• KOREAN JOURNAL OF CROP SCIENCE
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• v.51 no.4
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• pp.274-281
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• 2006

In Korea, rice cultivars have been changing to 'quality' rice rather than high yielding cultivars. However, more than 10% of paddy field has been changed to greenhouse in winter season for cropping of water-melon, oriental-melon, straw berry and et cetra. This experiment has been made to identify the usefulness of critical N and Si fertilization(SF) level to obtain high grain quality rice with reduced insect pest damage by N and SF combination. Before the experiment, watermelon-rice cropping system was maintained for three seasons by farmer from 1998 to 2001. The experiment of N and Si (silicate) fertilization levels was evaluated with Hwayoung-byeo (Oryza sativa L., medium-maturing variety) in 2002 and 2003 in Uiryeong, Korea. Nitrogen fertilization (NF) levels were three and five in 2002 and 2003, respectively, and three SF levels were compared for getting the valuable N/SF level in both years. TOYO-value was positively affected by Si application in N100% plot but it was negatively related with NF level. Normal grain percentage was positively related with TOYO-value and it was highest in 0N plot and Si plots in N100%. Other appearance qualities like powdered, damaged, and cracked grain, were decreased with increasing N fertilization level. SF improved appearance quality in N100% plots but no effects in other treatments. Leaf sheath related diseases were significantly decreased by SF but it was negatively related with NF. In conclusion, SF could be improve grain quality at the same yield levels of conventional fertilization and it also could be reduce the diseases damages of rice plant in all N treatments. NF treatment reduced grain quality and improved grain yield at N50% level, however NF above N50% could not get any kind of benefits. So, compared with conventional fertilizer, reduced NF level is recommended for high grain quality with reduced insect pest damage.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.411-417
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• 1998

The objective of this study was to dertermine the effects of oviductal fluid and oviductal conditioned medium on polyspermy and in vitro development of porcine oocytes. The addition of oviductal fluid and oviductal conditioned medium in the prefertilization and fertilization medium significantly decreased polyspermy rates and the mean number of spermatozoa in penetrated eggs(P＜0.05). The acrosome reaction rate significantly increased when spematozoa were exposed for 1.5, 3, 4.5h in oviductal fluid and oviductal conditioned medium(P＜0.05). When oocytes cultured for 192h, the percentage of oocytes that developed to the morula and blastocyst stage was higher in culture medium with oviductal fluid and oviductal conditioned medium than without oviductal fluid and oviductal conditioned medium(P＜0.05). These results indicated that the oviductal secreations will effectively reduce both the polyspermy rates and the mean number of spermatwa in penetrated eggs. And the presence of culture with oviductal fluid and oviductal conditioned medium promotes in vitro development of porcine oocytes.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.83-83
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• 2002

This study was to compare the characteristics of parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Mouse oocytes were recovered from superovulated 4wks hybrid F1 (C57BL/6xCBA/N) female mice. The oocytes were treated with 7% ethanol for 5 min and 5 ㎍/㎖ cytochalasin-B for 4 h. For IVF, the oocytes were inseminated with epididymal sperm of hybrid Fl male mice (1×10/sup 6//㎖). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count in blastocysts was carried out differential labelling using propidium iodide (red) and bisbenzimide(blue). (omitted)

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.251-256
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• 1995

Two experiments were conducted to study the production of in vitro fertilized bovine embryos and the viability of blastocysts cryopreserved by vitrification. In experiment 1, production rate of in vitro matured bovine oocytes after fertilization in medium containing bovine oviduct epithelial cells (BOEC), cumulus cells and granulosa cells to blastocysts were 18.4, 14.6 and 13.1%, respectively. Developmental percentages of blastocysts produced at day 6, 7 and 8 were 8.5, 10.6 and 15.2% respectively. Hatching rate of bovine embryos produced was 60.0%. In experiment 2, post-thawed surviving embryos in a vitrification solution consisting of 7.15M ethylene glycol, 2.5 mM ficoll and 0.3 M sucrose were 36.4% (56/154). Also, survival rate of bovine embryos after exposed to vitrification solution at 1, 2, 3, 4 and 5 min were 84.0, 88.0, 71.0, 48.0 and 24.0% respectively.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.161-169
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• 1997

This study was conducted to compare the insemination time of bovine oocytes and determine the effects of glucose(1.5 mM) on the development of bovine embryos at early cleavage stage. Oocytes were matured for 24 h, followed by exposure to sperm and cultured in modified Tyrode's media drops or with bovine oviduct epithelial cell monolayer prepared in TCM199(BOECM). Insemination time and culture system were varied in each experiment. In experiment 1, to investigate the developmental capacity of bovine embryos after different time of exposure to sperm, bovine ova and sperm were co-incubated for 18, 30 or 54 h, respectively. The development to blastocysts of 30 and 54 h insemination groups were significantly higher(P<0.05) than 18 h group, and in case of blastocysts of cleaved embryos, 30 h group were significantly higher(P<0.05) than other groups. In experiment 2, we investigated the effect of glucose on early bovine embryos. After 18 h insemination, in vitro fertilized oocytes were separated following 3 groups ; G+0, C+24 and C+48. Oocytes of G+0 group were cultured in glucose added Tyrode's medium after fertilization, oocytes in C+24 and C+48 groups were cultured in glucose free Tyrode's medium after fertilization. After 24 h culture, G+24 group was moved to glucose added medium. All oocytes of 3 groups were moved to BOECM after 48 h culture. The rates of cleavage and development to blastocysts in G+0 group were significantly lower than other groups. In experiment 3, we determined the effects of glucose exposure from 8 to 20 h after insemination on the cleavage and development of oocytes. The oocytes in glucose added group had high capacity of cleavage and further development. This study shows that in bovine oocytes, the optimal exposure to sperm is 30 h and glucose exposure to bovine one-cell embryos is detrimental to their first cleavage and further development in vitro but there has no evidence of detrimental effect of glucose(1.5 mM) exposure to bovine embryos over the two-cell stage in vitro.

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.394-400
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• 1998

Cryopreservation of embryos by vitrification is a simple method to preserve bovine embryos for subsequent embryo transfer, but embryonic viability after vitrification has been inconsistent and low compared with conventional slow freezing. The aim of the present study is to examine the effect of serum or serum albumin in a vitrification solution and epidermal growth factor(EGF) or fibroblast growth factor(FGF) on in vitro viability of bovine blastocysts frozen by vitrification. Bovine blastocysts were produced by in vitro maturation, fertilization of follicular oocytes and culture of embryos in a synthetic oviduct fluid medium(SOFM) containing BSA and 19 essential and nonessential amino acids. Blastocysts with excellent or good morphology were selected at 7 or 8 days after culture and utilized for vitrification. In experiment 1, blastocysts were vitrified in a solution containing semi-fetal calf serum(SFCS) or BSA(5 or 10mg/ml) and then their subsequent viabilities were examined by culturing thawed embryos in a SOFM containing BSA and 19 amino acids. Effect of EGF or FGF added to a SOFM containing polyvinyl alcohol(PVA) on the viability of vitrified-thawed blastocysts was investigated in experiment 2. BSA added at 5 or 10mg/ml to a vitrification solution showed significantly higher(p < 0.05) developmental rate to expanded and hatching blastocysts than SFCS, but there was no significant difference in the developmental rate to hatched blastocysts after thawing. Supplementation of a culture medium with EGF and/or FGF significantly increased(p < 0.05) embryo development to expanded blastocysts compared with control but showed no beneficial effect on the development to hatching or hatched blastocysts. Coculture of thawed embryos with granulosa cells in a TCM 199 containing 10% fetal calf serum(FCS) showed the highest developmental rate to expanded, hatching and hatched blastocysts among the groups tested. In conclusion, supplementation of a vitrification solution with BSA at 5mg/ml and culture of thawed blastocysts in a medium containing EGF and/or FGF can improve in vitro viability of bovine blastocysts frozen by vitrification.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.84-84
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• 2002

The advantages of the OPS techniques(Vajta G et al, Mol Reprod Dev 51: 53-58,1998) give 1) high survival rates of various types of eggs, 2) quick and simple process, 3) inexpensive equipment and reduced chilling injury. The efficiency of IVM/IVF technique in the porcine species is relatively lower than that obtained in other species such as ruminants. Two experiments were designed to investigate the effects of in-vitro fertilization of porcine oocytes matures using different OPS protocol for chilling and warming of vitrification. Porcine oocytes from ovaries collected at abattoir were matured for 44 hours in TCM199 Earle's salt supplemental with pyruvate, pff, L-cysteine, hormones and gentamycin. Oocytes were denuded and fertilized with frozen boar semen by common method. Porcine embryos produced routinely by in-vitro culture system of NCSU23 medium. The vitrification and the warming were conducted by OPS method with the glass micropipette instead of straw vessels and modified the protocol of G.Vajta(1999). In Exp 1, Chilling/Warming:Holding Medium(HM)+EG+DMSO/HM +sucrose Medium(SM) at 39$^{\circ}C$ warm stage. In Exp 2, : PBS+CS+EG+Ficoll+ Trehalose/PBS+Trehalose at 25$^{\circ}C$ stage. Filling, freezing, packing, thawing out and further culturing were performed to follow the basic protocol of G Vajta. During IVM-lVC and post-warming, fertilization parameter and developmental potential were compared to and statistically analysed. It was not significantly different from Exp 1 and Exp 2 but 25$^{\circ}C$ of stage was slightly higher on the morula/blastocyst forming rate and better atmosphere for worker than that at 39$^{\circ}C$ stage.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.1-6
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• 1994

This experiment was investigated the effect of presence of granulosa cells from follicles of different size on bovine oocyte maturation, cleavage and development to late stage. The nuclear and cytoplasmic maturation of oocytes in the IVM-IVF system are critical for subsequent embryo development. Granulosa cells when the co-cultured with oocytes may interact with cumulus-oocytes complexes and influence the development competence of the oocytes. Granulosa cells from medium (2~6 mm) and large(>1O mm) size follicles were recovered by aspiration, washed 3 times by centrifugation at 500 x g for 5 min. and used for co-culture at a concentration of 2~3 x 106 cells/mi. The oocytes were matured in vitro (IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g/ml FSH, 10 $\mu$g/ml LH, 1 $\mu$g/ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro (IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro (I VC) with bovine oviductal epithelial cells for 7 to 9 days. The assessment of maturation revealed that Grade J oocytes showed significantly(P

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.67-67
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• 2003

Early development of porcine oocytes fertilized in vitro was examined in different culture conditions. Porcine ovaries were collected from local slaughter-house. Cumulus-oocytes complexes were aspirated from 2 to 6 mm follicles. The collected oocytes were cultured for in vitro maturation in NCSU-23 medium with 5 mM hypotaurine, 0.57 mM cystein, 10% porcine follicle fluid, 10 IU/ml PMSG and 10 IU/ml hCG for 42~44 hrs. The frozen-thawed spermatozoa were washed by centrifigation 2 times at 1, 500 rpm in D-PBS with 5.56 mM glucose, 0.33 mM Na-pyruvate, 100 IU/ml penicillin, 1$\mu\textrm{g}$/ml streptomycin and 1ng/ml BSA. The fertilization medium used mTBM with 2 mM caffeine and 2 mg/ml BSA and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to 2.5$\times$10$^{6}$ cells/ml motile sperm during fertilization in vitro. At 8hrs h after insemination, the oocytes were transferred into NCSU-23 medium with 5.0 mM hypotaurine and 4 mg/ml BSA and cultured for 7 days. In first experiment, the mean numbers of oocytes collected from 20 ovaries were 674.4 oocytes, and 4.1(27.6), 12.5(84.0), 25.4(171.6) and 57.9%(390.8) for A, B, C and D grade in morphological classification. In the second experiment, when culture medium was supplemented with various concentrations of EGF, the proportions of oocytes cleaved were 56.9, 55.7, 61.9 and 54.7% for 0, 5, 10 and 20ng/ml EGF. The higher proportions(15.1%) of oocytes developed to morular stage were obtained at concentration of 10ng/ml than 0 and 5ng/ml EGF (P<0.05). However, the proportions of embryos developed to blastocyst stage were not significantly different among concentrations of EGF. In another experiment, when the medium supplemented with catalase was used, the proportions of oocytes cleaved were higher in the concentration of 0 unit (56.5%, 61/108) than 100 and 1, 000 unit/ml of catalase (P<0.05). Although the developmental capacity of embryos was improved by medium with 0 unit/ml compared with 100, 500 and 1, 000 units/ml of catalase in oocytes developed to morula and blastocyst stages, were not significantly different among concentrations of catalase.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.101-107
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• 2006

The objective of this study was to investigative the effects of retinol supplement to IVM and/or IVC medium on maturation, fertilization and development of pig oocytes. North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (pFF) was used as base medium. Each 1 uM, 5 uM and 10 uM concentration of retinol was supplemented to IVM and /or IVC medium. When the retinol was supplemented to maturation medium, the maturation rates were not different (p>0.05) among treatment groups ($66.7{\pm}6.0{\sim}69.2{\pm}5.3%$), but the developmental rate to blastocyst stage was higher (p<0.05) in $5{\mu}M$ group ($20.4{\pm}2.6%$) than in 0 uM ($13.6{\pm}2.1%$) and 10 uM groups ($9.7{\pm}1.7%$). Moreover, total cell number was significantly greater (p<0.05) in the 5 uM group ($37.0{\pm}1.6$) than in the other groups ($29.8{\pm}1.0{\sim}33.2{\pm}1.0$). Retinol supplement to maturation medium did not significantly affect the rates of fertilization and polyspermy (p>0.05). When the retinol was supplemented to culture medium or both maturation and culture medium, the rates of cleavage, and develop to morula and blastocyst stage were not affected, while those of 10 uM group were significantly decreased (p<0.05). These results indicate that 5 uM retinol supplement in maturation medium significantly stimulates embryo development, also improves the total cell number of blastocyst stage in pig.