One-step dilution and direct transfer would be a practical technique for the field application of frozen embryo. This study was to examine whether Jeju Black Cattle (JBC, Korean Cattle) can be successfully cloned from vitrified and one-tep diluted somatic cell nuclear transfer (SCNT) blastocyst after direct transfer. For vitrification, JBC-SCNT blastocysts were serially exposed in glycerol (G) and ethylene glycol (EG) mixtures〔10% (v/v) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.〕which is diluted in 10% FBS added D-PBS. And then SCNT blastocysts were loaded in 0.25 ml mini straw, placed in cold nitrogen vapor for 3 min. and then plunged into LN2. One-step dilution in straw was done in 25℃ water for 1 min, by placing vertically in the state of plugged- end up and down for 0.5 min, respectively. When in vitro developmental capacity of vitrified SCNT blastocyst was examined at 48 h after one-step dilution, hatched rate (56.4%) was slightly lower than that of control group (62.5%). In field trial, when the vitrified-thawed SCNT blastocysts were transferred into uterus of synchronized 5 recipients, a cloned female JBC was delivered by natural birth on day 299 and healthy at present. In addition, when the short tandem repeat marker analysis of the cloned JBC was evaluated, microsatellite loci of 11 numbers was perfectly matched genotype with donor cell (BK94-14). This study suggested that our developed vitrification and one-step dilution technique can be applied effectively on field trial for cloned animal production, which is even no longer in existence.

The objective of this work was to determine the effect of corpus luteum (CL) grade on pregnancy rate after embryo transfer in Korean cattle and we found that CL development was linked to pregnancy rate. The in vivo derived blastocyst-stage embryos were transferred to 15 recipients synchronized in the estrus cycles. Based on size and palpable characteristics, CLs were categorized into three grade. The grade three CL is not to be identified by rectal palpation. The pregnancy rates tended to increase with the increase in CL size of recipients. In grade one, two, and three, the pregnancy rates were 62.5%, 50.0%, and 0%, respectively. This result suggests that pregnancy rates after embryo transfer might be affected by the CL status of recipients.

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was performed to investigate whether oxygen consumption reflects morphological grade of in vivo derived bovine blastocyst-stage embryos (blastocyst). The oxygen consumption of in vitro produced blastocyst was compared to its total cell number. In addition, pregnant rate was measured after transplantation of in vivo blastocysts with different oxygen consumption. The quality of blastocyst collected on day 7 after artificial insemination was categorized as grade I and II (G I and G II) based on microscopic observation of the morphology. Oxygen consumption of blastocyst was measured using a scanning electrochemical microscopy (SECM) and total cell number of in vitro blastocyst was enumerated by counting cells stained by propidium iodide. Pregnancy of recipient cow was confirmed with rectal palpation after 60 days of embryo transfer. The oxygen consumptions of G I blastocysts were significantly higher than those of G II blastocysts ( versus , p<0.05). Total cell numbers of in vitro blastocysts were 74.8, 90.7, and 110.2 in the oxygen consumption of below 10.0, 10.0~12.0, and over respectively. Total cell number was significantly increased in embryos with high oxygen consumption (p<0.05). Pregnant rate in recipient cow was 0, 50, and 85.7% in the transplantation of embryo with the oxygen consumption of below 10.0, 10.0~12.0, and over , respectively. These results suggest that measurement of oxygen consumption may help increase the pregnant rate of bovine embryos.

In vivo embryo produced from Hanwoo donor cows were collected and transferred to Hanwoo recipients. Cows, at random stages of the estrous cycle, received Progesterone Releasing Intravaginal Device (CIDR-plus, InterAg, New Zealand) together with injection of 1 mg estradiol benzoate and 50 mg progesterone, and gonadotropin treatment began 4 day later. For superovulation, a total of 28 mg FSH was intramuscularly injected twice a day in the way of decreasing doses 4 day (5, 5, 4, 4, 3, 3, 2 and 2 mg). Twenty one Hanwoo donor cows were flushed on day 7 of estrus cycle with same FSH and artificial insemination by the same technicians. Embryos were recovered 7 days after the second insemination by flushing the uterus with Embryo Collection Medium. The results obtained were as follows: The rates of transferable embryos were 50.3%, and 78 fresh embryos at morulae and blastocysts stage were transferred into Hanwoo recipients on day 7 of estrus cycle. The pregnancy rates were first embryo transfer 55.6%, 2nd 62.9% and 3rd 57.9%, respectively. In conclusion, These results suggest that CIDR-based superovulation protocol may be effectively used for production of superior Hanwoo embryos. Also, since it seems the condition of recipient cows greatly affect pregnancy rate, it is very important to evaluate recipient for effective cattle production.

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.

Mongolia has 80% livestock of total agriculture industry, 170,000 farms are engaged, 2,500,000 of cows that were beef and dairy cows are raised. Despite of Mongolian has great application with milk, there are not clear differences between cow and dairy cattle, and the production of milk is also low. But the milk suppliers are varied (horse, sheep, goat, etc), so that the total milk production is 500 thousand ton per year. It's really considerable to improve the breed of owing to many problems with big differences among milk qualities. For carrying out for first year project, artificial insemination project was operated with 3rd grade Holstein semen that were imported from S. Korea, and initiation and field training were also carried out through appropriate AI technique we developed for Mongolia environment. Local information research and MOU conclusion were done with professor D. Altangerel in May , 2009, and development for AI technique and AI equipments were supplied for Mongolia breeding and natural environment in July in 2009. All cows were treated by synchronization for AI. To do this, injection were treated for luteal phase cow, if it wouldn't work, try again after 11 days. After confirmation of estrus, AI and AI training were carried out with sperm injection in the uterus or cervix by rectum-vagina method which is common worldwide, the most effective artificial insemination technique. If cows were return to next estrus cycle, second AI was carried out about approximately 21 days after artificial insemination. After 2 months, all cows not showing return estrus should be taken pregnancy test. Every pregnant cow will be cared thoroughly. Total 48 cows administrated by for synchronization and after 48 hours 45 cows (93.8%) showing estrus were detected and then artificial inseminate them within who 8 cows (27.8%) showed return estrus. Therefore, Using for synchronization is effective to use for Mongolia breeding conditions. There are possibility of base for food production after all, including increase of livestock production in Mongolia by improvement of breeding cow with AI and embryo transfer project.

This study was performed to investigate the possibility of repeated superovulation treatment at interval from 27 days to 41 days in Hanwoo cattle and to compare with superovulation effect between doses of FSH 200 mg and FSH 400 mg. Different doses of FSH (200 mg or 400 mg) were injected at Day 8 after controlled internal drug release (CIDR) treatment for superovulation of Hanwoo donors following CIDR treatment (Day 8 after the estrus). Superovulation was repeated four times for one donor and number of corpus luteum (CL), number of embryos, number of transferable embryos and pregnancy rate after embryo transfer (ET) were investigated. 5 cows were used for each FSH treatment (10 cows in total). Average number of CL were and for the donors treated with FSH 200mg and FSH 400mg, respectively. Average number of embryos collected were and for the donors treated with FSH 200 mg and FSH 400 mg, respectively. Average number of transferable embryos were and for the donors treated with FSH 200 mg and FSH 400 mg, respectively. The pregnancy rate following ET with embryos collected from 200 mg FSH treated donors and 400 mg FSH treated donors were 61.9% and 53.8% respectively. The numbers of embryos tended to be decreased as the numbers of repeat of superovulation were elapsed. These results indicated that superovulation treatment by about a month to Hanwoo donors is usable and 200 mg of FSH is preferable for simple FSH treatment following CIDR treatment.