Animal Use Protocol

James Madison University Institutional Animal Care and Use Committee Animal Use Protocol (AUP) (for Laboratory Animal Research or Teaching)

FOR OFFICE USE ONLY: Protocol #: Date Received:

1. Administrative Information (PLEASE SINGLE CLICK ON SHADED BOXES TO TYPE) Project Title: Project Dates: From: To: (Not to exceed 3 years minus 1 day) MM/DD/YYYY MM/DD/YYYY Principal Investigator: E-mail address: Phone Number: Lab Phone Number: Department: Address (MSC): Type of Project: Laboratory Animal Research Teaching If Teaching: Course # Course Title: Previously approved protocol number (if applicable): Use of recombinant DNA and synthetic nucleic acid molecule research: Yes No Institutional Biosafety If “Yes,” approval received: Yes No Pending Committee Review/Approval: IBC Protocol Number(s): Biosafety Level(s):

2. Project Funding (if applicable) Funding Source OSP Proposal # Start Date End Date Principal Investigator(s) VA- VA-

3. Experience and Training

List all persons working on this protocol. All individuals listed must satisfactorily complete all pertinent CITI training modules and enter the date below. In addition, face-to-face training of specific research and safety procedures must be provided by the Principal Investigator (PI) BEFORE students begin working with the animals. Those untrained in specific research and safety procedures must also be closely supervised by the Principal Investigator (PI) while learning these procedures.

Hands-on CITI Working Training (in the with the IACUC CITI Species SOP Training Date(s): Name: Lab): Training: Specific Training: 4. Project Summary for Layperson In lay language, briefly summarize the overall intent/objectives of the study. Remember, your target audience for this summary is a non-scientist. A suggestion is to write the summary on a high school grade reading level. Explain your project using terminology and concepts that are understandable to a non-scientist reader. Keep in mind your reader may not have background in specific procedures you are describing. Avoid terms and acronyms that are specific to your research field. Research protocols are commonly requested under the Freedom of Information Act (FOIA) and State Open Records. Should the university receive a request under FOIA or the state open record law, the summary ought to be written in such a way that the general public can understand.

Harm/Benefit Analysis: In 2-3 sentences and using lay language, compare the potential harm to participating animals with the potential benefits of the research for humans, animals and/or the advancement of science. 5. Animals Complete a separate column for each species or rodent strain to be used. If more than 3 species or strains are to be used, duplicate this page and insert appropriately. Please include all information that applies to the animals you propose to use in this protocol/proposed research.

NOTE: We recommend including enough animals to cover unforeseen circumstances, such as loss of animals.

B A C a. Species (common name) b. Strain

c. Gender M F M F M F d. Age range e. Weight range f. Number of animals/project year Year 1: Year 1: Year 1: Year 2: Year 2: Year 2: Year 3: Year 3: Year 3: g. Vendor/Source h. Building and room number where animals will be housed i. Building and room number where project will be performed j. If location of project is different from Yes No Yes No Yes No animal facility where housed, are animals housed for longer than 24 N/A N/A N/A hours in this area? k. Does this project require breeding Yes No Yes No Yes No of animals?*

*NOTE: If you frequently need to breed animals for multiple studies, or if breeding is a typical part of your research, you may want to submit a breeding protocol. Please use this template and indicate it is for breeding of animals for use on future studies.

6. Study Objectives a. In scientific language, provide a project summary describing the purpose of the study. Briefly explain the aim of the study and why the study is important to human or animal health. If this is a triennial resubmission, include an update of how far the project has proceeded up to this point and how the data will be expanded upon in the next 3 years.

b. List the specific aims or objectives for the project:

c. Please describe how animals will be monitored after normal work hours, weekends, and holidays: d. Does your project involve any of the following procedures? Please select the applicable boxes below and follow related instructions.

Yes Procedure Animal identification methods (e.g., ear tags, tattoos, collar, cage card, implant, etc.) Please specify:

Breeding Blood collection

Anesthesia

Non-survival surgery Animal pain or distress Prolonged restraint (physical restraint of a conscious animal lasting longer than 30 minutes)

Animal work done at another institution

Survival surgery (Please attach Form I: Surgery) Illness, induced disease, or pathological condition (Please attach Form II: Disease.) Special diets and/or food or water restriction (Please attach Form III: Dietary.)

Immunizations (Please attach Form IV: Immunization.)

Hazardous Materials (exogenous tumors, cells lines, hybridomas, biohazards, isotopes, etc.) (Please attach Form V: Hazardous.)

e. Provide a detailed description of the use of animals. All procedures to be employed in the study must be described. This description should allow the IACUC to understand the course of an animal from its entry into the project to the endpoint of the project. Cover all items checked above as instructed in the table. A best practice is to provide an acceptable range of the specific items described below to allow flexibility in the use of professional judgment and avoid non-compliance due to work conducted off protocol as a result of overly restricted parameters.

7. Justification for Animal Use a. Describe the number of animals to be used for each procedure in the table below. Procedure Animals per Groups per Procedures per Animals per Group Procedure Year Year Example: Oral gavage 10 10 1 100

*If this table does not fit with your study, you may create your own. However, the same information needs to be conveyed.

b. You arrived at these numbers by (select all that apply). Power analyses indicate that the proposed number of experiments is the lowest required for statistically valid tests of the hypothesis. The number of animals should be the minimum number required to obtain statistically valid results. Include justification for group size through a power analysis when possible: The experiments will compare the effects of several independent variables and therefore require many groups or cohorts. The outcome measures or phenomena being measured are variable and large sample sizes are necessary for statistically valid sampling. Differences from controls are expected to be small, and large sample sizes are necessary to distinguish differences reliably. The experiments are technically difficult and multiple attempts will be needed to obtain satisfactory data from each experiment. Other (explain): c. What is the rationale for using animals in this study? Select all that apply. This research requires behavioral measurements from living animals.

This research requires biological measurements or tissue samples from living animals.

Computer or other models cannot be used to replace animals in this research.

The research cannot be done in vitro.

This research is a direct extension of previous work on this species.

This research seeks to extend previous findings from other species specifically to this species.

Nothing is known about the physiological/behavioral phenomena of interest in this species.

More is known about related aspects of the physiological/behavioral phenomena of interest in this species than any other. This species represents the best compromise between the simplest (lowest) organism that can be used and the most relevant model system for human physiology/behavior. This species is the most cost-effective for the proposed research.

Other (explain): d. Search for alternatives to the use of live animals. A literature search for alternatives to the use of live animals is required. Please provide the details of your search below: d.i. List a minimum of 2 databases consulted (e.g. PubMed, Scopus, Toxline, Biological Abstracts, etc.). (1) (2) Additional databases: d.ii. Date of search: d.iii. Years covered by search: d.iv. Please provide a brief justification of date range used in search (e.g., anything past a certain date is outdated): d.v. Key words or search strategies used (e.g., models, in vitro, pilot, tissues, etc.): d.vi. Provide a brief summary of your search results: 8. Experimental Stress and Pain a. Prolonged Restraint a.i. Does this protocol/proposed research involve prolonged restraint (e.g. animals held in slings or small confinement apparatus)? Note: brief restraint, for the purpose of performing routine clinical or experimental procedures need not be described. Yes No

a.ii. If YES, indicate frequency and duration of restraint and describe the method of restraint and the rationale for its use. b. Research-related Stress: b.i. Will the animals be subjected to stressful conditions such as high intensity light or noise, water immersion of electrical shock as part of your project: Yes No

b.ii. Describe the stressor, its level and frequency, and the rationale for it use.

c. If conducting blood withdrawals, please describe the volume, frequency, withdrawal site(s), and methodology:

d. If conducting tail biopsies or tail bleeding, please describe procedure in detail:

e. Please provide a plan of action that will be followed in case of animal illness (i.e., initiate treatment, call investigator prior to initiating treatment, euthanasia):

f. Please describe the experimental endpoint criteria as defined in the SOP for Euthanasia (e.g., tumor size/appearance, percentage body weight gain/loss, inability to eat or drink, behavior abnormalities, clinical symptomatology, or signs of toxicity):

g. Indicate the appropriate pain and distress category(ies) and the number of animals in each. Sums should equal the total animals from Section 5 above. http://www.aphis.usda.gov/animal_welfare/downloads/Animal%20Care%20Inspection%20Guide.pdf, Policy # 11.

Pain and Distress Category Number of animals per year (based on USDA categories) Year 1 Year 2 Year 3 Totals Pain and distress category B – holding or breeding Pain and distress category C – minimal, transient, or no pain or distress Pain and distress category D – pain or distress relieved by appropriate measures Pain and distress category E* - unrelieved pain or distress *For category E animals, a scientific justification is required to explain why the use of anesthetics, analgesics, sedatives or tranquilizers during and/or following painful or distressful procedures is contraindicated. For category D and E animals, you must conduct a targeted literature search for alternatives to painful and distressful procedures. Please provide detailed results of your literature search below. g.i. List a minimum of 2 data bases consulted (e.g., PubMed, Toxline, Scopus, Biological Abstracts, etc.). (1) (2) Additional databases: g.ii. Date of search: g.iii. Years covered by search: g.iv. Please provide a brief justification of date range used in search (e.g., anything past a certain date is outdated): g.v. Key words or search strategies used (e.g., analgesics, reduce pain, non-invasive, training): g.vi. Provide a brief summary of your search results: 9. Euthanasia and Final Disposition of Animals a. If animals will not be euthanized, select disposition: Return to colony, flock, or herd

Adoption (http://www.jmu.edu/researchintegrity/iacuc/forms/adoption-release-form.doc)

Experimental animals may be transferred to another AUP for non-survival procedures. Breeders and non-experimental animals may be transferred to another AUP. Other:

b. If animals will be euthanized, check method(s) of euthanasia:

Carbon dioxide (CO2)-induced hypoxia followed by a secondary mechanical means of euthanasia If secondary, please specify: Exsanguination under anesthesia.

Perfusion under anesthesia.

Injectable agent overdose.

Decapitation. If used without prior anesthesia, you must provide scientific justification below*

Cervical dislocation. If used without prior anesthesia, you must provide scientific justification below* Other method of euthanasia. Please specify:

*Provide scientific justification for decapitation or cervical dislocation without anesthesia here: 10. Use of Anesthetic Agents/ Drug Enforcement Agency (DEA) Regulated Controlled Substances **Please see the Guidelines for Use of Non-Pharmaceutical Grade Drugs

a. Will this project involve any DEA regulated controlled Substance? Yes No If you answered YES: i. The PI will have a DEA license (or have applied for a license) BEFORE use of controlled substances under this AUP. Provide name on license and license number or the application confirmation number.

ii. The PI will include the proposed use of controlled substances at appropriate doses. iii. The PI will provide appropriate security (anchored cabinet with a minimum of 2 separately keyed doors and limited access to keys or lock combinations) for controlled substances. iv. The PI will be responsible for keeping records of controlled substance use.

b. Are the controlled substances to be used listed in either the anesthesia, euthanasia, or analgesia sections of this protocol? Yes No

Note: You must be individually licensed or have applied for a license with the DEA to use controlled substances at James Madison University. By signing this animal use protocol/proposed research you agree to abide by all JMU policies and procedures for use of controlled substances. Unauthorized use of DEA controlled substances may result in suspension of the protocol.

c. Specify in the table below the anesthetic agent for each procedure. Where anesthetic combinations are called for, list each drug separately. Researchers are encouraged to list potential drugs they may use in order to provide for flexibility.

Expected Duration of Procedure Drug Dose Route Anesthesia

d. Volatile (gas) anesthetic agents. d.i. Are you using a volatile anesthetic (e.g. isoflurane)? Yes No

d.ii. How will this be vented (e.g., fume hood) or scavenged (e.g. charcoal canister)? d.iii. List the location of the venting or scavenging equipment.

e. List who will administer the anesthesia and the qualifications of each person listed.

11. Principal Investigator’s Statement POLICY: It is University policy that the procurement, housing, care and use of animals should conform to the National Research Council Guide for the Care and Use of Laboratory Animals Eighth Edition (the Guide) or succeeding Editions and other relevant federal policies and procedures. The policy applies to all student/faculty research and teaching whether funded from external or internal sources. Please refer to the policy located at the following web address: http://www.jmu.edu/JMUpolicy/2202.shtml for additional guidance. I certify (select box) that the statements made in this request are accurate and complete and that the animal usage in this protocol/proposed research does not unnecessarily duplicate previous experiments. If I receive approval for this project, I agree to inform the IACUC in writing of any adverse events or unanticipated problems (http://www.jmu.edu/researchintegrity/iacuc/forms/iacuc- adverse-event.doc). I further agree not to proceed with the project until the problems have been resolved. I will not make minor or significant procedural changes to procedures involving animals without submitting a written amendment to the IACUC and will not undertake such changes until the IACUC has reviewed and approved them. I agree not to take visitors, including the media, into the animal facility without prior approval by the IACUC and my Department Chair. All photographs and videotapes of research animals and/or personnel will be for documentation of my research and for scientific purposes only. It is my responsibility to ensure that every person working with animals is appropriately trained.

I will not begin work on the procedures described in this protocol/proposed research until I receive notice of approval from the IACUC. I will keep a copy of this protocol/proposed research and all subsequent correspondence.

I have completed the Occupational Health Risk Assessment form and will ensure additional personnel also complete the Occupational Health Risk Assessment form. I agree to follow all laboratory safety procedures and wear the following protective gear when handling animals: lab coats, surgical gloves or other hand protection, and facemasks when appropriate. I agree to read and document that I have read and understood all applicable Standard Operating Procedures. I will ensure that all personnel will read and document that they have read and understood all applicable Standard Operating Procedures.

INVESTIGATOR SIGNATURE: To the best of my knowledge, I certify that the information provided in this Animal Care and Use Protocol is complete and accurate. I understand that approval must be renewed annually, that every third year the IACUC must perform a new review of my protocol, and that I might be required to complete a newer version of the Animal Care and Use Protocol and provide additional information at the time of the triennial review.

I also understand that IACUC approval must be obtained by an amendment to this protocol before I:  Use additional animal species, increase the number of animals used, or increase the number of procedures performed on individual animals;  Change procedures in any way that might be considered a minor or significant departure from the written protocol;  Perform additional procedures not described in this Institutional Animal Care and Use Protocol;  Allow other investigators to use these animals on other protocols, or use these animals on another of my IACUC-approved protocols. I further certify that:  No personnel will perform any animal procedures until they have been approved by the IACUC;  When new or additional personnel become involved in these studies, I will submit their qualifications, training, and experience to the IACUC and seek IACUC approval before they are involved in animal studies;  I will ensure that all personnel are aware of the institutional Occupational Health and Safety training prior to their contact with animals. The Standard Operating Procedures for Biology (http://www.jmu.edu/researchintegrity/iacuc/sops/index.shtml) or Standard Operating Procedures for the College of Health and Behavioral Studies (https://www.jmu.edu/researchintegrity/iacuc/sops- chbs/index.shtml) will fully explain the overall Occupational Health and Safety program and must be delivered face-to-face or be declined in writing to participate.  I will ensure that all personnel involved with the care of laboratory animals have received the proper on-the-job training in conjunction with the appropriate Standard Operating Procedures.  I will provide my after-hours telephone numbers to the animal care staff in case of emergency.

Principal Investigator: In signing this document I am ensuring that I have considered alternatives to animal models and alternatives to procedures that cause more than momentary pain or distress AND that I have paid careful attention to the appropriate species, the number of animals, and procedures used. All procedures involving animals will be performed under my direction. These activities do not unnecessarily duplicate previous procedures.

Name (Printed) Signature Date Co-Principal Investigator

Name (Printed) Signature Date Department Chairperson’s Endorsement: I have reviewed the proposal which is the basis of this Animal Use Protocol and endorse its submission.

Name (Printed) Signature Date Attending Veterinarian: I have reviewed this protocol in the context of provisions of the Animal Welfare Act, the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training and other statutes and regulations relating to animals.

Name (Printed) Signature Date When students are proposed in a protocol application, this certification must be obtained for all named students.

By signing below, the Responsible Student Researcher certifies that he/she is familiar with the University’s IACUC policy: http://www.jmu.edu/JMUpolicy/policies/2202.shtml. He/she further certifies that he/she has completed training regarding animal care and use protocols within the last three years and is familiar with the Standard Operating Procedures. Furthermore, by signing below, the Responsible Student Researcher acknowledges that he/she has read and understood this protocol.

Student Researcher(s) Name Signature of Student Researcher(s) Date

Submit an electronic version (in a Word document) of your ENTIRE protocol to [email protected]. Provide a SIGNED hard copy of the Animal Use Protocol (AUP) to: Office of Research Integrity, MSC 5738 820 Madison Drive Burrus Hall, Room # 110 Electronic signatures and scanned hard copies are accepted. If submitting electronically, a paper hard copy is not needed.