• Journal of Animal Reproduction and Biotechnology
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• v.35 no.2
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• pp.207-213
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• 2020

Cryopreservation is used for blastocyst preservation of most mammalian embryos and is an important technique for breeding. We aimed to compare the efficiency of the cryopreservation method using the standard Cryotop device and the ReproCarrier device, a domestic product manufactured in Korea. The efficacy of the two devices was analyzed based on the survival rate, intracellular levels of reactive oxygen species (ROS), and apoptosis of the vitrified bovine blastocysts. The survival rates of the vitrified-warmed blastocysts were similar between the ReproCarrier group (58.4 ± 17.7%) and Cryotop group (59.9 ± 14.1%). Intracellular ROS levels and apoptotic index were determined by DCFDA staining and TUNEL assay. Changes in intracellular ROS levels, number of total nuclei, and cellular apoptosis of vitrified blastocysts after cryopreservation were not significantly different between the two groups. These results indicate that the ReproCarrier device method is as effective as the standard Cryotop method for vitrification of bovine blastocysts in vitro.

• 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.

• Plant Biotechnology Reports
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• v.2 no.2
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• pp.123-131
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• 2008

Vitrification methods are convenient for cryopreserving plant specimens, as the specimens are plunged directly into liquid nitrogen (LN) from ambient temperatures. However, tissues and species with poor survival are still not uncommon. The development of vitrification solutions with high survival that cover a range of materials is important. We attempted to develop new vitrification solutions using bromegrass cells and found that VSL, comprising 20% (w/v) glycerol, 30% (w/v) ethylene glycol, 5% (w/v) sucrose, 10% (w/v) DMSO and 10 mM $CaCl_2$, gave the highest survival following cryopreservation, as determined by fluorescein diacetate staining. However, the cryopreserved cells showed little regrowth, for unknown reasons. To check its applicability, VSL was used to cryopreserve gentian axillary buds and the performance was compared with those of conventional vitrification solutions. Excised gentian stem segments with axillary buds (shoot apices) were two-step precultured with sucrose to induce osmotic tolerance prior to cryopreservation. Gentian axillary buds cryopreserved using VSL following the appropriate preculturing approach exhibited 78% survival (determined by the regrowth capacity), which was comparable to PVS2 and PVS1 and far better than PVS3. VSL had a wider optimal incubation time (20-45 min) than PVS2 and was more suitable for cryopreserving gentian buds. The optimal duration of the first step of the preculture was 7-11 days, and preculturing with sucrose and glucose gave a much higher survival than fructose and maltose. VSL was able to vitrify during cooling to LN temperatures, as glass transition and devitrification points were detected in the warming profiles from differential scanning calorimetry. VSL and its derivative, VSL+, seem to have the potential to be good alternatives to PVS2 for the cryopreservation of some materials, as exemplified by gentian buds.

• Journal of Embryo Transfer
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• v.4 no.1
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• pp.1-13
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• 1989

This paper reviews the most important steps that have generated consistent progress in principles and developmental progress of embryo cryopreservation, and also study on freezing procedure and its application by conventional method and current improved method for freezing procedure and its appilcation of embryo cryopreservation in farm animals. Four were of particular interest: 1.The transport of water across the ccli membrane (zona pellucida) during freezing and thawing accordinglyplays a role in determing whether the celi survives. This movement of water is controlied mainly by extracellular phase changes and by the nature and concentration of any cryoprotective agent present. Therates of cooling, freezing and warming, and the intervals over which they are applied are further decisi've factors in determining whether a cryopreservation procedure allows survival after thawing. 2.The first successful deep freezing experiments with sheep morula and blastocysts during the seventies were based on the early procedures used for mouse embryos.Current research during the eighties is developed with the aim of simplifying and improving current procedures such as one-step dilution and rapid or ultra-rapid cooling by using the model of laboratory animals. 3.The conventional method for the embryo cryopreservation is described. An alternative to this method which may result in high survival and also in reducing of the freezing and thawing time is done by combing a permeable cryoprotectant such as glycerol, DMSO or propanediol and a non-permeable compound such as sucrose, trehalose, raffinose or lactose. 4.Finally a different approach to the preservation of embryos, named vitrification, is introduced. This procedure depends upon the ability of concentrated solutions of cryoprotective agents such as glycerol and propanediol to supercool to very low temperature (-196$^{\circ}C$) during rapid cooling before solidifying without formation of ice. However, more complete data are necessary for successful vitrification of blastocysts.

• Development and Reproduction
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• v.3 no.1
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• pp.107-111
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• 1999

To find out the desirable cryoprotectant for cryopreservation of bivalve trochophores, four types of cryoprotectant were tested with trochophores of the pearl oyster (pinctada fucata martensii) generally used in the pearl production in Korea. Each cryoprotectant (dimethyl sulfoxide, ethylene glycol, glycerol and 1,2-propanediol) was mixed with 0.2 M sucrose to make final concentrations of 0.01, 0.1, 1.0 and 2.0 M. The trochophores were immersed in each preparation, waiting for 10 minutes to reach equilibration and cryopreserved in the liquid nitrogen (-l96$^{\circ}C$). Survival rate of trochophores thawed after cryopreservation increased as the media concentration increase. However, a few number of the trochophores seemed to be damaged with the efflux of cell inclusions. Our study rsults indicate that desirable cryoprotectants for cryopreservation of pearl oyster trochophores are 1.0~2.0 M dimethyl sulfoxide and ethylene glycol : 82.8~97.4% of the trochophores cryopreserved with these media survived after thawing.

• Journal of Embryo Transfer
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• v.33 no.2
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• pp.75-84
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• 2018

The cryopreservation has been extensively applied in many cells including spermatozoa (semen) during past several decades. Especially, the canine spermatozoa cryopreservation has contributed on generation of progeny of rare/genetically valuable dog breeds, genome resource banking and transportation of male germplasm at a distant place. However, severe and irreversible damages to the spermatozoa during cryopreservation procedures such as the thermal shock (cold shock), formation of intracellular ice crystals, osmotic shock, stress of cryoprotectants and generator of reactive oxygen species (ROS) have been addressed. According as a number of researches have been conducted to overcome these problems and to advance cryopreservation technique, several analytical methods have been employed to evaluate the quality of the fresh or cryopreserved canine spermatozoa in regards to the motility, morphology, integrity of membrane and DNA, mitochondrial activity, ROS generation, binding affinity to oocytes, in vitro fertilization potential and fertility potential by artificial insemination. Because the study designs with certain application of analytical methods are selective and varied depending on each experimental objective and laboratory condition, it is necessary to establish the normal reference data of the fresh or cryopreserved canine spermatozoa for each analytical method to monitor experimental procedure, to translate raw data and to discuss results. Here, we reviewed the recent articles to introduce various analytical methods for the canine spermatozoa as well as to establish the normal reference data for each analytical method in the fresh or cryopreserved canine spermatozoa, based on the results of the previous articles. We hope that this review contributes to the advancement of cryobiology in canine spermatozoa.

• Korean Journal of Plant Resources
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• v.15 no.2
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• pp.89-95
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• 2002

In woody plant germplasms, using prefrozen dormant buds for materials is one way to achieve successful cryopreservation. The protocol of cryopreservation for White birch (Betula platyphylla var. japonica) winter vegetative buds is the following. First, the branches of White birch were collected in January 20, when the vegetative buds were still in a state of quiescence. The winter buds with about 5㎜ of xylem tissue were removed from the branches. They were dehydrated to moisture contents about 44% by air dry treatment. The buds were prefrozen, with the temperature being decreased by 5∼-20$\^{C}$ and then transfered to the LN(liquid nitrogen) maintained below -l96$\^{C}$. After cryopreservation, the vegetative buds were rapidly thawed in a water bath at 40$\pm$5$\^{C}$. In this case, the cell survival rate of samples was about 86%. After sterilization, buds were then cultured on MS medium. These results demonstrate the feasibility for cryopreservation of winter vegetation buds of Betula platyphylla var. japonica.

• Biomaterials and Biomechanics in Bioengineering
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• v.3 no.2
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• pp.105-114
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• 2016

The purpose of this study is to evaluate the effects of cryopreservation on dental pulp-derived stem cells (DPSC) viability over a period of three years. Dental pulp-derived stem cells were isolated and cultured from thirty-one healthy teeth. DPSC isolates were assessed for doubling-time and baseline viability prior to cryopreservation and were assessed again at three time points; one week (T1), 18 months (T2), and 36 months (T3). DPSC can be grouped based on their observed doubling times; slow (sDT), intermediate (iDT), and rapid (rDT). Viability results demonstrated all three types of DPSC isolates (sDT, iDT and rDT) exhibit time-dependent reductions in viability following cryopreservation, with the greatest reduction observed among sDT-DPSCs and the smallest observed among the rDT-DPSC isolates. Cryopreserved DPSCs demonstrate time-dependent reductions in cellular viability. Although reductions in viability were smallest at the initial time point (T1) and greatest at the final time point (T3), these changes were markedly different among DPSC isolates with similar doubling times (DTs). Furthermore, the analysis of various DPSC biomarkers - including both intracellular and cell surface markers, revealed differential mRNA expression. More specifically, the relative high expression of Sox-2 was only found only among the rDT isolates, which was associated with the smallest reduction in viability over time. The expression of Oct4 and NANOG were also higher among rDT isolates, however, expression was comparatively lower among the sDT isolates that had the highest reduction in cellular viability over the course of this study. These data may suggest that some biomarkers, including Sox-2, Oct4 and NANOG may have some potential for use as biomarkers that may be associated with either higher or lower cellular viability over long-term storage applications although more research will be needed to confirm these findings.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.253-259
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• 1997

Objective: To investigate effects of cryoprotectant and cryopreservation on the chromosome of the human immature oocytes. Design: Intact cumulus-enclosed immature oocytes were collected from unstimulated ovaries and divided into three groups, such as no treatment as control (group 1), only 1,2-propanediol (PROH)-treated (group 2), and cryopreserved oocytes (group 3). Oocytes in group 1, 2, and survived oocytes after cryopreservation in group 3 were cultured for 48 hours. Setting: Infertility Medical Center at the CHA General Hospital, Seoul, Korea. Patients: Oocytes were obtained from Patients undergoing gynecological surgery. Main Outcome Measures: Maturation rate, abnormality in chromosomes by fluorescence in situ hybridization (FISH). Results: There was no effect of PROH only treatment on the chromosomal abnormalities in group 2 compared to control oocytes (41.4% and 31.8%, respectively). Whereas significantly increased abnormalities in chromosome (77.8%) were found in group 3. Conclusions: Human oocytes matured in vitro after cryopreservation at the germinal vesicle (GV) stage showed increased incidence of chromosomal abnormalities. These abnormalities may impair the capacity for further development of the embryos derived from frozen-thawed oocytes.

• YAKHAK HOEJI
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• v.50 no.6
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• pp.409-414
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• 2006

Although mammalian sperms are cryopreserved for in vitro fertilization a process of cryopreservation decreases the fertility. Acrosome reaction requires depolarization-induced Ca$^{2+}$ influx and Ca$^{2+}$ releases from the Ca$^{2+}$ stores. To examine whether the cellular Ca$^{2+}$ mobilization is altered by a sperm cryopreservation we compared cytosolic Ca$^{2+}$ signals between fresh and cryopreserved pig sperms using confocal Ca$^{2+}$ imaging. The magnitudes of depolarization induced Ca$^{2+}$ increases were significantly smaller in cryopreserved sperms. Exposures to 10 mM caffeine or 5 ${\mu}$M thapsigargin elicited less Ca$^{2+}$ increases in the cryopreserved sperms compared to fresh sperms. In addition, progesterone-trig-gered Ca$^{2+}$ rises, that are thought to enhance acrosome reaction, were completely abolished in the cryopreserved sperms. These results suggest that storage and(/or) release of Ca$^{2+}$ from the intracellular Ca$^{2+}$ stores in pig sperms are significantly impaired by the process of cryopreservation.