• 한국가축번식학회지
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• 제18권1호
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• pp.25-29
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• 1994

This study was carried out to investigate the in-vitro fertilization rate survival rate of rapidly frozen porcine immature embryos. The porcine embryos following dehydration by cryoprotectants containing sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water bath. Survival rate was defined as development rate on vitro culture or FDA test. The results are summarized as follows : 1. The in vitro fertilization rate of all frozen immature oocytes(6.7~26.7%) was very low, 35.0% of unfrozen oocytes and the rate of immature oocytes was very higher than that of mature oocytes. 2. The survival rate of all frozen immature oocytes(10.3~25.0%, 13.3~30.0%) was very low, 45% of unfrozen oocytes and the rate of immature oocytes was slightly higher than that of mature oocytes.

• 한국수정란이식학회지
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• 제27권3호
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• pp.163-169
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• 2012

The 3-isobutyl-1-methylxanthine (IBMX) is non-selective phosphodiesterase and is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte. The present study was conducted to analyze: (1) nuclear maturation (examined by the Hoechst staining), (2) whether cytoplasmic maturation (examined by the intracellular glutathione (GSH) concentration) of porcine oocytes is improved during meiotic arrest after prematuration (22 h) with IBMX. Before in vitro maturation (IVM), oocytes were treated with 1 mM IBMX for 22 h. After 22 h of pre-maturation, the higher rate of IBMX treated group oocytes were arrested at the germinal vesicle (GV) stage (42.3%) than control IVM oocytes (10.1%). It appears that the effect of IBMX on the resumption of meiosis has shown clearly. In the end of IVM, the reversibility of the IBMX effect on the nuclear maturation has been corroborated in this study by the high proportions of MII stage oocytes (72.5%) reached after 44 h of IVM following the 22 h of inhibition. However, intracellular GSH concentrations were lower in the oocytes treated with IBMX than the control oocytes (6.78 and 12.94 pmol/oocyte, respectively). These results demonstrate that cytoplasmic maturation in porcine oocytes pre-treated with IBMX for 22 h did not equal that of control oocytes in the current IVM system. These results indicate that pre-maturation with IBMX for 22 h may not be beneficial in porcine IVM system.

• 한국가축번식학회지
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• 제19권4호
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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.

• 한국가축번식학회지
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• 제20권3호
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• pp.315-322
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• 1996

The study was conducted to determine the optimal glucose, superoxide dimutase(SOD) and catalase levels during the in vitro culture of porcine oocytes matured and fertilized in vitro for morulae and blastocyst development. Oocytes were cultured for 0~8 days in TCM-199 medium supplemented with 20% FCS, different glucose, SOD and catalase levels. The results are summairzed as follows ; 1. The in vitro developmental rates of porcine oocytes cultured in TCM-199 medium containing 0.1, 0.3, 0.5, 1.0, 3.0 mM glucose levels 0~3 and 0~8 days after insemination were 22.8, 24.2, 21.9, 20.0, 12.1 and 21.9, 26.7, 25.0, 22.6, 16.7%, respectively. 2. The in vitro developmental rates of porcine oocytes cultured in TCM-199 medium containing 100, 200, 300, 500 $\mu\textrm{g}$/ml SOD levels 0~3 and 0~8 days after insemination were 16.7~23.3 and 16.7~25.0%, respectively. High levels of SOD(500 $\mu\textrm{g}$/ml) significantly reduced the rates of molurae and blastocysts stage(P<0.05). 3. The in vitro developmental rates porcine oocytes cultured in TCM-199 medium containing 100, 200, 300, 500 $\mu\textrm{g}$/ml catalase levels 0~3 and 0~8 days after insemination were 18.8~26.7 and 19.4~28.1%, respectively, and there was significant differences on the development to the molurae and blastocysts stage among the cumulus cells and glucose levels.

• 한국가축번식학회지
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• 제22권3호
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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.

• 한국가축번식학회지
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• 제26권4호
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• pp.361-368
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• 2002

The onset of pronucleus formation and DNA synthesis in porcine oocytes following the injection of porcine or murine sperm was determined in order to obtain insights into species-specific paternal factors that contribute to fertilization. After 44h in vitro maturation, spermatozoa was injected into the cytoplasm of oocytes. After injection, all oocytes were transferred to NCSU23 medium and cultured at 39'E under 5% CO2 in air. Similar frequencies of oocytes with female pronuclei were observed after injection with porcine sperm or with murine sperm. In contrast, male pronuclei formed 8 to 9 h following the injection of porcine sperm, and 6 to 8 h following the injection of murine sperm. After pronucleus formation maternally derived microtubules were assembled and appeared to move both male and female pronuclei to the oocyte center. A few porcine oocytes entered metaphase 22 h after the injection of murine sperm, but normal cell division was not observed. The mean time of onset of S-phase in male pronuclei was 9.7 h following porcine sperm injection and 7.4 h following mouse sperm injection. These results suggested that DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, and the sperm nuclear decondensing activity and microtubule nucleation abilities of the male centrosome are cell cycle dependent.

• 한국수정란이식학회지
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• 제12권3호
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• pp.315-324
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• 1997

This study was undertaken in an effort to develop a cryopreservation system of immature and mature porcine oocytes. For this aim, the experiments were designed to examine the effect of cryoprotectants and equdibration time on the viability of frozen-thawed oocytes by using trypan blue(TB) and fluorescene diacetate(FDA) test. The viability of frozen immature oocytes evaluated by TB test was slightly higher than that of frozen mature oocytes. The viability(25.O%) after IVM of frozen-thawed immature oocytes greatly decreased that(42.9%) of oocytes just after thawing, but it was higher than frozen-thawed mature oocytes(15.8%). When immature oocytes were equilibrated for 10, 20 and 30 minutes before freezing the oocyte viability was 20.0, 31.3 and 42.9%, respectively. There was a tendency for long equilibration before oocyte freezing to be more effective for the immature oocytes and a short equilibration time for mature oocytes. Although there was no difference in viability index of frozen oocytes hetween the viability test methods, the index of TB test was slightly higher than that of FDA test. The viability(FDA test) of frozen-immature oocytes with 3 different crtoprotectants was 22.2% for propylene glycol(PG), 9.3% for polyehtylene glycol(PEG) and 65.6% for PG+PEG, in which PG+PEG was more protective against freezing effect.

• 한국가축번식학회지
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• 제22권2호
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• pp.119-126
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• 1998

This study was conducted to find out the recovery rate of oocyte according to the different size of follicles from porcine ovaries, and the effect of in vitro maturation of porcine immature oocyte at the different transportation temperature of ovaries from slaughter house. The results obtained were summarized as follows : 1. The number of follicles per ovary was 22.5. The number of A-and B-typed oocytes(type A: cumulus-enclosed oocyte, type-B : corona-enclosed oocyte) per ovary was 2.4. The proportion of A-and B-typed oocytes was 29.6% of the total recovery oocytes. 2. When the immature oocytes were cultured for 36, 40, 44 and 48 h at 5$^{\circ}C$ transportation temperature of ovary, the germinal vesicle breakdown(GVBD) rates of porcine oocytes were 32.5, 28.2, 22.6 and 25.9% respectively. There were no significant differences between all the culture time for GVBD. Especially, most of oocytes were observed to arrest the development beyond germinal vesicle(GV) stage. 3. When the immature oocytes were cultured for 36, 40, 44 and 48 h at $25^{\circ}C$ transportation temperature of ovary, the GVBD rates were 81.0, 90.0, 91.7 and 92.9%, and the maturation (Met-II) rates were 51.2, 78.8, 76.2 and 78.6%, respectively. 4. When the immature oocytes were cultured for 36, 40, 44 and 48 h at 38$^{\circ}C$ transportation temperature of ovary, the GVBD rates were 93.9, 96.5, 96.5 and 95.3%, and the maturation rates were 62.2, 88.4, 84.7 and 86.0%, respectively. 5. The above results showed that the maturation rates of immature oocytes between $25^{\circ}C$ and 38$^{\circ}C$ transportation temperature of ovary did not differ significantly.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.221-225
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• 2011

This study was undertaken to evaluate the relationship between in vitro maturation and plasminogen activators (PAs) activity on porcine cumulus-oocytes complexes (COCs) exposed to oxidative stress. When COCs were cultured in maturation medium with hydrogen peroxide ($H_2O_2$), the proportion of the germinal vesicle breakdown (GVBD) and oocytes maturation were decrease with addition of $H_2O_2$, and were significantly (p<0.05) lower in medium with 0.1 mM $H_2O_2$ than control group. Also, the rate of degenerated oocytes was increased in as $H_2O_2$ concentration in eased. When COCs were cultured for 48 h, three plasminogen-dependent lytic bands were observed: tissue-type PA (tPA); urokinase-type PA (uPA); and tPA-PA inhibitor (tPA-PAI). PA activity was quantified using SDS-PAGE and zymography. When $H_2O_2$ concentration was increased, tPA and tPA-PAI activities also increased in porcine oocytes cultured for 48 h, but not uPA. In other experiment, embryos were divided into three groups and cultured in (1) control medium, (2) control medium with 1.0 mM $H_2O_2$ and (3) control medium with 1.0 mM $H_2O_2$ along with catalase in concentrations of 0.01, 0.1, and 1.0 mg/ml, respectively. $H_2O_2$ decreased the rate of GVBD and maturation in porcine COCs but catalase revealed protective activity, against oxidative stress caused by $H_2O_2$. In this experiment, tPA and tPA-PAI activities were higher in media with 1.0 mM $H_2O_2$ alone. Increasing concentration of catalase decreased tPA and tPA-PAI activities in porcine oocytes. These results indicate that the exposure of porcine follicular oocytes to ROS inhibits oocytes maturation to metaphase-II stage and increase the oocytes degeneration. Also, we speculated that increased ROS level may trigger tPA and tPA-PAI activities in porcine oocytes matured in vitro.

• Journal of Animal Science and Technology
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• 제66권5호
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• pp.905-919
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• 2024

Porcine oocytes undergo in vitro maturation (IVM) for 42-44 h. During this period, most oocytes proceed to metaphase and then to pro-metaphase if the nucleus has sufficiently matured. Forty-four hours is sufficient for oocyte nuclear maturation but not for full maturation of the oocyte cytoplasm. This study investigated the influences of extension of the IVM duration with rapamycin treatment on molecular maturation factors. The phospho-p44/42 mitogen-activated protein kinase (MAPK) level was enhanced in comparison with the total p44/42 MAPK level after 52 h of IVM. Oocytes were treated with and without 10 µM rapamycin (10 R and 0 R, respectively) and examined after 52 h of IVM, whereas control oocytes were examined after 44 h of IVM. Phospho-p44/42 MAPK activity was upregulated the 10 R and 0 R oocytes than in control oocytes. The expression levels of maternal genes were highest in 10 R oocytes and were higher in 0 R oocytes than in control oocytes. Reactive oxygen species (ROS) activity was dramatically increased in 0 R oocytes but was similar in 10 R and control oocytes. The 10 R group exhibited an increased embryo development rate, a higher total cell number per blastocyst, and decreased DNA fragmentation. The mRNA level of development-related (POU5F1 and NANOG) mRNA, oocyte-apoptotic (BCL2L1) genes were highest in 10 R blastocysts. These results suggest that prolonged IVM duration with rapamycin treatment represses ROS production and increases expression of molecular maturation factors. Therefore, this is a good strategy to enhance the developmental capacity in porcine oocytes.