• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.289-297
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• 1996

This study was conducted to investigate the effects of maturaton medium and porcine follicular fluid on in vitro maturation of porcine follicular oocytes. The results obtained are as follows; 1. When the oocytes were cultured for 42 hours, the maturation rate was significantly (P<0.05) higher in TCM-HEPES(75.5%) than Ham's F-10(60.9%) and mKRB(60.7%) medium. The optimal medium for the maturation of porcine oocytes in vitro was the TCM-HEPES medium. 2. When the oocytes were cultured for 42 hours in TCM-HEPES medium with 10, 20 and 30% of porcine follicular fluid(pFF), the maturation rates of porcine oocytes were 69.1, 65.6 and 63.3%, respectively. The maturation rate(51.4%) of oocytes cultured without pFF was significantly(P<0.05) lower than that of oocytes cultured with pFF. 3. The maturation rates of porcine oocytes cultured for 42 hours in TCM-HEPEs medium with 3 different porcine follicular fluid treatments were 68.6%(centrifused), 72.3%(filtered) and 73.1%(heat treated), respectively. The maturation rate(49.4%) of control group without pFF treatment was significantly(P<0.05) lower than that of oocytes cultured with pFF treatment.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.203-210
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• 2000

The objective of this study was to identify a follicular fluid ingredient inhibiting the cumulus oocyte complex (COC) expansion. Thus, follicular fluid or liquid chromatographic fractions of follicular fluid was supplemented in COC culture medium. And COCs were incubated for 48 hours to investigate about cumulus expansion and also the first polar body extrusion. The results obtained were as follows; 1. The fluid of medium follicle significantly inhibited the COC expansion. 2. The fluid of large follicle inhibited the COC expansion. 3. Follicular fluid showed six major fractions at retention volumes (RVs) 1.83, 1.91, 2.15, 2.34, 2.53 and 2.74 ml after separation with Superose 12 column. Of the major fractions, fractions RV2.15, RV2.34, RV2.53 and RV2.74 inhibited both COC expansion and polar body extrusion. Especially, fractions of RV2.15 and RV2.53 significantly inhibited COC expansion, oocyte denudation and polar body extrusion. In conclusion, porcine follicular fluid contained a COC expansion inhibiting ingredient (CEI) that may be contained largely in fractions RV2.15 and RV2.53. And CEI may inhibit oocyte maturation by inhibition of oocyte denudation and extrusion of the first polar body.

• Korean Journal of Animal Reproduction
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• v.15 no.3
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• pp.221-224
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• 1991

This study was undertaken to evaluate the effect of rabbit peritioneal fluid(rPF) on in vitro maturtion of porcine follicular oocytes. From does 20h after hCG injection, rPF was aspirated aseptically at laparatomy, and then centrifuged, filtrated, and preincubated immediately for 12h. Porcine follicular oocytes isolated from ovaries of slaughtered animals were incubated in TCM-HEPES＋10% FCS, TCM-HEPES＋rPF(v/v, 50/50), or rPE only and examined the nuclear maturation after aceto-orcein or hochest staining. After identifying the optimal incubation time, this experiment was repeated for 5 times. Under the TCM-HEPES containing hormones and serum codition, the time range of porcine follicular oocyte maturation was 38 to 44 hours and the optimal time of maturation of follicular oocyte in vitro was 42 hour cultivation, respectively. The maturatin rates(89.4% and 92.7%) of porcine follicular oocytes cultured in the media with 50% rPF or only rPF were signifciantly higher thanthat (84.6%) of oocytes cultured with TCM-HEPES, respectively. These results suggest that the unknown components(s) of rPF promoted in vitro maturation of porcine follicular oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.13 no.1
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• pp.39-51
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• 1986

This experiment has been done to evaluate the relationship between the follicular atresia and the protein patterns on electrophoreais of the follicular fluids in porcine ovary. The protein concentration of the follicular fluids was lower than that of serum, and gradually decreased as the follicle siae became larger. The number of protein bands of follicular fluid on electrophoresis was less than that of serum, and gradually increased as follicle size became larger. Three specific bands were detected on disc PAGE and one band(M W. 75,000) on SDS PAGE in the follicular fluids, while not in serum. One band (A) at ${\beta}$-globulin region on disc PAGE became heavier, as follicles became atretic. Two bands less than(M. W. 20,000) were detected only in the large follicular fluid. Another band(M. W. 43,000) was not detected in necrotic group, whereas all other groups showed it. It could be concluded that the component and composition of the proteins follicular fluids changes according to the follicular size during atresia. Therefore detection of the changing pattern of proteins in the follicular fluid can be used as a basic criterion for the identification of follicular atretic stage.

• Clinical and Experimental Reproductive Medicine
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• v.8 no.1
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• pp.1-12
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• 1981

It has been already suggested that specific macromolecules in follicular fluid produced by granulosa cells may play a role in suppressing further meiotic maturation of the oocytes. In general, the search for specific macromolecules in follicular fluid using immunological methods has not been rewarding. These studies were designed, by applying more effective immunological methods than conventionally employed, (l) to identify whether some unknown macromolecules are present in the porcine follicular fluid or not, and (2) to clarify the relationship between the oocytes and the specific macromolecules in the follicular fluid. The results obtained were as follows; (1) porcine follicular fluid contained two specific proteins, which were not present in pig plasma and serum. (2) each of two proteins showed electrophoretically fast alpha-globulin and beta-globulin mobilities. (3) these proteins seemed to have inhibitory effect on the maturation of mouse oocytes in vitro. From these results, it can be assumed that pig follicular fluid contains specific proteins which seem to be intra-follicular inhibitor(s) of oocyte maturation.

• Korean Journal of Animal Reproduction
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• v.19 no.4
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• pp.251-258
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• 1996

These studies were carried out to investigate the effect of gonadotropins (GTH), fetal calf serum (FCS), porcine follicular fluid (pFF) and FCS and pFF fractions obtained by the gel filtration on in vitro maturation of porcine follicular fluid. When the oocytes were cultured in TCM-199, the maturation rate was higher in pFF than in FCS in both with or without GTH and in pFF the maturation rate was higher in with GTH than in without GTH. In case of without GTH, pFF increased maturation rates in TCM-199, but not in Whitten's medium (WM). When the oocytes were cultured in WM supplemented with FCS fractions, the maturation rate(51.6%) of oocytes was significantly (P<0.05) higher in fraction B (about 30∼70 kDa) than in control, FCS and other fractions. When oocytes were cultured in WM supplemented with pFF fractions, fractions B (about 30∼70 kDa) and D (about 1∼10 kDa) were significantly (P<0.05) higher than in control, pFF and other fractions. In conclusiion, the addition of gonadotropins into the maturation media was effective for oocyte maturation. The addition of pFF was more effective than addition of FCS for maturation of porcine oocytes in vitro. And fraction B from FCS and fractions B and D from pFF was effective for oocyte maturation.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.103-111
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• 1993

These experiments were undertaken to establish the optimal culture systems for in vitro maturation, fertilization and subsequently embryonic development of porcine immature follicular oocytes isolated from the ovary of slaughtered pigs. 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) for 42hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were prepared forfertilizaing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${mu}ell$ of 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 culture media. The fertilization rates of in vitro matured follicular oocytes cultured in B. O., TCM-HEPES, m-KRB, and TALP-II media were 61.3%, 83.0%, 88.9% and 89.2%, respectively. In addition, the polyspermy rates were 60.7%, 66.5%, 53.8%, and 43.9%, respectively. These data indicated that the highest of fertilization and the lowest of polyspermy rate was shown in TALP-II medium. Spermatozoa capacitated by caffeine, heparin, and percoll density gradient treatment in the 4 different media, the fertilization rates were 33.0~57.2%, 39.9~90.2%, and 52.6~92.8%, respectively, showing the lowest rate in caffeine treatment. The development rate of follicular oocytes, fertilized with the spermatozoa capacitated by caffeine, heparin, and percoll gradient in the TALP-II medium, upto 2 to 4-cell stages were 32.6%, 74.5% and 70.9%, respectively. Finally, fertilization rates of follicular oocytes cultured with follicular fluid containing medium from 10 to 100% were 61.2~94.1% and the rates (90~94%) with 10~20% follicular fluids were significantly higher than those (85.3%) of cultured in the media without follicular fluid. In addition, the rates of pronucleus formation were also higher in follicular fluid treated group (73.1~83.0%) than those (64.7%) of oocytes cultured without follicular fluid. The highest fertilization and pronucleus formation rates was found in oocytes cultured with 10% follicular fluid. These results suggest that the addition of heparin or percoll density gradient method is better capacitation method. Furthermore, the addition of porcine follicular fluid to the fertilization medium may improve the fertilization rates and formation of pronucleus.

• Korean Journal of Veterinary Research
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• v.32 no.1
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• pp.135-142
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• 1992

This experiment was carried out to establish an effective technique of in vitro maturation of porcine follicular oocytes. Porcine ovaries were collected from an abbatoir and delivered to the laboratory in phosphate buffered saline in an hour. Immatured follicular oocytes were collected from the ovaries and divided into groups by the size of follicles and by the attachment of granulosa cells. The follicular oocytes were cultured in m-KRB solution supplemented with FCS(10%), follicular fluid(10%) or hormones of PMSG(10IU/ml), hCG(10IU/ml ) and $estradiol-17{\beta}(1{\mu}g/ml)$ for 48 hours at $39^{\circ}C$ under an atmosphere of 5% $CO_2$ in air. The results are as follows ; 1. The mean recoveration rate of follicular oocytes was 61.8%. 2. The maturation rate was significantly(p<0.05) higher when the oocytes were collected from large-sized follicles and under good state of granulosa cell attachment. 3. The maturation rate was significantly(p<0.01) promoted when the follicular oocytes were cultured in m-KRB solution supplemented with follicular fluid(74.8%) or hormones and fetal calf serum(70.6%).

• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.23-30
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• 1990

These studies were carried out to investigate the effects of fetal calf serum(FCS), estrous porcine serum(EPS), porcine follicular fluid(PFF), hormone and matured cumulus cell(MCC) on in vitro maturation and fertilization of porcine follicular oocytes. The ovaries and testes were obtianed from slaughtered Landrace sow and boars, respectively. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3~5 mm and the semen were prepared from boar's epididymal cauda. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, EPS, PFF and MCC for 48hrs. in a incubator with 5% CO2 in air at 36$^{\circ}C$ and then matured oocytes were again cultured for 18~20 hrs. with $1.5\times$106/ml motile capacitated sperm in the modified Tyroide solution containing 100$\mu\textrm{g}$/ml of heparin. The results obtained in these experiments were summarized as follows : 1. The maturation and fertilization rate of the follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS and PMSG＋HCG were 55.6~64.5% and 33.3~37.1%, respectively. 2. The maturation and fertilization rate of the follicular oocytes cultured in the TCM-199 medium supplemented with 20% EPS and PMSG＋HCG were 50.0~55.0% and 30.3~33.3%, respectively. 3. The maturation rate(59.0~64.2%) and fertilization rate(34.8~39.3%) of follicular oocytes cultured in TCM-199 medium supplemented 20% FCS and 50% PFF were higher than those of follicular oocytes cultured in TCM-199 medium supplemented with 5%, 10% and 15% FCS and 10% and 50% PFF. 4. The maturation rate(60.0%) and fertilization rate(40.0%) of follicular oocytes cultured in TCM-199 medium supplemented with 20% FCS and granulosa cell (1$\times$106/ml) were significantly higher than those of fiollicular oocytes cultured in TCM-199 medium supplemented with 5%, 10% and 15% FCS and granulosa cell.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2006.10a
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• pp.58-58
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• 2006