Animal Experiment Manual

Handling and Restraint

This section explains the purpose of restraining laboratory animals and the classification methods using hands and tools.

1. Purpose of Restraint

  1. 1

    Intraperitoneal injections

  2. 2

    Oral gavage

  3. 3

    Bleed

2. Classification of Restraint

  1. 1
    Using hands
    • Restrain the part of the tail closest to the body
    • Restrain the skin fold at the back of the neck using the thumb and index finger
    • Restrain the tail together with another area of skin
  2. 2
    Using tools
    • Method using a tube
    • Method using a restraint device

Oral Gavage

This section explains the recommended dose and method for intragastric administration of liquid diets, test substances, and drugs.

1. Purpose of Oral Gavage

  1. 1

    Feeding a liquid diet

  2. 2

    Administration of test substances

  3. 3

    Administration of drugs

2. Recommended Dose

  1. 1

    0.1 ml / 10 g

  2. 2

    Adjustment is required depending on the properties of the solution

3. Precautions

  1. 1
    Do not apply pressure
    • May cause perforation of the esophagus or trachea
    • Let the substance pass naturally by the weight of the syringe and gravity
  2. 2
    If resistance is felt, stop and try again
  3. 3
    Since the animal's movement may interfere with administration, accurate restraint is required

4. Method

Perform accurate restraint to enable oral gavage of the mouse

Measure the length between the mouse's nose and stomach using the gavage needle (sonde)

Insert the gavage needle toward the back of the oral cavity and keep the head positioned so that the body and the syringe are parallel

At the appropriate position, let it insert naturally by the weight of the syringe and gravity

Intraperitoneal Injection

This section explains the location and procedure for intraperitoneal administration of anesthetics and drugs.

1. Purpose of Intraperitoneal Injection

  1. 1

    Administration of anesthetics

  2. 2

    Administration of drugs

2. Recommended Dose

0.2 ml / 10 g

3. Precautions

  1. 1

    Take care not to damage the internal organs during administration

  2. 2

    Since the animal's movement may cause organ damage, accurate restraint is required

4. Method

When the abdomen of the restrained mouse is divided into four quadrants, select an appropriate position in the lower left quadrant

Insert into the peritoneal cavity at an angle of about 45°, pull the plunger back to check for air bubbles at the tip of the syringe, then proceed with administration

Subcutaneous Injection

This section explains the location for administering drugs under the skin and the method of forming a skin fold.

1. Purpose of Subcutaneous Injection

  1. 1

    Administration of anesthetics

  2. 2

    Administration of drugs

2. Recommended Dose

  1. 1

    Single site: do not exceed 1 ml (for a 25 g mouse)

  2. 2

    Multiple sites: 0.1 ml / 10 g (maximum 0.4 ml / 10 g)

3. Precautions

  1. 1

    Take care that the injection needle does not penetrate through

  2. 2

    Since the animal's movement may cause muscle damage, accurate restraint is required

4. Method

Pull the mouse's skin to form a fold

Pass the needle through the lower part between the skin folds and inject

If the injection site is on the back, use a surgical drape or towel to expose only the injection site

Pull the skin on the back to form a fold

Pass the needle through the lower part of the skin fold, then inject

Tail Vein Injection

This section explains the procedure for administering anesthetics, drugs, and cells via the tail vein.

1. Purpose of Tail Vein Injection

  1. 1

    Administration of anesthetics and drugs

  2. 2

    Administration of cells and proteins

2. Recommended Dose

Within 0.2 ml

3. Precautions

  1. 1
    Warm the tail before administration
    • Induces vasodilation
    • Isoflurane anesthesia also induces vasodilation
  2. 2
    Begin administration from the tip of the tail
    • Start from the point one-third from the distal end
    • If attempted from the body side and unsuccessful, blockage occurs

4. Method

Align the administration site straight with the lateral tail vein, then insert the needle

Confirm that blood appears at the tip of the syringe, then proceed with administration

Tail Vein Blood Collection

This section explains the procedure and precautions for collecting a small amount of blood for blood tests.

1. Purpose of Tail Vein Blood Collection

Collection of a small amount of blood for blood tests

2. Recommended Volume

Within 0.1 ml

3. Precautions

  1. 1
    Warm the tail before collection
    • Induces vasodilation
    • Isoflurane anesthesia also induces vasodilation
  2. 2
    Begin collection from the tip of the tail
    • Start from the point one-third from the distal end
    • If attempted from the body side and unsuccessful, blockage occurs

4. Method

Align the collection site straight with the lateral tail vein, then insert the needle

Confirm that blood appears at the tip of the syringe, then proceed with administration and collection

Anesthesia

This section explains the selection of injectable and inhalation anesthetics and the operation of the chamber.

1. Considerations for Anesthesia

  1. 1

    Minimize the pain and fear caused to the animal by the experiment

  2. 2

    A means to safely and easily handle, observe, measure, and operate on animals

2. Selection of Anesthetic

  1. 1
    Injectable anesthetics
    • The effect must be distinguished according to concentration
    • The intraperitoneal route is widely used
    • Intramuscular injection can reduce the dose but has lower reliability
  2. 2
    Inhalation anesthetics
    • No problem of dose-dependent side effects
    • Characterized by rapid induction and recovery

3. Method of Inhalation Anesthesia

  1. 1
    Injection anesthesia
    • Administer the injectable agent according to the method
    • SC, IP, IV
  2. 2
    Inhalation anesthesia
    • Check the equipment and connect the oxygen line
    • Place the animal in the chamber and adjust the oxygen concentration
    • Adjust the vaporizer and open the valve - when adjusting the vaporizer, always press the hook and increase the anesthetic gas concentration; the hook does not need to be pressed when locking the anesthetic gas; the concentration at introduction of the anesthetic gas is 5%
    • After anesthesia is sufficiently achieved within the chamber, lock the valve and adjust the oxygen and vaporizer concentrations - adjust the vaporizer concentration to 2–3%
    • Open the mask valve and position the mouse in the nose cone
    • After use, lock the vaporizer, lock the oxygen, and lock the valve
    • Clean the used chamber and organize the equipment

Euthanasia

This section explains the considerations and methods for a painless endpoint.

1. Considerations for Euthanasia

  1. 1

    Induce unconsciousness and death without accompanying pain, stress, or fear

  2. 2

    The emotional effect on the operator or observer

  3. 3

    As a principle, the time from the procedure until loss of consciousness should be as short as possible

  4. 4

    Perform euthanasia at the humane endpoint or to terminate the experiment

2. Method of Euthanasia

  1. 1

    Overdose of anesthetic; high-concentration pentobarbital anesthetic

  2. 2

    Use of CO₂

References

Patricia V T, et al. (2011)

Administration of Substances to Laboratory Animals: Routes of Administration and Factors to Consider. JAALAS 50: 600-613.

Hans Hedrich (2004)

The Laboratory Mouse. Academic Press. Chapter 31-34.

James G. F, et al. (2009)

Laboratory Animal Medicine, 2nd edition. OKVET. Elsevier 860-900.

Pain Category Classification

USDA Pain and Distress Categories - 4-level classification table.

USDA Pain and Distress Category Classification (B / C / D / E)
Category Examples
USDA Category B

No pain or distress
  • Animals (breeders, offspring that cannot be used because of improper genotype1 and/or gender, or other animals) being maintained without any research manipulation, prior to euthanasia or transfer to another protocol
  • Observation of animal behavior in the wild without manipulating the animal or its environment
USDA Category C

Slight or momentary pain or distress, or no pain or distress
  • Holding, weighing or transporting animals (relatively short distances under non-stressful conditions)
  • Injections (nonirritating), blood collection or catheterization of superficial vessels
  • Collection of body fluids or tissues post mortem
  • Tattooing animals
  • Ear punching of rodents
  • Routine physical examinations
  • Observation of animal behavior
  • Studies, which do not result in clinical signs of pain and/or distress
  • AVMA approved humane euthanasia procedures
  • Routine agricultural husbandry procedures
  • Observational studies and live trapping (traps must provide adequate shelter/food and be checked frequently to ensure survival)
USDA Category D

Pain or distress appropriately relieved by analgesia, tranquilization or anesthesia
  • Potentially stressful transportation of animals that requires tranquillization
  • Survival/terminal surgical procedures

The following clinical signs when relieved with analgesics (but are relieved with analgesics):

  • decreased appetite/activity level
  • adverse reactions to touch
  • open skin lesions
  • abscesses
  • lameness
  • conjunctivitis
  • corneal edema
  • photophobia
  1. Food or water deprivation beyond that necessary for normal presurgical preparation
  2. Noxious electrical shock that is not immediately escapable
  3. Paralysis or immobility in a conscious animal
USDA Category E

Unrelieved pain or distress

 -

1 If tail snips are collected to genotype the animals, Category B is not appropriate.

Major Drug Dosages (Antibiotics · Analgesics · Anesthetics)

Recommended doses and trade names based on mice.

Major Drug Dosages - 3 Anesthetics / 4 Analgesics / 2 Antibiotics (based on mice)
Treatment Anesthetics Analgesics Antibiotics
Drug Ketamine + Xylazine (Tiletamine + Zolazepam) + Xylazine Isoflurane Acetylsaliclic acid Acetaminophen Ketoprofen Meloxicam Gentamicin Cephalexin
Dosage 80–100 mg/Kg + 10 mg/kg 30 mg/kg + 10 mg/kg 2–5% induction, 0.25–4% maintenance 100–150 mg/kg 200 mg/kg 5 mg/kg 1–2 mg/kg 4–20 mg/kg 30 mg/kg
Route IP IP - PO PO SC, IM PO, SC SC, IM PO
Duration 20–30 min 40 min - 4 hours - 24 hours 24 hours 12 hours 12 hours
Trade name Ketara (50 mg/ml) + Rompun (23.32 mg/ml) Zoletil 50 (50 mg/ml) + Rompun (23.32 mg/ml) - Aspirin Tyrenol Ketoprofen (100 mg/2 ml) Metacam (5 mg/ml) Gentamicin sulfate (20 mg/2 ml) Methyl ocephalexin lysinate (500 mg/cap)
Dosage
(mouse, ml/10 g)		 0.02 ml/10 g + 0.004 ml/10 g, IP 0.006 ml/10 g + 0.004 ml/10 g, IP - - - 0.001 ml/10 g 0.004 ml/10 g 0.02 ml/10 g -
Comment - - - Nonsteroidal anti-inflammatory Nonsteroidal anti-inflammatory Nonsteroidal anti-inflammatory Nonsteroidal anti-inflammatory - -

* Source: Exotic Animal Formulary 3rd edition (James W. Carpenter), Laboratory Animal Medicine 2nd edition (James G Fox, Lynn C. Anderson).

Humane Endpoint Criteria (Mouse)

Slight / moderate / serious criteria by tumor, body weight, appearance, and clinical sign scale.

Humane Endpoint Criteria (Mouse) - Slight / Moderate / Serious
Mouse Scale Slight consideration Moderate consideration Serious consideration
Tumor Consider euthanasia when the tumor weight is about 10% of body weight, when the size is 1 cm³ or larger, or when the tumor shows ulceration or necrosis
Body weight Body weight Weight loss of 10% or more over 7 days Weight loss of 10–25% over 7 days Weight loss of 25% or more over 7 days
Food/water intake 40–75% intake over 3 days 40% or less intake over 3 days Anorexia over 3 days; only 40% of dietary intake over 7 days
Appearance Piloerection Partial Severe piloerection Severe piloerection + hunched posture
Posture Temporary hunching Intermittent hunching Continuous hunching
Clinical signs Respiration Normal Intermittent abnormal respiration Labored respiration
Tremors Temporary Intermittent Continuous
Convulsions None Intermittent (10 minutes or less) Continuous (euthanize if 10 minutes or more)
Exhaustion None Temporary (1 hour or less) Continuous (1 hour or more; euthanize if 2 hours or more)

Methods of Euthanasia

The following methods may be used according to the study protocol.

  1. 1
    Use of inhalation anesthetics or CO₂

    Euthanasia is performed by exposure to a higher concentration than that used to maintain anesthesia. Because unwanted revival may occur, it must be combined with cervical dislocation or diaphragmatic incision. If sufficient air is not supplied during the induction period, hypoxemia may be induced, causing the animal pain before it loses consciousness. Inhalation anesthetics or CO₂ are used for animals in which intravenous administration is difficult, such as rodents and rabbits weighing less than 1 kg.

  2. 2
    Exsanguination under anesthesia

    Under deep anesthesia (use 3–4 times the maintenance dose in the case of injectable anesthetics), exsanguination is performed to ensure the death of the unconscious animal. Because inducing hypovolemia is accompanied by pain, exsanguination must not be used as the sole method of euthanasia.

  3. 3
    Use of injectable agents

    This method can be used for animals in which intravenous access is easy (rabbits, dogs, cats, pigs, etc.). After inducing deep anesthesia through inhalation or injection anesthesia, KCl is slowly injected intravenously to bring about euthanasia. However, barbiturate-class drugs with a narrow margin of safety can achieve euthanasia by overdose alone, without the injection of KCl.

  4. 4
    Cervical dislocation and decapitation

    This is performed in special cases such as when tissues must not be chemically contaminated. In particular, decapitation is applied when undamaged brain tissue must be obtained for research purposes. When performed by a well-trained person, euthanasia can be achieved, but there is no scientific basis for standardizing such methods. Performing it under anesthesia is humane; otherwise, approval must be obtained from the IACUC regarding the reason these methods must be used.

※ In all cases, it must be confirmed with certainty that the heart has stopped.