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

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's National Zoo & 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

1. 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.
2. Prepare coral sperm activation concentrated stock solutions for CASA.
   NOTE: Disposable gloves should be worn when handling chemicals for preparation of the activation solution.
   1. 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 °C or by holding the tube in cupped hands, with periodic gentle swirling (do not shake vial).
   2. Once in solution, make up to the final volume (5 mL) using tissue culture water, label the tube with the date, and mark "30% BSA in H₂O".
   3. Store in the refrigerator (4 °C) for up to 2 weeks.
   4. Prepare 60 mM caffeine stock solution (25 mL) by dissolving 0.2913 g of caffeine in 24 mL of filtered sea water (FSW).
   5. Once in solution, make up to the final volume (25 mL) using FSW, label the tube with the date and mark "60 mM caffeine in FSW".
   6. Store in the refrigerator for up to 1 month.
      NOTE: The maximum solubility of caffeine in FSW is approximately 71 mM.
3. Prepare sperm activation working solution for sperm samples at ~10⁹ sperm/mL (0.9% BSA + 12 mM caffeine in FSW).
   1. 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.
   2. Label the tube with date and "sperm activation working solution – 0.9% BSA + 12 mM caffeine".
   3. Prepare on each day of use, store at room temperature (19-30 °C), and discard the unused solution at the end of the day.
4. Prepare cryoprotectant solutions (cryodiluents). Ensure that disposable gloves are worn when handling the cryoprotectant DMSO.
   ​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.
   1. Collect raw seawater from the tank system in which corals are held prior to spawning (~35 ppt salinity).
   2. Filter raw seawater using a bottle-top filtration system attached to a vacuum pump with a 0.2 µm non-pyrogenic membrane filter fitted.
   3. For a sperm concentration ≥2 × 10⁹ sperm/mL, prepare 20% DMSO in FSW by combining 10 mL of DMSO + 40 mL of FSW in a 50 mL tube.
   4. For a sperm concentration <2 × 10⁹ sperm/mL, prepare 30% DMSO in FSW by combining 15 mL of DMSO + 35 mL of FSW in a 50 mL tube.
   5. Label the tube with the date and DMSO concentration, i.e., 30% DMSO or 20% DMSO.
   6. Prepare on each day of use, store at room temperature (19-30 °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 °C for preparation of the cryodiluent to counterbalance this heat generation.
5. Prepare thermocouple datalogger.
   1. 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.
   2. 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.
   3. 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.
   4. Prepare fresh 10% DMSO in FSW and refill thermocouple vials daily.

2. Sperm preparation and assessment

1. 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).
2. 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. Hands should be washed thoroughly with fresh water, or gloves changed between each gamete sample to avoid cross-contamination.
3. 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.
4. 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).
5. 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.
6. Transfer the sperm sample to a 100 µm 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.
7. Label the filtered sperm sample with colony ID and move the sample to the sperm assessment workstation.
8. Open the coral biobanking auto-datasheet file (Supplementary File 1). In the sperm assessment tab, input the coral colony metadata (columns A-H).
9. Prepare sperm for CASA assessment.
   1. Mix the sperm sample well and remove a 10 µL aliquot to an empty 1.8 mL microcentrifuge tube.
   2. 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 × 10⁶/mL) if raw sperm concentration is ~2 × 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 × 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 µL aliquot of sperm sample added directly to it, then mixed well by inverting the tube 10× and flicking the tube lid prior to opening.
10. Assess sperm motility and concentration of activated sample using computer-assisted sperm analysis (CASA) as described in Zuchowicz et al.¹⁷ by placing 4 µL 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.
11. 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.
12. 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.

3. Sperm dilution and cryoprotectant equilibration

1. 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.
2. 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.
3. Check the auto-calculated column I for the required cryoprotectant concentration and column H for the volume of cryodiluent to be added (Table 1).
4. 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. 
5. Record cryodiluent addition time in column P.

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

1. 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.
2. Uncap sterile barcoded cryovials and set the caps on a clean/sterile surface.
3. Optional: Write the run # on each cryovial using a permanent marker; this can aid in quick sample identification for accessioning into the biobank.
4. 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.
5. Place one thermocouple vial and all full sample vials onto an empty cryo rack at room temperature. Ensure that the barcoded surface is facing outwards on all vials.
6. 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.
7. Enter the run number into the auto-datasheet (column U) and scan the cryo rack serial number (QR code) into column V.
8. 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.
9. While scanning, cross-check that data is being entered into the correct cell and that all cryovials have been scanned once.
10. Set aside loaded and scanned racks for the remainder of the cryoprotectant equilibration period and prepare the cooling vessel for cryopreservation.
11. Fill the cryo rack cooling vessel with liquid nitrogen to the appropriate pre-determined level necessary for cooling at approximately −20 °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.
12. 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.
13. Record the start time in column Q.
14. Once the thermocouple vial reads −80 °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.
15. Remove the cryovials from the rack and transfer them to a dry shipper for transport to the biorepository.