exotics test 2

What type of housing is used for mice? (3)

Shoebox housing, metabolic caging, and micro isolation cages

What is the purpose of shoebox caging?

Female mice can be housed together in pairs or groups as well as males of the same litter

What is the purpose of metabolic caging?

Used to accurately monitor food consumption and waste products of the animal

What is the purpose of micro isolation cages?

Prevents pathogens from entering or exiting the cages

What is barbering?

• Dominance behaviour where the dominant mouse pulls the hair from the other mice in the cage

• These mice should be separated to avoid this progressing into fighting

How to capture a mouse

• Mice are alert but often nervous animals with quick reactions

• They always try to avoid capture by running away 

  • When cornered, bite

• Grip the tail about midway down its length and lift it out of the cage

• Calm and deliberate movements induce less stress and excitement in the house and you are less likely to be bitten

How to restrain a mouse

• Need to alter grip

• Let mouse grip with its fore feet onto a course surface

  • E.g. the sleeve of your lab coat, or the cage top

• Fix the tail by crooking it in the last two or three fingers and let go with your thumb and fore fingers

• Use your now free thumb and forefingers to grasp the scruff of the neck, this needs to be down in one smooth and deliberate action, hesitation may frighten the animal into biting

• Enough loose skin must be gripped otherwise the mouse can twist its head and bite

  • If when taking the fold of skin you fail to take enough at the first attempt, then put the mouse down and restart

How do you sex a mouse?

• Mice are sexed by comparing the ano-genital distance

• It is always greater in the male but the prepuce is not always distinguished from the vulva

• Testes are often retracted

• It is easier to sex newborn puppies than 1 week old ones

How do you perform a SQ injection on a mouse?

• Made into folds of loose skin, most convenient site being over the lumbar area

• Restrain mouse by tail and let it grip the surface with its feet

• Push needle under skin from behind

  • Up to 1mL may be injected

How do you perform a IP injection on a mouse?

• Give intraperitoneal injections require restraining the animal in one hand with head lower than the hind legs 

  • So all the abdominal organs fall away the injection sites

• Place the tip of the needle slightly to side of the midline halfway between the rear edge of the breast bone and the front edge of the pelvis 

• Point the needle toward the centre of the abdomen

• Push it in with sharp movement but go no further than 2-3 mm into the abdomen

How do you perform a IV injection on a mouse?

• Tail veins are more clearly seen when the mouse is warmed for a few minutes

  • This is done by placing the cage under a heat bulb or a strong light

    • Albinos do not feel comfortable in strong light

• A special restrainer is an advantage

  • Lit slit for illuminating the tail from below while warming the animal

• Pull tail slightly towards you and rotate the tail 90 degrees to access either of the lateral veins

• Pull the point of the needle through the skin at a shallow angle, it will then slide easily into the vein

• Start injections at the caudal third of the tail, moving cranially for subsequent injections

• A plastic tube could be also used to fix the tail

  • Make a slit for the tail in the rubber prop

• SOME POINTS TO REMEMBER

  • Always inject in the lateral veins, not in the dorsal / ventral arteries

  • If the needle does not enter the vein then the injection is perivascular, and a diffuse white swelling occurs around the injection site

  • Use a syringe with a low resistance in the piston, so you can feel the difference between SQ (hard) and IV (no resistance to injection)

  • Start injecting as close to the tail tip as possible

    • It is easier to hit there, may closer to the tail base if you miss the first time

Methods for blood sampling of a mouse (3)

Saphenous vein, heart puncture, retro-orbital 

How and what vein do you use for blood sampling in a mouse?

• The best site from which a sample of adequate volume and quality can be taken is from the saphenous vein 

• Effective restraint is essential

  • A tube of approx 2cm diameter is used

    • Perforated 50ml lab tube

• Shave the leg along the saphenous vein from the heel to the knee

• The mouse will climb into the tube if you let the top of the tube turn upwards after the head is in the tube

• To enter the vein, the venous return must be blocked, this is done by tightening the skin over the thigh while extending the shaved leg

  • Shave with a scalpel

• Puncture the vein slightly above the heel with a needle about 1 mm deep

• The cutting edge must be along the vein to avoid severing the vessel

• Collect blood in a hematocrit tube or another collecting vessel

• When you have collected enough blood, pull off the tube and bleeding will stop

• Repeated sampling can be done by removing the clot (crust) by slight scrubbing with a swab

How do you perform a heart puncture for blood sampling on a mouse? How much blood can you take?

• 1-2ml blood can be collected from a mouse via cardiac puncture

• General anesthesia is mandatory use of injectable agents, CO2, nitrous or other inhalation anesthetic via chamber is common

• Although this is a terminal procedure, the longer the mouse’s heart is pumping, the larger the sample size that will be collected

• Larger needle used with cardiac collection will avoid destroying the fibrinogen, allowing clotting for serum analysis as well as better cell quality

• Locate the heartbeat with your index finger, about where the mouse’s left elbow touches the sternum, you may remove the skin with scissor for better visualization

• As soon as you penetrate the skin or chest wall, apply a negative pressure in the syringe by gently pulling the plunger, as soon as you are inside the ventricle, blood will flow

• Massaging the mouse body will increase venous return and give more blood

• Transfer blood into desired blood containers for analysis

How do you get a blood sample from a mouse retro orbitally? How much can you take?

• Can yield larger blood volume and higher quality samples than alternative methods in rodents

  • Generally used on mice and rats

• Anesthesia is mandatory, may be a terminal or recovery procedure dependent on the blood volume required

  • Maximum of 1% of animal’s body weight can be withdrawn

• Technique should be limited to once a week to a maximum of twice per eye as a recovery procedure

• Standard heparinized or non heparinized micro hematocrit capillary tubes as well as traditional needle and syringe can be used

• Animal is held by the back of the neck, and loose skin of head is tightened with the thumb and middle finger

• Tip of capillary tube is placed at the medial canthus of the eye under the nictitating membrane 

• A short thrust past the eyeball will enter the slightly resistant membrane of the retro orbital sinus, the eyeball should remain uninjured

• As soon as the sinus is punctured, blood enters the tubing by capillary action, it may be helpful to retract the tube slightly to facilitate blood flow

• When the allowable amount of blood is collected, the tube is withdrawn and slight pressure with a piece of gauze on the eyeball is used to prevent further bleeding

• Topical anesthetic can be administered to reduce the post procedural pain

How and why would you gavage tube a mouse?

• Accurate and reproducible oral dosing is done via a stomach tube

• A blunt ended gavage needle about 10 cm long and 1-2 mm in diameter is suitable in mice

• Start by measuring the distance from the nose to stomach/xyphoid process

• Try to straighten the neck before introducing the needle

• Wait for the pharyngeal reflex that brings the needle into the esophagus

  • It is not easy to get it into the trachea

• Once you are in the stomach, give approx 1 mL of fluid

  • Fasting the mouse for 12 hours before the gavage will allow for a slightly larger volume

How do you house rats?

• Generally housed in same manner as mine, but in larger cages

• Females are housed in groups of 2-4

• Mature males are housed individually

How do you sex a rat?

• Same as in mouse, distance between anal and genital orifices is greater in the male compared to the female

How do you capture a rat?

• Grasp the rat near the base of the nail and lift it from its cage

• Important to grasp close to the body, grasping too close to the tip may result in tail degloving

• Place the rat on your labcoat and pet to calm, rats are friendly and curious animals that can be quite cooperative when relaxed

How do you restrain a rat?

• Place your index and middle fingers down along the sides of the rat’s head for better control

• Use your thumb and other fingers to support the chest lightly, hold the tail and support the lower body with your other hand

• This method restricts the head movement but leaves an open pathway to the esophagus for gavaging

• If a MORE SESCURE METHOD of restraining the rat’s head is needed, grasp the rat by the tail and press it down on a surface with your index finger directly over the back of the skull to stabilise the head

• Slip your thumb and second finger under the rat’s forelegs, pushing them forward and parallel with the head

What is chromodacryorrhea in rats? What do you do if you notice is?

• Rats are generally friendly and docile

• Stress can be detected in a rat by the presence of porphyrin (“red tears”) secreted around the nostril and eye by the harderian gland

• Porphyrin may appear as tiny red dots (like petechiae) on the nose, and progress into red fluid coming out of the eyes and / or nose

• Flakes similar to flea dirt may be found in the coats of rats under chronic physical or environmental stress, as this is porphyrin that has been groomed away from the face

• If you notice, STOP HANDLING RAT

How do you perform an IP injection on a rat?

• Let the rat relax on the top of the lid

• Stretch the body of the rat by pulling up its tail and then cover the rat with a towel by your left hand

• Fold the corners of the towel under the rat from all directions

• Grasp up the left hind limb of the rat to expose the abdomen

• The injection site should be in the lower left quadrant of the abdomen, because vital organs are absent from this area

• Only the tip of the needle should penetrate the abdominal wall to prevent injection into the intestines

How do you collect blood from a rat? What vein?

• TAIL VEIN USED

• Optimal site of blood withdrawal in rats is around the distal ⅓ of the tail, since this part of the tails gives better visualization of the vessels

• Generally, requires two people if rat is conscious, as a separate restrainer will be needed

• Rotate the tail (the mouse)  90 degrees to access the lateral tail veins

• When the needles penetrates the epithelium of the tail, pull back the plunger a bit to create negative pressure inside the syringe, then push the needle in the vein slowly until you see flashback

• Using a scalpel to make a small wound on the tail is also an option for collecting blood from the tail vein, a pipette or hematocrit tube can then be used for collection

How do you gavage tube a rat?

• Stomach tubing in rats may occur with a metal gavage needle or a red rubber feeding tube, if the red rubber feeding tube, care must be taken to avoid the rat’s teeth, and the possible biting off of the tube

• Like the mouse, tube is measured from nose to the xiphoid process

• Restrain the shoulders of the rat with your thumb and index finger, then support the lower limbs with your palm

  • A towel wrap could aid in proper restraint 

• It is preferable to have another person restraining the rat for the procedure

• Introduce the ball tipped feeding needle from the pharynx into the esophagus when the rat is in the act of swallow, or slide the feeding tube from the left or right side of the mouth behind the teeth

• Placement may be confirmed by injecting a small amount to see if there is a cough response OR palpating the throat for the presence of 2 tubes

How do you house rabbits?

• Adult rabbits are housed individually shortly after weaning, fighting is common in both sexes even among littermates

• Coprophagy is a normal behaviour in rabbits (night feces)

  • Important that housing enables this behaviour 

• NOT METABOLIC CAGES THATS FOR DAMN SURE

How do you capture a rabbit?

• Rabbits are generally quiet, docile animals but they can be easily frightened as they are prey animals

• Approach the animal slowly, provide reassurance by stroking it along its back

• Grasp a handful of loose skin from the scruff of the neck and draw the rabbit towards you

How do you restrain a rabbit?

• NEVER LIFT A RABBIT BY ITS EARS

• ALWAYS SUPPORT THE HIND LEGS

• Now slide your hand under the belly and between the hind legs, use your forearm to take most of its weight as you lift

• A rabbit’s hind legs are very powerful, it is important to place your fingers and thumb firmly around each leg above the hock, with one finger between the legs so that you do not lose your grip

• When you life the rabbit out of the cage, supporting its weight on your arm and keeping a firm grip on the hind legs, it is advisable to tuck its head under your arm, which has a calming effect

How do you return a rabbit to its cage?

• Must go in backwards, if you put it in head first, it may leap forward and injure itself or you

• Much care is needed to support a rabbits hind legs and preventing them kicking out, as they may easily attain spinal damage if not handled correctly

How do you sex a rabbit?

• To sex a rabbit, keep your grip on the scruff and sit it upright on your knee, releasing the hindlegs, press with your forefingers in front of the genital opening

• In the male, the penis is extruded, but in the female, only a vulval slit is visible

• Testes are usually retracted in the male

• VERY HARD TO DO

How do you give a rabbit an SQ injection?

• Made into a fold of loose skin, most convenient being over the shoulder blades

• With one hand, lightly restrain the rabbit using gentle pressure over the shoulders

• The technique is the same as with a cat or dog

How do you give a rabbit an IP injection?

• A  restrainer is needed to position the rabbit with the head lower than the back legs

  • So abdominal organs fall away from injection site

• Assistant holds animal by the scruff with the dominant hand, and the hind legs with the non dominant hand, allowing its body to rest on the forearm of the hand scruffing

• Position the needle slightly to the side of the midline, halfway between the cranial edge of the pelvis

• Direct the needle toward the centre of the abdomen, push it in with a sharp motion to penetrate the muscle, advancing no further than 1 cm into the abdomen

How and where do you give IV injections?

• Usually given into an ear vein

• Those in ear are prominent, especially after local heating

• Lateral vessels are ear veins, central vessel is the artery

• Find it easier to inject intravenously if someone holds the rabbit for you

• Pluck or shave the injection site, then assistant “holds off” vein by restricting venous return at the base of an ear

  • Can use paperclip if alone

• Insert needle, bevel up, at a shallow angle, and slide it into the vein

• When the needle is in the vein, change your grip to fix the needle and ear between your finger and thumb

• Tell your assistant to release the vein before you inject

• If the needle has been positioned correctly, the blood in the vein will be washed away by the injection

• When you withdraw the needle, use digital pressure to prevent any bleeding

• Alternatively, use a paperclip half opened up and pushed halfway across the vein, BUT remember to remove it after a minute or two

How and where do you collect blood from a rabbit?

• Ear vessels are most commonly used for blood collection in rabbits

• An assistant, commercial restraint device or a towel wrap can be used to retrain

• Small blood samples can be collected from the lateral ear vein, shave or pluck the site and cut the vein longitudinally for about 1mm using a blood lancet or a small scalpel blade

• A drop of blood can be collected with a capillary tube sufficient for microscopic smears or a micro sample

• Small volumes can be collected with a needle and syringe

• Samples larger than 5 ml may be collected by the use of a vacutainer in the central artery

• Insert the needle with the bevel to the side, this prevents the arterial wall from occluding the opening to the needle

How do you insert a stomach tube into a rabbit and why?

• Oral dosing or nutritional support in rabbits is done via a stomach tube

• A gag prevents the rabbit from biting the tube, a cylindrical plastical device and about 1.5 cm in diameter works well

• A red rubber tube catheter 2-4 mm in diameter works well as a stomach tube in rabbits

• Estimate the length of the tube required to reach the stomach before insertion and mark it, using the measurement from the nose to the xiphoid process

• Slide the gag in the side of the mouth behind the incisor teeth so that the hole is central and allows easy entry for the stomach tube

• Moisten the tube with lubricant

• Push it through the gag and with gentle pressure, the animal will swallow reflexively

• Attach the syringe to the tube and give the solution slowly

• Tube placement can be validated by the palpation of tube tubes in the throat and no air exiting the tube on exhalation

Aquisition of specimen, how do we choose them? What kind of sources (2) are there?

• Animal models for human medical research are selected based on the similarity in the physiology and anatomy to a human in the applicable area

  • E.g. pigs are omnivorous and have similar GI function to humans - commonly used as models in gastroenterology research

• 3 R’s (replacement, reduction, refinement)  principle should always be applied when selecting a model, for this reason, small rodents used whenever possible

• Mice and rats also available with genetic alterations, creating a human disease state model

  • Transgenic (DNA added)

  • Knockout (genetic info removed)

• Two legal ways to source research animals

  • Purpose bred

  • Random source

What are purpose bred animals?

• The vast majority of lab research animals are purpose bred

• Born and raised under controlled conditions and may be genetically manipulated

• Purpose breeding of lab animals is becoming more and more common as researchers demand animals with particular genetic makeups

  • E.g. researchers investigating narcolepsy use dogs bred to be born with the condition

• Charles River Laboratories and Jackson laboratories are the largest source of purpose bred research animals in Canada and the US

What are random sourced animals?

• Live animals for research can also be purchased from random sources

• Random sources include dogs and cats obtained from animal shelters (once all legal obligations of pound have been fulfilled) as well as animals purchase from farms or breeders

• Random source animals must be legitimately / legally acquired and this process is outlined on the AUP

• Random source animals are preferable to purpose bred in the case that no genetic modification or SPF status is required as the cost is significantly lower, additionally not all species are available from a research breeding facility (sheep)

• Within the species, various breeds offer characteristics making them a more desirable research model for any particular experiment

• Attributes such as

  • litter size, life span, ease of handling as well as genetic modification availability need to be considered when selecting the appropriate breed

RATS (Rattus norvegicus)

Strain (W)

Synonym

Colour

Characteristics

Wistar

Bkl:Wistar

Albino

Good model for long term studies Easy to handle, docile Median life span - exceeds 50 months (long for rats)

RATS (Rattus norvegicus)

Strain (SD)

Colour

Characteristics

Sprague Dawley

Albino

Litter average; 10.5 Docile disposition Most widely used outbred rat in animal research  Excellent reproductive performance and maternal characteristics

RATS (Rattus norvegicus)

Strain (L)

Colour

Characteristics

Lewis

Albino

Litter average; 7.5 Docile disposition Inbred partner for several congenic strains Used in transplantation research, induced arthritis, and diabetes Sensitive to the development of a number of autoimmune disease

RATS (Rattus norvegicus)

Strain

Colour

Characteristics

Lister Hooded

Black and white

A little smaller breed than the other species used in research Vision is less sensitive than the albino breeds Susceptible to audiogenic seizures

MICE (Mus musculus)

Strain

Synonym

Colour

Characteristics

C57BL/6/Bkl

Black/6, B6, C57 Black

Black

Good reproductive performance  Used for transgenic / knockout model development, diet induced obesity, and immunology

MICE (Mus musculus)

Strain

Colour

Characteristics

BALB/c/Bkl

Albino

Emit certain pheromones which could have an effect on other strains of mice within a close distance  Used as a general purpose inbred strain in many different disciplines Used in hybridoma development (hybrid cell used to produce large amounts of antibodies for therapeutic or diagnostic use) Good breeding performance and long reproductive lifespan High intrastrain aggression especially the adult male Easy to handle, more docile than some other breeds

MICE (Mus musculus)

Strain

Colour

Characteristics

SCID (Severely Compromised Immunodeficiency Mutation)

Available as hairless for tumor growth

SCID (severely compromised immunodeficiency) mutation has been transferred onto a non obese diabetic mouse background Animals homozygous for the scid mutation are immunodeficient due to a block in T and B cell lymphocyte development

MICE (Mus musculus)

Strain

Colour

Characteristics

Nude mouse

Hairless

Mouse with a genetic mutation that has a deteriorated or removed thymus gland, resulting in an inhibited immune system due to a greatly reduced number of T cells Phenotype, or main outward appearance of the mouse is a lack of body hair, which gives it the “nude” nickname

GUINEA PIG (Cavia porcellus)

Strain

Colour

Characteristics

Hartley guinea pig

White

Very docile species, may stampede if frightened  May be housed in tubs as they cannot jump or climb

GUINEA PIG (Cavia porcellus)

Strain

Colour

Characteristics

IAF HAIRLESS GUINEA PIG

Hairless on albino background

Natural mutation in Hartley population identified in 1978 and bred Immunocompetent  Used in dermatology

GERBILS (Meriones unguiculatus)

Strain

Colour

Characteristics

Mongolian gerbil

Agouti with black

Litter average; 4.5 Docile disposition Low water requirement and highly concentrated urine makes it useful as a model in endocrine studies Susceptible to high serum and hepatic cholesterol levels, used extensively in lipid metabolism studies

HAMSTERS (Cricetus circetus)

Strain

Colour

Characteristics

Syrian hamster

Golden / tan

Litter average; 9.0 Excellent reproductive performance Relatively free from spontaneous disease, but c susceptible to induced pathogens Physiological characteristics of cheek pouches make it an ideal site for evaluating the carcinogenic potential of substances

RABBITS (Oryctolagus cuniculus)

Strain

Colour

Characteristics

New Zealand White (NZW)

Albino

Most commonly used research rabbit Gemera; multipurpose research model Bred originally for meat

RABBITS (Oryctolagus cuniculus)

Strain

Colour

Characteristics

New Zealand Red

Reddish brown

Non albino phenotype of NZW Better alternative if the albino variance presents an issue (light sensitivity, etc.) Less availability as a purpose bred research animal than the NZW

RABBITS (Oryctolagus cuniculus)

Strain

Colour

Characteristics

Californians 

Black and white

Raised for meat and fur Good mother with large litters (8 - 19 kits) Generally available as a random source model

RABBITS (Oryctolagus cuniculus)

Strain

Colour

Characteristics

Flemish giant

Non albino type, 7 different colour variations

Largest rabbit breed (15-22 lbs) Docile and easily handled Raised for meat, fur and as a domestic pet Generally available as a random source model

DOGS (CANIS LUPUS FAMILIARUS)

Strain

Colour

Characteristics

Beagle

Tricolour

Small enough to be easily handled and housed, large enough to easily perform procedures Due to long back ground raised as working hunting dogs, does very well in a kennel environment with conspecifics Used in both veterinary and human research when controlled breed to breed variables needed in a canine model precluding random source dogs Available as a purpose bred animal