• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.215-223
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• 2003

This study was to investigate the effects of fertilization time and culture medium of pig oocytes matured in-vitro by liquid boar sperm. The sperm rich fraction (30∼60 ml) was slowly cooled to room temperature (20∼23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min 800 ${\times}$ g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of the LEN diluent to provide 1.0${\times}$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$. The medium used for oocyte maturation was TCM-199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin B$_{12}$ , 25 mM HEPES, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9 h in 500 ${mu}ell$ mTBM fertilization media with 1.0${\times}$10$^{6}$ sperm/ml concentration, respectively. Thereafter, oocytes were transferred into 500 ${mu}ell$ NCSU-23, HEPES buffered NCSU-23, PZM-3 and PZM-4 culture media, respectively, for further culture of 6, 48 and 144 h. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times for 6 and 9 h than in those for 1 and 3 h. The rates of cleaved oocytes were higher in the fertilization times for 6 and 9 h (85.0 and 84.6%) than in those for 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time for 6 h (33.6%) than in that for 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9, 27.6, 26.3 and 24.4 in the fertilization times for 6, 9, 3 and 1 h, respectively. The rate of blastocyst from the cleaved oocytes and the number of cells per blastocyst were higher in HEPES buffered NCSU-23 culture medium than in NCSU-23, PZM-3 and PZM-4 culture media. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend the coincubation time of 6 h in 500 ${mu}ell$ TBM fertilization medium with 1${\times}$10$^{6}$ sperm/ml concentration and the HEPES buffered NCSU-23 culture medium for in-vitro fertilization of pig oocytes matured in-vitro.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.425-435
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• 1998

To improve the efficiency of nuclear transplantation in bovine, in this study the development in vitro of nuclear transferred (NT) embryos was compared by different activation regimens of the enucleated oocytes. The effect of developmental stage and culture system of donor nuclei on fusion and development in vitro of NT embryos were also evaluated. Oocytes were collected from Hanwoo ovaries obtained from slaughterhouse and matured in Ham's F-10 supplemented with hormones. After 20~22 h maturation, the oocytes were vortexed to be free from cumulus cells and subsequently their nucleus and the first polar body were removed. Enucleated oocytes were divided into 3 groups for activation; the oocytes of group I were activated with ionomycin for 5 min and subsequently incubated in 6-dimetylarninopurine (DMAP) for 4 h, Those of group II were treated with DMAP for 4 h at 39 h after onset of in vitro maturation (IVM) and those of group III were kept in room temperature ($25^{\circ}C$) for 3 h at 39 h after onset of IVM. After in vitro fertilization (IVF) the embryos for muclear donor were cultured either by group culture (20 embryos /50 ${mu}ell$ drop) or individually (1 embryo /50 ${mu}ell$ drop) for 4 day and 5 day. At day 4 and 5 after IVF, blastomeres were separated in calcium-magnesium free medium, and then classified into small (day 5: $\leq$ 38 ${\mu}{\textrm}{m}$, day 4: $\leq$ 46 ${\mu}{\textrm}{m}$) and large (day 5 : $\geq$ 38 ${\mu}{\textrm}{m}$, day 4 ; $\geq$ 46 ${\mu}{\textrm}{m}$). The separated blastomeres were replaced into enucleated and activated recipient cytoplasm. The blastomere-oocyte complexes were fused by electrically. The NT embryos were cultured in TCM-199 containing 10% FCS in 39$^{\circ}C$, 5% $CO_2$ incubator for 7 day. The results obtained were summarized as follows; There were no differences in fusion and development to blastocyst between groups as group I (68%, 10%), group II (75%, 14%) and group III (73%, 9%), respectively. However, the cell number in blastocyst of NT embryos in group III were significantly fewer than in the other groups (P＜0.05). No differences in fusion and development to blastocyst were found between individual or group cultured and between small or large blastomeres of day 4 and day 5 donor embryos. From these results, it was concluded that the combination of ionomycin and DMAP, or treatment of DMAP at 39 h after onset of IVM were useful for the efficient of production of NT bovine embryos, and the individual cultured embryos could be simply used as donor nuclei for NT bovine embryo.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.241-248
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• 2000

In this study, we examined the number and motility of sperms, and also observed the changes in testes weight, and histological changes of several organs after 5 days of a continuous administration of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) of per oral administration of a herb drug extracts, which were administerated altermate days, to elucidate the effects of the a herb drug extracts on reproductive toxicity of dioxin. 1. The sperm numbers of dioxin-administered groups were 90.7$\pm$3.6~l18.5$\pm$3.6$\times$10/suup 6/$m\ell$ and 67.3$\pm$4.1~88.2$\pm$3.3$\times$10$^{6}$ $m\ell$ for 10~20 $\mu\textrm{g}$/kg and 30~40 $\mu\textrm{g}$/kg dosages of dioxin-administerated groups, respectively. Each dioxin-administered group showed prominently lower value than that of control group's which was 119.3$\pm$3.4~120.2$\pm$4.7 $\times$ 10$^{6.}$$m\ell$. 2. The sperm motility of each dioxin-administered group's also showed lower value than that of control group's which was 93.6$\pm$3.8~94.9$\pm$3.4%. The sperm motility of each dioxin-administerated group were 77.0$\pm$4.7~89.5$\pm$3.6% and 66.5$\pm$3.3 ~79.9$\pm$3.8% for 10~20 $\mu\textrm{g}$/kg and 30~40 $\mu\textrm{g}$/kg dosages of dioxin-administerated groups, respectively. 3. The sperm numbers of each group, which was administered a herb drug extracts, were 77.4$\pm$3.2~90.9$\pm$3.4$\times$10$^{6}$ $m\ell$, 78.0$\pm$3.3~105.0$\pm$4.2$\times$10$^{6}$ $m\ell$, 76.2$\pm$2.8~84.4$\pm$3.5$\times$10$^{6}$ $m\ell$ and 75.4$\pm$3.3~80.2$\pm$3.3$\times$10$^{6}$ $m\ell$ for extracts of green leaf, red ginseng, Kugija and Oume-administered groups respectively. And the sperm motility of each group were 63.4$\pm$3.8~77.0$\pm$4.0%, 65.5$\pm$4.1~87.4$\pm$3.8%, 64.3$\pm$4.2~69.8$\pm$4.2%, 66.3$\pm$3.9~66.0$\pm$4.0% for extracts of green leaf, red ginseng, Kugija and Oume extracts-administered groups, respectively. 4. The number and motility of sperm of control group were 119.3$\pm$3.4~120.2$\pm$4.7$\times$10$^{6}$ $m\ell$ and 93.0$\pm$3.5~96.1$\pm$3.5%, respectively. Red ginseng extracts- administered group seemed to be recovered than my other groups, and the green leaf extracts-administered group was shown to be the next. The Kugija and Oume extracts-administered groups didn't show to be recovered much. 5. Most a herb drug extracts-administered group except the red ginseng-administered group displayed prominently lower values of testes weights than that of control group's which was 0.15$\pm$0.01~0.16$\pm$0.01g. The red ginseng extracts-administered group seemed to be recovered conspicuously. 6. After 5 days of a continuous administration of 30 $\mu\textrm{g}$/kg dosage of dioxin followed by 3 weeks of per oral administration of green leaf, red ginseng, Kugija, or Oume extracts, histological findings showed that the liver, spleen, and testis of most a herb drug extracts-administered groups were damaged severely. By the way, the testes of red ginseng extracts-administered group seemed to recover compared to the other group's.ompared to the other group's.

• The Korean Journal of Nuclear Medicine
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• v.32 no.1
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• pp.71-80
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• 1998

This study examines the levels of carbohydrate antigen 19-9(CA19-9) and cancer antigen 125(CA125) in serum and its related factors in healthy Korean population. Although CA19-9 and CA125 have been widely used tumor markers for gastroenteric cancers and ovarian cancer in Western countries, there are no information available on the serum levels of CA19-9 and CA125 in healthy population and the factors affecting the levels of these tumor markers in Korea. A cross-sectional study was performed to measure CA19-9 and CA125 among 76 healthy males and 95 healthy females in Korea. CA19-9 and CA125 were quantitated using solid-phase radioimmunoassay kits. Informations on the factors which might be related to the levels of these markers were collected by questionnaire(e.g., smoking, alcohol consumption, menstruation, oral pill use, breast-feeding history, etc.). There was no statistically significant difference in the mean of CA19-9 concentration between men(10.4 u/ml) and women(10.1 u/ml), whereas the mean of CA125 levels(11.2 u/ml) was higher in women than that(2.5 u/ml) in men. Although there was a statistically significant association between CA19-9 and average number of cigarette consumed per day(r=0.59, p=0.026) and total number of cigarettes consumed in women(r=0.74, p=0.003), the significance disappeared by multiple regression analysis after adjusting age and body mass index. Later age of menopause(p=0.035) and longer duration of breast-feeding(p=0.050) were significant predictors for CA125 levels in women by multiple regression analysis after adjusting age and body mass index. In conclusion, CA19-9 can be used as a stable tumor marker in clinical practices, however, menstruation and breast-feeding should be considered when CA125 is used in women.

• Korean Journal of Plant Taxonomy
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• v.46 no.2
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• pp.256-266
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• 2016

A total of 13,000 individuals of Dendrobium moniliforme (L.) Sw. artificially propagated in laboratories and greenhouses were restored in their natural habitat of Bogildo Island, Wandogun, in the southern part of Korea in June of 2013. The growing conditions of the individuals were monitored for two years. The parental individuals for the restoration were obtained from a wild population in southern Korea, from which seeds were produced via artificial crossings. These seeds were germinated and cultivated in growing media and two-year-old plants were then grown in greenhouse beds. The genetic diversity among the propagated individuals was confirmed by examining DNA sequences of five regions of the chloroplast genome and the nuclear ITS region. The diversity values were as high as the average values of natural populations. All propagated individuals were transplanted into two different sites on Bogildo by research teams with local residents and national park rangers. After restoration, we counted and measured the surviving individuals, vegetative propagated stems, and growth rates in June of both 2014 and 2015. There was no human interference, and 97% of the individuals survived. The number of propagules increased by 227% in two years. In contrast, the average length of the stems decreased during the period. In addition, different survival and propagation rates were recorded depending on the host plants and the restored sites. The shaded sides of rock cliffs and the bark of Quercus salicina showed the best propagation rates, followed by the bark of Camellia japonica. A few individuals of D. moniliforme successfully flowered, pollinated, and fruited after restoration. Overall, our monitoring data over two years indicate that the restored individuals were well adapted and vigorously propagated at the restored sites. In order to prevent human disturbance of the restored sites, a CCTV monitoring system powered by a solar panel was installed after the restoration. In addition, a human surveillance system is operated by national park rangers with local residents.

• Reproductive and Developmental Biology
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• v.31 no.1
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• pp.35-41
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• 2007

This study was conducted to examine the effects of energy substrates in different conoentration of carbohydrates in the human oviduct and uterus on the in vitro development of mouse 2-cell embryos. Two-cell embryos were collected from ICR female mice at $46{\sim}50\;hr$ after 5 IU hCG injection and cultured in three different media [control: 0 mM, Guoup A: glucose (G) 0.5 mM + pyruvate (P) 0.32 mM + lactate (L) 10.5 mM, Group B: G 3.15 mM + P 0.1 mM + L 5.83 mM] for 72 hr. Rates of morula formation of group A (72.3%) and B (56.6%) were significantly higher higher (p<0.05) than that of control (34.9%) at 24 hr. However, blastocyst rate was significantly higher (p<0.05) in control (51.8%) than group A (39.8%) and B (28.9%) at hr. At 72 hr, no differences were found in the number of zona-intact, zona-escape and total blastocysts among groups. Mean and ICM cell numbers were significantly higher (p<0.05) in group A (78.0, 13.4) and B (64.4, 11.8) than control (53.1, 5.7), respectively, The percent of ICM were significantly higher (p<0.05) in group A (22.9%) and B (23.7%) than control (14.2%). No differences were round in the TE cell numbers ($34.1{\sim}45.1$). The ICM:TE ratio was significantly higher $34.1{\sim}45.1$ in control (1:6.0) than group A (1:3.4) and B (1:3.4). This study shows that energy substrates added to culture media especially, the oviductal level of carbohydrates increase the developmental capacity of 2-cell mouse embryos.

• Korean journal of applied entomology
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• v.46 no.1 s.145
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• pp.51-56
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• 2007

The two species of rice bugs causing pecky rice, Cletus punctiger and Cletus schmidti are often observed coexisting in the rice fields of nearby fallow land. Direct feeding damage to rice by C. punctiger and C. schmidti can lead to a reduction in grain quality and quantity. These studies were carried out to investigate the development of C. punctiger and C. schmidti at various constant temperatures ranging from 20 to $30^{\circ}C$, 65% RH, and a photoperiod of 16L:8D. Egg hatchability of C. punctiger/C. schmidti at the temperatures of 20, 25 and $30^{\circ}C$ were 80.6/88.0, 91.7/96.3, 96.4/96.2%, respectively. The development periods of eggs of C. punctiger/C. schmidti at the temperatures of 20, 25, and $30^{\circ}C$ were 16.4/18.4, 9.4/10.2 and 6.4/7.3 days, respectively. Mean developmental periods of 1 st, 2nd, 3rd, 4th and 5th nymphs of C. punctiger/c. schmidti at $30^{\circ}C$ were 2.1/2.0, 3.5/4.0, 3.3/5.6, 3.2/4.8 and 5.8/6.9 days, respectively. Oviposition began 8.1 days after emergence at $25^{\circ}C$, and the longevity of female and male were 120.0 and 117.3 days, respectively. Total number of eggs through the life of female were 245.5 laying 2.2 eggs a day in average at $25^{\circ}C$. The development periods of egg and nymphs of C. punctiger were relatively shorter than those of C. schmidti. Availability of male had affected the egg hatchability greatly that laid at 30th day after 60 days period of oviposition in the presence of adult male of C. punctiger. The fertile eggs laid by the female together with male was 92.1% but those without male was only 9.6%.

• Development and Reproduction
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• v.6 no.2
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• pp.131-139
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• 2002

Since GnRH and its receptor genes are expressed in the ovary, it has been suggested that ovarian GnRH might be involved in the regulation of ovarian function and the apoptosis of ovarian cells. However, it was not known well on the expression and function of GnRH and its receptor in the corpus luteum. The present study was undertaken to investigate whether GnRH and its receptor are expressed in luteal cells and GnRH has any effect on the apoptosis of luteal cells. Luteal cells obtained from the pregnant rats were cultured and stained for GnRH and its receptor proteins. Cultured luteal cells showed distinct immunoreactivity against both anti-GnRH and anti-GnRH receptor antibodies. In addition, the presence of GnRH receptor protein in cultured cells was confirmed by Western blot analysis. To investigate the effect of GnRH on the apoptosis of luteal cells, luteal cells were cultured in the presence of 10$^{-6}$ M GnRH-agonist(GnRH-Ag) for 3, 8, and 12h. TUNEL assay showed that the number of cells undergoing apoptosis increased 12h after culture(P<0.05). DNA fragmentation analysis confirmed the results such that the cells treated for 12h showed the greatest increase of fragmentation(p<0.05). Further, Western blot analysis of cytochrome c in the mitochondrial and cytoplasmic fractions of the luteal cells showed that GnRH-Ag treatment increased the content of cytochrome c in cytoplasm. These results demonstrate that the luteal cells express GnRH and its receptor and GnRH-Ag treatment induces apoptosis of the luteal cells via mitochondrial release of cytochrome c. The present study suggest that the releasing of cytochrome c from mitochondria might be involved in the luteal cell apoptosis induced by GnRH-Ag.

• Development and Reproduction
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• v.4 no.2
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• pp.187-193
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• 2000

Gonad and blood samples were taken from the cultured female Korean sea bass, Lateolabrax japonicus from October to February between 1997 and 1999. Gonadosomatic index began to increase in November and reached the highest value in December (12.8$\pm$1.5) and January (14.8$\pm$3.5), and then decreased sharply in February (2.6$\pm$1.5, p<0.05). The ovarian oocytes developed to tertiary yolk stage and reached fully-Brown stage in December and January, and then underwent atresia without maturation and ovulation in February. The plasma estradio3-17 $\beta$ level increased from November, and reached the highest value in December (1,152.3$\pm$107.2 pg/ml) and January (1,315.4$\pm$99.5 pg/ml), after then decreased in February (P<0.05). The concentration of plasma 17 $\alpha$ ,20 $\beta$-dihydroxy-4-pregnen-3-one was not significantly changed at low levels (86.6$\pm$6.5∼93.8$\pm$2.8 pg/ml) during the experimental period (P<0.05). All the fish with fully-grown oocytes in the ovary were matured and ovulated by HCG injection. The number of floating eggs were 325,000$\pm$26,000 at HCG 1,000 luhg and 195,000$\pm$35,000 at 2,000 lUikg. There was no difference in fertilization rate and hatching rate of the eggs (P<0.05). Considering these results, we could infer that the ovarian oocyte of the cultured Korean sea bass were not matured and ovulated because of the lack of gonadotropin surge. Moreover, HCG injection could induce oocyte maturation and ovulation in the cultured fish, and the effective dose was 1,000 IU/kg.

• Development and Reproduction
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• v.10 no.1
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• pp.1-7
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• 2006

Ovarian follicular atresia in mammals is finely regulated by gonadotropins and sex steroid hormones. It is well known that granulosa cell pyknosis is a common cytological feature of atretic follicles in the ovary. The present study hypothesized that granulosa cell pyknosis during follicular atresia might be related to apoptotic process and associated with caspase-3 activation. Healthy (normal) and atretic follicles were isolated from porcine ovaries based on macro-morphological criteria. Isolated follicles were either processed for histological observation or used for collection of granulosa cells by aspiration. Hoechst 33258 staining of the cells showed a significantly higher number of fragmented nuclei, a typical morphological feature of apoptotic cell, in granulosa cells from atretic follicles than those from healthy follicles. In addition, the rate of cell death was significantly higher in granulosa cells from atretic follicles than healthy follicles, as measured by flow-cytometric cell cycle analysis. In situ detection of apoptotic cells by TUNEL revealed that apoptosis was mostly restricted to granulosa cells in follicles. Theca cells were TUNEL-negative. Finally, it has been shown by caspase-3 activity assay that granulosa cells from atretic follicles retain a higher caspase-3 activity compared to healthy follicles. Taken together, it is suggested that granulosa cell degeneration during folliclar atresia occurs by caspase-3-dependent apoptotic fashion.