Montana State University


Montana State University
P O Box 173085
Bozeman, MT 59717

Contact: Elizabeth Nicholas

Tel: (406) 994-6821
Fax: (406) 994-4790

Office of Research Compliance

Tel:(406) 994-6757


Montana State University Animal Care and Use Policies

Mechanism for Reporting issues of Non-compliance Or Misuse of Animals

Administrative Policies
Animal Use
Animal Transfers
Congruency between Grants to Protocols
Full Committee Review Process
Guidelines for Reporting to OLAW
Non-response on Protocols Pending Approval
Post-approval Monitoring
Protocol Modifications
Reporting Animal Concerns (Whistle-Blower Policy)
Research Projects using Insects
Research Projects using Chick Embryos
Tours, Videotapes and photographs
Vivarium Access

Single Housing of Rodents
Sanitation Frequency of Primary Enclosures for Rodents

Animal Procedure Policies

Access to Nonhuman Primate Housing Room
Escaped Rodents
Enrichment Policy
Food and Water Restriction
Humane Experimental Endpoints
Maximum Dosing Volumes
Mouse Housing Density
Non-terminal Blood Withdrawal Guidelines
Polyclonal Antibody Production
Complete Freund's Adjuvant
Stabilization Period after Animal Transport
Tail Snips
Weight Loss

Surgery and Anesthesia Policies

Anesthetic Cocktail Expiration
Multiple Major Survival Surgeries
Pain Medications
Rodent Surgical Guidelines
Survival Surgeries

Administrative Policies

 Adoption of Animals used in Research and Teaching

The Animal Resources Center and Montana State University recognizes the invaluable contribution of research animals in the advancement of biomedical knowledge. The Institutional Animal Care and Use Committee (IACUC) endorses and promotes the responsible, humane, and appropriate use of research animals while complying with the concept of “refinement, reduction, and replacement” in our use of research animals. The IACUC also recognizes that sometimes animals finish a research or teaching project in good health but are not suitable or needed for any other projects at the institution. In such circumstances, the Animal Resources Center may consider finding adoptive homes for the animals. It is understood and widely accepted that adoption programs not only enhance the quality of life for healthy research animals that are no longer needed by the University, but can also decrease stress and raise morale for both the research and animal care teams. We believe that responsible and compassionate researchers utilize animals only when necessary; therefore it follows that we should do our best to ensure good lives for those animals whose sacrifice is not required by science.

The purpose of this policy is to allow cats and other research animals that are no longer assigned to protocols to be adopted by individuals as companion pets. The IACUC recognizes that adoption of research and teaching animals raises the potential for human health and safety concerns. While it is impossible to reduce health and safety risks to zero, animals selected for adoption must be healthy and present minimal risk of transmitting zoonotic infection.

The following conditions must be met in order to place an animal for adoption:

Adoption privileges are granted only for Montana State University staff, faculty, and students.

An animal will be considered for adoption only if:

    • The purpose for which the animal was acquired no longer exists
    • The animal will not be needed in another IACUC approved study
    • The animal has been returned to the Animal Resources Center
    • An adopter has been identified to adopt the animal.

An animal may be placed for adoption only if considered clinically normal.

    • An animal must not have experienced any manipulation that has detrimentally altered its anatomical, physiological, metabolic, or locomotor function.
    • The animal must be free of detectable infectious disease or physical abnormality, which would threaten animal or public health.

Before being released for adoption, animals must be examined by the attending veterinarian and found to be in good health and of suitable temperament appropriate for a pet. Cats and rabbits must be neutered and receive all age-appropriate vaccinations, as is accepted by standard veterinary practice. Cats may be declawed if requested by the adopting party with the understanding that the cat will remain an indoor pet

The individual adopting an animal must sign a waiver which states the University is not liable for any injury or damage to persons or property by the adopted animal. The waiver further states that no warranties, guarantees, or promises of any kind have been made or can be made with regard to the adopted animal’s physical condition or temperament. The owner assumes all further responsibilities associated with responsible companion animal ownership.

An animal purchased from a Class A dealer (purpose-bred) may be eligible for adoption as long as the above conditions are met.

At the time of transfer of ownership, the adopter shall assume all financial responsibility for housing, care, and medication of that animal.

For each animal being adopted an “Animal Adoption Agreement” will be completed and signed by the attending veterinarian and the adopting party. This record should be maintained by the Animal Resources Center for a period of 3 years from the date of signature by the new owner.

Any deviations from the provisions of this policy must be approved by the Director of the Animal Resources Center and a simple majority of a quorum of the IACUC.

Animal Use According to Protocol Content

Animals ordered for a particular protocol must be used according to methods reviewed and approved by the IACUC for that protocol. Animals may be transferred between protocols following the “Animal Transfers” Policy.

Animal Transfers

The transfer of animals to other protocols is allowed if the animal’s condition is satisfactory and major survival surgery is not required. The Animal Transfer Request form must be completed and submitted to the ARC. If procedures have been performed on more than one protocol, the animal use will be accounted for under each protocol.

Congruency between Grants to Protocols

The MSU IACUC attempts to confirm congruency between PHS grant applications and IACUC protocols through three methods. First, it is the Primary Investigator’s responsibility to provide correct and accurate information to both the IACUC and the funding agency. As part of the animal care and use protocol form, the PI must sign an Assurance and Statement of Accuracy. They must certify that the information is complete and accurate and consistent with any submission to external funding agencies. Second, prior to grant submission, the MSU Office of Sponsored Programs requires the investigator to submit a Proposal Clearance Form. If the grant includes the use of animals, either the IACUC Chair or the Attending Veterinarian must review and sign off on the application. Third, the Director of the Office of Sponsored Programs is a member of the IACUC, further assisting in the comparison of these documents.

Full Committee Review Process

Protocols are reviewed by the full IACUC at each monthly meeting. The committee Chair or chosen designee selects a committee member (Primary Reviewer) to take responsibility for describing the proposal and answering questions about the proposal at the full committee meeting. Protocols are sent to committee members a week before the meeting so each member has time to review the proposal before presenting the project to the committee. The Primary Reviewer is responsible for contacting the Primary Investigator prior to the meeting if protocol clarifications are needed. The Veterinarian and Chair also perform a “pre-review” prior to distributing the protocol to the Committee. Protocols require approval by a quorum of the full committee.

The committee may review and approve, require modifications to secure approval, or withhold approval if significant changes are required. If modifications are required to secure approval, the questions are submitted to the Investigator via email. The response is reviewed by the Primary Reviewer, the IACUC Chair and the Attending Veterinarian. If the response is unanimously approved by this designated subcommittee, it is added to the protocol file and final approval is granted. If one or more subcommittee member(s) are not satisfied with the response, it returns to the full committee at the next meeting.

Guidelines on Prompt Reporting to OLAW under the PHS Policy

The MSU IACUC will follow the Guidelines issued by OLAW, Office of Extramural Research on February 24, 2005. This guidance is intended to assist IACUCs and Institutional Officials in determining what, when, and how situations of noncompliance should be reported under IV.F.3 of the PHS Policy on Humane Care and Use of Laboratory Animals.

PSH Policy, IV.F.3 requires that:

“The IACUC, through the Institutional Official, shall promptly provide OLAW with a full explanation of the circumstances and actions taken with respect to:

Any serious or continuing noncompliance with this Policy;Any serious deviation from the provisions of the Guide for the Care and Use of Laboratory Animals; or

Any suspension of an activity by the IACUC.”

These guidelines are available at:

Non-response on Protocols Pending Approval

If a there are questions on a submitted protocol and the PI fails to respond within 30 days of a written letter, the protocol will be administratively withdrawn.

Post-approval Monitoring

The Montana State University post-approval monitoring program is in place to ensure the well-being of animals involved in research activities, to make certain that activities are conducted in compliance with state and federal regulations and standards, and to ensure
that activities are consistent and conducted in compliance with IACUC approved protocols, as well as other institutional policies.

Since the animal care and use program at MSU is fairly small with only seven employees, there is no one person or institutional component responsible for performing post-approval monitoring. A multitude of individuals contribute to the post-approval monitoring program, including the IACUC, the research staff, the ARC staff, and the Attending Veterinarian. A variety of procedures are in place to assure compliance. These include:

The research program and facilities are reviewed on a semi-annual basis. Any issues of noncompliance are monitored for appropriate follow-up by the IACUC Coordinator to ensure resolution and to prevent recurrence.

Through initial orientation and periodic continuing education, all personnel involved in animal studies are aware of their responsibilities as they relate to animal activities. Protocols are available for review and all research personnel are expected to be familiar with the protocol
under which they are working.

Primary Investigators receive training on protocol submission and are aware of the methods available to change or modify protocols. IACUC policies are readily available on the IACUC web page.

Animals are housed under centralized conditions, with all mammals housed either at the Animal Resources Center or the Jutila Research Laboratory. The ARC staff is responsible for routine daily observations and monitoring of animal research activities.
The ARC Manager and three of the staff members are AALAS certified at the LATg level. In addition, they are intimately involved in assisting research staff with many of the research procedures and they communicate directly with the research staff on a daily basis. Each ARC staff member rotates through the IACUC on an annual basis and has ready access to all currently approved protocols.

All animal ordering is performed centrally by the ARC. A database is maintained which tracks the number of animals approved for each protocol and the number of animals ordered. This record-keeping system does not allow personnel to go beyond the number of animals approved on a protocol. If the investigator is close to the maximum number of animals allowed, an informational email is sent. As part of that email, they receive instructions for submitting a protocol modification.

All in-house breeding colonies are centrally maintained by the ARC staff. Animal numbers produced bythe breeding colonies are counted at the time of weaning and an “Animal Transfer Request” form is submitted. These numbers are entered into the database in the same way as animals ordered from outside sources.

The animal facility can be accessed only through the use of a keycard. This is used to limit access to individuals who are approved to work with animals. There are different access levels, including business hours only, ABSL1 access, ABSL2 access, and ABSL3 access.

Laboratories where animals are held longer than 12 hours (USDA covered species) or 24 hours (miceand rats) are reviewed as part of the semi-annual IACUC inspections.

MSU is small enough that we are able to maintain appropriate and short organizational reporting chains. The Director of the ARC reports directly to the Institutional Official and works closely with the ARC (six personnel) staff and research personnel.

A whistle-blower policy is in place to assure noncompliant issues are reported and animal welfare issues are resolved.

Annual renewals of protocols are reviewed by the full IACUC.

Protocol Modifications

Any proposed modifications of animal use methods must be communicated to the IACUC. Major modifications will be reviewed and approved by the full committee prior to implementation. Examples of major modifications include the following changes:

  • The objectives of the study
  • Non-survival to survival surgery
  • Increasing the invasiveness of a procedures or pain/distress to the animal
  • Change or addition of species
  • Increasing the number of animals used
  • Anesthetic or analgesic agents
  • Method of euthanasia
  • Status of biosafety, radiation safety, or environmental safety usage
  • Change in the Primary Investigator

Reporting Animal Concerns (Whistle-Blower Policy)

The use of animals in biomedical research is necessary and appropriate. Montana State University is committed to the humane treatment of animals and strives to ensure that institutional facilities and procedures adhere in all respects to NIH guidelines for care and use of laboratory animals.

The IACUC wants to be informed of any concerns that individuals of the campus community may have with respect to the care and use of laboratory animals and feels it is important that these concerns be addressed on an individual basis. Any individual who witnesses what he/she perceives to be inappropriate treatment of research animals can contact any member of the IACUC, or can file a concern anonymously by filling out an “Animal Concern Form” (posted at all locations where animals are housed) and submitting it to the IACUC. The identity of the person raising the concern will be held in confidence. Retaliation against a person who raises a good-faith concern will not be tolerated.

The IACUC will follow-up on all complaints and gather additional information as necessary and appropriate. If indicated, corrective action will be initiated and the results of the investigation will be communicated to the individual who raised the complaint.

Research Projects using Insects

Protocols utilizing insects are exempt from IACUC review.

Research Projects using Chick Embryos

Protocols utilizing chicken embryos up to day 15 of incubation are exempt from IACUC review.

Tours, Videotapes and Photographs

Tours of the ARC must be approved by the ARC Director or Manager. Requests will be reviewed on an individual basis, and if questionable, will be referred to the IACUC for approval. Any request for videotaping or photographing animals and animal procedures need to be approved by the IACUC. In addition, once approved, the IACUC can select to review the finished product before its release or publication. Cell Phones are prohibited from use in the ARC as the majority of cell phones have photography capabilities.

Vivarium Access

Both the Animal Resources Center and the Jutila Research Laboratory required keycard access. For the ARC, individuals must attend the orientation session and complete the AALAS Learning Library on-line course titled “Working with the IACUC” prior to gaining access to the facility. There are three levels of access that can be grants, and the access given is dependent on the request of the PI in whose lab the person will be working. Access options include business hours only, ABSL1 area only, and ABSL2 access. The Investigators are responsible for notifying the ARC Manager when someone is no longer working in the laboratory. At that time, the keycard is inactivated.

For the Jutila Research Laboratory, individuals must complete select agent requirements and access must be granted through the MSU Biosafety Officer and the Laboratory Director.

Single Housing of Rodents

The Guide for the Care and Use of Laboratory Animals (p. 37) states, “Consideration should be given to an animal’s social needs. The social environment usually involves physical contact and communication among members of the same species…..When it is appropriate and compatible with the protocol, social animals should be housed in physical contact with conspecifics.”

At Montana State University, every attempt will be made to socially house rodents. The IACUC recognizes that social housing is not always possible for experimental, health, and behavioral reasons. Some strains of rodents engage in barbering behavior and/or aggression which results in wounding of cage mates.

If individual housing is a required part of a research project, it should be discussed and justified within the animal care and use protocol. If single housing is required for health, behavioral, or breeding colony reasons it may be performed by the Animal Resources Staff using the appropriate professional judgment. Individual IACUC approval is not necessary for these cases.

Sanitation Frequency of Primary Enclosures for Rodents

The Guide for the Care and Use of Laboratory Animals (p. 43) states, “In general, enclosures and accessories, such as tops, should be sanitized at least every 2 weeks. Solid-bottom caging, bottles, and sipper tubes usually require sanitation at least once a week. Some types of cages and racking might require less-frequent cleaning or disinfection; these might include large cages with very low animal density and frequent bedding changes, cages that house animals in gnotobiotic conditions with frequent bedding changes, individually ventilated cages, and cages used for special circumstances.

The standard husbandry practice at the MSU Animal Resources Center is to sanitize rodent solid-bottom caging on a weekly basis. Under certain circumstances, sanitation may be performed every two weeks. Examples include rodents housed at a low density in individually ventilated cages, dams with new litters, and rodents housed in a high biocontainment environment.

The MSU IACUC understands that sanitation of primary enclosures for rodents is typically performed on a weekly basis, but under circumstances such as those described above, may be extended to every two weeks. Animal Resources Staff, utilizing professional judgment, will be allowed to determine the frequency of rodent cage sanitation not to exceed once every two weeks.

Animal Procedure Policies

Access to Nonhuman Primate Areas

Personnel who require access to the nonhuman primate areas are required to attend a nonhuman primate orientation session administered by the Director of the ARC. They must also complete the AALAS Learning Library course on nonhuman primates. Personnel who enter nonhuman primate areas are required to wear a dedicated long-sleeved lab coat, gloves, disposable bonnet, surgical mask, shoe covers, and eye protection. Long pants are required and open-toed shoes are not allowed.

Escaped Rodents

If a rodent escapes during the process of cage cleaning or handling and can be easily identified, it is placed back in the appropriate cage. If a rodent is found loose in an animal holding room and cannot be positively identified, it is placed in a separate cage and taken to the necropsy room for euthanasia. If a rodent is found loose within the facility outside an animal room, such as in the feed and bedding storage area, cage wash room, etc. the animal is captured, placed in a separate cage and taken to the necropsy room for euthanasia. Prior to euthanasia, the ARC staff will perform a health analysis on the rodent similar to that done for sentinel animals. Blood will be collected for serology and the animal will be examined for external and internal parasites.


When required for scientific data collection or clinical or operational requirements, animals will be humanely killed, as detailed in the approved protocol. Where applicable, a secondary method of euthanasia will be performed. Unintended recovery of animals after apparent death constitutes serious noncompliance with PHS Policy and serious deviation from the provisions of the Guide for the Care and Use of Laboratory Animals. MSU is obligated to report to the NIH any instances of animals awaking after intended death.

Public Health Policy (PHS) requires IACUC’s to determine that methods of euthanasia proposed in protocols meet criteria outlined in the 2007 Report of the AVMA Panel on Euthanasia. Primary Investigators (PI) are expected to follow the methods of euthanasia as outlined and approved in their protocols. All individuals who may perform or be expected to perform euthanasia should be familiar with the details in the protocol and the information outlined in this policy. Training in proper methods of euthanasia is part of the initial orientation and is provided by the ARC Director and Staff.

Acceptable methods for adult animals include: Carbon Dioxide (rodents only), Isoflurane, barbiturates, physical methods performed under anesthesia (cervical dislocation, exsanguination, decapitation), and MS222 (tricaine methane sulfonate) for aquatic species.  Physical methods of euthanasia without anesthesia are conditionally acceptable and must be scientifically justified and reviewed and approved by the IACUC. Personnel responsible for performing these techniques must be properly trained and the procedure will be reviewed by the ARC Director.

Carbon Dioxide Euthanasia The NIH Office of Laboratory Animal Welfare (OLAW) issued clarification regarding the use of carbon dioxide for euthanasia of small laboratory animals (July 17, 2002). The Animal Care Unit of the Animal and Plant Health Inspection Service, U.S. Department of Agriculture, has reviewed and has issued concurrence with the guidance provided by OLAW as it relates to USDA covered species.

MSU Guidelines for using CO2 include:

All personnel using CO2 for rodent euthanasia must have received appropriate training in its use. Training in the use of equipment and appropriate methods is provided by the ARC.

The only acceptable source of CO2 is compressed gas cylinders. It is NOT acceptable to use “dry ice” as a CO2 source.

Acute exposure of animals to high concentrations of CO2 is distressful and pre-filling the chamber with CO2 gas is therefore not recommended. The euthanasia chamber should contain only room air when placing the animals inside. The CO2 gas supply should then be turned on so that CO2 concentrations gradually rise.

Chambers must not be overcrowded. In this regard, it is important to also consider that mixing of incompatible animals in the same chamber may be distressful.

Death must be verified after euthanasia and prior to disposal. Unintended recovery must be avoided by the use of appropriate CO2 concentrations and exposure times or by other means. Animals subjected to CO2 euthanasia should be observed for cessation of respiratory motions and apparent death before removal for the chamber. It is best to administer CO2 for at least 3-5 minutes and allow the animals to remain in the chamber for an additional 3-5 minutes after the gas is turned off to determine that respirations have not resumed. Death may also be verified by cessation of a palpable heartbeat or ensured by performing cervical dislocation or t horacotomy.

Fetal and Neonatal euthanasia (Rodents) The AVMA Guidelines on Euthanasia (2007) do not provide specific recommendations for the euthanasia of prenatal and neonatal animals. These guidelines are based on NIH recommendations; Exceptions to these guidelines will be considered by the IACUC on a case-by-case basis.

Rodent Fetuses:

Fetuses up to 14 days in gestation: Neural development at this stage is minimal and pain perception is considered unlikely. Euthanasia of the mother or removal of the fetus should ensure rapid death of the fetus due to non-viability of fetuses at this stage of development.

Fetuses 15 days in gestation to birth: The literature on the development of pain pathways suggests the possibility of pain perception at this time. Whereas fetuses at this age are less sensitive to inhalant anesthetics, euthanasia may be induced by the skillful injection of chemical anesthetics. Decapitation with surgical scissors or cervical dislocation are acceptable physical methods of euthanasia. When chemical fixation or rapid freezing (immersion in liquid nitrogen) of the whole fetus is required, fetuses should be anesthetized prior to immersion in or perfusion with fixative solutions. Anesthesia may be induced by hypothermia of the fetus, by injection of the fetus with a chemical anesthetic, or by deep anesthesia of the mother with a chemical agent that crosses the placenta, e.g., pentobarbital. The veterinarian should be consulted for considerations of fetal sensitivity to specific anesthetic agents. When fetuses are not required for study, the method chosen for euthanasia of a pregnant mother must ensure rapid death of the fetus.

Rodent Neonates:

Up to 14 days of age: Acceptable methods for the euthanasia of neonatal rodents include: injection of chemical anesthetics (e.g., pentobarbital), decapitation, or cervical dislocation. Additionally, inhalant anesthetics (e.g., isoflurane used with appropriate safety considerations), may be used. However, neonates have a high tolerance for hypoxia, so exposure must be prolonged (>20 minutes) and death confirmed by a secondary means. Pups should be anesthetized prior to freezing with liquid nitrogen. Similarly, anesthesia should precede immersion or perfusion with chemical fixatives. Anesthesia may be induced by inhalant or injectable anesthetics; the veterinarian should be consulted for appropriate agents and dosages.

Older than 14 days: Follow guidelines for adults.

Enrichment Policy

Various terms are used to describe the welfare requirements of research animals, such as psychological well being, environmental enrichment, behavioral needs, etc. The 1985 amendments to the Animal Welfare Act require facilities to provide exercise for dogs and programs to promote the psychological well being of nonhuman primates. The Guide for the Care and Use of Laboratory Animals encourages “enriching the environment as appropriate to the species”.

The ARC is responsible for the Enrichment Program. The mission is to promote psychological well-being of all laboratory animals housed within the MSU research program through appropriate housing, enrichment, health care and activity, thereby allowing the animals to engage in species-typical behaviors.  Methods of enrichment include, but are not limited to: social housing; housing devices; manipulative devices; visual, auditory, and olfactory devices; food items. The goals of the program are to address the psychological well being of all species housed in the facility, provide interaction and self-initiated behaviors by allowing a degree of control over their environment, and to increase species-typical behaviors while decreasing pathological maladaptive behaviors.

The complete and detailed program is reviewed and approved by the IACUC and is available from the ARC.

Food and Water Restriction

Background:     Behavioral research often requires that an animal perform a task for which it receives food or fluid reward. This situation is not unlike conditions in the wild, in which animals must forage, travel distances, solve problems, or otherwise work to obtain food and water. Performing a task for rewards may also be behaviorally enriching for laboratory animals, especially nonhuman primates. However, a fundamental concern with studies that may involve food or water restriction is that animals are maintained in a healthy state and that they do not experience pain or distress. This document provides an overview of the issues and explains the IACUC’s current policy regarding food or water restriction.

The IACUC referenced the following documents in setting this policy:

NRC (1996). Guide for the Care and Use of Laboratory Animals. Washington DC: National Academy Press.

NRC (2003). Guidelines for the Care and Use of Mammals in Neurosciences and Behavioral Research. Washington DC: National Academy Press.

The federal Animal Welfare Regulations (Code of Federal Regulations, Title 9).

Toth, LA and Gardiner TW. Food and Water Restriction Protocols: Physiological and Behavioral Considerations. Contemporary Topics, 39(6). pg 9-17; 2000.


Standard for Food Intake: “Animals should be fed palatable, non-contaminated, and nutritionally adequate food daily or according to their particular requirements…..” (p. 38, “Guide for the Care and Use of Laboratory Animals,” National Research Counsel, 1996.)

Standard for Water Intake: “Ordinarily, animals should have access to potable, uncontaminated drinking water according to their particular requirements.” (p. 40, “Guide for the Care and Use of Laboratory Animals,” National Research Counsel, 1996.)

Restriction: Any deviation from the standards for food and water intake.

Fasting for surgical procedures: usually a period of less than 12 hours. This is not considered restriction and the following guidelines do not apply.


Individual animals may vary in their physiologic response to food or fluid restriction. Therefore it is imperative that animals be closely monitored on a daily basis to ensure that they are healthy, adapting normally and consume sufficient food and water to maintain their health status. Monitoring and if necessary, early intervention are the most important objectives of this policy.

It is the obligation of the investigator to demonstrate to the IACUC that food or water restriction is the only effective way to accomplish the scientific goals of the study. It is the investigator’s ongoing obligation to continue to attempt methods of positive reinforcement that do not involve food or water restriction and to use restriction only when other methods fail. The least restriction that will achieve scientific objective should be used.

The use of restriction to motivate behavior must be specifically discussed, adequately justified, and approved in each protocol in which it is used.

Criteria for monitoring animal health must be defined in the protocol (i.e. body weight, urine and fecal output, urine specific gravity, etc.). Criteria must be defined for temporary or permanent removal of an animal from the experimental protocol.

Investigators must maintain water consumption and food intake records in a form acceptable to the IACUC and readily available for inspection.

At the discretion of the IACUC, Investigators may be required to file updates or reports regarding the status of the animals on the restriction protocol.

At the discretion of the veterinary staff, a particular animal’s food or water ration must be increased if:

  • The animal becomes ill or requires medical treatment.
  • The animal exhibits weight loss of greater than 10-15% of its initial body weight.
  • The animal shows evidence of clinical dehydration.
  • A young animal fails to show reasonable weight increase during a time when it should be growing.
  • The animal undergoes any procedure requiring anesthesia.
  • Under any other circumstance deemed necessary by the veterinarian.

Humane Experimental Endpoints

The MSU IACUC is charged with evaluating and ensuring that research projects are conducted in accordance with the Animal Welfare Act and the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals. These regulations and policies require that any research, testing and teaching that uses animals must be performed in such a way as to minimize discomfort, distress and pain consistent with sound research design.

The IACUC recognizes that as a sequela of research or testing, laboratory animals may experience pain and/or distress. For certain studies the procedures may lead to the moribund condition or death. Therefore, the IACUC has developed guidelines for establishing humane experimental endpoints to assist investigators with the process of completing the UIC Protocol for Animal Use and to insure that animal welfare issues are appropriately addressed. Since the diversity of research and testing protocols preclude the establishment of a rigid set of guidelines, the ACC reserves the right to evaluate each protocol on an individual basis.


Humane Endpoint - The humane endpoint is the point at which pain and/or distress is terminated, minimized or reduced by taking actions such as euthanatizing the animal; terminating a painful procedure; or giving treatment to relieve pain and/or distress.

Moribund Condition - The moribund condition is a severely debilitated state that precedes imminent death.

Investigators should identify humane experimental endpoints that minimize pain, distress, or discomfort by choosing the earliest endpoint that is compatible with the scientific objectives of the research project. Whenever possible, the moribund condition should be used as an experimental endpoint instead of death, because it is assumed that euthanatizing a moribund animal will avoid or reduce terminal distress. The following specific sequela should also be considered:

The procedure should not interfere with the ability of the animal to ambulate, eat, drink, urinate and defecate.

The procedure should not result in a net weight loss of more than 20% of the body weight.

The procedure should be ended if the investigator has conclusive evidence that untreatable organ failure has occurred, and the animal exhibits signs associated with the failure of the organ system.

Animals should be euthanatized if unrelated health conditions develop that make their experimental use of no value to the investigator.

Obvious signs of illness should serve as alternatives to death as an experimental endpoint.

The use of death as an endpoint requires strong scientific justification in the Protocol and full committee review and approval by the IACUC.


Maximum Dosing Volumes


Adult Mice

Rats (>250 grams)

Intramuscular (IM)

0.1 ml

0.3 ml

Subcutaneous (SC)

3.0 ml

10 ml

Intraperitoneal (IP)

2 ml

5 ml

Intravascular (IV)

0.25 ml

2 ml

Oral (PO)

0.5 ml

4 ml



Non-terminal Blood Withdrawal


Maximum Blood Withdrawal


ml per 10 grams body weight


1 ml per 100 grams body weight


0.6 ml per 100 grams body weight


10 ml per kg

Nonhuman Primate

10 ml per kg

These stated volumes are the maximum blood to be withdrawn in a single bleed. At maximum, it can be repeated at two-week intervals. If more frequent bleeding is needed, the volume should be proportionally reduced. If more blood than this maximum volume is needed, the investigator must consult with the Veterinarian and receive IACUC approval.


Polyclonal Antibody Production

The production of antibodies to specific and nonspecific antigens is a tool utilized in nearly all fields of biomedical research. Today antibodies are routinely made to a vast array of proteins, carbohydrates, fats, and nucleic acids. In addition modern biochemical, biosynthetic, and recombinant techniques have created increasingly pure antigens, both natural and synthetic, in an effort to induce very specific immune responses. Many of these newer antigens are small and generally weak immunogens and it is necessary to augment their immunogenicity with adjuvants. One of these antigens is Freund’s Complete Antigen (FCA).

The NIH has published recommendations for the use of FCA. In these recommendations NIH stated that FCA is an "important biologic resource for investigators which should be used responsibly and with care to avoid or minimize the adverse effects of excessive inflammation." Recommendations are included on dosages and locations with specific mention of using caution with footpad and peritoneal inoculation. In addition, they recommend that the use of noninflammatory or less inflammatory adjuvants be considered when possible. Studies subsequent to the publication of the NIH Recommendations for the use of FCA indicate that FCA, when administered appropriately, may be pursued humanely in the production of polyclonal antibodies.

The MSU IACUC recognizes that FCA and other commercial adjuvants used in the production of polyclonal antibodies can cause granulomatous/inflammatory reactions. The IACUC also recognizes that FCA and other commercial adjuvants can be pursued humanely to produce polyclonal antibodies when administered in an appropriate manner by trained individuals familiar with the procedures. The following guidelines for the production of polyclonal antibodies in rabbits were developed to ensure consistency of technique and to minimize pain and distress.

All procedures related to production of polyclonal antibodies in rabbits using FCA will be performed by the ARC staff.

Commercial adjuvants should be administered in a manner consistent with the manufacturer’s recommendations.

It is recommended that FCA only be administered via the subcutaneous route using a 25 gauge needle. It should be administered over multiple sites with a maximum of 0.1ml dose at each site.

Gloves and lab coat should be worn when working with FCA. Individuals should be vigilant to prevent self-inoculation, which has been associated with granulamatous reactions in individuals that result in tuberculin skin test positive reactions.

Booster injections should be done with Freund’s Incomplete Antigen (FIA). Animals should not undergo more than four booster immunizations during the initial phase of antibody production.

Booster immunizations with FIA should be administered in the same manner as FCA. It is important that subsequent immunizations be administered in a different location than the previous immunizations.

Since some antigens are powerful immunomodulators, which can augment the reactive nature of FCA and commercial adjuvants, all animals administered adjuvants for production of polyclonal antibodies should be observed on a regular basis.

The administration of adjuvants for the production of polyclonal antibodies should only be performed by experienced individuals.

The Use of Complete Freund's Adjuvant in rabbits

Complete Freund’s adjuvant (CFA) is widely used and considered to be the most effective adjuvants available for consistently producing high titer antibodies to diverse antigens. It is irreplaceable and vital for immunology research and antibody production at the present time. The Institutional Animal Care and Use Committee (IACUC) has developed the following recommendations to permit the continued use of CFA while maximizing antibody titer and minimizing the adverse effects of excessive inflammation, arthritis and other toxic effects. CFA is a water-in-oil emulsion containing mycobacterial cell wall components that potentiate the humoral antibody response to injected immunogens. Adjuvant activity results from sustained release of antigen from the oily deposit and stimulation of a local immune response. The improper or unnecessary use of CFA may cause inflammation, induration, and/or necrosis in laboratory animals. The intention of the following guidelines is to minimize potential animal discomfort associated with the use of adjuvants in research. Unless specifically approved by the IACUC, antibody production using CFA at Montana State University must be done using these guidelines. Requests for deviations must be scientifically justified and will be considered on a case-by-case basis by the IACUC at the time of protocol review or in response to a request for protocol modification. Alternatives which reduce the number of animals (tissue cultures, chicken eggs, etc.) or utilize non-inflammatory adjuvants (Ribi, liposomes, incomplete Freund’s) must be considered.

CFA should be used only when necessary, such as small amounts of available antigen and weak antigens. The USDA has declared that the use of CFA may cause more than momentary pain or distress and an alternative must be considered. A written narrative description of the methods and sources used to search for alternatives must be provided in the animal care and use protocol. Key words should include CFA and alternatives.

CFA may be used only for the first (priming) dose. Subsequent immunizations should be with incomplete Freund’s or another adjuvant.

The inoculums must be free of extraneous microbial or other particulate contamination. When possible, sterilization by filtration through a low binding 0.22 micron filter should be performed.

Young adult rabbits (2.5-3.5 kg) should be used for primary immunization because of the vigorous antibody response. Immune function peaks at puberty and primary responses to new antigens decline with age.

Multiple injection sites must be separated from each other widely enough to insure continued blood supply. Anatomic sites which are weight bearing or used in restraint should be avoided (neck area in rabbits). It is strongly recommended to use sites close to the draining lymph nodes (ventral inguinal and axillary regions).

Fur must be clipped and the site aseptically prepared with the appropriate antiseptics.

Injections should be given subcutaneously. Any other route of administration requires additional scientific justification for IACUC approval.

Injections must be divided into fractions so that no more than 0.1 ml is injected per site, with no more than a total volume of 1 ml (10 sites).

Injection sites must be observed at least three times a week for four weeks after each injection. This should be properly documented in the animal record. If a lesion develops at any injection site, the Veterinarian must be notified so the animal can receive the appropriate treatment.

Stabilization Period after Animal Transport

All new animal arrivals, including mice, rats and other rodents, must undergo a minimum 48-hour stabilization period before experimental use. No component of the study may be initiated during this stabilization period. The stress associated with transportation has long been recognized to have profound physiological consequences. It is important to provide an adequate period before experimentation to allow animals to recover to a physiologically stable condition. This stabilization enhances both the validity of the experimental results and animal welfare.

Tail Snips

Rodents that are over 21 days of age must be anesthetized for a tail snip (amputation) procedure. Hemostasis must be ensured before the animal is returned to its cage.

Weight Loss

If weight loss reaches 15% of original body weight, the Veterinarian should be consulted to determine a course of action regarding the animal. In most cases the active use of the animal on study is suspended so the animal’s condition can be improved.

Surgery and Anesthesia Policies

Anesthetic Cocktail Expiration

Anesthetic Cocktail mixtures should be dated at the time of mixing and will be considered to expire 2 weeks later.

Multiple Major Survival Surgeries

Multiple major survival surgical procedures can be justified if they are related components of a research project, if they will conserve scarce animal resources, or if they are needed for clinical reasons. It is unacceptable to subject an animal to multiple unrelated survival surgeries simply to save money or because the animals are in short supply.

Pain Management

Investigators are required to administer appropriate analgesics to all animals associated with a procedure that would normally require pain medication in humans.  Questions concerning appropriate medication and dosage for a given species should be directed to Veterinarian.  Exemption from this regulation is possible only on a protocol–by-protocol basis after Full IACUC review and approval.

Rodent Surgical Guidelines


Surgery should be conducted in a disinfected, uncluttered area, which promotes asepsis during surgery.

Prepare the animal by removing hair from the surgical site. Perform this procedure in an area separate from where the surgery is to be conducted.

 Prepare the surgical site(s) with an appropriate skin disinfectant.

Surgeons should wash and dry their hands before aseptically donning sterile surgical gloves.


The animal must be maintained in a surgical plane of anesthesia throughout the procedure.

Begin surgery with sterile instruments (autoclave, chemical sterilant, ethylene oxide) for each animal and handle them aseptically. Glass bead sterilizers may be used to sterilize the tips of instruments between animals or procedures.

Instruments and gloves may be used for a series of similar surgeries provided they are maintained clean and disinfected between animals.

Monitor and/or maintain the animal's vital signs.

Close surgical wounds using appropriate techniques and materials.


Move the animal to a warm, dry area and monitor it during recovery. Return the animal to its routine housing only after it has fully recovered from anesthesia.

Provide analgesics as appropriate.

Generally, remove skin closures 10 to 14 days post-operatively.

Maintain a surgical record (e.g., annotate cage card with procedure and date).

Survival Surgery

Regulations pertaining to survival surgery are very specific and include the following definitions:

Survival Surgery includes all surgical procedures (even very minor ones such as vessel cut-downs) carried out on animals that are expected to recover from the anesthetic.

Major Survival Surgery is defined as survival surgery in which a major body cavity (abdomen, thorax, cranial cavity) is entered or which has the potential for causing substantial impairment of physical or physiologic function.

All survival surgery must be carried out following aseptic technique, including the use of sterile gloves and properly sterilized instruments.   This applies to all species including rats and mice. Major survival surgery on mammals other than rodents must be carried out in dedicated survival surgery facilities (ARC Room 111).  Major survival surgery on rodents must be done according to the Guidelines for Survival Rodent Surgery.