• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.1-6
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• 2004

This study was conducted to investigate the effects of donor cell type, individual, passage number and trypsinization time on the in vitro development of bovine somatic cell nuclear transfer embryos. Three cell types (skin, muscle and cumulus cells) and cells from 3 individuals were used for nuclear transfer. Cell were passaged by 5, 15 or 30 times, and cell were trypsinized for 1 or 3 min before injection. Nuclear transfer were performed by conventional fusion method. Development rates to the blastocyst stage were not significantly different among three cell types (16.5∼23.9%) and individuals (16.4∼19.5%). Blastocyst formation rate of cloned embryos reconstituted with cells at passage 30 (5.8%) was significantly lower than those of embryos reconstituted with 5- and 15-passaged cells (25.3 and 23.5%, respectively, P＜0.05). The rate of embryos developed to the blastocyst stage was higher in embryos reconstituted with cells trypsinized for 1 min (30.7%) compared to embryos reconstituted with cells trypsinized for 3 min (P＜0.05). The result of the present study indicates that different donor cell types and individuals used in this study did not affect the development of cloned bovine embryos. However, passage number and trypsinization time of donor cells affect the in vitro development of cloned bovine embryos.

• Journal of Embryo Transfer
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• v.25 no.2
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• pp.97-101
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• 2010

Intracytoplasmic sperm injection (ICSI) is one of the artificial fertilization methods when only a few sperm are available for insemination, and an important tool for the preservation of genetic materials of endangered animal species, especially the male is infertile. Different from other species such as mice and pigs, the conventional ICSI method which uses spiked pipette for injection (Spike-ICSI) is exhibited low success rates in cattle because the bovinesperm head membrane is hard to break during injection procedure. We chose piezo-assisted ICSI (Piezo-ICSI) for the improvement of the injection procedure including sperm head membrane rupture and efficient puncture of the plasma membrane of the oocytes. In this experiment, we compared the efficacy of the bovine ICSI embryo production between the Piezo-ICSI and Spike-ICSI. The second polar body extrusion, pronuclear formation, cleavage and blastocyst formation were evaluated after implementation of two different ICSI techniques. The Piezo-ICSI tended to show comparably higher rates of the second polar body extrusion (41.7%), the pronuclei formation (42.9%) and the two-cell cleavage (41.4%) than Spike-ICSI does (33.3%, 28.6% and 23.5%, respectively) although there is no statistic significance between two groups. In addition, the blastocysts were only obtained from the Piezo-ICSI group (10.3%). Our finding shows that the Piezo-ICSI may be used as an artificial fertilization method in cattle when in vitro fertilization is not applicable.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.9
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• pp.1260-1266
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• 2001

The effect of protein supplementation, $O_2$ concentration and co-culture on the development of embryos produced by nuclear transfer using cultured cumulus cell was investigated. Recipient oocytes and cumulus cells were obtained from the ovaries of the slaughtered Hanwoo cows. Donor cumulus cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum at 5% $CO_2$ in air at $38.5^{\circ}C$. The 1 to 6 passages of cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One $15{\mu}s$ pulse of 180 volts was applied to induce the fusion between karyoplast and cytoplast. The fused embryos were activated with $10{\mu}M$ calcium ionophore for 5 min and 2 mM 6-dimethylaminopurine for 3 h. To examine the effect of protein supplementation, nuclear transfer (NT) embryos were cultured in one of the following 4 treatments : 1) CR1aa + 3 mg/ml BSA for 7 days ; 2) CR1aa + 10% FBS for 7 days ; 3) CR1aa + 1.5 mg/ml BSA + 5% FBS for 7 days ; and 4) CR1aa + 3 mg/ml BSA for first 3 days and then CR1aa + 1.5 mg/ml BSA + 5% FBS for 4 days. Culture took place at 5% $CO_2$, 5% $O_2$ and 90% $N_2$ at $38.5^{\circ}C$. Although there were no significant differences in cleavage rate among different protein supplements, the rates of blastocyst formation were significantly different. When NT embryos were cultured in the medium supplemented with only BSA, they could develop to only morula not to blastocyst. However, when FBS was supplemented, NT embryos developed to blastocyst stage. In order to investigate the effect of $O_2$ concentration and co-culture, NT embryos were cultured in CR1aa + 1.5 mg/ml BSA + 5% FBS with or without cumulus cell co-culture at an atmosphere of 5% $CO_2$ in air (20% $O_2$) or 5% $CO_2$, 5% $O_2$, 90% $N_2$ (5% $O_2$) at $38.5^{\circ}C$ for 7 days. The percentage of blastocyst development was significantly higher when the NT embryos were cultured at an atmosphere of 5% $O_2$ than that of 20% $O_2$ (p<0.05). However, there was no significant difference between with and without cumulus cell co-culture at an atmosphere of 5% $O_2$ or 20% $O_2$. Fifty embryos were transferred to 25 recipients and 5 recipients were pregnant at 100 days. From 5 pregnant cows, only one cow was delivered of female twin. In conclusion, the embryos reconstructed by enucleation of metaphase II oocytes and introduction of the cycling and quiescent cumulus donor cells in Hanwoo had developmental potential to term after embryo transfer to recipient cows.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.163-172
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• 2001

The present study was carried out to examine the effects of $O_2$concentrations and culture media on in vitro maturation and embryo development of porcine follicular oocytes. The results were summarized as fellows : 1. The rates of GVBD and nuclear maturation in NCSU-23 and TCM-199 media with 10% PFF under the conditions of 5% and/or 20% $O_2$concentrations were not different among the each treatment groups(P>0.05). 2. The rates of polyspermy and mean numbers of penetrated sperm were significantly lower in NCSU-23 medium than in TCM-199 medium (P>0.05). However, the rates of polyspermy and mean numbers of penetrated sperm were not different between 5% and 20% $O_2$concentrations. 3. The rates of blastocyst formation at day 7 after in vitro fertilization were significantly higher in NCSU-23 medium under the condition of 20% $O_2$concentration than in TCM-l99 medium under the condition of 5% or 20% $O_2$concentrations. However, the rates of blastocyst formation and total cell numbers in blastocysts were not different between 5% and 20% $O_2$concentrations. In conclusion, the use of NCSU-23 medium under the condition of 20% $O_2$concentration was beneficial for porcine oocyte maturation and in vitro development.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.16 no.1
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• pp.365-370
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• 2015

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important hematopoietic growth factor and immune modulator. The aim of this study was to evaluate the effects of GM-CSF on the development and cell number of porcine parthenotes, as well as on their expression of implantation-related genes. In the present study, porcine parthenogenatic activated embryos were cultured in a protein-free culture medium in the absence or presence of 5, 10 and 20 ng/ml GM-CSF for 7 days. The percentage of blastocyst formation, total cell number and gene expressions were evaluated. The results showed that the addition of 20 ng/ml GM-CSF to protein-free culture medium significantly increased the blastocoel formation ($26.14{\pm}2.03%$ vs. $3.55{\pm}0.51%$, p < 0.05). In addition, the cell number also increased when they were cultured in the presence of 20 ng/ml GM-CSF ($43.51{\pm}3.6%$ vs. $30.68{\pm}5.51%$, p < 0.05). A real time reverse transcripts polymerase chain reaction (RT-PCR) showed that GM-CSF enhances mRNA expression of the interleukin-6, but does not influence the leukemia inhibitory factor (LIF) receptor mRNA expression in blastocyst stage parthenotes. These results suggest that GM-CSF may enhance the viability of porcine embryos developing in vitro in a defined culture medium.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.3
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• pp.201-207
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• 2002

Objective: To observe the capability of fertilization and embryo development including blastocyst formation of the oocytes in simple media after thawing of the cryopreserved cumulus-free mouse oocytes by vitrification method. Methods: Oocytes were collected from 5 to 6 weeks old ICR female mice, and were denuded from the cumulus cells by 0.1% hyaluronidase. Recovered mature oocytes in study group were cryopreserved by vitrification method using EM grid for $5{\sim}7$ days. In brief, oocytes were exposed in dPBS containing 1.5 M EG and 5.5 M EG+1 M sucrose for 2.5 minutes and 20 seconds each, and then executed vitrification by plunging in LN2 after loading on EM grid. Thawing treated by exposure of 1, 0.5, 0.25 and 0.125 M sucrose solution for 2.5 minutes each in order and used for experiments. Spermatozoa aspirated form the epididymis of 12 weeks old ICR male mice were used for insemination after capacitation. T6 media containing 0.4% BSA were used for fertilization and development. Results: Survival and fertilization rates after thawing were 76.9% and 79.6% respectively. Fertilization rate was lower (p<0.005) than that of control group (92.9%). There was no difference in embryo developmental rates from 2-cell to morula, however, the blastocyst formation rate and mean cell numbers of blastocysts in study group (63.3%, $58.9{\pm}9.2$) were lower compared with those of control group (76.1%, $63.5{\pm}8.9$). Conclusion: Vitrification is an effective method for mouse mature oocyte cryopreservation with high survival and fertilization rate after thawing. And in simple media, fertilization rates and embryo development of frozen-thawed mouse oocytes are satisfactory.

• Journal of Embryo Transfer
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• v.10 no.2
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• pp.163-169
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• 1995

In the experiment I for maas production of bovine early embryos, 18~20hpi fertilized eggs (756 eggs) and parthenogenic eggs (618 eggs) which were treated by 10% ethanol were cultured in both TGM and CZB. In the experiment II, suppiment effects each in CZB and CRlaa were tested by matured and fertilized oocytes which were after 18~20hpi. In the case of experiment I after 48hr, the cleavage rates of normally fertilized eggs were 66.6% in TCM treatment and 77.7% in CZB treatment, and after 240h the blastocysts were 7.5% in TCM and 14.1% in CZB. In the parthenogenic eggs, the deavage rates at 48hr were 39.6% in TCM and 57.5% in CZB, and at 240h, the blastocysts were 0.9% in TCM and 4.4% in CZB. These results showed that the effects of CZB on developmental ability to parthenogenic eggs as well as nomally fertilized eggs are relatively high. In experiment W, the effect of exposing the cleaved embryos to CZB for 30h on the blastocyst formation was examined. Similar rates of blastocyst formation were obtained both in TCM and CZB, suggesting that CZB exposure. during ealry development is critical. In experiments III ~ V, the effects of supplements were examined. The cleavage rates of CZB treatments at 48h were 83.8% in control, 78.1% in BSA+A.A+SIT, 75% in 5% FCS+A. A+SIT, 88.6% in BSA+A.A+SIT and not co-cultured BSA+A.A+SIT had 85.7% and in the case of 240h blastocysts showed 22.6, 0.0, fl.1, 6.5 and 0%, respectively. As a result, this study showed that CZB was effective culture system for in vitro development, and that CZB and CR$_1$aa had no significant differences and effects between them. It may be concluded that in the simple media containing supplements could replace the co-culture systems of bovine early embryo development.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.243-250
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• 2013

Pyracantha is a genus of thorny evergreen large shrubs in the family of Rosaceae, with common names Firethorn or Pyracantha. It's extract has also been used in cosmetics as a skin-whitening agent and functioning through tyrosinase inhibition. Recent studies have shown that pyracantha extract possesses antioxidant activities and may significantly improve lipoprotein metabolism in rats. Although the mode of action of Pyracantha extract is not fully understood, a strong relationship was observed between antioxidant and apoptosis in some types of cells. Thus, the aim of this study was to evaluated the effect of pyracantha extract on blastocysts formation and their quality of the porcine parthenogenetic embryos. After parthenogenetic activation by chemicals, presumptive porcine parthenogenetic embryos were cultured in PZM-3 medium supplemented with extracts of pyracantha leaf, stalk and root for 6 day (1, 5 and $10{\mu}g/ml$, respectively). In our results, the frequency of blastocyst formation in pyracantha root extract ($5{\mu}g/ml$) treated group had increased that of other groups. Furthermore, blastocysts derived from pyracantha root extract ($5{\mu}g/ml$) treated group had increased the total cell numbers and reduced apoptotic index. Blastocyst development was significantly improved in the pyracantha root extract ($5{\mu}g/ml$) treated group when compared with the $H_2O_2$ treated group (p<0.05). Subsequent evaluation of the intracellular levels of ROS in pyracantha root extract ($5{\mu}g/ml$) treated groups under $H_2O_2$ induced oxidative stress were decreased (p<0.05). In conclusion, our results indicate that treatment of pyracantha root extract may improve in vitro development of porcine parthenogenetic embryos through its antioxidative and antiapoptotic effects.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.5
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• pp.612-616
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• 2004

This study was carried out to compare the semen characteristics, frozen-thawed sperm viability and testosterone concentration and in vitro fertilization (IVF) and development of in vitro matured pig oocytes between two Yorkshire boars. Semen and blood samples were collected once per week from October to November 2002 from two adult Yorkshire boars at 18 months of age with 170 kg body weight. Sperm were deep frozen in 5 ml maxi-straws with lactose-egg yolk and N-acetyl-D-glucosamine (LEN) diluent and stored in liquid nitrogen. Blood samples were obtained at 10 a.m. by inserting a 21 gauge, hypodermic needle attached to 10 ml syringe into surface veins in the ear. The concentration of testosterone was determined by Competitive Enzyme Immunoassay. Ovaries were collected from prepubertal gilts at a local slaughter house. Cumulus oocyte complexes were aspirated from antral follicles (3 to 6 mm in diameter). The medium used for oocyte maturation was modified TCM 199. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at $38.5^{\circ}C$, 5% $CO_2$ in air. For IVF, one frozen 5 ml straw was thawed at $52^{\circ}C$in 40 sec and was diluted with 20 ml Beltsville thawing solution at room temperature. Sperm were washed 2 times in mTLP-PVA and inseminated without preincubation after thawing. Oocytes were inseminated with $2{\times}10^7$/ml sperm concentration. Oocytes were coincubated for 6 h in 500 ${\mu}$l mTBM fertilization medium. At 6 h after IVF, oocytes were transferred into 500 ${\mu}$l NCSU-23 culture medium for further culture of 48 and 144 h. There were no significant differences in the semen volume, motility, normal acrosome morphology and sperm concentration of raw semen between A and B of Yorkshire boar. However, motility and normal acrosome of boar A were higher than those of boar B at 0.5, 2, 3, 4, 5 and 6 h incubations of frozen-thawed sperm. Testosterone concentration (3.75 ng/ml) of boar A was higher than that (2.34 ng/ml) of boar B. The rate of blastocyst formation (15.1%) of boar A was higher than that (10.4%) of boar B. In conclusion, serum testosterone concentration of boar showed very important role for the frozen-thawed sperm viability and the blastocyst formation of pig oocytes matured in vitro.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.33-39
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• 2002

This study was conducted to investigate the effect of $Ca^{2+}$ concentration in fusion medium on the fusion, nuclear morphology and the development of bovine somatic cell nuclear transfer embryos. Bovine skin cells were transferred into an enucleated oocyte and fused with cytoplasm in the fusion medium containing with 0.05 to 1.0 mM Cacl$_2$. Nuclear transfer embryos were activated with a combination of A23187 and cycloheximide. Nuclear transfer embryos were fixed at 3 h after fusion or cultured for 7 ~8 days. Fusion rate was significantly (P＜0.01) increased by increasing the $Ca^{2+}$ concentrations in the fusion medium from 0.05 mM (56.6%) to 0.5 mM (50.1%) and 1.0 mM (84.3%). More than 80% of reconstituted embryos underwent premature chromosome condensation (PCC) with 0.05, 0.1 mM CaCl$_2$, whereas 54.5% and 59.3% of embryos formed pronucleus (PN) directly without PCC in the 0.5 and 1.0 mM CaCl$_2$, groups. Blastocyst formation rates were significantly (P＜0.05) different between 0.1 mM and 1.0 mM CaCl$_2$groups. From the present result, it is suggested that the elevated $Ca^{2+}$ concentrations in fusion medium can enhance the fusion and blastocyst formation rates of bovine nuclear transfer embryos.bryos.