• Journal of Embryo Transfer
• /
• v.10 no.3
• /
• pp.209-217
• /
• 1995

The present study was carried out to develop a cloning technology of mouse embryos by nuclear transplantation with electrofusion and to produce cloned offsprings by transfer of reconstituted embryos. A single nucleus from two- and eight-cell embryos was transplanted into the enucleated two-cell embryos by rnicromanipulation. The fusion of nucleus with recipient cytoplasm and the subsequent development of reconstituted embryos in vitro as well as in vivo to term were examined to determine the optimal electrofusion parameters for nuclear transplantation in mouse embryos. The successful enucleation of donor embryos was 84.9 and 83.3% in two- and eight-cell stage, respectively, and the successful injection of nucleus from two- and eight-cell donor embryos into the perivitelline space of enucleated two-cell embryos were 85.1 and 84.7%, respectively. No significant differences were found in enucleation or injection rate between the cell stages of donor embryos. When the blastomeres of intact two-cell mouse embryos were electrofused in 0.3 M mannitol medium(100 $\mu$sec., 3 pulses), the fusion rate was similarly 93.2, 92.2 and 92.0% in 1.0, 1.5 and 2.0 kV /crn, respectively, but in vitro development to blastocyst of the fused two-cell embryos was significantly(P<0.05) lower in 2.0 kV/cm (63.4%) than in 1.0 kV/cm (91.7%) or 1.5 kV/cm (82.4%). The development in vitro to eight-cell stage of the reconstituted embryos with nucleus from two-cell stage(45.5%) was significantly(P<0.05) higher than that from eight-cell stage blastomeres (16.7%). The number of blastomeres of the intact embryos at blastocyst stage was 50i0.6 and 55$\pm$2.4 in in vitro and in vivo cultured mouse embryos, respectively, but significantly(P<0.05) decreased to 35$\pm$0.7 in nuclear transplanted blastocyst embryos. The conception rate of mice following embryo transfer was 32.1% in the reconstituted two-cell embryos using two-cell donor nuclei, which was comparable to the fresh two-cell embryos(40.6%). However, the rate of development in vivo to term following embryo transfer of the reconstituted two-cell embryos using two-cell donor nuclei (23.5%) was significantly(P<0.05) lower compared with the percentage of two-cell fresh embryos(31.5%).

• Journal of Embryo Transfer
• /
• v.21 no.2
• /
• pp.129-135
• /
• 2006

This study was conducted to investigate the effect of in vitro maturation (IVM) duration of porcine follicular oocytes on maturation rate, polyspermic rate, and subsequent embryo development. The nuclear maturation rates of oocytes matured for 36, 38, 40, 42 and 44 hr were similar between 68.0, 78.0, 79.5, 73.8 and 81.8% respectively. There was no significant difference in the rates of polyspermy after in vitro feritilization (IVF). The cleavage rate in the group of 36 hr was significantly higher in than that of 40, 44 hr (p<0.05) but not to 38 and 42 hr. The development rate to blastocyst stage was significantly higher in the group of 38 hr (23.1%) than that in the group of 44 hr (15.6%) (p<0.05) but not to 36, 40 and 42 hr. These results suggest that the aged oocytes for 44 hr is not required for the production of bias to cysts derived from porcine IVF embryos.

• Journal of Embryo Transfer
• /
• v.24 no.1
• /
• pp.47-55
• /
• 2009

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 $10.16{\pm}3.85$ and $11.56{\pm}2.35$ for the donors treated with FSH 200mg and FSH 400mg, respectively. Average number of embryos collected were $8.85{\pm}4.05$ and $8.30{\pm}1.73$ for the donors treated with FSH 200 mg and FSH 400 mg, respectively. Average number of transferable embryos were $5.48{\pm}2.45$ and $4.58{\pm}2.23$ 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.

• Journal of Embryo Transfer
• /
• v.14 no.3
• /
• pp.203-210
• /
• 1999

In this stuyd, the effect of the dominant follicle aspiration for the superovulatory response in HanWoo was investigated. The criterion for the presence or absence of a dominant follicle based on their morphological examination. The dominant follicle was aspirated 48hr before the onset of superovulation treatment by 6.5MHz convex probe connected with a carrier and superovulation induced by FSH (Super-Ov Tyrer, Texas, U.S.A) adminstered twic a day s.c. over 4 day in a decreasing regimen. From 13 HanWoo scanned daily to determine the presence and growth of the dominant follicle, its an average diameter of 15.4mm was measured and an average diameter of corpora lutea was 18.7mm on day of follicular aspiration. In the experiment, a follicular remove by ultrasound-guided aspiration, the ovarian response was significantly enhanced when animals were superovulated in the aspiation of a dominant follicle compare with animals superovulated non-aspiration of a dominat follicle. In the aspiration of a dominant follicle donors yieleded more corpora lutea(14.4$\pm$4.7 vs 8.6$\pm$3.4) and transferable embryos(8.9$\pm$4.2 vs 5.4$\pm$2.7) than control. In cows in which the dominant follicle had been aspirated under sonographical control 2 days before superovuation, the number of corpus lutea and transferable embryos were significantly enhanced compared with animals superovulated in the presence of a dominant follicle (14.4$\pm$4.7 vs 6.9$\pm$2.7, ; 8.9$\pm$4.2 vs 3.3$\pm$1.6). After 7 days of artificial insemination, the embryos at 7 days were cllected by uterine flushing after dominant follicle insemination, the embryos at 7 days were collected by uterine flushing after dominant follicle aspiration and superovulation treatment, and evaluated their quality by morphological criteria. Sixteen embryos with excellent and good grade were transferred into 8 recipient cows. Six pregnancies were identified at 60 and 120 days of gestation by rectal palpations. In conclusion, the present study showed that 1) the presence or absence of a dominant follicle signficicnatly affects superovulatory responses, and 2) ultrasound-guided follicular aspiration of the dominant follicle and superovuation treatment provides an accurate and procedure to increase ovarian responses in HanWoo.

• Journal of Embryo Transfer
• /
• v.18 no.1
• /
• pp.27-33
• /
• 2003

The study was carried out to investigate the effects of morphology, reproductive cycle, incubation time and activation of oocytes in vitro maturation of canine oocytes and development of canine IVM/IVF embryos. The results were summarized as follows: 1. The developmental rates to 16 cells of fresh, salts and 4$^{\circ}C$-stored oocytes with and without cumulus cells were 14.3%, 5.0% and 7.5%, 2.8% and 5.7%, 0.0%, respectively. The rate of oocytes with cumulus cells(5.7%~14.3%) was higher than that of denuded oocytes(0.0%~5.0%). 2. The developmental rate to If cells of in vitro cultured oocytes recovered from ovaries collected at different stages of the reproductive cycle were 0.0%, 10.7%, 1.5%, respectively. 3. The developmental rate to 16 cells of fresh oocytes with cumulus cell cultured for 24, 32 and 48 hrs in $CO_2$ incubator were 0.0%, 5.3%, 11.8%, respectively. The rate of oocytes cultured for 48 hrs was higher than that oocytes cultured for 24 and 32 hrs. 4. The development to If cells treated activation and non-activation oocytes were 15.0%, 6.7%, respectively. The rate of oocytes treated activation was higher than that oocyte treat non-activation.

• Journal of Embryo Transfer
• /
• v.12 no.3
• /
• pp.259-267
• /
• 1997

To improve the efficiency of production of cloned embryos and animals by nuclear transplantation in the rabbit, the effect of cell cycle of donor nuclei and type of recipient cytoplasm on the in vitro developmental potential and production efficiency of offspring was determined. The embryos of 16-cell stage were collected from the mated does at 48h post-hCG injection and they were synchronized to G$_1$ phase of 32-cell stage. The oocytes collected at 14h post-hCG injection were freed from cumulus cells and then enucleated. One group of the enucleated cytoplasms was activated by electrical stimulation prior to injection of donor nucleus, and the other group was not pre-activated. The separated G$_1$phase blastomeres of 32-cell stage embryos were injected into the perivitelline space of recipient cytoplasms. After culture for 20h post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation and the fused nuclear transplant embryos were co-cultured for 120h and the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye and their blastomeres were counted. Some of the nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The electrofusion rate was similar between the types of donor nuclei and recipient cytoplasms used. However, the nuclear transplant embryos using G$_1$ phase donor nuclei were developed to blastocyst at higher rate(60.3%) than those using S phase ones(24.7%). Also, when non-preactivated oocytes were used as recipient cytplasms, the develop-mental rates of nuclear transplant embryos to blastocysts were significantly(P< 0.05) higher(57.1%) than those using preactivated ones(20.8%). The cell counts of nuclear transplant embryos developed to blastosyst stage were increased signficantly(P<0.05) more in the non-preactivated recipient cytoplasm(163.7 cells), as compared whit the preactivated recipient cytoplasm(85.4 cells), A total of 49 nuclear transplant embryos were tranferrid into 5 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer. these results showed that the blastomeres of G1 phase and non-preactivated oocytes might be utillzed efficiently as donor nuclei and recipient cytoplasms in the nuclear transplant procedure, thought the offspring production remained still low.

• Journal of Embryo Transfer
• /
• v.29 no.3
• /
• pp.283-287
• /
• 2014

Sexed semen is commonly used for the production of calves of the desired gender. Gender selection is important in animal production industries. For example, female cattle are required for the dairy industry while males are preferred in the beef cattle industry. The present study was to assess the in vivo embryo production efficiency using the semen separated according to sex during superovulation in Hanwoo. Seventy Hanwoo donor cows were flushed on day 7 of estrus cycle with same FSH and artificial insemination by the same technicians. Embryos were recovered on 7 days after the third insemination by flushing the uterus with embryo collection medium. KPN semen straws used artificial insemination contained 20 million sperm (total number 60 million per donor). Sex-sorted semen straws contained 4 million sperm (total number 12 million per donor). The results obtained were as follows: No differences were observed in the efficiency of superovulation rates on KPN semen 87%, and sexed semen 100%, respectively. The mean numbers of total embryos are each $12.58{\pm}8.31$ and $13.25{\pm}7.86$. The mean numbers of transferable embryos, sexed semen were significantly lower than KPN semen ($3.75{\pm}1.98$ vs. $8.23{\pm}6.07$, P<0.05). The rates of unfertilized embryos from superovulation using sexed semen were significantly higher than KPN semen (50% vs. 15%, P<0.05). The rate of degenerated 2-cell embryos from sexed and KPN semen was 60.87% and 11.11%, respectively (p<0.05). In conclusion, these results indicate that superovulation using sexed semen was useful, but efficient embryo production was important to reducing the damage caused by the Flowcytometer-based sperm sorting procedure.

• Journal of Embryo Transfer
• /
• v.29 no.3
• /
• pp.273-281
• /
• 2014

This study was carried out to establish the system of OPU derived embryo production, management of recipients as well as offspring production. OPU derived embryo production system was carried out of aspiration of immature oocytes 2 times per week, total 24 times for 3 months by an ultrasonographic guided follicular aspiration system and then produced in vitro-produced blastocysts by in vitro maturation, fertilization and culture system. This work was collected total 13,866 oocytes, average $8.2{\pm}4.5$ oocytes per session and 8,170 G1 + G2 grade oocytes, average 4.8 oocytes per session by 1,692 times session of total 71 donors for 4 years from 2010 to 2013. The rate of cleavage and blastocyst developmental competence were obtained 11,825 (85.3%) and 5,032 (36.3%) that was $7.0{\pm}3.8$ cleaved embryos and $3.0{\pm}2.5$ blastocysts per session. OPU derived embryo transfer were taken place in 2, 4, 6 and 7 local governments at 2010, 2011, 2012 and 2013 for 4 years and pregnancy rate were obtained 41.2, 43.9, 46.5 and 49.7% in each years. It means that pregnancy rate was continuously improved according of every year for 4 years. Pregnancy rate was significantly different according to individual local government in which was 62.7% in B, but 24.2% in F at 2012. Paternity identification was carried out total 26 offspring in C local government of 2012 and then confirmed 100% agreement of its analysis. In conclusion, the results obtained the possibility of mass production of elite cow embryos as well as offspring by OPU derived embryo production system, of which could be decreased the required time of genetic improvement.

• Proceedings of the Korean Aquaculture Society Conference
• /
• 2003.10a
• /
• pp.62-62
• /
• 2003

최근 연안 자원이 급격하게 감소하고 있고 새로운 양식 대상종으로 평가되고 있는 말쥐치의 대량 종묘생산을 위해서는 수정난 대량확보와 자어기 먹이생물 구명 등의 문제점이 해결되어야 한다. 본 연구자들은 우선 호르몬 처리에 의한 산란유도, 조기성숙유도에 의한 조기산란유도 및 수정난 확보방법을 개발하고, 위내용물 분석을 통한 자어의 초기먹이생물을 구명하고자 하였다. 산란유도를 위하여 hCG, LHRH-a 및 hCG+LHRH-a를 다양한 농도로 처리하여 실험한 결과 모든 실험구에서 67~76%의 수정률을 나타내었으나, 대량의 성숙란을 얻지는 못하였다. 따라서 본 종의 산란특성이 다회성인 점을 감안한다면 500 g 전후의 친어 200~300마리를 1개의 수조에 수용하여 자연산란된 수정란을 수거하는 것이 좋을 것으로 판단되었다. 평균 450 g 정도의 말쥐치 친어를 12월부터 6개월동안 천천히 수온을 상승시켜 자연산란을 유도한 결과, 자연산란기 (6월)보다 2개월 빠른 4월 15일(17$^{\circ}C$로 가온한 지 15일)에 첫 산란이 이루어졌고 1개월간 지속되었다. 초기 사육환경을 구명하기 위한 실험에서는 일반적으로 해산어 종묘 생산에 이용하는 클로렐라＋rotifer 실험군이 클로렐라 단독, 규조류 단독, 규조＋rotifer, 녹조＋copepods등이 번식된 환경보다 생존율에 있어 월등히 높은 결과를 나타내었다. 위 내용물 분석에 의한 사육기간 중 섭식한 주요 먹이생물종은 부화 후 5일부터 20일째까지는 rotifer egg, 부화 10일째는 rotifer egg와 small rotifer, 부화 20일째는 rotifer, Artemia sp., copepods, 부화 25일째부터는 copepods였다. 초기성장은 부화직후 전장 1.95 mm였던 것이 10일째는 2.96 mm, 20일째는 3.89 mm, 30일째는 12.9 mm, 50일째는 35.1 mm를 나타내었다. 그러나 말쥐치 자ㆍ치어가 성장함에 따라 발생하는 지속적인 폐사와 종묘의 수송시 발생하는 폐사를 줄일 수 있는 방법이 추후에 해결되어야 할 것으로 사료된다.

• Korean Journal of Animal Reproduction
• /
• v.25 no.1
• /
• pp.85-90
• /
• 2001

To study the signaling effect of growth hormone (GH) in vivo on animal physiology, transgenic mice containing GH Receptor (GHR) gene fused to metallothionein promoter were produced by DNA microinjection into one-cell stage embryos. Three founder mice were produced with transgenic mice with approximately 4~6 copies of GHR genes and transgene was transmitted into the progeny. The founder mice were mated with normal mice to produce F$_1$ mice, and intergation and transmission of transgene were checked by polymerase chain reaction and Southern blot methods. Transmission rate of GHR transgenic mice were 20~50% in F$_1$ generation and 50% in F$_2$ generation which means that some founder mice were mosaic and transgene in F$_1$ mice was transmitted to F$_2$ progeny with Mendelian ratio. Death rate of GHR transgenic mice after birth was about 10~30% in F$_1$ and F$_2$ progenies indicating that GHR gene may affect death of transgnenic progeny.