Study of Antioxidant Properties of Silymarin in Dealing with the Toxicity of Lithium Chloride in Ram Sperm

پژوهشهای علوم دامی ایران

Article 7, Volume 9, Issue 4 - Serial Number 32, December 2018, Pages 498-506   

Document Type: Other

DOI: 10.22067/ijasr.v9i4.59254

Authors

tahere choobineh¹; hamireza momeni¹; Mahdi Khodaei Motlagh^{* 2}; niloofar darbandi¹; atefeh khavari¹

¹arak

²Arak

Abstract

Introduction Lithium as a toxic and heavy metal and environmental contaminant could be a risk factor for male fertility. Lithium can induce male reproductive toxicity through damage in testes structure, sex hormones imbalance and decrease in testes and accessory sex organ weights as well as reduction in epididymal sperm count, normal morphology, viability and motility. Lithium is proposed to exert its cytotoxicity by free radicals generation and the activation of oxidative sensitive signaling pathways. Oxidative stress is metabolic and physiologic status caused by imbalance between free radical production and antioxidant defense of body. Therefore, the use of natural antioxidants could be a possible strategy for reducing oxidative stress in body. Silymarin is extracted from milk thistle (Silybum marianum) seeds. This compound is a polyphenolic flavonoid with a potent antioxidant property which not only acts as free radical scavenger but also increases the capacity of cell antioxidant enzymes. This study aimed to evaluate the harmful effects of lithium on sperm of Farahani’s ram and to know the protective effect of silymarin on protection against to toxic effects of lithium.
Materials and Methods In this experimental study, Farahani's ram testes were received from Arak slaughterhouse and transferred to the research laboratory under standard conditions. The experiment funding was approved by the ethical committee at Arak University. A few incisions were made in the caudal epididymis and spermatozoa were then washed into a sterile Falcon tube by Ham's F10 medium (Sigma, USA). Firstly sperm number and sperm motility were determined, according to World Health Organization protocol (WHO), to estimate sperm quality. High quality sperm samples were then used for experiments. The sperm samples were separated in eppendorf tubes as each tube contained 5×106 spermatozoa and divided into four groups to assess sperm viability. The MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay was used to assess viability. In brief, 10 μl of MTT (Sigma, USA) stock solution (5 mg/ml ham's F10) was added to each tube containing sperm suspension and incubated at 37PoPC in COR2R incubator for 1 hr. The tubes centrifuged at 6000 rpm for 6 min and the precipitate was dissolved in 200 μl dimethyl sulfoxide (DMSO). The solution was then centrifuged at 4000 rpm for 4 min. 100 μl of the purple solution were transferred into a 96-well plate and absorbance was measured using ELISA reader (SCO diagnostic, Germany) at 505 nm. Optical density of sample was then used for calculating sperm viability percentage Evaluation of sperm motility was done according to WHO guidelines. Minimum of five microscopic fields was evaluated to estimate sperm motility on at least 200 spermatozoa for each sample. The percentage of sperm motility was evaluated for following motion patterns: progressive motile sperm (PMS), non-progressive motile sperm (NPMS) and non-motile sperm (NMS). Evaluation of sperm motility was done according to WHO guidelines. In brief, 10 μl of sperm suspension was placed on semen analysis chamber. Minimum of five microscopic fields was evaluated to estimate sperm motility on at least 200 spermatozoa for each sample. The percentage of sperm motility was evaluated for following motion patterns: progressive motile sperm (PMS), non-progressive motile sperm (NPMS) and non-motile sperm (NMS). One-Way analysis of variance (ANOVA) followed by Tukey's test was used to assess data statistical significance. p

Keywords

Farahani ram sperm; Lithium chloride; Silymarin

References

1- Aziz, D. M., L. Ahlswede, and H. Enbergs. 2005. Application of MTT reduction assay to evaluate equine sperm viability. Theriogenology, 64: 1350-1356.

2- Brostrom, O. 1990. Prognosis in ulcerative colitis. Medical Clinics of North America, 74: 201-218.

3- Byun, J. W., S. H. Choo., H. H. Kim., Y. J. Kim., Y. J. Hwang, and D. Y. Kim. 2008. Evaluation of boar sperm viability by MTT reduction assay in Beltsville Thawing Solution extender. Asian-Aust. Journal of Animal Science, 4: 494-498.

4- Dalgleish, A. G, and K. O’Byrne. 2006. Inflammation and cancer: the role of the immune response and angiogenesis. Cancer Treat Research, 130: 1-38.

5- Dermar, D. A. 2001. The Review of Natural Product. Facts and Comparisons, 13: 405-409.

6- Dixit, D., S. Baboota., K. Kohli., S. Ahmad, and G. Ali. 2007. A review of pharmacological aspects and bioavailability enhancement approaches. Indian Journal of Pharmacology, 39: 172-179.

7- Reiter, R. J., D. Tan, and L. C. Manchester. 2001. Biochemical reactivity of melatonin with reactive oxygen and nitrogen species. Cell Biochemistry and Biophysics, 34: 237-256.

8- Evans, G., W. M. S. Maxwell, and T. Ahemen. 2013. Sperm quality and testicular morphometry of rabbits fed dietary levels of water spinach (Ipomoea aquatica) leaf meal. Agriculture and Biology Journal of North America, 4(3): 352-357.

9- Fadeel, B., S. Orrenius, and B. Zhivotovsky. 1999. Apoptosis in human disease: a new skin for the old ceremony?. Biochemical and Biophysical Research Communication, 266(3): 699-717.

10- Favier, A. E., J. Cadet., B. Kalyanaraman, and M. Fontecave. 1995. Analysis of free radicals in biological systems. Birkhauser Verlag, 38(2): 145-158.

11- Feher, A., I. Lang, and K. Nekam. 1990. Involvement and mechanism of lipid peroxidation in biological system. Biochemical Science, 15: 129-135.

12- Gibbons, B. H, and I. R. Gibbons. 1998. Lithium reversibly inhibits microtubule-based motility in sperm flagella. Nature, 309: 560-562.

13- Jackson, D. M., O. F. Jenkins, and B. Ross. 1988. The motor effects of bromocriptine- A review. Psychopharmacol, 95: 433- 46.

14- Jurjus, A. R, and N. N. Khoury. 2004. Animal models of inflammatory bowel disease. Journal of Pharmacological and Toxicological Methods, 50: 81-92.

15- Kang, J. S., Y. J. Jeon., S. K. Park, and K. H. Yang. 2004. Protection against lipopolysaccharide- induced sepsis and inhibition of interleukin - 1beta and prostaglandin E2 synthesis by silymarin. Biochemical and Pharmacology, 67(1): 175-81.

16- Kidd, P, and K. Head. 2005. Biochemical effect of the flavolignan silibin on RNA, protein and DNA synthesis in rat liver, Alternative Medicine Review, 10: 193-203.

17- Manna. S. K., A. Mukhopadhyay, and B. B. Aggarwal. 1999. Silymarin suppresses TNF-induced activation of NF-kappa B, c-Jun N-terminal kinase, and apoptosis. Journal of Immunology, 163(12): 6800-6809.

18- Radko, L, and W. Cybulski. 2007. Application of silymarin in human and animal medicine. Journal of Pre-Clinical and Clinical Research, 1(1): 022-026.

19- Rosa, H. J. D, and M. J. Bryant. 2003. Seasonality of reproduction in sheep. Small Ruminant Research, 48: 155-171.

20- Sanocka, D, and M. Kurpisz. 2004. Reactive oxygen species and sperm cells. Reproductive Biology and Endocrinlogy, 2: 12-19.

21- Srinivas, P., T. M. Vijaykran., H. V. Mahes, and H. Ganes. 2013. Evaluation of the protective effect of silymarin on doxorubicin induced chronic testicular toxicity in parts. International Journal of Pharmacology and Biology Sciences, 4(1): 473-484.

22- Toovey, S., E. Hudson, and W. F. Hendry. 1989. Sulfasalazine and male infertility. Reversibility and possible mechanism. Gut, 22: 445-451.

23- Watters, D, and M. Lavin. 2005. Signalling Pathways in Apoptosis. CRC Press, Boca Raton, Florida.

24- Wood, A. J. J. 1994. Lithium in the treatment of mood disorders. New England Journal of Medicine, 331(9): 591-8.

25- Zaneveld, L. J. D., R. T. Chatterton, and J. Wiley. 1982. Biochemistry of Mammalian Reproduction. J. Wiley Press, New York.