• Journal of Veterinary Science
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• 제23권2호
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• pp.40.1-40.13
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• 2022

Background: Somatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency. Objectives: This study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs. Methods: The viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer. Results: Most sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption. Conclusions: Sow urine-derived cells could be used to produce SCNT embryos.

• 한국수정란이식학회지
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• 제17권2호
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• pp.129-136
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• 2002

본 연구는 체내, 체외 소 배반포기 배의 GMP vitrification 후 활력도의 비교와 한우 수정란을 몽골 소에 수정란이식 후 산자생산 가능성을 조사하고자 실시하였다. 한우 수정란은 체외수정란 또는 과배란처리에 의한 체내수정란을 생산하여 GMP vitrification 방법으로 동결 후 몽고로 수송하였다. 수란우는 CIDR과 $PGF_2\alpha$ 처리에 의하여 동기화를 유도하였다 체내수정란생산을 위하여 7두를 과배란처리하였다. 총 64개의 배반포기를 회수하였다. ($9.1\pm2.94$per cow). 체외수정란생산은 80.1% 분할율(174／217)과 40.8% 배반포기 발달율(71／174)을 얻었다. 체내수정란(93.7%; 45／48)의 동결융해 후 생존율은 체외수정란(82.5%; 52／63)보다 유의적으로 높았다(P＜0.05). 8두의 몽골 소에 2개의 수정란을 이식하여 5두가 이식 후 60일째 임신이 확인되었으나, 그 중 1두는 240째 유산을 확인하였다. 그 중 2두의 수란우에서 2두의 산자를 275일째 생산에 성공하였다. 이러한 결과는 GMP vitrification 방법은 체내, 체외수정란의 동결보존방법으로 이용될 수 있을 뿐만 아니라 동결융해란의 몽골 소에 이식 후 한우를 생산할 수 가능성을 확인하였다.

• 한국가축번식학회지
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• 제25권2호
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• pp.191-198
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• 2001

본 연구는 체내, 체외수정란 및 배양체계가 세포막투과력 및 GMP vitrification후 생존성에 미치는 영향을 조사하고자 실시하였다. 체내수정란은 6마리 한우를 FSH와 PG $F_{2{\alpha}}$ 에 의한 과배란처리하여 생산하였다. 체외수정란은 난관상피세포 공배양 (OCS) 및 HECM-6 (DCS) 방법으로 생산하였다. 생산된 배반포기 배는 세포력투과력과 GMP vitrification 후 생존성의 조사를 위하여 사용되었다. 세포력투과력은 35$^{\circ}C$ 가온판과 0.5 M sucrose 용액에서 0, 2, 5 및 7분간의 노출시간에 세포질의 “가로 $\times$ 세로”의 직경을 조사하였다. 세포질의 용적은 조사한 직경을 4/3.$\pi$ $r^3$ 공식으로 계산하였다. 배반포의 동결보존은 GMP vitrification 방법으로 실시하였으며, 융해 후 0.25와 0.15 M sucrose 용액 및 TCM199에 각각 5분간 세척한 후 TCM199에 24 또는 48시간동안 배양하였다. 체내수정란의 0, 2, 5 및 7분 때의 용적변화(100, 37.1, 34.3 및 31.6%)는 OCS(100, 59.8, 48.9 및 47.9%)와 DCS(100, 57.2, 47.3 및 46.9%) 보다 유의적으로 높게 수축되었다(P＜0.05). 또한 체내수정란(93.6%)의 동결융해 후 생존성은 OCS 및 DCS (81.9 및 83.6%) 보다 유의적으로 높았다(P＜0.05). 현 배양체계에서 체외수정란의 형태는 체내수정란과 유사하였지만, 세포막투과력 및 응해 후 생존성 등의 질적인 면에서는 큰 차이를 보였다. 결론적으로 세포력 투과력 및 동결융해 후 생존성 등의 질적인 면에서 체내수정란은 OCS 또는 DCS 배양체계에서 생산된 체외수정란보다 우수하였다.

• 한국임상수의학회지
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• 제12권1호
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• pp.877-886
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• 1995

The recycling nuclear transplantation(NT) technique has the powerful potential of producing a large number of genetically identical embryos and offsprings from one embryo. Multiple generational cloning by this technique utilizes the NT embryo itself as the donor for the next generation of cloning. In this experiment, we have produced the third generational cloned embryos by recycling NT. Further we examined comparatively the electrofusion rate and in vitro developmental potential in the cloned embryos of the first second and third generations. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulberco's phosphate buffered saline containing 10 % fetal calf serum(FCS) at 47 hours after hCG injection. In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gl/S transition of 32-cell stage. The first and second generation NT embryos developed to 16-cell were used as donor nuclei for second and third generation. The recipient cytoplasms were utilized the oocytes collected at 14 hours after hCG injection, following revoming the nucleus and the first polar body by micromanipulation. The separated blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were fused by electrical stimulation. The electrofusion rate was seen to be 78.0, 88.0 and 90.3 % in the first second and third generation NT rabbit embryos, respectively. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10 % FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The in vitro developmental potential to blastocyst stage was significantly(P<0.05) decreased in the third(7.2 %) generation NT embryos compared to the first(53.1 %) and second(16.1 %) generation NT embryos. Following in vitro development to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The mean blastomere numbers and cell cycle numbers of NT embryos during the culture period were significantly(p<0.05) decreased in the second(93.9 cells and 6.55 cylces) and third(81.5 cells and 1.35 cylces) generation, compared to the first(189.9 cells and 7.55 cylces) generation.

• Asian-Australasian Journal of Animal Sciences
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• 제20권12호
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• pp.1820-1826
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• 2007

In this study, we conducted various experiments in order to develop enhanced cultural conditions for in vitro-produced porcine embryos. All embryos were produced by in vitro maturation (IVM) and fertilization (IVF) of immature oocytes from abattoir-derived ovaries. In experiment 1, we cultured IVF embryos in 4 different groups, namely, 0% bovine serum albumin (BSA), 3% BSA, 0.05% Polyvinyl alcohol (PVA), and 0.5% Polyvinylpyrrolidone (PVP) added to the basal fluid cultural medium, Porcine zygote medium 3 (PZM-3). The rates of embryo development were higher in the group where the PZM-3 media had been supplemented with 3% BSA than the other groups. While not statistically significant, the percent of blastocysts and hatched blastocytes were 6.9% and 25.0% in the 3% BSA group vs. 1.2-6.4% and 0-16.7% in the other groups, respectively. In experiment 2, we added 10% fetal bovine serum (FBS) to PZM-3 on day 0 of culture and observed the development rate of blastocysts per day of culture from days 0 to 5. The development rate of blastocysts was higher at 15.6% on day 4 than on any other day, and was significantly higher than on day 0 or day 1 (p<0.05). The development rate of hatched blastocysts was 26.7% on day 4, and was higher than on any other day. In experiment 3, we cultured IVF embryos with different fluid culture media, grouped as 1) PZM-3+0.3% BSA (day0-day7); 2) PZM-3+0.3% BSA${\rightarrow}$day-4) PZM-3+10% FBS; 3) PZM-3+0.3% BSA${\rightarrow}$PZM-3+0.3% BSA+(day-4) FBS 10%; and 4) PZM-3+0.3% BSA+10% FBS (day0-day7). The development rates of blastocysts and hatched blastocysts were 21.5% and 53.1% in group 3, respectively, which was significantly higher than group 4 with respect to blastocyst development (5.2%, p<0.05) but not hatched blastocysts (14.3%). The total cell number (TCN) of blastocysts in group 3 was higher at $37.8{\pm}16.1$ than the other groups at $16.8{\pm}4.4$ - $30.1{\pm}10.9$; however, this was not significantly different. The results of this study showed that PZM-3 containing 0.3% BSA and supplemented with FBS during the later stage of culture on day 4 resulted in better TCNs and an increased rate of hatched blastocysts.

• 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.

• Reproductive and Developmental Biology
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• 제33권1호
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• pp.13-18
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• 2009

The aim of this study was to examine the microtubule distributions of somatic cell nuclear transfer (SCNT) and parthenogenetic porcine embryos. Porcine SCNT embryos were produced by fusion of serum-starved fetal fibroblast cells with enucleated oocytes. Reconstituted and mature oocytes were activated by electric pulses combined with 6-dimethlyaminopurine treatment. SCNT and parthenogenetic embryos were cultured in vitro for 6 days. Microtubule assembly of embryos was examined by confocal microscopy 1 hr and 20 hr after fusion or activation, respectively. The proportions of embryos developed to the blastocyst stage were 25.7% and 30.4% in SCNT and parthenogenetic embryos, respectively. The frequency of embryos showing $\beta$-tubulins was 81.8% in parthenogenetic embryos, whereas 31.3% in SCNT embryos 1 hr after activation or fusion. The frequency of the embryos underwent normal mitotic phase was low in SCNT embryos (40.6%) compared to that of parthenogenetic ones (59.7%) 20 hr after fusion or activation (p<0.05). The rate of SCNT embryos with an abnormal mitosis pattern is about twice compared to that of parthenogenetic ones. The spindle assembly and its distribution of SCNT embryos in the first mitotic phase were not different from those of parthenogenetic ones. The result shows that although microtubule distribution of porcine SCNT embryos shortly after fusion is different from parthenogenetic embryos, and the frequency of abnormal mitosis 20 hr after fusion or activation is slightly increased in SCNT embryos, microtubule distributions at the first mitotic phase are similar in both SCNT and parthenogenetic embryos.

• 한국가축번식학회지
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• 제24권3호
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• pp.311-317
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• 2000

본 연구는 체외에서 한우 난포란의 성숙과 수정후 배의 체외발생에 있어서 온도충격의 영향이 처리온도 및 처리시간에 미치는 효과를 검토하였다. 1. 체외에서 생산된 4~8 세포기 배의 배반포로의 배발달율에 있어서 온도충격의 적정온도는 41$^{\circ}C$, 처리시간은 30초였다. 2. 체외성숙 후의 온도충격이 체외성숙 전의 온도충격보다 수정란의 분할율을 유의하게 상승시켰다(p<0.05). 3. 체외성숙 후와 배양 5일째 배에 각각 온도처리를 하였을 경우 비처리군보다 배반포의 배발달율이 향상되었다.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2004년도 제47차 추계학술대회
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• pp.103-103
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• 2004

• 한국수정란이식학회지
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• 제23권1호
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• pp.43-49
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• 2008

본 연구는 한우 체외 수정란의 성 감별과 신선란, 동결란 및 성 감별 수정란을 이식한 후 수태율, 분만율과 유산율, 생시 체중, 임신 기간을 조사하기 위하여 수행하였다 Aspiration과 punching법으로 biopsied한 수정을 24시간 배양 후 생존율은 각각 80.0%와 90.0%로 유의적인(p>0.05) 차이는 없었다. 수정란을 성 감별한 결과, 웅성 수정란과 자성 수정란의 비율은 각각 42.1%와 52.6%였으며, 5.3%는 수정란의 성을 판정하지 못하였다. hCG를 처리한 수란우의 수태율은 46.4%로서 무처리구(38.5%)에 비하여 높은 경향이었으나, 처리구간의 유의적인 차이는 없었다. 신선란과 동결란을 수정란 이식 후 수태율은 각각 41.3와 35.0%로서 유의적인 차이는 없었다. 성 감별된 수정란과 성 감별하지 않은 체외 수정란 이식 후 수태율은 각각 27.5와 42.1%로 유의적인 차이는 나타나지 않았다. 성 감별 된 수정란과 성 감별하지 않은 체외 수정란 유래 송아지의 분만율은 각각 85.0와 87.0%이었으며, 유산율은 각각 15.2와 13.3%로서 분만율과 유산율은 유의적인 차이는 없었다. 성 감별된 수정란과 성 감별하지 않은 체외 수정란 유래 송아지의 임신 기간은 각각 281.3일(female), 283.0일(male)과 288.2일(female), 282.3일(male)이었으며, 생시 체중은 각각 23.6(female), 24.6(남)과 25.0(female), 23.8 kg(male)로 유의적인 차이는 없었다. hCG 처리한 수란우는 한우 수정란 이식 후 수태율 개선 효과는 있는 것으로 판단된다. 성 감별 수정란 이식에 의해 송아지는 생산되었으나, 수정란의 biopsy 방법, 수정란 이식 후 수태율 개선과 같은 송아지 생산 효율을 개선해야 할 것으로 사료된다. 수정란 이식의 산업화가 정착하기 위해서는 동결란과 신선란 이식 후 수태율의 개선에 대한 노력이 필요할 것으로 사료된다.