Guidelines for Documenting Justification for Exceeding Cage Densities

Unit for Laboratory Animal Medicine
Aug 12, 2022 12:00 am

The Guide defines the maximum number of rodents allowed in a given cage space and is the regulatory source for the Guidelines on Mouse and Rat Breeding and Housing Management. This document is intended to provide guidelines for investigators who breed mice and would like to attain IACUC approval to house more than two adults and one litter, i.e. an exception to The Guide

The below steps must be taken to document problem breeding when housed within the Guideline requirements. If standard steps to optimize breeding efficiency do not improve problem breeding parameters, a protocol amendment can be submitted to provide scientific justification for a protocol amendment requesting overcrowded housing to the IACUC. If the IACUC approves the exception to overcrowd, performance standards must be fulfilled to demonstrate increased breeding performance in overcrowded cages, as well as ensure environmental conditions remain within Guide requirements. 

  • Responsibility

    1. Investigator and Lab Staff Managing Breeding: Responsible for generating and maintaining accurate and complete breeding records.
      1. If given approval to exceed maximum cage densities, lab must continue to collect breeding records to support continued need for overcrowding and work with ULAM staff to collect environmental performance data.
    2. Veterinary Faculty: Responsible to meet with the investigator or lab staff to review breeding records to assess for problem breeding and provide recommendations for optimizing breeding.
    3. IACUC: Responsible for reviewing amendments supported with scientific justification requesting Guide exceptions to house more than two adults and one litter per cage. Amendments may be approved or denied.
  • Glossary Definitions


    Recommended breeding strategy using one adult female and one adult male resulting in a single litter. Takes advantage of postpartum estrus. Pair breeding has been proven to produce significantly more pups per female when compared to trio breeding and may be less stressful for both pups and dams than trio breeding.a,b

    Trio Breeding

    Alternative breeding strategy in which two adult females and one adult male are co-housed . Unless otherwise approved, one of the adult females must be removed prior to parturition such that no more than two adults and one litter are in the cage at any given time.  Justification for overcrowding may be submitted to the IACUC and if approved, trio birthing may be used. Singly housed pregnant females or stud males should be labeled with a blue tab as described in the Identifying Single Housed Animals SOP.

    Trio Birthing

    Alternative breeding strategy in which two adult females and one adult male are co-housed and left together following the birth of pups, resulting in a maximum of two litters and three adults in a cage. To engage in trio birthing, justification for overcrowding must be submitted to and approved by the IACUC.


    Alternative breeding strategy in which three to four adult females are co-housed with one adult male resulting in multiple pregnancies. All but one female must be removed from the cage prior to the birth of any pups. Justification to house two adult females, one adult male, and two litters in the cage as described by the definition of trio birthing may be submitted to the IACUC. Singly housed pregnant females or stud males should be labeled with a blue tab as described in the Identifying Single Housed Animals SOP.

    Extended Wean

    Mouse pups must be weaned by 21 days of age. However some phenotypes result in runted pups that may benefit from extending their weaning age. If extended weaning required for a given strain or phenotype, it should be described in the approved protocol. Where extended weaning is used, post-partum breeding may not be utilized, as it will result in more than 1 litter in the cage. Co-housing of older and younger litters in one cage is detrimental to the health and survival of the younger litter.

    Breeding Record

    A cage specific record that may include:

    • Information about the breeders: birth dates of breeders (dam and sire), their genotypes, identifiers such as ear tag numbers, and what generation they are in the line (particularly if backcrossing or inbreeding)
    • Information about the breeding itself: the mate date (day the male and female/s were first co-housed)
    • Information about the resulting litters: birth dates of litters, number of pups born, number of pups noted to have died or become missing, and weaned, and weaning dates.
    • Digital records are useful to track breeding colony success over time. This can be done in excel or using an online mouse colony tracking software. A free online breeding tracking program is SoftMouse
  • Procedures

    1. Defining & Documenting Problem Breeders

    1. If a laboratory or investigator cannot produce enough pups for their research needs while remaining within Guide required cage densities, i.e. no more than two adults and one litter per standard mouse cage,  they must provide justification describing the rationale for overcrowding (i.e. for trio birthing.) 
    2. Problem breeding may be defined by demonstration of one or more of the following metrics in at least three cages of pair housed breeders:
      1. Greater than 90 days since the mate date and no pups have been born.
      2. 4 or less pups have been born in each of the last two litters.
      3. Of the last four litters born, two or less had any pups survive to weaning.
      4. Or as otherwise determined by a ULAM faculty veterinarian.
    3. Laboratory staff should contact the veterinary faculty for the lab's animal housing area to meet and review their rationale and any breeding records.

    2. Troubleshooting & Optimizing Breeding Efficiency Through Implementation of Best Practices

    1. The faculty veterinarian will discuss the strain of concern with the lab – including any alterations to normal care practices that may better support these animals. In addition, the lab will present any available breeding records. Assessment of the number of breeders set up, resulting pregnancies, pups and weanlings, as well as the age of the breeders can be very informative to determine a possible source of poor breeding.
    2. Faculty veterinarians may suggest one or more of the following best practices to improve breeding performance while within Guide required cage densities:
      1. Review Age of Breeders: Females should be less than 9 months of age, ideally less than 6 months of age, and males should be less than a year old and have produced at least 1 pregnancy in the last 3 months.
      2. Cage Placement in the Room: Breeding cages should be placed on the lowest shelf of their rack and their rack should be located away from areas of high traffic and light such as the door and flow hoods.
      3. Enrichment: Mice experiencing trouble breeding may benefit from both shredded paper, like Envirodry, and a nestlet. If available, a shelter may be beneficial as well.
      4. Proper Nutrition: Breeding females require a higher protein and fat diet and thus should be fed breeder diet (PicoLab 5058), Love Mash (Bio-Serv), or provided irradiated sunflower seeds. PicoLab 5058 is provided at no additional cost. The other two options may incur an additional cost if made part of the colony's standard care.
        1. Some strains or stocks prone to obesity may experience decreased fertility with increasing body weight. For these animals, a standard diet that prevents significant increases in weight may be desirable.
      5. Seasonal Changes: Breeding success has a natural cyclicity of decreasing in the winter months and improving in the summer months.
      6. Environmental Factors: Vibration, noise, or fluctuations in humidity can impact breeding success. Each should be minimized as much as possible.
      7. Discontinuing Use of Post-Partum Breeding: while biologically possible, gestating a new litter while nursing a current one can compromise the health of the dam, and may retard the growth and survival of the current litter, and result in smaller litter sizes. Allowing a dam to focus on rearing her current litter of pups rather than "gestating while lactating" may facilitate healthier litters.
    3. If the above changes do not improve breeding success consider changing caging or providing an "aunting" dam to reduce maternal stress. Note that there may be some increases in cost associated with these changes.
      1. Caging:
        1. Change from ventilated to static housing.
        2. Change from translucent to opaque caging.
      2. Providing "Aunting" Support to the Dam: Once pregnancy is confirmed, remove the male and add a non-pregnant experienced dam or if low milk yield is suspected, add a lactating dam (without additional pups), to support the pregnant dam.
        1. "Aunting" females should preferably be a stock or strain known for good mothering instincts such as the Swiss Webster or FVB/N and can either come from within the research colony or can be purchased as retired breeders or timed pregnant females, respectively, from approved rodent vendors.

    3. Submitting Justification for an Amendment to Breed in Cages Exceeding Maximum Mouse Cage Densities

    1. If the best practices described in Procedures section 2 improved breeding performance such that problem breeding is no longer a concern, the lab should continue to breed in cages within Guide required cage densities.
      1. If despite the improved breeding performances, the lab would still prefer to house more than 2 adults and 1 litter in a cage, they can pay for larger caging that will support this increased cage density in accordance with The Guide cage density requirements. Larger mouse caging is dependent on availability and space within the vivariums.
    2. If the best practices described in Procedures section 2 did not improve breeding parameters, justification can be submitted to the IACUC for housing more than 2 adults and 1 litter in a standard mouse cage.
      1. Justification must include a rationale for trio birthing in each strain of concern. Blanket justifications covering numerous strains may not be used.
    3. If the IACUC approves this exception to the Guide, a SToP Form documenting IACUC approved overcrowding must be placed in the housing room.
      1. Cages for which trio birthing is approved will be labeled with a yellow acetate. The strain name listed on the cage card must be consistent with those listed on the STOP form and in the associated protocol.

    4. Post-IACUC Approval

    1. Justification must be repeated with each three-year renewal of the associated protocol to maintain the exception to housing within Guide required cage densities.
  • Appendix A: Maximum Housing Densities for Rat and Mouse Cages

  • Appendix B: Rodent Breeding Record

  • Appendix C: Example of Completed Rodent Breeding Record

  • References

    1. Garner JP, Gaskill BN, Pritchett-Corning KR. (2016). Two of a Kind or a Full House? Reproductive Suppression and Alloparenting in Laboratory Mice. PLoS One. 11(5):e0154966. 
    2. Gaskill B, Prichett-Corning KR. (2015). The Effect of Cage Space on Behavior and Reproduction in Crl:CD1(Icr)and C5BL/6NCrl Laboratory Mice.  PLoS ONE 10(5): e0127875
Species: Rats Mice

If you have questions or comments about this document, contact ULAM Veterinary Staff (email or call 734-936-1696).

The Animal Care and Use Office (email or call 734-763-8028) can answer IACUC specific questions regarding committee review of scientific justification to support Guide exceptions.

The ULAM Training Core (email or call 734-763-8039) can be contacted to provide training in techniques at no charge.