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Experimental and Toxicologic Pathology 61 (2009) 339–346

www.elsevier.de/etp

Deleterious effects of cypermethrin on semen characteristics and testes of

dwarf goats (Capra hircus)

Maqbool Ahmada,�, Ijaz Hussaina, Ahrar Khanb, Najib-ur-Rehmana

aDepartment of Theriogenology, University of Agriculture, Faisalabad 38040, PakistanbDepartment of Pathology, University of Agriculture, Faisalabad 38040, Pakistan

Received 11 August 2008; accepted 6 October 2008

Abstract

This study was carried out on 30 male dwarf goats to determine the effects of cypermethrin (CY) on semencharacteristics and at tissue level in testes. Animals were divided randomly into five equal groups and each group wasdipped with 0%, 0.1%, 0.4%, 0.8% or 1.6% CY, on days 0 and 15. The semen was collected at day 0, then fortnightlytill 75 days and evaluated for physical characteristics, sperm morphology and concentration. None of the parametersstudied showed any statistical difference on days 0 and 15 of sample collection. From day 30, effect of CY on semencharacteristics started to appear and was more pronounced from days 45 to 75. Significantly (Po0.01) decreasedejaculatory volume, motility percentage, mass activity, and concentration of spermatozoa were observed in treatedgroups as compared to control. With the treatment of CY, pH of semen became more alkaline. Semen color changedfrom creamy (control) to milky white to straw color in treated bucks. Percentage of abnormal spermatozoa (tailless,bent tails, coiled tailed) was much higher in treated animals than in control. Dead spermatozoa increased significantly(Po0.01) in the treatment groups from days 30 to 75 as compared to the control group. All these changes were dosedependent, being less with low CY dose and more even extensive with high CY dose. Slight to moderate improvementin the above parameters was observed at day 75 nearly in all treated groups. Grossly decreased weight of testis andcyanotic epididymides were observed in bucks treated with 1.6% CY. Histopathologically, degenerative changes andloss of spermatogonia, spermatocyte, Sertoli cells, spermatids, and spermatozoa in seminiferous tubules were also dosedependent. It was concluded that CY caused dose-dependent effects on all parameters studied. High doses of CY(0.8% and 1.6% solution) affected the parameters on semen characteristics, though this effect seems to be transient asimprovement in these parameters was observed at day 75; however, CY in goats should be used with great care toavoid over-dosage.r 2008 Elsevier GmbH. All rights reserved.

Keywords: Cypermethrin; Dwarf goats; Semen characteristics; Testicular pathology

e front matter r 2008 Elsevier GmbH. All rights reserved.

p.2008.10.002

ing author. Tel.: +9241 9200161 170x3105;

0764.

ess: drmaqbool_ahmad@yahoo.com (M. Ahmad).

Introduction

Through scientific and technical advances in the 21stcentury, there has been a significant extension in lifeexpectancy and generally an enhanced quality of life. Atthe same time with industrial growth about 50–60thousand chemicals are in common use. Many new

ARTICLE IN PRESSM. Ahmad et al. / Experimental and Toxicologic Pathology 61 (2009) 339–346340

chemicals enter every year into this race. The firstaccident of toxicity of chemicals on the reproductivesystem was tragically accentuated by thalidomide in the1960s (Fabro, 1985). Many therapeutic agents have beenshown to be toxic to the male and female reproductivesystem and possibly capable of inflicting genetic damageto the germ cells (Barlow and SulliVan, 1982).

Various chemical compounds are used as ideal insecti-cides in agriculture, veterinary, and public health pro-grams. Pyrethroid insecticides have been used inagricultural and home formulations for more than 30years and account for approximately one-fourth of theworldwide insecticide market (Casida and Quistad, 1998)and their prevalence in insecticide formulations hasincreased particularly in the last decade (Wolansky et al.,2006). Cypermethrin (CY), a synthetic pyrethroid, is oneof the compounds extensively used for such purposes as ithas a wide margin of safety in mammals (Khan et al.,2008). In spite of a wide margin of safety, CY is not freefrom side-effects; hence toxicity of CY in farm animals canoccur through spray/dipping or ingestion or through theingestion of sprayed crops/fodders. The severity of theeffects of pyrethroids is influenced by route of exposure,vehicle, dosing volume (Soderlund et al., 2002; Shah et al.,2007), and route of exposure (Bradbury and Coats, 1989).Introduction of the compound into the brain is most toxic,followed by introduction into blood vessels, gut (intraper-itonium), oral, inhalation, and dermal (skin) exposure(Bradbury and Coats, 1989).

Cypermethrin not only produces toxicity in animalsbut also suppresses their cell-mediated as well ashumoral immunity (Tamang et al., 1988). Side-effectsof CY on male reproductive functions are so many(Song et al., 2008). Mutagenic risk, mutagenic activityinducing dominant lethal mutations in male germ cells,embryonic resorption, and fetal mortality and chromo-somal aberrations are the other side-effects reported(Shukla and Taneja, 2002; Giri et al., 2003).

CY is being used extensively in Pakistan for the control/eradication of ectoparasites, which may have health hazardto livestock and human beings as well. None of the pre-parations available in the market is locally manufactured,all are imported. Moreover, none of the products has beenlooked into what type of toxicity or lesions occur in animalspecies of the region. This study was, therefore, designedand executed to investigate whether CY (Eco-fleece,Bimeda, Ireland) is safe for use in male goats or what typeof toxic effect(s) it has on semen characteristics and testes ofdwarf male goats (Capra hircus).

Materials and methods

This experiment was designed and conducted takinginto consideration all the national legislation vis-a-vis

protection of animal welfare and following the guide-lines of the Advanced Studies and Research Board ofthe University.

Chemicals

Sodium chloride, formaldehyde (10% phosphatebuffered), paraffin, and xylene were purchased fromAppliChem (Germany). Cypermethrin (Ecofleeces,Bimeda, Ireland) was purchased from the local market.Injection Pentothel sodium (Pentobarbital sodium) waspurchased from Abbott Laboratories Ltd., Pakistan.Ethyl alcohol (rectified spirit) is being manufactured byShakarganj Sugar Mills Limited, Jhang, Pakistan, withabove 99.7% purity being procured and utilized. Eosinwas procured from Carl Roth (Germany). Harrishematoxylin was prepared by using hematoxylin (CarlRoth, Germany) 1 g, ethyl alcohol 10mL, ammoniumpotassium sulfate (Merck, Germany) 0.5 g, and distilledwater 200mL.

Experimental animals

Thirty healthy male dwarf goats of 112� 2 years

having similar weight were procured from a governmentfarm. All animals were kept under similar managementand feeding conditions throughout the experiment.Briefly, bucks of each group were housed together in ashed. Sheds were covered from three sides and one sidewas open; when felt necessary that was covered withcurtains to protect animals from cold. Animals were puton pasture (green fodder Trifolium alexandrinum;Barseem) early in the morning and returned in theevening. No concentrate or fodder was providedat night. Water was available ad libitum in theanimal shed. The animals are being vaccinated againstpleuropneumonia and enterotoxaemia and dewormedbiannually.

This experiment was conducted under completelyrandomized block design. Animals were randomlydivided into five equal groups (six animals in eachgroup) after 5 days of acclimatization. Cypermethrin0.1% solution is recommended for dipping. Therefore,the recommended level and the above levels wereselected for the treatment trials to assess CY side-effectsin male goats. All the animals in five groups were dippedon days 0 and 15 early in the morning with 0%, 0.1%,0.4%, 0.8% or 1.6% solution of CY. The group with0% CY solution served as control. A fresh aqueoussolution of CY was prepared in a tub for each animal ineach group keeping its mouth out and the animal wasdipped in prepared concentration until its body becamewet (1–2min).

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Semen collection and evaluation

Animals were properly cleaned before collectionof semen. The semen was collected with the helpof an artificial vagina at day 0 and then fortnightly.The duration of this study was 75 days. A femalegoat was used as a teaser. On each semen collec-tion day, one ejaculate was taken from each buck.Immediately after collection, each ejaculate was placedin a water bath at 37 1C. Then semen samples wereevaluated for physical characteristics viz. ejaculatoryvolume, color, pH, mass activity, motility percentage,live and dead spermatozoa, and sperm morphology(Salisbury and Van-Demark, 1978). The concen-tration of spermatozoa was estimated by the hemocyt-ometer method (Bane, 1952). Briefly, 20 mL ofsemen was diluted with 4mL of 3% sodium chloridesolution. A drop of diluted semen was placed in theNeubauer counting chamber and allowed to settle for4–5min. Sperm cells present in the four large cornersand fifth central square of counting chamber werecounted.

Table 1. The ejaculatory volume, pH, and mass activity of sem

concentrations of cypermethrin at different days of the experiment.

Days Dose (% solution) of cypermethrin

0 0.1

Ejaculatory volume (mL)

0 1.7070.65 1.9070.58

15 1.7070.63 1.8070.62

30 1.6870.53 1.8070.57

45 1.6870.48 1.5070.52

60 1.6770.56 1.4070.49

75 1.7070.47 1.7070.53

pH of semen

0 6.5570.10 6.5370.12

15 6.5870.12 6.6070.13

30 6.5870.12 6.5870.19

45 6.5870.11 6.9270.12*

60 6.5570.14 6.9770.12*

75 6.5570.14 6.7570.20*

Mass activity

0 3.6770.52 4.0070.00

15 3.8370.40 3.5070.55

30 3.8370.41 3.5070.55

45 3.6770.52 3.0070.00*

60 3.8370.41 2.5070.55*

75 3.8370.41 3.0070.00*

Each group consisted of six animals and was dipped on days 0 and 15 with

(Po0.05) difference compared to non-CY-treated control (0% CY) at a sam

Histopathology

At the end of the experiment, each animal waseuthanised by injecting an intravenous overdose ofpentobarbital sodium (90mg/kg b.wt). Testes of eachbuck were examined thoroughly for gross lesions within12h after euthanasia. Gross lesions were recorded and

testicular tissue from the middle of testis was fixed in10% buffered formalin immediately. The same processwas repeated for each animal. Fixed tissues wereprocessed by the routine method of dehydration andparaffin embedding. Sections of 4–5 mm thickness werecut and stained with hematoxylin and eosin (Bancroftand Gamble, 2008). Slides were analyzed for histo-pathological lesions by two persons; where differencesarose, a third person was consulted for opinion.Histopathological lesions were graded by allottingnumerics to the lesions. All the slides in each groupwere thrashed, average of the numerics was obtained,and an accumulative picture of each lesion in each groupwas drawn. Frequency of lesions in each group wasrecorded and incidence (%) of lesions was calculated.

en (Mean7S.D.) collected from bucks dipped with different

0.4 0.8 1.6

1.5070.41 1.7070.39 1.5070.47

1.4070.47 1.6070.40 1.4070.42

1.3770.43 1.5570.42 1.3070.35

1.2070.43 1.2570.38* 0.8070.25*

1.1570.45* 1.2070.32* 0.7070.18*

1.3070.41 1.4070.36 1.1070.17*

6.5570.10 6.5870.12 6.5770.12

6.5870.12 6.6070.13 6.5870.11

6.7370.18 6.7570.19 6.8570.10

7.0070.12* 7.1570.19* 7.3270.09*

7.1070.24* 7.3370.08* 7.3870.07*

6.8770.25* 7.1070.24* 7.3070.09*

4.0070.00 4.0070.00 3.9770.00

3.5070.55 3.5070.55 3.5070.55

3.5070.55 3.0070.00* 3.0070.00*

3.0070.00* 2.5070.53* 2.0070.00*

2.5070.55* 2.0070.00* 1.5070.50*

3.0070.00* 2.5070.50* 2.0070.00*

various concentrations of cypermethrin. Asterisk indicates significant

pling day.

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Data analysis

Data collected were subjected to the two-way analysisof variance (ANOVA) and means were compared byDuncan’s multiple range (DMR) test on a personalcomputer using the Minitab statistical software package.The significance level was Po0.05.

Fig. 1. Motility percentage of spermatozoa in semen collected

on various days of the experiment from treated and control

male dwarf goats. Each group consisted of six animals and was

dipped on days 0 and 15 with various concentrations of

cypermethrin. Asterisk indicates significant (Po0.05) differ-

ence compared to non-CY-treated control (0% CY) at a

sampling day.

Fig. 2. Concentration of spermatozoa in semen collected on

various days of the experiment from treated and control male

dwarf goats. Each group consisted of six animals and was

dipped on days 0 and 15 with various concentrations of

cypermethrin. Asterisk indicates significant (Po0.05) differ-

ence compared to non-CY-treated control (0% CY) at a

sampling day.

Results

None of the parameters studied showed any statisticaldifference on days 0 and 15 of sample collection.Significantly (Po0.01) decreased ejaculatory volumewas recorded at days 45–75 in animals treated with1.6% CY than in the control group. Animals treatedwith 0.4% CY showed decreased semen volume at day60 and at 0.8% CY concentration at days 45 and 60than in the control group. Dose-dependent decrease inthe volume of semen was recorded being less in highCY dose-treated animals than in the control group(Table 1).

Color of semen was creamy white in the controlgroup. Creamy white to milky semen color was observedin CY-treated bucks at day 30. The main effect of CYtreatment on semen color was observed at day 60(change in color from creamy to straw). The effect ofCY increased as the concentration increased from 0.1%to 1.6%.

The pH varied significantly (Po0.05) between varioustreatment groups on days 45, 60, and 75 as compared tothe control group. With the treatment of CY, pH ofsemen became more alkaline as compared to controlsemen samples. Mass activity started decreasing at day30 in high (0.8% and 1.6%) CY concentration and itwas significantly decreased in all treated animals at days45–75 as compared to the control group (Table 1).

At day 30, sperm motility decreased significantly inanimals treated with 0.4–1.6% CY as compared to thecontrol group. Similarly, at days 45–75 motilitypercentages showed significant difference in all treat-ment groups as compared to the control group. On day75, improvement in the motility percentage in treatmentgroups was observed, but significant variation from thecontrol group remained as such (Fig. 1). Concentrationof spermatozoa significantly (Po0.05) decreased at day30 in animals treated with 0.8% and 1.6% CY while atdays 45–75, spermatozoa concentration decreased sig-nificantly in all treatment groups as compared to thecontrol group except at day 75 with 0.1% CYconcentration (Fig. 2).

There was a slight effect of CY on the morphology ofspermatozoa in buck semen at day 15. At day 30,maximum numbers of tailless (25%) and bent tails (2%)spermatozoa were recorded in the high-dose group

(1.6% CY). At days 45 and 60, the highest tailless (25%and 35%), bent tail (8% and 12%), and coiled tailed(0% and 2%) spermatozoa were recorded in the high-dose group (1.6% CY), respectively. At day 75, thehighest number of tailless spermatozoa (12%) wasrecorded in the same group. Dead spermatozoa in-creased significantly in the treatment groups at days30–75 than in the control group. The increase in thedead spermatozoa was also dose dependent (Fig. 3). Atday 75, an improvement in the ejaculatory volume,semen color mass activity, and motility percentage wasobserved and dead spermatozoa also reduced in alltreatment groups.

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Fig. 3. Percentage of dead spermatozoa in semen collected on

various days of the experiment from treated and control male

dwarf goats. Each group consisted of six animals and was

dipped on days 0 and 15 with various concentrations of

cypermethrin. Asterisk indicates significant (Po0.05) differ-

ence compared to non-CY-treated control (0% CY) at a

sampling day.

Fig. 4. Photomicrograph of testis of dwarf goats showing

active spermatogenesis except for few seminiferous tubules

(arrow) that have lost spermatids, and proliferation of

connective tissue in between seminiferous tubules as a result

of cypermethrin treatment (0.1% solution dipping). H & E;

200� .

Fig. 5. Photomicrograph of testis of dwarf goats showing loss

of spermatids in most of the seminiferous tubules except in two

(arrows) and proliferation of connective tissue in between

seminiferous tubules as a result of cypermethrin treatment

(0.8% solution dipping). H & E; 100� .

Fig. 6. Photomicrograph of testis of dwarf goats showing loss

of spermatids in most of the seminiferous tubules (arrows),

marked reduction in germ cells (*), and proliferation of

connective tissue in between seminiferous tubules as a result of

cypermethrin treatment (1.6% solution dipping). H & E;

200� .

M. Ahmad et al. / Experimental and Toxicologic Pathology 61 (2009) 339–346 343

Grossly, bucks treated with 1.6% CY showedcyanotic epididymides. Histopathologically, spermato-genesis decreased with increase in the concentration ofCY. Testes of bucks treated with 0.1% CY showedactive spermatogenesis except in few seminiferoustubules where spermatids were lost (Fig. 4). However,loss of spermatids was extensive at 0.8 (Fig. 5) and 1.6%CY treatment (Fig. 6). Most of the seminiferous tubulesdid not show late spermatids and spermatozoa but thelarge number of immature spermatids along withdegenerative changes was evident in the testes of buckstreated with 1.6% CY. Two layers of germ cells wereseen in most of the seminiferous tubules while all othercells sloughed off or become degenerated in the buckstreated with 1.6% CY (Fig. 6). The testes of buckstreated with high doses showed a marked swelling of

tubular seminiferous cells. Proliferation of connectivetissue around the seminiferous tubules in between theinterstitial Lydig cells was also observed in all treatedgroups. Degenerative changes and loss of spermatogo-nia, spermatocytes, Sertoli cells, spermatids, and sper-matozoa in seminiferous tubules were dose dependent;with the increase in CY concentration, these parametersalso increased (Table 2).

Discussion

Semen quality, sperm concentration, and spermmotility are considered most important parameters inevaluating the fertilizing ability of sperm. Capability of

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Table 2. Frequency and incidence (%) of testis histopathological lesions observed in goats treated with different doses of

cypermethrin.

Lesions Dose (% solution) of cypermethrin

0.1 0.4 0.8 1.6

Fa Ib F I (%) F I (%) F I (%)

Fibrous tissue proliferation between seminiferous tubules + 50 + 50 + 100 + 100

Loss of

Spermatogonia � 0 � 0 + 16.6 + 16.6

Spermatocyte � 0 � 0 ++ 16.6 ++ 16.6

Sertoli cells � 0 � 0 ++ 16.6 ++ 33.3

Spermatids + 16.6 + 16.6 ++ 33.3 +++ 50.0

Spermatozoa � 0 � 0 ++ 16.6 ++ 33.3

Degenerative changes (pyknotic nuclei, condensation, etc.) + 16.6 ++ 16.6 ++ 33.3 +++ 66.6

Each group consisted of six animals and was dipped on days 0 and 15 with various concentrations of cypermethrin. Lesions were graded on the basis

of numerics allotted and then reported as no (�), mild (+), moderate (++), and severe (+++) lesions. No lesions were recorded in the control

group, therefore, not included in the table.aF ¼ Frequency.bI ¼ Incidence (%).

M. Ahmad et al. / Experimental and Toxicologic Pathology 61 (2009) 339–346344

sperm to fruitfully fertilize oocytes can be badly affectedby abnormal sperm motility and shape. Pyrethroidpesticides act as a neuropoison, which interfere ionicconductance by prolonging the sodium current (Clark,1997). It was well established that pyrethroid pesticidesare subjected to hydrolysis by plasma and tissueesterases (Xu et al., 2004). Cypermethrin absorbs fromthe skin through capillaries when applied by dipping. Itslipophilic nature enables it to come in systemic circula-tion and cross the blood–brain barrier. CY inducesalterations in the plasma membrane by producingincreased lipid peroxidation, which is the consequenceof increased free radicals, particularly superoxidedismutase released following pyrethroid treatment (Kaleet al., 1999). Reduction of flaccidity in the hydrophobicregion of the plasma membrane bilayer takes placewhere this pyrethroid is preferentially localized. Thedecrease of membrane flaccidity could induce changes ofactivities and conformation of proteins (Nasuti et al.,2003).

Changes in phospholipids, fatty acids, and cholesterolcontents modulate membrane flaccidity, which influ-ences the enzymatic activity and functionality ofreceptors and channels present in the plasma membranelevel. The commonly accepted mechanism of action ofpyrethroids is the prolongation of open-state voltage-sensitive sodium channels (VSSC) in nervous tissue(Soderlund et al., 2002). These altered sodium channelsresults in repetitive firing depolarizing block of theneuron, depending upon how long the channel openstate is prolonged (Narahashi, 2000). In addition, these

slow the rate of VSSC inactivation or closing; hence,sodium channels are held open longer, allowing moresodium ions to cross and depolarize the membrane(Wolansky et al., 2006). Cypermethrin was appliedthrough dipping in the present study. Due to itslipophilic nature, it also reaches the testes and acts onthe membrane of seminiferous tubules.

Another possibility of CY inducing male reproductivetoxicity could be due to a neuro-endocrine-mediatedphenomenon and a hormone-disrupting property(Yousef et al., 2003; Xu et al., 2004). Exposure to CYalso produces steroid alterations in testes and sera ofrats, resulting in the impairment of spermatogenesis andthe reduction of sperm output and sperm motility (Maniet al., 2002; Song et al., 2008).

The CY treatment causes reduction in ejaculatoryvolume at high doses as has also been reported (Salemet al., 1988; Yousef et al., 2003). The effect was dosedependent; with the increase in CY concentration,the effects became more severe in the present study.The severity of the effects of pyrethroids is influencedby route of exposure, vehicle, and dosing volume(Soderlund et al., 2002). The rather late appearanceof deleterious effects of CY observed in the presentstudy in male dwarf goat’s semen is also due to thefact that it takes about 40–50 days for spermato-genesis and about 1–2 weeks for ejaculation ofspermatozoa (Hafez, 1987). The highest deleteriouseffects of CY were recorded on the 60th day, as thesecond treatment enhanced the effect of the firsttreatment.

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In this study, the control group throughout theexperiment and the treatment groups from days 0 and15 had creamy white semen. The change in color ofsemen started from day 30 (creamy white to milky) andthe effect was more pronounced on days 45 and 60(straw color). This change in semen color could berelated to sperm concentration as it started to decreasefrom day 30 and continued to do so up to day 60 in thepresent study (Fig. 2).

Mass activity, motility percentage (Table 1), andsperm concentration (Fig. 2) were inversely related toCY dose in different groups. As in the publishedliterature, no report regarding the effect of CY on massactivity was available to support or negate the presentresults; this seems to be the first report of CY effect onmass activity. According to Kamijima et al. (2004),percentages of slow progressive and non-progressivemotile sperms in pyrethroid sprayers were twice as highand there was a decrease in progressively motile sperms.

In the present study, spermatozoa abnormalities anddead spermatozoa increased significantly in the treat-ment groups. Kumar et al. (2004) found that CYinduced adverse effects on sperm head shape in mice.Xia et al. (2004) also found that fenvalerate inducedmorphologic abnormality, genotoxic defects of sperma-tozoa, and reduction of some sperm motility parametersamong insecticide-exposed workers. DNA damageresulted in cell death or induction of mutations.

Dose-dependent histopathological changes were ob-served in male dwarf goats treated with CY in thepresent study. There were degenerative changes and lossof spermatogonia, spermatocytes, Sertoli cells, sperma-tids, and spermatozoa in seminiferous tubules at a highdose level (1.6% CY). Yousef et al. (1999) noted markedswelling of tubular seminiferous cells. The findings ofElbetieha et al. (2001) were also similar to the result ofthe present study, as they reported that there was asignificant accumulation of connective tissue surround-ing the seminiferous tubules, which contained a largenumber of immature spermatids. Cypermethrin hasbeen reported to cause significant reduction in thenumber of cell layers of the seminiferous tubules as wellas epididymal and testicular sperm counts, and dailysperm production (Elbetieha et al., 2001) and spermmotility (Mani et al., 2002).

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