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Effects of Meiotic Stages, Cryoprotectants, Cooling and Vitrification on the Cryopreservation of Porcine Oocytes

  • Huang, Wei-Tung (Laboratory of Marine Molecular Biology and Biotechnology, Institute of Zoology) ;
  • Holtz, Wolfgang (Institute of Animal Husbandry and Genetics, University of Goettingen)
  • Received : 2001.07.24
  • Accepted : 2001.11.27
  • Published : 2002.04.01

Abstract

Different factors may affect the sensitivity of porcine oocytes during cryopreservation. The effect of two methods (cooling and vitrification), four cryoprotectants [glycerol (GLY), 1, 2-propanediol (PROH), dimethyl sulfoxide (DMSO) or ethylene glycol (EG)] and two vitrification media (1 M sucrose (SUC)+8 M EG; 8 M EG) on the developmental capacity of porcine oocytes at the germinal vesicle (GV) stage or after IVM at the metaphase II (M II) stage were examined. Survival was assessed by FDA staining, maturation and cleavage following IVF and IVC. A toxicity test for different cryoprotectants (GLY, PROH, DMSO, EG) was conducted at room temperature before cooling. GV and M II-oocytes were equilibrated stepwise in 1.5 M cryoprotectant and diluted out in sucrose. The survival rate of GV-oocytes in the GLY group was significantly lower (82%, p<0.01) than that of the other group (92 to 95%). The EG group achieved a significantly higher maturation rate (84%, p<0.05) but a lower cleavage rate (34%, p<0.01) than the DMSO group and the controls. For M II-oocytes, the survival rates for all groups were 95 to 99% and the cleavage rate of the GLY group was lower than the PROH-group (21 vs 43%, p<0.01). After cooling to $10^{\circ}C$, the survival rates of GV-oocytes in the cryoprotectant groups were 34 to 51%, however, the maturation rates of these oocytes were low (1%) and none developed after IVF. For M II-oocytes, the EG group showed a significantly higher survival rate than those of the other cryoprotectant groups (40% vs 23-26%, p<0.05) and the cleavage rates of PROH, DMSO and EG group reached only 1 to 2%. For a toxicity test of different vitrification media, GV and M II-oocytes were equilibrated stepwise in 100% 8 M EG (group 1) and 1 M SUC + 8 M EG (group 2) or equilibrated in sucrose and then in 8 M EG (SUC+8 M EG, group 3). For GV-oocytes, the survival, maturation and cleavage rates of Group 1 were significantly lower than those in group 2, 3 and control group (p<0.05). For M II-oocytes, there were no differences in survival, maturation and cleavage rates between groups. After vitrification, the survival rates of GV and M II-oocytes in group 2 and 3 were similarly low (4-9%) and none of them matured nor cleaved after in vitro maturation, fertilization and culture. In conclusion, porcine GV and M II-oocytes do not seem to be damaged by a variety of cryoprotectants tested, but will succumb to a temperature decrease to $10^{\circ}C$ or to the process of vitrification, regardless of the cryoprotectant used.

Keywords

Cryopreservation;Cryoprotectants;Porcine Oocytes;Cooling;Vitrification

References

  1. Arav, A., B. Rubinsky, E. Seren, J. F. Roche and M. P. Boland. 1994. The role of thermal hysteresis proteins during cryopreservation of oocytes and embryos. Theriogenology 41:107-112. https://doi.org/10.1016/S0093-691X(05)80055-X
  2. Didion, B. A., D. Pomp. M. J. Martin, G. E. Homanics and C. L. Markert. 1990. Observation on the cooling and cryopreservation of pig oocytes at the germinal vesicle stage. J. Anim. Sci. 68:2803-2810. https://doi.org/10.2527/1990.6892803x
  3. Dobrinsky, J. R., C. N. Graves and L. A. Johnson. 1995. Effect of cryopreservation on the viability of porcine oocytes. Cryobiology 32:578-579(Abstr.).
  4. Fuku, E., L. Xia and B. R. Downey. 1993. Ultrastructure changes in bovine oocytes following vitrification. Cryobiology 30:634(Abstr.).
  5. Gustafsson, H., B. Larsson, M. Shamsuddin, U. Jaakma and U. Emanuelson. 2001. Factors affecting the survival of frozen thawed bovine in vitro produced blastocysts. Asian-Aust. J. Anim. Sci. 14:7-12. https://doi.org/10.5713/ajas.2001.7
  6. Huang, W. T., P. C. Tang, S. C. Wu, S. P. Cheng and J. C. Ju. 2001. Effects of levels and sources of follicular fluid on the in vitro maturation and development of porcine oocytes. Asian-Aust. J. Anim. Sci. 14:1360-1366. https://doi.org/10.5713/ajas.2001.1360
  7. Le Gal, F., P. Gasqui and J. P. Renard. 1994. Differential osmotic behavior of mammalian oocytes before and after maturation: a quantitative analysis using goat oocytes as a model. Cryobiology 31:154-170. https://doi.org/10.1006/cryo.1994.1019
  8. Nagashima, H., N. Kashiwazaki, R. J. Ashman and C. G. Grupen and M. B. Nottle. 1995. Cryopreservation of porcine embryos. Nature 374:416. https://doi.org/10.1038/374416a0
  9. Nagashima, H., N. Kashiwazaki, R. Ashman, C. Grupen, R. F. Seamark and M. Nottle. 1994a. Recent advances in cryopreservation of porcine embryos. Theriogenology 41:113-118. https://doi.org/10.1016/S0093-691X(05)80056-1
  10. Parks, J. E. and N. A. Ruffing. 1992. Factors affecting low temperature survival of mammalian oocytes. Theriogenology 37:59-73. https://doi.org/10.1016/0093-691X(92)90247-O
  11. Polge, C., I. Wilmut and L. E. A. Rowsons. 1974. The low temperature preservation of cow, sheep and pig embryos. Cryobiology 11:560(Abstr.). https://doi.org/10.1016/0011-2240(74)90195-3
  12. Polge, C., L. E. A. Rowson and M. C. Chang. 1966. The effect of reducing the number of embryos during early stages of gestation on the maintenance of pregnancy in the pig. J. Reprod. Fertil. 12:395-397. https://doi.org/10.1530/jrf.0.0120395
  13. Pursel, V. G., R. J. Wall, Jr., C. E. Rexroad, R. E. Hammer and R. L. Brinster. 1985. A rapid whole-mount staining procedure for nuclei of mammalian embryos. Theriogenology 24:687-691. https://doi.org/10.1016/0093-691X(85)90167-0
  14. Schellander, K., J. Peli, F. Schmoll and G. Brem. 1994. Effects of different cryoprotectants and carbohydrates on freezing of matured and unmatured bovine oocytes. Theriogenology 42:909-915. https://doi.org/10.1016/0093-691X(94)90114-X
  15. Vincent, C., V. Garnier, Y. Heyman and J. P. Renard. 1989. Solvent effects on cytoskeletal organization and in-vivo survival after freezing of rabbit oocytes. J. Reprod. Fertil. 87:809-820. https://doi.org/10.1530/jrf.0.0870809
  16. Le Gal, F. and A. Massip. 1999. Cryopreservation of cattle oocytes: effects of meiotic stages, cycloheximide treatment, and vitrification procedures. Cryobiology 38:290-300. https://doi.org/10.1006/cryo.1999.2172
  17. Whittingham, D. G. 1977. Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at - $196^{\circ}C$. J. Reprod. Fertil. 49:89-94. https://doi.org/10.1530/jrf.0.0490089
  18. Chen, L. R., W. Y. Huang, Y. S. Luoh and M. C. Wu. 1995. Cryopreservation of porcine oocytes before and after polar body formation by antifreeze protein type III. J. Taiwan Livestock Res. 28:169-179.
  19. Otoi, T., S. Tachikawa, S. Kondo and T. Suzuki. 1992. Development capacity of bovine oocytes cryopreserved after maturation in vitro and of frozen-thawed bovine embryos derived from frozen mature oocytes. Theriogenology 38:711-719. https://doi.org/10.1016/0093-691X(92)90033-N
  20. Parkening, T. A., Y. Tsunoda and M. C. Chang. 1976. Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozenthawed mouse eggs. J. Exp. Zool. 197:369-374. https://doi.org/10.1002/jez.1401970310
  21. Suzuki, T., A. Boediono, M. Takagi, S. Saha and C. Sumantri. 1996. Fertilization and development of frozen-thawed germinal vesicle bovine oocytes by a one-step dilution method in vitro. Cryobiology 33:515-524. https://doi.org/10.1006/cryo.1996.0055
  22. Hochi, S., T. Fujimoto, Y. H. Choi, J. Braun and N. Oguri. 1994. Cryopreservation of equine oocytes by 2-step freezing. Theriogenology 42:1085-1094. https://doi.org/10.1016/0093-691X(94)90856-7
  23. Martino, A., N. Songsasen and S. P. Leibo. 1996. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol. Reprod. 54:1059-1069. https://doi.org/10.1095/biolreprod54.5.1059
  24. Ruffing, N. A., P. L. Steponkus, R. E. Pitt and J. E. Parks. 1993. Osmometric behavior, hydraulic conductivity, and incidence of intracellular ice formation in bovine oocytes at different developmental stages. Cryobiology 30:562-580. https://doi.org/10.1006/cryo.1993.1059
  25. Graves, C. N., J. R. Dobrinsky and L. A. Johnson. 1995. Effect of cooling on the viability of porcine oocytes. Cryobiology 32:578(Abstr.).
  26. Chen, C. 1986. Pregnancy after human oocytes cryopreservation. Lancet 1:884-886. https://doi.org/10.1016/S0140-6736(86)90989-X
  27. Cheng, W. T. K. 1985. In vitro fertilization of farm animal oocytes. Institute of Animal Physiology. Cambridge England, Ph. D. Thesis. pp. 1-177.
  28. Dobrinsky, J. R., C. R. Long and L. A. Johnson. 1997. Stability of microfilaments during swine embryo cryopreservation. Theriogenology 47:343(Abstr.). https://doi.org/10.1016/S0093-691X(97)82470-3
  29. Nagashima, H., N. Kashiwazaki, R. J. Ashman, C. G. Grupen, R. E. Seamark and M. B. Nottle. 1994b. Removal of cytoplasmic lipid enhance the tolerance of porcine embryos to chilling. Biol. Reprod. 51:618-622. https://doi.org/10.1095/biolreprod51.4.618
  30. Otoi, T., S. Ogura, S. Tachikawa, S. Kitamura and T. Suzuki. 1993. Deep freezing of bovine oocytes using different cryoprotectants. Theriogenology 39:275(Abstr.). https://doi.org/10.1016/0093-691X(93)90130-W
  31. Miyamoto, H., E. Sato and T. Ishibashi. 1988. Attempts to cool pig oocytes. Japanese J. Zootech. Sci. 59:329-334.
  32. Wu, M. C. and H. M. Lee. 1996. Vitrification of porcine oocytes. J. Chinese Soc. Anim. Sci. 25:35-51.
  33. Leibo, S. P., A. Martino, S. Kobayashi and J. W. Pollard. 1996. Stage-dependent sensitivity of oocytes and embryos to low temperatures. Anim. Reprod. Sci. 42:45-53. https://doi.org/10.1016/0378-4320(96)01543-6
  34. Nakagata, N. 1993. Production of normal young following transfer of mouse embryos obtained by in vitro fertilization between cryopreserved gametes. J. Reprod. Fertil. 99:77-80. https://doi.org/10.1530/jrf.0.0990077
  35. Rubinsky, B., A. Arav and A. L. DeVries. 1992. The cryoprotective effect of antifreeze glycopeptides from antarctic fishes. Cryobiology 29:69-79. https://doi.org/10.1016/0011-2240(92)90006-N
  36. Lim, J. M., Y. Fukui, and H. Ono. 1992. Developmental competence of bovine oocytes frozen at various maturation stages followed by in vitro maturation and fertilization. Theriogenology 37:351-361. https://doi.org/10.1016/0093-691X(92)90193-U
  37. Niemann, H., A. Lucas-Hahn and C. Stoffregen. 1985. Sensitivity of pig morulae to DMSO/PVP or glycerol treatment and cooling to $10^{\circ}C$. Theriogenology 23:213(Abstr.). https://doi.org/10.1016/0093-691X(85)90119-0
  38. Al-Hasani, S., K. Kirsch, J. Diedrich, S. Blanke, H. Van der Ven and D. Krebs. 1989. Successful embryo transfer of cryopreserved and in vitro fertilized rabbit oocytes. Hum. Reprod. 4:77-79.
  39. Fuku, E., T. Kojima, Y. Shioya, G. J. Marcus and B. R. Downey. 1992. In vitro fertilization and development of frozen-thawed bovine oocytes. Cryobiology 29:485-492. https://doi.org/10.1016/0011-2240(92)90051-3
  40. Moor, R. M. and I. M. Crosby. 1985. Temperature-induced abnormalities in sheep oocytes during maturation. J. Reprod. Fertil. 75:467-473. https://doi.org/10.1530/jrf.0.0750467
  41. Arav, A., Y. Zeron, , S. B. Leslie, E. Behboodi, G. B. Anderson and J. H. Crowe. 1996. Phase transition temperature and chilling sensitivity of bovine oocytes. Cryobiology 33:589-599. https://doi.org/10.1006/cryo.1996.0062
  42. Rall, W. F. 1992. Cryopreservation of oocytes and embryos:methods and application Anim. Reprod. Sci. 28:237-245. https://doi.org/10.1016/0378-4320(92)90110-Y
  43. Rall, W. F. 1987. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 24:387-402. https://doi.org/10.1016/0011-2240(87)90042-3
  44. Nagashima, H., M. Kuwayama, C. G. Grupen, R. J. Ashman and M. B. Nottle. 1996. Vitrification of porcine early cleavage stage embryos and oocytes after removal of cytoplasmic lipid droplets. Theriogenology 45:180(abstr.). https://doi.org/10.1016/0093-691X(96)84653-X
  45. Wilmut, I. 1972. The low temperature preservation of mammalian embryos. J. Reprod. Fertil. 31:513-514. https://doi.org/10.1530/jrf.0.0310513
  46. Van Uem, J. F. H. M., E. R. Schuh, B. Siebzehnruebl, R. Koch, S. Trotnow and N. Lang. 1987. Birth after cryopreservation of unfertilized oocytes. Lancet 2:752-753. https://doi.org/10.1016/S0140-6736(87)90398-9
  47. 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

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