Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
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In Vitro Nuclear Maturation of Canine Oocytes Obtained from Differents Stage of Estrus Cycle

개의 발정주기가 난자의 체외성숙에 미치는 영향

  • Kim, Min-Kyu (College of Veterinary Medicine, Seoul National University) ;
  • Kim, Hye-Jin (College of Veterinary Medicine, Seoul National University) ;
  • Cho, Jong-Ki (College of Veterinary Medicine, Seoul National University) ;
  • Jang, Gu (College of Veterinary Medicine, Seoul National University) ;
  • Lee, Kyu-Seung (Division of Animal Science & Resources, Chungnam National University) ;
  • Kang, Sung-Geun (College of Veterinary Medicine, Seoul National University) ;
  • Lee, Byung-Chun (College of Veterinary Medicine, Seoul National University) ;
  • Hwang, Woo-Seok (College of Veterinary Medicine, Seoul National University)
  • 김민규 (서울대학교 수의과대학) ;
  • 김혜진 (서울대학교 수의과대학) ;
  • 조종기 (서울대학교 수의과대학) ;
  • 장구 (서울대학교 수의과대학) ;
  • 이규승 (충남대학교 농업생명과학대학 동물자원학부) ;
  • 강성근 (서울대학교 수의과대학) ;
  • 이병천 (서울대학교 수의과대학) ;
  • 황우석 (서울대학교 수의과대학)
  • Published : 2002.08.01
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Abstract

The aim of these experiments was to investigate in vitro nuclear maturation of canine oocyte collected from various stages of estrus cycles, Ovaries were obtained from 1 to 4 year-old mongrel bitch and minced for oocyte collection in phosphate buffered saline with 100 iu penicillin-G $m\ell$／sup -1／, 50 $\mu\textrm{g}$ streptomycin sulphate $m\ell$／sup -1／ and 0.1％ (w／v) polyvinyl alcohol. Cumulus-oocyte-complexes (COCs) were washed in HEPES buffered tissue culture medium (TCM)199 and in vitro matured in TCM-199 culture medium supplemented with sodium pyruvate 0.028mg/$m\ell$, L-glutamine 0.146mg／$m\ell$, penicillin G 10,000 IU／$m\ell$, streptomycin 0.031mg／$m\ell$ and 10％ (v／v) fatal calf serum. COCs were in vitro matured for 48～72 hrs at 39$^{\circ}C$ in humidified 5％ $CO_2$ in air atmosphere. In vitro matured oocytes were remove the cumulus cells using 0.2％ (v／v) hyaluronidase. After denuding, oocyte were placed in acetic acid : methanol : chlorform solusion (3:6 : 1.5 v／v) for 30 sec and acetic acid: ethanol(1:3 v／v) for 48hrs fixation. Nuclear maturation was classified to GV, GVBD, MI, MII and degenerate oocyte under microscopy after 1％ aceto-orcein stain. In vitro maturation rates at 48hrs were not significantly difference among the oocytes collected from different stage of estrus at 15.9％, 16.3％, 23.7％ and 18.2％ for anestrus, proestrus, estrus and diestrus. However, the oocytes maturation(36.6％) of collected from estrus ovaries were significantly different from oocytes derived from proesturs, diestrus and anestrus ovaries(30.8％, 17.5％ and 22.1％; p＜0.05). The overall in vitro maturation rates were significantly higher (p＜0.05) in 72hrs culture than 48hrs culture system. In summary, there was a tendency for higher in vitro maturation rates with the oocyte collected from estrus ovary than other stages of estrus. Also, for nuclear maturation, in vitro culture of oocyte for 72hrs was better than 48hrs culture.

본 연구는 개 난자의 체외성숙율 높이기 위해 개의 발정주기가 체외성숙에 미치는영향과 체외성숙율의 효율적 향상을 위해 실험을 실시하였으며, 다음의 결과를 얻었다. 1. Anestrus, proestrus, estrus와 diestrus의 발정주기로 구분하여 MII로의 체외성숙율을 48시간과 72시간 배양후 관찰한 결과 각각 15.9%, 16.3%, 23.7%와 18.2%와 22.1%, 30.8%, 36.6%와 17.5%로 나타났다. 2. 각 발정주기별로 72시간 배양한 후의 성숙율은 estrus 및proestrus기의 난자가 anestrus 난자보다 유의 적으로 높은 값를 보였다(p＜0.05). 3. 배양시간에 따른 체외성숙율은 72시간 배양한 성숙율이 48시간 배양 후보다 높은 결과를 나타내 었다(p＜0.05).

Keywords

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