산업화에 의해 발생되어진 여러 가지 오염물질 가운데 중금속인 니켈, 코발트, 수은은 연안 해역으로 유입 시 극히 미량일지라도 생체 내 축적되어 해양생물의 생리 및 발생에 장애를 일으키는 것으로 알려져 있으며 먹이연쇄 (food chain)를 통해 생물확대 (biomagnification)됨으로써, 해양생태계 전반에 영향을 미칠 수 있다. 본 연구는 유용 수산생물로써 조간대 암반 지역에 서식하는 말똥성게 (Hemicentrotus pulcherrimus)를 이용하여 니켈, 코발트, 수은의 생태 위해성을 조사하고자 한다. 성숙한 말똥성게 (H. pulcherrimus)에 0.5 M KCl를 주입하여 방란 및 방정을 유도한 후, 수집한 정자 및 난자를 니켈 (10, 25, 50, 100, 500 ppb), 코발트 (10, 100, 500, 1000, 2500 ppb) 그리고 수은 (10, 25, 50, 100, 500 ppb) 농도에서 10분간 인공수정 한 후, 수정 및 배아발생을 관찰하였다. 실험결과 수정률은 노출 된 각각의 니켈과 코발트 농도에 유의적인 영향을 받지 않았다. 수은은 농도가 증가할수록 농도 의존적으 로 감소하는 경향을 나타내었다. 배아 발생률은 니켈, 코발트 및 수은 농도가 증가 할수록 농도 의존적으 로 유의적인 감소를 나타내었다. 말똥성게 (H. pulcherrimus)의 배아 발생률에 대한 니켈, 코발트 및 수은 의 영향을 독성치로 나타냈을 때, 무영향농도 (NOEC)는 10 ppb로 모두 같은 값으로 나타났고, 최소영향 농도 (LOEC)는 각각 25 ppb, 10 ppb, 10 ppb로 나타났으며, 반수영향농도 (EC50)는 각각 34.19 ppb, 71.84 ppb, 144.66 ppb로 나타났다. 본 연구 결과, H. pulcherrimus의 배아 발생률을 이용한 생태독성 평가시, 니켈>코발트>수은 순으로 독성이 강한 것으로 나타났으며, 이들 중금속이 연안해역에 유입되어 10 ppb를 초과할 경우 연안 생태계 내에 서식하는 생물의 재생산에 유해한 영향을 미칠 것으로 판단된다.

The objective of this study was to evaluate the effects of co-culture of bovine oocytes with cumulus cells on in vitro maturation and development following in vitro fertilization in bovine oocytes. Bovine cumulus-oocyte complexes (COCs) and denuded oocytes (DO) were co-cultured with the cumulus cells in TCM199 for 20~22 hr, and evaluated the nuclear type of oocyte. After in vitro maturation, oocytes were coincubated for in vitro fertilization with frozen-thawed spermatozoa selected by 65% percoll in DM-Heparin and DM-Caffeine for 15~18 hr. Presumptive zygotes were cultured for 48 hr in CR1aa in vitro culture medium with 10% FBS, and evaluated the cleavage rates. The results confirmed that the highest percentage of metaphase II (M-II) stage was observed in COCs (30.1±3.5%, 24.2±1.8%) as compared to DO (7.1±1.3%, 17.4±13.9%) (p<0.05). In addition, the increased cleavage rates were obtained from COCs (69.6±2.1%, 75.6±2.9%) when compared to DO (21.6±7.5%, 29.5±12.6%) (p<0.05). In conclusion, this study suggested that cumulus cells secreted positive factors during in vitro maturation of oocytes and early embryonic development after in vitro fertilization of bovine oocytes.

Somatic cells such as oviduct epithelial cell, uterine epithelial cell, cumulus-granulosa cell and buffalo rat river cell has been used to establish an effective culture system for bovine embryos produced in in vitro. But nitric oxide (NO) metabolites secreted from somatic cells were largely arrested the development of bovine in vitro matured/ in vitro fertilized (IVM/IVF) embryos, suggesting that NO was induced the embryonic toxic substance into culture medium. The objective of this study was to investigate whether BOEC co-culture system can ameliorate the NO-mediated oxidative stress in the culture of bovine IVM/IVF embryos. Therefore, we evaluated the developmental rate of bovine IVM/IVF embryos under BOEC co-culture system in the presence or absence of sodium nitroprusside (SNP), as a NO donor, and also detected the expression of growth factor (TGF-p , EGF and IGFBP) and apoptosis (Caspase-3, Bax and Bcl-2) genes. The supplement of SNP over 5 uM was strongly inhibited blastocyst development of bovine IVM/IVF embryos than in control and 1 uM SNP group (Table 2). The developmental rates beyond morulae stages of bovine IVM/IVF embryos co-cultured with BOEC regardless of SNP supplement (40.4% in 5 uM SNP+ BOEC group and 65.1% in BOEC group) were significantly increased than those of control (35.0%) and SNP single treatment group (23.3%, p<0.05: Table 3). The transcripts of Bax and Caspase-3 genes were detected in all experiment groups (1:Isolated fresh cell (IFC), 2:Primary culture cell (PCC), 3:PCC after using the embryo culture, 4: PCC containing 5 uM SNP and 5: PCC containing 5 uM SNP after using the embryo culture), but Bcl-2 gene was not detected in IFC and PCC (Fig. 1). In the expression of growth factor genes, TGF-p gene was found in all experimental groups, and EGF and IGFBP genes were not found in IFC and PCC (Fig. 2). These results indicate that BOEC co-culture system can increase the development beyond morula stages of bovine IVM/IVF embryos, possibly suggesting the alleviation of embryonic toxic substance like nitric oxide.

호박벌(Bombus ignitus)에 대한 탄산가스의 휴면타파 효과를 검토하였다. 65%, 99% 농도의 탄산가스처리 결과, 산란율은 무처리구의 50%에 비하여 65%와 99%의 탄산가스에 접촉시킨 시험구에서 각각 75%와 77%로 증가하였고, 첫산란소요일수 또한 무처리구에서 30일이 걸린데 비하여 65%와 99%의 시험구에서는 각각 18일과 17일로 단축되어 뚜렷한 탄산가스 처리효과가 나타났다. 그러나 65%와 99%의 시험구 간에는 차이가 보이지 않아 65-99%범위에서는 어느 농도의 탄산가스를 사용해도 좋을 것으로 생각되었다. 또한 탄산가스 최적 처리시기를 구명하기 위하여, 교미 후 1일부터 4일째까지 탄산가스를 처리한 결과, 산란성과 봉세발달이 교미 후 2일째가 가장 우수하여 최적 시기로 판명되었다. 그러나 호박벌에 대한 탄산가스 처리는 산란성과 봉세발달에는 긍정적인 효과를 보였지만, 차세대 출현수가 월동 여왕벌보다 적어 탄산가스 처리만으로는 상품성 있는 호박벌 봉군을 연중 생산하는 방법으로 부적당한 것으로 판단되었다.

인공광하의 풍동 내에서 CO2 농도와 기류속도 제어가 플러그묘 개체군의 생육에 미치는 효과를 분석하고자 시도된 본 연구의 결과를 요약하면 다음과 같다. 인공광하에서 묘개체군의 줄기 길이 및 직경, 초장, 순광합성속도 등에 미치는 기류속도의 영향은 저농도의 CO2 조건보다는 고농도의 CO2 환경에서 분명하게 나타났다. 기류속도가 증가할수록 줄기 길이가 감소하였는 데, 이와 같은 결과는 CO2 농도가 높게 유지되는 조건에서 분명하게 나타났다. CO2 농도가 높은 조건에서 줄기 직경은 기류속도가 증가함에 따라 증가하는 것으로 나타났다. 묘개체군의 초장은 CO2 농도가 낮게 유지되는 조건에 비해서 CO2 농도가 높게 유지되는 경우에 작게 나타났다. 묘개체군의 순광합성속도는 0.7m.s-1의 기류속도에서 최대치가 나타났으며, 이러한 결과는 CO2 농도가 높게 유지될 때 더욱 분명하게 나타났다. CO2 농도가 950μmol.mol-1를 유지할 때 묘개체군의 순광합성속도는 310μmol.mol-1인 경우에 비해서 약 46% 정도 높게 나타났다. 그러므로 인공광하에서 묘소질이 우수한 플러그묘의 육묘에 CO2시용이 효과적일 것이다.

고정발생원으로 부터 배출되고 있는 이산화탄소를 분리하여 회수 및 재이용하는 기술개발이 에너지 보전 측면에서 뿐만 아니라 환경오염 문제 등을 해결할 수 있는 중요한 과제이다. 특히 내열성, 내식성 및 기계적 강도가 뛰어난 세라믹의 특성을 이용한 기체분리막을 응용한다면 고온으로부터 저온까지의 폭넓은 온도, 압력, 가스조성의 배기가스로부터 이산화탄소를 분리하는 것이 가능해 진다. 따라서 본 총설에서는 현재 일본에서 국책과제로 진행되고 있는 이산화탄소의 고온분리에 대한 연구개발(이하, 'CO2 프로젝트'로 약칭) 현황을 소개하고자 한다.

This study was conducted to investigate the effects of co-culture for the development rate to morula /blastocyst stages of early porcine embryos, derived from oocytes matured and fertilized in vitro, with porcine endometrial cell monolayers(PEM) in the two different media, respectively. The rates of embryos developed to 2-, 4-, 8~16-cell and morula /blastocyst stage were 49.6, 40.5, 28.2 and 15.3% in Ham's F-10 with PEM, and 55.3, 45.9, 32.7, and 17.6% in TCM-HEPES with PEM, respectively. The above development rates to morula /blastocyst stages were significantly higher than those of the embryos cultured in the Ham's F-10 and TGM-HEPES without PEM(P<0.05). The in vitro development rates to the morula /blastocyst stage of 1-cell embryos cultured in Ham's F-10 and TCM-HEPES without PEM were 0~1.2%. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. As shown in the above results, the co-culture of in vitro produced porcine embryos with PEM in the two different media enhanced the development of fertilized eggs to morula /blastocyst stages in vitro. However, we didn't find out any differences for the in vitro development to morula /blastocyst stages between Ham's F-10 and TcM-HEPES media.

This experiment was carried out to evaluate the effect of early mouse embryonic development in vitro by co-culture with bovine and porcine oviductal epithelial cells (BOEC and POEC). The 2-cell embryos were collected from the oviducts of the superovulated and mated cultured in D-PBS /15% FCS at 48 hours after hCG injection. The in vitro developmental rate of blastocyst formation in the embryos were examined under the fllowing treatments; 1) TCM 199 added 15% HCS, 2) Ham's F-10 added 15% HCS, 3) MediCult IVF medium, 4) TCM 199 added 15% HCS + BOEC, 5) TCM 199 added 15% HCS + POEC, 6) Ham's F40 added 15% HCS + BOEC, 7) Ham's F-10 added 15% HCS + POEC,8) MediCult IVF medium + BOEC, 9) MediCult IVF medium + POEC. For a comparative study of in vitro development for 96 hours after hCG injection, were cultured with oviductal epithelial cell and media only. The obtained results were 2-cell embryos developed to the blastocyst stage in TCM 199, Ham's F-10 and MediCult IVF medium at the rates of 84.4,83.2 and 81.6%. respectively. The higher developmental rates(91~97%) of blastocyst formation was appeared when the embryos were co-cultured with a monolayer of bovine or porcine oviductal epithelial cells in TCM 199 or Ham's F-10 and MediCult IVF media. No significant difference in developmental rates was shown between bovine and porcine oviductal epithelial cells but significant difference in co-culture system in comparison between media only system and co-cultures. In conclusions, oviductal epithelial cells, BOEC and POEC, when co-culture with mouse early embryos improved the rates of development, blastocyst and hatching. Therefore, it is suggested that co-culture system using oviductal epithelial cells improve early embryonic developtnent in mouse.

The present study was carried out to investigate the effects of co-culture cells and growth factors on in vitro culture of Korean native cattle(KNC) embryos fertilized in vitro. Two-eight cell embryos were cultured in vitro using 4 types of co-culture cells and 3 growth factors singly or in combination. The results were as follows, In the co-culture of 2~8 cell embryos with bovine oviductal epithelial cell(BOEC), granulosa cell(BGC), uterine epithelial cell(BUEC) and mouse embryonic fibroblast (MEF) monolayers, the developing rate to blastocysts was significantly(P<0.05) higher with BUEC(32.1%) than with MEF(15.3%), BGC(13.2%) and non co-culture control(11.6%). When the morula co-cultured with BOEC for 5 days following in vitro fertilization were co-cultured with BOEC continuously or with BUEC, respectively, the developing rate to blastocysts was higher with BUEC(73.9%) than with BOEC(56.0%). To examine the effects of growth factors on in vitro development of 2~8 cell embryos, epidermal growth factor(EGF), transforming growth factor-l(TGF-l) and insulin-like growth factor-1(IGF-1) were added singly or in combination to TCM 199 maturation medium with respective concentration. In a addition of each 10, 30 and SOng /rnl EGF, the developing rate to blastocysts was the highest in lOng /ml EGF(25.3%). In addition of each 1, 2 and Sng /mi TGF-1, the developing rate to blastocysts was the highest in lng /ml TGF-1(28.8%). In addition of each 50, 100ng/ml JGF-l, the developing rate to blastocysts was higher in 100ng/ml IGF-l(16.5%) than in SOng/mi IGF-1(12.9%). When lOng /ml EGF and lng /ml TGF-l was added singly or in combination, the developing rate to blastocysts was similar in groups added singly or in combination with EGF and TGF-l (23.l~24.6%), although higher than in control(16.7%). In the co-culture of 2~8 cell embryos Wth BOEC + each 10, 30 and 5Ong /rnl EGF, the developing rate to blastocysts was significantly(p<0.05) higher in BOEC + long /ml EGF(32.3%) than in BOEC + 3Ong /ml EGF(18.9%) and BOEC + song /ml EGF(9.7%). In the co-culture of 2~8 cell embryos with BOEC + each 1, 2, Sng /ml TGF-l the developing rate to blastocysts was higher in BOEC + Sng/rnl TGF-l(28.2%) than in BOEC + lng /ml TGF-l(21.7%) and BOEC + 2ng/ml TGF-l(21.4%). In summary, higher developing rate to blastocysts were obtained with co-culture of BUEC for co-culture system, with addition of lOng /ml EGF or lng /ml TGF-l for growth factor culture system, and with co-culture of BOEC + lOng /ml EGF or BOEC + Sng /ml TGF-l for co-culture + growth factor culture system.

The object of this study was to evaluate the effect of uterine epithelial cells on development of Korean native cattle(KNC) oocytes fertilized in vitro. Qocytes were collected from ovaries of slaughtered Korean Native Cows and matured in TCM199 with granulosa cells supplemented with 10% FBS, 5g/ml FSH, 10 JU/ml hCG, and 1g/ml estradiol-17 for 24 hrs. For co-culture of in vitro development of fertilized ova, oviductal epithelial cells (ll0˚cells /ml) obtained from slaughtered cow and uterine epithelial cells(110˚cells /ml) flushed from the superovulated holstein on Day 7 were incubated in 39, 5% , 95% air. Frozen-thawed KNC sperm was capacitated with BO(Brackett & Oliphant, 1975) medium supplemented with 10mM, 5mM-caffein. Matured oocytes were inseminated for 20 hrs. And then fertilized oocytes were washed with culture medium and transferred to oviductal epithelial cells for in vitro development and three days later a portion of embryos were transferred to uterine epithelial cells. Stastical methods of developmental rates on KNC-IVF oocytes was ANOVA-test. Developmental rates of KNC-IVF oocytes was significant higher(P<0.01) when co-cul-tured with uterine epithelial cells(25.2%) than oviductal epithelial cells. Blatocyst cul-tured for 7 to 9 days were frozen by automatic freezer with 1.4M glycerol-PBS. Survival rates of blastocyst was 40.0%. Fourteen frozen-thawed blastocysts were transferred to five holstein heifers on day 7 after natural estrus. Three recipients were observed twin and one recipient was single by ultra-sound systems on days 45 after embryo transfer.