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 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.

Allantoic Fluid-Based qPCR for Early Onset In Ovo Sexing

“…In birds, the sex chromosomes are represented by a ZZ chromosome pair in males and a ZW in females, 23 thereby allowing identification of female-specific sequences in the W chromosome. Previous reports have shown such detection using genes CDHZ/W 24 or HINTW 25,26 in different samples, e.g., blood, skin, or feathers. However, embryo blood and feather sampling is highly invasive and usually fatal.…”

“…The use of such conserved genes allows sexual sorting in other chicken lines and bird species due to their shared genetic background. 25,26,28 To accomplish our objectives, we developed novel primer pairs for the HINTW and DMRT-1 genes using the NCBI-BLAST engine. A comprehensive optimization process is carried out for the qPCR assay, utilizing synthetic DNA and genomic DNA (gDNA) extracted from blood samples collected on incubation day 14.…”