• Journal of Embryo Transfer
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• v.13 no.1
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• pp.11-19
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• 1998

This study was to determine the effect of ionomycin and 6-dimethylaminopurine (6-DMAP) and/or elcetrical stimulation on the oocyte activation and production of rabbit nuclear transplant embryos. The oocytes were collected from the oviduct of superovulated rabbits at 14 h post hCG injection and cultured in TCM-199 containing 10% FBS until 19 h post hCG injection. To determine the optimum concentration and exposure time of 6-DMAP, some oocytes were activated with 5 $\mu$M ionomycin for 5 min and then in 2.0 mM 6-DMAP for 0.5 to 3.0 h, or in 1.0 to 3.0 mM 6-DMAP for 2.0 h. Other control oocytes were stimulated electrically(3X, 1.25 kV/cm, 60 $\mu$sec) in 0.3 M mannitol solution supplemented with 100 $\mu$M CaCl$_2$ and MgCl$_2$. The nuclear donor embryos of 8-cell stage were synchronized to G$_1$ phase of 16-cell stage, and the recipient cytoplasms were obtained from removal of the first polar body and a portion of membrane bound cytoplasm of the oocytes collected at 15 h post hCG injection. A separated blastomere was injected into the perivitelline space of the enucleated oocytes. The oocytes injected with nucleus were cultured until 19 h post hCG and then electrofused and activated by electrical stimulation with or without ionomycin and 6-DMAP. These nuclear transplant embryos were cultured in TCM-199 containing 10% FBS in 39˚C, 5% CO2 incubator for 120 h. For the oncytes activated parthenogenetically with electrical stimulation with or with-out ionomycin and the various concentration of exposure time of 6-DMAP, the highest cleavage(92.3%) and development to blastocyst stage(41.0%) were resulted from the oocytes activated by ionomycin and 2.0 mM 6-DMAP for 2.0 h, which were found to be significantly(P<0.05) higher than the cleavage(45.2%) and developement to blastocyst stage(14.3%) from the oocytes activated with electrical stimulation. The significantly(P<0.05) more oocytes(71.4%) developed to 4 cell stage at 24 h post activation by ionomycin and 6-DMAP than those by electrical stimulation(18.9%). For the nuclear transplant embryos, the cleavage rate was similarly high in oocyte activation by electrical stimulation with(79.4%) or without ionomycin and 6-DMAP(70.5%). However, the embryo development to blastocyst stage was significantly(P<0.05) higher in oocyte activation by electrical stimulation with ionomycin and 6-DMAP(44.4%) than by electrical stimulation only(25.0%). The significantly(P<0.05) more nuclear transplant embryos(45.6%) developed to 4 cell stage at 18 h post activation by electrical stimulation with ionomycin and 6-DMAP than those by electrical stimulation only(10.6%). These results indicated that the supplemental oocyte activation by ionomycin and 6-DMAP with electrical stimulation enhanced and accelerated the preimplanted in vitro development of the rabbit nuclear transplant embryos.

• Journal of Sericultural and Entomological Science
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• v.47 no.1
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• pp.12-17
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• 2005

To develop technique for the production of P. tenuipes stromata on a large scale, the infection of P. tenuipes and the growth of stroma were investigated by silkworm (Bombyx mori) variety. Also, studied about biological activites of fruiting body formed on silkworm. Infection rate of the 5th instar larvae of the silkworm with P. tenuipes was the highest in Yangwonjam, followed by Hachojam, Baegokjam and Chilbojam in that order. Also, as the inoculation times was increased, infection rate tended to be raised. The rate of fruiting body formation of the silkworm pupae infected with P. tenuipes was the highest in Baegokjam, followed by Yangwonjam and Chilbojam in the order. But, actually the fruiting body formation of the 5th instar larvae of the silkworm tested was good in Chilbojam, followed by Yangwonjam and Baegokjam in that order in 3 times spraying inoculation. The fruiting bodies of Yangwonjam and Chilbojam infected with P. tenuipes had high amount of Mannitol, but Baegokjam and Hachojam had high concentration of Glucose on a dry weight basis. The mean content of total amino acid in the fruiting bodies of P. tenuipes was 1.03 ${\mu}mole/g$. The distribution rate of amino acid components decreased in the order of Arginine (12.2%)>Glycine (10.5%)> Proline (9.6)>Tyrosine (8.9%)>Serine>Leucine>Threonine. The most abundant amino acid in the fruiting bodies of the Baegokjam, Chilbojam and Hachojam infected with P. tenuipes was arginine, while Yangwonjam was Glycine. The most abundant fatty acid in P.tenuipes was Oleic acid on a dry weight basis. The unsaturated fatty acids such as Oleic acid, Linoleic acid and Linolenic acid accounted for more than 78% of the total fatty acids.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.13 no.4
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• pp.163-171
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• 1980

Recently, Bacillus has been identified as one of food poisoning bacteria especially in products of cereal foods in foreign countries. Therefore, the quantitative distribution of Bacillus cereus in market foods, its physiological characteristics, growth rate by temperature and heat resistance of its spore were examined. Thirty two samples of cooked rice, 20 samples of kimbab(cooked rice rolled with laver), 23 samples of rice cake, 13 samples of rice ana 13 samples of barley were collected from restaurents and food stores in Busan, Korea during the period from May to November in 1980. Forty samples of 101 samples submitted to the test appeared positive for Bacillus cereus showing abut $40\%$ in detection ratio. Detection ratio of Bacillus cereus was higher than $50\%$ in barley and rice, and about $30\%$ in rice products. Average Bacillus cereus content of in the samples was $2.6\times10^6/g$ in cooked rice, $2.3\times10^6/g$in kimbab, $4.9\times10^4/g$ in rice cake while that in rice and barley was about $10^3/g$. The result of biochemical tests of the bacterium was $100\%$ positive in catalase, egg yolk reaction, gelatin hydrolysis and glucose fermentation, $100\%$ negative in xylose, arabinose and mannitol oxidation, about $90\%$ positive in acetoin production, $80.0\%$ positive in nitrate reduction and citrate utilization and $55.0\%$ positive in starch hydrolysis test. Isolation ratio of Bacillus ceresus which showed haemolysis positive and starch hydrolysis negative results, was about $38\%$ in 40 strains examined. It is known that those strains has a close relation to food poisoning accident. Growth rate and generation time of Bacillus cereus isolated from the cooked rice were $0.34hr^{-1},\;2.02hr\;at\;20^{\circ}C,\;0.73hr^{-1},\;0.95hr\;at\;30^{\circ}C\;and\;0.49hr^{-1},\;1.44\;hr\;at\;40^{\circ}C$ respectively. Heat resistance value of Bacillus cereus spores suspended in phosphate buffer solution was $D_{90}=29.0min,\;D_{95}=8.7min,\;D_{98}=3.7\;min\;and\;D_{101}=2.3\;min(z=10.5)$.

• Korean Journal of Microbiology
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• v.42 no.1
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• pp.40-46
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• 2006

Galactose joined to glucose by a $\beta(1\rightarrow4)$ glycosidic bond makes lactose and this disaccharide is rich in milk. It is known that lacotse is hydrolyzed to each monomeric sugar by either lactase in human or $\beta-galactosidase$ in bacteria. Ingestion of milk by lactase-deficient persons causes a temporary diarrhea and subsequent chronic diarrhea results in colitis with chronic inflammation. We isolated a $\beta-galactosidase$ producing psycrotolerant strain AS-20 from near cattle shed and investigated the growth at various temperature conditions. Whereas Escherichia coli strains did not grow at $10^{\circ}C$, the AS-20 strain could grow well at this low temperature and showed optimal growth at $30^{\circ}C$. The isolated strain was identified as 97% Hafnia alvei by biochemical properties. This strain could ferment glucose, lacotse, maltose, mannitol, xylose, ONPG, rhamanose and L-arabinose, and decarboxylate lysin and ornithine. To confirm the identity of isolated strain we amplified 16S rDNA by PCR and searched similarity of the 1426 bp DNA sequcence with Genbank database. The strain AS-20 showed 99% similarity with Hafnia alvei. The activity of $\beta-galactosidase$ was 1.5 times higher when the cell was grown at 10 or $20^{\circ}C$ than at $30^{\circ}C$. The highest enzyme activity of AS-20 was also much higher than that of E. coli, which was grown at $30^{\circ}C$.

• Journal of fish pathology
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• v.20 no.3
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• pp.249-255
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• 2007

We investigated the phenotypic characteristics by using API20E, APIZYM and determined minimum inhibitory concentrations (MICs) of 7 antibiotics in motile aeromonads isolated from freshwater fishes in Korea and Japan, and 4 American Type Culture Collection (ATCC) strains. All isolates (n=7) were identified as motile Aeromonas species according to API20E test. Lysine decarboxylase activity and acid production from 4 different carbohydrates including mannitol, rhamnose, amygdalin and arabinose were observed in various strains. In enzymatic activities by APIZYM, all isolates showed negative reactions in valine and cystine arylamidases, α-chymotrypsin, α-galactosidase, β-glucuronidase, α-glucosidase, α-mannosidase and α-fucosidase. Although the intensities of each enzymatic activity were diverse in alkaline phosphatase, esterase-lipase, leucine arylamidase, β-galactosidase and N-acetyl-β-glucosaminidase, all isolates showed positive reactions. All isolates were resistant to ampicillin sodium (MIC>100㎍/ml), but sensitive to chloramphenicol (MIC≤1.6㎍/ml). However, recently isolated strains (AC9804, AC0202 and GMA0361) were commonly resistant to tetracycline (MIC=50㎍/ml). Furthermore, AC9804 was resistant to oxolinic acid (MIC=12.5㎍/ml). GMA0361 was resistant to kanamycin sulfate (MIC>100㎍/ml) and streptomycin sulfate (MIC>100 ㎍/ml).

• Korean Journal of Microbiology
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• v.49 no.1
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• pp.90-94
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• 2013

A new Bacillus strain designated as MP56 producing antimicrobial substance has been isolated from the mud flat of Korea. The strain MP56 was found to exhibit broad spectrum of antimicrobial activity against Gram-positive pathogenic microorganisms and MDR (multi drug resistant) strains. The 16S rRNA sequence revealed that the MP56 was closely related to Bacillus subtilis with 99.93% homology. The optimal medium composition for production of antimicrobial substance in the B. subtilis MP56 were 1% mannitol, 1% oat meal, 0.01% $CaCl_2$. Antimicrobial activity of the culture broth against different pathogenic strains was assessed using the antimicrobial spectrum. The result suggests that Bacillus strain MP56 produces high quality antimicrobial substance that might be very useful to control varieties of pathogenic microbial growth.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.319-327
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• 2011

This study investigated whether the addition of porcine sperm cytosolic factor (SCF) at fusion/activation affects in vitro development of porcine parthenogenetic(PA) and nuclear transfer (NT) embryos. To determine the optimum concentration of SCF, control group of oocytes was activated with 0.3M mannitol (1.0 mM $CaCl_2{\cdot}2H_2O$), other three groups of oocytes were parthenogentically activated with the fusion medium (0.1mM $CaCl_2{\cdot}2H_2O$) supplemented with 100, 200 or 300 ${\mu}$g/ml SCF, respectively. Matured oocytes were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}$sec. The activated embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. Oocytes activated in the presence of SCF showed a significantly higher blastocyst rate than control (p<0.05). Apoptosis rate was significantly lower in 100 ${\mu}$g/ml SCF group than other groups (p<0.05). Cdc2 kinase activity in control and SCF treatment group of oocytes was determined using MESACUP cdc2 kinase assay kit at 1, 5, 10, 15, 30, 45 and 60 min after activation. Cdc2 kinase activity was significantly decreased (p<0.05) in SCF group than MII oocytes or control within 5 min. For NT embryo production, reconstructed oocytes were fused in the fusion medium supplemented with 0.1 mM $CaCl_2{\cdot}2H_2O$ (T1), 1.0 mM $CaCl_2{\cdot}2H_2O$ (T2) and 0.1 mM $CaCl_2{\cdot}2H_2O$ with 100 ${\mu}$g/ml SCF (T3). Fused embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. Developmental rate to blastocyst stage was significantly higher in T3 than other groups (23.0% vs. 13.5 to 15.2%) (p<0.05). Apoptosis rate was significantly lower in T3 than T1 or T2 (p<0.05). The relative abundance of Bax-${\alpha}$/Bcl-xl was significantly lower in in vivo or SCF group than that of control (p<0.05). Moreover, the expression of p53 and caspase3 mRNA was significantly lower in in vivo or SCF group than that of control (p<0.05). These results indicate that the addition of SCF at fusion/activation might improve in vitro development of porcine NT embryos through regulating cdc2 kinase level and expression of apoptosis related genes.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.61-61
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• 2001

The main goal of this study was to produce transgenic piglets by the method of injection of sperm-mediated exogenous DNA. Spermatozoa (1$\times$106 sperm of final concentration) obtained from caudal epididymis were mixed with pBC1-hEPO (20 ng/${mu}ell$) or pcDNA3 LAC Z (20 ng/${mu}ell$), and followed by electroporation (500 V, 25 ㎌). Matured oocytes having the first polar body and dense cytoplasm were selected and centrifuged at 12,000g for 6 min. After sperm injection, the oocytes were activated electrically (1.7 ㎸/cm, 30 $\mu$ sec, single pulse) in 0.3 M mannitol solution. Eggs injected sperm were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air for 192 h. This study were comprised 3 experiments. Experiment 1 compared the developmental efficiencies between the sperm-injected oocytes (Group 1) and further activated electrically (Group 2). Experiment 2 compared the expression of pcDNA3 LAC Z in the embryos produced by Group 1 and Group 2. Finally, experiment 3 carried out transfer of embryos (1-8 cell stage) transfected with pBC1 -hEPO into surrogate recipients synchronized by injection of combination of PG600 with hCG. The rates of cleavage and development into blastocyst stage in Group 2 were significantly higher than those of Group 1 (71.3% and 28.1% vs. 43.3% and 10.3%, respectively, p<0.05). Thirty (24.2%) out of 124 embryos analyzed in Group 2 were positive by X-gal. Similarly, in Group 1, 16.3% (8/49) were positive. After transfer of 789 embryos to 7 recipient gilts, three out of them examined by ultrasound became pregnant. One recipient is in day 50 pregnancy. On day 54 of gestation, two were carried out uterotomy in order to confirm the pregnancy One had 7 and another had 2 fetuses. We conclude that injection of sperm-mediated gene transfer will be used as a valuable tool for the production of transgenic piglets.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.10
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• pp.1414-1420
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• 2005

The purpose of this research was to determine whether or not feeding induced hypovolemia (decreases in plasma volume) and decreases in plasma bicarbonate concentration caused by loss of $NaHCO_3$ from the blood, act to suppress feed intake and saliva secretion volumes during the initial stages of feeding in goats fed on dry forage. The animals were fed twice a day at 10:30 and at 16:00 for 2 h each time. Prior to the morning feeding, the collected saliva (3-5 kg) was infused into the rumen. During the morning 2 h feeding period (10:30 to 12:30), the animals were fed 2-3 kg of roughly crushed alfalfa hay cubes. At 16:00, the animals were fed again with 0.8 kg of alfalfa hay cubes, 200 g of commercial ground concentrate and 20 g of sodium bicarbonate. In order to compensate for water or $NaHCO_3$ lost through saliva during initial stages of feeding, a 3 h intravenous infusion (17-19 ml/min) of artificial mixed saliva (ASI) or mannitol solution (MI) was begun 1 h prior to the morning feeding and continued until the conclusion of the 2 h feeding period. The physiological state of the goats in the present experiment remained unchanged after parotid gland fistulation. Circulating plasma volume decreases caused by feeding (estimated by increases in plasma total protein concentration) were significantly suppressed by the ASI and MI treatments. During the first 1 h of the 2 h feeding period, plasma osmolality in the ASI treatment was the same as the NI (non-infusion control) treatment, while plasma osmolality in the MI treatment was significantly higher. In comparison to the NI treatment, cumulative feed intake levels for the duration of the 2 h feeding period in the ASI and MI treatments increased markedly by 56.6 and 88.3%, respectively. On the other hand, unilateral cumulative parotid saliva secretion volume following the termination of the 2 h feeding period in the ASI treatment was 50.7% higher than that in the NI treatment. MI treatment showed the same level as the NI treatment. The results of the present experiment proved that the humoral factors involved in the suppression of feeding and saliva secretion during the initial stages of feeding in goats fed on dry forage, are feeding induced hypovolemia and decrease in plasma $HCO_3^-$ concentration caused by loss of $NaHCO_3$ from the blood.

• Journal of Food Hygiene and Safety
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• v.6 no.2
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• pp.89-93
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• 1991

The present study was conducted to investigate the prevalence, the biochemical properties and the enterotoxin types of Staphylococcus aureus in healthy human and patient. A total of 61 S. aureus strains were isolated from 142 samples. The prevalence of S. aureus isolated from healthy human and patient were 17.7% and 14.0%, respectively. All the isolates showed the production of coagulase, lecithinase and hemolysis (${\alpha}-hemolysis;\;32.8%,\;{\beta}-hemolysis;\;67.2%$) on sheep blood agar. Coagulase type VII (38.4%) and type 111 (26.0%) were dominant among coagulase types I through VIII. Twenty-four (52.2%) of 46 strains tested produced one or more enterotoxin; enterotoxin A, Band C were produced by 3, 9 and 12 strains, respectively. Enterotoxins were produced by 100% of type 11 strains, 75% of type 111 and 39.1 % of type VII. Commonly, coagulase type II produced enterotoxin B or C, and type VII produce enterotoxin C.