SummarySex is a biological variable important to consider in all biomedical experiments. However, doing so in avian embryos can be challenging as sex can be morphologically indistinguishable. Unlike humans, female birds are the heterogametic sex with Z and W sex chromosomes. The female‐specific W chromosome has previously been identified in chick using a species‐specific polymerase chain reaction (PCR) technique. We developed a novel reverse transcription quantitative PCR (RT‐qPCR) technique that amplifies the W chromosome gene histidine triad nucleotide‐binding protein W (HINTW) in chick, quail, and duck. Accuracy of the HINTW RT‐qPCR primer set was confirmed in all three species using species‐specific PCR, including a novel quail‐specific HINTW PCR primer set. Bone development‐related gene expression was then analyzed by sex in embryonic lower jaws of duck and quail, as adult duck beak size is known to be sexually dimorphic while quail beak size is not. Trends toward sex differences were found in duck gene expression but not in quail, as expected. With these novel RT‐qPCR and PCR embryo sexing methods, sex of chick, quail, and duck embryos can now be assessed by either/both RNA and DNA, which facilitates analysis of sex as a biological variable in studies using these model organisms.

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 ofHINTWRT-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 novelHINTWRT-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.