• Reproductive and Developmental Biology
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• v.33 no.4
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• pp.257-262
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• 2009

This study was to investigate pregnancy rate of IVM/IVF/IVC Korean cattle (registered in government) embryos according to transport time course. For the production of embryos, oocytes recovered from slaughtered excellent grade cow and highly motile frozen-thawed bull semen (purchased from LIMC, KPN#497) was used. In vitro produced embryos were cultured in CR1aa medium for 8 days and some of them were frozen. The rate of average cleavage (>2-cell) was 83.0% (308/371) and blastocyst rate at day 8 was 34.7% (107/308). Among in vitro produced blastocyst embryos at day 8, most healthy embryos were freshly transferred on production day and some frozen embryos were direct transferred on appropriate day. These embryos were produced in a laboratory, embryo transfer (ET) was planned in 10 areas of the remote island (Jeju) from the laboratory by airplane. Thus, we examined the pregnancy rate in recipient cow according to embryo of transport time course before ET. From embryo transferred 44 recipient cows, overall pregnancy was 40.9% (18/44), these 18 cows were all calved [single, 94% (17/18); twin, 6% (1/18)] and total embryo implantation rate was 26% (19/66). Comparing transport time in the base of 6 hr, pregnancy rate in ET group required less 4 hr (60%, 9/15) was significantly higher than that required more 6 hr (26.3%, 5/19). In direct ET of freezing embryos, the pregnancy rate was 40% (4/10). However, it was difficult to find the meaning of temperature, pH and corpus luteum quality of recipients on comparison of pregnancy rate. When the cell death level of embryos according to storage time in thermos (straw container) before ET was measured by TUNEL staining, apoptotic index was increased with storage time-dependent. These results demonstrated that long distance transfer of IVM/IVF/IVC embryos is possible and the time of embryo transport is very important for the pregnancy rate on field trial.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.4
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• pp.169-174
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• 2015

Objective: To assess compliance with Korean guidelines for embryo transfer, the possible reasons for non-compliance, and multiple pregnancies according to each specific condition in compliant cycles. Methods: A single-institution, retrospective study was conducted of 256 fresh in vitro fertilization cycles during 2012-2014. To assess compliance with Korean guidelines, the maximum recommended number of embryos transferred (according to criteria of age, transfer day, and presence of favorable conditions) was compared with the actual number of embryos transferred. Clinical pregnancy rate (PR) was assessed as the percentage of pregnant women resulting from each set of transfer conditions, including the number of embryos transferred. The multiple pregnancy rate (MPR) was calculated as the percentage of pregnant women with a multifetal pregnancy. Results: The compliance rate with the Korean guidelines was 96.5% (247/256). Non-compliance occurred in nine cycles owing to poor embryo quality, repeated implantation failure, or hostile endometrium. In compliant cycles, the PR was 31.2% (77/247), and the MPR was 27.3% (21/77; 20 twins and one triplet). Higher MPR was noted in two types of transfer conditions: transfer of three cleavage embryos in women aged 35-39 years with favorable conditions (66.7%; primarily from those aged 35-37 years) and transfer of two blastocysts in women aged ${\geq}40$ years with favorable conditions (50%). Conclusion: Under the Korean guidelines, compliance rate was high in our center. Multiple pregnancies occurred primarily in group with favorable conditions. In high-risk groups for multiple pregnancies, reducing number of embryos transferred should be considered than suggested in the guideline.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.67-75
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• 2000

This study was conducted to establish the optimal conditions for in vitro embryo production using oocytes derived from follicles of slaughter-house ovaries. The ovaries of Hanwoo were obtained from a local slaughter-house. The oocytes were aspirated from visible follicles of 2~7mm in diameter. The recovered oocytes which were completely surrounded by at least 2 layers of cumulus cells and a homogeneous cytoplasmic pigmentation were used. The selected oocytes were washed 3 or 4 times with D-PBS containing 10% bovine calf serum (BCS) and matured in vitor (IVM) in Ham's F-10 supplemented with 10% BCS or 0.01 $\mu\textrm{g}$/ml epidermal growth factor(EGF) at 39$^{\circ}C$ under 5% CO2 in air for 24 hours. They were fertilizqed in vitro (IVF) with fresh sperm separated by Percoll density gradient or swim-up in TALP media. The zygotes were cultrued with or without bovine oviductal epitherial cells(BOEC) in media(HECM-6 supplemented with 11 amino acid and / or TCM-199 supplemented with 10% BCS) for 7 to 10 days. The results obtained were as follow: The cleavage rate and developmental rate to blastocyst after maturation and IVF were not significantly different between Ham's F-10 with EGF(76.0% vs. 44.0%) and BCS(75.9% vs. 43.6%)(P<0.05). The cleavage rate and development rate to blastocyst after fertilizing by swim-up or Percoll method were not signifciantly(P<0.05) different between swim-up (80.2% vs. 29.2%) and Percoll(81.9% vs. 26.5%) (P<0.05). The cleavage rate in TCM 199(80.5) was signficiantly higher than that in HECM-6 (72.0%) (P<0.05). However, developmental rate to blastocyst using TCM 199 following HECM-6 for 3 or 4 days (42.2%) was significantly higher than that in TCM-199 alone(26.7%)(P<0.05). The cleavage rate and development rate of embryos produced in vitro by exchange timing for HECM-6 media were not significantly different between in day 3(78.6% vs. 45.5%) and day 4(75.0% vs. 43.2%)(P<0.05). The cleavage rate and developmental rate to blastocysts according to co-culture system were not significantly different between with (74.2% vs. 41.4%) and without BOEC(73.95 vs. 43.5%) (P<0.05). The number of blastomere in blastocyst stage after co-culture with or without BOEC was not significantly different (106.7$\pm$5.1 and 96.6$\pm$4.0). In conclusion, the most transferable IVP embryos could be produced from Ham's F-10 medium for IVM, Percoll density gradient method for IVF sperm separation and in vitro culture in HECM-6 until day 3 or day 4, and then transferred into TCM-199 until day 9 within adequate embryo density in culture droplets after insemination.

• 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.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.10 no.2
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• pp.131-138
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• 1995

Essential and non-essential amino acids supplemented to culture medium stimulate mammalian embryo development in vitro. Amino acids such as glycine, taurine and alanine are concentrated in the lumen of oviduct and uterus and it can he thought that these amino acids may have physiological role on fertilization and embryo development. Our aim of this experiment was to investigate the effects of essential and non-essential amino acids, taurine or glycine supplemented to fertilization medium on the cleavage and subsequent in vitro development of bovine oocytes matured and fertilized in vitro. Immature oocytes were obtained from slaughtered Holstein cows and heifers and matured in TCM199 containing 10% fetal calf serum, 2.5 $\mu$g /mL of FSH and LH and 1 $\mu$g / mL of estradiol with granulosa cells in vitro. After maturation, oocytes were coincubated with sperm in fertilization medium supplemented with Minimum Essential Medium (MEM) essential and non-essential amino acids, taurine (3.75 mM) or glycine (10 mM) for 30 hours in vitro. Inseminated oocytes were cultured in synthetic oviduct fluid medium (SOEM) containing MEM essential, non-essential amino acids and 1 mM glutarnine up to 8 days after fertilization.Supplementation of fertilization medium with MEM essential and non-essential amino acids lowered significantly (p<0.05 and p<0.001) the cleavage rate after 30 hours of IVF (53.3%) and at Day 3 (62.7%: Day 0: the day of I VF) compared to control (64.3% and 77.3%, respectively). Subsequent developmental rates to morulae (Mo) and expanding blastocysts (ExBL) also significantly decreased (p<0.001 and p<0.05 for Mo and ExBL) when oocytes were coincubated with sperm in the medium containing MEM amino acids. Taurine added to fertilization medium have not increased the cleavage rate over the control, whereas glycine showed significantly lower (p<0.01) cleavage rate at Day 3 than that of taurine, but there was no significant difference in the developmental rates to Mo and ExBL of bovine embryos irrespective of the supplementation of taurine or glycine to fertilization medium. In conclusion, supplementation of fertilization medium with essential and non-essential amino acids, taurine or glycine has no beneficial effect on in vitro cleavage and development of bovine oocytes matured and fertilization in vitro.

• Korean Journal of Veterinary Research
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• v.29 no.4
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• pp.589-599
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• 1989

A total of 13 synchronized dairy cattle(Holstein) were used to determine pregnancy rates in relation to plasma progesterone, estradiol-$17{\beta}$ levels and serum chemical values on the day of last $PGF_{2{\alpha}}$ injection and day of frozen/thawed bovine embryo transfer. The pregnancy rate of recipients with 1.0~4.0ng/ml of progesterone levels at the day of last $PGF_{2{\alpha}}$ injection was higher than that of recipients with below 1.0ng/ml or above 4.0ng/ml of progesterone levels. On the day of transfer, optimal progesterone levels were between 1.0ng/ml and 4.0ng/ml coinciding with a pregnancy rate of 88.9%. Pregnancy rate decreased when progesterone levels were below 1.0ng/ml(33.3%) or above 4.0ng/ml(0%). Corpus luteum grade did not affect pregnancy rate and this result revealed that manual palpation of corpus luteum was not valid criterion of corpus luteum function. Progesterone levels as well as pregnancy rate did not significantly differ whether the corpus luteum was on the right($1.62{\pm}1.33ng/ml$; 63.5%) or left ovary($1.99{\pm}0.61ng/ml$; 85.0%). Estradiol-$17{\beta}$ levels were not significantly different between pregnant and nonpregnant recipients, but estradiol-$17{\beta}$ levels($82.2{\pm}13.5$ VS. $72.3{\pm}10.1pg/ml$) were higher at below 1.0ng/ml of progesterone, and pregnancy rates(33.3 VS. 80%) tended to be lower than above 1.0ng/ml of progesterone. Total cholesterol levels on the day of last $PGF_{2{\alpha}}$ injection and day of transfer did not affect pregnancy rate. Calcium and inorganic phoshorus levels belonged to normal range in most of the recipients. These range did not affect pregnancy rate. In reviewing above results, plasma progesterone levels(1.0~4.0ng/ml) at the time of transfer are diagnostic value for screening recipients prior to transfer of frozen/thawed bovine embryos.

• 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.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.17 no.3
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• pp.203-209
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• 2002

The aim of this study was to assess the developmental capacity of oocytes maturated in vitro after 10 days of culture when the preantral follicles were isolated from juvenile mice 10- and 20-day old, respectively, and to develop in vitro culture system that observed a view to morphology of follicles and nucleus maturation of oocytes. The antral-like cavities became formation after 6 days of culture in follicle isolated from 10- and 20-day old mice. The number of follicles were 21.5 and 33.3 in ovary isolated from 10- and 20-day old mice, respectively. The diameters of oocytes were 51.85 and 57.50 ${\mu}{\textrm}{m}$ before culture and were grew 55.95 and 63.11 ${\mu}{\textrm}{m}$ after culture for 10 days, in follicles isolated from 10- and 20-day old mice, respectively. The observation rates up to the M II and from GV to M II were 4.3 and 22.1%, and 14.5 and 61.1% after culture for 10 days in follicles isolated from 10- and 20-day old mice, respectively.