Mice: Basic Handling and Technique Workshop

University of North Carolina, Chapel Hill

First do no harm Greek Hippocratic Oath,
Great Watch Words of Medicine
Objectives:
1. Teach methods of safe, humane handling and restraint
2. Teach injection techniques: subcutaneous, intraperitoneal, intravenous and
intramuscular
3. Teach blood collection techniques
4. Teach rodent identification methods
5. Teach anesthesia administration and monitoring
6. Teach proper euthanasia methods

Basic Information about working with Mice:

Proper Personal Protection Equipment (PPE) is a requirement for working with animals.
In DLAM facilities, minimum requirements include disposable coveralls, shoe covers, head
bonnet, mask, and gloves. Please review requirements before entering any animal area!
Requirements may change from room to room so each door is posted with instructions.
The use of a face mask reduces your risk of allergy to animals. We strongly recommend that
you wear masks whenever you work with animals.

Training Information:
For additional training please contact the Training and Compliance Coordinators for the
Institutional Animal Care and Use Committee (IACUC) at 966-5569. We offer training in
both one on one and classroom settings.
For a look at IACUC Guidelines and dates of future classes, visit our website at
http://research.unc.edu/Offices/iacuc/index.htm as well as
http://research.unc.edu/Offices/NLAC/index.htm
If Bitten: DO NOT PUNISH THE MOUSE FOR ITS NATURAL RESPONSE
1.
2.
3.
4.

Calmly return the animal to its cage

Wash the wound with antibacterial soap and water
Bandage the wound
Notify your supervisor and contact the University Employee Occupational Health Clinic
(UEOHC) 966-9119

Mouse Psychology:
1. Mice are usually mild in temperament and easy to handle. They are not usually
aggressive, but can bite if frightened. There are some strains that are aggressive and
can inflict painful bites. Mice groom themselves almost constantly to maintain a smooth,
glossy haircoat.
2. Mice are nocturnal animals. Activities such as eating, drinking or mating are typically
done at night.

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3. Dominant mice exhibit a behavior called barbering. Barbering is the dominant mice
biting or chewing on the fur of a more subordinate mouse. Barbering should not be
confused with fur loss due to illness. Typically, barbering occurs around the face or back.
4. Male mice can be more aggressive and fight more often than females. Aggressive mice
should be housed individually to avoid severe injury to cage mates. Generally male
littermates may be housed together, but once separated, it is advisable to only house
males with females.
5. Mice are creatures of habit. Everyday events do not tend to stress or excite the mice.
However, handling and restraint can be stressful and result in the mouse being difficult
to work with. Conditioning the mice to such handling (so they do not associate handling
and restraint aversively) can make the animals much easier to work with.

Handling and Restraint:

When picking up adult mice, grasp them gently but firmly at the base or center of their tail. Do
not pick them up by the tip of the tail. Place the animal on a surface such as the wire cage top
or lid. It is best that the surface not be slick or smooth as mice will behave much more calmly if
they have firm footing. While still holding the tail near the base, with your other hand firmly
grasp the loose skin on the back starting near the ears using your thumb and first two fingers.
The tail can then be held by the last two fingers as shown. Your grip should be firm enough to
keep the mouse from struggling, but gentle enough for it to breathe comfortably.

For quick handling such as cage to cage transfers, it is acceptable to use forceps. Gently grasp
the loose skin on the back and quickly transfer them to the new cage. This technique is useful
for fractious or aggressive animals. Be sure to clean gloves or forceps with a disinfectant such
as Vircon between cages.
Gloves, either light leather, cloth or mesh greatly diminish sensitivity, increasing chance of injury
to mice. They also make it more difficult to perform delicate procedures, and mice can often bite
through them.

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Examples of Commercial Restrainers:

Snuggle: Lomir.com
Various styles and sizes available: See Page 21 for vendors

Sex Determination
Gender in mice is determined by comparing anogenital distance, or the distance between the
urogenital opening and the anus. Male mice typically have a larger anogenital distance when
compared with the females. Be aware there are variances in ano-genital distance among
strains. See diagrams below.

(a) Young Rodent

(b) Adult Rodent

Injections:

Use a fresh, sterile needle for each injection

To avoid excessive leaking, keep the needle in the needle tract for a few seconds
Always inject with the bevel of the needle facing up
Do not reuse needles between animals

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Injection Type

Ideal Needle
Size (Gauge)

Recommended Volume

Aspiration Required

Intraperitoneal (IP)

25-27G

0.1 mL/g body weight (ex.

2mLs for 20g mouse)

No

Subcutaneous (SQ,
SC)

25G

2-3 mL; scruff

Yes; inject if no blood in the

needle hub

Intramuscular (IM)

27G

50 100 L per site;

quadriceps

Yes; inject slowly if no blood

in the needle hub

Intravenous (IV)

26-28G

200 L; lateral veins

No; inject slowly

1. Intraperitoneal injections: Holding the mouse in dorsal recumbency, insert the needle in
a position below the bend of the knees; left or right of the midline. Avoid the midline to
prevent penetrating the bladder. Angle the needle approximately 45 to the body.

2. Subcutaneous injections: May be performed in any area of loose skin along the back or
flank. Tenting the skin between the shoulder blades or over the rump creates an
appropriate pocket for injection. Inserting the needle under the skin along the flank
results in the outline of the needle clearly visible when correctly situated.

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3. Intramuscular injections: Only to be used in instances where the other injections are not
appropriate, since this is potentially painful. The muscle mass running along the back of
the leg is used; with the needle angled parallel to the femur (avoiding the sciatic nerve).

4. Intravenous injections: Dilate the blood vessels by warming the mouse. Once warmed,
place the mouse in a restrainer. An additional method to dilate the veins is rubbing
alcohol on the tail. Locate one of the lateral veins. Insert the needle as low as possible
towards the tip of the tail, since the vein is very superficial at the tip. As you move up
toward the base of the tail the vein is located more deeply. The vein will clear from the
injection site to the base of the tail if properly situated, whereas ballooning around the
injection site will occur if the needle is not properly seated you will note ballooning at the
injection site.

Cross Section of Mouse Tail

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Various Helpful Tips for Injections:

1. When collecting blood from laboratory animals, the largest recommended
amount is 1.5% of the animals total body weight. Collection should not occur
again for two weeks. This will allow blood constituents to return to normal. If
blood is needed weekly, 0.5% body weight is a safe amount.
2. When giving substances intravenously, inject slowly to avoid shock.
3. Injecting slowly when giving substances intramuscularly will cause the least
amount of pain.
4. The volume of blood in an adult mouse is about 78-80 ml/kg. This is
approximately 10% of its body weight. Only about half of this can be recovered
in a terminal blood withdrawal procedure.
5. Check rodents teeth frequently. This will insure early detection of malocclusion.
If ma-occluded, teeth may become overgrown and interfere with eating.

Oral Gavage:
We recommend using stainless steel, ball tipped gavage needles. It is important to premeasure the needle before gavaging. The tube should measure the distance from the tip of the
nose to the last rib, so that the needle will pass down the esophagus into the stomach. If the
tube is too short, the injected fluid may be aspirated by the mouse causing possible pneumonia
and death. If the needle is too long, it may perforate the stomach.
The mouse should be firmly restrained. Insert the gavage needle into the mouth at one side.
Slide the needle down the back of the throat while tilting the mouses head back, so that the
neck is in a straight line. The needle should pass easily down the esophagus; with little to no
resistance. If the mouse struggles or resistance is met, stop, back up and start over. Improper
gavage technique can cause tearing of the esophagus or asphyxiation.
Observe the mouse carefully after the gavage is completed. No fluid should be coming from the
mouth or nose and the mouse should not show signs of distress. Oral dosing should not exceed
more than 10ml/kg.

The following table indicates appropriate needle sizes:

Mouse Wt (g)

Gauge

Length (inches)

Up to 14g
15-20g
20-25g
25-30g
30-35g

24
22
20
18
18

1
1 or1.5
1, 1.5 or 3
1, 1.5 or 2
2 or 3

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Ball Diameter
(mm)
1.25
1.25
2.25
2.25
2.25

Acknowledgements:
The University of North Carolina would like to thank:
The AALAS Learning Library
University of Minnesota, Research Animal Resources
University of Texas Medical Branch at Galveston

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Rodent Identification
There are several IACUC approved methods used to identify rodents. Each method has both
advantages and disadvantages. In long-term studies, it is important to choose a method that is
permanent and easily read.

Long Term Methods:

Ear Notching This method is frequently used in both mice and rats. There are several
tools that may be purchased to achieve this. Most resemble a hole puncher and are very
cheap. There are previously created maps that serve as a numbering system, or the
researcher may create a map. (See diagram below for universal numbering system that may
be used for identification via ear notching)
A) Advantages Ear notching can be done quickly while causing very little pain or
distress. The instruments are not costly and can be obtained easily.
B) Disadvantages This method can not be applied until the ears are fully developed.
This may be too late for those that use young rodents. This may not work with fractious
strains. Rips or tears caused by fighting may leave the pattern indiscernible.
Tools used to notch ears dull easily so must be replaced frequently.
Ear Tagging Ear tags can be purchased with numbers and/or letters. Correct placement
of the tag makes them fairly easy to read. (See images below of two commonly available ear
taggers, with proper tag placement).
A) Advantages Ear tags are inexpensive and are fairly easy to apply. This method does
not require the use of anesthesia. Tagging can be done quickly and does not seem to
cause pain and only minor distress.
B) Disadvantages Tags can fall out if not applied properly. They can also be lost if ears
are ripped or torn in strains that fight. Different sized tags are available for different
species. Tags are relatively heavy for weanlings and may cause young mice to tilt their
head even when the proper sized tag is applied. Some strains are prone to scratching
the tagged area which can lead to infection, hematomas, and granulomas. In a very
limited number of cases, a member of the DLAM veterinary team has seen ear tags
stimulate tumor growth.

Microchipping Microchips, electronic transponders, are safe and reliable.

A) Advantages Microchips may be applied without the use of anesthesia. Applying
microchips seems to cause little or no pain. Even though the chip may migrate to a
different area, they are not lost so prove to be a reliable method. Animals can be
identified without handling and removing them from the cage. Some microchips are
designed to provide other information such as core body temperature and heart rate.
B) Disadvantages The equipment used to read the chips is fairly expensive. Microchips
cost five to ten dollars each. Despite the manufacturers recommendation, chips can be
reused. In order to reuse the chips they must be sterilized by ethylene oxide. The
hospital will do this as a service for fee. If not implanted properly there is a slight risk of

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infection. In a very limited number of cases, a member of the DLAM veterinary team has
seen microchips stimulate tumor growth.
Micro tattooing This method seems to be growing in popularity. It is both
permanent and fairly easy to apply.

A) Advantages This method works in all strains, even the more fractious. The cost is
very reasonable after the initial expense. Tattoos can be applied to rodents of any age.
The markings are easily read, especially when applied to the tail of light colored rodents.
When placed in the proper area, it is not necessary to handle the animal to read the
tattoo. Tattooing causes only minor pain and distress and does not require the use of
anesthesia.
B) Disadvantages The identifying marks may be a little difficult to read in young
pigmented mice. This improves as the mice age. The initial cost is rather expensive.
There is a small chance of inducing infection if the tattoo is not applied correctly.

Toe Clipping Toe-clipping, as a method of identification of small rodents, should be

used only when no other individual identification method is feasible and should be
performed only on altricial neonates.1 The UNC Toe Clip Policy allows a maximum of four
toes and no more than two per foot. Do not cut the hallux (dew-claw or little toe) as this
may decrease the rodents grasping ability. This technique may only be performed on
rodents 10 days or less of age unless otherwise approved within protocol. The Toe Clip
technique must be described in the approved protocol before use.

A) Advantages Toe clipping can be done at a very early age, post natal day one. The
ideal time is between post natal day five and seven when the toe is large enough to work
with yet the bones are not calcified. The tissue can be used for genotyping. Toe clipping
may not be performed after post-natal day 10. No anesthesia is needed. This seems to
cause little or no pain when performed early enough. The young react to being removed
from their mother, but do not react to the clipping of the toe.
B) Disadvantages The young show signs of distress when removed from their mother
and siblings. This may cause a small amount of pain. The general public views this as a
form of brutal mutilation. There is a small possibility of infection. A reduction in the
number of toes may reduce the ability to grasp objects.

Short Term Methods:

Hair Clipping Trim patterns into the fur. Keep a record or picture to identify
rodents.
A) Advantages Causes no pain.
B) Disadvantages This is very temporary. The hair will grow back within ten days
and the clipping must be repeated.
Permanent Markers and Fur Dyes It is easy to apply marks or dyes to different
body parts.

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A) Advantages This method is non-invasive. It causes only minor stress due to

restraint.
B) Disadvantages This method can be time consuming, since it must be repeated
soon after the previous marking. If working with nursing pups, the mothers will
groom the neonates excessively and the markings may disappear overnight. This
could result in a loss of identity.

If you would like to inquire about equipment used in methods

discussed above, please email the OACU Training/Compliance Team.
You may send questions to the general IACUC email account
[email protected]
1

Guide for the Care and Use of Laboratory Animal Care and Use. National Research
Council.2011 National Academy of Sciences.

Ear Tagging:

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Ear Notching:

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ACCEPTABLE METHODS OF RODENT BLOOD WITHDRAWAL

Each laboratory must designate a Laboratory Animal Coordinator (LAC) who may train research
personnel in their laboratory in various animal-handling techniques, including blood collection.
The LAC must be certified by the Office of Animal Care and Use (OACU) or the Division of
Laboratory Animal Medicine (DLAM) and demonstrate proficiency before training others within
their lab. Alternatively, laboratory personnel may register for a hands-on techniques class here
or one on one session with the OACU Training and Compliance team (966-5569).
Chronic Blood Withdrawal: For sequential blood sampling (over a period of time), the
maximum survival blood withdrawal for most mammals is 1.5% of lean body weight
every 14 days.
Acute or Single Blood Withdrawal: The maximum survival amount of an acute blood
withdrawal is 1% of the lean body weight. [e.g.; For a 20 gram adult mouse, no more
than 4 X 50 ul micro capillary tubes (200 ul), may be withdrawn].
To facilitate blood collection, warm the rodent first. When using the tail veins or artery, you may
dip the tail in warm water (45C). The entire animal can be warmed with a carefully placed heat
lamp for 5-10 minutes or by placing the housing cage on a circulating water pad. Alternatively,
alcohol may be used initially as a vasodilator, but it should not be used on broken skin.
1) Submandibular:
A relatively simple way to obtain blood from a mouse is to puncture the area behind the hinges
of the jawbones. The superficial temporal vein is a large vessel positioned behind the eye,
which can be traced backward to the temporal vein, the maxillary vein, and finally, the jugular
vein.
Scruff the mouse and pierce the skin in the relevant area. A mouse bleeding lancet is strongly
recommended for use. However, an 18 gauge needle may also be used. Information on the
lancets and a video of this procedure may be seen by going to the following URL:
http://www.medipoint.com/html/animal_lancets.html
The submandibular bleed is one of the easiest methods of collecting blood from a mouse. Your
LAC may train in this technique or you can contact the OACU at 966-5569 to arrange for
training.
2) Saphenous Vein:
This method of obtaining blood is often used when a series of small samples is required. Place
the mouse in a conical tube and shave the caudal surface of the thigh. The saphenous vein can
be seen in this area. It is advantageous to apply a lubricant to prevent wicking. Place a
tourniquet above the knee and enter the vein with a 25 gauge needle. Micro-hematocrit and
microvette tubes work well to collect the blood. This method of blood withdrawal does not
require anesthesia, however, the method of restraint is cumbersome. For detailed instructions
and pictures of this procedure please visit http://www.uib.no/dyreavd/Vivarium-bloodsampling.pdf
3) Tail Artery / Vein (NICK):
Tail veins and artery can be used for serial bleedings. Use the central tail artery or lateral tail
veins. Anesthesia is not required for tail nick. Start midway up the tail and nick the artery or vein
(usually with a needle or lancet). You may collect blood with micro capillary tubes, a

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micropipette or various microtainer collection tubes. Move cranially 0.5 cm at a time applying
pressure after the bleed.

4) Tail Clip Bleed and/or Tail Biopsy for Genotyping:

Performed on un-anesthetized or anesthetized animals depending on amount of tissue
needed (see below):
Anesthesia is optional for the removal of up to 4mm from the tail tip. It is strongly
recommended that no more than 2mm be removed at a time. Anesthesia may be used
as a means of animal restraint and its use must be described in the approved animal
care application.
Harvesting greater than 4mm requires written permission from the IACUC. Depending
on the age of the animal, removal of greater than 4mm from the tail tip may involve
cutting into the vertebral column. Therefore, anesthesia is always required when
removing this much tail, irrespective of the age of the animal. The use of anesthesia
must be described in the approved animal care application. Requests to perform tail
biopsies or successive tail cuts totaling greater than 4mm without anesthesia must be
scientifically justified and must receive IACUC approval prior to implementation.
The IACUC has approved the tail cut method for both rats and mice to obtain blood and/or
tissue. This method must be described in the animal use application and approved by the
IACUC prior to use. See policy below.
1.
2.
3.
4.
5.
6.

7.
8.
9.

10.

Place animal in approved animal restrainer. (Experienced handlers may be able to

perform technique in habituated mice with little or no restraint).
Remove any bedding material or feces from the tail. The tail tip must be disinfected
with an approved disinfectant (i.e. Betadine)
Place the animal on a clean work surface.
Using a fresh scalpel blade or clean scissors, cut 1-2 mm of the distal tail at an angle
perpendicular to the work surface.
Apply gentle pressure proximal to the collection site to occlude venous return and
ease collection. Collect the blood in a suitable collection device.
Apply gentle digital pressure to the wound for 30-45 seconds with a clean gauze pad
to stop any hemorrhaging. For persistent bleeding, apply a silver nitrate stick, styptic
powder or a cautery pen to the wound to stop bleeding.
Return the animal to its cage only after bleeding has stopped.
Serial blood samples can be obtained over a short time frame by gently removing the
scab without performing an additional cut.
Only the fleshy portion of the tail tip should be cut. Cutting into the vertebrae is NOT
permitted. As only a small portion of the tail does not contain vertebrae, the use of
the tail cut procedure should be limited.
This procedure should be performed only by individuals trained and certified in the
technique and comfortable with rodent handling.

5) Retro Orbital Bleeding:

Retro-orbital or orbital sinus/plexus bleeding (permitted in rats, mice, gerbils, guinea pigs,
hamsters) must be proposed to and approved by the IACUC before implementation. The IACUC

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will permit orbital sinus bleeding when it is scientifically justified, performed with appropriate
technique and anesthesia. Veterinary staff experience indicates that this method may lead to
orbital damage, blindness and potentially death if not performed correctly. The IACUC
encourages the primary use of the submandibular, tail artery or veins; specifically the nick or cut
techniques. These methods are less likely to harm the animal and may be used repeatedly for
bleeding. LACs may not train in this technique so training and certification must be obtained
from OACU Training and Compliance team or DLAM veterinary services.
Alternating eyes for each bleeding is mandatory, and a week must separate each bleeding. A
maximum of two (2) bleedings per eye is permitted. Maximum volume withdrawn within a two
week period is 1.5% body weight. Orbital sinus bleeding requires training and must be
performed on anesthetized animals only with IACUC approval.
Following blood collection, the eyelids should be held closed for a few seconds to allow the
punctured blood vessel to clot. It is also common practice to place a small amount of
ophthalmic ointment into the eye following this procedure. excerpt from Laboratory Animal
Technician Training Manual
6) Cardiac Puncture: Always a terminal procedure conducted under anesthesia (or shortly
after death)!
Cardiac puncture as a method of blood withdrawal permitted in all species provided the
following conditions are met:
1. Animal is under a surgical plane of anesthesia when procedure is conducted.
2. Animal is NOT allowed to recover from anesthesia following the puncture.
3. If the animal is euthanized prior cardiac puncture, training and certification in the
technique is not required.
A needle is inserted into the heart and blood is extracted until a sufficient volume is collected or
the animal is exsanguinated. This procedure must be followed by a physical euthanasia method.
PDF format available on the IACUC website:
Click HERE

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INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE

MOUSE EUTHANASIA POLICY
Performing euthanasia correctly is an ethical imperative. Proper euthanasia is quick,
minimizes pain/distress and reliably causes death. Practical issues such as degree of
technical difficulty, time required to perform the procedure, readily available
equipment/resources to perform the procedure, as well as aesthetics and human
emotion must be considered. Standardized guidelines for humane euthanasia are
detailed in the June 2013 AVMA Guidelines on Euthanasia and available HERE.
In addition to Division of Laboratory Animal Medicine (DLAM) personnel, only trained
research personnel, listed in the IACUC approved Animal Care Application (ACAP),
may euthanize animals. All animals slated for euthanasia must be housed according to
UNC-Chapel Hill cage density standards, and should have access to food and water if
they are being housed for more than 3 hours prior to euthanasia. Unweaned animals
that are slated for euthanasia should stay with the lactating female until final
preparation(s) for euthanasia are complete. Cages marked for euthanasia should not
be overcrowded or stacked on top of each other, as this blocks air flow into the cage.
Euthanasia must follow the method(s) described in the approved ACAP. Euthanasia
must be confirmed by the physical method described in the approved ACAP. A
confirmation of death by a physical method is required for all animals, irrespective of
age.
The DLAM staff can, for a fee, perform euthanasia of research animals. When
requesting this DLAM service, research personnel must do the following:
1) Complete and submit a Request for Euthanasia in Animals form [available on
the DLAM website]. Ensure all euthanasia instructions are very clear (e.g. euthanize
dam and neonates or euthanize only the pre-weanling animals, not the dam);
2) Leave the animal(s) requiring euthanasia in the cage. All unweaned animals
should stay with the lactating female until the time of euthanasia.
3) Place a euthanasia card on the cage so that DLAM can readily identify the
animal(s) slated for euthanasia.
The investigator is responsible for ensuring proper documentation on a euthanasia
request form. DLAM is not responsible for errors on the form or miscommunications
that may occur during the euthanasia process. Do not make verbal arrangements with
DLAM staff.

Euthanasia of sick or injured animals

Sick or injured animals that cannot be successfully treated or relieved of pain and
distress should be euthanized promptly. Research personnel are responsible for
euthanizing sick, injured or moribund animals as soon as these conditions are noted.
These animals should not be held for later euthanasia by DLAM personnel. To
investigate unexpected illnesses, research personnel may contact Veterinary Services
to arrange for euthanasia and necropsy of the animals.
DLAM veterinarians have the authority to euthanize moribund animals, as well as
animals experiencing more than momentary or slight pain and/or distress. If the DLAM
veterinarian is unable to contact research personnel regarding the care or treatment of
a moribund animal, DLAM veterinarians or designated representatives are authorized

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to euthanize the animal. Ensure appropriate emergency contact numbers for all
research personnel are posted in the animal facility.

MOUSE EUTHANASIA
Section 1:Terms and Definitions
Secondary physical method to ensure death in order to confirm that animals are
dead, one of the following secondary physical methods must be performed on animals
that have been anesthetized with approved agents: 1) cervical dislocation; 2)
decapitation; 3) thoracotomy [open the chest cavity using sharp scissors or scalpel]; or
4) collection of vital organs.
Note: In addition to DLAM personnel, only research personnel who have been properly
trained and are listed on the approved ACAP, can perform these physical methods.
Un-anesthetized Physical Euthanasia Individuals who perform physical
euthanasia on un-anesthetized animals must first be trained and certified by IACUC
approved designees. Laboratory Animal Coordinators (LAC) may not certify personnel
in un-anesthetized physical euthanasia. Physical euthanasia on un-anesthetized
animals, irrespective of age, can only be done if the procedure is described in the
approved ACAP.
o Cervical Dislocation cervical dislocation in un-anesthetized neonatal and adult
rodents is permitted only if it is performed correctly by a trained person, its use is
scientifically justified, and it is described in an approved ACAP. Manual cervical
dislocation is a humane method of euthanasia when limited to rodents weighing less
than 200 grams. Personnel using cervical dislocation must be adequately trained,
demonstrate their technical proficiency, and must consistently apply this method
humanely and effectively.
o Decapitation decapitation in un-anesthetized neonatal and adult rodents is
permitted only if it is performed correctly by a trained person, its use is scientifically
justified, and it is described in an approved ACAP. When performed properly this
technique is nearly instantaneous and is considered humane. Guillotines that are
designed to accomplish decapitation in adult rodents in a uniformly instantaneous
manner are commercially available. Sharp scissors can be used to decapitate
neonatal rodents. Check guillotine and scissor blades frequently to ensure
sharpness. The equipment used to perform decapitation should be maintained in
good working order and serviced on a regular basis to ensure sharpness of blades.
The use of plastic cones to restrain animals appears to minimize stress from
handling, minimize the chance of injury to personnel, and improves positioning of
the animal in the guillotine. (2013 AVMA Guidelines on Euthanasia)
Note: The Physics Departments Instrument Shop, located in Phillips Hall 115A, will
sharpen blades
for a small fee [(919) 962-1183].
Gaseous Carbon Dioxide (CO2): must be supplied using a compressed gas tank. The
use of dry ice as a source of CO2 for euthanasia is not permitted. (Refer to section 2A
below.)

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Inhalant Anesthesia: anesthetic agent(s) delivered as a volatile gas to the respiratory

tract to induce anesthesia. Personnel should minimize their exposure to these agents
as some are considered chemical hazards. These agents should only be used in a
chemical fume hood, ducted biosafety cabinet or in a system with an active gas
scavenging device. (Refer to section 2B below.)
Injectable Anesthesia: chemical agent(s) administered by injection with a needle and
syringe to induce anesthesia. Common routes of injection include, but are not limited to,
intraperitoneal (IP), intramuscular (IM) or intravenous (IV). Injectable anesthetics are easy
to administer, require minimal equipment, and avoid safety concerns associated with
inhalants. (Refer to section 2CSection 2: Procedures

A. Gaseous Carbon Dioxide (CO2): The 2013 AVMA Guidelines on Euthanasia

recommends that the gradual displacement rate of CO2 into the euthanasia
chamber should be 10-30% to minimize pain and distress. All calculations
described below are for a DLAM shoe box style rat cage at 30% displacement.
Note: DLAM procedure rooms have dedicated CO2 euthanasia chambers
equipped with acceptable flow meters. Investigators who wish to perform CO2
euthanasia outside of DLAM facilities must adhere to all of the following
principals and must purchase the same equipment utilized by DLAM.
Appropriate flow meters must be purchased from VWR and can be found
through the UNC purchasing system, E-Pro, or at the following website:
https://us.vwr.com (part number: 89012-426). To purchase appropriately sized
euthanasia chambers, contact DLAM at (919-843-7992).
1. Place the Euthanex stainless steel lid over the plastic cage. The lid should be
connected to a CO2 tank via a plastic hose.
a. Make sure the two holes on the top of the lid are not blocked, as these holes
allow air to be pushed out by the heavier CO2.
b. Make sure the plastic cage does not have an automatic watering opening.
2. Remove each animal from the housing chamber and place into the euthanasia
chamber. Never place the housing chamber into the euthanasia chamber. Do not
place different animal species in the chamber at the same time. Do not overcrowd
the chamber. Each animal should have enough floor space available to lie down.
3. Turn on the valve located on top of the CO2 tank. Next, set the flow meter by
adjusting the regulator valve on the left side of the flow meter (see photo on the next
page):
a. Standard DLAM Shoebox style RAT cage: 8 liters per minute (lpm)
b. Standard DLAM Shoebox style MOUSE cage: 1.8 liters per minute (lpm)
c. Other CO2 Chambers: Use the following formula to calculate the appropriate
flow rate:
height x width x length = liters x .20 = flow rate/minute
61
4. Continue to allow CO2 to flow into the chamber for one minute after breathing stops
(approximately 6 minutes for mice and 8 minutes for rats). Young animals, certain

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strains of mice, and sick animals may require more time to become deeply
anesthetized.
5. Once animals are fully anesthetized, immediately perform a physical method of
euthanasia (i.e. cervical dislocation, thoracotomy, major organ harvest, or
decapitation) to confirm death.
6. Note: If a terminal procedure (i.e. cardiac puncture, tissue collection) must be
performed before the secondary physical method, ensure that animals remain
deeply anesthetized and that a physical method of euthanasia is performed
following the terminal procedure.
7. Place dead animals into a non-PVC containing bag. DLAM provides these bags in a
variety of sizes. Label the bag with the ACAP ID#. Seal the bag securely. Place the
bag with dead animal(s) into the DLAM carcass freezer available in each animal
facility. Please see the Policy on Rodent Carcass Disposal for more information.
8. Disinfect the euthanasia chamber bottom after each use.

Step 2: Set
Flow Meter to
appropriate
flow rate .

Step 1:
Turn on
valve on
top of the
CO2 tank

B. Inhalant Anesthetics (e.g. Isoflurane)

Induction chambers for inhalational anesthetics must allow animals appropriate floor
space without being too large. Chambers that are too large require increased volumes
of the anesthetic agent and may result in slow induction time. Where applicable,
induction chambers must prevent animals from coming into direct contact with an
anesthetic soaked material. The lid should fit snugly and the chamber must be used in
a fume hood, a ducted biosafety cabinet, or with a properly functioning active
scavenging system.
1. Pre-charge the anesthetic chamber by opening the vaporizer or placing two to three
pieces of absorbent material on the bottom of the chamber. Add approximately 3-5

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mls of isoflurane liquid to the absorbent material (amount of isoflurane is determined

by the size of the chamber). Close the lid and wait 5 minutes for the liquid to form a
volatile gas within the chamber.
2. Remove the lid of the chamber, quickly place the animals in the chamber, ensure
the absorbent material is not in direct contact with the animal, and immediately close
the lid.
3. The animals should become anesthetized in 2-5 minutes. Neonates require a longer
period of time to anesthetize and should remain in the chamber for at least five (5)
minutes.
4. When animals are completely recumbent and obviously deeply anesthetized,
remove them from the chamber.
5. Immediately perform a physical method of euthanasia. Isoflurane is highly volatile
and animals will quickly regain consciousness once removed from the chamber.
Therefore, it is imperative that physical euthanasia be performed immediately.
6. Note: If a terminal procedure (i.e. cardiac puncture, tissue collection) must be
performed before the secondary physical method, ensure that animals remain
deeply anesthetized and that a physical method of euthanasia is performed
following the terminal procedure.

C. Injectable Anesthetics
Injectable anesthetics can be effectively used to anesthetize animals prior to
performing physical euthanasia. The agent should be an anesthetic recommended for
the species, and the dosage used should be equal to or greater than the standard
published reference dose for anesthesia (e.g., a common dose of pentobarbital for
euthanasia is 100 mg/kg, which is approximately twice the anesthetic dose for rats and
mice). Once the injectable anesthetic is administered, allow sufficient time for the
animal to lose consciousness.
Injectable anesthetics intended for use in adult rodents may not have the desired effect
in neonates. In a pilot study conducted at UNC-Chapel Hill, few anesthetics were found
to be reliably effective in neonates. The drugs that provided the most effective
anesthesia are available only to veterinarians and as a result were considered
impractical for use by the scientific community. Contact a DLAM veterinarian for more
information about appropriate doses of injectable anesthetics.

Section 3: Euthanasia of Rodent Fetuses

Fetuses up to 14 days in gestation:
Neural development at this stage is minimal and pain perception is considered unlikely.
Euthanasia of the mother or removal of the fetus should ensure rapid death of the fetus
at this stage of development.

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Fetuses 15 days in gestation to birth:

The literature on the development of pain pathways suggests the possibility of pain
perception at this point in gestation. Whereas fetuses at this age are not sensitive to
inhalant anesthetics, anesthesia may be induced by injection of the fetus with a
chemical anesthetic, or by deep anesthesia of the mother with a chemical agent that
crosses the placenta, e.g., pentobarbital. Decapitation with sharp scissors and cervical
dislocation are acceptable physical methods of euthanasia when used by a trained
person. The specific technique(s) employed must be described in the approved ACAP.
When chemical fixation of the whole fetus is required, fetuses should be anesthetized
prior to immersion in or perfusion with fixative solutions. Consult with one of the
institutional veterinarians to learn more about fetal sensitivity to specific anesthetic
agents. When fetuses are not required for study, the method chosen for euthanasia of
a pregnant mother must ensure rapid death of the fetus.
PDF format available on the IACUC website:
Click HERE

EUTHANASIA REFERENCES
American Veterinary Medical Association (2013) AVMA Guidelines on Euthanasia.
http://www.avma.org/issues/animal_welfare/euthanasia.pdf.
Anden NE, Magnusson T & Stock G (1974) Effect of anesthetic agents on the
synthesis and disappearance of brain dopamine normally and after haloperidol, KCL or
axotomy. Naunyn-Schmiederbers Archiv fur Pharm 283(4), 409-418.
Bergstrom DA, Bromley SD & Walters JR (1984) Dopamine agonists increase pallidal
unit activity: attenuation by agonist pretreatment and anesthesia. Eur J Pharm 100(1),
3-12.
Bhathena SJ (1992) Comparison of effects of decapitation and anesthesia on
metabolic and hormonal parameters in Sprague-Dawley rats. Life Sciences 50(21),
1649-55.
Brown RE (1995) An Introduction to Neuroendocrinology. Cambridge.
Holson RR (1992) Euthanasia by decapitation evidence that this technique produces
prompt, painless unconsciousness in laboratory rodents. Neurotoxicology and
Teratology 14(4), 253-257.
Institute of Laboratory Animal Resources Commission on Life Sciences, National
Research Council (1996) Guide for the Care and Use of Laboratory Animals. National
Academy Press (65-66).
Malyapa RS, et al (1998) DNA damage in rat brain cells after in vivo exposure to 2450
MHz electromagnetic radiation and various methods of euthanasia. Radiation
Research 149, 637-45.

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Mantz J, Varlet C, Lecharny JB, Henzel D, Lenot P & Desmonts JM (1994) Effects of
volatile anesthetics, thiopental, and ketamine on spontaneous and depolarizationevoked dopamine release from striatal synaptosomes in the rat. Anesthesiology 80(2),
352-363.
NIH (2002) Guidelines for the Euthanasia of Rodent Feti and Neonates.

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Supply and Vendor Information

Isoflurane can be purchased from the UNC-CH Hospital Pharmacy Storeroom: Call 919966-1366 for more information or go to Ground Floor, room NG10B. A grant number
and department number is required for purchase.
Braintree Scientific
781-843-2202
www.braintreesci.com
Instruments, lab equipment, isothermal pads, tattoo paste
Fisher Scientific
800-766-7000
www.fishersci.com
Lab equipment, chemicals, instruments, pharmaceuticals
Henry Schein
800-872-4346
www.henryschein.com
Veterinary supplies, instruments, pharmaceuticals
Need Vet License or Researcher DEA license
JA Webster
800-225-7911
www.jawebster.com
Veterinary supplies, instruments, pharmaceuticals
Need Vet License or Researcher DEA license
Kent Scientific
888-572-8887
Surgical equipment, telemetry equipment

www.kentscientific.com

Med-Vet International
800-544-7521
www.shopmedvet.com
Veterinary supplies and instruments (discounted)
Need Vet License
National Band and Tag
ID tags, ear tags
Roboz

859-261-2035

www.nationalband.com

800-424-2984
Specialize in instruments

www.roboz.com

TW Medical
888-787-4487
Veterinary supply (Bill Forrester)

www.twmedical.com

UNC-CH Materials Management and Distribution

966-5671
Scientific Storeroom, General Storeroom, Chemical Storeroom
Veterinary Medical Supply 800-533-8674
Veterinary Supplies out of Zebulon, NC
Need Vet License
Southern Anesthesia
800-456-0757
http://www.sasvet.com/
This is a human source company that has a Veterinary division, will set up an account
without a vet license.
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The University of North Carolina at Chapel Hill

IACUC Training Record
Mice: Handling and Basic Techniques
Technique
needed?

Technique

Comments

Handling and Restraint

Intraperitoneal Injection
Subcutaneous Injection
Intramuscular Injection
Intravenous Injection
Oral Gavage
Retro-orbital Injection

*Individual appointment during class*

Retro-orbital Bleed

*Individual appointment during class*

Tail Nick Bleed

Tail Clip Bleed /Genotyping
Submandibular Bleed
Cardiac Puncture, terminal
Ear Notch (punch)
Ear Tag
Anesthesia Injectables
CO2 Flow meter w/ Phys. Euthanasia
Cervical Dislocation without Anesthesia

*Individual appointment during class*

Decapitation without Anesthesia

*Individual appointment during class*

*Provided during a 1-on-1 training
session.
Please call the OACU to schedule an
appointment
966-5569

Isoflurane Euthanasia / Anesthesia Drop Method

Inhalational Anesthesia Training-Vaporizer Machine

Training

Contact DLAM Vet Services at 966-2906

Proficiency Rating

I
II
III

Please add email address if you are a Lab Coordinator: ______________________________

Lab Phone #: _______________________________
I certify that I have received the above training:
Signature: ________________________________
PID:
______________
Print Name: _______________________________
PI:
______________
Instructor Signature: ________________________
Date: ______________

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