Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
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Effects of Development and Viability of Pig Oocytes Matured in Defined Medium Containing PVA, PVP and pFF

PVA, PVP 및 pFF를 첨가한 체외성숙 한정배지가 미성숙 돼지 난포란의 성숙과 배발달에 미치는 영향

  • 김인덕 (단국대학교 생명자원과학대학 동물자원학과) ;
  • 김세나 (단국대학교 생명자원과학대학 동물자원학과) ;
  • 한숙기 (단국대학교 생명자원과학대학 동물자원학과) ;
  • 석호봉 (단국대학교 생명자원과학대학 동물자원학과)
  • Published : 2004.12.01
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Abstract

This study was conducted to develop a serum-free, defined medium of IVM of pig oocytes. The TCM-199 with supplemented with polyvinylalcohol(PVA), polyvinylpyrrollidone(PVP) and porcine follicular fluid(pFF) were used as basal medium. The effects of the these additives on the rates of maturity and development under in-vitro fertilization and in vitro culture were examined and subsequently considered on the possibilities be sustituted for the bovine serum albumin(BSA). Maturation rate of pig oocytes in IVM media containing PVA(82.4％), pFF(89.4％) and BSA(90.0％) were significantly higher(P<0.05) than that of PVP(78.6％). Cleavage rate after IVF of PVP(64％) was significantly lower(P<0.05) than these of PVA(73％), pFF(77％) and BSA(73％) supplements. in vitro development rates to morulae and blastocyst on PVP(54％) were also significantly lower(P<0.05) than these of the supplements of PVA(63％), pFF(69％) and BSA(65％). In comparison of maturation and fertilization rates of pig oocytes in each supplements, the maturity rates of PVA(82.4％), pFF(89.4％) and BSA(90.0％) were significantly lower(P<0.05) than that of PVP(72.4％) and while, the fertilization rates of pFF(87.1％) and BSA(89.1％) were significantly higher(P<0.05) than these of PVA(78.0％) and PVP(70.6％). It may be concluded that PVA and pFF can be substituted far BSA in medium for culturing pig oocytes; however, it may be considered that PVP were limited to for BSA in the in vitro culture of the embryos.

본 연구는 체외 성숙액인 TCM-199 배지를 기초로 무혈청첨가물인 PVA, PVP 및 pFF의 한정배지를 이용하여 돼지 미성숙난자의 체외성숙, 수정 및 배양 후 난자의 수정율과 배발달에 미치는 영향과 나아가 BSA 대체물로서의 이용 가능성을 알아보았다. 1. 무혈청 첨가물을 이용하여 난포란의 체외 성숙을 유기한 결과 PVA, PVP, pFF, BSA의 전체 분할율은 각각 82.4%, 78.6%, 89.4%, 90.0%로 나타났으며, GV, MI-MII율은 PVA 첨가시 각각 15.1%, 84.9%, PVP는 각각 26.5%, 73.5%, pFF는 각각 11.8%, 88.2%, BSA는 각각 11.1%, 88.9%로 PVA 혹은 pFF 첨가시에는 모두 BSA와 유사한 결과를 보였으나 PVP와는 유의적인 차이를 나타내었다 (P<0.05). 2. 체외 성숙된 난자를 수정시킨 후의 배발달율을 확인한 결과 전체 난할율은 PVA가 73%, PVP는 64.1% , pFF가 77.2%, BSA가 73%로 PVA와 pFF는 BSA와 유사하게 나타났으나, PVP는 그 발달율이 다른 처리군 들과 비교하여 유의적(P<0.05)으로 낮은 결과를 보였다. 3. 각 처리구간의 morulae와 blastocyst의 합을 비교한 결과 체외성숙 첨가물 중 PVA와 pFF는 각각 63%, 69%로 BSA(65%)와 유사한 결과를 나타내었으나 PVP는 54%로 유의적으로 낮은 결과를 나타내었다(P<0.05). 4. 전체 성숙율과 수정율을 비교해 본 결과 성숙 율은 PVA, pFF, BSA가 82.4%, 89.4%, 90.0%인 반면 PVP는 72.4%로 유의적으로(P<0.05) 낮은 결과를 보였고, 수정율은 pFF, BSA가 각각 87.1%, 89.1%로 PVA, PVP의 78.0%, 70.6%에 비해 각각 유의적(P<0.05)으로 높았다. 이상의 연구결과 돼지 난자의 체외성숙 및 배양 배지에 있어 PVA, pFF 첨가는 BSA 대체물로서 이용이 가능하나 PVP는 그 이용에 제한이 따를 것으로 판단된다.

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. Abeydeera LR, Wang WH, Cantley TC, Rieke A, Murphy CN, Prather and Day BN. 2000. Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor. Theriogenology, 54:787-797 https://doi.org/10.1016/S0093-691X(00)00390-3
  3. Ashwood-Smith MJ and Warby C. 1971. Studies on the molecular weight and cryoprotective properties of polyvinylpyrrolidone and dextran with bacteria and erythrocytes. Cryobiology, 8:453-464 https://doi.org/10.1016/0011-2240(71)90036-8
  4. Ayoub MA and Hunter AG. 1993. Inhibitory effect of bovine follicular fluid on in vitro maturation of bovine oocytes. J. Dairy Sci., 76:95-100 https://doi.org/10.3168/jds.S0022-0302(93)77327-0
  5. Biggers JD, Summers MC and McGinnis LK. 1997. Polyvinyl alcohol and amino acids as substitutes for bovine serum albumin in culture media for mouse preimplantation embryos. Human Repord. Update 3(2):125-135 https://doi.org/10.1093/humupd/3.2.125
  6. Brackett B and Zuelke K. 1993. Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology, 39:43-64 https://doi.org/10.1016/0093-691X(93)90023-X
  7. Chatot CS, Klein NW, Clapper ML, Resor SR, Singer WD, Russman BS, Holmes GL, Mattson RH and Cramer JA. 1984. Human serum teratogenicity studied by rat embryo culture: epilepst, anticonvulasant druge and nutrition, Epilepsia, 25:205 https://doi.org/10.1111/j.1528-1157.1984.tb04177.x
  8. Chen L, Russel PT and Larsen WJ. 1993. Functional significance of cumulus expansion in the mouse:roles for the preovulatory syntheses of hyaluronic acid within the cumulus mass. Mol. Reprod. Dev., 34:87-93 https://doi.org/10.1002/mrd.1080340114
  9. Choi SH, Ryu IS, Kim IH, Park SB, Yeon SH, Jin HJ, Suh SW, Lee CS and Son DS. 1999. Effects of in vitro maturation condition on bovine IVF embryos development. Korean J. Emb. Trans., 14:113-119
  10. Choi SH, Yang BC, Kim IH, Son DS, Lee HJ, Lee KW, Park MK, Kim KN and Chung YC. 1997. Effects of serum on in vitro maturation of Korean native cattle oocytes. Theriogenology, 47(1):187 https://doi.org/10.1016/S0093-691X(97)82314-X
  11. Cholewa JA and Whitten WK. 1970. Development of two-cell mouse embryos in the absence of a fixed-nitrogen source. J. Reprod. Fert., 22:553-555 https://doi.org/10.1530/jrf.0.0220553
  12. Coy P, Martinez E, Ruiz S, Vazquez JM, Roca J, and Matas C. 1993. Sperm concentration influences fertilization and male pronuclear formation in vitro in pig. Therioginology, 40:539- 546 https://doi.org/10.1016/0093-691X(93)90407-V
  13. Coy P, Ruiz S, Romar R, Campos I and Gadea J. 1999. Maturation, fertilization and complete development of porcine oocytes matured under different systems. Theriogenology, 51:799-812 https://doi.org/10.1016/S0093-691X(99)00028-X
  14. Dekel N and Phillips DM. 1979. Maturation of the rat cumulus oophorus : a scanning microscopic study. Biol. Reprod., 21:9-18 https://doi.org/10.1095/biolreprod21.1.9
  15. Dumoulin JC, Bergers-Janssen JM, Pieters MH, Enginsu ME, Geraedts JP and Evers JL. 1994. The protective effects of polymers in the cryopreservation of human and mouse zonae pellucidae and embryos. Fertil Steril., 62:793- 798 https://doi.org/10.1016/S0015-0282(16)57006-X
  16. Eckert J and Niemann. 1995. In vitro maturation, fertilization and culture to blastocysts of bovine oocytes in protein-free media. 43:1211-1225 https://doi.org/10.1016/0093-691X(95)00093-N
  17. Edwards RG. 1965. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature, 208:349-351 https://doi.org/10.1038/208349a0
  18. Fukui Y and Sakuma Y. 1980. Maturation of bovine oocytes cultured in vitro : relation to ovarian activity. follicular size and the presence or absence of cumulus cells. Biol. Reprod., 22: 669-673 https://doi.org/10.1093/biolreprod/22.3.669
  19. Funahashi H, Stumpf TT, Terlouw SL, Cantley T, Rieke A and Day BN. 1994a. Developmental ability of pig oocytes matured and fertilized in vitro. Theriogenology, 41:1425-1433 https://doi.org/10.1016/0093-691X(94)90193-M
  20. Funahashi H, Cantley T, Stumpf TT, Terlouw SL, Rieke A, and Day BN. 1994b. In vitro development of in vitro-matured pig oocytes following chemical activation or in vitro fertilization. Biol. Reprod., 50:1072-1077 https://doi.org/10.1095/biolreprod50.5.1072
  21. Funahashi H, Kim NH, Stumpf TT, Cantley T and Day BN. 1996. presence of organic osmolytes in maturation medium enhances cytoplasmic maturation of pig oocytes. Biol. Reprod., 54: 1412-1419 https://doi.org/10.1095/biolreprod54.6.1412
  22. Gardner DK. 1994. Mammalian embryo culture in the absence of serum or somatic cell support. Cell Int., 18:1163-1179 https://doi.org/10.1006/cbir.1994.1043
  23. Hong JY, Yong HY, Lee BC, Hwang JM and Lee ES. 2004. Effects of amino acids on maturation, fertilization and embryo development of pig follicular oocytes in two IVM media., 62:1473-1482
  24. Iritani A, Niwa K and Imai H. 1978. Sperm penetration of pig follicular oocytes matured in culture. J. Reprod. Fert., 54:379-383 https://doi.org/10.1530/jrf.0.0540379
  25. Kane MJ. 1987. Minimal nutrient requirements for culture of one-cell rabbit embryos. Biol. Reprod., 37:775-778 https://doi.org/10.1095/biolreprod37.4.775
  26. Kikuchi K, Nagai T, Kashiwazaki N, Ikeda H, Nouguchi J, Shimada A, Soloy E, and Kaneko H. 1998. Cryopreservation and ensuing in vitro fertilization ability of boar spermatozoa from epididymides stored at 4$^{\circ}C$. Theriogenology, 50: 615-623 https://doi.org/10.1016/S0093-691X(98)00166-6
  27. Kim HS, Lee GS, Hyun SH, Lee SH, Nam DH, Jeong YW, Kim S, Kang SK, Lee BC and Hwang WS. 2004. Improved in vitro development of porcine embryos with different energy substrates and serum. 61:1381-1393 https://doi.org/10.1016/j.theriogenology.2003.08.012
  28. Kim KS, Mitsumizo N, Fujita K and Utsumi K. 1996. The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos. 45:787-799 https://doi.org/10.1016/0093-691X(96)00008-8
  29. Lee ES and Fukui Y. 1996. Synergistic effect of alanin and glycine on bovine embryos cultured in a chemically defined medium and animo acid uptake by in vitro-produced bovine morulae and blastocyst. Biol. Reprod., 55(6):1383-1389 https://doi.org/10.1095/biolreprod55.6.1383
  30. Leibfried ML and First NL. 1979. Characterization of bovine follicular oocytes and their ability to matured in vitro. J. Anim. Sci., 48:76-86 https://doi.org/10.2527/jas1979.48176x
  31. Long CR, Dobrinsky JR and Johnson LA. 1999. In vitro production of pig embryos: comparisons of culture media and boars. Theriogenology, 51: 1375-1390 https://doi.org/10.1016/S0093-691X(99)00081-3
  32. Marchal R, Feugang JM, Perreau C, Venturi M, Terqui M and Mermillod P. 2001. Meiotic and development competence of prepubertal and adult swine oocytes. Theriogenology, 56:17-29 https://doi.org/10.1016/S0093-691X(01)00539-8
  33. Mattioli M, Ealeati ML and Seren E. 1989. Developmental competence of pig oocyte maturation fertilized in vitro. Theriogenology, 31: 1201-1207 https://doi.org/10.1016/0093-691X(89)90089-7
  34. Motlik J and Fulka J. 1974. Fertilization of pig follicular oocytes cultivated in vitro. J. Reprod. Fert., 36:235-237 https://doi.org/10.1530/jrf.0.0360235
  35. Ogawa T, Ono T and Marrss RP. 1987. The effect of serum fractions on single-cell mouse embryos in vitro. J. Vitro Fertil. Embryo Transfer, 4:153-158 https://doi.org/10.1007/BF01555462
  36. Pincus G and Enzmann EV. 1935. The comparative behaviour of mammalian eggs in vivo and in vitro. J. Exp. Med., 62:655-657
  37. Pinyopummintr T and Bavister BD. 1994. Development of bovine embryos in a cell-free culture medium: Effects of type of surum, timing of its inclusion and heat inactivation. Theriogenology, 41:1241-1249 https://doi.org/10.1016/0093-691X(94)90481-W
  38. Romero A and Seidel GE Jr. 1994. Effects of bovine follicular fluid on maturation of bovine oocytes. Theriogenology, 41:383-394 https://doi.org/10.1016/0093-691X(94)90074-S
  39. Saito H, Berger T, Mishell DR Jr and Marrs RP. 1984. The effect of serum fractions on embryo growth. Fetil. Steril., 41:761-765 https://doi.org/10.1016/S0015-0282(16)47846-5
  40. Shamsuddin M, Larsson B, Gustaffsson H and Todriguez-Martinez H. 1994. A serum-free, cell-free culture system for development of bovine one-cell embryos up to blastocyst stage with improved viability. Theriogenology, 41: 1033-1043 https://doi.org/10.1016/S0093-691X(05)80026-3
  41. Sirard MA and Bilodeau S. 1990. Granulosa cells ingibit the resumption of meiosis in bovine oocytes in vitro. Biol. Reprod., 43:777-783 https://doi.org/10.1095/biolreprod43.5.777
  42. SAS/STAT. 1996. SAS user guide. release 6.12 edition, SAS Inst. inc., Cary NC., USA
  43. Takagi Y, Mori K, Tomizawa M, Takahashi T, Sugawara S and Masaki J. 1991. Development of bovine oocytes matured, fertilized and cultured in a serum-free, chemically defined medium. Theriogenology, 35:1197-1207 https://doi.org/10.1016/0093-691X(91)90366-L
  44. Yamashita S and Hoshi H. 1996. Bovine blastocyst formation from IVM/IVF produced zygotes in serum and serum free medium. Theriogenology, 46(1):197 abstract
  45. Yoshida M, Ishizaki Y and Kawagishi H. 1990. Blastocyst formation by pig embryos resulting from in vitro fertilization of oocytes matured in vitro. J. Reprod. Fertil., 88:1-8 https://doi.org/10.1530/jrf.0.0880001
  46. Vatzias G, Hagen DR. 1999. Effects of porcine follicular fluid and oviduct-conditioned media on maturation and fertilization of porcine oocytes in vitro. Biol Reprod., 60:42-48 https://doi.org/10.1095/biolreprod60.1.42
  47. Wang WH, Niwa K and Okuda K. 1991. In vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa. J. Reprod. Fertil., 93:491-496 https://doi.org/10.1530/jrf.0.0930491