• Title, Summary, Keyword: Cryoprotectants

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Effects of cryoprotectants and sucrose concentrations on the viability of aggregated mouse embryos frozen rapidly in liquid nitrogen vapour (동결보호제 및 Sucrose 농도가 급속동결한 마우스 집합배의 생존성에 미치는 영향)

  • Shin, Sang-tae
    • Korean Journal of Veterinary Research
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    • v.31 no.4
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    • pp.523-527
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    • 1991
  • The effects of ethylene glycol, DMSO and glycerol as cryoprotectants and the effect of concentrations(0, 0.25, 0.5 and 1.0M) of sucrose in the diluent on the viability of the aggregated morulae frozen rapidly in liquid nitrogen$(LN_2)$ vapour were examined. The morulae were produced by aggregation of ICR and CBA mice embryos at 8-cell stage. Before freezing the embryos were equilibrated in 1.5M cryoprotectants+0.25M sucrose in oae-step or in 3.0M cryoprotectants+0.25M sucrose in two-steps. The embryos were pipetted into the freezing medium fraction of 0.25ml plastic straws. The straws were frozeu by directly transfer into $LN_2$ vapour(about lcm above $LN_2$) for 2 minutes, and then plunged into $LN_2$. After thawing the cryoprotectants were diluted with 0, 0.25, 0.5 or 1.0M sucrose solution. The post-thawed in vitro viability of the aggregated embryos was significantly dependent on the type and concentration of cryoprotectants in the freezing medium and also on the concentration of sucrose in the diluent. When the aggregated embryos were equilibrated in 1.5M cryoprotectants +0.25M sucrose in one-step and diluted with 0.5M sucrose after thawing, the survival rate of the embryo5 was significantly(p<0.05) higher in DMSO(62.5%) or ethylene glycol(52.2%) than in glycerol(33.3 %). In the case that the concentration of the cryoprotectants was raised to 3.0M in two-steps, thc higher survival rate of the embryos was obtained in ethylene glycol or glycerol than in DMSO followed by diluting them with 0.5 or 1.0M sucrose after thawing(p<0.01).

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Preparation and Evaluation of Freeze-dried Solid Lipid Nanoparticles with Various Cryoprotectants

  • Li, Ri Hua;Seo, Seung-Yong;Eun, Jae-Soon;Lee, Mi-Kyung
    • Journal of Pharmaceutical Investigation
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    • v.40 no.1
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    • pp.39-43
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    • 2010
  • Solid lipid nanoparticles (SLNs) were freeze-dried to obtain a stable solid dosage form with the aid of various cryoprotectants such as trehalose, sucrose, glucose, fructose, and glycerol. Tricaprin(TC) and trilaurin(TL) were used as lipid matrices for SLNs and stabilizers were egg phosphatidylcholine and pegylated phospholipid. All cryoprotectants tested did not cause changes in mean particle size of SLNs when mixed with SLNs before freeze-drying. However, the mean particle sizes of reconstituted SLNs after freeze-drying were significantly different from those of the un-lyophilized original SLN dispersions depending on the types and concentration of cryoprotectants. Although the freeze-dried SLNs without any cryoprotectants were easily reconstituted by hand-shaking, the mean particle size drastically increased (> $8\;{\mu}m$ for TC SLNs and around $1\;{\mu}m$ for TL SLNs) compared to that of un-lyophilized original dispersion (97 nm for TC SLNs and 164 nm for TL SLNs). Trehalose and sucrose were the most effective additives to protect the SLNs during lyophilization. The reconstituted SLNs were physically stable for 24 hours when lyophilized with 12.5% trehalose, sucrose, glucose, fructose or glycerol.

Effect of Different Cryoprotectants on the Viability, Maturation and Development of In Vitro Bovine Oocytes (동결액 조성이 소 난자의 체외성숙, 발육능 및 생존성에 미치는 영향)

  • 류일선;양병철;연성홈;이동원;서국현;손동수;이병천;황우석
    • Journal of Embryo Transfer
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    • v.13 no.2
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    • pp.147-157
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    • 1998
  • This study was conducted to investigate the effects of in vitro fertilization, culture and embryo development according to in vitro maturation rate, protectant composition and equilibrium time after frozen /thawing of bovine immature oocytes. This results obtained in studies on the effect of different cryoprotectants on the viability, maturation and development of in vitro bovine oocytes were as follow: 1.The post-thawing of immature oocytes matured to metaphase II during culture time for 0 to 26 h, and those group (62~3%) were low than control group (76.7%). The optimal maturation time of frozen-thawed immature oocytes was at 24 h. 2.The viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants. The developmental competence of frozen4hawed oocytes was not affected by cryoprotectants. These results indicate that an optimal maturation time of frozen /thawed immature oocytes was at 24h. Furthermore the viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants and developmental competence of frozen /thawed oocytes.

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Effects on Viability of Different Cryoprotectants Treated Mouse Embryos after Quick Freezing (침투성 및 비 침투성 동결보호제를 이용한 생쥐 수정란의 급속동결에 따른 생존성에 관한 연구)

  • 김태영;남상규;석호봉
    • Journal of Embryo Transfer
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    • v.10 no.3
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    • pp.193-202
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    • 1995
  • In order to improve the cryopreservatory techniques of livestock embryos, the quick freezing method which is directly plunged in liquid nitrogen via prefreezing procedure without freezing machine was carried out for mouse embryos treated with permeable and nonpermeable cryoprotectants. The viability of frozen-thawed embryos were evaluated by FDA vital dye test. The results obtained was summaried as follows: 1. A total of 720 embryos were recovered from frozen embryos for viability test. Evalution of the fluorescein diacetate(FDA) vital dye test with mice embryos were resulted of 2.3 total mean score - evaluted in orderly higher mean grade of P3 453 (63%), P2 133(18%), P1 51(7%) and P0 83(12%). 2. An all-round evalution of these combination, the highest viability was showed in 3M ethylene glycol + 0. 25M trehalose treated with the copper prefreezing. 3. Effects of permeable and nonpermeable cryoprotectants combination were evaluated by means FDA score. 3M ethylene glycol + 0.25M trehalose showed the highest survival rates of 2.8 mean FDA score. 4. Effects of permeable cryoprotectants were evaluated by mean FDA score but the results were not significantly different each other. 5. In evalution of the nonpermeable cryoprotectants, 0. 25M trehalose obtalned higher mean FDA score than of 0.25M sucrose and it was significantly different(P<0.05). 6. There was no significantly difference between copper and stainless-steel in prefreezing procedures.

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Effects of the Improvement of Vitrification Solution and FDA-test on the Embryo Survival and Conception Rate by Ultrarapid Freezing I. Effects of the Combination of Cryoprotectants in Vitrification Solution on the Survival of Frozen-Thawed Mouse Embryos (초그속동결에 있어서 Vitrification Solution 개발과 FDA 생사판정이 수정란의 배양과 이식후 착상에 미치는 영향 I. Vitrification Solution내의 내동제 조합이 초급속동결 융해후 Mouse Morulae의 생존율에 미치는 영향)

  • 김종규;강민수;고경래;양병철
    • Korean Journal of Animal Reproduction
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    • v.16 no.3
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    • pp.269-276
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    • 1992
  • Studies were carried out to find the freezing media which gives no ice crystals in single(glycerol, ethylene glycol, dimethyl sulfoxide(DMSO)) and mixture solutions(glycerol+propylene glycol, glycerol+ethylene glycol) of permeable cryoprotectants in vitrification solution and to study effects of VS on the survival of vitrified mouse morulae. The results are summarized as follows: 1. In toxicity test of permeable cryoprotectants, 30% glycerol of single solution showed the highest FDA-score(4.1) in mouse morulae frozen compared among other single solutions. The FDA-score(4.1) of 30% glycerol was higher than 30% ethylene glycol(3.6) and DMSO(1.4( (P<0.05). 2. 20, 30 or 40% single solution of permeable cryoprotectants containing m-PBS with 10% sucrose and 20% BSA was not crystallized during cooling, but crystallized during warming. However, the 30% mixture solution of the two permeable cryoprotectants was not crystallized both during cooling and warming.3. When mouse morulae were frozen in 30% mixture solutions of two permeable cryoprotectants(glycerol and propylene glycol, glycerol and ethylene glycol), highest FDA-score(4.5) was obtained in a mixture solution of 20% glycerol and 10% ethylene glycol(20G10E) than other 30% mixture solutions(10G20E, 15G15E, 20G10P, 15G15P, 10G20P) and there was significant difference between 20G10E and 10G20E(P<0.05).

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Studies for Development of Freeze - Flow Process (Freeze - Flow Process 개발을 위한 기초연구)

  • Lee, Young-Chun;Shin, Dong-Bin
    • Korean Journal of Food Science and Technology
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    • v.17 no.6
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    • pp.495-499
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    • 1985
  • To develope a process by which liquid foods can be stored in the liquid state at the frozen storage temperature, suitable cryoprotectants were selected. Orange juice, chosen as an example of liquid foods, was stored with combined cryoprotectants at $-15^{\circ}C$, and quality changes of orange juice during storage were evaluated. Among 7 cryoprotectants tested, NaCl solution had lower initial freezing point than others, and initial freezing points of glucose, fructose, glycerol, propylene glycol and citric acid were close to each other. Considering flavor quality of orange juice, cryoprotectants suitable for reducing freezing point of orange juice were glucose, fructose, glycerol, and citric acid. Combined cryoprotectants for reducing freezing point of 3 and 4 folds concentrated orange juice to $-15^{\circ}C$ consisted of 10% glucose, 8% frutose, 4.6% glycerol and 3% citric acid, and 5.5% glucose, 4.5% fructose, 4.6% glycerol, and 3% cirtric acid, respectively. When destruction of ascorbic acid, sedimentation volume and sensory flavor score of orange juice stored with combined cryoprotectants at $-15^{\circ}C$ and the control stored at $-18^{\circ}C$ were compared, there were no significant differences. These results indicated that liquid foods with suitable combined cryoprotectants could be stored at $-15^{\circ}C$ or below in the liquid state without adverse effect on quality of the stored products.

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Effects of Cryoprotectants on the Textural Changes of Whole-coagulated Soybean Curd (Tofu) during Frozen Storage (비압착 냉동저장 두부의 조직감 변화에 미치는 항냉동제의 효과)

  • Chung, Sun-Hwa;Choi, Won-Seok;Son, Hye-Sook;Lee, Cherl-Ho
    • Korean Journal of Food Science and Technology
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    • v.31 no.4
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    • pp.957-963
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    • 1999
  • Effects of cryoprotectants on protein denaturation of soybean curd, tofu, during frozen storage were examined. A whole-coagulated non-press tofu was prepared by adding 2% of isolated soybean protein to soy milk in order to prevent loss of added cryoprotectants. The cryoprotectants added were glocose, glycerol, sorbitol, propylene glycol, and tripolyphosphate. The texture characteristics of soybean curds before and after frozen storage were measured by sensory evaluation and Texture analyzer, and the results were evaluated by response surface methodology (RSM). Glucose, glycerol, sorbitol, and sodium tripolyphosphate were effective as single cryoprotectant, and the mixtures of glucose and sodium tripolyphosphate, and sorbitol and propylene glycol were also effective in minimizing textural change during freezing. Overall, the mixture of cryoprotectants were more effective than single cryoprotectant. According to the RSM, the maximum effect of cryoprotectants in minimizing textural changes during freezing was obtained with the mixture of 2.1% glucose, 6.7% glycerol, 2.1% sorbitol, 0.4% propylene glycol, and 0.3% sodium tripolyphosphate. However, considering the sensory acceptability, the optimum use of cryoprotectants in frozen tofu was 1% glucose, 2% glycerol, 1% sorbitol, 0.2% propylene glycol, and 0.5% sodium tripolyphosphate.

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Studies on Effects of Kinds and Concentration of Cryoprotectants, Equilibration Time and Thawing Temperature on the Survival Rate of Rapidly Frozen Porcine Embryos (돼지 수정란의 급속동결시 내동제의 종류와 농도, 평형시간 및 융해온도에 다른 생존성에 관한 연구)

  • 오원진;오건봉;박병권;김상근;이규승
    • Korean Journal of Animal Reproduction
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    • v.18 no.1
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    • pp.15-23
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    • 1994
  • This study was carried out to investigate the effects of concentration, kinds of cryoprotectants, equilibration time, optimum thawing temperature on the survival rate of rapidly frozen porcine embryos. The porcine embryos following dehydration by cryoprotectants containing sucrose were directly plunged into liquid nitrogen and thawed in 30, 35 or 37$^{\circ}C$ water bath, Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows : 1. The high survival rate of porcine frozen embryos after rapidly thawed in freezing medium was attained 2.0M DMSO, 2.0M glycerol, 2.0M propanediol, 1.5M ethyleneglycol. 2. The high survival rate of porcine frozen embryos after rapidly thawed in freezing medium was obtained using single cryoprotectant(16.6~40.0%) than mixed cryoprotectants(12.5~33.3%). 3. The eqilibration time on the survival rate of rapidly thawed porcine frozen embryos was attained after short period of time(15.0~33.3%) in the freezing medium higher than long period of time(9.10~30.0%). 4. The thawing temperature on the survival rate of rapidly thawed porcine frozen embryos was attained at 3$0^{\circ}C$ of thawing temperature(33.3~40.6%) in the freezing medium higher than 25 or 37$^{\circ}C$ of thawing temperature.

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Effects of Cryoprotectants on Survival and Hatching of Black Seabream, Acanthopagrus schlegeli Embryos (감성돔, Acanthopagrus schlegeli 배의 생존과 부화에 미치는 동해방지제의 영향)

  • 임한규;장영진;조필규
    • Journal of Aquaculture
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    • v.16 no.4
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    • pp.262-266
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    • 2003
  • In order to develop a method for the cryopreservation of fish embryos, the determination of optimal concentrations of dimethyl sulfoxide (DMSO), ethylene glycol and glycerol as individual cryoprotectants was performed by using the early embryos of black seabream, Acanthopagrus schlegeli. Optimal concentrations of cryoprotectants were assessed in terms of effects on mortality, median lethal concentration and hatching rate of embryos. The mortality of black seabream embryos immersed in cryoprotectants was related to the concentrations of cryoprotectants and immersion times. The toxicity to embryos was lower in order of DMSO, < ethylene glycol, < glycerol. The results from the mortality, median lethal concentration and hatching rate evaluations suggest that DMSO was the most effective cryoprotectant for black seabream embryos followed by ethylene glycol, and suitable concentrations of DMSO and ethylene glycol were 2.0∼2.25M and 1.0∼1.78M, respectively, with 20 minutes of immersion time.

Effect of Protective Compounds on the Survival, Electrolyte Leakage, and Lipid Degradation of Freeze-Dried Weissella paramesenteroides LC11 During Storage

  • Yao, Amenan A.;Wathelet, Bernard;Thonart, Philippe
    • Journal of Microbiology and Biotechnology
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    • v.19 no.8
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    • pp.810-817
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    • 2009
  • The effect of cryoprotectants (maltodextrin+glycerol) and cryoprotectants+antioxidant [ascorbic acid and/or butylated hydroxytoluene (BHT)] mixtures on the survival, electrolyte leakage, and lipid degradation of freeze-dried Weissella paramesenteroides LC11 during storage was investigated and compared with that of the control (cells without additives) over a 90-day storage period at 4 or $20^{\circ}C$ in glass tubes with water activity ($a_w$) of 0.23. The survival, electrolyte leakage, and lipid degradation were evaluated through colony counts, electrical conductivity, and thiobarbituric acid reactive substances (TBARS) content, respectively. The fatty acids composition was determined by gas chromatography, in both the total lipid extract and the polar lipid fraction, and compared with that of the control after the 90-day storage period. As the storage proceeded, increases in leakage value and TBARS content, as well as a decrease in viability, were observed. After 90 days of storage, the major fatty acids found in both the total lipid extract and the polar lipid fraction were palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids. The survival, leakage value, TBARS content and 18:2/16:0 or 18:3/16:0 ratio were the greatest for the protected strain held at $4^{\circ}C$. Cells with the cryoprotectants+BHT mixture showed the highest percentage of survival and 18:2/16:0 or 18:3/16:0 ratio in both lipid extracts, as well as the lowest leakage value and TBARS content after the 90-day storage period. Drying cells with the cryoprotectants+BHT mixture considerably slowed down polar lipid degradation and loss of membrane integrity, resulting in improved viability during storage.