• Journal of Aquaculture
• /
• 제11권1호
• /
• pp.67-75
• /
• 1998

Experiments were performed to obtain cryopreservation techniques of black seabream (Acanthopagrus schlegeli) sperm. For sperm collection, brood stock reared in recirculating seawater system and fed with the commercial feed during experimental period. The results indicated that following cryopreservation method in block seabream sperm could be employed. Post-thaw survival rate of sperm revealed the highest value ($80{\pm}1.4$%) in 3% sodium citrate as a diluent for the cryopreservation. Cryopreserved sperm diluted with 5.4% glucose showed the highest fertilization rate to the ovulated eggs. Glycerol was a better cryoprotectant than dimethyl sulfoxide in sperm cryopreservation : survival rate and fertilizing capacity of cryopreserved sperm were decreased according to increase of glycerol concentration and varied in renges of 0.8~59.3% and 32.5~69.4% with 5~30% glycerol, respectively. A few of cryopreserved spermatozoa showed the enlarged head with granulated chromatin and ruptured plasma membrane by freezing and thawing injuries compared with unfrozen normal spermatozoa.

• Journal of Aquaculture
• /
• 제20권3호
• /
• pp.173-177
• /
• 2007

An experiment was performed to obtain cryopreservation techniques of starry flounder (Platichthys stellatus) sperm. Milt obtained from 24 males were cryopreserved using two diluents, artificial seminal plasma (ASP) and Stein's solution (SS) with three cryoprotectants, dimethyl sulfoxide (DMSO), methanol, and glycerol concentrated from 5% to 20%. Post-thaw sperm activity (motility and/or speed) revealed the highest in 10% DMSO and 15% methanol in ASP and SS as diluent. Motility and speed of cryopreserved sperm were decreased according to increase of glycerol concentration. To conclude, DMSO was a better cryoprotectant than methanol or glycerol for cryopreservation of starry flounder sperm. Glycerol was incongruent cryoprotectant because of toxic to starry flounder sperm. Most cryopreserved spermatozoa without cryoprotectant showed the enlarged head with granulated chromatin and ruptured plasma membrane by freezing and thawing injuries compared with unfrozen normal spermatozoa.

• Molecules and Cells
• /
• 제26권6호
• /
• pp.558-565
• /
• 2008

Although the fertilizing ability of spermatozoa is greatly reduced after freezing, complete understanding of alterations induced by cryopreservation has not been elucidated. The present study evaluates the effects of cryopreservation on the $Ca^{2+}$ handling of boar spermatozoa using several sperm activators. Intracellular $Ca^{2+}$ signals from single spermatozoa were measured using confocal $Ca^{2+}$ imaging of unfrozen samples and of other spermatozoa after having been frozen. Elevation of the external $K^{2+}$ concentration elicited a three times larger $Ca^{2+}$ increase in fresh spermatozoa than in cryopreserved spermatozoa. Caffeine elicited $Ca^{2+}$ transients with some oscillations in the fresh spermatozoa, but not in the thawed spermatozoa. Depletion of the $Ca^{2+}$ store with thapsigargin induced a rapid rise in $Ca^{2+}$ in the control but generated a smaller increase of $Ca^{2+}$ after thawing. Exposure to progesterone induced a biphasic rise of the $Ca^{2+}$ level in the fresh spermatozoa only. Sperm viability was reduced by cryopreservation. Resting $Ca^{2+}$ levels in fresh and cryopreserved spermatozoa were similar. Longer incubation (2.5 h) of thawed spermatozoa partly recovered the $Ca^{2+}$ response to the interventions. These results suggest that cryopreservation reduces the responsiveness of spermatozoa to depolarization, modulators of the internal $Ca^{2+}$ store and progesterone in terms of the $Ca^{2+}$ signal, thus providing a possible mechanism for reduced fertility observed in cryopreserved boar spermatozoa.

• Clinical and Experimental Reproductive Medicine
• /
• 제18권1호
• /
• pp.81-87
• /
• 1991

There studies were carried for evaluation of the efficiency of freezing of hamster oocytes for use in a human sperm penetration assay. The hamster oocytes fully equilibrated in various cryoprotectant agents and inseminated with human sperm. After insemination with hamster oocytes, there was no difference in penetrated rates. Cumulus free oocytes equilibrated in 1.5M various cryoprotective agents and slowely cooled to temperature $-30^{\circ}C$ before rapid cooling and storage in nitrozen tank. After rapid thawing, survival rates of frozen oocytes according to cryo-protective agents were examined and the human sperm penetration assay with zona free hamster oocytes was conducted. 1. Survival rates of oocytes after cryoprotectants exposure have no significant difference (range 88-91%) and peneration rate was 51.1%. 2. Recovery and survival rate of frozen-thawed oocytes were 85.1 and 66.8%. There was no significant difference on cryoprotective agents. 3. Penetration rates of the frozen-thawed and intact oocytes were 69.0 and 77.0%, respectively. 4. Hamster oocytes cryopreservation provides a convenient way of supplying and trans-porting hamster oocytes for the assessment of the fertilizing potential of human spermatozoa.

• Journal of Korean Tunnelling and Underground Space Association
• /
• 제8권3호
• /
• pp.237-247
• /
• 2006

The use of high-performance shotcrete lining is indispensable to improve long-term durability of a tunnel and to apply the single-shell tunnelling method. Among a lot of shotcrete admixtures, pozzolan materials such as silica fume have positive effects on increasing the strength and the durability of shotcrete. It is also well known that a cement-based accelerator is much faster in setting time and more eco-friendly than conventional accelerators. This study aimed to improve the properties of wet-mix shotcrete by incorporating with Metakaolin and the calcium aluminate based accelerator. To compare Metakaolin with silica fume, mixing ratios of each material were varied as 4% and 8% of cement weight. Moreover, Metakaolin was blended with silica fume, and their binder was also set to 4% and 8% of cement weight. At each mixing condition, setting time, compressive strength, flexural strength, permeability and freezing-thawing resistance were measured. From the experiments, it was revealed Metakaolin could be a substituting material for silica fume.

• Clinical and Experimental Reproductive Medicine
• /
• 제29권1호
• /
• pp.37-44
• /
• 2002

Objective : Heat shock protein family is related to protective mechanism of cells by environmental changes. This study was performed to evaluate the effect of cryopreservation on the heat shock protein 90 (Hsp90) expression in mouse ovarian tissue. Methods : Cryopreservation of mouse ovarian tissue was carried out by slow freezing method. The mRNA level of Hsp90 expression in both fresh and cryopreserved mouse ovarian tissue was analyzed by RT-PCR. The protein expression of Hsp90 was evaluated by Western blot analysis and immunohistochemistry. Results: The mRNA and protein of Hsp90 were expressed in both fresh and cryopreserved mouse ovarian tissue. The amount of Hsp90 mRNA was increased in cryopreserved ovarian tissue after 60 and 90 minutes after thawing and incubation. The amount of Hsp90 protein was increased in the cryopreserved ovarian tissue after 6 hours of the incubation in Western blot analysis. In immunohistochemical study, Hsp90 protein was localized in cytoplasm of oocytes and granulosa cells. Significant level of immunoreactive Hsp90 protein was detected in theca cells contrast to the weak expression in ovarian epithelial cells. Conclusion: This results showed the increase of Hsp90 expression in both mRNA and protein level in the cryopreserved mouse ovarian tissue. It can be suggested that Hsp90 may play a role in the protective or recovery mechanism against the cell damage during cryopreservaion.

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 한국수정란이식학회 2002년도 국제심포지엄
• /
• pp.84-84
• /
• 2002

The advantages of the OPS techniques(Vajta G et al, Mol Reprod Dev 51: 53-58,1998) give 1) high survival rates of various types of eggs, 2) quick and simple process, 3) inexpensive equipment and reduced chilling injury. The efficiency of IVM/IVF technique in the porcine species is relatively lower than that obtained in other species such as ruminants. Two experiments were designed to investigate the effects of in-vitro fertilization of porcine oocytes matures using different OPS protocol for chilling and warming of vitrification. Porcine oocytes from ovaries collected at abattoir were matured for 44 hours in TCM199 Earle's salt supplemental with pyruvate, pff, L-cysteine, hormones and gentamycin. Oocytes were denuded and fertilized with frozen boar semen by common method. Porcine embryos produced routinely by in-vitro culture system of NCSU23 medium. The vitrification and the warming were conducted by OPS method with the glass micropipette instead of straw vessels and modified the protocol of G.Vajta(1999). In Exp 1, Chilling/Warming:Holding Medium(HM)+EG+DMSO/HM +sucrose Medium(SM) at 39$^{\circ}C$ warm stage. In Exp 2, : PBS+CS+EG+Ficoll+ Trehalose/PBS+Trehalose at 25$^{\circ}C$ stage. Filling, freezing, packing, thawing out and further culturing were performed to follow the basic protocol of G Vajta. During IVM-lVC and post-warming, fertilization parameter and developmental potential were compared to and statistically analysed. It was not significantly different from Exp 1 and Exp 2 but 25$^{\circ}C$ of stage was slightly higher on the morula/blastocyst forming rate and better atmosphere for worker than that at 39$^{\circ}C$ stage.

• Journal of Korean Society on Water Environment
• /
• 제32권6호
• /
• pp.520-527
• /
• 2016

Phycocyanin (PC) is one of the water-soluble accessory pigments of cyanobacteria species, and its concentration is used to estimate the presence and relative abundance of cyanobacteria. In laboratory experiments, PC content of field data were determined using Sarada's freeze-thaw method in algal bloom season. The effectiveness of three selected extraction methods (repeated freeze-thaw method, homogenization, power control) for PC were determined. The extraction efficiency of phycocyanin was the highest (of the methods compared) when a single freezing-thawing cycle was followed by pre-sonication. Applying this optimized method to surface water of Korean inland waters, the average concentration distribution was estimated at $2.9{\sim}51.9mg/m^3$. It has been shown that the optimized pre-sonication method is suitable to measure cyanobacteria PC content for the characterization of inland waters. The approach and results of this study indicates the potential of effective methods for remote monitoring and management of water quality in turbid inland waters using hyperspectral remote sensing.

• Asian-Australasian Journal of Animal Sciences
• /
• 제22권4호
• /
• pp.492-499
• /
• 2009

A Silkie Bantam embryo fibroblast line (named SBF59 line) was successfully established by using direct explant culture and cryopreservation techniques. Cell morphology, viability, dynamic growth and contamination were tested and the karyotype and levels of isoenzymes of lactic dehydrogenase and malic dehydrogenase were analyzed. Four kinds of fluorescent protein extrogenes, including $pEGFP-N_3$, $pECFP-N_1$, $pEYFP-N_1$ and $pDsRed1-N_1$ were transfected into the cells. The results showed that the cells were healthy and possessed a fibrous structure without a change in morphology. The average viability of the cells was 96% before freezing and 90.5% after thawing. The growth curve appeared as typical "S" shape and the cell growth passed through a detention phase, a logarithmic phase and a platform phase; the estimated population doubling time (PDT) was 38.5 h; assays for the presence of bacteria, fungi, viruses and mycoplasmas were negative; the cell line showed no cross contamination when assessed by isoenzyme analysis; the chromosome number was 2n = 78 on more than 88% of occasions; four kinds of fluorescent protein extro-genes appeared to be expressed effectively with a high transfection efficiency between 18.3% and 42.3%. The cell line met the required quality control standard. It not only preserves the genetic resources of the important Silkie Bantam at the cellular level but also provides valuable materials for genomic, post-genomic, somatic cell cloning research and other applications.

• The Journal of the Korea Contents Association
• /
• 제16권4호
• /
• pp.114-122
• /
• 2016

Concrete is cost-benefit and high-durable construction material, however durability problem can be caused due to steel corrosion under chloride attack. Recently deicing salt has been widely spread in snowing season, which accelerates micro-cracks and scaling in surface concrete and the melted deicing salt causes corrosion in embedded steel. The previous governing equation of Fick's 2nd Law cannot evaluate the deteriorated surface concrete so that another technique is needed for the surface effect. This paper presents chloride penetration analysis technique for concrete subjected to deicing salt utilizing multi-layer diffusion model and time-dependent diffusion behavior. For the work, field investigation results of concrete pavement exposed deicing salt for 18 years are adopted. Through reverse analysis, deteriorated depth and increased diffusion coefficient in the depth are evaluated, which shows 12.5~15.0mm of deteriorated depth and increased diffusion coefficient by 2.0 times. The proposed technique can be effectively applied to concrete with two different diffusion coefficients considering enhanced or deteriorated surface conditions.