Management of Mouse Breeding Colonies The Principal Investigator

Management of Mouse Breeding Colonies

The Principal Investigator must have an approved IACUC protocol describing the breeding project and its relationship to other research projects. This protocol must detail the responsible chain of command for all activities related to the breeding program, including contact information for each individual.

The CMF staff monitors animal health daily but the research staff is responsible for management of rodent breeding colonies. He/she must check all breeding cages for pregnancy, births, pup size and quantity (recommended minimum of 3 times per week during breeding).

An investigator may request that CMF manage their breeding colony by completing a breeding colony management request form. This form can be found on the CMF web site. The form should be submitted to the CMF Veterinarian, Dr. Farshid Azarafrooz. The breeding management service will be charged on an actual time basis.

Breeding colonies under CMF management are housed in a locked room with limited investigator access. Contact the CMF Office for access. The location of the breeding room will depend on the health status of the colony for which breeding colony management is requested.

The standard mouse cage will accommodate one adult female mouse, one adult male mouse, and their litter. Harem breeding (one male and up to three females) is permitted with careful management. Pregnant females must be separated before they have delivered their litters and only one litter is allowed in a cage at one time; exceptions (e. g. breeding history of the strain, taking advantage of post partum estrus) must be justified in the breeding protocol.

Male mice that are placed with female(s) for breeding must not be put back into a cage with other males. They must be single housed to avoid fighting.

In general, litters must be weaned at 21 days of age. The day of birth is counted as day zero. Weaning may be delayed up to 28 days of age for strains whose pups are smaller than normal, as long as there is only one litter in the cage. When weaning, up to four adult mice of the same sex may be housed in a standard mouse cage. Keep male littermates together when weaning, not mixed with males from another litter. Any deviation from this policy must be detailed and justified in the IACUC protocol.

Research staff are responsible for completing the rodents weaned form and submitting to CMF on the day of weaning. This form will be used to account for animals in the IACUC approved protocol and to create bar coded cage cards. The form is available on the research portal.

Cage cards must not be reused. Substitute or handwritten cards can not be used in place of standard bar coded cage cards. When you separate or wean animals a form must be submitted to CMF through the research channel for new cage cards to be generated. Place a blue temporary cage card and a hand-written white card on the new cage. New white cards with bar codes will be generated and placed in the animal housing room. If the blue cards remain on the cage for more than 5 business days, a daily fee will be levied against your account. It is your responsibility to check the accuracy of the new cage card and to place it on the correct cage.

l Cages that are overcrowded will be tagged by CMF staff with a red “overcrowded cage” card. Cages must be separated by the research staff within 3 business days of the date written on the card. If the PI does not correct the situation CMF staff will separate the animals and a fee (currently $20. 00 per cage) will be charged. CMF has the right to separate animals immediately if animal welfare concerns exist.

DATE INVESTIGATOR DEPARTMENT IACUC # This cage is overcrowded and must be separated/weaned within 3 business days. If not done, the service will be performed by the CMF staff and a fee for service charge will be levied against your account.

Genotyping is recommended prior to weaning so appropriate cage cards can be generated and so that the animal numbers are deducted from the appropriate strain and pain category on the approved protocol. CMF staff is available to assist with weaning, sample collection and identification. Call the CMF administrative office or visit the CMF web site for a list of service charges.

Breeding Basics

l Sexual maturity. Laboratory mice reach sexual maturity at 28 – 42 days of age. Breeding onset for females is 6 – 8 weeks, for males 8 – 10 weeks. l Reproductive life span. Laboratory mice can breed for about seven to eight months, producing four or more litters. However, some strains produce only one or two litters. If a pair is breeding well beyond its expected reproductive life span, retain it until the female is not pregnant within 60 days of her previous litter’s birth.

l Gestation. The gestation period for laboratory mice is generally consistent within a strain but varies among strains from 18 -21 days. l Generation time in laboratory mice is about 12 weeks: ~three weeks gestation, three to four weeks suckling, and two to three weeks until sexual maturity.

l Litter size varies among strains, ranging from about two to three pups/litter in some poorly breeding substrains to 12 or more pups per litter. First time mothers may have a smaller litter size.

l Weaning age. Weaning refers to removing a pup from its home pen. Generally, laboratory mice are weaned between 21 and 28 days of age. Weaning age depends on weanling size and maturity. Although most strains are weaned when they are 21 days old, some benefit from being weaned when 28 days old.

Breeding performance of laboratory mice can be affected by many factors, including: • • • First Time Mother. Birth defects in the pups. Hybrid vigor. Hybrid mice tend to have more, larger, and healthier litters than inbred strains. Strain-specific behaviors. The aggressive behaviors of some strains and the poor mothering instincts of others affect breeding performance and pup survival. Mutations and transgene effects. Some mutations are embryonic lethal; some cause infertility or reduced fertility; some affect mammary gland function. The severity of such effects depends on strain background.

• Temperature and humidity. Laboratory mice breed best when the temperature is between 65 -75 o. F (~18 -23 o. C) and the humidity is between 40 -60%. If the temperature and humidity are uncomfortable to humans, they are probably uncomfortable to mice. • Light intensity and light cycle. Because mice generally breed at night, breeding performance is best when a consistent and uninterrupted light-dark cycle is maintained. Either a 14 hour lights on/10 -hour lights off cycle or a 12 -hour lights on/12 hours lights off cycle work well. CMF maintains a 12 -hour lights on/12 -hours lights off cycle. Requests for altered light cycles will be handled on a case-by-case basis.

• Noise and vibrations. Disturbances such as changes in noise and vibration levels can decrease breeding performance and may induce the mothers to cannibalize their pups. Construction-related noises and vibration are particularly problematic, but loud voices may also cause a problem. • Health. Laboratory mice may stop breeding if they are unhealthy.

• • • Handling. Laboratory mice respond best to calm and consistent handling. Pregnant mice, mice giving birth, wildderived strains and mice with new litters should be handled as little as possible. Odors. Noxious fumes, perfumes, and other strong odors can decrease breeding performance. Nutrition affects breeding performance. Some strains breed better when fed a higher fat diet. If your mice are not breeding well and you want to change the food, speak to the Operations Manager, CMF for alternative diets.

Feed. Some strains of mice are prone to malocclusions, no teeth, or other phenotypes that affect their ability to eat pelleted feed. These mice need special foods, such as ground or moistened pellets. If you notice overgrown incisors in your colony let CMF staff know so that they can clip their teeth (for a fee) and monitor the mice more closely. • Feed placement. Obese mice or older breeders sometimes cannot reach the food hoppers. Therefore, their food should be placed in a relatively low position in the cage or on the cage floor. •

Enrichment. Neslets, paper towels, Crink-l’Nest, and igloos provide security and/or nesting materials; these may alleviate stress and improve breeding. These items must be autoclaved prior to being placed in the cage. CMF will provide enrichment items.

To optimize the breeding performance of laboratory mice, observe the following practices: Replace breeders before their reproductive performance declines. Maintain breeder pairs of various ages by replacing a percentage of them monthly or weekly. A colony of mixed-aged breeders produces a more consistent number of pups than a colony of even-aged breeders. Replace non-productive breeders. The following signs indicate that breeders are non-productive: • They produce no litter within 60 days of mating (longer may be acceptable if delayed breeding is a strain characteristic). • They produce no litter within 60 days of their last litter and are not visibly pregnant. • They produce litters but the litters do not live to weaning for two to three litters.

Mate mice early. Mate mice when they are six-to-eight weeks old. Younger mice generally breed better than older ones. There can be a risk with very young, inexperienced mothers with their first litter. Use experienced males. Pairing young females with older, proven males often improves breeding performance.

Keep accurate breeding records. To evaluate the breeding performance of a mouse colony, maintain accurate records and review them regularly. The sooner a problem is detected, the sooner it can be corrected. • Investigate deviations in breeding performance and phenotype immediately. • Compare your colony’s breeding performance to that characterized by your supplier (call technical support). All mouse facilities are different; strains that breed well in one facility may not breed well in another. • Keep a colony’s environmental conditions suitable and stable. • Verify the genotypes of breeders, including those with a visible phenotype.

Litter fostering Females of some strains are poor mothers or cannot nurse. In such cases, litters may need foster care to survive. The foster mother must have a healthy and well-fed litter of her own that is within one or two days of age of the fostered pups. It is very helpful if her pups are of a different coat color than that of the fosterlings.

The foster litter should be no larger than the natural litter. If the foster litter is larger than six pups, divide it between two foster mothers. Remove the proposed foster mother and place her in a holding pen. Place the fosterlings in the foster mother’s home pen and cover them with some nest material or bedding so they acquire her scent and the scent of her pups. To be sure that the foster mother is feeding the pups, observe her and the pups carefully for a day or two. If litter survival is crucial, divide the litter among several foster mothers.

Mating numerous females simultaneously To induce numerous females to produce same-age pups, take advantage of the Whitten Effect. House four females together. The dense co-housing suppresses the females’ individualized estrous cycles. Then, induce them to resume their cycles simultaneously by exposing them to male androgen or shavings from a male cage. Mice have a four- to five-day estrous cycle and ovulate on the third day. Placing the females with a male on the third day of their cycle will result in the maximum number of pregnancies. For best results, house stud males individually for one to two weeks and then add females to the males cage.

You can tell that a female has mated in the last eight to 30 hours if her vagina contains a copulatory plug (a white or cream‑colored plug of solidified ejaculate). Because mice usually mate four to six hours into the dark cycle, look for a plug as early into the light cycle as possible. Otherwise, the plug may be dislodged or dissolved.

The nature and location of the vaginal plug can be a strain characteristic: it is superficially evident in some strains but deep in the vagina in others. The presence of a plug indicates only that the female has mated, not that she has conceived. Pregnancy may be verified by palpation on the eleventh day of gestation (day zero is the day a plug is found).

Basic principles for managing small mouse colonies: • • • Contemplate cryopreserving a strain in case breeding performance either declines, ceases, or a catastrophic event threatens your colony. Mary. Jo Johnson in the CMF office can provide you with a list of vendors who provide this service. Maintain a minimum of six breeding pairs representing different generations in your colony. Retain two generations of a strain, and do not eliminate one until the next one is producing.

• Keep the age range of your breeders between • • two and eight months old; older mice may not breed reliably. Monitor breeding performance closely; if performance declines, promptly take corrective measures. Consider backcrossing your strain approximately every 10 generations to prevent substrain divergence.

Maintaining Genetic Quality • • Employ proper nomenclature to describe your mouse models. Include a detailed description of the genetic background of the mice you use in all your communications.

• Obtain mice from a reliable breeding source. • Acquire new breeding stock from your supplier • periodically. Use a common genetic background when possible, so that your experiments can be replicated.

THE END

Contact Information Mary. Jo Johnson Karen Wielgus Procurement Ext. 69178 mjohns 5@luc. edu Breeding Colony Management Ext. 68316 kwielgu@luc. edu Farshid Azarafrooz Clinical Veterinarian Ext. 65162 fazarafrooz@luc. edu Tina Frese Lead Lab Animal Tech Ext. 64846 tfrese@luc. edu

Comparative Medicine Facility Web Site http: //hsd. luc. edu/comparative-medicine/