• Asian-Australasian Journal of Animal Sciences
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• 제12권1호
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• pp.22-27
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• 1999

Bovine oocytes with compact and complete cumulus cells were cultured in 6 groups for up to 24h in TCM199 buffered with 25 mmol/1 HEPES and supplemented with 10% FCS, 1 mg/ml $17{\beta}$-estradiol, 20 IU/ml hCG. Half of the oocytes at each group cultured in the presence of $25{\mu}g/ml$ cycloheximide at different times during maturation (0, 6, 12, 18, 20, 22 h) were fixed at 24 h of maturation to examine the nuclear progression. The rests of them were inseminated with frozen-thawed spermatozoa in medium BO with 10 mg/ml BSA and 10 mg/ml heparin and fixed after additional 18-20 h culture to evaluate the sperm head transformation. When a protein synthesis inhibitor was added at the onset of the maturation, the oocytes were prevented to proceed GVBD. A few of the oocytes (16%) were able to be penetrated and sperm head decondensation was inhibited either. Addition of cycloheximide after 6-12 h of culture resulted in an increasing percentage of GVBCD (more than 80%), but the oocytes became arrested in M-I (69.2%). More than half of the oocytes was penetrated with a decondensing sperm head. Formation of male pronucleus was first obtained at 12 h of culture in the presence of cycloheximide. When cycloheximide was added from 18 h of culture onwards, nuclear progression to M-II was increasingly restored (80.4-85.5%). The proportion of male and female pronuclear formation increased from 17.9% to 46.2%. It is concluded that protein synthesis is necessary not only for GVBD and development from M-I to M-II, but also for sperm head decendensation and male pronuclear formation in bovine oocytes.

• 한국수정란이식학회지
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• 제17권2호
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• pp.123-128
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• 2002

The present study aimed at determining the effective dose of Folltropin, a follicle timulating hormone (FSH), on superovulation in indigenous cows of Bangladesh. Fifteen regularly cycling 5～7 years old dry cows, weighing 200～250 kg with 2.5～3.0 body condition scores (BCS) were divided into three groups (n＝5). Individual groups were superovulated with 100, 200 or 300 mg of Folltropin per animal. The superovulation treatment was initiated at Day 10 or Day 11 of the estrous cycle (Day 0＝day of estrus). Alfaprostol (6 mg) was injected to each cow 72 h after the initiation of superovulation treatment to induce eestrus. After confirming standing estrus, the cows were inseminated 2～3 times, 12 h apart, depending on the duration of estrus. At Day 6 or Day 7, individual horns of the uterus were flushed with 150～200 $m\ell$ of phosphate buffered saline supplemented with BSA (0.2％), penicillin (100 IU／$m\ell$) and streptomycin (100 $\mu\textrm{g}$／$m\ell$) using a two-way foley catheter. The embryos were concentrated, removing the excess medium through an embryo filter, and identified under a stereomicroscope. The identified embryos were collected, washed four times, evaluated and graded as excellent, good, fair or poor. The excellent, good and fair embryos were considered as transferable quality embryos. The mean (range). numbers of embryos collected vs. transferable quality embryos far 100, 200 and 300 mg of Folltropin were 4.5 (1～10) vs. 3.5 (1～8); 2.5 (1～4) vs. 1 (0～2) and 0.0 (0～0) vs. 0.0 (0～0), respectively, Folltropin at a dose of 100 or 200 mg produced suitable ovarian stimulation for superovulation in indigenous zebu cows of Bangladesh. A dose of 300 mg or more Folltropin consistently caused preovulatory corpora lutea formation in the ovaries and resulted in zero embryo recovery.

• Journal of Nutrition and Health
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• 제28권8호
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• pp.737-748
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• 1995

Osteoblast(OBL) cells were isolated from ICR Swiss neonatal mouse calvarial tissues and cultured in a CO2 incubator with minimum essential medium (MEM) containing 0.25g BSA. The cells were cultured for 7 days and were treated with bovine parathyroid hormone (bPTH, 1-34) and calcitonin(CT). Enzyme activities related to mineral metabolism and other biochemical actions within the bone cells including protein phosphorylation were investigated. In other experiments using cultured calvarial bone tissues, hormones were treated for 24, 48, 72 or 96 hours. The activities of $\beta$-glucuronidase enzymes involved in bone collagen synthesis and mineral deposits were increased by 8% with bPTH and were inhibited with CT treatment, while those were 67% increase treated with bPTH and CT together. On the other hand, alkaline phophatase(AP) activities were inhibited by PTH hormone at all the time courses observed. Protein phosphorylation reaction in OBL was mediated by bPTH, cAMP and ionized Ca. Phosphorylation was observed in different cell fractions including homogenate, membrane and cytosol. The number of proteins phosphorylated by PTH, cAMP, and Ca were 10, 5, and 9, respectively. Most of the protein kinases(PKs) were existed in cytosolic compartment. In membrane fractions, two bPTH-dependent-PKs (70K, 50K Da) were observed of which 70K Da protein was also Ca-dependent. Most of the cAMP-dependent PKs were regulated via bPTH. 70K, 50K, 5K, 19K, 16K, 10.5K phosphoproteins regulated by Ca share the same pathways as those by bPTH-dependent proteins. Ca seems to regulate PK activities differently from cAMP.

• Asian-Australasian Journal of Animal Sciences
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• 제8권2호
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• pp.123-127
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• 1995

In vitro embryo production (IVP) is affected by various factors during in vitro maturation, fertilization, and development. In this experiment, the effect of ovary type, quality of follicular oocyte, medium used for fertilization, presence of hormone in medium, sperm concentration on in vitro maturation and fertilization were examined for effective IVP. In vitro maturation was carried out using TCM-199 supplemented with 15% FCS and hormones in 5% $CO_2$ incubator for 24h. In vitro fertilization was performed with frozen-thawed sperm in modified mTALP medium containing 0.3% BSA, $10{\mu}g/ml$ heparin, and 5mM/ml caffeine for 24h. The fertilized embryos were co-cultured on monolayer of cumulus cells in TCM-199. When oocytes were collected from functionally active and inactive ovaries, maturation rate was 76.9 and 7.7%, respectively. When oocytes were classified morphologically to good and poor grades, maturation rate was 75 and 58.8%, respectively. FSH + LH + $E_2$ (86.4%) showed higher maturation rate than control (53.0%) and FSH (73%). The fertilization rate was 28.2, 100 and 91.7% in $1.6{\times}10^5$, $5.0{\times}10^5$ and $10.0{\times}10^5$ sperm concentration per ml. When oocytes were fertilized in mTALP and BO media, fertilization and cleavage rates of oocytes in mTALP were higher (84.3 and 56.9%) than those (67.4 and 23.3%) in BO medium. In this experiment, in vitro maturation, fertilization and development of oocytes were affected by type of ovary, grade of oocyte, hormones, sperm concentration and fertilization medium.

• Asian-Australasian Journal of Animal Sciences
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• 제8권4호
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• pp.317-320
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• 1995

The effect of co-culture with cumulus cells and granulosa cells during maturation and development on in vitro developmental potency of follicular oocytes was examined. TCM-199 supplemented with 15% FCS and hormones was used as maturation medium. Sperm from frozen semen was capacitated in modified mTALP medium containing 0.3% BSA, $10{\mu}g/ml$ heparin and 5 mM/ml caffeine. The fertilized embryos were co-cultured on monolayer of cumulus cells or granulosa cells in TCM-199. The embryo co-cultured with cumulus cells showed higher percentage of embryo developed to morula and blastocyst (73.3%) than the embryo co-cultured without cumulus cells (30.8%). The percentage of oocytes developed to morula and blastocyst among cleaved oocytes was significantly (p < 0.05) higher in the oocytes co-cultured with cumulus cells during development (62.4%) than in the oocytes co-cultured with granulosa cells during maturation and with cumulus cells during development (52.3%), and in the oocytes co-cultured with granulose cells during development (52.8%). The results of this study indicate that co-culture of in vitro fertilized embryos with cumulus cells in the development medium increased the rate of embryos developed to morula and blastocyst among cleaved oocytes.

• Asian-Australasian Journal of Animal Sciences
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• 제16권9호
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• pp.1320-1325
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• 2003

The purpose of present study was to assess the effect of polyunsaturated fatty acids (PUFA) on the immune responses of laying hens. Three hundred and sixty hens at the age of 60 weeks were randomly assigned to ten diets, which contained no oil (CK), 1%, 3%, 5% fish oil (FO); 2%, 4%, 6% linseed oil (LO) and 2%, 4%, 6% corn oil (CO). After 5 weeks of feeding experimental diets, humoral and cellular immune responses were assayed. Laying hens were injected with Sheep Red Blood Cell (SRBC) and Bovine Serum Albumin (BSA) and antibody titers, which were measured on d6, d10, d14 after primary challenge and on d5, d9, d13 after secondary challenge. Concanavalin (ConA) and lipopolysaccharide (LPS) -stimulated proliferation of peripheral blood and spleen lymphocytes were assessed by [$^3$H] thymidine incorporation at the week age of 5 and 10, respectively. The results showed that antibody titers in FO-fed and LO-fed laying hens were higher than that in laying hens fed CO. The proliferation response to ConA was lower in laying hens that fed oils rich in n-3 fatty acids than that in laying hens fed CO. Higher level n-3 fatty acids can improve immune functions of laying hens. In conclusion, dietary fat source and level had a significant impact on immune responses of laying hens.

• Asian-Australasian Journal of Animal Sciences
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• 제27권3호
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• pp.414-421
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• 2014

This study was conducted to determine the effects of saturated long-chain fatty acids (LCFA) on cell proliferation and triacylglycerol (TAG) content, as well as mRNA expression of ${\alpha}s1$-casein (CSN1S1) and genes associated with lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows, and were passaged twice. Then cells were cultured with different levels of palmitate or stearate (0, 200, 300, 400, 500, and 600 ${\mu}M$) for 48 h and fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L). The results showed that cell proliferation tended to be increased quadratically with increasing addition of stearate. Treatments with palmitate or stearate induced an increase in TAG contents at 0 to 600 ${\mu}M$ in a concentration-dependent manner, and the addition of 600 ${\mu}M$ was less effective in improving TAG accumulation. The expression of acetyl-coenzyme A carboxylase alpha, fatty acid synthase and fatty acid-binding protein 3 was inhibited when palmitate or stearate were added in culture medium, whereas cluster of differentiation 36 and CSN1S1 mRNA abundance was increased in a concentration-dependent manner. The mRNA expressions of peroxisome proliferator-activated receptor gamma, mammalian target of rapamycin and signal transducer and activator of transcription 5 with palmitate or stearate had no significant differences relative to the control. These results implied that certain concentrations of saturated LCFA could stimulate cell proliferation and the accumulation of TAG, whereas a reduction may occur with the addition of an overdose of saturated LCFA. Saturated LCFA could up-regulate CSN1S1 mRNA abundance, but further studies are necessary to elucidate the mechanism for regulating milk fat and protein synthesis.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2002년도 추계국제학술대회
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• pp.169-169
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• 2002

The mobilizable amount of transition metals is a fraction of the total amount of the metal from urban particulate matter. Although the fraction is small, some metals (Fe, Cu) are the major participants in a reaction that generates reactive oxygen species (ROS), which can damage various biomolecules. Damaging effects of the metals can be measured by the single strand breakage (SSB) of X174 RFI DNA or the carbonyl formation of protein. In another study, we have shown that more metals are mobilized by PM2.5 than by PM10 in general. DNA SSB of >20% for PM2.5 and >15% for PM10 was observed in the presence of chelator (EDTA or citrate)/reductant (ascorbate), compared to the control (<3%) only with the chelator. The carbonyl formation by both PMs was very similar in the presence of the chelator, regardless of the kind of proteins. Compared to the control in the absence of chelator/reductant, 3.3 times and 4.9 times more carbonyl formation for PM2.5 and PM10, respectively, was obtained with BSA in the presence of chelator/reductant, showing that PM10 induced 33% more damage than PM2.5. However, 4.8 times and 1.9 times more carbonyl formation for PM2.5 and PM10, respectively, was observed with lysozyme in the presence of chelator/reductant, showing that PM2.5 induced 250% more damage than PM10. Although different proteins showed different sensitivities toward ROS, all these results indicate that the degrees of the oxidation of or damage to the biomolecules by the mobilized metals were higher with PM2.5 than with PM10. Therefore, it is expected that more metals mobilized from PM2.5 than from PM10, more damage to the biomolecules by PM2.5 than by PM10. We suggest that when the toxicity of the dust particle is considered, the particle size as well as the mobilizable fraction of the metal should be considered in place of the total amounts.

• 약학회지
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• 제58권5호
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• pp.294-299
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• 2014

In this study, we investigate anti-allergic and anti-inflammatory effects of Leonurus sibiricus seed (LSS) extract in basophilic leukemia RBL-2H3 cells. To identify anti-allergic actions of LSS, the degranulation was evaluated in IgE and DNP-BSA stimulated RBL-2H3 cells. At the concentration of $100{\mu}g/ml$ of methanol (MeOH) extract and Methylene chloride (MC) and Ethyl acetate (EtOAc) fractions, the degranulation was significantly inhibited 16.7%, 16.7% and 27.9% respectively. And then, to assess anti-inflammatory effects of LSS, IL-4 and IL-13 mRNA level were detected in PMA/ionomycin (PI)-induced RBL-2H3 cells and cell proliferation and IL-4 mRNA level in isolated splenocytes from Balb/c mice. LSS MeOH extract and MC and EtOAc fractions significantly decreased the level of IL-4 and IL-13 mRNA in PI-induced RBL-2H3 cells and showed inhibitory effects on cell proliferation and expression of IL-4 mRNA level in mouse splenocytes. Taken together, these results suggest that LSS has potential anti-allergic and anti-inflammatory effects and EtOAc fraction is the most effective in regulating immune responses.

• 대한임상검사과학회지
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• 제49권3호
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• pp.220-226
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• 2017

도파민과 세로토닌은 신경전달물질 및 호르몬으로서 미량이지만 체내에서 중요한 기능을 한다. 검체는 혈청과 24시간 뇨를 쓰며, 주로 HPLC-MS를 이용하여 검사한다. 본 연구에서는 항원-항체반응을 이용한 경쟁적 ELISA를 통해 도파민과 세로토닌을 검출하고자 하였다. $5{\mu}g/mL$ BSA conjugate를 96 well 표면에 고정시킨 뒤 각각 농도를 다르게 한 신경전달물질과 1차 항체를 넣어 반응시키고 HRP가 결합된 2차 항체와 TMB를 처리하여 흡광도를 측정하였다. 측정 결과를 토대로 회귀식과 $R^2$값을 구하여 신경전달물질에 대한 항체의 민감도와 신경전달물질 농도와 흡광도 사이의 상관관계를 판단하였다. 도파민은 1차 항체를 6,000배, 7,000배로 희석했을 때 $R^2=0.91$로 신경전달물질 농도와 흡광도의 상관관계가 가장 높게 나타났고, 세로토닌은 3,000배와 6,000배를 제외한 모든 희석배수에서 $R^2{\geq}0.90$의 높은 상관관계를 나타냈다. 도파민과 세로토닌 모두 $1.0{\times}10^{-7}M$ 이하의 저농도에서는 효과적으로 검출되지 않았기 때문에 참고치가 이보다 낮은 혈청 도파민 검출은 HPLC-MS 이용이 필요해 보이지만, 24시간 뇨 도파민과 혈청 및 24시간 뇨 세로토닌의 검출에는 competitive ELISA가 효과적일 것이라 사료된다. 보다 낮은 농도의 신경전달물질도 정확하게 검출하기 위해서는 추가 연구가 필요하다.