• Journal of Animal Reproduction and Biotechnology
• /
• v.36 no.2
• /
• pp.69-75
• /
• 2021

Cryopreservation is a widely-used efficient means of long-term sperm preservation. However, unlike other types of semen, cryopreserved boar semen has reduced fertility and the efforts continue to optimize post-thawing sperm recovery. In this study, we evaluated the effects of various washing solutions (Hulsen solution, lab-made DPBS and commercial DPBS) on post-thawing porcine sperm kinematics (CASA system), viability (SYBR-14/PI) and acrosome integrity (PSA/FITC). We also examined the effect of washing-centrifugation on frozen-thawed semen kinematics. The results indicate that type of washing solution and post-thawing centrifugation alters parameters linked to sperm quality (total motility, progressive motility, viability and acrosome integrity). Significantly higher (p < 0.05) motility and progressive motility were obtained when cryopreserved semen was processed with Hulsen solution. The post-thaw percentage of live and intact acrosomal sperm was significantly higher in group 1 (Hulsen solution) as compared to other groups. Following thawing-centrifugation, the results showed significantly higher motility and progressive motility in group 1 than other groups. However, the latter two DPBS groups did not differ statistically. Taken together, Frozen-thawed spermatozoa motility, acrosome integrity and viability can be affected by the type of washing solution used. Moreover, centrifugation of frozen-thawed semen has an unfavorable effect on total motility and progressive motility.

• Bulletin of the Korean Chemical Society
• /
• v.23 no.12
• /
• pp.1769-1772
• /
• 2002

Previous $^{19}F\;and\;^{1,2}H$ electron-nuclear double resonance (ENDOR) study of fluorometmyoglobin (MbF) in frozen-solution state provided sensitive tools sensing subtle structural changes of the heme that are not obtainable from X-ray. [Fann et al., J. Am. Chem. Soc. 1995, 117, 6019] Because of the intrinsic inhomogeneouse EPR line broadening effect of MbF in frozen-solution state, detection of the intrinsic inhomogeneouse EPR line broadening effect of MbF in frozen-solution state, detection of the electronic and geometrical changes of the heme ring itself and the proximal histidine by using $^{14}N$ CW ENDOR was interfered. In the present study, hyperfine-sensitive $^{14}N$ Mims ENDOR technique of pulsed-EPR was employed to probe the changes. With two different $\tau$ values of 128 and 196 ns, $^{14}N$ ENDOR signals of the heme and proximal histidine were completely resolved at $g'_{II}(=g_e=2)$. This study present that X-band $^{14}N$ Mims ENDOR sequence can sensitively detect the small changes of the spin densities and p orbital populations of the proximal and the heme nitrogens, caused by ligand and pH variation of the distal site.

• Journal of Navigation and Port Research
• /
• v.37 no.2
• /
• pp.129-135
• /
• 2013

Recently, waste water treatment system is developed in small and middle size to get more economic advantage. Freeze concentration system has high thermodynamic efficiency and low energy consumption, can re-use purified water and cold energy obtained from ice. This study was experimentally performed to investigate pollution containment in frozen layer by cooling wall temperature, air-bubble flow methods, initial ice-lining thickness of frozen layer in NaCl aqueous solution and the representative heavy metals, Pb and Cr aqueous solution. As the result, a decrease in the cooling wall temperature bring a higher growth rate of ice front and the more solute was involved in frozen layer. The method to inject directly air-bubble into ice-liquid interface through ring shape nozzle gave high purity of ice compared to indirect method. Ice lining in 5mm thickness resulted in frozen layer with higher purity than 1mm thickness.

• Journal of Veterinary Clinics
• /
• v.20 no.4
• /
• pp.482-488
• /
• 2003

Transplanted cortical bone grafts of freeze-dried bones also function as sustaining for defected bones, however, it has less strength and is fragile without rehydration. In this study, strength and stiffness of freeze-dried bone from bovine cortical bones were evaluated by three point bending test according to different time frames such as rehydration times of 0.5, 3, 6, 12 and 24 hrs in electrolyte solution and was compared with those of frozen bones. The strength and stiffness of frozen bone were $264.4\pm36.7$ MPa, $17.0\pm1.5$ GPa, respectively. The strength and stiffness of freeze-dried bone which fat was removed by treatments of chloroform-methanol solutions for 6 days, then was freeze-dried at $-80^{\circ}C$ and sterilized with ethylene oxide gas, were $224.9\pm27.6$ MPa, $19.2\pm2.8$ GPa, respectively. The strength and stiffness of feeze-dried bone were decreased 15.0% and increased 13.2% than these of frozen bone, respectively. The strength and stiffness of freeze-dried bone rehydrated for 6 hrs were restored to 96.0% strength and 99.2% stiffness of frozen bone. The rehydration time of freeze-dried bone which had the highest strength and stiffness was six hours and three hours, respectively. The results of the mathematica program for the variation of the strength and stiffness showed 3 hours and 30 minutes of rehydration time in electrolyte solution for the best condition in the strength and stiffness which was adequate to treat freeze-dried cortical bone.

• Asian Journal of Turfgrass Science
• /
• v.4 no.1
• /
• pp.42-48
• /
• 1990

Experiments were conducted to investigate the soil freezing depth and pattern with freezing measuring instruments during 1988-l989 winter season in Kangwon province. Freezing measuring instrument was made with acrylic pipes which were consisted of inner and outer parts. Inner pipe was filled with 0.01 % methylene blue solution and rubber hose to protect pipe breakdown by solution freezing. Freezing measurements were carried out by observing discoloration of methylene blue solution. Moisture content of evergreen trees and ground cover plants was also examined in the winter season. The observed results are as follows: 1.In the land of I OOM above sea level, soil freezing depth became deeper as the sum of Accumulated degree-days of temperature below 0˚C(0˚C . day) increased: Soil freezing depth was 30-40cm at l00˚C, 42-43cm at 150˚C, and 47cm at 200˚C day 2.Soil freezing with vinyl mulching was less developed by l3cm at l00˚C with sum of subzero temperature, by l7cm at 200˚C than that of the bare ground. Soil of rich hulls mulching with 4Ocm was not frozen until soil freezing at the bare ground was developed to 25cm depth. 3.Cashmeron mulching was more effective than felt mulching in the heat insulation of soil. 4.Thawing of soil was done from the lowest part of the frozen in the ground to upward in the beginning and after that it was done from the surface of frozen soil to downward. Finally thawing was completed at the middle of frozen soil.

• Korean Journal of Veterinary Research
• /
• v.38 no.2
• /
• pp.394-400
• /
• 1998

Cryopreservation of embryos by vitrification is a simple method to preserve bovine embryos for subsequent embryo transfer, but embryonic viability after vitrification has been inconsistent and low compared with conventional slow freezing. The aim of the present study is to examine the effect of serum or serum albumin in a vitrification solution and epidermal growth factor(EGF) or fibroblast growth factor(FGF) on in vitro viability of bovine blastocysts frozen by vitrification. Bovine blastocysts were produced by in vitro maturation, fertilization of follicular oocytes and culture of embryos in a synthetic oviduct fluid medium(SOFM) containing BSA and 19 essential and nonessential amino acids. Blastocysts with excellent or good morphology were selected at 7 or 8 days after culture and utilized for vitrification. In experiment 1, blastocysts were vitrified in a solution containing semi-fetal calf serum(SFCS) or BSA(5 or 10mg/ml) and then their subsequent viabilities were examined by culturing thawed embryos in a SOFM containing BSA and 19 amino acids. Effect of EGF or FGF added to a SOFM containing polyvinyl alcohol(PVA) on the viability of vitrified-thawed blastocysts was investigated in experiment 2. BSA added at 5 or 10mg/ml to a vitrification solution showed significantly higher(p < 0.05) developmental rate to expanded and hatching blastocysts than SFCS, but there was no significant difference in the developmental rate to hatched blastocysts after thawing. Supplementation of a culture medium with EGF and/or FGF significantly increased(p < 0.05) embryo development to expanded blastocysts compared with control but showed no beneficial effect on the development to hatching or hatched blastocysts. Coculture of thawed embryos with granulosa cells in a TCM 199 containing 10% fetal calf serum(FCS) showed the highest developmental rate to expanded, hatching and hatched blastocysts among the groups tested. In conclusion, supplementation of a vitrification solution with BSA at 5mg/ml and culture of thawed blastocysts in a medium containing EGF and/or FGF can improve in vitro viability of bovine blastocysts frozen by vitrification.

• Korean Journal of Animal Reproduction
• /
• v.26 no.4
• /
• pp.311-320
• /
• 2002

The objective of this study was to assess the developmental potential in vitro produced embryos frozen-thawed with the various containers, and also examined expression and localization of heat shock protein 70 at these embryos. For the vitrification, 2-cell, 8-cell and blastocyst stage embryos produced by in vitro fertilization (IVF) and nuclear transfer (NT) were exposed the ethylene glycol 5.5 M freezing solution (EC 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop, and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min. and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid and cryo-loop. However, survival rates by straw were relatively lower than other containers. The use of cryo-loop resulted in only survival of nuclear transferred embryos (43.7%). Also, there embryos after IVF or NT were analysed by semi-quantitive reverse transcription-polymerase chain reaction (RT- PCR) methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNh were higher thawed embryos than control embryos. Immunocytochemistry used to localize the hsp 70 protein in embryos. Two and 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some frozen-thawed embryos. However, in the control, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform In distribution. Therefore, this result suggests that the exploiting Hsp 70 in the early embryos may be role for protection of stress condition for increase viability of embryos within IVF, NT and there frozen-thawed embryos.

• Journal of Embryo Transfer
• /
• v.11 no.3
• /
• pp.309-316
• /
• 1996

The aim of this study is to elucidate sperm chemotaxis and to set up the optirnal condition for selection of motile and capacitated sperm from hovine frozen-semen. Thus, the effects of semen-washing after thawing, concentrations of progesterone (P4) and bovine serum albumin (BSA), and sperm-washing frequency on sperm selection were examined. For evaluating their effects, number, viability and acrosome reaction of sperm swim-up seperated from semen, which were incubated for 30 minutes at 36$^{\circ}C$ in the M2 solution containing P4 and BSA, were investigated. For frozen-semen just after thawing, sperm recovery and viability were not significantly different between P4-treated and -untreated semen. However, washing frozen-semen decreased the number of sperm and increased the viability of sperm that were recovered from semen treated with P4. Progesterone affected the recovery rate, the viability and the acrosome-reaction rate of sperm recovered from washed frozen-semen. Especially, number of motile and capacitated sperm were highest in semen treated with 50$\mu$g /ml among 0, 20, 50 and 100$\mu$g /ml of P4 concentrations. BSA affected the recovery rate and the viability of sperm recovered from washed frozen-semen that were treated with 50$\mu$g /ml of P4. Especially, the percentage of viable sperm were highest in semen treated with 4mg /ml among 0, 2, 4, and 6mg /ml of BSA concentrations. Repeatedly sperm-washing did not affect the recovery rate and the viability of sperm recovered from washed frozen-semen that were treated with 50$\mu$g /ml of P4 and 4mg /ml of BSA In conclusion, using progesterone and BSA could efficiently make the selection of motile and capacitated sperm from washed frozen-semen.

• Clinical and Experimental Reproductive Medicine
• /
• v.20 no.2
• /
• pp.165-176
• /
• 1993

These studies were carried out to investigate the optimal freezing protocol for 2 cell mouse embryos and to find the probability of quality control with 2-cell embryos frozen. The embryos showed the best survival by the protocol composed of a freezing solution with the cryoprotectants(1.5M propanediol + 0.1M sucrose), and a 2-steop thawing method(room temperature, 20 sec-37$^{\circ}C$, 20 sec). The developmental ability of frozen-thaw 2-cell embryos did not differ from that of fresh 2-cell embryos in m-KRB medium with 0.4% bovine serum albumin. But development of frozen-thaw embryos was depended on the supplements of the medium. In the albumin-free medium, the developmental rate(rate of blastocysts) was significantly reduced, compared with that in the medium with 0.4% BSA. Also, when frozen-thaw embryos were cultured in the meduim with human fetal cord serum(HCS), the developmental rate of frozen-thaw embryos was sligtly reduced, compared with that of fresh 2-cell embryos. Finally, frozen-thaw 2-cell mouse embryos were more sensitive to the toxic agent of disposable-plastic syringe. Therefore, toxicity of medium could be effectively detected by frozen-thaw 2-cell mouse embryos.

• Korean Journal of Food Science and Technology
• /
• v.28 no.5
• /
• pp.910-915
• /
• 1996

Ascidian muscle was blackened during the frozen storage, so the prevention against blackening was investigated. Low storage temperature and packaging in polyethylene bags delayed the blackening of ascidian muscle during the frozen storage. The blackening was prevented by dipping for $3{\sim}5$ minutes in 3% brine solution containing 0.3% citric acid, packaging in the polyethylene bag, freezing at $-45^{\circ}C$ for 5 hours and storing at $-20^{\circ}C$. Under this condition, the color and the quality of frozen ascidian muscle were nearly not changed for 200 days.