Real-time polymerase chain reaction for poultry sex genotyping

There is pressing demand for improved protocols to determine the sex of a chicken in the layer sector. Extensive efforts are being dedicated to avoiding day-old male chick culling by developing in-ovo sexing detection methods. Any established in-ovo detection method needs to be validated by embryo genotyping. As a result, there is growing demand for fast, inexpensive and precise methods for proper discrimination between males and females in the poultry scientific community.

The aim of the study, led by experts from the Ben Gurion University of the Negev in Israel and Syracuse University in the US, was to develop an accurate, high-throughput protocol for sex determination using small volumes of blood.

Processing large numbers

Primers were designed targeting the Hint-W gene within the W chromosome clearly distinguishing between males and females. They then took advantage of avian nucleated red blood cells to create a quick lysate that had enough gDNA for a direct PCR reaction without the need for DNA purification. They then eliminated the gel electrophoresis that can process fewer samples at a time and instead implemented qPCR which can process 96 samples per run.

The scientists say this method requires fewer steps than other chicken sexing protocols using PCR and has a lower cost per sample, which confirms it is ideal for processing large numbers of samples.

The Ct-score obtained in the qPCR reaction required for determining the sex of the chickens was then established and the researchers validated the accuracy of their method using established protocols and gonad phenotyping and tested their protocol with 4 different chicken breeds, day 9 embryos, day-old chicks and adult chickens.

*The article was published in the journal Livestock Genomics – Frontiers | Fast, accurate, and cost-effective poultry sex genotyping using real-time polymerase chain reaction.