Preserving and Banking Coral Figure 5. The effect of cooling rate for successful F. scutaria spermatozoa cryopreservation (no sperm exposed to freezing in any of these treatments). Two freezing ranges were examined and two cryoprotectant solutions (F. scutaria, n = 3–7 pooled sperm donors/cooling range and n = 16 egg donors) and then the influence of cooling rate on IVF success. Only the 10% DMSO at a cooling rate greater than 20uC produced reasonable post-thaw fertilization. Bars with the same letter were not different (P.0.05; ANOVA), whereas bars with different letters were different (P,0.05; ANOVA). doi:10.1371/journal.pone.0033354.g005 former reducing fertilization by ,27% compared to fresh controls (P.0.05, ANOVA, N = 16 egg donors and 14 to 20 sperm donors, F = 40.2, Fig. 6C). However, when the data were examined on a nightly basis, there was a night-of-sperm-collection effect on subsequent ability of spermatozoa from either species to survive cryopreservation (Fig. 6). On the basis of a nightly evaluation, 10% DMSO consistently produced the highest post-thaw IVF success in both species. Specifically for A. palmata, post-thaw fertilization success was 65% on the third night of spawning compared to 25% for counterparts collected on the first night, 0% on the second night and 3% on the fourth night (Fig. 6 B), all handled exactly the same way. A similar observation was made for 10% DMSO-frozen F. scutaria sperma- tozoa, with a fertilization success of 51% for the first night of collection compared to 13% for the second night (Fig. 6 D). In terms of survival of F. scutaria embryonic cells, ,50% of the dissociated cells were viable post-thawing, regardless of cryoprotec- tant type or concentration when the 0.5uC/min cooling rate was used (Fig. 7). Interestingly, the ability of these cells to withstand cold temperature also appeared dependent on night of collection. Within a coral-spawning interval of 3 days, the proportion of embryonic cells with intact membranes ranged from 50 to 80%. Using the knowledge generated in our earlier experiments and our findings that post-thaw functionality is achievable with 10% DMSO and cooling rates of 20 to 30uC/min for sperm and 0.5uC/min for embryonic cells, we have proceeded with the first ever, systematic, large-scale banking of coral sperm and embryonic cells. Our focus largely has been on A. palmata spermatozoa due to the now threatened status of this species. This effort to date has resulted in the storage of .450 A. palmata sperm samples and .500 billion spermatozoa. The donor sites for these collections have included individuals from over 2 linear km of the Tre´s Palmas Reserve (Rinco´n, Puerto Rico). Frozen and labeled cryovials stored in LN2 were transferred to LN2 dry shippers and transported (with proper permitting) to two animal germplasm repositories for long-term storage (i.e., National Animal Germ- plasm Program, Fort Collins, CO, USA and Omaha’s Henry Doorly Zoo, Omaha, NE). Additionally, although .100 F. scutaria sperm samples were successfully cryopreserved (based on post- thaw IVF and viability testing), these cells were not formally banked because this species is not threatened. Likewise, using the basic information generated in this project, we have created a frozen repository for 50 samples of coral embryonic cells (,107 cells/cryovial) from .60 F. scutaria individuals living in Kaneohe Bay that are managed in long-term LN2 storage at the Hawaii Institute of Marine Biology. Discussion Little was known historically about coral reproduction until about 30 years ago when massive spawns of multiple species were investigated on the Great Barrier Reef [45,46], including studies of larvae developmental biology after fertilization in vitro [47,48]. Therefore, it has been evident for decades that an admixture of fresh coral sperm and eggs can robustly produce viable embryos in culture. Our finding of .92% IVF success for fresh A. palmata sperm and eggs was consistent with earlier, recent reports for other Acroporids or members of the same family [21,49]; fertilization success for F. scutaria, which has not been studied before in this regard, was slightly lower, but consistently at .75%. Such high values are not normally observed in the ‘IVF world’, especially for traditionally-studied vertebrates (mostly mammals) [50]. There- fore, the consistently high levels of fertilization in vitro observed in coral gives the scientific community a valuable metric for examining the impact of a host of basic biological factors that might influence sperm/egg interaction and gamete/embryo form and function (e.g., inter-gamete attractants during spawning events; Morita et al. [51]). In the present study, we determined that IVF was especially useful for assessing the cryosensitivity of coral spermatozoa, helping us determine that significant sperm numbers from two distinctively different species survived freezing stress (to 2196uC) and produced developing embryos. Cryopreservation is a proven method for the long-term maintenance of genetic material for multiple marine species, PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e33354