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UNIVERSITY OF MIAMI ANIMAL CARE AND USE

GUIDELINES

Version: 3/07/04

 

 

CONTENTS

 

Overview

Responsibilities of the Principle Investigator

ACUC Membership and Functions

ACUC Records and Reporting Requirements

Reports to Institutional Official (IO)

Reports to Office for Laboratory Animal Welfare (OLAW)

Reports to United States Department of Agriculture (USDA)

ACUC Records

Personnel Qualifications and Training

Personnel Requiring Training

Training Program Content

Occupational Health and Safety of Personnel

Laboratory Inspections

Institutional Program for a Safe and Healthy Workplace

Role of the Office of Environmental Health and Safety (EHS)

            and Healthcare Provider

Role of Principal Investigators and Department Supervisors

Veterinary Care
Animal Use Protocols

Required Signatures and Scientific Merit Review

Justification for Using Animals

Justification for Using a Particular Species

Justification of Animal Numbers

Justification for Performing Potentially Painful Procedures

The Principal Investigator

Protocol Forms

Protocol Submission

Protocol Submission Deadlines

Protocol Review Process

Emergency Designated Reviews

Tissue Sharing
Reporting Animal Concerns

Animal Procurement and Housing

Ordering Animals

Animal Environment, Housing and Management

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

Animal Health and Procedure Records

Procedures

            Anesthesia/Euthanasia           

Gas Anesthesia and/or Euthanasia

Use of Carbon Dioxide for Euthanasia

Anesthesia for Rodent Pups

Hypothermia

Embryo Removal

            Pain Management

            Surgery

            Post-surgical Care

Food Deprivation

Animal Identification

Antibiotics

Avian Field Studies

Ascites Production

Use of Complete Freunds Adjuvant

Blood Draws, Volume and Frequency

Tail Biopsy

Tumor Burden

Expired Materials

 

Genetic Manipulations         
 

 

 

Overview

This document outlines guidelines drafted by the University of Miami Animal Care and Use Committee (ACUC). The guidelines 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 nominal position of the UM ACUC vis a vis the aforementioned agencies, and to provide investigators information to aid them in completing the ACUC animal use protocol forms. Deviations from these guidelines may be considered by the ACUC on a case by case basis.

 

Responsibilities of the Principle  Investigator

 

The privilege of using animals in research and teaching at the University of Miami is extended to the faculty with the proviso that such activities will be conducted in accord with all applicable policies, regulations and procedures of the PHS, the USDA and University of Miami.    Please review and check each of the following “Responsibilities of The PI”.

 

The Principal investigator is:

·        required to have a current animal use protocol, approved by the University of Miami ACUC, before any activities involving live or dead vertebrate animals is begun.

·        required to comply with all Federal regulations, laws and 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.

·        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.  PI’s must inform the IACUC when there are changes in personnel and funding.

·        responsible to assure that the work follows the approved protocol.   All 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 ACUC, 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 ACUC.   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.

As the PI, I agree to assume the responsibilities listed above.  Further, as the PI, I 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.

 

ACUC 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. Norman Altman.  The committee membership includes scientists affiliated with the University of Miami, at least one veterinarian, and at least one non-scientist or non-lab user.   In addition, the ACUC membership includes at least one person not affiliated with the University, in accordance with Federal regulations.  The ACUC:

1)          is responsible for oversight and evaluations of institution's animal care and research program,

2)          conducts semiannual evaluations of the Institutional Animal Care and Use (IACUC) program,

3)          conducts semiannual inspections of all institutional animal care and use facilities;

4)          reviews and investigates concerns about animal care and use at the university;

5)          establishes procedures for the review, approval and suspension of animal activities;

6)          establishes procedures for review & approval of significant changes to approved activities;

7)          establishes policies for the review of procedures (e.g. restraint, multiple survival surgery, fluid and food restriction, painful procedures) that require exemption from federal regulations, guidelines and policies.

8)          reports to the University of Miami Institutional Official.

Table of Contents

 

ACUC Records and Reporting Requirements

Reports to Institutional Official (IO)

The ACUC 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 ACUC views, if they exist.

 

Reports to Office for Laboratory Animal Welfare (OLAW)

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

 

Reports to United States Department of Agriculture (USDA)

The ACUC 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 ACUC is required to explain ACUC approved deviations from the AWA and USDA regulation.   Further the ACUC is required to report the details of any ACUC directed suspension of animal usage. 

 

ACUC Records

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

Table of Contents

 

Personnel Qualifications and Training

Personnel Requiring Training

The University of Miami has established and implemented a training program for professional/management/supervisory personnel, all animal care personnel, principal investigators, research investigators, instructors, technicians, trainees, and students.   In general, 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 (core training) prior to their involvement with animal research subjects.   Exceptions to this rule are undergraduate students, medical school students, and professionals undergoing clinical training/retraining in classroom and/or non-research settings.  Such individuals may, at the discretion of the ACUC, participate in a laboratory exercise under the immediate direct onsite supervision of DVR veterinarians and /or appropriately trained personnel. Satisfactory completion of the training course is assessed by evaluating performance on an objective exam.  Currently, core training is accomplished using a web based training module.   DVR employees must complete additional, task specific training provided by DVR supervisory personnel.  Other species or risk specific training (e.g., zoonotic diseases) may be required.

 

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.

Table of Contents

 

 

Occupational Health and Safety of Personnel

Laboratory Inspections

All areas where animal experimentation is performed 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. It’s content includes 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

3. Annual tuberculosis and allergy testing; semi-annual testing may be required for contact with some animal species

4. Baseline health assessment

5. Exposure incident evaluation and follow-up

6. Follow-up health assessment, as required

7. Phlebotomy for serum banking at the time the employee begins work involving recurrent animal contact

8. Pregnant woman counseling

9. Vaccinations, laboratory tests, and titers as required according to the need of the 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 Conversation Program

7. Monitor Respiratory Protection Program

8. Periodic tuberculosis screening

9. Phlebotomy for serum banking

10.   Pregnant woman counseling and consultation with employee’s obstetrician

11. Vaccinations as required

 

Role of Principal Investigators and Department Supervisors

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:

     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.

4. Names of employees who no longer have contact with animals.

5. Names of employees who work in areas that have been identified to require hearing protection

6. Names of employees who are required to wear a respirator during work duties.

7. Ensure proper completion of necessary forms.

8. Allow necessary time away from work duties to enable employees to participate in this program.

9. Ensure that covered employees’ participation in this program is at no cost to the employee.

10. Provide appropriate personal protective equipment.

11. Provide employee training in proper use of personal protective equipment.

12. Ensure that employees having contact with animals receive information regarding:

·      Health hazards associated with animal species with which they have contact.

·      Safe work practices.

·      Follow-up procedures for an exposure incident.

           

The Institutional Animal Care and Use Committee requires the following:

 

1. Principal Investigators to provide a listing of all employees who participate in each animal research protocol to IACUC.

2. 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

3. Notify Principal Investigators of the Occupational Health Program for Animal Research Personnel.

 

The employee shall:

 

1. Complete acknowledgement form for the occupational health program for animal research personnel.

2. Immediately report an exposure incident to their supervisor and EHS.

3. Use appropriate personal protective equipment as provided by their department and as trained.

Table of Contents

 

Veterinary Care

The University of Miami Division of Veterinary Resources (DVR) has full time veterinarians on staff with training and experience in laboratory animal medicine.  DVR veterinarians have access to all animals.  Provision for backup 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(s) has authority on all veterinary care aspects of the animal care and use program at the University of Miami.

 

Animal Use Protocols

Required Signatures and Scientific Merit Review

All work with animals requires written approval by the Animal Care and Use Committee prior to the start of the project.  Projects, which will not be subjected to peer review by an external funding agency, require verification of review for scientific merit by the Departmental Chairman, as signified by the presence of his/her signature on the protocol form.  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.   ACUC protocol review includes reviews for scientific merit and for compliance with OHRP Policy and 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.

 

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. 

Protocols must 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 study group size must be included. The rationale has to be based on previous (published) work or a recent statistical analysis of pilot data.  A modest allowance for attrition (model failure) may be acceptable if justified.   If such attrition is >20% explain what you will be doing to REFINE and 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

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 unless specifically and scientifically justified, with research references included where indicated on the protocol form.

 

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 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

A longform must be submitted:

·      For all new projects involving animal use (including "tissues only" projects).  

·      If it has been 3 years since the last ACUC approval on an existing project.

·      If there has been a substantial change in the methods or scope of an existing project.

 

A shortform should be completed:

·      For yearly renewals (required of all principal investigators),

·      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),

·      If the PI is resubmitting a grant proposal to the same funding agency with new funding intervals (dates), or

·      If the PI is making clerical "changes" in the original protocol, such as the Project Title, the Principal Investigator, Investigators or staff.

 

An addendum may be sufficient if:

·      The PI is requesting approval of minor methodological changes to an existing, approved protocol, or

·      If the PI is requesting additional animals/tissue on an existing, approved protocol.

 

Protocol Submission

Protocols are to be submitted in duplicate, or electronically (preferred method).  If submitted via floppy 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 ACUC office is 2 weeks prior to the meeting.  Meeting dates and protocol submission deadlines are published on the University of Miami ACUC website.

 

Protocol Review Process

Each protocol is assigned a number upon its receipt.  The ACUC Chair then assigns the protocol for preliminary review, prior to the monthly ACUC meeting. All reviews are conducted anonymously.  “Tissues only” protocols and addenda are reviewed by at least 3 ACUC members, including one veterinarian and two additional members.  Long form protocols are reviewed by 3 to 6 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 then has the opportunity to resolve reviewer concerns, in writing, before the protocol is added to the agenda of the monthly ACUC meeting for approval action.  Revisions in the protocol and/or written responses from the PI are then delivered to the reviewers for re-consideration.  If the reviewers approve the protocol, the protocol is placed on the agenda of the monthly ACUC meeting and discussed by the full committee.

 

Emergency Designated Reviews

Emergency designated reviews can be conducted by the ACUC at the direction of the ACUC Chair.  Consideration for an “expedited review” of a protocol will be entertained by the ACUC 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 expedited review is deemed justified, the protocol is assigned an ACUC number and submitted to reviewers as described above. After all concerns are addressed by the PI, the ACUC membership is poled to determine if there are any objections to granting an approval.  All committee members are provided access to the protocol.  If there are no objections from the ACUC membership, the protocol can be approved by the Chair.   If on the other hand, any of the ACUC members believe that the protocol should be discussed at the full ACUC meeting, the protocol cannot be approved until sufficient deliberation occurs at the next scheduled meeting of the ACUC.

Table of Contents

 

Tissue Sharing

In addition to the required forms, the ACUC encourages Investigators to fill out the Tissue Sharing Donor and Recipient Survey Form and return it via e-mail to the ACUC. Information from this form can be used to promote the Tissue Sharing Program.  This tissue-sharing program provides the opportunity to practice the concept of “Reduction”, a decrease in the number of animals used in research at the U of M.

 

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 ACUC.       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 to the DVR and/or to the University Animal Care and Use Committee, by contacting one of the following individuals:

 

Paul Braunschweiger, Ph.D., Chair, University of Miami Animal Care and Use Committee, 1600 NW 10th Avenue 1134A, Miami, FL  33101, (305) 243-2311, (305) 243-3922 fax; (305) 243-6650.  pbraunsc@med.miami.edu.

 

Bobby Collins, D.VM. Director, Division of Veterinary Resources, University of Miami, 1600 NW 10th Avenue, Miami, FL 33101 (305) 243-6802

 

Mary Eaton, Ph.D., Asst. Professor, The Miami Project to Cure Paralysis, University of Miami, 1095 14th Terrace, Miami, FL 33136, (305) 243-7136

 

Jennifer Daniels, The Office of Research, (305) 243-6415

 

Individuals reporting concerns should try to 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, the Division of Veterinary Resources should be contacted immediately (305-243-6802). If the problem remains unresolved, contact the University of Animal Care and Use Committee.

Table of Contents

 

 

Animal Procurement and Housing

Ordering animals

All vertebrate animals for approved protocols that do not involve collections of wild animals are ordered through the Division of Veterinary Resources.  All domesticated animals used in research programs must be obtained from commercial vendors. If the DVR fails to find a commercial source from which to obtain animals for an approved protocol, DVR will inform the PI and the ACUC of such, and alternative arrangements may be considered, at the discretion of the ACUC. 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 ACUC.

 

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

Transfer of animals (live or dead) to the U of M from external facilities requires prior approval from the DVR.    The attending veterinarian may require that the PI document the health status of the animals being brought into any U of M facility.  An approved animal use protocol must be on file with the ACUC before animals may be brought into the U of M.  Further, if animals are coming from other than a registered vendor, a letter from the ACUC of the “sending” institution indicating approval of the activity must also be filed with the U of M ACUC.

 

The transfer of animals (live or dead) from the U of M to other research institutions requires prior approval of the DVR and the ACUC.  The ACUC requires that the responsible individual at the receiving facilities have an approved protocol to conduct the proposed studies.

 

Animal Health and Procedure Records

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 ACUC.  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.

Table of Contents

 

Animal Procedures

Euthanasia - Euthanasia procedures must conform to the guidelines set forth by the AVMA panel on euthanasia (2000).   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. PI’s 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.   A copy of the report of the AVMA Panel on Euthanasia is available at the U of M ACUC website.


Anesthesia

Anesthesia methods must conform to the normal standards of veterinary care.    PI’s 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 U of M ACUC website.

 

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

 

Anesthesia for Rodent Pups

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 OC) before returning to the dam.

 

Hypothermia

Hypothermia may be an acceptable anesthesia for neonatal mice and rats.   Appropriate monitoring and rewarming following completion of the procedure is essential.   Hypothermia is not an acceptable method of anesthesia/analgesia for rodent dams, or reptiles.

 

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; Embryos will be decapitated at the time of removal, if possible, before removal from the dam.

 

Gas Anesthesia and/or Euthanasia

If a volatile anesthetic is used such as halothane (Fluothane~), or isoflurane (Forane~, AErrane~), 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 must be used with care because it is flammable and explosive. Ether is irritating to mucous membranes and causes excessive salivation in many species.  It should 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, 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.

 

Chloroform is 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 ACUC. 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 and 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 is acceptable for euthanasia. Compressed CO2 gas in cylinders is preferable to dry ice because the inflow to the chamber can be regulated precisely. If dry ice is used, animal contact must be avoided to prevent freezing or chilling. Carbon dioxide generated by other methods such as from a fire extinguisher or from chemical means (egg, Alka-Seltzer) is unacceptable. With an animal in the chamber, an optimal flow rate should displace at least 20% of the chamber volume per minute. Unconsciousness may be induced more rapidly by exposing animals to a CO2 concentration of 70% or more by prefilling the chamber. It is important to verify that an animal is dead before removing it from the chamber. If an animal is not dead, CO2 narcosis must be followed with another method of euthanasia. Larger animals, such as rabbits, cats, and swine, appear to be more distressed by CO2 euthanasia; therefore, other methods of euthanasia are preferable. In some animals, under deep carbon dioxide anesthesia, heartbeat can be maintained after visible respiration has ceased, and the animal might eventually recover. A thoracotomy is recommended to assure death of animals after CO2 exposure. Assessment of both respiratory and cardiac arrest prior to discarding the carcass is required.

 

Pain Management

The ACUC of the UM endorses a philosophy that promotes prevention and alleviation of research animal pain and suffering as an important and tenable therapeutic goal.  The ACUC 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 ACUC also recognizes that management of pain in many animals is problematic and may not be feasible under certain circumstances.  Nevertheless, it is ACUC 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 possible interference by analgesics, in the form of literature references, is required.

 

As a policy, if a procedure (and the post-operative period) is painful in human beings, then it is assumed to be painful in 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 ACUC 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 ACUC 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 ACUC 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's 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:

(1)   Analgesics might interfere with other interventions to be studied;

(2)   The increased number of animals to be used as the controls (+/- analgesics);

(3)   The likely difficulties with the interpretation of the results when analgesics are used; and finally,

(4)   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 the non-use of analgesics; each protocol will be CONSIDERED INDIVIDUALLY BY ACUC.  

In a situation where no data exists on the effects of analgesics on a particular lesion exists, it is the policy of the ACUC to encourage PI’s to include analgesia controls.  Such information should be made available to the ACUC to guide future reviews. 

 

During immediate post-operative recovery, sternal recumbency must be attained before an animal is left alone.  Exceptions: include procedures such as motor paralysis; such procedures MUST have description of post-op care and records of frequency/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.

 

Animals must be observed for an appropriate period (and frequency) post-operatively, watching closely for signs and symptoms of pain/distress, to evaluate the effectiveness of analgesics, and for post-surgical complications.  Observations and the use of analgesics will be recorded as part of that animal’s health record, and will be made available for inspection by the ACUC, if requested.

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

1.     Studies of therapeutic interventions for pain;

2.     The mechanisms of the initiation, development, and maintenance of acute,
tonic, or chronic pain;

3.     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; and

4.     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:

  1. The PI will provide ACUC a list of the categories of analgesics considered
  2. If analgesics are not used, provide scientific justification as to their absence;
  3. As part of the description of the procedures whose outcome is expected to cause pain/distress, a description of the timing of when analgesics might possibly be used; and
  4. Required documentation of normal grooming and weight gain in these untreated animals, unless these behavioral features are part of the model being studied.
  5. Criteria used to determine that animals should euthanitized prior to the scheduled completion of the study due to unexpected outcomes or complications.

 

Surgery 

In general, unless an exception is specifically justified as an essential component of the research protocol and approved by the ACUC, nonrodent 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, UM policy is that all major survival surgery on nonrodent species must be performed in approved surgical suites that meet all accreditation requirements.

 

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 (the responsibility of the PI) requires close monitoring of the organism 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 ACUC.

 

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, Animal Resources 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.

 

Food Deprivation

Food deprivation below 85% of ad libitum weight requires special justification to the ACUC.  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.

 

Animal Identification

Injection of microchips, and non-toxic tail or skin/tail marking 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 case-by-case basis.

 

Antibiotics

Invasive surgical procedures require the use of prophylactic antibiotics.  Justification for not using antibiotics must be documented with citations from the literature or data from the PI's laboratory.

 

Avian Field Studies

Birds 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 as 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 bord’s history, including date and place of capture, and signs of illness, hormone treatment, and date and place of release.  In addition, records of minimum and maximum temperatures are maintained.  Reports of captures and releases are submitted to the state and federal permit-granting agencies, as required.

 

Ascites Production

National Institute of Health guidelines require that in vitro technologies are the default method to produce monoclonal antibodies.  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 strong 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 ACUC, 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 volumes should 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 recommended that hybridomas be tested for the presence of adventitious viral and mycoplasma agents prior to use in animals. 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.
 
·             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, and weight loss, loss of body condition, inactivity, and difficulty in ambulation, tachypnea, and dyspnea. Any of the preceding signs of distress warrant notification of the veterinary staff or euthanasia of the animal.

 

Use of Complete Freunds Adjuvant

Improper or unnecessary use of Freund's adjuvant may cause inflammation, in duration, 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 ACUC maintains the following guidelines to eliminate or minimize animal discomfort associated with the use of this agent in research.

 

1.   Before using Freunds's complete adjuvant (FCA), or if you are already using

FCA, consider the use of Freund's incomplete adjuvant (FL4) or other types of

adjuvants or other commercial adjuvants on the market.

 

2.   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.

 

3.   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)

 

4.   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.

5.   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.

6    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

1.  Ten to 45 day intervals between immunizations are acceptable.

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

 

Blood Draws, Volume and Frequency

1.       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 ACUC.

 

 

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, followed by application of 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 ACUC protocol.

 

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 17mm in a 25g mouse or 35mm 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.

 

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 incrasing tumor burden); caliper measurements to determine tumor volume or mass; and inspection and palpation to locate the sites of tumor growth, as well as to assess distension, ulceration and compromised mobility.

 

Policy for the Use of Outdated and Expired Material

The use of expired antibiotics, analgesic and anesthetics is expressly prohibit by the Welfare Act and PHS policy.  Expired diagnostic reagents, drugs and devices may not be used at the U of M 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. 

 

 

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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 some evidence that animals may be at significant risk from transgenic research.  This will of course depend on the vectors and genes used, either in heterozygous/homozygous animals or those receiving gene transfer or cell transplants.  These risks include:

 

1) Pain and/or discomfort

2) physiological or morphological problems, including early death, internal bleeding, CNS/PNS dysfunctions and other system defects; and

3) 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 of evidence of animal suffering.  Such plans would, for instance, establish criteria for protocol termination that could be invoked in various experimental contexts. Additionally, the ACUC 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 ACUC 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.

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