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Efficient Cryopreservation of Porcine Blastocysts produced by In Vitro Fertilization

  • Received : 2016.02.16
  • Accepted : 2016.03.03
  • Published : 2016.03.31

Abstract

Cryopreservation has been applied successfully in many mammalian species. Nevertheless, pig embryos, because of their greater susceptibility to cryoinjuries, have shown a reduced developmental competence. The aim of this study was to evaluate the survival status of vitrified-warmed porcine embryos. Forced blastocoele collapse (FBC) and non-FBC blastocysts are vitrified and concomitantly cultured in culture media which were supplemented with/without fetal bovine serum (FBS). Porcine vitrified-warmed embryos were examined in four different methods: group A, non-FBC without FBS; group B, non-FBC with FBS; group C, FBC without FBS; group D, FBC with FBS. After culture, differences in survival rates of blastocysts derived from vitrified-warmed porcine embryos were found in group A~D (39.5 (A) vs 52.5 (B) and 54.8 (C) vs 66.7% (D), respectively, p<0.05). Reactive oxygen species (ROS) level of survived blastocysts was lower in group D than that of another groups (p<0.05). Moreover, total cell number of survived blastocysts was higher in group D than that of other groups (p<0.05). Otherwise, group D showed significantly lower number of apoptotic cells than other groups ($2.0{\pm}1.5$ vs $3.2{\pm}2.1$, $2.8{\pm}1.9$, and $2.7{\pm}1.6$, respectively, p<0.05). Taken together, these results showed that FBS/FBC improves the developmental competence of vitrified porcine embryos by modulating intracellular levels of ROS and the apoptotic index during the vitrification/warming procedure. Therefore, we suggest that FBS and FBC are effective treatment techniques during the vitrification/warming procedures of porcine blastocysts.

Keywords

References

  1. Abeydeera LR and Day BN. 1997. Fertilization and subsequent development in vitro of pig oocytes inseminated in a modified tris-buffered medium with frozen-thawed ejaculated spermatozoa. Biol. Reprod. 57:729-734. https://doi.org/10.1095/biolreprod57.4.729
  2. Choi J, Park SM, Lee E, Kim JH, Jeong YI, Lee JY, Park SW, Kim HS, Hossein MS, Jeong YW, Kim S, Hyun SH and Hwang WS. 2008. Anti-apoptotic effect of melatonin on preimplantation development of porcine parthenogenetic embryos. Mol. Reprod. Dev. 75:1127-1135. https://doi.org/10.1002/mrd.20861
  3. Crow JP. 1997. Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: Implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide : Biology and Chemistry/Official Journal of the Nitric Oxide Society 1:145-157. https://doi.org/10.1006/niox.1996.0113
  4. Cuello C, Gil MA, Alminana C, Sanchez-Osorio J, Parrilla I, Caballero I, Vazquez JM, Roca J, Rodriguez-Martinez H and Martinez EA. 2007. Vitrification of in vitro cultured porcine two-to-four cell embryos. Theriogenology 68:258-264. https://doi.org/10.1016/j.theriogenology.2007.05.039
  5. Cuello C, Sanchez-Osorio J, Alminana C, Gil MA, Parrilla I, Roca J, Vazquez JM, Martinez EA and Rodriguez-Martinez H. 2010. Superfine open pulled straws vitrification of porcine blastocysts does not require pretreatment with cytochalasin B and/or centrifugation. Reprod. Fert. Dev. 22:808-817. https://doi.org/10.1071/RD09160
  6. Du Y, Zhang Y, Li J, Kragh PM, Kuwayama M, Ieda S, Zhang X, Schmidt M, Bogh IB, Purup S, Pedersen AM, Villemoes K, Yang H, Bolund L and Vajta G. 2007. Simplified cryopreservation of porcine cloned blastocysts. Cryobiology 54:181-187. https://doi.org/10.1016/j.cryobiol.2007.01.001
  7. Fujino Y, Kojima T, Nakamura Y, Kobayashi H, Kikuchi K and Funahashi H. 2008. Metal mesh vitrification (MMV) method for cryopreservation of porcine embryos. Theriogenology 70:809-817. https://doi.org/10.1016/j.theriogenology.2008.05.045
  8. Funahashi H, Cantley TC, Stumpf TT, Terlouw SL and Day BN. 1994. In vitro development of in vitro-matured porcine oocytes following chemical activation or in vitro fertilization. Biol. Reprod. 50:1072-1077. https://doi.org/10.1095/biolreprod50.5.1072
  9. Gomez E, Rodriguez A, Munoz M, Caamano JN, Hidalgo CO, Moran E, Facal N and Diez C. 2008. Serum free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification. Theriogenology 69:1013-1021. https://doi.org/10.1016/j.theriogenology.2007.12.015
  10. Hao Y, Lai L, Mao J, Im GS, Bonk A and Prather RS. 2004. Apoptosis in parthenogenetic preimplantation porcine embryos. Biol. Reprod. 70:1644-1649. https://doi.org/10.1095/biolreprod.103.026005
  11. Hiraoka K, Hiraoka K, Kinutani M and Kinutani K. 2004. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Hum. Reprod. 19:2884-2888. https://doi.org/10.1093/humrep/deh504
  12. Khalil WA, Marei WF and Khalid M. 2013. Protective effects of antioxidants on linoleic acid-treated bovine oocytes during maturation and subsequent embryo development. Theriogenology 80:161-168. https://doi.org/10.1016/j.theriogenology.2013.04.008
  13. Kuwayama M. 2007. Highly efficient vitrification for cryopreservation of human oocytes and embryos: The Cryotop method. Theriogenology 67:73-80. https://doi.org/10.1016/j.theriogenology.2006.09.014
  14. Lee SY, Kim HJ, Park SJ, Yoon HJ, Yoon SH, Lee KH, Lee WD and Lim JH. 2006. Optimization of a dilution method for human expanded blastocysts vitrified using EM grids after artificial shrinkage. J. Assisted Reprod. Gene 23:87-91. https://doi.org/10.1007/s10815-005-9006-0
  15. Li L, Zhang X, Zhao L, Xia X and Wang W. 2012. Comparison of DNA apoptosis in mouse and human blastocysts after vitrification and slow freezing. Mol. Reprod. Dev. 79:229-236. https://doi.org/10.1002/mrd.22018
  16. Makarevich AV and Markkula M. 2002. Apoptosis and cell proliferation potential of bovine embryos stimulated with insulin-like growth factor I during in vitro maturation and culture. Biol. Reprod. 66:386-392. https://doi.org/10.1095/biolreprod66.2.386
  17. Men H, Agca Y, Critser ES and Critser JK. 2005. Beneficial effects of serum supplementation during in vitro production of porcine embryos on their ability to survive cryopreservation by open pulled straw vitrification. Theriogenology 64:1340-1349. https://doi.org/10.1016/j.theriogenology.2005.02.013
  18. Min SH, Lee E, Son HH, Yeon JY and Koo DB. 2013. Forced collapse of the blastocoel enhances survival of cryotop vitrified bovine hatching/hatched blastocysts derived from in vitro fertilization and somatic cell nuclear transfer. Cryobiology 66:195-199. https://doi.org/10.1016/j.cryobiol.2013.01.004
  19. Min SH, Song BS, Yeon JY, Kim JW, Bae JH, Park SY, Lee YH, Chang KT and Koo DB. 2014. A cathepsin B inhibitor, E-64, improves the preimplantation development of bovine somatic cell nuclear transfer embryos. J. Reprod. Dev. 60:21-27. https://doi.org/10.1262/jrd.2013-074
  20. Mito T, Yoshioka K, Noguchi M, Yamashita S, Misumi K, Hoshi T and Hoshi H. 2015. Birth of piglets from in vitroproduced porcine blastocysts vitrified and warmed in a chemically defined medium. Theriogenology 84:1314-1320. https://doi.org/10.1016/j.theriogenology.2015.06.024
  21. Mucci N, Aller J, Kaiser GG, Hozbor F, Cabodevila J and Alberio RH. 2006. Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification. Theriogenology 65:1551-1562. https://doi.org/10.1016/j.theriogenology.2005.08.020
  22. Mukaida T, Oka C, Goto T and Takahashi K. 2006. Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum. Reprod. 21:3246-3252. https://doi.org/10.1093/humrep/del285
  23. Neuber E, Chan AW, Dominko T, Simerly C and Schatten G. 2002. Cell allocation and cell death in blastocysts from nonhuman primates generated during in vitro fertilization and intracytoplasmic sperm injection. Fert. Steril. 77:1083-1085. https://doi.org/10.1016/S0015-0282(02)02996-5
  24. Pereira DC, Dode MA and Rumpf R. 2005. Evaluation of different culture systems on the in vitro production of bovine embryos. Theriogenology 63:1131-1141. https://doi.org/10.1016/j.theriogenology.2004.06.008
  25. Petters RM and Wells KD. 1993. Culture of pig embryos. J. Reprod. Fertil. Supplement 48:61-73.
  26. Son WY, Yoon SH, Yoon HJ, Lee SM and Lim JH. 2003. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum. Reprod. 18:137-139. https://doi.org/10.1093/humrep/deg029
  27. Sudano MJ, Paschoal DM, Rascado Tda S, Magalhaes LC, Crocomo LF, de Lima-Neto JF and Landim-Alvarenga Fda C. 2011. Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification. Theriogenology 75:1211-1220. https://doi.org/10.1016/j.theriogenology.2010.11.033
  28. Tareq KM, Akter QS, Khandoker MA and Tsujii H. 2012. Selenium and vitamin E improve the in vitro maturation, fertilization and culture to blastocyst of porcine oocytes. J. Reprod. Dev. 58:621-628. https://doi.org/10.1262/jrd.2012-064
  29. Vanderzwalmen P, Bertin G, Debauche C, Standaert V, van Roosendaal E, Vandervorst M, Bollen N, Zech H, Mukaida T, Takahashi K and Schoysman R. 2002. Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum. Reprod. 17:744-751. https://doi.org/10.1093/humrep/17.3.744