• Proceedings of the Korean Society of Developmental Biology Conference
• /
• 2003.10a
• /
• pp.72-72
• /
• 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
• /
• v.18 no.4
• /
• pp.417-420
• /
• 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.

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2002.11a
• /
• pp.103-103
• /
• 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.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.33 no.6
• /
• pp.597-599
• /
• 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.

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2003.10a
• /
• pp.72-72
• /
• 2003

• Development and Reproduction
• /
• v.17 no.4
• /
• pp.337-343
• /
• 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.

• The Korean Journal of Malacology
• /
• v.18 no.2
• /
• pp.77-82
• /
• 2002

This study was performed to find out the optimal kind of additive and concentration of ethylene glycol (EG) as the cryoprotectant on the D-shaped larvae of arkshell, Scapharca broughtonii. The larvae in straws was carried in the programed freezer set uo at 0$^{\circ}C$, frozen to -12$^{\circ}C$ by the freezing rate of -1$^{\circ}C$/min, held for 10 minutes, seeded at -12$^{\circ}C$, finally refrozen to -35$^{\circ}C$ by the freezing rate of -1$^{\circ}C$/min and then directly plunged into liquid nitrogen. The survival of larvae frozen in 1.0 M and 2.0 M ethylene glycol added with 0.5 M sucrose were high (50.5 ${\pm}$ 1.3% and 51.9 ${\pm}$ 1.7%, respectively) in the early D-shaped larvae cryopreserved in 1 M and 2 M ethylene glycol diluted with 0.2 M and 0.5 M fructose, glucose and sucrose. In the late D-shaped larvae were cryopreserved according to five concentrations of ethylene glycol added with 0.5 M sucrose, the survival of larvae frozen in 2.0 M ethylene glycol was the highest as 51.9 ${\pm}$ 1.7%. The morphological differences in cells between unfrozen and frozen-thawed D-shaped larvae were not found by the scanning and transmission electron microscopy.

• Development and Reproduction
• /
• v.11 no.2
• /
• pp.79-84
• /
• 2007

The freezing susceptibilities of two larval stages (D-shaped and umbo) of the pearl oyster (Pinctada fucata martensii) were evaluated by the electron microscopy (light, transmission electron and scanning electron). The morphological shapes were examined from each pre-frozen or frozen-thawed stage of the cryopreserved larvae in liquid nitrogen by using the cryoprotectant, dimethyl sulfoxide ($Me_2SO$) mixed with sucrose. Although a portion of the shell was damaged, the hinge and prodissoconch were intact and clearly visible after preservation in liquid nitrogen. In addition, the cytoplasm of the frozen-thawed larvae maintained the normal organelle integrities, e.g., endoplasmic reticula, lipid droplets, mitochondria, nucleus and microvilli. However, some of the frozen-thawed larvae showed irregularly arranged cilia, rough shell surfaces and round-lumped cilium heads. These results indicate that P. fucata martensii larvae are susceptible to freezing, at least at those two critical developmental stages (D-shaped and umbo), and suggest a new industrial investigation including reduction method of cell injury for preserving microbial starter cultures need to be developed.

• 한국해양학회지
• /
• v.23 no.2
• /
• pp.70-75
• /
• 1988

In order to set up a predictive model for an effective spat collection of ark shells, Anadara broughtonii, the survival rate and the time required for each developmental stage of planktonic larvae were investigated during the period from July 1 to October 30 in 1974, in one of the main ark shell seed collection areas, Chinhae Bay, in the southern part of Korea. The advent of D-shaped larvae ca. $94.3{\times}72.7{\mu}m$ long had three peaks during the surveyed period: August 25, August 31 and September 9, umbo-shaped larvae ca. $141.6{\times}108.4{\mu}m$ and full grown larvae ca. $269.3{\times}221.7{\mu}m$ long also showed three peaks: September 6, September 12 and September 20 for the former, and September 20, September 25 and October 5 for the latter, respectively. About 11 to 12 days was required for D-shaped larvae to develop to umbo-shaped stage. At this intermorphological stage, the daily survival rate was 0.93 with a total survival rate of 45% for the stage. The time required for umbo-shaped larvae to develop to full grown larvae varied from 13 to 15 days with a daily survival rate of 0.93 and with a total survival rate of 36% in the period. Twenty-five to twenty-six days were required for each peak group of the D-shaped larvae to reach a full grown stage, and their total survival rate was 16% during this developmental stage.

• Proceedings of the Korean Aquaculture Society Conference
• /
• 2003.10a
• /
• pp.145-145
• /
• 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.