Fig. 2

In treatment studies, pregnant females, and hence whole litters, are exposed to a substance (a, c, e). Litters are the experimental unit because they are randomized to the treatment. Offspring within a litter will be more similar to each other than to offspring from different litters and should be treated as subsamples or technical replicates. Similarly, in studies with mutant animals, individual animals coming from the same litter share prenatal and postnatal environments that render them more similar to each other than to animals from different litters (b, d, f). Most studies incorrectly ignore the litter and instead apply standard statistical tests to multiple animals per litter, thus failing to correct or control for relatively large differences between litters. Appropriate analysis can be conducted by a and b only using one animal of a given genotype and sex per litter (randomly selected), c and d using more than one animal per litter and averaging their values, or e and f using multiple animals per litter and applying a mixed-effects model for analysis. The mixed-effects model allows for each offspring to be used as a unit of analysis and treats the litter as a random variable in the ANOVA. Even though each offspring is used as a sample, the litter is still the statistical unit for exposure studies, as the dam was randomly assigned to the treatment condition. Sex of the offspring should be balanced across groups. It may be necessary to study male and female neonates separately, in which case a total of 1 male and 1 female per litter (and genotype if relevant), can be sampled