• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.61-69
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• 2007

This study was designed to evaluate effects of BSA, PVA, gonadotropins and follicle shell during IVM of porcine oocytes and subsequent development to the blastocyst stage after IVF. Cumulus oocyte complexes (COCs) were cultured in TCM-199 media containing 4 mg/ml BSA and 1 mg/ml PVA during IVM for 44 hr. To compare the effect of gonadotropins on oocyte maturation, COCs were cultured with FSH+LH, FSH, LH and FSH-LH-free media during IVM. respectively. Also, different number of follicle shells (0, 2, 4 and 6) was used to examine whether the presence of follicle shell in culture medium affects oocyte maturation. The percentages of fertilization and blastocyst formation, respectively, were higher in the medium containing the PVA (49.0 and 17.9%) than those containing the BSA (40.0 and 12.2%). Significantly higher rates of Mil oocytes were in the presence of FSH+LH and FSH (88.6 and 85.1 %) compared to other treatments (64.0 and 53.4% at LH and FSH-LH-free media). Co-culture with inverted follicle shells in 2 ml maturation medium enhanced the developmental competence of porcine oocytes. In conclusion, PVA could be used as a macromolecules instead of BSA, and FSH and follicle shell played important roles in maturation of porcine oocytes.

• Korean Journal of Animal Reproduction
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• v.19 no.2
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• pp.153-157
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• 1995

These studies were conducted to investigate the microbe control effect of antibiotics treatment in all media used for in vitro fertilization(IVF) embryo production. The bovine oocytes were recovered from follicles(2~5mm) and were cultrued for 22hrs at 38.5$^{\circ}C$ with 5% CO2 incubator. The contamination and development rate in vitro fertilized oocyte was evaluated everyday. The results were summerized as follow ; 1. Control, antibiotic-antimycotic solution(AAS, Gibco) 1%＋gentamycin, and AAS 1%＋kanamycin(Sigma, USA) treatment was contaminated with 72hrs. However Baytril and Kanamycin(Korea) added was not contaminated. 2. The blastocyst formation rate in Baytril supplementated 1, 2 and 3${mu}ell$/ml was 3.73, 1.28 and 0.00%, respectively. 3. The blastocyst formation in kanamycin added concentration of 50, 75 and 100$\mu\textrm{g}$/ml was 13.0, 9.4 and 3.49%, respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.2
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• pp.164-167
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• 2004

This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing $500{\mu}l$ mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to $24^{\circ}C$ over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were esuspended in a lactose-egg yolk and N-acetyl-Dglucosamine (LEN) diluent to contain $1{\times}10^{9}$ sperm/ml and cooled to $5^{\circ}C$ over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with $2{\times}10^{7}$ sperm concentration. Oocytes were coincubated for 6 h in $500{\mu}l$ mTBM fertilization. At 6 h after IVF, oocytes were transferred into $500{\mu}l$ NCSU-23 culture medium for further culture of 6 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF and developmental ability of oocytes at 48 h after IVF were evaluated. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium.

• Journal of Embryo Transfer
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• v.17 no.3
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• pp.239-249
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• 2002

Embryos derived from pig oocytes matured in mSOF are able to develop to blastocysts after IVF. Experiment 1 evaluated the effects of two maturation media (TCM-199 vs mSOF) on maturation rate, fertilization parameters, including penetration, polyspermy, male pronuclear formation, and the mean number of sperm penetrated per oocyte. Experiment 2 and Experiments 3 examined the effects of two maturation media on zona pellucida solubility and cortical granule distribution by transmissible electron microscopy, respectively. Experiment 4 assessed the effects of two maturation media on the in vitro embryo cleavage rate and development to blastocyst. Lastly, experiment 5 examined the cell number of blastocyst. An effect of media (P<0.05) was detected for mSOF on the mean number of sperm per oocyte. In TCM group, zona digestion time (196.5$\pm$15.5 vs 131.6$\pm$20.1 before IVF, 397.5$\pm$30.3s vs 185.3$\pm$16.4s after IVF, p<0.05) was higher in TCM-199 group. No significant effects of media was observed on cortical granule distribution between two groups by TEM. An effect (P<0.05) was observed on embryo development to blastocyst (16% vs 8%) but not on cleavage rates. No significant effects of media was observed on total cell number of blastocyst. We found that the high mean number of sperm penetrated per oocyte and the weaker zona pellucida on the basis of the digestion time was shown in pig oocytes matured in mSOF, however, porcine oocyte maturation with supplemented synthetic oviduct fluid medium (mSOF) resulted in blastocyst cell numbers comparable to those observed with Tissue Culture Medium 199.

• Clinical and Experimental Reproductive Medicine
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• v.20 no.1
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• pp.9-17
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• 1993

This study was carried out investigate the effect of water quality and the kind of media on the in vitro development of 1-cell and stage mouse embryos. $F_1$ hybrid mice were superovulated and timely mated. 1-cell stage and 2-cell stage mouse embryos were recruited and taken into Ham's F-10 or m-KRB media which was made of two of two kinds of water having different quality, highly purified water and tap water. 2-cell stage embryos grew up well in vitro to blastocyst or hatching blastocyst regardless of the composition of culture media, but 1-cell stage mouse embryo didn't develop well to blastocyst or hatching blastocyst in simple media like m-KRB. These results meant in vitro devleopment of 1-cell stage mouse embryo neded complex media like Ham's F-10 which contained abundant protein components. In case of quality control for water, in vitro fertilization program. observation of in vitro development of 2-cell mouse embryos up to blastocyst or hatching blastocyst media such as m-KRB would be efficatious in detecting the difference of water quality.

• Korean Journal of Animal Reproduction
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• v.16 no.1
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• pp.15-20
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• 1992

These experiments were undertaken to investigate the rate of in vitro fertilization of bovine follicular oocytes treated with glycosaminoglycans(GAGs). Bovine follicular oocytes were obtained from the ovary of slaughtered animal and matured in media containing the various concentrations of hydluronic acid, chondroitin sulfate or heparin for 26 hours. Epididymal spermatozoa were capacitated and insemination was made by introducing about 10~15 matured oocytes into the suspension of spermatozoa. Six hour after insemination the eggs were transferred to TCM-199 supplemented with FCS(10%) and then examined the embryo development. After in vitro insemination, percentages of ova fertilized were 61.3 or 48.3%, respectively, for the cumulus intact or removed in the percentages of GAGs. However, in case of cumulus-free oocytes treated with GAGs, the fertilization rates were 58.8, 62.1, 58.8, and 61.8%, respectively, showing significant effect compared to 48.3% in cumulus-free oocytes. Our findings suggest that cnondroitin sulfate and heparin are superior to hyaluronic acid in the fertilizatin and pronuclear formation of bovine oocytes.

• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.209-215
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• 1992

These studies were carried out to overcome 2-cell block and in vitro development to blastocysts in vitro fertilization of mouse embryos. The unfertilized ova were obtained by superovulation in ICR mice of 4 to 6 weeks old. Tyrode's 280 solution was used as basal media, and 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 embryos after 26hrs of incubation with preincubated sperm were evaluatated as in vitro fertilized ones. The results obtained were summarized as follows: 1. The optimal ranges of pH and osmolality of culture media and of sperm preincubation time for in vitro development of in vitro fertilized ova to blastocyst were pH 7.1 to 7.3, 250 to 350 mosmol and 60 to 180 min, respectively. 2. With the media of pH 7.1, 310 mOsm and sperm preincubation period of 120min in another experiment of large sample size, the in vitro fertilized ova was found 66.5% and the in vitro development of in vitro fertilized ova to blastocyst was found 35.8%. From the above results it was concluded that the optimal conditions of pH and osmolality of the media for mouse IVF and embryo culture, and the period of sperm preincubation might be 7.1, 310 mOam and 120min, respectively.

• Clinical and Experimental Reproductive Medicine
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• v.47 no.4
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• pp.284-292
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• 2020

Objective: This study investigated whether adding outer-well medium to inhibit osmotic changes in culture media in a dry-type incubator improved the clinical outcomes of in vitro fertilization-embryo transfer (IVF-ET) cycles. Methods: In culture dishes, the osmotic changes in media (20 µL)-covered oil with or without outer-well medium (humid or dry culture conditions, respectively) were compared after 3 days of incubation in a dry-type incubator. One-step (Origio) and G1/G2 (Vitrolife) media were used. Results: The osmotic changes in the dry culture condition (308 mOsm) were higher than in the humid culture conditions (285-290 mOsm) after 3 days of incubation. In day 3 IVF-ET cycles, although the pregnancy rate did not significantly differ between the dry (46.2%) and humid culture (51.0%) groups, the rates of abortion and ongoing pregnancy were significantly better in the humid culture group (1.5% and 49.5%, respectively) than in the dry culture group (8.3% and 37.8%, respectively, p<0.05). In day 5 IVF-ET cycles, the abortion rate was significantly lower in the humid culture group (2.2%) than in the dry culture group (25.0%, p<0.01), but no statistically significant difference was observed in the rates of clinical and ongoing pregnancy between the dry (50.0% and 25.0%, respectively) and humid culture groups (59.5% and 57.3%, respectively) because of the small number of cycles. Conclusion: Hyperosmotic changes in media occurred in a dry-type incubator by evaporation, although the medium was covered with oil. These osmotic changes were efficiently inhibited by supplementation of outer-well medium, which resulted in improved pregnancy outcomes.

• Journal of Embryo Transfer
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• v.11 no.1
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• pp.15-26
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• 1996

This study was conducted to improve the in vitro maturation(JVM), in vitro fertilization (IVF) and in vitro developmental capacity of oocytes derived from slaughtered Korean native cattle. The recoverd oocytes, obtained from a local slaughter house, were used completely surrounded by at least 3 layers of cumulus cells in combination with a homogeneous cytoplasmic pigmentation. In vitro maturation was induced in TCM-199 or Ham's F-10 supplemented with LH(1O $\mu$g/rnl), FSH(35 $\mu$g/ml), estradiol-17$\beta$(1 $\mu$g/ml) at 39$^{\circ}C$ under 5% $CO_2$ in air for 24 hours. Sperm from caudal epididyrnis and previously matured cumulus-oocytes complexes were cultured for 24 hours in 100 $\mu$l droplets of fertilization media under paraffin oil. The zygotes were cultured with media(TCM-199 with bovine oviductal epithelial cells or CRlaa) for 7 to 10 days. The cleavage rate of IVM-IVF oocytes was significantly (P<0.05) higher following maturation using Ham's F-10 (59.9%) than TCM-199 (51.6%). Development to the blastocysts among cleaved embryos was not signficantly different between maturation media: Ham's F-10 (16.0%) and TCM-199(11.9%). However, the hatching rate was affected significantly (P<0.05) on rnaturation media as 62.9% in Ham's F-10, compared with 41.2% in TCM-199. The cleavage rate of IVM-IVF oocytes was significantly (P<0.05) higher following IVF using m-TALP medium (80.1%) than BO medium (51.6%). The percentage of in vitro developed blastocysts among cleaved embryos was not signficantly different between fertimization media: BO (11.7%) and m-TALP (17.6%). The cleavage and the developmental rate to the blastocysts after IVF in m-TALP or condition medium(CM) with or without oviduct epithelial cell monolayer(OECM) was similar(80.1% and 17.6% in m-TALP, 83.8% and 19.4% in M-TALP with OECM. 82.9% and 18.9% in CM, 87.6% and 16.0% in CM with OECM, respectively). The percentage of in vitro developed blastocysts among cleaved embryos was significantly (P<0.05) higher in TCM-199 medium co-cul tured with bovine oviduatal epithelial cell monolayers(35.2%) than CRlaa medium(1.9%). These results stggest that the most transferable IVF embryos could be produced from Ham's F-10, m-TALP and TCM-199 medium with bovine oviductal epithelial cell monolayers for IVM, IVF and IVC, respectively.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.113-120
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• 1993

To provide the optimal culture conditions for the developm,ent of in vit개 produced embryos, we have been investigated various culture media as well as co-cultrue systems using porcine cumulus cells or mouse fetal fibroblast cells. Porcine ovaries were brought to the laboratory from local slaughter house within 1 hour after slaughtering and cumulus oocytes complexes were recovered from antral follicles(3~5mm) with 23 gauge needle. To maturate follicular oocytes, cumulus oocytes complexes were washed three times with TCM-199 containing 25mM HEPES and incubated(39$^{\circ}C$, 5% CO2 in air) in various maturation media for 42 hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were rpepared for fertilizing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${\mu}\ell$ fo capacitated spermatozoa containing 1~5$\times$105 sperm/ml to droplets. Eighteen to twenty-eight hours after sperm insemination, fertilized eggs were washed three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three different media, m-KRB, BECM and TCM-HEPES were 0~1.0%, showing extremely lower rates. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. The rates of embryos developed to 2-, 4-, 8-, 16-, 32-cell and morula or blastocyst stage in co-culture with porcine cumulus cells and mouse fetal fibroblast cells were 61.1~67.0%, 59.0~58.0%, 42.5~43.1%, 28.4~30.2% and 20.4~21.0%, respectively. These development rates upto morula or blastocyst stages were significantly higher than those of the embryos cultured in the basic culture medium(P<0.01). These findings suggest that co-culture of in vitro fertilized eggs with porcine cumulus cells or mouse fetal fibroblast cells enhance the development of fertilized eggs to morula or blastocyst stage in vitro.