Production of u 1-antitrypsin (AT) in transgenic cows has a great value in the field of medicine. The present study was conducted to determine the effect of chemically defined KSOM media on in vitro development of bovine transgenic nuclear transfer (NT) embryos. An expression plasmid for human AT was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human AT target gene into a pcDNA3 plasmid. Cumulus cells as donor nuclei in NT were collected from a Holstein cow and transfected by lipid-mediated method using FuGene6 (Roche Molecular Biochemicals, USA) as reagent. GFP expressed cumulus cells were introduced into recipient oocytes under DIC microscopy equipped with FITC filter set. After electrical fusion and chemical activation, reconstructed embryos were cultured in 1) SOF + 0.8% BSA, 2) KSOM + 0.8% BSA, 3) KSOM + 10% FBS and 4) KSOM +0.01% PVA for 192 h at 39 with 5% , 5% and 90% in humidified condition. The development of the embryos was recorded and the GFP expression in blastocyst was determined under FITC filter. The average fusion rate was 73.8% (251/340; n=8). The development rates to 2-4 cells, morula, blastocysts and expression rates in blastocysts varied from 70.3 to 76.5%, 30.2 to 33.8%, 25.4 to 33.8% and 11.8 to 15.6%, respectively. The difference in development and expression rates of embryos among 4 culture groups was not significant (P>0.05). This study indicates that chemically defined KSOM medium is also able to support development of bovine transgenic NT embryos at similar rate of SOF or KSOM supplemented with BSA or serum.

In vitro embryo culture techniques provide significant contributions not only for a basic research of fertilization and early embryogenesis, but also for a low cost mass production of bovine embryos for transfer, embryo diagnosis, nuclear cloning and the production of transgenic cows. This presentation introduces newly developed serum-free media (IVD101 and IVMD101) that are effective far high yields of transferable embryos of excellent quality from in vitro-matured and fertilized oocytes. Both serum-free media are superior to a conventional serum-containing medium on the increased rates of blastocyst formation, post-thaw embryo viability, and pregnancy after transfer. Furthermore, reduced risks of calf mortality and large calf syndrome are also observed for the serum-free-derived embryos. Serum-derived embryos contain a large number of lipid droplets and immature mitochondria in their cytoplasm that may account for the lower production of transferable embryos and poor embryo quality.

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

The objective of this study was to improve the efficiency of bovine embryo transfer by transferring of Hanwoo embryos into Hanwoo or Holstein recipients. The cryopreserved or fresh in vitro produced(IVP) embryos were transferred into uterine horn contralaterally or ipsilaterally to the corpus luteum. The recipients were inseminated by artificially on the next day of estrus. The pregnancy was diagnosed by rectal palpation at 60∼90 days after transfer of the embryos. The pregnancy rate by transfer of one or two embryos was 78%(7/9) and 74%(31/42), respectively. The pregnancy rates according to the grade of corpus lutea of recipients was 75% (20/27) and 82.0%(18/22) at the grade of A and B, respectively. Ten(67.0%) of 15 Holstein recipients transferred with IVP Hanwoo embryos and 5(42.0%) of 12 Holstein recipients transferred with frozen IVP Hanwoo embryos were pregnant. The single and twin calving ratio in Hanwoos was 77.0%(10/13) and 23.0%(3.13) in the recipients transferred with IVP embryos and 64.0%(7/10) and 27.0%(3/10) in the recipients transferred with frozen IVP embryos, respectively. Twenty-four pregnant cows following transfer of IVP embryos, 21(88.0%) calved the normal calves, and 2(8.3%) aborted. When the frozen IVP embryos were transferred, 16 pregnant cows calved 14(88.0%) normal calves and 2(13.0%) aborted. In conclusion, when one or two IVP bovine embryos were transferred into recipients, the A and B grade of corpus luteum resulted in high pregnancy rates. For the production of twin calves, transfer of the IVP or frozen IVP embryos could be suitable.

This study was carried out to determine the factors on achieving good viability of embryos biopsied fur sexing, to investigate pregnancy rate following embryo transfer(ET) with sexed embryos, and to confirm the accuracy for the calves bort following ET with sexed embryos by polymerase chain reaction(PCR). To investigate viability of Hanwoo embryos after biopsy for sexing, fresh and frozen/thawed embryos were biopsied according to different developmental day of blastocysts, different stage of blastocysts, and different biopsy grade and the embryos themselves were incubated for 2 hours in TCM199 after microsection to be evaluated morphologically for recovery as blastocyst. The results obtained were as follows : 1. The rate of oocytes cleaved in vitro and the rate of blastocyst of the cleaved oocytes were 52.5% and 21.6%, respectively. The rate of blastocyst on day 8 was 11.2%, denoting the highest rate during whole culture period posterior to in vitro fertilization(IVF) 2. After biopsy for sexing, the viability rate of blastocyst on day 7, 8 and 9 was 75.0%, 88.4%, and 100.0%, respectively and the viability of early, mid, and expanded blastocyst after biopsy was 75.0%, 88.9%, and 91.1%, respectively The viability rate of fresh and frozen/thawed embryos was 89.9%, 71.4%, respectively. And the viability of expanded, hatching, and hatched blastocyst of frozen/thawed embryos was : 75.0%, 75.0%, and 50.0%, respectively. The viability of embryos according to biopsy grade of 10∼20%, 21∼30%, and 31∼40% was 85.7%, 91.5%, and 71.4%, respectively. 3. Pregnancy rate after transfer with biopsied embryo between flesh and frozen/thawed embryos was 22.6% and 20.0%, respectively. 4. In comparison between sex by PCR method and sex of calves born after embryo transfer, the accuracy of sex deterimination was 92.3% (12/13).

This study was carried out to improve a technique of embryo transfer for twin calves production in Hanwoo cattle. Blastocysts for the donor of embryo transfer were classified into three criteria by accessment of morphology; early blastocyst, blastocyst and expanded blastocyst. Tow embryos were introduced transcervically into utrerine horn either of Hanwoo or Holstein by ipsilaterally or contralaterally to the corpus luteum. Thiry-six out of 57 recipients cows were inseminated by artificially on the next day of estrus, and followed by transfer of embryos into contralaterally. The pregnance rates of recipients following transfer of bovine embryos of day 7, 8 and 9 was 43.5, 18.2 and 8.3%, respectively. These results appeared that these was a significant (P<0.05) difference between on day-7 embryos and day-9 embryos, but not between on day-8 and day-9 embryos. Although there was not significant(P<0.05) difference in the pregnancy rates between the blastocysts(11/25, 44%) and expanded blastocysts(2/19, 10.5%) and between the blastocysts and early blastocysts(2/13, 15.4%), the embryos at blastocyst stage are more suitable than others for obtaining higher rate of pregnancy. There was no significant difference on pregnancy of the embryos transferred prior to presence(6/21, 29%) or absence (9/36, 25%) of artificial insemination. On pregnancy of Holstein, 2(15.4%) out of 13 recipients were pregnant in heifer. Similar Pregnancy rates were obtained between 1∼2 parities and 3∼4 parities by 30% (6/20) and 27.3%(3/11), respectively. Taken together, there was not significant difference in pregnancy rate due to small number of recipients used for this experiment. Both of Hanwoo and Holstein introduced the embryos by contralsterally to the corpus luteum were slightly higher pregnancy rate compare to by ipsilaterally (12/41, 29.3% vs, 3/16, 18.8%). The ratio of production of twin and single calves in Holstein was 20% (9/45) and 2.2% (1/45), respectively. However, in Hanwoo cows both of production of twin and single were similar as 8%. This result suggests that Holstein as recipients was superior to Hanwoo cows for production of twin calves. Out of all 15 pregnant, 12(80%) were produced a total of 22 normal calves in which the others composed of abnormal, as judging as 2(13.3%) for abortion and 1(6.6%) for stillbirth during the pregnant period.