• Journal of Embryo Transfer
• /
• v.13 no.2
• /
• pp.191-197
• /
• 1998

Immature nocytes and in VitrO matured Oocytes collected from the slaughtered Korean cattle were frozen slowly with 10% ethylene glycol+5% polyvinyl pyrolidine+0.05M trehalose (l0EPT), 10% ethylene glycol+5% ficoll+0.05M sucrose (1OEFS), or 10% ethylene glycol+5% ficoll+0.05M trehalose (l0EFT) by cell freezer (experiment 1). And also,They were ultra-rapidly frozen with 30% ethylene glycol+10% polyvinyl pyrolidine+0.5M trehalose (3OEPT) or 30% ethylene glycol+18% ficoll+0.5M sucrose (3OEFS) using electron microscope grid (experiment 2). In experiment 1, the cleavage rate was 23.0% when immature oocytes were frozen slowly using various cryoprotectants descrihed above, and 5.1% of cleaved oocytes developed to over morula stage after in Vitro fertilization (IVF). There were no significant differences among these groups. When matured oocytes were frozen slowly, the total cleavage rate was 19.7%, and over morula stage was 3.2%. lOEPT (4.8%) and EFS (4.4%) were slightly more effective than l0EFT (0.0%) for development in vitro. Only in l0EFT treated group, immature oocytes have higher developmental capacity than matured ones, when they were frozen slowly and IVF after thawing. In experiment 2, oocytes were ultra-rapidly frozen using the electron microscope grid with two kind of cryoprotectants described above. In immature oocyte group, the cleavage rate was 13.9% and 5.8% of cleaved oocytes developed to over morula stage after IVF, and in matured group, 25.7 and 7.6%, respectively. There were no significant differences between two kind of cryoprotectants, but in ultra-rapid freezing using electron microscope grid, the efficiency is slightly higher in matured oocyte group.

• Clinical and Experimental Reproductive Medicine
• /
• v.27 no.1
• /
• pp.23-29
• /
• 2000

Objective: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes in standard culture conditions, and that it is probably not a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. Methods: HAF was obtained from patients undergoing amniocentesis at $16{\sim}20$ weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at $48{\sim}52$ hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrs. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Fertilization rate was assessed in zona-intact or zona-free oocytes (denuded by trypsin). Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at $37^{\circ}C$ for 10 min. Results: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7%) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). Conclusions: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.

• Korean Journal of Organic Agriculture
• /
• v.26 no.4
• /
• pp.731-743
• /
• 2018

This study was conducted to evaluate the effects of green manures and complemental fertilization with oil cake or liquid fertilizer on growth and nitrogen use efficiency of Chinese cabbage cultivated in organi systems. Field experiments were carried out at the National Institute of Agricultural Science in Suwon, South Korea from 2012 to 2014. Two green manure crops, Crotalaria and Hairy vetch, was cultivated in summer and in winter, respectively. The application methods of the green manure consisted of three tillage systems (overall tillage, partial tillage and no tillage). Oil cake and liquid fertilizer were used as complemental fertilizer. The results showed that when used as covering material in the upland soil without tillage, green manure fertilization was more effective in increasing growth and yield of Chinese cabbage than when incorporated into soil. It was possible to grow and harvest Chinese cabbage in the spring season by the application of hairy vetch as winter green manure. The higher yield of Chinese cabbage with green manure application was caused by the lower incidence rate of soft rot and tip-burn. The yield of the Chines cabbage that received green manure applications over two consecutive seasons followed by the supplemental fertilization with oil cake was similar to that of the conventional chemical fertilization. Following a single season green manure application in summer, however, the yield of cabbage was only about 70% of the conventional treatment. Green manure cultivation with additional liquid fertilization produced a yield similar to the conventional fertilization treatment, soil inorganic nitrogen concentration remained stable and the nitrogen use efficiency increased in the green manure applied soil. In conclusion, the organic cultivation of Chinese cabbage in the autumn season could be outperformed in the upland soil receiving two seasons (winter and summer) of green manure fertilization followed by the supplemental fertilization with liquid fertilizer.

• Journal of Embryo Transfer
• /
• v.14 no.1
• /
• pp.23-29
• /
• 1999

The objectives of the study were to establish sperm separation method and duration of insemination for bovine IVF. Oocytes from slaughterhouse ovaries were matured and fertilized using general protocol. After 18 or 42 h of insemination, six to ten embryos were placed into a 30${mu}ell$ drop of each medium, and the embryos were examined 7~10d post in semination without medium renewal. First, we compared Percoll gradient will swim-up technique for sperm separation. There was no difference in cleavage rates between them, but the development rates over morula stage of oocytes fertilized with sperm separated by Percoll gradient was significantly higher than that sperm selected by swim-up technique (p<0.05). Second, we evaluated development of bovine embryos derived from the IVF procedure with different durations(18 vs 42 h) of fertilization. There was also no difference in cleavage rates, but the development to blastocyst stage of oocytes exposed in cleavage rates, but the development to blastocyst stage of oocytes exposed to sperm for 42 h was significantly higher than that exposed for 18 h (p<0.05). In conclusion, Percoll gradient can be used for sperm selecton, improving of embryonic development. Also, 42h of IVF may improve the development of bovine embryos.

• Clinical and Experimental Reproductive Medicine
• /
• v.47 no.2
• /
• pp.94-100
• /
• 2020

The inverse correlation between maternal age and pregnancy rate represents a major challenge for reproductive endocrinology. The high embryo ploidy error rate in failed in vitro fertilization (IVF) cycles reflects genetic misfires accumulated by older oocytes over time. Despite the application of different follicular recruitment protocols during IVF, gonadotropin modifications are generally futile in addressing such damage. Even when additional oocytes are retrieved, quality is frequently poor. Older oocytes with serious cytoplasmic and/or chromosomal errors are often harvested from poorly perfused follicles, and ovarian vascularity and follicular oxygenation impact embryonic chromosomal competency. Because stimulation regimens exert their effects briefly and immediately before ovulation, gonadotropins alone are an ineffective antidote to long-term hypoxic pathology. In contrast, the tissue repair properties (and particularly the angiogenic effects) of platelet-rich plasma (PRP) are well known, with applications in other clinical contexts. Injection of conventional PRP and/or its components (e.g., isolated platelet-derived growth factors as a cell-free substrate) into ovarian tissue prior to IVF has been reported to improve reproductive outcomes. Any derivative neovascularity may modulate oocyte competence by increasing cellular oxygenation and/or lowering concentrations of intraovarian reactive oxygen species. We propose a mechanism to support intrastromal angiogenesis, improved follicular perfusion, and, crucially, embryo ploidy rescue. This last effect may be explained by mRNA upregulation coordinated by PRP-associated molecular signaling, as in other tissue systems. Additionally, we outline an intraovarian injection technique for platelet-derived growth factors and present this method to help minimize reliance on donor oocytes and conventional hormone replacement therapy.

• 대한생식의학회:학술대회논문집
• /
• 2002.11a
• /
• pp.90-90
• /
• 2002

• Clinical and Experimental Reproductive Medicine
• /
• v.26 no.1
• /
• pp.117-121
• /
• 1999

Although anejaculation is a relatively uncommon occurrence in the general population, over 12,000 new cases are reported annually. Anejaculation may result from spinal cord injury, retroperitoneal lymph node dissection, diabetes mellitus, transverse myelitis, multiple sclerosis, or psychogenic disorders. At least 30% of men with this problem are or will be married and many will seek help to remedy their infertile state. The evolution of technique and instrumentation over the last 30 years has made electroejaculation an accessible and acceptable form of therapy. Recent successes in inducing ejaculation by means of rectal probe electrostimulation or vibratory stimulation combined with assisted reproductive techniques, such as zygote intrafallopian transfer (ZIFT), gamete intrafallopian transfer (GIFT), and in vitro fertilization (IVF), have provided these men means of producing their own biologic offspring. We have experienced a successful pregnancy with electroejaculation and in vitro fertilization in a infertile patient whose husband had an ejaculatory disturbance due to a spinal cord injury. So we report this case with a brief review of literatures.

• Applied Biological Chemistry
• /
• v.49 no.3
• /
• pp.175-179
• /
• 2006

This study was carried out to investigate the change of rhizosphere microflora in paddy soils of long-term application of same fertilizer by single cropping. Treatments consisted of no fertilizer, NPK, PK, NK, NP and NPK + compost and the same fertilizer has been applied to the soil of each treatment for thirty five years. Any remarkable difference of the microbial population structure was not detected from the rhizosphere of Hwayeongbyeo and that of Hwasambyeo. However, slight difference of dominant microbial species in the rhizosphere was recognized between different fertilization practice. The bacterial population in the rhizosphere was steadily reduced over time after rice plant setting out, while actinomycetal population was increased over time. The increase of actinomycetal population was the highest in the NPK + compost treatment, suggesting the effect of organic matters in the change of actinomycetal population. As for the aerobic culturable bacterial diversity, Bacillus megaterium, B. mycoides, B. licheniformis and B. subtilis were dominants in genus Bacillus, and Streptomyces spororaveus, S. canus, S. tauricus and S. galbus in genus Streptomyces and genus Micromonospora was another dominant in actinomycetes.

• Clinical and Experimental Reproductive Medicine
• /
• v.42 no.4
• /
• pp.156-162
• /
• 2015

Objective: To investigate fertilization and embryo quality of dysmorphic mature oocytes with specific morphological abnormalities obtained from intracytoplasmic sperm injection (ICSI). Methods: The fertilization rate (FR) and embryo quality were compared among 58 dysmorphic and 42 normal form oocytes (control 1) obtained from 35 consecutive ICSI cycles, each of which yielded at least one dysmorphic mature oocyte, performed over a period of 5 years. The FR and embryo quality of 441 normal form oocytes from another 119 ICSI cycles that did not involve dysmorphic oocytes served as control 2. Dysmorphic oocytes were classified as having a dark cytoplasm, cytoplasmic granularity, cytoplasmic vacuoles, refractile bodies in the cytoplasm, smooth endoplasmic reticulum in the cytoplasm, an oval shape, an abnormal zona pellucida, a large perivitelline space, debris in the perivitelline space, or an abnormal polar body (PB). Results: The overall FR was significantly lower in dysmorphic oocytes than in normal form oocytes in both the control 1 and control 2 groups. However, embryo quality in the dysmorphic oocyte group and the normal form oocyte groups at day 3 was similar. The FR and embryo quality were similar in the oocyte groups with a single abnormality and multiple abnormalities. Specific abnormalities related with a higher percentage of top-quality embryos were dark cytoplasm (66.7%), abnormal PB (50%), and cytoplasmic vacuoles (25%). Conclusion: The fertilization potential of dysmorphic oocytes in our study was lower, but their subsequent embryonic development and embryo quality was relatively good. We were able to define several specific abnormalities related with good or poor embryo quality.

• Asian-Australasian Journal of Animal Sciences
• /
• v.14 no.9
• /
• pp.1342-1351
• /
• 2001

Over the years, the embryo transfer industry has grown from the simple collection & transfer of embryos into an advanced field of embryo biotechnology. Currently a large demand exists for bovine oocytes and early embryos in both research and commercial settings. Bovine embryos can now be produced in-vitro. Primary oocytes collected from antral follicles of abattoir - obtained ovaries can be induced to undergo the maturation process. In-vitor maturation system, however must ensure that the resulting oocyte is capable of undergoing normal fertilization and yields a zygote competent of developing to term after embryo transfer. Sperm preparation for IVF has improved with the use of heparine. The use of co-culture system has proved beneficial in circumventing the developmental block in IVM/IVF bovine embryos.