• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.335-339
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• 2011

The objective of this study was to investigate the relationship between concentration of urea nitrogen, glucose, cholesterol and number of transferable embryos for the purpose of improving reproductive performance in blood of Hanwoo donors. Fifty five, at random stages of the estrous cycle, received a CIDR. Four days later the animals were superovulated with a total of 28AU FSH (Antorin, 2AU=1 ml) administered twice daily in constant doses over 4 days. On the 3th administration of FSH, CIDR was withdrawn and 25 mg $PGF_2$ ${\alpha}$ was administered. Cows were artificially inseminated twice after estrous detection at 12 hr intervals. The cows received 100 ${\mu}g$ GnRH at the time of 1nd insemination. Embryos were recovered 7 or 8 days after the 1st insemination. Cows with BUN < 10, 11~18 and ${\geq}$19 mg/dl had number of transferable embryos of $4.32{\pm}1.3$, $5.8{\pm}1.8$ and $4.7{\pm}2.1$ respectively. The mean numbers of total ova from < 10 and 10${\leq}$ of corpora lutea(CL) was 8.9 and 14.3, respectively. The number of transferable embryos differed between < 10${\leq}$ CL was 4.8 and 5.6, respectively.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.15-20
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• 2012

The objective of this study was to investigate the relationship between body weight, body condition score (BCS), blood urea nitrogen (BUN), glucose, cholesterol and number of transferable embryos for the purpose of improving reproductive performance in Hanwoo donors. Seventy five cows, at random stages of the estrous cycle, received a CIDR together with injection of 1mg estradiol benzoate and 50 mg progesterone, and gonadotropin treatment begann. Four days later, the animals were superovulated with a total of 28AU FSH (Antorin, 2AU = 1 ml) administered twice daily in constant doses over 4 days. On the 3rd administration of FSH, CIDR was withdrawn and 25 mg $PGF_2{\alpha}$ was administered. Cows were artificially inseminated twice after estrous detection at 12 hr intervals. The cows received $100{\mu}g$ GnRH at the time of 1st insemination. Embryos were recovered 7 days after the 1st insemination. In conclusion, cows with body weight < 400, 400~450 and > 450kg had number of transferable embryos of $4.2{\pm}1.7$, $6.1{\pm}2.7$ and $4.8{\pm}2.6$, cows with BCS <2.25, 2.25~2.75 and ${\geq}2.75$ had number of transferable embryos of $4.6{\pm}1.6$, $5.7{\pm}2.4$ and $5.1{\pm}2.7$ respectively. These data indicate that a body weight and BCS for superovulation of CIDR-treated Korean native cows does not affect the embryo yield.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.4
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• pp.483-491
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• 2009

In vitro produced porcine embryos have potential application in reproductive biotechnology. However, their development potential has been very low. This study evaluated the in vitro developmental ability and quality of cloned and parthenogenetic porcine embryos having 2-4 cells or 5-8 cells on Day 2 of in vitro culture. Analysis of results showed that 2 to 4 cell embryos had higher ability to form blastocysts than 5 to 8 cell embryos (p<0.05). Blastocysts produced from culture of 2 to 4 cell embryos also contained higher cell numbers and had lower BAX:BCLxL transcript ratio than those produced from 5 to 8 cell embryos (p<0.05), thereby suggesting 2 to 4 cell embryos have higher development potential. Further investigation revealed that 5 to 8 cell embryos had higher incidence (100${\pm}$0.0%) of blastomeric fragmentation than 2 to 4 cell embryos (15.2${\pm}$5.5% for parthenogenetic and 27.7${\pm}$7.1% for cloned embryos). This suggests that low development potential of 5 to 8 cell embryos was associated with blastomeric fragmentation. In conclusion, we have shown that morphological selection of embryos based on cell number on Day 2 of in vitro culture could offer a practical and valuable non-invasive means to select good quality porcine embryos.

• Journal of Embryo Transfer
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• v.27 no.4
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• pp.259-264
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• 2012

Solution of glycerol, ethylene glycol, sucrose, dextrose (GESD) and cryotop methods were carried out to investigate the survivability on vitrification of embryos. Embryos cultured in vitro were vitrified by GESD of 10 or 8 step and cryotop methods of 6 step, from cryopreservation step to frozen-thawed and culture step. Survival rate and ICM, TE cells of embryos were investigated after frozen-thawed 24 h. As a results, cryotop method was significantly (p<0.05) higher ($85.76{\pm}5.3$ vs. $66.71{\pm}2.4$, $44.80{\pm}2.1%$) than GESD 10 or 8 step methods on survivability. Also, In ICM cell number, cryotop method was significantly (p<0.05) higher to $45.67{\pm}4.7$ cells than GESD 8 step method. TE cell number was significantly (p<0.05) highest to $111.00{\pm}11.0$ cells in cryotop method. On the other hand, survival rate, TE and total cell number were all the significantly (p<0.05) high, except ICM in GESD 10 step method between GESD 10 step method and GESD 8 step method. In conclusion cryotop method was to be most effective, but it is considered necessary to study vitrification method for step-by-step freezing and thawing process.

• Journal of Embryo Transfer
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• v.12 no.1
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• pp.67-74
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• 1997

This study was carried out to increase the viability of bovine frozen4hawed in vitro produced (IVP) embryos and pregnancy rate by direct transfer method. Cumulus-oocyte complexes were aspirated from excised Hanwoo ovaries and matured in TGM 199 for 20~22 hours at 38.5$^{\circ}C$ in 2% $CO_2$ in air. Matured oocytes were fertilized with capacitated sperm for 6 hours and then co-cultured with cumulus cells for 9 days. 63% of the oocytes cultured was deaved and 29% out of them developed into blastocysts. Good or excellent grade of blastocysts on D 7 or 8 were frozen with 1.8M ethylene glycol as a cryoprotectant for direct transfer. Frozen embryos were thawed at 2$0^{\circ}C$ water for 10 sec following 4~5 second in air. For the survival assay of frozen4hawed lVP blastocysts, they were cultured in TCM 199 supplemented with 100$\mu$M $\beta$-mercaptoethanol and 20% FCS for 72 hours. The percentage of embryos developed to re-expanded or hatched after 72 hours culture was 95. 5 and 77.3%, respectively. When frozen-thawed Ivp embryos were transferred to 43 synchronized recipients by direct transfer method, eighteen recipients (41.8%) was pregnant. The highest pregnant was in naturafly synchronized recipients (71.4%), but induced estrus by using PRID(29.2%) and PGF$_2$$\alpha$(20.0%) was showed lower pregnancy rate. The pregnancy rate was higher in day 7 blastocysts(56.0%) than day 8 blastocysts(22.2%). (Key words: in vitro produced, blastocyst, frozen-thawed, direct transfer)

• 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 Embryo Transfer
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• v.22 no.4
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• pp.251-255
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• 2007

This study was carried out to examine on developmental competence of Hanwoo embryos cultured in vitro according to culture conditions and freezing methods. The in vitro developmental competence to blastocyst stage at Day 8 of culture in SOF was significantly (p<0.05) higher than that in CR1aa (30.3% vs. 18.4%). The in vitro developmental rate of morula and blastocysts cultured in group culture was significantly (p<0.05) higher than that in individual culture (41.4% and 36.0% vs. 21.1% and 10.5%, respectively). The cell number of Day 8 blastocysts in group culture was significantly (p<0.05) higher than that in the individual culture ($120.1{\pm}12.8\;vs.\;94.1{\pm}12.1$, respectively). The survival rates of frozen-thawed balstocysts that were exposed in 1.5 M ethylene glycol or 1.5 M ethylene glycol containing 0.1 M sucrose were 77.5% and 78.7%, respectively. The survival rates of blastocysts cultured for 48 h in slow freezing and vitrification was not significantly different (73.3 and 74.0%). In conclusion, in vitro developmental competence of bovine embryos was influenced on the culture medium (SOF) and culture method (Group culture). Survival rate of frozen-thawed of bovine embryos was not influenced on freezing solutions and freezing methods.

• Journal of Embryo Transfer
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• v.20 no.3
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• pp.311-315
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• 2005

This study was conducted to investigate the effects of disregarding of heat detection on embryo production in superovulation of Hanwoo cows. Donors which showed 1 or 2 times of normal heat and had no abnormality of reproductive tract were selected The superovulation was performed injection of 2.5 mL FSH (Antorin R-10, Japan) 2 times on 5 days before next heat and continuously with the reduction of dose to 0.5 mL of first injection 2 times in a day for next 3 days. Otherwise, the donors of CIDR group were inserted CIDR plus (with the capsule of estradiol benzoate 10 mg) on Day 10 from standing heat for 9 days. On 6 days from insertion of CIDR, FSH was injected above same manners. The response according to the natural heat and CIDR were $82.2\%,\;89.7\%$, respectively. There were no difference between both treatments. The recovery rates of embryos were 7.7, 10.5, respectively and transferable embryos were 3.4, 3.8. There showed significant difference between both treatments (p<0.05). These results suggested that disregarding of heat detection in superovulation could be produced transferable embryos for embryo transfer and preserve the donors from the excess hormonal administration and maintain the economical lift span of genetically available Hanwoo donors.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.9
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• pp.1260-1266
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• 2001

The effect of protein supplementation, $O_2$ concentration and co-culture on the development of embryos produced by nuclear transfer using cultured cumulus cell was investigated. Recipient oocytes and cumulus cells were obtained from the ovaries of the slaughtered Hanwoo cows. Donor cumulus cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum at 5% $CO_2$ in air at $38.5^{\circ}C$. The 1 to 6 passages of cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One $15{\mu}s$ pulse of 180 volts was applied to induce the fusion between karyoplast and cytoplast. The fused embryos were activated with $10{\mu}M$ calcium ionophore for 5 min and 2 mM 6-dimethylaminopurine for 3 h. To examine the effect of protein supplementation, nuclear transfer (NT) embryos were cultured in one of the following 4 treatments : 1) CR1aa + 3 mg/ml BSA for 7 days ; 2) CR1aa + 10% FBS for 7 days ; 3) CR1aa + 1.5 mg/ml BSA + 5% FBS for 7 days ; and 4) CR1aa + 3 mg/ml BSA for first 3 days and then CR1aa + 1.5 mg/ml BSA + 5% FBS for 4 days. Culture took place at 5% $CO_2$, 5% $O_2$ and 90% $N_2$ at $38.5^{\circ}C$. Although there were no significant differences in cleavage rate among different protein supplements, the rates of blastocyst formation were significantly different. When NT embryos were cultured in the medium supplemented with only BSA, they could develop to only morula not to blastocyst. However, when FBS was supplemented, NT embryos developed to blastocyst stage. In order to investigate the effect of $O_2$ concentration and co-culture, NT embryos were cultured in CR1aa + 1.5 mg/ml BSA + 5% FBS with or without cumulus cell co-culture at an atmosphere of 5% $CO_2$ in air (20% $O_2$) or 5% $CO_2$, 5% $O_2$, 90% $N_2$ (5% $O_2$) at $38.5^{\circ}C$ for 7 days. The percentage of blastocyst development was significantly higher when the NT embryos were cultured at an atmosphere of 5% $O_2$ than that of 20% $O_2$ (p<0.05). However, there was no significant difference between with and without cumulus cell co-culture at an atmosphere of 5% $O_2$ or 20% $O_2$. Fifty embryos were transferred to 25 recipients and 5 recipients were pregnant at 100 days. From 5 pregnant cows, only one cow was delivered of female twin. In conclusion, the embryos reconstructed by enucleation of metaphase II oocytes and introduction of the cycling and quiescent cumulus donor cells in Hanwoo had developmental potential to term after embryo transfer to recipient cows.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.349-353
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• 2011

The objective of this study was to investigate the relationship between estrous expression, body condition score (BCS), blood urea nitrogen (BUN) and number of transferable embryos for the purpose of improving reproductive performance in blood of Hanwoo donors. Sixty, at random stages of the estrous cycle, received a CIDR. Four days later, the animals were superovulated with a total of 28AU FSH (Antorin, 2AU=1 ml) administered twice daily in constant doses over 4 days. On the 3th administration of FSH, CIDR was withdrawn and 25 mg PGF2 ${\alpha}$ was administered. Cows were artificially inseminated twice after estrous detection at 12 hr intervals. The cows received 100 ${\mu}g$ GnRH at the time of 1nd insemination. Embryos were recovered 7 or 8 days after the 1st insemination. The estrous inducement rate and estrous expression rate were significantly lower for cows with BCS below 2.25 than for cows with BCS above 2.25. There was 50.0% of rate of mounting in cows with BCS below 2.25 whereas the rate of mounting was markedly increased in cows with BCS above 2.25 (94.1% and 89.5% for BCS 2.25~2.75 and BCS above 2.75 cows, respectively). Cows with BCS <2.25, 2.25~2.75 and ${\geq}$2.75 had number of transferable embryos of $4.5{\pm}0.7$, $5.9{\pm}1.8$ and $5.6{\pm}2.3$ respectively.