• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.72-72
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• 2003

The present study examined the possibility of cryopreservation of the D-shaped and umbo larvae of arkshell (Scapharca broughtonii), in terms of the survival rates after freezing and thawing. D-shaped and umbo larvae of arkshells were obtained from a shellfish farming on Yosu city. The average shell lengths were $93.3 \pm 10.1 \mu$m and $201.7 \pm 13.5 \mu$, respectively. Five cryoprotectants (CPAs), dimethyl sulfoxide (DMSO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol, were tested at the concentrations of 1.5, 2.0 and 2.5 M. After larvae suspended in CPAs, cryoprotectants were loaded in 0.5 ml straws at a larval density of 50-100 larvae per straw, and epuilibrated for 10 and 20 minute at room temperature ($23^{\circ}C$), repectively. Straws were cooled at a rate of $1^{\circ}C$/min from $0^{\circ}C$ to $-12^{\circ}C$, held for 5 min at $-12^{\circ}C$, and then cooled at $2^{\circ}C$/min to $-35^{\circ}C$ and equilibrated for 5 min followed by plunging in liquid nitrogen. After storage in liquid nitrogen for 1 day, straws were thawed in a $30^{\circ}C$ water. As soon as straws were observed to melt, larvae were diluted with an equal volume of ASW and then washed twice with a large volume of ASW at an interval of 2 min to unload the CPAs. The results showed that after equilibration for 10 and 20 minute at room temperature, no larvae survived using methanol as CPAs, and it was observed that larval shells all open slightly, and larval flesh broke down and slopped over the shells. The highest survival rates (D-shaped larvae: 77.6%, umbo larvae: 59.3%) were obtained with 2M DMSO, and 1.5M glycerol yielded survival rates of 53.8% for D-shaped larvae and 37.5% for umbo larvae. The surviving D-shaped larvae showed active rotary motion and perfect membrane integrity and cytoplasmic normality, and the vigorous movement of veliger cilia was observed inside the closed shells. The breakdown of tissue occurred in the abnormal larvae, and the isolated cell often run out of shells.

• Fisheries and Aquatic Sciences
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• 제18권4호
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• pp.417-420
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• 2015

We investigated the relationship between viability and IGF-1 receptor (IGF-1R) activity in D-shaped and umbo larvae of the surf clam Spisula sachalinensis after treatment with vitrification solution (VS) or freezing. In a toxicity assay, VS1, containing 5 M dimethyl sulfoxide (DMSO), was very harmful to D-shaped and umbo larvae. However, VS2, containing 5 M ethylene glycol (EG), was not harmful to either larval stage. Although VS2 had a promising toxicity test outcome, none of the larvae survived vitrification. After immersion into VSs and freezing, IGF-1R ${\beta}$-subunits were detected in all larvae; however, tyrosine phosphorylation of intracellular ${\beta}$-subunits was detected only in the control and live groups. These results suggest that activation of IGF-1R may influence surf clam larvae viability.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.103-103
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• 2002

This study was conducted to investigate cryopreservation of pearl oyster, Pinctada fucata martensii larvae. Four cooling rates (-0.25, -0.5, -0.75 and -1.0$^{\circ}C$/min.) were used to examine a proper cooling rate during cryopreservation of trochophores before seeding temperature (-12$^{\circ}C$). Seven developmental stages (early and late trochophores, early and late D-shaped larvae and early, middle and late umbo stage larvae) and different sugars (fructose, glucose and sucrose) were used to investigate optimal larval stage and effective sugar in cryopreservation of larvae. The survival rates of frozen-thawed trochophores increased at cooling rate of -1.0$^{\circ}C$/min. As larval developing, survival rate of frozen-thawed larvae increased, except umbo stage larvae, and especially late D-shaped larvae highly survived as 91%. Addition of sugar revealed positive effect on cryopreservation in this experiment and 0.2 M glucose and sucrose mixed with 2.0 M dimethyl sulfoxide significantly enhanced survival rate of larvae (P<0.05). The results of our study indicate that desirable cooling rate, developmental stages of larvae and effective sugar far cryopreservation of pearl oyster, P. fucata martensii larvae are -1$^{\circ}C$/min, late D-shaped larvae and 0.2 M glucose and sucrose, respectively.

• 한국수산과학회지
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• 제33권6호
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• pp.597-599
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• 2000

This study was performed to find out the cryoprotectants for cryopreservation of trochophores and early D-shaped larvae of surf clam, Spisula sachalinensis. Dimethyl sulfoxide (DMSO), ethylene glycol, 1,2-propanediol and methanol were used as cryoprotectant Each cryoprotectant was made to 1.0 M, 2.0 M, 3.0 M with dilution of 0.2 M fructose and 0.2 M sucrose. The trochophoers and early D-shaped larvae were immersed in each preparation for 10 minutes to reach equilibration and cryopreserved in liquid nitrogen. Survival rates of post-thawed trochophores and early D-shaped larvae in 2.0 M DMSO with 0.2 M sucrose were the highest as $91.4{\%}\;and\;78.9{\%}$, respectively.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2003년도 제3회 발생공학 국제심포지움 및 학술대회
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• pp.72-72
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• 2003

• 한국발생생물학회지:발생과생식
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• 제17권4호
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• pp.337-343
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• 2013

Post-thawed larval rearing in Pacific oyster Crassostrea gigas was performed to investigate the survival rate with time course in three kinds of larvae cryopreserved. The highest survival rate and larval activity index (LAI) of post-thawed larvae were obtained from the permeation in 0.2 M sucrose and 2.0 M ethylene glycol (EG) at $-1^{\circ}C/min$ in freezing speed showing the survival rates just after thawing of 63.8% in trochophore, 84.1% in D-shaped veliger and 56.3% in early umbo veliger. In post-thawed larval rearing with food supply, the larvae lasted their lives until 24 hours in trochophore, 75 hours in D-shaped veliger and 57 hours in early umbo veliger. The results suggested that each larval stage post-thawed revealed no more further development to subsequent respective stage.

• 한국패류학회지
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• 제18권2호
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• pp.77-82
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• 2002

피조개 D형 유생을 냉동보존하기 위한 적절한 첨가제 종류와 농도 및 동해방지제로 적용한 ethylene glycol 농도에 대한 냉동보존 효과실험을 실시하였다. 유생을 0$^{\circ}C$로 설정된 프로그램 냉동기로 옮겨 -1$^{\circ}C$/분의 냉동율로 -12$^{\circ}C$까지 냉동한 후 10분 동안 유지한 상태에서 식빙하고 -35$^{\circ}C$까지 재냉동한 다음, 즉시 액체질소통에 저장하였다. 1.0 M과 2.0 M ethylene glycol을 동해방지제로 사용하여 첨가제별로 D형 유생을 냉동한 결과, 0.5 M sucrose에서 생존율이 각각 50.5 ${\pm}$ 1.3%, 51.9 ${\pm}$ 1.7%로 가장 좋았다. 0.5 M sucrose를 첨가한 ethylene glycol의 농도별 냉동에서는 2.0 M이 생존율 51.9 ${\pm}$ 1.7%로 가장 높았다. 주사 및 투과전자현미경을 이용하여 유생을 관찰한 결과, 냉동 전, 후 유생의 외부형태 및 세포내 구조의 차이가 인정되지 않았다.

• 한국발생생물학회지:발생과생식
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• 제11권2호
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• pp.79-84
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• 2007

냉동보존이 진주조개(Pincata fucata martensii) 유생의 형태 및 구조에 미치는 영향을 알아보기 위하여 냉동전후 D형 및 각정기 유생을 광학 및 전자현미경으로 조사하였다. 동해방지제는 0.2 M sucrose를 첨가한 2.0 M $Me_2SO$를 사용하였다. 냉동후 유생은 일부 패각이 손상되긴 했지만 hinge와 prodissoconch가 뚜렷하게 나타났으며, 소포체, 지질 과립, 미토콘드리아, 핵 등을 포함한 세포내 소기관들이 고르게 분포되어 있었다. 또한, 섬모가 규칙적으로 배열되어 있었고, 섬모 아래 미토콘드리아와 지질과립이 위치해 있는 것이 관찰되었으나, 일부 해동된 유생에서 섬모의 불규칙적인 배열과 섬모환이 둥글게 뭉쳐져 있는 모습이 관찰되었다. 이러한 결과는 진주조개의 D형 유생과 각정기 유생이 냉동에 쉽게 영향을 받을 수 있다는 것을 보여준다. 따라서 냉동보존 시 세포의 손상을 감소시킬 수 있는 연구가 이루어져야 할 것으로 사료된다.

• 한국해양학회지
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• 제23권2호
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• pp.70-75
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• 1988

피조개(Anadara broughtonii)의 효율적(效率的)인 종묘생산기술개발(種苗生産技術開發)을 목적(目的)으로 1974년(年) 7월(月)부터 10월(月)까지 진해만내(鎭海灣內)의 가조도 앞바다에서 부유유생(浮游幼生)의 출현시기(出現時期)와 각(各) 유생단계별(幼生段階別) 생존율(生存率)을 조사(調査)하였다. 조사기간중(調査期間中) 크기가 약(約) $94.3{\times}72.7{\mu}m$인 D형유생(型幼生)은 8월(月) 25일(日), 8월(月) 31일(日) 및 9월(月) 9일(日)에 각각(各各) 가장 많은 개체가 출현(出現)하였으며, $141.6{\times}108.4{\mu}m$인 umbo형유생(型幼生)은 9월(月) 6일(日), 9월(月) 12일(日) 및 9월(月) 20일(日)에, $269.3{\times}221.7{\mu}m$의 성숙유생(成熟幼生)은 9월(月) 20일(日), 9월(月) 25일(日) 및 10월(月) 5일(日)에 각각(各各) 출현개체수(出現個體數)가 가장 많았다. D형유생(型幼生)에서 umbo형유생(型幼生)으로 되는데는 약(約) 11~12일(日) 걸렸으며, 일간생존율(日間生存率)은 0.93, 생존개체수(生存個體數)의 백분비(百分比)는 45%였다. umbo형유생(型幼生)에서 성숙유생(成熟幼生)으로 되는데는 약(約) l3~15일 걸렸으며 일간생존율(日間生存率)은 0.93, 생존개체수(生存個體數)의 백분비(百分比)는 36%였다. D형유생(型幼生)의 발생(發生)에서 채묘(採苗)의 대상(對象)이 되는 성숙유생(成熟幼生)으로 되는데는 25~26일(日)이 걸렸으며 이 기간중(期間中) 생존개체(生存個體)의 백분비(百分比)는 16%였다.

• 한국양식학회:학술대회논문집
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• 한국양식학회 2003년도 추계학술발표대회 논문요약집
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• pp.145-145
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• 2003

To evaluate the effect of PAHs on fertilization and larval development of the Pacific oyster, Crassostrea gigas via in vivo exposure for 30 days. Conditioned C. gigas adult brood stock was taken from oyster farm in May 2003 and raised in flow through tank at 20-22$^{\circ}C$ with feeding equivalently mixed marine microalgae: Chaetoceros simplex, C. gracilis, Isochrysis galbana and Tetrasemis tetrathele. The oysters were exposed to PAHs cocktail at 200ppb (10 species) until they are fully riped with an untreated group. Motility of sperm, Fertilization and The percent of D-shaped larvae was measured under microscope (Olympus BX-50). Fertilization capability using dry sperm and eggs from both non- and PAHs treated brood stocks was significantly reduced at the treated group (P<0.01). In addition to the origin effect, it was obvious that fertilization of eggs from both group were adversely suppressed in consistent with PAHs level (P<0.01). The percent of D-shaped larvae in treated group was significantly reduced comparing to non-treated. The percent was adversely correlated with PAHs level, which was more significantly reduced in treated group. Therefore, it was obvious that PAHs exposure on the Pacific oyster has adverse affects on the sperm as well as larvae in both maternal and acute source.