بررسی تأثیر افزودن رقیق‌کننده‌های حاوی ادیتات سدیم و پروپیلن گلیکول بر ماندگاری اسپرم منجمد قوچ قزل
محورهای موضوعی :
آسیب شناسی درمانگاهی دامپزشکی
پریسا شفاعتی
1
,
غلامعلی مقدم
2
,
حسین دقیق کیا
3
,
صادق علیجانی
4
1 - دانشجوی کارشناسی ارشد فیزیولوژی دام، گروه علوم دامی، دانشکده کشاورزی، دانشگاه تبریز، تبریز، ایران.
2 - استاد گروه علوم دامی، دانشکده کشاورزی، دانشگاه تبریز، تبریز، ایران.
3 - استاد گروه علوم دامی، دانشکده کشاورزی، دانشگاه تبریز، تبریز، ایران.
4 - دانشیار گروه علوم دامی، دانشکده کشاورزی، دانشگاه تبریز، تبریز، ایران.
تاریخ دریافت : 1397/10/05
تاریخ پذیرش : 1398/04/05
تاریخ انتشار : 1398/11/01
کلید واژه:
انجماد,
پروپیلن گلیکول,
قوچ قزل,
ماندگاری اسپرم,
ادیتات سدیم,
چکیده مقاله :
ادیتاتسدیم با کلات کردن کلسیم و ممانعت از ورود کلسیم خارج سلولی به داخل سلول و پروپیلن گلیکول با ممانعت از تشکیل بلورهای یخ داخل سلولی، از اسپرم در برابر فرآیند انجماد محافظت می کنند. مطالعه حاضر به منظور ارزیابی اثر افزودن سطوح مختلفادیتات و پروپیلن گلیکول بر کیفیت و ماندگاری اسپرم منجمد قوچ قزل انجام پذیرفت. پس از ارزیابی های اولیه منی 5 رأس قوچ نژاد قزل از لحاظ غلظت، حجم و رنگ نمونه، سلامت آکروزوم، حرکت موجی، تحرک کل و پیشرونده، درصد زنده مانی و درصد اسپرم های غیرطبیعی، نمونه های با غلظت 5/2 میلیارد اسپرم، حرکت پیشرونده ی بالای 70 درصد و حجم بیش از 5/0 میلی لیتر در گروه شاهد با مخلوط تریس، گلیسرول و زرده تخم مرغ و در گروه های تیمار با استفاده از 1، 5/1، 2 و 5/2 میلی مولار EDTA (ethylenediaminetetraacetate) و 1، 2، 3 و 7 درصد پروپیلن گلیکول رقیق شدند. سپس داخل پایت های 25/0 میلی لیتری کشیده شده و بعد از سردسازی در دمای 5 درجه سلسیوس به مدت 10-8 دقیقه، ابتدا در فاصله 5-4 سانتی متری بالای سطح نیتروژن مایع قرار گرفته و در نهایت در داخل آن غوطه ور شدند. خصوصیات کمّی اسپرم شامل زنده مانی، درصد تحرک کل، حرکت پیشرونده و درجا، تست یکپارچگی غشای پلاسمایی، درصد اسپرم های غیرطبیعی، سلامت آکروزوم در کل سلول ها و در سلول های مردهدر روزهای صفر، 20، 40 و 60 فرآیند انجماد بررسی شدند. تیمار 1درصد پروپیلن گلیکول با سایر گروه‌های تیمار پروپیلن گلیکول و گروه شاهد در میزان تحرک کل، درصد زنده مانی، آکروزوم سالم، تست یکپارچگی غشای پلاسماییو حرکت پیشرونده تفاوت معنی دار داشت (05/0p <). در بین تیمارهای EDTAنیز تیمار با 5/2 میلی مولار EDTA بهترین عملکرد را دارا بود ولی تفاوت معنی داری با سایر گروه‌های تیمارEDTAو گروه شاهد نداشت. بنابراین نتایج بررسی حاضر نشان داد که رقیق کننده های حاوی پروپیلن گلیکول و EDTA می توانند پارامترهای کیفی اسپرم قوچ را بهبود بخشند.
چکیده انگلیسی:
The present study was performed to evaluate the effect of adding different levels of EDTA and propylene glycol on quality and survival of Ghezel ram frozen semen. After primary evaluations on fresh semen for parameters including total motility, progressive motility, non-progressive motility, viability, abnormal sperm, and acrosome integrity in total cell and acrosome integrity in dead cells, samples with a concentration of 2.5 billion sperm and a progressive motility of over 70% and a volume greater than 0.5 ml were diluted with 1, 1.5, 2, 2.5 mM EDTA and 1, 2, 3% propylene glycol as an additive and propylene glycol 7% as a constitute. Straws (250 µl) were filled with dilution and were placed in a refrigerator for 1.5 to 2 hours to cool to reach 5°C. After cooling they are placed in 4-5 cm above nitrogen for 8-10 minutes and ultimately were immersed in liquid nitrogen. qualitative parameters of sperm included viability, total motility, progressive and non-progressive motility, hypo osmotic swelling test, percentage of acrosome integrity and acrosome health in dead cells were investigated in 0, 20, 40 and 60 days of freezing processes. The results of this experiment showed that diluent with 1% propylene glycol concentration in comparison with other group improved total and progressive motility, percentage of viability, acrosome integrity and HOST (p0.05). The results showed that dilutions of EDTA and propylene glycol could improve quantitative parameters of ram semen.
منابع و مأخذ:
Aisen, E.G., Alvarez, H.L., Grade, J.J. and Venturino, A. (1999). Effect of trehalose and EDTA on cryoprotective action of ram semen diluents. Theriogenolog, 53(5): 1053-1061.
Barri, A.B. and Anthony, V. (2001). Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential. Journal of Andrology, 22(6): 1061-1069.
Bittencourt, R.F., Bicudo, S.D., Biscarde, E.A., Chalhoub, M., Filho, A.D.L. and Oba, E. (2014). Trehalose and Calcium chelatorfor ram semen cryopreservation. Archives of Veterinary Science, 19(2): 66-77.
Bourinbaiar, A.S. and Lee, C. (1996). Synergistic effect of Gramicidin and EDTA in inhibiting sperm motility and cervical mucus penetration in vitro. Original Research Articale, 54(6): 367-372.
Buyukleblebici, S., Barbaros Tuncer, P., Ekin A., Bucak, M.N., Sariozkan, S., et al. (2014). Cryopreservation of bull sperm: Effect of extender supplemented with different cryoprotectants and antioxidants on sperm motility, antioxidant capacity and fertility results. Animal Reproduction Science, 150(3-4): 77-83.
Chi, H.J., Choi, S.Y., Chun, E.K., Chung, D.Y., Kim, J.H., Kim, M.H., et al. (2008). Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa. Human Reproduction, 23(5): 1023-1028.
Claudet, P.V., Natesan, M. and Selvakumar, N. (2016). Effect of cryopreservation and cooling rates on fertility potential of sperm in the giant freshwater prawn, Macrobrachium rosenbergii (De Man). Animal Reproduction Science, 171: 49-57.
Dolati Doorbash, P., Daghighkia, H., Moghaddam, G.H., Rafat, S.A. and Taghizadeh, A. (2015). The effect of adding raffinose concentrations in the diluents in semen cryopreservation of different breeds of ram at the reproductive seasone. Journal of Animal Science Researches (Agricultural Science), 25(2): 121-132. [In Persian]
Evans, G. and Maxwell, V.M.C. (1987). Salamon’s artificial insemination of sheep and goat. Butterworths Pty Limited, pp:125-132
Holt, W.V. (2000). Basic aspects of frozen storage of semen. Animal Reproduction Science, 62(1-3): 3-22.
Hu, J.H., Chen, X.Y., Li, G., Li, Q.W., Yang, H., Wang, L.I., et al. (2005). The cryopreservation effect on frozen-thawed semen of egg yolk low density lipoproteins. Asian-Aust. Journal of Animal Science, 19(4): 486-494.
Ieropoli, S., Do Espirito Santo, M., Masullo, P. and Sansone, G. (2004). Effect of extender composition, cooling rate and freezing on the fertilization viability of spermatozoa of the Pacific oyster (Crassostrea gigas). Cryobiology, 49(3): 250-257.
Karagiannidis, A., Alexopoulos, C., Amarantidis, I. and Varsakeli, S. (2000). Seasonal variation in semen characteristics of Chois and Feresian rams in Greece. Small Ruminant Research, 37(1-2): 125-130.
Keshtgar, S., Gharesi-Fard, B., Iravanpour, F. and Kazerooni, M. (2016). Combined effect of Trolox and EDTA on frozen-thawed sperm quality. Iranian Journal of Medical Sciences, 41(3): 230-237.
Li, Y.H., Cai, K.J., Kovacs, A. and Ji, W.Z. (2005). Effect of various extenders and permeating cryoprotectants on cryopreservation of Cynomolgus Monkey (Macaca fascicularis) spermatozoa. Journal of Andrology, 26(3): 387-394.
Moghaddam, G.H., Pourseif, M.M. and Rafat, S.A. (2012). Seasonal variation in semen quantity and quality traits of Iranian crossbred rams. Slovak Journal of Animal Science, 45(3): 67-75.
Mukherjee, K.P., Sahoo, A.K., Ray, K., Chattaraj, S. and Datta, U. (2016). Cryoprotective effect of EDTA, lactose, ascorbic acid and L-cysteine as an additives on garole ram (Ovis aries) semen. International Journal of Advanced Research in Biological Science, 3(7): 92-98.
Ozkavukcu, S., Erdemli, E., Isik, A., Oztuna, D. and Karahuseyinoglu, S. (2008). Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. Journal of Assisted Reproduction and Genetics, 25(8): 403-411.
Ping, S.H., Wang, C.Y., Yue, F., Yang, S.H., Si, W. and Luo, Y. (2012). Effect of Dimethyl Sulfoxide, Ethylene glycol, Propylene glycol and Glycerol on cryopreservation of wild tree shrew (Tupaia belangeri chinese) cauda epididimal sperm. Journal of Animal and Veterinary Advances, 11(19): 3568-3574.
Ranghraz Tavakoli, H., Daghighkia, H., Moghaddam, G.H. and Rafat, S.A. (2016). The effect of hCG injection on serum and seminal plasma testosterone in Ghezel ram. Journal of Animal Science Researches (Agricultural Science), 26(2): 131-139.
Silva, S.V., Soares, A.T., Batista, A.M., Almeida, F.C., Nunes, J.F. and Peixoto, C.A. (2013). Vitamin E (Trolox) addition to Tris-egg yolk extender preserves ram spermatozoon structure and kinematics after cryopreservation. Animal Reproductive Science, 137(1-2): 37-44
Wilhem, K.M., Graham, J.K. and Squires, E.L. (1996). Effect of phosphatidilserine and cholesterol liposomes on the viability, motility, and acrosomal integrity of stallion spermatozoa prior and after cryopreservation. Cryobiology, 33(3): 320-329.
World Health Organization. (2010). WHO laboratory manual for the examination and processing of human semen. 5th ed., pp: 149.
Zanganeh, Z., Mohammadi-Sangcheshmeh, A., Nabi, M.M., Najafi, A., Zare-Shahneh, A. and Zhandi, M. (2013). Does rosemary aqueous extract improve buck semen cryopreservation. Small Ruminant Research, 114(1): 120-125.
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Aisen, E.G., Alvarez, H.L., Grade, J.J. and Venturino, A. (1999). Effect of trehalose and EDTA on cryoprotective action of ram semen diluents. Theriogenolog, 53(5): 1053-1061.
Barri, A.B. and Anthony, V. (2001). Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential. Journal of Andrology, 22(6): 1061-1069.
Bittencourt, R.F., Bicudo, S.D., Biscarde, E.A., Chalhoub, M., Filho, A.D.L. and Oba, E. (2014). Trehalose and Calcium chelatorfor ram semen cryopreservation. Archives of Veterinary Science, 19(2): 66-77.
Bourinbaiar, A.S. and Lee, C. (1996). Synergistic effect of Gramicidin and EDTA in inhibiting sperm motility and cervical mucus penetration in vitro. Original Research Articale, 54(6): 367-372.
Buyukleblebici, S., Barbaros Tuncer, P., Ekin A., Bucak, M.N., Sariozkan, S., et al. (2014). Cryopreservation of bull sperm: Effect of extender supplemented with different cryoprotectants and antioxidants on sperm motility, antioxidant capacity and fertility results. Animal Reproduction Science, 150(3-4): 77-83.
Chi, H.J., Choi, S.Y., Chun, E.K., Chung, D.Y., Kim, J.H., Kim, M.H., et al. (2008). Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa. Human Reproduction, 23(5): 1023-1028.
Claudet, P.V., Natesan, M. and Selvakumar, N. (2016). Effect of cryopreservation and cooling rates on fertility potential of sperm in the giant freshwater prawn, Macrobrachium rosenbergii (De Man). Animal Reproduction Science, 171: 49-57.
Dolati Doorbash, P., Daghighkia, H., Moghaddam, G.H., Rafat, S.A. and Taghizadeh, A. (2015). The effect of adding raffinose concentrations in the diluents in semen cryopreservation of different breeds of ram at the reproductive seasone. Journal of Animal Science Researches (Agricultural Science), 25(2): 121-132. [In Persian]
Evans, G. and Maxwell, V.M.C. (1987). Salamon’s artificial insemination of sheep and goat. Butterworths Pty Limited, pp:125-132
Holt, W.V. (2000). Basic aspects of frozen storage of semen. Animal Reproduction Science, 62(1-3): 3-22.
Hu, J.H., Chen, X.Y., Li, G., Li, Q.W., Yang, H., Wang, L.I., et al. (2005). The cryopreservation effect on frozen-thawed semen of egg yolk low density lipoproteins. Asian-Aust. Journal of Animal Science, 19(4): 486-494.
Ieropoli, S., Do Espirito Santo, M., Masullo, P. and Sansone, G. (2004). Effect of extender composition, cooling rate and freezing on the fertilization viability of spermatozoa of the Pacific oyster (Crassostrea gigas). Cryobiology, 49(3): 250-257.
Karagiannidis, A., Alexopoulos, C., Amarantidis, I. and Varsakeli, S. (2000). Seasonal variation in semen characteristics of Chois and Feresian rams in Greece. Small Ruminant Research, 37(1-2): 125-130.
Keshtgar, S., Gharesi-Fard, B., Iravanpour, F. and Kazerooni, M. (2016). Combined effect of Trolox and EDTA on frozen-thawed sperm quality. Iranian Journal of Medical Sciences, 41(3): 230-237.
Li, Y.H., Cai, K.J., Kovacs, A. and Ji, W.Z. (2005). Effect of various extenders and permeating cryoprotectants on cryopreservation of Cynomolgus Monkey (Macaca fascicularis) spermatozoa. Journal of Andrology, 26(3): 387-394.
Moghaddam, G.H., Pourseif, M.M. and Rafat, S.A. (2012). Seasonal variation in semen quantity and quality traits of Iranian crossbred rams. Slovak Journal of Animal Science, 45(3): 67-75.
Mukherjee, K.P., Sahoo, A.K., Ray, K., Chattaraj, S. and Datta, U. (2016). Cryoprotective effect of EDTA, lactose, ascorbic acid and L-cysteine as an additives on garole ram (Ovis aries) semen. International Journal of Advanced Research in Biological Science, 3(7): 92-98.
Ozkavukcu, S., Erdemli, E., Isik, A., Oztuna, D. and Karahuseyinoglu, S. (2008). Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. Journal of Assisted Reproduction and Genetics, 25(8): 403-411.
Ping, S.H., Wang, C.Y., Yue, F., Yang, S.H., Si, W. and Luo, Y. (2012). Effect of Dimethyl Sulfoxide, Ethylene glycol, Propylene glycol and Glycerol on cryopreservation of wild tree shrew (Tupaia belangeri chinese) cauda epididimal sperm. Journal of Animal and Veterinary Advances, 11(19): 3568-3574.
Ranghraz Tavakoli, H., Daghighkia, H., Moghaddam, G.H. and Rafat, S.A. (2016). The effect of hCG injection on serum and seminal plasma testosterone in Ghezel ram. Journal of Animal Science Researches (Agricultural Science), 26(2): 131-139.
Silva, S.V., Soares, A.T., Batista, A.M., Almeida, F.C., Nunes, J.F. and Peixoto, C.A. (2013). Vitamin E (Trolox) addition to Tris-egg yolk extender preserves ram spermatozoon structure and kinematics after cryopreservation. Animal Reproductive Science, 137(1-2): 37-44
Wilhem, K.M., Graham, J.K. and Squires, E.L. (1996). Effect of phosphatidilserine and cholesterol liposomes on the viability, motility, and acrosomal integrity of stallion spermatozoa prior and after cryopreservation. Cryobiology, 33(3): 320-329.
World Health Organization. (2010). WHO laboratory manual for the examination and processing of human semen. 5th ed., pp: 149.
Zanganeh, Z., Mohammadi-Sangcheshmeh, A., Nabi, M.M., Najafi, A., Zare-Shahneh, A. and Zhandi, M. (2013). Does rosemary aqueous extract improve buck semen cryopreservation. Small Ruminant Research, 114(1): 120-125.
