Research Article: Simple, sensitive and robust chicken specific sexing assays, compliant with large scale analysis

Date Published: March 1, 2019

Publisher: Public Library of Science

Author(s): Liyan He, Priscila Martins, Joris Huguenin, Thi-Nhu-Ngoc Van, Taciana Manso, Therese Galindo, Flavien Gregoire, Lise Catherinot, Franck Molina, Julien Espeut, Ruslan Kalendar.


Chicken meat and eggs are important sources of food for the world population. The significant increase in food demand has pushed the food industry toward a rapid non-expensive production which in turn raises ethical issues. How chicken are cultivated and processed in food industry is no longer acceptable. Ethical and economical concerns emerging from chicken culling need to be solved in the near future. Indeed, in egg production industry, male chicken are killed at the age of 1-day post-hatching since they are not egg producers. A number of laboratory all over the world are looking for innovative non-invasive sexing methods to determine the sex of chicken in the early stages of the development before hatching. It will allow males’ chicken elimination before the pain-feeling stages. In order to evaluate the efficiency of these methods, the scientific community need a reliable, easy to use and cost-effective in-ovo invasive sexing method. In this report, we developed two new invasive assays based on PCR and Q-PCR techniques respectively, which fulfil the above mentioned requirements. In the same line with other groups, we exploited the differences betweed males (ZZ) and females (ZW) chicken sexual chromosomes. We identified two genes, SWIM and Xho-I, on chromosome W and DMRT gene on chromosome Z allowing a clear discrimination between the two sexes using PCR and qPCR respectively. These two new genomic markers and their corresponding methods not only increase the accuracy but also reduce time and cost of the test compared to previously developed sexing methods. Depending on the technology available in the lab, one can choose between the two techniques requiring different machines and expertise.

Partial Text

The chicken embryo is a well-established model in developmental biology studies since it allows direct manipulation of living embryo. Moreover, chicken meat and eggs are important sources of food for the global population. 118 million tons of meat and 1400 billion eggs were produced in 2017 (Source: Poultry Trends). In chicken eggs production industry, all males’ chicks are killed at the age of 1 day post-hatching, since only females are valuable. Each year, all over the world, around 6 billion male chicks are considered as useless products (Source: Food and Agriculture Organization of the United Nations). It raises ethical and economical concerns that must be fixed as soon as possible, mostly to avoid animal suffering. Many countries and companies around the world are running scientific programs in order to be able to determine the sex of living chicken embryos at the early stages of development. Once working, it will allow eliminating eggs with male embryos before pain feeling stages (there is a current consensus before day 11 of egg development). Up to date, various assays have been established to sex chicken embryos: specific DNA amplification by PCR or Q-PCR [1–11]; hormones detection [12]; infrared, fluorescence or Raman spectroscopy detection of sex specific signals following egg opening [13–15]; exalted odour analysis by gas chromatography coupled with mass spectrometry [16]. Among these approaches, odour analysis represents the sole non-invasive technique that does not require eggshell opening. However, up to now, this is not yet a reliable method for egg sexing. Other approaches raise a biological concern since egg sexing must be run under non-sterile environment i.e. hatcheries.

The scientific community and the poultry industry lack a reliable method to determine chicken embryo sex for which the performances have been evaluated and validated in a large scale manner. Indeed, benchmarking new technologies aiming to sex chicken eggs in a non-invasive way, will need a highly-reliable invasive method as reference (gold standard) to determine the sex of the embryo a posteriori that works at early stage of embryo development. Here, we developed two new simple alternative methods to sex chicken embryos and demonstrated their high performance for large-scale purpose. The 2 methods exhibit 100% of concordance and specificity for the sex determination of the 176 embryos tested in our studies. One of the methods is based on PCR technologies once the other one is based on RT qPCR. Depending on their resources and equipment, the users, in a scientific lab or in the poultry industry can choose between the 2 approaches. For users equipped with real-time PCR readers the RT qPCR will be more convenient since this approach requires fewer steps than PCR and the results analysis can be easily automatized. PCR will be more convenient for users that do not want to invest too much money in the equipment. Compared to the other technologies already developed to sex chicken, our testes combine high compliancy with large scale analysis, cost effectiveness and high sensitivity. Moreover, we showed that our testes are efficient on many different tissues of the embryo and can be used for different chicken breeds in a short-time scale since the 2 methods do not require any DNA purification step. Consequently, any laboratory from any speciality can easily implement our simplified and robust sexing assays using the Standard Operating Procedures (SOP) provided on ( The positive results from experiments with membrane samples (external and internal) from which the embryos continue to develop encourage a future development for a semi invasive in ovo sexing method.




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