TIPS FOR SUCCESSFUL BREEDING AND COLONY MANAGEMENT

Office of Lab Animal ResourcesUniversity of Colorado Denver AMCTracy Haney, CVT, RLATHolly Goold, CVT, RALAT

Environmental Factors

Drop in production (October-March) Even though mice are in a controlled environment

Light cycles, temperature, humidity, etc Smaller, less frequent litters.

External factors

Odor from staff (perfume/cologne, cigarette smoke)

Entering housing room after hours (8p-6a) Mice mate 5-6 hours after dark cycle starts

Conversations/music/phones in housing room Amount of traffic in housing room Shoving cages anywhere on rack Inexperienced /unconfident handling

of mice

Cage location

In room If possible, away from door and ATS

On rack Lowest row on rack, away from lights Keep separate from experimental mice

Less cage movement

Enrichment

Helps decrease stress/increase pup survival Manage their environment Mimics natural nest

Brown paper shred Mouse huts/paper towel rolls

Con Remove before cage wash if combined with nestlet Must use ~6-8 grams of shred Hard to visualize litters

Breeder Set-up

Setting Breeders Mice and Rats

Females: 5 weeks to 8-10 months Breed females before 3 months of age

better fertility Litter sizes decrease with age Genetic issues

Poor lactation in homozygous moms Males: 6 weeks to 12-18 months Set-up

Set in a clean cage Add enrichment/breeder chow “Charged” cage

Types of Matings

Continuous pair Pros

Post-partum estrous cycle Litters every 21 days

Cons Possibility of two litters in

the cage Non-Contiuous

Pros No double litters

Con Have to remove the male Less frequent litters

Triad Pros

Sister females, synced estrous cycle

Cons Unrelated females

1 Dominant female Requires protocol

approval Male rotated between

cages Miss females’ post-

partum estrous

Breeding Tips

Lithgow “Check for Pups/Wean” cards are provided in housing rooms

Track births, pups lost, and successful weaning Tracking fertility

Breeding Tips

DO NOT retire old breeding pairs until after confirming successful breeding of new pairs

Retiring old breeders Set new breeding pair one month before retiring old Ensure successful breeding before ending old pair

Stagger setup Insurance to always have mice

Maintaining a Litter

Avoid extra handling of litter < 3 days old

Reduce litter size when able One sex vs phenotype Genotype early: 7-10 days

Footpad tattoos/tail snips

Cage changing Anticipating pup birth Transfer half of the old nest with pups

Fostering Keep environmental noise levels “even”

Troubleshooting

Pup death Stress to female

Disturbing the cage frequently Removing male right before or after pup birth Separating females from cage right before or after pup birth Manipulating pups- touching pups within a week of birth

First-time mom (C57 background) Transgenic lines

*Please remember that you must remain compliant with the IACUC rodent overcrowding policy. If multiple litters are in the cage, you are obligated to separate them for animal welfare reasons regardless of the risk of cannibalism or maternal neglect. Therefore, it is essential that you separate mothers prior to the litter being born.

Breeding Specialists

For information on breeding services or training/help needed, please contact:

Tracy Haney for Barrier and R2: tracy.haney@ucdenver.edu, 303-724-3982

Holly Goold for RC-1:holly.goold@ucdenver.edu, 303-724-2237

Questions?

WHY DO MY MICE EAT THEIR BABIES?!?DIAGNOSING LACTATION PROBLEMS IN MICETOWN HALL MEETING, OCT. 24, 2013

Jenifer Monks, Ph.D.

Jenifer.monks@ucdenver.edu

How could my gene mutation be affecting lactation?!?Gene mutations may alter mammary function: off-target

Cell cycle, proliferation, cell death-> mammary development

Cell polarization, secretion, metabolism-> milk production

Neurological/behavioral-> mothering, stress, let-down, feeding behavior in pups

Embryonic

Pubertal

Adult

Pregnancy

Lactation

Involution

Developmental Stages of the Mammary Gland

…use wild-type, heterozygous or hemizygous females whenever possible

Indications the Dam may be having trouble feeding her pups

Normal Estrous Get pregnant, stay pregnant Normal delivery, normal litter size Pups scattered around cage Die peri-partum (shortly after birth) or are

cannibalized Failure to thrive Wean runty or with bald butts Only alternate litters survive

Tips for success:

1) Nesting material: thermoregulation, security, pheromones

2) “Do Not Disturb” pink cards peri-partum: reduce stress

http://med.stanford.edu/ism/2012/march/mice.html Making mice comfy leads to better science, researcher says

“The shape of the nest tells an experienced person whether the animals are too hot or too cold, whether they are sick or whether they are about to give birth,”

Do they have milk bands?

No? dead by day 3

You can inspect the cage without opening it

Estrous 4-5 daysGestation 18-21 daysLactation 21-28 days(if housed with male, remove weanling animals at 20 days old)

3) Careful record keeping

Tips for success:

Gestation time of your strain is known:

FVB18.7

C57BL6 19.6

Balb/c 20.1

12920.2

A/J20.5

Murray SA, Morgan JL, Kane C, Sharma Y, Heffner CS, et al. (2010) Mouse Gestation Length Is Genetically Determined. PLoS ONE 5(8): e12418.

Mouse weighing to track reproduction:Normal gestation is 19 days- Co-housed with male-implantation delay when concurrently pregnant and lactating

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ICR 9- reproductive cycle

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Dam-Litter body weights: non-invasive monitoring

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DAMLitter

mid-pregnancy resorption of litter

Lactation Failure and Early weaning by mother

**

n = 8 litters

Compromised milk production

CD-1/ICR & Black Swissmice make good foster dams

Timed matings of both strains

Remove transgenic pups from biological mother and give to foster dam as soon after birth as possible

Warm in hand if cold and scattered

Roll in soiled bedding of foster dam

Remove unneeded pups to normalize litter size-10 teats, match natural litter size

Place carefully in nest

Cross-fostering to improve breeding success

Timed mating of mice

Four stages of estrous in BALB/cByJ mice. The four stages of estrous are shown for an albino strain (proestrus (A), estrus (B), metestrus (C), diestrus (D)).Mouse estrous cycle identification tool and images. 2012. PloS one    

Photo courtesy Mouse Fancier websitehttp://www.fancymicebreeders.com/mousefancierforum/phpBB3/viewtopic.php?f=11&t=756

Why Care about breeding efficiency? Save time Save money: $0.83/day/cage Better animal care = better science

Tips for Increasing Efficiency and Cutting Costs

• Do not set up breeders without knowing their genotypes.• Know what genotypes you are trying to produce and the number you will

need.– Overproduction = wasted animals and $$$– Underproduction = wasted time– Account for seasonal variation and holiday schedules

• Keep a reserve of breeder-age animals.• Retire/replace unproductive breeders (record keeping, 2 consecutive lost

litters), unused experimental animals, and animals of useless genotypes.• Do not keep singly housed animals• Keep a calendar for breeders and experiments

• * Collaborate *Know which animals are Raptor SafeContact OLAR if you have unused animals that can be donated to training protocols

Still having trouble? Ask for help! Online resources-Jackson Laboratories OLAR: Care staff, vet techs, breeding

specialists Colleagues

…Andrew Lewis Toothless mice Mayonnaise milk

Jenifer.monks@ucdenver.edu

Website: www.medschool.ucdenver.edu/Transgenics

Email: Makeamouse@ucdenver.edu

Transgenic and Gene Targeting Core

Transgenic and Gene Targeting Corewww.medschool.ucdenver.edu/Transgenics

Core PersonnelPeter J. Koch, PhDProfessor of Dermatology and Cell & Developmental Biology, DirectorThe Charles C Gates Center for Regenerative Medicine and Stem Cell BiologyUniversity of Colorado

Saiphone Webb, BSSenior PRA, Laboratory ManagerEmbryo Manipulation ServicesEmbryonic Stem Cell Development and Production

Abby Zamora, CVT, LATPRAColony and Data ManagementEmbryo Manipulation Services

Abhilasha Jain, MSPRAEmbryonic Stem Cell Development and Production

How You Can Lose Your Mouse Line The transgene stopped working; loss of

phenotype (e.g. promoter methylation) There is a change in the phenotype of your

mice (e.g. gene drift) A disease outbreak Breeding has stopped and you’re left with

only few male and female mice.

Solutions• Order replacement mice from a vendor(if the

line is available)• Request the mice from a colleague working

at another University – (this will take time to breed up the mice for experiments)

• Start over and generate the line from scratch (this will take more time and money)

• Re-establish mouse line using previous frozen sperm(IVF) or embryos

Methods and Requirements For Cryopreservation and

Line Rescue ServicesEmbryo Cryo Embryo Cryo by

IVFSperm Cryo Ovary Transplant

• Very Reliable • Cryo of 2 cell

embryos (300-500*)

• Reliable • Cryo of 2 cell

embryos (300-500*)

• Reliable in most strains

• Cryo~20 straws of sperm aliquots

• The last resort !• Surgical

procedure

• 10-12 wild type or genetically modified females

• Equal number of stud males

• Multiple attempts are usually needed

• 2 proven males (8-15 wks old)

• 15-20 wild type females

• Strain dependent

• Usually one attempt is needed

• 2 proven males (8-15 wks old)

• Recovery is strain dependent

• Female mice only

• Slow to expand the colony

Transgenic and Gene Targeting Corewww.medschool.ucdenver.edu/Transgenic

makeamouse@ucdenver.eduSupported by: NIH (SDRC, CCTSI), Gates Center, SOM,

Dermatology Department, Service Fees