Lipopolysaccharide (LPS) is an endotoxin factor present in the cell wall of Gram-negative bacteria and induces various immune responses to infection. Recent studies have reported that LPS induces cellular stress in various cells including oocytes and embryos. Melatonin (N-acetyl-5-methoxytryptamine) is a regulatory hormone of circadian rhythm and a powerful antioxidant. It has been known that melatonin has an effective function in scavenging oxygen free radicals and has been used as an antioxidant to reduce the cytotoxic effects induced by LPS. However, the effect of melatonin on LPS treated early embryonic development has not yet been confirmed. In this study, we cultured mouse embryos in medium supplemented with LPS or/and melatonin up to the blastocyst stage in vitro and then evaluated the developmental rate. As a result of the LPS-treatment, the rate of blastocyst development was significantly reduced compared to the control group in all the LPS groups. Next, in the melatonin only treated group, there was no statistical difference in embryonic development and no toxic effects were observed. And then we found that the treatment of melatonin improved the rates of compaction and blastocyst development of LPS-treated embryos. In addition, we showed that melatonin treatment decreased ROS levels compared to the LPS only treated group. In conclusion, we demonstrated the protective effect of melatonin on the embryonic developmental rate reduced by LPS. These results suggest a direction to improve reproduction loss that may occur due to LPS exposure and bacterial infection through the using of melatonin during in vitro culture.

나도풍란의 경우 교배 후 화분이 발아하여 화분관 신장이 이루어지고 화분관은 수분 후 5-7일경 배주원기에 도착하였다. 어린배주 발달은 수정 후에야 비로소 진행되며 수분 후 14 일경에는 배주의 원기가 둥근 돌기형태이고 34일경에는 filament형태로 길어지며 Megaspore Mother cell 모습이 나타났다. 수분 후 40-50일 사이 배낭을 갖춘 성숙된 배주가 형성된다. 배주가 수정준비상태가 될 때까지 화분관 신장은 계속 활발히 진행되어 배주 주위를 완전 히 둘러싸고 있는 것으로 나타났다. 수분 후 54일경 화분관이 배낭을 침투하여 수정하는 모습이 관찰되었으며 보통 수정은 54에서 64일 사이에 일어나는 것으로 파악되었다. 수정 후 접합자세포는 가로로 한번 분열하여 윗부분의 작은 세포와 밑부분의 큰 세포로 나뉘어 지며 윗부분의 세포는 proembryo로 발달하며 밑부분의 세포는 다시 세로로 분열하여 두개 의 세포로 나뉘어지고 계속 분열하여 영양분을 공급하는 suspensor로 발달한다. 수분 후 110 일 경 배와 16개의 다리를 가진 suspensor가 부착 되어 있는 배 발달 양상이 관찰되었다. Proembryo는 계속 분열하여 140일경 종자로 성숙하는 것으로 나타났다. 나도풍란과 호접란 속간잡종을 얻기 위해 수분 후 발달하는 시기별로 꼬투리를 채취하여 인공배지에 발아시킨 결과, 수분 후 56일 이전의 배주로는 발아가 힘들었고 발아가능시점은 수분 후 69일로 분 석되었다.

Partheno Embryo's research is known to play a very important role in identifying the development of embryonic cells or analyzing the genetic mechanisms of embryonic development, but the information on apoptosis formed during the early stage of development on Partheno Embryo is very little. Therefore, this study analyzed whether the embryonic cell death of unit embryos can be inhibited by adding Scriptaid, one of HDACi, which plays a role in demethylation of histone proteins as a method of regulating the cell cycle in the early embryo development of Partheno Embryo. As a result, the differentiation rate was higher in the group that added Scriptaid and FBS, but the cellular development was higher in the group that added pregnant serum to Scriptaid. As a result of analyzing the expression of the gene through IF and PCR, the group with the addition of gestational serum increased the expression of BCL2 and PCNA, which affects the anti-Casp3 action in cell survival. In addition, it is interpreted that treatment of Scriptaid for 16 hours, rather than 24 h treatment lowers the expression of Casp-3, a representative factor of apoptosis, and also increases embryonic development, thus affecting early embryo development. Therefore, it is concluded that the 16-hour treatment of Scriptaid and the use of gestational serum will inhibit cell death in the early embryonic development and increase the development rate of the embryo.

This study was investigated to test whether the zygote recognized the topoisomerase II beta (TOP2B) mediated DNA fragmentation in epididymal spermatozoa or the nuclease degradation in vas deferens spermatozoa by testing for the presence of gammaH2AX (γH2AX). The γH2AX is phosphorylation of histone protein H2AX on serine 139 occurs at sites flanking DNA double-stranded breaks (DSBs). The presence of γH2AX in the pronuclei of mouse zygotes which were injected with DNA broke epididymal spermatozoa was tested by immunohistochemistry at 5 and 9 h post fertilization, respectively. Paternal pronuclei that arose from epididymal spermatozoa treated with divalent cations did not stain for γH2AX at 5 h. On the other hand, in embryos injected with vas deferences spermatozoa that had been treated with divalent cations, γH2AX was only present in paternal pronuclei, and not the maternal pronuclei at 5 h. Interestingly, both pronuclei stained positively for γH2AX for all treatments and controls at 9 h after sperm injection. In conclusion, the embryos recognize DNA that is damaged by nuclease, but not by TOP2B because H2AX in phosphorylated in paternal pronuclei resulting from spermatozoa treated with fragmented DNA from vas deferens spermatozoa treated with divalent cations, but not from epididymal spermatozoa treated the same way.

The purpose of this study was to analyze whether FSH and LH hormone treatment directly or indirectly affect embryo development in embryonic development. To determine this, we compared the development of embryonic cells through the expression pattern of MMPs. As a result, 33.8% of blastocysts were formed in FSH added group, 20.8% in LH added group and 10% in FSH + LH added group. In addition, the activity of MMP-9 was highly detected in the FSH-added group, and the expression of Casp-3 was much lower than that of the other groups. These results suggest that the addition of FSH seems to increase the activity of MMP-9 in embryonic cells, and that LH, on the contrary, may activate MMP-2 activity. In addition, the expression level of MMP-2 in the FSH-added group was high in the Trophoblast cell group and in the LH-added group, the hormone ideal secretion might affect the development of the embryonic cell.

Alpha-linolenic acid (ALA; n-3 18:3), a one of omega-3 fatty acid, is mainly contained in chloroplast of plant and ALA is an essential fatty acid, not synthesized in mammalian body, it must be supplied from foods. Polyspermy is especially high on in vitro fertilization (IVF) in pigs, which is a major obstacle to in vitro embryo production systems. In our previous study, when ALA was supplemented during in vitro maturation (IVM), the methaphase-II rate and gluthathione level was increased. The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) supplementation during IVM and subsequent of IVF in pigs. The cumulus-oocyte complexes (COCs) were submitted to IVM medium containing 0, 25, 50, and 100 μM ALA for 44 h. After 44 h of IVM, denuded oocytes were co-cultured with spermatozoa during 18 h. After 18 h of in vitro fertilization, oocyte were using aceto-orcein method, to evaluated penetration rate, monospermy (number of monospermy oocytes/total oocytes), and the IVF efficiency (number of monospermy/total penetrated oocytes). In results, 25 and 50 μM ALA groups were significantly increased on penetration rate compared with 100 μM ALA group (p<0.05). Similarly, monospermy rate were significantly increased 25 and 50 μM ALA groups than control group (p<0.05). IVF efficiency was no significant difference between control and ALA treatment groups. Our findings suggested that treatment of ALA supplementation during in vitro maturation (IVM) and subsequent of in vitro fertilization in pigs, ALA can increase IVF efficiency by effectively blocking polyspermy and increasing monospermy some mechanism in porcine oocytes. However, the study of mechanism by which ALA blocks polyspermy are needed, and this study suggests that ALA has a positive effect on in vitro production of porcine oocytes by decreasing polyspermy. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

The objective of this study was to determine the effect of fructose that was supplemented to a chemically defined in Vitro maturation (IVM) medium on oocyte maturation and embryonic development after parthenogenesis in pigs. The base medium for in Vitro maturation (IVM) was porcine zygote medium (PZM) that was supplemented with 0.05% (w/v) polyvinyl alcohol (PVA) or 10% (v/v) porcine follicular fluid (pFF). In the first experiment, when immature pig oocytes were matured in a chemically defined medium that was supplemented with 5.5 mM glucose or with 1.5, 3.0 and 5.5 mM fructose, 3.0 mM fructose resulted in a higher nuclear maturation (91.5%) than 1.5 and 5.5 mM fructose (81.9 and 81.9%, respectively) but showed a similar result with 5.5 mM glucose (94.2%). However, there was no significant differences among groups in the embryo cleavage (89.4-92.4%), blastocyst formation (37.5-41.1%), and mean cell number of blastocyst (30.8-34.2 cells). Fructose at the concentration of 3.0 mM (1.08 pixels/oocyte) resulted in a higher intra-oocyte glutathione (GSH) content than 1.5 and 5.5 mM fructose (1.00 and 0.87 pixels/oocytes, respectively) while the cumulus cell expansion was not influenced. In the second experiment, effect of individual and combined supplementation of a chemically defined maturation medium with 5.5 mM glucose or 3.0 mM fructose was examined. No significant effect was found in the nuclear maturation (86.3-92.6%). Embryo cleavage was significantly increased by the combined supplementation with glucose and fructose (95.2%) compared to that with 3.0 mM fructose only (85.7%) while blastocyst formation (37.3-42.8%) and embryonic cell number (33.3-34.1 cells) were not altered. Effect of supplementation of pFF-containing medium with glucose and fructose + glucose was examined in the third experiment. No significant effect by the supplementation with glucose and fructose or glucose alone was observed in the nuclear maturation of oocytes (90.7-94.1%) and blastocyst formation (51.0-56.5%). Our results demonstrate that 3.0 mM fructose was comparable to 5.5 mM glucose in supporting in Vitro oocyte maturation and embryonic development after parthenogenesis and could be used as an alternative energy source to glucose for in Vitro maturation of pig oocytes.

The oocyte undergoes various events during In vitro maturation (IVM) and subsequence development. One of the events is production of reactive oxygen species (ROS) that is a normal process of cell metabolism. But imbalances between ROS production and antioxidant systems induce oxidative stress that negatively affect to mammalian reproductive process. In vitro environments, In vitro matured oocytes have many problems, such as excessive production of ROS and imperfect cytoplasmic maturation. Therefore, In vitro matured oocytes still have lower maturation rates and developmental competence than in vivo matured oocytes. In order to improve the IVM and In vitro culture (IVC) system, antioxidants, vitamins were added to the IVM, IVC medium. Antioxidant supplementation was effective in controlling the production of ROS and it continues to be explored as a potential strategy to overcome mammalian reproductive disorders. Based on these studies, we expect that the use of antioxidants in porcine oocytes could improved maturation and development rates.

Maturation-promoting factor (MPF) is well-known as cell cycle regulator during oocyte maturation and fertilization. MPF activity maintains high levels and arrest the cell cycle progression until fertilization. After fertilization, Anaphase-promoting complex/cyclosome (APC/C) mediated degradation of cyclin B causes decrease of MPF activity. One of the cytostatic factor (CSF), Emi2 inhibits APC/C activity by binding to APC/C-cdc20, therefore blocks the proteolysis of cyclin B. Degradation of Emi2 requires phosphorylation by Polo-like kinase 1 (Plk1). Thus recognition and phosphorylation of Emi2 by Plk1 are essential step for meiotic cell cycle resumption. In our previous research, we found that two phosphorylated threonine regions at amino acid position 152 and 176 in Emi2 are respectively contributed for recognition by polo-box domain of Plk1. Peptidomimetics 103-8 can block the interaction between Plk1-PBD and Emi2, and therefore meiotic maturation and meiosis resumption via parthenogenetic activation were impaired. However, major drawback of 103-8 was the limitation of penetration through the cell membrane. We synthesized the new peptidomimetics and checked bioavailability in mammalian oocyte by injection and media treatment. Medium treatment with peptidomimetics C-4, meiotic maturation has significantly decreased and meiotic resumption via parthenogenetic activation has perfectly impaired. For the next experiment, we are preparing X-ray crystallography to identify the binding modes between Plk1-PBD and peptidomimetics C-4.

Three-dimensional (3D) culture system is useful technique for study of in vivo environment and it was used various experiments. This study was investigated to establish of embryo co-culture system and changes of PAs activity in 3D cultured endometrial cells of pigs. In results, growth of stromal cells into gel matrix were detected only with endometrial and myometrial cells. The most rapid growth of stromal cells were confirmed in 2.5x105cells/ml and gel matrix containing 15% FBS. Expression of urokinase-PA (uPA) after treatment of hCG (0.5, 1.0, 1.5 and 2.0 IU/ml) were higher than without hCG, but, there are not significant difference among the treatment. On the other hand, expression of uPA after treatment of IL-1β (0.1, 1, 10 and 100 ng/ml) were higher than without IL-1β, but, there are not significant difference. Expression of uPA after treatment of estrogen (0.2, 2, 20 and 200 ng/ml) were not difference, but PA activity was significantly decreased (p＜0.05). Blastocyst was producing in PZM-3 medium containing FBS and endometrial cells were grown in PZM-3 medium. When embryos development with cultured endometrial cells, cleavage rates were not significant difference and blastocyst were not produced in co-culture with stromal cells and 3D culture system. 3D culture system had similar activity to in vivo tissue and these features are very useful for study of in vivo physiology. Nevertheless 3D culture system was not proper in embryo co-culture system. Therefore, we suggest that 3D culture system with embryo co-culture need continuous research.

Humulus japonicus is an ornamental plant in the Cannabaceae family. Although the mode of action of Humulus japonicus is not fully understood, a strong relationship was observed between anti-inflammatory and anticancer in some types of cells. Recent studies also have shown that Humulus japonicus possesses anti-inflammatory activities and may significantly improve antioxidant potential in Raw 264.7 macrophage cells. Thus, the aim of this study was eva-luated the effect of Humulus japonicus extract on sperm motility and subsequent preimplantation developmental com-petence of the bovine embryos. After in vitro maturation, the oocytes with sperms were exposed in in vitro fertilization (IVF) medium supplemented with Humulus japonicus extract (0.01, 0.05, 0.1 μg/mL, respectively) for 1 day. In our results, exposure of IVF medium to Humulus japonicus extract did not affect sperm motility and percentage of pene-trated oocytes but ROS intensity was significantly decreased by 0.01 μg/mL compared with other groups (p< 0.05). Moreover, treatment with 0.01 μg/mL of Humulus japonicus extract was higher the frequency of blastocyst formation than the any other groups (p<0.05). Otherwise, treatment with 0.01 μg/mL of Humulus japonicus extract not increased the total cell number but reduced apoptotic-positive nuclei number. In conclusion, our results indicate that supple-mentation of Humulus japonicus extract in IVF medium may have important implications for improving early embryo-nic development in bovine embryos

본 연구에서는 1톤 용량의 배아미를 생산할 수 있는 중형 배아미 생산시스템의 설계, 개발 그리고 평가를 목표로 하였다. 개발된 배아정미기는 연삭과 마찰의 혼합방식으로 제조되었다. 배아미 생산시스템의 형태는 2대의 직렬 입형 배아정미기로 구성하였으며, 배아부착율, 백도, 싸래기율을 조사하였다. 또한, 연삭식, 마찰식, 연삭과 마찰의 혼합방식에 따라서 각각 배아미의 배아부착율, 백도, 싸래기율도 조사하였다. 본 시스템은 1단계 배아정미기에서는 연삭과 마찰의 혼합방식으로 미강을 깍은 후, 2번째 배아 정미기에서는 쌀의 배아가 떨어지지 않도록 미세 미강을 제거하면서 쌀의 백도를 높이도록 개발되었다. 배아정미기 시작기에서는 축 롤러 부분의 금강석 연삭돌을 3개, 스크린부에는 6개의 연삭돌을 설치하였고, 각각의 정미기 롤러축의 회전속도는 960 rpm과 780 rpm으로 하여 배아 부착율과 백도를 높였다. 그 결과, 약 20%의 배아 부착율을 증가시킬 수 있었다. 본 연구에서는 다음과 같이 요약할 수 있다. 첫째, 배아부착율은 현미의 함수율과 밀접한 관계가 있었다. 함수율 15.2±0.1%인 시료로 실험한 결과, 투입량 약 600 kg일 때 배아부착률은 약 70%를 나타내었다. 둘째, 배아미의 백도는 정미기 롤러축의 회전속도 960 rpm 과 780 rpm 조건으로 운전하였을 때 각각 35, 37 백도로 향상시킬 수 있었다. 셋째, 싸래기율은 본 시스템에서 1% 미만으로 나타냈다. 본 연구에서 개발된 연식마찰식 배아정미기를 평가해본 결과 배아부착율, 백도, 싸래기율을 효과적으로 개선할 수 있었고 30%의 에너지 이용을 절감할 수 있었다.

The present study assessed the effect of FSH and LH on oocyte meiotic, cytoplasmic maturation and on the expression level and polyadenylation status of several maternal genes. Cumulus-oocyte complexes were cultured in the presence of FSH, LH, or the combination of FSH and LH. Significant cumulus expansion and nuclear maturation was observed upon exposure to FSH alone and to the combination of FSH and LH. The combination of FSH and LH during entire IVM increased the mRNA level of four maternal genes, C-mos, Cyclin B1, Gdf9 and Bmp15, at 28 h. Supplemented with FSH or LH significantly enhanced the polyadenylation of Gdf9 and Bmp15; and altered the expression level of Gdf9 and Bmp15. Following parthenogenesis, the exposure of oocytes to combination of FSH and LH during IVM significantly increased cleavage rate, blastocyst formation rate and total cell number, and decreased apoptosis. In addition, FSH and LH down-regulated the autophagy gene Atg6 and upregulated the apoptosis gene Bcl-xL at the mRNA level in blastocysts. These data suggest that the FSH and LH enhance meiotic and cytoplasmic maturation, possibly through the regulation of maternal gene expression and polyadenylation. Overall, we show here that FSH and LH inhibit apoptosis and autophagy and improve parthenogenetic embryo competence and development.

말똥성게 (Hemicentrotus pulcherrimus)의 생식세포 및 pluteus 유생을 이용하여 중금속인 Arsenic (As)와 Chromium (Cr)이 정상 수정률 및 배아 발생률에 미치는 독성 영향을 조사하였다. H. pulcherrimus의 수정률 및 배아 발생률에 미치는 As와 Cr의 독성은 6.25, 12.5, 25, 50, 100 ppb의 농도에서 조사하였다. 0.5 M KCl 용액을 이용하여 방란 및 방정을 유도하였고, 정상 수정률 및 배아발생률은 수정 후 각각 10분 및 64시간째 관찰하였다. As와 Cr을 첨가하지 않은 대조구에서 정상 수정률과 배아 발생률은 각각 94%와 93% 이상을 나타냈다. 이들 중금속 첨가에 의해 수정률은 아무런 변화가 나타나지 않았지만 배아 발생률은 농도 의존적 감소하는 것으로 나타났으며, As의 첨가에 의해 배아 발생률은 6.25 ppb에서 유의적으로 감소하였으며 (P⁄0.01), Cr의 경우는 25 ppb에서 유의적인 감소를 나타냈다 (P⁄0.05). H. pulcherrimus의 정상 배아 발생률에 대한 LOEC는 As의 경우는 6.25 ppb를 Cr은 25 ppb를 나타냈다. 이들 연구결과로 해양생태계 내에서 As가 6.25 ppb, Cr이 25 ppb를 초과하는 농도일 때는 H. pulcherrimus와 같은 무척추동물의 정상부화율은 급격히 감소할 것으로 판단된다. 본 연구결과를 바탕으로, H. pulcherrimus의 정상 배아 발생률을 이용한 생물학적 평가방법은 중금속과 같은 유해물질에 대한 해양생태계의 영향을 판단하기 위한 시험방법으로 유용하게 이용될 수 있을 것으로 판단된다.

Biotechnologies for cloning animals and in vitro embryo production have the potential to produce biomedical models for various researches. Especially, pigs are a suitable model for xenotransplantation, transgenic production and various areas of reproductive research due to its physiological similarities to human. However, utilization of in vitro-produced embryos for transfer remains limited. Despite improvement over past few decades, obstacles associated with the production of good quality embryos in vitro still exist which limit the efficiency of cloning. One of major problems includes improper in vitro maturation (IVM) and culture (IVC). Oxidative stress caused from in vitro culture conditions contributes to inadequate IVM and IVC which leads to poor developmental competence of oocytes, failure of fertilization and embryo development. To reduce the oxidative stress, various antioxidants have been used to IVM and IVC system. However, limited information is available on the effects of resveratrol on livestock reproductions. Resveratrol is a polyphenolic natural product and well known as an antioxidant in foods and beverages (e.g. in grapes and red wine). Resveratrol is known to be cardioprotective, anticarcinogenic, anti-inflammatory, antioxidant and antiapoptotic. This paper will review the effects of resveratrol on in vitro maturation of oocytes and embryo development.

본 연구는 말똥성게(Hemicentrotus pulcherrimus)의 생식세포 및 pluteus 유생을 이용하여 중금속인 납(lead, Pb)과 아연(zinc, Zn)의 독성을 조사였다. H. pulcherrimus 배우자 및 배아에 미치는 Pb과 Zn의 독성은 각각 31, 63, 125, 250, 500 ppb 및 16, 31, 63, 125, 250 ppb의 농도에서 조사하였다. 0.5 M KCl 용액을 이용하여 방란 및 방정을 유도하였고, 수정률 및 정상 배아발생률의 조사는 수정 후 각각 10분 및 64시간째 관찰하여 시행하였다. Pb 노출 시 수정률은 대조군과 비교하여 유의적인 변화가 없었다. 그러나 정상 배아발생률은 농도가 높을수록 농도의존적으로 유의적인 감소를 보였다. Zn을 노출시켰을 경우 수정률과 정상 배아발생률은 농도가 높을 수록 농도의존적인 유의적 감소를 나타냈다. H. pulcherrimus의 정상 배아 발생에 대한 독성치는 각각 Pb (반수영향농도 (EC50) 45.13 ppb, 95% Cl 40.12~50.05 ppb), Zn (반수영향농도(EC50) 19.82 ppb, 95% Cl 18.26~21.31 ppb)로 나타났다. 또한 Pb과 Zn의 무영향농도(NOEC)는 각각 ⁄31.25 ppb 및 ⁄15.63 ppb로 나타났고, 최소영향농도(LOEC)는 31.25 및 15.63 ppb로 나타났다. 본 연구 결과, H. pulcherrimus의 초기 배아발생 과정은 Pb과 Zn 등의 중금속에 높은 민감성을 보인다. 따라서 H. pulcherrimus는 해양생태계 위해 평가를 위한 시험생물로서 사용이 가능하다고 사료된다.

The objective of this study was to investigate the effects of NEAA and leptin supplemented to in vitro culture medium on the developmental competence of porcine embryos after intracytoplasmic sperm injection (ICSI), and to modify the culture condition to improve the quality and the development of ICSI-derived porcine embryos in vitro. After ICSI, the putative zygotes were then cultured in PZM-3 medium with/without NEAA or leptin. The proportion of embryos that developed to the blastocyst stage significantly increased when 1% NEAA (24.62%) was added to the medium compared with 2% NEAA and no NEAA group (17.24% and 20.24%, respectively, p<0.05). The effect of different concentration of leptin (0, 10, 100, 500 ng/ml) was evaluated on the development of porcine ICSI embryos cultured in vitro. In case of blastocyst formation, 100 ng/ml group (27.05%) showed significantly higher rate than 10, 500 ng/ml, and control group (23.45%, 17.99%, and 19.68%, respectively, p<0.05). We also evaluated the effects of different NEAA and leptin treatment time on the development of porcine embryos after ICSI. Among groups of embryos cultured in the presence of NEAA or leptin for whole 7 days (D 1-7), first 4 days (D 1-4), the subsequent 3 days (D 5-7), both NEAA (27.13%, 21.17 %, and 17.56%, respectively, p<0.05) and leptin (25.60%, 20.61%, and 16.53%, respectively, p<0.05) showed that supplementation for whole 7 days significantly increased the blastocyst formation rate compared with the other groups of D1-4 and D5-7. We further evaluated the combination effect of 1% NEAA and 100 ng/ml leptin compared with the effect of each supplementation with 1% NEAA or 100 ng/ml leptin or no supplementation on development of embryos. For blastocyst formation, combination group of NEAA and leptin (24.78%) showed significantly higher rate than other three groups (18.37%, 20.44 %, and 13.27%, respectively, p<0.05). We further evaluated the expression of proapoptosis genes such as BAX and BAK and anti-apoptosis genes, BCL-XL and BCL-2 in blastocysts cultured in the presence of 100 ng/ml leptin. RT-PCR analysis revealed that leptin supplementation significantly decreased the expression of pro-apoptosis genes as well as increased the expression of anti-apoptosis genes. These results of present study demonstrate that NEAA and leptin could improve the in vitro development of ICSI- derived porcine embryos with optimal concentration of each reagent. Furthermore, the optimal culture condition could increase the quality of ICSI-derived embryos in vitro.

In mammal, oocytes are arrested at the metaphase Ⅱ until fertilization. However, unfertilized oocytes that remain in the oviduct or under in vitro culture, which is called "oocyte aging". Asynchrony negatively affects fertilization, pre- and post-implantation embryo development. Caffeine is known to phosphodiesterase inhibitor that rescues oocyte aging in several species. Nevertheless, the effect of caffeine was not clear in bovine aging oocytes. In this study investigated the cytoskeleton distribution in aged oocytes and the embryo development ability of aged oocytes from treated with or without caffeine during maturation. The cumulus and oocyte complexes (COCs) were cultured in 10% FBSTCM199 for up to 22h at 38.5℃ in 5% CO₂. For oocyte aging study, the COCs were cultured in 10% FBS-TCM199 supplemented with or without 10 mM caffeine for 40hs. And then oocytes underwent in vitro fertilization using highly motile sperm recovered from frozen and than thawed bull semen. As a result normal cytoskeleton percentage of caffeine treatment group more than the aging group (67.57%±4.11 VS 44.61%±6.40) and no significantly different compared to control group. Aged oocytes derived from addition of caffeine to the in vitro maturation medium significantly increased the percentage of 2- cell that developed to the blastocyst stage compared to the aging group. Blastocysts derived from caffeine treatment group significantly increased the total cell number compare aging (90.44%±10.18 VS 67.88%±7.72). Apoptotic fragmenting of genomic DNA was measured in individual embryos using the TUNEL assay. Blastocyst derived from caffeine treatment group significantly decereased the apoptotic index compared to blastocyst derived from aging group. In conclusion, we inferred that the caffeine treatment during oocytes aging periode can improved the develpmental rate and quaility in bovine embryos developing in vitro.