• Journal of Aquaculture
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• v.8 no.3
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• pp.195-207
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• 1995

In order to study the embryonic development and hatching of wild long shanny, Stichaeus grigorjewi, were caught with the gill nets in the East Sea of Korea, and stocked at indoor tanks to induce natural spawning in February 25, 1994 and February 16 to 24, 1995. They were already matured when stocked, and average body length (50.66 cm) and body weight (1,192.74 g) of 57 females and average body length (48.62 cm) and body weight (612.58g) of 43 males were recorded. Before stocking, they were inserted with identification tags(ID tags) in the dorsal muscle, and spawning was traced by the portable reader (Destron/lDl Ltd.) Forty females among 57 spawned successfully in the average of 4 days after stocking. Females spawned almost all eggs contained in the ovaries at one time in the form of an egg mass and averaging 227,200 eggs Per egg mass. The egg mass was oval in shape, translucent milky in color, 20.32cm long axis and 14.57cm short axis in size, and 803.7g in weight. Male parents guarded their egg masses and circulated water with the tail part of the body. Fertilized egg was spherical in shape, and their average diameter was 1.54 mm. Each egg had a containing single oil globule, and it's average diameter was 0.37 mm. The average water temperature was $13.2^{\circ}C$ and incubation times after fertilization were 5 hours 25 minutes up to 2-cell stage, 13 hours up to morula stage, and 66 hours 35 minutes up to embryo formation stage. Hatching rate was approximately 10 percent in 368 hours 50 minutes after fertilization, and approxionateoly 90 percent of eggs were hatched in 425 hours 30 minutes after fertilization.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.139-146
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• 1997

This experiment was carried out to evaluate the effect of mouse early embryonic development in vitro by co-culture with bovine and porcine oviductal epithelial cells(BOEC and POEC). The 2-cell embryos were collected from the oviducts of the superovulated and mated does with D-PBS/15% FCS at 48 hours after hCG injection. The in vitro developmental rate of blastocyst formation and the number of nuclei in the embryos were examined. For a comparative study of in vi패 and in vitro development, the fresh blastocyst which developed in vivo for 120 hours after hCG injection was collected from the uterus, and their numbers of nuclei were also counted. The higher developmental rates of blastocyst formation was a, pp.ared from 91% to 97% when the embryos were co-cultured with a monolayer of bovine or porcine oviductal epithelial cells in TCM 199 or Ham's F-10 and MediCult IVF media. No significant difference in developmental rates was shown between bovine and porcine oviductal eptithelial cells. The number of nuclei in the embryos cultured for 72 hours under each conditions was significantly reduced it than blastocyst in vitro conditions. The number of nuclei in embryos cultured in TCM 199, Ham's F-10 and Medicult IVF medium were counted 68.1$\pm$6.00, 67.3$\pm$4.49, 66.4$\pm$5.64, and 94.3$\pm$8.61, 92.5$\pm$7.60, 92.1$\pm$6.10 with BOEC and 93.3$\pm$5.80, 92.9$\pm$6.53, 92.3$\pm$7.35 with POEC coculture, respectively. These numbers were lowered than 107.2$\pm$7.43 in vivo conditions. In conclusions, the coculture between the mouse early embryos, and oviductal epithelial cells of BOEC and POEC give to improve the developmental and hatching rates of blastocyst but in vivo culture systems for the growth of nuclei were ineligible than in vitro conditions.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.347-353
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• 1997

This study was to test whether in vitro/in vivo survival of vitrified mouse blastocysts was influenced by culture conditions and ET method. Mouse blastocysts were obtained from in vitro fertilization and cultured for 4 days in M16 medium, and they were vitrified in EFS40 which contained 40% ethlyene glycol, 18% Ficoll and 0.5 mol sucrose in PBS. In experiment I, in vitro and in vivo survival rate of these embryos were evaluated in different culture condition after thawing. When thawed embryos were cultured in M16 medium as a control, m-CR1 medium contained 20 amino acids (2% BME amino acis and 1% MEM non-essential amino acids solution) and 4 mg/ml BSA and cumulus monolayer cell co-cultured condition in mCR1 medium (10% FBS), their in vitro survival at 24 hr after thawing was not affected by culture condition (75.6, 83.1, 82.4%). However, in vivo survival rates of implantation in m-CR1 medium (80.4%) were significantly higher than those of M16 medium (51.2%), co-culture (57.1%) condition, although there was no difference in live fetuses rates on day 15 gestation (39.0, 49.0, 38.1%). In experiment II, the in vivo development potential of embryos by ET methods was examined. When blastocysts were transferred to the day 2, 3 pseudopregnant recipient without culture soon after thawing, no pregnant recipient was obtained on the day 2 pseudopregnancy, and 50% of pregnancy rates and 15.4% of live fetus rates were obtained on the day 3 pseudopregnant recipients. These results were significantly lower than those of transferred group (day 3 pseudopregnant recipients) after culture for 16 hr post thawing (73.5, 57.1%) (p<0.05). In experiment III, to elevate usability of delayed embryos in vitro/in vivo survival of vitrified embryos (day 4 early, day 5 early and expanding blastocyst) were examined. in vivo survival rates (live fetus, total implantation) were higher in day 4 early blastocysts (33.3, 66.7%) than in day 5 expanding blastocysts (29.0, 38.7%), although the highest in vitro survival rates were obtained in the day 5 expanding brastocysts (78.3%). Therefore, these results suggest that the in vitro/in vivo survival rates of vitrified embryos could be improve by the culture condition and ET method and that the in vivo development rates of delayed embryos were decreased with longer culture duration in vitro. It means that more effective cryopreservation was obtained in day 4 early blastocysts than in day 5 expanding blastocysts.

• Journal of Aquaculture
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• v.6 no.4
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• pp.235-253
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• 1993

The freshwater crab, Eriocheir japonicus inhabits from sub-tropical to temperate zone in Asia. This species belongs to a large size group among freshwater crabs. Common size of this crab is 5-6cm in carapace length and occasionally 7cm in carapace length. This species of crab used to inhabit in estuaries, rivers and inland waters in Korea. However, natural population recently has been rapidly decreased because of pollution and lost their habitats by suburban development. Therefore, development of proper methods of seedling production to increase natural stock became necessity. As parts of achieving this goal, duration from mating to spawning, egg incubation period, and egg development of this species were studied. The influence of temperatures and salinities on the egg incubation and hatching was also investigated. It took 2-8 hours from mating to egg spawning and the spawning lasted 3-9 hours from the first spawning. Egg numbers per female (6cm in carapace length) were 380,000­410,000. Optimum temperature for egg incubation was $17\~23^{\circ}C$ and optimum salinity, $14.0\~31.5\%o$. Incubation period of the eggs at $14^{\circ}C,\;17^{\circ}C,\;20^{\circ}C,\;26^{\circ}C,\;and\;28^{\circ}C$ was 42, 28, 21, 15, and 14 days. respectively. Relation between temperature (X) and incubation days (Y) was LogY = Log 2764.267 - 1.608 LogX. A female can spawn 4-6 times per year by manipulation of environmental conditions. Under the conditions of $18^{\circ}C\;and\;24.5\%o$, it took 6 days up to embryo formation, 18 days up to compound eye formation, 22 days up to abdominal movement, and 25 days up to hatch out as zoea larvae.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.265-274
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• 1995

가토 20마리의 혈액중 혈소판의 수를 수정 전후로 조사한 결과, 수정전과 비교하여 수정후 1일부터 유의하게 감소하였으며 (26.84%, P<0.01), 이러한 감소는 수정후 5일까지 계속되었다. 수정후 5일동안 혈소판의 감소율과 혈소판의 최저치를 수정란의 수와 비교한 바, 이들간의 유의한 상관관계(r=0.5032)가 있었다. 가토수정란 (2세포기) 각각 30∼40개를 5마리의 비장적출수란가토의 난관에 이식한 다음, 혈소판수의 변화를 조사한 결과, 이식전에 비해 이식후 90분 (P<0.025)과 135∼270분 (P<0.025)에 유의한 감소를 확인하였따. 특히 수정란 이식후 180분경에 가장 크게 감소하였다 (P<0.001). 60∼70개의 2세포기 가토수정란을 24시간 배양한 다음, 배양액을 3마리의 비장적출가토에 주사한 후 혈소판의 변화를 조사하였을 때, 주사후 120분경부터 혈소판의 수가 유의하게 감소하기 시작하였다. 이들 배양액을 동결, 해동한 다음 주사한 실험에서도 유사한 결가를 얻었다. 또한 이 배양액을 비장적출생쥐에 주사한 경우에도 가토에서와 마찬가지의 혈소판 촉진현상이 나타났다. 혈소판 촉진인자(PAF)의 길항제인 kadsurenone이 첨가된 배양액에서 24시간동안 가토수정란을 배양한 다음, 이 배양액을 4마리의 비장적출생쥐에 주사한 결과, 혈소판수의 변화가 일어나지 않았다. 또한 kadsurenone이 첨가된 배양액에서는 2세포기 가토수정란의 상실기와 배반포기까지 발달율은 각각 4와 7%로 대조구의 7과 49%보다 유의하게 낮았다. 따라서 본 연구에서 가토의 초기배는 가토나 생쥐의 혈소판수를 감소시키고, 특히 길항제 처리는 이러한 혈소판 감소현상을 억제시킬 뿐만 아니라 가토 초기배발달을 억제한다는 것을 확인하였다. 결론적으로 가토초기배에서는 PAF 또는 수정란 유래의 PAF가 분비된다는 것을 알 수 있었으며, 이러한 인자는 동결처리에서도 그 기능은 전혀 변하지 않는다고 본다. 이후에 있어서 mouse LIF의 첨가는 돼지의 수정란을 배반포 이후의 단계에까지 발달시킬 수 있었다. 있어서 더 적합한 것으로 판단되었다. 5. 개발된 모형은 논 관개의 물리적 측면과 관리목표 모두를 고려한 것으로 계산된 효율은 벼, 생육 각 단계에서의 효율 비교에 양호한 방법임을 알 수 있다.은 Sharpsburg 점질양토에 대한 S.C.S 한계허용치 10ton/ha/year 이내로 나타났다. 비처리구에서의 토양유실량은 평균 2.56ton/ha/year로 높게 나타난 반면 3개의 서로 다른 추리구인 비수구, 초생수로구 및 Bromegrass구에서는 각각 0.152, 0.192 및 0.290ton/ha/year로 낮은 결과를 가져왔다. 6. 평균 침전량에 대한 L.S.D. 검정 걸과 전시험구중 비처리구가 고도의 유의차를 나타낸 반면 비수구, 초생수로구 및 Bromegrass 목초구 간에는 아무런 유의차가 인정되지 않았다. 7. 농지보전 처리구인 배수구와 초생수로구는 비처리구에 비해 낮은 침두 유출량과 낮은 토양유실량을 나타내었다.구보다 14% 절감되는 것으로 나타났다.작용하는 것으로 사료된다.된다.정량 분석한 결과이다. 시편의 조성은 33.6 at% U, 66.4 at% O의 결과를 얻었다. 산화물 핵연료의 표면 관찰 및 정량 분석 시험시 시편 표면을 전도성 물질로 증착시키지 않고, Silver Paint 에 시편을 접착하는 방법으로도 만족한 시험 결과를 얻을 수 있었다.째, 회복기 중에 일어나는 입자들의 유입은 자기폭풍의 지속시간을 연장시키는 경향을 보이며 큰 자기폭풍일수록 현저했다. 주상에서 관측된 이러한 특성은 서브스톰 확장기 활동이 자기폭풍의 발달과 밀접한 관계가 있음을 시사한다.se that were all low in two aspects, named "the Nonsignificant group". And the issues were high risk perception in general

• Korean Journal of Veterinary Research
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• v.9 no.1
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• pp.23-62
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• 1969

Throughout the studies the following experimental results were obtained and are summarized: 1. Multiplication of agents in primary cell cultures of both GF classical and CR-64 acute strain of Marek's disease infected chicken kidneys was accompanied by the formation of distinct transformed cell foci. This characteristic nature of cell transformation was passaged regularly by addition of dispersed cell from infected cultures to normal chicken kidney cell cultures, and also transferred was the nature of cell transformation to normal chick-embryo liver and neuroglial cell cultures. No cytopathic changes were noticed in inoculated chick-embryo fibroblast cultures. 2. The same cytopathic effects were noticed in normal kidney cell monolayers after the inoculation of whole blood and huffy coat cells derived from both forms of Marek's disease infected chickens. In these cases, however, the number of transformed cell foci appearing was far less than that of uninoculated monolayers prepared directly from the kidneys of Marek's disease infected chickens. 3. The change in cell culture IS regarded as a specific cell transformation focus induced by an oncogenic virus rather than it plaque in slowly progressing cytopathic effect by non-oncogenic viruses, and it is quite similar to RSV focus in chick-embryo fibroblasts in many respects. 4. The infective agent (cell transformable) were extremely cell-associated and could not be separated in an infective state from cells under the experimental conditions. 5. The focus assay of these agents was valid as shown by the high degree of linear correlation (r=0.97 and 0.99) between the relative infected cell concentration (in inoculum) and the transformed cell foci counted. 6. No differences were observed between the GF classical strain and the CR-64 acute strain of Marek's disease as far as cell culture behavior. 7. Characterization of the isolates by physical and chemical treatments, development of internuclear inclusions in Infected cells, and nucleic acid typing by differential stainings and cytochemical treatments indicated that the natures of these cell transformation agents closely resemble to those described fer the group B herpes viruses. 8. Susceptible chicks inoculated with infected kidney tissue culture cells developed specific lesions of Marek's disease, and in a case of prolonged observation after inoculation (5 weeks) the birds developed clinical symptoms and gross lesions of Marek's disease. Kidney cell cultures prepared from those inoculated birds and sacrificed showed a superior recovery of cell transformation property by formation of distinct foci. 9. Electron microscopic study of infected kidney culture cells (GF agent) by negative staining technique revealed virus particles furnishing the properties of herpes viruses. The particle was measured about $100m{\mu}$ and, so far, no herpes virus envelop has been seen from these preparations. 10. No relationship of both isolates to avian leukosis/sarcoma group viruses and PPLO was observed.

• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.85-92
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• 1999

This study was designed to determine effect of cryoprotectant kinds and cell stages on OPP vitrification method in mouse embryos. The freezing speed, cryoprotectants and cell stage could affect of embryo viability following various vitrification methods. The vitrification solution used were consisting of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll, 0.3 M sucrose solution in holding medium (D-PBS supplemented with 5% FCS: HM) (EFS) or 16.5% ethylene glycol , 16.5% dimethyl sulfoxide, 0.5 M sucrose in HM (EDS). The embryos were collected from oviduct at 18 h after hCG injection and then washed and cultured in mHTF medium until use. In experiment 1, the blastocysts were vitrified by OPP straw to determine the optimal vitrification solution of EFS or EDS. The post-thaw survival rates at re-expanded stage rates were significantly different between EFS and EDS (95.0 vs 100%), but at hatching stage was not different between EFS and EDS (90.0 vs 95.0%). respectively. In experiment 2, zygotes, 2-, 4-cell, morula and blastocysts were vitrified by OPP method to determine the acceptable of early stage embryos. The development rates to expanded blastocyst in zygote (70.0%) were significantly lower rather than those in 2-, 4- 8-cell, compacted morula or blastocyst (89.7, 90.0, 92.8, 97.6 or 97.5%), respectively. However, the cell number of post-thaw developed to expanded blastocyst in blastocyst and control blastocyst stage (39.6$\pm$2.81, 35.7$\pm$2.98) were significanty higher than those in zygote, 2-, 4-, 8-cell, compacted morula (29.8$\pm$3.21, 31.3$\pm$3.83, 29.3$\pm$3.58, 28.9$\pm$3.21 or 30.8$\pm$2.93). In experiment 3, the zygotes were exposed in VSl for 1, 2, and 3 min to the optimal exposed time. The cleavage rates (91.6, 88.5, 88.9%) and develop mental rates to blastocyst (83.3, 74.3 and 69.4%) depends on the exposed time in VSl were not significantly different among 1, 2, or 3 min, respectively. The cell number also were not significantly different among exposed time in VS1. respectively. These results indicate that OPP method could be useful for vitrification either EFS or EDS vitrification solution. The post-thaw survival rates at zygote were significantly lower than those at 2-, 4-, 8-cell, morula or blastocyst, respectively. The zygote stage were more sensitive rather than late stage embryos. The exposing time in VS1 for 1 min was better than that for 2 or 3 min, even it was not significantly different. The OPP vitrification method could be useful of mouse embryos either with EFS or EDS vitrification solution.

• Journal of Embryo Transfer
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• v.25 no.1
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• pp.15-20
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• 2010

This study was performed to identify the optimal timing for oocyte donor replacement during OPU procedure. OPU was carried out to collect oocytes from every donor at an interval of $3{\sim}4$ days (2 times a week). The collected oocytes were matured in vitro in TCM-199 supplemented with 10% FBS, 10 mg/ml of FSH and 1 mg/ml of estradiol for 24 h. After 24 h of exposure to sperm, the presumptive zygotes were cultured in CR1aa medium supplemented with 4 mg/ml of BSA for 3 days before being changed to CR1aa medium with 10% of FBS for another $3{\sim}4$ days. The mean numbers of retrieved oocytes were remained constantly up to 3 months ($6.0{\pm}0.5$, $6.2{\pm}0.7$, $5.2{\pm}0.6$), but significantly decreased at over 4 to 6 months ($3.7{\pm}0.5$, $2.8{\pm}0.4$, $1.2{\pm}0.2$) (p<0.05). The blastocyst development potential was also very similar rate from 1 to 3 months (37.2%, 40.4% and 44.6%), but significantly decreased from 4 to 6 months (24.8%, 29.3% and 28.6%, respectively) (p<0.05). The production of OPU derived embryos in periods of 1 to 3 months ($2.2{\pm}0.3$, $2.5{\pm}0.3$ and $2.3{\pm}0.4$) were significantly higher than those in 4 to 6 months ($0.9{\pm}0.2$, $0.8{\pm}0.2$ and $0.3{\pm}0.2$, respectively) (p<0.05). In conclusion, the efficient periods for the production of OPU derived embryos was until 4 months, twice per week to produce over 64 transferable embryos and then replace new donor after 3 months use. The best replacement time is 3 months and could be maximized production of OPU derived embryos.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.41-51
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• 2002

The present study was carried out to examine the effect of cysteamine in vitro maturation (IVM) of porcine oocytes and development of porcine IVM/IVF Embryos. The results were summarized as follows : 1. The rates of nuclear maturation, penetrated oocytes, pronuclear formation, polyspermic oocytes and mean numbers of the penetrated sperm were not different in NCSU23 maturation medium with 0, 25, 50 and 100 $\mu$M cysteamine (P〉0.05). 2. The rates of blastocyst formation at day 7 after in vitro fertilization in 0, 25, 50 and 100 $\mu$M cysteamine were 17.9$\pm$6.1, 17.4$\pm$6.3, 24.2$\pm$1.9 and 16.9$\pm$2.0%, respectively. And the total cells were 30.7$\pm$2.4, 34.9$\pm$2.8, 39.6$\pm$2.3 and 36.8$\pm$3.6, respectively. Fifty $\mu$M cystealnine group was significantly higher than those of any other treatment groups (P＜0.05). 3. The ratios of ICM/total cells in 20~40% category were 20.5, 41.6, 19.5 and 31.5%, respectively. Twenty five $\mu$M cysteamine group was higher than those of other groups. 4. The rates of blastocyst formation at day 7 in the NCSU-23 culture medium of porcine IVF-produced embryos with 0, 25, 50, and 100 $\mu$M cysteamine were 16.0$\pm$0.2, 13.6$\pm$1.7, 25.0$\pm$0.8 and 15.7$\pm$4.5%, respectively. And the total cells were 27.0$\pm$3.7, 36.1$\pm$4.8, 34.0$\pm$3.8 and 25.2$\pm$4.4, respectively. Fifty $\mu$M cysteamine group was significantly higher than those of any other treatment groups (P＜0.05). 5. The ratios of ICM/total cells in 20~40% category were 53.8, 30.0, 16.6 and 11.1%, respectively. The addition groups of cysteamine were lower than those of control group. In conclusion, these results suggested that the addition of 50 $\mu$M cysteamine in the IVM medium and 25~50 $\mu$M cysteamine in IVC medium were effective on the blastocyst formation and total cells of blastocysts.

• Korean Journal of Ecology and Environment
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• v.35 no.3 s.99
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• pp.198-212
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• 2002

A numerical variation and abnormalities were studied on egg bags and embryos of Korean salamander, Hynobius leechii from agricultural habitat. The teratogenic and toxic effects of fungicide benomyl were also investigated with early embryos from non-agricultural habitat. We collected 144 egg bags from agricultural region, and 3418 of early embryos were contained. The lengths of egg bags were varied from 10 to 23 cm and the most frequent length was 19 cm. The number of embryos was varied from 7 to 43, and the most frequent range was 22 to 26. Spontaneous abnormalities were occurred in 406 embryos among 116 egg bags, and 24 kinds of external abnormalities were found. Individuals showing severe external defect were histologically studied and they showed optic dyspalsia, thyroid carcinoma, somatic muscular dysplasia, partial biaxial structure, decrease of red blood cells in the heart, cephalic degeneration and intestinal dysplasia. 385 embryos from non-agricultural region were exposed to 200 nM${\sim}$ 1 ${\mu}$M of benomyl at blastula or gastrula for 12 days. All embryo were dead in the concentration of 1 ${\mu}$M (LD$_{100}$) and 75% of embryos were dead in 800nM of benomyl. Speciflc effect due to benomyl was acrania or cephalic dysplasia and this restult suggests that the benomyl inhibit stongly to the development of neural tissue. These abnormal developments may be caused by antimitotic action, inhibition of tubulin complex, destruction of microtubule, inhibitions of neurulation and closing of neural fold, and by the inhibition of the movement of neural crest cells.