- Open Access
Effect of Thymus vulgaris leaf extract on cadmium-induced testicular toxicity in rats
Bulletin of the National Research Centre volume 45, Article number: 125 (2021)
Cadmium (Cd) is a known metallohormone which mimics the action of steroid hormones with adverse effect on testicular function. It is highly toxic and a prevalent environmental contaminant with no conventional antidote. This study investigates the possible ameliorative effects of Thymus vulgaris extract on testicular toxicity induced by Cd in male rats.
The testicular and epididymal weights, serum concentration of follicle stimulating hormone, luteinizing hormone, and testosterone were significantly (p ≤ 0.05) lower in the cadmium-treated group compared to the control. Necrosis of germ cells of the seminiferous tubules was observed in the testicular tissues of the cadmium-treated group. Administration of extract showed mild but non-significant (p ≥ 0.05) protective effect on the cadmium-induced decrease in sex hormones and sperm count as well as oxidative stress and histological changes.
Thymus vulgaris leaf extract had weak ameliorative effect on cadmium-induced testicular injury in rats but with promising antioxidant activity.
Cadmium (Cd) is a highly toxic environmental contaminant resulting from industrial activities. It is a heavy metal which is broadly utilized in industry but adversely affects animal and human health through occupational exposure, contaminated food and water or smoking (El-Demerdash et al. 2004; de Souza et al. 2010). This toxic metal enters and accumulates in different organs of the body in animals and man to cause severe tissue damage ranging from cellular degeneration, inflammation to cancers. Some of these organs include the kidney, liver, testicles, pancreas, thyroid, salivary glands, bone and brain (Thompson and Bannigan 2008; Ognjanović et al. 2010). However, the primary target organ for cadmium toxicity is the male reproductive organ. In the testes, a single non-carcinogenic dose of Cd is known to cause significant testicular atrophy and calcification following blood-testis barrier disruption, inflammation, germ cell loss and haemorrhage (Acharya et al. 2008; Ola-Mudathir et al. 2008; Deng et al. 2010). Previous reports have associated the toxic effects of Cd to its induction of oxidative stress and alteration in the antioxidant defence system in several tissues leading to a decrease in the activity of antioxidant enzymes and a change in cell membrane structure through lipid peroxidation (Bagchi et al. 1997; Zikic et al. 1998; Siu et al. 2009). This testicular oxidative stress and its associated cellular damage have been established as a major cause of severe male infertility due to Cd toxicity (Tremellen 2008; Turner and Lysiak 2008). Thus, heavy metal poisonings like Cd toxicity are suspected to be one of the major reasons for the recent declining fertility associated with reduced sperm count and testicular function in men in developed countries (Siu et al. 2009), as animals and humans that inhabit industrial areas where Cd is used for manufacturing certain products are vulnerable to accidental Cd exposure. However, modern research has shown that phytogenic compounds or bioactive compounds of plant origin are rich in antioxidants, such as flavonoids, phenols, terpenes, polysaccharides, saponins, alkaloids, vitamins, and trace elements (Miliauskas et al. 2004; Gouthamchandra et al. 2010; Chaves et al. 2020). These antioxidants directly or indirectly exert their effects on the body’s antioxidant system by eliminating excessive free radicals and thus protecting the body (Chaves et al. 2020). Thymus vulgaris L. (Lamiaceae) is an indigenous perennial herb in Africa, Asia, central and southern Europe, that is known to be rich in essential oils and phenolic substances (WHO 1999). In folk medicine, it is widely used for the treatment of diseases such as gastroenteric and bronchopulmonary disorders and as an anthelmintic (Rustaiyan et al. 2000). It is also known to have immunomodulatory, anti-inflammatory, antioxidant and free radical scavenging effects (Vigo et al. 2004; El-Nekeety et al. 2011). It contains potent antioxidants such as carvacrol, linalool and thymol (Satyal et al. 2016). Therefore, this study was designed to evaluate the effect of Thymus vulgaris extract (TVE) on cadmium-induced testicular toxicity using male albino rats as they are genetically similar to human.
Cadmium chloride (CdCl2—99%) was obtained from Sigma Aldrich Chemicals Co. (St. Louis, Mo, USA). All chemicals and reagents utilized were obtained from commercial suppliers.
Extraction of plant material
The leaves of T. vulgaris were purchased from Ogige market in Nsukka and identified by Mr. A. Ozioko, a botanist at the Department of Plant Science and Biotechnology, University of Nigeria, Nsukka, and the voucher specimen was deposited at the University of Nigeria Herbarium museum, with herbarium number UNNH648. The plant material was dried under shade for 10 days and ground to powder using a grinding machine. The powdered material was soaked in 70% methanol for 3 days after which it was filtered through Whatman paper (No. 1) and concentrated using a rotary evaporator (Daud et al. 2017). The T. vulgaris extract (TVE) obtained was stored at 4 °C and dissolved in 2% tween 80 in distilled water prior to administration (Onoja et al. 2020).
Acute toxicity (LD50) study
Acute toxicity (LD50) of the TVE extract was determined according to the Organization of Economic Cooperation and Development (OECD) guideline 423 (OECD 2003). Adult male rats were administered 625, 1250, 2500, and 5000 mg/kg doses of the extract orally and observed for clinical signs of toxicity and mortality. No mortality or signs of toxicity were observed in animals administered 5000 mg/kg of the extract. Hence, the median lethal dose (LD50) was considered to be greater than 5000 mg/kg body weight in rats.
Thirty two healthy male albino rats about 10–12 weeks old, weighing between 160 and 180 g were obtained from the Experimental Animal Unit of the Zoological Garden, University of Nigeria, Nsukka. They were housed in standard metal cages with wood shavings as bedding in the Experimental Animal Unit of the Faculty of Veterinary Medicine, University of Nigeria, Nsukka and housed at a temperature of 25 ± 4 °C and relative humidity of 65 ± 5% with an alternating 12 h light and dark cycle. The animals had free access to food and water daily, and were acclimatized for one week before the commencement of the study.
The experiment was conducted in compliance with the National Institutes of Health guidelines on the care and use of laboratory animals (National Research Council 2011) as approved by the Faculty of Veterinary Medicine, Institutional Animal Care and Use Committee (IACUC), University of Nigeria, Nsukka (FVM No.20/20/11/7). The rats were randomly (randomized controlled trial) divided into four groups of eight rats each. Group A was administered 0.5 ml of 2% tween 80 in distilled water orally and a single subcutaneous dose of phosphate buffered saline (PBS); Group B was administered a single subcutaneous (SC) dose of cadmium at 3 mg/kg in PBS only; Group C was administered 500 mg/kg TVE in 2% tween 80 in distilled water orally, daily for 3 weeks while Group D was administered single (SC) dose of cadmium (SC) at 3 mg/kg + 500 mg/kg TVE in 2% tween 80 in distilled water orally, daily for 3 weeks. The group allocation and treatment were concealed from the laboratory technologist and pathologist involved in sample analysis and interpretation. The effective dose of Cd and the extract used and the duration of study were based on previous reports (Ponnusamy and Pari 2011; Onoja et al. 2020).
At the end of the experiment, the rats were fasted overnight and 2 mL of blood was collected via the retro-orbital plexus into plain sample bottles after euthanasia by intraperitoneal injection of 90 mg/kg body weight ketamine hydrochloride and 5 mg/kg body weight xylazine (Zarei and Shahrooz 2019). Blood in the plain sample bottles was allowed to clot, centrifuged (3000 rpm for 10 min) and serum was collected for hormonal assay. Thereafter, the rats were dissected and the epididymis was collected for sperm count while the testes were harvested for antioxidant enzyme activity, lipid peroxidation assay and histopathology.
The testicular and epididymal weights were determined using a sensitive Mettler weighing balance (manufactured by Mettler Toledo, Switzerland) while the epididymal sperm count was assessed using the standard haemocytometric method (Obembe and Ige 2016).
Enzyme-linked immuno-absorbent assay (ELISA) kit was used for the quantitative determination of testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentration according to the manufacturer’s protocols.
Lipid peroxidation and antioxidant assay
Lipid peroxidation biomarker, malondyaldehyde (MDA) was measured by spectrophotometric method as described previously (Ohkawa et al. 1979), while catalase (CAT) and superoxide dismutase (SOD) activity was estimated according to standard methods (Nishikimi et al. 1972; Hadwan 2018).
Testes from the different groups were dehydrated in graded concentrations of ethanol, cleared in xylene, and embedded in paraffin wax. Five-micrometer thick sections were cut, mounted on glass slides, and stained with haematoxylin and eosin for light microscopy (Bancroft and Gamble 2008).
Statistical analyses of data were carried out by one way analysis of variance (ANOVA) using the Statistical Package for Social Sciences (SPSS) version 22.0. The mean differences were considered significant at p ≤ 0.05.
Body, testicular and epididymal weights
Table 1 shows the body weights, testicular and epididymal weights of rats. Compared with control, there was no significant difference in body weight between the CdCl2-treated rats and control group. It was observed that CdCl2-treated rats showed a significant decrease in testes and epididymal weights when compared with the control group. However, the testis and epididymal weights of rats administered CdCl2 and treated with TVE were not significantly (p ≥ 0.05) different compared to the control.
Epididymal sperm counts
The epididymal sperm counts were significantly (p ≤ 0.05) lower in the CdCl2-treated group compared to the control (Fig. 1). Although, the CdCl2 + TVE group showed an increase in sperm count, this was not significantly (p ≥ 0.05) different from the CdCl2-treated group.
Serum testosterone, LH and FSH
The hormonal profile in the experimental groups showed a significantly (p ≤ 0.05) lower serum FSH, LH and testosterone in CdCl2-treated rats when compared to the control (Table 2). However, the serum FSH, LH and testosterone levels in the rats administered CdCl2 and treated with TVE were comparable to the group administered CdCl2 only.
Testicular oxidative stress and antioxidant markers
The administration of Cd to rats in group B led to a significant (p ≤ 0.05) decrease in SOD and CAT activity with increased MDA concentration in the testis compared to the control group A and TVE treated group C. However, the co-administration of Cd and TVE in group D increased activity of CAT and SOD with a significant decrease in the MDA level in testicular tissues when compared to group B (Cd only) as shown in Table 3.
Histopathological evaluation (Fig. 2) revealed normal seminiferous tubular epithelium with spermatogenic cell lines and lumen filled with numerous spermatids in the control group. The CdCl2-treated group showed severe testicular damage which included degeneration, coagulative necrosis of seminiferous tubular epithelium with increased interstitial space, mononuclear cells infiltration and Sertoli cells-only pattern of tubules, while rats administered Cd and treated with TVE had mild restoration in testicular architecture.
Human exposure to CdCl2 usually occurs through occupational contact in factories and consumptions of contaminated food and drinking water (El-Demerdash et al. 2004; de Souza et al. 2010). Cd can cause blood-testis barrier disruption, germ cell loss, testicular oedema, haemorrhage and necrosis leading to impaired reproductive physiology and irreversible infertility (Zikic et al.1998; Takiguchi and Yoshihara 2006; Blanco et al. 2007; Acharya et al. 2008; Deng et al. 2010; Oguzturk et al. 2012). As seen in the present study, the testicular and epididymal weights were decreased in the CdCl2-treated group compared to the control. It is well known that the weight of the testis depends on the mass of undifferentiated spermatogenic cells (Ponnusamy and Pari 2011) and can also serve as the primary indicator of a possible alteration in androgen status (Biswas et al. 2001). This was observed in the serum FSH, LH and testosterone levels in CdCl2-treated group which showed a substantial decrease when compared to the control. Cd exerts its known toxic effects on organs of the body like the testis through its induction of oxidative stress (Dzobo and Naik 2013; Kumar et al. 2019).
In experimental models, Cd exposure is known to affect testis weight and induce pathology leading to reduced sperm count which adversely affects male fertility (Biswas et al. 2001; Yang et al. 2006). This was also confirmed by the severe necrosis of the seminiferous tubules and interstitial inflammation of the testis, as seen in the CdCl2-group. This study also showed that administration of TVE did not elicit meaningful amelioration of cadmium-induced testicular damage. However, the consistent tendencies for increase in testicular and epididymal weights, epididymal sperm counts, serum FSH and testosterone, and mild reduction in the severity of interstitial inflammation in the group that was administered CdCl2 and treated with TVE is promising. Hence, it can be stated that at higher doses and/or longer duration of study, TVE can reverse or ameliorate the testicular toxicity induced by Cd which is in contrast with previous studies (El-Newary et al. 2017; Onoja et al. 2020) where TVE was shown to protect against hepatotoxicity induced by Cd. The discrepancies were partly, attributed to differences in sensitivity of the organs to TVE. However, the antioxidant enzyme activity in the testes of rats administered CdCl2 and treated with TVE increased but was relatively lower compared to those reported in the aforementioned studies. This may further account for the observed low potency of TVE on cadmium-induced testicular injury.
This study shows that Thymus vulgaris leaf extract has weak ameliorative effect on cadmium- induced testicular damage in rats and also high antioxidant activity.
Availability of data and materials
All data analysed and generated in this study are mentioned in this article.
Animal Research Reporting of In Vivo Experiments
Thymus vulgaris Extract
Phosphate buffered saline
- CdCl2 :
Lethal dose 50
Follicle stimulating hormone
Acharya UR, Mishra M, Patro J, Panda MK (2008) Effect of vitamins C and E on spermatogenesis in mice exposed to cadmium. Reprod Toxicol 25:84–88. https://doi.org/10.1016/j.reprotox.2007.10.004
Bagchi D, Vuchetich PJ, Bagchi M, Hassoun EA, Tran MX, Tang L, Stohs SJ (1997) Induction of oxidative stress by chronic administration of sodium dichromate (chromium VI) and cadmium chloride (cadmium II) to rats. Free Rad Biol Med 22:471–478. https://doi.org/10.1016/s0891-5849(96)00352-8
Bancroft JD, Gamble M (2008) Theory and practice of histological techniques, 6th edn. Churchill Livingstone, London, pp 273–292
Biswas NM, Sen Gupta R, Chattopadhyay A, Choudhury GR, Sarkar M (2001) Effect of atenolol on cadmium-induced testicular toxicity in male rats. Reprod Toxicol 15(6):699–704. https://doi.org/10.1016/s0890-6238(01)00184-8
Blanco A, Moyano R, Vivo J, Flores-Acuña R, Molina A, Blanco C, Agüera E, Monterde JG (2007) Quantitative changes in the testicular structure in mice exposed to low doses of cadmium. Environ Toxicol Pharmacol 23(1):96–101. https://doi.org/10.1016/j.etap.2006.07.008
Chaves N, Santiago A, Alias JC (2020) Quantification of the antioxidant activity of plant extracts, analysis of sensitivity and hierarchization based on the method used. Antioxidants (basel) 9(1):76. https://doi.org/10.3390/antiox9010076
Daud D, Fekery NFM, Hashim N (2017) Reproductive health of rats with benign prostatic hyperplasia following Cosmos caudatus ethanolic extracts consumption. J App Pharm Sci 7(6):202–205. https://doi.org/10.7324/japs.2017.70630
de Souza PF, Diamante MA, Dolder H (2010) Testis response to low doses of cadmium in Wistar rats. Int J Exp Pathol 91:125–131. https://doi.org/10.1111/j.1365-2613.2009.00692.x
Deng X, Xia Y, Hu W, Zhang H, Shen Z (2010) Cadmium-induced oxidative damage and protective effects of N-acetyl-l-cysteine against cadmium toxicity in Solanum nigrum L. J Hazard Mater 180:722–729. https://doi.org/10.1016/j.jhazmat.2010.04.099
Dzobo K, Naik YS (2013) Effect of selenium on cadmium-induced oxidative stress and esterase activity in rat organs. S Afr J Sci 109(5–6):1–8. https://doi.org/10.1590/sajs.2013/965
El-Demerdash FM, Yousef MI, Kedwany FS, Baghdadi HH (2004) Cadmium-induced changes in lipid peroxidation, blood hematology, biochemical parameters and semen quality of male rats: protective role of vitamin E and beta-carotene. Food Chem Toxicol 42:1563–1571. https://doi.org/10.1016/j.fct.2004.05.001
El-Nekeety AA, Mohamed SR, Hathout AS, Hassan NS, Aly SE, Abdel-Wahhab MA (2011) Antioxidant properties of Thymus vulgaris oil against aflatoxin-induce oxidative stress in male rats. Toxicon 57(7–8):984–991. https://doi.org/10.1016/j.toxicon.2011.03.021
El-Newary SA, Shaffie NM, Omer EA (2017) The protection of Thymus vulgaris leaves alcoholic extract against hepatotoxicity of alcohol in rats. Asian Pac J Trop Med 4:361–371. https://doi.org/10.1016/j.apjtm.2017.03.023
Gouthamchandra K, Mahmood R, Manjunatha H (2010) Free radical scavenging, antioxidant enzymes and wound healing activities of leaves extracts from Clerodendrum infortunatum L. EnvironToxicol Pharmacol 30:11–18. https://doi.org/10.1016/j.etap.2010.03.005
Hadwan MH (2018) Simple spectrophotometric assay for measuring catalase activity in biological tissues. BMC Biochem 19(1):7. https://doi.org/10.1186/s12858-018-0097-5
Kumar A, Pandey R, Siddiqi NJ, Sharma B (2019) Oxidative stress biomarkers of cadmium toxicity in mammalian systems and their distinct ameliorative strategy. J Appl Biotechnol Bioeng 6(3):126–135
Miliauskas G, Venskutonis PR, van Beek TA (2004) Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Food Chem 85:231–237. https://doi.org/10.1016/j.foodchem.2003.05.007
National Research Council (2011) Committee for the update of the guide for the care and use of laboratory animals, guide for the care and use of laboratory animals, 8th edn. National Academies Press, Washington, DC
Nishikimi M, Appaji N, Yagi K (1972) The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Biophys Res Commun 46(2):849–854. https://doi.org/10.1016/S0006-291X(72)80218-3
Obembe OO, Ige AO (2016) Sperm parameters of male Wistar rats treated with Anacardium occidentale L. leaf extract. Res J Health Sci 4(1):83–89
OECD Test No. 423 (2003) Acute oral toxicity—acute toxic class method. Organization for Economic Cooperation and Development Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris
Ognjanović BI, Marković SD, Ethordević NZ, Trbojević IS, Stajn AS, Saicić ZS (2010) Cadmium-induced lipid peroxidation and changes in antioxidant defense system in the rat testes: protective role of coenzyme Q(10) and vitamin E. Reprod Toxicol 29:191–197. https://doi.org/10.1016/j.reprotox.2009.11.009
Oguzturk H, Ciftci O, Aydin M, Timurkaan N, Beytur A, Yilmaz F (2012) Ameliorative effects of curcumin against acute cadmium toxicity on male reproductive system in rats. Andrologia 44(4):243–249. https://doi.org/10.1111/j.1439-0272.2012.01273.x
Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95(2):351–358. https://doi.org/10.1016/0003-2697(79)90738-3
Ola-Mudathir KF, Suru SM, Fafunso MA, Obioha UE, Faremi TY (2008) Protective roles of onion and garlic extracts on cadmium-induced changes in sperm characteristics and testicular oxidative damage in rats. Food Chem Toxicol 46:3604–3611. https://doi.org/10.1016/j.fct.2008.09.004
Onoja RI, Chukwudi CU, Emejuo NT, Ugwuanyi HE, Ugwueze EU (2020) Possible ameliorative effects of hydromethanol extract of Thymus vulgaris on cadmium-induced hepato-renal toxicity in rats. Not Sci Biol 12(3):568–577. https://doi.org/10.15835/nsb12310765
Ponnusamy M, Pari L (2011) Protective role of diallyl tetrasulfide on cadmium-induced testicular damage in adult rats: a biochemical and histological study. Toxicol Ind Health 27(5):407–416. https://doi.org/10.1177/0748233710387633
Rustaiyan A, Masoudi S, Monfared A, Kamalinejad M, Lajevardi T, Sedaghat S, Yari M (2000) Volatile constituents of three Thymus species grown wild in Iran. Planta Med 66(2):197–198. https://doi.org/10.1055/s-0029-1243136
Satyal P, Murray BL, McFeeters RL, Setzer WN (2016) Essential oil characterization of Thymus vulgaris from various geographical locations. Foods 5(4):70. https://doi.org/10.3390/foods5040070
Siu ER, Mruk DD, Porto CS, Yan Cheng C (2009) Cadmium-induced testicular injury. Toxicol Appl Pharmacol 3:240–249. https://doi.org/10.1016/j.taap.2009.01.028
Takiguchi M, Yoshihara S (2006) New aspects of cadmium as endocrine disruptor. Environ Sci 13(2):107–116
Thompson J, Bannigan J (2008) Cadmium: toxic effects on the reproductive system and the embryo. Reprod Toxicol 25:304–315. https://doi.org/10.1016/j.reprotox.2008.02.001
Tremellen K (2008) Oxidative stress and male infertility-a clinical perspective. Hum Reprod Update 4:243–258. https://doi.org/10.1093/humupd/dmn004
Turner TT, Lysiak JJ (2008) Oxidative stress: a common factor in testicular dysfunction. J Androl 29:488–498. https://doi.org/10.2164/jandrol.108.005132
Vigo E, Cepeda A, Gualillo O, Perez-Fernandez R (2004) In vitro anti-inflammatory effect of Eucalyptus globulus and Thymus vulgaris: nitric oxide inhibition in J774A.1 murine macrophage. J Pharm Pharmacol 56(2):257–263. https://doi.org/10.1211/0022357022665
WHO (1999) WHO monographs on selected medicinal plants, vol.1. World Health Organization, Geneva
Yang HS, Han DK, Kim JR, Sim JC (2006) Effects of alpha-tocopherol on cadmium-induced toxicity in rat testis and spermatogenesis. J Korean Med Sci 21(3):445–451. https://doi.org/10.3346/jkms.2006.21.3.445
Zarei L, Shahrooz R (2019) Protective effects of Cornus mas fruit extract on methotrexate-induced alterations in mice testicular tissue: evidences for histochemical and histomorphometrical changes in an animal model study. Vet Res Forum 10(4):307–313. https://doi.org/10.30466/vrf.2019.69516.1955
Zikic RV, Stajn AS, Ognjanovic BI, Saicic ZS, Kostic MM, Pavlovic SZ, Petrovic VM (1998) The effect of cadmium and selenium on the antioxidant enzyme activities in rat heart. J Environ Pathol Toxicol Oncol 1:259–264
The assistance of the technical staff of the Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, is appreciated.
No funding was obtained for this study.
Ethics approval and consent to participate
This study was approved by the Faculty of Veterinary Medicine Institutional Animal Care and Use Committee University of Nigeria, Nsukka (FVM2020117) in compliance with ARRIVE guidelines.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Onoja, R.I., Chukwudi, C.U., Ugwueze, E.U. et al. Effect of Thymus vulgaris leaf extract on cadmium-induced testicular toxicity in rats. Bull Natl Res Cent 45, 125 (2021). https://doi.org/10.1186/s42269-021-00583-1
- Thymus vulgaris