• Journal of Embryo Transfer
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• v.22 no.4
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• pp.223-227
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• 2007

This study was carried out to investigate the effect of morphology of oocytes, kinds of media, cysteine and myo-inositol supplementation on IVM rate of porcine oocytes. Cumulus- enclosed oocytes were incubated in maturation NCSU-23 and TCM-199 medium with supplementation with 3, 5, 10, 20 mM myo-inositol and 0.05, 0.1, 0.5, 1.0 mM cysteine. 1. When classified by morphology, excellent, good and fair of cumulus-enclosed oocytes were incubated for 48 hrs and the IVM rate were $14.2{\pm}3.7%{\sim}58.7{\pm}4.0%$, respectively. The rate were greater in oocytes with excellent cumulus cells than those without cumulus cells. 2. The IVM rate of oocytes cultured in TCM-199 and NCSU- 23 medium supplementation or non-supplementation with 1.0 mM myo-inositol were $7.5{\pm}4.5%,\;45.0{\pm}4.8%\;and\;4.4%,\;42.5{\pm}4.2%,\;18.0{\pm}5.2%$, respectively. Supplementation with myo-inositol significantly increased the IVM rate of oocytes. 3. The IVM rate of oocytes cultured in NCSU-23 medium supplementation of 3, 5, 10, 20 mM myo-inositol for 48 hrs were $47.5{\pm}4.5%,\;57.5{\pm}4.2%,\;62.5{\pm}4.9%,\;50.0{\pm}5.2%$, respectively. The IVM rate of oocytes in NCSU-23 medium supplemented with 10 mM myo-inositol were significantly increased compared to control ($42.5{\pm}4.0%$). 4. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 media supplement with 0.3, 0.5, 1.0, 2.0 mM myo-inositol were $50.0{\pm}4.5%,\;62.5{\pm}4.2%,\;52.5{\pm}4.9%,\;45.0{\pm}4.2%$, respectively. The IVM rate of oocytes in NCSU-23 medium supplemented with 10 mM cysteine were significantly increased compared to control ($42.5{\pm}4.0%$).

• Journal of Embryo Transfer
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• v.23 no.2
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• pp.87-91
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• 2008

This study was carried out to investigate the effects of the supplementation of glutamine, glucosamine and glutathione on the porcine oocytes on IVM rates. Cocs were incubated in NCSU-23 supplemented with at $2.0{\sim}10.0\;mM$ glucosamine, $0.5{\sim}4.0\;mM$ glutamine and $0.1{\sim}1.0\;mM$ glutathione for 48 hrs. Oocytes were transferred to 50 ul drops of maturation medium covered with mineral oil and cultured in a $CO_2$ incubator ($38^{\circ}C$, 5% $CO_2$, 95% air). The IVM rates of oocytes cultured in NCSU-23 supplemented with 0.5, 1.0, 2.0 and 4.0 mM glutamine for 48 hrs were $46.0{\pm}4.5%$, $52.0{\pm}4.8%$, $50.0{\pm}4.2%$ and $44.0{\pm}4.5%$, respectively. The IVM rates of oocytes cultured in NCSU-23 supplement with 2.0, 5.0, 7.0, 10.0 mM glucosamine for 48 hrs were $44.0{\pm}4.5%$, $42.0{\pm}4.5%$, $38.0{\pm}4.6%$ and $24.0{\pm}4.8%$, respectively. The IVM rates of oocytes cultured in NCSU-23 supplemented with glucosamine were no significantly increased compare to the control ($42.5{\pm}4.0%$). The IVM rate of oocytes cultured in NCSU-23 supplemented with 3.0, 5.0, 7.0, 10.0 mM glutathione for 48 hrs were $40.0{\pm}3.2%$, $54.0{\pm}4.2%$, $48.0{\pm}4.5%$, $44.0{\pm}4.8%$, respectively. The IVM rate of oocytes cultured in NCSU-23 supplemented with glutamine and glutathione were significantly increased co~pared to those control ($42.5{\pm}4.0%$). Glucosamine did not affect the IVM rates of oocytes. IVM rates of oocytes cultured in NCSU-23 medium for 48 hrs were significantly increased compared to the cultured for 40 hrs.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.357-365
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• 2020

The aim of present study was to investigate regulatory mechanism of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation of porcine oocytes. Basically, immature cumulus-oocyte complexes (COCs) were incubated for 22 h in IVM-I to which hormone was added, and then further incubated for 22 h in IVM-II without hormone. As a result, relative cumulus expansion was increased at 22 h after IVM and it was enhanced by treatment of ALA compared with control group (p < 0.05). During IVM process within 22 h, cAMP level in oocytes was decreased at 6 h (p < 0.05) and it was recovered at 12 h in ALA-treated group, while oocytes in control group recovered cAMP level at 22 h. In cumulus cells, it was reduced in all time point (p < 0.05) and ALA did not affect. Treatment of ALA enhanced metaphase-I (MI) and MII population of oocytes compared with oocytes in control group at 22 and 44 h, respectively (p < 0.05). Intracellular GSH levels in ALA group was increased at 22 and 44 h after IVM (p < 0.05), whereas it was increased in control group at 44 h after IVM (p < 0.05). In particular, the GSH in ALA-treated oocytes during 22 h of IVM was higher than control group at 22 h (p < 0.05). Lipid amount in oocytes from ALA group was higher than control group (p < 0.05). Treatment of ALA did not influence to absorption of glucose from medium. Cleavage and blastocyst formation of ALA-treated oocytes were enhanced compared with control group (p < 0.05). These findings suggest that supplementation of ALA could improve oocyte maturation and development competence through increasing GSH synthesis, lipid storage, and regulation of cAMP accumulation during early 22 h of IVM, and these might be mediated by cumulus expansion.

• Journal of Embryo Transfer
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• v.22 no.4
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• pp.219-222
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• 2007

These study was carried out to investigate the effects of the collection time, culture time and activation of canine oocytes on in vitro maturation rates. The activated oocytes were cultured in 10% FCS+TCM-199 media containing hormonal supplements (10 IU/ml HCG, 10 IU/ml PMSG, 10 ug/ml gonadotropin) at 5% $CO_2$, 95% air, $38^{\circ}C$. 1. IVM rate of in vitro cultured cumulus-attached oocytes recovered from ovaries that collected at follicular and luteal stages of the reproductive cycles were 11.4% and 5.7%, respectively. IVM rate of oocytes recovered from ovaries that collected at follicular stages of the reproductive cycles was significantly higher than that of luteal stage (p<0.05). 2. When IVM was carried out at different periods of 40, 48, and 70 hrs, the IVM rates of oocytes matured in vitro were 2.9%, 8.6%, 5.7%, respectively. These results indicate that the IVM time between $48{\sim}70$ hrs gives the highest maturation rate for the oocytes matured at the different stages. 3. IVM rate of oocytes matured in vitro for 10 hrs after single and combined activation treatment by ET, IP and CH and Ca+DMAP, CH+DMAP, ET+CH were $11.5{\pm}1.2%,\;10.8{\pm}1.0%,\;9.6{\pm}1.2%\;and\;12.4{\pm}1.5%,\;11.8{\pm}1.5%,\;11.2{\pm}1.4%$ respectively. This was higher than that in both single and combined stimulated groups compared to control group ($6.2{\sim}7.2%$).

• Korean Journal of Animal Reproduction
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• v.21 no.4
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• pp.355-362
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• 1997

This study was undertaken in an effort to product embryos through in vitro maturation(IVM), in vitro fertilization(IVF) and in vitro culture(IVC) after cryopreservation of immature and mature porcine oocytes. The experiments were conducted to investigate IVM rate of oocytes frozen with 3 different cryoprotectants and to examine IVF and IVC of frozen-thawed oocytes. The CEI(cumulus cells expansion index) after IVM of frozen-thawed immature oocytes was higher in oocytes frozen with PG＋PEG(propylene glycol plus polyethylene glycol) than those frozen with single cryoprotectant and this index was almost 90% of unfrozen oocyte's index(2.39 vs. 2.66). The IVF rate of all frozen oocytes was very low(68% of unfrozen oocytes) and the IVF rate of frozen immature oocytes was slightly higher than that of frozen mature oocytes(39.0% vs. 34.4%), but polyspermic penetration was higher in frozen immature oocytes(21.9% vs. 19.1%). The cleavage rate after IVF of frozen-thawed oocytes was 9.3% for frozen mature oocytes and 11.3% for frozen immature oocytes and this rate was significantly lower(P<0.05) than that of control(60.7%). The development to 8-cell stage was greatly lower in frozen mature oocytes than in frozen immature oocytes. The results indicate that the use of PG plus PEG as cryoprotectant may be very effective for vitrification of porcine oocytes and the frozen-thawed immature porcine oocytes can be used fro in vitro embryo production based on IVM, IVF and IVC system.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.241-248
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• 1996

This study was conducted to improve the production efficiency of in vitro produced (IVP) embryos in Korean Native cows. The optimal conditions and procedures for in vitro maturation(IVM), in vitro fertilization(IVF) and in vitro culture(IVC) of bovine follicular oocytes and IVP embryos were evaluated. Immature follicular oocytes were collected fiom the follicles of bovine ovaries obtained from abattoirs. The oocytes of Grade I and II for IVM were cocultured with monolayered bovine oviductal epithelial cells(BOEG) or granulosa cells in TCM-199 solution supplemented with follicle stimulating hormone, lutenizing hormone, estradiol-17$\beta$ and heat inactivated fetal calf serum at 39$^{\circ}C$ under 5% $CO_2$ in air for 14 to 24 hours. Most of the oocytes(93%) matured to metaphase II in 24 hours. The cocultured IVM oocytes were fertilized in vitro at significantly(P<0.05) higher rate with BOEC(83.8%) and with granulosa cells(84.6%) than the non-cocultured IVM oocytes(73.6%). The IVM-IVF embryos developed to morula and blastocyst at significantly(P<0.05) higher rate in coculture with BOEC(41.2%) than with granulosa cells(23.1%) or conditioned medium(23.4%).

• Journal of Embryo Transfer
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• v.27 no.3
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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.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.475-478
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• 2011

In the present study, the effect of cysteine and NT or bisphenol A(BP) on in vitro aturation(IVM) of porcine oocytes were examined. COCs was cultured in NCSU-23 medium supplement with 10% FCS which had previously been covered with mineral oil and equilibrated in a humidified atmosphere of 5% $CO_2$ and 95% air at $38^{\circ}C$. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 0.5~10.0 mM cysteine were $34.0{\pm}3.2%$, $36.0{\pm}3.5%$, $48.0{\pm}3.8%$, $22.0{\pm}3.2%$, respectively. The IVM rate of oocytes cultured in NCSU-23 medium supplement with 0.5~5.0mM NT for 48 hrs were $24.0{\pm}4.2%$, $18.0{\pm}4.9%$, $8.0{\pm}2.2%$, respectively. NT affects oocyte in vitro maturation rate in a dose-dependent. This result were significantly lower than the control group. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 1.0 mM NT+5.0 mM cysteine($38.0{\pm}4.3%$) were significantly higher than that of NT treatment. The IVM rate of oocytes cultured in NCSU-23 medium supplement with 0.05~5.0 mM BP for 48 hrs were $20.0{\pm}4.7%$, $10.0{\pm}5.3%$, $6.0{\pm}3.2%$, respectively. The IVM rate of oocytes cultured in NCSU-23 medium supplement with BP was significantly lower cultured non supplement of BP ($44.0{\pm}3.5%$). BP affects porcine oocyte maturation rate in a dose-dependent manner. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 1.0 mM BP+5.0 mM cycteine ($32.0{\pm}3.2%$) were increased than that of BP treatment.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.2
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• pp.79-84
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• 2017

Objective: Optimizing in vitro maturation (IVM) media to achieve better outcomes has been a matter of interest in recent years. The aim of this prospective clinical trial was to investigate the effects of different media on the IVM outcomes of immature oocytes at the germinal vesicle (GV) stage. Methods: A total of 400 immature oocytes at the GV stage with normal morphology were retrieved from 320 infertile women aged $31{\pm}4.63years$ during stimulated intracytoplasmic sperm injection (ICSI) cycles. They were divided into groups of homemade IVM medium (I, n = 100), cleavage medium (II, n = 100), blastocyst medium (III, n = 100), and Sage IVM medium (IV, n = 100) and cultured for 24 to 48 hours at $37^{\circ}C$. ICSI was performed, and the rates of fertilization and embryo formation were compared across the four groups. Results: In the 400 retrieved GV oocytes, the total maturation rates showed significant differences in groups I to IV (55%, 53%, 78%, and 68%, respectively, p<0.001). However, there were no significant differences in the fertilization, embryo formation, or arrest rates of metaphase II oocytes across these groups. In all groups, GV maturation was mostly completed after 24 hours, with fewer oocytes requiring 48 hours to mature (p<0.01). Moreover, the rate of high-quality embryos was higher in group IV than in the other groups (p=0.01). Conclusion: The quality of the IVM medium was found to affect clinical IVM outcomes. Additionally, blastocyst medium may be a good choice in IVM/ICSI cycles as an alternative IVM medium.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.71-77
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• 2012

The objective of this study was to examine the effect of thymidine treatment during $in$ $vitro$ maturation (IVM) of porcine follicular oocytes on blastocyst development. Porcine oocytes were treated with thymidine (10 mM, 20 mM and 30 mM) for 2 or 6 hr in the preiods of IVM I and/or II. The survival rates of the blastocysts in the 6 hr treatment groups of 10 mM and 20 mM during IVM I period were significantly higher than those of control group ($p$<0.05). However, the survival rate of the blastocysts in the 2 hr treatment group of 20 mM during IVM II period was significantly higher than control group ($p$<0.05). Furthermore, the survival rate of the blastocysts in the 6 hr treatment group of 30 mM during IVM II period was significantly lower than control group ($p$<0.05). Consistent with the previous result, blastocyst development of both IVM I and II treatment group was also showed as similar pattern. Total and apoptotic cell numbers of blastocysts derived from thymidine treated porcine oocytes were examined by using Tunel assay. The results showed that there was no significant differences in total cell number of blastocysts between thymidine treated and untreated groups. However, apoptosis-positive cells in the thymidine treated group (6 hr IVM I) were significantly lower than those of other groups ($p$<0.05). Taken together, these results indicate that high quality oocytes were selected by DNA synthesis mechanism according to high concentration thymidine treatment during porcine oocyte maturation. Therefore, we concluded that presumptive selected oocytes by thymidine treatment during maturation periods improved the further embryo development and embryonic quality of IVF embryos by decreasing the incidence of apoptosis in preimplantation porcine embryos.