اثر روش‌های مختلف یخ گشایی بر فراسنجه های اسپرم منجمد قوچ قزل

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه علوم دامی دانشکده کشاورزی دانشگاه تبریز

2 گروه علوم دامی دانشگاه تبریز

3 استاد دانشگاه تبریز

چکیده

سابقه و هدف: امروزه عمل تلقیح مصنوعی حایگاه ویژه ای در بهبود راندمان تولید و اصلاح و بهبود پیشرفت ژنتیکی بازی می کند. به منظور تحقق بخشیدن به بسیاری از مزایای بالقوه تلقیح مصنوعی، ذخیره سازی منی برای بلند مدت یک امر ضروری محسوب می شود. این امر توسط فرایند انجماد محقق می شود. مراحل انجماد از جمله فرایند یخ گشایی منجر به تغییرات مورفولوژیکی، آسیب به عملکردهای طبیعی اسپرم و در نهایت کاهش باروری می شود. بنابراین، بهبود روش یخ گشایی منی قوچ با شاخص‌های کیفی اسپرم ارتباط داشته و از اهمیت قابل‌توجهی برخورداراست. هدف از این مطالعه مقایسه اثرات دو نرخ یخ گشایی بر پارامترهای میکروسکوپی و فعالیت های آنزیمی منی قوچ پس از فرایند انجماد - یخ گشایی بود.

مواد و روش ها :در این مطالعه، نمونه‌های منی از پنج رأس قوچ قزل، با استفاده از مهبل مصنوعی، دو بار در هفته جمع آوری شد. برای از بین بردن اثرات فردی دام‌های نر، نمونه های منی در هر بار اسپرم گیری با هم مخلوط شدندو سپس مورداستفاده قرار گرفت. نمونه ها با رقیق کننده تریس حاوی 5/1% لستین سویا و 7% گلیسرول، رقیق شدند. نمونه‌های رقیق شده در معرض بخار ازت منجمد گردیده و نهایتا تا زمان ارزیابی در تانک حاوی ازت مایع ذخیره شدند. پایوت های حاوی منی با حجم ml 25/0 در حمام آب گرم توسط دو پروتکل (i)ºc 37 بمدت s30 و (ii)ºc 60 بمدت s6 یخ گشایی شدند. بدنبال فرایند انجماد-یخ گشایی، اثرات زمان و دمای یخ‌گشایی بر ویژگی‌های جنبایی (CASA)، زنده‌مانی، یکپارچگی غشاء پلاسمایی (HOS test)، ناهنجاری‌های مورفولوژیکی، غلظت مالون دی آلدهید (MDA)، فعالیت‌های آنتی اکسیدانی (GPx ،SOD) و ظرفیت تام آنتی اکسیدان منی (TAC) ارزیابی شدند.آزمایش به صورت طرح کاملا تصادفی انجام شد و مدل آماری شامل اثر دما و زمان یخ گشایی است. تجزیه و تحلیل داده ها داده‌ها به کمک رویه Proc GLM انجام شد و میانگین‌ها به روش توکی مورد مقایسه قرار گرفتند. اختلاف معنی‌داری در سطح پنج درصد(P<0/05) گزارش شد.

یافتهها: نتایج نشان داد که فراسنجه های حرکتی اسپرم همچون درصد تحرک پیش‌رونده (PM)، سرعت در مسیر مستقیم (VSL)، سرعت در مسیر منحنی (VCL) در دمای ºc37 بهبود یافت، اما این افزایش از نظر آماری معنادار نبود. درصد زنده‌مانی و یکپارچگی غشاء پلاسمایی در ºc37 بمدت s30 نسبت ºc60 بمدت s6 بهبود یافت اما این افزایش نیز غیرمعنی‌دار بود. همچنین میزان اسپرم‌های ناهنجار در نرخ یخ گشایی بالا نسبت به نرخ پایین بهبود نیافت. علاوه براین نتایج حاصل از مقایسه پراکسیداسیون لیپید (MDA)، فعالیت آنزیم گلوتاتیون پراکسیداز GPx))، سوپر اکسید دیسموتاز (SOD) و ظرفیت آنتی‌اکسیدانی کل ((TAC در دو نرخ یخ گشایی معنی دار نبود.

نتیجه گیری :با توجه به نتایج بدست آمده از روش‌های یخ گشایی، نرخ یخ‌گشایی ºc60 بمدت s6 نمی تواند جایگزین مناسبی برای نرخ یخ گشایی ºc37 بمدت 30 ثانیه باشد.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Effect of different thawing procedures on frozen semen quality of Ghezel rams

نویسندگان [English]

  • Hossein Daghigh Kia 1
  • S Vatankhah 1
  • M Ebrahimi 2
  • Gholamali Moghaddam 3
1 ِDepartment of Animal Science, Faculty of Agriculture, University of Tabriz
2 Department of Animal Science, University of Tabriz
3 University of Tabriz
چکیده [English]

Background and objectives: Today, artificial insemination plays an important role in improving production efficiency and improving genetic progress. In order to realize many of the potential benefits of artificial insemination, semen storage for a long-term is considered a necessary step by freezing process. The freezing process, including the freezing process, leads to morphological changes, damage to the natural functions of the sperm and ultimately reduced fertility. Therefore, improving the thawing method of ram semen is related to the sperm quality indicators and is of considerable importance. The purpose of this study was to compare the effects of two thawing rates on microscopic parameters and enzyme activity of ram semen after freeze-thawing process.

Materials and methods: In this study, semen samples were collected from five Ghezel ram using an artificial vagina twice a week. To eliminate the individual effects of animals, semen samples were pooled after each sampling. Then diluted with Tris diluent. The diluted semen samples were frozen in nitrogen vapor and eventually stored in a tank containing liquid nitrogen until evaluation. The straws containing 0.25 ml semen were thawed at water bath temperatures at (i) 37ºC for 30s and (ii) 60ºC for 6 s. Following the freeze-thawing process, the effects of time exposure and thawing temperature on the kinematic features (CASA), viability, plasma membrane integrity (HOS test), morphological abnormalities, malondialdehyde (MDA) concentration, antioxidant activity (GPx, SOD) and total antioxidant capacity (TAC) were evaluated. The experiment was conducted as a completely randomized and the statistical model includes the effect of temperature and thawing time. The data were analyzed by the general linear model (GLM) procedure and statistical differences between the various group means were determined by Tukey's test. Significant differences were reported at the level of (P<0/05).

Results: The results showed that sperm motility parameters such as PM, VSL, VCL were improved at 37°C, but this increase was not statistically significant. The viability and plasma membrane integrity was improved at 37°C for 30 s compared 60°C for 6 s, but it was no significant. Also, total abnormality did not improve at high thawing rates than low rates. In addition, the results of the comparison of lipid peroxidation (MDA), glutathione peroxidase (GPX), superoxide dismutase (SOD) and total antioxidant capacity (TAC) were no significant at two thawing rates.

Conclusion: According to the results obtained from thawing methods, the thawing at 60°C for 6s cannot be a suitable alternative for semen thawing at 37°C for 30 seconds.

کلیدواژه‌ها [English]

  • Thawing rates
  • Motility parameters
  • Frozen semen
  • Ram

مراجع

1. Aamdal, J., and Andersen, K. 1968. Freezing of ram semen in straws. Int Congr Anim Reprod Artif Insemination.
Anim Reprod Sci, Paris, France. 977-
980.
2. Aboagla, E.M.E., and Terada, T. 2004. Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa. J. Theriogenology. 62: 1160-1172.
3. Ahmad, K., 1984. Effect of thaw rates on survival of buffalo spermatozoa frozen in straws. J. Dairy. Sci. 67: 1535-1538.
4. Andersen, K., and Aamdal, J. 1972. Artificial insemination with frozen semen in sheep in Norway. Reprod Domest Anim. 26:27–30.
5. Andersen, V.K., Aamdal, J., and Fougner, J. 1973. Intrauterine and deep cervical insemination with frozen semen
in sheep. Reprod Domest Anim. 8: 113-118.
6. Athurupana, R., Ioki, S., and Funahashi, H. 2015. Rapid thawing and stabilizing procedure improve postthaw survival and in vitro penetrability of boar spermatozoa cryopreserved with a glycerol-free trehalose-based extender. J. Theriogenology. 84(6): 940-947.
7. Athurupana, R., Ioki, S., and Funahashi, H. 2015. Rapid thawing and stabilizing procedure improve post-thaw survival and in vitro penetrability of boar spermatozoa cryopreserved with a trehalose-based extender. J. Reprod Dev. 61: 205–10.
8. Barranco, I., Tvarijonaviciute, A., Perez-Patiño, C., Parrilla, I., Ceron, J.J., Martinez, E.A., Rodriguez-Martinez, H. and Roca, J. 2015. High total antioxidant capacity of the porcine seminal plasma (SP-TAC) relates to sperm survival and fertility. Sci. Rep. 5: 18538.
9. Bearden, H.J., Barranco, I., Tvarijonaviciute, A., Perez-Patiño, C., Parrilla, I., Ceron, J.J., Martinez, E.A.,
Rodriguez-Martinez, H., and Roca, J. 2015. High total antioxidant capacity of the porcine seminal plasma (SP-TAC) relates to sperm survival and fertility. Sci. Rep. 5: 18538.
10. Barranco, I., Tvarijonaviciute, A., Perez-Patiño, C., Parrilla, I., Ceron, J.J., Martinez, E.A., RodriguezMartinez, H., and Roca, J. 2015. High total antioxidant capacity of the porcine seminal plasma (SP-TAC) relates to sperm survival and fertility. Sci. Rep. 5: 18538.
11. Bearden, H. J., and Fuquay, J.W., Applied animal reproduction, Reston Publishing Company, Inc. 1980. Reston Publishing Company, Inc, Reston,Virginia. 337 .
12. Bilodeau, J.F., Chatterjee, S., Sirard, M.A., and Gagnon, C. 2000. Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Mol Reprod Dev. 55(3): 282-288.
13. Blackshaw, B.A. 1955. Factors affecting the revival of bull and ram spermatozoa after freezing to—79 C. J. Aust Vet. 31: 238-241.
14. Borah, B.K.D., Deka, B.C., Biswas, R.K., Chakravarty, P., Deori, S., Sinha, S., and Ahmed, K. 2015. Effect of thawing methods on frozen semen quality of yak (Poephagus grunniens L.) bulls. Vet World. 8 (7): 831.
15. Chatterjee, S., and Gagnon, C. 2001. Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing. Mol Reprod Dev. 59: 451-458.
16. Dandekar, S.P., Nadkarni, G.D., Kulkarni, V.S., and Punekar, S. 2002. Lipid peroxidation and antioxidant enzymes in male infertility. J. Postgraduate Medicine. 48(3): 186.
17. Deka, B., and Rao, A. 1987. Effect of extenders and thawing methods on post thawing preservation of goat semen.Vet World. 64: 591-594.
18. Dodaran, H.V., Zhandi, M., Sharafi, M., Nejati-Amiri, E., Nejati-Javaremi, A., Mohammadi-Sangcheshmeh, A., Shehab-El, M.A., and Shakeri, M. 2015 .
Effect of ethanol induced mild stress on
post-thawed bull sperm quality. J. Cryobiology. 71: 12-17.
19. Eriksson, B.M., and Rodriguez-Martinez, H. 2000. Effect of freezing and thawing rates on the post-thaw viability of boar spermatozoa frozen in FlatPacks and Maxi-straws. Anim Reprod Sci. 63: 205-220.
20. Fiser, P., and Fairfull, R. 1989. The effect of glycerol-related osmotic changes on post-thaw motility and acrosomal integrity of ram spermatozoa. J. Cryobiology. 26: 64-69.
21. Fiser ,P., Ainsworth, L., and Fairfull, R. 1987. Evaluation of a new diluent and different processing procedures for cryopreservation of ram semen. J. Theriogenology. 28: 599-607.
22. Gadea, J., Sellés, E., Marco, M.A., Coy, P., Matás, C., Romar, R., and Ruiz, S .
2004 . Decrease in glutathione content in boar sperm after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders. J. Theriogenology. 62: 690-701.
23. Grøtte, O., Graffer, T. and Olesen, I., Artificial insemination with frozen ram semen in Norway, Proc. 12th Int. Congr. Anim Reprod, 1992, pp. 1557-1559.
24. Hashemi, A., Farhoomand, P., Pirmohammadi, R., Nayebpor, M., and Razzaghzadeh, S. 2007. Effect of extender and thawing methods on post thawing preservation. J. Anim. Vet Adv. 6: 1337-1339.
25. Holt, W., Head, M., and North, R. 1992. Freeze-induced membrane damage in ram spermatozoa is manifested after thawing: observations with experimental cryomicroscopy. Biol Reprod. 46: 1086-1094.
26. Jainudeen, M., and Das, S. 1982. Effect of level of glycerol, rates of freezing and thawing on the survival of buffalo spermatozoa in straws. Asian-Australas J. Anim. Sci. Serdang, Malaysia. Penerbit University, Kuala Lumpur, Malaysia. 409-411.
27. Jeyendran, R., Van der Ven, H., Perez-Pelaez, M., Crabo, B., and Zaneveld, L. 1984. Development of an assay to assess
the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod Fertil. 70: 1.219-228.
28. Knox, R., Ringwelski, J., McNamara, K., Aardsma, M., and Bojko, M. 2015. The effect of extender, method of thawing, and duration of storage on in vitro fertility measures of frozen–thawed boar sperm. J. Theriogenology. 84: 407-412.
29. Lasso, J.L., Noiles, E.E., Alvarez, J.G., and Storey, B.T. 1994. Mechanism of superoxide dismutase loss from human sperm cells during cryopreservation. Int J. Androl. 15: 255-265.
30. Lector ,C. 1996. Oxidative stress and role of antioxidants in normal and abnormal sperm function. Front Biosci. 1: 78-86.
31. Lyashenko, A. 2015. Effect of different thawing procedures on the quality and fertility of the bull spermatozoa. Asian. Pac. J. Reprod. 4: 17-21.
32. Marshall, C. 1984. Considerations for cryopreservation of semen. Z Biol. 3: 343-356.
33. Masoudi, R., Shahneh, A.Z., Towhidi, A., Kohram, H., Akbarisharif, A., Sharafi, M., Zhandi, M., and Shahab-El-Deen, M. 2017. Cervical dilation and improvement of reproductive performance in fat-tailed ewes via cervical dilator treatments. Asian. Pac. J. Reprod. 6: 93.
34. Mazur, P. 1985. Basic concepts in freezing cells. Deep freezing of boar semen. Uppsala, Sweden. Agric. Sci. 37–60.
35. Mehdipour, M., DaghighKia, H., Nazari, M., and Najafi, A. 2017. Effect of lecithin nanoliposome or soybean lecithin supplemented by pomegranate extract on post-thaw flow cytometric, microscopic and oxidative parameters in ram semen. J. Cryobiology. 78: 34-40.
36. Mehdipour, M., Daghigh Kia, H., Najafi, A., Dodaran, H.V., and García-Álvarez, O. 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. J. Cryobiology. 73: 297-303.
37. Nair, S.J., Brar, A., Ahuja, C., Sangha, S., and Chaudhary, K. 2006. A comparative study on lipid peroxidation, activities of antioxidant enzymes and viability of cattle and buffalo bull spermatozoa during storage at refrigeration temperature. Anim Reprod Sci. 96: 1-2.21-29.
38. Najafi, A., Daghigh Kia, H., Mohammadi, H., Najafi, M.H., Zanganeh, Z., Sharafi, M., Martinez-Pastor, F. and Adeldust, H. 2014. Different concentrations of cysteamine and ergothioneine improve microscopic and oxidative parameters in ram semen frozen with a soybean lecithin extender. Cryobiology. 69: 1.68-73.
39. Najafi, A., Najafi, M., Zanganeh, Z., Sharafi, M., Martinez‐Pastor, F. and Adeldust, H. 2014. Cryopreservation of ram semen in extenders containing soybean lecithin as cryoprotectant and hyaluronic acid as antioxidant. Reprod Domest Anim. 49: 6.934-940.
40. Najafi, D., Taheri, R.A., Najafi, A., Rouhollahi, A.A. and Alvarez-Rodriguez, M. 2018. Effect of Achillea millefolium-loaded nanophytosome in the post-thawing sperm quality and oxidative status of rooster semen. Cryobiology. 69: 1.68-73.
41. Nur, Z., Ileri, I.K. and Ak, K. 2006. Effects of different temperature treatments applied to deep stored bull semen on post-thaw cold shocked spermatozoa. B Vet Ipulawy. 50: 1.79.
42. Nur, Z., Segirkaya, H., Dogan, I., Soylu, M., Ak, K. and Ileri, I. 2005. Effect of low temperature thawing procedure and post-thaw cold shock on frozen bull semen. Med Weter. 9:
43. Ollero, M., Perez-Pe, R., Muino-Blanco, T. and Cebrian-Perez, J. 1998. Improvement of ram sperm cryopreservation protocols assessed by sperm quality parameters and heterogeneity analysis. Cryobiology. 37: 1.1-12.
44. Paulenz, H., Söderquist, L., Ådnøy, T., Nordstoga, A., Gulbrandsen, B. and Berg, K.A. 2004. Fertility results after
different thawing procedures for ram semen frozen in minitubes and mini straws. Theriogenology. 61: 9.1719-1727.
45. Peña, A., and Linde-Forsberg, C. 2000. Effects of Equex, one-or two-step dilution, and two freezing and thawing rates on post-thaw survival of dog spermatozoa. J. Theriogenology. 54: 859-875.
46. Perry, E.J. 1955. Artificial insemination of farm animals, Rutgers University Press: NB. 368.
47. Pontbriand, D., Howard, J., Schiewe, M., Stuart, L., and Wildt, D. 1989. Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa . J. Cryobiology. 26: 341-354.
48. Rastegarnia, A., Shahverdi, A., Topraggaleh, T.R., Ebrahimi, B., and Shafipour, V. 2013. Effect of different thawing rates on post-thaw viability, kinematic parameters and chromatin structure of buffalo (bubalus bubalis) spermatozoa. J. Cell (Yakhteh). 14: 306.
49. Rijsselaere, T., Pope, E., Wirtu, G., Filliers, M., Maes, D., Dresser, B., and Van Soom, A. 2007. Effect of thawing temperature on survival of cooled, cryopreserved epididymal cat spermatozoa, Proceedings of the 16th Congresso Nacional and the 5th Annual Symposium of EVSSAR (Estoril, Portugal). 106-106.
50. Robbins, R., Saacke, R., and Chandler, P. 1976. Influence of freeze rate, thaw rate and glycerol level on acrosomal retention and survival of bovine spermatozoa frozen in French straws. J. Anim. Sci. 42: 145-154.
51. Salamon, S., and Maxwell, W.M.C. 1995. Frozen storage of ram semen I. Processing, freezing, thawing and fertility after cervical insemination. J. Anim. Sci. 37: 185-249.
52. Salamon, S., and Maxwell, W. 2000. Storage of ram semen. J. Anim. Sci. 62: 77-111.
53. Salisbury, G.W., VanDemark, N., and Lodge, J.R., 1978. Physiology of reproduction and artificial insemination
of cattle.WH Freeman and Company. No.Ed. 798 .
54. Sarangi, A., Verma, A., Patel, R.N., Rath, A.P., Sahu, S., Virmani, M., and Devi, P . 2018. Vitamin E and Gluthathion as Antioxidant in Liquid Preservation of Semen: A Review. Int. J. Curr Microbiol. App. Sci. 7: 1680-1684.
55. Sawyer, D.E., Mercer, B.G., Wiklendt, A.M., and Aitken, R.J. 2003. Quantitative analysis of gene-specific DNA damage in human spermatozoa. Mutat. Res. 529: 21-34.
56. Shah, S., Andrabi, S., and Qureshi, I. 2016. Effect of equilibration times, freezing, and thawing rates on post‐thaw quality of buffalo (Bubalus bubalis) bull spermatozoa. J. Andrology. 4: 972-976.
57. Söderquist, L., Madrid-Bury, N., and Rodriguez-Martinez, H. 1997. Assessment of ram sperm membrane integrity following different thawing procedures. J. Theriogenology. 48: 1115-1125.
58. Sukhato, P., Thongsodseang, S., Utha, A., and Songsasen, N. 2001. Effects of cooling and warming conditions on
post-thawed motility and fertility of cryopreserved buffalo spermatozoa. J. Anim. Sci . 67: 69-77.
59. Surai, P., Blesbois, E., Grasseau, I., Chalah, T., Brillard, J.-P., Wishart, G., Cerolini, S., and Sparks, N. 1998. Fatty acid composition, glutathione peroxidase and superoxide dismutase activity and total antioxidant activity of avian semen. Comparative Biochemistry and Physiology Part B: Bio. Mol. Educ. 120: 527-533.
60. Vazquez, J., Martinez, E., Martinez, P., Garcia-Artiga, C., and Roca, J. 1997. Hypoosmotic swelling of boar spermatozoa compared to other methods for analysing the sperm membrane. J. Theriogenology. 47: 913-922.
61. Verma, A., and Kanwar, K. 1999. Effect of vitamin E on human sperm motility and lipid peroxidation in vitro. Asian. J. Androl. 1: 151-154..
62. Wai-Sum, O., Chen, H., and Chow, P. 2006. Male genital tract antioxidant enzymes—their ability to preserve sperm DNA integrity. Mol Cell Endocrinol. 250: 80-83.
نشریه پژوهش‌ در نشخوار کنندگان
دوره 6، شماره 2
شهریور 1397
صفحه 117-132

فایل ها

هم رسانی

ارجاع به این مقاله

آمار