Numerous species of animals serve as subjects for education demonstration and research. However, the laboratory rat seems to be the most used subject. The rat's small size and excellent health make it an superior choice for experimentation. The following presents information about anatomy, general physiology, general care, and breeding of rats as well as euthanasia techniques used on rats.

Anatomy

Many texts of rat anatomy are presently available. These range from early line drawings of gross anatomy and morphology (Wills, 1964; Smith and Calhoun, 1968) to a recent color plate atlas of rat gross anatomy (Olds and Olds, 1979). The latter atlas is a particularly useful atlas of the peripheral anatomy of the rat. In addition, several atlases describe the central nervous system of the rat in stereotaxic coordinates. These include the early line-drawn atlas of deGroot (1959) and the photographically enlarged plates of Konig and Klippel (1963), Pellegrino and Cushman (1967), Skinner (1971) and Thompson (1978).

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Physiological Data for the Rat

Table 1 presents average values for several physiological variables of the adult rat. These average values are intended as general information about the rat; most important among these are the values for body weight, food intake, sexual maturity and body temperature.

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Housing

Several organizations have developed explicit guidelines to regulate the care and housing of rodents used in the laboratory (Author, 1978; NIH Guidelines, 1984).. According to the National Academy of Sciences (National Research Council), rats should be housed in spacious, ventilated, and dry cages constructed of either plastic or stainless steel. Bedding (wood shavings, sawdust or commercial litter such as Beta-Chip) should be changed frequently (2-3 times weekly) to minimize odor and to reduce the possibility of disease in the colony. Cages should be sterilized frequently with steam or chemical disinfectants. Humidity within the colony room should be maintained between 40 and 60 percent whereas the temperature should be kept between 70 and 74 degrees F. Lighting should be diffuse throughout the colony and of an intensity (75-125 footcandles) sufficient to allow laboratory procedures to be carried out. Light schedules should be diurnal (i.e. 12 hr/12 hr day/night schedule) because continuous lighting schedules may produce partial retinal degeneration in rats. Tap water and nutritionally complete feed such as Purina Rat & Mouse Diet should be freely available from water bottles and feeders suspended outside of the cage except where inconsistent with the experimental procedures. While it is recognized that not all rat facilities can meet these stringent guidelines, you should be aware of their existence and strive to meet this standard of care.

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Handling

Rodents that are individually housed over a long period frequently display increased emotionality (urination, defecation and, perhaps, aggression) upon handling. Each rat should be weighted to the nearest gram on a balance (scale) daily to assess its general health and to provide daily handling. You should be aware that rapid weight losses (e.g. 10-20 grams overnight) are frequently the first indicator of disease. To remove a rat from a cage, grasp the rat with your thumb and forefinger around the neck preventing biting movements by the rat. If the rat grasps the cage floor with its paws as you attempt to remove it, do not exert excessive pull to remove the rat as a toenail may be torn. Practice picking up the rat, holding it and then replacing it into the home cage. Body handling is the preferred method of handling rats. If tail handling is done, you should pick up the rat by the base of the tail (that part of the tail closest to the body). This method is not recommended for novices, however, as grasping the tip of the tail will frequently shear off the flesh surrounding the tail tip.

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Animal Identification

Although rats are frequently group housed for reasons related to available space and expense, experimenters must keep track of individual animal data. Some method, therefore, must be used to identify individual rats. The simplest technique is to place a numbered metal tag in the ear (National Band and Tag Co., 721 York Street, Newport, KY 41071). Other investigators prefer to use an ear notching system in which a punch is used to notch the rats ears using the system illustrated in Figure 1. In this system, one ear is used to represent single digits whereas the other ear is used to represent 10's. A particular number is represented by where on the ear a notch or hole is made. Dyes may be used to identify individual rats. These include India ink on the palmar surface or the inner surface of the ear. In addition, either picric acid (yellow) or carbolfuchsin (red) or an indelible felt-tipped pen can be used to stain the fur on the rat's back. The dyes are typically prepared as 1-5% solutions in 70% alcohol and are applied to the fur using dye-soaked cotton tips.

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Breeding

The Classic work of Long (1922) described the estrous cycle of the female rat and its clinical characteristics. The rodent estrous cycle is approximately 4 days in length and consists of 4 stages: proestrus, estrus, metestrus and diestrus. Maximal sexual receptivity of the female accompanies estrus, which in the rat occurs 24 hours into the cycle and is indicated by a dry vagina and swollen vulva. Because the estrus cycle is associated with regular changes in the cell type found within the vaginal fluids, cervical smears can be taken daily to estimate the occurrence of estrus. To do so, insert the blunt tip of a disposable pipette containing 1.0 ml of saline into the vagina of the female to be examined. Expel the saline and then a few minutes later, reinsert the pipette and withdraw .25 ml of vaginal fluid. This sample can be smeared on a microscope slide, dipped into 100% alcohol, air-dried and then stained by dipping into a 5% solution of Giemsa stain (Sigma Chemical). Clear the slide by dipping into distilled water, air-dry and then examine the cells on the slide using a light microscope. Using this method, estrus is indicated by the presence of large cornified cells in the vaginal smear (See Figure 2). Other clinical signs of estrus include an ear quiver response induced by stroking the head and back (Farris and Griffith, 1949) or the lordosis response (arched back) induced by manual stimulation of the vulva using a cotton swab.

Placement of an adult female rat into a cage with one or more adult male rats for a 6 day period will result in detection of sperm in the vaginal tract and pregnancy (Baker, 1980). Sperm can be detected in the vaginal smear (using the technique described above without staining the vaginal smear) or one can examine the bedding of the rat cage in search of the so-called vaginal plug (a dried mass of sperm and vaginal secretions) that is dislodged from the vagina after successful copulation. To prevent cannibalism of the offspring by the male, the pregnant female should be isolated in a large cage provided with adequate amounts of food, water and bedding. Gestation in the rat is approximately 21-23 days. The abdomen of a pregnant female rat is distinctly swollen at 13 days of gestation. This is most easily observed by suspending the rat vertically by the tail. Litter size is approximately 8-14. The number of male and female pups are approximately equal in most litters. If the litter size is large (12 or more pups), the litter size should be reduced or culled to 8-10 pups. The pups that are to be euthanized are those that are smallest for their sex. To determine pup sex, one can use the ano-genital distance as an indicator of sex (Myer, 1971). In general, male pups exhibit a larger distance between the anus and the genitals than do females (see Table 2). Moreover, female pups may display rudimentary nipples at about 9-15 days post-partum. Litters should be weaned (i.e. removed from the mother) between 23 and 28 days post-partum with pups placed into either individual or group cages with chow and water freely available. If facilities are not available for breeding, commercial breeders supply rodents of either sex and a given weight range. Moreover, special surgical procedures (i.e. ovariectomy, hypophysectomy etc) are often available from the breeder for a nominal charge.

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Rat Diseases

Great advances in the production of disease-free laboratory rats have been made in the last two decades. Commercial breeders often derive their stock using barrier techniques in which a litter is delivered by caesarean section and raised in a germ free environment. Such rats are remarkably free from diseases of the respiratory and digestive tracts. Upon arrival in the laboratory, however, rats obtained from commercial suppliers should be placed in quarantine for a 7 day period. If skin sores indicating lice or mites are observed, commercially available powders such as Dichlorovose or Equigard (the latter is placed on the cage bedding) will control lice and mites. Viral or mycoplasmal infections may produce upper respiratory difficulties in rats, often indicated by a chattering or wheezing sound. Such infections are highly contagious. Because antibiotic treatments do not readily reverse these conditions, infected animals ( or whole colonies ) should be euthanized (killed) and the colony housing cages should be disinfected with steam or chemical disinfectant. Another common disease observed in rats is labyrinthitis, a bacterial infection of the middle ear. Infected rats display a marked twisting of the body when suspended by the tail. No therapy is available for this disease; infected animals should be euthanized and the colony disinfected. If you are concerned about the health of animals in your care, you should alert your laboratory instructor.

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Animal Euthanasia

Care should be taken during the course of an experiment that animals are not subjected to unnecessary pain or discomfort. Rats that undergo surgical procedures must be rendered incapable of feeling pain via suitable anesthesia. Certainly, the same principle applies to animals that are to be sacrificed or euthanized at the end of a study. The term euthanasia means "good death". Methods of euthanasia should result in a rapid, painless, and humane death for an experimental subject. An accepted technique of euthanasia for rats is lethal injection of pentobarbital (80 mg/ml/kg, intraperitoneal). Such injections rapidly produce unconsciousness and then death. Although this technique may not be suitable for experiments in which biochemical samples are to be collected after death, it is the most humane. Other techniques are described below. They vary in terms of method (physical vs inhalant) and rapidity of death.

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Decapitation

A physical euthanasia procedure is that of decapitation using one of several commercially available guillotines. The rat's head is carefully introduced between the guillotine blades and then separated from the trunk using a rapid movement of the guillotine arm. This procedure may be used to collect large (approximately 5 ml) blood samples or to collect tissue samples in a way that is not compromised by chemically-induced euthanasia. It should be noted, however, that decapitation is not a routine euthanasia technique. Moreover, recent electroencephalic data suggests that decapitation may not produce death as rapidly as once thought (Mlkeska and Klemm, 1975). 

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Carbon Dioxide

Placement of rats into a chamber containing highly concentrated carbon dioxide will result in unconsciousness and then death. This technique is often used to euthanize large numbers of rats and is thought to be more humane than decapitation. Inexpensive carbon dioxide euthanasia chambers are described in the literature (Myers, 1971; Kraus, 1980).

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Other Inhalant Gases

Ether, chloroform, halothane and metofane have been used in high concentrations to euthanize rats. Ether and chloroform, although inexpensive, are rather caustic to the lungs of the rat and may result in a painful death. In contrast, halothane and metofane are not caustic, are somewhat rapid but can be prohibitively expensive. Moveover, these gases may be harmful to hepatic function in laboratory personnel that are repeatedly exposed to these gases.

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Carcass Disposal

Death should be verified (absence of heart rate, cool body and rigidity of the body) in any euthanized animal prior to its disposal. Again, if you are in doubt, consult your instructor. Each carcass should be double-wrapped in plastic bags and tagged as to their source. Disposal of carcasses should be carried out according to IACUC recommended practices.

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Table 1. Normative physiological values for the adult rat.

Table adapted from: H.J. Baker, J.R. Llndsey and
S.H. Weisbroth (Eds). (1979) The Laboratory Rat, New York: Academic Press.

Table 2. Average ano-genital distance (mm's) in rat pups.

Table adapted from: Myers, R.D. (Ed), (1971) Methods in Psychobiology, Volume 1, New York: Academic Press.

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