On Thin Ice: Cryopreservation of Coral Sperm for Reef Conservation & Restoration

In Summer 2019, I was fortunate to join Dr. Mary Hagedorn and colleagues at the Hawai’i Institute of Marine Biology to learn all about novel techniques for cryopreserving coral sperm. I am now integrating these exciting methods into my own coral reproduction research.

Read on to learn why cryopreservation is an especially promising tool to help conserve and restore reefs:

Stony corals are ecosystem engineers; they build reefs that support one quarter of all marine organisms and contribute billions of dollars to global economies each year through tourism, coastline protection, and fisheries (Spurgeon et al., 1992; Jones et al., 1994; Wells et al., 2006). However, coral reefs are declining globally at an alarming rate, which threatens the critical ecosystem services they provide (Pandolfi et al., 2003; Wild et al., 2010). The combined and accelerating effects of multiple local and global stressors – including ocean warming, ocean acidification, nutrient pollution, coastal development, overfishing, and predator and disease outbreaks – may outpace corals’ natural capacity to adapt and evolve (Hughes et al., 2003; Palumbi et al., 2014).

If coral reefs are to persist into the “Anthropocene” - the current geological age during which human activity has been the dominant influence on climate and the environment - interventions are urgently needed. 

  Cryopreservation is the long-term storage of biological materials such as proteins, cells, organs, and tissues at extremely low temperatures  - usually -196°C, the temperature of liquid nitrogen. Under such cold conditions, all enzymatic and biochemical activity essentially ceases, including processes leading to DNA degradation and cell death. Free from deterioration, this material can (at least in theory) be thawed at some point in the future and remain alive and viable. As such, cryopreservation is a powerful tool for maintaining diverse genetic repositories of existing species that are endangered in the wild and preventing further loss of genetic variation as populations decline. Further, thawed gametes (the collective term for eggs and sperm) can be used to rescue dwindling or even extinct populations in the future when conditions are more favorable for their survival. 

These techniques have already been utilized successfully for the conservation of endangered species. Since 1976, San Diego Zoo has maintained a genetic repository with frozen cells and tissues from more than 10,000 individual animals representing over 1,000 species of birds, reptiles, amphibians, and mammals. Called the “Frozen Zoo”, this gene bank not only stores genetic material from these species, but has also been thawed and used to introduce new genetic material into endangered populations of pheasants, rhinos, and snakes.

Similarly, the University of Nottingham’s “Frozen Ark” (www.frozenark.org) stores thousands of tissue, cells, and DNA samples from endangered animals including the scimitar horned oryx, which is now extinct in the wild. A particular focus of cryopreservation has been freezing gametes (eggs and sperm), due to their ability to generate offspring and pass preserved genetic material directly to a new generation when thawed and fertilized. The ability to store high-quality biological samples long-term has facilitated genetic exchanges between captive areas, zoos, and research centers for the purposes of artificial insemination, gamete micromanipulation, in vitro culture, and grafting.

To date, relatively limited work has been done on the cryobiology of gametes from aquatic species, perhaps because of the very different physical and chemical conditions that they face compared with terrestrial animals. However, the conservation applications of cryopreservation are increasingly relevant and urgently needed for threatened aquatic species, particularly tropical reef-building corals.

Why is cryopreservation necessary for corals?

Cryopreservation of coral genetic material, particularly sperm, may be extremely beneficial to prevent further loss of coral cover and diversity and boost recovery of damaged populations:

First, this can serve as a valuable gene bank, allowing scientists to store genetic material from a range of individuals that are threatened by rising sea temperatures, increasing ocean acidity, disease outbreaks, pollution, and other stressors driving coral mortality. It is estimated that coral cover has already been reduced by 80% in parts of the Caribbean (Gardner et al. 2003), meaning the genetic material from 80% of corals has likely also been lost. To prevent further loss of genetic variation that may prove important in the face of current or future conditions, efforts should be made to freeze gametes from as many species and as many locations as possible. 

Second, cryopreserving coral sperm can help restoration practitioners selectively breed gametes of parent colonies that may not be able to mate in nature because they are too far from one another – a process termed “assisted gene flow”. Recently, Dr. Mary Hagedorn and colleagues succeeded in fertilizing fresh Acropora palmata (A. palmata) coral eggs from Curaçao with cryopreserved coral sperm from Curaçao, Florida, and Puerto Rico (2018). Each treatment resulted in viable, healthy larvae that grew into a total of 4,700 juveniles from cryopreserved sperm.

This study proved that 1) gametes from distant A. palmata populations that cannot breed in nature are reproductively compatible and 2) assisted gene flow is possible throughout the Caribbean. This may become a promising approach to help boost genetic diversity by injecting genes from more distant populations while local ones lose diversity. This could also become a useful tool to selectively breed for specific traits by crossing gametes from colonies deemed to have some fitness advantage (such as resistance to bleaching or disease) in order to create offspring with these desirable traits.

Third, nature’s best recovery strategy – sexual reproduction, or the introduction of new genetic diversity by mixing male and female gametes to generate new individuals – is breaking down on reefs due to dwindling coral populations and environmental changes (Kaniewska et al. 2015, Teo & Todd 2018, Shlesinger & Loya 2019). Unlike humans, corals cannot choose to reproduce at any time during the year. For most species, their spawning is restricted to just a few nights each year, when all the colonies on a reef carefully synchronize the release of their gametes in hopes they will meet and fertilize one another. Unfortunately, as coral populations decline worldwide, a negative feedback loop forms: the fewer corals in a given area, the lower their collective reproductive success becomes. This is due to the “Allee effect”, a decline in individual fitness at low population density. As coral cover decreases and the distance between colonies increases, it will become increasingly unlikely that their gametes will meet in high enough concentrations to achieve adequate fertilization (Levitan & McGovern 2005, Teo & Todd 2018). 

Furthermore, even for colonies that remain close enough together for their gametes to mix, environmental changes such as increasing ocean temperature may be throwing off their reproductive timing (Baird et al. 2009, Shlesinger & Loya 2019). Corals have evolved to spawn with careful precision, utilizing cues from the lunar cycle, water temperature, day length, and water motion to release their gametes with incredible precision (Babcock et al. 1986, Oliver & Babcock 1992, Levy et al. 2007). However, as conditions change due to human impacts, corals may not be able to “read” environmental cues with enough precision. Peak fertilization occurs when spawning is well-synchronized (Levitan et al. 2007), so any shift in timing poses a major threat to the successful formation of offspring.

Finally, heat waves have become increasingly frequent and severe on reefs around the world, triggering what is known as coral “bleaching” (Hughes et al. 2017). Stony corals depend on tiny, single-celled algae called Symbiodiniaceae that live within their tissues and use sunlight to make energy for their coral hosts. When corals experience stressful conditions such as high seawater temperatures, they eject the algae from their tissues, becoming white in appearance (hence the term “bleaching”). Without Symbiodiniaceae, corals lose the primary source of nutrition they need to survive and grow. Although corals can recover by re-acquiring Symbiodiniaceae if the stressful conditions subside, these events damage the reproductive potential of reefs. With depleted energetic resources, colonies that have recently experienced severe bleaching often fail to spawn (Fisch et al. 2019), and their reduction in reproductive output may last years after a disturbance (Levitan et al. 2014). Few reefs on Earth have avoided bleaching, and the time between severe bleaching events is diminishing (Hughes et al. 2018). If corals do not have sufficient time to recover after disturbances, reproduction and recruitment on impacted reefs may totally collapse and prevent the ecosystem from bouncing back. 

For all these reasons, it is urgent that scientists and stakeholders test a variety of interventions to halt the rapid decline of coral reefs worldwide. Without these immensely diverse ecosystems, we could lose over 25% of all species in the oceans, many of which human beings rely on for food, fisheries, and pharmaceuticals. Cryopreservation is a low-risk strategy that can prevent further loss of genetic diversity and help mitigate the intensifying problems of scarce parent colonies and gametes (Hagedorn & Carter 2016). If sperm is collected and frozen from male colonies whenever they spawn, it can be thawed and used to fertilize fresh eggs any time females spawn, even if that is hours, days, months or years apart from the males in question. On shorter time scales, this can help minimize the negative impacts of desynchronized spawning and still enable managers to generate offspring (and new genetic diversity). 

Progress in corals

While cryopreservation techniques have been used for decades in threatened terrestrial animals such as rhinos (and also in humans), they are only starting to be applied to coral conservation. Studies by Dr. Mary Hagedorn and colleagues have pioneered the use of cryopreservation for coral spermatozoa. To date, her team has successfully frozen ~30 species of corals, primarily from Hawai’i and the Great Barrier Reef, and frozen sperm from these species are being stored long-term with the USDA’s Animal Germplasm Program (Fort Collins, CO, USA).

In 2012, Hagedorn and colleagues published the results of a study that generated Acropora tenuis and Acropora millepora larvae from frozen sperm, the first known confirmation that cryopreserved coral sperm could be thawed and remain reproductively viable. They have since shown no difference in fertilization success depending on the length of time that sperm has been frozen (ranging from a few hours to 2 years), illustrating the promise of this approach for long-term storage of viable sperm for use in conservation efforts (Hagedorn et al. 2017).

Hagedorn and her colleagues are beginning to publish more on their sperm freezing and thawing techniques to disseminate the knowledge to scientists and restoration practitioners around the world, in hopes that anyone with access to coral spawning events can incorporate cryopreservation into their efforts. 

Recently, Hagedorn and colleagues demonstrated the potential for gametes from distant populations of the same species to successfully fertilize one another after cryopreservation and result in viable, settled offspring (Hagedorn et al. 2018). Using the threatened Caribbean species Acropora palmata (staghorn coral), this study succeeded in fertilizing fresh eggs from Curaçao with frozen sperm from Curaçao, Florida, and Puerto Rico, and all crosses resulted in swimming larvae and settled recruits. These results demonstrated that (1) distant conspecific populations can remain reproductively compatible, and (2) genetic diversity can be transferred from one population to another for the purposes of assisted gene flow in corals (Hagedorn et al. 2018)!

Limitations and knowledge gaps

Despite these promising applications, coral cryopreservation has a number of limitations and remaining knowledge gaps. First, freezing sperm almost always decreases its motility, often by about 50% (Hagedorn et al. 2012). For instance, fresh sperm with 90% motility prior to freezing will likely have ~45% motility after it is thawed. Therefore, it is essential to obtain robust, highly motile sperm to use for freezing (Hagedorn et al. 2017), because high motility yields better fertilization.

Furthermore, a portion of sperm cells will die as a result of the freezing and thawing, which decreases the concentration of sperm and thus decreases the rate of fertilization it is likely to achieve. The process of freezing and thawing any biological material is fraught with risk, as forming ice crystals threaten to cause lethal damage and changing concentrations of water and salts threaten to disrupt the structure and/or function of proteins, organelles, cells, and tissues (Hagedorn et al. 2006, Benson et al. 2012). As such, it is important to maintain a relatively high sperm-to-egg ratio when attempting to fertilize fresh eggs with cryopreserved sperm (Hagedorn et al. 2017). 

Another limitation is the failure to successfully freeze and thaw coral eggs. With a larger size and much more complex structure than sperm cells, eggs are more susceptible to damage during the extreme temperature changes. To date, no one has been able to thaw coral eggs without ice crystals rupturing and killing them during some part of the process. If we can only freeze sperm, we cannot freeze gametes from female colonies. Although the majority of corals are hermaphrodites, many species are gonochoric, producing either eggs or sperm (Shlesinger et al. 1998). Without female gametes, we lack the genetic material from female corals, and thus cannot capture the full scope of diversity in gonochoric populations. In addition, our ability to artificially aid in reproduction and selectively breed corals is restricted without  cryopreserved eggs. Using frozen sperm, we are still only able to create offspring when fresh eggs become available, which only happens on a few nights a year for any given species.

One possible solution to increase the availability of fresh eggs is spawning corals in captivity. The Horniman Museum in London and the Florida Aquarium have tackled this challenge with facilities that mimic environmental conditions to artificially induce spawning in corals at various times throughout the year, enabling captive coral spawning research. Inducing corals to spawn at different times during the year can minimize the challenges of narrow reproductive timing in corals, and thus increase the amount of sperm available for freezing and/or fresh eggs for fertilizing. 

Next steps

Sexual reproduction in wild corals seems to be failing for a variety of reasons. A lack of sexual reproduction means a devastating dearth of new genetic diversity and new recruits reaching reefs that desperately need them to help with recovery following disturbances. Cryopreservation may begin to help managers with this problem. If too few individuals spawn at a given time in a given area, we can freeze the sperm that is released for later use when fresh eggs become available. We can pool sperm from as many individual colonies as possible – locally, or even on a larger, regional scale – to artificially boost the diversity of crosses made when exposing frozen sperm to fresh eggs. 

These promising methods have only been trialed on a few dozen stony coral species to date, with most frozen samples coming from Hawai’i and the Great Barrier Reef, meaning that they still have not been tested on hundreds of species from many regions. In the near future, it should be a priority for coral researchers and conservationists to attempt to freeze material from as many species and sites as possible, in order to quickly preserve the maximum amount of coral diversity as populations dwindle.

We can also prioritize freezing sperm from colonies that survive bleaching events or disease outbreaks; these gametes may possess a level of resistance to these stressors somewhere in their genes that could result in more resistant, “fit” offspring. For instance, corals in the Persian Gulf survive some of the hottest temperatures in the oceans, often failing to bleach even at 34 – 36°C (significantly past the normal bleaching threshold). Although many of the species here are the same as those found throughout the Indo-Pacific, these corals have evolved some mechanism of tolerating extreme heat that are not seen in other regions. If we can successfully cryopreserve sperm from heat-tolerant Persian Gulf corals, we can transport and use it to inject heat-tolerance genes into new generations of at-risk populations of Indo-Pacific corals. In this way, assisted gene flow could soon become a powerful tool for rapidly propagating desirable traits within and among populations. With active intervention, cryopreservation of gametes may assist with creating diverse, viable, and even “climate-smart” offspring even while coral populations are increasingly unable to do so in nature.

This technology may serve as a particularly crucial intervention for species on the brink of extinction. Dendrogyra cylindrus, the pillar coral, is a Caribbean reef-building species that is highly endangered throughout most of its historical range (Neely et al. 2018). It is highly susceptible to Stony Coral Tissue Loss Disease (SCTLD) and bleaching, and has experienced dramatic population declines in recent decades. With their high sensitivity to environmental stress, species like these may have a very hard time responding positively to other restoration initiatives like coral gardening. As such, in order to preserve what is left of this beautiful species, we should consider concentrating our cryopreservation efforts on genetically banking material from the most at-risk coral populations.

Hagedorn and colleagues are currently developing methods to cryopreserve coral larvae. They have succeeded in doing so with the Indo-Pacific coral Lobactis scutaria (Daly et al. 2018), but have had trouble replicating the technique between species, years and even larval batches. While the sperm freezing and thawing protocols require minimal technical equipment and can be performed for relatively low cost, the existing larval thawing protocol requires a specific type of laser to warm the larvae at an extremely rapid rate (Daly et al. 2018). This equipment is very specialized and expensive, and thus this method may not be as readily scaled up and performed by scientists all over the world. 

The next frontier for coral cryopreservation is freezing and thawing entire fragments of live coral tissue, a technique that members of the Hagedorn Lab are testing. Tissue fragments would be important resources for reviving entire genotypes of corals if, once thawed, they could continue to grow and form colonies once again. 

Finally, cryopreservation techniques for Symbiodiniaceae, the algae that live in coral tissues and provide the majority of their energy for growth and calcification, remain elusive but hold promise for conservation. Most corals cannot survive for very long or grow to build reefs if they do not have a stable partnership with these algae, and in many cases this partnership is highly specific (Douglas 1998, Weis et al. 2001, Fabina et al. 2012).

Although Symbiodiniaceae are a very diverse family of algae (LaJeunesse et al. 2018) and most corals are capable of hosting a variety of types (Silverstein et al. 2012), some coral species almost exclusively associate with a single algal species. For instance, Dendrogyra cylindrus, the highly threatened Caribbean reef-builder mentioned above, only hosts Breviolum dendrogyrum (Chan et al. 2019). As such, if at some point in the future we intend to “revive” Dendrogyra by cryopreserving and eventually thawing it, we need to ensure we have its obligate algal partner ready to infect it with, otherwise it may not survive. Therefore, cryopreservation of a wide variety of Symbiodiniaceae types will be important to ensure that future coral populations have access to diverse algal populations in order to aid their calcification and reef building. 

Dr. Chiahsin Lin and colleagues recently published the first report of successful cryopreservation and thawing of Symbiodiniaceae cells. They froze algal cells from the thermotolerant genus Durusdinium for 2 and 10 days in liquid nitrogen, then thawed and cultured them for 2 months with continued growth and proliferation. These are exciting results, and should be replicated for additional genera/species to begin genetically banking these important algae.

Conclusion

In summary, cryopreservation offers a wealth of potential for coral restoration, but needs to be applied more widely and trialed rapidly with as many species as possible. I personally believe in  a “fast fail” approach, where any interested scientists with access to reproductive material can learn the basics of freezing and thawing sperm and try it whenever they can with the species they work with. Ideally, this will help to quickly build a body of knowledge, identify species that “work” given our current methods and species that require more research, and create a global community of stakeholders contributing material to genetic banks and generating knowledge about this novel mode of coral conservation. 

Back to Current Research