Role of Ancillary Variables in the Design, Analysis, and Interpretation of Animal Experiments

Abstract

During the course of an experiment using animals, many variables (e.g., age, body weight at several times, food and water consumption, hematology, and clinical biochemistry) and other characteristics are often recorded in addition to the primary response variable(s) specified by the experimenter. These additional variables have an important role in the design and interpretation of the experiment. They may be formally incorporated into the design and/or analysis and thus increase precision and power. However, even if these variables are not incorporated into the primary statistical design or into the formal analysis of the experiment, they may nevertheless be used in an ancillary or exploratory way to provide valuable information about the experiment, as shown by various examples. Used in this way, ancillary variables may improve analysis and interpretation by providing an assessment of the randomization process and an approach to the identification of outliers, lead to the generation of new hypotheses, and increase generality of results or account for differences in results when compared across different experiments. Thus, appropriate use of additional variables may lead to reduction in the number of animals required to achieve the aims of the experiment and may provide additional scientific information as an extra benefit. Unfortunately, this type of information is sometimes effectively discarded because its potential value is not recognized. Guidelines for use of animals include, in addition to the obligation to follow humane procedures, the obligation to use no more animals than necessary. Ethical experimental practice thus requires that all information be properly used and reported.

Effects of Sodium Chloride-Rich Mineral Water on Intestinal Epithelium. Experimental Study

“…The animals were divided into three groups of five rats each. The animal distribution was at random and auxiliary variables such as sex and weight were previously taken into account to confer minimal variations to the groups according to the literature [29,30]. In the first group used as control (CG), the diet was standard laboratory food (SAFE ® A04, Augy, France) and water (tap water) ad libitum.…”

Guidance for the Description of Animal Research in Scientific Publications

“…In addition, numerous husbandry, nutritional, and environmental factors strongly influence body weight, often through incompletely understood interactions (Haseman et al 1997(Haseman et al , 2003Keenan et al 1999;Laroque et al 1997). Because weight correlates with many biological outcomes (Gaines Das 2002) it is important to include it in the animal description together with age (Klimentidis et al 2010).…”

Fundamental steps in experimental design for animal studies

“…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 13 . Preferentially, isogenic strains should be used.…”

“…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 13,14 .…”

“…Blocking refers to direct manipulation to control one or more independent variables and thus, avoid variation 4,14 . Ancillary variables such as sex and weight may be firstly manipulated to confer minimal variation between the groups 13 . The investigator may divide the animals that are comparable in cages and then randomize them to the groups 1,14 .…”