• 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.

• Clinical and Experimental Reproductive Medicine
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• v.18 no.2
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• pp.201-208
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• 1991

Recently the application of gonadotropin-releasing hormone (GnRH) agonist to superovulation in previous poor responders has resulted in the improved outcomes after in vitro fertilization (IVF) outcome. However, poor responders with poor estradiol $(E_2)$ rise or single dominant follicle are a particularly challenging group. Recent reports have also shown that patients with higher basal serum follicle stimulating hormone (FSH) level, result in poorer ovarian response and lower pregnancy rate. Analysis of the differences of superovulation outcomes according to the different protocols of GnRH agonist, long (L, n = 18) and short (S, n = 16) protocols, in patients with high basal FSH levels (>20mIU/ml) were undertaken at Seoul National University Hospital from June to October 1990. The administration of GnRH agonist was begun on day 21 of the cycle in long protocol, and on day 2 in short protocol. Ages of patients and husbands, basal FSH and luteinizing hormone (LH) levels and FSH/LH ratio did not differ significantly. Types and causes of infertility were evenly distributed. Whereas the duration of stimulation and the amounts of gonadotropins administered were significantly reduced in short protocol, the numbers of oocytes retrieved and cleaved, the cleavage rate and the number of embryos transferred were higher in long protocol without statistical signifieance. The pregnancy rate per ET was 16.7% (2/12) in short protocol, and 17.6% (3/17) in long protocol. These data suggest that both protocols result in the similar superovulation outcomes in patients with higher basal serum FSH levels.

• Journal of Animal Science and Technology
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• v.58 no.3
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• pp.11.1-11.6
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• 2016

Background: This study was investigated the effects of culture media conditions on production of eggs fertilized in vitro of embryos from ovaries of high grade Korean native cow, Hanwoo. Methods: The IVMD 101 and IVF 100 were used for in vitro maturation of selected Hanwoo oocytes and In vitro embryo culture after in vitro fertilization, respectively. The IVMD 101 and IVD 101 were used for in vitro culture and completely free of serum. Results: The cleavage rates of 2-cell embryos in reference to Hanwoo oocytes were 86.7, 92.9, and 90.1 % in the control group, IVDM101 medium and IVD101 medium, respectively which indicates that the IVDM101 medium and IVD101 medium may result favorable outcomes. The in vitro development rates of blastocysts were 12.4, 38.4 and 32.4 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. For hatched blastocysts, it was 5.3, 33.9, and 28.6 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. Hence, more favorable results were expected for the hatched blastocysts in which the IVMD101 medium and IVD101 medium were used than the control group. Average cell numbers of blastocysts were 128.3, 165.7, and 163.6 in the groups of TCM-199 + 10 % FBS medium, IVMD 101 medium, and IVD 101 medium, respectively which clearly show that the IVMD 101 and IVD 101 medium consequence significantly higher cell numbers compared to the control group (i.e., TCM-199 +10 % FBS medium). Pregnancy rate after embryo transfer was 39.6 % when the serum free medium was used which is higher than that of the medium supplemented with serum (32.8 %). In addition, stillbirth rates were 4.9 % in the group of serum free medium whereas it was 13.6 % in the serum supplemented medium (13.6 %). Conclusions: Taken altogether, serum free media, the IVMD 101 and IVD 101 represented more favorable results in the embryo development rate of embryos, cell numbers of blastocyst, and pregnancy rate. Of note, the IVMD 101 medium showed better outcomes hence, it might be a better option for future applications for in vitro culture of bovine embryos.

• Clinical and Experimental Reproductive Medicine
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• v.17 no.2
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• pp.173-184
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• 1990

At infertility clinic, department of Obstetrics and Gynegology, Kyung Hee Medical Hospital, 80 patients who underwent IVF-ET from January to July, 1989 were evaluated for the prediction of endogenous LH Surge and its effects on outcome of controlled ovarian hyperstimulation (COH) were compared among LH Surge group without hCG given (N=18), with hCG given (N=5), and no-LH Surge group with hCG given (N=57). LH Surge were occurred in 23(28.7%) out of 80 patients studied. Serum E2 levels on Day-1, Day 0, Day+1, were no significant different among three groups. When basal serum LH/FSH ratio is above 1.0, the possibility of endogenous LH Surge is much higher (56.3% in LH Surge group without hCG given). Serum P4 levels on Day 0 were significantly increased in LH Surge group without hCG given. Cycles which serum P4 level is higher than l.0ng/ml were 70.6% of LH Surge group without hCG given. But there was no significant interrelationship between endogenous LH Surge and serum P4 rising rate as an efficient predictor of the occurrence of endogenous LH Surge in COH for IVF. There was no significant differences in number of follicles, follicular size on Day-1, Day 0, Day+1, and number of oocyte collected per cycle. The oocyte fertilization rate of No-LH surge group with hCG given was significantly higher than LH Surge group without hCG given. There was no significant difference in oocyte cleavage rate among three groups.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.305-315
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• 2001

In this study, the production of transgenic embryo was attempted by microinjection or round spermatid cultured with foreign DNA. At first, the expression of haploid spermatids specific gene, mTP1 in mouse and hPrm2 in hamster spermatids were investigated by RT-PCR method in testes of young mice and hamster testis. The specific gene expression first appeared at 18 days post partum (dpp) in mice spermatid and 20 dpp in hamster spermatid. Therefore, the round spermatids isolated from 17 dpp mice and 19 dpp hamster were used for the introduction of foreign EGFP gene into haploid round spermatids. For the introduction of EGFP gene haploid round spermatids suspended in medium including EGFP gene were treated with a different electric field strength at 0.11, 0.18 and 0.44 ㎸/cm. After electrical stimulation, viability of testicular sperm cells and 67.6%, 66.4% and 49.9%, in mice and 62.6%, 57.9% and 27% in hamster, respectively. These values were significantly lower than those of non-treated control groups 80.5% in mouse and 69.1% in hamster After 72 hrs culture, the highest expression rate of EGFP gene, 28.5% in mice and 32.1% in hamster were obtained from tile spermatogenic cells electroporated by the field strength or 0.18 ㎸/cm. Then, the ability of fertilization and embryonic development of haploid spermatids transfected with foreign EGFP gene were estimated by the microinjection of spermatids into hamster oocytes. The Irate pronuclear formation rate (77.5%) was lower than non-treated control (80%), and the cleavage rate of the treated group (58.8%) was lower than control (65%). To prove the foreign EGFP integration in hamster embryos, 2-cell stage hamster embryos were subjected to the observation under the fluorescence microscope, and the PCR analysis. As a result, about 44% of 2-cell embryos were showed the integration of EFGP gene into their genome. Therefore, These results suggest the possibility to produce transgenic hamsters by microinjection of haploid spermatid transfected with foreign DNA.

• Journal of Animal Science and Technology
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• v.44 no.6
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• pp.701-710
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• 2002

Studies on the Viability of In Vitro-Matured Bovine Oocytes Vitrified by Microdrop and Straw Method To establish vitrification method for bovine oocytes, mature bovine oocytes were vitrified by microdrop (MD) or straw (Straw) method and the viability of vitrified oocytes with or without cumulus cells (CC) were examined by several methods; a) parthenogenetic activation; b) pronuclear formation after in vitro fertilization (IVF); and c) embryonic development after IVF. The survival rate of vitrified oocytes by MD was significantly higher than by Straw (92.50 vs. 74.19%, p<0.05). Most of the oocytes survived from vitrification using the MD methods. Cleavage and blastocyst development of parthenogenetically activated oocytes were higher in MD (45.05% and 10.81%, respectively; p<0.05)) than those in Straw method (27.17% and 6.52%, respectively; p<0.05). Male and female pronuclear formation of vitrified-thawed oocytes with or without cumulus cells (CC) after IVF were examined, respectively. The survival rate of vitrified oocytes by MD without CC was no difference between MD and Straw (80.368.14% vs. 67.31%). Normal fertilization (2PN) rates were not different among groups (Fresh; 54.55% vs. MD; 42.22% vs. Straw; 37.14%, p>0.05). While no fertilization (<1PN) rates were significantly different between fresh and vitrified-thawed groups (Fresh; 32.47% vs. MD; 57.78% and Straw 62.86%, p<0.05). The polyspermy (3PN) was appeared in the fresh (12.99%), but no appeared in the vitrified-thawed groups. In the without CC, normal fertilization (2PN) rates were significantly different between fresh and vitrified-thawed oocytes (Fresh; 59.38% vs. MD; 17.31% and Straw; 30.43%, p<0.05). Moreover, no fertilization (<1PN) rates were significantly different between fresh and vitrified-thawed groups (Fresh; 23.44% vs. MD; 73.08% and Straw 58.70%, p<0.05). The polyspermy (3PN, >4PN) was appeared not only fresh but vitrified-thawed groups. After IVF, two-cell developmental rates of vitrified oocytes with CC by MD and Straw were significantly low compared to fresh oocytes (Fresh; 81.76% vs. MD; 22.22% and Straw; 11.36%, p<0.05). Blastocyst developmental rates of vitrified oocytes also were significantly low compared to fresh oocytes (Fresh; 28.38 vs. MD; 1.71% and Straw 0%, p<0.05). In the without CC, two-cell developmental rates were no difference between Fresh and MD (27.59% vs. 19.25%, p<0.05), while blastocyst rates were difference between Fresh and MD or Straw (4.31% vs. 0.62% and 0%, respectively; p<0.05). In conclusion, the results indicate that the vitrified bovine oocytes have the ability to develop to the blastocyst stage after IVF.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.219-227
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• 2004

This study was conducted to develop a serum-free, defined medium of IVM of pig oocytes. The TCM-199 with supplemented with polyvinylalcohol(PVA), polyvinylpyrrollidone(PVP) and porcine follicular fluid(pFF) were used as basal medium. The effects of the these additives on the rates of maturity and development under in-vitro fertilization and in vitro culture were examined and subsequently considered on the possibilities be sustituted for the bovine serum albumin(BSA). Maturation rate of pig oocytes in IVM media containing PVA(82.4％), pFF(89.4％) and BSA(90.0％) were significantly higher(P<0.05) than that of PVP(78.6％). Cleavage rate after IVF of PVP(64％) was significantly lower(P<0.05) than these of PVA(73％), pFF(77％) and BSA(73％) supplements. in vitro development rates to morulae and blastocyst on PVP(54％) were also significantly lower(P<0.05) than these of the supplements of PVA(63％), pFF(69％) and BSA(65％). In comparison of maturation and fertilization rates of pig oocytes in each supplements, the maturity rates of PVA(82.4％), pFF(89.4％) and BSA(90.0％) were significantly lower(P<0.05) than that of PVP(72.4％) and while, the fertilization rates of pFF(87.1％) and BSA(89.1％) were significantly higher(P<0.05) than these of PVA(78.0％) and PVP(70.6％). It may be concluded that PVA and pFF can be substituted far BSA in medium for culturing pig oocytes; however, it may be considered that PVP were limited to for BSA in the in vitro culture of the embryos.

• Journal of Embryo Transfer
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• v.21 no.4
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• pp.345-351
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• 2006

The objective of this study was to investigate the effects of preservation period of porcine liquid semen on bacterial contamination and in vitro production of embryo. Extended liquid semen was prepared by three mixture of boar's ejaculates from each farm without antibiotics, and were kept in $17^{\circ}C$ semen preservation incubator until use. Sperm motility was significantly (p<0.05) decreased as semen preservation time goes by (78.7$\pm$2.4% for 1 day vs. 71.1$\pm$2.4 and 64.8$\pm$2.4% for 3 and 5 days of presentation, respectively). Quantitative of bacteria in semen was significantly (p<0.05) higher in 5 days ($57.8\pm105.2\times10^4$ Cfu) compared to 0 and 3 days ($32.1\pm76.8\times10^4$ and $26.9\pm46.6\times10^4$ Cfu, respectively) of preservation. In terms of development of in vitro fertilization of porcine embryos inseminated by preserved semen, the rate of normal fertilization (2PN) was significantly (p<0.05) decreased in 5 days (56.0$\pm$2.6%) compared to 1 and 3 days (66.0$\pm$2.7 and 64.0$\pm$2.7%, respectively) of preservation. Cleavage rate was also significantly (p<0.05) affected by preservation period (75.0$\pm$4% for 1 day, 70.0$\pm$0.3 and 71.0$\pm$0.3% for 3 and 5 days, respectively). The in vitro developmental rate of blastocyst stage embryo was significantly (p<0.05) affected by semen preservation period (15.0$\pm$1.0% for 1 day vs. 11.0$\pm$0.9 and 8.0$\pm$0.9% for 3 and 5 days, respectively). It is concluded that more than 3 days of liquid semen preservation without antibiotics increased the quantity of bacteria resulted in detrimental effect on sperm motility and decreased both normal insemination rate and the developmental rate of blastocyst stage embryo.

• Journal of Embryo Transfer
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• v.20 no.1
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• pp.1-8
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• 2005

This experiment was carried out to investigate the effects of saccharide in the lactose-egg yolk(LEY) extender for freezing of boar semen on the viability, normal acrosome, fertilizable of in vitro or in vivo oocyte after thawed. Normal acrosome post-thawed spermatozoa was higher when increasing of glucose concentration in LEY extender with 3 or $4\%$ glycerol, but viability was not significant. Viability of the post-thawed spermatozoa was higher when fructose or fructose and glucose were added to LEY extender with $3\%$ glycerol than glucose and sucrose or fructose, glucose and sucrose(P<0.05). Rate of normal acrosome of post thawed spermatozoa was higher when both fructose and glucose$(81.4{\pm}2.3\%)$ were added to the LEY extender than saccharide not added$(41.6\pm0.6\%)$ to it(P<0.001). The percentage of fertilization, cleavage and development to blastocyst of oocytes fertilized with post-thawed spermatozoa from freezing by LEY extender were $70.8\~80.7\%$, $44.6\~45.7$ and $13.6\~16.0\%$, respectively. Conception rate by artificial insemination with frozen boa. semen was higher$(83.1{\pm}0.3\%)$ than commercial frozen semen from SGI company$(50.0{\pm}0.1\%,\;P<0.05)$, but litter size were no significant differences between frozen by LEY extender$(9.4{\pm}1.7\~10.4{\pm}0.7head/sow)$ and SGI semen$(8.0{\pm}1.1 head/sow)$.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.193-201
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• 2001

This study was designed to examine the factors affecting in fertilization and development of embryos in vitro, and to examine whether zone drilling by laser irradiation can improve the hatching rate of IVF embryos from DNA marker-proved Hanwoo. DNA markers related to marbling score were identified using DNA fingerprinting with Ml3 probe and restriction enzyme Hae III. Oocytes were aspirated from immature ovarian follicles using a combined method of rectal ovarian-palpation and transvaginal ultrasound-guidance(6.5MHz) under local anesthesia. The aspirated oocytes were washed twice with fresh D-PBS containing 5% FBS and were rewashed 4 to 5 times with TCM-199 containing 5% FBS. A morphological grade of I to IV was assigned to each oocyte. Data were analyzed using the GLM procedure of SAS. Sperm separation methods did not have any significant effect on cleavage or developmental abilities of IVF embryos. Significantly(P<0.05) higher cleavage rate was observed in embryos from GI(60.0%, 3/5), GII(69.2%, 18/26) and GIII(62.1%, 59/95) compared to embryos from GIV oocytes(36.2%, 25/69). And the developmental rate to blastocyst stage was higher(P<0.05) in embryos from GI(33.3%, 1/3) and GII oocytes(38.9%, 7/18) than those from GIII(16.9%,10/59) and GIV oocytes(4.0%, 1/25). There was no significant difference in development of IVF embryos to blastocyst by media for in vitro culture. Proportion of hatched blastocyst was significantly(P<0.05) higher in embryos received zona drilling by laser than those of non-drilled.