산소 소비량에 의한 한우 수정란 평가에 미치는 영향 = Evaluation of Hanwoo Embryos by Oxygen Consumption Rate

다국어 초록 (Multilingual Abstract)

Oxygen consumption rate has been regarded as a useful indicator for assessment of mammalian embryo quality. However, there was no standard criterion to measure the oxygen consumption rate of embryos.
Here, we measured oxygen consumption rate of bovine embryos at various developmental stages was measured using a Scanning electrochemical microscopy (SECM). We found that the oxygen consumption rates significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2cell stage to morula stage), indicating that oxygen consumption rates reflects the cell number(5.2~7.6×1014/mol s-1, p<0.05). In the morula stage embryos, the oxygen consumption rates of in vivo derived embryos was significantly higher than that of in vitro produced embryos(4.0×1014/mol s-1 versus 2.4×1014/mol s-1, p<0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro derived bovine blastocyst stage embryos (4.7×1014/mol s-1 versus 1.0×1014/mol s-1, p<0.05).
And performed to investigate whether oxygen consumption rate reflects morphological grade of in vivo derived bovine blastocyst stage embryos (blastocyst). The oxygen consumption rates of in vitro produced blastocyst was compared to its total cell number. In addition, pregnant rate was measured after transplation of in vivo blastocyst with different oxygen consumption rates. The quality of blastocyst collected on day 7 after artificial insemination was categorized as grade Ⅰ and Ⅱ(GⅠand GⅡ) based on microscopic observation of the morphology. Oxygen consumption rates of blastocyst was measured using a SECM and total cell number of in vitro blastocyst was enumerated by counting cells stained by propidium iodide.
Pregnancy of recipient cow was confirmed with rectal palpation after 60 days of embryo transfer. The oxygen consumption rate or GⅠ blastocysts were significantly higher than those of GⅡ blastocysts (10.2×1015/mol s-1 versus 6.4×1015/mol s-1, p<0.05). Total cell numbers of in vitro blastocysts were 74.8, 90.7 and 110.2 in the oxygen consumption rates of below 10.0, 10.0~12.0 and over 12.0~ 1015/mol s-1, respectively. Total cell number was significantly increased in embryos with high oxygen consumption rate(p<0.05). Pregnant rate in recipient cow was 0, 60 and 80% in the transplantation of embryo with the oxygen consumption rates of below 10.0, 10.0~12.0 and over 12.0 × 1014/mol s-1, respectively.
The expression of anti-oxidant enzyme, apoptosis, and pluripotent gene was determined using Real-time PCR by extracting RNA according to the oxygen consumption rates (1014/mol s-1) of in vivo embryo. Although in anti-oxidant enzyme over 10.0 group showed higher level than below 10.0 group, there was no significant difference. Also, in apoptosis gene, there was no significant difference between over 10.0 group and below 10.0 group. In OCT-4, pluripotent gene, there was a significant difference(p<0.05) between the below 10.0 (0.98±0.1) and over 10.0 (1.79±0.2).
In conclusion, it can be useful for assessing the quality of embryo by measuring the oxygen consumption rate of Hanwoo embryo with SECM. The data confirms the assumption that when the embryo is alive and has a high number of cells then the oxygen consumption rate will increase. With a consistent condition of the embryo transfer operator’s skill and health of recipient, implanting a embryos with high oxygen consumption rate is expected to increase Pregnant rate. It was also shown that more oxygen consumption rate in OCT-4, pluripotent gene, leads to higher content of cellular regulatory factor with multipotency.

목차 (Table of Contents)

• Ⅰ. 서 론 1
• Ⅱ. 연 구 사 3
• 1. 수정란의 산소 소비량 3
• 2. 수정란의 유전자 발현 4
• Ⅲ. 재료 및 방법 7
• 1. 수정란의 산소 소비량 측정 및 수정란이식 7
• (1) 공시동물 7
• (2) 체내수정란의 생산 7
• (3) 체외수정란의 생산 8
• (4) 수정란의 산소 소비량 측정 9
• (5) 수정란의 총세포수 조사 10
• (6) 수정란이식 11
• (7) 임신 진단 11
• (8) 통계 분석 11
• 2. 수정란의 산소 소비량 측정 및 유전자 발현 12
• (1) 공시재료 12
• (2) Total RNA 추출 및 cDNA 합성 12
• (3) Real-time PCR에 의한 정량적인 유전자 분석 13
• Ⅳ. 결 과 15
• 1. 수정란의 산소 소비량 측정 및 수정란이식 15
• (1) 체외수정란의 발달 단계별 산소 소비량 변화 15
• (2) 체내외 수정란의 산소 소비량 비교 17
• (3) 동결융해 수정란 생존여부에 따른 산소 소비량 비교 19
• (4) 체내수정란의 등급별 산소 소비량 비교 21
• (5) 체외수정란의 산소 소비량과 총세포수 비교 23
• (6) 체내수정란의 산소 소비량에 따른 수정란이식 수태율 25
• 2. 수정란의 산소 소비량에 따른 유전자발현 28
• (1) Anti-oxydant Enzyme 28
• (2) Apoptosis Gene 30
• (3) Pluripotent Gene 32
• Ⅴ. 고 찰 34
• Ⅵ. 결 론 37