• 한국가축번식학회지
• /
• 제15권2호
• /
• pp.103-107
• /
• 1991

This study was carried out to investigate the survival rate in vitro culture after frozen-thawed to used DMSO(dimethyl sulfoxide), glycerol and ethylene glycol of cryoprotective agents at the zona pellucida removed and encased into alien bisected embryo of the mouse early embryos. The results obtained from this study were as follows : 1. The survival rate of in vitro culture after frozen-thawed to used cryoprotective agents of three kinds at the zona pellucida removed bisected morula was 46.6%, 35.8% and 27.3%, total or mean were 36.6%, respectively. 2. The survival rate of in vitro culture after frozen-thawed to used cryoprotective agents of three kinds at the encased into alien bisected morula was 70.6%, 65.3% and 66.4%, total or mean were 67.4%, respectively. 3. The survival rate of in vitro culture after frozen-thawed to used cryoprotective agents of three kinds at the zona pellucida removed bisected blastocysts was 50.4%, 36.7% and 30.4%, total of mean were 39.2%, respectively. 4. The survival rate of in vitro culture after frozen-thawed to used cryoprotective agents of three kinds at the encased into alien bisected blastocysts was 71.1%, 66.7% and 63.9%, total or mean were 67.2%, respectively.

• 한국가축번식학회지
• /
• 제15권2호
• /
• pp.97-101
• /
• 1991

This study was carried out to investigate the survival rate of in vitro culture after frozenthawed, to used DMSO(dimethyl sulfoxide), glycerol and ethylene glycol of cryorpotective agents at the zona pellucida removed and intact on the morulae and blastocysts. The results obtained from this study were as follows : 1. The survival rate of in vitro culture after frozen-thawed to used cryoprotective agents of three kinds at the morulae was 86.0%, 87.1% and 83.3%, total or mean were 85.5%, respectively. 2. The survival rate of in vitro culture after frozen-thawed to used cryoprotective agents of three kinds at the zona pellucida removed morulae was 53.2%, 42.3% and 37.5%, total or mean were 44.3%, respectively. 3. The survival rate of in vitro cultrue after frozen-thawed to used cryoprotective agents of three kinds at the blastocysts was 89.4%, 86.2%, total or mean were 86.7%, respectively. 4. The survival rate of in vitro culture after frozen-thawed to used cryoprotective agents of three kinds at the zona pellucida removed blastocysts was 55.8%, 51.6% and 40.6%, total or mean were 49.3%, respectively.

• Journal of Microbiology and Biotechnology
• /
• 제34권5호
• /
• pp.1135-1145
• /
• 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.

• Integrative Medicine Research
• /
• 제6권1호
• /
• pp.12-18
• /
• 2017

Cryopreservation is a process that preserves organelles, cells, tissues, or any other biological constructs by cooling the samples to very low temperatures. The responses of living cells to ice formation are of theoretical interest and practical relevance. Stem cells and other viable tissues, which have great potential for use in basic research as well as for many medical applications, cannot be stored with simple cooling or freezing for a long time because ice crystal formation, osmotic shock, and membrane damage during freezing and thawing will cause cell death. The successful cryopreservation of cells and tissues has been gradually increasing in recent years, with the use of cryoprotective agents and temperature control equipment. Continuous understanding of the physical and chemical properties that occur in the freezing and thawing cycle will be necessary for the successful cryopreservation of cells or tissues and their clinical applications. In this review, we briefly address representative cryopreservation processes, such as slow freezing and vitrification, and the available cryoprotective agents. In addition, some adverse effects of cryopreservation are mentioned.

• 한국수정란이식학회지
• /
• 제7권1호
• /
• pp.13-19
• /
• 1992

This study were carried out to investigate the effective concentration of cryoprotective agents and sucrose by one-step straw method, and to determine the optimum thawing temperature and equilibration time of frozen porcine embryos. The porcine embryos foflowing dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival rate was defined by FDA test. The results are sunnnarized as follows : 1. The survival rates of porcine embryos after ultrarapid frozen4hawing in the freezing medium with a various concentration of glycerol, DMSO and propanediol added 0.25M sucrose were higher survival rate than those of sucrose concentration of 0.50M. 2. The survival rates of porcine embryos after ultrarapid ftozen4hawing in the freezing medium added 0.25M and 0.SOM sucrose were higher survival rate than those of sucrose concentration of 0.75M and 1.00M. 3. The temperature thawed at 2$0^{\circ}C$ and 3$0^{\circ}C$ resulted in a significantly higher embryos survival rate after 72 hrs in culture than did at 35$^{\circ}C$. 4. The equilibration time on the survival rate of porcine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time(10~20 min.).

• Clinical and Experimental Reproductive Medicine
• /
• 제18권1호
• /
• pp.81-87
• /
• 1991

There studies were carried for evaluation of the efficiency of freezing of hamster oocytes for use in a human sperm penetration assay. The hamster oocytes fully equilibrated in various cryoprotectant agents and inseminated with human sperm. After insemination with hamster oocytes, there was no difference in penetrated rates. Cumulus free oocytes equilibrated in 1.5M various cryoprotective agents and slowely cooled to temperature $-30^{\circ}C$ before rapid cooling and storage in nitrozen tank. After rapid thawing, survival rates of frozen oocytes according to cryo-protective agents were examined and the human sperm penetration assay with zona free hamster oocytes was conducted. 1. Survival rates of oocytes after cryoprotectants exposure have no significant difference (range 88-91%) and peneration rate was 51.1%. 2. Recovery and survival rate of frozen-thawed oocytes were 85.1 and 66.8%. There was no significant difference on cryoprotective agents. 3. Penetration rates of the frozen-thawed and intact oocytes were 69.0 and 77.0%, respectively. 4. Hamster oocytes cryopreservation provides a convenient way of supplying and trans-porting hamster oocytes for the assessment of the fertilizing potential of human spermatozoa.

• 한국수정란이식학회지
• /
• 제9권1호
• /
• pp.65-71
• /
• 1994

This study were carried out to investigate the effective concentration of cryoprotectant agents and sucrose by one-step straw method, and to determine the optimum thawing temperature and equilibration time of frozen bovine embryos. The bovine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 3O$^{\circ}C$ water. Survival rate was defined by FDA test. The results are summarized as follows : 1. The survival rate of bovine embryos thawed after rapid freezing in the freezing medium containing a various kinds of cryoprotective agents added 0.25M and O.50M sucrose were 28.6% and 25.0%, 35.1% and 31.6%, 32.4% and 24.4%, 34.2% and 28.2%, 18.9% and 17.6%, 14.7.% and 21.6%, respectively. 2. The survival rate of bovine embryos thawed after rapid freezing in the freezing medium containing a various concentration of sucrose added 1.5M and 2.OM glycerol, i.5M and 2.OM DMSO and 1.5M and 2.OM propanediol were 22.9~37.8%, 2O.7~31. 3%, 19.2~30.0% and 17.2~25.0%, respectively. 3. The temperature thawed at 2$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 3$0^{\circ}C$ and 35$^{\circ}C$. 4. The equilibration time on the survival rate of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (1O~20 min.).

• 한국가축번식학회지
• /
• 제15권2호
• /
• pp.133-139
• /
• 1991

This stduy was carried out in order to investigate the effects of concentration and equilibration time of cryoprotective agents on survival rate of slow and ultrarapidly frozen in vitro fertilized bovine embryos. In vitro fertilized bovine embryos, following dehydration by cryoprotective agents and sucrose, were slowly freezed(from 2$0^{\circ}C$ to -7$^{\circ}C$/-1$^{\circ}C$/min., from -7$^{\circ}C$ -35$^{\circ}C$/-0.2$^{\circ}C$/min. from -35$^{\circ}C$ to -38$^{\circ}C$/-0.3$^{\circ}C$/min.) by cell freezer and directly plunged into liquid nitrogen and thawed in 38$^{\circ}C$ water. Survival rate was defined by development rate to the morula and blastocyst stage after in vitro cultured and FDA test. The results are summarized as follows : 1. The survival rates of in vitro fertilized bovine embryos after slow frozen-thawing in the freezing medium of 0.25M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋2.0M propanediol were 84.3%, 85.9%, 77.8%, 74.3%, respectively. 2. The survival rates of in vitro fertilized bovine embryos after slow frozen-thawing in the freezing of 0.50M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋2.0M propanediol were 83.8%, 85.1%, 71.4%, 74.6%, respectively. 3. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing of 0.25M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋3.0M propanediol were 69.3%, 70.8%, 63.2%, 67.1%, respectively. 4. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing of 0.25M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋2.0M propanediol were 69.4%, 70.1%, 62.3%, 63.5%, respectively. 5. The survival rates of in vitro fertilized bovine embryos after slow and ultrarapid fromthawing in the freezing medium of sucrose added cryoprotective agents were not significant difference between 5min. and 10min. of equilibration time.

• 한국발생생물학회지:발생과생식
• /
• 제15권4호
• /
• pp.301-308
• /
• 2011

결빙억제제(CPA)의 종류, 농도 및 침지시간이 참전복 Haliotis discus hannai 발생배의 생존활성에 미치는 영향을 조사하였다. 4세포, 담륜자 및 피면자를 dimethyl sulfoxide, ethylene glycol, propylene glycol 농도별로 10분, 20분 및 30분간 침지하였다. CPA 침지 후 각 발생배의 발생진행률은 각 CPA 농도가 낮을수록, CPA 침지시간이 짧을수록 높았다. 또한, 피면자에서 비틀림전까지의 발생진행률은 4세포에서 담륜자까지의 것보다 높았다. CPA 침지 후 발생진행률 및 유생활성지수는 ethylene glycol에서 가장 높았다. 참전복 발생배의 냉동보존 효과를 높일 수 있는 CPA 및 농도는 ethylene glycol 2.0 M, 평형시간은 30분인 것으로 추정된다.

• Fisheries and Aquatic Sciences
• /
• 제24권2호
• /
• pp.63-77
• /
• 2021

The aim of this research was to investigate different factors, including cryoprotective agents (CPAs), diluents, dilution ratios, equilibrium times, freezing rates, and thawing methods to optimize cryopreservation protocols for large yellow croaker (Larimichthys crocea). The parameters evaluated were sperm motility, sperm activity index (SAI), survival rate, and DNA damage. Different types of CPAs, such as dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), methanol, and glycerol, were tested for sperm preservation. The highest motility, SAI, and survival rate were observed when EG was used. Different diluents such as Stein's solution, Hank's balanced salt solution, marine fish Ringer's solution, artificial seminal plasma (ASP) of small yellow croaker, and Cortland solution were investigated. The highest post-thaw motility was observed upon using ASP as the diluent. Different concentrations of EG were then mixed with ASP to identify the optimal EG concentration. Experimental results showed that the motility (70.33 ± 1.20%), SAI (5), and survival rate (78.30 ± 0.42%) of post-thaw sperm were optimum when 10% EG and ASP were used as the CPA and diluent of cryopreservation, respectively. Post-thaw sperm motility was high at equilibration times below 150 s and at an optimum dilution ratio of 1:1 (sperm: CPA + diluent) and was not significantly different compared with fresh sperm motility. The freezing rate was found to be slow below -10℃/min. The thawing temperature of 45℃ was identified as ideal. The percentage of tail DNA in post-thaw sperm at 10% EG and ASP was also investigated and was found to have more significant DNA damage than that in fresh sperm but significantly lower damage than that in post-thaw sperm at EG concentrations of 5%, 15%, and 20% (p < 0.05). The cryopreservation protocols obtained in this study will be useful in large yellow croaker hatcheries.