Instructions to Complete Animal Use Protocol
General Instructions
About the Form
The form is in Microsoft Word and is unprotected. We are experimenting with more sophisticated formats.
The idea with the form is to try to make information retrieval easy. You will notice that there are
hyperlinks throughout the form connecting with these instructions. The instructions also include examples
and suggestions. Where possible, hyperlinks to IACUC, USDA, or NIH policies are included. The IACUC
considers this form to be a work in progress and appreciates any comments you have about the format
and instructions. There are many new additions, most having to do with classification of pain and stress
levels. These sections were added to assist data retrieval for annual reports and to be compliant with
new animal use regulations.
Common Misconceptions:
Many principal investigators (PI’s) have the mistaken impression that this form is only intended to provide
animal use information to their TJU colleagues and Office of Animal Resources (OAR) and thus believe
that their expertise should be known and they should be trusted to “do the right thing”. The information on
the Animal Use Protocol (AUP) form is not just for TJU but will be read by outside agencies such as the
USDA and AAALAC-I who probably will not know who you are or know your reputation. This AUPl may
be accessed by the general public through the Freedom of Information Act. The information must be
understandable to the agency inspectors and all information directly related to animal care must be
included in the form making each AUP a “stand-alone” document. This is why journal format is
inappropriate. The AUP is not a grant submission and is not ranked on its scientific content but is
approved or not approved based on adherence to animal care and use principles and guidelines. Ideally,
the AUPl should be understandable by a person with an USA 8th grade education, at least in general. Not
everyone who deals with this form is a scientist. In many, if not most, cases, the inspectors, and the
IACUC reviewers are not experts in your field, nor should they need to be in order to understand the
procedures performed on animals or to understand the general purpose of these experiments.
Organization
Background
Title, PI information, Pre-reviewer, and Protocol Information
Species/ Strain
Checklist
Number of Animals Involved
Funding Information
Section 1: Support Personnel
Section 2: Animal Suppliers
Purpose and Procedures
Section 3: Lay Explanation of Study Objective and Impact
Section 4: Procedures and Experiments
Section 5: Justification of Animal Numbers
Section 6: Restraint Devices
Section 7: Pain/Stress Classifications
Section 8: Survival, Non-Survival, and Multiple Surgical Procedure(s) Summary
Section 9: Animal Monitoring Plan
Section 10: Euthanasia
Hazardous and/or Regulated Materials and Procedures
Section 11: Hazard Identification
Section 12: Hazardous Material Approval
Section 13: Use of Non-pharmaceutical Materials in Animals
Section 14: Other regulated materials or procedures
Animal Housing
Section 15: Animal Housing and Location where Procedures are Performed
Justifications and Assurances
Section 16: Search for Alternatives to Animal Use, Alternate Species, Less Painful/Distressful
Procedures, and for Research Duplication.
Adding Rows:
Insert the cursor (i.e. place the pointer over the row and left-click) anywhere in the row to be duplicated.
On the Table drop-down menu, go to Insert  Row Above or Row Below, and left-click
A new row should appear.
Abbreviation
AUP
Definition
Association for Assessment and Accreditation of Laboratory Animal Care
International
Animal Use Protocol
BLSB
Coll./Curtis
DoD
EHS
EPA
FDA
Bluemle Life Sciences Building
College-Curtis Building
Department of Defense
Environmental Health and Safety -- TJU
Environmental Protection Agency
Food and Drug Administration
IACUC
JAH
MD
NIH
OLAW
Institutional Animal Care and Use Committee
Jefferson Alumni Hall
Doctor of Medicine
National Institutes of Health
Office of Animal Resources -- Manages TJU Animal Facilities, does not
oversee or manage the IACUC
Office of Laboratory Animal Welfare (NIH)
PhD
PI
SOP
TJU
USDA
Doctor of Philosophy
Principal Investigator – person responsible for the Animal Use Protocol
Standard Operating Procedure
Thomas Jefferson University
United States Department of Agriculture
AAALAC-I
OAR
For Office Use Only
- For office use only.
Background
- This information is essential to enter the information into databases, complete annual reports, and to
provide a quick reference for important sections.
Protocol Title
- Check with the granting agency to determine if the Animal Use Protocol must have the same title as the
corresponding grant.
- Brevity is appreciated, but accuracy is essential.
Principal Investigator
- That’s you, or the person in whose name you are writing the protocol. There can be only 1 Principal
Investigator (PI). The Principal Investigator (PI) must be a faculty member of Jefferson Medical
School/Thomas Jefferson University. Post-doctoral fellows, adjunct appointments, graduate students,
visiting scientists, etc. are not acceptable PI’s on a TJU Animal Use Protocol form. The PI is
responsible for all personnel and work conducted under the PI’s protocol(s). If the PI does not
have an RO-4A form on file, please fill one out now. RO-4A forms are found at Animal Use Forms.
Department, Address, E-mail Address
- self explanatory –
- If you are a new faculty member and have not yet physically moved to TJU, fill in as much as possible
with your TJU department and location. HOWEVER, use your current E-mail, and phone number.
Please update the form when you arrive and are settled.
Pre-reviewer
To increase the likelihood that the form is filled out correctly and will be approved by the IACUC, it is
required that the form be pre-reviewed by a current committee member. The pre-reviewer does not
sign your application. Revisions and Triennial Renewals also require a pre-review. NOTE: The
pre-review does not count as a full review. Some IACUC members only scan the form to make sure the
appropriate sections are filled out and to catch obvious errors while others pre-review as though they
were an assigned reviewer. Comments made, or not made, by the pre-reviewer are not considered in
the final assessment of the proposal.
See: List of IACUC members.
Emergency Contacts
- If this section is left blank, you are ceding authority to the OAR to make whatever decision they believe
is best regarding your animals without notifying you. The primary contact is either the PI or someone
designated by the PI as the person able to make immediate decisions regarding the disposition of the
animals. In case of an emergency where an animal may need medical attention or need to be
euthanized, the Contact should have intimate knowledge of the experiment and be able to authorize
veterinary treatment or euthanasia. If you want to list your pager, in addition to your TJU and Off
Campus phone numbers, repeat your name on the next row and in the TJU column write PAGER and
then put the pager’s number in the Off Campus column. You may be reluctant to supply your OffCampus phone number. These forms are considered confidential and access is limited. We prefer
more than one contact, but one will be accepted. Please be informed that the Attending Veterinarians
have the authority to take whatever veterinary action they believe is best for the health and well-being
of the animal including euthanasia, the attending veterinarians have this authority at all times, and do
not need approval from any investigator to perform necessary treatment. This regulation cannot be
revoked by the PI. Remember that all animals on campus are TJU property.
Protocol Type
New protocol and Triennial renewal are self-explanatory. Revised is used when extensive changes or
additions will be made to an existing protocol prior to its Triennial renewal. Please remember to include
the protocol number for Triennial and Revised protocols.
Category
- Teaching means that the procedures are for training purposes. For example, microsurgical techniques
may be taught, and microsurgery residents may practice using an animal model. Teaching support
personnel to perform a technique described in the Animal Use Protocol, does not qualify as a Teaching
Protocol. If a protocol is both research and teaching, check both and explain in the Procedures and
Experiments section (section 4). An example of this would be where an animal is used to train
residents in a sophisticated surgical technique, but modifications to that technique are also tried and
compared to the standard procedure (research), or organs are harvested after the animal is euthanized
to provide cells for in vitro experiments.
Species
- Use the common name for the species, such as, mouse, rat, cat, dog, rabbit, primate, etc. There can be
only 1 species per form.
Age: Indicate the age, or age range to be used. Example: Birth to 2 years-old
Gender: Can use “M” for male and “F” for female. Can also enter “Both” or “M&F” if both sexes are
used.
Strain(s)
- List the strains to be used, such as; New Zealand White Rabbit, Syrian Hamster, Rottweiler, Bengal
Tiger, Macaque, C57BL/6, etc. (NOTE: The species name is the general name for all strains of that
animal. Using the strains in the first sentence as examples, the corresponding species are; Rabbit,
Hamster, Dog, Tiger, Non-Human Primate, Mouse, etc., respectively.) The box will increase in size to
accommodate multiple strains, as is common with rodent protocols. Please be accurate and complete
when listing these strains. For example, indicating C57 (a mouse strain) is not sufficient as there are
several C57 strains (C57BL/6; C57BL/10, etc.). If you are using multiple strains of this species, you
may alternatively list the number of strains here, and then identify them more fully in the protocol
description and animal number justification sections. For example, if 10 transgenic mouse lines and
two wild-type strains are to be used then enter “10 transgenic mouse lines; and 2 wild-type strains
C57BL/6J and 129S3/ImJ”.
CHECKLIST
Place an “X” (or symbol of choice) in boxes to indicate situations applicable to this proposal. This
section serves as a quick check for reviewers and also provides OAR and LAS personnel with readily
accessible information when emergencies arise. Please make sure this checklist is correct.
Number of Animals Involved in Protocol
- This is the total number of animals to be used on this protocol, including all those for breeding and
experimental purposes. The answer for any 1 year must be a whole number. Do not provide a
range of numbers (such as, 100-150) or break down the total (Example: 50 – Purchased; 200 – Bred;
50 – Experiments). List the total number of animals by year, and then add these together to derive the
total for the Protocol’s three year span. The numbers must match the numbers in Section 5,
Justification of Animal Numbers. One of the most common mistakes is that there are inconsistencies
between the number of animals justified (Section 16), and the number listed here.
FUNDING INFORMATION
Funding level or status has no effect on the IACUC review of the Proposal. This section is for
ORA to facilitate matching Protocols and Grants.
- Funding Agency/Source: List the funding agency or agencies, if applicable. Only agency
abbreviations listed in the abbreviation table at the beginning of this form and NIH standard
abbreviations for institutes can be used. Please provide the full name for all other agencies. If internal
funds are used, then the Agency is TJU or your department.
- Submission Date: The date on which the grant application was submitted or the grant submission
deadline date.
- Start Date: The date on which the grant has started, will definitely start, or may start if the submitted
grant application is successful.
- IACUC Approval Deadline: Indicate the date by which IACUC approval is required. Acquiring or
submitting a grant does not guarantee IACUC approval. At least 1 month (application submitted on
the month’s application due date), is required to obtain approval, assuming there are no problems with
the AUP. Expedited reviews are possible, but this practice is strongly discouraged and will only be
allowed when justified. “JUST IN TIME” DOES NOT MEAN “AT THE LAST MOMENT”. There is no
guarantee that the IACUC review will be finished by the deadline. Animal use problems must be
addressed and resolved before the IACUC can grant approval. The IACUC cannot grant partial
approval, or phased approval. The IACUC cannot break or bend these rules to accommodate
someone’s schedule. It is strongly recommended that the AUP be submitted immediately upon
receipt of the ranking score if the score is in the fundable, or almost fundable, range (contact
the executive secretary).
1. SUPPORT PERSONNEL
NOTE: As of January 1, 2007 all TJU personnel, including the PI, listed on an Animal Use
Protocol must have completed their Web-based training or the Protocol will not be reviewed.
This section lists all the individuals (technicians, students, volunteers, faculty, etc.) that have a role in
the project. LAS animal caretakers are not listed. The TJU clinical veterinarian, veterinary technicians,
or other LAS personnel that have a role in the protocol can be listed if they have agreed to assist on the
project and have permission from the OAR director to do so. All Support Personnel must complete (or
have filed) an RO-4A or RO-4B form. Non-TJU personnel can also be listed as Support Personnel, and
must provide verification from their institution’s IACUC that they are sufficiently trained to perform the
activities described in the protocol. If such verification cannot be obtained, the non-TJU personnel can
take the appropriate web-based training modules, and link it to TJU, and their Certificate(s) of
Completion can be submitted to the IACUC Office. For non-TJU personnel, indicate their affiliation in
the Position column (i.e. Investigator – Johns Hopkins Univ.). Accreditation agencies (AAALAC-I,
USDA, etc.) state that academic or professional rank does not indicate animal use proficiency.
Link to Web-based Training Information
Link to RO-4A/ B Forms.
Example:
1. SUPPORT PERSONNEL (Add rows as needed; Boxes expand to fit text)
Indicate any non-TJU Personnel that are involved in the Position column by listing their affiliation after their
position (Example: Technician – Univ. of Penn.)
RO-4B
Name
Position
Attached
On File
x
Dr. Tom Jefferson
investigator
Dr. Ben Franklin
collaborator (Univ. of Penn.)
Dr. Betsy Ross
investigator
x
Fred Lafayette
technician
x
Comments: Dr. Ben Franklin is a collaborator from the University of Pennsylvania. A letter from the Univ. of
Penn. IACUC confirming Dr. Franklin’s training has been submitted to the IACUC Office.
2. ANIMAL SUPPLIER(S)
Approved Vendor:
Contact OAR for list of approved vendors. DO NOT ASSUME THAT AN INSTITUTE, UNIVERSITY,
OR COMPANY IS AN APPROVED VENDOR. CHECK FIRST!
TJU Source:
There are 3 common TJU sources:
1) One of your protocols – usually a breeding protocol on which animal lines are bred and then
transferred to other protocols for experiments.
2) Another TJU investigator – either they will supply you with animals to start your own colony or
supply enough animals for these experiments. In the animal number justification section,
indicate which, and how many animals, will be supplied by another TJU investigator. The
animals you receive are to be included in your animal use totals, and also must be included in
the total number of animals bred on the supplier’s Annual Review (supplier must also have a
sufficient number of animals on their breeding protocol to accommodate your needs and must
submit a modification if they need to increase animal numbers.). If more than 1 TJU PI is
contribution animals to this project, list all the protocol numbers in the Protocol # box and
then place the protocol number after the PI’s name in the PI box (see example below).
For TJU sources #1 and #2 above, enter the number of the approved TJU protocol and the
PI, if it is someone other than you. If there is a breeding component on the current form (the
form being submitted), then in the box labeled “Protocol #” enter “This Protocol”. If this
protocol has an animal breeding component, and animals will be obtained from another
investigator or core, then enter “This Protocol” and then the other relevant protocol number
(Example: This protocol & #1313A), and/or fill in the Core information. Boxes will expand to
accommodate the text.
3) A TJU Core Facility, such as, the Kimmel Cancer Center Mouse Facility or the Alcoholic Rat
Core. Note: Indicate TJU core facilities only if they are a current source of animals. Do not
list core facilities, that may be used in the project, such as the Bioimaging or Microarray Core
Facilities, but do not provide animals. If you have already obtained the animals from a core
facility, and will not acquire any more animals from that core facility for the duration of this
AUP, then that core should not be listed as a current animal supplier. Note that all TJU
Core Facilities are administratively independent of the IACUC and having an IACUC
approved protocol does not obligate any Core Facility to comply with your needs. You must
make arrangements with the core director/manager to use the core. Provide pertinent
protocol numbers and PI’s name for the core (see example below).
Other:
List source and justify why a non-approved vendor or source must be used. OAR approval is required
to import animals from non-approved sources and the animals must be imported according to OAR
guidelines. (See Animal Importation Form). Please remember that a justification is required. The most
common reason for using an “Other” source is importation of mouse strains. In this example, the
justification would be; “This proposal will use (STRAIN NAME) strain(s) of mice. Currently, these mice
can only be obtained from (SOURCE’S NAME) at (SOURCE’S LOCATION) and is (are) not available
from OAR-approved commercial vendors.”
Example:
2. ANIMAL SUPPLIER(S) (Check all boxes that apply and provide appropriate information)
Approved Vendor
List vendor(s) – Consult with OAR for list of approved vendors.
The Jackson Laboratory
TJU Source
Indicate Protocol Number and PI (if PI on Protocol is other than you.)
Protocol #
TJU Core Facility
Other
PI
Dr. L. Pasteur (#2323A)
Dr. R. Koch (#4589C)
Mouse Tg Animal Facility (2335K – Dr. A. Lathrop)
List source and justify why a non-approved vendor or source must be used.
(See Animal Importation Form) (Box expands to fit text)
Transgenic strain B6:129 Tju-/-LStm1 is being imported from Dr. L. Silver (Univ.
of Maine) per OAR. Dr. Silver is the only source for this strain.
Purpose and Procedures
General Comments
This protocol,
#2323A; #4589C
In this Animal Use Protocol (AUP) version similar material is grouped together. This part will have
sections for the Lay Statement, Description of Procedures and Experiments, Restraint Devices,
Pain/Stress Classifications, Surgical Summary, Post-Operative Monitoring Plan, and Euthanasia.
Some sections may seem, and indeed are, redundant. IT IS NECESSARY TO ACCURATELY
COMPLETE ALL APPROPRIATE SECTIONS. It is possible to limit repetition if the form is first reviewed
to determine what information is required in which sections. The governing and oversight organizations
(AAALAC-I, USDA, OLAW) prefer if they can find specific information rapidly without having to read a
detailed protocol. This information may also save valuable time in case of a medical emergency.
Phrases such as, “See Procedures and Experiments, Section 4” will not be accepted.
3. LAY EXPLANATION OF STUDY OBJECTIVES AND IMPACT
Important Considerations
- Write the statement so that it can be understood by a lay person, assuming an education level of an
average eighth grade student. One of the most common queries raised in the IACUC review is that the
Lay Explanation is too complex.
- This section must be complete within itself as it may be excerpted for various uses. Do not refer to other
sections of the form.
- Use common words and avoid jargon. For example, instead of ischemia say “blockage of blood flow”.
- Avoid abbreviations unless described in this section. Common abbreviations, such as DNA, RNA, HIV,
AIDS, MRI, can be used as these are often seen in the lay literature.
- Describe not only the proposed research, but also its possible impact on the layman and/or how it will
extend our knowledge.
- The reason this section is placed here is so that it may serve as an introduction for the Procedures and
Experiments section.
Example – Layman’s Statement:
Our goal is to determine if a new drug, compound XYZ, will improve the survival of animals after a heart
attack. Heart attacks are often caused by a blockage in one of the arteries supplying blood to the heart
muscle resulting in damage to the heart. The heart muscle cells do not die immediately after blood
deprivation, and there is a complex process controlling cell death. Compound XYZ is a new drug thought
to limit the amount of cell death if administered quickly after a heart attack. We will utilize mice in our
experiments. To simulate a heart attack, the chest is opened and a blood vessel supplying blood to the
heart is tied off limiting blood flow to the heart. Compound XYZ will be administered, by injection into the
blood stream through the tail vein of the mouse, at various times after the blood vessel has been tied off.
The mouse will be allowed to recover, if possible, from the procedure and we will monitor how long it
survives after the simulated heart attack and how well the heart functions by monitoring blood pressure,
beats per minute, imaging of the heart and blood flow on living animals, and finally dissection of the heart
from euthanized experimental animals.
If effective in mice, compound XYZ will be tested in more complex animal models of heart attack.
The ultimate goal is to determine if compound XYZ will improve the survival rate and decrease the
recuperation time of an animal after heart attack. If compound XYZ is effective in animals, we can then
try it in humans applying the knowledge of dose and time after heart attack to try to increase the
survivability of humans undergoing heart attacks. This will be of great benefit to humans as heart attack
is one of the leading causes of death and/or debilitation.
Note: Words such as myocardial infarction, ischemia, ligation, apoptosis, agonist/antagonist etc. were not
used.
4. PROCEDURES AND EXPERIMENTS:
Important Considerations:
- In this section, write for someone with an eighth grade, non-science oriented, education. For some
descriptions this may be impossible, but the overall purpose should be evident. For example, the
reader will know that surgery will be performed on an animal, and why surgery will be performed, but
may not know all the identified body parts or terminology.
- IDENTIFY ALL ABBREVIATIONS THE FIRST TIME THEY ARE USED OR INCLUDE A TABLE OF
ABBREVIATIONS AT THE BEGINNING OF THIS SECTION.
- Avoid unnecessary jargon – Suggestions: Do not use medical terms that are not in the on-line version
of Stedman’s Medical Dictionary. Especially for medical and anatomical nomenclature, do not show off
by using every possible synonym for the same subject – be consistent.
- It is permissible to “cut and paste” from another application or grant. HOWEVER, MAKE SURE THAT
THE CUT AND PASTED SECTIONS MAKE SENSE IN THIS PROTOCOL AND DO NOT INCLUDE
REFERENCES TO JOURNAL ARTICLES, SPECIFIC AIMS, OR MATERIALS THAT ARE NOT
INCLUDED IN THIS APPLICATION.
- A recommended outline with three major points is provided for your convenience, but alternate formats
can be used. For example, it may be more convenient to write the entire section incorporating the
experimental design, description of procedure, and endpoints as a single narrative. However, the 3
points, A. Purpose, B. Experimental Design with Endpoint(s), and C. Description of Procedures must be
clearly addressed.
- IT IS RECOMMENDED TO EXAMINE THE FOLLOWING SECTIONS BEFORE WRITING THIS
SECTION. For example, you may opt to briefly describe points, such as, restraint devices in this
section and go into more detail in the specific section addressing that issue.
A. Purpose
The Layman’s Statement may be adequate as the purpose, in which case, it is appropriate to write:
“Purpose described in Layman’s Statement.” However, you may want to provide more detail. The
purpose does not need to be long, nor highly detailed ias required in a grant. The purpose should
inform the reviewers of what to expect in the protocol and familiarize them with particular systems.
In these examples, the names of investigators, drugs, procedures, and results,
may be fictitious.
Example: Scenario A: Purpose for a simple, straightforward experiment:
The experimental drug, TJU007, has been tested using a cell migration assay in vitro and has been
shown to inhibit migration of metastatic cancer cells. The purpose of this experiment is to determine
the efficacy of TJU007 to inhibit metastasis in vivo using a mouse model of liver cancer.
-This would be a sufficient purpose statement. The reviewer now knows to expect a brief description
of drug TJU007, administration of the drug, and description of the mouse liver cancer model. The
reviewer will also expect pain/stress issues to be addressed as this is a tumorigenesis model and to
see a Search for Alternatives for the tumorigenesis model.
Example: Scenario B: Purpose for a more involved experiment.
The proposed experiments will examine the role of a novel nuclear transcription factor (NNTF) in a
mouse heart infarction model. NNTF is a DNA-binding protein that functions as a regulator of gene
transcription and has been shown to have vital roles in apoptosis. Expression of NNTF has been
shown to be anti-apoptotic (pro-survival) in many systems. NNTF consists of 2 members of the
NNTF protein family either as heterodimers or homodimers. The NNTF protein family has 3 known
members. Our approach is to determine the effect of deficiency of various NNTF proteins using
transgenic mice lacking a NNTF protein family member. The transgenic mice will supply fibroblasts
and myocytes for an in vitro system to test the ability of various drugs to activate transcription of a
promoter/reporter construct with NNTF binding sites in the promoter, and production of a specific
NNTF protein(s), using standard biochemical analysis. Next, interesting compounds will be tested for
their effect on cell survival in vitro by treating the cells with a compound known to induce apoptosis
and then using novel NNTF activating drugs to determine if they activate NNTF and can counteract or
limit the apoptosis. Compounds that are protective against apoptosis will then be tested using a
mouse-based in vivo heart attack model.”
- From this description, the reviewer has learned something about the NNTF gene regulatory system,
at least enough to understand that various strains of transgenic mice will be employed. The reviewer
also expects to see a tissue-harvest protocol for fibroblasts and myocytes and expects that the
Procedures and Experiments or Animal Number Justification section will describe experimental
groups. Compounds will be tested in an in vivo model in mice that involves infarction. The reviewer
will expect an infarction model description; can anticipate that there will be pain/stress issues, and a
search for alternatives.
B. Experimental Design with Endpoints:
Discuss how the experiment(s) involving animals will be performed. Consider Scenario B above. The
IACUC would not require, and indeed would discourage, an extensive and detailed descripton of the in
vitro biochemical assays for drug testing using tissue culture cells. Also, the IACUC does not require
detailed information about the cell isolation and culture procedures from harvested tissues. However,
the protocol should include detailed descriptions of all procedures to be used on the animals. This
includes such standard practices as mouse tail clips and blood collection.
This section includes:
i. Variables to be tested. This is essential in order to understand the experiment. In some cases,
such as, teaching or breeding protocols, there is no experiment, and thus, no variables to be
tested.
ii. Experimental groups. The required experimental groups follows naturally when the variables are
identified. At a minimum, there is a control group and an experimental group.
iii. The order in which the procedures will be performed must be provided either in this part of the
Procedures and Experiments section or in the next part.
iv. Expected results, especially those pertaining to pain and/or stress. This response does not need
to be highly detailed. For example, in Scenario A above it would be sufficient to write: “We expect
to see a decrease in the number of tumors in the organs of the mouse.” You do not need to
provide a detailed mechanism why the drug inhibits metastasis.
iv. ENDPOINT(S) and Analysis. Endpoints are commonly overlooked in an experimental design
description. An endpoint is euthanasia or removal from the study. The IACUC also needs to
know what happens to the animal at the endpoint. For example, will the animal be used in other
procedures or protocols (removal from study), will it undergo a dissection, will organs be
harvested for tissue-culture experiments, or will it be disposed? The IACUC does not need
extensive information of how the animal is used after it has died. However, the IACUC does need
to know why the animal had to die (for analysis, tissue harvest, etc.) and what will be done to the
carcass. DEATH AS AN ENDPOINT IS STRONGLY DISCOURAGED AND NEEDS A STRONG
SCIENTIC JUSTIFICATION. Most, if not all, animals can sense that they are in a terminal state
and this causes stress. Some procedures may involve a painful death, for example, toxicology or
tumorigenesis studies. Consider if an indicator, such as, weight loss, tumor size, blood analysis,
etc., can be used to determine if the animal is terminal.
IMPORTANT POINT: The endpoints do not need to be based on a specific time point, but
can be based on biological criteria. For example, suppose the experiment is to study
leukemia. The PI has an animal model in which leukemia can be induced and normally the
animal’s white blood cell (WBC) count escalates rapidly within 48 hours after induction, thus the
PI proposes to euthanize the animal 48 hours after induction. However, when the PI performs the
experiment, the WBC count does not reach the expected level by 48 hours and the PI is
concerned that the induction may be delayed and take longer than 48 hours. According to the
protocol, and especially if USDA covered species are involved, the PI is required to euthanize the
animal at the time stated and requires a modification to extend the endpoint. If the PI wrote the
protocol so that euthanasia occurs when the WBC has reached a certain level, then they can
legally wait another 48 hours, or even longer. However, if biological criteria are used as
endpoints, the PI must specifiy the maximum amount of time allowed for the biological
criteria to be reached before sacrificing the animal.
Note: Confusion over temporal versus biological endpoints causes problems for PI’s and the IACUC.
For example, consider the following statement:
Endpoint Example 1:
“The tumor will reach 1 cubic cm two weeks after the modified tumor cells are injected. At this point
the animal is euthanized and the tumor is harvested for histological and biochemical analysis.”
What is the actual endpoint? Is the endpoint two weeks after the tumor cells were injected, or when
the tumor cells form a tumor of 1 cubic cm? If there are two conflicting endpoints, a temporal and a
biological, then the PI is in risk for non-compliance as the endpoint statement could be interpreted
differently from what they intended (they needed a tumor or 1 cubic cm). The endpoint statement in
this example is not acceptable because the endpoint is ambiguous.
Endpoint Example 2:
“Two weeks after the tumor cells are injected, the animal will be euthanized and the tumor harvested
for histological and biochemical analysis.”
Suppose that the tumor cells do not form a usable tumor by 2 weeks post cell injection and the PI
would like to allow the animal to live until the tumor does reach an acceptable size. According to the
endpoint statement, the PI is obligated to euthanize the animal two weeks after the tumor cells were
injected regardless of tumor size. The PI cannot wait until the tumor reaches a usable size which is
what they intended. (The PI can submit a modification to the IACUC, but in a timely manner.) In
cases where the occurrence of a biological event is required, it may be preferable to have a
biologically based endpoint. However, it is not acceptable to wait an unreasonably long time for the
biological event to occur; some maximum temporal endpoint is needed. The endpoint statement in
this example can be accepted by the IACUC, as the endpoint is at a definite time point, and a query
about the endpoint may not appear on the Review.
Endpoint Example 3:
Tumor size will be checked twice weekly (Tuesday and Friday) and when the tumor reaches
approximately 1 cubic cm in size the mouse will be euthanized and the tumor will be harvested. We
will wait a maximum of 6 weeks after cancer cell injection for a usable tumor to form, at which time
the mouse will be euthanized, regardless of tumor size.
This endpoint statement clearly indicates that the endpoint is when the tumor reaches 1 cubic cm, or
6 weeks after cancer cell injection, whichever happens first. This statement is acceptable to the
IACUC as it specifies a definite maximum endpoint (6 weeks post-injection) and accurately reflects
what the PI requires. Note that the endpoint also states what will happen to the mouse when the
endpoint is reached (euthanasia). Suppose the PI had merely written: “Tumor size will be checked
twice weekly (Tuesday and Friday) and when the tumor reaches approximately 1 cubic cm in size the
mouse will be euthanized and the tumor will be harvested. We will wait a maximum of 6 weeks for a
tumor to form.” Note that the PI does not indicate what happens to a mouse that received tumor cells,
but did not form a tumor by 6 weeks post-injection. It is unknown what will happen to this mouse – will
the PI attempt another cancer cell injection using the same mouse, will it be euthanized, or what?
Ambiguity concerning the final disposition of an animal is unacceptable.
Example of Experimental Design with Endpoints: Let’s consider Scenario (A) described above
dealing with experimental anti-metastasis drug, TJU007. The variables might be whether or not
TJU007 has an effect in vivo, and over what time period it is effective.
“The experiment utilizes a mouse liver cancer model (see procedure below). Two variables
will be tested, one is TJU007 treatment and the other is time after TJU007 administration. Liver
tumors are induced by injection of compound LTP#9 which is known to cause liver tumors
approximately 1 week after administration and is commonly used in mice to produce a liver
tumor model. Mice, of the strain LOC233/J, 8 to 9 weeks-old, (NOTE, the age of the mice is
specified) will be used in this study and receive a unique numeric identifier using ear tags
(Monel #1; National Band and Tag Co., Newport KY). A Random Number Table (RNT) (NOTE:
Defined RNT as Random Number Table) is then used to divide the mice into two groups, one
group receiving LTP#9 (Tumor) and one untreated (Tumor Free) group. The LTP#9-treated
group is then further subdivided into TJU007 treated and TJU007 untreated using the RNT.
Thus, there will be 3 main cohorts, 1) Tumor Free Placebo, 2) Tumor-TJU007, and 3) TumorPlacebo. (NOTE: At this point the reviewer knows what variables will be tested and knows the
the strain and age of the mice, knows how the mice will be chosen for each group, and was
introduced to the liver tumor model.)
One may question the use of a Tumor Free Placebo group in the experiment as mouse
strain LOC233/J has a low rate, of less than 1 in 10,000, of spontaneous tumor formation.
However, this low rate is obtained only when the mice are kept in carcinogen free
environments. LOC233/J is particularly sensitive to environmental carcinogens, especially if
ingested, which is why LTP#9 is so effective at producing liver tumors in these mice. To rule
out tumor formation arising from environmental sources and the solvent used to dissolve
TJU007, it is necessary to have a control group of LOC233/J mice that have not received
LTP#9. (NOTE: This paragraph explains why a particular group, the Tumor Free group, is
included in the experiment as this may not be obvious to everyone. This could also have been
placed in Section 16 for the Animal Number Justification.)
The Tumor-TJU007 group will also be subdivided, using a RNT, into 3 groups receiving
dosages of 100 mg/kg, 250 mg/kg, and 500 mg/kg. (NOTE: This investigator did not mention
how TJU007 will be delivered. They have opted to put this information into the “Description of
Procedures” section.) Each of these dosage groups will be further subdivided into 4 time points
of; 0, 1 week, 5 weeks, and 10 weeks after TJU007 administration. The 0 time point is
immediately before administration of TJU007. At each time point, mice to be euthanized for
analysis are selected using a RNT. The Tumor-Placebo group will also consist of mice with
liver tumors that will receive a placebo consisting of the solvent without TJU007 and
administered exactly as TJU007. As this is a placebo,and the injection volumes are the same
for the 3 TJU007 dosage groups, there is no point in dividing the Tumor-Placebo cohort into
dosage groups, but this group will be divided into 4 subgroups, using a RNT, corresponding to
the four time points of 0, 1, 5, and 10 weeks after placebo administration. The same design
rationale applies to the Tumor-Free- Placebo group. All other experimental procedures will be
the same for the, Tumor-TJU007, Tumor Free-Placebo, and Tumor-Placebo groups.
(NOTE: The endpoints have been defined. At 10 weeks after the administration of TJU007,
all mice will have been euthanized and at least the animal portion of the experiment is
completed. Note that the PI opted for a temporal endpoint and not a biological endpoint.
Scientifically, one might wonder why the PI did not use bioimaging to first confirm that the LPT9
did induce liver tumor growth and then to monitor the decrease in tumor size over time. There
may be many reasons why this is not possible, whether technical, cost, scheduling, animal
housing between scans, past experience with bioimaging, etc. The IACUC reviewer can
suggest bioimaging as a means that may decrease animal numbers and could ask if
bioimaging was considered and why it is not being used. However, the IACUC cannot force the
PI to use bioimaging.
Further, the PI has presented the experimental design. All the subgroups will also be
treated in the same manner from this point so the investigator does not need to refer to all the
major groups and subgroups individually.)
At the time points mentioned above, mice in that time group will be euthanized and
necropsied with removal of all major organs. Gross observations are recorded for each organ
and then each organ will be histologically analyzed to determine the number of tumors present.
If TJU007 has an anti-metastasis effect, it is expected that there will be fewer tumors in organs
other than the primary organ (Liver-there may be a decrease in the liver as well) when
compared to data from the Tumor-Placebo group. It is expected that the Tumor-Free Placebo
group will not develop any tumors as the published incidence (The Jackson Laboratory;
Phenome Database) is one mouse in 10,000 develops spontaneous tumors by 1 year of age.
However, if a significant number of mice develop tumors in this group during this experiment,
the experiment will have to be terminated and all mice will be euthanized. We will have to find
the source of the environmental carcinogen before proceeding with a repeat experiment. In this
event, the IACUC will be notified of our findings and we will work with the IACUC and OAR in
order to find the environmental carcinogen as this carcinogen could affect the experiments of
other investigators. We have used the LOC233/J mouse strain in previous experiments at TJU
and have not had problems with environmental carcinogens.
(NOTE: Euthanasia is mentioned but not in detail as the investigator will provide euthanasia
details in Section 9 – Euthanasia. This section discusses the expected results. There is a
description of what happens to the animal after death, a brief description of what data will be
collected, how data will be collected, and analyzed. More detail about the organ harvest will be
described in the “Description of Procedures” section, but details of the histological examination
are not provided, and are not required, as this occurs after the animal is euthanized. The
IACUC is primarily concerned with procedures using live or dead animals. The IACUC
review is not a grant or publication peer review. The investigator has also discussed the
approach if the Tumor-Free group, which is the control for environmental carcinogens, develops
tumors.)
One of the goals is to determine the biological half-life of TJU007 Thus at intervals of 5
days until euthanasia, blood is colleced from each mouse by a retro-orbital bleed. Blood is
then analyzed hematologically and also for TJU007 levels.”
(NOTE: This paragraph introduces blood collection from the experimental animals, the method
for collection, interval of collection, and explains why the blood is collected. The details of the
method will be given in the next section. The IACUC reviewer could suggest that the PI also
perform lymph node biopsies as metastatic cells are usually seen in lymph nodes, but cannot
demand that they do so.)
Tissue Harvest: An experimental design for cells harvested from animals for use in vitro does need
to be described, however, it does not need to be as as detailed as an experiment performed
using live animals. The IACUC does need to know how, and why, animals are used. The in
vitro experimental design is also important to justify animal use. Most tissue harvest
procedures are performed using dead animals. In cases of non-survival surgery for organ
harvest (i.e. perfusion with PBS to remove blood from the organ or proteinase perfusions), the
non-survival surgery (perfusion) needs to be described in detail, but not the cell culture.
Example – Scenario B:
“The goal is to determine the effects of various AAD’s using cells isolated from mice
deficient in either one or more of the NNTF protein family. An AAD that is effective in wildtype cells will be tested in NNTF-A and NNTF-B deficient cells to determine if the drug is
specific for one, or both, subunits. Mice deficient in either NNTF-A or –B are viable and will
be used in these experiments. NNTF-C deficient mice perish in utero at embryonic day 14
because the heart fails to form properly. However, NNTF-C levels decrease dramatically
after birth coincident with an increase in NNTF-A and –B. In a mouse deficient in either
NNTF-A or –B, the heart forms in utero, but then develops slowly. NNTF-A or –B deficient
mice are runted, survive to 3 weeks-old, and usually perish between 5 to 6 weeks. In mice
heterozygous for both NNTF-A and –B ( NNTF-A/+; NNTF-B/+), the mice do survive to
adulthood, and are fertile, but are smaller than their littermates. Mice deficient in both
NNTF-A and –B perish shortly after birth. Hence it is believed that NNTF-C is essential for
heart development, but not post-natally. For these reasons, only NNTF-A and –B deficient
mice will be used for tissue harvest and in vitro drug testing.
Mice deficient in NNTF-A or -B protein, and wild-type mice, 3 weeks-old, are
euthanized and the heart and leg muscles are then immediately harvested. The removed
tissue is dissociated into single cells that are placed in culture.”
Note: The PI does not need to describe how the heart and leg muscles are harvested (other
than it is harvested after euthanasia of the animal), how the cells are cultured, or how the
AAD test is performed. However, the IACUC knows that the harvested cells are being
used in experiments appropriate to this protocol and knows the relevance of these
experiments. In the animal number justification, the PI can indicate the experimental
groups and can then justify how many animals will be needed to provide enough cells for
the tissue culture.
C. Description of Procedure(s):
Detailed descriptions of all procedures to be performed on live animals are required. The final
disposition of dead animals (i.e. discarded, dissected, used for cell harvest) must also be stated.
This section of the AUP contains detailed descriptions of:
 Injections or inoculations – injection or inoculation of any material, including but not limited to,
drugs, chemicals, infectious agents, adjuvants, etc.
Provide:
o Dose (use metric units)
o Sites – sometimes this is implied, such as, intraperitoneal (IP -- abdomen) and
subcutaneous (SC – dorsal neck region) but if an alternate site is used, then specify the
location. For example, IV administration could be by a number of veins and
intramuscular injection could use several different muscles.
o Volume (use metric units)
o Route – check the table of abbreviations for acceptable abbreviations for routes of
administration (i.e. IV for intravenous). If the abbreviation is not on the list, then use the
full term.
o Schedules – If no schedule is provided, a single administration is assumed. For some
compounds it may be necessary to change the site of administration for subsequent
dosings. In circumstances, such as adimistration of analgesics, where the animal’s
welfare is at risk, the dosing schedule can be changed with approval by the attending
veterinarian. The change must be documented, and if the change is to be permanent,
then the AUP must be modified. Note that post-procedure analgesic schedule(s) must be
included in the Animal Monitoring section so can be briefly mentioned in this section.
o Expected Effect and Possible Side-effects of Experimental Material – The IACUC
does not expect a list of every possible side-effect, just the most likely under the
experimental conditions. It is important to describe side-effects so that animal care
personnel, vet-techs, and veterinarians, will know whether the animal’s symptoms are
due to the experimental material, or might be due to some other condition. Remember to
address treatment for likely side-effects in the Monitoring Plan, Section 8. Listing side
effects is not required for the use of anesthesia, analgesics, or common
pharmaceuticals used for their manufactured, or FDA approved, purpose and
administered according to manufacturer’s or veterinary guidelines, unless these
compounds are used in a manner other than that intended by the manufacturer.
 Blood withdrawals -- include; volume, frequency, withdrawal sites, and methodology.
 Surgical procedures – see detailed instructions below
 Use of Radiation or Irradiation: if administering radionuclides, include dosage data as above for
injections. For irradiation, include dosage and schedule. (Note: irradiation dosage for X-ray
imaging does not need to be stated, but the location of the X-ray machine and who will perform
the X-ray must be provided.). IACUC no longer requires proof of RSO approval for irradiation.
RSO has informed the IACUC that use of irradiation equipment by unauthorized personnel is a
federal crime and thus is stringently controlled and monitored.
 Restraint Methods -- indicate use of restraint devices in the procedure description but
description, frequency, length of restraint, and justification must be specified in Section #5,
Restraint Devices.
 Other procedures -- tail biopsies, behavior monitoring, biophysical monitoring, etc.
 Resultant effects, if any, that the animals are expected to experience, especially regarding pain
and stress.
 Other potential stressors (e.g., food or water deprivation)
 Monitoring -- Animal monitoring is described in Section 8. Monitoring can be mentioned and
described here, but must be described in Section 8.
- Survival Surgery Protocols:
Also examine Section 7. Survival, Non-Survival, and Multiple Surgical Procedure(s) Summary.
Policies Addressing Surgical Procedures:
104.01: Guidelines for Anesthesia, Analgesia, and Tranquilization
104.02: Guidelines for Survival Surgical Procedures in Rabbits and Higher Mammals
104.03: Postoperative Monitoring of Research Animals
104.04: Surgery and Postsurgical Care (S.O.P.)
104.05: Surgical Suite Policy; (Also see Veterinary Services)
104.06: Guidelines for Survival Surgical Procedures and Post-Operative Care in Rodents
Surgery is any procedure requiring a skin incision or opening a body cavity. Implantation of
subcutaneous pumps and other devices is surgery if the skin is cut, and not surgery if a needle is
used to insert the device. Needle biopsies are not surgery unless an incision is required to
visualize the target organ.
TAIL CLIPS, RETRO-ORBITAL BLEEDING, AND TAIL VEIN INJECTIONS INVOLVING MICE ARE
NOT CONSIDERED AS SURGERY. The use of anesthesia to facilitate animal handling does not
constitute surgery.
1) Describe pre-surgical procedures, such as fasting, adminstration of biological tracers, etc. Presurgical fasting guidelines for some animals are provided in Policy 104.02. Consult the
veterinarian regarding pre-surgical fasting regimens. Deviations from the policy must be
described and justified.
2) Anesthesia. The anesthetic agent should be stated here, but details must be provided in
Section #7. “Survival, Non-Survival, and Multiple Surgical Procedures Summary”. However, if
anesthesia delivery involves intubation, an IV delivery system, or any similar method, a
description of the procedure must be given here.
3) Surgical Site Preparation. For survival surgery, the surgical site must be disinfected, and
surgical drapes may be required depending on the site, surgical procedure, and animal (Policy
104.02). For non-survival surgery, disinfection is discretionary. Often the animal is first
anesthetized and then the hair is removed by shaving or depilitation (Example; Nair™). The
exposed skin is disinfected using Betadyne or similar disinfectant. Ethyl alcohol or isopropanol
are often used but are not as effective as Betadine.
NOTE: The surgical site preparation procedure does not need to be described in detail if policy
104.02 is followed.
Example:
”The incision site is prepared using hair clippers to remove hair. The exposed skin is thoroughly
swabbed with Betadine and a surgical drape is used in accordance with the guidelines in
Policy 104.02.”
SAFETY NOTE: If 100% oxygen gas is used to deliver a volatile anesthetic (such as isoflurane)
using a nosecone, especially on a small animal, or where the surgical site is close to the
nosecone, never use an alcohol, or any volatile, flammable material as a disinfectant in surgical
procedures that use a source of ignition, such as, electrical equipment that may spark or
produce heat (example: cauterizers, electro-surgical instruments). The oxygen and alcoholic
fumes combination is extremely flammable. Under these conditions, consider using a nonvolatile, non-flammable disinfectant or an injectable anesthetic. Use extreme care when using
flammable anesthesia, such as, nitrous oxide around any source of heat or ignition (electrical
spark). Nitrous oxide is rarely used in animal surgery.
4) Describe the surgical procedure. Keep the description basic and try to avoid excessive use of
obscure medical terms that only MD’s understand, especially if the word is not in the on-line
version of Stedman’s Medical Dictionary. If you wish to use such terms, please define them the
first time they are used. Do not forget that a lay person should understand, at least generally,
what is involved in the surgery. Minute detail is not required regarding surgical instruments and
supplies. For example, the IACUC does not need to know the blade size or shape of a scalpel.
Example: Embryo transfers. Embryos are transferred to pseudopregnant female recipients
via an aseptic (per Policy 104.06) laparoscopic surgery. Female mice in estrus are mated
with vasectomized males (obtained from a vendor) and mating (i.e. ovulation stage) is
determined visually by noting the presence of a vaginal plug the next morning (plugged).
Plugged females (pseudopregnant) are used as recipients for embryos. A pseudopregnant
female is anesthetized using isoflurane. The incision site is on the dorso-lateral side
approximately midway between the hip and the last rib and is prepared by removing the hair
using hair-clippers and the exposed skin is wet with 70% alcohol (per Policy 104.02). An
incision (approximately 0.5 cm) is made perpendicular to the vertebral column approximately
midway between the hip and the last rib using sterile scissors and forceps. The animal is
then wiped once firmly in a head-to-tail direction with a sterile alcohol wipe to remove loose
hair. A second pair of sterile forceps is used to grasp the abdominal wall muscle and a 3 – 6
mm incision is made using a second pair of scissors, parallel to the first incision, in order to
expose the ovarian fat pad. The fat pad is then grasped with a pair of forceps and
exteriorized, drawing with it the ovarian capsule, oviduct, and the tip of the adjacent uterine
horn. A serrafine clip (small clamp) is attached to the fat pad to prevent the organs from
being pulled back into the abdominal cavity, and the exteriorized organs lay across the back
of the mouse so that the ovary is facing the surgeon. The bursa (membrane) covering the
end of the oviduct by the ovary is torn with 2 pairs of watchmaker’s forceps to expose the
infundibulum (open end) of the oviduct. A micropipette holding the embryos is inserted into
the infundibulum and the embryos are expelled into the oviduct. The serrafine is then
removed, the organ returned to the abdominal cavity, and the skin incision is closed with a
wound clip. Buprenex will be administered SC, immediately after surgery. The animal is
returned to a warm cage to prevent hypothermia during anesthetic recovery. The animal is
monitored post-surgery to assure that it is eating, drinking and behaving normally and that
the wound is healing properly (details in Section 8, Animal Monitoring Plan). The wound clip
is removed three days post-surgery.
5) Describe the closing method.
Example: From the description of embryo transfer provided above, The serrafine is then
removed, the organ returned to the abdominal cavity, and the skin incision is closed with a
wound clip.
6) Describe post-operative procedures, such as, suture removal, bandaging, etc. There is a
separate section for post-operative monitoring of the animals (Section 8 – Animal Monitoring
Plan) so details will be provided in that section. Here it would be advisable to mention postoperative analgesic use and that there is a monitoring plan. The reason for splitting out the
Animal Monitoring Plan into a separate section is that a significant number of PI’s were omitting
this important section. Another important reason for a separate Animal Monitoring section is so
that OAR personnel can quickly find this information in an emergency.
Example: From the description of embryo transfer provided above. Buprenex will be
administered SC, immediately after surgery. The animal is returned to a warm cage to
prevent hypothermia during anesthetic recovery. The animal is monitored post-surgery to
assure that it is eating, drinking and behaving normally and that the wound is healing
properly (see Section 8, Animal Monitoring Plan). The wound clip is removed three days
post-surgery.
Points to Remember:
For multiple surgeries on the same animal, state the time interval between surgeries. This is
needed to assess that adequate recovery time is allowed between procedures.
Justification for multiple surgeries is required in Section 7. Survival, Non-Survival, and
Multiple Surgical Procedure(s) Summary.
- Non-Survival Surgery:
Non-survival surgery is defined as any surgical procedure on a living animal in which the animal does
not regain consciousness before euthanasia. If the animal regains consciousness, even very
briefly, then it is survival surgery. Euthanasia, followed by harvest of organs is not considered to
be non-survival surgery as the animal is dead before the harvest procedure begins. Perfusion of
an animal while anesthetized, with subsequent or concurrent euthanasia, is non-survival surgery.
Adequate anesthesia and/or analgesia are required for non-survival surgery, or a strong scientific
justification why it cannot be provided must be given in Section 15; Lack of Non-Painful, Nonstressful Alternatives. Include points 1, 2, 3, and 4 from the above Survival Surgery section. Even
though the animal will not live past the surgery, it is not acceptable for the animal to experience unnecessary pain and stress during the procedure. Aseptic technique and sterile instruments are not
required for non-survival surgery, but constitute good practice.
- Other Types of Procedures:
Describe any and all procedures that are to be performed on a live animal, even if you may think of it
as a minor procedure.
Examples of Procedures Using Live Animals: (this is not an all inclusive list)
- any blood collection method (via vein, cardiac puncture, retro-orbital, etc.)
- tail clips on mice
- tattooing, implantation of tracking chip, or any other identification method other than earnotching
- any data collection method that requires restraint, sedative, or anesthesia
- administration of any material, regardless of the method of administration
Procedures performed on dead animals need to be stated (i.e. dissection with removal of organs for
histological analysis, removal of tissue for in vitro tissue culture, etc.) but do not require the same
level of detail for procedures on live animals. The IACUC needs to know why an animal was
euthanized and what the final disposition of the animal is.
Examples: Procedures performed on a dead animal.
“At 3 weeks after administration of cancer cells, the animal is euthanized and the brain is harvested
for histological examination for tumor growth and the rest of the carcass is appropriately
discarded.”
“The animal is euthanized and the lungs are harvested to provide cells for in vitro primary cell
culture experiments. These in vitro experiments will consist of testing three viral gene delivery
systems, retrovirus, adenovirus, and adeno-associated virus for the ability to express the
recombinant surfactant protein. The in vitro experiments should be described in the
experimental design section (section B). Again, great detail is not needed, but in order to justify
the need to culture cells, one must describe a need for those cells.
Common procedures performed to maintain the health and well-being of the animal do not need to be
described unless they are directly related to the experiment.
Examples:
Clipping dog toe nails.
Weaning mouse pups
Enrichment programs need to be described if required by USDA regulations. The PI may state that
the TJU primate enrichment program will be followed.
Obviously, unexpected medical emergency procedures cannot be described in the AUP. In cases of
medical emergencies involving USDA covered species, the attending veterinarian must be
immediately contacted. All emergency medical procedures and drug administration must be
documented in the animal’s records.
5. JUSTIFICATION OF ANIMAL NUMBERS
The NIH requires an accounting of all animals used in experiments including those
animals obtained by breeding but not used in experiments due to genotype or other
considerations, as well as, those that may be used only to maintain the strain.
A common mistake in this section is to provide only the rationale for the number of experimental
groups based on the experimental design. What is most often overlooked, but is an essential, if not
THE essential part of the animal number justification, is the justification for the number of animals
in each group. For example, if you are testing a new drug, and in the absence of any prior data, it is
justifiable to use several groups of animals to determine the most effective dose as well as a control
group receiving a placebo. The PI can readily justify the need for the experimental groups receiving
different drug dosages and a control group receiving no drug. The mistake is only saying “Each group will
consist of 5 animals.” The number of animals per group needs to be justified.!!!! Why are 5 animals
per group needed? Why not 3 or 4 animals per group? This is where statistical analysis can provide a
strong justification.
The best justification for the number of animals per group is based on the need to produce
statistically significant data. Thus, statistical analysis can be used to determine the minimal number of
animals required for an experiment. Power Analysis is one way to determine group size, but is not the
only way. Consultation with a biostatistician is recommended. More animals per group than determined
by statistical analysis can be requested if justified. For example, the mortality rate of the procedure may
be sufficiently great to warrant the use of more animals per group to ensure sufficient animals remain at
the end of the experiment to obtain statistically significant data. Data should support the justification for
more than the statistically predicted minimum number of animals. For example, if the survival rate for a
procedure is known to be 80%, and Power Analysis indicated a group size of 10 animals, then requesting
13 animals per group (10/0.8 = 12.5, round off to 13) is reasonable, but requesting 20 animals per group
is not as reasonable. Merely stating that
“Some animals may die, so we are requesting twice the number of animals as determined by Power
Analysis.” is not acceptable.
Tissue or organs may be harvested from animals to be used for in vitro or ex vivo experiments.
In these cases, the number of animals needed is based on the amount of material obtained per animal,
as well as any genotypic considerations. A pilot experiment may be warranted to determine the amount
of material that can be obtained per animal and then submitting a Request to Increase Animal Numbers
once this data is obtained.
Often for experiments involving transgenic mice, breeding is required to produce the animals for
the experiments. Animal breeding protocols must be approved by the IACUC, even though these
protocols usually do not involve pain or distress issues. For animal breeding protocols, the IACUC
requires a reasonable animal use estimate, demonstrating thought and planning, and based on genetic
inheritance, known characteristics of the strains, and experience. An exact animal use number for
breeding protocols is not expected and is not realistic. This statement does not mean that the approved
animal number on a breeding protocol can be exceeded without prior IACUC approval. Nor should it be
interpreted to mean that accurate records of animals produced and/or used in breeding are not needed.
NIH requires that accurate breeding records be maintained that include all animals produced by
breeding, even those animals that will not be used in experiments due to genotype, are in excess, or
were accidentally generated.
Avoid the use of phrases that convey an indefinite amount, such as, “at least” or “will probably
need”. If indefinite phrases are used, the IACUC must interpret these phrases in a definitive manner, for
example, meaning “no more than” and “will need”, respectively.
Always check the Protocol Approval Notices (i.e. the letter you receive from the IACUC when the
protocol is approved) that the allowable number of animals to be used per year is the number of animals
that you requested.
6. RESTRAINT DEVICES
Physical restraint is the limitation of the natural movements and postures of an animal, either partially or
in total, by physical or manual means. Prolonged restraint is discouraged unless scientifically justified.
Restraint devices should not be used simply as a convenience but can, and should, be used to improve
the safety of hazardous procedures or to protect the handler from injury (i.e. primate bites). The period of
restraint should be minimized. In protocols requiring the repeated use of restraint, especially those
involving USDA covered species, it is recommended to train the animal to adapt to the restraint device,
personnel, and technique. If restraint is to be used for a prolonged period of time, animal monitoring
procedures and parameters must be described (use the Monitoring Plan Section). Here, describe the
device, frequency of restraint, duration of restraint, and provide a reasonable scientific justification for the
restraint.
7. PAIN/STRESS CLASSIFICATIONS
This is the USDA classification system for pain and distress. The reason the IACUC is asking for a
classification of the procedures into these classes and accounting of the animals involved in experiments
in Class D and E is that this information must be submitted to the USDA in our annual report. NIH policy
also requires accounting of all animals undergoing painful and/or distressful procedures.
A) Procedure Classification. List all procedures (breeding is a procedure – Class B) to be
performed and the pain/distress class by checking the appropriate box. (Add rows as needed,
boxes expand to fit text)
All that is required is a list of the procedures. Procedure details and descriptions must be in Section #4,
Procedures and Experiments. The comments column is optional. It is used if you wish to explain why a
certain pain/distress classification was assigned to a procedure. These should be brief comments. For
lengthy comments, place the comment in Section 4, with the procedure description.
Painful/Distressful Phenotypes:
Please note that the phenotype of an animal, whether transgenic or naturally occurring malady,
may place it into class D or E if pain or distress is associated with the phenotype even if no
painful/distressful procedures will be performed on these animals. In this case, state in the
procedure column “Phenotype – (Strain)”.
Class B is only for breeding procedures!
Procedure
B
Tail clip on pre-weaning mice
Phenotype – Abc null mice
Comments:
E
x
Implantation of Alzet pumps
Breeding
Class
C D
Adults will not be tail clipped
x
x
x
B) Number of Animals in Class D or E
List the total number of animals in these categories by year, and then add these together to derive the
total for the term of the AUP.
C) Veterinary Consultation: If animals in class D or E are USDA covered species (USDA Animal),
the attending veterinarian must be consulted before submission of this form.
This is a new USDA guideline. Provide the date for this consultation in the box using the
month/day/year format.
USDA Definition of “Animal”
“Animal: any live or dead dog, cat, nonhuman primate, guinea pig, hamster, rabbit, or any other
warm blooded animal, which is being used, or is intended for use for research, teaching, testing,
experimentation or exhibition purposes, or as a pet. This term excludes: birds, rats of genus
Rattus and mice of the genus Mus bred for use in research and horses not used for research
purposes and other farm animals, such as, but not limited to livestock or poultry, used or intended
for use as food or fiber, or livestock or poultry used or intended for use for improving animal
nutrition, breeding, management, or production efficiency, or for improving the quality of food or
fiber. With respect to a dog, the term means all dogs, including those used for hunting, security,
or breeding purposes.” Taken from: Animal Care Resource Guide, Research Facility Inspection
Guide, “Definitions” 2.1.2; 4/01.
8. SURVIVAL, NON-SURVIVAL, AND MULTIPLE SURGICAL PROCEDURE(S)
SUMMARY
Policies Addressing Surgical Procedures:
104.01: Guidelines for Anesthesia, Analgesia, and Tranquilization
104.02:
104.03:
104.04:
104.05:
104.06:
Guidelines for Survival Surgical Procedures in Rabbits and Higher Mammals
Postoperative Monitoring of Research Animals
Surgery and Postsurgical Care (S.O.P.)
Surgical Suite Policy; (Also see Veterinary Services)
Guidelines for Survival Surgical Procedures and Post-Operative Care in Rodents
Regulatory Definition of Surgery:
Surgery is any procedure requiring a skin incision or opening a body cavity. The use of anesthesia
does not constitute a surgical procedure if no incision is made to enter a body cavity or for tail clips, ear
notching, or tattooing rodents. HOWEVER, some non-surgical procedures require the use of
anesthesia and the use of anesthesia in these procedures must be documented here.
For example, anesthesia is used to perform retro-orbital eye bleeds, but this procedure is not considered
surgery. If an incision is made to implant subcutaneous pumps, or other material, this does count as
surgery (subcutaneous is considered as “entering a body cavity”.), however the use of a needle to implant
material subcutaneously, or elsewhere, is not considered surgery if no incision is made. If the skin over
the skull is cut and a hole is bored in the skull to allow injection of some material into the brain or
placement of a probe, this is considered surgery.
Terminal, non-survival surgery is considered a “surgery” and thus is considered in the multiple
surgery section.
a) Survival Surgery and Use of Anesthesia:
Survival surgery is any surgery where the animal regains consciousness, even if for a brief time. Provide
the procedure, anesthesia dose and route of administration. HOWEVER, some non-surgical
procedures require the use of anesthesia and the use of anesthesia in these procedures must be
documented here. For surgical procedures where more than 1 anesthesia is to be used (i.e. one for
induction and a different one for maintenance), or alternative anesthsia may be used, just list the
procedure for each type of anesthesia and explain in the Comments box. For alternative anesthesias,
state the criteria under which each would be used and which is the primary choice (availability is a
legitimate criterion). If multiple survival surgical procedures are performed and all use the same
anesthesia, then enter the phrase “All Surgeries” in the Surgical Procedure box. Post-operative or postprocedure analgesic use is described in Section 8: Animal Monitoring Plan. Survival surgery without
anesthesia will not be performed unless an extremely compelling scientific justification can be provided,
and even then, may not be allowed.
A) Survival Surgery and Use of Anesthesia
List procedures in which anesthsia is administered, whether the procedure is surgical or not, using 1 row per
anesthetic and procedure. Add rows as needed. Boxes expand to fit text.
Procedure
Anesthetic & Concentration
retro-orbital eye bleed
isoflurane
coronary ligation
ketamine/xylazine
coronary ligation
isoflurane
Dose
~ 3%, delivered to
effect
100 mg/kg ketamine;
10 mg/kg xylazine
~ 3%, delivered to
effect
Route/Device
inhalation/
vaporizer
IP
inhalation/
vaporizer
Comments: (Procedures are to be described in Section 4)
For coronary ligation, ketamine/xylazine is used for induction and isoflurane is used to maintain unconsciousness
during the procedure.
A) Survival Surgery and Use of Anesthesia
List procedures in which anesthsia is administered, whether the procedure is surgical or not, using 1 row per
anesthetic and procedure. Add rows as needed. Boxes expand to fit text.
Procedure
Anesthetic & Concentration
Dose
retro-orbital eye bleed
isoflurane
~3% to effect
retro-orbital eye bleed
pentobarbital
50-90 mg/g
Route/Device
inhalation/
vaporizer
IP
Comments: (Procedures are to be described in Section 4)
Isoflurane is the anesthsia of choice. If vaporizers are not available pentobarbital will be used.
b) Non-Survival Surgery
Non-survival surgery is defined as a surgical procedure performed on a living, anesthetized, animal that
does not regain consciousness before euthanasia. Non-survival surgery without anesthesia will not be
performed unless an extremely compelling scientific justification can be provided, and even then, may not
be allowed.
c) Multiple Surgeries
For Animal Use, multiple surgery is defined as two or more surgical procedures that occur at different
times on the same animal. If two or more surgical procedures are performed consecutively or
concurrently such that the animal does not regain consciousness between procedures this is counted as
a single surgery. For human patients, multiple surgery is defined as “per incision”, but this is not the case
for research animals.
Terminal, non-survival surgery is considered a “surgery” and thus is considered in the multiple
surgery section. For example, suppose the first surgery is performed to implant tumor cells under the
kidney capsule of a mouse. The PI then treats the animal with some anti-cancer compound for several
days and then performs a terminal surgery in which the animal is anesthetized and perfused. The
terminal surgery is a second surgical procedure (i.e. the animal is anesthetized for the perfusion and is
alive when the incision is made) and thus the PI is performing multiple surgeries.
However, if the PI were to first euthanize the animal and secondly perfuse the dead animal, this is not
surgery (i.e. the animal is dead before any incision is made) and would not count as a second “surgery”
and thus would not be considered as multiple surgeries for that animal.
Example: An animal undergoes surgery to generate coronary vessel damage, and at the same time, a
leg vein is harvested to provide cells for in vitro culture, these two surgical procedures count as only one
surgery as the animal does not regain conscious between surgeries. If two months later, that same
animal undergoes surgery to implant gene-modified endothelial cells into the injured coronary artery, that
is considered a second surgery and qualifies as multiple surgery.
Note that this applies only to surgical procedures and not non-surgical procedures, even if the same
animal is used. In the example above, if the gene-modified endothelial cells had been introduced by
intravenous injection (IV), that did not require surgery, then obviously the IV injection does not count as a
second surgery.
There is a limit to the number of surgeries permitted per animal, and yes, this limit is not clearly
defined. While one goal is to reduce the number of animals involved in experiments, that goal should not
be interpreted that it is better to allow excessive pain/stress to occur to a few animals rather than more
animals experiencing less pain/stress. In other words, it is not considered more humane to subject a
single animal to many occurances of pain/stress than to subject more animals to a only one occurance of
pain/stress. Unfortunately, there is no universally accepted method to score pain and distress that
defines a humane upper limit.
Example: Consider the above scenario where the animal underwent surgery to damage the coronary
artery and harvest a vein. That same animal undergoes a second surgery for implantation of genemodified endothelial cells. In this example, the PI is also interested in vein graft procedures and proposes
that 2 months after the implantation of gene-modified cells they will remove the damaged blood vessel
and also harvest a vein from the animal’s other leg to graft in place of the removed coronary artery
section. Two months later they propose to perform yet another surgery on the same animal to remove
the grafted portion of the artery and replace it with a synthetic blood vessel. This is excessive pain and
distress for one animal, unless there is a compelling scientific justification why this should occur. It is not
considered as more humane to use 1 animal for all 3 experiments compared to using 3 animals each
undergoing 1 experiment.
9. ANIMAL MONITORING PLAN
Policy 104.03
The provided examples may be fictitious and may not be accurate regarding drug
dosages, symptoms, or other information.
It may be difficult to predict pain/distress in an animal model or procedure. In this case, consider
that if the procedure were performed on humans is it likely to cause pain/distress. If it is likely
that the average human would experience pain/distress, or if the procedure is perceived as
causing pain/distress, then one must assume that the animal will experience pain/distress.
It is important to consider the “average” human, not just yourself. For example, when having a minor
cavity in a tooth repaired by a dentist using non-laser techniques, some patients opt to forego the
Novocain and will put up with the minor to moderate pain rather than go through the needle-stick and
subsequent facial numbness. In the same situation, other patients will opt for the Novocain, and others
prefer to be completely anesthetized.
Thus, for any procedure performed on an animal, you must consider that the animal cannot make a
personal preference known or signal that it is in pain, so the investigator must assume the animal will feel
pain and will benefit from analgesic/anesthesia.
General Points:
- Avoid using indefinite terms, such as, “may”, “possibly”, “might”, “should” and replace them with
definite terms, such as, “will” or “must”. The IACUC automatically interprets indefinite terms as
definite unless it is clear that a choice or decision is to be made. If a decision, or choice, is to be
made regarding post-procedure care, then decision criteria, and who will make the decision, must
be provided.
- “Greater than” or its symbol (>) is interpreted to mean “greater than or equal to”, or “at least”. For
example, if it is stated that euthanasia will be performed if animals lose > 15% of their starting
weight, then at the first instance when the animal’s weight has decreased by an amount greater
than or equal to 15%, the animal would be euthanized.
Definitions:
- “Daily” means every day, including weekends and holidays. If the animal(s) will not be monitored
by your group on weekends or holidays, do not use the word “Daily”. DO NOT ASSUME THAT THE
LAS AUTOMATICALLY MONITORS ANIMALS ON WEEKENDS. Depending on the species, LAS
may only check on food or water and not examine individual animals. Contact OAR if monitoring is
needed on weekends and make these arrangements before starting the experiment.
Criterion (pl. criteria) – In this context, criterion is a statement defining when an action will occur. For
example, “…when weight loss is equal to or greater than 15%, the animal will be euthanized.” is a
criterion because an action is stipulated to occur when certain conditions are achieved. There are
two basic types of criteria and parameters, subjective and objective.
Parameter – In this context, parameter is a variable, quantity, or quality to be examined as defined by the
criterion (criteria). For example, “…when weight loss is equal to or greater than 15%, the animal will
be euthanized.” -- weight is the parameter.
Subjective means that a direct measurement cannot be made and the monitor’s experience is used to
make the assessment. For example, “hunched appearance and lethargy” could be interpreted
differently by monitors. What one monitor considers “hunched and lethargic” warranting euthanasia,
another monitor may not consider severe enough to warrant euthanasia. It is recommended to
establish an assessment scale for subjective criteria, such as is used in clinical practice, to
standardize the health assessments.
Objective means that a direct measurement can be made that will be the same, within experimental
error, between monitors. An example of an objective criterion is weight loss because two monitors
weighing the same animal, at approximately the same time, should derive the same weight for an
animal within the standard error of the balance. Thus, a definitive point can be set for the criteria,
such as “…an animal is euthanized if its weight loss equals or exceeds, 20% from the animal’s weight
at the start of the experiment…”. So, if the animal weighed 1000 g at the start of the experiment, it
will be euthanized as soon as its weight drops to 800 g or less.
Each example presented below is independent of other examples.
The provided examples may be fictitious and may not be accurate regarding drug
dosages, symptoms, or other information and are intended solely for
instructional purposes.
A) Post-Procedure Analgesia: Class D procedures (One row per procedure: Add rows as needed; boxes expand to fit text.)
Procedure
Embryo Transfer
Analgesic & Concentration
Buprenex (0.3 mg/ml; 4 ul/g)
Dose
0.5 mg/kg
Route/Device
SC
This section addresses Class D procedures in which analgesic is provided to alleviate pain or distress.
Sometimes the analgesia intended for pain-relief post-surgery is administered during or before
the surgical procedure. Include those analgesias here, even though they not administered “Postprocedure”.
Note: If a specific analgesic is listed, that analgesic must be used. It is not permissible to
substitute a similar analgesic, or generic, without prior IACUC approval. It is permissible to list
more than one analgesic for the same procedure, but then criteria for the choice of analgesia must
be provided in the Comments box. These criteria may be very straightforward, such as, drug
availability.
The PI must maintain a readily accessible (to the monitors) analgesic supply to be used for any
procedures that are likely to result in pain and/or distress. For example, it is unacceptable to have
an animal experience pain and distress due to lack of analgesia when the procedure was known to,
or likely to, result in pain/distress. The PI is responsible for maintaining an analgesic stock and
may not perform the procedure (or surgery) if the post-procedure analgesic is not immediately
available.
B) Pain/Stress Criteria: List the criteria that determine pain/stress -- Classes D & E. (Box expands to fit text)
Pain will be assessed by whining, rapid breathing, lethargy, guarding of incision, nervous behaviors
such as constant licking, aggression, wound sensitivity, or weight loss of 5% from the previous weight
but not yet at the weight loss criterion for euthanasia.
Consult the listed policies for assistance to describe criteria for pain and distress assessments. It
should be recognized that different species have vastly different responses to pain/stress. For
example, a dog in pain may whimper or whine, but rodents rarely vocalize unless in extreme pain.
One possible explanation is that the dog is a pack animal and vocalization notifies the pack
members that it needs help, while rodents are often prey and vocalization would attract predators.
Policy 102.03 Pain and Distress
Policy 104.01 Guidelines for Anesthesia, Analgesia, and Tranquilization
This Example could also be presented in table format:
Pain Criteria:
 whining
 rapid breathing
 lethargy, sad expression
Incision Site
Examined for closure and healing
Incision infection assessed by:
 swelling





guarding of incision
nervous behaviors such as constant licking
aggression
wound sensitivity
weight loss – 5% of previous weight





exudates (pus)
redness
foul odor
fever
C) Euthanasia Criteria: List criteria that will result in euthanasia of the animal or removal of the animal from the
study – Classes D & E (Box expands to fit text.)
If any of the pain criteria in Section B, above, are not relieved in 24 hrs, the animal will be euthanized.
If weight loss of 15% or greater, relative to the weight of the dog at the start of the experiment, is
observed, or the animal does not eat for 24 hours, and attending veterinarian believes that this
condition cannot be resolved, the animal will be euthanized.
During the monitoring schedule provided below, if a condition develops such that the attending
veterinarian believes that the animal is in a state from which pain cannot be relieved, or satisfactory
health cannot be achieved, upon the advice of the veterinarian the animal will be euthanized.
This section is similar in format and content, to Section B above and section B criteria can also be used
here, however, these criteria determine euthanasia.
D) Monitors: List animal monitors and their decision authorization regarding use of analgesics and euthanasia to
relieve pain/stress – Classes D & E (Add rows as needed, boxes expand to fit)
Decision Authorization
Name
George Washington
Comments:
Analgesia
Euthanasia
x
x
The reason the IACUC wants to know the authority level of the monitors is to assess if adequate care
can be provided. The rationale is that for rodents and some other animals it is impossible for the
LAS or OAR staff to examine each cage every day and therefore, we must rely on the expertise
and integrity of the investigators to assess pain/distress. If only the PI can make decisions
regarding analgesic administration and euthanasia, and is unavailable for several days (i.e.
attending a symposium), and if the situation is not brought to the attention of the LAS, these
animals could suffer needlessly when they could either receive analgesics or be euthanized. Some
investigators distinguish between “removal from study” and euthanasia, so if this is the case, then
indicate this special case in the Comments box.
If LAS personnel are to be involved in animal monitoring, arrangements must be made with LAS
directors or the OAR director to ensure that LAS personnel are adequately informed and trained to
provide the necessary monitoring. Even though LAS personnel routinely check on the animal’s
health, one should not rely on this routine examination, especially in the case of rodent colonies.
E) Monitoring Plan: Provide the monitoring plan. Include analgesia (for Class D) schedule and other postprocedure components (suture removal, additional care, etc.) – Classes D & E -- (Box expands to fit text)
The animal will be examined twice daily for 3 days post-surgery, and then once daily for the remaining
17 days of the experiment before the experimental endpoint euthanasia, except as altered as
described below by adverse events.
Sutures in the skin are removed 7 days post-procedure.
The incision site will be treated topically with Neosporin, twice per day, for 3 days post-surgery. The
incision site will be examined for closure, swelling, exudates (pus), foul odor, or redness associated
with infection. Body temperature will also be monitored for fever. Should infection be apparent, a
topical antibiotic cream (Neosporin) will be immediately applied and the attending veterinarian will be
immediately consulted and treatment guidelines will be followed. Incision site infection has not been
observed in the 12 operations performed to date.
Buprenex (0.3 mg/ml stock; 4 µl/g) will be administered SC, every 12 hours starting immediately after
the incision is closed and continuing for 72 hours post-surgery. If the animal is still in pain at 84
hours (12 hrs after last Buprenex injection), another injection will be given and the attending
veterinarian will be consulted within the next 12 hour period to determine if it is likely that the pain
cannot be relieved and the animal should be euthanized or if a long-term analgesic (fentanyl patch)
or other analgesic regimen is appropriate.
The attending veterinarian will also be immediately contacted if it appears that the Buprenex treatment
is insufficient to control pain and the veterinarian’s recommendations will be followed.
In the 12 operations performed to date, the animal was pain-free by 84 hours post-procedure.
- Do not commit to more than what you intend to do.
- “Daily” means every day, including weekends and holidays.
- Adhere to all monitoring and analgesic schedule commitments described in the Animal Use
Protocol and make sure that all support personnel understand what is required of them to meet
those commitments.
- Maintain up-to-date monitoring records. The records may need to be made available to LAS or
veterinary staff. The monitoring records must be readily available to LAS/OAR staff during the
monitoring period (a location within the animal room is appropriate). It is recommended that the
records be archived after the experiment. Link for How to Document Animal Monitoring
Monitoring Plan – Examples of Anesthesia/Analgesia Administration Schedules:
Consider the following statements:
1) Buprenex (0.3 mg/ml stock; 4 µl/g) will be administered SC, every 12 hours starting after the incision
is closed and continuing for 72 hours post-surgery.
Notice the wording in this dosage schedule. It says “will be administered” and “continuing for 72
hours post-surgery”. That means that the last Buprenex injection occurs at 72 hours post surgery
and not 60 hours post-surgery (in this example, the Buprenex is effective for 12 hours). Also, do
not forget that it also means that the analgesic will be administered every 12 hours. If one
injection is given at 10 hours, and the next 14 hours later, that would be a violation of the
guidelines. Rewording that statement to “approximately every 12 hours” provides some leeway, but
it must be reasonable. For example, if 13 hours lapse between analgesic administration, that would
probably be marginally acceptable, but 16 hours between administrations is unacceptable.
2) Buprenex (0.3 mg/ml stock; 4 µl/g) will be administered SC, immediately after the incision is closed
and then provided as needed on an approximately 12 hour schedule for a maximum of 72 hours
post-surgery. At 84 hours post-surgery, if the animal is still in pain that cannot be alleviated, it will
be sacrificed.
In this statement, analgesia will be administered immediately after the surgery and subsequent
administration is based on the criteria in Section f. Remember that if any pain assessment
criteria are reached, the analgesic must be administered immediately.
Another Example Response:
The dog will receive buprenorphine (Buprenex) (0.01-0.02 mg/kg, SC), immediately after the
procedure and then approximately every 12 hours (± 1 hr; not to exceed 13 hours between
injections) for the next 48 hours. After the injection at the 48 hour time point, the dog will be
monitored twice daily for at least an additional 7 days. The first daily observation will be
performed between 7 and 9 AM and the second observation between 4 and 6 PM. After the
additional 7 days of twice-daily-monitoring (9 days post-surgery), the monitoring will be
decreased to once a day, Monday to Friday. Observations will include body temperature,
examination of the incision site, and pain assessment (Section f). The dog must have a normal
body temperature, be pain-free for at least 2 days, and the incision must be healing with no
infection, preceding the switch to the once-a-day monitoring schedule. Arrangements have
been made with the LAS staff to provide weekend monitoring during the once-a-day monitoring
phase.
If infection is suspected at the incision site (Section f), topical Neosporin Plus Pain Relief Antibiotic
Cream™ (Pfizer: Cream consists of Neomycin, 3.5 mg/g; Polymyxin B, 10000 U/g; Pramoxine
Hydrochloride, 10 mg/g) will be applied and the veterinarian will be immediately notified for a
consultation.
If any of the pain criteria are met (Section B) after the post-op, 48 hr injection, the buprenorphine
injections will continue as described above and the veterinarian will be consulted regarding
long-term analgesic options.
If fever, as first indicated by a warm, dry nose and confirmed by a body temperature in excess of
103 degrees F (39.4 degrees C), is detected, the veterinarian will be consulted. If a body
temperature of 105 degrees F (40.56 degrees C), or more, is obtained, the veterinarian will be
immediately consulted.
Example of Animal Monitoring Forms
F) Class E Justification: Provide a justification for Class E procedures: (Box expands to fit text)
Phrases, such as those below, but not limited to those below, are not acceptable justifications:
“may interfere” – it must be known that the analgesic will interfere with the experiment, or a reasonable,
scientifically based, explanation as to why all analgesics may interfere, must be provided. The
IACUC takes the view, in the absence of any further scientific justification, that it is just as likely that
analgesia may not interfere with the experiment and thus there is no reason to withhold analgesia.
“administration of analgesia may result in unnecessary death” – is only acceptable if it is known,
meaning that a reference must be provided and that all analgesics are known to increase mortality.
“based on our experience, the animal does not suffer pain or distress” – this is somewhat acceptable
but will require that pain/distress criteria are stated (section d) and the PI must have previous postprocedure records indicating that the stated pain/distress criteria were examined and that
pain/distress symptoms were not evident. The previous post-procedure records may be examined
during a IACUC laboratory inspection, and/or by the USDA and AAALAC.
To provide a strong justification, include specific citations stating that analgesic use is contraindicated
for the proposed procedure or experiment. The PI must consider all analgesics in the justification.
For example, if Buprenex (buprenorphine) is known to interfere with the experiment, does that also
mean that ibuprophen will interfere?
10. EUTHANASIA:
Euthanasia must be performed according to the American Veterinary Medical Association (AVMA)
guidelines. Conditionally acceptable methods, such as cervical dislocation or decapitation without
anesthesia, must be scientifically justified. Justifications such as, “we have always used this method”,
or “drugs or gases may interfere with the experiment”, or “we are leaders in this field and know what
we are doing” are not acceptable.
If the concern is that a drug or gas will interfere with the experiment, scientific data or a citation must be
provided to substantiate this statement, or a scientifically plausible explanation must be provided
describing how all drugs or gases used for euthanasia would interfere with the experiment. In this
case, the PI is encouraged to perform a pilot study to determine if drugs or gases (no, you are not
expected to test all of them) used for euthanasia would interfere with the experiment.
If using an anesthetic overdose as a means of euthanasia, usually 3 times the anesthetic dose is
sufficient to cause death. However, some anesthetics, such as avertin, have a broad therapeutic
range and death should be confirmed. With anesthetics that are prepared in the laboratory and not
obtained from a pharmaceutical supplier, such as avertin, it is always possible that there may be
variations in strength between batches.
The assurance of euthanasia is a new question in this section. This question was added in response to
PHS policy NOT-OD-02-062 (http://grants2.nih.gov/grants/guide/notice-files/NOT-OD-02-062.html).
The concern arose over incidents where supposedly euthanized animals recovered in the animal
disposal areas. In these cases, most scientists have discounted the possibility of resurrection and
proposed that the euthanasia method was not effective. Although the PHS policy specifically
addresses euthanasia with carbon dioxide, it is practical to confirm euthanasia from any chemical,
drug, or gas. Physical methods, such as, decapitation are usually definitive and do not need to be
assured. For perfusion, if a perfusate such as 10% formaldehyde is used, then it can be reasonably
assumed the animal is dead if the viscera have blanched (i.e. turned a pale color) and/or a major
organ, such as the heart, or blood vessel, such as, the aorta is removed or severed. However, care
should be exercised when exsanguination, without perfusion or subsequent removal or organs, is
performed to ensure that a sufficiently large blood volume has been removed. In these cases, a
physical method, such as cervical dislocation, removal of the heart, or cutting a major blood vessel,
such as the aorta, can be easily applied.
Perfusion is usually terminal and hence a form of euthanasia. THE PERFUSION PROCEDURE IS
DESCRIBED IN SECTION 4. Here indicate that perfusion will be used, the anesthesia, the perfusate,
and how euthanasia is assured.
Example:
10. Euthanasia
Euthanasia methods must be in accord with the AVMA Panel on Euthanasia for adult animals and/or NIH
guidelines for rodent embryos and neonates. For chemical agents and gases, provide dosages and route of
administration. For physical methods include anesthesia (dose, route). Perfusion and exsanguination are also
euthanasia methods. Boxes expand to fit text.
Method(s):
For brain tissue harvest for mRNA harvest: cervical dislocation while under anesthesia; isoflurane, 3-4% to
effect; or cervical dislocation without anesthesia as justified below.
Perfusion for organ harvest for histology: Anesthesia is Pentobarbital; IP; 50 mg/kg – Perfusate is 10%
formaldehyde.
For euthanasia of un-needed or excess animals from the breeding protocol, carbon dioxide to effect delivered
from a gas cylinder per AVMA guidelines will be used.
How is euthanasia assured?
Carbon Dioxide -- Continue gas exposure for 5 minutes after breathing has stopped or cervical dislocation on
supposedly dead animals.
Perfusion – blanching of internal organs
Justification for Conditionally Acceptable Euthanasia Methods
Provide a scientific justification for the use of any conditionally acceptable methods of euthanasia, as defined by
the AVMA (examples: decapitation or cervical dislocation without anesthesia), (Box expands to fit text)
Cervical dislocation without anesthesisa will only be used if we observe that the sample (brain mRNA) degrades
rapidly, or is altered, when anesthesia is used. If this is the case, only those animals from which these samples
must be collected will be euthanized by cervical dislocation without anesthesia. We have described a small pilot
experiment to determine the best euthanasia method in Section 4.
REMEMBER that you may have to provide documented evidence to a regulatory agency to
substantiate any claims.
Hazards
11. HAZARD IDENTIFICATION
Indicate the use of hazards in experiments by placing a checkmark in the “Yes” box next to that hazard
and then identify the hazardous material in the “Specify” box. Superscripted numbers refer to the
footnotes at the bottom of the table. These footnotes contain information regarding the necessary
approval for the use of these hazards in animals and clarifications of what is considered a hazard. Please
note that approval to use hazardous materials in animals must be obtained from the appropriate
committee(s) before the IACUC can approve the protocol.
Carcinogen
Approval Form
Teratogen
Approval Form
Chemical
Approval Form
Toxin
Approval Form
Irradiation
RSO Registration*
Radioisotope
Approval Form
Human Origin Tissue
Approval Form
Recombinant DNA
Approval Form
Biohazard
Approval Form
*RSO requires that you register to use irradiation equipment. IACUC does not need proof of this
registration as it is a federal crime for unauthorized personnel to use irradiation equipment and access to
this equipment is tightly controlled.
12. HAZARDOUS MATERIAL APPROVAL:
Either attach the approval letter or provide the appropriate IBC approval number or RSO license number.
NOTE THAT THE APPROVAL MUST BE FOR THE USE OF THE HAZARDOUS MATERIAL IN
ANIMALS. For example, even if an isotope is listed on your RSO license, you will still need to obtain
RSO approval for the use of that isotope in animals and there is a separate form for this (RO 13).
The most common mistake PI’s make is to forget to attach copies of their Hazardous Materials
approval letters (whether from IBC, EHS, or RSO) to Triennial Renewals and Revisions. Some
believe that if this information is provided once, that should be enough. While that is definitely logical, it
does not work because the reviewer does not have ready access to the files and cannot confirm that the
letter is indeed in the file. Also, changes may have been introduced into the protocol that change the
approval status and/or IACUC may not be aware that the PI’s hazard approval was suspended or
revoked. It could occur that the approval letter is the wrong type for the proposed experiments and the
administrators may not notice this discrepancy. Note that in the certification section at the end of the
protocol, the first statement is that all information provided is true and accurate. By requiring
resubmission of the approval letters, the IACUC shifts responsibility onto the PI to update the committee
regarding their approval status. At some point in the future, the University may establish an
administrative database available to the various regulatory committees that will contain information on the
approval status of any PI. However, until that happens, the IACUC will need to rely on the copies of
approval letters.
13. USE OF NON-PHARMACEUTICAL AND BIOLOGICAL MATERIALS IN ANIMALS
This section replaces Section 8, Use of Biological Material in Animals section of the old form. It was
disconcerting to the committee to see the number of PI’s who do not know what a biological is
considering many have advanced degrees in some branch or biology or medicine. A common mistake
was to designate viruses, bacteria, extracts from living organisms, etc. as non-biologicals. The USDA
also requires justification for the use of any non-pharmaceutical material in animals. Hence, the
committee changed this section to Use of Non-pharmaceutical Material in Animals.
NOTE: The use of out-of-date (expired) pharmaceuticals (i.e. drugs) or medical devices (i.e. catheters,
disposable surgical equipment, etc.) in animals in survival procedures is unacceptable unless a
justification can be provided and cost is usually not acceptable as the primary justification. Out-of-date
pharmaceuticals can be used in non-survival, terminal procedures, but IACUC approval must be
obtained. Please consider that the use of out-of-date anesthetics or analgesics, even for terminal
procedures, may be inappropriate due to loss of efficacy.
NOTE: Chemical and biotechnology companies, such as, Sigma-Aldrich, Chemicon, etc., are not
pharmaceutical companies and may not certify their chemicals for use in vivo.
From the Animal Use Protocol Form
Non-pharmaceutical/biological material is any compound whether biological (e.g. protein, peptide,
DNA, recombinant virus, cells, cell extract, etc.), or chemical not certified for in vivo use. Also
includes use of out-of-date pharmaceuticals.
A) Identify the source of the material (Manufacturer or production system, such as, bacteria, tissueculture cells, plants, etc). For cells or tissues, including cells used to produce recombinant virus
or any other biological material, identify species, cell type (also primary culture or cell line), and
source (supplier).
The following examples will be used consistently in all three sections (A, B, C). Thus Example
1 for section A refers to the same situation in Example 1 for sections B and C.
Example 1: Avertin (Many investigators use Avertin as an anesthetic agent, however, Avertin
is not sold commercially and must be made in the laboratory by combining chemicals
obtained from chemical companies.)
Avertin stock (100%) is made by combining 10 ml tert-amyl alcohol (Sigma) and 10 g 2,2,2,
tribromoethanol (Sigma) and is stored in the dark at 4oC. The stock is diluted 1:40 to give a
2.5% working solution.
Example 2: Recombinant Virus
Recombinant adenovirus is produced in our laboratory using tissue culture techniques. The
cells used to produce the recombinant adenovirus are of NIH3T3 cells of mouse origin,
which is the same species as the recipient animal.
Example 3: DNA
Plasmid DNA to be used is produced in our laboratory from bacteria (E. coli).
B) Justify the use of non-pharmaceutical grade chemicals, drugs, or similar compounds.
Example 1: Avertin
Avertin is not produced by a pharmaceutical company yet it is a commonly used anesthetic
in mice. Avertin is known to be relatively safe, often requiring huge overdoses to cause
death. The chemicals used to make Avertin are commonly available and not associated
with illegal drug use, such as is the case for the anesthetic ketamine. We are aware that
avertin stock and working solutions must be carefully monitored for discoloration that is
associated with a toxic breakdown product. Avertin stocks will be stored in the dark and
replaced every month. We will also use the purest sources of these chemicals. There is
information indicating that use of Avertin results in intestinal adhesions. As the mice will be
anesthetized only once in our experiments, and then euthanized a few days later, we do
not believe this would present a problem, and the animals will be monitored as described in
the Animal Monitoring Plan (section 8). If avertin use becomes associated with morbidity or
mortality, then we will modify the procedure to use a different anesthetic, with IACUC
approval.
Example 2: Recombinant Virus
There is no pharmaceutical company that produces the recombinant adenoviral strain
proposed in these experiments. Adenovirus can be concentrated to a high titer ensuring
that a high percentage of the target cells will be infected. Strong immune reactions have
often been associated with adenoviral use in vivo, however, this strain was developed to
minimize the immunological problems seen with the first generation vectors. Our in vitro
experiments have demonstrated that adenoviral vectors are the most efficient viral vectors
for the cell type to be transduced in vivo.
Example 3: DNA
No company provides pharmaceutical grade plasmid DNA. There is no known method to
produce substantial quantities of plasmid DNA that does not involve plasmid replication in a
host organism, such as, bacteria. Plasmid DNA will be combined with various liposomal
transfection reagents and used for in vivo transfection experiments. Our purpose is to
compare the efficiency of the new liposomal agents in comparison to other methods to
transfect or transduce cells in vivo. Plasmid DNA is relatively safe to use and should not
present any immunological reactions or chromosomal integration events that plague other
methods.
C) Indicate how the non-pharmaceutical material is sterilized or determined to be pathogen-free. For
animal cells, tissues, or organs, and/or derivative material, including, but not limited to, viral
vectors, antibodies, proteins, etc., MAP or IMPACT testing may be required. Attach the test
result(s) and date(s) of most recent test(s). Provide a justification if biological material is not
sterilized or MAP or IMPACT tested.
Example 1: Avertin
Avertin stock (100%) is diluted to 2.5% v/v using sterile 0.9 % saline and then filter
sterilized through 0.2 micron tissue culture grade filters into a sterile, dark, polypropylene
tubes using aseptic technique.
Example 2: Recombinant Virus
We have discussed this with the OAR and have had the cells IMPACT tested. IMPACT
tests determined the cells to be pathogen-free using the pathogen panel specified by the
OAR. Please see attached IMPACT data sheets. During culture, the cells are examined
microscopically for bacterial and fungal contamination. Mycoplasma testing is performed
yearly and to date, the cells are mycoplasma free. All tissue culture and subsequent virus
concentration methods will be performed using BL2 procedures and aseptic technique.
Example 3: DNA
The DNA is isolated from the bacteria using an Endotoxin-Free DNA plasmid kit from
Eppendorf. This kit uses alkaline lysis and filtration methods that efficiently remove any
bacterial cells and endotoxins. After purification, the DNA is further concentrated using
isopropanol precipitation and the isopropanol is also an anti-bacterial agent. Sterile
solutions are used to resuspend the DNA and to make the liposomes. The DNA is stored
in sterile microfuge tubes and handled aseptically. As a further test for bacterial
contamination, an aliquot of the final DNA solution is used to inoculate Luria-broth (a
bacterial growth media used to culture the plasmid containing bacteria) without antibiotics
and this is cultured for 24 hours at 37 degrees C and then inspected for microbial growth.
14. OTHER REGULATED MATERIALS OR PROCEDURES
A. Will neuromuscular blocking agent(s) be used?
Neuromuscular blocking agents are paralytics, in other words, they cause temporary paralysis of muscles
preventing movement. Neuromuscular blocking agents are often used in surgery on humans as an
anesthesia adjunct or for intubations. The use of neuromuscular blocking agents on animals is
strongly discouraged and must be scientifically justified. The reason for this is that the human patient is
often under a general anesthesia, or is conscious and can either signal or tell the physician that they are
in pain; however, the same is often not true for paralyzed animals in pain. In animals, anesthesia can
often be used in place of neuromuscular blocking agents for intubation. Consult with the veterinary staff
before use of any neuromuscular blocking agents.
Example of a neuromuscular blocking agent is curare and its derivatives, such as, Atracurium,
Alcuronium, Cisatracurium, Dimethyl tubocurarine iodide, Doxacurium, Gallamine, Mivacurium,
Pancuronium, Pipecuronium, Rapacuronium, Rocuronium, Tubocurarine, and Vecuronium.
B. Will hybridomas/ascites be produced?
The major concern is the production of ascites fluid in an animal, usually mice. Production of
ascites fluid involves injection of a hybridoma cell line into mice and subsequent harvest of the fluid which
contains high quantities of the monoclonal antibody (mAb). Specific TJU IACUC guidelines can be found
in Policy 104.09; Guidelines for the utilization of rodents in experimental neoplasia and ascites
production. Other information can be found at: http://grants2.nih.gov/grants/olaw/references/dc9801.htm#resources , and the following are excerpted from that website;
“…IACUCs must determine that (i) the proposed use is scientifically justified, (ii) methods that avoid or
minimize discomfort, distress, and pain (including in vitro methods) have been considered, and (iii) the
latter have been found unsuitable. Fulfillment of this three-part IACUC responsibility, with appropriate
documentation, is considered central to an institution's compliance with its Animal Welfare Assurance and
the PHS Policy.” (underline is my notation).
Please note that there is an emphasis placed on demonstrating that in vitro methods have been found to
be unsuitable. Therefore, justifications such as, “the way we have always done it”, “in our experience”,
and “best method known” cannot be accepted as justifications.
While justifications citing prohibitive cost are usually not accepted, for ascites production this
justification may be acceptable. Recommendation #1 of the Committee on Methods of Producing
Monoclonal Antibodies, Institute for Laboratory Animal Research, National Research Council (Monoclonal
Antibody Production; National Academy Press, Washington DC, 1999) stated:
“Recommendation 1: There is a need for the scientific community to avoid or minimize pain and
suffering by the animals. Therefore, over the next several years, as in vitro systems are further
developed, in vitro methods for the production of monoclonal antibodies should be adopted as
the routine method unless there is a clear reason why they cannot be used or why their use would
represent an unreasonable barrier to obtaining the product at a cost consistent with the realities
of funding of biomedical research programs in government, academe, and industry. This could be
accomplished by establishing in vitro production facilities in institutions.” (underline is my
addition)
HOWEVER, THE EASE OF PURIFICATION, HIGHER mAb YIELD, AND LOWER COST ARE NOT
ACCEPTABLE UNLESS CAREFULLY AND PROPERLY JUSTIFIED.
Justification Considerations:
1) Demonstrate that the mAb is not commercially available. (i.e. perform web-based search and provide
parameters of search, search of companies specializing in mAb’s, etc.)
2) Cell line did not adapt to tissue culture conditions
If this is used as a justification, the PI must have data indicating that mAb production in vitro had
been attempted and was unsuccessful using different culture conditions. Demonstration that
either insufficient mAb yield was achieved, inactivation of the antibody, or failure of cells to grow
must be documented and should be supplied with the form. If insufficient mAb yield is cited as a
justification, then a rationale for larger quantities of mAb must be provided. There must be data
that the PI considered common causes of low mAb production, such as, the media, contamination
with mycoplasma, and mAb purification methods.
3) In vitro produced mAb did not perform as well as in vivo produced mAb
If this is used as a justification, the PI must have data indicating that a comparison was conducted
between in vitro produced mAb and in vivo produced mAb.
4) Consider using an outside facility, a PHS Approved Animal facility, for in vitro or in vivo production of
mAb’s. This may be better and more efficient than trying to develop the in vitro methods in your
laboratory or in vivo production in our facility. Note that you will still need TJU IACUC approval to
have the mAb produced in vivo by a company and that the same regulations apply regarding this
form. Use of an outside facility does not obviate the need for IACUC approval.
List of PHS assured institutions/companies can be found at:
http://grants.nih.gov/grants/olaw/assurance/300index.htm
(Note: This is not a list of companies that provide mAb services, merely a list of assured
institutions/companies. Therefore, first pick a company and second, determine if they have PHS
Approved Animal Assurance.
4) If “prohibitive cost” is used as a justification, the following considerations must be documented.
a) prohibitive cost of purchasing mAb from outside sources using either in vitro or in vivo methods
(price quotes are recommended)
b) prohibitive cost estimates of in vitro production in the PI’s laboratory should include: cost of
equipment and supplies (usage may be estimated, but the cost per item should be citable – i.e. a
vendor’s price), cost of maintaining and housing animals, labor, and mAb purification from media
versus ascites fluid.
5) If using an outside facility, please include:
a) Name of company or organization producing the mAb
b) Organization’s USDA registration number (should be supplied by organization)
c) AAALAC-I accreditation status (should be supplied by organization or can check using website
below)
http://www.aaalac.org/accreditedorgs/index.cfm
C. Will any adjuvant(s) be used?
All adjuvants must be listed, even if they are not Complete Freund’s Adjuvant (CFA). Consult the
following IACUC policies:
Policy 104.08 Guidelines for the production of polyclonal and monoclonal antibodies in rodents and
rabbits
In the description of adjuvant use, any deviation from the policy must be noted and justified.
The use of CFA, and any adjuvants that are known to, or are likely, to cause severe inflammation that can
lead to necrosis must be described, whether or not severe inflammation or necrosis was ever observed
using your technique. The description must include:
a) the adjuvant to be used
b) adjuvant formula (what is in it) or company supplier
c) amount of antigen to be used
d) amount of adjuvant/antigen to be injected
e) location(s) of injection
f) frequency of injections
Also describe the use of any subsequent adjuvants.
Justification must be provided for the use of CFA and adjuvants that could cause significant inflammation
that can lead to necrosis.
Additional information on the use adjuvants can be found at the website address provided below.
http://www.nal.usda.gov/awic/pubs/antibody/
Animal Housing
15. ANIMAL HOUSING AND LOCATION WHERE PROCEDURES ARE
PERFORMED:
PLEASE NOTE THAT THIS SECTION DOES NOT RESERVE ANIMAL SPACE FOR YOUR
USE. THERE IS A SEPARATE FORM TO REQUEST ANIMAL SPACE. THE OAR
HANDLES ANIMAL SPACE REQUESTS AND THE IACUC DOES NOT HAVE CONTROL
OVER THE ASSIGNMENT OR USE OF ANIMAL SPACE.
Two common mistakes are forgetting to describe the transport of dead animals and transport within
the same building in which the animals are housed. The IACUC and other regulatory concerns are often
the same regardless of whether the animal is alive or dead. These sections apply even if the animal is
euthanized in the animal facility and is taken to a laboratory for harvest of tissues or necropsy.
A. Indicate where animals will be housed:
If animal space has not yet been assigned, then enter “Assignment Pending” in the box for the
Building name.
B. Describe special animal husbandry requirements:
Special animal husbandry requirements include the use of custom animal feeds, housing, bedding,
or other non-standard LAS materials. Custom feeds include feeds containing antibiotics or
analgesics. The use of wire-bottom cages or metabolic cages, special bedding requirements,
special lighting needs, etc. should be listed here. Do not rely on the Animal Use Protocol to obtain
special animal husbandry, but also discuss this with the OAR and facility managers.
C-1. Identify the procedure(s) and location(s) where procedures are performed on living or dead animals
on the TJU/TJUH Campus, including procedures performed in your laboratory.
If all procedures are performed in the same place, then enter “All Procedures” in the List
Procedures column instead of listing them separately.
Indicate whether the procedure room is in the animal facility or outside of it. NOTE: if at anytime in
the transportation of animals, a public corridor, elevator, or public space is part of the transportation
route, this counts as leaving the animal facility even if the two locations (animal housing room and
procedure room) are in the same building.
Your laboratory is probably a non-animal-facility location.
For USDA-Covered Species: All surgery and procedures involving USDA covered
species must be conducted within the Animal Facilities unless the use of other
facilities (such as your laboratory) is justified. Note that the USDA regulations
cover both living and dead animals.
The IACUC is concerned about the transport of animals, whether living or dead, between buildings
on campus or to non-animal facility locations. In order to provide better animal care, the use of
space within the Animal Facilities is required for all procedures involving USDA covered species.
However, this section must be completed even if non-USDA covered species are involved. This
also includes animals that are dead or will be euthanized after arriving at their destination.
C-2. Identify the procedure(s) and the location(s) for procedures involving living or dead
animals that are performed off of the TJU/TJUH Campus.
The IACUC is concerned about the transport of animals, whether living or dead, to off-campus
locations. For animals from TJU arriving at an off-campus facility, permission may be required from
the off-campus facility or their IACUC. Include a letter from the receiving institution indicating that
they can receive the animals. This section must be completed even if non-USDA covered species
are involved.
D. Justification for use of non-Animal Facility, and/or off-campus location(s): (Box expands to fit
text)
Justification based on convenience is unacceptable (removal for dissection may be an exception to
be determined by the IACUC). The most readily accepted justifications are based on the need to
use equipment (i.e. biological hoods for tissue harvest and need to process material quickly, use of
large or specialized equipment such as, a CT or PET scanner, etc.) that is either not located within
the facility or that cannot be used within the animal facilities.
E. Describe route to be taken and time during which transport may occur: (Box expands to fit text)
Do not forget that this is a two part question; both the route and time of transport must be
described. The route should be definitive but occasionally construction or other obstacles may alter
the route. It is recommended to keep alert to these obstructions and notify OAR if deviation from
the defined route is necessary. The OAR has defined routes between most buildings. Transport
within a building must also be described if the animals leave the animal facility at any point.
Concern is over the use of public elevators, corridors, or other public areas as the public may be
distressed observing research animals. The time period need not be limited to an exact time and it
is acceptable to give a time range, such as, “…between 8:00 AM to 12:00 Noon…”. Consult with
the OAR if transport is to an off-campus site as there are often local or state regulations that apply
to animal transport. Use of a personal vehicle to transport animals for short distances must be
approved by the OAR and may also need to be approved by the receiving facility and/or their
IACUC. If the TJU OAR will handle the transportation, then enter “Transport by OAR” in the answer
box.
F. Describe container/caging used to transport the animals. Only OAR approved containers/cages
may be used for transport of animals. (Box expands to fit text)
IACUC concern involves safety for the animal during transport, security of the animal, and shielding
from public view. This question must also be answered for transport within a TJU building. If OAR
will handle the transportation and provide animal containers, then enter “Transport by OAR” in the
answer box.
G. How long will live animals be held outside the animal facility?
The TJU IACUC uses the USDA time limit of 12 hours even though NIH allows up to 24 hours. Thus,
it is usually not possible to house animals in your laboratory overnight so carefully plan the
experiment to be finished in 12 hours. A justification is required in the “Comments Regarding
Animal Transport” answer box for holding live animals for more than 12 hours outside of the TJU
animal facility, including off-campus sites. Also indicate how long the animals are to be housed at an
off-campus site and indicate where the animals will be housed off-campus (an approved animal
facility, a laboratory, etc.). It is unacceptable to not know how long the animals are to be housed
outside of the animal facility, including housing at off-campus sites. If necessary, provide a time
range. The concern is over proper care of the animal regarding access to food and water, proper
bedding or cage changes, monitoring and security of the animals, and the environmental conditions of
the site (temperature, light, etc.).
H. Will live animals be returned to TJU animal facilities?
Returning rodents to the TJU animal facilities is discouraged and requires IACUC and OAR approval.
The concern is that the animals may be exposed to pathogens when outside of the animal facility and
then introduce these pathogens to other animals within the facility leading to an epidemic that could
result in the loss of a colony or colonies. There is less concern in this regard when larger animals
are involved as these animals are often not maintained under pathogen-free conditions, do not breed
at TJU, and the colony size is much smaller. Infection can be more readily detected in larger animals
and can often be treated effectively, whereas, in rodents the infection is often quickly lethal or
predominately lethal to neonates (Mouse Hepatitis Virus) or immune compromised animals, and can
go undetected until other animals or the sentinels are affected.
I. Will living or dead animals enter or travel through patient care areas?
There are at least two concerns here. One is that the animals may contaminate a patient area with
pathogens, cause an allergic reaction, or soil the equipment, such that a patient’s health could be
affected. Remember that some patients are immune compromised either because of the drug
treatment, their condition, or advanced age. Second, patients or other people in patient care areas
may become distressed upon seeing research animals being transported or worked on within patient
care areas. Thus, there are serious regulatory concerns not only by the IACUC but also the clinic and
hospital. Remember, patient care areas also include corridors, elevators, and stairs. Permission is
required from the Epidemiological Officer for the use of, or transport through, patient care areas and
to provide proper disinfection instructions for any equipment or areas (bench tops) that are also for
human use.
Justifications and Assurances
This section addresses the 3 R’s of Animal Use Alternatives: Reduction, Refinement, and
Replacement. In this version of the Animal Use Protocol, all the points requiring justification, such as,
the use of animals, alternatives to painful/stressful procedures, animal numbers, and research duplication
have been grouped together.
IMPORTANT NOTE CONCERNING JUSTIFICATIONS: Many investigators will use justifications,
such as, “…we have always performed the experiment this way…”, or “…we are leaders in the
field and therefore we must be correct…”, or “…I am a MD and know this stuff…”, etc. This type
of justification is not appropriate. Some investigators are offended if the IACUC asks them to
substantiate such claims or justifications with scientific data as it is interpreted that the IACUC
does not trust them. Remember, the protocol is not written for the IACUC members but for the
general public and regulatory agencies. In most, if not all, cases, the general public or agencies
will not know who you are or know of your reputation. It is also clearly stated in the guidelines
that a person’s rank, funding level, or degree earned do not confer proof of animal use knowledge.
16. SEARCH FOR ALTERNATIVES TO ANIMAL USE, ALTERNATE SPECIES, LESS
PAINFUL/DISTRESSFUL PROCEDURES AND RESEARCH DUPLICATION.:
You do not need to answer this section if no painful/stressful procedures, animal models, or phenotypes
are involved. For example, if the Animal Use Protocol is written to breed mice, and does not involve any
painful or stressful procedures, then the search for alternatives is not needed. (However, a search may
still be needed to address the concern for research duplication – see below). Painful/stressful procedures
include, but are not limited to, any survival or non-survival surgery, toxicity studies, tumorigenesis, sepsis,
application of noxious stimuli, tail clipping of adult rodents, and injection of agents that induce, or may
induce, inflammation or necrosis such as Complete Freund’s adjuvant. This concern also applies to
animals whose phenotype, whether natural or induced, results in pain or stress.
You might wonder why the TJU IACUC requires that the search for alternatives be performed for all
animals when the USDA does not regulate some species, such as, most murine strains. One reason is
that the list of USDA regulated species may include sub-species of the excluded species or nondomesticated animals captured from their environment. Secondly, the NIH prefers to see this regulation
extended to all vertebrate species. TJU decided that it is best to include all species to avoid trouble.
Further, the rules are changing and it may well happen that the USDA will regulate all research species.
This point was discussed in the web-based training module “Working with the IACUC”
General Comment on Justifications:
A common error in justifications is the use of phrases, such as, “commonly used in this type of
experiment”, “widely known”, “at other institutions”, “common knowledge”, “discussions with
colleagues”, “standard procedure”, etc. The mistake is that if any of these phrases are used, they
must be readily verifiable. All statements concerning a fact that is commonly known, or is a standard
procedure, should be confirmed by reference citations. For statements concerning other institutions, the
institution must be identified and a reference (IACUC policy, university website etc.) must be given so that
the statement can be verified. For statements concerning colleagues and meetings, see the section
below concerning types of searches.
Policy 102.03; Pain and Distress
The three R’s
Reduction, -- consider alternatives that would result in a reduction in the number of animals required for
the experiments. This may include consulting a statistician to determine the best statistical analysis
method or deriving a more efficient breeding strategy.
Refinement, -- consider if an invasive procedure could be replaced by a more efficient procedure
resulting in greater chance of survival, or a reduction in the time duration for pain or stress. Refinement
should also consider the use of anesthesia and analgesics. For example, if one analgesic group of drugs
would complicate the results of the experiment, might there be another group that is compatible with the
experiment.
Replacement, -- consider replacements for the use of animals, and consider if there are less
painful/distressful replacements for the procedures that are proposed.
A) The Search for Alternatives
Policy 102.05; Alternatives
Policy 102.06; AWIC Tips for Searching for Alternatives to Animal Research and Testing
http://grants2.nih.gov/grants/olaw/references/dc98-01.htm
The site listed above is supported by OLAW and contains
1) requirements for the search for alternatives regarding monoclonal antibody production in animals
(ascites)
2) laws governing the search for alternatives
3) several helpful websites to assist in the search for alternatives.
The USDA requires that a search be performed for alternatives to animal use and for all procedures
that may cause more than momentary pain and/or distress as described in USDA Policy #12.
One type of search must be performed, but it is preferable if more than one type were performed.
Types of searches:
Literature search – this is the most commonly used search as many scientists are familiar with the
PubMed and Ovid databases and is recommended by the USDA. The USDA requires the following
information regarding a discussion on alternatives:
1) Database and search engine used. (Examples: PubMed, Ovid, Scopus, MedLine)
2) Date search was performed -- must be within 3 months of the Animal Use Protocol submission.
3) Period covered by the search (dates searched); must go back at least 5 full years from present, but
going further back is recommended.
4) Keywords used (at least 5 words – suggestions: can also include words such as, “pain”, “distress”,
“alternative”, the painful/stress procedure(s), and can also include closely related species in the search
for alternatives)
5) Discussion of results. -- You should not include a list of all articles that were found, but inclusion of
relevant citations, that will be discussed, is permitted. It is expected that you will examine the list of
articles and address those, either in a general sense or specifically, that may apply to your research.
REMEMBER, THIS SEARCH ALSO PROVIDES PROOF THAT THIS RESEARCH DOES NOT
UNNECESSARILY DUPLICATE PREVIOUS WORK (PART E). So consider including a statement that
research duplication was considered.
The form now has separate entry boxes for points 1 through 4 to remind the PI to provide this data.
Example of Results Discussion: The literature search returned several hundred articles. The
keywords were used in combinations to reduce this number to a manageable group. Over 300
journal article titles were examined, approximately 20 abstracts were examined, and 2 journal
articles were read. We considered replacement of animals, replacement of painful or distressful
techniques, refinement of techniques to reduce pain and distress, if a reduction in animal
numbers was possible and to confirm that these projects do not unnecessarily duplicate previous
work. Results addressing specific points are discussed in the sections below.
The search also substantiated that these experiments are not an un-necessary duplication of
research.
Web search – a web search engine, such as Google, Yahoo, or Ask.com can be used to search the
world-wide-web for alternatives. Describe the web search using the same points as in a literature search
with the exception of the period covered by the search (unless the website provides the period it covers).
Specific Web Site Search – there are websites containing information on alternatives, addressing both the
use of animals and painful/distressing procedures. The USDA Animal Welfare Information Center and
the Johns Hopkins University website altweb, alternatives to animal testing are good websites containing
links to more databases. Keywords used in the web search, and the web pages that were read; must be
listed in the response.
Meetings, Colleagues: -- other professionals in the same, or similar, research field can be used as a
source for the search for alternatives. This may be the most reasonable resource if the proposed
procedure(s) is novel and it would be unlikely that any of the other resources would contain information
on the subject.
If you are citing a scientific meeting include:
a) the date of the meeting,
b) location,
c) organizing entity (i.e. the society, institution, etc. that organized the meeting – not the “sponsors” which
are usually corporations that donated money and advertise at the meeting),
d) the particular workshop/talk that addressed the issue
e) the person(s) who organized the workshop or gave the presentation(s), their affiliation(s), and
credentials.
If you wish to cite a pertinent discussion held with colleagues concerning alternatives, include:
a) name and qualifications of the person(s) taking part in the discussion
b) date of the discussion
c) location of discussion (includes time and type of discussion – E-mail, telephone, letter)
NOTE: The same 5 year time limit used in literature searches also apply to meetings and
discussions used in justifications.
B. Justify the use of animals for these experiments
This addresses why an in vitro system, computer modeling, or any other non-animal system, that would
replace the use of animals cannot be used. Obviously, if such a system were found and adopted you
would not be filling out this form.
Example: Our goal is to study how the immune system eliminates aberrant cell types, such as
cancer, from the body. The immune system is extremely complex involving several cell types,
multiple organs, and numerous epitopes or antigens. There is no in vitro or computer model that
can accurately reproduce the complexity of the immune system. Only a complex animal model
can supply all the components necessary to address our research question.
C. Justify why this particular species, and sex if limited to one sex, was chosen
You need to consider:
i) replacing the proposed animal model with a lower phylogenetic species (i.e. replacing a dog model with
a mouse model)
ii) if the proposed animal is a common pet species (cat, dog), or a non-human primate, consider
replacement with a common agricultural species that is not commonly used as a pet (pig, sheep). This
consideration is based on the public’s perception and is not really based on whether one species suffers
less than another species. For example, the public may be more concerned about the use of a dog to
train physicians in a microsurgical technique, as they think of the dog as a pet to which they form
emotional ties, than the use of a pig which many perceive as being raised for food production and hence
would usually be killed before its natural demise.
iii) the concern about the use of only one sex needs to be addressed on NIH grant submissions, and
hence, this is applied to all protocols whether or not they are funded by NIH (the university stated in its
NIH Assurance Statement that NIH regulations would be applied to all sources of funding).
D. Provide justification(s) if less painful/stressful alternatives for any of the
painful/stressful procedures described are known but not used.
This question was probably one of the most misunderstood questions on the old form. This section deals
only with procedures and the justifications regarding the replacement of animals in the experiment
(section B) and other species (section C) should have been discussed in the prior sections.
--The search for alternatives may reveal a less painful/distressful technique. If the less painful/distressful
procedure cannot be used, then the use of the more painful/stressful technique must be scientifically
justified. Justifications such as, “…the alternative may confound the experiment…” or “…we have always
performed the experiment using a standard technique and believe that replacing it with a newer, refined,
technique lessening pain and distress may produce differences from previous data…” by themselves are
not acceptable as the IACUC takes the view that it is just as likely that the less painful/stressful procedure
may not confound or complicate the experiment. A reasonable, scientific rationale must be provided
explaining why the less painful/stressful procedure cannot be used.
-- Consider the use of more humane endpoints.
-- Describe if an alternative was found that can be used and is likely to result in a reduction in pain/stress.
Example: An alternative was found for the proposed surgical biopsy method. The literature
search revealed 5 articles dealing with the use of needle biopsies for tumor analysis. One of
these articles specifically dealt with mammary tumors which are the proposed model. The
surgical biopsy procedure was chosen over the needle biopsy because the surgical biopsy
removes approximately 0.5 cm3 of tissue that we have found sufficient for our multiple
experimental needs. The needle biopsy removes only a small sample of the tumor, about
0.1cm3 or less of tissue that is appropriate if one is only performing limited histological or PCR
analysis. In our experiments, we perform extensive histological, biochemical, and genetic tests
and hence require more tissue.
Example: Alternatives to the use of tail clips for the isolation of DNA for genotype analysis are
known and include PCR using DNA isolated from a small ear flap sample, oral or anal swabs,
or hair bulbs. Unfortunately, there are several pseudogenes in the genome rendering the PCR
results unreliable. This situation dictates the use of DNA blots that require greater amounts of
DNA, and hence more tissue must be collected, to accurately genotype the animals.
Example: In the past, death was used as an endpoint in the Kaplan-Meier survival analysis for
this type of cancer treatment experiment. A journal article was found (Anim. Res. Guide 2005,
v16, pg 25) demonstrating that the onset of anemia leads inexorably to death in 4 days. Thus,
we have incorporated this observation into our experiments and will collect blood and perform
red blood cell counts to determine onset of anemia. Anemia onset is now the criteria for
euthanasia and is a more humane endpoint than death.
E. Research Duplication
The key word in this question is “un-necessary”. Previous experiments can be duplicated if scientifically
justifiable, such as, the need to verify published results, training, controls, etc. It is extremely rare for
an investigator to un-necessarily duplicate previous work.
I certify that:
There is nothing new here. This section is to inform you what responsibilities are assumed by you with
the submission of this AUP. Please note that all people involved on this protocol must read the final
version of this protocol, or be provided with a complete copy, or have ready access to a copy and
required to read it.
Attachments:
List all attachments, whether submitted electronically (PDF, Word Document) or as a paper copy.
Indicate how the attachment was submitted (electronic or paper copy).
Example:
University of Pennsylvania IACUC certification of training for our collaborator, Dr. B.
Franklin (electronic – Word document)
TJU IBC approval for the use of recombinant viral vectors in mice (paper copy)
IACUC Members (As of January, 2007; Check OAR Website for List of Current
Members)
Christopher Adams, Ph.D.
Orthopaedic Surgery
501 Curtis
215-955-8754
Christopher.Adams
@jefferson.edu
Oliver Dutton
Non-affiliated Member
American College of Physicians
6th and Race Streets
215-351-2562
oliverd@acponline.org
Fax: 215-351-2501
Stephen E. McIlhenny
Ph.D. Graduate Student,
Tissue Engineering and
Regenerative Medicine
Department of Surgery
215-955-4438
smcilhenny@gmail.com
David Anderson, Ph.D.
Pathology, Anatomy and Cell
Biology
511 JAH
215-503-5108
david.anderson@jefferson.edu
Sue Gotta
Biological Safety Officer
Environmental Health & Safety
Suite 1630 Edison Bldg.
215-503-7422
sue.gotta@jefferson.edu
Michael Oshinsky, M.D., Ph.D.
Neurology
398 JAH
215-503-0433
michael.oshinsky
@jefferson.edu
Raffaele Baffa, M.D.
Chairperson
Urology
1102A College
215-955-6961
Raffaele.Baffa@jefferson.edu
Jeffrey Joseph, DO
Anesthesiology/Cardiovascular
524 Main
215-955-1593
Jeffrey.Joseph@jefferson.edu
Vladimir M. Popov
Student Member
Department of Cancer Biology
1032 BLSB
215-503-9342
vladimir.popov@jefferson.edu
Steven Benowitz
Marketing/Communication/Writers
Non-Scientist Member
211 S. 9th Street, Suite 310
215-955-5291
steven.benowitz@jefferson.edu
Carlisle P. Landel, Ph.D.
Co-Chairperson
Director, Transgenic & Gene
619 BLSB
215-503-4539
Carlisle.landel@jefferson.edu
Troy Wilkins
IACUC Compliance Monitor
Office of Research Administration
304 Martin
215-503-4745
Troy.Wilkins@jefferson.edu
Alan Cahill, Ph.D.
Pathology, Anatomy and Cell
Biology
242 JAH
215-955-0630
alan.cahill@jefferson.edu
Cynthia Lang, D.V.M.
Veterinarian
Associate Director, Office of
Animal Resources
1150D BLSB
215-503-6167
cynthia.lang@jefferson.edu
Guang-Xian Zhang
Research Assistant Professor
Department of Neurology
900 Walnut Street, Suite 200
215-955-9425
guan-xian.zhang@jefferson.edu
Tung Chan, Ph.D.
Department of Medicine
813 College
215-955-4231
tung.chan@mail.jci.tju.edu
Linda Lowney
Non-affiliated Member
Town & Country Kennel
215-752-4781
TCKennel@voicenet.com
Fax: 215-752-7109
Judy Daviau, D.V.M.
Director, Office of Animal Resources;
Veterinarian
M-28, JAH
215-503-5885
Judith.Daviau@jefferson.edu
Stephen E. McIlhenny
Ph.D. Graduate Student,
Tissue Engineering and
Regenerative Medicine
Department of Surgery
215-955-4438
smcilhenny@gmail.com
How to Document Animal Monitoring
It is not required to attach a blank copy of the monitoring record sheet to the Animal Use Protocol,
however a copy may be required for the Annual Report of Animal Usage, and these next paragraphs
provide guidelines and suggestions on how to document the animal monitoring.
- Regardless of whether subjective or objective criteria are used, if the monitoring plan states a specific
parameter in analgesic/anesthetic administration criteria, or that euthanasia will be performed, that
parameter must be documented at each inspection, or the monitoring plan must describe when
such determinations will be made. You can specify on the monitoring form what “normal” means or
use a checklist listing the parameters and criteria.
- Analgesia/Anesthesia administration must be documented in the animal monitoring records.
- Monitoring records must be maintained up-to-date for the full period specified in the Animal Use
Protocol, even if the animals are apparently healthy and pain/stress–free before the monitoring
period ends. Even if the animal appears normal, a record of the examination must still be made and
the entry can be as simple as “Animals checked – no problems”. HOWEVER, if an animal is in
pain/stress at the end of the monitoring period, and the Attending Veterinarian agrees that the pain is
being successfully managed so that administration of analgesic (or other methods) should extend
beyond the period specified in the AUP, then the monitoring period must be extended for the duration
of such treatment, or modified as directed by the Attending Veterinarian. If it now becomes likely that
an extended monitoring period is needed, a modification describing the new monitoring plan and the
reason for the change must be submitted to the IACUC. The Attending Veterinarian can provide
guidance whether IACUC notification regarding this change to the monitoring plan is required.
- If an unexpected adverse event occurs, record the administration of any drugs, or interventions,
provided by the Attending Veterinarian, or any instructions provided by the Attending Veterinarian. It
is permissible, and often required from a regulatory perspective for the attending veterinarian to
provide medication or such treatment as is appropriate to maintain the animal’s health and well-being,
even if these medications and/or procedures not described in the Animal Use Protocol (i.e.
administered in response to unforeseen complications or accidents). Therefore, you must keep the
attending veterinarian informed if disease symptoms are expected to develop as part of the
experiment. Maintain a timed record (date and time) including a record of any symptom or
complication, the time action was taken (calling the veterinarian, administration of a drug, etc.), and
any subsequent additional monitoring beyond what was described in the Animal Use Protocol. The
record should also include who was involved.
For example, -- a dog undergoes a surgical procedure on Friday is monitored on the following Saturday
morning and the monitor observes unusual behavior that they think may be a sign of pain. The monitor
then contacts the Attending Veterinarian (AV) for a consultation. The AV tells the monitor to increase the
analgesic dosage and then to check the animal 1 hour later and report. The monitor must administer the
analgesic as directed by the AV and then record the time of the phone call to the AV, the name of the AV,
the instructions given by the AV and their (the monitor’s) subsequent actions. The monitor must also
record the re-examination one hour later, their observations, and subsequent actions.
Example of entry for the above situation:
Date: 3/5/06
Time: 8:00 AM
Monitor: I. M. Curtis
(Records all parameters as specified in the Animal Use Protocol)
Observation: The dog was observed to be unusually aggressive when I tried to examine the incision site.
Called Dr. Herriot at 8:30 AM and was told to increase the analgesic dosage to 0.03 mg/kg and observe
the dog 1 hour later to see if it is acting normally. Provided analgesic as instructed at 9:00 AM. Dog did
not enjoy receiving injection and the LAS caretaker R.O.T. Weiler provided assistance by restraining the
dog.
Date: 3/5/06
Time: 10:00 AM
Monitor: I. M. Curtis; Sam Gross (Note: Sam Gross is also listed as Support Personnel on the Animal
Use Protocol)
(There is no need to reassess all the parameters again as the observation at 8:00 AM fulfilled that
requirement as stipulated in this fictitious Animal Use Protocol.)
Observation: Sam Gross accompanied me in case the dog became violent. Dog was resting peacefully,
but was alert and allowed me to examine the incision. The incision appeared normal with minimal
swelling and redness. Do not think the wound is infected, but applied Neosporin topical cream just in
case and also because this Neosporin cream has the analgesic Pramoxine Hydrochloride which may also
help to alleviate any pain.
Example of Monitoring Record Sheet:
Date:
Time:
Monitor:
Check one
Comments:
Normal Abnormal
Subjective
Parameters
Overall appearance
Hunched
Significant hair loss
Skin pinch test*
Activity
Stool Appearance
Objective Parameters
Measurement
Comments:
Weight
Tumor Size
*Skin pinch test is used to assess hydration. Pinch the skin to make a skin fold on a normal animal and the skin
quickly flattens back into shape but a dehydrated animal’s skin will take longer to flatten out.
Analgesic Administered
Dosage
Comments:
Criteria for Euthanasia
Hunched appearance with dehydration.
Lethargic, no response to stimuli, that continues for more than 24 hours
Weight loss equals or exceeds 15% from starting weight
Tumor volume equals or exceeds 2 cubic cm or there is necrosis resulting in open wound
Bloody stool or constipation exceeding 1 day
Self mutilation that continues for more than 1 day or damage to appendage so it cannot be used -loss of toes -- wound on body equals or exceeds 0.5 square cm or exposes peritoneum.
Criteria for Analgesic Administration
Weight loss equals or exceeds 5% from previous weight but does not yet meet euthanasia criteria
Hunched appearance without significant dehydration
Guarding or chewing at wound/incision site
Self-mutilation
Self-barbering