한국수정란이식학회지 (Journal of Embryo Transfer)

  • 제18권3호
  • /
  • Pages.195-201
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  • 2003
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  • 2508-755X(pISSN)
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  • 2288-0178(eISSN)
한국수정란이식학회 (The Korean Society of Embryo Transfer)
권호 표지

개 정자의 동결보존에 있어서 Glycerol 농도, 동결 및 융해 속도가 정자의 생존율 및 운동성에 미치는 영향

Influence of Glycerol Concentration, Freezing Rate and Thawing Rate on Survival of Canine Spermatozoa Frozen

  • 이제협 (대구대학교 식품.생명.화학공학부) ;
  • 박향 (대구대학교 식품.생명.화학공학부) ;
  • 박흠대 (대구대학교 식품.생명.화학공학부) ;
  • 김재명 (포천중문의과대학교 생리학교실)
  • Lee, Je-Hyub (Division of Food, Biological and Chemical Engineering, Daegu University) ;
  • Park, Hyang (Division of Food, Biological and Chemical Engineering, Daegu University) ;
  • Park, Heum-Dae (Division of Food, Biological and Chemical Engineering, Daegu University) ;
  • Kim, Jae-Myung (College of Medicine, Pochon CHA University)
  • 발행 : 2003.12.01
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초록

본 실험은 실험견의 정액 동결 시 희석 액에 첨가되는 Glycerol 농도, 동결속도, 동결보존액 첨가후 평형시간, 융해온도와 시간에 따라 정자의 생존성과 운동성을 조사하여 최적의 동결조건을 확립하기 위해 실시하였다. 1. 각기 다른 Glycerol 농도를 함유한 동결보존액에서 동결보존 후 융해하였을 때 4%의 Gly-cerol 농도에서 각각 68.8$\pm$7.4%, 73.2$\pm$8.3%로서 다른 군보다 유의하게 높은 생존율과 운동성 나타냈다(P<0.05). 2. 희석 액 I 을 첨가한 정액을 상온에서 4$^{\circ}C$까지 각각 30, 60, 120분간 속도로 하강 시켜 동결보존 후 융해하였을 때 60분간의 속도로 하강시킨 군에서 생존성은 70.6$\pm$6.8%, 운동성은 72.8$\pm$8.4%로서 다른 군보다 유의하게 높은 생존율과 운동성을 나타냈다(P<0.05). 3. 희석 액 I 을 상온에서 4$^{\circ}C$까지 60분의 속도로 하강시킨 후 희석 액 II를 첨가하여 각각 30, 60, 120분 동안 평형을 유지시킨 후 동결보존 하였을 때 60분 동안 평형을 유지시킨 군에서 생존성은 71.4$\pm$7.3%, 운동성은 74.6$\pm$7.5%로서 다른 군보다 유의하게 높은 생존율 및 운동성을 나타냈다(P<0.05). 4. 정액을 동결함에 있어 액체질소의 표면에서 각각 3, 5, 7, 9cm의 높이에서 정치시킨 후 동결을 실시하였을 때 액체질소의 표면 5cm의 높이에서 동결을 실시한 군에서 융해 후 생존성과 운동성은 각각 77.0$\pm$7.0%, 80.2$\pm$6.3% 로서 다른 군보다 유의하게 높은 생존율과 운동성을 나타냈다(P <0.05). 5. 일정기간 보존된 동결정액을 융해 시 융해온도에 따른 정자의 생존성과 운동성은 37$^{\circ}C$의 온도에서 120초 동안의 기간에 녹였을 때 각각 77.0$\pm$7.0%, 80.2$\pm$6.3%로서 다른 군보다 유의하게 높은 생존율을 나타냈다(P<0.05).

This study was carried out to establish most suitable freezing condition, to evaluate the different condition of freezing and thawing rates on the viability and motility of frozen canine spermatozoa. The ejaculated semen was added to obtained 200∼400 ${\times}$ 10$^{6}$ /$m\ell$ with extender I and was cooled to 4$^{\circ}C$ over 30, 60 and 120 minutes. And then, semen was diluted with extender II containing 4, 6 and 8％(v/v) glycerol for 60 min, respectively and packaged in 0.5$m\ell$ straw, equilibrated far 30, 60 and 120 min at 4$^{\circ}C$ and cryopreserved in liquid nitrogen vapor at different distance(3, 5, 7 and 9 cm, respectively), plunged into nitrogen tank. Samples were thawed by placing straws into 27, 37, 47, 57$^{\circ}C$ water bath for 120, 20 and 12 sec, respectively. The results were as follows; 1. The survival and motility rate immediately post-thawing was significantly higher in samples frozen in 4％ glycerol than 6 or 8％ glycerol(P< 0.05). 2. According to equilibration time at 4$^{\circ}C$, the survival and motility rate immediately post-thawing was significantly higher in samples frozen after 60 min equilibration than 30 or 120 min equilibration(P<0.05). 3. Freezing in distance of 5 cm from liquid nitrogen yield better survival and motility rate than the others(3, 7 or 9 cm)(P<0.05). 4. The effect of thawing rate on sperm survival were higher when the thawing was done at 37$^{\circ}C$ for 120 sec(P<0.05).

키워드

참고문헌

  1. Abdelhakeam AA, Graham EF, Vazquez IA and Chaloner KM. 1991. Studies on the absence of glycerol in unfrozen and frozen ram semen : Development of an extender for freezing : Effects of osmotic pressure, egg yolk levels, type of sugars, and the method of dilution. Cryobiology, 28: 43-49 https://doi.org/10.1016/0011-2240(91)90006-A
  2. Andersen K. 1975. Insemination with frozen dog semen based on a new insemination technique. Zuchthyg, 10:1-4 https://doi.org/10.1111/j.1439-0531.1975.tb00275.x
  3. Aslam M, Ahmad KM, Ahmad M and Gill SA. 1992. Additive effects of carbohydrates in tris as bull semen extenders equilibration for three od five fours. Pakistan Vet. J., 12:174-177
  4. Battista M, Parks J and Concannon P. 1988. Canine sperm' post-thaw survival following freezing in straws or pellets using PIPES, lactose, tris or TEST extenders. Proc. XI Int. Cong. Anim. Reprod. Artif. Insem., 3:229 (abstr)
  5. Dobrinski I, Lulai C, Barch AD and Post K. 1993. Effects of four different extenders and three different freezing rates on post-thaw viability of dog semen. J. Reprod
  6. Foote RH. 1964. Extenders of for freezing dog semen. Am. J. Vet. Res., a 25:37-39
  7. Foote RH. 1964 The effects of electrolytes, sugars, glycerol, and catalase on survival of dog sperm stored in buffered-yolk mediums. Am. J. Vet. Res., b 25:32-36
  8. Gill HP, Kaufman CF, Foote RH and Kirk RW. 1970. Artificial insemination of beagle bitches with freshly collected, liquid-stored and frozen semen. Am. J. Vet. Res., 31:1807-1813
  9. Ivanova-Kicheva MG, Bobadov N and Somlev B. 1997. Cryopreservation of canine semen in pellets and 5ml aluminium tubes using three extenders. Theriogenology, 48: 1343-1349 https://doi.org/10.1016/S0093-691X(97)00375-0
  10. Marzur P, Leibo SP, Farrant J, Chu EHY, Hanna MG and Smith LH. 1970 Interactions of cooling rate, warming rate and protective additive on the survival of mammalian cells. In : Wolstenholme GEW O Connor M. (eds.). The frozen cell. CIBA Foundation symposium. 69-88
  11. Molinia FC, Evans G and Maxwell WMC. 1994. In vitro evaluation of zwitter ions buffers in dilutents for freezing ram speratozoa. Reprod. Nutr. Dev., 34:491-500 https://doi.org/10.1051/rnd:19940510
  12. Morton DB. 1988. Artificial insemination with frozen semen in the dog : principles of 'DNA fingerprinting'. In: Jones DE and Joshua JO (eds), Reproductive clinical problems in the dog. Bristol : Wright and Sons Ltd. 169-186
  13. Oettle EE and Soley JT. 1986. Ultrastructural changes in the acrosome of human sperm during freezing and thawing : a pilot trial. Arch. Androl., 17:145-150 https://doi.org/10.3109/01485018608990187
  14. Olar TT. 1984 Cryopreservation of dag spermatozoa. MS Thesis, Colorado University, Fort Collins, 158
  15. Pena AI, Barrio F, Quintela LA and Herradon PG. 1998. Effect of different glycerol treatment on frozen-thawed dog sperm longevity and aerosomal integrity. Therigenology, 50: 163-174 https://doi.org/10.1016/S0093-691X(98)00122-8
  16. Pena AI, Barrio F, Quintela LA and Herradon PG. 1998. Effects of sodium dodecyl Sulphate on post-thaw dag semen quality during in vitro incubation at 39°C and 22 °C. Reprod. Dam. Anim., 33:393-398 https://doi.org/10.1111/j.1439-0531.1998.tb01377.x
  17. Pena A, Johannisson A and Linde-Forsberg C. 1999. Post-thaw evaluation of dog spermatozoa using a new triple fluorescent staining and flow cytometry. Theriogenology, 52:965-980 https://doi.org/10.1016/S0093-691X(99)00186-7
  18. Pena A and Linde-Forsberg C. 2000. Effect of equex, one-or two-step dilution, and two freezing and thawing rates on post-thaw survival of dog spermatozoa. Theriogenology, 54:859-875 https://doi.org/10.1016/S0093-691X(00)00397-6
  19. Rota A, Strom B, Linde-Forsberg C and Rodriguez- Martinez H. 1997. Effects of Equex STM Paste on in vitro viability of frozen-thawed dog spermatozoa subjected to a thermoresistance test. Theriogenology, 47:1093-1101 https://doi.org/10.1016/S0093-691X(97)00066-6
  20. Rota A, Iguer-Ouada M, Verstegen J and Lindeforsberg C. 1999. Fertility after vaginal or uterine deposition of dog semen frozen in a tris extender with or without equex stm paste. Therigenology, 51:1045-1058 https://doi.org/10.1016/S0093-691X(99)80010-7
  21. Smith FO. 1994. Cryopreservation of Canine Semen: Technique and Performance. Proc 10 th Int. Congr. Anim. Reprod., 2:216. (abstr)
  22. Seager SWJ. 1969. Successful pregnancies utilizing frozen dog semen. AI Digest.,17:6-7
  23. Strom B, Rota A and Linde-Forsberg C. In vitro characteristics of canine spermatozoa subjected to two methods of cryoperservation. Theriogenology, 1997, 48:247-256 https://doi.org/10.1016/S0093-691X(97)84072-1
  24. Szasz F, Gabor G and Solti L. 2000. Comparative study of different methods for dog semen cryopreservation and testing under clinical conditions. Acta. Vet. Hung., 48:325-333 https://doi.org/10.1556/AVet.48.2000.3.9
  25. Yubi AC. 1984. Investigations of dog semen with particular reference to freezing techniques. MVM thesis, Faculty of Veterinary Medicine. University of Glasgow
  26. 이영락, 이성림, 강태영, 최상용. 2003. 개 정자의 동결융해 후 생존성 및 첨체의 변화. 한국수정란이식학회지, 18: 51-59
  27. 유대중, 정수룡, 오인석, 김홍률, 이계웅, 조성균, 배인휴, 양철주, 공일근. 2002. Tris-buffer에 첨가되는 당의 종류가 동결. 융해정자의 운동성에 미치는 영향. 한국수정란이식학회지, 17:137-143
  28. 정정란, 유재규, 양성열, 여현진, 박종식, 예은하, 노규진, 최상용. 2001. 개정자의 보존방법에 따른 첨체 및 생존성의 변화 ll. 동결 보존에 따른 효과 한국수정란이식학회지, 16:133-138