Preserving and Banking Coral Figure 2. Adult and larval forms of A. palmata and F. scutaria. A) Adult and developing A. palmata larvae (inset) at the cornflake stage at ,24 h. Scale bar = 50 mm. Adult photo by R. Williams, Smithsonian Institution. B) Adult and developing F. scutaria larvae at the swimming stage. Scale bar = 50 mm. Embryos that reached these stages were scored as successfully developed. doi:10.1371/journal.pone.0033354.g002 27 to 29uC (for A. palmata) and at 23 to 26uC (for F. scutaria) and then assessed in the IVF assay. While various cryoprotectants and concentrations were tested per species (Table 2), ultimately we selected a total of four treatments for subsequent studies: 5 and 10% dimethyl sulfoxide (DMSO) and propylene glycol (PG). Experiment III: Sperm and embryonic cell freezing sensitivity This experiment used the collective findings of Experiments I and II to assess and apply cryopreservation methods for cells from both species. There were four components: 1) determining optimal cooling/freezing rate for A. palmata and F. scutaria spermatozoa; 2) evaluating the impact of freeze-thawing spermatozoa from both species; 3) examining the efficacy of cryopreserving dissociated F. scutaria embryonic cells; and 4) implementing systematic cryobank- ing of spermatozoa (A. palmata) and embryonic cells (F. scutaria). Identifying an optimal cooling rate for cryopreservation, or rate at which cells are frozen, is often an iterative process due to the wide range of potentially perturbing factors (e.g., natural species differences, volume of the sample, packaging, type and concentra- tion of cryoprotectant, among others). For this reason, most cryo- studies begin by examining the influence of cooling rate as well as cryoprotectant type and concentration. Therefore, we first conduct- ed a preliminary trial of A. palmata spermatozoa using 10% DMSO versus PG, two common cryoprotectants used in marine cryobiology studies [43]. Early findings (data not shown) demonstrated that both of these cryoprotectants at this concentration resulted in post-thaw motility after sperm were cooled/frozen at a rate of 20 to 30uC/min. This information was used as the foundation for developing the more complicated comparative studies reported below. Specifically, for each species we compared two concentrations of DMSO or PG (5% versus 10%) and the original cooling rate of 20 to 30uC/min with a slower rate of 8 to 15uC/min (A. palmata, n = 3–7 pooled sperm donors/cooling range and n = 28 egg donors; F. scutaria, n = 3–7 pooled sperm donors/cooling range and n = 16 egg donors). To achieve the faster cooling rate, a liquid nitrogen (LN2) field apparatus was used (described below), while the slower cooling rate was achieved using an electronic, controlled-rate freezer (for A. palmata, a Grant Asymptote EF600; for F. scutaria, a Planar Freezer Kryo 360). When either the LN2 field box or the controlled-rate freezer was used, the sample temperature was lowered from 25 to 29uC to 280uC at the specific cooling rate, then plunged into LN2 for at least 10 min before thawing. Motility and viability of the F. scutaria sperm treated with 5 and 10% DMSO and PG also were assessed with phase microscopy and flow cytometry (see above) before and after thawing (n = 10 females, n = 3–7 pooled sperm donors each of 3 nights). It is challenging to cryopreserve living biomaterials in the field, so robust methods that suited coral cells were developed. Our simple field freezing apparatus consisted of a Styrofoam box (27.5 cm long622.5 cm wide620.5 cm high) partially filled with LN2 fitted with a lid (to maintain the vapor) that allowed 20 cryovials to float ,1 cm above the LN2 vapor. The float consisted of a frame of floating polyresin ‘flip-flop material’ (Ben Franklin Craft Store) with the frame opening covered with a rectangle of aluminum mesh. Two sections of aluminum cryocanes (Thermo Fisher) bent into a v-shape were snuggly attached to the mesh. With this apparatus, a typical freezing run consisted of exposing spermatozoa to either 5 or 10% DMSO or PG and then cooling at 20 to 30uC/min. For each species, we evaluated pooled sperm samples from three to seven sperm donors on each collection night. Each pooled sample was evaluated for initial cellular motility and sperm count (as described above), pooled and then diluted in FSW to 26109 cells/ml (A. palmata) or 26108 cells/ml (F. scutaria). A double-strength concentration of either 10 or 20% DMSO or PG was added to permit the process of equilibration to begin over 20 min at 27 to 29uC (A. palmata) or 23 to 26uC (F. scutaria). Each 1 ml sample then was pipetted into a 2 ml cryovial, affixed to the v-shaped holder that was quickly (,5 sec) placed onto the LN2 surface (3 to 5 cm below the top of the box) of the Styrofoam freezing box, ensuring ,1 cm gap between the cryovial and the LN2 fraction. The lid was replaced, the samples held in the vapor for 5 to 6 min until the cryovials reached 280uC and then the whole apparatus plunged into LN2 (2196uC) for at least 10 min or longer before analysis or transfer to a dry shipper (for short term storage and transportation from the field). Preliminary testing with a thermocouple (Omega HH147 RS-232 Data Logger Thermometer, Stamford, CT) indicated that the 1 ml content within each cryovial was being exposed to a freezing rate of 20 to 30uC/min rate. To examine the slower cooling rate (8 to 15uC/ min), cells were diluted with cryoprotectant, loaded into the 2 ml cryovials, placed into the control-rate freezer, cooled to 280uC, and then individual cryovials were plunged into LN2 (2196uC) for at least 10 min before analysis. For both species, a subset of frozen aliquots was thawed the same day to evaluate the effect of cooling rate and cryoprotectants on IVF success as well as sperm motility and viability (only F. scutaria). This process involved removing the cryovials from LN2 which then were exposed to 30uC in a water bath for 2 min. A 50 ml aliquot of each sample was pipetted immediately into each of the vials containing 30 to 50 eggs so that the amount of viable sperm in the vial was ,16106 cells/ml (while maintaining a low and negligible cryoprotectant concentration in the vial). An additional 100 ml of the thawed sperm sample was used to PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e33354