Protocols must be submitted at least six (6) weeks prior to a scheduled meeting. Submit the protocol electronically to ResearchNow@stcloudstate.edu or it can also be emailed to the IACUC Administrator.
Research, including data collection, cannot begin until you receive an approval letter from the IACUC.
Minor changes to an approved protocol may be requested using the Change in Protocol Form. Minor changes to an existing protocol may include but are not limited to:
Significant changes require the submission of a new protocol for review and approval. Final determination of whether the change is minor or significant will be made by the IACUC and may follow either the full committee or designated member review process. Significant changes to an existing protocol may include but are not limited to:
Protocols can initially be approved for one year with up to two one-year renewals if a Continuing Review Report is submitted to the IACUC and approved. After three years or when the project is complete, whichever comes first, a Final Report must be completed and submitted to IACUC. Submit reports electronically to the IACUC Administrator at ResearchNow@stcloudstate.edu:
Any live vertebrate animal used or intended for use in research, research training, experimentation, testing or for related purposes. This includes:
The USDA AWA regulations stipulate that the number of animals used in research and teaching by an institution must be reported annually to the USDA. The animals must be placed by species into one of four USDA pain/distress categories. To help collect accurate information, investigators must categorize the animals requested using the same system.
To do this properly, you must understand how animals are assigned to the four USDA pain/distress categories. The category labels (B through E) come from the column labels used on the USDA annual report form. The categories will be discussed in order from no pain/distress (B) to most pain/distress (E).
A simple yet useful definition of a painful or distressful procedure on an animal is, "a procedure that would cause pain or distress in a human."
It is important to understand that if multiple procedures will be performed on an animal, the animal is placed in the category appropriate for the most painful/distressful procedure. One animal cannot be placed in multiple categories.
Category B animals are those that are being "bred, conditioned, or held for use in teaching, testing, experiments, research, or surgery but not yet used for such purposes." These animals have not been used for any research procedure, however minor. Category B is the place to put breeders and other animals that are not undergoing any experimental procedures.
Category C animals are not subjected to procedures that involve pain or distress or would require the use of pain-relieving drugs. Routine procedures such as injections and blood sampling from veins that produce only mild, transient pain or discomfort are reported in this category. Another example of category C procedures is an observational study of animal behavior. Animals that are euthanized before tissue collection or other manipulations are also commonly placed in this category, if no other procedures are to be performed that put them in a higher pain/distress category.
Category D animals are those subjected to potentially painful procedures for which anesthetics, analgesics, or tranquilizers will be used. The important concept is that animals are given appropriate anesthesia and/or pain relief to limit their pain and distress as much as possible.
Examples of category D procedures are:
Category E animals are those that are subjected to painful or stressful procedures without the use of anesthetics, analgesics, or tranquilizers. Withholding of anesthetics, analgesics, or tranquilizers can only be allowed if it is scientifically justified in writing and approved by the IACUC. Examples of category E procedures are lethal dose studies (e.g., LD50 studies) that allow animals to die without intervention, pain studies that would not be possible if pain-relieving agents were administered, and psychological conditioning experiments that involve painful stimuli such as a noxious electrical shock that cannot immediately be avoided by an animal.
Category E studies are given increased scrutiny by IACUCs because they must be satisfied that less painful or stressful alternatives are not available, or that less painful/stressful endpoints cannot reasonably be used. By law, the institution must annually report all category E procedures to the USDA and include a scientific justification supporting the IACUC's decision to approve them. Often, the justification given by the researcher on the animal forms submitted to the IACUC is used for the annual report.
It is important for information on category E procedures to be complete and accurate. Once submitted to the USDA, this information will likely be available to the public through a Freedom of Information Act request.
USDA Policy #3 (Veterinary Care) states the following regarding the use of non-pharmaceutical grade compounds in research involving live vertebrate animals:
Investigators are expected to use pharmaceutical-grade medications whenever they are available, even in acute procedures. Non-pharmaceutical-grade chemical compounds should only be used in regulated animals after specific review and approval by the IACUC for reasons such as scientific necessity or non-availability of an acceptable veterinary or human pharmaceutical-grade product. Cost savings is not a justification for using non-pharmaceutical grade compounds in regulated animals.
The Guide for the Care and Use of Laboratory Animals (The Guide, 8th ed.) states the following regarding the use of non-pharmaceutical grade compounds in research involving live vertebrate animals:
The use of pharmaceutical-grade chemicals and other substances ensures that toxic or unwanted side effects are not introduced into studies conducted with experimental animals. They should therefore be used, when available, for all animal-related procedures. The use of nonpharmaceutical-grade chemicals or substances should be described and justified in the animal care and use protocol and be approved by the IACUC; for example, the use of a nonpharmaceutical grade chemical or substance may be necessary to meet the scientific goals of a project or when a veterinary or human pharmaceutical-grade product is unavailable. In such instances, consideration should be given to the grade, purity, sterility, pH, pyrogenicity, osmolality, stability, site and route of administration, formulation, compatibility, and pharmacokinetics of the chemical or substance to be administered, as well as animal welfare and scientific issues relating to its use.
When reviewing animal care and use protocols involving non-pharmaceutical grade compounds, the following factors should be considered:
Although the potential animal welfare consequences of complications resulting from the use of a non-pharmaceutical grade compound are less evident in non-survival studies, the scientific issues remain the same, and the principles and need for professional judgment described above still apply.
The use of non-pharmaceutical grade compounds must be described thoroughly and justified scientifically in the animal care and use protocol and approved by the IACUC. Any such compounds used in survival studies must be sterile, maintained in a sterile container, and labeled with
The Principal Investigator, in consultation with the Attending Veterinarian, is responsible for determining the “shelf life” of the compound after it is dissolved in solvent. If a shelf life cannot be determined, it is recommended that a fresh solution of the compound be prepared each day on which it is to be used. Compounds must be properly disposed of per university procedures and appropriate timeframe as determined by the shelf life.
TBE is an injectable anesthetic agent used in rodents. It was once manufactured specifically for use as an anesthetic by Winthrop Laboratories under the trade name Avertin, but this product is no longer commercially available. Investigators who wish to use TBE as an anesthetic must make their own solutions from a non-pharmaceutical grade chemical.
Please note that this anesthetic may not be used unless an investigator has described it in an animal care and use protocol, provided scientific justification for using this anesthetic rather than a pharmaceutical-grade anesthetic, and received IACUC approval for its use. See Use of Non-Pharmaceutical Grade Compounds in Research Involving Live Vertebrate Animals.
TBE is appropriate for short procedures involving laboratory rodents, especially surgical procedures. It is best used in situations where it will be administered only once to a particular animal subject. Repeated use of TBE on an animal can be associated with an increase in morbidity and mortality1,2.
Two chemicals are needed to produce a solution of TBE.
Mix the contents of a container by swirling prior to administration. The solution is administered by IP injection at a dose of 250 mg/kg body weight. Induction requires only a few minutes and the righting reflex returns approximately 40-90 minutes. Surgical anesthesia lasts for 15-45 minutes with a sleep time of 60-120 minutes.
Do not administer non-sterile solutions, outdated solutions, more concentrated solutions, or higher doses than recommended above. Store the solutions under refrigeration and in the dark. Containers should be wrapped in foil. Replace refrigerated TBE solutions at least every 14 days.
A TBE solution may not be used more than 14 days after it has been prepared.
Animals are normally euthanized at the end of a study for the purpose of sample collection or postmortem examination. Animals may be euthanized if they are experiencing pain or distress. Euthanasia is defined as a pain-free or stress-free death. The IACUC has approved selected methods for humanely killing animals which meet the definition of euthanasia. The appropriateness of the method varies from species to species and depends in part on the experimental endpoint of the study for which an animal is being used. These guidelines are adapted from the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition.
An animal may only be euthanized by the method(s) described in the IACUC-approved animal use and care protocol for which the animal is being used. A change in euthanasia method, including dose or route of administration, is a significant change in protocol and must be reviewed and approved by the IACUC before implementation.
Below are a set of standard acceptable euthanasia methods for the species identified. Please contact the vivarium manager if you have any questions about or would like training in the use of these methods.
Mice and Rats
Amphibians and Fish
Instructions for Use of CO2 Chamber for Euthanasia of Rodents
The standard chamber for CO2 asphyxiation is a clean, empty 10-gallon aquarium. The CO2 chamber is located in ISELF 316, the post-operative room of the vivarium surgical suite.
The animal is not dead if:
If the animal is not dead, place it back in the chamber, and begin euthanasia procedure at Step 2 above.
NOTE: Neonates and fetuses are resistant to carbon dioxide euthanasia. See NIH Guidelines for the Euthanasia of Rodent Fetuses and Neonates for guidance.
The following guidelines are suggested to assist Animal Care and Use Committees at the NIH in reviewing proposals which involve the use of rodent fetuses or neonates. In all cases, the person performing the euthanasia must be fully trained in the appropriate procedures.
The AVMA Guidelines for the Euthanasia of Animals, 2013 Edition states that “Scientific data indicate that mammalian embryos and fetuses are in a state of unconsciousness throughout pregnancy and birth.” It also states that “The precocious young of guinea pigs remain insentient and unconscious until 75% to 80% of the way through pregnancy and remain unconscious until after birth due to chemical inhibitors” and “embryos and fetuses cannot consciously experience feelings such as breathlessness or pain. Therefore, they also cannot suffer while dying in utero after the death of the dam, whatever the cause.”1
Maturation of nociceptors and the development of excitatory and inhibitory receptor systems occur during the period just prior to birth and into the second week of postnatal life.7-11 Resistance to hypoxia at this age results in a prolonged time to unconsciousness when CO2 is used as a euthanasia agent.1,3,12 A secondary physical method of euthanasia is recommended to ensure death (e.g. cervical dislocation, decapitation, bilateral pneumothorax). Death must be verified after euthanasia and prior to disposal.11
The following guidance is regarding the safety for personnel preparing solutions of MS-222, appropriate dosages for Xenopus laevis, and disposal procedures for MS-222 solutions. All use of MS-222 for animal anesthesia or euthanasia must be approved by the St. Cloud State University IACUC. All activities described below must be performed by the Principal Investigator (PI) or co-PI(s) named on the IACUC-approved animal use protocol that requires their use. In the absence of these individuals, only the IACUC attending veterinarian or the manager of the animal facility from which the frogs originated may perform these activities.
The PI named on an IACUC-approved animal use protocol that requires use of MS-222 is responsible for ensuring the safe use of this compound by personnel and students who engage in any animal use activity involving frogs exposed to MS-222. Personnel who use MS-222 should be familiar with the Safety Data Sheet (SDS) for this compound, which is included as an addendum to this document. MS-222 is a skin, eye, and respiratory irritant.
Solutions of MS-222 should be prepared in a fume hood to minimize risk of inhalation of its powdered form. Examination gloves must be worn at all times when handling powdered MS-222, aqueous solutions of the compound at any concentration, or animals that have been exposed to MS-222. Balances, spatulas, beakers or other laboratory devices and materials used to prepare a solution of MS-222 must be cleaned thoroughly and immediately after use by the individual(s) preparing the solution.
Preparation and Use of MS-222 Solutions
Only ultrapure deionized water may be used for preparation of MS-222 solutions. Deionized water is available in the Biology stockroom (WSB 281) or from any of the deionized water faucets located throughout the ISELF building (e.g., ISELF 320-330 Integrated Research Suite).
Solutions of MS-222 MUST be prepared fresh on the day on which they are to be used for anesthesia or euthanasia. NO EXCEPTIONS. Since MS-222 can acidify the water in which it is solubilized, all MS-222 solutions MUST be buffered to a pH of 7.0 with sodium bicarbonate before use. Prepare ONLY as much MS-222 solution as is needed on a single day.
Anesthesia and Euthanasia Guidelines
For anesthesia and euthanasia details, please refer to the approved Guidelines for the Preparation and Use of MS-222 (Tricaine Methanesulfonate) for Anesthesia and Euthanasia in the African Clawed Frog (Xenopus laevis)
Disposal of MS-222 Solution
All solutions of MS-222 must be discarded on the day on which they are prepared. A solution of MS-222 may not be used on more than one day.
A solution of MS-222 prepared at either of the concentrations described above may be discarded via a sanitary sewer. At a sink, open both valves on the faucet (maximum flow of tap water) and pour the MS-222 solution slowly into the sink to allow the solution to be flushed with copious water.