• Journal of Embryo Transfer
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• v.31 no.2
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• pp.117-121
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• 2016

Although piglets have been delivered by embryo transfer (ET) with in vitro produced (IVP) embryos and blastocysts, a success rate has still remained lower level. Unlike mouse, human, and bovine, it is difficult to a production of piglets by in vitro fertilization (IVF) because of an inappropriate in vitro culture (IVC) system in pig. Therefore, the present study was conducted to investigate whether minimized exposure time in IVC can improve the pregnancy and delivery rates of piglets. Immediately after IVM, the oocytes were denuded and co-incubated with freshly ejaculated boar semen for 3.5 to 4 hours at $38.5^{\circ}C$ under 5% $CO_2$ in air. To avoid long-term exposure to in vitro state, we emitted IVC step after IVF. After that the presumptive zygotes were transferred into both oviducts of the surrogate on the same day or 1 day after the onset of estrus. Pregnancy was diagnosed on day 28 after ET and then was checked regularly every month by ultrasound examination. The 3 out of 4 surrogates were determined as pregnant (75%) and a total of 5 piglets (2 females and 3 males) were delivered at $118.3{\pm}2.5$ days of pregnancy period. In conclusion, a short-term exposure time may be an important factor in the production of IVP-derived piglets. It can be apply to the in vitro production system of transgenic pig by IVF, cloning, and pronuclear microinjection methods.

• Journal of Embryo Transfer
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• v.24 no.1
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• pp.47-55
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• 2009

This study was performed to investigate the possibility of repeated superovulation treatment at interval from 27 days to 41 days in Hanwoo cattle and to compare with superovulation effect between doses of FSH 200 mg and FSH 400 mg. Different doses of FSH (200 mg or 400 mg) were injected at Day 8 after controlled internal drug release (CIDR) treatment for superovulation of Hanwoo donors following CIDR treatment (Day 8 after the estrus). Superovulation was repeated four times for one donor and number of corpus luteum (CL), number of embryos, number of transferable embryos and pregnancy rate after embryo transfer (ET) were investigated. 5 cows were used for each FSH treatment (10 cows in total). Average number of CL were $10.16{\pm}3.85$ and $11.56{\pm}2.35$ for the donors treated with FSH 200mg and FSH 400mg, respectively. Average number of embryos collected were $8.85{\pm}4.05$ and $8.30{\pm}1.73$ for the donors treated with FSH 200 mg and FSH 400 mg, respectively. Average number of transferable embryos were $5.48{\pm}2.45$ and $4.58{\pm}2.23$ for the donors treated with FSH 200 mg and FSH 400 mg, respectively. The pregnancy rate following ET with embryos collected from 200 mg FSH treated donors and 400 mg FSH treated donors were 61.9% and 53.8% respectively. The numbers of embryos tended to be decreased as the numbers of repeat of superovulation were elapsed. These results indicated that superovulation treatment by about a month to Hanwoo donors is usable and 200 mg of FSH is preferable for simple FSH treatment following CIDR treatment.

• Journal of Embryo Transfer
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• v.19 no.1
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• pp.1-10
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• 2004

These studies were carried out to improve the reproductive efficiency through embryos transfer of Hanwoo IVM/IVF embryos. Following routine IVM/IVF procedure, oocytes and zygotes were cultured far 40 to 44 h in CRlaa medium with BSA. Then 2 to 8-cell embryos were removed the cumulus cell and were cultured in CRlaa medium containing 10% fatal bovine serum and 2.5 mM taurine in 5% $O_2$ and 5% $CO_2$ at 38.5$^{\circ}C$. The fresh embryos of the morulae and blastocysts cultured for 6 to 9 days in vitro or the frozen-thawed embryos were transferred into recipients. The pregnancy rates of the blastocyst produced for 6, 7, 8, and 9 days in vitro culture were 59.4, 68.2, 66.0 and 100%, respectively. In the developmental stage, pregnacy rates of early blastocysts (61.1%), blastocysts(64.7%) and expanded blastocysts(69.5%) were higher than that of morulae stage(20.0%). The pregnancy rates according to the corpus luteum grades of A, B and C in recipients were 73.6, 62.9 and 50.0%, respectively. Effects of donor-recipients synchrony of after day 2, 1 and 0, before day 1 and 2 on the pregnancy rates were 35.7, 65.5, 72.6, 67.9 and 60.0%, respectively. Pregnancy rates of the body condition score of recipients $\leq$2(71.3%) were higher than those of $\geq$3.0 score(40.0%). The pregnancy rates according to the parity of recipients when embryo was transferred to cow(70.6%) was higher than in heifer(59.1%). The pregnancy rates according to hormone treatment before embryo transfer were 69.9% in hCG + GnRH administration group and 63.0% in control group. Fresh and frozen-thawed embryos on the pregnancy rates were 70.6 and 36.4%, respectively. Pregnancy rates in single and AI+single was 90.0% and 64.8%. Pregnancy rates in twin induction was better than in single. These results indicate that pregnancy rates after transfer were affected on the embryo ages, donor-recipient synchrony, body condition score of recipients, corpus luteum status, parity and hormone treatment to recipients.

• 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.

• 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 Embryo Transfer
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• v.24 no.1
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• pp.33-37
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• 2009

Multiple ovulation and embryo transfer (MOET) has the potential to increase the rates of genetic improvement in cattle. Thus this study was performed to investigate several factors influencing in vivo embryo production in Holstein cattle under field conditions. The donors were superovulated with Folltropin-V and $PGF_2{\alpha}$ combination method. From Day 10 onward, donors were superovulated by i.m., twice daily, administration of 400mg Folltropin-V given in a series of decreasing doses over a 4-day period: on the first day, 3.5ml; on the second day, 3.0ml; on the third day, 2.0ml; and on the fourth day, 1.5ml (20ml in total, equivalent to 400mg of NIH-FSH-P1). Estrus was induced by i.m. administration of 25mg prostaglandin $F_2{\alpha}$ on the sixth and seventh of FSH treatment. Estrus detection was performed twice daily beginning 24h after the first prostaglandin $F_2{\alpha}$ injection. Donor cows were artificially inseminated 12 and 24 h after first standing estrus with semen from a proven Holstein sire. Embryos used in this study were recovered Day 7.5 of the cycle (Day 0: first standing estrus). From 195 superovulated dairy cows, 2,104 eggs were recovered, of which 1,172 were classified as transferable embryos based on morphological evaluation of quality. The results are summarized as follows: 1. The numbers of recovered and transferable embryos did not significantly differ among the capacity of milk production that were < 10,000kg/305days (group 1), $10,000{\sim}12,000\;kg$/305days (group 2) or > 12,000kg/305 days (group 3) (p>0.05, Table 1). 2. No differences in the numbers of recovered and transferable embryos were found among the donor's postparient days (p>0.05, Table 2). 3. Also, the numbers of recovered and transferable embryos of each superovulation seasons did not significantly differ among the four groups (p>0.05, Table 3).

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.335-340
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• 2015

The objective of this study was to evaluate the initial detection time and development of the fetal genital structures using ultrasound in twelve pregnant small bitches. The initial detection time of the fetal genital structures was as follows: genital tubercle at days 32.6; os penis at days 45.2; labia at days 45.7; scrotum at days 47.5. Ultrasonograms of fetal genital structure according to gestational stage were as follows: Undifferentiated stage (before day 35), the genital tubercle was observed to have a small elevation and just a hyper-echogenic structure in the midline between the umbilical cord and the tail in male and female fetus. Migration stage (between day 35~45), the genital tubercle was observed as a hyper-echogenic, bilobular, oval shaped and the genital tubercle began to migrate from the initial position toward the umbilical cord in males, and toward the tail in females. Differentiated stage (after day 46), the penis and os penis were observed to stand out in the abdominal wall and the scrotum was observed toward the perineal region in male fetuses. The labia was detected at the base of the tail in female fetuses. These results indicate that ultrasound of fetal genital structures could be useful for fetal gender determination and a completely prepartum evaluation of the canine fetus.

• Journal of Embryo Transfer
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• v.17 no.3
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• pp.211-218
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• 2002

These studies were carried out to investigate the effects of the addition of amino acids and FBS as source of exogenous nitrogen fixation added to medium on in vitro production of blastocyst derived from bovine follicular oocytes. The base medium was TCM-199 solution for in vitro maturation(IVM) of bovine follicular oocytes and Fer-TALP solution for in vitro fertilization(IVF) and YS solution for in vitro culture(IVC). IVC used the fertilized oocytes of 24-hr culture (day 1) after IVF. Rmbryos were cultured in drop-culture that contained 25 embryos per 10 ${mu}ell$. The results obtained are as follows: 1 The developmental rates of fertilized oocytes to blastocyst that developed from YS solution with NEAA derived from MEM alone were higher than those of YS solution without NEAA. 2. The developmental rates of fertilized oocytes to blastocyst that developed from YS solution with EAA derived RPMI 1640 alone were significantly higher than those of YS solution without EAA (p<0.05). 3. When added to EAA on day 5 after NEAA supplementation on day 1, the developmental rates of hatched blastocyst and blastocyst to hatched blastocyst were improved. 4. When removed to EAA on day 3, day 4 and day 5 from medium after NEAA and EAA supplementation on day 1. the developmental rates of blastocyst to hatched blastocyst were reduced. 5. When added to FBS as source of exogenous nitrogen fixation, the developmental rates of blastocyst and hatched blastocyst that developed from the later culture higher(day 5) than those of the early culture.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.185-190
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• 2005

This study was performed to investigate the effects of multiple superovulation and parity on embryo production in Hanwoo cows. Donors were superovulate 4 times $1\~2$ months interval and inserted CIDR plus (with the capsule of estradiol benzoate 10mg) on Day 10 from standing heat for 9 days and injected 2.5ml FSH (Antorin R-10) 2 times in a day on 6th day to 10th day from insertion of CIDR and the doses of FSH were decreasing 0.5ml on every 2 times. On 3th day of FSH injection, 25ml $PGF_2{\alpha}$ were injected i.m. and on 4th day, CIDR was removed. After 2 days from removing CIDR, AI was performed 2 times 12 hour apart with 2 straws of Korean Proved frozen Semen and simultaneously 200ug/ml GnRH was injected and embryos were recovered on 7th day from Al. The response rates of superovulated donors were $85.7\%,\;90.5\%,\;62.5\%,\;100\%$ from 1 to 4 times of superovulation, respectively. There were significant differences among No. of superovulation times (P<0.05). The results of transferable embryos were 3.7, 3.4, 3.4, 5.7 from 1 to 4 times of superovualtion, respectively. There were no differences among No. of superovulation times. The results of transferable embryos were 2.5, 3.0, 5.3, 3.0, 3.4 form heifer, first born to 4 the born, respectively. There were significant differences among the parities of donors (P<0.05). These results suggested that even 4 times of superovulations of Hanwoo donors could be able to recover transferable embryos, it might be used the donors maximally and improved the adaptation of embryo transfer to farms safely.

• Journal of Embryo Transfer
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• v.17 no.3
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• pp.195-201
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• 2002

This study was to assess the developmental capacity of oocytes matured in vitro after 20, 15, 10, 5 and 0 days of organ culture when ovaries were isolated from juvenile mice at 0-, 5-, 10-,15- and 20-day old, respectively, and to develop in vitro culture system that observed a view to morphology of ovaries and nucleus maturation of oocytes. The size of ovaries decreased 35.9%, 8.7%, 1.2% and 14.4% after 20, 15, 10, 5 days of organ culture when the ovaries were isolated from 0-, 5-, 10 and 15-day old mice, respectively. After organ culture, the recovery rates, diameters of oocytes and the number of oocytes progressed from GV to MII were increased as increasing age of mice.