We thank the Traditional Owners of Konomie, the Woppaburra people, for permission to trial the system described in this paper during on-country spawning in November 2022, and the Konomie Environmental Education Centre for use of their facilities. We would also like to acknowledge the support of the Australian Institute of Marine Science staff and scientists who facilitated the collection and spawning of colonies within the National Sea Simulator. This work was undertaken as an activity of the Cryopreservation sub-program (RRAP-CP-01) for the Reef Restoration and Adaptation Program, a partnership between the Australian Government&#39;s Reef Trust, and the Great Barrier Reef Foundation, with additional support from Taronga Conservation Society Australia, the Taronga Conservation Science Initiative and other philanthropists supporting the Taronga Foundation.

The methods described herein can be used to process and cryopreserve sperm from both hermaphroditic and gonochoric coral species. A general introduction and primer on coral spawning and gamete collection for sperm cryopreservation for both reproductive modes can be found at the Smithsonian&#39;s National Zoo &#38; Conservation Biology Institute Coral Cryopreservation Training Course19. The described methods were primarily developed using gametes from Acropora millepora colonies collected from the Keppel Island group on Woppaburra Sea Country and from Davies Reef on Bindal Sea Country, on the Great Barrier Reef in Australia. All collection and use of corals and gametes were undertaken with the free prior and informed consent of the Traditional Custodians of the relevant Sea Countries. The reagents and equipment used for this study are listed in the Table of Materials.
1. Pre-spawning - system checks and preparation of sperm activation solution and cryodiluent
<ol>
	<li>Confirm that the barcode scanner is charged, active, and set to enter spreadsheet data as horizontal cell entry. Consult the barcode reader user manual to customize this setting.</li>
	<li>Prepare coral sperm activation concentrated stock solutions for CASA. 
	NOTE: Disposable gloves should be worn when handling chemicals for preparation of the activation solution.
	<ol>
		<li>Prepare 30% bovine serum albumin (BSA) stock solution (5 mL) by dissolving 1.5 g of BSA in 4 mL of sterile purified tissue culture water with the aid of a tube warmer set to 37 &#176;C or by holding the tube in cupped hands, with periodic gentle swirling (do not shake vial).</li>
		<li>Once in solution, make up to the final volume (5 mL) using tissue culture water, label the tube with the date, and mark &#34;30% BSA in H2O&#34;.</li>
		<li>Store in the refrigerator (4 &#176;C) for up to 2 weeks.</li>
		<li>Prepare 60 mM caffeine stock solution (25 mL) by dissolving 0.2913 g of caffeine in 24 mL of filtered sea water (FSW).</li>
		<li>Once in solution, make up to the final volume (25 mL) using FSW, label the tube with the date and mark &#34;60 mM caffeine in FSW&#34;.</li>
		<li>Store in the refrigerator for up to 1 month. 
		NOTE: The maximum solubility of caffeine in FSW is approximately 71 mM.</li>
	</ol>
	</li>
	<li>Prepare sperm activation working solution for sperm samples at ~109 sperm/mL (0.9% BSA + 12 mM caffeine in FSW).
	<ol>
		<li>For 10 mL working solution, combine 2.0 mL of 60 mM caffeine stock solution + 0.3 mL of 30% BSA stock solution + 7.7 mL of FSW.</li>
		<li>Label the tube with date and &#34;sperm activation working solution - 0.9% BSA + 12 mM caffeine&#34;.</li>
		<li>Prepare on each day of use, store at room temperature (19-30 &#176;C), and discard the unused solution at the end of the day.</li>
	</ol>
	</li>
	<li>Prepare cryoprotectant solutions (cryodiluents). Ensure that disposable gloves are worn when handling the cryoprotectant DMSO. 
	&#8203;NOTE: FSW is prepared with varying cryoprotectant concentrations depending on the initial sperm concentration within a sample and the desired final sperm concentration of cryopreserved aliquots. When preparing the cryodiluent, ensure that FSW is aliquoted into the 50 mL tube prior to the addition of DMSO.
	<ol>
		<li>Collect raw seawater from the tank system in which corals are held prior to spawning (~35 ppt salinity).</li>
		<li>Filter raw seawater using a bottle-top filtration system attached to a vacuum pump with a 0.2 &#181;m non-pyrogenic membrane filter fitted.</li>
		<li>For a sperm concentration &#8805;2 &#215; 109 sperm/mL, prepare 20% DMSO in FSW by combining 10 mL of DMSO + 40 mL of FSW in a 50 mL tube.</li>
		<li>For a sperm concentration &#60;2 &#215; 109 sperm/mL, prepare 30% DMSO in FSW by combining 15 mL of DMSO + 35 mL of FSW in a 50 mL tube.</li>
		<li>Label the tube with the date and DMSO concentration, i.e., 30% DMSO or 20% DMSO.</li>
		<li>Prepare on each day of use, store at room temperature (19-30 &#176;C), and discard the unused solution at the end of each day. 
		NOTE: Mixing of DMSO and FSW creates an exothermic reaction, and the cryodiluent should be made up well in advance of use to allow cooling to room temperature. FSW can be stored at 4 &#176;C for preparation of the cryodiluent to counterbalance this heat generation.</li>
	</ol>
	</li>
	<li>Prepare thermocouple datalogger.
	<ol>
		<li>To measure the approximate cooling rate of each cryopreservation run, place a cryovial containing 10% DMSO in FSW with a thermocouple probe inserted through the lid in a sample slot of the cryopreservation rack alongside the sperm samples.</li>
		<li>To make a thermocouple vial, poke or melt a small hole in the cap of the barcoded cryovial and insert a K-type or T-type thermocouple through the opening. Push the thermocouple probe tip down to a level such that it will sit in the middle of the sample when the vial is filled, keeping the probe tip centered in the vial to avoid contact with the inside vial wall. Seal the cap and hold the thermocouple probe in place with hot glue.</li>
		<li>Wearing disposable gloves, fill the barcoded cryovial with a volume of 10% DMSO in FSW equal to the volume of the sperm sample to be cryopreserved.</li>
		<li>Prepare fresh 10% DMSO in FSW and refill thermocouple vials daily.</li>
	</ol>
	</li>
</ol>
2. Sperm preparation and assessment
<ol>
	<li>For hermaphroditic species (e.g., Acropora species), collect gamete bundles from the surface of the water using a 3 mL transfer pipette and place them into a labeled 50 mL tube at a ratio of 5 mL of gamete bundles over 5 mL of seawater (10 mL total volume).</li>
	<li>For gonochoric species, collect sperm from close to the polyp mouth using a 3 mL transfer pipette and place the sample into a labeled 50 mL tube, then proceed directly to step 2.8. 
	NOTE: Disposable gloves are not necessary for the collection and handling of gamete samples but may be worn if preferred.&#160;Hands should be washed thoroughly&#160;with fresh water, or gloves changed between each&#160;gamete sample to avoid cross-contamination.</li>
	<li>For hermaphroditic species, agitate the sperm-egg bundles by gently swirling the sample by hand until the bundles have broken apart. 
	NOTE: Some Montipora species have a toxin in the eggs, so they must be allowed to fall apart slowly with minimal agitation to avoid damaging sperm.</li>
	<li>Let the sample sit for 2 min in a tube rack to allow eggs to float to the surface and sperm to settle (eggs will form a pink/brown layer at the surface, and individual eggs will be visible; see Figure 1i).</li>
	<li>To separate the sperm and remove contaminating eggs, gently aspirate the sperm from the bottom of the tube using a clean 3-mL transfer pipette.</li>
	<li>Transfer the sperm sample to a 100 &#181;m cell strainer sitting atop a new sterile 50 mL centrifuge tube. The sample should flow easily through the filter. Tap the filter to encourage sample flow, and if necessary, a clean transfer pipette can be used to collect any residual sample from the underside of the filter. Remove the filter, then cap the tube loosely to allow air exchange.</li>
	<li>Label the filtered sperm sample with colony ID and move the sample to the sperm assessment workstation.</li>
	<li>Open the coral biobanking auto-datasheet file (Supplementary File 1). In the sperm assessment tab, input the coral colony metadata (columns A-H).</li>
	<li>Prepare sperm for CASA assessment.
	<ol>
		<li>Mix the sperm sample well and remove a 10 &#181;L aliquot to an empty 1.8 mL microcentrifuge tube.</li>
		<li>Immediately add 0.390 mL of sperm activation working solution dropwise over 10-20 s, gently mixing sperm into the solution. This gives a dilution ratio of 1:39 (sperm: solution, v/v) (or dilution factor of 40), which provides an appropriate concentration for CASA assessment (~50 &#215; 106/mL) if raw sperm concentration is ~2 &#215; 109 sperm/mL. Invert the tube 5 times and flick the tube lid prior to opening to settle the solution into the bottom of the tube. 
		NOTE: Sperm concentration for CASA should be in the range of 8-50 &#215; 106/mL to permit accurate sperm tracking17. Additional sperm activation working solution can be added to the raw sperm aliquot to reduce the concentration if needed. Recalculate the dilution rate and enter this value into the spreadsheet. In cases where many samples must be processed simultaneously, the 1.8 mL microcentrifuge tubes can be pre-loaded with 0.390 mL of sperm activation working solution with the 10 &#181;L aliquot of sperm sample added directly to it, then mixed well by inverting the tube 10&#215; and flicking the tube lid prior to opening.</li>
	</ol>
	</li>
	<li>Assess sperm motility and concentration of activated sample using computer-assisted sperm analysis (CASA) as described in Zuchowicz et al.17 by placing 4 &#181;L of activated sperm suspension on a Makler counting chamber20 or by visual assessment and hemocytometer count under phase contrast microscopy as described in Hagedorn et al.4. 
	NOTE: The Makler chamber and coverslip must be cleaned well with 70% ethanol, then distilled water, and wiped with a lint-free tissue wipe between samples.</li>
	<li>Input the sperm dilution factor used for CASA assessment and enter CASA outputs for sperm concentration (million/mL), total motility (%), and progressive motility (%) into the auto-datasheet. 
	NOTE: Additional CASA metrics, including average path velocity (VAP), straight-line velocity (VSL), and curvilinear velocity (VCL) can optionally be included in the auto-datasheet or retrieved from saved CASA files later.</li>
	<li>Write the sperm concentration on the filtered sperm sample tube and transfer the sample to the cryopreservation workstation in the same order as of bundle break-up and processing.</li>
</ol>
3. Sperm dilution and cryoprotectant equilibration
<ol>
	<li>At the cryopreservation workstation, open the same coral biobanking auto-datasheet file that the sperm assessment workstation is using and select the Cryopreservation tab. Check the sperm sample tube label and identify the corresponding entry by checking Colony ID (column C). If not accessing the same auto-datasheet file simultaneously between workstations, manually enter data into the Colony ID (column C) and sperm concentration (column F) in the auto-datasheet.</li>
	<li>Using a serological pipette, measure the sperm sample volume by drawing the sample into the pipette and expelling it back into the same tube; enter the value in column E.</li>
	<li>Check the auto-calculated column I for the required cryoprotectant concentration and column H for the volume of cryodiluent to be added (Table 1).</li>
	<li>Start a timer for 10 min and, using a serological pipette, immediately begin adding the cryodiluent, dropwise, with constant gentle mixing of the sample, ensuring that disposable gloves are worn for DMSO handling.&#160;</li>
	<li>Record cryodiluent addition time in column P.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sperm concentration</td>
			<td>Cryodiluent</td>
			<td>Dilution ratio (sperm:cryodiluent)</td>
			<td>Final DMSO concentration in sperm sample (%)</td>
		</tr>
		<tr>
			<td>&#60; 2 &#215; 109/mL</td>
			<td>30% DMSO in FSW</td>
			<td>2:1</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>&#8805; 2 &#215; 109/mL</td>
			<td>20% DMSO in FSW</td>
			<td>1:1</td>
			<td>10%&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Cryodiluent concentration and dilution ratios for cryopreservation of coral sperm, based on assessment of total sperm concentration in the sample to be cryopreserved.
4. Sperm cryopreservation
<ol>
	<li>Read column K to determine how many cryovials to fill. This is auto-calculated from the total volume of sperm and the desired volume per vial (1 mL in most cases). During the 10 min incubation period, proceed through steps 4.2 to 4.6.</li>
	<li>Uncap sterile barcoded cryovials and set the caps on a clean/sterile surface.</li>
	<li>Optional: Write the run # on each cryovial using a permanent marker; this can aid in quick sample identification for accessioning into the biobank.</li>
	<li>Using a serological pipette and an aliquoting pipettor set to the desired sample volume (1 mL), aliquot samples into uncapped barcoded cryovials and recap vials.</li>
	<li>Place one thermocouple vial and all full sample vials onto an empty cryo rack&#160;at room temperature. Ensure that the barcoded surface is facing outwards on all vials.</li>
	<li>If necessary, place additional dummy cryovials containing 10% DMSO in FSW in any empty spaces on the cryo rack to ensure that all spaces are occupied.</li>
	<li>Enter the run number into the auto-datasheet (column U) and scan the cryo rack serial number (QR code) into column V.</li>
	<li>Place the cursor in the correct cell of column Z and scan in the thermocouple vial, followed by all cryovial tubes that are loaded on the rack (column AA onwards). Avoid tilting the rack on its side to ensure the sample does not enter the thread of the vial.</li>
	<li>While scanning, cross-check that data is being entered into the correct cell and that all cryovials have been scanned once.</li>
	<li>Set aside loaded and scanned racks for the remainder of the cryoprotectant equilibration period and prepare the cooling vessel for cryopreservation.</li>
	<li>Fill the cryo rack cooling vessel with liquid nitrogen to the appropriate pre-determined level necessary for cooling at approximately &#8722;20 &#176;C/min. 
	NOTE: Nitrogen should only be used in well-ventilated areas as there is a risk of asphyxiation. Closed-toe shoes, safety eyewear/face shields, and cryo gloves must be worn when handling liquid nitrogen. Refer to the institutional guidelines for specific information on liquid nitrogen handling and use.</li>
	<li>At the end of the 10 min cryoprotectant equilibration period, connect the thermocouple probe to the datalogger and begin recording, then gently place the full cryo rack into the cooling vessel and apply the lid.</li>
	<li>Record the start time in column Q.</li>
	<li>Once the thermocouple vial reads &#8722;80 &#176;C or below on the datalogger, quench the samples in liquid nitrogen by transferring the cryo rack insert to a liquid nitrogen bath in a separate vessel such as a polystyrene box.</li>
	<li>Remove the cryovials from the rack and transfer them to a dry shipper for transport to the biorepository.</li>
</ol>

Optimizing Coral Sperm Cryopreservation from Threatened Coral Species

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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. 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The authors report there are no competing interests to declare.

The semi-automated processing pathway described in this protocol permits the efficient processing and cryopreservation of coral sperm to secure genetics from threatened species and support reef restoration and adaptation efforts. The motivation for the development of this protocol was the lack of existing systems suited to the throughput requirements of coral sperm cryopreservation and the use of cryovials, since high-throughput processing systems for sperm cryopreservation are typically based on sample packaging in 0.25 mL or 0.5 mL French straws21,22. By comparison, cryovials are generally either used at a small scale for low throughput processing (e.g., cryopreservation of laboratory samples for research23,24), or in high-throughput processing systems for bulk samples using expensive, non-portable equipment (e.g., cell culture processing for industry18,25). We also investigated the potential for using an auto-decapping system to streamline the removal and replacement of cryovial caps, but systems were only available for individual cryovials or for entire cryovial racks, so they did not provide a cost-effective solution. Currently, there are several groups globally who are using the cryopreservation protocol devised by Hagedorn et al.4&#160;to secure the genetic diversity of corals, and it is important that this work continues to expand to more reefs around the world. Therefore, a major consideration in the development of the current protocol was the need to utilize cost-effective and accessible technologies that could be readily implemented by these other groups, and which would not be cost-prohibitive for new groups wanting to begin coral sperm cryopreservation.
A key component of the described protocol is the improved handling of sample metadata via linked spreadsheets in Microsoft Excel. Data input is generally straightforward, but it must be noted that editing information in the auto-datasheets should only be done by deleting and re-entering information, as quick functions (e.g., Ctrl + C, Ctrl + V) to edit data will potentially affect simultaneous inputs by other users and can cause problems with data linking between spreadsheets. An important metadata component is a unique identifier (namely, colony ID) that is linked to the donor colony and that is attached to the sample at all stages of the processing pathway. It is essential that sample tubes be clearly labeled with the colony ID at the time of bundle collection, and that this information be accurately transcribed onto any new tubes into which the sample is transferred during preparation (e.g., during filtering to separate eggs and sperm, or for cryodiluent addition). Although the protocol enables the automatic transfer of sample quality information from the CASA operator to the cryopreservation workstation, issues can be encountered when internet or Wi-Fi coverage is poor. If data transfer delays are encountered, it is recommended that the two workstations work offline and maintain separate auto-datasheets that can be reconciled afterward. The key information required by the cryopreservation operator is the colony ID and sample concentration, so it is recommended that the CASA operator write the sample concentration on the sample tube as a backup to ensure that this information is at hand for cryopreservation preparation if there is a delay in the upload or download of metadata between computers.
The choice of barcode scanner and barcoded cryovials used for this protocol can be varied to suit budget and product availability; however, there are some key elements that should be considered in their selection. The barcode scanner should have the capacity for customized settings, specifically the ability to change the data input specifications and direction of data entry. The auto-datasheets used for this protocol use horizontal entry, but on some occasions (e.g., accessioning into the biobank or for other laboratory uses) vertical entry may be required, so it is important that this feature be customizable. While the protocol can be used with both 1D and 2D barcodes, it is recommended that the cryovials selected have a human-readable component (e.g., 1D barcodes typically include a unique number) to permit cross-checking of sample entry during cryopreservation. In addition to sample entry, the barcode scanner can be used to automate the input of some metadata fields by creating and printing out QR codes for information that is repeated across samples (e.g., species names, dates, and reef locations) prior to spawning. This information can then be quickly and easily entered into the auto-datasheets by scanning with the barcode scanner. Moreover, by attaching serial number barcodes to each cryo rack and thermocouple probe, it is also possible to link each cryopreservation run with a specific set of equipment within the database, which is useful for quality control and to identify components that require repair or replacement.
The limiting factors in processing are often the time spent waiting for bundles to break up and the time required to filter and separate the sperm from the eggs prior to CASA and cryopreservation. Where possible, it is recommended to process samples in the order that bundles break up; however, further gains in efficiency can be made through the strategic management of sample processing orders. For example, if there are 5 or fewer cryovials per colony due to low sample volumes (i.e., less than 3 mL sperm per colony) or low sperm concentration requiring a 2:1 dilution with cryodiluent (i.e., less than 2 &#215; 109/mL), then it is better to add cryodiluent to two colony samples at the same time so that they can be run together on the same cryo rack (total # slots available = 11), rather than running them separately with dummies filling the empty spaces. Additionally, it is possible to run multiple cryo racks simultaneously (for samples &#62;6 mL volume) provided that care is taken to ensure that all processes can be completed within the 10 min cryodiluent equilibration time, which can further increase sample throughput. However, when running multiple cryo racks or combining multiple colonies on a single cryo rack, care must be taken to ensure that cryopreservation metadata is assigned to the correct sample in the auto-datasheet, especially if samples are cryopreserved in a different order to their CASA assessment.
In addition to the development of the semi-automated workflow, the present protocol description also provides two methodological comparisons related to sperm concentration that aim to improve sperm analysis and cryopreservation outcomes. In general, collecting 5 mL of gamete bundles over 5 mL of seawater (total volume 10 mL) will result in a sperm concentration at or above 2 &#215; 109 cells/mL, but there are occasions when the sperm concentration may be lower due to species differences or bundles breaking up during collection. The use of a higher concentration DMSO cryodiluent (30% v/v) reduces the amount by which the sperm are diluted to help minimize batch-to-batch variation in the concentration of sperm in the cryovial. Importantly, the use of 30% DMSO to achieve the final DMSO concentration does not impact post-thaw concentration or motility parameters, as shown by the representative data in Figure 3. The second methodological comparison provides an alternative to the single-use fixed-coverslip chamber slides typically used for CASA. The main challenge with using commercially available slides for the analysis of coral sperm is that they can impact the accuracy of motility assessment due to sperm sticking to the slide coating. The use of the activation solution overcomes this issue in many but not all samples, so it is still recommended to perform a separate CASA analysis for motility using a plain slide to ensure reliability and consistency. The use of a Makler counting chamber overcomes the need to analyze concentration and motility separately, and potentially improves the accuracy of concentration measurements (Figure 2), so it is recommended for use with the current protocol. Given this discrepancy in concentration measurements, a finding that has been reported previously20, it is important always to record slide details in the database alongside sperm quality data and, wherever possible, to be consistent in the type of chamber slide used to minimize batch-to-batch variation and help to ensure reliable calculations of sperm: egg ratios for fertilizations.
The semi-automated process described herein provides a standardized and efficient pathway for cryopreservation and biobanking of sperm from threatened coral species while retaining sample biosecurity and quality. The protocol described is easily transferrable and relatively inexpensive to implement in programs around the world that are working to secure existing coral diversity using cryopreservation, which will be essential to prevent extinctions and maximize the resources available for reef restoration efforts now and in the future.

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.

<table><tbody><tr><td>Ovation ALI-Q 2 VS Pipette Controller - Aliquotting pipette</td><td>Vistalab</td><td>2100-1005</td><td>Fluid handling &amp;ndash; measuring sperm volume, addition of cryoprotectant solution, aliquoting samples into cryovials</td></tr><tr><td>5 mL serological pipettes (bulk)</td><td>Thermo Scientific&amp;nbsp;</td><td>Nunc 170355</td><td>Fluid handling &amp;ndash; 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&amp;nbsp; 170356</td><td>Fluid handling &amp;ndash; measuring sperm volume, addition of cryoprotectant solution, aliquoting samples into cryovials</td></tr><tr><td>P2 0.2&amp;ndash;2 &amp;micro;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&amp;ndash;10 &amp;micro;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&amp;ndash;20 &amp;micro;L pipettor</td><td>Gilson</td><td>&amp;nbsp;F144056M</td><td>Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment</td></tr><tr><td>P200 20&amp;ndash;200 &amp;micro;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&amp;ndash;1000 &amp;micro;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-&amp;micro;m filter discs, mixed cellulose esters&amp;nbsp;</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>&amp;ndash;</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-&amp;micro;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-&amp;micro;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&amp;reg; 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>&amp;ndash;</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>&amp;ndash;</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>&amp;ndash;</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>&amp;ndash;</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>&amp;ndash;</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 &amp;ndash; 1.5 Litre 18/8 Stainless Steel Double-Wall Vacuum Food Container</td><td>Isosteel</td><td>&amp;nbsp;VA-9683&amp;nbsp;</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>&amp;ndash;</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 &amp;ndash; 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 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.

The steps outlined in this protocol build on the original methods for coral sperm cryopreservation described by Hagedorn et al.4 and subsequent refinement by Zuchowicz et al.6, providing key improvements to increase the efficiency of sperm processing for cryopreservation and biobanking. The use of barcoded cryovials and an aliquoting auto-pipettor simplifies the sperm packaging procedure by removing the need to print and hand-label cryovials, and reduces the strain of hand-pipetting large numbers of samples. Importantly, these improvements also reduce the time needed for sample preparation for cryopreservation, from around 8-10 min to under 5 min. Together with the use of CASA, these improvements reduce the number of personnel required for efficient biobanking of coral sperm, from 4 to 6 in the original methods, down to as few as two people using the current protocol. Additionally, the use of barcoded cryovials means that each tube has a unique identifier, so each can be tracked within the database with greater resolution than the previous method using batch-printed labels.
Field testing of the protocol across two spawning events during the 2022 Great Barrier Reef spawning season in Australia resulted in the cryopreservation and biobanking of 389 samples from 57 colonies across 5 species by two operators (one person for CASA, one person for cryopreservation), with one additional person assisting with bundle break-up and sperm separation as needed. During the November 2022 spawning event on Konomie (North Keppel Island) in Queensland, Australia, 150 samples from 24 colonies of Acropora millepora were cryopreserved by two operators over a 2 h period. The efficient processing and cryopreservation of this volume of samples from such a large number of coral colonies was enabled primarily by the efficiencies gained in sample preparation time and the simplified transfer of metadata between workstations.
Further testing of the workflow during spawning in December 2023 determined that, for coral sperm, the Makler counting chamber provided more accurate motility and concentration data compared to commercially available fixed-coverslip single-use chamber slides commonly used with CASA systems. The Makler counting chamber was validated for CASA using commercially available latex microbeads at a known concentration with a 4 &#181;L sample volume and the standard CASA settings17 used for coral sperm. These analyses showed consistent counts with low variability (44.5 &#177; 0.7 million/mL) that fell within the expected concentration range (46.0 &#177; 7.0 million/mL)&#160;provided by the manufacturer (Figure 2). A comparison of CASA data for Acropora millepora collected using the Makler counting chamber and the fixed-coverslip chamber slides found a significant difference in sperm concentration counts between the two chamber types (P = 0.002; Figure 2), with the fixed-coverslip chamber slides registering less than half of the concentration determined using the Makler counting chamber. This trend was also observed in sperm samples from two other coral species (data not shown). Testing during December 2023 also found no significant difference in the post-thaw concentrations of total, motile, or progressively motile sperm in samples cryopreserved using either a 1:1 dilution with 20% DMSO or a 1:2 dilution (sperm: cryodiluent) with 30% DMSO (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66233/66233fig01.jpg" /> 
Figure 1: The workflow used for semi-automated processing of coral sperm for cryopreservation and examples of mobile equipment used in the field. (A) Coral gamete bundles are collected at a ratio of 5 mL bundles over 5 mL of FSW and broken up to separate eggs and sperm (i). Colony metadata are entered into the auto-datasheet (ii), and the isolated sperm sample is assessed using computer-assisted sperm analysis (CASA) (iii). Sperm quality parameters are added to the auto-datasheet to calculate the addition of either 20% or 30% DMSO in FSW (iv), and the diluted sample is aliquoted into barcoded cryovials (v). The cryovials are loaded onto the cryo rack and scanned into the auto-datasheet (vi), then cooled at a rate of &#8722;20 &#176;C/min to &#8722;80 &#176;C (vii). The samples are then quenched in liquid nitrogen and transferred to dry shippers for transport to the Taronga CryoDiversity Bank (viii). (B) Example of the CASA (left) and cryopreservation (right) stations set up at a remote field site in the classroom at the Konomie Environmental Education Centre. (C) Example of the&#160;lab-based high-capacity&#160;cryopreservation station set up to run multiple cryo racks at the Australian Institute of Marine Science National Sea Simulator. <a href="https://www.jove.com/files/ftp_upload/66233/66233fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66233/66233fig02.jpg" /> 
Figure 2: Comparison of two different chamber slide options for measurement of coral sperm concentration. (A) Comparison of total sperm concentration measurements in Acropora millepora (n = 3 individuals) using the Makler counting chamber and commercially available fixed coverslip chamber slides. Columns show mean &#177; SEM.; asterisks indicate a significant difference (P = 0.002). (B) Validation of the Makler counting chamber using commercially available latex microbeads at a known concentration and standard CASA settings for coral sperm. <a href="https://www.jove.com/files/ftp_upload/66233/66233fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66233/66233fig03.jpg" /> 
Figure 3: Post-thaw concentrations of total, motile, or progressively motile sperm in cryopreserved samples. Post-thaw comparisons of total, motile, and progressively motile sperm concentrations using either 20% or 30% DMSO in FSW to achieve the final 10% DMSO concentration for cryopreservation. Samples from n = 3 individuals were split into 2 aliquots each, with one aliquot cryopreserved with a 1:1 addition of 20% DMSO and the second aliquot diluted to &#60;2 &#215; 109/mL using FSW for cryopreservation with a 1:2 addition (sperm: cryodiluent) of 30% DMSO. Columns show mean &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/66233/66233fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Coral biobanking auto-datasheet file. <a href="https://www.jove.com/files/ftp_upload/66233/Coral biobanking auto datasheets_TEMPLATE.zip" target="_blank">Please click here to download this File.</a>

Author Spotlight: Developing Efficient Cryopreservation and Biobanking Technologies for Global Reef Restoration

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.

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

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 ...

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642 Views.  Taronga Conservation Society Australia. 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.

Video: Author Spotlight: Developing Efficient Cryopreservation and Biobanking Technologies for Global Reef Restoration

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid cooling in an &#34;open&#34; system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable &#181;S-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-&#181;m ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (&#181;S-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified &#181;S-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the &#181;S-VTF procedure has not affected its successful application, as high survival rates (&#62; 95%) and pregnancy rates continue today.

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid ...

Developmental Biology

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

Environment

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

Metabolite Profiling of Hard Coral Samples Using GC-MS

Gas Chromatography-Mass Spectrometry-Based Targeted Metabolomics of Hard Coral Samples

Gas chromatography-mass spectrometry (GC-MS)-based approaches have proven to be powerful for elucidating the metabolic basis of the cnidarian-dinoflagellate symbiosis and how coral responds to stress (i.e., during temperature-induced bleaching). Steady-state metabolite profiling of the coral holobiont, which comprises the cnidarian host and its associated microbes (Symbiodiniaceae and other protists, bacteria, archaea, fungi, and viruses), has been successfully applied under ambient and stress conditions to characterize the holistic metabolic status of the coral.
However, to answer questions surrounding the symbiotic interactions, it is necessary to analyze the metabolite profiles of the coral host and its algal symbionts independently, which can only be achieved by physical separation and isolation of the tissues, followed by independent extraction and analysis. While the application of metabolomics is relatively new to the coral field, the sustained efforts of research groups have resulted in the development of robust methods for analyzing metabolites in corals, including the separation of the coral host tissue and algal symbionts.
This paper presents a step-by-step guide for holobiont separation and the extraction of metabolites for GC-MS analysis, including key optimization steps for consideration. We demonstrate how, once analyzed independently, the combined metabolite profile of the two fractions (coral and Symbiodiniaceae) is similar to the profile of the whole (holobiont), but by separating the tissues, we can also obtain key information about the metabolism of and interactions between the two partners that cannot be obtained from the whole alone.

Author Spotlight: Separation of Coral Host Tissues and Algal Symbionts and Analyzing Their Metabolites 

Here, we present the extraction and preparation of polar and semi-polar metabolites from a coral holobiont, as well as separated coral host tissue and Symbiodiniaceae cell fractions, for gas chromatography-mass spectrometry analysis.

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

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Chapters in this video

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

Semi-automated Optical Heartbeat Analysis of Small Hearts

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Fish Sperm Assessment Using Software and Cooling Devices

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Gas Chromatography-Mass Spectrometry-Based Targeted Metabolomics of Hard Coral Samples