• 농업과학연구
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• 제38권2호
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• pp.269-275
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• 2011

The objective of this study was to develop a rapid and reliable PCR method for sexing of morula or blastocyst stage bovine embryo. BOV97M and bovine 1.715 satellite DNA sequences were selected for amplification of male and bovine specific DNA, respectively. But the unbalanced number of copies of these two repetitive sequences required some modification of PCR method. Karyotyping of blastomeres were carried for the confirmation of sex determination in bovine embryos. The coincidence rate of sex between biopsied-single blastomere and matched blastocyst was 80.0%. When in vivo- and in vitro- derived embryos were compared, 61.8% and 56.7% were male in in vitro- and in vivo-derived embryos, respectively. In vivo-derived embryos showed better hatching rate than in vitro-derived embryos following biopsy of blastomeres. In conclusion, rapid and effective PCR could be applied to sexing of bovine preimplantation embryos using single blastomere. The sensitivity of this assay may eliminate the need for biopsy of more than one nucleated blastomere and reduce trauma to the embryos derived from biopsy procedure.

• 한국가축번식학회지
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• 제26권2호
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• pp.173-182
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• 2002

포유동물의 체외수정란 치적발달조건을 수립하기 위하여 난자의 성숙, 수정 및 배양시 형태학적인 관찰만으로는 불충분하여 각 세포내 구성물 및 핵상 등의 변화를 조사하여 개별 수정란의 품질평가를 통하여 수정란 발달 능력을 향상시키는 방법의 개발이 필요하다. 최근 체외생산된 수정란이 발달율 및 착상율이 현저히 감소되는 원인을 조사한 결과 이러한 수정란에서 할구의 파편화가 보다 더 진행되는 것을 관찰하였다. 이에 본 실험의 목적은 체외수정과 핵치환 유래 소 배반포에서 세포사멸 기전으로 알려진 apoptosis 조절 유전자로써 Bcl-2와 Bax 유전자의 전사체 발현량을 비교 조사함으로써 착상전 수정란 발달과정에서의 역할을 확립하고자 하였다. Apoptosis의 분석은 TUNEL 방법으로 수행하였다 체외수정과 핵치환유래 배반포에서 Bcl-2와 Bax 유전자의 발현량은 RT-PCR로 확인하였다. 핵치환유래 배반포에 있어 TUNEL 표식으로 확인된 비율은 체외수정 된 배반포보다 유의하게 높다 (P<0.001). 체외수정된 배반포의 Bcl-2의 발현량은 핵치환유래 배반포보다 높다. 반대로 체외수정된 배반포의 Bax 발현량은 핵치환 유래 소 배반포보다 낮았다 (P<0.05). 이러한 결과는 체외수정보다 핵치환 유래 소의 배반포에서 좀더 많은 파편화를 초래함을 보여준다. 또한, 핵치환 유래 수정란의 발달 정지의 증가는 apoptosis와 같은 핵의 파편화로써 야기될 수 있다고 사료된다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.39-39
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• 2002

The mechanisms underlying the visual assessment and resulting in optimum embryonic development following in vitro maturation, fertilization, and culture are unclear. It is known that in vitro produced embryos show more frequent occurrence of fragmentation, which result in poor developmental potential and decreased implantation rate. The objective of this study was to investigate the apoptotic rates in in vitro fertilization (IVF) and nuclear transferred (NT)bovine blastocyst. (omitted)

• 한국수정란이식학회지
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• 제5권1호
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• pp.21-27
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• 1990

The technique for maturation of follicular oocyte has been devised to provide such a low cost and in ptentifut number supply of bovine embryo. Some of problems concerning production of bovine embtyo in vitro were discussed in this paper. Bovine follicular oocytes cultured in vitro achieved normal fertilization but cleavage rates to blastocyst were low compared to the oocyte matured in vivo. It has been concluded that a deficient cytoplasmic maturation occurs in the oocytes matured in vitro. These results indicate that the studies for maturation of bovine follicular oocytes in vitro need improvement of culture conditions and to define the characteristics that might be indicative of healthy oocyte.

• 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.

• 한국수정란이식학회지
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• 제26권3호
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• pp.171-180
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• 2011

In this study I report that in vitro development rates of bovine nuclear transfer embryos activated either with boar sperm cytosolic factor (SCF) or with ionomycin followed by cycloheximide (CHX) and subsequent in vivo developmental rates after embryo transfer are related to blastocyst quality as evaluated by apoptosis analysis. SCF was extracted from porcine semen then purified for post-activation injection after nuclear transfer. The optimal timing for SCF injection was determined to be at least 22 h post-IVM for parthenogenetic activation of bovine oocyies. A total of 364 oocytes were successfully enucleated and 268 (73.6%) fused and were injected with SCF. The survival rate of fused and injected embryos was 109/113 (96.5%) after 2 h. The cleavage rates of nuclear transfer embryos after 3 d of culture in the ionomycin/CHX treated group were significantly higher than those of the SCF-activated group (93.3% vs 81.7%, p<0.01, respectively). However, at 7 d and 9 d there was no significant difference between the total developmental rates to blastocyst for either treatment group. Total blastocyst cell numbers were also not significantly different between the two activation treatments (ionomycin/CHX: 149.5${\pm}$7.7 vs. SCF: 139.3${\pm}$4.4 cells). In contrast, the apoptotic levels in the SCF blastocysts were higher than those produced after the chemical treatment (28.2${\pm}$5.1% vs. 8.8${\pm}$0.6%, respectively). A total of 18 expanded or hatching blastocysts was transferred to nine synchronized recipients in each activation group; 5/9 (55.5%) and 2/9 (22.2%) were pregnant at 40 d in the ionomycin/CHX treatment and SCF activated group, respectively. However, only one went to term in the ionomycin/CHX treatment while none of the pregnancies from the SCF group were maintained by 90 d. In conclusion, these results suggest that SCF derived from different species is a limited activator to be used for activation after bovine nuclear transfer in lieu of a chemical activation protocol.

• 한국가축번식학회지
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• 제27권4호
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• pp.339-347
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• 2003

The purpose of this study was to investigate the development of bovine nuclear transfer (NT) embryos cultured in serum-free conditions. Bovine NT embryos cultured in various culture conditions were compared blastocyst development, total cell number and apoptosis using TUNEL assay. In experiment 1, blastocyst rates of NT embryos were significantly higher (P<0.01) in FBS (22.0％) and BSA (26.6％) groups than in PVA (6.3％) group. Total cell number was significantly higher in FBS (78.4$\pm$19.4) and BSA (90.9$\pm$29.1) groups than in PVA group (46.0$\pm$0.0). Apoptotic cell number was significantly fewer in FBS (3.1$\pm$1.4) and BSA (1.7$\pm$1.4) groups than in PVA group (7.0$\pm$20.0) However, all of results were not different between the FBS and BSA group. In experiment 2, blastocyst rates of NT embryos were significantly higher (P<0.05) in fatty acid free-BSA (FAF-BSA) group (26.8％) than in fraction V-BSA group (11.2％). Total cell number were somewhat higher in FAF-BSA group (89.8$\pm$30.7) than in fraction V-BSA group (88.1$\pm$19.3). Apoptotic cell number were somewhat fewer in FAF-BSA (1.7$\pm$1.5) group than in fraction V-BSA group (4.2$\pm$2.9). These findings suggest that serum free condition were effective for the in vitro development of bovine NT embryos. Therefore, we concluded that fatty acid free-BSA has beneficial effect in development bovine NT embryos and can be use as a serum substitute.

• 한국동물생명공학회지
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• 제38권3호
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• pp.143-150
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• 2023

Background: The successful production of superior or transgenic offspring from in vitro produced embryos in cattle relies heavily on the quality of blastocyst stage embryos. In order to enhance the developmental competency of these embryos, a novel culture method was devised. Methods: This study utilized stem cell culture medium (SCM) from hESCs as a supplement within the culture medium for bovine in vitro produced embryos. To gauge the efficacy of this approach, in vitro fertilized embryos were subjected to culture in CR1aa medium enriched with one of three supplements: 0.3% BSA, 10% FBS, or 10% SCM. Results: The blastocyst development and hatching rates of one-cell zygotes cultured in CR1aa medium supplemented with SCM (23.9% and 10.2%) surpassed those cultured in CR1aa medium supplemented with BSA (9.3% and 0.0%) or FBS (3.1% and 0.0%) (p < 0.05). Furthermore, post-zygotic gene activation, cleaved embryos cultured in CR1aa medium supplemented with SCM (57.8% and 34.5%) exhibited notably higher rates (p < 0.05) compared to those cultured with BSA (12.9% and 0.0%) or FBS (45.7% and 22.5%) supplementation. Furthermore, the microinjection of SCM into the cytoplasm or pronucleus of fertilized zygotes resulted in elevated blastocyst development and hatching rates, particularly when the microinjected embryos were subsequently cultured in CR1aa medium supplemented with SCM from the 8-cell embryo stage onwards (p < 0.05), in contrast to those cultured with FBS supplementation. Conclusions: In conclusion, this study conclusively demonstrated that the incorporation of SCM into the culture medium significantly enhances the developmental progress of preimplantation embryos.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.229-229
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• 2004

The identification of embryo-specific genes would provide insights into early embryonic development. However, the current methods employed to identify the genes that are expressed at a specific developmental stage are labor intensive and suffer from high rates of false positives. Here we employed a new and accurate reverse transcription-polymerase chain reaction (RT-PCR) technology that involves annealing control primers (ACPs) to identify the genes that are specifically or prominently expressed in bovine early blastocysts and hatched blastocysts produced in vitro. (omitted)

• 한국가축번식학회지
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• 제9권1호
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• pp.36-39
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• 1985

This experiment was carried out to investigate the morphology of bovine embryos thawed after deep freezing at -196$^{\circ}C$ and the development of frozen-thawed embryos after in vitro culture in Ham's F-10 medium with 10% NBCS. The results obtained were summarized as follows: 1. The propotion of embryos which a, pp.ared mophologically normal was averaged 77.5% (79/102). 2. The morphologically normal rate of frozen-thawed blastocyst (78.6%) was higher than that of morula (76.7%), but there was no significant difference. 3. Normal development was observed in 20 of 68 embryos cultured for 24-72hr in medium and overall survival rate was 29.4%. 4. Survival rate fo blastocyst (33.3%) was higher than that of morula (25.7%).