• Journal of Embryo Transfer
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• v.9 no.3
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• pp.235-241
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• 1994

The study was conducted to determine the optimal hormone and glucose levels during the in vitro culture of bovine oocytes matured and fertilized in vitro for blastocyst development. Oocytes matured in TCM 199 + 10% FCS + hormones and glucose were fertilized in vitro in a TALP medium with swim up separated and heparin-treated epididymal cauda spermatozoa. Oocytes were cultured for 2~5 days in synthetic oviduct fluid medium (SOFM) supplemented with 10% FGS and with different hormone and glucose levels, and further cultured 5 days same medium in SOFM. The results are summarized as follows : The in vitro maturation and penetration rates of porcine oocytes cultured in TCM 199 media containing PMSG, hCG, PMSG + hCG, hCG + $\beta$ estradiol, PMSG + $\beta$ estradiol 0 to20 hours after insemination were 88.0% and 81.8%, 82.6% and 68.4%, 80.0% and 75.0%, 80.0% and 65.0%, 77.3% and 64.7%, respectively. The in vitro maturation and penetration rates of porcine oocytes cultured in TCM 199 media containing PMSG, hCG, PMSG + hCG, hCG + $\beta$ estradiol, PMSG + $\beta$ estradiol 20 to 40 hours after insemination were 92.0% and 87.0%, 92.0% and 82.6%, 91.3% and 81.0%, 85.2% and 73.9%, 87.5% and 81.0%, respectively. The cleavage and in vitro developmental rates to blastocyst of porcine oocytes cultured in TCM 199 media containing 0.05 mM, 0.10 mM, 0.30 mM, 0.50 mM, 1.00 mM, and 3.00 mM glucose lelvels 0~3 days after insemination were 31.5~48.1% and 10.0~16.7%, respectively. The cleavage and in vitro developmental rates to blastocyst of porcine oocytes cultured in TCM 199 media containing 0.05 mM, 0.10 mM, 0.30 mM, 0.50 mM, 1.00 mM, and 3.00 mM glucose levels 4~8 days after insemination were 30.0~53.8% and 8.7~19.2%, respectively. The cleavage and in vitro developmental rates to blastocyst were higher in TCM 199 media containing various glucose levels 0~3 days after insemination than 4~8 days.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.251-256
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• 2013

The present study was performed to investigate the effect of Hh-Ag1.5, a small-molecule chemical agonist of SMOothened receptor, on the in vitro maturation and development of in vitro fertilized (IVF) embryos in pigs. Oocytes or fertilized embryos were cultured in a maturation or embryo culture medium supplemented with 0 (control), 25, 50 or 100 nM of Hh-Ag1.5, respectively. Although the maturation rate were not different among treatment groups, the blastocyst formation rate in the group treated with 25 nM Hh-Ag1.5 was significantly increased compared to other groups (P<0.05). While the highest dose of Hh-Ag1.5 (100 nM) did negatively affect to the embryo development and cell number in blastocysts compared to other groups (P<0.05), the apoptotic cell index in blastocysts was significantly lower in 25 and 50 nM groups than in control and 100 nM groups (P<0.05). The mRNA expression of the proapoptotic gene Bax and the ratio of Bax/Bcl-XL decreased in among treatment groups compared to control (P<0.05). The embryo quality related genes, Tert and Zfp42, were significantly decreased in 50 and 100 nM groups compared with control and 25 nM groups (P<0.05). In conclusion, the addition of 25 nM Hh-Ag1.5 to in vitro maturation and culture medium can enhance the developmental potential as well as quality of IVF embryos in pig.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.2
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• pp.119-141
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• 2020

The Dromedary camel (Camelus dromedaries) is an important species because of its ability to produce good quality meat, milk, and fibers under harsh environmental conditions. Camels are also crucial for transportation, racing, and as draft animals in agriculture. Therefore, dromedary camels play a critical role in the economy for millions of people living in the arid part of the world. The inherent capability of camels to produce meat and milk is highly correlated with their reproductive performance. Compared with other domestic species, the reproductive efficiency in camelids is low. Although recent reproductive technologies such as in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) have been successfully applied to camelids and the birth of live offspring following these technologies has been reported; in vitro embryo production (IVP) has lagged in this species. The development of the IVP system for dromedary camels may be a useful tool for the genetic improvement of this species. IVP in farm animals includes three main steps; in vitro maturation (IVM) of an oocyte, IVF of a matured oocyte, and in vitro culture (IVC) of fertilized oocyte up to the blastocyst stage. This review aims to summarize various factors that influence oocyte quality, IVM, and in vitro embryo development in dromedary camel.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.9
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• pp.1152-1158
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• 2010

In differentiating human embryonic stem (d-hES) cells there are a number of types of cells which may secrete various nutrients and helpful materials for pre-implantation embryonic development. This study examined whether the d-hES could function as a feeder cell in vitro to support mouse embryonic development. By RT-PCR analysis, the d-hES cells revealed high expression of three germ-layered differentiation markers while having markedly reduced expression of stem cell markers. Also, in d-hES cells, LIF expression in embryo implantation-related material was confirmed at a similar level to undifferentiated ES cells. When mouse 2PN embryos were cultured in control M16 medium, co-culture control CR1aa medium or co-cultured with d-hES cells, their blastocyst development rate at embryonic day 4 (83.9%) were significantly better in the d-hES cell group than in the CR1aa group (66.0%), while not better than in the M16 group (90.7%)(p<0.05). However, at embryonic days 5 and 6, embryo hatching and hatched-out rates of the dhES cell group (53.6 and 48.2%, respectively) were superior to those of the M16 group (40.7 and 40.7%, respectively). At embryonic day 4, blastocysts of the d-hES cell group were transferred into pseudo-pregnant recipients, and pregnancy rate (75.0%) was very high compared to the other groups (M16, 57.1%; CR1aa, 37.5%). In addition, embryo implantation (55.9%) and live fetus rate (38.2%) of the d-hES cell group were also better than those of the other groups (M16, 36.7 and 18.3%, respectively; CR1aa, 23.2 and 8.7%, respectively). These results demonstrated that d-hES cells can be used as a feeder cell for enhancing in vitro and in vivo developmental potential of mouse pre-implantation embryos.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.147-151
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• 2017

Commercial applications of OPU/IVP were to produce embryos and calves from high genetic cows. The aim of this present study was to compare the number of recovered oocytes and cultured In vitro produced embryos from Ovum Pick-up (OPU). OPU derived embryo production was carried out of oocytes by ultrasonographic guided follicular aspiration and then produced in vitro produced blastocysts by IVP culture system. In result, the rate of recovered oocytes was obtained 612 (57.2%) and 451(73.7) G1+G2 grade oocytes. No difference of recovered rate (51.1~62.1%) was seen in six donor. The rate of cleavage and blastocyst development were obtained 320 (70.9%) and 78 (24.4%) that was $3.3{\pm}0.4$ cleaved embryo and $0.9{\pm}0.2$ blastocysts per session. Cleavage rate of OPU oocytes in No. 6 donor was 90.6%, significantly (P<0.05) higher than that in the other donors, However, blastocysts was similar (25.8~30.0%). In conclusion, limited numbers of OPU oocytes had competent development when cultured in SOF culture medium.

• Journal of Embryo Transfer
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• v.8 no.1
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• pp.31-36
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• 1993

The post-thaw survival of mouse embryos of the various developmental stages was determined after cryopreservation by vitrification in a solution containing ethylene glycol, Ficoll and sucrose (EFS). All the embryos were equilibrated for 2 minutes just prior to freezing. The number of blastomeres during in vitro development was counted by nuclei higher rates of post-thaw survival were obtained from the embryos of 2-cell(92.2%), 8-cell(77.2%) or morula stage(90.0%) than those of blastocyst stage(62.7%). The number of blastomeres per embryo following in vitro culture for 24 hours was significantly(P<0.05) smaller as 66.0f22.3 in vitrified and thawed morulae than fresh morulae(91.7$\pm$12.2).

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.217-222
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• 2008

In previous studies, we reported that sow which was transferred OPS-freezing embryos not able to deliver a piglet (Kim et al, 2004). This study was conducted to investigate a possibility of gilt as recipients which produce piglets after transfer of OPS-freezing embryos. All transferred embryos were prepared by in vitro production (IVP) system. In vitro culture (IVC) medium used glucose-free NCSU23 supplemented with 5mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at $39^{\circ}C$. From day 3 of IVC, 10% fetal bovine serum albumin was added to the culture medium. In preparing of freezing embryos, embryos were treated with 7.5 $\mu g/ml$ cytochalasin-B for 30 min and centrifuged at $13,000{\times}g$ for 13 min. And then, embryos were exposed sequentially to an ethylene glycol (EG) solution, aspirated into open pulled straw (OPS), and plunged or thawed into the liquid nitrogen. In embryo transfer (ET), we used two kinds of type (surgical method vs. non-surgical method). In surgical method of embryo transfer, $55\sim65$ embryo were transferred in both uterine horn of two recipient gilts by plastic straw. Non-surgical method which is like artificial insemination was performed on three gilts. Each 140 frozen embryos were transferred to two gilts and 40 fresh embryos to one gilt. Pregnancy establishment was shown one recipient at 45 days after ET. However, the one recipient was also aborted at 58 days after ET. These results suggest that gilts can be considered as a candidate of recipients for OPS-freezing embryo transfer.

• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.239-244
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• 2015

Calanthe striata f. sieboldii Decne. ex Regel is a terrestrial orchid with beautiful flowers arranged in racemose inflorescences. This species is threatened due to over-collection and loss of suitable habitats. Asymbiotic germination is useful in the conservation efforts to re-establish plants in the wild, and for commercial propagation. In this study, we investigate the effects of sodium hypochlorite (NaOCl), three types of culture media (Phytomax Orchid Maintenance - POM, Seed Germination Maintenance - SGM, and Murashige and Skoog 1962 - MS), and plant growth regulators on embryo swelling, protocorm formation, and embryo diameter of C. striata f. sieboldii. Treatment with 1% NaOCl for 30 min greatly enhanced embryo swelling (28.3%), embryo diameter ($205.8{\mu}m$), and embryo protocorm formation (54.8%) compared to seeds without NaOCl treatment (embryo swelling 8.5%, embryo diameter $14.6{\mu}m$, and protocorm formation 13.4%) on POM medium. Protocorm formation on POM medium supplemented with 1.0 mg/L N6-benzyladenine (BA) (95.6%) was better than the control (54.5%). Additionally, the effects of activated charcoal (AC) and sucrose on seedling growth in in vitro culture were examined. The protocorm converted into healthy plants with well-developed shoot primordia on the POM medium with AC and sucrose. The most suitable conditions for seedling growth after 10 weeks of culture were the POM medium with AC or sucrose. These results show effective asymbiotic seed germination and in vitro seedling growth of C. striata f. sieboldii.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.241-245
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• 2005

Porcine oviduct epithelial cells (POEC) are widely used in co-culture experiments to improve early embryonic development, in vitro fertilization in embryo transfer programs for domestic animals and in vitro maturation of immature germ cells. POEC were mechanically isolated and cultured in tissue culture medium 199. Cells grew continuously, and confluent monolayers were formed after 7 days. After forming confluent monolayer of epithelial cells, supernatant was collected as the condition medium for maturing round spermatids in vitro. Round spermatids were also separated mechanically and cultured in the POEC condition medium. In this study we observed that $20\%$ of round spermatid cultured were matured into elongating spermatid after 24 h, and about $10\%$ of round spermatid cultured showed complete elongation (elongated spermatid) within $24\~48$ h of in vitro culture. No further development was observed within $50\~72$ h and transformed cells lost their viability after 72 h. These preliminary findings suggest that the condition medium from POEC may be possible to overcome the round spermatid block by improving the milieu of culture system.