• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.1-9
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• 1998

This study was carried out to confirm whether the developmental capacity of bovine mature oocytes frozen ultra-rapidly using electron microscopic(EM) grids and EFS30 can be obtained, and whether the cryoprotectants and the freezing method used in this study effect detrimentally to the bovine oocytes by indirect immunocytochemistry. 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. The results obtained in this experiment were summarized as follows: When the effects of cryoprotectant and freezing procedure on the microtuble, micrfilament and chromatin morphology of oocytes were evaluated using indirect immunocytochemistry, the results of freezing as well as exposure group were not different with that of the control oocytes. When the fertilization abnormality after ultrarapid freezing of bovine mature oocytes was examined by Hoechst staining, the rates of total penetration(96.7, 9.0%), normal two pronuclei formation(74.6, 68.9%) and mean number of sperm / oocyte(1.50, 1.44) were not different between control and freezing group. In addition, when the developmental capacity of frozen-thawed of oocytes(85.5%) was survived, 74.5% of them were cleaved and 31.4% of cleaved embryos were developed to blastocyst. These data were similar to those of the control(76.0%, 34.6%) and exposure(74.5%, 33.0%) except survival rates. Also, when the total cell number of blastocysts produced from the each treatment at day after IVF was examined by hoechst staining, there were not different among groups. There results demonstrate that developmental capacity of frozen-thawed bovine mature oocytes can be successfully obtained by ultra-rapid freezing method using EM grid and EFS30 solution.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.4
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• pp.329-338
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• 2010

Objective: This study was carried out to know whether cryopreservation of sibling 2PN zygotes could increase the cumulative delivery rates in the patients who had less than 10 fertilized zygotes. Methods: A retrospective analysis was performed in 138 in vitro fertilization-embryo transfer (IVF-ET) cycles with less than 10 fertilized zygotes during January 2003 to December 2007 in Cheil General Hospital. These cycles were divided into two groups. In Group I (n=86), all fertilized embryos were cultured to transfer on day 3 without cryopreserved embryos at the 2PN stage. In Group II (n=52), among fertilized zygotes, some sibling zygotes were frozen at the 2PN stage, the remainder were cultured to transfer. Clinical outcomes in fresh ET cycles and cumulative ongoing pregnancy rates after subsequent frozen-thawed (FT)-ET cycles were compared. Results: There were no significant differences in female mean age, number of retrieved oocytes and total fertilized embryos between two groups, Number of cultured embryos was significantly lower in Group II ($5.2{\pm}0.5$) than in Group I ($8.4{\pm}0.7$) (p<0.01). Also, number of transferred embryos was significantly lower in Group II ($3.3{\pm}0.6$) compared with Group I ($3.6{\pm}0.6$) (p<0.01). ${\beta}$-hCG positive rates and delivery rates (51.2 vs. 46.2 % and 41.9 vs. 34.6 %, respectively) after fresh ET were slightly higher in Group I than in Group II. However, the differences were not statistically significant. Also, the cumulative delivery rates after subsequent FT-ET cycles were not significantly different between Group I (48.8%) and Group II (50.0%). Conclusion: This study showed that cryopreservation of sibling 2PN zygotes from patients who had less than 10 zygotes in the fresh ET cycles did not increase cumulative delivery outcomes. But, it could provide an alternative choice for patients due to offering more chance for embryo transfers if pregnancy was failed in fresh IVF-ET cycles.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.197-205
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• 2013

This study established a method for preserving chicken primordial germ cells (PGCs) that enables long-term storage in liquid nitrogen ($LN_2$) for preservation of the species. The purpose of this study was to compare the effects of Ethylene Glycol (EG) and Propylene Glycol (PG) on viability of cryopreserved PGCs with vitrification in Korean Native Chicken (Ogye), and to fine should be find or to the optimal protocol for PGCs freezing. One of the important components of cryopreservation process is cryopreservation medium that plays a vital role in preventing cellular injury during freeze-thawing. Cryoprotective agents have been known to improve cell viability after freeze-thawing. PGCs obtained from the germinal gonade of 5.5~6 day (stage 28) chick embryos, using the MACS method were suspended in a freezing medium containing a freezing and protecting agents. Gonads were harvested from stage 28 chick embryos and pooled in groups of 10E embryos, contributing gonads to the cell suspension. The gonadal cells, including PGCs, were then frozen in 1 of the following cryoprotectant treatments: 2.5% EG, 5% EG, 10% EG, 2.5% PG, 5% PG, 10% PG, and 0% cryoprotectant as a control. Effects of exposure to vitrification solution and vitrification, with different concentrations of the cryoprotectant solution, were examined. After freezing and thawing, survival rates of the frozen-thawed PGCs from the 0, 2.5, 5, 10 and 15% EG plus FBS treatment were 44.24%, 64.51%, 85.63%, 80.51% and 73.52% (p<0.05), respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% PG + FBS treatment (p<0.05)(85.63% vs 66.81%). Therefore, these systems may contribute in the improvement of cryopreservation for a scarce species in birds preservation. This study established a method for preserving chicken PGC that enables systematic storage and labeling of cryopreserved PGCs in liquid N at a germplasm repository and ease of entry into a database. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.275-289
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• 2002

The objective of this study was to develop an in vitro assessment of sperm fertilizing capacity of bulls and investigate the factors influencing sperm function and characteristics of frozen-thawed bovine spermatozoa. in vitro fertilization (IVF), the evaluation of motility and normal morphology, HOST (hypoosmotic swelling test), Ca-ionophore induced acrosome reaction, luminol and lucigenin-dependent chemiluminescence for the measurement of reactive oxygen species (ROS), the measurement of malondialdehyde formation for the analysis of lipid peroxidation (LPO), and the evaluation of DNA fragmentation using the method of 747-mediated nick end labelling (TUNEL) by flow cytometry were performed in frozen-thawed bovine spermatozoa. Correlations between the rates of fertilization, blastocyst formation after IVF and the values of respective assays were investigated. 1. IVF rate and blastocyst formation rate averaged 64.4% and 34.3% for spermatozoa from high -fertility bull group and averaged 18.5% and 6.2% for spermatozoa from low-fertility bull group, respectively. There were significantly different between two bull groups. Sperm motility and percentage acrosome reaction averaged 79.0% and 66.2% for spermatozoa from high-fertility bull group and averaged 40.7% and 22.9% for spermatozoa from low-fertility bull group, respectivitely. There were not different between two bull groups. 2. Luminol depenent chemiluminescence, LPO and DNA fragementation averaged 6.4, 2.0 nmol and 2.6% from spermatozoa from high-fertility bull group and averaged 6.5, 3.1 nmol and 7.4% for spermatozoa from low-fertility bull group, respectively. There were significantly different between two bull groups. There was no significant difference in lucigenin dependent chemiluminescence between two bull groups. 3. Fertilization rate was positively correlated with motility and the rate of Ca-ionophore induced acrosome reaction, but negatively correlated with the frequency of luminol-dependent chemiluminescence, the rate of LPO, and the percentage of sperm with DNA fragmentation. There was no correlation between fertilization rate and the percentage of swollen spermatozoa, normal morphology, and the frequency of lucigenin-dependent chemiluminescence. 4. Blastocyst formation rate was positively correlated with the rate of Ca-ionophore induced acrosome reaction, but negatively correlated with the frequency of luminol-dependent chemiluminescence, the rate of LPO, and the percentage of sperm with DNA fragmentation. There was no correlation between blastocyst formation rate and motility, the percentage of swollen spermatozoa, normal morphology, and the frequency of lucigenin-dependent chemiluminescence. In conclusion, these data suggest that ROS significantly impact semen quality. The assays of this study may provide a basis fur improving in vitro assessment of sperm fertilizing capacity.

• Parasites, Hosts and Diseases
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• v.28 no.4
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• pp.241-252
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• 1990

In a basic attempt to develop the prophylactic and therapeutic measures on intestinal giantcystic disease of the Israel carp, C), prinks carpio nudum, the effects of physical and chemical factors on viability or survival of the spores of Thelchcnellus kiteuei were checked in vitro by means of extrusion test on the polar filament. When the fresh spores suspended with 0.45% and 0.9% scdium chloride solution and distilled water were laid at $5^{\circ}C$ and $28^{\circ}C$ for short terms, the extrusion rates increased until the 3rd day, meanwhile when son;e of them were suspended with Tyrode's solution at $-70^{\circ}C$ the rates increased gradually until the 8th day. Viabilities of the spores suspended with 0.9% saline and added antibiotics to the suspension at $5^{\circ}C$ for long terms lasted for 997 days and 1, 256 days (presumed values) at maximum, respectively. The spores suspended with distilled water at $28^{\circ}C$ for long terms survived 152.4 days, but the spores suspended with Tyrode's solution at $-70^{\circ}C$ for long terms showed almost the same viable pattern as early freezing stages up to 780 days. The spores suspended with Tyrode's solution, frozen at $-70^{\circ}C$ and thawed at $5^{\circ}C$, showed the highest rate of extrusion of the polar filament. In the case of frozen spores, the extrusion rates during heating tend to become higher in accordance with the increase of frozen period, and the critical points of 180 day-frozen spores to be killed were generally 78.5 hr. at $60^{\circ}C$, 23.4 hr. at $70^{\circ}C$, 189.1 min. at $80^{\circ}C$ or 10.5 min. at $90^{\circ}C$. The longer the spores were frozen, the more time was needed for the death of spores after thawing; 20 days-17.4 days, 100 days-33.2 days, and 400 days-37.8 days. The longer the spores were frozen, the more time was needed for the death of spores at a conventional when they were dried air drying condition, 540 days-23.5 days, 160 days-21.0 days, and 20 days-14.4 days. On the other hand, the longer the spores were frozen, the more spores were dead rapidly when they were irradiated with 10W UV-ray; 100 days-26.0 hr, 300 days-21.9 hr, and 540 days-13.9 hr. The time needed for killing 200 days-frozen spores by various disinfectants at 1, 000 ppd was 5.2 min. by calcium oxide, 10.4 min. by potassium permanganate, 27.8 min. by malachite green and 14.3 hr. by formalin. Transient inhibitory effects of the extrusion of the polar filament were observed by various antiprotozoal and antifungal agents in the descending order of ketoconazole. metronidasole and dapsone. The above results presume that full drying, followed by spraying CaO and maintaining sunny condition for a few days on the concrete bottoms of knish farm may be an effective method for the prevention of intestinal giant.cystic disease.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.9
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• pp.251-258
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• 2020

This study investigated the effect of Zardaverine supplementation in freezing extender, on kinetic characteristics of post-thawed boar sperm. Cryopreservation of boar sperm is an important technique of assisted reproductive technology and genetic resource banking. Although this technique is particularly useful, freeze-thaw cycles associated with sperm cryopreservation significantly reduce sperm quality. Semen from mature Duroc boars were collected and cryopreserved in freezing extenders (LEY) treated with varying concentrations of Zardaverine (0, 20, 50, 75, 100 𝜇M). The time-dependent kinetic characteristics of post-thawed spermatozoa were determined after thawing by applying computer-assisted sperm analysis (CASA). We observed that the motility immediately after thawing was significantly higher in 20 𝜇M stocks than in control (0 𝜇M) and the other treatments (p<0.05). Curvilinear velocity (VCL) in 0 𝜇M and 20 𝜇M stocks were significantly higher than the other treatment groups, except 75 𝜇M (p<0.05). Higher average path velocity (VAP) was obtained at 20 𝜇M as compared to 100 𝜇M, whereas amplitude of head lateral displacement (ALH) was significantly higher at 20 𝜇M than 50 𝜇M and 100 𝜇M (p<0.05). No differences were obtained for Straight-line velocity (VSL) and Linearity (LIN). In conclusion, our results indicate that Zardaverine improves the motility, VCL, VAP, and ALH of post-thawed boar sperm.

• Korean Journal of Food Science and Technology
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• v.30 no.4
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• pp.862-870
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• 1998

This study shows the application of Ci-ELISA method for monitoring the denaturation of myosin by the frozen treatment in order to differentiate thawed beef from chilled. Hanwoo M.Semitendinosus (n=25) was treated under the two different frozen process as follows; simple frozen treatment (Exp-1) at 4 different temperatures, -10, -20, -50 and $-80^{\circ}C$, respectively, and repeated thawing-refreezing treatment (Exp-2) stored at 4 different temperatures, -10, -20, -50 and $-80^{\circ}C$, respectively. Antibodies (Abs) were produced from rabbits immunized with myosin whole molecule (MWM) isolated from beef round, heavy meromyosin S-1 (S-1) and light meromyosin (LMM) prepared by digestion of MWM. Each immunoglobulin G (IgG) was separated from antiserum. At 6 month storage, IA of anti-MWM IgG for myosin was decreased to 32.67, 32. 23, 51.52 and 34.27% in Exp-1 and to 14.82, 15.61, 25.3 and 23.7% in Exp-2 at -10, -20, -50 and $-80^{\circ}C$, respectively (P<0.05). In Exp-1, the reactivities of anti-LMM IgG were decreased to 25.12, 21.42, 49.05 and 28.96%, and those of Exp-2 were to 11.88, 9.56, 20.63 and 12.64% at -10, -20, -50 and $-80^{\circ}C$, respectively, at 6 times thawing (P<0.05). Conclusively, myosin was denaturated by freezing treatment and LMM or myosin rod part might have suffered from more extreme demage than HMM S-1, and samples at $-50^{\circ}C$ were slightly injured less than others by freezing treatment.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.221-226
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• 2016

The aim of the present study was to assess the embryo development and survivability of post-thawed bovine embryos produced in vitro by addition of cysteine. The rates of metaphase II formation were not differed significantly among three groups(TCM199 73.8%, TCM199 with 0.3% cysteine 76.9%, TCM199 with 0.5% cysteine 83.8%, respectively). No difference of cleavage rate(70.6~74.6%) was seen among three culture medium(TCM199 70.6%, CR1aa 71.3%, SOF 74.6%) with 0.5M cysteine. however, Significantly(P<0.05) higher development rate into blastocyst stage by 0.5M cysteine addition was obtained in SOF medium(35.6%) than in TCM199(27.6%) or CR1aa(26.6%), however no significant differences in the cleavage rates were among three culture medium. After frozen the blastocysts cultured with 0.5M cysteine, The re-expansion rates were 61.3%~86.4% among groups, and hatching rates were 26.3%~46.9% among groups, the rates of re-expansion and hatching were significantly(P<0.05) higher in SOF medium(86.4% and 46.9%) than those in TCM199(61.3% and 26.3%) and CR1aa medium(87.1 and 44.4%). After thawing, the blastocyst re-expansion rate was significantly(P<0.05) higher in in vivo (87.1%) and in vitro (70.3%) embryos. In conclusion, our results demonstrate that supplementation of IVM and IVC medium with 0.5M cysteine improved the quality of in vitro production embryo and post- thawed embryo. Future studies comparing these media systems in well-designed trials should be performed.

• Journal of Animal Science and Technology
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• v.49 no.2
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• pp.287-294
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• 2007

The present study was to conducted the sexing efficiency and accuracy of bovine embryo by LAMP (Loop-mediated isothermal amplification) method, the development of the biopsied embryos into re- reformation and the freezability of these blastocysts by slow-freezing and vitrification. In vivo embryos were superovaluted with gonadotropin(Antorin R-10) for 4 days combined with progestrone releasing intravaginal(CIDR) insertion in Hanwoo donors, and in vitro embryos were used blastocyst embryos at Day 7 or Day 8 after post-insemination in vitro. The biopsy of bovine embryo was carried out in a 80μl drop with Ca2+-Mg2+ free D-PBS and the viability of biopsied embryos were evaluated in IVMD medium at over 12 h culture time in 5% CO2 incubator.For embryo sexing, about five or seven blastomeres were isolated from in vitro and in vivo embryos at blastocysts with microblade. and were then subjected to LAMP. The survivability of biopsied embryos were no difference in the development rate to re-formation of blastocysts between in vivo and in vitro embryos(100% and 90% respectively). The rates of sexed embryos were compared according to two groups, the female rate was lower than that the male in the in vivo and in vitro embryos(46% vs, 54% and 40% vs, 60%, respectively). However, there were no difference in the overall sexing ratio between the two groups. The survivability of frozen-thawed sexed embryos were lower in the in vitro than in vivo embryos in the slow-freezing(Group 1) and vitrification method(Group 2), (41.7% vs. 58.8% and 57.1% vs, 77.8. respectively).

• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.121-129
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• 2001

Objective: The aim of this study is to compare the efficiency of a method for the cryopreservation of mouse blastocyst.. Methods: Mouse embryos were obtained at 2-cell stage and cultured to blastocyst stage in T6 medium supplemented with 10% fetal bovine serum. Morphologically normal blastocysts were collected and randomly divided to one control and four experimental groups. In control group, blastocysts were cultured in vitro continuously for additional two days. In group 2, blastocysts were exposed to vitrification solution (ethylene glycol) only without cryopreservation (exposure only group). In group 3, 4 and 5, blastocysts were cryopreserved by slow-freezing procedure with glycerol (slow-fteezing group) or by vitrification procedure using EM grids (EM grids group) and cryoloop (cryoloop group), respectively. Frozen blastocysts were thawed and cultured for additional two days. Twenty four hours after thawing, some blastocysts were fixed and stained with Hoechst 33342 (bisbenzimide) and the number of nuclei in each blastocysts were counted to confirm the survival of bias to cysts in experimental groups. Results: Survival rate and hatching rate of the blastocysts in slow-freezing group (24 h: 72.4% and 66.0%, 48 h: 63.2% and 64.6%) and EM grids group (24 h: survival rate 77.3%, 48 h: 70.1% and 71.4%) were significantly lower ($X^2$-test p<0.05) than those of control group (24 h: 93.4% and 86.0%, 48 h: 88.5% and 90.7%). In contrast, the survival rate and hatching rate of the blastocysts in cryoloop group (24 h: 84.1% and 84.1%,48 h 79.3% and 87.7%) is well compared with those in the control group. The mean (${\pm}SD$) cell number of blastocyst in the exposure only ($89.2{\pm}11.5$), EM grids ($85.0{\pm}10.3$) and cryoloop ($89.0{\pm}11.0$) groups, except slow-freezing group ($79.0{\pm}10.0$), were not significantly different from that of control group ($93.1{\pm}13.9$) 24 h after thawing (Student's t-test). Conclusion: This study demonstrates that higher survival rate of vitrified-thawed mouse blastocyst can be obtained using cryoloop as the embryo container at freezing rather than slow-freezing or vitrification using EM grids. The results of this study suggest that vitrification using cryoloop (with ethylene glycol) may be a preferable procedure for mouse blastocyst cryopreservation and could be applied to the human blastocyst cryopreservation.