• 한국가축번식학회지
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• 제13권3호
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• pp.164-170
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• 1989

These experiments were carried out to develop the practical technique for the production of identical twins in cattle. Morula and blastocyst stage embryos collected from superovulated donors were bisected into halves by micromanipulation. The resulting demi-embryos were transferred to the uterine horn ipsilateral to the corpus luteum of synchronous recipients. The viability of demi-embryos after splitting was also evaluated by culturing demi-embryos with and without a zona-pellucida. The results obtained in these experiments were summarized as follows : 1. Of total 132 embryos collected by superovulation from 29 donors, 37 embryos were morular and 30 at blastocyst stages. 2. Total 111 demi-embryos were produced from 67 embryos by bisection and 98% of those were normal in morphology. 3. The viability of the demi-embryos cultured with zona-pellucida ranged from 70 to 76.5% and that of the demi-embryos without from 53.8 to 69.2%. 4. The viability of demi-embryos obtained from morula was 63.6% and that of demi-embryos from blastocyst was 73.3%, respectively. 5. 35 demi-embryos were transferred to 21 recipients, 7 of which were confirmed to be pregnant by rectal palpation at 55∼60 days after embryo transfer. One of them produced a calf and 6 are still on pregnancy.

• 한국수정란이식학회지
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• 제9권1호
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• pp.43-48
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• 1994

This study was carried out to produce monozygotic twin calves by transfer of bisected embryos. Four Korean native cattle donors were superovulated with FSH and flushed to collect embryos on day 6 or 7 of the estrus cycle. Morula and early blastocyst embryos showed 1 or 2 grade were bisected with microblade and each set of demi-embryos without zona pellucida were transferred nonsurgically to 10 recipients respectively. The results obtained were as follows; 1. Twenty four demi-embryos (92.3%) were separated from 13 original embryos and among them 20 demi-embryos (83.3%) had normal appearance without severe damage. 2. Four sets of fresh demi-embryos were transferred to 4 recipients and one recipient was twin pregnant 3. Six sets of frozen-thawed demi-embryos were transferred to 6 recipients. Two recipients were pregnant, one of them twin.

• 한국가축번식학회지
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• 제21권2호
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• pp.85-93
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• 1997

These experiments were carried out to examine the development capacity of sexed and then bisected embryo from 8-cell to morula stage. Antisera to histocompatibility-Y(H-Y) antigen were prepared in inbred SD female rat by repeated immunization of spleen cell or testis supernatant from males of same strain. Male and female embryos were separated by delaying development of embryos against H-Y antibody. After sexing, rabbit embryos were bisected and aggregated. The results obtained from the these experiments were summuerized as follows: 1. When mouse and rabbit 8-, 16-cell and morular embryos were culature in H-Y antiserum, the ratio of embryo which has developed to hatching blastocyst was 53.4, 46.3 and 57.4% in mouse embryos, and 49.0, 52.0 and 61.0% in rabbit embryo, respectively. The ratio of mouse and rabbit embryos developed to hatching blastocyst showed nearly natural sex rate(50%), except rabbit mourla showed a little higher ratio(61.0%) as compared to natural sex ratio. 2. When rabbit demi-embryos from 8-, 16-cell embryo and morula were cultured, the percentage of demi-embryos was 70, 68 and 58% without zona pellucida removed, and 62, 69 and 51% with zona pellucida. The rate of aggregation was higher in 8- and 16-cell demi-embryos than in morula demi-embryo. 4. When sexed-demi-embryo was aggregated with another demi-embryo with demi-embryo with same sex, the rate of embryo developed to blastocyst was 60, 50 and 25%, respectively. Eight-cell demi-embryo showed highest rate. In conclusion, it showed that H-Y antiserum which was made by rat spleen cell enabled sexing rabbit embryos. And when rabbit sexed 8-, 16-cell and morula demi-embryo were aggregated, they were developed to eu-blastocyst which suggested the potential of sexed embryo manipulation.

• 한국가축번식학회지
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• 제21권3호
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• pp.287-292
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• 1997

This study was carried out to investigate the effects of concentration and kinds of cryoprotectants, equilibraction time, thawing temperature and time, sucrose concentration on the survival rates of frozen bovine demi-embryos. The bovine demi-embryos following dehydration by cryoprotectants a various concentration of sucrose were freezed by cell freezer and thawed in 3$0^{\circ}C$ water bath. Survival and in vitro developmental rates was defined as development rates on in vitro culture or FDA-test. The results are summarized as follows : 1. The high survival rates of demi-embryos after frozen-thawing in freezing medium was attained 2.0M glycerol. The high survival rates of demi-embryos after frozen-thawing in freezing medium was obtained using single cryoprotectant(25.0~30.0%) than mixed cryoprotectants(16.7~19.0%). 2. The survival rates of demi-embryos after frozen-thawing in freezing medium added 1.5M, 2.0M glycerol＋0.25M sucrose(37.5~33.3%) were higher survival rates than those of sucrose concentration of 0.50, 0.75M(12.5~26.7%). 3. The equilibration time on the survival rates of demi-embryos was attained after short period of time(30.0~35.0%) in the freezing medium higher than long period of time(21.1%). 4. The thawing temperature on the survival rates of demi-embryos was attained at 3$0^{\circ}C$ of thawing temperature(26.7~40.0%) higher than $25^{\circ}C$ or 37$^{\circ}C$ of thawing temperature(13.3~20.0%). 5. The thawing time on the survival rates of demi-embryos was attained at 1~5 minutes of thawing time(26.7~33.3%) in the freezing medium higher than 10 minutes of thawing time(13.3~18.8%).

• 한국수정란이식학회지
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• 제4권1호
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• pp.28-34
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• 1989

These experiments were carried out to determine the effect of cell stage in embryo bisection on the sub-Sequent in vitro and in vivo development in mouse. The embryos of ICR mouse were microsurgicaily bisected at 2-cell, 4-cell, 8-cell, morula and blastocyst stage using a microsurgical blade attached a micromanipulator. These demi-embryos without zona pellucida were cultured up to blastocyst stage and transferred to pseudopregnant mice, and the development of these demi-embryos was compared with the results of intact embryos of the corresponding cell stage. The successful rate of mouse embryo bisection at 4-cell stage (59.0%) was significantly (p <0.05) lower than those at 8-cell (75.6%), 2ce11 (80.7%) or morula stage (84.8%), and highest at blastocyst stage (95.7%). When the bisected embryos without any damage from microsurgery were cultured in vitro up to blastocyst,the in vitro de'velopment of demi-embroys bisected at morula to blastocyst was 91.6 to 95.3%, which was similar to the culture result of intact embryos of corresponding stage. However, the in vitro development of demi-em-bryos bisected at 2- to 8-cell stage was signiflcantiy (p <0.05) lower.The post-transfer implantation rate of demi-embryos developed in vitro to eu-blastocyst were 19.6 and 25.4% in demi-embryos bisected at morula and blastocyst stage,respectively and not significantly (P <0.05)different from the result of intact embryos of the same stage. However, the implantation rates of demi-embryos bisected at 2- or 8-cell stage were significantly (P <0.05) lower than the result from the intact embryos of the corresponding stage.

• 한국가축번식학회지
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• 제18권1호
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• pp.31-37
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• 1994

This study carried out to investigate the effects of cryoprotectants in the medium on the survival rate of rapidly frozen porcine bisected demi-embryos. The porcine bisected demi-embryos following dehydration by cryoprotectants containing sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water bath. Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows : 1. The survival rates of without-zona pellucida embryos and 2 blastomeres porcine embryos were 10.0 and 7.1%, respectively. The rate of unfrozen blastomeres (20.00%) was significantly higher than that of non-frozen oocytes. 2. The survival rates of with and without-zona pellucida of bisected porcine embryos by micromanipulator were 20.0 and 14.3%, respectively. 3. The developmental rates of with and without-zona pellucida of bisected poricine embryos by micromanipulator were 13.3 and 7.1%, respectively.

• 대한수의학회지
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• 제29권2호
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• pp.123-128
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• 1989

Demi-embryos were successfully produced by bisection of ICR mouse embryos at preimplantation stages. They were microsurgically bisected using a microsurgical blade attached to a micromanipulator after pretreatment with 0.5% pronase in PBS for two minutes or not. Embryos with softened zona pellucida were more easily bisected and less damaged than intact embryos. The highest success rate in bisection has been achieved by selecting blastocysts(94.1% in success rate with intact blastocysts and 100% in success rate with zona softened blastocysts). Demi-embryos without zona pellucida were cultured in D-PBS or M-16 medium at $37^{\circ}C$, 5% $CO_2$ in air for 72 hours for 2-cell stage embryos, 48 hours for 4-to 8-cell stage embryos, 24 hours for morula stage embryos and 6~12 hours for blastocyst stage embryos. For the in vitro culture of 2-cell stage embryos, $100{\mu}M$ 2Na-EDTA was added to the media. M-16 medium was better for the in vitro development of mouse embryos than PBS, and PBS is not considered to be suitable for long-term culture of embryos, especially at early stage of cleavage. In M-16 medium, developing rate of demi-embryos of which pair underwent development to form eublastocysts was 15.8% at 2-cell stage, 16.8% at 4-cell stage, 38% at 8-cell stage, 89.6% at morula stage and 94.4% at blastocyst stage, respectively. The more rapid and efficient production of demi-embryos and higher viability after bisection can be expected by softening zona pellucida with pronase and by selecting morulae or blastocysts rather than embryos at early stage of cleavage.

• 한국수정란이식학회지
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• 제15권2호
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• pp.175-180
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• 2000

The possible use of micromanipulative biopsy and PCR of the biopsied embryonic cells was tested to produce sexed bovine embryos in practical terms. By micromanipulation and PCR techniques, higher survival rate and accurate sexing of demi-embryos were btained. Bovine oocytes matured and fertilized in vitro were co-cultured with bovine oviductal epithelial cell (BOEC) monolayer in USU-6 medium supplemented with 15% FBS, and the embryos of 37% (327/885) were developed to blastocysts. Among 111 blastocysts produced by invitro, only 7 (6.3%) embryos were found unable to determine their sex, probably due to the loss of cells, since no PCR product was found from those cells. All the remaining 104 (93.7%) demi-embryos survived micromanipulation and demonstrated male-specific product or bovine-specific product alone suggesting that correct sexing of the sample. Forty-three point one percent(25/58) of manipulated and cryopreserved demi-embryos after thawing were survived. Final verification of the sexed embryos is necessary to make sure the same sex in fetus and newborn calf upon embryo transfer. The established sexing method on a large number of bovine embryos from previous and this study suggests that this a could be used practically in the field.

• 한국수정란이식학회지
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• 제11권3호
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• pp.283-289
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• 1996

This study was carried out to determine the sex of genomic and embryonic DNA using polymerase chain reaction(PCR). Bovine specific(216bp) and Y chromosome speicific DNA primers(l4lbp) were synthesized and tested for sexing. Bovine embryos used in this study were produced by in vitro fertilization. Few blastomeres for PCR were bisected by nicromanipulator and demi -embryos were cultured in TCM 199 medium containing 0.1% of solcoseryl. The results obtained were as follows; 1. Average optical density of genomic DNA extracted from blood of Hanwoo was 1.79$\pm$ 0.14. 2. 2. The ratio of the demi-embryos developed to blastocyst was 62.1 and 81.9% in morula and blastocyst, respectively. 3. When DNA of 2~4, 5~10 and more than 11 blastomeres was amplified with Y chromosome specific DNA primer by PCR, appreance rate of Y specific DNA band was 16.7, 46.2 and 40.0%, respectively. At least 5 to 10 blastomeres were required to determine the sex of embryos. 4. The rate of demi-embryos developed to blastocyst was 73.3% in TCM 199 medium supplemented with 0.1% solcoceryl. but 55.6% in control.

• Current Research on Agriculture and Life Sciences
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• 제6권
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• pp.145-156
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• 1988

본(本) 연구(硏究)는 생쥐에 대한 과배란반응(過排卵反應), 회수(回收)된 수정란(受精卵)의 양분율(兩分率), 양분(兩分)된 분할구(分割球)의 체외배양(體外培養) 및 이식후(移植後) 산자(産仔)로의 발달율(發達率)을 조사하기 위하여 CBA 및 C57BL 계통(系統)의 공시(供試)생쥐에서 얻어진 수정란(受精卵)을 0.5% protease로 투명대 제거 및 미세유리봉으로 양분(兩分)하여 얻어진 분할구(分割球)를 blastocyst로 배양(培養)하여 BALB/C 계통(系統)의 수란(受卵)생쥐에 이식(移植)하여 다음과 같은 결과(結果)를 얻었다. hCG 주사후(注射後) 36~42시간(時間), 48~54시간(時間), 62~66시간(時間), 72~78시간(時間)에 수정란(受精卵)을 회수(回收)한 결과(結果) 각각(各各) 2세포기(細胞期), 4세포기(細胞期), 8세포기(細胞期) 및 상실배기(桑實胚期)의 수정란(受精卵)이 대부분 회수(回收)되었으며 수당(首當) 평균(平均) 17.0개(個)의 난자(卵子)가 회수(回收)되었다. 회수후(回收後) 양분(兩分)한 8세포기(細胞期) 수정란(受精卵)의 양분율(兩分率)은 83.6%로서 상실배(桑實胚)의 65.5%에 비(比)해 유의적으로 높았으나 blastocyst로의 분할구(分割球)의 발달율(發達率)은 상실배(桑實胚)에서 얻어진 분할구(分割球)가 76.5%로서 8세포기(細胞期) 분할구(分割球)의 60.9%에 비(比)해 유의적으로 높았다. 한편 배양(培養)된 분할구(分割球)와 intact embryo에 있어서 세포기(細胞期)에 따라 각각(各各) 수란(受卵)생쥐당(當) 2개(個), 10개(個), 15개(個)씩 이식(移植)하였을 때 공(共)히 15개(個)씩 이식(移植)한 경우 수태율(受胎率)이 가장 높았으나 세포기(細胞期)에 따른 수태율(受胎率)에 있어서 유의적인 차이는 없었다. 배양(培養)된 분할구(分割球)와 intact embryo의 이식(移植)후 새깨생산율(生産率)에 있어서 8세포기(細胞期)에서는 intact embryo를, 상실배(桑實胚)에서도 intact embryo를 이식(移植)하였을때 새끼 생산율(生産率)은 각각(各各) 37.6% 및 43.4%로서 각각(各各) 배양(培養)된 분할구(分割球)를 이식(移植)하였을때 보다 새끼생산율(生産率)이 유의적으로 높았다.