4b. Confounders

What to write

Describe the strategy used to minimise potential confounders such as the order of treatments and measurements, or animal/cage location. If confounders were not controlled, state this explicitly.

Explanation

Ensuring there is no systematic difference between animals in different groups apart from the experimental exposure is an important principle throughout the conduct of the experiment. Identifying nuisance variables (sources of variability or conditions that could potentially bias results) and managing them in the design and analysis increases the sensitivity of the experiment. For example, rodents in cages at the top of the rack may be exposed to higher light levels, which can affect stress¹.

Reporting the strategies implemented to minimise potential differences that arise between treatment groups during the course of the experiment enables others to assess the internal validity. Strategies to report include standardising (keeping conditions the same, e.g., all surgeries done by the same surgeon), randomising (e.g., the sampling or measurement order), and blocking or counterbalancing (e.g., position of animal cages or tanks on the rack), to ensure groups are similarly affected by a source of variability. In some cases, practical constraints prevent some nuisance variables from being randomised, but they can still be accounted for in the analysis (see Item 7. Statistical methods).

Report the methods used to minimise confounding factors alongside the methods used to allocate animals to groups. If no measures were used to minimise confounders (e.g., treatment order, measurement order, cage or tank position on a rack), explicitly state this and explain why.

Examples

‘Randomisation was carried out as follows. On arrival from El-Nile Company, animals were assigned a group designation and weighed. A total number of 32 animals were divided into four different weight groups (eight animals per group). Each animal was assigned a temporary random number within the weight range group. On the basis of their position on the rack, cages were given a numerical designation. For each group, a cage was selected randomly from the pool of all cages. Two animals were removed from each weight range group and given their permanent numerical designation in the cages. Then, the cages were randomized within the exposure group’².

‘… test time was between 08.30am to 12.30pm and testing order was randomized daily, with each animal tested at a different time each test day’³.

‘Bulls were blocked by BW into four blocks of 905 animals with similar BW and then within each block, bulls were randomly assigned to one of four experimental treatments in a completely randomized block design resulting in 905 animals per treatment. Animals were allocated to 20 pens (181 animals per pen and five pens per treatment)’⁴.

Training

The UK EQUATOR Centre runs training on how to write using reporting guidelines.

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References

1.

Ishida A, Mutoh T, Ueyama T, et al. Light activates the adrenal gland: Timing of gene expression and glucocorticoid release. Cell Metabolism. 2005;2(5):297-307. doi:10.1016/j.cmet.2005.09.009

2.

El‐Agroudy NN, El‐Naga RN, El‐Razeq RA, El‐Demerdash E. Forskolin, a hedgehog signalling inhibitor, attenuates carbon tetrachloride‐induced liver fibrosis in rats. British Journal of Pharmacology. 2016;173(22):3248-3260. doi:10.1111/bph.13611

3.

Carrillo M, Migliorati F, Bruls R, et al. Repeated witnessing of conspecifics in pain: Effects on emotional contagion. Gallup AC, ed. PLOS ONE. 2015;10(9):e0136979. doi:10.1371/journal.pone.0136979

4.

Del Bianco Benedeti P, Paulino PVR, Marcondes MI, Maciel IFS, Silva MC da, Faciola AP. Partial replacement of ground corn with glycerol in beef cattle diets: Intake, digestibility, performance, and carcass characteristics. Pas MFW te, ed. PLOS ONE. 2016;11(1):e0148224. doi:10.1371/journal.pone.0148224