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In vitro development of Korean native goat embryos was investigated in 2 different culture systems with and without goat oviduct epithelial cells (GOEC). Estrus was synchronized by inserting intravaginal progestagen-impnegnated sponge (Veramix) containing 60 mg medroxyprogesterone acetate (MAP) for 14 d. Superovulation was induced with follicle stimulating hormone (FSH). Goat ova were surgically obtained by retrograde flushing the oviducts of does at 66 to 68 h after MAP removal. Mean number of recovered ova per doe was 7.28 +/- 3.91, and the proportion of fertilized embryos in recovered ova was 66.5% (121/182 ). Fertilized embryos were cultured for 9 d in CR1aa medium supplemented with 10% estrous goat serum (EGS) at 38.5 degrees C, 5% CO(2) in air. There was no difference in development of the embryos to the morula stage between the 2 culture systems (84.4 and 84.0%, respectively). However, developmental rate to blastocysts (65.6%) of the embryos co-cultured with GOEC was significantly higher than of those (12.0%) cultured without GOEC (P < 0.001). Goat zygotes were injected with bovine beta-casein/human lactoferrin cDNA fusion gene (pBL1). When the DNA-injected embryos were co-cultured with GOEC, developmental rates of the embryos to the morula and blastocyst stages were 82.9 and 36.6%, respectively. The results obtained in this study indicate that "blocking" of in vitro development of Korean native goat embryos appears to occur at the morula stage, but can be overcome to some extent by co-culture with GOEC. In the co-culture system, DNA-injected goat embryos could successfully develop to normal hatching blastocysts.

In vitro matured and fertilized bovine ova were microinjected with pBL1, which consisted of the bovine beta-casein gene promoter, human lactoferrin cDNA and SV40 polyadenylation signal. Of the 2931 zygotes injected, 2505 (85.5%) survived 1 h after DNA injection and were cultured in 50-microl drops of CR1aa medium containing 3 mg/ml BSA under mineral oil at 39 degrees C, 5% CO2 in air. Cleaved (2- to 8-cell) embryos were selected at approximately 48 h after DNA injection and then cultured further in 50-microl drops of CR1aa medium supplemented with 10% (v/v) FBS. Blastocysts were classified into 4 quality grades and 3 developmental stages by morphological criteria. Then all but poor quality blastocysts were nonsurgically transferred to the uterus of heifers 7 to 8 d after natural estrus. Following transfer, the recipients were observed for signs of estrus, and pregnancy was confirmed by palpation per rectum at approximately 60 d of gestation. Although 72.0% (1804/2505 ) of the DNA-injected zygotes reached 2- to 8-cell stages only 5.2% (131/2505) developed to blastocysts. A total of 75 DNA-injected, in vitro cultured blastocysts were transferred to 59 recipients. When 2 blastocysts were transferred to a single recipient, only the better quality embryo was counted. The overall pregnancy rate was 30.5% (18/59 ) and reflected 1) an apparent correlation between the quality of embryos and the pregnancy rate. However, the difference was not statistically significant. 2) expanded blastocysts had a higher pregnancy rate (50.0%, 11/22 ) than early (13.3%, 2 15 ) or mid (22.7%, 5/22 ) blastocysts with a significant difference between expanded and early blastocysts (P0.05). 3) the pregnancy rate of DNA-injected blastocysts was higher when they were transferred at Day 7 (34.5%, 10/29 ) or 8 (36.8%, 7/19 ) than at Day 6 (9.0%, 1/11 ). The results indicate that the developmental stage of DNA-injected bovine embryos may be one of contributing factors in improving the pregnancy rate after transfer, although the effects of the quality and culture period of the embryos may not be inconsequential.

We have developed a transgenic female goat harboring goat beta-casein promoter/human granulocyte colony stimulating factor (G-CSF) fusion gene by microinjection into fertilized one-cell goat zygotes. Human G-CSF was produced at levels of up to 50 microg/ml in transgenic goat milk. Its biological activity was equivalent to recombinant human G-CSF expressed from Chinese hamster ovary (CHO) cell when assayed using in vitro HL-60 cell proliferation. Human G-CSF from transgenic goat milk increased the total number of white blood cells in C57BL/6N mice with leucopenia induced by cyclophosphamide (CPA). The secreted human G-CSF was glycosylated although the degree of O-glycosylation was lower compared to CHO cell-derived human G-CSF.

During the breeding season in Korea (September 1997 to April 1998), a goat embryo recovery and transfer program using a Korean native strain (Capra hircus aegagrus) was performed for the production of transgenic goats. Donors were synchronized with norgestomet implants and superovulated by a combined treatment with FSH and hCG. The treatment regime induced a consistent and efficient superovulation rate of 90% from donors with an ovulation rate of 12.1 0.5. 50.5% of the recovered oocytes/embryos were fertilized and most of them were at the 1-cell stage. After DNA microinjection, a total of 188 embryos were immediately transferred to naturally cycling or hormonally synchronized recipients with two or three embryos per animal. There was a tendency for the pregnancy rate of naturally cycling recipients to be higher (38.9%) than that of hormonally synchronized recipients (25.7%). When the recipients were classified into a two or three embryo-received group, the embryo viability was markedly decreased from 58.3% in two embryo-received group to 35.3% in three embryoreceived group, without an increase in the kidding rate. This resulted from a high occurrence of abortions or stillbirths from the multiple-pregnant recipients which had received three embryos. This indicated that the transfer of two embryos per recipient is highly recommendable for an optimal embryo survival in Korean goats. Altogether, 188 embryos were transferred to 71 recipients and two transgenic Korean goats were produced from 25 offspring. # 2000 Elsevier Science B.V. All rights reserved.

In vitro development of Korean native goat embryos was investigated in 2 different culture systems with and without goat oviduct epithelial cells (GOEC). Estrus was synchronized by inserting intravaginal progestagen-impnegnated sponge (Veramix) containing 60 mg medroxyprogesterone acetate (MAP) for 14 d. Superovulation was induced with follicle stimulating hormone (FSH). Goat ova were surgically obtained by retrograde flushing the oviducts of does at 66 to 68 h after MAP removal. Mean number of recovered ova per doe was 7.28 +/- 3.91, and the proportion of fertilized embryos in recovered ova was 66.5% (121/182 ). Fertilized embryos were cultured for 9 d in CR1aa medium supplemented with 10% estrous goat serum (EGS) at 38.5 degrees C, 5% CO(2) in air. There was no difference in development of the embryos to the morula stage between the 2 culture systems (84.4 and 84.0%, respectively). However, developmental rate to blastocysts (65.6%) of the embryos co-cultured with GOEC was significantly higher than of those (12.0%) cultured without GOEC (P0.001). Goat zygotes were injected with bovine beta-casein/human lactoferrin cDNA fusion gene (pBL1). When the DNA-injected embryos were co-cultured with GOEC, developmental rates of the embryos to the morula and blastocyst stages were 82.9 and 36.6%, respectively. The results obtained in this study indicate that "blocking" of in vitro development of Korean native goat embryos appears to occur at the morula stage, but can be overcome to some extent by co-culture with GOEC. In the co-culture system, DNA-injected goat embryos could successfully develop to normal hatching blastocysts.

To clarify the developmental capacity of frozen two-cell blastomeres, we investigated in vivo and in vitro viabilities of blastomeres that were frozen ultrarapidly after separation from two-cell mouse embryos. Two-cell embryos obtained from superovulated F1 hybrid females were denuded by treatment with 0.5% pronase solution and then induced to separate into two single blastomeres by gentle pipetting. The blastomeres were cryopreserved by an ultrarapid freezing method.The preimplantation developmental rate of two-cell embryos frozen in 3.0 M DMSO was significantly higher than the rate of those frozen in 15 and 4.5 M DMSO (at least P0.05). The in vitro developmental rate of the ultrarapidly frozen-thawed blastomeres separated from two-cell embryos (75.0%) was similar to that of nonfrozen blastomeres (76.0%). When eight pairs of blastocysts that developed from frozen two-cell mouse blastomeres were transferred to pregnant ICR recipients on Day 3, four live singletons were born.Thus, the results indicate that two-cell mouse blastomeres can be frozen by the ultrarapid freezing method.

Phosphatidylinositol-3-kinases (PI3K) play pivotal roles in the meiotic progression of oocytes from metaphase I to metaphase II stage. This study evaluated the effect of 3-methyladenine (3MA), a specific inhibitor of Class III PI3K, on the meiotic progression of porcine oocytes. Immature porcine oocytes (n = 4744) retrieved from abattoir-derived oocytes were cultured in the absence or presence (10 mM) of 3MA for 22 h and evaluated for meiotic progression by florescent Hoechst 33342 staining. Data were analysed by chi-square test or ANOVA using SPSS software, and differences at P 0.05) from nontreated control oocytes with respect to their ability to fertilize, cleave (74.1 ± 1.2 v. 72.7 ± 2.8%), and form blastocyst (15.4 ± 1.5 v. 12.7 ± 0.6%) upon IVF or parthenogenetic activation (cleavage rate: 89.8 ± 1.7 v. 84.6 ± 5.1%; blastocyst rate: 44.3 ± 12.4 v. 45.1 ± 7.6%). These data suggest that 3MA reversibly blocks and synchronizes the meiotic progression of porcine oocytes at GV stage without affecting their ooplasmic maturation in terms of post-fertilization/activation in vitro embryonic development. Our data also provide indirect evidence for the likely participation of Class III PI3K in meiotic maturation of porcine oocytes beyond GV stage. This work was supported by grants (Code #200901FHT010305191 and #20070401034017) from BioGreen 21 program of RDA, Republic of Korea.