본 연구는 돼지 배아의 유리화 동결 시 CB 처리군과 CB 비처리군의 영향을 비교하였다. CB의 농도별, 처리 시간별로 처리한 뒤 유리화 동결 융해 후 정상적인 형태 회수율과 생존률을 각각 조사하였다. 1. CB를 전 처리한 군과 비처리군의 정상적인 형태율이 각각 84.2%, 81.9%로 유의한 차이가 없었으나, 융해 후 72시간 째 생존률의 결과에서는 각각 60.5%, 32.8%로 유의성(p<0.1)이 검증되었다. 2. CB 농도별 생존률에 미치는 영

This study evaluated the efficiency and compared with different materials of loading vessels for vitrification-plastic/glass, copper grid and nylon. The loading method, vitrification, cryop-reservation and warming method of the oocytes were examined. The loading samples prepared in manual or company-made and sterilized, loaded the COCs selected on each samples and cultured for maturation during 40 hours, and then exposed sequentially to ethylene glycol solution. Thawing method was reversely treated and exposed for warmed oocytes. After oocytes were thawed, fertilized and cultured in vitro for 3-4 hours, rates of development and morphological appearance were examined. The results were as summarized: ㆍOPS from company-made or hand-made of the hematocrit micropipettes, NLS from fishing line and EMG from company-made for EM were used for loading oocytes, respectively. ㆍThe efficiency of freezing method and loading convenience were orderly higher in OPS, NLS and EMG. The optimal capacity per vessel was orderly lowered in NLS, EMG and OPS, respectively. ㆍAfter oocytes were warmed, the recovery rate, morphology and rate of development were orderly higher in OPS, NLS and EMG, respectively. ㆍIn conclusion, OPS has the advantages of achieving a little more survival and preserving results than other two loading methods.

Human embryonic stem (hES) cell lines have been derived from human blastocysts and are expected to have far-reaching applications in regenerative medicine. The objective of this study is to improve freezing method with less cryo-injuries and best survival rates in hES cells by comparing various vitrification conditions. For the vitrifications, ES cells are exposed to the 4 different cryoprotectants, ethylene glycol (EG), 1,2-propanediol (PROH), EG with dime-thylsulfoxide (DMSO) and EG with PROH. We compared to types of vehicles, such as open pulled straw (OPS) or electron microscopic cooper grids (EM grids). Thawed hES cells were dipped into sequentially holding media with 0.2 M sucrose for 1 min, 0.1 M sucrose for 5 min and holding media for 5 min twice and plated onto a fresh feeder layer. Survival rates of vitrified hES cells were assessed by counting of undifferentiated colonies. It shows high survival rates of hES cells frozen with EG and DMSO (60.8％), or EG and PROH(65.8％) on EM grids better than those of OPS, compared to those frozen with EG alone (2.4％) or PROH alone (0％) alone. The hES cells vitrified with EM grid showed relatively constant colony forming efficiency and survival rates, compared to those of unverified hES cells. The vitrified hES cells retained the normal morphology, alkaline phosphates activity, and the expression of SSEA-3 and 4. Through RT-PCR analysis showed Oct-4 gene expression was down-regulated and embryonic germ layer markers were up-regulated in the vitrified hES cells during spontaneous differentiation. These results show that vitrification method by using EM grid supplemented with EG and PROH in hES cells may be most efficient at present to minimize cyto-toxicity and cellular damage derived by ice crystal formation and furthermore may be employed for clinical application.

본 연구는 미성숙, 성숙 단계의 돼지 난포란이 유리화 동결에 의한 동결보존이 가능한지를 조사 하고자 실시하였다. 난포란은 세포질 내 지방구를 분극시키기 위해 원심분리를 실시하였고, 미세조작기를 이용하여 지방구를 제거하였다. 돼지 난포란을 CB 처리하여 원심분리 후 지방구를 제거한 지방제거구(Delipated), CB 처리 후 원심분리만 하여 지방구를 분극시킨 원심분리구(Centrifuged), 아무처리도 하지 않은 대조구(Control)를 EM grid

본 연구에서는 vitrification방법을 사용하여 돼지 미수정란의 동결-융해시 난자생존능력에 대한특정 동해방지제 사용과 superoxide dismutase(SOD) 첨가의 영향을 검토하고자 수행하였다. 그 결과 미성숙 난자를 ethylene glycol과 DMSO 노출 후 성숙율(M-I에서 19.9%)이 glycerol과 DMSO 노출 후 성숙율(M-I에서 6.5%)보다 더 높았으며 ethylene glycol에 노출 후에는 M-I기로 성숙발달한

The advantages of the OPS techniques(Vajta G et al, Mol Reprod Dev 51: 53-58,1998) give 1) high survival rates of various types of eggs, 2) quick and simple process, 3) inexpensive equipment and reduced chilling injury. The efficiency of IVM/IVF technique in the porcine species is relatively lower than that obtained in other species such as ruminants. Two experiments were designed to investigate the effects of in-vitro fertilization of porcine oocytes matures using different OPS protocol for chilling and warming of vitrification. Porcine oocytes from ovaries collected at abattoir were matured for 44 hours in TCM199 Earle's salt supplemental with pyruvate, pff, L-cysteine, hormones and gentamycin. Oocytes were denuded and fertilized with frozen boar semen by common method. Porcine embryos produced routinely by in-vitro culture system of NCSU23 medium. The vitrification and the warming were conducted by OPS method with the glass micropipette instead of straw vessels and modified the protocol of G.Vajta(1999). In Exp 1, Chilling/Warming:Holding Medium(HM)+EG+DMSO/HM +sucrose Medium(SM) at 39 warm stage. In Exp 2, : PBS+CS+EG+Ficoll+ Trehalose/PBS+Trehalose at 25 stage. Filling, freezing, packing, thawing out and further culturing were performed to follow the basic protocol of G Vajta. During IVM-lVC and post-warming, fertilization parameter and developmental potential were compared to and statistically analysed. It was not significantly different from Exp 1 and Exp 2 but 25 of stage was slightly higher on the morula/blastocyst forming rate and better atmosphere for worker than that at 39 stage.

The purpose of this study was to investigate the warming temperature and exposed time on the post-thaw survival rate and viability of bovine blastocyst cryopreserved by GMP vitrification. Groups of three bovine IVP blastocysts were sequentially placed into vitrification solution before being loaded into the GMP straws and immersed into LNwithin 20 to 25 sec. The warming rate was increased 2 times of warming temperature for improvement of post-thaw survival rates. The frozen embryos were warmed either at 35 or 70 for 1 or 2 sec and then diluted in sucrose solution. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in holding medium (HM: TCM199 supplemented with 10% FCS) and TCM-199 for each 5 min, respectively, and then cultured in TCM199 for 24 h. The rate of re-expanded blastocyst was significantly different fer 35 and 70 warming temporature (76.4 vs. 89.3%; P<0.05). The rate of re-expanded blastocyst at 70 for 1 sec was significantly higher than that for 2 sec (91.1 vs. 70.9%; P<0.05). The number of nuclei counted were significantly different among control, 35 and 70 (1218.5 vs. 10411.7 vs. 11410.3; P<0.05). These results indicated that the increasing of warming rate can provide high survival rates of bovine IVP blastocysts. Especially, the best viability of post-thaw blastocyst could be thaw at 70 for 1 sec. The warming temperature and exposed time far warming was considered to be limiting factors to the viability of bovine IVP embryos. he purpose of this study was to investigate the warming temperature and expose.

본 연구는 소형견의 불임 해결을 위해 미숙난자를 보존 후 이용할 수 있는지의 여부를 판명하기 위하여 미성숙 난포란을 시간별로 배양한 뒤 vitrification 동결 융해후 체외수정율과 생존율을 조사하였다. 1 미숙난포란을 회수 후 1, 6, 12, 24시간 성숙 배양 후vitrification동결 융해 후 체외수정 시켰을때 수정율은 각각 31.4％. 22.5％, 11.9％ 및 5.3％로서 대조군의 수정율 60.0％에 비해 낮은 성적이었고, 회수 후 시

한우 미성숙 난자의 vitrification 동결시 발생단계별 생존성과 체외발생율을 알아보고자 난자를 0, 10, 14 및 20시간 성숙배양시킨 후 vitrification 동결 융해 후의 체외발생율을 조사하였다 본 연구에서 나타난 결과를 요약하면 다음과 같다. 1. 0, 10, 14 및 20시간 성숙배양시킨 난자를 vitrification 동결보존 후 MR 단계로의 발생율은 각각 33.3%, 55.0%, 68.3% 73.3%였으며, diploid로의

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.

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.