• Journal of Embryo Transfer
• /
• v.23 no.3
• /
• pp.223-227
• /
• 2008

The cryopreservation of Hanwoo embryos has become an integral part of assisted reproduction in animal. The objective of this study was to assess the effect of The objectives of this study were: (1) to evaluate the influence of bovine embryo developmental stage on in vitro embryo development after freezing, (2) to study the efficiency compared with conventional freezed embryos at different embryo source. For conventional slow-freezing, day 7 or 8 expanded blastocysts were collected. The standard freezing medium was 1.8 M ethylene glycol (EG). Embryos were equilibrated in 1.8 Methylene glycol(EG) with 0.1 M sucrose in Dulbecco's phosphate-buffered saline (D-PBS) supplemented with 0.5% bovine serum albumin. Embryos were then loaded individually into 0.25 ml-straw and placed directly into cooling chamber of programmable freezer precooled to $-7^{\circ}C$, after 2 min, the straw was seeded, maintained at $-7^{\circ}C$ for 8 min, and then cooled to $-35^{\circ}C$ at $0.3^{\circ}C$/min, plunged and stored in liquid nitrogen for at least 3 days. For thawing, the straw containing embryos were warmed in air for 10 see and exposed to $37^{\circ}C$ water for 20 sec. Straws were then removed from $37^{\circ}C$ water. Rates of blastocyst survive and hatched were evaluated at 12 to 48h post-warming. The re-expansion and hatched rates of morula embryos were significantly lower than those obtained for blastocysts and expansion blastocysts (31.6%, 10.5% vs, 68.9%, 22.2% vs, 73.7%, 53.6%, respectively). No differences in re-expansion rates were found between in vivo and in vitro blastocysts. whereas hatched rates was significantly higher (51.2%) in vivo compared with in vitro embryos (18.6%). in conclusion, demonstrate that conventional freezing can be used successfully in cryopreservation of in vitro and in vivo bovine embryos, and that it might be considered for use in commercial programs and embryo preservation.

• Korean Journal of Animal Reproduction
• /
• v.8 no.1
• /
• pp.22-28
• /
• 1984

This experiment was carried out to investigate effects of DMSO or ethylene glycol as a cryopotectant and of freezing methods on survival rate of forozen-thawed rat 2-cell embryos by morphological observation. 2-cell embryos were recovered from oviducts of Sprague Dawley females mated with males of same strain on day 2 of pregnancy after inducing superovulation by intrapertioneal injection of PMSG and HCG. In slow freezing and thawing groups, embryos were frozen to -79$^{\circ}C$ or -196$^{\circ}C$ in a glass test tube containing 0.2ml PBI with 1.5M DMSO or 1.2M ethylene glycol at a rate of 0.3-1.0C/min. and thawed slowly. When samples were frozen to -79$^{\circ}C$, higher survival rate was obtained in the medium containing DMSO (43.9%) than ethylene glycol (41%). And similar result was obtained (32.5% in DMSO vs. 31.4% in ethylene glycol) when samples were frozen. In rapid freezing and thawing groups, embryos were frozen to -79 or -196$^{\circ}C$ in a glass test tube containing 0.2ml of PBI with 1.5M DMSO or 1.2M ethylene glycol by rapid cooling, and thawed rapidly. When samples were frozen to -79$^{\circ}C$, 1.5M DMSO (13.2%) was more effective than 1.2M ethylene glycol (6.1%). When the storage temperature was -196$^{\circ}C$, survival rates were 9.8% in 1.5M and 5.4% in 1.2M ethylene glycol.

• Journal of Bio-Environment Control
• /
• v.21 no.4
• /
• pp.354-361
• /
• 2012

This study was conducted to evaluate on the freezing tolerance of introduced blueberry cultivars in Korea and to investigate availability of portable timber moisture meter for simple and rapid diagnosis of blueberry-shoot damage by freezing during wintering. Frost tolerance of blueberry cultivars showed big difference that rates of blueberry-shoot death were widely distributed from about 0% to 100% after wintering. Optical density in TTC reduction of blueberry twig treated low temperature was low in order of $-40^{\circ}C$ < $-21^{\circ}C$ < $4^{\circ}C$. Hardiness evaluation of visible injury in the cross-sectional surface color did not agree with that of rates of blueberry-shoot death during wintering. Lowest water content of blueberry stem measured by timber moisture tester during wintering was about 15%. During wintering, water contents of blueberry stems were higher at lower part of tree, but were low at end part of stems, and then when the blueberry grew again for spring, the water content gradually increased to 20~40%. Water content of blueberry stem with freezing injury during wintering decreased to under 5% by desiccation. Therefore it is assummed that the moisture content of blueberry stem injured by freezing during wintering was about under 14%, and it is expected that portable timber moisture meter could be available for rapid diagnosis of blueberry freezing injury in field.

• Journal of Embryo Transfer
• /
• v.6 no.1
• /
• pp.13-24
• /
• 1991

The objective of this experiment was to study some possibilities to simplify freezing, thawing and transfer procedure of one-step straw method comparing with the conventional methods using bovine embryos. The previous work are also designed to investigate the thawing effect by development stage and its quality using the embryos. Results obtained were summarized as follows: 1. A total of 87 embryos from 14 donor cows were frozen-thawed and an average of frozen embryo/donor was 6.2. 2. The survival rates of morula stage(65.4%) were higher than those of blastocyst stage(57.l%) and vice versa in rate of morphological recovery (80% vs 95.4%). However. no significant difference was denoted between them. 3. In difference between the groups of good quality and poor quality. good quality was resulted in a significantly higher embryo survival rate(75%) and recovery rates(95%) than poor quality(P<0.0l). 4. In effects of non-permeable sugar dilution in added to l.0M glycerol. higher survival rates were orderd in sucrose. lactose, raffinose and xylose. But lactose-raffinose, sucrose-trehalose and xylose in added to 2.OM glycerol. 5. The highest survival rates were obtained by direct plunge into the liquid nitrogen with 3.OM concentration both of glycerol and trehalose. 6. The survival rates in vitro condition of one-step and direct plunge methods(75%-87.5%) were significantly higher than those of multiple steps (21.4-52.6%) in in vitro (P<0.0l). However, the results of single-step were critical in comparing to other steps of final pregnant conformation.

• Transactions of the Korean hydrogen and new energy society
• /
• v.35 no.2
• /
• pp.240-248
• /
• 2024

The purpose of this study is to investigate the freezing phenomena in printed circuit heat exchanger (PCHE) for cryogenic liquid hydrogen vaporizer. Local freezing phenomena in hot channels should be avoided in designing PCHE for cryogenic liquid hydrogen vaporizer. Hence, the flow and thermal characteristics of PCHE is experimentally investigated to figure out the conditions under when freezing occurs. To conduct lab-scale PCHE experiment, liquid nitrogen is used as a working fluid in cold channels instead of using liquid hydrogen. Glycol water is used as a working fluid in hot channels. Based on the experimental data, ratio between mass flow rates of cold channels and that of hot channels is proposed as contour map to avoid the freezing phenomena in PCHE.

• Korean Journal of Animal Reproduction
• /
• v.20 no.2
• /
• pp.207-214
• /
• 1996

To examine the developmental capacity of manipulated embryos after ultrarapid refreezing and thawing, mouse embryos were biopsied at 4-cell stage, frozen twice at 4-cell and morula stages, respectively, and then transferred to rec-ipients. Single blastomeres were biopsied from 4-cell embryos by a modified aspiration method. Biopsied 4-cell embryos were equilibrated into freezing medium at room temperature for 2.5 min, loaded into 40 $\mu$I of freezing medium in 0.25 ml plastic straw and then directly immersed into liqiud nitrogen. Freezing medium for 4-cell embryos consisted of 4.0 M ethylene glycol and O.25 M sucrose in dPBS supplemented with 6 mg/lm BSA. Morulae were frozen into freezing medium containing 5.0 M glycerol instead of ethylene glycol. Thawing was conducted by agitating each straw in 3TC water for 20 sec. The c content of each straw was expelled into 0.5 ml of dilution medium, which consisted of 0.25 M sucrose and 3 mg/ml BSA in dPBS. The thawed embryos were rehydrated in dilution medium for 10 min, washed 3 times with dPBS and then cultured in M16 medium at 37$^{\circ}C$, 5% CO$_2$ in air. Blastocysts that developed from frozen or refrozne biopsied embryos were transferred to recipients on Day 3 of pseudopregnancy, respectively. In vitro and in vivo developmental rates of the biopsied and intact 4~cell embryos after freezing and thawing were 78 (10l/130) and 25% (10/40), and 91 (114/125) and 30% (12/40), respectively. Although the rates of in vitro development of biopsied and intact embryos to blastocyst stage were significantly different after freezing and thawing (P

• International Journal of Highway Engineering
• /
• v.12 no.2
• /
• pp.99-106
• /
• 2010

Until now, the thickness design of anti-freezing layer has been empirically conducted using the frost depth determined from the freezing index. This approach cannot consider the structural properties of anti-freezing layer, which can cause the over-design of pavement structure. This paper presents results of structural evaluation of anti-freezing layer using the Falling Weight Deflectormeter (FWD) deflections. The FWD testing was directly conducted on top of the subbase layer located at the embankment, cutting, and boundary area of each section. It is observed from this testing that the center deflections of pavement structure with anti-freezing layer are smaller than those without anti-freezing layer. The deflection reduction rates are 15~55% in the embankment, 11~64% in the cutting, and 2~38% in the boundary, respectively. It was also found that the use of antifree zing layer enables to reduce the Surface Curvature Index (SCI) values up to 24 percent. Fatigue lives show that pavement structure with antifreezing layer are about two times higher than the those without anti-freezing layer. This fact indicates that the anti-freezing layer should be considered as a structural layer in the asphalt pavement system.

• Clinical and Experimental Reproductive Medicine
• /
• v.27 no.1
• /
• pp.59-65
• /
• 2000

Objective: The purpose of this study was to determine the important factors affecting survival and pregnancy rate in frozen-thawed embryo transfer cycles. Methods: we performed retrospective analysis in 738 cycles of frozen-thawed embryo transfers, in relation to the insemination methods, the freezing stage of embryo, patient's age, infertility factors and the origin of injected sperm in ICSI cycles. After conventional IVF or ICSI, the supernumerary PN stage zygotes or multicellular embryos were cryopreserved by slow freezing protocol with 1,2-propanediol (PROH) as a cryoprotectant. Results: The survival rates of thawed embryos were 69.3% (1585/2287) in conventional IVF group and 71.7% (1645/2295) in ICSI group. After frozen-thawed embryo transfers, 27.0% (92/341) and 32.0% (109/341) of pregnancy rates were achieved in conventional IVF and ICSI group, respectively. There were no significant difference in the survival and pregnancy rates according to the insemination methods, the freezing stage and patient's age. However, the pregnancy rate (36.2%) of male factor infertility was significantly higher than the tubal (27.2%) and other female factor infertility (22.9%). In ICSI group, the origin of injected sperm did not affect the outcome of frozen-thawed embryo transfer cycles. Conclusion: The present study demonstrates that acceptable clinical outcomes can be achieved after the transfer of frozen-thawed embryos regardless of the stage of embryos for freezing, the patient's age and the origin of injected sperm.

• Clinical and Experimental Reproductive Medicine
• /
• v.25 no.1
• /
• pp.71-76
• /
• 1998

This study was carried out to examine whether the developmental capacity of bovine immature oocytes frozen ultra-rapidly using electron microscope (EM) grids and EFS30 can be obtained. As freezing solution, we used EFS30 which consisted of 30% ethylene glycol, 0.5 M sucrose, 18% ficoll and 10% FBS added in D-PBS. As criterior of oocyte viability, the rates of maturation, fertilization and embryonic development were determined. The results obtained in this experiment were summarized as follows: When ultra-rapidly frozen immature oocytes were thawed, 43.2% of them were survived. The rates of maturation (84.1%) and normal 2 pronuclei formation (57.5%) of frozen immature oocytes were not significantly different when compared to those of control (92.5, 65.0%). In addition, the rates of $\geq2$-cell (65.0%) and blastocyst formation (30.8%) of freezing group were not significantly different when compared to those of control (73.7, 35.7%). These results demonstrate that developmental capacity of frozen-thawed bovine immature oocytes can be successfully obtained when survived from the ultra-rapid freezing method using EM grid and EFS30.

• Journal of Embryo Transfer
• /
• v.26 no.1
• /
• pp.13-17
• /
• 2011

Research in the area of equine artificial insemination (AI) has led to its increased application in field trials. However, procedures for equine semen collection, cooling and freezing of semen and artificial insemination need further improvement. In experiment 1, we investigated the percentage of total motility (TM) and progressive motility (PM) of sperms at after-collection, cooled-diluted, cooled-transported or frozen-thawed semen. In experiment 2, mares were inseminated with either cooled-diluted, cooled-transported or frozen-thawed semen. In experiment 3, we examined the effect of buffer (skim-milk extender), which was infused into the uterus at the time of AI with frozen-thawed semen. In experiment 4, we compared AI pregnancy rates for mares ovulating spontaneously versus after treatment with hCG. In experiment 1, the average percentage of TM was decreased from 75.3% to 14.4% at the stage of after-collection to frozen-thawed semen (p<0.05). The average percentage of PM was 58.2% and 59.6% at after-collection and cooled-diluted, but it was significantly increased 71.7% after frozen-thawed (p<0.05). In experiment 2, the pregnancy rates after AI using cooled-diluted, cooled-transported and frozen-thawed semen were 60%, 50% and 37.5%, respectively, and similar among treatments. In experiment 3, the pregnancy rate of mares infused with buffer at AI was 40% which was higher than that with no buffer (10%). In experiment 4, the pregnancy rates of mares were similar between ovulated spontaneously (25%) and ovulated with hCG (50%). The results suggest that equine semen that has been cooled-diluted, cooled-transported or frozen can be successfully used to establish AI, pregnancy and foal production. Also, the pregnancy rates after AI can be increased by infusing buffer into the uterus at AI or by inducing ovulation with hCG, but further study is need.