International Peer Reviewed Journal

Soybean-Genistein as Endocrine
Disruptor on Estrous Cyclicity

and Ovarian Follicular Development
in Albino Rats (Rattus norvegicus)

GERALDINE C. SANCHEZ
ORCID NO.: 0000-0003-4628-227X

gengsanchez923@gmail.com

RONALDO D. DIZON

ARIS F. MICLAT
ORCID NO.: 0000-0003-3749-8570

aris.miclat@gmail.com
Institute of Veterinary Medicine and Zootechnics

Pampanga Agricultural College
Magalang, Pampanga

MARIA FE S. BULAO
ORCID NO.: 0000-0002-6049-3885
mariafesimbulanbulao88@gmail.com

JULIA A. EGGERT
ORCID NO.: 0000-0003-1350-8420

Jaegger@clemson.edu
College of Health, Education and Human Development,

Edwards Hall, Clemson University, Clemson,
South Carolina U.S.A. 29631

Vol. 12 · March 2013
Print ISSN 2012-3981 • Online ISSN 2244-0445
doi: http://dx.doi.org/10.7719/jpair.v12i1.209

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by PAIR, an ISO 9001:2008 QMS certified

by AJA Registrars, Inc.

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ABSTRACT

Endocrine disruptors are chemicals that interfere with the body’s endocrine system
and produce adverse developmental, reproductive, neurological, and immune effects
in both humans and wildlife. One example of endocrine disruptor is phytoestrogen
which is a group of naturally occurring compounds that have been reported to cause
fertility problems in animals. The major phytoestrogen in soy products is genistein,
which has potent estrogenic activity both in vitro and in vivo. Previous findings have
demonstrated that the control of primordial follicle development and subsequent
folliculogenesis appears to be mediated by local production and action of specific
paracrine factors. Preliminary studies also have shown that steroid hormones like
estrogen play a critical role in the onset of primordial follicle assembly. These findings
led us to further look into the effects of genistein on estrous cyclicity and ovarian
folliculogenesis specifically on pre-antral and antral follicular development including
their possible effects on ovarian morphometry of sexually matured female albino rats.
The objectives of the study was to determine the effects of genistein on estrous
cyclicity and ovarian folliculogenesis specifically on pre-antral and antral follicular
development including their possible effects on ovarian and uterine morphometry
of sexually matured female albino rats. Furthermore, the study elucidated its effect
on the apoptosis of granulose and theca cells resulting to follicular atresia. A total of
24 female albino rats approximately 2-3 months of age of almost the same size were
used in this study. The treatments were: Control (T0) distilled water, 8 mg/kg body
weight genistein (T1), 12.5 mg/kg body weight genistein (T2) and 16 mg/kg body
weight genistein (T3). Estrous cyclicity was determined using vaginal cytology. The
experimental animals were sacrificed after five weeks and their ovaries and uterus were
collected. Ovarian tissues were subjected to Paraffin technique for tne microscopic
examination. All data gathered were subjected to One-Way Analysis of Variance
(ANOVA) and significant differences among treatments were analyzed using Least
Significant Difference (LSD). Results showed an increased length of proestrus and
estrus period in treated rats, metestrus on the first week of treatment and diestrus on
the second week of treatment period. In terms of antral and preantral follicles, rats
treated with genistein have greater mean number compared with the control and the
mean number of non-atretic follicles was high in the control group and T4 . Genistein
treated rats at 12.6 and 16 mg/kg body weight have greater mean number of pre-
antral and antral follicles as compared with those treated at 8 mg/kg body weight
and the control. Genistein in soybean has endocrine disruption effect by altering
estrous cyclicity and ovarian folliculogenesis but it has no adverse effect on heart and
respiratory rates as well as on body temperature.

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Keywords - Antral Follicle, Estrous Cycle, Genistein, Ovary and Soybean

INTRODUCTION

There is a considerable concern on endocrine disruptors which are chemicals
that interfere with the body’s endocrine system and produce adverse developmental,
reproductive, neurological, and immune effects in both humans and wildlife (www.
niehs.nih.gov,2000)[2]. One example of endocrine disruptors is phytoestrogen which
is a group of naturally occurring compounds that have been reported to cause fertility
problems in animals. Of particular concern is genistein, the major phytoestrogen in
soy products, which has potent estrogenic activity both in vitro and in vivo (Diel,
2001). It can bind to the estrogen receptor to induce estrogen-like effects in animals,
humans and cultured cells (Liu, 2006).

The possibility that some chemicals may disrupt the endocrine systems in
humans and animals has received considerable attention in the scientific and public
community. Endocrine disruption is on the agenda of many experts’ groups, steering
committees and panels of governmental organizations, industry, and academia
throughout the world. Because the disturbance of the endocrine system is a very
sensitive topic, scientific findings or observations are often controversially discussed
among scientists, environmentalists, and authorities (Lintelmann, 2003).

Soy and products derived from soy, such as soya milk, tofu, tempeh, soy flour, soy
sauce, taho and isoflavone supplements, are being consumed in increasing quantities
by humans. Similarly, high quantities are used as a feed ingredient for laboratory,
companion and food animals (Brown, 2001).

Although enormous progress has been made in understanding the events and
regulation of the later stages of ovarian follicular development, the early stages of
development, to a large extent and particularly in large mammals, remain a mystery.
Mechanisms that regulate the initiation of follicular growth and the ensuing growth
and differentiation of preantral follicles are of considerable interest, since their
elucidation is a prerequisite to use of the primordial pool to enhance reproductive
efficiency in domestic animals, humans, and endangered species (Fortune [15]2003).

The study of Kouki (2003) on the effects of neonatal treatment with
phytoestrogens, genistein and daidzein, on sex difference in female rat brain function
obtain findings in genistein treated groups that ovaries were smaller and contained no
corpora lutea. Ovaries from daidzein treated females were also small.

Moreover, the study of Flyn (2000) on the effect of genistein in sexually dimorphic
behavior of rats revealed findings of decreased in average weight per live pup at birth.

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High amounts of isoflavones given to newborn rats resulted in disturbance of
estrous cyclicity at a very early stage of life (Delclos, 2009). Experimental data from
cell cultures and whole animal studies showed that such concentrations had strong
estrogenic effects (Mueller, 2004).

Previous research and preliminary studies have demonstrated that the control
of primordial follicle development and subsequent folliculogenesis appears to be
mediated by local production and action of specific paracrine factors involving theca
cells, granulose cells, and the oocyte. Preliminary studies also have shown that steroid
hormones like estrogen play a critical role in the onset of primordial follicle assembly
(Skinner[30] 2008).

These findings led us to further look into the effects of genistein on estrous
cyclicity and ovarian folliculogenesis specifically on pre-antral and antral follicular
development including their possible effects on ovarian morphometry of sexually
matured female albino rats. Also, the effect of genistein on cellular changes
particularly on apoptosis of granulose and theca cells resulting to follicular atresia
was investigated.

OBJECTIVES OF THE STUDY

The general objective of the study was to elucidate the effects of genistein on
estrous cyclicity and ovarian follicular development using rats as the animal model.

Specifically, it aimed to determine genistein’s effects on:

1. different stages of the estrous cycle;
2. body temperature, heart and respiratory rates;
3. gross changes in the heart, liver, lungs and
kidney;
4. body weights;
5. uterine morphometry (horn length and width);
6. ovarian morphometry (length, width, weight)
7. antral and pre-antral follicles
8. atretic and non-atretic follicles

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MATERIALS AND METHODS

Experimental Animals

A total of 24 female albino rats approximately 2-3 months of age of almost the
same size were used in this study. They were caged individually in 6 x 6 x 8 inches
cage and were given one-week acclimatization period prior to the conduct of the
study. The experimental rats were housed at the Animal Research Laboratory of
the Institute of Veterinary Medicine and Zootechnics at room temperature with a
relative humidity of at least 30 % and not exceeding 70 % measured with the use of a
digital thermometer (CDR- KING, 2010). The heart rate, respiratory rate and rectal
temperature were taken every other day to avoid additional stress on the rats. Body
weights were taken using a triple beam balance (OHAUS).

Preparation and administration of genistein

Genistein was purchased from Xi’an Feida Bio-Tech Co., Ltd, China with a purity
of 95 %. In preparing dosages of genistein powder, the manufacturer’s prescription
was used. Moreover, the dosage used by Zhou (2008) which is 12.5 mg/kg body
weight were adopted in the study to serve as baseline for dosage determination.
Genistein powder was dissolved in normal saline solution and was administered to
rats subcutaneously using a glass syringe.

Monitoring of Estrous Cycle

In determining the estrous cyclicity of rats, the protocol of Reyes (2006) was
adopted. Data on estrous cycle was taken two weeks before the start of the study (D0)
until the rats were sacrificed. Daily monitoring of estrous cycle was done through
microscopic examination of sample vaginal smear. The estrus stage was determined
by the presence of abundant cornified cells in the smear. The rats were sacrificed
through cervical dislocation a week after the two (2) weeks treatment period when
they were at the diestrus stage.

Collection, gross examination and processing of ovaries, uterus and other
visceral organs

The experimental animals were sacrificed after five weeks and their ovaries and
uterus were collected. The ovaries, uterus, heart, lungs, liver and kidney were observed

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for gross changes. With the use of a digital weighing scale, the ovaries were weighed
and the length and width were measured. The collected ovaries, uterus and lungs with
gross lesions were then preserved in ten percent (10%) formalin solution and were
brought to Histopathology Laboratory of University of the Philippines, Los Baños,
Laguna for processing using the Paraffin Technique. The thickness of the ovarian
section used in this study was 5 microns (Sanchez, 2005) and the processed tissues
were stained using hematoxylin and eosin (H & E).

Microscopic examination of processed tissues

Processed ovarian, uterine and lung tissues were examined using a microscope
at 100 x magnification. Pre-antral, antral, atretic, and non-atretic follicles were
identified and quantified on the ovaries. The uterine and lung tissues were observed
for histopathological lesions.

Experimental design and treatments

A Complete Randomized Design was used in this study. The experimental animals
were randomly distributed in four treatments with six replications per treatment by
means of draw lots. The treatments were as follows:

T1 – distilled water (control)
T2 – 8 mg/kg body weight genistein
T3 – 12.5 mg/kg body weight genistein
T4 – 16 mg/kg body weight genistein

RESULTS AND DISCUSSION

Length of proestrus period

The proestrus stage is composed of nucleated epithelial cells and can be seen for a
period of 12 - 14 hours in a 5-day estrous cycle. This could be increased or decreased
depending on several factors such as stress and hormonal problems. And at this stage
the female rat make acceptance to the male at the end of the phase (www.lssu.edu)[3]

As reflected in Table 1, the proestrus stage was observed on the average, for
a period of once a week for the control. Those treated with genistein showed an
increased in their proestrus stage especially during the second week of treatment
period. It also appears that although T2 received a dose of 8mg/kg body weight of

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genistein only, it had an increase of 0.17 days proestrus stage during the first week of
treatment period. On the second week of treatment, T2 maintained its proestrus stage
of 1.17 days however, for T3 which have received a dose of 12.5 mg/kg body weight
of genistein and T4 with 16 mg/kg body weight of genistein, showed an increase of
0.6 and 0.67 days, respectively. On the withdrawal of genistein, all of the treatments
resume on their once a week average proestrus stage.

The increased in the proestrus stage during the second week of treatment best
describes that genistein is highly absorbed and elicited its effect to prolong the estrous
cycle by increasing this stage on T2, T3 and T4.

Since the monitoring of estrous cycle was done only on a per day basis, the exact
proestrus length in terms of hours (hr) was not observed by the researcher.

Table 1. Length of proestrus during pre-treatment, first week of treatment,
second week of treatment and post-treatment period (days)ns

Treatment Pre-treatment 1st week 2
nd

week
Post-

Treatment
T1 1.00 1.00 1.00 1.00
T2 1.00 1.17 1.17 1.00
T3 1.00 1.00 1.60 1.00
T4 1.00 1.00 1.67 1.00

ns- not significantly different (P>0.05)

Length of estrus period

Estrus stage is characterized by the presence of 75% nucleated epithelial cells
and 25 % cornified cells on the vaginal smear. This could be observed for a period
of 12 – 27 hours in a 5-day estrous cycle. Estrus can be detected when the vulva
becomes slightly swollen and the vagina becomes dry in contrast to the usual moist
pink. Female rats in heat are hyperactive and brace themselves when touched. The
ears quiver when they are stroked on the head or back, and touching the pelvic region
induces a posture termed lordosis, in which the head and rump are raised and the
back is arched downward.

As shown in Table 2, there were differences in the length of estrus period of
rats treated with genistein compared with the control (T1). On post treatment, all
treatments showed significant difference showing longer estrus for T3. Treatment 1
showed longer estrus in the first week only but not on the second week. T2 exhibited
an increase in estrus length during the first and second week of treatment with

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genistein compared during the pre-treatment period .On the withdrawal of genistein,
a slight increase in estrus length was also noted.

The effect on T3 exhibited more during the post treatment period which exhibited
longer estrus than the other treatments. For the Control (T1), it could be noted that
a slight increase in estrus length from 1 at pre-treatment to 2 days on the first week
of treatment period as shown in Table 2, was observed during the time that the
other rats were treated with genistein. This could be explained by the presence of the
vomeronasal organ present in rats which primarily detect pheromones that specialize
in non-volatile chemicals found in the urine and other secretions. The introduction
of genistein on the other rats by means of the vomeronasal organ of the Control
rats shoot up a separate pathway to the accessory olfactory bulbs, and from there to
the amygdala, then to both the preoptic area and the hypothalamus, which are areas
known to be involved in reproductive behavior (Brennan, 2001).

Based on the findings of this study, an increased in estrus length as shown in
Treatments 1, 2 and 3 during the first and second week of treatment is due to the
action of genistein, which mimicks the hormone estrogen in the body. This finding is
significant in prolonging the estrus period on the average by 1.5 days longer than the
normal estrus length of 1.0 day only in female rats. Thus, soybean-genistein prolongs
and enhances sexual receptivity and sexual activity. This breakthrough could help in
addressing fertility problems both in animals and human beings. Women who are
on their menopausal stage may have exogenous source of estrogen as in this case, the
genistein present in soybeans which could help in the prevention of vaginal dryness
thus improving sexual activity. Further study on this aspect is therefore necessary.

Table 2. Length of estrus during pre-treatment, first week of treatment,
second week of treatment and post-treatment period (days)

Treatment Pre-treatment 1st week 2nd week Post-Treatment
T1 1.00 2.00 1.00 1.00d

T2 1.00 2.16 1.83 2.67c

T3 1.00 1.50 1.67 3.17a

T4 1.00 2.50 2.17 2.83b

Means having different superscripts are significantly different at (P<0.05)

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Length of metestrus period

The metestrus stage is characterized by the presence of many leukocytes with
nucleated and cornified cells in a vaginal smear. It can be observed for a period of 21
hours in a 5-day estrous cycle and the female rat is observed with no male acceptance.

As revealed in Table 3, there was an increased in the length of metestrus stage
of treated rats. During the first week of treatment period, T2, T3 and T4 gained an
increase of 0.83, 1.17 and 1, days, respectively. On the second week of treatment
and post treatment periods, there was a general decrease in the length of metestrus
compared during the first week.

The increase on the metestrus stage during the first week of treatment can be
associated to the effect of genistein administration. However, the increase of 0.5 day
metestrus stage on the control (T1) during the second week of treatment period was
just normal to meet the normal 5-7 days length of estrous cycle and can be compared
to a slight decrease on the metestrus stage of treated rats as affected by genistein.
Also, the long metestrus period during the post-treatment period of treated rats is
correlated to the increase in average estrus length of treated rats on the same period.

Table 3. Length of metestrus during pre-treatment, first week of treatment,
second week of treatment and post-treatment period (days)

Treatment Pre-treatment 1st week 2nd week Post-Treatment
T1 1.00 1.00d 1.00 1.00
T2 1.00 1.83c 1.17 1.00
T3 1.00 2.17a 1.60 1.00
T4 1.00 2.00b 1.67 1.00

Means with different superscript in the same column are significantly different
(P<0.05)

Length of diestrus period

Diestrus stage is characterized by the presence of leukocytes in a vaginal smear
and this can be observed for a period of 57 hours in a 5-day estrous cycle. During this
stage, the female rat has no male acceptance.

As shown in Table 4, the length of diestrus period prior to genistein treatment (Pre-
treatment period) revealed same length of all treatments with a mean of 4. However,
on the 1st and second week of treatment, there was a marked decrease in diestrus

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length of treated rats as compared with the control (T1) and the whole pre-treatment
period except for T3 which showed longer diestrus of 4.4 days. Furthermore, on the
post-treatment period, an increase of 0.67 day was again observed in the control (T1)
while a decrease of 0.33 day on T1, 1.73 on T2 and 0.33 day on T3. This could be
explained by an increase in the proestrus, estrus and metestrus stage of rats will result
to a decrease on the length of diestrus stage. A decrease on the proestrus, estrus and
metestrus stage also result to an increase on the length of diestrus stage of rats and
vice versa.

Table 4. Length of diestrus during pre-treatment, first week of treatment,
second week of treatment and post-treatment period (days)

Treat-ment Pre-treatment 1st week 2nd week Post-Treatment
T1 4.00 3.00a 3.33 4.00a

T2 4.00 1.83ab 3.00 2.67ab

T3 4.00 2.17ac 4.40 2.67ab

T4 4.00 1.50ad 2.83 2.5ad

Means with different superscript are different at (>0.05).

Mean body temperature

As revealed in Table 5, the rectal temperature values obtained from Treatment
1, 2 and 3 were within the normal temperature of rats which indicate absence of
hyperthermia post genistein treatment.

Table 5. Mean body temperature (°C)ns

Treatment 1st week 2nd week
T1 36.7 36.9
T2 36.8 36.9
T3 36.7 36.9
T4 36.5 36.9

ns- not significantly different at (P>0.05).

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Effects on heart rate and gross appearance

As shown in Table 5, there is a decreased in the heart rate of rats during the 2nd
week of treatment although their values fall on the normal range. In the study of Al-
Nakkash (2010), genistein had no effect on the weights of heart and heart-to-body
ratio. Moreover, fat pad significantly decreased heart rate and pulse pressure. No
pathological gross lesions were found in the heart.

Table 5. Mean heart rate (beat/minute)ns

Treatment 1st wk 2nd wk
T1 346 298
T2 346 296
T3 348 292
T4 349 280

ns- not significantly different at (P<0.05).

Effects on respiratory rates, gross and microscopic appearance of the lungs

As revealed in Table 6, there was no significant difference noted among treatments
during the first and second week of treatment although it appears that during the
second week, the respiratory rates fall until the termination of the study. However,
the values obtained are still within the normal range. Pathological gross lesions were
found on the lungs of T1 and T2 wherein they were pale, hard and granulomatous.
Sneezing was also observed on rats during the second week of treatment period.

Microscopically, the lungs have numerous neutrophils, hemorrhage and
congestion which is suggestive of pulmonary emphysema. The study of Yellayi
(2002) determined that genistein treatment suppressed immune system function.
Genistein-treated mice might have produced lower amounts of antibodies following
administration and it might have contributed to the gross lesions found in the lungs.

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Table 6. Mean respiratory rate (breaths/minute)ns

Treatment 1st Week 2nd Week
T1 91.0 89.2
T2 91.0 88.7
T3 91.0 89.4
T4 92.0 88.4

ns-not significantly different at (P>0.05)

Body weights

Shown in Table 8 is the mean body weight of mice. During the first week, there
was no significant difference noted among treatments but on the second week, there
was a reduction in the body weight of treated group and the untreated remained to
have the heaviest weight. This suggests that genistein influence the body weight of
rats.

Table 8. Mean body weights (grams)

Treatment 1st Week 2nd Week
T1 244 261a

T2 253 242ab

T3 222 209bc

T4 222 210b

Means with different superscripts in the same column are significantly different
at (P<0.05) level.

Length of uterine horn, gross and microscopic appearance

Reflected in Table 9 is the length of the uterine horn. Statistical analysis revealed
that T3 exhibited the longest left horn. Generally, the increased in the uterine length
of treated rats as compared to the Control is suggestive of uterine hypertrophy. Also
on microscopic examination , treated rats exhibited hemorrhages, congestion and
hypercellularity of the myometrium and endometrium.

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Table 9. Length of the uterine horn (mm)

Treatment Right horn Left horn
T1 27.5 23.7bd

T2 30.7 26.0b

T3 32.5 29.0a

T4 29.0 25.5bc

Means with different superscripts in the same column are significantly different
at P (< 0.05) level.

Width of the uterine horns

As shown in Table 10, there is no difference on the width of the left and right
uterine horns among treatments.

Table 10. Width of the uterine horns (mm)ns

Treatment 1st week 2nd wk
T1 2.33 2.33
T2 2.67 2.67
T3 3.00 3.00
T4 2.67 2.67

ns-not significantly different at (P>0.05)

Mean ovarian length

Revealed in Table 11 are the length of the right and left ovaries showing no significant
difference among the treatment groups.

Table 11. Mean ovarian length(mm)ns

Treatment Right ovary Left ovary
T1 6.5 6.0
T2 5.8 5.5
T3 5.2 4.7
T4 5.5 4.8

ns-not significantly different at (P>0.05)

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Mean ovarian width

Table 12 presents the mean width of the right and left ovaries. It appears that
there was no significant difference among treatments in the right ovary but there was
among the left showing a reduction in the width of the treated group especially the
one that received the highest amount of genistein. At (P<0.01).

Table 12. Mean ovarian width (mm)ns

Treatment Right ovary Left ovary
T1 5.83 5.67a

T2 5.33 5.00ab

T3 4.67 4.17b

T4 5.00 3.67c

Means with different superscripts in the same column are significantly different
at (P<0.05) level.

Mean ovarian weight

Shown in Table 13 is the mean ovarian weight. There was no significant difference
among the treatments in the right ovary but there was in the left ovary at (P<0.01). All
treated animals manifested much lower ovarian weight which implies that genistein
affects ovarian weight.

Table 13. Mean ovarian weight (grams)

Treatment Right ovary Left ovary
T1 0.063 0.059b

T2 0.057 0.054ab

T3 0.050 0.045a

T4 0.054 0.035a

Means with different superscripts in the same column are significantly different
at (P<0.05) level.

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Mean preantral and antral follicles

Shown in Table 14 are the mean pre-antral and antral follicles showing significant
difference (P < 0.05) in the mean number of pre-antral follicles of treated groups
compared with the control. The highest number of pre-antral follicles was observed
in T4 followed by T3, T2 and T1.In terms of antral follicles, there was no significant
difference among treatments which implies that genistein affects only growing
follicles.

Zhuang (2010) in their study in genistein treated rats showed a higher percentage
of primordial follicles by 4 months of age and a greater number of surviving follicles
at 15 months of age compared to a control group (P < 0.05). In addition, vaginal
cytology showed that age-dependent cessation of regular estrus was delayed for
2 months in genistein-treated group than control group which suggest that genistein
alters rat ovarian follicular development and increases the number of surviving
follicles, which may prolong ovarian reproductive life.

Table 14. Mean pre-antral and antral follicles (grams)

Treatment Pre-antral Antral
T1 1.83cd 11.5
T2 1.80c 10.4
T3 6.17b 12.0
T4 8.17a 12.5

Means with different superscripts in the same column are significantly different
at (P<0.05)

Mean Number of Atretic and Non-atretic follicles

As reflected in Table 15, untreated rats had the least number of pre-antral follicles
that underwent apoptosis of granulose and theca cells. However, among the treated
rats, findings revealed that T3 had the highest mean number of 1.83 followed by
T1 with mean number of 1.4 and T4 with mean number of 1.33. Furthermore, T3
had the highest mean ratio of atretic to antral follicles of 0.15 followed by T2 with
a mean ratio of 0.13 and T3 with mean ratio of 0.11. This indicates that for every
100 antrals, 15 became atretic in T3, 13 in T2 and 11 in T4. The Control (T1) got
the least with 10 atretic follicles for every 100 antrals. Statistical analysis revealed no
significant at difference at (P<0.05). In terms of the mean number of non-atretic

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antral follicles, there was also no difference noted among treatments showing T4 to
contain the most number of non-atretic follicles.

The results of this study show that genistein alters the rat ovarian follicular
development resulting to higher number of surviving follicles. Pubertal genistein
treatment can possibly lead to follicular atresia. However, genistein treatment at old
stages of rats increases the number of surviving follicles as obtained from the results
of the study of Zhuang (2010) because at this stage, estrogen level is triggered by
different factors.

Table 15. Mean number of atretic and non-atretic follicles

Treatment Atretic Non atretic
T1 1.00 10.50
T2 1.40 9.00
T3 1.83 10.20
T4 1.33 11.17

CONCLUSIONS

Based on the findings of the study, the following are concluded:

1. Genistein exerted effects even at the lowest dosage of 8 mg/kg body weight
of rats.

2. Genistein altered the estrous cyclicity of rats. The length of estrus was
increased in treated rats while that of diestrus decreased.

3. Genistein causes atrophy of the ovaries.
4. Genistein has weight reducing effects.
5. There are no adverse effects on the vital signs.
6. Rats treated with genistein have greater mean number of pre-antral and

antral follicles as compared with the control.

RECOMMENDATIONS

a. Do hormonal assays specifically on the level of endogenous estrogen present
per rats

b. Do clinical trial trials in livestock as well as human beings particularly those
with fertility problems.

c. The effects of genistein on cholesterol level

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d. The actual amount of genistein absorbed and metabolize by body tissues
e. The frequency of vaginal discharge collection in order to evaluate the exact

length of estrus stage in terms of hours
f. The effects of genistein on different ages of rats
g. The effects of genistein in male reproductive tract.

LITERATURE CITED

Al-Nakkash L, Markus B, Batia L, Prozialeck W and Broderick L.
2010 “Genistein Induces Estrogen-Like Effects in Ovariectomized Rats but Fails to
Increase Cardiac GLUT4 and Oxidative Stress.”Jmed food (6):1369-75

BP
2000 ‘’Endocrine Disruptors’’ Human Reproduction.Yahoo Appl [ Internet]

National Institute of Environmental Health Sciences – National Institute
of Health. [cited 2010 August 21].Available from: http://www.niehs.nih.
gov/health/topics/agents/endocrine/index.cfm

BP
2000 ‘’The Laboratory Rat’’. Google Appl [Internet]. Iowa State University.
Available from: http://www.lssu.edu/faculty/jroese/AnimalCare/Rat/

OccHealth.htm

BP
2000 ‘’The Ovarian Process’’. Google Appl [Internet].Available from: http://www.

siumed.edu.html, cited August 17

Brennan P.
2001 “The vomeronasal system’, Google Appl [Internet]. [cited 2010 December

28].Cell Mol Life Sci. 58(4):546-55,2001

Brown N and Setchell K.
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