• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.395-403
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• 1998

In this study we determined fertilization processes and developmental ability of porcine oocytes following injection of round spermatid in the presence and absence of artificial activation. Electrical stimulation at 2 h before spermatid injection significantly increased the incidence of normal fertilization as compared to those following injection without stimulation or with stimulation immediately after injection. The incidences of two pronuclear formation and apposition were not different in oocytes between following intracytoplasmic spermatid and spermatid nucleus injection. Indirect immunocyto chemistry and laser scanning confocal microscopy study revealed that microtubuels were organized from the oocyte cortex following round spermatid injection, and this seemed to move both male and female pronuclei into the oocyte center. At 6 days following injection blastocoele formation was seen in the eggs following round spermatid (25%) and round spermatid nucleus injection (27%). However, none of oocytes developed to the blastocyst stage at 6 days following sham injection. The average cell numbers of blastocysts at 8 days following injection of spermatid and spermatid nucleus were 87 to 99. These results suggested that either round spermatid or it's nudeus can be used to produce viable embryos by injection into unfertilized oocytes in the pig.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.107-115
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• 2001

Development of effective activation protocols is of great importance for improving the success of cloning and subsequent transgenic. Three methods for oocyte activation, including 5$\mu$M ionomycin (5 min) alone, ionomycin + 1.9 mM 6-dimetylaminopurine (DMAP, 3 hrs) and ionomycin + 10 $\mu\textrm{g}$/ml cycloheximide (CHX, 3 hrs) were compared for their effects of pronuclei (PN) formation, development, developmental velocity and ploidy of parthenotes to IVF control in bovine. In group of ionomycin + DMAP, the oocytes having more 3PN were significantly (P<0.05) higher than in groups of ionomycin alone and of ionomycin + CHX (45.5% vs. 0 and 0%, respectively). Activation with the ionomycin alone, ionomycin + DMAP and ionomycin + CHX resulted in cleavage rates of 30, 85.5 and 57.9%, respectively. The blastocysts rate of parthenotes activated by ionomycin + DMAP treatment was significantly higher (12.3%. p<0.05) than those of other treated groups. Chromosome analysis shows that ionomycin + DMAP treatment greatly enhances the incidence of chromosomal abnormality of the parthenotes. From the results, we may conclude that DMAP treatment to the oocytes accelerates developmental velocity resulting in both the higher incidence of chromosome abnormality and of PN formation, and strongly suggest that CHX combined with ionomycin is better than DMAP for the purpose of successful nuclear transplantation. Developmental velocity of parthenotes activated by ionomycin + DMAP treatment was significantly (P<0.05) faster than others.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.2
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• pp.177-182
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• 1994

ICR female mice aged 3 to 4 weeks, were stimulated with 7.5 IU PMS injection. At 48-52h post-PMS injection, ovaries were dissected out and oocytes-cumulus complexes(OCCs) were divided into three groups, cumulus-free oocytes(O), cumulus-free oocyte cocultured with cumulus cells(O+C) and OCC. The oocyte were cultured in TCM199 containing various protein sources, FCS, BSA or PVP with gonadotropins(Gns) for 24h. Spermatozoa were collected from cauda epididymis and capacitated in T6 + BSA for 2h. After oocyte maturation in vitro(IVM) in different experimental groups, matured oocytes were inseminated with the capacitated spermatozoa in T6 + BSA for 6h. In the groups of IVM in TCM + BSA or PVP, fertilization(IVF) did not occur efficiently. However, increased fertilization was found in TCM+ FCS group. The oocytes groups, with cumulus cells showed decreased polyspermy in FCS group (O; 31.8 %, O + C; 12.2 %, OCC; 16%), the addition of Gns did not prevent polyspermy in all three groups. The rates of fertilization increased in zona-free oocytes in PVP group. This results showed that culture system for IVM and IVF could be improved. Furthermore, PVP can be used for the substitution of protein source during maturation, and its low rate of fertilization has been found due to zona hardening which occurred in FCS-free medium.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.163-169
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• 2001

OSBA(oocytes-sperm binding assay) is a tool developed for rapid test of optimal condition of IVF medium and protein source by binding ability of mouse sperm and egg. Mouse oocyte-cumulus complexes were prepared by removing of the cumulus cells with 0.1% hyaluronidase. 10$\pm$2 oocytes per 30 ${mu}ell$ medium drop were inseminated with 3 ${mu}ell$ sperm suspension and were cultured f3r 3 hours and 24 hours, respectively. And the oocytes were recovered gently and the No. of sperm bound on oocytes were counted. In the Exp. 1, the ratio of oocytes bound with one sperm at least were 60.2%(50/83), 2%(2/77) and 100%(79/79) in the medium with no protein, FBS(15%, v/v) and BSA(0.4%. w/v), respectively, Fetal bovine serum(FBS) seriously inhibited sperm binding on oocyte, although bovine serum albumin(BSA) promoted the binding ability. The inhibiting effect of FBS was dependent on the concentration of FBS. The sperm binding ability according to oocyte maturity was tested in the Exp. 2. There was no significant difference between Met. II (mature) and Met. I (intermediate mature) oocytes in the number of oocytes bound with sperm and the number of sperm bound on oocytes. Finally, in Exp. 3, two batches of Ham's F10 medium with good and poor quality by OSBA were tested (The ratios of embryos developed from PN 1-cell stage to hatched blastocyst; 25% vs. 70%). In the medium with good quality, sperm binding ability was significantly increased (P ＜ 0.05). The ratio of oocytes bound with one sperm at least was 66% and 90% in the medium with poor and good quality, respectively. Conclusively, It was possible to test IVF medium condition rapidly and easily by OSBA.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.67-75
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• 2000

This study was conducted to establish the optimal conditions for in vitro embryo production using oocytes derived from follicles of slaughter-house ovaries. The ovaries of Hanwoo were obtained from a local slaughter-house. The oocytes were aspirated from visible follicles of 2~7mm in diameter. The recovered oocytes which were completely surrounded by at least 2 layers of cumulus cells and a homogeneous cytoplasmic pigmentation were used. The selected oocytes were washed 3 or 4 times with D-PBS containing 10% bovine calf serum (BCS) and matured in vitor (IVM) in Ham's F-10 supplemented with 10% BCS or 0.01 $\mu\textrm{g}$/ml epidermal growth factor(EGF) at 39$^{\circ}C$ under 5% CO2 in air for 24 hours. They were fertilizqed in vitro (IVF) with fresh sperm separated by Percoll density gradient or swim-up in TALP media. The zygotes were cultrued with or without bovine oviductal epitherial cells(BOEC) in media(HECM-6 supplemented with 11 amino acid and / or TCM-199 supplemented with 10% BCS) for 7 to 10 days. The results obtained were as follow: The cleavage rate and developmental rate to blastocyst after maturation and IVF were not significantly different between Ham's F-10 with EGF(76.0% vs. 44.0%) and BCS(75.9% vs. 43.6%)(P<0.05). The cleavage rate and development rate to blastocyst after fertilizing by swim-up or Percoll method were not signifciantly(P<0.05) different between swim-up (80.2% vs. 29.2%) and Percoll(81.9% vs. 26.5%) (P<0.05). The cleavage rate in TCM 199(80.5) was signficiantly higher than that in HECM-6 (72.0%) (P<0.05). However, developmental rate to blastocyst using TCM 199 following HECM-6 for 3 or 4 days (42.2%) was significantly higher than that in TCM-199 alone(26.7%)(P<0.05). The cleavage rate and development rate of embryos produced in vitro by exchange timing for HECM-6 media were not significantly different between in day 3(78.6% vs. 45.5%) and day 4(75.0% vs. 43.2%)(P<0.05). The cleavage rate and developmental rate to blastocysts according to co-culture system were not significantly different between with (74.2% vs. 41.4%) and without BOEC(73.95 vs. 43.5%) (P<0.05). The number of blastomere in blastocyst stage after co-culture with or without BOEC was not significantly different (106.7$\pm$5.1 and 96.6$\pm$4.0). In conclusion, the most transferable IVP embryos could be produced from Ham's F-10 medium for IVM, Percoll density gradient method for IVF sperm separation and in vitro culture in HECM-6 until day 3 or day 4, and then transferred into TCM-199 until day 9 within adequate embryo density in culture droplets after insemination.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.155-163
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• 2004

The aims of this study are 1) to test oocytes and embryos collected from in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to Funahashi et al (1994). Glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at $39^{\circ}C$, and 10% fetal bovine serum albumin was added to the culture medium thereafter. Embryos were treated with 7.5 ${\mu}g/ml$ cytochalasin-B for 30 min, centrifuged at 13,000 rpm for 13 min and then exposed sequentially to an ethylene glycol(EG) vitrification solution, aspirated into OPS, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three dornors after AI for control group. Forty-nine embryos were washed 3 times in mPBS + 10% FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients were transferred individually with 100, 100 frozen embryos derived from abattoir and 34 fresh embryos by surgically, and another three recipients were transferred individually with 150, 150 frozen embryos and 100 fresh embryos by nonsurgically, respectively. all recipient sows exhibited delayed returns to estrus. To our knowledge, theses results suggest that required an improved techniques, more vigorous embryos preparation and substitute to gilt with cleaner uterous condition.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.183-190
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• 2006

These studies were conducted to monitor developmental competence of follicular oocytes collected from the carcass of the high meat quality in Korean native cattle using each individual protocol of IVM, IVF and IVC. The follicular oocytes that were collected from the ovaries of the cow yielded 1, $1^+\;and\;1^{++}$ meat quality were matured, fertilized and cultured using each individual protocol of IVM, IVF and IVC. As results, the number of follicular oocytes collected from individual fundamentally-registered cows yielded 1, $1^+\;and\;1^{++}$ meat grade were 28.9, 28.8 and 29.6 per head, respectively. The rates of blastocyst formation after IVM, IVF and IVC were 27.2, 28.7 and 32.9% in the cows yielded 1, $1^+\;and\;1^{++}$ meat quality, respectively. The rate of blastocyst formation was 8.4 per head. The number of follicular oocytes collected from pedigree registered cows yielded 1, $1^+\;and\;1^{++}$ meat quality were 25.8, 27.1 and 27.0 per head, respectively. The rates of blastocyst formation were 23.0, 33.7 and 42.6% in the meat quality of 1, $1^+\;and\;1^{++}$ after in vitro-manipulation, respectively (p<0.05). The rate of blastocyst formation was 8.5 per head. In conclusion, these results suggest that in vitro embryo production system using individual culture system including IVM, IVF and IVC can make good use of the gene from the carcass of the high meat quality in Korean native cattle.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.167-176
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• 1997

This study was carried out to evaluate the potential for preselection of transgenic embryos prior to transfer into recipient animals. In these experiments, I used a 3.2 kb transgene which contained the neomycin resistance gene (neo) and lac Z gene driven by the $\beta$ actin promoter (iacZ Ineo). Oocytes were aspirated from abattoir ovaries, matured in TCM-199 supplemented with 10% fetal bovine serum (FBS), 5 ${\mu}\textrm{g}$/ml LH, 0.5 ${\mu}\textrm{g}$/ml FSH, 100 unit/ml penicillin, and 100 ${\mu}\textrm{g}$/ml streptomycin for 22 to 24 hrs then inseminated with a sperm suspension of 1 X 10$^6$ sperm/ml containing 5 ${\mu}\textrm{g}$/ml of heparin. At 18-20 hrs after insemination, cumulus cells were removed by vortexing and pronuclei of centrifuged zygotes microinjected with the lacZ/neo construct (3 ng/$\mu$l). All cultures were carried out in CR1aa with transfected BRL monolayers containing 3 mg/ml BSA, 20 $\mu$/ml NEM amino acids and 40 $\mu$I/ml BME amino acids. To identify the appropriate concentration of G418 for selection, non-microinjected zygotes were cultured in the presence of 0, 50, 100 and 200 $\mu$g/ml of G418. After 8 days of culture in these treatments, 44/145 (30.3%), 13/150 (8. 7%), 1/151 (0.7%) and 0/134 (0.0%) devel-oped to the blastocyst stage in 0, 50, 100 and 200 $\mu$g/ml of G418, respectively. A total of 1,127 zygotes were microinjected and placed into culture (without G418) and subsequently 710 (63.0%) cleaved. At 48 hrs post-injection, embryos ($\geq$2-cell) were randomly assigned to control (medium alone) or G418 (100 ${\mu}\textrm{g}$/ml) treatments. A control culture of 740 non-microinjected embryos from the same replicates of embryos were also placed into control medium. After 8 days in culture, 54/343 (15.7%) and 22/367 (6.0 %) of the microinjected embryos developed to the blastocyst stage in control and G418 media, respectively. A total of 151/740 (27.2%) of the non-microinjected embryos placed in the control medium developed to the blastocyst stage. The blastocysts in the control treatment had a mean of 70.7 ${\pm}$ 4.7 cells of which 23.1 ${\pm}$ 2.6 (32.7%) stained for $\beta$-Gal activity. B1astocysts in the G418 treatment had a mean of 48.8${\pm}$7.5 cells of which 40.3 ${\pm}$ 4.1 (82.6%) stained for $\beta$-Gal ac tivity (P<0.05). In the control treatment 26 of 30 (87.0%) blastocysts had some cells with $\beta$-Gal activity while all of the blastocysts in the G418 treatment had some cell with $\beta$-Gal ac tivity (P<0.05). However, ICM colonies in either control or G418 treatments were not expressed any epiblast cell except of trophetoderm celIs. The $\beta$-actin promoter/lacZ gene may not be e expression or silence expression in epiblast cells These results clearly show an enrichment of blastocysts expressing the transgene in the majority of their cells after culture in the presence of G418. The exogeneous gene was not express a and silence in ICM colonies especiallly epiblast cells except of trophectederm cells. Even though the higher rate cell number expressed of exogeneous gene in the G418 treatments, a total cell number was decrease significantly (P<0.05) of which might be also drop of the establishment of ES like-cell colonies and production of transgenic animals. However, futher studies need to determine the viability of these selected embryos and the avability of production of transgenic offspring.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.61-68
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• 2002

The present study was examined effects of aesculetin and $O_2$concentrations on in vitro development of Hanwoo (Korean Native Cattle) embryos derived from in-vitro matured and fertilized (IVM-lVF) oocytes. The oocytes were cultured for the first 40~44 h after in vitro fertilization, then embryos of 2 to 8 cell stages were cultured under the different culture conditions for another 6 days. In experiment 1, the higher rates of morulae and blastocysts were produced in 5% $O_2$, than in 20% $O_2$(P＜0.05). There was significantly (P＜0.05) higher in embryos cultured with 1 $\mu\textrm{g}$/$m\ell$ than with 0, 5 and 10 $\mu\textrm{g}$/$m\ell$ of aesculetin. In experiment 2, the proportions of embryo developed with blastocysts and morulae plus blastocysts in 5% $O_2$, again was significantly (P＜0.05) higher in 20% $O_2$, during the culture with aesculetin and/or taurine. In the 5 and 20% $O_2$atmosphere, the inclusion of 1 $\mu\textrm{g}$/$m\ell$ aesculetin or 2.5mM taurine increased significantly (P＜0.05) the percentages of blastocysts and morulae plus blastocysts. In experiment 3, in medium with aesculetin plus PDGF and taurine plus EGF than other treatment groups, significantly (P＜0.05) higher developmental rates were obtained. Number of blastomere in balstocyst stage were also higher in medium with that than without aesculetin. However, there were no significant differences in all culture conditions. In experiment 4, the proportions of embryo developed to the morulae and blastocyst stages were significantly (P＜0.05) higher rates in medium with natural and commercial aesculetin than in control medium. No significant differences, however, were observed in between natural (71%) and commercial (70.0%) aesculetin. Number of blastomere in blastocyst stage were also higher in medium with natural and commercial aesculetin than in control medium. However, there was no effect on the number of blastomeres by these treatment. These data indicate that preimplatation embryos are very sensitive to condition that can cause oxygen concentration and show that efficiency role of aesculetin for improving bovine embryo development in vitro.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.19-28
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• 2001

The present study was undertaken to investigate the effects of cooling rate and equilibration time on the survival, in vitro maturation and development to embryos of frozen-thawed bovine immature oocytes(Germinal Vesicle Stage). The cryoprotectants are used 10% ethylene glycol(EG) as permeating cryoprotectant and 0.05M soc.ose(S) or trehalose(T) as low molecular weight nonpermeating cryoprotectants and 5% ficoll(F) or polyvinylpyrrolidone(PVP) as high molecular weight nonpermeating cryoprotectants. Four freezing solution were uysed in this experiment(EFT: 10% EG + 5% F + 0.05M T, EFS: 10% EG + 5% F + 0.05M S, EPT: 10% EG + 5% P + 0.05M T, EPS: 10% EG + 5% P + 0.05M S). The best equilibration time and freezing solution was 15 min in EPT(83% survival rate of frozen-thawed bovine immature oocytes). When frozen-thawed bovine oocytes were cultured following IVM and IVF, there was no significant difference in cleavage and development rates among the EFT, EFS, EPT and EPS solutions. When 9 blastocysts derived from frozen bovine oocytes were transferred to 6 recipients, two recipients were pregnant. And one was aborted at 45 days of pregnancy and the other had a stillbirth.