• Korean Journal of Agricultural Science
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• v.34 no.1
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• pp.19-35
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• 2007

The present study was carried out to examine the effect of EGF and IGF-I in vitro maturation (IVM) of porcine oocytes and development of porcine IVM/IVF embryos. The results were summarized as follows : 1. The rates of nuclear maturation, penetrated oocytes, pronuclear formation, polyspermic oocytes and mean numbers of the penetrated sperm were not different in NCSU-23 maturation medium with 0, 1, 5 and 10 ng/ml EGF and IGF-I (P>0.05). 2. The rates of blastocyst formation at day 7 after in vitro fertilization in 0, 1, 5 and 10 ng/ml EGF groups were $11.2{\pm}1.5%$, $15.0{\pm}8.3%$, $16.8{\pm}2.8%$ and $21.4{\pm}2.0%$, also 0, 1, 5 and 10 ng/ml IGF-I groups were $11.2{\pm}1.5%$, $15.0{\pm}8.3%$, $16.8{\pm}2.8%$ and $21.4{\pm}2.0%$, respectively. In the total cells case, EGF groups were $22.8{\pm}3.7$, $25.7{\pm}5.5$, $26.0{\pm}4.2$ and $35.1{\pm}4.7$, also IGF-I groups were $21.5{\pm}3.7$, $25.2{\pm}2.8$, $26.2{\pm}2.9$ and $33.2{\pm}3.6$, respectively. Both 10 ng/ml EGF group and 10 ng/ml IGF-I group were significantly higher than those of other treatment groups (P<0.05). 3. The rates of blastocyst formation at day 7 in the NCSU23 culture medium of porcine IVF-produced embryos with 0, 1, 5, and 10 ng/ml EGF groups were $14.0{\pm}1.7%$, $16.2{\pm}1.4%$, $16.9{\pm}1.2%$ and $23.1{\pm}1.6%$, also 0, 1, 5, 10 ng/ml IGF-I groups were $13.6{\pm}1.7$, $15.7{\pm}4.5$, $16.0{\pm}0.2$ and $25.0{\pm}0.8$, respectively. And in the total cells case, EGF grups were $21.8{\pm}2.9$, $25.2{\pm}2.8$, $39.7{\pm}2.7$ and $46.2{\pm}3.6$, also IGF-I groups were $20.7{\pm}2.9$, $26.2{\pm}2.9$, $24.6{\pm}2.4$ and $46.1{\pm}3.5$, respectively. Both 10 ng/ml EGF group and 10 ng/ml IGF-I group were significantly higher than those of any other treatment groups (P<0.05). In conclusion, these results suggested that the addition of 10 ng/ml EGF and IGF-I were effective on the blastocyst formation and total cells of blastocysts.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.129-135
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• 2006

This study was conducted to investigate the effect of in vitro maturation (IVM) duration of porcine follicular oocytes on maturation rate, polyspermic rate, and subsequent embryo development. The nuclear maturation rates of oocytes matured for 36, 38, 40, 42 and 44 hr were similar between 68.0, 78.0, 79.5, 73.8 and 81.8% respectively. There was no significant difference in the rates of polyspermy after in vitro feritilization (IVF). The cleavage rate in the group of 36 hr was significantly higher in than that of 40, 44 hr (p<0.05) but not to 38 and 42 hr. The development rate to blastocyst stage was significantly higher in the group of 38 hr (23.1%) than that in the group of 44 hr (15.6%) (p<0.05) but not to 36, 40 and 42 hr. These results suggest that the aged oocytes for 44 hr is not required for the production of bias to cysts derived from porcine IVF embryos.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.119-126
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• 2004

The present study was carried out to examine the effect of four different media (NCSU (North Carolina State University)-23, PZM (Porcine Zygotes Medium)-3, PZM-4 and TCM (Tissue Culture Medium)-l99) and two oxygen concentrations (39 , 5% $O_2$, 5% $CO_2$ and 90% $N_2$, 5% $CO_2$ in air) on in vitro production of porcine IVM/IVF embryos. The results were summarized as follows: The rates of GVBD and nuclear maturations were not significantly different (p>0.05) for 44 hours of culture with four media in two oxygen concentrations. The rates of polyspermy, penetrated sperm(s) and male and female prouclei formation were not significantly different (p>0.05). among four media in two oxygen concentrations. The cleavage rates were not significantly different (p>0.05) among four media in two oxygen concentrations. At day 7 under gas atmosphere of 5% $O_2$, 5% $CO_2$ and 90% $N_2$, the blastocyst formation was significantly higher (p<0.05) in PZM-3 (19.9$\pm$2.4) than other media. Also, NCSU-23 medium gave high rate of blastocyst formation at day 7 under gas atmosphere of 5% $CO_2$ in air (p<0.05). Based on the result of differential staining of porcine blastocyst at dat 7, inner cell mass cell and total cell numbers were not significantly different (p>0.05) among four media in two oxygen concentrations. However, the observed total cell number was higher in PZM-3 medium (36.8$\pm$6.5) than other madia. In conclusion, these results suggested that in vitro production of porcine embryos in PZM-3 medium under a gas atmosphere of 5% $O_2$, 5% $CO_2$ and 90% $N_2$ was effective on the blastocyst formation rate and total blastocyst cell number.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.199-205
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• 2009

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 ${\mu}g/ml$ porcine FSH, 0.5 ${\mu}g/ml$ equine LH, 1.0 ${\mu}g/ml$ 17 $\beta$-estradiol ($E_2$) and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ${\mu}l$) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.173-183
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• 2004

This study was carried out to examine the effects of epidermal growth factor (EGF) and the number of cumulus-oocyte complexes (COCs) on in vitro maturation (IVM) of porcine immature oocytes, and on subsequent in vitro fertilization (IVF) and development (IVD). COCs were collected from antral follicles of porcine ovaries collected from abattoir, and were maturated in modified NCSU-23 (mNCSU-23) with 10% pFF, 0.6 mM cysteine, 50 ${\mu}mM{\beta}-mercaptoethanol$, 1 mM dbcAMP, 10 IU/mL PMSG and 10 IU/mL hCG, which was supplemented with or without 10 ng/mL EGF and into which 50 or 15 COCs per droplet was put. Oocytes matured in vitro, were fertilized in vitro in modified Tris-buffered medium (mTBM) with the final motile sperm concentration of 1${\times}$105 sperm/mL, and subsequently putative embryos were developed in vitro in NCSU- 23. The results are as follows. 1.In the result of IVM, 10 ng/mL EGF supplement duplicated the percentage of C4 group of COCs(41% vs 81%). But the rate of germinal vesicle breakdown (GVBD) and of nuclear maturation were not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet, and there was not a significant interaction between the two factors, either. 2. In the result of IVF, there was not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet, or was not a significant interaction between the two factors, in the rate of sperm penetration, in the percentage of oocytes with male pronucleus (MPN), and in the rate of polyspermy. 3. In the result of IVD, there was not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet in the percentage of cleaved oocytes. There was not significantly different between the number of COCs per culture droplet, but between control and EGF supplemented (p<0.01) in the percentage of blastocysts, the number of inner cell mass (ICM), trophectoderm (TC) and total cells. There was no significant interaction between the two factors anywhere. These results suggested that 10 ng/mL EGF supplement into mNCSU-23 for IVM was effective in the production of more as well as better blastocysts during IVD through increasing the number of cells in those.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.3
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• pp.340-345
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• 2008

Oxidative stress is one of the major causes of failure of in vitro storage of boar semen. Reactive oxygen species (ROS) are one of the important mediators of oxidative stress during in vitro storage of boar semen. Our study examined the effects of taurine on sperm characteristic and on in vitro developmental embryos during in vitro storage of boar semen for 7 days. Semen was randomly aliquoted into 3 centrifuge tubes and treated with different concentrations of taurine (25-100 mM). The characteristics of boar sperm were analyzed for motility by light microscopy, viability by using a Makler counting chamber and membrane integrity by a hypoosmotic swelling test (HOST). The percentages of motile spermatozoa in taurine groups after 5 days were significantly higher compared to the control. Sperm viability in the control was lower than in taurine groups after 7 days irrespective of different taurine concentration. In the hyoosmotic swelling test (HOST), significantly higher results were obtained in taurine groups after 3 days. Also, the developmental rates of IVM/IVF porcine embryos from semen treated with pyruvate and taurine were significantly increased when compared with the control (p<0.05). These results indicate that supplementation of taurine as an antioxidant in boar semen extender can improve the semen quality.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.121-128
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• 2006

In this study, we examined the effects of molecular weight, concentrations and treat the duration of polyvinylpyrrolidone (PVP) in vitro maturation (IVM) medium (Experiment 1), and the effect of PVP in IVM, in vitro fertilization (IVF) and in vitro culture (IVC) medium on the development and cell number of porcine embryos (Experiment 2). The base mediums were NCSU 23 solution for IVM, mTBM solution for IVF and PZM3 solution for IVC. In experiment 1, the development rates to 2 cell and blastocyst stage were not differ from the different molecular weight (MW), concentration and duration of PVP in IVM medium. However, the hatching rate of blastocyst was significantly higher in the group of MW 40,000, 0.5% and $0{\sim}44hr$ than in the other groups (p<0.05). In experiment 2, the results of IVM, IVF and IVC medium with (W) or without (W/O) 0.5% MW 40,000 PVP are follows. The development rate to 2 cell stage was highest in the group of W-W/O-W (p<0.05). The development rate to blastocyst and hatching rate was higher in the group of W-W/O-W and W-W/O-W/O than that of other treatments (p<0.05).

• Journal of Embryo Transfer
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• v.20 no.3
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• pp.289-295
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• 2005

This study was performed to investigate the relationship between Heat shock protein 70 (HSP70) gene polymorphism and in vitro fertilization(IVF) embryo development in the pigs. The single strand conformation polymorphism(SSCP) genotypes from HSP70 K1, K3 and K4 PCR products were detected different patterns. In cleavage rate of oocyte fertilized in vitro, HSP70 K1-AA genotype($73.1\%$) and K1-AB genotype($62.3\%$) showed significantly higher oocyte cleavage rate than HSP70 K1-BB genotype($49.3\%$)(p<0.05). And HSP70 K3-AA genotype ($72.4\%$) and K3-AB($62.2\%$) also showed significantly higher oocyte cleavage rate than HSP70 K3-BB genotype($49.1\%$)(p<0.05). The IVF embryo development of 2-cell stage according to HSP70 genotypes of sperm and pig breeds also showed a significant difference. The number of embryos developed to 2-cell stage in Landrace(28.8) and Duroc(29.8) were significantly higher than in Yorkshire(10.9)(p<0.05). And also HSP70 K4-AB genotype group(29.6) higher than HSP70 K4-AA genotype group(10.6)(p<0.05). However, the number of embryos developed to blastocyst stage did not showed significant differences among breeds as well as HSP70 genotypes. These resrults suggest that in vitro development in porcine early embryos may be affected by HSP70 genotypes and breeds.

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.267-272
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• 2010

The present study was to investigate the source of contamination during semen processing for in vitro uses. In the present study, frozen semen was prepared from liquid semen in our laboratory for in vitro fertilization (IVF) experiments due to lack of fresh semen. Antibiotics were added in the frozen semen extender (kanamycin and gentamicin) and in vitro culture (IVC) medium (gentamicin) for further inhibiting growth of microorganisms. Nevertheless, proliferations of microorganisms were observed in IVC culture drop during culturing of IVF embryos using frozen semen. Randomly 3 samples were taken from the liquid semen, frozen semen and egg yolk. Contaminated IVC medium, frozen-thawed semen, liquid semen and egg yolk were cultured in de Man, Rogosa and Sharpe (MRS) agar medium. Whitish colonies were detected in contaminated IVC drop, frozen-thawed semen samples and egg yolk but no colonies were formed in liquid semen samples. Gram-negative and rod-shaped identical bacteria were found in both frozen-thawed semen sample and contaminated IVC drop and egg yolk samples. Enterobacter cloacae were confirmed by API 20E kit according to manufacturer's instruction with identification value (% ID) 94.3% and T index 0.88. Antibiotic susceptibility tests were done according to Clinical and Laboratory Standards Institute (CLSI) by using ampicillin, amikacin, cephalothin, gentamicin, kanamycin, tetracycline, oxytetracycline, sulfamethoxazole trimethoprim, norfloxacin and ciprofloxacin test. Among them Enterobacter cloacae were resistant to ampicillin, amikacin, cephalothin, gentamicin, kanamycin but susceptible to tetracycline, oxytetracycline, sulfamethoxazole trimethoprim, norfloxacin and ciprofloxacin. From these findings it could be suggested that this contamination sources might be from egg yolk.

• Journal of Embryo Transfer
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• v.22 no.3
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• pp.155-160
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• 2007

During in vitro culture of mammalian oocytes and embryos, the cells are exposed to the risks that cause cell injury or death. Numerous studies have been reported that the cell injury may be induced by the action of free radicals generated by auto-oxidation. This study was undertaken to investigate the optimal culture condition system for in vitro culture of porcine embryos. We first evaluated the effect of culture media on the porcine embryo development. NCSU-23 and PZM-5, culture medium tested, were failed to produce significant difference on the rate of blastocyst formation. In NCSU-23, the developmental rate was slightly higher than that in PZM-5. During in vitro maturation (IVM), fertilizaton (IVF), and culture (IVC) under 5 or 20% oxygen ($O_2$), the rates of cleavage and development were insignificantly different from each other under our culture condition (20% $O_2$, in NCSU-23), the mean cell number per blastocyst was $40{\pm}10$. These results showed that medium and $O_2$ concentration had no significant effect on the development of porcine embryos.