Analytical and Bioanalytical Chemistry

, Volume 409, Issue 5, pp 1185–1194 | Cite as

In ovo sexing of chicken eggs by fluorescence spectroscopy

  • Roberta Galli
  • Grit Preusse
  • Ortrud Uckermann
  • Thomas Bartels
  • Maria-Elisabeth Krautwald-Junghanns
  • Edmund Koch
  • Gerald Steiner
Research Paper

Abstract

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.

Graphical abstract

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

Keywords

Optical spectroscopy Fluorescence Raman scattering Chicken embryo Sexing In ovo 

References

  1. 1.
    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.Google Scholar
  2. 2.
    Tierschutz-Schlachtverordnung - TierSchlV (BGBl. I S. 2982). 20-12-2012.Google Scholar
  3. 3.
    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.CrossRefGoogle Scholar
  4. 4.
    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.CrossRefGoogle Scholar
  5. 5.
    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.CrossRefGoogle Scholar
  6. 6.
    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.CrossRefGoogle Scholar
  7. 7.
    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.CrossRefGoogle Scholar
  8. 8.
    Tran HT, Ferrell W, Butt TR. An estrogen sensor for poultry sex sorting. J Anim Sci. 2010;88(4):1358–64.CrossRefGoogle Scholar
  9. 9.
    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.Google Scholar
  10. 10.
    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.CrossRefGoogle Scholar
  11. 11.
    Clinton M, Haines L, Belloir B, McBride D. Sexing chick embryos: a rapid and simple protocol. Br Poult Sci. 2001;42(1):134–8.CrossRefGoogle Scholar
  12. 12.
    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.CrossRefGoogle Scholar
  13. 13.
    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.CrossRefGoogle Scholar
  14. 14.
    Yilmaz-Dikmen B, Dikmen S. A morphometric method of sexing white layer eggs. Braz J Poult Sci. 2013;15(3):203–10.Google Scholar
  15. 15.
    Webster B, Hayes W, Pike TW. Avian egg odour encodes information on embryo sex, fertility and development. Plos One. 2015;10(1):e0116345.CrossRefGoogle Scholar
  16. 16.
    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.CrossRefGoogle Scholar
  17. 17.
    Wei D, Chen S, Liu Q. Review of fluorescence suppression techniques in Raman spectroscopy. Appl Spectrosc Rev. 2015;50(5):387–406.CrossRefGoogle Scholar
  18. 18.
    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.CrossRefGoogle Scholar
  19. 19.
    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.Google Scholar
  20. 20.
    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.CrossRefGoogle Scholar
  21. 21.
    Fridolfsson AK, Ellegren H. A simple and universal method for molecular sexing of non-ratite birds. J Avian Biol. 1999;30(1):116–21.CrossRefGoogle Scholar
  22. 22.
    Schart C. Thesis: Entwiklung einer Steuerung für die Fokussierung und das Tracking embryionaler Blutgefäße im Hühnerei. Hochschule für Technik und Wirtschaft Dresden. 2015.Google Scholar
  23. 23.
    Baumann R, Meuer HJ. Blood-oxygen transport in the early avian embryo. Physiol Rev. 1992;72(4):941–65.Google Scholar
  24. 24.
    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.CrossRefGoogle Scholar
  25. 25.
    Johnston P. Hematocrit values for the chick embryo at various ages. Am J Physiol. 1955;180(2):361–2.Google Scholar
  26. 26.
    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.Google Scholar
  27. 27.
    Berezin MY, Achilefu S. Fluorescence lifetime measurements and biological imaging. Chem Rev. 2010;110(5):2641–84.CrossRefGoogle Scholar
  28. 28.
    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.CrossRefGoogle Scholar
  29. 29.
    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.CrossRefGoogle Scholar
  30. 30.
    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.CrossRefGoogle Scholar
  31. 31.
    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.CrossRefGoogle Scholar
  32. 32.
    Tagirov M, Golovan S. Sexual dimorphism in the early embryogenesis of the chicken (Gallus Gallus domesticus). Mol Reprod Dev. 2015;82(5):332–43.CrossRefGoogle Scholar
  33. 33.
    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.Google Scholar
  34. 34.
    Abdi H, Williams LJ. Principal component analysis. WIREs Comput Stat. 2010;2(4):433–59.CrossRefGoogle Scholar
  35. 35.
    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.CrossRefGoogle Scholar
  36. 36.
    Talari ACS, Movasaghi Z, Rehman S, Rehman I. Raman spectroscopy of biological tissues. Appl Spectrosc Rev. 2015;50(1):46–111.CrossRefGoogle Scholar
  37. 37.
    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.CrossRefGoogle Scholar
  38. 38.
    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.Google Scholar
  39. 39.
    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.CrossRefGoogle Scholar
  40. 40.
    Brereton RG, Lloyd GR. Support vector machines for classification and regression. Analyst. 2010;135(2):230–67.CrossRefGoogle Scholar
  41. 41.
    Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morphol. 1951;88(1):49–92.CrossRefGoogle Scholar
  42. 42.
    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.CrossRefGoogle Scholar
  43. 43.
    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.CrossRefGoogle Scholar
  44. 44.
    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.CrossRefGoogle Scholar
  45. 45.
    Aleksandrowicz E, Herr I. Ethical euthanasia and short-term anesthesia of the chick embryo. Altex-Altern Tierexp. 2015;32(2):143–7.Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Roberta Galli
    • 1
  • Grit Preusse
    • 1
  • Ortrud Uckermann
    • 2
  • Thomas Bartels
    • 3
  • Maria-Elisabeth Krautwald-Junghanns
    • 3
  • Edmund Koch
    • 1
  • Gerald Steiner
    • 1
    • 4
  1. 1.Faculty of Medicine, Anesthesiology and Intensive Care Medicine, Clinical Sensoring and MonitoringTechnische Universität DresdenDresdenGermany
  2. 2.Faculty of Medicine, University Hospital Carl Gustav Carus, NeurosurgeryTechnische Universität DresdenDresdenGermany
  3. 3.Faculty of Veterinary Medicine, Clinic for Birds and ReptilesUniversity of LeipzigLeipzigGermany
  4. 4.Faculty of PhysicsVilnius UniversityVilniusLithuania

Personalised recommendations