Effects of Embryo Developmental Stage and Superoxide Dismutase on the Survival of Frozen-Thawed Porcine Embryos by Open Pulled Straw (OPS) Method

배 발달단계와 Superoxide Dismutase가 Open Pulled Straw(OPS) 방법에 의해 동결-융해한 수정란의 생존성에 미치는 영향

  • Lee Sang-Young (Biotechnology Division, Gyeongsangnam Province Advanced Swine Research Institute) ;
  • Yu Jae-Suck (Biotechnology Division, Gyeongsangnam Province Advanced Swine Research Institute) ;
  • Sa Soo-Jin (College of Animal Life Sciences, Kangwon National University) ;
  • Park Choon-Keun (College of Animal Life Sciences, Kangwon National University)
  • 이상영 (경상남도첨단양돈연구소 생명공학과) ;
  • 유재숙 (경상남도첨단양돈연구소 생명공학과) ;
  • 사수진 (강원대학교 동물생명과학대학) ;
  • 박춘근 (강원대학교 동물생명과학대학)
  • Published : 2006.03.01

Abstract

This study was performed to investigate the effects of embryo developmental stage and superoxide dismutase (SOD) on the survival of frozen-thawed porcine embryos by open pulled straw(OPS) method. Porcine IVF blastocysts were frozen-thawed by OPS method and cultured for 48 h under the existence of SOD. There are no significant differences in the proportions of normal morphology among the early, mid- and expanded blastoryst stages $(30.8{\sim}38.6%)$. After culture of embryos, the developmental rates to the expanded blastocyst stage(38.7%) were significantly higher than those of other stages (P<0.05). The proportions of expanded and hatched embryos were higher in medium with 1 unit/ml SOD than 0 and 10 units/ml of SOD. The result indicates that OPS method can use for the pig embryo cryopreservation, especially for the late stage blastocysts. SOD may can reduce the demage of frozen-thawed porcine embryos.

본 연구는 OPS 기법에 의한 돼지 수정란의 동결-융해 시 수정란의 발달 단계와 superoxide dismutase (SOD)가 수정란의 생존능력에 미치는 영향을 검토하였다. 돼지 체외수정 배반포는 OPS방법에 의해 동결 후 융해하여 $0{\sim}10units/ml$의 SOD 존재 하에 48시간 체외배양하였다. 동결-융해 후 형태학적으로 정상적인 수정란의 비율은 초기, 중기 및 확장배반포간에 유의적인 차이는 인정되지 않았다$(30.8{\sim}38.6%)$. 그러나 발육단계가 높을수록 형태적으로 정상인 수정란의 비율이 높은 경향을 나타냈다. 수정란의 융해 후 48시간 추가 배양했을 때, 발육이 진행된 수정란은 후기배반포기에 동결한 수정란이 38.7%로 유의적으로 높았으며(P<0.05), 1 unit/ml의 SOD를 첨가한 경우 비교적 높은 생존율을 나타내었다. 본 연구의 결과로부터, 수정란의 OPS방법에 의한 동결-융해 후 생존성의 향상을 위해서는 후기배반포기 단계에 동결하는 것이 유리하며, SOD의 첨가는 수정란의 손상을 어느 정도 방지할 수 있을 것으로 사료된다.

Keywords

References

  1. Berthelot F, Martinat-Botte F, Locatelli A, Perreau C, Terqui M (2000): Piglets born after vitrification of embryos usung the open pulled starw method. Cryobiology 41:116-124 https://doi.org/10.1006/cryo.2000.2273
  2. Corsby IM (1988): Control of protein synthesis during cleavage of sheep embryos. J Reprod Fertil 82: 769-775 https://doi.org/10.1530/jrf.0.0820769
  3. Dobrinsky JR (2001): Cryopreservation of swine embryos: A Chilly past with a vitrifying future. Theriogenology 56:1333-1344 https://doi.org/10.1016/S0093-691X(01)00634-3
  4. Dobrinsky JR, Pursel VG, Long CR, Johnson LA (2000): Birth of piglets after in transfer of embryos eryopreserved by cytoskeletal stabilization and vitrification
  5. Hotamisligil S, Toner M, Powers RD (1996): Changes in membrane intergrity, cytoskeletal structure, and developmental potential of murine oocytes after vitrification in ethylene glycol. BioI Reprod 55:161-168 https://doi.org/10.1095/biolreprod55.1.161
  6. Kaidi S, Donnay I, Lambert P, Dessy F, Massip A (2000): Osmotic behavior of in vitro produce bovine blastocysts in cryoprotectant solutions as a potential predictive test of survival. Cryobiology 41 :106-115 https://doi.org/10.1006/cryo.2000.2272
  7. Kim MS, Kim SW, Cheong HT, Lee SY, Yang BK, Kim CI, Park CK (2002): Effect of superoxide dismutase and cryoprotectants on viability of frozenthawed porcine oocytes by vitrification method. Korea J Emb Trans 16:117-126
  8. Kim SW, Park CK, Cheong HT, Yang BK, Kim CI (2001): Survival ability of porcine oocytes frozenthawed by open pulled straw method. Korea J Emb Trans 16:117-126
  9. Lane M, Gardner DK (2001): Vitrification of mouse oocytes using a nylon loop. Mol Reprod Dev 58:342-347 https://doi.org/10.1002/1098-2795(200103)58:3<342::AID-MRD13>3.0.CO;2-X
  10. Lee SY, Park YH, Chung DS, Park CK (2003): Survival ability of pig embryos frozen-thawed by open pulled straw methods. Proc 3rd Int Dev Engineering Cong p. 108
  11. Legge M, Sellens MH (1991): Free radical scavengers ameliorate the 2-cell block in mouse embryo culture. Hum Reprod 6:867-871 https://doi.org/10.1093/oxfordjournals.humrep.a137442
  12. Lewis IM, Lane MW, Vajta G (1999): Pregnancy rate following transfer of in vitro produced bovine embryos vitrified by the open pulled straw(OPS) method. Theriogenology 51:168(Abstr) https://doi.org/10.1016/S0093-691X(99)91727-2
  13. Li J, Foote RH, Simkin ME (1993): Development of rabbit zygotes cultured in protein-free medium with catalase, taurine orsuperoxide dismutase. BioI Reprod 48:33-39 https://doi.org/10.1095/biolreprod48.1.33
  14. Martino A, Songsasen N, Leibo SP (1996): Development into blastocysts of bovine oocytes cryopreserved by ultrarapid cooling. BioI Reprod 54:1059-1069 https://doi.org/10.1095/biolreprod54.5.1059
  15. Murray RK, Granner DK, Mayes PA, Rodwell VA (1990): Lipid peroxidation is a source of free radicals in vivo. Harper's Biochem(eds.) pp. 142-143
  16. Nakagata N (1989): High survival rate of unfertilized mouse oocytes after vitrification. J Reprod Fertil 87:479-483 https://doi.org/10.1530/jrf.0.0870479
  17. Nasr-Esfahani MH, Aitken RJ, Johnson MH (1990): Hydrogen peroxide levels in mouse oocytes and early cleavage stages embryos developed in vitro or in vivo. Development 103:501-507
  18. Park SE, Chung CJ, Son WY, Chung HM, Lee SH, Lee WS, Ko JJ, Yoon TK, Cha KY (1997): Chromosome configurations of human oocytes matured in vitro following cryopreservation at the germinal vesicle stage. Korean J Fertil SteriI 24:253-259
  19. Park SP, Kim EY, Kim DI, Park NH, Won YS, Yoon SH, Chung KS, Lim JH (1999): Simple, efficient and successful vitrification of bovine blastcysts using electron microscope grids. Hum Reprod 14:2838-2843 https://doi.org/10.1093/humrep/14.11.2838
  20. RaIl WF, Fahy GM (1985): Ice-free cryopreservation of mouse embryos at $-196^{\circ}C$ by vitrification. Nature 313:573-575 https://doi.org/10.1038/313573a0
  21. Suzuki T, Boediono A, Takagi M, Sha S, Sumantri C (1996): Fertilization and development of frozenthawed germinal vesicle bovine oocytes by a onestep dilution methods in vitro. Cryobiology 33: 515-524 https://doi.org/10.1006/cryo.1996.0055
  22. Vajta G, Booth PJ, Holm P, Greve T, Callessen H (1997a): Successful vitification of early stage bovine in vitro produced embryos with the open pulled starw (OPS) method. Cryo-Letter 18:191-195
  23. Vajta G, Greve T, Callesen H (1997b): Vitrification of porcine embryos using the open pulled straw (OPS) method. Acta Vet Scand 38:349-352
  24. Vajta G, Lewis IM, Kuwayama M, Greve T, Ca-llesen H (1998): Sterile application of the open pulled straw (OPS) vitrification method. Cryo-Letters 19:389-392
  25. Whittingham DG, Leibo SP, Mazur P (1972): Survival of mouse embryos frozen to $-196^{\circ}C$ and $-269^{\circ}C$. Science 187:411-414 https://doi.org/10.1126/science.11643261
  26. 이상영, 박영호, 도은희, 김형주, 정대석 (2003): 돼지에서 open pulled straw(OPS) 방법에 의해 동결-융해한 수정란의 생존능력. 제19차 한국축산기술협의회 학술발표 논문집 59-74