# Justification of Numbers of Animals Used for Research, Teaching, and Outreach Activities

Justification of the number of animals used in research, teaching, and outreach activities is a requirement for projects submitted for approval to the North Carolina State University Institutional Animal Care and Use Committee (IACUC). It is consistent with consideration of the “3R’s” - replacement, reduction, and refinement – which is a recommendation of both USDA and NIH. The underlying goal is for the least possible number of animals to be used without compromising the validity of research or educational objectives. There are a variety of different types of animal use activities at NC State. The following is intended to provide guidance to Principal Investigators (PI) with regard to important information that should be considered when providing a justification for numbers of animals requested.

## Hypothesis-Driven Research Studies

Hypothesis-driven research studies typically are those for which cause-and-effect conclusions are the end goal. There typically are different treatment groups included in the experimental design, one of which is a control group that represents a normal or unaltered condition of the animal. With these types of studies, the assumption is made that there are previous data available, either conducted by the PI or published in peer-reviewed literature, from which a Power Analysis can be conducted. A Power Analysis is the calculation used to determine the smallest number of animals needed per treatment based upon the desired significance level (p-value); desired statistical power (1 – β); and the magnitude or size of the effect (difference among treatments). There are a number of different approaches for conducting Power Analyses for both continuous (analysis of variance, regression analyses, etc.) and categorical (chi-square, non-parametric analyses, etc.) data. Examples of how to conduct a Power Analysis for a variety of different types of studies can be found on-line or in most statistic’s textbooks. **The decision with regards to which one of these to use is at the discretion of the PIs and not dictated by the IACUC.**

Most Power Analyses require PIs to select or choose the desired significance level (p-value); statistical power (1 – β); and effect size (difference among treatments) for their studies. **These also are left up to the discretion of PIs and are not dictated by the IACUC **However, for most animal-related experiments, significance levels of less than or equal to 0.1 and statistical powers of greater than or equal to 0.80 are considered highly powered studies and typically are required for publication in peer-reviewed journals.

The other component that is required to conduct a Power Analysis is some estimate of the normal variation of the dependent (response) variable in the animal population being studied. The most common measure of this is the standard deviation. In many situations, PIs know or can calculate the standard deviation from their own data or obtain a reasonable estimate from peer-reviewed publications. Data in peer-reviewed publications typically are presented as the treatment mean plus/minus the associated standard error and include the number of units used in these calculations. The mathematical relationship between the standard error and the standard deviation is as follows:

*Standard error = standard deviation / square root of the sample size.*

This equation can be modified mathematically to obtain an estimate of the standard deviation by multiplying each side by the square root of the sample size as follows:

*Standard deviation = standard error x square root of the sample size*

Inclusion of the results from the Power Analysis in the justification of animal numbers is a requirement for IACUC approval of hypothesis-driven research studies. The most common language that is used to do this is as follows:

“Results of the power analysis indicates that

of animals per treatment will allow the detection of a(number)difference among treatments at a significance level of(percentage or other units)with a statistical power of(p-value).”(1 – β)

This statement provides only a justification for the number of animals per treatment. The number of treatments in the study and the number of times the study is going to be repeated need to be included in the justification, as well. In other words, the IACUC needs to be able to determine how PIs obtain the total number of animals requested from the results of the Power Analysis. This can be done with the following equation:

*Number of animals per treatment (from Power Analysis) x number of treatments in study x number of times study will be replicated*.

For example, if the results of the power analysis determined that 10 animals were needed per treatment for an experiment with 5 different treatments and the experiment needed to be replicated once per year for 3 years, then the total number of animals requested should be as follows:

10 animals/treatments x 5 treatments/study x 3 replicates = 150 animals

Most studies typically involve collection of data associated with more than one dependent (response) variable and many of these may differ in terms of the normal variation within the population being studied**. In this situation, PIs only need to conduct a single Power Analysis and should use the dependent (response) variable that has the largest variation within the population being studied. **For example, if a proposed experiment involves monitoring both weight gain and circulating levels of various hormones as response variables and weight gain has a higher coefficient of variation *([standard deviation/mean] x 100) *compared with the hormone levels, then the power analysis should be conducted using weight gain as the dependent variable.

It is also important for PIs to anticipate and plan for any experimental losses of animals – animals that have to be removed before the study is completed. The most common way to do this is to add the anticipated experimental losses after the total number of animals needed has been calculated. For example, if it is anticipated that 10% of the animals might need to be removed from the experiment prior to completion of data collection and the total animals needed is 150 (based on the power analysis), then addition of 15 animals *(150 x 0.1 = 15)* would be appropriate and PIs should increase their total number requested to 165.

Careful consideration by PIs should be given to their experimental treatments when estimating potential experimental losses. For some studies, there may be one treatment that, for whatever reason, might result in a disproportionate number of experimental failures compared with other treatments. In these cases, it is advisable to calculate the anticipated experimental losses for each treatment separately. For example, a study might contain three experimental treatments: one that has increased calcium levels; one that has decreased calcium levels and one with calcium levels at the recommended (maintenance) levels. This could lead to a higher percentage of animals being removed in the reduced calcium treatment in a long-term study due to lameness, etc. If the normal experimental losses for this species of animal is 10%, but the PI expects this to increase to 20% in the reduced calcium treatment these adjustments need to made after the numbers of animals per treatment are determined from the Power Analysis. Assuming that results from the Power Analysis indicated that 10 animals were needed per treatment, then the number of animals needed in the control and high calcium treatments would 11 *(10 x 0.1=1; 10 + 1)* and 12 for the low calcium diet *(10 x 0.2 = 2; 10 + 2 = 12)*. If the experiment was to be replicated 3 times, then the total animal request would be 103 *(11 + 11 + 12 = 34; 34 x 3)*.

### Summary

- Inclusion of results from Power Analyses are required in the justification of the number of animals requested for all hypothesis-driven studies.
- Power analyses determine the minimum number of animals per treatment based on significance level; statistical power; detectable difference among treatments; and response variable variance.
- Selection of the methodology used to conduct Power Analyses is at the discretion of the PI as well as the significance level; statistical power; and differences among treatments that are needed.
- When more than one dependent (response) variable is being studied, then the one with the most variance within the population being studied should be used in the calculation of the number of animals per treatment in the Power Analyses.
- Total number of animals per treatment from the Power Analyses needs to be adjusted for the number of treatments per experiment; number of replications of experiment; and experimental losses.

## Pilot or Descriptive Research Studies

The IACUC recognizes that there are situations in which it is not possible for PIs to perform a valid Power Analysis. These types of studies still require IACUC approval and are classified as pilot or descriptive studies. Lack of a good estimate of means and/or the normal variation for a given dependent variable or limited knowledge of how an animal will respond to a novel treatment or therapy are the most common situations in which PIs may not have the necessary information to conduct a Power Analysis. **The main purpose of a pilot or descriptive study is to collect sufficient information so that a Power Analysis eventually can be performed in order to plan a hypothesis-driven research study.**

Justifications for pilot or descriptive research studies need to acknowledge that it is not possible to conduct a valid Power Analysis and state the reasons why – insufficient estimates of population variance or means; insufficient data with regards to treatment responses, etc. – and then provide the PI’s best estimate of the number of animals required to obtain this information. The latter can be based on work in a related breed or species; similar studies in different geographic regions or environments; or observations by PIs themselves. It also is recommended that “Pilot Study” be inserted at the end of the title for the project. This facilitates the IACUC review process.

### Summary

- Pilot or descriptive research studies are ones in which a valid Power Analysis cannot be performed.
- A brief description of why a valid Power Analysis cannot be performed should be included in the justification along with the best estimate of the number of animals required to obtain this information.
- “Pilot Study” should be inserted at the end of the title of the application.

**In Vitro Research Studies that Require Cells or Tissues from Animals**

The IACUC understands that there are research studies in which the experimental unit does not involve live animals but rather cells of tissues obtained from live animals that are treated or manipulated *in vitro*. Some common examples include dissected ligaments or tendons; white blood cells; cultured liver or lung cells; and spermatozoa, ova or embryos. **It is not necessary to conduct a power analysis for the number of animals requested for these types of studies. Instead, the number of animals requested should be based upon the number of animals necessary to obtain an adequate number of cells or tissues to conduct the in vitro experiments.**

Justifications for these types of studies need to describe the amount of animal tissue needed for each experiment and the number of animals typically required to obtain this amount of tissue. The “yield” of suitable cells or tissue from a particular animal species typically is based on previous experience by the PI or, sometimes, based on published information. In the review process, it is helpful for the IACUC to have some idea with regards to the number of *in vitro* treatments associated with each experiment and the number of times each experiment needs to be replicated. This provides information with regards to the total number of cells or tissues needed for the entire study, which, in turn, assists with an objective evaluation of the total number of animals requested. The most common language used in the justification of total number of animals requested for in vitro studies is something similar to the following:

“The experimental unit for this study is a culture well (or plate; tissue; etc.) containing

of(number). Typically, each experiment contains(type of cell)of treatments that is replicated(number)times. Previous work indicates that the number (or percentage yield) of viable cells (or tissue) obtained from a single animal averages(number). Therefore,(actual number or percentage yield)of animals are needed for this study”(number)

The IACUC recognizes that the harvest, isolation, and preparation of cells and tissues can vary considerably over time and among individual animals for a number of valid reasons. Therefore, it is advisable for PIs to assume or use the lowest yield percentages when determining the number of animals requested. This helps avoid the need for PIs having to submit amendments to increase the number of animals needed before the completion of a study. It also is important for PIs to recognize that there are animals that may not provide any suitable cells or tissues for *in vitro* study and essentially are experimental failures. This tends to occur more frequently in studies involving embryos, ova or spermatozoa. Total number of animals requested for *in vitro* studies should also be adjusted for anticipated experimental failures.

### Summary

- A Power Analysis is not required for studies in which animals are needed to obtain cells or tissues that will be manipulated
*in vitro*. - A brief description of the number of cells or tissues needed to complete the study and the typical yield of usable cells or tissue from a single animals needs to be mentioned in the justification for total number of animals requested.
- Experimental failures or animals that may not provide any useable cells or tissue for
*in vitro*study need to be considered and total number of animals requested should be adjusted accordingly depending on anticipated frequencies of their occurrence.

## Teaching and Outreach Activities

Teaching and outreach activities are important aspects of the educational mission of NC State. If the activity involves university-owned animals or NC State. faculty using privately-owned animals on-campus as part of a class, workshop, demonstration, etc. then IACUC approval likely is required. **If PIs are unsure of whether their teaching or outreach activity requires IACUC approval then they should contact the IACUC office.** If it is determined that IACUC approval is not needed, then they will be asked to complete a form and receive an IACUC waiver.

Most teaching and outreach activities typically can be placed into one of two categories in terms of requesting animals – those that are either dependent or independent of the number of students (participants). Some examples of a teaching/outreach activities that are dependent on the number participants would be laboratories in which the goals are either to teach students how to obtain a blood sample via venipuncture or how to correctly trim feet on sheep/goats. For these types of activities, PIs need to provide an estimate of the number of students (participants) enrolled in the activity and the ratio of students (participants) to animals needed to meet the educational objectives. For some, such as teaching students how to obtain blood samples, it is reasonable to expect that students may need to bleed more than a single animal, whereas for others, such as learning how to trim feet, one animal many only be needed for every four students. **The decision with regards to how many animals are needed per student (participant) is left up to the discretion of PIs and typically is based upon their previous experience.**

It is important for PIs to recognize that enrollment in both classes and outreach activities vary over time and that for some activities, such as blood sampling, there is the possibility of “teaching failures” – animals on which students attempt to perform the activity but are not successful. When appropriate, these aspects need to be considered in the justification of numbers of animals requested**. The IACUC recognizes that the previous experience of the PIs provides the best estimate for teaching failures.** The most common language used in the justification of these types of teaching/outreach activities is similar to the following:

“The largest anticipated enrollment in the laboratory (outreach activity) is

of students. It is anticipated from previous experience of the PI that(number)of students enrolled would attempt(number or percentage)and that(the activity)of animals are needed per(number)of students enrolled. This results in a total of(number)animals being used for this activity.”(number)

In the case of learning how to bleed pigs via venipuncture the previous statement might read something similar to the following for a class that has two laboratory sections that is taught 2 semesters per year:

“Maximum enrollment in each laboratory section is

students. It is anticipated from previous years that only30of the students enrolled in each laboratory section attempt to bleed pigs via venipuncture.80%pig is used forOnestudent. Therefore, the total number of animals requested per laboratory section iseach(30 x 0.80). There are24laboratory sections2so the total number of animals requested over theper semester and 2 semesters per yearyear period is3(24 per laboratory x 2 laboratories per semester x 2 semesters per year x 3 years).”288

Examples of teaching/outreach activities that are independent of the number of students (participants) would be laboratories in which the goal is to teach students proper techniques involved when assisting animals that are giving birth or teaching students how to administer vaccinations to an entire herd or flock**. In these situations, there usually is a group, herd, or flock that is being used for the activity and the associated number of animals is determined by normal management practices or the production schedule of the farm or unit.**

Justification of animal numbers for educational activities that are independent of the number of students (participants) should include the typical group size for the animals being used. The most common language used in the justification of these types of teaching/outreach activities is similar to the following:

“The herd (flock, group, etc.) size on which students will learn how to

is(activity)of animals. All the animals in the herd (flock, group, etc.) need to be(number)at this time as part of recommended management practices.”(activity)

For a laboratory whose educational objective is to teach students proper techniques associated with assisting sows during farrowing on a farm whose production group size is 24 animals the previous statement might read similar to something as follows, assuming the course is taught once per semester for 3 years:

“A farrowing group at the unit consists of

sows. All animals that are farrowing during the laboratory in a farrowing group will be used to teach students24. The class is taughtproper techniques associated with the manual removal of pigletsso the total number of animals requested isonce per semester144(24 sows/group x 2 semesters per year x 3 years).”for the 3 year period

Alternatively, a laboratory whose educational objective is to teach students proper techniques for administering yearly vaccinations per year over a 3 year period might read similar to something as follows:

It is advisable for PIs to assume that new animals will be used each year, even though it is likely that many of the animals in the herd or flock remain in production over extended periods of time and for more than a year. This prevents PIs from having to file amendments to increase the numbers of animals used during the 3-year approval period.

Herd/ flock justification examples:

“The herd that is going to be used to teach students

consists ofproper techniques for administering vaccinationsanimals. Animals need to be vaccinated yearly so the total number of animals that may be used over the50would be3 year period(50 animals/year x 3 years).”150

The numbers of Category C animals is calculated based on the number of animals owned by Reedy Creek Equine Farm throughout the previous protocol duration (126) plus an additional 10% per year (36 total) to cover foals born and any new acquisitions.

All horses owned by NC State have the potential to be used in any of the listed activities including all courses, non-student teaching, and any other teaching, so we request that all animals be included in this protocol. A herd of this number is sufficient to allow us to teach these skills with adequate rest periods.

The total number of birds requested allows for rotation of individual animal use, to minimize the amount of handling time and stress encountered by each individual bird. Typically, one bird is handled by teams of 2-4 students in each of the laboratory sessions in the various courses. Most of the courses have 6-12 students per course; in the Fall, we teach 4 lab sessions with 25 students/session. We anticipate the maximum number of birds used in a single teaching lab will be 20 chickens and 10 cockatiels. Most birds will be used in teaching laboratory sessions less than once every two weeks; in the Fall semester they are used twice weekly for a total of 4 labs.

It is unlikely that the maximum number of birds (chickens and psittacine) will be used, as a smaller number are available in the animal facility as part of our permanent teaching collection and are suitable for use in different teaching labs. In reality, we are unlikely to require more than 60 chickens and 30 psittacine during a 3-year period, unless we have unexpected health problems among the permanent collection. If we lose birds due to health problems, replacement birds will be obtained. We will either purchase an entirely new flock of chickens if we have to depopulate due to a serious flock health problem. Loss of individual parrots or the entire flock in the event of health problems will be handled by purchasing replacement birds, and quarantining those new birds before addition to the flock.

### Summary

- Educational activities conducted with animals owned by NC State or conducted by NC State. faculty using privately owned animals as part of an NC State-sponsored event likely requires IACUC approval. If PIs are not sure whether their planned activity requires approval, then they should contact the IACUC office.
- For educational activities that are dependent on the number of students (participants), the justification needs to include the maximum number of participants expected; participant to animal ratio required; and the frequency of the activity over the 3-year approval period.
- For educational activities that are independent of number of students (participants), the justification needs to include the number of animals in the herd or group being used and the frequency of the activity over the 3-year approval period.

## Research Holding, Routine Herd (Flock) and Breeding Colony Maintenance Activities

Maintenance of animals that are available for use in research, teaching, and outreach activities is an important aspect of the educational mission of land-grant universities such as NC State. These animal-use activities require IACUC approval and are referred to as a “Holding” or “Maintenance” protocol. **Justification of animal numbers for maintenance protocols typically is based on previous experience of PIs with regards to numbers of breeding animals and the resultant offspring that are necessary to meet research, teaching and outreach activities.** Moreover, specific types of transgenic animals often are needed and have to be produced by PIs from founder animals that are maintained in breeding colonies. These are often referred to as “purpose-bred” animals. Often with purpose-bred animals, only a certain percentage contain or express the trait of interest needed for subsequent studies. In these situations, all animals produced, even the ones not possessing the desired characteristics need to be included in the justification of animal numbers by the PI. **The IACUC recognizes that the previous experience of PIs or information in peer-reviewed literature provide the best estimates for the proportion of animals possessing desired characteristics in these situations.**

Justification of numbers of animals for maintenance protocols should include the maximum number of animals PIs expect to be present at any given time. **Adult breeding animals, newborns, young developing animals, and animals selected as breeding replacements need to be considered, when appropriate.** An example of the common language used in the justification of a maintenance or holding protocol for a herd of beef cattle (50 cows) owned by NC State. would be as follows:

“A beef cattle herd of 50 cows and 5 mature bulls is maintained to provide sufficient numbers of animals for research, teaching and outreach requests. Cows produce one calf per year and those not needed for educational activities typically are sold through commercial channels when they are between 12 and 15 months old. Annual replacement rate for cows and bulls in this herd is 20%. As a result, the maximum annual inventory for this herd would consist of 50 cows, 5 bulls, 50 calves, 50 yearlings, 10 replacement heifers and 1 replacement bull.”

Stock dogs housed under category

: We will have a maximum of 30 stock animals at any one time. The maximum number of animals adopted out or otherwise undergoing disposition is not expected to exceed 15/year. Therefore, 30 + 15 + 15 yields 60 dogsD

An example of the common language used in the justification of a maintenance or holding protocol for a purpose-bred mouse colony would be as follows:

“Previous experience indicates that 1000 mice per year from each of 3 different genetic lines are needed for studying mechanisms associated with pregnancy failure: line A (wild type); line B (gene B knockout); and line C (gene C knockout). For line A, 50 female mice and 50 male mice are needed to produce 1000 wildtype mice per year. For lines B and C, only 25% of the offspring possess the desired phenotype needed for further study. As a result, 200 female and 200 male mice in each of these lines are needed in order to produce 1000 mice with the desired phenotypes. Therefore, total numbers requested on an annual basis is 9,900 (Line A – 100 adults; 1000 pups; Line B – 400 adults and 4000 pups; and Line C – 400 adults and 4000 pups).”

For many justifications of this nature, it is easier (and preferred) to include a table containing numbers of animals requested within each genetic line (or group) along with the appropriate assumptions and calculations, especially when a number of different phenotypes are needed for the proposed activity.

### Summary

- Animals maintained by PIs for the purpose of producing offspring that are used in educational activities are required to be covered by a holding or maintenance protocol.
- Justification of animal numbers for holding or maintenance protocols typically is based on previous experience of PIs and should include an inventory of all animals at a given period of time – adult breeding animals, newborns, yearling, replacement females and males, etc. – when appropriate.
- For purpose-bred animals, all offspring, including those without the desired phenotype, need to be included in the justification.

## Special Considerations for Wildlife/Trapping Educational Activities

Educational activities associated with studying population densities and migratory activities of wildlife that involve trapping or capturing animals for short periods of time require IACUC approval. For many of these types of activities, animal species not being studied by PIs are also captured. These typically are referred to as a “non-target species”. PIs are required to provide an estimation of the number of animals for all the non-target species that might also be caught in their traps in their justification of total animals requested even though they will be released and not manipulated in any way. This estimation can be based on previous experience or published data with regard to population densities of non-target species. For situations in which neither of these may be available, then a reasonable approach would be to consider the study as a Pilot study from which these data could be obtained.

IACUC approved guidance 8/17/2023