Using Chicken Embryos for Teratology Studies

Diane S. Henshel1, Jamie DeWitt1, Andrea Troutman1

1 Indiana University, Bloomington, Indiana
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 13.4
DOI:  10.1002/0471140856.tx1304s14
Online Posting Date:  February, 2003
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This unit describes methods for injecting, incubating, handling and analyzing domestic chicken embryos used in teratology studies. It also includes a discussion of caveats and special handling issues as well as some discussion of statistical analyses that differentiate working with chicken embryos from working with clutches of eggs or litters of pups. As an example of potential data, preliminary data from a study of abnormalities in early embryos and hatchling chicks exposed to chlordane are presented.

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Table of Contents

  • Basic Protocol 1: Egg Injections and Incubation
  • Basic Protocol 2: Embryo Removal and Handling
  • Basic Protocol 3: Handling Hatchlings
  • Support Protocol 1: Sacrificing Chicks
  • Support Protocol 2: Treatment of Organs
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Egg Injections and Incubation

  • Eggs (any commercial or university farm; fertile and free from chemical contamination)
  • Egg cleaning agents (e.g., 20% povidone iodine, see recipe)
  • 70%, 95% and 100% ethanol
  • Injection vehicle (see annotation to step ; also see Critical Parameters)
  • Agent to be injected mixed with the vehicle
  • Uncontaminated vehicle kept separate from injection agent or injection agent vehicle dilution quantity
  • 4% paraformaldehyde or 10% formalin
  • Kimwipes (or other lintless wipes)
  • Melted paraffin and a small paintbrush for sealing injection holes
  • Probe: a small sharp metal object such as an awl or a compass point, sterilized by soaking in 70% alcohol for 15 min, then air dried, for piercing a small hole in the injection site
  • Syringes for injecting (e.g., Hamilton brand 700 series microliter syringes with cemented needles)
  • Circulated air incubator with a 99.5°F dry bulb and 87° to 88°F wet bulb

Basic Protocol 2: Embryo Removal and Handling

  • Hexamethyldisilazane or silane
  • PBS‐azide (see recipe)
  • 10% neutral buffered formalin or 4% paraformaldehyde prepared in pH‐neutral buffer
  • Sharp‐edged file or a diamond‐tipped glass cutter
  • Pasteur pipets
  • Plastic beaker filled with lintless wipes, to hold eggs in fashioned “nest”
  • Curved and pointed forceps (no. 5 with a 45° angle)
  • Small dissecting scissors (e.g., Moria curved, blunt‐tipped scissors, available from most fine instrument suppliers)
  • Pipets, disposable
  • Oval “donuts” made out of filter paper, ∼2‐cm × 1‐2–cm external diameter and 1.5‐cm × 0.5‐cm internal diameter (for 48‐ and 72‐hr embryos only)
  • Metal scooper (normally used for measuring out dry chemicals)
  • Petri dishes or other small containers to hold PBS‐azide
  • Pre‐labeled vials for storing embryos, filled with 10% neutral buffered formalin

Basic Protocol 3: Handling Hatchlings

  • Chicks
  • Sodium pentobarbitol (Somnotol or equivalent; see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.75% avian saline (see recipe)
  • Fixative (heparin; see recipe)
  • Blood collection vial, i.e., heparinized or EDTA‐treated vacutainer tube
  • Scalpel
  • Large shears (e.g., sewing shears)
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Literature Cited

Literature Cited
   Brunstrom, B. 1983. Toxicity in chick embryos of three commercial mixtures of chlorinated paraffins and of toxaphene injected into eggs. Arch. Toxicol. 54:353‐357.
   Brunstrom, B. 1988. Sensitivity of embryos from duck, goose, herring gull, and various chicken breeds to 3,3′,4,4′‐tetrachlorobiphenyl. Poult. Sci. 67:52‐57.
   Brunstrom, B. and Darnerud, B. 1983. Toxicity and distribution in chick embryos of 3,3′4,4′‐tetrachlorobiphenyl injected into the eggs. Toxicology. 27:103‐110.
   Brunstrom, B. and Lund, J. 1988. Differences between chick and turkey embryos in sensitivity to 3,3′4,4′‐tetrachloro‐biphenyl and in concentration/affinity for the hepatic receptor for 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin. Comp. Biochem. Physiol. C Comp. Pharmacol. Toxicol. 91:507‐512.
   Casanova, M., You, L., Gaido, K.W., Archigeque‐Engle, S., Janszen, D.B., and Heck, H.A. 1999. Developmental effects of dietary phytoestrogens in Sprague‐Dawley rats and interactions of genistein and daidzein with rat estrogen receptors alpha and beta in vitro. Toxicol. Sci. 51:236‐244.
   Davies, W.J. and Freeman, S.J. 1995. CHick Embryotoxicity Screening Test (CHEST I and II). Methods Mol. Biol. 43:307‐310.
   Firestone, D. 1973. Etiology of chick edema disease. Environ. Health Perspect. 5:59‐66.
   Fox, L.L. and Grasman, K.A. 1999. Effects of PCB 126 on primary immune organ development in chicken embryos. J. Toxicol. Environ. Health 58:233‐244.
   Grasman, K.A. and Whitacre, L.L. 2001. Effects of PCB 126 on thymocyte surface marker expression and immune organ development in chicken embryos. J. Toxicol. Environ. Health 62:191‐206.
   Hamburger, V. 1992. The stage series of the chick embryo. Dev. Dyn. 195:273‐275.
   Hamburger, V. and Hamilton, V.L. 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88:49‐92.
   Hamburger, V. and Hamilton, V.L. 1992. A series of normal stages in the development of the chick embryo. 1951. Dev. Dyn. 195:231‐272.
   Henshel, D.S. 1993. LD50 and teratogenicity studies of the effects of TCDD on chicken embryos. Soc. Environ. Toxicol. Chem. Abstr. 14:652.
   Henshel, D.S., Hehn, B., Vo, M.T., and Steeves, J.D. 1993. A short‐term test for dioxin teratogenicity using chicken embryos. In ASTM STP #1216: Environmental Toxicology and Risk Assessment, 2nd Volume (W. Landis, J. Hughes, and M. Lewis, eds.) pp. 159‐174. American Society for Testing and Materials, Philadelphia.
   Henshel, D.S., Allen, C.A., Lam, Y., and Benson, K. 1996. A comparison of the early embryo effects of ethanol, 1,1,1‐trichloroethane, atrazine, 2,4‐D, methyl mercury, lead, and 2,3,7,8‐TCDD. Fundam. Appl. Toxicol. 30:197.
   Hoffman, D.J. 1975. Physiological effects of hypoxia and trypan blue in 17‐day chick Embryos. Teratology. 12:57‐60.
   Hoffman, D.J. and Campbell, K.I. 1978. Embryotoxicity of irradiated and nonirradiated automotive exhaust and carbon monoxide. Environ. Res. 15:100‐107.
   Hoffman, D.J. and Ramm, G.M. 1972a. Physiological effects of trypan blue on chick embryos. J. Exp. Zool. 182:227‐232.
   Hoffman, D.J. and Ramm, G.M. 1972b. Effects of hyperoxia on chick embryos injected with trypan blue. Teratology. 5:315‐318.
   Jelinek, R. and Marhan, O. 1994. Validation of the Chick Embryotoxicity Screening Test (CHEST). A comparative study. Funct. Dev. Morph. 4:317‐323.
   Ludwig, J.P., Kurita‐Matsuba, H., Auman, H.J., Ludwig, M.E., Cummer, C.L., Goesy, J.P., Tillitt, D.E., and Jones, P.D. 1996. Deformities, PCBs, and TCDD‐equivalents in double‐crested cormorants (Phalacrocorax auritis) and caspian terns (Hydroprogne caspia) of the upper Great Lakes 1986‐1991: Testing a cause‐effect hypothesis. J. Gt. Lakes Res. 22:172‐197.
   McLaughlin, J. Jr., Marliac, J.P., Verrett, M.J., Mutchler, M.K., and Fitzhugh, O.G. 1963. The injection of chemicals into the yolk sac of fertile eggs prior to incubation as a toxicity test. Toxicol. Appl. Pharmacol. 5:760‐771.
   Noel, S. and Henshel, D.S. 1996. Effects of in ovo exposure to TCDD on heart development in chickens. Independent research study (L490), Department of Biology, Indiana University, Bloomington, IN.
   Powell, D.C., Aulerich, R.J., Meadows, J.C., Tillitt, D.E., Giesy, J.P., Stomberg, K.L., and Bursian, S.J. 1996a. Effects of 3,3′,4,4′,5‐pentachlorobiphenyl (PCB 126) and 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) injected into the yolks of chicken (Gallus domesticus) eggs prior to incubation. Arch. Environ. Contam. Toxicol. 31:404‐409.
   Powell, D.C., Aulerich, R.J., Stromborg, K.L., and Bursian, S.J. 1996b. Effects of 3,3′,4,4′‐tetrachlorobiphenyl, 2,3,3′,4,4′‐pentachlorobiphenyl, and 3,3′,4,4′,5‐pentachlorobiphenyl on the developing chicken embryo when injected prior to incubation. J. Toxicol. Environ. Health 49:319‐338.
   Purves, D. and Lichtman, J.W. 1985. Principles of Neural Development. Sinauer Associates, Inc. Sunderland, MA.
   Romanoff, A.L. 1960. The Avian Embryo: Structural and Functional Development. The Macmillan Company, New York.
   Romanoff, A.L. and Romanoff, A.J. 1972. Pathogenesis of the Avian Embryo; An Analysis of Causes of Malformations and Prenatal Death. Wiley‐Interscience, New York.
   Sanes, J.R. 1992. On the republication of the Hamburger‐Hamilton stage series. Dev. Dyn. 195:229‐230.
   Verrett, M.J. 1976. Investigation of the toxic and teratogenic effects of halogenated dibenzo‐p‐dioxins and dibenzofurans in the developing chicken embryo. In Memorandum Report, U.S. Food and Drug Administration, Washington, D.C.
   Walker, N.E. 1967. Distribution of chemicals injected into fertile eggs and its effect upon apparent toxicity. Toxicol. Appl. Pharmacol. 10:290‐299.
Key References
   See Hamburger and Hamilton, .
  Contains the first comprehensive overview of chicken embryonic development, with both text and photos, detailing anatomical changes from day 0 to day 21 of development.
   Henshel, D.S., Sparks, D.W., Allen, C.A., Benson, K., Fox, C., Lam, Y., Sobiech, S.A., and Wagey, R. 1997. Preliminary results using the early embryo teratogenesis assay: A comparison of early embryo abnormalities with late embryo and hatchling teratogenic changes. In ASTM STP #1317: Environmental Toxicology and Risk Assessment: Modeling and Risk Assessment, 6th Volume (F.J. Dwyer, T.R. Doane, and M.L. Hinman, eds.) pp. 391‐401. American Society for Testing and Materials, Philadelphia.
  Contains an overview of the Early Embryo Assay and its comparison to older embryos and hatchling chicks.
  See Henshel et al., .
  Contains an overview of the Early Embryo Assay using dioxin as a model toxicant.
  See Romanoff,
  Similar to the Hamburger and Hamilton () book, chronicles the development of the chicken embryo. Chapters divided by system.
  See Romanoff and Romanoff,
  This book chronicles the types of effects that may result in chicken embryos exposed to a variety of events and agents.
Internet Resources
  The Web site of the University of Illinois (Champagne‐Urbana) Extension, Incubation and Embryology. This Web site provides a basic primer on chicken eggs, how to incubate them, and how to preserve the embryos. The site is aimed at pre‐college teachers, and so some of the information is not necessarily relevant to a researcher. However, it has some good practical advice on incubation, including a nice troubleshooting page.
  B.R. Speer's, University of California Berkeley, 1995, Web site on the Introduction to the Amniota. This Web site provides a description of the structure of the egg as well as a nice diagram of a basic egg. A simple, but useful “atlas” of the amniote egg.
  M. Hill's, University of New South Wales, Chicken Development, Web site contains a series of lecture notes and figures utilized for a college course on embryological development. Includes an abbreviated table of the Hamburger and Hamilton () stages of chicken embryonic development, as well as references to other atlases.
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