본 실험에서는 외인성 효소 첨가제 및 혼합 세균 배양을 통한 고상발효(Solid-state fermentation, SSF)가 채종박(Rapeseed meal, RSM)의 체외건물소화율(In vitro dry matter digestibility, IVDMD) 및 단쇄지방산(Short chain fatty acid) 생성에 미치는 영향을 조사하기 위해 수행되었다. 외인성 효소 칵테일(첨가 및 미첨가) 및 RSM에 대한 SSF(발효 및 비발효)를 나타내는 2 x 2 요인 설계가 적용되었다. 3-step 돼지 소화율 모델을 적용하여 채종박의 건물 소화율을 분석하였으며, 72시간 대장발효 후 상층액을 수집하여 단쇄지방산 생성량을 분석한 후 칼로리 단위로 변환하여 가소화에너지 소화율을 분석하였다. 소장 (IVDMDh) 및 전장 (IVDMDt) 건물소화율에서는 고상발효된 채종박이 더 높게 나타났다 (각각 p < 0.01). 마찬가지로, 외인성 효소 첨가제 처리구에서 채종박의 소장 소화율(IVDMDh)이 증가하는 경향을 나타냈다(p = 0.06). Acetic acid 및 butyric acid의 생산은 대조구에 비해 고상발효 처리 시 유의하게 더 생산되었으며 (각각 p < 0.01), 이는 총 단쇄지방산의 생산 증가 경향을 나타냈다(p = 0.09). 에너지 소화율에서는 채종박의 고상발효 및 외인성 효소제 첨가가 유의적으로 높게 나타났다 ( p < 0.01). 그러므로 채종박의 고상발효 처리는 단백질 이용성을 비롯한 영양적 가치를 향상시키는데 효과적이라고 사료된다.

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.

Chicken embryonic stem (ES) cells have great potential and provide a powerful tool to investigate embryonic development and to manipulate genetic modification in a genome. However, very limited studies are available on the functional characterization and robust expansion of chicken ES cells compared to other species. Here, we have developed a method to generate chicken embryonic stem cell-like cells under pluripotent culture conditions. The chicken embryonic stem cell-like cells were cultivated long-term over several passages of culture without loss of pluripotency in vitro and had the specific expression of key stem cell markers. Furthermore, they showed severe changes in morphology and a significant reduction in pluripotent genes after siRNA-mediated NANOG knockdown. Collectively, these results demonstrate the efficient generation of chicken embryonic stem cell-like cells from EGK stage X blastoderm-derived singularized cells and will facilitate their potential use for various purposes, such as biobanking genetic materials and understanding stemness in the fields of animal biotechnology.

인삼은 다양한 국가에서 과거로부터 민간의약적으로 사용해 온 유용한 한약자원으로, 동북아시아와 북아메리카에서 서식하고 있다. 인삼의 학명은 Panax ginseng C. A. Meyer로 Araliaceae에 속하고 약용 부위는 곁뿌리를 제거한 뿌리 부분이다. 본 연구에서는 전세계에 분포하는 인삼의 종류와 명칭을 정리하고, 동북아시아 공정서 내 인삼의 기원들을 알아보았다. 다양한 인삼종의 전초의 형태를 비교하였고, 여러 문헌 정보를 바탕으로 각국 인삼종의 약리학적 효능을 정리하였다. 현재 한국에서 재배되고 있는 고려인삼의 품종은 약 15종이며, 이들 중 재래종(자경종), 천풍, 연풍, 금풍, 산양삼의 형태학적 특징을 비교하였다. 한약재 활용 가능한 인삼을 수확하기 위해서는 오랜 기간 재배해야 하며, 그 조건도 까다롭다. 약리학적으 로 가치가 있는 인삼종의 시장 수요를 충족하기 위한 대량증식 방법으로 기내조직배양이 활용될 수 있다. 따라서, 각 인삼종의 종자를 수집하여 크기를 측정하고 형태를 비교하였고, 종자를 횡단면으로 절단해 배의 형태를 관찰했다. 각 인삼종의 종자를 활용하여 기내배양을 진행하였고, MS+GA 1.0mg/L 조건에서 발아율은 미국삼이, 생존율은 금풍이 가장 우수한 것으로 확인 되었다. 인삼의 한 종류인 산양삼은 자연에서 성장 속도가 느리고, 재배조건이 까다롭다는 특징을 가지고 있다. 따라서, 산양삼을 수경 재배해, 해당 인삼종에서 발견되는 다양한 미생물종을 동정하였다. 기내배양을 통한 4종의 인삼종 종자 발아 실험에서도 다양한 미생물종이 확인되었고, rDNA 염기서열 분석을 통해 동정하였다. 본 연구는 한국한의학 연구원이 제공하는 전통의학정보포탈(KIOM Oasis Portal)과 농업진흥청 원예특작과학원의 인삼특작부에서 각 인삼종의 종자를 분양받아 진행되었 다. 해당 논문은 기내배양을 통한 인삼의 대량생산 체계 구축 연구의 초석이 될 것으로 사료된다.

체외 환경에서 생산되는 배아 (Embryo)는 활성산소종 (Reaction oxygen species, ROS) 수준이 일정 수준을 초과함에 따라 산화적인 손상을 받게 된다. 선행연구에 따르면, 항산화제는 ROS를 감소시켜주는 효과를 가지기 때문에 ROS로부터 오는 배아의 단백질, DNA의 손상, 세포 자멸사를 방지하여 배아의 발달률을 향상시킨다. 이전연구에 따르면 항산화제로써 엘라그산 (Ellagic acid, EA)은 ROS를 효과적으로 제거하고, 난자의 산화스트레스를 방지하는 효과를 가지고 있다고 보고되었다. 그리하여, 본 연구를 통해 우리는 소의 수정란 배양체계 중 in vitro culture (IVC) 단계에서 EA의 농도 (0, 5, 10 μM) 별 첨가가 소의 수정란 발달률과, 질적 수준에 미치는 영향을 조사하고자 실험을 진행하였다. 결과적으로, 배반포의 단계별 발달 수준에서 cleavage 형성률은 EA첨가군과 대조군 간의 차이를 발견할 수 없었으나 배반포 형성률에서는 모든 EA 첨가군들이 대조군보다 높았고 EA 첨가군 중에 5 μM 첨가군이 가장 높았다 (p < 0.05). 생산된 배반포의 총 세포 수는 5 μM EA 첨가군이 대조군과 10 μM EA 첨가군 보다 유의적으로 높았으며, 대조군과 10 μM EA 첨가군 사이의 유의적 차이는 없었다 (Control vs. 5 μM vs. 10 μM; 137 ± 7.90 vs. 163.2 ± 7.42 vs. 138.8 ± 6.67, p < 0.05). 세포 자멸사 세포 수는 모든 EA 첨가군들이 대조군보다 유의적으로 낮았다 (Control vs. 5 μM vs. 10 μM; 22.65 ± 4.08, 9.61 ± 1.55, 6.14 ± 0.90, p < 0.05). ROS 수준에서 모든 EA 첨가군들과 대조군 간의 유의적 차이는 없었다 (Control vs. 5 μM vs. 10 μM; 6.81 ± 1.31, 3.86 ± 0.23, 4.11 ± 0.18, p < 0.05). qRT-PCR 실험 결과에서 Nrf2 gene expression은 대조군과, 5 μM 첨가군에서 유의적 차이가 없었으나, 10 μM 첨가군에서는 유의적으로 상향 조절된 것을 관찰하였다. Keap1 gene expression은 5 μM 첨가군에서 유의적으로 하향 조절된 것을 관찰하였다. 하지만 EA의 농도가 10 μM으로 높아짐에 따라 발현 수준이 증가한 것을 관찰할 수 있었다. CAT gene expression은 5 μM 첨가군에서 유의적으로 상향조절 되었으나 10 μM 첨가군에서는 유의적인 차이를 보이지 않았다. SOD1 gene expression은 대조군과 5 μM 첨가군은 유의적인 차이를 보이지 않았으나 10 μM 첨가군에서는 유의적으로 상향 조절된 것을 관찰하였다.

The evergreen oak tree, Quercus myrsinaefolia Blume, is not only economically important for wood, medicine, landscape trees, etc., but also becoming more important in terms of ecology due to climate change. However, asexual reproduction was difficult, so this study was conducted to establish the optimum conditions for micropropagation by shoot multiplication. The surface sterilized seeds of Q. myrsinaefolia were successfully germinated in WPM basal medium. BAP (1.0 mg/L) treatment was most effective for inducing multiple shoots. The highest induction rates of adventitious roots from the multiple shoots was shown in the treatment of 1.0 mg/L NAA. Both MS and WPM medium were most effective for growth of multiplied plantlets. For ex vitro acclimatization, the survival rates of multiplied plantlets were 100% in vermiculite and commercial soil. The results of this study can be used for proliferation and supply, and establishment of ex situ conservation of Q. myrsinaefolia elite trees.

In the early development of parthenogenetic embryo, cytoplasm and nucleic acid fragmentation may be a cause of lower embryo development. The purpose of this study was to evaluate whether embryonic development and apoptosis factors can be reduced by controlling the in-vitro culture environment by the addition of hormones, pregnancy serum and uterine milk. Our study showed that the activity of Casp-3 increased within the cytoplasm when artificially used hormones to induce the incubation environment, and PCNA's manifestation was low. However, the addition of pregnant serum appeared to lower the Casp-3 activity compared to the other groups. In addition, MMP-9 activity was increased and early embryo development and cytoplasmic fidelity were also increased. Therefore, the results of the present study showed that the use of gestational serum in the development of parthenogenetic embryo inhibit apoptosis and increases cytoplasmic reorganization by natural environmental control in in vitro culture.

The establishment of porcine embryonic stem cells (ESCs) from porcine somatic cell nuclear transfer (SCNT) blastocysts is influenced by in vitro culture day of porcine reconstructed embryo and feeder cell type. Therefore, the objective of the present study was to determine the optimal in vitro culture period for reconstructed porcine SCNT embryos and mouse embryonic fibroblast (MEF) feeder cell type for enhancing colony formation efficiency from the inner cell mass (ICM) of porcine SCNT blastocysts and their outgrowth. As the results, porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days showed significantly increased efficiency in the formation of colonies, compared to those for 7 days. Moreover, MEF feeder cells derived from outbred ICR mice showed numerically the highest efficiency of colony formation in blastocysts produced through in vitro culture of porcine SCNT embryos for 8 days and porcine ESCs with typical ESC morphology were maintained more successfully over Passage 2 on outbred ICR mice-derived MEF feeder cells than on MEF feeder cells derived from inbred C57BL/6 and hybrid B6CBAF1 mice. Overall, the harmonization of porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days and MEF feeder cells derived from outbred ICR mice will greatly contribute to the successful establishment of ESCs derived from porcine SCNT blastocysts.

Lilium dauricum is a rare and endangered species belonging to the family Liliaceae. The species contains several bioactive compounds used as functional foods and medicinal agents in Northeast Asia. This study aimed (1) to establish an in vitro bulblet culture using an air-lift bioreactor and callus culture for the conservation of L. dauricum and obtaining its bioactive compounds; (2) investigate the plant phenolic compounds from both cultures system. The highest bulblet production with 12.5-fold increase in growth rate was obtained using MS medium supplemented with 0.5 g L-1 BA and 3% sucrose. Addition of 7% sucrose facilitated bulblet enlargement, with approximate 2.5- and 7-fold increases in diameter and fresh weight, respectively. The highest rate of callus (100%) was obtained using a combination of 1.0 mg L-1 picloram and 0.5 mg L-1 Kinetin. The callus proliferation occurred on MS medium supplemented with 1.0 mg L-1 picloram, 0.25 mg L-1 kinetin, and 0.25 g L-1 casein hydrolysate. There was a significant difference in the total phenolic compound content of callus, which was 1.5-fold higher than that in the bulblets. These findings indicate a suitable system for optimizing both bulblet and callus culture of L. dauricum, therefore, providing useful bio-materials for industrial purposes and contributing to the conservation of this species.

Although somatic cell nuclear transfer (SCNT)-derived embryonic stem cells (ESCs) in pigs have great potential, their use is limited because the establishment efficiency of ESCs is extremely low. Accordingly, we tried to develop in-vitro culture system stimulating production of SCNT blastocysts with high performance in the colony formation and formation of colonies derived from SCNT blastocysts for enhancing production efficiency of porcine ESCs. For these, SCNT blastocysts produced in various types of embryo culture medium were cultured in different ESC culture medium and optimal culture medium was determined by comparing colony formation efficiency. As the results, ICM of porcine SCNT blastocysts produced through sequential culture of porcine SCNT embryos in the modified porcine zygote medium (PZM)-5 and the PZM-5F showed the best formation efficiency of colonies in α-MEM-based medium. In conclusion, appropriate combination of the embryo culture medium and ESC culture medium will greatly contribute to successful establishment of ESCs derived from SCNT embryos.

The objective of this study was to establish an in vitro culture system for ovarian preantral follicles of B6D2F1. First, we optimized the in vitro preantral-follicle culture by culture duration, follicle stimulating hormone (FSH) type, and activin A concentration. Duration of in vitro culture for 9, 11, and 13 days was sufficient for the normal development of preantral follicles to antral follicles. Formation of cumulus cell–oocyte complex (COC) was induced by treatment with human chorionic gonadotropin (hCG; 2.5 IU/mL) and epidermal growth factor (EGF; 5 ng/mL). In addition, metaphase II (MII) oocytes formed during this in vitro culture of preantral follicles. In vitro preantral-follicle culture for 9 days showed higher rates of growth and maturation, thus yielding a greater number of antral follicles, and there were significant differences (p < 0.05) in the number of MII oocytes (that formed from these preantral follicles via differentiation) between the 9-day culture and 11-day or 13-day culture. The follicles cultured for 9 days contained a tightly packed well-defined COC, whereas in follicles cultured for 11 days, the COC was not well defined (spreading was observed in the culture dish); the follicles cultured for 13 days disintegrated and released the oocyte. Second, we compared the growth of the preantral follicles in vitro in the presence of various FSH types. There were no significant differences in the growth and maturation rates and in differentiation into MII oocytes during in vitro culture between preantral follicles supplemented with FSH from Merck and those supplemented with FSH from Sigma. To increase the efficiency of MII oocyte formation, the preantral follicles were cultured at different activin A concentrations (0 to 200 ng/mL). The control follicles, which were not treated with activin A, showed the highest rate of differentiation into antral follicles and into MII oocytes among all the groups (0 to 200 ng/mL). Therefore, activin A (50 to 200 ng/mL) had a negative effect on oocyte maturation. Thus, in this study, we propose an in vitro system of preantral-follicle culture that can serve as a therapeutic strategy for fertility preservation of human oocytes for assisted reproductive medicine, for conservation of endangered species, and for creation of superior breeds.

본 연구는 한국에 자생하는 대표적인 식충식물인 끈끈이주 걱(Drosera rotundifolia)에 있어서 기내 대량번식 체계를 확립 하기 위해 잎 부위별로 절편체(엽신, 사각형으로 자른 엽신, 엽병, 엽신+엽병, 엽신+엽병의 중앙에 칼집)를 달리하여 신초 증식 및 재분화에 적합한 배지농도(1/4MS, 1/2MS, 1MS, 2MS) 및 배양방법(고체 및 액체배지 그리고 암 4주후 명 1주, 암 2 주후 명 3주, 명 5주처리)을 구명하고자 하였다. 고체배지의 경우, 식물체 재생율과 신초수는 1/4MS~1/2MS배지에서 사각 형으로 자른 엽신 절편(100%, 1.8개)과 엽신+엽병 절편(100%, 1.6~1.8개)에서 가장 높게 나타났다. 액체배지의 경우, 식물체 재생율은 1/2MS배지의 사각형으로 자른 엽신 절편에서 100%, 신초수는 1/4MS와 1/2MS배지의 사각형으로 자른 엽신과 엽 신+엽병(중앙-칼집) 절편에서 2.6~2.7개로 높게 나타났다. 고 체배지와 액체배지를 비교해 볼 때, 액체배지에서 5주간 배양 한 결과가 고체배지에서 8주간 배양한 결과보다 좋은 것으로 나타나 끈끈이주걱의 재분화를 위해서는 액체배양이 효율적 인 것으로 판단된다. 배양조건에서는 암 4주 후 명 1주 처리 와 암 2주 처리 후 명 3주 처리의 1/4MS배지에서 신초수가 각각 3.5개, 3.6개로 가장 높았으나 생장상태를 고려한다면 암 2 주처리후 명 3주처리의 1/4MS배지가 가장 좋은 것으로 판단 된다. 본 실험에서는 오히려 cytokinin이 첨가된 배지보다는 첨가되지 않은 배지에서 신초 및 뿌리생장이 양호한 것으로 나타났다.

The osmolarity of a medium that is commonly used for in vitro culture (IVC) of oocytes and embryos is lower than that of oviductal fluid in pigs. In vivo oocytes and embryos can resist high osmolarities to some extent due to the presence of organic osmolytes such as glycine and alanine. These amino acids act as a protective shield to maintain the shape and viability in high osmotic environments. The aim of this study was to determine the effects of glycine or/and alanine in medium with two different osmolarities (280 and 320 mOsm) during IVC on embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. To this end, IVC was divided into two stages; the 0-2 and 3-7 days of IVC. In each stage, embryos were cultured in medium with 280, 320, or 360 mOsm and their combinations with or without glycine or/and alanine according to the experimental design. Treatment groups were termed as, for example, "T(osmolarity of a medium used in 0-2 days of IVC)-(osmolarity of a medium used in 3-7 days of IVC)" T280-280 was served as control. When PA embryos were cultured in medium with various osmolarities, T320-280 showed a significantly higher blastocyst formation (29.0%) than control (22.2%) and T360-360 groups (6.9%). Glycine treatment in T320-280 significantly increased blastocyst formation (50.4%) compared to T320-280 only (36.5%) while no synergistic was observed after treatment with glycine and alanine together in T320-280 (45.7%). In contrast to PA embryonic development, the stimulating effect by the culture in T320-280 was not observed in SCNT blastocyst development (27.6% and 23.7% in T280-280 and T320-280, respectively) whereas the number of inner cell mass cells was significantly increased in T320-280 (6.1 cells vs. 9.6 cells). Glycine treatment significantly improved blastocyst formation of SCNT embryos in both T280-280 (27.6% vs. 38.0%) and T320-280 (23.7% vs. 35.3%). Our results demonstrate that IVC in T320-280 and treatment with glycine improves blastocyst formation of PA and SCNT embryos in pigs.

In vitro culture (IVC) can be used for a variety of assisted reproductive technologies. However, IVC in dog has been low efficient compared to other mammalian. It is believed that an embryo developmental block in IVC embryos is cause of low production efficiency. There is no study of embryo developmental block in dog yet. In this study, we attempted to estimate the beneficial role of EDTA in canine parthenogenic (PA) embryos development to overcome embryo developmental block. The PA embryos were divided into EDTA treated and non-treated groups. Embryo developmental efficiency was measured by activating chemically parthenote. After EDTA induction, PA embryos were evaluated for embryonic development, ROS activity, mitochondrial integrity, ATP production and genomic activation. The EDTA treated PA embryos showed significantly higher survival rate and improved cavity formation compared to non-treated. Moreover, cytoplasmic ROS level was mitigated and mitochondrial membrane potential was found significantly higher in EDTA treated group followed by higher ATP production. Furthermore, major embryonic genomic activation specific markers/factors were also elevated in EDTA treated group. Conclusively, these results indicated that EDTA showed substantially positive effect to overcome embryo developmental block in canine.

Generally, in vivo, primary oocytes are grown and matured into secondary oocytes in the ovarian follicles. Quality of the oocytes matured in vivo is higher than that of oocytes matured in vitro, indicating the importance of materializing the microenvironment of ovarian follicles for production of high quality oocyte. Therefore, we tried to mimic the stiffness of ovarian follicles using an agarose as a biocompatible natural polymer. Unfortunately, to date, there are no many reports on whether the quality of porcine oocytes can be increased effectively under the soft matrix. Accordingly, we tried to evaluate the effects of IVM using different mechanical properties of agarose substrate on developmental competence of porcine oocytes. Agarose substrate was constructed and cumulus-oocyte-complexes (COCs) retrieved from porcine medium antral follicles were matured on non-coated (control) culture dish or dishes coated with 1% and 2% (w/v) agarose substrate. Then, cumulus expansion, embryonic development after parthenogenetic activation, and gene expression level were analyzed and compared. As the results, significant increase in blastocyst formation and cumulus expansion were detected in COCs matured on 1% (w/v) agarose substrate compared with control. Moreover, oocytes of COCs matured on 1% (w/v) agarose substrate showed significantly higher BMP15 expression level compared with control. Pro-apoptotic gene BAX expression was significantly increased in oocytes of COCs matured on 2% (w/v) agarose substrate compared with control. In the glycolytic enzyme phosphofructokinase (PFKP) gene expression, cumulus cells of COCs matured on agarose substrate showed significantly higher PFKP expression than control while they showed significantly lower BAX expression than control. These results demonstrated that quality of porcine oocytes could be increased efficiently by the IVM of immature oocytes on the soft culture matrix using agarose.

This study was conducted to evaluate the effects of insulin and epidermal growth factor (EGF) in a in vitro growth (IVG) medium on oocyte growth, in vitro maturation (IVM) and embryonic development of pig oocytes derived from small antral follicles (SAF) less than 3 mm in diameter. SAF oocytes were cultured for 2 days to induce IVG in alpha-minimal essential medium supplemented with 1 mM dbcAMP and 15% (v/v) fetal bovine serum. After IVG culture, oocyte maturation was induced by culturing IVG oocytes in IVM medium for 44 h. IVM oocytes that extruded the first polar body were selected and induced for parthenogenesis (PA) by applying electric stimulus. SAF oocytes cultured under the insulin treatment showed a significantly increased (P < 0.05) nuclear maturation (73.8%) compared to those cultured with insulin and EGF (59.8%). After PA, the proportions of blastocysts based on the number of metaphase II oocytes were significantly higher (P < 0.05) in oocytes that were cultured for IVG with insulin, EGF, and insulin + EGF (32.4%, 35.2%, and 34.8%, respectively) than in control (22.9%). IVG oocytes treated with insulin showed an increased oocyte diameter (116.3 μm) compared to those treated with insulin and EGF (114.0 μm) (P < 0.05). Intra-oocyte GSH content significantly increased (1.07 pixels/oocyte) by insulin treatment during IVG compared to that of oocytes treated with insulin + EGF (0.78 pixels/oocyte). These results demonstrate that IVG culture of SAF oocytes under insulin or/and EGF treatment supports oocyte maturation and improves embryonic development to the blastocyst stage after PA in pigs.

Mammalian fetal ovaries contains numerous primordial germ cells, however fewer ones can yield mature oocytes due to apoptosis and follicle atresia. Successful in vitro reconstitution of primordial germ cells has recently had a significant effect in the field of assisted reproductive technologies. However, the regulatory mechanisms underlying oogenesis remain unknown and recapitulation of oogenesis in vitro remains unachieved. Therefore, development of methods for obtaining mature oocytes by culturing the fetal ovaries in vitro could contribute to clarify these mechanisms. We adapt an in vitro system for culturing mouse fetal ovaries that support successful follicle assembly and improve oocyte growth and maturation. Ovarian tissues from 12.5 days postcoitum (dpc) fetal mice were cultured in vitro and the matured oocytes were differentiated from primordial germ cells after a 31 days culture period. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with artificial ovarian cells, and that oocytes within the growing follicles are able to mature normally in vitro. Taken together, this in vitro culture system is expected to aid in the development of new strategies to identify the reasons behind failure of follicle assembly and offer a platform for innovative research into preservation of female germ cells and conservation of endangered species.

Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for animal cloning. Until now, SCNT has been inefficient, especially in dog. It is believed that an embryo developmental block in SCNT embryos is cause of low production efficiency. However, no studies have been performed on canines for embryo developmental block. In this study, we attempted to evaluate the beneficial role of EDTA in canine parthenogenic (PA) embryos development to overcome embryo developmental block. The PA embryos were divided into 0.01 mM EDTA treated and non-treated groups. Embryo developmental efficiency was measured by activating chemically parthenote. After EDTA induction, PA embryos were evaluated for embryonic development, Reactive Oxygen Species (ROS) activity, mitochondrial integrity, ATP production and genomic activation. The EDTA treated PA embryos showed significantly higher survival rate and improved cavity formation compared to non-treated. Furthermore, cytoplasmic ROS level was mitigated and mitochondrial membrane potential was found significantly higher in EDTA treated group followed by higher ATP production. Moreover, major embryonic genomic activation specific markers/factors were also elevated in EDTA treated group. Conclusively, we elucidated that EDTA showed substantially positive effect to overcome embryo developmental block in canine.

An efficient method for in vitro propagation and growth of the wild garlic(Allium ochotense Prokh.) was established. Bulbs of wild garlic were collected from Ullung Island, Korea, and the growth pattern of plantlets on various culture media was observed. High growth of shoot was obtained on LP, NN and N6 medium. The growth media supplemented with 3%(w/v) sucrose was found optimal for shoot growth. After 10 weeks multiple shoots were observed in the plantlets growing on the medium containing 1.0 mg/l of zeatine and 0.1 mg/l NAA. Roots were induced directly at the base of the shoot in all treatments. The medium with 2.0 mg/l of IBA proved to be the best rooting medium. The studies of this kind may be used to develop strategies for large-scale propagation of elite wild garlic.