Ethical committee form

8/7/2019 Ethical committee form

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Application for permission for Animal Experiments

Application to be submitted to send either to the CPCSEA (address in form A

above) or Institutional Animal Ethics Committee (IAEC).

Para A

*1. Name and address of establishment

*2. Registration number and date of registration

3. Name, address and registration number of breeder from whom animals acquired (or to

be acquired) for experiments mentioned in parts B and C

CCSHAU Farm (Dept. of Animal Breeding). If not available, than from localfarmer.

4. Place where the animals are presently kept (or proposed to be kept)

Animal House, Dept. of Vety. Pathology, COVS, CCSHAU, Hisar.

5. Place where the experiment is to be performed

Dept. of Vety. Pathology, COVS, CCSHAU, Hisar.

6. Date on which the experiment is to commence and duration of experimentIn month of March, 2009 or just after getting permission.

Duration of Experiment: Approx. 2-3 months

Duration of Experiment:

(The appropriate protocols form for the research proposal - Part B in the case ofexperiments using animals other than non human primates, part C for use of non-

human primates to be duly filled in, signed and appended to this form)

Signature

Name and Designation of Chief Investigator

Date

Place

________________________________________________________________________


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*Applicable only for application to be submitted to CPCSEA

Part B

Protocol form for research proposal to be submitted to the Committee/Institutional

Animal Ethics Committee, for new experiments or extensions of ongoing

experiments using animals other than non-human primates.

1. Project Title: Pathobiological and Immunological Studies on

Chlorpyrifos Toxicity and its Interaction withSalmonella infection in Broiler Chickens

2. Chief Investigator:

a. Name: Dr. R.P. Gupta

b. Designation: Professor

c. Deptt./Div./Lab.: Veterinary Pathology

d. Telephone number: 289254

3. List of names of all individuals authorized to conduct procedures under this

proposal

1. Dr. R.P. Gupta

2. Dr. Vikas Nehra (Ph,D. student)3. Dr. S.K. Mishra

4. Funding Source: State Scheme Plan

5. Duration of the project:

a. Number of months: Approx. 2-3 months

b. Date of initiation: In March, 2009 or just after getting permission

c. Date of completion: October, 2009 (Including laboratory work)

d. 6. If date by which approval is needed is less than six weeks from dates of

submission, justification for the same:

Student wants to start at the earliest.

7. Study Objectives {The Aims of Study (and why they are important) to beexplained briefly using non-technical terms as far as possible}


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Pathobiological and Immunological Studies on Chlorpyrifos Toxicity and its

Interaction with Salmonella infection in Broiler Chickens. Haematological,

Biochemical, Immunological and Pathological studies will be undertaken.


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8. Animal required:

a. Species: Broiler chicken

b. Age/Weight/Size: Day-old

c. Gender: Either sex

c. Numbers to be used (Year-wise break-ups and total figures needed to be given):

260

d. Number of days each animal will be housed: Approx. 8-9 weeks

9. Rationale for animal usage:

a. Why is animal usage necessary for these studies?Use of birds is necessary to investigate Pathobiological and Immunological

Studies on Chlorpyrifos Toxicity and its Interaction with Salmonella infection in

Broiler Chickens.b. Why are the particular species selected required?

Organophosphate insecticides have been reported to cause immunosuppression in poultry and

thus their exposure may increase susceptibility to opportunistic organisms. So in vivo study on

broiler chickens is required.

c. Why is the estimated number of animals essential?

Plan of the work is enclosed which includes experimental design.

d. Similar experiments conducted in the past. If so, the number of animals used and results

obtained in brief

No

e. If yes, why new experiment is required? N.A.

f. Have similar experiment(s) been made by any other organization/agency? If so, their

results in your knowledge

In my knowledge, no.

10. Description of procedures to be used

(List and description of all invasive and potentially stressful non-invasive procedures that animals will be subjected to in the course ofthe experiment, indication of the frequency for all procedures where appropriate. The following specific issues are also to be addressed

when relevant-injections (substances, doses, sites and volumes), blood withdrawal (volumes and sites), radiation (dosage and

schedules), all anaesthetics and/or analgesics (dosage and routes), mechanical methods of restraint, animal identification methods,methods of non-survival surgical procedures and experimental endpoint criteria (required when pathological changes are expected to be

caused).

Tentative protocol for the research project is enclosed. Withdrawal of blood from heart

at regular intervals and sacrificing of 5 birds at each interval from each subgroup.


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11. Does the protocol prohibit use of anaesthetic or analgesic for the conduct of painful

procedures (any which cause more pain than that associated with routine injection, orblood withdrawal)?

If yes, explanation and justification: Yes, Anaesthetics/and analgesic drugs will

effect the results.

12. Will survival surgery be done? If yes, the following to be described: No

a. List and description of all such surgical procedures (including methods of asepsis):

N.A.

b. Names, qualifications and experience levels of operators:

N.A.

c. Description of post-operative care: N.A.

d. Justification if major survival surgery is to be performed more than once on a single

individual animal:

N.A.

13. Methods of disposal post-experimentation:

Rehabilitation/Euthanasia (in case of euthanasia, justification for not undertakingrehabilitation and drug dosage and route for anaesthesia, where appropriate, as well asmethods of carcass disposal):

Naturally dead and sacrificed birds will be disposed off properly away from

post-mortem room. Birds will be euthanized by dislocation of atlanto-occipital joint.

14. Animal transportation methods if extra-institutional transport is envisaged:

Through well ventilated card board boxes.

15. Use of hazardous agents (use of recombinant DNA-based agents or potential human pathogensrequires documented approval of the Institutional Biosafety Committee (IBC). For each category, the

agents and the biosafety level required, appropriate measures and the mode of disposal of

contaminated food, animal, wastes and carcasses must be identified).

No


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e. Any other (give name): -

Copy of IBC approval to be attached in case hazardous agents are used.

d. Recombinant DNA: -

Investigators declaration1. I certify that I have determined that the research proposal herein is not unnecessarily duplicative of

previously reported research.

2. I certify that all individuals working on this proposal, and experimenting on the animals, have been

trained in animal handling procedures.

3. For procedures listed a. Radionuclides: -

b. Biological Agents: -

c. Hazardous chemicals or drugs: -

under item 11, I certify that I have reviewed the pertinent scientific literature and have found no valid

alternative to any procedure described herein which may cause less pain or distress.4. I will obtain approval from the IAEC/CPCSEA before initiating any significant changes in this study.

5. Certified that performance of experiment will be initiated only upon review and approval of scientific

intent by appropriate expert body {institutional scientific Advisory Committee/funding agency/other

body (to be named)}.

6. Institutional Biosafety Committees (IBC) certification of review and concurrence will be taken(Required for studies utilizing DNA agents of human pathogens).

7. I shall maintain all the records as per form (Form D).

Signature

Date: Name of Investigator

______________________________________________________________________________________

(for IAEC/CPCSEA usage)Proposal number

Date first received

Date received after modification (if any)

Date received after second modification (if any)

Approval date

Expiry date

Name of IAEC/CPCSEA Chairperson

Date Signature


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Department of Veterinary Pathology

6. Title of the Project: Pathobiological and Immunological Studies onChlorpyrifos Toxicity and its Interaction with

Salmonella infection in Broiler Chickens.

TECHNICAL PROGRAMME OF WORK

Experimental birds:

Two hundred and sixty, day old broiler chicks will be reared in the departmental animal

house under strict hygienic conditions providing feed and waterad libitum. From this, thirty birdswill be used for calculation of absolute lethal dose (ALD50).

Determination of ALD50:

A pilot experiment on thirty birds will be conducted to calculate absolute lethal dose

(ALD50) of chlorpyrifos as per the method of Reed and Muench (1938).

Experimental design:

Two hundred and thirty (230) broiler chicks at the age of 14 days will be dividedrandomly into three groups (group A, B and C) of 80, 80 and 70 chicks respectively in each

group. All the birds of group A will be given feed mixed with 1/10th of ALD50 of Chlorpyrifos and

that of group B will be given feed mixed with 1/20th of ALD50 of Chlorpyrifos. Each bird of

group C will receive plain feed. At the age of 21 days these three groups will be further divided

into two subgroups (group A into A1 and A2, group B into B1 and B2 and group C into C1 and C2)

of 40 birds each in subgroup A1, A2, B1, B2 and C2, group C1 will be having 30 birds. Each bird of

group A2, B2 and C2 will be injected with infective dose (ID50)ofSalmonella infection. Blood andSerum samples will be collected from five birds of each group on 0th, 3rd, 7th, 14th, 21st and 28th day

post infection for haematological, biochemical and immunological studies. After collection of

blood and serum, these birds will be sacrificed. They will be subjected to thorough post-mortem

examination. Representative pieces of liver, heart, lungs, brain, kidneys, intestine, spleen, thymusand Bursa of Fabricius will be collected in 10 per cent buffered formalin for histopathological

studies.

Observations:

1. Body weight gain: Birds will be weighed on days 14, 17, 21, 28, 35, and 42 of theage.

2. Clinical signs and mortality: Birds will be closely observed daily for clinical signsand mortality, if any.

3. Haematological studies: Blood samples collected at different intervals will be

analysed for the following parameters.a. Haemoglobin concentration (Hb).

b. Packed cell volume (PCV).

c. Total erythrocyte count (TEC).

d. Total leucocyte count (TLC).

e. Differential leucocyte count (DLC).

f. Mean corpuscular volume (MCV) and Mean corpuscular haemoglobinconcentration (MCHC).


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4. Biochemical studies: Serum samples collected at different intervals will be analysedfor the following parameters.

a. Total protein and Albumin concentrations.

b. Serum sodium and potassium concentration.

c. Serum Aspartate transaminase activity.d. Serum Alanine transaminase activity.

e. Serum Creatine phosphokinase activity.f. Serum Alkaline phosphatase activity.g. Serum Acetylcholine esterase activity.

5. Viable bacterial cell count in Salmonella infection: Viable bacterial cells will becounted in broiler chicken at 0, 3, 7, 14, 21 & 28 days post infection.

6. Pathological studies

a. Gross changes: Gross lesions, if any, will be recorded during post mortem

examination of the birds sacrificed or died naturally.b. Histopathological changes: The formalin fixed tissues will be processed for

paraffin embedding technique. The sections will be stained with haematoxylin

and eosin for histopathological interpretation (Luna, 1968).

7. Immunological studies: Blood serum will be collected at 0, 3, 7, 14, 21 & 28 dayspost infection from at least 5 birds and immune response will be studied as follows:-

A. Study of humoral mediated immune response (HMI):- Humoral immune response

will be studied against Salmonella infection treated with chlorpyrifos by indirectELISA.

B. Study of cell mediated immune response (CMI):-

a. The CMI will be studied by lymphocyte proliferation assay at each of the six timeintervals as in humoral immune response. The birds will be bled and their

lymphocyte will be separated by Ficoll-Hypaque. CMI will be studied forSalmonellainfection treated with chlorpyrifos and mitogen induced lymphoproliferation

response by MTT based lymphoproliferative assay.

b. The population of different T-cell markers i.e. CD 4+, CD8+ will be analyzed usingmonoclonal antibodies (mAbs) against these T-cell markers by flow

cytometry/immunoperoxidase technique as per the method of Sharma et al. (1990).

Ethical committee form

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