Document Type : Original article


1 Department of Basic Science, Veterinary Medicine Faculty, Tabriz University, Tabriz, Iran.

2 Department of Basic Science, Faculty of Veterinary Medicine, Islamic Azad Branch, University of Shushtar, Khuzestan, Iran.


Introduction: Today, smoking has become a common habit, and researchers have largely been concerned with the adverse health effects of smoking. Some approaches have been proposed to minimize these effects. Nicotine is an alkaloid, which is considered to be a detrimental agent in smokers. The present study aimed to investigate the protective effects of astaxanthin against the adverse effects of nicotine.
Methods: In this study, 42 BALB/c male mice were purchased from Mashhad University in Mashhad, Iran and randomly divided into six groups. Group one received one milliliter of normal saline daily, group two received nicotine (1.5 mg/kg), group three was administered with astaxanthin (25 mg/kg), group four also received astaxanthin (50 mg/kg), group five was administered with astaxanthin (25 mg/kg) and nicotine (1.5 mg/kg), and group 6 was administered with astaxanthin (50 mg/kg) and nicotine (1.5 mg/kg). After the experiments, the epididymis was collected, and the motility, viability, and count of the sperms were evaluated.
Results: Nicotine at the dose of 1.5 mg/kg decreased the count, viability, and motility of sperm. In contrast, astaxanthin at the doses of 25 and 50 mg/kg was observed to diminish the destructive effects of nicotine.
Conclusion: According to the results, astaxanthin is a potent antioxidant for the protection of the reproductive system against nicotine-induced toxicity.


In recent decades, the outbreak of infertility has increased partly due to increased exposure to harmful agents (1-4). Infertility is a major health concern, affecting 10-15% of couples worldwide. It is defined as the inability to conceive after 12 months of unprotected intercourse (5,6). Evidence suggests that the rate of male infertility is higher compared to female infertility (7). Smoking is considered to be a significant contributing factor to infertility. Cigarettes contain numerous toxic agents, including nicotine, tar, and carbon monoxide (8).
Nicotine is a toxic agent found in cigarettes and exerts various detrimental effects on human health (9). Nicotine is an alkaloid with a high concentration in Nicotiana tabacum, where it acts as an insecticide (10). This active pharmacological agent has detrimental effects on the reproductive system and fertility in men (11,12).
There are red or yellow pigment carotenoids in animals and plants, which have antioxidant, anti-inflammatory, antitumor, and immunomodulatory properties (13,14). Among the carotenoids found in microalgae and seafood (e.g., salmon, trout, and shrimp), astaxanthin has been reported to have the highest antioxidant activity (15). A study in this regard investigated the antioxidant activity of astaxanthin and vitamin C, reporting the antioxidant activity of astaxanthin to be approximately 600-fold higher than vitamin C (16). Therefore, astaxanthin is extensively applied in the production of cosmetics and dietary supplements.
The effects of astaxanthin are exerted through several mechanisms, such as the scavenging of singlet oxygen via conjugated double bonds, inhibition of lipid peroxidation, and suppression of nuclear factor-kappa B (NF-kB) (17-19). Moreover, astaxanthin could be transferred to the brain tissue and protect the brain vessels against cerebrovascular diseases, such as cerebral ischemia and subarachnoid hemorrhage (20,21).
Oxidative stress is involved in the deterioration of various diseases due to the overgeneration of reactive oxygen species (ROS), superoxide radical, hydroxyl radical, and hydrogen peroxide (H2O2). According to reports, astaxanthin could ameliorate oxidative stress (22). Among various carotenoids (e.g., β-carotenoid), astaxanthin has been reported to be comparatively more beneficial, especially in the elimination of singlet oxygen (23).
The structure of astaxanthin consists of conjugated double bonds and terminal ring sections, which are associated with the enmeshing of radicals on membrane surfaces and internal membranes (24) (Figure 1).
The present study aimed to investigate the protective effects of astaxanthin against the adverse effects of nicotine on the motility, morphology, and viability of sperm.

Experimental Animals
In this study, 42 BALB/c male mice weighing 25-30 grams were purchased from the School of Animal Sciences at Mashhad University in Mashhad, Iran. The animals were housed in a room with the temperature of 22±2°C under controlled environmental conditions within a 12-hour light/dark cycle and had free access to water and food. The ethical and humane principles were observed during the experiments (4, 25).

Experimental Design
The animals were randomly divided into six groups of seven. Group one received one milliliter of normal saline daily, group two received nicotine (1.5 mg/kg), group three was administered with astaxanthin (25 mg/kg), group four was also administered with astaxanthin (50 mg/kg), group five received astaxanthin (25 mg/kg) and nicotine (1.5 mg/kg), and group six received astaxanthin (50 mg/kg) and nicotine (1.5 mg/kg). Astaxanthin was administered via intraperitoneal injection twice per week, and nicotine was injected intraperitoneally once a day. The treatment period was four weeks.

Sample Collection for Sperm Analysis
After the separation of cauda epididymis from the testes, it was divided into small pieces on one milliliter of Ham’s F10 culture medium. Hemocytometer with light microscopy with the magnification of 400* was used to evaluate the epididymal sperm count. In addition, phase contrast microscopy with the magnification of 400* was applied to assess sperm motility, showing 10 microscopic fields. The average counted sperms was expressed as sperm motility in each rat. In order to evaluate sperm viability, 20 milliliters of sperm suspension was combined with 20 milliliters of 0.05 Eosin-Y, and the slides were observed via bright-field microscopy with the magnification of 400*.

Statistical Analysis
Data analysis was performed using one-way analysis of variance (ANOVA) and Dunnett’s test, and the data were expressed as mean and standard error of the mean (SEM) at the significance level of P<0.05.

The sperm count decreased more significantly in group two compared to group one. On the other hand, supplementation with astaxanthin significantly increased the sperm count, and the maximum sperm count was observed in group four (Figure 2).
Sperm motility reduced more significantly in group two compared to group one. However, astaxanthin administration significantly increased sperm motility, and the maximum sperm motility was observed in group four (Figure 3).

According to the findings, sperm viability decreased more significantly in group two compared to group one. On the other hand, supplementation with astaxanthin significantly increased sperm viability, and the maximum viability was observed in group four (Figure 4).

Infertility is a significant health issue, which adversely affects the personal, social, and economic aspects of life. Evidence suggests that male factors account for 40% of infertility cases (26). Reduced sperm count, sperm immaturity and abnormality, and lack of sperm motility are associated with sperm dysfunction (27). Over the past decades, special attention has been paid to the toxicity of various agents, and researchers have focused on finding compounds to decrease the adverse effects of these compounds (1-4,6,28-33).
According to reports, agents containing nicotine reduce sperm count and motility (34). Nicotine could readily cross the cell membrane and interact with the tubulin protein present in the cytoplasm of multiplying cells, thereby leading to cell division disorders (35). In a study in this regard, Racowsky and Kaufman (2008) demonstrated that nicotine leads to damages in the sperm membrane and DNA, stimulating apoptosis in the interstitial cells in the testes (36). The results of the present study confirmed the adverse effects of nicotine exposure on sperm count, motility, and viability. Accordingly, the intraperitoneal injection of astaxanthin increased sperm motility, count, and viability. Therefore, it could be concluded that the high antioxidant activity of astaxanthin could enhance the antioxidant defense of the body and decrease lipid peroxidation.
In another research, Aitken (1995) claimed that sperm motility may be considered a significant influential factor in fertility, and sperm motility is the main cause of various fertilities (37). Furthermore, nicotine has been reported to improve the activity of cannabinoids, resulting in decreased sperm motility via activating the CB1 receptors in mature sperm (38). According to the current research, nicotine could increase the motility of sperms, which could be due to the inhibition of cannabinoid activity by astaxanthin.
In general, there is a negative association between smoking and fertility (39), and nicotine has been shown to impair the reproductive system in men and women (40). The present study particularly investigated the effects of nicotine on the male reproductive system. Nicotine exposure may lead to decreased gametogenesis, reduced steroidogenesis, and inhibited secretion of gonadotropins (41-44). Several studies have indicated that nicotine use reduces testosterone release, estradiol, follicle-stimulating hormone, and luteinizing hormone. Furthermore, nicotine could adversely affect the count, motility, survival, and morphology of sperms. Nicotine has also been reported to reduce the weight of the testes and body, as well as the libido. In addition, nicotine exerts alterations in the pituitary-hypothalamic axis, thereby stimulating the release of cortisol, vasopressin, and oxytocin and increasing oxidative stress. Nicotine exposure could also increase the risk of apoptosis in the reproductive system (45-53).
Oxidative stress is caused by the overproduction of oxygen radicals or dysfunction of the antioxidant enzymes, which are highly involved in the etiology of male infertility (54,55). Appropriate levels of ROS are vital to the human body, required for the sperm to undergo capacitation and acrosome reaction (56-58). The adverse effects of nicotine on sperm quality could be attributed to the increased levels of oxidative stress, and astaxanthin could suppress oxidative stress through its high antioxidant activity.

According to the results, astaxanthin could be a potential candidate for protection against the adverse effects of nicotine as it increased the indices of sperm count, motility, and viability.


Conflict of Interest
The authors declare no conflict of interest.

  1. Ahmadi Z, Ashrafizadeh M. Downregulation of Osteocalcin Gene in Chickens Treated with Lead Acetate II. Int Biol Biomed J. 2018;4.
  2. Ashrafizadeh M, Rafiei H, Ahmadi Z. Histological Changes in the Liver and Biochemical Parameters of Chickens Treated with Lead Acetate II. Iran J Toxicol. 2018;12:1-5.
  3. Rafiei H, Ahmadi Z, Ashrafizadeh M. Effects of Orally Administered Lead acetate II on Rat Femur Histology, Mineralization Properties and Expression of Osteocalcin Gene. Int Biol Biomed J. 2018;4:149-155.
  4. Rafiei H, Ashrafizadeh M. Expression of Collagen Type II and Osteocalcin Genes in Mesenchymal Stem Cells from Rats Treated with Lead acetate II. Iran J Toxicol. 2018;12:35-40.
  5. Mosher WD. Fecundity and infertility in the United States. Am J Public Health. 1988;78:181-182.
  6. Abdollahzadeh Soreshjani S, Ashrafizadeh M. The Effects of the Exercise on the Testosterone Level, Heat Shock Proteins and Fertility Potential. Rev Clin Med. 2018;5:12-15.
  7. Ibrahim SF, Osman K, Das S, et al. A study of the antioxidant effect of alpha lipoic acids on sperm quality. Clinics (Sao Paulo). 2008;63:545-550.
  8. Hammond D, Fong GT, Cummings KM, et al. Cigarette yields and human exposure: a comparison of alternative testing regimens. Cancer Epidemiol Biomarkers Prev. 2006;15:1495-1501.
  9. Seema P, Swathy SS, Indira M. Protective effect of selenium on nicotine-induced testicular toxicity in rats. Biol Trace Elem Res. 2007;120:212-218.
  10. Da Silva FR, Erdtmann B, Dalpiaz T, et al. Effects of dermal exposure to Nicotiana tabacum (Jean Nicot, 1560) leaves in mouse evaluated by multiple methods and tissues. J Agric Food Chem. 2010;58:9868-98674.
  11. Aydos K, Güven MC, Can B, et al. Nicotine toxicity to the ultrastructure of the testis in rats. BJU Int. 2001;88:622-626.
  12. Kavitharaj NK, Vijayammal PL. Nicotine administration induced changes in the gonadal functions in male rats. Pharmacology. 1999;58:2-7.
  13. Chan KC, Mong MC, Yin MC. Antioxidative and anti‐inflammatory neuroprotective effects of astaxanthin and canthaxanthin in nerve growth factor differentiated PC12 cells. J Food Sci. 2009;74:H225-231.
  14. Khan SK, Malinski T, Mason RP, et al. Novel astaxanthin prodrug (CDX-085) attenuates thrombosis in a mouse model. Thromb Res. 2010;126:299-305.
  15. Yan T, Zhao Y, Zhang X, et al. Astaxanthin inhibits acetaldehyde-induced cytotoxicity in SH-SY5Y cells by modulating Akt/CREB and p38MAPK/ERK signaling pathways. Mar Drugs. 2016 10;14. pii: E56. 
  16. Nishida Y, Yamashita E, Miki W. Quenching activities of common hydrophilic and lipophilic antioxidants against singlet oxygen using chemiluminescence detection system. Carotenoid Science. 2007;11(6):16-20.
  17. Zhou XY, Zhang F, Hu XT, et al. Depression can be prevented by astaxanthin through inhibition of hippocampal inflammation in diabetic mice. Brain Res. 2017;1657:262-268.
  18. Kishimoto Y, Yoshida H, Kondo K. Potential anti-atherosclerotic properties of astaxanthin. Mar Drugs. 2016;14. pii: E35.
  19. Jiang X, Chen L, Shen L, et al. Trans-astaxanthin attenuates lipopolysaccharide-induced neuroinflammation and depressive-like behavior in mice. Brain Res. 2016;1649:30-37.
  20. Wu Q, Zhang XS, Wang HD, et al. Astaxanthin activates nuclear factor erythroid-related factor 2 and the antioxidant responsive element (Nrf2-ARE) pathway in the brain after subarachnoid hemorrhage in rats and attenuates early brain injury. Mar Drugs. 2014;12:6125-6141.
  21. Pan L, Zhou Y, Li XF, et al. Preventive treatment of astaxanthin provides neuroprotection through suppression of reactive oxygen species and activation of antioxidant defense pathway after stroke in rats. Brain Res Bull. 2017;130:211-220.
  22. Wu J, Hua Y, Keep RF, et al. Oxidative brain injury from extravasated erythrocytes after intracerebral hemorrhage. Brain Res. 2002;25;953:45-52.
  23. Fukuzawa K. Singlet oxygen scavenging in phospholipid membranes. Methods Enzymol. 2000;319:101-110.
  24. Goto S, Kogure K, Abe K, et al. Efficient radical trapping at the surface and inside the phospholipid membrane is responsible for highly potent antiperoxidative activity of the carotenoid astaxanthin. Biochim Biophys Acta. 2001;1512:251-258.
  25. Rafiei H, Ahmadi Z, Ashrafizadeh M. Effects of Orally Administered Lead acetate II on Rat Femur Histology, Mineralization Properties and Expression of Osteocalcin Gene. Int Biol Biomed J. 2018;4:149-155.
  26. Razzak AH, Wais SA. The infertile couple: a cohort study in Duhok, Iraq. East Mediterr Health J. 2002;8:234-238.
  27. Araoye MO. Epidemiology of infertility: social problems of the infertile couples. West Afr J Med. 2003;22:190-196.
  28. Ahmadi Z, Ashrafizadeh M. Down Regulation of Osteocalcin Gene in Chickens Treated with Cadmium. Iran J Toxicol. 2019;13:1-4.
  29. Hassanzadeh Davarania F, Ashrafizadeh M, Saberi Riseh R, et al. Antifungal nanoparticles reduce aflatoxin contamination in pistachio. PHJ. 2018;1:25-33.
  30. Mohammadinejad R, Dadashzadeh A, Moghassemi S, et al. Shedding light on gene therapy: carbon dots for the minimally invasive image-guided delivery of plasmids and noncoding RNAs. J Adv Res. 2019;18:81-93.
  31. Sobhani B, Roomiani S, Ahmadi Z, et al. Histopathological Analysis of Testis: Effects of Astaxanthin Treatment against Nicotine Toxicity. Iran J Toxicol. 2019;13:41-44.
  32. Mohammadinejad R, Ahmadi Z, Tavakol S, et al. Berberine as a potential autophagy modulator. J Cell Physiol. 2019 Feb 15. doi: 10.1002/jcp.28325.
  33. Abdollahzadeh Soreshjani S, Ashrafizadeh M. Effects of Exercise on Testosterone Level, Heat Shock Protein, and Fertility Potential. Rev Clin Medi. 2018;5:141-145.
  34. Saleh RA, Agarwal A, Sharma RK, et al. Effect of cigarette smoking on levels of seminal oxidative stress in infertile men: a prospective study. Fertil Steril. 2002;78:491-499.
  35. Gorrod JW. The mammalian metabolism of nicotine: an overview. In: Nicotine and related Alkaloids. Dordrecht:Springer; 1993 p.31-43.
  36. Racowsky C, Kaufman ML. Nuclear degeneration and meiotic aberrations observed in human oocytes matured in vitro: analysis by light microscopy. Fertil Steril. 1992;58:750-755.
  37. Aitken RJ. Free radicals, lipid peroxidation and sperm function. Reprod Fertil Dev. 1995;7:659-668.
  38. Rossato M, Ion Popa F, Ferigo M, et al. Human sperm express cannabinoid receptor Cb1, the activation of which inhibits motility, acrosome reaction, and mitochondrial function. J Clin Endocrinol Metab. 2005;90:984-991.
  39. Lagunov A, Anzar M, Sadeu JC, et al. Effect of in utero and lactational nicotine exposure on the male reproductive tract in peripubertal and adult rats. Reprod Toxicol. 2011;31:418-423.
  40. Mohammadghasemi F, Jahromi SK. Melatonin ameliorates testicular damages induced by nicotine in mice. Iran J Basic Med Sci. 2018;21:639-644.
  41. Carvalho CA, Favaro WJ, Padovani CR, et al. Morphometric and ultrastructure features of the ventral prostate of rats (Rattus norvegicus) submitted to long‐term nicotine treatment. Andrologia. 2006;38:142-151.
  42. Gu Y, Xu W, Nie D, et al. Nicotine induces Nme2-mediated apoptosis in mouse testes. Biochem Biophys Res Commun. 2016;472:573-579.
  43. Jana K, Samanta PK, De DK. Nicotine diminishes testicular gametogenesis, steroidogenesis, and steroidogenic acute regulatory protein expression in adult albino rats: possible influence on pituitary gonadotropins and alteration of testicular antioxidant status. Toxicol Sci. 2010;116:647-659.
  44. Mohammadghasemi F, Jahromi SK, Hajizadeh H, et al. The protective effects of exogenous melatonin on nicotine-induced changes in mouse ovarian follicles. J Reprod Infertil. 2012;13:143-150.
  45. Abd El-Aziz GS, El-Fark MO, Hamdy RM. Protective effect of Eruca sativa seed oil against oral nicotine induced testicular damage in rats. Tissue Cell. 2016;48:340-348.
  46. Kim KH, Joo KJ, Park HJ, et al. Nicotine induces apoptosis in TM3 mouse Leydig cells. Fertil Steril. 2005;83:1093-1099.
  47. Nesseim WH, Haroun HS, Mostafa E, et al. Effect of nicotine on spermatogenesis in adult albino rats. Andrologia. 2011;43:398-404.
  48. Nie D, Zhang D, Dai J, et al. Nicotine induced murine spermatozoa apoptosis via up-regulation of deubiquitinated RIP1 by Trim27 promoter hypomethylation. Biol Reprod. 2016;94:31.
  49. Oyeyipo IP, Raji Y, Bolarinwa AF. Antioxidant profile changes in reproductive tissues of rats treated with nicotine. J Hum Reprod Sci. 2014;7:41-46.
  50. Oyeyipo IP, Raji Y, Bolarinwa AF. Nicotine alters male reproductive hormones in male albino rats: the role of cessation. J Hum Reprod Sci. 2013;6:40-44.
  51. Patil SR, Ravindra, Patil SR, et al. Nicotine induced ovarian and uterine changes in albino mice. Indian J Physiol Pharmacol. 1998;42:503-508.
  52. Petrik JJ, Gerstein HC, Cesta CE, et al. Effects of rosiglitazone on ovarian function and fertility in animals with reduced fertility following fetal and neonatal exposure to nicotine. Endocrine. 2009;36:281-290.
  53. Reddy A, Sood A, Rust PF, et al. The effect of nicotine onin vitro sperm motion characteristics. J Assist Reprod Genet. 1995;12:217-223.
  54. Gharagozloo P, Aitken RJ. The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy. Hum Reprod. 2011;26:1628-1640.
  55. Moazamian R, Polhemus A, Connaughton H, et al. Oxidative stress and human spermatozoa: diagnostic and functional significance of aldehydes generated as a result of lipid peroxidation. Mol Hum Reprod. 2015;21:502-515.
  56. de Lamirande E, Leclerc P, Gagnon C. Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization. Mol Hum Reprod. 1997;3:175-194.
  57. de Lamirande E, Tsai C, Harakat A, et al. Involvement of reactive oxygen species in human sperm arcosome reaction induced by A23187, lysophosphatidylcholine, and biological fluid ultrafiltrates. J Androl. 1998;19:585-594.
  58. Lambert H, Overstreet JW, Morales P, et al. Sperm capacitation in the human female reproductive tract. Fertil Steril. 1985;43:325-327.