Complete article

Comparison of the protective effects of
garlic (Allium sativum L) extract, vitamin
E and N acetyl cystein on testis structure
and sperm quality in rats treated with lead
acetate
R. ASADPOUR1, A.A. SHAHBAZFAR2*, D. KIANIFARD3, M. AZARI1, N. ZABOLI1
Department of Clinical sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz. IRAN. Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz. IRAN. 3
Department of Basic sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz. IRAN. 1
2
*Corresponding author: [email protected]
SUMMARY
RESUME
The deterioration of male reproductive function is one of the major
manifestations of lead exposure. For exploring the protective effects of some
antioxidants (garlic extract, vitamin E and N acetyl cystein) on testicular
lead toxicity, 25 healthy adult male rats were divided into 5 equal groups:
the first group was reserved as the not exposed and not treated group
(negative control) whereas the second group served as positive control (lead
acetate administration in drinking water (1000 ppm) for 28 days), and rats
from the 3 last groups were intoxicated with lead acetate but treated with
aqueous garlic extract (500 mg/kg/day by gavage), vitamin E (350 ppm in
the ration) and N acetyl cystein (800 ppm in the ration), respectively. Sperm
quality, testis stereologic, morphometric and histological changes were
assessed on day 28. Lead poisoning has significantly affected sperm quality
(reduction in epididymal sperm count, in sperm motility and viability),
reduced seminiferous tubules characteristics (histological proportions,
epithelium height and volume density) and induced necrotic changes
mainly in secondary spermatocytes. Sperm viability was significantly
increased with garlic or vitamin E treatment and epididymal sperm count
with N acetyl cystein treatment. Significant increases in the seminiferous
tubule epithelium height and volume density as well as in the proportions of
seminiferous tubules within testis (only with the vitamin E) were observed
with antioxidant co-treatments. However, histological lesions were still
present albeit diminished. These results show that testis lead toxicity was
mediated by oxidative stress and that garlic extract may act as an antioxidant
such as vitamin E and N acetyl cystein, partially preserving sperm quality
and tubule histological organisation.
Comparaison des effets protecteurs de l’extrait d’ail (Allium sativum L),
de la vitamine E et de la N acétyl cystéine sur la structure du testicule et
sur la qualité du sperme chez les rats traités avec de l’acétate de plomb
Key-words: Male rat, garlic, sperm quality, testis,
stereology, morphometry, histology, spermatocyte,
vitamin E, N-acetyl cystein
La baisse de la fonction reproductive chez le mâle est une des manifestations
majeures d’une exposition au plomb. Afin d’explorer les effets protecteurs
de différents antioxydants (extrait d’ail, vitamine E et N acétyl cystéine) sur
la toxicité testiculaire du plomb, 25 rats mâles adultes ont été répartis en 5
groupes égaux : le premier groupe a servi de témoin négatif (pas d’exposition
et pas de traitement), le second de témoin positif (administration d’acétate
de plomb (1000 ppm) dans l’eau de boisson pendant 28 jours) et les rats des
3 autres groupes ont été exposés au plomb et traités oralement par l’extrait
aqueux d’ail (500 mg/kg/jour par gavage), la vitamine E (350 ppm dans
la ration) ou la N acétyl cystéine (800 ppm dans la ration). La qualité du
sperme et les paramètres stéréologiques, morphométriques et histologiques
des testicules ont été évalués le 28ème jour. L’exposition au plomb a
significativement diminué la qualité du sperme (réduction du nombre de
spermatozoïdes dans l’épididyme, de la motilité et de la viabilité du sperme),
altéré les caractéristiques des tubes séminifères (diminution des proportions
relatives au sein du tissu testiculaire, réduction de la hauteur et de la densité
volumique de l’épithélium des tubes séminifères) et a induit des lésions
nécrotiques affectant principalement les spermatocytes secondaires. La
viabilité du sperme a été significativement augmentée par un traitement par
l’extrait d’ail ou par la vitamine E, et le nombre de gamètes dans l’épididyme
par la N acétyl cystéine. Des augmentations significatives de la hauteur et
de la densité volumique de l’épithélium des tubes séminifères ainsi que de
la proportion des tubes séminifères au sein du testicule (seulement avec
la vitamine E) ont été observées lors de l’administration des antioxydants.
Cependant, les lésions histologiques sont restées présentes quoique
diminuées. Ces résultats montrent que la toxicité testiculaire du plomb
résulte de l’existence d’un stress oxydatif et que l’extrait d’ail agit comme un
antioxydant comme la vitamine E et la N acétyl cystéine en préservant au
moins partiellement la qualité du sperme et l’organisation structurale des
tubes séminifères.
Mots-clés : Rat mâle, ail, qualité du sperme, testicule,
stéréologie, morphométrie, histologie, spermatocyte,
vitamine E, N acétyl cystéine.
Introduction
Lead (Pb) is one of the well-known ubiquitous nonessential metals, poisoning the environment. Thus, it is
obvious that lead exposure is implicated in serious health
hazards in animals and humans [18]. The deterioration of
Revue Méd. Vét., 2013, 164, 1, 27-33
male reproductive health is one of the major manifestations
of occupational and/or environmental exposure to lead [21].
NAHA et al. [25] suggested that men working in lead based
factories showed poor sperm production in terms of quality
and density. BISWAS and GHOSH [6] demonstrated that
lead exposure reduces the activities of testicular steroidogenic
28
enzymes in rats. Recent studies showed that lead inhibits
activities of antioxidant enzymes, including glutathione
peroxidase, catalase and superoxide dismutase [12].
Garlic (Allium sativum L.) is a versatile vegetable often
used as ingredient in many dishes for flavour, aroma and
taste enhancement [31]. It is a good source of dietary
phytochemicals with proven antioxidant properties and
ability to modulate the detoxification systems [26]. Some
researchers isolated and identified several flavonoids
and sulphur-containing compounds (diallyl sulphide,
trisulphide and allyl-cystein) in garlic [23]. These are likely
to play an important role in the widely demonstrated
biological effects of garlic, which include anti-tumour [10],
hypolipidemic, hypocholesterolemic, antiatherosclerotic,
antioxidant [8] and immunomodulatory [7] effects.
Many previous investigations demonstrated that garlic
organo-sulphur compounds prevent toxicity induced
by cyanide, sodium nitrite [11], carbon tetrachloride
[22], ethanol [19] and sodium arsenate [8]. The toxicity
mechanism for all of them entails oxidative stress and
impairment in the antioxidant defence systems. Based
on this fact, the following discussion will provide the
therapeutic potential of A. sativum in prevention of lead
acetate induced toxicity on various systems. This study was
carried out to investigate the possible protective properties
of A. sativum extract on lead-induced toxicity in rat testis.
Materials and Methods
AQUEOUS GARLIC EXTRACT, ANIMALS, PROTOCOL DESIGN
Fresh garlic was collected in August 2010 from the
University plant garden, Tabriz, Iran. It was recognized and
authenticated by agricultural engineer and the voucher kept
in the herbarium of university. Peeled garlic cloves (5 g)
were crushed mechanically in a mortar-pestle for 1 minute,
together with 10 mL distilled water. The crushed material was
carefully decanted by pressing through cheesecloth (yield
was 500 mg/mL). The aqueous garlic extract (AGE) was
freshly prepared for the experiment and diluted accordingly.
A total of 25 healthy adult male albino Wistar rats
weighing 210 ± 10 g, obtained from the University of Tabriz
animal house was housed in individual stainless steel cages
at 23 ± 1ºC and exposed to 12 hours light/dark cycle. They
had access to a standard rodent laboratory diet and drinking
water ad libitum throughout the whole experimental period.
After one-week long acclimatization period, they were
randomly divided into 5 equal groups (containing each 5
animals) according to the dietary treatments applied for 4
weeks: rats from the group 1 served as untreated controls
and were fed with the standard diet; rats from the other 4
groups were orally treated with lead acetate (1000 ppm
in drinking water) and animals in the groups 3, 4 and 5
were additionally treated with the aqueous garlic extract
ASADPOUR (R.) AND COLLABORATORS
(500 mg/kg/day) by gavage, vitamin E (350 ppm mixed
with the daily ration) and N-acetyl cystein (800 ppm
mixed with the daily ration), respectively. The animals
were observed daily for signs of toxicity. Food intake and
body weights were recorded daily during the experimental
period. At the end of experimental period (on the day 28),
animals were given rest overnight and then on the next
day, they were sacrificed under light ether anaesthesia.
ASSESSMENT OF TESTICULAR FUNCTIONS
Epididymal sperm concentration and sperm motility:
The epipidymal sperm count and sperm progressive
motility were evaluated by the following method [17]:
Epididymal spermatozoa were obtained by mincing the
epididymis with anatomical scissors in 5 ml of Ham’s F12
medium and incubated at 32ºC for 2 minutes. An aliquot
of this solution was placed in Neubauer haemocytometer
and motile sperms were counted by using light microscope
at x 400 magnification. Non-motile sperm numbers were
first determined, followed by counting total sperm. Sperm
motility was expressed as a percent of motile sperm from
the total sperm counted. Percentage of morphologically
abnormal spermatozoa was determined by the method
described by EVANS and MAXWELL [13]. According to this
method, slides were prepared with Wells and Awa stains for
morphological examination and 1% eosin B and 5% nigrosine
in 3% sodium citrate dehydrate solution for live / dead
ratio was used. A total of 400 sperm cells were counted on
each slide under light microscope at x 400 magnification.
Testicular stereology and histopathology:
One of the testes of each animal was fixed in neutral
buffered 10% formalin and then microscopic slides were
made of them using haematoxylin and eosin staining.
Testicular stereology (seminiferous tubules diameter,
seminiferous epithelium height, volumetric proportion
of tubules and quantification of Sertoli and Leydig cells
per gram of testis) was evaluated by light microscopy. To
obtain the height and tubular diameter of the seminiferous
epithelium, 30 histological cross sections of the seminiferous
tubules were analyzed, contoured as circular as possible, and
randomly selected at x 400 magnification per animal. The
volume density of testicular components was determined
with a 441- intersection grid, where 20 fields were analyzed
for each animal, at x 400 magnification, totalling 8820
points of assessment. The percentage occupied by each
element in testicular parenchyma and the total testicular
volume were used to obtain the volume of testicular
components (seminiferous epithelium, lumen, Leydig cells).
As the testicular density is very close to 1 (1.03-1.04), the
testicular weight was considered to be equal to its volume.
Revue Méd. Vét., 2013, 164, 1, 27-33
EFFECTS OF GARLIC, VITAMIN E AND N ACETYL CYSTEIN ON LEAD TESTICULAR TOXICITY IN RATS
Cell quantification:
STATISTICAL ANALYSIS
The calculation of the total length of the seminiferous
tubules (LST) was conducted based on total volume of
the seminiferous tubule (TSV) according to the formula
provided by ATTAL and COUROT [3]: LST = TSV/πR2.
In 20 cross sections of seminiferous tubules, the number
of nucleoli of Sertoli cells was quantified. With the LST and
number of nucleoli of Sertoli cells (S) per seminiferous tubule
cross section, it was possible to calculate the total number
of Sertoli cells (TNSC) per testis using the formula provided
by HOUCHERAU-DE-REVIERS and LINCOLN [16]:
TNSC = LST × n0 corrected from S per transverse section/
thickness section. To find TNSC / testis, the calculated value
was divided by the testis weight. To quantify the total number
of Leydig cells in testis, the method proposed by PAULA
et al. [28] was used. With the volume of a Leydig cell and
the total volume of these cells in testis, it was possible to
obtain their total number per testis and per gram of testis.
TSI
ESC (106/mL)
Sperm motility (%)
Sperm viability (%)
29
Data are expressed as means ± standard deviations
(SD). Differences among the experimental groups
were assessed by one-way ANOVA followed by Tukey
test using SPSS-version 19 –Software. Values were
considered statistically significant when p < 0.05.
Result
No clinical signs were recorded in rats exposed to lead
poisoning. No significant differences in body weight, weight
gains and food intake were evidenced between groups.
As shown in Table I, although the relative testis weights
have not significantly differed among groups, the sperm
quality was significantly altered in lead exposed rats: the
epididymal sperm count, the sperm motility and the sperm
viability were significantly depressed compared to the not
exposed controls (p < 0.01). When rats were treated with
Lead exposition
Negative
Control
Positive controls
+ AGE
+ Vit. E
+ NAC
0.57 ± 0.04
33.8 ± 2.1a
72.7 ± 1.5a
90.8 ± 1.0a
0.59 ± 0.04
19.3 ± 1.4c
62.3 ± 0.7c
73.2 ± 1.2c
0.61 ± 0.08
25.2 ± 1.3bc
66.7 ± 0.8bc
82.5 ± 1.5b
0.66 ± 0.03
24.1 ± 0.8bc
69.8 ± 1.8ab
80.2 ± 1.3b
0.57 ± 0.08
27.8 ± 1.5ab
64.8 ± 1.2bc
78.7 ± 2.4bc
AGE: aqueous garlic extract; NAC: N-acetyl-cystein; TSI: testicular-somatic index given with the formula: testes weight / total body weight; ESC: Epididymal sperm count.
Different superscripts a,b,c in the same row indicate significant differences (p < 0.05 or more) among groups.
Table I: Changes in sperm quality and testes weight in rats orally exposed to lead (1000 ppm in drinking water) and eventually orally treated with aqueous
garlic extract (500 mg/kg/day), vitamin E (350 ppm mixed with the daily ration) and N-acetyl-cystein (NAC, 800 ppm mixed with the daily ration) for 28 days
compared to rats not exposed to lead (negative controls). Results are expressed as means ± standard deviations.
Negative
Control
PST (%)
STD (mm)
STEH (mm)
STEVD (%)
Sertoli cells (%)
Leydig cells (%)
87.7 ± 6.4a
265.4 ± 20.6
74.3 ± 3.6a
55.5 ± 4.1a
1.3 ± 0.6a
1.1 ± 0.5
Lead exposition
Positive
controls
+ AGE
+ Vit. E
+ NAC
76.3 ± 5.1c
255.3 ± 12.9
58.8 ± 2.1c
46.0 ± 1.0c
1.5 ± 0.5b
0.8 ± 0.3
80.4 ± 0.9bc
257.5± 11.7
66.5 ± 3.5b
51.5 ± 2.4b
1.5 ± 0.3b
0.8 ± 0.5
81.6 ± 2.2b
261.3 ± 12.6
68.5 ± 3.7b
52.5 ± 1.4b
1.5 ± 0.2b
0.9 ± 0.7
79.2 ± 0.6c
256.9 ± 10.4
66.1 ± 4.0b
51.4 ± 2.1b
1.5 ± 0.3b
0.8 ± 0.4
AGE: aqueous garlic extract; NAC: N-acetyl-cystein; PST: proportions of seminiferous tubules expressed from the total testis mass; STD:
seminiferous tubule diameter; STEH: seminiferous tubule epithelium height; STEVD: seminiferous tubule epithelium volume density.
Different superscripts a,b,c in the same row indicate significant differences (p < 0.05 or more) among groups.
Table II: Stereologic and morphometric values of testis tissue in rats orally exposed to lead (1000 ppm in drinking water) and eventually orally treated with
aqueous garlic extract (500 mg/kg/day), vitamin E (350 ppm mixed with the daily ration) and N-acetyl-cystein (NAC, 800 ppm mixed with the daily ration) for
28 days compared to rats not exposed to lead (negative controls). Results are expressed as means ± standard deviations.
Revue Méd. Vét., 2013, 164, 1, 27-33
30
garlic, vitamin E or N acetyl-cystein, the sperm quality
was slightly improved but remained globally lower than in
negative controls (not exposed to lead). In garlic treated rats,
the sperm viability was significantly increased compared
to rats only exposed to lead (p < 0.01) but still remained
significantly lower than the control value (p < 0.05) and
the epididymal sperm count and the sperm motility, albeit
numerically increased, were not significantly different to
values observed in lead exposed rats. In rats treated with
vitamin E, while the epididymal sperm count remained low,
the sperm motility has significantly increased compared
to poisoned rats (p < 0.05) and reached values closely
related to the control value and the sperm viability was also
significantly higher than in positive controls (p < 0.01) but
has remained depressed compared to the not exposed rats
(p < 0.05). With N acetyl dietary supplementation, sperm
motility and viability remained significantly low compared
to the negative controls (p < 0.05) and did not significantly
differ from values recorded in poisoned rats and only the
epididymal sperm count has significantly increased (p < 0.05).
ASADPOUR (R.) AND COLLABORATORS
Figure 2: Karyorrhexis in secondary spermatocytes (arrows) in a rat exposed
to lead (1000 ppm in drinking water) and vitamin E (350 ppm mixed
with the daily ration). Haematoxylin-eosin, X 200.
Stereologic and morphometric criteria measured in testis
tissue were summarized in Table II. It was observed that lead
caused marked decreases in all measured parameters although
differences in seminiferous tubule diameter and in Leydig
cell percentages between the poisoned rats and the negative
controls were not significant because of high value dispersion.
Oral treatments with garlic, vitamin E and N acetyl cystein
have significantly improved stereologic and morphometric
testis parameters compared to the lead exposed rats (except
for the proportions of seminiferous tubules with garlic and N
acetyl cystein treatments) (p < 0.05) but have not completely
restored the cellular organization of testis (differences
with the negative controls were significant: p < 0.05).
Hyperaemia and oedema in interstitial tissue (figure
1), decrease in germinal epithelial cell number, single cell
necrosis, partial necrosis in some tubules with karyorrhexis
Figure 3: Necrotic detached cells with picnotic nuclei in the lumen of a
seminiferous tubule (arrows) in a rat exposed to lead alone (1000 ppm
in drinking water). Haematoxylin-eosin, X 800.
specially in secondary spermatocytes (figure 2), tissue
debris in some tubules (figure 3), vacuolar degeneration
(figure 4) and germinal epithelium detachment from
basement membrane (figure 5) were observed. Secondary
spermatocytes were the most vulnerable cells among all
cell classes. These lesions were most prominent in rats
only exposed to lead but were also observed in exposed
animals treated with garlic, vitamin E or N acetyl cystein.
Discussion
Figure 1: Hyperaemia (white arrow) and oedema (black arrows) in testicular interstitium in a rat exposed to lead (1000 ppm in drinking water)
and N-acetyl cystein (800 ppm mixed with the daily ration). Haematoxylin-eosin, X 80.
The results of the present research showed that the lead
exposure in drinking water caused significant reductions
in epididymal sperm concentration, suppression of sperm
progress motility, and live/dead sperms ratio. Many studies
on reproductive system of male animals have documented
lead as a toxicant for testicular tissue and functions [4, 29]
such as significant reductions in the number of spermatozoa
Revue Méd. Vét., 2013, 164, 1, 27-33
EFFECTS OF GARLIC, VITAMIN E AND N ACETYL CYSTEIN ON LEAD TESTICULAR TOXICITY IN RATS
Figure 4: Vacuolar degeneration and necrosis in the germinal epithelium
of a seminiferous tubule (arrows) in a rat exposed to lead (1000 ppm in
drinking water) and aqueous garlic extract (500 mg/kg/day) by gavage.
Haematoxylin-eosin, X 200.
Figure 5: Germinal epithelium detachment from basement membrane of a
seminiferous tubule (arrows) in a rat exposed to lead alone (1000 ppm
in drinking water). Haematoxylin-eosin, X 200.
within the epididymis in mice received lead acetate (0.25%
and 0.50%) in drinking water [34]. One possible explanation
is that these compounds may be toxic to testicular histological
structure. Second explanation is that lead may have a direct
influence on sperm quality. Moreover, the decrease in
sperm motility can be due to indirect effects of lead, like
increase of ROS (reactive oxygen substances) generation
in sperm cells. By causing lipid oxidation, ROS alter the
integrity and the fluidity of cellular membrane structures,
particularly of cell membrane which is essential for sperm
motility, structural integrity, and ultimately for sperm
viability [1, 5, 35]. This finding is in agreement with other
reports who reported that administration of lead acetate
resulted in an obvious decline in sperm quality [20]. In
another study, where lead acetate (10 mg/kg of body weight)
was administered to rats once a week for 6 and 9 weeks, a
decrease in sperm counts and absolute concentration of
motile sperms was described in the latter period [17].
Revue Méd. Vét., 2013, 164, 1, 27-33
31
In the current study, treatments with garlic or vitamin E
resulted in a significant improvement in sperm parameters
in rats exposed to lead. In this regard, NUUTILA et al. [26]
hypothesized that constituents of garlic extract and vitamin
E inhibited lipid peroxidation and preventing reduced
glutathione depletion. The postulated roles of garlic organosulphur compounds in prevention of lead toxicity can be
explained by firstly their ability in free radical scavenging and
secondly the prevention of GSH depletion [30]. Moreover,
it can be suggested that the ameliorative potential of garlic
was probably due to the combined effects both on metal
absorption and excretion from the body. HANAFY and
SOLTAN demonstrated that vitamin E sustained the sperm
motility in rats exposed to lead more efficiently than in
those exposed to cobalt or mercury [15]. Vitamin E could
also improve the sperm motility in humans: treatment of
astheno-spermic patients with oral vitamin E significantly
decreased the lipid peroxidation in sperm and improved
sperm motility [32]. Vitamin E allows free radicals to abstract
a hydrogen atom from the antioxidant molecule rather than
from polyunsaturated fatty acids, thus breaking the chain of
free radical reactions, resulting in a marked decrease in the
reactivity of free radicals [27]. Therefore, protective effect
of vitamin E supplementation for the organism from toxic
agents and free radical damages is a time consuming process.
Some studies indicate that the length of seminiferous
tubules of testis is directly proportional to sperm production
[24]. AL-OMAR et al. [2], HAMADOUCHE et al. [14] and
CORPAS et al. [9] have reported that lead causes decrease
in seminiferous tubules diameter in adult rats. Although the
seminiferous tubule diameter was not significantly affected
in the present study, the presented histological analysis
indicated statistically significant decreases in seminiferous
epithelium height and volume density, leading to significant
reduction in the proportions of seminiferous tubules inside
testis in lead poisoned rats. By contrast, oral treatment
with antioxidants (vitamin E, garlic and N acetyl cystein
at a lesser extend) succeeded in partly counteracting the
deleterious effects of lead on seminiferous tubule dimensions
by markedly increasing the tubule epithelium height and
volume density and less evidently (for garlic and N acetyl
cystein) the proportions of seminiferous tubules in testis.
Lead exposure produced pronounced testicular
damages evidenced by histological alternations in testis
include degeneration of seminiferous tubules, germ cell
necrosis and vacuolar degeneration especially in secondary
spermatocytes [33]. These findings correspond with the
observations that showed lead acts as a spermicidal agent
in the case of high exposure [33], leading to a dose-related
suppression of spermatogenesis and serum testosterone
concentration in the young adult male rats [14]. According
to the presented results, secondary spermatocytes were
the most affected germ cells in the treatment groups. Oral
administrations of antioxidants (garlic, vitamin E and N
acetyl cystein) have partly alleviated lead induced histological
changes in testis but have not totally prevented them.
32
As a conclusion, it is evident that oral treatments
with antioxidants such as the aqueous Allium sativum
extract, vitamin E or N acetyl cystein at a lesser extend,
partially protected spermatogenesis and sustained sperm
quality in animals chronically intoxicated with lead
acetate by limiting oxidative stress but did not completely
prevent necrotic histological alterations in testes.
Acknowledgment
The authors are thankful to the authorities of
University of Tabriz for providing support to this study.
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