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.
