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.
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Turner J. Animal reproduction, human control. In: Bekoff M, editor. Encyclopedia of animal rights and animal welfare. 2nd ed. Santa Barbara: ABC-CLIO, LLC; 2010. p. 30–6.
Tierschutz-Schlachtverordnung - TierSchlV (BGBl. I S. 2982). 20-12-2012.
Bruijnis MRN, Blok V, Stassen EN, Gremmen HGJ. Moral “Lock-In” in responsible innovation: the ethical and social aspects of killing day-old chicks and its alternatives. J Agric Environ Ethics. 2015;28(5):939–60.
Harz M, Krause M, Bartels T, Cramer K, Rosch P, Popp J. Minimal invasive gender determination of birds by means of UV-resonance Raman spectroscopy. Anal Chem. 2008;80(4):1080–6.
Steiner G, Bartels T, Krautwald-Junghanns ME, Boos A, Koch E. Sexing of turkey poults by Fourier transform infrared spectroscopy. Anal Bioanal Chem. 2010;396(1):465–70.
Steiner G, Preusse G, Zimmerer C, Krautwald-Junghanns ME, Sablinskas V, Fuhrmann H, et al. Label free molecular sexing of monomorphic birds using infrared spectroscopic imaging. Talanta. 2016;150:155–61.
Steiner G, Bartels T, Stelling A, Krautwald-Junghanns ME, Fuhrmann H, Sablinskas V, et al. Gender determination of fertilized unincubated chicken eggs by infrared spectroscopic imaging. Anal Bioanal Chem. 2011;400(9):2775–82.
Tran HT, Ferrell W, Butt TR. An estrogen sensor for poultry sex sorting. J Anim Sci. 2010;88(4):1358–64.
Phelps P, Bhutada A, Bryan S, Chalker A, Ferrell B, Neuman S, et al. Automated identification of male layer chicks prior to hatch. World Poult Sci J. 2003;59(1):33–8.
Weissmann A, Reitemeier S, Hahn A, Gottschalk J, Einspanier A. Sexing domestic chicken before hatch: a new method for in ovo gender identification. Theriogenology. 2013;80(3):199–205.
Clinton M, Haines L, Belloir B, McBride D. Sexing chick embryos: a rapid and simple protocol. Br Poult Sci. 2001;42(1):134–8.
Jensen T, Mace M, Durrant B. Sexing of mid-incubation avian embryos as a management tool for zoological breeding programs. Zoo Biol. 2012;31(6):694–704.
Porat N, Bogdanov K, Danielli A, Arie A, Samina I, Hadani A. Direct detection of chicken genomic DNA for gender determination by thymine-DNA glycosylase. Br Poult Sci. 2011;52(1):58–65.
Yilmaz-Dikmen B, Dikmen S. A morphometric method of sexing white layer eggs. Braz J Poult Sci. 2013;15(3):203–10.
Webster B, Hayes W, Pike TW. Avian egg odour encodes information on embryo sex, fertility and development. Plos One. 2015;10(1):e0116345.
Galli R, Preusse G, Uckermann O, Bartels T, Krautwald-Junghanns ME, Koch E, et al. In ovo sexing of domestic chicken eggs by Raman spectroscopy. Anal Chem. 2016;88:8657–63.
Wei D, Chen S, Liu Q. Review of fluorescence suppression techniques in Raman spectroscopy. Appl Spectrosc Rev. 2015;50(5):387–406.
Gosnell ME, Anwer AG, Mahbub SB, Menon PS, Inglis DW, Adhikary PP, et al. Quantitative non-invasive cell characterisation and discrimination based on multispectral autofluorescence features. Sci Rep. 2016;6:23453.
Rudbeck L, Dissing J. Rapid, simple alkaline extraction of human genomic DNA from whole blood, buccal epithelial cells, semen and forensic stains for PCR. Biotechniques. 1998;25(4):588–90.
Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. Touchdown PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991;19(14):4008.
Fridolfsson AK, Ellegren H. A simple and universal method for molecular sexing of non-ratite birds. J Avian Biol. 1999;30(1):116–21.
Schart C. Thesis: Entwiklung einer Steuerung für die Fokussierung und das Tracking embryionaler Blutgefäße im Hühnerei. Hochschule für Technik und Wirtschaft Dresden. 2015.
Baumann R, Meuer HJ. Blood-oxygen transport in the early avian embryo. Physiol Rev. 1992;72(4):941–65.
Sheng G. Primitive and definitive erythropoiesis in the yolk sac: a bird’s eye view. Int J Dev Biol. 2010;54(6–7):1033–43.
Johnston P. Hematocrit values for the chick embryo at various ages. Am J Physiol. 1955;180(2):361–2.
Campbell GL, Weintraub H, Mayhall BH, Holtzer H. Primitive erythropoiesis in chick embryogenesis. 2. Correlation between Hemoglobin Synthesis and the Mitotic History. J Cell Physiol. 1971;50(3):669–81.
Berezin MY, Achilefu S. Fluorescence lifetime measurements and biological imaging. Chem Rev. 2010;110(5):2641–84.
Hirsch RE. Heme-protein fluorescence. In: Lakowicz JR, editor. Topics in fluorescence spectroscopy, Vol. 6 Protein Fluorescence. New York: Kluwer Academic Publishers; 2002. p. 221–47.
Chaiken J, Finney WF, Yang X, Knudson PE, Peterson K, Weinstock RS, et al. Progress in the noninvasive, in vivo, tissue modulated Raman spectroscopy of human blood. P SPIE. 2001;4254:216–27.
Chaiken J, Goodisman J, Deng B, Bussjager RJ, Shaheen G. Simultaneous, noninvasive observation of elastic scattering, fluorescence and inelastic scattering as a monitor of blood flow and hematocrit in human fingertip capillary beds. J Biomed Opt. 2009;14(5):050505.
Lee JY, Ji HS, Lee SJ. Micro-PIV measurements of blood flow in extraembryonic blood vessels of chicken embryos. Physiol Meas. 2007;28(10):1149–62.
Tagirov M, Golovan S. Sexual dimorphism in the early embryogenesis of the chicken (Gallus Gallus domesticus). Mol Reprod Dev. 2015;82(5):332–43.
Morita VS, Boleli IC, Cargnelutti A. Hematological values and body, heart and liver weights of male and female broiler embryos of young and old breeder eggs. Braz J Poultry Sci. 2009;11(1):7–15.
Abdi H, Williams LJ. Principal component analysis. WIREs Comput Stat. 2010;2(4):433–59.
Bonnier F, Byrne HJ. Understanding the molecular information contained in principal component analysis of vibrational spectra of biological systems. Analyst. 2012;137(2):322–32.
Talari ACS, Movasaghi Z, Rehman S, Rehman I. Raman spectroscopy of biological tissues. Appl Spectrosc Rev. 2015;50(1):46–111.
Bankapur A, Zachariah E, Chidangil S, Valiathan M, Mathur D. Raman tweezers spectroscopy of live, single red and white blood cells. Plos One. 2010;5(4):e10427.
Cuadros MA, Coltey P, Carmen NM, Martin C. Demonstration of a phagocytic cell system belonging to the hemopoietic lineage and originating from the yolk sac in the early avian embryo. Development. 1992;115(1):157–68.
McIntyre BA, Alev C, Tarui H, Jakt LM, Sheng G. Expression profiling of circulating non-red blood cells in embryonic blood. BMC Dev Biol. 2008;8:21.
Brereton RG, Lloyd GR. Support vector machines for classification and regression. Analyst. 2010;135(2):230–67.
Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morphol. 1951;88(1):49–92.
Speksnijder G, Ivarie R. A modified method of shell windowing for producing somatic or germline chimeras in fertilized chicken eggs. Poult Sci. 2000;79(10):1430–3.
Fineman RM, Schoenwolf GC, Huff M, Davis PL. Causes of windowing-induced dysmorphogenesis (neural tube defects and early amnion deficit spectrum) in chicken embryos. Am J Med Genet. 1986;25(3):489–505.
Kaleta EF, Redmann T. Approaches to determine the sex prior to and after incubation of chicken eggs and of day-old chicks. World Poult Sci J. 2008;64(3):391–9.
Aleksandrowicz E, Herr I. Ethical euthanasia and short-term anesthesia of the chick embryo. Altex-Altern Tierexp. 2015;32(2):143–7.
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.
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.
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). https://doi.org/10.1007/s00216-016-0116-6