This study was to analyse the usability of morphological evaluation of vitrified-thawed oocyte before somatic cell nuclear transfer (SCNT) using Oosight imaging system to show spindle. For the vitrification, in vitro matured bovine MII oocytes were treated by two-step freezing medium without (control group) or with 5 ug/ml cytochalasin-b (CCB group). In Exp. 1, after thawing, recovered oocytes in each treatment group were assessed by live image using Oosight imaging system or/and cytoskeletal protein image using immunostaining. In Exp. 2, in each treatment group the in vitro developmental potential of frozen-thawed bovine oocytes post evaluation using Oosight imaging system and then SCNT was investigated. The SCNT embryos were cultured in CR1aa medium supplemented with 10% FBS, 1 mM EGF and 1 mM IGF at 38.5 C in 5% O2 and 5% CO2 in air for 8 days. In Exp.1, the rates of in vitro survival, morphological good grade and spindle normality of CCB treatment group (91.1%, 54.2% and 55.5%) were better than those of control group (86.1%, 48.5% and 48.5%). After SCNT using vitrified-thawed oocyte, the rates of fusion, reconstructed embryos and blastocyst development were also high in CCB treatment group (66.6%, 36.4% and 3.0%) than control group (60.0%, 27.3% and 0%). These results demonstrated that the identification of morphological spindle image of the vitrified-thawed bovine oocytes using Oosight imaging system helps to predict the SCNT embryo quality.

This study was to investigate the effect of flavonoid treatment on in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos, and their pregnancy and delivery rate after embryo transfer into recipient. In experiment 1, to optimize the flavonoid concentration, parthenogenetic day 2 (≥ 2-cell) embryos were cultured in 0 (control), 1, 10 and 20 μM flavonoid for 6 days. In the results, in vitro development rate was the highest in 10 μM flavonoid group (57.1%) among treatment groups (control, 49.5%; 1 μM, 54.2%; 20 μM, 37.5%), and numbers of total and ICM cells were significantly (p<0.05) higher in 10 μM flavonoid group than other groups. We found that 10 μM flavonoid treatment can significantly (p<0.05) decrease the apoptotic index and derive high expression of anti-oxidant, anti-apoptotic, cell growth and development marker genes such as Mn-SOD, Survivin, Bax inhibitor, Glut-5, In-tau, compared to control group. In experiment 2, to produce the cloned Jeju Black Cattle, beef quality index grade 1 bull somatic cells were transferred into enucleated bovine MII oocytes and reconstructed embryos were cultured in 10 μM flavonoid added medium. When the in vitro produced day 7 or 8 SCNT blastocysts were transferred into a number of recipients, 10 μM flavonoid treatment group presented higher pregnancy rate (10.2%, 6/59) than control group (5.9%, 2/34). Total three cloned Jeju Black calves were born. Also, two cloned calves in 10 μM flavonoid group were born and both were all healthy at present, while the one cloned calf born in control group was dead one month after birth. In addition, when the result of short tandem repeat marker analysis of each cloned calf was investigated, microsatellite loci of 11 numbers matched genotype between donor cell and cloned calf tissue. These results demonstrated that the flavonoid addition in culture medium may have beneficial effects on in vitro and in vivo developmental capacity of SCNT embryos and pregnancy rate.

This study was to investigate pregnancy rate of IVM/IVF/IVC Korean cattle (registered in government) embryos according to transport time course. For the production of embryos, oocytes recovered from slaughtered excellent grade cow and highly motile frozen‐thawed bull semen (purchased from LIMC, KPN#497) was used. In vitro produced embryos were cultured in CR1aa medium for 8 days and some of them were frozen. The rate of average cleavage (>2‐cell) was 83.0% (308/371) and blastocyst rate at day 8 was 34.7% (107/308). Among in vitro produced blastocyst embryos at day 8, most healthy embryos were freshly transferred on production day and some frozen embryos were direct transferred on appropriate day. These embryos were produced in a laboratory, embryo transfer (ET) was planned in 10 areas of the remote island (Jeju) from the laboratory by airplane. Thus, we examined the pregnancy rate in recipient cow according to embryo of transport time course before ET. From embryo transferred 44 recipient cows, overall pregnancy was 40.9% (18/44), these 18 cows were all calved [single, 94% (17/18); twin, 6% (1/18)] and total embryo implantation rate was 26% (19/66). Comparing transport time in the base of 6 hr, pregnancy rate in ET group required less 4 hr (60%, 9/15) was significantly higher than that required more 6 hr (26.3%, 5/19). In direct ET of freezing embryos, the pregnancy rate was 40% (4/10). However, it was difficult to find the meaning of temperature, pH and corpus luteum quality of recipients on comparison of pregnancy rate. When the cell death level of embryos according to storage time in thermos (straw container) before ET was measured by TUNEL staining, apoptotic index was increased with storage time‐dependent. These results demonstrated that long distance transfer of IVM/IVF/IVC embryos is possible and the time of embryo transport is very important for the pregnancy rate on field trial.