• Journal of Embryo Transfer
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• v.22 no.1
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• pp.1-7
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• 2007

This study was conducted to examine the role of intact cumulus cells during in vitro fertilization (IVF) on sperm penetration, male pronuclear (MPN) formation and subsequent embryo development of oocytes matured and fertilized in vitro. Cumulus-oocyte complexes obtained from the slaughtered gilt ovaries were matured for 44 h in TCM199 containing 10% porcine follicular fluid, epidermal growth factor and hormones. After maturation culture, denuded oocytes or oocytes with intact cumulus cells were coincubated with frozen-thawed boar semen for 8h in a modified tris-buffered medium containing 5mM caffeine and 10mM calcium chloride. Putative zygotes were fixed and examined for sperm penetration and MPN formation (Experiments $1{\sim}3$), or cultured in North Carolina State University-23 medium fo. 156 h (Experiment 3). In Experiment 1, sperm penetration was examined after insemination of denuded oocytes and oocytes with intact cumulus cells at the concentration of $7.5{\times}10^5$ sperm/ml. Optimal sperm concentration for IVF of cumulus-intact oocytes was determined in Experiment 2 by inseminating intact oocytes with $2{\sim}5{\times}10^6$ sperm/ml. In Experiment 3, denuded or intact oocytes were inseminated at the concentrations of $7.5{\times}10^5$ and $4.0{\times}10^6$ sperm/ml, respectively, and in vitro embryo development was compared. Sperm penetration was significantly (p<0.01) decreased in cumulus-intact oocytes compared to denuded oocytes (35.2% vs. 77.4%). Based on the rates of sperm penetration and normal fertilization, the concentration of $4.0{\times}10^6$ sperm/ml was optimal for the IVF of intact oocytes compared to other sperm concentrations. The presence of intact cumulus cells during IVF significantly (p<0.05) improved embryo cleavage (48.8% vs. 58.9%), blastocyst (BL) formation (11.0% vs. 22.8%) and embryo cell number $(22{\pm}2\;vs.\;29{\pm}2\;cells)$ compared to denuded oocytes. In conclusion, these results suggest that intact cumulus cells during IVF inhibit sperm penetration but improve embryo cleavage, BL formation and embryo cell number of porcine embryos produced in vitro.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.41-51
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• 2002

The present study was carried out to examine the effect of cysteamine 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 NCSU23 maturation medium with 0, 25, 50 and 100 $\mu$M cysteamine (P〉0.05). 2. The rates of blastocyst formation at day 7 after in vitro fertilization in 0, 25, 50 and 100 $\mu$M cysteamine were 17.9$\pm$6.1, 17.4$\pm$6.3, 24.2$\pm$1.9 and 16.9$\pm$2.0%, respectively. And the total cells were 30.7$\pm$2.4, 34.9$\pm$2.8, 39.6$\pm$2.3 and 36.8$\pm$3.6, respectively. Fifty $\mu$M cystealnine group was significantly higher than those of any other treatment groups (P＜0.05). 3. The ratios of ICM/total cells in 20~40% category were 20.5, 41.6, 19.5 and 31.5%, respectively. Twenty five $\mu$M cysteamine group was higher than those of other groups. 4. The rates of blastocyst formation at day 7 in the NCSU-23 culture medium of porcine IVF-produced embryos with 0, 25, 50, and 100 $\mu$M cysteamine were 16.0$\pm$0.2, 13.6$\pm$1.7, 25.0$\pm$0.8 and 15.7$\pm$4.5%, respectively. And the total cells were 27.0$\pm$3.7, 36.1$\pm$4.8, 34.0$\pm$3.8 and 25.2$\pm$4.4, respectively. Fifty $\mu$M cysteamine group was significantly higher than those of any other treatment groups (P＜0.05). 5. The ratios of ICM/total cells in 20~40% category were 53.8, 30.0, 16.6 and 11.1%, respectively. The addition groups of cysteamine were lower than those of control group. In conclusion, these results suggested that the addition of 50 $\mu$M cysteamine in the IVM medium and 25~50 $\mu$M cysteamine in IVC medium were effective on the blastocyst formation and total cells of blastocysts.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.17-23
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• 2002

The present study was carried out to investigate the effects of the maturation media such as a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium on penetrability of pig oocytes by liquid boar sperm. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. When immature pig oocytes were cultured in mTCM-199, mWaymouth MB 752/1 and NCSU-23 maturation media for 44 h in 5% $CO_2$, in air at 38.5$^{\circ}C$, the germinal vesicle breakdown (CVBD) rates of the oocytes were 95.6, 94.1 and 94.9%, respectively, and the maturation rates (metaphase II) of oocytes were 92.5, 90.1 and 91.1%, respectively. No differences were observed among the maturation media. The spermrich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ for 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes, cumulus cells were removed and oocytes (30~40) coincubated far 6 h in 0.5 $m\ell$ mTCM-199 and mTBM fertilization media with 2$\times$1061$m\ell$ sperm concentration. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ mTCM-199 and NCSU-23 culture media for further culture 6 or 42 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF, and developmental ability of oocytes at 48 h after IVF were evaluated. The oocytes in combination with NCSU-23 medium for maturation and mTBM medium for IVF increased male pronuclear formation (48.0%) compared to those in combination with mTCM-199 media for maturation and IVF, and mWaymouth MB 752il medium for maturation and mTCM-199 medium far IVF. The rates of cleaved embryos (2~4 cell stage) at 48 h after IVF were 24.1% in combination with mTCM-199 media for maturation, IVF and culture, 43.6% in combination with mWaymouth MB 75211 medium fur maturation and mTCM-199 media for IVF and culture, and 71.2% in combination with NCSU-23 medium for maturation, mTBM medium for IVF and NCSU-23 medium for culture. In conclusion, we found out the oocytes matured in vitro were fertilized by liquid boar sperm stored in BTS extender at 17$^{\circ}C$ for 5 days. We recommend the simple defined NCSU-23 medium for nuclear maturation, mTBM medium and liquid boar sperm for IVF, and NCSU-23 medium for embryo culture.

• Journal of Animal Science and Technology
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• v.44 no.6
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• pp.701-710
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• 2002

Studies on the Viability of In Vitro-Matured Bovine Oocytes Vitrified by Microdrop and Straw Method To establish vitrification method for bovine oocytes, mature bovine oocytes were vitrified by microdrop (MD) or straw (Straw) method and the viability of vitrified oocytes with or without cumulus cells (CC) were examined by several methods; a) parthenogenetic activation; b) pronuclear formation after in vitro fertilization (IVF); and c) embryonic development after IVF. The survival rate of vitrified oocytes by MD was significantly higher than by Straw (92.50 vs. 74.19%, p<0.05). Most of the oocytes survived from vitrification using the MD methods. Cleavage and blastocyst development of parthenogenetically activated oocytes were higher in MD (45.05% and 10.81%, respectively; p<0.05)) than those in Straw method (27.17% and 6.52%, respectively; p<0.05). Male and female pronuclear formation of vitrified-thawed oocytes with or without cumulus cells (CC) after IVF were examined, respectively. The survival rate of vitrified oocytes by MD without CC was no difference between MD and Straw (80.368.14% vs. 67.31%). Normal fertilization (2PN) rates were not different among groups (Fresh; 54.55% vs. MD; 42.22% vs. Straw; 37.14%, p>0.05). While no fertilization (<1PN) rates were significantly different between fresh and vitrified-thawed groups (Fresh; 32.47% vs. MD; 57.78% and Straw 62.86%, p<0.05). The polyspermy (3PN) was appeared in the fresh (12.99%), but no appeared in the vitrified-thawed groups. In the without CC, normal fertilization (2PN) rates were significantly different between fresh and vitrified-thawed oocytes (Fresh; 59.38% vs. MD; 17.31% and Straw; 30.43%, p<0.05). Moreover, no fertilization (<1PN) rates were significantly different between fresh and vitrified-thawed groups (Fresh; 23.44% vs. MD; 73.08% and Straw 58.70%, p<0.05). The polyspermy (3PN, >4PN) was appeared not only fresh but vitrified-thawed groups. After IVF, two-cell developmental rates of vitrified oocytes with CC by MD and Straw were significantly low compared to fresh oocytes (Fresh; 81.76% vs. MD; 22.22% and Straw; 11.36%, p<0.05). Blastocyst developmental rates of vitrified oocytes also were significantly low compared to fresh oocytes (Fresh; 28.38 vs. MD; 1.71% and Straw 0%, p<0.05). In the without CC, two-cell developmental rates were no difference between Fresh and MD (27.59% vs. 19.25%, p<0.05), while blastocyst rates were difference between Fresh and MD or Straw (4.31% vs. 0.62% and 0%, respectively; p<0.05). In conclusion, the results indicate that the vitrified bovine oocytes have the ability to develop to the blastocyst stage after IVF.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.51-57
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• 2014

The objectives of this study were to investigate the effects of trehalose as a cryoprotectant for porcine freeze-dried spermatozoa, to find the optimal freeze-drying time and storage periods of freeze-dried spermatozoa, and to find out pronuclear formation rates, cleaved rates, and embryo development through intracytoplasmic injection of freeze-dried spermatozoa on porcine oocytes. The survival rates of spermatozoa after freeze-drying with trehalose treatment were significantly higher than those of them without trehalose treatment (p<0.05). The highest survival rates were found at 75 mM trehalose treatment. The longer storage periods after freeze-drying seemed to have a lower survival rates. Development in culture of pig by ICSI with trehalose treatment were significantly higher than those of them without trehalose treatment (p<0.05). Shorter freeze-drying time of spermatozoa was resulted in the highest cleaved rates and 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.

• Korean Journal of Animal Reproduction
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• v.24 no.2
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• pp.143-154
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• 2000

Most eggs initiated the fertilization processes but arrested at specific stages. The stages included failure of the oocyte to exit from the meiotic metaphase-II with or without sperm penetration, failure of appropriate sperm aster formation, inability to form proper male and female pronuclei, failure of suitable pronuclear apposition, and failure to form proper number of either male or female pronuclei. Various images of defective microtubule organization and chromatin configuration during IVF and ICSI procedures were observed. We discussed the data with previous research results during normal fertilization in humans and other mammals. In conclusion, various aberrant patterns in microtubule assembly and chromatin configuration, which were assessed in the present study, could be used as criteria to improve assisted reproductive technology in clinics. However, further cellular and molecular characterization is needed to clarify these aberrant patterns of cytoskeletal assembly.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.193-201
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• 1999

In this study we determined fertilization processes and developmental ability of porcine oocytes following injection of round spermatid in the presence of artificial activation. Electrical stimulation at 3 h before spermatid injection significantly increased the incidence of normal fertilization as compared to those following injection without stimulation or with stimulation immediately after injection. The incidences of two pronuclear formation and apposition were not different in oocytes between following intracytoplasmic spermatid and spermatid nucleus injection. Indirect immunocytochemistry and laser scanning confocal microscopy study revealed that micro tubules were organized from the oocyte cortex following round spermatid injection, and this seemed to move both male and female pronuclei into the oocyte center. Paternal mitochondria which are introduced with spermatid have been observed up to 4-cell. Our study indicated that either round spermatid or it's nucleus can be used to produce viable bovine embryos by injection into unfertilized oocytes.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.63-63
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• 2003

This study was carried out to investigate the effects of liquid boar sperm stored at 4$^{\circ}C$ on sperm motility, normal acrosome, and in-vitro fertilization and culture of pig oocytes matured in-vitro. The sperm-rich fraction (30~60 ml) of ejaculate was collected into an insulated vacuum bottle. Semen was slowly cooled to room temperature (20~23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min at 800$\times$g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of lactose, egg yolk and N-acetyl-D-glucosamine (LEN) diluent to provide 1.0$\times$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$ and preserved for 5 days to examine sperm motility and normal acrosome. The medium used for oocyte maturation was modified tissue culture medium (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. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 6 h in 500 ${mu}ell$ mTBM fertilization media with 0.2, 1, 5 and 10$\times$10$^{6}$ /ml sperm concentration, respectively. At 6 h after IVF, oocytes were transferred into 500 ${mu}ell$ Hepes-buffered NCSU-23 culture medium for further culture of 6, 48 and 144 h. There were significant differences in sperm motility and normal acrosome among preservation days and incubation times, respectively. The rates of sperm penetration and polyspermy were higher in 5 and 10$\times$10$^{6}$ sperm/ml than in 0.2 and 1$\times$10$^{6}$ sperm/ml. Male pronuclear formation was lower in 0.2$\times$10$^{6}$ sperm/ml than in 1, 5 and 10$\times$10$^{6}$ sperm/ml. Mean numbers of sperm in penetrated oocyte were highest in 10$\times$10$^{6}$ sperm/ml compared with other sperm concentrations. The rate of blastocysts from the cleaved oocytes (2~4 cell stage) was highest in 1$\times$10$^{6}$ sperm/ml compared with other sperm concentrations. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend 1$\times$10$^{6}$ ml sperm concentration for in-vitro fertilization of pig oocytes.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.305-315
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• 2001

In this study, the production of transgenic embryo was attempted by microinjection or round spermatid cultured with foreign DNA. At first, the expression of haploid spermatids specific gene, mTP1 in mouse and hPrm2 in hamster spermatids were investigated by RT-PCR method in testes of young mice and hamster testis. The specific gene expression first appeared at 18 days post partum (dpp) in mice spermatid and 20 dpp in hamster spermatid. Therefore, the round spermatids isolated from 17 dpp mice and 19 dpp hamster were used for the introduction of foreign EGFP gene into haploid round spermatids. For the introduction of EGFP gene haploid round spermatids suspended in medium including EGFP gene were treated with a different electric field strength at 0.11, 0.18 and 0.44 ㎸/cm. After electrical stimulation, viability of testicular sperm cells and 67.6%, 66.4% and 49.9%, in mice and 62.6%, 57.9% and 27% in hamster, respectively. These values were significantly lower than those of non-treated control groups 80.5% in mouse and 69.1% in hamster After 72 hrs culture, the highest expression rate of EGFP gene, 28.5% in mice and 32.1% in hamster were obtained from tile spermatogenic cells electroporated by the field strength or 0.18 ㎸/cm. Then, the ability of fertilization and embryonic development of haploid spermatids transfected with foreign EGFP gene were estimated by the microinjection of spermatids into hamster oocytes. The Irate pronuclear formation rate (77.5%) was lower than non-treated control (80%), and the cleavage rate of the treated group (58.8%) was lower than control (65%). To prove the foreign EGFP integration in hamster embryos, 2-cell stage hamster embryos were subjected to the observation under the fluorescence microscope, and the PCR analysis. As a result, about 44% of 2-cell embryos were showed the integration of EFGP gene into their genome. Therefore, These results suggest the possibility to produce transgenic hamsters by microinjection of haploid spermatid transfected with foreign DNA.