j o ur na l ho me pag e: www.elsev ier .com/ locate /aquatox
ffect of glyphosate on the sperm quality of zebrafish Danio rerio
ernanda Moreira Lopesa,b, Antonio Sergio Varela Juniora,c,e, Carine Dahl Corcinib,d,e,lessandra Cardoso da Silvae, Vitória Gasperin Guazzelli d,eorgia Tavaresd, Carlos Eduardo da Rosaa,b,∗
Instituto de Ciências Biológicas, Universidade Federal do Rio Grande, Av. Itália km 8, 96203-900 Rio Grande, RS, BrazilPrograma de Pós-Graduac ão em Ciências Fisiológicas – Fisiologia Animal Comparada, BrazilPrograma de Pós-Graduac ão em Biologia de Ambientes Aquáticos Continentais, Universidade Federal do Rio Grande, Av. Itália km 8,6203-900 Rio Grande, RS, BrazilFaculdade de Veterinária, Universidade Federal de Pelotas, Campus Universitário, Caixa Postal 354, 96001-970 Pelotas, RS, BrazilPrograma de Pós-Graduac ão em Medicina Veterinária, Universidade Federal de Pelotas, Campus Universitário, Caixa Postal 354,6001-970 Pelotas, RS, Brazil
r t i c l e i n f o
rticle history:eceived 6 May 2014eceived in revised form 2 July 2014ccepted 4 July 2014vailable online 12 July 2014
eywords:erbicideanio rerioeproduction
a b s t r a c t
Glyphosate is a systemic, non-selective herbicide widely used in agriculture worldwide. It acts as aninhibitor of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase by interrupting the synthesis ofessential aromatic amino acids. This pathway is not present in animals, although some studies haveshown that the herbicide glyphosate can affect fish reproduction. In this study, the effect of glyphosateon sperm quality of the fish Danio rerio was investigated after 24 and 96 h of exposure at concentrationsof 5 mg/L and 10 mg/L. The spermatic cell concentration, sperm motility and motility period were mea-sured employing conventional microscopy. The mitochondrial functionality, membrane integrity andDNA integrity were measured by fluorescence microscopy using specific probes. No significant differ-ences in sperm concentration were observed; however, sperm motility and the motility period were
perm motilityNA damage
reduced after exposure to both glyphosate concentrations during both exposure periods. The mitochon-drial functionality and membrane and DNA integrity were also reduced at the highest concentrationduring both exposure periods. The results showed that glyphosate can induce harmful effects on repro-ductive parameters in D. rerio and that this change would reduce the fertility rate of these animals.
Glyphosate [N-(phosphonomethyl) glycine] formulations areidely used in rice fields in southern Brazil and present a poten-
ially harmful effect on aquatic life, considering water drainagerom the plantations coincides with the breeding season of fish
Giesy et al., 2000; Primel et al., 2005). This herbicide is a non-elective, systemic herbicide used to control a wide variety ofnnual, biennial and perennial herbs (Alonzo and Corrêa, 2008). It
∗ Corresponding author at: Instituto de Ciências Biológicas, Universidade Federalo Rio Grande, Av. Itália km 8, 96203-900 Rio Grande, RS, Brazil.el.: +55 53 32935175.
is presented under different names and commercial formulationsand acts as an inhibitor of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase, which is responsible for the synthesis of anintermediate in the biosynthesis of various aromatic amino acidsin plants. Although it is important for plant growth, this pathwayis not present in animals (Costa, 2008).
Studies have shown that glyphosate, or its commercial formu-lation (Roundup), may affect reproduction in animals. It has beendemonstrated that female fish exposed to these herbicides pre-sented alterations in their sex hormone profile (Soso et al., 2007)and a reduction in egg production and embryo viability (Websteret al., 2014). In male rabbits, Yousef et al. (1995) noted that chronicexposure to the herbicide glyphosate resulted in a reduction insperm concentration, accompanied by the increase of abnormal
or dead sperm cells. The authors suggest that this may be due todirect effects on spermatogenesis and/or indirect effects via thehypothalamus-pituitary-testis axis. Cavalli et al. (2013) stated thatfertility in rats can be affected by Roundup and glyphosate by the
nduction of cell death in Sertoli cells, which are responsible forhe maintenance of spermatogenesis. Additionally, as proposed by
alsh et al. (2000), glyphosate is able to disrupt the steroidogeniccute regulatory (StAR) protein expression in cultured tumoral Ley-ig cells. This leads to a reduction of steroidogenesis, eliciting thendocrine disruption role of this agrichemical. These results are ingreement with the findings that Roundup alter the sperm pro-uction in rats (Romano et al., 2012) as it has been demonstratedhat glyphosate and Roundup negatively affect Leydig cells, caus-ng apoptosis in the first hours of exposure (Clair et al., 2012). Theseffects would contribute to inefficient sperm cell functionality and,onsequently, to problems in fecundity. In fact, Harayashiki et al.2013) demonstrated negative effects of the commercial formula-ion Roundup on sperm functionality in the fish Poecilia vivipara.
Although the commercial formulation represents the main formf the application, and therefore has a higher relevance in termsf ecotoxicology, the effects of glyphosate on sperm quality andunctionality have not been evaluated in fish. It is important tomphasize that due to the presence of inert compounds, the com-ercial formulations presented higher toxicity when comparedith the active principle (Giesy et al., 2000). Considering that
hese parameters are key factors to the reproductive success of fishpecies, the objective of the present study was to evaluate the effectf glyphosate herbicide on sperm quality in the fish Danio rerio after4 and 96 h of exposure.
. Materials and methods
.1. Animals and treatment
The handling and maintenance of zebrafish were in complianceith the Zebrafish Book (www.zfin.org). Adult zebrafish (D. rerio)ales were obtained from commercial distributors and maintained
n tanks containing dechlorinated and aerated water at 28 ± 2 ◦C, pH.0, 0 ppm of nitrite, under a photoperiod of 12 h light: 12 h dark.ish were fed ad libitum with a commercial fish food twice a dayTetra ColorBits).
After three weeks of acclimation, the animals (total length:7.9 ± 0.6 mm, weight: 0.541 ± 0.2) were randomly divided intohree experimental groups (n = 27 per group). The experimentsere performed in 2 L aquariums (three fishes per aquarium) withine replicates. The water conditions during the experimentaleriod were the same as during the maintenance period. The con-rol group was kept only in water and the exposed groups receivedlyphosate (N-(phosphonomethyl)glycine, Sigma–Aldrich) solu-ions at final concentrations of either 5 mg/L or 10 mg/L. Theoncentrations were chosen based on previous studies that usedhese concentrations for the commercial formulation (Modesto and
artinez, 2010). Animals were exposed for 24 h or 96 h (wateras renewed after 48 h, to keep water quality). It is important toote that no mortality was observed in the test groups during thexposure period. At the end of the exposure period, the animalsere sacrificed, and the pair of testes of each fish were excised andlaced in tubes containing 100 �L of Beltsville Thawing SolutionBTS) for subsequent analysis (Varela Junior et al., 2012). The tubesere shaken for the release of spermatozeugmatas (sperm bun-les). Sperm was released by gently and repeatedly disrupting thepermatozeugmatas with a 10-�L pipette tip. The sperm suspen-ion was used for the analyses described below. In all subsequentpplications, two hundred cells per animal were analyzed.
.2. Sperm concentration, sperm motility and the motility period
The sperm concentration, sperm motility and motility periodere evaluated using a phase-contrast microscope (BX 41 Olympus
logy 155 (2014) 322–326 323
América, Inc., São Paulo, SP, Brazil), with 200× magnification, aftercombining 1 �L of sperm diluted in BTS solution and 99 �L of con-trol water (28 ◦C) in order to active the spermatozoa. The spermconcentration was measured using a Neubauer chamber, and theresults were expressed as sperm per milliliter of semen. Spermmotility and the motility period were accessed on a slide coveredwith a coverslip (Varela Junior et al., 2012). Sperm motility wasexpressed as the percentage of progressive motile spermatozoa10 s after activation, and the motility period was comprised of thetime (in s) between sperm activation and the absence of progressivemovement (straight line movement).
2.3. Mitochondrial functionality
The mitochondrial functionality was evaluated according to Heand Woods (2004), adapted by Varela Junior et al. (2012), usinga rhodamine 123 probe, which accumulates only in functionalmitochondria. Five microliters of sample were incubated with20 �L of the rhodamine 123 solution (13 �M) at 20 ◦C for 10 min.After incubation, the cells were counted using an epifluorescencemicroscope (Olympus BX 51, América, São Paulo, SP, Brazil) at 400×magnification. The mitochondria were considered functional whensperm presented positive rhodamine 123 staining (green fluores-cence) and nonfunctional when sperm presented no fluorescence.The results are expressed as the percentage of sperm with func-tional mitochondria compared to total sperm.
2.4. Sperm membrane integrity
The membrane integrity of the sperm was examined follow-ing the methodology of Harrison and Vickers (1990). For thatgoal, 5 �l of sample were diluted in 20 �l of saline solution with1.7 mM formaldehyde, 20 �M carboxyfluorescein diacetate (CFDA)and 7.3 �M propidium iodide (PI). Fluorescence was verified at400× magnification using an epifluorescence microscope (OlympusBX 51, América, São Paulo, SP, Brazil). When the spermatozoa mem-brane was intact, CFDA accumulation occurred. After the hydrolysisof CFDA, carboxyfluorescein was generated along with a corre-sponding green fluorescence. Sperm with damage in the membraneincorporated PI and emitted a red or red and green fluorescence.The percentage of sperm viability was determined by the propor-tion of sperm emitting green fluorescence compared with the totalnumber of sperm (green, red or red and green).
2.5. DNA integrity
Sperm DNA integrity was evaluated using the acridine orangemethod described by Varela Junior et al. (2012), where themetachromatic colorant acridine orange emits green fluorescencein reaction to double-stranded DNA and an orange or red fluores-cence in reaction to single-stranded DNA, identifying a break in theDNA. The sperm sample (45 �L) was diluted in 50 �L TNE (0.01 MTris–HCl; 0.15 M NaCl; 0.001 M EDTA; pH 7.2). After 30 s, 200 �Lof Triton solution 1× were added, and 30 s later, 50 �L of acridineorange were added (2 mg/mL in deionized H2O). The evaluation wasconducted after 5 min without exceeding 1 min of slide exposure.The sperm presenting with green fluorescence contained intactDNA, and those presenting with red or orange fluorescence con-tained denatured DNA. The rate of DNA integrity was determinedby the proportion of sperm emitting green fluorescence comparedwith the total number of sperm analyzed (green, red or orange).
2.6. Statistical analysis
The results were presented as the means ± standard error ofthe means (SEM). The statistical analyses were made using the
Fig. 1. Sperm concentration in Danio rerio exposed for 24 h and 96 h to glyphosate(m(
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3
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24 hours 96 hours0
100
200
300
400
500
Control
5 mg/L
10 mg/L
a
b
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Mo
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Fig. 3. The motility period, in s, in Danio rerio exposed for 24 h and 96 h to glyphosate(5 mg/L and 10 mg/L) and the respective control groups (0.0 mg/L). The values
5 mg/L and 10 mg/L) and the respective control groups (0.0 mg/L). The values areeans ± SEM (n = 24–27). There was no significant difference among the treatments
p < 0.05).
tatistix 9.0 Analytical Software (Tallahassee, FL, USA) (Statistix,008). The normality of the samples and the homogeneity of theariances were tested using the Shapiro–Wilk test and the Leveneest, respectively. The parameters considered normally distributednd homoscedastic were tested by analysis of variance (ANOVA),omparing the three groups of animals exposed to glyphosate0 mg/L, 5 mg/L and 10 mg/L), with comparisons of the means doney Tukey’s test HSD with a significance level of 5%. The parametersithout normal distribution were submitted to the Kruskal–Wallis
nalysis of variance for non-parametric data, followed by theruskal–Wallis all-pairwise comparisons, using a significance levelf 5%.
. Results
.1. Sperm concentration, sperm motility and motility period
No significant differences were observed in sperm concentra-ion between groups at any duration of exposure (Fig. 1).
Glyphosate exposure significantly reduced (p < 0.05) spermotility and the motility period after both exposure periods and
t both herbicide concentrations. The control group had a spermotility of 90.9% (±2.5) after 24 h and 91.4% (±2.7%) after 96 h
f exposure. Treatment with 5 mg/L of glyphosate resulted in aecrease of 62.8% and 51.4%, at 24 and 96 h, respectively. At a con-entration of 10 mg/L, there was a decrease of 62.6% after 24 h and
7.1% after 96 h (Fig. 2). No significant differences were observed
n these parameters between glyphosate-exposed groups at theifferent exposure periods.
24 hours 96 hours0
20
40
60
80
100
Control
5 mg/L
10 mg/La
b
b
a
b b
Sp
erm
mo
tili
ty (
%)
ig. 2. Sperm motility in Danio rerio exposed for 24 h and 96 h to glyphosate5 mg/L and 10 mg/L) and the respective control groups (0.0 mg/L). The valuesre means ± SEM (n = 24–27). The different letters represent significant differencesmong treatments at the same exposure periods (p < 0.05).
are means ± SEM (n = 24–27). The different letters represent significant differencesamong treatments at the same exposure period (p < 0.05).
Concerning the sperm motility period, a 2.5-times reduction wasobserved with the 5 mg/L glyphosate treatment, and a 2.9-timesreduction was observed with the 10 mg/L glyphosate after 24 h.After 96 h of exposure, there was a decrease of 2 times in the 5 mg/Lgroup and 4 times in the 10 mg/L group (Fig. 3). Again, no significantdifferences were observed between groups exposed to glyphosate(p > 0.05).
3.2. Mitochondrial functionality
Mitochondrial functionality was significantly (p < 0.05) affectedby glyphosate exposure (p < 0.05). Reductions of 20% (±6.6%) and35% (±7.7%) in mitochondrial functionality after 24 and 96 h ofexposure, respectively, were observed in fishes exposed to 10 mg/Lof glyphosate when compared with the control group (Fig. 4).
3.3. Sperm membrane integrity
A significant decrease (p < 0.05) in the sperm membraneintegrity of fish exposed to 10 mg/L of glyphosate was observed(Fig. 5). After 24 h of exposure, the membrane integrity was 75.8%(±3.7%) in this group, while in the control group and the groupexposed to 5 mg/L, it was 90.5% (±1.8) and 88.9% (±2%), respec-tively. The same was observed after 96 h. The animals exposed to10 mg/L of glyphosate presented a significant (p < 0.05) reduction
in membrane integrity to 57.7% (±5.6) compared with the controlgroup (85.7 ± 1.9%) and the group exposed to 5 mg/L (76.2 ± 4%).
24 hours 96 hours0
20
40
60
80
100
Control
5 mg/L
10 mg/La
ab
b
a abb
Mit
och
on
dri
al
fun
cti
on
ali
ty (
%)
Fig. 4. Sperm mitochondrial functionality in Danio rerio exposed for 24 h and 96 hto glyphosate (5 mg/L and 10 mg/L) and the respective control groups (0.0 mg/L).The values are means ± SEM (n = 24–27). The different letters represent significantdifferences among treatments at the same exposure period (p < 0.05).
F.M. Lopes et al. / Aquatic Toxico
24 hou rs 96 hours0
20
40
60
80
100
Control
5 mg/L
10 mg/Laa
b
a a
b
Sp
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teg
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(%
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Fig. 5. Sperm membrane integrity in Danio rerio exposed for 24 h and 96 h togvf
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lyphosate (5 mg/L and 10 mg/L) and the respective control groups (0.0 mg/L). Thealues are means ± SEM (n = 24–27). The different letters represent significant dif-erences among treatments at the same exposure periods (p < 0.05).
.4. DNA integrity
The DNA integrity was significantly compromised comparedith the control group animals after 24 h of exposure to 10 mg/L
f glyphosate. A reduction of 14% was observed compared to theontrol group at this exposure period. After 96 h, the DNA integrityn the animals exposed to 10 mg/L of glyphosate was 78.3% (±3.5),
hich was significantly different from both the control group ani-als (94.7 ± 0.9%) and the group exposed to 5 mg/L of glyphosate
92.6 ± 1.9%) (Fig. 6).
. Discussion
This is the first study that demonstrated the harmful effects oflyphosate per se on the components (membrane, mitochondriand DNA) of fish spermatozoa, without the interference of surfac-ants and other inert compounds in its commercial formulations.lyphosate can interfere with the fertilization rate, which is directly
inked to the quality of the gamete, hindering reproductive successLinhart et al., 2000). In the present study, the reproductive param-ters of D. rerio males were evaluated after 24 and 96 h of exposureo glyphosate. In summary, the biochemical parameters and cellerformance analysis were affected by acute glyphosate exposuret both analyzed concentrations (5 and 10 mg/l) within the first 24 hf exposure.
A chronic study in rabbits exposed to Roundup demonstratedhat male fertility was reduced. Reductions in body weight, libido,jaculate volume, sperm concentration, semen fructose and semen
smolality were observed. The authors suggested that this mayave been due to direct effects on spermatogenesis or indirectffects via the hypothalamus-pituitary-testis axis (Yousef et al.,
24 hours 96 hours0
20
40
60
80
100
Control
5 mg/L
10 mg/L
a a
ba ab
b
DN
A i
nte
gri
ty (
%)
ig. 6. DNA integrity in Danio rerio exposed for 24 h and 96 h to glyphosate5 mg/L and 10 mg/L) and the respective control groups (0.0 mg/L). The valuesre means ± SEM (n = 24–27). The different letters represent significant differencesmong treatments at the same exposure periods (p < 0.05).
logy 155 (2014) 322–326 325
1995). In the present study, the results showed no significant dif-ferences in sperm concentration; however, the sperm motility andthe period of motility were significantly lower in the groups treatedwith glyphosate. Corroborating the present study, the sperm motil-ity of the fish P. vivipara was reduced when exposed to Roundupfor 96 h at 0.1 and 0.7 mg/L of glyphosate, although no signifi-cant decrease in the period of motility was observed (Harayashikiet al., 2013). Sperm motility is one of the most important char-acteristics to be examined to evaluate sperm quality because itis a pre-requisite for fertilization (Rurangwa et al., 2004). Thus, itcould be suggested that the fertilization rate of animals treated withglyphosate would be lower than that of the control group, consid-ering that motility refers to the ability of the sperm to move towardthe oocyte. This idea can still be maintained by the fact that, in ourresults, there was a reduction of mitochondrial functionality at thehigher concentration of glyphosate after both exposure periods.
The mitochondrion is essential for energy generation duringsperm movement. If mitochondrial functionality is reduced, thiswill lead to a reduction in sperm motility. Harayashiki et al.(2013) also observed a reduction in mitochondrial functionality inother fish species when exposed to 0.1 and 0.7 mg/L of Roundup(glyphosate equivalent) after 96 h. In our study, this parameter wasaffected even after an acute exposure of 24 h. In another study withisolated mitochondria from rat liver, glyphosate presented no effecton mitochondrial bioenergetics at concentrations ranging from84.535 mg/L to 845.35 mg/L, although the commercial formulation,Roundup, affected mitochondrial bioenergetics by inducing non-selective membrane permeabilization at the same concentrations(Peixoto, 2005). This difference may be due to other substancespresent in the commercial formulation that promote increased her-bicide penetration into biological membranes (Giesy et al., 2000),or glyphosate could be affecting mitochondrial function indirectly.Despite these differences, it was demonstrated that sperm mito-chondrial functionality is affected by glyphosate exposure. Becausefertilization depends on sperm motility, which needs high energyexpenditure maintained by aerobic metabolism, this process wouldalso be seriously affected.
The sperm membrane integrity was affected after exposure to10 mg/L of glyphosate, in both exposure periods. It is important toemphasize that membrane integrity is essential for the penetra-tion of sperm into oocytes. The integrity was lower after exposureto 10 mg/L when compared with the animals of the control groupand animals exposed to 5 mg/L. These results showed that thereproduction of these fish can be affected by the lack of viablesperm. Decreases in sperm membrane integrity were observedin P. vivipara exposed for 96 h to Roundup at 0.1 and 0.7 mg/L(glyphosate equivalent) (Harayashiki et al., 2013); however, Akchaet al. (2012) found no effect in oyster sperm when cells wereexposed in vitro to glyphosate (200 �g/L) or Roundup (200 �g/Lglyphosate equivalent). We suggest an indirect effect on membraneintegrity may exist, such as via lipid peroxidation, because it hasbeen demonstrated that exposure to these compounds causes lipidperoxidation in other tissues of fish and rats (Lushchak et al., 2009;Modesto and Martinez, 2010; El-Shenawy, 2009). In this sense, anup-regulation of the mRNA of antioxidant enzymes in the gonads ofzebrafish males exposed to glyphosate (10 mg/L) has been demon-strated, suggesting possible oxidative stress in the gonads (Websteret al., 2014).
Several authors have shown that glyphosate, as well as its com-mercial formulation, could alter the levels of antioxidant defensein different organisms, eventually leading to a state of oxidativestress (Langiano and Martinez, 2008; Contardo-Jara et al., 2009;
Ferreira et al., 2010). In general, an excess of reactive oxygen speciescan be harmful to the cell, causing lipid peroxidation, the oxida-tion of amino acids, the inactivation of enzymes, the oxidation ofco-factors, and DNA damage (Brooker, 2011). Redox impairment
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26 F.M. Lopes et al. / Aquatic
nd oxidative stress could be the mechanism responsible for lipidamage as well as DNA damage caused by glyphosate.
DNA integrity decreased significantly at the higher concentra-ion of glyphosate (10 mg/L) in both analyzed exposure periods.uilherme et al. (2012) observed DNA damage in the liver andills of the fish Anguilla anguilla when exposed to 58 �g/L and16 �g/L (18 and 36 �g/L of glyphosate equivalent, respectively).e and Woods (2004) showed that any alteration in the structurer functionality of the membrane and mitochondria, as well as theeduction of sperm motility, were critical factors to the fertiliza-ion process of teleost fish. Additionally, some studies have shownhat sperm with damaged DNA present a lower rate of fertilityr other problems after fertilization, such as issues in embryonicleavage, low hatching rate or abnormal development (Agarwallnd Allamaneni, 2004; Bakos et al., 2007; Benchaib et al., 2003).
. Conclusion
The results showed that glyphosate may induce harmful effectsn the reproductive parameters of D. rerio males, such as damagingperm DNA, reducing the integrity of the mitochondrial membranend functionality, and decreasing sperm motility and the motilityeriod. The alterations at the molecular level, such as reduction
n DNA integrity and damage to membranes and mitochondrialunctionality, are the causes of the cellular functionality impair-
ent observed in terms of motility and the motility period. Takenogether, these alterations would dramatically reduce the fertilityate of these animals, hindering reproductive success.
cknowledgements
Fernanda Moreira Lopes is a post-graduation student financedy CAPES (Coordenac ão de Aperfeic oamento de Pessoal deível Superior - Process Number 42004012008P9). The authorsould like to thank the National Institute of Science and
echnology–Aquatic Toxicology (INCT-TA) from CNPq (Conselhoacional de Desenvolvimento Científico e Tecnológico - Processumber 573949/2008-5).
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