1. By Kalani M PhD candidate in Immunology What kind of animals? – Rattus Norvegicus – Rabbit (Oryctolagus cuniculus) – Hamster (syrian) – Guinea pig

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In the name of God

LABORATORY ANIMALS HANDLING AND TECHNIQUES

ByKalani M

PhD candidate in Immunology

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What kind of animals?

– Rattus Norvegicus– Rabbit (Oryctolagus cuniculus) – Hamster (syrian)– Guinea pig–Mus domesticus domesticus (musculus)

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1. Abundance of literature published

2. Ease of handling

3. High fertility rate

4. Short gestation period

5. Low maintenance

6. Disease model for various human disorders

NOD, Nude, EAE, CIA, NZB/NZW

Why animals?

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Mice Restraining

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Mechanical Restrainers

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Injections

Intramuscular (IM)

Subcutaneous (SC)

Intraperitoneal (IP)

Intravenous (IV)

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Blood Collection

Tail

Orbital sinus

Saphenous vein

Heart

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Intramuscular Injections

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Intramuscular Injections cont.

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Subcutaneous Injections

12Pick up a nude mouse and spin it’s tail to put it in a faint condition

Subcutaneous Injections cont.

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Grasp the loose skin on the back of the mouse from ears along the legs and restrain the legs with your ring finger and little finger


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After disinfect the surface area, insert the needle in the lateral side of the abdominal wall and push upwards to the armpit of the mouse


15Inject the substance slowly


16A lump of injection substance can be seen through the skin after injection


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Intraperitoneal Injections

18Place a mouse on a cage lid and grasping the loose skin behind the ears with your thumb and forefinger

Intraperitoneal Injections cont.

19As soon as the mouse’s head is restrained, the mouse can be picked up and the tail secured within your ring finger and little finger


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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 intestine.


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Intravenous Injections

• Warm the mouse under a heat lamp or other heating device

• being sure not to

OVERHEAT the animal.

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Placing a mouse on a cage lid and grasping the loose skin behind the ears by the thumb and forefinger

Blood collection from Tail

23Push the mouse into the restrainer

Blood collection from Tail cont.

24Leave the tail of the mouse outside the cover of the restrainer


25Amputate the tip of the mouse tail by scissors


26Massage the tail and collect blood by sampler


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• Lateral Tail Veins


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Blood Collection From Orbital Sinus in Mouse

• Should apply anesthetic before blood withdraw• A convenience and easy apply method for blood

collection in mouse• Collect amount up to 0.5 ml

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Blood Collection From Orbital Sinus cont.

30Anesthetize a mouse by intraperitoneal injection of Hypnorm


31Use a sharp end glass capillary tube to penetrate the orbital conjunctiva and rupture the orbital sinus


32Collect blood with a vial


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Tools for blood collection from Saphenous vein in mice

• 75% alcohol cotton ball for surface disinfection

• 50 ml syringe tube with small holes at the end as restrainer

• a scalpel and shaver for remove of hair

• 24 G “ needle for release of blood

• tips and pipetteman for blood collection

34Placing a mouse on a cage lid and grasping the loose skin behind the ears with your thumb and forefinger

Blood collection from Saphenous vein cont.

35Place the mouse in the restainer


36Pull out the leg and removed the hair by a assistant


37Hair can also be shaved by using a small scalpel


38The saphenous vein is seen on the surface of the thigh


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Apply vaseline after disinfect the surface area to reduce clotting and coagulation during blood collection.


40Use a 24 G 1” needle to puncture the vein and release blood from the saphenous vein


41Use a Microvette or a pipetteman with tip to collect blood from the saphenous vein


42Approximate 100 microliters can be collected


43Flex the foot of the mouse to reduce the flow of blood back to the puncture site


44A cotton ball is applied to the puncture site to stop further bleeding


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Blood Collection From Cardiac Puncture in Mouse

• For collect up to 1 ml of blood within a short period of time

• Must be performed under general anesthetic

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Tools for Cardiac puncture in Mouse

• 75% alcohol cotton ball for surface disinfection• Hypnorm used as anesthetic• 27G needle with 1 ml syringe for injection• 24G needle with 3 ml syringe for blood withdraw

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Blood Collection From Cardiac Puncture cont.

48Anesthetize a mouse by intraperitoneal injection of Hypnorm


49Disinfect the thorax area with 75% alcohol cotton ball


50Search for the maximum heart palpitation with your finger


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Insert a 24G 1” needle through the thoracic wall at the point of maximum heart palpitation


52Withdraw blood slowly by your right hand


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Administration volumes considered good practice (and possible maximal dose volumes)

Oral s.c. i.p. i.m. i.v. (bolus) i.v. (slow)

Mouse 10 (50) 10 (40) 20 (80) 0.5b (0.1)b 5 (25)Rat 10 (40) 5 (10) 10 (20) 0.1b (0.2)b 5 (20)

Rabbit 10 (15) 1 (2) 5 (20) 0.25 (0.5) 2 (10)

Species route and volumes (mL kg-1) Journal of Applied Toxicology

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Species Injection SiteSC IM IP IV

Feline Scruff, back,21 – 23G

Quadriceps, 23G 21 – 23G Cephalic vein,21 – 25G

Canine Scruff, back,21 – 23G

Quadriceps, 21 - 23G

21 – 23G Cephalic vein,21 – 25G

Ferret Scruff, 21 – 23G


21 – 23G Cephalic vein,21 – 25G

Guinea pig Scruff, back,23 – 25

Quadriceps, 25G 23 – 25G Ear vein,Saphenous vein, 25 – 27G

Hamster Scruff, 25G

Quadriceps, 25G 23 – 25G Femoral or jugular vein,25 – 27G

Mouse Scruff, back,25G

Quadriceps, 27G 25 – 27G Lateral tail vein,26 – 28G

Primate(small)

Scruff, back,23 – 25G


21 – 25G Lateral tail vein,21 – 25G

Primate(large)

Scruff, 21 – 25G

Quadriceps, triceps,23 - 25G

21 – 23G Cephalic vein, recurrent tarsal vein, jugular vein21 – 25G

Rabbit Scruff, flank,21 – 25G

Quadriceps, lumbar muscles, 25G

21 – 23G Marginal ear vein,23 – 25G

Rat Scruff, 25G

Quadriceps, 25G 23 – 25G Lateral tail vein,21 – 23G

Bird Pectoral, interscapular or inguinal fold 1 – 3% BW bid or tid,<21 G

Pectoral/per site 0.2 ml/<100g BW 0.2-0.5ml/100-500g BW 0.5-1.0ml/>500g BW <25G

Not applicable Cutaneous ulnar vein, <25G, short bevel

Needle Sizes and Recommended Injection Sites; Adapted from: Formulary for Laboratory Animals, 3rd Ed., Hawk, Leary, Morris, 2005

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Identification methods

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Euthanasia

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Thanks for your attention

Documents

1. By Kalani M PhD candidate in Immunology What kind of animals? – Rattus Norvegicus – Rabbit (Oryctolagus cuniculus) – Hamster (syrian) – Guinea pig