TIPS FOR SUCCESSFUL BREEDING AND COLONY MANAGEMENT Office of Lab Animal Resources University of...
Transcript of TIPS FOR SUCCESSFUL BREEDING AND COLONY MANAGEMENT Office of Lab Animal Resources University of...
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: [email protected], 303-724-3982
Holly Goold for RC-1:[email protected], 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.
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
Website: www.medschool.ucdenver.edu/Transgenics
Email: [email protected]
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
[email protected] by: NIH (SDRC, CCTSI), Gates Center, SOM,
Dermatology Department, Service Fees