체외 환경에서 생산되는 배아 (Embryo)는 활성산소종 (Reaction oxygen species, ROS) 수준이 일정 수준을 초과함에 따라 산화적인 손상을 받게 된다. 선행연구에 따르면, 항산화제는 ROS를 감소시켜주는 효과를 가지기 때문에 ROS로부터 오는 배아의 단백질, DNA의 손상, 세포 자멸사를 방지하여 배아의 발달률을 향상시킨다. 이전연구에 따르면 항산화제로써 엘라그산 (Ellagic acid, EA)은 ROS를 효과적으로 제거하고, 난자의 산화스트레스를 방지하는 효과를 가지고 있다고 보고되었다. 그리하여, 본 연구를 통해 우리는 소의 수정란 배양체계 중 in vitro culture (IVC) 단계에서 EA의 농도 (0, 5, 10 μM) 별 첨가가 소의 수정란 발달률과, 질적 수준에 미치는 영향을 조사하고자 실험을 진행하였다. 결과적으로, 배반포의 단계별 발달 수준에서 cleavage 형성률은 EA첨가군과 대조군 간의 차이를 발견할 수 없었으나 배반포 형성률에서는 모든 EA 첨가군들이 대조군보다 높았고 EA 첨가군 중에 5 μM 첨가군이 가장 높았다 (p < 0.05). 생산된 배반포의 총 세포 수는 5 μM EA 첨가군이 대조군과 10 μM EA 첨가군 보다 유의적으로 높았으며, 대조군과 10 μM EA 첨가군 사이의 유의적 차이는 없었다 (Control vs. 5 μM vs. 10 μM; 137 ± 7.90 vs. 163.2 ± 7.42 vs. 138.8 ± 6.67, p < 0.05). 세포 자멸사 세포 수는 모든 EA 첨가군들이 대조군보다 유의적으로 낮았다 (Control vs. 5 μM vs. 10 μM; 22.65 ± 4.08, 9.61 ± 1.55, 6.14 ± 0.90, p < 0.05). ROS 수준에서 모든 EA 첨가군들과 대조군 간의 유의적 차이는 없었다 (Control vs. 5 μM vs. 10 μM; 6.81 ± 1.31, 3.86 ± 0.23, 4.11 ± 0.18, p < 0.05). qRT-PCR 실험 결과에서 Nrf2 gene expression은 대조군과, 5 μM 첨가군에서 유의적 차이가 없었으나, 10 μM 첨가군에서는 유의적으로 상향 조절된 것을 관찰하였다. Keap1 gene expression은 5 μM 첨가군에서 유의적으로 하향 조절된 것을 관찰하였다. 하지만 EA의 농도가 10 μM으로 높아짐에 따라 발현 수준이 증가한 것을 관찰할 수 있었다. CAT gene expression은 5 μM 첨가군에서 유의적으로 상향조절 되었으나 10 μM 첨가군에서는 유의적인 차이를 보이지 않았다. SOD1 gene expression은 대조군과 5 μM 첨가군은 유의적인 차이를 보이지 않았으나 10 μM 첨가군에서는 유의적으로 상향 조절된 것을 관찰하였다.

Embryo produced in vitro environments can be oxidatively damaged as the level of the reaction Oxygen Species (ROS) exceeds a certain level. According to previous studies, antioxidants have the effect of reduction ROS to a low level, thus preventing damage to the protein and DNA of embryos from ROS and apoptosis of cells, thereby improving the development rate of embryos. Previous studies have shown that ellagic acid (EA) as an antioxidant can effectively remove ROS and prevent oxidative stress of oocyte. Thus, though this study, we conducted an experiment to investigate the effect of the addition of EA by concentration (0, 5, 10 μM) on the development rate and quality of blastocyst in the in vitro culture stage of bovine. To investigate the effect of EA addition, we measured the developmental level of blastocysts, total number of cells, number of apoptosis cells, ROS production level, and mRNA expression level of Nuclear factor erythroid 2-related factor 2 - Kelch-like ECH-associated protein 1 (Nrf2-Keap1) pathway-related genes (Nrf2, Keap1, Catalase (CAT), Superoxide dismutase 1 (SOD1). The Nrf2-Keap1 pathway is an antioxidant-related mechanism. Through the addition of EA, we want to observe the expression of CAT and SOD1 genes that are related to the downstream of Nrf2-Keap1 pathway as well as Nrf2 and Keap1 genes that are regulated antioxidant mechanisms. As a result, no difference was found in cleavage rate between EA and control group (Control vs. 5 μM vs. 10 μM; 91.42 ± 1.52 vs 93.77 ± 1.45 vs 94.27 ± 1.18), but in blastocyst formation rate, all EA treat group were higher than in the control group, and 5 μM of EA group was the highest (Control vs. 5 μM vs. 10 μM; 41.09 ± 2.80 vs 53.63 ± 2.44 vs 45.27 ± 1.86, p < 0.05). The total cell number of the blastocyst was significantly higher in the 5 μM of EA treat group than in the control group and the 10 μM of EA treat group, and there was no significant difference between the control group and the 10 μM of EA treat group (Control vs. 5 μM vs. 10 μM; 137 ± 7.90 vs. 163.2 ± 7.42 vs. 138.8 ± 6.67, p < 0.05). The number of apoptosis cells was significantly lower in all EA treat groups than in the control group, and significantly higher in the treat concentration, the lower the apoptosis (Control vs. 5 μM vs. 10 μM; 22.65 ± 4.08, 9.61 ± 1.55, 6.14 ± 0.90, p < 0.05). There was no significant difference between all EA treat groups and control groups at the ROS level (Control vs. 5 μM vs. 10 μM; 6.81 ± 1.31, 3.86 ± 0.23, 4.11 ± 0.18). In the qRT-PCR experimental results, there was no significant difference in Nrf2 gene expression between the control group and the 5 μM treat group, but significantly upregulated in the 10 μM treat group (Control vs. 5 μM vs. 10 μM; 1.01 ± 0.04, 0.94 ± 0.05, 1.45 ± 0.06, p < 0.05). The Keap1 gene expression was significantly down-regulated downward in the 5 μM treat group. However, it was observed that the expression level increased as the 10 μM treat group (Control vs. 5 μM vs. 10 μM; 1.01 ± 0.05, 0.61 ± 0.03, 1.64 ± 0.16, p < 0.05). CAT gene expression was significantly upregulated in the 5 μM treat group, but there was no significant difference in the 10 μM treat group (Control vs. 5 μM vs. 10 μM; 1.04 ± 0.08, 1.93 ± 0.27, 1.65 ± 0.16, p < 0.05). SOD1 gene expression, there was no significant difference between the control group and the 5 μM treat group, but it was observed that it was significantly up- regulated in the 10 μM treat group (Control vs. 5 μM vs. 10 μM; 1.03 ± 0.07, 1.08 ± 0.11, 2.63 ± 0.21, p < 0.05).

초록
Abstract
서론
재료 및 방법
    1. 공시동물과 시약
    2. 실험 디자인
    3. 미성숙 난자 채란 및 체외성숙
    4. 체외수정 및 엘라그산을 첨가한 체외배양
    5. TUNEL Assay
    6. mRNA 추출 및 cDNA 합성
    7. qRT-PCR 분석
    8. H2DCFDA Assay (ROS Detection)
    9. 통계처리
결과 및 고찰
    1. 엘라그산 농도 설정
    2. 엘라그산 첨가된 배아의 수정률 및 발생률
    3. 엘라그산 첨가한 8일 차 소 배아의 총 세포 수
    4. 유전자 발현 분석
    5. 소 배아의 세포질 내 ROS 수준에 EA가 미치는 효과
References

• 강선민(경상국립대학교 축산생명학과) | Seon-Min Kang (Department of Animal Science, Gyeongsang National University, Jinju, 52828, Korea)
• 주명돈(경상국립대학교 응용생명과학부 (BK21 Four)) | Myeong-Don Joo (Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Korea)
• 공일근(경상국립대학교 축산생명학과, 농업생명과학연구원, 경상국립대학교 응용생명과학부 (BK21 Four)) | Il-Keun Kong (Department of Animal Science, Gyeongsang National University, Jinju, 52828, Korea, Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Korea, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Korea) Corresponding autho