• Korean Journal of Veterinary Research
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• v.31 no.4
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• pp.523-527
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• 1991

The effects of ethylene glycol, DMSO and glycerol as cryoprotectants and the effect of concentrations(0, 0.25, 0.5 and 1.0M) of sucrose in the diluent on the viability of the aggregated morulae frozen rapidly in liquid nitrogen$(LN_2)$ vapour were examined. The morulae were produced by aggregation of ICR and CBA mice embryos at 8-cell stage. Before freezing the embryos were equilibrated in 1.5M cryoprotectants+0.25M sucrose in oae-step or in 3.0M cryoprotectants+0.25M sucrose in two-steps. The embryos were pipetted into the freezing medium fraction of 0.25ml plastic straws. The straws were frozeu by directly transfer into $LN_2$ vapour(about lcm above $LN_2$) for 2 minutes, and then plunged into $LN_2$. After thawing the cryoprotectants were diluted with 0, 0.25, 0.5 or 1.0M sucrose solution. The post-thawed in vitro viability of the aggregated embryos was significantly dependent on the type and concentration of cryoprotectants in the freezing medium and also on the concentration of sucrose in the diluent. When the aggregated embryos were equilibrated in 1.5M cryoprotectants +0.25M sucrose in one-step and diluted with 0.5M sucrose after thawing, the survival rate of the embryo5 was significantly(p<0.05) higher in DMSO(62.5%) or ethylene glycol(52.2%) than in glycerol(33.3 %). In the case that the concentration of the cryoprotectants was raised to 3.0M in two-steps, thc higher survival rate of the embryos was obtained in ethylene glycol or glycerol than in DMSO followed by diluting them with 0.5 or 1.0M sucrose after thawing(p<0.01).

• Korean Journal of Fisheries and Aquatic Sciences
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• v.49 no.5
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• pp.628-634
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• 2016

We investigated the characteristics and rate of development of allotriploid embryos derived from a cross between female olive flounder Paralichthys olivaceus and male starry flounder Platichthys stellatus. The allotriploidy was induced by cold shocking fertilized eggs three minutes post-fertilization at 3°C for 45 minutes. The average cellular DNA content of the allotriploid embryos was 2.06±0.03 pg/cell, which is equal to the sum of the cellular DNA content of a diploid olive flounder (1.42 pg/cell) and a haploid starry flounder (0.66 pg/haploid cell). The first cleavage, midblastula, gastrula and Kupffer's vesicle appearance stages of the allotriploid eggs began at 1.5, 8, 13 and 26 hours after cold shocking at 18°C, respectively. The developmental rate of allotriploid eggs was equivalent to that of diploid and triploid olive flounder eggs at 10, 14 and 18°C. However, the hatching times of allotriploid eggs, 7 h at 10°C, 5 h at 14°C and 4 h at 18°C, were earlier than those of diploid and triploid olive flounder.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.4
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• pp.189-196
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• 2019

Objective: We aimed to evaluate the effects of different oxygen conditions (20% [high O₂], 5% [low O₂] and 5% decreased to 2% [dynamic O₂]) on mouse pre- and peri-implantation development using a novel double-channel gas supply (DCGS) incubator (CNC Biotech Inc.) to alter the oxygen concentration during in vitro culture. Methods: The high-O₂ and low-O₂ groups were cultured from the one-cell to the blastocyst stage under 20% and 5% oxygen concentrations, respectively. In the dynamic-O₂ group, mouse embryos were cultured from the one-cell to the morula stage under 5% O₂ for 3 days, followed by culture under 2% O₂ to the blastocyst stage. To evaluate peri-implantation development, the blastocysts from the three groups were individually transferred to a fibronectin-coated dish and cultured to the outgrowth stage in droplets. Results: The blastocyst formation rate was significantly higher in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. The total cell number was significantly higher in the dynamic-O₂ group than in the low-O₂ and high-O₂ groups. Additionally, the apoptotic index was significantly lower in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. The trophoblast outgrowth rate and spread area were significantly higher in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. Conclusion: Our results showed that a dynamic oxygen concentration (decreasing from 5% to 2%) had beneficial effects on mouse pre- and peri-implantation development. Optimized, dynamic changing of oxygen concentrations using the novel DCGS incubator could improve the developmental competence of in vitro cultured embryos in a human in vitro fertilization and embryo transfer program.

• Reproductive and Developmental Biology
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• v.31 no.4
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• pp.273-278
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• 2007

This study was conducted to examine the protein kinase inhibitors, 6-dimethylaminopurine (DMAP) and cycloheximide (CHXM) on the development and chromosome constitution of porcine parthenogenetic embryos. In vitro matured oocytes were activated by electric stimuli (ES) or a combination of ES with culture in 2 mM DMAP or $10{\mu}g/ml$ CHXM for 4 hr. Activated oocytes were cultured in PZM-3 for 6 days. Some 1-cell embryos and blastocysts were fixed by air dry method to analyze the chromosome constitutions and/or total cell number. Blastocyst development of DMAP-treated group (26.7%) was significantly higher (p<0.05) than those of CHXM-treated and ES control groups. Ploidy in 1-cell stage embryos was not different among groups (77.3 to 81.0%), however, proportion of diploid chromosome constitutions was high in DMAP-treated group (61.9%, p<0.05). In the blastocyst stage, proportion of diploid chromosome plates was significantly high in DMAP-treated group (64.2%, p<0.05), and proportion of abnormal chromosome plates was higher in CHXM-treated group (36.6%, p<0.05) than DMAP-treated group (28.3%,). Proportion of embryos with abnormal chromosome constitutions was slightly increased by DMAP (40.0%) and CHXM (42.1%) treatment due to the increasing of mixoploid (47.4 and 52.0%). The present study shows that the DMAP treatment increase the development of porcine parthenotes. However, parthenogenetic activation by ES or combined treatment with ES and DMAP or CHXM detrimentally affects the chromosome constitutions of porcine parthenotes during early embryonic development, leads to increased abnormal ploidy in the blastocyst stage.

• Journal of Embryo Transfer
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• v.27 no.4
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• pp.259-264
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• 2012

Solution of glycerol, ethylene glycol, sucrose, dextrose (GESD) and cryotop methods were carried out to investigate the survivability on vitrification of embryos. Embryos cultured in vitro were vitrified by GESD of 10 or 8 step and cryotop methods of 6 step, from cryopreservation step to frozen-thawed and culture step. Survival rate and ICM, TE cells of embryos were investigated after frozen-thawed 24 h. As a results, cryotop method was significantly (p<0.05) higher ($85.76{\pm}5.3$ vs. $66.71{\pm}2.4$, $44.80{\pm}2.1%$) than GESD 10 or 8 step methods on survivability. Also, In ICM cell number, cryotop method was significantly (p<0.05) higher to $45.67{\pm}4.7$ cells than GESD 8 step method. TE cell number was significantly (p<0.05) highest to $111.00{\pm}11.0$ cells in cryotop method. On the other hand, survival rate, TE and total cell number were all the significantly (p<0.05) high, except ICM in GESD 10 step method between GESD 10 step method and GESD 8 step method. In conclusion cryotop method was to be most effective, but it is considered necessary to study vitrification method for step-by-step freezing and thawing process.

• Korean Journal of Poultry Science
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• v.26 no.2
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• pp.119-129
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• 1999

In recent years, numerous researches have been carried out in author's laboratory to develop several kinds of methods for producing transgened chicken, leaving a lot of new findings. Some of them are very useful to search for new approaches necessary to improve the efficiency of hatchability and the survival rate of developing trasgened embryos. The results obtained hitherto might be summarized as follows: (1) foreign gene(Lac Z/ Miw Z) introduced into blastodermal cells of developing embryos was successfully transferred to embryos, leading to the production of primordial germ cells(PGCs) carrying foreign DNA. However, hatched hickens failed to show the incorporation of introduced gene into the gonads. (2) When foreign gene was introduced into germinal crescent region (GCR), the gene was also efficiently incorporated into germ cells, resulting in the production of transgened chickens(offspring) which produced fruther offspring having foreign gene in the gonads. In this case, 2nd and 3rd generations of chickens were obtained through the reproduction of transgened birds. (3) In another way, the gene was injected into blood vessels of developing embryos at stage 13∼15, creating PGCs having foreign gene, and produced some transgened chickens. In this work, the PGCs were transfered between embryos, resulting in the production of transgenic chickens. (4) in these experiments, PGCs were effectively employed for producing transgenic birds, developing some kinds of chimeric chickens from homo- or hetero-sexual transfer of the PGCs from embryos. This means that the gonads from donor PGCs developed in some degree to the stage of hatching. However, these gonads showed slightly abnormal tissues similar to ovotestis like organs through histological examination. (5) Avian Leukosis Virus(ALV) induced B cell line(DT40) successfully carried foreign genes into chicken embryos, suggesting the possibility of the cells as a vector in this field of study in the future. (6) Inter-embryonic transfer of the PGCs also gave us some.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.4
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• pp.455-461
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• 2001

The effect of exogenous gonadotrophins on superovulation in rabbits was examined. One hundred and sixteen sexually mature California, Chinchilla and New Zealand White rabbits were randomly allocated to control (100 IU hCG), PMSG-treated (100 IU HCG following 150 IU PMSG) and FSH-treated groups (0.3 mg/head /12 h for 3 days followed by 100 IU hCG). All does were mated after hCG injection and were sacrificed or laparotomized within 1 to 4 days postcoitus for counting the number of ovulation points. The number of ovulations was higher in FSH-treated animals than in the control and PMSG-treated groups (37.2 vs. 10.4 and 14.5, p<0.05). Follicle haemorrhagicum was observed in many cases in the PMSG-treated group. No significant difference in ovulation number was observed between left and right ovaries regardless of gonadotropin treatment. In another experiment, 2-cell stage embryos were collected at 26 h postmating and blastomeres were separated by mechanical pipetting or gentle pressure with a fine glass needle. Aggregated or chimeric embryos were produced from two single blastomeres from two breeds, New Zealand White and Chinchlla, with different coat colors. All the embryos were cultured in Ham's F-10 medium supplemented with 1.5% BSA (bovine serum albumin fraction V) and 10% PRS (pregnant rabbit serum), and incubated in a humidified atmosphere with 5% $CO_2$ at $38^{\circ}C$. After development to morula or early blastocyst, the embryos were transferred into the oviducts of recipient does. Results showed that 7 out of 10 does (70%) receiving intact embryos (control) became pregnant and 41 kits were delivered. However, no pregnancy was obtained from the recipient of either denuded demi- or aggregated embryos. It is suggested that embryos without zona pellucida could not develop to term in rabbits.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.2
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• pp.177-185
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• 2005

Objective: This study was conducted to find an optimal condition for the vitrification of mouse morulae and expanded blastocysts. Materials and Methods: Mouse embryos were obtained at 2-cell stage and cultured to morula and expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% Serum Substitute Supplement (SSS). The vitrification solutions used were EFS30, EFS35 and EFS40 that contains 30%, 35% and 40% ethylene glycol, respectively, with 18% ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% SSS. The vitrification procedure was performed in EFS solution with three steps, followed by thawing in 6 steps with 0.5 M sucrose, and then survival and hatching-hatched rate per embryos recovered were compared among six groups. Results: After 24 h culture in different vitrification and thawing solution, the survival rate of morula embryos was 94.1%, 85.4% and 59.7% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of morula embryos after 72 h culture was 30.6%, 25% and 11.3% for EFS30, EFS35 and EFS40 group, respectively. The survival rate of expanded blastocyst embryos after 24 h culture was 90.4%, 98.5% and 100% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of expanded blastocyst embryos after 48 h culture was 46.2%, 57.6% and 64.3% for EFS30, EFS35 and EFS40 group, respectively. Conclusion: The EFS30 solution was the best for vitrification of mouse morulae. The EFS40 solution was the best for vitrification of mouse expanded blastocysts. The mouse expanded blastocyst was better than mouse morula for vitrification of mouse embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.3
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• pp.358-363
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• 2008

The intracytoplasmic sperm injection (ICSI) procedure has recently been utilized to produce transgenic animals and may serve as an alternative to the conventional pronuclear microinjection in species such as pigs whose ooplasm is opaque and pronuclei are often invisible. In this study, the effects of sperm membrane disruption and electrical activation of oocytes on in vitro development and expression of transgene green fluorescent protein (GFP) in ICSI embryos were tested to refine this recently developed procedure. Prior to ICSI, sperm heads were treated with Triton X-100+NaCl or Triton X-100+NaCl+NaOH, to disrupt membrane to be permeable to exogenous DNA, and incubated with linearized pEGFP-N1 vector. To induce activation of oocytes, a single DC pulse of 1.3 kV/cm was applied to oocytes for $30{\mu}sec$. After ICSI was performed with the aid of a micromanipulator, in vitro development of embryos and GFP expression were monitored. The chemical treatment to disrupt sperm membrane did not affect the developmental competence of embryos. 40 to 60% of oocytes were cleaved after injection of sperm heads with disrupted membrane, whereas 48.6% (34/70) were cleaved without chemical treatment. Regardless of electrical stimulation to induce activation, oocytes were cleaved after ICSI, reflecting that, despite sperm membrane disruption, the perinuclear soluble sperm factor known to mediate oocyte activation remained intact. After development to the 4-cell stage, 11.8 (2/17, Triton X-100+NaCl+NaOH) to 58.8% (10/17, Triton X-100+NaCl) of embryos expressed GFP. The expression of GFP beyond the stage of embryonic genome activation (4-cell stage in the pig) indicates that the exogenous DNA might have been integrated into the porcine genome. When sperm heads were co-incubated with exogenous DNA following the treatment of Triton X-100+NaCl, GFP expression was observed in high percentage (58.8%) of embryos, suggesting that transgenic pigs may efficiently be produced using ICSI.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.105-112
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• 2005

The present study was conducted to examine some factors affecting in vitro development of oocytes from somatic cell nuclear transfer (SCNT) in Korean native goats. Recipient oocytes were surgically collected after superovulation by using CIDR and FSH, PMSG, hCG and estrous synchronization in Korean Native goats. For nuclear transfer, the fibroblasts from caprine ear cells and fetal fibroblasts were surgically harvested and were cultured in vitro until cell confluency in serum-starvation condition (TCM-199 + $0.5\%$ FBS) for 3 to 5 days. The zona pellucidae of matured oocytes were partially drilled by laser irradiation. A single somatic cell was individually transferred into each enucleated oocyte. The reconstructed oocytes were then electrically fused and activated. Activated NT embryos were cultured in mSOF medium supplemented with $0.8\%\;BSA\;6\~7\;day\;at\;39^{\circ}C,\;5\%\;CO_2,\;5\%\;O_2,\;90\%\;N_2$ in air. There were no significant difference in the number of embryos cleaved and 4-cell development between the fibroblast nuclei from mature ear cells and fetal cells, but the rate of 8-cell development was higher (P<0.05) in ear cells $(40.5\%)$ than in fetal cells $(55.5\%)$. However, the embryo development to morula or blastocyst was not significantly different between both the groups$(6.7\%\;vs\;16.0\%)$, respectively. The number of embryo cleaved $(79.0\%)$ were higher (P<0.05) in the oocytes activated with ionomycin+6-DMAP than in the oocytes activated electrically $(9.5\%)$. The development of fused embryos to morula or blastocyst was found $15.6\%$ in ionomycin+6-DMAP, but no morula or blastocysts were developed in electrical stimulation. The development rate of SCNT embryos to morula or blastocyst was love. (P<0.05) in SCNT embryos $(19.0\%\;vs\;0.0\%)$ than that in parthenotes $(66.1\%\;vs\;59.1\%)$. In the parthenotes, the cleavage rate and development to morula or blastocyst were significantly higher (P<0.05) as $86.8\%\;and\;50.0\%$ in ovulated oocytes than in follicular oocytes $(69.0\%\;vs\;23.6\%)$, respectively. These results suggest that some factors Including superovulation treatment, oocyte source, maturation of follicular oocytes, activation method and culture condition may affect in vitro developmental capability of embryos produced by somatic cell nuclear transfer in Korean Native goats, and the fusion rate be greatly low compared with other species.