• Development and Reproduction
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• v.4 no.2
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• pp.161-173
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• 2000

The aim of this study was to assess the effect of the frequency of the L$N_2$ infusion on the ultrastructure, metabolism and development of the embryo after freezing and thawing by computerized cell freezer. Two-cell embryos of ICR mouse were randomly allocated into fresh (control), high-frequency freezing (group 1) and low-frequency freezing (group 2). For fresh and frozen-thawed intact 2-cell embryos, total ceil number in the blastocyst was counted by fluorescent microscope after Hoechst 33258 staining. Relative amount of $H_2O$$_2$ was measured by DCHFDA. Intracellular location and membrane potential of the mitochondria were evaluated by staining with rhodamine 123 and JC-1. The structure of actin filament was also evaluated by confocal microscope. DNA fragmentation was assessed by TUNEL method after development into blastocyst. The survival rate of intact embryo was higher in group 1 than group 2 (50.7％ vs. 34.6％ respectively, p<0.05). The blastocyst developmental rate was significantly low in group 2 (86.7％, 76.7％ vs. 44.0％ for control, group 1 and group 2 respectively, p<0.05). Total cell number in the blastocyst was also significantly lower in group 2 than control (79.5$\pm$12.9, 71.6$\pm$8.0, and 62.5$\pm$4.7 for control, group 1 and group 2 respectively, p<0.05). The relative amount of $H_2O$$_2$ was higher in group 2 than other groups (15.3$\pm$3.0, 16.6$\pm$1.6 vs. 23.4$\pm$1.8, p<0.05). After JC-1 staining, relative intensity of mitochondria with high membrane potential was significantly lower in group 2 than control and group 1 (17.2$\pm$3.8, 17.4$\pm$1.3 vs. 13.2$\pm$2.0, p<0.05). In group 2, partial deletion and aggregation of the actin filament was found. DNA fragmentation rate was also hieher for group 2 versus other groups (30.8％, 36.0％ vs. 65.6％, p<0.05). The frequency of the L$N_2$ infusion is an important factor for the development of frozen-thawed mouse embryo. High-frequency infusion may prevent damages of cytoskeleton and mitochondria in the embryo probably by preventing the temperature fluctuation during dehydration phase. We speculate that the application of high-frequency infusion method in human embryo may be promising.

• Clinical and Experimental Reproductive Medicine
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• v.16 no.1
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• pp.9-14
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• 1989

For the cryopreservation of human embryos this study was accomplished as a preliminary experiment. The purpose of this study is to obtain optimal cryoprotectant, addition and dilution method of cryoprotectant and cooling rate for raising survival of frozon and thawed 2-cell mouse embryos. Seeding was done at $-7^{\circ}C$ and the straw contained embryos was plunged at $-30^{\circ}C$ when the slow cooling was ended. Embryos those developed normally to blastocyst after in vitro culture for over 96 hours were regarded as survival ones. The survival was the rate of number of survival embryos against the recovered embryos. The results are followed : 1. The survivals were 6.3, 71.2 and 67.4% respectively, when Glycerol, DMSO and 1,2-Propanediol were used as cryoprotectant. 2. When sucrose was added in freezing solution, the survival was 69.0%. That was higher than the survival of embryos frozen without sucrose in freezing solution. The difference was not significant. 3. Addition and dilution of cryoprotectant by 4 stepwise raised the survival than by direct, but that was not significant. 4. When embryos were frozen by -0.3, -0.5 and $-1^{\circ}C/min$ before plunged into $LN_2$, the survivals were 67.9, 78.0 and 37.0% respectively. The differnce was significant.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.231-235
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• 2009

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. However, there was no standard criterion to measure the oxygen consumption of embryos. Here, we measured oxygen consumption of bovine embryos at various developmental stages was measured using a scanning electrochemical microscopy (SECM). We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell-stage to morula-stage), indicating that oxygen consumption reflects the cell number ($5.2{\sim}7.6{\times}10^{14}/mol\;s^{-1}$ versus $1.2{\sim}2.4{\times}10^{14}/mol\;s^{-1}$, p<0.05). In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos ($4.0{\times}10^{14}/mol\;s^{-1}$ versus $2.4{\times}10^{14}/mol\;s^{-1}$, p<0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro-derived bovine blastocyst-stage embryos (p>0.05). In the frozen-thawed blastocyst-stage embryos, live embryos showed significantly higher oxygen consumption than dead embryos ($4.7{\times}10^{14}/mol\;s^{-1}$ versus $1.0{\times}10^{14}/mol\;s^{-1}$, p<0.05). These results indicate that the measuring oxygen consumption by SECM can be used to evaluate bovine embryo quality.

• 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 Embryo Transfer
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• v.23 no.4
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• pp.269-274
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• 2008

The purpose of this study was to determine the effects of Taxol pre-treatment to in vitro matured bovine oocytes, and sucrose and trehalose added to vitrification solution on spindle morphology and embryonic development following cryopreservation. Bovine oocytes were collected from ovaries and matured in tissue culture medium 199 (TCM 199) supplemented with 10% Fetal Bovine Serum (FBS), 0.05ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone and $1{\mu}g/ml$ estradiol for 22h in $39^{\circ}C$, 5% $CO_2$, TCM 199-HEPES containing 20% FBS was used as basic medium (BM) to prepare vitrification solution. Oocytes were pre-treated with $1\;{\mu}M$ Taxol in maturation medium for 15 min prior to vitrification. Oocytes were exposed to 1.6 M ethylene glycol (EG) and 1.3M dimethyl sulfoxide (DMSO) in BM and then were exposed to 3.2 M EG, 2.6 M DMSO and 0.5 M sucrose in BM or 3.2 M EG, 2.6 M DMSO and 0.5 M trehalose in BM. Oocytes with cumulus cells and oocytes without cumulus cells were considered as control 1 and control 2, respectively and held in TCM 199-HEPES at $39^{\circ}C$. Oocytes were frozen using modified solid surface vitrification and were stored in cryotubes in liquid nitrogen for more than 1 week. Frozen oocytes were thawed in TCM 199-HEPES containing 0.5 M, 0.25 M and 0.1 M sucrose in BM for 2 min, respectively or 0.5 M, 0.25 M and 0.1 M trehalose in BM for 2 min, respectively. Immunoflurorescence staining of oocytes was performed to assess spindle morphology and chromosome configuration of oocytes. The rates of cleavage and blastocyst were examined following in vitro fertilization. Normal spindle morphology rate of oocytes pre-treated with Taxol prior to vitrification was not higher than that of other vitrified groups. Taxol pre-treatment did not increase cleavage and blastocyst formation rates, although control groups showed significantly higher rates (p<0.05). Percentages of normal spindle and embryonic development were not significantly different among vitrified groups regardless of type of sugar. In conclusion, Taxol pre-treatment of oocytes before cryopreservation did not reduce the damage induced by vitrification and subsequently did not improve embryonic development following vitrification. Trehalose may be used as an alternative non-permeating cryoprotectant in vitrification solution.

• Journal of Embryo Transfer
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• v.19 no.1
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• pp.19-25
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• 2004

The objective of this study was to optimize the selection of sperm, optimal culture system of in vitro derived porcine embryos. The results obtained were summarized as follows: 1. When oocytes were inseminated with liquid sperm and frozen-thaw sperm, the cleavaged rate of liquid sperm (46.2%) was higher than that of frozen-thaw sperm (39.7%), however there were not show significant different each other. The blastocyst rates of liquid sperm (15.8%) was significantly higher than that of frozen-thawed sperm (9.3%)(P< 0.05). 2. When oocytes were inseminated with epididymal sperm after 1, 2 and 3 day storage, the cleavaged rate of epididymal sperm after 1, 2 and 3 day storage was 60.5, 61.0 and 56.8% respectively. The morulae (17.4, 19.9 or 17.3%) and blastocyst (8.7, 15.4, 11.3%) rate of epididymal sperm after 1, 2 and 3 day storage was no significantly respectively(P< 0.05). 3. In vitro developed to cleavaged rate of G1.3/G2.3 media used for culture was significantly(P< 0.05) higher as 62.1% compared with the results using the media NCSU23(52.8), however in vitro developed to blastocyst rate of NCSU23(11.6%) media was significantly(P< 0.05) higher than that'of G1.3/G2.3(4.7%). 4. When the fertilized oocytes were cultured with NCSU23 in addition to 1 mM glutathione(GSH), the cleavaged rate of treated groups of GSH(62.3%) was significantly higher than that of control(53.5%) respectively(P< 0.05). And in vitro developed to blastocyst rates of treated groups of GSH(15.6%) was higher than that of control(12.6%) however, there was no significant difference(P< 0.05).

• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.75-84
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• 1999

This study assessed development in vitro of pronuclear(PN) stage embryos cryopreserved by the method of either vitrification or slow freezing, by using of different cryoprotectants, and equilibration and cooling rate, in rabbit. Ethyleneglycol- ficoll- sucrose(EFS) or ethyleneglycol- polyvinylpyrrolidone - galactose- (EPG-I) for vitrification, and EPG- II for slow freezing as cryoprotectant were used. The pronuclear embryos were exposed to EFS for 0 to 5 min and diluted with D-PBS and/or pre-dilution with 0.5 M sucrose. To examine the viability of frozen-thawed embryos, PN embryos were co-cultured with bovine oviductal epitherial cell(BOEC) for 5 days to hatching blastocyst stage in 39 $^{\circ}C$ 5% $CO_2$incubator. The results obtained were as follows: The dilution with 0.5 M sucrose and D-PBS after the exposure to EFS for 1.0 min resulted in no significant(P＜0.05) decrease in the development of PN embryos to hatching blastocyst(72.0%), compared with controls. The development of PN embryos cryopreserved to hatching blastocyst was not significantly (P＜0.05) different between EFS for 1.0 min(72.0%), EPG-I for 1.0 min(72.0%) and EPG-II for 30 min(66. 7%). The post-thaw development of PN embryos to hatching blastocyst was similarly very low as 6.1% and 11.5% in vitrification with EFS and slow freezing with EPG-II, respectively. The incidence of post-thaw zona-crack in PN embryos cryopreserved by slow freezing with plunging to liquid nitrogen at －35$^{\circ}C$ was signicantly(P＜0.05) higher(25.0%), compared with －85$^{\circ}C$ (1.9%). These results indicated that the rabbit PN embryos could be cryopreserved with either vitrification or slow freezing procedure, and frozen PN embryos could be successfully developed in vitro to haching blastocyst. but the post-thaw development of cryopreserved PN embryos was still very low under the present conditions.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.77-83
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• 2011

One-step dilution and direct transfer would be a practical technique for the field application of frozen embryo. This study was to examine whether Jeju Black Cattle (JBC, Korean Cattle) can be successfully cloned from vitrified and one-tep diluted somatic cell nuclear transfer (SCNT) blastocyst after direct transfer. For vitrification, JBC-SCNT blastocysts were serially exposed in glycerol (G) and ethylene glycol (EG) mixtures [10%, (v/v) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.] which is diluted in 10% FBS added D-PBS. And then SCNT blastocysts were loaded in 0.25 ml mini straw, placed in cold nitrogen vapor for 3 min. and then plunged into $LN_2$. One-step dilution in straw was done in $25^{\circ}C$ water for 1 min, by placing vertically in the state of plugged-end up and down for 0.5 min, respectively. When in vitro developmental capacity of vitrified SCNT blastocyst was examined at 48 h after one-step dilution, hatched rate (56.4%) was slightly lower than that of control group (62.5%). In field trial, when the vitrified-thawed SCNT blastocysts were transferred into uterus of synchronized 5 recipients, a cloned female JBC was delivered by natural birth on day 299 and healthy at present. In addition, when the short tandem repeat marker analysis of the cloned JBC was evaluated, microsatellite loci of 11 numbers was perfectly matched genotype with donor cell (BK94-14). This study suggested that our developed vitrification and one-step dilution technique can be applied effectively on field trial for cloned animal production, which is even no longer in existence.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.6
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• pp.739-747
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• 2000

This experiment was conducted to evaluate the influence of dimethyl-sulfoxise (DMSO, 0, 5, 50, 100 and $500{\mu}M$) on the motility and acrosome reaction of the frozen-thawed spermatozoa from 3 different bulls (Bull A, Band C). Also we evaluated the developmental capacity of bovine embryos fertilized in a medium containing DMSO at various concentrations. DMSO had negligible effects on the sperm motility and acrosome reaction in all three bulls. However, the development rates from 2 to 16 cells stage on the 3rd day after insemination with 50, 100 and $500{\mu}M$ DMSO in Bull-B, and up to the blastocyst stage fertilized with 5, 50, 100 and $500{\mu}M$ in Bull-A were significantly higher (p<0.05) than those of control ($0{\mu}M$ DMSO) group from each bull. Furthermore, the rates of blastocysts per cleaved embryos of 5 to $500{\mu}M$ DMSO group in Bull-A and of 5 to $100{\mu}M$ DMSO in Bull-C were also significantly higher (p<0.05) than those for their $0{\mu}M$ groups, respectively. These results indicate that DMSO at micromol level used for in vitro fertilization might stimulate the development of embryos for some bulls.

• Korean Journal of Veterinary Research
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• v.33 no.1
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• pp.179-188
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• 1993

The developmental capacity of bovine oocytes under three different culture systems was investigated in this experiment ; One was culture in TCM199 with bovine oviductal epithelial cells(BOEC) for in vitro culture, another was culture in TCM199 with BOEC for 2 days and then transfer of 4~8cell embryos to rabbit oviduct(RO) and the other was transfer of 1 or 2cell embryos to RO for in vivo culture. And the other concern of this experiment was to investigate the effect of culture period and transfer site on recovery. Immature bovine oocytes were cultured in TCM199 with granulosa cells for 22-24hrs and then fertilized in vitro using frozen-thawed semen treated with BO-caffine and BO-BSA. Fifteen to 18hrs after in vitro fertilization oocytes were cultured in TCM199 with BOEC or transferred to RO for 5 days. The rate of development to the morula or blastocyst was higher in transfer of 1 or 2cell embryos to RO(23.1%) than culture in TCM199 with BOEC(11.7%). But, there was no difference between transfer of 1 or 2cell embryos and transfer of 4~8cell embryos to RO(12.8%). Recovery under different culture periods in RO was significantly higher in 90~95hrs(70.1%) than 122~125hrs(50.9%, p<0.05) and recovery significantly increased when oocytes were transferred deeper in RO(2.5cm>, 47.7% ; 2.5~4.5cm, 63.9% ; 4.5cm<, 77.3%, p<0.05). The results show that transfer of 1 or 2cell embryos to RO is an effective means of supporting the further development of in vitro matured and fertilized bovine oocytes than culture in TCM199 with BOEC or transfer of 4~8cell embryos to RO, and recovery from RO increases when oocytes are transferred deeper and incubated shorter in RO.