important advances have been made Wildlife, Harry Crockett and Matt Nicholl as well as Ted Smith, since the publication of the ACAP, progress is slow and available Hatchery Manager, and the staff at the Native Aquatic Species reports on gamete cryopreservation remain limited compared to Restoration Facility in Alamosa, Colorado. Special thanks to Tom other taxonomic groups. Mix, Joshua Houghtaling, David Westerman and Noel Heredia for There are several studies on diverse species of amphibians assisting with Natalie’s experiments during her 2014 and 2015 exploring the use of exogenous hormones for the collection of stays at NASRF. Thanks to our partners in the Mountain Yellow spermatozoa from live animals, where hormones, doses, concen legged program including the United States Fish and Wildlife Ser trations and results vary distinctively among species vice, California State Department of Fish and Wildlife, United States [18,21e23,32,34,37e46]. The most utilized hormones include Forest Service, United States Geological Survey, the Los Angeles and GnRH A [des Gly10, D Ala6, Pro NHEt9], Human Chorionic Gonad the Omaha’s Henry Doorly Zoo. Lachlan Howell would like to thank otropin (hCG) and cocktails including GnRH and a dopamine the University of Newcastle acknowledging their financial support antagonist such as metoclopramide, domperidone or pimozide in awarding his PhD scholarship. John Clulow acknowledges sup [18,23,32,34,41e43,47e53]. port from the University of Newcastle. In appendix A, one of the broad goals outlined in the ACAP Captive Breeding Program action suggests collection and cryo preservation of germplasm from wild specimens for later use in Appendix A. Research Objectives CBP’s to increase gene diversity. However, before this can take place, protocols for the collection of SU for the species of interest Objective 1: Determine optimal model species that represent a range should be first developed in captivity. Alternatively, the collection of amphibian orders and then, through systematic studies, of “wild” samples would involve testicular spermatozoa, which determine fundamental reproductive strategies for each requires euthanasia of valuable wild specimens, but this may be undesirable depending on the conservation status of the species in In recent years, the term “model species” or “model organism” question. Furthermore, collection of germplasm from unstudied, has become confusing and controversial. Due to the emergence of living animals should always involve prior research on hormonal new research questions and needs, existing classical systems, might stimulation in the same or closely related species before it is not be appropriate because there was a lack of consideration for applied to wild studies. phylogenetic traits and variation at the time of their establishment [54,55]. Historically, model organisms have been used to under stand and gather information on biological processes that are then Acknowledgements translated to human biology and/or medicine [56]. In the past de cades, the “model organism” concept was formalized by the Na The authors appreciate the support of the Secretaría Nacional de tional Institutes of Health (NIH) and the National Science Ciencia y Tecnología de Panama (Senacyt) and the Sistema Nacional Foundation (NSF) restricting it to a very limited number of species de Investigacion de Panama (SNI). Gina Della Togna thanks Dr. for which complex infrastructure and support systems have been Matthew Larsen, Dr. Brian Gratwicke, Dr. Pierre Comizzoli, Dr. built [54,57]. According to NIH: “model organisms are a small group Roberto Ibanez, Dr. Oris Sanjur and Jorge Guerrel from the Smith of research organisms that serve as a proxy for understanding the sonian Institution (National Zoo and Tropical Research Institute) for biology of humans” [58]. These include well known species such as their amazing support during her scientific career. Collectively, Drosophila, C. elegans, Arabidopsis, M. musculus, zebrafish and Xen Natalie Calatayud, Ruth Marcec and Cecilia Langhorne would like to opus, and a few others. Thus, the formal definition of the concept is thank Memphis Zoo and Mississippi State University. Natalie Cal restricted to its use and application in understanding human atayud would also like to thank San Diego Zoo Institute for Con biology while excluding other species. servation Research for supporting the post doctoral fellowship in According to Bolker (2014), “a model acts as a representation of particular, Dr. Barbara Durrant, Dr. Ron Swaisgood, Dr. Alison or an analogy for something else, either as a direct proxy, or as an Alberts and Dr. Debra Shier. Thanks to Exploradora de Immuebles, exemplary representative of a broader group”. Model organisms S.A. (EISA) for awarding financial aid for her first two years as a should follow four key principles: similarity, practicality,

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418 G. Della Togna et al. / Theriogenology 150 (2020) 412 431 accessibility and generalization. Depending on the research ques evanesco responds poorly to treatment with hCG, but spermiation is tion, the model should be similar enough to what it represents to be stimulated with GnRH and Amphiplex (Della Togna, unpublished worth studied, simple, easy to grow, reproduce and maintain, and data). Variability in the time of response to hormone treatments is conclusions drawn from the model should support generalizations also observed among species. A study by Kouba et al., 2012, in to other systems or organisms [55,59,60]. Existing amphibian Anaxyrus americanus showed no clear peak of sperm production models, in the strict sense of the word, are very limited although between 3 and 24 h post injection of GnRH A, while hCG stimu highly valuable for scientific research. Anuran species such as lation produced a peak between 3 and 9 h post injection [74]. In Xenopus laevis and Silurana tropicalis are classic model organisms A. zeteki, GnRH A reached a 1 h long sperm production peak after that have been widely studied and have contributed greatly to the 2.5 h post injection, while Amphiplex reached a 1 h long sperm fields of developmental biology, immunology, molecular and cell production peak after 3.5 h post injection. Furthermore, GnRH A biology, toxicology, endocrinology and physiology [56,60]. Sala elicited a more rapid response 30 min post injection compared to manders, especially Ambystoma mexicanum, have been the focus of Amphiplex [18]. Species specificity is also observed in the response research on regeneration, although they have also served as models to hormonal treatments for the stimulation of ovulation in females, for studies in embryology, evolution, electrophysiology, genetics even in standard model species such as Xenopus laevis. Protocols and genomics [61e63]. Other species of amphibians such as Rana originally developed for Rana pipiens required significant modifi catesbeiana, Rhinella marina, Hymenochirus boettgeri, Bombina ori cations to be functional in X. leavis [75]. entalis, Lithobates pipiens, Necturus maculosus, Notophthalmus vir Additional evidence on the difficulty of establishing model idescens, Triturus sp. and Ambystoma tigrinum have been used as species for the application of ART’s, specifically GRB’s, is the di non traditional models for retinal and renal research studies, versity of responses to cryoprotectant agents (CPA’s) in the recov physiology, cardiac regeneration, embryology, agro toxicology and ery of viable cells. In 2009, Kouba et al., reported no successful pharmacology [64e66]. recovery of viable cells using already developed cryopreservation Although this limited number of amphibians has served as protocols for testicular spermatozoa of Xenopus sp., Anaxyrus models to understand well conserved biological mechanisms americanus, Rhinella marina and Eleutherodactylos coqui to the across different taxa, it would be difficult to justify their role as cryopreservation of hormonally induced SU of A. fowleri, standard models to understand and generalize reproductive as A. americanus, A. boreas and A. baxteri [22]. In Clulow at al., 2019 a pects across the amphibian class itself. Objective 1 of the ACAP’s summary of amphibian cryopreservation studies showed that Appendix A suggests the identification of model species that different species require different CPA’s and concentrations for represent a range of orders for the development of GRB’s, which recovery of functional cells [76]. One of many examples shows 5% may not be easy or even feasible. Due to their evolutionary history, dimethylformamide (DMFA) þ 10% trehalose as effective for the the three amphibian orders are highly divergent from each other, recovery of motile SU cells in A. zeteki (Della Togna, 2015), whereas and there is variability within orders as well. Therefore, the prin 10% DMFA þ 10% sucrose was effective for the recovery of viable SU ciples of similarity and generalization may not be met by many cells of Epidalea calamita, both species belonging to the Bufonidae species [61]. Amphibians are known as the group of animals with family [77]. the highest diversity of reproductive strategies [19,67e69] The number of CBP’s for endangered amphibians has grown in (Supplemental Fig. 2). These strategies are the result of quite recent years. Many of these are established for the conservation of intricate interactions between physiological, morphological and species that have not been previously studied or for which there is environmental factors, specific to species or even to different too little information available, as is the case for Craugastor evan populations of a single species, highly influenced by natural se esco. Shortly after its description in 2010 [78], the species was lection, with the sole goal of producing viable offspring [68]. incorporated into the Panama Amphibian Rescue and Conservation Gametogenesis and hormonal cycles are essential aspects of the Project CBP [79]. Husbandry practises and breeding protocols have physiological characteristics related to reproductive strategies. In had to be developed since no prior information on its natural his addition, reproductive strategies seem to influence aspects such as tory was readily available. Reproductive success for this species is gamete morphology [70], as is the example of Salamandrina sp. and poor in captivity and the application of ARTs is necessary. With Triturus sp., where spermatozoa differ in size of the midpiece and examples such as C. evanesco and the immense variability found in structure of the tail, the former presenting a larger midpiece due to amphibians, many species are incapable of adhering to the princi the higher viscosity of the swimming fertilization medium ples of similarity and generalization that “animal models” should compared to the more aqueous one of the latter [71]. This variation satisfy. Directly transferring protocols that have been already is also evident in anurans as egg size and sperm length are bigger in developed for other species, without taking into consideration terrestrial breeders [72]. factors such as reproductive strategies, phylogenetics and ende Physiological variation within the amphibian class is also micity, could result in a waste of the already scarce resources, evident in the diversity of responses to hormonal treatments which precious time and valuable specimens, impacting directly the differ among species (Supplemental Fig. 1A C)[73]. In 1998, Wag success of these CBP’s and the safekeeping of species. gener and Carroll treated eight species of anurans with GnRH, Therefore, we recommend developing species specific protocols successfully obtaining spermatozoa from Ceratophrys ornata, for ART’s following a standard “model process” while considering Ceratophrys cranwelli, Pysicephalus adsperus, Lithobates pipiens, what has been developed for other species as a guide or reference Xenopus laevis, Lepidobatrachus llanensis and Lepidobatrachus laevis only. The objective of hormone stimulation protocols is to promote but not from Ceratophrys cornuta. Comparatively, Pseudophryne reproductive behaviours or to obtain viable gametes for their use in corroboree responds positively to treatment with both GnRH and research or in other techniques. For the scope of this objective, we hCG, but GnRH stimulates the production of significantly higher will centre our recommendations on the processes of collecting and numbers of sperm than hCG [41]; while in Anaxyrus fowleri treat cryopreserving viable gametes. Most protocols involving hormonal ment with hCG is more effective than GnRH (Browne et al., 2006). stimulation and cryopreservation have been developed empirically, Atelopus zeteki produces sperm after stimulation with GnRH, hCG replicating or adapting what has reportedly been successful in and Amphiplex (GnRH A þ Metoclopramide), with the latter other species [22,76]. However, it is important to note that in the stimulating the production of the highest sperm concentrations, current literature, positive results rarely describe further explora followed by GnRH and hCG [18]. Contrary to A. zeteki, Craugastor tion as to the physiological ‘why’ or ‘how’ a given treatment has

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G. Della Togna et al. / Theriogenology 150 (2020) 412 431 419 worked or if there are other more effective protocols for a species in involves the selection of reproductive individuals from which particular. samples are going to be collected. After application of the already The development of a hormonal stimulation protocol for a given developed hormonal stimulation protocol, samples should be species should first initiate with the selection of hormones. Here is collected during the concentration peak time points and stored at where previous studies could serve as a reference starting point, 4 C, while a sperm quality assessment is conducted. This step in always considering aspects such as phylogenetics and reproductive volves the evaluation of sperm concentration, osmolality, pH, per strategies between species. Protocols for gamete induction will centage motility and percentage FPM prior to cryopreservation to differ greatly depending on whether the researcher requires sperm ensure the sample is worth freezing. Low concentration or quality or eggs (Fig. 1)[22]. Nevertheless, to assess the effectiveness of the samples will not usually show good post thaw recovery. Here is treatments for the collection of either spermatozoa or eggs, wide where previously determining the sperm concentration peaks ranges of hormone concentrations should be considered and tested. plays a key role given that using highly concentrated samples for Then, the method of administration should be tried and selected on cryopreservation purposes is essential. We recommend freezing the basis of higher yield of high quality samples, effective working hormonally induced samples showing concentrations equal or concentrations and time of response to stimulation. We recom above 106, motility and FPM higher than 70%. Other sources of mend administering the treatments in volumes of 10 mL/gbw to sperm samples include testicular spermatozoa from fresh carcasses avoid causing osmotic imbalance. In the case of sperm collection or euthanized animals. This usually involves the surgical removal of protocols, SU collection is accomplished by catheterization or the testes followed by maceration in amphibian ringer solution stimulation of urination or spermatophore production by gentle (ARS). For quality assessment of testicular samples, we suggest abdominal massage, positioning the specimen above a petri dish or activating an aliquot by lowering its osmolality below 120 mOsm/ any other similar container to avoid losing the valuable sample Kg in water. Regardless of the source of the sample, aliquots should through spillage [18,70]. In many salamander species, the sper be fixed for further morphology and DNA fragmentation assess matophore is emitted as a thick semen like substance and it is ment prior to freezing. recommended that this is pipetted directly from the cloaca to avoid Stage 2 or cryotoxicity stage (Fig. 4) first involves the identifi loss of sample [80]. A number of post stimulation collection time cation and selection of penetrating and non penetrating cryopro points should be previously set, regardless of the administration tective agents (CPA’s) and concentrations for each [84]. Ideally, all method, starting as early as 30 min post stimulation and possible combinations should be tested. As mentioned above for continuing with 1 h intervals, for as long as 24e48 h post hormonal stimulation protocols, previous cryopreservation studies stimulation. To gain a better understanding of the effect of the could serve as a reference starting point, nevertheless, it is worth hormones through time, sperm concentration, osmolality, pH, considering that most studies do not present data on cryotoxicity percentage motility and percentage forward progressive motility prior to freezing. Once the CPA’s have been selected and prepared, it (FPM) should be assessed for each time point. Motility parameters is important to determine the CPA addition rate to the sample, can be assessed either visually through microscopy or with the use which is related to osmotic shock and its effect on cell viability [84]. of automated programs such as CASA system. Similarly, a small After applying fast or stepwise addition of the CPA, aliquots of the aliquot should be fixed at each time point for later morphology and sample should be activated with water and assessed for quality and DNA fragmentation analysis. The next step involves determining morphology. CPA addition rates yielding samples with motility and the peaks of sperm concentration. We suggest following the forward progressive motility higher than 70% should be considered method originally developed by Mouttham et al., 2011 [81], for the most appropriate. The next step involves analyzing and iden elephants, later adapted for the collection of anuran sperm by Della tifying the best equilibration time of the samples in the CPA prior to Togna et al., 2017 [18]. freezing. Equilibration time allows the CPA to penetrate the cell to The development of hormonal treatments for the collection of avoid or reduce ice formation during freezing. However, pene eggs has been more complicated than those of sperm collection trating CPA’s are usually toxic to cells, therefore, overly long ex since they usually require the administration of priming doses, posures will lead to cell death while an insufficient exposure time possible due to the more complex hormonal cycles present in fe would not allow the CPA to enter the cell, resulting in ice crystals males [49,76,82,83]. After the selection of the hormones and formation and cell death during freezing [84]. We recommend administration method, the assessment of response time with and selecting several exposure times, for example, starting with 5 min, without priming doses should be explored, considering combina continuing with 10, 15 and 20 min, after which, activation for tions of different hormones and concentrations for priming and quality and morphology analysis should follow. The successful ovulation doses. Collection of eggs after stimulation can be completion of this stage should yield information on the most accomplished by the female ovipositing eggs by itself or by strip suitable CPA, its addition method and equilibration time for the ping. After collection, eggs should be assessed for quality, consid samples prior to freezing. ering aspects such as number of eggs/clutch, size and color of eggs The first step of stage 3, the freezing and thawing stage (Fig. 5), and normal morphology. Fixing and staining a sample of eggs is requires the selection of the freezing container in which the sam recommended for the evaluation of morphology (e.g. number of ples will be cryopreserved (usually straws or cryovials). Sample jelly layers and appearance) (Fig. 2). The application of this process volume and density, sterility and sealing are important factors to for the development of hormonal stimulation protocols should, in consider when selecting the appropriate container [84]. Samples the end, yield information related to which are the most effective should be kept at 4 C during the entire process. Selecting the hormones, concentrations and doses, hormone administration freezing mode is highly dependent on finding the optimum cooling methods, sample collection methods, time of response to treat rate for the samples. Freezing modes include: controlled rate ments, peaks for sperm concentration, time of ovulation and the freezing (slow programmable freezers and temperature ramping in effect of the treatments on sperm and egg quality over time after styrofoam boxes), passive cooling devices ( 80 C freezers) and stimulation for each species. vitrification (directly plunging the sample into LN2). This step is After finding the optimal hormonal stimulation method for highly dependent on the equipment and resources available. We sperm collection for a single species, the intricate process of recommend testing different freezing modes and cooling rates for developing an effective cryopreservation protocol for the estab each species. Thawing can be accomplished by two methods: wet lishment of a GRB follows. Stage 1 or pre freezing stage (Fig. 3), and dry thawing. Both methods involve testing different

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420 G. Della Togna et al. / Theriogenology 150 (2020) 412 431 temperatures and exposure times, the former in water baths, the compounds into a biological system. latter in pellets or through air drying. The effect of a given freezing Using a similar approach, Silla et al. (2018) topically applied 25 mode and cooling rate on the quality of a frozen sample cannot be or 50 mg/g GnRH diluted in 100 mL of distilled water and dripped assessed without thawing and activating the sample first. Thawing onto the ventral side of male and female Pseudophryne pengilleyi conditions greatly affect the quality of samples as well. This is the [87]. Both 25 or 50 mg/g GnRH resulted in the amplectic pairs reason why both, freezing and thawing conditions should be releasing eggs and both treatment groups showed similar cleavage assessed concurrently. This particular stage includes multiple var rates. Once again, topical administration of GnRH had to be much iables, one or more of which could affect post thaw quality of the greater than IP injection to elicit spawning, with the lowest con sample. Manipulation of one variable at a time, while maintaining centration injected (4 mg) being the most efficacious in inducing the rest of the variables unchanged, is required to identify the spawning. Furthermore, eggs deposited by animals treated with optimal freezing and thawing conditions. For example, testing two lower concentrations had higher cleavage rates and this may be different cooling rates while keeping the same thawing conditions related to over maturation of eggs in the higher concentration for both or testing two or more thawing conditions while main groups. taining the same cooling rates. In 2019, Julien et al., tested the efficacy of GnRH when admin istered nasally by dripping diluted hormones into the nasal cavity Objective 2: Develop safe, non invasive methodologies for of Anaxyrus fowleri [88]. The authors found that GnRH concentra recovering viable sperm tions of 5, 10 and 20 mg were all relatively successful in eliciting spermiation. However, compared to injection in this species [89], Very few studies have been reported in the literature that sperm concentrations were low regardless of the doses used explore truly non invasive strategies for sperm induction using (Table 1). Compared to those induced by injection, nasal treatments hormones (Table 1). In addressing this objective, we take ‘non induced sperm concentration of a magnitude of 105 instead of 106. invasive’ to refer specifically to procedures that do not involve the In cases where sperm collections are to be used for AF this may not death of the animal, surgery or even injectable administration of be an issue as the concentrations may be more than adequate for hormones. Notably, out of a total of four studies, three have fertilising a known number of eggs. However, if sperm concentra explored the effects of topical application of hormones for sperm tions are low this type of induction may not be enough to properly induction using GnRH (also in its other commercially available fertilize a large number of eggs or provide an appropriate sample form, LHRH, p Glu His Trp Ser Tyr D Ala Leu Arg Pro NHEt). The for cryopreservation. The authors emphasized that sperm motility first study by Obringer et al. (2000) pre dates the ACAP objectives may be a better indicator of sperm quality than concentration itself. but since it is only one of four studies, we considered it worth However, other quality parameters such as cell viability or DNA mentioning in this review. integrity were not considered. Obringer et al. (2000) tested the capacity of GnRH to induce A possible disadvantage to using alternative methodologies to spermiation when applied topically both ventrally and dorsally to injections is that these approaches require the application of much Anaxyrus americanus and Anaxyrus baxteri [85]. Four mg GnRH higher hormone concentrations [85e88]. This is possibly indicative treatments were diluted in saline and results showed that of insufficient treatment absorption resulting in an unknown, compared to subcutaneous (SC) and intra peritoneal (IP) injection, probably lower, effective concentration. In searching for non topical administration had a low rate of induction, 3/9 males invasive methodologies, the ACAP objective also failed to reiterate compared to 7/9 (IP) and 9/9 (SC). In this instance, low induction of the importance of complementary plasma or urinary hormone sperm from topically treated animals may have related to the low assays to validate physiological responses to these treatments, effective concentration absorbed. Later, studies would show that whether administration occurs through injections or other higher concentration of hormones are required if administration is methods. Additional measurements of circulating plasma hor to occur through absorption. mones would help assess the efficacy of the methods and hormone Rowson et al. (2001) explored the efficacy of 1, 10 and 100 mgof concentrations. Although Obringer et al. (2000) used a hormone GnRH applied topically to the abdominal seat region of Anaxyrus assay validation, only changes in plasma hormones were measured americanus and Anaxyrus valliceps [86]. Results were statistically for animals stimulated with SC and IP injections, not for those similar for both species with 75% of males responding when GnRH treated topically [85]. was diluted in water. However, when GnRH was diluted in 40% (vol/ Furthermore, the underlying implication of objective 2 is that vol) DMSO (vol/vol) the concentration of hormone required to non invasive methodologies are safer than invasive treatments induce spermiation was reduced to 10 mg with a higher proportion such as injections. However, with proper training, SC or IP in of toads responding, 100% Anaxyrus americanus and 91% Anaxyrus jections are minimally invasive and more effective than topical or valliceps. Finally, this study analysed oral dispensation of the hor nasal treatments. In our 10 years of experience, IP injections have mone using prey items. When mealworms were injected with 1, 10 not caused a single adverse event or endangered the survival of and 100 mg GnRH, and fed to toads, 3/9 males from the 100 mg GnRH animals in any way (Della Togna & Calatayud, personal communi group produced SU. cation). Additionally, from the researcher’s perspective, this As Rowson et al. (2001) discussed in their paper, dermal drug or objective does not contemplate occupational health and safety hormone administration might offer several benefits over in (OH&S) standards required for the handling and use of hormones jections [86]. Dermal administration was more effective than oral for sperm induction. Regardless of the method of administration, administration presumably due to the fact that it bypassed the some level of training is required to ensure the safety of those gastrointestinal and hepatic system [86]. A possible disadvantage to delivering and receiving hormones. using alternative methodology to injections is that it requires much Added to non invasiveness, other advantages these methods higher concentrations of hormones. However, compared to provide are a reduction in waste, lessened disposal issues and Obringer, Rowson was able to lower concentrations of hormones reduced stress caused to the animal by handling or the injection when the latter was diluted into a solvent such as DMSO. In medical itself. Furthermore, it may provide another approach to hormone research, DMSO is often used as a drug vehicle in vivo and in vitro dosing when animals are small, and injecting may be awkward or experiments, and as a topical analgesic penetrating the skin and complicated. One important consideration not discussed by any of other membranes without damaging them and carrying the authors of the studies mentioned above is whether animals

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G. Della Togna et al. / Theriogenology 150 (2020) 412 431 421 displayed signs of discomfort when hormones were applied nasally determined to have low CPA cryotoxicity when equilibrated for or topically. It is important to verify that substances applied cuta 10 min at 2 C[112]. In 2002, Browne et al., conducted a cryotoxicity neously should be measured for pH and assessed for their irrita study on R. marina testicular spermatozoa after exposure to pene bility potential [90]. Nonetheless, the selection of the trating CPA’s (15 and 20% DMSO or glycerol) mixed with diluents administration route is the researcher’s decision. Topical and nasal (ARS or 10% sucrose) at 0 C for 6 days before freezing. They found induction of spermiation do offer captive breeding programs higher recovery of motility after exposure to 15% DMSO with either alternate treatment routes. These few studies are important and diluent. Browne et al., 2018 suggest equilibrating samples for a provide sound, alternative methodologies which need much more period of 10 min at low temperatures (close to or at 0 C) prior to attention in the future. freezing to allow penetration of CPA’s into the cells [32]. Della Togna, 2015, assessed the cryotoxic effect of exposure to dime Objective 3: Increase our understanding of cryosensitivity of thylformamide (5 and 10% DMFA) for 5, 10, 15 and 20 min before amphibian spermatozoa motility activation in Atelopus zeteki spermatozoa [25]. Higher motility recovery was achieved after exposure to both CPA’s for Cryoinjury results from physiological and/or morphological 5 min, while longer exposures significantly decreased motility. changes caused by the freezing process leading to substantial Upton et al., 2018, adapted the protocol developed by Browne et al., damage or cell death. Characteristically, cryoinjury is identified 1998 for the cryopreservation of testicular spermatozoa of Litoria through cell membrane damage, morphological abnormalities, fallax resulting in the first study to report the production of sexually sperm motility impairment, decreased fertilization capacity or DNA competent individuals through artificial fertilization. In their pro fragmentation, among others [91]. Motility is considered a common tocol, researchers included a pre freezing exposure period of and important sperm quality indicator since it involves the normal 10 min to CPA’s (15% DMSO þ 1% sucrose) at 2 C[113]. Results development and correct functioning of several cellular systems showed no apparent difference between unfrozen and cry working together to achieve motion, forward progression and opreserved samples in the production of offspring. finally, fertilization [25]. Therefore, motility impairment could Most studies on amphibian sperm cryopreservation have result from one or more damaged structural systems or molecular measured cryosensitivity in terms of post thawed sperm motility pathways. Activation of sperm motility has also been associated recovery, while others have considered assessing additional pa with other critical elements such as changes on the plasma mem rameters such as cell viability (sperm lysis, DNA integrity or brane potential and lipid composition, extracellular and intracel membrane integrity), fertilization capacity, embryo development, lular pH and ion concentration, and the temperature of the larvae survival and development to adulthood (for further refer extracellular environment [92e96]. Consider that in the majority of ence see Clulow at al., 2019) [76]. Thirty five percent of reported spermatophore producing caudate species, sperm is typically studies have assessed sperm cryosensitivity by measuring per immotile until it comes in contact with egg jelly and motility is centage motility recovery, 19% sperm viability, 16% embryo devel considered a poor indicator of sperm quality in these cases [97e99]. opment, 12% fertilization capacity, 9% development to adulthood Osmotic shock and response to osmolality is still poorly understood and 2% embryo development. Despite these results, interpreting in spermatophore producing caudates, therefore other quality pa the efficacy of the aforementioned studies is difficult due to a lack rameters such as cell viability should be considered [32,80,100]. of consistency with respect to processes of assessment. Most Osmotic shock can cause damage in other cell compartments studies show incredible variation in their validation of protocols [101]. Exposure to hypo osmotic environments has been related to with respect to cryoinjury not always considering appropriate an increase in the production of Reactive Oxygen Species (ROS) in viability assessments. Figs. 3e5 suggest a standard process for the the skeletal muscle, fibroblasts, and spermatozoa of some species. assessment of sperm cryosensitivity at different stages in the Moderate amounts of ROS in spermatozoa, as in most cell types, are development of the cryopreservation protocol. Systematic appli believed to play an important role in the metabolic and physio cation of this process will lead to a better understanding of cry logical molecular pathways of these cells [102e105]. Nevertheless, oinjury considering motility recovery, morphology and DNA increasing concentrations of ROS can cause DNA fragmentation and integrity as the basic assessment parameters. cell damage [102,105,106], where oxidized bases can lead to DNA breakages [107,108]. Objective 4: Develop ‘field friendly’ sperm cryopreservation One of the major obstacles for successful cryopreservation is the technologies and tools for assessing the viability/functionality of cytotoxic effect of penetrating CPA’s[109]. Cytotoxicity occurs thawed sperm when exposure to a CPA causes a detrimental or damaging effect on cell membrane structure, cellular metabolism, signaling pathways, In order to retain extant genetic diversity and periodically mitochondrial activity, DNA integrity, enzyme activity, sperm reinvigorate the genetics of a captive population we must develop motility, structure and function of macromolecules, among other standardized collection methods, evaluation and storage of germ consequences [110]. Besides cytotoxicity itself, other factors such as plasm from wild founders. The ability to increase the carrying ca CPA concentration, addition rate, equilibration time and tempera pacity and genetic diversity of a CBP without increasing the captive ture and elution, influence the effect of CPA’s on sperm viability. population size would be hugely advantageous and eliminate the Most amphibian sperm cryopreservation reports have not need to remove free living individuals from the wild. Most considered the cytotoxic effects of CPA’s or explored equilibration amphibian sperm cryopreservation studies have been conducted in conditions on the evaluation of post thaw viability. Beesley et al., the lab with ready access to equipment and facilities but the 1998, used non frozen suspensions of R. sylvatica, L. pipiens and methodologies described generally use relatively cost effective A. americanus spermatozoa (CPA’s DMSO, methanol, glycerol, or materials and basic freezing techniques [34,46,53,98] that could ethylene glycol þ supplements) only as control samples to compare be adaptable to a field setting. For example, a closed Styrofoam cell lysis effect of frozen/thawed sperm exposed to the same sus freezing box containing LN2 with a straw holding platform situated pensions, but did not specify the pre freezing equilibration time in LN2 vapour will deliver a cooling rate that can be customised by [111]. Browne et al., 1998, assessed the recovery of motility and adjusting the height of the platform. Ideally, SU should be pro fertilization potential of frozen spermatozoa of R. marina using cessed and cryopreserved within 1 h to ensure optimal sample several concentrations of DMSO and glycerol previously quality. However, site location and available resources might

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422 G. Della Togna et al. / Theriogenology 150 (2020) 412 431 necessitate the temporary storage of samples for several days or sperm collection site and host facility is not feasible and/or the hours prior to cryopreservation. Short term cold storage can vehicle is unsuitable for transporting LN2. maintain sperm motility over several days [44,75,114e116] offering 3. Field kit only. This set up works in circumstances where the a viable method of sample collection from remote field locations sperm collection site is accessible only by foot very remote and/ and where immediate access to LN2 is limited. These cold storage or access to a mobile lab or host facility is limited. Remote field techniques coupled with the development of SU collection pro sites create logistical problems in the transportation of equip tocols (allowing donor males to be released back to their capture ment meaning that only the necessary equipment for the locations on completion of sperm harvest) offer a viable strategy for collection and temporary cold storage of sperm samples can be non lethal in situ collection and cryopreservation of sperm from transported to the collection site with sample processing taking free living cohorts. This field oriented technique has successfully place several hours or even days later. been applied to the boreal toad [46] and to several fish species, with similar modes of reproduction [117,118]. Equipment required for in situ sperm cryopreservation: However, field gene banking efforts must be preceded by complementary ex situ measures, such as the prior development of Cold stored hormones, syringes and collection catheters and an optimized hormone stimulation protocol for the species. Un tubes. derstanding the reproductive response to hormone treatment is A portable cooler maintained between ~0 and 4 C with key to ensuring that sperm is collected during peak production and temperature monitoring (ie HOBO logger) device to store at appropriately timed collection points, thereby allowing multiple samples prior to cryopreservation. collections from the same donor male. In addition, successful Styrofoam box containing liquid nitrogen and straw platform for cryopreservation protocols should be established in advance, cooling of samples. where possible, using captive bred counterparts. Gamete cryo Freezing straws and straw sealer. preservation techniques often require modification at a species Thermocouple. level [118] and some studies in fish using a Styrofoam freezing box Appropriate liquid nitrogen container. have shown low reproducibility under field conditions due to Dry shipper for temporary storage of frozen samples. variability in freezing rates [117]. Therefore, future research ob Portable generator. jectives should focus on the development of a standardized Microscope. portable freezing unit to circumvent variability in cooling rates. Slides, coverslips, pipettes and tips. Magyray et al. (1995) describe a self sustained portable kit for in Cell fixation method or live/dead cell staining kit that fixes situ gamete collection in fish in which cooling rates could be samples on site for future analysis. regulated by an adjustable straw platform [118]. Further in vestigations of alternative freezing methods that would eliminate Finally, it is essential to follow biosecurity protocols when col the need to transport LN2 into the field (i.e. in the vapour of a fully lecting gametes from the wild. Although there are no reports of charged dry shipper) should also be explored. disease transmission through gametes or ART’s, methods such as The practicality of in situ sperm banking depends on a number swabbing, PCR and sample isolation are recommended before using of factors related to the availability of resources and facilities and the samples in captive breeding programs. We suggest these the location and accessibility of the field site. The proximity of the samples are stored in separate containers until test results confirm field location to the sperm processing site determines the length of existence or lack of disease. Basic protocols such as chlorine, 100% time that samples are maintained in temporary cold storage prior ethanol or heat, should be applied for the disinfection of all con to freezing. We propose three potential modes of operation for in tainers used in the field. situ gamete collection, which are adaptable based on the avail ability of resources and facilities and the location and accessibility Objective 5: Establish methods for recovering viable spermatozoa of the field site: from fresh carcasses

1. Fully independent mobile lab facility (i.e. trailer/bed of a truck) The recovery of amphibian spermatozoa has been accomplished utilising a generator and all necessary sperm analysis, process through the application of two methods: testicular excision and ing and cryopreservation equipment. This set up assumes that spermic urine collection [19]. Testicular sperm sampling is usually the on site collection location is proximal to the mobile lab fa achieved by euthanasia of captive or wild animals, followed by cility and that sperm samples are held in cold storage for a processing of the testes, sperm analysis and storage (either refrig limited time prior to cryopreservation. This is the optimal field erated or cryopreserved) for its immediate or later use. However, gene banking scenario, allowing for cryopreservation of freshly another source of testicular sperm are recently deceased animals. collected samples. This situation would also be applied in cir To our knowledge, there are only two reports on the recovery of cumstances where the sample collection site is proximal to the spermatozoa from recently deceased amphibian carcasses. The host facility but access to host facility resources, e.g. for bio first, by Roth and Obringer, (2003), assessed the percentage motility security reasons, is restricted. and integrity of the mitochondrial vesicle in sperm obtained from 2. Reduced capacity mobile lab (i.e. use of a generator but no ac refrigerated carcasses of Anaxyrus baxteri. They found very low cess to LN2). This set up utilises all necessary sperm analysis and percentages of motile sperm and intact mitochondrial vesicles processing equipment without access to LN2. In this situation, (<10%) 12e36 h after death. Additionally, the same authors report sperm parameter analysis (motility counts, concentration etc.) no recovery of motility from Andrias japonicus spermatozoa 6e18 h can be conducted in the field but cryopreservation is only after death. The second report, by Shishova et al., 2013, explored the possible at the host facility. The main disadvantage of this quality and viability of sperm collected from carcasses of Rana arrangement is the length of time that samples are held in cold temporaria [120]. Percentage motility, percentage FPM, cell mem storage prior to freezing, however, it does allow for field col brane integrity, DNA integrity and fertilization capacity were lections over several days prior to returning to the host facility. assessed in testicular sperm samples refrigerated for up to 7 days. This scenario would be applied when daily travelling between Percentage motility and FPM decreased over time, from 55% motility and 6% FPM at day 1e10% motility and less than 5% FPM

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G. Della Togna et al. / Theriogenology 150 (2020) 412 431 423 after 7 days of refrigeration. Cell membrane integrity remained Collections (Smithsonian tropical Research Institute (STRI)) and the above 70% while DNA fragmentation increased over 50% after the NMNH Biorepository (National Museum of Natural History) located same time (7 days). Fertilization capacity also decreased over time, in the US. Additionally, CryoArks Biobank, in the UK, is a consortium with only 17% of oocytes fertilized after 4 days of storage and of institutions that include the Natural History Museum, The Frozen almost no fertile spermatozoa after 7 days. Comparatively, Browne Ark Project, the Zoological Society of London (ZSL), the Royal et al., found that testicular spermatozoa from Rhinella marina, Zoological Society of Scotland (RZSS), and National Museums macerated and kept in ARS, retained high motility and fertilization Scotland (NMS) as well as partnerships with the IUCN (Interna capacity for up to 10 days after refrigeration at 0 C when compared tional Union for Conservation of Nature) through its Conservation to 4 C. In comparison to testes macerates, spermatozoa recovered Genetics Specialist Group. The Smithsonian Conservation Biology from whole testes refrigerated for the same time did not show good Institute in the US as well as other partners from Ireland, Germany, quality parameters, but had higher motility and fertilization ca Denmark, Norway, the USA, Colombia, South Africa, India, Vietnam, pacity after refrigeration at 0 C compared to 4 C. We recommend Malaysia, South Korea, Australia and New Zealand are also part of the methodology detailed in Browne et al., 2001, for the storage and the CryoArks Biobank consortium. The Conservation Biobank in recovery of testicular spermatozoa. Denmark, the Centro Nacional de Recursos Genomicos in Mexico, Considering the yielding of high quality sperm from deceased the Fraunhofer Institute for Biomedical Engineering IBMT in Ger animals requires the rapid detection and processing of the carcass, many; to name a few, have also been established with the purpose CBP’s must implement efficient protocols that allow daily moni of storing genomic resources. Nevertheless, exotic species are still toring and detection of sick or deceased animals. Where samples poorly represented in the biobanking world and certain taxonomic may be collected opportunistically due to the death of a captive groups (mammals in particular) are favored due to anthropomor animal, a high degree of coordination between the various de phic interest. Despite the extinction crisis, amphibians are still partments involved (e.g. reproductive biology and pathology) is highly neglected by the exotic species biobanking community and required for the timely processing and successful testicular sperm the development of ART’s poorly researched or funded compared to recovery. In some cases, institutional policies (e.g. pathological other groups. As such, the corresponding protocols required for examination) influence the speed at which necropsy samples are cryopreservation, have lagged behind those reported for other processed and available for use, delaying and potentially compro taxonomic groups. Positively, the recent proposal to launch mising the quality of the sperm sample. Future ACAP recommen Amphibia Bank, suggests that a network of cell culture and tissue dations should address the need for timely coordinated repositories with the goal of collecting cell samples (blood, cell institutional policies surrounding sample collection, particularly in cultures, tissues, spermatozoa, and in the future, eggs and embryos) the case of critically endangered species. of amphibian species will change this in the near future. A pilot project of this effort is focused initially on collecting North Amer Objective 6: demonstrate the biological competence of ican salamander species [129]. cryopreserved spermatozoa through the production of healthy Ironically, frog sperm was one of the first to be used in cooling offspring and cryopreservation studies. The earliest experiments date back to 1776, when Lazzaro Spallanzani (1729e1799), an Italian priest and Amphibian biobanking has lagged behind achieving the proof of scientist, discovered he could keep frog sperm viable after cooling concept reported in other taxa. Popularly, human gamete cryo in snow and re warming [130]. It would take another two centuries preservation, IVF and production of infants has become common before Luyet & Hodapp first used sucrose to dehydrate frog sperm practise in medicine and has blossomed into a multi million dollar, prior to immersion in liquid nitrogen ( 192 C) and achieved 60% global enterprise run both privately and publicly [119e121]. Bio motility recovery [131]. To date, amphibian cryopreservation re banking has expanded to include other species, primarily domestic mains limited to sperm because of the large size, constitution and plants and animals, that are considered economically valuable to volume of eggs and embryos. Historically, “successful” cryopreser human existence [122]. The biomedical industry has relied on vation in amphibians has been reported in relation to post thaw biobanking for several decades to maintain research species, recovery of sperm motility and function. Few studies detail the specialized breeds, strains and genetically modified research long and multifaceted procedure detailed previously in this models. Strangely, despite numerous literary references to the manuscript. All these parameters, (cryopreservative selection, application of cryopreservation and biobanking to wildlife conser sperm preparation and dilution, cryotoxicity, freezing and post vation, establishment and maintenance of long standing bio thawing analysis of functional capability, and the numerous sub repositories for preservation of exotic species are as few as are the steps in each), if not properly determined, can have adverse ef reports of successful use of frozen sperm for the breeding of exotic fects on the functionality of spermatozoa [132], and this is impor species. When it comes to biorepositories, plants receive far more tant because consistency in procedure must be ensured if multiple attention than animals and biobanking of exotic animals have species are to be incorporated into a biobank. popularly focused on charismatic mammals [123e126]. The most All things being equal, the final measure of success or gold notable success story for cryopreservation in conservation is that of standard, as with any other species, is the production of offspring the black footed ferret Mustela nigripes [126e128]. So, despite its that develop normally and become reproductively viable. The obvious application, cryopreservation and biobanking still go number of species currently reported with successful cryopreser largely ignored by the broader conservation community, who vation protocols can be grouped into those that report successful favour more ecologically based methodologies along with the for recovery through sperm viability assessments (motility, function mation of CBP’s, as sufficient approaches to species conservation. ality tests, membrane and acrosomal integrity) and those that The longest standing exotic species biobank is the San Diego resulted in successful fertilization (AF, egg hatching, larvae survival Zoo’s Frozen Zoo® which has been in operation since 1972 (https:// and production of healthy, reproductively viable offspring). Since institute.sandiegozoo.org/resources/frozen zoo%C2%AE). Other the ACAP was published in 2007, there have been some advances in exotic species biorepositories include the Pan Smithsonian Cryo sperm cryopreservation citing 28 species in the literature, (anurans Initiative (PSCI) which includes the Genome Resource Bank for and salamanders) and 12 species opportunistically collected and Global Conservation (National Zoological Park/Smithsonian Con stored in the San Diego Zoo’s Frozen Zoo, USA, and University of servation Biology Institute), the Molecular Evolution Frozen Newcastle, Australia respectively (personal communication Nicole

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Ravida and Rose Upton respectively) (Supplemental Fig. 2). Sperm categories would actually suggest that cryopreservation still holds from another six species have been collected after hormonal some promise which, when used in conjunction with other stimulation and banked at the Panama Amphibian Rescue and methods, would increase conservation outcomes. Funding has also Conservation Project (PARC) (Della Togna personal communication) been a problem and the unequal global investment in conservation (Supplemental Fig.1). Four of the 40 biobanked species known to us projects is, as with most economical situations, biased to the more represent salamanders, and include Cryptobranchis alleganensis, developed countries. The financial aspect of the ACAP biobanking and the subspecies of Cryptobranchis alleganensis bishopi [133], objectives is explored later in this manuscript. Ambystoma mexicanum [32,100,134], Ambystoma tigrinum [36,80,135] and Andrias davidianus [36](Supplemental Fig. 2). As Objective 7: conduct the necessary computer modelling required to previously mentioned, the majority of reported protocols for determine the optimal number of individuals to be banked cryopreservation end with motility or viability assessments. Only a handful report post thaw artificial fertilization as proof of concept Storage of recoverable genomes as live cells and tissues in [46,80,113,135e137] and truly demonstrate the biological compe genome resource banks serves three main purposes: 1) catastrophe tence of frozen amphibian sperm with the production of viable F1 insurance against extinction, 2) facilitation of assisted reproduction individuals. when gamete supply is asynchronous, and 3) a resource to manage In 2015 and 2016, Langhorne and Marcec respectively, had a the genetics of captive and wild populations [27,76]. These objec series of successes producing viable offspring from anuran and tives have different requirements in terms of the logistics of salamander species including Lithobathes sevosa, Anaxyrus boreas genome resource banking, particularly with respect to types of boreas, Anaxyrus fowleri [82] and Abystoma tigrinum [80]. From genomes required and the numbers of individuals banked. these studies, offspring were produced and successfully meta Catastrophe insurance requires that materials are capable of morphosed for three species, Anaxyrus boreas boreas, Lithobates generating whole organisms, so banking whole embryos would be sevosa [46] and Ambystoma tigrinum [80]. In 2018, Vance et al., optimal. Storage of only one type of gamete or somatic cells might reported a follow up study in an abstract for Cryobiology, in which also achieve recovery of an extinct species, but only indirectly the authors demonstrated that offspring created by Marcec and through chimeras [141e143], nuclear transfer [144 ,145] or repeated Langhorne had not only reached sexual maturity but were capable back crossing [146,147]. The objective of assisted reproduction can of producing viable gametes [138]. Of twenty one A. tigrinum F1 be met where asynchronous gamete release is a problem by storing produced in 2015, three individuals were hormonally induced, and the gametes of one sex. Numbers banked for these two objectives their gametes used in AF experiments. A single F1 female was are likely to be determined by circumstances. According to genetic ultrasounded to confirm egg development and her eggs fertilized theory, about 95% of the genetic diversity of a randomly outbred using sperm collected from a non cryogenically produced male. population would be sampled by banking 10 individuals [147]. Out of 550 viable eggs, 18% developed to blastula. In contrast, sperm Hence, for catastrophe insurance, a limited number of animals collected from F1 cryogenically created males yielded 42% embryo banked may meet the genetic objectives. Where gamete storage is a development to blastula when fertilising eggs from a non tool to facilitate assisted reproduction in a CBP, the number of in cryopreserved female. The survivability of F2 generated animals’ dividuals banked will be determined by the number of adults in the past metamorphosis was less than 10%. In the same year, Upton program. et al. (2018a) reported the successful cryopreservation of sperm The scenario is more complicated when the objective is to taken from testicular macerates of an endangered Australian capture the genetic diversity of a founder population with the aim anuran, Litoria aurea [137]. Later that year, Upton et al. (2018b) were of maintaining the genetic diversity of a captive population at a the first to report the production of sexually mature offspring in level close to that of the source population. A similar problem oc another Australian Hylid, Litoria fallax [113]. Their results showed curs where the aim is to restore the genetic diversity of a small, that similar developmental progression was observed in control increasingly inbred wild population to a level of heterozygosity frogs versus those produced using cryopreserved sperm. Moreover, present at an earlier generation. For amphibians, this is most likely the males produced by cryopreserved sperm reached sexual to be achieved by the use of banked spermatozoa that are thawed maturity and were capable of sperm production [139]. and used in AF with living females from a captive colony or wild Though undeniable proof exists that ART’s can be utilized to population. This is because, as discussed elsewhere in this review, produce viable amphibians it has taken more than a decade to cryopreservation of spermatozoa has been achieved for a growing reach the goal of this objective. Proving the methodology effective number of species, but the cryopreservation of eggs and embryos enough to incorporate into CBP standard practise will require has not. further studies and extensive training and capacity building to The principles of quantitative genetics [146,147] allow estima make cryopreservation more accessible to a diversity of conserva tion or modelling of the required numbers of individuals to be tion programs. Furthermore, the amphibian conservation commu banked to achieve desired outcomes under different scenarios. A nity needs to open itself up to the potential that biobanking has as useful benchmark to aim for is the retention of 90% of the initial an additional approach. In a report by ‘Conservation evidence’, source population heterozygosity of a captive colony over a 100 Smith et al. (2019) reviewed biobanking as a conservation strategy year period [13,147,148]. The same criteria could be applied as a and suggested that its overall effectiveness was “unlikely to be target for managing the genetic diversity of a small wild population beneficial”. This ranking system based on criteria modelled by the facing inbreeding. The use of cryopreserved founder spermatozoa authors included measures of ‘effectiveness’, ‘certainty’ and ‘harm’ in backcrossing with live females in a captive colony or small wild [140]. However, the problems arising from this publication include population is one way to achieve this, and it is particularly relevant 1) the outdated nature of the literary reports used to formulate the in the case of amphibians (an approach applied successfully to a assessments, 2) the indisputable fact that production of live young mammal, the black footed ferret, in a real world scenario) [128]. A from natural breeding will always be preferable to the production number of parameters will determine how many individuals need of a species through cryopreservation and 3) that this system of to be banked to achieve these objectives including the mean gen classification does not take into account that none of the other eration interval, the effective population size of the target species categories cited here (Supplemental Table 1) would likely be (Ne) and its ratio to population census number (Nc), and the fre effective when worked as individual strategies. Taken together the quency of back crossing to founders. The impact of this on the

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G. Della Togna et al. / Theriogenology 150 (2020) 412 431 425 number of live animals that must be maintained in a captive colony 7. Access exists to collection sites using local knowledge and or small wild population to retain the 90% heterozygosity/100 year expertise, taking into account that many species are located in target is quite dramatic. The Ne required to achieve the 90%/100 or near to indigenous communities and protected areas, each year target is approximately Ne 475/L (where L generation with particular restrictions. interval [147], with the number of live animals dramatically 8. Country policies on access to genetic resources allow such large increased when the Ne/Nc ratio is taken into account (this ratio may scale operations. often be in the order of 0.10) [147]. For a hypothetical amphibian population with a generation length of 2 years, and an Ne/Nc ratio of Points 2 through 8 are beyond the scope of this review. There 0.1, implies that a colony size of 2500 animals would be required to fore, we focus our analysis on examples of current capacity building meet the 90%/100 year target. However, if live females in the as a first step towards the future systematic collection and pres captive colony were backcrossed [146] to founder males using ervation of global amphibian biodiversity. Multidisciplinary col cryopreserved spermatozoa, that target could be reached with a laborations are key to providing financial support and training much smaller colony. There is a need to assess and model the po opportunities to regional or internationally managed research fa tential application of this approach in depth, but in theory, at least, cilities, particularly for those that currently lack the expertise or the potential to achieve genetic management goals while dramat resources to contribute to a global GRB. ically decreasing the costs of maintaining large, live captive col Regionally, inter capacity training, knowledge transfer and the onies is considerable. As Clulow et al. [27,76] noted, the global exchange of technology should be promoted through inter resources available to manage the large number of threatened institutional partnerships and collaborations (e.g. zoos and uni amphibian species requiring captive management to avoid extinc versities and research facilities). Such partnerships and/or col tion is insufficient. The approach of combining banked founder laborations have already been exemplified in the US where sperm and assisted breeding in colony management could both organizations such as the Smithsonian Conservation Biology dramatically reduce costs, achieve genetic management targets, Institute, San Diego, Memphis and Fort Worth Zoos and Mis and allow many more species through captive breeding with the sissippi State University (MSU) have, on different occasions, available resources to avoid potential extinction. collaborated on the exchange of sperm collection protocols and cryopreservation techniques resulting in the successful produc tion of offspring from multiple species including, Anaxyrus boreas Objective 8: implement and maintain an inventory and database for boreas, Anaxyrus fowleri, Rana sevosa, Peltophryne lemur, Atelopus effective management of cryopreserved samples zeteki, to name a few. In the case of Anaxyrus boreas boreas, capacity building was Maintaining databases or an inventory of frozen samples will be extended to include other organizations such as Colorado Fish and at the discretion of the user and the institution. A variety of soft Wildlife’s Native Aquatic Species Restoration Facility (NASRF). ware already exists that can be modified or customized to suit the NASRF houses the largest captive breeding colony of Anaxyrus b. cataloguing needs of each GRB. It is impossible to expect that boreas in the US and exogenous hormones are often used to pro standardization of a common product will be implemented across mote reproduction in the colony. Through collaborative efforts institutions. However, programs such as Access (Microsoft™), between MSU, Memphis Zoo and San Diego Zoo, post doctoral and Oracle, Structured Query Language (SQL), Filemaker© (Claris In PhD researchers were able to develop and optimize protocols that ternational Inc.), among others, could all fulfil the user’s cata assisted in the deposition of eggs and collection of sperm for later loguing needs. These programs must be used to define, manipulate, use in AF. Furthermore, in 2014 and 2015, San Diego Zoo re retrieve and manage data and its record structure in a systematic, searchers trained facility staff to administer hormones, collect replicable and accountable manner. Independently of each in gametes, perform AF and characterize embryo development. stitution’s database needs and choice, the building of a common Though long term survival of offspring produced by AF was low in platform for global sharing and usage will be of higher impact and the facility, tadpoles were released as part of the joint effort pose greater challenges to the scientific community. spearheaded by MSU, Memphis and San Diego Zoos in partnership with Colorado Parks and Wildlife. Other examples in the US include Objective 9: Increase scientific capacity in country through training collaborations between Memphis Zoo, MSU and the Smithsonian to routinely allow large scale and safe collection and Institute which conducted cryopreservation trials on Atelopus zeteki cryopreservation of germplasm from free living and captive sperm. In 2013, post doctoral and PhD staff from MSU and Mem amphibians phis Zoo in collaboration with FortWorth Zoo developed exogenous hormone protocols for Peltophryne lemur [149]. In 2017, the Panama In the authors opinion, objective 9 should only address Amphibian Rescue and Conservation Project of the Smithsonian increasing scientific capacity in country through training. The in Tropical Research Institute and Smithsonian Conservation Biology clusion of establishing routine, large scale collection and cryo Institute trained staff from Memphis Zoo for the optimization of preservation of germplasm from wild and captive animals is not cryopreservation protocols using four Atelopus species: A. limosus, realistic at this time since it implies and requires that: A. varius, A. certus and A. glyphus. Collectively, the examples mentioned above have resulted in important advances for the 1. Scientific capacity and well trained human resources are in conservation of endangered amphibian species and in the transfer place. of key knowledge and techniques. 2. CBP’s are well established in each of the range countries. 3. There is well established knowledge on existing wild pop Objective 10: Explore the possibility of establishing satellite (back ulations for each species. up) locations for storage of bio materials 4. Well established gamete collection and cryopreservation pro tocols are already developed for each species (see Figs. 1e5). Various amphibian GRB’s exist globally, the San Diego Zoo’s 5. National laws and policies allow and facilitate the collection, Frozen Zoo, the Frozen Ark in the UK, the Ian Potter Australian transportation and storage of biological materials. Wildlife Biobank, all of which collect and store genetic material 6. Sustainable funding for these operations exists. from global threatened fauna (including amphibians). However,

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426 G. Della Togna et al. / Theriogenology 150 (2020) 412 431 these exist in a fragmented sector as standalone institutions (the have been no studies on the economics of amphibian biobanking. same phenomenon occurs domestically). A detriment of this frag Contributing to the lack of progress in developing a funding mented sector is that critical genomes for amphibian species are mechanism for the management of cryopreserved amphibian often stored entirely in one location. While we recognise the samples, is a lack of understanding of economic costs required to detailed fail safe procedures and insurance measures possessed by establish and manage long term biobanking infrastructure. The GRB’s, including alarms, back up power and strict specimen literature in this area is dominated by biobanking economics in a handling procedures, we also recognise that there is risk with any human biomedical context where similar funding challenges facility. Recognising this risk, having critical genomes stored exist [156]. Funding mechanisms are clearly defined for entirely in one location, poses a management risk. One way to biomedical biobanks, including detailed itemization of the full manage this risk is duplication of specimens across multiple facil range of fixed overhead and recurring variable costs. Start up ities as back up, ideally in a managed and catalogued network. For costs are reported as initial equipment purchase, space plan example, the San Diego Zoo’s Frozen Zoo maintains multiple bio ning and GRB construction costs, as well as inventory manage repositories located at different sites which house replicate sam ment and computers. Recurrent operating costs include ples of any tissue, cell or gamete collected by staff at the employee payroll, consumables, vendors and consultants, as well organization. as additional equipment purchase, maintenance and replace Satellite (back up) locations for bio materials would ideally be ment. Activity costs are on going and comprise variable labour low cost alternatives isolated from the main amphibian GRB and equipment costs incurred through biobank usage and are infrastructure and be limited in usage to the deposit of bio reported broadly as building biobank inventory (frozen samples materials. Withdrawal is unlikely unless access to back up mate and data), sample collection, service provision, administration, rials is necessary. We recommend that future ACAP thinking should facility operations and maintenance and quality management consider two options for developing these back up locations. [157]. Many of these costs are common to GRB infrastructure in other sectors and are not unique to biomedical GRB’s. As with 1. Hold back up samples in GRB infrastructure of other sectors much of the traditional conservation infrastructure (particularly (biomedical biobanks, zoological storage facilities, natural his zoological CBP’s), biomedical biobank operating costs are not tory museums, agricultural biobanks). To our knowledge’ these generally publicly available, so actual investment across these partnerships have not been generated for the interdisciplinary broad cost categories is largely unknown. Qualitative approaches sharing and storing of frozen biomaterials across sectors. ACAP were required to generate detailed funding mechanisms and objectives should include qualitative research by amphibian itemize these cost categories for biomedical biobanks. This in GRB practitioners in range countries to identify potential formation was provided through surveys of biobank operators cooperating institutions and generate these partnerships. Back and practitioners [157], to our knowledge no similar approaches up materials will likely require minimal space and result in have been conducted to itemize operating costs of amphibian minimal deviation from current operating costs for cooperating GRB’s. Greater economic transparency and qualitative research institutions. (involving communication with conservation GRB practitioners 2. Low cost additional infrastructure could involve using off site and zoological institutions containing this infrastructure), would floor space in cooperating institutions (zoological institutions, enable the development of publicly available funding models and universities). Generating this basic infrastructure for back up funding mechanisms based on clearly, itemized real amphibian storage would be a viable low cost option, requiring dewars, GRB costs. freezers and liquid nitrogen (>$14,000 Howell, unpublished) The original ACAP objectives proposed a short term funding (see objective 11 for further detail). mechanism for establishing an amphibian GRB. Adjusted for infla tion, these costs in 2019 are broadly as follows; year one infra structure costs at US$84,206, research at US$531,587 across 3 years, Objective 11: Establish a funding mechanism to manage the personnel and staffing at US$501,525 and minor equipment and cryopreserved samples (including personnel, equipment, liquid consumables at US$123,833 across 5 years. These costs represent a nitrogen, storage space) specific scenario where a GRB is constructed, staffed and operated for 5 years as additional infrastructure to a zoological institution. Establishment of GRB’s as viable and commonplace infrastruc The adjusted budget is adequate for the specific criteria and pro ture for amphibian conservation is being held back severely by posed scenario; however, the funding mechanism is narrow in constrained resources [150e152], as well as funding neglect for ex scope and aspects are not optimal or sustainable. The budget also situ conservation techniques. Amphibians represent an ideal taxon neglects some of the logistical cost categories listed above. Eco for which to establish GRB’s. Comparatively low ex situ conserva nomic planning for biomedical biobanks usually gives close tion costs [153] and various life history traits are favourable for consideration to the type and scale of biobank and different usages, advances in the ART’s which would underpin biobanking technol where specific operational characteristics require unique funding ogy. Despite limited overall funding, various flagship amphibians mechanisms. It is unclear whether these types of biomedical bio have been able to draw significant public and private investment banks are similar to potential amphibian GRB’s in usage and scale for ART’s. Significant investment in CBP infrastructure, often leads and whether they could provide model funding mechanisms. In the to in situ outcomes globally for amphibian species facing extinc absence of this detailed knowledge we propose amphibian GRB’s tion. Some well known examples include the Southern Corroboree could likely occur in three broad forms which differ considerably in frog, Pseudophryne corroboree [41], the Wyoming toad, (Anaxyrus usage, scale and cost: baxteri)[49], the Mississippi Gopher frog, (Lithobates sevosus)[154], and the Puerto Rican Crested Toad (Peltophryne lemur)[155]. 1. Low cost: utilising existing institutions which store frozen ma Establishing GRB’s as viable infrastructure to deliver similar out terials (natural history museums, zoos, universities) and utilis comes for amphibian conservation will depend on consistent and ing existing infrastructure or providing basic infrastructure for sustainable public and private investment, leveraged using eco long term storage of biomaterials, including dewars, freezer nomic incentives and clearly defined funding mechanisms. and liquid nitrogen (>$14,000) (Howell, unpublished). Under Since the ACAP, biobanking objectives were derived there this approach it is assumed that all costs (outside of potential

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user pays costs) are absorbed into already existing operating become self sustaining after initial short term and often significant costs. This scenario would likely be restricted to the deposit of investment for project start up and infrastructure costs. This likely biomaterials (unlikely to be used for withdrawal unless represents a future challenge for amphibian biobanks given accessing back up material) and provides a low cost back up stretched resources and the limited periods of funding under storage solution (see objective 10), as well as providing low cost conventional grant mechanisms. This issue has already been potential storage of critically endangered species with minimal observed for wildlife biobanks in Australia, where start up costs individuals remaining. This scenario is also ideal for: a) storage have been covered and infrastructure has been developed; how of species for which use of frozen biomaterials in ART’sisnotyet ever, programs have stalled (consider the Animal Gene Storage viable, b) as rapid response to stochastic events, and c) as Resource Centre of Australia, established in 1995 and on hold and temporary infrastructure in range countries with lower research volunteer maintained since 2009 https://theconversation.com/ and development capacity which often face the challenge of australias frozen zoo and the risk of extinction 565). global geographic bias in research and development as well as in In the biomedical sector, various funding mechanisms have infrastructure [158]. emerged to address these issues and generate self sustaining GRB’s 2. Moderate cost: additional GRB infrastructure for storage incor [160]. Cost recovery [159,160], public perception and acceptance porated into existing ex situ conservation facilities (zoos, uni [160], analysis of impact and value studies [160,161], online eco versities) ideally providing access to live colonies. Under this nomic planning tools [157], user pays systems [162], commerciali approach it is assumed that a portion of overhead costs are sation [163], as well as business strategies and thinking to develop absorbed into already existing operating costs. Economic cases based on cost efficiency, have all emerged as potential finan models are required to suggest the extent of deviation from cial solutions. The ability of these funding mechanisms to provide current operating costs for cooperating stakeholders, however, long term sustainable biomedical biobanking infrastructure has been they would not be expected to be significant. For example, Zoos mixed, and their applicability for establishing and maintaining Victoria’s proposed start up costs of AU$22,000 to incorporate amphibian GRB’s is unexplored. Therefore, the challenge is not basic GRB infrastructure into an extensive native species estimating short term start up budgets and itemizing costs (re breeding program, which includes iconic amphibian species: flected in previous ACAP objectives); however, it is to develop the Northern Corroboree frog (Pseudophryne pengilleyi), and funding mechanisms to maximise the chance of long term mainte Southern Corroboree frog, (Pseudophryne corroboree), the nance and usage of amphibian GRB’s. If we adopt the goal of 90% Spotted Tree Frog (Litoria spenceri), and the Stuttering Barred retention of genetic diversity in captive populations across 100 years frog (Mixophyes balbus)[159]. Embedding amphibian GRB’sin (although the infrastructure is likely maintained indefinitely), then public and private conservation infrastructure may provide a this time frame should be the minimum lifecycle when considering cost effective solution and establish biobanking technology as a future funding mechanisms. Future ACAP objectives should draw on key component in ex situ conservation programs. perspectives from the biomedical biobanking sector and seek to 3. High cost: dedicated infrastructure generated from the ground explore the applicability of funding to amphibian GRB’s. This up with the intention of deposit and withdrawal of stored ma approach would enable a range of strategies, outlined above in the terials for use in amphibian ART programs (ideally involving full aim, to generate a framework for amphibian GRB sustainability. freezing and storage infrastructure, as well as laboratory and Also underpinning progress towards a funding mechanism for CBP infrastructure for live colonies). This scenario would require amphibian GRB’s is a lack of understanding of the complete the establishment of dedicated live colonies and would not be financial life cycle of biobanking infrastructure (Fig. 6). Costs limited to species in current CBP’s. However, establishing occurring in the construction stage can be easily predicted and are additional CBP’s requires its own unique start up and recurring itemized in the original ACAP budget for the particular scenario costs ($148,600 to establish additional programs based on 2007 presented. Based on perspectives from the biomedical sector, ACAP costings for captive programs adjusted to 2019 dollars). amphibian biobanks are unlikely to become self sustaining within this maintenance period. Budget planning for amphibian bio Future research and ACAP objectives should endeavour to pro banking has largely neglected this period and whilst cheaper than vide detailed itemized costings and funding mechanisms for these construction costs these recurring costs would be significant and three broad amphibian GRB scenarios. In addition to cost estimates, open ended. A particular challenge is the inability to predict future qualitative research is required to develop a network of committed usage of amphibian GRB’s and to factor this into funding mecha participants and stakeholders (scenario 1 and 2) to host additional nisms. Funding for amphibian GRB’s will be conditional on their amphibian GRB infrastructure and collaborate on ART’s programs. value, however, considerable uncertainty exists regarding future Considerations should be given to qualitative approaches to itemize usage value of amphibian GRB’s. Unforeseen use of biobanking costs for these scenarios, in particular survey studies of conserva infrastructure (likely delivered by advances in the amphibian ARTs tion GRB and CBP practitioners to inform detailed, accurate and and potential to use cryopreserved samples) may influence the transparent funding mechanisms for developing amphibian GRB’s. return on investment of original planning and construction fund Also lacking is knowledge on the potential cost efficiency for both ing, long after the funding has been delivered, based on potential biobanking practitioners and cooperating stakeholder institutions cost effectiveness, increased value and unforeseen potential con under each of the above scenarios, as well as the return on in servation outcomes. A better understanding of this uncertainty and vestment in conservation outcomes for amphibian species. Future unforeseen usage value could build the case for the initial funding ACAP thinking should seek to quantify and estimate the usage and communicate with policy makers before construction. Future benefits for all stakeholders under these broad scenarios. ACAP objectives should seek to generate predictive economic Additional considerations should be made beyond itemizing models required to account for this uncertainty, with emphasis on start up costs for amphibian GRB’s and objectives should look to potential advances which may influence the overall cost providing long term sustainable funding mechanisms to ensure the effectiveness and value of programs. For example, the potential of maintenance and usage of infrastructure indefinitely. Based on the backcrossing with frozen sperm withdrawn from GRB’s and sup proposed ACAP start up and 5 year budget, it is largely unclear how plemented into a live captive colony of amphibians using AF could infrastructure (and programs) would be financially maintained into reduce the number of live individuals required to be kept in the future. It is important to note that biomedical biobanks rarely traditional CBP’s and provide genetic and conservation outcomes

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428 G. Della Togna et al. / Theriogenology 150 (2020) 412 431 for endangered amphibians (see objective 7). This may lead to cost ART’s, cryopreservation and biorepositories should become an savings for cooperating stakeholders leading to increased capacity additional priority tool. to develop new programs for other at risk amphibian species. The economic justification for this approach is poorly developed and is Appendix B. Supplementary data largely based on anecdotal arguments and only preliminary data exists [162] (L. G. Howell, R. Frankham, J. C. Rodger, R. R. Witt, S. Supplementary data related to this article can be found at Clulow, R. M. O. Upton, and J. Clulow, unpublished). 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