Fig. 13.1
Developing embryos. (a) Fresh and (b) cryopreserved sperm were used to produce developing coral larvae. Regardless of whether fresh or frozen sperm was used to fertilize fresh eggs, both groups developed, grew, settled and absorbed their symbiotic algae (brown spots in the tentacles) in a similar manner. Photo Credit: Emily Howells
Cells that are cryopreserved and banked properly can retain viability for years, or even centuries, without DNA damage. The greatest challenges facing this critical conservation effort are the time and resources to train individuals to form their own banks. However, the successful accomplishment of worldwide capacity building would create an insurance population for reefs, securing their biodiversity and helping to maintain or bolster their related economies.
2 Assisted Reproductive Technologies (ART)
2.1 The Fundamentals of Cryobiology
Cryopreservation (the study of cells under cold conditions) is an extremely effective conservation tool for maintaining genetic diversity. In this approach (see Box 13.1), cells are frozen in sugar-like compounds called cryoprotectants, frozen to −80 °C and placed into liquid nitrogen where they can remain frozen, but alive, for decades in a genetic bank. Most technological innovations in the field of germplasm cryopreservation arose from a sound understanding of the mechanisms of cryodamage and cryoprotection (Mazur 1970; 1984). Successful cryopreservation of cells, germplasm and tissue must address intrinsic biophysical properties (e.g., water and cryoprotectant permeability, osmotic tolerance limits, intracellular ice nucleation, etc.) to maximize survival (Rall 1993). A similar systematic approach is vital to improving post-thaw survival of coral and its associated organisms.
Box 13.1 Cryopreservation Primer
1.
Slow Freezing Cryopreservation: Uses extracellular ice to dehydrate cells, slowly dehydrating and freezing cells over minutes to hours.
Advantage: amenable to most cells.
Disadvantage: some cells are damaged by a slow reduction in temperature.
2.
Vitrification: Uses high concentrations of cryoprotectants and ultrafast freezing temperatures to form a glass instead of ice.
Advantage: good for chill sensitive cells or organisms.
Disadvantage: solutions can be toxic, thawing must use very fast warming temperatures to prevent ice formation.
3.
Slow Vitrification: Increases cryoprotectant concentration slowly over time to prevent ice crystal formation.
Advantage: good for chill sensitive cells or organisms, no ice crystals form, no need for rapid thawing.
Disadvantage: more complicated handling process.
Conventional cryopreservation of many types of cells relies upon cryoprotectants and slow freezing to dehydrate and shrink the cell. Cryoprotectants that enter the cell, such as dimethyl sulfoxide, propylene glycol, or glycerol, are effective, yet their mechanisms of action are not completely understood. They depress the freezing point of solutions in and around the cells and may directly alter membrane bilayers or interact with bound proteins on the external cell surface (Hammerstedt et al. 1990). Too little entering the cell before cooling reduces effectiveness and may lead to damaging intracellular ice formation (Taylor et al. 1974); too much entering the cell causes osmotic swelling and rupture during thawing and dilution (Levin and Miller 1981). Often, these procedures must be tailored for each type of cell, based upon a thorough understanding of its properties.
Preventing intracellular ice formation is essential to successful cryopreservation. During slow cooling, extracellular fluid freezes before intracellular fluid, pulling pure water out of the cell, leading to osmotic dehydration of the cells as they supercool. If ~90 % of the intracellular water can be removed before lethal intracellular ice forms, then many cells will survive thawing and dilution (Mazur 1984).
However, certain cells can be damaged by the slow-freezing process because a sudden reduction in temperature can cause cold shock injury (or chilling sensitivity), often resulting in severe membrane damage. It is common in some mammalian sperm cells, such as in pigs (He et al. 2001) and aquatic oocytes, embryos and larvae (Hagedorn et al. 1997). Vitrification, whereby cell water is converted to a glass rather than undergoing a damaging phase transition to ice, may prove to be a more viable technique for aquatic cells. Vitrification entails the use of: (1) highly concentrated cryoprotectants (5–6 M), which cause dehydration before cooling; and (2) rapid cooling of the cell suspension, forming a transparent glass-state. Vitrification permits rapid cryopreservation with improved survival in some cells (Rall and Fahy 1985).
If a tissue is chill sensitive, yet too large or too sensitive to the toxic cryoprotectants used for vitrification, a “slow vitrification” method can be used (Farrant 1965; Pegg et al. 2006). Generally, cytotoxicity of the cryoprotectant decreases with temperature, because of the reduced permeability and metabolism of the cryoprotectant. During slow vitrification the concentration of cryoprotectant is slowly increased at sub-zero temperatures (instead of at room temperature for vitrification). Slow vitrification reduces toxicity and the necessity for fast cooling and thawing rates.
2.2 Current Status of the Cryobiology of Reef Organisms
Storage of important coral and related cells through cryopreservation will profoundly advance basic research in embryology, genetics, systematics and molecular biology, as well as enhance management strategies for reef restoration. Although cryopreservation is a proven method for long-term maintenance of genetic material, current protocols for coral and associated organisms are not fully developed, and so the associated programs that could employ these important genetic resources have not reached their full potential. An important point, however, is that once the material is frozen, a great deal of research can be done to determine how it might best be used in the future. In the past 10 years, we have characterised some of the fundamental cryobiology for coral sperm, larvae and associated symbionts (Hagedorn et al. 2006a, b, 2010, 2012).
2.2.1 Coral Sperm Cryopreservation (Successful)
The sperm from eight different coral species (Caribbean: Acropora palamata (threatened), Hawaii: Fungia scutaria, Great Barrier Reef: Acropora millepora, Acropora tenuis, Acropora loripes, Platygyra lamolina, Platygyra daedalea, Goniastrea aspera Fig. 13.2) has been successfully cryopreserved, using the same standardised cryopreservation protocol and preserved in banks around the world (Hagedorn et al. 2012).
The general cryopreservation method for coral germplasm and embryonic cells has been described in detail in Hagedorn et al. (2012). Briefly, the sperm was collected and held in a concentrated form (approximately 2 × 109 cells/ml). Sub-samples were diluted either 1:10 or 1:100 in filtered seawater, counted with a hemocytometer and their motility assessed on a phase microscope approximately 30–45 min after collection. This standardized process was important because some Acroporid species only reach full motility 20–30 min after they have been released from their bundle (Hagedorn et al., unpublished data). Sperm samples with 50 % motility or greater were pooled across males and prepared for cryopreservation. The sample was diluted 1:1 with 20 % dimethyl sulfoxide in filtered seawater. Aliquots (1 ml) were loaded into 2 ml cryovials held at 26–28 °C. After a 10 min exposure to the cryoprotectant, the cells in the vials were frozen at 20 °C/min, quenched in liquid nitrogen, and then placed into a dry shipper for transport to permanent storage. A single sample from each freezing trial was thawed to examine post-thaw motility and fertilization success with fresh eggs.
2.2.2 Assessment of Sperm Viability and Use of the Frozen Bank
Frozen-thawed sperm have been used to fertilize conspecific eggs released in the same spawn and from successive spawns (Hagedorn et al. 2012). While variability remains across species and even within individuals on different nights of the same spawn these sperm have reached fertilization success of 60 % (Hagedorn et al. 2012). These small-scaled in vitro experiments demonstrated how to improve the cryopreservation process in developing larvae up to 12 h. In recent preliminary experiments, however, larvae produced from frozen/thawed Acropora clathrata sperm have developed, settled and assimilated symbionts over a 8 week period (Hagedorn et al, unpublished; Fig. 13.1). These longer-duration small-scaled experiments suggested that larger-scale grow outs would be possible to examine the effects of cryopreservation on the growth and maturity of coral over several years. In 2013, tens of thousands of Acropora tenuis embryos were generated with (1) sperm collected immediately after spawning; (2) this same sperm frozen for 1 h and then thawed, and; (3) sperm that been frozen for 1 year and then thawed. These coral are growing and maturing in the SeaSim facility at the Australian Institute of Marine Science. These studies will help guide future usage of these invaluable frozen resources, because one day they may be needed to help expand and diversify shrinking coral populations worldwide.
Fig. 13.2
Examples of coral currently cryopreserved and stored in banks around the world. Photo Credits: Acropora palmata, Raphael Ritson-Williams, Smithsonian Institution; Fungia scutaria, Ginnie Carter, Smithsonian Institution; Acropora cervicornis, Eric Borneman, University of Houston; Acropora tenuis, Andrew Heyward, Australian Institute of Marine Sciences
2.2.3 Coral Larvae and Oocytes (Not Yet Successful)
No coral larvae have yet been successfully cryopreserved because of their chilling sensitivity. With less than 1 min of exposure to 0 °C, 100 % of all tested F. scutaria larvae disintegrated (Hagedorn et al. 2006a). Oocytes have never been cryopreserved either because of chilling sensitivity (Lin et al. 2011, 2012).
2.2.4 Dissociated Coral Embryonic Cells (Successful)
Using modified embryonic stem cells protocols, dissociated larval cells were successfully cryopreserved from eight different species (Caribbean: Acropora cervicornis (threatened), Hawaii: Fungia scutaria, Great Barrier Reef: Acropora millepora, Acropora tenuis, Acropora loripes, Platygyra lamolina, Platygyra daedalea, Goniastrea aspera) and demonstrated 50–90 % post-thaw viability (Hagedorn et al. 2012) and Hagedorn et al. (unpublished data).
The pluripotent nature of 8-cell coral cells has been clearly defined by Heyward and Negri (2012). We have concentrated our embryo cell cryopreservation efforts on this stage of embryo to maximize the potential of the bank. To preserve embryonic cells, approximately 1 ml of 8-cell embryos was placed in a 15-ml tube with 0.1 % Bovine Serum Albumin in filtered seawater. This was diluted 1:1 with 20 % DMSO in filtered seawater. This sample was placed into a glass tissue homogenizer to create a homogenous cell suspension with a targeted cell concentration of approximately 5 × 106 cells/ml. Aliquots (1 ml) of the cell suspension were loaded into 2 ml cryovials, placed into a passive freezing device, such as the Biocision Coolcell®, placed in a 80° freezer for at least 4 h, and then quenched in liquid nitrogen prior to being loaded into a dry-shipper for shipment to the repository. At least one sample in each group of samples was stained with the Live/Dead Viability Stain (Invitrogen) assessed on a fluorescent microscope or run on a flow cytometer to determine cell integrity post-thaw. The viability of these cells in culture, and the potential for them to develop to maturity has not been measured due to the lack of robust, well-defined culture methods for coral, but given the steady advancement of human stemcell culture, these cells have enormous future potential for conservation and coral disease work.
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