Towards responsible management and a reduction in laboratory mouse breeding

Responsible management and Reduction of laboratory mouse colonies

For over 50 years, the mouse has been a key animal model for biomedical research, largely due to the ability to manipulate its genome with precision. The mouse reference genome is now annotated with 50,763 genes, including 22,198 coding genes (NCBI GRCm39). A variery of genetic tools exist the targeted modification or control of their expression. This approach facilitates cell tracking, the selective removal of specific cell populations, and tissue-specific or inducible gene activation or inactivation. For instance, this can be achieved through the administration of an inducer to the animal.

The use of genetically modified mice for scientific purposes often requires the establishment and maintenance of complex colonies, presenting significant challenges in terms of planning, traceability and controlling the number of animals produced. Each breeding line must be regarded as a long-term, cross-disciplinary project, involving coordination between research teams, animal care staff, the lead vet, with the central aim of limiting the production of animals with no direct scientific utility.

In France, as in many countries, the mouse is the most frequently used animal model for scientific purposes: it accounts for 72.4 per cent of reported uses in 2024. Maintaining colonies of genetically modified mice also constitutes a major component of national statistics, with 588,473 reported uses in 2024.

However it should be noted that this figure does not correspond to the total number of mice produced or maintained in breeding colonies. It reflects only those animals falling within the scope of annual reporting, notably due to a harmful phenotype or a reportable procedure such as certain invasive genotyping techniques. Therefore, it provides only an incomplete picture of all the animals involved in colony management. This includes breeding stock, animals whose genotype is inferred from the breeding scheme, animals available in insufficient numbers or at the wrong time to form an experimental cohort, or animals reared or killed outside an experimental procedure, such as for the collection of organs and tissues.

The management of mouse colonies is therefore a key strategy for Reducing the number of animals used in research, both within the regulatory scope of declared uses and, more broadly, in the production of animals required to maintain the strains. Optimising breeding programmes, anticipating experimental needs, minimising surpluses, monitoring strains over time and using cryopreservation where appropriate all help to reduce the number of animals produced without direct scientific purpose.

‘Responsibility’ in the management of genetically modified mice

Working with genetically modified mice is becoming increasingly challenging. This is due to the increasing complexity of genotypes, the ever-growing number of strains, and the various regulatory, ethical, scientific and economic requirements. Colony management is a planned and structured activity. It aims to ensure the availability of animals useful for research while minimising the number of animals produced and killed unnecessarily. This approach is founded on the principles of the 3Rs (Replacement, Reduction, Refinement) and shared responsibility among all stakeholders involved in the design, production, monitoring and use of mouse strains ( Mesbah, K and Ayadi, A. ).

There are now numerous methods for producing genetically modified animals, including additive transgenesis, homologous recombination (knockout and knock-in), genome editing using CRISPR-Cas9-type molecular scissors, random physical mutagenesis (ionising radiation) or chemical mutagenesis (mutagenic agents such as ENU), and spontaneous natural mutations that can result in a striking or, in some cases, silent phenotype.

The combination of multiple mutations within a single strain can present signifiant challenges in the breeding process. Mendelian laws and initial heterozygosity, as well as constraints related to sex, genetic background or the viability of certain genotypes, can significantly reduce the proportion of animals with the desired experimental genotype. These cross-breeding programmes therefore entail significant costs, not only in terms of the number of animals produced that are of no direct use to the experimenter, but also in terms of the time taken to produce them, the workload involved and the use of housing capacity. In complex situations, generating an experimental cohort can take at least six months, and often well over a year.

One of the most important strategies to address this challenge is the optimisation of mating schemes. The objective is to design matings in a manner that ensures the production of the highest possible proportion of useful animals, and, where relevant, their controls. In the case of lines carrying multiple genetic modifications, it may be preferable to fix certain mutations in the homozygous state, provided this is compatible with the animals' viability, fertility and phenotype. This strategy reduces the number of genotypes generated in each generation and increases the proportion of animals that are directly usable. Conversely, heterozygote x heterozygote crosses may produce a high proportion of animals that do not match the desired genotype. In order to ensure optimal efficiency and cost-effectiveness, it is advisable to reserve these for situations where their use is unequivocally required.
In cases where experimental design allows, more targeted crosses may be preferable. For example, heterozygote x homozygote crosses could be used if the heterozygous animals can be used as a relevant control group. Alternatively, mutant homozygous lines and wild-type lines could be maintained in parallel, scaled according to experimental requirements, provided that the genetic background is controlled and comparable. This rationalisation of matings helps to limit the production of surplus animals, reduce the time taken to obtain cohorts and decrease the total number of mice produced.

Coordinated Breeding Management

In many institutions, management is centred on available space: researchers submit their requests directly to animal care staff, who then arrange matings within the limits of available cages and racks. In order to move away from this approach, large institutions are setting up a 'breeding coordination team'. This team will be separate from researchers and animal carers. This team serves as the primary liaison between animal users and the animal facility services. It is responsible for managing requests, discussing specific requirements (such as exact genotype, sex, age, and number of animals required), monitoring the experimental timetable, setting colony targets, and determining the opening or closing of breeding pairs. The coordinators assess the scientific relevance and the overall capacity of the animal facility. This organisational change will enable us to drastically reduce unnecessary fluctuations in colony size and avoid unplanned surpluses of animals.

© S.Gomez FC3R

Digitals Tools fof Colony Management

Computer tools can be used for categorising and forecasting colonies, for example, a colony and cohort rearing calculator. Strains are classified according to their production level, enabling a target number of cages and breeding pairs to be assigned to each category. Based on this classification, a 'breeding team' can make a fairly reliable medium-term estimate of the number of cages required for all projects, anticipate activity peaks and advise researchers on when to open or close colonies. Computerised colony management systems and databases detailing production levels (maintenance, low, medium and high) support this planning. These databases include information on each animal's identification number, genotype, pedigree, health history and samples taken.

Colony management software plays an important role in Reduction and Refinement. They provide automated reports that enable breeding practices to be assessed objectively, including the number of cages per strain, the performance of breeding pairs (litter size and viability, mating success), the rapid identification of low-productivity strains, and the detection of isolated or elderly animals with no experimental value, as well as recurring surpluses of excess animals. While this information does not replace the expertise of the teams, but it provides a useful basis for adjusting mating schemes, reducing surpluses, comparing practices between teams, deciding whether to discontinue or cryopreserve certain strains, and guiding teams towards more responsible management.

Cryopreservation and assisted reproduction

Another key aspect of improving practices is the use of gamete and embryo cryopreservation technologies, which are commonly used in mice. These technologies enable a genotypes to be frozen and preserved over time, while also providing a backup of the genetic resources in the event of health-related contamination of breeding colonies or genetic drift.

Although sperm cryopreservation is less costly, it involves a loss of homozygosity and, more importantly, a risk of losing the genetic pool, since the male gametes will be used to fertilise freshly ovulated oocytes, sometimes years later. Only cryopreserving embryos from the single-cell stage to the blastocyst stage preserves the genetic pool and the complexity of a set of mutations or modified alleles.

In vitro fertilisation (IVF) may be more effective than natural mating for producing cohorts within a shorter timeframe and can enable the number of animals produced to be adjusted according to actual needs.

Optimised genotyping of colonies

Genotype the animals as early as possible, entering the results into the database immediately. This will enable animals with an undesirable genotype to be identified promptly, allowing their cages to be removed or reallocated. Non-invasive approaches minimise pain and stress for the animals while providing sufficient quantities of high-quality DNA.

Combining genotyping of semen and embryos with these technologies further optimises the processes. Monitoring the genetic quality of frozen semen stocks and assessing the success of gene-editing strategies directly on cultured embryos avoids the production of live animals. Similarly, analysing the sperm of chimeric males created through stem cell engineering allows the selection of those most likely to pass on the mutation. This reduces the number of matings required and the number of wild-type pups.

The ethical dimension of animal breeding and the importance of training

Training for researchers and staff involved in colony management is a key factor. Dedicated training programmes, combined with simple educational materials and tools to assist in determining colony size, alongside clear internal policies, can help minimise the duplication of lines, encourage cryopreservation when keeping animals alive is no longer justified, and ensure rigorous genetic traceability.

Rigorous colony management, underpinned by well-considered breeding schemes, advanced technologies, refined genotyping methods, robust digital systems, and clear organisation of responsibilities, enables improved data quality, reduced project timelines and costs, a decreased number of animals used, and enhanced animal welfare and staff wellbeing, in line with the 'culture of care'.