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تأثیر بوتیلیتد هیدروکسی آنیزول (BHA) و بوتیلیتید هیدروکسی تولوئن (BHT) بر فراسنجه‌های کیفی اسپرم قوچ پس از فرآیند انجماد و یخ گشایی

نوع مقاله: مقاله پژوهشی

نویسندگان

1 دانشجوی سابق کارشناسی ارشد فیزیولوژی دام، دانشگاه آزاد اسلامی، واحد کرج

2 دانشجوی دکتری فیزیولوژی دام، پردیس کشاورزی ومنابع طبیعی دانشگاه تهران، کرج

3 استادیار، گروه دامپزشکی، واحد ساوه،دانشگاه آزاد اسلامی، ساوه، ایران

4 استادیار، پردیس کشاورزی ومنابع طبیعی دانشگاه تهران، کرج

چکیده

هدف از انجام این پژوهش، بررسی تأثیر غلظت­های مختلف بوتیلیتد هیدروکسی تولوئن (BHT) و بوتیلیتد هیدروکسی آنیزول (BHA) بر فراسنجه­های کیفی اسپرم قوچ پس از فرآیند انجماد-یخ‌‌گشایی است. به این منظور، از چهار رأس قوچ بالغ و سالم نژاد سافولک (4–5/2 سال) به مدت سه هفته و هر هفته دو نوبت اسپرم­گیری شد. پس از ارزیابی اولیه، نمونه­های منی با هم مخلوط و بر پایۀ غلظت مورد نیاز به هشت قسمت تقسیم شدند. سپس نمونه­ها با تیمارهای آماده‌شده [شامل: شاهد منفی (بدون پاداکسنده یا آنتی­اکسیدان و حلال)، شاهد مثبت (بدون پاداکسنده با افزودن حلال) و سطوح 5/0، 1 و 2 میلی‌مول از پاداکسنده­های BHA و BHT، هرکدام در قالب یک تیمار)] رقیق‌ و به مدت 150 دقیقه در دمای 4 درجۀ سلسیوس به تعادل رسیده و به نی (پایوت)­های ویژه 25/0 میلی­لیتری کشیده شدند. پس از آن نی­ها منجمد و برای ذخیره­سازی به تانک نیتروژن منتقل شدند. پس از یخ­گشایی، فراسنجه­های کیفی اسپرم شامل جنبایی، زنده‌مانی و یکپارچگی غشای پلاسمایی ارزیابی شدند. نتایج به‌دست‌آمده از این آزمایش نشان می­دهد، افزودن 2 میلی­مول BHA به رقیق­کنندۀ اسپرم قوچ سبب بهبود جنبایی کل نسبت به گروه­های شاهد مثبت و منفی شده است (01/0>P). همچنین، تیمار 2 میلی­مول BHA سبب افزایش جنبایی پیش­رونده و یکپارچگی غشای اسپرم نسبت به دیگر گروه­ها شده است (05/0>P)، اما زنده‌مانی تیمارهای حاوی سطوح مختلف BHA و BHT تفاوت معنی­داری با گروه­های شاهد مثبت و منفی نداشت (05/0<P). بنابراین، نتیجه­گیری می­شود که افزودن 2 میلی­مول پاداکسندۀ BHA به رقیق­کننده، سبب بهبود فراسنجه­های پس از انجماد اسپرم قوچ می­ شود.

کلیدواژه‌ها


عنوان مقاله [English]

Effect of Butylated Hydroxy Anisole (BHA) and Butylated Hydroxy Toluene (BHT) on semen quality of Ram semen after freeze thawing Process

نویسندگان [English]

  • Ahmad Akbari Kalesar 1
  • Afshin Seifi-Jamadi 2
  • Jafar Yadi 3
  • Hamid Kohram 4
1 Former M. Sc. Student in Animal Physiology, Islamic Azad University, Karaj Branch, Iran
2 Ph.D. Candidate in Animal Physiology, University College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran
3 Assistant Professor, Department of Veterinary, Saveh Branch, Islamic Azad University, Saveh, Iran
4 Assistant Professor, University College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran
چکیده [English]

The aim of this study was to evaluate the effects of different concentration of Butylated hydroxy toluene (BHT) and Butylated hydroxyanisole (BHA) on freezing capacity of Rams semen. Semen of four healthy mature ram (2.5 - 4 years) was collected, twice a week for three weeks. Collected semen samples were pooled after primary evaluation and divided into 8 groups. Then samples were diluted with one of the treatments [ including negative control (without antioxidants and DMSO), positive control (without antioxidants, with DMSO), BHA-0.5 (containing 0.5 mM BHA), BHA-1 (containing 1 mM BHA), BHA-2 (containing 2 mM BHA), BHT-0.5 (containing 0.5 mM BHT), BHT-1 (containing 1 mM BHT) and BHT-2 (containing 2 mM BHT)] and were equilibrated at 4 ºC for 150 min. Afterward the cooled semen were loaded into 0.25 mL straws and were frozen in liquidnitrogen vapor. Motion characteristic (using CASA: Computer Assisted Sperm Analysis), viability and membrane integrity were assessed after thawing. The results of this study showed that addition of 2mM BHA to the extender could significantly increase total motility compared to the negative and positive control treatments (P>0.01).Also addition of 2 mM BHA significantly improved the progressive motility and membrane integrity compared to rest of the groups (P>0.05). However, different levels of BHA and BHT have not shown any significant difference in viability of ram sperm (P>0.05). So it is concluded that the addition of 2 mM BHA to the extender could improves the freezability of Ram semen.

کلیدواژه‌ها [English]

  • Freezabiliy
  • ram semen
  • intergrity
  • Membrane
  • motion
  1. Agarwal, A. & Sekhon, L. H. (2010). The role of antioxidant therapy in the treatment of male infertility. Human Fertility, 13, 217-225.
  2. Cemeli, E. & Anderson, D. (2011). Mechanistic investigation of ROS-induced DNA damage by oestrogenic compounds in lymphocytes and sperm using the comet assay. International Journal of Molecular Sciences, 12, 2783-2796.
  3. Farshad, A., Khalili, B. & Jafaroghli, M. (2010). Effects of Butylated Hydroxytoluene on Freezability of Ram Spermatozoa. Asian-Australasian Journal of Animal Sciences, 23, 1276-1281.
  4. Gacitua, H. & Arav, A. (2005). Successful pregnancies with directional freezing of large volume buck semen. Theriogenology, 63, 931-938.
  5. Hinneburg, I., Dorman, H. D. & Hiltunen, R. (2006). Antioxidant activities of extracts from selected culinary herbs and spices. Food Chemistry, 97, 122-129.
  6. Holt, W. (2000). Basic aspects of frozen storage of semen. Animal Reproduction Science, 62, 3-22.
  7. Ijaz, A., Hussain, A., Aleem, M., Yousaf, M. & Rehman, H. (2009). Butylated hydroxytoluene inclusion in semen extender improves the post-thawed semen quality of Nili-Ravi buffalo (Bubalus bubalis). Theriogenology, 71, 1326-1329.
  8. Jeong, S.-H., Kim, B.-Y., Kang, H.-G., Ku, H.-O. & Cho, J.-H. (2005). Effects of butylated hydroxyanisole on the development and functions of reproductive system in rats. Toxicology, 208, 49-62.
  9. Leboeuf, B., Guillouet, P., Batellier, F., Bernelas, D., Bonne, J., Forgerit, Y., Renaud, G. & Magistrini, M. (2003). Effect of native phosphocaseinate on the in vitro preservation of fresh semen. Theriogenology, 60, 867-877.
  10. Leboeuf, B., Restall, B. & Salamon, S. (2000). Production and storage of goat semen for artificial insemination. Animal Reproduction Science, 62, 113-141.
  11. Masoudi, R., Sharafi, M., Shahneh, A. Z., Towhidi, A., Kohram, H., Zhandi, M., ... & Shahverdi, A. (2016). Effect of dietary fish oil supplementation on ram semen freeze ability and fertility using soybean lecithin–and egg yolk–based extenders. Theriogenology, 86(6), 1583-1588.
  12. Michael, A., Alexopoulos, C., Pontiki, E., Hadjipavlou-Litina, D., Saratsis, P. & Boscos, C. (2007). Effect of antioxidant supplementation on semen quality and reactive oxygen species of frozen-thawed canine spermatozoa. Theriogenology, 68, 204-212.
  13. Neagu, V., García, B. M., Sandoval, C. S., Rodríguez, A. M., Ferrusola, C. O., Fernández, L. G., Tapia, J. & Pena, F. (2010). Freezing dog semen in presence of the antioxidant butylated hydroxytoluene improves postthaw sperm membrane integrity. Theriogenology, 73, 645-650.
  14. Naijian, H. R., Kohram, H., Shahneh, A. Z., Sharafi, M. & Bucak, M. N. (2013). Effects of different concentrations of BHT on microscopic and oxidative parameters of Mahabadi goat semen following the freeze–thaw process. Cryobiology, 66, 151-155.
  15. Ogbuewu, I., Aladi, N., Etuk, I., Opara, M., Uchegbu, M., Okoli, I. & Iloeje, M. (2010). Relevance of Oxygen Free Radicals and Antioxidants in Sperm Production and Function. Research Journal of Veterinary Sciences, 3, 138-164.
  16. Pankaj, P., Raina, V., Roy, B., Mohanty, T. & Mishra, A. (2009). Effect of antioxidant preservative on cold protection ability of low grade riverine buffalo (Bubalus bubalis) bull spermatozoa. Asian-Australasian Journal of Animal Sciences, 22, 626-635.
  17. Pena, F., Johannisson, A., Wallgren, M. & Martinez, H. R. (2003). Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate. Animal Reproduction Science, 78, 85-98.
  18. Purdy, P. (2006). A review on goat sperm cryopreservation. Small Ruminant Research 63, 215-225.
  19. Pursel, V. (1979). Effect of cold shock on boar sperm treated with butylated hydroxytoluene. Biology of Reproduction, 21, 319-324.
  20. Quinn, P. J., Chow, P. Y. & White, I. G. (1980). Evidence that phospholipid protects ram spermatozoa from cold shock at a plasma membrane site. Journal of Reproduction and Fertility, 60, 403-7.
  21. Rahmatzadeh, M., Kohram, H., Zare Shahneh, A., Seifi‐Jamadi, A. & Ahmad, E. (2017). Antioxidative effect of BHA in soya bean lecithin‐based extender containing Glycerol or DMSO on freezing capacity of goat semen. Reproduction in Domestic Animals, 00, 1–7.
  22. Revell, S. & Mrode, R. (1994). An osmotic resistance test for bovine semen. Animal Reproduction Science, 36, 77-86.
  23. Roca, J., Gil, M. A., Hernandez, M., Parrilla, I., Vazquez, J. M. & Martinez, E. A. (2004). Survival and fertility of boar spermatozoa after freeze‐thawing in extender supplemented with butylated hydroxytoluene. Journal of Andrology, 25, 397-405.
  24. Rossi, T., Mazzilli, F., Delfino, M. & Dondero, F. (2001). Improved human sperm recovery using superoxide dismutase and catalase supplementation in semen cryopreservation procedure. Cell and Tissue Banking, 2, 9-13.
  25. Saleh, R. A. & Agarwal, A. (2002). Oxidative stress and male infertility: from research bench to clinical practice. Journal of Andrology, 23, 737.
  26. Seifi-Jamadi, A., Kohram, H., Zareh-Shahne, A., Dehghanizadeh, P. & Ahmad, E. (2016). Effect of various concentrations of butylated hydroxyanisole and butylated hydroxytoluene on freezing capacity of Turkman stallion sperm. Animal Reproduction Science, 170, 108-113.
  27. Shoae, A. & Zamiri, M. (2008). Effect of butylated hydroxytoluene on bull spermatozoa frozen in egg yolk-citrate extender. Animal Reproduction Science, 104, 414-418.
  28. Soobrattee, M. A., Neergheen, V. S., Luximon-Ramma, A., Aruoma, O. I. & Bahorun, T. (2005). Phenolics as potential antioxidant therapeutic agents: mechanism and actions. Mutation Research/ Fundamental and Molecular Mechanisms of Mutagenesis, 579, 200-213.
  29. Strzezek, J. (2002). Secretory activity of boar seminal vesicle glands. Reproductive Biology, 2, 243-266.
  30. Uysal, O. & Bucak, M. (2007). Effects of oxidized glutathione, bovine serum albumin, cysteine and lycopene on the quality of frozen-thawed ram semen. Acta Veterinaria Brno, 76, 383-390.
  31. Watson, P. (2000). The causes of reduced fertility with cryopreserved semen. Animal Reproduction Science, 60, 481-492.
  32. Watson, P. & Anderson, W. (1983). Influence of butylated hydroxytoluene (BHT) on the viability of ram spermatozoa undergoing cold shock. Journal of Reproduction and Fertility, 69, 229-235.
  33. World Health Organization. (2010). WHO laboratory manual for the Examination and processing of human semen. Switzerland.