Number of Mice Maintained in a Breeding Cage

Purpose

To provide guidance on the number of mice that can be maintained in a standard breeding cage, and establish expectations for what information needs to be included in the IACUC protocol for variances to these standards.

Background

Space allocations for breeding mice are described in the Eighth Edition of the “Guide for the Care and Use of Laboratory Animals” (2011). 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. 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 University of Washington (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.

At the UW, 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 and approval of this system would require justification.

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).

Policy

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):

a) 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.

b) 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.

c) 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.

d) 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 breeding:

a) Use of this breeding scheme must be justified on the IACUC protocol.

b) Females must be separated after pregnancy is detected and prior to delivery. If pregnancy is not detected, then mice must be separated when the pups are between 3 and 5 days old (best practice is to remove non-lactating females from the cage 3-5 days after the litter is born).

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:

a) 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.

b) Breeding scheme and maximum number of adults and litters that will be in the cage.

c) 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 pups per dam, should include:

a) An explanation of the benefit to the breeding and weaning efficiency of trio breeding for this strain (including past data) and strain(s) involved.

b) Breeding scheme and maximum number of pups that will be in the cage.

c) 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 ensuring compliance will be as follows:

Principal 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. The posted variance must contain:

a. PI name
b. Protocol number
c. Protocol expiration date
d. Language of approved variance as outlined above

Department of Comparative Medicine (DCM):

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 https://depts.washington.edu/compmed/billing/rates.html.

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.

Approval/Review Dates

Originally Approved: 03/22/2011
Last Reviewed/Revised by the IACUC: 03/17/2022