National Workshop on Ethics, Selection, Handling and

Immunization of Laboratory Small animals

7th to 9th March 2019

Conveners
Prof. M. Sivanandham, Secretary, SVEHT & Professor of Biotechnology

Prof. E. Nakkeeran, HoD/ Biotechnology

Organizing Secretary
Dr. S. Prabhu, Associate Professor/ Biotechnology

Coordinators

Mr. P. K. Praveen Kumar, Assistant Professor/ Biotechnology

Mr. J. Hariharan, Assistant Professor / Biotechnology

Mr. D. Suresh Kumar, Assistant Professor / Biotechnology

Organized By

Department of Biotechnology
Sri Venkateswara College of Engineering (Autonomous)
Sriperumbudur Tk-602117
 National Workshop on Ethics, Selection, Handling and Immunization of
Laboratory Small Animals

7th - 9th March 2019

Organized
By
Department of Biotechnology
Sri Venkateswara College of Engineering
Sriperumbudur-602117

Programme Schedule
Day I: 7th March, 2019
Time Events
8.30 AM - 9.30 AM Registration
9.30 AM - 9.35 AM Prayer Song
Welcome address
by
Prof. E. Nakkeeran, Ph. D
9.35 AM - 9.40 AM
Professor & Head
Department of Biotechnology
Sri Venkateswara College of Engineering
Patron Address
by
Prof. S. Ganesh Vaidyanathan, Ph. D
9.40 AM - 9.45 AM
Principal
Sri Venkateswara College of Engineering

Chief Patron address

by
Prof. M. Sivanandham
9.45 AM - 9.50 AM
Secretary,
Sri Venkateswara Educational & Health Trust, Kotturpuram.

Welcoming the Chief guest with Bouquet

9.50 AM- 9.53 AM Lighting the Kuthuvillakku & Releasing the Workshop manual by Chief
guest
Introducing the Chief guest
by
Dr. S. Prabhu
9.53 AM -9.55 AM
Associate Professor
Department of Biotechnology
Sri Venkateswara College of Engineering

2
 Inaugural Address
By
Chief guest
9.55 AM-10.30 AM
Prof. P. Tensingh Gnanaraj
Registrar
Tamilnadu Veterinary and Animal Sciences University
Madhavaram Milk Colony, Chennai- 51
10.30 AM – 10.45 AM Tea Break
A Technical Talk on
Immunization and Monoclonal Antibody Production
By
10.45 AM – 11. 30 AM Prof. P. Kumaraswamy
Controller of Examinations
Tamilnadu Veterinary and Animal Science University,
Madhavarm Milk Colony, Chennai-51

A Technical Talk on
Maintenance of Laboratory Small animals
By
Dr. Y. Yasotha
11.30 AM - 12.15 PM
Assistant Professor
Department of Veterinary Pathology
Madras Veterinary College
Madras Veterinary College, Vepery, Chennai- 7
12.15 PM - 01.00 PM Lunch
01.00 PM – 02.00 PM Hands on Session – I
Batch - I Selection, Ethical Handling of Mice
Training by
02.00 PM - 03.00 PM Mr. J. Hariharan
Batch - II Assistant Professor
Department of Biotechnology, SVCE
Hands on Session – II
01.00 PM – 02.00 PM Selection, Ethical Handling of Rats
Batch - II Training by
Dr. S. Prabhu
02.00 PM - 03.00 PM Associate Professor
Batch - I Department of Biotechnology, SVCE
03.00 PM- 3.15 PM High Tea
Day 2: 08th March, 2019
Time Events
09.00 AM - 09.45 AM A Technical Talk on
Handling of Laboratory Small Animals
By
Dr. P.C. Prabhu
Assistant Professor
Department of Veterinary Pathology
Madras Veterinary College, Vepery, Chennai- 7
09.45 AM- 10.30 AM A Guest Talk on
Welfare on Animal Care
By
3
 Mr. D. Shegar
Honourable Animal Welfare Officer, Animal Welfare Board of India
Ballabgarh Bear, New Delhi, India
&
Honourable Joint Secretary
The Massachusetts Society for the Prevention of Cruelty to Animals
Velachery, Chennai
10.30 AM – 10.45 AM Tea Break
10.45 AM – 12.00 PM Hands on Session – III
Batch - I Selection and Ethical handling of Rabbits
Training by
12.45 PM –02.00 PM Prof. R. B. Narayanan
Batch - II Dean and Professor
Department of Biotechnology, SVCE
12.00 PM- 12.45 PM Lunch Break
10.45 AM – 12.00 PM Hands on Session – IV
Batch - II Immunization of Mice
Training by
12.45 PM –02.00 PM Mr. J. Hariharan
Batch - I Assistant Professor
Department of Biotechnology, SVCE
02.00 PM - 2.15 PM Tea Break
02.15 PM – 3.15 PM A technical Talk on Poly clonal Antibody Production
Day 3: 09th March 2019
Time Events
09.00 AM - 09.45 AM A Technical Talk on
Pharmacological and Toxicological Studies in Laboratory Small animals
by
Dr. P. Sriram
Professor and Head
Department of Veterinary Pharmacology and Toxicology
Madras Veterinary College
Madras Veterinary College, Vepery, Chennai- 7
A Technical Talk on
Ethical Breeding of laboratory small animals
By
09.45 AM - 10.30 AM Dr. T. Sarath
Assistant Professor
Department of Animal Reproduction, Gynaecology and Obstetrics
Madras Veterinary College, Vepery, Chennai- 7

10.30 AM – 11.30 AM Hands on Session – V

Immunization of Rats
Training by
Dr. S.Prabhu
Associate Professor
Department of Biotechnology, SVCE
11.30 AM – 12.30 PM
Lunch Break
4
 Hands on Session – IV
Immunization of Rabbits
Training by
12.30 PM – 01.30 PM
Prof. R. B. Narayanan
Professor & Dean
Department of Biotechnology, SVCE
01.30 PM- 02.00 PM Tea Break
TIME Valedictory Function
02.00 PM- 02.05 PM Welcoming the Chief guest with Bouquet
Introducing the Chief guest
by
Mr. J. Hariharan
02.05 PM -02.10 PM
Assistant Professor
Department of Biotechnology
Sri Venkateswara College of Engineering
Valedictory address
by
Chief guest
by
02.10 PM-02.20 PM
Dr. A.Ramesh, Ph.D.,D.Sc.
Director & Member - Test Facility Management
International Institute of Biotechnology and Toxicology
Padappai-601 301, Kancheepuram District, Tamil Nadu, India
02.20 PM-02.40 PM Certificate distribution to the participants by Chief guest
Feedback from the participants
by
Prof. E. Nakkeeran, Ph. D
02.40 PM –02.50 PM
Professor & Head
Department of Biotechnology
Sri Venkateswara College of Engineering

Vote of Thanks
by
Dr. S. Prabhu
02.50 PM – 02.55 PM
Associate Professor
Department of Biotechnology
Sri Venkateswara College of Engineering

02.55 PM – 03.00 PM NATIONAL ANTHEM

Experiment No: 1

Animal Ethics - Experimenting on animals

Hands on Training

5
 By
Prof. R. B. Narayanan, Professor & Dean
Dr. S. Prabhu, Associate Professor
Mr. J. Hariharan, Assistant Professor
Department of Biotechnology

Aim: To give awareness about the animal ethics.

Animal experimentation

Animal experiments are widely used to develop new medicines and to test the safety of other
products. Many of these experiments cause pain to the animals involved or reduce their
quality of life in other ways. If it is morally wrong to cause animals to suffer then
experimenting on animals produces serious moral problems. Animal experimenters are very
aware of this ethical problem and acknowledge that experiments should be made as humane
as possible. They also agree that it's wrong to use animals if alternative testing methods
would produce equally valid results.

Two positions on animal experiments

In favour of animal experiments:

Experimenting on animals is acceptable if (and only if):

1. Suffering is minimised in all experiments human benefits are gained which could
not be obtained by using other methods

Against animal experiments:

Experimenting on animals is always unacceptable because:

1. It causes suffering to animals

2. The benefits to human beings are not proven

3. Any benefits to human beings that animal testing does provide could be
produced in other ways

Harm versus benefit

The case for animal experiments is that they will produce such great benefits for humanity
that it is morally acceptable to harm a few animals.
6
The equivalent case against is that the level of suffering and the number of animals involved
are both so high that the benefits to humanity don't provide moral justification.

The three R’s

The three Rs are a set of principles that scientists are encouraged to follow in order to reduce
the impact of research on animals.

The three Rs are: Reduction, Refinement, Replacement.

Reduction:
Reducing the number of animals used in experiments by:
 Improving experimental techniques

 Improving techniques of data analysis

 Sharing information with other researchers

Refinement:
Refining the experiment or the way the animals are cared for so as to reduce their suffering
by:
 Using less invasive techniques

 Better medical care

 Better living conditions

Replacement:

Replacing experiments on animals with alternative techniques such as:

 Experimenting on cell cultures instead of whole animals
 Using computer models

 Studying human volunteers

 Using epidemiological studies

Drug safety

Animal experiments and drug safety

Scientists say that banning animal experiments would mean either

1. An end to testing new drugs or

2. Using human beings for all safety tests
Animal experiments are not used to show that drugs are safe and effective in human beings -
they cannot do that. Instead, they are used to help decide whether a particular drug should be
tested on people.

7
Animal experiments eliminate some potential drugs as either ineffective or too dangerous to
use on human beings. If a drug passes the animal test it's then tested on a small human group
before large scale clinical trials.

Animal experiments and animal rights

The issue of animal experiments is straightforward if we accept that animals have rights: if an
experiment violates the rights of an animal, then it is morally wrong, because it is wrong to
violate rights.

The possible benefits to humanity of performing the experiment are completely irrelevant to
the morality of the case, because rights should never be violated (except in obvious cases like
self-defence).

And as one philosopher has written, if this means that there are some things that humanity
will never be able to learn, so be it.

This bleak result of deciding the morality of experimenting on animals on the basis of rights
is probably why people always justify animal experiments on consequentialist grounds; by
showing that the benefits to humanity justify the suffering of the animals involved.

Justifying animal experiments

Those in favour of animal experiments say that the good done to human beings outweighs the
harm done to animals.

This is a consequentialist argument, because it looks at the consequences of the actions under
consideration.

It can't be used to defend all forms of experimentation since there are some forms of suffering
that are probably impossible to justify even if the benefits are exceptionally valuable to
humanity.

Ethical arithmetic
Animal experiments and ethical arithmetic

The consequentialist justification of animal experimentation can be demonstrated by

comparing the moral consequences of doing or not doing an experiment.

8
This process can't be used in a mathematical way to help people decide ethical questions in
practice, but it does demonstrate the issues very clearly.

The basic arithmetic

If performing an experiment would cause more harm than not performing it, then it is
ethically wrong to perform that experiment.

The harm that will result from not doing the experiment is the result of multiplying three
things together:

1. The moral value of a human being

2. The number of human beings who would have benefited

3. The value of the benefit that each human being won't get

The harm that the experiment will cause is the result of multiplying together:

1. The moral value of an experimental animal

2. The number of animals suffering in the experiment

3. The negative value of the harm done to each animal

But it isn't that simple because:

1. It's virtually impossible to assign a moral value to a being

2. It's virtually impossible to assign a value to the harm done to each individual

3. The harm that will be done by the experiment is known beforehand, but the
benefit is unknown

4. The harm done by the experiment is caused by an action, while the harm
resulting from not doing it is caused by an omission

Certain versus potential harm

In the theoretical sum above, the harm the experiment will do to animals is weighed against
the harm done to humans by not doing the experiment.

But these are two conceptually different things.

1. The harm that will be done to the animals is certain to happen if the
experiment is carried out

9
 2. The harm done to human beings by not doing the experiment is unknown
because no-one knows how likely the experiment is to succeed or what
benefits it might produce if it did succeed

So the equation is completely useless as a way of deciding whether it is ethically acceptable

to perform an experiment, because until the experiment is carried out, no-one can know the
value of the benefit that it produces.

And there's another factor missing from the equation, which is discussed in the next section.

Acts and omissions

The equation doesn't deal with the moral difference between acts and omissions.

Most ethicists think that we have a greater moral responsibility for the things we do than for
the things we fail to do; i.e. that it is morally worse to do harm by doing something than to do
harm by not doing something.

For example: we think that the person who deliberately drowns a child has done something
much more wrong than the person who refuses to wade into a shallow pool to rescue a
drowning child.

In the animal experiment context, if the experiment takes place, the experimenter will carry
out actions that harm the animals involved.

If the experiment does not take place the experimenter will not do anything. This may cause
harm to human beings because they won't benefit from a cure for their disease because the
cure won't be developed.

So the acts and omissions argument could lead us to say that

1. It is morally worse for the experimenter to harm the animals by experimenting on

them

2. Than it is to (potentially) harm some human beings by not doing an experiment

that might find a cure for their disease.

And so if we want to continue with the arithmetic that we started in the section above, we
need to put an additional, and different, factor on each side of the equation to deal with the
different moral values of acts and omissions.

10
Proposed EU directive

In November 2008 the European Union put forward proposals to revise the directive for the
protection of animals used in scientific experiments in line with the three R principle of
replacing, reducing and refining the use of animals in experiments. The proposals have three
aims:

1. To considerably improve the welfare of animals used in scientific procedures

2. To ensure fair competition for industry

3. To boost research activities in the European Union

The proposed directive covers all live non-human vertebrate animals intended for
experiments plus certain other species likely to experience pain, and also animals specifically
bred so that their organs or tissue can be used in scientific procedures.

The main changes proposed are:

1. To make it compulsory to carry out ethical reviews and require that experiments
where animals are used be subject to authorisation

2. To widen the scope of the directive to include specific invertebrate species and
foetuses in their last trimester of development and also larvae and other animals used
in basic research, education and training to set minimum housing and care
requirements

3. To require that only animals of second or older generations be used, subject to

transitional periods, to avoid taking animals from the wild and exhausting wild
populations

4. To state that alternatives to testing on animals must be used when available and that
the number of animals used in projects be reduced to a minimum

5. To require member states to improve the breeding, accommodation and care measures
and methods used in procedures so as to eliminate or reduce to a minimum any
possible pain, suffering, distress or lasting harm caused to animals

11
References

The above article is adopted from the following site

1. http://www.bbc.co.uk/ethics/animals/using/experiments_1.shtml

Experiment No: 2

Criteria for Selecting Experimental Animals

Hands on Training
By
Dr. S. Prabhu, Associate Professor
Prof. R. B. Narayanan, Professor & Dean
Mr. J. Hariharan, Assistant Professor
Department of Biotechnology

Aim: To provide guidelines to select an experimental animal

12
Factors to be considered while selecting experimental animal

The goal of any experiment is to determine the effects of defined experimental variables in a
controlled environment. When working with biologic systems variability between
experimental animals can confound the interpretation of results. By selecting appropriate
animals an investigator will reduce experimental variability. This will result in the use of
fewer animals and less work to produce significant results.

Scientists who are planning experiments evaluate both animal and non-animal approaches. If
there are no suitable alternatives to the use of live animals, the appropriate species is selected
on the basis of various scientific and practical factors, including the following:

 Which species will yield the most scientifically accurate and interpretable results?

 According to critical review of the scientific literature, which species have provided
the best, most applicable historical data?

 On which species will data from the proposed experiments be most relevant and
useful to present and future investigators?

 Which species have special biologic or behavioral characteristics that make them
most suitable for the planned studies?

 Which species have features that render them inappropriate for the planned studies?

 Which species present the fewest or least severe biologic hazards to the research
team?

 Which species require the fewest number of animals?

 Which species that meet the above criteria are most economical to acquire and house?

Selection of required number of animals based on experimental design

An experiment in which laboratory animals arc used should be designed carefully, so that it
produces unequivocal information about the questions that it was designed to address. The
two most important requirements of proper experimental design in that connection are as
follows:

13
 1. Animals in different groups should vary only in, the treatment that the experiment is
designed to evaluate. So that the experimental outcome will not be confounded by
dissimilarities in the constitution of the groups or in how they are treated or measured.
2. Each treatment should be given to enough animals for the experimental outcome to be
attributed confidently to treatment difference and not merely to chance.

The best way to ensure that groups of experimental animals are comparable is to draw them
from a Single homogeneous pool and to assign them randomly to treatment groups. Choosing
Animals of the same age, sex, and inbred strain for all treatment groups and even assigning
littermates randomly to different treatment groups can eliminate factors that might partially
account for group-to-group differences in experimental outcome. Once animals are assigned
to groups, they should be handled identically, except for the treatment differences that the
experiment is designed to evaluate. Food, water, bedding, and other features of animal
husbandry should be the same. For long-term experiments, cages should be rotated to
minimize group differences caused by cage position. For invasive experimental treatments,
sham or placebo procedures should be performed in comparison groups; for example, animals
given treatment by gavage should be compared with controls given the vehicle by gavage,
animals treated surgically should be compared with animals that undergo sham surgical
operations, and animals exposed to treatment by inhalation should be compared with animals
placed in inhalation chambers that circulate only air. Following those precautions will ensure
that differences in outcome between groups can be attributed to the experimental treatment
itself and not to ancillary differences associated with the administration of the treatment.
Finally, wherever possible, the outcome of interest should be measured by people who are
unaware of which treatment each animal received, because such knowledge can magnify or
even create Observed treatment differences. It is particularly important to carry out “blind”
studies when the outcome is to be evaluated subjectively (e.g., by grading of disease
severity), rather than measured quantitatively (e.g., by measuring concentrations of serum
constituents).

The number of animals needed in each group will depend on many features of the
experimental design, including the following:

1. The goals of the study;

2. The primary outcome measure that will be compared;
3. The number of groups that will be compared;
4. The expected number of technical failures or usable end points;
14
 5. The number and type of comparisons that will be made;
6. The expected animal-to-animal and measurement variability in the outcome;
7. The statistical design and analysis that will be used;
8. The magnitude of the differences between control and treatment groups that it is
desirable to detect;
9. The projected losses; and
10. The maximal tolerable chance of drawing erroneous conclusions.

The more variable an outcome measure is, either because outcomes in identically treated
animals vary substantially or because there is a high degree of measurement variability, the
more animals will be needed in each group to distinguish between group differences caused
by treatment and those caused by chance. How outcome measurement variability, treatment
difference to be detected, and tolerable chance of drawing an erroneous conclusion affect the
required sample size depends on the measurement to be made, the type of group comparison
to be made, and the statistical analysis to be used. Tables and formulas for comparing
proportions among two or more groups have been published (Gan et al.. 1986), as has useful
information for other types of outcomes (Mann et al., 1991). For most experiments, it in
highly desirable to collaborate with a statistician through-out, beginning with the design
stage, so that appropriately defined groups of sufficient size will be available for a proper
statistical analysis.

The following are some of the factors which contribute to the increase in the
variability in experimental results.

Factors that can increase variability in experimental results:

1. Use of outbred animals or animals from varying backgrounds or sources.

2. Infection with pathogenic microorganisms.
3. Inflammation or infection associated with wounds.
4. Extravasation of irritating substances such as chemotherapeutics. 
5. Animals with ulceration, necrosis and or infection of a tumor.
6. Behavioral changes in animals can signal a difference. For example the mouse that
constantly runs in circles in its cage is not the same as the one who does not.

15
To reduce experimental variability:

1. Use inbred animals from the same source when possible.

2. Do not use animals with open wounds or other signs of illness or injury.

3. Do not use animals that display abnormal behavior.

4. Protect animals from infection with potential pathogenic microorganisms.

5. Select endpoints that will not introduce additional variables. Complete the study
before animals become moribund or develop ulcerated, necrotic or infected tumors.

6. Use aseptic technique when preparing for and performing surgery. Shave the fur
and scrub the surgery site. Select an appropriate method of sterilizing instruments for
surgery.

References:

The above article is adopted from the following sites

1. https://www.ncbi.nlm.nih.gov/books/NBK236591/
2. https://www.nap.edu/read/2119/chapter/4
3. https://www.research.psu.edu/arp/experimental-guidelines/selecting-appropriate-
animals.html

Experiment No: 3. Handling of Laboratory Small Animals: Mice, Rat & Rabbits

Hands on Training
By
Mr. J. Hariharan, Assistant Professor
Prof. R. B. Narayanan, Professor & Dean
Dr. S. Prabhu, Associate Professor
Department of Biotechnology

Aim: To get practice in selection, handling of small laboratory animals (mice, rats and
rabbits) to carryout subsequent animal experiments

Introduction:

16
 The animal species like rat, mice and rabbit are the most favourite choices of lab
animals for immunological experiments. These animals are used in immunological
experiments because of their genetic variation, different size, high fertility rate, short
gestation period, easy maintenance under lab conditions and resistance to many infections.

Materials required:

2 rats –Wistar albino rats (for each student -one male or one female rats), 2 mice –Swiss
albino or Balb/c (for each student-one male or one female mouse) and 2 rabbits- Wild
domestic (one male or one female), cages, water bottles, animal feed, husk, staining markers
and cage labels.

Procedure:

Identification and numbering of experimental animals:

Marking experimental animals for identification is prime importance in laboratory

studies. The individual animals are identified by cage marking, staining the skin with picric
acid or Indian ink or saffronin. The animal cages are labelled using tag labels to show all
details of the animals

Proper restraint and handling techniques are essential for reducing stress to laboratory
animals and the handler.  Animals become much easier to handle if they are trained and
accustomed to handling.  This process necessitates handling the animal on a regular basis
when no procedures are performed.  Most rodents will attempt to bite when handled. Since
rodent bites are painful and can become infected, care and proper technique in handling
rodents is essential.  Restraint devices or chemical restraint should be considered for
prolonged or potentially painful procedures.

Handling of Mice

Mice can be safely restrained by grasping firmly at the base of the tail.  This form of
restraint is suitable for moving the mouse over a short distance, animal identification and
weighing.  For greater control such as during examination, or injections, place the mouse on a
surface it can grasp.  A suitable surface is a wire cage top or a towel.  Apply a slight tension
to the base of the tail so the mouse grasps onto the surface. Gently but firmly place your free
hand over the shoulders and quickly grasp the scruff of the neck close to the base of the skull
between the thumb and forefinger.  Restrain the tail by your little finger.
17
Handling of Rats

Rats/Mice are picked at the base of tail. However, care is taken to hold the tail at the
base, close to the rats/mouse body. One hand is placed over the animal back with the thumb
and forefinger gently by firmly pressing its forelegs towards its head. Rats can be restrained
by grasping firmly at the base of the tail.  Holding the tail distal to the base can result in a
degloving (stripping off the skin) injury.  This restraint is suitable for moving the rat over a
short distance or a cursory examination. To calm a rat place it on your lab coat.  Providing a
place to hide such as under a towel will also help to calm the rat. For a firmer restraint grasp
the whole body, with the index and middle fingers along the sides of the head and the thumb
and remaining fingers under the axilla. Alternatively, circle your thumb and index fingers
under the jaw to control the head while the rest of your finger support the chest behind the
forelegs. Use your other hand to support the lower body and hold the tail. Both methods
restrict head movement while allowing access to the facial area. Applying too much pressure
to the head or chest can result in struggling and injury to the animal.  It may also increase the
tendency to bite. Some rat strains are more aggressive than others.  If your rats are difficult to
handle you may want to wear loose cotton work glove. Remember that the rat may still be
able to bite through the glove.

Do not attempt to grasp rats at the nape of the neck.  Unlike mice and hamsters, rats
object strongly to being restrained by the scruff.  Rats can inflict painful bites with their
incisors.

Fig: 1: Handling of Mice

18
 Fig: 2: Handling of Rats

Handling of Rabbits

Rabbits are docile, timid and tend to panic easily. Some aggressive rabbits will bite. They can
inflict painful scratches with their hind legs.  It is a good idea to assess the attitude of the
rabbit before opening the cage door. Rabbits are highly susceptible to lumbar spinal luxation,
resulting in paralysis.  Hence, extreme care must be taken when handling rabbits to avoid
sudden movement.  It is necessary to support the animal's hindquarters at all times.  Rabbits
are characteristically timid and excitable. Occasionally they resist handling. Rabbits are
19
restrained physically by manual or mechanical technique using restraining cafer. The rabbits
are gently restrained to relax and stop struggling. Rabbits are removed from a cage, and then
hold gently near the neck with one hand by supporting the hindquarters and back with the
other hand.

One way of lifting a rabbit is by grasping the skin over the shoulder with one hand and gently
lifting it with the other arm cradling the body, the head nestled in the crook of your arm. 
Another is to hold it upright by the scruff of the neck with one hand while the other hand
supports it hind quarters.  A towel wrapped around the body can also be used for restraint.
The towel may also be draped over the face.  Do not lift a rabbit by it ears.

For longer restraint for blood collection or fluid administration, an approved restraint
box or cat bag may be used or chemical restraint may be indicated.

20
 Fig: 3. Handling of Rabbits

References: National Research Council (US) Committee for the Update of the Guide for the
Care and Use of Laboratory Animals. Guide for the Care and Use of Laboratory Animals. 8th
edition. Washington (DC): National Academies Press (US); 2011.

Experiment No. 4: Immunization of Laboratory Small animals

Hands on Training

21
 by
Prof. R. B. Narayanan, Professor & Dean
Dr. S. Prabhu, Associate Professor
Mr. J. Hariharan, Assistant Professor
Department of Biotechnology, SVCE

Aim: To provide training for students on various routes of antigen immunization in mice,
rats and rabbits and about the method used for production of polyclonal/Monoclonal
antibodies.

Introduction:

Immunizations in laboratory animals are performed for a wide variety of reasons.

Primary purposes include (1) induction of specific B cells for the generation of hybridomas,
(2) production of PAbs and MAbs, (3) development and quality control of immunobiological
products, (4) fundamental immunological studies, and (5) induction of specific disease
models. Clearly, these studies have been integral to scientific breakthroughs that have
occurred in many areas of biomedical research. One such critical breakthrough has been the
development of highly effective vaccines. In other cases, products such as PAbs and MAbs,
which are obtained from immunization procedures, have become essential reagents in the
laboratory and are being applied in diagnostic testing, in cancer therapy, and in numerous
other ways.

Mice, rats, or hamsters are given biweekly injections of a purified antigen, cultured
cells, or cDNA. Adjuvants (Freund's, Ribi, Hunter's TiterMax, ImmunEasy, or Alum) are
mixed with the immunizing antigen for the first two immunizations only. Complete Freund's
adjuvant is only used with the first immunization. Subsequent immunizations are performed
in phosphate-buffered saline (PBS) or normal saline, with or without Incomplete Freund's
adjuvant. The choice of adjuvant is dependent on the subclass of immunoglobulin required.
The gauge of the needle is kept as small as possible (usually 23 gauge) to minimize
discomfort of the animal. Over the course of the 6-wk immunization schedule, each mouse
usually receives a total of six injections (three subcutaneous and three intraperitoneal).

Antigen can be introduced into an animal body by a variety of routes and antiserum
(polyclonal antibody) is produced. Polyclonal antibodies are antibodies which bind with more
than one type of epitope, whereas Monoclonal antibody (MAb) is a single type of antibody
that is directed against a specific antigenic determinant (epitope). It is interesting that
immortal monoclonal antibody producing cells to exist in nature. They are found in the

22
patients suffering from a disease called multiple myeloma (a cancer of B-lymphocytes). It
was in 1975, George Kohler and Cesar Milstein (Nobel prize, 1984) achieved large scale
production of monoclonal antibodies. They could successfully hybridize antibody-producing
B-lymphocytes with myeloma cells in vitro and create a hybridoma. The result is that the
artificially immortalized B-lymphocytes can multiply indefinitely in vitro and produce
monoclonal antibodies. The hybridoma cells possess the growth and multiplying properties of
myeloma cells that secrete antibody of B-lymphocytes. The production of monoclonal
antibodies by hybrid cells is referred to as hybridoma technology. These monoclonal
antibodies have wide variety of application in diagnostics and therapeutics.

Requirement:

Animals: Mice or Rats or Rabbits

Other Requirements: BSA or Human IgG, Adjuvant (peanut oil) or Freund’s Complete
Adjuvant, Rectified sprit, Sterile 2ml syringe, Electric shaver, Cotton wool, Collecting tubes
and Restraining café

1: Procedure: Routes of immunization

1.1. Intradermal:

This route is generally used for injecting viscous and slowly dispersing forms of antigen.
E.g. Antigen emulsified with Freund’s adjuvant. It provides rapid access to the lymphatics.
The area to be inoculated is carefully shaved in order to avoid any abrasion and clean with
rectified sprit. The syringe is filled with solution to be injected and a fold of skin between
forefinger and thumb is hold. The point of the needle just below the surface is placed and the
skin fold is released by holding the needle firmly at its point of entry. A maximum volume of
0.5 ml antigen is inoculated. The needle is withdrawn slowly by compressing simultaneously
with forefinger and thumb along the track of the needle. Swelling at the site of injection
indicates the true indradermal injection.

1.2. Subcutaneous

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 This route is suitable for emulsions, precipitates and viscous materials and in this
route of injection the antigens are spreading a little more as compared to intradermal mode of
injection. The mouse is held against the wire grid and restrained by holding the tail with right
hand. A fold of skin is grasped over the back near the neck region between the palm and 4 th
and 5th finger of the right hand. The tail is released and the mouse lifted in the left hand. The
belly skin is cleaned rectified sprit and the fold of the skin is hold between thumb and finger.
The needle is inserted into the pocket of skin lying behind the skin fold and care is taken not
to pierce the peritoneum. After the desired volume is injected, the needle is withdrawn and
the needle track is pinched to avoid antigen loss due to package.

1.3. Intramuscular:

It is one of the most frequently adopted group of immunization suitable for alum
precipitated and adsorbed antigens. Normally the antigen is deposited in the muscular layer
e.g. thigh muscles. The needle is inserted in the rear, at right angle to the skin surface at the
point halfway along the femur, so that the point lies within the muscle. After the inoculation
is made, the needle is withdrawn and gentle massage is done at the site.

1.4. Intraperitonial:

This route is generally used for injecting viscous forms of antigens. The animal is
placed on a wire surface and the tail is grasped with little forefinger of the left hand around it.
The animal will anchor on the wire and pull in opposite direction. The animal is picked up by
grasping the skin over the nape of neck with the index finger and thumb of the left hand. The
palm is turned to expose the animal in the supine position. The fur is wet slightly at the centre
of the abdomen using alcohol and the small skin area is exposed by brushing aside the wet
fur.

The syringe is pointed upward by standing in front of the animal and the needle is
inserted in the peritonial cavity. The abrupt of the cessation of resistance against the needle
indicates the penetration. The needle is refracted slightly inorder to reduce the possibility of
injection into the intestine. The material is injected quickly and the syringe is withdrawn
immediately.

1.5. Intravenous:

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 The intravenous route of delivery is the most efficient means of delivering substances
to animals because it bypasses the need for solute absorption. With this method, substances
are administered as a bolus or infusion directly into blood vessels on either an acute or
chronic basis (Figure 3). Precision electronic infusion pumps equipped with alarms to
indicate flow interruptions and microdrop infusion sets are used to ensure accurate chronic
intravenous delivery of many substances; however, less expensive precision and spring-
operated disposable pumps have become available for this purpose in recent years and may
represent a more economical alternative for experimental intravenous substance delivery,
depending on the nature of the material to be administered and the duration of treatment.

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Fig 1: Different routes of skin administration of substances. Depicted are intramuscular
(IM), intravenous (IV), subcutaneous (SC), and intradermal (ID) routes. Illustration
courtesy of Gianni Chiappetta
Table: 1 Dosage of Antigen for Immunization

2: Procedure: Immunisation

Prepare an emulsion of 0.5% BSA or human IgG in equal volume of peanut or

paraffin oil. 0.5 ml of emulsion is injected into mice or 1 ml in to rats or 2 ml in to rabbits by
using intraperitonial or intramuscular or intradermal or subcutaneous methods. The final
dosage is injected after seven days. The animal are bled and the serum is collected to assess
the antibody against BSA or human IgG.

Fig.2. Immunization method

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 Fig.3. Immunization of Mice for production antiserum

References: National Research Council (US) Committee for the Update of the Guide for the
Care and Use of Laboratory Animals. Guide for the Care and Use of Laboratory Animals. 8th
edition. Washington (DC): National Academies Press (US); 2011.

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