• Journal of Marine Life Science
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• v.1 no.1
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• pp.70-78
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• 2016

Microalgae are of significant importance for future biotechnological applications. Many microalgae banks or laboratories attempt to maintain various microalgae for further research purposes. Cryopreservation has been preferred to reduce a labor-intensive and costly routine sub-culturing. Cryopreservation can also diminish the genetic drift risk. However, cryopreservation as a long term storage of microalgae method are still in developing progress because it cannot be generalized for all microalgae. Microalgae types, cryoprotectant agents (CPAs) types, freezing and thawing methods are the most important factors that should be considered for cryopreservation. In this short review the basic principles and the current advanced of microalgae cryopreservation methods are discussed with a suggested starting parameters for microalgae cryopreservation.

• Journal of Plant Biotechnology
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• v.45 no.4
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• pp.400-408
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• 2018

Biodiversity has continued to degrade in the $21^{st}$ century due to global warming occasioned by destruction of the environment around the world.. The Nagoya protocol places Korea in a unique position to effectively develop and protect its domestic genetic resources. Microalgae under study in this research contains large amount of antioxidant substances such as beta carotene and astaxanthin, that can be used as biological resource owing to the large amounts of biomass that can be secured through photosynthesis. However, it is difficult to preserve it since cryopreservation method used for long-term preservation is yet to be developed. A basic study for long term cryopreservation was carried out on Nizschia frustulum and Nitzschia amabilis which belong to marine diatoms. As cryoprotectants (CPAs), glycerol, DMSO, and methanol which penetrate into cells were prepared at 5%, 10%, and 15% concentrations each, in case of methanol, it was tested at concentrations of 5%, 10% and 12% by its nature. Two kinds of microalgae, N. frustulum and N. amabilis, were diluted with $10^2$, $10^3$ and $10^4cells\;ml^{-1}$, respectively. The highest survival rate was shown at12% concentration of methanol, and the figures were $6.94{\pm}0.31%$ in N. frustulum and $8.85{\pm}0.16%$ in N. amabilis. As a result of 3 weeks cultivation of thawed microalgae after freezing, the result is shows that N. frustulum increased about 10 times faster and N. amabilis increased about 12 times the original concentration.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.3
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• pp.328-335
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• 2009

This study was undertaken to investigate the influence of exposure and removal of four different cryoprotectants (CPAs) on the ATP content of cumulus cell-enclosed (COs) and cumulus cell-denuded (DOs) immature porcine oocytes. The in vitro nuclear maturation of the COs, exposed to and removed from the CPAs was also assessed. Both COs and DOs were exposed to 1.5 M concentrations of each CPA (ethylene glycol (EG); propylene glycol (PG); dimethyl-sulfoxide (DMSO); and glycerol (G)) for durations of 5, 15, and 30 minutes at room temperature ($23.5{\pm}1.5^{\circ}C$), and immersed in physiological saline supplemented with 20% (v/v) fetal bovine serum for 5 minutes ($39^{\circ}C$) to remove each CPA. Before, during and after exposure to each CPA, the ATP content of both the COs and the DOs was measured. After removal from each CPA an aliquot of the COs was matured for 44${\pm}$2 h, and their nuclear maturation rates were measured up to the metaphase stage of the second meiotic division (the M-II stage). The maturation rates up to the M-II stage were not significantly different between all the groups that were exposed to each CPA for 5 minutes. For 15 and 30 minute exposures, the maturation rates of the COs exposed to PG, DMSO and EG were almost the same as those of the control groups; however, the rates of G group exposed for 15 and 30 minutes were significantly lower (p<0.05) than the control group. These groups were also found to have a decrease in the ATP content of COs and DOs during and after exposure for the same periods (p<0.05). In addition, although the ATP contents of the COs after exposure to EG for any period were the same as the controls, the ATP content of the DOs exposed to EG for any period were significantly lower (p<0.05) than those of the controls. When the ATP content of the COs and DOs of each CPA were compared, the DOs exposed to PG were found to have a significantly greater level (p<0.05) than DOs exposed to G for any duration. In addition, the ATP content of DOs exposed to PG for 30 min and removal was also higher (p<0.05) than when exposed to DMSO for the same period. These findings indicate that PG may be a useful CPA for the cryopreservation of immature porcine oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.1
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• pp.24-30
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• 2011

Objective: The objectives of this study were to analyze efficacy of immature and mature mouse oocytes after vitrification and warming by applying various combinations of cryoprotectants (CPAs) and/or super-rapid cooling using slush nitrogen ($SN_2$). Methods: Four-week old ICR female mice were superovulated for GV- and MII-stage oocytes. Experimental groups were divided into two groups. Ethylene glycol (EG) only group: pre-equilibrated with 1.5 M EG for 2.5 minutes and then equilibrated with 5.5 M EG and 1.0 M sucrose for 20 seconds. EG+dimethylsulfoxide (DMSO) group: pre-equilibrated with 1.3 M EG+1.1 M DMSO for 2.5 minutes and equilibrated with 2.7 M EG+2.1 M DMSO+0.5 M sucrose for 20 seconds. The oocytes were loaded onto grids and plunged into $SN_2$or liquid nitrogen ($LN_2$). Stored oocytes were warmed by a five-step method, and then their survival, maturation, cleavage, and developmental rates were observed. Results: The EG only and EG+DMSO groups showed no significant difference in survival of immature oocytes vitrified after warming. However, maturation and cleavage rates after conventional insemination were greater in the EG only group than in the EG+DMSO group. In mature oocytes, survival, cleavage, and blastocyst formation rates after warming showed no significant difference when EG only or EG+DMSO was applied. Furthermore, cleavage and blastocyst formation rates of MII oocytes vitrified using $SN_2$ were increased in both the EG only and EG+DMSO groups. Conclusion: A combination of CPAs in oocyte cryopreservation could be formulated according to the oocyte stage. In addition, $SN_2$ may improve the efficiency of vitrification by reducing cryoinjury.

• YAKHAK HOEJI
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• v.37 no.2
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• pp.143-148
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• 1993

The objective of this study was to evaluate the sedative activity of four aporphine alkaloids (APA) and nine cyclopeptide alkaloids(CPA), which had been isolated from the seeds (sanjoin) of Zizyphus vulgaris var. spinosus, and the fruits and stem bark of Zizyphus jujuba var. inermis. The assessment of sedative activity was carried out, employing a hexobarbital-induced sleeping time method in mice. When the relative sedative potency of sanjoinine-A(CPA) was given as one unit, those of nuciferine (APA), lysicamine (APA), chlorpromazine (positive control), and sanjoinine -Ahl (an epimer of sanjoinine-A) were 13, 6.5, 5, and 3, respectively. The sedatvie activities of other CPAs were much lower than those of sanjoinine-A and -Ahl, and other APAs were not active. On heat treatment, nuciferine and lysicamine were degraded into some artifacts which exhibited no sedative activity, while sanjoinine-A was converted into sanjoinine-Ahl which showed more potent sedative activity. These results suggested that nuciferine and sanjoinine-A were major sedative components of native sanjoin, and that sanjoinine-A and its epimeric artifact, sanjoinineAhl were the active principles of roasted sanjoin. It provides a scientific basis for heat-processing (roasting) of this Oriental medicine.

• Journal of the Society of Naval Architects of Korea
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• v.56 no.4
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• pp.352-360
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• 2019

International Maritime Organization (IMO) regulates an emission of greenhouse gases by creating an Energy Efficiency Design Index (EEDI) to reduce environmental pollution. In propulsion system field, studies are under way on Energy Saving Device (ESD), which can improve propulsion efficiency with the propeller, to reduce the EEDI. Among the studies, the study of Pre-Swirl Stator (PSS) has been actively conducted from long time ago. Recently the variable pith angle type pre-swirl stator has been studied to improve the propulsion efficiency in non-uniform flow fields of the Stern. However, for traditional design methods, no specific design method has been established on the blade or location of radius. In this study, proper design method is proposed for each blade or location for radius according to hydrodynamic pitch angle.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.47 no.1
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• pp.39-44
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• 2014

This study aimed to find an optimal diluent and cryoprotective agent (CPA) during cryopreservation of common Korean bitterling Acheilognathus signifier sperm. The bitterling is an endangered species in Korea, and this study will enable conservation and further technical development of artificial seed production. We tested the effects of cryopreservation and toxicity by the type of diluent and CPA. The optimal combination of diluent and CPA for cryopreservation was 300 mM glucose+10% methanol, resulting in a survival rate and sperm activity index (SAI) of $96.3{\pm}1.5%$, and $3.0{\pm}0.0$ respectively, and no significant difference compared to fresh sperm. The survival rate and SAI of post-thawed sperm was $9.7{\pm}1.5%$, $0.8{\pm}0.3$ respectively, which was significantly higher than had been achieved with other diluents and CPAs.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.44 no.6
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• pp.665-670
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• 2011

The rotifer Brachionus plicatilis is one of the most important food organisms in aquaculture. The resting eggs produced by mictic female rotifers are easily stored and hatched, making them useful as the starter for the mass culture of rotifers in marine larval culture. This study examined the optimum preservation method for resting eggs to ensure a high hatching rate. To produce resting eggs, the marine rotifer B. plicatilis was cultured with Nannochloris oculata (KMMCC 16). The resting eggs were harvested and cryopreserved using 5% and 10% methanol (MeOH), dimethylsulfoxide (DMSO), and glycerol as cryoprotectant agents (CPAs). The cryopreservation comprised slow or rapid freezing and the resting eggs were stored for one month in liquid nitrogen ($-196^{\circ}C$). The resting eggs were also dried at different temperatures (30, 40, and $50^{\circ}C$) and for different times (1, 2, and 3 h). In general, the hatching rates of the resting eggs preserved with CPA were higher than those without CPA and the slow freezing method was better than the rapid freezing method. However, the optimum CPA concentration for the hatching rate of the resting eggs varied with the freezing method and kind of CPA, and the CPA also affected the viability of the resting eggs. Dried resting eggs had a high, rapid hatching rate over 80%. The moisture content of the resting eggs cryopreserved in liquid nitrogen affected the hatching rate. Drying at $30^{\circ}C$ for 1 hour resulted in a high hatching rate of the resting eggs. In conclusion, drying at $30^{\circ}C$ for 1 hour and preservation in liquid nitrogen with the slow freezing method, without CPA, is recommended for a high hatching rate (ca. 95%) of rotifer resting eggs.

• Journal of Convergence for Information Technology
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• v.11 no.1
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• pp.128-135
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• 2021

The possibility and effectiveness of cryopreservation was determined to assess survival rates and improve stock management of thawed embryos of Manila clam, Ruditapes philippinarum. The ideal freezing rates were designed and tested to allow cryoprotectants to equilibrate across the membrane during freezing. Survival rates ranging from 0 to 64.3% were obtained using a stepwise freezing protocol compared with 82.3% control rates. Embryos of Ruditapes philippinarum were equilibrated in 2 CPAs plus sea water for 10 min at 25℃ and then cooled at -1℃/min from 20℃ to -12℃. Straws containing more than 100 embryos were held at 12℃ for 5 min allowing equilibration after seeding and slowly cooled at 2℃/min. to -35℃ for 30 min for equilibration before quenching in liquid nitrogen. Dimethyl sulfoxide (DMSO) is the best cryoprotectant indicated for embryos of R. philippinarum with a survival rate of 64.3±3.28% in the presence of 2.0 M DMSO.

• Journal of Microbiology and Biotechnology
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• v.34 no.5
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• pp.1135-1145
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• 2024

When cells are exposed to freezing temperatures, high concentrations of cryoprotective agents (CPA) prevent ice crystal formation, thus enhancing cell survival. However, high concentrations of CPAs can also cause cell toxicity. Exopolysaccharides (EPSs) from polar marine environments exhibit lower toxicity and display effects similar to traditional CPA. In this study, we sought to address these issues by i) selecting strains that produce EPS with novel cryoprotective activity, and ii) optimizing culture conditions for EPS production. Sixty-six bacteria producing mucous substances were isolated from the Ross Sea (Antarctic Ocean) using solid marine agar plates. Among them, Pseudoalteromonas sp. RosPo-2 was ultimately selected based on the rheological properties of the produced EPS (p-CY02). Cryoprotective activity experiments demonstrated that p-CY02 exhibited significantly cryoprotective activity at a concentration of 0.8% (w/v) on mammalian cells (HaCaT). This activity was further improved when combined with various concentrations of dimethyl sulfoxide (DMSO) compared to using DMSO alone. Moreover, the survival rate of HaCaT cells treated with 5% (v/v) DMSO and 0.8% (w/v) p-CY02 was measured at 87.9 ± 2.8% after freezing treatment. This suggests that p-CY02 may be developed as a more effective, less toxic, and novel non-permeating CPA. To enhance the production of EPS with cryoprotective activity, Response Surface Methodology (RSM) was implemented, resulting in a 1.64-fold increase in production of EPS with cryoprotective activity.