• Development and Reproduction
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• v.17 no.4
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• pp.337-343
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• 2013

Post-thawed larval rearing in Pacific oyster Crassostrea gigas was performed to investigate the survival rate with time course in three kinds of larvae cryopreserved. The highest survival rate and larval activity index (LAI) of post-thawed larvae were obtained from the permeation in 0.2 M sucrose and 2.0 M ethylene glycol (EG) at $-1^{\circ}C/min$ in freezing speed showing the survival rates just after thawing of 63.8% in trochophore, 84.1% in D-shaped veliger and 56.3% in early umbo veliger. In post-thawed larval rearing with food supply, the larvae lasted their lives until 24 hours in trochophore, 75 hours in D-shaped veliger and 57 hours in early umbo veliger. The results suggested that each larval stage post-thawed revealed no more further development to subsequent respective stage.

• Magazine of the Korean Society of Agricultural Engineers
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• v.44 no.1
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• pp.116-124
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• 2002

Natural resources fur the construction materials such as good soil, sand, and coarse aggregates have been encountered to be short due to excessive use by human. Even though some soil has been found to be unsuitable for construction materials, soil with reinforcement can naturally be an answer to these alternatives. According to recently published papers on fiber mixed soil, fiber mixed with soil can improve shear strength, compressive strength and post-peak load strength retention. In this study, a series of tests were performed to clarify the characteristics of fiber mixed soil and to give basic data for design and construction and their engineering properties, that is, unconfined compressive strength, splitting tensile strength, shear strength, crack by drying, freeze-thaw, creep and Poisson\`s ratio, were investigated and analyzed. It has been shown that fiber mixed soil is one of good alternatives fur the civil and building construction materials.

• Geomechanics and Engineering
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• v.18 no.2
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• pp.189-203
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• 2019

Brine leakage is a common phenomenon during construction facilitated by artificial freezing technique, threatening the stability of frozen wall due to the continual thawing of already frozen domain. This paper takes the frequently encountered soft clay in Wujiang District as the study object, and remolded specimens were prepared by mixing calcium chloride solutions at five levels of concentration. Both the deformation and pore water pressure of frozen specimens during thawing were investigated by two-stage loading tests. Three sections were noted from the changes in the strain rate of specimens during thawing at the first-stage load, i.e., instantaneous, attenuated, and quasi-stable sections. During the second-stage loading, the deformation of post-thawed soils is closely correlated with the dissipation of pore water pressure. Two characteristic indexes were obtained including thaw-settlement coefficient and critical water content. The critical water content increases positively with salt content. The higher water content of soil leads to a larger thaw-settlement coefficient, especially at higher salt contents, based on which an empirical equation was proposed and verified. The normalized pore water pressure during thawing was found to dissipate slower at higher salt contents, with a longer duration to stabilize. Three physical indexes were experimentally determined such as freezing point, heat conductivity and water permeability. The freezing point decreases at higher salt contents, especially as more water is involved, like the changes in heat conductivity. The water permeability maintains within the same order at the considered range of salt contents, like the development of the coefficient of consolidation. The variation of the pore volume distribution also accounts for this.

• The Korean Journal of Blood Transfusion
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• v.29 no.3
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• pp.291-300
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• 2018

Background: The use of a functionally closed system for the glycerolization and deglycerolization of red blood cells (RBCs) allows for prolonged post-thaw storage for more than 24 hours. The aim of this study was to assess glycerolization and deglycerolization processing for RBCs using a high glycerol method in the automated, closed system provided by Haemonetics ACP 215. Methods: Thirty-five packed RBCs were glycerolized using the ACP 215 to a final concentration of 40% (wt/vol). The units were either frozen as such (n=30) or excess glycerol was removed (n=5) before freezing. After storage at $-80^{\circ}C$, the units were thawed, deglycerolized and resuspended in SAG-M. The frozen-thawed RBCs were stored at $4^{\circ}C$, and analyzed for their stability and in vitro quality. Results: No prefreeze excess glycerol removal units showed significantly less potassium leakage during post-thaw storage compared to the prefreeze excess glycerol removal units. All measurements of the stability and in vitro quality of thawed RBCs prepared from frozen RBCs without the prefreeze removal of excess glycerol during post-thaw storage at $4^{\circ}C$ for 7 days were acceptable to the American Blood Bank Association's standards and European standards. Conclusion: RBCs frozen without prefreeze removal of excess glycerol and the ACP 215 simplifies cryopreservation procedure and increases the stability of frozen-thawed RBCs. This increases the practical applicability of cryopreserved RBCs in blood transfusion practice.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.3
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• pp.318-325
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• 2010

Two experiments were designed to evaluate the effects of the amino acids glycine and cysteine on cryopreservation of ram spermatozoa. After primary evaluation of collected ejaculates, the semen samples were pooled and diluted 1:4 before cooling (experiment 1) and freezing (experiment 2) with Tris-Citrate-Fructose-Yolk (TCFY) extender supplemented with different concentrations of glycine and cysteine (5, 10, 15 and 20 mM). As the control, semen was diluted and frozen in the extender without amino acids. Motility, viability and membrane integrity were assessed as the parameters for semen quality in the first experiment. In the second experiment, motility, progressive motility, viability, membranes and acrosome integrity were evaluated after the freezing-thawing process. The results of the first experiment indicated that the addition of 10 and 15 mM cysteine compared to the control (basic) extender significantly increased (p<0.01) the motility, viability and membrane integrity of spermatozoa after cooling. However, further increasing these amino acids up to 20 mM had a significant negative effect (p<0.05). Our results showed no significant differences (p>0.05) between 5 mM glycine compared to 5 mM cysteine and between 20 mM glycine and 20 mM cysteine. The results of experiment 2 showed that the amino acids significantly improved post-thaw motility, progressive motility, viability, membranes and acrosome integrity of ram spermatozoa. These positive effects were observed at concentrations between 5 to 15 mM of glycine and cysteine, with the best results at 15 mM. Further increasing of amino acid concentrations significantly decreased the post-thaw characteristics of spermatozoa, but the results showed that cysteine was better than glycine and control extenders. The data indicated that addition of glycine or cysteine to the freezing extender can be recommended for cryopreservation of ram spermatozoa. However, further studies are still needed to determine the effect of such addition on fertility in farm animals.

• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.1989-1996
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• 2015

Ice-binding proteins (IBPs) can inhibit ice recrystallization (IR), a major cause of cell death during cryopreservation. IBPs are hypothesized to improve cell viability after cryopreservation by alleviating the cryoinjury caused by IR. In our previous studies, we showed that supplementation of the freezing medium with the recombinant IBP of the Arctic yeast Glaciozyma sp. (designated as LeIBP) could reduce post-thaw hemolysis of human red blood cells and increase the survival of cryopreserved diatoms. Here, we showed that LeIBP could improve the viability of cryopreserved mammalian cells. Human cervical cancer cells (HeLa), mouse fibroblasts (NIH/3T3), human preosteoblasts (MC3T3-E1), Chinese hamster ovary cells (CHO-K1), and human keratinocytes (HaCaT) were evaluated. These mammalian cells were frozen in dimethyl sulfoxide (DMSO)/fetal bovine serum (FBS) solution with or without 0.1 mg/ml LeIBP at a cooling rate of -1℃/min in a -80℃ freezer overnight. The minimum effective concentration (0.1 mg/ml) of LeIBP was determined, based on the viability of HeLa cells after treatment with LeIBP during cryopreservation and the IR inhibition assay results. The post-thaw viability of mammalian cells was examined. In all cases, cell viability was significantly enhanced by more than 10% by LeIBP supplementation in 5% DMSO/5% FBS: viability increased by 20% for HeLa cells, 28% for NIH/3T3 cells, 21% for MC3T3-E1, 10% for CHO-K1, and 20% for HaCaT. Furthermore, addition of LeIBP reduced the concentrations of toxic DMSO and FBS down to 5%. Therefore, we demonstrated that LeIBP can increase the viability of cryopreserved mammalian cells by inhibiting IR.

• Clinical and Experimental Reproductive Medicine
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• v.18 no.1
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• pp.55-61
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• 1991

In order to increase the pregnancy rate by means of cryopreservation of the excess oocytes in IVF-ET program, the survival rate of the frozen-thawed oocytes of mouse was examined according to the stages of maturation, cryoprotectants and their treatment. The results were summarized as follows. First, during the continuous treatment with cryoprotectant media, the survival rate of oocytes was higher in DMSO than in PROH, and higher at low temperature($4^{\circ}C$) than at room temperature($25^{\circ}C$). Second, as regard with the maturation of immature(GV-intact) oocytes after treatment with cryoprotectant media, the rate of maturation in DMSO-treated group(52%) was higher than in PROH-treated group(35%). Third, according to the treatment of cryoprotectant media, the survival rate of frozen-thawed oocytes in DMSO-treated group (45%) was higher than in PROH-treated group(29%), and that of oocytes in DMSO 4-step treated group was higher than any other groups. Finally, in the post-thaw oocytes frozen at various stage of maturation, the survival rate of immature oocytes with GV was the highest in all groups. These results suggest that in the cryopreservation of mouse oocytes, DMSO was better than PROH as cryoprotectant, in treatment of cryprotectant the multi-step treatment was better than single-step, and the post-thaw survival rate of oocytes was closely related to the maturity of oocytes. It is assumed that the highest survival rate of mouse oocytes with GV is due to the stability of the structures in nucleus and intracelluar organelles, and of physiological function.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.7
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• pp.1021-1028
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• 2017

Objective: The objective of this study was to evaluate the effects of ageing time of lamb loins prior to freezing on technological characteristics and oxidation stability of coarse ground lamb loin sausage using in a model system. Methods: Lamb loins (M. longissimus lumborum, n = 25) were aged at $-1.5^{\circ}C$ for 0, 1, 2, 3, and 8 wk and then frozen for the remaining days (a total of 30 wk). The aged/frozen/thawed lamb loins were ground, and model sausages were formulated with 75% aged/frozen/thawed lamb loin, 25% water, 1.5% sodium chloride (NaCl) and 0.3% sodium tripolyphosphate. The pH and thaw/purge loss of aged/frozen/thawed lamb loins were evaluated, and protein functionality (protein solubility and emulsifying capacity), water-holding capacity and textural properties of model sausages were determined. Cooked model sausages were vacuum-packaged in a plastic bag and displayed under continuous fluorescent natural white light ($3^{\circ}C{\pm}1^{\circ}C$). Colour and lipid oxidation of the cooked model sausages were evaluated on 0 and 21 d of display storage. Results: Ageing prior to freezing had no impact on pH and purge/thaw loss of lamb loins and the colour of cooked sausages (p>0.05) made from the loins. Lamb loins aged for at least 3 wk prior to freezing numerically improved total and myofibrillar protein solubilities (p>0.05) and emulsion activity index (p = 0.009) of meat batter, but decreased cooking loss (p = 0.003) and lipid oxidation (p<0.05) of model sausages. Conclusion: This study suggests that post-mortem ageing of raw meat prior to freezing could improve water-holding capacity and lipid oxidative stability of sausage made from the meat.

• Korean Journal of Veterinary Research
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• v.35 no.3
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• pp.635-641
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• 1995

The purpose of this study was to investigate the effects of developmental stage and equilibration time on survival of rabbit embryos following freezing by vitrification. Adult New Zealand White female rabbits were superovulated with PMSG and hCG. The 8-cell stage embryos were collected from 40 to 45 hours after hCG injection by flushing oviducts with Dulbecco's phosphated buffered saline and in vitro cultured in TCM-199 containing 10% fetal calf serum(FCS). Each embryos developed in vitro to 16-cell, compact morula and blastocyst was cryopreserved and cultured following thawing to examine their developmental potential to expanded blastocyst stage in vitro. The frozen-thawed-cultured embryos were stained with Hoechst 33342, and their nuclei were counted using a fluorescence microscope. On the toxicity test of EFS solution as cryopreservation, the survival rates of 8-cell stage embryos was decreased in reverse to increasing of exposure time over 5 minutes. The post-thaw survival rates of embryos on equilibration times was significantly(P<0.05) higher for 2 min. than for 5 or 10 minutes. From morula to blastocyst of rabbit embryos was more suitable than 8-cell stage for cryopreservation by vitrification. The higher post-thaw survival rate of embryos can be achieved by keeping the cryoprotectant at $4^{\circ}C$ than at $20^{\circ}C$. The mean number of nuclei per embryo following freezing by vitrification and in vitro culture to expanded blastocyst at compacted morula and blastcyst was not significantly differ from fresh blastocyst.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.130-135
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• 2022

Epididymal sperm cryopreservation provides a potential method for preserving genetic material from males of endangered species. This pilot study was conducted to develop a freezing method for tiger epididymal sperm. We evaluated post-thaw sperm condition using testes with intact epididymides obtained from a Siberian tiger (Panthera tigris altaica) after castration. The epididymis was chopped in Tyrode's albumin-lactate-pyruvate 1x and incubated at 5% CO₂, 95% air for 10 min. The Percoll separation density gradient method was used for selective recovery of motile spermatozoa after sperm collection using a cell strainer. The spermatozoa were diluted with modified Norwegian extender supplemented with 20 mM trehalose (extender 1) and subsequent extender 2 (extender 1 with 10% glycerol) and frozen using LN₂ vapor. After thawing at 37℃ for 25 s, Isolate^® solution was used for more effective recovery of live sperm. Sperm motility (computerized assisted sperm analysis, CASA), viability (SYBR-14 and Propidium Iodide) and acrosome integrity (Pisum sativum agglutinin with FITC) were evaluated. The motility of tiger epididymal spermatozoa was 40.1 ± 2.0%, and progressively motile sperm comprised 32.7 ± 2.3%. Viability was 56.3 ± 1.6% and acrosome integrity was 62.3 ± 4.4%. Cryopreservation of tiger epididymal sperm using a modified Norwegian extender and density gradient method could be effective to obtain functional spermatozoa for future assisted reproductive practices in endangered species.