• Journal of Embryo Transfer
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• v.20 no.2
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• pp.169-176
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• 2005

Loop-mediated isothermal amplification (LAMP) is novel DNA amplification methods that amplifies a target sequence specifically under isothemal condition. The present study was to assess the in vitro viability afier biopsy and sexing rate of different types of embryo biopsied. In vivo compact morulae and blastocyst embryos were obtained from Korean Native Cow (KNC) superovulated with FSH (Antorin, R-10) on 7 Day after artificial insemination. in vitro compact morulae and blastocyst embryos were obtained with KNC or Holsteins that were gained on 6, 7 or 8 day after in vitro fertilization(IVF) with frozen semen. Biopsy of bovine embryo was carried out in a $80{\mu}l$ drop with $Ca^{2+}-Mg^{2+}$ free D-PBS and the viability of biopsied embryos were evaluated in IVMD (IFP, Japan) medium at 12 hrs culture time. The sex ratio of biopsied Hanwoo embryos were male vs. female of $43.5\%\;vs.\;56.5\%$ in vivo and $33.9\%\;vs.\;49.2\%$ in vitro respectively, and male rate of biopsied Holstein embryos were significantly higher than female $(70.8\;vs.\;29.2\%)$. and indefinite rate of in vitro embryos was $16.9\%$ and in vivo was not. The degeneration rate of biopsied embryo, in vitro embryos were significantly higher than in vivo $(13.2\%\;vs,\;0.0\%,\;p<0.05)$. The survivability of in vivo embryo were between biopsied following punching method was significantly (P<0.05) higher than bisection method produced embryos $(100\%\;vs.\;83.3\%)$ and in vitro had no difference. However, the degeneration rate of biopsied embryo by bisection method was significantly higher than punching methods between in vivo and in vitro $(16.7\;vs.\;22.6\%,\;respectively,\;p<0.05)$. In conclusion, these results indicate that punching method was optimal and survivability after embryo biopsy was useful for reducing the damage caused by the embryo biopsy procedure for LAMP-based embryo sexing.

• 월간낙농육우
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• v.36 no.6
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• pp.125-128
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• 2016

"원유의 질과 소의 체형도 중요하지만, 결과적으로 우리 기후와 풍토, 조사료에 맞는 종모를 만들어 목장에 돈을 벌어주는 종축을 생산해야 한다고 생각합니다." 경기 연천군 전곡읍 늘목리에 위치한 늘목목장의 남군희 대표(62세)는 지난 2009년 '제11호 청정육종농가'로 발탁되며 7년간 수정란 송아지 100여 두를 생산했다. 현재 '한국형 종모'를 생산하겠다는 자부심으로 후계자인 남상용 씨(37세)와 목장을 이끌어 가고 있는 남군희 대표를 만나봤다.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.451-455
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• 2000

Gene targeting are very useful tools for the research on the gene function in vivo, mass production of foreign materials and biomedical approach of therapeutic process. But this process is very complicated and necessary highly skilled technique, because it is very different from ES cell origin, genetic background of embryo, and experimental conditions. We investigated the productivity ability of chimeric mouse after aggregation with TT2 ES cells. Increse of ES cell density caused gradual decrease in embryo development in vitro and in th $\varepsilon$ production of chimeric mice in vivo. One million ES cell density for the aggregation was very efficient to produce high percentage chimeric mice in their coat color. These results suggested that appropriate cell density plays a key role in the development and production of chimeric mice by a 8-cell aggregation method.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.73-78
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• 2001

This study was carried out to investigated developmental ability of IVM/IVF derived hanwoo embryos after DNA microinjection. Microinjected hanwoo embryos were cultured fur 7 days. The cleavage rates of DNA injected embrys(36.3%) was significantly lower than those of non-injected embryos(67.4%; p<0.05). The percentage of injected embryos reaching to the morulae and blastocyst was significantly lower than those of non-injected embryos(p<0.05). When injected embryo were cultured contaning L-ascorbic acid and $\alpha$-tocopherol for 168 hrs, the morulae and blastocyst rates were significantly higher than control(p<0.05). These results suggested that the addition of L-ascorbic acid and $\alpha$-tocopherol can enhanced development to the morulae and blastocyst of microinjected embryos and improved culture condition increased the transgenic hanwoo embryos.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.163-168
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• 2005

The objective of this study was to produce superior cows by sexual distinction of embryos collected from the donor with pedigree information. Collected embryos distinguished by biopsy using punching or bisection methods and Loop-mediated isothermal amplification of sex-determined DNA for sexing. Six embryos predicted as female were transplanted to recipients and then 2 pregnant cows were normally delivered of calves at 278 and 285 days after embryo transfer. Birth weights of calves named Barani and Borani were 18kg and 25kg, respectively. Adjusted body weights for 90 days were 61.1kg and 88.8kg, respectively. Average daily gains were 0.48kg and 0.71kg, respectively.

• Journal of Aquaculture
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• v.1 no.1
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• pp.25-39
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• 1988

The flounder reared in captivity matured and spawned when the water temperature gradually increased to $14\~15^{\circ}C$ from $12^{\circ}C$ during winter and the diurnal photoperiod changed from 10L/14D in winter season to 14L/10D in spring. The eggs spawned naturally by reared spawners in captivity during the first half of a spawning period were superior in quality to those spawned during the later half. It would be better to use the eggs of the first half for the mass production of the seedlings.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.73-73
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• 2002

돼지 난포란의 체외성숙, 수정 및 배양에 관한 연구는 생명공학 기술을 도입하기 위한 기반 기술로 최근까지 계속 진행되고 있으나, 돼지 난포란을 이용한 효율적인 수정란 생산은 불규칙적인 웅성전핵 형성율과 높은 다정자 침입율 그리고 체내에서 발달된 배반포에 비하여 적은 세포수는 돼지 체외수정 체계에 있어서 끊임없는 문제점으로 제기되고 있다. 따라서 본 연구에서는 난포란을 이용한 보다 효율적인 수정란 생산을 위하여 돼지난자의 체외수정시 정자의 adenylate cyclase 활성을 조절하여 수정능획득 및 자발적인 첨체반응을 조절하여 정상적인 수정을 돕는 것으로 알려진 ADP를 체외수정 배양액에 첨가했을 때 수정율, 다정자 침입율, 배발달율 및 배반포의 세포수에 미치는 영향을 조사하였다. (중략)

• Proceedings of the Korean Aquaculture Society Conference
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• 2003.10a
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• pp.64-64
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• 2003

대구 인공종묘 생산 기술 개발의 일환으로 수정란에 기생하여 폐사를 유발하는 세균 및 세균 감염 경로를 파악함으로써 수정란의 생존율과 부화율 향상을 위하여 본 실험을 실시하였다. 세균 감염 경로를 파악하기 위하여 난소와 정소 자체 세균 감염 여부를 조사하였다. 일반배지 (BHIA)와 비브리오 선택배지 (TCBS)를 사용하여 세균검사를 실시하였다. 난소의 세균 검사는 생식소 내부를 일부 절개한 후 멸균 roop를 찔러 세균을 검사하였고, 정소는 채정하기 전 복부를 절개하여 멸균 roop로서 적출하여 배지에 도말한 후 배양하였다. 수정란 내 세균 수는 수정란을 각각 20개씩 샘플하여 난 외부에 기생하는 세균을 제거하기 위하여 난소독제로 이용되고 있는 benzalkonium 0.1%로 1분간 소독한 후 멸균 생리식염수로 3회 세척하여 호모게나이즈하여 검사하였다. 호모게나이즈한 액 중 100${\mu}\ell$를 pipetting하여 일반배지(BHIA)에 도말하여 인큐베이터에서 배양하였다. 난소와 정소 내에서 세균이 검출됨에 따라 수정 시 세균 감염을 억제하기 위하여 자외선 살균해수와 일반해수를 수정액으로 사용하여 발생율을 비교 시험하였다. 수정 시간은 1분으로 동일하게 적용하였으며, 수정 용기는 멸균 처리된 일회용 100$m\ell$ 플라스틱 용기를 사용하였다. 수정 후 과다한 정자를 제거하기 위한 세란은 1$\ell$ 멸균 비이커에서 5회 30분 가량 실시하여 정자를 제거하였고 멸균 봉으로 저어주면서 수정란의 점착력을 제거하였다. 수정란은 100$\mu\textrm{m}$ 그물망으로 수정란 유실을 방지한 플라스틱 용기에 수용하여 유효수량 270$\ell$ FRP 수조에 수용하여 3$\ell$/min 환수하였고, 수온은 자연수온 1$^{\circ}C$로 유지하였다. 발생율은 만능투영기로 3회 측정하였다. 수정 후 세균과 기생충에 의한 수정란의 폐사를 억제하기 위하여 수정 직후, 수정 후 1일, 2일째 oxytetracycline과 iodine 처리에 따른 발생율 변화를 조사하였다. 발생율은 만능투영기로 조사하였고, 시험구별로 3회 측정하였다. 경과 일수별로 약제 처리는 약제 미처리 수정란 중 정상적인 발생이 이루어지고 있는 것을 선별하여 조사하였다. 약제 처리에 따른 배체 발생 단계는 수정 후 1일째는 상실기, 2일째는 포배기였다. 수정란 및 대구 자어에서 분리된 V. splendidus 에 의한 폐사를 예방하기 위해 in vivo에서 oxytetracycline 외 5종의 항생제를 대상으로 96well plate에서 최고 농도 250ppm부터 2 fold로 단계 희석하여 $25^{\circ}C$에서 48시간 배양하여 MIC를 조사하였다. 세균 감염경로 파악을 위하여 난소, 정소 및 정자에서 세균을 분리한 결과 일반배지 및 비브리오 선택배지에서 모두 균이 검출되었고, 균 동정 결과 터봇 자어에서 검출된 것으로 보고된(Gatesoupe et al., 1999) V. splendidus로 나타났다. 수정액과 정자 및 미수정란의 세균 분리 결과 일반배지에서 3$\times$10/ml ~ 7$\times$10/ml로 균이 검출되었다. 수정액을 일반해수와 자외선 살균 해수를 사용하여 발생율을 비교한 결과 수정 후 3일째 발생율은 자외선 살균해수 72.3%, 일반해수 52.7%였으며, 수정 후 7일째 40.9%와 25.1%로 자외선 살균해수가 유의적으로 높게 나타났다. 수정 후 경과 일수별로 oxytetracycline과 Iodine을 처리한 결과 수정 직후 처리한 시험구는 7일째 19.8%와 18.9%로 대조구 23.1%와 유의적인 차이가 없는 것으로 나타났다. 수정 후 1일째 처리한 시험구는 54.5%와 56.8%, 수정후 2일째 처리구는 47.9%와 50.6%로 두 시험구 모두 대조구와 수정 직후 처리구보다 유의적으로 높게 나타났다. 수정란 및 대구 자어에서 분리된 V. splendidus 에 의한 폐사를 예방하기 위해 in vivo에서 항생제 종류별 MIC 조사 결과 oxytetracycline이 0.48ppm으로 가장 효과가 높은 것으로 나타났다.

• Journal of Embryo Transfer
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• v.19 no.1
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• pp.1-10
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• 2004

These studies were carried out to improve the reproductive efficiency through embryos transfer of Hanwoo IVM/IVF embryos. Following routine IVM/IVF procedure, oocytes and zygotes were cultured far 40 to 44 h in CRlaa medium with BSA. Then 2 to 8-cell embryos were removed the cumulus cell and were cultured in CRlaa medium containing 10% fatal bovine serum and 2.5 mM taurine in 5% $O_2$ and 5% $CO_2$ at 38.5$^{\circ}C$. The fresh embryos of the morulae and blastocysts cultured for 6 to 9 days in vitro or the frozen-thawed embryos were transferred into recipients. The pregnancy rates of the blastocyst produced for 6, 7, 8, and 9 days in vitro culture were 59.4, 68.2, 66.0 and 100%, respectively. In the developmental stage, pregnacy rates of early blastocysts (61.1%), blastocysts(64.7%) and expanded blastocysts(69.5%) were higher than that of morulae stage(20.0%). The pregnancy rates according to the corpus luteum grades of A, B and C in recipients were 73.6, 62.9 and 50.0%, respectively. Effects of donor-recipients synchrony of after day 2, 1 and 0, before day 1 and 2 on the pregnancy rates were 35.7, 65.5, 72.6, 67.9 and 60.0%, respectively. Pregnancy rates of the body condition score of recipients $\leq$2(71.3%) were higher than those of $\geq$3.0 score(40.0%). The pregnancy rates according to the parity of recipients when embryo was transferred to cow(70.6%) was higher than in heifer(59.1%). The pregnancy rates according to hormone treatment before embryo transfer were 69.9% in hCG + GnRH administration group and 63.0% in control group. Fresh and frozen-thawed embryos on the pregnancy rates were 70.6 and 36.4%, respectively. Pregnancy rates in single and AI+single was 90.0% and 64.8%. Pregnancy rates in twin induction was better than in single. These results indicate that pregnancy rates after transfer were affected on the embryo ages, donor-recipient synchrony, body condition score of recipients, corpus luteum status, parity and hormone treatment to recipients.

• Korean Journal of Poultry Science
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• v.35 no.3
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• pp.241-245
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• 2008

Lentiviral vector system is efficient vehicles for the delivery of exogenous genes, and it is generally used in the generation of transgenic chickens. In this study, we used recombinant lentiviral vectors to generate transgenic chicks that express the human superoxide dismutase-3 gene driven by the chicken ovalbumin promoter. It is well known that superoxide dismutases(SODs) are believed to play a crucial role in protecting cells against oxygen toxicity. There are three forms of SOD proteins: cytosolic Cu-Zn SOD, mitochondrial Mn SOD, and extracellular SOD(SOD-3). The recombinant lentivirus containing the human SOD-3 gene was injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. From 341 injected embryos, the 78 chicks hatched after 21 days incubation. The hatched chicks were screened for the human SOD-3 gene by using PCR. Two of 47 male chickens that survived to sexual maturity contained the human SOD-3 gene in their semen. These results showed that our transgenic chicken generation system was completely established.