Preserving and Banking Coral Figure 3. Species-specific sperm concentrations were not necessary for successful in vitro fertilization. A) Regardless of the A. palmata sperm concentration used (106 to 108 cells/ml), a successful in vitro fertilization success of .92% was observed regardless of whether the eggs were exposed to the sperm for 5 min (grey bars) or overnight (black bars) (P.0.05; ANOVA). B) Both sperm concentrations for F. scutaria produced uniform IVF results (P.0.05; Mann-Whitney). doi:10.1371/journal.pone.0033354.g003 scutaria, these individual variations did not affect the fertilization success of the pooled sperm samples (P.0.05, Kruskal-Wallace test, N = 16 egg donors and 7 to 10 sperm donors); insufficient data were available for individual A. palmata to evaluate statistically. Thus, for IVF there was no preferred sperm concentration for either species, and A. palmata eggs fertilized just as efficiently with a 5 min versus 12 h sperm exposure. Experiment II: Determining cryosensitivities Fresh spermatozoa from A. palmata were not sensitive to cryoprotectant concentrations. Regardless of the 10 cryoprotectant exposure treatments tested (Table 2), cryoprotectant-exposed fresh sperm fertilized .85% of inseminated eggs at a rate no different from controls (93%) (P.0.05, Kruskal-Wallis test, N = 4 egg donors, 3 to 7 sperm donors). Because there was no difference among cryoprotectants and we wanted to develop a single, effective cryopreservation protocol for both species, all subsequent A. palmata experiments evaluated only 5 and 10% DMSO and PG solutions. By contrast, fresh F. scutaria spermatozoa were sensitive to cryoprotectant treatments with both sperm motility (P,0.05, ANOVA, F = 3.5, N = 10 egg donors and 3 to 7 sperm donors, Fig. 4A) and IVF success (P,0.05, ANOVA, F = 14.8, N = 10 egg donors and 3 to 7 sperm donors, Fig. 4C) adversely affected by higher (10%) compared to lower (5%) DMSO and PG concentrations. One of the most interesting aspects of F. scutaria sperm physiology was its variable response in motility to cryoprotectants from night-to-night within a spawning season (Fig. 4B), a variation that was obscured when data were averaged (Fig. 4A). For example, sperm unexposed to cryoprotectant had a 90% motility rating on each of the three spawning nights. By contrast, fresh sperm treated with 10% DMSO varied from 20 to 85% motility on different nights. F. scutaria also differed from A. palmata in that IVF success for the former was reduced by 30 to 40% compared to controls in the presence of 10% DMSO or 10% PG (P,0.05, ANOVA, F = 14.8, N = 10 egg donors and 3 to 7 sperm donors, Fig. 4C). The adverse influence was lost when the DMSO and PG concentrations was lowered to 5%. Thus, a higher concentration of either of these cryoprotectants had a toxic influence on IVF success of sperm from F. scutaria but not A. palmata. Experiment III: Producing a successful cryopreservation protocol A singular, effective cryopreservation method for A. palmata and F. scutaria sperm and embryonic cell freezing was achieved using 10% DMSO at varying cooling rates. PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e33354