This study was performed to investigate the effect of laser-assisted hole in the zona pellucida (ZP) of frozen-thawed ICR mouse embryos on the process of hatching that is critical for expanded blastocysts to implant into endometrium, Vitrification medium, composed of ethylene glycol and sucrose supplemented with 7.5% (w/v) PVP, was used to freeze cell stage embryos recovered from oviducts of superovulated and mated female mice before storing them in . Right after thawing them, a laser beam was shot to make a hole in ZP followed by culturing in KSOM for and examining development to blastocyst and hatching every 12 hr. Laser-treated embryos showed significantly higher hatching rate compared to control (92.9% vs. 22.1%, p<0.05). From around Day 4, blastocysts developed from laser-treated embryos started hatching while the blastocysts of control group failed to hatch showing a lot of shrinkage. This study shows that a laser-assisted hole in ZP improves the hatching rate of blastocysts developed from frozen-thawed, in vitro cultured ICR mouse embryos.

본 연구는 OPS 기법에 의한 돼지 수정란의 동결-융해 시 수정란의 발달 단계와 superoxide dismutase (SOD)가 수정란의 생존능력에 미치는 영향을 검토하였다. 돼지 체외수정 배반포는 OPS방법에 의해 동결 후 융해하여 0~10unitsml의 SOD 존재 하에 48시간 체외배양하였다. 동결-융해 후 형태학적으로 정상적인 수정란의 비율은 초기, 중기 및 확장배반포간에 유의적인 차이는 인정되지 않았다(30.8~38.6%). 그러나 발육단계가 높을수록 형태적으로 정상인 수정란의 비율이 높은 경향을 나타냈다. 수정란의 융해 후 48시간 추가 배양했을 때, 발육이 진행된 수정란은 후기배반포기에 동결한 수정란이 38.7%로 유의적으로 높았으며(P<0.05), 1 unit/ml의 SOD를 첨가한 경우 비교적 높은 생존율을 나타내었다. 본 연구의 결과로부터, 수정란의 OPS방법에 의한 동결-융해 후 생존성의 향상을 위해서는 후기배반포기 단계에 동결하는 것이 유리하며, SOD의 첨가는 수정란의 손상을 어느 정도 방지할 수 있을 것으로 사료된다.

The role of heat shock proteins in shielding organism from environmental stress is illustrated by the large-scale synthesis of these protein by the organism studied to date. However, recent evidence also suggests an important role for heat shock protein in fertilization and early development of mammalian embryos. Effects of elevated in vitro temperature on in vitro produced bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (HSP70) by control and frozen-thawed after in vitro fertilization (IVF) or nuclear transfer (NT). The objective of this study was to assess the developmental potential in vitro produced embryos with using of the various containers and examined expression and localization of heat shock protein 70 after it's frozen -thawed. For the vitrification, in vitro produced embryos at 2 cell, 8 cell and blastocysts stage after IVF and NT were exposed the ethylene glycol 5.5 M freezing solution (EG 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min, and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid, cryo-loop. However, survival rates by straw were relatively lower than other containers. Only, nuclear transferred embryos survived by using cryo-loop. After IVF or NT, in vitro matured bovine embryos 2 cell, 8 cell and blastocysts subjected to control and thawed conditions were analysed by semiquantitive reverse transcription polymerase chain reaction methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNA were higher thawed embryos than control embryos. Immunocytochemistry used to localization the hsp70 protein in embryos. Two, 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed frozen-thawed. However, under control condition, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform in distribution.

Bovine oocytes surrounded with compact cumulus cells were cultured for 20 to 22 hours(, 5% ) in modified TCM-199 medium supplemented with 5% superovulated cow serum(SCS) and inseminated by in vitro capacitated spermatozoa. Day 7 to 8 embryos were equilibrated for 10 minutes in 1.3M methyl cellosolve(MC) <1.1M diethylene glycol(DEG), 1.8M ethylene glycol(EG), 1.6M propylene glycol(PG) and 1.1 M 1,3-butylene glycol(BG) solutions. They were then loaded into 0.25ml straws, placed into an alcohol bath freezer at , cooled from to at /minute, seeded, held for 10 minutes, and stored in liquid nitrogen. After thawing in water, the embryos wee rehydrated in TCM-199 medium and then cultured for 48 hours in TCM-199 plus 5% SCS. Embryos were considered viable if they progressed to later developmental stages with a good morphology. Some of the embryos frozen in each cryoprotectant were thawed and transferred non-surgically without removing the cryoprotectant. Hatched embryos survived freezing and one-step dilution as follows : EG(50.0%), MC(53.6%), DEG(56.9%), PG(58.0%) and BG(11.5%). The survival rate of embryos cooled at vs. /minute was not significantly different(P<0.05), however, blastocysts hatched most often (P<0.01) in vitro when cooled at a rate of /minute(64.6%), 31/48) than at /minute(22.6%, 12/53). Pregnancy rates resulting from embryos frozen in the different cryoprotectants were as follows : MC(48%, 10/21); DEG(30%, 3/10); EG(74%, 20/27); and PG(40%, 4/10). These results indicate that MC, DEG, EG and PG have utility as cryoprotectants for the freezing and thawing of IVF Bovine embryos.

This study was carried out to establish the techniques for producing the calves of genetically superior Korean Native cattle by transfer of frozen-thawed embryos. The effects of some factors related to embryo recovery following superovulation and pregnancy rate following transfer of frozen-thawed embryos were evaluated. Also calving state was investigated. The results obtained were as follows ; The mean number of total and transferrable embryos recovered per superovulated cow was 8.72 and 4.90, respectively, from a total of 72 superovulations using 34 donor cows. There were no significant differences in the number of total or transferrable embryos recovered per superovulated cow between products of follicle stimulating hormone (FSH), years, seasons, and collection numbers. The pregnancy rate was found 44.44% following transfer of frozen-thawed embryos of Korean Native cattle to a total of 180 recipient cows including 82 Angus, 27 Charolais, 62 Hereford and 9 Korean Native cows. The pregnancy rate was significantly (P<0.05) higher in the transfer of excellent (42.99) and good embryos (40.17%), compared with fair (5.90%) grade embryos. And the pregnancy rate was significantly (P<0.05) higher in the transfer of embryos of morula stage (43.86%) than blastocyst stage (15.51%). But there were no significant differences in pregnancy rates between natural and induced estrus estrus asynchrony of 1 days, breeds, and parities of recipient cows. The normal calving rate of 80 pregnant cows following transfer of frozen4hawed em-bryos was 87.5% and the other 10 pregnant cows showed abortion during the period from pregnancy diagnosis at 50~60 days to calving. The average gestation length of normally delivered recipients was 288.50 days and the average birth weight of 70 calves born was 24.22 kg. The gestation length was significantly (P<0.05) shorter in the recipients delivering female calves (286.70 days) than males (289.39 days). But there were no significant differences in gestation tength and birth weight of calves born between the recipient breeds.

The present study was carried out to investigate the effects of cryoprotectants, equilibration step, freezing rate, culture condition following in vitro fertilization, and age and development stage of embryo by freezing with conventional slow freezing and vitrification on survival of frozen-thawed Korean native cattle(KNC) blastocysts produced in vitro. The KNC blastocysts produced in vitro were equilibrated in 1.8M ethylene glycol or 1.4M glycerol and cooled from -6 to -35 at -0.3 or -O.6 /minute. When equilibrated in 1.8M ethylene glycol, survival rate of fiozen4hawed blastocysts was sarne in both -0. 3 /min and -0.6 /min cooling rate(71.4%). With the equilibration in 1.4M glycerol, survival rate was higher in -0.3 /min(63.6%) than in -0.6 /min cooling rate(53.8%). For vitrification of the KNC blastocysts produced in vitro, they were equilibrated in 2-step or 3-step exposure to vitrification solution(25% ethylene glycol + 25% glycerol). Survival rate was sirilar in both 2-step(45.0%) and 3-step exposure(47.4%). According to culture condition following in vitro fertilization, higher survival rate was obtained for blastocysts co-cultured with bovine oviductal epithelial cell(BOEC, 77.3%) than for those cultured with epidermal growth factor(EGF, 65.7%) or for those co-cultured with BOEG + EGF (54.8%). According to embryo age and development stage, higher survival rate was obtained for 7-day ernbryos(70.0%) than 8-day(56.8%) or 9-day(20.0%) for blastocyst stage and obtained for 8-day embryos(74.3%) than 7-day(62.5%) or 9-day(42.9%) for exponded blastocyst. In surnmary, higher survival rate of frozen4hawed KNC blastocysts produced in vitro were obtained by using ethylene glycol for cryoprotectant and -0.3 /min for cooling rate. And higher survival rate were obtained with co-culture with BOEC for culture condition following in vitro fertilization and with 7-day blastocyst or 8-day expanded blasto cyst for embryo age and development stage.

This study investigated full-term development potential of ultrarap idly frozen and thawed mouse 2-cell embryos. Mouse 2-cell embryos, dehydrated by exposure to freezing medium, were directly immersed into liquid nitrogen and thawed in 37 water. The embryos that were frozen and thawed were cultured in uitro and transferred to foster mothers to examine there developmental potential. As a result, the frozen-thawed 2-cell embryos developed to blastocysts in vitro as a similar rate as control 2-cell embryos did(in vitro 2-cell, 86.4%; in vivo 2-cell, 90.9%; solution control, 89.9%; control, 89.7%). Normal live young were obtained from transfer of frozen-thawed embryos to the oviduct and uterus of pseudopregnant recipients (3l.4~56.7%).

The objective of this experiment was to study some possibilities to simplify freezing, thawing and transfer procedure of one-step straw method comparing with the conventional methods using bovine embryos. The previous work are also designed to investigate the thawing effect by development stage and its quality using the embryos. Results obtained were summarized as follows: 1. A total of 87 embryos from 14 donor cows were frozen-thawed and an average of frozen embryo/donor was 6.2. 2. The survival rates of morula stage(65.4%) were higher than those of blastocyst stage(57.l%) and vice versa in rate of morphological recovery (80% vs 95.4%). However. no significant difference was denoted between them. 3. In difference between the groups of good quality and poor quality. good quality was resulted in a significantly higher embryo survival rate(75%) and recovery rates(95%) than poor quality(P<0.0l). 4. In effects of non-permeable sugar dilution in added to l.0M glycerol. higher survival rates were orderd in sucrose. lactose, raffinose and xylose. But lactose-raffinose, sucrose-trehalose and xylose in added to 2.OM glycerol. 5. The highest survival rates were obtained by direct plunge into the liquid nitrogen with 3.OM concentration both of glycerol and trehalose. 6. The survival rates in vitro condition of one-step and direct plunge methods(75%-87.5%) were significantly higher than those of multiple steps (21.4-52.6%) in in vitro (P<0.0l). However, the results of single-step were critical in comparing to other steps of final pregnant conformation.

The purpose of this is to investigate the effects of vitrification in open pulled straws (OPS) on in vitro survival of porcine embryos. Blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed boar semen, and subsequent culture on granulosa cell monolayer. After frozen-thawing, embryos were culture in NCSU-23 medium with 5 mM hypotaurine, 4 mg/ BSA and 10 ng/ for 48 hrs to survival tests. When blastocysts were frozen-thawed by OPS methods, the embryos with normal morphology were 32.1, 34.5 and 38.9 % in early blastocyst, blastocyst and expanded blastocyat stages. The rates of partial damaged embryos were significantly (P<0.05) higher in early biastocysts than expanded blastocysts. In another experiment, the embryos frozen by OPS methods were cultured for 48 hrs for survival and developmental rates in vitro. The proportions of embryos hatched were 11.8, 20.2 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. On the other hand, The proportions of embryo with normal morphology after culture were 23.5, 25.0 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. These finding indicate the possible broader application for OPS methods that this procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages.