• Reproductive and Developmental Biology
• /
• v.28 no.4
• /
• pp.221-226
• /
• 2004

The present study was performed to investigate the effects of in vitro maturation (IVM) and in vitro fertilization (IVF) duration on the development of Korean Native Cattle embryos. The time of blastocyst formation and the quality of blastocysts based on cell numbers were examined. The cleavage rate increased with the length of IVF duration in the groups of 18-hr IVM, but was constant in the groups of 24-hr IVM. The development rate to the 8-cell stage was significantly higher in the IVM 18: IVF 20 group than in the IVM 24: IVF 24 group. The development rate to the blastocyst stage was highest in the IVM 18: IVF 20 group, significantly different from that of the IVM 18: IVF 16, IVM 24: IVF 20 and IVM 24: IVF 24 group. The time of blastocysts formation tended to be shorter when IVM and IVF duration were decreased. The number of inner cell mass, trophoblast and the total cells were significantly higher in the IVM 18: IVF 16 group than in the IVM 24: IVF 24 group (P<0.05). These results demonstrated that the IVM and IVF duration should be adequate for the efficient production of bovine embryos, and it might particularly be essential to determine the proper combination of IVM and IVF duration.

• Journal of Embryo Transfer
• /
• v.22 no.4
• /
• pp.223-227
• /
• 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 ($42.5{\pm}4.0%$).

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

• Journal of Embryo Transfer
• /
• v.22 no.4
• /
• pp.219-222
• /
• 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 ($6.2{\sim}7.2%$).

• Journal of Embryo Transfer
• /
• v.33 no.1
• /
• pp.1-11
• /
• 2018

Cellular cyclic adenosine-3' 5'-monophosphate (cAMP) modulator is known as meiotic inhibitor and can delays spontaneous maturation in IVM experiment. Among many cAMP modulators, the role of Pituitary adenylate cyclase activating polypeptide (PACAP) on IVM isn't known. The purpose of this study is to improve the maturation of oocytes derived from follicles ${\leq}3mm$ in diameter through PACAP as meiotic inhibitor during pre-in vitro maturation (pre-IVM). First, we checked PACAP and its receptors in cumulus cells and, to establish the optimal phase and concentration of PACAP for pre-IVM, we conducted chromatin configuration assessments. As a result, the rate of GV (Germinal Vesicle) according to duration of pre-IVM was significantly decreased 12 h and 18 h after IVM (87.1 and 84.1%, respectively) compared to 0 h (99.4%). When COC was cultured for 18 h, the GV rate in the $1{\mu}M$ of PACAP treatment group (82.1%) was significantly higher than any other PACAP treatment groups (60.5, 64.1, 74.4 and 69.9 %, respectively). So, we divided into four groups as follows; MF (the conventional IVM group, obtained from follicle from 3 to 6 mm in diameter), SF (the conventional IVM group, obtained from follicle ${\leq}3mm$ in diameter), Pre-SF(-)PACAP (IVM group including 18 h pre-IVM without $1{\mu}M$ of PACAP, obtained from follicle ${\leq}3mm$ in diameter) and Pre-SF(+)PACAP (IVM group including 18 h pre-IVM with $1{\mu}M$ of PACAP, obtained from follicle ${\leq}3mm$ in diameter). To examine the effect of PACAP during pre-IVM, we investigated analysis of nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels. In cumulus cells, PACAP receptors, ADCYAP1R1 and VIPR1 were detected but were not detected in oocytes. After IVM, the Pre-SF(+)PACAP had the highest Metaphase II rate (91.7%) among all groups (P<0.05). The GSH levels in the MF and Pre-SF(+)PACAP were significantly higher than in the other groups (P<0.05) and ROS levels was no significant difference among all groups. In conclusion, these results indicated that even though the oocytes were derived from SF, pre-IVM application of PACAP improved meiotic and cytoplasmic maturation by regulating intracellular oxidative stress.

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

• Animal Bioscience
• /
• v.37 no.6
• /
• pp.1007-1020
• /
• 2024

Objective: We increased the nuclear maturation rate of antral follicle derived oocytes by using a pre-in vitro maturation (IVM) culture system and improved the developmental potential of these porcine pathenotes by supplementing with melatonin. Furthermore, we investigated the expression patterns of genes involved in cumulus expansion (HAS2, PTGS2, TNFAIP6, and PTX3) derived from small and medium antral follicles before and after oocyte maturation. Methods: Only the cumulus oocyte-complexes (COCs) derived from small antral follicles were induced with [Pre-SF(+)hCG] or without [Pre-SF(-)hCG] the addition of human chorionic gonadotropin (hCG) during the last 7 h of the pre-IVM period before undergoing the regular culture system. The mature oocytes were investigated on embryonic development after parthenogenetic activation (PA). Melatonin (10^-7 M) was supplemented during in vitro culture (IVC) to improve the developmental potential of these porcine pathenotes. Results: A pre-IVM culture system with hCG added during the last 7 h of the pre-IVM period [Pre-SF(+)hCG] effectively supported small antral follicle-derived oocytes and increased their nuclear maturation rate. The oocytes derived from medium antral follicles exhibited the highest nuclear maturation rate in a regular culture system. Compared with oocytes cultured in a regular culture system, those cultured in the pre-IVM culture system exhibited considerable overexpression of HAS2, PTGS2, and TNFAIP6. Porcine embryos treated with melatonin during IVC exhibited markedly improved quality and developmental competence after PA. Notably, melatonin supplementation during the IVM period can reduce and increase the levels of intracellular reactive oxygen species (ROS) and glutathione (GSH), respectively. Conclusion: Our findings indicate that the Pre-SF(+)hCG culture system increases the nuclear maturation rate of small antral follicle-derived oocytes and the expression of genes involved in cumulus expansion. Melatonin supplementation during IVC may improve the quality and increase the blastocyst formation rate of porcine embryos. In addition, it can reduce and increase the levels of ROS and GSH, respectively, in mature oocytes, thus affecting subsequent embryos.

• Journal of Embryo Transfer
• /
• v.11 no.3
• /
• pp.241-248
• /
• 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%).

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

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