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Slide 1 - UMC - IACUC


									IACUC Animal Protocol
 Writing Workshop 101
   How to Write an Effective and
 Successful Animal Activity Protocol:
         Hands On Training
            A Good Sources of Information
   Working with the IACUC: Writing an Animal Protocol
    AALAS Publication: American Association for Laboratory Animal Science “Bookstore” link

   How to Write an Application Involving Research Animals

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

    ILAR Publication: Institute for Laboratory Animal Research
                 Protocol Form

6 sections
   General information
   Number of animals
   Surgical and non-surgical procedures
   Alternative considerations and literature search
   Statement of Assurances
   IACUC actions
 1.           Principal Investigator

Name           Susan Warren, Ph.D.
                 Associate Professor

            Must be full-time UMC faculty
              (Instructor – Professor)
                        Other Personnel
Name        Eddie Perkins           Name
Title        grad student           Title         Post Doc
Dept         Anatomy/Neurosurgery   Dept
Ext/pager    X4-1671                Ext/pager
email                               email

Name          Olga Golanov, MD      Name
Title        research tech          Title       undergraduate student
Dept         Anatomy                Dept
Ext/pager     X4-1662               Ext/pager
email                               email

 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.
      Laboratory Personnel Training

Have all the individuals listed above submitted their
completed training requirements registration form?
Yes    No

If no, you must complete
the IACUC Mandated online training requirement
And complete the IACUC Training Registration Form
              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
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 years
        Needs 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)?
  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?
  YesNo      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

Survival 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
Species     Strain/      Sex         Source      Total for   Average
            Stock                                Project     daily census
rat         SD           male                    213         13

mouse       C57BL/6      female                  113         21

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


•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
                                                                Yes       No     Pending

A   Chemical toxins in bedding/cages/carcasses?

    Reviewed by Risk Mgmt?

B   Radioisotopes in bedding/cages/carcasses?

    Reviewed by Radiation Safety?

C   Infectious agents or recombinant DNA usage?

    Reviewed by Biohazards Committee?

        If YES, provide specific details of specialized animal husbandry, care,
        cleaning, or decontamination procedures, especially identifying responsible
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
                 Standard   Non Standard

Feeding                     High/low sodium
                            chow; beef fat

Watering                    sweetener/
Caging/Housing              Metabolic cages/
                            single housing
                            following surgical
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 water
  limited quantities of water
  feeding specialized diets (e.g., high fat, High/Low
  feeding specialized amounts
  “unconventional” housing (metabolic cage, single
  behavioral manipulation of environment (e.g.,
     changes in light-dark cycle)
13.                           Housing

Will animals be housed outside of the LAF for greater than 12 hours?
 Yes    Where?
Note:    If yes, provide complete explanation and justification for any 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
14. Objectives in lay terminology
In non-technical/lay terminology, what is the
objective of the experiments proposed in this Animal
Activity Protocol? (i.e. Response should be written in
non-scientific language, as though explaining the study
to a high school student.

Why   are the experiments proposed?

What   knowledge do you hope to achieve?

Generally,single sentence explanations for these
types of questions will suffice
14. Objectives in lay terminology
Important to write response in non-technical, lay
language. Imagine this response presented to a local
news media. Ideally, this response will highlight a
particular human/animal problem that the study
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 rationale for using the particular
animal species noted in #9?
  This question seeks to understand why a particular animal is
noted in #9. Literature searches provide numerous examples of
appropriate animal models for specific types of studies. This
response should support the animal species noted in #9. Cost
alone should not be a reason for choosing a particular animal
B. What is the rationale for using animals rather
than using non-animal models?
This question seeks to understand if all in vitro, inanimate models
have been considered. In many cases, in vitro methodologies may
exist that could support the study, yet will not provide the level of
understanding required to meet the objectives. If in vitro methods
are being used in conjunction with animal, state that also.
16.          Brief Outline
Provide a general description of the animal
procedures included in the experimental design.

Brieflyoutline the proposed animal manipulations
and provide a time-line of events.

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

Verbatim descriptions from a grant submission
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

                              2 weeks      Surgical
                     Diet 1
  6 week                                 manipulations
old rabbits                                  4 weeks
                    Diet 2    2 weeks
                                           sample              Sacrifice
                  Example Answer

   In this response, #16 would include information about the diet,
    especially if potential nutrient deficiencies were anticipated. General
    information about the surgical procedure would be included.
    Likewise, any information about urine/fecal collection methods
    would need to be a part of the response (metabolic cage,
    catheterization, litter pan retrieval, etc).
   A probable time-line might be:
   Experimental diet (4 weeks) →Telemetry implant (1 day) → Monitor
    (4 weeks) → behavioral test (forced swim) → euthanasia
   Additionally, the following appendices must be included:
         Appendix C [Surgery]
         Appendix D [Biological samples]
         Appendix F [Administration Test Compound (diet)]
Appendix A         Environmental Enhancement/Enrichment
Appendix B         Breeding Programs
Appendix C         Surgery & Management of Surgical Pain & Distress
Appendix D         Collection of Biological Samples
Appendix E         Antibody Production
Appendix F         Administration of Drugs/Test Compounds
Appendix G         Prolonged Physical Restraint
Appendix H         Multiple Major Surgical Procedures
Appendix I         Food and /or Fluid Restriction
Appendix J         Animal Pain and/or Distress
Appendix K         Progress Report
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
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?
Note: Secondary methods are recommended to ensure
death. (Consult Section VII of the LAF Training and
Procedural Manual for appropriate methods of
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?

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

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.
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
Appendix A: Environmental Enhancement/

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

Describe the above techniques

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

Justification for exemption          YES    NO

If YES, provide complete justification for this exemption
       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.

1. Description
Provide 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

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

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

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

7. Final Disposition of Offspring
What is the final disposition of any offspring not utilized in the experimental
                   Appendix C
     Surgery & Management of Surgical Pain and
1.   A Complete description of surgical procedure

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

Surgical approach               A midline incision will extend from brow ridge to the
                                occipital protuberance. The skin and underlying muscle will
                                be reflected. A bilateral craniotomy will be made 3 mm from
                                the midline using a hand-held drill equipped with a ¼” burr.
Any unique or special            An ICV cannula, having an internal diameter of 0.25 mm,
techniques                      will be implanted via a craniotomy into the underlying
                                lateral ventricle and cemented to the skull using dental
                                acrylic and stainless steel screws.
Wound closure method, suture     Wound will be closed in layers. Vicryl suture will be used
materials, and suture removal   for muscle. Silk sutures will be used for skin. Skin sutures
plan                            will be removed 7-10 days post-operatively
        Appendix C Item 2A: Formulary
                 Agent              Dose      Route      Frequency/Duration
Pre-anesthetic   ketamine HCL                   im        once for cage
                 Atropine sulfate                        removal
Anesthetics      sodium                        iv,        1 time for
                 pentobarbital                inhalant   induction; 2-5
                 Isoflurane                              hours
Analgesics       Sensorcaine                  im, sub q 1x for 2-3 days;
                 Carprofen                              1x every 12 hours

Fluid/blood      5% lactated
replacement      ringers
                Appendix C: Anesthesia

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

2c. Anesthesia criteria
What 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/training
Describe 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 procedures
Where 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 Anesthesia

   Respiration rate
   Heart rate
   ECG
   Toe pinch
   Tail pinch
   Corneal reflexes
   Color of mucous membranes
   Muscle relaxation
   Other
       Pulse oximeter
       respirometer
        Appendix C: Post-operative Care and

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 responsible
Indicate 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
      Appendix D: Collection of Biological Samples
                 from the Live Animal

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

       Agent                     Dose                       Route                     Frequency/Duration
  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

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

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
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).

  1. Provide the Following Information

  Agent                  Dose                  Volume                Vehicle                Route          Frequency

     WGA-HRP             20% solution          0.01-0.05 ul             sterile water                         one time

     BDA                  4% solution            0.1-0.5 ul             sterile water                         one time



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.
         Appendix G: Prolonged Physical Restraint

Justify the need for prolonged physical restraint.
Why you need to employ restraint – safety of the animal during procedure;

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/day
Long 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 H: Multiple Major Survival Surgical

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

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

                    FREQENCY OF CHECKS
Body weight
Urine output
Fecal output
Food intake
     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
       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     No
        If NO, explain and justify
       Appendix J: Animal Pain and/or Distress

1.   Justify the scientific need to withhold appropriate drugs or induce the

2.   What is the duration of time that an animal may experience this

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
 Appendix J: Animal Pain and/or 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)?
              Criteria used to Assess
   Loss of appetite
   Loss of weight
   Restlessness
   Abnormal resting posture
   Licking, biting, scratching, shaking
   Failure to show normal patterns of inquisitiveness
   Failure to groom
   Guarding
              Criteria used to Assess

   Loss of mobility
   Red stain around eyes (rats)
   Unresponsiveness
   Self-mutilation
   Labored breathing
   Other
   Appendix K: Progress Report
This appendix must accompany every 3 year
              Full Submission

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