Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
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Effects of Some Factors on In Vitro Production of Embryos from Antral Follicle-Derived Porcine Oocytes III. Effects of Fertilization Media and the Sperm Concentration during Fertilization on In Vitro Fertilization and Development

돼지 난포란 유래 체외수정란 생산에 대한 제요인의 영향 III. 체외수정배양액과 정자농도가 체외수정 및 체외발달에 미치는 영향

  • 연성흠 (농촌진흥청 축산연구소 가축유전자원시험장) ;
  • 손동수 (농촌진흥청 축산연구소 가축유전자원시험장) ;
  • 진현주 (농촌진흥청 축산연구소 가축유전자원시험장) ;
  • 최선호 (농촌진흥청 축산연구소 가축유전자원시험장) ;
  • 김인철 (농촌진흥청 축산연구소 가축유전자원시험장) ;
  • 박창식 (충남대학교 동물자원학부) ;
  • 이규승 (충남대학교 동물자원학부)
  • Published : 2004.12.01
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Abstract

This study was carried out to examine the effects of fertilization media and sperm concentration on in vitro fertilization (IVF) and development (IVD) of porcine oocytes matured in vitro. Cumulus-oocyte complexes (COCs) were collected from antral follicles of porcine ovaries collected from abattoir, and were matured in vitro in modified NCSU-23 (mNCSU-23) supplemented with 10％ porcine follicular fluid (pFF). After the fertilization by experimental scheme, putative embryos were developed in vitro in NCSU-23. The results are as follows. When the oocytes were fertilized in vitro in modified TBM or modified TLP-PVA by 1 ${\times}$10$^{5}$ sperm/$m\ell$, all of the fertilization parameters were not significantly different between two media. Subsequently, as these putative embryos were developed in vitro in NCSU-23, the percentage of oocytes cleaved and of blastocysts were not different between two media, either. When the oocytes were fertilized in vitro in mTBM by 5${\times}$10$^4$, 1${\times}$10$^{5}$ or 5${\times}$10$^{5}$ sperm/$m\ell$, all of the fertilization parameters were significantly (P<0.05 or P<0.01) increased as sperm concentration was elevated. Subsequently, as these putative embryos were developed in vitro in NCSU-23, the percentage of oocytes cleaved and of blastocysts were significantly boosted (P<0.01) as sperm concentration at fertilization was elevated from 5${\times}$10$^4$ to 1${\times}$10$^{5}$ sperm/$m\ell$, but were not different between 1${\times}$10$^{5}$ and 5${\times}$10$^{5}$ sperm/$m\ell$.

본 연구는 mNCSU-23에서 체외성숙시킨 난자를 이용하여 돼지 체외수정시 적절한 배양액과 정자농도를 구명하고자 수행하였다. 정액은 액상정액을 이용하였고 체외수정배양액으로 mTBM과 mTLP-PVA, 정자농도는 운동정자수 5${\times}$$10^4$, 1${\times}$$10^{5}$ , 5${\times}$$10^{5}$ sperm/$m\ell$로 체외수정 한 다음 NCSU-23에서 체외발달을 유도한 결과는 다음과 같다. 1. 돼지 체외성숙란을 mTBM 또는 mTLP-PVA에서 운동정자수 1${\times}$$10^{5}$ sperm/$m\ell$로 체외수정시킨 다음 체외발달시킨 결과, 정자침투율, 웅성전핵형성율, 다정자수정발생율, 난분할율, 배반포 발달율 등 어느 것도 두 배양액간 차이를 나타내지 않았다. 2. 체외성숙란을 mTBM에서 운동정자수 5${\times}$$10^4$, 1${\times}$$10^{5}$ , 5${\times}$$10^{5}$ sperm/$m\ell$l로 체외수정시킨 다음 체외발달시킨 결과, 정자침투율, 웅성전 핵형성율, 다정자수정발생율 등은 모두 정자 농도간 유의적인 차이를 보였고(P<0.05), 난 분할율과 배 반포발달율은 5${\times}$$10^4$이 1${\times}$$10^{5}$ , 5${\times}$$10^{5}$ sperm/$m\ell$보다 유의 적으로 낮았으나(P<0.01), 1${\times}$$10^{5}$ 와 5${\times}$$10^{5}$ sperm/$m\ell$ 간에는 차이가 없었다. 따라서, 액상정액을 이용한 돼지 체외성숙란의 체외수정배양액으로 mTBM과 mTLP-PVA간 차이는 없으며, mTBM에서 체외수정시킬 때 정자농도는 운동정자수로 1${\times}$$10^{5}$ sperm/$m\ell$가 적절할 것으로 사료된다.

Keywords

References

  1. Abeydeera LR. 2002. In vitro production of embryos in swine. Theriogenology, 57:256-273
  2. Abeydeera LR and Day BN. 1997. In vitro penetration of pig oocytes in a modified Tris- buffered medium: effect of BSA, caffeine and calcium. Theriogenology, 48:537-544 https://doi.org/10.1016/S0093-691X(97)00270-7
  3. Abeydeera LR, Funahashi H, Kim NH and Day BN. 1997. Clortetracycline fluorescence patterns and in vitro fertilization of frozen-thawed boar spermatozoa incubated under various bicarbonate concentrations. Zygote, 5: 117-125 https://doi.org/10.1017/S0967199400003798
  4. Abeydeera LR, Wang WH, Cantley TC, Prather RS and Day BN. 1998. Presence of $\beta$-mercaptoethanol can increase the glutathione content of pig oocytes matured in vitro and the rate of blastocyst development after in vitro fertilization. Theriogenology, 50:747-756 https://doi.org/10.1016/S0093-691X(98)00180-0
  5. Boquest AC, Abeydeera LR, Wang WH and Day BN. 1999. Effect of adding reduced glutathione during insemination on the development of porcine embryos in vitro. Theriogenology, 51: 1311-1319 https://doi.org/10.1016/S0093-691X(99)00075-8
  6. Byun TH, Lee SH and Song HB. 1991. Development of a rapid staining method of the oocytes from domestic animal. Kor. J. Anim. Sci., 33: 25-31
  7. 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
  8. Day BN, Abeydeera LR, Johnson LA, Welch GR, Wang WH, Cantley TC and Rieke A. 1998. Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y bearing spermatozoa stored by high speed flow cytometry. Theriogenology, 49:360(abstr.) https://doi.org/10.1016/S0093-691X(98)90713-0
  9. Florman HM, Tombes RM, First NL and Babcock DF. 1989. An adhesion-associated agonist from the zona pellucida activates G protein-promoted elevations of internal Ca2+ and pH that mediate mammalian sperm acrosomal exocytosis. ev. Biol., 135:133-146
  10. Fraser LR. 1995. Ionic control of sperm function. Reprod. Fertil. Dev., 7:905-925 https://doi.org/10.1071/RD9950905
  11. Harrison RA, Mairet B and Miller NG. 1993. Flow cytometric studies of bicarbonate-mediated Ca2+ influx in boar sperm populations. Mol. Reprod. Dev., 35:197-208 https://doi.org/10.1002/mrd.1080350214
  12. Iritani A, Niwa K and Imai H. 1978. Sperm penetration in vitro of pig follicular oocytes matured in culture. J. Reprod. Fertil., 54: 379-383 https://doi.org/10.1530/jrf.0.0540379
  13. Kidson A, Colenbrander B, Verheijden JHM and Bevers MM. 2001. Polyspermia in the pig is dependent on both IVF medium and sperm dose during fertilization in vitro. Proc. 6th Int. Conf. Pig Reprod., 75(abstr.)
  14. Lee MA and Storey BT. 1989. End point of first stage of zona pellucida-induced acrosome reaction in mouse spermatozoa charac- terized by acrosomal H+ and $Ca^2^+$ permeability: Population and single cell kinetics. Gamete Res., 24: 303-326 https://doi.org/10.1002/mrd.1120240307
  15. 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
  16. Marchal R, Feugang JM, Perreau C, Venturi E, Terqui M and Mermillod P. 2001. Meiotic and developmental competence of prepubertal and adult swine oocytes. Theriogenology, 56:17-29 https://doi.org/10.1016/S0093-691X(01)00539-8
  17. Martinez-Madrid B, Dominguez E, Alonso C, Diaz C, Garcia P and Sanchez R. 2001. Effect of IVF medium and sperm concentration on fertilization parameters. Proc. 6th. Int. Conf. Pig Reprod., 74(abstr.)
  18. Mattioli M, Bacci ML, Galeati G and Seren E. 1989. Developmental competence of pig oocytes matured and fertilized in vitro. Theriogenology, 46:1201-1207
  19. Motlik J and Fulka J. 1974. Fertilization of pig follicular oocytes cultivated in vitro. J. Reprod. Fertili., 36:235-237 https://doi.org/10.1530/jrf.0.0360235
  20. Nagai T and Moor RM. 1990. Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilized in vitro. Mol. Reprod. Dev., 26:377-382 https://doi.org/10.1002/mrd.1080260413
  21. Parrish JJ, Susko-Parrish JL and Graham JK. 1999. In vitro capacitation of bovine spermatozoa: role of intracellular calcium. Theriogenology, 51:461-472 https://doi.org/10.1016/S0093-691X(98)00240-4
  22. Parrish JJ, Susko-Parrish JL, Uguz C and First NL. 1994. Differences in the role of cyclic adenosine 3',5'-monophosphate during capacitation of bovine sperm by heparin or oviduct fluid. Biol. Reprod., 51:1099-1108 https://doi.org/10.1095/biolreprod51.6.1099
  23. Petters RM and Wells KD. 1993. Culture of pig embryos. J. Reprod. Fertil., 48: 61-73
  24. Rath D. 1992. Experiments to improve in vitro fertilization techniques for in vivo-matured porcine oocytes. Theriogenology, 37:885-896 https://doi.org/10.1016/0093-691X(92)90050-2
  25. Sirard MA, Dubuc A, Bolamba D, Zheng Y and Coenen K. 1993. Follicle- oocyte-sperm interactions in vivo and in vitro in pigs. J. Reprod. Fertil., 48(suppl):3-16
  26. Tulsiani DRP, Abou-Haila A, Loeser CR and Pereira BMJ. 1998. The Biological and functional significance of the sperm acrosome and acrosomal enzymes in mammalian fertilization. Exp. Cell Res., 240:151-164 https://doi.org/10.1006/excr.1998.3943
  27. Uguz C, Vredenburgh WL and Parrish JJ. 1994. Heparin-induced capacitation but not intracellular alkalinization of bovine sperm is inhibited by Rp-adenosine-3',5'-cyclic monophosphorothioate. Biol. Reprod., 51:1031-1039 https://doi.org/10.1095/biolreprod51.5.1031
  28. Visconti PE and Kopf GS. 1988. Regulation of protein phosphorylation during sperm capacitation. Biol. Reprod., 59:1-6
  29. Walensky LD and Snyder SH. 1995. Inositol 1,4,5- trisphosphate receptors selectively localized to the acrosomes of mammalian sperm. J. Cell Biol., 130:857-869 https://doi.org/10.1083/jcb.130.4.857
  30. Xu X, Ding J, Seth PC, Harbison DS and Foxcroft GR. 1996a. In vitro fertilization of in vitro matured pig oocytes: Effects of boar and ejaculate fraction. Theriogenology, 45:745-755 https://doi.org/10.1016/0093-691X(96)00004-0
  31. Xu X, Seth PC, Harbison DS, Cheung AP and Foxcroft GR. 1996b. Semen dilution for assessment of boar ejaculate quality in pig IVM and IVF systems. Theriogenology, 46:1325-1337 https://doi.org/10.1016/S0093-691X(96)00313-5
  32. Yoshida M, Mizoguchi Y, Ishigaki K, Kojima T and Nagai T. 1993. Birth of piglets derived from in vitro fertilization of pig oocytes matured in vitro. Theriogenology, 39:1303-1311 https://doi.org/10.1016/0093-691X(93)90232-T
  33. 연성흠, 손동수, 한만희, 위미순, 최선호, 이규승. 2004. 돼지 난포란 유래 체외수정란 생산에 대한 제요인의 영향. Ⅱ. 체외성숙배양시 EGF와 COC의 수가 체외성숙, 체외수정 및 체외발달에 미치는 영향. 한국수정란이식학회지, 19: 173-183