Text Entry: Try to provide your responses in the grey boxes. If you are copying and pasting from another source, copy the text from its source, then paste it into a grey area by using “Paste Special…” “Unformatted Text” from the Edit menu in Word. You may have to touch up any special characters, but it would be best if you spelled out all special characters.
Tables: If you have more items for a table than the table has rows, you can add a row by placing the cursor at the end (outside) the last row and hitting return. Alternatively, you may place the cursor in the table, select Table > Insert > Row above (or below) from the menu.
Please use brief, one or two word phrases for the descriptions.
Provide only one number for each category rather than a number for each description or each strain. An animal should be included in the table only once, in the category that represents the highest pain level it will experience. For example, if you have 10 animals that will receive injections, but 5 of them will also have recovery surgery, enter 5 in category C and 5 in category D for a total of 10.
Category descriptions are given below the table.
Example: Table for Animal Numbers
Year 1 |
||
Category |
Number |
Description of Use |
C |
200 |
Breeding, injections, behavioral studies |
D |
100 |
Recovery surgery |
E |
0 |
|
Total |
300 |
|
Year 2 |
||
Category |
Number |
Description of Use |
C |
100 |
Injections, blood sampling |
D |
50 |
Terminal procedure |
E |
10 |
Unavoidable shock |
Total |
160 |
|
Year 3 |
||
Category |
Number |
Description of Use |
C |
100 |
Radiation |
D |
100 |
Imaging (under anesthesia) |
E |
50 |
Death as an endpoint |
Total |
250 |
|
Category C: Animals that will not experience pain, discomfort, or distress.
Category D: Animals that may experience pain, discomfort or distress but will be administered appropriate anesthetic, analgesic or tranquilizing drug(s) to alleviate these effects.
Category E*: Animals will experience pain, discomfort or distress for which anesthetics, analgesics or tranquilizing drugs would customarily be given but will not be administered because their use would adversely affect experimental results.
Written approval from Environmental Health and Safety (EH&S), Radiation Safety (RS), or the Institutional Biosafety Committee (IBC) regarding the safety aspects of the protocol must be received by UCAR before an Animal Use Protocol can be activated. Each group, EH&S, RS, or IBC, will notify UCAR and the PI of its requirements for hazard use in animals.
Examples of hazardous agents include MPTP, DMBA, BrDU, investigational drugs, antineoplastic agents, toxins of biological origin, human pathogens (viruses, bacteria, fungi, parasites, prions, etc.), recombinant DNA molecules, viral vectors, DNA used to generate transgenic animals, human or non-human primate tissue, human or non-human primate blood, human or non-human primate cells / cell lines, and radioactive materials. Antibiotics and injectable anesthetics do NOT need to be listed as hazardous agents.
Call Environmental Health and Safety at extension 5-3241 or visit the appropriate web pages if you have questions about the human hazard potential of any substance(s) you plan to administer to animals.
Animal pathogens that are not also human pathogens do NOT need to be listed as a hazardous agent unless they are modified in some way or are recombinant molecules, or are on the USDA list of restricted pathogens. However, you must indicate on the Procedure Checklists that you are using an animal pathogen.
Animal cells or animal cell lines do NOT need to be listed as a hazardous agent unless they have been modified in some way. However, you must check the appropriate box in the Procedures Checklist and provide information about MAP or IMPACT testing of cell lines.
Environmental Health and Safety: http://www.safety.rochester.edu/
Institutional Biosafety Committee: http://www.safety.rochester.edu/ibc/
Radiation Safety: http://intranet.urmc.rochester.edu/depts/radiationsafety/
According to federal regulations the UCAR needs assurance that: 1) a sufficient number of animals is used to answer satisfactorily the scientific questions posed, and 2) an excessive number of animals is not used.
Provide the justification for all animals mentioned in the protocol, including fetuses, even though fetuses don’t “count” for question A1. You do not need to break the justification down by year. The justification is for the total number of animals requested. Do include all animals that you will have, not just those used for experiments. This is particularly important if you must breed many more animals than you can use, either because you require a particular genotype for your experiments, or because you must have a specific age and cannot use all the pups in a litter.
The type of justification required depends on the animal work being done. Several examples are given below. In general, summary information and tables are best way to convey this information. Please do not provide the entire Experimental Design section of your grant.
Parametric studies: In order for reviewers to evaluate animal numbers for these kinds of studies, you must provide them with the following:
An outline of the proposed experiments involving animals. Generally the information should include a listing of experimental and control groups, treatments, time points, etc. – in other words, a description of the experimental design (not procedural details)
A statement of how group size was determined. Power analysis is the gold standard here, and should be described if possible. Consultation with the Department of Biostatistics (x5-2484) may be helpful in addressing this section, especially in consideration of the estimated sample size (by power analysis) required for a given level of statistical significance in a study. If a power analysis has not been done, the basis for your decision about group size should be discussed. This might include previous experience with this type of measurement, published literature, limits on the number of samples that can be handled at a time, etc. The procedural details (e.g. the doses, routes of administration, etc.) should be provided in the procedure description, not here.
Example:
The proposed studies investigate the effects of CNS Bozoamine receptor blockers on the executive behavior (measured by the Enron behavior battery) of old and young male and female mice. The study will be done in duplicate so that tissue can be taken for chemical assays and histology. In all cases, group size will be 8 (estimating the variability of behavioral measures and similar behavioral studies in the literature).
n |
Male |
Female |
8 weeks |
18 months |
Doses (0.1 – 100 mg/Kg) |
8 |
+ |
+ |
6 |
||
8 |
+ |
+ |
6 |
||
8 |
+ |
+ |
6 |
||
8 |
+ |
+ |
6 |
||
= 384 |
Alternately:
8n X 2 sexes X 2 ages X 6 doses X 2 assays = 384 mice
If you have a number of different experiments, a summary or table for each experiment may be the clearest way to provide the information. If you have a number of experiments, all with the same design, you might describe the design, then discuss how many different experiments will be done. Using the above example, you might state that the same design would be repeated for each of 8 different receptor blockers: 384 mice/ blocker X 8 blockers = 3072 mice.
Transgenic mice and studies done in a single sex: Studies done with transgenic mice often require the investigator to breed many more animals than are needed for the study in order to get the required number of mice with the correct genotype. This information should be included in your justification. Breeding pairs must also be included.
Example:
The proposed studies examine the effects of Bozoamine receptor knockout on the executive behavior in male mice. Only males will be used because previous studies have determined that executive behavior is dependent on Bozoamine binding in males but not females.
Genotype |
n |
Assays |
Total |
Breeding Ratio |
BoRKO |
8 |
2 |
16 |
1:4 |
Wild type |
8 |
2 |
16 |
|
Total |
32 |
A total of 32 experimental animals will be required, of which 16 will be BoRKO males. The ratio of BoRKO:wt is 1:4, and males 1:2 therefore we expect approximately 1 of every 8 mice to have the appropriate genotype and sex. We must breed
16 (# transgenics needed) X 8 (4x2) = 128 mice
to obtain the required experimental animals. This breeding will also produce the required number of controls. Estimating 8 mice/litter, we will require 16 litters for a total of 128 pups + 16 breeding pairs (32 mice) = 160 mice.
Numbers determined by amount of tissue needed:
Often the determining factor for the number of animals needed is the amount of tissue or the number of cells that can be obtained from an animal. Such a justification would include;
Estimate of the number of experiments/assays
Estimate of how many animals would be required to produce tissue for each assay
Example:
We propose to isolate and culture Bozoamine expressing cells from the brains of wild type male mice for studies on transcription factors that regulate the expression of Bozoamine synthase. These dose response assays are run in triplicate and require 1x106 cells/measurement. One brain yields 5x105 cells or 2 brains/measurement.
2 brains/measurement x 6 transcription factors x 10 doses/factor x 3 repeats = 360 male mice needed for assays. Note that if mice are being purchased, the justification is complete, and the number requested would be 360. If animals are being bred, you would have to estimate the litter size, number/litter and add the breeders:
We estimate that each litter will produce 10 mice, of which 5 will be male, so to produce 360 male mice we would require 360/5=72 litters (720 mice). We anticipate that each breeding pair will produce 6 litters, so require 72/6=12 breeding pairs for a total of 744 mice (720 mice bred + 12 pairs).
An alternative approach might be
The proposed studies use mice as a source of cells for a large series of in vitro experiments associated with the expression and regulation of the onno gene. We anticipate doing 2 assays per week over the course of 3 years for a total of approximately 300 assays. Each assay requires cells from 1 neonatal (P01) mouse brain (2 per week). Because cells will last in culture only a few days, we will require a new litter each week. We will begin with 8 breeding pairs, then use mice not used for experiments to replace the retired breeders. 8 pairs + (150 litters x 8 mice/litter) = 1216 mice.
General principles:
When you are providing your justification keep in mind that it must be clear enough so that reviewers can follow your thinking. Tables and summaries help. Leave out experimental and procedural details. This is an outline that should give the reviewers a brief overview of the kinds of procedures the animals will experience and how many animals will experience them. You should make it clear how you determined the number of animals (show your work!). In addition, it is important to state how the size of each group was determined.
Do not put your procedure descriptions in the notes column of the checklist. It will be deleted during protocol processing by UCAR. All procedures must be described in A5. Refer to the notes column of A4 to determine what information to include in your discussion.
You may list recovery surgeries by name, but do not provide surgical details in section A. These must be described in detail in section B. Non-recovery/terminal procedures (not including euthanasia) should be discussed in A5.
Selection of the method of euthanasia is dependent upon the animal species involved, objective of the procedure and skill of personnel. It is essential that proper physical control over the animal be maintained prior to euthanasia and that fear and apprehension be minimized. Noxious stimuli induce various responses including vocalization, struggling, escape, aggression, salivation, urination, defecation, pupillary dilation, tachycardia, sweating, shivering, tremors and spasms. Not only are these responses undesirable from an aesthetic and humane point of view, they are usually undesirable complications of research where variation in baseline levels of cellular or extracellular biological values must be minimized.
Euthanizing agents terminate life by one of three basic methods: direct or indirect hypoxia, depression of vital neurons, or physical damage of brain tissue. Regardless of the method, it is essential to induce unconsciousness as rapidly as possible if euthanasia is to be aesthetically and scientifically successful.
When using these or any other method of euthanasia, it is important to take adequate measures to insure animals are dead and have no chance to revive or regain consciousness at any later time, especially when using anesthetics.
All methods must be recommended by the AVMA Guidelines on Euthanasia.
You may use decapitation or cervical dislocation of rodents without prior sedation/analgesia, but must provide a scientific justification for doing so. For example, you can provide a statement that analgesia/sedation will affect the end point measurements you make. You should also provide a reference supporting your justification. In addition, you must provide a statement that the persons performing the euthanasia technique are appropriately trained and are using proper equipment. You should specifically indicate who will be performing euthanasia in the Associates section of the protocol.
Procedures such as CO2 euthanasia or anesthetic overdose on rodents, where death may not be immediately evident, must include a secondary physical method for ensuring death. These include decapitation, pneumothorax by opening the thoracic cavity, cervical dislocation of rodents under 200 g, complete severance of the spine just below the base of the skull using a dorsal approach, or perfusion of a histological fixative via the major blood vessels.
There are special considerations for euthanizing rodent embryos, fetuses and neonates. Please see the UCAR policy on Euthanasia for Rodent Embryos, Fetuses and Neonates on the UCAR website.
Approved Euthanasia Dosage and Techniques
Rodents
1. Sodium Pentobarbital 100 mg/kg body weight IV or IP (followed by physical method)
2. Carbon Dioxide Inhalation Chamber according to DLAM SOP (followed by physical method)
3. Cervical dislocation for rats weighing less than 200 grams and all mice after sedation. Cervical dislocation without prior sedation must be scientifically justified and approved by UCAR.
4. Decapitation with guillotine after the animal has been sedated. Decapitation without prior sedation must be scientifically justified and approved by UCAR.
5. Cardiac perfusion or exsanguination under deep plane of surgical anesthesia.
Rabbits, nonhuman primates, dogs, cats, swine
1. Sodium Pentobarbital 100 mg/kg IV
2. Cardiac perfusion or exsanguination under deep plane of surgical anesthesia.
Your protocol must describe what will be done with animals that experience pain, distress or impairment, whatever the cause. Routine handling, and procedures that may result in momentary pain or stress (e.g., routine injections) when done properly need not be discussed. These are procedures for which you would not routinely provide anesthesia. However, you must discuss all procedures for which you anesthetize animals because you are using the anesthesia to minimize pain and distress.
In many cases, the investigator does not expect the procedures to cause more than momentary pain or distress, but there is the possibility that animals might become ill (e.g. injection of substances for which effects are not know, production of small tumors, irradiation, surgery). UCAR policy requires that any animal exhibiting significant weight loss (e.g. 10%), inability to ambulate, inability to maintain food or water intake, and clinical signs of pain, including ruffled fur, hunched posture, vocalization, and guarding behavior, be removed from the study, euthanized or provided with supportive care. Therefore, you must describe in your protocol symptoms that your animals might develop based on the treatments they will receive, and state what will be done with animals that meet UCAR clinical endpoints. If such animals must be maintained without intervention, you must provide a scientific justification for doing so.
Experimental pain, distress or impairment may include any physical, physiological or pathological damage (temporary or permanent) resulting from treatment of animals with
a hazardous agent,
radiation,
physical or chemical restraint longer than a few minutes duration to facilitate either substance administration or specimen collection,
aversive conditioning (e.g., food/water scheduling or sensory deprivation),
use of adjuvants;
chronic insertion or attachment of a foreign object (e.g., osmotic pump or electrodes), or
recovery surgery.
For transgenic, knockout, or mutant animals, there may be known or predicted phenotypic consequences of the genetic manipulations to the animals that impair normal physiological functions.
Examples of procedures that result in pain distress or impairment include:
Muscle Paralysants (paralytic agents): See instructions in the Procedure Checklist, and discuss the use of paralytic agents with a DLAM veterinarian before submitting your protocol.
Aversive Conditioning, Food or Water Scheduling: If any form of aversive conditioning (e.g., footshock) is proposed, describe the aversive condition qualitatively and quantitatively. If access to food or water is restricted in any manner, scientific justification is required. Describe in detail the criteria you propose to use to determine that each animal, so restricted, receives adequate dietary and fluid intake. DLAM veterinarians have developed an acceptable record-keeping procedure that places the responsibility on the PI or on Vivarium animal care technicians. A daily feeding or watering log for each individually housed animal must be kept. All water restricted animals must be weighed at least once a week and weight records must be maintained by the PI. A statement indicating that these procedures will be followed must appear in the protocol. Consultation with a DLAM clinical veterinarian or Vivarium Supervisory personnel in developing and implementing any restricted or scheduled food or water regimen is highly recommended. If the creation of caloric or other specific nutritional deficiency is an intrinsic part of the proposed study, you must provide a rationale and description for it in the protocol.
Sensory deprivation: If any form of sensory deprivation is proposed, scientifically justify and describe in detail the duration, extent and known or anticipated effect of the sensory deprivation.
Harmful physical agents: Harmful or potentially harmful physical agents include irradiation, microwaves, radio frequency waves, thermal injury, physical injury, environmental injury, such as high levels of sound or ultrasound, high or low temperature, high or low barometric pressure, ultrasound, high intensity light or exposure to potentially damaging wavelengths of light. Please scientifically justify the use of such physical agents and describe the conditions of exposure, the amount (in units appropriate to the agent), duration and frequency of exposure, and the expected effects of such exposure.
Experimental endpoint studies: Such endpoints may include tumor size, percentage body weight gain or loss, inability to eat or drink, behavioral abnormalities, clinical symptomatology, or signs of toxicity.
Death-as-an-endpoint studies
are discouraged and must always be scientifically justified. This justification must include a discussion stating why endpoints short of death cannot be used.
For transgenic, knockout or mutant animals, describe any special care or monitoring that the animals will receive. Examples include increasing the frequency of monitoring, euthanasia of animals expressing premature lethality, providing supportive care such as soft food to toothless mice, delaying weaning time for pups with stunted growth, or providing foster moms to replace dams with abnormal mammary gland development.
Procedures to minimize pain/distress may include; analgesics or other drugs to prevent or reduce perception of painful stimuli, physical or environmental changes to reduce discomfort or injury, and general anesthesia. If general anesthesia is used, describe the methods used to assess a surgical plane of anesthesia.
Methods of assessing a surgical plane of anesthesia include:
All species
· Toe or tail pinch/pedal reflex: For a surgical plane of anesthesia, the foot should not withdraw after gently pinching the toe with your fingers. There should not be a reaction to a tail pinch in rodents.
· Palpebral reflex: For a surgical plane of anesthesia, a blink should not be elicited after gently touching the eyelid.
· Respiratory rate (depth and character): Respirations should be of regular rate and depth to maintain pink mucous membranes. A rapid increase in respiration may indicate a light plane of anesthesia. A rapid decrease may indicate a dangerous depth.
Non-rodent regulated species (e.g. primate, dog, cat, ferret, sheep, goat, pig, and rabbit)
· Heart rate and rhythm: The heart rate should be regular and relatively unchanged throughout the procedure to maintain a pink mucous membrane color. A rapid decrease or increase in heart rate indicates that the anesthetist should check other parameters/reflexes to determine if the animal is too lightly or too deeply anesthetized.
· Ear pinch: For a surgical plane of anesthesia in rabbits and ruminants, there should be no reaction when the ear is gently pinched.
· Jaw tone: For a surgical plane of anesthesia, the jaw should be relaxed as you try to open the mouth. An increase in jaw tone indicates a light plane of anesthesia.
· Corneal reflex: For a very deep plane of surgical anesthesia (e.g., orthopedic procedures) a blink should not be elicited after gently touching the cornea. Loss of this reflex may indicate a dangerously deep plane of anesthesia.
Other useful references include: UCAR manual – Anesthesia and Table 1 – Recommended Anesthetics, UCAR Manual – Analgesia and Table 1 – Recommended Analgesics
1. Explain why you must use animals for your studies and provide a justification for your choice of species, strain(s) and animal models.
2. Describe your search for alternatives. Alternatives are usually thought of as non-animal models and approaches, such as cultured cells, and computer modeling. However, most of the questions being studied at the U or R clearly have no such alternatives. As a result, it is important to address the 3 Rs (Reduction, Refinement and Replacement). You may be reducing the number of overall animals needed for a study by doing your pilot work in culture, but you may also reduce the number by using a more sensitive assay that does not require pooling of samples. You might reduce the number of animals needed by refining your blood sampling methods so that you can obtain repeated samples from the same animal. You may be able to replace your main species of choice for some of the work (e.g. replace a non-human primate with a rat for doing pilot studies on neuronal tracer effectiveness or staining techniques).
The Miner Library offers assistance with literature searches for animal testing alternatives. The search can be done for you on Medline and some additional databases without charge. If you prefer to do your own search, assistance with search strategy design is available. Miner also provides tips about subject headings/keywords/databases, and a saved search strategy at http://www.urmc.rochester.edu/hslt/miner/resources/researchers/Animal_Testing_Alternatives.cfm. For further information contact Margaret Chretien in the Reference Department at 53399 or margaret_chretien@urmc.rochester.edu. The Animal Welfare Information Center at the USDA National Agricultural Library can also be of assistance. Phone: (301) 504-6212 Web site: http://www.nal.usda.gov/awic
Although the literature search is the gold standard for documenting the existence of alternatives, you should also consider mentioning other sources such as conference attendance, committee membership, journal and grant reviewing, etc.
After you have described your search, be sure to comment on any alternatives found and why they are not appropriate for your studies.
Federal regulations govern the duration of animal housing in laboratories. For regulated species (e.g., nonhuman primates, dogs, cats, guinea pigs and hamsters) animals may be kept in laboratories no more than twelve hours. For laboratory bred rats and mice, periods of up to twenty-four hours are allowed. If longer periods are necessary, one must establish a "Study Area" as defined by the federal regulations and the Public Health Service. To do this, you must submit a request for a satellite facility to UCAR. Contact the UCAR Office for further information.
Updated 2/23/06