Number of Mice Maintained in Breeding Cages

Number of Mice Maintained in Breeding Cages

(Also see the FAQ for this policy.)

Approved March 22, 2001    Revised August 16, 2012    (Download) 

Background: Space allocations for breeding mice have changed in the Eight Edition of The “Guide for the Care and Use of Laboratory Animals” (2011). The University’s assurance of adherence to the PHS Policy and of AAALAC Accreditation status is based, in part, on compliance with these guidelines. The “Guide” provides the recommendation that a female and litter require approximately 51 in2 of floor space within their cage and 67 in2 is allocated for a female, male, and litter. As the “Guide” does not clearly indicate that these floor spaces are additive, The NIH Office of Laboratory Animal Welfare (OLAW) has interpreted this guidance with caution and recommends a performance based approach to evaluating the appropriate number of mice in a breeding cage. Performance measures typically used to evaluate breeding mice include number of mice in the litter, weaning ratio, weaning weight, and reproductive efficiency. Other indicators of behavioral abnormalities including fighting or stereotypical behavior can also be evaluated.

Most mouse cages at the UW have 75 in2 of floor space, which would only allow for one breeding pair (one dam and one sire) and litter to be present in the cage, which is in contrast to our previous IACUC policy that allowed two dams, a sire, and two litters per cage. There are two less commonly used caging systems that have smaller floor space (60 in2). This caging is being gradually phased out by the Department of Comparative Medicine, but it will take some time before they have all been replaced.

There is significant variation in mice in gestation length, litter size and reproductive efficiency (time to first litter, inter-litter interval). The gestation period in mice typically ranges from 19-21 days, but varies by strain. Outbred mice and a few inbred mouse strains with high breeding efficiency have larger average litter sizes of 10-12 pups per litter (e.g., CD-1) and short inter-litter interval where a new litter is born as soon as 20 days following the first litter (Pritchett and Taft, 2007). Conversely, many inbred strains (C57BL/6J, BALB/cByJ) have small average litter sizes of 4-6 pups per litter and longer inter-litter intervals. These inbred strains can also have pups that grow slower and are smaller.

One other factor influencing breeding efficiency is the timing of estrus. Generally mice are in estrus once every 4 days, however, estrus usually ceases during periods of peak lactation. Thus, mice in breeding situations where the male is present, will come into estrus and breed within the first few days after parturition. If this window is missed (as no male is present), then the females of most strains will not return to estrus until closer to weaning or after weaning.

At the University of Washington, pair breeding (one male and one female) is commonly used for outbred strains of mice, inbred strains that are prolific breeders, or in situations where the precise parentage of every mouse must be known. Trio breeding systems (2 females and one male) are commonly used with less efficient breeders and less viable pups as this allows multiple dams to rear the litter providing increased grooming and care (Heiderstadt and Blizzard, 2011; Bronchi, 2009). The growth rate of pups is greater if there are more dams present. Breeding systems with three females and one male are used more rarely, primarily when there is a valuable and rare breeding male or if larger caging is available.

The recommendations provided by the “Guide” represent a generally accepted standard, although the performance of different strains of mice in the current rodent cages can be considered to justify exceptions to these recommendations. The IACUC approves the following policy in order to allow trio breeding to continue for strains with small litter size and poor breeding efficiency and to clarify separation procedures for other breeding schemes. If investigators have other scientific reasons to vary from this policy based on the unique characteristics of the strains of mice they have, then these instances must be outlined in their IACUC-approved protocol as a “variance” (see below for what to include in the variance).


  1. Standard (pair) breeding: one standard mouse cage can house one adult male, one adult female, and one litter until weaning.
  2. Trio breeding (two females and one male):
    • Can be used only for inbred strains or genetically modified mice where small litters (average 6 pups or less per litter) or poor breeding efficiency is seen.
    • If unusually large litters (more than 12 pups, 2 females and 1 male) are born, one of the two females with her litter must be separated to a new cage before the pups reach two weeks of age.
    • If a third litter is born prior to weaning of the previous litters (thus new pups in the cage with older pups present), older pups must be separated immediately.
    • When pups of very different ages (i.e., 19-20 days old versus newborn) are present in the cage, the older pups must be either weaned or separated with the dam.
  3. Three females and one male: Females need to be separated after pregnancy is detected, prior to delivery. If pregnancy is not detected, then females with litters must be separated before the pups are 10 days old (typically non-lactating females are removed from the cage soon after the litter is noted).
  4. Animals will be weaned by the research group on or before 21 days of age unless an IACUC variance is approved.

Variances from this standard policy as part of the approved IACUC protocol:

  1. An IACUC variance for weaning at an age greater than 21 days of age shall include:
    • Reason for late weaning (Please note that the “Guide” and current federal regulations do not allow cost alone as justification for departures from the Guide) and strain(s) involved.
    • Breeding scheme and maximum number of adults and litters that will be in the cage.
    • An assurance that if a new litter is born in the cage prior to weaning of the old litter (pups greater than 19 days of age), the old litter will be weaned or separated immediately. Conversely, the variance can provide an explanation (with data) as to why this is not needed.
  2. An IACUC variance for breeding in trios (two females and one male) when average litters are larger than 6 per dam, should include:
    • An explanation of the benefit to the breeding and weaning efficiency of trio breeding for this strain (including past data) and strain(s) involved.
    • Breeding scheme and maximum number of pups that will be in the cage.
    • An assurance that if a new litter is born in the cage prior to weaning of the old litter (pups greater than 19 days of age), the old litters will be weaned or separated immediately. Conversely, the variance can provide an explanation (with data) as to why this is not needed.


The responsibilities for insuring compliance will be as follows:

Primary Investigator: For all breeding schemes, PI must place a breeding card on the cage that estimates (within 1-2 days) the birth dates of each litter in the cage and must separate animals in accordance with the policy above.

Variances approved by the IACUC must be indicated clearly in the room. It is the PI’s responsibility to post these variances.

Department of Comparative Medicine: If animals have not been weaned by 21 days of age, and no threat to health is seen, DCM personnel will place a “Please Wean” card on the cage. If the cage is not weaned within 2 days, DCM personnel will separate animals and charge the PI for time and materials at the technical assistance rate.

If a cage is out of compliance with the above policy and animals need to be weaned or separated immediately as detailed above, DCM personnel will wean or separate animals and charge the PI for time and materials at the technical assistance rate.

Current rates can be found at: Variances approved by the Institutional Animal Care and Use Committee must be clearly posted in the room.

Selected References:

Heiderstadt KM, Blizard DA. 2011. Increased juvenile and adult body weights in BALB/cByJ mice reared in a communal nest. J Am Assoc Lab Anim Sci. 50(4):484-7.

Branchi I. 2009. The mouse communal nest: investigating the epigenetic influences of the early social environment on brain and behavior development. Neurosci Biobehav Rev. 33(4):551-9.

Pritchett, K.R. and R.A. Taft. 2007. Reproductive Biology of the Laboratory Mouse In The Mouse in Biomedical Research, Normative Biology, Husbandry and Models. J.G. Fox, M.T. Davisson, F.W. Quimby, S.W.Barthold, C.E. Newcome, A.L. Smith. eds., Academic Press. Burlington. pp.103-121.