• Clinical and Experimental Reproductive Medicine
• /
• v.27 no.3
• /
• pp.283-289
• /
• 2000

Objective: The study was performed to compare the survival rate and the development of day 2 mouse embryos which had freezing procedures done. Methods: We used three different vitrification solutions (EFS, VS14, DPS) and a ultrarapid freezing solution (UFS) for cryopreservation of day 2 mouse embryo. Results: We tested toxicity by exposing embryos to vitirification solutions and a ultrarapid freezing solution. The survival rates are 100%, 97.8%, 95.6% and 100% (EFS, VS14, DPS and UFS). After cultured for 96 hours, hatching rates of each group are 93.5% (no freezing), 95.6% (EFS), 86.4% (VS14), 93.0% (DPS), and 93.0% (UFS). There is no significant differences among groups. The survival rates after thawing cryopreserved embryos are 80.2%, 91.7%, 69.5%, 0% and 91.8% (slow freezing, EFS, VS14, DPS and UFS). Also cultured for 96 hours, the hatching rates are 93.5% (no freezing), 84.1% (slow freezing), 93.9% (EFS), 48.5% (VS14) and 70.1% (UFS). Conclusion: The survival rates of vitrification in EFS solution and ultrarapid freezing are higher than slow freezing (p<0.05). The hatching rate of vitrification in EFS solution cultured for 96 hours is highest, so vitrification of day 2 mouse embryos in EFS solution considered as more effective for cryopreservation.

• Food Science of Animal Resources
• /
• v.41 no.6
• /
• pp.1012-1021
• /
• 2021

This study evaluated the effect of freezing rate on the quality characteristics of pork loin to establish an objective standard for rapid freezing. To generate various freezing rates, three air flow rates (0, 1.5, and 3.0 m/s) were applied under three freezing temperatures (-20℃, -30℃, and -40℃). Based on the results, freezing rates ranged from 0.26-1.42 cm/h and were graded by three categories, i.e, slow (category I, >0.4 cm/h), intermediate (category II, 0.6-0.7 cm/h) and rapid freezing (category III, >0.96 cm/h). Both temperature and the air flow rate influenced the freezing rate, and the freezing rate affected the ice crystal size and shear force in pork loin. However, the air flow rate did not affect thawing loss, drip loss or the color of pork loins. In the comparison of freezing rates, pork belonging to category II did not show a clear difference in quality parameters from pork in category I. Furthermore, pork in category III showed fresh meat-like qualities, and the quality characteristics were clearly distinct from those of category I. Although the current standard for rapid freezing rate is 0.5 cm/h, this study suggested that 0.96 cm/h is the lowest freezing rate for achieving meat quality distinguishable from that achieved with conventional freezing, and further increasing the freezing rate did not provide advantages from an energy consumption perspective.

• Korean Journal of Animal Reproduction
• /
• v.21 no.3
• /
• pp.287-292
• /
• 1997

This study was carried out to investigate the effects of concentration and kinds of cryoprotectants, equilibraction time, thawing temperature and time, sucrose concentration on the survival rates of frozen bovine demi-embryos. The bovine demi-embryos following dehydration by cryoprotectants a various concentration of sucrose were freezed by cell freezer and thawed in 3$0^{\circ}C$ water bath. Survival and in vitro developmental rates was defined as development rates on in vitro culture or FDA-test. The results are summarized as follows : 1. The high survival rates of demi-embryos after frozen-thawing in freezing medium was attained 2.0M glycerol. The high survival rates of demi-embryos after frozen-thawing in freezing medium was obtained using single cryoprotectant(25.0~30.0%) than mixed cryoprotectants(16.7~19.0%). 2. The survival rates of demi-embryos after frozen-thawing in freezing medium added 1.5M, 2.0M glycerol＋0.25M sucrose(37.5~33.3%) were higher survival rates than those of sucrose concentration of 0.50, 0.75M(12.5~26.7%). 3. The equilibration time on the survival rates of demi-embryos was attained after short period of time(30.0~35.0%) in the freezing medium higher than long period of time(21.1%). 4. The thawing temperature on the survival rates of demi-embryos was attained at 3$0^{\circ}C$ of thawing temperature(26.7~40.0%) higher than $25^{\circ}C$ or 37$^{\circ}C$ of thawing temperature(13.3~20.0%). 5. The thawing time on the survival rates of demi-embryos was attained at 1~5 minutes of thawing time(26.7~33.3%) in the freezing medium higher than 10 minutes of thawing time(13.3~18.8%).

• Journal of Food Hygiene and Safety
• /
• v.3 no.1
• /
• pp.19-26
• /
• 1988

Most of meat spoilage bacteria area Gram negative, which are very sensitive to freezing ; for instance , 90% of E. coli cells are killed or sub-lethally injured by freezing at -3$0^{\circ}C$, and the freeze-injury rate is dependent upon freezing rate. Since the injured bacterial cells are sensitive to selective agents, they fail to multiply in selective media. Injured bacterial cells are, however, capable of spontaneous repair at appropriate environmental and nutritional conditions . Enumeration of injured bacterial cells involves artificial induction of repair at these conditions. Cubic beef samples(3$\times$3$\times$3cm) were frozen at -6$0^{\circ}C$, -4$0^{\circ}C$, or -18$^{\circ}C$. The samples frozen at each temperature were thawed at 4$^{\circ}C$, 2$0^{\circ}C$, or by microwave . After these respective freezing an thawing treatments, the percentage of sub-lethally injured total coliforms out of total surviving ones was measured and compared. The results were as follows: 1. The interaction between freezing and thawing on injury rate was not significant. 2. The injury rates(as means of all three thawing treatments post-freezing) by freezing at -6$0^{\circ}C$, -4$0^{\circ}C$, or -18$^{\circ}C$ were 32.2$^{\circ}C$ and 19.2$^{\circ}C$ respectively . 3. The injury rates(as means of all three freezing treatments)by thawing at 4$^{\circ}C$, 2$0^{\circ}C$, or by microwave were 49.3%, 11.7% and 21.0% respectively. The highest injury rate was caused by freezing at -6$0^{\circ}C$ and subsequent thawing at 4$^{\circ}C$. However since the injury rates by freezing treatment were not significantly different, freezing at -18$^{\circ}C$ and subsequent thawing at 4$^{\circ}C$ can also be recommended , from an economic perspective.

• Korean Journal of Animal Reproduction
• /
• v.21 no.2
• /
• pp.95-102
• /
• 1997

The studies on the carried out to investigate the effective concentration of cryoprotectant agents and sucrose by one-step straw method of bovine embryos. The follicular oocytes were cultured in TCM-199 medium containing 10 IU/ml PMSG(Sigma, USA), 10 IU/ml hCG(Sigma, USA), 1$\mu\textrm{g}$/ml $\beta$-estradiol(Sigma, USA) and 10% FCS for 24~48 hrs in incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation of heparin. The bovine embryos following dehydration by cryoprotective agents and various concentrations of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival and in vitro developmental rate was defined as devellpmental rate on in vitro culture or FDA-test. The results are smmarized as followes : 1. The high in vitro developmental rates of bovine frozen embryos after rapidly thawed in freezing medium was attained 2.0M glycerol, 2.0M DMSO, 1M or 2.0M propanediol. 2. The high in vitro developmental rates of bovine frozen embryos after rapidly thawed in freezing medium was obtained single cryoprotectant(6.7~17.4%) than mixed cryoprotectants(6.7~16.7%). 3. In vitro developmental rate of bovine embryos after rapid frozen-thawing in the freezing medium added 0.25M and 0.50M sucrose were higher cleavage rate than those of sucrose concentration of 0.75M and 1.00M. 4. The freezing methods on in vitro developemental rates of bovine embryos was attained slow freezing method(9.70~15.6%) higher than rapid freezing method(9.4~13.3%).

• Korean Journal of Food Science and Technology
• /
• v.20 no.3
• /
• pp.447-452
• /
• 1988

In order to study effect of freezing rates on the quality changes such as pH, TBA value, free fatty acids and protein extractability, cylindrical chopped beef logs with 10cm of diameter and 10cm of height were frozen at three freezing rates(0.97cm/hr, 2.05cm/hr, 3.71cm/hr)using air blast freezer. Physicochemical changes of frozen meat were investigated during forzen storage at $-20^{\circ}C$ for 16weeks. Results on pH change showed $0.1{\sim}0.2unit$ increase at the 16th week of the frozen storage and the change was smaller with the increasing freezing rates. Free fatty acids content and TBA value also were increased during forzen storage, but they were minimal at 3.71cm/hr freezing rate. Correlation coefficient between TBA value and free fatty acids content were highly significant(r=0.804). After 16weeks of storage, extractibilities of salt soluble protein were decreased by 17.7%, 6.1% and 1.6% at freezing rates of 0.97, 2.05 and 3.71cm/hr, respectively. On the other hand, extractabilities of water soluble protein were decreased by 26.0%, 21.2% and 18.5%, respectively. The effect of freezing rates on the protein extractability appeared to be greater in salt soluble protein than in water soluble protein, but freezing denaturation was more rapid in water soluble protein.

• Journal of Embryo Transfer
• /
• v.29 no.1
• /
• pp.13-19
• /
• 2014

This study was carried out to study the survival rate of thawed Hanwoo embryos frozen by the slow-rate freezing or the cryotop vitrification method. Hanwoo cumulus-oocyte complexes were recovered from ovaries at a slaughter house, matured for 20~22 hours, fertilized with Hanwoo semen for 5~6 hours, and cultured for 7~9 days in $38.5^{\circ}C$, 5% $CO_2$ incubator. For freezing, Day 7~9 blastocysts were collected. Embryos for the slow-rate freezing were equilibrated in 1.8 M ethylene glycol (EG) with Dulbecco's phosphate-buffered saline (D-PBS). Programmable cell freezer was precooled down to $-7^{\circ}C$, and the straw was seeded during 8 minutes-holding time, and was cooled to $-35^{\circ}C$ at the cooling rate of $0.3^{\circ}C/min$, and then was plunged and stored in liquid nitrogen. Embryos for the cryotop vitrification were treated in TCM199 with 0.5 M sucrose, 16% EG, 16% dimethylsulfoxide (DMSO). Embryos were then loaded individually onto cryotop and plunged directly into liquid nitrogen. The survival rates of embryos frozen by these two freezing methods were evaluated at 12 to 24h post-thawing. The survival rates of frozen/thawed Hanwoo embryos by the cryotop vitrification method ($56.86{\pm}26.53%$) were slightly higher than those by the slow-rate freezing method ($55.07{\pm}26.43%$) with no significant difference. Using the cryotop vitrification and the slow-rate freezing of Hanwoo blastocysts on Day 7 following in-vitro fertilization (IVF) treatment, the survival rates of frozen/thawed Hanwoo embryos were $72.65{\pm}18.3%$ and $79.06{\pm}17.8%$, respectively. The survival rates by the cryotop vitrification were higher than those by the slow-rate freezing on both Day 8 and 9 with significantly higher survival rate on Day 9 (p<0.05). Using the cryotop vitrification and the slow-rate freezing of Hanwoo embryos to compare between three different blastocyst stages, the survival rates of the blastocyst stage embryos were $66.22{\pm}18.8%$ and $45.76{\pm}12.8%$, respectively with higher survival rate by the vitrification method (p<0.05). And the survival rate of expanded blastocysts was higher than those of early blastocysts and blastocysts in two freezing methods with significantly higher survival rate by the slow-rate freezing method (p<0.05).

• Korean Journal of Poultry Science
• /
• v.14 no.2
• /
• pp.145-151
• /
• 1987

In order to select the optimum freezing condition for the minimization of physico -chemical changes such as protein denaturation, lipid oxidation and pH change, the effect of freezing rates on the poultry meat quality changes was studied during frozen storage at -20$^{\circ}C$. Results obtained from the experiments are as fellows. When chicken breast and leg meat were frozen at above -3cm/hr or the freezing rate, pH change during frozen storage was minimal Although TBA value and free ratty acids were increased during frozen storage, the effect of freezing rates was different depending on muscle types. In terms of protein extractability, the extractability of salt soluble protein and water soluble protein were the highest at above -3cm/hr of the freezing rate during frozen storage. This trend was more obvious with breast meat than leg meat. Considering the above - described results, above -3cm/hr of the freezing rate seemed to be the optimum freezing condition for chicken meat because or the least pH change, low TBA value and high protein extractability.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.47 no.2
• /
• pp.126-133
• /
• 2023

Genetic resources of mulberry trees are commonly preserved as trophosomes, which are vulnerable to environmental factors, such as natural disasters, diseases, and pests. This study establishes a basic protocol for ultra-low temperature cryopreservation of mulberry trees using a two-step freezing process. The procedure was established using the "Daeshim" variety and then tested on genetic resources from 24 other mulberry varieties. Samples were first dried to a moisture content of 33-43% in a low-temperature forced-air chamber at -5 ℃, then slowly frozen from -5 ℃ to -20 ℃, and preserved in liquid nitrogen (-196 ℃). To determine the regeneration rate, isolated dormant buds were inoculated into MS basal medium, and grown shoots were grafted onto 1-year-old rootstock via chip budding and then cultured. After freezing in liquid nitrogen, the "Daeshim" variety exhibited a survival and regeneration rate of more than 70% and 50%, respectively. Applying the two-step freezing process to genetic resources from 24 mulberry species yielded average survival and regeneration rates of 85.3% and 75.5%, respectively. Morus alba showed survival and regeneration rates of 100%, confirming the efficacy of the two-step freezing method. These results indicate the high feasibility of ultra-low-temperature cryopreservation through two-step freezing of dormant buds from mulberry genetic resources. Additional research is required into the variations in regeneration rates with freezing period in liquid nitrogen.

• Korean Journal of Animal Reproduction
• /
• v.16 no.2
• /
• pp.125-131
• /
• 1992

This study was carried out to investigate the effects of concentration and equilibration time of cryoprotective agents on the survival rate of slowly and ultrarapidly frozen porcine embryos. The porcine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 38$^{\circ}C$ water bath. Survival rate was defined as development rat to the morula and blastocyst stage after in vitro culture or by FDA test. The results are summarized as follows : 1. The survival rates of porcine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0, 2.5, 3.0, 3.5, or 4.0M glycerol was 65.3, 61.8, 64.3, 59.4 or 39.4%, respectively. Addition of 0.25M sucrose into the freezing medium containing 2.0M glycerol showed higher survival rate than those of 2.5~4.0M glycerol. 2. The survival rates of porcine embryos after ultraradpid frozen-thawing in the freezing medium of 0.25M sucroese added 2.0, 2.5, 3.0, 3.5 or 4.0M DMSO was 65.6, 67.6, 68.6, 60.6 or 23.6%, respectively. However, addition of 0.25M sucrose into the freezing medium containing 3.0M DMSO showed higher survival rate than those of 2.0, 2.5, 3.5 or 4.0M DMSO. 3. The survival rates of porcine embryos after ultrapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0, 2.5, 3.0, 3.5 or 4.0M propanediol was 63.2, 60.3, 62.1, 52.3 or 24.3%, respectively. Addition of 0.25M sucroese into the freezing medium containing 2.0M propanediol showed higher survival rate than those of 2.5~4.0M glycerol. 4. The survival rates of porcine embryos after ultrarapid frozen-thawing the freezing medium of 2.0M glycerol added 0.10, 0.25, 0.50 or 0.75M sucrose was 61.8, 70.8, 67.6 or 52.2%, respectively. Addition of 2.0M glycerol into the freezing medium containing 0.25M sucreose showed higher survival rate than that those of 0.10, 0.50 or 0.75M sucrose. 5. The higher suvival rate of porcine embryos were attained at short period of equilibration time 92.5~5min.) in the freezing medium added 0.25M sucreose and 3.0M compared to those of 10 or 20min. equilibration time in the same condition.