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TIPS FOR SUCCESSFUL BREEDING AND COLONY MANAGEMENT Office of Lab Animal Resources University of Colorado Denver AMC Tracy Haney, CVT, RLAT Holly Goold,

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Presentation on theme: "TIPS FOR SUCCESSFUL BREEDING AND COLONY MANAGEMENT Office of Lab Animal Resources University of Colorado Denver AMC Tracy Haney, CVT, RLAT Holly Goold,"— Presentation transcript:

1 TIPS FOR SUCCESSFUL BREEDING AND COLONY MANAGEMENT Office of Lab Animal Resources University of Colorado Denver AMC Tracy Haney, CVT, RLAT Holly Goold, CVT, RALAT

2 Environmental Factors  Drop in production (October-March)  Even though mice are in a controlled environment Light cycles, temperature, humidity, etc  Smaller, less frequent litters.

3 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

4 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

5 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

6 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 months Set-up Set in a clean cage Add enrichment/breeder chow “Charged” cage

7 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

8 Breeding Tips  Lithgow “Check for Pups/Wean” cards are provided in housing rooms Track births, pups lost, and successful weaning Tracking fertility

9 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

10 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”

11 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.

12 Breeding Specialists For information on breeding services or training/help needed, please contact: Tracy Haney for Barrier and R2: Holly Goold for RC-1:

13 Questions?

14 WHY DO MY MICE EAT THEIR BABIES?!? DIAGNOSING LACTATION PROBLEMS IN MICE TOWN HALL MEETING, OCT. 24, 2013 Jenifer Monks, Ph.D.

15 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

16 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

17 Tips for success: 1) Nesting material: thermoregulation, security, pheromones 2) “Do Not Disturb” pink cards peri-partum: reduce stress 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,”

18 Do they have milk bands? No? dead by day 3

19 You can inspect the cage without opening it

20 Estrous 4-5 days Gestation days Lactation days (if housed with male, remove weanling animals at 20 days old) 3) Careful record keeping Tips for success:

21 Gestation time of your strain is known: FVB 18.7 C57BL Balb/c A/J 20.5 Murray SA, Morgan JL, Kane C, Sharma Y, Heffner CS, et al. (2010) Mouse Gestation Length Is Genetically Determined. PLoS ONE 5(8): e12418.

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

23 Dam-Litter body weights: non-invasive monitoring mid-pregnancy resorption of litter Lactation Failure and Early weaning by mother ** n = 8 litters Compromised milk production

24 CD-1/ICR & Black Swiss mice 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

25 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 PloS one Photo courtesy Mouse Fancier website erforum/phpBB3/viewtopic.php?f=11&t=756

26 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 Safe Contact OLAR if you have unused animals that can be donated to training protocols

27 Still having trouble? Ask for help!  Online resources-Jackson Laboratories  OLAR: Care staff, vet techs, breeding specialists  Colleagues  …Andrew Lewis  Toothless mice  Mayonnaise milk

28 Website:

29 Transgenic and Gene Targeting Core

30 Peter J. Koch, PhD Professor of Dermatology and Cell & Developmental Biology, Director The Charles C Gates Center for Regenerative Medicine and Stem Cell Biology University of Colorado Saiphone Webb, BS Senior PRA, Laboratory Manager Embryo Manipulation Services Embryonic Stem Cell Development and Production Abby Zamora, CVT, LAT PRA Colony and Data Management Embryo Manipulation Services Abhilasha Jain, MS PRA Embryonic Stem Cell Development and Production

31  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.

32 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

33 Embryo Cryo Embryo Cryo by IVF Sperm CryoOvary Transplant Very Reliable Cryo of 2 cell embryos ( *) Reliable Cryo of 2 cell embryos ( *) Reliable in most strains Cryo~20 straws of sperm aliquots The last resort ! Surgical procedure wild type or genetically modified females Equal number of stud males Multiple attempts are usually needed 2 proven males (8-15 wks old) 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

34 Transgenic and Gene Targeting Core Supported by: NIH (SDRC, CCTSI), Gates Center, SOM, Dermatology Department, Service Fees

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