Korean Journal of Poultry Science (한국가금학회지)

The Korean Society of Poultry Science (한국가금학회)
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Effect of Ethylene Glycol(EG) and Propylene Glycol(PG) on the Viability of Frozen-thawed Primordial Germ Cells(PGCs) on Korean Native Chicken(Ogye) by Vitrification

한국재래닭(오계)의 유리화 동결 시 생존율에 미치는 Ethylene Glycol(EG)과 Propylene Glycol(PG)의 영향

  • Kim, Hyun (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Kim, Dong Hun (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Han, Jae Yong (WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University) ;
  • Choi, Sung Bok (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Ko, Yeoung Gyu (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Do, Yoon Jung (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Seong, Hwan-Hoo (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Kim, Sung Woo (Animal Genetic Resources Station, National Institute of Animal Science, RDA)
  • 김현 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 김동훈 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 한재용 (서울대학교 동물자원과학과) ;
  • 최성복 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 고응규 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 도윤정 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 성환후 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 김성우 (농촌진흥청 국립축산과학원 가축유전자원시험장)
  • Received : 2013.08.06
  • Accepted : 2013.09.03
  • Published : 2013.09.30
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Abstract

This study established a method for preserving chicken primordial germ cells (PGCs) that enables long-term storage in liquid nitrogen ($LN_2$) for preservation of the species. The purpose of this study was to compare the effects of Ethylene Glycol (EG) and Propylene Glycol (PG) on viability of cryopreserved PGCs with vitrification in Korean Native Chicken (Ogye), and to fine should be find or to the optimal protocol for PGCs freezing. One of the important components of cryopreservation process is cryopreservation medium that plays a vital role in preventing cellular injury during freeze-thawing. Cryoprotective agents have been known to improve cell viability after freeze-thawing. PGCs obtained from the germinal gonade of 5.5~6 day (stage 28) chick embryos, using the MACS method were suspended in a freezing medium containing a freezing and protecting agents. Gonads were harvested from stage 28 chick embryos and pooled in groups of 10E embryos, contributing gonads to the cell suspension. The gonadal cells, including PGCs, were then frozen in 1 of the following cryoprotectant treatments: 2.5% EG, 5% EG, 10% EG, 2.5% PG, 5% PG, 10% PG, and 0% cryoprotectant as a control. Effects of exposure to vitrification solution and vitrification, with different concentrations of the cryoprotectant solution, were examined. After freezing and thawing, survival rates of the frozen-thawed PGCs from the 0, 2.5, 5, 10 and 15% EG plus FBS treatment were 44.24%, 64.51%, 85.63%, 80.51% and 73.52% (p<0.05), respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% PG + FBS treatment (p<0.05)(85.63% vs 66.81%). Therefore, these systems may contribute in the improvement of cryopreservation for a scarce species in birds preservation. This study established a method for preserving chicken PGC that enables systematic storage and labeling of cryopreserved PGCs in liquid N at a germplasm repository and ease of entry into a database. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

동결 닭 원시생식세포의 생식계열 키메라를 이용한 생체에의 복원을 실용화하기 위해서는, 닭 원시생식세포의 동결보존기술의 향상에 의해 동결 및 융해 후의 많은 생존세포를 확보하는 것이 반드시 필요하다. 닭 원시생식세포는 배양 5.5일령의 닭 원시생식선으로부터 채취하고, MACS 방법에 의해서 순수 닭 원시생식세포를 분리했다. 15% 각각의 EG를 동결보호제로 사용한 처리군이 각 군의 농도에 상관없이 유의적(p<0.05)으로 PG 처리군보다 동결 및 융해 후의 세포의 생존율이 높음을 확인하였다. 특히, 10% EG 처리군에서 85.63%로 동일한 농도의 PG 처리군(66.81%)보다 유의적(p<0.05)으로 가장 높은 생존율을 보였다. 한편, 상업용 닭(한협3호)에서도 오계와 비슷한 경향의 결과를 확인하였다. 이상의 결과들로부터 유리화 동결에 있어서 가장 높은 생존율을 보인 10% EG이 최적의 동결 보호제로서 사용 가능함을 확인하였다.

Keywords

References

  1. Arav A, Yavin S, Zeron Y, Natan D, Dekel I, Gacitua H 2002 New trends in gamete's cryopreservation. Mol Cell Endocrinol 187:77-81. https://doi.org/10.1016/S0303-7207(01)00700-6
  2. Bakst MR, Sexton TJ 1979 Fertilizing capacity and ultrastructure of fowl and turkey spermatozoa before and after freezing. J Reprod Fertil 55:1-7. https://doi.org/10.1530/jrf.0.0550001
  3. Bautista. JA, Dela Pena EC, Katagiri S, Takahashi Y, Kanagawa H 1998a In vitro viability of mouse oocytes vitrified in an ethylene glycol-based solution. Jpn J Vet Res 46:13-18.
  4. Bautista JA, Kanagawa H 1988b Current status of vitrification of embryos and oocytes in domestic animals : ethylene glycol as an emerging cryoprotectant of choice. Jpn J Vet Res 45: 183-191.
  5. Boutron P 1984 A more accurate determination of the quantity of ice crystallized at low cooling rates in the glycerol and 1,2-propandiol aqueous solutions : Comparison with equilibrium. Cryobiology 21:183-191. https://doi.org/10.1016/0011-2240(84)90210-4
  6. Bryant G 1995 DSC measurement of cell suspensions during successive freezing runs: implications for the mechanisms of intracellular ice formation. Cryobiology 32:114-128. https://doi.org/10.1006/cryo.1995.1011
  7. Chi HJ, Koo JJ, Kim MY, Joo JY, Chang SS, Chung KS 2002 Cryopreservation of human embryos using ethylene glycol in controlled slow freezing. Hum Reprod 17:2146-2151. https://doi.org/10.1093/humrep/17.8.2146
  8. Cocero MJ, Sebastian AL, Barragan ML, Picazo RA 1996 Differences on post thawing survival between ovine morulae and blastocysts cryopreserved with ethylene glycol or glycerol. Cryobiology 33:502-507. https://doi.org/10.1006/cryo.1996.0053
  9. Donnay I, Anquier P, Kaidi S, Carolan C, Lonergan P, Mermillod P 1998 Vitrification of in vitro produced bovine blastocysts : methodological studies and developmental capacity. Anim Reprod Sci 52:93-104. https://doi.org/10.1016/S0378-4320(98)00098-0
  10. Emiliani S, Van den Bergh M, Vannin AS, Biramane J, Englert Y 2000 Comparison of ethylene glycol, 1,2 propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4 cell embryos and blastocysts. Hum Reprod 15:905-910. https://doi.org/10.1093/humrep/15.4.905
  11. Freshney RI 2005 Culture of Animal Cells : A Manual of Basic Technique. 5th Edn Wiley-Liss Inc Hoboken.
  12. Friedler S, Giudice LC, Lamb EJ 1988 Cryopreservation of embryos and ova. Fertil Steril 49:743-764. https://doi.org/10.1016/S0015-0282(16)59879-3
  13. Gardner DK, Lane M 2000 Embryo culture systems In "Handbook of in vitro Fertilization". 2nd ed CRC Press, Boca Raton, FL 205-64.
  14. Gee GF, Bakst MR, Sexton TJ 1985 Cryogenic preservation of semen from the greater sandhill crane. J Wildl Manage 49:480-484. https://doi.org/10.2307/3801557
  15. Gilmore JA, McGann LE, Liu J, Gao DY, Peter AT, Kleinhans FW 1995 Effect of cryoprotectant solutes on water permeability of human spermatozoa. Biol Reprod 53:985-995. https://doi.org/10.1095/biolreprod53.5.985
  16. Hamburger V, Hamilton HL 1951 A series of normal stage in the development of the chick embryo. J Morphol 8:49-92.
  17. Han JY, Park TS, Hong YH Jeong DK, Kim JN, Kim KD, Lim JM 2002 Production of germline chimeras by transfer of chicken gonadal primordial germ cells maintained in vitro for an extended period. Theriogenology 58:1531-1539. https://doi.org/10.1016/S0093-691X(02)01061-0
  18. Hey JM, MarFarlane DR 1996 Crystallization of ice in aqueous solutions of glycerol and dimethly sulphoxide. Cryobiology 33:205-216. https://doi.org/10.1006/cryo.1996.0021
  19. Jiang JY, Umezu M, Sato E 1999 Vitrification of two cell rat embryos derived from immature hypothyroid rdw rats by in vitro fertilization in ethylene glycol based solutions. Cryobiology 38: 160-164. https://doi.org/10.1006/cryo.1999.2151
  20. Kasai M, Hamaguchi Y, Zhu SE, Miyake T, Sakurai T, Machida T 1992 High survival of rabbit morulae after vitrification in an ethylene glycol based solution by a simple method. Biol Reprod 46:1042-1046. https://doi.org/10.1095/biolreprod46.6.1042
  21. Kasai M, Ito M, Edashige K 2002 Morphological appearance of the cryopreserved mouse blastocyst as a tool to identify the type of cryoinjury. Hum Reprod 17:1863-1874. https://doi.org/10.1093/humrep/17.7.1863
  22. Kasai M, Komi JH, Takakamo A, Tsudera T, Sakurai T, 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
  23. Kim JN, Kim MA, Park TS, Kim DK, Park HJ, Ono T, Lim JM, Han JY 2004 Enriched gonadal migration of donorderived gonadal primordial germ cells by immunomagnetic cell sorting in birds. Mol Reprod Dev 68:81-87. https://doi.org/10.1002/mrd.20051
  24. Kino K, Pain B, Leibo SP, Clark ME, Etches RJ 1997 Production of chicken chimeras from injection of frozenthawed blastodermal cells. Poultry Sci 76:753-760. https://doi.org/10.1093/ps/76.5.753
  25. Kotobuki N, Hirose M, Machida H, Katou Y, Muraki K, Takakura Y, Ohgushi H 2005 Viability and osteogenic potential of cryopreserved human bone marrow-derived mesenchymal cells. Tissue Eng 11:663-673. https://doi.org/10.1089/ten.2005.11.663
  26. Kuleshova LL, MacFarlane DR, Trounson AO, Shaw JM 1999 Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes. Cryobiology 38:119-30. https://doi.org/10.1006/cryo.1999.2153
  27. Lee DR, Yoon TK 2009 Effect of slush-nitrogen on the cryopreservation of oocytes and embryos using vitrification. Korean J Reprod Med 36:1-7.
  28. Lovelock JE, Bishop MWH 1959 Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature 183:1394-1395. https://doi.org/10.1038/1831394a0
  29. Maeda T, Terada T, Tsutsumi Y 1984 Morphological observations on frozen and thawed Muscovy spermatozoa. British Poultry Sci 25:409-413. https://doi.org/10.1080/00071668408454881
  30. Meryman HT 2007 Cryopreservation of living cells : Principles and practice. Transfusion 47:935-945. https://doi.org/10.1111/j.1537-2995.2007.01212.x
  31. Miyake T, Kasai M, Zhu SE, Sakurai T, Machida T 1993 Vitrification of mouse oocytes and embryos at various stages of development in an ethylene-glycol based solution by a simple method. Theriogenology 40:121-134. https://doi.org/10.1016/0093-691X(93)90346-7
  32. Mukaida T, Wada S, Takahashi K, Pedro PB, An TZ, Kasai M 1998 Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Hum Reprod 13:2874-2879. https://doi.org/10.1093/humrep/13.10.2874
  33. Natio T, Tajima A, Tagami T, Yasuda Y, Kuwana T 1994 Preservation of chick primordial germ cells in liquid nitrogen and subsequent production of viable offspring. J Reprod Fertil 102:321-325. https://doi.org/10.1530/jrf.0.1020321
  34. Naito M 2003 Cryopreservation of avian germline cells and subsequent production of viable offspring. Journal of Poultry Sci 40:1-12. https://doi.org/10.2141/jpsa.40.1
  35. Park TS, Jeong DK, Kim JN, Song GH, Hong YH, Lim JM, Han JY 2003 Improved germline transmission in chicken chimeras produced by transplantation of gonadal primordial germ cells into recipient embryos. Biol Reprod 68:1657-1662. https://doi.org/10.1095/biolreprod.102.006825
  36. Petitte JN 2006 Avian germplasm preservation: embryonic stem cells or primordoial germ cells. Poultry Sci 85:237-242. https://doi.org/10.1093/ps/85.2.237
  37. Polge C 1951 Functional survival of fowl spermatozoa after freezing at - $79^{\circ}C$. Nature 167:949-950.
  38. Rall WF, Fahy GM 1985 Ice-free cryopreservation of mouse embryos at - $196^{\circ}C$ by vitrification. Nature 313:573-5. https://doi.org/10.1038/313573a0
  39. Rall WF 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
  40. Rall WF, Wood MJ, Kirby C, Whittimgham DG 1987 Development of mouse embryos cryopreserved by vitrification. J Reprod Fertil 80:499-504. https://doi.org/10.1530/jrf.0.0800499
  41. Rezazadeh VM, Eftekhari-Yazdi P, Karimian L, Hassani F, Movaghar B 2009 Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos. J Assist Reprod Genet 26:347-354. https://doi.org/10.1007/s10815-009-9318-6
  42. Saha S, Otoi T, Takagi M, Boediono A, Sumantri C, Suzuki T 1996 Normal calves obtained after direct transfer of vitrified bovine embryos using ethylene glycol, trehalose, and polyvinylpyrrolidone. Cryobiology 33:291-299. https://doi.org/10.1006/cryo.1996.0029
  43. Scheffen B, Vanderzwalmen P, Massip A 1986 A simple and efficient procedure for preservation of mouse embryos by vitrification. Cryo Letters 7:260-269.
  44. Shaw JM, Ward C, Trounson AO 1995 Evaluation of propanediol, ethylene glycol, sucrose and antifreeze proteins on the survival of slow cooled mouse pronuclear and 4 cell embryos. Hum Reprod 10:396-402. https://doi.org/10.1093/oxfordjournals.humrep.a135951
  45. Takase K, Sawai M, Yamamoto K, Yata J, Takasaki Y, Teraoka H, Tsukada K 1992 Reversible G1 arrest induced by dimethyl sulfoxide in human lymphoid cell lines : kinetics of arrest and expression of the cell cycle marker proliferating cell nuclear antigen in Raji cells. Cell Growth Differ 3:515-521.
  46. Watanabe M, Kinutani M, Naito M, Ochi O, Takashima Y 1992 Distribution analysis of transferred donor cells in avaian blastodermal chimeras. Development 114:331-338.
  47. Yasuda Y, Tajima A, Fujimoto T, Kuwana T 1992 A method to obtain germ-line chimaeras using isolated primordial germ cells. J Reprod Fertil 96:521-- 528. https://doi.org/10.1530/jrf.0.0960521
  48. Yavin S, Aroyo A, Roth Z, Arav A 2009 Embryo cryopreservation in the presence of low concentration of vitrification solution with sealed pulled straws in liquid nitrogen slush. Hum Reprod 24:797-804.
  49. Yoon TK, Lee DR, Cha SK, Chung HM, Lee WS, Cha KY 2007 Survival rate of human oocytes and pregnancy outcome after vitrification using slush nitrogen in assisted reproductive technologies. Fertil Steril 88:952-956. https://doi.org/10.1016/j.fertnstert.2006.12.071
  50. 김현 양보석 고응규 김재환 최성복 김성우 2011 한국재래닭의 계통별 번식능력 비교. 한국동물번식학회지 35:393-394.