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

نوع مقاله : علمی پژوهشی- سایر

نویسندگان

1 دانشگاه تبریز

2 تبریز

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


عنوان مقاله [English]

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

نویسندگان [English]

  • Zahra Blooki 1
  • Hossein Daghigh Kia 2
  • Abozar Najafi 2
  • Mahdieh Mehdipour 2
1 University of Tabriz
2 University of Tabriz
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
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