References and resources for this tutorial
Jacoby, Robert O., Fox, James G., Davisson, Muriel, "Biology and Diseases of Mice," Chapter 3 in Laboratory Animal Medicine, 2nd Edition James G. Fox, et. al., editors (New York: Academic Press, 2002)
"Rodentia" in The UFAW Handbook on the Care and Management of Laboratory Animals, 7th Edition (Malden: Blackwell Science, 1999)
Suckow, Mark A., Danneman, Peggy, Brayton, Cory, The Laboratory Mouse, a volume in the Laboratory Animal Pocket Reference Series (Boca Raton: CRC Press, 2001)
Laboratory Animal Management: Rodents (online book from National Academies Press, 1996)
Overview
Mice have been used for research since the 17th century but with the rise of biological and genetic experiments in the 1900s, they have become the most widely used animal in research. Some of the factors for this are their small size, high reproductive rate in a short period of time, handling ease as well as the massive amount of knowledge about them. They are the model of choice for genetic research. Given the current technology for transgenic work with mice, they are currently the “most widely and heavily used experimental animal.” (Fox, op. cit. p. 36) (e.g. see the Trans-NIH Mouse Initiative, or the Mouse Genome Database) Mice are ubiquitous, either in the wild or as guests in households, both urban and rural. Given their high numbers, quick reproduction capacity and the lack of sentimentality about them, it might seem that concerns about their inherent moral value do not make much sense.
But, what is worth considering is the ease with which we consider them a scientific tool. (See the Whole Mouse Catalog, for example, the Mouse Information Resources page from the Jackson Laboratory, or the Rodent Surgery Services from Charles River Laboratories.) Since most of us do not have the emotional attachment to mice that we might have to other species, we can look more objectively at some of the welfare and ethical issues about using a living creature as a biological instrument. This is particularly true with the advent of recent mouse genome discoveries. Are there any limits to considering the mouse as a mere precursor to human beings? How should we think about welfare issues for some of the transgenic and knock-out mice? How do monitoring for pain and distress initiatives shift when the animal has been bred to become ill in a specific way?
Mice are probably the easiest animal to maintain in a laboratory setting. Their small size makes them “cost-effective” and given the large amount of information about them, they are easy to fit into a laboratory protocol. Since they have been so utilized, there are usually precedents to follow in their use and technicians are generally skilled in taking care of them.
In general, they are timid and non-aggressive, sociable with each other although the males will fight after maturity. They are usually group housed at weaning; males from the same litter, housed as weanlings, usually will not fight, although this may be a problem in some strains. Although fighting over territory may occur briefly, mice will form hierarchical structures; in some cases the dominant mouse will chew and nibble on the hair of subordinates, resulting in specifically patterned hair loss called barbering. Thus, there needs to be some attention to their caged behavior as part of the maintenance. In their natural state mice are nocturnal; in the laboratory they are relatively quiet in the day, if undisturbed. They eat throughout the day and food is provided ad lib, usually in pellet form. They are usually housed in clear plastic boxes covered with a wire lid with a food hopper extending along the lid; the mice will climb up the underside of the lid. The solid pen contains bedding materials to absorb urine and fecal pellets and to provide for normal burrowing and nesting behavior.
Comfortable Quarters for Mice in Research Institutions, an article from the Animal Welfare Institute, is cross listed by the The Animal Welfare Information Center. This 17 page document covers husbandry, handling and behavior for mice. The Electronic Zoo/NetVet is another basic resource for information. Raising Mice, from Case Western Reserve University, has details about managing a mouse colony, including breeding and handling information.
The mouse and science
The history of the use of mice in the laboratory can be seen as a microcosm of the rise of the scientific research establishment in the United States. During the early decades of the 20th century, research in America was not yet a big business or a well established career path. In 1914, research into mice genetics was just beginning; for example, Little and Tyzzer were just begging the first work with Japanese waltzer/DBA crosses in mice, following up on their ideas that Mendelian genetics was a promising area to inspect for relevance to human diseases, cancer in particular.
The Jackson Lab in Bar Harbor Maine, founded in 1929 by Clarence Cook Little (1888-1971), was the first American institution to join the disciplines of genetics and medical research with the idea of the laboratory animal as a tool par excellence. Karen A. Rader, in her essay, “The Multiple Meanings of Laboratory Animals; Standardizing Mice for American Cancer Research 1910-1950 (Animals in Human Histories, 2002) notes, “By virtue of the founding of the Jackson Lab and the development of its inbred mouse sales and production enterprise, inbred mice had become standardized social and scientific commodities. …Thus defined, inbred mice were the methodologically responsible tools for investigating a socially responsible research problem—the causes of human cancers.” (Rader, p. 411) Rader makes the point that the mouse was not intrinsically a laboratory tool, but was turned into one by both literal genetic manipulation and a culture that saw scientific research as an enterprise of high moral calling—she calls this the “Scientizing of the mouse.” (op.cit. 409) We can see a current example of this, the mouse as a scientific commodity, in the listing for mouse strain C57BL/6NCrlBR on the website for another major laboratory resource for mice, the Charles River Laboratories.
“The bioengineered mouse,” Donna Haraway recently wrote, “is simultaneously a metaphor, a technology, and a beast living out its many layered life as best it can.” (Rader, 419)
Where the ethical questions become very complex is in the emerging area of xenotransplantation. Some cutting edge work involves pigs and mice, both as instruments for biomedical advances planned to benefit human beings. How do we think about the issue of inherent value of an animal in these cases? Quoting from Laboratory Animal Medicine, 2nd edition, “In a mouse model, inhibition of xenoantibody production was accomplished by retroviral transfer into mouse bone marrow of a gene encoding the enzyme that synthesizes swine carbohydrate antigens.” (Laber, et.al.) The September 1999 issue of the newsletter ANZCCART News contains articles on both xenotransplantation and “Ethical and welfare implications associated with transgenic animals.” Scroll through the newsletter for this article.
The Animal Welfare Information Center has posted an online bibliography, Housing, Husbandry and Welfare of Rodents, which contains abstracts covering a wide range of topics, from housing to procedures to protocols.
Case Western Reserve University has an extensive set of informational links with information on knockout mice in particular.
Biology
The University of Iowa’s Animal Research: Institutional Animal Care and Use Committee has posted a basic review of the mouse, “Biomethodology of the Mouse,” covering biology, husbandry and routine medical procedures.
One of the benefits of using the mouse is their genetic standardization which helps assure experimental reproducibility. As the breeding and nomenclature for mice has becoming increasingly complex, we will not focus on this here except to note that they are identified by both strain and breeding system. For example, a CH3 mouse indicates an inbred strain (20 generations of brother-sister matings, or full-sib matings) resulting in a group of 98% genetically homozygous animals. An F1 hybrid mouse is the first generation of the cross of two inbred strains. Generally, a hybrid animal is thriftier than an inbred one; mating of this F1 sort assures a uniformly heterozygous genetic heritage. An outbred strain is genetically heterogeneous; sometimes the term random bred is used.
For detailed information about breeding systems see the Mouse Genome Database.
Mice have an extremely high metabolic rate; they utilize large amounts of oxygen for their size, have a rapid respiratory rate, a short tracheal-bronchus distance, have a high blood carrying capacity for oxygen and a high blood sugar concentration. You can see from this why sufficient food and water need to be consistently available as they have a smaller range for error than a larger animal.
Mice have incisors and molars only; since their incisors grow continuously they must be checked for malocclusion. Their gastrointestinal flora contains up to 100 species of bacteria and thus there is a complicated internal balance resulting in a resistance to some pathogens, vitamin production. The biological trait of having a complex microbiological ecosystem has been used to advantage in research. It is common practice for some work to derive or purchase “germ-free” mice and then colonize their gut with a selected set of gastrointestinal flora.
As do human beings, mice have a four chambered heart. Interestingly, an increase in body temperature does not lead to an increase in blood pressure. Heart rates and blood pressures vary widely from strain to strain; Fox et. al. report a heart rate range from 310-840 per minute between different strains.
Mice void only a drop or two of urine at a time. Their urine is very concentrated and contains a large amount of protein. This is one reason that high levels of ventilation are important in mouse rooms; people susceptible to allergic reactions can have trouble with the high protein levels in mouse urine. For the mouse, this concentration is part of the territorial marking system. Thus, a balance needs to be maintained in cage cleaning and bedding changes to be sure the levels of urine proteins in the air and pen do not adversely affect either respiration or behavior since a certain level of urinary pheromones is necessary.
As we have said, the fast reproductive rate is one of the reasons for the popularity of mice as research subjects. Sexual maturity varies between strains and is sensitive to environment; for instance, noise, light, diet and population density all have an affect on the hormonal system and thus on reproductive rate. Females are often group housed so as to synchronize their estrous cycles before being paired with males. Males can be bred between 49-70 days; the females between 45-65 days. Gestation takes 19-21 days and the average litter size ranges from 1 to 12 pups depending on the strain. Although mice can live up to two years and some strains can reproduce for that long, most female breeders are retired after one year and most inbred strains are only active breeders for six months. The females nurse their young for 3 weeks; their milk production peaks at 12 days postpartum and slowly lessens until normal weaning at three weeks. Antibody protection is in the colostrum; due to their thin skin, milk is visible in the neonate’s stomachs. This is a good indication of their health. When monitoring breeding colonies inspecting the litters for equal amounts of milk in their bellies is a good practice.
Nutritional requirements
For detailed information on mouse nutrition you can access the digital edition of Nutrient Requirements of Laboratory Animals (National Academies Press, 1996) and read the 22 page chapter, Nutrient Requirements of the Mouse. Mice are commonly fed a standardized pellet diet from a reputable vendor; it is important to store feed away from direct sunlight and monitor the storage area for signs of insect or mold infestations.
Water
Adult mice need between 6-7 ml of water per day and decreased water intake leads to decreased eating. Mice have a high ration of body surface to mass and therefore they have great sensitivity to water deprivation. Since sick mice drink less water, it is not advisable to put medications into the water. But, it is a common practice to put prophylactic antimicrobials in the drinking water of immunodeficient mice.
Several common illnesses of mice
Commercial vendors provide disease free animals, but the practice of sharing mice between labs can lead to disease outbreaks. We will summarize the six diseases of mice noted in the chart, “Typical Core Agents Monitored in Research Facilities” from the National Research Council’s book Laboratory Animal Management: Rodents, published by National Academies Press and available online as well as several others that can occur in mouse colonies.
The National Academies Press has an online book Infectious Diseases of Mice and Rats.
The University of Missouri, College of Veterinary Medicine, has an extensive online training site: access their Mouse Diseases page for detailed descriptions and photographs.
Some of the more common viral diseases to be aware of are:
Tyzzers Disease disease is an important bacterial disease not only in rodents but can infect other lab animals. It occurs most often in weanling animals, presenting with watery diarrhea, anorexia, dehydration, and lethargy. It can have a short course, with death after a day or two, or go on to become a chronic state, with weight loss. The route is oral, via spores. A high level of sanitation is critical to help prevent this illness; preventing stress is important as well. Again, be sure your source for the mice is able to assure disease-free animals.
Helicobacteriosis, caused by Helicobacter species, has been the subject of increasing study. Helicobacter spp. colonize crypts in the lower bowel. Depending both on the individual bacterial species and the immune status of the host, Helicobacter colonization can remain nonpathogenic or cause liver disease, forms of hepatitis, or various sorts of gatrointestinal disease--inflammatory bowel disease, colitis, proctitis, for example. Immuno-deficient mice are most at risk. "Recent surveys and anecdotal evidence sutggest that helicobacteriosis is widespread among conventional and barrier maintained mouse colonies." (Jacoby, et. al. "Biology and Diseases of Mice," in Fox, et.al. 2002, p.88)
Two of the parasites most likely to infect mice in the laboratory are pinworms or fur mites.For more detail on diseases in mice, you can access the National Academies Press’s 400 page book, Infectious Diseases of Mice and Rats; the pathologies are organized via the organ systems and there are chapters on Health Surveillance Programs, as well as Research Complications of Infectious Diseases.
Husbandry and welfare considerations
Due to their small size and high proportion of body surface to mass, they are highly sensitive to water loss and do not do well with wide fluctuations in room temperatures. They do not pant and have no sweat glands; they mainly deal with higher room temperatures with decreasing their metabolism or increasing their body temperature. They are also poor at adapting to lower temperatures—their main coping mechanism to conserve body heat is by burrowing into bedding. Neonates do not develop internal temperature regulatory control before 20 days. Thus, environmental temperatures need to be steady and constant; bedding must be provided for breeding mice. This inability to compensate for outer temperature shifts is one reason transport is so stressful for mice. It is also important not to decrease room temperatures at night.
They can play with water bottles, particularly when nesting, pushing bedding materials up into the water spout, thus flooding the cage: new bedding materials have been developed to avoid this problem but frequent monitoring is essential.
The National Academies Press Recognition and Alleviation of Pain and Distress in Laboratory Animals (1992) is available online. This 138 page book talks about these issues in general terms that relate to all laboratory animals, without focusing on individual species. “Rodents That Require Special Consideration,” a chapter in the NRC’s Rodents (op.cit). focuses on mice with particular needs due to alterations in their genome to facilitate research such as immunodeficient rodents and transgenic mice.
Indications of pain in mice (list from Suckow, et. al. op.cit, p. 108)
One of the difficulties of deciding to use pain medication is that there will likely be accompanying stress from the increased handling—either injection or gavage—many medications will need to be repeated every one to five hours. It is important to make any environmental changes minimal, although sometimes weakened mice need to be protected from aggression from cage-mates and housed singly until recovery. It can be helpful to moisten the food with a little water; most mice do better with surgery recovery in a slightly warmer than normal room.
Handling and Procedures
Although they are generally tractable when handled, they can bite (surprisingly painfully) at the moment of restraint, so training in restraint techniques is helpful. Mice are often handled with forceps, gripping them at the base of the tail. If restraining by hand, catch them at the base of the tail, placing them on a rough surface for traction. They will attempt to escape and this extends their body so that you can grasp them by the loose fur around the scruff of the neck. Some mice will attempt to twist around and bite (they have a surprisingly sharp bite). Once they sense you are experienced at restraint—i.e. they feel comfortably held-- they generally are docile.
The University of Minnesota’s Research Animals Resources has a page on handling of specific species: after accessing the home page, click on “animal handling” at the left and then scroll through for diagrams and text for the various species until you reach the section on mice.
Monitoring
All laboratory animals, as well as their environment should be monitored on a daily basis: the University of Minnesota, Research Animal Resources, has produced an excellent set of guidelines for this entitled, Housing and Husbandry Guidelines for Laboratory Animals, which is available electronically.
Monitoring Checklist:
With all animals, it is best to decide on a formal repeatable examination pattern that with practice, will quickly review the normal and single out the abnormal. Since mice generally are group housed, it is practical to have an examination pattern that takes in the group first and then focuses on the individuals that differ from the rest. With practice, a technician can quickly focus on the “abnormal” animal.
For example:
First check each pen overall; look for normal activity levels. Be alert to unusual clumping around a water bottle since that can be indicative of problems. Check for lethargic or hunched mice: a hunched animal can indicate illness or discomfort. If possible, quickly check each pen for a water bottle and check that food hoppers all contain similar amounts of food.
Then, walk between the cage rows and do a quick check of the animals; with practice, your eye will see the “different” mouse from the rest of the group.
Overall: coat should be clean, dry, shiny, well groomed, no obvious weight gain or loss, no hair loss or lesions, normal activity level, expression BAR (bright, alert, responsive). A rough or “staring” coat/a non-groomed animal can indicate a problem. Dehydration can show as a ruffled coat, hunched posture. Comparing size to others is useful: an unusually small mouse can indicate some developmental or sub-clinical difficulty, e.g. malocclusion.
Breathing: normal breathing is rapid. With some respiratory diseases you can hear “chattering” which indicates difficulty breathing.
Nose: pink, dry
Mouth: check that teeth occlude correctly, gums should be pink, moist, not overly wet or dry.
Eyes bright, eyelids clean, not swollen
Ears: clean, not reddened.
Tail/Anus: clean, no evidence of mucous or feces or diarrheal staining.
Genitalia: clean, no exudates
Legs: no stiffness, damage or swelling
Housing and Husbandry
The amount of time laboratory animals spend undergoing a procedure of some sort is usually minor compared to their time in their quarters. Husbandry is thus of central importance to them. In “The Ill-Effects of Uncomfortable Quarters", William M. S. Russell talks of the necessity for healthy, unstressed animals in research.
When checking the mice, it is important to check the room for appropriate temperature, humidity and ventilation. (Data from Suckow, et. al., op.cit.)
| Room temperature | 64-79 degrees Fahrenheit (minimize variability) |
| Humidity | 30-70% (45-60% reported as optimal, anecdotally (Suckow, et. al. p. 21) |
| Ventilation | 10-15 air exchanges per hour individual pens can contain their own filter top |
Traditionally, mice are group housed in clear plastic boxes with metal grid lids where the food and water are placed. Space requirements as set out in “the Guide:” (See Table 2.2)
| Weight (g) | Floor area/animal (”squared) | Height (in) |
| Less than 10 | 6
|
5
|
| Up to 15 | 8
|
5
|
| Up to 25 | 12
|
5
|
| Greater than 25 | 15
and greater
|
5
|
Current research using mice
As models
Dr. Jim Pearce has posted an essay (with photographs and diagrams) “Mice and Men: Making the Most of Our Similarities” that describes some of the current research using mice as models for diseases such as diabetes, obesity and sickle cell anemia. “The Mouse in Science: Why Mice?” is posted by the University of California, Center for Animal Alternatives (UCCAA), or access the Whole Mouse Catalog and then click on “other stuff” and scroll through those hyperlinks until you get to this article. In a very general sense, there are two categories of the mouse as model in research; 1) cancer research and 2) genetics research (which includes specific genetic components of disease). The Alternatives section of this website gives many resources for the 3R's in general: for a specific data base for protocol refinement for mice see Refinement of Research Methods with Mice. The National Academies Press has an online book, Immunodeficient Rodents: a Guide to Their Immunobiology, Husbandry and Use (1989).
Cancer research
The Mouse Models of Human Cancers Consortium is a website run by the National Cancer Institute. The Mouse in Science: Cancer Research is another posting from the UCCAA.
Genetic research
The mouse has certainly been used for genetics research, but the real growth in their use has been in transgenics. Currently, transgenic and knockout models are being used in almost every area of biomedical research, rather than for actual genetics research. For example, see the Database of Gene Knockouts, or the BioMedNet Research Mouse Knockout and Mutation Database.
The Rat
The University of Iowa’s Animal Research: Institutional Animal Care and Use Committee has posted a basic review of the rat, “Biomethodology of the Rat,” covering biology, husbandry and routine medical procedures. Comfortable Quarters for Rats in Research Institutions is a useful overview. Also see the Electronic Zoo/NetVet. For genetic information, see The Rat Genome Database.
RATLIFE.ORG feautures an online film, "Shot as a wildlife documentary over several months, this 27 min film follows the lives of domestic rats after being released in a large outdoor enclosure where they have to compete, like their wild cousins, for food, shelter and mates. As we witness the emergence of a complex and structured society which soon thrives in this wild environment, the film demonstrates how studies ranging from physiology to psychology have uncovered a number of features which, despite generations of domestification, remain ready to be expressed when given the opportunity. We may have taken the rat out of the wild, but have we taken the wild out of the rat?"
The Guinea Pig
The University of Iowa’s Animal Research: Institutional Animal Care and Use Committee has posted a basic review of the guinea pig, “Biomethodology of the Guinea Pig” covering biology, husbandry and routine medical procedures. Also see Comfortable Quarters for Guinea Pigs in Research Institutions and the Electronic Zoo/NetVet.
The Hamster
See Electronic Zoo/NetVet. and Comfortable Quarters for Hamsters in Research Institutions.
Study Questions
1. Rodents have very strong hierarchical behavior patterns: it has been reported that low ranking mice will not urinate in the presence of higher ranking peers, leading to chronic urinary retention, distended bladders leading to interstitial nephritis, and even death. (Stevens-Larson, 1985) Also see, Social Defeat in C57Bl/6 mice. Reviewing Comfortable Quarters for Mice in Research Institutions, what sorts of arrangements might you provide to reduce dominent individuals from stressing subordinates? In what ways might environmental enrichment provide lower ranking individuals with security and on the other hand, in what ways might it exacerbate behavioral hierarchies? Which seems to you to be the "best" system of caging given this normal hierarchical behavior, single, pair or group housing? How could you tell?
2. In a special issue of the ILAR Journal, Humane Endpoints for Animals Used in Biomedical Research and Testing, Jerrold Tannenbaum (ILAR 40: 97-110) asks whether it is more ethical in a research project that will involve pain and discomfort to use fewer animals, subjecting each one to more extensive research, or to use more animals, thus giving each individual a lesser degree of distress. Which approach would you prefer and why? Does the availability of large numbers of mice impact on your decision? Should it?
Discussion Question
The 3R policy suggests that animals of lower rank in the neurological complexity scale (e.g. rodents) be substituted for higher ranking ones (primates, dogs, cats, pigs.) Burn research is one such area where this sort of "replacement" is used. Does this sort of "reduction" by "replacement" makes sense to you? Why or why not? What does such replacement imply about the "moral standing" and/or "inherent worth" of rodents? Do you think this is a serious concern or not? If you were asked to make a decision about which rodent species to choose for burn research, rats, mice, guinea pigs, hamsters or gerbils, which one would you choose and what does this decision imply?