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ORIGINAL ARTICLE
Aflatoxin B1 effects on ovarian follicular growth and atresia
in the rat
Shapour Hasanzadeh &Saeedeh Amani
Received: 13 August 2011 /Accepted: 16 February 2012
#Springer-Verlag London Limited 2012
Abstract For this investigation, 28 female healthy adult
Wistar rats were selected. The animals were divided into
four groups (n07 per group): control, test group 1, test
group 2 and test group 3. Each rat in test groups 1, 2 and
3, received 0.8 ppm, 1.6 ppm and 3.2 ppm aflatoxin B1
(AFB1), respectively, via gavage for a period of 25 days.
The control group received distilled water only. All tissue
specimens were processed for routine paraffin embedding
and serial cross-sections cut at 5–7μm and stained with
haematoxylin–eosin. Both histomorphologic and histomor-
phometric analysis was performed under light microscopy.
An increase in the concentration of AFB1 resulted in a
reduction in the population of healthy primordial, primary,
secondary and tertiary follicles. The greatest reduction was
in seen in group 3 (with 3.2 ppm AFB1/day). In all test
groups, due to an increase in AFB1 concentration, in both
the right and left ovaries, all types of atretic follicles, in-
cluding primordial, primary, secondary, and tertiary atretic
follicles were significantly increased (P<0.01). In conclusion,
AFB1 is toxic for all type of ovarian follicles, including non-
growing and growing follicles and exerts an atretogenic effect
on all types of ovarian follicles. The atretogenic effect of
AFB1 is dose dependant. Due to its toxic effects (gametotox-
icity), the resting pool ofovarian follicles (primordial follicles)
decreases significantly. The ovulatory follicular population
either decreases or is completely depleted.
Keywords Aflatoxin B1 .Follicular growth .Follicular
atresia .Rat
Introduction
The storage of cereals in silage, in tropical and sub-tropical
regions generally where ambient temperature and humidity are
high, fungi such as Pencillium,Fusarium and Aspergillus
species have a tendency to grow (Peraica et al. 1999).
Aflatoxins B
1
,B
2
,G
1
and G
2
are mycotoxins that may be
produced by three moulds of the Aspergillus species:
Aspergillus flavus,Aspergillus parasiticus and Aspergillus
nomius, which contaminate plants and plant products.
Aflatoxins M
1
and M
2
, the hydroxylated metabolites of afla-
toxin B
1
and B
2
, may be found in milk or milk products
obtained from livestock that has been ingested in contaminated
feed. Aflatoxin M1 is also seen in human breast milk (El-
Nezami et al. 1995). To date, 17 types of aflatoxins have been
identified, of these, aflatoxin B
1
(AFB1) is the most frequent
one present in contaminated samples; aflatoxins B
2
,G
1
and G
2
are generally not reported in the absence of AFB
1
. Most of the
toxicological data relates to AFB
1
. Dietary intake of aflatoxins
occurs mainly from contamination of maize and groundnuts
and their products (Clarke et al. 1987; Ostrwski-Meissner
1983).
The toxins of these moulds cause considerable economic
burden in the animal industry (Diekman and Green 1992).
The low levels of aflatoxins which are consumed alongside
S. Hasanzadeh (*)
Department of Basic Science, Histology and Embryology
Sections, Faculty of Veterinary Medicine, Urmia University,
Urmia, Iran
e-mail: shasanzadehs@yahoo.com
S. Amani
Department of Veterinary Basic Sciences, Histology and
Embryology Sections, Faculty of Veterinary Medicine,
Urmia University,
Urmia, Iran
e-mail: Saeedeh_amani@yahoo.com
Comp Clin Pathol
DOI 10.1007/s00580-012-1446-1
foods may not produce noticeable physiological symptoms
but may cause subtle damage to the reproductive capability
of these animals resulting in economic loss (Jones et al.
1982).
Aflatoxins enter the body either directly through consump-
tion of contaminated food or indirectly by animal products
(Kovacs 2004). These toxins are generally heat and light
resistant. After entering the body, they exert their deleterious
effects on different organs such as the gonads. Their carcino-
genic impact has been documented for decades but the anti-
fertility effects of these toxins are still under investigation.
The ovarian follicles of mammals are distributed within the
cortical area of the ovaries, beneath the tunica albuginea. Two
types of follicles are present in the ovaries: non-growing or
quiescent (90–95% of the ovarian follicles) and growing
follicles. The primordial follicles (PMFs) are the resting pool
of the follicles, from which follicles are recruited for growth,
thus the PMFs are the basic units of an ovary (Erickson 1986).
The PMFs are in turn produced from primordial germ cells
before birth. All PMFs enter prophase of meiotic division
before birth, and with increasing age, the number of these
follicles undergoes a steady decline whereby at the end of the
reproductive period very few remain.
The growing follicles are primary follicles (PrFs; approxi-
mately 40 μm), secondary follicles (SFs; 200 μm) and mature
follicles (MFs; 10 mm; Wasserman 1988). The MFs have
ellipsoidal fibroblasts which are present at the theca interna
which grow in size and covert in to multihedral cells called
epithelioid cells in which the nuclear chromatin is lighter than
in elliptical cells. Epitheliod cells, especially those in MFs,
increase in number during prooestrus and early oestrus and in
the early stage of follicular atresia and degeneration. Most
ovarian follicles are committed to atresia and death and are
finally removed by phagocytic cells (Erickson 1986).
It has been suggested that a point mutation of gene P53
caused by AFB
1
is responsible for most hepatocellular carci-
nomas of the liver. In order to better understand this concept,
current studies are focused on the interference between AFB
1
and DNA and the mechanism by which AFB
1
connects to
oligodeoxynucleotide 2(ATG CAT)2 (Gopal et al. 1980;
Hafez et al. 1982a,b;Gopalakrishnanetal.1989; Preston
and Williams 2005). Combinations with DNA, prevention of
DNA synthesis and over all DNA damage are important
mechanisms for AFB
1
function (Larsson and Tjalve 1995;
Wang and Groopman 1999).
Entry of aflatoxin into the body may present as clinical
symptoms of aflatoxicosis in the animal. Two types of
aflatoxicosis are predictable, i.e. acute aflatoxicosis which
leads to liver damage, and consequently disease and death,
whereas chronic aflatoxicosis which leads to nutritional
(Ibeh et al. 1994), immunological and other minor patho-
logic anomalies (Silvotti et al. 1997; Sur and Celik 2003;
Faridha et al. 2007).
Embryotoxicity, (Geissler and Faustman 1988;Ibehand
Saxena 1997a,b; Wangikar et al. 2004,2005; Turner et al.
2007), gametotoxicity (Hafez et al. 1982a,b; Agnes and
Akbarsha 2003; Ibeh and Saxena 1997a,b), genotoxicity
(Shimada et al. 1987; Matthiaschk et al. 1990), hypophysio-
toxicity (Abdel-Haq et al. 2000;Clarkeetal.1987), cytotox-
icity (Miele et al. 1996), haematological toxicity (Dietert et al.
1983; Bababunmi and Bassir 1982) and delayed reproductive
development (Doerr and Ottinger 1980) resulting from afla-
toxicosis are the most important chronic effects of AFB
1
.The
individual's sensitivity to aflatoxin is greatly dependant on
species, age, sex and nutritional status; young animals show
a greater sensitivity to the toxin.
In this study, we investigated the deleterious effects of
AFB
1
on the reproductive system of female rats following
exposure to different doses and the consequent effect on
oogenesis in the ovaries. Numerous changes occur in ovar-
ian tissues after impact by AFB
1
, all of which are due to
type 2 aflatoxicosis and lead to a reduction in reproductive
potential of the animal (Sharlin et al. 1980).
Aflatoxins are very potent toxins affecting the growth of all
animals (Abdel-Wahhab et al. 1999) specifically delayed pri-
mary growth and development, failure in locomotor coordi-
nation and learning (Kihara et al. 2000), delay in genital
system growth (Hafez et al. 1982a,b), reduction in fertility
and hatchability in poultry (Howarth and Wyatt 1976), abor-
tion (Ray et al. 1986), high disturbances in oestrus cycle,
reduced pregnancy rate and number of live new born, failure
in nidation and intrauterine death of the foetus.
The toxic effects of aflatoxins are not only seen on the
oocytes, but also on spermatozoa causing reduced sperm mo-
tility and increased anomalies (Ibeh et al. 2000). Infertility in
men is an important detrimental effect of aflatoxins (Ibeh et al.
1994).
It is possible to investigate changes in the gonads of
animals using histological and histometric analysis. In this
study, our principal aim was to detect degenerative changes
in the different categories of ovarian follicles in adult female
rats.
The majority of previous studies have concentrated on the
deleterious effects of these toxins on different body systems
and little attention has been paid to the reproductive system; if
studies exist, they have concentrated on the male reproductive
system. Therefore, in this study, we established a model in
which different doses of AFB1 and duration of the exposure
coincided with ovarian follicular cycle. The effects of the
AFB1 on different types of ovarian follicles (primordial, pri-
mary, secondary tertiary and mature) were distinguished in this
study. The results will help to standardise the destructive con-
sequence of AFB1 on the reproductive system of female rats.
The result may also be extrapolated and provide useful infor-
mation on the potential impact on humans and other mamma-
lian species.
Comp Clin Pathol
Materials and methods
In this study, we evaluated the chronic effects of oral ad-
ministration of AFB1 on ovarian follicles in adult female
rats.
Twe nty- e ight Wi star female rats, weighing 200 ± 15 g
were selected for this study. They were kept in 12-h light/
12-h dark period, 20–30°C temperature and 50–60% rela-
tive humidity conditions. The rats were allocated randomly
in to four equal groups: control, test groups 1, 2, and 3 (C,
G1, G2 and G3), then caged separately. The animals were
fed on standard pellet diet (ad libitum). AFB1 was obtained
from Supleco chemical company (Supleco Park, Bellefate,
PA 168230048). Toxin doses were prepared in distilled
water and groups G1, G2 and G3 received AFB1 at levels
of 0.8, 1.6, 3.2 ppm/1 cc distilled water/animal/day respec-
tively for 25 days by gavage. Control group (group C) was
gavaged with 1 cc distilled water/animal/day for same peri-
od. At the end of the experiment, the animals were anaes-
thetised with xylazine/ketamine and then sacrificed.
The ovaries were dissected and fixed in 10% formal saline
solution and, after complete tissue fixation, the specimens
processed through routine paraffin embedding and serial sec-
tions cut at 5–7μm and stained with Haematoxylin–Eosin.
Both histo-morphological as well as histometric studies were
performed.
Data were analysed by SPSS software. ANOVA and
Duncan's tests were adopted. The histograms and graphs are
prepared using Graphpad prism 4. For the logarithmic behav-
iours of graphs, MATLAB software was used. Significant
differences were set at a level of P≤0.5–0.01.
Results
Histo-morphological observations In the control group, the
surface epithelium of ovaries consisted of simple squamous
epithelial cells or rarely simple cuboidal, the sub-epithelial
tissue was loose connective tissue and beneath this the
tunica albugina. The PMFs were located in the loose con-
nective tissue of the ovarian cortex separately or in clusters.
Different categories of growing healthy, as well as atretic,
follicles including PrFs, SFs TFs and GFs in the cortices of
the ovaries were observed. In the majority of the ovaries, the
corpora luteum was seen in different activity phases.
On histo-morphological observations of the ovaries in rats
which received 0.8 ppm (G1), 1.6 ppm (G2) and 3.2 ppm
(G3), the following changes were seen: (1) Hyperaemia in
ovarian tissues in G3, (2) increase in signs of follicular atresia
with increasing dose of AFB1. (3) Increase in the population
of the PrFs with precautious antrum formation in G3. (4)
Hyperthecosis, increase in antral vesicles near the granulose
cell layer, increase in granulose cells pyknosis and (5) ovarian
interstitial tissue oedema (Fig. 1illustrates the different histo-
pathology signs in the different groups of rats). With an
increase in AFB1 amount, the intensities of follicular atresia
increased accordingly.
Histometric observations Data analysis revealed that signifi-
cant (p<0.01) differences exist between the control and all the
test groups in the populations of healthy PMFs. In all groups
which received AFB1, a reduction in the population of healthy
PMFs in both the right and left ovaries was seen in comparison
to control (Figs. 2a, e and 3a, e), and this condition was also
seen in cases of primary (Figs. 2b, e and 3b, e), secondary
(Figs. 2c, e and 3c, e) and tertiary (Figs. 2d, e and 3d, e)
follicles. The populations of different types of atretic follicles
in the right as well as left ovaries increased significantly (p<
0.01) in test groups in comparison to controls (Figs. 4a,b,c,d
and e and 5a,b,c,dande). As the results indicate, with an
increase in the dose of AFB1 in group G3, the population of
the atretic SFs significantly increased in the right ovaries,
whereas, in the left ovaries differences between G1, G2 and
controls, were not significant, but in G3 a significant difference
was observed between these follicles.
The behaviour pattern of normal ovarian follicles in the
three test groups in comparison to the control group are shown
for both right and left ovaries in Figs. 2e and 3e,respectively.
The numeric distances in normal follicles in the right as well
as left ovaries are relatively identical, and progressive decline
in the population of such follicles are obviously associated
with the increase in the amount of toxin received; the largest
change is seen in group G3.
The comparative analysis of the tertiary atretic follicles
revealed that there is a significant increase in the population of
these follicles in both the right and left ovaries in groups G2 and
G3, but not in group G1. With an increase in dose of the AFB1,
the atresia of the different types of follicles increased accordingly.
The behaviour pattern of ovarian atretic follicles in all
three test groups in comparison to the control group are
shown for the right and left ovaries in Figs. 4e and 5e and
the numeric distances in the atretic follicles in the right
ovaries were reduced, but in the left ovaries, were increased
and were also seen in group G3
Discussion
Reproduction is fundamental for the continuation of all
species. Any factor which causes a disturbance in reproduc-
tion is of biological importance. In medical, veterinary and
biological sciences, reproduction is of prime importance. In
animal and veterinary sciences, research is focused on factor
(s) which have a negative effect on reproduction and have an
impact on economic loss.
Comp Clin Pathol
The synthesis and mechanistic study on potent aflatoxins
(AFs) has revealed that these are highly oxygenated hetero-
cyclic difuranocumarin compounds (Faridha et al. 2007).
Contaminations of food stuffs by AFB
1
is a main prob-
lem in different region of the world including the Middle
East, China, India, Sub-Sahara in Africa and USA (Clarke et
al. 1987; Williams et al. 2004).
The pathogenesis of this toxin was revealed in 1961
(Dafalla et al. 1987). These toxins cause gametoxicity (Ibeh
and Saxena 1997a,b; Agnes and Akbarsha 2003) and embry-
otoxicity (Matthiaschk et al. 1990). These compounds are
carcinogenic (Ward et al. 1975) and especially hepatocarcino-
gen (Preston and Williams 2005). AFs cause immune defi-
ciency in animals and this is more profound in broiler poultry
(Qureshi et al. 1998). AFs can pass through the foeto-uterine
barrier and appear in the foetal circulation and exert their
different noxious effects (Ibeh et al. 2000). A reduction in
egg production is a profound sign of food poisoning with AFs
in poultry (Clarke et al. 1986). It has been reported that AFs
have deleterious effects on oocytes and spermatozoa (Ibeh et
al. 2000). AFB1 was reported to exert deleterious effects on
the reproductive capacity of lab and domestic female animals
(Ibeh et al. 2000;Oliveiraetal.2002; Ogido et al. 2004;
Abdelhamid and Dorra 1990; Abdelhamid et al. 2007).
According to previous investigations, their effects included
decreases in ovarian and uterine size, increases in foetal
resorption, implantation loss and intrauterine death in
aflatoxin-exposed female rats (Ibeh and Saxena 1997a,b
I, II). Histopathological examinations of the ovaries in aflatoxin-
treated mature domestic fowl showed follicular atresia, accom-
panied by cessation of egg production during the whole feed-
ing period (Hafez et al. 1982a,b).
Female factors were thought to be the reason behind all
fertility problems. Whereas, it is now recognised that female
infertility accounts for approximately 40% of all infertility
cases, ovulatory disorders are a predominant cause for wom-
en not being able to conceive and accounts for 25% of
female infertility (Paul and Lauren 2004). There are numer-
ous causes of infertility, such as sexually transmitted dis-
eases, parasitic diseases, physiological and genetic defects
and toxic agents. One of the least understood among these
factors seems to be the impact of toxic agents, including
mycotoxins, on the reproductive performance of humans
(Uriah et al. 2001).
Fig. 1 a Early primary follicle displaying atretic signs. Vacuoles are
present within the ooplasm (arrows) and granulosa cells pycnosis is
obvious (white arrow). H & E, ×400. bAtretic tertiary follicle showing
signs of atresia in ooplasm (black arrows) and in the granulose cell
layer (white arrow) H & E, ×400. cAn oocyte of an atretic secondary
follicle (white arrow), Vacuolization of ooplasm degenerating nucleus
and narrow and weak zona pleucida (black arrow) of oocyte. H & E,
×400. dTertiary follicle (white arrow) which has two degenerating
oocytes floating in its antrum. Large numbers of degenerating granu-
lose cells with pycnotic nuclei are also floating in antrum. In this figure
a primary follicle with precautious antrum formation is seen in the right
distal corner (black arrow) H & E, ×160. ePrimary follicle with
apparently healthy oocyte (arrow) and precautious antrum formation
(asterisk) H & E, ×250. fPrimary follicle with oocyte (arrow) and
precautious antrum formation (cross) H & E, ×160. gPrimary follicle
with double layer of granulose cells degenerating oocyte (asterisk) and
precautious antrum formation (arrow) H & E, ×160. hCortical region
of an ovary showing congested blood vessels (black arrows) and a
primary growing follicle (white arrow) H & E, ×100. iTertiary follicle
with a degenerating oocyte. H & E, ×100
Comp Clin Pathol
Infertility in men is one of the noxious effects of AFB1
(Ibeh et al. 1994). The toxic effects of AFs at different levels
on the growth pattern of different ovarian follicles, i.e. PMFs,
PrFs, SFs, TFs and mature follicles at different dosages has
not been studied. This study investigated what amount and to
what extent AFB1 exerts its deleterious effect on different
types of ovarian follicles. In this study, two important param-
eters, i.e. amounts of toxin (0.8 ppm, 1.6 ppm and 3.2 ppm/
day) through an oral route, in three different test groups were
considered to verify, what amount(s) of this toxin is/are exert-
ing atretogenic effect on the ovarian follicles and which type
(s) of follicle(s) is/are more sensitive to toxin. We set the time
of the toxin reception based on the duration of normal folli-
culogenesis in the rat, which is 25 days. The noxious effects of
AFB1 on the right and left ovaries of each animal were carried
out separately. The statistical analyses revealed that an in-
crease in the amount of AFB1, i.e. 0.8, 1.6 and 3.2 ppm/rat/
day for a period of 25 days, brought about a progressive
reduction in the population of PMFs in both of the right and
left ovaries proportionate to the toxin quantities. This order
was factual in the case of the PrFs, SFs and TFs in both of the
right and left ovaries. This study revealed that with an increase
in the amount of AFB1 all of the ovarian healthy follicle
categories diminished (Figs. 2and 3).
Fig. 2 a Comparative disposition of the normal primordial follicles (N.
Prel.F.R), bnormal primary follicles (N.Pr.F.R), cnormal secondary
follicles (N.S.F.R), dnormal tertiary follicles (N.T.F.R), ecomparative
behaviours of different normal follicles (Rright, Nnormal, Ffollicle,
PL primordial, Pr primary, Ssecondary and Ttertiary) in the right
ovaries of control and test groups 1,2 and 3 (Con, G1, G2 and G3).
*P<0.001
Comp Clin Pathol
For verification of AFB1 effects, an assessments of
the different types of atretic follicles in both the right and left
ovaries were also considered, and from the results of both the
right and left ovaries in all test groups when compared to
controls, an increase in the amount of AFB1 resulted
in an increase in the different types of the atretic follicles
(Figs. 4and 5).
As the results of this study indicate, AFB1 has deleteri-
ous effects on all categories of ovarian follicles, but this
effect differed in the quiescent as well as different types of
growing follicles and is more likely to be due to differences
in the sensitivity of the different categories of ovarian fol-
licles to noxious effects of AFB1.
Apoptosis of granulosa cells during follicular atresia is
preceded by oxidative stress, partly due to a drop in antiox-
idant glutathione (GSH). Under oxidative stress, GSH re-
generation is dependent on the adequate supply of NADPH
by glucose-6-phosphate dehydrogenase (G6PD). Lower
G6PD activity in large follicles indicates a higher suscepti-
bility to oxidative stress associated with apoptosis progres-
sion in follicle atresia. The lower G6PD activity in the large
follicles is associated with a greater susceptibility to oxida-
tive stress, which produces a higher degree of protein oxi-
dation during follicular atresia. The induction of apoptosis
of granulosa cells during atresia in small follicles is associ-
ated with GSH depletion probably due to a low NADPH
Fig. 3 a Comparative disposition of the normal primordial follicles (N.
Pl.F.L), bnormal primary follicles (N.Pr.F.L), cnormal secondary
follicles (N.S.F.L), dnormal tertiary follicles (N.T.F.L), ecomparative
behaviours of different normal follicles (Lleft, Nnormal, Ffollicle, PL
primordial, Pr primary, Ssecondary and Ttertiary) in the left ovaries of
control and test groups 1, 2 and 3(Con, G1, G2 and G3). *P<0.001
Comp Clin Pathol
supply caused by a decrease in G6PD activity (Kitamura et
al. 2002; Ortega-Camarillo et al. 2009).
Most authors hypothesise that aflatoxin may affect the
reproductive system by its toxic effect on the liver, leading
to the desquamation of the membranes of hepatocytes, the
mitochondria, the cytosol and the endoplasmic reticulum.
This cellular damage could include inhibition of enzyme
synthesis and/or enzyme activity or inhibition of lipid metab-
olism or fatty acid synthesis, which may derail the capacity of
the hepatocytes to handle the conversion of intermediate bio-
molecules, such as precursor molecules for gonadal as well as
gonadotropic hormones, e.g. FSH, luteinizing hormone (LH)
oestradiol, testosterone and progesterone. Depression or ab-
sence of normal hormone levels could cause a wide range of
degenerative changes in sexual organs (Shen et al. 1995;
Handan and Güleray 2005). Aflatoxin may also affect the
reproductive system by causing lysis of germ cells.
Evidence indicates that AFB1 influences the reactive oxy-
gen species (ROS) generation and chemotaxis of human poly-
morphonuclear leukocytes (Ubagai et al. 2008). In living cells,
ROS are formed continuously as a consequence of both bio-
chemical reactions, e.g. within the mitochondrial respiratory
chain and external factors. ROS induce lipid peroxidation,
structurally and functionally alters protein and DNA, promotes
Fig. 4 a Comparative disposition of the atretic primordial follicles (A.
pl.F.R), batretic primary follicles (A.Pr.F.R), catretic secondary fol-
licles (A.S.F.R), datretic tertiary follicles (A.T.F.R) and ecomparative
behaviours of different atretic follicles in right ovaries (Rright, A
atretic, Ffollicle, PL primordial, PR primary, Ssecondary and T
tertiary) in control and test groups 1, 2 and 3 (Con, G1, G2 and G3)
*P<0.001
Comp Clin Pathol
apoptosis and contributes to the risk of chronic diseases like
cancer and heart disease via effects on the redox status and/or
redox-sensitive signalling pathways and gene expression
(Ames et al. 1993). Evidence from in vitro, animal model
and clinical studies suggests that ROS plays a role in the
aetiology of adverse reproductive events in both women and
men (Sharma and Agarwal 1996;Jozwiketal.1999;Duruet
al. 2000; Shen and Ong 2000; Vural et al. 2000; Walsh et al.
2000; Acevedo et al. 2001; Sikka 2001). ROS occurs when
the generation of radical species exceeds scavenging by anti-
oxidants as a result of excessive production of ROS and/or
inadequate intakes or increased utilisation antioxidants.
Antioxidants (such as vitamins C and E) and antioxidant
cofactors (such as selenium, zinc and copper) are compounds
that are capable of disposing, scavenging or suppressing the
formation of ROS.
Oocyte ROS exposure is associated with decreased fer-
tilisation and blastocyst development (Takahashi et al.
2003). Increased ROS is associated with decreased steroido-
genesis and their cyclic productions contribute to a decline
ovarian function (Kodaman and Behrman 2001).
Oocyte maturation occurs with the second meiotic divi-
sion (MII), which arises in response to an increase in pre-
ovulatory LH (Thibault et al. 1987). The process is sus-
pended in metaphase and does not resume unless fertilisa-
tion occurs following ovulation of the mature oocyte. In
both humans and rats, granulosa and luteal cells respond
negatively to ROS and adversely affect MII progression,
Fig .5 a comparative disposition of the atretic primordial follicles (A.
Pl.F.R), batretic primary follicles (A.Pr.F.R), catretic secondary fol-
licles (A.S.F.R), datretic tertiary follicles (A.T.F.R) and ecomparative
behaviours of different atretic follicles in left ovaries (Lleft, Aatretic, F
follicle, PL primordial, PR primary, Ssecondary and TTertiary) in
control and test groups 1, 2 and 3 (Con, G1, G2, and G3) *P<0.001
Comp Clin Pathol
leading to diminished gonadotrophin and antisteroidogenic
actions, DNA damage and inhibited protein ATP production
(Behrman et al. 2001). GSH, a non-protein sulphydryl tri-
peptide and key cellular antioxidant, has also been identified
as critical for oocyte maturation, particularly in the cyto-
plasmic maturation required for pre-implantation develop-
ment and formation of the male sperm pronucleus (Yoshida
et al. 1993; Eppig 1996).
In conclusion, (1) AFB1 is toxic for all types of ovarian
follicles and exerts atretogenic effect. (2) Increasing amounts
of toxin entering the body cause increasing follicular destruc-
tion. (3) Owing to follicular atresia, caused by AFB1, the
follicular pool in the ovary reduces. (4) The populations of
the quiescent as well as developing follicles are significantly
reduced and this condition may lead to either permanent or
explicit infertility or sterility in the rat.
References
Abdelhamid AM, Dorra TM (1990) Study on effects of feeding laying
hens on separate mycotoxins (aflatoxins, patulin, or citrinin)-
contaminated diets on the egg quality and tissue constituents.
Arch Tierernahr 40(4):305–316
Abdelhamid AM, Salem MFI, Mehrem AI, El-Shaarawy MAM (2007)
Nutritious attempts to detoxify aflatoxic diets of tilapia fish: fish
performance, feed and nutrient utilization, organs indices, resi-
dues and blood parameters. Egypt J Nutr Feeds 10(1):205–223
Abdel-Haq H, Giacomelli S et al (2000) Aflatoxins inhibit prolactin
secretion by rat pituitary cells in culture. Drug Chem Toxicol 23
(2):381–386
Abdel-Wahhab MA, Nada SA et al (1999) Effect of aluminosilicates
and bentonite on aflatoxin-induced developmental toxicity in rat.
J Appl Toxicol 19(3):199–204
Acevedo CG, Carrasco G, Burotto M, Rojas S, Bravo I (2001) Ethanol
inhibits L-arginine uptake and enhances NO formation in human
placenta. Life Sci 68:2893–2903
Agnes VF, Akbarsha MA (2003) Spermatotoxic effect of aflstoxin B1
in albino mouse. Food Chem Toxicol 41(2):119–130
Ames BN, Shigenaga MK, Hagen TM (1993) Oxidants, antioxidants,
and the degenerative diseases of aging. Proc Natl Acad Sci U S A
90:7915–7922
Bababunmi EA, Bassir OA (1982) Delay in blood clotting of chickens
and ducks induced by aflatoxin treatment. Poult Sci 61(1):166–
168
Behrman HR, Kodaman PH, Preston SL, Gao S (2001) Oxidative
stress and the ovary. J Soc Gynecol Invest 8(Suppl 1 Proceed-
ings):S40–S42
Clarke RN, Doerr JA et al (1986) Relative importance of dietary
aflatoxin and feed restriction on reproductive changes associated
with aflatoxicosis in the maturing White Leghorn male. Poult Sci
65(12):2239–2245
Clarke RN, Doerr J, Ottinger MA (1987) Age related changes in
testicular development and reproductive endocrinology associated
with aflatoxicosis in the male chicken. Biol Reprod 36(1):117–
124
Dafalla R, Yagi A, Adam SE (1987) Experimental aflatoxicosis in
hybro-type chicks. Sequential changes in growth and serum con-
stituents and histopathological changes. Vet Hum Toxicol 29
(3):222–226
Diekman MA, Green ML (1992) Mycotoxins and reproduction in
domestic livestock. J Anim Sci 70(5):1615–1627
Dietert RR, Bloom SE et al (1983) Hematological toxicology follow-
ing embryonic exposure to aflatoxin-B1. Proc Soc Exp Biol Med
173(4):481–485
Doerr JA, Ottinger MA (1980) Delayed reproductive development
resulting from aflatoxicosis in juvenile Japanese quail. Poult Sci
59:1995–2001
Duru NK, Morshedi M, Schuffner A, Oehninger S (2000) Semen treat-
ment with progesterone and/or acetyl-L-carnitine does not improve
sperm motility or membrane damage after cryopreservation-
thawing. Fertil Steril 74:715–720
El-Nezami HS, Nicoletti G et al (1995) Aflatoxin M1 in human breast
milk samples from Victoria, Australia and Thailand. Food Chem
Toxicol 33(3):173–179
Eppig JJ (1996) Coordination of nuclear and cytoplasmic oocyte
maturation in eutherian mammals. Reprod Fertil Dev 8:485–489
Erickson GF (1986) Endocrinology and metabolism, the ovary: basic
principles and concepts, 2nd edn. University of California, San
Diego, pp 2–65
Faridha A, Faisal K, Akbarsha MA (2007) Aflatoxin treatment brings
about generation of multinucleate giant spermatids (symplasts)
through opening of cytoplasmic bridges: light and transmission
electron microscopic study in Swiss mouse. Reprod Toxicol
24:403–408
Geissler FE, Faustman M (1988) Developmental toxicity of aflatoxin
B1 in the rodent embryo in vitro: contribution of exogenous
biotransformation systems to toxicity. Teratology 37(2):101–111
Gopal T, Oehme FW, Liao TF, Chen CL (1980) Effects of intratestic-
ular aflatoxin B1 on rat testes and blood estrogens. Toxicol Lett
5:263–267, Issues 3–4
Gopalakrishnan S, Byrd S, Stone MP, Harris TM (1989) Biochemistry
28:726
Hafez AH, Megalla SE, Abdel-Fattah HM, Kamel YY (1982a) Aflatoxin
and aflatoxicosis. II. Effects of aflatoxin on ovaries and testicles in
mature domestic fowl. Mycopathlogica 77(3):137–139, 9
Hafez AH, Megalla SE et al (1982b) Aflatoxin and aflatoxicosis. II.
Effects of aflatoxin on ovaries and testicles in mature domestic
fowls. Mycopathologia 77(3):137–139
Handan U, Güleray A (2005) Selenium protective activity against
aflatoxin B1 adverse affects on Drosophila melanogaster. Braz
Arch Biol Technol 48:2
Howarth B, Wyatt RD (1976) Effect of dietary aflatoxin on fertility,
hatchability, and progeny performance of broiler breeder hens.
Appl Environ Microbiol 31(5):680–684
Ibeh IN, Saxena DK (1997a) Aflatoxin B1 and reproduction. I. Repro-
ductive performance in female rats. Afr J Reprod Health 1(2):79–84
Ibeh IN, Saxena DK (1997b) Aflatoxin B1 and reproduction. II.
Gametoxicity in female rats. Afr J Reprod Health 1(2):85–89
Ibeh IN, Uriah N et al (1994) Dietary exposure to aflatoxin in human
male infertility in Benin City, Nigeria. Int J Fertil Menopausal
Stud 39(4):208–214
Ibeh IN, Saxena DK et al (2000) Toxicity of aflatoxin: effects on
spermatozoa, oocytes, and in vitro fertilization. J Environ Pathol
Toxicol Oncol 19(4):357–361
Jones FT, Hagler WH, Hamilton PB (1982) Association of low levels
of aflatoxin in feed with productivity losses in commercial broiler
operations. Poult Sci 61:861–868
Jozwik M, Wolczynski S, Szamatowicz M (1999) Oxidative stress
markers in preovulatory follicular fluid in humans. Mol Hum
Reprod 5:409–413
Kihara T, Matsuo Tet al (2000) Effects of prenatal aflatoxin B1 exposure
on behaviors of rat offspring. Toxicol Sci 53(2):392–399
Kitamura A, Yoshimura Y, Okamoto T (2002) Changes in the popula-
tions of mitotic and apoptotic cells in white follicles during atresia
in hens. Poult Sci 81(3):408–413
Comp Clin Pathol
Kodaman PH, Behrman HR (2001) Endocrine-regulated and protein
kinase C-dependent generation of superoxide by rat preovulatory
follicles. Endocrinology 142:687–693
Kovacs M (2004) Nutritional health aspects of mycotoxins. Orv Hetil
145(34):1739–1746
Larsson P, Tjalve H (1995) Extrahepatic bioactivation of aflatoxin B1
in fetal, infant and adult rats. Chem Biol Interact 94(1):1–19
Matthiaschk R et al (1990) Embryotoxicity and mutagenicity of myco-
toxins. J Environ Pathol Toxicol Oncol 10(1–2):1–7
Miele M, Donato F et al (1996) Aflatoxin exposure and cytogenetic
alterations in individuals from the Gambia, West Africa. Mutat
Res 349(2):209–217, Mutate Res.8:167–81
Ogido R, Oliveira CA et al (2004) Effects of prolonged administration
of aflatoxin B1 and fumonisin B1 in laying Japanese quail. Poult
Sci 83(12):1953–1958
Oliveira CFA, Rosmaninho JF, Butkeritis P et al (2002) Effects of low
levels of dietary aflatoxins B1 on laying Japanese quail. Poult Sci
81:976–980
Ortega-Camarillo C, González-González A et al (2009) Changes in the
glucose-6-phosphate dehydrogenase activity in granulosa cells
during follicular atresia in ewes. Reproduction 137:979–986
Ostrwski-Meissner HT (1983) Effect of contamination of diets with
aflatoxins on growing ducks and chickens. Trop Anim Health
Prod 15(3):161–168
Paul DS, Lauren AR (2004) Management of ovulatory dysfunction in
the infertile couple. Gunderson Lutheran Med J 3:21–25
Peraica M, Radic B et al (1999) Toxic effects of mycotoxins in
humans. Bull World Health Organ 77(9):754–766
Preston RJ, Williams GM (2005) DNA-reactive carcinogens. Mode of
action and human cancer hazard. Crit Rev Toxical 35:673–683
Qureshi MA, Brake J et al (1998) Dietary exposure of broiler breeders
to aflatoxin results in immune dysfunction in progeny chicks.
Poult Sci 77(6):812–819
Ray AC, Abbitt B et al (1986) Bovine abortion and death associated
with consumption of aflatoxin-contaminated peanuts. JAm Vet
Med Assoc 188(10):1187–1188
Sharlin GS, Howarth B Jr, Wyatt RD (1980) Effect of dietary aflatoxin
o reproductive performance of mature white leghorn males. Poult
Sci 59(6):1311–1315
Sharma RK, Agarwal A (1996) Role of reactive oxygen species in
male infertility. Urology 48:835–850
Shen H, Ong C (2000) Detection of oxidative DNA damage in human
sperm and its association with sperm function and male infertility.
Free Radic Biol Med 28:529–536
Shen HM, Ong DN, Shi CY (1995) Involvement of reactive oxygen
species in aflatoxin B
1
-induced cell injury in cultured rat hepato-
cytes. Toxicology 99:115–123
Shimada T, Nakamura S et al (1987) Genotoxic and mutagenic activa-
tion of aflatoxin B1 by constitutive forms of cytochrome P-450 in
rat liver microsomes. Toxicol Appl Pharmacol 91(1):13–21
Sikka SC (2001) Relative impact of oxidative stress on male reproduc-
tive function. Curr Med Chem 8:851–862
Silvotti L, Petterino C et al (1997) Immunotoxicological effects on piglets
of feeding sows diets containing aflatoxins. Vet Rec 141(18):469–472
Sur E, Celik I (2003) Effects of aflatoxin B1 on the development of the
bursa of Fabricius and blood lymphocyte acid phosphatase of the
chicken. Br Poult Sci 44(4):558–566
Takahashi T, Takahashi E, Igarashi H, Tezuka N, Kurachi H (2003)
Impact of oxidative stress in aged mouse oocytes on calcium
oscillations at fertilization. Mol Reprod Dev 66:143–152
Thibault C, Szollosi D, Gerard M (1987) Mammalian oocyte matura-
tion. Reprod Nutr Dev 27:865–896
Turner PC, Collinson AC et al (2007) Aflatoxin exposure in utero
causes growth faltering in Gambian infants. Int J Epidemiol 36
(5):1119–1125
Ubagai T, Tansho S, Ito T, Ono Y (2008) Influences of aflatoxin B1 on
reactive oxygen species generation and chemotaxis of human
polymorphonuclear leukocytes. Toxicol in Vitro 22(4):1115–1120
Uriah N, Ibeh IN, Oluwafemi FO (2001) A study of the impact of
aflatoxin on human reproduction. Afr J Reprod Heal 5:106–110
Vural P, Akgul C, Yildirim A, Canbaz M (2000) Antioxidant defense in
recurrent abortion. Clin Chim Acta 295:169–177
Walsh SW, Vaughan JE, Wang Y, Roberts LJ (2000) Placental isoprostane is
significantly increased in preeclampsia. FASEB J 14(10):1289–1296
Wang JS, Groopman JD (1999) DNA damage by mycotoxins. Mutat
Res 8:167–181
Wangikar PB, Dwivedi P et al (2004) Effect in rats of simultaneous
prenatal exposure to ochratoxin A and aflatoxin B1. I. Maternal
toxicity and fetal malformations. Birth Defects Res B Dev Reprod
Toxicol 71(6):343–351
Wangikar PB, Dwivedi P et al (2005) Effects of aflatoxin B1 on embryo
fetal development in rabbits. Food Chem Toxicol 43(4):607–615
Ward JM, Sontag JM et al (1975) Effect of lifetime exposure to
aflatoxin B1 in rats. J Natl Cancer Inst 55(1):107–113
Wasserman PM (1988) Zone Pellucida glycoproteins. Ann Rev Bio-
chem 57:415–442
Williams JH, Phillips TD, Jolly PE, Stiles JK, Jolly CM, Aggarwall D
(2004) Human aflatoxicosis in developing countries: a review of
toxicology, exposure, potential health consequences, and inter-
ventions. Am J Clin Nutr 80:1106–1122
Yoshida M, Ishigaki K, Nagai T, Chikyu M, Pursel VG (1993) Gluta-
thione concentration during maturation and after fertilization in
pig oocytes: relevance to the ability of oocytes to form male
pronucleus. Biol Reprod 49:89–94
Comp Clin Pathol
