• Reproductive and Developmental Biology
• /
• v.29 no.2
• /
• pp.117-120
• /
• 2005

This study was conducted to establish the optimal temperature condition before oocyte activation in B6m F1 mouse. In experiment 1, two embryo culture media (CZB vs KSOM) were evaluated for the development of activated mouse oocytes. Parthenogenetic embryos cultured in KSOM showed better blastocyst development than ones cultured in CZB $(56.2\%\;vs\;81.0\%\;p<0.01)$. Two-hour of pre-incubation before activation significantly reduced the number of hatched blastocysts in KSOM $(22.0\%\;versus\;8.8\%\;p<0.05)$. In experiment 2, recovered oocytes were pre-incubated at different temperature conditions before activation. The experimental groups were divided by 5 as follows. Group A: pre-incubation for 120 min at $37^{\circ}C$, Group B: pre-incubation at $37^{\circ}C$ for 90 min then at $25^{\circ}C$ for 30 min, Group C: pre-incubation at $37^{\circ}C$ for 60 min then at $25^{\circ}C$for 60 min, Group D: pre-incubation at $37^{\circ}C$ for 30 min then at $25^{\circ}C$ for 90 min, and Group E: pre-incubation at $25^{\circ}C$ for 120 min before activation. Group A $(67.6\%)$ and B $(66.7\%)$ showed better development to the blastocyst stage than other groups $(Group\;C:\;50.0\%\;Group \;D:\;49.2\%\;Group\;E:\;33.3\%,\;p<0.05)$. The present study indicates that the temperature before activation affects the development of B6D2 F1 mouse parthenogenetic oocytes and exposure to room temperature should be limited to 30-min when the oocytes are left in HEPES-buffered medium for micromanipulation.

• Journal of Embryo Transfer
• /
• v.15 no.2
• /
• pp.121-128
• /
• 2000

This study was conducted to enhance the efficiency of embryo transfer and embryos produced in vitro according to select good quality blastocyst among in vivo or in vitro produced embryos. After middle blastocys(MB) and late blastocyst(LB) stages embryos that developed in vitro for 7∼8 days were treated hypertonic solution(380 mOsm or 600 mOsm) and the rate of development to hatched blastocyst(HB) was examined. The results obtained were summarized as follows: The proportion of embryos developed to HB was higher than that of control regardless of hypertonic solution treatment. However, it was related to culture time in hypertonic solution The propotrion of embryos developed to HB of morphologically recovered embryos in 380 nOsm solution ws higher than that of morphologically shrinked embryos. However, treatment time was not significantly different the rates of HB development. Especially, the proportion of embryos developed to HB of morphologically recovered embryos of the 30-min-treated group was significantly higher than that of control (p<0.05). The results suggest that hypertonic solution treatment should enhance the efficiency for criterion of transferable blastocyst quality.

• Journal of Embryo Transfer
• /
• v.31 no.1
• /
• pp.27-32
• /
• 2016

This study was conducted to evaluate the effects of different volume ($100{\mu}l$ vs. 2 ml) of microdrop culture on B6D2F1 mice oogenesis. In the present study, B6D2F1/CrljOri $F_1$ mice were utilized in order to maximize oogenesis. Also we used TCM-199, Dulbecco's medified Eagle's medium (DMEM), embryo culture medium (Fertilization medium, Cleavage medium, Blastocyst medium), G series medium and One step medium. Blastulation rate was not different between groups ($58.4{\pm}2.9%$ vs. $61.2{\pm}4.8%$). Zona hatched rate ($38{\pm}15.4%$ vs. $27{\pm}3.4%$) and attached rate ($55{\pm}13.9%$ vs. $46{\pm}3.9%$) did not differ by the volume of culture media. Total cell numbers ($59.8{\pm}9.7$ vs. $70.3{\pm}8.7$), ICM cell numbers ($15.8{\pm}0.6$ vs. $16.8{\pm}1.5$), TE cell numbers ($44.0{\pm}9.7$ vs. $53.6{\pm}7.3$), % ICM ($26.4{\pm}2.9%$ vs. $23.8{\pm}3.3%$) and ICM:TE ratio ($1:2.8{\pm}0.4$ vs. $1:3.2{\pm}0.6$) were not different between groups (i.e., $100{\mu}l$ vs. 2 ml). These results show that the capacity of the culture medium did not effect the cell numbers of B6D2F1 mice blastocysts. In summary, these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice.

• Journal of Embryo Transfer
• /
• v.29 no.2
• /
• pp.101-109
• /
• 2014

Matrix Metalloproteinases (MMP)-2 and -9 are participated in embryo development, implantation, remodeling of epithelial cell and ovulation. The objective of this study is to evaluate an impact of MMP2 and MMP9 on embryonic developmental competence as well as gene expression profiles of in vitro-produced bovine embryos. After in vitro fertilization, embryos of all groups were transferred into IVC-2 medium treated with MMP2 and MMP9 to check the optimum concentration on the basis of embryo development competence and cell numbers. The optimum concentrations for MMP2 and 9 were 1,200 ng/ml and 300 ng/ml. The blastocyst development competence was not different among 1,200 ng/ml of MMP2 vs. 300 ng/ml of MMP9 vs. combined MMP2 + 9 vs. control groups ($41.46{\pm}10.66$ vs. $37.73{\pm}8.92$ vs. $45.11{\pm}11.41%$ vs. $41.59{\pm}11.88$, respectively). Furthermore, the developmental competences to hatching and hatched blastocysts were not also different among the same groups ($79.84{\pm}12.63$ vs. $83.3{\pm}17.46$ vs. $78.55{\pm}14.48%$ vs. $72.02{\pm}14.09$). In addition, total cell number was significantly (p<0.05) greater in blastocyst treated with MMP9 300 ng/ml among all treatment groups. On the other hand, there was no significant difference of ICM vs. TE ratio in all groups. The expression of five out of six genes (i.e., MMP2, MMP9, IFNt, SSLP1 and HNRNPA2B1) was different among the groups. The expression of IFNt and HNRNPA2B1 genes was significantly greater in MMP9 (p<0.05), but there was no difference of MMP9 expression between MMP2 and MMP9 group (p>0.05). The normalized expression of MMP2 and SSLP1 was greater in MMP2 than other groups (p<0.05). In conclusion, MMPs treatment during IVC-2 medium was remarkably effected on blastocyst developmental competence and gene expression profiles that are related to embryo quality and implantation.

• Korean Journal of Animal Reproduction
• /
• v.20 no.1
• /
• pp.19-26
• /
• 1996

In the present study, we investigated devel-opmental ability and transgene expression of IVM/IVF derived porcine embryos following microinjection with SV40-LacZ. A total of 412 IVM/IVF derived embryos were used to examine developmental ability and transgene expression following DNA microinjection. After centrifugation, pronuclei were visible in 60.3% when examined between 18~21h after IVF. Development and transgene expression were assessed after 9 days in culture. The percentages of injected embryos reaching to the morula and blastocyst were significantly lower (P<0.05) than those of non-injected control embryos. However, the percentages of DNA microinjected embryos and non-injected embryos that developed to the blastocyst or hatched blastocyst stage in dual culture systems (NCSU23 and EMEM) were significantly higher (P<0.05) than those in NCSU23 medium alone. As the resuIt of X-gal staining, the proportion of positive embryos was 40~43% in morula and blastocyst stage embryos, however, mosaicism has been observed in the most putative transgenic morulae and blastocysts. In the PCR analysis, the percentages of embryos integrated gGH gene were 45.0 and 44.4% in morula and blastocyst stage, respectively. These results suggest that improved IVM /IVF system and culture condition increased the embryo viability and ex-pression of a microinjected transgene.

• Korean Journal of Animal Reproduction
• /
• v.25 no.2
• /
• pp.131-138
• /
• 2001

This experiment was designed to evaluate effects of nonspecific immunostimulator(NIS) Barodon-FX(equation omitted), anionic alkali mineral complex and far-infrared radiation solution on in vivo-produced mouse and in vitro-produced bovine embryos to blastocyst development. Proportion of mouse embryos developing into blastocyst was not greater in BSA- and Barodon-added medium than in BSA-control, but there was signifcantly different(P ＜ 0.05) in hatching and hatched blastocyst development between 0.25% Barodon-and PVP-contained medium(54.7%) than PVP-control(32.5%). BOEC and GC resulted in higher proliferation rate(24∼40% and 17∼22%, respectively) in 0.25∼0.5% Barodon-added medium than in controls, but proliferation of GC and CC greatly decreased in 1∼2% Barodon-added medium. Effect of Barodon on cell proliferation greatly varied among somatic cells. Proportion of early bovine embryos developing into morula and blastocyst was significantly greater(P ＜ 0.05) in 0.5% Barodon-added medium(50% and 63.6%) than in control(31.6% and 27.4%) under co-culture with BOEC and GC, but developmental rate was not different between other Barodon treatments and control. These data indicate that effect of Barodon on cell proliferation significantly varied between somatic cells and that addition of 0.5% Barodon in BOEC-coculture system may further improve blastocyst development in early bovine embryos.

• Korean Journal of Animal Reproduction
• /
• v.22 no.3
• /
• pp.287-292
• /
• 1998

The objective of this study was to test whether ZP drilling using a 1.48$\mu$m diode laser beam on mouse IVF embryos becomes effective the hatching and normal in vivo development, as a preliminary test for obtaining the additional proof that the 1.48$\mu$m diode laser could be used safely for human applications. The results obtained in this experiment were as follows: when the hatched rates of mouse embryos by laser ZP drilling according to the embryonic stage were examined until 72 hr (in case of blast tocyst: day 4 after IVF) or 120 hr (in case of 4-cell: day 2 after IVF) after treatment, the d data of laser drilled blastocysts (81.8%) was significantly higher than those of control (hatching blastocyst: day 4 after IVF) (54.2%) and laser drilled 4-cell embryos (45.5%) (p<0.05). When the effect of laser drilling on implantation rates following embryo transfer in day 3 synchronized pseudopregnant recipients was examined, the l laser drilled group (48.7%) was slightly higher than that of control group (43.6%). In addition, when the several pregnant mice delivered in two groups were analysed their chromosomal normality and tested reproductive ability, all p pups were presented normal chromosomal number (n=40) and showed normal growth and reproductive ability. Therefore, these results dem-onstrated that ZP drilling using a 1.48$\mu$m diode l laser can increase the embryo hatching and ind duce the normal pregnancy of mouse embryos.

• Clinical and Experimental Reproductive Medicine
• /
• v.47 no.4
• /
• pp.312-318
• /
• 2020

Objective: The objective of the study was to compare the effects of long-term and short-term embryo culture to assess whether there is a correlation between culture duration and clinical outcomes. Methods: Embryos were divided into two study groups depending on whether their post-warming culture period was long-term (20-24 hours) or short-term (2-4 hours). Embryo morphology was analyzed with a time-lapse monitoring device to estimate the appropriate timing and parameters for evaluating embryos with high implantation potency in both groups. Propensity score matching was performed to adjust the confounding factors across groups. The grades of embryos and blastocoels, morphokinetic parameters, implantation rate, and ongoing pregnancy rate were compared. Results: No significant differences were observed in the implantation rate or ongoing pregnancy rate between the two groups (long-term culture group vs. short-term culture group: 56.3% vs. 67.9%, p=0.182; 47.3% vs. 53.6%, p=0.513). After warming, there were more expanded and hatching/hatched blastocysts in the long-term culture group than in the short-term culture group, but there was no significant between-group difference in embryo grade. Regarding pregnancy outcomes, the time to complete blastocyst re-expansion after warming is shorter in women who became pregnant than in those who did not in both culture groups (long-term: 2.19±0.63 vs. 4.11±0.81 hours, p=0.003; short-term: 1.17±0.29 vs. 1.94±0.76 hours, p=0.018, respectively). Conclusion: The outcomes of short-term culture and long-term culture were not significantly different in vitrified-warmed blastocyst transfer. Regardless of the post-warming culture time, the degree of blastocyst re-expansion 3-4 hours after warming is an important marker for embryo selection.

• Korean Journal of Animal Reproduction
• /
• v.23 no.4
• /
• pp.293-301
• /
• 1999

This study was to examine whether Hanwoo IVM/IVF/IVC blastocyst can be successfully survived in vitro/in vivo after vitrification and one-step dilution. For vitrification, blastocysts were serially exposed in glycerol (G) and ethylene glycol(EG) mixtures［10% (v Iv) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.］ which is diluted in 10% FBS added D-PBS. And then they were loaded in the straw, placed in cold nitrogen vapor for 3 min. and plunged into L$N_2$(－196$^{\circ}C$). One-step dilution within the straw was done in $25^{\circ}C$ and 36$^{\circ}C$ water for about 5 min. and 3 min., respectively. Recovered embryos after one-step dilution were cultured in cumulus cell mono-layered drop for 48 h or were transferred into recipient cows. When the embryo survival in vitro was assessed to re-expanded and hatched rates at 24 hand 48 h after one-step dilution, the results of vitrified group (85.4, 43.8%) was high, although these results were significantly lower than normal development (100.0, 63.3%) of control group, respectively (P＜0.001, P＜0.05). When in vitro survival of vitrified groups according to developmental stage was compared, the results of fast developed embryos (expanded blastocyst and early hatching blastocyst stage) were significantly higher than those of delayed developed one (early blastocyst stage) after one-step dilution (early hatching: 88.0, 48.0%: expanded: 81.1, 45.3%; early: 66.7, 14.3%) (P＜0.05). Also, in case of in vitro survival of vitrified groups according to embryo age (day 7, 8 and 9), when embryo age was younger, in vitro survival was significantly higher (day 7: 67.3, 34.5%; day 8: 76.9, 40.7%; day 9: 60.9, 23.9%)(P＜0.05). Finally, when in vivo development potential of vitrified and one-step diluted Hanwoo blastocysts was examined, 4 of 8 recipient (50%) cows became confirmed pregnant. These results demonstrated that our vitrification and one-step dilution technique can be applied easily and effectively on field trial without the equipment and embryological skills required for conservative dilution and transfer.

• Journal of Embryo Transfer
• /
• v.21 no.3
• /
• pp.255-262
• /
• 2006

The purpose of this study is to investigate the effects of vitrification in open pulled straws (OPS) methods on in vitro survival ability of porcine embryos. For in vitro maturation of immature oocytes, the porcine ovaries were collected from local slaughter-house. The cumulus-oocytes complexes were aspirated from 2 to 6 mm follicles. The collected oocytes were cultured for in vitro maturation in NCSU-23 medium with 5 mM hypotaurine, 0.57 mM cysteine, 10% porcine follicle fluid, 10 IU/ml PMSG and 10 IU/ml hCG for $21{\sim}22$ hrs. Then, the oocytes were more cultured $21{\sim}22$ hrs in vitro maturation in medium removed hormones. The frozen-thawed spermatozoa were washed by centrifugation 2 times for 10 min at 1,500 rpm in D-PBS with 5.56 mM glucose, 0.33 mM Na-pyruvate, 100 IU/ml penicillin, $100 {\mu}g/ml$ streptomycin and 4 mg/ml BSA. The fertilization medium used mTBM with 2 mM caffeine and 2 mg/ml BSA and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to $2.5{\times}10^6$cells/ml motile sperm during fertilization in vitro. At 8 hrs after insemination, the oocytes were transferred into NCSU-23 medium with 5.0 mM hypotaurine, 4 mg/ml BSA and 10 ng/ml EGF and cultured for 7 days. When the blastocysts of different stages were frozen-thawed by OPS methods, the proportions of embryos with normal morphology were significantly (p<0.05) higher in embryos frozen-thawed at expanded blastocyst stage (38.9%) than in early blastocyst stage (28.3%). On the other hand, the proportions of embryos damaged after frozen-thawing were significantly (p<0.05) higher in embryos frozen at early blastocyst stages than in expanded blastocyst stage. In another experiment, the normal embryos morphology after frozen- thawing were further cultured for 48 hrs. After culture, the proportions of embryos hatched were 6.7, 20.0 and 33.3% for embryos frozen-thawed at early blastocyst, mid-blastocyst and expanded blastocyst stages. These finding indicate the possible broader application for OPS methods, as frozen-thawed embryos may be accompanied by developmental stage according to requirements of the survival ability after freezing of blastocyst stage in the pig.