• Korean Journal of Animal Reproduction
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• v.27 no.1
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• pp.87-93
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• 2003

This study was conducted to investigate the effects of embryo development by fusion condition on the nuclear transfer with brindle coated cow's ear cells. Ear cells were transferred into an enucleated oocyte and fused with cytoplasm in the fusion condition with 1.9kv/cm, 2.0kv/cm, 2.1kv/cm each 10 and 20ug duration Nuclear transfer embryo were activeted with a combination of 5ug/ml and 1.9mM 6-DMAP (4min, 4h). Fusion rate was 51∼68% range among fusion condition (1.9, 2.0, 2.1kv/cm; 10, 20us). But, cytoplasm lysis rate was increased by higher electric condition (0∼51.8% range). Each parameter's cleavage and blastocyst formation rate were 1.9kv/cm for 10us (75.8 and 19.5%), 20us (69.8 and 48.6%), 2.0kv/cm for 10us (76.9 and 20.0%), 20 us (68.5 and 40.9%), 2.1kv/cm for 10us (70.5 and 44.2%), 20 us (68.5 and 27.0%). We compared the effectiveness of cloning for between brindle coated cow's ear cells and Hanwoo fetal fibroblast cells. There was no significant differences in the fusion rate and developmental rate to the blastocyst stage. After transfer of blastocysts derived from nuclear transfer embryos, pregnancy rates of the Hanwoo fetal fibroblast cells and brindle coated cow's ear cells were checked pregnant on day 60 as assessed by ultrasonography, 40% (2/5) and 15.8% (3/19), respectively. This studies conclude that brindle coated cow's ear cells have the developmental potentiality to term by nuclear transfer. These results demonstrate that the increased the field strength was to be profitable for development of blastocyst or reduce of cytoplasm's damage than increasing the pulse duration.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.213-221
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• 2001

This study was conducted to examine in vitro developmental ability of porcine embryos after somatic cell nuclear transfer. The porcine ear fell was cultured in vitro for confluency in serum-starvation condition(TCM-199 + 0.5% FBS) far 3~6 days of cell confluency. The zona pellucida of IVM oocytes were partially drilled using laser system. Single somatic cell was individually transferred into enucleated oocytes. And the reconstructed embryos were electrically fused(single DC 1.9kv/cm, 30$\mu$ sec) with 0.3M mannitol. After electrofusion, embryos were activated(single AC 5v/mm, 5sec) and cultured in HCSU-23 medium containing 10% FBS at 39$^{\circ}C$, 5% $CO_2$ in air for 6 to 8 days. The fusion rate of donor cells was 45.6, 36.8 and 46.1% in 3~4, 5~6 days of serum starvation and non serum starvation(N-S), and were 52.7. 53.0 and 51.7% in 1~2. 5~6 and 13~14 passages of donor cell culture, respectively. No significant difference was found in the fusion rate of donor cells by the duration of serum starvation treatment or the number of donor cell passages. By the size of donor cells, however, the fusion rate was significantly higher(P<0.05) for reconstructed embryos derived from 25r $\mu$m $\geq$ site of donor cells (65.3%) than that of 25~30$\mu$ m(42.5%) or 30$\mu$ m(45.5%)$\leq$ cells. The cleavage rate was significantly (P<0.05) higher in 3~4 darts of serum starvation treatment(67.1%) than that in N-S (50.7%) or 5~6 days of starvation(57.1%). The activation rate by the size of donor cells in fused oocytes was 56.5, 68.8 and 58.5%, respectively, and was not significant.

• Journal of Embryo Transfer
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• v.22 no.3
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• pp.155-160
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• 2007

During in vitro culture of mammalian oocytes and embryos, the cells are exposed to the risks that cause cell injury or death. Numerous studies have been reported that the cell injury may be induced by the action of free radicals generated by auto-oxidation. This study was undertaken to investigate the optimal culture condition system for in vitro culture of porcine embryos. We first evaluated the effect of culture media on the porcine embryo development. NCSU-23 and PZM-5, culture medium tested, were failed to produce significant difference on the rate of blastocyst formation. In NCSU-23, the developmental rate was slightly higher than that in PZM-5. During in vitro maturation (IVM), fertilizaton (IVF), and culture (IVC) under 5 or 20% oxygen ($O_2$), the rates of cleavage and development were insignificantly different from each other under our culture condition (20% $O_2$, in NCSU-23), the mean cell number per blastocyst was $40{\pm}10$. These results showed that medium and $O_2$ concentration had no significant effect on the development of porcine embryos.

• Journal of Embryo Transfer
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• v.26 no.1
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• pp.65-70
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• 2011

This study was conducted to investigate the survival rate of AndroMed and Tris-egg yolk extender for cryopreservation of Korean Native Bull Semen (Chick Cow). Semen was collected from a Korean Native Bull Semen over 3 year's old. The semen was diluted 1:1 by AndroMed and Tris-egg yolk extender. The pellet was diluted to final sperm concentration of $5{\times}10^7$ cell/ml by doubling in every 10 minutes at $4^{\circ}C$ cold chamber. The semen was equilibrated for 1 hrs at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm of liquid nitrogen for 5 minutes. And then the frozen straw was plunged to LN2. The presented straws were examined the viability and motility after thawed at $37^{\circ}C$ water bath. The survival rates was significantly higher (p<0.05) in Tris-egg yolk extender than AndroMed extender ($89.7{\pm}19.8$ vs. $73.4{\pm}11.2$). However, motility was no significant differences ($78.4{\pm}18.7$ vs. $67.9{\pm}14.6$). Survival rate in time of equilibration between visual and CASA program had higher in 2 h ($86.33{\pm}9.4$ vs. $92.32{\pm}12.4$) than in 5 h ($78.20{\pm}7.8$ vs. $88.28{\pm}13.1$) 15 h ($65.24{\pm}6.6$ vs. $76.48{\pm}17.3$) 20 h ($56.26{\pm}4.6$ vs. $67.73{\pm}18.4$). The development rates to cleavage was higher in Tris-egg yolk extender than AndroMed extender (82.2% vs. 81.7%). Similarly, the development rates to blastocyst was significantly higher (p<0.05) in Tris-egg yolk extender than AndroMed extender (42.3% vs. 29.6%). In conclusion, the obvious impact of this study will be its practical application to improve viability and fertilizing ability of cryopreserved spermatozoa used for in vitro fertilization (IVF) and AI, Which in turn will be beneficial to animal genetic resources conservation.

• Journal of Embryo Transfer
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• v.14 no.1
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• pp.17-22
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• 1999

To investigate the maturational and development competece of porcine oocytes of different diameter groups, oocytes were obtained by aspiration from slaughterdhouse ovaries. After washing three times in NCSU23 medium, each cumulus-oocyte complex was transferred into a $8{mu}ell$ drop of the maturation medium (one oocyte per drop) under paraffin oil. The diameter without zona pellucida of oocytes was measured with micor-calibrator (Mikrometer, E. Leitz) on a screen connected to a VCR on an inverted microscope $(200\times)$. After being measured, the oocytes were divided into 6 groups according to their diameter size : <105, 105 to < 110, 110 to < 115, 115 to < 120, 120 to < 125 and > $125{\mu}{\textrm}{m}$, and in vitro maturation (IVM), fertillzation (IVF) and production (IVP) of oocytes / embryo was performed. The rates of in vitro maturation on oocytes in the greater 105 ${\mu}{\textrm}{m}$ size groups(91.8~100%) were significantly (P<0.05) higher than in the < 105 ${\mu}{\textrm}{m}$ group(66.7%). The rates of sperm penetration were significantly (P<0.05) low in < $105{\mu}{\textrm}{m}$ group (50.0%) than others groups (81.6~85.5%). But the plyspermic fertilization rate was significantly (P<0.05) higher in < $110{\mu}{\textrm}{m}$ oocytes groups than in the $110\leq{\mu}{\textrm}{m}$ size groups. The rates of cleavage and development to blastocysts rose as oocytes diameter increased, however, while oocytes over $120{\mu}{\textrm}{m}$ in diameter failed to develop to blastocysts. There results suggest that porcine oocytes have acquired full meiotic competece at a diameter of $105{\mu}{\textrm}{m}$ but not yet attended full development competence to blastocyst and that oocytes have acquired full development competence at a diameter of $110{\mu}{\textrm}{m}$.

• Korean Journal of Weed Science
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• v.8 no.2
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• pp.187-198
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• 1988

The second leaves from 30 days old seedlings of two rice cultivars which were selected as tolerant (cv. Chokoto) and susceptible (cv. Weld pally) cultivar were soaked in the concentration of $10^{-6}$, $10^{-5}$, $10^{-4}$ and $10^{-3}M$ of oxyfluorfen for 10, 15 and 22hrs and anatomical characteristics were abserved. Dipping to the solutions were carried out either directly to the attached leaves or to the seperated leaves. Development of any symptoms in epidermis, bundle sheath, mesophyll cells and bulliform cells were microscopically inspected. Both cultivars showed reductionin leaf thickness, but the susceptible ones was more sensitive than the tolerant. The degradation and disappearance of epidermal cell layer, breakage of bundle sheath cells, shrinkage of mesophyll cells and disappearance of bulliform cells were general response as affected by oxyfluorfen treatment. The susceptible cultivars showed such responses at the concentration of $10^{-5}M$ for 10 hrs while tolerant ones $10^{-3}M$, for 10 hrs. Those treatments were more effective in seperated leaves than in attached ones. The epicuticular wax layer of leaves treated as above for 20 hrs was inspected by SEM. Weld pally, the susceptible cultivar (Weld pally) showed rapid cleavage of wax layer under $10^{-5}M$ concentration while the tolerant (Chokoto) showed only minor damage on wax layer at the concentration of $10^{-3}M$.

• Journal of Embryo Transfer
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• v.22 no.2
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• pp.89-95
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• 2007

The present study was conducted to examine some factors affecting in vitro development and fecundity of embryos recloned with somatic cell nuclear transfer (SCNT). Fibroblast cells retrieved from the ear of a 3-week-old, cloned Korean goat (Jinsoonny) were used as karyoplast donors and serum-starvation was conducted in tissue culture medium (TCM)-199 supplemented with 0.5% FBS. Recipient oocytes were surgically collected by flushing the oviducts 35 h after hCG injection following FSH priming. The zonae pellucidae of the oocytes were partially perforated with a laser drill and a donor cell was transferred into an enucleated oocyte. The couplets were electrically fused and activated by ionomycin (5 min) and 6-DMAP (4 h). The reconstructed embryos were cultured in mSOF medium containing 0.8% BSA at $39^{\circ}C$ in an atmosphere of 5% $CO_2$, 5% $%O_2$, 90% $N_2$ for 12 to 15 h. Re-cloned embryos (2- to 4-cell stages) were surgically transferred into the oviducts of the recipients and pregnancy was subsequently diagnosed by progesterone assay and ultrasound on Days 21 and 63 of pregnancy. The fusion rate following 1st fusion pulse was higher (p<0.05) in 2nd cloning (65.9%) compared to 1st cloning (51.0%), but it was not different in the other groups. The rate of cleavage after fusion was significantly higher (p<0.05) in 1st (77.7%) than in 2nd cloning (56.0%). A total of 175 re-cloned embryos were transferred into 28 recipients. On day 21 and 60 after transfer, 11 (39.3%) and 4 recipients (17.4%) were pregnancy, respectively. In comparison of pregnancy rate by estrous synchronization, a total of 66 and 109 re-cloned embryos were transferred into 11 recipients in natural estrus and 17 recipients in induced estrus, respectively. Five (45.4%) and 2 recipients (18.2%) in natural estrus were pregnant on days 21 and 63 while 6 (35.3%) and 2 (11.8%) recipients in induced estrus were pregnant, respectively. These results show that recloning of goat can be achieved by SCNT and estrous synchronization between donor and recipient animals may be one of the major factors affecting success rate.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.45-53
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• 2002

The main goal of this study was to produce transgenic porcine embryos by direct injection of sperm-mediated exogenous DNA. Spermatozoa (6$\times$10$^{6}$ sperms of final concentration) were mixed with pcDNA LAC Z (20 ng/$\mu$l) and subjected into electroporation (300~750 volts, 25 $\mu$F, 0.4 cm electrode). After sperm injection, the oocytes were activated electrically (1.7 KV/cm, 30$\mu$sec, single pulse) in 0.3 M mannitol solution or not. The sperm injected eggs were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air fur 144 h. The rates of cleavage and development into blastocyst stage in activation group were significantly higher than those of non-activation group (79.6% and 24.1% vs. 46.3% and 14.4%, respectively, p<0.05). Control oocytes and shame injection were developed to blastocysts low (2.5%). Sixty five (27.1%) out of 240 embryos observed in activation and non-activation groups were showed positive by X-gal staining. However, all embryos in both groups were expressed partial or mosaic pattern. These results suggested that electrical stimulation far oocytes activation after sperm injection enhances the incidence of both fertilization and development fellowing sperm injection in the pig. Our study also suggested that sperm-mediated transfer of exogenous DNA by ICSI would be used as a valuable tool for the production of transgenic porcine embryos.

• Korean Journal of Environmental Agriculture
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• v.14 no.2
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• pp.186-201
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• 1995

In order to elucidate the fate of the residues of the pyrethroid acaricide-insecticide, acrinathrin in soil, maize plants were grown for one month on the specially-made pots filled with two different types of soils containing fresh and one-month-aged residues of [$^{14}C$]acrinathrin, respectively. The mineralization of [$^{14}C$]acrinathrin to $^{14}CO_2$ during the one-month period of aging and of maize cultivation amounted to $23{\sim}24%$ and $24{\sim}33%$, respectively, of the original $^{14}C$ activities. At harvest after one-month growing, the shoots and roots contained less than 0.1% and 1% of the originally applied $^{14}C$ activity, respectively, whereas the $^{14}C$ activity remaining in soil was $65{\sim}80%$ in both soils. Three degradation products with m/z 198(3-phenoxybenzaldehyde), m/z 214(3-phenoxybenzoic acid), and m/z 228(methyl 3-phenoxybenzoate) besides an unknown were identified from acetone extracts of both soils without and with maize plants after treatment of [$^{14}C$]acrinathrin, by autoradiography and GC-MS, and those with m/z 225(3-phenoxybenzaldehyde cyanohydrin) and m/z 198 (3-phenoxybenzaldehyde) from acetone extract of the Soil A treated with 50 ppm acrinathrin and grown with maize plants for 30 days were identified by mass spectrometry. These results suggested that the hydrolytic cleavage of the ester linkage adjacent to the $^{14}C$ with a cyano group, forming 3-phenoxybenzaldehyde cyanohydrin. The removal of hydrogen cyanide therefrom leads to the formation of 3-phenoxybenzaldehyde as one of the major products. The subsequent oxidation of the aldehyde to 3-phenoxybenzoic acid, followed by decarboxylation would evolve $^{14}CO_2$. Solvent extractability of the soils where maize plants were grown for 1 month and/or [$^{14}C$]acrinathrin was aged for 1 month was less than 31% of the original $^{14}C$ activity and over 95% of the total $^{14}C$ activity in soil extracts was distributed in the organic phase. Accordingly, acrinathrin turned out to be degraded rapidly in both soils and be bound to soil constituents as well, not being available to crops.

• Korean Journal of Environmental Agriculture
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• v.14 no.2
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• pp.202-212
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• 1995

In order to elucidate the degrading characteristics of the pyrethroid acaricide-insecticide, acrinathrin in two different types of soils, Soil A(pH, 5.8; organic matter, 3.4%; C.E.C., 115 mmol(+)/kg soil; texture, sandy loam) and Soil B(pH, 5.7; organic matter, 2.0%; C.E.C., 71 mmol(+)/kg soil; texture, sandy loam), residualities of the non-labeled compound under the field and laboratory conditions, extractability with organic solvents and formation of non-extractable bound residues, and degradabilities of [$^{14}C$]acrinathrin as a function of aging temperature and aging period were investigated. The half lives of acrinathrin in Soil A treated once and twice were about 18 and 22 days and in Soil B about 13 and 15 days, respectively, in the field, whereas, in the laboratory, those in Soil A and B were about 36 and 18 days, respectively, suggesting that the compound would be non-persistent in the environment. The amounts of $^{14}CO_2$ evolved from [$^{14}C$]acrinathrin in Soil A and B during the aging period of 24 weeks were 81 and 62%, respectively, of the originally applied $^{14}C$ activity, and those of the non-extractable soil-bound residues of [$^{14}C$]acrinathrin were about 70% of the total $^{14}C$ activity remaining in both soils, increasing gradually with the aging period. Degradation of [$^{14}C$]acrinathrin in both soils increased with the aging temperature. Three degradation products of m/z 198(3-phenoxy benzaldehyde), m/z 214(3-phenoxybenzoic acid), and m/z 228(methyl 3-phenoxybenzoate) as well as an unknown were detected by autoradiography of acetone extracts of both soils treated with [$^{14}C$]acrinathrin and aged for 15, 30, 60, 90, 120, and 150 days, respectively, and the degradation pattern of acrinathrin was identical in both soils. Acrinathrin in soil turned out to be degraded to 3-phenoxybenzaldehyde cyanohydrin by hydrolytic cleavage of the ester linkage adjacent to the $^{14}C$ with a cyano group, the removal of hydrogen cyanide therefrom led to the formation of 3-phenoxybenzaldehyde as one of the major products, and the subsequent oxidation of the aldehyde to 3-phenoxybenzoic acid, followed by decarboxylation would lead to the evolution of $^{14}CO_2$.