A novel qPCR-based technique for identifying avian sex: an illustration within embryonic craniofacial bone

Abstract

Sex is a biological variable important to consider in all biomedical experiments. However, analyzing sex differences in avian models can be challenging as the sexes are morphologically indistinguishable in most avian embryos. Unlike humans, female birds are the heterogametic sex with a Z and W chromosome. The female-specific W chromosome has previously been identified using species-specific polymerase chain reaction (PCR) techniques. We developed a novel quantitative real-time PCR (RT-qPCR) technique which amplifies the W chromosome gene histidine triad nucleotide binding protein W (HINTW) in chick, quail, and duck. We confirmed the accuracy of the single set of HINTW RT-qPCR primers in all three species using species-specific PCR. Bone development-related gene expression was then analyzed by sex in embryonic lower jaws of duck and quail, as duck beak size is known to be sexually dimorphic while quail beak size is not. Trends towards sexual dimorphism were found in duck gene expression but not in quail, as expected. Our novel HINTW RT-qPCR technique to identify the sex of avian embryos is a useful tool for including sex as a biological variable in analysis of a variety of tissues and cells used in developmental biology research.