LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II ...

LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES IIRATS AND MICE:Care and ManagementV-9042David M. Moore, MS, DVM, Diplomate ACLAMOffice of Animal ResourcesVirginia Polytechnic Institute and State Universityand theVirginia-Maryland Regional College of Veterinary MedicineBlacksburg, VirginiaThe Laboratory Animal Medicine and Science - Series II - has been developed by the AutotutorialCommittee of the American College of Laboratory Animal Medicine (ACLAM): G. L. Van Hoosier,Jr., DVM, Chair G. L. Borkowski, DVM; K. Boschert, DVM; J. F. Harwell, Jr., DVM; J. M. Linn,DVM; C. W. McPherson, DVM; A. F. Moreland, DVM; G. Otto, DVM.Instructional development and production assistance provided by Barbara Macfadden.The development of this program is supported by a grant fromthe DuPont Merck Pharmaceutical Company.Laboratory Animal Medicine and Science Series IIis produced by theHealth Sciences Center for Educational ResourcesUniversity of Washington

2 LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES IIUniversity of WashingtonHealth Sciences Center for Educational ResourcesBox 357161Seattle, WA 98195-7161 206/685-1156ISBN: 1-55910-052-4Copyright © 2000by the University of Washington Health Sciences Center forEducational Resources and the American College of LaboratoryAnimal MedicineAll rights reservedPrinted in the United States of America

V-9042 RATS AND MICE: Care and Management 3PRIMARY AUDIENCEVeterinary and biomedical students, animal care and researchtechnicians, and veterinariansSECONDARY AUDIENCEGraduate students, research veterinarians, and investigatorsGOALTo familiarize the viewer with the requirements and methods for theproper care and management of laboratory rats and mice.OBJECTIVESWhen you complete this program, you should be able to:1. Describe appropriate criteria for housing rats and mice.2. Describe the environmental parameters which need to bemonitored and maintained for their health.3. Describe the requirements for and problems associated withproviding food, water, and bedding for rats and mice.4. Describe good sanitation procedures.5. Describe several acceptable methods for handling, restraint, andtransport of rats and mice.6. Describe breeding and reproductive management of rats andmice.7. List and describe several acceptable methods of euthanizing ratsand mice.

4 LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES IIPRETESTPrior to viewing Rats and Mice: Care and Management, take time to assess your currentknowledge of care and management principles for rats and mice by answering the followingquestions. A post-test is available at the end of the program for review once you have completedthis autotutorial.1. Identify environmental parameters which need to be monitored and maintained, anddescribe adverse healtheffects when not maintained.2. List two major types of animal facility design.3. Describe the major types of rodent caging, giving advantages and disadvantages of each.4. List three types of bedding and describe potential health problems of each.5. Describe the basic food and water requirements of rats and of mice.6. Describe sanitation procedures and practices for rooms, caging, and equipment.7. Discuss methods to control or eliminate invertebrate and vertebrate pests and why this isimportant.8. Describe at least one method of restraint of rats and mice.9. Describe the ideal criteria or conditions for shipment of “clean” laboratory rodents.10. List problems with transporting animals from a primary holding area to a distant laboratory.11. Discuss the necessity for quarantine and daily observation of animals.12. Describe the estrous cycle and a method for monitoring mating.13. Discuss the effect of pheromones on reproduction in laboratory mice.14. Describe the three general types of euthanasia methods.

6 LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II6. Caging7. Cage size—miceCages for rats and mice are designed to includethree important characteristics: provide comfortableliving space, protect the occupants from airbornepathogens or chemicals, and be fabricated frommaterials that can be efficiently cleaned andsanitized. Mice are typically housed in plastic“shoebox-size” cages that have solid sides andbottoms, require absorptive bedding, and can beplaced on shelves or suspended from racks. Ratscan be housed in either plastic shoebox or wirecages. Suspended wire caging, preferablyconstructed of stainless steel, separate animals fromtheir excreta. Wire caging provides better ventilationthrough greater air exchange but has limited valuefor preventing airborne exposure to aerosolizedmicrobes or chemicals. Solid bottom cages withbedding are warmer for small animals and providemore protection from airborn contaminants.Floor areas listed in this image are based on bodyweight and illustrate the minimum space requirementsrecommended in the Guide for mice to livecomfortably. The recommended height of 5 in. (12.70cm) allows mice to sit up and make other normalpostural adjustments and excludes space occupied byfeeders built into cage lids (2).8. Social groups—mice A special consideration for housing is that male mice caged togethermay demonstrate aggression and will often fight and mutilate oneanother. When injuries or fighting are noticed during daily healthobservations, aggressors should be removed from the cage andhoused separately to prevent further injury or death to cagemates.9. Cage size—ratsThis image shows the minimum floor spacerequirements for laboratory rats. Currently, the interiorheight of all cages, excluding lid space, is 7 in. (17.78cm) (2). In general, the minimum floor space increaseswith every 100 g increase in body weight over 100 g.10. Spread of diseases Rats and mice are susceptible to a wide range of diseases that can bespread by direct contact, through poor hand washing practices bycaretakers, by contaminated caging or equipment, and by aerosolizedparticles and dust. Diseases introduce unwanted experimentalvariables; thus, procedures, practices, and specialized equipment areutilized to minimize or eliminate the spread of diseases.

V-9042 RATS AND MICE: Care and Management 711. Types of facilities Rats and mice have traditionally been housed in facilities where stableenvironmental temperature, humidity, and lighting are maintained inanimal rooms. The current trend, however, is to enhance these“conventional housing” facilities by the addition of specializedequipment, such as laminar airflow (LAF) hoods, individually ventilatedcaging systems, and cages with air filters built into the lids. Thisequipment increases the level of health protection for the animals bycreating barriers against the entry of pathogens. The “barrier” conceptis taken a step further by the design of buildings that utilize separatecorridors for moving clean and dirty equipment to minimize crosscontamination between work areas. In barrier facilities, supplies arechemically or steam sterilized, and personnel are required to showerand don sterile clothing prior to entry into the building or into animalrooms. The effectiveness of these barrier facilities has been proven intheir frequent utilization for maintaining specific pathogen-free (SPF) orvirus antibody-free (VAF) animals required for support of sophisticatedresearch projects.12. LAF pattern13. Filter bonnetsAnother way to exclude pathogens in bothconventional and barrier facilities is to minimize thedeposition of contaminated dust particles in cages byutilizing a laminar air flow (LAF) system. Air passesthrough High Efficiency Particulate Air (HEPA) filtersand is directed at high velocity in a uniform laminarflow pattern over an individually ventilated cage,through a hood, or across an entire room. Propermanagement procedures must be established andfollowed, however, because the benefits of thesesystems will be compromised if staff members do notpractice good personal hygiene or if equipment is notproperly sanitized.“Shoebox” cages can be equipped with filter covers toprevent microbial contamination by aerosols orfomites. Although there are various types of filterbonnets manufactured, they all limit air and gasexchange, allowing heat, carbon dioxide, moisture,and ammonia to build up within the cage, unless theyare individually ventilated.

8 LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II14. “Micro-barrier” cages15. IsolatorsThe principle of the filter cover has been improvedupon with the manufacture of a special “micro-barrier”small cage that provides a pathogen-free internalenvironment. The plastic top is fitted with a filter insert,a removable feeder/water bottle rack is installed as aninside lid, and the top and bottom halves of the cage fittightly together to provide a sealed environment. Tomaximize the protection afforded by these cages:_ caging, food, water, and bedding should beautoclaved,_ gloves or tongs used to handle animals and theoutside of the cage should be disinfected each timethe cage is opened, and_ cages must be opened in a disinfected laminar air flow hood.This image shows three types of isolators: rigid,flexible, and room-sized. Isolators can be used in bothbarrier and nonbarrier facilities to house eithergermfree animals or to maintain SPF or VAF animals.Traditional isolators can be relatively small, housingapproximately 9 rodent cages, or up to the size of aroom containing 100 or more cages. Incoming air isHEPA-filtered, and high air flow rates are required toprevent entry of contaminants should a small leakoccur in the glovebox. Animals are introduced into theisolator through chemical immersion tanks or insterilized cages, and all other materials must be eithergas sterilized or autoclaved prior to introduction.16. Section title BEDDING, FOOD and WATEROther materials in the animals' housing environment also play a critical role in ensuring theirhealth and well-being.

V-9042 RATS AND MICE: Care and Management 917. BeddingThis image illustrates a variety of bedding materialsused in lab animal facilities. Solid bottom cagesrequire the use of absorbent bedding to maintain acomfortable dry environment. Absorbent beddingoffers rats and mice the opportunity to make nestsand provides hiding places. Commercially availablecontact bedding includes a variety of absorptivematerials: hardwood chips, hardwood and softwoodshavings, processed peanut hulls, and pelletedcorncobs. Noncontact bedding, usually placed in pansunder suspended wire cages, includes: cageboard (acardboard material), multilayered absorptive cellulosepaper, or any of the contact bedding listed above.18. Complications Various types of bedding have occasionally been associated withrespiratory, digestive, skin, trauma, and biochemical complications. Forexample, aromatic hydrocarbons given off by softwood shavings, suchas cedar chips or pine bedding, can affect liver enzymes, therebyaltering an animal’s response to anesthesia or its metabolism of drugsor test agents. Contamination of natural product bedding withpesticides, herbicides, or other chemical contaminants prior toprocessing may introduce an unwanted variable into toxicity studies,necessitating analysis of the bedding prior to use. Exceptionally dustybedding can cause respiratory problems in humans and animals, andairborne particles can facilitate transport of microbial agents.19. Ammonia contaminationThis graphic demonstrates a complication associatedwith a plastic cage and absorbent bedding—thebuildup of ammonia. Urine in bedding can be brokendown by urease bacteria to produce ammonia. At highconcentrations ammonia can impede or eliminate thefunction of the mucociliary apparatus in the lowerrespiratory passages and predispose animals torespiratory disease. The respiratory tracts of rats andmice are adversely affected at 5 ppm, while thehuman nose cannot detect ammonia until it reacheslevels of 25 ppm. Frequent bedding changes andadequate ventilation of rooms and cages shouldminimize ammonia buildup. Since filter tops on cagesminimize air exchange, there is an increasedrequirement for cleanliness and adequate ventilationwhen using them.

10 LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II20. Watering21. DietWater should be provided to rats and mice ad libitum,by water bottles equipped with sipper tubes or by theuse of an automatic system with drinking manifolds.Rats consume approximately 10 ml/100 g of bodyweight per day; mice consume approximately 1.5ml/10 g of body weight per day. Water bottles, sippertubes, and stoppers should be washed and sanitizedeach time they are removed for refilling. On automaticwatering systems, animals can contaminate themanifolds with oral microbes while drinking, and theinfectious organisms can be spread throughout thesystem. Water lines must be flushed periodically withwater or appropriate antibacterial agents followed bythorough rinsing to remove chemicals.Hyperchlorination or acidification of the water supplycan also be used to deter the build-up of bacteria inthe lines. These treatments can, however, adverselyaffect immune parameters and act as unwantedexperimental variables.Rats and mice require a low fiber diet (5%) and aretypically fed a commercially pelleted diet referred to as“rodent chow” (in feeder) (3). Rats consumeapproximately 5 g per 100 g of body weight in food perday and mice 12 g per 100 g of body weight per day.The adequacy and availability of food can affect thehealth and well-being of rats and mice and can impactexperimental results if the quality is inappropriate.22. FeedingThree methods for food presentation or delivery areshown in this image:_ in feeders pressed into wire grid tops of shoeboxcages,_ in stainless steel feeders hung inside the cage, or_ in special feeding cups designed for powdered(mash) feed.Rats and mice will dig powdered feed out of cups,making it difficult to assess food consumption. Asshown here, special grids or lids are placed on or inthe cups to minimize wastage and aid record keepingfor research purposes.

V-9042 RATS AND MICE: Care and Management 1123. Section title ENVIRONMENTChanges in environmental parameters such as temperature, relativehumidity, and light intensity are known to cause changes in thephysiological reactions of animals and could show up as unwantedexperimental variables in research data. Thus, environmentalparameters should be kept as stable as possible within normal ranges.24. Physical The environmental parameters in a room (macroenvironment) may bevery differentconditions from those within an animal cage (microenvironment). Thus, the designand use of caging—suspended wire versus shoebox cage with filtertop, the number of animals per cage, the total animal population withina room, the humidity within the cage and the room, and the number ofair changes per hour can affect environmental conditions.25. Temperature The Guide recommends room temperatures for rats and mice in arange of 18 to26_C (64-79_F) (2). Room temperature can be monitored and adjustedby sophisticated electronic monitoring systems or evaluated by use ofmaximum-minimum thermometers located in each animal room.Environmental temperatures outside these accepted ranges can affectreproductive performance, food consumption, and metabolism of testagents. Rats and mice can produce significant heat depending onventilation within the cage and the cage population, so cagemicroenvironmental temperatures can exceed recommended ranges.Where LAF or other systems with high rates of air exchange areutilized, temperatures within and around the cages may decrease, andchilling of animals, especially nude mice, may occur.26 HumidityRecommended relative humidity (RH) for rats and miceshould be maintained in a range of 30 to 70% (2). Asillustrated in this image, RH can be measuredelectronically and monitored centrally in facilities withmodern air handling systems, or it can be monitoredwith a hygrothermograph chart recording device placedin a room. Undesirably high humidity may be caused byexcessively wet bedding, rooms containing racks withautoflush waste pans, and low room air exchange rates.High air flow rates and heated air during winter monthscan cause the RH to drop below desired levels.27. High or low RH High humidity, coupled with an elevated room temperature, can lead toheat stress and reduced resistance to infections in rats and mice (4).Low humidity results in increased accumulation of dust in animal cagesand rooms--dust that can transport viral and bacterial agents. Lowhumidity can also decrease the function of the mucociliary apparatus inthe respiratory passages, allowing particulate matter to accumulate inthe lungs, predisposing the animal to pneumonia and other respiratoryillnesses.

12 LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II28. RingtailThis image shows a rat with the pathological conditionknow as “ringtail.” Ringtail is manifested ascorrugated, constricted, annular rings around the tailwhich may decrease vascular circulation and lead tosloughing of the tail distal to the constricted region.The condition is most likely to occur in preweaningrats and is associated with low humidity (

V-9042 RATS AND MICE: Care and Management 1332. Recommended schedulesThis image shows recommended frequencies forsanitization of cages and racks.Pre-washing with detergents can help loosen andremove organic debris and particulate matter,which must be removed thoroughly, because theycan inhibit the action of disinfectants. Effectivedisinfection can be achieved with wash water at143_F and rinse water at 180_F. Detergents andchemical disinfectants enhance the effectivenessof hot water but need to be thoroughly rinsed off(2). Rooms and hallways should be dampmopped daily with a disinfectant solution. Roomexhaust air filters should be vacuumed orotherwise cleaned as needed. Periodic microbialsampling can assure that quality sanitation istaking place.33. Sanitation equipmentProper sanitation of cages and bottles is facilitated byequipment such as the tunnel washer shown on theright. The tunnel washer occupies 2 rooms. The dirtyitems are placed in the washer in the unsanitary roomand removed clean and sterile in the sanitary room.This example shows an optional automatic beddingdispenser.Larger items such as racks are cleaned and sterilizedin the rack washer shown on the left. This piece ofequipment also is accessed from an unsanitary and asanitary room.34. Vermin control Insects (cockroaches and flies) and vertebrate pest species (wild orferal rodents) can contaminate animal housing facilities and transmitdiseases. The best method for controlling pests combines preventingtheir entry into the facility and maintaining good sanitation practices toeliminate their opportunities to live and breed. Seal cracks andcrevices and install sweeps on the bottoms of outside doors. Purchasehigh quality food and bedding and test regularly to ensure they are freefrom contamination by pests. Pesticides can induce toxic effects onresearch animals and interfere with experimental procedures, so theyshould be used in animal facilities only when necessary.

14 LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II35. Section title HANDLING, RESTRAINT, and IDENTIFICATIONRats and mice are routinely handled during cage changeouts, genderdetermination, clinical and experimental observations, and forexperimental or technical procedures. Proper techniques for handlingand restraining rats and mice minimize distress to the animals, lessenthe risk of injury to either the animals or their handlers, and preventtransmission of disease.36. “Gentling”Frequent handling of animals to familiarize andaccustom them to human contact is referred to as“gentling.” This is performed prior to experimentalmanipulations and will minimize or eliminate stress,struggling, or the tendency to bite. Most routine andresearch manipulations occur during the daily lightphase, when these nocturnal animals would typicallybe resting or asleep. By talking quietly to a rodentwhen approaching and opening a cage and movingone’s hand slowly around the inside of the cagebefore grasping it, will decrease the likelihood ofstartling it. A startled animal will react by biting ordefensive behavior.37. Short term restraintRats and mice may be picked up by the base of thetail, using the thumb and forefinger, for short periodsonly; for example, moving them quickly from one cageto another. The animals must not be picked up by thetip of their tails because they may twist about andinjure the tip which may slough off. Mice, if heldsuspended by the tail for longer periods, will “crawlup” their own body and tail in an attempt to bite theirhandler. Some strains of rats (e.g., Fischer rats) andmice are aggressive and difficult to restrain, requiringgreat skill and patience on the part of their handlers.38. Scruff hold39. Two-handed holdA scruff hold is one method for restraining both rats andmice. To scruff a mouse, grasp its tail and place its frontpaws on a rough surface, such as the wire top of acage. This allows it to grip the wire and diverts itsattention. Then grasp the skin on the scruff of the neckwith the thumb and forefinger of the other hand andsecure the tail between the little finger and palm of thehand. To scruff a rat, hold it in place by the base of itstail. With the other hand, press down gently and firmlyover the back of the animal and gather up the loose skinover the neck and back. “Scruffing” may pull the skintight over the thorax and prevent adequate expansion ofthe chest and lungs in both species, so handlers shouldmonitor animals for signs of respiratory distress.

V-9042 RATS AND MICE: Care and Management 15Another method for restraining a rat is shown here. Thehandler uses thumb and forefinger to encircle thethorax, taking care to place them behind the rat’s elbow.This hold should cause the forearms to cross andprotect the handler's fingers from being bitten. The otherhand is used to grasp the base of the tail, furtherstabilizing the animal’s hindquarters. The sameconcerns regarding restriction of chest expansion andhindrance of breathing apply for this technique.40. Other methods41. Restraint devicesBlunt thumb forceps may be used to pick up mice bythe scruff of the neck when concerns for sterility andworker safety exist. Forceps are dipped in adisinfectant solution before each use, therebyreducing or eliminating the likelihood of diseasetransmission from one animal or one cage to another.If chainmail or leather gloves are worn, care should betaken to avoid excessive squeezing of the animalsand to prevent injury to the incisor teeth should theanimal bite the glove.This image shows one example from the variety ofcommercial restraint products designed to facilitatesome procedures, such as tail vein injections or bloodpressure measurements. Animals should be held inthese restraints for the minimum amount of timenecessary to perform a procedure (2).

16 LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II42. IdentificationThis image shows an ear punch (on left) and ear tagswith their application pliers—two of the methods usedfor individual identification of mice and rats. Othermethods include fur dyes, tattooing, micro-chipimplantation, and cage cards. Toe clipping is usuallyconsidered unacceptable because suitablealternatives are available. Permanent identificationmethods are required for studies that must complywith the Good Laboratory Practices (GLP).43. Section title TRANSPORT AND HEALTH ASSESSMENTHealth assessment of animals following receipt in animal facilities isimportant to ensure their well-being and in minimizing and eliminatingvariables which could effect experimental integrity.44. Shipping cartonTransport cartons of the type seen in this image areused to ship animals. The cartons should be crushproofand, because of the potential for contaminationduring shipments, they should have filter materialcovering the ventilation openings. Boxes should beexamined for evidence of damage to the box or thefilters, and personnel should reject the shipment ifthere is any damage. Many facilities use chemicaldisinfectants to sanitize the exterior of the cartonbefore removing animals to minimize the likelihood ofcontamination of the animals.45. Moving animals The use of cages with filter tops when animals are transported to andfrom a laboratory will minimize the likelihood of contamination of theanimals. If animals are transported between buildings in inclementweather, they should be transported in climate controlled vehicles tominimize heat or cold stress. Familiarizing animals with transport willminimize stress.46. Quarantine Rats and mice should be purchased from vendors who havedemonstrated that their animals are free of diseases through the use ofeffective animal health and genetic quality control examinations.Quarantine can last as little as 24 hours but is usually for a week. Thisallows the animals to stabilize after transport. More importantly, itallows the veterinary and technical staff to observe the animals forsigns of illness brought on by the stress of shipment and to awaitresults of health assessment tests.47. Daily observation Research staff and animal care technicians should observe eachanimal at least once a day, preferably during the morning hours, toassess its health and activity. Abnormal activity, abnormal behavior, orevidence of ill-health can be brought to the attention of the facilitymanager and the laboratory animal veterinarian for additionalassessment, diagnosis, or action.

V-9042 RATS AND MICE: Care and Management 1748. Health surveillanceThe health status of incoming animals and long termcolony residents can be assessed through serologicand microbiologic testing, as well as necropsy andhistopathologic examination. Specimens for laboratoryexamination can be obtained from incoming animalsat the time of delivery or from sentinel animals housedwithin long-term animal holding rooms. Sentinelanimals do not receive experimental treatments, butshould be handled at least as frequently as testanimals. Placing samples of soiled bedding fromseveral cages into the sentinel cages facilitatesexposure to latent infections that might be present inthe room. For examples of serologic panels for virusesin rats and mice and a discussion of viral healthsurveillance, see V-9044: Rats and Mice-ViralDiseases in this series.49. Section title BREEDING50. Gender determinationGender can be determined by comparing the distancebetween the genital papillae and the anus. Thisanogenital distance is greater in males than infemales. A vaginal closure membrane is present inyoung rat and mouse females, which usually opensaround 35 days of age in mice and around 72 days inrats.51. Mating systems There are two types of mating systems: monogamous—where onemale is paired with one female, and polygamous—where severalfemales are housed in a cage with one or more males. Monogamousmating provides ease of recordkeeping and maximizes opportunitiesfor postpartum breeding. This method requires maintenance of largenumbers of males and has a lower productivity than polygamousmating. Monogamous mating is commonly used for inbred strains andless commonly for outbred stocks. Polygamous mating systems areused primarily for production of outbred stocks.52. Breeding systems There are three major types of breeding systems:∞ inbreeding—to produce inbred strains,∞ outbreeding—to produce outbred stocks,∞ selective breeding—choosing breeding partners based uponspecific physical, behavioral, or physiological parameters.

18 LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II53. Creating strains To decrease variation from animal to animal within a species(maximizing isogenesity), animal breeders produce inbred strains ofrats and mice. A strain is designated or identified only after >20consecutive generations of brother x sister matings or youngest parentx offspring matings. The Committees on Standard Nomenclature forInbred Strains of Rats and Mice ensure that existing and newlydeveloped strains are uniformly identified. Researchers should becautioned that strains from different vendors may have slightdifferences, and strains from the same vendor should be usedthroughout an experiment to ensure consistency and validity of results.54. Maintain stocks Rats and mice can be outbred in an attempt to maintain diversity(heterogenesity) between animals of the same species yielding anoutbred stock of animals. Stringent procedures are employed tominimize inbreeding. The International Nomenclature System forOutbred Stocks of Laboratory Animals ensures uniform identification ofoutbred stocks of rats and mice.55. Sexual maturity The age at puberty varies between the species, strain, or stock of ratsand mice. In general, rats reach puberty around 50 to 60 days of age,while mice reach puberty around 28 to 49 days of age. Althoughfemale mice can have their first estrus as early as 39 days, their firstfertile mating generally occurs around 7 to 10 weeks of age. The usefulbreeding period for inbred female mice lasts through about 10 to 12months of age, although breeding could continue for several monthslonger. Rats are usually bred after 9 weeks of age and have a usefulbreeding period through about 45 weeks of age.56. Estrous cycleThis graph shows the regular estrous cycle of rats andmice. The cycle lasts approximately 5 days in the ratand 4 to 5 days in the mouse. Estrus or “heat,” theperiod of sexual receptivity of the female, usuallyoccurs on day 3 of the cycle (based on vaginalcytology) and lasts for 12 to 14 hours, typically atnight. Ovulation occurs within 8 to 12 hours after theonset of estrus. Rat and mouse females arepolyestrus, breeding throughout the year.57. Monitoring the estrous cycleThe stages of the estrous cycle can be determined byperforming daily vaginal flushing or lavage usingsterile saline (shown) and microscopically examiningthe cell types during the cycle (shown are methyleneblue stained cornified epithelial cells indicating estrus).A sterile eyedropper can be used for the flushing andcollection of the fluid. This procedure facilitatesbreeding of animals on the day of ovulation, producingtimed-pregnant animals of known gestational age.

V-9042 RATS AND MICE: Care and Management 1958. Mating For mating, females may be caged with males, but if housedseparately, female(s) should be placed in the male’s cage for breedingpurposes. Mating or copulation usually occurs at night, when thesenocturnal animals are most active. Receptive females will exhibitlordosis, a rigid posture with the hindquarters elevated when downwardpressure is applied to the pelvis. Females will breed during apostpartum estrus that occurs between 14 and 28 hours afterparturition.59. Pheromonal influences The following phenomena are pheromone mediated:_ Whitten Effect⎯Synchronization of estrous cycles may be inducedby the introduction of any male mouse into a room or cage offemale mice._ Bruce Effect⎯A similar introduction of an unfamiliar male into acolony of females 4 days after they have been bred will oftenresult in the termination of any pregnancies._ Lee-Boot Effect⎯Another influence on the estrous cycle in whichthe absence of males causes irregular estrous cycles andsuppresses estrus.60. Detection of matingMating in rats and mice is detected normally by thepresence of a vaginal plug in the females or byvaginal lavage and examination for the presence ofsperm. The plug, as shown in these pictures, isformed from the mixing of vesicular and coagulatinggland secretions at the time of ejaculation. Vaginalplugs may be retained in mice for 16 to 48 hours, butoften fall out of rats within hours after breeding. Directobservation of the vaginal plug in the vagina orpresence of the plug in the waste pan beneathsuspended wire caging is a good indication thatmating took place.61. GestationThe length of the gestation period may vary with thestrain or stock of animal. The range in rats is from 21to 23 days (usually averaging 21 days) and 18 to 21days in mice (19 days average). Pups are usuallydelivered at night, with all pups delivered within 1 to 4hours after the onset of parturition.62. Parturition Periodic stretching and extension of the hindlegs signals impendingparturition; and for delivery, females stand in a semi-crouched position.Fetuses are delivered with either head or breech presentation. Thefemale usually eats the placenta, then turns her attention to the pups.Delivery of all pups may take between 1 to 4 hours.

20 LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES II63. Section title EUTHANASIASeveral techniques are available for euthanasia, the rapid, painless,and minimally distressing killing of rats and mice. The NAS Guidemandates that individuals performing euthanasia procedures beadequately trained and experienced and use approved techniques andagents in accordance with guidelines established by the 1993 Reportof the American Veterinary Medical Association Panel onEuthanasia (1, 2).64. Euthanasia methods Three general methods of euthanasia used in rats and mice are:1. Inhalant agents (carbon dioxide, halothane, isoflurane, etc.)sufficient to cause respiratory depression and death;2. Injectable drugs, such as an overdose of barbituric acidderivatives or highly concentrated pentobarbital agents;3. Physical methods, such as cervical dislocation, decapitation, orfocused microwave irradiation. Physical methods are utilized incases where other agents would contaminate or interfere withresearch results. If this is not a concern, physical methodsshould be performed while the animal is sedated oranesthetized.65. Inhalant methodsThis image shows a mouse in an inhalation chamber.One or more animals may be placed in an inhalationchamber, precharged with gaseous anesthetic agentsor carbon dioxide, and held there until death.Overcrowding in the chamber should be avoided, as itmay produce distress in the animals and prolong theonset of anesthetic activity. When soaked materials ordry ice are used, there should be a grid or separationbetween these and the animal to prevent distressassociated with dermal contact. Chloroform should notbe used because of the risk of toxicity to workers andother animals. Ether should not be used because of itsexplosive potential. When using gaseous agents, ahood or scavenging system should be utilized toprovide protection to the technician. Carbon dioxideexposure, while rapid and effective in adult rats andmice, requires prolonged exposure times in neonatesto ensure death.66. Physical methodsThere are several physical methods of euthanasia.One method is cervical dislocation (shown) whichinvolves placing a rod or thumb and forefinger on theanimal’s neck, immediately behind the skull, as shownin this image. While holding the base of the tail, pullbackwards and up, separating the cervical vertebraefrom the skull. This severs the spinal cord and disruptsblood flow to the brain, resulting in death. Otherphysical methods include decapitation or microwaveirradiation. Commercially available focused microwaveirradiation instruments direct their energy at the brain ofthe rodent undergoing euthanasia. Microwave ovensare not acceptable.

V-9042 RATS AND MICE: Care and Management 2167. Confirmation Death of an animal after euthanasia should be confirmed beforecarcass disposal. This can be done by checking for respiration a fewminutes after a procedure is completed and ensured byexsanguination, or by opening the chest cavity to collapse the lungs.Carcasses of animals treated with radioactive agents, infectiousagents, ether, or toxic chemical agents usually require special handlingand disposal.68. Conclusion This image concludes this autotutorial session on the biology and usein research of rats and mice. There are seven programs in this series:• V-9039 RATS AND MICE: Introduction and Use in Research,Part 1• V-9040 RATS AND MICE: Introduction and Use in Research,Part 2• V-9041 RATS AND MICE: Biology• V-9042 RATS AND MICE: Care and Management• V-9043 RATS AND MICE: Bacterial and Mycotic Diseases• V-9044 RATS AND MICE: Viral Diseases• V-9045 RATS AND MICE: Parasitic Diseases69. ACLAM creditsThis program was developed for theAmerican College of Laboratory Animal Medicine.G. L. Van Hoosier, Jr., DVM, ChairG. L. Borkowski, DVMK. Boschert, DVMJ. F. Harwell, Jr., DVMJ. M. Linn, DVMB. Macfadden, BAC. W. McPherson, DVMA. F. Moreland, DVMG. Otto, DVMInstructional development, editing, and production managementprovided by Barbara Macfadden, BA,andgraphic illustrations designed by B. J. Meredith.The development of this program was supported by a grant from theDuPont Merck Pharmaceutical Company.70. HSCER creditsProduced by theHealth Sciences Center for Educational ResourcesUniversity of WashingtonSeattle WA 98195-7161(206) 685-1156Fax: (206) 543-80512000

22 LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES IIREFERENCES1. American Veterinary Medical Association. 1993. Report of the AVMA panel oneuthanasia. J. Am. Vet. Med. Assoc. 202(2):229-249.2. National Academy of Sciences. 1996 (in press). Guide for the Care and Use of LaboratoryAnimals. NIHPublication No. 86-23. National Institutes ofHealth, Bethesda, Md.3. National Academy of Sciences and National Research Council. 1978. Nutrientrequirements of laboratoryanimals, 3rd ed. National Academy of Sciences,Washington, D.C.4. Pakes, S. P., Y. Lu, and P. C. Meunier. 1984. Factors that complicate animal research, p.649-665. In J. G. Fox, B. J. Cohen, and F. M. Loew (ed.),Laboratory animal medicine. Academic Press, Inc., New York.5. Potkay, S., N. L. Garnet, and J. G. Miller. 1995. Laboratory animals: laws, regulations andguidelines [images,videotape]. American College of LaboratoryAnimal Medicine, University of Washington, Seattle, Wa.6. Public Health Service. 1996 reprint. Public Health Service Policy on Humane Care and Useof LaboratoryAnimals. Office for Protection from ResearchRisks, National Institutes of Health, Bethesda, Md.BIBLIOGRAPHYBaker, D. E. J. 1979. Reproduction and breeding, p. 153-168. In H. J. Baker, J. R. Lindsey, andS. H. Weisbroth (ed.), The laboratory rat, vol. I. Academic Press, Inc., New York.Baker, H. J., J. R. Lindsey, and S. H. Weisbroth. 1979. Housing to control research variables,p. 169-192. In H. J. Baker, J. R. Lindsey, and S. H. Weisbroth (ed.), The laboratory rat, vol. I.Academic Press, Inc., New York.Besch, E. L. 1980. Environmental quality within animal facilities. Lab. Anim. Sci. 30:440-450.Burek, J. D., et al. 1980. Considerations in the selection and use of chemicals within the animalfacility. Lab. Anim. Sci. 30:414-421.Canadian Council on Animal Care. 1980. Guide to the care and use of experimental animals,vol. I. Canadian Council on Animal Care, Ottawa, Ontario.Clough, G. 1987. The animal house: design, equipment, and environmental control, p. 108-143.In T. Poole (ed.), The UFAW handbook on the care and management of laboratory animals.Longman Scientific & Technical, Essex, England.Farris, E. J, and J. Q. Griffith. 1962. The rat in laboratory investigation. Hafner PublishingCompany.Foster, H. L. 1980. Gnotobiology, p. 43-57. In H. J. Baker, J. R. Lindsey, and S. H. Weisbroth(ed.), The laboratory rat, vol. II. Academic Press, Inc., New York.Green, E. L. 1981. Breeding systems, p. 91-104. In H. L. Foster, J. D. Small, and J. G. Fox (ed.),The mouse in biomedical research, vol. I. Academic Press, Inc., New York.Hafez, E. S. E. 1970. Reproduction and breeding techniques for laboratory animals. Lea &Febiger, Philadelphia.Hessler, J. R., and A. F. Moreland. 1984. Design and management of animal facilities, p. 505-526. In J. G. Fox, B. J. Cohen, and F. M. Loew (ed.), Laboratory animal medicine. AcademicPress, Inc., New York.Kraft, L. M. 1980. The manufacture, shipping, receiving, and quality control of rodent beddingmaterial. Lab. Anim. Sci. 30:366-376.

V-9042 RATS AND MICE: Care and Management 23Loew, F. M., and J. G. Fox. 1983. Animal health surveillance and health delivery systems, p. 69-82. In H. L. Foster, J. D. Small, and J. G. Fox (ed.), The mouse in biomedical research, vol. III.Academic Press, Inc., New York.Melby, E. C., and M. W. Balk. 1983. The importance of laboratory animal genetics, health, andthe environment in biomedical research. Academic Press, Inc., Orlando, Fla.Small, J. D. 1983. Environmental and equipment monitoring, p. 83-100. In H. L. Foster, J. D.Small, and J. G. Fox (ed.), The mouse in biomedical research, vol. III. Academic Press, Inc., NewYork.Small, J. D. 1984. Rodent and lagomorph health surveillance - quality assurance, p. 709-723. InJ. G. Fox, B. J. Cohen, and F. M. Loew (ed.), Laboratory animal medicine. Academic Press, Inc.,New York.Snell, G. D., (ed.). 1956. Biology of the laboratory mouse. Dover Publications, Inc., New York.U.S. Food and Drug Administration. 1992. Good laboratory practice standards. Food and DrugAdministration, Office of Regulatory Affairs, Rockville, Md.Whittingham, D. G., and M. J. Wood. 1983. Reproductive physiology, p. 137-164. In H. L.Foster, J. D. Small, and J. G. Fox (ed.), The mouse in biomedical research, vol. III. AcademicPress, Inc., New York.

24 LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES IIAPPENDICESAPPENDIX 1POST-TEST1. What adverse health effects may occur when proper environmental parameters forlaboratory rats and mice arenot maintained in a facility?2. What are the advantages and disadvantages of filter bonnets?3. Describe a method of delivering water, food, and bedding to rats and mice and a specificpotential problemassociated with your choice that must beaddressed in a laboratory facility.4. Describe a method for handling a rat or mouse.5. Why is it important to control vermin in animal facilities?6. Why is it necessary to quarantine and daily observe new animals?7. What is the preferred method for monitoring estrous cycle in rats and mice?8. What two types of mating systems are used with rats and mice?9. Discuss the effect of pheromones on reproduction in laboratory mice.10. List three primary concerns when choosing a method of euthanasia.

V-9042 RATS AND MICE: Care and Management 25APPENDIX 2Answers to posttest:1. Adverse temperature affects reproductive performance, food consumption, and metabolismof test agents.Adverse humidity: High RH can lead to stress and reduce resistance to infections. Low RHcan lead torespiratory diseases and ringtail.2. Advantages: prevent microbial contaminationDisadvantages: limit air and gas exchange. See #13.3. Review the section on “bedding, food, and water”, p. 7-8, to verify your answer. Thebibliography also offersadditional resources.4. Review #37, p. 11.5. Vermin can transmit diseases.6. Allows animals to stabilize after transport and allows facility staff to assess health andactivity while waiting fortest results.7. Vaginal lavage and microscopic examination of cells.8. Monogamous and polygamous9. See #59, p. 14.10. The method should be rapid, painless, and minimally distressing

26 LABORATORY ANIMAL MEDICINE AND SCIENCE - SERIES IINOTES