The Laboratory Rat
CLASS: Mammalia
ORDER: Rodentia
FAMILY: Muridae
GENUS: Rattus
SCIENTIFIC NAME: Rattus norvegicus
Rat is one of the most commonly and vastly used class of laboratory animals. Its popularity as laboratory animal
is next only to that of mice. Just as the mice, rat’s availability spreads over the whole world specially in places
of human habitation. Rattus norvegicus adapts readily to breeding and living in the laboratory conditions. It is
belived that about 3 to 5 million rats are used yearly in laboratories all over the world, which account for use of
10-15% of the total number of laboratory animals of different species. Rats are used mainly in the research of
toxicity, nutrition, behaviour and cancer. A smaller number in proportion is also used in physiological
investigations as well as for teaching.
Over last few decades, the laboratory rat has been developed from the wild brown rat, known as the ‘Norwegian
rat’. Three well-knows groups of strains as under have been developed and these groups have served as the
source for the numerous substrains of rats.
Strains of Rat
1. The Wistar Albino
This strain was developed originally at Wistar Institute, Phile. Delphia, U.S.A. It is a prolific strain and is now
maintained in various laboratories all over the world. It is rather resistant to infections and has a low incidence
of spontaneous tumors.
2. The Sprague-Dawley Albino Rat
The strain was developed at Sprague-Dawley Farms, Madison, Wisconsin, U.S.A. The animals grow more
rapidly and are more prolific than the Wistar. It is less resistant to infection, especially respiratory infections.
3. The Long-Evans Rat
This strain is comparatively smaller in size than the Wistar and the Sprague-Dawley strains. It has a black hood
over the head, black at the back of the neck and has black lines over the back. They are known also as “hooded-
rats”.
The Wistar albino strain has been selected as the representatives of the species for discussion.
Exterior Features of Rat
The Wister rat possesses a big sized head and long ears. The length of the tail is always shorter than the body-
length. Its body is of long cylindrical stature with long thin tail and very short legs. The head has a pointed
snout with two slit-like nostrils, a narrow mouth with short lower jaw and split upper lip, two small round and
glistening eyes-set so that they look diagonally forward and side ways two rounded pinnac and good number of
whiskers (long stiff hairs near the mouth). It has a short neck. The trunk is shortly wider than the head. The anus
is at the base of the tail. In the female the urinary and genital apertures are anterior to the anus on the ventral
surface of the hind and of the abdomen. The female has ten/twelve nipples, situated in two rows on either side
of belly. In the male the common urogenital aperture is at the tip of the short retractile penis, behind which are
two very large scrotal sacs. The body weight of an adult rat stands around 250-450 gms or more. It is about 16
cms; in length from the snout to the base of the tail which is about 12-14 cms in length.
Anatomical Peculiarities of Rat
Anatomically rats are nearly similar to mice. They can also produce gnawing due to their peculiar teeth. They
do not have any vomiting centre [absence of CTZ] tonsil or gall bladder. They have extremely diffuse pancreas.
The stomach shows an obvious division into two parts by a prominent white somewhat curved transverse ridge.
The upper two-fifth non-secretary portion (rumen) is translucent and thinner than the lower three-fifth glandular
secretory portion. The antrum is roughly semicircular area straddling the lesser curvature below the limiting
ridge. It is relatively thin and translucent, and has a slightly irregular inner lining. The remainder of the lower
two-third comprises the body of the stomach which straddles the entire greater curvature below the ridge.
Except for a small area above the pylorus. This is the thick portion of the stomach with mucosa thrown into
folds. This part of the stomach is analogous to the body of the stomach in man both anatomically and
functionally.
Housing of Rat
The rats can be housed in the cages in the laboratory, cages being made of plastic, galvanized iron, anodised
aluminium or stainless steel. The base floor of the cage can be either solid or screen bottomed. The solid
bottomed cages are either placed on the shelf or suspended whereas a screen bottomed cage is always
suspended. The thickness of the wire used for mesh of the screen bottom should be 0.0359 inch in diameter (20
guage) and the mesh should have 2 × 2 openings per square inch to afford maximum comfort to the animal
placed on it. When screen bottomed cages are used, a shallow tray is placed underneath the cages to facilitate
the collection of urine and faecal droppings. These trays can be provided with some materials which absorbs
moisture to reduce bacterial growth and the resultant smell. The trays are subjected to thorough washing and
disinfection everyday. Usually, some material (saw dust or rice husk is) provided as bedding inside the solid
bottom cage. This provide warmth and comfort to the animal inside the cage, besides absorbing urine and
moisture from the faeces. In order to ward off any chance of out break of disease, the cage and the bedding
should be replaced at quick and regular intervals with provision of fresh cages with bedding.
The size of the cage will depend u”on the age, size and the number of animals to be housed. The minimum
recommended floor space per adult rat is 258 sq. cm and the minimum height of the cage should be 17.3 cm. A
cage with a floor area of 850 sq. cm can be conveniently used to house a female with as many as 14 young with
her. A breeding male and six adult females can be accommodated in a cage with a floor area of not less than
1700 sq. cm. But as the animals grow older and bigger, they will need more space for breeding.
The density of rats in a cage has a marked effect on the weight-gain of weaning rats. The young animals lose
heat very rapidly if exposed in a large airy cage with too few companions for huddling. On the other hand, rats
which are older than 3 weeks will not tolerate too many companions in a cage, mainly because of excessive heat
production.
The Colony of Rat
For laboratory use, it is better and convenient to maintain a rat colony, although in some localities rats are
adequately available for purchase from local dealers. It is advisable to begin with a good stock of rats for
selection of best progency for breeders. While attaining age of six months, rats will have reached nearly their
full size. Wood savings are more or less satisfactory for bedding and the cages should be cleared weekly with
hot water and with 5% DDT solution once a month to prevent infections.
Requirement of External Environment of Rat
An ideal temperature for rats ranges between 20°C to 25°C but rapid and frequent fluctuations have to be
avoided to prevent any injury. When the animals are caged in open wire cages, the ambient temperature should
be more cautiously controlled, than if they are housed in solid walled plastic cages. The relative humidity (RH)
is very critical for rats. It should be maintained anywhere between 50-55%. If RH falls below 45% and the
temperature is also low, an out break of ring tail may crop up. Ring tail occurs in a colony where the relative
humidity falls below 45% and the temperature is less than 20°C. Under this condition the new-born rats develop
reddened tails that become swollen, and constricted with corrugated rings. The tail and occassionally the toes
blacken and drop off. During winter season the ring tail usually occurs. A very effective ventilation system
should be provided in housing since the rats are very susceptible to lung infections. In case of air-conditioned
colony, twelve to fifteen change of air per hour in the animal rooms are recommended. Twelve hours of light
and twelve hours of darkness should ensure optimum breeding activity. The intensity of light inside the animal
rooms should be 350 – 400 lux, one metre above floor level.
Handling of Rat
Rats are more inclined to human handling, unlike the mice, rats can be tamed by soft and gentle handling. They
will rarely bite unless startled or hurt. Like mice, small rats may be picked up by tail. Bigger animals can also
be similarly handled for short periods, but proximal half, preferably root of the tail must be grasped. A heavy rat
should not be held by its tail for longer time, since the tail skin may tear. While, however, handling the rat by
tail, the animal should not be dangling in the air, as it may create panic to the animal.Larger rats, especially the
pregnant females should not be picked up, but only caught, by the tail. The weight should be taken by a hand,
either underneath the body or by grasping the rat over the shoulder and chest. A rat will not show annoyance at
if being grasped from above, but it will be nervous if driven into a corner and can attempt to bite.
Rats, who are not accustomed to handling may bite, but a rat with his mouth shut cannot bite. Therefore, it
should be caught with thumb and index finger around and below the lower jaw (not around the throat). The
body or throat should not be squeezed and obviously it will struggle to escape. It is easy to pick up rats by tail,
which incidentally does them no harm, because they will pull away and can be picked up while straining. They
cannot be held along suspended by their tails because they will climb up and attempt to bite. Gloves can be
worn but this is not convenient and also not necessary if proper handling technique is adopted.
Nutritional Requirement of Rat
Like mice, rats are omnivorous and infact, used to devour all things and are experimental feeders. Unfamiliar
foods are consumed by the rats with great care and caution and return to such foods only if they have not
suffered any ill effects. Their nutritional requirements would be adequately satisfied by many combinations of
food stuffs. For all practical purposes, the nutritional requirements are the same as those of mice. An adult rat
consumes about 12-15 gms of diet and drinks about 35 ml of water per day. Diets of highly purified ingredients,
prepared in dried checker or cube form, can surely promote excellent growth and reproduction in rats. Such type
of diet is formulated from food values measured in terms of proteins, fats, carbohydrates, vitamins, minerals and
water. The cube method affords certain advantages. It is economical and simple to feed and handle, does not
spoil, and permits ease in cage cleaning. The diet becomes actually monotonous as noticed that the rats have
interest in other preferential types of food, if available for supply in cages.
Breeding of Rat
The laboratory rat is sexually mature at the age of 6 ½ to 7 ½ weeks. The female rats obtains her first estrus at 6
to 7 weeks of age. The testes of the male descend at about this time, but they are retractable throughout life.
Rats are introduced for the first mating when they are between 90 – 120 days of age. Males and females used
for mating should be approximately of the same size and age.
Estrus Cycle of Rat
The estrus cycle of the rat occurs usually every 4 to 5 days and each is characterized by periodic histological
changes in the epithelium of the uterus and vagina.
The estrus cycle of the rat is divided into four stages according to the cell types found in the vaginal smear,
These are pro-estrus, estrus, met estrus and di-estrus. The estrus phase is characterized as the period of sexual
receptivity when copulation is welcomed by the female.
Estrus cycle does not occur when the rat is in pregnancy, pseudopregnancy or is lactating. However, soon after
delivery, the female rats come into estrus, which is known as the post-partum estrus. The ‘estrus, is followed by
a period of met-estrus (which lasts for about 21 hours), di-estrus (the longest of the phases, about 57 hours) and
pro-estrus (about 12 hours duration) in the order.
Changes of Vaginal Smear during an Estrus Cycle of Rat
The estrus cycle is followed by observing the changes in types of cells in the vagina, the so called “Vaginal
Smear”. In order to elicit this smear, tufts of cotton are wrapped tightly around the ends of toothpicks, the swab
moistened with saline and gently inserted and slightly rotated within the vagina. Thereafter swab pressed in a
drop of saline on a glass slide and examined under low power microscope. From day to day appearance of the
cells in the smear noting as well as recording is made During an anestrus portion of the cycle the smear consists
preponderantly of leucocytes with an occasional cornified epithelial cell. The first appearance of the estrus is
marked by mucification of vagina, rapidly followed by the complete or nearly complete, disappearance of
leucocytes and their replacement by good numbers of round, nucleated, epithetical cells. These cells are about
three times as large as the leucocytes and once seen, cannot be mistaken. At this stage, after about 12 hours, the
animal will breed, but at no other time. Desquamation of the epithelial cells now occurs and white cheesy
masses of disintegrating squamous cells are noticed in the smear. Leucocytes now again make their appearance
and the cycle is repeated. The whole cycle in the rat last for about 5 days, being divided roughly into 3 days of
anestrus and 2 days spent in the manifestation of estrus just described. The cycle appears at puberty which
reaches in rats at the age of 60-90 days.
Mating Technique of Rat
Both monogamous and polygamous methods of mating (pairing with another of opposite sex) are practised in
rats. With a view to having collection of a large numbers of young, two males and six females are
accommodated together in one cage. The female rats remain there till pregnant. Each female rat is examined in
the cage twice a week for signs of pregnancy. The presence of a droplet of blood at the vaginal opening by day
6-10th of pregnancy or a marked increase in weight approximately 25-50 gms by days 12 to 18, is indicative of
pregnancy. Enlargement of abdomen and nipples is evident by 14th day of gestation. All female rats burdened
with pregnancy are segregated in individual cages and remain there until the litters are born and weaned. Paper
cuttin54
The rat gives birth to 8-16 pups per delivery. The litter size may very from strain to strain depending on parity.
Usually the earlier litters are large. The litter size may gradually decrease after the 6 ^ (th) litter. A female will
bread successfully till about 14 to 16 months of age when menopause may occur. This is indicated by irregular
cycles and frequent bursts of activity during the transition. During this period a maximum of litters can be
produced per female breeder.
Breeding Nucleus (Foundation Stock)
It is essential to have a breeding nucleus in order to maintain a breeding colony of rats. The breeding nucleus
(foundation stock) normally consists of 6 to 8 monogamous pairs of breeders. The animals for the foundation
stock are selected either from existing stock or obtained from other animal colonies maintaining good quality
animals of known strain. Animals for breeding nucleus are selected from litters with the following qualities,
varieties or criteria
(a) Large litter size (Animals from litters having lees number of pups than average litter size are avoided).
(b) Good birth weight (above average) and more or less equal sex Ratio.
Least mortality between day of birth and weaning (least pre- weaning mortality).
(c) No cannibalism by mother.
No abnormal pups in the litter.
(f) Good weight at weaning (above average).
(g) Consistent litter size (hence usually the animals for breeding are selected from 2 ^ (nd) or 3 ^ (rd) litter).
(h) Tameness (animals which have a tendency to bite or are aggressive are avoided from breeding stock).
(i) Early maturity (puberty and sexual maturity).
Most of the strains of rats attain sexual maturity around 90 days of age. The breeding nucleus or foundation
stock animals are breed by inbreeding (brother x sister) in monogamous pairs. From each pair (usually from
second or third litter) animals are chosen for next generation. The excess animals are passed on to the
production colony. In the production colony animals can be breed rampantly by polygamous method. A group of
4-6 females can be mated with one male. The advanced pregnant females are separated to individual clean cages
to give birth to pups. Pregnant females have to be provided with sufficient diet, water (ad libitum) and clean
quarters. On the day of delivery the pups aregs duly sterilized are spread in the cage as nesting materials.
The average periodicity of gestation is 21 days, and the maximum number of deliveries generally taking place at
22 days of gestation.
Timed Pregnancies
By dint of monitoring the estrus cycle through vaginal smears, only females in heat can be mated. Another
method is to examine the females introduced for mating for the presence of copulatory plug or vaginal plug
every morning. In rats, unlike in mice, the plug may fall off easily and hence may not be observed. In such cases
a vaginal swab can be taken and the smear can be examined for the presence of sperms in the smear. The day on
which the copulatory plug or the sperm is observed in the smear is counted as day zero of pregnancy.
Seized and average weight at birth (for males and females separately) is recorded. If in any particular litter the
number of pups born are less than average (say less than 8) excess pups from some other litter (born in the same
day or previous day) can be nourished, so that all suckling mothers will have uniform number of pups. This will
ensure more or less uniform weight at the time of weaning.
The young ones are weaned on day 21 post-partum. Each animal is weighed and the weights are recorded. They
are seized and housed in groups of 3-4 in each cage, ready to be supplied for experimental work, depending
upon the experimental requirement such as age and or weight- range. After weaning, each breeder is rested for
4-6 days and reintroduced for mating. In case a large number of rats are required, the females can be mated
post-partum. The rats will come into estrus soon after delivery. If a male is present in the cage, then she will get
mated soon after delivery (post-partum mating) thereby lactation and gestation will run concurrently.
The females have to be provided with additional dietary supplements to cope with additional stress and
nutritional requirement. Generally skimmed milk powder is supplemented in the diet (2-3% extra).
By means of practising post-partum (intensive breeding), it would be possible to get a birth in course of every
21 days, But it is likely that the pups will not be as healthy as those raised from non-intensive breeding. In the
process of non-intensive breeding it may be possible to get atleast 5 6 litters per breeder during its active
breeding span. There may be a reduction in litter size subsequent to 4 ^ (th) or 5 ^ s litter. Such litters are
usually called off. Whenever a pregnant female is separated from the breeding group, the group size can be kept
constant by introducing another female to the group. The animals weaned from the production colony are kept
In the holding room on holding area, awaiting despatch for the experimental work. A schematic representation
of the animals in foundation stock, production colony and the holding room is indicated hereunder:
Life Span of Rat
The life span of the albino rat is about 2-3 years. A great majority of laboratory rats develop chronic respiratory
disease, which results in progressive damage to the lungs, so that by the time the animal is 1.5 to 2 years, old, a
large proportion of the lungs tissue is damaged.
Anaesthesia Techniques for Rat
Usually in rat the same anaesthetic is used for induction and for maintenance and is given by injection.
Experiments in which the animal recovers, however, it may be more convenient and safer to use a volatile
anaesthetic. For minor surgical procedures of short duration such as cardiac puncture, carbondioxide or
anaesthetic ether can be used to produce anaesthesia. Either solid carbondioxide or ether is placed in the bottom
of a glass desiccator, covered with a perforated platform and the rat put in the vessel which is then closed. The
animal is removed when it has lost consciousness and the operation procedure is quickly carried out. For blood
sampling by means of cardiac puncture, carbondioxide anaesthesia is preferable since it causes the least change
in blood chemistry.
For anaesthesia of rat, urethane is frequently used. It is convenient to give half the anaesthetic dose by the
intraperitoneal (IP), route and when the animal is partly anaesthetized, to give reminder subcutaneously (SC). It
may be necessary to wait 20 to 30 minutes before the rat is fully anaesthetized. This method combines the
relatively rapid onset of the IP injection with more prolonged duration of SC injection. It has been found that
less satisfactory results are obtained when the entire dose is given by one route.
Ether can be used for experiments of longer duration, although it does Cause troublesome and copious salivary
and bronchial secretions, and in Addition is highly inflammable. The volatile non-inflammable anaesthetic
halothane is much more suitable and with it depth of anaesthesia can be easily controlled, Recovery is very
rapid without any of the side effects associated with ether anaesthesia.
Pentobarbitone sodium is quite a satisfactory alternative of urethane anaesthetic in rats for short procedures.
Sodium hexobarbitone can be administered by IP, or IV route to produce short acting anaesthesia. Male rats
metabolize barbiturates more rapidly than female rats and so will require even more frequent maintenance
doses.Some preparation (e.g. the pithed rat) must be ventilated artificially. In other experiments (e.g.
neuromuscular blocking agents) artificial respiration might be necessary because the animal's breathing has
become inadequate. The rate and volume of respiration are most important and the following are recommended:
400ml / k * g / m * in
During anaesthesia it is usually necessary to warm the animal because its temperature-regulating mechanism is
not functioning normally. The operating table itself may be placed on a small electric blanket. Alternatively the
animal may be heated by an overhead infra-red lamp. Whatever method is used, it is important to check the
rectal temperature of the animal and to see that it is maintained within normal limits i. e. 37.3 deg plus/minus
0.5 deg * C Ideally a feedback circuit is used so that the animals temperature control the heat source and when
the animal gets too cold the heat is switched on.
Collection of Blood from Rat
The composition of a blood sample will depend on the method of sampling. In long term experiments therefore,
it is imperative that the same method should be adopted throughout for both control and experimental animals.
Analytical methods which will enable the volume of the blood sample withdrawn, to be kept to a minimum,
should be chosen.
Small blood samples can be obtained from a tail vein by snipping the tip of the tail. The animal including the
tail, should be kept warm by means of a lamp and gently restrained. The tip of the tail is cleansed with alcohol
and when this has dried the tip is snipped with a clean scalpel and the blood collected in a pipette or directly on
to a slide or tube, after discarding the first few drops. The tail must not be massaged which can be diluted with
tissue fluids.Large blood samples should be collected by cardiac puncture. The anaesthetized rat is placed on its
back and the heart located under the 5 ^ (th) or 6 ^ (th) rib by palpation with the index finger of the left hand,
the left thumb being held on the rat’s right side. A 2-5 ml capacity syringe and a 19-25 mm 24-26 guage needle
is suitable for the purpose. The needle is inserted at 45° to the long axis of the body into the thoracic cavity upto
the point where the heart is felt throbbing against the needle. The needle is then pushed into the ventricle and
blood sample is drawn. The syringe and needle can contain an anticoagulant (Heparin) if necessary. Large
samples of blood can be collected from anaesthetized rats by orbital sinus with a haematocrit tube. In rat the
total blood volume is about 6 - 7% of the whole body weight. The maximum safe volume that can be collected
at one bleeding is 5.5ml / k * g body-weight.
Administration of Drugs to Rats
Drugs can be administered to rat by oral (PO), subcutaneous (SC). intramuscular (IM), intraperitoneal (IP) and
intravenous (IV) routes. Volumes for injections should be kept between 0.2-0 3 m * 1/100 gms of body weight.
The dorsal tarsal vein is useful for injection as also the saphenous vein where it crosses the leg above the hock.
IV injections are best given in the tail vein using a 12.7 mm * 24 guage needle. Tail veins are not readily seen as
in mouse. For visibility of the veins, the tail should be dipped in saturated sodium sulphide solution for 2
minutes.PO injection is made with a syringe fitted with a long hypodermic needle with a bulb soldered on to the
top end. This needle is inserted into the mouth and gently slid over the tongue and pushed downwards. It should
slide easily into stomach passing through oesophagus. If there is any resistance the needle should be withdrawn
and another attempt is made to find the oesophageal opening. It is helpful to lift the rat’s head slightly to align
the oesophageal and stomach orifices.
Common Diseases of Rat
In general rat is quite resistant to infections. However, some diseases of bacterial, viral and parasitic origin are
known to appear, even in well maintained rat colonies. These infections can be eliminated by early detection. In
practice, a disease contracted animal should be destroyed. Rat is very much susceptible to respiratory infections.
An affected rat will start losing weight and produce characteristic wheezing or chattering noise. The hair coat
will be staring and there will be increased respiration.
Euthanasia of Rat
Ether or carbondioxide in a jar can be used to kill a rat without pain or discomfort. Sodium pentobarbitone
injected intraperitoneally at three times the anaesthetic dose can also be applied.
Experimental uses of Laboratory Rat
1) Study of antiulcer activity of a drug using NSAID using wistar albino rats
2) Antiallergic activity by mast cell stabilisation assay using rats
3) Determination of acute oral toxicity of drug
4) Effect of atropine on dose response curve of acetylcholine using rat ileum
5) Bioassay of oxytocin using RAT uterine horn by interpolation method
6) Bioassay of serotonin using rat fundus strip by three point bioassay
7) Bioassay of acetylcholine using rat colon by four point bioassay
8) study of diuretic activity of drugs using rat
9) Anti inflammatory activity of drugs using carrageenan induced paw edema model
References
Rodents for Pharmacological Experiments-Dr Tapan Kumar Chatterjee
