Tips for Successful Breeding Colony OLAR Breeding Core University of Colorado Denver AMC Tips for Successful Breeding Colony

Breeder Facts Breeder Facts: General for Mice & Rats Ages for setting up breeders (vary depending on strain) General for Mice & Rats Females: ~5 weeks and can continue to ~8-10 months. Litter sizes will decrease as female ages around 6 months Females not mated before 4 months of age may have decreased fertility Males: ~6 weeks and continue until ~12-18 months.

Estrus Cycle Both mice and rats can start estrous around 35 days, cycling every 4-5 days with estrus lasting about 12 hours. Visual of cycle Diestrus Proestrus Estrus Metestrus

Estrous Cycle Vaginal swabs smeared onto slide under microscope Visual of cycle

Breeding Setup New breeding pairs should be set in a clean cage or female(s) may be placed in singly housed male’s cage Pheromones in cage can place female in estrous quicker Provide Breeder Chow 2919 Add Enrichment Brown paper shred Toilet paper roll Hut Place “Check for Pups” Card on Cage

Lithgow Cards Lithgow “Check for Pups” cards are provided in housing rooms On the back of these cards you can track births, pups lost, and successful weaning. A female can be retired by tracking the decrease in her fertility (indicated by increasing time between litters and / or decrease in average litter size)

Types of Mating's Continuous pair mating Pros Cons Post-partum estrous cycle, litters every 21 days Cons Overcrowded cages, possibility of two litters in the cage Non-Continuous mating Better pup survival for difficult to breed strains Miss female’s post-partum estrous

Types of Mating's Continued Triad mating 2 females with 1 male Pros If females are sisters, synced estrous cycle Two females can remain together to help raise each others litters Male has to be removed If more than 14 pups you must contact a vet tech Cons If females are not related, usually 1 dominate female gets pregnant Must remove male from cage Male can be rotated between females in different cages Requires protocol approval Harem mating 3-4 females with 1 male Only allowed if pups are used before live birth

Plug checks Timed Pregnancies Set pairs day before Check for plugs early next morning (6-7am) Day plug found equals day 0 of pregnancy Separate male from cage if plug found Finding a plug does NOT confirm pregnancy only that the mice mated Maybe cheaper and less time consuming to order timed pregnant females from approved vender

Things to Know About Your Housing Room Light cycle, temperature and humidity are controlled in housing rooms Breeders should be placed in areas where there is the least amount of disturbance- if possible away from doors/ATS’s and on the bottom row of racks Avoid dark cycle interruptions. Mice usually mate 5-6 hours after dark cycle begins No access to rooms 8p to 6a 2919 (Breeder Chow) offered at no additional charge Increased protein and fat for nursing moms Available upon request Flag your cages with an “Extruded Diet” Lithgow card and mark “Breeder chow”

Enrichment Enrichment helps decrease stress and increase pup survival Mouse huts/paper towel rolls Con Female(s) can hide from male Can also use to attack other mice Brown paper shred Manage their environment Mimics natural nest Must dispose of before cage wash Harder to see litters/health check **Available upon request**

Breeding Concerns Many rodent colonies experience a drop in production during winter months (October-March) May result in smaller litter sizes, irregular estrous cycles, and lower conception/wean rates If your colonies regularly experience this drop, you should set up additional breeding pairs around September/October to ensure a continuous supply of younger breeders during these months Non-productive breeders Remove from colony if: No litter produced within 60 days from original mating 90 days for “slow starters” No new litter 60 days from last born date No weanlings survive from 2 consecutive litters

Breeding Tips To retire old breeders, set new breeding pairs a month prior to ensure successful breeding before ending old pairs DO NOT retire old breeding pairs until you have new litters Staggering your setup of breeders can help to ensure that you always have a pair producing New Breeders should be setup when your current breeders are about 6-7 months old Old breeders should be retired between 8-10 months

How to Maintain a Line Set-up 2-3 breeding pairs Non-continuous Set-up new breeders using weanlings New pairs should be set when pups are 6-8 weeks old Keep one cage of 4-5 males and one cage of 5 females for future breeders Remove retired breeders and any extra mice from the colony Maybe more cost effective to cryopreserve Gates.Bioengineering@ucdenver.edu

Maintaining a Litter Reduce litter size Limit competition by: Sex Phenotype Early genotyping Avoid any disturbance of litter < 4 days old Cage changing Transfer at least half of the old nest with the pups Keep environmental noise levels “even” Consider fostering litters for difficult strains

Cross-Fostering Litters Donor and recipient litters should be within 2 days of age, and neither litter should be more than 4 days of age, for best results If possible cross-foster strain to strain or use a recipient strain with a different coat color from the donor strain, for easier identification of the cross-fostered pups Outbred strain such as CD-1, ICR, or Swiss, tend to make the best foster moms Remove the recipient mom from her cage When you pick her up, try to get her to urinate on the pups

Cross-Fostering Litters cont. Remove some (or most) of the recipient’s pups if she has a large litter After cross-fostering, the total number of pups in her cage should be approximately the same as original litter Mingle the transferred pups with the recipient’s pups and rub them with dirty bedding from the recipient’s cage Return the recipient mom to her cage and place it back on the rack Observe the recipient cage 24 hours after cross fostering

Troubleshooting Stress to females can cause pup death by neglect or cannibalism. Some examples are: Disturbing the cage(s) frequently Vibrations and loud noises 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. *Please remember that you must remain compliant with the IACUC rodent overcrowding policy. * Do you know the most common reason why mice do not breed? Same sexes

External Factors Problems with breeding can be due to external factors such as: Odor from staff (heavy perfume/cologne, cigarette smoke, cats) Volume of conversations/music in the housing room Shoving cages into cage slots anywhere on the rack Place experimental mice on non-breeding racks if possible The number of lab personal working in the room daily Inexperienced /unconfident handling of the mice Entering the housing room after hours (8p-6a)

OLAR Breeding Core Our Group The Breeding Core is a group of 8 team members 2 Breeding Specialists (BC2’s) that help the labs setup and maintain their breeding colonies 4 Assistant Breeding Specialists (BC1’S) that maintain their own housing rooms and complete the breeding work in their rooms and a student worker A Manager that meets with new labs and discusses breeding needs and concerns

What We Do Set-up new breeders and advise the labs when to replace old breeders Wean, tag and provide snips to labs for genotyping Footpad tattoos and snips up to P9 Provide breeding updates weekly Provide a monthly census on animals 90 days and younger Euthanasia's Breeding core animals Retired Breeders

OLAR Breeding Barrier R2 Suite 4 A shower in suite only accessible to the Breeding Core Quiet Low foot traffic One designated BC1 per breeding room Transfers to RC1 on Thursdays No additional testing or paperwork needed

OLAR Breeding Core Cost Breeding cost: $30 per mating/successful litter If triad mating is requested, each female will get an individual mating number and be charged a $30.00 mating fee Includes: Setup of breeders Weekly monitoring of colony and database management Managing breeders includes Enrichment, +/- separating male and weaning pups Detailed record keeping Euthanasia of any mice handled by the core Mating cards are placed on the cage Pups are weaned and ear tagged, their ear tag number, DOB, and breeding number are recorded on an ID card

Example OLAR Breeding Summary

Example OLAR Census

Special Requests and Breeding Services Weaning, ear tagging, paw tattooing, snips for genotyping and plug checks $36.32/hr (Tech Time) Additional tissue sample taken from ear General training on breeding colony management FREE Troubleshooting for breeding problems Rodent breeding information resources Other services as requested

Rodent Breeding Information Resources Vendors Jackson Lab, Charles River Lab, Taconic, and Envigo Colony management websites SoftMouse.net Mousecolony.com OLAR Breeding Core Olarbreedingcore@ucdenver.edu 303-724-8350

OLAR Breeding Core

Special Thanks BC1s Student worker OLAR management Our labs Arthur Vigil Jenette Skees Michelle Mangette Brittany Lebere Student worker Sarah Railsback OLAR management Our labs

Questions For information or questions on breeding services or training/help needed please contact: tracy.young@ucdenver.edu, 303-724-3982 cassandra.levens@ucdenver.edu, 303-724-2237 scott.sand@ucdenver.edu, 303-724-8350 OLARbreedingcore@ucdenver.edu