pakistan journal of biological sciences 18 (3): 107-114, 2015
issn 1028-8880
© 2015 asian network for scientific information asian network for scientific information ans.net
research article open access
doi: 10.3923/pjbs.2015.107.114
toxicity effect of aflatoxin b1 on reproductive system of albino male rats
abeer fauzi murad, shaima ahmed and shaima abead
department of biology, college of science for women, babylon university, iraq
a r t i c l e i n f o
article history:
received: january 02, 2015
accepted: may 06, 2015
corresponding author:
abeer fauzi murad
department of biology,
college of science for women,
babylon university, iraq
a b s t r a c t
aspergillus flavus and aspergillus parasiticus are two important molds that produce
aflatoxin(af). humans and animals can be exposed to aflatoxins by consuming
foods contaminated with the products of these fungi. aflatoxin b1 (afb1) is one
of the most common mycotoxins found in human foods. in general, acute toxicity
by is afb1 less likely to occur than chronic toxicity. the principal target organ for
aflatoxins is liver which may lead to hepatocytes and necrosis and also can be
carcinogenic and cause immunosuppression. afb1 could be toxic to the male
reproductive system in man as well as wild and domestic animals. according to this
study the afb1 effect on testis and sperm of male rats which revealed pathological
changes in testis and epididymis, with the high dosage of afb1 the edematous fluid
inside the tumbles increases while there are significant (p>0.05) decreases in the
number of leydig cells, the height of seminiferous, as well as there was a significant
decreasing in the number of spermatogenesis, spermatocytes and spermatids.
key words: toxicity, aflatoxin b1, albino rats, epididymis, spermatogenesis
introduction
aflatoxins are secondary metabolites principally produced
by aspergillus flavus and aspergillus parasiticus
(jacobsen et al., 2007) . although twenty types of aflatoxins
have been identified, only aflatoxin b1, b2, g1 and g2 are
usually found together in feeds and feedstuffs in various
proportions. however, afb1 is the most toxic one. direct
consequences consumption of aflatoxins-contaminated feed
include decreased feed intake, feed rejection, poor feed
conversion (akande et al., 2006), decreased body weight,
increased disease incidence due to immune-suppression
(meissonnier et al., 2006) and reduced reproductive capacities
(el-saad and mahmoud, 2007) which leads to economic losses
in agriculture and domestic animals (wu, 2006). also it can be
carcinogenic, mutagenic, teratogenic, tremorgenic and
hemorrhagic. it also cause damage in the central nervous
system, liver or kidneys (wangikar et al., 2005 akande et al.,
2006). aflatoxins lead to acute toxicity with symptoms such
as fever, oedema, vomiting, abdominal pain, in appetence and
liver failure. during aflatoxicosis in rabbits, histopathological
examination revealed vascular congestion, leucocytic
infiltration and degenerative changes in the affected organs
during the initial stage of toxicosis. at its terminal stage, a
coagulative necrosis, perivascular and per ductal fibro cellular
reactions along with mononuclear-cellular infiltration and
distortion of the hepatic chords were observed in the liver
(lakkawar et al., 2004).
exposure to aflatoxins is known to produce male
reproductive toxic effects with several events which have been
reported in previous studies. the target organs in causing male
reproductive toxicity are the testicles and various aspects of
spermatogenesis (richburg, 2000). however, the epididymis
can also be targeted by such reproductive toxicants
(akbarsha et al., 2000 krausz and forti, 2000).
the seminiferous tubules show degeneration of the
epithelium and reduction in the number of mature spermatids
in aflatoxin treated rabbits (lakkawar et al., 2004). generation
of multinucleate giant spermatids or simplistic spermatids as
one of the more symptoms of aflatoxicosis (agnes and
akbarsha, 2003 faridha et al., 2007) were additionally
observed.
this study shows the effect of aflatoxin b1 on male
reproductive system in albino rats, it also focus on the
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pak. j. biol. sci., 18 (3): 107-114, 2015
histological changes on testicles tissue and epididymis and
some aspects of spermatogenesis and the consequent effects on
cell counts and cell numbers.
material and methods
animals: the experiment evaluated the chronic effects of oral
administration of afb1 on testis of adult male rats. twenty
adult male rats with a body weight of 250±30 g were used.
these rats were provided by the center of animal housing of
the faculty of science\kufa university. the animals were
placed in standard cages (2 animals per cage) for two weeks
under 12 h light-dark cycle with 23-25°c room temperature for
acclimation before beginning the study. all animals received
standard laboratory animal’s food and water ad labium during
the whole period of experiment.
animals were divided randomly into two groups: control
group (c) and experimental group (e) each containing 5 rats:
group (e): the experimental group (e) was subdivided into
three subgroups, each containing 5 rats as the
following
group (e1): animals in this group received (15 ?l of af
b1/kg three times in the week) for 40 days
group (e2): animals in this group received (30 ?l of af
b1/kg three times in the week) for 40 days
group (e3): animals in this group received (45 ?l of
afb1/kg three times in the week) for 40 days
group (c): normal and apparently healthy rats that did not
receive any type of treatment. all groups left
for two weeks before killing
after oral administration of afb1 which were obtained
from sigma chemical company, toxin doses were prepared in
distilled water. along with these dosages, a portion of testis
and epididymis tissue from each group was preserved in a 10%
formaldehyde solution for 24 h. paraffin blocks were sectioned
at 5 ?m thickness by rotary microtome for histopathological
and histophotometric studies. hemotoxylin and eosin were
used for staining and later the microscopic slides of the testis
and epididymis were photographed at a magnification
of (x40, x10).
morphometric examination: microscopic examination of
stained testicular and epididymis tissue sections was done for
all groups. the following parameters were measured using a
pre-calibrated ocular micrometer with stage micrometer to
evaluate the extent of testicular and epididymis damage and
the whole process was recorded through a list of figures, these
parameters include:
c diameter of the round seminiferous tumbles. thirty
tubular profiles that were round or nearly round were
chosen randomly and measured for each animal
c height of the germinal epithelium was obtained in the
same tubule sections utilized to determine tubular
diameter
c diameter of the epididymis tubules of caput epididymis
were measured at 40x magnification
c diameter lumen of epididymis tubules of caput
epididymis were measured at 40x magnification
c height of the germinal epithelium of caput epididymis
were measured at 40x magnification
c diameter lumen of epididymis tubules of caudal
epididymis were measured at 10x magnification
cell counts and cell numbers: to evaluate the efficiency of
spermatogenesis, 50 seminiferous tubules selected from each
group were evaluated for spermatogonia, spermatocytes and
spermatids. the sertoli cell counts and the leydig cell numbers
were evaluated in the interstitial tissue. the average of the
different cell counts of each animal was used for the analysis.
the evaluation of the all samples was performed at a constant
magnification 40x with light microscopy. the sertoli cell
index was calculated by dividing the sum of the counted
spermatogonia and spermatocytes via the sertoli cell numbers
per tubule (russell et al., 1990).
results
the clinical signs recorded in afb1 treated animals
include hair fall, loss of appetite and marked decrease in body
weights. however, the degeneration of spermatogonial cells
lining seminiferous tubules, edema associated with marked
atrophy of seminiferous tubules as well as focal hemorrhage
and disturbed process of spermatogenesis as shown in fig. 1.
the study concludes that afb1 causes pathological changes
in epididymis including degeneration and necrosis of epithelial
cells of sperm tubules and reduces the number of sperms inside
the cavity of tubules as in the case of high concentration of
toxin there was no sperm in the cavity. the changes at the top
of epididymis include necrosis of epithelium cells of tubules
and edema and the presence of edematous fluid inside the
tumbles. these changes increased with high dosage of afb1
fig. 2 and 3.
as shown in fig. 4a and b there is a significant (p>0.05)
decrease in the numbers of leydig cells and the height of
seminiferous tubules in animals treated with (15, 30, 45 ppm)
afb1 in comparison with the control group. there was a
significant reduction (p>0.05) in the number and the index of
sertoli cells as a result of afb1 dosage when compared with
control group fig. 4c and d. the number of spermatogenesis,
spermatocytes as well as spermatids is significantly (p>0.05)
reduced with the increasing of toxin concentration as seen in
fig. 5a-c. with the increase in the afb1 dosage concentration,
the diameter of caput epididymis were significantly (p>0.05)
decreased while the height of caput epididymis also highly
reduced in the 30 and 45 ppm concentration of afb1
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pak. j. biol. sci., 18 (3): 107-114, 2015
(a) (b)
(c) (d)
(a) (b)
(c) (d)
fig. 1(a-d): cross section in testicular male rats treated with afb1 (200x), (a) control treatment, (b) 15 ppm concentration notes
the starting of degeneration of spermatogenesis, (c) 30 ppm concentration shows high degree of degeneration of
spermatogenesis cells and (d) 45 ppm concentration shows focal hemorrhage and disturbed process of
spermatogenesis
fig. 2(a-d): cross-section of epididymis tail of male rats treated with afb1 (140x), (a) control treatment, (b) 15 ppm
concentration shows degeneration of epithelial cells and epithelializes seminiferous tubules to the tail of the
epididymis, (c) 30 ppm concentration shows necrosis of the epithelium and decrease in sperm number and (d)
45 ppm concentration shows increasing necrosis of the epithelium and decrease in sperm number
treatment as shown in fig. 6a-d indicate that afb1 causes
significant (p>0.05) reduction in the diameters rate of lumen
caput epididymis (head cavity) and the diameters rate of lumen
cauda epididymis with the increase in toxin concentration.
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pak. j. biol. sci., 18 (3): 107-114, 2015
(a ) (b)
(c) (d)
20
18
16
14
12
10
8
6
4
2
0
number of leydig cells
(a) 7
6
5
4
3
2
1
0
height of seminiferous tubules
(b)
16
14
12
10
8
6
4
2
0
sertoli cell s index
control c1 c2 c3
(c) 25
20
15
10
5
0
no. of sertoli cells
control c1 c2 c3
(d)
afb1 concentration afb1 concentration
fig. 3(a-d): cross-section at the top of epididymis of male rats treated with afb1 140x. (a) control treatment,
(b)15ppmconcentration shows necrosis in tubule epithelium and there is no sperm, (c) 30 ppm concentration revealed
necrosis of tubules epithelium and the presence of edematous fluid inside tubule and (d) 45 ppm concentration, in
addition to the previous symptoms notes spread of edematous liquid inside the tubules the absence of the sperm inside
the tubules
fig. 4(a-d): comparison of (a) no. of leydig cells, (b) height of seminiferous tubules, (c) index and (d) p>0.05 no. of sertoli cells
with afb1 concentration at p>0.05, c1:15 ppm,c2:30 ppm, c3:45 ppm
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pak. j. biol. sci., 18 (3): 107-114, 2015
100
80
60
40
20
0
rate of spermetogensis number
control c1 c2 c3
(a) 100
80
60
40
20
0
no. of spermatocytes
control c1 c2 c3
(b)
120
100
80
60
40
20
0
no. of spermatids
control c1 c2 c3
(c)
afb1 concentration afb1 concentration
afb1 concentration
7
6
5
4
3
2
1
0
25 (b)
20
15
10
5
0
(a)
control c1 c2 c3 control c1 c2 c3
afb1 concentration afb1 concentration
fig. 5(a-c): comparison of (a) rate of spermatogenesis number, (b) rate number of spermatocytes and (c) no. of spermatids with
afb1 concentration at p>0.05, c1:15 ppm of afb1, c2:30 ppm afb1, c3:45 ppm afb1
fig. 6(a-d): continue
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25
20
15
10
5
0
(c) 35
30
25
20
15
10
5
0
(d)
control c1 c2 c3 control c1 c2 c3
afb1 concentration afb1 concentration
fig. 6(a-d): comparison of (a) diameter of caput epididymis tubules, (b) height of caput epididymis, (c) diameters rate of lumen
caput epididymis and (d) diameters rate of lumen cauda epididymis, with afb1 concentration at p>0.05,
c1:15 ppm, c2:30 ppm, c3:45 ppm
discussion
as it was explained in the result, afb1 causes
pathological changes in testicles and epididymis including
degeneration and necrosis of epithelial cells of sperm tubules
and reduces the number of sperms. one of the earliest studies
indicating morbidness of reproductive efficiency due to af
toxicity was done by maryamma and sivadas (1975) who
reported that continuous feeding of a diet containing 0.7 ppm
af produces testicular degeneration in male goats. there have
been other reports which indicate that afb1 causes delay in
physiological and behavioral sexual maturation and also delays
testicular development in japanese quail (doerr and
ottinger, 1980). it has been additionally discovered that afb1
decreases semen volume and testis weight and leads to the
disruption of the germinal epithelium in mature male white
leghorn chicks (sharlin et al., 1980).
the testicular changes in this study are harmonious with
the result of earlier studies (lakkawar et al., 2004
faridha et al., 2007). faridha et al. (2007) for example fed
adult male rats with aflatoxin diet for prolonged period,
showed regressive changes of different intensity in the
germinal epithelium of the seminiferous tubules resulting in a
severe dystrophic alteration of the spermatogenic epithelium
along with edematous changes in the interstitial tissue.
another study by el-shewy and ebrahem (2004) revealed the
degeneration of lining epithelium of seminiferous tubules and
congestion of testicular blood vessels with interubular oedema
in the rats treated with afb1. moreover, coagulative necrosis
of entire lining epithelium of some seminiferous tubules
replaced by homogenous eosinophilic debris in their lumina
was also noticed.
in a similar study, agnes and akbarsha (2003) reported
that the prevalence of cell ruins in the sperm suspension of
mice treated for 35 and 45 days with afb1 proves that this
toxin affects the albino mouse epididymal sperm
concentration. another study suggested that the decreased
sperm count might indicate severe impact of afb1 on
spermatogenesis. thus, aflatoxin has direct effect on sperm
(salem et al., 2001). tajik et al. (2007) showed that aflatoxin
has direct spermatotoxic effects on ejaculatory and epididymal
ram sperm.
the present experiment also showed that afb1 reduces
significantly the numbers of leydig cells, the height of
seminiferous tubules, the number and the index of sertoli cells,
the diameter of caput epididymis and lumen caput epididymis.
the number of spermatogenesis, spermatocytes as well as
spermatids is also reduced.
according to a study by nair and verma (2000),
following the intraperitoneal injection of afb1 at the rate of
25 and 50 mg/mice/day for a duration of 48 days, the
population of germ cells in the seminiferous tubules was
disrupted, decreased considerably and became disorganized.
in these animals, degenerative changes in leydig cells were
also observed. moreover, significant changes in sperm count
at the tail of epididymis were noticed along with a reduction in
sperm motility. this study confirms that toxicity with afb1
causes adverse histological changes in seminiferous tubules
and these changes involve both the spermatogenic cell series
and interstitial tissue (leydig cells). in a similar experiment
that involved roosters treated with af at 5, 10 and 20 ppm
concentrations in the diet for 8 weeks, the testis were
atrophied, the incidence of abnormal spermatozoa increased
and the epithelium was desquamated. no spermatogenesis
occurred in several birds. the size and thickness of the
generative layer and the level of plasma testosterone
also decreased (ortatatli et al., 2002). disruption of androgen
synthesis and sperm function was observed in male
mice treated afb1 at 50-60 mg kgg1 body weight/day for
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pak. j. biol. sci., 18 (3): 107-114, 2015
30 days (egbunike, 1979, 1982 egbunike et al., 1980
ikegwuonu et al., 1980). in a study done on mice by
faridha et al. (2006) histopathological changes were noted in
the testis appearance of small to large vacuoles in the
epithelium including decrease or absence of elongating
spermatids. due to the effect of afb1-treatment on the leydig
cells, there were regression of testis, morbidness of
spermatogenesis and loss of germ cells. continuous feeding on
a diet containing 0.7 ppm aflatoxin caused regression of testis,
impairment of spermatogenesis, premature loss of germ cells
and pathological changes in the leydig cells (ortatatli et al.,
2002). agnes and akbarsha (2003) have similarly reported the
effects of afb1 on albino mouse epididymis sperm
concentration.
the clinical signs recorded in animals treated with afb1
include hair fall, loss of appetite and marked decrease in body
weights. this result agrees with another study done by
lakkawar et al. (2004).
conclusion
aflatoxins are extremely potent mycotoxins which
produce serious health hazards in different animal species. it
has also been reported that aflatoxins have a deletingrious effect
on the reproductive systems of a wide spectrum of domestic
animals. this study shares the opinion that the presence of
afb1 in feed and feedstuff at graded doses induced severe
toxicity and damage in the testis in male rats and causes
different histopathological changes in testis tissue and
epididymis and also reduce the number of spermatogenesis,
spermatocytes, spermatids, leydig cells and sertoli cells.
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