• Korean Journal of Veterinary Research
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• v.29 no.2
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• pp.123-128
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• 1989

Demi-embryos were successfully produced by bisection of ICR mouse embryos at preimplantation stages. They were microsurgically bisected using a microsurgical blade attached to a micromanipulator after pretreatment with 0.5% pronase in PBS for two minutes or not. Embryos with softened zona pellucida were more easily bisected and less damaged than intact embryos. The highest success rate in bisection has been achieved by selecting blastocysts(94.1% in success rate with intact blastocysts and 100% in success rate with zona softened blastocysts). Demi-embryos without zona pellucida were cultured in D-PBS or M-16 medium at $37^{\circ}C$, 5% $CO_2$ in air for 72 hours for 2-cell stage embryos, 48 hours for 4-to 8-cell stage embryos, 24 hours for morula stage embryos and 6~12 hours for blastocyst stage embryos. For the in vitro culture of 2-cell stage embryos, $100{\mu}M$ 2Na-EDTA was added to the media. M-16 medium was better for the in vitro development of mouse embryos than PBS, and PBS is not considered to be suitable for long-term culture of embryos, especially at early stage of cleavage. In M-16 medium, developing rate of demi-embryos of which pair underwent development to form eublastocysts was 15.8% at 2-cell stage, 16.8% at 4-cell stage, 38% at 8-cell stage, 89.6% at morula stage and 94.4% at blastocyst stage, respectively. The more rapid and efficient production of demi-embryos and higher viability after bisection can be expected by softening zona pellucida with pronase and by selecting morulae or blastocysts rather than embryos at early stage of cleavage.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.335-341
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• 1997

The aims of this study are to establish a stable isolation method of blastomeres from bovine early embryos and examine their developmental potential in vitro Early embryos were produced by maturation and fertilizaion in vitro of bovine follicular oocytes. Blastomeres were isolated from 2~8-cell embryos in $Ca^2$+-, $Mg^2$+-free PBS+EDTA after removing the zonae pellucidae Isolated blastomeres were cultured in CZB containing BOEC for upto 240 hpi. Cleavage rates of them were 18.5%(10 /54) in 1 /2 blastomeres, 33.3%(16/48) in 1/4 blastomeres and 34.2%(14 /41) in 1/8 blastomeres, respectively. The rates of blastocystic vesicle formed were 8.7%(4 /46) in 1/2 blastomeres, 26.6% (17/64) in 1/4 blastomeres and 10.3%(8 /78) in 1/8 blastomeres, respectively. Blastomeres developed into various types of blastocystic vesicles and trophoblastic vesicles as evidenced by the Hoechst 33258 staining and morphology. This results suggest that the isolation method used and subsequent culture of isolated blastomeres from bovine early embryos should be useful to obtain extra embryonic cells for various analyses such as PCR and putative ES cell culture.

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.394-400
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• 1998

Cryopreservation of embryos by vitrification is a simple method to preserve bovine embryos for subsequent embryo transfer, but embryonic viability after vitrification has been inconsistent and low compared with conventional slow freezing. The aim of the present study is to examine the effect of serum or serum albumin in a vitrification solution and epidermal growth factor(EGF) or fibroblast growth factor(FGF) on in vitro viability of bovine blastocysts frozen by vitrification. Bovine blastocysts were produced by in vitro maturation, fertilization of follicular oocytes and culture of embryos in a synthetic oviduct fluid medium(SOFM) containing BSA and 19 essential and nonessential amino acids. Blastocysts with excellent or good morphology were selected at 7 or 8 days after culture and utilized for vitrification. In experiment 1, blastocysts were vitrified in a solution containing semi-fetal calf serum(SFCS) or BSA(5 or 10mg/ml) and then their subsequent viabilities were examined by culturing thawed embryos in a SOFM containing BSA and 19 amino acids. Effect of EGF or FGF added to a SOFM containing polyvinyl alcohol(PVA) on the viability of vitrified-thawed blastocysts was investigated in experiment 2. BSA added at 5 or 10mg/ml to a vitrification solution showed significantly higher(p < 0.05) developmental rate to expanded and hatching blastocysts than SFCS, but there was no significant difference in the developmental rate to hatched blastocysts after thawing. Supplementation of a culture medium with EGF and/or FGF significantly increased(p < 0.05) embryo development to expanded blastocysts compared with control but showed no beneficial effect on the development to hatching or hatched blastocysts. Coculture of thawed embryos with granulosa cells in a TCM 199 containing 10% fetal calf serum(FCS) showed the highest developmental rate to expanded, hatching and hatched blastocysts among the groups tested. In conclusion, supplementation of a vitrification solution with BSA at 5mg/ml and culture of thawed blastocysts in a medium containing EGF and/or FGF can improve in vitro viability of bovine blastocysts frozen by vitrification.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.11
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• pp.1541-1546
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• 2011

This study was carried out to examine the influence of minimum essential medium (MEM) vitamins supplementation to in vitro maturation medium and in vitro culture medium on the development of porcine embryos. Porcine embryo development was investigated following cultivation in both in vitro maturation and culture medium with the supplementation of MEM vitamins (0, 0.1, 0.2 and 0.4%) using immature oocytes collected from the ovary of prepubertal gilts. Embryo development was observed and the total cell number in each blastocyst generated under the culture conditions was quantified following supplementation of the medium. The embryonic development rate of the blastocyst and hatched blastocyst was higher, but not significantly so, when 0.4% MEM vitamins were supplemented to the in vitro maturation medium of the porcine oocyte. Interestingly, the total number of cells in the blastocyst was significantly higher in the in vitro maturation MEM vitamins supplemented group compared to either the untreated group or the group which had MEM vitamins supplemented to both in vitro maturation and in vitro culture medium simultaneously (p<0.05). Therefore, the supplementation of 0.4% MEM vitamins to the in vitro mature medium has a beneficial effect on the embryonic development of in vitro produced blastocysts derived from the immature porcine oocytes.

• 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.

• Korean Journal of Animal Reproduction
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• v.27 no.4
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• pp.309-315
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• 2003

DNA methylation at CpG sites, which is a epigenetic modification, is associated with gene expression without change of DNA sequences. During early mouse embryogenesis, dynamic changes of DNA methylation occur. In this study, DNA methylation patterns of porcine embryos produced in vivo and in vitro were examined at various developmental stages by the immunocytochemical staining method. Interestingly, active demethylation was not observed on the paternal pronucleus of porcine zygotes. However, differences were detected in the passive demethylation process between in vivo and in vitro embryos. There was no change in the DNA methylation state until the blastocyst stage of in vivo embryos, whereas partial demethylation was observed in several blastomeres from a 4 cell stage to a morula stage of in vitro embryos. The whole genome of inner cell mass (ICM) and trophectoderm (TE) cells in porcine blastocysts were evenly methylated without de novo methylation. Our findings demonstrate that genome-wide demethylation does not occur in pig embryos during preimplantation development unlike murine and bovine embryos. It indicates that the machinery regulating epigenetic reprogramming may be different between species.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.1
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• pp.25-32
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• 2010

The present study aimed to examine the effects of ${\gamma}$-linolenic acid (GLA) supplementation to in vitro culture (IVC) medium on in vitro developmental competence, freezability and morphology of in vitro matured and fertilized bovine embryos. In vitro produced (IVP) bovine zygotes were cultured in IVC medium supplemented with 0 (negative control), 15, 31, 62, 125, 250, 500 or 1,000 ppm GLA, 250 ppm linoleic acid albumin (LAA) and without any supplement as a control. Day 6 blastocysts derived from culture control were cultured in IVC medium containing either 62, 250 GLA or 250 LAA for 24 h, and at Day 7 were subjected to freezing or morphological examination by electron microscope. GLA 15 showed a tendency to have a higher cleavage rate at Day 2 (70.3%) than other groups. The hatching rate at Day 9 in LAA (38.2%) was significantly higher than the control and all treatment groups (p<0.05), while the blastocyst rate in LAA (32.4%) did not differ from those of 15 (30.5%), 31 (27.1%), and 62 GLA (33.1%) or the control (35.1%). GLA in concentrations of 125, 250, 500, and 1,000 ppm had significantly detrimental effect on the blastocyst rate compared to 15, 31 and 62 ppm GLA, LAA, and control groups (p<0.05). In contrast, the highest post-thaw survival rate (100%) was observed in the control group (p<0.01). Large lipid droplets were observed in the cytoplasm of trophoblastic cells, even in the control, but were abundant in GLA groups. Taking the results of the study into consideration, the addition of GLA to the culture medium for IVP bovine embryos at the dose of 15 ppm increased the developmental competence of zygotes and enhanced the cleavage rate up to Day 2. However, blastulation rate and post-thaw survival were not increased when GLA was added to the culture media.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2003.10b
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• pp.2-3
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• 2003

A method for rapid propagation of mature Jack fruit was developed. Four types of explants (mature embryos, apical meristems of young seedlings, apices from mature plants and nodal segments) were used. It has been found 88% of young apical meristems produced shoots in Campbell and Durzan (CD) medium compared to 60% in Murashige and Skoog (MS) medium. Only 1/3 of them produced multiple shoots. Shoot initiation from nodal segments was very rare. Mature apices produced callus. Although removal of the sheathing cover around mature buds enhanced the shoot initiation but success rate was low in growth regulator free medium. Embryos respond to the CD medium but not to the MS medium. Embryos from seeds soaked in water for 24 hours produced shoots after 8 weeks of incubation and the success rate was 70% while embryos from dry seeds only produced roots. There was no significant effect of cold storage (refrigeration) for 7 days on shoot initiation from mature embryos (65%) but the ability for shoot induction declines with storage time (55% after 21 days of cold storage). Mature axillary buds were established in Modified Campbell and Durzan (CD) medium supplemented with 0.5mg/1 and IBA. There was a significant difference in the growth performance of shoots according to the period of the year in which explants were collected. Highest (60%) was observed in November-January period. It was only 30% when the explants were collected in February-April or May-July and decreased to 20% in August-October. The shoots produced in November-January showed a higher vigor than those produced in other months. Since Jak fruit show seasonal changes in fruit bearing and shedding of leaves, it can be suggested that the difference in growth performances of tissues cultured in artificial culture media would have been affected by endogenous rhythms.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2003.10a
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• pp.9-18
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• 2003

A method for rapid propagation of mature Jack fruit was developed. Four types of explants (mature embryos, apical meristems of young seedlings, apices from mature plants and nodal segments) were used. It has been found 88% of young apical meristems produced shoots in Campbell and Durzan (CD) medium compared to 60% in Murashige and Skoog (MS) medium. Only 1/3 of them produced multiple shoots. Shoot idtiation from nodal segments was very rare. Mature apices produced callus. Although removed of the sheathing cover around mature buds enhanced the shoot initiation but success rate was low in growth regulator free medium. Embryos respond to the CD medium but not to the MS medium. Embryos from seeds soaked in water for 24 hours produced shoots after 8 weeks of incubation and the success rate was 70% while embryos from dry seeds only produced roots. There was no significant effect of cold storage (refrigeration) for 7 days on shoot initiation from mature embryos (65%) but the ability for shoot induction declines with storage time (55% after 21 days of cold storage). Mature axillary buds were established in Modified Campbell and Durzan (CD) medium supplemented with 0.5mg/1 and IBA. There was a significant difference in the growth performance of shoots according to the period of the year in which explants were collected. Highest (60%) was observed in November-January period. It was only 30% when the explants were collected in February-April or May-July and decreased to 20% in August-October. The shoots produced in November-January showed a higher vigor than those produced in other months. Since Jak fruit show seasonal changes in fruit bearing and shedding of leaves, it can be suggested that the difference in growth performances of tissues cultured in artificial culture media would have been affected by endogenous rhythms.

• Korean Journal of Animal Reproduction
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• v.21 no.4
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• pp.335-343
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• 1997

The effect of thiol compounds on development and intracellular glutathione(GSH) concentrations of bovine embryos produced by in vitro maturation and in vitro fertilization(IVM/IVF) was examined in CRlaa medium with or without $\beta$-mercaptoethanol(0, 10, 25 and 50$\mu$MME) and cysteamine(0, 25, 50 and 75 $\mu$M). Numbers of cells comprising blastocysts were also counted using double fluorescence stain and the total glutathione levels(oxidized and reduced form) of morula and blastocyst embryos were than measured by an enzymatic method. Following routine IVM/IVF procedures oocytes and zygotes were cultured for 40 to 44h in CRlaa medium. Then 2 to 8-cell embyos had cumulus cell removed and were allotted randomly to the experimental medium. In Experiment 1, the proportion of embryos developing to and beyond morulae stages in 0, 10, 25 and 50 $\mu$M $\beta$-ME was 42.9%, 50.0%, 53.7% and 65.6%, respectively. Fifty $\mu$M $\beta$-ME group was significantly higher than those of any other groups (P<0.05). In Experiment 2, the percentages of embryos developed beyond morulae stages in 0, 25, 50 and 75 $\mu$M cysteamine was 42.9%, 40.4%, 60.0% and 59.2%, respectively. Fifty and 75$\mu$M cysteamine groups were significantly higher than in 0 and 25 $\mu$M cysteamine groups, but all of culture medium containing cysteamine(52.6%) was not significantly difference in control group(42.9%). In Experiment 3, the intracellular GSH concentrations of morulae and blastocyst embryos in 0 and 50 $\mu$M $\beta$-ME was 42.4 pM and 44.9 pM, 49.5 pM and 67.8 pM, respectively. Morulae embryos were not difference, but blastocyst embryos were significantly difference between treatments(P<0.05). In Experiment 4, the intracellular GSH concentrations of morulae in CRlaa with or without cysteamine were 39.8 pM and 45.6 pM, and blastocysts were 59.3 pM and 66.8 pM, respectively. Cell numbers of blastocysts were similar to in all experimental groups. These experiments indicate that thiol compounds can increase the proportion of embryos that developing to and beyond morulae stage and the intracellular GSH concentrations.