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The Effect of Chronic Exposure with Imidacloprid Insecticide on Fertility in Mature Male Rats

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Background: This study was conducted to evaluate the effect of chronic exposure to imidacloprid (IM) insecticide on male testicular tissue, sperm morphology and testosterone levels in the serum of mature male rats. Materials and Methods: Animals were divided into and control-sham groups. The test group was subdivided into two groups of rats which were administered doses of 225 and 112 mg/kg IM per group. Each test group received the designated oral dose of IM once daily, for 60 days while the control-sham group received corn oil (0.2 ml/day) for the same time period. Results: Clinical observations demonstrated decreased movement, staggering gait, occasional trembling, diarrhea and spasms in the test groups. No clinical signs were seen in control-sham rats. Light microscopic analyses revealed increased thickness of tunica albuginea, obvious edema in the sub-capsular and interstitial connective tissue, atrophied seminiferous tubules, arrested spermatogenesis, negative tubular differentiation and repopulation indexes, decreased Leydig cells/ mm 2 of interstitial tissue, hypertrophy and cytoplasmic granulation of the Leydig cells, vasodilation and thrombosis, elevated death, as well as immature and decreased immotile sperm velocity. Hormonal investigations showed significant (p<0.05) decrease in serum testosterone levels. No hormonal changes were seen in the testosterone levels of the control-sham group. Conclusion: The current data provide inclusive histological feature of chronic IM exposure in two doses with an emphasis on reproductive disorders including a histological adverse effect on testicular tissue, spermatogenesis, sperm viability, velocity and abnormality which potentially can cause infertility.
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The Effect of Chronic Exposure with Imidacloprid
Insecticide
on Fertility in Mature Male Rats
Golamreza Najafi, D.V.Sc.
1
, Mazdak Razi, Ph.D.
2
*, Aref Hoshyar, D.V.M.
3
,
Simineh Shahmohamadloo, D.V.M.
1
, Sajad Feyzi, Ph.D.
1
1. Anatomy Department, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
2. Comparative Histology and Embryology Department, Faculty of Veterinary Medicine, Urmia University,
Urmia, Iran
3. Basic Science Department, Razi Research Institute, Tehran, Iran
Abstract
Background: This study was conducted to evaluate the effect of chronic exposure to imidacloprid
(IM) insecticide on male testicular tissue, sperm morphology and testosterone levels in the serum
of mature male rats.
Materials and Methods: Animals were divided into and control-sham groups. The test group was
subdivided into two groups of rats which were administered doses of 225 and 112 mg/kg IM per
group. Each test group received the designated oral dose of IM once daily, for 60 days while the
control-sham group received corn oil (0.2 ml/day) for the same time period.
Results: Clinical observations demonstrated decreased movement, staggering gait, occasional
trembling, diarrhea and spasms in the test groups. No clinical signs were seen in control-sham
rats. Light microscopic analyses revealed increased thickness of tunica albuginea, obvious edema
in the sub-capsular and interstitial connective tissue, atrophied seminiferous tubules, arrested
spermatogenesis, negative tubular differentiation and repopulation indexes, decreased Leydig cells/
mm
2
of interstitial tissue, hypertrophy and cytoplasmic granulation of the Leydig cells, vasodilation
and thrombosis, elevated death, as well as immature and decreased immotile sperm velocity.
Hormonal investigations showed significant (p<0.05) decrease in serum testosterone levels. No
hormonal changes were seen in the testosterone levels of the control-sham group.
Conclusion: The current data provide inclusive histological feature of chronic IM exposure in
two doses with an emphasis on reproductive disorders including a histological adverse effect on
testicular tissue, spermatogenesis, sperm viability, velocity and abnormality which potentially can
cause infertility.
Keywords: Imidacloprid, Testes, Spermatogenesis, Atrophy, Testosterone
Original Article
Introduction
Imidacloprid (IM) is a neonicotinoid compound,
which is a class of neuro-active insecticides and
manufactured after synthetic nicotine. It is widely
used in pest control, seed treatment, termite con-
trol, flea control, as an insecticide spray and a sys-
temic insecticide (1). According to the WHO and
United States Environmental Protection Agency
this compound is categorized as a “moderately
toxic” Class II or III requiring a Warning or Cau-
tion labels on marketed products (2). Animal tox-
icities of IM are similar to that of toxicities in the
parent compound, nicotine. Such toxicities as fa-
tigue, twitching, cramps, and weakness leading to
asphyxia are seen (2). The oral LD
50
of IM is 450
mg/kg body weight in rats and 131 mg/kg in mice
(3). IM is rapidly and almost completely absorbed
from the gastrointestinal tract, and eliminated via
urine and feces, in the case of chronical adminis-
tration within 48 hours, 70-80% and 20-30% of
compound respectively will be absorbed via urine
and feces. The most important metabolic steps in-
clude the degradation to 6-chloronicotinic acid, a
compound which affects the nervous system (4).
Like other neonicotinoid compounds, IM is re-
lated to nicotine in its structure and action at the
nicotinic acetylcholine receptor (5). IM has mul-
tiple agonist and antagonist effects on neuronal
nicotinic acetylcholine receptor channels of clonal
rat phaeochromocytoma cells (6). There are some
reports that show IM has an adverse effect on the
reproductive tract (7), also this compound has been
identified as having teratogenic (8), mutagenic (9),
carcinogenic (10) effects in animals and humans.
9
Royan Institute
International Journal of Fertility and Sterility
Vol 4, No 1, Apr-Jun 2010, Pages: 9-16
Received: 30 Sep 2009, Accepted: 21 Feb 2010
* Corresponding Address: P.O.Box: 1177, Comparative Histology
and Embryology Department, Faculty of Veterinary Medicine, Urmia
University, Urmia, Iran
Email: Mazdak_razi22@yahoo.com
Therefore, the health risks to humans of this class
of insecticides have attracted the attention of many
investigators. Histopathological changes have been
widely used as significant biological markers for
environmental toxicity (11, 12). Thus the purpose
of this study was to investigate the effect of IM on
testicular tissue and to evaluate the effect of this
compound on the quality, quantity and morphology
of sperm content in chronic exposed mature male
rats as a laboratory model for humans.
Materials and Methods
Animals
In this study, 42 mature male Wistar rats, 8 weeks
old and weighing 200 ± 14 g were used. The rats
were purchased from the Animal Resources Center
of the Faculty of Veterinary Medicine at Urmia
University, Iran. Rats were acclimatized in an en-
vironmentally controlled room (20-23°C, and a
12 hours light/12 hours dark cycle). Special plates
containing tap water were given to all group ani-
mals. In this study all experiments conducted on
the animals were in accordance with the guidance
of the Ethics Committee for Research on Labora-
tory Animals at Urmia University.
Experimental design
Following an acclimation period of one week, the
animals were assigned to three groups (n=10) as con-
trol and two test groups. All animals were weighed
prior to the onset of treatment as well as following
treatment to evaluate any increase in body weight
gain (BWG). Animals in the control group were ad-
ministered corn oil (0.2 ml/day) and animals in the
test groups were gavaged with IM, 225 and 112 mg/
kg per body weight, once daily a total of 60 days.
Testicular weight determination
Following anesthesia with Ketamine (5%, 40 mg/
kg, i.p.) on days 10, 20, 30, 40, 50, 60 all rats in
each test group (n=6) were euthanized by using
CO
2
gas in a special device and immediately fol-
lowing weighting of total body weight the testi-
cles were excised free of surrounding tissues and
weighed on a Mattler Basbal scale (Delta Range,
Tokyo).
Serum sampling and hormonal analysis
On days 10, 20, 30, 40, 50 and 60 blood samples
from the corresponding animals were collected di-
rectly from the heart and the serum samples sepa-
rated by centrifugation. The collected serum sam-
ples were subjected to hormonal analysis. Serum
testosterone levels were measured by radioimmu-
noassay. The limit of detection (LOD) was 0.12 ng/
ml for testosterone. The intra-assay and inter-as-
say coefficient of variances for testosterone were
determined to be 4.8 and 9.9 (both for 10 times),
respectively.
Histopathological analyses
All specimens were fixed in 10% formalin fixative
for histological investigations and subsequently
embedded in paraffin. Sections (5-6 μm) were
stained with iron-weigert for histopathological
assessment of germinal cell nuclei in the testes.
All specimens were studied by multiple magnifi-
cations (× 400 and × 1000). For the quantification
of cells and their dimensions, a 100 μm morpho-
metrical lens - device was used. The dimensions
were expressed in μm.
Epididymal sperm content, quantitative sperm
mortality and morphology
The epididymis was separated carefully from the
testicle under × 10 magnification with the use of
a stereo zoom microscope (model TL2, Olympus
Co., Tokyo, Japan). The epididymis was divided
into three segments: head, body and tail. The epidi-
dymal tail was trimmed and minced in 5 ml ham’s
F10 medium (Sigma Co.) for 20 minutes, 5% CO
2
,
37°C in a CO
2
incubator (Model LEEC, England).
After 20 minutes, the epididymis was removed
from the medium, 10 drops from medium were
used for analyzing the percentage of sperm viabilty.
Sperm with stained cytoplasms in the head, neck
and tail pieces were considered nonviable. For this
reason, the eosin-necrosin staining technique was
conducted; moreover, sperm that contained any cy-
toplasmic droplets in the head, neck and tail pieces
were labeled as immature. The proportion of non-
viable spermatozoa was determined by counting
100 squares in a randomly selected field from ten
smeared slides, for each case (13).
Tubular differentiation index (TDI) determination
To estimate TDI, the percentage of seminiferous tu-
bules (STs) with greater than three layers of differenti-
ated germinal cells from spermatogonia type A, in 200
sections (6μm) were prepared. STs with greater than
three layers were considered to be TDI positive.
Repopulation index (RI) calculation
To determine RI, the ratio of active spermatogo-
nia (spermatogonia type B with light nuclei as
seen by iron-weigert staining) to inactive sper-
matogonia (spermatogonia type A with dark nu-
clei as seen by iron-weigert staining), in STs was
calculated in 200 prepared sections, as mentioned
earlier (14).
Naja et al.
IJFS, Vol 4, No 1, Apr-Jun 2010 10
Statistical analyses
Statistical analyses was performed on all data us-
ing the paired t test to compare quantitative param-
eters referring to paired organs within a group and
one-way analyses followed by the Bonferroni test,
using Graph Pad Prism 4.00, Graph Pad Software.
All values were expressed as the mean± SD. P<0.05
was considered to be statistically significant.
Results
Both IM and corn oil administration had no ef-
fect on food and water consumption in the test
and control-sham cases, respectively. Corn oil did
not exert any significant effect on BWG in the
control-sham group, while administration of IM
reduced body weight in the test group. Testicles
decreased in size and weight in the IM adminis-
trated rats (Table1).
Also all animals in the test groups were observed
to have decreased movement, staggering gait, oc-
casional trembling, diarrhea and spasms.
Histological investigations revealed that the tunica
albugina increased in thickness after day 30 in the
high dose and day 40 in the low dose test rats. The
control-sham group showed normal tunica thick-
ness. Sub-capsular and perivascular edema was
demonstrated in both test groups, which increased
with time (Fig 1A, B, C). Considerable vasodila-
tation associated with remarkable thrombosis was
demonstrated in both the right and left testicles af-
ter days 20 and 30 in both the high and low dose
test rats, respectively.
Infiltration of the immune-mononuclear cells in the
interstitial connective tissue was elevated in the test
groups in comparison to the control-sham group.
After day 30 in the high dose and day 40 in the low
dose cases, 74.52 ± 2.396 and 64.073 ± 0.874 per-
cent of Leydig cells demonstrated severe hyper-
trophy and cytoplasmic granulation in both test
groups, respectively.
Effect of Chronic Exposure to IM on Fertility
Table 1: Effect of IM on testicular weight (T.W), body weight (B.W), germinal epithelium height (G.E.H), Sts diameter (STs.D)
in high dose (H.D) and low dose (L.D) test rats (Mean±SD).
6050403020 10 Con
T.W (gr)
0.63 ± 0.10
*
0.63 ± 0.09
*
0.65 ± 0.10
*
0.68 ± 0.06
*
0.74 ± 0.07
*
0.88 ± 0.10
*
0.89 ± 0.106
H.D
0.64 ± 0.10
*
0.66 ± 0.08
*
0.66 ± 0.08
*
0.69 ± 0.10
*
0.76 ± 0.08
*
0.89 ± 0.10*0.89 ± 0.106
L.D
B.W(gr)
157.24 ± 2.33
*a
162.37 ± 2.07
*a
166.19 ± 3.48
*
175.94 ± 1.99
*a
192.65 ± 1.41
*a
197.44 ± 1.68
*a
208.64 ± 5.64
H.D
157.17 ± 1.37
*a'
165.33 ± 2.61
*a'
169.61 ± 4.20
*
178.51 ± 1.26
*a'
195.37 ± 1.04
*a'
200.91 ± 1.78
*a'
208.64 ± 5.64
L.W
G.E.H (μm)
47.98 ± 2.33
*b
46.47 ± 1.72
*b
42.60 ± 2.06
*b
41.91 ± 0.78
*b
54.41 ± 2.64
*b
59.10 ± 0.63
*b
64.88 ± 1.98
H.D
49.09 ± 2.55
*b'
50.31 ± 2.55
*b'
47.41 ± 0.95
*b'
42.51 ± 3.48
*b'
54.30 ± 1.87
*b'
59.85 ± 1.43
*b'
64.88 ± 1.98
L.D
STs.D (μm)
196.87 ± 6.17
*c
191.10 ± 1.61
*c
184.28 ± 0.66
*c
190.01 ± 5.27
*c
208.82 ± 1.96
*c
213.59 ± 1.36
*c
224.80 ± 1.07
H.D
196.87 ± 6.17
*c'
203.93 ± 3.80
*c'
199.32 ± 2.98
*c'
197.99 ± 2.00
*c'
210.65 ± 3.00
*c'
216.80 ± 2.48
*c'
224.80 ± 1.07
L.D
Different superscript letters indicate signicant differences (p<0.05) between the high and low dose test rats in the same col-
umn. The stars represent signicant differences between all test and control-sham groups in the same rows.
11
Table 2: Mean average for immune-mononuclear cells (IMN) and Leydig cells (L .Cells) (number/mm
2
of the interstitial
connective tissue) in high dose (H.D) and low dose (L.D) test rats (Mean±SD)
6050403020 10 Con
I.MN. Cells (NO)
31.11 ± 3.25
*
32.33 ± 1.33
*
30.11 ± 2.147
*
25.55 ± 1.01
*
22.66 ± 1.50
*a
3.11 ± 1.45
*a
7.88 ± 1.16
H.D
29.88 ± 3.75
*
29.33 ± 1.58
*
27.44 ± 1.33
*
23.44 ± 1.94
*
17.77 ± 0.97
*a'
8.11 ± 1.05
*a'
7.88 ± 1.66
L.D
L.Cells (NO)
4.8 ± 0.83
*
2.6 ± 0.54
*
3.6 ± 0.54
*
4.4 ± 0.89
*
6.20 ± 1.33
*
8.80 ± 0.83
*
9.40 ± 0.14
H.D
5.00 ± 1.00
*
3.6 ± 1.14
*
3.8 ± 0.83
*
4.8 ± 0.83
*
7.20 ± 1.33
*
9.00 ± 0.70
*
9.40±0.14
L.D
Different superscript letters indicate signicant differences (p<0.05) between the high and low dose test rats in the same
column. The stars represent signicant differences between all test and control-sham groups in the same rows.
No histological changes were observed in the
control-sham group (Figs. 2A, B). The data for im-
mune and Leydig cells are presented in table 2.
Light microscopic analyses showed severe atrophy
Fig 3: (A & B) Histological architecture of the testis in test groups. A) Note the arrested spermatogenesis with severe STs de-
pletion in advanced stages of exposure to IM. B) In this gure STs present with a negative tubular differentiation index and
repopulation index in the earlier stages of exposure to IM. Note the weak spermayogenesis. C) Histological architecture of
the testis in the control-sham group. The spermatogenesis and spermayogenesis processes are normal, seminiferous tubules
present with a high proportion of spermatozoa, Iron-weigert staining technique (× 100).
IJFS, Vol 4, No 1, Apr-Jun 2010 12
Naja et al.
Fig 1: (A&B) Histological architecture of the testis in test groups. A) Edema (E) in interstitial connective tissue with high inl-
tration of immune-mononuclear cells (arrows) is noted, vascular thrombosis (T), depleted seminiferous tubules (S). B) Note the
advanced edema with increased inltration of the immune-mononuclear cells (E), remarkable seminiferous tubule depletion. C)
Histological architecture of the testis in the control-sham group. STs with no histological changes in germinal cell proportion and
edema in the interstitial tissue are present. Iron-weigert staining technique (× 100).
of the STs. Degenerated germinal epithelium was
seen in 90% of the STs in both test groups after
day 30. Accordingly this situation progressed in
the high dose administered rats. It should be men-
Fig 2: A) Histological architecture of the testis in test groups. The Leydig cells present with cytoplasmic granula-
tion, hypertrophy and aggregation. B) Histological architecture of the testis in the control-sham group. Leydig
cells have a normal appearance. Iron-weigert staining technique, (× 400).
13
Effect of Chronic Exposure to IM on Fertility
tioned that spermatozoa presentation decreased
in the lumen of the STs and the spermatogenesis
process was arrested in both test groups. Histologi-
cal investigations demonstrated arrested sperma-
togenesis in the test groups(This is the same as the
previous sentence.). Decreased height (2-3 layers)
was seen in the germinal epithelium of 90% of the
STs after days 40 and 50 in high and low dose ad-
ministrated rats (negative TDI), respectively (Figs.
3A, B, C).
In both test groups increased spacing between the
germinal cells and the association between Sertoli
and germinal cells was also disrupted (Fig 4A, B).
Fig 4: (A) Histological architecture of the testis in test
groups. Note the germinal cells that are dissociated in STs.
(B) Histological architecture of the testis in the control-sham
group, Sts are normal and no histological changes are seen
in the gure B, iron-weigert staining technique, (× 100).
No histological changes were observed in the con-
trol-sham group. Prior to day 50 in the high dose
and 40 in the low dose test groups, the percentages
of spermatogonia type B decreased in compari-
son to spermatogonia type A when compared to
the control-sham group (negative RI; Fig 5). This
weakly improved on days 50 and 40 in the high
and low dose cases, respectively.
The eosin-necrosin staining technique showed in-
creased abnormal sperm velocity with decreased
sperm viability. The motile sperm velocity was
reduced in the test groups in comparison to the
control-sham rats (Fig 6A, B, C).
Fig 5: Repopulation index seen in different groups. There
are signicant differences (p<0.05) between data for sper-
matogonia A (S.A) and spermatogonia B (S.B) in the low
(L) and high (H) dose test groups when compared with the
control-sham group).
% of normal sperms
80
70
60
50
40
30
20
10
0
Con 10 20 30 40 50 60
DAYS
(A)
High Dose Low Dose
a
a
*
% of normal sperms
80
70
60
50
40
30
20
10
0
Con 10 20 30 40 50 60
DAYS
(B)
High Dose Low Dose
a
a
*
% of sperm motility
90
80
70
60
50
40
30
20
10
0
Con 10 20 30 40 50 60
DAYS
(C)
High Dose Low Dose
a
a
*
Fig 6: A) Normal sperm content, B) viability of sperm con-
tent and, C) motile sperm content in the control-sham and
test groups. Star indicates signicant differences between
test and control-sham groups. Letters indicate signicant
differences between the two doses. (p0.05).
[% of spermatogonia A&B]
80
70
60
50
40
30
20
10
0
Con 10 20 30 40 50 60
DAYS
S.A.L
S.B.L
S.A.H S.B.H
S
.A.
L
S
.B.L
S
.A.
H
S
.B.
H
IJFS, Vol 4, No 1, Apr-Jun 2010 14
Naja et al.
Meanwhile these parameters insignificantly im-
proved after days 50 in both test groups, when
compared to prior to day 50 Hormonal analyses
showed significantly (p<0.05) reduced testoster-
one levels in both test groups until day 50, fol-
lowed by a slight increase on day 60 in both test
groups (Fig 7).
ng/ml
7
6
5
4
3
2
1
0
Con 10 20 30 40 50 60
DAYS
High Dose Low Dose
a
b
a'
b'
*
Fig 7: Testosterone levels in the control-sham and test
groups. Star indicates signicant differences between test
and control-sham groups. Letters indicate signicant differ-
ences between the two doses. (p0.05).
Discussion
Different insecticides are in wide use worldwide,
of which 5% of the world’s population (mainly
agro-workers) are directly exposed to these insec-
ticides. According to recent reports, this popula-
tion is calculated to be 2.6 million persons (15).
IM is a chlorinated analog of nicotine, which be-
longs to the class of neonicotinoid insecticides. IM
has low vapor pressure and the technical product
(94.0% IM) has a moderate order of toxicity with
respect to ingestion in the rat, but appears to be less
toxic when absorbed by the skin or inhaled. IM
may cause minimal redness to the eyes but is non-
irritating to the skin (16). In agreement with oth-
er reports; in the present study no dermal lesions
and irritations were seen in rats were exposed to
IM. All animals were observed to have decreased
movement, staggering gait, occasional trembling,
diarrhea and spasms - clinical findings which were
similar to those reported by a Smith Corporation
report in 1999 (17). In the present study, all animals
in the test groups showed significant decreases in
body weight gain in comparison to the control rats.
Testicular weight and size. Insecticides and pesti-
cides act as reproductive toxicants in male rats and
histologically induce severe focal necrosis of the
germinal cells in STs associated with tubular atro-
phy (18-20).
In accordance with previous findings, light micro-
scopic analyses of the current study showed that the
adverse effect of IM was not limited to cellular de-
generation in the germinal epithelium of STs, but
also affected TDI and RI of the germinal epithelium
by causing severe inflammation in the STs.
The importance of androgens for normal sperma-
togenesis has been previously well documented.
Previous studies indicated that most insecticides
inhibit the non-specific esterase activity in leydig
cells that, in turn, result in reduced testosterone
production (20, 21). Testosterone, through modula-
tion of P-mod-S in the peritubular cells, could af-
fect Sertoli cell function (22, 23). Any functional
damage in sertoli cells could lead to germinal cells
degeneration and dissociation. In light of the previ-
ous findings, our histological investigations demon-
strated decreased numbers/mm
2
of Leydig cells of
the interstitial connective tissue in the test groups.
On the other hand, hormonal analyses showed tes-
tosterone reduction in animals administered IM.
Spermatogenesis depends on testosterone produc-
tion by Leydig cells in response to stimulation by
follicular stimulation hormone (FSH) and luteiniz-
ing hormone (LH) (23). Therefore, it would be more
logical to hypothesize that IM-exposure resulted in
leydig cell degeneration, lead to reduction in blood
testosterone level and altimetry caused sertoli cell
dysfunction. Consequently, sertoli cell dysfunction
in turn could be able to result in germinal cell de-
generation and dissociation in STs of test animals.
Similar results were obtained for bromopropane
(an organophosphorus compound), endosulfan,
malathion, and methomyl (insecticide compounds)
when rats were chronically exposed to these com-
pounds (20, 24-26).
Some authors reported a significant reduction in
the number of motile sperm with a considerable
increase in the percentage of dead sperm in the
cases with chronic insecticide exposure (20, 25,
26). Researchers attributed the dramatic dysfunc-
tion of the testicular tissue to the direct toxic effect
of the insecticides on testicular tissue. Insecticides
deplete the renewing type A spermatogonia, which
essential for proliferation in spermatogenesis (20).
Thus, we can conclude that any degeneration event
by IM in the germinal epithelium was able to lead
interruption in the mitotic activity of type A sper-
matogonia cells, which in turn could arrest sper-
matogenesis and spermyogenesis processes. Ac-
cording to previous reports, chronic exposure to
insecticides (for example carbendazim) increased
the death, abnormal and immature sperm ratios
(27, 28).
Reactive oxygen species (ROS) are known to
mediate many toxin-induced testicular injuries
(30). There are some reports concerning the as-
sociation between organophosphate compounds
15
Effect of Chronic Exposure to IM on Fertility
(as well known pesticides and/or insecticides) and
ROS that cause injuries to different organs (31,
32). A variety of components in the male genital
system are capable of generating ROS, including:
morphologically abnormal spermatozoa, precursor
germ cells, leukocytes, particularly those which
are peroxidase-positive, and degenerated cells
in the spermatogenesis series (29). Exposing the
sperm to artificially produced ROS causes DNA
damage in the form of modification of all bases,
and leads to production of base-free sites, dele-
tions, frame shifts, DNA cross-links, and chromo-
somal rearrangements (30). In the present study
such evidence including; elevated abnormal sperm
content, increased infiltration of immune-mononu-
clear cells/mm
2
of the interstitial connective tissue,
degenerated germinal cells in both test groups, all
of which indicate a probable major role of imbal-
anced oxidative stress in the generation of various
disorders. According to this finding, it is possible
that IM has exerted oxidative stress in the testicles
of the test group rats and consequently it possibly
increases nonviable sperm velocity (because of se-
vere DNA damage).
Levels of ROS production by sperm correlate nega-
tively with sperm quality in the original semen (34).
The link between poor sperm quality and increased
ROS generation lies in the retention of excess resid-
ual cytoplasm (cytoplasmic droplets) in abnormal
spermatozoa. When spermatogenesis is impaired,
the cytoplasmic extrusion mechanisms are defec-
tive (35). In the present study all animals in the test
groups manifested with impaired spermatogenesis
and the sperm morphological study showed an el-
evated immature sperm content with cytoplasmic
droplets in different components of the sperm. Thus
we can conclude that possibly the direct effect of
toxin on testicles and androgens secretion increased
all the pathological characteristics that were able
to cause oxidative stress and, in the second phase,
generated an oxidative condition that enhanced dys-
function of the testicular tissue.
Insecticides generally affect the liver as a probable
target. According to a California Environmental
Protection Agency report in 1992, the rats that were
chronically exposed to varying doses of different in-
secticides showed pathological changes to the liver,
as seen under light microscopic analyses, which in-
cluded: tumors, hypertrophy and malignancy (30).
Repeated doses of methomyle (an insecticide) were
found to induce inhibition of B6-dependent ky-
urinine hydrolase and kyurinin aminotransferase
activities in the mouse liver (24). The liver, kidneys,
thyroid, heart, lungs, spleen, adrenal, brain, and go-
nads have been reported as affected organs for IM
toxicity (16). It is well known that IM rapidly ab-
sorbs via the gastrointestinal tract (4) and the liver is
the main organ to metabolize this compound. Thus,
physiologically the liver will be affected directly by
this toxin in the different period of consumption. Ac-
cording to our results, rats in the test groups showed
insignificant improvement for all of the pathologi-
cal characteristics in the testes, sperm content and
testosterone levels. This can suggest that possibly
the pathological changes that occurred in the liver
and its enzyme activities in the test groups stopped
IM metabolism. Therefore, this deficiency lead to a
decrease in the main toxic metabolite of IM in the
circulatory system. Therefore our results showed an
insignificant improved condition in germinal cells
series, abnormal, immature and death sperm veloc-
ity in the test groups. On the other hand, more than
89% of the IM induced rats showed clinically severe
diarrhea after days 30 in the high and 40 in the low
dose administrated rats. It was possible that the di-
arrhea itself participated in reducing IM absorption
in the test groups. Physiologically if the absorption
rate of IM reduced, the proportion of IM would be
decreased in the total blood circulation.
Conclusion
According to our results male reproductive tract
can be consider as a target for IM in the case if ani-
mals which expose to this compound chronically
and it can cause histological damage on testicular
tissue, sperm mortality, morphology and decreased
testosterone level in mature male rats. Thus this
compound can cause infertility problems in chron-
ically induced cases.
Acknowledgments
The authors acknowledge no conflict of interest
in this study. The authors wish to thank Mr. Ali
Karimi and the Department of Comparative His-
tology and Embryology for their assistance. This
manuscript has not been supported with any or-
ganization.
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... In the present study IM caused a significant reduction in body weight in high dose IM treated rats following 4 weeks treatment, whereas it was evident in low dose following 8 weeks treatment, thus reduction in the body weight was more drastic with increase in the dose and duration of treatment. Our results are in agreement to the observations of (48,11,12) in rats treated with IM and of (9) in Swiss albinos mice treated with IM. Adverse effect on body growth is the most common clinical symptom of IM in treated animals (73) . ...
... These results reveal a dose and duration dependent action of IM in the present study in rats. These results are similar to the observations of (30) in rats treated with organophosphate moncrotophos, (7) in albinos mice treated with cypermethrin, (50) in rats treated with dimethoate and of (46,48) in rabbits and rats treated with imidacloprid. The observed reduction in reproductive organ weights may be due to a degeneration of the seminiferous tubule and germ cells necrosis with depletion of spermatogenic cells and degeneration of the interstitial connective tissue as well as the decrease in bioavailability of antiandrogenic activities, which were observed in this study. ...
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Left varicocele detrimentally affects spermatogenesis and the epididymal sperm matura- tion process bilaterally. We analyzed the effect of antioxidants on sperm production and quality in rats which were varicocelized. Non-varicocelized rats served as controls (group A). Left varicocele was done in 5 groups (B, C , D , E and F) of rats of the same age. Antioxidants taurine, catalase or superoxide dismutase were administered intra- abdominally twice a week in groups C, D or E, respectively. Group F received taurine, catalase and superoxide dismutase. Groups A and B received a vehicle dose of saline equally. Eight weeks after varicocele induction bilateral epididymal caudal sperm con- tent, motility and fertilizing ability were analyzed. All the parameters were significantly lower in group B than in groups A, C, D, E and F, although the values in group A were higher than in the other groups. Bilateral testicular weight and testosterone responses to human chorionic gonadotropin stimulation were significantly higher in groups A, C, D, E and F than in the group B. There were no significant differences in serum testos- terone basal profiles among groups A, B, C, D, E and F. These results indicated the effectiveness of antioxidants for the preservation of testicular function and the epididy- mal sperm maturation process in varicocelized subjects.
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A series of cage and flight pen trials evaluated the responses of male red-winged blackbirds (Agelaius phoeniceus) to rice seed treated with imidacloprid, a systemic insecticide. In two-cup trials, when both the treated and untreated seed was either dyed or undyed, individually caged redwings consistently avoided rice seed treated with 833 and 2500 mg kg−1 imidacloprid but not 278 mg kg−1. When birds had a choice between undyed, untreated rice and dyed imidacloprid-treated rice, consumption of treated seed was suppressed at all levels. In a one-cup trial, rice consumption during the 4-day test phase was reduced relative to that during the 4-day pretest period at 833 and 2500 mg kg−1 of imidacloprid but not at 0 and 278 mg kg−1. During 4-day trials in a 0.2 ha flight pen, six different ten-bird flocks removed an average of 41.1% of the untreated rice seed on sample quadrats compared with 8.8% lost from plots having 2500 mg kg−1 imidacloprid-treated seed. On the basis of residues from whole seeds and from hulls of rice seed eaten by test birds, it is estimated that redwings ingested 13–16% of the imidacloprid initially present on the seed. Thus, even at 2500 mg kg−1, red-winged blackbirds feeding at an average rate of six seeds min−1 acquired only a fraction of the imidacloprid calculated to be a lethal dose. Imidacloprid appears to be an effective bird repellent seed treatment with minimal avian hazard.
Article
1 The reproductive effects of 2-bromopropane (2-BP) in sexually mature and immature male Sprague-Dawley rats were investigated. The animals were randomly divided into three treatment groups and one control group each of which comprised six mature and six immature rats. The treated groups were injected s.c. 200, 600 and 1800 mg/kg of 2-BP on 5 days a week for 5-7 weeks and the control group received the vehicle.2 The absolute and relative testis weights were significantly reduced in 600 and 1800 mg/kg b.w. dose groups in both mature and immature rats. The absolute epididymis, prostate, seminal vesicle, and pituitary weights and the relative epididymis weights, however, were significant only at the highest dose level used in both mature and immature rats.3 The sperm concentration and sperm viability in epididymal duct decreased and the percentage of abnormal sperm increased in a dose-dependent manner in both mature and immature rats. Additionally, serum testosterone level was significantly decreased in all dose groups in mature rats, and was significantly reduced only in the group treated with the middle and highest dose in immature rats.4 In both mature and immature rats treated with 200 and 600 mg/kg, the seminiferous tubules were atrophied and all types of germ cells were decreased in number. At the highest dose level, the effect was more marked showing severely atrophied seminiferous tubules and a complete loss of all types of germ cells.5 The mating, pregnancy and fertility indices were significantly reduced in the 600 and 1800 mg/kg groups. Additionally, at the highest dose group the number of implantations and viable fetuses per litter were reduced and the resorption rate was increased significantly.6 In the mature rats, the b-LH gene expression increased significantly in the 1800 mg/kg group when compared to the control group.7 It can be concluded that 2-BP induces alterations in both neuro-endocrine axis and the reproductive tract under the present experimental conditions. The no observed adverse effect level (NOAEL) in this study could be estimated to be lower than 200 mg/kg/b.w. based on the alteration in testicular morphology as well as on sperm parameters observed at the dose level of 200 mg/kg.
Article
Imidacloprid insecticide, applied as a seed treatment, singly or in combination with fungicides (carboxin-thiram, triadimenol-captan, and tebuconazole-thiram), protected growing plants of wheat and barley from developing infestations of Russian wheat aphid, Diuraphis noxia (Mordvilko) for 27-85 d after planting based on four greenhouse and four field trials. There were no compatibility problems in insecticide performance by combining imidacloprid with fungicides on the seed. Imidacloprid significantly reduced plant damage by D. noxia in all trials and increased yields in three of three field trials where plots were entirely infested. As a seed treatment, imidacloprid offers an environmentally safer approach to the control of D. noxia than is possible with wide-spectrum aerial or in-furrow granular insecticide treatments.
Article
The coccinellid predator Coleomegilla maculata (DeGeer) is found throughout the central and eastern United States and is a potential biological control agent for interiorscapes. Currently, the systemic insecticide imidacloprid is widely used in interiorscape and landscape integrated pest management. Effects of imidacloprid on the fitness and behavior of C. maculata were examined by confining groups of adults with inflorescences of treated sunflower, Helianthus annus L. `Big Smile'; chrysanthemum, Chrysanthemum morifolium Ramat. `Pelee'; and dandelion, Taraxacum officinale Wiggers. Confinement with inflorescences from imidacloprid-treated plants significantly decreased the general mobility of C. maculata in each plant system. The magnitude of the reduction in mobility varied with plant species. In the sunflower bioassay, survivorship was lower for beetles exposed to imidacloprid at the label rate and twice the label rate (38.3 ± 6.60 and 20.0 ± 6.71% survival, respectively) than for beetles confined to untreated controls (97.5 ± 2.50% survival). Also, in the sunflower bioassay, beetles exposed to the label rate and twice the label rate of imidacloprid took longer to produce their 1st eggs (9.78 ± 2.01 and 14.00 ± 2.10 d after bioassay respectively) than beetles from untreated controls (2.56 ± 0.50 d after bioassay). The results reported here indicate that the use of imidacloprid may not be compatible with the coccinellid predator C. maculata.
Article
Imidacloprid insecticide was applied in pelleted seed dressing at sugar beet sowing in March 1993. The field was divided into plots. On some plots, one cow manure treatment (50 tons ha) had been applied either 18 or 6 months before sugar beet sowing. On other plots, two cow manure treatments (2 × 50 tons ha) had been applied: either at 18 and 6 months, or at 18 and 1 months before sugar beet sowing. During the first two crop months period, the imidacloprid soil half‐lives in these plots were 40, 85, 112 and 121 days, respectively. The single cow manure treatment applied 18 months before sugar beet sowing almost had no more influence on the rate of imidacloprid soil biodegradation. The cow manure treatments applied either 6 or 1 months before sowing slowed down the imidacloprid soil biodegradation with similar intensities. To the greater imidacloprid soil concentrations corresponded greater imidacloprid concentrations in the leaves of sugar beet. After the first two months crop period, the rates of imidacloprid soil biodegradation increased significantly. The imidacloprid soil residues became very low, and was similar for all the cow manure treatments.
Article
Objective: To evaluate the effects of oxidative stress on DNA and plasma membrane integrity of human spermatozoa. Design: Prospective cohort study. Setting: University-based, tertiary-care infertility center. Patient(s): Men (n = 10) undergoing infertility investigation. Intervention(s): Purified populations of sperm with high motility were separated using Percoll density gradients. Then, spermatozoa were incubated with 0, 10, 100, and 200 microM hydrogen peroxide (H(2)O(2)) under capacitating conditions. Main outcome measure(s): Motion parameters were assessed by computer analysis. Genomic integrity was examined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay. Plasma membrane integrity was evaluated by the annexin V-binding assay, a measure of phosphatidylserine translocation. Result(s): Under basal conditions, there was a significant and negative relationship between sperm motility and the percentages of sperm with DNA fragmentation and membrane translocation of phosphatidylserine. After a 2-h incubation, there was a significant, dose-dependent effect of H(2)O(2) on motion parameters (decrease) and DNA fragmentation (increase). The percentage of annexin V(-) live (normal) cells declined significantly as the level of oxidative stress increased. Although the percentages of annexin V(+) live cells (sperm depicting translocation of phosphatidylserine) and necrotic cells increased at the highest H(2)O(2) levels, these changes were not significant. Conclusion(s): In vitro sperm incubation with H(2)O(2) induces DNA fragmentation in a dose-dependent fashion. The sublethal effects of oxidative stress on motion parameters were not significantly associated with membrane translocation of phosphatidylserine.