Defining the experimental unit. Pregnant females are the experimental units because they are randomised to the treatment (e.g. valproic acid) or control conditions and therefore n = 6 in this example. The three offspring within a litter will often be more alike than offspring from different litters and multiple offspring within a litter can be thought of as subsamples or “technical replicates”, even though these are the scientific unit of interest. Only the mean of the within-litter values are important when comparing treated and control groups. Using all of the offspring without averaging will result in an inflated sample size (pseudoreplication) with standard statistical analyses. Instead of averaging, one could randomly select only one animal from each litter, or use a mixed-effects model to appropriately partition the different sources of variation. The only way to increase sample size, and thus power, is to increase the number of litters used.