RESEARCH Open Access

Protective effects of Camellia sinensis onSyzygium aromaticum- or chlorpyrifos-induced reproductive toxicity in maleWistar ratsDamola V. Akinwande1, Joseph A. Adeyemi1* , Solomon T. Olawuyi2, Busuyi K. Akinola2 and Chris O. Adedire1

Abstract

Background: The potential toxicity of clove, Syzygium aromaticum, notwithstanding its beneficial health effect tohuman health remains a critical issue.

Purpose: This study was designed to assess the effects of oil extracts of clove (S. aromaticum) on reproductiveparameters in Wistar rats. The ameliorative effect due to co-administration with green tea, Camellia sinensis, was alsodetermined.

Methods: Adult rats were exposed via oral gavage to mineral oil (negative control), 5% green tea (GT), 12.5 mg/kg/daychlorpyrifos (CHL, positive control), 360 mg/kg/day clove oil (CO), green tea + chlorpyrifos (GT+CHL), or green tea +clove oil (GT+CO) for 3 weeks, after which the animals were sacrificed and the following sperm parameters: total spermcount, sperm motile count, sperm progressive assessment, and sperm morphology were determined. The serum levelsof reproductive hormones, testosterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH), weredetermined. The histological sections of the testes were also performed.

Results: The results revealed that S. aromaticum treatment resulted in significant damage to the sperm morphologyespecially at the neck and tail regions with only a marginal change to the total sperm count, sperm motile count, andsperm progressive assessment. The levels of testosterone, FSH, and LH decreased significantly in rats treated withS. aromaticum. Histopathological analyses revealed significant disruption of normal testes structure in rats thatwere treated with either clove oil or chlorpyrifos.

Conclusion: Overall, the results of this study show that the co-administration with C. sinensis has the potential toameliorate the clove-induced reproductive toxicity in rats.

Keywords: Plant-based pesticide, Chlorpyrifos, Syzygium aromaticum, Sperm parameters, Sex hormones, Histopathology

BackgroundIn recent times, there is strong advocacy for the use ofeco-friendly plant-based pesticides as alternatives to thenon-eco-friendly synthetic pesticides (Dubey, Srivastava,& Kumar, 2008; Kedia, Prakash, Mishra, Singh, & Dubey,2015). The literature is rich on the toxic effects of syn-thetic pesticides on man and other living organisms;these toxic effects include developmental impairments,

DNA fragmentation, compromised immunity, organtoxicity, endocrine disruption, neuro-degeneration, oxida-tive stress, and reproductive abnormality (BenAbdallah,Slima, Dammak, Keskes-Ammar, & Mallek, 2009; Joshi,Mathur, gajraj, & Sharma, 2003; Sakr, & Al-Amoudi, 2012).Plant-based pesticides are considered as better alternativesto conventional synthetic pesticides for obvious reasons;they tend to have a broad-spectrum activity, they arehumanly safe and biodegradable and are easy to processand use (Dinesh, Kimara, Kumar, & Das, 2014).Several plant materials have been reported to possess

insecticidal properties. Examples are Azadirachta indica,

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made.

* Correspondence: joseph.adeyemi@gmail.com1Department of Biology, School of Sciences, Federal University ofTechnology, P.M.B. 704, Akure, Ondo State, NigeriaFull list of author information is available at the end of the article

The Journal of Basicand Applied Zoology

Akinwande et al. The Journal of Basic and Applied Zoology (2019) 80:48 https://doi.org/10.1186/s41936-019-0122-2

Nicotiana sp, Derris sp, Schoenocaulon officinale, Syzy-gium aromaticum Clerodendrum infortunatum, Dennet-tia tripetala, Anchomanes difformis, etc. (Adedire &Akinkurolere, 2005; Dimetry, 2012; Dinesh et al., 2015;Mikami & Ventura 2008). In spite of the wide advocacyfor their use as pesticides, little is currently known aboutthe potentially toxic effects that may arise from theiruse. The treatment of albino rats with an aqueous solu-tion of nicotine, a compound derived from Nicotianatabacum, caused oxidative stress and damage to the liver(BenSaad et al., 2018; Chowanski et al., 2016). Recently,we showed that oil extracts of clove, S. aromaticum alsocaused oxidative stress and liver damage in male Wistarrats (Adeyemi et al., 2018). These and other studies areevidence of potential toxicity of plant-based pesticidesto mammals.The health benefits of drinking green tea are quite

enormous; they include reduced risk of cardiovasculardiseases, degenerative diseases, and cancer. Also, green teahas been shown to result in improvement of asthenia,diarrhea, bronchitis, asthma, hyperlipidemia, cellulitis, andabscesses as well as weight reduction (Kao, Chang, Lee, &Chen, 2006; Shula, 2007; Schönthal, 2011; Wolfram, 2007). More importantly, green tea has been shown to improvethe reproductive health in humans (Ly, Yockell-Lelievre,Ferraro, Arnason, & Gruslin, 2015; Roshdy et al., 2013).The therapeutic ability of green tea has been closely linkedto the presence of polyphenols such as epigallocatechingallate (EGCG), epigallocatechin (EGC), epicatechingallate (ECG), epicatechin (EC), catechin gallate, and andgallocatechin gallate (Chen et al., 2003; Harold & Graham1992; Henning et al., 2003).Reproductive impairments such as low sperm counts,

damaged sperm and testes, and alteration in hormonallevels are often associated with exposure to syntheticchemical pesticides (Hu et al., 2013; Li, Pan, Hu, Li, &Xu, 2013), so the likelihood of plant-based pesticides toas well cause reproductive defects is worth investigating.This study is therefore designed to investigate the re-productive toxicity of oil extract of clove, S. aromaticum,in male Wistar rats. The ameliorative effect of green tea,Camellia sinensis was equally investigated.

Materials and methodsExtraction of essential oils from S. aromaticumThe essential oil was extracted from clove flower budsfollowing the procedures of Ileke and Ogungbite (2015).Dried flower buds of S. aromaticum were obtained froma local market within Akure metropolis and pulverizedinto a fine powder using a blender. Acetone extracts ofS. aromaticum were obtained using a cold extractionmethod. This was done by soaking 100 g of the powderin an extraction bottle containing 300 ml of acetone.

The mixture was stirred occasionally with a glass rod,and extraction was terminated after 72 h. The extractwas filtered through Whatman filter paper (pore size;0.7 μm). The extraction solvent was evaporated usinga rotary evaporator set at 56 °C. The resulting extractwas air-dried in order to remove traces of solvent.

Experimental animalsAdult male Wistar rats weighing approximately 200 gwere obtained from a commercial farm within Akuremetropolis and were placed individually in poly-propylene cages, with laboratory grade pine shavingsas bedding. Rats were allowed to acclimatize toexperimental room conditions for 2 weeks prior tocommencement of experiments. Rats were fed withrat chow and tap water ad libitum, throughout theperiod of the experiment.

Experimental designThe animals were randomly allocated into six groups(n = 5 per group) and were exposed through oral ga-vages to one of the following treatments: mineral oil(OIL, vehicle for extracted clove oil, thus serving asnegative control), 5% green tea (GT), 12.5 mg/kg/daychlorpyrifos (CHL, positive control), 360 mg/kg/dayextracted clove oil (CO), green tea + chlorpyrifos(GT+CHL), and green tea + extracted clove oil(GT+CO). Experimental treatment was done everyday and lasted for 3 weeks. At the end of the thirdweek of treatment, animals were sacrificed using cer-vical dislocation.

Total sperm count and motility assessmentThe total sperm count and motility assessment weredone following the procedure described by Yokoi,Uthus, and Nielsen (2003) with minor modification.The caudal epididymis was removed and minced withsurgical blade in 1 ml of normal saline, placed in arocker for 5–10 min, and incubated at 37 °C for about2min. The solution was diluted 100-folds with sodiumbicarbonate/formalin solution. The total sperm count wasdone by placing 10 μl of the aliquot into the Neuber’scounting chamber. The sample was allowed to standfor 5 min and then observed under the microscope at ×400 magnification. Sperm motility progressive assess-ment was assessed by placing a drop of the spermaliquot on the slide, covered with coverslip and thenobserved under the microscope at × 400 objectives. Asperm was considered motile when it exhibited distinctflagella activity. Sperm motility was determined byassessing at least five microscopic fields to classify 100spermatozoa. Motile sperms were further categorizedinto either fast or slow based on recommendations ofthe WHO (1999).

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Sperm morphologyThe morphology of the spermatozoa was determinedon the same sperm dilution that was used for motilityassessment using the procedures described by Rezvanfaret al. (2008). In detail, a drop of sperm suspension wasadded into an equal volume 1% eosin-y 5% nigrosin,which was then mixed together and smeared on pre-warmed clean glass slides and air-dried. A total of200 sperm cells were examined at × 400. The mor-phology of the sperm cells was categorized based onthe presence of one or more abnormal features intotail defects (short, irregular, coiled or multiple tail),neck and middle piece defects (distended, irregular,bent middle piece, abnormally thin middle piece), andhead defects (round head, small or large size, doubleor detached head).

Histology of the testisThe excised testes from the rats were processed forhematoxylin and eosin staining as described by Bancroftand Stevens (1996). The testes were clearly dissectedout, sliced, and fixed in neutral buffered 10% formalinsolution for 48 h after which they were transferred into70% ethanol for storage until further histological pro-cedures were performed. Fixed tissues were dehydratedin ascending series of ethanol, cleared in methyl benzo-ate, and embedded in paraffin wax. About 7-μm-thicksections of the samples were cut with microtome andstained with hematoxylin and eosin dyes. Sections werelater observed under light microscope and photomicro-graphs were taken.

Determination of serum hormonal levelsBlood samples were collected into plain bottles throughcardiac puncture and were allowed to clot to get thesera. The sera were then pipetted into plain samplebottles and stored at − 20 °C until assayed for hormonallevels. The levels of testosterone, luteinizing hormone(LH), and follicle-stimulating hormone (FSH) weredetermined using enzyme-linked immunosorbent assay(ELISA) kits obtained from Cayman Chemical, USA.The assays were performed following the instructions ofthe manufacturer.

Statistical analysisAll statistical analyses were performed using StatisticalPackage for Social Sciences (SPSS version 15.0) soft-ware. Analysis of variance (ANOVA) and post hoctests were used to analyze the data. Tukey’s multiplecomparisons was used to test for statistically signifi-cant difference between control and experimentalgroups. Results are presented as the mean ± standarderror of mean (SEM). Results were considered signifi-cant at p < 0.05.

ResultsTotal sperm countThe total sperm counts are presented in Fig. 1. The totalsperm counts differed significantly among the groups(F5, 24 = 10.99; p < 0.0001). The total sperm count waslowest in the positive control group (CHL) in which thesperm count was 45 ± 4.47 × 106 while the group thatwas treated with green tea (GT) had the highest spermcount of 99.80 ± 6.60 × 106. The animals that weretreated with the extract of clove oil had a sperm countof 61.25 ± 10.08 × 106. The co-administration withgreen tea resulted in increased total sperm count inthe chlorpyrifos (CHL) and the clove oil-treated animals.

Motility assessmentThe motile count differed significantly among thegroups (F5, 24 = 9.348; p = 0.0001). The motile countwas lowest in the positive control group (CHL) in whichthe motile count was 23 ± 1.23 × 106 while the groupthat was treated green tea (GT) exhibited the highestmotile count of 75.2 ± 6.25 × 106. The animals thatwere treated with the extract of clove oil showed amotile count of 41.25 ± 10.08 × 106. The co-administrationwith green tea resulted in increased sperm motilityin the chlorpyrifos (CHL) and the clove oil-treatedanimals (Fig. 2).

Sperm progressive assessmentThe sperm progressive assessment (slow and fast) ispresented in Fig. 3. There was a significant differencein the percentage fast (F5, 24 = 6.119; p = 0.0014) andslow (F5, 24 = 5.556; p = 0.0019) sperms among the treat-ment groups. The percentage of slow sperm was signifi-cantly higher in both the positive control (CHL) and the

OIL GTCHL

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Fig. 1 Total sperm count (× 106) in Wistar rats treated with mineraloil (OIL), green tea (GT), chlorpyrifos (CHL), extract of clove oil (CO),green tea and chlorpyrifos (GT+CHL), and green tea and extract ofclove oil (GT+CO)

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clove oil-treated animals. The percentage of slow-movingsperms for the positive control (CHL) and the cloveoil-treated animals was 68.60 ± 11.67 and 53.00 ±10.16, respectively. The values for the mineral oil- andgreen tea-treated groups were 44.20 ± 3.338 and 27.00 ±2.49, respectively. The co-administration with green tearesulted in an increased number of fast-moving spermsin the chlorpyrifos (CHL) and the clove oil-treated

animals especially those that were treated with clove oiland green tea.

Sperm morphologyThe data on sperm morphology were summarized in Fig. 4.The percentage of sperm with normal morphology was sig-nificantly higher (p < 0.05) in the green tea- and mineraloil-treated animals in comparison with those treatedwith clove oil or chlorpyrifos or those that received co-administration (F5, 24 = 6.757; p = 0.0005). The percentageof sperm with normal physiology in mineral oil- and greentea-treated groups was 56.20 ± 5.652 and 73.40 ± 2.676,respectively, while the percentage in those that weretreated with clove oil and chlorpyrifos were 46.20 ± 9.378and 27.20 ± 11.04, respectively.The co-administration with green tea resulted in

increased percentages of sperms with normal morphologyin the chlorpyrifos (CHL) and the clove oil-treated ani-mals. The percentage of sperm with tail deformity mor-phology was significantly (p < 0.05) lower in the greentea- and mineral oil-treated animals in comparison withthose treated with clove oil or chlorpyrifos or those thatreceived the co-administration treatment (F5, 24 = 10.95;p < 0.0001). The percentage of sperms with tail defor-mity was 9.60 ± 1.939, 17.67 ± 1.453, 22.25 ± 3.425,32.25 ± 4.029, 26.25 ± 2.394, and 14.40 ± 1.122 for GT,OIL, CO, CHL, GT+CHL, and GT+CO, respectively.The percentage of sperm with neck deformity was sig-

nificantly lower in the green tea-and mineral oil-treatedanimals in comparison with those treated with clove oil orchlorpyrifos or those that received the co-administrationtreatment (F5, 24 = 6.067; p = 0.0010). The percentageof sperm with head deformity was significantly lower(p < 0.05) in the green tea- and mineral oil-treatedanimals in comparison with those treated with clove oil orchlorpyrifos or those that received the co-administrationtreatment (F5, 24 = 4.336; p = 0.0063).

Serum hormonal levelsThe serum testosterone levels decreased significantly (p< 0.05) in rats that were treated with chlorpyrifos andclove oil in comparison to those treated with green tea,mineral oil, or the co-administration treatment (F5, 24 =6.442; p = 0.0010). The levels of testosterone for thepositive control (CHL) and the clove oil-treated animalswere 0.0638 ± 0.0119 and 0.1194 ± 0.0222 ng/ml re-spectively. The values for the mineral oil- and green tea-treated groups were 0.1586 ± 0.0155 and 0.2005 ±0.0178 ng/ml respectively. The co-administration withgreen tea increased the levels of testosterone in thechlorpyrifos (CHL) and the clove oil-treated animals.The levels of follicle stimulating hormone (FSH) differedsignificantly among the groups (F5, 24 = 153; p < 0.0010).The levels of follicle stimulating hormone (FSH) were

OIL GTCHL

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GT+CO

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01 X( s t

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Fig. 2 Sperm motile counts (× 106) in Wistar rats treated with mineraloil (OIL), green tea (GT), chlorpyrifos (CHL), extract of clove oil (CO),green tea and chlorpyrifos (GT+CHL), and green tea and extract ofclove oil (GT+CO)

OIL GTCHL CO

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tsaf%

Fig. 3 Sperm progressive assessment (%) in Wistar rats treated withmineral oil (OIL), green tea (GT), chlorpyrifos (CHL), extract of cloveoil (CO), green tea and chlorpyrifos (GT+CHL), and green tea andextract of clove oil (GT+CO)

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9.824 ± 0.560, 2.748 ± 0.328, 1.678 ± 0.1295, 1.018 ±0.1245, 6.284 ± 0.1636, and 1.238 ± 0.0963 μg/ml for GT,OIL, CO, CHL, GT+CHL, and GT+CO, respectively. Thelevels of luteinizing hormone (LH) differed significantlyamong the groups (F5, 24 = 98.31; p < 0.0010). The levelsof luteinizing hormone (LH) were 7.586 ± 0.5614, 1.544 ±0.08548, 1.404 ± 0.1463, 0.7560 ± 0.1282, 4.656 ± 0.2902,and 1.062 ± 0.0887 μg/ml for GT, OIL, CO, CHL,GT+CHL, and GT+CO, respectively (Fig. 5).

Histology of testesThe changes in the histological sections of the testeswere summarized in Table 1 and shown in Plate 1. Over-all, there was no obvious damage to the seminiferous tu-bules of the testes in rat groups that were treated withmineral oil, green tea, green tea + chlorpyrifos, andgreen tea + clove oil. However, the structural architec-ture of the testes was disrupted in rats that were treatedwith chlorpyrifos and extracts of clove oil. The observedhistological changes included loss of both the cellularand tubular constituents of the seminiferous tubules,derangement of the seminiferous tubules, infiltration offats, and outright loss of the seminiferous tubules.

DiscussionThe present study investigated the potential spermicidaleffects of oil extract of clove and chlorpyrifos on maleWistar rats and the ameliorating effect of the green tea.Results did show that both the clove oil and chlorpyrifos

had adverse effects on certain sperm parameters likemotility, total sperm count, and sperm progressive assess-ments. The decreased sperm count in rats that weretreated with oil extract of S. aromaticum could be anindication that this plant material has the potential to alterthe processes of spermatogenesis by evoking eitherhormonal imbalance or damage to important testicularcells such as Sertoli cells (Bretveld, Brouwers, Ebisch, &Roeleveld, 2007; Mehrpour, Karrari, Zamani, Tsatsakis, &Abdollahi, 2014). ATP is the energy currency of the celland very important to cellular activities including motility.The decrease in sperm motility and viability in rats thatwere treated with extracts of clove oil and chlorpyrifosmay be due to non-availability of adenosine triphosphate(ATP) for metabolism as a result of damage of spermplasma membrane architecture by clove oil and chlorpy-rifos (Chaki & Misro, 2002; Chaudhury, Bhattacharyya, &Guha, 2004). Similarly, the morphological analyses ofsperm revealed that rats that were treated with clove oil orchlorpyrifos had a higher frequency of sperm abnorma-lities especially at the tail and neck regions. Although themechanistic explanations for these abnormalities areconjectural, however, the abnormalities could have beendue to abnormal chromosome, minor alteration in testicu-lar DNA, or errors during the process of spermatogenesis(Giri, Prasad, Giri, & Sharma, 2002; Otitoloju, Obe,Adewale, Otubanjo, & Osunkalu, 2010).Aside from sperm parameters, hormonal assays quantify-

ing the levels of important hormones such as testosterone,

Fig. 4 Sperm deformity (%) in Wistar rats treated with mineral oil (OIL), green tea (GT), chlorpyrifos (CHL), extract of clove oil (CO), green tea andchlorpyrifos (GT+CHL), and green tea and extract of clove oil (GT+CO)

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FSH, and LH are useful biomarkers in reproductive toxico-logy (Rockett & Kim, 2005). In the present study, the serumlevels of testosterone, FSH, and LH were significantly lowerin the rats that were treated with clove oil and chlorpyrifosin comparison to other treatment groups. Since thesehormones play important roles in the process of spermato-genesis, rats with reduced level of these hormones areprone to developing abnormal sperm and also reducedsexual activity. This could explain the reason for a higher

frequency of abnormal sperm in rats that were treated withclove oil and chlorpyrifos (Bretveld, Brouwers, Ebisch, &Roeleveld, 2007; Mehrpour, Karrari, Zamani, Tsatsakis, &Abdollahi, 2014).The reproductive toxicity of clove oil and chlorpyrifos

was further substantiated by histopathological analysis ofthe testes. The treatment of Wistar rats with clove oiland chlorpyrifos resulted in disruption of the normalarchitecture of the testes, e.g., loss of both the cellularand tubular constituents of the seminiferous tubules,derangement of the seminiferous tubules, fatty infiltration,and outright loss of the seminiferous tubules. The resultsof this study are consistent with the findings of Sebastianand Raghavan (2015) in which Wistar rats that wereexposed to endosulfan, an organochlorine pesticide deve-loped testicular atrophies. Similarly, Joshi, Mathur, andGulati (2007) in another but similar study also reportedthat the treatment of Wistar rats with chlorpyrifos for30 days at various dosage levels caused degeneratedseminiferous tubule having decreased number of sper-matogenic elements in exposed rats. The loss of cells ofthe seminiferous tubules in rats that were exposed to cloveoil and chlorpyrifos could have been due to increasedinduction of reactive oxygen species leading to apoptosisin the testicular cells (Sebastian & Raghavan, 2015).In this study, the potential of green tea to ameliorate

the reproductive toxicity induced by clove oil andchlorpyrifos was investigated since there are reportsthat green tea has the potential to improve the ferti-lity of male rats (Abdelrazek, Helmy, Elsayed, Ebaid,& Mohamed, 2016; Ghafurniyan, Azania, Nabiuni, &Karimzadeh, 2015). The data from the present study sup-ported the assertion that treatment of rats with extracts ofgreen tea has the potential to improve the reproductivefitness of rats. The rats treated with extracts of green teaeither alone or in combination with clove oil or chlorpy-rifos had higher total and motile sperm counts, thepercentage of fast sperm was higher and the frequency ofdeformed sperms was low compared to the groups thatwere treated with clove oil or chlorpyrifos. Also, the serumlevels of reproductive hormones were higher in rats thatwere treated with extracts of green tea either alone or incombination with clove oil or chlorpyrifos.The mechanisms through which the green tea per-

forms these functions are still speculatory but itsantioxidant properties may be involved. One of the pro-posed mechanisms through which the sperm quality instressed organisms could be compromised is oxidativestress through excessive production of reactive oxygenspecies (Sanocka & Kurpisz, 2004; Schulte, Ohl, Sigman,& Smith, 2010). Green tea possesses a good number ofantioxidant compounds such as catechin which has thepotential to inactivate free radicals (Al-Wafai, 2013;Burits and Bucar, 2000).

Fig. 5 Serum hormonal levels in Wistar rats treated with mineral oil(OIL), green tea (GT), chlorpyrifos (CHL), an extract of clove oil (CO),green tea and chlorpyrifos (GT+CHL), and green tea and extract ofclove oil (GT+CO)

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ConclusionIn conclusion, the data obtained from this study showedthat S. aromaticum significantly affected the spermmorphology: a higher percentage of tail-, neck- andhead-deformed sperms in rats while the effects on spermtotal counts, sperm motile counts, and sperm progres-sive assessment were merely marginal. Also, hormonalassays showed that S. aromaticum has the tendency to

cause hormonal imbalance in rats. The histopathologicalanalyses of sections of the testes of stressed rats revealedapparent damage to the structure of the testes; loss oftubular and cellular constituents, fatty infiltration,derangement of seminiferous tubules, etc. The treat-ment of rats with C. sinensis alone or in combinationwith S. aromaticum resulted in improved sperm para-meters: higher sperm total counts, sperm motile counts,

Table 1 Histological changes in testes of Wistar rats

A B

C D

E

Plate 1 Representative micrograph of sections through the testes of Wistar rats showing A) normal structure of seminiferous tubules, B) loss oftubular constituents of seminiferous tubules, C) derangement of seminiferous tubules, D) loss of cellular constituents of seminiferous tubules, andE) fatty infiltration of seminiferous tubules

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higher percentage fast sperm, less percentage deformedsperms, higher levels of reproductive hormones, and lessdamage to the testes. From the foregoing, S. aromaticumis potentially toxic to mammals; caution must, therefore,be applied to ensure that a low level of the plant materialwas used as a pesticide to avoid potential negative effectson mammals.

AbbreviationsANOVA: Analysis of variance; LH: Luteinizing hormone; FSH: Folliclestimulating hormone; ELISA: Enzyme-linked immunosorbent assay;ATP: Adenosine triphosphate; SEM: Standard error of the mean

AcknowledgmentsThe authors declare that there is no source of funding to be acknowledged.

Authors’ contributionsThe authors JAA and COA designed the study. DVA, STO, and BKAperformed the experiments. JAA and STO performed the statistical analysisof the data. DVA and BKA wrote the draft of the manuscript. JAA and COAedited the final draft of the manuscript. All authors approved the final draftof the manuscript.

FundingNot funded

Availability of data and materialsAll data sets, on which the conclusions of the manuscript rely on, arepresent in the results section in the manuscript.

Ethics approvalThe experimental procedures were in conformity with national andinternational standards on the use of laboratory animals. Also, the study wasapproved by institutional committee on the care and use of animal forexperiments.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Department of Biology, School of Sciences, Federal University ofTechnology, P.M.B. 704, Akure, Ondo State, Nigeria. 2Department of Anatomy,School of Health Sciences, Federal University of Technology, P.M.B. 704,Akure, Ondo State, Nigeria.

Received: 11 April 2019 Accepted: 14 June 2019

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