Final Report: Critical Stages in Avian Development: Estrogen Hazards to Altricial and Precocial Birds

EPA Grant Number: R825294
Title: Critical Stages in Avian Development: Estrogen Hazards to Altricial and Precocial Birds
Investigators: Fry, D. Michael , Millam, James R.
Institution: University of California - Davis
EPA Project Officer: Aja, Hayley
Project Period: November 1, 1996 through October 31, 2000
Project Amount: $521,971
RFA: Endocrine Disruptors (1996) RFA Text |  Recipients Lists
Research Category: Endocrine Disruptors , Environmental Justice , Human Health , Safer Chemicals


The overall objective of this research project was to determine the critical exposure periods in the development of precocial and altricial birds for estrogenic disruption of differentiation of the reproductive system and sexual dimorphic areas of the brain.

Specific objectives of the research project were to:

· Determine the differences in critical exposure periods in embryos and neonatal chicks of Japanese Quail and Zebra Finches to estradiol exposure. These studies focused on anatomical changes in gonads, oviducts, and brain nuclei in embryos exposed in ovo, and newly hatched chicks exposed by oral dosing with estradiol. Additionally, the sexually dimorphic nuclei of songbirds were evaluated to compare the neuroanatomical changes with the gonadal changes, and with changes in reproductive behavior.

· Determine the efficacy of xenobiotic estrogens and masculinize the song control nuclei of female Zebra Finches and demasculinize mounting behavior in male Zebra Finches. These studies focused on neurochemical, anatomical, and behavioral differences between adult male and female birds after postnatal exposure to environmental estrogens. The estrogenicity of these compounds was compared to that of estradiol benzoate, which served as a positive control for the gender altering effects.

· Determine the reproductive success of Zebra Finch males and females exposed to estrogenic substances as chicks. These experiments assessed the extent to which exposure to environmental estrogens impaired fecundity, and the correlation of persistent adult effects with anatomical changes demonstrated in hatchling birds exposed in ovo or as neonates. Several components of reproductive performance were assessed, including: incidence of pair formation and egg laying, clutch size, eggshell thickness, fertility, hatchability, fledging success, and incidence and severity of altered gonadal morphology.

· Utilize cell culture systems to determine the sensitivity of hypothalamic slice cultures to sex steroids in the regulation of brain-derived neurotrophic factor levels. This steroid hormone regulation could play a critical role in the modulation of programmed cell death and synaptic maturation during sexual dimorphic development of the avian brain.

Summary/Accomplishments (Outputs/Outcomes):

Throughout our investigation, we discovered that: (1) orally administered estrogens act like parenterally administered estrogens (Quaglino, et al., 1999); (2) posthatch treatment of females produces a dose-response increase in the size of song control nuclei in adult females (Quaglino, et al., 1999) and singing behavior (Erichsen, et al., 1999); (3) posthatch treatment of male finch alters adult reproductive behaviors in a doselike manner, demasculinizing copulatory behavior (males do not mount) and increasing nesting behavior (Erichsen, et al., 1999); (4) the reproductive performance of adults treated as chicks with estrogens is severely impaired in sex and dose-specific manners (reduced egg production; increased incidence of cracked/broken or missing eggs; reduced percentage of candled fertility; reduced number of hatched chicks), (Millam, et al., 2000); (5) treatment with octylphenol increases embryo mortality; (6) posthatch estradiol benzoate (EB)-treatment severely compromises adult reproductive competence through an array of behavioral and physiological changes. It is not a question of whether posthatch estrogen treatment compromises reproduction (it very clearly does); it is rather a matter of dose and time of exposure. The results of our experiments provide answers on where and when to look in birds to detect effects of early estrogen exposure; and (7) in hypothalamic slice cultures, we showed that neuronal activity, induced by glutamate agonists and potassium chloride, can regulate brain-derived neurotrophic factor messenger RNA levels. This regulation could play a critical role in the modulation of programmed cell death and synaptic maturation during development of the hypothalamus. This regulation was not influenced by either acute or chronic treatments with 10 and 100 nM 17b-estradiol or 10 nM testosterone, providing evidence that sex steroids are not regulators of steady-state level of this growth factor in hypothalamic slices in culture.


The results present a consistent and profound picture: posthatch exposure of an altricial passerine to orally administered estrogenic substances can completely disrupt adult reproductive performance. Our results using oral administration are fully consistent with earlier reports using parenterally administered estradiol. Brain nuclei of females are masculinized, resulting in the ability to sing, whereas copulatory behavior of males is demasculinized. Our studies went further than previous studies in assessing the influence of EB on reproductive performance. In our studies, the combination of reduced reproductive performance of males and females led to complete reproductive failure when both sexes were treated as chicks. Estrogen treatment of female finch reduced mean eggs per clutch and increased the incidence of egg breakage, while posthatch estrogen treatment of males reduced fertility. The data suggest that female fertility (in addition to increased egg breakage) also may be affected by estrogen treatment. When both sexes were estrogen-treated, there were significant declines in mean eggs per clutch; candled fertility and significant increases in egg-breakage, leading to a complete reproductive failure: no eggs were hatched. What is not clear is the extent to which the reductions in fertility and hatchability were due to altered reproductive behavior (a failure to mate and/or incubate), reduced gamete viability (e.g., reduced sperm number and/or viability), and/or impaired zygote/embryo viability (e.g., due to eggshell thinning). Our observation of increased eggshell breakage is consistent with impaired embryo viability through eggshell thinning, but disruption of the regularity of the mammillary layer and consequent influence on the palisade layers of the shell (without eggshell thinning) also is a tenable hypothesis to explain these results.

Posthatch estrogen exposure impairs reproduction. It is only a matter of dose. Whether birds would likely be exposed to concentrations of xenobiotic estrogens that would cause such effects is now a separate question. Nonetheless, the reproductive potential of more than 4,000 species of passerines and other altricial avian species could be impaired by posthatch exposure to xenoestrogens.

The dose of octylphenol (OP) could likely be encountered by birds in highly contaminated environments. As a point of reference, the 100 mM dose of OP used represents a daily intake of about 70 to 200 µg OP for growing finch between 3.5 and 9 g body mass. Benni (1999) summarized several reports recording OP concentrations of 200 to 5,000 µg/g in such substrates as raw sewage, STP final effluent, or chemical plant effluent. Servos (1999) reported bioaccumulation and/or bioconcentration factors from one to several hundred, and in the case of certain marine animals, several thousand for alkylphenol and alkylphenol polyethoxylates in a variety of organisms. Although more trophic studies are needed to determine exposure threats in terrestrial environments, it seems reasonable that nestlings fed biota grown in highly contaminated environments could easily encounter the loads of OP used in our studies.

Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other project views: All 14 publications 3 publications in selected types All 3 journal articles
Type Citation Project Document Sources
Journal Article Millam JR, Craig-Veit CB, Quaglino AE, Erichsen AL, Famula TR, Fry DM. Post-hatch oral estrogen exposure impairs adult reproductive performance of Zebra Finch in a sex-specific manner. Hormones and Behavior 2001;40(4):542-549. R825294 (Final)
not available
Journal Article Quaglino AE, Craig-Veit CB, Viant MR, Erichsen AL, Fry DM, Millam JR. Oral estrogen masculinizes female Zebra Finch song system. Hormones and Behavior 2002;41(2):236-241. R825294 (Final)
not available
Journal Article Viant MR, Millam JR, Delany ME, Fry DM. Regulation of brain-derived neurotrophic factor messenger RNA levels in avian hypothalamic slice cultures. Neuroscience 2000;99(2):373-380.
abstract available   full text available
R825294 (Final)
not available

Supplemental Keywords:

toxics, endocrine disruptors, environmental exposure, risk, human health, environmental chemistry, adverse outcomes, animal models, biological effects, estrogen receptors, estrogen response, fertility, industrial chemicals, bird, ecological effects, ecosystem protection, ecological indicators, pesticides, fetal development, xenoestrogen, reproduction, female, in vivo model, male, reproductive processes, cell culture, avian development, chickens, developmental processes, Zebra Finch, Japanese Quail, steroid., RFA, Health, Scientific Discipline, Toxics, Environmental Chemistry, pesticides, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Biochemistry, Children's Health, Biology, Endocrine Disruptors - Human Health, altricial and precocial birds, avian development, xenobiotic, endocrine disrupting chemicals, exposure studies, developmental biology, animal models, Japanese Quail, estrogen response, reproductive processes, brain development, neonatal chicks

Relevant Websites: Exit

Progress and Final Reports:

Original Abstract
  • 1997
  • 1998
  • 1999