1. Colony Management & Breeding

2. Topics Colony Management CLAR responsibilities PI responsibilities Breeding Planning Genotyping Documenting (breeding log) Breeding systems Weaning No later than 28 days Fighting Cage density Overcrowding Corrective action for violations Animal Identification (SOP 3.16) Tattooing (SOP 7.12) Ear notching Animal Transfers (SOP 1.11) Animal Numbers Billing Communication

3. Basic Breeding Responsibilities CLAR Provide housing materials (cages, rooms, environmental monitoring/controls) Provide all bedding, food, water, nesting materials, etc. Maintaining all documentation on the care and use of the animals in CLAR PIs Provide genetics information Submit timely requests

4. Who Does the Colony Management? PIs: May do their own breeding and colony management – They must monitor the breeders, create breeding cages and wean animals per CLAR standards – Ensure cages do not become overcrowded – Provide accurate and thorough documentation of breeding and weaning CLAR: Will provide breeding and colony management assistance – All genetics of the colony breeders must be provided prior to breeding for specific genetics is attempted – Requests for breeding must be submitted in a timely manner – Charges will be accrued for technician time devoted to managing your colony ($25 per hour) Chargeable time includes: emails, maintenance of records, breeding, weaning, etc. *Note: Investigators may choose to do as little or as much colony management as they wish. Please communicate exactly what you wish to do and what you wish for CLAR to do.

5. Colony Maintenance Identify breeders: – Males are sexually mature 7-8 weeks old. – Females are sexually mature 6-7 weeks old. – Generally, breeders are set up when mice are 8-10 weeks old.

6. Colony Maintenance (cont.) Set up breeding cage – Breed 1 male with 1 or 2 females. – The male is placed in a cage by himself and allowed 1-24 hours to mark the cage with his scent. The male is NEVER taken to the females’ cage. – When mice are bred a breeding card is used which provides information about the genotype, generation, dob, identification of mouse, species, strain, sex, parents of the breeder and protocol number.

7. Colony Maintenance (cont.) Confirm breeding receptivity – Check the vulva of the female for redness and swelling, this is a good indication she is in estrus and will be receptive to the male. The estrus cycle is 4-5 days and the female should become pregnant 4-5 days after being placed with the male. – The female is placed in the male’s cage. – Daily observation of the breeding cage is necessary to check for fighting, injuries, and health of the breeders. If the female is pregnant she will show obvious weight gain as early as 2 weeks post breeding. – The female is removed when pregnancy is first observed, usually 2-3 weeks post bred date, means the breeding cage should be checked daily for compatibility and health of breeders during week 1-2 and for pregnancy during week2-3. – Record on the females’ breeding card the date she was removed from the male. If the female has not given birth within 21 days post removal from the males’ cage she was not pregnant. This practice is helpful when obese females are breed.

8. Colony Maintenance (cont.) Weaning – The pups stay with the mother for 3+ weeks (21-24 days) and are weaned (no later than 28 days) according to gender. – Weaning should occur at the beginning of the week, no later than Wednesday. – Give a water bottle, and put food on floor of weanling cage. – Daily observation of weanlings is mandatory for a minimum of 1 month post weaning. Check for eating and drinking (solid and liquid waste can be observed in the cage). – Make sure mice are not hanging from the wire bar lid. – DNA collection is done between 21-28 days old.

9. Mouse Breeding Data Gestation Time: 19-21 days Age at weaning: 3 weeks (21-28 days) Age at sexual maturity: 6-8 weeks Approximate weight at birth: 1g Approximate weight at weaning: 8-12g Approximate weight of adults: 30 - 40g (male > female) Lifespan in laboratory: 1.5-2.5 years Average litter size: 6-8 Number of litters per breeding female: 4-8 Useful breeding life of females: 6-8 months may vary with genotype. Useful breeding life of males: 18-24 months may vary with genotype.

10. Breeding Log

11. Breeding Log (cont.)

12. Breeding Systems Pair Breeding – 1:1 male to female ratio, stable, long-term pair. Recommended for foundation colony perpetuating and to provide breeders to the pedigreed expansion colony. No more than 12 pairs are required per line. The method produces the maximum number of litters per female in the shortest time and simplifies recordkeeping. This system requires more males than polygamous mating systems. There is a significant increase in labor, space and equipment to service this breeding population. Polygamous Mating – 1:2 to 1:3 male to female ratio. There are three types of polygamous mating systems, however, only two can be used due to cage size limitations.  Colony mating or Trio system – One male and two females are housed together continuously. Females are not removed after parturition only the young are removed at weaning. The post partum estrus is utilized thus, very frequent checks (sometimes daily depending on size) of the colony are needed to record births and litter size. Early genetic identification of pups and the culling will help to lessen the likelihood of overcrowding.

13. Breeding Systems (cont.)  Harem mating – one male and 2 or 3 females are housed together. The females are removed from cage just prior to parturition. This allows for more young to be weaned per litter. This method does not utilize the post partum estrus, thus the number of litters produced will be lower. The labor and equipment cost are increased. There is also the possibility of male exhaustion resulting in psuedopregnancies. Rotation systems are a variation of the harem mating. The male is moved between cages of females. This system, like harem mating, will reduce female productivity, because the postpartum estrus is not utilized.

14. Guidelines for Detecting Fighting Mice Adult male mice WILL fight and they usually start at 7-8 weeks of age and older. Signs : Mice embracing, kicking up bedding, rolling around in the cage and making high pitched noises. One or more mice in cage may appear normal. Blood on fur Rough hair coat. Hair missing on back and or found in cage. What to do: SEPARATE the animals (one per cage) before they have open wounds on back or are castrated.

15. Overcrowding In breeding trios, the following are guidelines that should be followed in order to prevent overcrowding of breeding mice in cages. Monogamous pairs  The pair may stay together until the pups are weaned (no later than 28 days).  No more than one litter should be in the cage at any given time.  The oldest litter of animals must be weaned before the expected delivery date of the female. Trio breeding  The litter must be weaned prior to the delivery of a second litter by the same female.  One of the two females should be removed to a separate cage when observed to be pregnant. This facilitates compliance with housing standards and permits lineage documentation.  It is permissible to keep the male, two females and the pups together in a ventilated rack system ONLY IF the oldest litter reaches 10 days of age and there are no more than 12 pups in the cage.  Harem ratios of 1:3 or 1:4 are not allowed unless some of the pregnant females are removed once noted to be pregnant to restore a ratio of 1:1 or 1:2 prior to parturition.  Date of birth for the litters must be recorded on the cage card by the person conducting the breeding.

16. Overcrowding (Cont.) The following are conditions that will be considered overcrowding of breeding mice in cages:  A cage is overcrowded if a new litter is born before the older litter is weaned  Two litters from separate females coexist in the cage beyond 14 days.  A litter remains with the female beyond 28 days of age or if the animals are clearly robust and self sufficient after 21 days of age.  Weaning beyond 28 days of age is a special circumstance and requires either written veterinary approval (if done sporadically for health reasons) or prior IACUC approval (if done routinely by the laboratory).

17. Cage Density Requirements (9.5 in x 5 in= 48 in 2 caging) (10 in x 4 in = 40 in 2 caging) Body Weight: gMaximum Number Mice per Cage: <101 mother with pups 10-156 15-254 >253 Body Weight: gMaximum Number Mice per Cage: <104 10-152 15-251 >251

18. Animal Transfers/Moves (Cont.) Moving Animals within the Facility All animals moved within the facility must be moved within an approved transport cage. All cages must be covered while in transport (mouse filter top or rat filter bonnet) and all cages must be covered to prevent the public from viewing the animals. Transfers within the building but outside of the animal facility must utilize the route with the least amount of human foot traffic despite an increase in travel time if necessary. Documentation Upon removing animals from their housing rooms an Animal Relocation Card must be filled out and placed within the empty slot from which the housing cage was removed. Document the following: – Date of relocation – Time of removal – Number of animals removed – Protocol number of the study – Expected time of return – List of procedures completed and/or any other necessary information These cards are located in each room. Once animals have returned, the card must be cleared of all information and returned to the appropriate place within the room. It is only necessary to fill these cards out when animals will be returned.

19. CLIP SYSTEM WHITE CLIP: BREEDING RED CLIP: PREGNANT YELLOW CLIP: 0-14 DAYS OLD GREEN CLIP: 15 DAYS OLD-WEANING BLUE CLIPS: SERVE A DUAL PURPOSE & MUST REMAIN ON CAGE CARDS FOR A MINIMUM OF ONE (1) MONTH POST WEANING. Monitoring the mouse’s ability to maintain hydration after weaning. Blue clip right side of cage card ▼ : 1-14 days post weaning keep water bottle. Blue clip right side of cage card ◄ : 14-31 days post weaning remove water bottle and observe hydration. * Blue clip left side of cage card ▼ : DO NOT remove water bottle if on automatic water rack. Monitoring the mouse’s general health. Overgrown teeth, normal growth and activity hydrocephalus, hanging from wire bar lid, etc… Black clip= sick/dead /observe * Remove water bottles on Monday and Tuesday only when changing all cages.

20. Animal Numbers Number of animals produced under a single protocol are counted towards the total number allotted. This is assessed post weaning Closely monitor the number of animals produced. Not adhering to the approved animal numbers is considered a reportable offense to OLAW and could jeopardize all the research at MSM. Non-utilized animals may be transferred from one investigator to another (protocol transfer form). – The animal numbers will be subtracted from the receiving PI’s animal numbers. – The animal numbers will also be subtracted from the donating PI’s animal numbers if done post weaning. – Non-utilized animals are those that have not been experimentally manipulated.

21. Billing Tech time: $25/hr, minimally 1 hr charged Time charged includes:  communication of breeding needs  record keeping  maintaining the breeding cages  weaning  genotyping Weaning must be done by 28 days. Penalties post 28 days for overcrowded caging will be charged to the investigators. Repeated violations could result in reporting to OLAW. Animal numbers will be assessed upon weaning. Per diems are charged once animals are weaned.

22. Billing (cont.)

23. Communication Colony management worksheet Desired genetics (must have the breeding line’s genetic information first) Timely (no less than a week) Document (if maintaining your own breeding)

24. Colony Management Worksheet