A Quantitative Polymerase Chain Reaction Protocol for Sex Identification of Zebra Finch Embryos Using Blood Samples

Investigating sex differences in avian developmental biology research is increasingly relevant for clinical and ecology-based studies. Songbirds like zebra finches (Taeniopygia guttata), like most birds, are not sexually dimorphic at hatching. Their plumage is indistinguishable as embryos, complicating experiments where sex is not yet visually evident, but biological differences between sexes are expected (e.g., gene expression studies). The current methods for zebra finch embryo genetic sex identification where only a single W locus in analyzed are prone to false identification because of genetic variation or technical failure of amplification. As a result, any further validation steps of the amplified product such as gel electrophoresis adds additional steps, only to receive inaccurate results. Thus, there is a need for an efficient and accurate method for time-sensitive developmental and behavioral studies. Here, we developed an approach that targets two different W loci, using SYBR-based quantitative PCR to analyze amplification curves. We applied this method to determine sex of 30 zebra finch embryos (embryonic day 13) and subsequently confirmed genetic sex in all cases by brain transcriptome sequencing. Using this multi-marker approach, we also identified primer sets that are effective for sex determination in chickens. Overall, our findings contribute to the field of avian development biology by offering an efficient and accurate method for genetic sex determination. The CSV files attached have (1) qPCR DNA Absorbance (A260/280 ratios) and DNA Yield (ng/µL) data from E13 zebra finch embryo's trunk blood, and (2) raw data from the qPCR quantification amplification and melt curve analyses.