Basic Steps of Embryo Cryopreservation and Rederivation
- Collecting Embryos
- Freezing Embryos
- Long-Term Storage
- Embryo Transfer Surgery
- Quality Assurance
- Cost Projections
- Forms
- Animal Protocol Issues
Collecting Embryos
The investigator shall supply the embryo donor mice. A minimum of twenty donor females and ten donor males is desired for cryopreservation and half that number for embryo transfer rederivation. Ideally, the strains should be bred to homozygosity before harvesting embryos. Accommodations can be made if this quantity or genotype of donor mice is not available, please contact Dr. Donna M. Bouley () for advice.
Donor females will be given two intraperitoneal injections of exogenous gonadotropins 48 hours apart to induce follicular development and to control the expected time of ovulation. These mice will be mated (1F:1M) immediately after the second injection. Reproductive tissues (oviducts, uterus) will be harvested from female mice the following morning or on the third day after mating to collect preimplantation stage embryos for rederivation and cryopreservation, respectively. Optimal response to this superovulation protocol is achieved in prepubescent (4 weeks of age) mice of common inbred strains (e.g. C57BL/6, 129); however, some strains of mice respond poorly (e.g. BALB/c) or not at all (e.g. A, AKR) to this exogenous gonadotropin treatment. Moreover, prepubescent mice may be in limited supply necessitating the use of older females that generally ovulate fewer oocytes in response to hormone priming. To determine the requirements for your particular strain, please contact Dr. Donna M. Bouley () for advice.
Freezing Embryos
Preimplantation stage embryos are frozen in 0.25 mL plastic straws according to a two-step equilibrium freezing method (Theriogenology 21:767-90, 1984). Ten to twenty embryos are frozen per straw. Using this method Dr. Bouley successfully frozen and thawed embryos from >50 strains.
Long-Term Storage
Cryopreserved embryos can be stored long-term in liquid nitrogen (-196ºF) without any detrimental effects. Upon completion of a cryopreservation job the frozen embryos will be returned to the PI for long-term storage. If the PI cannot provide for this long-term storage, then we will maintain a frozen embryo bank for a minimal fee. Ideally, the stock of frozen embryos should be split between at least two storage vessels. This redundancy will minimize the potential for catastrophic loss.
Embryo Transfer Surgery
Zona pellucida-intact embryos that are free of adherent materials are transferred through a minimum of ten successive washes in sterile antibiotic-containing culture medium with each washing step achieving at least a 1:100 dilution (Manual of the International Embryo Transfer Society, 3rd Edition). This is done to prevent the transmission of pathogens between donor and recipient mouse. These washed embryos are surgically implanted in the oviduct of the foster mouse.
Quality Assurance
We will determine the survival rate of cryopreserved embryos from each batch by thawing a small percentage (5-10%) of the frozen embryos and culturing them to the blastocyst stage. This survival rate determination will provide valuable quality control data that is required to provide assurance that a sufficient number of embryos have been cryopreserved and that mice can be reconstituted from this frozen embryo stock. Generally, our survival rates have been >80%.
We will collect serum from the foster mouse at the time of weaning to verify the absence of mouse pathogen contamination by serology. In other words, we will use the foster mouse as a contact sentinel to determine the health status of the rederived litter.
Cost Projections
These services depend heavily on: 1) the reproductive fecundity of the embryo donor strain, 2) the response of the female mouse to superovulation treatments, 3) the number of donor mice available and their genotype, the embryo cryopreservation survival rate, and 4) the embryo transfer success rate. See the VSC Rates page for a complete list of rates.
Cryopreservation: The cost of the embryo cryopreservation service is based on collecting and freezing preimplantation stage embryos from up to 20 donor females. Typically up to 10 embryo donors are processed per batch per week. Therefore, a minimum of two weeks is required to complete a job. We will also thaw a few embryos to determine the cryopreservation survival rate. However, surgically implanting thawed embryos into a foster mice to produce live mice is not included in the cost of this service. If the 20 donor females do not produce a sufficient number of embryos, then more embryos many need to be collected on a pro-rated basis. Moreover, depending on the embryo genotype and the cryopreservation survival rate additional embryos may need to be frozen to ensure that a sufficient number of mice of the appropriate genotype can be reconstituted.
The following are examples of cost projections using federal rates for cryopreserving embryos from homozygous donor mice with a good response to superovulation treatments (e.g. C57BL/Ka), and homozygous donor mice with a poor response to superovulation treatments (e.g. C.B-17 SCID).
| C57BL/Ka | C.B-17 SCID | |
| # Embryos produced per donor | 18 | 5 |
| # Donor Females | 20 | 40 |
| # Donor Males | 10 | 10 |
| # Embryos Collected (total) | 360 | 240 |
| Per-Diem Charges (12 cages x 14 or 28 days) | $85.51 | $171.02 |
| Cost of Service (Cryopreservation) | $1,987.00 | $1,987.00 |
| Total Charges | $2,072.51 | $2,158.02 |
Embryo Transfer Rederivation: The cost of the embryo transfer rederivation service is $1,332.45 (federal rate) to collect, aseptically wash, and surgically implant embryos from up to5 donor mice. In addition, the investigator will be billed for each pseudopregnant recipient mouse (~$30), and for the per-diem charges to house these mice. The following is an estimated cost projection using federal rates for a typical embryo transfer rederivation service.
| Typical Cost | |
| Foster Mouse (1) | $30.00 |
| Per-Diem Charges (1 cage x 60 days) | $30.54 |
| Cost of Service (Embryo Transfer) | $1,332.45 |
| Total Charges | $1,392.99 |
Forms
Please submit a Cryopreservation Request Form (also found on the "VSC Forms" page of this web site) for each mouse strain that you want to cryopreserve.
Animal Protocol Issues
The PI must have an approved animal protocol to use mice for experimental purposes. If you do not, then you must submit a protocol to the Administrative Panel on Laboratory Animal Care (A-PLAC).
