• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.12
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• pp.1647-1651
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• 2004

The follicles (1.8 to 7.8 mm in diameter) were recovered from the ovaries in marketed pigs and the number of granulosa cells, the diameter of oocytes obtained from different development stages of the follicles and follicular fluid levels were determined. Correlations between size measurements and cell counts as well as the diameter of antral follicles and oocytes were also investigated. The results indicated that, while expanding in size, follicle numbers decreased with a greater atretic proportion. Granulosa cells increased in numbers continuously and remained unchanged beyond the size of 200 ${mm}^3$ in non-atretic follicles, whereas a sudden drop of granulosa counts was observed in atretic follicles. Follicular fluid, on the other hand, linearly increased its volume with follicle size and differed little between those of non-atretic and atretic follicles. Diameters of oocytes in non-atretic follicles increased to its maximum when follicles expanded to 150 ${mm}^3$ and maintained its size during later follicular expansion. It is concluded that, for in vitro culture, the optimal size of porcine follicle should be between 150 to 180 ${mm}^3$if they are collected from pre-pubertal gilts of marketing size slaughtered in an abattoir.

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.323-331
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• 1999

The effects of insulin-like growth factor-I (IGF-I) and cumulus cells during in vitro maturation in porcine oocytes were examined. When follicular oocytes were cultured in medium with different concentrations of IGF-I, maturation rates were 60, 61 and 62 and 72% for 0, 15 and l0ng/$m\ell$ IGF-I. In medium with 10ng/$m\ell$ IGF-I, maturation rates were not significantly difference between oocytes with (68%) and without (52%) cumulus cells during the culture. In medium with-out IGF-I, however, the maturation rates in oocytes with cumulus cells (63%) was significantly (P＜0.05) higher than oocytes without cumulus cells (32%). On the other hand, when IGF-I was added for first 24 h period or later 24 h period of culture, maturation rates were higher in oocytes with (61 and 49%) that than without (49 and 45%) cumulus cells. In experiment used medium without fetal calf serum (FCS) and porcine follicular fluid (PFF), the maturation rates in oocytes with cumulus cells for 48 h (48 and 67%) or first 24 h (46 and 63%) period after culture were significantly (P＜0.01) higher than in oocytes without cumulus cells (16 and 18%) in the presence or absence of IGF-I. These results indicated that cumulus cells is essential on maturation in vitro in porcine oocytes, but IGF-I can promote oocytes maturation of oocytes without cumulus cells in medium with FCS and PFF.

• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.85-91
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• 2009

In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups of oocytes were cultured in TCM 199 supplemented with 10% FBS, 10% pFF or 5% FBS + 5% pFF, respectively. After 44 h maturation, oocytes with the first polar body were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}sec$. Also, matured oocytes of four groups were reconstructed and fused. Reconstructed embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. The oocytes matured in the medium supplemented with FBS or/and pFF showed significantly higher maturation rates (64.0 vs. 73.9 to 85.2%). In PA embryos, cleavage rates (89.7 vs. 77.1 to 86.6%) and blastocysts rates (30.0 vs. 16.2 to 26.2%) were significantly higher in pFF group (p<0.05). In NT embryos, there was no difference among treatments in cleavage rate, but the blastocyst rates (28.5 vs. 15.5 to 24.6%) were significantly higher in pFF group (p<0.05). The apoptosis rate was significantly higher (p<0.05) in the control than other groups (10.8 vs. 4.9 to 8.2% for PA, 3.1 vs. 0.5 to 1.3% for NT). In order to select the comparable oocyte to in vivo oocytes, each group of oocytes was stained with Brilliant cresyl blue (BCB) after 42h maturation. The matured oocytes were separated according to color of cytoplasm; stained group (BCB+) and unstained group (BCB-). The oocytes matured in the presence of FBS or/and pFF showed significantly higher staining rates (70.3 to 72.7 vs. 35.1%) (p<0.05). To verify the fact that the supplementation of FBS or/and pFF can increase the maturation rates, cdc2 kinase activity, the catalytic subunit of MPF, was determined. The cdc2 kinase activity of the oocytes matured in the medium supplemented with FBS or/and pFF was significantly higher than control group (6.7 to 9.3 vs. 3.8). In conclusion, the supplementation of FBS or/and pFF can support in vitro maturation rate of porcine oocytes through the increment of cdc2 kinase activity level in the cytoplasm.

• Korean Journal of Animal Reproduction
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• v.24 no.2
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• pp.155-161
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• 2000

The morphological changes on nuclear phase of the germinal vesicle of porcine follicular oocytes during in vitro culture were examined. The high rates (75~77%) of the oocytes collected from follicles of 1~2mm or 6~100mm in diameter were at the GV-I to GV-II stages. When oocytes with or without cumulus cells after collection from follicles of 2~6mm in diameter were cultured for 5 h, the rates of oocytes at GV-IV to GV-Ⅵ stages were higher in oocytes with (52%) than in oocytes without (30%) cumulus cells. After 1 h of oocyte culture, there was no differences in the distribution of GV-IV to GV - Ⅵ stages in the media with or without catalase, xanthine and catalase＋xanthine. After 5 h of culture, however, the distribution of GV-IV to GV-Ⅵ stages were 46, 69, 69 and 70% for medium with none, catalase, xanthine and catalase＋xanthine. The highest rate of GVBD was also observed in the medium with catalase＋xanthine (6%). These results indicate that exposure of porcine follicular oocytes to catalase＋xanthine excels maturation to GV stage and enhances oocyte nuclear maturation.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.205-211
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• 2019

In this study, we investigated the possibility of using mouse embryonic stem cell conditioned medium (ESCM) and embryonic stem cell medium (ESM) for in vitro maturation in the efficient in vitro production of blastocysts from porcine follicular oocyte. Depending on the concentration of supplement of ESCM added to the NCSU-23 solution did not affect 2-cell development rates and blastocysts development. However, in particular, the survival rate (10 days of culture) of blastocyst was significantly higher than that of the control group as the additive concentration (30%) increased (p < 0.05). The survival rate of blastocysts showed a similar tendency even with addition of ESM (30%) alone. On the other hand, the duration of the addition of these additives during IVM (0-44 h) was that the IVM I period (0-22 h) were more effective than the IVM II period (22-44 h). Thus, the effect of these additives is probably due to the combination of the various physiologically active substances of ESCM or the appropriate amino acids and vitamins of ESM. In particular, these additives were more effective during the first half (IVM I) of in vitro maturation. In summary, optimization of ESCM or ESM supplementation may improve in vitro maturation of porcine oocyte and affect developmental competency. Therefore, if more efficient methods of adding ESCM or ESM to basal culture medium can be developed during in vitro maturation of porcine follicle oocytes, high quality blastocysts will be developed from low porcine follicular oocyte compared to other domestic animals.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.4
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• pp.322-322
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• 2019

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.7
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• pp.950-955
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• 2012

This study was conducted to determine how the isolation method of the porcine preantral follicles influenced the following follicular growth in vitro. Mechanical and enzymatical isolations were used for retrieving the follicles from prepubertal porcine ovaries, and in vitro-growth of the follicles and the expression of folliculogenesis-related genes were subsequently monitored. The enzymatic retrieval with collagenase treatment returned more follicles than the mechanical retrieval, while the percentage of morphologically normal follicles was higher with mechanical retrieval than with enzymatic retrieval. After 4 days of culture, mechanically retrieved, preantral follicles yielded more follicles with normal morphology than enzymatically retrieved follicles, which resulted in improved follicular growth. The mRNA expression of FSHR, LHR Cx43, DNMT1 and FGFR2 genes was significantly higher after culture of the follicles retrieved mechanically. These results suggest that mechanical isolation is a better method of isolating porcine preantral follicles that will develop into competent oocytes in in vitro culture.

• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.121-125
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• 2005

The present study was performed to determine the ability of canine oocytes to achieve nuclear maturation according to oocyte diameter and different culture environments. All of the collected oocytes were classified by grade 1 to 3 and by their diameters such as $<100{\mu}m,\;<100{\mu}m\;to\;<110{\mu}m,\;<110{\mu}m,\;to\;<120{\mu}m,\;>120{\mu}m,$. Oocytes were cultured in culture medium supplemented with $10\%\;FBS,\;0.4\%\;BSA,\;10\%$ porcine follicular fluid (pFF), $10\%$ canine serum (CS), or $10\%$ canine estrus serum (CES). The mean number of oocytes recovered from estrus status ovaries was significantly higher than that of anestrus status ovaries (p<0.01). The maturation rate of grade 1 oocytes $(>120{\mu}m)$ was significantly higher than that of the other groups (p<0.05). Nuclear maturation to MI to MII in diameter of $>110{\mu}m$ groups was significantly higher than that in $<100{\mu}m$ group (p<0.05). The oocytes cultured in $10\%$ FBS­supplemented group were significantly higher rate of GVBD compared to the other supplemented groups (p<0.05), and oocytes maturation to MI to MII in $10\%$ FBS-, $0.4\%$ BSA-, and $10\%$ pFF-supplemented groups were significantly higher than those in $10\%$ CS-supplemented group (p<0.05). Based on these results, the estrus status and the size of oocyte affect positively to improve nuclear maturation of canine immature oocytes in vitro. Among several protein sources, porcine follicular fluid was the most effective supplementation to culture medium to achieve higher in vitro maturation rate.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.237-242
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• 1995

Porcine follicular oocytes matured in culture were inseminated with frozen-thawed spermatozoa. When the oocytes were inseminated in the medium with oviductal epithelial cell monolayer, the penetration rates higher in those with (4.1, 31.7, 45.1, 54.5 and 69.4%) than without cells (0, 17.1, 34.8, 45.2 and 58.9%) at 4, 8, 12, 16 and 20 h after insemination. The proportions of polyspermy in penetrated oocytes in medium with or without cells increased with time of examine. In another experiment, the penetration rate was higher without (57.6%) than with (19.6~24.1%) preincubation of spermatozoa for 1~4 h in medium. However, when the oocytes were inseminated with spermatozoa preincubated for 1~2 h, the penetration rates significantly higher (P<0.05) in those with (65.6 and 55.9% for 1 and 2 h) than without (24.1 and 20.6% for 1 and 2 h) oviductal epithelial cell monolayer. On the other hand, the proportions of polyspermy decreased with time of spermatozoa preincubation. These results indicate the significant advantages of the spermatozoa preincubation with oviductal epithelial cell monolayer for 1 and 2 h to maintain penetration potential during in vitro fertilization in the porcine.