A. palmata F. scutaria A. palmata F. scutaria 4 – 4 10 3 to 7*** – 3 to 7*** 3 to 7*** 1Sigma-Aldrich, St Louis, MO, USA. 2Acros Organics, Fair Lawn, NJ, USA. *Thirty to 50 fresh eggs were used per pooled sperm sample. ***An inseminate concentration of 106 sperm/ml was used for each species. doi:10.1371/journal.pone.0033354.t002 evaluate post-thaw motility and cellular viability (as described above for F. scutaria). For embryonic cell cryopreservation, we examined the cryo- sensitivity of these cells, specifically those of F. scutaria, because only this species was in proximity to our laboratory in Hawaii that had the required equipment. Based on the sperm experiments, we tested DMSO and PG, both at a 5 and 10% concentration. Methods were modified from Falciatori et al. [44] who had studied mouse spermatogonial stem cell lines. Since there was a lack of information on coral stem cells or the optimal time to dissociate such embryos, we collected thousands of F. scutaria embryos at ,12 h post-fertilization, a time when cells were beginning to differentiate into various lineages (Hagedorn, unpublished data). Embryos were dissociated simultaneously by placing into a 6% pronase (10 mg/ml) solution in FSW for 2 min followed by mechanical disruption by pressing through a cell-sorting basket with 40 mm mesh screen (Falcon, Fisher Scientific) using the rounded end of a plastic spatula. The resulting cell suspension (,107 cells/ml) was collected into 1 ml Eppendorf tubes and then vortexed to produce a consistent suspension that was confirmed by examining microscopically (400 to 8006) for uniform cellular dispersion. After allowing cell settlement for 30 to 60 sec, the pronase solution was gently aspirated and discarded. Sufficient FSW was added to the admixture to produce a final 500 ml volume that was supplemented with 150 ml of 10% bovine serum albumin in FSW. This solution was centrifuged (5,9006g, 5 min; Eppendorf Centrifuge 5415D) and the supernatant removed. A 500 ml aliquot of bovine serum albumin (4% in FSW) was added, the cell pellet re-suspended with a pipettor, and then the 500 ml volume transferred into a 2 ml cryovial. Then, 500 ml of a double- strength cryoprotectant was added to each cryovial (e.g., of 10 or 20% DMSO or PG) and mixed using a pipette. Eighteen vials were placed into the slots of a 4uC alcohol freezer (Mr. Frosty Freezers, Nalgene, Fisher Scientific) that was held for 20 min at 4uC (to equilibrate the cryoprotectant), and then the alcohol freezer was transferred into a 280uC freezer for at least 8 h to produce a cooling rate of 0.5uC/min. From the time of placing cryovials into the alcohol container through the 280uC freezer exposure, at least one vial was monitored with the Omega thermocouple to generate a temperature profile. After reaching 280uC, the vials were quenched in LN2 for at least 10 min to reach 2196uC and then either stored long-term in a 35 L LN2 Preserving and Banking Coral 1. Preliminary Screening Experiments 10 separate test cryoprotectants: 5 and 10% dimethyl sulfoxide (DMSO)1, 5 and 10% propylene glycol (PG)1, 5% glycerol1, 5% methanol1, 5% ethylene glycol1, 5% methylene glycol1, 5% 2-methyl-2,4-pentanediol2, 5% 1-methoxy-2 propanol2 2. Tests With Candidate Cryoprotectants 4 separate tests cryoprotectants: 5 and 10% dimethyl sulfoxide (DMSO)1, 5 and 10% propylene glycol (PG)1, 4 separate test cryoprotectants: 5 and 10% dimethyl sulfoxide (DMSO)1, 5 and 10% propylene glycol (PG)1, dewar or thawed in a 30uC water bath for 2 min and assessed for viability (as described above). Each of the four cryoprotectant treatments was evaluated simultaneously four times over the course of 3 different days. Once the fundamental protocols were developed, we systemat- ically banked spermatozoa from A. palmata, first by holding in a dry shipper (for transportation from the field) to permanent storage in LN2 dewars. A similar approach was used for embryonic cells of F. scutaria that were maintained in 35 L LN2 tanks in Hawaii. Data analysis All statistical evaluations were performed using Graphpad Prism 5.0 (San Diego, CA) and Microsoft Excel (version 2007). Percentage data were arcsine-transformed, and significance was recognized at P,0.05 (for all tests). Results among groups were evaluated statistically by one-way analysis of variance (ANOVA), and an F value reported. For nonparametric assessments, a Kruskal-Wallis or a Mann Whitney test was used. Results Experiment I: Optimizing IVF for coral Overall, fertilization rates in vitro were consistently high in both species using fresh spermatozoa, .85% IVF success for A. palmata and .75% for F. scutaria. There were no differences in fertilization success in either A. palmata or F. scutaria on the basis of fresh sperm inseminate concentration or duration of the insemination interval (for A. palmata). For A. palmata, .92% of eggs fertilized whether in the presence of 106, 107 or 108 sperm cells/ml or when incubated from 5 min and then rinsed or 12 h and not rinsed (P.0.05, ANOVA, F = 70, N = 8 egg donors and 3 to 7 sperm donors, Fig. 3A). Due to being expelled directly into the water, it was not possible to produce a highly concentrated solution of F. scutaria spermatozoa. For this species, .75% of eggs fertilized whether in the presence of 105 or 106 sperm cells/ml (P.0.05, t-test, N = 16 egg donors and 7 to 10 sperm donors, Fig. 3B). The incidence of self- fertilization was negligible in both species (A. palmata, 2.0%; F. scutaria, 0%). Although we dealt mostly with pooled samples, we did examine fresh motility prior to pooling. Individual male motility from both species varied considerably from 25% to .90% and appeared to be night-dependent during an individual spawning period. In F. Table 2. Experiment II: In vitro fertilization after exposing fresh sperm to various cryoprotectant treatments. Species No. individual egg donors* No. pooled sperm donors Treatment PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e33354