Embryo Cryopreservation

Speed cryogenics is the most common method used for embryo cryopreservation projects as hundreds of embryos can be generated by IVF in a single day.

Mice Needed

• Two males between eight weeks to eight months of age will be used as sperm donors initially.  Additional males may be needed if it is necessary to repeat the IVF.
• Homozygous, hetero/hemizygous males, or both can be used as sperm donors.
• They should be singly housed for two weeks prior to the procedure for optimal sperm production.  TTML staff will contact you in approximately two weeks prior to beginning your project to confirm the schedule date and have you separate a male.
• Wild-type females are purchased and used as a source of oocytes.

IVF and Cryopreservation Procedure

• On the day of IVF, sperm is collected from two males, pooled and immediately incubated with freshly isolated oocytes.
• After a five-hour incubation period, embryos are collected from the IVF droplet, washed through fresh media and cultured overnight.
• The next day, healthy two-cell stage zygotes are collected, loaded into straws and frozen at a rate of -0.5°C/minute to -30°C, then rapidly frozen in liquid nitrogen.

Expected Results

A minimum of 150-200 embryos are frozen for each line for strains with normal reproductive performance.

• If fewer than 150 embryos are obtained in the first two IVF sessions, a third and final session will be performed, and the project will be completed regardless of the number of embryos frozen.
• A test straw of cryopreserved embryos from each freeze is thawed to determine the success of the procedure. Thawed embryos are assessed for viability and the ability to continue to develop in vitro.

The genotype of the frozen embryos will depend on the genotype of the original males used for IVF.

• If homozygous males were used as sperm donors, the genotype of all the cryopreserved embryos will be hetero/hemizygous
• If hetero/hemizygous males were used as sperm donors, the embryos will be a mix of both hetero/hemizygous and wild-type.

To cryopreserve homozygous embryos, it is essential to used both homozygous females and homozygous males to generate the embryos.

Due to our current staffing and animal room health status, the only way at this time to freeze homozygous embryos through the TTML requires that your lab maintains the single housed males and hormonally treats/mates the females. The TTML provide the hormones and information on preparation, dosage and timing. Working together, we have successfully frozen homozygous embryos for several investigators to date.

• Your lab will be responsible for maintaining 6-10 singly housed homozygous males for the duration of the project.
• Your lab will be responsible for hormonally treating, mating and plug checking homozygous females. The TTML will provide the hormones and information on the preparation, dosage and timing of hormones.
• Your lab will be responsible for delivering the 0.5 dpc mated females to our lab.
• We will isolate, culture and cryopreserve all healthy two-cell stage embryos.
• Test thaws are performed for each freeze.
• The process will be repeated until your lab feels enough embryos have been successfully frozen.

Test straws from each freeze are thawed and cultured in vitro to the blastocyst stage to assess viability and developmental capacity.

If the test thaw is inconclusive, a straw will be thawed and transferred into recipient female to confirm embryo viability.

Depending on these results and the number of embryos frozen, we will contact you to either schedule another session or confirm that the project is complete.

Cryopreserved embryos from each line are stored in two separate liquid nitrogen dewars in two different buildings.

There is a modist annual fee for each line maintained in our dewars.

Embryos can be cryorecovered at any time simply by contacting our lab.

Embryos can also be shipped worldwide from our lab.