Potential Feminization of Japanese Quail (Coturnix

japonica) Embryos

Due to In Ovo Exposure to Genistein

Steven Piquette

Supervisor: Dr. Dave Hackett

Co-Supervisor: Dr. Reehan Mirza

Nipissing University

April 28, 2011

Acknowledgements

I would like to acknowledge Dr. Dave Hackett for all the help,

time and guidance he has put forth in the development of this

study. I would like to thank Alison Jackson for the extremity of

hours spent in the lab to help with the care and the experimental

protocol. As well I would like to give thanks to Dr. Tony Parks

for letting me use his camera and his time in the process. I

would also want to show appreciation to Geatan Piquette, Matt

Jones, Tina Piché and David Piquette who all helped out with the

experimental process for this study. Lastly, I would like to

recognize the support from the Department of Biology and

Chemistry at Nipissing University and the permission from the ACC

((PR) 2009-04-11-5) to conduct this study.

II

Table of Contents

Acknowledgements……………………………………………………………………………….IITable of Contents………………………………………………………………………………...IIIList of Figures……………………………………………………………………………………IVAbstract…………………………………………………………………………………………...V

Introduction………………………………………………………………………………………..1Material & Methods……………………………………………………………………………….5

Treatments…………………………………………………………………………………5Euthanasia and Preservation………………………………………………………………7Dissection and Evaluation…………………………………………………………………7Statistical Analysis………………………………………………………………………...8

Results……………………………………………………………………………………………..8Sex Ratio…………………………………………………………………………………..8

III

Feminization………………………………………………………………………………9Effects of Increased Exposure…………………………………………………………...10Minimum Concentration of Genistein Causing

Feminization……………………...........11Other Effects of Genistein……………………………………………………………….14

Discussion………………………………………………………………………………………..15Sex Ratio…………………………………………………………………………………15Feminization……………………………………………………………………………..16Effects of Increased Exposure…………………………………………………………...17Minimum Concentration of Genistein Causing

Feminization…………………………...18Other Effects of Genistein……………………………………………………………….19Conclusion……………………………………………………………………………….21

Literature Cited…………………………………………………………………………………..24Appendix A………………………………………………………………………………………28Appendix B………………………………………………………………………………………29

List of Figures

Figure 1: Comparison of Japanese quail (Coturnix japonica) mortality in various treatment

groups……………………………………………………………………………………...8Figure 2: Total individual sex count ratio of male and females Japanese quail (Coturnix japonica) between treatment groups………………………………………………………9

IV

Figure 3: % reproductive system development types of Japanese quail (Coturnix japonica) embryo between treatment groups………………………………………………………...9Figure 4: Graphical representation of the lowest concentration ofgenistein to have a minimal effect on Japanese quail (Coturnix japonica) embryos…………………………………..10Figure 5: A) Visual comparison of the % of normal male developed and % of feminized males Japanese quail (Coturnix japonica) exposed to all treatment groups. B) Visual comparisonof the % of normal female developed and % of over-feminized females exposed to all treatment groups………………………………………………………….11Figure 6: Distribution of Japanese quail (Coturnix japonica) embryosreproductive development between treatment groups)……………………………………………………………….13Figure 7: Comparison of the mean weight (g) of developed Japanesequail (Coturnix japonica)

……………………………………………………………………………………………15Figure A1. Distribution of Japanese quail (Coturnix japonica) embryos with teratogenic deformities between treatment groups……………..………………….…………………28 Figure B1. Example of normal male reproductive anatomy for Japanese quail (Coturnix japonica)………………………………………………………………………………... 29Figure B2. Example of feminized treated male reproductive anatomyfor Japanese quail (Coturnix japonica….

…………………………………………………………………....29Figure B3. Example of feminized treated male reproductive anatomyfor Japanese quail (Coturnix japonica)……………………………………………………………...……….30Figure B4. Example of feminized treated male reproductive anatomyfor Japanese quail (Coturnix japonica)………………………………………………………………………30Figure B5. Example of normal female Japanese quail (Coturnix japonica) reproductive anatomy……………………………………………………………………………….….30Figure B6. Example of over-feminized Japanese quail (Coturnix japonica) female exposed to

treatments………………………………………………………………………..……….30

V

Abstract

Many environmental contaminants such as genistein (40, 5, 7-trihydroxyisoflavone) have the potential to disrupt thevertebrate endocrine system. For humans, it has become anincreasingly popular pharmaceutical and has been increased in ourdiets. Typically found in soybeans and soy products manly used asanimal feed, it is consumed by livestock and can be transferredor accumulated into the resulted animal products, such as meatand eggs. The Japanese quail (Coturnix japonica) egg provides aperfect closed system test organism in which to test suchendocrine disrupting chemicals, due to its embryonic sensitivityand the fact that the presence of estrogen determines thedifferentiation of sexual dimorphism. In this study, genisteinwas examined to determine the minimum concentration forfeminization as an endocrine disrupter. Genistein wasadministered in ovo dissolved in a corn oil vehicle. At the lowconcentration of 0.01µg/egg, genistein feminized 31% of the malesand 6% of the females. The higher concentrations of 0.1µg/egggenistein feminized 58% of males and 15% of females. 1.0µg/egg ofgenistein resulted in 71% male and 23% female feminizationbetween all developed embryos. These results suggest thatgenistein can cause endocrine disruption triggering feminizeddevelopment, where the concentration of 0.1µg/egg resulted inbeing the lowest dose needed to have significant feminization.However, the lowest concentration of 0.01µg/egg genistein stillshowed a noteworthy amount of feminization; implying that the

VI

isoflavones found in meat and eggs can still lead to potentialendocrine disrupting effects to humans if consumed.

VII

In recent years, there has been strong evidence that some

industrial pollutants such as plasticiser contaminants

(xenoestrogens) and natural substances like antioxidant

flavonoids (environmental estrogens) can be accumulated in the

environment (Waring and Harris 2005). Endocrine disrupting

chemicals (EDCs) are defined as “exogenous substances that change

endocrine function and cause adverse effects at the level of the

organism, its progeny, and of populations of the

organisms”(EDSTAC 1998); they have the capability of impacting

endocrine function, which guides the proliferation and

differentiation of cells in many species, including human beings.

Endocrine disrupters (EDs) are compounds that are estrogen or

estrogen-like molecules that alter normal hormone regulation

within the body even in low concentrations (Waring and Harris

2005; Panzica et al. 2009).

Compounds such as genistein (40, 5, 7-trihydroxyisoflavone)

are estrogen mimicking and are the simplest isoflavonoid known as

phytoestrogens. They are produced in legumes, particularly from

soybeans and soy products which are used mainly as animal feed

(Dixon and Ferreira 2002). Its benefit to human beings has made

1

it an increasingly popular pharmaceutical as it has antimicrobial

activity and has been proposed to prevent cancer and

cardiovascular diseases, as well as to relieve post menopausal

difficulties (Dixon and Ferreira 2002). Efforts have been made to

integrate soy base products within human diets because of the

awareness of potential benefits of isoflavones. However, soy is

most consumed as feed for livestock, where it can be transferred

or accumulated into the resulted animal products, such as meat

and eggs (Chapman 1998.). The isoflavones found in meat and eggs

can still lead to potential advantages and disadvantages to

humans if consumed (Lin et al. 2004).

Genistein has the potential to induce endocrine disrupting

effects (Dixon and Ferreira 2002). Genistein and other similar

phytoestrogens can exert both estrogenic and antiestrogenic

activity by competing with estradiol for receptor binding

(Bramlett et al. 2001). Recent studies have shown that genistein

can have adverse effects on other vertebrates including rats,

mice and quail (Panzica et al. 2007). In birds, genistein has

been shown to induce oviduct growth in broiler chicks (Berry et

2

al. 1999) and zebra finches (Millam et al. 2002). Genistein has

been released in the environment via several point and non-point

sources which include sewage treatment plant effluents (Green and

Kelly 2009), agricultural feed, agricultural runoff (Burnison et

al. 2003; Kiparissis et al. 2003), as well as landfill leachate.

Genistein may disrupt endocrine development in wildlife if

ingested over time in sources such as freshwater, marine and

terrestrial food products (Colborn et al. 1993).

According to Herbst and Bern (1981), exposure to estrogenic

substances during critical periods of development can have

adverse consequences on differentiating reproductive systems of

many animals. Several EDCs may interact with estrogen or

androgen receptors (Mura et al. 2009). The most common hormones

guiding this process are the gonadal hormones, such as 17β-

estradiol (E2) or androgens, which play key roles in the

development of primary and secondary sex characteristics in

higher vertebrates (Panzica et al. 2009). Hormonal interactions

with these receptors are needed for sexual determination or

alteration for many species during prenatal periods of embryo

development. For birds, sexual determination of the avian

3

reproductive system is dependent on estrogen levels, which

regulates production of the female phenotype (Waring and Harris

2005). Early studies indicate that estrogen to produce

feminization of the gonads; the removal of the left ovary from

immature chickens resulted in the right gonad developing into a

testis (Benoit 1926). Therefore, if estrogen synthesis is

inhibited during gonadal sex differentiation, genetic females

will develop a male phenotype (Elbrecht and Smith 1992). Early in

life, both male and female bird embryos have pairs of

undifferentiated gonads and Müllerian ducts (MDs), which

differentiate in a sex-dependent manner during gonadogenesis

(Romanoff 1960.). The resulting sex differentiation is identified

asymmetrically when the left gonad and Müllerian duct of the

chick embryo develops into a functional ovary and oviduct, while

the right gonad and duct regress to produce a female bird. In

males, gonads and Wolffian ducts will develop into a symmetrical,

bilateral reproductive system, while the MDs will regress (Berg

et al. 1999). Berg et al. have stated that exposure to estrogen

during early quail development induces dose-dependent

malformation of the Müllerian ducts of both sexes and a

4

feminization of the left testis, which is transformed into an

ovotestis (Berg et al. 1999).

As described above, it is clear that estrogenic compounds

affect the reproductive organs of many species which later affect

their population growth due to reproductive difficulties

(Rochester et al., 2009). The Japanese quail (Coturnix japonica)

offers several advantages as an experimental test subject because

it shares many features with other avian species. One advantage

is that exposure in ovo can be carried out under well-controlled

conditions and at different stages of embryonic development. The

egg offers an enclosed environment for the embryo and controlled

doses of chemicals are easily administered. Other advantages to

using quail include the relatively small sizes of the adults and

eggs. Also, in spite of its small size, the embryo is large

enough for experimental studies (Halldin et al. 2005). Quail are

considered to be a model bird species for experimentation and

they have the advantage of being more representative of wildlife

fauna than other common test subjects, such as chickens

(Abdelnabi et al. 2000).

5

Studies have shown that in rats, a dietary supplement of

genistein given to a pregnant mother was shown to cause a

decrease in food consumption as well as weight in both the mother

and the litter (Flynn et al. 2000). In Japanese quail, genistein

was associated with feminizing dimorphic portions of the brain

(Panzica and Melcangi 2008). Few studies have shown the minimal

amount of phytoestrogens needed to produce estrogenic effects.

Accordingly, this study will focus on whether or not estrogen

mimicking compounds such as genistein can show the effect of an

endocrine disrupter causing feminization. . It is hypothesed that

increasing concentrations of genistein will result in more

pronounced feminization; where the male:female groups of japanese

quail eggs will be skewed from 50:50 towards showing signs of

over-feminization of female embryos and any recognizable males

will have feminized reproductive organs.

The objectives of this experiment will be: I) to determine

whether or not in ovo exposure to genistein will result in a

skewed sex ratio (favouring females) and feminization of male

embryos, II) to determine whether or not the above effects

increase with exposure to greater concentrations of genistein,

6

and III) to determine the minimum concentration of genistein that

has an observable effect on quail embryos. Lin et al. (2004) have

illustrated that dietary genistein is readily transferred from

the mother to the egg and accumulates in the yolk. This implies

dangers of releasing genistein into the environment since it

could easily be ingested by birds such as quail and transferred

to their embryos, causing detrimental effects in wild

populations.

Material and Methods

335 Fertilized Japanese quail eggs were obtained from a

local breeder at Cro Quail Farms Incorporated in St. Anns,

Ontario, Canada. Once received, the eggs were left to aclimate to

room temperature. Afterwards, they were examined for cracks and

other damages. Any organic matter found on the shell was

carefully removed; then eggs were submerged in a diluted 10%

7

provadine solution and rinsed in deionized water for

disinfection. The eggs were incubated at 37.5 oC and at 60%

relative humidity and turned every 3 hours via a Brinsea Ova-Easy

190 Advance digital cabinet incubator with a programmable

automatic egg turning system, along with an adaptable humidity

pump (Berg et al. 2001). After three days of incubation, the eggs

were candled to confirm fertilization and 180 healthy eggs were

chosen to be randomly assigned to 6 intermixed colour coded

treatment groups (n=30 for each treatment group) that were

randomly placed into 6 egg trays (30 eggs/tray).

Treatments

Genistein (4, 5, 7 trihydroxyisoflavone, CAS No. 446-72-0,

purity >99 %) was ordered from LC Laboratories, Woburn, MA, USA;

β-estradiol ((17 β)-Estra-1, 3, 5 (10)-rienne-3, 17-diol, CAS No.

50-28-2) was ordered from Tocris Bioscience, Ellisville, MO, USA;

MAZOLA 100% pure corn oil, used as a carrier for the test

chemicals, was purchased from a department store. The day before

treatment, genistein and β -estradiol were diluted with corn oil

8

to appropriate concentrations for administration (Utsumi and

Yoshimura 2009).

On Day 3 of incubation, the fertilized eggs were removed

from the incubator and placed blunt-side up in egg holders; the

blunt ends were then sandpapered to facilitate adhesion of the

glue and swabbed with 70% ethanol for sterilization. A small hole

was drilled through the outer shell membrane using a Dremel

drill, fitted with a 1/16 inch engraving cutter bit that was

sterilized in 70% ethanol. Eggs were then administered to 1 of 6

treatment group. Treatment 1 which was the control group,

received no injection; Treatment 2 the vehicle control group,

received an injection of 10µl/egg of corn oil; Treatment 3

received 0.01µg/egg of genistein; Treatment 4 received 0.1µg/egg

of genistein; Treatment 5 received 1.0µg/egg of genistein and

finally Treatment 6 received an estrogen dose of 0.01µg/egg of β

-estradiol to produce examples of eggs feminized by estrogen.

A typical quail egg weighs approximately 10g (Berg et al.

1998; Heinz 2006). The injection volume for each egg was 1.0µl/g;

therefore a total solution volume of 10µl was injected into the

air space of each quail egg receiving treatment (Heinz 2006). Ten

9

micro litters of test solution with their respective treatments

were injected into the air chamber through the exposed shell

membrane of each egg using a micropipette (Kamata et al. 2006).

After injection, each hole was sealed with a dab of glue from a

hot glue gun (Mattsson et al. 2008). Using a randomized number

table, the treated and untreated eggs were randomly placed back

in 1 of 6 egg trays and returned to the incubator.

Euthanasia and Preservation

Japanese Quail have an incubation period of approximately

17-18 days until they hatch (Chickscope 1998). The embryos were

dissected on Day 15, 2 days before anticipated hatching for quail

embryos (Berg et al. 2001). Euthanasia is accomplished

instantaneously by injecting an overdose of sodium pentobarbital

(240mg/ml) into the pleuroperitoneal cavity of the quail (CCAC

Guidelines; Close 1997). The euthanized quail embryos were then

fixed in Bouin’s (for about 4 hours). The Bouin’s solution was

rinsed and the fixed quails were preserved in sealed containers

with 70% ethanol for later analysis (Tokita 2003). The preserved

birds were later dissected to determine the gender and degree of

10

feminization, along with size and weight and any other

teratogenic physical deformities or malformations.

Dissection and Evaluation

Each developed quail embryo was then dissected and examined

macroscopically with the aid of a dissecting microscope. The

reproductive organs were inspected to sex the embryos, with

normal males showing a pair of symmetrical testis along with

Wolffian ducts or vas deferens (figure 10). Normal females showed

to have a left ovary and a right vestigial gonad with a left

Müllerian duct and vestigial right Müllerian duct (figure 14).

Feminizations of the quail embryos were shown to have abnormal

Müllerian or Wolffian duct malformation, such as increased length

of right Müllerian duct (longer than 5 mm) or partially retained

Müllerian ducts. Ovotestis formation in males or hyper

feminization of every individual bird was noted (Berg et al.

1999).

Statistical Analysis

11

A descriptive analysis was initiated to assess the

normality and mean of the data. A one way ANOVA was run to

analyze mean weights of quail embryos followed by Tukey’s post

hoc between groups test. A chi-square of independent samples was

run in conjunction with the Pearson chi-square to determine

whether or not there was a significant difference in the data as

a whole. Subsequent pair wise comparisons using the same tests

were run between test groups along with chi-square Goodness-of-

Fit to determine where significant differences exist (>0.05 has

no significant difference, <0.05 has a significant difference).

Version 17 of SPSS was used to run all statistical procedures.

Results

Total hatchability of the fertilized eggs was 67%. The

percent mortality was analyzed between all treatment groups.

Percent mortalities consisted of undeveloped quail eggs. Figure 1

demonstrates that these mortality rates are relatively low and

are well within the normal range of mortality in vehicle control

embryos observed in our laboratory.

12

0204060

0%

37%57%

37% 30%40%

Treatment Group

% Mo

rtal

ity

Figure 1 : Comparison of Mortality of Japanese quail (Coturnix japonica) in various treatment groups. Each treatment group consist of n=30 quail eggs.

Sex Ratio

Later the birds were dissected an analysized for the sex

ratio between male and female chick embryos between each

treatment groups. Using a Pearson Chi-Square it was determined

that there was not a significant difference in the overall sex

ratio of male and female chick embryos between all treatment

groups (x²=5.846, df=5, p= 0.321; Figure 3). Using a Chi-square

Goodness-of-Fit test it was determined that there was a

significant difference between the amount males to females within

the 1µg/egg genistein (x²=3.857, df=1, p= 0.050; Figure 3).

13

There is no significant difference in the amount of males and

females in the following treatments: Control, vehicle Control,

0.01µg/egg genistein, 0.1µg/egg genistein and the 0.01µg/egg β-

estradiol (p > 0.05).

Control

Vehicle Control

0.01ug Genistein

0.1ug Genistein

1ug Genistein

0.01ug β-estradiol

0612

MaleFemale

Treatment Group

Number of Chick Embryos

Figure 2: Total individual sex count ratio of male and females Japanese quail (Coturnix japonica) between all treatment groups. Each treatment group consist of n=30 quail eggs.

Feminization

The overall feminization between treatment groups was

analyzed as a whole to determine if a significant difference

existed between treatment groups. A Pearson Chi-Square was used

to analyze the data. A significant difference was found in the

feminization of quail embryos in comparison to embryos that were

14

not feminized between all treatment groups (x²=39.829, df = 15,

p≤ 0.0001; Figure 4).

020406080

Normal MaleFeminized MaleNormal FemaleOver-Feminized FemaleNot Developed

Treatment Group

% of

Chi

ck D

evel

opme

nt

Figure 3: % reproductive system development types of Japanese quail (Coturnix japonica) embryo between treatment groups. Each treatment group consist of n=30 quail eggs.

Effects of Increased Exposure

A visual comaprison was conducted to determine whether or

not the effects of feminization would increase with exposure to

greater concentrations of genistein (Figure 6).

15

04080

28% 38%74%

95%

Treatment Group

% Fe

mini

zati

on

Figure 4: Graphical representation of the lowest concentration of genistein to have a minimal effect on Japanese quail (Coturnix japonica) embryos, showing the difference of the feminizations of bother gendersover the tested concentrations.

According to Figure 5a., there is a visual increase in the

percente of feminization in male embryos exposed to an increase

in genistein concentrations, However, in comparison to the

vehicule control, the feminised males still show a gradual

increase. To solidify the observation, a comparative analysis was

done between the normal male and feminized male developed between

treatment groups. A Pearson chi-square was used to determine

whether or not a significant difference existed between the two

types of development. It was determined that a significant

difference was present between the amount of normally developed

male and feminized males (x²=18.090, df=3, p≤ 0.0001; Figure 5a).

In Figure 7b, the over-feminized females show an increase in

percente feminisation as the concentration of genistein increase;16

however, in comparison to the vehicle control, the feminization

of female quails does not show a significant increase in the

lower concentration of genistein, but shows an increase at the

highest concentration. A second comparitive analysis was

conducted on the development of normal females and over-feminized

females between all treatment groups. No significant difference

was found between the two development types (x²=7.615, df=3, p=

0.055; Figure 5b). A significant difference was found in the

degree of feminization of quail embryos in correlation to

increased concentrations of Genistein found in both male and

female genders (x²=19.672 df=10, p=0.033).

0

20

40

60

80

Normal MaleFeminized Male

Treatment Group

% of

Qua

il D

evel

opme

nt

01020304050

Normal FemaleOver-Feminized Female

Tireatment Group

% of

Qua

il D

evel

opme

nt

Figure 5: A) Visual comparison of the % of normal male developed and %of feminized males of Japanese quail (Coturnix japonica) exposed to all treatment groups. B) Visual comparison of the % of normal female

17

developed and % of over-feminized females exposed to all treatment groups.

Minimum Concentration of Genistein Causing Feminization

A visual comparison between the normal development and

feminization that occurred in each test group can be seen in

Figure 3, from which the following pair-wise comparisons using a

Pearson chi-square were run. Statistically, there was no

significant difference between the control group and the vehicle

control group (x²=3.402 , df=3, p=0.334; Figure 6a, b). There was

no statistical difference between the vehicle control group and

0.01µg/egg genistein group (x²=5.201 , df=3, p=0.158; Figure 6b,

c). Yet there was a significant difference between the vehicle

control group and 0.1µg/egg genistein group (x²=10.503 , df=3,

p=0.015; Figure 6b, d). The vehicle control group to the 1µg/egg

genistein also had a significant difference (x²=21.840, df=3,

p≤0.0001; Figure 6b, e). ). Between the vehicle control and

0.01µg/egg β-estradiol group there was a significant difference

(x²=8.850 , df=3, p=0.031; Figure 6c, f). In comparing the

0.01µg/egg β-estradiol group to the various concentrations of

genistein it was determined that there was no difference

18

(x²=0.549 , df=3, p=0.908; Figure 6c, d, e, f). The lowest

concentration to have significant feminization is the 0.1µg/egg

genistein group shows and about the same feminizing effect as

0.01ug/egg β-estradiol group. Meaning that genistein has 1/10th

the effect of estradiol.

19

Normal Males

Feminized Males

Normal Females

Feminized Females

0612

Type of Developement

Number of Chick

Embryos

A)

Normal Males

Feminized Males

Normal Females

Feminized Females

048

Type of Developement

Number of Chick

Embryos

B)

Normal Males

Feminized Males

Normal Females

Feminized Females

036

Type of Development

Number of Chic

k Embryos

C)

048

12

Type of Development

Numb

er o

f Ch

ick

Embr

yos

D)

Normal Males

Feminized Males

Normal Females

Feminized Females

0612

Type of Development

Number of Chick

Embryos

E)

Normal Males

Feminized Males

Normal Females

Feminized Females

048

Type of Development

Number of Chick

Embryos

F)

Figure 6: Distribution of Japanese quail (Coturnix japonica) embryos reproductive development between treatment groups. a) Control, b) Vehicle Control, c) 0.01 µg/egg Genistein l, d) 0.1 µg/egg Genistein, e) 1 µg/egg Genistein, f) 0.01 µg/egg β-estradiol.Other Effects of Genistein

20

An analysis was conducted to determine whether or not

exposure to an increasing concentration of genistein would have a

significant difference in the amount of different teratogenic

deformities found between all treatments groups. A Pearson Chi-

Square analysis was run and identified that a significant

difference was present in teratogenic deformities of quail

embryos exposed to all treatment groups (p=82,085, df = 25,

x²≤0.0001; Appendix A). Further comparisons have determined that

the control group and the vehicle control are significantly

different from one another, with the vehicle control group having

more deformities (p=16.458, df =3, x²=0.001; Appendix A). The

vehicle control in comparison to the 0.01 µg/egg β-estradiol has no

significant difference (p=4.309, df =5, x²=0.506; Appendix A). The

vehicle control to the 0.01 µg/egg Genistein group has no

significant difference (p=3.135, df =3, x²=0.371; Appendix A).

The vehicle control in comparison to the 0.1 ug/egg genistein

group has a significant difference in the number of teratogenic

deformities (p=12.220, df =5, x²=0.032; Appendix A). As well the

vehicle control in comparison to the 1ug/egg genistein group shows

21

a significant difference in teratogenic deformities (p=22.289, df =5,

x²≤0.0001; Appendix A).

The developed quails were submitted to a mean weight

comparison to determine whether or not different exposure to

treatments would have a significant effect on the body weight of

the Japanese quail embryos. Using a one way ANOVA it was

determined that there was a significant difference in the

bodyweight of the Japanese quail embryos between all treatment

groups (F=5.308, df =5, p ≤ 0.0001; Figure 7). The post-hoc

analysis used by Tukey HSD, showed 1.32 times higher embryo

bodyweight of the control in comparison to 0.01 µg/egg β-

estradiol (p≤0.0001), and 1.26 times more than 0.01ug/egg

Genistein (p=0.008), as well as 1.25 times more than 0.1ug

Genistein (p=0.006) however, there was no difference in

bodyweight between the control and the vehicle control group

(p=0.051), as well as the vehicle control and the treated birds.

22

4.5

5.5

6.5

Treatment Group

Mean

Wei

ght

(g)

Figure 7: Comparison of the mean weight (g) of the developed Japanese quail (Coturnix japonica) embryos after being treated with test solutions.Each treatment group consist of n=30 quail eggs.

Discussion

Sex ratio

For birds, the male is the non-hormonal sex and the presence

of estrogen results in the differentiation of sexual dimorphism

(Lyons 2007). Therefore, estrogen is most important for sex

differentiation in female birds and, without estrogen, female

embryos will develop male sex characteristics (Berg et al. 1998).

This study concluded that there was no difference in the sex

ratio of male and female quail embryos between all treatments

23

groups. However, a dose dependant trend is observed, where higher

production of males versus female quail is noted at

concentrations of 0.1 µg/egg genistein, 1 µg/egg genistein and

0.01 µg/egg β-estradiol. At the 1 µg/egg genistein a significant

difference was found, where there was a greater number of males

than female quails. The increased proportions of male and

intersex individuals in this study could have resulted from the

dual role of genistein as not only an estrogen agonist but also

as an antagonist blocking estrogens action (Green and Kelly

2009). It is possible that the dietary genistein used in our

study resulted in greater numbers of phenotypic males due to the

compound’s weak estrogenic properties, blocking of estrogen

receptors, and inhibition of aromatase and the resulting

decreases in estradiol synthesis (Green and Kelly 2009). This

then suggest that genistein at the tested concentrations in this

study does not act as a significant estrogen mimic, where gender

differentiation would have had a higher frequency of normally

developed females against male development.

Feminization

24

Phytoestrogens are secondary plant compounds, which can act

to mimic estrogen and cause the disruption of estrogenic

responses in organisms (Rochester and Millam 2009) and

phytoestrogens exposure during development may alter adult

reproduction (Millam et al. 2002). The results confirm that males

and females of Japanese quails show feminized or over-feminized

traits in the presence of genistein. Confirming that genistein

can act as an endocrine disrupter causing feminization. which can

lead to behviroural and reproduction difficulties, which may vary

across species. Importantly, disturbances in normal behaviour may

influence the individual fitness and, therefore, assume a real

biological significance in both animal and human ecosystems

(Panzica et al. 2007). Ecologically, plants high in phytoestrogens

have the ability to organizationally disrupt reproductive

endpoints in Japanese quail chicks, because constitute a large

part of the diet of quail and other granivorous wild birds. It is

probable that birds are encountering the levels tested in this

study of estrogenic plant containing genistein in the wild

(Rochester and Millam 2009). According to Rochester and Millam

(2009) showed the effect of seasonal temperatures, such as

25

drought and rainfall would reduce or increase the genistein

concentration produce in these phytoestrogenic plants that would

be regularly be part of Japanese quail wild diet. They then go to

suggest that these plants use phytoestrogens as a chemical

defence against herbivory, which therefore controls the quail and

other wild birds population by effecting their reproduction

endpoints by feminization, behavioural, deformities or just

reduce their fitness as a whole (Rochester and Millam 2009).

Effects of Increased Exposure

Upon further analysis a significant difference was

determined in the amount of normal males in comparison to

feminized males. The normal males regressed as concentration of

genistein became higher, while the amount of feminization

increased. Normal female to over-feminized female had no

statistical difference however, at the highest concentration of

genistein, the effect of feminization are similar with the β-

estradiol group. As one both genders demonstrates an exponential

trends of increased feminization with higher concentration of

genistein. Concentrations of 0.1 µg/egg of genistein yielded

26

similar percentage of overall feminization for both genders than

did 0.01µg/egg β-estradiol, a natural estrogen. Meaning that

genistein is still behaving like a weak form of β-estradiol in

promoting feminizing effects. This study can support the

statement of Herbst and Bern (1981), exposure to estrogenic

substances during critical periods of development can have

adverse consequences on differentiating reproductive systems of

many animals. In correlation to increased concentrations of

genistein, a significant difference was found in the degree of

feminization of quail embryos. More pronounced feminization was

observed as concentration of genistein increase. This tells us

that higher levels of genistein exposure will result in more

harmful endocrine disrupting effects, leading to multiple issues

concerning animal and human health if consumption and

environmental exposure continues to rise through environmental

waste, animal feeds, or other soy products available as the

markets for soy products continue to expand.

Minimum Concentration of Genistein Causing Feminization

27

The developmental trends for both the control group and

vehicle control group typically displayed normal embryo

development. There was no significant difference between them,

which suggests that the vehicle control worked properly. The

occurrence of a multiple feminized chick in both the control

group and vehicle control group are likely a result of avian

farming practices, where there is inherent inbreeding from a

limited gene pool. Such an occurrence was also seen in a study by

Berg et al. (1998) where there was the occurrence of some

feminization in the control group. The 0.01µg/egg β-estradiol

test group was significantly different to the vehicle control

group. It acted primarily as a feminization control to have

something to compare the genistein treatment groups to in terms

of their developed feminized reproductive anatomy. The feminized

development amongst all three genistein treatment groups appeared

to impact both sexes, where few studies have shown the minimal

amount of phytoestrogens needed to produce estrogenic effects.

The lowest concentration 0.01 µg/egg of genistein yielded no

significant results. Exposure to this concentration would be

biologically significant but would not interfere ecologically

28

with reproduction and breeding. Concentration of 0.1 µg/egg and 1

µg/egg of genistein both showed a significant difference in their

feminization of the quail embryos. Lin et al. (2004) showed that

dietary genistein is transferred from the mother to the egg and

accumulates in the yolk. The highest amount of genistein in the

egg yolk obtained in the study was less than 3 µg /egg yolk,

which is low as compared to the amount of supplementation (100

mg/day). This accumulation of genistein within the egg yolk

exceeds the concentration of observed effect for feminization

found in this study. This implies dangers of releasing

genistein into the environment since it could easily be ingested

by birds such as quail and transferred to their embryos, causing

detrimental effects in wild populations.

Other Effects of Genistein

Mortality and body weight mesurements of quail embryos were

not aspects of this study set forth in the objectives. However,

the percent mortality and body weight of quail of every treatment

group were gathered. As expected the control group had no

mortalities and higher mortalities were expected in those groups

29

that received in ovo treatments since they had a hole drilled

into their air cell and a foreign substance introduced to them.

Mortality percentage between all treatments groups remained

practically level with the vehicle control. Thus, identifying

that the major cause of quail mortality while in embryonic

development was the process of which a hole was drilled into the

air cell of the treated eggs.

Exposure to genistein also demonstrated an increase of

teratogenic deformities and a decrease in normal development.

This implies breeding difficulties towards deformed quail, when

time comes for courtship. Courting patterns in Japanese quail

for male birds consiste at least five components: 1) they would

have an increased neck and body tonus, where the posterior is

elevated and the neck is thrust forward and slightly downward.

The male circles the female with head cocked inward toward the

hen. 2) Leg action: the legs straighten and stiffen with the

body being brought up and forward. The leg is stiff during the

strut. 3) Toe walking: the

Bird raises itself on its toes beyond a normal stance and struts

about the female. 4) Vocalization: the courting call is a

30

subdued, hoarse, vibrating call given by the male only when

courting. It is a two-syllable squawk sound which lasts for

several seconds. 5) Feather puffing: most of the body feathers

from the neck downward are fluffed (Farris 1967). With the

occurrences of deformities, males quail will greatly reduce their

individual fitness which is associated in the proper display of

the courting patterns which may not be successful.

To further inspect the treatment protocol the body weight of

the chick embryos were dissected and weighed 2 Days before their

anticipated hatching, yielding a significant difference between

all treatment groups. Further analysis of the trend found in

figure 7 demonstrates what was expected throughout the treatment

groups with a decrease in body weight in treatments where holes

were drilled in the air cell and injected with corn oil., as well

notice that there is a slight increase in body weight at1 µg/egg

of genistein and a decrease in the 0.01µg/egg β-estradiol test

group. This shows us that the 0.01µg/egg β-estradiol group adds

to the effect of drilled hole and an injection of corn oil in the

eggs. However the slight increase in body in the 1 µg/egg of

genistein group can be explained by perhaps laboratory error.

31

We can conclude that corn oil used as a vehicle for genistein did

not increase or reduce the body weight of the quail embryos

rather that the drilling of a hole in the air cell was the

difference in the weight and the increase of mortality in

comparisons to the control group.

Conclusion

Naturally occurring compounds such as genistein is the

isoflavones found in soybeans and soy products and act as weak

estrogens (Warning & Harris 2005). Thus, the impact of genistein

as an endocrine disrupter will vary depending upon a variety of

factors, including the doses that subjects are exposed to, when

in the life cycle of an organism exposure occurs, as well as the

duration of the exposure. Particularly the developmental stages

are typically more vulnerable to disruption than adult stages and

the consequences of foetal exposure may be drastically different

from those of adult exposure (Panzica 2009). Compounds with

endocrine disrupting effect such as genistein can affect any

species and can potentially affect human beings (Warning & Harris

2005).Genistein has been proposed as being protective against

32

breast cancer and peri-menopausal symptoms with many other

benefits. However, they may not necessarily be beneficial to

humans. Consequently, their ingestion and absorption could cause

an increase in local estrogen levels. Where most breast cancers

are, at least initially, estrogen dependent and a surge in levels

would be expected to promote growth (Warning & Harris 2005).

What this study has pursued is the need for tests involving the

effects of environmental relevant genistein whether found through

natural portals or agricultural. Many previous studies have

shown that dietary genistein can be accumulated in areas such as

eggs, mammary milk and other areas in which would be sensitive to

endocrine disrupting effects at early stages of gonadal

development in many different species. This study investigated

the need to find the minimal level at which such a compound would

be able to have a significant endocrine disrupting effect that

would accumulate in Japanese quail eggs in the environment or in

captivity from their exposure to phytoestrogens rich foods.

Future studies, might look at possibly hatching the eggs, to

let the reproductive system developed further, which would permit

33

easier dissections and identification of feminized traits. Having

an assistant identify the reproductive system of quail embryos

while being unaware with which treatment they have been exposed

to and compare results from observation made when knowing which

embryo were exposed to specific treatment. Examining the

behaviour of hatchlings would also give greater insight on the

effect of phytoestrogens, such as genistein in quails. A further

problem is that we do not know whether mixtures of different

endocrine disrupters (the so-called ‘cocktail effect’) are

synergistic and possibly causing further effect of those exposed

at critical stages of development (Warning & Harris 2005). As

well because morphological sex differentiation is a critical

period in early development and is clearly sensitive to the

dietary genistein concentrations (Green and Kelly 2009) future

studies should look in to determining the minimal concentration

of dietary genistein that would produce the concentration of

observed effect found in this study.

Our studies and others have shown that (1) plant-produced

estrogenic chemicals can disrupt reproductive endpoints in birds;

34

(2) dietary (and possibly environmental) levels of phytoestrogens

have disruptive effects on avian reproduction; These facts,

combined with the estrogenic effects documented in our study,

suggest that we can rule out the hypothesis that male-female

groups of Japanese quail eggs will be skewed from 50:50; However,

we can confirm that males and females of Japanese quails will

show feminized or over-feminized traits in the presence of

genistein and that higher concentrations of genistein will result

in more pronounced feminization. Genistein is a highly effective

estrogen mimic, due to the fact that it can cause endocrine

disrupting effects at such low environmental concentrations. But

can also act as an antagonist blocking estrogen action.

Therefore, plant phytoestrogens can exert reproductive effect on

wildlife, especially on developing vertebrates.

Consequently, we can concluded that estrogen mimicking

compounds such as genistein can confirm its effects of as an

endocrine disrupter, causing feminization and that higher

concentrations of this compound will result in a higher frequency

and more pronounced feminization of quail embryos.

35

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Appendix A

42

Figure A1: Distribution of Japanese quail embryos with teratogenic deformities between treatment groups. a) Control, b) 0.01ug Genistein ,c) Vehicle Control, d) 0.1ug Genistein, e) 0.01ug β-estradiol, f) 1ug Genistein. Each treatment group consist of n=30 quail eggs.

43

Appendix B

Comparative gonadal observations between normal and feminized embryo development: The appearance of treated and control gonads in genetically male embryos are illustrated in Figures B1, B2, B3, B4. Whereas in untreated and corn oil-treatedembryos, left and right gonads appeared to be symmetrical (FigureB1). After various treatments, feminized chicks showed to have areduced size of the right gonad, while the comparatively much larger left gonad assumed the general appearance of an ovary (ovotestis) (Figure B2). It had a more rugose outline than did the control gonads. Wolffian ducts were usually thicker and

signs of an incipient ovarian cortex could be made out along its lower edge. Some chicks exposed to estradiol and 1 µg/egg genistein treatment was observed to have two swollen testis (ovotestis) and retained Müllerian duct (Figure B3). Higher concentration of genistein also produced deformed testis with ovarian like features, such as thicker Wolffian duct, swollen vasdeferens and distorted and flattened testis (Figure B4).

*Note in interpreting the gonads of reproductive anatomy. The figures are described as if the chick is standing facing away from you. During dissections, the bird is on its back, therefore

44

you must reverse these (so left is actually right). There are also different degrees of feminization, and many embryos that

underwent feminized development had subtler degrees of feminization. The figures demonstrated the extremes of feminization along with examples of normal reproductive system for quail embryos.

Most of all of the genetically female embryos had a left ovary and a vestigial right gonad, along with a left Müllerian duct andvestigial right Müllerian duct (Figure B5). After exposure to various treatments, some quails showed to have been over-

45

Figure B1: Example of normal male reproductive anatomy for Japanese quail (Coturnix japonica) showing symmetrical testis and a pair of Wolffian ducts control treated chick. See note below (*).

Figure B2: Example of feminized treated male reproductive anatomy exposed to genistein demonstratinga small right testis and ovotestison the left along with a thicken Wolffian duct and signs of an

Figure B3: Example of feminized treated male reproductive anatomy exposed to genistein. Showing a pair of swollen testis (ovotestis) and retained left Müllerian duct. See note above (*).

Figure B4: Example of feminized treated male reproductive anatomy exposed to genistein. Showing a pair of distorted testis with thickWolffian duct and signs of an incipient ovarian cortex could be

feminized, where the left ovary and its Müllerian duct will become overly enlarged and the right side a developed retained Müllerian duct (Figure B6).

Figure B5: Example of normal femalereproductive anatomy, demonstratingthe left ovary, the right vestigialgonad, the left Müllerian duct and

Figure B6: Example of over-feminized female exposed to treatments, showing an enlarged left ovary with its vestigial gonadand right retained Müllerian duct

46