Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
권호 표지

Effects of Kinds and Concentrations of Cryoprotectants, PVP on Survival Rate of Vitrified Porcine Embryos

내동제의 종류와 농도, PVP 첨가가 돼지 수정란의 Vitrification 동결 융해 시 생존율에 미치는 영향

  • Lim J. G. (College of Agriculture and Life Science, Chungnam National University) ;
  • Quan J. H. (Colege of Veterinary Medicine, Chungnam National University) ;
  • Lee K. S. (College of Agriculture and Life Science, Chungnam National University) ;
  • Kim S. K. (Colege of Veterinary Medicine, Chungnam National University)
  • Published : 2005.08.01
Download PDF
⟨ Previous Next ⟩

Abstract

The present study examines the effects of kinds and concentrations of cryoprotectants, PVP and sucrose and trehalose on the survival rate of vitrified porcine embryos. 1. The developmental stages for the embryos used in vitrification were $245(23.0\%)$ for 2 cell stage, $256(24.1\%)$ for the blastocyst, $234(22.0\%)$ for the early blastocyst $221(20.8\%)$ from the expanded blastocyst and $107(10.1\%)$ from hatching blastocyst out of 1,063 embryos. 2 The survival rate of morula, early blastocyst and expanded blastocyst vitrification-thawed with EDT and EGS were $69.1\%,\;70.3\%,\;69.8\%\;and\;62.5\%,\;61.7\%,\;63.6\%$, respectively. The expanded blastocyst treated with EDS showed the highest survival rate compared with the other cryoprotectants. 3. The survival rate of early blastocyst, expanded blastocyst and hatching blastocyst vitrification-thawed with EDS diluted in $medium + 10\%$ FCS were $61.1\%,\;27.8\%,\;16.7\%$, respectively. This result were love. than the control of group $(92.3\%,\;71.2\%,\; 55.8\%)$. 4. The survival rate of embryos vitrified with EDS and EDT supplemented with $10\%\;and\;20\%$ PVP were $74.3\%,\;77.5\%\;and\;79.4\%,\;71.1\%$, respectively. The survival rate of vitrified embryos cultured for $24\~48$ hours were $37.1\%,\; 40.0\%\;and\;35.3\%,\;31.6\%$ which were significantly lower than that of non-cultured embryos. The survival rate of embryos vitrified with EDS and EDT supplemented between $10\%\;or\;20\%$ PVP did not have a significant difference. 5. The survival rate of embryos vitrification-thawed with EDS to morula, early blastocyst, expanded blastocyst and hatching blastocyst were $58.2\%,\; 36.4\%,\;14.5\%$ to morula, $62.5\%,\;45.8\%,\;20.8\%$ to early blastocyst, $74.1\%,\;61.1\%,\;29.6\%$ to expanded blastocyst and $60.0\%,\;40.0\%,\;14.0\%$ to hatching blastocyst.

본 연구는 돼지 수정란의 동결에 있어서 vitrification 동결 융해 후 내동제의 종류완 농도, PVP 및 sucrse와 trehalose의 첨가가 생존율에 미치는 영향을 조사하고자 수행하였다. 1 Vitrification동결에 이용된 각 발생단계의 체외수정란은 1,063개 중 2세포기는 245$(23.0\%)$개, 배반포는 $256(24.1\%)$, 초기 배반포는 $234 (22.0\%)$, 확장 배반포는 221개 $(20.8\%)$, hatching 배반포는 107개 $(10.1\%)$이었다. 상실배, 초기 배반포 및 확장배반포를 EDS와 ETS로 희석 후 vitrification동결 융해했을 때 생존율은 각각 $69.1\%,\;70.3\%,\;69.8\%$ 및 $62.5\%,\;61.7\%,\;63.6\%$로서 EDS군에서 확장 배반포군에서 가장 높은 생존율을 나타냈다. 2. 각 발생단계의 수정란을 vitrification동결 융해했을 때 생존율은 초기 배반포는 $61.1\%$, 확장 배 반포는 $27.8\%$, hatching 배 반포는 $16.7\%$로서 대조군의 $92.3\%,\;71.2\%,\;55.8\%$에 비해 낮았지만 높은 생존율을 나타냈다. 3. 수정란을 EDS와 EDT내동제에 $10\%$ 및 $20\%$ PVP 액을 첨가하석 희석 후 vitrification 동결 융해했을 때 정상적 발생을 나타내는 수정란은 $74.3\%,\;77.5\%$ 및 $79.4\%$ 및 $71.1\%$였다. 동결 융해한 수정란을 $24\~48$시간 배양했을 때 $37.1\%,\;40.0\%$ 및 $35.3\%,\;31.6\%$로서 생존율이 현저하게 감소하였다. 수정란에 EDS와 EDT와 $10\%$ 및 $20\%$의 PVP를 첨가한 내동제를 이용하여 동결 응해했을 때 PVP농도간의 생존율은 유의한 차이가 없었다. 4. 각 발생단계의 수정란을 EDS 내동제로 vitrification 동결 융해 후 배양했을 때 발생율은 상실배는 $58.2\%,\;36.4\%,\;14.5\%$였고, 초기 배반포는 $62.5\%,\;45.8\%,\;20.8\%$였고, 확장 배반포는 $74.1\%,\;61.1\%,\;29.6\%$였고, hatching 배반포는 $60.0\%,\;40.0\%,\;14.0\%$였다.

Keywords

References

  1. Candy CJ, Wood M, Whittingham DG, Merriman JA and Choudhury N. 1994. Cryopreservation of immature mouse oocytes. Hum. Reprod., 9:1738-1742 https://doi.org/10.1093/oxfordjournals.humrep.a138785
  2. Clark P, Rahy GM and Karow Jr AM. 1984. Factor influencing renal cryopreservation. II. Toxic effects of three cryopreservation in combination with three vehicle solutions in non-frozen rabbit cortical slices. Cryobiology, 21:260-273 https://doi.org/10.1016/0011-2240(84)90322-5
  3. Cuello C, Sntonia MG, Parrilla I, Tomei J, Vazquez JM, Roca J, Berthelot F, Martinat-Botte F and Martinez EA. 2004. Vitrification of porcine embryos at various development stages using different ultra-rapid cooling procedures. Theriogenology, 62:353-361 https://doi.org/10.1016/j.theriogenology.2003.10.007
  4. Dinnyes A, Wallace GA and Rail WF. 1995. Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification of slow freezing methods. Mol. Reprod. Dev., 40:429-435 https://doi.org/10.1002/mrd.1080400406
  5. van der Elst JC, Nerinckx SS and van Steirteghem AC. 1993. Slow and ultrarapid freezing of fully grown germinal vesicle-stage mouse oocytes: optimization of survival rate outweighted by defective blastocyst formation. J. Assist. Reprod. Gene, 10:202-212 https://doi.org/10.1007/BF01239222
  6. Fahy GM, MacFarlane DR, Angell CA and Meryman HT. 1984. Vitrification as an approach to cryopreservation. Cryobiology, 21:407-426 https://doi.org/10.1016/0011-2240(84)90079-8
  7. Holm P, Vajita G, Machaty Z, Schmidt, Prather RS and Grve T. 1999. Open pulled straw (OPS) vitrification of porcine blastocysts; simple procedure yielding excellent in vitro survival, but so far no piglets following transfer. Cryo-Letters, 20:307-310
  8. Ishimori H, Saeki K, Inai M, Naga Y, Itasaka J, Miki Y, Seike N and Kainuma H. 1993. Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide. Therigenology, 40:427-433 https://doi.org/10.1016/0093-691X(93)90279-E
  9. Iwasaki S, Yoshikane Y, Watanabe S and Nakahara T. 1994. Effect of freezing of bovine preimplantation embryos derived from oocytes fertilized in vitro on survival of their inner cells. Mol. Reprod. Dev., 37:272-275 https://doi.org/10.1002/mrd.1080370305
  10. Kasai M, Komi JH, Takakamo A, Tssnoda H,. Sakurai T and Machida T. 1990. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J. Reprod. Fertil., 89:91-97 https://doi.org/10.1530/jrf.0.0890091
  11. Kuwayama M, Holm P, Jacobsen H, Greve T and Callesen H. 1997. Successful cryopreservation of porcine embryos by vitrification. Vet. Rec. Absr., 4:365
  12. Lane M, Forest KT, Lyons EA and Bavister BD. 1999. Live births following vitrification of hamster embryos using a novel containerless techniques. Theriogenology Abst., 51:167 https://doi.org/10.1016/S0093-691X(99)91726-0
  13. Leibo SP and Oda K. 1993. High survival of mouse zygote and embryos cooled rapidly or slowly in ethylene glycol plus polyvinylpyrrolidone. Cryo-Letters, 14:133-144
  14. Mahmoudzadeh AR, van Soom A, van Vlaenderen I and de Kruif A. 1993. A comparative study of the effect of one-step addition of different vitrification solutions on in vitro survival of vitrified bovine embryos. Theriogenology, 39: 1291-1302 https://doi.org/10.1016/0093-691X(93)90231-S
  15. Massip A, Mermilod P, Wilis C and Dessy F. 1993. Effects of dilution procedure and culture conditions after thawing on survival of frozen bovine blastocysts produced in vitro. J. Reprod. Fert., 97:65-69 https://doi.org/10.1530/jrf.0.0970065
  16. Rall WF and 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
  17. Rall WF and Wood MJ. 1994. High in vitro and in vivo survival of day 3 mouse embryos vitrified or frozen in a non-toxic solution of glycerol and albumin. J. Reprod. Fertil., 101:681-688 https://doi.org/10.1530/jrf.0.1010681
  18. Saha S, Otoi T and Suzuki T. 1996. The efficiency of ethylene glycol, trehalose and polyvinylpyrrolidone for successful vitrification of IVF bovine embryos. J. Reprod. Dev., 42:163-169 https://doi.org/10.1262/jrd.42.163
  19. Saito N, Imai K and Tomizawa M. 1994. Effect of sugar-addition on the survival of vitrified bovine blastocysts produced in vitro. Theriogenology, 41:1053-1063 https://doi.org/10.1016/S0093-691X(05)80028-7
  20. Schilling E, Niemann H and Smidt D. 1982. Evaluation of fresh and frozen cattle embryos by fluorescence microscopy. Cryobiology, 15:245-248 https://doi.org/10.1016/0011-2240(78)90034-2
  21. Sutton RL. 1992. Critical cooling rates for aqueous cryoprotectants in the presence of sugars and polysaccharides. Cryobiology, 29:585-598 https://doi.org/10.1016/0011-2240(92)90063-8
  22. Tachikawa S, Otoi T, Kondo S, Machida T and Kasai M. 1993. Successful vitrification of bovine blastocyst, derived by in vitro maturation and fertilization. Mol. Reprod. Dev., 34:266-271 https://doi.org/10.1002/mrd.1080340306
  23. Takai M, Otoi T, Boediono A, Saha S and Suzuki T. 1994. Viability of frozen-thawed bovine IVM/IVF embryos in relation to aging using various cryoptotectants. Theriogenology, 41:915-921 https://doi.org/10.1016/0093-691X(94)90507-F
  24. Toth TL, Jones HW, Baka SC, Muasher S, Veeck LL and Lanzendorf SE. 1994. Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil. Steril., 61:891-894 https://doi.org/10.1016/S0015-0282(16)56702-8
  25. Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen A, Greve T and Callesen H. 1998a. Open pulled straw(OPS) vitrification : a new way to reduce cryoinjuries of bovine ova and embryos. Mol. Reprod. Dev., 51:53-58 https://doi.org/10.1002/(SICI)1098-2795(199809)51:1<53::AID-MRD6>3.0.CO;2-V
  26. Vajta G, Lewis IM, Kuwayama M, Greve T and Callesen H. 1998b. Sterile application of the open pulled straw(OPS) vitrification method. Cryo-Letters, 19:389-392
  27. Utsumi K, Hochi S and Iritani A. 1982. Cryopretective effect of polyols on rat embryos during two-step freezing. Cryobiology, 29:332-341 https://doi.org/10.1016/0011-2240(92)90034-Y
  28. Voelkel SA and Hu YX. 1992. use of ethylene glycol as a cryoprotectant for bovine embryos allowing direct transfer of frozen thawed embryos to recipient females. Theriogenology, 37:687-697 https://doi.org/10.1016/0093-691X(92)90148-K