The Design and Statistical Analysis of Animal Experiments

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The resource is aimed at scientists who use animals in their research. Benefits include advice and feedback on the experimental plans, along with a range of functionalities providing support with the randomisation and blinding of the experiment, as well as sample size calculation.

It equips researchers with practical information and knowledge, allowing them to determine the most efficient design for their experiment and understand the implications of choosing a particular design.

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A central feature of the EDA is the use of a formal, diagrammatic notation to describe experimental plans and analyses. This is an approach that has been adopted by many technical disciplines to improve communications. It allows the design of an experiment to be recorded clearly and unambiguously and EDA diagrams help convey experimental plans efficiently. The EDA is not designed to replace specialist statistical advice. For researchers who have limited access to statistical support, the feedback and advice provided by the system will be particularly pertinent, as it will provide users with information, which is specific to the experiment they are planning.

For all scientists involved in the research process, the EDA is also extremely useful as a communication tool, for example, between students and their supervisors, or with colleagues and collaborators. These visual representations are far more explicit than the cursory text description traditionally included in grant applications, ethical review submissions or journal publications. Our goal is to integrate the EDA into the scientific process to facilitate better peer review of experimental plans. We look forward to hearing what you think about the EDA.

The feedback has been fantastic but this is a very new and novel system, which has to evolve according to the needs of the research community. Categories: Research , Resources. Your email address will not be published. We want to make our website, and the services we provide, useful and reliable. This sometimes involves placing small amounts of information called cookies on the device you used to access the internet. During literature search, investigators should pay attention to both new and pioneer studies on the field of the planned research.

Relevant reports coming from the same country and especially from the same university or research center are imperative to be referred. Scientific method. The most important point of the experimental design is its adherence to the scientific method. The scientific method could be synthesized in four phases: 1 observation and description of a scientific phenomena, 2 formulation of the hypothesis and the questions, 3 prediction of the results on the formulated hypothesis, and 4 implementation of the procedures to test the hypothesis by using appropriated methods 1,4.

The objective of the study should be clearly stated. Moreover, the potential application of the findings arising from the experiment should be related to either human or animal conditions. The formulation of the hypothesis should predict two events: the null and the alternate hypothesis.

The null hypothesis should assert that the treatment tested is not different from the control.

In contrast, the alternate hypothesis should predict that the treatment tested actually differs from the controls 4,9. Both the definition of the question to be answered by the experiment and the formulation of the hypothesis are vital at this stage of the project. The next phase of the experiment is the choice of the animal model followed by the pilot study. Animal models. A good literature search should be performed to find out the adequate animal model.

However, new investigators will find in good centers sufficient knowledge to follow practical advices from either tutors or senior staff. The key purpose of using animals in basic and applied research is to discover biologic phenomena and to model physiologic processes that are likely comparable to those that occur in humans. Thus, a good animal model should be reliable, practical and useful. An animal model must have some characteristics that resemble to the desired correlation animal to humane extrapolation, for instances 9.

For example, insulin was tested in rabbits because it was solidly established that insulin decreases blood glucose in rabbits as in humans though rabbits differs from humans in many ways. Ethically, the animals should be chosen by its lowest phylogenic scale 7 , adequacy to the planned procedures, and similarity to both humane anatomy and physiology. Of course, the costs should be considered 8. Even the life span of an animal should be considered in long studies. In this context, the advise of an experienced veterinary is quite important at this point.

Animals models can be classified into: 1 spontaneous models, 2 experimental models, 3 genetically modified models, 4 orphan models, and 5 negative models 4, Spontaneous animals models are those that exist in the nature with similarity to human condition or disease. Asthma in felines and the occurrence of natural killers cells NK cells in rats are good examples. Experimental models are those that need to be created to attend the desired conditions to be tested.

Surgical models are examples of experimental or induced models. Molecular biology has permitted modifying the animal genoma to attain the aims of some studies. Genetically modified models are therefore created for that reason. Insertions of an alien DNA and the knock-out models are good examples of these animal models. Negative model or non-model is the term used for species, strains, or breeds in which a certain disease or condition does not develop.

Negative models thus include animals that demonstrate a lack of reactivity to a particular stimulus. Their main application is in studies on the mechanism of resistance that seek to gain insight into its physiological basis. Why dogs do not develop arteriosclerosis is one of this questions that may be answered by a negative model. Contrarily, an orphan model is used to investigate either a condition or disease that occur in some species but not in humans. It may be important to recognize diseases that may affect humans in the next future. The mad-cow disease is the example of the day Variation control.

After choosing the animal model, the investigator should control variation. Uncontrolled variations may jeopardize the experiment in a way that all data may be invalidated The chosen model must be sensible to the treatment showing minimal variation of outcomes. Ancillary variables as gender, age and weight of the animal should be similar among the groups Preferentially, isogenic strains should be used. The laboratory environment may affect the course of an experiment. There is substantial evidence showing that many important biochemical data may vary with room temperature, and whether they were collect during day light or at night for instances Therefore, the experimental design must state the laboratory conditions and the data must be collected in the same period to reduce variation 1,4,9, Special attention should be paid to the diet of animals.

When testing different nutrients, the diets in all groups must be isocaloric and isonitrogenous. Personnel collecting data should be unchanged, qualified and trained in the procedures used in the project. If there are surgical procedures involved preferentially only one surgeon should perform all operations to avoid variation as well 9, Each experiment may contain a number of small experiments. When planning stratification of animals in groups, the assignment of animals at random to different groups and sub-groups may be improved by dividing animals in blocks to achieve minimal variation This technique is called "blocking" and allows less variation during randomization.

For instances, if an experiment is aimed at investigating the anastomotic healing in two different time points in two groups, it will be then four treatment sub-groups. Thus, it will be better to allocate multiples of four similar animals per cage and then randomize them to receive the four treatments. Blocking in this way assures more balance between animals and minimizes variation in ancillary variables as weight and diet for example 13, Important steps in experimental designs.

During the preparation of the study design, investigators should hold his attention in planning important steps before starting the study. These include the number of animals to be used, pilot studies, randomization, blinding, control groups, type of variables collected, and the statistical methods 1,4,9. Although of prime concern in a research design, the statistical methods are beyond the scope of this paper and will not be considered here.

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The number of animals assigned to the experimental groups and sub-groups is vital. In this matter, a statistical staff may be important 1. However, calculations can be easily done by on-line internet sites that investigators may consult. Computer softwares may be also used to calculate the figures. Most of times, mathematical formulas are used. Power analysis if possible must be calculated to ascertain the number of animals per group. The first two factors are unique for the experiment and the other two are conventional.

Dell et al. Before the beginning of the experiment, a pilot or a series of pilot studies should be carried out. This is an important step to assure that the entire experiment will work out fine. Frequently, a few number of animals is required though large number may be necessary. In experiments that an induced animal model is needed intestinal anastomosis studies for example the pilot study is the moment for the investigator to become skilled in the procedures involved.

Moreover, pilot studies are also important to give an idea and calculate the size of the experiment, i. Sometimes the original design is changed during the course of the pilot study due to the outcomes. Therefore, pilot studies should be minded by the investigator as a useful weapon for the strategy of the project 9. Randomization is another valuable topic in the experimental design The allocation of animals to different groups of treatment should be at random: 1 to avoid biases, 2 to guarantee that groups have the same probability to receive a treatment, and 3 to control variation 9, The method used to randomize should be clearly stated.

Dices, envelopes containing pieces of papers with codes, and tables with random numbers are examples of some frequent methods used for randomization 4. Experiments with either completely randomized groups or randomized blocking designs are all correct 1. Blocking refers to direct manipulation to control one or more independent variables and thus, avoid variation 4, Ancillary variables such as sex and weight may be firstly manipulated to confer minimal variation between the groups The investigator may divide the animals that are comparable in cages and then randomize them to the groups 1, Investigators should abort any temptation in rationalizing this step.

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  • In some occasions, investigators may be tempted to collect data in one group of treatment and after in other due to either technical or economical reasons. Imagine that is all set to collect data in only one of a two groups experimental study. It may appear to the investigator that it would be both rational and time saving to carry on and collect data in one of the groups firstly.

    However, this choice would jeopardize the whole experiment for the lack of randomization. Note that data should be collected in all groups at the same time, especially when operations are involved because skill in surgical procedure is progressive acquired.


    The second group may benefit of a more skilled surgeon! When two or more treatments are being compared, the investigator must diminish the occurrence of bias. Thus, the experiment should be done "blind" to diminish the possibility of a subjective effect in collecting data. Along the entire study, the main investigator may need collaborators. It is important to code animals, specimens, and samples, and also not to inform any coworker responsible for registering data about both the aims and groups of treatments investigated in the experiment 4,9.

    Control groups should be planned with care. They are fundamental in experimental designs and should be preferred than historical comparison 4.

    Determination and Justification of Animal Numbers

    There are many types of control groups though the most important are the positive, negative, sham and comparative controls 9. Positive controls are those in whom an effect or changes are most expected. They are necessary to estimate alterations that a condition may cause and then detect what the investigated treatment may modify. For example, the effect of two different diets on the recovery of malnutrition in rats must be compared with a positive control group studied during malnutrition.

    In a negative control no changes are expect. It is like a mirror of the positive control. In the above example, a negative control would be a group maintained with laboratory rat chow during all the experiment. Sham controls are used to mimic a procedure or treatment. For example, in a rat model of enterectomy, two treatments may be compared with a sham group submitted to the same laparotomy but instead of performing an enterectomy the bowel is only handled with care in the same site of resection of the resected group. Comparative control is a type of positive control in which it is used a known treatment to contrast with the new investigated treatment 4,9.

    An experimental study should use the same endpoints of a clinical study whenever is possible Surrogates of a clinical effect however are commonly used. During the construction of the study design investigators should attempt to the dependent variables. A variable can be continuous, ordinal, and categorical 4,16, Continuous variables are those expressed by numbers serum glucose level, anastomotic bursting pressure, heart frequency, etc.

    When it is considered an effect that may or may not occur such as death, infection or the occurrence of peritonitis for instances, the variable is said categorical. Whenever is possible the investigator should use continuous variables because an effect may be noticed earlier with few animals 4,9, Final considerations.

    It was emphasized the need of improvement in animal research.