• Development and Reproduction
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• v.13 no.4
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• pp.217-226
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• 2009

Effects of sera from several fish species on monolayer formation, viability and functions of catfish hepatocytes were investigated to establish a primary hepatocyte culture system for screening endocrine disruptors. Hepatocytes of Korean catfish (Silurus asotus) were attached and formed monolayer using the media supplemented with their own serum or sera from eel and tilapia, but not with fetal bovine serum (FBS). The amount of fish sera (0.5~3%) for monolayer culture of the catfish hepatocytes was less than 1/10 of FBS (5~20%) that is commonly used for primary culture of hepatocytes of other species. The results indicate that FBS can be replaced with sera from some fish species and the fish sera are more effective than FBS in maintaining the shape and functions of the hepatocytes. The primary culture of catfish hepatocytes was maintained monolayer with fish sera for at least 10 days, which makes possible to be used for screening the activities of endocrine disruptors. In conclusion, the primary culture system of hepatocytes with fish sera in the present study could be a useful tool for screening and studying endocrine disruptors.

• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.53-61
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• 1999

The present study was designed to investigate the effects of gonadotrophins on in vitro growth and maturation in mouse preantral follicles. Ovaries were removed from 12-day-old ICR mice. Follicles were dissociated enzymetically in Leibovitz L-15 medium containing 1 mg/$m\ell$ collagenase and 0.2 mg/$m\ell$ DNase I. The follicles were cultured on Transwell-COL membrane inserts in six well cluster dishes for 10 days. The culture medium was $\alpha$MEM medium supplemented with 5% fetal bovine serum and FSH or HMG. After 10 days of growth in vitro, follicles were allowed to mature for 18~20 hr in medium supplemented with 1.5 IU/$m\ell$ hCG. The oocytes were then denuded of their cumulus cells and assessed maturation status. Concentrations of oestradiol and progesterone were measured with a radioimmunoassay. Oocyte diameter was determined with an ocular micrometer. The survival and Metaphase II rates of oocytes were significantly higher in FSH treatment groups than in control group (P＜0.001), but there were no differences among the groups of treated FSH concentration. The survival and Metaphase II rates of oocytes in HMG treatment group (60.9 and 40.6%) were higher than in FSH treatment group (76.6 and 48.2%) and control group (49.2 and 7.1%). The survival and Metaphase II rates of oocytes on both FSH and LH treatment groups were no differences among the ratios of FSH and LH. Diameter of oocyte was no differences among the treatment groups, but smaller than compared to in vivo grown oocyte. Through the entire culture period, secretions of oestradiol and progesterone were significantly less in control group than in HMG and FSH treatment groups. These results suggest that gonadotrophins playa key role in in vitro culture of mouse preantral follicles. Especially, addition of FSH and LH should be more effective than FSH alone.

• Reproductive and Developmental Biology
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• v.35 no.2
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• pp.151-157
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• 2011

Clinical arthritis is typically divided into rheumatoid arthritis (RA) and osteoarthritis (OA). Arthritis-induced muscle weakness is a major problem in aged people, leading to a disturbance of balance during the gait cycle and frequent falls. The purposes of the present study were to confirm fiber type-dependent expression of muscle atrophy markers induced by arthritis and to identify the relationship between clinical signs and expression of muscle atrophy markers. Mice were divided into four experimental groups as follows: (1) negative control (normal), (2) positive control (CFA+acetic acid), (3) RA group (CFA+acetic acid+type II collagen), and (4) aging-induced OA group. DBQA/1J mice (8 weeks of age) were injected with collagen (50 ${\mu}g/kg$), and physiological (body weight) and pathological (arthritis score and paw thickness) parameters were measured once per week. The gastrocnemius muscle from animals in each group was removed, and the expression of muscle atrophy markers (MAFbx and MuRF1) and myosin heavy chain isoforms were analyzed by reverse transcription-polymerase chain reaction. No significant change in body weight occurred between control groups and collagen-induced RA mice at week 10. However, bovine type II collagen induced a dramatic increase in clinical score or paw thickness at week 10 (p<0.01). Concomitantly, the expression of the muscle atrophy marker MAFbx was upregulated in the RA and OA groups (p<0.01). A dramatic reduction in myosin heavy chain (MHC)-$I{\beta}$ was seen in the gastrocnemius muscles from RA and OA mice, while only a slight decrease in MHC-IIb was seen. These results suggest that muscle atrophy gene expression occurred in a fiber type-specific manner in both RA- and OA-induced mice. The present study suggests evidence regarding why different therapeutic interventions are required between RA and OA.

• Analytical Science and Technology
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• v.25 no.3
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• pp.190-196
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• 2012

The human exposure of lead has usually detected the amount of lead in the whole blood, however, this method has a shortcoming to give the information on the short-term exposure to lead. In that sense, it is desirable to estimates the level of lead in plasma to draw the chronic bio-marker of lead exposure even though it is difficult to measure lead of several ng/L. An inductively coupled plasma-mass spectrometry (ICP-MS) method was developed for determining lead in plasma as the chronic bio-marker of lead of workers. To minimize the contamination of lead from the environment, we constructed class 1,000 clean room and compared the amount of floating dust before and after the operation of the clean room. The limit of detection (LOD) and the limit of quantification (LOQ) of lead in fetal bovine serum were 4.3 ng/L and 12.2 ng/L by NIOSH method (statistical calculation method) and 7.0 ng/L and 22.1 ng/L by signal/noise ratio, respectively. The accuracy was in a range of 92.3-101.3%, and the precision of the assay was less than 4% in the samples spiked in the concentration of 20 ng/L and 2,000 ng/L. The method was simple, reproducible and sensitive enough to permit reliable analysis of lead to the ng/L level in plasma and/or serum. The method was also useful for the biological monitoring of chronic exposure to lead.

• Applied Microscopy
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• v.33 no.4
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• pp.261-266
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• 2003

The growth patterns of primary culture of bovine brain microvessel endothelial cells (BBMECs) were studied using electron microscopy when grown on $3.0{\mu}m$ and $0.4{\mu}m$ pore Transwell. The capillary fragments and isolated endothelial cells grew on collagen coated culture plate and Transwell membrane. The BBMECs grew only on the upper surface of membrane of $0.4{\mu}m$. But BBMECs on $3.0{\mu}m$ pore membrane migrated through the pore and grew on the opposite side of the membrane. In summary, BBMECs isolated by enzyme digestion could migrate through $3.0{\mu}m$ pore membrane but not through $0.4{\mu}m$ pore membrane. So $0.4{\mu}m$ pore membrane instead of $3{\mu}m$ pore membrane should be used for drug transport experiment or transendothelial electrical resistance measurement.

• Journal of Veterinary Clinics
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• v.19 no.2
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• pp.228-235
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• 2002

Mastitis is the most costly disease results in lost milk production, decreased milk quality, milk discard, early culling of cows, drug costs and labor costs in dairy cow. Until now, a antibiotic administration at the end of lactation, dry cow therapy has been known the most effective and widely used mastitis control method. However, dry cow therapy do not control a new infection in the late dry and prepartum period because dry cow products have only persistent activity in the early dry period. Therefore, this study was conducted to evaluate clinical effect of sustained released biodegradable cephalexin microsphere using PLGA in bovine mastitis control during dry period. PLGA has been approved as controlled drug release system because of non-toxic, non-tissue reactive and bioerodible characteristics. This study revealed that cephalexin microsphere had a spherical shape with characteristic porous structure on the surface. Also, in vitro drug release studies are clearly observed that the release rate of cephalexin from PLGA microsphere decrease during the first 21 days after initial burst and then increase again between 3 and 4 weeks showing pulsatile releasing pattern. On the other hand, as tried in field the new infection rate, cure rate and mean SCC after parturition in cephalexin microsphere infused group were significantly differenced as compared to the control group. Accordingly, a sustained release of cephalexin from a biodegradable microsphere could make dry cow therapy more efficiently by preventing a new infection and decreasing the number of existing infection of mammary gland during dry period.

• Journal of Veterinary Clinics
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• v.32 no.3
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• pp.219-223
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• 2015

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis or Johne's disease, an intestinal granulomatous infection in domestic and wild animals. The study aimed to develop and evaluate a panel of multiplex quantitative real-time polymerase chain reaction (mqPCR) assay for simultaneous detection of three MAP-specific genes (IS900, F57 and ISMAP02 genes). The analytic sensitivity (i.e., limit of detection, expressed as cells per 1 ml) was 150 for IS900, 1500 for F57, and 50 for ISMAP02. The specificity of the method was determined by testing 152 bovine fecal samples. Based on the test, it showed that the assay simultaneously detected the target genes in short period of time and at lower cost compared to laboratory routine tests. The test agreement between the assay and routine test was 94%. The discrepancy in the results was due to samples that were tested positive by the panel but negative by the routine tests, suggesting that the assay has higher sensitivity than the routine tests. In conclusion, the mqPCR assay could be a rapid and accurate testing tool for investigating paratuberculosis or Johne's disease cases in domestic and wild animals.

• Toxicological Research
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• v.22 no.4
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• pp.417-422
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• 2006

Cytogenetic and hematological analysis was performed in peripheral blood obtained from cattle bred in the high background radiation areas (HBRA, Goesan-gun, Cheongwon-gun, Boeun-gun) and a control area. The frequencies of gamma-ray induced micronuclei (MN) in the cytokinesis-blocked (CB) lymphocytes at several doses were measured in 3 cattle. An estimated dose of radiation was calculated by a best fitting linear-quadratic model based on the radiation-induced MN formation from the bovine lymphocytes exposed in vitro to radiation over the range from 0 mGy to 1,969 mGy. The measurements performed after irradiation showed dose-related increases in the MN frequency in each donors. The results were analyzed using a linear-quadratic model with a line of best fit of $y=(0.0583{\pm}0.0137)D+(0.0366{\pm}0.0081)D^2+(0.0093{\pm}0.0015)$ (y=number of MN/CB cells and D=irradiation dose in Gy). MN rates per 1,000 CB lymphocytes of cattle from the Goesan-gun, Cheongwon-gun, Boeun-gun and the control area were $6.50{\pm}2.72,\;9.00{\pm}4.50,\;10.89{\pm}4.23\;and\;9.60{\pm}4.70$, respectively. The MN frequencies of CB lymphocytes from cattle bred in 4 areas mean that the values are within the background variation in this experiment. The MN frequencies and hematological values were similar regardless of whether the cattle were bred in the HBRA or the control area.

• Korean Journal of Veterinary Research
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• v.29 no.1
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• pp.69-73
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• 1989

In vitro antifungal susceptibility test was carried out on 53 strains of Candida spp isolated from milk of dairy cows with subclinical mastitis and teat cups of milking machines, Nystatin, clotrimazole, miconazole, econazole, 5-fluorocytosine, cycloheximide, haloprogin and griseofulvin were tested by the agar dilution method. The 84.8% to 98.2% of Candida strains were inhibited by clotrimazole, econazole and miconazole at $${\leq_-}25{\mu}g/ml$$, and clotrimazole was most active. Interspecies differences of antifungal susceptibility were recognized and these were as follows. C albicans was most sensitive to clotrimazole (GM-MIC, $5.49{\mu}g/ml$) followed by 5-fluorocytosine, econazole and miconazole. C pseudotropicalis and C guilliermondii were notably sensitive to haloprogin, clotrimazole, miconazole, cconazole, 5-fluorocytosine, and haloprogin (GM-MIC, $0.17{\sim}0.19{\mu}g/ml$) was most active. C krusei was most sensitive to cycloheximide (GM-MIC, $0.54{\mu}g/ml$) followed by clotrimazole, haloprogin, miconazole and econazole. C parapsilosis was somewhat sensitive to econazole, cycloheximide, clotrimazole, and econazole (GM-MIC, $7.26{\mu}g/ml$) was most active. C tropicalis showed very low sensitivity to all tested drugs (GM-MIC, $${\geq_-}20.32{\mu}g/ml$$).

• Korean Journal of Veterinary Research
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• v.36 no.3
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• pp.665-680
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• 1996

Bovine Pneumonic Pasteurellosis는 수송열(輸送熱)로 일반적으로 알려져 있는 질병으로서, 여러가지 요인의 복합적(複合的)인 작용에 의해 발병하는 것으로 알려져 있으나, Pasteurella haemolytica A1이 가장 주요(主要)한 인자(因子)로 밝혀져 있다. P haemolytica A1은 leukotoxin(LKT), lipopolysaccharide(LPS), capsular polysaccharide 등 여러가지의 병원성인자(病原性因子)을 생성한다. 이들 인자중 LKT가 가장 중요한 병원성인자로 밝혀져 있다. 이에 본 실험은 P haemolytical A1의 LKT 유전자를 Bacillus subtilis에서 발현(發現)시킴으로서 LPS에 오염(汚染)되지 않은 LKT을 대량으로 생산할 목적으로 실시되었다. 실험의 첫 단계(段階)로서 pLKT52 plasmid을 Sau3 A1의 제한효소을 이용하여 부분소화(部分消化)시킨 후 이 부분 소화(消化)된 유전자들로부터 3~5kb 크기의 유전자들을 순수분리하여 pUC18와 결합시킨 후 E coli NM522에 형질전환(形質轉換)시켰다. 이때 형질전환된 균주들은 LKT에 대한 단크론 항체인 MAb601을 이용하여 colony blot 법에 의해서 LKT 유전자 보유 및 발현여부(發現與否)을 조사하였다. 이들 양성 clone들은 제한효소분석(制限酵素分析), 염기서열분석(鹽基序列分析) 및 Western blot 등에 의해서 재확인(再確認)하였다. 총 9개의 양성 clone중 위의 방법에 의해서 한 clone을 선택(選擇)하여 lktCA insert를 재분리하여 shuttle vector에 subcloning 하였다. Subcloning된 LKT 유전자들은 shuttle vector의 종류(種類)(pHPS9, p602/20, pHPS9-Sac)와 각기(各其) 다른 종류(種類)의 B subtilis(spoO12A, BR121, WB3O, Raj1105) 숙주내(宿主內)에서 발현정도를 Western blot 법에 의해서 비교(比較)하였다. 이때 최적발현조건(最適發現條件)은 p602/20와 pBL1의 dual plasmid system을 이용하여 B subtilis spoO12A에서 2시간동안 IPTG로 발현을 유도(誘導)하는 것이었다. B subtilis에서 발현된 LKT을 visual 법과 neutral red uptake 법을 이용하여 소 폐포(肺胞) 대식구(大食求)에 대한 biological activity를 확인하였다. 발현된 LKT에 대한 LPS 오염은 LKT을 SDS-PAGE 후 silver stain에 의해서 확인하였다. 본 실험을 통해서 볼 때에 lktCA 유전자를 보유(保有)하고 있는 p602/20는 B subtilis에서 매우 불안정(不安定)하였고, 발현된 LKT는 세균자체(細菌自體)에서 생성되는 protease들에 의해서 파괴(破壞)됨으로서 농도(濃度)가 매우 낮았다. 이러한 문제점들은 다음 단계(段階)의 실험에서 해결되어야할 문제들이다.