• Journal of Embryo Transfer
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• v.22 no.3
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• pp.195-198
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• 2007

This study was carried out to investigate the effects of hormones treated (progesterone, and combination of progesterone and estradiol) on the reproductive performance of dairy cattle. The intravaginal CIDR was inserted in the vagina of cow on day 14 (P-7, PE-7), 16 (P-5, PE-5) and 18 (P-3, PE-3) post-insemination to stimulate progesterone secretion with (PE) and without estradiol(P). The CIDR was removed on 7 (P-7, PE-7), 5 (P-5, PE-5) and 3 (P-3, PE-3) days after. The cows for control group were not treated with CIDR or hormones. Conception rate, the duration needed for conception after AI and the number of AI services needed for conception were measured. Conception rate in control, CIDR without estradiol (P-7, P-5 and P-3) and CIDR treated (PE-7, PE-5 and PE-3) cows were 15.8%, 40.0%, 31.5%, 28.6%, 62.5%, 50.0% and 0%, respectively. The days needed for conception after AI in control, P-7, P-5, p-3, PE-7, PE-5 and PE-3 were 64.9, 63.0, 59.1, 8.0, 0.0, 18.9 and 83.3 days, respectively. The days needed for conception in cows treated with CIDR (PE-7) has shown longer than control and PE-3 (p<0.05). The number of AI services needed for conception in control, CIDR (PE-7, PE-5 and PE-3) and CIDR without estradiol treated cows (P-7, P-5 and P-3) were 27, 2.3, 1.9, 1.3, 1.0, 1.7 and 2.8 times, respectively. The number of AI services needed for conception have shown significantly lower in PE-7 compared to control and PE-3.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.201-208
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• 2004

This study was performed to examine the effects of catalase and $\beta$-mercaptoethanol ($\beta$ME) on the establishment of clonal lines from porcine fetal fibroblast cells. Fibroblasts derived from a pig fetus (Day 50) were passaged two times before use. A single cell was seeded in 96-well plates and cultured in medium supplemented with or without catalase or $\beta$ ME. Cell colonies were passaged two times into 4-well dish. Cell lines with proliferating potential were classified as an established clonal cell line. In experience 1, the establishment efficiencies were examined by addition of catalase (100ng/$m\ell$) or $\beta$ME (100 uM) in culture medium. The establishment efficiency of $\beta$ME-added group (8.3％) was significantly higher than that of control group (3.2％, P<0.05). However, catalase did not have a positive efffct on the establishment efficiency. In experience 2, the establishment efficiencies were examined by addition of different concentrations of catalase (0-1,000 ng/$m\ell$) in culture medium. However, establishment efficiencies were not different among the different concentrations of catalase (0-2.6％). In experience 3. the establishment efficiencies were examined by addition of different concentrations of $\beta$ME(0-1,000 uM) in culture medium. The establishment efficiency was significantly higher in 100 uM $\beta$ME-added group (9.4％) compare to others (0-1.6％). The result of present study shows that the establishment efficiency of clonal cell lines can be enhanced by the culture in media supplemented with 100uM $\beta$ME. However, catalase did not have a positive effect on the establishment efficiency.

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.61-68
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• 2003

Considerable attention has been focused on the cryopreservation of semen and estrus induction in dog, as consequence of poor productivity caused by long anestrus period, in order to enhance the productivity of youngs and to preserve the breeds. The objectives of this study were to improve reproductive efficiency of artificial insemination with fresh- and frozen-semen following estrus induction in dog. Fifty infertilie dogs (age 2~3 years) were selected fur the study and divided into three different estrus induction treatment groups. Group 1 : dogs (n=15) were given clomifene (0.1 mg/kg) orally f3r five days at 12 hr intervals. Croup 2: dogs (n=15) were given bromocriptine (50 $\mu$g/kg) orally for five days at 12 hr intervals, followed by single injection intravenously of 500 IU GnRH (Croup 3, n=20) when pro-estrus occurred. After being treated, the dogs were evaluated fur the rates of estrus induction and time interval lapses from treatment to beginning of the pro-estrus. The rates of pregnancy in estrus inducted dogs mated naturally compared to those inseminated artificially with ejaculated fresh semen and frozen-thawed semen. Estrus detection was performed using the method of vaginal smear and confirmed by the plasma progesterone assay. Pregnancy was confirmed by ultrasonograpy on day 25, 35 and 55 post insemination. The ejaculated semen was exposed to a mixture of Tris extender with cryoprotectant (Trisma, 81 mM; TES, 209 mM; citric acid, 6 mM; glucose, 5 mM; glycerol, 8%) and cryopreserved gradually by slow-cooling at 17 co above the surface of liquid nitrogen (L$N_2$) for 23 min. The use of fresh semen, the pregnancy rates were observed 66.6, 66.6, 75.0 and 83.3% in natural estrus, clomifene induced, bromocriptine induced and a combination of GnRH and bromocriptine, respectively. The use of frozen-thawed semen, the pregnancy rates were observed 66.6, 33.3, 50.0 and 60.0% in natural estrus, clomifene induced, bromocriptine induced and a combination of GnRH and bromocriptine, respectively. No difference was observed in the number of offspring produced among natural estrus and treated groups inseminated with fresh or frozen-thawed semen. In conclusion, there was no significant differences in the pregnancy rate of dogs between group treated with a combination of GnRH and bromocriptine and group treated clomifene or bromocriptine only. However, frozen-thawed semen can be used successfully fur artificial insemination in dog.

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.16 no.2
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• pp.205-210
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• 1989

To investigate the effects of ovarian cysts on the controlled ovarian hyper-stimulation cycles, 16 patients with 16 paired cycles for IVF-ET were analyzed. These patients had taken both type of cycles, i.e., with cyst(cyst group) and without cyst(control group). Mean diameter of ovarian cysts in cyst group was 18.2mm. There were no significant differences in hormone levels in early follicular phase between two groups. No significant differences were found in total dosage of hMG(IU) administered during the ovarian stimulation $843.8{\pm}123.0$ vs $891.0{\pm}129.8$, serum estradiol level (pg/ml) on the day of hCG administration($1542.8{\pm}1100.6$ vs $1567.5{\pm}1193.0$), the number of aspirated follicles $10.0{\pm}3.4$ vs $11.2{\pm}4.3$ and oocytes $5.3{\pm}3.3$ vs $6.2{\pm}3.1$, the fertilization rate(51.2 % vs 57.2 %) and the cleavage rate(40.5 % vs 52.0 %). Serum estradiol terminal patterns during COH in one group tended to be repeated in the other group. In conclusion, this study suggests that small ovarian cysts do not adversely impact on the controlled ovarian hyperstimulation parameters in IVF - ET program and the presence of small ovarian cyst without concomitant high basal serum estradiol level is not an indication of the cancellation of the controlled ovarian hyperstimulation for IVF-ET.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.3
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• pp.169-176
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• 2004

Objective: This study was performed to evaluate and compare the embryonic developmental capacity and pregnancy rates in conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with ejaculated sperm or testicular sperm cycles. Materials and Methods: Fertilization was examined in the following morning after IVF (group I), ICSI (group II) or TESE-ICSI cycles (group III). Fertilized oocytes were co-cultured with Vero cells until embryo transfer (ET). On day 2 and $5{\sim}7$, grades of embryos (<4- or $\geq$4-cell) and blastocysts (BG1, 2, 3 or early) were evaluated. Clinical pregnancy rate was determined by detecting G-sac with transvaginal ultrasonogram. We analyzed the results by $X^2$ and Student's t-test and considered statistically significant when P value was less than 0.05. Results: Fertilization rate was significantly higher (p<0.05) in group I ($79.0{\pm}21.2%$) than in group II and III ($56.8{\pm}21.6%$ and $36.7{\pm}25.3%$). Cleavage and blastulation rate of group I ($95.8{\pm}13.8%$ and $59.5{\pm}25.3%$) were significantly higher (p<0.05) than those of group III ($83.4{\pm}18.6%$ and $40.4{\pm}36.5%$). Clinical pregnancy rate was significantly higher (p<0.05) in group I and II (40.7% and 41.7%) than that in group III (12.5%). No differences were found in the rates of multiple pregnancy and abortion among three groups. Embryonic implantation rate was higher in group I ($15.1{\pm}20.2%$, p<0.05) and II ($14.7{\pm}20.6%$, NS) than that in group III ($5.1{\pm}15.6%$). However, embryonic implantation rate was increased in ET with blastocyst(s) among three groups. Conclusions: Fertilized oocytes obtained from TESE-ICSI were harder to be successfully cultured to blastocyst stage for 5$\sim$7 days than that from IVF cycles. However, all blastocyst(s) ET increased the embryonic implantation rate equally in IVF, ICSI and TESE-ICSI cycles.

• Parasites, Hosts and Diseases
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• v.27 no.4
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• pp.231-248
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• 1989

The growth and development of the metacercariae of F. seoulensis cultivated in vitro or on the chick chorioallantois were assessed by comparison with the optimum process of maturation in albino rats and new born chickens. The process of maturation was divided for convenience into six stages: Stage 1 ; cell multiplication, Stage 2; body shaping, Stage 3; separation of genital anlagen, Stage :1 organogeny, Stage 5; gametogony, and Stage 6: oviposition. In Hank's and Tyrode's .solutions, the metacercariae were alive up to 200 days or more at $4^{\circ}C$ without any development. The in vivo maturation process in rats or chicks was as follows: stage 1 from 6 hours; stage 2 from 24 hours; stage 3 from 48 to 72 hours; stage 4 from 3 to 4 days; stage 5 from 4 to 5 days; and stage 6 from 5 to 8 days. Despite unsuccessful infection of the metacercariae to 12 day old chicks, fully mature worms of stage 5 or 6 were recovered from new born chicks (1 to 2 days old), The metacercariae of F. seoulensis grown in vitro were up to stage 3 and no further maturation was observed. Of various media employed, the medium NCTC 109 (Gibco) or NCTC 135(Gibco) supplemented with 20% egg yolk or 20% whole egg macerate or 0.5% yeast was basically required for the earlier development of the fluke. It took 16.1 days(in average) to reach the stage 3 after cultivation. The metacercariae cultivated on the chorioallantoic membranes of 6∼13 day old chick embryo at 37∼38℃ showed their full development up to stage 5 or 6. However, the worms were in general remarkably retarded, compared with those grown in rats or chickens. In the experiments of worm transplant, although the transfer was failed from in vitro culture to in vivo of rats(Per os), the transplants from in vitro culture to the chorioallantois and from the choriollantois to in vivo of rat host were successful with or without development of the transferred worms. In the present study, it was observed that the metacercariae of F, seoulensis can be maintained in vitro media with poor development as well as fully matured in 1 to 2 day-old chicks or on the chorioallantois at a very low rate.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.251-260
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• 1998

It is well known that the clinical test for responsibility of accurate fertilization capacity in male partners is very important to diagnose and treat the infertility. However, it has been reported that the traditional semen analysis cannot accurately predict fertilization and pregnancy potential. The present study was performed to evaluate the acrosomal reaction to ionophore challenge (ARIC) test as a prognostic indicator for fertilization of sperm and oocyte in an in vitro fertilization and embryo transfer (IVF-ET) program. From March 1996 to Februry 1997, 30 couples undergoing IVF program were allocated to this study group. All female partners in the study group were 35 years old or less and their serum level of basal follicle stimulating hormone (FSH) and estradiol $(E_2)$ were normal. All the male partners have normal parameters of semen analysis. The ARIC tests were performed on the day of ovum pick up and in vitro insemination in all the male partners. The controlled ovarian hyperstimulation (COH) using luteal long protocol of gonadotropin releasing hormone (GnRH) agonist was used in all couples for IVF-ET. The acrosomal reaction with $10{\mu}l$ of 10% DMSO was induced spontaneously in $10.1{\pm}9.8%$, and acrosomal reaction with calcium ionophore A 23187 was induced in $27.4{\pm}18.1%$, and the ARIC value was $17.4{\pm}16.2%$. There were no significant correlation between the ARIC value and the fertilization rate ($r^2$=0.044, p=0.268). There were also no significant correlation between the ARIC value and the percentage of the grade I, II embryos ($r^2$=0.046, p=0.261). On the basis of above results, it was suggested that ARIC test might not be a useful prognostic indicator for fertilization in IVF-ET in male partners with normal parameters of conventional semen analysis. We guessed that IVF-ET could be performed to the patients primarily without universal appilcation of ARIC test to all male partenrs, and if fertilization failure occurs, the micro assisted fertilization (MAF) such as intracytoplsmic sperm injection (ICSI) might be used as an alternative mode of treatment with acceptable success rate.

• Korean Journal of Plant Tissue Culture
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• v.26 no.3
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• pp.189-195
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• 1999

This study was carried out to clarify several factors affecting embryogenesis from anther culture of nine cultivars in Brassica oleracea L. var. italica and to investigate the characteristics of plants derived from anther culture. Androgenesis from anther culture was elevated on the B5 medium supplemented with 0.1mg/L NAA, 0.1mg/L 2.4-D and 10% sucrose. Embryo production in liquid medium was five-fold higher than solid medium. High temperature treatment at 35$^{\circ}C$ for one day before transfer to culture room maintained at $25^{\circ}C$ had effective to induce embryogenesis of cultured anthers but extended treatment at 35$^{\circ}C$ decreased significantly the percent of embryogenesis. Frequency of embryogenesis from cultured anthers exhibited significant difference from 2.8% in 'Green Valiant' to 21% in 'Haisi' as affected by genotypes. Percent of spontaneously dihaploid among regenerated plants from anther culture was ranged from 62 to 74% as affected by the genotypes. Characteristic in relation to plant height, number of leaves and branches, and size of head from anther-derived plants showed differential variation in 'Rokguray' and 'Haisi'. Among these charaters obtained from two cultivars, five lines were selected for early maturity, long plant height and large head. Selected lines were used as breeding meterials for F$_1$ hybrid.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.281-287
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• 2013

A major barrier to progress in pig to primate organ transplantation or cell therapy is the presence of terminal ${\alpha}$-1,3-galactosyl epitopes on the surface of pig cells. Therefore, the purpose of this experiment was to establish and cha- racterize mesenchymal stromal/stem cells (MSCs) derived from ${\alpha}$-1,3-galactosyltransferase (GalT) knock out (GalT KO) pig to confirm their potential for cell therapy. Bone marrow (BM)-MSCs from GalT KO pig of 1 month old were isolated by Ficoll-Paque PLUS gradient and cultured with A-DMEM + 10% FBS on plastic dishes in 5% $CO_2$ incubator at 38.5. GalT KO BM-MSCs were analyzed for the expression of CD markers ($CD45^-$, $29^+$, $90^+$ and $105^+$) and in vitro differentiation ability (adiopogenesis and osteogenesis). Further, cell proliferation capacity and cell aging of GalT KO BM-MSCs were compared to Wild BM-MSCs by BrdU incorporation assay (Roche, Germany) using ELISA at intervals of two days for 7 days. Finally, the cell size was also evaluated in GalT KO and Wild BM-MSCs. Statistical analysis was performed by T-test (P<0.05). GalT KO BM-MSCs showed fibroblast-like cell morphology on plastic culture dish at passage 1 and exhibited $CD45^-$, $29^+$, $90^+$ and $105^+$ expression profile. Follow in ginduction in StemPro adipogenesis and osteogenesis media for 3 weeks, GalT KO BM-MSCs were differentiated into adipocytes, as demonstrated by Oilred Ostaining of lipid vacuoles and osteocytes, as confirmed by Alizarinred Sstaining of mineral dispositions, respectively. BrdU incorporation assay showed a significant decrease in cell proliferation capacity of GalT KO BM-MSCs compared to Wild BM-MSCs from 3 day, when they were seeded at $1{\times}10^3$ cells/well in 96-well plate. Passage 3 GalT KO and Wild BM-MSCs at 80% confluence in culture dish were allowed to form single cells to calculate cell size. The results showed that GalT KO BM-MSCs($15.0{\pm}0.4{\mu}m$) had a little larger cell size than Wild BM-MSCs ($13.5{\pm}0.3{\mu}m$). From the above findings, it is summarized that GalT KO BM-MSCs possessed similar biological properties with Wild BM-MSCs, but exhibited a weak cell proliferation ability and resistance to cell aging. Therefore, GalT KO BM-MSCs might form a good source for cell therapy after due consideration to low proliferation potency in vitro.