• Fisheries and Aquatic Sciences
• /
• v.15 no.1
• /
• pp.77-81
• /
• 2012

We assessed the toxicity of cryoprotectant agents (CPAs) to gametophytic thalli of red alga Porphyra yezoensis at room temperature. The CPAs used were: dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol (GC), 1,2-butanediol (1,2-BD), 1,3-butanediol (1,3-BD), 2,3-butanediol (2,3-BD), 1,3-propanediol (1,3-PD) and propylene glycol (PG). CPA concentrations of 10, 15, 20, 25, 30, 35, 40, 45, and 50% were employed with 30 or 60 s immersion. The toxicity of the eight CPAs to gametophytic thalli of P. yezoensis was in the order: 1,3-BD < DMSO ${\approx}$ 2,3-BD ${\approx}$ PG ${\approx}$ EG < GC < 1,3-PD ${\approx}$ 1,2-BD. All thalli were more sensitive to high CPA concentrations, and most (>75%) thalli survived exposure to 10-25% CPA for 60 s. These data will facilitate selection of the optimal cryoprotectant concentration for cryopreservation of P. yezoensis thalli.

• 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.

• Journal of Plant Biotechnology
• /
• v.45 no.4
• /
• pp.400-408
• /
• 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
• /
• v.22 no.3
• /
• pp.328-335
• /
• 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
• /
• v.38 no.1
• /
• pp.24-30
• /
• 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.

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

• Clinical and Experimental Reproductive Medicine
• /
• v.37 no.4
• /
• pp.307-319
• /
• 2010

Objective: Vitrification requires a high concentration of cyroprotectant (CPA) and an elevated cooling speed to avoid ice crystal formation. We have evaluated the effect of different combinations of cooling rate and CPA on embryonic integrity (developmental competence) in order to increase the efficiency of vitrification without impairing embryo viabilit. We hypothesized that the combination of CPA or the increase of cooling rates can reduce the concentration of toxic CPA for vitrification. As consequently, we performed experiments to evaluate the effect of various composition of CPA or slush nitrogen ($SN_2$) on the mouse embryonic development following vitrification using low CPA concentration. Methods: Vitrification of mouse embryos was performed with EM grid using liquid nitrogen ($LN_2$) or $SN_2$ and different composition of CPAs, ethylene glycol (EG) and dimethylsulfoxide (DMSO). After vitrification-warming process, their survival and blastocyst formation rates were examined. For analyzing long-term effect, these blastocysts were transferred into the uterus of foster mothers. Results: Survival and blastocyst formation rates of vitrified embryos were higher in EG+DMSO group than those in EG only. Furthermor, the group using $SN_2$ with a lower CPA concentration showed a higher survival of embryos and developmental rates than group using $LN_2$. Conclusion: The combination of EG and DMSO as CPAs may enhance the survival of mouse embryos and further embryonic development after vitrification. $SN_2$ can generate high survival and developmental rate of vitrified/warmed mouse embryos when a lower concentration of CPA was applied. Therefore, these systems may contribute in the improvement of cryopreservation for fertility preservation.

• Journal of Marine Life Science
• /
• v.8 no.2
• /
• pp.115-120
• /
• 2023

This study aims to find out a suitable extender and cryoprotective agent (CPA) for cryopreservation and its optimum concentration in order to conduct planned artificial seed production of Korean bullhead Pseudobagrus fulvidraco and to preserve superior sperm. Experiments were designed to investigate the effects of the different combinations of three extenders (I: 300 mM glycose, II: Kurokura extender, III: Li extender), four cryoprotectants (dimethyl sulfoxide, ethylene glycol, methanol and glycerol) and four concentrations (5, 10, 15, 20%) on the cryopreservation of Korean bullhead sperm. Postthawed sperm survival rate and sperm activity index (SAI) were detected to evaluate the effects of sperm cryopreservation. The optimal combination of extender and CPA for cryopreservation of Korean bullhead sperm was extender III + 10 and 15% methanol, resulting in a survival rate and SAI of 66.9 ± 8.7, 67.3 ± 13.1% and 2.6 ± 0.4, 2.6 ± 0.5 respectively, which was higher than had been achieved with other extenders and CPAs.