• Journal of Embryo Transfer
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• v.31 no.3
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• pp.185-190
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• 2016

During the ovary preservation in low temperature, the cumulus oocyte complexes(COCs) lose their developmental competences after in vitro fertilization. We used phosphate-buffered saline (PBS) as a basic solutions of at various temperatures (25, 15 or $5^{\circ}C$) and supplemented them with 1mM glucose and 0.5mM glutamine as a source of carbohydrate metabolites. After recovery of COCs and in vitro fertilization, a significantly higher number of oocytes developed into blastocysts. The developmental competence of embryos that were originated from ovaries preserved at $15^{\circ}C$ was increased compared to those of 25 or $5^{\circ}C$. The maturation rate of oocytes was not differed between 24 and 36 h at $15^{\circ}C$ but showed lower than control group (71% versus 78%). In vitro-fertilized oocytes from ovaries stored at $25^{\circ}C$ for 24 h or at $5^{\circ}C$ for 24 h had a significantly decreased developmental potentials, but at $15^{\circ}C$ did not (27% versus 29% of blastocysts to develop into day 8). With these results, bovine ovaries can be preserved at $15^{\circ}C$ for 36 h without decreasing developmental capacity of in vitro-fertilized oocyte at least to the blastocyst stage. This information provides valuable information of preserving ovaries for embryo transfer or in vitro embryo production.

• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.199-208
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• 1992

These studies were carried out to investigate optimal physological conditions for in vitro fertilization (IVF) of mouse ova. The unfertilized ova were obtained by superovulation from ICR mice of 4 to 6 weeks old. Tyrode's 280 solution was used as basal media, and pH and osmolality of basal media were adjusted with the supplementation of sodium bicarbonate and sodium chloride, respectively. The optimal pH, and osmolality of culture media and the optimum period of sperm preincubation were examined in fertilization in vitro of mouse ova and the subsequent culture in vitro of embryos. The pH range of media examined was designed from 6.5 to 7.5 with 0.2 interval and the range of osmolality from 250 to 370 mOsm with 20 interval, and the period of sperm preincubation examined was 30, 60, 120, and 180 minutes. The ova developed to 2-cell embryosafter 26hrs. of incubation with preincubated sperm were evaluated as in vitro fertilized ones. The results obtained were summarized as follows: 1. The percentage of in vitro fertilized ova was highest (64.7%) in media of pH 7.1 and lowest (38.0%) in pH 6.7. No significant difference in % fertilized ova was found from the media of pH 7.1 to 7.5. Compared with the result from pH 7.1 medium, the pollyspermy was increased signifciantly (p<0.05) in the media of pH over 7.5 and below 6.9;, and the % degenerated ova was significantly (p<0.05) increased in the media of pH below 6.9. 2. The percentage of in vitro fertilized ova was highest (69.4%) in media of osmolality 330 mOsm and lowest (47.9%) in osmolality 250 mOsm. No significant difference in % fertilized ova was found from the media of osmolality 310 to 350 mOsm. Compared with the result from osmolality 330 mOsm in medium, the polyspermy aws increased significantly(p<0.05) in the media of osmolality over 350 mosmol and blow 290 mOsm, and the % degenerated ova was significantly (P<0.05) increased in the media of osmolality below 290 mOsm. 3. The percentate of in vitro fertlilized ova was highest (62.7%) in media of period sperm preincubation 180 min. and lowest (40.4%) in sperm preincubation 30 minutes. No significant difference in % fertilized ova was found from the media of sperm preincubation 120 to 180 minutes. Compared with the result from sperm preincubation 180 minutes in medium, the polyspermy was low differ no significantly(P<0.05) in the media of period sperm preincubation, and the % degenerated ova was signifciantly(P<0.05) increased in the media of sperm presincubation below 60 minutes.

• Korean Journal of Organic Agriculture
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• v.26 no.2
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• pp.281-296
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• 2018

This study was conducted to evaluate the effect of different processing of rice on rumen fermentation in in vitro and in situ experiments. Different processing treatments (extruding, roasting, and steaming) were used in this study and all treatments were ground through a cyclone mill (Foss, Hillerød, Denmark) fitted with a 1 mm screen. Non-treated rice was considered to a control substrate. Then, all treatments were used in in vitro and in situ experiments. Total gas production and dry matter digestibility in control were lower than any other treatment at all incubation times (P<0.01). The lowest ammonia nitrogen ($NH_3-N$) concentration was observed in control among treatments at 6, 12, and 24 h incubation (P<0.01). Extruding had a highest total volatile fatty acids (VFA) concentration at 6, 12 h incubation (P<0.01) and Steaming exhibited a highest total VFA at 24 h (P<0.01). The lowest total VFA concentration was observed in control at 6, 12, and 24 h (P<0.01). In an in situ, The highest value of soluble fraction, degradation rates, effective degradability was observed in extruding (P<0.01). It was considered that feed processing increased dry matter digestibility, total VFA concentration, and decreased pH as well as $NH_3-N$ concentration indicating that processing may increase nutrient degradation of rice in the rumen.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.39-46
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• 1994

This experiment was carried out to develop an in vitro culture system for rabbit embryos. The zygotes or 2-cell embryos were collected from the oviducts of the superovulated and mated does with D-PBS/10% FCS at 24 hours after hCG injection. The in vitro developmental rate of blastocyst formation and the number of nuclei in the embryos were examined under the following treatments; 1) TCM-199 with 10% FCS, 2) EBSS with 10% FCS, 3) rabbit vitreous humor(VH), 4) TCM-199 with 10% FCS＋BOEC, 5) TCM-199 with 10% FCS＋ROEC, 6) EBSS with 10% FCS＋BOEC and 7) EBSS with 10% FCS＋ROEC. For a comparative study of in vivo and in vitro development, the fresh blastocysts, which were developed in vivo for 96 hours after hCG injection, were collected from the uterus and their numbers of nuclei were counted. 1. The zygotes or 2-cell embryos developed to the blastocyst stage in TCM-199, EBSS and VH at the rates of 93, 92 and 89%, respectively. 2. The higher developmental rates 95~98% of blastocyst formation was achieved when the embryos were co-cultured with a monolayer of bovine or rabbit oviductal epithelial cells in TCM-199 or EBSS. No significant difference in developmental rates was shown between bovine and rabbit oviductal epithelial cells. 3. In a comparative study of in vivo and in vitro development, the total numbers of nuclei were significantly less in the in vitro cultured embryos(104~224) than the in vivo developed embryos(1, 0090 at 96 hours after hCG injectin. 4. The mean cell cycle numbers in the embryos cultured for 72 hours in TCM-199 with 10% FCS, EBSS with 10% FCS, TCM-199 with 10% FCS＋BOEC, TCM-199 with 10% FCS＋ROEC, EBSS with 10% FCS＋BOEC and in vivo was 7.38, 6.63, 7.76, 7.69, 7.01 and 9.92, respectively. From these results, it can be suggested the optimal culture system for in vitro culture of rabbit embryos is a co-culture system with bovine or rabbit oviductal epithelial cells in TCM-199 with 10% FCS. Considering the significant reduction in total numbers of nuclei in the in vitro cultured embryos, the advanced research on development of in vitro culture system for rabbit embryos is expected.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.269-273
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• 2012

The present study was to assess the in vitro viability and sexing rate of bovine embryos. Blastocysts were harvested on day 7~9 day after insemination(in vitro and in vivo), and the sex of the embryos was examined using the LAMP method. Embryo cell biopsy was carried out in a $80{\mu}l$ drop $Ca^{2+}$, $Mg^{2+}$ free D-PBS and, biopsied embryos viability were evaluated after more 12 h culture in IVMD culture medium. The formation of recovered embryo to expanded and hatching stages had ensued in higher of sexed embryo in vivo than in vitro (100% vs. 89%, p<0.05), and in vitro, the rates of degeneration after sexing were significantly (p<0.05) higher in vitro than in vivo(11% vs. 0.0%). The rates of the predicted sex were female 61% vs. 56%, and male 39% vs. 44% in vivo and in vitro, respectively. The rates of survival following different biopsy methods were seen between punching and bisection method in vivo and in vitro (100% vs. 89% and 100% vs, 78% respectively). Biopsy method by punching was significantly (p<0.05) higher than bisection between produced embryos in vivo and in vitro. The present data indicate that with microblade after punching for embryo sexing results in high incidence of survivability on development after embryo biopsy. It is also suggested that LAMP-based embryo sexing suitable for field applications.

• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.1-8
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• 1990

These experiments were carried out to investigate the effects of cumulus cells for in vitro fertilization and development of bovine follicular oocytes matured in vitro. The bovine ovaries were obtained at a slaughter house and the follicular oocytes surrounded by cumulus cells were collected by puncturing follicles with 2~6 mm of diameter. Bovine oocytes were matured in vitro for 24~26 hours in a CO2 incubator with 5% CO2 in air at 39$^{\circ}C$. The medium used for maturation was TCM-199 supplemented with hormones, pyruvate, FCS and antibiotics. Epididymal spermatozoa were capacitated by in vitro culture for 2~3 hours in BO solution containing BSA(5mg/ml) and caffeine(2.5mM). Insemination was made by introducing about 10~15 matured oocytes into the suspension of capacitated spermatozoa. Six hour after insemination the eggs were transferred to TCM-199 supplemented with FCS(10%) and HEPES(25mM), cultured for 7~8 days with 10~15 eggs/well in 4-well multidishes(Nunc Co.) forming cumulus cell monolayer. The results obtained in these experiments were summarized as follows ; 1. The majority of the follicular oocytes with compacted cumulus cells existed in GV stage while those with dispersed or denuded cumulus cells existed GVBD and M II stage. 2. After 24~26 hours maturation, the maturation rates of the follicular oocytes cultured in TCM-199 containing hormones were slightly higher than those of oocytes cultured in medium without hormones, and the frequency of cumulus compacted or denuded oocytes reaching M II stage cultured in medium containing hormones was 75.7% or 51.7%, respectively(P<0.05). 3. After 20 hours in vitro insemination, percentages of ova fertilized were 61.4% or 51.4%, respectively, for cumulus oophorus intacted or removed, and increased frequency of ova with both male and female pronuclei was found when cumuli were present(P<0.05). 4. The rates of embryos developed to 2-, 4-, 8-, 16-cell and morula or blastocyst stage after cocultured with cumulus cells were 65.0%, 45.3%, 34.7%, 28.0% and 22.7%, respectively. The results for momla or blastocyst stage were significantly higher than those of the embryos cultured in the basic medium(P<0.05).

• Molecular & Cellular Toxicology
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• v.3 no.3
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• pp.177-184
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• 2007

As a possible feasibility of the extrapolation between in vivo and in vitro systems, we investigated the global gene expression from both mouse liver and mouse hepatic cell line treated with hepatotoxic chemical, acetaminophen (APAP), and compared between in vivo and in vitro genomic profiles. For in vivo study, mice were orally treated with APAP and sacrificed at 6 and 24 h. For in vitro study, APAP were administered to a mouse hepatic cell line, BNL CL.2 and sampling was carried out at 6 and 24 h. Hepatotoxicity was assessed by analyzing hepatic enzymes and histopathological examination (in vivo) or lactate dehydrogenase (LDH) assay and morphological examination (in vitro). Global gene expression was assessed using microarray. In high dose APAPtreated group, there was centrilobular necrosis (in vivo) and cellular toxicity with the elevation of LDH (in vitro) at 24 h. Statistical analysis of global gene expression identified that there were similar numbers of altered genes found between in vivo and in vitro at each time points. Pathway analysis identified glutathione metabolism pathway as common pathways for hepatotoxicty caused by APAP. Our results suggest it may be feasible to develop toxicogenomics biomarkers or profiles by comparing in vivo and in vitro genomic profiles specific to this hepatotoxic chemical for application to prediction of liver toxicity.

• Korean Journal of Animal Reproduction
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• v.14 no.2
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• pp.101-106
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• 1990

These mxperiments were carried out to investigate the effect of rabbit anti-bovine cumulus cell antibodies on in vitro fertilization and following development of bovine follicular oocytes matured in vitro. The bovine ovaries were obtained at a slaughter house and the follicular oocytes surrounded by cumulus cells were collected by puncturing follicles with 2~6mm of diameter. Bovine oocytes were matured in vitro for 24~26hrs in a CO2 incubator with 5% CO2 in air at 39$^{\circ}C$ and subsequently cultured in medium containing cumulus cell antibody for 1 hour. The medium used for maturation was TCM-199 supplemented with hormones, pyruvate, FCS and antibiotics. Epididymal spermatozoa were capacitated by in vitro culture for 2~3 hrs in BO solution 10~15 matured oocytes into the suspension of capacitated spermatozoa. Six hour after insemination the eggs were transferred to TCM-199 supplemented with FCS(10%) and then cultured for 7 days. The results obtained in these experiments were summarized as follows : 1. When the follicular oocytes matured in vitro were treated with antibody to intact cumulus cells, the fertilization rate of cumulus intact and removed oocytes was ranged to 45.0 to 53.7%. These value is slightly lower than that(64.3%) of follicular oocytes not treated with the antibody, and increased frequency of both male and female pronuclear formation was found in cumulus intact oocytes cultred in medium without the antibody(p<0.05). 2. The fertilization rate of cumulus intact and removed oocytes treated with antibody to solubilized cumulus cells was ranged 45.0 to 52.5%, significantly lowre than that(62.8%) of oocytes cultured in antibody free medium, and increased frequency of ova with male and female pronuclei was found when cumulus cells were present(p<0.05). 3. The rates of cumulus cell intact and removed oocytes developed to 8-, 16-cell and morula or blastocyst after treatment of intact and solubilized cumulus cell antibody were ranged 7.1 to 14.5, 2.9 to 5.9 and 1.5 to 2.9%, respectively, slightly lower than 18.6, 10.0 and 8.6% of cumulus intact oocytes cultured in medium without the antibody. The results of this stduy indicate that cumulus cells promote not only normal fertilization with proper pronuclear formation, but embryo development and that the beneficial effect of cumulus cell to the pronuclear formation and embryo development is blocked by the action of antibody to cumulus cell.

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.95-99
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• 1998

Three experiments were conducted with follicular oocytes, to compare some somatic cells, growth factors and media for in vitro maturation of bovine oocytes. In the first experiment, the type of somatic cells had no effects on in vitro maturation of bovine follicular ooctyes. In the second experiment, oocytes were matured in TCM199 su, pp.emented with growth factors on IVM of bovine follicular oocytes, then all were co-cultured with cumulus cells. The proportion of used oocytes that developed to expanding blastocysts was 22.2%, 20.2%, 17.7%, 22.2%, 24.4% and 20.2% after maturation in TCM199 su, pp.emented with control, insulin, IGF-I, IGF-Ⅱ, FGF and EGF, respectively. In the third experiment, oocytes were matured in BO, Ham's F10 and TCM199, then all were fertilized in BO, and embryos cultured in BO, Ham's F10 and TCM199, respectively. Cleavage rates in BO were 90%, had higher than in Ham's F10(80%) or in TCM199(64%). But production of expanding blastocysts in TCM199(21%) or Ham's F10(20.6%), had higher than in BO(4.6%).

• Korean Journal of Animal Reproduction
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• v.22 no.3
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• pp.253-264
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• 1998

Porcine follicular oocyte, collected from antral follicles (2~5 mm in diameter) of gilt ovaries were matured in vitro porcine follicular fluid (pFF) with PMSG (pFF-PMSG) buffer with at 37$^{\circ}C$ under 5% CO2 in air their ability of maturation promoting factor (MPF), of GV and GVBD formation was examined followed during time after in vitro culture. Formation of second metaphase was observed in 57.6% and 71.2% of matured in with pFF-PMSG buffer to 45 and 50 hours after invitro. Porcine oocytes cultured in pFF-PMSG for various periods of up to 30 hours were stained with Hoechst-33342 and classified according to maturation before assaying. Histone H1 kinase (H1K) activity was assayed during meiotic maturation in porcine oocytes matured in pFF-PMSG buffer in vitro. In oocytes matured in pFF-PMSG, H1K activity was at the 30 hours after culture and increased about 15 fold than at the germinal vesicle stage with before at the cultured in vitro. This pattern is similar to those reported in non-mammalian species and su, pp.rts the concepts that H1K is ubiquitous in eukaryotes and controls the meiotic cell cycle in mammals. These results suggest that the maturation pFF-PMSG buffer used influences the fluctuation pattern of H1K activity and biological characteristics of porcine oocytes cultured in vitro.