• Development and Reproduction
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• v.8 no.2
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• pp.119-122
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• 2004

It is well known that unidentified factors in sera, hormones and growth factors promote the proliferation of granulosa cells and nuclear maturation of bovine COCs in vitro. Attemps had been developed the simple composition of culture media and similar system to in vivo conditions has been applied. In the present study, we investigated the effect of TGF-${\beta}$ on in vitro maturation and in vitro development of Hanwoo COCs. When the COCs were matured in TCM 199 containing 0.1, 1 or 10 ng/ml TGF-${\beta}$ for 24 hrs, metaphaseⅡ of COCs were obtained 95.8%, 100% of matured COCs, respectively and there were no differences among the concentrations of TGF-${\beta}$. Matured COCs with TGF-${\beta}$ cultured in maturation medium after in vitro fertilization, developmental rate to blastocyst were 0~0.8%. Matured COCs with TGF-${\beta}$ were cultured in TCM 199+10% FBS, 0.8% BSA, 0.1% PVA, blastocyst formation were showed in 12.4%, 12.8%, 8.5% of those and cultured in IVMD or IVD without serum were 38.4%, 34.8%, respectively. There were significant differences among the media (P<0.05). TGF-${\beta}$ is available for i vitro maturation of bovine COCs, but further investigation would be need for finding the synergistic autocrine/paracrine fashion of other growth factors in early bovine development.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.191-197
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• 2016

Historically, Korea old cattle had been consisted with various lines of coat color brindle, black and white-brown breeds or more. The two rare lines of black and white coat color are maintained for animal resources and preserved critically. The present study was carried out to evaluate potential usage of cysteamine supplementation during in vitro matration (IVM) and in vitro culture/production of embryo (IVP) by transvaginal ultrasound-guided follicle aspiration (Ovum Pick-Up: OPU) for the establishment of cryo-banking system. Immature slaughterhouse-derived cumulus-oocyte complexes (SL-COCs) were matured in IVM medium supplemented with 0, 0.1, 0.3 or 0.9 mM cysteamine, and then cultured in mSOF-BAS for 8 days after in vitro fertilization. The treatment of 0.1 mM cysteamine on SL-COCs showed higher rate of blastocyst, so OPU-derived COCs from rare breeds were matured in TCM media supplemented with or without 0.1 mM cysteamine, FSH and 5% FBS. The embryos were evaluated their developmental stages on day 8. During IVM, cysteamine treatment significantly increased the embryo production rate of slaughterhouse-derived COCs (19.6% vs. 30.5%). The presence of cysteamine during IVM of OPU-derived COCs from rare Korean cattle breeds (albino white and black line) also increased embryo production rates than those from SL-COCs (27.4% vs. 41.9% and 36.4%). With these results, cysteamine treatment during IVM is one of key factors IVP of blastocysts to establish banking system of endangered rare Koarean cattle with OPU derived transferable blastocysts.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.327-333
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• 2015

The objectives of the present study were to select an effective basic medium including its hormone and protein supplementation for IVM of oocytes of indigenous zebu cows. The ovaries of cows were collected from slaughter house and the follicular fluid was aspirated from 2 to 8 mm diameter follicles. The COCs with more than 3 cumulus cell layers and homogenous cytoplasm were selected for maturation. The oocytes were matured in media for 24 hrs at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The maturation of oocytes was evaluated by examining the presence of first polar body under microscope. An efficient basic medium was determined after culturing COCs in either TCM 199 or SOF medium in Experiment 1. An efficient hormone supplementation was determined after culturing COCs in either FSH or gonadotrophin supplemented TCM 199 in Experiment 2. An efficient protein supplementation was determined after culturing COCs in either FBS or Oestrous cow serum (OCS) supplemented TCM 199 in Experiment 3. The oocyte recovery rate per ovary was 3.35. The overall rate of IVM was 74.6%. The maturation rate was $75.5{\pm}3.9$ and $62.2{\pm}20.2%$ in TCM and SOF medium, respectively (P>0.05). The maturation rate of oocytes was significantly higher ($76.6{\pm}13.2%$) in FSH supplemented medium than gonadotrphin supplemented counterpart ($69.7{\pm}10.8%$) (P<0.05). The maturation rates of oocytes were $81.7{\pm}12.9$ and $85.7{\pm}12.7%$ in medium supplemented with FBS and OCS, respectively (P>0.05). In conclusions, both TCM 199 and SOF supplemented with either FBS or OCS, and FSH may be used as medium for IVM of indigenous zebu oocytes in Bangladesh.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.141-145
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• 2012

The present study was conducted to develop a simple method for porcine oocyte maturation without $CO_2$ regulation. In experiment 1, we evaluated that the effect of $CO_2$ non-supplement on porcine oocyte maturation. Cumulusoocyte complexes (COCs) were collected from 2~6 mm follicles and divided into three groups (Control, tube-$CO_2$, and tube-non-$CO_2$). For control, COCs were cultured in 4-well multidish in a $CO_2$ incubator. For tube-$CO_2$, COCs were cultured in a round-bottom tube in a $CO_2$ incubator, and for tube-non-$CO_2$, COCs were cultured in a round-bottom tube sealed tightly without $CO_2$ supplement in a dry incubator. The proportion of oocytes reached to metaphase II (M-II) was not significantly different among three groups (87.9% to 91.4%). In experiment 2, we evaluated the effect of $CO_2$ non-supplement during oocyte maturation on development of embryos. Oocytes with a polar body were divided into two groups (Control and tube-non-$CO_2$) and applied 1.1 kV/cm or 1.2 kV/cm voltages for parthenogenetic activation. After activation, embryos were cultured for 6 days and examined the development. The proportion of embryos cleaved was not significantly different among treatment (86.3% to 91.5%). The proportion of embryo reached to blastocyst stage was not significantly different among treatment (13.9% to 25.2%). The cell number of blastocysts was not significantly different among treatment (29.0 to 32.4). In conclusion, oocytes cultured in a dry incubator without $CO_2$ supplement have enough competence to development after parthenogenetic activation. These results would be useful for transporting oocytes or embryos a long distance.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.177-182
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• 2014

Feline ovulation time after LH surge have not been defined because its LH surge is occurred by several times of coital vaginal induction and cat has relatively longer time between LH surge and ovulation compared with other mammalian species. This study was performed to investigate the feline ovulation time after LH surge that was induced by hCG injection for superovulation with PMSG. For superovulation, all cats were received an initial injection of PMSG (200 IU, i.m.) followed 80 hrs later with an injection of hCG (200 IU, i.m.). And then, sampling of both ovaries was surgically performed at each 6 different times (10, 18, 22, 26, 29, and 32 hrs) after hCG injection. Cumulus-oocyte-complexes (COCs) were collected from 2 sides of oviducts and ovaries were fixed for ovarian histology. Total 38 COCs were collected only at hCG 32 hrs and no COCs were shown at earlier 5 times. However, in the ovarian histology, corpus haemorrhagicum or corpus luteum was not shown in all groups including ovary at hCG 32 hrs that COCs were collected. In conclusion, it was suggested that feline ovulation was occurred at 29~32 hrs after LH surge and taken relatively long time for CL formation after ovulation.

• Development and Reproduction
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• v.17 no.1
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• pp.63-72
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• 2013

A specific inhibitor of RNA polymerase II, ${\alpha}$-amanitin is broadly used to block transcriptional activities in cells. Previous studies showed that ${\alpha}$-amanitin affects in vitro maturation of cumulus-oocyte-complex (COC). In this study, we evaluated the target of ${\alpha}$-amanitin, and whether it affects oocytes or cumulus cells (CCs), or both. We treated ${\alpha}$-amanitin with different time period during in vitro culture of denuded oocytes (DOs) or COCs in comparison, and observed the changes in morphology and maturation status. Although DOs did not show any change in morphology and maturation rates with ${\alpha}$-amanitin treatment, oocytes from COCs were arrested at metaphase I (MI) stage and CCs were more scattered than control groups. To discover causes of meiotic arrest and scattering of CCs, we focused on changes of cumulus expansion, gap junctions, and cellular metabolism which to be the important factors for the successful in vitro maturation of COCs. Expression of genes for cumulus expansion markers (Ptx3, Has2, and Tnfaip6) and gap junctional proteins (Gja1, Gja4, and Gjc1) decreased in ${\alpha}$-amanitin-treated CCs. However, these changes were not observed in oocytes. In addition, expression of genes related to metabolism (Prps1, Rpe, Rpia, Taldo1, and Tkt) decreased in ${\alpha}$-amanitin-treated CCs but not in oocytes. Therefore, we concluded that the transcriptional activities of CCs for supporting suitable transcripts, especially for its metabolic activities and formation of gap junctions among CCs as well as with oocytes, are important for oocytes maturation in COCs.

• Reproductive and Developmental Biology
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• v.34 no.4
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• pp.275-281
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• 2010

The aim of this study was to investigate what protein(s) of porcine epididymal fluid (pEF) are able to enhance the nuclear maturation of porcine germinal vesicle (GV) oocytes in vitro. Proteins of pEF were fractionated by affinity, ion exchange, and gel filtration chromatography. Porcine cumulus-oocytes complexes (COC) from follicles were cultured in tissue culture medium (TCM 199) containing various fractions obtained by chromatography. Porcine COCs were also cultured in TCM 199 containing various meiosis inhibitors and pEF. After 24 or 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. When porcine COCs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than 80% of oocytes were unable to resume meiosis. However, porcine COCs supplemented with pEF were able to overcome the inhibitory effect of dbcAMP and Fo. Maturation rate of oocytes was significantly (p<0.05) increased in the media supplemented with cationic protein(s) during in vitro maturation than in those with anionic protein(s) (44.1% vs 20.0%). When oocytes were cultured in the TCM 199 with fractions obtained by gel filtration, the maturation rate of oocytes was significantly (p<0.05) higher in fraction 11 containing 18 kDa than other fractions. The present study suggests that 1) dbcAMP and Fo prevent the spontaneous maturation of oocyte after isolation from follicles, and that pEF contain a substance(s) that improves meiosis resumption in vitro of porcine COCs, 2) cationic 18 kDa protein(s) are responsible for promotion of Mil stage.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.113-119
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• 1999

This study was performed to improve the development of the in vitro fertilized bovine embryos by the condition of in vitro maturation. COCs were matured in TCM 199 supplemented with 0.1% PVA, 10ng/ml EGF, Hormones (5$\mu\textrm{g}$/ml FSH, 10 IU hCG, 1 $\mu\textrm{g}$/ml estradiol 17-$\beta$) or granulsa cell+Hormones atmosphere 39$^{\circ}C$, 5% CO2, 95% air for 24hrs. Matured oocytes were fertilized with frozen-thawed semen capacitated with 5mM caffein in BO medium for 20 hrs. IVF embryos were cultured in TCM 199 containing with hormones(same as matured medium), 10% FBS and co-culture with bovine oviduct epitherial cells. Maturation rates of COCs were showed 73.8%, 78.5%, 83.2% and 87.6% respectively, and were significant differences between PVA, EGF, and Hormones, GC+Hormones(p<0.05). The cleavage rates of IVF embryos were revealed 72.5%, 78.4%, 82.3% and 84.2% and showed same tendency as maturation rates(p<0.05). The blastocysts matured by above maturation condition and cultured for 7~10 days after fertilization had 34.4, 43.6, 52.3 and 59.3 cells had no differences among the treatments. These results suggest that high molecules as a substitutes of serum and growth factor may induce nuclear resumption of COCs but we need more study to produce transferable IVF blastocysts by use of that agents.

• Clinical and Experimental Reproductive Medicine
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• v.41 no.1
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• pp.15-20
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• 2014

Objective: Combined oral contraceptives (COCs) have some adverse effects on the serum lipid profile. Because hyperlipidemia is one of the risk factors in cardiovascular diseases, lipid abnormalities should be evaluated in women consuming COCs. Vitamins E and C are known to have beneficial effects on serum lipid profiles. Therefore, in this study, we evaluated the effects of vitamins E and C on serum lipids in women using COCs. Methods: The study compared changes in lipid parameters with and without vitamin therapy in women consuming COCs compared to those of a control group (40 non-contraceptive users or NCU) for 4 weeks. Total cholesterol and triglyceride, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) levels along with HDL/LDL ratios were measured for all participants. Results: COC users experienced significantly higher increases in the levels of triglycerides and LDL than non-users (p<0.05). However, no significant differences were noted in the total cholesterol and HDL levels. In the treated COC group receiving vitamins E and C, the HDL level and the HDL/LDL ratio increased and the LDL and triglycerides levels decreased significantly compared with those of the other groups. Conclusion: The results of our study indicate that supplementation with antioxidant vitamins E and C restores a normal lipid profile in COC users.

• Journal of Animal Science and Technology
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• v.63 no.2
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• pp.272-280
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• 2021

Cumulus-oocyte complexes (COCs), which contain immature oocytes, are matured in vitro for in vitro embryo production. Oocyte and cumulus cells are then separated using hyaluronidase. To date, there have only been a few reported cases of the toxic effects of hyaluronidase on porcine oocytes. The aim of this study was to compare the effects of bovine testis-derived hyaluronidase and recombinant human hyaluronidase on oocyte denudation and quality. Porcine COCs were matured for 44 h and denuded using different hyaluronidase concentrations and exposure times. Then, oocytes were activated by electrical parthenogenesis. In experiment 1, COCs were denuded using bovine-derived, ovine-derived (Hirax), and human recombinant (ALT-BC4) hyaluronidases for 10 and 20 min. In experiment 2, bovine-derived and human recombinant (ALT-BC4 and ICSI Cumulase®) hyaluronidases were used to denude the COCs for 2 and 20 min. In both experiments the oocytes were all completely denuded, and there was no degeneration. Rate of embryo development was significantly increased in group treated ALT-BC4 for 2 min and not significantly different in other treatment groups. In general it slightly decreased with longer exposure times. These results have confirmed that different sources of hyaluronidase do not have detrimental effects on the quality of porcine oocytes and suggest that the human recombinant hyaluronidase ALT-BC4 is suitable for oocyte denudation with an increased blastocyst rate.