Archiving best practice

When choosing an archiving approach it is important to consider technical requirements, financial costs and the number of animals required for each method, as well as the time it takes to recover a cryopreserved strain. All archiving methods have well established protocols (see Cryopreservation protocols on the Infrafrontier website) and both embryos and sperm can be easily transported in a liquid nitrogen dry shipper (although frozen sperm can also be sent on dry ice).

Considerations for selecting an archiving method

Sperm cryopreservation and recovery

• Number of animals required: two to four males.
• Cost and equipment/techniques required:
  • Freezing sperm is low cost – simple and cheap freezing method with no special equipment required and minimal training.
  • Recovery of live animals from sperm is high cost – IVF requires a specialised incubator, followed by embryo transfer.
• Efficiency: high efficiency – fast and generates large volumes of embryos on recovery.
• Genotype recovered: heterozygous or wild-type only.
• Recovery method: IVF followed by embryo transfer (or on occasion artificial insemination).
• Special requirements: oocyte donor.
• Quality control:
  • Before freeze:
    • Confirmation of genotypes of stock animals before cryopreservation.
    • Check for sperm motility and volume at point of cryopreservation.
  • After freeze:
    • Test thawed sperm in IVF with one female.
    • Ideally genotype resulting embryos.
    • Freeze remaining embryos if possible.

Embryo cryopreservation using superovulation with natural mating

• Number of animals required: 10 – 20 females plus three to five stud males.
• Cost and equipment/techniques required: 
  • Freezing embryos can be high cost – this requires a controlled-rate freezer. Embryo vitrification is a lower cost alternative.
  • Recovery of embryos is low cost – involves embryo transfer of thawed embryos.
• Efficiency: low efficiency – use of large number of animals, some strains do not superovulate well.
• Genotype recovered: any genotype.
• Recovery method: embryo transfer (surgical or non-surgical).
• Special requirements: breeding to generate stock for cryopreservation.
• Quality control:
  • Before freeze: confirmation of genotypes of stock animals before cryopreservation.
  • After freeze: 
    • Test thaw one straw and culture to blastocyst stage (for strains known to have issues recovering animals post cryopreservation, an embryo transfer and live pups can be obtained to confirm viability, but this is not recommended as routine practice).
    • Genotype embryos after thawing.

Embryo cryopreservation using superovulation with IVF*

• Number of animals required: six to 10 females plus one to two males for sperm.
• Cost and equipment/techniques required: 
  • Freezing embryos can be high cost – requires specialised incubator and controlled-rate freezer. Embryo vitrification is a lower cost alternative.
  • Recovery of embryos is low cost – involves embryo transfer of thawed embryos.
• Efficiency: medium efficiency – six to 12 animals are needed on average to preserve a strain.
• Genotype recovered: any genotype.
• Recovery method: embryo transfer (surgical or non-surgical).
• Special requirements: breeding to generate stock for cryopreservation.
• Quality control:
  • Before freeze: confirmation of genotypes of stock animals before cryopreservation.
  • After freeze: 
    • Test thaw one straw and culture to blastocyst stage (for strains known to have issues recovering animals post cryopreservation, an embryo transfer and live pups can be obtained to confirm viability, but this is not recommended as routine practice).
    • Genotype embryos after thawing.

*New technologies/reagents are becoming increasingly available to increase the efficiency of superovulation, for example the use of inhibin antisera to increase the number of eggs (hyperovulation). These technologies could reduce the number of mice needed to produce enough embryos for IVF/freezing, however there can be associated welfare concerns. 

Age of animals

• Males should be 10 – 24 weeks of age ideally. Males can be older but there is a decreased chance of obtaining good quality sperm. Sperm can be frozen from eight weeks of age, but sperm volume is often lower, especially in more complex crosses and some backgrounds.
• Females should be three to six weeks of age ideally. Females can be used up to ~24 weeks, but fertilisation/recovery of embryos will decrease significantly with age, requiring the use of more animals.

Record keeping

• It is important to keep accurate and comprehensive strain notes on allele, background and phenotype (see Mouse passport guidelines for recommendations).
• Keep a record of the freeze/thaw method used, as this may change over time.
• Consider freezing some tissue in case you want to genotype in future.

When to freeze

• On arrival/creation of a strain, freeze sperm from one to two males.
• When backcrossing a strain, freeze sperm from one to two males, every three to five generations.
• For complex breeding consider the value of freezing intermediate breeding steps.
• Once the final product is ready (e.g. multi-allelic, backcrossed), freeze embryos and sperm (see Timelines for archiving methods).

Storage

• Ensure your samples are split across two banks, and preferably two sites.
• Liquid phase is recommended for storage given the small volume in straws.

Quality control measures

• It is important to always perform quality control checks before and after freezing, following the recommendations in Considerations for selecting an archiving methods.

See below for examples** of the number of males and embryos generally recommended to safely archive a strain without keeping any animals in a live colony. It should be noted that freezing sperm will allow the recovery of most strains and alleles, but that this approach requires longer to recover the strain in its experimental state for multi-allelic strains.

**Examples are based on C57BL/6J substrains; requirements and timelines may vary for other backgrounds.

Animal numbers and expected timelines for archiving different types of GA strains

Single allele GA strain – heterozygous

• Number of embryos: none
• Number of males: two
• Estimated time for cryopreservation: if a supply of wild type females is available, then time to freezing is only dependent on the age of the males. Freezing is possible when males reach 10+ weeks of age.
• Estimated time for recovery of adults from sperm: adults available in 10 weeks.
• Estimated time for recovery of adults from embryos: not applicable.

Single allele GA strain – homozygous 

• Number of embryos: 50 – 100 
• Number of males: two
• Estimated time for cryopreservation: six to 10 weeks to breed sufficient females for IVF/natural mating, or longer if a male needs to age for IVF or repeat rounds of breeding are required.
• Estimated time for recovery of adults from sperm: 20 weeks minimum – very dependent on the genotyping protocol, likely closer to 30 weeks.
• Estimated time for recovery of adults from embryos: adults available in 10 weeks (if homozygous embryos frozen).

Multiple allelic strain – heterozygous

• Number of embryos: 50 – 250 

• Number of males: two to four 

• Estimated time for cryopreservation: if a supply of wild type females is available, then time to freezing is only dependent on the age of the males.
• Estimated time for recovery of adults from sperm: adults may be available in 10 weeks, although obtaining all alleles in one animal may take up to 20 weeks.
• Estimated time for recovery of adults from embryos: adults available in 10 weeks.

Multiple allelic strain – homozygous

• Number of embryos: 100 – 250 
• Number of males: two to four 
• Estimated time for cryopreservation: potentially up to 20 weeks to breed sufficient females for multiple rounds of IVF. (More complex strains and those on other backgrounds often respond poorly to superovulation).
• Estimated time for recovery of adults from sperm: dependent on strain complexity, this could take up to a year.
• Estimated time for recovery of adults from embryos: adults available in 10 weeks.