L-Citrulline Supplementation Enhances Reproductive Functions of Lead Acetate Induced Testicular Toxicity in Male Sprague Dawley Rats

Main Article Content

Tolunigba Abisola Kolawole
Beatrice Olatundun Oluwatayo
Ogadinma Ilochi
Elizabeth Umoren
Datonye Victor Dapper


Introduction: Lead (Pb) is a transition metal and a known male reproductive toxicant that induces its effects mainly through oxidative stress. This study investigated the effects of L-Citrulline (Cit) supplement on reproductive functions and antioxidant activities in Lead acetate treated male rats. Materials and Methods: Twenty  male rats (180-200 g) were grouped into four  and treated as follows; Group 1 (Control), given distilled water, Group 2 was given Pb acetate (2.25mg/kg), Group 3 was co-treated with Pb acetate (2.25mg/kg) and Cit (900mg/kg) and Group 4 was given Cit (900mg/kg) only. All administrations were done orally for thirty days. Caudal sperm, serum hormone levels and testicular antioxidant activities and Nitric oxide levels were evaluated at the end administrations.

Results: Results showed decrease (p<0.05) in sperm morphology, count, viability, motility, FSH, LH, Testosterone, Catalase, Nitric Oxide and Super oxide dismutase in Group 2(Pb treated rats), when compared to Group 1(Control).However, in Group 3 (Pb acetate co-administered with Cit) the effect was significantly reversed (p<0.05) compared with Group 2 and significant increase was observed in Group 4 (Cit only group) compared with Group 2.

There was an increase (p<0.05) in Malondialdehyde level in Group 2 compared with (Group 1) Control while a significant decrease (p<0.05) was observed in Groups 3 and 4 compared with Group 2.

Conclusion: The results of this study suggest that L- Citrulline supplement has ameliorating capacity on the toxic effect of Lead acetate on sperm parameters.

L- Citrulline, lead acetate, sperm parameters, reproductive hormones, antioxidants, rats.

Article Details

How to Cite
Kolawole, T. A., Oluwatayo, B. O., Ilochi, O., Umoren, E., & Dapper, D. V. (2020). L-Citrulline Supplementation Enhances Reproductive Functions of Lead Acetate Induced Testicular Toxicity in Male Sprague Dawley Rats. Journal of Advances in Medical and Pharmaceutical Sciences, 22(12), 22-35. https://doi.org/10.9734/jamps/2020/v22i1230208
Original Research Article


Curis E, Nicolis I, Moinard C, Osowska S, Zerrouk N, Benazeth S, Cynober L. Almost all about citrulline in mammals. Amino Acids. 2005;29:177–205.

Bahareh Barkhidarian, Masoud Khorshidi, Sakineh Shab-Bidar,Baran Hashemi.Effect of L-Citrulline supplementation on blood pressure: Asystematic review and meta-analysis. Avicenna Journal of Phytomedicine. 2019;9(1):10-20.

Schwedhelm E, Maas R, Freese R, Jung D, Lukacs Z, Jambrecina A, Spickler W, Schulze F, Böger RH. Pharmacokinetic and pharmacodynamic properties of oral L‐citrulline and L‐arginine: Impact on nitric oxide metabolism. Br J Clin Pharmacol. 2008;65:51–59.
[PMC free article] [PubMed] [Google Scholar]

Morita M, Hayashi T, Ochiai M, Maeda M, Yamaguchi T, Ina K, Kuzuya M. Oral supplementation with a combination of L-citrulline and L-arginine rapidly increases plasma L-arginine concentration and en hances NO bio availability. Bio chem Bio phys Res Commun. 2014;454:53–57.
[Pub Med] [Google Scholar]

Raghavan SA, Dikshit M. L-citrulline mediated relaxation in the control and lipo polysaccharide-treated rat aortic rings. Eur J Pharmacol. 200;43:61–69.

Mori A, Morita M, Morishita K, Sakamoto K, Nakahara T, Ishii K. L-Citrulline dilates rat retinal arterioles via nitric oxide-and prostaglandin-dependent pathways in vivo. J Pharmacol Sci. 2015;127:419–423.
[PubMed] [Google Scholar]

Wong A, Alvarez-Alvarado S, Jaime SJ, Kinsey AW, Spicer MT, Madzima TA, Figu eroa A. Combined whole-body vibration training and L-citrulline supplementation improves pressure wave reflection in obe se postmenopausal women. Appl Physiol Nutr Metab. 2015;41:292–297.
[PubMed] [Google Scholar]

Wong A, Chernykh O, Figueroa A. Chronic l-citrulline supplementation improves car diac sympathovagal balance in obese postmenopausal women: A preliminary report. Auton Neurosci. 2016;198:50.
[PubMed] [Google Scholar]

Gonzales JU, Raymond A, Ashley J, Kim Y. Does l‐citrulline supplementation impro ve exercise blood flow in older adults? Exp Physiol. 2017;102:1661–1671.
[PMC free article] [Pub Med] [Google Scholar]

Sanchez-Gonzalez MA, Koutnik AP, Ramirez K, Wong A, Figueroa A. The effects of short term L-citrulline supple men tation on wave reflection responses to cold exposure with concurrent isometric exercise. Am J Hypertens. 2012;26:518–526.
[PubMed] [Google Scholar]

Orozco-Gutiérrez JJ, Castillo-Martínez L, Orea-Tejeda A, Vázquez-Díaz O, Valdes pino-Trejo A, Narváez-David R et al. Effect of L-arginine or L-citrulline oral supplementation on blood pressure and right ventricular function in heart failure patients with preserved ejection fraction. Cardiol J. 2010;17:612–618.
[PubMed] [Google Scholar]

Figueroa A, Alvarez-Alvarado S, Jaime SJ, Kalfon R. L-Citrulline supplementation attenuates blood pressure, wave reflection and arterial stiffness responses to metaboreflex and cold stress in overweight men. Br J Nutr. 2016;116:279–285.
[PubMed] [Google Scholar]

Figueroa A, Sanchez-Gonzalez MA, Wong A, Arjmandi BH. Watermelon extract supplementation reduces ankle blood pressure and carotid augmentation index in obese adults with prehypertension or hypertension. Am J Hypertens. 2012;25: 640–643.
[PubMed] [Google Scholar]

Figueroa A, Alvarez-Alvarado S, Ormsbee MJ, Madzima TA, Campbell JC, Wong A. Impact of L-citrulline supplementation and whole-body vibration training on arterial stiffness and leg muscle function in obese postmenopausal women with high blood pressure. Exp Gerontol. 2015;63:35–40.
[PubMed] [Google Scholar]

TainYL, Hsieh CS, Lin IC, Chen CC, Sheen JM, Huang LT. Effects of maternal L-citrulline supplementation on renal function and blood pressure in offspring exposed to maternal caloric restriction: The impact of nitric oxide pathway. Nitric Oxide. 2010;23:34–41.
[PubMed] [Google Scholar]

Seema Tiwari IP, Tripathi HL Tiwari. Effects of Lead on Environment International Journal of Emerging Research in Management and Technology. 2013;2(6).
ISSN: 2278-9359.

Mohammed Abdulrazzaq Assi, Mohd Noor Mohd Hezmee, Abd Wahid Haron, Mohd Yusof Mohd Sabri, Mohd Ali Rajion. The detrimental effects of lead on human and animal health. Vet World. 201;9(6):660-671.

Järup L, Åkesson A. Current status of cadmium as an environmental health problem. Toxicol. Appl.Pharmacol. 2009; 238:201–208.
[CrossRef] [PubMed]

Casas SJ, Sordo J. Lead chemistry, analytical aspects, environmental impact and health effects; Elsevier: Amsterdam, The Netherlands; 2006.
ISBN: 9780444529459.

Satarug S, Garrett SH, Sens MA, Sens DA. Cadmium, environmental exposure, and health outcomes. Environ. Health Perspect. 2010;118:182–190.
[Cross Ref] [PubMed]

WHO action is needed on chemicals of major Public Health Concern. Public Health Environmental. 2010;1–4.
Accessed on 26 September 2018.

ATSDR Substance Priority List ATSDR.
accessed on 26 September 2018.

Carlsen E, Giwercman A, Keiding N, Skakkebaek NE. Evidence for decreasing quality of semen during past 50 years. BMJ. 1992;305:609-13.

Jørgensen N, Asklund C, Carlsen E, Skakkebaek NE. Coordinated European investigations of semen quality: Results from studies of Scandinavian young men is a matter of concern. Int J Androl. 2006; 29:54-61.

Jensen TK, Jørgensen N, Asklund C, Carlsen E, Holm M,Skakkebaek NE. Fertility treatment and reproductive health of male offspring: A study of 1,925 young men from the general population.Am J Epidemiol. 2007;165:583-90.

Ten J, Mendiola J, Torres-Cantero AM, Moreno-Grau JM, Moreno-Grau S, Roca Met al. Occupational and lifestyle exposures on male infertility: A mini review. TORSJ. 2008;1:16-21.

Gosselin M, Bouquegneau JM, Lefèbvre F, Lepoint G, Pergent G, Pergent-Martini Cet al. Trace metal concentrations in Posidoniaoceanica of North Corsica (north western Mediterranean Sea): Use as a biological monitor? BMC Ecol. 2006;6:12.

Kumar R, Pant N, Srivastava SP. Chlorinated pesticides and heavy metals in human semen. Int J Androl. 2000;23: 145-9.

Ghaffari MA, Motlagh B. In-vitro effect of lead, silver, tin, mercury, indium and bismuth on human sperm creatine kinase activity:A presumable mechanism for men infertility. Iran Biomed J. 2011;15:38-43.

Meeker JD, Rossano MG, Protas B, Dia mond MP, Puscheck E, Daly D, et al. Cadmium, lead, and other metals in relation to semen quality: Human evidence for molybdenum as a male reproductive toxicant. Environ Health Perspect. 2008; 116:1473-9.

Umeyama T, Ishikawa H, Takeshima H, Yoshii S, Koiso K. A comparative study of seminal trace elements in fertile and infertile men. Fertil Steril. 1986;46:494-9.

Kaur P, Bansal MP. Effect of experimental oxidative stress on steroidogenesis and DNA damage in mouse testis. J Biomed Sci. 2004;11(3):391-397.

Mahaneem M, Sulaiman SA, Jaafar H, Sirajudeen KNS, Ismail ZIM, Islam MN. Effect of honey on testicular functions in rats exposed to cigarette smoke. J Api Prod Api Med Sci. 2011;3(1):12-17.

Narayana K, Prashanthi N, Nayanatara A, Kumar HH, Abhilash K, Bairy KL. Effects of methyl parathion (o, o-dimethyl o-4-nitrophenyl phosphorothioate) on rat sperm morphology and sperm count, but not fertility, are associated with decreases ascorbic acid level in the testis. Mutat Res. 2005;588(1):28-34.

Cheesbrough M. District laboratory practice in tropical countries. Cambridge University Press, London. 2006;131-32.

Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite and [15N] nitrate in biological fluids. Anal Bio chem. 1982;126:131–138.
[PubMed] [Google Scholar]

Wileman SM, Mann GE, Baydoun AR. Induction of L-arginine transport and nitric oxide synthase in vascular smooth muscle cells: Synergistic actions of pro-inflammatory cytokines and bacterial lipopolysaccharide. Br. J. Pharmacol. 1995;116:3243–3250.
[PMC free article] [PubMed] [Google Scholar]

Rice-Evans C, Omorphos CS, Baysal E. Sickle cell membrane and oxidative damage. Biochem. J. 1986;237:265-269.

Misra HP, Fridovich I. The generation of superoxide radical auto-oxidation of haemoglobin. J. Biol. Chem. 1972;247: 6960-6962.

Aebi H. Catalase in-vitro. Methods in Enzymology. 1984;105:121-126.

Kolawole TA, Dapper DV, Ojeka SO. Ameliorative effects of the methanolic extract of the rind of Citrullus lanatus on lead acetate induced toxicity semen parameters and reproductive hormones of male albino wistar rats. Eur J Medi Plants. 2014;4(9):1125-1137.

Ragan HA, Mast TJ. Cadmium inhalation and male reproductive toxicity. Rev Environ Contam Toxicol. 1990;114:1-22.

Aruldhas MM, Subramanian S, Sekar P, Vengatesh G, Chandrahasan G, Govindarajulu Pet al.Chronic chromium exposure-induced changes in testicular histoarchitecture are associated with oxidative stress: Study in a non-human primate (Macaca radiata Geoffroy). Hum Reprod. 2005;20:2801-13.

Massanyi P, Lukac N, Slivkova J, Kovacik J, Makarevich AV, Chrenek Pet al. Mercury-induced alterations in rat kidneys and testes in-vivo. J Environ Sci Health A Tox Hazard Subst Environ Eng. 2007; 42:865-70.

Al-Attar AM. Antioxidant effect of vitamin E treatment on some heavy metals-induced renal and testicular injuries in male mice. Saudi J Biol Sci. 2011;18:63-72.

Li Y, Wu J, Zhou W, Gao E. Effects of manganese on routine semen quality parameters: Results from a population-based study in China. BMC Public Health. 2012;12:919.

Ponnapakkam TP, Bailey KS, Graves KA, Iszard MB. Assessment of male repro ductive system in the CD-1 mice following oral manganese exposure. Reprot Toxicol. 2003;17:547-51.

Subramanian S, Rajendiran G, Sekhar P, Gowri C, Govindarajulu P,Aruldhas MM. Reproductive toxicity of chromium in adult bonnet monkeys (Macaca radiata Geof frey). Reversible oxidative stress in the semen. Toxicol Appl Pharmacol. 2006;215: 237-49.

Yousef MI, El-Demerdash FM, Kamil KI, Elaswad FA. Ameliorating effect of folic acid on chromium (VI)-induced changes in reproductive performance and seminal plasma biochemistry in male rabbits. Reprod Toxicol. 2006;21:322-8.

Zhai XW, Zhang YL, Qi Q, Bai Y, Chen XL, Jin LJet al. Effects of molybdenum on sperm quality and testis oxidative stress. Syst Biol Reprod Med. 2013;59:251-5.

Johansson L, Pelliciari CE. Lead-induced changes in the stabilization of the mouse sperm chromatin. Toxicology. 1988;51: 11–24.

Apgar J. Zinc and reproduction: An update. J Nutr Biochem.1992;3:266-78.

Egwurugwu JN, Ifedi CU, Uchefuna RC, Ezeokafor EN, Alagwu EA. Effects of zinc on male sex hormones and semen quality in rats. Niger J Physiol Sci. 2013;28: 17-22.

Sudha Srivastava, Prashant Desai,Evans Coutinho, Girjesh Govil. Mechanism of action of l-arginine on the vitality of spermatozoa is primarily through increased biosynthesis of nitric oxide. Biology of Reproduction. 2006;74:954–958.

Kisa U, Basar MM, Ferhat M, Yilmaz E, Basar H, Caglayan O, Batislam E. Testicular tissue nitric oxide and thiobarbituric acid reactive substance levels: Evaluation with respect to the pathogenesis of varicocele. Urol Res. 2004;32:196–199.

Scibona M, Meschini P, Capparelli S, Pecori C, Rossi P, Fabris GFM. Arginine and male infertility. Minerva Urol. Nefrol. 1994;46:251–253.

Aydin S, Inci O, Alagol B. The role of arginine indomethacin and kallikrein in the treatment of oligospermia. Int. Urol. Nephrol. 1995;27:199–202.

Radany EW, Atherton RW, Forrester IT. Arginine uptake by rabbit spermatozoa. Arch. Biochem. Biophys. 1981;210:770–774.

Patel AB, Srivastava S, Phadke RS, Govil G. Arginine activates glycolysis of goat epididymal spermatozoa: An NMR study. Biophys J. 1998;75:1522–1528.

Patel AB.Srivastava S.Phadke RS.Govil G. Argininine acts as a protective and reversal agent against glycolytic inhibitors in spermatozoa. Physiol Chem Phys Med NMR.1999;31:29-40.

Pandey R, Singh SP. Effects of molybdenum on fertility of male rats. Bio metals. 2002;15:65-72.

Fossato da Silva DA, Teixeira CT, Scarano WR, Favareto AP, Fernandez CD, Grotto D, et al. Effects of methylmercury on malereproductive functions in Wistar rats. Reprod Toxicol. 2011;31:431-9.

Liu XF, Zhang LM, Zhang Z, Liu N, Xu SW, Lin HJ. Manganese-induced effects on testicular trace element levels and crucial hormonal parameters of Hyline cocks. Biol Trace Elem Res. 2013;151: 217-24.

Pires VC, Gollücke AP, Ribeiro DA, Lungato L, D’Almeida V, Aguiar O Jr. Grape juice concentrate protects reproductive parameters of male rats against cadmium-induced damage: A chronic assay. Br J Nutr. 2013;110:2020-9.

Zeng Q, Zhou B, Feng W, Wang YX, Liu AL, Yue J et al. Associations of urinary metal concentrations and circulating testosterone in Chinese men. Reprod Toxicol. 2013;41: 109-14.

Pillai P, Pandya C, Bhatt N, Gupta SS. Biochemical and reproductiveeffects of gestational/lactational exposure to lead and cadmium with respect to testicular steroidogenesis, antioxidant system, endogenous sex steroid and cauda-epididymal functions. Andrologia. 2012;44: 92-101.

Reddy A, Sood A, Rust PF, Busby JE, Varn E, Mathur RS, et al. The effect of nicotine on in-vitro sperm motion characteristics. J Assist Reprod Genet. 1995;12(3):217-223.

Didia BC, Dapper DV, Fawehinmi HB. The effect of Metakelfin on ovulation and the oestrus cycle in cyclic rats. W Afr J Pharmacol Drug Res. 2000;16(1&2):14-18.

Aydos K, Güven MC, Can B, Ergün A. Nicotine toxicity to the ultra-structure of the testis in rats. BJU Int. 2001;88(6):622–626.

Demirezen D, Kadiriye U. Comparative study of trace elements in certain fish, meat and meat production. Meat Sci. 2006; 74:255–60.

Xienia U, Foote GC, Van S, Devreotes PN, Alexander S, Alexander H. Differential developmental expression and cell type specificity of dictyostelium catalases and their response to oxidative stress and UV light. Biochim Biophys Acta. 2000;1492: 295–310.

Foyer CH, Noctor G. Oxygen processing in photosynthesis: Regulation and signaling. New Phytol. 2000;146:359–88.

Govil G, Phadke RS, Srivastava S. In: Lipid soluble antioxidants: Biochemistry and clinical applications Ong ASH, Packer L, (Eds.) Birkhauser Verlag Basel/ Switzer land. 1992;27–46.

Srivastava S, Desai P, Coutinho E, Govil G. Protective effect of L-arginine against lipid peroxidation in goat epididymal spermatozoa. Physiol. Chem. Phys. and Med. NMR. 2000;32:127–135

Kaore SN, Kaore NM. Chapter 53—Citrulline: Pharmacological perspectives and role as a biomarker in diseases and toxicities. In Biomarkers in Toxicology; Gupta RC, Ed; Academic Press: Cam bridge, MA, USA. 2014;883–905.

Palmer RMJ, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature. 1988;33:664 –668.

Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: Physiology pathophysiology and pharmacology. Pharmacol. Rev. 1992;43: 109–142.

Brown GC. Nitric oxide and mitochondrial respiration. Biochim. Biophys. Acta. 1999;1411:351–369.

Lipton SA. Neuronal protection and destruction by NO. Cell Death Differ. 1999;6:943–951.

Wu G, Meininger CJ. Arginine nutrition and cardiovascular function. J Nutr. 2000;130: 2626–2629.
[PubMed] [Google Scholar]