Rodent Breeding: Mice
Boston University is committed to observing federal policies and regulations and AAALAC international standards and guidelines for the humane care and use of animals.
Mice have short gestation times and large litters. Therefore, cages may become overcrowded quickly if the individual responsible for managing the breeding colony and separating animals at the proper times does not do so in a timely fashion.
When overcrowding of cages happens, the animals become uncomfortable and stressed. Pups can die from being trampled. In static micro barrier cages, the air quality quickly deteriorates with a high density of animals and may predispose them to respiratory disease.
Overcrowding of cages is an animal welfare concern and can have a deleterious effect on research.
This policy provides guidelines for mouse breeding colonies to define standards and responsibilities for mouse housing and mouse breeding cages. These policies assign responsibility for action when mouse breeding cages are overcrowded.
Investigators are responsible for:
- Coordinating with Boston University Animal Science Center (BU ASC) for space allocation of their mouse breeding colonies.
- Managing their own breeding colonies unless arrangements for technical support have been made with BU ASC.
- Designating a colony manager (someone who has received specific training on managing mouse breeding colonies, and who will be the primary contact person for the lab). More than one colony manager may be designated by the PI, but it is preferable that this number be minimized to facilitate communication between the BU ASC and the mouse users.
The designated colony manager (research staff or BU ASC staff) is responsible for separating animals according to allowed cage space as described in the Breeding Schemes, Weaning Pups, and Numbers of Mice Per Cage sections, below.
BU ASC staff and investigators
Both the BU ASC staff and investigators managing their own mouse breeding colonies must abide by procedures as outlined in this policy.
Any recurring problems with mouse breeding colony management will be brought to the attention of the IACUC.
Breeding Schemes Procedures
Two different breeding schemes are acceptable. In either, the designated colony manager (research staff or BU ASC staff) is responsible for carefully monitoring pregnancies.
A. Monogamous pairs
Postpartum estrus occurs within 24 hours of parturition: If the male is left in the cage, the female is likely to become pregnant again while lactating and nursing the new litter.
One male (only one male per cage is allowed) and one female are housed together for mating. Nesting material is provided in the cage. The mice are not separated when the female becomes pregnant or delivers the pups. This model takes advantage of postpartum estrus and allows the female to become pregnant and nurse at the same time. Litters are born approximately 21 days apart. The three-week-old litter must be weaned prior to the birth of the new litter.
B. Harem mating
This method houses two, or at most three, females in a cage with one male (only one male per cage is allowed).
- During routine health/breeding checks, each noticeably pregnant female is removed and placed in her own individual cage. When the pregnant female is separated from the harem cage, she is given nesting material in her delivery cage to make a nest for her pups. The female delivers her pups and nurses them for 21 days (or up to 28 days, with BU ASC approval, following the procedure below in Weaning Pups). Only one nursing female and litter is allowed per cage. After the pups are weaned, the female may be returned to a harem cage.
- Alternatively, when one or more females are noticeably pregnant, the male is removed and housed singly in a fresh cage. The singly housed male is given environmental enrichment according to BU ASC standard operating procedures. Females deliver their pups and nurse them for 21 days (or up to 28 days, with BU ASC approval, following the procedure below in Weaning Pups). Housing two nursing females with litters in one cage requires prior BU ASC approval. After the pups are weaned, the female(s) may be returned to a harem cage.
C. Checking Pregnancy and Birth
The designated colony manager has primary responsibility for checking for pregnancy and birth and for recording these events on the cage card(s). When the litter is born, the cage is flagged with a New Litter Card and the date of birth (DOB) and projected weaning date is documented. However, if BU ASC staff find births of litters when checking and changing cages, they will place a New Litter Card and the DOB.
D. After Birth
After pups are born, the cage is left undisturbed for at least three days except for replenishing of food and water as needed. If the bedding gets very dirty or wet and the cage must be changed sooner, the following procedure will be followed:
- The female is transferred first
- Then the litter, plus a small amount of the dirty bedding (so the smell in the clean cage will be familiar) is scooped up altogether with a gloved hand and transferred to the new cage.
- The same procedure is followed until the pups start moving around the entire cage.
Required Documentation and Cage Cards
Cage Card Required Information
- Principal investigator (name)
- Account # (when required)
- IACUC protocol (ID #)
- User name/colony manager
- Contact information
- Strain or stock and specific GEM or mutant nomenclature
- Vendor (“in-house” for animals bred in-house)
- Number of animals and sex
- Date of birth (DOB)
- Date of arrival (DOA)*
*Not always applicable
Breeding Card Required Information
- Principal investigator (name)
- IACUC protocol (ID #)
- User/colony manager
- Contact information
- Litters born and DOB
New Litter Card Required Information
So that the card is visible, place it vertically behind the Cage Card
- Projected weaning date of new litter
A. Age of pups at weaning
The weaning age for mouse pups is routinely 21 days of age. In the case of some inbred, genetically modified, or mutant strains, it may be advantageous to allow the pups to remain with the female for 28 days.
- To extend nursing time past the 21-day standard, BU ASC must be notified by placing a note on the New Litter Card. The colony manager must communicate via email to BUASC@bu.edu or LACFemail@example.com when all pups in a given breeding line will be routinely weaned at 28 days.
- User writes “extended weaning,” “date to wean,” and “dam (mother) not pregnant” on the cage card.
- Allowing a three-week-old litter to stay in the cage with a lactating female who also has a newborn litter is not permitted.
B. Monogamous pairs
Assuming the lactating mother is pregnant, pups are weaned at 20–21 days of age, just before the new litter is born. This will prevent trampling of newborn pups by the weanling pups, and prevent the cage from being overcrowded.
C. Harem-mated females
- If a singly housed lactating female is alone in a cage with her litter, weaning is less urgent than with monogamous pairs. However, mouse pups are routinely weaned at 21 days of age unless an exception has been approved by the BU ASC.
- If two females raise their pups together in one cage, a procedure which must be pre-approved by BU ASC, attention must be paid as the pups grow older so that the cage does not become overcrowded. Each female with litter must be singly housed no later than two weeks after birth of pups.
D. Separation of sexes at weaning
- Male and female pups are separated at weaning, mice of each sex being placed in a separate cage.
- If a litter contains only one pup of a given sex, provisions must be made to house this pup with others of the same sex. Newly weaned pups must not be housed singly. Possible housing options include:
- A single female pup may remain with the mother.
- A single male pup may be placed with other male pups from a different litter of the same age.
- If the parents are a monogamous pair, a single male pup may be housed with the father, both being separated out into a new cage.
- A single male pup may be housed with female siblings up to six weeks of age (adulthood).
- More than one male pup may NOT be housed with female siblings.
- It is recommended that sexing of the pups be verified one week later.
E. Feeding of weaned pups
At the time of weaning, a small amount of chow (only a few pellets so they don’t get moldy) must be provided on the cage floor for the next seven days.
Number of Mice Per Cage
Cage sizes vary in the different facilities; the colony manager is encouraged to ask BU ASC staff about the size of the cages in which their mice are housed.
All mouse cages
- Unless the PI is willing to weigh the mice weekly to ascertain that the actual weight is 25g and document these data, mice are classified as adults at 6 weeks of age and older.
- When mice are weaned, they are weaned to adult specifications to avoid needing to separate them and re-house them in 2–3 weeks.
- Five adult female mice or four male mice or one lactating female with nursing litter are allowed per shoebox cage.
- One adult male and one adult female with or without nursing litter (monogamous pair).
- Occasionally when females have small litters or do not lactate well it may be beneficial to house two lactating females together in one 60- or 75-square-inch cage so they can raise their litters cooperatively. This must be pre-approved by BU ASC, noted on the Cage Card, and communicated to BUASC@bu.edu or LACFfirstname.lastname@example.org.
- One adult male and two or maximum three adult females without litters (harem cage).
B. 75-square-inch cage
Same as above, except five males may be housed in one cage.
Defining Responsibility for Separating and Weaning Mice
Designated colony manager
The designated colony manager (research staff or BU ASC staff) is responsible for cage card documentation and for separating and weaning according to the above guidelines.
The PI or research staff separates non-breeding experimental mice, unless technical support has been arranged with BU ASC in advance.
BU ASC Actions in Cases of Overcrowding
- The BU ASC staff checks for overcrowding (O/C) and pregnancy when performing daily health checks and when changing cages. Any cages that are overcrowded according to standards defined above are marked with a Problem Notification—O/C Card, dated, and initialed.
- When overcrowding is noted, the responsible individual, the designated colony manager, is given up to 48 hours to correct the problem, depending on the severity of the overcrowding. Note: Weekends and holidays count as days and are not exempt.
- If overcrowding is not addressed within the allotted time, BU ASC staff separates the mice and Special Service charges will be incurred at a minimum of 15 minutes.
- Males that are fighting or have fight wounds: ASC staff will separate promptly.
- When a harem-housed mouse is noticeably pregnant (usually 14 days gestation), 48 hours. However, if a female seems to be about to give birth, she is promptly separated.
- When two litters, one newborn and one previous litter, are in one cage, separation is performed as soon as possible. In such a case, the BU ASC staff separates the older pups into separate cages and gives a few food pellets on the cage floor. Female and newborn pups are left in the breeding cage. The cage is marked with a Problem Notification—O/C Card and any other information necessary to identify the mice, dated, and initialed.
- Any time a cage is significantly O/C and the welfare of the animals is at stake (Emergency O/C), the animals are promptly separated into acceptable group sizes.
- Adding/updating the number of cages is documented on the census sheet by the colony manager or the person who separates the mice.
- When the overcrowding is corrected, the O/C Card is removed.
In all cases where ASC staff are performing separations, there will be a Special Service charge for a minimum of 15 minutes.
- The Guide for the Care and Use of Laboratory Animals. 1996. NRC ILAR. P.27. Table 2.1. Recommended Space for Commonly Used Group-Housed Laboratory Rodents.
- BU ASC Standard Operating Procedures.
- UMDNJ New Jersey Medical School Comparative Medicine Resources Rodent Breeding Policy and Standard Operating Procedures.
- The Boston University Mouse User Advisory Committee.
BU IACUC Approved October 2010, Revised January 2014, Reviewed June 2019