Rodents accounted for 81% of animals used in research in the UK in 20108, with mice comprising over 72% of animals used. Mice are used in studies including genetics, oncology, toxicology, immunology and developmental biology. Mice are used because they are small, have a high fertility rate and short gestation period, and are easy to maintain. They are also susceptible to many genetic diseases that afflict humans. Rats are used in studies including nutritional research and neuroscience.
Rodents have a number of distinguishing features. Despite their success, all members of the order are small: the largest rodent, the Capybara, weighs only 30–50 kg and is about the size of a small pig.
The word rodent derives from Latin, meaning ‘to gnaw’. This apt description relates to the extremely sharp, chisel-like incisor teeth. Rodents have a single pair of incisors in upper and lower jaws. These are open-rooted, and grow throughout life. For example, an adult rat’s upper incisors may grow 2.2 mm each week, (0.31–0.32 mm per day), and the lower incisors about 2.8 mm per week (0.4 mm per day)9. The teeth are worn away by chewing, and by bruxing (tooth grinding). The incisor teeth have enamel only on the labial (outer) surface, with softer dentine on the inner surface which wears away more quickly, ensuring the teeth always have a sharp edge. The jaw can move both sideways and forwards and backwards, to facilitate gnawing and bruxing.
Nutrition and digestion
Rodents have adapted to feed on a variety of foodstuffs, including seeds and grains, roots, leaves or fruits, as well as invertebrates and even small fish. Laboratory species eat mostly vegetable matter and cereals. Rodents have a simple stomach, with glandular and non-glandular parts, and cannot vomit. A fold of muscular tissue at the gastro-oesophageal junction forms a physical barrier, and they don’t have the oesophageal muscle strength to overcome this barrier. Also they appear to lack the ability to coordinate the different muscles required for vomiting. Rodents, like other herbivores, require a microbial fermentation chamber in which digestion of cellulose from plant material can occur. This takes place in the caecum, located between the ileum and the colon. However, since this is at the end of the digestive tract, the digested matter has to be re-ingested in order for nutrients to be absorbed. During the dark period, pellets of digested material from the caecum are coated with mucus, and these caecotrophs are ingested directly from the anus, allowing the nutrients to be absorbed. This is known as coprophagy. Undigested material that has been through the digestive tract twice is expelled as dry pellets, which can be seen in the environment.
Rodents in the laboratory are usually fed ad libitum with a complete cereal-based pelleted diet, from hoppers suspended above the floor to prevent faecal contamination. These should be cleaned weekly, but the cleaning routine will depend on factors, such as the housing system, stocking density, or presence of newborn litters. Disease states and pregnancy affect the food requirements.
Water is required for lubrication of the food as well as hydration, particularly in mice, and if insufficient fluid is available mice have difficulty eating. It may be supplied in automated systems or bottles. Water provided in bottles becomes contaminated by saliva and faecal matter, and frequent cleaning is required. For some strains, acidification or chlorination of the water may be needed to prevent disease arising in the animals from contaminated water. Automated systems ensure that there is a constant supply available, but when used with solid-bottomed cages there can be a risk of flooding, as animals may play with the sipper tubes or push bedding material into the tube, causing it to drip. When ill, mice drink very little and rapidly dehydrate. Medicines administered in the water may be ineffective, and care must be taken to ensure that adequate quantities of fluid are administered by other means.
Rodents are mostly social animals, living in groups and defending territories from intruders. A considerable amount of rodent behaviour is directed towards marking and maintaining their territory. Most rodents are crepuscular (active at dawn and dusk), or nocturnal. They often exhibit burrowing behaviour if presented with a suitable substrate, and this can be used to assess welfare10.
Senses and communication
Scent marking is particularly important in rodents. It helps maintain a familiar and secure environment, attracts mates and repels intruders, and helps the animal orientate itself. Removal of scent marks, such as occurs during cage cleaning, can lead to insecurity and increased aggression and leads to increased marking. Animals often form a dominance hierarchy, which helps to regulate conflict and reduce aggression and injury. However, fighting can and does occur, particularly between similarly matched individuals. If the territory comes under threat, rodents will mark and defend the territory, fighting if necessary. If an animal perceives the scent of an intruder but cannot then identify them, this can lead to stress.
Rodents have little or no colour vision, being nocturnal. They hear in the ultrasound range, mice detecting frequencies up 100 kHz (compared to about 20 kHz in humans), and are most sensitive in the 15–20 kHz range and around 50 kHz. They communicate both in the human audible range with squeaks (for long-distance warnings), and in the ultrasound range (for short-distance communication).
Rodents can be housed in conventional units where the pathogen status may be unknown, but more frequently are kept in barrier units with a defined and monitored health status. Cages are often of the shoebox type made of plastic. Rodent housing should have solid floors, with environmental enrichment, nesting and bedding material, and refuges, allowing them to exhibit a range of natural behaviours. Most species are sociable and can be housed in groups of compatible animals, although adult male mice may fight, and are sometimes housed alone, and hamsters are solitary by nature. Females of all species with litters will defend their young and are best separated while nursing.
To comply with the requirements of EU Directive 2010/6311, rodents require temperatures between 20 and 24°C, humidity between 45 and 65% and 12 h of daylight daily. The light intensity should be 350–400 lux, except for albino animals. For these, it should be less than 60 lux to avoid damage to the retina. Rodents also hear in the ultrasound range (the wild mouse has an upper frequency limit of 92 kHz)12, and care should be taken to monitor and reduce ultrasound in facilities, since many pieces of equipment and everyday activities such as running a tap may produce ultrasound.
Anaesthesia in small laboratory animals is associated with significant risk. Their small size makes access to blood vessels difficult and increases heat loss during anaesthesia, resulting in hypothermia. Anaesthesia should only be induced in healthy, stress free animals. Small animals need to be kept warm during and after anaesthesia, to prevent hypothermia, and may need supplementary fluids to prevent dehydration and encourage return to normal physiology in the post-anaesthetic period.
Anaesthesia can be induced by inhalation or injection, and inhalation is the preferred method for small-animal anaesthesia. It has a very high safety margin for all of the laboratory species and is rapidly excreted from the tissues via the lungs, which results in rapid recovery. There is virtually no biotransformation, making it the most suitable agent for studies that require maintenance of normal drug-metabolising ability.
Many anaesthetic agents are available (see Chapter 9). Drug doses are intended only as a guide and will have to be adjusted to take account of varying responses of different animals and strains.
The mouse, Mus musculus, is the most commonly used laboratory animal. Many well-defined inbred strains and outbred stocks are available, for which the karyotypes are known. More is known about the genome of the mouse than any other species. An international project has undertaken to sequence the entire mouse genome, and there are numerous centres working on producing genetically modified mice that model human diseases. There are many types of genetically modified mouse available that are useful models for specific disease entities (see Chapter 6).
Mice are active, social animals, which naturally live in harmony in groups with one male and several females and their young once their hierarchy is established. They live in a variety of places near food sources, and construct nests from various soft materials. They exhibit a wide range of behaviours including exploring, foraging, climbing, grooming and resting. Mice usually run on all fours, but when eating, fighting or exploring they stand on their hind legs, supported by the tail. Mice are good jumpers, climbers and swimmers. Opportunities to perform as many of these behaviours as possible should be provided in the laboratory. Failure to provide an adequate variety of enrichment may lead to stress and abnormal behaviour, confounding experimental results.
Mice are territorial, and dominant males respect each other’s territory, venturing into another’s territory only if it is vacant. If two or more males are held together in a cage they will often show aggression unless they have been raised together from birth.
Mice are afraid of rats, which may kill and eat mice, so these species should not be held in the same air space.
Mice use pheromones to communicate, and it is vital to understand this communication in order to manage these animals successfully in the laboratory. Some of the effects of pheromones are listed below:
- stress in one mouse causes dispersal of other mice,
- female mice attract male mice, and vice versa,
- pheromones from lactating females attract the young,
- foreign females stimulate aggression by other females,
- foreign males provoke aggression from other male mice, and may cause recently mated females to abort (Bruce effect),
- group-housed females become anoestrus, and resume cycling if a male is introduced (Whitten effect),
- co-existing males emit pheromones to reduce aggression within the group, but which cause foreign males to avoid the territory.
Communication in mice through scent marking has been extensively studied. Two types of information are carried in scent marks: genetic information (fixed), and information on current status (variable; depending on diet etc.). Scents are detected by the main olfactory system, and via the vomeronasal organ, which detects involatile compounds. This is very finely tuned to specific compounds such as pheromomes.
Scents are composed of two components: airborne volatile compounds and small peptides, and major urinary proteins (MUPs), which are specialised communication proteins. Most MUPS are excreted in urine, but some are also excreted by other routes. MUPs in scent marks signal the genetic identity of the mouse to other mice, for example their sex and whether they are a relative. Individuals have different proteins, allowing animals to recognize each other and avoid mating with their relatives13.
Airborne volatiles allow the scent source to be identified from a distance. If the scent is known, the animal will tend to ignore it. If not, they will try to identify the source of the scent and investigate the MUP pattern, to determine the exact identity of the scent marker; that is, remote sensing via the airborne volatile compounds leads to contact sensing of the MUP pattern. The mouse then learns an association between the airborne volatile scent and the MUP pattern. The next time it encounters the airborne volatile it will be recognised and the MUP may not need to be investigated14.
Female mice can distinguish between dominant and weaker males by how fresh and dense a male scent mark is. Dominant individuals mark more than others, so leave more dense markings, and they also leave the most recent scent marks. Subordinate males scent mark to a lesser extent. Females are attracted to the airborne scent of familiar males and choose males with the most recent scent marks15.
When mice meet, the dominant male allows the intruder to sniff him before he attacks; this gives the intruder a chance to recognise him and run away, thus avoiding aggressive encounters.
Implications for laboratory mice
All laboratory mice have been shown by mitochondrial DNA to have come from a single female, and they therefore have very little variation in their MUP16. Thus they share common genetic signals: this reduces aggression between individuals. However, animals also recognise each other through non-genetic factors influencing the airborne volatiles, and changes in these airborne volatile signals can lead to aggression. Some experimental treatments may influence the airborne volatile signals, and can therefore increase aggressive encounters.
If mice are only exposed to airborne scents without finding the source for contact sensing, mice will eventually ignore it (this happens on racks of conventional cages). However, if they are then exposed to the contact scent of unfamiliar animals, this may lead to an aggressive response. Thus placing unfamiliar animals into shared equipment without cleaning between them can lead to aggressive behaviour and stress. Pheromones are used to maintain stability in the colony and if they are removed each time the cage is cleaned fighting will ensue and subordinate mice will be barbered (where the dominant mouse chews the whiskers of subordinates), or possibly injured (Figure 11.2).
Keeping mice in compatible groups, providing environmental enrichment such as tubes, objects to climb on and refuges, and leaving a little of the soiled bedding in the new cage to reduce the need to keep re-establishing the dominance hierarchy and territory marking all help to reduce aggression and stress.
There are strain differences in behaviour. For example, male BALB/c mice are particularly aggressive, and fight wounds are common.
Estimating the quantitative nutrient requirements for mice is difficult, since different strains have different requirements, and there is little published information on nutrient requirements for maintenance and production in mice17. Different environments also affect nutritional requirements: animals from conventional, specific-pathogen-free or germ-free environments have different intestinal flora, and these influence nutrient requirements. Temperature, diet composition, physiological status, microbiological status and genetic background influence maintenance energy requirements: obese mice have a lower energy requirement than lean mice, and females a lower requirement than males. The daily metabolisable energy (or ME) requirement for maintenance is approximately 160 kcal (670 kJ)/BWkg0.75/day in mice with no genetic or stress-induced abnormalities17. Diets for laboratory mice may be pelleted (compressed or expanded), powdered or in gel form. Diets for maintenance typically contain 4–5% lipid and 12–14% protein, and diets for growth and reproduction contain 7–11% lipid and 17–19% protein18. Mice will eat approximately 3.5–6.2 g of pelleted diet daily depending on strain and physiological status17.
Mice have a large surface area to volume ratio and lose heat rapidly, and so are sensitive to changes in ambient temperature. Much energy is expended in maintaining body temperature, and they cannot tolerate a reduction in room temperature. Mice are also susceptible to water loss. They cannot afford to sweat or pant to lose heat, as this would cause dehydration. In the wild they use behavioural mechanisms, such as burrowing, to keep cool. Therefore, maintenance of the correct environmental conditions and provision of bedding and shelters to allow the animals to manipulate their own microenvironment is vital.
Male mice reach puberty at approximately 7 weeks, and females at 6 weeks. Females then cycle every 4–5 days. This is affected by photoperiod and the presence of others, a period of 12–14 h light daily is needed to maintain oestrous cycles18. Oestrus, mating and ovulation tend to occur during the dark phase of the light cycle. The Whitten effect can be used for synchronising oestrus or for timed matings. Mating results in the formation of a vaginal plug, which can be detected to confirm mating. Fetuses can be palpated from day 7–10. Gestation lasts 19–21 days and 1–15 pups may be born, depending on the strain. Females have a post-partum oestrus within 24 h of giving birth, and if kept with the male will be mated again at this time.
Mice may be bred using a harem system, with one male for two to six females, pregnant females being removed from the group to give birth, or may be kept together in a monogamous system. With the latter system, the young are removed before the next litter is born. Mice will breed until they are 12–18 months old, although the economic breeding life of most strains is around 6 months.
Mouse pups are dark purplish red at birth, and may have a visible milk spot in the abdomen. Their eyes and ears are closed and there is no hair coat. They become lighter in colour over the first few days, and in coloured mice pigment may be visible by day 3. By day 4 the ear flaps are starting to separate from the head, and this is complete by day 5. Hair appears from day 7 and covers them fully by day 10. From day 11, teeth begin to erupt and the eyes begin to open. These are fully open by day 12, and they start nibbling solid food19 (see Figure 11.3). They become increasingly active and increase their intake of solid food until weaning takes place at about 21 days. Weaning should only be carried out if the animals are large enough, and for slower growing strains this may take up to 28 days. Strains vary dramatically in their rate of growth, with some strains growing twice as fast as others17. In general, outbred stocks grow much faster than inbred strains.
Mice are generally easy to restrain, but their small size makes them vulnerable to physical injury. Some mice are also very active and may attempt to jump away from the handler. Movements must be quick and decisive. Handling mice for procedures is potentially a significant source of stress. The most common method used to capture and handle laboratory mice is to pick up and restrain the mouse by its tail; however, this induces a high level of anxiety, and mice do not habituate to handling by this method20. Picking mice up by either placing a tunnel in the home cage for the animal to crawl into or by cupping in the open hands causes less stress, and these methods should be used where possible. Approach the animal after first removing the lid of the cage. Otherwise, they may hide beneath the food hopper, which makes them harder to catch and also increases the risk of being bitten.
If it is necessary to restrain the animal for injection, grasp the base of the tail gently but firmly and lift the mouse. Place the mouse down on a non-slip surface, such as the top of the cage, without releasing the tail. The animal may then be sexed by lifting the tail to expose the perineum. The ano-genital distance in the male is approximately twice that seen in the female. Pull back slightly so the mouse grips the surface. Slide the thumb and index finger of the other hand up the animal’s body and grasp the scruff of the neck to restrain the head. The animal is then secure and may be examined or injected safely. Extra restraint may be achieved by holding the tail with the fourth and fifth fingers (see Figure 11.4).
To handle newborn mice, transfer the mother to a separate cage first, to prevent aggression from her. The pups can then be gently rolled into the palm of the hand. To avoid subsequent cannibalism by the mother rub the hands in soiled bedding material to acquire pheromones before handling the pups, and rub the young with nest material after handling before replacing the mother. From 10 days of age they can be handled as adults, but the mother should still be removed first.
Pain and stress recognition
Familiarity with normal appearance and behaviour is required in order to detect when all is not well. There will be normal variation between healthy animals, due to strain differences, age, diet or cyclical changes in the female due to oestrus, etc. Signs of normal appearance and behaviour include:
- alert, inquisitive and interacting with others in the group,
- well groomed with a glossy coat,
- relaxed with a normal gait: no lameness or ataxia, and not hunched or prone,
- bright and clear eyes,
- normal respiration, not shallow and fast, or deep and laboured,
- colour of the extremities (pink, pale or deep red),
- signs of eating and drinking,
- normal faeces and urine,
- teeth not overgrown and no malocclusion,
- moderate degree of body fat over the backbone,
- normal breeding performance with low neonatal mortality.
Signs of pain and distress include:
- increased sleep time,
- weight loss,
- piloerection and hunched appearance,
- isolated from the rest of the group,
- squeals on handling or pressure on affected area,
- may become more docile (or sometimes more aggressive),
- may eat bedding or neonates,
- abdominal writhing.
Common diseases and health monitoring
Conventionally housed mice may be carrying a number of commensal and potentially pathogenic organisms, and barrier-reared animals can develop infections if there is a breakdown in the barrier. Many infections do not produce clinical signs in adult animals. However, they may cause disease in immunocompromised animals, or interfere with research data. Respiratory infections increase the risks associated with anaesthesia and surgery. Regular health screens should be performed to ascertain the health status or the colony, and to ensure that there are no potentially problematic organisms in the colony (see Chapter 5). Table 11.1 lists some of the pathogens to be included in health-screening protocols. This list is not exhaustive, and more information can be found in reference 21.
|Type of infectious agent||Examples|
|Zoonoses||Hantaan virus, lymphocytic choriomeningitis (LCM) virus, leptospirosis|
|Disease-causing agents||Sendai virus, pneumonia virus of mice, Pasteurella pneumotropica, Staphylococcus aureus, mouse hepatitis virus (MHV), mouse norovirus, mouse rotavirus (epidemic diarrhoea of infant mice EDIM), Theiler’s murine encephalomyelitis virus (TMEV), Clostridium piliformis (Tyzzer’s disease), Mycoplasma spp., endoparasites (e.g. Syphacia obvelata, Aspicularis tetraptera, Tritrichomonas and coccidia) and ectoparasites (e.g. Myobia musculi and Mycoptes musculinus)|
|Diseases affecting research||Minute virus of mice (MVM), lactate dehydrogenase elevating virus (LDHV), Helicobacter spp.|
Biological data and useful reference data
Biological data and useful reference data are given in Table 11.2.
Rats account for approximately 8% of animals used in research, and 293 905 rats were used in 2010 in the UK8. Rats used in research are mainly derived from the brown or Norwegian rat, Rattus norvegicus. Outbred and inbred strains are available. Commonly used outbred strains include the albino Wistar and Sprague–Dawley varieties, and the hooded Lister and Long–Evans strains. Inbred strains include the Lewis, Dark Agouti (DA) and Brown Norway strains. Some transgenic rat strains are also available.
Rats are social animals. In the wild, rats are territorial, and live in social groups in burrows. They usually live in groups of one dominant individual with several subordinates, and they establish and maintain their dominance hierarchies through agonistic behaviours, such as chasing and neck grooming. Usually the subordinate rat flees, and fighting is rare. Rats are usually friendly and easy to handle, although there are strain differences, and males tend to be friendlier than females. They become friendlier with more frequent handling. Rats are nocturnal or crepuscular, being active in the dark period. Their eyesight is poor, and blind rats behave as if perfectly normal. See www.ratlife.org for details of rat behaviour.
Rats are curious and intelligent creatures and need an enriched environment. They should be group housed in stable social groups, with solid floors and nesting material to allow them to create an appropriate microenvironment11. Tubes, houses, shelves or other structures allow the animals to divide their environment and make use of the three dimensional space in the cage. Paper-based commercial bedding, wood shavings or corn cobs may be used. Rats like to stand erect, and so cages with high lids are required. The minimum cage height specified in EU Directive 2010/63 is 18 cm, although the UK standard has been 20 cm and this is preferred.
The rat is omnivorous, eating a wide variety of seeds, grains and other plant matter as well as invertebrates and small vertebrates22. The digestive tract is similar to other omnivorous rodents, and coprophagy occurs. Rats do not have a gall bladder. Nutritional requirements are better understood than for many laboratory rodents but still vary considerably with physiological status and developmental stage, strain and sex17. They can be fed ad libitum on a complete pelleted rodent diet, containing 20–27% protein and 5% fat. Higher protein levels than this may reduce reproduction efficiency. Rats are cautious eaters and will reject strange food. Rats will eat 5–10 g of feed per 100 g of body weight daily. This equates to 15 g/day for maintenance in young and adult rats, 15–20 g/day in pregnancy and 30–40 g in lactation. As rats age, it may be beneficial to restrict their food intake to 80% of ad libitum intake: this increases their life span and reduces the incidence of some types of neoplasia23.
Water may be provided by sipper tubes or by automated watering systems. The water may need to be acidified or chlorinated to reduce contamination, particularly for immunocompromised rats. Rats drink approximately 5–10 ml of water per 100 g of body weight daily.
Rats should be kept between 20 and 24°C. Young rats have much brown fat to assist in thermogenesis, and this reduces with age. The humidity should be 45–65%. Rats are less sensitive to temperature changes than mice, but low humidity can lead to ringtail, in which an annular lesion appears around the tail, potentially leading to necrosis and sloughing of the tail. A 12 h light period is adequate for rats but bright light is deleterious, particularly for albino rats, and results in retinal degeneration. The level should be less than 400 lux, or 100 lux for albinos. Photoperiod affects the oestrous cycle, and 12–16 h light is best for optimal breeding. Ventilation is particularly important for rats, as many of their pathogens are aerosol-borne11.
Rats become sexually mature at about 2 months, females maturing slightly earlier than males. Breeding is usually carried out from 3 months, when females weigh 250 g and males 300 g, and rats breed until they are 12–18 months old. Oestrus occurs every 4–5 days. The Whitten effect is less pronounced in rats than mice (synchronisation of oestrus in females by exposure to male pheromones), but does occur. Mating usually occurs at night, and a copulatory plug of gelatinous material is left in the vagina for 12–24 h, which then falls out and can be detected to confirm that mating has occurred. Gestation lasts 21–23 days, and can be detected from day 15 by palpation23. The female will start nest building in the later stages of gestation, so appropriate nest building material should be provided. A litter of 6–12 pups is born, often overnight. Pups are very sensitive to rearing conditions, which can influence physiological and psychological development23. In addition, if a female is disturbed during the post-partum period she may destroy her young, so it is very important to minimize disturbances and take extreme care cleaning cages during this time.
Polygamous mating systems are preferred. In these systems one male is housed with two to six females. Pregnant females are removed prior to parturition and returned after weaning. Females in this system produce more milk and have larger litters. Females produce 1–12 litters per year, and if held in a colony females may nurture their young collectively. Alternatively, with monogamous systems, males and females are housed in pairs. The female is mated at the post-partum oestrus, and the young are removed at weaning. This produces the maximum number of litters, but the male may interfere with the young. He can be removed at parturition, and returned to the female after the young are weaned. If the female is lactating during gestation then implantation can be delayed, leading to a 3–7 day increase in the length of gestation. In a variation on this system, a single male is moved between singly housed females, spending a week with each. One male is used for every seven females.
Growth and development
The pups are born pink and hairless, with eyes and ears closed. They begin to eat solid food from 11 days, and within 14 days the hair coat develops and the eyes open. Weaning occurs from 21 days. Rats grow rapidly in the first 3 months, reaching approximately 50 g at weaning. Adult weight is reached by about 15 weeks, although male rats exhibit prolonged growth, and bones do not become fully ossified until their second year. Male rats can become obese if fed ad libitum with insufficient space for exercise, reaching in excess of 500 g. Inbred and outbred rats differ slightly in their rates of growth, outbred strains tending to be larger.
After weaning, young rats should be group housed with plenty of space and enrichment to allow them to play, which has been shown to be essential in the development of normal adult behaviour23.
In general, rats are amenable animals, which rarely bite if approached correctly, particularly if they are routinely handled using appropriate techniques, although there are strain and sex differences. Approach the animal confidently and restrain it gently but firmly. Animals will typically only bite if stressed or in pain.