• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.75-81
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• 2000

Objective: The objective of this study is to influence of sequential embryo transfers in an invitro fertilization was examined. Method: After in vitro fertilization, a maximum of 6 fertilized oocytes was enrolled in this study. At day 3 after an oocytes retrieval, embryos with good quality were transferred (mean 4.9), remaining embryos (mean 2.0/cycle) were cryopreserved at blastocyst stage (Group 1). At day 5 after oocytes collection, second a embryo transfer (mean 1.2/cycle) was performed, if one of these embryos had reached the blastocyst stage (Group 2) using P1 supplemented with 10 SSS and 30% Follicular fluid. No statistical difference in the pregnancy rate could be seen between the group without a second embryo transfer (n=21; 28.6%) and the group with a second transfer (n=52; 28.8%). Results: The incidence of multiple pregnancy rate per embryo transfer was not statistically different between both group and no high-rank multiple pregnancy (greater than triplete) were observed (0.9%, 15.4%, respectively, p=0.74, ${\chi}^2$). Out of 114 cycles (506 embryos) cultured embryos in group 2, 52 cycles (159 embryos, 29.8%) reached the blastocyst stage. Conclusion: The second transfer did not have a significant effect on the pregnancy rate. The most important factor for the pregnancy seems to be the quality of the embryos transferred on day 3 following oocyte retrieval. We recommend embryo transfer is performed only one, day $2{\sim}3$ or D5.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.7
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• pp.969-976
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• 2009

Use of oocytes from prepubertal animals for in vitro embryo production holds potential application for reducing generation intervals and increasing genetic progress through embryo transfer. The objective of these studies was to compare the effect of three sperm pretreatments (prior to in vitro fertilization) and seven embryo culture protocols on fertilization rate and (or) subsequent development of in vitro fertilized embryos derived from oocytes harvested from ovaries of 1-6 month old prepubertal Boer goats in China. Cleavage rates were highest for embryos fertilized with heparin-treated versus calcium ionophore- or caffeine-treated sperm. Similar rates of blastocyst development were observed using heparin- and ionophore-treated sperm, which were higher than obtained with caffeine-treated sperm. No differences in cleavage or blastocyst rates were observed following embryo culture in basal medias (synthetic oviductal fluid (SOF), Charles Rosenkrans 1 (CR1) or tissue culture medium-199 (TCM-199)) containing 10% fetal bovine serum (FBS). Cumulus or oviductal cell co-culture did not enhance cleavage or blastocyst rates relative to culture in SOF+10% FBS. Replacement of FBS in SOF medium with 0.3% BSA increased cleavage rates, but did not increase rates of blastocyst development. Sequential culture in SOF+0.3% BSA followed by SOF+10% FBS increased blastocyst yield versus continuous culture in SOF+10% FBS and tended to increase blastocyst yield versus continuous culture in SOF+0.3% BSA. These results demonstrate a pronounced effects of sperm pretreatments and in vitro embryo culture systems on rates of blastocyst development and provide a potential protocol (sperm pretreatment with heparin and sequential embryo culture in SOF+0.3% BSA followed by SOF+10% FBS) for generation of the significant numbers of in vitro produced blastocysts from oocytes of prepubertal Boer goats necessary for application of embryo transfer in rural regions of China for distribution of Boer goat genetics.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.186-192
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• 2011

Objective: Since IVF program was first established, various types of media and culture systems have been developed either in-house or commercially. The aim of this study was to compare the efficacy of in-house Maria Research Center (MRC) media to that of commercially available Sydney IVF media in human day 3 embryo transfer cycles. Methods: Three hundred sixty nine couples were included in this prospective, randomized, and comparative study. All couples undergoing IVF treatment at the Maria Fertility Hospital were randomly assigned to either Sydney IVF (n=178) or MRC (n=191) media. Results: No difference was observed between the MRC media and Sydney IVF media groups with respect to fertilization rate (74.4% vs. 75.5%). The clinical pregnancy and implantation rates of MRC media (47.1% and 20.0%, respectively) were also similar to those of Sydney IVF media (44.4% and 19.4%, respectively). However, the proportion of embryos with good quality on day 3 was significantly higher in the MRC media group than the Sydney IVF media group (50.2% vs. 43.2%) ($p$ <0.05). Conclusion: MRC media were as effective as Sydney IVF media for sustaining embryo development and pregnancy rates. The present study implies that MRC media can be a suitable alternative to commercially available media for human IVF-ET program.

• Clinical and Experimental Reproductive Medicine
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• v.40 no.3
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• pp.122-125
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• 2013

Objective: The majority of embryo transfers (ETs) to date have been performed on day 3 to reduce the potential risk of developmental arrest of in vitro cultured embryos before ET. Development of sequential media has significantly improved culture conditions and allowed blastocyst transfer on day 5. While day 5 ET provides higher clinical pregnancy outcomes with reduced risks of multiple pregnancies, it still has potential risks of developmental arrest of IVF embryos. The aim of this study was to evaluate the clinical outcomes of day 4 ETs and compare the efficacy of day 4 ET with day 5 ET. Methods: From 2006 to 2009, a total of 747 fresh IVF-ET cycles were retrospectively analyzed (day 4, n=440 or and day 5, n=307). The cycles with any genetic factors were excluded. The rates of matured oocytes, fertilization, good embryos, and clinical pregnancy of the two groups were compared. The chi-square test and t-test were used for statistical analysis. Results: There were no significant differences between the two groups with respect to the mean age of the females and rates of matured oocytes. The pregnancy outcomes of day 4 ET (40.7%) were similar to those of day 5 ET (44.6%). The implantation rate of day 5 ET (24.2%) was significantly higher than that of day 4 ET (18.4%) (p=0.003). Conclusion: Day 4 ET can be chosen to avoid ET cancellation in day 5 ET resulting from suboptimal circumstances in the IVF laboratory, but the decremented quality of embryos for transfer and the decreased pregnancy rate must be taken into consideration.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.175-181
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• 2008

This study was conducted to investigate an effective recipient oocyte and culture system for producing of Hanwoo (Korean native cattle) somatic cell nuclear transfer (SCNT) embryos. Hanwoo ear skin fibroblasts were used as donor cells. In vitro matured Hanwoo or Holstein oocytes were enucleated, and single donor cells were transferred into the perivitelline space of the enucleated oocytes. The couplets were subsequently fused and activated. The reconstructed embryos were cultured in a conventional or sequential culture system. In the former, embryos were cultured in CR2aa medium for eight days; in the latter, embryos were cultured in modified CR2aa-A (mCR2-A) for three days and then further cultured in modified CR2aa-B (mCR2-B) for five days. In the experiment with the recipient oocyte, the rate of embryo development to the blastocyst stage was significantly (p<0.05) higher in Hanwoo recipient oocytes than in Holstein ones (48.8% vs 38.9%). BIastocysts derived from Hanwoo recipient oocytes contained significantly (p<0.05) higher numbers of total cells than those derived from Holstein recipient oocytes ($156.0{\pm}68.2$ vs $134.7{\pm}54.8$). There was no difference in the mean proportion of apoptotic cells in blastocysts between the sources of recipient oocytes. In the experiment with the embryo culture system, the blastocyst rate was somewhat higher in sequential system than in conventional system (50.0% vs 43.5%), though there was no significant difference. The numbers of total ($160.0{\pm}69.0$ vs $156.7{\pm}68.4$) and apoptotic cells ($14.0{\pm}10.4$ vs $11.8{\pm}6.4$) were not different between the culture systems. In conclusion, the present study demonstrated that Hanwoo recipient oocytes and the sequential culture system were more effective in supporting the production of Hanwoo SCNT embryos.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.235-240
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• 2014

Ethylene glycol (EG) has been successfully used as a cryoprotectant for vitrification of mammalian embryos (including human embryos) due to its low formula weight and high permeation into cells compared with other cryoprotectants, including propylene glycol (PROH). Cryopreservation is able to store the surplus pre-embryos for freezing and furthermore thawing and transfer in a subsequent cycle. This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Female ICR mice (6~8 weeks old) were induced to superovulate by sequential intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48 h apart. Mouse embryos were collected according to its developmental stage after the injection of hCG. Embryos were cryopreserved not only during cryoprotectant step (1~4 step) but also in a variety of media (HTF, IVF medium, D-PBS) and cell stage. The results were as follows : There is no clear advantage in these freezing media of rapid method, but 4 cell and 8 cell of slow method (2, 3 and 4 step) have advantage in D-PBS. The development of embryos according to cell stage become greater in 8 cell stage. In the treatment steps of cryopreservation, the development of embryo to blastocyst was similar among rapid method, but the development of 4 cell and 8 cell embryos to blastocyst according to slow method was better than rapid method.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.130-138
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• 2019

Although somatic cell nuclear transfer (SCNT)-derived embryonic stem cells (ESCs) in pigs have great potential, their use is limited because the establishment efficiency of ESCs is extremely low. Accordingly, we tried to develop in-vitro culture system stimulating production of SCNT blastocysts with high performance in the colony formation and formation of colonies derived from SCNT blastocysts for enhancing production efficiency of porcine ESCs. For these, SCNT blastocysts produced in various types of embryo culture medium were cultured in different ESC culture medium and optimal culture medium was determined by comparing colony formation efficiency. As the results, ICM of porcine SCNT blastocysts produced through sequential culture of porcine SCNT embryos in the modified porcine zygote medium (PZM)-5 and the PZM-5F showed the best formation efficiency of colonies in α-MEM-based medium. In conclusion, appropriate combination of the embryo culture medium and ESC culture medium will greatly contribute to successful establishment of ESCs derived from SCNT embryos.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.297-304
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• 2018

The purpose of the present study was to determine the elaborate characteristics of ovarian changes including follicles and corpus luteum, and hormonal patterns of gonadotropin surge mode secretions during the normal consecutive estrous cycle in three dairy cows. Non-lactating and multiparous Holstein cows (n=3) used as experimental animals. The cows were assigned to examine the relationship among ovarian changes (follicle, corpus luteum), ovarian steroids (estradiol, progesterone) and gonadotropin (LH, FSH) surge mode secretion during the successive estrous cycles by rectal palpation, ultrasonography and hormonal assay. The mean length of the estrous cycle for the three cows was $23.1{\pm}1.44days$ (${\pm}SEM$), with a range of 20-28 days. In six estrous cycles, the number of two follicular waves, three follicular waves and four follicular waves was 2, 3 and 1, respectively. The sequential ultrasonographic monitoring showed that the corpus luteum with ${\geq}10mm$ in diameter detected from Day 2 (Day 0 is ovulation) in six estrous cycles of all cows. Preovulatory increases in estradiol concentration reached $10.36{\pm}1.10pg/ml$ on the 2 days before ovulation of the last dominant follicle. All cows exhibited a preovulatory rise in estradiol concentration followed by a typical preovulatory LH and FSH surge. The mean interval from the peak of LH/FSH surge to ovulation of the last dominant follicle was $31.3{\pm}1.76h$ (${\pm}SEM$). In these results, each dairy cow showed that ovarian morphological changes and gonadotropin surge mode secretion will be regulated by various environmental factors including age, breeds, nutrition, breeding conditions, etc.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.129-137
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• 2018

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig ($GalT^{-MCP/-MCP}$). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain $GalT^{-MCP/-MCP}/CD39/CD73$ pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from $GalT^{-MCP/-MCP}$ pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate $GalT^{-MCP/-MCP}/CD39/CD73$ pig expressing CD39 and CD73 at endothelial cells.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.127-135
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• 1999

Cryopreservation is able to store the surplus pre-embryos for freezing and furthermore thawing and transfer in a subsequent cycle. Cryopreserving cells which are maintaining their viability are the very complex process. This study has been carried out in order to find the effects of cryopreservation steps, freezing media and embryonic stages on the rates of viability and development of cryopreserved mouse embryos. Female ICR mice ($6{\sim}8$ weeks old) were induced to superovulate by sequential intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48h apart. Mouse embryos were collected according to its developmental stage after the injection of hCG. Embryos were cryopreserved not only by cryoprotectant step (1 step${\sim}$4 step) but also in a variety of media (HTF, IVF medium, D-PBS) and cell stage. The results were as follows: There is no clear advantage in these freezing media of rapid method, but 4 cell and 8 cell of slow method (2, 3, 4 step) have advantage in D-PBS. The development of embryos according to cell stage become greater in 8 cell stage. In the treatment steps of cryopreservation, the development of embryo to blastocyst was similar among rapid method, but the development of 4 cell and 8 cell embryos to blastocyst according to slow method was better than rapid method.