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.
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.
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 −20 °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 >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.
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
2. Sperm preparation and assessment
3. Sperm dilution and cryoprotectant equilibration
Sperm concentration | Cryodiluent | Dilution ratio (sperm:cryodiluent) | Final DMSO concentration in sperm sample (%) |
< 2 × 109/mL | 30% DMSO in FSW | 2:1 | 10% |
≥ 2 × 109/mL | 20% DMSO in FSW | 1:1 | 10% |
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
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 µL sample volume and the standard CASA settings17 used for coral sperm. These analyses showed consistent counts with low variability (44.5 ± 0.7 million/mL) that fell within the expected concentration range (46.0 ± 7.0 million/mL) 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).
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 −20 °C/min to −80 °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 lab-based high-capacity cryopreservation station set up to run multiple cryo racks at the Australian Institute of Marine Science National Sea Simulator. Please click here to view a larger version of this figure.
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 ± 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. Please click here to view a larger version of this figure.
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 <2 × 109/mL using FSW for cryopreservation with a 1:2 addition (sperm: cryodiluent) of 30% DMSO. Columns show mean ± SEM. Please click here to view a larger version of this figure.
Supplementary File 1: Coral biobanking auto-datasheet file. Please click here to download this File.
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 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 × 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 >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 × 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.
The authors have nothing to disclose.
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'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.
Ovation ALI-Q 2 VS Pipette Controller – Aliquotting pipette | Vistalab | 2100-1005 | Fluid handling – measuring sperm volume, addition of cryoprotectant solution, aliquoting samples into cryovials |
5 mL serological pipettes (bulk) | Thermo Scientific | Nunc 170355 | Fluid handling – measuring sperm volume, addition of cryoprotectant solution, aliquoting samples into cryovials |
10 mL serological pipettes (bulk) | Thermo Scientific | Nunc 170356 | Fluid handling – measuring sperm volume, addition of cryoprotectant solution, aliquoting samples into cryovials |
P2 0.2–2 µL pipettor | Gilson | F144054M | Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment |
P10 1–10 µL pipettor | Gilson | F144055M | Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment |
P20 2–20 µL pipettor | Gilson | F144056M | Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment |
P200 20–200 µL pipettor | Gilson | F144058M | Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment |
P1000 100–1000 µL pipettor | Gilson | F144059M | Preparation of sperm activation solutions and sperm sample handling for concentration and motiliy assessment |
Vacuum pump | Millipore | WP6122050 | Preparation of filtered sea water for solution preparation |
Reusable bottle-top filtration system | Thermo Scientific | DS0320-5045 | Preparation of filtered sea water for solution preparation |
0.22-µm filter discs, mixed cellulose esters | Merck Millipore | GSWP04700 | Preparation of filtered sea water for solution preparation |
Filtered sea water | N/A | – | Base medium for sperm activation and cryoprotectant solutions |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D4540 | Cryoprotectant chemical used at a final concentration of 10% v/v in filtered seawater for sperm cryopreservation |
Caffeine | Sigma-Aldrich | C0750 | Used to activate sperm motility |
BSA heat shock fraction | Sigma-Aldrich | A9647 | Used to minimise sperm adherance to CASA well slides |
15-mL tubes – racked | Thermo Scientific | 339651 | Preparation of sperm activation solution |
50-mL tubes racked | Thermo Scientific | 339653 | For collection of gamete bundles and filtered sperm samples |
Transfer pipettes | Thermo Scientific | Samco 202PK | To aid collection of gamete bundles from the water surface |
100-µm filter baskets | Fisher Scientific | 22363549 | To exclude eggs during separation of the sperm sample |
Eppendorf racks | Interpath | 511029 | Dilution and activation of sperm for concentration and motiliy assessment |
Eppendorf 1.5-mL tube | Eppendorf | 30120086 | Dilution and activation of sperm for concentration and motiliy assessment |
Glass coverslips 18×18 mm | Brand | 4700 45 | Assessment of sperm concentration and motility using phase microscopy |
Plain glass slides, precleaned, 75×25 mm | Corning | 2947 | Assessment of sperm concentration and motility using phase microscopy |
Haemocytometer | Hausser Scientific | 1492 | Assessment of sperm concentration and motility using phase microscopy |
CASA slides (Leja 20-µm 4 chamber, SC-20-01-04-B) | IMV Technologies | 025107 | Assessment of sperm concentration and motility using Computer Assisted Sperm Analysis (CASA) |
Makler sperm counting chamber (CASA) | IVFStore | SM-373 | Assessment of sperm concentration and motility using Computer Assisted Sperm Analysis (CASA) |
accu-bead® counting beads | Hamilton-Thorne | 710111 | Assessment of sperm concentration and motility using Computer Assisted Sperm Analysis (CASA) |
CASA system + laptop | Hamilton Thorne | Ceros II | Assessment of sperm concentration and motility using Computer Assisted Sperm Analysis (CASA) |
Safety Glasses | Generic | – | Personal protective equi[pment for use when handling DMSO and liquid nitrogen |
Lab coat | Long sleeve, full length | – | Personal protective equi[pment for use when handling DMSO and liquid nitrogen |
Cryogloves (pair) | Tempshield | Mid-Arm | Personal protective equi[pment for use when handling DMSO and liquid nitrogen |
Medium forceps | Generic | – | For removing cryopreserved samples from the cryo racks and manipulating samples in liquid nitrogen |
Barcode scanner (2D compatible) | Zebra | DS2278 | For reading 1D and 2D barcodes on cryovials for sample management |
2.0-mL CryoStorage Vial, external thread, pre-capped, 2D SafeCode (DataMatrix/ECC200), linear and human readable | Eppendorf | 30079434 | Barcoded cryovials for cryopreservation of sperm samples |
Cryovial rack | Simport | T315 | Rack to hold cryovials, with locking base to allow for one hand de-capping and capping |
Freezing racks | Custom | – | 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) |
Freezing rack lid | Custom | – | 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) |
Freezing Thermos – 1.5 Litre 18/8 Stainless Steel Double-Wall Vacuum Food Container | Isosteel | VA-9683 | 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) |
Lab timers | Generic | – | For timing of cryoprotectant equilibration prior to cryopreservation |
Nitrogen bath 9L | BelArt | M16807-9104 | For quenching samples during cryopreservaton and holding samples during sorting and handling |
Thermocouple data logger- multichannel | Omega | HH520 | Temperature monitoring during cryopreservation to determine freezing rate and end point |
Thermocouple probe – Type K | Omega | 5SC-TT-K-30-36 | Temperature monitoring during cryopreservation to determine freezing rate and end point |
Cryo pens/coloured permanent pen | Staedtler Lumocolor | 318 | Optional for marking cryovial lids to assist with sample management |
Dry Shipper – charged | Taylor Wharton | CXR100, or CX500 | For transfer of cryopreserved samples from field/collection sites to the biorepository for storage |