Preserving and Banking Coral Figure 4. F. scutaria sperm were sensitive to cryoprotectants (no sperm exposed to freezing in any of these treatments). A) If the prefreeze motility data (N = 7) for several spawning periods were averaged across the test cryoprotectants, there was no clear indication which cryoprotectant solution might impact the motility the least, except DMSO solutions might be slightly preferable. For analysis, the % motility was measured in quartiles, which were converted into numbers from 1 (25% or less motile) to 4 (.90% motile). Bars with the same letters were not different (P.0.05; Kruskal-Wallis test), but bars with different letters were different (P,0.05; Kruskal-Wallis test). FSW controls included fresh sperm with no cryoprotectant. B) However, if the effect of the cryoprotectants on F. scutaria sperm motility for one individual spawning period in the month of July was examined each day, there was a variability pattern in sperm motility each night. Note on Day 1 and 2, the toxicity of 10% DMSO was high (low motility), whereas on Day 3 it was low (high motility). C) In contrast, 10% DMSO and PG solutions caused a 30 to 40% decrease in fertilization success for fresh F. scutaria, whereas the 5% solutions did not. 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.g004 Preliminary cooling rate studies were conducted with A. palmata, suggesting that sperm cooled at 20 to 30uC/min in 10% DMSO had up to 75% post-thaw motility, whereas rates from 8 to 15uC/ min using either 5 or 10% DMSO or PG resulted in no IVF success. For F. scutaria, a cooling rate of 20 to 30uC/min using 10% DMSO produced the highest IVF success (28%), whereas 8 to 15uC/min and the other solution reduced IVF success (6% or lower) (P,0.05, ANOVA, F = 49; N = 20 egg donors and 12 sperm donors, Fig. 5). Therefore, a cooling rate of 20 to 30uC/min was chosen for cryopreserving sperm from both species. Once the freezing rate range was established, a more detailed examination of how the two cryoprotectants affected IVF success was undertaken. Freezing the sperm from these coral species always reduced IVF success compared to fresh counterparts (Fig. 6A, C). For A. palmata, either the 10% DMSO and 10% PG treatment produced similar IVF success (P.0.05, ANOVA, N = 16 egg donors and 14 to 20 sperm donors, F = 100.5; Fig. 6A); however, both reduced fertilization in vitro by ,78% compared to fresh controls. By contrast, 10% DMSO was advantageous compared to PG for F. scutaria spermatozoa, the PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e33354