First, think about what the unit of replication is. This is not a
trivial as it sounds (a cage with 3 animals in it is not necessarily n = 3!). A
common mistake is to have all members of (for example) genetic strain 1 in one cage, and all members
of genetic strain 2 (or non-carrier siblings) in another cage. The unit of replication
in this case would be cage, not individual animal (i.e., one cage would constitute n = 1!). The reason this is important is
because the variability within each
cage will usually be lower than the
variability between each cage. So,
any effect that you observe might be an effect of cage, rather than an effect
of (for example) your knocked-out allele.
Steps
to follow to ensure balanced allocation into comparison groups:
1.
Groups should be
age/sex matched as far as is possible. In addition, for genetic mutation studies, the comparison group should always be wild-type siblings of the same strain. At the very least, ensure that there is no source effect (in other words, the mutants came from supplier X, and the comparison group from in-house stock, or supplier Y).
2.
Groups should be
formed from multiple families (or original batches of animals) to increase variability.
3.
If possible,
members of each group (e.g., 1 x genetic strain + 1 x wild-type sibling, etc.) should share a
cage. Clearly, this is sometimes not possible owing to local or government legislation for work with genetic breeds, but talk to your local ethical adviser for clarification. The benefit of adopting this method is to refine and reduce animal usage, and this is something that most animal health and welfare officals are interested in promoting.
4.
If you need to individually identify your animals (e.g., they are kept in large groups) take care when
choosing marking substrates as some may interfere with behaviour. If possible, identify
individuals by natural markings.
5.
If possible,
include enrichment devices in cages, as overly standardized environments can adversely
affect the outcome of experiments.
6.
During the
experiment, ensure that all strains and treatment groups have equal exposure to handling/husbandry.
4.
Finally, it is a good idea to plan to carry out at least one (preferably more than one) independent replication of the study.