HUSBANDRY · CONTROLLED DOCUMENT
AE-SOP-0230 — Cryo-Banking & Gamete Preservation
To preserve gametes and embryos in the cryo-bank as a genetic insurance line against the loss of wild and captive populations, so that diversity banked today can rebuild a lineage decades from now. The procedure guarantees that every deposit is screened, controlled-rate frozen, and split across independent dewars, that the bank's holdings and genetic coverage are catalogued against the registry, and that no sample is ever the only copy — because a single dewar failure must never erase a lineage.
ActiveHusbandryRev B
Doc Control AE-SOP-0230Revision BEffective 2090-04-28Next Review 2091-04-28Owner Breeding Program LeadApprover Marine ScienceStatus ActiveClass RESTRICTED // TIDEWATER-EYES-ONLY
1. Purpose
To preserve gametes and embryos in the cryo-bank as a genetic insurance line against the loss of wild and captive populations, so that diversity banked today can rebuild a lineage decades from now. The procedure guarantees that every deposit is screened, controlled-rate frozen, and split across independent dewars, that the bank's holdings and genetic coverage are catalogued against the registry, and that no sample is ever the only copy — because a single dewar failure must never erase a lineage.
2. Scope
All cryo-preservation of gametes and embryos in the genetic bank.
- In scope: Gamete collection, screening, controlled-rate freezing, split-dewar storage, inventory, and thaw-viability verification.
- Out of scope: Live larval rearing of thawed and fertilised material, which follows AE-SOP-0102; registry cataloguing of banked lineages, which follows AE-SOP-0220.
3. Definitions
| Term | Definition |
|---|
| Cryoprotectant | The medium that protects cells from ice damage during controlled-rate freezing. |
| Controlled-Rate Freezing | The programmed cooling curve that vitrifies a deposit without lethal ice crystal formation. |
| Split-Dewar Storage | Dividing every deposit across at least two independent dewars so no single failure loses a lineage. |
| Thaw Viability | The verified post-thaw survival fraction confirming a banked deposit remains usable. |
| Genetic Coverage | The breadth of distinct lineages held, catalogued against the broodstock diversity record. |
4. Responsibilities
| Role | Responsibility |
|---|
| Breeding Program Lead (Owner) | Owns the bank; approves deposits and signs any withdrawal for fertilisation. |
| Cryo Technician | Collects, screens, freezes, and splits deposits; maintains dewar inventory and levels. |
| Marine Science | Sets cryoprotectant and freezing protocol per species and confirms lineage identity. |
| NEREID | Tracks dewar fill levels and inventory and flags any lineage held in only one dewar or any dewar trending low. |
5. Cryo-Preservation Parameters
| Deposit Type | Cool Rate | Storage | Split / Viability |
|---|
| Sperm / milt | −1 °C/min to seed, then plunge | ≤ −196 °C vapour-phase LN₂ | ≥ 2 dewars, viability ≥ 50 % |
| Oocytes | Programmed vitrification curve | ≤ −196 °C liquid-phase LN₂ | ≥ 2 dewars, viability ≥ 40 % |
| Embryos / larvae | Species-specific curve | ≤ −196 °C vapour-phase LN₂ | ≥ 2 dewars, viability ≥ 35 % |
| Larval tissue (genetic) | Snap freeze | ≤ −150 °C ultra-low freezer | ≥ 2 locations |
Note. No deposit is ever held in a single dewar; a lineage flagged as single-copy is split before any other work proceeds, and a deposit below its thaw-viability floor is re-collected, never relied upon.
6. Materials & Equipment
- Controlled-rate freezing unit with programmable cooling curves
- Species cryoprotectant media
- Liquid-nitrogen dewars with level monitoring, ≥ 2 independent banks
- Health-screen panel for pre-freeze deposits
- Thaw-viability assessment station
- Cryo-inventory catalogue linked to the registry
7. Procedure
7.1 Collection & Screen
- Collect gametes or embryos and confirm lineage identity.
- Health-screen each deposit before freezing; never bank a positive screen.
- Load into cryoprotectant per the species protocol.
7.2 Freeze & Split
- Run the controlled-rate freezing curve for the deposit type.
- Split every deposit across at least two independent dewars.
- Catalogue the deposit and its lineage against the registry.
7.3 Inventory & Verify
- Monitor dewar fill levels and top up before any low-level flag.
- Verify thaw viability on a sample schedule and re-collect below the floor.
- Flag and split any lineage found held in only one dewar.
8. Records
- Cryo-deposit catalogue with lineage and location (retained permanently)
- Pre-freeze health-screen results
- Controlled-rate freezing curve logs
- Dewar level and inventory log
- Thaw-viability verification record
9. References
10. Revision History
| Rev | Date | Author | Summary |
|---|
| A | 2089-02-20 | Breeding Program Lead | Initial issue; controlled-rate freezing and split-dewar storage established. |
| B | 2090-04-28 | Breeding Program Lead | Added thaw-viability floors, single-copy flagging, and registry-linked catalogue. |