• Journal of Embryo Transfer
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• v.22 no.4
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• pp.235-243
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• 2007

Our goal was to examine the effects of early denudation on the enucleation efficiency and developmental competence of embryos following somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA). Oocytes were denuded following 30 h of in vitro maturation (IVM) and then cultured with (D+) or without (D-) their detached cumulus cells for additional $10{\sim}14$ h. Control oocytes were denuded after $40{\sim}44$ h of IVM. The size of the perivitelline space was larger at 40 h of IVM ($11.7{\sim}11.8{\mu}m$) than at 30 h ($8.9{\mu}m;$ p<0.01). The distances between the metaphase II (M II) plates and the polar bodies (PBs) were shorter in D+ ($19.4{\mu}m$) and D- oocytes ($18.9{\mu}m$) than in control oocytes ($25.5{\mu}m;$ p<0.01). Enucleation rates following blind aspiration at 40 h of IVM were higher (p<0.01) in D+ (92%) and D- oocytes (93%) compared to controls (82%). Early denudation did not affect oocyte maturation or the in vitro development of SCNT and PA embryos. When SCNT embryos from D+ oocytes were transferred to four gilts, pregnancy was established in two pigs, and one of them farrowed three live piglets. In conclusion, early denudation of oocytes at 30 h of IVM could improve the enucleation efficiency by maintaining the M II plate and the PB within close proximity and support the in vivo development of SCNT embryos to term.

• Development and Reproduction
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• v.21 no.2
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• pp.205-213
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• 2017

The aim of this study was to determine the effect of additional alpha-linolenic acid (ALA) supplementation during in vitro maturation (IVM) and culture (IVC) on nucleic maturation and embryo development of pigs. Cumulus-oocyte complexes (COCs) were incubated in IVM medium containing different concentration of ALA (25, 50 and $100{\mu}M$) for 44 h. After in vitro maturation, nuclear maturation of oocytes were evaluated by aceto-orcein stain. Mature oocytes with $50{\mu}M$ ALA were fertilized and cultured in IVC medium with ALA (25, 50 and $100{\mu}M$) during early-embryogenesis (48 hours after fertilization). Then, embryos were cultured with $25{\mu}M$ ALA during early embryogenesis and/or late embryogenesis (120 hours after early-embryogenesis). In results, oocyte maturation were significantly increased by $50{\mu}M$ ALA treatment groups compared with control groups (p<0.05). Treatment of $25{\mu}M$ ALA during early-embryogenesis enhanced cleavage rate of embryo compared with other groups (p<0.05), whereas formation and total cell number of blastocyst had no significant difference. Similarly, cleavage rate of embryos were increased by $25{\mu}M$ ALA supplement during early- or late-embryogenesis than ALA treatment both stage of embryogenesis (p<0.05), but did not influence to blastocyst formation. Interestingly, total cell number of blastocyst were enhanced in ALA treatment group during early-embryogenesis. These findings indicated that ALA supplement enhance the nuclear maturation of oocyte and embryo development, however, excessive ALA could negatively influence. Therefore, we suggest that ALA is used for improvement of in vitro production of mammalian embryo and further study regarding with functional mechanism of ALA is needed.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.77-82
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• 2007

This study was conducted to investigate the effects of oocyte maturational age and activation condition on in vitro development of porcine parthenogenetic embryos (parthenotes). Porcine follicular oocytes were matured in vitro for 30 to 44 hr. Maturation rate was examined during in vitro maturation (IVM) every 2 hr interval. The cdc2 kinase activity was measured at 36 and 44 hr of IVM. Some oocytes were activated at 36 or 44 hr of IVM by three different conditions; 1) single electric stimulation (1.5 kV/cm for $30{\mu}sec$; ES), 2) double electric stimulations (1.5 kV/cm for $30{\mu}sec$, followed by 1.0 kV/cm for $50{\mu}sec$ after 1 hr; ES+ES) or 3) ES+ES followed by culture in 6-dimethlyaminopurine (6-DMAP) for 4 hr (ES+ES+D), and cultured for 6-7 days. Maturation rate was significantly increased as culture period was increased to 36 hr (66.9%, p<0.05), and then gradually increased to 87.1% at 44 hr of IVM. The cdc2 kinase activity was decreased (p<0.05) with culture period prolonged from 36 hr to 44 hr. Lower blastocyst formation rate (4.3%, p<0.05) were obtained by ES in 36 hr-matured oocytes compared to other treatments (16.5 and 20.5%) in the same age and the same treatment in 44 hr-matured oocytes (15.0%). High blastocyst formation rate (23.6%) was obtained by ES+ES+D in 44 hr-matured oocytes (p<0.05). These results demonstrate that porcine oocyte activation and in vitro development of parthenotes can be affected by interactions between oocyte maturational age and activation condition.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.73-83
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• 1994

The objective of this study was to develop an effective in vitro production system capable of obtaining more porcine embryos from immature oocytes. These experiments were thus conducted to examine the effect of oocytes type and maturation time on the in vitro maturation(IVM) and fertilization(IVF) of oocytes and the in vitro development (IVD)of IVF embryos. 1. The degree of oocyte maturation based on cumulus expansion index(GEI) did not differ for A- and B-typed oocytes but the index of oocyte type C was lower(P<0.05) than that of other oocyte types. 2. When the oocytes of type A and B were matured for 36, 42 and 48hrs, the GEl was not different between the 36- and 42-h maturation but the GEl after 48hrs was greatly lower(P<0.05) than that of other maturation times. 3. The highest cleavage rate(48.6%) of IVF oocytes was obtained from A typed oocytes and 42-h maturation but the developmental potential based on cleavage index was the highest when B-typed oocytes were matured for 42hrs.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.5
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• pp.693-696
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• 2001

The effect of replacement of in vitro maturation medium completely with the buffalo follicular fluid (buFF) on in vitro oocyte maturation of buffalo oocytes was studied. 5 to 8 buffalo cumulus oocyte complexes were cultured in a single drop with each of the eight media studied i.e., M199+steer serum (10% v/v), M199+steer serum (10% v/v)+PMSG, M199+buFF (10% v/v), M199+buFF (10% v/v)+PMSG, M199+buFF (50% v/v), M199+buFF (50% v/v)+ PMSG, buFF (100%) and buFF+PMSG at $39^{\circ}C$ and 5% $CO_2$ in air for 24 h. Supplementation of M199 with Steer serum alone resulted in IVM rate of 35% only. When the above medium was supplemented with PMSG, the maturation rate rallied to 82%. Significant increase in the maturation rates were observed when M199 was supplemented with increasing levels of buFF. A further increase in the maturation rate was also obtained when PMSG was incorporated into the medium of M199 supplemented with buFF. The rate of maturation was to the tune of 91% when oocytes were matured in buFF alone which was increased non significantly on the addition of PMSG. Highest maturation rate (97%) obtained with M199+buFF (50%v/v)+PMSG did not differ significantly from that obtained by either M199+buFF (10%v/v)+PMSG or buFF+PMSG. It is suggested that buFF alone without any supplementation can form the effective in vitro maturation medium for buffalo oocytes.

• Korean Journal of Veterinary Research
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• v.57 no.2
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• pp.89-95
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• 2017

This study was conducted to determine the effects of biophoton treatment during in vitro maturation (IVM) and/or in vitro culture (IVC) on oocyte maturation and embryonic development in pigs. An apparatus capable of generating homogeneous biophoton energy emissions was placed in an incubator. Initially, immature pig oocytes were matured in the biophoton-equipped incubator in medium 199 supplemented with cysteine, epidermal growth factor, insulin, and gonadotrophic hormones for 22 h, after which they were matured in hormone-free medium for an additional 22 hr. Next, IVM oocytes were induced for parthenogenesis (PA) or provided as cytoplasts for somatic cell nuclear transfer (SCNT). Treatment of oocytes with biophoton energy during IVM did not improve cumulus cell expansion, nuclear maturation, intraoocyte glutathione content, or mitochondrial distribution of oocytes. However, biophoton-treated oocytes showed higher (p < 0.05) blastocyst formation after PA than that in untreated oocytes (50.7% vs. 42.7%). In an additional experiment, SCNT embryos produced from biophoton-treated oocytes showed a greater (p < 0.05) number of cells in blastocysts (52.6 vs. 43.9) than that in untreated oocytes. Taken together, our results demonstrate that biophoton treatment during IVM improves developmental competence of PA- and SCNT-derived embryos.

• Development and Reproduction
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• v.21 no.2
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• pp.131-138
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• 2017

This study was conducted to investigate stimulatory effect of epidermal growth factor (EGF) on nuclear maturation and the expression level of EGF-receptor (EGFR), GM-130 (a marker of Golgi apparatus), transport protein Sec61 subunit beta ($Sec61{\beta}$), and coatomer protein complex subunit gamma 2 (COPG2) in porcine oocytes. The cumulus-oocyte complexes were collected from follicle with 3-6 mm in diameter. They were incubated in medium with/without EGF for 22 h (IVM I) and subsequently incubated hormone-free medium with/without EGF for 22 h (IVM II). Nuclear maturation state was checked by aceto-orcein stain. Protein expression of EGFR, GM-130, $Sec61{\beta}$, and COPG2 were measured by immunofluorescence. In results, nuclear maturation of oocytes in EGF non-treated oocytes were significantly lower than EGF-treated groups at IVM I or IVM II stage (P<0.05), whereas maturational rate in EGF treatment groups at both of IVM stage was higher in among the all treatment groups (P<0.05). EGFR, GM-130, $Sec61{\beta}$ and COPG2 were expressed in the cytoplasm of oocytes. Especially, GM-130 and EGFR were strongly expressed, but $Sec61{\beta}$ and COPG2 were weakly expressed in cortical area of cytoplasm. The protein level of GM-130, $Sec61{\beta}$, and COPG2 were significantly higher in the EGF-treated groups (P<0.05). However EGFR was no difference between non EGF-treated groups and control. In conclusion, EGF plays an important role in the systems for oocyte maturation with endoplasmic reticulum and Golgi apparatus. In addition, the protein levels of $Sec61{\beta}$ and COPG2 could be changed by EGF in the porcine oocytes during maturation.

• Clinical and Experimental Reproductive Medicine
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• v.43 no.2
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• pp.126-132
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• 2016

Objective: The purpose of this study was to identify useful clinical factors for the identification of patients with polycystic ovary syndrome (PCOS) who would benefit from in vitro maturation (IVM) treatment without exhibiting compromised pregnancy outcomes. Methods: A retrospective cohort study was performed of 186 consecutive patients with PCOS who underwent human chorionic gonadotropin-primed IVM treatment between March 2010 and March 2014. Only the first IVM cycle of each patient was included in this study. A retrospective case-control study was subsequently conducted to compare pregnancy outcomes between IVM and conventional in vitro fertilization (IVF) cycles. Results: Through logistic regression analyses, we arrived at the novel finding that serum $anti-M{\ddot{u}}llerian$ hormone (AMH) levels and the number of fertilized oocytes in IVM were independent predictive factors for live birth with unstandardized coefficients of 0.078 (95% confidence interval [CI], 1.005-1.164; p=0.037) and 0.113 (95% CI, 1.038-1.208; p=0.003), respectively. Furthermore, these two parameters were able to discriminate patients who experienced live births from non-pregnant IVM patients using cut-off levels of 8.5 ng/mL and five fertilized oocytes, respectively. A subsequent retrospective case-control study of patients with PCOS who had serum AMH levels ${\geq}8.5ng/mL$ showed that IVM had pregnancy outcomes comparable to conventional IVF, and that no cases of ovarian hyperstimulation syndrome were observed. Conclusion: Serum AMH levels are a useful factor for predicting pregnancy outcomes in PCOS patients before the beginning of an IVM cycle. IVM may be an alternative to conventional IVF for PCOS patients if the patients are properly selected according to predictive factors such as serum AMH levels.

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.97-104
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• 2009

The objective of this study was to examine the effect of macromolecule in a maturation medium on nuclear maturation, intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were cultured in maturation medium that was supplemented with each polyvinyl alcohol (PVA), pig follicular fluid (pFF) or newborn calf serum (NBCS) during the first 22 h and the second 22 h. Oocyte maturation was not influenced by the source of macromolecules during in vitro maturation (IVM). Embryo cleavage and cell number in blastocyst after PA was altered by the source of macromolecule but no difference was observed in blastocyst formation among treatments. Oocytes matured in PVA-PVA medium showed lower rates of oocyte-cell fusion (70.4% vs. 77${\sim}$82%) and embryo cleavage (75% vs. 86${\sim}$90%) after SCNT than those matured in other media but blastocyst formation was not altered (13${\sim}$27%) by different macromolecules. pFF added to IVM medium significantly increased the intracellular GSH level of oocytes compared to PVA and NBCS, particularly when pFF was supplemented during the first 22 h of IVM. Our results demonstrate that source of macromolecule in IVM medium influences developmental competence of oocytes after PA and SCNT, and that pFF supplementation during the early period (first 22 h) of IVM increases intracellular GSH level of oocytes.

• 대한생식의학회:학술대회논문집
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• 2005.11a
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• pp.22-35
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• 2005