Locally, reefs are damaged by pollution, nutrients and sedimen- tation from poor land-use, fishing and mining practices [3]. Globally, increased levels of greenhouse gases are warming and acidifying oceans, which is making corals more susceptible to stress, bleaching and newly emerging diseases [4,5,6]. The coupling of climate change and anthropogenic stressors has caused a widespread reef crisis [4,5,6,7,8,9]. The status of Caribbean reefs is dire, with elkhorn (Acropora palmata) and staghorn (Acropora cervicornis) corals, once the foundation species for this region, showing widespread population declines [10,11,12]. For example, recent surveys have revealed that 80 to 99% of these populations have been extirpated [13,14], leading to the dubious distinction of being the first of this group to be listed as threatened under the Endangered Species Act [15,16,17]. In general, corals can reproduce sexually (via planktonic or floating larvae) or asexually (by fragmentation and resettlement). The former can generate genetic diversity, whereas the latter maintains the status quo of the species. Caribbean A. palmata and A. cervicornis appear to most commonly propagate via asexual breakage and then re-attachment of branches, in contrast to recruitment of sexually-produced larvae [18,19,20]. When sexual reproduction does occur, larvae are produced once annually when hermaphroditic colonies release eggs and sperm (spawning) and fertilization occurs. Because (1) occurrence and spawn time can be unpredictable in Caribbean Acroporids and (2) A. palmata and A. cervicornis do not generally self-fertilize [21], reproductive success can be sporadic and encumbered in the isolated, small populations common in the Caribbean. In certain areas in the Florida Keys, genotypic diversity can be so low (e.g., only one clone per reef) that sexual reproduction is all but impossible [20]. Such reproductive failures contribute to the continuing decline of Caribbean Acroporids [22,23]. Additionally, young recruits are being adversely affected by increasing macroalgal cover on reefs [24,25]. Together, these factors increase the likelihood of elkhorn and staghorn coral extinctions within the next 5 to 20 years [26,27]. Historically, ex situ populations of wildlife species have been important as ambassadors for public awareness (i.e., zoos and aquaria), and can serve as a hedge against extinction, to generate species-specific biological knowledge impossible to collect in situ and for producing offspring for reintroduction into native habitats [28]. Some of the best practices from ex situ wildlife management programs have application to conserving coral. First, advances in coral husbandry now allow for maintenance of ex situ populations. The coral conservation consortium of aquarists and scientists, called SECORE (SExual COral REproduction, www.secore.org), successfully collected A. palmata egg-sperm bundles, used in vitro fertilization (IVF) and reared and transported larvae so that there are now .3,000 young adults in public zoos and aquaria around the world [29,30]. Second, preliminary findings suggest that biomedical reproductive methods can be applied in corals [31]. Similar to many wildlife groups [28], assisted reproductive technologies could be relevant to propagating wild coral populations. Especially advantageous is the field of cryobiology – low temperature biology – and understanding the mechanisms that allow the successful cooling, freezing and thawing of germplasm or embryos. Because cells that are frozen and banked properly can retain viability for years (or even centuries) without DNA damage, this is a means to safeguard all existing species and gene diversity [32,33,34]. More specifically, these ‘genome resource banks’ offer (1) large samples of preserved and protected gene pools that can be used to ‘seed’ shrinking populations, including those with heterozygosity, or re-populate depauperate Preserving and Banking Coral habitats, (2) easy and inexpensive transport of genetic materials among living populations, (3) extended generation intervals and (4) vast improvement of access to biomaterials for scholarly research [35,36,37]. We believe these benefits also have relevance to coral conservation, including applying a suite of tools related to ex situ husbandry and cryomethodologies. Our studies focus on the in vitro reproduction and cryobiology of two coral species, Acropora palmata and Fungia scutaria. The goal was to examine commonalities in reproductive biology and cryophy- siology that might lead to a singular, effective cryopreservation procedure broadly applicable to diverse coral species. Our comparative approach was important because our target study species differ in mode of sexual reproduction (hermaphroditic versus gonochoristic or separation of the sexes in different individuals) and occupy different ecological niches (reef-building coral versus isolated). A. palmata is a large colonial species occupying only shallow water, with hermaphroditic polyps producing positively buoyant egg/sperm bundles that develop into free-swimming larvae [1]. In contrast, F. scutaria is a solitary, small-sized species that lives in a variety of reef zones as gonochoric individuals that produce negatively buoyant gametes. In an earlier study, we offered preliminary evidence that coral spermatozoa can survive cryopreservation [31] based largely on post-thaw motility. However, to date, the functional viability of such cells for fertilization has not been shown. The objectives of this investigation were to examine (1) the in vitro reproductive characteristics and the influence of cryoprotectants, chilling and freezing on the motility and fertilization capacity of A. palmata and F. scutaria spermatozoa for creating a successful cryopreservation protocol, (2) the cryosensitivity and post-thaw viability of dissociated embryonic cells from 12 h F. scutaria and (3) the feasibility of large-scale, field banking to begin preserving coral bio- and genetic diversity. Methods Due to limited gamete availability from naturally brief spawning durations, collection periods for both species were short. A. palmata studies were conducted in Puerto Rico over 1 wk (,4 nights), whereas F. scutaria investigations were done in Hawaii over 4 mo (,16 nights). Although we report the findings made during a single annual breeding cycle for both species, 4 years of preliminary studies were performed to establish our field and laboratory practices. In terms of general methods, we studied two species with different types of reproduction. A. palmata produced egg/sperm bundles which were collected from several specimens at once and then separated into eggs and sperm. Spermatozoa were pooled, counted, diluted and checked for motility, and then pooled sperm was subjected to various cryopreservation testing for toxicity and sensitivity to freezing. Each sample, then was, thawed and used to inseminate fresh, conspecific eggs in vitro on that same night. For F. scutaria, sperm and eggs were expelled from different male and female animals, so were collected separately. However, all subsequent processing and evaluation procedures were the same as with A. palmata. Gamete collection Recovery of coral reproductive cells was performed with the appropriate permits from the Departments of Land and Natural Resources in both Puerto Rico and the State of Hawaii. A. palmata eggs and sperm were collected during the annual spawn from Tre´s Palmas Reserve (Rinco´ n, Puerto Rico) in August, 2009. Egg-sperm bundles were collected with specially designed fine mesh nets PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e33354