Embryo & Sperm Cryopreservation and Revival Services
Introduction
Sperm or pre-implantation stage embryos can be isolated from mice and treated with a cryoprotective agent that allows the germplasm or pre-implantation embryos to be stored indefinitely at ultra-low temperature (i.e. in liquid nitrogen or its vapor phase). Using appropriate thawing techniques, germplasm or pre-implantation embryos stored in this manner can be used to regenerate live mice.
Embryos can be cryopreserved at all stages of pre-implantation development. The most robust method of embryo cryopreservation uses 8-cell stage embryos that are infused with a cryoprotectant, slowly lowered to –35C, and then rapidly submerged in, and stored in, liquid nitrogen (-196C). Sperm cryopreservation involves harvesting the cauda epididymis from euthanized males, releasing the sperm into a cryoprotective solution, loading the sperm suspension into semen straws, incubating the straws in the vapor phase of liquid nitrogen, plunging them into the liquid phase, and storing them in liquid nitrogen.
Historically, the revival of mice from cryopreserved sperm has suffered from large strain-to-strain variations in efficiency. Sperm from popular strains such as C57BL/6J produced very low yields of fertilized embryos after thawing and performing in vitro fertilization (IVF). Improvements from Kumamoto University (Takeo and Nakagata, Biology of Reproduction 85, 1066 (2011)) have been shown to make sperm cryopreservation as reliable as embryo cryopreservation (although the latter may still be necessary for some strains).
The cryopreservation of mouse strains serves two main functions:
- When mouse strains are not immediately needed for experimental purposes, but may be needed at a later date and cannot be readily obtained from vendors, it may be more economical to store the strain as frozen embryos or sperm, rather than maintaining a live colony.
- Mouse strains that are difficult to replace should be cryopreserved to allow reconstitution in the event of a pathogen outbreak, natural disaster, or other occurrence that may result in the loss of all existing breeding stock.
Comparison of Embryo and Sperm Cryopreservation
A complete cycle of cryopreserving a strain and reconstituting it from frozen material involves the same biological steps whether one uses embryos or sperm. In both cases, the production of fertilized embryos is the most expensive step. With embryo cryo, the fertilization step takes place before freezing, whereas with sperm cryo it is done after thawing. Cryopreservation is generally used as insurance against disaster, and reconstitution is a relatively uncommon event. Therefore, the overall costs of sperm cryo are likely to be significantly lower than for embryo cryo.
| Biological material being frozen: | Embryos | Sperm |
|---|---|---|
| Number of mice typically needed for freezing: | ~30 (15 females and 15 males - the males can be re-used) | 2 males |
| Number of sessions needed per strain: | 2 or more, on average | Usually 1 |
| Cost to freeze: | Higher | Lower |
| Cost to reconstitute live mice: | Lower | Higher |
| Time needed for freezing: | 1-2 weeks | 1 day |
| Time needed for reconstitution: | 6-7 weeks | 6-7 weeks |
| Potential number of offspring from a typical cryopreserved strain: | dozens | hundreds |
Sperm cryopreservation has three significant biological disadvantages compared to embryo cryopreservation:
- Since a haploid genome is being frozen, more breeding of reconstituted mice may be necessary if more than one mutation and/or transgene is involved, or if homozygosity is needed.
- Some mutations must be maintained on a mixed or otherwise unusual background to maintain the desired phenotype, and oocyte donors with that background may not be available at the time of reconstitution.
- As mitochondria are inherited exclusively from the maternal lineage, cryopreservation of sperm cannot be used to preserve strains of mice with mtDNA polymorphisms.
Also, despite recent improvements in the efficiency of reconstitution from frozen sperm, a very small percentage of strains are still extremely difficult to revive from frozen sperm. Thus, in some cases, embryo cryopreservation may still be necessary.
Ordering
To order services involving embryo or sperm cryopreservation or thawing, please request a Service Request Form from the TMF. Signed forms should be returned to the TMF via e-mail. In addition to the Service Request Form, we must also receive a completed copy of our Strain Data Sheet for each strain. In particular, we need to know the precise formal strain name before we can schedule the cryopreservation. A complete list of rules for naming mouse strains and alleles is available on the Mouse Genome Informatics website.
Materials Required from the Investigator
Embryo Cryopreservation
For embryo cryopreservation the client must provide the embryo donors and stud males. We recommend providing at least 15 donors per strain, depending on how many embryos the client wishes to freeze and assuming an average response to superovulation and normal fertility.
If sufficient stud males are available, it may be to the client’s advantage to order wildtype females of the same background for use as embryo donors. This is a convenient way of ensuring all females are of the optimum age for superovulation. Superovulation is generally most efficient using females that are either 3-4 weeks of age, or 8-10 weeks of age. Females older than 10 weeks can be used, but the average embryo yield can diminish quickly with age.
The maximum number of embryo donors we can process per session is 30. Each donor should be mated with a separate male. A flat fee is charged for each cryopreservation session, regardless of how many donors are supplied. Donors from more than one strain can be processed in a single session.
Normally, the embryo donors will be superovulated and mated by the client, using hormones supplied by the TMF, and the mice will remain in the client's mouse room until the females are euthanized. If TMF staff must perform the hormone injections, we prefer to have someone from the client’s lab present during the superovulation to help identify the mice.
Sperm cryopreservation
For sperm cryo, two fertile males are generally sufficient to cryopreserve a strain. We recommend that the client supply proven breeders between 3 and 8 months of age.
Turnaround Times
The TMF's ability to schedule a cryopreservation job will, of course, depend on prior work committments. Embryo cryopreservation usually requires about a week, once the embryo donors and stud males are designated by the client, and a freeze date has been scheduled. Sperm cryo usually requires one day. In vitro fertilization requires a minimum of 2 days.
Reconstituting live mice from frozen embryos or sperm requires about 6-7 weeks from the day of thawing to produce weaned pups ready for transfer to the client. If the frozen embryos or sperm are from an outside institution, the foster mothers will have to be tested for pathogens before the weaned pups can be released, adding 1-2 weeks to the turnaround time.
Performance Guarantees
The number of embryos that are cryopreserved depends on several factors, including the number of embryo donors supplied by the client, their age and response to superovulation, and the fertility of the stud males. Due to inherent variation, no guarantee can be provided as to how many embryos will be frozen in any given session.
Generally about 40% of thawed embryos develop into live pups, but there can be large strain-to-strain variations in embryo survival and developmental potential.
If enough embryos are obtained, some of them (usually ~20) will be thawed shortly after freezing and transferred to foster mothers to demonstrate the ability to recover live mice.
Sperm cryo yields 18 straws of frozen sperm from one male, with each straw holding enough sperm to fertilize the oocytes from up to 12 females. 16 straws are reserved for client use with two straws being reserved for TMF use in QC. Yields from in vitro fertilization (IVF) with thawed sperm can exhibit large variations from strain to strain. One straw of frozen sperm from each cryopreservation will be used to perform IVF to demonstrate the ability to produce fertilized embryos. If at least 20% of the oocytes are fertilized and develop into embryos, we will consider the strain to be successfully frozen. If fewer than 20% of the oocytes are fertilized and develop into embryos, we will transfer the embryos to foster mothers and, if live pups of the desired genotype are produced, we will consider the strain to be successfully frozen. If neither condition is met, we will request 2 more males from the client and repeat the entire process at no additional charge. For biosecurity, frozen sperm or embryos are routinely split into two batches with each batch being stored in one of two liquid nitrogen freezers in different buildings on campus.
Service Description
Our embryo cryopreservation service includes:
- Superovulation or hyperovulating and mating embryo donors
- Harvesting fertilized embryos from donors
- Freezing embryos
- Thawing some embryos and implanting them into foster moms to demonstrate recovery of live mice, if enough embryos are available
- Long-term storage in liquid nitrogen
Reconstitution of live mice from frozen embryos includes:
- Generating pseudopregnant foster moms
- Transferring thawed embryos to foster moms
- Monitoring pregnancies
- Weaning pups
Our sperm cryopreservation service includes:
- Harvesting and freezing sperm from 2 males per strain
- Thawing one straw of sperm and performing IVF to demonstrate the ability to produce fertilized embryos
- Long-term storage in liquid nitrogen
Reconstitution of live mice from frozen sperm includes:
- Performing IVF with oocyte donors of the appropriate strain
- Generating pseudopregnant foster moms
- Transferring fertilized embryos to foster moms
- Monitoring pregnancies
- Weaning pups