• Development and Reproduction
• /
• v.11 no.2
• /
• pp.79-84
• /
• 2007

The freezing susceptibilities of two larval stages (D-shaped and umbo) of the pearl oyster (Pinctada fucata martensii) were evaluated by the electron microscopy (light, transmission electron and scanning electron). The morphological shapes were examined from each pre-frozen or frozen-thawed stage of the cryopreserved larvae in liquid nitrogen by using the cryoprotectant, dimethyl sulfoxide ($Me_2SO$) mixed with sucrose. Although a portion of the shell was damaged, the hinge and prodissoconch were intact and clearly visible after preservation in liquid nitrogen. In addition, the cytoplasm of the frozen-thawed larvae maintained the normal organelle integrities, e.g., endoplasmic reticula, lipid droplets, mitochondria, nucleus and microvilli. However, some of the frozen-thawed larvae showed irregularly arranged cilia, rough shell surfaces and round-lumped cilium heads. These results indicate that P. fucata martensii larvae are susceptible to freezing, at least at those two critical developmental stages (D-shaped and umbo), and suggest a new industrial investigation including reduction method of cell injury for preserving microbial starter cultures need to be developed.

• Journal of Embryo Transfer
• /
• v.13 no.2
• /
• pp.173-178
• /
• 1998

The objective of this study was to evaluate the embryonic development ability and the appearance of blastocysts of bovine in vitro fertilized oocytes cultured in different culture media, and also to evaluate survival rate after thawing of frozen embryos by using 1.5 or 1.8M ethylene glycol(EG) with sucrose or trehalose. Fertilized oocytes were divided into three groups; i ) monolayer of cumulus /granulosa cell prepared by TGM 199+5% calf serum(TGM199), ii)GRlaa+5% CS, iii)SOF+5% CS, and they were cultured after insemination for 9 days, at 39˚C, under 5% $CO_2$ in air, but SOF+5% CS was cultured at 39˚C, under 5% 02, 5% GO2, 99% N2. Blastocysts derived from GRlaa + 5% CS on day 7~8 after insemination were frozen by using 1.5M EG or 1.8M EG with/without 0.2M sucrose or O.1M trehalose. The development rate of blastocysts on day 7 after insemination in SOF+5% CS was significant higher than in TCM199 or CR1aa(P<0.05). The appearance rate of blastocysts on day 7-8 after insemination was higher than in TCM199, when fertilized oocytes were cultured in GRlas or SOF. The survival rate of frozen blastocysts after thawing tended to increase, when blastocysts were frozen by using 1.8M EG with 0.2M sucrose or O.1M trehalose. These results indicated that SOF or CRlaa media with amino acids was superior to TCM199 with monolayer in terms of blastocyst development in culturing of in vitro fertilized bovine nocytes, and sucrose or trehalose was supposed to prevent embryos from the freezing shock.

• Asian-Australasian Journal of Animal Sciences
• /
• v.14 no.1
• /
• pp.7-12
• /
• 2001

Factors Affecting the Survival of Frozen Thawed Bovine In Vitro Produced Blastocysts. The effect of some factors on the post-thaw survival of a total of 240 in vitro produced bovine blastocysts was investigated using logistic regression analysis. The explanatory variables tested were: type of culture medium before freezing (TCM 199 supplemented with BSA, BSAITS (BSA+insulin+transferrin+selenium), ECS (estrous cow serum) with or without BOEC (bovine oviductal epithelial cells), age of the blastocyst (Day 7, Day 8+9), morphological appearance before freezing (distinct=Q1 or indistinct=Q2 inner cell mass) and type of cryoprotectant (glycerol, 1.0 M or ethylene glycol, 1.6 M). The survival after thawing based on the post-thaw quality and the development after co-culture with BOEC for 24 and 48 hours. Day 7 blastocysts had an almost three times better chance of survival than Day 8+9 blastocysts. Q1, Day 8+9 blastocysts had higher odds to survive after 48 hours in culture than Q2 blastocysts (p<0.05). Blastocysts produced in BSAITS medium had the best chances of survival; however, the odds were not always significant. Blastocysts frozen in glycerol had a better post-thaw quality rating than those frozen in ethylene glycol; however, the difference in post-thaw development at culture was not significant. The relationship between post-thaw quality and post-thaw development at culture was significant (p<0.05). The developmental stage and/or age of the embryo and culture medium where development up to blastocyst takes place affect the post-thaw survival of the bovine embryos.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.18 no.1
• /
• pp.44-51
• /
• 1985

The changes of the get forming ability of common carp (Cyprinus carpio) and conger eel (Astroconger myriaster) meat during ice and frozen storage were investigated in connection with the other quality indices, such as pH, K-value, VBN, viable cell count and sensory evaluation. The shelf-life of iced common carp and conger eel as the raw materials for fish jelly product was considered to be about 16 and 13 days, respectively. Little change in gel forming ability of two species was found during frozen storage at $-30^{\circ}C$ for 3 months. From the results obtained in the examination on the gel forming abilities of the two species stored at $-30^{\circ}C$ for 3 months were superior to those stored in ice for 3 to 4 and 4 to 6 days individually. Fish jelly from the common carp was much more elastic than that of conger eel meat. The overall qualities of two species held in frozen storage as the raw material for fish jelly product were excellent.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.56 no.6
• /
• pp.818-825
• /
• 2023

The surge in online seafood consumption has increased parcel delivery, leading to a need to implement effective preservation methods. As the cold chain system is not fully established in Korea, styrofoam boxes and cold packs are commonly used for low-temperature seafood distribution. The impact of cold packs on product preservation depends on the number utilized. Herein, the freshness of vacuum-packed frozen fish fillets (Paralichthys olivaceus and Scomber japonicus) stored at 25±0.5℃ for up to 84 h was measured. Chemical (pH and volatile base nitrogen), microbiological (viable cell count), and physical (odor intensity) properties were assessed using 2 or 4 cold packs in a styrofoam box. Four cold packs yielded lower values, indicating superior freshness, and extended fish freshness by approximately 12 h compared with two cold packs. Therefore, it is recommended to use a minimum of 4 cold packs (-350 g/cold pack) in a styrofoam box for distributing approximately 300 g of frozen fish fillets at room temperature during the summer, considering an average delivery period of 2 days in Korea.

• Clinical and Experimental Reproductive Medicine
• /
• v.28 no.3
• /
• pp.183-190
• /
• 2001

Objective: Recently, microdissection of tissue sections has been used increasingly for the isolation of morphologically identified homogeneous cell populations, thus overcoming the obstacle of tissue complexity for the analysis cell-specific expression of macromolecules. The aim of the present study was to establish the minimal conditions required for the RNA extraction and amplification from the cells captured by the laser captured microdissection. Methods : Mouse ovaries were fixed and cut into serial sections (7 im thickness). Oocytes were captured by laser captured microdissection (LCM) method by using PixCell $II^{TM}$ system. The frozen sections were fixed in 70% ethanol and stained with hematoxylin and eosin, while the paraffin sections were stained with Multiple stain. Sections were dehydrated in graded alcohols followed by xylene and air-dried for 20 min prior to LCM. All reactions were performed in ribonuclease free solutions to prevent RNA degradation. After LCM, total RNA extraction from the captured oocytes was performed using the guanidinium isothiocyanate (GITC) solution, and subsequently evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Results: With the frozen sections, detection of the GAPDH mRNA expression in the number of captured 25 oocytes were not repeatable, but the expression was always detectable from 50 oocytes. With 25 oocytes, at least 27 PCR cycles were required, whereas with 50 oocytes, 21 cycles were enough to detect GA PDH expression. Amount of the primary cDNA required for RT-PCR was reduced down to at least 0.25 $\grave{i}$ l with 50 oocytes, thus the resting 19.75 il cDNA can be used for the testing other interested gene expression. Tissue-to-slide, tissue-to-tissue forces were very high in the paraffin sections, thus the greater number of cell procurement was required than the frozen sections. Conclusion: We have described a method for analyzing gene expression at the RNA level with the homogeneously microdissected cells from the small amount of tissues with complexity. We found that LCM coupled with RT-PCR could detect housekeeping gene expression in 50 oocytes captured. This technique can be easily applied for the study of gene expression with the small amount of tissues.

• The Korean Journal of Cytopathology
• /
• v.7 no.2
• /
• pp.177-184
• /
• 1996

Intraoperative cytologic examination of intracranial tumors using crush preparation provides useful information in operative decision making. The diminutive nature of many biopsy specimens, particularly those obtained by stereotactic neurosurgical procedures emphasizes the importance of combining the cytologic smear method with conventional frozen section interpretation. The great advantage of the cytologic smear method resides in its suitability for the study of minute fragments of tissue, allowing retention of the majority of the specimen for optimal processing. We present the cytologic features of 3 cases of intracranial germ cell tumors(2 germinomas and 1 endodermal sinus tumor), using crush preparation during intraoperative diagnosis and compare them with histologic findings. The cytologic features of the germ cell tumors were similar to those of the respective gonadal counterparts. The cytologic differential diagnosis of both types of germ cell tumors is described.

• Korean Journal of Animal Reproduction
• /
• v.15 no.2
• /
• pp.133-139
• /
• 1991

This stduy was carried out in order to investigate the effects of concentration and equilibration time of cryoprotective agents on survival rate of slow and ultrarapidly frozen in vitro fertilized bovine embryos. In vitro fertilized bovine embryos, following dehydration by cryoprotective agents and sucrose, were slowly freezed(from 2$0^{\circ}C$ to -7$^{\circ}C$/-1$^{\circ}C$/min., from -7$^{\circ}C$ -35$^{\circ}C$/-0.2$^{\circ}C$/min. from -35$^{\circ}C$ to -38$^{\circ}C$/-0.3$^{\circ}C$/min.) by cell freezer and directly plunged into liquid nitrogen and thawed in 38$^{\circ}C$ water. Survival rate was defined by development rate to the morula and blastocyst stage after in vitro cultured and FDA test. The results are summarized as follows : 1. The survival rates of in vitro fertilized bovine embryos after slow frozen-thawing in the freezing medium of 0.25M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋2.0M propanediol were 84.3%, 85.9%, 77.8%, 74.3%, respectively. 2. The survival rates of in vitro fertilized bovine embryos after slow frozen-thawing in the freezing of 0.50M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋2.0M propanediol were 83.8%, 85.1%, 71.4%, 74.6%, respectively. 3. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing of 0.25M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋3.0M propanediol were 69.3%, 70.8%, 63.2%, 67.1%, respectively. 4. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing of 0.25M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋2.0M propanediol were 69.4%, 70.1%, 62.3%, 63.5%, respectively. 5. The survival rates of in vitro fertilized bovine embryos after slow and ultrarapid fromthawing in the freezing medium of sucrose added cryoprotective agents were not significant difference between 5min. and 10min. of equilibration time.

• Korean Journal of Animal Reproduction
• /
• v.16 no.2
• /
• pp.117-123
• /
• 1992

This Study was carried out ot investigate the effects of concentration and equilibration time of cryoprotective aagents on survival rate of slowly and ultrarapidly frozen porcine embryos. The porcine embryos following dehydration by cryoprotective agents and 0.25M sucrose were slowly freezed(from 2$0^{\circ}C$ to -7$^{\circ}C$/-1$^{\circ}C$/min., from -7$^{\circ}C$ to -35$^{\circ}C$/-0.2$^{\circ}C$/min., from -35$^{\circ}C$ to -38$^{\circ}C$/-0.3$^{\circ}C$/min.) by Cell Freezer and directly plunged into liquid nitrogen and thawed in 38$^{\circ}C$ water bath. Survival rate was defined as development rate 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 slow frozen-thawing in the freezing medium of 0.25M sucrose added 2.0M glycerol, 3.0M DMSO, 2.0M propanediol or 2.0M glycerol＋2.0M propanediol was 80.6, 84.7, 75.0 or 78.8%, respectively. 2. The survival rates of porcine embryos after slow frozen-thawing in the freezing medium of 0.50M sucrose added 2.0M glycerol, 3.0M DMSO, 2.0M propanediol or 2.0M glycerol＋2.0M propanediol was 80.9, 82.4, 73.1 or 77.1%, respectively. 3. The survival rates of porcine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucroese added 2.0M glycerol, 3.0M DMSO, 2.0M propanediol or 2.0M glycerol＋2.0M propanediol was 65.3, 68.6, 63.2 or 59.9%, respectively. 4. The survival rates of porcine embryos after ultrapid frozen-thawing in the freezing medium of 0.50M sucrose added 2.0M glycerol, 3.0M DMSO, 2.0 propanediol or 2.0M glycerol＋2.0M propanediol was 67.5, 62.9, 56.9, or 62.8%, respectively. 5. The higher survival rate of porcine embryos was attained at the short period ofequilibration time(5min.) in the freezing medium added 0.25M sucrose and 3.0 DMSO compared to those of 10 or 20min. equilibration time in the same condition.

• Journal of Animal Science and Technology
• /
• v.55 no.5
• /
• pp.417-425
• /
• 2013

We sought to provide a method for freezing and preserving primordial germ cells, or an avian germ cell of a bird, as a material for developmental engineering or species preservation. The aim of this study was to compare the efficacy of slow freezing with a vitrification method for the cryopreservation of chicken primordial germ cells (PGCs). PGCs obtained from the germinal gonad of day 5.5-6 day (stage 28) cultured chick embryos, using the MACS method, were classified into two groups: slow freezing and vitrification. We examined the viability of PGCs after Cryopreservation. Four freezing methods were compared with each other, including the following: Method 1: The PGCs were frozen by a programmed freezer in a plastic straw, including 2.0 M ethylene glycol (EG) as cryoprotective additive (slow freezing) Method 2: The PGCs were vitrified in a plastic straw, including 8.0 M EG, plus 7% polyvinylpyrrolidone (PVP) (rapid freezing). Method 3: The slow freezing was induced with a cryotube including 2.0 M EG Method 4: The PGCs were frozen in a cryotube including 10% dimethyl suloxide (DMSO) (rapid freezing). After freezing and thawing, survival rates of the frozen-thawed PGCs from Method 1 to 4were 76.4%, 70.6%, 80.5% and 78.1% (p<0.05), respectively. The slow freezing ($-80^{\circ}C$ programmed freezer) method may provide better survival rates of frozen-thawed PGCs than the vitrification method for the cryopreservation of PGCs. Therefore, these systems may contribute to the cryopreservation of a rare avian species.