• Journal of Embryo Transfer
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• v.19 no.3
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• pp.265-273
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• 2004

This study was carried out to examine the effects of fertilization media and sperm concentration on in vitro fertilization (IVF) and development (IVD) of porcine oocytes matured in vitro. Cumulus-oocyte complexes (COCs) were collected from antral follicles of porcine ovaries collected from abattoir, and were matured in vitro in modified NCSU-23 (mNCSU-23) supplemented with 10％ porcine follicular fluid (pFF). After the fertilization by experimental scheme, putative embryos were developed in vitro in NCSU-23. The results are as follows. When the oocytes were fertilized in vitro in modified TBM or modified TLP-PVA by 1 ${\times}$10$^{5}$ sperm/$m\ell$, all of the fertilization parameters were not significantly different between two media. Subsequently, as these putative embryos were developed in vitro in NCSU-23, the percentage of oocytes cleaved and of blastocysts were not different between two media, either. When the oocytes were fertilized in vitro in mTBM by 5${\times}$10$^4$, 1${\times}$10$^{5}$ or 5${\times}$10$^{5}$ sperm/$m\ell$, all of the fertilization parameters were significantly (P<0.05 or P<0.01) increased as sperm concentration was elevated. Subsequently, as these putative embryos were developed in vitro in NCSU-23, the percentage of oocytes cleaved and of blastocysts were significantly boosted (P<0.01) as sperm concentration at fertilization was elevated from 5${\times}$10$^4$ to 1${\times}$10$^{5}$ sperm/$m\ell$, but were not different between 1${\times}$10$^{5}$ and 5${\times}$10$^{5}$ sperm/$m\ell$.

• Journal of Embryo Transfer
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• v.25 no.1
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• pp.1-7
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• 2010

The objective of this study was to examine the effect of vitamin B (pantothenic acid, folic acid, and myo-inositol) that was supplemented to embryo culture medium on in vitro development of parthenogenetically activated (PA) pig embryos. Cumulus-oocyte complexes derived from slaughtered ovaries were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (hCG and eCG) for the first 22 h and then further cultured in hormone-free medium for an additional 22 h. After maturation culture, metaphase II oocytes that extruded 1st polar body were electrically activated and treated with $5.0\;{\mu}g/ml$ cytochalasin B for 4 h. Then, PA embryos were cultured for 7 days in a modified NCSU-23 that was supplemented with pantothenic acid, myo-inositol, or folic acid at different concentrations ($3{\sim}300\;{\mu}M$) according to the experimental design. Myo-inositol added to culture medium did not show any beneficial or inhibitory effects on embryo cleavage and blastocyst formation. However, $300\;{\mu}M$ pantothenic acid significantly inhibited blastocyst formation compared to control (no addition) (24% vs. 36%, p<0.05). Folic acid ($300\;{\mu}M$) significantly (p<0.05) increased blastocyst formation (56%) compared to control (41%). Our results demonstrated that in vitro development of PA embryos was significantly influenced by vitamin B and addition of $300\;{\mu}M$ folic acid to culture medium improved in vitro development of pig PA embryos.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.42-42
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• 2001

The objective of this study was to assess the development of porcine follicular oocytes fertilized by ICSI. Cumulus-oocyte-complexes (COCs) were collected by aspiration from follicles of 2-7 mm in diameter from a local slaughterhouse ovaries. Oocytes matured for 40-44 h were centrifuged at 12,000g for 6 min and then injected with sperm prepared by swim-up procedure in the presence or absence of 5 mM dithiothreitol (DTT). Injected oocytes were cultured in NCSU 23 medium during 6 to 8 days. IVF controls were compared to those of resulting embryos. The results obtained were as. follow: 1, The rates of cleavage and development rates into blastocyst by ICSI were not significantly (P<0.05) different between with (53.0% and 19.7%) or without (48.3% and 23.8%) centrifugation, respectively. 2. The cleavage and developmental rates to blastocyst after ICSI with or without 5mM DTT treated-sperm were not significantly (P<0.05) different (60.4% vs 16.4% and 48.5% vs 22.2%, respectively). 3. The cleavage and the developmental rates to blastocyst were not significantly (P<0.05) different between the zygotes obtained by IVF (51.8% vs. 22.4%) and ICSI (51.4% vs. 21.6%). 4. The number of blastomere in blastocyst stages after IVF or ICSI was not significantly different (46.7 $\pm$2.9 and 41.9$\pm$4.6).

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.55-62
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• 2010

Matrix metalloproteinases (MMP) play important roles in extracellular matrix (ECM) remodeling during ovarian follicular development, oocytes development and ovulation. In an attempt to investigate the effect of MMP activation in development cumulus-oocytes complexes, we examined the localization and expression of MMP, and monitored MMP expression profile. Cumulus-oocytes complexes were collected and matured in vitro for 24 hr, 36 hr and 48 hr. A mRNA expression of MMP-2, MMP-9, TIMP-2 and TIMP-3 was detected in all culture medium regardless of CC, DC and CDCs. Activity of MMP-2 in the DC progressively was increased from 24 hr to 48 hr. But MMP-9 was not detected in all culture medium. The localization of MMP-2 was also measured by immunohistochemistry analysis. The MMP-2 and TIMP-2 was detected in cumulus cell and oocyte zone pellucida. Expression of MMP-2 protein in the COCs was progressively increased from 24 hr to 48 hr. However, MMP-9 protein was progressively decreased from 24 hr to 48 hr. And TIMP-2 protein was most highly expressed in the CDCs 36 hr. Expression of TIMP-3 protein in the CDCs was progressively increased from 24 hr to 48 hr. In conclusion, these results suggest that MMP-2 plays a role in maintaining normal maturation and development by controlling the ECM inhibitor concentration on cumulus cell and oocytes.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.169-174
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• 2005

This study was comducted to examine the effects of culture medium, and the osmolarity and osmotic change of the culture medium on in vitro maturation of porcine oocytes and developement of porcine parthenogenetic embryos. In Experiment 1, cumulus-oocyte complexes were matured in NCSU-23, mWM and mKRB, respectively, There was no difference in maturation rate($62.1\~71.3\%$) among groups. In Experiment 2, matured oocytes in each medium were activated and cultured for 6 days in the same media. Blastocyst formation rate was higher in NCSU-23($22.9\%$) than those of others($0\~0.6\%$, P<0.05). In Experiment 3, parthenogenetic embryos were cultured for 6 days in NCSU-23 with different osmolarity(300, 280 and 256 mOsmols) adjusted by NaCl. There were no differences in development rates to the blastocyst stage($11.0\~14.4\%$) among groups. In Experiment 4, activated oocytes were cultured for 2 days in NCSU-23 with 300, 280 and 256 mOsmols and then transferred to increased or decreased osmotic condition. Blastocyst formation rate was higher in a group which was transferred from the higher osmotic condition to the lowe. osmotic condition($21.0\%$) than a contrary group( $11.8\%$, (P<0.05). This result shows that the culture medium and the osmolarity of the culture medium affect the development of porcine parthenogenetic embryos, and the change of osmolarity from the higher condition to the lower condition at a certain developmental stage can enhance the development of porcine parthenogenetic embryos.

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.187-194
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• 1998

In order to determine suitable conditions for rapid freezing of porcine embryos, the kind and concentration of cryoprotectants, sucrose concentrations, equilibration time and thawing temperature in freezing medium were examined in relation to the survival of frozen-thawed oocyte and embryos. The results obtained are as follows : 1. The suitable concentrations of cryoprotoctant in the freezing medium which consisted of TCM-199+20% FCS were 1.5M for glycerol, 2.0M for DMSO, 2.5M for ethylene glycol, and 2.0M for propanediol. The sucrose concentration of 0.25M in the medium was found to optimal because the survival rate was markedly higher at this concentration when compared to the others. The survival rate was relatively high when the frozen embryos were thawed at 30$^{\circ}C$ in the freezing medium containing 2.5M cryoprotectants. The equilibration periods of 2.0 and 5.0 minutes revealed the higher survival in the media containing 1.5 or 2.1M glycerol when compared to 10 and 15 minutes. 2. The fertilization rates of frozen-thawed follicular oocytes which matured in vitro for 1, 12, 24 and 48 hours were 6.7~26.7% depending on the maturation time, and the rates were relatively high for those matured for a short period of time. The survival rates of frozen-thawed oocytes which matured in vitro for certain periods and fertilized were 10.0~30.0% depending on the maturation time.

• Korean Journal of Agricultural Science
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• v.26 no.2
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• pp.19-24
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• 1999

This study was conducted to investigate the influence of media and hormones on in vitro maturation and development of porcine follicular oocytes. Basic media were used to TCM-199, Waymouth MB751/1 and BMOC-II, and hormones were used to hCG and FSH in each medium. The results obtained were summarized as follows : 1. The maturation rates of oocytes cultured in TCM-199 medium containing hCG, FSH and hCG+FSH were 78.05, 72.50 and 67.50%, respectively. The maturation rates of oocytes with hormones were significantly (P<0.05) higher than those of oocytes cultured without hormone. However, the cleavage rate(hCG 46.88%, FSH 31.04%. hCG+FSH 37.04%) of embryo cultured in TCM-199 containing hormone was significantly(P<0.05) lower than that(89.47%) of oocytes cultured without hormone. 2. The maturation rates of oocytes cultured in Waymouth MB751/1 medium containing hCG. FSH and hCG+FSH were 69.77, 71.43 and 80.00%, respectively. The maturation rates of oocytes with hormones were significantly(P<0.05) higher than those of oocytes cultured without hormone. However. the cleavage rate(hCG 46.67%. FSH 36.00%, hCG+FSH 35.71%) of embryo cultured in Waymouth MB751/1 containing hormone was significantly(P<0.05) lower than that(60.00%) of oocytes cultured without hormone. 3. The maturation rates of oocytes cultured in BMOC-II medium containing hormone were 66.67(control). 66.67(hCG). 91.89(FSH) and 81.82(hCG+FSH)%. respectively. showing the highest rate in FSH treatment. And, the cleavage rates of oocytes cultured in BMOC-II medium containing hormone were 81.82 (control, 79.17(hCG), 50.00(FSH) and 66.67(hCG+FSH)%, respectively.

• Current Research on Agriculture and Life Sciences
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• v.8
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• pp.45-50
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• 1990

This study was carried out ot investigate the effects of fetal calf serum (FCS) and gonadotropins supplemented to the medium on maturation and fertilization in vitro of porcine follcular oocytes. Ovaries were obtained from gilts at local slaughter-house. Oocyte-cumulus complexes were recovered by puncturing the ovarian follicles(3~5 mm in diameter). The complexes from individual ovaries were pooled in a $0.4m{\ell}$ droplet of medium covered with paraffin oil, then washed twice in fresh droplet and cultured for 36hrs in culture media according to experimental conditions. Boar epididymal spermatozoa were capacitated by preincubation for 4hrs in m-KRB medium and the preincubated spermatozoa were insemenated in the fertilization medium containing the cultured oocytes. The results obtained in this study are summarized as follows: 1. The maturation rates of oocytes cultured in m-KRB and m-KRB supplemented to 10% FCS were 82 and 37%, respectively. When PMSG, hCG. and PMSGt hcG($10Iu/m{\ell}$) were added to the media supplemented to 10% FCS, the maturation rates were 66, 58 and 68%, respectively. 2. Expansion of cumulus cells was not occured in m-KRB and m-KRB supplemented to 10% FCS. However, when PMSG, hCG and PMSG+hCG($10Iu/m{\ell}$) were added to m-KRB supplemented to 10% FCS, the expansion rates of cumulus cell layers were 92, 13 and 91%, respectively. 3. When oocytes were mltured in m-KRB, the rates of penetration and formation of male pronucle: were 93 and 7%, respectively. By adding FCS and gonadotropin to m-KRB, the penetration and formation of male pronuclei were 100 80%, respectively.

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.1-7
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• 2007

This study was conducted to examine the role of intact cumulus cells during in vitro fertilization (IVF) on sperm penetration, male pronuclear (MPN) formation and subsequent embryo development of oocytes matured and fertilized in vitro. Cumulus-oocyte complexes obtained from the slaughtered gilt ovaries were matured for 44 h in TCM199 containing 10% porcine follicular fluid, epidermal growth factor and hormones. After maturation culture, denuded oocytes or oocytes with intact cumulus cells were coincubated with frozen-thawed boar semen for 8h in a modified tris-buffered medium containing 5mM caffeine and 10mM calcium chloride. Putative zygotes were fixed and examined for sperm penetration and MPN formation (Experiments $1{\sim}3$), or cultured in North Carolina State University-23 medium fo. 156 h (Experiment 3). In Experiment 1, sperm penetration was examined after insemination of denuded oocytes and oocytes with intact cumulus cells at the concentration of $7.5{\times}10^5$ sperm/ml. Optimal sperm concentration for IVF of cumulus-intact oocytes was determined in Experiment 2 by inseminating intact oocytes with $2{\sim}5{\times}10^6$ sperm/ml. In Experiment 3, denuded or intact oocytes were inseminated at the concentrations of $7.5{\times}10^5$ and $4.0{\times}10^6$ sperm/ml, respectively, and in vitro embryo development was compared. Sperm penetration was significantly (p<0.01) decreased in cumulus-intact oocytes compared to denuded oocytes (35.2% vs. 77.4%). Based on the rates of sperm penetration and normal fertilization, the concentration of $4.0{\times}10^6$ sperm/ml was optimal for the IVF of intact oocytes compared to other sperm concentrations. The presence of intact cumulus cells during IVF significantly (p<0.05) improved embryo cleavage (48.8% vs. 58.9%), blastocyst (BL) formation (11.0% vs. 22.8%) and embryo cell number $(22{\pm}2\;vs.\;29{\pm}2\;cells)$ compared to denuded oocytes. In conclusion, these results suggest that intact cumulus cells during IVF inhibit sperm penetration but improve embryo cleavage, BL formation and embryo cell number of porcine embryos produced in vitro.