Preserving and Using Germplasm and Dissociated Embryonic Cells for Conserving Caribbean and Pacific Coral

Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (∼5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had∼50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (−196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems

Preserving and using germplasm and dissociated embryonic cells for conserving Caribbean and Pacific coral.

Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (∼5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had∼50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (-196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems

Cryopreservation of coral sperm (all sperm exposed to freezing in these treatments).

<p><b>A</b>) <i>A. palmata</i> sperm were cryopreserved at cooling rates 20 to 30°C/min using 10% DMSO and PG, and IVF success was assessed and averaged over the single spawning period. This averaged graph revealed no difference between the two cryoprotectants and a mean fertilization success of ∼18%. <b>B</b>) However, if the <i>A. palmata</i> fertilization success during the spawning period was graphed by day, a 65% fertilization success occurred on Day 3 with 10% DMSO, whereas it was 25, 0 and 3% on Day 1, 2 and 4, respectively. For the first 3 nights, the control with fresh sperm held at ∼90%, then fell to 76% on the fourth evening. <b>C</b>) <i>F. scutaria</i> sperm were cryopreserved at rates 20 to 30°C/min using 10% DMSO versus PG, and fertilization success was assessed and averaged over two spawning periods (July and August 2010). Averaging indicated that 10% DMSO was the preferred cryoprotectant, and (as in <i>A. palmata</i>) there was no variability in time in terms of physiological responses during a spawning season. <b>D</b>) Variability in <i>F. scutaria</i> IVF success after cryopreservation over two nights of a single representative month (August 2010). Fresh sperm IVF success held steady at 65%, but sperm cryopreserved with 10% DMSO varied from 52 to 13% on the two evenings. Bars with the same letters were not different (<i>P</i>>0.05; ANOVA), whereas bars with different letters were different (<i>P</i><0.05; ANOVA).</p

Embryonic <i>F. scutaria</i> cells after cryopreservation (all cells exposed to freezing in these treatments).

<p>Mean post-thaw viability of <i>F. scutaria</i> cells was ∼50% for all cryoprotectants tested. Ten thousand events were measured for each sample. Controls (the three left bars) were live-stained and unstained cells and 100% dead cells that produced control data for the flow cytometer (i.e., 100% intact versus 100% dead). Bars with the same letters were not different, whereas bars with different letters were different (P<0.05; Kruskal-Wallis test).</p

Species-specific sperm concentrations were not necessary for successful <i>in vitro</i> fertilization.

<p><b>A</b>) Regardless of the <i>A. palmata</i> sperm concentration used (10⁶ to 10⁸ cells/ml), a successful <i>in vitro</i> 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) (<i>P</i>>0.05; ANOVA). <b>B</b>) Both sperm concentrations for <i>F. scutaria</i> produced uniform IVF results (<i>P</i>>0.05; Mann-Whitney).</p

Adult and larval forms of <i>A. palmata</i> and <i>F. scutaria</i>.

<p><b>A</b>) Adult and developing <i>A. palmata</i> larvae (inset) at the cornflake stage at ∼24 h. Scale bar = 50 µm. Adult photo by R. Williams, Smithsonian Institution. <b>B</b>) Adult and developing <i>F. scutaria</i> larvae at the swimming stage. Scale bar = 50 µm. Embryos that reached these stages were scored as successfully developed.</p

Experiment II: <i>In vitro</i> fertilization after exposing fresh sperm to various cryoprotectant treatments.

1<p>Sigma-Aldrich, St Louis, MO, USA.</p>2<p>Acros Organics, Fair Lawn, NJ, USA.</p>*<p>Thirty to 50 fresh eggs were used per pooled sperm sample.</p>***<p>An inseminate concentration of 10⁶ sperm/ml was used for each species.</p

<i>F. scutaria</i> sperm were sensitive to cryoprotectants (no sperm exposed to freezing in any of these treatments).

<p><b>A</b>) 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 (<i>P</i>>0.05; Kruskal-Wallis test), but bars with different letters were different (<i>P</i><0.05; Kruskal-Wallis test). FSW controls included fresh sperm with no cryoprotectant. <b>B</b>) However, if the effect of the cryoprotectants on <i>F. scutaria</i> 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). <b>C</b>) In contrast, 10% DMSO and PG solutions caused a 30 to 40% decrease in fertilization success for fresh <i>F. scutaria</i>, whereas the 5% solutions did not. Bars with the same letters were not different (<i>P</i>>0.05; ANOVA), whereas bars with different letters were different (<i>P</i><0.05; ANOVA).</p

Experiment I: Fresh sperm treatments for <i>in vitro</i> fertilization.

*<p>Thirty to 50 fresh eggs were used per pooled sperm sample.</p>**<p>Eight replicates per female for each sperm concentration.</p

Scientific SECORE divers placing several collection nets on a large <i>A. palmata</i> colony prior to spawning in the evening.

<p>A) <i>A. palmata</i> setting, note the pink egg/sperm bundles resting on the surface of the brown colony prior to release into the water column. B) Scientific divers placing the specially-designed nets on a colony to collect egg/sperm bundles. C) Egg/sperm bundles were collected in the cup at the top of the net. Photos contributed courtesy of the Pittsburgh Zoo & PPG Aquarium, Photo ©2010 Paul A. Selvaggio.</p