• PROCEDURES

TAIL VEIN BLOOD SAMPLE COLLECTION

Requirements include animal, rodent handling gloves, towel, cotton, sample collection
tube and animal warming chamber. This method is recommended for collecting a large
volume of blood sample (up to 2ml/withdrawal).
•

o STEP 1: The animal is made comfortable in a restrainer while

maintaining the temperature around at 24 to 27°C.
o STEP 2: The tail should not be rubbed from the base to the tip as it will
result in leukocytosis. If the vein is not visible, the tail is dipped into
warm water (40°C).
o STEP 3: Local aesthetic cream like lidocaine must be applied on the
surface of the tail 30 min before the experiment.
o STEP 4: A 23G needle is inserted into the blood vessel and blood is
collected using a capillary tube or a syringe with a needle. In case of
difficulties, 0.5 to 1 cm of surface of the skin is cut open and the vein is
pricked with bleeding lancet or needle and blood is collected with a
capillary tube or a syringe with a needle.
o STEP 5: Having completed blood collection, pressure/silver nitrate
ointment/solution is applied to stop the bleeding.
o STEP 6: If multiple samples are needed, temporary surgical cannula may
be used.
o STEP 7: Restrainer is washed frequently to
avoid/prevent pheromone (chemicals secreted that may cause
behavioral changes in animals of the same specie) induced stress or
cross infection

BLOOD COLLECTION-3
Taking a blood sample is one of the most common procedures performed on laboratory
animals:

• for analysis of biochemical, metabolic, toxicological or immunological

parameters,
• for examination or culture of micro-organisms, and
 • for production of antibodies.

Using a technique appropriate for the purpose and the species, performed by a trained
and competent member of staff, is essential to ensure that any pain, distress or discomfort
is kept to a minimum. Avoiding adverse effects is important for scientific as well as ethical
and legal reasons, as they can cause biological changes which may affect the blood sample
and hence the validity and reproducibility of the research results.

The blood collection techniques for mice and rats are classified into three:

• Surgical techniques
• Non-surgical techniques
• Terminal techniques

The method of blood collection to be used largely depends on two factors: the frequency
of blood collection and the volume of blood needed to be extracted.

Here are the common blood collection techniques based on their classification.

• Surgical techniques:
o Blood vessel cannulation
▪ considered when repeated samples are required
▪ can be used to take blood from the carotid artery, vena
cava and femoral vein
▪ surgery is required and appropriate anaesthesia and
analgesia should be used to minimise any pain caused
o Tail snip method
▪ considered when repeated samples are required
▪ can be used to take blood from the carotid artery, vena
cava and femoral vein
▪ surgery is required and appropriate anaesthesia and
analgesia should be used to minimise any pain caused

• Non-surgical techniques:
o Tail vein
 ▪ a crude method of sampling and should be avoided as it
involves the removal of soft tissue from the tip of the tail
using a scalpel
▪ resulting in permanent damage to the tail and pain to the
mouse
▪ limitation includes contamination, with the sample
containing tissue fluid as well as blood

•
o Saphenous vein
▪ relatively quick method of obtaining blood samples from
all strains of mice
▪ does not require the animal to be warmed (exposed to a
warmer lamp to increase blood circulation) for sample
collection
▪ blood is collected from the lateral saphenous vein which
runs dorsally and then laterally over the tarsal joint

• Terminal techniques:
o Retro-orbital sinus
▪ should only be used under general anaesthesia
▪ conducted only by staff members competent (practiced) in
the technique
▪ a capillary tube/pipette is inserted medially, laterally or
dorsally from the venous sinus in the eye
o Cardiac puncture
▪ suitable technique to obtain a single, large, good quality
sample from a euthanised mouse or a mouse under deep
terminal anaesthesia
▪ taken preferably from the ventricle, which can be
accessed either via the left side of the chest, through the
diaphragm, from the top of the sternum or by performing
a thoracotomy
o Axillary bleed
▪ make a deep incision in the axilla (armpit) at the side of the
thorax and hold the skin at the posterior part of the
incision using forceps to create a cupped area
▪ incise the blood vessels in the area with a scalpel or
straight edge razor and collect blood as it pools
▪ it may be important to consider that tissue fluids will
contaminate the blood sample
▪ 1-2 mL of blood can be collected
 GENERAL GUIDELINES FOR BLOOD COLLECTION:
These general rules are based on the recommended UK GUIDELINESLinks to an external
site. in blood collection on a healthy adult laboratory animal. The health status of
laboratory animal used should be maintained to guarantee the validity of experimental
results. Animals that are too young, stressed, have already undergone manipulations
(procedures) or suffering from cardiac or any other disease should not be used in a
research study.
Circulating blood volume is around 5.5-8% of an animals weight, so if a rabbit used
is 4kg having a mean blood volume of 56mL/kg the total circulating blood would be 224
mL. Blood collection (non-terminal) should be 10% of the total circulating blood which
means that 22.4 mL of blood can be collected from the rabbit once every two
weeks without fluid replacement.
The blood collection for mice and rats would also be 10% of the total volume, this
can be safely removed every 2 to 4 weeks, 7.5% every 7 days, and 1% every 24 hours.
Volumes greater than recommended should be scientifically justified and appropriate
fluid and/or cellular replacement provided. Approximate blood sample volumes for a
range of body weights are included in the table below:

BODY 7.5% CBV 10% CBV

1% CBV
WEIGHT CBV in mL every 7 every 2-4
every 24hrs
IN g days weeks
0.082-
20 1.10-1.40 0.011-0.014 0.11-0.14
0.105

30 1.65-2.10 0.017-0.021 0.12-0.16 0.17-0.21

40 2.20-2.80 0.022-0.28 0.16-0.21 0.22-0.28

200 11.00-14.00 0.11-0.14 0.82-1.05 1.10-1.40

300 16.50-21.00 0.17-0.21 1.20-1.60 1.70-2.10

CBV= CIRCULATING BLOOD VOLUME

COMMON SITES FOR BLOOD COLLECTION

SPECIE RECOMMENDED SITES & CONDITION:
 Superficial temporal vein (a.k.a., "submandibular" or "facial"),
MOUSE saphenous vein, tail vein, retro-orbital (anesthetized), cardiac
(anesthetized, terminal)
Tail vein, saphenous vein, superficial temporal vein (a.k.a.,
RAT "submandibular" or "facial"), cardiac (anesthetized, terminal),
sublingual, jugular
RABBIT Marginal ear vein, cardiac (anesthetized, terminal)

EUTHANASIA-3
Euthanasia is the act of inducing humane death in an animal by a method that
induces rapid loss of consciousness and death with a minimum of pain, discomfort or
distress.

Types of Euthanasia
“Acceptable methods”
As defined by the American Veterinary Medical Association or AVMA, it renders an
animal unconscious and insensitive to pain and distress as quickly as possible, followed by
cessation of all respiratory and circulatory functions and brain activity.

• Barbiturates
• Carbon dioxide – bottled gas only (at least 40 up to 99% concentration)
• Inhalant anesthetics
• 70% Ethanol
• Ether – inexpensive but flammable
• Microwave irradiation
• Tricaine methane sulfate (TMS, MS22)
• Captive penetrating bolt

“Acceptable methods with conditions”

These methods are considered “Acceptable” only if specific conditions are met, as
indicated in the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition.

• Cervical dislocation
 • Decapitation
• Pithing
• Exsanguination
• Air the IV route

Unacceptable methods:

• Chloral hydrate, chloroform, cyanide

• Decompression
• Neuromuscular blockers
• Dry-ice generated CO2

Moribund and Morbid Conditions

Moribund - in a dying state; dying; at the point of death; defined as a clinically irreversible
condition leading inevitably to death.
Clinical signs of a moribund condition:

• impaired ambulation
• evidence of muscle atrophy or other signs of emaciation
• any obvious severe illness including such signs as lethargy, anorexia, bleeding,
difficulty breathing, CNS disturbance, or chronic diarrhea
• inability to remain upright
• loss of consciousness
• drop in body temperature below 28°C for a prolonged period

Morbid - pertaining to, affected with, or inducing disease; diseased; animals begin to
exhibit clinical signs of disease

The Different Euthanasia Techniques

Barbiturates
• most commonly used and most acceptable method
• sodium pentobarbital, Euthasol, Eutha 6, Fatal Plus
 • moves rapidly to the heart and then into the brain, where it quickly and
painlessly depresses all vital life functions
• may be administered IV, IP or IC

Inhalation of CO2
• the most common method of euthanasia used at National Institutes of Health
or NIH for mice, rats, guinea pigs and hamsters
• although CO2 is generally considered an acceptable euthanasia agent, its
acceptability is predicated on a number of critical factors as described in the
AVMA Guidelines for the Euthanasia of Animals
• death by CO2 should be induced as painlessly and quickly as possible
• compressed CO2 gas in cylinders is the only recommended source as it allows
the inflow of gas to the induction chamber to be controlled
• dry ice as a source of CO2 or pre-filled chambers are not acceptable

Inhalant Anaesthetics
• work on the principle of displacing oxygen with an alternate gas
• according to the American Veterinary Medical Association or AVMA,
halothane is considered the most effective and humane inhalant anaesthetic
• other inhalant anaesthetics like isoflurane are less desirable because their
pungent odor may cause the animal to initially hold his or her breath
• chloroform and ether are not acceptable inhalants for use in euthanasia in part
because of the risk of bodily harm they pose to humans
• liquid inhalant anaesthetics must never be allowed to directly contact animals
because they can cause irritation; rather, the liquid should be poured onto
cotton balls, gauze, or other absorbent materials and placed with the animal in
an airtight container

Ethanol
• intraperitoneal (IP) injection of 70-100% ethanol
• this method has been shown to result in unconsciousness and ultimately
euthanasia in less than 7 minutes in adult mice, however, is suspected to be
unreliable in rats and in mice less than 28 days of age (Allen-Worthington)
Ether
• ether is highly flammable and explosive liquid
• it only when no reasonable alternatives exist
• must be used only in facilities designed and approved for its use (with fume
hoods) and in a manner which will avoid the known hazards. Acute
toxicity by inhalation in high concentrations may include inebriation, sedation,
unconsciousness and respiratory paralysis. Diethyl ether is irritating to the
eyes, respiratory system and skin but these effects are usually reversible on
removal of exposure.

Microwave Irradiation
• used primarily by neurobiologists fix brain metabolites in vivo while
maintaining the anatomic integrity of the brain
• designed for use in laboratory mice and rats
• death occurs in < 1 sec., although the instrument is expensive and only useful
for laboratory rats and mice

TMS (tricaine methane sulfonate)

• MS 222, tricaine methane sulfonate
• used on amphibians and fish
• a concentration 250 mg/L is recommended and fish should be left in this
solution for at least 10 minutes following cessation of opercular movement

Captive Penetrating Bolt

• euthanasia of ruminants, horses, swine, laboratory rabbits, and dogs
• concussion and trauma to the cerebral hemisphere and brainstem
• captive bolt guns are powered by gunpowder or compressed air and must
provide sufficient energy to penetrate the skull of the species on which they are
being used
 • the penetrating captive bolt is an effective method of euthanasia for use in
slaughterhouses, in research facilities, and on the farm when use of drugs is
inappropriate

Cervical Dislocation
• a technique that has been used for many years and, when performed by well-
trained individuals, appears to be humane
• used to euthanise poultry, other small birds, mice, and immature rats and
rabbits
• the thumb and index finger are placed on either side of the neck at the base of
the skull or, alternatively, a rod is pressed at the base of the skull
• with the other hand, the base of the tail or the hind limbs are quickly pulled,
causing separation of the cervical vertebrae from the skull
• the animal is stretched and the neck is hyperextended and dorsally twisted to
separate the first cervical vertebra from the skull

Decapitation
• used to euthanise rodents and small rabbits in research settings
• provides a means to recover tissues and body fluids that are chemically
uncontaminated
• also provides a means of obtaining anatomically undamaged brain tissue for
study
• guillotines that are designed to accomplish decapitation in adult rodents and
small rabbits in a uniformly instantaneous manner are commercially available

Exsanguination
• can be used to ensure death subsequent to stunning, or in otherwise
unconscious animals
• because anxiety is associated with extreme hypovolemia, exsanguination must
not be used as a sole means of euthanasia
• animals may be exsanguinated to obtain blood products, but only when they
are sedated, stunned, or anesthetized
Pithing
• pithing is used as an adjunctive procedure to ensure death in an animal that has
been rendered unconscious by other means
• for some species, such as frogs, with anatomic features that facilitate easy
access to the central nervous system, pithing may be used as a sole means of
euthanasia, but an anesthetic overdose is a more suitable method

Air Embolism
• the injection of a large amount of air into the circulatory system

Stunning
• animals may be stunned by a blow to the head, by use of a non-penetrating
captive bolt, or by use of electric current

Electrocution
induces death by cardiac fibrillation, which causes cerebral hypoxia; however, animals do
not lose consciousness for 10 to 30 seconds or more after onset of cardiac fibrillation