استفاده از آنتی‌اکسیدان الاژیک اسید برای بهبود کیفیت اسپرم انجماد-یخ‌گشایی شده در قوچ نژاد قزل

نوع مقاله : علمی پژوهشی- سایر

نویسندگان

• زهرا بلوکی
• حسین دقیق کیا
• ابوذر نجفی
• مهدیه مهدی پور

گروه علوم دامی، دانشکده کشاورزی، دانشگاه تبریز، تبریز، ایران

10.22067/ijasr.v10i1.58308

چکیده

هدف از پژوهش حاضر، بررسی تأثیر آنتی‌اکسیدانی الاژیک اسید بر کیفیت اسپرم قوچ در طول فرآیند انجماد-یخ‌گشایی بود. در این مطالعه از 5 قوچ نژاد قزل در 5 تکرار (2 بار در هفته) بوسیله واژن مصنوعی اسپرم‌گیری شد. بمنظور حذف اثرات فردی هر دام نر، نمونه‌های منی پس از ارزیابی اولیه و در صورت داشتن شرایط یکسان، با هم مخلوط شدند. تیمارهای آزمایشی عبارت بودند از: الاژیک اسید در چهار سطح 25/0، 5/0، 1 و 5/1 میلی‌مولار و گروه شاهد (بدون آنتی‌اکسیدان). رقیق‌کننده بر پایه تریس-لستین مورد استفاده قرار گرفت. انجماد نمونه‌های منی بدنبال دو ساعت سردسازی و تعادل‌سازی دما تا 5 درجه سانتی‌گراد صورت گرفت. یخ‌گشایی در دمای 37 درجه سانتی‌گراد به مدت 30 ثانیه انجام شد. پارامترهای جنبایی‌کل، زنده‌مانی، یکپارچگی‌غشاء، میزان اسپرم‌های غیرطبیعی، پارامترهای جنبایی، میزان مالون‌دی‌آلدهید، ظرفیت‌تام آنتی‌اکسیدانی اسپرم، فعالیت آنزیم‌های گلوتاتیون پراکسیداز و سوپراکسید دیسموتاز بعد از یخ‌گشایی اندازه‌گیری شدند. آنالیز داده‌ها به کمک نرم‌افزار SAS انجام گرفت. یافته‌های این تحقیق بیانگر بهبود زنده‌مانی، تحرک اسپرم‌ها و ظرفیت تام آنتی‌اکسیدانی در چهار سطح 25/0، 5/0، 1 و 5/1 میلی‌مولار نسبت به گروه شاهد بود. فعالیت آنزیم‌های گلوتاتیون پراکسیداز، سوپراکسید دیسموتاز، غلظت مالون‌دی‌آلدهاید و یکپارچگی غشاء پلاسمایی در هیچ یک از سطوح تفاوت معنی-داری با گروه شاهد نداشت. میزان اسپرم غیرطبیعی در سطح 5/0 میلی‌مولار کاهش یافت و در صفات سرعت در مسیرمنحنی، سرعت در مسیرمستقیم و خطی‌بودن حرکت اسپرم با گروه شاهد تفاوت معنی‌داری داشت. بطورکلی، سطح 5/0 میلی‌مولار نسبت به سایر گروه‌های تیماری مورد مطالعه، سبب بهبود کیفیت منی قوچ بعد از فرآیند انجماد-یخ‌گشایی شد.

کلیدواژه‌ها

• اسپرم
• الاژیک اسید
• انجماد
• قوچ

20.1001.1.20083106.1397.10.1.1.4

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عنوان مقاله [English]

Use of the Ellagic Acid Antioxidant to Improve Quality of Freeze-Thawed Ghezel Sperm

نویسندگان [English]

• Zahra Blooki
• Hossein Daghigh Kia
• Abozar Najafi
• Mahdieh Mehdipour

Department of Animal Sciences, Faculty of Agriculture, University of Tabriz, Tabriz, Ira

چکیده [English]

Introduction The highest damage to the sperm is caused by oxidative stress due to the production of reactive oxygen species (ROS). Semen cryopreservation causes some structural, biochemical and functional changes, which leads to various problems in sperm transport, survival and fertility rate in domestic animals. Also, sperm metabolism produces ROS, which is potentially harmful to the sperm plasma membrane integrity loss of motility .High concentrations of ROS have negative effects on sperm quality and increased degradation of DNA, lipid peroxidation and oxidative stress which inhibits sperm motility and changes in sperm infrastructure and finally the reduction of fertility.
Seminal plasma contains enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) which have an important role in the inhibition of the deleterious effects of ROS. Lipid peroxidation may be due to lack of coordination between SOD, GPX, and CAT in seminal plasma or deficiency of total antioxidant capacity of the cell. Ellagic acid is a polyphenol compound that can be found in green tea and other natural resources such as pomegranate, strawberry, raspberry, walnut and eucalyptus bark. Ellagic acid showed antioxidant and anti-apoptotic effects, it can delay or prevent cellular oxidation; ultimately reduce oxidative stress.Ellagic acid increases the activity of enzymes SOD, CAT and GPx by preventing free radical attack to cells. This research was conducted to evaluate the antioxidant effect of different levels of Elligic acid on microscopic, biochemical parameters, antioxidant enzyme activities and total antioxidant capacity of ram semen after freezing-thawing process.
Materials and Methods Five Ghezel rams were used. Semen samples were collected twice a week using artificial vagina. In order to remove individual effects, the semen samples were pooled together. Different levels of ellagic acid (0.25, 0.5, 1, 1.5 mM) were added in diluent of tris-lecithin based. After processing and freezing, the samples were stored in liquid nitrogen until the time of evaluation. After thawing of semen samples, sperm motility parameters were evaluated using CASA system, viability by with Eosin-nigrosin staining, membrane integrity with a solution of hypo-osmotic, sperm abnormalities using a solution of Hancock, lipid peroxidation by measuring malondialdehyde and the seminal plasma antioxidant enzymes of glutathione peroxidase, superoxide dismutase and total antioxidant capacity using the RANDOX Laboratories kit. The data were analyzed with SAS (9.1.3) software using GLM procedures.
Results and Discussion The results showed that the levels of 0.25, 0.5, 1 and 1.5 mM ellagic acid improved survival and total motility parameters (P< 0.05). Addition of 0.5 mM ellagic acid reduced abnormal sperm, but improved the linearity motion, straight line velocity and average path velocity of sperm (P< 0.05). Addition of 0.25 mM level improved the curvilinear velocity compared to the control group. Total antioxidant capacity was improved in all the treated compared to the control group (P< 0.05). Increasing TAC level in seminal plasma may have a significant role in sperm normality. No significant difference was found in the glutathione peroxidase activity and superoxide dismutase enzymes and malondialdehyde levels in treatments containing ellagic acid compared to the control group. Sperm have multiple mechanisms of defense against reactive oxygen species. These mechanisms include catalase, uric acid, taurine, thiols, ascorbic acid and α-tocopherol, but the superoxide dismutase, glutathione peroxidase and glutathione reductases are the most important ones. Apart from the increase in the activity of superoxide dismutase and glutathione peroxidase enzymes, ellagic acid probably had an impact on other factors to increase the total antioxidant capacity. All levels of ellagic acid non-significantly increased the plasma membrane integrity compared to the control group. The loss of sperm plasma membrane activity due to the lipid peroxidation may lead to loss of membrane fluidity and cell activity (Aitken and Sawyer, 2003). For this reason, addition of cryoprotectants and antioxidants to the extender can reduce the effects of cold shock on sperm.
Conclusion The findings of this study showed that the diluent containing 0.5 mM ellagic acid significantly improved sperm parameters compared to other levels, after freezing-thawing process.

کلیدواژه‌ها [English]

• Cryopreservation
• Ellagic acid
• Ram
• Sperm

مراجع

1- Agarwal, A. and R. A. Saleh. 2002. Role of oxidants in male infertility: rationale, significance, and treatment. Urologic Clinics of North America, 29(4):817-827.

2- Aitken, R.J. 1994. Pathophysiology of human spermatozoa. Current Opinion in Obstetrics and Gynecology, 6(2): 128–135.

3- Aitken, R. J., H. M. Fisher, N. Fulton, E. Gomez, W. Knox, B. Lewis, and S. Irvine. 1997. Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine. Molecular reproduction and development, 47(4):468-482.

4- Aitken, R. J. and D. Sawyer. 2003. The human spermatozoon–not waving but drowning. Advances in Experimental Medicine and Biology, 518: 85–98.

5- Al-Daraji, H. J. 2015. The use of pomegranate juice for counteract lipid peroxidation that naturally occurred during liquid storage of roosters' semen. Pharmacogn Commu, 5(1): 70-76.

6- Alvarez, J. G. and B. T. Storey. 1995. Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa. Molecular Reproduction and Development, 42(3): 334–346.

7- Amini Rad, O., M. A. Khalili and H. R. Soltani Gord Faramarzi. 2009. Influence of pomegranate juice on sperm parameters and fertility in mice. Medical Journal of Hormozgan University, 13(3): 182-188.

8- Bailey, J. L., J. F. Bilodeau and N. Cormier. 2000. Semen cryopreservation in domestic animals: A damaging and capacitating phenomenon. Journal of Andrology, 21(1): 1-7.

9- Bansal, A. K. and G. S. Bilaspuri. 2008. Effect of ferrous sulphate and ascorbic acid on motility, viability and lipid peroxidationof crossbred cattle bull spermatozoa. Animal, 2(1): 100–104.

10- Bilodeau, J. F., S. Blanchette, C. Gagnon and M. A. Sirard. 2001. Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen. Theriogenology, 56(2): 275-86.

11- Bondet, V., W. Brand-Williams and C. Berset. 1997. Kinetics and mechanisms of antioxidant activity using the DPPH free radical method. Food Science and Technology, 30(6): 609-615.

12- Ceribasi, A. O., G. Turk, M. Sonmez, F. Sakin and A. Atessahin 2010. Toxic effect of cyclophosphamide on sperm morphology, testicular histology and blood oxidant–antioxidant balance, and protective roles of lycopene and ellagic acid. Basic Clinical Pharmacology and Toxicology, 107(3): 730–736.

13- Çeribaşı A. O., F. Sakin, G. Türk, M. Sönmez and A. Ateşşahin. 2012. Impact of ellagic acid on adriamycin-induced testicular histopathological lesions, apoptosis, lipid peroxidation and sperm damages. Experimental and Toxicologic Pathology, 64(7-8): 717–724.

14- Cocuzza, M., S. C. Sikka, K. S. Athayde, and A. Agarwal. 2007. Clinical relevance of oxidative stress and sperm chromatin damage in male infertility: an evidence based analysis. International Brazilian Journal of Urolology, 33(5):603-621.

15- Daghigh Kia, H., R. Shahbaz zadeh and I. Ashrafi. 2015. Antioxidant effect of Macrantha Satureja extraction on microscopic and biochemical parameters of bull sperm after freeze -thawing process. Animal Science Journal, 28(108): 101-112.

16- Dominguez-Rebolledo, A. E., M. R. Fernandez-Santos, A. Bisbal, J. L. Ros-Santaella, M. Ramon, M. Carmona, F. Martinez-Pastor and J. Garde. 2010. Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments. Reproduction, Fertility, and Development, 22(5): 856–70.

17- Dorai, T. and B. B. Aggarwal. 2004. Role of chemopreventive agents in cancer therapy. Cancer letters, 215(2): 129-140.

18- El-Sheshtawy, R. I., G. A. El-Sisy and W. S. El-Nattat. 2016. Effects of pomegranate juice in Tris-based extender on cattle semen quality after chilling and cryopreservation. Asian Pacific Journal of Reproduction, 5(4): 335-339.

19- Evans, G., W. M. C. Maxwell and S. Salamon. 1987. Salamon’s Artificial Insemination of Sheep and Goats. Butterworths, Sydney, Boston.

20- Farhadi, R., H. Daghigh Kia and I. Ashrafi. 2015. The effect of salvia sahendica ethanolic extract as natural antioxidant on quality parameters of cryopreserved Holstein bull sperm. Research on Animal Production, 6(12): 79-86.

21- Halliwell, B. and J. M. C. Gutteridge. 1998. Free Radicals in Biology and Medicine (3rd ed.) Oxford University.

22- Hassoun, E. A., J. Vodhanel and A. Abushaban. 2004. The modulatory effects of ellagic acid and vitamin E succinate on TCDD-induced oxidative stress in different brain regions of rats after subchronic exposure. Journal of Biochemical and Molecular Toxicology, 18(4): 196–203.

23- Jones, R., T. Mann, and R. Sherins. 1979. Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides, and protective action of seminal plasma. Fertility and sterility, 31(5):531-537.

24- Khosrowbeygi, A. and N. Zarghami. 2007. Fatty acid composition of human spermatozoa and seminal plasma levels of oxidative stress biomarkers in subfertile males. Prostaglandins, Leukotrienes and Essential Fatty Acids, 77(2):117-121.

25- Kumar, H. and S. Mahmood. 2001. The use of fast acting antioxidants for the reduction of cow placental retention and subsequent endometritis. Indian Journal of Animal Sciences, 71: 650–653.

26- Lenzi, A., L. Gandini, M. Picardo, F. Tramer, G. Sandri, and E. Panfili. 2000. Lipoperoxidation damage of spermatozoa polyunsaturated fatty acids (PUFA): scavenger mechanisms and possible scavenger therapies. Frontiers in Bioscience, 5(1):1-15.

27- Maia Mda, S., S. D. Bicudo, C. C. Sicherle, L. Rodello and I. C. S. Gallego. 2010. Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen cryopreserved in extenders with antioxidants. Animal Reproduction Science, 122(1-2): 118–23.

28- Mansour, S. W., S. Sangi, S. Harsha, M. A. Khaleel and A. R. Ibrahim. 2013. Sensibility of male rats fertility against olive oil, Nigella sativa oil and pomegranate extract. Asian Pacific Journal of Tropical Biomedicine, 3(7): 563-568.

29- Mehdipour, M., H. Daghigh kia, A. Najafi, H. Vaseghi Dodaran and O. Garcia-Alvarez. 2016. Effect of green tea (Camellia sinensis) extract and pre-freezing equilibration time on the post-thawing quality of ram semen cryopreserved in a soybean lecithin-based extender. Cryobiology, 73, 297-303.

30- Mertens-Talcott, S. U. and S. S. Percival. 2005. Ellagic acid and quercetininteract synergistically with resveratrol in the induction of apoptosis and cause transient cell cycle arrest in human leukemia cells. Cancer letters, 218(2): 141-151.

31- Miller, J. K., E. Brzezinska-Slebodzinska and F. C. Madsen. 1993. Oxidative stress, antioxidants, and animal function. Journal of Dairy Science, 76(9): 2812–2823.

32- Najafi, A., H. Daghigh Kia, H. Mohammadi, M. H. Najafi, Z. Zanganeh, M. Sharafi, F. Martinez-Pastor and H. Adeldust. 2014. Different concentrations of cysteamine and ergothioneine improve microscopic and oxidative parameters in ram semen frozen with a soybean lecithin extender. Cryobiology, 69: 68-73.

33- Omur, A. D. and K. Coyan. 2016. Protective effects of the antioxidants curcumin, ellagic acid and methionine on motility, mitochondrial transmembrane potential, plasma membrane and acrosome integrity in freeze-thawed Merino ram sperm. Veterinarni Medicina, 61(1): 10–16.

34- Omur, A., K. Coyan, C. Ozturk, S. Gungor and M. Bucak. 2014. The effects of curcumin, ellagic acid and methionine on post-thawed Merino rams sperm parameters. The FASEB Journal, 28(1): supplement 759.1.

35- Osawa, T. 1994. Novel natural antioxidants for utilization in food and biological systems. In Postharvest Biochemistry of plant Food-Materials in the Tropics; Uritani, I, Garcia, V, Mendoza V. Japan Scientific Societies Press: Tokyo, Japan; 241−251.

36- Placer, Z. A., L. L. Cushman and B. C. Johnson. 1966. Estimation of product of lipid peroxidation (malonyldialdehyde) in biochemical systems. Analytical Biochemistry, 16(2): 359–364.

37- Priyadarsini, K. I., S. M. Khopde, S. S. Kumar and H. Mohan. 2002. Free radical studies of ellagic acid, a natural phenolic antioxidants. Journal of Agricultural and Food Chemistry, 50(7): 2200–6.

38- Revell, S. G. and R. A. Mrode. 1994. An osmotic resistance test for bovine semen. Animal Reproduction Science, 36(1-2): 77–86.

39- Rice-Evans, C. A., N. J. Miller, P. G. Bolwell, P. M. Bramley and J. B. Pridham. 1995. The relative antioxidant activities of plant derived polyphenolic flavonoids. Free Radical Research, 22(4): 375–383.

40- Salamon, S. and W. M. Maxwell. 2000. Storage of ram semen. Animal Reproduction Science, 62(1-3): 77-111.

41- Schäfer, S. and A. Holzmann. 2000. The use of transmigration and Spermac stain to evaluate epididymal cat spermatozoa. Animal Reproduction Science, 59(3-4): 201–211.

42- Seeram, N. P., L. S. Adams, S. M. Henning, Y. Niu, Y. Zhang, M. G. Nair and D. Heber. 2005. In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice. The Journal of Nutritional Biochemistry, 16(6): 360–7.

43- Sikka, S. C. 1996. Oxidative stress and role of antioxidants in normal and abnormal sperm function. Frontiers in Bioscience, 1.e78–e86.

44- Tavilani, H., M. T. Goodarzi, A. Vaisi-Raygani, S. Salimi, and T. Hassanzadeh. 2008. Activity of antioxidant enzymes in seminal plasma and their relationship with lipid peroxidation of spermatozoa. International Braz Journal Urology, 34(4):485-491.

45- Türk, G., A. Ateşşahin, M. Sönmez, A. O. Ceribaşi and A. Yüce. 2008. Improvement of cisplatin-induced injuries to sperm quality, the oxidant-antioxidant system, and the histologic structure of the rat testis by ellagic acid. Fertility and Sterility, 89(5): 1474–148.1.

46- Turk, G., S. M. Sonmez, M. Aydin, A. Yuce, S. Gur, M. Yukselm E. H. Aksu and H. Aksoy. 2008. Effects of pomegranate juice consumption on sperm quality, spermatogenic cell density, antioxidant activity and testosterone level in male rats. Clinical Nutrition, 27(2): 289–296.

47- Vattem, D. A. and K. Shetty. 2003. Ellagic acid production and phenolic antioxidant activity in cranberry pomace (Vaccinium macrocarpon) mediated by Lentinus edodes using solid-state system. Process Biochemistry, 39(3): 367-379.

48- Verma, A., K. C. Kanwar. 1999. Effect of vitamin E on human sperm motility and lipid peroxidation in vitro. Asian Journal of Andrology, 1(3): 151-4.

49- Woolliams, J. A., G. Wiener, P. H. Anderson and C. H. Murray. 1983. Variation in the activities of glutathione peroxidase and superoxide dismutase and in the concentration of copper in the blood in various breed crosses of sheep. Research in Veterinary Science, 34 (3): 253-256.

50- Zalata, A., T. Hafez, and F. Comhaire. 1995. Evaluation of the role of reactive oxygen species in male infertility. Human Reproduction, 10(6):1444-1451.