IACUC Animal IACUC Animal Protocol Protocol

Writing Workshop Writing Workshop 101101How to Write an Effective How to Write an Effective

and Successful Animal and Successful Animal Activity Protocol: Hands On Activity Protocol: Hands On

TrainingTraining

A Good Sources of InformationA Good Sources of Information

Working with the IACUC: Writing an Animal ProtocolWorking with the IACUC: Writing an Animal ProtocolAALAS Publication: American Association for Laboratory AALAS Publication: American Association for Laboratory Animal ScienceAnimal Sciencewww.aalas.orgwww.aalas.org “Bookstore” link “Bookstore” link

How to Write an Application Involving Research AnimalsHow to Write an Application Involving Research Animalswww.niaid.nih.gov/ncn/clinical/researchanimals/tutorial/www.niaid.nih.gov/ncn/clinical/researchanimals/tutorial/index.htmindex.htm

Guidelines for the Care and Use of Mammals in Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral ResearchNeuroscience and Behavioral Research

ILAR Publication: ILAR Publication: IInstitute for nstitute for LLaboratory aboratory AAnimal nimal RResearchesearchwww.national-academies.org/ilarwww.national-academies.org/ilar

Protocol FormProtocol Form

6 sections6 sections General informationGeneral information Number of animalsNumber of animals Surgical and non-surgical Surgical and non-surgical

proceduresprocedures Alternative considerations and Alternative considerations and

literature searchliterature search Statement of AssurancesStatement of Assurances IACUC actionsIACUC actions

1. Principal Investigator

NameName   Susan Warren, Ph.D.Susan Warren, Ph.D.TitleTitle           Associate ProfessorAssociate ProfessorDeptDept           AnatomyAnatomyExt/pager Ext/pager

          X4-1671X4-1671emailemail

          swarren@anatomy.umsmed.eduswarren@anatomy.umsmed.edu

Must be full-time UMC faculty (Instructor – Professor)

Other Personnel

NameName Eddie Perkins     Eddie Perkins      NameName           

TitleTitle   grad student    grad student     TitleTitle           Post DocPost Doc

DeptDept     Anatomy/NeurosurgeryAnatomy/Neurosurgery DeptDept           

Ext/pager Ext/pager   X4-1671    X4-1671     Ext/pager Ext/pager           

emailemail    emailemail           

NameName         Olga Golanov, MDOlga Golanov, MD NameName           

TitleTitle     research tech   research tech    TitleTitle   undergraduate student    undergraduate student    

DeptDept   Anatomy    Anatomy     DeptDept           

Ext/pager Ext/pager       X4-1662  X4-1662   Ext/pager Ext/pager           

emailemail            emailemail           

Identify all persons associated with the animal work. This information assists the LAF and IACUC in contacting knowledgeable persons to answer questions/concerns relative to the animal experimentation.

For access to the animal facility, all personnel working on this protocol must be listed.

Have Have all all the the individualsindividuals listed above listed above submitted their completed training submitted their completed training requirements registration form?requirements registration form?

YesYes No No

If noIf no, you , you must completemust complete the IACUC Mandated online training the IACUC Mandated online training

requirement (requirement (www.researchtraining.orgwww.researchtraining.org))And complete the IACUC Training And complete the IACUC Training

Registration FormRegistration Form

Laboratory Personnel Training Certification

Project Title

          

This title will be used in the IACUC database to identify the project. (may be the same as the grant title)

The title should briefly state procedures to be carried out and the animal species to be used.

Project Title - Examples

          

Tissue Harvest in Rats

Health Monitoring of Guinea Pigs

Production of Transgenic and Gene Knockout Mice

Evaluation of Drugs for Osteoporosis in Squirrel Monkeys

Breeding protocol for AXt-1a knockout mice

4. Proposal is

New - designation of a de novo submission

Revision of Existing Protocol - designation of amendments/revisions to the protocol. Any “revised” information should be easily identified [highlighted, bold, italics, colored font]

3 year Full Submission designation of a previously approved protocol

that must be re-written after three yearsNeeds to include Appendix K

5. Outside Contracts?

Will any “outside” contracts be used in this study that involve live animals (antibody production, toxicology screening, subcontracts with other facilities, etc)? No Yes (provide information on the level of involvement)Mark YES if any non-UMC entity will be involved in any animal care and use activities. Examples include housing your animals at another institution for specialized procedures, use of a contract toxicology research lab, antibody production techniques by another lab, etc.     

6. Funding Source

This question aids the IACUC and LAF in allocation of resources.

In times of lean animal housing space, the LAF will further give top priority to those extramurally-supported protocols.

Generally, the IACUC does not attempt to provide scientific review of a protocol, yet is obligated to provide some level of scientific review for those departmentally-funded protocols lacking any peer review.

6. Funding Source

Title:  Grant Application Title    Grant Funding Agency: NIH, NSF, American

Heart, etc    Covered Dates: 11-01-04 thru 1-31-07

Departmental Funds    Has this proposal received any peer review? Yes No Pending

7. Procedure Category

Acute (anesthesia without recovery or euthanasia)Acute procedures not only refer to an anesthesia without recovery, but also those studies where no advance forms of animal manipulations have occurred.

Examples include animal sacrifice tissue collection terminal surgical procedure

7. Procedure Category

SurvivalSurvival procedures not only include survival surgical procedures but any animal manipulations that may be conducted on the animal.

Examples include any type of dosing (oral, feeding, injections,

topical, gavage, etc) sample collection (blood, urine, CSF) behavioral experimentation (swim test, radial arm maze, single unit recording)

8. Dates of Study

A. Anticipated start date of study:  2-23-04  thru 1-31-07

B. Study Duration (maximum 3 years): 2 years

Response assists the LAF in planning/coordinating space for the study.

9. Animal Requirements

List all animals required to complete the study. Animal numbers must be calculated for a period of three years. Complete all columns of the chart.

If using nonhuman primates, must complete Appendix A.

If using time pregnant animals, must complete Appendix B. If using animals from an in-house breeding program, must complete Appendix B.

SpeciesSpecies Strain/Strain/

StockStockSexSex SourceSource Total for Total for

ProjectProjectAverage Average daily daily censuscensus

ratrat SDSD malemale 213213 1313

mousemouse C57BL/6C57BL/6 femalefemale 113113 2121

10. Breeding Program

Will animals be involved in a breeding program at UMMC or will time-pregnant animals be used?

•No

•Yes (provide information in Appendix B)Mark YES for any study proposal involving animal mating or for proposals utilizing time-pregnant animals. Must also include Appendix B.

11. Potential Hazards

YesYes NoNo PendingPending

AA Chemical toxins in Chemical toxins in bedding/cages/carcasses?bedding/cages/carcasses?

Reviewed by Risk Mgmt?Reviewed by Risk Mgmt?

BB Radioisotopes in Radioisotopes in bedding/cages/carcasses?bedding/cages/carcasses?

Reviewed by Radiation Safety?Reviewed by Radiation Safety?

CC Infectious agents or recombinant DNA Infectious agents or recombinant DNA usage?usage?

Reviewed by Biohazards Committee?Reviewed by Biohazards Committee?

If If YESYES, provide specific details of , provide specific details of specialized animal specialized animal husbandry, care, cleaning, or decontamination husbandry, care, cleaning, or decontamination proceduresprocedures, especially identifying responsible parties., especially identifying responsible parties.

          

11. Potential Hazards

Specific check boxes are included for chemical, radioisotopes, and/or infectious agent exposures in research animals. Boxes should be checked where appropriate and the proposal must be reviewed by the responsible oversight group. IMPORTANTLY, any YES answers must also include specific details of any specialized animal care procedures to be used, paying particular attention to occupational safety of animal caretakers. The explanation should state type of exposures, personal protective equipment to wear, decontamination procedures, and any other relevant information.

12. Animal Husbandry

StandardStandard Non StandardNon Standard

FeedingFeeding High/low High/low sodium chow; sodium chow; beef fat beef fat

WateringWatering sweetener/ sweetener/ alcohol/water alcohol/water restrictionrestriction

Caging/HousingCaging/Housing Metabolic Metabolic cages/ single cages/ single housing housing following following surgical surgical implantsimplants

12. Animal Husbandry

Standard: Standard methods for UMC include

• ad lib access to water• LAF feeding of commercial diets• conventional housing

12. Animal Husbandry

Nonstandard: Examples include

providing limited access to waterlimited quantities of waterfeeding specialized diets (e.g., high fat, High/Low

sodium)feeding specialized amounts“unconventional” housing (metabolic cage, single

housing)behavioral manipulation of environment (e.g.,

changes in light-dark cycle)

13. Housing

Will animals be housed outside of the LAF for greater Will animals be housed outside of the LAF for greater than 12 hours? than 12 hours?

NoNo

Yes Where?     Yes Where?     

Note:Note: If yes, provide complete explanation and If yes, provide complete explanation and justification for any justification for any de-centralized animal housing.de-centralized animal housing.          

Mark YES for any proposal that requires an animal to be maintained in a laboratory/testing location in excess of 12 hours. Any location maintaining animals for greater than 12 hours will be considered as a housing location and must be maintained in accordance with the USDA, OLAW, and AAALAC. The IACUC will need to provide approval for such sites.

14. Objectives in lay terminology

In In non-technicalnon-technical/lay terminology/lay terminology, what is the , what is the objective of the experiments proposed in this Animal proposed in this Animal Activity Protocol? (i.e. Response should be written in Activity Protocol? (i.e. Response should be written in non-scientific language, as though explaining the study non-scientific language, as though explaining the study to a high school student.to a high school student.

Why Why are the experiments proposed?are the experiments proposed?

What knowledgeWhat knowledge do you hope to achieve? do you hope to achieve?

Generally, Generally, single sentence explanationssingle sentence explanations for these for these types of questions will sufficetypes of questions will suffice

14. Objectives in lay terminology

Important to write response in Important to write response in non-technical,non-technical, lay lay language. Imagine this response presented to a local language. Imagine this response presented to a local news media. Ideally, this response will news media. Ideally, this response will highlight a highlight a particular human/animal problem that the study particular human/animal problem that the study seeks to investigate.seeks to investigate.

•How will the use of animals help you investigate this particular problem?

•What answers do you anticipate from the proposal?

15. Rationale

A. What is the A. What is the rationale for using the particular rationale for using the particular animal speciesanimal species noted in #9? noted in #9?

    This question seeks to understand why a particular animal is noted This question seeks to understand why a particular animal is noted in #9. Literature searches provide numerous examples of in #9. Literature searches provide numerous examples of appropriate animal models for specific types of studies. This appropriate animal models for specific types of studies. This response should support the animal species noted in #9. Cost response should support the animal species noted in #9. Cost alone should not be a reason for choosing a particular animal alone should not be a reason for choosing a particular animal species.species.

B. B. What is the What is the rationale for using animals rather rationale for using animals rather than using non-animal modelsthan using non-animal models? ? This question seeks to understand if all This question seeks to understand if all in vitroin vitro, inanimate models , inanimate models have been considered. In many cases, have been considered. In many cases, in vitroin vitro methodologies may methodologies may exist that could support the study, yet will not provide the level of exist that could support the study, yet will not provide the level of understanding required to meet the objectives. If in vitro methods understanding required to meet the objectives. If in vitro methods are being used in conjunction with animal, state that also. are being used in conjunction with animal, state that also.           

16. Brief Outline

Provide a Provide a general descriptiongeneral description of the animal of the animal procedures included in the experimental design. procedures included in the experimental design.

Briefly outlineBriefly outline the proposed animal manipulations the proposed animal manipulations and provide a time-line of events. and provide a time-line of events.

Note that specific details about methods and Note that specific details about methods and procedures will be required in the appropriate procedures will be required in the appropriate appendix (see list below)appendix (see list below)

Verbatim descriptions from a grant submission Verbatim descriptions from a grant submission are not acceptable and will not be reviewed. are not acceptable and will not be reviewed.

16. Brief Outline

Appropriate responses to this question will briefly outline all animal manipulations. This question also contains a check list of related appendices (A through K). Investigators will note that many specific details of surgical procedures, sample collection, administration of test compounds, antibody production, etc will necessitate completion of the appropriate appendices.

An important consideration for inclusion is a time-line of events. This time-line helps the IACUC clarify the proposed work.

16. Brief Outline

For example, a study seeks to feed an experimental diet to rabbits then surgically instrument each animal with telemetry devices. Following the surgery, the animals will be monitored for 4 weeks and intermittent urine/fecal samples will be collected.

6 week old rabbits

Diet 1

Diet 2

Surgical manipulations

Urine/fecal sample Sacrifice

2 weeks

2 weeks

4 weeks

Example AnswerExample Answer

In this response, #16 would include information about the diet, In this response, #16 would include information about the diet, especially if potential nutrient deficiencies were anticipated. especially if potential nutrient deficiencies were anticipated. General information about the surgical procedure would be General information about the surgical procedure would be included. Likewise, any information about urine/fecal collection included. Likewise, any information about urine/fecal collection methods would need to be a part of the response (metabolic cage, methods would need to be a part of the response (metabolic cage, catheterization, litter pan retrieval, etc). catheterization, litter pan retrieval, etc).

A probable time-line might be:A probable time-line might be: Experimental diet (4 weeks) →Telemetry implant (1 day) → Monitor Experimental diet (4 weeks) →Telemetry implant (1 day) → Monitor

(4 weeks) → behavioral test (forced swim) → euthanasia(4 weeks) → behavioral test (forced swim) → euthanasia Additionally, the following appendices must be included:Additionally, the following appendices must be included: Appendix C [Surgery]Appendix C [Surgery] Appendix D [Biological samples]Appendix D [Biological samples] Appendix F [Administration Test Compound (diet)] Appendix F [Administration Test Compound (diet)]

Appendix AAppendix A Environmental Enhancement/EnrichmentEnvironmental Enhancement/Enrichment

Appendix BAppendix B Breeding ProgramsBreeding Programs

Appendix CAppendix C Surgery & Management of Surgical Pain & DistressSurgery & Management of Surgical Pain & Distress

Appendix DAppendix D Collection of Biological SamplesCollection of Biological Samples

Appendix EAppendix E Antibody ProductionAntibody Production

Appendix FAppendix F Administration of Drugs/Test CompoundsAdministration of Drugs/Test Compounds

Appendix GAppendix G Prolonged Physical RestraintProlonged Physical Restraint

Appendix HAppendix H Multiple Major Surgical ProceduresMultiple Major Surgical Procedures

Appendix IAppendix I Food and /or Fluid RestrictionFood and /or Fluid Restriction

Appendix JAppendix J Animal Pain and/or DistressAnimal Pain and/or Distress

Appendix KAppendix K Progress ReportProgress Report

Appendices

Check and complete only those appendices that apply to the animal manipulations in your experimental design.

17. Justification of Animal Number

The total number of animals needed to complete item #16 should be explained in this response. Likewise, this number should match the requested number provided in #9.

It is often beneficial to include a flow diagram or chart/table that defines the animal use.

Break numbers down into number of animals/group, number of groups, number of replicates, etc.

Any animals needed for pilot work or training new technical personnel should be accounted for.

Responses should have some statistical merit.

18. Location and Transportation

          A. Indicate room(s) where animal procedures will be conducted.

This response is noted in the IACUC database as the experimental area.

Semi-annual program reviews will include this location into subsequent inspections.

Likewise, affirmative answers to #13 (Housing) should also be listed in this response.

18. Location and Transportation

          B. Studies involving animal transportation to locations other than the housing area must identify the animal transport device, the nature of shrouds used to cover the transport device, and describe the route of transport

Numerous laboratories are required to remove their animals from the LAF environment to their respective labs. The method and route of transport should be courteous to Medical Center visitors/patients, conscious of occupational exposures, and respectful of those opposing the use of animals in research. All animal movement must occur in appropriate restraint boxes/cages and with a concealment shroud/drape. Consult LAF SOPs for descriptions of routes of appropriate animal transportation.

19A. Euthanasia

A. At what point in the proposed experiments will animals normally be euthanized (experimental end-points)? Or at what point will any individual animal be euthanized?

This response would correlate with the experimental end points described in #16. The question seeks to clarify the IACUC’s understanding of when any particular animal will conclude the study.

19B. Euthanasia

B. What criteria will be used to determine if an animal is to be euthanized prior to, rather than at, the anticipated end-point of an experiment? Note: Contact LAF, ext.4-1385, for recommendations on the assessment criteria.

The appropriate response will articulate potential adverse consequences or effects of the experiment. Investigators should list potential surgical complications, adverse effects of drug/diet administration, or failure to manage pain and distress. Specific, objective criteria may also be incorporated (e.g., 20% loss in body weight, hematocrit falls below 25%, creatinine levels above 2.5 mg/dl, etc) to note when an animal is removed from the study.

19C. Euthanasia

          

C. If proposing a natural death (or death due to manipulations), explain and justify. This type of scenario is described as “death as an endpoint”. These studies must firmly justify the scientific need to allow an animal to advance through a potentially painful/distressful terminal event. Unless information to the contrary is known, it is assumed that all “death as an endpoint” studies will result in some level of pain and/or distress to the animal while in the moribund state. Ideally, some definitive, objective criteria can be used to “measure” when an animal can be removed. For example, in a model of renal failure, perhaps BUN levels >100 coupled with Cr values >3.5 are indicative of end-stage kidney disease and will provide the necessary endpoints. Likewise, in an oncology study, perhaps tumor growth beyond a specific size could be used.

20. Euthanasia Procedures

What procedures will be used to euthanize the animals? What procedures will be used to euthanize the animals? Note: Note: Secondary methodsSecondary methods are recommended to ensure are recommended to ensure death. death. (Consult Section VII of the (Consult Section VII of the LAF Training and LAF Training and Procedural ManualProcedural Manual for appropriate methods of for appropriate methods of euthanasia.)euthanasia.)

Procedures must be consistent with the 2000 Report of the AVMA Panel on Euthanasia [copy located in the LAF Training & Procedural Manual]. Likewise, secondary methods should be proposed to ensure and/or guarantee death. Secondary methods are typically those involving exsanguination, pneumothorax, or tissue harvest.

Institutional requirement for all persons to be registered and known by the health services organization.

Have all personnel received a medical evaluation from UMC Student/Employee Health?

Have all personnel become familiar with the LAF Training & Procedural Manual?

Assurances

The LAF Training and Procedural Manual is jointly produced by the LAF and the IACUC. The manual is designed to answer many commonly asked questions and address particular areas related to animal-based research programs at UMC.

Each Principal Investigator is issued a copy of the manual. The manual should be available to all research personnel in the laboratory in an effort to foster a better understanding of animal care and use programs.

Assurances

To comply with the USDA’s Animal Welfare Act [9 CFR, part 2, Section 2.31 (d)(1)(ii) and (iii)] you must complete a written NARRATIVE of the sources you consulted to determine whether or not alternatives exist to procedures that may cause pain and distress. Likewise, this search will ensure that the proposed studies are not unnecessarily duplicative.

Assurances

Review of the available resources and previous experiments have determined that the proposed activity is not unnecessarily duplicative of previously reported activities?

The question seeks to prompt the investigator to conduct a literature review and assure the IACUC that the proposal does not duplicate existing work.

Assurances

Appendix A: Environmental Enhancement/ Enrichment

          

Enrichment Techniques

Are there any enrichment forms/techniques that are included in this protocol? YES NO

Description

Describe the above techniques

No Enrichment?

Are there any forms of enrichment/enhancement that should not be used in this study? YES NO

Justification for exemption YES NO

If YES, provide complete justification for this exemption

          

          1. DescriptionProvide a specific description of the type of breeding program to be utilized (harem, monogamous pair, etc).

2. Personnel Responsible Identify personnel responsible for the breeding program.

3. Records Identify personnel responsible for maintaining breeding program records.

Appendix B: Breeding Programs

Complete Appendix B for all proposals planning on establishing a breeding colony or for those studies utilizing time-pregnant animals. Studies incorporating breeding programs or offspring from time-pregnant animals will be required to report annual production (number of offspring used) at the time of IACUC protocol annual renewal.

Appendix B: Breeding Programs

4. AdultsHow many adults will be utilized in this breeding program?

5. Final Disposition of AdultsWhat is the final disposition of these adults at the conclusion of their breeding program?

6. OffspringHow many offspring are anticipated from each breeding or time-pregnancy?

7. Final Disposition of OffspringWhat is the final disposition of any offspring not utilized in the experimental program?

Surgical site preparationSurgical site preparation    The surgical site will be shaved, cleansed with soap, The surgical site will be shaved, cleansed with soap, swabbed with alcohol and bathed in providine solution   swabbed with alcohol and bathed in providine solution   

Surgical approachSurgical approach A midline incision will extend from brow ridge to the A midline incision will extend from brow ridge to the occipital protuberance. The skin and underlying muscle will occipital protuberance. The skin and underlying muscle will be reflected. A bilateral craniotomy will be made 3 mm be reflected. A bilateral craniotomy will be made 3 mm from the midline using a hand-held drill equipped with a ¼” from the midline using a hand-held drill equipped with a ¼” burr. burr.

Any unique or special Any unique or special techniquestechniques

    An ICV cannula, having an internal diameter of 0.25 mm, An ICV cannula, having an internal diameter of 0.25 mm, will be implanted via a craniotomy into the underlying will be implanted via a craniotomy into the underlying lateral ventricle and cemented to the skull using dental lateral ventricle and cemented to the skull using dental acrylic and stainless steel screws.acrylic and stainless steel screws.

Wound closure method, suture Wound closure method, suture materials, and suture removal materials, and suture removal planplan

  Wound will be closed in layers. Vicryl suture will be used Wound will be closed in layers. Vicryl suture will be used for muscle. Silk sutures will be used for skin. Skin sutures for muscle. Silk sutures will be used for skin. Skin sutures will be removed 7-10 days post-operatively   will be removed 7-10 days post-operatively   

Appendix C Surgery & Management of Surgical Pain and

Distress

1. A Complete description of surgical procedure

AgentAgent DoseDose RouteRoute Frequency/DurationFrequency/Duration

Pre-anestheticPre-anesthetic ketamine HCL ketamine HCL Atropine sulfateAtropine sulfateAcepromazineAcepromazineXylazineXylazine

10mg/kg  10mg/kg           im im    once for cage once for cage removal    removal    

AnestheticsAnesthetics sodium sodium pentobarbitalpentobarbitalIsofluraneIsoflurane

             iv, iv, inhalantinhalant

    1 time for 1 time for induction; 2-5 induction; 2-5 hourshours

AnalgesicsAnalgesics SensorcaineSensorcaineCarprofenCarprofenbuprenorphine buprenorphine   Ibuprophin  Ibuprophin  

           im, sub qim, sub q 1x for 2-3 days;1x for 2-3 days;1x every 12 hours  1x every 12 hours  

Fluid/blood Fluid/blood replacementreplacement

5% lactated 5% lactated ringersringers

                                

AntibioticsAntibiotics                                            

Appendix C Item 2A: Formulary

Appendix C: Anesthesia

2b. Anesthesia Who will conduct the anesthesia procedure(s)?List Individual(s) by name

2c. Anesthesia criteriaWhat criteria will be used to assess anesthetic depth and how will this be monitored? toe pinch, reduced/absent corneal reflex, withdrawal reflex

3. Experience/training Describe the training/experience, including species of animals, of personnel conducting the anesthetic procedure?

List individual named in #2B and indicate level of training, number of years actively involved in procedure, and what species he/she has worked

Appendix C: Surgical Procedures

4. Surgical procedure Who will conduct the Surgical procedure(s)?List individual(s) by name

5. Experience/trainingDescribe the training/experience, including species of animals, of personnel conducting surgical procedures?

List individual named in #4 and indicate level of training, number of years actively involved in procedure, and what species he/she has worked.

6. Location of proceduresWhere will the surgical procedures be conducted?Indicate Room in which dedicated space for surgical procedures is located, e.g., LAF procedure room, R-717, N-412.

Criteria for Assessing Level of AnesthesiaCriteria for Assessing Level of Anesthesia

Respiration rateRespiration rate Heart rateHeart rate ECGECG Toe pinchToe pinch Tail pinchTail pinch Corneal reflexesCorneal reflexes Color of mucous membranesColor of mucous membranes Muscle relaxationMuscle relaxation OtherOther

Pulse oximeterPulse oximeter respirometerrespirometer

Appendix C: Post-operative Care and Emergencies

7. Experience/training Who will conduct and document post-procedural animal care? (post-op analgesia, nursing care, etc.) Include a plan of monitoring frequency, duration and intervals of post-op analgesia, nursing care, etc.?List Individual responsibleIndicate a plan for monitoring - twice daily for the first three days; three times per week for the duration of the recovery period

8. Emergency Contacts Emergency contact (pager/phone number) for evenings or weekends concerning post-operative complications.Provide a named individual(s) and appropriate phone/pager numbers

AgentAgent DoseDose RouteRoute Frequency/Frequency/DurationDuration

                                           

                                           

                                           

                                           

Biological samples include blood collection, urine collection, ascites, cerebrospinal fluid, biopsy, etc. Appendix D is completed for all sample collections from live animals (excludes sample collection at necropsy.

Indicate the body fluid or material to be collected.      

Indicate the method and site of collection.   Indicate the volume of fluid or material to be collected.      Indicate the frequency of collection.      Will the animal(s) be anesthetized or sedated during this procedure? YES NO

Appendix D: Collection of Biological Samples from the Live Animal

Appendix E: Antibody Formation / Tumor Induction / Hybridoma & Ascites

Indicate what antigen will be used.      Indicate what vehicle/adjuvant will be used:      initial immunization:     subsequent immunizations:     anticipated complications/side effects:     Indicate sites for immunization:      Indicate route of administration:      What is the total and per site injection volume?       What is the frequency/duration of immunization (e.g., 1 injection every 2 weeks for 3 injections)?      

Appendix E: Antibody Formation / Tumor Induction / Hybridoma & Ascites

 ASCITES PRODUCTIONFluid accumulation associated with ascites/hybridomas should not become greater than 10% of body weight. Animals should be euthanized if they become moribund.

Indicate the maximum volume of ascites fluid to be collected per sampling (ml/mouse) and the method of collection (skin prep, gauge needed, gravity vs suction, etc)      Indicate the number of fluid collections and anticipated frequency of collection.     Describe procedures used to care for and monitor the health of animals with ascites and the point of euthanasia.

Appendix F: Administration of Drugs/Test Compounds

AgentAgent DoseDose VolumeVolume VehicleVehicle RouteRoute FrequencyFrequency

          WGA-HRPWGA-HRP   20% solution20% solution 0.01-0.05 0.01-0.05 ul     ul     

          sterile sterile waterwater

                     one timeone time

          BDABDA       4% 4% solution  solution  

        0.1-0.5 ul 0.1-0.5 ul            sterile sterile waterwater

                     one timeone time

          BDNFBDNF                                                       

          MK801MK801                                                       

2. Describe any potential adverse side effects that may result from the administration of this material. If agents are unknown or their potential side effects are not documented, provide a reasonable estimate of the effects of the general class of chemicals (e.g., cmpd may have sedative properties, cmpd will likely produce diarrhea, etc). No adverse affect anticipated. We will consult with the veterinary staff should unanticipated problems arise.      3. What special procedures do animal care personnel and veterinary caregivers need to assume when working with these animals or their bedding/cages? These compounds pose no threat to the health and welfare of the animal care staff.      

1. Provide the Following Information

All agents listed in Animal Activity Protocol question #11 (Potential Hazards to personnel or other animals) must be listed in this section. Additionally, other experimental (Test) compounds used in the study should be noted. This section is not for the listing of veterinary pharmaceuticals (antibiotics, anesthetics, analgesics).

Justify the need for prolonged physical restraint. Why you need to employ restraint – safety of the animal during procedure; training      Describe the restraint device: Tether, harness, primate chair     Describe how the animal(s) will be adapted to the restraint device. Detail the time frame for adaptation to device; detail any rewards/encouragements used to achieve compliance     What is the duration of a restraint period? Daily – indicate the total numbers of hours/dayLong term - weeks/ months     

Appendix G: Prolonged Physical Restraint

Are animals monitored during the restraint period? Yes No If YES, describe who and document procedures How often?     

Are there any anticipated problems as a result of the restraint device (e.g., skin lesion from harness, moist dermatitis, etc) Indicate plan for monitoring and detail procedure for corrective measures

Appendix G: Prolonged Physical Restraint

A major surgical procedure is defined as a surgical intervention that penetrates or exposes a body cavity (peritoneal, thoracic, cranium) or produces substantial impairment of physical or physiologic function. Multiple major procedures are those whereby an animal will regain consciousness after each procedure. Procedures must be described in Appendix C. A major surgery followed by a second procedure where the animal is sacrificed is not considered in multiple major surgical procedures.

Justify the need for multiple major surgical events in a single animal. Need for sequential administration of tracer materials; need to instrument animal proir to manipulations     

What is the time interval between the surgical events? Day to weeks to months

Appendix H: Multiple Major Survival Surgical Procedures

METHODMETHODFREQENCY OF CHECKSFREQENCY OF CHECKS

Body weightBody weight           

Urine outputUrine output           

Fecal outputFecal output           

BUNBUN           

HctHct           

Food intakeFood intake           

Appendix I: Food and/or Fluid Restriction

1. Will FOOD or FLUIDS be restricted? Justify the need to restrict food and/or fluid.      2. Check all methods that will be used to ensure adequate nutritional intake and hydration

Appendix I: Food and/or Fluid Restriction

3. Restriction protocols typically base the restriction amount relative to a baseline, (free-choice consumption) parameter (body weight, intake amount). What will this restriction amount use as the baseline?   What is the maximum % restriction for any animal?     4. Growing animals must be frequently re-assessed to ensure normal

growth patterns. What provisions will be made for these animals to assure that their

nutritional needs are maintained?     5. The Animal Welfare Act states that if “water is not continually available, it must be

offered to them as often as necessary to ensure their health and well-being, but no less than twice daily for at least 1 hour each time, unless otherwise required by the

attending veterinarian”. Will animals be provided unrestricted access to water/fluids twice

daily for at least 1 hour each time? Yes NoIf NO, explain and justify

1. Justify the scientific need to withhold appropriate drugs or induce the pain/distress.     2. What is the duration of time that an animal may experience this pain/distress.     3. Describe non-pharmaceutical means to alleviate pain/distress (soft bedding,

social housing, supplemental heat, etc).      

4. Describe situations where an animal may be removed prematurely from a study.    

Appendix J: Animal Pain and/or Distress

NON-SURGICAL PAIN/DISTRESS

5. Describe those proposals whereby animals are likely to experience more than momentary pain or distress as a result

of manipulations or procedures (noxious injections, tumor growth, sequelae to compound administration, etc).      

6. Will any anesthetics, analgesics, or tranquilizing drugs be used to reduce this pain or distress?     

7. What non-pharmaceutical measures will be used to minimize discomfort, distress, pain, or suffering (e.g., fluids, supplemental heat, soft bedding, etc)?

Appendix J: Animal Pain and/or Distress

Criteria used to Assess Criteria used to Assess Pain/Distress/DiscomfortPain/Distress/Discomfort

Loss of appetiteLoss of appetite Loss of weightLoss of weight RestlessnessRestlessness Abnormal resting postureAbnormal resting posture Licking, biting, scratching, shakingLicking, biting, scratching, shaking Failure to show normal patterns of inquisitivenessFailure to show normal patterns of inquisitiveness Failure to groomFailure to groom GuardingGuarding

Criteria used to Assess Criteria used to Assess Pain/Distress/DiscomfortPain/Distress/Discomfort

Loss of mobilityLoss of mobility Red stain around eyes (rats)Red stain around eyes (rats) UnresponsivenessUnresponsiveness Self-mutilationSelf-mutilation Labored breathingLabored breathing OtherOther

Appendix K: Progress Report

This appendix must accompany every 3 year Full Submission