Donahue13 project protocol

Reproductive performance of 13 CXB recombinant inbred strains of mice   (2011)

Donahue L


Project protocol — Contents
Workflow and sampling
Equipment and supplies
Reagents and solutions
Additional information

Workflow and sampling

    Procedure done
    Data collected
    JAX breeding performance data was collected from Repository Production Colonies from which JAX® Mice are shipped.
    October 8, 2010 to December 10, 2011
    female reproductive/ breeding performance
    Males and females were pair-mated
    October 8, 2010 to December 10, 2011
    Cages were monitored by JAX Special Mouse Strains Resource (SMSR) caretakers for the duration of the study.
    October 8, 2010 to December 10, 2011
    The number of pups born, date of birth, and the number and sex of surviving pups were recorded at the time of weaning (usually 3-4 wks of age).
    October 8, 2010 to December 10, 2011
    litter/pups parameters

Equipment and supplies

  • Vivarium for breeding mice
  • Essentials for recording female mice breeding/reproductive performance
  • Gloves, masks, laboratory gown, and head wear
  • Large forceps

Investigator Notes: "All mouse colonies (even within the same facility) are different and breeding performance may vary. Thus, the breeding performance data in this study should be used as a guideline to indicate strain breeding potential and to help identify potential breeding problems."

Reagents and solutions

Alcohol for disinfection


  • Breeder mice are maintained in small colonies. No set rotation schedule is applied. Age at mating and at retirement varies.
  • All mice are pair-mated with siblings.
  • Pups are weaned at approximately 3 wks of age.

For Breeding Strategies for Maintaining Colonies of Laboratory Mice go here.

For convenience we have made the following information publicly available:
Table 1. Strategies for maximizing productivity of a breeding colony.
Table 2. Reproductive characteristics for most inbred strains of laboratory mice.
Table 3. Environmental factors that can affect breeding.
Table 4.
Breeding problems and possible resolutions.

To browse the Special Mouse Strains Resource (SMSR) for other information.

Additional information from The Jackson Laboratory Handbook on Genetically Standardized Mice

Table 1. Strategies for maximizing productivity of a breeding colony.
Action to promote successful breeding Comments
When to set up breeding
• Mate mice when they are 6–8 weeks old. • Note that it is not unusual for the first litter to be smaller than the second or third, which are typically the largest.
When to foster pups
• Foster pups
- if the mother does not "nest" the pups right away but leaves them scattered around the cage , or
- if milk spots do not appear in the pups by the time they are 24-hours old.
• If you need offspring from a female who is a poor mother, plan ahead to have foster mothers ready when she gives birth.
• Housing 2 females together will often allow them to work together and successfully raise 2 litters.
When to replace breeding pairs for optimal performance
• Replace breeding pairs as their reproductive performance declines (typically 6–8 months of age; see Table 2.) • A colony of mixed-age breeders produces a more consistent quantity of pups than does a colony with mice of the same age.
• For a large colony, maintain a stable supply of breeding pairs at various ages by replacing a specific percentage on a weekly or monthly basis.
When to replace individual female breeders
• Replace female breeders when they
- do not produce a litter within 60 days of pairing (unless this delay is normal for the strain), or
- do not produce a litter within 60 days of their last
- produce litters but pups do not survive for 2–3 litters.
When to replace individual male breeders
• Replace male breeders
- when they reach 1 year of age, or
- if they are infertile with a young, fertile female.
When to cull a litter
• Reduce the number of pups in a litter if the mother is having problems feeding the pups. • Some mothers may be unable to provide milk for more than a few pups.
• For segregating strains, remove unwanted pups as soon as they can be phenotyped or genotyped.
How to improve breeding behavior
• With young females, use experienced males. • Males mature later than females; therefore age at first litter for same-aged breeding pairs is often determined by the male.
• Isolate males for 2 weeks before pairing.
• Rotate males within a strain among cages.


Table 2. Reproductive characteristics for most inbred strains of laboratory mice.
Characteristic Normal range Comments
Sexual maturity 4–8 wks of age Males usually reach sexual maturity by 6 weeks, females by 4–6 weeks.
Estrous cycle 4-day or 5-day cycle The normal cycle can be interrupted by mating, pheromones, vibration, noise, and other environmental stresses.
Postpartum estrus 6- to 8-hr period This is the estrous period females enter within a few hours after giving birth. If a male is in the cage, mating and pregnancy are likely.
Ovulation rate 4-12 ova per estrus Strain dependent.
Litter size 2–12+ pups (aver. 6–8 pups) Strain dependent. Pattern is for litter size to increase, reach a peak at about 3–4 months of age, then start decreasing.
Fertility rate 50–100% With some strains, all pairs of mice are fertile; with others, as few as 50% are.
Gestation length 18–21 days Gestation length is typically 18–19 days for most strains; may be longer if litters are smaller (4 or fewer pups) or mothers are older.
Time between litters 22–50 days Typically the shortest time between litters occurs between 3–4 months of age (between the first and second litter), then progressively increases.
Generation time About 10 wks Strain dependent. Conception-to-conception time is determined as follows: 3 weeks gestation, 3–4 weeks suckling, 1–3 additional weeks until sexual maturity. But, generation time for the first litter is often longer.
Weaning age 21–28 days In husbandry context, the age at which pups are removed from their mother, not when they start eating solid food. Varies among strains; depends on weanling size and maturity. Most strains are weaned at 21 days, some at 28 days. Do not remove pups from mother before 17 days unless they are transferred to a foster mother.
Total litters 2–8 Total litter number varies by strain. A total of 4+ litters is typical, but some strains produce as few as 1 or 2 litters.
Reproductive lifespan (female) Terminates at 6-12 mo. of age Strain dependent. For inbred strains, fecundity usually begins to decrease with the 3rd litter. By 6–8 months, some females become infertile, and litter size for most females of most strains has diminished to the point that it is more economical to set up a new breeding pair.
Reproductive lifespan (male) Terminates at 12-14 mo. of age Strain dependent. Fertility in some strains decreases starting at 10 months.
Delayed implantation n/a Implantation normally occurs during the fifth day after conception. However, if a female is nursing, implantation may be delayed for more than a week, during which time embryonic development is held in suspension.
Seasonal breeding fluctuations n/a Some strains are susceptible to changes in breeding behavior based on season.


Table 3. Environmental factors that can affect breeding
Action to promote successful breeding Comments
Location of cages

• Avoid locating cages near a heavily trafficked or noisy area, such as near a door or sink or a loudspeaker used for paging.

• Changes in levels of noise and vibration can decrease breeding performance and may induce mothers to resorb litters, cannibalize their pups, or stop breeding.

• Place cage racks several inches from the wall to prevent building vibrations from transferring to the cages. • Construction-related noise and vibrations may be especially problematic.

• Maintain a single, consistent, lighting cycle. The most common cycles are 14:10 (on:off) and 12:12.

• Disruption of the light cycle may have an adverse effect on breeding.
• For wild-derived mice, use less intense lighting.
Barometric pressure
• Maintain a stable barometric pressure. • Falling barometric pressure can cause some strains to become hyperactive, which reduces breeding performance.
Temperature and humidity

• Keep the temperature at 16–26°C (64–79°F)

• Thermoneutral temperature for mice is around 27–28°C (80–82°F). In contrast, for humans, it is about 22–23°C (72–74°F).
• Keep the humidity at 40–60%.
Air quality and odors

• Keep the room air fresh, free from strong odors.

• Mice are extremely sensitive to strong or noxious odors, but they like their own scent to be strong in their environment.

• Establish a "no perfume" policy for caretakers, technicians, or anyone who works with the mice. • Noxious fumes may reduce breeding performance.
• Use clean forceps or clean (or new) gloves for each cage.

• Work gently, slowly, and quietly when handling breeding mice.

• Laboratory mice respond best to calm, consistent handling.

• Try to regularly assign caretakers to the same cages so that the mice receive consistent care with familiar handling and odors.

• Avoid changing cages more often than once a week; if females are ready to give birth, skip cage changing until pups are 2 days old or you can see milk spots. • Often, mice do not like new technicians.
• Minimize handling and checking on the mice, especially if mice are pregnant or giving birth, or have new litters. • Wild-derived mice are especially sensitive to handling stress.
• Unless absolutely necessary, do not handle pups until they are 3 days old.
Bedding and nesting material
• Provide nesting materials in the cage, especially if bedding (such as pellets from corn cob) does not lend itself to nesting.

• The addition of material such as Kimwipes® or Nestlets® can promote nesting.

• Nesting behavior is strain dependent.
• Provide food with a dietary fat content of 4–11% fat w/w. • The optimal percentage of fat in the diet, with respect to fecundity, is strain dependent. Optimal fat content ranges from 4–12% w/w; a commercially available breeder diet with 9% fat w/w works well for most strains of mice.
• If mice have bad teeth, broken teeth (e.g., from gnawing or malocclusion), no teeth, or other phenotypes that affect their ability to eat normal mouse food, provide an alternate food supply, perhaps either ground or dampened food.
• If mice cannot reach the "normal" water or food supply, provide access to both on the cage floor.


Table 4. Breeding problems and possible resolutions.
Problem Possible resolution
Mice are not breeding.
Make sure you have male and female breeders.
Add nesting material specifically designed for that purpose.
Try a different diet, for example, one with more or less fat.
Minimize stress—including human contact with the mice and activity and noise in the room. Construction-related vibrations, even outside the building, can disrupt breeding. If possible, move mice to a quieter area.
Try a 14:10 light cycle.
Make sure mice are healthy and able to breed (for example, not too obese or too old). Try healthier, younger animals.
If you never see vaginal plugs, try a new male.
Females are not getting pregnant.
Determine whether the problem is the male or female: pair a proven breeder female with the male and a proven breeder male with the female.
Surgically evaluate the reproductive tract. If the female appears normal, try low dose gonadotrophins, ovarian transplant, IVF; if the male appears normal, try IVF.
Research the strain reproductive characteristics. For a transgenic, check the effect of the transgene on breeding. For a strain carrying a mutation, check the effect of the mutation. Adjust the breeding strategy accordingly.
Mice get pregnant but you never see pups (females are resorbing fetuses).
D (above)
The most common cause of fetal resorption is stress.
Individual breeders are not producing an expected number of pups.
B, C, D, E, F, H, J (above)
Females give birth, but don’t raise their pups.
D, J (above) K
If you introduced a male to a cage with pups, he may have killed them. Wait to add a male until all pups have been weaned and removed.
Pups disappear or do not survive.
Colony productivity has dropped.
Check for environmental factors that might have changed. Consider room conditions (temperature, vibrations, building construction, odors, etc.), caretaker, and diet. If more than one cage, area, or strain is affected, expand the search. If possible, restore environment to previous conditions.

Definitions and calculations

DOB: Date of birth

Breeder ID: The pedigree numbers of the breeding female(s) and male

Breeder Unit:  Mice were either pair-mated (1 female and 1 male) or trio-mated (2 females and 1 male).  All matings were between siblings.

Dam Age at Mating (wks): The age of the female(s) on the day she was mated: = Mating Date - Breeders DOB/7

Dam Age at Birth of First Litter (wks): The age of the female(s) on the day her first litter was born, in weeks:  = Litter DOB - Breeders DOB/7

Dam Age at Retirement (wks): The age of the female(s) on the day she was retired:  = Date Retired - Breeders DOB/7

Litter Number: This number indicates the birth order of all the litters from a given breeding unit.

Latency to 1st litter/Days to First Litter (wks): The amount of time that elapsed between the mating date of a breeder unit and the birth of the first litter: = Mating Date - Litter DOB/7

Litter size born: The number of mice born in a given litter

Litter size weaned: The number of mice weaned in a given litter

Breeding Interval (wks): The amount of time elapsed between consecutive litters in pair-mated breeder unit:  = first litter litter DOB - second litter litter DOB/7

F Weaned: The number of female pups in the litter that survived to weaning age

M Weaned: The number of male pups in the litter that survived to weaning age

Total Weaned:  The total number of pups in the litter that survived to weaning age: = M Weaned + F Weaned

Wean to born ratio:  The ratio of pups weaned to pups born: = Total Pups Weaned / Total Pups Born

Fecundity index: = mean litter size / mean litter interval

Data & information collected

Data determined or derived

  • percent females at weaning
  • litter characteristics
    • interval between litters
    • number born
    • number weaned
    • wean to born ratio
  • fecundity index (mean litter size divided by mean litter interval)

Supplemental parameters

  • dam age at mating
  • dam age at retirement
  • breeding span (duration breeding from mating to retirement)
  • number of litters
  • breeding period (time breeding since current litter)