In ovo exposure quail assay for risk assessment of endocrine disrupting chemicals

Although there are in vivo assays using various organisms for the risk assessment of chemicals with endocrine disrupting properties, effective experimental methods for avian species are still under debate. We have developed an in ovo exposure assay using Japanese quail eggs, aimed at assessing disrupting effects on avian reproductive development and function. Hybrid eggs from Brazilian Brown male and White Egg female quails, which can be genetically sexed by their plumage color after hatching, were prepared, and test materials dissolved in olive oil were injected into the air-chamber on day 10 of incubation. After sexual maturation of hatched chicks, we observed egg production by females and the egg quality and male-typical reproductive behavior, and then examined reproductive system morphology and serum steroid concentrations in both sexes. Treatment with a synthetic estrogen, diethylstilbestrol (DES, 0.5–50 ng/g egg), dose-dependently reduced the eggshell thickness and strength of eggs. A few females treated with 5 ng/g DES per egg produced soft-shelled/unmarked eggs, and all laying females treated with 50 ng/g egg produced eggs completely lacking shells. DES also induced shortening of the left oviduct and abnormal development of the right oviduct in a dose-dependent manner, while testis weight was reduced symmetrically. In addition, 2,2′,4′,6′-tetrachlorobiphenyl-4-ol (10–1,000 ng/g egg), which previously showed relatively high estrogenic activity in vitro, caused dose-dependent shortening of the left oviduct and reduction in testis weight. The methods for evaluating endocrine disrupting effects and preparing experimental birds proposed in the present study are expected to facilitate assays for avian reproductive toxicology.     

Mechanisms of estrogen-induced effects in avian reproduction caused by transovarian application of a xenoestrogen,diethylstilbestrol

To clarify breeding failure in avian species caused by the estrogenicity of chemicals, alterations in the reproductive systems of Japanese quail exposed in ovo to a xenoestrogen were investigated. An injection of diethylstilbestrol (DES) into the yolk before incubation decreased, after sexual maturation, egg-laying performance of female quails, which accompanied inducing abnormal development of the oviducts. All females treated with 50 ng DES/g of egg did not lay eggs, while 0.5–5 ng DES/g reduced egg weight and eggshell strength and thickness. In the uterus (shell gland), the mRNAs for calcium regulating factors, osteopontin and calbindin D28 K, were reduced dose-dependently by DES. Scanning electron microscopy showed that shell thinning was pronounced in the mammillary and cuticular layers of the eggshell, regions where osteopontin proteins are reportedly located. These indicate that transovarian exposure to xenoestrogens causes malformation and dysfunction of the oviducts, where calcium regulating molecules could play key roles in eggshell thinning.     

Distribution of bisphenol A and tetrabromobisphenol A in quail eggs, embryos and laying birds and studies on reproduction variables in adults following in ovo exposure

In a previous study, we showed that bisphenol A (BPA) had oestrogen-like effects in bird embryos, causing malformations of the oviducts in Japanese quail (Coturnix japonica) and feminisation of the left testis in chicken (Gallus domesticus). In this study, uptake and distribution of BPA and tetrabromobisphenol A (TBBPA) in embryos and laying quail were examined as well as variables related to reproduction in adult quail following administration of the compounds into the yolk of embryonated eggs. The uptake of radiolabelled BPA, TBBPA and the reference compound diethylstilboestrol (DES) was studied in the embryos using beta-spectrometry. Autoradiography was employed to examine distribution in egg and embryo after yolk sac injection of BPA or TBBPA and in laying birds, following intravenous and oral administration. Following embryonic exposure to BPA or TBBPA, sexually mature male birds were examined for reproductive behaviour and testis morphology, and females were examined for egg laying and oviduct morphology. Neither BPA (200 microg/g egg) nor TBBPA (15 microg/g egg) caused any significant oestrogen-like effects on the variables studied, although effects on the female oviducts after BPA exposure were indicated. Embryonic exposure to DES is known to cause profound effects on male sexual behaviour and female oviduct morphology at doses 3-5 orders of magnitude lower than the BPA and TBBPA doses used in the present study. The proportions of BPA and TBBPA taken up by the embryos after yolk sac injection were similar to the proportion of DES taken up. Differences in bioavailability, therefore do not account for any major part of the potency differences between DES and the two bisphenol A compounds. The concentration of radioactivity in the embryo, as revealed by autoradiography, was low compared with that in the yolk at all stages studied (days 6, 10 and 15). Pronounced labelling of the bile and the allantoic fluid was observed, however, indicating that both compounds were readily metabolised and excreted. Radiolabelled BPA and TBBPA administered to laying quail were largely excreted via the bile and 9 days after oral dosing, only small amounts of the labelled compound remained within the body. Maternal transfer of labelled BPA and TBBPA to the egg was low.     

Endocrine disrupting effects of low dose 17 β-estradiol (E2) on the Japanese quail (Coturnix japonica) were detected by modified one-generation reproduction study

Previously, we investigated endocrine disrupting effects of 17 β-estradiol (E(2)) on Japanese quail (Coturnix japonica) in the avian reproduction test according to the testing guidelines, in which new endpoints such as blood vitellogenin (VTG) concentration in parent quails and pathology of F(1) chicks were added, and consequently these additional endpoints suggested to be sensitive markers for detecting any impacts of endocrine disrupting effects (Shibuya et al., 2005b). In the present study, to investigate low dose effects of estrogenic endocrine disrupting chemicals in birds, the avian reproduction study of E(2) at low dose levels was conducted using Japanese quail with additional endpoints such as observations of F(1) chicks until 10 weeks of age, histopathology of F(1) chicks at 14 days and 10 weeks of age and blood VTG concentration in parent quails. Sixteen pairs of 10-week-old quails were fed a low phytoestrogen diet containing E(2) at 0 (control), 0.3, 3, and 30 ppm for 6 weeks, and parent quails, eggs and offspring were examined. F(1) chicks were maintained up to 14 days or 10 weeks of age. Serum E(2) and VTG concentrations in males of the E(2) 3- and 30-ppm groups and in females of the E(2) 30-ppm groups were significantly elevated. In the E(2) 30-ppm group, two parent females died, and toxic changes such as suppression of body weight gain, decrease in food consumption and atrophic and degenerative changes of the reproductive organs were observed in parent quails. In the same group, the number of eggs laid and the fertility rate of eggs were significantly decreased. In addition, the viability of F(1) chicks in the E(2) 30-ppm group were significantly decreased at 10 weeks of age. On the other hand, no abnormalities described above were observed in any parent quails, eggs and F(1) chicks in the E(2) 3- and 0.3-ppm groups, although the fertility rates of eggs in both groups were decreased and the body weight gain of F(1) females in the E(2) 3-ppm group was significantly suppressed. In the histopathological examination of F(1) chicks maintained up to 10 weeks of age, persistent right oviduct and atrophy of the oviduct gland were observed in females of E(2)-treatment groups with significantly high incidences. Moreover, cystic dilatation and tubular degeneration of the seminiferous tubules and atrophy of the cloacal gland were also observed in males of the E(2)-treatment groups. Thus, the dietary treatment of low dose E(2) (even 0.3 ppm) to parent quails resulted in decreased viability and induction of abnormalities in the oviduct, testis and cloacal gland in F(1) chicks maintained up to 10 weeks of age. These results suggest that additional endpoints such as observations of F(1) chicks until 10 weeks of age, histopathology of F(1) chicks at 14 days and 10 weeks of age and blood VTG concentration in parent quails would be useful and sensitive endpoints for evaluating estrogenic endocrine disrupting effects in the avian reproduction study.     

CHANGES IN EGG COMPOSITION OF AMERICAN KESTRELS EXPOSED TO DIETARY POLYCHLORINATED BIPHENYLS

Changes in the quality of eggs of birds exposed to polychlorinated biphenyls (PCBs) have been described, but have never been directly attributed to PCBs. Polychlorinated biphenyl residues in eggs have been associated with reduced reproductive success and embryonic deformities in wild birds. Egg size and composition, specifically the amount of albumen, yolk, and water in an egg, also influence the growth and viability of embryos and hatchlings, and consequently the reproductive success of birds. To deter mine whether PCB exposure of adult birds affected the size and composition of their eggs, 25 pairs of captive American kestrels (Falco sparverius) were fed a mixture of PCB-spiked (1248:1254:1260) food to give an approximate exposure of 7 mg/kg body weight/d, beginning 1 mo prior to pairing, and continuing throughout the courtship, egg-laying, and incubation periods. This dietary level in the adult female kestrels resulted in mean total PCB residues in the eggs of 34.1 µg/g wet weight (geometric mean), which is environmentally relevant. PCB residues in eggs increased with the time of female exposure to the contaminated diet and laying date. Variation in egg size within PCB clutches was significantly greater than within control clutches, although absolute egg mass and volume did not differ markedly by treatment. Only infertile eggs and only one egg per clutch were used for egg composition analysis. Yolks in the PCB-contaminated eggs were heavier, with less wet and dry albumen relative to control eggs. Water content and eggshell thickness were not significantly affected by PCB exposure. These results suggest that eggs from the PCB treatment have relatively more lipid and less protein available for embryonic development. Changes in egg composition were not associated with egg size, lay date, ambient temperature, humidity, or precipitation, which are factors known to affect these variables in bird eggs. The PCB-induced changes in egg composition described here provide insight into possible mechanisms contributing to reduced reproductive performance in wild birds exposed to PCBs.     

Changes in egg composition of American kestrels exposed to dietary polychlorinated biphenyls

Changes in the quality of eggs of birds exposed to polychlorinated biphenyls (PCBs) have been described, but have never been directly attributed to PCBs. Polychlorinated biphenyl residues in eggs have been associated with reduced reproductive success and embryonic deformities in wild birds. Egg size and composition, specifically the amount of albumen, yolk, and water in an egg, also influence the growth and viability of embryos and hatchlings, and consequently the reproductive success of birds. To deter mine whether PCB exposure of adult birds affected the size and composition of their eggs, 25 pairs of captive American kestrels (Falco sparverius) were fed a mixture of PCB-spiked (1248:1254:1260) food to give an approximate exposure of 7 mg/kg body weight/d, beginning 1 mo prior to pairing, and continuing throughout the courtship, egg-laying, and incubation periods. This dietary level in the adult female kestrels resulted in mean total PCB residues in the eggs of 34.1 microg/g wet weight (geometric mean), which is environmentally relevant. PCB residues in eggs increased with the time of female exposure to the contaminated diet and laying date. Variation in egg size within PCB clutches was significantly greater than within control clutches, although absolute egg mass and volume did not differ markedly by treatment. Only infertile eggs and only one egg per clutch were used for egg composition analysis. Yolks in the PCB-contaminated eggs were heavier, with less wet and dry albumen relative to control eggs. Water content and eggshell thickness were not significantly affected by PCB exposure. These results suggest that eggs from the PCB treatment have relatively more lipid and less protein available for embryonic development. Changes in egg composition were not associated with egg size, lay date, ambient temperature, humidity, or precipitation, which are factors known to affect these variables in bird eggs. The PCB-induced changes in egg composition described here provide insight into possible mechanisms contributing to reduced reproductive performance in wild birds exposed to PCBs.     

Photoperiods and age as factors modifying the effects of kepone in Japanese quail

Immature Japanese quail (Coturnix coturnix japonica) of both sexes fed 200 ppm of the insecticide Kepone, when exposed to 16 hr of light per day, had significantly enlarged reproductive organs, livers and adrenal glands. After a reduction in the photoperiod high mortality occurred in only the birds previously fed Kepone. Administration of Kepone to adult females while egg-laying did not affect the weight of reproductive organs or livers. In adult males of the same age testes and livers were greatly enlarged. In both sexes of adult birds, the adrenals were enlarged. Subsequent exposure to reduced photoperiod of the Kepone-fed adult quails of both sexes produced no mortality. In males fed Kepone, testicular regression was recorded much earlier than in controls. Previous ingestion of Kepone affected neither the cessation of egg-laying nor the regression of the ovary. Kepone exerted its effects primarily on the immature quail of both sexes and to a lesser degree on adults. An estrogenic action of Kepone was indicated by increased oviduct weight of young females on a photoperiod of 6 hr light and 18 hr dark per day. In adult males the major estrogenic effect was increased liver weight.     

Response of Japanese quail fed seed meal from sunflowers grown on a municipal sludge-amended soil: elevation of cadmium in tissues

Sunflowers were grown on soil amended with 224 metric tons/ha of municipal sewage sludge from Syracuse, N.Y. The yield of sunflower seeds was reduced by 47.2% by the sludge addition. The harvested seeds contained 1.71 ppm dry weight of cadmium. Deoiled seed meal was incorporated as 25 and 50% of semipurified diet and fed to male and female Japanese quail. The concentrations of cadmium were higher in kidney, liver, muscle, and eggs of birds fed the sludge-grown seed meal as compared to control quail. Tissue concentrations of cadmium increased with increasing dietary levels of sludge-grown seed meal. No significant differences (p greater than 0.05) were observed between dietary treatments in the activity of hepatic microsomal p-nitroanisole O-demethylase or aminopyrine N-demethylase in the male birds. Additionally, no mutagenic activity, either direct or with metabolic activation, was found in quail eggs. No observable changes in tissue ultrastructure were observed under electron microscopy in any of the treatment groups. There were no significant (p greater than 0.05) differences among the dietary treatment groups in feed intake, growth rate, egg production, or egg hatchability.     

Pathological effects of formalin (37% formaldehyde) feeding in female Japanese quails (Coturnix coturnix japonica)

The aim of this study was to determine the pathological effects of formalin on female quails. Seventy-five 1-day-old female Japanese quails (Coturnix coturnix japonina) were divided into five equal groups (A-E) and fed formalin daily at dose levels of 20, 10, 5, 2.5 and 0 mL/ kg feed, respectively. All birds were slaughtered at the end of the experiment -- the eighth week. No clinical signs were observed in quails fed 2.5 mL formalin/kg feed. Depression, dullness and anorexia were prominent in quails fed 20 mL formalin/kg feed. Feed intake, body weight, egg production and egg weight together with absolute and relative weight of organs, erythrocyte and leukocyte counts, hemoglobin concentration and hematocrit were decreased at higher doses, i.e., 10 and 20 mL formalin/kg feed. Total serum proteins and globulin significantly increased (P <0.05) in all formalin fed quails compared to control birds. No gross lesions were observed in the 2.5 mL formalin/kg fed group. At higher doses (10 and 20 mL formalin/kg feed) hemorrhages on the thigh muscles, decreased weight and reduction in area and folds of different segments of oviduct were recorded. Reduced size of liver, heart and kidneys was recorded in quails fed 20mL formalin/kg feed compared to controls and other birds. The histopathological changes in oviduct consisted of degeneration of mucosal glands characterized by vacuolation of nuclei of cells. It was concluded from the study that formalin feeding to female quails at 2.5 mL/kg feed is without harmful effects, however, higher doses are not without health risks.     

Effects of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) exposure on reproduction and hatchling development in Northern bobwhite quail

Adult Northern bobwhite quail (Colinus virginianus) were exposed via food to octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), an energetic compound found in soils at military training installations. Depuration of HMX into eggs was examined in an initial study, and effects on egg production, hatching, growth, development, and survival of chicks were examined in a follow-up study. HMX was readily and rapidly transferred from female quail into eggs. Marked weight loss was observed in quail exposed to 125 and 250 mg/kg HMX in food, likely due to reductions in food intake rather than a toxic mechanism. In the second study, significant alterations in body mass occurred among quail at concentrations >52.5 +/- 9.3 mg/kg but not at 12.3 +/- 1.1 mg/kg in food. Treatment-related reductions in food consumption and decreases in egg laying rates were observed. No HMX-related effects were found in chick growth or survival. Quail inhabiting HMX-contaminated sites could possibly be exposed to HMX and therefore deposition of HMX into eggs is also possible. However, results of these studies further suggest that the potential for reproductive toxicity of HMX to birds is low.     

Reassessment of ecotoxicities of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane in Japanese quail under the OECD draft new avian one-generation reproduction test guideline

Ecotoxicological hazards of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p'-DDT) were investigated by a one-generation reproduction study using Japanese quail (Coturnix coturnix japonica) under an Organization for Economic Co-operation and Development (OECD) draft new test guideline 206 following acute and subchronic toxicity studies. In the subchronic feeding toxicity study, tremors, convulsions, and deaths were observed with a clear sex difference, males being more susceptible than females. The estimated total number of sperm tended to decrease in a dose-dependent manner at the end of 6-week treatment. In the one-generation reproduction study conducted at dose levels of 0, 6, 30, and 150 ppm, the estimated total number of sperm tended to decrease in a dose-dependent manner with a significant difference at 150 ppm. Tremors were observed in the majority of hatchlings in the 150 ppm group and at lower incidences in the 30 ppm group. Significantly high mortality rate in chicks persisted from treatment week 3-6 in the 150 ppm group and at treatment weeks 4 and 5 in the 30 ppm group. Despite of these severe adverse effects of p,p'-DDT on hatchlings and chicks, fertilization, egg laying, eggshell thickness or embryonic development was hardly impaired by p,p'-DDT or its metabolites. From these results, it appears that the OECD draft new avian one-generation reproduction test guideline is effective for ecological hazard assessment of chemicals.     

The Chinese rare minnow (Gobiocypris rarus) as an in vivo model for endocrine disruption in freshwater teleosts: a full life-cycle test with diethylstilbestrol

Chinese rare minnow (Gobiocypris rarus), a freshwater teleost, was exposed to diethylstilbestrol (DES) at 0.05, 0.5, 1 and 5 microg/L from fertilized eggs for up to mature period under flow-through condition. Several endpoints that related to development, reproductive fitness and transgenerational effects were evaluated. It was found that body length and body weight were significantly reduced and vitellogenin (Vtg) levels were significantly increased for fish exposed to DES. Histological examination showed that the sex ratios of F0 fish skewed to female and about 2% of the fish exposed to 0.05 microg/L DES developed testes-ova. The reproductive success, as determined from data on egg production, was reduced in female fish exposed to 0.05, 0.5, 1 and 5 microg/L DES. The lowest-observed-effect concentrations (LOEC) for changes of sex ratios, reproductive success and histology alteration of F0 are 0.05 microg/L. In the offspring, transgenerational effects on egg hatching rate, egg fertilization and Vtg levels of juvenile individuals were not observed. However, survival of F1 generation fry significantly declined. The analysis of sex steroid levels revealed a significant decrease of testosterone (T) in the whole body homogenates (WBH) of male progeny and somewhat elevation of estradiol (E2) in the WBH of female offspring. These findings indicate that exposure to DES causes a variety of developmental, reproductive and transgenerational effects.     

Reproduction study in Japanese quail fed hexachlorobutadiene for 90 days

Adult male and female Japanese quail were fed diets containing 0.3, 3, 10, or 30 ppm of hexachlorobutadiene (HCBD) or 20 ppm of hexachlorobenzene (HCB) for 90 days. HCBD at all dose levels had no effect on body weight, demeanor, food consumption, egg production, the percent fertility and hatchability of eggs, the survival of hatched chicks, and egg-shell thickness. The birds showed no evident signs of toxicity during the study. In addition, there were no gross or histopathologic changes evident in the organs or tissues of birds at the termination of the study which could be related to treatment. Ingestion of a diet containing 20 ppm HCB resulted in decreased survival of chicks hatched during the study and an increase in the liver weight of adult birds at the end of the study. No other treatmentrelated changes were observed.     

Diethylstilbestrol at environmental levels affects the development of early life stage and target gene expression in Japanese Medaka (<Emphasis Type="Italic">Oryzias latipes</Emphasis>)

In this study, the biologic effects of DES on the early life and adult life stages of Japanese medaka (Oryzias latipes) were evaluated. At the early life stage, the fertilized eggs were exposed to 1–1000 ng/L diethylstilbestrol (DES) for 15 days and the hatched larvae were continually exposed to the same concentrations for an additional 25 days. Significant adverse effects on hatchability, time to hatching and mortality rate occurred at DES concentrations of 100 and 1000 ng/L, while the abnormality (scoliosis and abdominal swelling) rate was significantly increased at 10 ng/L and above. After exposure, the fish were maintained in charcoal-dechlorinated tap water for a further 30 days. Only the male gonadosomatic index (GSI) at 1000 ng/L was significantly increased. At concentrations greater than 1 ng/L, estrogen receptor α (ERα) mRNA in both sexes and vitellogenin-I (Vtg-I) mRNA in males were significantly down-regulated; while Vtg-I mRNA in females was significantly up-regulated. When sexually mature medaka were exposed to 10 and 1000 ng/L DES for 21 days, only the GSI in females was significantly decreased at 1000 ng/L. At 10 and 1000 ng/L, ERα mRNA in both sexes was significantly down-regulated, while Vtg-I mRNA in males was significantly up-regulated. These findings showed that DES at the environmental concentration of 10 ng/L can affect the early life stage development of medaka and alter liver ERα and Vtg-I gene expression. Therefore, if we only focused on these sensitive toxicity endpoints such as ERα and Vtg-I mRNA expression, DES has a strong estrogenic effect on Japanese medaka.     

Adverse effects of fipronil on avian reproduction and development: maternal transfer of fipronil to eggs in zebra finch Taeniopygia guttata and in ovo exposure in chickens Gallus domesticus

Two studies were carried out to examine the impact of maternal fipronil exposure on embryonic and offspring development. In the first study, breeding female zebra finches were orally dosed with single sublethal levels of fipronil (1, 5, and 10 mg/kg body weight) to determine behavioural and developmental consequences on chicks following maternal pesticide exposure. Significant levels of fipronil and fipronil-sulfone residues were detected in eggs laid by females in all dosed groups, however, these were undetectable in eggs laid 13 days after treatment. The level of sulfone detected in eggs was consistently higher than that of the parent fipronil compound. Of the seven eggs laid in the treatment groups, only one (14%) chick hatched and this was from the lowest dose group. This chick was severely underdeveloped at 10 days of age in comparison to control chicks and fiproles were detected in brain, liver, and adipose tissues collected following euthanasia of this individual. In contrast, there was 100% hatchability of control group eggs and all chicks fledged nests on schedule. In the second study, domestic chicken eggs were injected with 5.5, 17.5, and 37.5 mg/kg egg weight of fipronil directly into the yolk sac on day 12 of incubation. Treatment did not affect hatching success, however, behavioural and developmental abnormalities were observed in hatchlings from the highest dose group. These chicks also demonstrated reduced feeding rates, as indicated by reduced body mass at 48 h period post hatch. Both fipronil and fipronil-sulfone residues were detected in brain and liver tissue of hatchlings at all pesticide dose levels tested.     

Disrupted carbonic anhydrase distribution in the avian shell gland following in ovo exposure to estrogen

Eggshell thinning among wild birds has been an environmental concern for almost half a century and the underlying mechanisms are still not fully understood. Previously we showed that exposure of quail embryos to ethynylestradiol (EE2) caused disorganization of the tubular glands in the shell gland of adult birds. In this study, we have examined the effect of in ovo exposure to EE2 on carbonic anhydrase (CA) localization, especially in the shell gland, because CA is required for shell formation. In the control birds, CA was localized in the cell membranes of the tubular gland cells of the shell gland, whereas the surface epithelium was always devoid of CA. In ovo treatment with 20ng EE2/g egg resulted in a loss of CA activity in the tubular glands while the surface epithelium showed strong induction of both membrane bound and cytoplasmic CA activity in 49+/-1% of the cells. The dose 2ng EE2/g egg resulted in partial loss of tubular gland CA and strong induction of CA activity in 2.5+/-0.5% of the surface epithelial cells and weaker induction in 22+/-2% of the epithelial cells. In conclusion, this study shows that embryonic exposure to a xenoestrogen disrupts CA distribution in the adult shell gland. We propose that eggshell thinning in avian wildlife could reflect a functional malformation in the shell gland, already induced by xenoestrogen during embryonic development rather than being caused solely by exposure of the adult bird.     

Metal stress in zooplankton diapause production: post-hatching response

Aquatic organisms commonly respond to harsh conditions by forming diapausing stages, which enable populations to survive adverse periods forming egg banks. Production of diapausing eggs is frequently observed in monogonont rotifers, previously changing from asexual to partial sexual reproduction (mixis). In despite that zooplankton are frequently used in ecotoxicological assessment because of their sensitivity to various toxicants and their important role in the ecosystems, toxicity evaluations often consider the directly exposed population produced by parthenogenetic reproduction, exclusively. We assessed experimentally effects of exposure to metals on mixis delay and fitness of hatchlings of the rotifer Brachionus plicatilis obtained from a brackish water lagoon with high metal content, especially copper. We show that sub-lethal concentrations of copper affected traits related to sexual reproduction and diapausing egg production in the rotifer. Copper addition did not delay the start of mixis, suggesting that rapid initiation of mixis is promoted in risky environments, according to the hypothesis of mixis as an escape strategy. Higher investment in mixis was obtained when individuals were exposed to metal. Addition of copper negatively affected the hatching success of diapausing eggs and performance of hatchlings. Nevertheless, these effects were greater for individuals formed in non-metal conditions, suggesting an adaptive advantage of populations from natural sediments exposed to copper. These results highlight the ecological and evolutionary consequences of the presence of metals in freshwater environments by modulating diapause adaptive efficacy and the selective process in egg banks.     

Dietary ferulic acid,biochanin A,and the inhibition of reproductive behavior in Japanese quail (Coturnix coturnix)

A study was conducted to determine the effects of two dietary compounds, biochanin A and ferulic acid, on the reproductive performance of Japanese quail, Coturnix coturnix. These compounds may be present in the diet of wild birds during dry years or at the end of the growing season during normal years. Four treatment groups, each fed a diet supplemented with biochanin A or ferulic acid, and a control group were tested for morphological and behavioral effects before, during and after treatment. Fertility and hatchability data were collected during treatment only. Ferulic acid was shown to inhibit male copulatory behavior during treatment. Biochanin A inhibited male copulatory behavior during and after treatment. Neither drug affected female copulatory behavior though egg-laying was inhibited by the low dose of biochanin A. Ferulic acid significantly reduced the hatchability of eggs. Biochanin A increased the fertility of males and the hatchability of eggs fertilized by them. A correlation between results of this study and natural conditions of reproduction is discussed. It is concluded that both biochanin A and ferulic acid exert inhibiting influences on the reproductive behavior of Coturnix.     

Effects of 3-methyl-4-nitrophenol on the reproductive toxicity in female Japanese quail (Coturnix japonica)

We previously showed that 3-methyl-4-nitrophenol (4-nitro-m-cresol, PNMC), a component of diesel exhaust particles and a degradation product of the insecticide fenitrothion, has reproductive toxicity in adult male and immature female Japanese quail (Coturnix japonica). Here we investigated effects of PNMC on the reproductive toxicity of mature female Japanese quail. The experiment consists of 3 periods of pretreatment, treatment, and post-treatment for 5 d each. The birds were reared, bred naturally for 1 week, and after 5 d of pretreatment, then injected intramuscularly with PNMC at doses 1, 10, or 100 mg/kg body weight daily for 5 d. Body weight, egg weight, and hatchability did not differ among the observation periods. However, at all doses of PNMC, the egg-laying rate showed a modest decrease during the treatment period, with recovery during the post-treatment period. Plasma concentrations of luteinizing hormone (LH) and estrodiol-17beta, were significantly decreased (p<0.05), and plasma concentrations of progesterone significantly increased (p<0.05) in birds treated with 10 and 100 mg/kg PNMC. These results suggest that PNMC have acute toxicity, and inhibited LH secretion, disturbing egg-laying in mature female quail. Our findings indicate that PNMC induces endocrine malfunction at the central level and subsequently disrupts reproductive processes in mature female quails.     

Effects of perfluorooctane sulfonate on mallard and northern bobwhite quail exposed chronically via the diet

Adult mallard ducks and northern bobwhite quail were exposed to 0, 10, 50, or 150mg perfluorooctane sulfonate (PFOS)/kg in the diet for up to 21 weeks. Adult health, body and liver weight, feed consumption, gross morphology and histology of body organs, and reproduction were examined. Due to mortality, birds exposed to 50 or 150mg PFOS/kg feed were terminated by Week 7. In quail, the lowest observable adverse effect level (LOAEL) was 10mg PFOS/kg feed based on decreased survivorship of 14-day-old quail offspring. For adult female quail fed 10mg/kg feed, there was a slight but statistically significantly PFOS-related increase in liver weight when compared to controls. When liver weight was normalized to body weight, the statistically significant differences were still observed indicating that PFOS affected liver size. However, no other pathological effects were observed livers of quail from this treatment group which suggests that this enlargement may have been an adaptive response. For adult mallards, no treatment-related effects on feed consumption, body or liver weight, growth, or reproductive performance were observed. There was a slightly greater incidence of small testes (length) in adult male mallards and quail exposed to 10mg PFOS/kg, feed when compared to controls. However, spermatogenesis was not affected and there was no effect on the rates of egg fertilization. Due to transfer to eggs, concentrations of PFOS measured in the liver and blood at study termination were greater in male birds than female birds.