Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
권호 표지

Transfer, Cryopreservation and Production of Bovine Embryos Cultured in Serum-Free System

Serum-Free Medium에서 배양한 한우 배의 내동성과 이식

  • Im, Y.-J. (Division of Applied Life Science, Gyeongsang National University, ET Biotech) ;
  • Kim, J.-H. (Division of Applied Life Science, Gyeongsang National University) ;
  • Song, H.-B. (Division of Applied Life Resources, Daegu University) ;
  • Jung, Y.-G. (ET Biotech)
  • 임여정 (국립경상대학교 응용생명과학부, 이티바이오텍) ;
  • 김진희 (국립경상대학교 응용생명과학부) ;
  • 송해범 (대구대학교 생명자원학부) ;
  • 정연길 (이티바이오텍)
  • Published : 2004.08.01
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Abstract

Serum-contain is commoly used for the production of in vitro-derived bovine embryos. However, were biological activity of serum varies from lot to lot, time consuming to choose better serum with good quality and risks of virus, bacteria and mycoplasma infection. This study established serum-free culture systems of in vitro embryo development to efficiently obtain a large number of blastocysts from ovaries of Hanwoo and oocytes maturation, cell number, tlerance of cryopreservation. Secondly, serum-contain medium is suspected of contributing to the large calf size, dystocia, cersarean sections, calf mortality and confirmed these blastocysts are high quality in terms of cyotolerance, high rates of pregancy and normal birth. For these reasons, Culture media (IVMD101 and IVD101) designed specifically for the preimplantation bovine embryo are rather simplistic, being based on salt solutions with additional energy substrates and growth factors. An improved serum-free medium (IVMD101) was developed for bovine oocytes maturation in vitro. Proportions of embryos developing to the blastocyst stage cultured in both IVD101(32.4%) and IVD101(34.5%) serum-free media were higher than in TCM199+10% FBS(12.4%) serumcontaining medium. Futhermore, the cell numbers per blastosyst obtained in the serum-free media were superior to those of blastocysts developed in serum-supplemented medium. Also, cell numbers of blastocysts obtained in the serum-free media were similar with blastocysts derived in vivo. Survival rate blastocysts after 24 hr incubation after thawing, the blastocysts cultured in both IVD101(94.5%) and IVD101(95.8%) serum-free media were higher than in TCM199+10% FBS (52.5%) serum-containing medium. After 72 hr incubation after thawing, hatching rates of blastocysts developed in IVD101(78.4%) and IVMD101(83.7%) were sighnificantly higher than that developed in the serum-supplemented medium(32.0%). The pregnancy rates almost not different between fresh blastocysts(38.2%) and frozen blastocysts(34.9%). The results suggested that the improved serum-free media(IVMD101 and IVD101) offer several advantages over culture in serum-cotaining medium, including increased rates of blastocyst formation and high cel numbers. Additionally, the survival and hatching rates of embryos product in serum-free media after post-thaw culture were superior to those of embryos produced in the serum-containg medium and useful for the production of high quality bovine embryos for cryo-preservation. These improved serum-free media are beneficial not only for the study of the mechanisms of early embryogenesis but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis.

난자의 체외성숙 및 체외배양에는 일반적으로 동물의 혈청을 사용하고 있다. 그러나, 채취한 소의 상태에 따라서 혈청의 질에 차이가 있어 실험데이터가 일정하지 않을 수 있고, 그것으로부터 바이러스, 세균, 마이코플라즈마 등에 오염될 가능성이 있다. 따라서, 본 실험에서는 완전 무 혈청 배양액에서 난자의 성숙, 배 발생율, 세포 수, 동결성을 검토하였다. 다음으로, 근래 혈청배지로 생산한 체외 배양 수정란은 과체중의 산자 생산, 초기 산자 사망률, caesarean section, dystocia 같은 발병으로 문제가 지적되고 있다. 그러나 무 혈청 배지로 생산한 체외 배양 수정란은 이러한 현상을 개선할 수 있다는 보고가 있어 본 실험에서는 완전 무혈청 배양액에서 생산된 동결란과 신선란을 젖소에 이식하여 그 결과를 검토하였다. 무혈청 배양액에는 에너지원과 세포성장인자가 첨가된 IVD101과 IVMD101, 대조군인 혈청 배양액에는 TCM199 + 10% FBS가 사용되었다. IVMD101에 의해 체외성숙이 이루어 졌고, 혈청이 첨가된 TCM199 + 10% FBS에서는 12.4% 배 발생율을 보인데 반해, 무혈청 배양액 IVD101과 IVMD101이 각 32.4%, 34.5%로 훨씬 높은 배 발생율을 보였다. 더욱이, 세포 수에 있어서도 무혈청 배양액에서 발생된 배반포의 세포수가 혈청이 첨가된 배양액에서 발생된 배반포의 세포수보다 월등하였으며, 이는 체내란과 비슷한 세포 수를 보이고 있다. 수정란의 내동성을 보면, 융해하고 24시간 배양기 정치 후에 IVD101과 IVMD101은 94.5%, 95.8%의 생존율을 보인 반면 TCM199 + 10% FBS는 52.5%에 불과하다. 융해하고 72시간후 탈출 배반포율이 IVD101과 IVMD101는 78.4%, 83.7%였는데 반해 TCM199 + 10% FBS는 32.0%였다. 마지막으로, 무혈청 배양액에서 발생한 동결란과 신선란을 젖소에 이식한 후에 임신율에 있어서는 유의적 차이는 없었지만, 무혈청 배지에서 생산된 수정란의 내동성의 탁월함이 증명되었다. 이러한 결과들로 종합해 보면, 무혈청 배양액 (IVD101, IVMD101)에서 발생된 수정란이 혈청 배양액에서 발생된 수정란보다 배발생율, 세포수, 동결성, 임신율에 있어서 우수함을 알 수 있었다. 이러한 무혈청 배양액은 배발생 과정의 연구뿐만 아니라 수정란 이식을 위한 고품질의 체외 수정란의 대량 생산, 복제, 형질전환 동물 생산 등에도 유익할 것으로 사료된다.

Keywords

References

  1. Abe H, Yamashita S, Satoh T and Hoshi H. 2002. Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media. Mol. Reprod. Dev., 61:57-66 https://doi.org/10.1002/mrd.1131
  2. Abe H, Yamashita S, Satoh T and Hoshi H. 1999. Ultrastructure of bovine embryos developed from in vitro-matured and -fertilized oocytes: comparative morphological evaluation of embryos cultured either in serum-free medium or in serum-supplemented medium. Mol. Reprod. Dev., 53:325-335 https://doi.org/10.1002/(SICI)1098-2795(199907)53:3<325::AID-MRD8>3.0.CO;2-T
  3. Abe H, Otoi T, Tachikawa S, Yamashita S, Satoh T and Hoshi H. 1999. Fine structure of bovine morulae and blastocysts in vivo and in vitro. Anat Embryol., 199:519-527 https://doi.org/10.1007/s004290050249
  4. Aoyagi K, Ito H, Saitou M, Kanauchi T, Kobayashi M and Hoshi H. 1999. In vitro development and production of offspring from boving nuclear transfer embryos cultured in a serum- free medium. J. Reprod. Dev., 45:129-134 https://doi.org/10.1262/jrd.45.129
  5. Bavister BD. 1995. Culture of preimplantation embryos:facts and artifacts. Human Reprod. Update, 1:91-143 https://doi.org/10.1093/humupd/1.2.91
  6. Behboodi E, Anderson GB, BonDurant RH, Cargill SL, Kreuscher BR, Medrano JF and Murray JD. 1995. Birth of large calves that developed from in vitro-derived bovine embryos. Theriogenology, 44:227-232 https://doi.org/10.1016/0093-691X(95)00172-5
  7. Brackett BG, Younis AI and Fayrer-Hosken RA. 1989. Enhanced viability after in vitro fertilization of bovine oocytes matured in vivo with high concentration of lutenizing hormone. Fertil. Steril., 52:319-324 https://doi.org/10.1016/S0015-0282(16)60862-2
  8. Brackett BG and Oliphant G. 1975. Capacitation of rabbit spermatopza in vitro. Biol. Reprod., 12: 260-274 https://doi.org/10.1095/biolreprod12.2.260
  9. Evans EP. 1987. Karyotyping and sexing of gametes, embryos and fetuses and in situ hybridization to chromosomes. In. Mammalian Development.(Ed. H. Monk.) pp. 93-114.(IRL Press: City)
  10. Eyestone WH and First NL. 1989. Co-culture of early cattle embruos to the blastocyst stage with oviductal tissue or in conditioned medium. J. Reprod. Fertil., 85:715-720 https://doi.org/10.1530/jrf.0.0850715
  11. Farin P and Farin C. 1997. Perspectives on large calves following transfer of embryos produced in vitro. Embryo Transfer Newslett, 15:15-19
  12. Farin PW and Farin CE. 1995. Transfer of bovine embryos produced in vivo or in vitro：survival and fetal development. Biol. Reprod., 52:676- 682 https://doi.org/10.1095/biolreprod52.3.676
  13. Fukuda Y, Ichikawa M, Naito K and Toyoda Y. 1990. Birth of normal calves resulting from bovine oocytes matured, fertilized, and cultured with cumulus cells in vitro up to the blasto-cyst stage. Biol. Reprod., 42:114-119 https://doi.org/10.1095/biolreprod42.1.114
  14. Fukui Y. 1989. Effect of sera and steroid hormones on development of bovine oocytes matured and fertilized in vitro and co-culture with bovine oviduct epithelial cells. J. Anim. Sci., 67:1318-1323 https://doi.org/10.2527/jas1989.6751318x
  15. Gandolfi F. 1994. Autocrine, paracrine and environmental factors influencing embryonic development from zygote to blastocyst. Theriogenology, 41:95-100 https://doi.org/10.1016/S0093-691X(05)80053-6
  16. Greve T, Avery B and Callesen H. 1993. Viability of in-vivo and in-vitro produced bovine embryos. Reprod Dom Anim., 28:164-169 https://doi.org/10.1111/j.1439-0531.1993.tb00113.x
  17. Hasler JF, Henderson WB, Hurtgen PJ, Jin ZQ, McCauley AD, Mower SA, Neely B, Shuey LS, Stokes JE and Trimmer SA. 1995. Production, freezing and transfer of bovine IVF embryos and subsequent calving results. Theriogenology, 43:141-152 https://doi.org/10.1016/0093-691X(94)00020-U
  18. Hayakawa T, Yamashita K, Tanzawa K, Uchijima E and Iwata K. 1992. Growth-promoting activity of tissue inhibitor of metalloproteinase-1 (TIMP-1) for a wide range of cells. A possible new growth factor in serum. FEBS Lett., 298:29-32 https://doi.org/10.1016/0014-5793(92)80015-9
  19. Hayakawa T, Yamashita K, Ohuchi E and Shinagawa A. 1994. Cell growth-promoting activity of tissue inhibitor of metalloproteinase-2 (TI- MP-2). J Cell. Sci., 107:2373-2379
  20. Hiroyuki ABE and Hoshi H. 2003. Evaluation of bovine embryos produced in high performance serum-free media, The Journal of reproduction and development 49:193-202 https://doi.org/10.1262/jrd.49.193
  21. Hochi S, Semple E and Leibo SP. 1996. Effect of cooling and warning rates during cryopreservation on survival of in vitro-produced bovine embryos. Theriogenology, 45:837-847
  22. Holm P, Walker SK and Seamark RF. 1996. Embryo viability, duration of gestation and birth weight in sheep after transfer of in vitro matured and in vitro fertilized zygotes cultured in vitro or in vivo. J Reprod Fertil., 107:175- 181 https://doi.org/10.1530/jrf.0.1070175
  23. Hoshi H. 1996. In vitro production of bovine preimplantation embryos in a serum-free medium. In: Gene expression and nutrition in animals: From cells to whole body (Ed. T. Muramatsu). Research Singpost, Trivandrum. India. pp. 59-70
  24. Hoshi H, Satoh T, Yamashita S, Abe H, Sata R, Tsujii H and Goto T. 1999. Production and embryo trasfer of preimplantation bovine embryos cultured in serum-free medium-comparative evaluation of developmental ability and quality of embryos obtained in serum-free and serum-supplemented medium. Tiss. Cult. Res. Commun., 18:163-172
  25. Hoshi H. 2003. In vitro production of bovine embryos and the application for embryo transfer. Theriogenology, 59:675-685 https://doi.org/10.1016/S0093-691X(02)01247-5
  26. Kikuchi M, Sendai Y, Yamashita S, Satoh T and Hoshi H. 1996. Expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and collagenase genes in preimplantation bovine embryos. J. Reprod. Dev., 42:55-60 https://doi.org/10.1262/jrd.42.55
  27. Kobayashi K, Takagi Y, Satoh T, Hoshi H and Oikawa T. 1992. Development of early bovine embryos to the blastocyst stage in serum-free conditioned medium from bovine granulosa cells. In vitro Cell & Dev. Biol., 28A:255-259 https://doi.org/10.1007/BF02634241
  28. Kobayashi K, Yamashita S and Hoshi H. 1994a. Influence of epidermal growth factor and transforming growth factor-$\alpha$ on in vitro maturation of cumulus cell-enclosed bovine oocytes in a defined medium. J. Reprod. Fertil., 160:439-446
  29. Kobayashi K, Yamashita S, Stoh T and Hoshi H. 1994b. low oxygen amd glucose improves the development of fertilized bovine oocyte in defined mediun without somatic cells. In vitro Cell. Cev. Biol., 30A:556-558 https://doi.org/10.1007/BF02631251
  30. Kruip TAM and den Daas JHG. 1997. In vitro produced and cloned embryos: effects on pregnancy, parturition and offspring. Theriogenology, 47:43-52 https://doi.org/10.1016/S0093-691X(96)00338-X
  31. Leibfried-Rutledge ML, Crister ES and First NL. 1986. Effects of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster oocyte complexes. Biol. Reprod., 35:850-857 https://doi.org/10.1095/biolreprod35.4.850
  32. Leibo SP and Loskutoff NM. 1993. Cryobiology of in vitro-derived bovine embryos. Theriogenology, 39:81-94 https://doi.org/10.1016/0093-691X(93)90025-Z
  33. Martin KL, Hardy K, Winston RML and Leese HJ 1993. Activity of enzymes of energy metabolism in single human preimplantation embryos. J. Reprod. Fertil., 99:259-266 https://doi.org/10.1530/jrf.0.0990259
  34. Massip A, Mermillod P and Dinnyes A. 1995. Morphology and biochemistry of in vitro produced bovine embtyos : implications for their cryopreservation. Human Reprod., 10: 3004-3011 https://doi.org/10.1093/oxfordjournals.humrep.a135837
  35. Moor RM, Dai Y, Lee C and Fulka J. 1998. Oocyte maturation and embryonic failure. Human Reprod. Update, 4:223-236 https://doi.org/10.1093/humupd/4.3.223
  36. Pinyopummintr T and Bavister BD. 1991. In vitro- matured/in vitro-fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined protein-free culture media. Biol. Reprod., 45:736-742 https://doi.org/10.1095/biolreprod45.5.736
  37. PollardJW andLeibo SP. 1993. Comparative cryobiology of in vitro and in vivo derived bovine embryos. Theriogenology, 39:287(abstract) https://doi.org/10.1016/0093-691X(93)90142-R
  38. Rieger D, Loskutoff NM and Betteridge KJ. 1992. Developmentally related changes in the uptake and metabolism of glucose, glutamine and pyruvate by cattle embryos produced in vitro. Reprod Fertil Dev., 4:547-557 https://doi.org/10.1071/RD9920547
  39. Rexroad CE. 1989. Co-culture of domestic animal embryos. Theriogenology, 31:105-114 https://doi.org/10.1016/0093-691X(89)90568-2
  40. Sata R, Tsujii H, Abe H, Yamashita S and Hoshi H. 1999. Fatty acid composition of bovine embryos cultured in serum-free and serum- containing medium during early embryonic development. J. Reprod. Dev., 45:97-103 https://doi.org/10.1262/jrd.45.97
  41. Satoh T, Kobayashi K, Yamashita S, Kikuchi M, Sendai Y and Hoshi H. 1994a. Tissue inhibitor of metalloproteinases (TIMP-1) produced by granulosa cells and oviduct cells enhances in vitro development of bovine embryo. Biol. Reprod., 50:835-844 https://doi.org/10.1095/biolreprod50.4.835
  42. Satoh T, Kobayashi K, Yamashita S and Hoshi H. 1994b. Bovine tissue inhibitor of metalloproteinase-1(b TIMP-1) improves the frequency and quality of bovine embryo in serum-free medium. Cytotechnology. 14(suppl-1)(Abstr. 7. 37)
  43. Schmidt M, Greve T, Avery B, Beckers JF, Sulon J and Hansen HB. 1996. Pregnancies, calves and calf viability after transfer of in vitro produced bovine embryos. Theriogenology, 46: 527-539 https://doi.org/10.1016/0093-691X(96)00174-4
  44. Schultz GA and Heyner S. 1993. Growth factors in preimplantation embryos. Oxf. Rev. Reprod. Biol., 15:43-81
  45. Semple ME, Betteridge KJ and Leibo SP. 1995. Cryopreservation of in vitro-derived bovine embryos produced in a serum-free culture system. Theriogenology, 43:320(Abstr.) https://doi.org/10.1016/0093-691X(95)92474-N
  46. Shamsuddin M, Lsrsson B, Gustafsson H and Rodriguez-Martinez H. 1993. In vitro development up to hatching of bovine in vitro- matured and fertilized oocytes with or without support from somatic cells. Theriogenology, 39:1067-1079 https://doi.org/10.1016/0093-691X(93)90007-R
  47. Sinclair KD, McKvoy TG, Carolan C, Maxfield EK, Maltin CA, Young LE, Wilmut I, Robinson JJ and Broadbent PJ. 1998. Conceptus growth and development following in vitro culture of ovine embryos in media supplemented with bovine sera. Theriogenology, 49: 218(abstract) https://doi.org/10.1016/S0093-691X(98)90571-4
  48. Staigmiller RM and Moor RM. 1984. Effect of follicle cells on the maturation and developmental competence of ovine oocytes matured outside the follicle. Gamete Res., 9: 221-229 https://doi.org/10.1002/mrd.1120090211
  49. Takagi 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 cryoprotectants. Theriogenology, 41: 915-921 https://doi.org/10.1016/0093-691X(94)90507-F
  50. Takahashi Y and First NL. 1992. In vitro development of bovine one-cell embryos: influence of glucose, lactate, pyruvate, amino acids, and vitamins. Theriogenology, 37:963-978 https://doi.org/10.1016/0093-691X(92)90096-A
  51. Tervit HR, Wittingham DG and Rowson LEA. 1972. Successful culture in vitro of seep and cattle ova. J. Reprod. Fertil., 30:493-497 https://doi.org/10.1530/jrf.0.0300493
  52. Thompson JGE and Simpson AC, Pugh PA, Donnelly PE and Tervit H. R. 1990. Effect of oxygen concentration on in-vitro development of preimplantation sheep and cattle embtyos. J. Reprod. Fertil., 89:573-578 https://doi.org/10.1530/jrf.0.0890573
  53. Thompson JG, Gardner DK, Pugh PA, McMillan WH and Tervit HR. 1995. Lamb birth weight is affected by culture system utilized during in vitro pre-elongation development of ovine embryos. Biol. Reprod., 53:1385-1391 https://doi.org/10.1095/biolreprod53.6.1385
  54. Ushijima H, Yamakawa H and Nagashima H. 1999. Cryopreservation of bovine pre-morula-stage in vitro matured/in vitro fertilized embryos after delipidation and before use in nucleus transfer. Biol Reprod., 60:535-539
  55. Van Langendonckt A, Donnay I, Schuurbiers N, Auquier P, Carilan C, Massip A and Dessy F. 1997. Effects of supplementation with fetal calf serum on development of bovine embryos in synthetic oviduct fluid medium, Journal of reproduction and fertility 109: 87-93 https://doi.org/10.1530/jrf.0.1090087
  56. Vanderhyden BC and Armstrong DT. 1989. Role of cumulus cells and serum on the in vitro maturatuion, fertilization, and subsequent development of rat oocytes. Biol. Reprod., 40: 720-728 https://doi.org/10.1095/biolreprod40.4.720
  57. Voelkel SA and Hu YX. 1992. Effect of gas atmosphere on the development of one-cell bovine embryos in two culture systems. Theriogenology, 37:1117-1131 https://doi.org/10.1016/0093-691X(92)90109-5
  58. Wright RW and Bondioli KR. 1981. Aspects of in vitro fertilization and embryo culture in domestic animals. J. Anim. Sci., 53:702-729 https://doi.org/10.2527/jas1981.533702x
  59. Walker SK, Hartwich KM and Seamark RF. 1996. The prodution of unusually large offspring following embryos manipultion: concepts and challenge. Theriogenology, 45:111-120 https://doi.org/10.1016/0093-691X(95)00360-K
  60. Yamashita S, Abe H, Itoh T, Satoh T and Hoshi H. 1999. A serum-free culture system for efficient in vitro production of bovine blastocysts with improved viability after freezing and thawing. Cytotechnology, 31:1-9 https://doi.org/10.1023/A:1008198829774
  61. Yazawa S, Aoyagi Y, Konishi M and Takedomi T. 1997. Characterization and cytogenetic analysis of Japanese Black calves produced by nuclear trasnfer. Theriogenology, 48:641-650 https://doi.org/10.1016/S0093-691X(97)00280-X
  62. Zhang L and Barry DM. Denniston RS, Bunch TD, Godke RA. 1993. Birth of live calves after transfer of frozen-thawed bovine embryos fertilized in vitro. Vet. Rec, 132:247-279 https://doi.org/10.1136/vr.132.10.247