• Reproductive and Developmental Biology
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• v.41 no.4
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• pp.65-70
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• 2017

Alpha-linolenic acid (ALA) is one of n-3 polyunsaturated fatty acids and found mainly in the chloroplasts. Many studies have been reported that intracellular reactive oxygen species (ROS) in mammalian oocytes were reduced by supplementation of ALA in in vitro maturation (IVM) medium. Based on these reports, we expected that ALA acts as an antioxidant during IVM of porcine oocytes. Therefore, the objective of this study was to investigate the antioxidant effect of ALA supplementation during IVM in porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated in IVM medium containing $200{\mu}m$ $H_2O_2$ or $H_2O_2$ with $50{\mu}m$ ALA for 44 h. Nuclear maturation stage of oocytes was evaluated using aceto-orcein method. For measurement of oxidative stress state, intracellular ROS and glutathione (GSH) levels were measured using carboxy-DCFDA and cell tracker red, respectively. In results, oocytes in metaphase-II (MII) stage development was significantly reduced in $H_2O_2$ group compared to non-treated control group $61.84{\pm}1.42%$ and 80.00%, respectively; p<0.05) and it was slightly recovered by treatment of ALA ($69.76{\pm}1.67%$; p<0.05). The intracellular GSH levels was decreased in $H_2O_2$ groups compared with control groups, but it was enhanced by ALA treatment (p<0.05). On the contrary, $H_2O_2$ treatment increased intracellular ROS level in oocytes and $H_2O_2$-induced ROS was decreased by treatment of ALA (p<0.05). Our findings suggested that ALA treatment under oxidative stress condition improve oocyte maturation via elevated GSH and reduced ROS levels in oocytes. Therefore, these results suggest that ALA have an antioxidative ability and it could be used as antioxidant in in vitro production system of porcine embryo.

• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.36-42
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• 1990

The effect of preincubation on in vitro maturation and fertility were investigated using preovulatory oocytes with and without cumulus cells obtained from superovulated ouot-bred ICR mice. Oocytes were recovered from fully grown folicle at 10 hr after hCG administration. A part of oocytes recovered were treated with the solution of 0.1% hyaluronidase to remove cumulus cells. Both intact and treated oocytes were then incubated for 0 to 6hr in mT6 medium containing 0.3% BSA. After incubation for various times, a part of oocytes were subjected to the investigation of nuclear maturation and the remaining oocytes were used fro the induction of in vitro fertilization by adding them into medium containing capacitated mice epididymal spermatozoa. Above all, the percentage of preovulatory oocytes at the stage of metaphase II after preincubation for 0, 2, 4 and 6hr was 15.8, 36.4, 47.5 and 66.7%, respectively, suggesting the in vitro maturation of oocytes during their incubation. On the other hand, fertilizatin rate of oocytes preincubated for 0, 2, 4 and 6hr with and without cumulus cells were 41.0, 58.7, 68.7 and 75.6%, and 50.0, 45.1, 37.8 and 39.2%, respectively. No significant differences in fertilization rate between preovulatory oocytes preincubated for 6hr with cumulus cells and naturally ovulated were observed. These results suggest that cumulus cells take very important role in maturtion of oocytes in vitro. The precentage of preovulatory oocytes developed to 2-cell stage in vitro fertilization following preincubation for 0 to 6hr with and without cumulus cells ranged from 48.5 to 82.4% and 36.9 to 56.1%, respectively. Also, the rates of oocytes developed to blastocyst in vitro fertilization after preincubation for 0 to 6hr with and without cumulus cells were 28.1, 39.3, 42.5 and 44.0% and 12.5, 32.6, 24.4 and 15.5%, respectively. From these results, it could be said that fertility of preovulatory oocytes with cumulus cells could be improved to the level of that of naturally ovulated oocytes by adquate preincubation in vitro. Cumulus cells may, therefore, affect in vitro maturation, fertilization and following development of oocytes by influencing zona hardening.

• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.233-240
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• 2003

This study was conducted to examine the effects of oocyte maturation period, phytohemagglutinin-P (PHA-P) treatment and activation agent on the enucleation, fusion, activation or in vitro development of bovine nuclear transfer embryos. Bovine oocytes were enucleated at 16∼24 h of in vitro maturation (IVM). Adult ear skin cells treated or non-treated with PHA-P were transferred into enucleated oocytes. Reconstituted oocytes treated or non-treated with PHA-P were fused by a pulse of 1.5 kV/cm for 30 $\mu$sec. Fused oocytes were activated with a combination of calcium ionophore (A23187) and cycloheximide (CHXM) or dimethylaminopurine (DMAP), and cultured in vitro for 7∼9 days. Enucleation rate was significantly increased when oocytes were matured for 16∼18 h (70.2∼92.3%, P<0.05) compared to that of oocytes were matured for 20∼24 h (44.3∼53.4%). The location of metaphase-II plate was far off from the 1st polar body as maturation time was increased. PHA-P treatment of donor cells or reconstituted oocytes significantly improved fusion rate (P<0.05). Cleavage and blastocyst formation rates were significantly increased after activation with a combination of A23187 and DMAP (78.6% and 32.9%, respectively) compared to those of embryos activated with a combination of A23l87 and CHXM (48.5 and 15.2%, respectively). From the present result, it is suggested that high enucleation efficiency can obtained by using oocytes matured for 18 h. It also shows that PHA-P treatment can improve the fusion rate, and activation with a combination of A23187 and DMAP can enhance the embryo development.

• Korean Journal of Medicinal Crop Science
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• v.13 no.3
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• pp.118-121
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• 2005

Karyotypes were established in seven Angelica species cultivated in Korea. The somatic chromosome numbers were 2n = 2x = 22 with the basic number of x = 11 in all Angelica plants examined. Their metaphase chromosomes ranged from 3.56 ${\mu}M$. to 8.91 x. in length. Distinctive Karyotypes were found in two species, A. tenuissima with all metacentries, K(2n) = 2x = 22m, and A. genuflexa with all subtelocentrics, K(2n) = 2x = 22st. Karyotype formulas of A. gigas, A. acutiloha, A. sinensis, A. decursiva and A. dahurica were K(2n) = 2x = 20m + 2sm, K(2n) = 2x = 12m + 10sm, K(2n) = 2x = 16m + 6sm, K(2n) = 2x = 18m + 4sm and K(2n) = 2x = 10m + 10sm + 2st, respectively. Cytological data showed that chromosomal polymorphisms within species were observed in Angelica plants compare to other regions.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.65 no.4
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• pp.399-405
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• 2020

The objective of this study was to identify the effect of zebularine soaking on the early growth stage of barley (Hordeum vulgare L.). Hence, root elongation was measured daily according to the different concentrations of zebularine (1.0, 2.5, 5.0, and 10.0 μM) for 4 days. On the first day, root length at 2.5 and 5.0 μM was significantly longer than that in the non-treated control. On the second day, root length was not significantly different among all concentrations and controls. On the third day, root elongation was suppressed by the effect of zebularine, except at 2.5 μM. The treatment time of zebularine accounted for the largest proportion of the variation in root elongation. After transplanting, plant growth velocity was similar to that of the control; however, plants at 2.5 μM showed faster growth velocity than that of the other concentrations and the control. In the metaphase of mitosis, most chromosomes of cells under zebularine treatment were ordinary regardless of the concentration, while some cells with short chromosomes were investigated at around 2%. The short chromosome showed a centromere. In addition, it showed short and long arms based on the centromere. The lengths of the short and long arms were different for each short chromosome. It is necessary to study the effect of the short chromosome as a chromosomal function on plant growth and phenotype through investigation of meiosis and fertilization at the chromosome level.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.51-61
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• 2001

This study was conducted to determine developmental ability of reconstructed embryos by nuclear transfer using somatic cell of Korean bull with high genetic value. Fibroblast cells obtained from ear biopsy of the bull were cultured in Dulbecco's Modified Eagle's medium (DMEM) at 37$^{\circ}C$ in air containing 5% $CO_2$. The cummulus-oocyte complexes were collected from slaughterhouse and were matured in vitro for 20 h in TCM 199 culture medium and the oocytes were then enucleated in modified phosphate buffered saline with cytochalasin B. Matured bovine oocytes were enucleated by aspirating the first polar body and metaphase chromatin using a beveled pipette in modified phosphate buffered saline. The ear fibroblast cells were fused into enucleated oocyte by electrical stimulation. The reconstructed oocytes were activated with ionomycine and 6-dimethylaminopurine, and then cultured in CR1aa medium for 7.5 days. Out of 524 bovine eggs reconstructed by nuclear transfer 65.6%(277/422) embryos were cleaved, and 30.7% (85/277) cleaved embryos were developed to the morula to blastocysts. There was no difference of developmental ability in vitro of reconstructed embryos regardless of donor cell passages. In order to determine fate of foreign mitochondria of donor nucleus, the Mito Tracker stained cells were fused into enucleated oocytes. The donor mitochondria were detected early stage of embryos, but disappeared rapidly. The developmental ability of reconstructed embryos was not impaired by Mito Tracker treatments. The results indicate that viable reconstructed embryos can be producted by nuclear transfer using somatic cell of Korean bulls.bulls.

• Reproductive and Developmental Biology
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• v.38 no.4
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• pp.147-158
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• 2014

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic status after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Dai et al., 2010), use of m-carboxycinnamic acid bishydroxamide (CBHA) as a histone deacetylase inhibitor during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA treatment on the in vitro preimplantation development of porcine SCNT embryos and on the acetylated status of histone H3K9 on cloned embryos at the zygote stage. We performed the three groups SCNT: SCNT (NT), CBHA treatment at the porcine fetus fibroblast cells (PFFs) used as donor cells prior to SCNT (CBHA-C) and CBHA treatment at the porcine SCNT embryos during the in vitro early culture after oocyte activation (CBHA-Z). The PFFs were treated with a $15{\mu}M$ of CBHA (8 h) for the early culture and the porcine cloned embryos were treated with a $100{\mu}M$ concentration of CBHA during the in vitro early culture (10 h). Cleavage rates and development to the blastocyst stage were assessed. No significant difference was observed the cleavage rate among the groups (82.6%, 76.4% and 82.2%, respectively). However, the development competence to the blastocyst stage was significantly increased in CBHA-Z embryos (22.7%) as compared to SCNT and CBHA-C embryos (8.6% and 4.1%)(p<0.05). Total cell numbers and viable cell numbers at the blastocyst stage of porcine SCNT embryos were increased in CBHA-Z embryos as compared to those in CBHA-C embryos (p<0.05). Signal level of histone acetylation (H3K9ac) at the zygote stage of SCNT was increased in CBHA-Z embryos as compared to SCNT and CBHA-C embryos. The results of the present study suggested that treatment with CBHA during the in vitro early culture (10 h) had significantly increased the developmental competence and histone acetylation level at the zygote stage.

• Journal of Embryo Transfer
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• v.23 no.4
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• pp.263-267
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• 2008

The purpose of this study was to determine toxic effect of sucrose and trehalose prior to cryopreservation on nuclear maturation and embryonic development in immature bovine oocytes. All cryoprotectant was prepared in tissue culture medium 199-HEPES (TCM 199-HEPES) with 10% fetal bovine serum (FBS). Immature oocytes were exposed to 1.2M ethylene glycol (EG) and 0.1M sucrose or 1.2M EG and 0.1M trehalose for 3 min and then were exposed to 3.2 M EG and 0.25 M sucrose or 3.2 M EG and 0.25 M trehalose for 1 min. Oocytes treated with cryoprotectants were exposed to 0.25 M sucrose or 0.25 M trehalose for 5 min and then 0.1 M sucrose or 0.1 M trehalose for 5 min. Depending on type of sugar added to cryopreservation solution, oocytes were allocated to sucrose group and trehalose group, respectively. Oocytes exposed to TCM 199-HEPES with 10% FBS were considered as control. Oocytes were cultured in TCM 199 supplemented with 10% FBS, 5 ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone, and $1\;{\mu}g/ml$ estradiol for 24 h in $39^{\circ}C$, 5% $CO_2$. Nuclear maturation was assessed by staining oocytes with 1% aceto-orcein. Oocytes were fertilized in vitro and were cultured in TCM 199 supplemented with 10% FBS, 5 mM sodium pyruvate, and antibiotics in $39^{\circ}C$, 5% $CO_2$. The rates of cleavage and blastocyst, and cell number in blastocyst were assessed. Metaphase II rates were not different among experimental groups regardless of type of sugar. The cleavage rate of trehalose group (73.3%) was significantly higher (p<0.05) than those of sucrose group (62.8%) and control group (60.8%). The blastocyst rate was significantly higher in trehalose group (p<0.05). Mean cell number in blastocyst were not different among experimental groups, although cell number of blastocyst in trehalose group was significantly higher on day 7 (p<0.05). In conclusion, sucrose and trehalose were not toxic to immature bovine oocytes prior to cryopreservation. In particular, trehalose was more effective on embryonic development.

• Korean Journal of Poultry Science
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• v.37 no.3
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• pp.247-253
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• 2010

Telomeres are nucleoprotein structures at the ends of chromosomes consisting of DNA sequences arranged in tandemly repeated units $(TTAGGG)_n$. However, this hexamers can also occur at non-telomeric sites in some avians and vertbrate. This study was carried out to present the distribution of telomeric DNA sequences in Korean Native Chicken chromosomes. The fluorescence in situ hybridization technique using a telomeric DNA probe was performed to the metaphase spreads of chicken early embryonic cells. Telomeric DNA signals were detected at the ends of chromosomes including macrochromosomes and microchromosomes. In chicken, surprisingly, chromosome 1 showed very distinct interstitial telomeric DNA hybridization patterns which located two interstitial sites in the p-arm at 1p11 and 1p23, and one in the q-arm at 1q32. In chromosome number 2 and 3 also displayed interstitial telomeric signals (ITS) in the long arms at 2q24 and 3q32, respectively. The pattern of telomeric DNA distribution in Korean Native Chicken chromosomes was in agreement with a previously reported in Gallus domesticus. The relative amount of telomeric DNA sequences in each macrochromosomes ranged from 4.6% to 16.3%. Distribution of telomeric DNAs at the end of p-arm was much more than that of q-arm in almost chicken chromosomes. The distribution of ITS in chicken chromsomes implicate to tandem chromosome fusions that might have occurred during the process of karyotype evolution.

• Parasites, Hosts and Diseases
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• v.26 no.2
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• pp.107-111
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• 1988

As a series of systematic classification of paramphistomes, the worms in the rumen and reticulum of 310 Korean cattle slaughtered at Chonju abattoir were collected from February 1986 to June 1987 and were classified by morphology of the worms. Afterwards, the karyotype of Fischoederius cobboldi (Poirier, 1883), which is a very rare species in Korean cattle, was studied with germ cells of the worm by means of modified air-drying method. The chromosome numbers in the haploid and diploid cells of 315 F. cobboldi were n=9 and 2n=18, respectively. The meiotic divisions were observed frequently; 1,904 haploid and 49 diploid cells were recognized. Nine pairs of mitotic chromosomes were homologous in the metaphase stage and the chromosomes were composed of seven medium-sized metacentrics (m) or submetacentrics (sm) and two small-sized submetacentrics (sm). While, meiotic metaphases were composed of seven medium and two small·sized chromosomes. The 3rd, 4th, 2nd and 5th pairs of chromosomes was metacentric having centromere indices of 40.4%, 40.0%, 39.7% and 38.9%, respectively, and the remaining ones were submetacentric with centromere indices from 32,4% to 36.2%. As a series of C-banding method, C-band was shown in centromeric region from all of the haploid germ cells, except chromosome No. 1 which included heterochromatin at the tip region. Chromosomes No, 4, 6 and 9 showed remarkable C-band distinguished from others.