수정란 이식 기술의 응용

임현주,손준규,윤호백,백광수,최창용,김시동,권응기 韓國受精卵移植學會 2013 한국동물생명공학회지 Vol.28 No.3

Embryo transfer (ET) technology is of high importance in modern cattle breeding programs. ET is one step in the process of removing one or more embryos from the reproductive tract of an outstanding donor female and transferring them to one or more recipient females. Embryos also can be produced in the laboratory via techniques such as in vitro fertilization (IVF). But the actual transfer of an embryo is only one step in a series of processes that may include some or all of the following: superovulation and insemination of donors, collection of embryos, isolation, evaluation and short-term storage of embryos, micromanipulation and genetic testing of embryos, freezing of embryos and embryo transfer. Cryopreservation and direct transfer of frozen-thawed embryos is common-place with pregnancy rates near that of fresh embryos. Polymerase chain reaction (PCR) technology is currently being used for sexing embryos, and this technology will be used for “embryo diagnostics” and “embryo genomics” in the future. Although, many limitations and problems remain to overcome, these and other new technologies promise to change livestock breeding drastically in the next decade.

Embryo sexing methods in bovine and its application in animal breed

Shelema Kelbessa Bora 한국동물생명공학회(구 한국동물번식학회) 2022 Journal of Animal Reproduction and Biotechnology Vol.37 No.2

The ability to determine the sex of bovine embryos before the transfer is advantageous in livestock management, especially in dairy production, where female calves are preferred in milk industry. The milk production of female and male cattle benefits both the dairy and beef industries. Pre-implantation sexing of embryos also helps with embryo transfer success. There are two approaches for sexing bovine embryos in farm animals: invasive and non-invasive. A non-invasive method of embryo sexing retains the embryo’s autonomy and, as a result, is less likely to impair the embryo’s ability to move and implant successfully. There are lists of non-invasive embryo sexing such as; Detection of H-Y antigens, X-linked enzymes, and sexing based on embryo cleavage and development. Since it protects the embryo’s autonomy, the non-invasive procedure is considered to be the safest. Invasive methods affect an embryo’s integrity and are likely to damage the embryo’s chances of successful transformation. There are different types of invasive methods such as polymerase chain reaction, detection of male chromatin Y chromosomespecific DNA probes, Loop-mediated isothermal amplification (LAMP), cytological karyotyping, and immunofluorescence (FISH). The PCR approach is highly sensitive, precise, and effective as compared to invasive methods of farm animal embryonic sexing. Invasive procedures, such as cytological karyotyping, have high accuracy but are impractical in the field due to embryonic effectiveness concerns. This technology can be applicable especially in the dairy and beef industry by producing female and male animals respectively. Enhancing selection accuracy and decreasing the multiple ovulation embryo transfer costs.

젖소 동결수정란의 비외과적 이식에 있어서 수정란의 상태 및 이시조건이 수태율에 미치는 영향

이은송,조충호,황우석,Lee, Eun-song,Jo, Choong-ho,Hwang, Woo-suk 대한수의학회 1989 大韓獸醫學會誌 Vol.29 No.3

This study was performed to investigate the effects of stage and quality of embryo, synchrony between donor and recipient and difficulty of transfer on pregnancy rate following non-surgical transfer of frozen-thawed bovine embryos. The results were as follows; 1. The overall pregnancy rate of this experiment was 63.4% and that of heifers(73.1%) was higher than that of cows(46.7%). 2. The pregnancy rates of recipients transferred with morulae, early blastocysts and blastocysts were 50.0%, 64.7% and 71. 4%, respectively. 3. The pregnancy rate of recipients transferred with good embryos(67.9%) was higher than that of recipients transferred with fair embryos(53.8%). 4. The pregnancy rates of embryos transferred to left and right uterine horn were 63.2% and 63.6%, respectively. 5. The pregnancy rate of recipients in estrous synchrony 0(76.2%) was higher than those of recipients in synchrony -1(55.6%) and +1(44.4%). 6. The pregnancy rate of recipients transferred with 2 embryos (71. 4%) was higher than that of recipients transferred with 1 embryo(61.8%). 7. The pregnancy rate of embryos transferred to uterine tip (72.0%) was higher than that of embryos transferred to uterine base(50.0%). 8. Ease of transfer was ranked to a scale of one to three on the basis of increasing difficulty. Transfers ranked as ease score 1 accounted for 77.8% of pregnancies and had higher pregnancy rate than ease score 2(66.7%) or 3(45.5%). 9. The pregnancy rate of recipients with excellent corpus luteum(CL) (70.0%) was higher than those of recipients with good CL(61.1%) or fair CL(61.5) %. In reviewing above results, it was considered that the factors such as embryo stage, embryo quality, estrous synchrony, corpus luteum quality, transfer site within uterus, recipient's parity and ease score affected the pregnancy rate after non-surgical transfer of frozen-thawed bovine embryos.

공법 : 배아와 인간존엄

방승주 ( Seung Ju Bang ) 한양대학교 법학연구소 2008 법학논총 Vol.25 No.2

As a result of the remarkable developments in biotechnology and biomedicine, it has become an important issue whether an embryo in vitro should be afforded human dignity and the right of life. In Korea, ``The Law of Bioethics and Safety`` was enacted to resolve conflicts between bioethics and biotechnology. Since section 17 of the law holds that supernumerary embryos, which elapsed more than 5 years of preservations-period or fewer than 5 years of this period, if the parents give consent, can be used in research for the treatment of sterility and contraceptive measures, muscle dystrophy, or a rare or incurable disease. Two embryos, their parents and other people (law professor, philosophers, doctors, lawyer etc.) have brought constitutional complaint against the law in the Constitutional Court, for the reason why the section violates their human dignity and the embryos` right of life. In this case, the most important points are whether an embryo could be the subject of human dignity and the right of life on the one hand, and whether sections 16 and 17 of the law violate human dignity and the right of life, as the embryos and other people including their parents articulated, on the other hand. There are many theories about when human dignity and the right of life begin: from the time of fertilization, implantation, building of the brain, development of the human shape, or birth, among other time points. In my opinion, the embryo is the subject of human dignity from the time of fertilization, even before it is implanted in the mother`s womb (fertilization`s theory). Nevertheless, from the perspective of the protect-intensity of human dignity, embryos before implantation could be treated differently than the embryo after implantation. In other words, the human dignity of the embryo before implantation could be restricted by the fundamental rights of the mother like the rights of self-decision (Article 10 Constitution). 배아와 인간존엄 37 The conclusions that follow from the recognition of embryos as the subject of human dignity and the possibility of restricting dignity-protection, which the author formed in this article, are as follows: First, except for the purposes of childbirth, supernumerary embryos in vitro should not be created. Second, nevertheless, in the case of creating supernumerary embryos according to the current law, the woman should not be obligated to implantation, if the woman no longer desires pregnancy. Third, if the woman dies or has no intention for childbirth while the supernumerary embryos are being preserved, the state should search the chances of implantation like a surrogate mother, for the purpose of protecting human dignity and the right of life. If not, the state should preserve the embryos permanently in principle until it finds the possibility of implantation. Fourth, the embryo not only should not be abolished, but also should not be used for research, even if the 5-year period of preservations has passed, lest the creation of embryos for research should be legalized in practice. Fifth, so-called "somatic cell nuclear transfer embryos" are also the subject of human dignity, because they could develop into a human being, on the condition that they would be transplanted into the wombs. Therefore, the creation of somatic cell nuclear transfer embryos should not be permitted, even if they are made for therapeutic purposes. In conclusion, sections 16 and 17 of the law, which prescribe that embryos should be abolished after 5 years or less period, if the parents consent to it, to be preserved, so that they may be used for research, are unconstitutional, because they violate the human dignity clause and the rights of life for embryos in vitro.

Embryo sexing methods in bovine and its application in animal breed

Shelema Kelbessa Bora 사단법인 한국동물생명공학회 2022 한국동물생명공학회지 Vol.37 No.2

The ability to determine the sex of bovine embryos before the transfer is advantageous in livestock management, especially in dairy production, where female calves are preferred in milk industry. The milk production of female and male cattle benefits both the dairy and beef industries. Pre-implantation sexing of embryos also helps with embryo transfer success. There are two approaches for sexing bovine embryos in farm animals: invasive and non-invasive. A non-invasive method of embryo sexing retains the embryo’s autonomy and, as a result, is less likely to impair the embryo’s ability to move and implant successfully. There are lists of non-invasive embryo sexing such as; Detection of H-Y antigens, X-linked enzymes, and sexing based on embryo cleavage and development. Since it protects the embryo’s autonomy, the non-invasive procedure is considered to be the safest. Invasive methods affect an embryo’s integrity and are likely to damage the embryo’s chances of successful transformation. There are different types of invasive methods such as polymerase chain reaction, detection of male chromatin Y chromosome specific DNA probes, Loop-mediated isothermal amplification (LAMP), cytological karyotyping, and immunofluorescence (FISH). The PCR approach is highly sensitive, precise, and effective as compared to invasive methods of farm animal embryonic sexing. Invasive procedures, such as cytological karyotyping, have high accuracy but are impractical in the field due to embryonic effectiveness concerns. This technology can be applicable especially in the dairy and beef industry by producing female and male animals respectively. Enhancing selection accuracy and decreasing the multiple ovulation embryo transfer costs.

생쥐 수정란의 분할조작후 생존성 향상에 관한 연구

이효종,박희성,김택석,최상용,박충생,Lee, Hyo-jong,Park, Hee-sung,Kim, Taeg-seog,Choe, Sang-yong,Park, Choong-saeng 대한수의학회 1989 大韓獸醫學會誌 Vol.29 No.2

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.

Rapid in vitro Germination of Zygotic Embryos via Endosperm Removal in Eleutherococcus senticosus

You Xiang-Ling,Choi Yong-Eui,Yi Jae-Seon The Korean Society of Plant Biotechnology 2005 Plant molecular biology and biotechnology research Vol.7 No.1

Eleutherococcus senticosus (also called Acanthopanax senticosus), belonging to Araliaceae family, has been used as an important medicinal woody plant. Mature seeds of Eleutherococcus senticosus have rudimentary (extremely immature) zygotic embryos and require a long-term stratification for about 18 months to induce germination. Here, through the methods of endosperm removal and other exogenous treatments, we investigated the factors for inducing rudimentary embryos by in vitro culture, Rudimentary zygotic embryos in seeds were at globular to heart-shaped stage at about $250{\mu}m$ in length just after harvest of fruits. When the seeds without testa were cultured on 1/2 MS (Murashige and Skoog 1962) medium, they did not germinate regardless of medium and sucrose concentrations but the removal of endosperm tissue markedly stimulated the growth of rudimentary zygotic embryos. The embryo reached ear-lier maturation, once when the endosperm surrounding the rudimentary embryos was removed. Rudimentary zygotic embryos developed cotyledons within 3 weeks of culture after endosperm emoval. However, post-mature zygotic embryos failed to germinate though they were morphologically normal, indicating another dormancy of embryos. $GA_3\;(2.0\;\cal{mg/L})$ and/or charcoal ($0.2\%$) treatment rapidly enhanced the germination of zygotic embryos. These results suggest that E. senticosus seeds have double dormancy; i. e. morphological rudimentary dormancy influenced by surrounding endosperm and physiological dormancy after post-maturation of zygotic embryos. Based on the above findings, we established the rapid germination of rudimentary zygotic embryos by in vitro culture of excised seeds with endosperm removal and $GA_3$ treatment.

Improved pregnancy rate and sex ratio in fresh/frozen in vivo derived embryo transfer of Hanwoo (Bos taurus coreanae) cows

Jihyun Park,Wonyou Lee,Islam M. Saadeldin,Seonggyu Bang,Sang Hoon Lee,이준구,Jong Ki Cho 한국축산학회 2023 한국축산학회지 Vol.65 No.4

This study aimed to assess the effects of embryonic developmental stage, quality grade, and fresh or frozen/thawed conditions on the pregnancy rate and sex ratio of live offspring in Hanwoo (Bos taurus coreanae) cows. The quality and developmental stage of in vivo-derived (IVD) transferred embryos were evaluated using the standard criteria of the International Embryo Technology Society. The recipient cows were synchronized using conventional (estradiol benzoate and progesterone) protocols before embryo transfer. Embryos were transferred to 297 cows, and pregnancy was monitored for 60–70 days after embryo transfer. The pregnancy rates of fresh and frozen/thawed embryos were 56.90% and 52.49%, respectively. Pregnancy rates varied according to embryo quality (56.18% for grade 1 vs. 36.67% for grade 2). Pregnancy rates also varied by developmental stage and cryopreservation (67.86% vs. 63.49% for stage 4-1, 64.00% vs. 54.72% for 5-1, and 50.00% vs. 47.83% for 6-1, in fresh embryos vs. frozen/thawed embryos, respectively). For stage 7-1, the pregnancy rates were 72.73% for fresh embryos and 20.00% for frozen/thawed embryos. In 66 fresh embryos, the sex ratio of live offspring was 5:5, whereas it was 4(female):6(male) for frozen/thawed embryos among the 95 frozen/thawed embryos. The miscarriage rate was approximately 3% higher for frozen/thawed embryos than for fresh embryos (18.1% for fresh vs. 21.1% for frozen). Seasonal fertility rates were 33.3% in spring, 55.67% in summer, 52.8% in autumn, 60.0% in winter. The following male-to-female ratios were observed in different seasons: 6.7:3.3 in spring, 4.0:6.0 in summer, 5.5:4.5 in autumn, and 3.3:6.7 in winter. The current data revealed no significant differences in pregnancy rates between fresh and frozen/thawed IVD embryos. However, there was a lower pregnancy rate with advanced-stage frozen/thawed embryos (stage 7-1). The current study provides comprehensive results for the better optimization of embryo transfer in Hanwoo cattle to obtain the desired fertility rate, pregnancy rate, and sex ratio of calves. These results provide important insights into the factors that influence the viability and success of IVD embryo transfer in Hanwoo cows and may have practical applications for improving breeding programs and reducing production costs.

대두 체세포배에서 전형성층 분화와 경단분열조직의 발달

최필선,권석윤 한국식물생명공학회 2013 식물생명공학회지 Vol.40 No.1

Immature embryos of Glycine max L. was cultured on Murashige and Skoog’s (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D). After 6 to 8 weeks of culture, immature embryos produced somatic embryos. Of somatic embryos, two cotyledonary embryo (14%), one cotyledonary embryo (37%), fused cotyledonary embryo (43%), and stunted globular embryos (6%) were observed. The procambial strand of cotyledons originated from circular procambial tissues of lower hypocotyl. The circular procambial tissues were independently divided into one or two procambial strand at the edge of cotyledonarynode,and then connected to each cotyledon to form somatic embryos with one or two cotyledons. When cotyledon was a fused type, the circular procambial strand in lower hypocotyl was continuously connected to the cotyledon. Also, somatic embryos with two cotyledons developed a functional shoot apex with the tunica-corpus structure. In contrast, somatic embryos with one or fused cotyledon formed an abnormal shoot apex without the tunica-corpus structure or with non-dome shape in the inter-cotyledonary area. These results indicated that the variation of cotyledon in somatic embryos is closely related to procambial differentiation and shoot apical meristem development.

Comparison of Sexing Analysis between Karyotyping and Blasomere-PCR in Bovine embryos

Chang Suk-Min,Lee Jong-Ho,Park Joong-Hoon,Park Wha-Sik,Park Chang-Sik,Jin Dong-Il 한국발생생물학회 2003 한국발생생물학회 학술발표대회 Vol.2003 No.1

Accurate analysis of nuclear status is needed when biopsied-blastomeres are used for embryo sexing. In this study, the nuclear status of blastomeres derived from 8- to 16-cell stage IVF bovine embryos was analyzed to evaluate the representative of single blastomere for embryo sexing. When 55 embryos were analyzed by PCR following biopsy, the coincident rate of sex determination between biopsied-single blastomere and matched blastocyst by PCR was 80 %. Karyotyping of biastomeres in 8- 16-cell stage bovine embryos was conducted to assess chromosome status of IVF embryos. To establish karyotyping of blastomeres, concentrations of vinblastine sulfate and duration of exposure time for metaphase plate induction with 8- to 16-cell stage bovine embryos were tested. The most effective condition for induction of metaphase plate (>45%) was 1.0 ug/ml vinblastine sulfate treatment for 15 h. In 22 embryos under the condition, only 8 embryos out of ten that had a normal diploid chromosome complement showed a sex-chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four out of the other 11 embryos having a mixoploid chromosomal complement contained haploid blastomere with wrong sex chromosome (18.2%). These results suggested that morphologically normal bovine embryos derived from IVF had considerable proportion of mixoploid and sex-chromosomal mosaicism which could be the cause of discrepancies of the sex between biopsied-single blastomere and matched blastocyst by PCR analysis.