• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.257-257
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• 2004

The study was carried out to investigate the effects of morphology, reproductive cycle, incubation time and activation of oocytes on in vitro maturation of cat oocytes and development of IVM/IVF embryos. 1. When recovered from ovaries collected at different stages of the reproductive cycle (inactive, follicular and luteal stage), the developmental rates of oocytes to GV and MI stage were 72.5% and 27.5%, 57.5% and 7.5%, 62.5% and 17.5%, respectively. (omitted)

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.165-172
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• 2004

This study was carried out to examine the effects of maturation media on in vitro maturation (IVM) of porcine immature oocytes, and on subsequent in vitro fertilization (IVF) and development (IVD). Cumulus-oocyte complexes (COCs) were collected from antral follicles of porcine ovaries collected from abattoir, and were matured in vitro in modified NCSU-37 (mNCSU-37), modified NCSU-23 (mNCSU-23), or TCM-199 supplemented with 10% porcine follicular fluid (pFF). Oocytes matured in vitro, were fertilized in vitro in modified Tris-buffered medium(mTBM) with the final motile sperm concentration of 1${\times}$105 sperm/mL, and subsequently putative embryos were developed in vitro in NCSU-23. The results are as follows. 1. In the result of IVM, the rate of germinal vesicle breakdown (GVBD) and of nuclear maturation were not significantly different among the media, though numeric value of them were slightly lower in TCM-199 than in mNCSU-37 or in mNCSU-23. 2. In the result of IVF, though the rate of sperm penetration was not significantly different among the maturation media, the percentage of oocytes with male pronucleus (MPN) of ones matured in mNCSU-37 (88.0%) was significantly higher than in TCM-199 (71.1%) (p<0.05). 3. In the result of IVD, the percentage of cleaved oocytes of ones matured in mNCSU-37 (52.3%) or in mNCSU-23 (53.7%) was significantly higher than in TCM-199 (43.1%) (p<0.05), but the rate of blastocysts at day 6 was not significantly different among the maturation media, though putative embryos from oocytes matured in mNCSU-37 or in mNCSU-23 were developed more than in TCM-199. These results suggested that mNCSU-37 or mNCSU-23 was more appropriate than TCM-199 as IVM medium for porcine immature oocytes.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.173-183
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• 2004

This study was carried out to examine the effects of epidermal growth factor (EGF) and the number of cumulus-oocyte complexes (COCs) on in vitro maturation (IVM) of porcine immature oocytes, and on subsequent in vitro fertilization (IVF) and development (IVD). COCs were collected from antral follicles of porcine ovaries collected from abattoir, and were maturated in modified NCSU-23 (mNCSU-23) with 10% pFF, 0.6 mM cysteine, 50 ${\mu}mM{\beta}-mercaptoethanol$, 1 mM dbcAMP, 10 IU/mL PMSG and 10 IU/mL hCG, which was supplemented with or without 10 ng/mL EGF and into which 50 or 15 COCs per droplet was put. Oocytes matured in vitro, were fertilized in vitro in modified Tris-buffered medium (mTBM) with the final motile sperm concentration of 1${\times}$105 sperm/mL, and subsequently putative embryos were developed in vitro in NCSU- 23. The results are as follows. 1.In the result of IVM, 10 ng/mL EGF supplement duplicated the percentage of C4 group of COCs(41% vs 81%). But the rate of germinal vesicle breakdown (GVBD) and of nuclear maturation were not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet, and there was not a significant interaction between the two factors, either. 2. In the result of IVF, there was not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet, or was not a significant interaction between the two factors, in the rate of sperm penetration, in the percentage of oocytes with male pronucleus (MPN), and in the rate of polyspermy. 3. In the result of IVD, there was not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet in the percentage of cleaved oocytes. There was not significantly different between the number of COCs per culture droplet, but between control and EGF supplemented (p<0.01) in the percentage of blastocysts, the number of inner cell mass (ICM), trophectoderm (TC) and total cells. There was no significant interaction between the two factors anywhere. These results suggested that 10 ng/mL EGF supplement into mNCSU-23 for IVM was effective in the production of more as well as better blastocysts during IVD through increasing the number of cells in those.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.129-135
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• 2006

This study was conducted to investigate the effect of in vitro maturation (IVM) duration of porcine follicular oocytes on maturation rate, polyspermic rate, and subsequent embryo development. The nuclear maturation rates of oocytes matured for 36, 38, 40, 42 and 44 hr were similar between 68.0, 78.0, 79.5, 73.8 and 81.8% respectively. There was no significant difference in the rates of polyspermy after in vitro feritilization (IVF). The cleavage rate in the group of 36 hr was significantly higher in than that of 40, 44 hr (p<0.05) but not to 38 and 42 hr. The development rate to blastocyst stage was significantly higher in the group of 38 hr (23.1%) than that in the group of 44 hr (15.6%) (p<0.05) but not to 36, 40 and 42 hr. These results suggest that the aged oocytes for 44 hr is not required for the production of bias to cysts derived from porcine IVF embryos.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.201-206
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• 2014

The objectives of the study were to determine an effective culture dish, culture duration and protein supplementation in medium for in vitro maturation (IVM) of oocytes of native zebu cows in Bangladesh. The ovaries of cows were collected from local slaughterhouse followed by aspiration of follicular fluid. The cumulus-oocyte-complexes (COCs) with more than 3 compact cumulus cell layers were cultured in tissue culture medium (TCM) 199 for maturation. The maturation of oocytes was determined by observing polar body under microscope. To determine an effective culture dish, 130 COCs derived from 48 ovaries in a well of 4-well dish and 102 COCs derived from 36 ovaries in drops covered with mineral oil within 35 mm petri dish were cultured for 24 hours. The rate of maturation of oocytes did not vary between 4-well dish ($51.3{\pm}15.0%$) and drops in petri dish ($52.4{\pm}11.6%$). To determine the effective culture duration, 185 COCs derived from 62 ovaries were cultured in drops for 18, 21, 24 and 27 hours. The rate of maturation of occytes ranged from $51.9{\pm}9.4%$ (18 hours) to $59.0{\pm}17.1%$ (27 hours) and the difference in maturation rate among different culture durations was not significant (P>0.05). To determine an effective protein supplementation, 63 oocytes from 19 ovaries were cultured separately in TCM 199 supplemented with either fetal bovine serum (FBS) or bovine serum albumin (BSA). The rate of maturation was significantly (P<0.01) higher in medium supplemented with FBS ($55.63{\pm}16.19%$) than that of BSA ($14.82{\pm}9.36%$). In conclusion, COCs of native zebu cows can be cultured for IVM either in 4-well culture dish or droplets in petri dish for 18 to 27 hours in medium supplemented with FBS.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.467-472
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• 1997

This study was conducted to determine the optimal DC voltage for NT of in vivo donor embryo nuclei and investigate the effect of donor embryo guality on fusion and in vitro development of NT embryos . Recpient oocytes were enucleated 25~27h after IVM and further cuitured for 18~20h prior to fusion for oocyte aging. Donor embryos of molura stage were recovered from superovulated heifers and classified l into good and low quality group. Their nuclei were transferred in to the emucleated oocytes 42~44h post-IVM and fused 43-45h post-IVM with a single 0.75kV /cm or 1.0kV /cm DC voltage for 70${\mu}\textrm{A}$sec. The fusion rate of oocytes was not different between two DC voltages. However, the cleavage and M + B developmnent was more high at 1.0kV /cm DC voltage and the proportion of M+B was 19.0% at 0.75kV /cm DC and 29.4% at 1.0kV /cm DC voltage. Donor embryo qualtiy did not greatly affect the fusion and cleavage of NT oocytes, but none of NT embryos derived from low embryo quatity reached the morula stage. The results indicate that the most suitable DC v voltage for electrofusion of in vivo donor muclei was a single 1.0kV /cm DC voltage and donor embryo quality was an important factor affecting the development in vitro of NT embryos.

• Journal of Embryo Transfer
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• v.10 no.1
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• pp.53-63
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• 1995

The effects of different protein sources (serum vs bovine serum albumin), growth factors (EGF and PDGF) and co-culture with various type of somatic cel1s (BOEC, MEF and BRL) on the in vitro development of in vitro matured / in vitro fertilized bovine oocytes were examined, and the viability of frozen/thawed embryos derived from IVM /IVF was examined. Cell numbers of blastocysts were also counted. In Experiment 1, CR$_1$aa with serum was superior to CR$_1$aa with BSA in producing morulae plus blastocysts from IVM /IVF oocytes(24.4% vs 30.4%, p>0.05). In Experiment 2, more morulae plus blastocysts(42.3%) were produced in CR$_1$aa containing long /ml EGF than in the control CR$_1$aa(33.3%). In Experiment 3, 2- to 8-cell embryos derived from IVM /IVF oocytes were randomly allotted to one of 4 culture groups : a) CR$_1$aa ; b) CR$_1$aa + ing /ml PDGF ; CR$_1$aa + Sng /ml PDGF ; CR$_1$aa + lOng /ml PDGF ; culture resulted in 21.3, 51.2, 41.4 and 45.9%(p<0.05), respectively, developing into morulae and blastocysts. In Experiment 4, 0 and Sng /ml PDGF added to CR$_1$aa coculture with BRL or BOEC yielded 47.5, 42.5, 33.8 and 41.6% morulae and blastocysts, respectively. In Experiment 5, the proportion of embryos into morulae and blastocysts was highest in CR$_1$aa with MEF coculture group(50.9%) compared to any other group(CR$_1$aa, 22.3%; CR$_1$aa+BRL, 32.9%; CR$_1$aa+BOEC, 33.8%, p>0.05). In Experiment 6, survival rate of blastocysts produced by in vitro fertilization when cryoprotectant was removed in 0.7M glycerol+0.7M sucrose and 0.7M sucrose solution for 10 min. after thawing at 2$0^{\circ}C$ (Exp. H, 58.8%) was slightly higher than when cryoprotectant was removed 10%, 6.7% and 3.3% glycerol for 10 min. after thawing at 37$^{\circ}C$ (Exp. I, 54.3%). These study indicate that growth factors and somatic cell co-culture can increase the proportion of embryos that develop into morulae and blastocysts without an increase in the cell number and frozen /thawed method employed this experiment was not different.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.7
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• pp.956-965
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• 2019

Objective: The main goal of this study was to provide a morphological indicator that could be used to select high-quality oocytes of appropriate meiotic and developmental capabilities in pig. The higher quality of immature oocytes, the higher success rates of in vitro maturation (IVM) and in vitro fertilization (IVF). Thus, prior to the IVM culture, it is important to characterize oocytes morphologically and biochemically in order to assess their quality. Two of the largest indicators of oocyte quality are the presence of cumulus cells and status of chromatin. To investigate the effects of porcine oocyte chromatin configurations on the developmental capacity of blastocysts, we assessed oocyte chromatin status according to follicle size and measured the developmental potency of blastocysts. Methods: To sort by follicle size, we divided the oocytes into three groups (less than 1 mm, 1 to 3 mm, and more than 3 mm in diameter). To assess chromatin configuration, the oocytes were assessed for their stages (surrounded nucleolus [SN] germinal vesicle [GV], non-surrounded nucleolus [NSN] GV, GV breakdown, metaphase I [MI], pro-metaphase II [proMII], and metaphase II [MII]) at different maturation times (22, 44, and 66 h). To assess the development rate, oocytes of each follicle size were subjected to parthenogenetic activation for further development. Finally, GV oocytes were grouped by their chromatin configuration (SN, SN/NSN, and NSN) and their global transcriptional levels were measured. Results: SN GV oocytes were more suitable for IVF than NSN GV oocytes. Moreover, oocytes collected from the larger follicles had a greater distribution of SN GV oocytes and a higher developmental capacity during IVM, reaching MII more quickly and developing more often to blastocysts. Conclusion: Porcine oocytes with high-level meiotic and developmental capacity were identified by analyzing the relationship between follicle size and chromatin configuration. The porcine oocytes from large follicles had a significantly higher SN status in which the transcription level was low and could be better in the degree of meiotic progression and developmental capacity.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.1
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• pp.17-21
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• 2005

The effect of melatonin on in vitro embryo development and the expression of antioxidant enzyme gene in preimplantation porcine embryos was determined by modified semi-quantitative single cell RT-PCR. Porcine embryos derived from in vitro maturation /in vitro fertilization were cultured in 5% $CO_2$ and 20% $O_2$ at $37^{\circ}C$ in NCSU23 medium. Melatonin was added to medium at concentration of 1nM, 5 nM, and 10 nM. When treated with 1nM (39.0%) of melatonin, the developmental rate of embryos beyond the morula stage were higher than that of control group (31.0%) (p<0.05). Number of inner cell mass and tropectoderm cell in control (23.0${\pm}$0.5 and 17.3${\pm}$0.8), 1 nM (23.6${\pm}$0.6 and 19.0${\pm}$0.5), and 5 nM (23.3${\pm}$1.1 and 16.3${\pm}$0.8) treated with melatonin were higher than in 10 nM (20.0${\pm}$0.5 and 13.3${\pm}$0.8) treated with melatonin (p<0.05). To develop an mRNA phenotypic map for the expression of catalase, bax and caspase-3, single cell RT-PCR analysis were carried out in porcine IVM/IVF embryo. Catalase was detected in 0, 1 and 5 nM supplemented with melatonin, but bax and caspase-3 were detected in 10 nM treated with melatonin.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.443-451
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• 1997

These studies were conducted to determine the sex of preimplantation Hanwoo embryos produced in vitro using polymerase chain reaction(PCR). Y chromosome specific and bovine speicific DNA primers were synthesized and tested for embryo sexing. Bovine IVF embryos were produced in TCM 199 and CR1aa medium, and classified by developmental stages on Day 7 to 9. The effects of developmental rates to bovine IVF blastocysts on sex ratio were also investigated using PCR methods. The results obtained in this study were as follows; 1. Developmental rates to blastocyst from IVM/IVF embryos in TCM 199 and CR1aa medium for 9 days were 23.5 and 30.2%, respectively, and there was significant difference between the media(P<0.05). 2. Male to female ratio of early, mid, expanded and hatching balstocyst produced on Day 7 were 0.7:1, 1.4:1, 2.2:1, and 2.5:1, respectively, and male embryos was significantly higher proportion in expanding and hatching blastocysts(P<0.01). 3. On Day 8, male to female ratio of early, mid, expanded and hatching blastocysts were 0.6:1, 1:1, 2.5:1, and 2.7:1, respectively. Both expanded and hatching blastocysts obtained a significantly higher proportion of males(P<0.01). 4. The male : female ratio of early, mid, expanded and hatching blastocyst produced on Day 9 was 0.6:1, 0.8:1, 1:1, and 2.2:1, respectively. Hatching blastocysts had a significantly higher ratio of males(P<0.01). The developmental rate of IVM/IVF embryos to blastocyst for 9 day culture was higher in CR1aa than that in TCM 199 medium. For the sex ratio by developmental stages of IVF embryos, male ratio was higher in expanded blastocyst but female in early blastocysts.