Embryo and Sperm Cryopreservation
To see background, more information is available here.
Resources and recommendations
Embryo cryopreservation: For strain cryopreservation, 10-12 homozygous or heterozygous stud males will be mated with superovulated wildtype females. Approximately 250-300 eight cell stage embryos will be collected and frozen. Roughly 20-30 embryos will be stored per straw, which can then be stored in liquid nitrogen in the Core’s freezers. Freezing of 8-cell embryos is the most stable stage for embryo cryopreservation and storage.
Sperm cryopreservation: This is the fastest and least expensive option for cryopreservation of mouse strains. Frozen sperm can be stored in LN2 indefinitely, and reconstitution of live mice can generally be achieved using IVF. Following collection of epididymal sperm, 10 samples will be frozen and stored in liquid nitrogen and kept in two sperate locations.
IVF embryo cryopreservation (Speed Cryo): The freshly collected sperm from 3-4 transgenic males is used to fertilize the eggs of superovulated females of the same strain background via in vitro fertilization and then cryopreservation of 2 cell or 6-8 cell stage embryos is done. Simultaneously we also freeze 10 samples of sperm from these males. Thus, your strain will be cryopreserved and stored as both embryos and as well as sperm.
Despite the existence of several approaches for cryopreservation we recommend to employ Speed Cryo for C57BL/6 as well as for other mouse lines.
Quality control: Each cryopreserved strain can be checked to ensure overall the overall quality and viability of embryos or sperm using one of two methods:
- Following standard or Speed Cryo freezing, one straw will be thawed, and the embryos will be placed in culture for development to blastocysts where a >75% development to blastocysts will be considered a successful cryopreservation.
- Following standard or Speed Cryo freezing, one straw will be thawed, and the embryos will be placed in culture for development to blastocysts followed by a uterine transfer into a pseudopregnant recipient. The generation of live pups displaying the appropriate genotype (genotyping to be done by the customer) will be considered a successful cryopreservation.
- After sperm cryopreservation one straw will be thawed and a quality of the spermatozoa will be visually evaluated under a microscope.
Long-term storage of germplasm: The frozen embryos and sperm are stored in liquid nitrogen in two freezers located at two geographically distinct sites: one located on the OHSU Main Campus LBRB build and the other at OHSU Main Campus RJH building.
Responsible parties and timeline for embryo cryopreservation
| Step | Timeframe | Protocol | Responsible party |
|---|---|---|---|
| 1 | Week 1 | Provision of mutant stud males | Customer |
| 2 | Week 2 | Embryo cryopreservation | TMM |
| 3 | Week 3 | Thawing of embryos and quality control via transfer to foster mom | TMM |
| 4 | Week 6 | Transfer newborn offspring to customer | TMM |
| 5 | Variable | PCR genotyping of newborn offspring | Customer |
Service ordering
To initiate a freezing project, please fill a Project Request Form. In addition please provide the following materials:
- For standard cryopreservation of 8-cell stage embryos provide 10-12 stud males at the age of 2-9 months.
- For Speed Cryo provide 3-4 stud males at the age of 2-9 months.
- For Sperm Cryopreservation provide 3-4 stud males at the age of 3-9 months.
Standard embryo cryopreservation service fees include
- Purchase of 3 week old C57BL6/NJ or B6xDBA-2F1 females (donors of oocytes).
- Cryopreservation of 250-300 eight -cell embryos.
- Cryostorage of frozen embryos for 1 month.
Speed cryo cryopreservation service fees include
- Purchase of virgin C57BL6/J or B6xDBA-2F1 females (donors of oocytes).
- Cryopreservation of 250-300 eight cell embryos.
- Cryopreservation of 10 samples of sperm.
- Cryostorage of frozen embryos and sperm for 1 month.
Sperm cryopreservation service fees include
- Cryopreservation of 10 samples of sperm.
- Cryostorage of frozen sperm 1 month.
Service fees do not include
- Control of frozen germplasm. This is very important, therefore we recommend to order, and pay the small additional fee.
- Test thaw of control embryos.
- Test thaw Speed Cryo frozen embryos.
- Test thaws of frozen sperm and IVF.
- Transfer of embryos into pseudopregnant recipient females.
- Long term storage of embryos and sperm.
- Shipping charges.
Additional service
According to customers’ request, TMM can purchase and use other mouse lines for the cryopreservation of genetically modified mice. These additional services will be charged further fees, including the extra costs of the cage(s) per diem.