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Reduction Alternatives
Experimental design and analysis
To properly implement reduction, experiments using animals should be planned with careful consideration
of the following aspects of experimental design.
Control of variation
"Fixed effects" variation can result from differences in species, sex, strain, age, experimental conditions,
bedding and diet of experimental animals, for example. These can be controlled directly by the investigator
and, if desired, can be deliberately varied as part of the experiment. "Random effect" variation results
from inter-individual variation, non-systematic measurement error, and variation associated with time and
location, for example. This type of variation may be minimized with choice of experimental subject (e.g.,
use of inbred strains of animal) and design. To minimize variation, the use of isogenic strains of rats and
mice should be considered due to their genotypic and phenotypic uniformity.
Choice of formal experimental design
Choice of formal experimental design involves consideration of the following:
- the experimental unit that will be used (e.g., individual animal, litter, cage);
- the process for randomization that will be used to ensure each experimental unit has a known and
equal probability of receiving a given treatment;
- whether blinding to treatments should be used to avoid bias, particularly when subjective elements
are part of the assessment of results;
- the use of pilot studies to test the logistics of a proposed experiment and/or give an indication of
likely response that can then be used in a power analysis to determine sample size; and
- the type of design that will be used (e.g., completely randomized, randomized block, factorial,
Latin square, crossover, repeated measures, spilt-plot, incomplete block, or sequential designs).
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Selection of sample size
Justification of sample size and number of animals to be used is an important component of animal
care protocols. An experiment that uses too few samples may not detect biologically important
effects, while a sample size that is unnecessarily large will waste animals. Sample sizes can be
best selected using one of two statistical approaches:
- Power Analysis (requiring knowledge about the effect size of interest and standard deviation
from previous experiments or a pilot study); or
- Resource Equation Method (when there is no information about standard deviation or effect size).
Statistical analysis
The general aim of statistical analysis is to extract all the useful information in the data, and to
either identify patterns or test a pre-stated hypothesis. Steps involved in statistical analysis
include examination of the raw data and application of the appropriate method of analysis.
Presentation of results
Clear presentation of results is the final step of good experimental design and statistical analysis.
It includes accounting for every experimental subject, appropriate measures of variance, and discussion
of the biological relevance of any statistically significant and non-significant results.
This section has been adapted from Festing & Altman, 2002.
For more information on experimental design and analysis, the following resources may be useful:
Online articles
- Festing M, & Altman D. (2002) Guidelines for the design and statistical analysis of experiments
using laboratory animals. ILAR Journal 43:244-258.
- Garner J. (2005) Stereotypies and other abnormal repetitive behaviors: potential impact on validity,
reliability, and replicability of scientific outcomes. ILAR Journal 46:106-117.
- Howard B. (2002) Control of variability. ILAR Journal 43:194-201.
- Kilkenny C., Browne W.J., Cuthill I.C., Emerson M. and Altman D.G. (2010) Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research. PLoS Biology 8(6), (accessed on 2010-09-14).
- Kilkenny C., Parsons, N., Kadyszewski E., Festing M.F.W., Cuthill I.C., Fry D., Hutton J. & Altman D.G. (2009) Survey of the Quality of Experimental Design, Statistical Analysis and Reporting of Research Using Animals. PloS ONE 4(11): e7824. doi:10.1371/journal.pone.000782443:194-201.
- The National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) (2006)
Why do a pilot study?
- Shaw R., Festing M.F.W., Peers I. & Furlong L. (2002) Use of factorial designs to optimize animal
experiments and reduce animal use. ILAR Journal 43: 223-232.
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Online resources
Other publications
- Horgan G. (2005) Interpretation of two-stage experiments in animal studies. Laboratory Animals 39:75-79.
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