<meta charset="utf8"></head><body>אופטימיזציה של שימור זרע אלמוגים בהקפאה ממיני אלמוגים בסכנת הכחדה

<ol>
	<li>Eakin, C. M., Sweatman, H. P. A., Brainard, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2014-2017+global-scale+coral+bleaching+event:+Insights+and+impacts.">The 2014-2017 global-scale coral bleaching event: Insights and impacts.</a> Coral Reefs. 38 (4), 539-545 (2019).</li><li>Anthony, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+interventions+are+needed+to+save+coral+reefs.">New interventions are needed to save coral reefs.</a> Nature Ecology &amp; Evolution. 1 (10), 1-3 (2017).</li><li>Wildt, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+resource+banking+for+wildlife+research,+management,+and+conservation.">Genome resource banking for wildlife research, management, and conservation.</a> ILAR journal / National Research Council, Institute of Laboratory Animal Resources. 41 (4), 228-234 (2000).</li><li>Hagedorn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+and+using+germplasm+and+dissociated+embryonic+cells+for+conserving+Caribbean+and+Pacific+coral.">Preserving and using germplasm and dissociated embryonic cells for conserving Caribbean and Pacific coral.</a> PLoS ONE. 7 (3), e33354 (2012).</li><li>Hagedorn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+frozen+repository+for+the+Great+Barrier+Reef+coral+created.">First frozen repository for the Great Barrier Reef coral created.</a> Cryobiology. 65 (2), 157-158 (2012).</li><li>Zuchowicz, N., Daly, J., Lager, C., Williamson, O., Hagedorn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freezing+on+the+beach:+A+robust+coral+sperm+cryopreservation+design.">Freezing on the beach: A robust coral sperm cryopreservation design.</a> Cryobiology. 101, 135-139 (2021).</li><li>Hobbs, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+coral+biobanking+science+in+Australia+-+transitioning+into+applied+reef+restoration.">A decade of coral biobanking science in Australia - transitioning into applied reef restoration.</a> Frontiers in Marine Science. , 9 (2022).</li><li>Daly, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+can+assist+gene+flow+on+the+Great+Barrier+Reef.">Cryopreservation can assist gene flow on the Great Barrier Reef.</a> Coral Reefs. 41, 455-462 (2022).</li><li>Hagedorn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assisted+gene+flow+using+cryopreserved+sperm+in+critically+endangered+coral.">Assisted gene flow using cryopreserved sperm in critically endangered coral.</a> Proceedings of the National Academy of Sciences of the United States of America. 118 (38), e2110559118 (2021).</li><li>Daly, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+cryopreservation+of+coral+larvae+using+vitrification+and+laser+warming.">Successful cryopreservation of coral larvae using vitrification and laser warming.</a> Scientific Reports. 8, 1-10 (2018).</li><li>Daly, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+proof+of+concept+demonstration+of+nanowarming+in+coral+tissue.">The first proof of concept demonstration of nanowarming in coral tissue.</a> Advanced Sustainable Systems. , (2023).</li><li>Powell-Palm, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+and+revival+of+Hawaiian+stony+corals+via+isochoric+vitrification.">Cryopreservation and revival of Hawaiian stony corals via isochoric vitrification.</a> Nature Communications. 14, 4859 (2023).</li><li>. Reef Restoration and Adaptation Program Available from: <a target="_blank" href="https://gbrrestoration.org">https://gbrrestoration.org</a> (2023)</li><li>. Florida Coral Rescue Available from: <a target="_blank" href="https://myfwc.com/research/habitat/coral/disease/rescue">https://myfwc.com/research/habitat/coral/disease/rescue</a> (2023)</li><li>Harrison, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Coral reefs: An ecosystem in transition. , 59-85 (2011).</li><li>Hagedorn, M., Carter, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryobiology:+Principles,+species+conservation+and+benefits+for+coral+reefs.">Cryobiology: Principles, species conservation and benefits for coral reefs.</a> Reproduction, Fertility and Development. 28 (8), 1049 (2016).</li><li>Zuchowicz, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+coral+sperm+motility.">Assessing coral sperm motility.</a> Scientific Reports. 11, 61 (2021).</li><li>Thomas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+an+automated+cryovial+dispensing+system+for+cell+banking.">Evaluation of an automated cryovial dispensing system for cell banking.</a> Genetic Engineering &amp; Biotechnology News. 37 (17), 22-23 (2017).</li><li>. Coral cryopreservation training course Available from: <a target="_blank" href="https://nationalzoo.si.edu/center-for-species-survival/coral-cryopreservation-training-course">https://nationalzoo.si.edu/center-for-species-survival/coral-cryopreservation-training-course</a> (2023)</li><li>Bailey, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+sperm+counting+methods+using+limits+of+agreement.">Validation of sperm counting methods using limits of agreement.</a> Journal of Andrology. 28 (3), 364-373 (2007).</li><li>Hu, E., Yang, H., Tiersch, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+cryopreservation+of+spermatozoa+of+blue+catfish+(Ictalurus+furcatus):+Establishment+of+an+approach+for+commercial-scale+processing.">High-throughput cryopreservation of spermatozoa of blue catfish (Ictalurus furcatus): Establishment of an approach for commercial-scale processing.</a> Cryobiology. 62 (1), 74-82 (2011).</li><li>Pickett, B. W., Berndtson, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+bovine+spermatozoa+by+freezing+in+straws:+A+review.">Preservation of bovine spermatozoa by freezing in straws: A review.</a> Journal of Dairy Science. 57 (11), 1287-1308 (1974).</li><li>Westbrook, C. E., Daly, J., Bowen, B., Hagedorn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+the+collector+urchin+embryo,+Tripneustes+gratilla.">Cryopreservation of the collector urchin embryo, Tripneustes gratilla.</a> Cryobiology. 15, 104865 (2024).</li><li>Maria, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+cryotubes+for+the+cryopreservation+of+Tambaqui+fish+semen+(Colossoma+macropomum).">Use of cryotubes for the cryopreservation of Tambaqui fish semen (Colossoma macropomum).</a> Cryobiology. 70 (2), 109-114 (2015).</li><li>Fuchs, Y. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+biobanking:+Employing+a+robotic+system+for+automated+mononuclear+cell+isolation.">Next-generation biobanking: Employing a robotic system for automated mononuclear cell isolation.</a> Biopreservation &amp; Biobanking. 21 (1), 106-110 (2023).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Ovation ALI-Q 2 VS Pipette Controller - Aliquotting pipette</td>
			<td >Vistalab</td>
			<td >2100-1005</td>
			<td >Fluid handling &#8211; measuring sperm volume, addition of cryoprotectant solution, aliquoting samples into cryovials</td>
		</tr>
		<tr >
			<td >5 mL serological pipettes (bulk)</td>
			<td >Thermo Scientific&#160;</td>
			<td >Nunc 170355</td>
			<td >Fluid handling &#8211; measuring sperm volume, addition of cryoprotectant solution, aliquoting samples into cryovials</td>
		</tr>
		<tr >
			<td >10 mL serological pipettes (bulk)</td>
			<td >Thermo Scientific</td>
			<td >Nunc&#160; 170356</td>
			<td >Fluid handling &#8211; measuring sperm volume, addition of cryoprotectant solution, aliquoting samples into cryovials</td>
		</tr>
		<tr >
			<td >P2 0.2&#8211;2 &#181;L pipettor</td>
			<td >Gilson</td>
			<td >F144054M</td>
			<td>Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment</td>
		</tr>
		<tr >
			<td >P10 1&#8211;10 &#181;L pipettor</td>
			<td >Gilson</td>
			<td >F144055M</td>
			<td>Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment</td>
		</tr>
		<tr >
			<td >P20 2&#8211;20 &#181;L pipettor</td>
			<td >Gilson</td>
			<td >&#160;F144056M</td>
			<td>Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment</td>
		</tr>
		<tr >
			<td >P200 20&#8211;200 &#181;L pipettor</td>
			<td >Gilson</td>
			<td >F144058M</td>
			<td>Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment</td>
		</tr>
		<tr >
			<td >P1000 100&#8211;1000 &#181;L pipettor</td>
			<td >Gilson</td>
			<td >F144059M</td>
			<td>Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment</td>
		</tr>
		<tr >
			<td >Vacuum pump</td>
			<td >Millipore</td>
			<td >WP6122050</td>
			<td >Preparation of filtered sea water for solution preparation</td>
		</tr>
		<tr >
			<td >Reusable bottle-top filtration system</td>
			<td >Thermo Scientific</td>
			<td >DS0320-5045</td>
			<td >Preparation of filtered sea water for solution preparation</td>
		</tr>
		<tr >
			<td >0.22-&#181;m filter discs, mixed cellulose esters&#160;</td>
			<td >Merck Millipore</td>
			<td >GSWP04700</td>
			<td >Preparation of filtered sea water for solution preparation</td>
		</tr>
		<tr >
			<td >Filtered sea water</td>
			<td >N/A</td>
			<td >&#8211;</td>
			<td >Base medium for sperm activation and cryoprotectant solutions</td>
		</tr>
		<tr >
			<td >Dimethyl sulfoxide (DMSO)</td>
			<td >Sigma-Aldrich</td>
			<td >D4540</td>
			<td >Cryoprotectant chemical used at a final concentration of 10% v/v in filtered seawater for sperm cryopreservation</td>
		</tr>
		<tr >
			<td >Caffeine</td>
			<td >Sigma-Aldrich</td>
			<td >C0750</td>
			<td >Used to activate sperm motility</td>
		</tr>
		<tr >
			<td >BSA heat shock fraction</td>
			<td >Sigma-Aldrich</td>
			<td >A9647</td>
			<td >Used to minimise sperm adherance to CASA well slides</td>
		</tr>
		<tr >
			<td >15-mL tubes - racked</td>
			<td >Thermo Scientific</td>
			<td >339651</td>
			<td >Preparation of sperm activation solution</td>
		</tr>
		<tr >
			<td >50-mL tubes racked</td>
			<td >Thermo Scientific</td>
			<td >339653</td>
			<td >For collection of gamete bundles and filtered sperm samples</td>
		</tr>
		<tr >
			<td >Transfer pipettes</td>
			<td >Thermo Scientific</td>
			<td >Samco 202PK</td>
			<td >To aid collection of gamete bundles from the water surface</td>
		</tr>
		<tr >
			<td >100-&#181;m filter baskets</td>
			<td >Fisher Scientific</td>
			<td >22363549</td>
			<td >To exclude eggs during separation of the sperm sample</td>
		</tr>
		<tr >
			<td >Eppendorf racks</td>
			<td >Interpath</td>
			<td >511029</td>
			<td>Dilution and activation of sperm for concentration and motiliy assessment</td>
		</tr>
		<tr >
			<td >Eppendorf 1.5-mL tube</td>
			<td >Eppendorf</td>
			<td >30120086</td>
			<td>Dilution and activation of sperm for concentration and motiliy assessment</td>
		</tr>
		<tr >
			<td >Glass coverslips 18x18 mm</td>
			<td >Brand</td>
			<td >4700 45</td>
			<td>Assessment of sperm concentration and motility using phase microscopy</td>
		</tr>
		<tr >
			<td >Plain glass slides, precleaned, 75x25 mm</td>
			<td >Corning</td>
			<td >2947</td>
			<td>Assessment of sperm concentration and motility using phase microscopy</td>
		</tr>
		<tr >
			<td >Haemocytometer</td>
			<td >Hausser Scientific</td>
			<td >1492</td>
			<td>Assessment of sperm concentration and motility using phase microscopy</td>
		</tr>
		<tr >
			<td >CASA slides (Leja 20-&#181;m 4 chamber, SC-20-01-04-B)</td>
			<td >IMV Technologies</td>
			<td >025107</td>
			<td >Assessment of sperm concentration and motility using Computer Assisted Sperm Analysis (CASA)</td>
		</tr>
		<tr >
			<td >Makler sperm counting chamber (CASA)</td>
			<td >IVFStore</td>
			<td >SM-373</td>
			<td >Assessment of sperm concentration and motility using Computer Assisted Sperm Analysis (CASA)</td>
		</tr>
		<tr >
			<td >accu-bead&#174; counting beads</td>
			<td >Hamilton-Thorne</td>
			<td >710111</td>
			<td >Assessment of sperm concentration and motility using Computer Assisted Sperm Analysis (CASA)</td>
		</tr>
		<tr >
			<td >CASA system + laptop</td>
			<td >Hamilton Thorne</td>
			<td >Ceros II</td>
			<td >Assessment of sperm concentration and motility using Computer Assisted Sperm Analysis (CASA)</td>
		</tr>
		<tr >
			<td >Safety Glasses</td>
			<td >Generic</td>
			<td >&#8211;</td>
			<td>Personal protective equi[pment for use when handling DMSO and liquid nitrogen</td>
		</tr>
		<tr >
			<td >Lab coat</td>
			<td >Long sleeve, full length</td>
			<td >&#8211;</td>
			<td>Personal protective equi[pment for use when handling DMSO and liquid nitrogen</td>
		</tr>
		<tr >
			<td >Cryogloves (pair)</td>
			<td >Tempshield</td>
			<td >Mid-Arm</td>
			<td>Personal protective equi[pment for use when handling DMSO and liquid nitrogen</td>
		</tr>
		<tr >
			<td >Medium forceps</td>
			<td >Generic</td>
			<td >&#8211;</td>
			<td >For removing cryopreserved samples from the cryo racks and manipulating samples in liquid nitrogen</td>
		</tr>
		<tr >
			<td >Barcode scanner (2D compatible)</td>
			<td >Zebra</td>
			<td >DS2278</td>
			<td >For reading 1D and 2D barcodes on cryovials for sample management</td>
		</tr>
		<tr >
			<td >2.0-mL CryoStorage Vial, external thread, pre-capped, 2D SafeCode (DataMatrix/ECC200), linear and human readable</td>
			<td >Eppendorf</td>
			<td >30079434</td>
			<td >Barcoded cryovials for cryopreservation of sperm samples</td>
		</tr>
		<tr >
			<td >Cryovial rack</td>
			<td >Simport</td>
			<td >T315</td>
			<td >Rack to hold cryovials, with locking base to allow for one hand de-capping and capping</td>
		</tr>
		<tr >
			<td >Freezing racks</td>
			<td >Custom</td>
			<td >&#8211;</td>
			<td >Cryopreservation system custom designed for coral sperm, utilising 3D-printed and readily available components. Parts list and assembly instructions are available in Zuchwicz et al., 2021 (doi:10.1016/j.cryobiol.2021.04.005)</td>
		</tr>
		<tr >
			<td >Freezing rack lid</td>
			<td >Custom</td>
			<td >&#8211;</td>
			<td >Cryopreservation system custom designed for coral sperm, utilising 3D-printed and readily available components. Parts list and assembly instructions are available in Zuchwicz et al., 2021 (doi:10.1016/j.cryobiol.2021.04.005)</td>
		</tr>
		<tr >
			<td >Freezing Thermos &#8211; 1.5 Litre 18/8 Stainless Steel Double-Wall Vacuum Food Container</td>
			<td >Isosteel</td>
			<td >&#160;VA-9683&#160;</td>
			<td >Cryopreservation system custom designed for coral sperm, utilising 3D-printed and readily available components. Parts list and assembly instructions are available in Zuchwicz et al., 2021 (doi:10.1016/j.cryobiol.2021.04.005)</td>
		</tr>
		<tr >
			<td >Lab timers</td>
			<td >Generic</td>
			<td >&#8211;</td>
			<td >For timing of cryoprotectant equilibration prior to cryopreservation</td>
		</tr>
		<tr >
			<td >Nitrogen bath 9L</td>
			<td >BelArt</td>
			<td >M16807-9104</td>
			<td >For quenching samples during cryopreservaton and holding samples during sorting and handling</td>
		</tr>
		<tr >
			<td >Thermocouple data logger- multichannel</td>
			<td >Omega</td>
			<td >HH520</td>
			<td >Temperature monitoring during cryopreservation to determine freezing rate and end point</td>
		</tr>
		<tr >
			<td >Thermocouple probe &#8211; Type K</td>
			<td >Omega</td>
			<td >5SC-TT-K-30-36</td>
			<td >Temperature monitoring during cryopreservation to determine freezing rate and end point</td>
		</tr>
		<tr >
			<td >Cryo pens/coloured permanent pen</td>
			<td >Staedtler Lumocolor</td>
			<td >318</td>
			<td >Optional for marking cryovial lids to assist with sample management</td>
		</tr>
		<tr >
			<td >Dry Shipper - charged</td>
			<td >Taylor Wharton</td>
			<td >CXR100, or CX500</td>
			<td >For transfer of cryopreserved samples from field/collection sites to the biorepository for storage</td>
		</tr>
	</tbody>
</table>

a semi-automated workflow for the cryopreservation of coral sperm to support biobanking and aquaculture

Coral reefs globally are experiencing a loss of coral species, populations, and genetic diversity due to ocean warming and acidification caused by climate change, diminishing the viability of these critical habitats and impacting the species that they support1,2. The most effective approach to secure genetics long-term is cryopreservation and biobanking, which permits the frozen storage of living samples at cryogenic temperatures in liquid nitrogen indefinitely3. The development in 2012 of methods for the cryopreservation of coral sperm4 enabled the biobanking of genetics from these species for the first time and led to the development of the first biorepository for coral genetics in 20125. Since then, this cryopreservation protocol has been further refined6 and used to secure the genetics of over 50 species of coral globally, with 30 of those species coming from the Great Barrier Reef in Australia7. Cryopreserved coral sperm can generate healthy corals that develop normally and have been used to facilitate assisted gene flow experiments in Australia8 and the Caribbean9. While technologies are currently under development to enable the cryopreservation of complex tissue types such as larvae10 and adult coral tissues11,12, cryopreservation of coral sperm is currently the most established tool available for routine biobanking of coral genetics.
As the impacts on coral populations have increased, several countries have initiated large-scale programs to support reef restoration and adaptation (e.g., the Reef Restoration and Adaptation Program [RRAP] in Australia13) or to secure remaining imperiled coral populations (e.g., Florida Coral Rescue in the USA14). In the context of these programs, cryopreservation can be thought of as an enabling technology, supporting research and large-scale coral production in addition to securing existing genetic diversity and preventing extinctions. Sperm cryopreservation can enable greater control over reproduction between populations that are physically or temporally separated and can permit genetic management of broodstock to select for desirable traits such as thermal tolerance or disease resistance8. To date, coral sperm biobanking has been undertaken on a relatively small scale in support of biodiversity management7, so some level of upscaling will be required if such biobanking is to meet its potential within these larger reef restoration programs. As with all reef restoration efforts based on coral reproduction, the main impediment to increasing biobanking efforts is the limited period during which coral gametes are available, since spawning in most reef-building species coincides with the full moon in late spring and early summer15, meaning that gametes are only available for cryopreservation and biobanking on a few nights each year. Moreover, in regions where mass spawning occurs, e.g., the Great Barrier Reef, there are typically multiple species spawning simultaneously or within a few hours of each other on the same night15. Enhancements to the sperm cryopreservation pathway are therefore required to increase the scale and efficiency of processing to maximize biobanking capacity during these brief annual spawning windows while ensuring the integrity of samples and associated metadata.
Cryopreservation and biobanking of coral sperm involve several key steps, from the collection of gametes during spawning to the accession of samples into the biorepository and database (Figure 1). The process begins with the separation of sperm from eggs (in hermaphroditic species) or the collection of sperm from the water column (in gonochoric species), followed by the assessment of sperm motility and concentration. Sperm are then combined with cryoprotectant (10% v/v final concentration, dimethyl sulfoxide, DMSO) in filtered seawater (FSW) and cooled at &#8722;20 &#176;C/min in a custom-designed cooling device4. Early iterations of this process relied on visual assessment of sperm motility and concentration via phase-contrast microscopy and hemocytometer counts, printing, and labeling tubes as samples were processed for cryopreservation, hand-pipetting sperm samples into cryovials, and cooling on a specially designed floating rack16. The application of computer-assisted sperm analysis (CASA)17 and the development of a 3D-printed cooling device6 have improved the efficiency and reliability of coral sperm cryopreservation, but the sperm sample processing pathway has remained largely the same since its inception. While this approach is suitable for processing samples from a moderate number of colonies and species on a spawning night, for large volumes and numbers of colonies (e.g., individual samples from &#62;10 colonies at a time), there are bottlenecks in the cryopreservation pathway that impede processing of the samples in a timely manner (i.e., within 2 h of sperm release) without degradation of sample fertility potential. Although large-scale fluid handling systems for cryovials are available (e.g., Thomas et al.18), they are typically designed for large batch processing (i.e., multiple racks of cryovials) and are not suitable for transport and use in the field, so they are not cost-effective for this application. Therefore, the present study aimed to improve the efficiency of coral sperm cryopreservation by introducing portable, inexpensive automation equipment and methods at key steps in the processing pathway to maximize the number of samples that could be effectively cryopreserved on a spawning night.

<meta charset="utf8"></head><body>מחבר זרקור: פיתוח טכנולוגיות יעילות לשימור בהקפאה וביו-בנקאות לשיקום השונית הגלובלית

זרימת עבודה חצי-אוטומטית לשימור בהקפאה של זרע אלמוגים לתמיכה בביו-בנקאות ובחקלאות ימית

Our research program aims to develop and apply cryopreservation and biobanking technologies to help secure the genetic diversity and biodiversity of corals in Australia and globally. Cryopreservation of coral sperm has been possible for a decade, but coral spawning is limited to just a few nights each year, which provides us a narrow opportunity to bank the genetics of threatened species. We aim to enhance these protocols to improve cryopreservation efficiency during coral spawning events.Semi-automated processing pathway that we've developed helps to streamline the assessment handling and cryopreservation of coral sperm to improve the efficiency of sample processing and metadata management in the field. This helps us to maximize the number of samples that can be cryopreserved during those limited biobanking opportunities. So the protocol developed here is easily transferable and relatively inexpensive to implement.So it can be used by scientists and reef managers around the world to help manage reproduction and genetics in coral populations, and help to support restoration programs to prevent the extinction of coral species.

Coral reefs are facing a crisis as the frequency of bleaching events caused by ocean warming increases, resulting in the death of corals on reefs around ...

a-semi-automated-workflow-for-cryopreservation-coral-sperm-to-support

Research

JoVE Journal

Biology

A Semi-Automated Workflow for the Cryopreservation of Coral Sperm to Support Biobanking and Aquaculture

478 Views.  Taronga Conservation Society Australia. תוכנית המחקר שלנו שואפת לפתח וליישם טכנולוגיות שימור קריוגני וביו-בנקאות כדי לסייע באבטחת המגוון הגנטי והמגוון הביולוגי של אלמוגים באוסטרליה ובעולם. שימור בהקפאה של זרע אלמוגים אפשרי כבר עשור, אך השרצה של אלמוגים מוגבלת ללילות ספורים בלבד בכל שנה, מה שמ?...

Video: מחבר זרקור: פיתוח טכנולוגיות יעילות לשימור בהקפאה וביו-בנקאות לשיקום השונית הגלובלית

Coral reefs are facing a crisis as the frequency of bleaching events caused by ocean warming increases, resulting in the death of corals on reefs around the world. The subsequent loss of genetic diversity and biodiversity can diminish the ability of coral to adapt to the changing climate, so efforts to preserve existing diversity are essential to maximize the resources available for reef restoration now and in the future. The most effective approach to secure genetics long-term is cryopreservation and biobanking, which permits the frozen storage of living samples at cryogenic temperatures in liquid nitrogen indefinitely. Cryopreservation of coral sperm has been possible since 2012, but the seasonal nature of coral reproduction means that biobanking activities are restricted to just a few nights per year when spawning occurs. Improving the efficiency of coral sperm processing and cryopreservation workflows is therefore essential to maximizing these limited biobanking opportunities. To this end, we set out to optimize cryopreservation processing pathways for coral sperm by building on existing technologies and creating a semi-automated approach to streamline the assessment, handling, and cryopreservation of coral sperm. The process, which combines computer-assisted sperm analysis, barcoded cryovials, and a series of linked auto-datasheets for simultaneous editing by multiple users, improves the efficiency of both sample processing and metadata management in the field. Through integration with cross-cutting research programs such as the Reef Restoration and Adaptation Program in Australia, cryopreservation can play a crucial role in large-scale reef restoration programs by facilitating the genetic management of aquaculture populations, supporting research to enhance thermal tolerance, and preventing the extinction of coral species. The described procedures will be utilized for coral cryopreservation and biobanking practitioners on reefs worldwide and will provide a model for the transition of cryopreservation technologies from research laboratories to large-scale applications.

This protocol describes a semi-automated pathway to improve the efficiency and capacity of processing and cryopreservation of sperm from threatened coral species, aiming to secure genetic diversity and support reef restoration efforts.

Computer Assisted Coral Sperm Analysis and Cryopreservation for Biobanking

After collecting gamete bundles from hermaphroditic and sperm from gonochoric coral species, place them into labeled 50-milliliter tubes. Place four microliters of activated sperm suspension on a Makler counting chamber. To assess sperm motility, add concentration of the activated sample.Open the coral biobanking auto-datasheet file and input the sperm dilution factor used for the computer-assisted sperm analysis assessment. Then, enter computer assisted sperm analysis outputs for sperm concentration, total motility, and progressive motility. Write the sperm concentration on the filtered sperm sample tube and transfer the tube to the cryopreservation workstation.Open the shared coral biobanking auto-datasheet and select the Cryopreservation tab. After checking the sperm sample tube label, identify the corresponding entry by checking colony ID.Using a serological pipette, measure the volume of the sperm sample by drawing and expelling it back into the same tube. Enter the value in column E.Check the auto calculated column 1 for the required cryoprotectant concentration and column H for the cryodiluent volume to add.Start a timer. And using a pipette, dropwise add the cryodiluent DMSO with constant gentle mixing of the sample. Record the time of the cryodiluent edition in column P.Read column K to determine the number of cryo vials to fill.Uncap sterile barcoded cryo vials, and arrange the caps on a sterile surface. Using a serological pipette and an aliquoting pipetter, aliquot one milliliter of samples into barcoded cryo vials and recap vials. Place one thermocouple vial and all filled sample vials into an empty cryo rack insert at room temperature.Then, place the dummy cryo vials containing 10%DMSO in filtered seawater in empty slots on the cryo rack. Enter the run number into column U of the auto-datasheet and scan the cryo rack serial number into column V.Place the cursor in the correct cell of column Z and scan the thermocouple vial, followed by all cryo vial tubes. Set aside loaded and scanned racks for the remainder of the cryoprotectant equilibration.Fill the cryo rack cooling vessel with liquid nitrogen to the necessary level for cooling at approximately 20 degrees Celsius per minute. Following the 10-minute cryoprotectant equilibration period, connect the thermocouple probe to the data logger and start recording. Gently place the full cryo rack into the cooling vessel and apply the lid.Record the start time in column Q.Once the thermocouple vial indicates 80 degrees celsius or lower on the data logger, transfer the cryo rack insert into a liquid nitrogen bath to quench the samples. Remove the cryo vials from the rack and transfer them to a dry shipper for transport to the biobank. In Acropora millepora, fixed cover slip chamber slides showed less than half of the sperm concentration counts compared to the Makler counting chamber.Validating the Makler counting chamber with commercially available latex microbeads at a known concentration resulted in consistent counts with low variability within the expected range. No significant difference was observed in post thaw concentrations of total, motile, or progressively motile sperm in samples cryopreserved using 20%or 30%DMSO as the cryodiluent.