Biomethodology of the Mouse - Animal Research

The genus and species of the laboratory mouse is mus musculus and is of the order Rodentia. The laboratory mouse has been domesticated by man for many generations and is in general a very docile and easily handled animal. Other notable biological characteristics are their very acute hearing, well developed sense of smell, poor vision, small size and short generation interval. Mice are by far the most common laboratory animal used for research.

Genetic and Microbial Categories:

There are two common methods by which to characterize laboratory mice; Genetics and Microbial flora. Common genetic categories are "random-bred" mice which are managed to maintain genetic diversity by mating unrelated mice; "Inbred" mice which are managed to maintain genetic homozygosity by breeding siblings; "F1 hybrid mice" in which two inbred strains are crossbred for one generation; "knockout mice" in which part of the genome has been removed or inactivated; "transgenic mice" in which specific genetic material has been introduced into the genome of another inbred mouse strain; "mutant" mice which are inbred mice that have developed genetic mutations. The microbial flora of mice can be used to group mice: Specific Pathogen Free Mice (SPF) mice are free from known bacterial, viral, and parasitic mouse pathogens, as opposed to "conventional" mice, which are not known to be free of pathogens. Most mice purchased at the University of Iowa are SPF mice but the standard housing (i.e. conventional housing) cannot totally prevent the introduction of mouse pathogens. To maintain the SPF microbial status of mice requires that animals be housed in more stringent conditions that prevent the introduction of rodent pathogens (i.e. barrier housing). This type of housing is available at the University of Iowa. Other less common microbial groups are axenic mice (i.e free from all microbial organisms) and gnotobiotic mice which have a known microbial flora.

Sources and Ordering:

Inbred, outbred, hybrid, knockout, transgenic or mutant mice are available from many commercial sources. Office of Animal Resources personnel are familiar with many of the commonly available strains and will assist in locating animals available for purchase. Typically, for commercially available mice, animals ordered by Thursday 12 am will arrive the following week. Most animals will recover from the stress of shipping within 48 hours. To order animals the investigator must provide the protocol number of the approved "Animal Care and Use Form".

Behavior:

The laboratory mouse is a docile animal and can be easily handled. Animals can be grouped soon after weaning usually coexist peacefully. However, some strains of mice (i.e. BALB/cJ, SJL/J, HRS/J) will begin to fight even if grouped at weaning. Breeding males that have been removed from breeding cages and caged together will usually fight. Wounds on the tail are a good sign of aggression between cagemates.

Biological Characteristics and Data:

Mice, like most species have a circadian rhythm. Investigators should be aware that this may affect biological data and should standardize the time of day that samples/measurements are taken to avoid this affect. The standard light/dark cycle at the University of Iowa is 12/12. This light cycle can be modified upon the request of the investigator in special circumstances.

The adult mouse weighs approximately 40 grams and this small size and resulting large surface area/body weight ratio makes them susceptible to changes in environmental conditions. The core body temperature is easily affected by small changes in temperature which may modify the physiologic responses of the animal. ¹⁷ The acute hearing of mice makes them highly sensitive to ultrasounds and high pitched noises inducing a stress response that has been empirically related to cannibalism of pups by their dams. The well developed sense of smell is used to detect pheromones used in social interactions. The poor vision of mice makes them unable to detect color and red light is often used to observe animals during the dark cycle.

Basic Biological Data

Adult body weight: male	20-40gm⁵
Adult body weight: female	20-40gm⁵
Body surface area	10.5(wt. in grams)2/3⁵
Life Span	1.5-3 years⁵
Food consumption	15 gm/100 gm/day⁵
Water consumption	15 ml/100 gm/day⁵
Breeding onset:male	50 days⁵
Breeding onset:female	50-60 days⁵
Gestation Period	19-21 days⁵^,²⁴
Body Temperature	36-37C²¹
	36.5-38.0C⁵
Heart rate	500-600 beats per minute²¹
	325-780 beats per minute⁵
Respiratory Rate	84-230 per minute²¹
	60-220 per minute⁵

Basic Husbandry:

Most mice are housed in shoebox cages composed of polycarbonate material (picture) with a wire bar lid used to hold the water bottle and feed. Bedding is placed directly into the shoe box cage allowing the absorption of urine and the animal to burrow and/or den. This type of cage will hold 4-5 adult mice depending on the size of the cage. The majority of mice are housed in ventilated racks which supply filter sterilzed air. Water is frequently supplied by an automatic watering system rather than a water bottle. When removing the lid from this type of cage it is important to remove the water bottle (if present) to prevent spillage. If the cage is to be transported the bottle should be turned sipper tube up to (picture) prevent spillage during transport. However, you should remember to turn the bottle back over to allow access to water after transport. The caretaking staff, change the cages every one to two weeks, thereby providing the animal a clean cage with new bedding, food and water. Water bottles and feed hoppers are checked daily by caretakers to insure the provision of water. Some mice are housed on wire mesh bottom cages to allow collection of feces and urine or to prevent contact with bedding. This type of housing is not preferred and is used only when dictated by experimental design. Many cages provided with automatic watering which is supplied by a nipple valve (i.e. lixit) located in the back of the cage that is operated by animal contact. When replacing a suspended cage that is provided with automatic watering it is important to push the cage fully into the rack care to insure that the lixit fully extends into the cage, allowing the animal access to water. Occasionally a mouse will jam the lixit open resulting in a constant dripping of water from the lixit, saturating the bedding material. Upon observation the bedding will appear much darker. This problem should be reported to husbandry personnel immediately to allow correction.

Pelleted natural ingredient diets are used to feed all rodents and are composed primarily of cereal grains which are supplemented with additional protein, vitamins and mineral. Due to the nature of this type of diet the exact composition can vary substantially from lot to lot. The water provided to animals is chlorinated tap water.

Some mice are housed to prevent the acquisition of rodent pathogens. Rodent pathogens often do not produce clinical signs in affected animals but often have an immunomodulating effect. Therefore, this type of housing is important for studies that involve the immune system. Immunocomprimised animals, which are sensitive to opportunistic agents, are also housed in this fashion to allow for their long term survival. These animals are housed in sterilized cages and are provided sterile food and water. Access to this housing area is limited to prevent inadvertent fomite transmission of rodent pathogens by personnel.

Identification:

Cage cards are utilized to identify the strain of mouse, sex, number, principal investigator, and research protocol. Cage cards should not be removed from the cage to avoid misidentification of the animals. Temporary identification of individual mice can be accomplished by pen marks on the tail, hair clipping or dyeing the fur. Pen marks will only last 1-2 days whereas hair clipping may last up to 14 days. Tail and toe clipping are not recommended. Ear punch identification can be utilized but may be obliterated by fighting between individuals.

Handling and Restraint:

When handling mice it is advisable to wear gloves to prevent the development of allergies due to direct contact with animal allergens. Mice are usually caught and lifted by the tail. The tail should be grasped between its midpoint and the mouse's body (picture). The tail may be grasped with the thumb and forefinger or by the used of smooth-tipped forceps. With this simple method of holding, they may be transferred to another cage, a balance, identified, examined casually or sex may be determined. Pregnant mice or very obese mice may be handled by this method but they should be supported by use of the second hand placed under their feet (future picture). However, such restraint is not sufficient for treatment and close examination. For more effective control, the mouse may be held by the tail and placed on a table or other surface, (preferably one such as a wire cage lid that the mouse can grasp) and the loose skin over neck and shoulders grasped with thumb and fingers (picture, mp2a.jpg, video). It is necessary to perform this maneuver expeditiously, or the mouse may turn and bite. Once the mouse is grasped correctly, the head is adequately controlled. Restraint is improved if the tail or the tail and rear legs are held by the third and little fingers of the same hand or with the other hand (picture). Mice should not be dropped into the cage, which may result in spinal fracture, but should be lowered into the cage and released upon contact with the bedding.

Mice less than two weeks of age can be handled by grasping the loose skin over the neck and shoulder with thumb and forefinger or smooth tipped forceps (picture). Handling neonatal mice should be avoided especially during the first few days after birth to avoid cannibalism or litter abandonment. If it is necessary to handle the litter, remove the dam to a separate cage and handle the neonates using plastic gloves to avoid contamination with human scent. Multiparous females are less likely to cannibalize if they have been successful mothers and should be chosen if litter manipulation is necessary.

Numerous types of restraint devices are commercially available to restrain mice. Quality devices prevent the animal from turning around yet allow easy access to the tail or legs (picture). Devices should also be easy to clean and provide adequate ventilation. For tail vein injections a wire box cage top can be turned over and the tail gently passed through the wire bars preventing the mouse from turning (future picture).

Transport:

Mice that are moved indoors can be transported in their cage after removing the water bottle (if present) and placing it upright in the cage lid to prevent spillage (picture). It is recommended that a permeable drape be placed over the cages to darken the cage and prevent over arousal of the mice during transport. Mice cannot be transported out of doors or by vehicles by other than husbandry personnel. Investigators can request transfer to other buildings by contacting the husbandry staff (335-7985).

Sexing:

Male and female mice can be differentiated by observing the distance from the anus and genital papilla which is greater in males (picture). This difference is also present in neonatal mice (future picture).

Breeding:

Mouse colonies tend to be very labor intensive and are discouraged for the production of commercially available strains of mice. The cost of breeding mice for research use far outweigh the cost of purchasing from commercial vendors. Also, it is very difficult to maintain the genetic heterozygosity required for outbred mice in small colonies and to prevent subline divergence when breeding inbred mice. For strains not available commercially or in instances where neonatal or fetal animals are required, breeding colonies are maintained. Investigators requiring the establishment of breeding colonies should consult with the Office of Animal Resources to assure proper management of the colonies. Breeding animals have different nutritional requirements, which if not provided, will have a detrimental effect on reproductive performance. Light cycles are important in breeding mice and are provided with 12 hours of light and 12 hours of dark. Deviations from this cycle will effect reproductive performance.

Suggested References available in the Office of Animal Resources Library:

Textbooks:

Poole TB 1987, The UFAW Handbook on the Care and Management of Laboratory Animals, 6th edition. Longman Scientific & Technical: England

Harkness JE, Wagner JE 1989, The Biology and Medicine of Rabbits and Rodents; 3rd Edition, Lea and Febiger, Philadelphia

Green EL 1981, Biology of the Laboratory Mouse, 2nd edition. Dover publications: New York

Foster HL, Small JD, Fox JD (eds) 1981, 1982, 1983, The Mouse in Biomedical Research, Volume I-IV. Academic Press: New York

Altman PL, Katz DD 1979 1979, Inbred and genetically defined strains of laboratory animals, Biological Handbooks III, Part I. Mouse and Rat. Federation of American Societies for Experimental Biology, Betheseda: Md

Videotapes:

Brooks D, Timm K, Porte W,Jahn S, 1987,Practical Methodology: Humane Handling and Laboratory Techniques for the Mouse. University of California; Davis, CA

General Biology: