In ovo sexing and genotyping using PCR techniques: a contribution to the 3R principles in chicken breeding

in-ovo-sexing-and-genotyping-using-pcr-techniques:-a-contribution-to-the-3r-principles-in-chicken-breeding
In ovo sexing and genotyping using PCR techniques: a contribution to the 3R principles in chicken breeding

References

  1. Bahr, J. M. in Sourcebook of Models for Biomedical Research (ed P. Michael Conn) 161–167Humana Press, (2008).

  2. Beacon, T. H. & Davie, J. R. The chicken model organism for epigenomic research. Genome 64, 476–489. https://doi.org/10.1139/gen-2020-0129 (2021).

    Google Scholar 

  3. Kaplan-Arabaci, O., Dančišinová, Z. & Paulsen, R. E. The Chicken Embryo: An Alternative Animal Model in Development and Disease. Heliyon (2024). https://doi.org/10.2139/ssrn.4724622

  4. Burt, D. W. Emergence of the chicken as a model organism: implications for agriculture and biology. Poult. Sci. 86, 1460–1471. https://doi.org/10.1093/ps/86.7.1460 (2007).

    Google Scholar 

  5. Stern, C. D. The chick; a great model system becomes even greater. Dev. Cell. 8, 9–17. https://doi.org/10.1016/j.devcel.2004.11.018 (2005).

    Google Scholar 

  6. Sarnella, A. et al. The Chicken Embryo: An Old but Promising Model for In Vivo Preclinical Research. Biomedicines 12 (2024). https://doi.org/10.3390/biomedicines12122835

  7. Davey, M. G. & Tickle, C. The chicken as a model for embryonic development. Cytogenet. Genome Res. 117, 231–239. https://doi.org/10.1159/000103184 (2007).

    Google Scholar 

  8. Gruber, F. P. & Hartung, T. Alternatives to animal experimentation in basic research. ALTEX (2004).

  9. Davey, M. G. M., Mike, J., Holmes & Tana A Scientific Case for Revisiting the Embryonic Chicken Model in Biomedical Research. SSRN (2024). https://doi.org/10.2139/ssrn.5012603

  10. Weiss, L. et al. Nocicepton in chicken embryos, Part I: Analysis of cardiovascular responses to a mechanical noxious stimulus. Anim. (Basel). 13. https://doi.org/10.1101/2023.04.14.536899 (2023).

  11. Kollmansperger, S. et al. Nociception in chicken embryos, Part II: Embryonal development of electroencephalic neuronal activity in ovo as a prerequisite for nociception. bioRxiv https://doi.org/10.1101/2023.04.14.536947 (2023).

    Google Scholar 

  12. Süß, S. C. et al. Nociception in chicken embryos, Part III: Analysis of movements before and after application of a noxious stimulus. Animals 13 https://doi.org/10.1101/2023.04.20.537674 (2023).

  13. Bruijnis, M. R. N., Blok, V., Stassen, E. N. & Gremmen, H. G. J. Moral Lock-In in Responsible Innovation: The Ethical and Social Aspects of Killing Day-Old Chicks and Its Alternatives. J. Agric. Environ. Ethics. 28, 939–960. https://doi.org/10.1007/s10806-015-9566-7 (2015).

    Google Scholar 

  14. Corion, M. et al. Trends in in ovo sexing technologies: insights and interpretation from papers and patents. J. Anim. Sci. Biotechnol. 14, 102. https://doi.org/10.1186/s40104-023-00898-1 (2023).

    Google Scholar 

  15. Xu, S. et al. Egg characteristics assessment as an enabler for in-ovo sexing technology: A review. Biosyst. Eng. 249, 41–57. https://doi.org/10.1016/j.biosystemseng.2024.11.008 (2025).

    Google Scholar 

  16. Di Concetto, A., Morice, O. & Corion, M. & Monteiro Belo dos Santos, S. Chick and Duckling Killing: Achieving an EU-Wide Prohibition (European Institute for Animal Law & Policy, 2023).

  17. Code rurale de la pêche maritime; Partie réglemetaire. Journal officiel de la République française, Livre, I. I. & Chapitre, I. V. Section  2, Sous-Sect.  1, R214-217 (2022).

  18. Code rurale de la pêche maritime; Partie réglementaire Journal officiel de la République française, Livre, I. I. & Chapitre, I. V. Section  4, Sous-Sect.  3, R214-278 (modifié par Décret n°2022-2137-Art.2021) (2022).

  19. Animals (Scientific Procedures) Act. London Gazette, c. 14 (2013). (1986).

  20. (German Animal Welfare Act & Tierschutzgesetz ). BGBl. I, S 1826, § 4c (2021).

  21. Animal Welfare Regulation Governing Experimental Animals (TierSchVerV). BGBl. I S. 1308, Sect.  2, § 14. (2024).

  22. Bacon, L. D., Hunt, H. D. & Cheng, H. H. A review of the development of chicken lines to resolve genes determining resistance to diseases. Poult. Sci. 79, 1082–1093. https://doi.org/10.1093/ps/79.8.1082 (2000).

    Google Scholar 

  23. Ballantyne, M. et al. Direct allele introgression into pure chicken breeds using Sire Dam Surrogate (SDS) mating. Nat. Commun. 12, 659. https://doi.org/10.1038/s41467-020-20812-x (2021).

    Google Scholar 

  24. Lengyel, K. et al. Unveiling the critical role of androgen receptor signaling in avian sexual development. Nat. Commun. 15, 8970. https://doi.org/10.1038/s41467-024-52989-w (2024).

    Google Scholar 

  25. Jansen, S. et al. Relationship between Bone Stability and Egg Production in Genetically Divergent Chicken Layer Lines. Anim. (Basel). 10. https://doi.org/10.3390/ani10050850 (2020).

  26. Henderson, L., Okuzaki, Y., Marcelle, C., McGrew, M. J. & Nishijima, K. I. Avian bioresources for developmental biology: Chicken and quail resources in the United Kingdom, France, and Japan. Dev. Biol. 521, 1–13. https://doi.org/10.1016/j.ydbio.2025.02.001 (2025).

    Google Scholar 

  27. Milchevskaya, V. et al. Group size planning for breedings of gene-modified mice and other organisms following Mendelian inheritance. Lab. Anim. (NY). 52, 183–188. https://doi.org/10.1038/s41684-023-01213-1 (2023).

    Google Scholar 

  28. Buch, T. et al. Reducing surplus experimental animal generation. Lab. Anim. 56, 305–305. https://doi.org/10.1177/00236772221096054 (2022).

    Google Scholar 

  29. Wewetzer, H., Wagenknecht, T., Bert, B. & Schonfelder, G. The fate of surplus laboratory animals: Minimizing the production of surplus animals has greatest potential to reduce the number of laboratory animals. EMBO Rep. 24, e56551. https://doi.org/10.15252/embr.202256551 (2023).

    Google Scholar 

  30. Morinha, F. et al. High-resolution melting analysis for bird sexing: a successful approach to molecular sex identification using different biological samples. Mol. Ecol. Resour. 13, 473–483. https://doi.org/10.1111/1755-0998.12081 (2013).

    Google Scholar 

  31. Cordeiro, C. D. et al. Fast, accurate, and cost-effective poultry sex genotyping using real-time polymerase chain reaction. Front. Vet. Sci. 10, 1196755. https://doi.org/10.3389/fvets.2023.1196755 (2023).

    Google Scholar 

  32. He, L. et al. Simple, sensitive and robust chicken specific sexing assays, compliant with large scale analysis. PLoS One. 14, e0213033. https://doi.org/10.1371/journal.pone.0213033 (2019).

    Google Scholar 

  33. Rosenthal, N. F. et al. High-throughput applicable genomic sex typing of chicken by TaqMan real-time quantitative polymerase chain reaction. Poult. Sci. 89, 1451–1456. https://doi.org/10.3382/ps.2010-00638 (2010).

    Google Scholar 

  34. Clinton, M., Haines, L., Belloir, B. & McBride, D. Sexing chick embryos: a rapid and simple protocol. Br. Poult. Sci. 42, 134–138. https://doi.org/10.1080/713655025 (2001).

    Google Scholar 

  35. Jensen, T., Mace, M. & Durrant, B. Sexing of mid-incubation avian embryos as a management tool for zoological breeding programs. Zoo Biol. 31, 694–704. https://doi.org/10.1002/zoo.20433 (2012).

    Google Scholar 

  36. V.D. Hofstadt, M. et al. Molecular sexing of chick embryos by LAMP and RPA assays: a step toward in ovo egg sexing. PREPRINT (Version 1) available Res. Square. https://doi.org/10.21203/rs.3.rs-5772672/v1 (2025).

    Google Scholar 

  37. Weissmann, A., Reitemeier, S., Hahn, A., Gottschalk, J. & Einspanier, A. Sexing domestic chicken before hatch: a new method for in ovo gender identification. Theriogenology 80, 199–205. https://doi.org/10.1016/j.theriogenology.2013.04.014 (2013).

    Google Scholar 

  38. Weissmann, A. et al. In ovo-gender identification in laying hen hybrids: Effects on hatching and production performance. Europ Poult. Sci. https://doi.org/10.1399/eps.2014.25 (2014).

    Google Scholar 

  39. Turkyilmaz, M. K., Karagenc, L. & Fidan, E. Sexing of newly-hatched chicks using DNA isolated from chorio-allantoic membrane samples by polymerase chain reaction in Denizli chicken. Br. Poult. Sci. 51, 525–529. https://doi.org/10.1080/00071668.2010.502521 (2010).

    Google Scholar 

  40. Seleggt. https://www.seleggt.com/

  41. PLANTegg. Accessed October 28, (2025). https://www.plantegg.de/

  42. Monteiro Belo Santos, S., Corion, M., De Ketelaere, B., Lammertyn, J. & Spasic, D. Allantoic Fluid-Based qPCR for Early Onset In Ovo Sexing. J. Agric. Food Chem. https://doi.org/10.1021/acs.jafc.3c09418 (2024).

    Google Scholar 

  43. Respeggt. Accessed October 28, (2025).

  44. Jia, N. et al. A Review of Key Techniques for in Ovo Sexing of Chicken Eggs. Agriculture 13 https://doi.org/10.3390/agriculture13030677 (2023).

  45. Xie, C., Tang, W. & Yang, C. A review of the recent advances for the in ovo sexing of chicken embryos using optical sensing techniques. Poult. Sci. 102, 102906. https://doi.org/10.1016/j.psj.2023.102906 (2023).

    Google Scholar 

  46. Woodcock, M. E. et al. Reviving rare chicken breeds using genetically engineered sterility in surrogate host birds. Proc. Natl. Acad. Sci. U. S. A. 116, 20930–20937 (2019). https://doi.org/10.1073/pnas.1906316116

  47. Panda, S. K. & McGrew, M. J. Genome editing of avian species: implications for animal use and welfare. Lab. Anim. 56, 50–59. https://doi.org/10.1177/0023677221998400 (2022).

    Google Scholar 

  48. Percie du Sert. Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0. PLoS Biol. 18, e3000411. https://doi.org/10.1371/journal.pbio.3000411 (2020).

    Google Scholar 

  49. Percie du Sert. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol. 18, e3000410. https://doi.org/10.1371/journal.pbio.3000410 (2020).

    Google Scholar 

  50. Mangir, N., Dikici, S., Claeyssens, F. & MacNeil, S. Using ex Ovo Chick Chorioallantoic Membrane (CAM) Assay To Evaluate the Biocompatibility and Angiogenic Response to Biomaterials. ACS Biomater. Sci. Eng. 5, 3190–3200. https://doi.org/10.1021/acsbiomaterials.9b00172 (2019).

    Google Scholar 

  51. Cloney, K. & Franz-Odendaal, T. A. Optimized ex-ovo culturing of chick embryos to advanced stages of development. J. Vis. Exp. 52129 https://doi.org/10.3791/52129 (2015).

  52. Hamburger & Hamilton A series of normal stages in the development of the chick embryo. J Morphol 88(1): 49–92. (1951).

  53. Hosono, S. et al. Unbiased whole-genome amplification directly from clinical samples. Genome Res. 13, 954–964. https://doi.org/10.1101/gr.816903 (2003).

    Google Scholar 

  54. Dean, F. B. et al. Comprehensive human genome amplification using multiple displacement amplification. Proceedings of the National Academy of Sciences 99, 5261–5266 (2002). https://doi.org/10.1073/pnas.082089499

  55. Suriyaphol, G., Kunnasut, N., Sirisawadi, S., Wanasawaeng, W. & Dhitavat, S. Evaluation of dried blood spot collection paper blotters for avian sexing by direct PCR. Br. Poult. Sci. 55, 321–328. https://doi.org/10.1080/00071668.2014.925087 (2014).

    Google Scholar 

  56. Bailes, S. M., Devers, J. J., Kirby, J. D. & Rhoads, D. D. An Inexpensive, Simple Protocol for DNA Isolation from Blood for High-Throughput Genotyping by Polymerase Chain Reaction or Restriction Endonuclease Digestion. Poult. Sci. 86, 102–106. https://doi.org/10.1093/ps/86.1.102 (2007).

    Google Scholar 

  57. Dierks, C., Altgilbers, S., Weigend, A., Preisinger, R. & Weigend, S. Sexing assay for chickens and other birds for large-scale application based on a conserved sequence variant in CHD1 genes on W and Z chromosomes. Anim. Genet. 53, 235–237. https://doi.org/10.1111/age.13176 (2022).

    Google Scholar 

  58. Fridolfsson, A. K. & Ellegren, H. A simple and universal method for molecular sexing of non-ratite birds. J. Avian Biol. 30, 116–121 (1999).

    Google Scholar 

  59. Wragg, D. et al. Endogenous retrovirus EAV-HP linked to blue egg phenotype in Mapuche fowl. PLoS One. 8, e71393. https://doi.org/10.1371/journal.pone.0071393 (2013).

    Google Scholar 

  60. Altgilbers, S., Dierks, C., Klein, S., Weigend, S. & Kues, W. A. Quantitative analysis of CRISPR/Cas9-mediated provirus deletion in blue egg layer chicken PGCs by digital PCR. Sci. Rep. 12, 15587. https://doi.org/10.1038/s41598-022-19861-7 (2022).

    Google Scholar 

  61. Tona, K. et al. Effects of egg storage time on spread of hatch, chick quality, and chick juvenile growth. Poult. Sci. 82, 736–741. https://doi.org/10.1093/ps/82.5.736 (2003).

    Google Scholar 

  62. Boerjan, M. L. in Avian and Poultry Biology Reviews.4 edn 237–238.

  63. Chen, L. et al. Dynamic 3D morphology of chick embryos and allantois depicted nondestructively by 3.0T clinical magnetic resonance imaging. Poult. Sci. 102, 102902. https://doi.org/10.1016/j.psj.2023.102902 (2023).

    Google Scholar 

  64. Baggott, G. K. Development of Extra-embryonic Membranes and Fluid Compartments. Avian Biol. Res. 2, 21–26 (2009).

    Google Scholar 

  65. Simkiss, K. Water and Ionic Fluxes inside the Egg. Am. Zool. 20, 385–393 (1980).

    Google Scholar 

  66. Everaert, N., Willemsen, H., Willems, E., Franssens, L. & Decuypere, E. Acid-base regulation during embryonic development in amniotes, with particular reference to birds. Respir Physiol. Neurobiol. 178, 118–128. https://doi.org/10.1016/j.resp.2011.04.023 (2011).

    Google Scholar 

  67. Rideout, B. A. Investigating embryo deaths and hatching failure. Vet. Clin. North. Am. Exot Anim. Pract. 15, 155–162. https://doi.org/10.1016/j.cvex.2012.02.005 (2012).

    Google Scholar 

  68. Romanoff, A. L. Critical Periods and Causes of Death in Avian Embryonic Development. Auk 66, 264–270. https://doi.org/10.2307/4080357 (1949).

    Google Scholar 

  69. Martinez-Rodero, I. et al. Blastocoel fluid aspiration improves vitrification outcomes and produces similar sexing results of in vitro-produced cattle embryos compared to microblade biopsy. Theriogenology 218, 142–152. https://doi.org/10.1016/j.theriogenology.2024.01.042 (2024).

    Google Scholar 

  70. Powers, L. & Huntersville, N. (2021).

  71. Zendehboudi, M. & Vesal, N. Comparison of cardiopulmonary effects of propofol, ketamine-propofol and isoflurane anesthesia in the domestic chicken (Gallus gallus domesticus). Vet. Anaesth. Analg. 51, 449–457. https://doi.org/10.1016/j.vaa.2024.06.005 (2024).

    Google Scholar 

  72. Horr, M., Sommerfeld, S., Silva, M. V. & Fonseca, B. B. A fast and simple protocol to anaesthesia in chicken embryos. Exp. Anim. 72, 294–301. https://doi.org/10.1538/expanim.22-0133 (2023).

    Google Scholar 

  73. Zumbrink, L., Brenig, B., Foerster, A., Hurlin, J. & Wenzlawowicz, M. v. Electrical anaesthesia of male chicken embryos in the second third of the incubation period in compliance with animal welfare. Eur. Poult. Sci. 84, 1–11. https://doi.org/10.1399/eps.2020.315 (2020).

    Google Scholar 

  74. Aleksandrowicz, E. & Herr, I. Ethical euthanasia and short-term anesthesia of the chick embryo. ALTEX 32, 143–147. https://doi.org/10.14573/altex.1410031 (2015).

    Google Scholar 

  75. Hatt, J. M., Kreyenbuhl, K. & Kummrow, M. [Methods of analgesia and euthanasia in backyard poultry]. Schweiz. Arch. Tierheilkd. 165, 503–511. https://doi.org/10.17236/sat00398 (2023).

    Google Scholar 

  76. Schlegel, L., Kleine, A. S., Doherr, M. G. & Fischer-Tenhagen, C. How to see stress in chickens: On the way to a Stressed Chicken Scale. Poult. Sci. 103, 103875. https://doi.org/10.1016/j.psj.2024.103875 (2024).

    Google Scholar 

  77. Dierks, C., Simianer, N. T. H. H., Cavero, D. & Preisinger, R. S. Weigend. in 70th Annual Meeting of the European Federation of Animal Science, 26–30 August. (ed Scientific Committee). (ed Scientific Committee). (2019).

  78. Dierks, C., Simianer, N. T. H. H., Andersson, B., Cavero, D. & Preisinger, R. S. Weigend. in XI European Symposium on Poultry Genetics, 23–25 October 2019. (ed S. Weigend P. Trefil).

Download references