1.
Introduction
Glyphosate is the active ingredient of all the glyphosate-based
herbicides which are broad spectrum herbicides that mediate their phytotoxic
action by inhibiting the synthesis of protein through inhibition of shikimic
acid pathway[1]. In mammals, the mechanism of toxic action
of glyphosate is stillnot clear and may have several enzymatic effects [2,3], however, some agricultural workers using glyphosate-based
herbicides were reported to have pregnancy problems [4]and reduced respiratory control ratio, enhanced ATPase
activity and stimulated oxygen uptake rate were observed in liver- mitochondria
of rats given glyphosate and these toxicological effects were said to be
primarily as a result of uncoupling of oxidative phosphorylation[5].
Oxidative stress was reported to be involved in toxicity
of glyphosate at molecular level[6]. The body reacts to oxidative stress by evoking the
enzymatic defense system in the body [7]. There is paucity of information on the reproductive
effects of glyphosate and the few available literatures are at variance with
one another. Laboratory studies have found that glyphosate and roundup®
formulations may be linked to endocrine disruption in animals and human cell
lines [8-11]with effects recorded at concentrations below
those used in agriculture. Whether or not such effects could be occurring in
wildlife after field application of glyphosate has not yet been established and
glyphosate is not currently included on lists of confirmed endocrine disrupting
chemicals (UHPPDB) [12]whereas SERA[13]documented that, tests on the potential effect of
glyphosate on the endocrine system have been conducted and all of these tests
reported no effect and they deduced that, conclusion on glyphosate not been an
endocrine disruptor is re-enforced by epidemiological studies that have
examined relationships between occupational exposures to glyphosate
formulations and risk of spontaneous miscarriage, fecundity, sperm quality and
serum reproductive hormone concentrations and the studies have not found
positive associations between exposure to glyphosate formulation and
reproductive or endocrine outcomes.
Living organisms contain a complex network of antioxidant
metabolites and enzymes that work synergistically to avoid oxidative damage to
cellular components such as DNA, proteins and lipids within the body [14,15]. The antioxidant systems generally either prevent
reactive oxygen species formation or eliminate them before they become
detrimental to important cell components [14,15].The reactive oxygen species play a vital role in
cellular signaling and therefore, their function is not to remove oxidants
entirely but rather to keep them at optimum level [16].
Zinc is an essential trace element for a number of animal
species [17-19]. Zinc performs its antioxidant role through
two essential mechanisms as follows: The first mechanism is the protection of
sulfhydryl groups from oxidation and this it does by preventing intramolecular
disulfide formation [20]. The second mechanism is by the prevention
of free radicals (-OH and -O2) formation by transition metals [21]. Since oxidative stress was reported to be involved in
glyphosate toxicity [6], it was inferred that an antioxidant will
play an important role in mitigating the pathological changes if any following
chronic exposure to graded doses of glyphosate in this study.
2.
Materials
and Methods
2.1. Research
Animals: Acquisition, Acclimation, Housing and Feeding
Eighty adult male Westar rats used for this experiment
were purchased from the National Institute for Trypanosomiasis and Onchocerciasis
Research, Vom Office, Jos Plateau State. They were kept in the animal room of
the Department of Veterinary Pathology, Ahmadu Bello University-Zaria, Nigeria
for two weeks for acclimatization before the commencement of the research. The
rats were dewormed during the two weeks using albendazole at the dose rate of
10 mg/kg body weight as reported by Teruel [22]. The rats were fed using standard rat chow and water was
given ad libitum.
2.2. Compliance with Ethical Standards
This work was carried out after approval was obtained
from the ethical committee on the care and use of animals in research of Ahmadu
Bello University, Zaria, Nigeria.
2.3. Chemicals and Test Kits Sources
Glyphosate (Bushfire®) which contains 360g glyphosate/liter in the form of
441g/liter potassium salt, distilled water, hematoxylin and eosin stain, Sudan
black stain, chloroform and zinc chloride (BDH Chemical Ltd; Poole, England) is
a white deliquescent granule with minimum assay 98.0 %, maximum limits of
impurities: Acid-insoluble matter 0.005 %, zinc oxide 1.2 %, sulphate 0.002 %,
cadmium 0.0005 %, calcium 0.001 %, copper 0.0005 %, iron 0.001 %, lead 0.001 %,
magnesium 0.001 %, potassium 0.001 % and sodium 0.001 % were purchased from a
reputable chemical store in Zaria. Enzyme immunoassay kits for follicle
stimulating hormone, luteinizing hormone and testosterone were purchased from
the manufacturers (AccuBind Inc., Lake Forest, USA).
2.4.
Experimental Design
2.4.1.
Chronic toxicity study
The eighty adult male Wistar rats were randomly divided
into eight groups of ten Wistar rats as detailed below:
·
Group I (DW): served as the control and
received 2mL / kg of distilled water daily.
·
Group II (Z): received zinc at the dose rate
of 50 mg/kg body weight [23].
·
Group III (GC): received glyphosate at 14.4
mg / kg body weight (2% concentration in 2 mL distilled water).
·
Group IV (G): received 375 mg / kg body
weight glyphosate (10% of the LD50)[24].
·
Group V (G1): was administered with glyphosate (20% of the LD50), 750 mg / kg body weight as reported by Tizhe [24].
·
Group VI (Z + GC): received zinc at 50 mg /
kg for 1 hour + glyphosate at 14.4 mg / kg body weight (2% concentration in 2
mL distilled water).
·
Group VII (Z + G): received zinc at 50mg/kg
for 1 hour + glyphosate 375 mg/Kg (10% of the LD50).
·
Group VIII (Z + G1): received zinc at 100mg/kg for 1 hour + glyphosate 750
mg/Kg (20% of the LD50).
The dose regimens were administered per so by gavage once daily for a period of 36
weeks as recommended by OECD[25]. The rats were weighed weekly for
appropriate dosing using electronic digital balance (Hangzhou Gongheng,
Electronic Weighing Scale, China).
2.5. Determination of semen characteristics
2.5.1. Sperm viability (life and death ratio)
Live and dead sperm cells were distinguished by adding
one drop (10 - 15µL) of eosin nigrosine stain to one drop of
semen at room temperature, for 1-2 min, and smearing the mixture on a clean
grease free slide and then examined microscopically using x10 and then x40
objectives to count the percentage of viable and non-viable cells [26]. Viable spermatozoa remained unstained while non-viable
spermatozoa stain red. Two hundred (200) cells were counted and the average
taken and expressed as percentage (%).
2.5.2. Sperm Motility
This was evaluated by the method of Sonmez [27]. The fluid obtained from the left caudal epididymis was
diluted with pipette to 0.5 mL with Tries buffer solution. A slide was placed
on light microscope with heater table, an aliquot of this solution was on the
slide and percentage motility was evaluated visually at a magnification of
x400. Motility estimates were performed from three different fields in each
sample. The mean of the three estimates was used as the final motility score.
The percentage of sperm motility was calculated using the number of live sperm
cells over the total number of sperm cells, both motile and non-motile. The
sperm cells that were not moving at all were considered non motile, while the
rest, which displayed some movements were considered to be motile.
2.5.3. Epididymis sperm concentration
Spermatozoa in the right epididymis were counted by a
modified method of Yokoi and Mayi [28]. Briefly, the epididymis was minced with scissors in 5
mL physiologic saline, placed in a rocker for 10 min and allowed to incubate at
room temperature for 2 min.After incubation, the supernatant fluid was diluted
1:100 with solution containing 5g sodium bicarbonate and 1 mL formalin (35%).
Total sperm number was determined by using the new improved Neuber’s counting
chamber (haemocytometre). Approximately, 10µL of the diluted sperm suspension was transferred to each
counting chamber of the haemocytometre and was allowed to stand for 5 min. This
chamber was then placed under a binocular light microscope using an adjustable
light source. The ruled part of the chamber was then focused and the number of
spermatozoa counted in five (5) 16-celled squares. The sperm concentration was
then calculated and multiplied by five (5) and expressed as [X] x 106mL-1, where “X” is the number of spermatozoa in a 16-celled
square.
2.5.4. Sperm Morphology
The sperm cells were evaluated with the aid of light
microscope at x400 magnification. Caudal sperm cells were taken from the
original dilution for motility and diluted 1:20 with 10% neutral buffered
formalin. Five hundred (500) sperm cells from the sample were scored for
morphological abnormalities [29].
Briefly, in wet preparations using phase contrast optics, spermatozoa were
categorized. In this study, a sperm cell was considered abnormal
morphologically if it had one or more of the following features: rudimentary
tail, round head and detached head and was expressed as a percentage of
morphologically normal sperm[29].
2.5.5. Determination of testicular sialic
acid
Semen samples were obtained from the epididymis of the
testes after the Wistar rats were sacrificed under light chloroform
anaesthesia. The semen samples were allowed to liquefy for 20 min at room
temperature and the sialic acid concentrations were determined according to the
method outlined by Warrren[30].
2.5.6. Determination of concentration of
follicle-stimulating hormone, luteinizing hormone and testosterone
The concentrations of serum FSH, LH and testosterone were
assayed using their Enzyme Immune Assay (EIA) kits (Monobind Inc. Lake Forest,
CA92630, USA).
2.5.7. Histopathological
examination
Samples measuring at most 0.5 cm in diameter of testes
were taken for histopathological sections preparation and examination after
chloroform anaesthesia which was used only at the end of the study so as to
prevent pain in the rats. The samples collected were fixed in 10% neutral
buffered formalin; they were processed for histopathological assessment using
the method outlined by Baker [31] and
viewed under light microscope;histochemical features of the testes were also
studied by usingSudan black stain after sectioning the10% buffered formalin
fixed samples as outlined by Bancroft and Gamble [32].
3.
Results
3.1. Effects of
Treatments on Reproductive Parameters
3.1.1. Effects of treatments on sperm concentration
There was a significant increase (p< 0.05) in sperm
concentration recorded in Z + G1 group as compared to the sperm concentration in G1 group; however, there was relative increase in groups Z
+ Gc (1%), Z (1.7%) and Z + G (27.6%) when compared to the sperm concentration
in DW group. On the other hand, there was relative decrease in the sperm
concentration in groups G (7.7%), G1 (16.2%) and Gc (16.6%) when
compared to the sperm concentration in DW group as shown on (Table
1).
3.1.2 Effects of
treatments on live sperm count
The result of this study showed no significant difference
in the live sperm count between the groups; however, there were relative
increase in the live sperm count in groups Z + Gc (5.7%), Z + G (10.3%) and Z +
G1 (14.5%) when compared to the live sperm
count in DW group. Conversely, there were relative decrease in the live sperm
count in groups Z (2.9%), G (5.7%), Gc (13.4%) and G1 (21.7%) when
compared to the live sperm count in the DW group as shown on (Table
1).
3.1.3.
Effects of treatments on dead sperm count
The result of this study showed no significant difference
in the dead sperm count between the groups; however, there were relative
decrease in dead sperm count in groups Z + Gc (17.8%), Z + G (32.2%) and Z + G1 (45.2%) when compared to DW group. Conversely, there
were relative increase in the dead sperm count in groups G (17.8%), Z (57.5%),
Gc (67.2%) and G1 (67.5%) when compared to the dead sperm
count in DW group as shown on (Table 1).
3.1.4. Effects of treatments on normal sperm
morphology
There was a significant decrease (p< 0.05) in the
normal sperm concentration in this study in group G1 when compared to that of DW group; however, there were
relative decrease in the normal sperm morphology in groups Z + G1 (3.5%), Z (6.5%), Z + G (10%), Z + Gc (11.7%), Gc
(12.3%) and G (14.2%) when compared to the normal sperm morphology in DW group
as shown on (Table 1).
3.1.5. Effects
of treatments on abnormal sperm morphology
The result of this study showed a significant increase
(p< 0.05) in the abnormal sperm morphology in G1group when compared to that of DW group; however, there
were relative increase in the abnormal sperm morphology in groups Z (12.5%), Z
+ G1 (22.5%), Z + G (65%), G (70.1%), Z + Gc
(76.3%) and Gc (119.4%) when compared to the abnormal sperm morphology recorded
in DW group as shown on (Table 1).
3.1.6. Effects
of treatments on sperm head abnormality
There was a very highly significant increase (p <
0.001) in head abnormality recorded in G1 group as compared to the
head abnormality seen in DW group; however, there were relative increase in the
head abnormality recorded in groups Z + G1(31.3%), Z (87.5%), G (106.3%), Z + G (120.3%), Gc
(129.7%) and Z + Gc (167.2%) when compared to the sperm head abnormality
observed in DW group as shown in (Table 1).
3.1.7. Effects
of treatments on mid-piece abnormality
There was no change in the mid-piece abnormality recorded
in G1 group when compared to DW group; however, there were relative
decrease in the mid-piece abnormality recorded in groups G (33.3%), Z + G (50%)
and Z + Gc (100%) when compared to the mid-piece abnormality seen in DW group.
conversely there were relative increase in the mid-piece abnormality in groups
Z + G1 (33.3%), Z (166.7%) and Gc (175%) when
compared to the mid-piece abnormality recorded in DW group as shown on (Table
1).
3.1.8. Effects
of Treatment on Sperm Tail Abnormality
The result of this study showed no significant difference
in the tail abnormality between the groups; however, there were relative
increase in the tail abnormality observed in groups Z + G1 (14.3%), Z (19%), Z + G (23.8%), Z + Gc (32.1%), G
(33.3%), G1 (64.3%) and Gc (71.4%) when compared to DW group as
shown on (Table 1).
3.1.9. Effects
of Treatment on Sperm Motility
There was no significant difference in sperm motility
recorded in this study between the groups; however, there was a relative
increase in sperm motility observed in group Z +G1 (2.4%) when compared to the sperm motility recorded in
the DW group. On the other hand, there were relative decrease in sperm motility
in groups Z + Gc (1.2%), Z (10.5%), Gc (11.8%), G1 (15.7%), Z + G (16.5%) and G (16.9%) when compared to
the sperm motility observed in DW group as shown on (Table
1).
3.1.10. Effects of treatments on spermatozoan
progressive movement
There was a significant decrease (p< 0.05) and a
highly significant decrease (p< 0.01) in spermatozoan progressive movement
in G1 and Z + G groups respectively when compared to the
spermatozoan progressive movement observed in DW group; however, there were
relative decrease in the spermatozoan progressive movement in groups Z + G1 (18.7%), Z (22%), Gc (23.2%), Z + Gc (27.5%) and G
(31.8%) when compared to the spermatozoan progressive movement in DW group as
shown on (Table 1).
3.1.11. Effects of treatments on spermatozoan
non-progressive movements
There was a significant increase in the spermatozoan
non-progressive movement (p< 0.05) in G1 and Z + G groups,
however, there were relative increase in the spermatozoan non-progressive
movement in groups Gc (58.5%), Z (60%), G (74.4%), Z + G1 (131.4%) and Z + Gc (151.3%) when compared to the
spermatozoan non-progressive movement in DW group as shown on (Table
1).
3.1.12. Effects of treatments on spermatozoa
non-motility
There was no significant difference in the spermatozoan
non-motility in this study between the groups; however, there was a relative
decrease in the spermatozoan non-motility in Z + G1 (9.8%) group when compared to the spermatozoan
non-motility observed in DW group. On the other hand, there was relative
increase in the spermatozoan non-motility in groups Z + Gc (5.9%), G1
(41.2%), Gc (50%), Z + G (67.7%), Z (68.6%) and G (82.4%) when compared to the
non-motility observed in the spermatozoa in DW group as shown on (Table
1).
3.1.13.
Effects of treatments on testicular sialic acid concentration
There was no significant difference in the sialic acid
concentration between the groups; however, there was a relative increase in the
sialic acid concentration in Z+G group (3.5%) when compared to the sialic acid
concentration in DW group. On the other hand, there were relative decrease in
the sialic acid concentration in groups Z (0.4%), Z + G1 (1.8%), G1 (5.7%), G (16.5%), Gc (22.3%) and
Z+Gc (25.8%) when compared to DW group as shown on (Table
1).
3.1.14.
Effects of treatments on follicle stimulating hormone (FSH) concentration
There was no change observed in the FSH concentration in
groups Z, Z + G, Z + G1, Gc and G1 when compared to the
FSH concentration in DW group; however, there were relative increase in the FSH
concentration in groups G (0.1%) and Z + Gc (0.1%) when compared to the FSH
concentration in DW group as shown in (Figure 1).
3.1.15. Effects of
treatments on LuteinizingHormone (LH) concentration
There was no change observed in the LH concentration in
groups Z, Z + Gc, Z + G and Gc when compared to the LH concentration in DW
group; however, there were relative increase in LH concentration in groups Z +
G1 (0.03%), G (0.03%) and G1 (0.03%) when compared to the LH concentration in DW group
as depicted in (Figure 1).
5.1.16. Effects of treatments on testosterone
concentration
There was no significant difference in the testosterone
concentration between the groups; however, there was a relative increase in the
testosterone concentration in Z (0.7%) group when compared to the testosterone
concentration in DW group. Conversely, there was a relative decrease in the
testosterone concentration in groups Z + G1 (0.6%), Gc (0.8%), Z +
Gc (1.1%), Z + G (1.2%), G (2.6%) and G1(7%) when compared to the testosterone concentration in
DW group as shown in (Figure 1).
4.
Histopathological findings
The testes of rats in group I (DW) showed no observable
lesion as represented on (Plate Ia).
Similarly, the testes of rats in groups II (Z), VI (Z +
Gc), VII (Z + G) and VIII (Z + G1) showed no observable lesion
similar to those of group I (DW), however, vacuolations in the seminiferous
tubules, degeneration of spermatogenic cells and decreased number of spermatids
in the seminiferous tubules were observed in the testes of rats in group III
(Gc) as shown on (Plate IIa).
The testes of rats in group IV (G) revealed vacuolations
in the seminiferous tubules and degeneration of the spermatogenic cells as
depicted on (Plate IIIa).
Vacillations in the seminiferous tubules, degeneration of
spermatogenic cells, fragmented and coiled spermatids which were detached from
the basement membrane were observed in the testes of rats in group V (G1) as shown on plate IVa.
Histochemical investigation of testes of Wistar rats in
this study using Sudan Black (SB) stain showed normal histoarchitecture in the
testes of Wistar rats in groups DW, Z, Z + Gc, Z + G and Z + G1 as
shown on (Plate Ib)
while the testes of Wistar rats in groups Gc, G and G1
revealed vacuolations in the seminiferous tubules as represented on (Plate
IIb, IIIb and IVb)respectively.
5.
Discussion
Chronic glyphosate exposure in this study showed decrease
in sperm concentration in all the groups treated glyphosate alone which might
be as a result of increased generation of the Reactive Oxygen Species (ROS) in
the testes which might have impaired spermatogenesis in the exposed rats. The
induction of oxidative stress in testicular cells by Roundup®, a glyphosate based herbicide, was said to be evident by
decreased glutathione reductase levels accompanied by increased ThiobarBituric Acid
Reactive Substance (TBARS) levels by Roundup® in rat’s testes thus
linking ROS over-generation and oxidative damage [33]. Spermatozoa on their own are highly susceptible to
oxidative damage by excessive ROS due to high concentration of PolyUnsaturated Fatty
Acid (PUFA) within their plasma membrane [34]. Reduction in epididymis sperm count following exposure
to organophosphate, chlorpyrifos, was reported by earlier researchers to be
caused by low levels of scavenging enzymes and glutathione as well as high production
of free radicals, resulting from mitochondrial respiration and deficient DNA
repair mechanisms and thus providing unfavorable condition for spermatogenesis
in the seminiferous tubules [22,35,36].
The slight increase in the sperm concentration in the zinc group, Z, might
underscore the antioxidant effect of zinc as evident by increased sperm
concentration in the group when compared to the sperm concentration recorded in
DW group. Similarly, pretreatment with zinc in zinc supplemented groups, Z+Gc,
Z+G and Z+G1caused apparent increase in sperm
concentration in the said groups when compared to the sperm concentration in
the DW group and a significant increase in the sperm concentration was obtained
in Z + G1 group when compared to that of G1 group probably
mediated by the antioxidant effect of zinc which might have mitigated the
probable impaired spermatogenesis observed in the G1 group sequel to glyphosate exposure. Zinc had been
reported to play an important role in maintenance of structure and function of
biological membrane by scavenging free radicals [37]. Protective role of antioxidant (vitamin C) in
increasing sperm count following pretreatment in chronically
chlorpyrifos-exposed rats had been reported by Shittu [38].
Live sperm count in this study decreased in the
glyphosate exposed groups when compared to the live sperm count in DW group.
The decreased live sperm count associated with chronic glyphosate exposure in
this study might be due to the level of the oxidative damage in the testes of
the exposed rats. Razi [39]in a sub-chronic study on glyphosate exposure
in rats reported decreased sperm viability alongside other alterations in
reproductive parameters in the glyphosate exposed rats. Zinc administration
caused a relative decrease in the live sperm count which might be associated
with slight proxidant effect of the zinc on the sperm viability in the rats in
the Z group. Proxidant effect of zinc was reported earlier by Abdallah and
Samman [40].On the other hand, pretreatment with zinc in
the zinc supplemented groups revealed apparent protective effect of zinc in the
mentioned groups as evident by the apparently higher live sperm count in all
the zinc supplemented groups when compared to the live sperm count in the DW
group possibly due to the antioxidant role of zinc in the zinc supplemented
groups. Zinc plays a structural role in the maintenance of the integrity of Cu
- Zn superoxide dismutase as a cofactor [41]and it is also known to regulate glutathione that is vital
to cellular antioxidant defense [42].
The dead sperm count in this study increased in all the
glyphosate exposed groups and the increase in the percentage of dead sperm
count might be as a result of high level of oxidative damage caused by
oxidative stress in the testes of the exposed rats. Increased dead sperm count
has been reported in a sub-chronic toxicity study on reproduction in rats by
Razi [39]. A relatively high dead sperm count was also
recorded in the rats administered zinc alone in Z group possibly due to
proxidant effect of the zinc in the rats in that group as reported by Abdallah
and Samman [40] since the rats were not exposed to any
environmental toxicant. Zinc supplementation in the zinc pretreated rats,
however, ameliorated the lethal oxidative damage of the glyphosate on the sperm
by apparently reducing the dead sperm count to levels below that of the DW
group in all the rats in zinc supplemented groups which were likely brought
about by the antioxidant effect of zinc. Apart from its direct antioxidant role
by occupying iron and copper binding sites on lipids, proteins and DNA as
reported by Prasad and Kucuk [43], zinc
also plays a structural role in the maintenance of the integrity of Cu - Zn
superoxide dismutase as a cofactor as well as maintaining the structure and
function of biological membranes [37,41]. Normal sperm morphology in this study decreased in a
dose-dependent fashion in the glyphosate exposed groups with a significant
decrease observed in the highest glyphosate exposed group which might have been
occasioned by increased generation of ROS causing oxidative damage to the
normal architecture of the spermatozoa. Morphologically abnormal spermatozoa,
precursor germ cells, leucocytes and at last degenerated cells in
spermatogenesis series are the components of the male genital system capable of
generating Reactive Oxygen Species (ROS) as reported by Chapin [44]. A dose-dependent reduction in morphologically normal
spermatozoa which was said to be the possible cause of the decreased sperm
concentration was also recorded in a dose-dependent fashion in chlorpyrifos
toxicity study as earlier reported by Olorunshola [45]. A relative decrease in the normal sperm morphology
recorded in the group administered with zinc alone, Z group, might be as a
result of proxidant effect of zinc in the absence of oxidative stress in the
rats in Z group as reported by Tizhe[46]. Zinc pretreatment in the zinc supplemented groups,
showed variable ameliorative effect by increasing the percentages of the normal
spermatozoa in the zinc supplemented groups to near normal when compared to
those of the glyphosate exposed groups. The ameliorative effect of antioxidant
on reduction in the sperm morphology in chlorpyrifos toxicity study was earlier
reported by Olorunshola [45].
Abnormal sperm morphology in this study was shown to
significantly increase in G1group which is the highest glyphosate exposed group and
apparent increase in the abnormal sperm morphology were recorded in Gc and G
groups and the increased abnormality observed in all the glyphosate exposed
groups might be attributed to the oxidative damage caused by probable increased
generation of free radicals in the testes. Razi [39] documented elevated abnormal sperm content with
different characteristics in glyphosate exposed groups which they attributed to
the probable major role of imbalanced oxidative stress in generating various
disorders. Zinc administration in Z group caused relative increase in the
abnormal sperm morphology observed in the rats in Z group possibly as a result
of proxidant effect of zinc in the rats since they were not exposed to any environmental
toxicant. Proxidant effect of zinc was earlier documented by Abdallah and
Samman [40]. Pretreatment with zinc in groups which were
supplemented with zinc showed ameliorative effect by decreasing the levels of
the abnormal sperm morphology in the said groups when compared to the
glyphosate exposed groups which might have been caused by the protective role
of zinc in the maintenance of morphology of the sperm cells. Zinc was reported
to protect the testes against degenerative changes [47]. Bettger and O’Dell [37] reported the role of zinc in maintenance of structure
and function of biological membranes.
Sperm head abnormality in this study was shown to be
apparently increased in the glyphosate-exposed groups when compared to DW group
which might possibly be sequel to increased level of oxidative damage to the
sperm cells. Increased abnormal sperm content with different characteristics
such as elongated head, pyriform head, bent head, cytoplasmic droplets and
degenerated germ cells were reported by Raji[39]. Zinc administration in Z group caused apparent increase
in the sperm head abnormality possibly mediated by the pro-oxidant effect of
zinc as reported by earlier researchers [40,46]. Zinc supplementation in Z + Gc and Z + G did not ameliorate
the increased abnormal sperm head recorded in the said groups, however, an
apparent ameliorative effect was recorded in Z + G1 group which was the highest glyphosate exposed group
that was supplemented with zinc at high concentration; it is therefore,
possible that the increased sperm head abnormality recorded in Z + Gc and Z + G
which were not ameliorated might be ameliorated with increased zinc
supplementation similar to that used for Z + G1 group. The role of
zinc as antioxidant to protect the cell membrane and nuclear chromatin of
spermatozoa was earlier reported by Chvapil [48]. Various changes were observed in sperm mid-piece
abnormality following glyphosate exposure in this study ranging from apparent
increase in the sperm mid-piece abnormality in Gc group to a decrease in the
mid-piece abnormality in G group and no noticeable difference in the sperm
mid-piece abnormality in G1 when compared to DW group. The changes
recorded in the sperm mid-piece abnormality in this study suggested that most
of the abnormal changes seen were associated with the head and the tail of the
sperm cells but the reason for the varying changes recorded is not known for
certain especially considering the similar pattern of changes observed in the
sperm head abnormality and the sperm tail abnormality in this study. Zinc
administration in Z group caused apparent increase in the sperm mid-piece
abnormality similar to that seen in the sperm head abnormality in this study
following zinc administration in Z group probably associated with pro-oxidant
effect of zinc in the rats in the group. Pro-oxidation in animals following
zinc administration was reported in earlier study by Abdallah and Samman [40]. Zinc supplementation in Z + Gc and Z + G groups
effectively ameliorated the sperm mid - piece abnormality in the rats in those
groups but ameliorative effect was not observed in the sperm mid - piece
abnormality recorded in Z + G1 group and the reason for that is not clear but might be
as a result of the highest level of oxidative damage in the group.
Zinc can counteract oxidation by binding suphydryl groups
in proteins and by occupying binding sites for iron and copper in lipids,
proteins and DNA [44,49] and to substantiate these antioxidant effect
of zinc, evidence was found for oxidative damage of proteins lipids and DNA in
zinc-deficient rat and mice [50,51].
Sperm tail abnormality in this study increased in all the
glyphosate exposed groups, likely due to increased level of oxidative damage in
the sperm cells of the rats in the aforementioned groups. Sperm abnormalities
were documented in earlier studies on organophosphates toxicity in rats [39,45]. Zinc administration in Z group in this study caused relative
increase in the sperm tail abnormality when compared to DW group which might be
as a result of the pro-oxidant effect of the zinc similar to those of head and
mid - piece abnormality in the sperm cells observed in this study. Abdallah and
Samman[40]have documented the prooxidant effect of
zinc. Zinc supplementation in the zinc pretreated groups caused ameliorative
effect by the decreased sperm tail abnormality to near normal probably due to
the antioxidant role of zinc. Zinc salts have been shown to protect against
oxidative damage and glutathione depletion in mice [51]. Decreased sperm motility was recorded in glyphosate -
exposed groups, in this study which might be attributed to the possible
oxidative damage in the testes caused by glyphosate exposure in the affected
rats. Choudhary [52] documented that sperm motility is affected
by altered enzymatic activities of oxidative phosphorylation. Full ATP pool is
essential for normal spermatozoan movement and a slight deprivation of ATP
leads to reduction in sperm motility which may cause infertility[53]. Another factor which may cause decrease in sperm
motility may be androgen deprivation effect of the pesticide[52]. A positive correlation between testosterone and
motility and fertilizing capacity of the spermatozoa has been reported and the
compounds of seminal vesicle secretion also act as energy source for sperm
motility [54]. Lipid peroxidation, had been documented to
destroy the structure of lipid matrix in the membranes of spermatozoa, and it
is associated with loss of motility [55-57]. Decreased sperm motility in organophophates toxicity in
rats had been documented by earlier researchers [39,45]. Decrease in sperm motility has been documented to be
caused principally by defective spermatozoa [58]. Zinc administration in Z group caused decreased sperm
motility possibly due to pro-oxidant role of zinc in the absence of oxidative
stress in the rats. Zinc pretreatment in zinc supplemented groups, apparently
increased the rate of motility in the sperm cells in the rats in the said
groups. Decreased sperm motility was documented to be ameliorated following the
use of an antioxidant (Vitamin C) in a previous study by Olorunshola [45].
Decrease in sperm progressive movement were recorded in
all the glyphosate exposed groups, which was more pronounced in G1 group where a significant decrease in the sperm
progressive movement was recorded. The decline in the sperm progressive
movement observed following glyphosate exposure in this study might be
associated with some oxidative damage in the viability of the sperm and/or the
testosterone level in the rats. Decrease in sperm motility has been documented
to be caused principally by defective spermatozoa[58]. The epididymis spermatozoa are highly dependent on
testosterone and epididymis protein for their final maturation and development
of progressive motility and fertilizing capacity [59]. Zinc administration in Z group showed apparent decrease
in the sperm progressive movement which might be associated with pro-oxidant
effect of zinc in the rats since they were not under oxidative stress.
Proxidant effect of zinc was documented in earlier studies [40,60]. Zinc supplementation in Z + Gc and Z + G did not show
any protective effect but instead it further decreased the sperm progressive
movement in the aforementioned groups, however, zinc supplementation in Z + G1
group has greatly ameliorated the sperm progressive movement in the rats in
that group, possibly due to the high dose of zinc used for supplementation in
that group. Olorunshola [45] has documented the ameliorative role of an
antioxidant, vitamin C, in decreased sperm motility observed in their study on
chlopyrifos toxicity in rats.
Non-progressive movement in spermatozoa in this study
apparently increased in a dose dependent fashion in the different doses of
glyphosate used for the study and the decrease was statistically significant in
G1 group which was the highest glyphosate exposed group. The changes
recorded might be due to the increased level of oxidative stress in the testes
or due to decrease in the testosterone level in the rats. Vawda and Davies [59]documented that epididymis spermatozoa are greatly
dependent on testosterone and epididymis protein for their final maturation and
development of progressive motility and fertilizing capacity. The increased non-progressive
motility recorded followingglyphosate exposure in this study might also be as a
result of increased deprivation of ATP to the spermatozoa since full ATP was
said to be crucial for the normal spermatozoon movement and any slight
deprivation of ATP leads to reduction in spermatozoan movement and may cause
infertility (Bedford, 1983). Zinc administration in Z group caused apparent
increase in the non - progressive movement of sperm in this study probably
mediated by the pro-oxidant effect of zinc since the rats in Z group were not
under oxidative stress. Pro-oxidant effect associated with zinc was documented
in earlier studies [40,46,60]. Zinc supplementation in zinc pretreated
groups did not show ameliorative effect but instead further increased the sperm
non - progressive movement in a similar pattern of negative effect seen in the
sperm progressive movement in this study and the non-progressive sperm movement
in this study is significant in Z+G group, however, ameliorative effect was
apparently seen in Z + G1 similar to that recorded for sperm
progressive movement in this study probably caused by the antioxidant effect of
zinc in the rats since the zinc was in high dose in that group and might
therefore, account for the ameliorative effective recorded in Z+G1
group which was not seen in Z+Gc and Z+G group. Vitamin C as an antioxidant was
used in a previous study by Olorunshola [45]to mitigate various sperm abnormal changes recorded in
the study.
Glyphosate exposure in a chronic study in this research
has been shown to cause a dose dependent apparent increase in the number of
non-motile sperm cells observed in the glyphosate exposed groups, probably
caused by oxidative damage in the testis or alteration in the testosterone
levels in the Wistar rats. Lipid peroxidation was said to destroy lipid matrix
in the membranes of spermatozoa, and it is associated with loss of motility[55-57]. Complete ATP pool is necessary for normal spermatozoan
movement and slight deprivation of ATP leads to reduction in spermatozoan
movement which may cause infertility (Bedford)[53]. Zinc administration in Z group caused an apparent
increase in the number of non-motile sperm cells observed in the Z group
possibly due to the pro-oxidant effect of zinc as earlier reported by Abdallah
and Samman [40]. Zinc supplementation in zinc pretreated
groups ameliorated the detrimental effect of the glyphosate by reducing the
number of the non-motile sperm cells in the rats pretreated with zinc in the
said groups probably due to the antioxidant role of zinc as documented by
Olorunshola [45]who used vitamin C as an antioxidant to
ameliorate the adverse effect of chlorpyrifos in the spermatozoa of the
chlorpyrifos - exposed rats.
Testicular sialic acid concentration decreased in all the
glyphosate exposed groups, in this study likely due to the increased oxidative
stress in the testes of the rats in the aforementioned groups. In a similar
trend, Choudhary [52]reported a significant decline in the
contents of sialic acid among other reproductive parameters they studied
following exposure of male rats to malathion. Zinc administration in Z group
did not have negative effect on the testicular sialic acid concentration in
this study, possibly because of the antioxidant effect of the zinc on the
testicular sialic acid and zinc supplementation in the zinc pretreated groups
showed a noticeable ameliorative effect by restoring the sialic acid
concentration to near normal in the zinc supplemented groups. Several studies
have demonstrated the protective role of zinc on organophosphates compounds
toxicity[22,24,46,61-63].
In this study, however, chronic exposure to glyphosate in
male Wistar rats caused a dose- dependent decrease in testosterone
concentration in the glyphosate exposed groups which might be linked to the
possible oxidative damage to the testes of the glyphosate exposed rats. Most
earlier researches on organophosphate compounds recorded decrease in
testosterone concentration which was said to be perhaps as a result of the
inhibitory effect of organophosphates on the secretion of pituitary
gonadotropins (FSH and LH), which are involved in testosterone biosynthesis,
direct damage to the leading cells or inhibition of testosterone metabolism or
testosterone synthesis [23,45,64-66]. Conversely, chronic exposure to sub-lethal
concentration of a glyphosate-based herbicide (3.6mg/L) in female Jundia showed
increased pattern of testosterone secretion which was similar to the
progression of the vitellogenesis. Zinc administration in the Z group caused
relative increase in the serum testosterone concentration which might
underscore an antioxidant role of zinc in increasing serum testosterone
concentration. Zinc had been reported to protect sulphydryl group against
oxidation thereby preventing protein from oxidation, hence stabilizing the
cellular thiol pools [67]. On the other hand, pretreatment with zinc
as an antioxidant restored the testosterone concentration to near normal in Z+G
and Z+G1 groups thus, underscoring the possible
involvement of oxidative mechanism in the pathology of glyphosate in rats.
Previous studies on the use of antioxidants to ameliorate the decreased
testosterone levels following exposure to organophosphates have been documented
[23,45]. Conversely, pretreatment with zinc in Z+Gc
group did not ameliorate the relative decrease in serum testosterone level and
the reason for that is not known especially considering the relative increase
in the level of serum testosterone in Z group and the apparent ameliorative
effect recorded following pretreatment with zinc inZ+G and Z+G1 groups.
Chronic glyphosate exposure in this study showed no
difference in the FSH concentration between the glyphosate treated groups Gc
and G1 when compared to the DW group, perhaps due
to minimal effect or no toxic effect of the glyphosate on FSH because the only
glyphosate exposed group that showed a negligible difference is G group which
showed a slight increase in the FSH concentration when compared to DW group and
that is negligible to be linked only to glyphosate because minor difference in
concentration can be seen even within normal animals. The result of this study
is at variance with most studies on organophosphate compounds effect on FSH
concentration in animals who documented that organophosphates cause decrease in
the serum levels of FSH probably due to the ability of the organophosphate
compounds to suppress the gene involved in gonadotrophins synthesis or
interfere with steroidogenesis [8,23,68].
Zinc administration in Z group also did not cause any noticeable change in the concentration
of serum FSH in the rats in that group likely because zinc as an antioxidant
has no toxic effect on FSH concentration in animals even on long term exposure.
On the other hand, pretreatment with zinc in Z + G and Z + G1 groups
also caused no noticeable difference in the serum FSH concentration when
compared to DW and those of glyphosate exposed rats alone, however,
pretreatment with zinc in Z + Gc group also caused a negligible increase in FSH
concentration. Zinc had been reported to play an important role in the
structure and function of biological membranes[37].
The result of this study showed a very negligible
increase in LH concentration in glyphosate exposed groups G and G1 and no difference in the LH concentration was recorded
in Gc group when compared to the serum LH concentration in DW group and that
might be due to the low glyphosate exposure in that group. This result suggests
that chronic glyphosate exposure has little or no pathologic and/or toxic
effect on serum LH concentration which differs from most previous studies on
the serum LH concentration in animal’s sequel to organophosphate exposure who
documented decreased serum LH concentration which they attributed to be likely
due to their effects on Hypothalamo-pituatory endocrine function [8,35,69-71]. In tandem with the finding of this study, increase in
serum level of LH following organophosphate exposure had been documented and
were said to be detrimental to the germinal cells of the testes and hence
capable of disrupting spermatogenesis. Zinc administration in the Z group did
not cause any change in the serum level of LH when compared to DW group and
pretreatment with zinc in Z + Gc and Z + G also did not cause any change in the
serum concentration of LH when compared to DW, however, pretreatment with zinc
in Z + G1 group caused a negligible increase in the
serum level of LH in Z + G1 group
similar to that recorded in G and G1groups and the reason for that is not known for certain
but mightbe due to the high exposure to the glyphosate in G1 and therefore difficult to mitigate the increase in the
serum concentration of the LH even when high zinc supplementation was used. The
antioxidant role of zinc in organophosphate toxicity had well been documented
in previous studies [23,46,61-63].
Histomorphological examination of testes in the study
showed vacuolation in the seminiferous tubules, degeneration of spermatogenic
cells, fragmented and coiled spermatids with H & E stains, and when stained
with Sudan black due to the presence of vacuoles observed when stained with H
& E, the sections were still found to have vacuolations in the seminiferous
tubules which are most likely fat vacuoles and that might have deleterious
effect on spermatogenesis in the glyphosate exposed groups where they were
observed. Similar to this finding, severe degeneration in seminiferous tubules
and dissociation of germinal cells and arrested spermatocytogenesis,
spermatogenesis and spermatogenesis with severe depletion of seminiferous
tubules which was accompanied by huge infiltration of inflammatory cells were
documented in diazinon exposed rats [72]. There are several independent studies which indicated
that following severe inflammation, elevated oxidative stress causes apoptosis
in spermatogenesis series characterized by remarkable cellular depletion in
seminiferous tubules [73,74]. Gradual decrease in paired testicular
weight and seminiferous tubular diameter in association with progressive degenerative
changes in seminiferous epithelial cells were found to occur in relation to
various doses and durations of pesticide treatment [75]. Zinc administration in Z group and zinc supplementation
in zinc pretreated groups showed no observable microscopic lesions in the
testes of the rats in those groups, probably due to the ameliorative effect of
zinc as an antioxidant which ameliorated the histopathological changes in the
testes of rats in the zinc pretreated groups as evident following examination
of both the H & E and Sudan black-stained sections. Antioxidant mineral,
selenium, had been documented to mitigate the detrimental effects of diazinon
in the seminiferous tubules of diazinon exposed rats [72].
Note: There is no
conflict of interest among the authors
Contributor
ship Statement
Dr. Uchendu
Chidiebere, department of Veterinary Pharmacology, Physiology and Biochemistry
helped us with the data analysis after the research work. Prof. N.D.G Ibrahim,
Prof. M.Y Fatihu, Prof. S.F Ambali and Prof. I.O Igbokwe were very instrumental
in the design, supervision and editing the manuscript. I.J. Gosomji Dr. U.
Delia and DR. E.V Tizhe contributed in the design, literature review, carrying
out the research work and wrote the manuscript for publication.