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Biomethodology of the Mouse
· Behavior
· Biological Characteristics and Data
· Occupational Health/Zoonosis
A. Nomenclature and Breeds
· The laboratory mouse (Mus musculus) is a mammal of the order Rodentia. The laboratory mouse has been domesticated by man for many generations and is in general fairly easy to handle.
· Other notable biological characteristics are their very acute hearing, well developed sense of smell, poor vision, small size and short generation interval. While mice are often considered to be the most common laboratory animal used for research, studies utilizing rats are more frequently cited in the scientific literature.
· "Out-bred" mice are managed to maintain genetic diversity by mating unrelated mice.
· "Inbred" mice are managed to maintain genetic homozygosity by breeding siblings for more than 20 generations.
· "F1 hybrid mice" involve crossbreeding two inbred strains for one generation.
· "Transgenic mice" are mice in which specific genetic material has been introduced into the genome of another inbred mouse strain. "Knockout mice" are mice in which specific genetic material has been removed from the genome.
· "Mutant" mice are inbred mice that have developed spontaneous genetic mutations.
B. Unique Biological Characteristics
· The mouse does not have an appendix.
· The urine of mice is normally clear, yellow and very concentrated. Large amounts of protein are normally excreted in the urine.
· Dark pigmentation of the spleen is a non-pathogenic condition in C57Bl mice. This pigmentation may be focal.
· Barbering is the removal of hair and/or whiskers and can be seen when animals are group housed. The area of hair loss is usually well demarcated and dominant mice “barber” subordinate mice. There are normally no skin wounds associated with barbering.
· Mice are nocturnal (most active at night), however substantial activity can also be seen during the day. 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 in the DLAR is 12/12. This light cycle can be modified upon the request of the investigator in special circumstances.
· The adult mouse weighs approximately 20-40 grams. Their small size, and resulting large surface area/body weight ratio, makes them highly susceptible to changes in environmental conditions (i.e. hypothermia or hyperthermia). The core body temperature is easily affected by small changes in temperature that may modify the physiologic responses of the animal.
· Mice have a high ratio of evaporative surface to body mass and is therefore more sensitive to water loss. The biological half time turnover for water in the mouse is 1.1 days. Mice use surface evaporation to compensate for increases in body temperature. They can easily dehydrate leading first to shock, then death.
· Mice can compensate better to cold than hot ambient temperatures.
· 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.
· Cancer research
· Monoclonal antibody production
· Immunology
· Molecular genetics
· Dermatology
· Toxicology
· Inbred, outbred, hybrid, transgenic, knockout or mutant mice are available from many commercial sources. The DLAR office staff is familiar with many of the commonly available strains and will assist in locating animals available for purchase.
· Commercially available mice from Charles River and Taconic must be ordered by Tuesday at 9:00 a.m. for delivery on Thursday of that week. Mice from Jackson Laboratory must be ordered by Monday at 12:00 p.m. for delivery on Wednesday of that week.
· Animals should be held for 48 hours before experimental use to allow recovery from the stress of shipping.
· The laboratory mouse is a docile animal and can be easily handled. Animals can be grouped soon after weaning usually coexist peacefully.
· Some strains of mice (i.e. BALB/cJ, SJL/J, HRS/J) are prone to fighting and will begin to fight even if grouped at weaning. Housing males from one litter or pairing males from different litters prior to weaning may help to diminish fighting.
· 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 cage mates. You can normally determine which mouse is the aggressor because they will be the animal without skin lesions. This animal should be separated into another cage, however, once he is removed, another male may take on the aggressor role. In some cases when fighting is a problem, all males must be singly housed.
<DIV align=center>
|
<TBODY> Adult body weight: male |
20-40gm1 |
|
Adult body weight: female |
20-40gm1 |
|
Body surface area |
10.5(wt. in grams)2/31 |
|
Life Span |
1.5-3 years1 |
|
Food consumption |
15 gm/100 gm/day1 |
|
Water consumption |
15 ml/100 gm/day1 |
|
Breeding onset:male |
50 days1 |
|
Breeding onset:female |
50-60 days1 |
|
Gestation Period |
19-21 days1,3 |
|
Body Temperature |
36-37C2 |
|
36.5-38.0C1 |
|
|
Heart rate |
500-600 beats per minute2 |
|
325-780 beats per minute1 |
|
|
Respiratory Rate |
84-230 per minute2 |
|
60-220 per minute1 </TBODY> |
· Male and female mice can be differentiated by observing the distance from the anus and genital papilla which is greater in males. This difference is also present in neonatal mice.
· Breeding systems can be monogamous (one male and one female) or polygamous (one male to 2-3 females). Female mice have a postpartum estrous.
· The mouse is polyestrous and the female cycles every 4 to 5 days.
· Evidence of mating is normally detected in the female by the presence of a copulatory plug.
· Gestation in the mouse is 19-21 days, depending on strain. Litter size varies greatly between strains of mice and litter size in an individual female will increase, plateau and then dimish over her reproductive life. Genetics, environment and embryonic mortality can also influence litter size. Pups are generally weaned at 21 days of age.
· Mice can be grouped by their microbial colonization.
- Specific Pathogen Free Mice (SPF) mice are free from specified bacterial, viral, and parasitic mouse pathogens. To insure the maintenance of the SPF microbial status requires that mice be housed in more stringent conditions that prevent the introduction of rodent pathogens (i.e. maximum isolation housing).
- Conventional mice are not known to be free of pathogens. Standard housing (i.e. conventional housing) cannot prevent the introduction of mouse pathogens.
- Other less common categories are axenic mice (i.e. free from all microbial organisms) and gnotobiotic mice which have a known microbial flora. Axenic mice are normally housed within germfree isolaters and gnotobiotic animals may be housed in germfree isolaters or under maximum isolation conditions.
· Choosing the proper housing conditions for mice is very important. Rodent pathogens often do not produce clinical signs in affected animals but often have immunomodulating effects. Conventionally housed animals are exposed to room air and therefore may readily contract viral, bacterial and/or parasitic diseases.
· Immunocompromised animals (i.e. athymic, SCID, transgenic, knockout mice) are very sensitive to opportunistic agents, and must be housed under maximum isolation conditions to allow for their long term survival. Valuable animals and animals that may need to be transported to other institutions should also be housed under maximum isolation conditions.
· Conventional Housing
- Most mice are housed in shoebox cages composed of polypropylene (opaque) or polycarbonate material (clear) 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.
- When removing the lid from this type of cage it is important to remove the water bottle to prevent spillage. If the cage is to be transported, the bottle should be turned sipper tube up toprevent spillage during transport. However, you should remember to turn the bottle back over to allow access to water after transport.
- It is important to replace the wire bar lid securely on the top of the cage after removing mice or changing mouse cages. Mice will readily climb out of a cage. Also, since mice climb readily, be sure not to crush the mouse when replacing the wire bar lid.
- The husbandry staff changes cages once per week (singly housed) or twice per week (multiply housed), thereby providing the animal a clean cage with new bedding, food and water. Water bottles and feed hoppers are checked daily by animal caretakers. Water bottles should always be checked to insure free water flow (i.e. no air locks).
· Maximum Isolation Housing
- Mice housed under maximum isolation conditions are housed in sterilized, polycarbonate cages with microisolater bonnets. Bedding is sterilized within the cage and allows the absorption of urine and the opportunity for the animal to burrow. This type of cage will hold 3-4 adult mice depending on the size of the cage.
- Animals are provided irradiated food and hyperfiltrated water.
- Personnel are required to wear protective clothing (sterile gowns, gloves, mask, head covers and foot covers) while working in the room.
- All experimental work and husbandry care must be conducted within a laminar flow hood.
- If animals are removed from the housing area and taken to the investigators laboratory, they may not be returned (terminal procedure only).
- Access to maximum isolation housing areas is limited to prevent inadvertent fomite transmission of rodent pathogens by personnel.
- All research personnel must be trained by the DLAR Assistant Director before working in this area.
· Suspended cages – conventional housing
- 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 (i.e. toxicological research).
- Most suspended cages have water bottles but are occasionally provided with automatic watering. Typically a nipple valve (i.e. lixit) is 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.
- A mouse will sometimes jam the lixit valve open, resulting in constant dripping of water, which saturates the bedding material. Upon observation the bedding will appear much darker. The cage should be immediately changed, either by research personnel or by a husbandry staff member since mice can die due to hypothermia from chilling.
- 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.
· Environmental Parameters
- Room temperature 64-79 F°
- Humidity 30-70%
- Ventilation 10-15 air changes per hour
· 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.
· Permanent identification methods can be achieved by tail tattooing which can be performed by veterinary personnel upon request (future picture). Ear tag or ear punch identification can also be utilized but may be obliterated by fighting between individuals. Subcutaneous microchip transponders are easy to implant and work well but are expensive. Transponders can, however, serve a dual function since they can be utilized to monitor body temperature.
· Tail and toe clipping are not generally recommended.
· 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. The tail may be grasped with the thumb and forefinger or by the use of smooth-tipped forceps. With this simple method of holding, they may be transferred to another cage, identified, examined 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. 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. 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.
· 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. Handling neonatal mice should be avoided especially during the first few days after birth to avoid cannibalism or litter abandonment by the mother. 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.
· When handling mice remember not to squeeze the thoracic cavity because this can cause the mouse to have decreased respiratory efficiency and trauma can occur to the chest and lungs.
Restraint Devices
· 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. 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).
· Mice that are moved indoors can be transported in their cage after removing the water bottle and placing it upright in the cage lid to prevent spillage. A clean mouse cage can be obtained from the cage wash area and one half inch of bedding material added. If you cannot locate a clean cage, ask a supervisor or caretaker in the area for assistance.
· A wire bar lid should be placed over the cage to secure the animal during transport. If the animal is going to be in the laboratory for more than an hour a water bottle should also be obtained.
· During transport the water bottle should be placed upright in the cage lid to prevent spillage.
· Transport always results in some stress to the animal, however, animals should recover from indoor transport within their own cage within an hour. No recovery time may be needed if the animals are moved with care and have become accustomed to routine transport.
· 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.
· Only DLAR husbandry or veterinary staff members can transport mice to other buildings or to other animal facilities. Investigators can request animal transportation by contacting the business office at 444-2194. Transport requests should be made 48 hours in advance to allow scheduling of staff.
· Group housing, to allow social interaction, is a primary source of environmental enrichment. All mouse cages also have an additional shredded paper material (Enviro-dri ™) added to their cage to encourage nest building.
· Frequent handling by personnel is important and may increase the ease of working with these animals.
· If animals cannot have environmental enrichment opportunities because of the nature of the research, please contact the DLAR Director or the Clinical Veterinarian (444-2194).
· Zoonoses are infectious diseases that can be passed from animals to humans.
· Potential zoonotic diseases associated with mice include:
- Hantavirus infection – Humans that acquire this infection have severe hemorrhagic disease with involvement of the kidneys. Humans may experience fever, respiratory symptoms and myalgia. The virus is most commonly seen in wild rodents and is shed in the saliva, urine and feces. Mice show no clinical sign of disease. Mice from commercial vendors should be free of Hanatavirus, however, since the possibility of infection always exists, personnel should wear gloves and a face mask when working with mice.
- Lymphocytic choriomeningitis (LCM) – Humans that acquire this disease have variable symptoms ranging from mild flu-like symptoms to central nervous system symptoms. The virus can be transmitted by direct contact with urine and feces or from a bite wound. Commercial colonies are normally free of LCM virus, however transplantation of tumors into mice may serve as a reservoir for the agent. Mice show no signs of disease. Hamsters may also carry this virus.
- Tapeworms – The tapeworm Hymenolepis nana rarely infects laboratory bred mice and may infect humans.
· Mice can inflict sharp, deep bites that can easily become infected. As always, it is important to clean the wound promptly and seek medical attention, when appropriate.
· A tetanus vaccination is required for all personnel working with animals.
· Allergy to mouse dander and urine is not uncommon and usually develops within a few years of working with the animals. Sensitive personnel should wear personal protective equipment such as face masks and/or respirators (properly fitted), gloves and a lab coat. Personnel should alert Occupational Medicine during their annual health risk assessment and if allergy is a problem, obtain advice and treatment from the Occupational Medicine physician.
A. Monitoring
· Investigators should monitor their animals and animal colonies routinely for common signs of illness, such as:
- loss of appetite
- weight loss
- diarrhea
- nasal or ocular discharge
- lethargy
- unkempt appearance
- decreased reproductive efficiency
- increased neonatal mortality
- skin lesions
- changes in experimental results (i.e. immunological, behavioral)
- sudden deaths
· The frequency of monitoring of the animal by the investigator is stated in their approved IACUC protocol. Early endpoints are also defined in the protocol and the investigator is responsible for euthanize their animals when these early endpoints are reached.
B. Reporting Sick Animals
· If an animal is identified with either experimental or non-experimentally related illness, the investigator should notify the DLAR veterinary staff at 444-2194 between the hours of 8:00 a.m. and 5:00 p.m., Monday through Friday.
· If the investigator needs to contact a veterinarian on the weekend, the veterinary “on-call” list is posted in the main entry area of all DLAR facilities and provides the name, home phone number and pager number of the veterinarian on duty.
· When contacting the veterinarian, please provide the following information:
- Investigators name/ your name
- Species of animals and animal ID number
- Location of the animal (building, room #, rack or cage #)
- Signs of illness
- Description of the experimental manipulations performed, if any.
- Phone number where you can be reached.
· In emergency situations, if an animal needs immediate veterinary care, call the main DLAR office at 444-2194. The DLAR office will send an emergency (911) page or radio the veterinarian. If the emergency occurs after hours, call the veterinarian directly.
1) Harkness JE, Wagner JE. The Biology and Medicine of Rabbits and Rodents; 3rd Edition. Lea and Febiger, Philadelphia, 1989.
2) The UFAW Handbook on the Care and Management of Laboratory Animals; 6th Edition; editor Poole, TB. Longman Scientific & Technical, England, 1986.
3) Jacoby RO, Fox JG. Laboratory Animal Medicine, Academic Press, New York, New York, 1984.
4) Suckow MA, Danneman P, Brayton, C. The Laboratory Mouse. CRC Press, New York, New York, 2001.