Guidelines for Troubleshooting Breeding Difficulties in Mice

Investigators and laboratory staff members managing breeding

1. Problem breeding may be defined by demonstration of one or more of the following metrics in at least three cages of pair housed breeders of the same strain or stock:
   1. Greater than 90 days since the mating date and no pups have been born.
   2. 4 or fewer pups born in each of the last two litters.
   3. Of the last four litters born, two or fewer had any pups survive to weaning.
   4. Or as otherwise determined by a ULAM Faculty Veterinarian.
2. When attempting to determine which strains or stocks are difficult to breed, accurate breeding records are essential.

1. The faculty veterinarians are an invaluable resource when attempting to troubleshoot breeding concerns. If breeding efficiency is unsatisfactory, contact the area faculty veterinarian for a consultation.
2. Faculty veterinarians may suggest one or more of the following best practices to improve breeding performance:
   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: Place breeding cages on the lowest shelf of their rack, and locate their rack away from areas of high traffic and light such as the door and flow hoods.
   3. Enrichment: Strains or stocks with poor breeding efficiency may benefit from both shredded paper, like Envirodry, and a nestlet. If available, a shelter may be beneficial as well.
   4. Nutritional support: Breeding females require a higher protein and fat diet. A diet formulated for breeding animals such as (PicoLab 5058), Love Mash (Bio-Serv), or the addition of irradiated sunflower seeds may improve the nutritional plane of breeding females. 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. Planning for this fluctuation can help maintain a stable population of target animals.
   6. Environmental factors: Vibration, noise, or fluctuations in humidity can impact breeding success. Minimized these factors when 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. This may 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, the next tier of intervention may include changing caging type or providing an “aunting” dam to reduce maternal stress. Note that there may be increases in per diem associated with these changes.
   1. Caging:
      1. Change from ventilated to static housing. (May require increased cage changing frequency to maintain sanitary standards).
      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. Use stocks or strains known for good mothering instincts (Swiss Webster or FVB/N) for “aunting”. These animals can come from within the research colony, or can be purchased as retired breeders or as timed pregnant females, as required, from approved rodent vendors.

1. If the best practices described in Procedures section 2 improve breeding performance such that problem breeding is no longer a concern, continue to breed in cages within Guide-required cage densities.
   1. If despite the improved breeding performances, more than 2 adults and 1 litter housed in one cage is desired, contact the husbandry supervisor to request larger caging (at an increased per diem rate) 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 vivaria.
2. If the best practices described in Procedures section 2 did not improve breeding parameters, submit justification to the IACUC via a protocol amendment to house 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, request a SToP Form documenting IACUC approved overcrowding to be placed in the housing room.
   1. Label cages for which trio birthing is approved 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.

1. Ensure justification is included with each three-year renewal of the associated protocol to maintain the exception to housing within Guide-required cage densities.

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.
   1. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154966
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
   1. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127875