Preserving and Banking Coral Figure 6. Cryopreservation of coral sperm (all sperm exposed to freezing in these treatments). A) A. palmata sperm were cryopreserved at cooling rates 20 to 30uC/min using 10% DMSO and PG, and IVF success was assessed and averaged over the single spawning period. This averaged graph revealed no difference between the two cryoprotectants and a mean fertilization success of ,18%. B) However, if the A. palmata fertilization success during the spawning period was graphed by day, a 65% fertilization success occurred on Day 3 with 10% DMSO, whereas it was 25, 0 and 3% on Day 1, 2 and 4, respectively. For the first 3 nights, the control with fresh sperm held at ,90%, then fell to 76% on the fourth evening. C) F. scutaria sperm were cryopreserved at rates 20 to 30uC/min using 10% DMSO versus PG, and fertilization success was assessed and averaged over two spawning periods (July and August 2010). Averaging indicated that 10% DMSO was the preferred cryoprotectant, and (as in A. palmata) there was no variability in time in terms of physiological responses during a spawning season. D) Variability in F. scutaria IVF success after cryopreservation over two nights of a single representative month (August 2010). Fresh sperm IVF success held steady at 65%, but sperm cryopreserved with 10% DMSO varied from 52 to 13% on the two evenings. Bars with the same letters were not different (P.0.05; ANOVA), whereas bars with different letters were different (P,0.05; ANOVA). doi:10.1371/journal.pone.0033354.g006 especially agriculturally important groups, including mollusk sperm, oocytes and embryos [52,53,54] and fish sperm (e.g., .200 species, especially salmonids; [43,55,56]). The primary justifications for using this technology in these cases have been to maintain gene diversity in distinctive species, subspecies or races, while providing an ‘insurance policy’ in the case of a catastrophe affecting either free-living or ex situ populations. One of the most interesting findings of the present work was that corals (at least the two species studied here) defied conventional wisdom for most other previously studied species and biomaterials. Conventional wisdom states that cryosensitivity is highly species- and cell- specific, with differing taxa and cells generally requiring highly explicit freezing and thawing protocols [57,58]. By contrast, we determined that spermatozoa from A. palmata and F. scutaria both had high IVF success after cryopreservation as did embryonic cells from F. scutaria that expressed post-thaw viability, all from using a simple, 10% solution of DMSO in FSW and a cooling rate of 20 to 30uC/min (sperm) and 0.5uC/min (embryonic cells). These results insinuated that sperm from a diversity of corals may be effectively cryopreserved using a single approach. In fact, these same sperm cryopreservation techniques have been used successfully on two additional Acroporid species (Hagedorn et al, unpublished data). While conclusive experiments on more species are necessary, these initial observations suggested potential broad application to male gametes from Scleractinia (i.e., the stony coral). Although the basic 10% DMSO and 20 to 30uC/min cooling protocol was effective for spermatozoa from both A. palmata and F. scutaria, there were some detectable, physiological variations between species, including traits that could ultimately influence field application. Spermatozoa from both species were unaffected by exposure to 5% solutions of DMSO and PG, a concentration considered to be inadequate for safeguarding against intracellular lysis damage during freezing [57,58]. During our quest to reach the more protective 10% level, we discovered that A. palmata sperm motility and fertilization in vitro were unaffected by elevated concentrations of both cryoprotectants. By contrast, F. scutaria sperm motility and IVF success were highly sensitive to rising cryoprotectant levels. This was important as such a species PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e33354