• Korean Journal of Animal Reproduction
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• v.21 no.4
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• pp.389-396
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• 1997

Arrest in embryo development during in vitro culture has been reported in various mammals. Although some cause of the arrest have been suggested, little is known of the way that can overcome the arrest using in vitro culture system. The antioxidant, $\beta$-mercaptoethanol($\beta$-ME), has been shown to play an important role in embryo development. This study was designed to examine the effect of $\beta$-ME on the developing boving embryos produced in vitro by IVM and IVF. To select a, pp.opriate concentration of $\beta$-ME during whole culture period (7 days), various concentrations (10, 50 and 100$\mu$M) of $\beta$-ME were added to the CZB medium and their effects was significantly higher in 100$\mu$M of $\beta$-ME. The effects on development of embryos cultured with and without somatic cells to blastocyst stage were greater in FCS treatment (56.6 and 29.3%) than in BSA treatment(25.5 and 12.8%). We also evaluated the effects of $\beta$-ME addition on the blastocyst formation when embryos at different stages were exposed to 100$\mu$M $\beta$-ME. $\beta$-ME promoted increased development of embryo to blastocyst stage and the effect was greater in 6-cell to morula embryos than in embryos fewer than 4-cells at the initiation of treatment. The results suggested that $\beta$-ME can improve bovine embryo development by overcoming the arrest in early development.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.145-147
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• 2005

Autocrine or paracrine mediators released by the early embryo are implicated in the support of embryonic development. Their mechanisms and optimal embryo density in the medium, however, are uncertain. This study was conducted to establish the optimal embryo density and culture medium volume in mouse parthenogenetic embryo culture. In experiment 1, culture of parthenogenetirally activated oocytes at a concentration of $2{\~}4$ embryos/${\mu}L$ significantly improved development to the blastoryst stage ($72{\%}{\leq}$) compared with culture at the lower ($0.2{\~}1$e mbryos/${\mu}L,\;0\~37.5\%$) and the higher ($5{\~}6$ embryos/${\mu}L,\;30\~53\%$) concentration for 120 h when the oocytes were cultured in a 5 ${\mu}L$ drop under mineral oil In experiment 2, the embryos cultured at a concentration of $2{\~}4$ embryos/${\mu}L$ in a 10 ${\mu}L$ drop ($81.1{\%}$) showed significantly higher blastocyst rates than those in a 5 ${\mu}L$ drop ($68.5{\%}$). This study optimizes in vitro culture condition by modifying embryo density and the volume of culture medium It may give appropriate level of autocrine and/or paracrine factors to enhance viability and subsequent normal development of mouse parthenogenetic embryos in vitro.

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.171-176
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• 1999

This study was carried out to investigate the effect of co-culture system(bovine oviduct epithelial cells; BOEC) and defined culture system (modified TALP ; mTALP) on the conception of embryos transferred, and pregnancy and twin birth rates after transfer of fresh or frozen-thawed bovine blastocysts produced in vitro were also evaluated. Oocytes from the slaughterhouse ovaries were matured and fertilized using general protocol. The results obtained were as the following. The pregnancy rate after transfer was higher in co-culture group than in mTALP group, but was not signficantly different, and there is no difference between fresh embryo group and frozen-thawed embryo group in conception rate. The conception rate was not different whether 3∼4 blastocysts or 2 blatocysts transferred into a recipient, but the production rate of twin calves was significantly higher (p<0.05) when 3∼4 embryos transferred. The average birth weight of twin calves(24.38kg) was numerically, but not significantly lighter than that of single calves(26.68kg).

• Journal of Forest and Environmental Science
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• v.37 no.4
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• pp.323-330
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• 2021

Zygotic embryo culture was performed to propagate evergreen oak, Quercus myrsinifolia, which has recalcitrant seeds and is difficult to propagate by cuttings. Zygotic embryos appeared in WPM medium after 14 days, and after 56 days, they developed into complete plants with cotyledons and roots. The medium suitable for zygotic embryo culture was 1/4 WPM medium, showing a shoot growth of 2.43 cm and root growth of 8.7 cm after 8 weeks of culture. As a result of investigating the effect of GA₃ on the growth of plants germinated from zygotic embryos through GA₃ treatment, the best growth was shown in 0.5 mg/l GA₃ treatment. The in vitro rooting and growth of IBA-treated zygotic embryo-derived plants were good in the 0.5 mg/l IBA treatment and rooting and shoot growth were not observed at higher concentrations. And the callus induction rate also increased as the concentration of IBA increased. Plants grown in vitro were transferred to a plastic pot containing artificial soil and acclimatized in a greenhouse for about 4 weeks, resulting in more than 90% survival. As a result of this study, the zygotic embryo culture method was confirmed to be effective for mass propagation of Q. myrsinifolia. The results of this study are expected to contribute significantly to the mass propagation of elite Q. myrsinifolia.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.1-5
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• 2014

Recent 2 decades, including in vitro maturation (IVM), assisted reproductive technologies (ARTs) achieved noteworthy development. However the efficiency of ARTs with in vitro matured oocytes is still lower than that with in vivo oocytes. To overcome those limitations, many researchers attempted to adapt co-culture system during IVM and consequently maturation efficiency has been increased. The beneficial effects of applying co-culture system is contemplated base on communication and interaction between various somatic cells and oocytes, achievement of paracrine factors, and spatial effects of extracellular matrix (ECM) from somatic cell surface. The understanding of co-culture system can provide some information to narrow the gap between in vitro and in vivo. Here we will review current studies about issues for understanding cu-culture system with various somatic cells to improve in vitro maturation microenvironment and provide bird view and strategies for further studies.

• Journal of Embryo Transfer
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• v.11 no.2
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• pp.125-135
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• 1996

The present study was carried out to investigate the effects of cryoprotectants, equilibration step, freezing rate, culture condition following in vitro fertilization, and age and development stage of embryo by freezing with conventional slow freezing and vitrification on survival of frozen-thawed Korean native cattle(KNC) blastocysts produced in vitro. The KNC blastocysts produced in vitro were equilibrated in 1.8M ethylene glycol or 1.4M glycerol and cooled from -6$^{\circ}C$ to -35$^{\circ}C$ at -0.3$^{\circ}C$ or -O.6$^{\circ}C$ /minute. When equilibrated in 1.8M ethylene glycol, survival rate of fiozen4hawed blastocysts was sarne in both -0. 3$^{\circ}C$ /min and -0.6$^{\circ}C$ /min cooling rate(71.4%). With the equilibration in 1.4M glycerol, survival rate was higher in -0.3$^{\circ}C$ /min(63.6%) than in -0.6$^{\circ}C$ /min cooling rate(53.8%). For vitrification of the KNC blastocysts produced in vitro, they were equilibrated in 2-step or 3-step exposure to vitrification solution(25% ethylene glycol + 25% glycerol). Survival rate was sirilar in both 2-step(45.0%) and 3-step exposure(47.4%). According to culture condition following in vitro fertilization, higher survival rate was obtained for blastocysts co-cultured with bovine oviductal epithelial cell(BOEC, 77.3%) than for those cultured with epidermal growth factor(EGF, 65.7%) or for those co-cultured with BOEG + EGF (54.8%). According to embryo age and development stage, higher survival rate was obtained for 7-day ernbryos(70.0%) than 8-day(56.8%) or 9-day(20.0%) for blastocyst stage and obtained for 8-day embryos(74.3%) than 7-day(62.5%) or 9-day(42.9%) for exponded blastocyst. In surnmary, higher survival rate of frozen4hawed KNC blastocysts produced in vitro were obtained by using ethylene glycol for cryoprotectant and -0.3$^{\circ}C$ /min for cooling rate. And higher survival rate were obtained with co-culture with BOEC for culture condition following in vitro fertilization and with 7-day blastocyst or 8-day expanded blasto cyst for embryo age and development stage.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.2
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• pp.169-173
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• 1999

Studies were made to evaluate the specific and combined effects of different fatty acids on the in vitro development of 8-cell rat embryo in culture media with and without carbohydrate substrate. Palmitic, oleic, linoleic and arachidonic acids were added singly and in combination to media which contained fatty acid-free BSA. Cell numbers in blastocysts cultured in the media were counted and compared with cell numbers in blastocysts at the corresponding stage collected from the uterus. Oleic, linoleic and arachidonic acids promoted the rat embryo development from 8-cell to the blastocysts. especially in the absence of carbohydrate substrates. Among these three, oleic acid was the most effective but embryo development was not accelerated by the addition of palmitic acid in either the presence or the absence of carbohydrate substrates. Addition of the mixture of four fatty acids was more effective for rat embryo development than single treatment with any of fatty acids tested. Cell numbers per blastocyst in the presence and absence of carbohydrate substrate were similar, and did not differ from those for blastocysts obtained from the uterus.

• 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.24 no.4
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• pp.281-287
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• 2009

As a simple and economical method for in vitro produced embryos, we have used BSA instead of serum for the production and embryo transfer of Hanwoo in vitro fertilized (IVF) embryos and obtained the following results: 1) When using serum (FBS; fetal bovine serum) or BSA-containing culture media as the initial culture media for immature oocytes, it is regarded as inappropriate to add only BSA to the culture solutions from maturation of the immature oocytes to development stage culture, but serum still needs be added though there is no significant difference in the concentration, with a change from 5% to 10%. 2) The results of culturing IVF embryos after development (4 cell stage) in the Medium199 solutions containing BSA instead of serum (FBS) showed that 0.3% BSA concentration is not optimal and 0.5% or higher BSA concentration has no significant difference among 0.5%, 0.7%, 1% and 2% (p > 0.05). 3) The post-freezing survival ratio after development in 5% FBS-Medium199 showed that 1% BSA concentration of the culture solution is the most suitable in the BSA concentrations of 0.3% (51%), 0.5% (67%), 0.7% (69%), 1% (77%) and 2% (75%). 4) The pregnancy rates of the transplanted fresh(not frozen) blastocyst had no significant concentration dependency (p > 0.5), and the average pregnancy rate was 63.8%. 14% of overweight calves were found among the calves given birth to by the transfer of IVF blastocysts cultured in the serum-added culture solution, but none was found in the experimental groups in which BSA was added instead of serum.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.94-101
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• 2020

In the early development of parthenogenetic embryo, cytoplasm and nucleic acid fragmentation may be a cause of lower embryo development. The purpose of this study was to evaluate whether embryonic development and apoptosis factors can be reduced by controlling the in-vitro culture environment by the addition of hormones, pregnancy serum and uterine milk. Our study showed that the activity of Casp-3 increased within the cytoplasm when artificially used hormones to induce the incubation environment, and PCNA's manifestation was low. However, the addition of pregnant serum appeared to lower the Casp-3 activity compared to the other groups. In addition, MMP-9 activity was increased and early embryo development and cytoplasmic fidelity were also increased. Therefore, the results of the present study showed that the use of gestational serum in the development of parthenogenetic embryo inhibit apoptosis and increases cytoplasmic reorganization by natural environmental control in in vitro culture.