• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.1-9
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• 1998

This study was carried out to confirm whether the developmental capacity of bovine mature oocytes frozen ultra-rapidly using electron microscopic(EM) grids and EFS30 can be obtained, and whether the cryoprotectants and the freezing method used in this study effect detrimentally to the bovine oocytes by indirect immunocytochemistry. As freezing solution, we used EFS30 which consisted of 30% ethylene glycol, 0.5 M sucrose, 18% ficoll and 10% FBS added in D-PBS. The results obtained in this experiment were summarized as follows: When the effects of cryoprotectant and freezing procedure on the microtuble, micrfilament and chromatin morphology of oocytes were evaluated using indirect immunocytochemistry, the results of freezing as well as exposure group were not different with that of the control oocytes. When the fertilization abnormality after ultrarapid freezing of bovine mature oocytes was examined by Hoechst staining, the rates of total penetration(96.7, 9.0%), normal two pronuclei formation(74.6, 68.9%) and mean number of sperm / oocyte(1.50, 1.44) were not different between control and freezing group. In addition, when the developmental capacity of frozen-thawed of oocytes(85.5%) was survived, 74.5% of them were cleaved and 31.4% of cleaved embryos were developed to blastocyst. These data were similar to those of the control(76.0%, 34.6%) and exposure(74.5%, 33.0%) except survival rates. Also, when the total cell number of blastocysts produced from the each treatment at day after IVF was examined by hoechst staining, there were not different among groups. There results demonstrate that developmental capacity of frozen-thawed bovine mature oocytes can be successfully obtained by ultra-rapid freezing method using EM grid and EFS30 solution.

• Korean Journal of Animal Reproduction
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• v.21 no.4
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• pp.345-353
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• 1997

Antioxidants and antioxidants with somatic cell co-culture, bovine oviduct epithelial cells(BOEC) and buffalo rat liver cells(BRLC), were studied as a mean of increasing the development and intracellular glutathione(GSH) concnetrations of bovine embryos derived from in vitro matured(IVM) and in vitro fertilized(IVF) oocytes. Cell numbers and intracellular GSH concentrations of blastocysts were also counted. The developmental rate beyond morula stages in CRlaa containing taurine(2.5mM), superoxide dismutase(SOD, 600U) and catalase(250U) were 1%, 75.0%, 64.8% and 62.3%, respectively. The developmental rate in antioxidant groups was significantly higher than in control(P<0.05). The intracellular GSH concentrations of blastocysts cultured in 0, 2.5mM taurine, 600U SOD and 250U catalase were 33.8pM, 39.3pM, 42.3pM and 54.8pM, respectively. This result indicated that the developmental rates and intracellular GSH concentrations of catalase group was significantly higher than any other groups(P<0.05). The developmental capacity in CRlaa plus various antioxidants co-cultured with BOEC were 55.3%(control), 74.1%(2.5mM taurine), 66.7%(600U SOD) and 70.7%(250U catalase) and in CRlaa plus various antioxidants co-cultured with BRLC in control, 2.5mM taurine, 600U SOD and 250U catalase were 63.8%, 75.5%, 71.0% and 73.5%, respectveily, the intracellular GSH concentrations of blastocyst embryos co-cultured with BOEC and BRLC in CRlaa with 0.25mM taurine, 600U SOD and 250U catalase were 73.4pM and 64.4pM, 79.9pM and 67.5pM, 82.3pM and 71.7pM, and 83.0pM and 80.0pM, respectively. Cell numbers of blastocysts were not difference in all experimental groups. These studies indicate that andtioxidants and antioxidant with somatic cell co-culture can increase the proportion of embryo that developed into morula and blastocysts, and the intracellular GSH concentrations of blastocyst embryos.

• Reproductive and Developmental Biology
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• v.38 no.4
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• pp.147-158
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• 2014

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic status after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Dai et al., 2010), use of m-carboxycinnamic acid bishydroxamide (CBHA) as a histone deacetylase inhibitor during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA treatment on the in vitro preimplantation development of porcine SCNT embryos and on the acetylated status of histone H3K9 on cloned embryos at the zygote stage. We performed the three groups SCNT: SCNT (NT), CBHA treatment at the porcine fetus fibroblast cells (PFFs) used as donor cells prior to SCNT (CBHA-C) and CBHA treatment at the porcine SCNT embryos during the in vitro early culture after oocyte activation (CBHA-Z). The PFFs were treated with a $15{\mu}M$ of CBHA (8 h) for the early culture and the porcine cloned embryos were treated with a $100{\mu}M$ concentration of CBHA during the in vitro early culture (10 h). Cleavage rates and development to the blastocyst stage were assessed. No significant difference was observed the cleavage rate among the groups (82.6%, 76.4% and 82.2%, respectively). However, the development competence to the blastocyst stage was significantly increased in CBHA-Z embryos (22.7%) as compared to SCNT and CBHA-C embryos (8.6% and 4.1%)(p<0.05). Total cell numbers and viable cell numbers at the blastocyst stage of porcine SCNT embryos were increased in CBHA-Z embryos as compared to those in CBHA-C embryos (p<0.05). Signal level of histone acetylation (H3K9ac) at the zygote stage of SCNT was increased in CBHA-Z embryos as compared to SCNT and CBHA-C embryos. The results of the present study suggested that treatment with CBHA during the in vitro early culture (10 h) had significantly increased the developmental competence and histone acetylation level at the zygote stage.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.3
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• pp.258-264
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• 2020

This study investigated the effect of variation in the number of somatic-cell-cloned embryos and their developmental stage at transfer on pregnancy, as well as the influence of the estrus status of recipient pigs on in vivo development of cloned porcine embryos after embryo transfer. For somatic cell nuclear transfer (SCNT), fibroblast cells were obtained from a male porcine fetus. Recipient oocytes were collected from prepubertal gilts at a local abattoir and then cultured. After SCNT, reconstructed embryos of different numbers and developmental stages were transferred into recipient pigs. The developmental stage of the cloned embryos and the number of transferred embryos per surrogate showed no significant differences in terms of the resulting cloning efficiency. However, the pregnancy rate improved gradually as the number of transferred cloned embryos was increased from 100-150 or 151-200 to 201-300 per recipient. In pre-, peri-, and post-ovulation stages, pregnancy rates of 28.6%, 41.8%, and 67.6% and 16, 52, and 74 offspring were recorded, respectively. The number of cloned embryos and estrus status of the recipient pig at the time of transfer of the cloned embryo affect the efficiency of pig production; therefore, these variables should be particularly considered in order to increase the efficiency of somatic cell pig cloning.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.2
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• pp.190-197
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• 2011

The objective of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co-culture on developmental capacity of bovine IVM/IVF embryos and to determine whether or not melatonin acts as an antioxidant in BOEC culture and subsequent embryo development. These studies examined the effects of melatonin against NO-induced oxidative stress on cell viability, lipid peroxidation (LPO) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl-2, Caspase-3 and Bax) during BOECs culture. We also evaluated the developmental rates of bovine IVM/IVF embryos with BOEC co-culture, which were pre-treated with melatonin ($1,000\;{\mu}M$) in the presence or absence of sodium nitroprusside (SNP, $1,000\;{\mu}M$) for 24 h. Cell viability in BOECs treated with SNP (50-$2,000\;{\mu}M$) decreased while melatonin addition (1-$1,000\;{\mu}M$) increased viability in a dose-dependent manner. Cell viability in melatonin plus SNP ($1,000\;{\mu}M$) gradually recovered according to increasing melatonin addition (1-$1,000\;{\mu}M$). The LPO products were measured by thiobarbituric acid (TBA) reaction for malondialdehyde (MDA). Addition of melatonin in BOEC culture indicated a dose-dependent decrease of MDA, and in the SNP group among BOECs treated with SNP or melatonin plus SNP groups MDA was significantly increased compared with SNP plus melatonin groups (p<0.05). In expression of apoptosis or antioxidant genes detected by RT-PCR, Bcl-2 and antioxidant genes were detected in melatonin or melatonin plus SNP groups, while Caspase-3 and Bax genes were only found in the SNP group. When bovine IVM/IVF embryos were cultured for 6-7 days under the BOEC co-culture system pre-treated with melatonin in the presence or absence of SNP, the highest developmental ability to blastocysts was obtained in the $1,000\;{\mu}M$ melatonin group. These results suggest that melatonin has an anti-oxidative effect against NO-induced oxidative stress on cell viability of BOECs and on the developmental competence of bovine IVM/IVF embryo co-culture with BOEC.

• Korean Journal of Ecology and Environment
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• v.50 no.3
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• pp.305-313
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• 2017

We investigate that the impact of freshwater organism exposed to the salinity environment by the frequent rainfall following climate change. To evaluate the stress response following salinity exposure, we assessed the survival rate, molting success rate, the developmental period and mouthpart deformities in Chironomus riparius. In addition, we measured the molecular responses of biomarker gene, gene expression of heat shock protein 70 (HSP70) in C. riparius exposed to salinity after 96 hour. The C. riparius survival rates were showed on time dependent manner and not observed survival organisms above 15 psu at day 4. The pupation and emergence of C. riparius were not seen above 15 psu, and the molting success rate was less than 20% at 10 psu. The developmental retardation of C. riparius was well observed in the pupation and emergence period and was delayed by 4 days at 10 psu compared to the control and 5 psu. The mouthpart deformities after salinity exposure at 96 or 72 hour were observed at 10 psu and 15 psu. The expression of C. riparius HSP70 level was significantly increased exposure to 5 psu and 10 psu. Thus, salinity has been caused to be various ecotoxicological and molecular stress responses on freshwater organisms similar to harmful substances such as EDCs and so on.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.35-40
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• 2006

This study was performed to investigate the effects of embryo developmental stage and superoxide dismutase (SOD) on the survival of frozen-thawed porcine embryos by open pulled straw(OPS) method. Porcine IVF blastocysts were frozen-thawed by OPS method and cultured for 48 h under the existence of SOD. There are no significant differences in the proportions of normal morphology among the early, mid- and expanded blastoryst stages $(30.8{\sim}38.6%)$. After culture of embryos, the developmental rates to the expanded blastocyst stage(38.7%) were significantly higher than those of other stages (P<0.05). The proportions of expanded and hatched embryos were higher in medium with 1 unit/ml SOD than 0 and 10 units/ml of SOD. The result indicates that OPS method can use for the pig embryo cryopreservation, especially for the late stage blastocysts. SOD may can reduce the demage of frozen-thawed porcine embryos.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.65-71
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• 2017

The elevated temperature and high humidity has been known as main reason for heat stress on animals and cause detrimental effects on productivity of organisms and physiological conditions of normal bioactivities. The aims of this study were to evaluate the relationship between time of heat shock simulation during in vitro maturation and developmental competence of subsequent embryo after in vitro fertilization. Heat shocked cumulus-oocyte complexes (COCs) of Korean native cattle were subjected to normal conditions for 22, 21, 18 and 12 h respectively and transferred to heat stress inducing condition at $40.5^{\circ}C$ in other incubator for 0 (control), 1 and 4 h. After maturation for 22 h, the oocytes were fertilized and cultured in mSOF media for 8 d and examined the developmental capacity of embryos. There were no differences in maturation and cleavage rates between 0, 1 and 4 h heat socked oocytes, but blastocysts formation were lower in the 4 h heat stressed oocytes. The apoptotic cells of developed blastocysts were also increased in at day 8 with 4 h heat shocked oocytes. These results indicate that heat shock on oocytes during maturation could cause negative effects on the developmental competence of embryos.

• Korean Journal of Organic Agriculture
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• v.22 no.3
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• pp.445-455
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• 2014

The pear sucker (Cacopsylla pyricola) is the most important insect pest in Korea. The hatching rates of overwintered adults laid eggs were observed at 10, 13, 18, and $22^{\circ}C$. The liner model was draw as Y=0.00277X+0.00146(Y=developmental rate, X=temperature) about temperature and developmental rate to eggs. The developmental threshold temperature to eggs was assumed about $-0.83^{\circ}C$. The egg hatching timing over 50% as the accumulated temperature by day maximum temperature $6^{\circ}C$ over from 1st February in 2008, 2009 and 2011 was 429.7, 417.6, and 424.3 degree $^{\circ}C$, respectively, was 3 to 7days before full blooming in pear orchard. On the other hand, the abamectin 1.8EC, lime sulfer, machine oil and 13 kinds of eco-friendly materials were not shown the control effect to the eggs. To mixed stage of pear sucker, the eco-friendly materials over 90% control value were 4 kinds such as a mixture of Azadiractin A+B, Nimbin, Salanin, Meliantriol and Vepol after twice application as 21st May and 5th June in pear growing season, in 2012.

• Reproductive and Developmental Biology
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• v.31 no.1
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• pp.29-33
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• 2007

This study was conducted to examine the development of in vitro fertilized (IVF) and nuclear transfer (NT) embryos following vitrification IVF and NT embryos developed to the blastocyst stage were equilibrated by 3 steps, vitrified and thawed, and their survival and hatching rates were examined. In IVF embryos, higher survival (82.1%, 96/117) and hatching rates (64.1%, 75/117) were obtained respectively after thawing and culture in expanded blastocysts compared to blastocysts (p<0.05). High survival and hatching rates were also obtained by vitrification of NT blastocysts, especially in expanded and hatching blastocysts (81.1 and 78.3%, respectively). The result of this study shows that IVF and NT blastocysts, especially late stage blastocysts, are successfully cryopreserved by vitrification.