• Asian-Australasian Journal of Animal Sciences
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• 제17권12호
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• pp.1652-1656
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• 2004

The aim of this study was to obtain mature ova or embryos at a single cell stage, which can be used in avian transgenesis and nuclear transfer through multiple ovulations, in vitro fertilization and culture. Chicken anterior pituitary extract (CAPE) or acetone-dried chicken anterior pituitary extract (ACAPE) was used to induce multiple ovulations in hens pretreated with pregnant mare' serum gonadotrophin (PMSG). In vitro fertilization of the multiple ovulated ova was performed by inseminating sperm onto the germinal disks in m-Ringer' solution and incubating the ova at 41$^{\circ}C$, 5% $CO_2$ for 10 h in DME-F12 medium containing 20% liquid albumen. The in vitro fertilization process was observed using an environmental scanning electron microscope. When normal laying hens (white Leghorn) were administered daily with PMSG (100 IU), egg laying ceased in most hens within 3 to 8 days. Ovulation began to occur about 7.5 h after injection of CAPE and ACAPE. The number of ovulated ova was 1.00${\pm}$0.00, 2.33${\pm}$0.52 and 2.20${\pm}$0.45, respectively, after receiving 100, 200 and 300 mg CAPE. The number of ovulated ova was 2.00${\pm}$0.00, 2.86${\pm}$0.69 and 3.00${\pm}$1.22, respectively, after receiving 10, 15 and 20 mg ACAPE. The fertilized and cultured ova were able to develop into embryos up to the 32 cell stage. The present experiments demonstrated that multiple ovulations can be induced by CAPE and ACAPE successfully, and the ova resulted from the treatment retained the capability for further fertilization and embryonic development. These data provide new information to support the establishment of an in vitro culture system for future avian transgenesis studies.

• Reproductive and Developmental Biology
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• 제28권2호
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• pp.101-105
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• 2004

This study was carried out to evaluate the effects of glucose and sodium phosphate on in vitro development of porcine oocytes matured and fertilized in vitro. When the culture medium was supplemented with various concentrations of glucose, the higher proportions (23 and 26%) of oocytes developed to morular or blastocyst stages were at the concentrations of 2.78 and 5.56 mM than 0 (9%; P<0.05) and 11.12 mM (18%). In experiment to evaluate effect of sodium phosphate during in vitro development of porcine oocytes, a significantly (P<0.05) higher proportions of embryos developed to morular or blastocyst stages was obtained with sodium phosphateof 0.28 (25%) and 0.53 (27％) mM than 0 (15%), 1.05 (19%) and 2.10 (10%) mM. On the other hand, when oocytes were cultured in medium with (0.53 mM) sodium phosphate, the proportions of developed embryos were significantly (P<0.05) higher in medium without (29%) that than with (14%) 5.56 mM glucose. However, a higher proportion of embryos developed to morular or blastocyst stages were obtained in medium with (23%) that than without (8%) glucose (P<0.05). The minimum essential medium (MEM) added to the culture medium were higher regardless of presence of sodium phosphate and glucose on the development of embryos. Although sodium phosphate and glucose could support morular and blastocyst development to a limited extend (10∼24%), significantly higher proportion (36%) at morular or blastocyst stages was obtained by MEM adding in the medium with sodium phosphate and glucose. These results suggest that the early development of in vitro fertilized porcine oocytes can be maintained efficiently by glucose and sodium phosphate when they were cultured in medium with MEM.

• 한국수정란이식학회지
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• 제31권2호
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• pp.117-121
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• 2016

Although piglets have been delivered by embryo transfer (ET) with in vitro produced (IVP) embryos and blastocysts, a success rate has still remained lower level. Unlike mouse, human, and bovine, it is difficult to a production of piglets by in vitro fertilization (IVF) because of an inappropriate in vitro culture (IVC) system in pig. Therefore, the present study was conducted to investigate whether minimized exposure time in IVC can improve the pregnancy and delivery rates of piglets. Immediately after IVM, the oocytes were denuded and co-incubated with freshly ejaculated boar semen for 3.5 to 4 hours at $38.5^{\circ}C$ under 5% $CO_2$ in air. To avoid long-term exposure to in vitro state, we emitted IVC step after IVF. After that the presumptive zygotes were transferred into both oviducts of the surrogate on the same day or 1 day after the onset of estrus. Pregnancy was diagnosed on day 28 after ET and then was checked regularly every month by ultrasound examination. The 3 out of 4 surrogates were determined as pregnant (75%) and a total of 5 piglets (2 females and 3 males) were delivered at $118.3{\pm}2.5$ days of pregnancy period. In conclusion, a short-term exposure time may be an important factor in the production of IVP-derived piglets. It can be apply to the in vitro production system of transgenic pig by IVF, cloning, and pronuclear microinjection methods.

• 농업생명과학연구
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• 제43권6호
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• pp.67-75
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• 2009

본 연구는 한약재 (결명자, 계피, 산초, 감초) 추출물의 반추위내 발효와 미생물 활성에 대한 효과를 구명하기 위하여 수행되었다. In vitro 건물소화율은 계피와 산초 추출물 첨가구에서는 0시간대, 감초 추출물 첨가구에서는 3시간대에 대조구에 비해 현저히 (P<0.05) 낮았다. 한약재 추출물 첨가에 따른 발효 시간대별 휘발성 지방산 조성의 변화는 3시간대에서만 처리간 유의성이 인정되었는데, acetate 비율은 대조구가 천연 추출물 첨가구보다 유의적으로 높았으나, butyrate, isobutyrate, isovalerate 및 valerate은 대조구에서 가장 낮았다 (P<0.05). 미생물 성장율은 발효 3시간대에서 결명자 첨가구를 제외한 한약재 첨가구에서 대조구에 비해 유의적 (P<0.05)으로 높았으나 다른 발효 시간대에서는 차이가 없었다. 이상의 결과로부터 한약재로 사용되고 있는 계피, 산초 및 감초 추출물을 in vitro 반추위 배양액에 첨가하였을 때, 발효초기에 반추위 미생물의 활성을 억제하는 경향은 있었으나 미생물 성장에 대한 억제 효과는 없는 것으로 나타났다.

• 한국한의학연구원논문집
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• 제9권2호
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• pp.45-54
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• 2003

Obesity is caused by unbalance of energy intake and expenditure, which results in extra accumulation of adipose tissue. Obesity is directly related to metabolic diseases such as diabetes, hyperlipidemia, fatty liver and so on. To investigate the anti-obesity effects of Scutellariae Radix, 70% EtOH extract and water extract of it were tested by in vitro and in vivo studies of fat accumulation. 3T3-L1 preadipocyte cell line was used in a in vitro study of fat accumulation. After 3T3-L1 cells were induced to differentiate into adipocytes, S. radix extract were added and fat accumulation was measured by oil red O staining. In vivo study showed that weight and epididymal/ retro-peritoneal adipose tissues were significantly reduced in mice fed Scutellariae Radix extract compared with control group. Especially, mice fed Scutellariae Radix extract showed reduced serum triglyceride and glucose levels. When adipose tissues were analyzed by microscope, mean adipocyte size was significantly reduced in Scutellariae Radix extract-fed mice. Therefore, this study showed inhibitory effects of Scutellariae Radix on in vitro and in vivo fat accumulation.

• Advances in Traditional Medicine
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• 제9권2호
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• pp.174-181
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• 2009

Pterocarpus marsupium, Clitoria ternatea, and Sanseveiria cylindrica are some of the important and endangered medicinal plant species of India. Despite of medicinal properties, antibacterial potential of the plants have not yet been explored. The present study was designed to optimize the in vitro technique for micropropagation and to screen the extracts from leaves and in vitro raised calli for antibacterial properties. Excised leaf-explants from the parent plants were surface sterilized and cultivated on Murashige & Skoog's (MS) medium containing $N^6$-benzyladenine (BA) in concentrations of 1, 2, 5, and $10{\mu}M$. Optimal growth of calli was noticed at a concentration of $5{\mu}M$, therefore the extracts from calli grown at this concentration were further studied for antibacterial activity. Both alcoholic and aqueous extracts from leaves of respective plants, and their in vitro raised calli were tested for antibacterial activity by agar well diffusion method against a range of Gram-positive and Gram-negative bacteria. Aqueous extracts showed antibacterial activity against limited number of bacterial species; notably the extracts of C. ternatea which showed antibacterial activity against Streptococcus pyogenes, Bacillus subtilis and Bacillus cereus. Alcoholic extracts of all three plants showed antibacterial activity against a wider range of bacteria. Among the Gram-positive bacteria, extracts from C. ternatea showed strong antibacterial activity against Bacillus spp., whereas the extracts of S. cylindrica showed good antibacterial potential for Staphylococcus aureus, S. epidermidis and S. pyogenes. The extracts from all three plants showed antibacterial activity against Gram-negative bacteria, including, Salmonella spp. and Shigella dysenteriae; organisms causing enteric fever and dysentery. In most of the cases, the extracts from respective calli showed comparable, and in some cases better, result in comparison to the extracts from parent leaves. To the best of our knowledge this is the first preliminary report on antibacterial potential, especially through calli extracts, of these plants; and in vitro cultivation of the explants may be used to obtain phytotherapeutic compounds.

• 생명과학회지
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• 제29권10호
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• pp.1159-1163
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• 2019

남성호르몬 감소와 관련된 남성갱년기에 대한 관심이 고조되고 있지만, 남성호르몬의 정량을 위해 항체를 이용하는 고가의 kit가 이용되고 있다. 본 연구에서는 in vitro 전사 활성 시험법을 이용하여 남성 스테로이드호르몬의 활성 혹은 농도를 검증하는 시스템을 구축하였다. 테스토스테론-AR (androgen receptor) 복합체와 반응하는 ARE-AdE1bTATA 염기서열이 삽입되고 리포터로 luciferase를 발현하는 테스토스테론 유사활성 검증 리포터 플라스미드인 pGL2-Neo-ARE-AdE1BTATA를 제조하고, 인체 전립선암 세포인 LNcap-LN3 세포에 stable transfection을 실시하였다. 구축된 LNcap-LN3/pGL2-Neo-ARE-AdE1BTATA TCD (testosterone compound detection) 시스템은 표준물질인 테스토스테론의 $10^{-13}{\sim}10^{-8}M$ 범위에서 농도 증가에 비례하는 정량성을 보였다. 이 연구에서 확립된 in vitro TCD 시스템을 이용하면 천연물 유래 테스토스테론 유사물질 및 테스토스테론 저하물질의 대량 탐색 등이 가능할 것이므로, 건강기능성 식품이나 의약품 신소재의 개발에 기여할 것이다.

• 한국수정란이식학회지
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• 제10권1호
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• pp.65-72
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• 1995

The purposes of this study were to produce cloned rabbit embryos and offsprings by nuclear transplantation(NT) using in vitro matured oocytes as nuclear recipient cytoplasm and to determine the effect of frozen nuclei donor embryos on the production efficiency of cloned embryos. The 8cell embryos were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline containing 10% fetal calf serum(FCS) at 40 hours after hGG injection. A portion of collected embryos were preserved at 4$^{\circ}C$ for 24 hours and a portion of them were frozen by vitrification method. The embryos used for donor nuclei were synchronized in the phase of Gi /S transition. The in vitro matured oocytes were used as recipient cytoplasm following removing the nucleus and the first polar body. The synchronized blastomeres from fresh, cooled or frozen embryos were injected into the enucleated oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.0 W /cm in 0.28 M mannitol solution. 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. Following in vitro culture of the NT embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The results obtained were summarized as follows: 1. The fusion rates of the blastomeres from fresh, cooled and frozen embryos with the in vitro matured and enucleated oocytes were 100, 95.8 and 64, 3%, respectively. 2. Development in vitro to blastocyst was significantly(p<0.05) different between the cloned embryos with the blastomeres from fresh, cooled or frozen embryos as 39.0, 20. 9 and 15.7%, respectively. 3. The mean numbers of cell cycle per day during in vitro culture of cloned embryos blastomeres from fresh, cooled or frozen embryos was 1.31, 1.29 and 1.16, respectively. 4. A total of 77 nuclear transplant embryos were transferred into 6 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer.

• 한국식품영양과학회지
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• 제42권4호
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• pp.497-504
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• 2013

본 연구에서는 4종의 천연칼슘소재를 이용하여 칼슘 이온화 특성 및 in vitro 칼슘 이용률을 조사하였다. 천연칼슘소재는 8.0%(w/v) 첨가농도까지 칼슘용해량과 칼슘이온 함량은 증가하였으나 이상의 농도에서는 큰 변화는 없었다. 또한 이온화율은 약 90%로, 칼슘소재와 첨가농도에 따른 큰 차이는 나타나지 않았다. 칼슘의 이온화에 용해온도는 큰 영향이 없었으며, 용해 18시간째 가장 높은 칼슘이온 함량을 나타났다. 칼슘액 중 BS의 in vitro 칼슘 이용률은 67.3%로 가장 높게 나타났으며, AS는 62.4%, DS는 57.9%, CS는 57.5%로 시판 칼슘제 및 천연칼슘소재에 비해서 약 2배 정도 높게 나타났다. 시판 우유, 두유 및 오렌지 주스의 in vitro 칼슘 이용률을 조사한 결과 탄산칼슘보다 이온화 칼슘액을 첨가한 구간에서 2배 이상 높게 나타났다. 따라서 천연칼슘소재의 이온화 칼슘은 생체 이용율이 높은 다양한 식품소재로 활용이 기대된다.

• 한국동물생명공학회지
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• 제35권1호
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• pp.94-101
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• 2020

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.