University of Miami Animal Care and Use Policies and Procedures

Universityof MiamiAnimal Care and Use Policies and Procedures

Updated: 7-25-09

Contents

I. Overview
II. Responsibilities of the Principal Investigator

III. IACUC Membership and Functions
IV. IACUC Records and Reporting Requirements

a. Reports to Institutional Official (IO)
b. Reports to Office for Laboratory Animal Welfare (OLAW)
c. Reports to United States Department of Agriculture (USDA)
d. IACUC Records

V. Personnel Qualifications and Training

a. Who requires training
b. Training Program Content

VI. Occupational Health and Safety of Personnel

a. Laboratory Inspections
b. Institutional Program for a Safe and Healthy Workplace:
c. Role of the Office of Environmental Health and Safety (EHS), and Healthcare Provider
d. Role of Principal Investigators and Department Supervisor

VII. Veterinary Care

VIII. Animal Use Protocols

a. Required Signatures and Review by the Departmental Chair
b. Justification for Using Animals
c. Justification for Using a Particular Species
d. Justification for the number of animals requested
e. Justification for Performing Potentially Painful Procedures
f. The Principal Investigator
g. Protocol Forms

IV. Reporting Animal Concerns

a. Investigating Allegations of Non-Compliance
b. The Role of the IACUC in instances of alleged Non-Compliance
c. Gathering Information
d. Assessment of Information by the IACUC
e. Formulating an Action Plan

X. Animal Procurement and Housing

a. Ordering animals
b. Animal Environment, Housing and Management
c. Animal Transfer To/From Facilities Outside of the University of Miami
d. Animal health and procedure record
e. Animal Transport Guidelines

XI. Guidelines for use of Zebra fish embryo/larvae use

XII. Policy for the Use of Avian Embryos

XIII. Policy for Use of “Biomedical Products” (i.e., dead animals, tissues, whole blood, etc) that are not owned by UM

XIV. Policy for Tissue Sharing

XV. DVR Training Policy

XVI. Photography in Animal Facilities and Laboratories

XVII. Animal Procedures

a. Amphibian oocyte collections
b. Anesthesia
c. Hypothermia anesthesia for rodent pups
d. Avertin (tribromoethanol) usage
e. Animal restraint
f. Animal Identification
g. General guidelines and procedures on the care and handling of reptiles, amphibians, and fish:
h. Avian Field Studies:
i. Antibiotics
j. Ascites Production
k. Complete Freund’s Adjuvant
l. Blood Draws, Volume and Frequency
m. Death as an Endpoint:
n. Embryo Removal
o. Environmental and Social Enrichment
p. Euthanasia Policy
q. Food Deprivation
r. Gas Anesthesia and/or Euthanasia
s. Pain Management
t. Surgery
u. Tail Biopsy

XVIII. Cell Line Testing

XVIIII. Tumor Burden

XX. Policy for the Use of Outdated and Expired Material

XXI. Genetic Manipulations

Glossary of Terms

I. Overview

The University of Miami is committed to high quality laboratory animal care and sensitive to the changing animal welfare landscape. This document, adopted by the University of Miami Institutional Animal Care and Use Committee (IACUC), provides guidance for the use of laboratory animals in research and teaching. The guidelines and policies described in this document were formulated using The Animal Welfare Act(AWA), Guide for the Care and Use of Laboratory Animals(National Research Council, National Academy Press 1996), and the regulations set forth by the United States Department of Agriculture(USDA). The intent of this document is to clarify the oversight position of the University of Miami IACUC, with respect to the aforementioned agencies, and to provide investigators with information to aid them in completing the IACUC animal use protocol forms. Deviations from these guidelines will be considered and may be approved by the IACUC on a case by case basis. The policies described herein mirror the Federal regulations.

The University of Miami considers it a privilege to use laboratory animals in research and teaching activities. The University expects that faculty and staff will diligently follow all Federal regulations and University policies. The intentional mistreatment of laboratory animals by any University of Miami employee and/or faculty member will be taken seriously and handled with an appropriate University investigation and actions that include notification of the funding agency, notification of Federal authorities, revocation of privileges to use animals in research and teaching at the University and/or termination/dismissal from the University in keeping with the Disciplinary/Professional Conduct Policyfor staff and the University Employee Handbookand the Faculty Manual for faculty.

II. Responsibilities of the Principal Investigator

The use of laboratory animals by the faculty at the University of Miami, in research and teaching is a privilege. As a condition of this privilege, the PI must agree to assume the responsibilities listed below, and further, the PI must agree to adhere to the PHS Policy on Humane Care and Use of Laboratory Animals, the ILAR Guide for the Care and Use of Laboratory Animals, USDA regulations as defined in the Animal Welfare Act and it’s amendments and all applicable policies of the University of Miami.

The Principal investigator is:

Responsible for all aspects of the project.
Required to have a current animal use protocol, approved by the University of Miami IACUC, before any activities involving live or dead vertebrate animals is begun. . This includes the timely submission of yearly renewal forms to prevent a lapse in protocol approval and suspensions that must be reported to USDA and OLAW.
Required to comply with all Federal regulations, laws and policies (see PHSand AWA Policies).
Required to assure that all participants on this protocol will follow the policies set forth in the Guide for the Care and Use of Laboratory Animals (The Guide).
Required to comply with all University of Miami Policies regarding the humane care and use of animals in research and teaching.
Responsible for the conduct of the work and to assure that all co-investigators, technical staff, students and visiting fellows or collaborators comply with all federal regulations and Institutional policies.
Required to provide all members of the research team the opportunity to participate in the UM occupational health program for laboratory animal users. This would include TB testing as appropriate.
Responsible for assuring that all participants in animal research studies under their supervision are properly trained in the humane care and use of animals in research and in the procedures used in the current model system according to Institutional policy.
Responsible for keeping the animal use protocol accurate and up to date. PIs must inform the IACUC when there are changes in personnel and funding.
Responsible to assure that the work follows the approved protocol. All significant changes in the protocol or procedures must be reviewed and approved before the procedure can be implemented.
Responsible for maintaining animal records in a secure but accessible location. Records must be available at all time for review by the attending veterinarian, the IACUC, the USDA and/or PHS.
Responsible for assuring that all pharmaceuticals, reagents and devices used in animal research are appropriate for the research and are within the manufacturer’s expiration date. The use of expired reagents or devices must be approved in advance by the IACUC. Under no circumstances can expired anesthetics, analgesics or antibiotics be used in either survival or non-survival surgery. All expired reagents should be clearly marked “expired - do not use” and returned to the DVR for disposal.
Responsible for assuring that all controlled substances are stored in a safe and secure manner.
Responsible for assuring that all animals in the protocol are properly identified and described using DVR cage cards.
Responsible for obtaining approvals from Radiation Safety, Occupational Health and Safety and/or the Recombinant DNA Committee prior to beginning animal activities regulated by these campus agencies.
Responsible for all personnel being familiar with and having access to the approved protocol.

III. IACUC Membership and Functions

The University of Miami Animal Care and Use Committee consists of at least 5 members appointed by the University of Miami Institutional Official (IO), Dr. Richard Bookman. The committee membership includes scientists affiliated with the University of Miami, at least one veterinarian, and at least one ethicist or non-lab animal user. In addition, the IACUC membership includes at least one person not affiliated with the University, in accordance with Federal regulations. The IACUC:

Is responsible for oversight and evaluations of institution's animal care and research program,
Conducts semiannual evaluations of the Institutional Animal Care and Use (IACUC) program,
Conducts semiannual inspections of all institutional animal care and use facilities;
Reviews and investigates concerns about animal care and use at the university;
Establishes procedures for the review, approval and suspension of animal activities;
Establishes procedures for review & approval of significant changes to approved activities;
Establishes policies for the review of procedures that require exemption from federal regulations, guidelines and policies (e.g. restraint, multiple survival surgery, fluid and food restriction, painful procedures).
Reports to the University of Miami Institutional Official.

IV. IACUC Records and Reporting Requirements

Reports to Institutional Official (IO):

The IACUC transmits reports of the semiannual program review & facility inspections to the IO. These reports describe departures from the Guide or PHS Policy, reasons for departure, distinguish between significant and minor deficiencies, and include plans and or schedules for correction of each deficiency. Each report to the IO includes minority IACUC views, if they exist.

Reports to Office for Laboratory Animal Welfare (OLAW):

The IACUC submits annual reports to Office of Laboratory Animal Welfare(OLAW) that document program changes & dates of IACUC semiannual reviews and inspections; advises OLAW of serious/ongoing Guide deviations or PHS Policy noncompliance; advises OLAW of any suspension of activity by the IACUC, and such reports include any minority IACUC views.

Reports to United States Department of Agriculture (USDA):

The IACUC submits an annual report to the USDA, which in addition to required information, details species -specific animal usage tabulated according to the level of pain and distress. The IACUC is required to explain IACUC approved deviations from the AWA and USDA regulation. Further the IACUC is required to report the details of any IACUC directed suspension of animal usage.

IACUC Records:

The IACUC maintains copies of the minutes of IACUC meetings and semiannual reports for 3 years. IACUC protocol review documentation is maintained for 3 years after end of approved study.

V. Personnel Qualifications and Training

Overview:

Who requires training:

The University of Miami has established and implemented a training program for professional/management/supervisory personnel, principal investigators, research investigators, instructors, technicians, trainees, and students. All DVR animal care personnel participate in the ALAT training program with the exception of personnel that have ACLAM certification and/or veterinarian with significant training in laboratory animal medicine. The default IACUC Policy is that all personnel having routine or significant contact with animals must satisfactorily complete formal training in the basics of care and use of animals in research (“Working with the IACUC”) prior to their involvement with animal research subjects. Exceptions to this rule are:

1. Professionals undergoing surgical training/retraining in classroom and/or non-research settings.

Such individuals may, at the discretion of the IACUC, participate in a laboratory exercise under the immediate direct onsite supervision of DVR veterinarians and /or appropriately trained personnel.

Satisfactory completion of “Working with the IACUC” is assessed by evaluating performance on an objective exam. Currently, core training is accomplished using a web-based training program (www.citiprogram.org). In addition, training on the specific type of animal used by investigators must also be completed. It is the responsibility of the investigator or staff to fulfill all training requirements. The IACUC office staff will monitor training status and protocols will not be approved until ALL members of the research team have their training requirements up to date. A refresher course or approved meeting/seminar must be taken every four years. Additionally, the online Occupational Health and Safety course must be updated every four years. Those individuals who will be observing animals, without contact, will be given an orientation by certified trainers. Documentation of said orientation must be provided to the IACUC office.

Training Program Content:

The content of the animal use training program includes humane practices of animal care (e.g. housing, husbandry, handling), humane practices of animal use (e.g. research procedures, use of anesthesia, pre- & post-operative care), research/testing methods that minimize animal pain and distress, and the use of hazardous agents. In addition, training on the specific animal model used must also be completed.

VI. Occupational Health and Safety of Personnel

Laboratory Inspections:

The default IACUC Policy is that all areas where animal experimentation is performed and where animals are housed are required to have a current (annual) approval from the Office of Environmental Health and Safety before IACUC protocols involving those areas will be activated.

Institutional Program for a Safe and Healthy Workplace:

The University of Miami has established an Occupational Health and Safety Program for all personnel performing work duties with animals. The program is designed to comply with the National Institutes of Health Office for Human Research Protections recommendations. The program is based on hazard identification and risk assessment. Its content include personnel training (e.g. zoonoses, hazards, pregnancy/ illness/ immunosuppression precautions), personnel hygiene procedures (e.g. work clothing, eating/ drinking/ smoking policies, specific procedures for personnel protection (e.g. shower/change facilities, injury prevention). It is the policy of the University of Miami to offer the following occupational health services to covered employees:

1. Annual audiogram for employees in a hearing conservation program.
2. Annual respiratory protection training and fit testing for employees who are authorized to wear respirators (includes personnel working with certain species or hazards).
3. Annual tuberculosis and allergy testing; semi-annual testing is required for all DVR personnel, Physical plant and security personnel, and other personnel with NHP contact.
4. Baseline health assessment.
5. Exposure incident evaluation and follow-up.
6. Follow-up health assessment, as required.
7. Pregnant woman counseling.
8. Vaccinations, laboratory tests, and titers maybe required according to the need of the experimental protocol, individual, and the animal species for which there is contact.

Role of the Office of Environmental Health and Safety (EHS), and Healthcare Provider:

The Office of Environmental Health and Safety (EHS), and the designated healthcare provider, shall provide the following functions:

1. Baseline health assessment
2. Exposure incident evaluation and follow-up
3. Follow-up health assessment
4. Laboratory tests as required
5. Maintenance of medical records
6. Monitor Hearing Conservation Program
7. Monitor Respiratory Protection Program
8. Periodic tuberculosis screening
9. Phlebotomy for serum banking
10. Pregnant woman counseling and consultation with an obstetrician
11. Vaccinations as required
12. Training in Chemical and Biological Hazards

Role of Principal Investigators and Department Supervisor:

It is the policy of the University of Miami that Principal Investigators and Department supervisors assure compliance with the following:

1. Inform covered employees of this occupational health program
2. Notify IACUC and EHS with the names and identification numbers of all employees participating in any activity involving recurrent contact with animals
3. Provide EHS with the following:
4. Employee’s name, work phone number, social security or employee identification number, department name and the animal species with which each employee will have contact.
5. Names of employees who no longer have contact with animals.
6. Names of employees who work in areas that have been identified to require hearing protection
7. Names of employees who are required to wear a respirator during work duties.
8. Ensure proper completion of necessary forms.
9. Allow necessary time away from work duties to enable employees to participate in this program.
10. Ensure that covered employees’ participation in this program is at no cost to the employee.
11. Provide appropriate personal protective equipment.
12. Provide employee training in proper use of personal protective equipment.
13. Ensure that employees having contact with animals receive information regarding:
14. Health hazards associated with animal species with which they have contact.
15. Safe work practices.
16. Follow-up procedures for an exposure incident.

The Institutional Animal Care and Use Committee require:

Principal Investigators to provide a listing of all employees who participate in each animal research protocol to IACUC.
Principal Investigators to provide EHS with the employee’s name, work phone number, employee identification number, department name and the animal species with which each employee will have contact .

The employee shall:

Complete acknowledgement form for the occupational health program for animal research personnel.
Immediately report an exposure incident to their supervisor and EHS.
Use appropriate personal protective equipment as provided by their department and as trained.

VII. Veterinary Care

The University Of Miami Division Of Veterinary Resources (DVR, 305 243-2310) has full time veterinarians on staff with training and experience in laboratory animal medicine. DVR veterinarians have access to all animals. Provision for emergency veterinary care is provided. Veterinarians provide guidance on handling, immobilization, sedation analgesia, anesthesia, and euthanasia. Veterinarians also provide guidance/oversight on surgery programs, and oversight of post-surgical care. The DVR attending veterinarian has exclusive authority on all veterinary care aspects of the animal care and use program at the University of Miami.

VIII. Animal Use Protocols

Required Signatures and Review by the Departmental Chair:

The IACUC policy is that all work with laboratory animals requires written approval by the Animal Care and Use Committee prior to the start of the project. In addition, all new animal use protocols require review and approval by the departmental chairman. If the Departmental Chairman is either the principal or a co-investigator on the study, scientific merit of the study should be verified by the Vice Provost for Research. Only an original signature of the appropriate individual is acceptable. IACUC protocol review includes reviews for significance, experimental procedures, pain and distress, scientific merit and for compliance with all OLAW Policies and all USDA regulations.

Justification for Using Animals:

Requests for the use of animals will only be considered if non-animal alternatives (e.g., cell lines) have been carefully evaluated and rejected. The scientific basis for rejecting the use of non-animal alternatives must be explicitly described in the protocol. This is particularly the case where the procedures to be used in the animal model will have the potential for causing pain and distress.

Justification for Using a Particular Species:

Use of a particular species of animal must be justified in the animal use protocol form. For example, Investigators may choose a particular species due to a) the extensive knowledge base concerning that species that is critical for the proposed research; b) a similarity between the biological systems of that species and the analogous human system, etc. Lower financial cost is not an acceptable justification for the use of one species or another.

Justification for the number of animals requested:

PIs must scientifically justify the number of animals requested, by describing the proposed study groups, the numbers of animals per group and replicate groups. The methods (statistical, non-statistical) for determining sample size must be included. The rationale must be based on previous (published) relevant work in the same animal model or a recent statistical analysis (e.g., power analysis). A modest allowance for attrition (model failure) may be acceptable, if justified. If such attrition is >20%, the PI will be asked to rigorously justify and explain what will be done to REFINE the model so that the number of animal subjects may be REDUCED in the future. New work, for which previous data do not exist, may be proposed as a small pilot study.

Justification for Performing Potentially Painful Procedures:

If the proposed studies will cause pain and distress to the animal subjects, PHS Policy and USDA Regulations require the administration of analgesics to minimize pain and distress. It is required that a narrative be provided in the protocol form that describes all potentially painful procedures to be used. In addition, the PI is required to provide a description of less painful alternative procedures, if they exist. If less painful procedures do exist for the study, the PI must clearly describe why the less painful procedures cannot be used. The IACUC is very rigorous in its review of the section.

Unrelieved pain/distress with chronic damage to tissue that is innervated, even if such pain/distress is unknown (pilot work), anticipated, expected or predicted, requires prophylactic analgesia. This requirement may be waived by the IACUC, if withholding analgesia relief is scientifically justified. Such justification must be specific for the proposed methods and models. A review of alternative analgesic strategies should accompany a request for a waiver of analgesia. The burden is on the PI to provide a convincing case to the IACUC that there is sufficient justification to grant the waiver. The IACUC will rigorously review the waiver request and make determinations on a case by case basis after careful consultation with pain specialists and the veterinary staff.

The Principal Investigator:

It is the policy of the University of Miami that only full time faculty of the University may serve as principal investigators (PI) on animal research projects, teaching or demonstration projects. Any other individual must identify a member of the full time faculty who agrees to be his/her sponsor and will accept responsibility for the training of the personnel, conduct of procedures, and all other issues related to the use of animals in compliance with federal regulations and NIH animal welfare policy.

Protocol Forms:

All activities with laboratory animals at the University of Miami must be reviewed and approved by the IACUC. Three types of animal request forms are provided for the convenience of the faculty. Careful completion of the forms will usually provide all the information needed by the IACUC to adequately review and approve the proposed activity.

A "longform" must be submitted:

i. For all new projects involving animal use (including "tissues only" projects).
ii. If it has been 3 years since the last IACUC approval on an existing project (e.g., year 04 of a 5 year project.)
iii. If there has been a substantial change in the methods or scope of an existing project.

A "shortform" should be completed:

i. For yearly renewals (required of all principal investigators).
ii. If the PI has previously completed a longform protocol application, and is resubmitting the same grant proposal to a different funding agency with new funding intervals (dates).
iii. If the PI is resubmitting a grant proposal to the same funding agency with new funding intervals (dates).
iv. If the PI is making clerical "changes" in the original protocol, such as the Project Title, the Principal Investigator, Investigators or staff.

Modifications to Approved Protocols:

We have has provided guidance to help define a "significant change." They include, but are not limited to:

i. Changes in the objectives of a study.
ii. Proposals to switch from non-survival to survival surgery.
iii. A change from a minor to a more invasive major surgical procedure.
iv. Additional sampling intervals.
v. Changes in the pain category.
vi. Changes in species or in the approximate number of animals used.
vii. Change in the PI.
viii. Changes in anesthetic agent(s).
ix. The use or withholding of analgesics.
x. Changes in the methods of euthanasia.
xi. Changes in the duration, frequency, or number of procedures performed on an animal.

Since the University of Miami IACUC recognizes that it is often difficult to determine the difference between minor and significant changes, the default IACUC Policy is that all changes in procedures, minor or significant, require review and approval by the IACUC.

The only exception to this Policy is the approval of minor changes limited to:

1. Editorial changes to the text of the protocol.
2. Signatures of the PI, Chair or IO are required.
3. Changes in contact information for people listed on the protocol.
4. The deletion of an investigator from the protocol.
5. The approval of tabled protocols when IBC or EH&S approvals are the only outstanding issues and they are received in the IACUC Office.

Thus, all changes (except those noted above) to the protocol must be approved by either (1) full committee review or by (2) the Emergency Designated Review (EDR) process. This requirement does not, however, preclude the veterinary staff deviating from approved procedures to appropriately respond to urgent medical emergencies.

4. An addendum may be used:

a. To request the approval of proposed changes to a protocol that do not alter the scope or objectives of the original protocol. Such changes may include, but not be limited to:
b. Minor changes in a surgical procedure.
c. Changes in housing or husbandry.
d. Addition of a mouse strain.
e. Changes to the protocol that result in a decreased ethical cost.
f. Minor changes in treatment strategy.
g. The addition of supplemental sampling and/or study intervals.
h. The addition of supplemental drug doses or different routes of administration.
i. The addition of drugs / devices of a similar class or mode of action.
j. Addition of new study group that will have procedures identical or similar to those already approved.
k. A change in anesthesia or analgesia strategy.
l. Request for additional animals.

The IACUC Policy is that any IACUC member may, during the Addenda review process, determine that the PI’s proposed modifications to an existing protocol represent a significant change in scope from the original protocol and recommend that a new "long form" protocol be submitted. The Chair and IACUC Office staff will review the recommendation and determine if the proposed changes represent a change in scope or objectives. If a new long form is required, the Chair will inform the PI prior to the meeting. A change in the scope or objectives of a study might be concluded if a short form or addenda proposed to do any of the following:

· Change the disease or injury model.
· Change from "tissues only study" to a survival surgery study.
· Change from non-survival to survival surgery.
· Change from purchasing animals from an approved vendor to creating and breeding transgenic animals.
· Change the pain category from B to C or from C to D.
· Change from rodents to a covered species.
· Markedly increase the number of animals.
· Change or withhold analgesia.
· Change the number of survival surgeries conducted on an animal.
· Restrain animals more than momentarily.
· Add the restriction of food and water to the protocol.

Protocol Submission:

Protocols are to be submitted electronically. If submitted via email or disk, a signed original must be on file in the Office of Animal Care and Use prior to approval of the protocol.

Protocol Submission Deadlines:

The deadline for submission of protocols to the IACUC office is 2 weeks prior to the meeting. Meeting dates and protocol submission deadlinesare published on the University of Miami IACUC website.

Protocol Review Process:

PHS Policy and USDA Regulations permit 2 type of protocol review. Protocols may be reviewed and approved by either the designated review (DR) procedure and at a duly convened meeting of the Full Committee review (FCR).

The IACUC Chair assigns 2-4 IACUC members to review the protocol, prior to the monthly IACUC meeting. All reviews are conducted anonymously. “Tissues only” protocols and addenda are reviewed by at least 2-3 IACUC members, including one veterinarian and 1-2 additional members. Long form protocols are reviewed by 3 to 5 members, including at least one veterinarian. Comments from the reviewers are collected, collated, and returned to the PI along with a letter summarizing the reviewer’s comments, concerns, and queries. The PI must resolve reviewer concerns in writing before the protocol is added to the agenda of the monthly IACUC meeting for approval action. Revisions in the protocol and/or written responses from the PI are then delivered to the reviewers for reconsideration. If the reviewers recommend approval of the protocol, it is placed on the agenda of the monthly IACUC meeting and discussed by the full committee.

The IACUC Chair reviews the list of protocols and selects the protocols that, due to their nature, will be discussed by the full IACUC. In accordance with PHS Policy and USDA Regulations that provide the opportunity for any IACUC member to call for a FCR of any protocol, the Chair distributes a list to the membership of the protocols to be reviewed and approved by the designated review process before the meeting. If, for any reason, an IACUC member calls for a FCR of a specific protocol, it is added to the meeting agenda and discussed by the full IACUC.

Emergency Designated Reviews:

The IACUC policy is that emergency designated reviews (EDR) can be conducted by the IACUC at the direction of the IACUC Chair. Consideration to conduct an EDR of a protocol will be entertained by the IACUC Chair upon receipt of written justification from the PI. Emergency designated reviews are not routinely granted and are conducted only under extremely extenuating circumstances. If an EDR is deemed justified, the protocol is assigned an IACUC number and submitted to reviewers as described above. The chair simultaneously polls the IACUC membership to seek permission to invoke the EDR procedure. All committee members are provided access to the protocol. If there are no objections to the EDR procedure from the IACUC membership, the protocol can be approved by the Chair when all concerns are met by the PI. If on the other hand, any IACUC member believes that the protocol should be discussed at a convened IACUC meeting, the EDR stops and the protocol cannot be approved until sufficient deliberation occurs at the next convened meeting of the IACUC.

Holding protocol policy after a protocol expires:

The National Research Council’s Guide to the Care and Use of Laboratory Animals requires that all animals used in research, testing, or education must be covered under a protocol approved by an Institutional Animal Care and Use Committee (IACUC). Conducting animal activities – including housing, breeding, interventions/experimental manipulations, and euthanasia – after a protocol has expired is a violation of NIH grants Policy as well as the PHS Policy. The Office of Laboratory Animal Welfare (OLAW) views such activity as a serious violation of the PHS Policy and these instances must be promptly reported to the OLAW by the Institutional Official.

In the event that a protocol expires while animals are still in-house, the IACUC has developed the following policy to handle animal care and disposition of these animals. It is expected that a new protocol be submitted and approved as soon as possible.

Principal Investigators (PI) are notified by the IACUC office prior to expiration of their IACUC protocol (see IACUC website for more details). If a new protocol application is not approved prior to the expiration of the old protocol, the PI and his/her department chairperson will be notified that all animals associated with the protocol will be immediately transferred to the DVR holding protocol the day after the protocol expires.

The animals will be maintained in the DVR facilities and cared for by DVR husbandry personnel.

Once animals are transferred to the Holding Protocol, the PI cannot perform any procedures on these animals. This includes data collection, sample collection, surgery, administration of test substances or any other procedures that may have been previously approved. (Breeding of animals to maintain critical transgenic lines may continue with permission from IACUC).

DVR per diem rates for animal maintenance will continue to be charged to the PI and account of the previously approved protocol.

Animals can be transferred off the holding protocol when the new protocol is finally approved or if they are transferred to another approved protocol which covers that species/strain and experiments.

Animals can be held on the Holding Protocol for a period not to exceed three IACUC review cycles (e.g., 3 months).

If after a period of two IACUC review cycles a new protocol (or request for transfer) has not been approved, the animals will either be euthanized or, if suitable, offered to transfer to other investigators with approved protocols. The institutional official will be notified.

Please note that notification to NIH funding agencies may be made if NIH funded animal protocols expire while animals are still present.

IX. Reporting Animal Concerns:

The humane care and use of animals in research and teaching at the U of M is of paramount importance to the IACUC. Concerns regarding the health and welfare of animal subjects used at the U of M can be reported anonymously and without fear of reprisal. Concerns should be reported via the University of Miami Compliance Hotline at 866-YOUR CALL or to the DVR and/or to the University Animal Care and Use Committee.

THE CONTACTS FOR REPORTING CONCERNS ARE:

The Office of the IACUC at 305-243-2311
Paul Braunschweiger, Ph.D., Chair, University of Miami Animal Care and Use Committee, 904 Sewell Building, Miami, FL 33136, (305) 243-3922 fax; (305) 243-6650. pbraunsc@med.miami.edu.
Linda Waterman, DVM. Director, Division of Veterinary Resources, University of Miami, 1600 NW 10th Avenue, room 1077, Miami, FL 33136 (305) 243-5396 lwaterman@med.miami.edu.
Mercy Dominguez-Nunez, the Office of Research, (305) 243-6415 mnunez@med.miami.edu.

Individuals reporting concerns should be as specific as possible and include the date, time, species, specific animal identification numbers, and the names of any University personnel involved. Routine animal husbandry concerns should be discussed with the animal caretaker supervisor of the facility. If the concern is not resolved promptly or reoccurs, contact the Operations Manager of DVR (Tom Beaty 305-243-2316) or DVR office (305-243-2310). If the problem remains unresolved, contact the University of Animal Care and Use Committee.

Investigating Allegations of Non-Compliance:

Institutional Animal Care and Use Committees (IACUCs) are sometimes faced with the failure of investigators to comply with animal research standards, including provisions of the Public Health Service (PHS) Policy and USDA Regulations. Incidents of noncompliance, reported to the IACUC, will be evaluated on a case by case basis. In order to help investigators be in compliance with IACUC policies and procedures, the IACUC provides oversight through the activities of the post-approval protocol monitor.

Investigator noncompliance may include, but not be limited to:

1. Conducting animal research procedures without IACUC review and approval.
2. Failure of personnel to adhere to IACUC policies and procedures.
3. Failure to follow direct instructions from veterinary staff.
4. Performing animal experiments without federally mandated animal welfare training.
5. Using outdated pharmaceuticals, dressings or devices unless specifically approved to do so.
6. Failure to adhere to aseptic technique.

The Role of the IACUC in instances of alleged Non-Compliance:

Frequently, the DVR veterinarians, animal care personnel, and the investigator can work together to prevent or correct minor compliance issues. However, the IACUC Policy is that serious or repeated problems always require the involvement of the IACUC. All allegations must remain confidential to the extent possible to protect all concerned. Those against whom the complaint is addressed will have an opportunity to explain the situation and mitigating circumstances before a formal investigation is initiated. Allegations of serious and/or ongoing non-compliance must have sufficient substance before the Chair will launch a formal investigation. (Reporting Animal Concerns).To evaluate and manage instances of investigator non-compliance, the IACUC Policy is that the process will consist of gathering information, assessing the information and formulating an action plan.

Gathering Information:

When investigator non-compliance is alleged, factual information about the event (e.g. date, time, participants, species, procedure performed, and adverse effects) and the type of incident (e.g. disapproved procedure, housing violation, and lack of skill/training) is collected and presented to the Chair of the IACUC for initial review.

The IACUC Chair will convene a subcommittee of the IACUC to hear the allegations and determine if there is sufficient cause to proceed with a formal investigation.

If the Chair decides that there may be sufficient justification to begin a formal investigation of alleged noncompliance, the Chair will convene a meeting with the PI and at least one IACUC member (e.g. attending veterinarian) to inform the PI of the nature of the noncompliance and that an investigation by the IACUC will be forthcoming. At this meeting with the PI, additional information from the PI will be obtained about the incident.

After all parties have had the opportunity to discuss the situation, the Chair will report the incident and the results of the PI interview to the membership at the next IACUC meeting. The Institutional Official will also be informed.

The PI will be informed that the incident will be reported to the IACUC and will be invited to provide a letter, documents, or records to the IACUC explaining the circumstances, any mitigating factors, and in some cases, actions that the PI will take to ensure that the noncompliance is not repeated.

Assessment of Information by the IACUC:

At the next meeting of the IACUC, the Chair will present the issues of noncompliance and any documents or information provided by the PI. Serious noncompliance could justify an emergency meeting of the IACUC to affect immediate actions.

The instance of noncompliance will be carefully reviewed by the IACUC. The Committee may empower a subcommittee to investigate the issues in more depth and report back at a later date. In addition, the IACUC may at this time vote to suspend all activity on any or all protocols being conducted by the PI while the investigation is underway. If a protocol is suspended by the IACUC, the Committee is obligated to inform the Institutional Official, OLAW, USDA (if covered species are involved), and the funding agency in a timely fashion.

The IACUC or subcommittee members should utilize a standardized set of questions to evaluate an instance of alleged noncompliance. These questions may consist of, but not be limited to:

· What experimental procedures were performed?
· What were the adverse consequences to the animals?
· Would such consequences have been prevented by prior IACUC review?
· Was veterinary medical intervention required?
· Was the investigator aware that IACUC approval was required before performing experimental procedures?
· What is the history of the investigator’s relationship with the IACUC?

Formulating an Action Plan:

Once the results of an investigation are reported to the IACUC, the Committee will vote to deliver one of the following:

· A written warning to the investigator, without notifying the investigator’s department chairperson or dean.
· A written warning to the investigator, with a copy sent to the investigator’s department chairperson or dean.
· Suspension of any or all previously approved projects.
· All resolutions and any recommendations for additional actions will be reported to the Institutional Official for appropriate action.

X. Animal Procurement and Housing:

Ordering animals:

All vertebrate animals for approved protocols that do not involve the collection of wild animals are ordered through the Division of Veterinary Resources (DVR). All domesticated animals used in research programs must be obtained from commercial vendors. If the DVR fails to find a commercial source for the requested animals for an approved protocol, DVR will inform the PI and the IACUC of such, and alternative arrangements will be considered with the approval of the IACUC and DVR. Investigators proposing to collect wild animals must justify these collections in the appropriate section of the protocol form.

Animal Environment, Housing and Management:

Minimum space requirements and details of housing must conform to the “Guide for the Care and Use of Laboratory Animals”. Proposed temporary deviations from this standard must be approved by the IACUC.

Animal Transfer To/From Facilities Outside of the University of Miami:

The IACUC policy is that transfer of animals (live or dead) between (to or from) UM and external facilities require prior approval from the DVR. The attending veterinarian may require that the PI document the health status of the animals being brought into any UM facility. An approved animal use protocol must be on file with the IACUC before animals may be brought into UM. Further, if animals are coming from other than a registered vendor, paperwork from the “sending” institution indicating approval of the activity must be filed with DVR and/or the UM IACUC. All animals arriving at UM from non-approved vendors or from collaborating investigators must be quarantined for an appropriate period upon arrival. The transfer of animals (live or dead) from UM to other research institutions requires prior approval of the DVR.

Animal Health and Procedure Record:

Records of animal health and observations must be maintained and made available for inspection. Animal records are to be kept in the animal colony in order to facilitate health monitoring, unless alternative arrangements are explicitly otherwise permitted by DVR and the IACUC. Important records would include, but not be limited to, surgery records, anesthesia records, drug administration records, observation times and analgesia administration records. Records must be readily available for review by the attending veterinarian and USDA inspectors.

Animal Transport Guidelines:

When animals are being transported within any of the University of Miami campuses (Coral Gables, Medical, Marine School or South Campus) human walkways and elevators should be avoided if at all possible. Transport between buildings should be done either very early or late in the day to avoid contact with the public. Animals should be concealed for discretion and for the health of the animals and other people. The method of transport should be appropriate to the size of animal and their animal biosafety level. Unless for acute or non-survival procedures, all USDA covered animals shall be moved by DVR personnel after receipt of a completed transfer request form.

· Rodents and guinea pigs should be moved in a clean transport box or clean DVR cage and covered with drape material or other porous, opaque cloth. The cage can be hand carried or placed on a cart.

· Larger animals such as rabbits, guinea pigs, dogs, cats, pigs and monkeys are transported in an appropriate transport box/carrier that should also be covered.

· Larger animals (e.g., pigs) should be sedated and covered for transport. Drapes or some form of containment of excrement should be used to prevent inadvertent contamination of public spaces during transport.

· Monkeys should always be sedated or anesthetized while in transport and they must be in a nonhuman primate transport box.

· Exotic species (such as amphibians, reptiles, birds) should be transported in closed containers which do not compromise them from extremes of temperature, moisture or overcrowding. Often, dark enclosures or bags are the least stressful on the animal.

· Animals that are part of studies which utilize biological or chemical hazards should be transported in a hepa-filtered cart or contained in a sealed apparatus or bag.

· If animals need to travel distances that require the use of a motor vehicle, in all circumstances the animals will be moved only by DVR personnel.

If there are any questions about transport procedures, please contact a DVR supervisor, veterinarian or office administrator.

DVR policy on bringing outside animals into animal facilities

· It is University of Miami policy that no animals (other than seeing-eye/working dogs) are allowed in any campus building. This policy refers to domestic or wild animals. DVR supports this policy and specifies that under no circumstances are any outside animals (other than from approved vendor or quarantine) allowed into any DVR animal facility areas. This policy is to protect the health of laboratory animals.

XI. Guidelines for use of Zebra fish embryo/larvae use:

Guidelines for Use of Newly-Spawned Zebrafish (Danio rerio)

Zebrafish are quickly becoming a popular choice for many biomedical research studies. They reproduce quickly and offer the investigator the opportunity to observe many critical developmental changes due to a relatively clear egg. Many transgenic lines are already commercially available and more are produced every year. With any breeding program, animal accounting issues can become challenging. This document serves as a guideline for when newly-spawned zebrafish are to be considered animals and no longer larvae or embryos.

Significant morphogenesis and growth occur from 10 hours through 4 days post-fertilization. Kimmel et al describe the larval period in “Stages of Embryonic Development of the Zebrafish¹”.

“By day 3 the hatched larva has completed most of its morphogenesis, and it continues to grow rapidly. Prominent changes during the next day include the inflation of the swim bladder and the continued anterior-dorsal protrusion of the mouth…” “Whereas during the hatching period the embryo is usually at rest, the early larva gradually begins to swim about actively, and moves its jaws, opercular flaps, pectoral fins, and eyes.”

Based upon these changes, the University of Miami has determined that on day 5 post-fertilization, the zebrafish is no longer an embryo and is considered a fish. Therefore, all zebrafish 5 days and older will be accounted for in the same manner. Exceptions to this guideline are for studies that involve alterations in the fish’s environment such as temperature or toxin studies. These studies can be granted exemption by the University of Miami IACUC.

This guideline is only meant to be in relation to zebrafish (Danio rerio). Guidelines for using other fish species will be determined on an individual basis.

1. Kimmel C, Ballard W, Kimmel S, et al. “Stages of Embryonic Development of the Zebrafish.” Developmental Dynamics. 1995 Jul;203(3):253-310.

XII. Policy for the Use of Avian Embryos:

The use of vertebrate animals in research, teaching and testing is regulated by the University of Miami Institutional Animal Care and Use Committee (IACUC). Avian embryos are not considered “live vertebrate animals” under the U.S. regulatory system. However, if an egg hatches intentionally or unintentionally they become live vertebrate animals and are subject to oversight by the IACUC. There is also increasing scientific evidence that avian embryos greater than two thirds of the way to hatching can experience pain. As a result, the University of Miami IACUC has developed the following guidelines. The guidelines address chicken embryos and should be adjusted for other avian species according to hatching time.

Investigators using avian embryos must inform the IACUC by means of the “Notice of Intent of Use Avian Embryos” form. If embryos will be sacrificed before embryonic day 15, the research will not be subject to IACUC protocol review, unless specifically requested by the investigator.

Chicken embryos 15 days or older are subject to normal IACUC policy for vertebrate animals and must be reviewed.

Chicken embryos from embryonic day 15 and older can experience pain and should be euthanized by decapitation or other rapid humane method.

Chicken embryos younger than embryonic day 15 are assumed to be unable to experience pain. It is recommended that embryonic day 14 or younger be euthanized by hypothermia, typically by placing the eggs in a -20º C freezer.

The IACUC recognizes that inadvertent hatching may occur. In these cases only AVMA approved euthanasia methods may be used and investigators are asked to describe their method for humane euthanasia of hatchlings.

These guidelines were developed based on the recommendations of the AVMA 2007 Panel on Euthanasia.

XIII. Policy for Use of “biomedical products” (i.e., dead animals, tissues, whole blood, etc) that are not owned by UM:

This policy concerns the purchase or acquisition of biomedical products, dead animal cells or tissues, from non-UM owned animals. Potential sites to obtain these tissues include, but aren’t limited to, slaughterhouses or other non-experimental sources (e.g., tissue banks, etc). Example tissue types include various organs, embryos, specific tissues, and whole blood (serum, plasma, or antibodies are excluded).

This policy does not apply to the following:

proposals involving animals to be euthanized for the purpose of using their tissues
proposals involving project-specific manipulation prior to euthanasia
proposal involving live animals at any point

Use of biomedical products (as defined above) is permitted with the stipulation that the vendor is known and DVR approved. DVR has a list of currently approved vendors for animal tissues and invertebrates from which investigators can order products. If the vendor they desire is not on the list, then they must consult with DVR prior to importation.

XIV. Policy for tissue sharing:

Policy for sharing of tissues and cells “tissue sharing”

Tissue sharing is defined as collection and use of tissues or samples following euthanasia of animals in the course of otherwise approved animal protocols from another PI in the institution (owned by UM). Tissue sharing supports and fulfills the 3R’s (Reduce, refine, replace) and is encouraged by the institution as it decreases the total number of animals used in research.

Procedures:

If tissues are requested for use by a PI, both the donor PI and the recipient PI must complete the Tissue Sharing portion of the protocol (or complete and submit it as an addendum). The procedures to be conducted on the tissues should be listed in the recipient PI’s protocol (or amended to it).

If data (other than pilot data) will be generated from tissue sharing, then the PI receiving tissues should be placed on the donor’s protocol and these experiments described.

XV. DVR training policy:

All investigators and research staff utilizing animals in research must undergo training. In addition to the required IACUC modules, personnel must be trained in the specific species and/or techniques to be performed. These modules will be required at the start of research prior to animal use and must be repeated every four years. In order to facilitate uniformity of current regulations and standard practices, all investigators will be required to take these modules regardless of prior experience. These modules will be available as didactic session or as on-line modules.

XVI. Photography in Animal Facilities and Laboratories:

The IACUC policy is that photography (video, digital, film) is not allowed in University of Miami animal facilities and locations where animal research takes place, without specific written IACUC approval or approval given via the Institutional Official. This also applies to anyone entering these animal facilities. Violation of this policy will lead to confiscation of your photographic equipment by university security as well as potential disciplinary actions. All photography related to protocols must be approved by the IACUC prior to the event. Questions may be directed to Paul Braunschweiger, IACUC Chairman, Richard Bookman, Institutional Official or Tony Artrip, Director of Security.

XVII. Animal Procedures

a. Amphibian oocyte collections

Amphibian oocytes are used in studies of embryology, biochemistry and molecular biology. Oocytes are collected by surgical laparotomy. Multiple surgeries in single animals is common, but must be scientifically justified.

These criteria should be followed:

i. Up to a maximum of 4 survival surgeries will be permitted with the 5^th being terminal
ii. The total number of surgeries shall be dependent on the health of the animal.
iii. Surgeries should be performed by trained personnel using appropriate anesthesia. Tricaine methane-sulfonate (MS-222) is appropriate and most commonly used.
iv. Frogs should be fasted up to 12 hours before surgery.
v. Surgeries shall be done using aseptic technique appropriate for amphibians
vi. Frogs will be monitored during the post-operative period
vii. Adequate time must be allowed between laparotomies. Investigators should alternate between collection of the left and right ovaries. In addition, investigators should rotate frogs allowing an adequate amount of time between surgeries.
viii. As stated in Green, 2003, additional studies are needed to establish the safety and efficacy of traditional analgesics on Xenopus, as well as the potential impact on oocyte production.

References

Green, SL. Postoperative analgesics in South African Clawed frogs (Xenopus laevis) after surgical harvest of oocytes. Comp Med. 2003 Jun; 53(3) 244-7.

Oocyte Harvesting in Xenopus Laevis. 2001 revision. ARAC Guidelines. http://oacu.od.nih.gov/ARAC/index.htm

b. Anesthesia Policy

Anesthesia methods must conform to the accepted standards of veterinary care. PIs should consult with the DVR staff to make the appropriate choice in anesthesia for their own purpose. Information on acceptable agents and dose for specific species is available at the University of Miami DVR web site.

Withholding anesthesia and analgesia for procedures which have the potential to cause more than momentary pain and distress in animal subjects requires rigorous scientific justification before the procedure will be approved by the IACUC.

c. Hypothermia Anesthesia for Rodent Pups

Hypothermia may be an acceptable anesthesia for neonatal mice and rats. Appropriate monitoring and re-warming following completion of the procedure is essential. Anesthesia for neonate rodent pups (less than 10 days after birth) will be immersion in ice until unresponsive to external stimuli. Surgical procedures must be performed before responsiveness returns (usually 5-10 min). No pup with hair will be anesthetized by this method; other anesthetics will be required. Pups will be re-warmed on heating blanket (37°C) before returning to the dam. Hypothermia is not an acceptable method of anesthesia/analgesia for rodent dams or reptiles.

d. Avertin (tribromoethanol) usage

Tribromoethanol (TBE) produces short-term surgical anesthesia with good muscle relaxation and moderate respiratory depression. TBE is not commercially available as a pharmaceutical drug, and can cause peritonitis and ileus in mice and rats, and the risk of peritonitis increases with each time it is used. Post-procedural analgesia has not been demonstrated, so use of another analgesic is generally required (eg., adding an alpha-2 adrenergic agonist such as xylazine or medetomidine (0.5 mg/kg) have sedative, muscle relaxant and analgesic properties).

Unless strongly justified in the animal care and use protocol, use of avertin is restricted to mice and rats, for a single survival anesthesia plus terminal/acute use. Justification for the use of TBE vs. a commercially available anesthetic must be provided.

These criteria should be followed:

· Avertin must be carefully prepared in the laboratory under aseptic conditions. This should include filtration through a 0.2 micron sterile filter.
· It must be stored in a dark bottle or foil covered container at a temperature of 4C.
· The solution should be tested to be certain the pH is greater than 5. It should not be used if the solution becomes discolored.
· Solutions are best prepared fresh for use, or stored for no more than one week.
· Avertin is not to be used twice in one animal on a survival basis (if used a second time that use should be terminal/acute). Where possible, inhalants should be used in place of Avertin.

References

Gopalan, Hegade, Bay, Brown, Talcott. Tribromoethanol-medetomidine combination provides a safe and reversible anesthetic effect in Spraque-Dawley rats. Contemp Top Lab Anim Sci 2005. Jan, 44(1): 7-10.

Liegi, CC, Artwohl, JE, Laszczynski, JK, Rodriguez, NA, Fickbohm, BL, Fortman, JD. Efficacy and safety of stored and newly prepared tribromoethanol in ICR mice. Contemp Top Lab Anim Sci. 2005 Jan:44(1)17-22.

Meyer, RE, Fish RE. A review of tribromoethanol anesthesia for production of genetically engineered mice and rats. Lab Animal. 2005 Nov;34(10)47-52.

Administration of replacement fluids (i.e. 1-2 ml of saline, subcutaneously) should be considered when large volumes of ascitic fluid are harvested.

Post-paracentesis mice should be monitored for 30 minutes for the development of circulatory shock.

Roughened hair coat, hunched posture, inactivity, pallor of the ears and eyes, tachypnea and dyspnea are compatible with shock. The presence of these signs warrants fluid administration. Persistence of these signs warrants notification of the veterinary staff or euthanasia of the animal.

Daily monitoring of each animal administered intraperitoneal hybridoma cells is required (including weekends and holidays). Personnel should look for hunched posture, roughened hair coat, anorexia, dehydration, weight loss, loss of body condition, inactivity, difficulty in ambulation, tachypnea, and dyspnea. Any of the preceding signs of distress warrant notification of the veterinary staff or euthanasia of the animal.

e. Animal Restraint

Animals may be restrained for procedural or handling purposes either chemically (sedatives, anesthesia) or physically. Physical restraint involves manual or mechanical means to limit some or all of an animal’s normal movement.

It is permissible to physically restrain animals briefly (usually minutes) for the purposes of sample collection, drug administration, therapy or experimental manipulation. Prolonged restraint pertains to procedures that require restraint of anaesthetized animals for longer periods of time and must be approved by the IACUC committee.

Restraint devices used should:

· Be suitable in size, design and operation to minimize discomfort or injury to the animal
· Be used for the minimum time required to satisfy the research objectives
· Allow clear observation of the animal at all times
· Not be considered as a normal method of housing
· Be fully justified by the PI in the animal protocol prior to use

Restrained animals should:

· Receive acclimation and/or training to novel restraining devices (with documentation)
· Be provided veterinary care for lesions or illnesses associated with restraint
· Be continuously monitored by trained personnel while restrained

Whenever possible, less-restrictive systems that do not limit an animal’s ability to make normal postural movements or ambulation should be used over more restrictive systems.

Exceptions to this policy require rigorous justification by the PI and approval from the IACUC committee prior to protocol approval.

i) Handling Non-Human Primates Without Sedation

Small and/or young non-human primates may be handled without sedation for minor procedures such as gavage or blood collection. A training period of at least 2 weeks is required to acclimatize unsedated animals to the procedures. Training of personnel must also occur before the actual procedures can begin. An animal behaviorist will be available for consultations. Once an animal reaches 3.5 kg and or has adult canine teeth, they may not be handled without sedation in order to protect the handlers, prevent harm to the animal and/or reduce the level of distress that the animal may experience. Should a non-human primate larger than 3.5 kg, or one with adult canine teeth, need to be handled without sedation or anesthesia for a research intervention, the following must be in place:

ii) Adequate methodology by which to safely restrain the animal:

This will most likely be via use of a pole and collar and chairing device. All other methods of restraint must be carefully reviewed and approved by the IACUC as well as the veterinary core.

Specific scientific justification must be provided if nonhuman primates over 3.5 kg and/or who have adult canine teeth will be restrained without sedation. Scientific justification must also be provided if animals will be restrained with some method other than the use of pole/collar/chair restraint if sedation is withheld.

iii) Appropriate training and acclimation:

Appropriate Training and acclimation of the animal to the restraint device, as well as training of personnel, must occur before the new methodology is put in place for a research use. A veterinarian must certify that the animals, as well as the individuals on the protocol performing restraint, have been adequately trained.

Pole and collar restraint of non-human primates:

Pole and collar restraint of non-human primates, as well as chairing of these animals must be monitored by a veterinarian on a weekly basis initially (for the first 4 weeks) and then monthly thereafter.

iv) Cage cleaning:

It is understood that husbandry staff transfer animals from cage to cage during room cleaning without chemical sedation.

f. Animal Identification:

Injection of microchips, and non-toxic tail or skin marking (e.g., tattooing) is preferable to toe clipping, tail clipping, or ear punching. If a PI proposes to use any of these methods scientific justification must be provided and approval will be considered on a case-by-case basis.

Toe Clipping for Identification of Rodents:

The Guide for the Care and Use of Laboratory Animals limits the use of toe clipping as a means of identification in rodents to well justified circumstances. This method involves removal of phalangeal bones of one or more limbs. The pattern of toe removal is the means of identificaton. According to the Guide, toe-clipping "should be used only when no other individual identification method is feasible and should be performed only on altricial (hairless) neonates." The IACUC has adopted the following policy in accordance with these guidelines.

Guidelines:

· Investigators must demonstrate that they have considered alternative means of identification (other than toe clipping).
· The Investigator must provide the IACUC with a justification of why toe clipping is necessary for identification of rodents.
· Toe clipping without anesthesia is limited to rodents within the first week of life and must be limited to one digit per extremity.

Information on alternative means of rodent identification is available from the Division of Veterinary Resources. One such alternative is the use of a micro-tattooing device which can be used on altricial neonates, including those with dark skin.

g. General guidelines and procedures on the care and handling of reptiles, amphibians, and fish:

All animal rooms housing reptiles, amphibians, or fish should have DVR room door sheets which allow for the daily logging of room temperatures, humidity, time of room checks, and cleaning procedures. Mortality information should also be recorded on this sheet or on its own log.

A temperature and humidity monitoring device (including recording of minimum and maximum daily temperature) must be present and in operating order in each room.

Any special ambient room temperature requirements (initiated by the PI and depending on the species) should be clearly documented on the room door and inside the room. This information should also be clearly stated in the PI’s IACUC protocol.

Water quality measurements (eg., temperature, pH, dissolved oxygen, etc) required for each room will vary according to the investigator, species and experiment. These requirements should be documented in SOP’s created by each investigator specific for their experiment and should be clearly posted in the animal room. Copies of these SOP’s should be on file with DVR and updated every 3 years with IACUC protocol renewal or sooner as needed.

Records of these water quality measurements are routinely collected and maintained by the investigator and their staff, but must be either located in the animal room or easily available to the veterinary staff upon request.

Names and contact information for DVR building supervisors and veterinary staff should be updated and clearly posted within each animal room to provide easy access to investigators in case of any animal health or facility emergency.

Investigator name, contact information (including an off-campus emergency contact number), protocol number and protocol expiration date should be clearly documented in each room either via individual DVR cage cards on each tank or on a single sheet of paper (laminated) and posted within each room. For the latter case, DVR cage cards should be presented upon request by the investigator or be on file in the animal room.

For situations where animals are not purchased from approved vendors by DVR, DVR cage cards may not be present. PI’s must create individual cage cards with all the pertinent information for placement on each tank.

All animal rooms must contain census records documenting the total number of animals present in each room. Census should be taken at least twice weekly by the investigator and/or their staff. A copy of the census sheets should be sent to the DVR main office (RMSB 1113) monthly.

For RSMAS and Cox Science buildings: Census numbers, especially when new animals are obtained, are required to document and monitor animal usage on the IACUC protocol. Individualized census sheets will be available for these buildings.

As for all animal rooms, these animal rooms should be maintained in clean, neat and orderly fashion. Surfaces that are not sanitizable (eg., unsealed or unpainted wood) are not permitted. If wood perches are required to maintain a proper environment for the animal, then a written SOP detailing the frequency of disinfecting and/or disposal of the wood perch is required to be in place inside the animal room. Rust is also not permitted on any surface inside animal rooms and must be addressed and removed as soon as detected.

Multiple survival surgical procedures:

A maximum number of 3 survival surgery procedures and 1 non-survival surgical procedure (with a minimum of two weeks in between surgeries) can be performed on any one amphibian. Most commonly, this surgical procedure is oocyte removal. For other species and other procedures, multiple survival surgeries will need justification and approval in the IACUC protocol on a case-by-case basis.

All fish, amphibians, reptiles, and birds are subject to the same animal care and use regulations and conditions (AWA, PHS guidelines) as are mammalian species. All procedures should be documented clearly in the approved IACUC protocol. Safety requirements (including appropriate PPE, bite kits, instructions, and emergency numbers in case of injury, MSDS forms, etc.) must be clearly documented and risk assessments must be in place where needed.

h. Avian Field Studies:

Birds captured in the wild and housed in cages should be observed at least once per day for signs of distress or illness. The following are taken as evidence of illness:

· Ruffled feathers or a “puffy” appearance when the body plumage is partly erect.
· Head tucked under wing or eyes closed when the bird would normally be awake.
· General inactivity, sitting on the floor of the cage, or difficulty standing on a perch.
· Drooping wings or a low stance while perching.
· Nasal discharges, sneezing or difficulty breathing.
· Pasting of feces around the vent and/or feet.

Centralized records are kept of each bird’s history, including date and place of capture, and signs of illness, any treatments, and date and place of release. In addition, records of minimum and maximum ambient temperatures are maintained. Reports of captures and releases are submitted to the state and federal permit-granting agencies, as required.

i. Antibiotics

The Default IACUC policy is that invasive surgical procedures require the use of prophylactic antibiotics. Exemptions to this policy can be made at the discretion of the veterinary staff.

j. Ascites Production

National Institute of Health guidelines require that in vitro technologies are the default method to produce monoclonal antibodies. The University of Miami IACUC Policy is consistent with these guidelines. Requests to use in vivo ascites production methods (intraperitoneal hybridoma ascites tumors) will be approved only if clear, sound scientific justification is provided by the PI. It is the responsibility of the PI to demonstrate that in vitro methods will be unsatisfactory for the specific application proposed. A rigorous scientific justification would include data to support the PI’s argument. Any proposed use of the ascites method should also include measures the PI will take to reduce pain and suffering (e.g., the use of analgesics) in the tumor bearing animals.

Upon prior approval from IACUC, hybridomas may be prepared according to the following general guidelines. These guidelines are designed to insure that animals experience minimal pain and distress, yet provide sufficient quantities of antibody for research use.

Immunization volumesshould not exceed 0.2 ml when administered subcutaneously or 0.5 ml when administered by intraperitoneal injection. In vitro immunization procedures have been reported to be beneficial when antigen is in limited supply and previous attempts at in vivo immunization have failed. Other advantages of in vitro immunization are the requirement for fewer animals and the immunization period is reduced to 4-5 days.

Pristane priming: The volume of pristane should not exceed 0.2 ml. Freund's incomplete adjuvant has been demonstrated to be as good or better than pristane as a priming agent.

It is required that hybridomas be tested for the presence of adventitious viral and mycoplasma agents prior to use in animals according the current cell line testing policy. Viral contamination is common in murine leukemia and transplantable tumor specimens. Inoculation may result in infection of the animal and spread of disease through the vivarium. Also, lymphocytic choriomeningitis virus has been isolated from tumor cell lines, which is capable of producing illness in humans.

Abdominal Paracentesis: It is recommended that the number of abdominal taps should be limited to two taps. The second tap should be performed after the mouse has been euthanized. However, in more aggressive cell lines which cause significant morbidity it may be necessary to limit taps to less than this and vice versa for cell lines that are more slowly progressive and cause minimal morbidity, more than two taps may be appropriate.

Ascites pressure should be relieved when visible abdominal distention becomes evident, and prior to the development of marked abdominal distention with associated clinical signs of pain or distress. The undesirable side effect of painful abdominal distention can be avoided by daily monitoring, relieving ascites pressure, or terminating the procedure. If paracentesis does not relieve abdominal distention the abdomen of the mouse should be gently palpated to determine if distention is due to intra-abdominal solid tumor growth. Confirmation of intra-abdominal solid tumor growth warrants euthanasia of the animal. To minimize bacterial contamination, preparation of the paracentesis site with an antiseptic is recommended. It is recommended that a large gauge needle (20 to 18 gauge) be utilized for paracentesis. Ascitic fluid is often viscous and a large gauge will allow more rapid collection thereby reducing the period of restraint required.

k. Complete Freund’s Adjuvant

Improper or unnecessary use of Freund's adjuvant may cause inflammation, induration, or necrosis in animals. Disseminated granulomas have been reported in lungs, liver, kidney, heart, lymph nodes, and skeletal muscle after subcutaneous or intravenous injection in rabbits and mice, with similar results in hamsters, mice, and guinea pigs. Humans accidentally injected have suffered long-term, painful abscesses. The IACUC maintains the following guidelines to eliminate or minimize animal discomfort associated with the use of this agent in research.

Before using Freund's complete adjuvant (FCA), or if you are already using FCA, consider the use of Freund's incomplete adjuvant (IFA) or other types of adjuvants or other commercial adjuvants on the market.

FCA should be used only for the first (priming) antigenic dose. Using more doses of FCA is rarely warranted and must be justified on your Animal Use Protocol Form. If more than one dose of FCA must be used, an interval of three weeks should be allowed between doses.

The recommended sites and volumes for rabbit immunizations are:

· IM 0. 5 cc (in two 0. 25 ml aliquots)
· SQ 1. 0 cc (in six to ten 0. 10 ml aliquots)
· ID 0. 5 cc (in five 0. 10 ml aliquots)

There is confusion over the amount of distress caused by injecting FCA in these routes. If abscesses or lameness occur, other routes and doses must be considered. Using footpad and intravenous injections are not recommended. With the injection of any material, the inoculum should be free of extraneous microbial contamination. Millipore filtration [0. 45 um or 0. 22 um filter] of the antigen before mixing with adjuvant is recommended when possible. Injection sites should be cleaned and free of debris. Injection sites become infected if the solution is contaminated or the site is not clean.

Frequency and Total Number of Immunizations:

Ten to 45 day intervals between immunizations are acceptable.

Two immunizations are the minimum number usually given. Four to eight are suggested maximums that usually produce adequate titers.

l. Blood Draws, Volume and Frequency

Maximum Blood Volume to be drawn in repeated collections is 1% of the body weight (i.e. 10 ml per kg body weight) every 2 weeks. Larger volumes and/or shorter intervals are allowable ONLY if the animals are monitored weekly for a drop in hematocrit.

Requests for closer intervals and/or larger volumes in injections for antigen only (no adjuvant) injections will need to be approved by the IACUC.

Blood Draw by Retro-orbital Venous Plexus:

Retro-orbital blood sampling can be used in both rats and mice (though usually not a method of choice in the rat) by penetrating the retro-orbital plexus/sinus with a glass capillary tube. The DVR guideline is that in the hands of a skilled operator, retro-orbital bleeding conducted under general anesthesia is a humane procedure that produces minimal and transient pain/distress.

Care must be taken to ensure adequate hemostasis following the procedure.

m. Death as an Endpoint:

The default policy of the IACUC is that spontaneous death of a study animal, without analgesic relief, is inhumane, unethical and can only rarely be justified. Further, it is IACUC policy that laboratory animals should be humanely euthanized before they exhibit a moribund condition. Since alternative, less painful /distressful endpoints are almost always available to the investigator, death as an endpoint can only be considered if all available alternatives are rejected after rigorous IACUC review. The IACUC may consider an exemption under some circumstances. An exemption from this policy can only be granted if the investigator provides a rigorous, convincing argument, based in documented scientific fact, that:

· There are no alternative less painful endpoints to assess the disease, treatment or intervention.
· Euthanasia at the onset of the moribund state is scientifically inappropriate.
· To permit animals to enter and experience the moribund condition to a spontaneous death is ethically and scientifically justified.

The IACUC will review all "death as an endpoint" studies on a case by case basis.

n. Embryo Removal

Removal of embryos from a rodent dam requires that the dam be adequately anesthetized the depth of anesthesia checked by non-responsiveness to noxious stimuli, before removal of embryos. The dam will them be exsanguinated before disposal. Hypothermia is an unacceptable means of anesthetization/analgesia for dams. The dam will be anesthetized with CO₂.

o. Environmental and Social Enrichment

Enrichment and Exercise Policy for Dogs

All dogs are permitted to run freely in the animal room during cage changing/cleaning either with other dogs or alone if they are not socialized.

Dogs are provided with a resting board and given toys to play with.

Additional human interaction with animal care, veterinary and investigator technicians is encouraged.

Dogs that are housed without conspecifics are required to have human contact daily.

Exemption from any of these environmental enrichment basics requires scientific justification by the investigator and must be approved by the IACUC.

For example, if a post-operative dog requires exercise restriction due to surgery, this must be justified by the type of surgery and the length of time such restriction remains in effect.

Environmental Enrichment (EE) & Housing for the Psychological Well-being of Nonhuman Primates

Background:

The assessment of the psychological well-being of an animal is based on several indices:

· The animal’s ability to cope with changes in its’ environment
· The animal’s ability to engage in beneficial species-specific activities
· The absence of maladaptive or pathological behaviors that may result in self-injury or other undesirable consequences
· The presence of balanced temperament with the absence of behavioral or physiologic signs of distress.

Guidelines:

According to the “Final Report on Environmental Enhancement to Promote the Psychological Well-Being of Nonhuman Primates” (USDA, 1999), there are five critical elements that need to be satisfied in the environment of nonhuman primates to promote their psychological well-being:

· Social grouping and contact with conspecifics
· Social needs of infants or weaning at an appropriate age
· Adequate structure and substrate of the enclosure including perches or nesting boxes
· Foraging opportunities by use of complex foods or foraging boards or toys
· Manipulanda or the opportunity to handle items with the hands.

If a nonhuman primate is deprived of any of these elements, then its psychological well-being is not being supported by the institution. Therefore, any investigator that requires that any critical E.E. element be eliminated due to research protocol restrictions MUST justify why it is necessary. In addition, the investigator must describe a plan whereby additional measures will be taken to compensate the animal for the lack of this critical part of its well-being. This plan should include how the psychological well-being of the animal is going to be evaluated and must be recorded in the medical record.

Addition of sensory stimulation and increasing the complexity of an animal’s environment can also be used to compensate for certain critical elements:

· Foraging opportunities can still be provided to animals with dietary restriction by the use of specialized foraging boards and specialized dietary treats are commercially available.
· Singly housed animals housed in the same room should be positioned to see other conspecifics or be able to see itself in a mirror.
· Inanimate Environmental Enrichment (e.g., playback of tropical forest sounds/soft music and viewing of appropriate TV/videos) can reduce boredom/stress and enhance psychological well being.

Requests and justifications for exemptions must be submitted to the IACUC for approval:

Other exemptions:

Minimizing species appropriate structures in the animal enclosure is appropriate when medically dictated by the condition of the animal. The lack of available equipment is not justification.

The presence of toys in and on the cage can only be exempted if the animal may cause harm to itself secondary to a psychological aberration. Interference with the squeeze back cage mechanism is not a justification, as tools are available for toy safe removal and many toys are available that do not interfere with this mechanism.

Nonhuman primate housing guidelines:

Individual housing of non-human primates (not pair or socially housed) MUST be scientifically justified. Requests and justifications for exemptions must be submitted to the IACUC for approval.

The inability to find a suitable partner after several attempts would be considered justification based on the unsociable nature of an animal. Attempts at pairing must be documented in the medical records of all involved animals.

The early removal of an infant from its mother can be done without justification only if the animal is either rejected or is at risk of harm by remaining with its mother. Attempts to foster an infant must be made and documented.

Intervention/Documentation of Stereotypes:

Captive primate may exhibit stereotypes or other abnormal deleterious behaviors (e.g. over-grooming, self-biting, etc) that can be mediate through the use of the environmental enrichment methods described above. All deleterious behaviors and method use to alleviate the condition should be recorded in the animal’s records.

Rodent Enrichment:

Enriching the environment for rodents has been suggested to ensure the highest quality of health and well-being among animals used for research and teaching purposes.. When in agreement with the objectives of the protocol, the structural and social environments of rodents will be enhanced to encourage species-typical behavior, improve animal well-being and prevent maladaptive behaviors.

Background Information

From the ILAR Journal, 2005, vol 26(2): Environmental Enrichment for Laboratory Rodents

Eric Hutchinson, Anne Avery, and Sue VandeWoude

Regulatory agencies in the United States and Europe have also been increasingly concerned about this issue relative to laboratory animal husbandry. However, little is known about the influence such husbandry modificationsmay have on biological parameters. Because introduction of enrichment schemes involves addition of experimental variables, therefore potentially decreasing the power of the experimental design, the implications of altered experimental outcome associated with addition of enrichment to a rodent husbandry scheme must be considered.

In addition, the composition of comfort and “enrichment” for rodents may not always be intuitively obvious. Rodent species used in biomedical research range from captive wild animals to strains bred thousands of generations in a laboratory setting, sometimes with spontaneous or induced genetic alterations. Devising enrichment techniques that might be applied safely and easily across such diverse backgrounds is a daunting task. Finally, it is also necessary to factor the practical considerations of rodent enrichment (e.g., ease of use and safety) into the cost:benefit equation. Therefore, what first appears to be a simple and logical act quickly becomes much more problematic when the scientific and practical aspects of providing an enrichment program for rodents are considered.

Laws and Regulations Governing Environmental Enrichment (EE) of rodents

The Animal Welfare Act (AWA1) currently contains no provisions for laboratory-reared rats and mice, nor does it explicitly mandate EE standards for other rodents. However, given the attention of other agencies to this topic, and the flurry of recent legal challenges to the AWA such as Alternative Research and Development Foundation v. Veneman (2001), laboratory rodents are likely to be addressed more specifically in this primary regulatory statute at some time in the future. In the Guide for the Care and Use of Laboratory Animals (the Guide1) (NRC 1996), natural behaviors are highlighted as a crucial measure of success for an animal program. The discussion of proper housing begins with the statement, “Animals should be housed with the goal of maximizing species specific behaviors and minimizing stress induced behaviors” (p. 22). It is also suggested that readers consider housing the animals in “natural environments,” and an entire section on behavioral management stresses animals’ structural, social, and activity needs. As a result of this suggestion, the Public Health Service and the Association for Assessment and Accreditation of Laboratory Animal Care International more closely emphasize/scrutinize the use of enrichment in laboratory animal facilities for all species, including rodents.

DVR Policy &Standard Practices

· EE is currently not required for rodents housed at University of Miami.
· The Principal Investigator may request EE in writing after discussion with the veterinarians to ensure that enrichment devices and materials are compatible with the objectives of the protocol. This request then should go to the building supervisor.
· The Supervisor will direct staff to implement enrichment in a uniform and comprehensive manner in accordance with this procedure.
· The use of enrichment devices and materials will be uniform and involve all animals of a study or colony unless instructed otherwise by the PI to ensure that devices and materials do not introduce a variable that may confound the interpretation of protocol data.
· At the time of cage change, an autoclaved nestlet or igloo is placed into the clean rodent VAF cage or a clean nestlet or igloo is placed into the clean conventional rodent cage.
· Enrichment materials are removed and sanitized or disposed of with the dirty bedding at the time of cage change.

p. Euthanasia Policy

Euthanasia procedures must conform to the guidelines set forth by the AVMA panel on euthanasia(2007). Procedures must accomplish a swift unconsciousness and painless death. The panel describes euthanasia methods as approved, conditional approval and non-approved. Non-approved methods are unacceptable. PIs that want to use a conditionally approved method must provide clear scientific justification for using the method. Decapitation is not an acceptable means of euthanasia unless anesthesia is provided. Clear scientific justification must be provided to withhold anesthesia prior to decapitation and verification of training/experience in decapitation methods must be approved by the IACUC.

A copy of the report of the AVMA Panel on Euthanasia is located at http://www.avma.org/resources/euthanasia.pdf.

q. Food Deprivation

The IACUC default Policy is that food deprivation below 85% of ad libitum weight requires special justification to the IACUC. It is suggested that for each group of animals ordered, one animal be used as a free food ad lib control in order to more accurately judge the deprivation state of the food deprived subjects.

r. Gas Anesthesia and/or Euthanasia

If a volatile anesthetic is used such as isoflurane, an appropriate method (acceptable to Environmental Health and Safety) to scavenge the waste gas must be in place.

Although diethyl ether is an effective anesthetic, it is not recommended for use because it is flammable and explosive. Ether is irritating to mucous membranes and causes excessive salivation in many species. It MUST not be used in survival surgery procedures. The use of ether as an anesthetic agent will require clear scientific justification and will be reviewed on a case-by-case basis. For all inhalation anesthetics, an adequate scavenging system must be in place. All animals should be "air washed" under an appropriate hood prior to being transported to the DVR for disposal.

Chloroformis a known hepatotoxin, a suspected carcinogen, and is unacceptable as a euthanasia agent according to the AVMA. Its use must be justified and will require more rigorous review by the IACUC. Although chloroform is non-explosive, its use in the presence of a flame may result in the production of phosgene gas. Because of its significant hazards to human beings, work with chloroform must be performed in a working chemical fume hood, with sash lowered to the rated face velocity mark. Gloves must be worn during the procedure. Following the procedure, the animal carcasses and euthanasia apparatus (i.e., container, nose cone, gloves, etc.) must be left in the hood for a sufficient amount of time (approximately one hour) to allow the chloroform to volatilize.

Use of Carbon Dioxide for Euthanasia of Laboratory Animals
Carbon dioxide from compressed CO₂ gas in cylinders is acceptable for euthanasia of rodents. Carbon dioxide generated by other methods such as from a fire extinguisher, dry ice or from chemical means (e.g. Alka-Seltzer) is unacceptable. With an animal in the chamber, an optimal flow rate (of 100% CO₂) should displace at least 20% of the chamber volume per minute. It is important to verify that an animal is dead before disposal of the carcass. CO₂ narcosis must be followed with another method of euthanasia to ensure death (e.g., thoracotomy or decapitation).

Currently established exposure times vary with the age of mice: (adapted from Comparative Medicine, 2005, vol 55(3))

Age 0-6 days old, need 60 minutes exposure

Age 7-13 days old, need 20 minutes exposure

Age 14-20 days old, need 10 minutes exposure

Age >21 days old, need 5 minutes exposure

Larger animals, such as rabbits, cats, and swine, appear to be more distressed by CO₂ euthanasia; therefore, other methods of euthanasia are preferable.

s. Pain Management

The Policy of the University of Miami IACUC is to promote the prevention and alleviation of research animal pain and suffering as an important and tenable therapeutic goal. The IACUC acknowledges that complete elimination of pain in individual animals may not be obtainable or desirable (see: exceptions). Rather than being aimed at eliminating all pain, therapeutic strategies should be aimed at improving an animal’s ability to cope with pain, thereby decreasing suffering. Treatment of pain can be considered successful if the degree of pain does not prevent an animal from engaging in relatively normal activities, such as eating, sleeping, ambulating, grooming, and interacting with other members of its species or its care givers.

The IACUC also recognizes that management of pain in many animals is problematic and may not be feasible under certain circumstances. Nevertheless, it is IACUC policy that every attempt will be made to prevent or alleviate pain in animals unless there are compelling scientific reasons to withhold relief. Normally, documentation for interference in study endpoints by analgesics, in the form of personal experience or specific literature references, is required to justify withholding pain/distress relief.

The default IACUC policy is that if a procedure (and the post-operative period) is painful in human beings, then it is assumed to be painful in laboratory animals. It may be useful to draw parallels between people and animals, but the severity of pain produced by various procedures is not always similar. Because it is difficult to compare the experience of pain in animals to that in people, it is IACUC policy to require administration of analgesics preemptively if there is any question that a procedure will induce pain in an animal. As a matter of policy, the IACUC assumes that all surgical procedures (involving tissue/bone damage) will result in at least a temporary period of pain (varies with the procedure). It is IACUC Policy that animals will be prophylactically treated with appropriate analgesics for all surgical/traumatic procedures to research animals, unless justified in the protocol as an exception (described below). Many procedures create long-lasting damage so that some level of spontaneous/evoked pain is created. Each procedure/protocol/animal must be examined individually.

It is recognized that with the current research interest in developing strategies and treatments for chronic pain and preventing/decreasing the outcome of traumatic injury in humans, that research animals might be required to remain untreated for post-surgical pain.

A number of possible problems may be encountered with ordinary post-operative analgesics:

· Analgesics might interfere with other interventions to be studied
· The increased number of animals to be used as the controls (+/- analgesics)
· The likely difficulties with the interpretation of the results when analgesics are used
· The difficulty in comparing the data from such analgesic-animals with that found in the literature.

None of these problems could alone serve as criteria for withholding analgesia; the IACUC will consider each protocol on a case by case basis.

In a situation where no data exist on the effects of analgesics on a particular lesion, it is the policy of the IACUC to encourage PIs to include analgesia controls. Such information should be made available to the IACUC to guide future reviews. During immediate post-operative recovery, sternal recumbency and normothermic body temperature must be attained before an animal is left alone. Animals must be observed for the appropriate period and frequency during the post operative period to:

· Detect signs and symptoms of pain and distress
· Evaluate the efficacy of analgesics
· Detect and remedy post operative complications

Exceptions from this policy may include procedures such as motor paralysis, but, such procedures MUST have a full description of post-op care and records of the frequency and type of care provided. Post-operative analgesia must be provided, unless clearly justified in the protocol (see below). The time period for analgesics (and antibiotics) will be determined by the procedure. Observations and the use of analgesia willl be recorded as part of the animal's health record and will be made available for inspection by the IACUC if requested.

Protocols that might be considered as exceptions to the above policies include:

· Studies of therapeutic interventions for pain
· The mechanisms of the initiation, development, and maintenance of acute, tonic, or chronic pain
· Where analgesics will cause changes in the animal at the cellular level or in the intervention being studied, e.g., cell transplants or gene therapy
· Studies where injuries or interventions require an untreated (with analgesics) animal.

A guideline for what should be included in such protocols, and will function as criteria are:

· The PI will provide the IACUC with a list of the categories of analgesics considered.
· If analgesics are not used, the PI must provide scientific justification for withholding relief.
· A description of the timing of when analgesics might possibly be used should be included as part of the description of the procedures whose outcome is expected to cause pain/distress.
· Documentation of normal grooming and weight gain in unrelieved animals.
· Criteria used to determine that animals should be euthanized prior to the scheduled completion of the study due to unexpected outcomes or complications.

t. Surgery

Major surgery penetrates and exposes a body cavity or produces substantial impairment of physical or physiologic function. In general, unless an exception is specifically justified as an essential component of the research protocol and approved by the IACUC, non-rodent aseptic surgery should be conducted only in facilities intended for that purpose. Functional components of aseptic surgery include surgical support, animal preparation, surgeon's scrub, operating room, and postoperative recovery. In compliance with the Guide, all major survival surgery on non-rodent species must be performed in approved surgical suites that meet all accreditation requirements.

Modified Aseptic Rodent Technique:

In regard to rodent surgery, the Guide for the Care and Use of Laboratory Animals states, “Some characteristics of common laboratory-rodent surgery, smaller incision sites, fewer people in the surgical team, manipulation of multiple animals at one sitting and briefer procedures, as opposed to surgery in larger species, can make modifications in standard aseptic techniques necessary or desirable.” Any deviation for rodents or any other species from the UM Policy on Experimental Animal Surgery must be described and justified in the animal protocol and approved by the IACUC.

Standard Practices

Surgery must be conducted in a dedicated area that is not used for any other purpose during the time of surgery. Traffic in the surgery area should be limited during the procedure. Surgery on rodents does not require a dedicated facility but must be performed using aseptic procedures as described in the UM Policy for Experimental Animal Surgery.

· The surgical table or surface must be wiped with a disinfectant before and after use and/or covered with a clean drape.
· Hair should be removed from the surgical site with clippers or a depilatory.
· The site must be prepared with an antiseptic scrub (chlorhexidine or betadine) and rinsed with solution. The scrub-wipe routine must be repeated three times.
· All instruments and implants must be sterilized, but the method of choice may be determined by the surgical instruments or devices being used. Sterile tip instruments must be placed on a sterile drape to reduce contamination while waiting for the next rodent to be prepared. A new sterile pack should be used after four or five individual rodents.
· Sterile suture material must be used.
· The surgeon must wear clean scrubs or a lab coat. The surgeon must perform a surgical scrub with an antiseptic surgical scrub preparation and then put on sterile gloves. Gloves must be changed if they are perforated or become contaminated. The surgeon must wear a surgical mask and cap.
· Aseptic technique requires aseptic manipulation of instruments and any material that contacts the surgical site as well as gentle tissue handling and minimal dissection of tissue.
· Multiple surgeries for rodents present a special situation. After the first surgery, the sterilized instruments may be kept in a cold sterilant or immersed in a hot bead sterilizer. Sterile gloves must be changed between surgeries.
· Antibiotics must be given routinely unless justified in the investigator’s protocol and approved by the IACUC.

Multiple major survival surgery:

Multiple major survival surgical procedures on a single animal are discouraged, but, may be permitted if scientifically justified by the user and approved by the IACUC. For example, multiple major survival surgical procedures can be justified:

· If they are related components of a research project.
· If they will conserve scarce animal resources.
· If they are needed for clinical reasons.

If multiple major survival surgery is approved, the IACUC should pay particular attention to animal well-being through continuing evaluation of outcomes. Cost savings alone is not an adequate reason for performing multiple major survival surgical procedures.

All protocols involving multiple major survival surgery must come to full committee review and are not eligible for Emergency Designated Review (EDR).

Post-surgical Care:

According to the Guide, appropriate facilities and equipment should be available for post surgical care. An important component of post-surgical care is observation of the animal and intervention as required during recovery from anesthesia and surgery.

Adequate care is the responsibility of the PI and requires close monitoring of the animal during the acute postoperative period. All animals should be observed continuously until they are conscious (arousable to an alert and responsive state), able to maintain sternal recumbency, and are normothermic. Monitoring is critical for identifying immediate complications such as hemorrhage, dehiscence of surgical incisions, neuropraxia, myositis, shock, pain, etc.

The immediate postoperative periods should also be used to assess comfort and identify those animals requiring postoperative analgesics. At a minimum, temperature, pulse, mucous membrane color and refill time, and respiratory rate should be monitored at least every 15 minutes until the animal is conscious.

Thereafter, animals must be observed daily for at least 7 days following any surgical procedure. Animals may be monitored more frequently and for a longer postoperative period as the situation requires, and at the discretion of DVR veterinarians and/or the IACUC.

It is the responsibility of the investigator to provide and document postoperative care during the recovery period. A postoperative treatment form should be filled out and attached to the animal’s clinical record. The form should contain, at minimum, the following information: animal ID, date, surgical procedure or behavioral manipulation, observation notes, weight. Upon request, DVR may be able to provide monitoring and treatment during the recovery period. Investigators, or their staff, may provide their own monitoring during recovery as long as the monitoring is continued until the animal meets the conditions described above and the appropriate records are maintained. However, failure to provide proper care or maintain complete records will lead to loss of this privilege at which time Animal Resources personnel will provide proper care and maintain adequate records at the standard charge.

u. Tail Biopsy

Ideally, mice should be 10-21 days of age. For mice this age, local anesthesia to the tail is used prior to clipping. Local anesthesia can be achieved through tail immersion in ice cold ethanol for 10 seconds, or by disinfecting the tail with 70% ethanol, allowing it to dry, and applying ethyl chloride spray. For mice greater than 21 days old, a local or general anesthetic is required. In general, less than 5mm of tail should be excised. Repeated tail biopsies require general anesthesia and must be justified in the IACUC protocol.

XVIII. CELL LINE TESTING

The current DVR Policy is that all cell lines and biologic materials currently used in rodents must be tested for the presence of adventitous agents that can impact the health of our rodent colonies. New cell lines are to be tested prior to the initiation of all new studies. Cell lines are to be tested for comprehensive panel of pathogens through DVR. Any cell lines found to be positive for rodent pathogens must not be used until cleared of the agent.

XVIIII. Tumor Burden

No precise quantitative guide can be given as to the acceptable upper limit of tumor burden, since the adverse effects on the host will depend on the biology of the tumor, the site and mode of growth, and the nature of associated treatments. However, tumor burden should not usually exceed 5% of the host animal’s normal body weight in the case of animals being used for routine tumor passage, or 10% in animals involved in therapeutic experiments. The latter size (10%) would typically represent a mean subcutaneous flank tumor diameter of 14 mm (~1.5cm³) in a 25g mouse or 35mm (~20cm³ in a 250g rat. Calibration curves relating tumor weight to measured diameters should be established as part of the initial characterization of any new tumor system. Consideration should be given to variation in measurement between individual experimenters. Although the sizes given above may serve as a maximum guideline, it should be emphasized that problems may arise with much smaller tumor burdens and the clinical condition of the individual animal will always be the over-riding consideration.

Experimental protocols and severity limits should specify early experimental or humane end points requiring appropriate intervention. Criteria for such endpoints should be determined before the study commences.

The following clinical signs may be useful:

Persistent anorexia or dehydration
Consistent or rapid weight loss of 20% maintained for 72 hours
Unable to maintain an upright posture or to move
Muscle atrophy or emaciation
Moribund, lethargic or failure to respond to gentle stimuli
Hypothermia
Unconscious or comatose
Bloodstained or mucopurulent discharge from any orifice
Labored respiration – particularly if accompanied by nasal discharge or cyanosis
Enlarged lymph nodes or spleen
Anemia
Ulcerated tumors
Significant abdominal distension
Incontinence or prolonged diarrhea
The frequency with which animals must be inspected for signs of pain and distress and the extent of each examination will be dictated by:
The known biology of the tumor and/or the effects of the inducing agent.
The effect of any associated techniques.
The changing clinical status of the animal.

Rapidly growing or invasive tumors will require more frequent attention, and greater care will be required as the tumor burden increases. As a minimum, every tumor bearing animal should be inspected daily and additional, more detailed, examinations undertaken as appropriate. The frequency of the latter should be increased during critical periods where the potential for animal suffering may be anticipated. The experimental design should ensure that these do not occur when staff are absent. Particular attention should be given to animals in poor health, especially on weekends.

Appropriate assessment techniques will include:

Evaluation of overall clinical condition, including appearance, posture, body temperature, behavior and physiological responses; assessment of food and water intake
Weighing to determine changes in body weight (both positive and negative changes compared to controls can be associated with increasing tumor burden)
Caliper measurements to determine tumor volume or mass
Inspection and palpation to locate the sites of tumor growth, as well as to assess distension, ulceration and compromised mobility.

XX. Policy for the Use of Outdated and Expired Material

The use of expired antibiotics, analgesics and anesthetics is expressly prohibited by the Animal Welfare Act and PHS policy. Expired diagnostic reagents, drugs and devices may not be used at UM without prior approval by the IACUC. If you plan to use any outdated or expired reagents, materials or devices please provide a clear concise justification for their use in the protocol form.

XXI. Genetic Manipulations

Genetic manipulations such as placing genetic material from animals of one species in animals of another species, or knocking out specific proteins via genetic transformations, can be powerful research techniques. However, there is evidence that some genetic modifications may cause pain and distress in laboratory animals depending on the vectors and genes used, either in heterozygous/homozygous animals or those receiving gene transfer or cell transplants.

These risks include:

Physiological or morphological problems, including early death, internal bleeding, CNS/PNS dysfunctions and other system defects.
Behavioral problems, including lack or loss of nurturing behaviors, disorientation, aggressiveness, and extraordinary fearfulness.

These possibilities suggest the need to consider special justification for creating the animals and attending to welfare issues. Because it is not always possible to predict the outcome of transgenic/knockout manipulations, or other genetic manipulations such as gene transfer or cell therapy, investigators must take special precautions to have clearly defined contingency plans in case there is evidence of animal suffering. Such plans would, for instance, establish criteria for protocol termination that could be invoked in various experimental contexts. Additionally, the IACUC must be notified in case of animal morbidity due to transgene/knockout manipulations.

Of particular importance is whether an investigator is used to working with a species in general or a specific -strain (e.g. of mice) whose members might behave differently than those they are used to. In ways analogous to current requirements for documentation of investigator experience, the IACUC may want to consider requesting evidence of experience in transgenic research, or require the use of previously characterized strain of genetically manipulated animals. The use of gene transfer or cell transplants to manipulate and animal will require the prediction of the manipulation to the final phenotype of the host animal, especially regarding possible pain/distress that such a manipulation might cause. Such statements should be included in the research protocol.

Glossary of Terms:

Baseline Health Assessment means an initial health evaluation performed by a qualified licensed healthcare professional designated by the University and occurs at the commencement of the employee’s work duties that involve recurrent contact with animals.

Covered Employee means any employee performing work duties that involve recurrent contact with animals.

EHS means the Office of Environmental Health and Safety.

Exposure Incident means a) skin, eye, mucous membrane, or parental contact with blood or potentially infectious materials, b) bites, c) scratches, or d) other wounds that may result from the performance of an employee’s duties.

Follow-up Health Assessment means a health evaluation by a qualified licensed healthcare professional designated by the University and occurs during the employee’s employment in which work duties involve recurrent contact with animals.

IACUC means Institutional Animal Care and Use Committee.

PI means Principal Investigator

Zoonosis means an infection or infestation shared in nature by humans and other animals that are the normal or usual host; a disease of humans acquired from an animal source.