• Journal of Embryo Transfer
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• v.24 no.2
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• pp.89-95
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• 2009

Successful in vitro embryo production heavily relies on the normal maturation and fertilisation of oocytes. We examined the normal and abnormal fertilisation of zebu cattle oocytes matured in vitro. Immature cumulus oocyte complexes (COCs) from zebu cattle ovaries at slaughter were matured in vitro (IVM) for 24 h. The oocytes were either fixed, stained and examined for nuclear changes or fertilised in vitro (IVF) with Percoll-separated, heparintreated spermatozoa (1.0 ${\times}$ $10^6$/mL) of zebu (n = 7) and crossbred bulls (n = 7). After 18 h of sperm-COCs co-incubation at $39^{\circ}$C with 5% $CO_2$ in humidified air, the presumptive zygotes were fixed, stained and examined for pronuclei. The number of oocytes retrieved per ovary was 5.4 ${\pm}$ 0.7. The percentage of matured oocytes was 73.0. The difference in motility of spermatozoa before and after Percoll seperation was significant (p<0.001). The percentages of normal and abnormal fertilisation (polyspermia and oocytes with one pronucleus) varied significantly depending on individual bulls (p<0.05). A protocol for IVF of IVM oocytes in Bangladeshi zebu cattle is developed. A future study may elucidate the capacity of such IVM-IVF oocytes to develop to the blastocyst stage for transfer to surrogate mother.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.361-368
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• 1997

A total of 55 patients with various etiologies of infertility particitated in a study comparing two regimens of controlled ovarian hyperstimulation (COH) with GnRH agonists and gonadotropins. Nineteen patients were given an ultra-short stimulation protocol when the agonist was administered for 3 day from Day 2 of the cycle. The remaining 36 patients were given a long stimulation protocol when the agonist was administered from the mid-luteal phase of the cycle preceding the stimulation cycle. The mean number of gonadotropins used per patient was not different between two groups. No significant differences were found in the mean number of oocytes recovered, fertilization rate and embryo cleavage rate between two groups. Pregnancy and delivery rates were higher in ultra-short protocol than in long protocol, but these were not significant. These results suggest that an ultra-short protocol is as effective as a long protocol in in-vitro fertilization and embryo transfer.

• 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.

• 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%).

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.47-47
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• 2003

The present study was conducted to determine the expression of the antioxidant enzyme(CuZn-SOD, Mn-SOD and GPX and apoptosis gene(caspase-3) for in vitro culture in in vitro maturation and in vitro fertilization(IVM/IVF) embryos in porcine. Porcine embryos derived from IVM/IVF were cultured in NCSU23 medium under 5% $CO_2$ in air at 38.5$^{\circ}C$. The patterns of gene expression for several antioxidant enzyme and apoptosis genes during preimplantion porcine embryo development were examined by the modified semi-quantitative single cell reverse transcriptase- polymerase chain reaction (RT-PCR). Preimplantation porcine embryos produced by IVM/IVF have expressed mRNAs for CuZn-SOD and GPX, whereas transcripts for Mn-SOD have not detected at any developmental stages. Expression of caspase-3 mRNA was detected at 2 cell, 8 cell, 16 cell and morula stages. The fas ligand transcripts were detected in porcine blastocyst. These results suggest that various antioxidant enzymes and apoptosis genes play crucial roles in in vitro culture of porcine IVM/IVF embryos.

• Journal of Embryo Transfer
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• v.12 no.1
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• pp.111-116
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• 1997

The present study was carried out to produce in vitro fertilized embryos with immature follicular oocytes collected by transvaginal aspiration from Holstein cows. A simple aspiration apparatus consists of two stainless steel tubes, an inner tube (needle holder; 1.2cmdiameter, 55cm long) and an outer tube (1.5cm diameter, 4Scm long), and a hand-operated vacuum pump was used. Under epidural anesthesia, the needle guide was passed into the vagina of the cow to a point next to the cervix. An ovary was placed against the wall of the vagina over the end of the aspiration needle by rectal manipulation. As the needlepassed into the ovary, an assistant was asked to apply vacuum(l00mrnHg) and the ovary was manipulated back and forth in all directions over the needle. When all sites of the ovary was aspirated, the needle was withdrawn and the needle guide was moved to the other side of ovary and the procedure was repeated. When the oocyte aspiration procedure was finished, collected fluid was transported to laboratory. Oocytes surrounded with at least 1 layer of cumulus cells were matured, fertilized and cultured in vitro. The results were as follows; Ninety seven oocytes were collected by transvaginal aspiration from seventeen Holstein cows(5.7 /head). The number of oocytes surrounded with at least 1 layer of cumulus cells were 60(61.9%). Following in vitro maturation, fertilization and culture, the cleavage and development rate to morula+blastocyst were 83.3% and 30.0%, respectively. From this study, transferable in vitro fertilized embryos could be produced with imma- ture follicular oocytes collected by transvaginal aspiration from Holstein cows using a simple aspiration apparatus.

• 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.24 no.1
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• pp.71-76
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• 2009

It is not easy for porcine embryos produced by in vitro systems to develop into blastocysts with high quality. To solve this problem, many researchers have developed novel culture methods. However, the formation of blastocysts with high quality is still low. In this study, we aimed to produce piglet following transfer of in vitro produced early embryos ($2{\sim}4$ cell stage embryos) or morula and blastocyst. The $2{\sim}4$ cell stage embryos were transferred to five estrus-synchronized recipients (200 embryos per recipient). One of the five sows farrowed three piglets, which contain two live piglets and one dead piglet, 114 days after embryo transfer. However, two recipients transferred with morula and blastocysts did not farrow. Microsatellite analysis confirmed that the genomic DNA of two live piglets were not genetically identical to that of the recipient. These results indicate that it is possible to obtain piglets by transfer of early embryos produced by in vitro production (IVP) systems.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.28-28
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• 2001

The objective of this study was to the effect on subsequent development of EGF present in defined medium during bovine 1)oocyte maturation or 2)embryo culture. The presence of EGF during IVM, irrespective of concentration(1, 10, 100ng/$m\ell$), stimulated cumulus expansion and significantly increased the proportion of oocytes attaining metaphaseII, the rate of cleavage, and develop to blastocyst. 1. In the group of EGF-added medium(1, 10, 100ng/$m\ell$), nuclear maturation rate for in vitro maturation was 91% to 92% but was not significantly higher than control group(87%). 2. For in vitro maturation, in the group of EGF-added medium(1, 10, 100ng/$m\ell$)the rate of cumulus cell expansion degree 2 ranged from 81% to 87%, which was significantly higher than the control group(medium with EGF not added). The rate of in vitro fertilization, developing to 2-to 4- cell stage, was 76% to 80%, which was also significantly higher(p<0.05)than control group(62%). 3. For in vitro maturation, in the group of EGF added in medium(1, 10, 100ng/$m\ell$)the development rate to blastocyst was 24.3% to 27%, which was significantly higher than control group(13.7%). The total cleavage rate in the group of EGF-added medium was 77% to 82%, which was higher than control group. 4. The development rate to blastocyst for 6 days of cultivation and the hatching blastocyst were 30.6% and 59.1%, respectively, in the group of 100ng/$m\ell$ of EGF, which were significantly higher(p<0.05)than control group(14.0% and 24%, respectively), The numbers of cells in blastocyst were 140.2 and 148, respectively, in 10ng/$m\ell$ and 100ng/$m\ell$ of EGF-added medium, which were higher than 108.5 in control group. 5. The development rate of in vitro fertilized embryos to blastocyst in 10ng/$m\ell$ of EGF-added medium co-cultured with somatic cell was 28%, which was significantly higher(p<0.05)than control group(11.8%). The numbers of cells in blastocyst were 141.6 for EGF-added medium and 145 for EGF+co-culture group, which were higher than control(101.6)and medium co-cultured with somatic cells(110.6). These results showed that in vitro maturation and fertilization, EGF was found a significant effect of increase of development rate to blastocyst and cell number.

• Journal of Embryo Transfer
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• v.7 no.2
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• pp.89-96
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• 1992

This research was conducted to investigate the interrelationship among methods of injection of PMSG-hCG to the number of ovulated eggs, percentage of matured oocytes and in vitro fertilization using out-bred ICR mice. The results obtained are as follows, 1) The optimurn dose was 5 IU for both PMSG and hCG, while the number of ovulated eggs was 42$\pm$8, percentage of M II was 73% and in vitro fertilization rate was 81 %. 2) The optimum injection interval of PMSG-hCG was 48 hours, while the number of ovulated eggs was 48 $\pm$ 8, percentage of M II was 80% and in vitro fertilization rate was 81%. 3) The optimum time for collecting eggs was between 16 and 18 hours after hCG injection, while the numbers of ovulated eggs were 44$\pm$8, 42$\pm$7 and 43$\pm$7 in 14,16 and 18 hours after hCG injection respectively, and percentages of M II were 79 and 81 %, and in vitro fertilization rates were 81 and 80% in 16 and 18 hours after hCG injection, respectively. 4) The repeat of superovulation decreased with the number of ovulated eggs, percentage of M II and in vitro fertilization rate, than in control. But it was recovered by increasing the repeat interval.