Date:

STUDY OF ROUTES OF DRUG ADMINISTRATION IN EXPERIMENTAL

ANIMALS

ANIMAL CARE HANDLING AND SEX DETERMINATION:

Precaution to be taken before handling the animals;

Before restrain. first soothe the animal by slow deliberate movements on their body.
Overcrowding near the animal cage should be avoided.

Noise should be kept to a minimum as much as possible.

Don't hold anilnal too hard, it may face difficulty in breathing and die too. Never agitate the animal; it may
become violent föl self protection.

MOUSE:
One can handle with the help of blunt forceps by grasping the skin behind the nick/body. This technique is often
used to transfer mice from one cage to another. Grasp the base of the tail with one hand and with other grasp the
loose skin behind its neck.

Hold the complete body bygrabbing back of the neck by using all fingers. RAT:
Lift rat out of the cage by grasping the base of the tail and place on a soft surface.

Place your index and middle fingers alongside the rat's and your thumb and ring fingers under its forelegs. Use your
index and middle fingers to secure its head and the remaining fingers to support the body.

Hold the complete body by grabbing the back by using complete palm.

GUINEA PIG:
By using both hands, calmly grasp it with one hand under the chest and use your other hand to support its
hindquarters.

Handle guinea pig with one hand by holding its hind quarter.

RABBIT:
By using single hand, hold the pelvic region. This technique is mainly used to transfer rabbits from one cage to
another.

By using both the hands, hold the complete hindquarter.

By using the both hahds, calmly grasp it with one hand supporting back of neck and the other hand supporting its
hindquarters.
SEX DETERMINATION IN RODENTS:

MOUSE / RAT:
Restrain the mouse/rat and lift the base of the tail. Sex is most easily determined by anogenital distance.

Males normally have a greater distance between the anus and urogenital openings. Male mice also have a larger genital
papilla. Scrotum can be easily palpable in mice.
Females normally have a lesser distance between the anus and urogenital openings.

GUINEA PIG:
Both male and female guinea pig display similar anogenital distances. The female detectable pink nipple at either side, a
separate urethal orifice, a vaginal membrane, a perineal sac and an anus, whereas tnale has penis, a larger perineal sac and an
anus.
ROUTE OF DRUG ADMINISTRATION IN EXPERIMENTAL ANIMALS:
Feeding or Oral gavaoe:
Hold the rodent in a hand carefully.
Measure the tube length from the nose to the last rib of the rodent and mark it.
Give a gentle tight grip at the back of neck, so that it opens its mouth widely.
Push the rodent head slightly upward and back to straighten the esophagus and then either from right or left side of
teeth, insert the tube by gentle rotation to avoid the resistance ( do not force the tube in esophagus, this may injure
mucous membrane). Slowly pass the tube observing for the swallowing reflex and when desired length of tube has
been inserted, inject solution with the help of syringe. ORAL ROUTE:

The Oral administration in rats and mice is done by using an oral feeding needle devised
for this purpose. The tip of such a needle is either blunted or is covered with smooth
rubber tube to avoid injury to the During oral administration it is
expected that the drug is delivered in stomach ( gastric lavage). The administered material
should not enter into the respiratory tract. Accidental entry of the material in respiratory
tract is traced by appearance of material in nasal cavity and violent striving by the animal.
Unsterilized substances and their solution / suspensions can be administered by this route.
Oral administration results into slow and incomplete bioavailability of substances. The
effect of orally administered drugs appears slowly due to delayed absorption. By oral
about 5-7 ml of liquid material can be administered.
the Select correct-sized gavage needle, ensuring that there is a metal ball on the end to
prevent the tip from being sharp. Never use a hypodermic needle for oral gavage.
Measure the needle length against thc mouse's body; the needle should be no longer than
from the nose to the last rib. If the needle is longer, take care to only insert the
appropriate length to prevent damaging the stomach. Shorter gavage needles can be used;
but if injecting acidic compounds, ensure that the needle fits adequately into the stomach
to prevent damage to the esophagus.
Fill the syringe with the appropriate amount of article to be dosed,
Restrain the mouse by scuffing.
Place the tip of the needle in the mouseDs mouth.
Slide the tip down the back of the mouth, moving it toward the front in one
fluid motion.
Take your time; any resistance felt indicates improper placenlent, in which case remove the needle and start again.
Do not force the needle, as it may enter the trachea and damage the epiglottis. The needle should slide down into
the esophagus easily.
Once the needle is properly placed, administer the inject ion article.
Remove the needle carefully so as not to damage the esophagus.
Note: Recommended in mice and rats but is not preterable in guinea pig bcoz they have a
small palatal ostium which gets easily dainagecl.
 4,
INJECTION SITE AND TECHNIQUES:
Following precaution should be taken before injection;

Injection sites should be cleaned with a suitable disinfectant / antiseptic (isopropyl alcohol, ethanol etc.).

Sterile syringes and needles must be used for any type of injections.
Always select the smallest possible gauge (G) needle to limit tissue trauma and injection
discomfort. Ex: 25-27G needle is preferrecl in adult mice for the injection in tail vein.
Aspiration technique is always an important aspect betöre pushing the injection solution
at the site.
Note: Aspiration is a technique of creating vacuum at the site of injection by pulling
piston back to check the right placement of the needle,
INTRAPER!TONEAL INJECTION (1.1))
By this route sterile drug solutions are injected into the petitoneal cavity in the gap between the wall of peritoneum visceral
organs. This site provides a broader surfåce iör absorption of drug. For 1.1) administration, the animal is held in one hand in
such way that its abdominal portion is accessible with a needle and animal remains immobile during the injection. The site of
injection is disinfected with 70% ethanol. Needle of 23-26G size can be used for this route. The needle is inserted into the
abdominal cavity .at 900 angle to the abdominal wall. Plunger of syringe is pulled back to check whether the needle has
entered into any visceral organ. Appearances of air bubble in the syringe assure that the needle is in the peritoneal cavity. By
IP route up to 2.0 to 4.0 ml solution can be injected to rats and upto 2.0 ml solution can be injected to a mouse.

l . Restrain the mouse by scuffing.

Prepare the area with an alcohol swab to disinfect the skin (this should be routinely done before all injections/blood collections).

Position the animal so that its head is lower than its body to allow any internal -organs to move out of
the way.
nsert the needle into the lower left/right quadrant of the abdomen at a 30 0 angle. 5. Aspirate the syringe to ensure proper
placement. Any sign of blood in the syringe indicates improper placemcnt, in which case the needle needs to be repositioned.
f other fluids are seen in the syringe upon aspiration, such as a yellow/clear colour (indicating puncture of the urinary bladder) or
green/brown colour (indicating puncture ofthe intestines/ caecum), discard the needle and syringe and start again.
Administer in a steady, fluid motion.
INTRAVENOUS INJECTION (I.V)
Drug administered by IV route instantly exerts it effect and is 100% bioavailability. For administration of a drug by IV
route, it must be sterile, clear solution (not containing particulate mater) non-irritant nature. Care is taken to adjust the pH of
the solution to about 7.4. The animal is restrained and the tail is cleaned and disinfected with 70% ethanol. To make the tail
veins appear prominent, either tail is dipped for 4-5 min in warm water or cotton swab dipped in spirit is rubbed on the tail.
A needle of 26-30G can be used for intravenous administration care is taken to avoid air bubble in the syringe. The held
with one hand in such way that it lies just near the site of injection and helps to support the syringe during injection. Upto 5-
8mm of the needle is inserted in the tail vein at an pngle of 30-400 and pulling back the plunger, it is confirmed that the
needle has enter the lumen of the vein. This confirmed by appearance of blood in the syringe. After this, the plunger is
slowly pushed forward till required amount of drug solution is injected. By this route, upto 1.0ml drug solution can be
injected in rats and mice.

Place the mouse into a plastic restraint device or anaesthetize it.

Prepare the tail with an alcohol swab to disinfect the skin.
Ensure that you can visualize the lateral tail veins. This can be assisted with the use of a heated lamp or by placing
the animal in a cage warmer or on top of a warming plate for a féw minutes prior to inje

With the tail under tension, insert the needle approximately parallel to the vein.
Ensure proper needle placement by inserting the needle at least 3 mm into the lumen of the vein. Once in the lumen,
the needle should feel smooth and there should be no resistance upon injection. Intravenous injection utilizing
lateral tail veins.
Administer in a slow, fluid motion to avoid rupture of the vessel. There is clearing of the lumen as the injection
article replaces the blood in the vein
 INTRAMUSCULAR INJECTION (I.M)
The drug administered by 1M route is slowly absorbed into systemic circulation and exerts a prolonged action. In rats the
drugs can be administered by intramuscular route into muscle hind limb. The hind limb of the rat is held between thumb and
forefinger of one hand. The area on the limb muscle is disinfected with 70% ethanol. The injection is made with a fine
needle of 26-30G size. This inflicts pain to animal and hence a volume more than 0.1 to 0.2 ml should not be injected by this
route. For administration of fine suspension of less water soluble drugs can be administered by this route. During
experimental procedure, this route is used for administration of anesthetic or analgesic substances.

SUBCUTANEOUS INJECTION (S.C)

The drugs which are highly active and are expected to get slowly absorbed are administered by this route. The small volume
of drug solution not more than 0.2-0.3 ml is administered by ihis route. For injecting the drug below skin, a fold is created in
the skin by pressing it between the thumb and fore finger of one hand and needle is inserted at the base of this fold at 15-20 0
angle. When drug solution is injected subcutaneously, bubble is formed at the site Of injection.

l . Fill the syringe with the solution to be administered.

Restrain the mouse by scruffing. or use an appropriate anesthesia.
Swab the area with an alcohol to disinfect the skin.

Insert the needle at the base of the skin fold between your thumb and forefinger, keeping the needle
straight because if there is an angle to the needle, it may pierce the muscle or go through the skin
and into your finger.
Aspirate the syringe to ensure proper placement. Any sign of blood •in the syringe indicates improper placement, in which case
the needle needs to be repositioned.
Administer the solution in a steady, fluid motion. As you inject, you can feel the injection article creating a bulbous under the
skin betvseen your fingers.

7. The location is to inject into the flank, between the hind leg and the front leg. This is also
the preferred location for injecting tumour cells.
lib

INTRADERMAL INJECTION:
 In this of type of drug administration, the drug solution is injected into the skin layers. Very small amouiåt Of drug can be
administered by this route. Administration this route is painful and maximum amount that can be injected is lesser that 0.05
ml. This route is rarely used in experimental pharmacology for inducing sensitization to antigens. Very fine needle of 30G
can be used for intradermal administration. The needle is inserted at 10-15 0 angle to achieve intraderaml administration. A
bulge at injection site confirms intradermal administration.

I . Intradermal injection is not typically carried out in mice, apart from the administration of
certain compounds via the footpad or ear pinna.
Intradermal injection must be performed under anaesthesia.
When injecting on the back of the mouse, take the scalpel holder and scalpel carefully in one hand' and extend the Skin between
the fingers of the other hand. With the scalpel remove the hair. Visualise the skin after injection.

Prepare the area with an alcohol swab to disinfect the skin (this should be routinely done before all
injections/blood collections).
Insert the needle carefully through the dermis at a 300 angle.

Aspirate the syringe to ensure proper placement. Any sign of blood in the syringe indicates improper
placement, in hich case the needle needs to be repositioned. 7. Administer slowly, with a
maximum volume of 50 ytl„ for footpad and ear pinea.
 INTRACARDIAC INJECTION

BLOOD COLLECTION:
Volume

As a rough guide, up to 10% of the total blood volume can be taken on a single occasion from a normal, healthy
animal on an adequate plane of nutrition with minimal adverse effects; this volume may be repeated after 3-4 weeks. For
repeat bleeds at shorter intervals, a maximum of 1.0% of an animal's total blood volume .can be removed every 24 hours;
the effects of stress, site chosen and anaesthetic used, must be carefillly considered.

As a general rule, total blood volume can generally be estimated as 55 - 70 ml/kg body weight. However, care
should be taken in these calculations as the percentage of total blood will be lower (-15%) in obese and older animals.

Exceeding this guideline may result in a physiologic coinpensatory response that may increase experimental
variability.
Sites for blood collection in the Mouse

Mice are considered to have around 58.5 ml of blood per kg of bodyweight. A mouse

x 0.025 kg 1.46 ml.

Retro-orbital sinus
tral jaw (mandible)

diac puncture
tral tail artery
henous vein
llary bleed
Sites for blood collection in the Rat
ARP personnel are available to provide training and discuss the advantages and disadvantages for each blood collection
technique.
ro-orbital plexus
diac puncture
eral tail vein
tral tail artery

Jugular vein
Blood withdrawal utilizing retro-orbital sinus for large-volume blood collection:
l . Retro-orbital bleeds must be performed under anaesthesia.
Anaesthetise the mouse. After the mouse is anaesthetised, proceed.
Place the haematocrit tube or Pastkur pipette at the medial canthus of the eye.
With a rotating motion, apply gentle pressure to insert the tube through the membrane.
Continue rotating the tube on the back of its orbit until blood flows.
Collect the blood in an appropriate vessel.
Upon completion, ensure good haemostasis before returning the animal to the cage by closing the eyelids and placing a
gauze pad over the eye until bleeding stops

Number of samples: It is recommended that only one sample be taken.

Sample volume: Up to 0.2 ml with recovery; Up to 0.5 ml non-recovery. Equipment: A
glass capillary tube or Pasteur pipette.
Blood withdrawal utilizino lateral tail veins for small-volume blood collcction:
The lateral tail vein is usually used and 50 ul to 0.2 ml of blood can be obtained per sample
depending on the size of the animal and specific requirements. The tail may need to be washed with diluted Hibiscrub (1%)
in order to see the blood vessel.

Sample Volume: 50 HI to 0.2 ml

Equipment: 25G - 27G needle or lance.

l . Restrain the mouse using a plastic restraint device or anaesthetize it. Prepare the tail with an alcohol swab to
disinfect the skin.
Needle placement should be no closer to the body than half the length of the tail.
Ensure that you can visualise the lateral tail veins. This can be assisted with the use of a heated lamp or by placing the
animal in a cage warmer or on top of a warming plate for a few minutes prior to injection. The lateral tail vein
runs along either side of the tail and can be visualized easily in albino mice. In non albino strains, it is more
important to warm the tail or Éalpate the vein to find the correct location.

With the tail under tension, insert the needle approximately parallel to the vein.
Ensure proper needle placement by inserting the needle at least 3 mm into the lumen of the vein.

Once blond •tart s to flow into the hub of the needle, place the haematocrit tube into the
needle hub or remove the needle to allow the blood to collect directly into a suitable
collection tube.
Upon completion, ensure good haemostasis before returning the animal to the cage by placing the gauze pad over the
blood collection site and applying pressure until bleeding stops.
Number of samples: No mole than föur blood smnples should be taken within any 24-hour period.
Sample volume: Up to 0.15 ml for a single sample, which can usually be repeated at 2-week intervals without disturbances
to haematological status.
TABLE CONTAINING DETAILS FOR BLOOD SAMPLING
• = Femoral artery and vein, carotid artery, jugular vein, dosral aorta

Sampling Volume Equipment No. of Samples

Mouse Rat Rabbit Mouse Rot Rabbit Mouse rut Rabbit

Blood vessel 0.1 - 0.2 236

connulo Up to 6
connulotion• ml samples in 2-
hour or UP
to 20
samples in
24hrs

Temporary 0.1 - 2.0 22G, Upto 6

connulation of ml 0.90 mm samples in
the lateral toil i.v. o 2-hour, or
vein catheter 8 samples
in o 24-
hours

Toil Vein 0.1 - 2 ml I or 2 in

24hrs No more
21G - 23G
0.2 - thon 8
needle or
samples in
27G needle butterfly
ml any 24-
needle
h0U( period.

Sophenous vein Up to 0.1 5 Up to 0.2 256 - 27G 23G needle No more than
(nonsurgical) needle No more
ml ml 4 samples in
than 4
24 hrs
samples in
24 hrs

Jugular vein 0.1 -2 m! 23G needle No more

(nonsurgical) thon 8
samples in
ony 24 hrs

Up to 0.2 ml one one sample

with Up to 4 ml sample per per 2 week
gloss gloss
recovery; Up with 2 week
copillory copillory
recovery;
to 0.5 ml tube or tube or
410 ml
non- nonrecover
Pasteur Pasteur
recovery pipette. pipette.
y

Up to I ml Up to 10ml 25G needle 19 - 216 One One

Abdominol/thorocic
blood vessel needle
(terminol)
Cordioc puncture Up to 1 ml Up to 15m! 196 - One One One
(terminal)
60
216 19 216
200
needle
ml needle needle

Morginoj ear 0.5 . Upto 8

veirvortery 10
196 . samples
ml in
needle 24hrs

REPORT:
Studied the various route of administration on experimental anilnals and
denu»nstrated.