NC State Rodent Cage Density and Breeding Standard

Principal Investigators and their staff are responsible for adhering to this standard by ensuring appropriate colony management, accurate record-keeping and prompt weaning of breeding animal cages. Researchers must ensure cages are appropriately identifiable at all times, including at the time of weaning when an animal facility cage card may be unavailable. A temporary card or note may be used and must include at minimum the PI name, protocol number, date of birth, sex and the date of separation. Labs may be able to request colony management services for a fee, depending on the facility and availability of staffing. 

Animal care technicians (ACT) will monitor cage density as part of routine husbandry duties. Lab-managed colonies with cages that exceed the above housing density limits will be flagged either as an “overcrowded cage” or “wean cage.” The IACUC requires that overcrowded cages be corrected within 48-hours. Responsibility to take action falls on the lab, communication/notice will be per facility policies/procedures.

The maximum number of adult rodents in a single cage varies according to the cage type used in the facility. Researchers must be familiar with and adhere to the space guidelines established by the facility where work is conducted. The appendix in this document provides caging density guidelines for most rodent caging sizes utilized at NC State. For any caging that falls outside of these measurements, please follow the caging densities established by the facility. Note, per the Guide, larger animals may require more space. Space requirements for breeding configurations depend on the number of adults and litters, as well as the size and age of the litters. Researchers must follow the guidelines established by the respective facility where the animal colony is housed. 

Humane and experimental endpoints should be described in the approved IACUC protocol. Animals should not be held for prolonged periods without scientific justification and a plan for future use.

Rats maintained longer than 6 months should be weighed at least monthly by the lab to ensure adherence to this standard. Documentation should be available for IACUC review.

When breeding inbred strains, backcrossing to the inbred control strain is recommended every 5-10 generations to mitigate the impact of genetic drift on your mutant and transgenic mouse strain.  Breeding in house should be reserved for non-commercially available strains in order to promote reproducibility and prevention of strain drift.  Scientific justification is required for breeding commercially available strains. 

Breeding must be described in the approved IACUC protocol. Researchers are responsible for maintaining breeding records and accurately accounting for the number of animals used on the approved IACUC protocol. These records should be available for IACUC review. The PI must include the expected number of mice that will be needed in total on the protocol, including genotypes that may not be needed for research.   

Setting up breeding cages, separating breeders, and weaning pups within 21-23 days of birth is the responsibility of the research team unless other arrangements have been made with the animal facility.  Date of birth must be recorded at the cageside level to accurately project the anticipated weaning date. Pregnant females must be provided with nesting material prior to parturition. 

Age of retirement of breeders should be described in the IACUC protocol.

Breeding schemes will depend on the characteristics of the strain and research needs. Investigators using trio and harem breeding strategies must be aware of the increased responsibilities of ensuring adequate cage space. 

• Pair breeding (Mouse or Rat) – one female/one male
  • Pair separated once female confirmed pregnant (breeding plug or palpable pregnancy)
  • Required for strains that need extended weaning (see Weaning section)
  • Pairs with litters near weaning must be monitored closely for the birth of the second litter. If a new litter is born, the older litter must be weaned. 
• Monogamous pairs continuous pair breeding (Mouse only) – one female/one male. 
  • Offspring density is unrestricted provided pups are less than 21 days old.
  • Birth of a second litter requires immediate removal of the oldest litter. 
• Trio (Mouse or Rat)/Harem (Mouse only) Breeding: Two (trio) or 3-4 (harem) females/one male.
• Continuous trio breeding (Mouse only): 
  • Females and the male are not removed from the breeding cage during the postpartum estrus 
  • Scientific justification and completion of the “exception to standard” must be included in the approved IACUC protocol. 
  • This scheme is not appropriate for animals needing a weaning extension or for strains with large litters/pups. 
  • Please see the appendix at the end of this document for acceptable densities based on caging size.
  • IVC cages ≥ 140 in² do not have a pup limit for up to 2 litters, but weaning must be done by 21-23 days as described below. 
• Pregnant females must be moved to a separate cage prior to parturition   to avoid the birth of litters in the breeding cage.
• No offspring may be born in the breeding cage.

Weaning must occur by 21-23 days of age in normally developing mice. Extended weaning (P24-P28) may be requested on the IACUC protocol as an exception to standard for strains that consistently require delayed weaning. The request must include scientific justification. If approved, the researcher must separate the female from the male during pregnancy to avoid breeding at the postpartum estrus. 

Rodents prone to fighting should not be placed in the same cage. This includes males not weaned together or males that are not littermates, and certain strains that are known to be more prone to fighting (Kappel et al., 2017). Incompatible animals must be separated as soon as they are identified and any injuries reported to appropriate personnel to ensure adequate veterinary care.  

These caging sizes represent different cages currently used across the NC State campus for mice, including main campus animal housing facilities and the CVM. The allowed number of mice or rats per cage is based on the recommended amount of floor space described in the Guide.  If you are unsure of your caging size, please contact your facility manager. Any deviations from the below densities require an IACUC reviewed and approved exception to standard. 

Maximum housing density for mouse cages

IVC Duplex cage

• 3 adult mice 
• One female with litter (no male)

Static and IVC standard size cage, 68-72 in² 

• 4 adult mice 
• Up to 2 adults with a single litter  

IVC caging 77-84 in² 

• 5 adult mice 
• Up to 3 adults with a single litter 

IVC caging 110-115 in² 

• 7 adult mice 
• Up to 3 adults with a single litter 
• 2 females with 2 litters (male removed) 

IVC caging ≥ 140  in²

• 9 adult mice 
• Up to 3 adults with 2 litters 

Maximum housing density for rat cages 

Static or IVC caging ≥ 140  in²

• 1 rat > 500-1000 grams* 
• 2 rats 400-499 grams 
• 3 rats 300-399 grams 
• 4 rats 200-299 grams 
• 5 rats 100-200 grams 
• 1 adult rat with 1 litter of pups until weaning age

*Exemption for single housing must be described in the approved IACUC protocol.

Health Research Extension Act of 1985 and Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals. 

National Research Council Guide for the Care and Use of Laboratory Animals, Eighth Edition. National Academy of Sciences, 2011. 

Jackson Laboratories. (2021). Jax Mice pup appearance by age. NIH OFFICE OF ANIMAL CARE AND USE. Available at https://oacu.oir.nih.gov/system/files/media/file/2021-02/jaxpupsposter.pdf. Accessed February 5, 2024. 

Kappel S, Hawkins P, Mendl MT. To Group or Not to Group? Good Practice for Housing Male Laboratory Mice. Animals (Basel). 2017 Nov 24;7(12):88. 

DOI: 10.3390/ani7120088

Office of Laboratory Animal Welfare FAQ. 2023. Can performance standards be used in determining rodent housing practices including management of rodent breeding colonies? Available at 

https://olaw.nih.gov/faqs#/guidance/faqs?anchor=50373. Accessed February 5, 2024. 

Trio Breeding (AAALAC FAQ). AAALAC 2022. Available at 

https://www.aaalac.org/accreditation-program/faqs/#Ctrio, accessed February 5, 2024. 

NIH. 2012 .Guidelines for the Establishment and Use of Mouse Breeding Groups. Animal Research Advisory Committee, Office of Animal Care and Use, NIH. Available at https://oacu.oir.nih.gov/system/files/media/file/2022-03/b18_mouse_breeding_groups.pdf; accessed February 5, 2024.