individual 50 ml conical plastic tubes. By contrast, eggs released from the mouth and visualized as a particulate white cloud were collected from the bottom of the bowl using a disposable pipette. There was no opportunity for gamete interaction, and all eggs were immediately washed three times in ,100 ml FSW and then placed into a glass beaker to count and dilute at a temperature of 23 to 26uC for IVF. Meanwhile, spermatozoa were maintained at the same temperature in raw seawater (1,000 mOsm) for later processing. Generally, gametes were cleaned and separated as rapidly as possible, and always ,1 hr post-recovery. Sperm remained concentrated in FSW until density was determined, whereas eggs remained in FSW until placed in vials for reproductive assessments. Two teams worked in parallel, one processing, evaluating and cryopreserving spermatozoa with the other separating eggs. Assessments of sperm concentration and motility Sperm motility estimates for each collection were made immediately by placing a ,10 ml aliquot onto a glass slide, adding a cover-slip (only for A. palmata) and visualizing the sample via phase optics (2006) on a Zeiss Student or Olympus BX41 microscope. Specifically, a qualitative quartile method was used with the slide moved to assess a minimum of three full frames. The collective fields then were estimated as having sperm expressing ,25, 50, 75 or .90% progressive or forward motility. The process was repeated with two additional 10 ml samples, and an overall motility average calculated. Sperm concentration in each diluted sample was determined using a standard hemocytometer method [40]. For both species, spermatozoa were pooled from at least three males (most commonly 5 to 7 donors) on a given collection night, usually into a total volume of 25 to 50 ml. This strategy also increased total sperm available for each cryopreservation trial, but also was considered as a useful future approach for ensuring heterozygosity in thawed inseminates (especially important in A. palmata where there are barriers to self-fertilization [21]). Assessment of cell viability Viability of thawed embryonic cells was defined as those cells with intact cell membranes. We used a standard propidium iodide (Invitrogen) assay that exposed cells to a fluorescent dye that intercalates with DNA nucleotides and is generally excluded by intact cells [41]. Stained cells were analyzed by flow cytometry (Accuri C6, Accuri Cytometers, Inc. Ann Arbor, MI USA), and 10,000 events were analyzed per sample. This instrument measured total number of cells in a sample, and propidium iodide revealed the number of damaged cells. In brief, coral cells were exposed to a 2.4 mM stock solution of propidium iodide (1 ml stock solution/250 ml of cells) and incubated for 10 min at 24uC. Control (dead) samples were created by subjecting cells to three cycles of freezing to 2196uC and thawing at 30uC (without Preserving and Banking Coral cryoprotectant) before staining with propidium iodide and flow cytometry analysis. Data were evaluated using Accuri software (CFlow Plus, Ver. 1.0.202.1). Experiment I: Sperm viability and IVF validation This experiment determined the optimum concentration of fresh spermatozoa needed to achieve fertilization in vitro. This was essential because all subsequent cryosensitivity experiments evaluated sperm survival post-treatment based on the capacity of these cells to interact with and fertilize eggs from the same species in culture [21]. For both species and all trials, IVF was conducted in 20 ml scintillation vials (Thermo Fisher Scientific), each containing 5 ml of FSW, 30 to 50 eggs and no (control) or fresh spermatozoa of varying concentrations. For A. palmata, eggs were exposed to spermatozoa for 5 min and then serially rinsed three times in 5 ml of FSW or not rinsed at all, with fertilization success scored 12 hr later (see below). For F. scutaria, eggs were exposed to sperm for 12 hr according to Krupp [38] and Schwarz et al. [39]. For A. palmata, we examined a shorter fertilization time (5 min), because we were concerned about bacterial contamination of a 12 hr long incubation from decaying, large, yolk-filled eggs and the high sperm concentrations. Initially and for validation purposes, we determined optimal concentration of fresh spermatozoa needed to initiate fertilization (defined below). Table 1 lists the treatments tested for A. palmata and F. scutaria in these initial trials. After adding spermatozoa (at the test concentration), the original vials were maintained at 27 to 29uC (without agitation) and developmental progression assayed at 12 hr post-insemination. Fertilization was based on numbers of developing larvae (versus un-inseminated eggs) quantified under a Wild dissecting microscope (506). More specifically, for A. palmata, we calculated the percentage of larvae that developed to the ‘cornflake’ or ‘prawn-chip’ stage by12 hr of culture at 27 to 29uC, described as fertilized by Okubo and Motokawa [42] (Fig. 2A). For F. scutaria, we determined fertilization as the proportion of larvae developing to a motile state by 12 hr at 27uC (Fig. 2B) according to Krupp [38] and Schwarz et al. [39]. Experiment II: Cryoprotectant toxicity The purpose of this study was to identify candidate cryopro- tectants by examining the effect of cryoprotectant toxicity on the ability of fresh sperm to fertilize eggs from A. palmata and F. scutaria. All cryoprotectant solutions described in this paper were prepared originally at double-strength concentration (vol/vol) and diluted 1:1 with 0.2 mm FSW to produce the final test concentration. This eventually facilitated our fieldwork as this approach allowed quick mixing of one-part cryoprotectant and one-part seawater with cells to achieve our final target concentration. Spermatozoa were exposed to each cryoprotectant (protocols and gamete donors are summarized in Table 2 for each cryoprotectant trial) for 20 min at Table 1. Experiment I: Fresh sperm treatments for in vitro fertilization. Species A. palmata F. scutaria F. scutaria F. scutaria 8 8 16 16 *Thirty to 50 fresh eggs were used per pooled sperm sample. **Eight replicates per female for each sperm concentration. doi:10.1371/journal.pone.0033354.t001 No. individual egg donors* No. pooled sperm donors Fresh sperm concentrations tested 0 0 3 to 7** 7 to 10 None added (control) 106, 107, 108 cells/ml None added (control) 105, 106 cells/ml PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e33354