Animal Handling

EXPERIMENTAL ANIMALS INTRODUCTION Animals are essential in scientific research, medicines development and safety testing. They are necessary to understand the body in health and disease, and to develop new and improved medical treatments. But their use is not undertaken lightly. Both the potential scientific and medical benefits of the research, and the possible suffering of the animals used, are weighed up carefully before any animal research project can proceed. No-one wants to use animals in research, and no one would use them unnecessarily. Animal research is considered a last resort, to be used only when there is no alternative method. In the UK, strict regulations and a licensing system mean that animals must be looked after properly and may not be used if there is any other way of doing a piece of research. Pharmacological research encompasses a wide range of molecular, chemical and biological techniques. The use of animals in drug discovery is an essential component of the research and is carried out as part of a broad range of studies in which alternative experimental approaches are used where possible. While the majority of pharmacological and toxicological experiments can be and are carried out using alternatives to animals (e.g. isolated cells, purified enzymes, computer modelling, pharmacologists must integrate knowledge from in vitro and in vivo systems, which makes it essential to evaluate the effects of drugs on complex whole animal physiology and behaviour. Most procedures are conducted on common laboratory species especially rodents (mice and rats), although some require the use of dogs, cats and non-human primates, such as marmosets and monkeys. Non-human primates are used in a small number of procedures where they have been determined, through ethical consideration and cost/benefit analysis, by both the host institute and the Home Office, to be the most appropriate species for evaluating medicines designed for human and veterinary use.

ANIMAL IDENTIFICATION METHODS 1. Multicolored animals may be identified by their natural markings. 2. Ear tags.

3. Ear notching: May be accomplished by the following method: a. Restrain animal (Refer to Restraint Techniques of individual species). b. Place ear punch in desired location. c. Firmly and quickly punch ear to avoid an incomplete cut. 4. Ear tattoos: Use a cutting edge suture needle or electric tattooer. Use black ink on white ears and green ink on pigmented ears. May also be done on the tail. 5. Fur Dyes a. Used temporarily for up to 6 weeks. b. Clean fur with 70% alcohol to remove fur grease and allow to dry. c. Use any of the following solutions, made up in 70% alcohol: 1. Saturated picric acid (yellow) 2. 3-5% acidic, basic, or carbol-fuchsin (red) 3. 3-5% methyl or gentian violet (purple) 4. 3-5% brilliant, ethyl, or malachite green 5. 3-5% trypan blue 6. Implantable Microchips This system uses a computer microchip which is injected subcutaneously into the animal. A portable programmable computer scanner is then used to identify the animal.

BASIC INFORMATION ABOUT WORKING WITH MICE INTRODUCTION Mice are common experimental animals in biology and psychology primarily because they are mammals, and so share a high degree of homology with humans. They are the most commonly used mammalian model organism, more common

than rats. The mouse genome has been sequenced, and virtually all mouse genes have human homologs. They can also be manipulated in ways that would be considered unethical to do with humans (note Animal Rights). A knockout mouse is a genetically engineered mouse that has had one or more of its genes made inoperable through a gene knockout. There are other reasons for why mice are used in laboratory research. Mice are small, inexpensive, easily maintained, and can reproduce quickly. Several generations of mice can be observed in a relatively short period of time. Mice are generally very docile if raised from birth and given sufficient human contact. However, certain strains have been known to be quite temperamental. (Mice and rats) have the same organs in the same places, just different proportions. RESEARCH USES Mice of various varieties have provided very useful models in many different areas of biomedical research (Cancer research, Monoclonal antibody production, Immunology, Molecular genetics, Dermatology, Toxicology etc.). BIOLOGY GENERAL CHARACTERISTICS The genus and species of the laboratory mouse is mus musculus (order Rodentia). The laboratory mouse has been domesticated by man for many generations. Other notable biological characteristics are their very acute hearing, well developed sense of smell, poor vision, small size and short generation interval. Mice are by far the most common laboratory animal used for research. BEHAVIOR The laboratory mouse can be easily handled with appropriate training. Animals that grow up together or those grouped at weaning usually coexist peacefully. However, some strains of mice (i.e. BALB/CJ, SJL/J, HRS/J) may begin to fight even if grouped at weaning. Breeding males that have been removed from breeding cages and then caged together will usually fight. Wounds on the tail or along the back are a common sign of aggression between cage mates.

BIOLOGICAL CHARACTERISTICS AND DATA Mice, like most species, have a circadian rhythm. Investigators should be aware that this may affect biological data, and it is best to standardize the time of day that samples/measurements are taken to avoid this effect. The standard light/dark cycle in LARC (Laboratory Animal Research Center) animal rooms is 12/1 2. This light cycle can be modified upon request by the investigator.

The adult mouse weighs approximately 25-30 grams. The small size and relatively large surface area/body weight ratio makes mice susceptible to changes in environmental conditions. The core body temperature is easily affected by small changes in ambient temperature which may modify the physiologic responses of the animal. 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. Mice have poor vision and are unable to detect color. Red light is often used to observe animals during the dark cycle.

Table: BASIC BIOLOGICAL DATA Adult body weight: Male

20-40gm

Adult body weight: Female Body surface area (cm2) Life Span Food consumption Water consumption Breeding onset: male Breeding onset: female Gestation Period Body Temperature Heart rate Respiratory rate

20-40gm 10.5(wt in grams) 1.5-3 years 15 gm/100gm/day 15 ml/100gm/day 50 days 50-60 days 19-21 days 36.5-38.0 C 325-780 beats per minute 84-230 per minute

NUTRITION Mice are commonly fed commercial pelleted mouse diet. These diets are nutritionally complete, but they still need a large variety of vegetables. Food intake is approximately 15 g (0.53 oz) per 100 g (3.5 oz) of body weight per day; water intake is approximately 15 ml (0.53 imp fl oz/0.51 US fl oz) per 100 g of body weight per day. REPRODUCTION Breeding systems can be monogamous (one male and one female) or polygamous (one male to 2-3 females). Female mice have a postpartum estrous. Breeding onset is at about 50 days of age in both females and males, although females may have

their first estrus at 25-40 days. Mice are polyestrous and breed year round; ovulation is spontaneous. The duration of the estrous cycle is 4-5 days and estrus itself lasts about 12 hours, occurring in the evening. Vaginal smears are useful in timed matings to determine the stage of the estrous cycle. Mating is usually nocturnal and may be confirmed by the presence of a copulatory plug in the vagina up to 24 hours post-copulation. The presence of sperm on a vaginal smear is also a reliable indicator of mating. Female mice housed together tend to go into anestrus and do not cycle. If exposed to a male mouse or the pheromones of a male mouse, most of the females will go into estrus in about 72 hours. This synchronization of the estrous cycle is known as the Whitten effect. The exposure of a recently bred mouse to the pheromones of a strange male mouse may prevent implantation (or pseudopregnancy), a phenomenon known as the Bruce effect. The average gestation period is 20 days. A fertile postpartum estrus occurs 14-24 hours following parturition, and simultaneous lactation and gestation prolongs gestation 3-10 days owing to delayed implantation. The average litter size is 10-12 during optimum production, but is highly strain dependent. As a general rule, inbred mice tend to have longer gestation periods and smaller litters than outbred and hybrid mice. The young are called pups and weigh 0.5–1.5 g (0.018–0.053 oz) at birth, are hairless, and have closed eyelids and ears. Cannibalism is uncommon, but females should not be disturbed during parturition and for at least 2 days postpartum. Pups are weaned at 3 weeks of age; weaning weight is 10–12 g (0.35– 0.42 oz). If the postpartum estrus is not utilized, the female resumes cycling 2-5 days postweaning. IDENTIFICATION Cage cards are utilized to identify the strain of mouse, sex, number, principal investigator, and research protocol. Cage cards should not be removed from the cage to avoid misidentification of the animals. Temporary identification of individual mice can be accomplished by pen marks on the tail, hair clipping or dyeing the fur. Pen marks will only last 1-2 days whereas hair clipping may last up to 14 days. Tail and toe clipping are not recommended. Ear punch identification can be utilized but may be obliterated by fighting between individuals. GENDER DIFFERENTIATION 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.

In addition, one can usually determine gender by looking for the presence of testicles. However, one must realize that rodents have the ability to retract their testicles into the abdominal cavity (thus the apparent absence of testicles does not necessarily mean the mouse is female). NOTE- The vagina will be open if the animal has recently mated (the opening may be small or large) or closed if the animal has never mated or has mated, but not recently.

Female

Male

Female

Male

Fig: GENDER DIFFERENTIATION (Anogenital distance) HUSBANDRY HOUSING AND ENVIRONMENT Most mice are housed in shoe box cages composed of a plastic (polycarbonate) material with a stainless steel 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. The bedding also allows the animal to burrow and/or den. 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 to prevent spillage during transport. However, you should remember to turn the bottle back over to allow access to water after transport. Pelleted natural ingredient diets are used to feed all rodents and are composed primarily of cereal grains supplemented with additional protein, vitamins and minerals. The water provided to the mice is municipal tap water. For mice housed under sterile conditions, the water is autoclaved.

Two levels of barrier housing of mice are available. One level involves the housing of mice in a cage as described above. However, in addition, the cage is fitted with a filter top (microisolater top). This filter top allows pathogen exclusion and containment. LARC also maintains ventilated cage racks which provide HEPA filtered supply and exhaust air to each individual cage. This type of housing also provides for pathogen exclusion and containment. Depending upon the needs of the investigator, a HEPA filtered change-out hood can be placed in the animal room to allow for pathogen containment and exclusion when the filter top is removed for cage change-outs or animal manipulations. Also, arrangements can be made to have the entire cage and its contents autoclaved to allow for a sterile environment in which the mice can live.

MANAGEMENT The animal care staff change the cages on a fixed schedule (frequency depends upon the type of housing), thereby providing the animal a clean cage with new bedding, food, and water. Water bottles and feed hoppers are checked daily by caretakers to insure the provision of food and water and to monitor for health or other problems. A health surveillance program is in place utilizing sentinel animals to detect the presence of rodent pathogens. Rodent pathogens often do not produce clinical signs in affected animals but their presence serves as an unwanted research variable. TRANSPORTATION Mice that are moved indoors can be transported in their cage after removing the water bottle (if present) and placing it upright in the cage lid to prevent spillage. It is recommended that a permeable drape be placed over the cages to darken the cage and prevent over arousal of the mice during transport. Mice cannot be transported out of doors or by vehicles by other than husbandry personnel.

HANDLING AND RESTRAINT When handling mice it is advisable to wear gloves to prevent the development of allergies due to direct contact with animal allergens. Mice are usually caught and

lifted by the tail. The tail should be grasped between its midpoint and the mouse's body. The tail may be grasped with the thumb and forefinger or by the used of smooth-tipped forceps. With this simple method of holding, they may be transferred to another cage, a balance, identified, or casually examined. 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 (see “handling and restraint” section). 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 (see “handling and restraint” section). Mice should not be dropped into the cage as this may result in spinal fracture. Rather, they 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 triggering cannibalism or litter abandonment by the dam. 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 historically been successful mothers. Numerous types of restraint devices are commercially available to restrain mice. Quality devices prevent the animal from turning around yet allow easy access to strategic parts of the mouse. Devices should also be easy to clean and provide adequate ventilation. There are some techniques A. Mouse Restraint Technique I - For removal from caging Materials: Disposable gloves Procedures: 1. Grasp mouse near base of tail (grasping near the tip of the tail may induce a “de-gloving” injury in which the skin on the tip of the tail is removed). 2. Lift animal out of cage and place in new caging or on firm surface. 3. DO NOT suspend mouse by the tail for a prolonged time period.

B. Mouse Restraint Technique II - For technical manipulation Materials: Disposable gloves Procedures: 1. Grasp mouse near base of tail. 2. Lift animal out of cage and place on firm surface. 3. Grasp nape of neck with opposite hand. 4. Place tail between fingers to secure and control animal. 5. Mouse is now ready for technique manipulations.

Mouse Restraint

Fig: Mouse Restraint Technique II B. Mouse Restraint Technique III - For technical manipulation using mechanical restraint Materials: Disposal gloves, Plexiglas restraint box Procedures: 1. Restrain mouse by grasping near base of tail. 2. Grasp nape of neck with opposite hand. 3. Place mouse’s head into opening of the restraint box. 4. Release hold on neck while maintaining grasp on tail. 5. Place securing block on appropriate slot for necessary restraint.

Fig: Mouse Restraint Technique III (a)

Fig: Mouse Restraint Technique III (b)

INSTRUCTIONS A. Wear a minimum of a clean laboratory coat and gloves. The use of surgical masks or respirators may assist in reducing allergen exposure. B. Keep records of each procedure performed on each mouse or group of mice on the Laboratory Animal Care Record located in the animal room. C. If Bitten: • Don’t punish the mouse for its natural response! Calmly return the animal to its cage. • Wash the wound thoroughly with an antiseptic soap and water. • Cover the wound with a bandage. • Notify your immediate supervisor of the bite so that procedures appropriate to the injury can be followed.

D. Mouse psychology: • Mice respond positively to quiet, gentle handling. They are normally not aggressive (except for some strains), but if frightened or distressed can inflict painful bites. • Like any animal, mice are creatures of habit. Everyday events do not tend to stress or excite the mice. However, out of the ordinary events such as being picked up, handled, and restrained are stressful and can result in the mice being fractious. Conditioning the mice to handling and restraint will prevent the mice from associating being handled with “negative” things (like being stuck with a needle) and often makes the animals much easier with which to work. • Work quietly among the animals, and try to avoid performing procedures in the animal housing room. This will minimize the excitement experienced by the mice from smells and noises, and will allow you to perform your tasks on a more tractable, less stressed animal.

BASIC INFORMATION ABOUT WORKING WITH RAT INTRODUCTION

The laboratory rat was the first animal where the primary reason for domestication was for use in scientific endeavors. The laboratory rat is one of the most extensively using animals used in biomedical research. Because of their high availability and easy maintenance rat becomes one of the most preferable choice of research animal. Rats have several unique biological characteristics. The acute hearing of rats makes them sensitive to ultrasounds and high pitched sounds. The vision of rats is very poor and they are unable to detect color and are blind to long-wave (red) light. The tail of the rat is the principal organ for heat exchange. The common laboratory rat Rattus norvegicus is an ideal experimental animal for several reasons: abundance of literature published pertaining to them, ease of handling, high fertility rate, short gestation period, low maintenance and disease model for various human disorders and diseases. RESEARCH USES

Mice of various varieties have provided very useful models in many different areas of biomedical research (Oncology, Aging studies, Toxicology etc.). BIOLOGY GENERAL CHARACTERISTICS · · · · · · ·

The acute hearing of rats makes them sensitive to ultrasounds and high pitched sounds. The vision of rats is very poor and they are unable to detect color and are blind to long-wave (red) light. The tail of the rat is the principal organ for heat exchange. Rats are nocturnal. Rats have no tonsils, water taste receptors, sweat glands or gall bladders. Rats do not vomit. Malocclusion (mal-alignment of the teeth) may occur in rats due to trauma, genetics or feeding a soft diet that prevents proper wearing of teeth.

BEHAVIOR · · ·

The rat can become accustomed to handling providing they are not upset by the experience. Rats will bite without warning, but usually not repeatedly. Males tend to be more aggressive than females. Unlike mice, groups of the same sex can be housed together without fighting.

· · ·

Rats are active primarily during the night at which time they feed; the light hours are used primarily for rest, sleep and digestion. Handling animals during the night phase can be more difficult due to this increase in activity. The diurnal rhythm can be changed by a 12 hour shift in the light cycle. It takes approximately two weeks for rats to adjust to this shift. Rats are coprophagic (ingest their own feces).

BIOLOGICAL CHARACTERISTICS AND DATA • • • •

Most active at night (nocturnal) Curious and investigative behaviour Poor vision, acute sense of hearing and smell Social animals, adult males may require separation if aggressive

Body temperature Respiratory rate Heart rate Daily water consumption Daily food consumption Estrous cycle Duration of estrus Litter size Gestation Birth weight Weaning age Sexual maturity Breeding duration Male adult weight Female adult weight Life span

35.9-37.5 70-115 breaths/min 250-450 beats/min 10-12 ml/100 g body weight 10 g/100 g body weight 4-5 days 12 hours 6-12 21-23 days 5-6 g 21 days 65-110 days 12 - 16 months or 7-12 litters commercially 450-550 g 250- 300 g 2.5-3.5 yrs

REPRODUCTION · · · · ·

Breeding systems can be monogamous (one male and one female) or polygamous (one male and one to two females). Gestation is 21-23 days. Litter size varies widely with stock/strain, ranging from 3-18 pups. Birth weights are normally 5-6 grams. Pups are weaned at 21 days of age. Female rats have a post-partum estrus. Male and female rats can be differentiated by observing the distance from the anus and genital papilla (anogenital distance) which is greater in males. This difference is also present in neonatal rats.

IDENTIFICATION Cage cards are utilized to identify the strain of rat, 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 rats can be accomplished by pen marks on the tail, hair clipping or dyeing the fur. Pen marks will only last 1-2 days whereas hair clipping may last up to 14 days. Tail and toe clipping are not recommended. Ear punch identification can be utilized but may be obliterated by fighting between individuals. Rat can be identified by the following methods without anaesthesia 1. Ear punch/notch (Fig 6) 2. Tail tatoo (all ages, including neonates) 3. Ear tag 4. Electronic identification (subcutaneous microchip) 5. Toe clipping (in animals up to 14 days old). After 2 weeks with anaesthesia and analgesia (and only with an approved protocol ) 6. Marker (temporary) always ensure it’s a non toxic marker. 2 2

1

5

10

2

4

40

50

2

20

6

60

3 30

100

200

Fig: Ear notch/punch code. These codes are used in various combinations to produce the desired number. GENDER DIFFERENTIATION

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. Sexing the rat is based upon anogenital distance: males have a greater distance between the anus and urogenital opening than females. An opposite sex comparison is advisable initially. The testicles can be retracted into the abdomen; therefore, it may be easier to sex a mature male by holding its head up vertically. The genital papilla is more prominent in males than females. The scrotum and testes are obvious in the male rat.

In addition, one can usually determine gender by looking for the presence of testicles. However, one must realize that rodents have the ability to retract their testicles into the abdominal cavity (thus the apparent absence of testicles does not necessarily mean the mouse is female). HUSBANDRY HOUSING, ENVIRONMENT AND ENVIRONMENT

Most rats are housed in shoebox cages composed of polypropylene (opaque) (future picture) 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. This type of cage will hold 1-3 adult rats 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 to prevent spillage during transport. However, you should remember to turn the bottle back over to allow access to water after transport. The care taking staff changes the cages twice per week, thereby providing the animal a clean cage with new bedding, food and water. Water bottles and feed hoppers are checked daily by caretakers to insure the provision of food and water. Some rats 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. Suspended cages are occasionally provided with automatic watering. Typically a nipple valve is located in the back of the cage which can be operated by contact. When replacing a suspended cage that is provided with automatic watering it is important to push the cage fully into the rack care to insure that the lixit fully extends into the cage, allowing the animal access to water. Occasionally a rat will jam the nipple open resulting in a constant dripping of water from the nipple, saturating the bedding material. Upon observation the bedding will appear much darker. This problem should be reported to husbandry personnel immediately to allow correction. Pelleted natural ingredient diets are used to feed all rodents and are composed primarily of cereal grains which are supplemented with additional protein, vitamins and minerals. Due to the nature of this type of diet the exact composition can vary substantially from lot to lot. The water provided to animals is chlorinated tap water. Some rats are housed to prevent the acquisition of rodent pathogens. Rodent pathogens often do not produce clinical signs in affected animals but often have an immunomodulating effect. Therefore, this type of housing is important for studies that involve the immune system. Immunocomprimised animals, which are sensitive to opportunistic agents, are also housed in this fashion to allow for their long term survival. These animals are housed in sterilized cages and are provided sterile food and water. Access to this housing area is limited to prevent inadvertent fomite transmission of rodent pathogens by personnel. TRANSPORTATION Rats 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. 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 rats during transport. Rats cannot

be transported out of doors or by vehicles by other than Office of Animal

Resources husbandry personnel. HANDLING AND RESTRAINT Before removing rats from their cages, check the cage to determine the number and location of rats. Mothers with litters may be more aggressive than usual. The skin of the rat’s tail is fragile and easily stripped from the underlying tissue. Great care should be used when handling a rat by its tail. The tail should be grasped at the base only and only for brief manipulations. A rat is capable of curling up and biting you when it is suspended by its tail. It is important to support the body of heavier rats when manipulated. Three methods are commonly used 1. The handler’s thumb is thrust between the forelegs and under the chin, and the index finger is clasped around the neck in front of the forelegs. For more control, the index finger may be positioned on the top of the head to create a vise-like action with the thumb.

Fig: Handling rat by Tail 2. Place your first and second fingers on either side of the head, grasping the head with your knuckles and positioning the forelegs of the rat between your adjacent fingers. Rats are usually lifted by grasping the thorax. The grip must be firm without compromising the animal’s respiration.

Fig: Lifted by grasping the Thorax Never pick a rat up by the loose skin on the back of the neck. 3. You can wrap a larger more aggressive rat in a towel and this method has the advantage of controlling the hind limbs which prevents you from being scratched (the most common injury afflicted by rats). Note: Scruffing a rat like a mouse is not a recommended technique

Fig: Wrapping the rat in a Towel RESTRAINT DEVICES Restraint devices are available in different sizes and different materials (e.g. plexiglass, plastic) and can be used when performing technical manoeuvres such as subcutaneous, Intraperitoneal and intramuscular injections. The animal should not be able to turn around or stand up, however, its respirations must be unimpeded at all times. Never allow the animal to overheat.

Figure: From left to right, only the first three restrainers are suitably sized for rats. (A flexible plastic restrainer is in the background)

SEX DIFFERENTIATION Sex differentiation in rat is same as that for mice. BASIC INFORMATION ABOUT WORKING WITH RABBIT INTRODUCTION RABBIT (Oryctolagus cuniculus): Rabbit are also docile animals with large ears. Usually Newzealand white rabbits are used. It has huge caccum and large appendix. The enzyme atropine esterase is present in rabbit liver and plasma, so it can tolerate large does of belladona (atropine). Cardioaortic nerve forms a separate depressive nerve. Vasodilator nerves are absent and so vasomotor reversal phenomenon can not be demonstrated. Histamine causes increase in blood pressure. Ovulation is related to the release of luteinizing hormone and occurs 10 hours after coitus. EXPERIMENTAL USE- Adult weight 1.5-3.0 Kg. age suitable for experiment 5-6 months. 1. Pyrogen testing. 2. Bio assay of antidiabetics, curareform drugs and sex hormones. 3. Screening of agents affecting capillary permeability. 4. Irritancy tests. 5. Screening of antitoxic agents and teratogens. 6. Studies related to reproduction (antifertility agents). 7. Isolated preparation like heart, dudenum, ileum, Finkleman preparation. 8. Study of local anesthetics (surface anaesthesia). 9. Study of miotics & mydriatics. Research Uses The New Zealand White (albino) is the most widely used breed in Canadian laboratories. Their medium size and very docile nature make these rabbits particularly easy to maintain, handle and restrain, while their large, unpigmented ears facilitate repeated venous injections and bleeding. Unfortunately, though classed as a medium sized breed, these rabbits, with age, usually become very heavy. This results in their tending to be awkward to handle and too large for the standard cage; they also tend to consume excessive amounts of food and to develop sore feet. Rabbits of various varieties have provided very useful models in many different areas of biomedical research (embryology, toxicology, virology, etc.), and are also widely used in safety testing (pyrogen, teratogenicity, etc.). They are universally and routinely used in serology because of the readiness with which they produce serum antibodies in response to a wide variety of antigenic stimuli. Two major disadvantages of rabbits as research animals are the difficulties encountered with anesthesia, and the fact that they are plagued by a large number of spontaneous diseases. BIOLOGY

GENERAL CHARACTERISTICS Of the many breeds of the domesticated European rabbit (Oryctolagus cuniculus), the albino New Zealand White is the most common breed utilized in biomedical research. The visibility of the peripheral vasculature in albino rabbits is advantageous to the biomedical use of this rabbit. Non-albino breeds such as the black and white Dutch belted rabbit tend to be preferred where pigmentation is required (i.e. ophthalmological research). Rabbits like rodents have two large upper incisors and two large lower incisors. Unlike rodents, however, an additional pair of incisors is located caudal to the large upper incisors (peg teeth). This additional pair of upper incisors is greatly reduced in size and is why rabbits are members of the order Lagomorpha rather than the order Rodentia. Rabbits have several unique anatomical characteristics. As previously mentioned the long slender ears of the albino New Zealand White breed facilitate phlebotomy procedures. The long backbone predisposes rabbits to spinal fractures, if not captured and restrained properly. Rabbit teeth continue to grow throughout life. Malocclusion occasionally occurs in rabbits preventing normal tooth wear resulting in severe overgrow of the teeth inhibiting normal mastication. The neutrophil of the rabbit resembles an eosinophil due to the numerous intracytoplasmic eosinophilic granules. Rabbits can be grouped by their microbial colonization: "Specific Pathogen Free" (SPF) rabbits are free from several common pathogenic organisms such as Pasteurella , Encephalitzoonois, and Coccidiosis. SPF rabbits are bred specifically for research use. "Conventional" rabbits tend to be colonized with the preceding organisms which can occasionally be detrimental to their health and longevity. Conventional rabbits are bred primarily for food and fur. Unlike SPF rabbits, conventional rabbits are widely available and if appropriate for the project can represent a substantial savings upon purchase. The investigator must weigh these considerations when choosing the type of rabbit for purchase. Numerous genetic mutations have been noted in the rabbit and several inbred rabbit strains have been produced. The rabbit's life expectancy in the laboratory or breeding colony will rarely exceed four to five years, although under natural conditions they may, particularly in the case of males, live at least twice that long. Rabbits are alert, docile, timid animals that adapt readily to cage housing. In the wild state they are nocturnal. The dental formula of the adult rabbit is: Incisors 2/1, canines 0/0, premolars 3/2, molars 3/3. These teeth are peculiar in that they all exhibit continuous growth. Growth of the incisors is particularly rapid, up to 12 cm/yr. The incisors are worn down by normal chewing provided that occlusion is normal. Average body temperature is 39.5oC (range 38.5-40oC) which usually fluctuates considerably with excitement caused by handling, even when other signs of disturbance are not obvious. Rabbit urine is normally thick and cloudy, and contains crystalline material. Urinary diment tends to accumulate on the excreta trays.

BIOLOGICAL CHARACTERISTICS AND DATA Adult body weight: male

2-5 kg

Adult body weight: female

2-6 kg

Life span

5-6 up to 15 years

Food consumption

5 g/100 g/ day

Water consumption

5-10 ml/100 g/day

Rectal Temperature

38.5-39.5C

Heart rate

205-235/minute

Respiratory rate

30-60 per minute

Normative value of rabbit Parameters Body weight Life span Water consumtion Food consumption Urine excretion Oestrus cycle Gestation period Breeding life Litter size Body temprature Respirayory rate Heart rate Blood pressure Blood volume

Value 2000-4000gm 4-5yrs 200-300ml/d 100-150g/d 300ml/d Spontaneous ovulator 32d 3yrs 4-8 38.3C 35-60b/min 205-308beat/min 45-70ml/kg

NUTRITION Rabbits are herbivorous and may, under normal conditions, be very adequately maintained on any one of the numerous complete pelleted rations widely available commercially. Fresh water should be provided daily and ad libitum. Hay supplements may be provided. Reingestion of feces (coprophagy) is a normal practice amongst rabbits and is important, indeed essential, to maintain adequate nutrition and normal intestinal physiology. Nutrient requirements of rabbits are well

established and have been published; they should be consulted when semi-purified or purified diets are to be prepared. REPRODUCTION Spontaneous ovulation does not occur in the female rabbit (doe). Coitus is normally required to induce ovulation. Because of this, there is no defined estrus cycle in this species. Under optimal conditions the mature doe will remain ready to accept the male (buck) ndefinitely, and will have a constant but fluctuating number of ripe (graafian) follicles ready to rupture if coitus occurs. Whether or not the doe is actually ready to mate can be determined by the state of her vulva, which in heat should be enlarged and somewhat red, and by her immediate behavior when placed with a buck. Generally, the doe is taken to the buck's cage for breeding. Semen collection and artificial insemination techniques have been described. The pregnant doe should be housed individually in a quiet area, and provided with an enclosed nesting box in which she will make a hairlined nest for her naked born litter. GENDER DIFFERENTIATION (PALPATION) Palpation of the genital area with eversion of the penis of the male to determine the sex of the animal may be undertaken at any age. However, if this is undertaken on the newborn, the doe should be removed until after the young settle down from the disturbance. In older animals, restraint for this procedure may be accomplished by grasping the loose skin over the shoulders in one hand, and the hind legs in the other hand. The rabbit is then turned over in the lap of the handler, with the hind legs pressed against its chest. In this way the genital area is exposed for examination. The small, slit-like vulva of the doe and the rounded, papilla-like penis of the buck, with its urethral opening positioned immediately adjacent to the anus, can be readily visualized when the skin of the perianal region is stretched. Palpation for pregnancy should be done carefully and gently with the female sitting in a normal position on a table. The rabbit’s urogenital region is examined by lifting the rabbit by the scruff while supporting the hindquarters. To avoid being scratched, approach the genitals from behind the hocks. Apply gentle pressure against the external genitals. Male: The penis will evert slightly, exposing the rounded urethral opening. The typical scrotal sack is not always obvious, especially in young males. Male rabbits also have open inguinal rings, which allow the testicles to retract into the abdomen. Female: A slit like vulva will project while the caudal portions remain closely attached near the anus.

Fig - GENDER DIFFERENTIATION HUSBANDRY HOUSING AND ENVIRONMENT Adult rabbits are housed individually in metal, preferably stainless steel, cages with wire mesh floors, with excreta collection trays below. The latter should periodically be treated with acidic compounds to remove the sedimentary accumulation that is peculiar to rabbit urine. It must be emphasized that adequate, free ventilation is vital to maintaining rabbits free of respiratory disease. Routinely, a 12-14 hour light schedule will prove satisfactory for the research colony. Breeding colony females should be provided with 14-16 hours light, with 8-10 hours recommended for the males. Sudden switching on of lights without prior warning should be avoided during the dark period as it may cause consternation and even spontaneous ovulation and occasional self injury. Similarly, sudden, loud noises should be guarded against, particularly in the breeding colony where they may interfere with estrus and maternal behavior. MANAGEMENT The caging should be washed at least once a week. Regular cleaning and sanitizing of racks, equipment and room surfaces should also be regularly undertaken. Excreta trays should be cleaned as often as necessary to minimize ammonia buildup in the rooms. Disposal of dirty bedding and cleaning of excreta trays should not be done inside the animal room. Fresh food and water should be provided daily. All animals should be observed at least once daily, with food and water consumption and nature of excrements being noted. Sick or dead animals should be removed immediately, with the latter being handled in accordance with the provisions of the experimental protocol.

ACQUISITION PROCUREMENT Rabbits that are to be introduced into a laboratory colony, either for research use or as replacement breeding stock, should always be ones of known and assured genetic quality. This requirement necessitates dealing with reputable breeders who maintain proper records on such matters as the origin of breeding stock, mating system(s) used animal dentification, and other information pertinent to maintaining a genetically defined population. The supplier's facilities, caging, husbandry and management should be consistent with the principles laid down by the CCAC. The rabbits should be healthy, and free from overt signs of disease or parasitism. It is always advisable, insofar as it may be possible, to use a single, reliable supplier for all replacement stock. TRANSPORTATION Newly acquired rabbits should have been transported to the research facility in disposable containers with sufficient space to allow the animals to stand, lie down and turn around. A source of water and food, sufficient for the duration of transportation, should have been provided; carrots are often used for this. Generally speaking, rabbits tend not to travel well over long distances and periods of time; thus it is often advantageous to use local suppliers. QUARANTINE AND CONDITIONING Newly acquired rabbits should be held in quarantine for at least three weeks, and examined regularly for disease, for which they should be treated as necessary. The quarantine period may also serve as a conditioning period to acclimatize the animals to the institution's facilities, husbandry and feeding (dietary) practices. All animals dying during this period should be subjected to complete postmortem examinations. HANDLING AND RESTRAINT HANDLING When removing a rabbit from a cage, or picking it up, the loose skin over the shoulders can be grasped with one hand, the other hand also grasping the loose skin along the back (in large rabbits) or supporting the abdomen. Rabbits should never be picked up by the ears as these are easily hurt and injured; the ears are sensitive organs that play a role in body temperature regulation, as well as hearing, in animals of this family. Rabbits can safely be carried in the crook of the arm with the rabbit's head tucked in behind the elbow, while the forearm supports the body and the hand is placed around the rabbit's rump. Improperly handled rabbits may

struggle furiously, and in doing so may injure themselves or the handler. It should be remembered that the bones of the rabbit are extremely light and brittle and consequently fracture easily. Traumatic injury to the spine of the rabbit may occur during violent struggling, resulting in a broken back (usually lumbar vertebrae) with posterior limb paralysis and urinary and bowel dysfunction. Such rabbits should be humanely killed as soon as possible. The strongly muscled hind limbs and sharp claws can inflict deep scratches on the hands and arms of the handler, and it is recommended that long sleeved laboratory coats be worn while handling rabbits.

Rabbits are especially susceptible to the effects of stress and should always be approached in a calm and confident manner.

. The handler is restraining the rabbit firmly by the scruff with the other hand ready to support the animal’s hindquarters

The rabbit should be held its head tucked under the handler’s arm and with the back and hindquarters supported by the handler’s forearms. PHYSICAL RESTRAINT A variety of restraint devices (rabbit boxes, rabbit boards) are available commercially for rabbit restraint while injecting, bleeding or performing other nonpainful manipulations. These restraint devices must be used with care on rabbits not accustomed to them; however, the acclimatization period is usually short, particularly for the more docile breeds. For short holding and simple procedures, rabbits can often be restrained by wrapping a towel snugly around the animal's body, ensuring that the legs are contained. A state of "hypnosis" or tonic immobility may be induced in rabbits placed in dorsal recumbency and held in that position. Animals so immobilized exhibit reduced responses to stimuli. Because it is unclear whether there is a reduced response to pain during "hypnosis", it should not be used as an alternative to adequate anesthesia.

Rabbits may also be restrained for injection by wrapping the animal in a drape or towel. If the rabbit is securely wrapped, it will not struggle. HAZARDS A variety of hazards can arise through the use of laboratory animals, whether supposedly healthy or experimentally infected. The hazards arising from the latter are considered in the section on biological hazards. The risks with healthy animals are two-fold; the animals can inflict wounds and can carry unsuspected infection -

possibly transmissible to man. Although it is convenient to consider these risks separately, they can interact, since a bite or scratch may provide the route of entry for infection. If you are intending to perform experiments on living animals then you must have a valid Home Office Licence. A suitable Code of Practice must also be in force in each Department, which includes contingency plans for countering the diseases below, and what to do in the event of unexplained animal deaths, especially of primates. Staff at the Veterinary Hospital are available to advise on the health of animals. Animals must be kept in suitable, regularly cleaned, accommodation to which only authorized persons have access. New stock must be quarantined before being released for use. Apart from the more obvious physical hazards of handling animals such as bites, scratches etc. a serious problem can be the zoonoses, which are diseases transmissible among animals and men. The most important include · · · · · · ·

Mice - Salmonellosis, ringworm, tapeworm Rats - Salmonellosis, Leptospirosis, ratbite fever, ringworm. Guinea pigs - Listeriosis, brucellosis, pneumonia. Rabbits - Pasteurellosis, listeriosis. Cats - Ringworm, cat-scratch fever, Toxocara spp., rabies. Dogs - Ringworm, rabies, Toxocara spp., tapeworm. Reptiles - Salmonellosis Amphibia, Hens.

Bites from animals must be noted and an Internal Accident Report Form completed. Indeed it is wise to report any injury, even those associated with non-infected animals. Stock animals must be kept in a separate room from experimental animals and regularly inspected for signs of illness. Sick animals must be isolated and treated. Carcasses and litter must be incinerated. Cages must be regularly sterilised and rooms washed with a suitable disinfectant. The use of protective clothing and personal hygiene practices is the same as that adopted for working in microbiological laboratories, so no eating or storing of food is allowed. Animal house dust can carry particles of excreta and skin containing microorganisms which may cause allergic reactions, such as nasal congestion or sneezing. Simple face masks are generally ineffectual and half-face respirators are recommended; medical advice should be sought at the first signs of an allergic reaction.