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However, research on mammalian systems has shown that this oversight is no longer supportable and that both sexes should be considered in an experimental design: the bias towards including only male animals or male cells results in a failure to identify sex differences relevant to downstream clinical studies and may contribute to a lack of reproducibility in many studies [Clayton and Collins, 2014].

As regards non-mammalian vertebrates, we have recently shown that chicken somatic cells possess an inherent sex identity and that male and female cells and tissues respond differently to extracellular stimulation [Zhao et al., 2010].

For procedures involving live embryos, a small volume (∼0.5 µl) of blood was collected, diluted to 10 µl with water, and heated at 95°C for 5 min.After cooling, 7.5 µl was used in the Hologic Invader® sexing assay.Five independent cultures of male and female chicken embryo fibroblasts (CEF) were prepared [Hernandez and Brown, 2010] from embryos at day 16 of development.Embryo sex was determined by visual examination of the gonads.Manipulations can be performed at different developmental stages via a small window cut in the egg shell, and the egg can then be sealed and re-incubated, allowing further embryonic development - an approach that is extremely difficult with placental vertebrates.

In common with other vertebrate model systems, the majority of chicken studies fail to consider the sex of embryos.

A small volume of diluted blood (equivalent to 0.063-2.0 µl of whole blood) was adjusted to 7.5 µl with water, heated to 95°C, and used in the Hologic Invader® sexing assay.

Blood collected from 5 male and 5 female adult birds was used to prepare genomic DNA by standard procedures [Sambrook, 1989].

For analysis, water (100 µl) was added to the tissue and the sample was heated at 95°C for 5 min.

Without further processing, 7.5 µl was transferred to a 96-well plate for assaying.

Benefits of this model include: (a) a shelled egg that is independent of the hen, allowing easy access for observation and experimental manipulation; (b) the ability to synchronise the incubation of large numbers and isolate embryos at precise stages of development; (c) the comparatively large size; (d) a planar development that shows significant similarities to that of the early human embryo; and (e) the fact that fertilised eggs are both inexpensive and readily available.