In ovo sexing of chicken eggs by fluorescence spectroscopy


Culling of day-old male chicks in production of laying hen strains involves several millions of animals every year worldwide and is ethically controversial. In an attempt to provide an alternative, optical spectroscopy was investigated to determine nondestructively in ovo the sex of early embryos of the domestic chicken. The extraembryonic blood circulation system was accessed by producing a window in the egg shell and the flowing blood was illuminated with a near-infrared laser. The strong fluorescence and the weak Raman signals were acquired and spectroscopically analyzed between 800 and 1000 nm. The increase of fluorescence intensity between 3.5 and 11.5 days of incubation was found to be in agreement with the erythropoietic stages, thus enabling to identify hemoglobin as fluorescence source. Sex-related differences in the fluorescence spectrum were found at day 3.5, and principal component (PC) analysis showed that the blood of males was characterized by a specific fluorescence band located at ∼910 nm. Supervised classification of the PC scores enabled the determination of the sex of 380 eggs at day 3.5 of incubation with a correct rate up to 93% by combining the information derived from both fluorescence and Raman scattering.

The fluorescence of blood obtained in ovo by illumination of embryonic vessels with a IR laser displays spectral differences that can be employed for sexing of eggs in early stage of incubation, before onset of embryo sensitivity and without hindering its development into a healthy chick

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The authors gratefully acknowledge Andrea Büchner for performing molecular genetic analyses and Lohmann Tierzucht GmbH (Cuxhaven, Germany) for providing eggs. Special thanks to Prof. Rudolf Preisinger and Dr. Anke Förster (Lohmann Tierzucht GmbH, Cuxhaven, Germany) for the insightful discussions about hatchery practice.

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Corresponding authors

Correspondence to Roberta Galli or Gerald Steiner.

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This work was financially supported by the German Federal Ministry of Food, Agriculture, and Consumer Protection (BMELV) through the Federal Office for Agriculture and Food (BLE), grant no. 511–06.01-28-1-33.010-07.

Conflict of interest

Patent applications for in ovo sexing with the methods described in this paper are pending.

Additional information

Dedicated to Professor Reiner Salzer on the occasion of his 75th birthday. Professor Reiner Salzer is an internationally recognized leader in analytical chemistry and pioneered spectroscopic methods.

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Galli, R., Preusse, G., Uckermann, O. et al. In ovo sexing of chicken eggs by fluorescence spectroscopy. Anal Bioanal Chem 409, 1185–1194 (2017).

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  • Optical spectroscopy
  • Fluorescence
  • Raman scattering
  • Chicken embryo
  • Sexing
  • In ovo