• Journal of Embryo Transfer
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• v.19 no.2
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• pp.133-145
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• 2004

Serum-contain is commoly used for the production of in vitro-derived bovine embryos. However, were biological activity of serum varies from lot to lot, time consuming to choose better serum with good quality and risks of virus, bacteria and mycoplasma infection. This study established serum-free culture systems of in vitro embryo development to efficiently obtain a large number of blastocysts from ovaries of Hanwoo and oocytes maturation, cell number, tlerance of cryopreservation. Secondly, serum-contain medium is suspected of contributing to the large calf size, dystocia, cersarean sections, calf mortality and confirmed these blastocysts are high quality in terms of cyotolerance, high rates of pregancy and normal birth. For these reasons, Culture media (IVMD101 and IVD101) designed specifically for the preimplantation bovine embryo are rather simplistic, being based on salt solutions with additional energy substrates and growth factors. An improved serum-free medium (IVMD101) was developed for bovine oocytes maturation in vitro. Proportions of embryos developing to the blastocyst stage cultured in both IVD101(32.4%) and IVD101(34.5%) serum-free media were higher than in TCM199+10% FBS(12.4%) serumcontaining medium. Futhermore, the cell numbers per blastosyst obtained in the serum-free media were superior to those of blastocysts developed in serum-supplemented medium. Also, cell numbers of blastocysts obtained in the serum-free media were similar with blastocysts derived in vivo. Survival rate blastocysts after 24 hr incubation after thawing, the blastocysts cultured in both IVD101(94.5%) and IVD101(95.8%) serum-free media were higher than in TCM199+10% FBS (52.5%) serum-containing medium. After 72 hr incubation after thawing, hatching rates of blastocysts developed in IVD101(78.4%) and IVMD101(83.7%) were sighnificantly higher than that developed in the serum-supplemented medium(32.0%). The pregnancy rates almost not different between fresh blastocysts(38.2%) and frozen blastocysts(34.9%). The results suggested that the improved serum-free media(IVMD101 and IVD101) offer several advantages over culture in serum-cotaining medium, including increased rates of blastocyst formation and high cel numbers. Additionally, the survival and hatching rates of embryos product in serum-free media after post-thaw culture were superior to those of embryos produced in the serum-containg medium and useful for the production of high quality bovine embryos for cryo-preservation. These improved serum-free media are beneficial not only for the study of the mechanisms of early embryogenesis but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis.

• Development and Reproduction
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• v.9 no.1
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• pp.49-52
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• 2005

The effects of cytochalasin B was studied for the cleavage and development of in vitro matured porcine follicular oocytes. The follicular oocytes were collected from slaughtered pig ovaries and matured for 65 hours. The matured oocytes were activated by 7% ethanol(v/v) in DPBS and the activated oocytes were subjected to cytochalasin B concentrations of 2.5, 5.0 and $7.5\;{\mu}g/mL$ for 3, 5 and 7 hours, and then the treated oocytes were cultured in NCSU23 with 0.4% BSA for 7 days. The cleavage rates were not different significantly in each treatment. However, the oocytes treated with $5.0\;{\mu}g/mL$ for 5 hours yielded a significantly higher morula rate(19.7%) than oocytes treated with $2.5\;{\mu}g/mL$ for 3 and 5 hours(9.4%). The sum rate of $2.5\;{\mu}g/mL$ concentration(10.5%) by hour was also significantly lower than those of 5.0(18.0%) and $7.5\;{\mu}g/mL$ concentration(14.6%). The blastocyst rate in oocytes treated with $5.0\;{\mu}g/mL$ for 3(9.4%) and 5 hours(9.0%) was significantly higher than the rate in oocytes treated with $2.5\;{\mu}g/mL$ for 3 hours(0%). The sum rate of $5.0\;{\mu}g/mL$ concentration also significantly higher than those of 2.5 and $7.5\;{\mu}g/mL$ concentration. The results demonstrated that the treatment of oocytes with cytochalasin B of $5.0\;{\mu}g/mL$ for $3{\sim}5$ hours was the optimal concentration and duration for parthenogenetic activation and blastocyst formation of in vitro matured porcine oocytes.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.245-255
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• 2004

In recent years, an increasing number of studies on pig in vitro maturation(IVM) and in vitro fertilization(IVF) have been separated. the wide range of new technologies, including that in applied molecular genetics, has increased this interest. the production of viable porcine embryos in vitro is a prerequisites for the successful production of transgenic pigs to date. The efficiency of IVM/IVF techniques in the porcine is lower than that obtained in other species such as cattle and mouse. The several problems are generally thought to be the cause of poor results: the low rate of MPN formation derived from inadequate IVM of oocytes, the high incidence of polyspermy after IVF and cell blocking at 4 cell during embryos culture. For there reasons overcoming, many studies have been conducted to improve in vitro embryo-genic competence of oocytes. In the last several years, many maturation culture media have been evaluated and various exogenous factors such as hormones and grows factors have been tested to improve the efficiency of porcine in vitro system. In the study several antioxidants have been examined to improve in vitro fertilization and development of porcine oocytes. In this study, several antioxidants were examined to determine the effects on the development of oocytes to the cleavage, morula and blastocyst stage when added at the maturation(IVM) or in vitro fertilization(IVF) or in vitro culture(IVC) of porcine embryos. Porcine oocytes were matured, fertilized and embryos were cultured in defind conditioned medium in vitro with or without supplementation with the antioxidents of cysteine, catalase and glutathione. 1. Significant improvement of blastocyst rate (27.2％ versus 15.4％, p<0.05) were achieved when catalase(500U/$m\ell$) were added to TCM-199 medium and morula rate(72.0％ versus 53.9％, p<0.05) were significantly higher when glutathione(1.0mM/$m\ell$) were added to TCM-199 medium than those of control. 2. In mTBM medium for oocytes fertilization, the addition of cysteine, catalase and glutathione had no positive effect on embryonic development. glutathione had no positive effect on embryonic development. In conclusion, this study shows that addition of catalase, gluththione during IVM improved the rate of porcine embryo development.

• Journal of Embryo Transfer
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• v.30 no.1
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• pp.7-15
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• 2015

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM ($61.1{\pm}1.5%$,$64.7{\pm}2.0%$) of nicotinic acid than other groups (0 mM, $52.1{\pm}2.3%$; 20 mM, $47.8{\pm}5.1%$, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid ($26.1{\pm}1.8%$, $24.9{\pm}1.5%$) than other groups (0 mM, $35.3{\pm}0.8%$; 20 mM, $36.5{\pm}1.9%$, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM ($84.2{\pm}3.6%$, $88.4{\pm}2.3%$) of nicotinic acid than other groups (0 mM, $77.3{\pm}4.4%$; 20 mM, $73.3{\pm}3.6%$, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM ($17.0{\pm}1.3%$) of nicotinic acid than other groups (0 mM, $9.4{\pm}0.5%$; 5mM, $12.6{\pm}0.8%$; 20 mM, $5.0{\pm}1.0%$, P<0.05). Moreover, total cell number was higher in 5 and 10 mM ($53.6{\pm}2.9%$, $57.9{\pm}2.8%$) of nicotinic acid than other groups (0 mM, $41.0{\pm}1.4%$; 20 mM, $23.2{\pm}2.8%$, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid ($0.7{\pm}0.1%$) than other groups (0 mM, $1.0{\pm}0.1%$; 10mM, $0.9{\pm}0.0%$; 20 mM, $1.4{\pm}1.0%$, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.131-138
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• 2017

In most mammals, metaphase II (MII) oocytes having high maturation promoting factor (MPF) activity have been considered as good oocytes and then used for assisted reproductive technologies including somatic cell nuclear transfer (SCNT). Caffeine increases MPF activity in mammalian oocytes by inhibiting p34cdc2 phosphorylation. The objective of this study was to investigate the effects of caffeine treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after SCNT in pigs. To this end, morphologically good (MGCOCs) and poor oocytes (MPCOCs) based on the thickness of cumulus cell layer were untreated or treated with 2.5 mM caffeine during 22-42, 34-42, or 38-42 h of IVM according to the experimental design. Caffeine treatment for 20 h during 22-42 h of IVM significantly inhibited nuclear maturation compared to no treatment. Blastocyst formation of SCNT embryos was not influenced by the caffeine treatment during 38-42 h of IVM in MGCOCs (41.1-42.1%) but was significantly improved in MPCOCs compared to no treatment (43.4 vs. 30.1%, P<0.05). No significant effects of caffeine treatment was observed in embryo cleavage (78.7-88.0%) and mean cell number in blastocyst (38.7-43.5 cells). The MPF activity of MII oocytes in terms of p34cdc2 kinase activity was not influenced by the caffeine treatment in MGCOCs (160.4 vs. 194.3 pg/ml) but significantly increased in MPCOCs (133.9 vs. 204.8 pg/ml). Our results demonstrate that caffeine treatment during 38-42 h of IVM improves developmental competence of SCNT embryos derived from MPCOCs by influencing cytoplasmic maturation including increased MPF activity in IVM oocytes in pigs.

• Development and Reproduction
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• v.8 no.2
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• pp.119-122
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• 2004

It is well known that unidentified factors in sera, hormones and growth factors promote the proliferation of granulosa cells and nuclear maturation of bovine COCs in vitro. Attemps had been developed the simple composition of culture media and similar system to in vivo conditions has been applied. In the present study, we investigated the effect of TGF-${\beta}$ on in vitro maturation and in vitro development of Hanwoo COCs. When the COCs were matured in TCM 199 containing 0.1, 1 or 10 ng/ml TGF-${\beta}$ for 24 hrs, metaphaseⅡ of COCs were obtained 95.8%, 100% of matured COCs, respectively and there were no differences among the concentrations of TGF-${\beta}$. Matured COCs with TGF-${\beta}$ cultured in maturation medium after in vitro fertilization, developmental rate to blastocyst were 0~0.8%. Matured COCs with TGF-${\beta}$ were cultured in TCM 199+10% FBS, 0.8% BSA, 0.1% PVA, blastocyst formation were showed in 12.4%, 12.8%, 8.5% of those and cultured in IVMD or IVD without serum were 38.4%, 34.8%, respectively. There were significant differences among the media (P<0.05). TGF-${\beta}$ is available for i vitro maturation of bovine COCs, but further investigation would be need for finding the synergistic autocrine/paracrine fashion of other growth factors in early bovine development.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.205-214
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• 2002

There are many methods to introduce exogenous DNA into embryo to produce transgenic animals. Exogenous gene can be integrated into oocyte by sperm vector. In this study, sperm was used as a vector for a transgene, which is encoding enhanced green fluorescent protein (EGFP). The objective of this study was to investigate the expression of exogenous gene in bovine embryos after injection of spermatozoa cocultured with EGFP DNA fragment. Spermatozoa were plunged into liquid nitrogen and thawed several times or shook in 0.2% Triton X-100 to remove sperm membrane followed by DTT treatment. The injected oocytes were co-cultured with vero cells in CR1aa, and expression of EGFP gene was observed under fluorescent microscope. Blastocyst formation rates of oocytes injected with sperm treated with DTT, DTT-freezing or DTT-Triton X-100 were 34.7, 39.4 and 31.9%, respectively. The rates of EGFP expression in oocytes injected with 54 ng DNA after DTT-treated, DTT-freezing and DTT-Triton X-100-treated sperm were 0, 19.1 and 13.9%. On the other hands, expression rate of oocytes injected with sperm cocultured with 13.5, 27 and 63.5 ng of EFGP DNA were 6.7, 9.0 and 5.1%, respectively. When intact sperm was mixed with 63.5 ng/${mu}ell$ EGFP DNA fragment, and then electroporated before injection, the expression rate of injected oocyte was 2%. Unexpectedly, electro-poration could not increase the expression rate. These results suggest that sperm can be used as a transgene vector, even if the efficiency was low (19.1%).

• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.233-240
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• 2003

This study was conducted to examine the effects of oocyte maturation period, phytohemagglutinin-P (PHA-P) treatment and activation agent on the enucleation, fusion, activation or in vitro development of bovine nuclear transfer embryos. Bovine oocytes were enucleated at 16∼24 h of in vitro maturation (IVM). Adult ear skin cells treated or non-treated with PHA-P were transferred into enucleated oocytes. Reconstituted oocytes treated or non-treated with PHA-P were fused by a pulse of 1.5 kV/cm for 30 $\mu$sec. Fused oocytes were activated with a combination of calcium ionophore (A23187) and cycloheximide (CHXM) or dimethylaminopurine (DMAP), and cultured in vitro for 7∼9 days. Enucleation rate was significantly increased when oocytes were matured for 16∼18 h (70.2∼92.3%, P<0.05) compared to that of oocytes were matured for 20∼24 h (44.3∼53.4%). The location of metaphase-II plate was far off from the 1st polar body as maturation time was increased. PHA-P treatment of donor cells or reconstituted oocytes significantly improved fusion rate (P<0.05). Cleavage and blastocyst formation rates were significantly increased after activation with a combination of A23187 and DMAP (78.6% and 32.9%, respectively) compared to those of embryos activated with a combination of A23l87 and CHXM (48.5 and 15.2%, respectively). From the present result, it is suggested that high enucleation efficiency can obtained by using oocytes matured for 18 h. It also shows that PHA-P treatment can improve the fusion rate, and activation with a combination of A23187 and DMAP can enhance the embryo development.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.89-96
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• 1998

Anti-phospholipid antihodies (aPL) have important roles in various pregnancy complications such as recurrent miscarrige, growth retardation, placental abruption and stillbirth. However, their biological actions on preimplantation development of oocytes are still unclear. In this study, we investigated whether either aPL containing sera or phospholipids could affect in vitro fertilization and development of mouse oocytes. Sera used in this study were collected from three patients and the presence of aPL in the sera was confirmed by enzymatic-linked immunosorbent assay. When mouse oocytes were cultured in a serum-free, Chatot, Ziomek and Bavister (CZB) medium (Experiment 1), addition of aPL-containing sera (10%) to CZB medium did not. significantly (P>0.05) influence sperm penetration of oocytes. However, development to the blastocyst stage was significantly (P<0.05) inhibited by serum addition, and formation of morulae (16-23% vs. 58%) and blastocysts (0-4% vs. 21%) was markedly reduced compared with no addition (Experiment 2). In Experiment 3, pronuclear stage embryos were cultured for 96 h in GZB medium supplemented with 1 $\mu$g /ml phosphatidyl ethanolamine, 1 $\mu$g/ml phosphatidyl inositol or 1 $\mu$g /ml phosphatidyl choline. No increase in embryo development was found after addition of the phospholipids to CZB medium. These results suggest that 1) aPL have an inhibitory role in preimplantation development of mouse embryos, and that 2) the action of aPL may be related to a specific phospholid (s) rather than the tested phospholipids in the present study.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.173-179
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• 2004

This study was conducted to investigate the toxi-cological effects of different vitrification solution on development of immature porcine oocytes in vitro. Oocytes were exposed to EFS solution [40% ethylene glycol (EG) + 18% Ficoll + 0.3M sucrose], ES solution (5.5M EG + 1.0M sucrose) or GE solution [10% glycol (G) + 20% EG], and these oocytes were transferred to sucrose solution directly. Maturation rates were significantly (P<0.05) higher in the ES solution (44.5%) and control (57.6%) than in the EFS solution (38.8%) and GE solution (22.4%). No differences among three solution were found in fertilization rates. Cleavage rates was significantly (P<0.05) higher in the ES solution (47.1%) and control (65.9%) than in the EFS solution (21.9%) and GE solution (19.0%), but no difference among three solutions was found in the blastocyst formation rates. These results indicate that combination of EG and sucrose solutions had effects on development of immature porcine oocytes.