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8-cell embryo suitable for cryopreservation

Introduction

The maintenance of mouse colonies can be expensive and time consuming. Embryo cryopreservation offers the researcher an alternative to maintenance breeding to preserve a mouse line that is not in current use. Embryo cryopreservation also provides a back-up to preserve the line in the unlikely event that the colony becomes pathogen compromised, has a fertility issue or background/genotype is compromised.

When deciding to cryopreserve a line, the client will need to choose if embryo or sperm freezing is best suited to the strain. Click here to view a table showing the pros and cons of freezing embryos vs freezing sperm.

Embryos will be stored in 2 separate cryopreservation vessels/sites.

The number of embryos stored is determined by the mating strategy used to produce the embryos for freezing.

  • Homozygous X Homozygous = 250 embryos- all re-derived progeny will be homozygous.
  • Homozygous X Het or Wild type = 250 embryos- re-derived progeny will have varying genotype, therefore will need to be screened for genotype.
  • Heterozygous X Heterozygous = 300 embryos- re-derived progeny will have varying genotype, therefore will need to be screened for genotype
  • Heterozygous X Wild type = 500 embryos- re-derived progeny will have varying genotype, therefore will need to be screened for genotype

All strains cryopreserved at TASQ remain the property of the Client and will not be distributed without the permission of the Client.

Embryo Production

Embryos for cryopreservation will be produced using hormone priming and IVF. This method on average produces a higher number of embryos per female than conventional superovulation and mating.

Mice Needs

The number of females required per strain to produce the predetermined number of embryos will vary between strains. There are many factors affecting the success of embryo production, including the age of the females, the strain background and fertility of the males. Therefore it is not possible to accurately predict the exact number of embryos that will be produced per female. 

Females must be between 8 and 16 weeks of age. At this age the females are more receptive to the super-ovulation regime, thereby producing more viable eggs. This will reduce the overall number of females required.  A minimum of 20 females will be required.

If the required number of embryos has not been reached after the initial 20 females are used, TASQ will discuss with the Client the number of additional females that may be needed to reach the required number of embryos.

Wild type females can be purchased on the Client’s behalf.

Males must be of proven fertility. A minimum of 3 males will be required. Stud males should be aged between 2 and 6 months for best results. Ex-breeders can also be used. If no proven males are available, fertility testing can be done by UQBR at an additional cost to the Client.

If these numbers are not available a different strategy may be required. Please contact TASQ for consultation.

Embryos are frozen by rapid cooling of the 4-8 cell embryo.

Costs

Internal $992.25 per 20 females used
External $1576.58 per 20 females used

This includes:

  • Superovulation of 20 female mice
  • IVF
  • Embryo culture
  • Embryo freezing
  • Test thaw
  • Tissue sampling of test thaw progeny
  • Administration costs
  • Ethics reporting

Additional Costs

  • Courier costs to UQ animal facility (if applicable).
  • Animal agistment of stud males and donor females during the cryopreservation service. Agistment is the weekly charge applied by the animal facilities for the basic care of the animals (contact TASQ for charges).
  • Any wild type females needed for super-ovulation and mating.

How to start

  • Contact TASQ (refer to Contacts page).
  • Health reports from the animal facility where the donor mice reside will need to be sent to TASQ (if applicable)
  • Download and complete a registration form (UQ Internal or External to UQ) and return to TASQ
  • Once health reports have been reviewed, TASQ will liaise with your Animal Facility Manager to have the mice shipped to the necessary UQ animal facility.
  • Embryo freezing will be scheduled, with TASQ notifying the Client of the starting date.

Additional Notes

  • TASQ will freeze all viable embryos collected. 
  • TASQ will schedule IVF runs until all females supplied have been used. TASQ will notify the Client of the viable embryo numbers frozen and await instructions depending on the number of embryos obtained. If females supplied run longer than a month TASQ will update the Client at the end of each calendar month with a summary file of all embryo freezing. If more females are required to reach the embryo numbers needed TASQ will request a further shipment of females.
  • TASQ will perform a test thaw of one straw. This will involve the thawing and culturing of the embryos to assess viability. Cultured embryos will then be transferred to a pseudopregnant female to assess the viability of live birth. TASQ can not guarantee that 100% of embryo transfers will result in live births. TASQ will notify the Client of the success of the test thaw. TASQ cannot guarantee all mouse strains’ embryos will tolerate and survive the freeze/thaw procedure.
  • Once the embryo numbers have been reached, the Client will be notified of the completion of freezing. At this time the Client will be asked if the donor and stud stocks held at the UQ animal facility can be euthanised. TASQ must have this confirmation in writing. Stocks must not be euthanised at the Client’s facility until discussion with TASQ regarding further needs for the embryo freezing program have taken place.
  • A TASQ registration form must be completed prior to service commencement.
  • A current AEC certificate and OGTR number must be supplied prior to commencement of service.
  • Download the “UQBR Cryopreservation and Rederivation Services Information and Protocol Booklet” which has additional information for Clients.

Cryopreservation of mouse embryos section

Cryopreservation Comparison Table - Embryo vs Sperm

The following table demonstrates basic differences between cryopreservation of sperm and embryos.

Embryo Freezing

Gamete (sperm & ovary)

Initial labour

Can take a few months to acquire all the embryos for storage.

Work is done in one day

Initial costs

As charges are dependant on the number of females used, costs will vary for each strain. Is more expensive than gamete freezing

Set cost.

Cheaper than embryo freezing

Numbers cryostored

Het X WT matings= 500

Het x Het matings=300

Homo X WT= 250

Homo X Homo= 250

Collect sperm from 3-5 males, so there are millions

Test thaw

Thaw assesses viability to live births.

Thaw assesses only motility. After freezing/thaw need an IVF session to determine live birth viability. More expensive than embryo freezing

Rederivation

Set cost for rederivation per round. Involves embryo thaw and culture, then an embryo transfer. Cheaper and less labour intensive

Set cost for Mouse IVF per round. Requires donor priming, embryo collection, IVF and culture, then embryo transfer. More expensive and more labour intensive

Genotype/phenotype

When rederived the genotype and phenotype of the resulting progeny will be exactly same as when freezing done.

Only cryopreserves half of genetic make-up of the original strain/background. Future rederivation can only produce a Het (due to IVF with WT eggs).