The effects of climate change on our oceans imperil the health and resilience of coral reef systems around the globe. Addressing drivers of global climate change, while concurrently implementing restoration interventions at a large scale, provides the best chance for the continued existence of these ecosystems and their essential function and services (Anthony et al., 2017; Hoegh-Guldberg et al., 2019; Knowlton et al., 2021; Vardi et al., 2021). In Australia, the Great Barrier Reef (GBR) has experienced an increase in the frequency of climate-change-driven mass bleaching events over the last decade, including in its previously untouched southern range (Hughes et al., 2018; Australian Institute of Marine Science, 2021; Great Barrier Reef Marine Park Authority et al., 2022). These unprecedented heat stress events have caused a decline in coral cover and have reduced reproductive output (Hagedorn et al., 2016; Howells et al., 2016; Daly et al., 2022), consequently depressing larval supply (Cheung et al., 2021) and recruitment (Hughes et al., 2019).

In addition to action on climate change and improved local management practices, active reef restoration approaches to repair and re-seed reefs will likely be required for many sites to maintain or restore biodiversity and support or accelerate adaptation (Anthony et al., 2017; van Oppen et al., 2017; Suggett and van Oppen, 2022). Current restoration efforts primarily use asexual propagation methods (colony fragmentation) to generate new material for out-planting (Young et al., 2012; Boström-Einarsson et al., 2020); however, this approach has limitations for upscaling to a system as large as the GBR and it only generates clonal material. There is increasing interest in the sexual production of coral juveniles for out-planting (e.g., Randall et al., 2020), which is more amenable to upscaling, has the added advantage of helping to maintain population genetic diversity, and has the ability to strengthen or introduce adaptive traits to populations through selective breeding (Quigley et al., 2020; Hagedorn et al., 2021).

The sexual reproduction of corals ex situ is commonly achieved either by allowing individual colonies to naturally spawn in the same system or by controlling pairwise matches by selectively mixing freshly washed oocytes with optimized concentrations of fresh sperm (commonly termed ‘in-vitro fertilization’, or IVF). The relatively recent development of sperm cryopreservation technologies for corals (Hagedorn et al., 2006) enables selective breeding to also occur using sperm samples that are spatially and temporally removed from fresh oocyte supply. Cryopreservation refers to the controlled cooling of living cells and tissues to ultra-low temperatures using techniques that maintain the structure and function of cells when samples are brought back to physiological temperatures, allowing them to be stored for decades, possibly centuries, if maintained correctly. Biobanking of cryopreserved samples, when combined with assisted reproductive technologies such as IVF, can support species conservation by preserving genetically distinct specimens over long time periods. The potential application of biobanking to wildlife conservation was recognized in the 1970s (Watson, 1978; Benirschke, 1984) and has progressed over ensuing decades with zoos emerging as global leaders in the establishment of wildlife biobanks (Comizzoli and Wildt, 2017; Hagedorn et al., 2019; Hobbs et al., 2019; Hagedorn et al., 2021).

In 2011, scientists from the Taronga Conservation Society and Zoo (Sydney, Australia) and the Smithsonian Institution (United States of America) initiated a program with collaborators from the Australian Institute of Marine Science (AIMS) to apply coral sperm cryopreservation methods to species from the Great Barrier Reef. Cryopreservation techniques proved directly transferable to Australian Acropora species (Acropora tenuis, A. millepora), leading to the establishment of Australia’s first frozen coral biorepository (Hagedorn et al., 2012a; Hagedorn et al., 2012b). Moderate upscaling of IVF methods for the generation of live offspring (larvae and juveniles) using cryopreserved material was successfully demonstrated over the first three years of the program (Hagedorn et al., 2017). Subsequent improvements to the specifically designed cryopreservation apparatus (Zuchowicz et al., 2021) have aided in the up-scaling of time-sensitive sperm sample processing. Biobanking of gametes from GBR corals by the Taronga and Smithsonian teams has continued annually during spawning, utilizing wild coral colonies held temporarily at the AIMS National Sea Simulator (Cape Ferguson, Queensland, Australia). These achievements have resulted in the integration of coral biobanking via cryopreservation as an enabling technology within the largest research and development program for coral reefs globally, the Reef Restoration and Adaptation Program (RRAP ¹ ). A decade from its conception, we conducted a review and quality assurance check of Taronga’s CryoDiversity Bank coral inventory. This review aimed to generate census data on the existing collection to understand the quantity and scope of sampling from bioregions and species, and to identify gaps in quality assessments and metadata that need to be addressed. As the Taronga CryoDiversity Bank transitions from a relatively small-scale opportunistic sperm sample collection to a more targeted approach, we will use this information to direct future resource investment, inform collection management and ensure that the collection remains relevant to the needs of the coral conservation and restoration communities. It is hoped that the information and experiences of the Taronga CryoDiversity Bank will be useful to other coral biorepositories around the world that will likely face similar considerations as they seek to support coral conservation and restoration efforts.

Data associated with Taronga’s CryoDiversity Bank, in Sydney on Cammeraigal Country and in Dubbo on Wiradjuri Country, are managed using Microsoft Excel and stored on a user-restricted server. Prior to conducting the inventory review, a quality assurance check of the metadata was performed. An example of the metrics and metadata collected from each colony, and the evolution of these data fields over the 10+ year program, are included in Supplementary Material ( Figure S1 ). New metadata fields were added as new technologies (e.g. computer assisted sperm analysis; CASA) were incorporated into best practices and metadata needs for future restoration were refined by collaborators (e.g. GIS data).

An inventory review was conducted of the data associated with physical sperm samples collected from corals of the GBR and cryopreserved between November 2011 and February 2022.

To obtain estimates of colony numbers and quantity of material from species, reefs, and regions, data were handled as follows:

As of March 2022, Taronga’s CryoDiversity Bank contains 2614 cryovials of cryopreserved sperm sourced from:

Between 2011 and 2014 a total of 446 cryovials from 7 species were cryopreserved and banked ( Figure 1A ) consisting of pooled sperm samples only ( Figure 1B ). In 2016, the first medium-scale cryopreservation of coral sperm specifically for conservation biobanking was undertaken at the National Sea Simulator (AIMS) adjacent to the central GBR. This single collection event increased the total number of cryovials preserved, the number of colonies from which sperm were sampled, and the number of species represented by approximately 100%, to a cumulative total of 1088 cryovials from 123 colonies across 11 species. This was also the first year that sperm samples were collected and cryopreserved from single colonies, creating samples of individual genetic units ( Figure 1B ). Between 2016 and 2020, an average of 415 cryovials of cryopreserved sperm from 5 species and 60 new colonies were added to the bank each year.

Eleven of the 31 species represented in Taronga’s CryoDiversity Bank had sperm samples collected and cryopreserved across more than one year ( Figure 2 ), five of which were sampled across six or more years. Eighteen species have been sampled only on one occasion.

Zoos are global leaders in the establishment of wildlife biobanks both for fundamental research and as a tool to support genetic management of ex situ breeding programs (Comizzoli and Wildt, 2017; Hagedorn et al., 2019; Hobbs et al., 2019; Traylor-Holzer et al., 2019; Holt & Comizzoli, 2021). Alongside biobanking, development of reproductive technologies across a range of taxa (Comizzoli, 2015; Herrick, 2019) has seen these tools more frequently integrated into breed-for-release programs and species recovery plans in recent years (Swanson et al., 2007; Howard et al., 2016; O'Brien et al., 2016; Della Togna et al., 2020; Hagedorn et al., 2021; Hobbs et al., 2021). Biobanking has been championed by these organisations for decades as a viable conservation tool that does not detract from, and should not be to the exclusion of, other conservation strategies.

Through collaborative programs over the last decade, Taronga’s CryoDiversity Bank coral collection has become the largest single biorepository of cryopreserved coral sperm ^{2 , 3} , in both number of species represented and quantity of material. Crucially, this collection includes sperm samples that pre-date the recent mass-bleaching events on the GBR (e.g., 2016, 2017, 2020 and 2022) along with samples from colonies sourced from targeted resilient populations that have the demonstrated ability to produce heat-tolerant offspring (Quigley and van Oppen, 2022). The CryoDiversity Bank therefore provides an important reservoir of genotypic and phenotypic biodiversity that could become a unique resource for reef restoration practices, especially those that incorporate adaptation and adaptive management into their strategies (Quigley et al., 2022; Shaver et al., 2022). Our recent review showed that the Bank contains sperm samples from over 380 individual colonies of 30 identified species of scleractinian corals collected from a wide geographic area of the GBR Marine Park. Over the lifetime of Taronga’s Reef Recovery Project³, most sperm samples have been collected opportunistically from colonies prioritized and sourced by our collaborators conducting their own research on a small number of coral species year-to-year. Hence, the collection is dominated (70% of samples) by five species (A. hyacinthus, A. loripes, A. millepora, A. sarmentosa and A. tenuis) and by reefs in close proximity to the National Sea Simulator on the Central GBR.

Sampling strategies have been suggested to support ex situ propagation of genetically diverse coral populations (e.g., Shearer et al., 2009; Baums et al., 2019; Quigley et al., 2019), which are directly applicable to the sampling of coral donors for sperm biobanking. One such target is the sampling of ≥30 genets per species, which captures approximately 95% of allelic diversity of an average population of unknown genetic heritage (Shearer et al., 2009). It is important to undertake genotyping of all banked samples, particularly for those species with less resolved population genetics, to confirm genetic diversity prior to potential future use of cryopreserved sperm for conservation breeding and to inform ongoing collection targets. To this end, we recommend that a fragment, or voucher sample, be taken from each coral colony and banked alongside cryopreserved sperm samples to permit future genotyping and species identification (Voolstra et al., 2021). The most highly represented species in Taronga’s biobank, A. tenuis, easily surpasses this target with sperm samples preserved from 94 colonies. However, only six colonies have been sampled from the southern GBR where A. tenuis is known to have higher genetic diversity compared to northern populations (Lukoschek et al., 2016) so future effort will be focussed on bolstering genets collected from this region. For other species with fewer samples, a target of 30 genets per species and population will prioritized. Sourcing rare and poorly represented species will remain a challenge on the GBR as the majority of its reefs are tens of kilometres from the mainland; to reach them requires significant resourcing. Engagement with Traditional Owner and other community groups to prioritize culturally significant and biodiverse sites and species will help to ensure that limited resources have the most impact for people and communities.

At present the most effective cryopreservation methodology for coral is sperm preservation, as cryopreservation technologies for other coral cell sample types are not yet sufficiently developed for biobanking. Taronga’s CryoDiversity Bank and similar groups working in Hawaii, Florida, Mexico, the Red Sea, and the Caribbean have therefore focused their acquisitions on cryopreservation of the male germline (Hagedorn et al., 2006; Grosso-Becerra et al., 2021; Zuchowicz et al., 2021). Approximately 63% of coral species surveyed are broadcast spawners (Baird et al., 2009) so the capture of sperm for most species should be possible, albeit with some notable challenges such as gonochores (e.g., Porites species) and species for which reproductive timing remains unknown (Baird et al., 2021). Coral sperm cryopreservation can help diversify shrinking populations through selective breeding, but currently relies on availability of fresh oocytes; we also need additional strategies to mitigate species extinctions by preserving whole organisms, or somatic cells. Cryopreservation and laser-warming of coral larvae (Daly et al., 2018) is a technology with promise but may be too complex for large-scale or field application. Newer, simpler cryopreservation technologies may provide additional opportunities for coral; for example, cryo-grid technology can easily be used to cryopreserve large numbers of Drosophila embryos and pancreatic cells from various species (Zhan et al., 2021; Zhan et al., 2022). Taronga’s CryoDiversity Bank coral acquisitions have only recently expanded to include experimental preservation of other cell types (e.g., embryonic cells: Hagedorn et al., 2012b), life history stages (e.g., larvae: Daly et al., 2018; Cirino et al., 2019), and the microalgal endosymbionts of corals (Symbiodiniaceae: Hagedorn et al., 2010; Hagedorn and Carter, 2015; Lin et al., 2019; Kihika et al., 2022). While cryopreservation of sperm should remain a priority in the near term, the inclusion of new sample types into existing biorepositories and restoration programs will be rapidly adopted as the technologies mature and become ready for broader application.

The most efficient and effective use of cryopreserved sperm samples for reef restoration will likely be for genetic management of brood-stock within ex situ coral aquaculture systems, where sexually produced juveniles are generated under controlled conditions (Hagedorn et al., 2017; Randall et al., 2020) and microbiome manipulations are possible (Buerger et al., 2020; Santoro et al., 2021; Maire and van Oppen, 2022) prior to deployment onto selected reefs (e.g., Quigley et al., 2021; Randall et al., 2021). The use of cryopreserved sperm is a cost-effective way to increase the effective breeding population (e.g., Howell et al., 2021), providing greater control over brood-stock and genetic management for trait selection via selective crosses among individual colonies. Alternatively, pooled samples enable “batch” multi-colony fertilisation mixtures to maximise the number and diversity of larvae produced, and fit with the planned automation of ex situ coral spawning activities to generate restoration-scale quantities of material. Although the Taronga CryoDiversity Bank has tended towards cryopreservation of sperm from individual colonies in recent years, future collections should aim to achieve a mix of both individual and pooled samples.

Cryopreserved sperm could also be used to overcome temporal differences in spawning between colonies. Importantly, cryopreserved sperm can easily be moved between institutions and populations to facilitate assisted gene flow (e.g., Hagedorn et al., 2021; Daly et al., 2022), reducing the cost and risk of disease transfer associated with moving coral colonies. An example of where this approach may be important is the Florida Coral Rescue program ⁴ , which has brought over 2300 colonies from 20 species of coral into the care of 27 facilities managed by 28 partners (including zoos and aquaria) in 15 states across the USA in response to extensive coral mortality in the wild from stony coral tissue loss disease (SCTLD). To manage population genetics within this ex situ breeding program and permit genetic crosses amongst institutions, cryopreserved sperm could be utilized to produce new corals for reef restoration and for biobanking to ensure that founder population genetics are retained (B. Firchau, pers. comm.).

Depending on the scale to which fertilization using cryopreserved sperm can be expanded, it may be possible to generate larvae directly for settlement and deployment using biobanked sperm samples. In vitro fertilization methods for some coral species are well established and typically involve a standard fertilization ratio of 10 eggs per ml exposed to 1×10⁶ sperm/ml, corresponding to approximately 1×10⁵ sperm per egg (Pollock et al., 2017; dela Cruz and Harrison, 2020). In the case of bulk fertilization of freshly collected gametes, fertilization volumes can reach up to 200 L for large mixed-batch cultures, generating hundreds of thousands of embryos (Negri and Heyward, 2000). Cryopreservation methods were developed to preserve 1 mL aliquots of sperm at a 1×10⁹ sperm/mL, with a predicted efficiency of 50% sperm survival and consequently a reduction in overall motility (Hagedorn et al., 2017). Existing IVF production methods using cryopreserved coral sperm (Hagedorn et al., 2021; Daly et al., 2022) are capable of producing tens of thousands of coral larvae, suitable for the genetic maintenance of brood-stock or for small-scale restoration activities (e.g. Grosso-Becerra et al., 2021), especially if combined with advanced microfragmentation and husbandry techniques (Page et al., 2022). Current protocols for sperm cryopreservation and use may therefore already be suitable for many restoration applications; however, to support the production of millions of coral larvae, different approaches to sperm cryopreservation, sample packaging, and fertilisation methods, such as those used in the agriculture and aquaculture industries, may be required.

Reef recovery programs developing and applying reef restoration practices at the scale required to help large reef ecosystems (e.g., Bay et al., 2019; National Academies of Sciences, Engineering, and Medicine, 2019) will necessitate a prioritization of species and populations; this is especially true for the GBR, which encompasses hundreds of species across thousands of individual reefs along thousands of kilometres of coastline. Taronga’s coral cryo-collection will, in part, support the production needs of an established aquaculture program (Gibbs et al., 2019; Worley Parsons Services and Australian Institute of Marine Science, 2019) whose species priorities will be informed by restoration modelling and species selection tools (Madin et al., 2021). Large-scale aquaculture of this type will likely target a smaller number of species to produce large quantities of coral recruits for direct deployment onto reefs, potentially leading to a further increase in sampling from species that are already well represented in the bank (e.g., A. tenuis and A. hyacinthus). Of equal importance, however, will be the need to establish a more diverse collection that provides a reservoir of biodiversity from which to draw once initial coral cover is restored. This strategy will warrant continued collection from lesser represented species and regions, which will be supported by competitive funding streams and philanthropy in partnership with Traditional Owners and various community groups. Striking the balance between securing broad biodiversity and storing large volumes of prioritized species to support aquaculture will be an important logistical challenge for all coral biobanking programs going forward. Modelling efforts aimed at prioritizing the array of suggested interventions (e.g., Condie et al., 2021) will also be required before we can fully understand the scale at which cryopreservation will integrate with coral production in Australia and therefore the quantities of cryopreserved material that will be required.

Coral reefs are complex ecosystems to conserve; ongoing applied research and modelling will be needed to improve our understanding of the necessary interventions, their scale, and their design (Sivapalan and Bowen, 2020). Based on our experience, biobanking activities should ensure that: each colony is uniquely identified and either genotyped or vouchered; collection and cryopreservation of sperm is prioritized and new sample types are introduced as technologies become available; and collections target a mix of individual and pooled samples matching production strategy and restoration goals. To achieve this will require increased resourcing for infrastructure to support the required biobanking capacity, along with continued refinement of metadata and database management to ensure that these collections can be maintained in perpetuity.

Aquaculture and biobanking on the scale required for coral conservation worldwide will require strategic partnerships between science, industry, restoration specialists, government, and community. Taronga scientists, along with scientists from other institutions utilizing coral cryopreservation strategies globally, are collaborating to refine coral cryopreservation technologies and build capacity through groups such as the Coral Restoration Consortium (CRC) Cryopreservation and Biobanking Working Group and the Coral Biobank Alliance. It is hoped that these initiatives will help to achieve the shared goal of minimizing the loss of biodiversity on coral reefs globally.

Taronga Conservation Society commits to working in a way that respects, recognizes and includes First Nations people. Taronga will consult with Traditional Owners to ensure that the proposed cryopreservation interventions are socially and culturally safe, including that: consent is obtained to collect material from sea Country; movement of cryopreserved material to Taronga’s biobank facilities is performed in collaboration with Traditional Owners of sea Country and Cammeraigal (North Sydney, NSW) and Wiradjuri (Dubbo, NSW) people; and access to biobanked material only occurs within the consent provided by the relevant Traditional Owner group. Moreover, Taronga recognizes the current and ongoing heritage and spiritual connection of Traditional Owners to their sea Country and will permanently track Traditional Custodianship within the CryoDiversity Bank electronic database. Recognition of the Traditional Custodians of living cells within the biobank into perpetuity will be vital to the long-term stewardship of these samples and harmonizes with the integrated and inclusive approach to species conservation being undertaken by Taronga and RRAP.

The prospects of the Taronga CryoDiversity Bank to support reef restoration are promising; however, the reality is that the causes of climate change progress unabated. Recent fine-scale modelling (Dixon et al., 2022; Kalmus et al., 2022) predicts that an elevated temperature of 1.5°C could see <1% of coral surviving by the mid-2030s. Securing biodiversity through continued sperm cryopreservation, and the development of simple, field-ready, technologies for coral embryo cryopreservation, will be crucial to stop-gap species loss in the coming years and decades. We hope the results of our work and efforts may give future generations options for healthier, more diverse reef ecosystems.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

RH compiled and analysed the data, wrote the manuscript and prepared the figures. JD did the main manuscript revision and editing. MH, RS and JO’B contributed significantly to the establishment and ongoing management of the Taronga CryoDiversity Bank coral collection and reviewed the manuscript. All other authors listed have made a substantial, direct, and intellectual contribution to the work over the life of Taronga’s reef recovery program, reviewed the manuscript and approved the manuscript for publication.

Taronga’s CryoDiversity Bank infrastructure and biobanking collection trips were co-funded by Taronga Conservation Society Australia, the Taronga Conservation Science Initiative and other philanthropists supporting the Taronga Foundation and we acknowledge the Australian Institute of Marine Science which provided laboratory space and facilities within the National Sea Simulator. Development, refinement and training in enabling technologies (MH, VC, NZ, JD) were co-funded through the Smithsonian Conservation Biology Institute, the Hawaii Institute of Marine Biology, the Australian Institute of Marine Science, the Paul M. Angell Family Foundation, the Smithsonian’s Women’s Committee, the Roddenberry Foundation, the Seaver Institute, the William H. Donner Foundation, the Barrett Family Foundation, the Skippy Frank Translational Medicine and Life Sciences Fund, the Compton Foundation, the Cedar Hill Foundation and the Anela Kolohe Foundation. From 2020, co-funding was provided by the Taronga Foundation and the Reef Restoration and Adaptation Program, a partnership between the Australian Government’s Reef Trust and the Great Barrier Reef Foundation.

The authors would like to acknowledge the Traditional Owner groups of the sea Country where coral colonies have been collected from. The authors thank those Traditional Owners who have given permission to preserve and store samples cryopreserved from their corals and commit to continuing consultation with all Traditional Owners regarding both historic and future collection of material for biobanking from sea Country. We would also like to acknowledge the support of The Australian Institute of Marine Science staff and scientists who facilitated the collection and spawning of colonies within the National Sea Simulator and the Great Barrier Reef Legacy who enabled access to corals from the North MA to be preserved. This work was undertaken as an activity of the Cryopreservation sub-program (RRAP-CP-01) for the Reef Restoration and Adaptation Program, a collaboration of leading experts working to create a suite of innovative measures to help preserve and restore the Great Barrier Reef. Funded by the Australian Government, partners include: the Australian Institute of Marine Science, CSIRO, the Great Barrier Reef Foundation, Southern Cross University, The University of Queensland, Queensland University of Technology and James Cook University.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.