Purpose

In order to ensure appropriate use of Complete Freund’s Adjuvant (CFA), the UConn Health’s Institutional Animal Care and Use Committee (IACUC) has implemented a policy on the use of CFA.

Background

CFA is an oil-in-water emulsion containing mycobacterial cell wall components. It is used as an agent to potentiate antibody response to injected immunogens by eliciting a local inflammatory response. CFA should be used only when absolutely necessary (e.g., weak antigens, induction of auto-immune response) and may only be used for the initial immunization. Incomplete Freund’s Adjuvant (IFA) or other adjuvants that produce less intensive inflammatory responses (e.g., RIBI^® or TiterMax^®) should be used whenever possible. Improper use of Freund’s adjuvant can cause undesirable and painful side effects such as excessive inflammation and swelling, abscess formation, ulceration, and tissue necrosis.

Any proposed deviation from these guidelines must be fully explained and justified to the Animal Care Committee in the research proposal.

Occupational Health and Safety

Due to the fact that CFA contains killed mycobacteria, accidental inoculation into personnel handling this agent can result in subsequent positive tuberculin titers and/or local inflammatory lesions, which have poor response to antibiotic therapy. These lesions can be severe and result in granulomas, abscesses, and/or tissue necrosis. Extreme caution should be taken when using CFA.

Personnel Protective Equipment (PPE), especially gloves and eye protection, must be worn when handling CFA. Needles should not be recapped, Luer lock syringes should be used, and anesthesia or sedation of animals being injected is recommended to minimize the risk of accidental exposures to personnel.

Action

Procedure

CFA may be used only for the first (priming) immunization. IFA should be used for any subsequent, booster immunizations. Inoculum should be free of extraneous microbial or chemical contamination. Filtration of the antigen before mixing with adjuvant is recommended.

Sites of administration of the CFA should be chosen so as to avoid anatomic sites used for handling or restraint of the animals being inoculated. Intravenous administration of CFA is prohibited. Intramuscular, intradermal, and footpad administrations (rodents only, one footpad only) are permitted only with scientific justification and approval of the IACUC.

Injection sites should be clean and free of debris as contamination is likely to result in local infections. Clipping of hair and surgical preparation of the injection sites will reduce the potential for the development of infection and/or abscess formation. Sterile Luer lock syringes and needles should be utilized. Needles should be 25 to 31 gauge in size. The number of injection sites should be limited to the minimum necessary to accommodate the volume of material being administered. Injection sites should be sufficiently separated to prevent coalescing of any inflammatory responses from different injection sites.

Animals may receive booster immunizations of IFA or another adjuvant, as required, to develop the desired immune response as long as no adverse effects from prior immunizations are present. There should be a minimum interval of two weeks between initial and subsequent immunizations.

The following is a list of acceptable routes of administration and volumes of CFA for the indicated species:

Species	Subcutaneous	IM	SubQ via Footpad	ID	IP
Mouse	0.1 ml/site 4 sites maximum	NR (Not Recommended)	Requires Scientific Justification	0.05 ml/site 4 sites maximum	0.25 ml maximum
Rat	0.2 ml/site 4 sites maximum	NR	Requires Scientific Justification	0.05 ml/site 4 sites maximum	0.5 ml maximum
Rabbits	0.25 ml/site 4 sites maximum	0.25 ml/site 4 sites maximum Requires Scientific Justification	NR	0.05 ml/site 1.0 ml total	Not permitted

Animals must be closely monitored for complications following CFA administration. Daily observation of injection sites must be documented in written records for the initial 2 days post immunization and a minimum of once weekly thereafter. Injection sites should be closely monitored for the development of severe lesions. When the adjuvant has been administered intraperitoneally in rodents, the animals must be monitored closely for excessive abdominal distension.

References

1. ARAC Guidelines: Recommendations for Consideration in the Research Use of Inflammatory Agents. NIH Office of animal Care and Use. May, 1996.

2. Jackson, LR and Fox, JG. Institutional Policies and Guidelines on Adjuvants and Antibody Production. ILAR Journal, Vol. 37(3): 141-152, 1995.

Effective Dates:     December 7, 2023 through December 31, 2026

Purpose

In order to comply with NIH-PHS government regulations which state that all vertebrate animal procedures described in a PHS grant must be approved by the institutional animal care and use committee, the University of Connecticut Health’s Institutional Animal Care and Use Committee (IACUC) has implemented a policy regarding new animal care and use protocol submissions.

Action

1. All animal care and use protocol submissions must be submitted electronically through the use of the InfoEd lab animal module.  The InfoEd electronic Research Administration (eRA) portal is web-based tool available at https://ovpr.uchc.edu/services/rics/animals/iacuc/protocol/.

2. Protocols must be submitted by deadlines which are on the IACUC website.  Only complete protocols will be reviewed by the IACUC; simply submitting a protocol by the deadline does not mean that the protocol is ready for committee review.  If your protocol is returned for any reason, you must resubmit the revised protocol by the deadline date to be considered at that month’s meeting.  Extensions of deadlines must be approved by the IACUC Chair.

3. Training on the use of InfoEd is highly recommended.  Information on how to receive training can be obtained from the Animal Care Committee office.

Effective Dates:     December 7, 2023 through December 31, 2026

Purpose

UConn Health’s Institutional Animal Care and Use Committee (IACUC) is committed to the humane care and use of laboratory animals. To ensure that laboratory animals receive humane care and use or treatment in accordance with the highest ethical standards, laws, regulations, and policies governing animal research, the IACUC must review and, if warranted, address any animal-related concerns by the public or UConn Health employees. The following will outline the established procedure to ensure that concerns are communicated to the IACUC. The IACUC must review each concern in a timely and systematic manner and, when necessary, take prompt and appropriate corrective action.

Reports of animal welfare concerns may be made anonymously, if desired. However, if the complainant would like to know the resolution of the investigation, he must provide a name. All reports will be handled confidentially, although anonymity cannot be guaranteed.

This policy is subject to the UConn Health Center Whistle Blower’s Protection Policy.

Action

1. Letter-sized posters outlining how to report animal care and use concerns are available and posted throughout the health center: in the main department offices, and on each floor where animal rooms are located. Concerns may be sent to the following:

• Non-compliance Report Line, UCHC Service, 888-685-2637
• Office of the Institutional Animal Care and Use Committee, 860-679-4129
• Center for Comparative Medicine, 860-679-2731
• IACUC chair, 860-679-8199

2. When alleged incidences of non-compliance are reported to the IACUC, these incidences will be investigated with the utmost concern for confidentiality and due process, without compromising the welfare of the animals.

3. If the incidence directly involves animal health and safety, a Center for Comparative Medicine (CCM) veterinarian will assess the health and well-being of the animal(s) and will verify the concern. If there is animal suffering, the veterinarian will take immediate action, including supportive care or euthanasia. While a good faith effort will be made to contact the principal investigator (PI), the immediate animal welfare situation may necessitate that immediate action be taken without the PI’s knowledge.

4. Once the animal(s)’s welfare is assessed and addressed (or in those situations where animals are not directly involved), the alleged concern or issue will be evaluated by the IACUC Chair, the Attending Veterinarian, the IACUC Administrator, and/or the Institutional Official (IO) generally within 5 working days of receiving a report of an animal welfare concern.

• If they determine a complaint does not warrant further investigation, the IACUC Chair will inform the IACUC of the incident and its resolution at the next convened meeting.
• If they determine a complaint warrants investigation, the IACUC Chair will inform the Principle Investigator and appoint a subcommittee of IACUC members to lead a formal investigation of the complaint.  Once the investigation is complete, a report will be generated for the full IACUC to evaluate.
• The IO will then, if a reportable non-compliance has occurred, inform the NIH Office of Laboratory Animal Welfare (OLAW) to comply with the OLAW requirement for prompt reporting (NOT-OD-05-034).  The IO will inform the affected PI(s) of this notification.

5. It is the responsibility of the IACUC subcommittee to review the concern and determine the appropriate requirements for the investigation in a timely manner (generally within 5 working days of the formation of the subcommittee) and communicate with the PI involved in the complaint.

6. Once the subcommittee informs the IACUC of the results of the investigation, and it is reviewed and agreed by the full IACUC, then the IACUC will inform the PI and the IO of the results of the investigation.

7.  A final report of reportable non-compliance will be sent to OLAW by the IO.  If the non-compliance activity requires reporting to the USDA Animal and Plant Health Inspection Service, this will also be done at this time.  Concurrent with notification to federal agencies, AAALAC will also be informed of the non-compliant event.

8.  If the complainant disclosed his name, he will then be contacted by the IACUC Administrator with the results of the investigation.

9.  If allegations of misuse of laboratory animals or non-compliance with federal, state, or institutional policies or regulations have been established by the investigation, the potential consequences to the involved individual(s) may include:  mandatory retraining, suspension of animal protocol(s), termination of animal protocol(s), or permanent withdrawal of IACUC approval to use laboratory animals.

Effective Dates:  December 2, 2021 through December 30, 2024

Purpose

In order to comply with governmental regulations and to simplify the review process, the IACUC has established a policy on the ability to use the Designated Member Review (DMR) on new protocol submissions and modification submissions.

Action

1. New protocols and modifications will be reviewed by the IACUC coordinator to see if they meet the review criteria for a Designated Member Review (DMR). (Table 1)

2. If a protocol or modification meets these criteria, it will generally be assigned a primary (scientific), a veterinary, a safety reviewer, and a regulatory reviewer. These individuals will act as agents of the IACUC and can require and perform any function of the IACUC with the sole exception of withholding approval of a protocol. Only a full committee vote may do that.

3. All members of the committee will be assigned in InfoEd as a “Call for FCR or not” reviewer to allow for a call for a full committee review by the specified deadline.  In addition, primary reviewers will be assigned in InfoEd as “Primary review” with a deadline for review.

4. If no one calls for a FCR, then the primary reviewers will complete the review.  The review will be sent via InfoEd to the Principal Investigator for any required changes.  Subsequent changes will be reviewed by the primary reviewer without requiring a call for FCR by the Committee.

5. 3-year renewals will be processed as DMR regardless of pain category and species used.

Exceptions require the approval of the IACUC Chair.

Table 1: Criteria for Designated Member Review versus Full Committee Review

	Designated Member Review (DMR)	Full Committee Review (FCR)
Pain/Distress Level	B and/or C	D and/or E
Species	Non-USDA regulated	USDA regulated
Invasiveness	Euthanasia only (tissue harvest) Breeding/holding protocols Injections Routine blood collections Minor surgery Non-survival surgery	Survival surgery Death as an Endpoint Prolonged restraint Food/fluid restriction Any exceptions to the regulations
Protocol Type	3 year renewal protocols (regardless of species or pain category)

Effective Dates:   June 1, 2023 through June 30, 2026

This policy has been approved by a majority vote of the IACUC members.

Joseph Lorenzo, M.D., Chair, Institutional Animal Care and Use Committee

Purpose

The Animal Welfare Act, Animal Welfare Regulations, and PHS Policy require institutions to provide training for all personnel appointed to the Institutional Animal Care and Use Committee (IACUC) at an institution. The UCHC IACUC is charged with ensuring that all personnel who are appointed to the IACUC are adequately trained.

Action

1. All new individuals who are being considered for an appointment to the IACUC will meet with the IACUC Chair, coordinator, and the Institutional Official. This meeting will cover topics including, but not limited to, the following: the function of the IACUC, logistical aspects of appointment (meeting dates, times, location, etc.), and the responsibilities of the member related to the committee position they will hold (e.g., scientific member, community member, etc.).

2. If the individual agrees to an oral invitation to the committee by the Institutional Official, an appointment letter will be sent to them by the Executive Vice President for Health Affairs.

3. Once the individual is appointed, they will meet with the IACUC coordinator. In this discussion, the new committee member will be introduced to the federal and state laws and regulations which govern the use of laboratory animals as well as institutional policies and procedures.

4. The new committee member will be given a copy of the following:

• The IACUC Committee Member Handbook
• The Guide for the Care and Use of Laboratory Animals
• Public Health Service Policy on Humane Care and Use of Laboratory Animals (This document includes the Health Research Extension Act of 1985 and the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training.)
• Animal Care (USDA) Policies
• AVMA Panel on Euthanasia Guidelines
• Institutional Animal Care and Use Committee Guidebook

5. The new committee member will be required to complete two training modules through CITI Training. These include “Working with the IACUC” and “Essentials for IACUC Members”. The new committee member will be required to pass the examinations associated with these training modules.

6. The new committee member will be trained on how to access InfoEd and access the IACUC shared folder, if required, by the IACUC coordinator.

7. The IACUC office will be responsible for documenting all training activities of IACUC members here at the Health Center.

8. Any exemptions to the training requirements require the written consent of the IACUC Chair.

Effective Dates:  June 6, 2024 thru June 30, 2027

Purpose

The Animal Welfare Regulations (9 CFR, Ch. 1, 2.31 (c)(8)) and PHS Policy (IV.B.8) charge the IACUC (Institutional Animal Care and Use Committee) with the authority to suspend an activity involving animals in accordance with the specifications set forth in the Animal Welfare Act and/or PHS Policy or because it has determined that the activity is not being conducted in accordance with the description of that activity as provided by the principal investigator in the animal care application. The following will outline the established procedure which will be used to notify Principle Investigators (PI) of the decision of the IACUC to suspend protocols.

Action

There are three ways a protocol can be suspended:

1. The decision to suspend a protocol may be reached by a majority vote of a convened meeting of a quorum of the IACUC.

a. The PI will be notified by phone or email by the chair of the ACC within 24 hours of this action being taken by the IACUC.

b. The PI will also receive a letter from the IACUC stipulating the actions taken by the IACUC.

2. In exceptional circumstances where there is a clear threat to animal health, the Attending Veterinarian (AV) has the authority to suspend animal activities on a protocol. This action needs to be followed by a convened meeting of the IACUC as soon as possible to review the cause of the suspension of animal activity.

a. The PI will be notified by phone or email by the AV within 24 hours of this action being taken by the AV.

b. The PI will also receive a letter from the IACUC stipulating the actions taken by the AV and/or a description of actions prospectively to be taken as a result of the IACUC meeting following the suspension of activities by the AV.

3. The Institutional Official (IO) may suspend an activity that the IACUC has previously approved in the case of serious or continuing non-compliance with IACUC approved protocol or governmental law or regulation, misuse, or mistreatment of research laboratory animals, or previously unanticipated risk to the laboratory research personnel. This action needs to be followed by a convened meeting of the IACUC as soon as possible to review the cause of the suspension of the animal activity.

a. The PI will be notified by phone or email by the IO within 24 hours of this action being taken by the IO.

b. The PI will also receive a letter from the IACUC stipulating the actions taken by the IO and/or a description of actions prospectively to be taken as a result of the IACUC meeting following the suspension of activities by the IO.

4. All further actions of investigating animal welfare issues, complaints, and cases of non-compliance with animal care and use protocols are outlined in the IACUC Policy on Reporting Animal Welfare Concerns.

Effective Dates:  December 1, 2022 through December 31, 2025

Purpose

The IACUC has an obligation to ensure compliance with federal regulations regarding the use of laboratory animals. PHS policy states that animals must be counted in the protocol without regard to the age of the animals. If animals are bred for the purpose of utilizing the animals for research, testing, or training purposes, the animals must be counted toward the protocol even if the animals are not “used” for an experimental, testing, or training procedure.

Action

1. If a pre-weaned animal is genotyped, found to be inappropriate for an experiment and subsequently euthanized, the animal must be counted as being used toward the protocol.

2. If an animal is euthanized prior to weaning and was bred for the purpose of being used for research, testing, or training activities, the animal must be counted as being used toward the protocol.

3. If a pre-weaned animal has any other experimental procedures performed other than genotyping- including behavioral studies- that animal must be counted toward the total number of animals used on the protocol.

4. If an animal is not used experimentally, but is euthanized post-weaning (e.g., the genotyping was performed post-weaning), it must be counted toward the number of animals used in the protocol.

5. At the next cage change after a litter is born, the Center for Comparative Medicine (CCM) will provide a weaning card with the number of animals that were born in the litter.  Research personnel must then enter this number into InfoEd as an order for unused pups.  This will automatically delete this number of animals from the authorized amounts for each protocol.

 

Effective Dates:    June 1, 2023 through June 30, 2026

This policy has been approved by a majority vote of IACUC members.

Purpose

In order to comply with government regulations (PHS and USDA) and the American Association for the Accreditation of Laboratory Animal Care (AAALAC) the UConn Health’s Institutional Animal Care and Use Committee (IACUC) has implemented a policy stating that the ACC will use the recommendations and standards set forth by The Guide for the Care and Use of Laboratory Animals, 8th Edition (NRC, 2011) in determining appropriate standards of laboratory animal care.

Action

Any protocols or situations which require a deviation from the Guide must be submitted to the IACUC in writing with a justification of why the deviation is required. This justification must be scientifically sound and not for the convenience of the investigator or for monetary reasons.

Effective Dates:    December 7, 2023 through December 31, 2026

Purpose

In order to ensure adequate review of animal care and use protocols, the UCHC Institutional Animal Care and Use Committee has established a policy on protocol reviews.

Action

1. All protocols to be reviewed at an IACUC meeting must be submitted through the InfoEd eRA Portal Animal Use / IACUC module by the 10th of the month in which the protocol will be reviewed to guarantee a review at that month’s IACUC meeting.  Anything submitted after the 10th of the month may be considered for that month’s meeting, but a review is not guaranteed.

2. The principal investigator (PI) will typically be notified of the results of the IACUC review of their protocol within 5 working days of the date of the IACUC meeting in which the review occurred. This is sent to the PI through the InfoEd system by email to the PI’s outlook email address.

3. If the IACUC review was MODIFICATIONS REQUIRED, SUBSEQUENT REVIEW BY FULL COMMITTEE REVIEW:

• The PI will need to respond to all comments outlined by the IACUC
• The PI will need to submit a revised protocol
• The protocol will have to be reviewed by the full IACUC
• The PI will have 3 months to respond to an IACUC review
• If no response is received in 3 months, the protocol will be considered withdrawn unless the PI writes a letter to the committee requesting an extension

4. If the IACUC review was MODIFICATIONS REQUIRED, SUBSEQUENT REVIEW BY DESIGNATED MEMBER REVIEW:

• The PI will need to respond to all comments outlined by the IACUC
• The PI will need to submit a revised protocol
• The PI will have 3 months to respond to an IACUC review
• If no response is received in 3 months, the protocol will be considered withdrawn unless the PI writes a letter to the committee requesting an extension

5. If the IACUC review was APPROVED:

• No further action is required by the PI

6. If the IACUC review was DISAPPROVED:

• The IACUC will include a statement of the reasons for its decision
• The PI will have the opportunity to respond in writing to the disapproval
• If the chair allows a re-submission, it must be a brand new protocol submission.

Effective Dates:  December 1, 2022 through December 31, 2025

Purpose

In order to comply with government regulations governing the use of vertebrate animals and to ensure the well-being of animals at UConn Health, the Institutional Animal Care and Use Committee (IACUC) has implemented a policy regarding the collection of samples for genomic analysis from genetically engineered mice and rats.

Background

To determine if genetically-engineered mice and rats carry a gene of interest, tail biopsies are commonly performed to obtain samples for DNA analysis. DNA for polymerase chain reaction (PCR) analysis can also be obtained from ear punches during identification procedures, blood samples, or saliva swabs. To obtain larger amounts of DNA for Southern Blot testing, tail biopsy is usually performed. Tail biopsy is a safe and humane procedure when it is performed correctly.

Tail Biopsy:

Tail biopsy is recommended to be performed between 10-21 day old mice as the tissue is soft, and the bones have not yet been calcified.  Further, the yield of DNA is the highest.

Procedure for Mice and Rats Between 10 and 21 Days of Age (or at weaning)

1. The tail biopsy procedure should be described in the approved animal care and use protocol.

2. At this age, the tail is still soft and the tail vertebrae have not yet calcified.

3. Local anesthetics are recommended, but not required.

4. Plastic restrainers are useful to hold the mouse.

5. The tail biopsy procedure must be performed using clean gloves and a sterile sharp scalpel, sharp scissors, or razor blade.

6. Prior to biopsy, the tip of the tail may be numbed with ice or immersed in ice-cold isopropyl alcohol for 10 seconds.

7. Do not use iodine solutions because they may interfere with DNA analysis.

8. Tail skin should be disinfected with alcohol prior to incising the tip. Make one clean cut through the tail. The length of the biopsy cannot exceed 5mm.

9. Following the biopsy procedure, bleeding should be controlled using local pressure. After releasing the animal back into the cage, it should be observed to make certain that the bleeding has stopped. The presence of blood in the cage may cause aggression between cagemates.

10. If bleeding occurs, it may be necessary to cauterize the tip of the tail with silver nitrate.

11. Please consult the Attending Veterinarian for advice when using cauterizing agents, as they may be toxic if they are ingested by the animal.

Procedure for Mice Older than 21 Days of Age

1. The procedure should be described in the approved animal care and use protocol. The rationale for testing older mice should be explained.

2. The use of a general or local anesthetic is required. Appropriate local anesthetics can include EMLA or lidocaine cream. Appropriate general anesthetics can include ketamine/xylazine, ketamine/medetomidine, or isoflurane.

3. Please consult the surgery guidelines for general anesthetic details.

4. Steps 5 to 10 should be followed as described above.

5. For rats > 35 days of age, the use of a general anesthetic is required.

Note: Removal of more than 5 mm of tail in rodents is only allowed in exceptional cases, and it must be approved by the IACUC. If repeat sampling is necessary, anesthesia must be used and only small amounts of tissue may be taken.

Ear Biopsy/Punch:

1. It can be done during the ear punch identification procedure.

2. The ear punching technique does not require anesthesia if performed by skilled staff. The appropriate time for ear punch is between 15 and 17 days of age.

3. The animals should be manually restrained and the sample should be collected aseptically.

4. Bleeding after an ear punch is uncommon and the animal can be released directly into the cage.

References

1. Blickman, A. and C. Vogelweid. ISUM IACUC Guidelines, February, 2003.

2. The Guide for the Care and Use of Laboratory Animals. National Research Council, 2010.

3. Tissue Collection for Genetic Identification of Rodents. Division of Laboratory Animal Resources, University of Kentucky Medical Center.

Effective Dates:  June 2, 2022 through June 30, 2025