  Background: Animals from the same litter are often more alike compared with
animals from different litters. This litter-to-litter variation, or "litter
effects", can influence the results in addition to the experimental factors of
interest. Furthermore, an experimental treatment can be applied to whole
litters rather than to individual offspring. For example, in the valproic acid
(VPA) model of autism, VPA is administered to pregnant females thereby inducing
the disease phenotype in the offspring. With this type of experiment the sample
size is the number of litters and not the total number of offspring. If such
experiments are not appropriately designed and analysed, the results can be
severely biased as well as extremely underpowered.
  Results: A review of the VPA literature showed that only 9% (3/34) of studies
correctly determined that the experimental unit (n) was the litter and
therefore made valid statistical inferences. In addition, litter effects
accounted for up to 61% (p <0.001) of the variation in behavioural outcomes,
which was larger than the treatment effects. In addition, few studies reported
using randomisation (12%) or blinding (18%), and none indicated that a sample
size calculation or power analysis had been conducted.
  Conclusions: Litter effects are common, large, and ignoring them can make
replication of findings difficult and can contribute to the low rate of
translating preclinical in vivo studies into successful therapies. Only a
minority of studies reported using rigorous experimental methods, which is
consistent with much of the preclinical in vivo literature.
