• Clinical and Experimental Reproductive Medicine
• /
• v.38 no.4
• /
• pp.222-227
• /
• 2011

Objective: To evaluate the ability of serum anti-M$\ddot{u}$llerian hormone (AMH), FSH, and age to clinically predict ovarian response to controlled ovarian hyperstimulation (COH) in IVF patients with endometriosis. Methods: We evaluated 91 COH cycles, including 43 cycles with endometriosis (group I) and 48 cycles with male factor infertility (group II) from January to December, 2010. Patients were classified into study groups based on their surgical history of endometriosis-group Ia (without surgical history, n=16), group Ib (with a surgical history, n=27). Results: The mean age was not significantly different between group I and group II. However, AMH and FSH were significantly different between group I and group II ($1.9{\pm}1.9$ ng/mL vs. $4.1{\pm}2.9$ ng/mL, $p$ <0.01; $13.1{\pm}7.2$ mIU/mL vs. $8.6{\pm}3.3$ mIU/mL, $p$ <0.01). Furthermore, the number of retrieved oocytes and the number of matured oocytes were significantly lower in group I than in group II. In group II, AMH and FSH as well as age were significant predictors of retrieved oocytes on univariate analysis. Only the serum AMH level was a significant predictor of poor ovarian response in women with endometriosis. Conclusion: Serum AMH may be a better predictor of the ovarian response of COH in patients with endometriosis than basal FSH or age. AMH level can be considered a useful clinical predictor of poor ovarian response in endometriosis patients.

• Korean Journal of Animal Reproduction
• /
• v.26 no.3
• /
• pp.235-243
• /
• 2002

This study was designed to examine the ability of the bovine (MII) oocytes cytoplasm to support several mitotic cell cycles under the direction of differentiated somatic cell nuclei of bovine, porcine, mouse and human. Bovine GV oocytes were matured in TCM-199 supplemented with 10% FBS. At 20h after IVM, recipient oocytes were stained with 5 $\mu\textrm{g}$/$m\ell$ Hoechst and their 1st polar body (PB) and MII plate were removed by enucleation micropipette under UV filter. Ear skin samples were obtained by biopsy from an adult bovine, porcine, mouse and human and cultured in 10% FBS added DMEM. Individual fibroblast was anlaysed chromosome number to confirm the specificity of species. Nuclear transferred (NT) units were produced by electrofusion of enucleated bovine oocytes with individual fibroblast. The reconstructed embryos were activated in 5 $\mu$M ionomycin for 5 min followed by 1.9 mM 6-dimethylaminopurine (DMAP) in CR1aa for 3 h. And cleaved NT embryos were cultured in CR1aa medium containing 10% FBS on monolayer of bovine cumulus cell for 8 days. Also NT embryo of 4~8 cell stage was analysed chromosome number to confirm the origin of nuclear transferred somatic cell. The rates of fusion between bovine recipient oocytes and bovine, porcine, mouse and human somatic cells were 70.2%, 70.2%, 72.4% and 63.0%, respectively. Also, their cleavage rates were 60.6%, 63.7%, 54.1% and 62.7%, respectively, there were no differences among them. in vitro development rates into morula and blastocyst were 17.5% and 4.3% in NT embryos from bovine and human fibroblasts, respectively. But NT embryos from porcine and mouse fibroblasts were blocked at 16~32-cell stage. The chromosome number in NT embryos from individual fibroblast was the same as chromosome number of individual species. These results show that bovine MII oocytes cytoplasm has the ability to support several mitotic cell cycles directed by newly introduced nuclear DNA.

• Korean Journal of Animal Reproduction
• /
• v.26 no.2
• /
• pp.153-163
• /
• 2002

In vitro matured porcine oocytes have very small volume of perivitellinspace (PVS). In these respect, the effect of sucrose and polybrene on the efficiency of gene transfer was investigated. As a gene (hGH) transfer vehicle, vesicular stomatitis virus glycoprotein pseudotyped retroviral vector (VSV-G) was used. Sucrose treatment has no detrimental effect on the rates of cleavage and resulted in the enlargement of PVS for the efficient introduction of retroviral vector stocks. Introduction rates of retrovirus in 0.5, 1, 2, 3 % sucrose treatment group were higher than that of the non-treatment group (39.3, 43.3, 35.7, 40.7 % vs. 8.3 %), respectively. In addition, we observed that sucrose pretreatment during injection procedure significantly reduce the frequency of polyspermy. In general, polybrene is a polycation essential for retrovirus transduction. The groups with the addition of 0.5, 5, 50$\mu\textrm{g}$/$m\ell$ polybrene exhibited a significant effect on gene transfer compared to that of the non-addition group (56.5, 50.0, 57.1 % vs. 34.6 %), respectively But, when the oocytes were co-injected with retrovirus and 50$\mu\textrm{g}$/$m\ell$ polybrene, the rates of cleavage and blastocyst development were 43.3 and 4.6%, respectively. This rates were lower than those of the non-addition group (70.0 and 17.3 %). In conclusion, sucrose pretreatment have increased efficiency of retroviral mediated gene transfer in porcine oocytes with no damage on in vitro fertilization and embryo development. In addition, sucrose pretreatment was beneficial in polyspermy inhibition. Presence of polybrene during microinjection showed a beneficial effect on the gene transfer in porcine oocytes, in low concentration. And these results will provide an useful tool for production of transgenic pigs by retroviral mediated gene transfer.

• Korean Journal of Animal Reproduction
• /
• v.27 no.2
• /
• pp.153-161
• /
• 2003

This study has evaluated effect of the spermatozoa incubation on the glycosidase activity and fertilizing ability in vitro in the pig. To identify sperm glycosidases specific for sugar residues found in the zona pellucida of pig oocytes, the spermatozoa were treated experimentally and assayed for activities of $\alpha$-L-fucosidase, $\alpha$-D-mannosidase, $\beta$-D-galactosidase and N-acetyl-$\beta$-D-glucosaminidase ($\beta$-GlcNAc'ase). The glycosidases activity were higher in spermatozoa incubated for 2h than without incubation. The $\beta$-GlcNAc'ase activity was at least two-fold higher than other glycosidase regardless of spermatozoa incubation. In the same glycosidases, the activity had a tendency to increase as time of spermatozoa incubation was prolonged, but there were no differences in spermatozoa incubated during the various periods (4~24h). The percentages of spermatozoa that reached acrosome reaction were affected by glycosidases in the medium (P<0.05, for mannosidase), and were higher in spermatozoa with that than without incubation. On the other hand, the spermatozoa motility were decreased with incubation periods, but no effects by different glycosidases on the change of sperm motility during the various periods of incubation. In other experiment, the binding and penetration of pig spermatozoa were tested with oocytes matured in vitro in the presence of various glycosidase. The penetration rates were decreased with incubation of spermatozoa when oocytes were inseminated in medium with different glycosidases. These rates were higher in spermatozoa non-incubated than with incubation for 2h (P<0.05 for GlcNAc'ase; P<0.01 for control group). The sperm-zona binding rate in control group were higherthan in medium with glycosidases. In addition, the highest binding rate were obtained in medium with GlcNAc'ase. In all glycosidases, the sperm-zona binding rate in spermatozoa without incubation were higher than incubation for 2h. The significant differences were obtained in spermatozoa treated with $\alpha$-D-mannosidase (P<0.05). These results suggest that $\beta$-GlcNAc'ase is present mainly in the plasma membrane of pig spermatozoa. It was also shown that the glycosidase activity were increased in all glycosidases in spite of low sperm-zona binding rate and penetration rates by spermatozoa incubation.

• Asian-Australasian Journal of Animal Sciences
• /
• v.30 no.4
• /
• pp.585-592
• /
• 2017

Objective: The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. Methods: Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts. Results: The competence to develop into blastocysts was not significantly different between the sexes. The mean cell number of female and male cloned blastocysts obtained by in vivo culture ($143.8{\pm}10.5$ to $159.2{\pm}14.8$) was higher than that of in vitro culture of somatic cell nuclear transfer (SCNT) groups ($31.4{\pm}8.3$ to $33.4{\pm}11.1$). After embryo transfer, 5 pregnant gilts from each treatment delivered 15 female and 22 male piglets. The average birth weight of the cloned piglets, gestation length, and the postnatal survival rates were not significantly different (p<0.05) between sexes. Conclusion: The present study found that the sex difference of the nuclear donor does not affect the developmental rate of porcine SCNT embryos. Furthermore, postnatal survivability of the cloned piglets was not affected by the sex of the donor cell.

• Korean Journal of Animal Reproduction
• /
• v.22 no.4
• /
• pp.425-435
• /
• 1998

To improve the efficiency of nuclear transplantation in bovine, in this study the development in vitro of nuclear transferred (NT) embryos was compared by different activation regimens of the enucleated oocytes. The effect of developmental stage and culture system of donor nuclei on fusion and development in vitro of NT embryos were also evaluated. Oocytes were collected from Hanwoo ovaries obtained from slaughterhouse and matured in Ham's F-10 supplemented with hormones. After 20~22 h maturation, the oocytes were vortexed to be free from cumulus cells and subsequently their nucleus and the first polar body were removed. Enucleated oocytes were divided into 3 groups for activation; the oocytes of group I were activated with ionomycin for 5 min and subsequently incubated in 6-dimetylarninopurine (DMAP) for 4 h, Those of group II were treated with DMAP for 4 h at 39 h after onset of in vitro maturation (IVM) and those of group III were kept in room temperature ($25^{\circ}C$) for 3 h at 39 h after onset of IVM. After in vitro fertilization (IVF) the embryos for muclear donor were cultured either by group culture (20 embryos /50 ${mu}ell$ drop) or individually (1 embryo /50 ${mu}ell$ drop) for 4 day and 5 day. At day 4 and 5 after IVF, blastomeres were separated in calcium-magnesium free medium, and then classified into small (day 5: $\leq$ 38 ${\mu}{\textrm}{m}$, day 4: $\leq$ 46 ${\mu}{\textrm}{m}$) and large (day 5 : $\geq$ 38 ${\mu}{\textrm}{m}$, day 4 ; $\geq$ 46 ${\mu}{\textrm}{m}$). The separated blastomeres were replaced into enucleated and activated recipient cytoplasm. The blastomere-oocyte complexes were fused by electrically. The NT embryos were cultured in TCM-199 containing 10% FCS in 39$^{\circ}C$, 5% $CO_2$ incubator for 7 day. The results obtained were summarized as follows; There were no differences in fusion and development to blastocyst between groups as group I (68%, 10%), group II (75%, 14%) and group III (73%, 9%), respectively. However, the cell number in blastocyst of NT embryos in group III were significantly fewer than in the other groups (P＜0.05). No differences in fusion and development to blastocyst were found between individual or group cultured and between small or large blastomeres of day 4 and day 5 donor embryos. From these results, it was concluded that the combination of ionomycin and DMAP, or treatment of DMAP at 39 h after onset of IVM were useful for the efficient of production of NT bovine embryos, and the individual cultured embryos could be simply used as donor nuclei for NT bovine embryo.

• Korean Journal of Animal Reproduction
• /
• v.24 no.1
• /
• pp.69-76
• /
• 2000

This study was carried out to improve of effective culture system on development of IVM/IVF/IVC bovine embryos. The cumulus-oocyte-complexes (COCs) collected from Korean cattle ovaries harvested at a local abattoir were matured in 50 ${mu}ell$ of TCM199 supplemented with 10% fetal bovine serum (FBS) and hormones (35 $\mu\textrm{g}$/$m\ell$ FSH, 10 $\mu\textrm{g}$/$m\ell$ LH, 1$\mu\textrm{g}$/$m\ell$ estradiol 17 $\beta$ under paraffin oil at 39$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. At 24 hrs after culture, matured oocytes were fertilized in vitro for 22~24 hrs with motile semen in which obtained by centrifugation of a frozen thawed semen on Percoll-density gradients (45% vs. 90%) at 500 g for 20 min. The presumptive zygotes were divided into three experimental groups. Single egg (Group 1), 25 (Group 2) or 50 eggs (Group 3) were cultured on cumulus cell in 50 ${mu}ell$ TCM199 supplement with 10% FBS for 6~9 days after fertilization. In vitro developmental rates into the blastocysts in the groups 2 and 3 were significantly (P＜0.05) higher than those of group 1 (37,27 vs. 6%, respectively). Cell number of blastocysts obtained in groups 2 and 3 at day 8 were significantly (P${mu}ell$) resulted in higher developmental competence and cell number of bovine blastocysts produced in vitro than those the culture of single embryos with cumulus cells.

• Korean Journal of Agricultural Science
• /
• v.33 no.1
• /
• pp.35-41
• /
• 2006

This study was conducted to investigate the developmental ability of interspecies cloned embryos after nuclear transfer of goat fetal fibroblast cells into porcien oocytes. Recipient porcine and goat oocytes were obtained from slaughterhouse and matured in vitro according to established protocols. Enucleation was accomplished by aspirating the first polar body and cytoplasm and a single donor cell was individually microinjected into vitelline space of the enucleated oocyte. The reconstructed oocytes were electrically fused with 0.3M mannitol fusion medium. After electro-fusion, interspecies reconstituted embryos were cultured in PZM-3 for 7 days. In porcine interspecies nuclear transfer with goat fetal fibroblast cells, the cleavage rate of reconstituted embryos were 58.9% which was no significant different from that in porcine nuclear transfer embryos (67.4%). However, the developmental rate into blastocyst stage was 5.4% in interspecies nuclear transfer which was significantly lower than that in porcine intraspecies nuclear transfer (13.6%). When the developmental ability of porcine interspecies nuclear transfer with goat cells was compared with goat intraspecies nuclear transfer, the cleavage rate of embryos were 59.2% and the developmental rate into morular and blastocyst stage was 13.6% in interspecies nuclear transfer which were significantly lower than those in intraspecies nuclear transfer embryos. This result indicated that porcine interspecies nuclear transfer with goat fetal fibroblast cells showed the developmental potential in vitro with lower cleavage and developmental rate compared with intraspecies nuclear transfer.

• Reproductive and Developmental Biology
• /
• v.35 no.1
• /
• pp.105-113
• /
• 2011

The present study investigated the nuclear remodeling, development potential with telomerase activity and transcription level of X-linked genes (ANT3, HPRT, MeCP2, RPS4X, XIAP, XIST and ZFX) in the bovine somatic cell nuclear transfer (SCNT) embryos using two different fusion and activation methods. Female adult fibroblasts were injected into perivitelline space of in vitro matured oocytes. The oocyte-nucleus complexes were fused and followed by immediately either activated (Group 1), or activated at 1 h post-fusion (hpf) (Group 2), respectively. The incidence of normal premature chromosome condensation (PCC) at 1 hpf was slightly increased in the Group 2, compared to those of Group 1, but there was no significant (p<0.05) difference. The incidence of normal pronucleus (PN) and chromosome spread at 5 and 18 hpf were significantly (p<0.05) higher in the Group 2 than those of Group 1. The cleavage rate to 2-cell stage, developmental rate to blastocyst stage, and the mean number of total and ICM cell numbers were significantly (p<0.05) higher in the Group 2, compared to those of Group 1. Level of telomerase activity was significantly (p<0.05) higher in the SCNT blastocysts of Group 2, compared to those of Group 1. Transcript levels of HPRT, MeCP2 and XIST were not significantly (p<0.05) different between blastocysts of Group 1 and 2. However, transcript level of ANT3, RPS4X, XIAP and ZFX were significantly (p<0.05) up-regulated in the SCNT blastocysts of Group 2, compared to those of Group 1. Taken together, it is concluded that oocyte activation at 1 hpf induces the enhanced developmental potential by efficient nuclear remodeling and subsequent facilitation of the nuclear reprogramming of bovine SCNT embryos.

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