• 한국수정란이식학회지
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• 제27권4호
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• pp.265-270
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• 2012

In canine, oocytes are ovulated at the GV (germinal vesicle) stage and they have to fulfill maturation phase before reaching metaphase II stage. The efficiency of in vitro maturation is still very low. Therefore, the aim of this study was to investigate the effect of in vitro maturation on nuclear changes of immature canine oocytes recovered from different reproductive stages ovaries and different culture conditions. The oocytes were cultured in TCM-199 with supplement at 5% $CO_2$ and $38.5^{\circ}C$ for 72 h. The nuclear maturation of canine oocytes was evaluated with Hoechst 33342 stain under fluorescence microscope (Fig. 1). The results of this study detected differences in in vitro maturation rate between oocytes recovered from follicle status and non-follicle status ovaries. However, these differences were not significant as indicated in Table 1 and Fig. 2. In regard to the effect of culture condition with supplements, we did not found significant differences compared with control group (Table 2, Table 3). One of the reasons for this data could be the conditions that ovaries were exposed during slaughtering process or the long distant transportation of the ovaries. Although these data have not shown clearly significant differences results compared with control, furthermore the different reproductive status ovaries was beneficial for maturation of oocytes in vitro and can be a basic part of knowledge to improve in vitro maturation of canine oocytes.

• 한국수정란이식학회지
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• 제30권4호
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• pp.315-317
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• 2015

In vitro culture of murine embryos is an important step for in vitro production systems including in vitro fertilization and generations of genetically engineered mice. M16 is widely used commercialized culture media for the murine embryos. Compared to other media such as potassium simplex optimization medium, commercial M16 (Sigma) media lacks of amino acid, glutamine and antibiotics. In the present study, we optimized M16 based embryo culture system using commercialized antibiotics-glutamine or amino acids supplements. In vivo derived murine zygote were M16 media were supplemented with commercial Penicillin-Streptomycin-Glutamine solution (PSG; Gibco) or MEM Non-Essential Amino Acids solution (NEAA; Gibco) as experimental design. Addition of PSG did not improved cleavage and blastocyst rates. On the other hand, cleavage rate is not different between control and NEAA treated group, however, blastocyst formation is significantly (P<0.05) improved in NEAA treated group. Developmental competence between PSG and NEAA treated groups were also compared. Between two groups, cleavage rate was similar. However, blastocyst formation rate is significantly improved in NEAA treated group. Taken together, beneficial effect of NEAA on murine embryos development was confirmed. Effect of antibiotics and glutamine addition to M16 media is still not clear in the study.

• Reproductive and Developmental Biology
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• 제28권4호
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• pp.235-240
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• 2004

In vitro maturation of denuded porcine immature oocytes can be enhanced by co-incubation with spermatozoa even before fertilization. This study was to determine whether the addition of spermatozoa into the culture medium could influence the nuclear maturation of porcine cumulus-enclosed germinal vesicle (GV) oocytes. Cumulus-oocyte complexes (COCs) were collected from follicles of 3- to 5-mm diameter. Porcine COCs were cultured in tissue culture medium containing spermatozoa. After 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. The proportion of oocytes reaching at metaphase II was significantly (P < 0.05) increased in the oocytes cultured in media containing spermatozoa compared to those in media without spermatozoa (52.3% vs 12.5%). No difference in the percentage of metaphase II was observed among the different periods of spermatozoa exposure and among the spermatozoa from different species. The proportion of oocytes reaching metaphase II was significantly different between high and low concentrations of spermatozoa. The present study suggests that manunalian spermatozoa contain a substance(s) that improves nuclear in vitro maturation of porcine cumulus-enclosed GV oocytes. Enhancing effect of spermatozoa for in vitro maturation of oocytes is a highly dose-dependent.

• 한국초지조사료학회지
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• 제33권3호
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• pp.206-212
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• 2013

This study was conducted to investigate the effect of biological membrane transfer modifier, lysophospholipd (LPLs) on the parameters from in vitro rumen simulated fermentation. Commercially available LPLs product (Lipidol$^{TM}$) was supplemented into experimental diets which consisted of orchard grass and concentrate diet (60:40) in different levels (0.1%, 0.3% and 0.5%). Then in vitro rumen simulated fermentation was performed. Although, a declining trend of pH was found in treatments, all pH values were detected in a range relevant to normal rumen fermentation. Gas production, ammonia nitrogen and total VFA production were greatly influenced by the supplementation of LPLs. All parameters were increased along with increased levels of LPLs in diet. As a result, 0.1% of Lipidol$^{TM}$ is recommended based on the determined in vitro rumen fermentative parameters in this study.

• 한국수정란이식학회지
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• 제24권2호
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• pp.121-125
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• 2009

In the present study, effects of concentration of cryoprotectant solutions on the nuclear maturation of vitrifiedthawed bovine oocytes were examined. Also, the developmental capacity of vitrified-thawed immature oocytes following ICSI was investigated. Oocytes were cultured in TCM-199 medium supplemented with 5% FBS at $38^{\circ}$C in 5% $CO_2$ and air. The in vitro maturation rate of vitrified oocytes was 24.5 ${\pm}$ 4.2%. The in vitro maturation rate of vitrified oocytes was lower than that of the control (72.0 ${\pm}$ 3.5%, p<0.05). The in vitro maturation rate of vitrified${\sim}$thawed oocytes incubated in TCM-199 medium supplemented with 1.0${\sim}$5.0 ug CB were 26.7 ${\pm}$ 3.2%, 35.7 ${\pm}$ 3.2%, 54.0 ${\pm}$ 3.0%, 42.5 ${\pm}$ 3.6%, respectively. The in vitro maturation rate (57.0 ${\pm}$ 3.0%) of the vitrified-thawed oocytes treated with 3.0 ${\mu}$g CB for 20 min was the highest of all vitrification groups, although the maturation rate were significantly (p<0.05) lower than those of fresh oocytes. The in vitro maturation rates of the vitrified-thawed (with EDS and EDT) oocytes were 53.8 ${\pm}$ 3.4%, 51.1 ${\pm}$ 3.5%, respectively. This results were lower than the control group (72.0 ${\pm}$ 3.0%). The in vitro developmental rates of the vitrified-thawed oocytes following ICSI were 28.6 ${\pm}$ 4.5%, 25.6 ${\pm}$ 4.3%, respectively. This results were lower than the control group (40.0 ${\pm}$ 4.0%).

• Asian-Australasian Journal of Animal Sciences
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• 제11권1호
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• pp.13-16
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• 1998

A $2{\times}2{\times}4$ factorial study was conducted to examine the possibility of improving estimates of in vitro digestibility, using untreated (UBS) and ammonia-treated (ABS) barley straw, through homogenization of rumen fluid (RF) and by additions of urea (U) and casein (C) as N sources and Xylose + Glucose (XG) as energy sources into artificial saliva. Digestibiltiy of ABS was significantly greater than that of UBS (p < 0.001). There was a significant decrease in digestibility when additions (U, UC, UCXG) were compared with the control (p < 0.001). A 2-way interaction between RF and straw type was significant (p < 0.05) for dry matter digestibility (DMD). Homogenization of RF increased DMD of ABS (p < 0.05) whereas it decreased DMD of UBS (p > 0.05). The study showed that addition of N alone or in combination with energy sources was not better than control, rather the reverse, digestion was inhibited by a combination of U and casein (UC). It was concluded that sufficient N and branched chain fatty acids were supplied in the inoculum from sheep fed high protein grass cubes to support the growth of cellulolytic microbes during in vitro incubation. Further studies are, however, required to explore the potential of homogenization in improving the in vitro method to estimate digestibility of cereal straws.

• Molecular & Cellular Toxicology
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• 제2권2호
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• pp.106-113
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• 2006

Phthalate analogues are a plasticizer and solvent used in industry. Phthalates were classified in the category of "suspected" endocrine disruptors. The purpose of our study was to screen and elucidate the endocrine disrupting activity of seven phthalate analogues. E-screen assay was performed in MCF-7 human breast cancer cells with seven phthalate analogues. In this cell proliferation assay, benzyl butyl phthalate (BBP) and dibutyl phthalate (DBP) showed high estrogenic activity. Their relative proliferation efficiencies (RPE) were 109 and 106%, respectively. In vitro estrogen receptor (ER) binding assay, BBP, di-n-octyl phthalate (DOP) and dinonyl phthalate (DNP) showed weak relative binding affinity (RBA: 0.02%) compared to $17{\beta}-estradiol\;(E2)$ (RBA: 100%). In uterotrophic assay, E2 produced a significant increase, whereas four tested phthalate analogues had potential estrogenic effects in vitro did not increased in uterus weight in immature rats. From these results, we demonstrated that phthalate analogues exhibit weak estrogenic activity in vitro assays at high concentrations. Although phthalates induced an increase in MCF-7 cell proliferation by an estrogenic effect, they could not induce a uterus weight increase in vivo. From these, we may suggest that these phthalate analogues are easily metabolized to inactive forms in vivo. Further investigation in other in vitro and in vivo experimental systems might be required.

• 한국가축번식학회지
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• 제26권2호
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• pp.133-142
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• 2002

본 연구의 목적은 체외성장된 생쥐 preantral follicle 내에 존재하는 난자의 성숙율, 수정율, 배발달율을 조사하는 것이었으며, 그리고 이러한 결과들을 체내 성장된 난자와 비교하는 것이었다. Preantral follicle은 생후 12일령된 생쥐로부터 분리하였으며, 분리된 preantral fo11ic1e은 Transwell-COL membrane insert에서 배양을 실시하였다. 체외성장 및 성숙 후, 제 1극체를 방출한 metaphaseII 난자는 72.5%로서 체내성장된 난자의 70.5%에 비하여 차이가 없는 것으로 나타났다. 그러나 난자직경은 체외성장군 (69.6$\pm$2.l$\mu$m)이 체외성장군(73.3$\pm$3.0$\mu$m)에 비하여 작은 것으로 나타났다. 체외수정율은 체외성장군 (76.5%)이 체내성장군(90.2%)에 비하여 유의하게 낮았지만, 다정자 수정된 난자의 비율은 두 군간에 차이가 없었다. 배반포까지의 배발달율은 체외성장군 (14.4%)이 체내성장군 (56.6%)에 비하여 유의하게 낮았으며, 또한 배반포의 세포수에 있어서도 체외성장군 (39.0$\pm$10.8)이 체내성장군 (60.5$\pm$12.5)에 비하여 유의하게 작은 것으로 나타났다. 체외성장 및 성숙 유래의 2-세포기 수정란을 이식한 결과, 산자 생산을 확인할 수 있었다. 결론적으로 이러한 결과는 체외성장된 난자는 체내성장된 난자와 같은 발생능력을 갖지 못함을 보여주고 있다.

• 대한화장품학회지
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• 제40권1호
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• pp.1-10
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• 2014

현재, 기능성 화장품 성분의 피부흡수에 대한 연구 보고가 부족하며, 인체에서의 in vivo 피부 흡수는 거의 보고된 바 없다. 본 연구에서는 in vivo 라만 분광 피부 측정기를 이용하여 대표적 기능성 화장품 8종 성분의 인체 표피 투과 데이터를 수집 및 분석하고, 이를 in vitro 결과와 비교하여 주요 성분의 피부 흡수 자료를 확보하였다. 그리고 본 연구에 사용한 성분의 피부 투과도 측정한 결과를 보면, in vitro 평가에서 ascorbyl-2-glucoside, retinol, retinyl palmitate, 그리고 kojic acid가 우수한 피부 투과율을 보였으며, in vivo 평가에서는 retinol, vitamin C, 그리고 arbutin 이 우수한 것으로 나타났다. 반면, In vitro 평가와 in vivo 평가에서 가장 우수한 투과율을 보인 성분은 각각 ascorbyl-2-glucoside와 retinol이었다. 이러한 차이점은 in vitro 평가의 경우 무모생쥐 모델의 피부를 모두 관통하는 물질을 평가하였고, 라만 분광 피부 측정기의 경우는 표피의 각질층을 평가하였으므로 물질의 특성에 따라서 표피 및 진피층의 영향에 민감할 수 있기 때문인 것으로 판단된다. 그리고 대부분 물질의 투과 장벽은 각질층이므로 각질층에서 물질 이동을 살펴보는 것이 의미 있다고 생각된다. 결론적으로, 본 연구에서는 라만 분광 피부 측정기를 이용하여 대표적 기능성 화장품 성분의 피부 흡수에 대한 기초적인 자료를 확보하였으며, 따라서 이들 자료는 추후 많은 연구에 활용될 수 있을 것으로 생각된다.

• Journal of Forest and Environmental Science
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• 제35권1호
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• pp.54-60
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• 2019

Plant tissue culture techniques offer quick methods of regeneration of plants of medicinal importance but the survival chances of such plants are always questionable when shifted to the in vivo conditions. The present study enumerates the micromorphological developments in the leaves of in vitro regenerated and field transferred plantlets of Oldenlandia umbellata. The leaves developed in vitro after $4^{th}$ subcultures of multiplication phase and after 6 weeks of field transferred plants were used. Statistically significant differences in the number of stomata, veins, raphides, crystals and trichome density per square mm were observed. The improvements in stomatal apparatus and density (decreased from 41.85 to 32.20), developments in leaf architectural parameters and emergence of defense mechanism through increased numbers of raphides (8 to 15), crystals and trichomes (13.5 to 18.2) proved acclimation of tissue culture raised plantlets from in vitro to the in vivo environments lead to 100 % success in field establishment of the plantlets. The in vitro induced foliar abnormalities (changes in stomata, venation pattern, vein density, trichomes, crystals etc.) were repaired while hardening of plantlets in the greenhouse and finally in the field. The observed micromorphological response of leaves under altered environmental conditions could help in determination of proper stage of field transfer and prediction of survival percentage of in vitro regenerated O. umbellata plantlets.