• Integrative Medicine Research
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• v.6 no.1
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• pp.12-18
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• 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.

• 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.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.4
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• pp.486-494
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• 2006

In order to protect the spermatozoa against cold shock, hen egg yolk is widely used as a cryoprotective agent in semen freezing extenders for domestic animals. The protective action of yolk is largely presumed to be due to low density lipoproteins (LDL). The effects of LDL on sperm quality of bull and northern pike (Esox lucius) after freezing-thawing have been reported, but no study has been made to evaluate the effect of LDL on boar sperm motility and other characteristics. The experiment was carried out to investigate the effect of LDL on the freezing of boar sperm in 0.25 ml straws. The aim was to evaluate the quality of boar spermatozoa cryopreserved in the presence of LDL. Motility of semen cryopreserved in LDL was analyzed and compared to semen cryopreserved with Tris-citric acid-glucose (TCG) and Tris-citric acid-fructose (TCF), two basic freezing extenders containing egg yolk. Similarly, acrosome and plasma membrane integrity were also evaluated and compared to semen cryopreserved with TCG and TCF. Analysis of sperm quality after freeze-thaw showed that the motility, acrosome and plasma membrane integrity were improved with LDL in the extender, as compared to the TCG and TCF. The highest post-thaw integrity of acrosome and plasma membrane and motility were obtained with 9% LDL (w/v). Consequently, the optimum LDL concentration in the extender was 9%. It is also suggested that the concentration of LDL addition is important for the effect on boar sperm protection during freezing and thawing. The percentage of motile spermatozoa was significantly higher after freezing in 9% LDL than in TCG and TCF 54.4% versus 30.4% and 30.1% (p<0.05), respectively. The integrity of acrosome and plasma membrane were also significantly higher at 70.3% and 50.5% respectively with semen frozen in 9% LDL extender compared to TCG at 37.8% and 30.3% and TCF at 36.4% and 29.9%, respectively (p<0.05),. In conclusion, we propose that extender containing LDL extracted from hen egg yolk could be used as a cryoprotective media with a better efficiency than TCG and TCF. LDL improved boar semen quality, allowing better spermatozoa motility, acrosome and plasma membrane integrity after the freeze-thaw process. Furthermore, we found out that the extender with 9% LDL concentration significantly enhanced motility, acrosome and plasma membrane integrity of boar sperm after freezing and thawing.

• Journal of Embryo Transfer
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• v.4 no.1
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• pp.35-40
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• 1989

The effects of cryoprotectants (glycerol, DMSO and ethylene glycol) and the concentrations (0, 0, 25, 0.5and 1.0 M) of sucrose in the diluent on the is vitro survival of mouse morulae froaen rapidly in liquid nitrogenvapour were examined. When the embryos were equilibrated in 1.5 M cryoprotectants +0.25 M sucrose in one-step or in 3.0 M cryoprotectants +0.25 sucrose in two-step and diluted with 0, 0.25, 0.5, or 1.0 M sucrose solution after thawing, high survival rates were obtained in ethylene glycol (48.0% to 88.2 %) or in glycerol (35.0 % to 77.8 %). These results show that 1.5 M ethylene glycol is a highly efficient cryoprotective agent for the rapid freezing of mouse morula embryos and 0.5 M sucrose was optimal concentration in the diluent after thawing.

• Journal of Life Science
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• v.24 no.11
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• pp.1252-1257
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• 2014

While dimethyl sulfoxide (DMSO) is the most commonly used cryoprotectant agent in the cryopreservation of cultured mammalian cells, it has been reported to cause differentiation of some cell lines by DNA methylation and associated histone modifications. To avoid the side effects of DMSO in cryopreservation, other agents might be more appropriate for maintaining the stable differentiation of cultured cell phenotypes through cryopreservation. All cryoprotectants should be highly soluble in water and display low cell toxicity. Cryoprotective agents have been shown to be effective in animal sperm preservation, and eight types of amides were examined in the cryopreservation of cultured mouse endothelial cells. Among the amides examined, acetamide and lactamide were effective cryoprotectants for cultured mammalian cells. The most effective concentration of lactamide, 1.5 M, had an even lower cryoprotective ability than 1M DMSO. Because successful cryopreservation of cultured cells is hampered by osmotic stress, the adequate ionic concentration was determined by diluting phosphate-buffered saline (PBS) in the 1.5M lactamide solution. The most effective concentration was $0.4{\times}PBS$, which minimized osmotic stress during the cryopreservation of cultured cells. As the addition of high molecular weight materials in cryopreservation media improves the viability of cells, the effects of bovine serum albumin (BSA), hydroxyethyl-starch (HES), and dextran were examined. The best combination of lactamide-based media for cryopreservation was found to be 1.5 M lactamide in $0.4{\times}PBS$ with 1% BSA.

• The Korean Journal of Malacology
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• v.29 no.3
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• pp.251-258
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• 2013

This study aims to find out a suitable cryoprotective agent (CPA) for cryopreservation and its optimum concentration in order to conduct planned artificial seed production of Pacific oyster, Crassostrea gigas and to preserve superior sperm. For this, we tried to understand toxicity and the effect of cryopreservation by CPA type and concentrations first and then looked into cell damage of the sperm after thawing. Toxicity analysis on the sperm of Pacific oyster according to different CPA and immersion time shows that dimethyl sulfoxide (DMSO) comes first when it comes to survival rate and mobility followed by ethylene glycol (EG), glycerol and methanol. To identify the optimum CPA and its level, filtered seawater was used as a diluent before cryopreservation for 30 days. As a result, cryopreserved sperm of Pacific oyster with 15% of DMSO showed the highest survival rate and activation. Also, we observed the cryopreserved and thawed sperm with Scanning electron micrographs by CPAs and concentrations. Consequently, DSMO showed the lowest cell damage followed by EG, methanol, glycerol and the level was 15, 20, 10, 5% respectively. In a nutshell, it is proven that the optimum CPA and its level is 15% of DMSO.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.12
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• pp.1721-1727
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• 2008

Four experiments were conducted to study the following: i) the influence of different concentrations of sucrose (0.15, 0.3 and 0.5 M with osmolality of 308, 500 and 760 mOsm/kg, respectively) in diluents and control diluent (370 mOsm/kg) on intensity of motility and progressive motility of goat sperm without rehydration and freezing step in four incubation periods (0, 0.5, 2 and 4 h after dilution); ii) the influence of gradual dilution (in 3 steps) on improvements in ascertained results of the first experiment; iii) cryoprotective effects of different concentrations of sucrose (0.15, 0.22, 0.29 and 0.37 M with osmolality of 450, 560, 740 and 920 mOsm/kg, respectively) plus 7% glycerol and 20% egg yolk in basic diluent (Tris-Citric acid-Fructose) and iv) the effect of two concentrations of sucrose (0.15 and 0.22 M) with and without glycerol (7%). In experiment 1, we obtained better results for control diluent, 0.15 and 0.3 M sucrose supplemented diluents with 0 and 0.5 h incubation periods. In experiment 2, apart from a slight improvement, similar tendencies to experiment 1 were observed. In experiment 3, we obtained the best result for diluent with 0.22 M sucrose with regard to intensity of motility, progressive motility, live sperm and normal acrosomes ($40{\pm}4%$, $3.1{\pm}0.2$, $37{\pm}4%$ and $37{\pm}4%$, repectively). In experiment 4, we obtained the best result for diluent with 0.22 M sucrose plus 7% glycerol in regard to intensity of motility, progressive motility and live sperm ($39{\pm}3%$, $3.6{\pm}0.4$ and $41{\pm}4%$, respectively). The characteristic normal acrosomes in diluents without glycerol, i.e. diluents with 0.15 and 0.22 M sucrose showed better results ($39{\pm}8$ and $42{\pm}6%$ respectively). With regard to the release of hyaluronidase enzyme there were no significant differences between diluents (p>0.05). The results of the diluents with 0.15 and 0.22 M sucrose without glycerol were slightly lower than those with glycerol ($69{\pm}11$ and $70{\pm}11$ vs. $72{\pm}11$ and $70{\pm}11{\times}120{\times}10^6$ units $ml^{-1}$, respectively). In conclusion, the use of concentrated sucrose solutions showed that goat sperm can tolerate osmolality up to 560 mOsm (0.22 M) in the freezing period. In addition, glycerol proved to be a necessary cryoprotective agent in the cryopreservation of goat sperm, particularly for intensity of motility, progressive motility and live sperm.

• Development and Reproduction
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• v.16 no.2
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• pp.77-85
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• 2012

Various cryoprotective agents (CPA) were tested to establish the best conditions for the cryopreservation of sperm from black porgy Acanthopagrus schlegeli acclimated and raised in freshwater (BFW). Survival rates of frozen/thawed sperm from BFW were higher in the order of dimethy sulfoxide (DMSO), glycerol, ethylene glycol (EG) and methanol. Sperm motility was higher in the order of glycerol, DMSO, EG and methanol. These effects were the same in thawed sperm from black porgy raised in seawater (BSW). Thus, optimum CPA for sperm cryopreservation of BFW and BSW were DMSO and glycerol where the highest survival rates and sperm motility were found at the concentration of 10%. In particular, the survival rates and motility of thawed sperm from BFW and BSW after cryopreservation using 10% DMSO were better than when cryopreserved using 10% glycerol. On the other hand, for the thawed sperm from both BFW and BSW, the longer the preservation period was, the lower the survival rates and sperm motility were. Notably, the higher the concentration of CPA was, the lower the survival rates and sperm motility were.

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