본 연구는 돼지 난포란의 체외성숙에 적합한 배양액을 조사하고, 아울러 산소농도가 돼지 난포란을 이용한 체외수정란의 생산에 미치는 영향을 구명하고자 실시하였다. 본 연구에서 얻어진 결과를 요약하면 다음과 같다. 1. NCSU-23과 TCM-199 배양액에 10% PFF를 첨가하여 5% 및 20% 산소조건하에서 체외성숙을 유기한 결과, 배양액 및 산소조건에 따른 난핵붕괴률 및 핵성숙률에는 유의적 (P>0.05)인 차이가 없었다. 2. NCSU-23이 TCM
본 연구는 돼지 난포란의 체외성숙배양액에 superoxide dismutase(SOD)를 첨가배양하여 체외성숙과 수성이후 배발달에 미치는 영향과 산소농도 및 SOD가 체외생산된 돼지초기수정란치 배발달에 미치는 영향을 규명하고자 실시하였다. 본 연구에서 얻어진 결과를 요약하면 다음과 같다. 1. 돼지 난포란을 체외성숙배양액인 NCSU-23에 SOD를 각각 0, 100,500 및 1,000uni1s/m1 첨가하여 성숙시킨 다음, 체외수정을 실시한 결과, 핵
본 연구는 고품질육의 DNA marker가 규떵된 한우로부터 초음파유노 난포란의 연속적 채취를 통하여 능력이 우수한 한우 수정란온 대량생산하는 방법의 확립과 이를 한우농가에 응용하고자 초음파 난자채취기를 이용하여 등지방층두께, 일당증체량, 근내지방도 및배최장근 단면적에 연관된 DNA marker를 보유하고 있는 한우 5두로부터 개체및 난포수, 채취방법, 회수한 난포란의 등급 등을 조사하였다. 한우 5두의 개체별 난포수는 6, 10, 5, 4 및 11회
본 연구에서는 DNA marker가 검정된 한우로부터 생산한 체외수정란을 이식하여 육질 및 육량의 유전적 능력이 우수한 한우를 대량생산하여 고품질 한우 쇠고기 생산 시스템을 구축하기 위한 전단계로서 DNA marker 검정 한우로부터 초음파유도 난포란을 채란하여 체외수성 및 수정란의 체외 발달에 미치는 각종 요인들과 배반포기 수정란의 부화율 개선을 위하여 투명대를 laser로 drilling을 실시하여 부화율을 조사하였다. 초음파유래 체외수정란의 분할률
성간별에 이용할 돼지 수정란을 체외수정 방법으로 생산하기 위하여 돼지 난소에서 채취한 난자를 NCSU 23 배지에서 eCG, hCG를 첨가한 상태에서 22시간, 첨가하지 않은 상태에서 22시간 체외성숙을 시킨 후 mTBM을 이용하여 여러가지 정자농도(5, 2.5 , 6.0그리고 10.0 )에 따라 6시간 수정시켰고, NCSU 23 배지에서 배양시켰다. 배양 후 44시간에 수정란의 분할률을 관찰하였고, 144시간에 배반포 형성율을 확인하였다. 성감별 방법
The success of nuclear transplantation with mammalian oocytes depends critically on the potential of oocytes activation, which mainly caused to prevent the re-accumulation of maturation promoting factor (MPF). This study was conducted to compare the effect of combined treatment of lonomycin with a Hl-histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on activation of bovine oocytes. In vitro matured bovine oocytes with the first polar body (PB) and dense cytoplasm were assigned to 3 experimental groups. For activation treatment, oocytcs were exposed to 5 M lonomycin for 5 min (Group 1), and followed by 1.9 mM dimethylaminopurine (DMAP) for 3 h (Group 2) or followed by 2 mM sodium pyrophosphate (SPP) for 3 h (Group 3). The activation effects in the three treatments and the control group (untreated) were judged by the extrusion of the second PB and formation of a pronucleus (PN). Differences among groups were analysed using one-way ANOVA after arc-sine transformation of proportional data. All three treatments led to high activation rates (90% to 95%), with significant difference from the control. However, the extrusion of the second PB and the rate of PN formation differed remarkably among treatments. In Group I and 3, about 95% of the oocytes had extruded the second polar body, but one PN had formed in a higher proportion of oocytes in Group 3 than in Group 1 (90% vs. 5%). In experiment 2, the rates of cleavage and development into blastocysts in Group 1 were significantly lower than those of Group 2 and 3 (8.7% and 0% vs. 50.5% and 11.6%, and 44.6% and 7.2%, respectively, P<0.05). In experiment 3, ~80% of parthenotes in Group 1 were developed with haploid chromosomal sets. However, when ionomycin was followed immediately by DMAP (Group 2). only 20% of parthenotes were haploid. In Group 3, combined treatment with ionomycin and SPP, the appearance of abnormal chromosomal tracts was significantly (P〈0.05) reduced and the proportion of haploid parthenotes was increased to 85% (17/20) than in Group 2. These results demonstrate that SPP acted as a cdc2 kinase inhibitor and formed the haploidy in oocyte activation. Thus, the present study suggests that cdc2 kinase inhibitor, such as sodium pyrophosphate, may have an effective role in oocyte activation for the production of cloned embryos/animals by nuclear transplantation.
To develop an effective vitrification method, we examined the use of a conventional straw as vessel fur vitrification of mouse oocytes, and to compare the post-thaw survival and chromosome configuration of these oocytes with those vitrified in grids. Intact cumulus-enclosed oocytes were vitrified with DPBS with 5.5 M ethylene glycol and 1.0 M sucrose, and loaded into straws and onto eletron microscopic copper grid fur storing in liquid nitrogen. Intact vitrified and thawed oocytes were karyotying for chromosome. The rates of post-thawed survival were 88.5% in vitrified oocytes with straws, and 83% in vitrified ooctyes with grids. Vitrified and thawed oocytes with straws and grids were increased chromosomal abnormality (31.4% and 30.9%) compared with fresh oocytes (17.8%). The conventional straws can be used as vessel for vitrification to prevent of inflection in liquid nitrogen.
Selection of oocyte cryopreservation method is a prerequisite factor for developing an effective bank system. Compared with slow freezing method, the vitrification has various advantages such as avoiding intracellular ice crustal formation. In our previous, we attempted to employ a vitrification method using ethylene glycol and an electron microscope grid for cryopreservation of mouse oocytes. However, A high incidence of spindle and chromosome abnormalities was detected in thawed oocytes after vitrification. We examined whether the addition of a cystoskeleton stabilizer Taxol , to the vitrification solution could promote the post-thawed survival and subsequent development of stored oocytes. More oocytes developed to the 4-cell (44.7% vs. 69.7%), 8-cell (31.8% vs. 64.2%), morula (24.7% vs. 54.3%), and blastocyst (20.3% vs. 49.2%) stages after the addition of Taxol to the cryoprotectant than after no addition. 21 and 26 mouse pups were born after transfer of blastocyst derived from oocytes vitrified without and with Taxol. The addition of Taxol to vitrification solution greatly promoted post-thaw preimplantation development of ICR morose oocytes.tes.
This study was performed to evaluate whether vitrification method using ethyle glycol and eletron microscopic (EM) grid could be used far the cryopreservation of human oocytes in ART program. Surplus oocytes were obtained from consented IVF patients. These surplus human oocytes were frozen with our vitrification method, Oocytes were exposed to 1.5M ethylene glycol (EG) in DPBS far 2,5 minutes, followed by 5.5M EG plus 1.0M Sucrose in DPBS for 20 seconds. Then oocytes were transferred onto the EM grid and the grid was plunged into LN2 for storage. For thawing, oocytes containing EM grid were sequentially transferred in 1.0M, 0.5M, 0.25M, 0.125M and 0 M sucrose in DPBS solution at the intervals of 2.5 minutes. Thawed and survived oocytes were provided for ICSI. Embryos from vitrified oocytes were transferred to uterus of the patient on 4 to 5 days after ovulation in natural cycles of on 15 to 17 day of hormone replacement cycles. A total of 370 oocytes from 26 patients were thawed and 159 (43.0%) of them survived. One hundred thirty four oocytes (84.3%) were fertilized normally and 126 pre-embryos were transferred to 26 patients, resulting in 5 clinical pregnancies. The pregnancy rate per transfer was 19.2% and implantation rate was 4.0%. Among the five pregnant, 4 patients delivered 4 healthy babies and the one patient was 32-week ongoing pregnancy. From this results, vitrification using ethylene glycol as cryoprotectant and EM grid is a rapid and simple method that can be effectively applied for the cryopreservation of human oocytes in ART program.