• Journal of Embryo Transfer
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• v.30 no.3
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• pp.161-170
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• 2015

These studies were conducted to establish the practical Hanwoo (Korean native cattle) improvement system through the combining of embryo transfer technology and confirming individual Hanwoo oocyte culture system and to investigate that correlation of Hanwoo carcass classification (intramuscular marbling) and oocyte donor for blastocyst production in vitro. In case of Hanwoo, the carcass meat quality grades were divided to grade $1^{{+}{+}}$, $1^{+}$, 1, 2, and 3 depends on fat distribution of longest muscle cross-sectional surface. As results, the numbers of follicular oocytes collected from individual fundamentally-registered Hanwoo yielded $1^{{+}{+}}$, $1^{+}$, 1, 2 and 3 meat quality were 9.5, 9.4, 8.5, 8.8 and 8.8 per ovary, respectively. The numbers of retrieval oocyte from follicles were significantly higher in the cattle yield $1^{{+}{+}}$, $1^{+}$ meat quality than in the cattle yield 1, 2 and 3 meat quality (p<0.05). The rates of blastocyst formation were 18.2, 21.3, 29.4, 30.9, and 31.5% in the cattle yield $1^{{+}{+}}$, $1^{+}$, 1, 2 and 3 meat quality of after in vitro maturation, respectively. It was significantly lower in the cattle yield $^{{+}{+}}$ and $1^{+}$ meat quality than in the cattle yield 1, 2 and 3 meat quality (p<0.05). In order to evaluate embryos quality, TUNNEL assay was conducted for each meat quality grade using blastocyst stage embryo on day 8. The results showed that apoptosis cell number was higher tendency in the cattle yield $1^{{+}{+}}$and $1^{+}$ meat quality (81 and 79, respectively) than in the cattle yield 1, 2 and 3 meat quality (51, 48 and 50, respectively) but there was no statistical significance in each group. After embryo transfer, the conception rate of recipient was 53.5 (23 out of 43), 52.1 (38 out of 73) and 58.0% (58 out of 100) in the meat quality of 1, $1^{+}$ and $1^{{+}{+}}$, respectively. These results showed that the conception rate was significantly higher in the cattle yield 1 meat quality than in the cattle yield $1^{{+}{+}}$, $1^{+}$, 2, and 3 meat quality (p<0.05). In summary, these results indicate that the application of confirming Hanwoo individual oocyte culture system and embryo transfer technology can make good use of the genetic resources conservation and improvement of Hanwoo. Relevance of the meat quality grade and reproductive ability of carcasses of Hanwoo will be considered to be one of the effective means for the associated research with obesity and reproduction.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.20 no.9
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• pp.510-516
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• 2019

This study examined the effects of fetal bovine serum (FBS), goat blood serum (gBS), and poly-vinyl alcohol (PVA) on the in vitro development and embryo quality of goats for an improvement of embryo production. For the experiment, an in vitro fertilized embryo culture medium was supplemented with 10% FBS, 10% gBS, and 10% PVA to determine their effects on the embryo development efficiency and blastocyst quality. The results showed that the non-serum supplementation group showed significantly lower cleavage rate and blastocyst formation. On the other hand, the gBS and PVA supplementation groups showed a significant increase in the cleavage rate and better blastocyst formation than the control and FBS supplementation group. Furthermore, a TUNEL assay performed to confirm the blastocyst quality showed the same pattern as the embryo development experiment. These results showed that the supplemented gBS or PVA was more efficient in enhancing the in vitro development efficiency of goats than the supplementation of FBS or non-serum. On the other hand, considering the risk of an unidentified factor in gBS, PVA appears to be safer and more efficient in the in vitro development of goat embryos.

• Korean Journal of Animal Reproduction
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• v.10 no.1
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• pp.76-82
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• 1986

This experiment was conducted to determine the effects of trophoblastic vesicles (TV) and estradiol-17$\beta$ on the development in vitro of rabbit embryos. Thirty matured female rabbits were treated with PMSG followed by HCG injection and mating. Embryos were recovered with D-PBS (Dulbecco's Phosphate Buffered Saline) after superovulation, and normally developed to two-to four-cell embryos were used in the subsequent in vitro culture. Basal medium was Medium-199 su, pp.emented with 1.5% bovine serum albumin. Embryo on Day 5 after mating (Day 0) was cut into two or three pieces to remove the embryonic disc. Each piece of tissue was cultured for 24 hours at 37$^{\circ}C$ in 0.5 mlMedium-199 in 5% CO2. During culture, peices of trophoblastic tissue changed into spherical vesicles which were used for co-culture. These spheres were called trophoblstic vesicles. Two-to four-cell embryos were cultured for 4 days in Medium-199 in the absence or presence of trophoblastic vesicle, and two-to four-cell embryos cultured with varing concentration (0, 0.1, 1, 10ng/ml) of estradiol-17$\beta$ for 4 dyas. Culture vessels used were watch glass for coculture with trophoblastic vesicles and micortube for estradiol-17$\beta$ infusion. Compared with the Medium-199 alone as basal culture medium, more blastocysts (46.7% vs 15.1%; P<0.01) and morulae (84.4% vs 56.6%; P<0.05) were developed in the co-culture with trophoblastic vesicles. Estradiol-17$\beta$ infused in culture medium was not effective for embryo development to blastocysts (78.3% in control, 50.0% in 0.1ng/ml, 61.5% in 1ng/ml and 64.4% in 10ng/ml) and also to morulae (91.3% in control, 84.2% in 0.1ng/ml, 92.3% in 1ng/ml and 91.1% in 10ng/ml). Compared with the watch glass culture mehotd, more (P<0.01) blastocysts were developed in microtube culture (78.3% vs 56.6%) and more (P<0.01) morulae in microtube culture (91.3% vs 56.6%).

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.167-173
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• 2008

Somatic cells such as oviduct epithelial cell, uterine epithelial cell, cumulus-granulosa cell and buffalo rat river cell has been used to establish an effective culture system for bovine embryos produced in in vitro. But nitric oxide (NO) metabolites secreted from somatic cells were largely arrested the development of bovine in vitro matured/ in vitro fertilized (IVM/IVF) embryos, suggesting that NO was induced the embryonic toxic substance into culture medium. The objective of this study was to investigate whether BOEC co-culture system can ameliorate the NO-mediated oxidative stress in the culture of bovine IVM/IVF embryos. Therefore, we evaluated the developmental rate of bovine IVM/IVF embryos under BOEC co-culture system in the presence or absence of sodium nitroprusside (SNP), as a NO donor, and also detected the expression of growth factor (TGF-$\beta$, EGF and IGFBP) and apoptosis (Caspase-3, Bax and Bcl-2) genes. The supplement of SNP over 5 uM was strongly inhibited blastocyst development of bovine IVM/IVF embryos than in control and 1 uM SNP group (Table 2). The developmental rates beyond morulae stages of bovine IVM/IVF embryos co-cultured with BOEC regardless of SNP supplement (40.4% in 5 uM SNP+ BOEC group and 65.1% in BOEC group) were significantly increased than those of control (35.0%) and SNP single treatment group (23.3%, p<0.05: Table 3). The transcripts of Bax and Caspase-3 genes were detected in all experiment groups (1:Isolated fresh cell (IFC), 2:Primary culture cell (PCC), 3:PCC after using the embryo culture, 4: PCC containing 5 uM SNP and 5: PCC containing 5 uM SNP after using the embryo culture), but Bcl-2 gene was not detected in IFC and PCC (Fig. 1). In the expression of growth factor genes, TGF-$\beta$ gene was found in all experimental groups, and EGF and IGFBP genes were not found in IFC and PCC (Fig. 2). These results indicate that BOEC co-culture system can increase the development beyond morula stages of bovine IVM/IVF embryos, possibly suggesting the alleviation of embryonic toxic substance like nitric oxide.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.147-157
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• 1998

This study was conducted to investigate the effects of in vitro fertilization, culture and embryo development according to in vitro maturation rate, protectant composition and equilibrium time after frozen /thawing of bovine immature oocytes. This results obtained in studies on the effect of different cryoprotectants on the viability, maturation and development of in vitro bovine oocytes were as follow: 1.The post-thawing of immature oocytes matured to metaphase II during culture time for 0 to 26 h, and those group (62~3%) were low than control group (76.7%). The optimal maturation time of frozen-thawed immature oocytes was at 24 h. 2.The viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants. The developmental competence of frozen4hawed oocytes was not affected by cryoprotectants. These results indicate that an optimal maturation time of frozen /thawed immature oocytes was at 24h. Furthermore the viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants and developmental competence of frozen /thawed oocytes.

• Plant Biotechnology Reports
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• v.3 no.4
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• pp.347-353
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• 2009

Procedures that induce microspore embryogenesis have been described for a range of Brassica species, but embryo yield remains low for a number of genotypes. We have carried out experiments with the microspores from a weakly responsive line of B. napus to determine the culture conditions that optimize their in vitro embryogenesis by treating them with effectors of ethylene synthesis and action. The results revealed that isolated microspores subjected to an initial heat stress in a medium supplemented with inhibitors of ethylene synthesis such as AVG and $CoCl_2$ exhibited significantly increased embryo yields. This suggested that regulatory effects are exerted by the ethylene produced by the isolated microspores on the early processes of gametogenesis. As a consequence, treatment of microspores with SAM, an ethylene synthesis precursor, or with the ethylene-releasing agent ethephon, led to decreases in embryo yield. A special response to ethylene during the early stages of microspore development was finally shown to occur through experiments where isolated microspores were treated for increasing periods of time with $CoCl_2$. Collectively, our data demonstrated that the induction of embryogenesis induced by heat stress can be enhanced by inhibitors of ethylene biosynthesis.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.113-121
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• 2005

This study was conducted to investigate the effects of different hexoses (glucose, mannose, galactose and fructose) on in vitro development of parthenogenetic embryos in the pigs. When the parthnogenetic embryos were cultured in medium with concentrations of 5mM glucose or 1mM galactose, the rates of embyos developed to morula and blastocyst stages were significantly higher than those in another culture conditions (P<0.05). However, high concentration of galactose inhibited development to morula and blastocyst stages. Addition of hexoses at early stage of porcine parthenogenetic embryos were effective for in vitro development. Especially, the embryos cultured in medium with glucose at early stage were effective for development to 2-cell $(72\%)$ and blastocyst $(19\%)$ stages compared with embryo cultured without glucose. From the present results, it is suggested that development of porcine parthenogenetic embryos can improve in medium with 5mM glucose. The concentration of 1mM galactose was also effective for development of porcine parthenogenetic embryos. It also show that parthenogenetic embryos cultured with glucose at early stage can improve in vitro development.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.93-97
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• 1999

The development potential of bovine somatic cells was evaluated using nuclear transfer. A single donor cell derived from fetus of HanWoo(Korean Native Cattle) was selected and deposited into perivitelline space of each enucleated oocyte before electrical fusion and activation. Nuclei of donor cells starved for 7 days (37%) tended to support the development of reconstitute embryo the blastocyst stage better than those of donor cells starved 3, 14 and 30 days. The cleavage rate was significantly lower(P<0.05) in reconstitute embryos derived from large size donor cells(51.2%), than those from small medium size donor cells(76.6 and 73.5, respectively). The developmental rate to blastocyst of reconstructed embryos from medium size donor cells was higher than those from small and medium size donor cells. This study demonstrates that an appropriate culture period for induction into quiescent stage and the size of donor cells effect on the efficiency of nuclear transfer using cultured bovine cells.

• Journal of Environmental Health Sciences
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• v.31 no.4 s.85
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• pp.254-259
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• 2005

Permethrin, a synthetic pyrethroid insecticide, has been widely used to protect domestic animals and the public health, as well as in agriculture against a variety of pests, which provides potential for environmental exposure. Permethrin is classified as possible human carcinogen and endocrine disrupting chemical by many international authorities. However, its developmental effects have been rarely studied. This study investigated the effects of permethrin during embryo-genesis. Developmental toxicity of permethrin was evaluated using short-term in vitro battery system. Gestation day 9.5 rat embryos (organogenesis) were cultured with permethrin (0.1,0.4 and 0.8 mg/ml) for 48 hours using whole embryo culture system. All the treatments exhibited significant decreases in the total morphological score. Permethrin induced significant growth retardation and the developmental abnormality at doses of 0.4 and 0.8 mg/ml. Moreover, the DNA and protein contents of embryos decreased in dose-dependent manner. These observations suggest that permethrin contributes to toxicity on embryonic developments in rats.

• 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.