Microstructural examination of the Nb-Si-B alloys at Nb-rich compositions is performed. The Nb-rich corner of the Nb-Si-B system is favorable in that the constituent phases are Nb (ductile and tough phase with high melting temperature) and T2 phase (very hard intermetallic compound with favorable oxidation resistance) which are good combination for high temperature structural materials. The samples containing compositions near Nb-rich corner of the Nb- Si-B ternary system are prepared by spark plasma sintering (SPS) process using T2 and Nb powders. T2 bulk phase is made in arc furnace by melting the Nb slug and the Si-B powder compact. The T2 bulk phase was subsequently ballmilled to powders. SPS is performed at 1300oC and 1400oC, depending on the composition, under 30 MPa for 600s, to produce disc-shaped specimen with 15 mm in diameter and 3 mm high. Hardness tests (Rockwell A-scale and micro Vickers) are carried out to estimate the mechanical property.

The nature of molecular mechanisms governing embryonic cell block is largely unknown, but recent reports have demonstrated that proper execution of programmed cell death is crucial for this process. The main objective of this study is to determine effects of programmed cell death on porcine oocytes development in vitro after parthenogenesis. Among the blastocysts matured in 3MA, MAP1LC3A and ATG5 RNA gene expression level increased in the order of Cyst < 3MA < RP. However, Casp-3 and TNF-r RNA gene expression level decreased in the order of RP < 3MA < Cyst. Expression of mTOR within the RP-cultured blastocyst was the most highly to the inner cell mass, while 3MA-cultured blastocyst showed very lowest expression in inner cell mass. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in Cyst group. When the enzymatic activity of MMP-2 and MMP-9 was assessed in culture, the level of active MMP-9 was higher expression in the medium of each RP treatment group, with the level of another treatment group being relatively higher. Analyses of TIMP-2 and TIMP-3 revealed that their expression was higher in groups that did not receive RP treatment. More specifically, the level of TIMP-2 was not affected by Cyst treatment, while the level of TIMP-3 was higher in 3MA and RP treatment group. There was highly cell division activation efficiency of parthenogenesis on cultured system of RP supplement IVC medium. Therefore, these results suggest that embryo development was significantly increased in conditional culture medium with active autophagy as compared to common cultured condition. Further investigation of this distinction may enable the development of innovative improvements for the production of porcine somatic cell nuclear transfer.

The main purpose of this study is to estimate the effect of adding Tea-N-Tris (TES) to the freezing buffer for miniature pig sperm. In particular, we attempted to identify the association between the MMPs expression and the fertility and viability of frozen sperm from each extender (LEY (Lactose Egg-Yolk), TLE (TES + LEY), TFGE (TES + Fructose + Glucose Egg-Yolk)). In accordance with this, Hypoosmotic Swelling Test (HOST) respond test was the lowest among sperms frozen in LEY while the highest HOST respond was observed among sperms frozen in TLE. Furthermore, we observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of MMP-9 and MMP-2 was the highest in sperms frozen in LEY, Meanwhile, sperms from the TFGE and TLE group showed lower level of MMP-9 and MMP-2 expression in the order of TLE being the lowest. LEY group showed lower rate of blastocyst development than the TES supplement group, although the difference was not statistically significant. Meanwhile the rate of blastocyst development appeared similar when sperms from TLE and TFGE group were used for IVF. Together, these results indicate that adding Tea-N-Tris to the sperm freezing buffer only suppresses MMPs protein activation but also maximize in-vitro fertility, providing a means to improve the success rate in the in vitro manipulation of miniature pig sperm.

To describe the macroscopic anatomy and ovarian-physiological difference of the genital organs of the female Korean water deer, organs from captured animals in a wild area of Korea were dissected. The ovary of estrus group was about 1.10 ± 0.02 mm long and weighed about 0.50 ± 0.02 g. And pregnant group was about 1.3 ± 0.10 mm long and weighed about 0.40 ± 0.05 g. And the crowns of corpora lutea were found in the estrus group, but we couldn't find crowns at the pregnant group. Especially, the estrus ovaries tended (p=0.04) to be heavier than the ovaries during pregnancy. The MMP-9 activity was higher at the Graafian follicles of pregnant group than that in estrus group. However, with regard to follicles of estrus group, MMP-2 level was higher than that in pregnant group. Furthermore, apoptosis detection marker (Casp-3) was highly expressed in Graafian follicle of the pregnant group and the corpora lutea of estrus group. Thus, the differential expression of MMPs observed in this study suggests that the reflected the mechanisms underlying of monovulatory in estrus and/or pregnancy. Our results may be very useful as it provides with information that may be considered for the development of reproductive biotechnologies in endangered animals.

Bovine coat color is decided by the melanocortin receptor 1 (MC1R) genotype mutation and melanogenesis. Specially, in the various cattle breeds, dominant black coat color is expressed by dominant genotype of ED, red or brown is expressed in the frame shift mutation of recessive homozygous e by base pair deletion and wild type of E+ is expressed in various coat colors. However, not very well known about the effected of MC1R genotype mutation on the coat color through family lines in KBC. Therefore, this study were to investigate effect of MC1R genotype mutation on the coat color, and to suggest mating breed system in accordance with of MC1R genotype for increased on brindle coat color appearance. Parents (sire 2 heads and dam 3 heads) and offspring (total : 54 heads) from crossbreeding in KBC family line with the MC1R genotype and phenotype records were selected as experimental animals. The relationship between melanocortin 1 receptor (MC1R) genotypes expression verified by PCR-RFLP, and brindle coat color appearance to the family line of the cross mating breed from MC1R genotype pattern was determined. As a result, 4MC1R genetic variations, E+/E+ (sire 1), E+/e (sire 2 and dam 3), E+/e with 4 bands of 174, 207 and 328 bp (dam 1) and E+/e with 3 bands of 174, 207, 328 and 535 bp (dam 2) from parents (sire and dam) of KBC. However, 3 genetic variations, e/e (24%), E+/E+ (22%) and E+/e (56%) were identified in offspring. Also, brindle coat color expressrated was the e/e with the 0%, E+/E+ with 67% and E+/e with 77% from MC1R genotype in offspring on the cross mating of KBC. Furthermore, when the sire had E+/e genotype and the dam had E+/E+ with the 3 bands or E+/e genotype, and both had whole body-brindle coat color, 62% of the offspring had whole body-brindle coat color. Therefore, the seresults, the mating system from MC1R genotype patterns of the sires (E+/e) and dams (E+/E+ with the 3 bands or E+/e) with brindle coat color may have the highest whole body-brindle coat color expression in their offspring.

The present study was conducted to compare on embryo survival rates by blastomere isolation methods, and establish the optimal PCR procedure for perform the sexing of bovine blastocysts produced by IVF. IVF embryos used in the study was used the Bisected or Sliced methods for blastomere isolation, and the survival rates of blastocyst with rapid way of sexing PCR was assessed. In the present study for survival rates in blastocyst was the total cleavage rate was 75% and a blastocyst development among cleaved embryos was 40%. Survival rate of embryos treated with intact, bisected or sliced method was 100, 63.3 or 81.3%, respectively. Therefore, survival rate of embryos treated with sliced method was higher compared to that of embryos treated with bisected method. The sexing rate of female or male was not significantly different between S4BFBR primer and BSY + BSP primer (1.75 : 1 vs. 1.43 : 1), respectively. Because of the PCR amplification using the S4BFBR primer was simpler method than multiplex PCR amplification method. Furthermore, the accuracy of sexing rate and reduction of PCR work time between 2-step and 3-step of PCR methods was 98.0% / 1.5 hr and 97.0% / 3.5 hr, respectively. Based on these results, it can be suggested that the sliced and PCR methods we developed was very effective method to reduce time consuming and procedure of PCR amplification for sexing with the increase of survival rate on the blastocyst.

The main purpose of this study is to estimate the effect of adding Tea-N-Tris to the freezing buffer for miniaturepig sperm. In particular, we attempted to identify the association between the MMPs expression and the survival and viability of sperms. Prior to freezing, sperms in LEY without Tea-N-Tris showed 40.3±2.8% viability and 60.3±1.3% acrosome intact rate at 4℃. After freezing, sperms stored in LEY (lactose+Egg yolk) with Tea-N-Tris (=TLE) showed the highest viability (57.4±1.8%) and acrosome intact rate (65.6±4.6%). In accordance with this, DNA fragmentation was the highest among sperms frozen in LEY while the lowest fragmentation was observed among sperms frozen in TLE. When these sperms were used for in vitro fertilization (IVF), the LEY group showed lower rate of blastocyst development, although the difference was not statistically significant. Meanwhile the rate of blastocyst development appeared similar when sperms from TLE and TFGE (Tea-N-Tris+Fructose+ Glucose+Egg yolk) group were used for IVF. We observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of MMP-9 and MMP-2 was the highest in sperms frozen in LEY, Meanwhile, sperms from the TFGE and TLE group showed lower level of MMP-9 and MMP-2 expression in the order of TLE being the lowest. Together, these results indicate that adding Tea-N-Tris to the sperm freezing buffer would not only suppress MMPs expression but also minimize DNA fragmentation, providing a mean to improve the success rate in the in vitro manipulation of miniaturepig sperms. * This work was supported by BioGreen 21 Program (No. PJ008029). Rural Development Administation, Republic of Korea.

The purpose of this thesis is to examine the effect of hormone treatment in blastocyst development of in vitro cultured porcine oocyte. Oocytes used in the study was matured in vitro in the presence of 10% FBS or 10% pFF, and treated with FSH, LH or FSH+ LH, and the rate of blastocyst development was assessed based on the expression of autophagic genes. There was no significant differences in blastocyst development between oocytes maturaed in 10% FBS or 10% pFF. In vitro matured oocytes treated with FSH+LH showed blastocyst development rate as high as that of untreated oocytes, while groups treated with LH only showed a decrease in blastocyst development. About the expression of cell death assosiated factors, mRNA levels of autophagy and apoptosis genes were increased in oocytes matured in 10% FBS and treated with LH. Oocytes that did not receive hormone treatment showed low expression of most cell death genes except ATG5. When oocytes were matured in 10% pFF, ATG5 expression was the highest in FSH treated group, while LC3 showed strong expression in all hormone treated groups. On the other hand, the expression level of mTOR and caspase-3 did not show significant differences between groups. We also examined the protein level of apoptotic genes in the blastocyst. The amount of caspase-3 protein was similar between groups matured in 10% FBS and 10% pFF, but was the highest when treated with LH. Blastocysts treated with FSH and FSH+LH showed similar level of caspase-3 protein, while the level was the lowest when hormone treatment was not given. Within the blastocyst, caspase-3 was mostly expressed in trophoblast cells when matured in 10% FBS, while maturation in 10% pFF caused expression of this protein in the inner cell mass (ICM). Expression of MAP1LC3A was higher in groups matured in 10% pFF than groups matured in 10% FBS in all types of hormone treatment. Among the blastocysts matured in 10% pFF, MAP1LC3A level increased in the order of untreated < FSH < FSH+ LH. Expression of MAP1LC3A within the FBS-matured blastocyst was concentrated to the trophoblast, while pFF-matured blastocyst showed expression in both trophoblast and ICM. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in 10% FBS group without hormone treatment. In both FBS and pFF group, and in all three combinations of hormone treatment, mTOR expression was ovserved mostly in ICM. Together, these results indicated that hormone treatments tend to induce expression of genes associated with programmed cell death. We suggest that proper induction of programmed cell death by FSH and LH treatment would increase the rate of blastocyst development. * This work was supported by BioGreen 21 Program (No. PJ008029). Rural Development Administation, Republic of Korea.

The objective of this study was to evaluate the effect of Tea-N-tris medium on the sperm viability and acrosomal morphology for semen of normal and miniaure pig by type of freezing extender. The present study was to determine of Tea-N-tris (0.02 g/ml) effect to freezing extender LEY(Lactose 11% + Egg yolk 20%) and FGE(Fructose 3%+Glucose 7%+Egg yolk 20%) for the spermatozoa viability, acrosomal morphology and DNA fragmented analysis from normal and miniature pig semen, were evaluated freezing extender TFGE, TLE and LEY during thawing at 37℃ for 45 sec and 75℃ for 5 sec, respectively. Interestingly, the result that sperm after addition of Tea-N-tris extender(TFGE, TLE) during 15 4℃ cooling significantly increased the viability(p<0.05), as compared to than of sperm cooling in LEY extender, but lower the percentage of AR(acrosome reacted spermatozoa) pattern than LEY extender. The sperm viability and AR pattern after freezing was appeared like sperm cooling method pattern. And treatment spermatozoa during freezing after addition of Tea-N-tris extender significantly (p<0.05) increased the viability and AR to miniature pig sperm, than normal pig sperm, but most highly percentage of viability and AR pattern to normal pig sperm during freezing in LEY extender. Chromosomal DNA fragmentation increased from LEY extender to sperm of normal and miniature pig, but decreased from the Tea-N-tris extender. Therefore, suggest that Tea-N-tris freezing extender method for freezing of miniature pig sperm is required for increasing viability. This Study was supported by Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

This study was conducted to detect the specific fragment genes by using RAPD-PCR and RFLP method in the Korean Tiger cattle and Korean Native cow. And then, the specific fragment gene was investigated by the analysis of the genes for detection significance according to the expressing pattern. We found the specific expression gene by the RAPD-PCR analysis in Korean Tiger cattle. It were a detected the differences of the species in the colour and external section. The Korean Tiger cattle were vary low compare to the Korean Native cattle by analysis result of polymorphism and distribution. And the polymorphism over 500bp into the size classification detected was highly pattern and it could be utilize by the resource of the specific gene. There was a found the specific gene by sequencing in the 1855bp gene fragment of Korean Tiger cattle. And the sequencing result of the R9B was different between Korean Native cow and Holstein cattle. Thus, this gene can be apply as the specific gene in the Korean Tiger cattle.

In this study, we analyzed expression patterns of apoptotic and autophagic gene products in culture follicular cells of normal and miniature pigs to assess the effect of hormones on the choice for programmed cell death. Autophagic activity progressively increased from control cultures to luteinizing hormone (LH)-treated cultures of follicular cells of normal pigs, but decreased from the LH to follicle stimulating hormone (FHS) +LH-treated cultures. Expression of Casp-3 protein in follicular cells was highest in LHtreated cultures, but the activity of Casp-3 decreased in the control, FSH-treated, and FSH+LH-treated cultures. The activity of the apoptosis protein was highly expressed in the control, LH-treated, and FSH+LH-treated follicular cells of miniature pigs, but autophagy- associated proteins were expressed at low levels in all treatments groups of the miniature pig. The expression of autophagy and apoptosis proteins appeared similar in control and rapamycin-treated cells. In addition, stimulation with FSH triggered the activation of autophagy in the follicular cells of normal pigs, but induced apoptosis in the follicular cells of miniature pigs. A similar effect was obtained when LH was applied. These results suggest that the autophagy process and FSH stimulation is more effective for stable and innovative follicular cell development.

Matrix metalloproteinases (MMP) play important roles in extracellular matrix (ECM) remodeling during ovarian follicular development, oocytes development and ovulation. In an attempt to investigate the effect of MMP activation in development cumulus-oocytes complexes, we examined the localization and expression of MMP, and monitored MMP expression profile. Cumulus-oocytes complexes were collected and matured in vitro for 24 hr, 36 hr and 48 hr. A mRNA expression of MMP-2, MMP-9, TIMP-2 and TIMP-3 was detected in all culture medium regardless of CC, OC and COCs. Activity of MMP-2 in the OC progressively was increased from 24 hr to 48 hr. But MMP-9 was not detected in all culture medium. The localization of MMP-2 was also measured by immunohistochemistry analysis. The MMP-2 and TIMP-2 was detected in cumulus cell and oocyte zone pellucida. Expression of MMP-2 protein in the COCs was progressively increased from 24 hr to 48 hr. However, MMP-9 protein was progressively decreased from 24 hr to 48 hr. And TIMP-2 protein was most highly expressed in the COCs 36 hr. Expression of TIMP-3 protein in the COCs was progressively increased from 24 hr to 48 hr. In conclusion, these results suggest that MMP-2 plays a role in maintaining normal maturation and development by controlling the ECM inhibitor concentration on cumulus cell and oocytes.

This study was conducted to investigate the specific expression genes in the cloned bovine tissues. Donor cells, cloned tissues were analysed by RAPD-RFLP method. The results were detected three genes (CH-U7B, CH-U7M and CH-U7P) in the cloned fetus. It was found a single copy genes by southern hybridization. Sequence analysis of CH-U7M gene was shown 99% homology to a previously reported EST from a cloned bovine fetus. The putative ORF was encode a protein of hydrophobicity index 0.03. Semi-quantitative RT-PCR by using the CH-LS001 specific primer was remarkably detected in the lung tissue of cloned fetus. Further investigation of these genes may provide one of the key information to explain the early death, abnormal fetus, large off-spring and the low pregnancy rate in the production of cloned bovine.

Pregnancy-associated plasma protein-A (PAPP-A) is a 200 kDa metalloprotease identified as an IGFBP-4 protease and likely an important regulator of IGF bioavailability. PAPPA mRNA is detected in bovine granulosa and theca cells and the PAPP-A protein is also found in follicular fluid. Proteolytic activity supposed to be due to the PAPP-A in bovine follicular fluid is induced by follicle stimulating hormone (FSH) treatment and PAPP-A-like activity appears concomitantly with increased E2 during follicular development. However, the effects of FSH, E2 and progesterone on the expression of PAPP-A in bovine corpus luteum of pregnancy have been evaluated in a limited number of studies. This study was performed to expression of PAPP-A and progesterone during early pregnancy in bovine corpus luteum. To determine the function of PAPP-A gene during early pregnancy, we collected the bovine pregnancy corpus luteum samples on 30, 60 and 90 day of pregnancy. The mRNA expression of PAPP-A, progesterone-receptor, VEGF and IGFBP4 gene was conducted by real-time PCR. And proteins expression of PAPP-A and progesterone antibody was detected by Immunohistochemistry and ELISA. The VEGF and PAPP-A mRNA expression was progressively increased on day 90 in the pregnancy corpus luteum. The mRNA expression of progesterone-receptor, IGFBP4 in the corpus luteum progressively was increased from 30 to 60 day, but decreased on 90 day of pregnancy. The proteins expressions of progesterone and PAPP-A were similar pattern in mRNA expression. Our results indicate that the IGFBP4 protease role of PAPP-A increases in response to pregnancy 90 day corpus luteum and suggest that progesterone is an connected for the expression of PAPP-A genes in luteal cells. Therefore, we suggest that PAPP-A stimulated with progesterone play a crucial role for IGF-I system in bovine early pregnancy. And could be used to predict the condition of normal pregnancy.