Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
권호 표지

Studies on the In Vitro Fertilization and In Vitro Development of Porcine Embryos in Different Culture System

여러 가지 배양조건에서 돼지 난포란의 체외수정 및 체외발달에 관한 연구

  • Kim, Jae-Young (Infertility Medical Center of CHA General Hospital) ;
  • Park, Hyang (Infertility Medical Center of CHA General Hospital) ;
  • Kim, Jae-Myung (College of Medicine, Pochon CHA University) ;
  • Lee, Jung-Hyung (College of Medicine, Pochon CHA University) ;
  • Park, Heum-Dae (Division of Food, Biological and Chemical Engineering, Daegu University)
  • 김재영 (차병원 여성의학연구소 불임의학연구소) ;
  • 박향 (차병원 여성의학연구소 불임의학연구소) ;
  • 김재명 (포천중문의과대학교) ;
  • 이정형 (포천중문의과대학교) ;
  • 박흠대 (대구대학교 식품.생명.화학공학부)
  • Published : 2004.04.01
Download PDF
⟨ Previous Next ⟩

Abstract

The objective of this study was to optimize the selection of sperm, optimal culture system of in vitro derived porcine embryos. The results obtained were summarized as follows: 1. When oocytes were inseminated with liquid sperm and frozen-thaw sperm, the cleavaged rate of liquid sperm (46.2%) was higher than that of frozen-thaw sperm (39.7%), however there were not show significant different each other. The blastocyst rates of liquid sperm (15.8%) was significantly higher than that of frozen-thawed sperm (9.3%)(P< 0.05). 2. When oocytes were inseminated with epididymal sperm after 1, 2 and 3 day storage, the cleavaged rate of epididymal sperm after 1, 2 and 3 day storage was 60.5, 61.0 and 56.8% respectively. The morulae (17.4, 19.9 or 17.3%) and blastocyst (8.7, 15.4, 11.3%) rate of epididymal sperm after 1, 2 and 3 day storage was no significantly respectively(P< 0.05). 3. In vitro developed to cleavaged rate of G1.3/G2.3 media used for culture was significantly(P< 0.05) higher as 62.1% compared with the results using the media NCSU23(52.8), however in vitro developed to blastocyst rate of NCSU23(11.6%) media was significantly(P< 0.05) higher than that'of G1.3/G2.3(4.7%). 4. When the fertilized oocytes were cultured with NCSU23 in addition to 1 mM glutathione(GSH), the cleavaged rate of treated groups of GSH(62.3%) was significantly higher than that of control(53.5%) respectively(P< 0.05). And in vitro developed to blastocyst rates of treated groups of GSH(15.6%) was higher than that of control(12.6%) however, there was no significant difference(P< 0.05).

본 연구는 돼지 체외수정란의 생산에 있어서 체외배양체계를 확립하기 위해 정액의 차이와 보관시간에 따른 체외성숙난자의 체외수정 및 체외발달율을 조사하였고, NCSU23와 G1.3/G2.3 배양액으로 체외배양 시 분할율과 체외발달율을 조사하였고, 체외배양액에 GSH를 첨가 시 배발달율을 조사하였다. 본 연구에서 얻은 결과는 다음과 같다. 1. 액상정액과 동결정액을 사용하여 정액에 따른 체외수정율은 각각 46.2 및 39.7%로써 액상정액을 이용한 것이 수정율이 높았지만 유의적(P<0.05)인 차이는 없었다. 한편 배반포로의 발달율은 액상정액이 동결정액보다 유의적으로 높았다.(P< 0.05) 2. 정소상체미부 정액을 day 1, 2, 3 보관후 체외수정을 유도하였을 때 수정율은 각각 60.5, 61.0 및 56.8%로서 2일 동안 보관후 사용하였을 때가 높았지만 유의적(P<0.05)인 차이가 없었다. 또한 상실배와 배반포기까지의 배발달율에서도 2일 동안 보관 후 사용하였을 때가 높았지만 유의적(P<0.05)인 차이는 없었다. 3. NCSU-23와 G1.3/G2.3으로 나눠 배양 시 분할율과 상실배기까지의 배발달율은 각각 52.8, 62.1%와 16.0, 28.9%로서 G1.3/G2.3에서 유의적(P<0.05)인 차이로 높았다. 그러나 배반포기로의 배발달율을 각각 11.6와 4.7%로서 NCSU-23에서 유의적으로 높았다.(P<0.05) 4. NCSU-23을 기본 배양액으로 하여 1 mM GSH가 첨가된 군의 분할율은 62.3%로서 GSH가 첨가되지 않은 군 53.5%보다 유의적(P<0.05)인 차로 높았지만, 상실배나 배반포기까지의 배발달율은 GSH가 첨가되지 않은 군보다 높았지만 유의적(P<0.05)인 차는 존재하지 않았다.ne을 처리하는 것이 수태율을 향상시키는 것으로 나타났다.25^{\circ}C$에서 발현되었고 이 중 79.1%가 3일령, 4일령과 5일령에 집중적으로 나타났다. 계분의 경우 $25^{\circ}C$ 처리구에서 35.12%(56.95mg/kg), $37^{\circ}C$에서 45.89%(74.40mg/kg)로 나타나 주로 $25^{\circ}C$ 이상에서 발현한 것으로 특징지어졌다. 우분의 경우 $10^{\circ}C$ 처리구에서 28.21%(43.86mg/kg), $25^{\circ}C$에서 49.30% (76.66mg/kg)로 나타나 주로 $25^{\circ}C$ 이하에서 발현한 것으로 나타났다. 3. 배양온도에서 검지 된 뷰틸산의 량은 6일 동안 돈분에서 1,463.87mg/kg, 계분에서 96.72mg/kg, 우분에서 129.18mg/kg이 발현되었으며 돈분의 경우 93.31%(1,365.95mg/kg)가 $25^{\circ}C$에서 발현되었고 이 중 87.92%가 3일령, 4일령과 6일령에 집중적으로 나타났다. 계분의 경우 $37^{\circ}C$ 처리구에서 76.60%(74.09 mg/kg)로 발현되었고 이 중 88%가 1일령, 2일령과 5일령에 집중적으로 나타났다. 우분의 경우 61.55%(79.51mg/kg)가 $25^{\circ}C$에서 발현되었고 이 중 89.6%가 1일령, 3일령과 4일령에 집중적으로 나타났다. 4. 배양온도에서 검지된 이소밸릭산의 량은 6일 동안 돈분에서 6,885.99mg/kg, 계분에서 307.47mg/kg,

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. Bormann CL, Ongeri EM and Krister RL. 2000. Effects of vitamins on maturation of caprine oocytes and their subsequent developmental capacity in vitro. Biol. Reprod. Abst., 62(Suppl 1):166
  3. Calvin HI, Grosshans K and Blake EJ. 1986. Estimation and manipulation of glutathione levels in prepuberal mouse ovaries and ova: relevance to sperm nucleus transformation in the fertilized egg. Gamete Research, 6:107-114
  4. Edwards RG. 1965. Maturation in vitro of mouse, cow, pig, rhesus monkey and human ovarian oocytes. Nature, 208:349-351 https://doi.org/10.1038/208349a0
  5. Iritani A, Niwa K and Imai H. 1978. Sperm penetration of pig follicular oocytes matured in culture. J. Reprod. Fertil., 54:379-383 https://doi.org/10.1530/jrf.0.0540379
  6. Jarrell VI, Day BN and Prather RS. 1991. The transition from maternal to Zygotic control of development occurs during the 4-cell stage in the domestic pig, Suscrofa: Quantitative aspects of protein synthesis. Biol. Reprod., 44:62-68 https://doi.org/10.1095/biolreprod44.1.62
  7. Krisher RL, Lane M and Bavister BD. 1993. Developmental competence and metabolism of bovine embryos cultured in semi definded and defind culture media, Biol. Reprod., 60:1345-1352 https://doi.org/10.1095/biolreprod60.6.1345
  8. Kosower NS and Kosower EM. 1978. The glutathione status of cells. Int. Rev. Cytol., 54:109-160 https://doi.org/10.1016/S0074-7696(08)60166-7
  9. Lafleur MVM, Hoorweg JJ, Westmijze EJ and Retel J. 1994. The ambivalent role of glutathione in the protection of DNA against singlet oxygen. Free. Radic. Res., 21:9-17 https://doi.org/10.3109/10715769409056550
  10. Luvoni GC, Keskintepe L and Brakett BG. 1996. Improvement in bovine embryo production in vitro by glutathione-containing culture media. Mol. Reprod. Dev., 43:437-443 https://doi.org/10.1002/(SICI)1098-2795(199604)43:4<437::AID-MRD5>3.0.CO;2-Q
  11. Mattioli M, Ealeati ML and Seren E. 1989. Developmental competence of pig oocyte mature on fertilized in vitro. Theriogenology, 31:1201-1207 https://doi.org/10.1016/0093-691X(89)90089-7
  12. Meister A and Anderson ME. 1983. Glutathione. Annu. Rev. Biochem., 52:711-760 https://doi.org/10.1146/annurev.bi.52.070183.003431
  13. Nagai T, Takahasi T, Masuda H, Shioya Y, Kuwayama M, Fulushima M, Iwasaki S and Hanada A. 1988. In vitro fertilization of pig oocytes by frozen boar spermatozoa. J. Reprod. Fettil., 84:585-591 https://doi.org/10.1530/jrf.0.0840585
  14. Ongeri EM, Bormann CL, Butler R, Mellican D, Schaller E, Blash S, Behboodi E, Krisher RL. 2000. Biol. Reprod. Abst., 62(Suppl 1):322
  15. Schoenback RA, Peters MS, Rickords LF, Stumpf TT and Prather RS. 1992. Characterization of deoxyribonucleic acid synthesis and the transition from maternal to embryonic control in the 4-cell porcine embryos. Biol. Reprod., 47:1118-1125 https://doi.org/10.1095/biolreprod47.6.1118
  16. Swain JE, Bormann CL and Krisher RL. 2001. Development and viability of in vitro derived porcine blastocysts cultured in NCSU23 and G1.2/G2.2 sequential medium. Theriogenology, 56:459-469 https://doi.org/10.1016/S0093-691X(01)00577-5
  17. Yoshida M and Kojima Y 1989. Male pronuclear formation by boar spermatozoon with hairpin curved tail in zona-free hamster egg. Jpn. J. Vet. Sci., 51(2):428-430 https://doi.org/10.1292/jvms1939.51.428
  18. Zheng YS, Fiser SP and Sirard MA. 1992. The use of ejaculated boar semen after freezing in 2 or 6% glycerol for in vitro fertilizationof porcine oocytes matured in vitro. Theriogenology, 38: 1065-1075 https://doi.org/10.1016/0093-691X(92)90120-G