• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.217-224
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• 1992

These studies were carried out ot investigate on the transferred embryo development following ultrarapid frozen for 8-cell and morula of in vitro fertilization mouse embryos. The post-thaw embryo survival was evaluated and compared by cell stage of embryos and by equilibration time before ultrarapid freezing. The results obatined were summerized as follows: 1. The effects of equilibration time of 3 vs. 6 minutes before ultrarapid freezing and after thawing on the morphological survival and the viability of 8-cell and morulas embryos were not significant. 2. When the ultrarapid frozen-thawed 32 eight-cell and 33 morula embryos, and 30 fresh blastocysts were transferred to pseudopregnant recipient mice, the number of normal offsprings produced were 9(28.1%), 14(42.4%) and 18(60.0%), respectively. From the above resutls, it was concluded that the optimal conditions of pH osmolality of the media for mouse IVF and embryo culture, and the period of sperm preincubation might be 7.1, 310 mOsm and 120 min., respectively,a nd somewhat high conception rate might be resulted from transfer of frozen embryos of morula stage and fresh embryos of blastocyst stage.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.169-176
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• 2005

Loop-mediated isothermal amplification (LAMP) is novel DNA amplification methods that amplifies a target sequence specifically under isothemal condition. The present study was to assess the in vitro viability afier biopsy and sexing rate of different types of embryo biopsied. In vivo compact morulae and blastocyst embryos were obtained from Korean Native Cow (KNC) superovulated with FSH (Antorin, R-10) on 7 Day after artificial insemination. in vitro compact morulae and blastocyst embryos were obtained with KNC or Holsteins that were gained on 6, 7 or 8 day after in vitro fertilization(IVF) with frozen semen. Biopsy of bovine embryo was carried out in a $80{\mu}l$ drop with $Ca^{2+}-Mg^{2+}$ free D-PBS and the viability of biopsied embryos were evaluated in IVMD (IFP, Japan) medium at 12 hrs culture time. The sex ratio of biopsied Hanwoo embryos were male vs. female of $43.5\%\;vs.\;56.5\%$ in vivo and $33.9\%\;vs.\;49.2\%$ in vitro respectively, and male rate of biopsied Holstein embryos were significantly higher than female $(70.8\;vs.\;29.2\%)$. and indefinite rate of in vitro embryos was $16.9\%$ and in vivo was not. The degeneration rate of biopsied embryo, in vitro embryos were significantly higher than in vivo $(13.2\%\;vs,\;0.0\%,\;p<0.05)$. The survivability of in vivo embryo were between biopsied following punching method was significantly (P<0.05) higher than bisection method produced embryos $(100\%\;vs.\;83.3\%)$ and in vitro had no difference. However, the degeneration rate of biopsied embryo by bisection method was significantly higher than punching methods between in vivo and in vitro $(16.7\;vs.\;22.6\%,\;respectively,\;p<0.05)$. In conclusion, these results indicate that punching method was optimal and survivability after embryo biopsy was useful for reducing the damage caused by the embryo biopsy procedure for LAMP-based embryo sexing.

• Korean Journal of Animal Reproduction
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• v.7 no.1
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• pp.19-23
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• 1983

Present studies were conducted to investigate the developmental stage and the location of embryos in the reproductive tract at various times after ovulation, the morphologically normal after thawing of embryos preserved in liquid nitrogen, and the survival after transferring frozen-thawed embryos. The results obtained were as follows: 1. Embryo stage and location in the reproductive tract after hCG administration. For the investigation of embryo stage and location in the reproductive tract after ovulation, rabbits were laparotomized at 24, 40, 48, 72 and 120 hrs post hCG injection, simultaneously with mating. the oviducts and uteri were flushed out with PBS medium containing 50% rabbit serum, respectively. 1) Most of embryos was remained in the oviduct within 48 hrs, with the lapse of time, embryos were started to move to uterus and shifted in uterus at 72 hrs after hCG injection. 2) The representatives of embryos stage collected at 24, 40, 48, 72 and 120 hrs were 1-cell(60.4%), 8-cell to early morula (52.3, 39.3%), late blastocyst (95.5%) stages, respectively. 2. Morphological normality and survival of the frozen-thawed embryos. For the evalution of the quality and viability on the frozen-thawed embryos, immediately after thawing, embryos were assessed by morphologically normal under a dissecting microscope, and a further test of frozen-thawed embryos was made by transferring the morphologically normal embryos to the uteri of recipient rabbit induced pseudopregnancy by the injection of hCG at the time of hCG injection in donor rabbits. 1) The propotions of embryos which a, pp.ared morphologically normal was higher when 8-cell (85.7%) and morula(90.5%) were used for freezing than when 4-cell (66.7%) and blastocyst (75.8%) were used. 2) Preganacies were observed at Day 15 after transfer of frozen-thawed 8-cell (7/13), morula (19/42) and blastocyst (3/19) but not after transfer of embryos at 4-cell stage.

• Journal of the Korean Applied Science and Technology
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• v.36 no.1
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• pp.258-268
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• 2019

The extracts of AS contain in alloxanthin, halocynthiaxanthin, astaxanthin and 13 kinds of carotenoids. The aim of the study was to assess the anti-oxidant activity and cell viability of AS. The anti-oxidant activity was determined by using DPPH radical inhibition activity and superoxide dismutase (SOD)-like activity. The results of cell viability assay showed that the extracts from AS were cytotoxic at concentrations above $5.0mg/m{\ell}$. This study was designed to examine inflammation induced by LPS, protection effect by UVB and the toxicity of Ascidian shell extract(ASE) as a functional cosmetic ingredient. Evaluation of embryo toxicity resulted in embryo coagulation and mortality when treated at 5.0, 10.0, $20.0mg/m{\ell}$. At the lowest concentration of $1.0mg/m{\ell}$, hatchability resulted in 100.0 % rate. The results of arrhythmia measurement in larvae showed similarity to the evaluation of embryo toxicity. This result demonstrated that toxicity is present at concentrations greater than $5.0mg/m{\ell}$. The protective effect of ASE on LPS and UVB-induced in the zebrafish was investigated. Intracellular reactive oxygen species(ROS) generated by the exposure of zebrafish to LPS, UVB-radiation were significantly decreased after treatment with ASE at $0.1mg/m{\ell}$. As a result, ASE similarly reduced UVB-induced ROS generation and cell death in live zebrafsih. Therefore, it is suggested that ASE has anti-Inflammatory effects and can possibly be used as a functional substance for skin protection in the future.

• 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 Veterinary Research
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• v.31 no.4
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• pp.523-527
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• 1991

The effects of ethylene glycol, DMSO and glycerol as cryoprotectants and the effect of concentrations(0, 0.25, 0.5 and 1.0M) of sucrose in the diluent on the viability of the aggregated morulae frozen rapidly in liquid nitrogen$(LN_2)$ vapour were examined. The morulae were produced by aggregation of ICR and CBA mice embryos at 8-cell stage. Before freezing the embryos were equilibrated in 1.5M cryoprotectants+0.25M sucrose in oae-step or in 3.0M cryoprotectants+0.25M sucrose in two-steps. The embryos were pipetted into the freezing medium fraction of 0.25ml plastic straws. The straws were frozeu by directly transfer into $LN_2$ vapour(about lcm above $LN_2$) for 2 minutes, and then plunged into $LN_2$. After thawing the cryoprotectants were diluted with 0, 0.25, 0.5 or 1.0M sucrose solution. The post-thawed in vitro viability of the aggregated embryos was significantly dependent on the type and concentration of cryoprotectants in the freezing medium and also on the concentration of sucrose in the diluent. When the aggregated embryos were equilibrated in 1.5M cryoprotectants +0.25M sucrose in one-step and diluted with 0.5M sucrose after thawing, the survival rate of the embryo5 was significantly(p<0.05) higher in DMSO(62.5%) or ethylene glycol(52.2%) than in glycerol(33.3 %). In the case that the concentration of the cryoprotectants was raised to 3.0M in two-steps, thc higher survival rate of the embryos was obtained in ethylene glycol or glycerol than in DMSO followed by diluting them with 0.5 or 1.0M sucrose after thawing(p<0.01).

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.307-313
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• 1997

Experiments were conducted to assess the effect of quality and viability of bovine blastocysts derived from in-vitro culture(IVC) of in vitro matured and fertilized(IVM-IVF) oocytes during their transport 2 hours. Follicular oocytes were collected form ovaries obtained at a slaughterhouse and were cultured for 24 hours in TCM-199. The IVM oocytes were fertilized in vitro with caudal epididymis spermatozoa. Fertilized oocytes were cultured for 7 to 9 days, and embryos that developed to the blastocyst stage were used for the experiment. The blastocysts, packed in straws with storage medium that consisted TCM-199 with HEPES equilibratd in air and supplemented with 10% FCS were transported at 39~(2.0 h). The quality of blastocysts was assessed and ranked as A(excel-lent), B(Good), fair or poor after transportation. The percentages of A and B grade blastocysts after transport duration for < 1 hours(97.7%) were similar to the result from transport duration for 1~2 hours (92.9%) and 2~3 hours(89.6%), but significantly(P<0.05) higher than transpot duration for 3~4 hours(76.3%). The percentages of A and B grade blastocysts after transport duration for two hours from developed blastocyst at 7day(100%) and 8day(85.0%) were higher 9day(96.6%) and >9day (40.0%). And early to expanded blastocyst produced in vitro were transferred to recipient cow by additional embryos at 7 and 8th day after AI. Three of them were pregnant to term and produced four twin calves, and two calves was premature birth. The gestation lengths of male to female and female to female twin were 282 and 281 days, respectively. And birth weight of twin calves were male to female(22.Skg) and female to female twin(20.3Okg), respectively.

• Journal of Embryo Transfer
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• v.24 no.4
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• pp.253-257
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• 2009

To enhance the embryo preservation technology and better application of embryo transfer technique to the field (dairy science or animal reproduction. etc.), we examined the viabilities of bovine embryos produced in vitro and in vivo after cryopreservation according to their developmental stage and thawing temperature. Bovine embryos from in vivo/vitro fertilization (Hanwoo) were examined at day 7, 8, and 9. Survival rates and total cell numbers of in vivo fertilized embryos were as follows: morulae 68.8% and $67\;{\pm}\;6.0$; blastocysts 80.5% and $120\;{\pm}\;10$; expanded blastocysts 77.4% and $138\;{\pm}\;9.7$, respectively. Rates of embryo development for blastocysts and expanded blastocysts after thawing were significantly higher than that of morula stage embryos (p<0.05). While survival rates of in vitro fertilized embryos according to developmental stage showed no significant difference among groups (morula 67.9%; blastocyst 74.3%; and expanded blastocyst 79.4%), total cell numbers were significantly lower than those of other groups (morula $64\;{\pm}\;5.9$; blastocyst $116\;{\pm}\;8.7$; and expanded blastocyst $135\;{\pm}\;9.1$) For the viability according to thawing temperature, survival rate was higher in $37^{\circ}C$.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.195-199
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• 2011

The objective of this study was to evaluate the efficiency of the conventional slow freezing and vitrification methods for cryopreserving in vivo and in vitro-produced bovine embryos. Morphology of post-thawed embryos was evaluated and normal embryos were used for successive culture for 72 h. In experiment I, In embryo viability, There was no significant differences in blastocyst re-expansion rates were found between in vivo and in vitro embryos(89.6% vs. 81.5%). whereas hatched-BL and total cell number rates was significantly higher (p<0.05) for in vivo-derived embryos (76.9%, 136${\pm}$3.6 vs. 43.4%, 107${\pm}$3.8). In experiment II, There was no significant differences in blastocyst re-expansion and Expansion-BL rates were found between in slow freezing and vitrification methods (91.3% vs. 85.7% and 71.4% vs. 75.0%, respectively). in conclusion, These results suggested that the field application for bovine embryo transfer is in part supported by improvements of technologies in embryo conventional slow freezing and vitrification cryopreservation.

• Journal of Embryo Transfer
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• v.22 no.4
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• pp.245-249
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• 2007

The aim of present experiment was to examine hatching rate as in vitro indicator of viability of porcine embryos before early stage embryo transfer such as zygotes or 2-cell stage embryos. Cumulus-oocyte complexes (COCs) collected from ovaries were matured in North Carolina State University 23 (NCSU-23) containing 10% porcine follicular fluid (pFF), 10 ng/ml epidermal growth factor (EGF), $10{\mu}g/ml$ follicle stimulating hormone (FSH), $35{\mu}g/ml$ luteinizing hormone (LH), and 1mg/ml cysteine. After 24 hours, the COCs were transferred to the same medium without hormones. After 65h of maturation, oocytes were exposed to phosphate buffered saline (PBS) with 7% ethanol (v/v) for 7 minutes, and then the oocytes were washed and cultured in tissue culture medium (TCM) 199 containing 5 ug/ml cytochalasin B for 5h at $38.5^{\circ}C$ in an atmosphere of 5% $CO_2$ and 95% air with high humidity. After cytochalasin B treatment, the presumptive parthenotes were cultured in porcine zygote medium (PZM)-5 and cleavage of the parthenotes was assessed at 72h of activation, Normally cleaved parthenotes were cultured for an additional 8 days to evaluate their ability to develop to blastocyst and hatching stages. The fetal bovine serum (FBS) were added at Day 4 or 5 with concentrations of 2.5, 5 or 10%. The blastocyst rates were ranged within $39.1{\sim}70%$ in each treatment. However hatching rate was dramatically decreased in non-addition group. In this experiment, embryo viability in female reproductive tract may be estimated before embryo transfer with in vitro culture adding FBS by hatching ability.