• Journal of Embryo Transfer
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• v.19 no.2
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• pp.133-145
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• 2004

Serum-contain is commoly used for the production of in vitro-derived bovine embryos. However, were biological activity of serum varies from lot to lot, time consuming to choose better serum with good quality and risks of virus, bacteria and mycoplasma infection. This study established serum-free culture systems of in vitro embryo development to efficiently obtain a large number of blastocysts from ovaries of Hanwoo and oocytes maturation, cell number, tlerance of cryopreservation. Secondly, serum-contain medium is suspected of contributing to the large calf size, dystocia, cersarean sections, calf mortality and confirmed these blastocysts are high quality in terms of cyotolerance, high rates of pregancy and normal birth. For these reasons, Culture media (IVMD101 and IVD101) designed specifically for the preimplantation bovine embryo are rather simplistic, being based on salt solutions with additional energy substrates and growth factors. An improved serum-free medium (IVMD101) was developed for bovine oocytes maturation in vitro. Proportions of embryos developing to the blastocyst stage cultured in both IVD101(32.4%) and IVD101(34.5%) serum-free media were higher than in TCM199+10% FBS(12.4%) serumcontaining medium. Futhermore, the cell numbers per blastosyst obtained in the serum-free media were superior to those of blastocysts developed in serum-supplemented medium. Also, cell numbers of blastocysts obtained in the serum-free media were similar with blastocysts derived in vivo. Survival rate blastocysts after 24 hr incubation after thawing, the blastocysts cultured in both IVD101(94.5%) and IVD101(95.8%) serum-free media were higher than in TCM199+10% FBS (52.5%) serum-containing medium. After 72 hr incubation after thawing, hatching rates of blastocysts developed in IVD101(78.4%) and IVMD101(83.7%) were sighnificantly higher than that developed in the serum-supplemented medium(32.0%). The pregnancy rates almost not different between fresh blastocysts(38.2%) and frozen blastocysts(34.9%). The results suggested that the improved serum-free media(IVMD101 and IVD101) offer several advantages over culture in serum-cotaining medium, including increased rates of blastocyst formation and high cel numbers. Additionally, the survival and hatching rates of embryos product in serum-free media after post-thaw culture were superior to those of embryos produced in the serum-containg medium and useful for the production of high quality bovine embryos for cryo-preservation. These improved serum-free media are beneficial not only for the study of the mechanisms of early embryogenesis but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.245-252
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• 2002

This study was to evaluate the in vitro survival of bovine enucleated MII (eMII) oocytes according to minimum volume cooling (MVC) freezing method and activation timing, and their in vitro development after somatic cell nuclear transfer (SONT). in vitro matured bovine oocytes for 20 h were stained with 5 $\mu\textrm{g}$/$m\ell$ Hoechst, and their 1st polar body and MII plate were removed by enucleation micropipette under UV filter. Also, eMII oocytes were subjected to activation after (group II) and before (group III) vitrification in 5 ${\mu}{\textrm}{m}$ ionomycin added CRlaa medium for 5 min. For vitrification, eMll oocytes were pretreated with EG10 for 5 min, exposed to EG30 for 30 sec and then directly plunged into L$N_2$. Thawing was taken by 4-step procedures at 37$^{\circ}C$. Survived eMII oocytes were subjected to SONT with cultured adult bovine ear cells. Reconstructed oocytes were cultured in 10 $\mu\textrm{g}$/$m\ell$ of cycloheximide and 2.5 $\mu\textrm{g}$/$m\ell$ of cytochalasin D added CRlaa medium for 1 h, and then in 10 $\mu\textrm{g}$/$m\ell$ of cycloheximide added CRlaa medium for 4 h. Subsequently, the reconstructed oocytes were incubated for 2 days and cleaved embryos were further cultured on cumulus-cell monolayer drop in CRlaa medium for 6 days. Survival rates of bovine vitrified-thawed eMII oocytes in group II (activation after vitrification and thawing) and III (activation before vitrification) were 81.0% and 84.9%, respectively. Fusion rates of cytoplasts and oocytes in group II and III were 69.0% and 70.0%, respectively, and their results were not different with non-frozen NT group (control, 75.2%). Although their cleaved rates (53.4% and 58.4%) were not different, cytoplasmic fragment rate in group II (32.8%) was significantly higher than that in group III (15.6%)(P<0.05). Also, subsequent development rate into >morula in group II (8.6%) was low than that in group III(15.6%). However, in vitro development rate in group III was not different with that in control (24.8%). This result suggested that MVC method was appropriate freezing method for the bovine eMII oocytes and vitrified eMII oocytes after pre-activation could support in vitro embryonic development after SONT as equally well as fresh oocytes.

• Journal of Chest Surgery
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• v.38 no.1 s.246
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• pp.38-43
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• 2005

Matrix metalloproteinase-2 (MMP-2) is a class of proteolytic enzymes that digest collagen type IV and other components of the basement membrane. It plays a key role in the local invasion and the formation of distant metastases by various malignant tumors. The aim of this study was to evaluate the activity of MMP-2 and its significance as a prognostic marker in resected stage I non-small cell lung cancer (NSCLC). Material and Method: In this study we obtained fresh-frozen samples of tumor and non-tumor tissues from 34 patients with stage I NSCLC who underwent resection without preoperative radiotherapy or chemotherapy. After the extraction of total protein from tissue samples, MMP-2 activities were assessed by gelatin-substrate-zymography. The activities were divided into the higher or lower groups. Result: The MMP-2 activities were higher in tumor tissues than in non-tumor tissues. The MMP-2 activity of non-tumor tissues in recurrent group was higher than in non-recurrent group (p<0.01). Also the patients with higher MMP-2 activity of non-tumor tissues showed poor 5 year survival (p<0.01). Conclusion: This result indicates that the higher level of MMP-2 activity in the non-tumor tissue is associated with the recurrence and survival after the resection of stage I NSCLC. Therefore, MMP-2 activity in the non-tumor tissue could be used as a potential prognostic marker for the resected stage I-NSCLC.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.10 no.4
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• pp.189-197
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• 1977

In present study, the effects of various factors including the solvent concentration, extraction time and temperature, the ratio of sample vs extraction solvent, (w/v) and pH upon the extractability of the NaCl and alcohol soluble proteins of marine algae were investigated. Eight species of fresh algae, the major ones in consumption as food, namely Porphyra suborbiculata, Undarie pinnatifida, Hizikia fusiforme, Sargassum, fulvellum, Enteromorpha linza, Sargassum kjellmanianum, Codium coarctatum, and Ulva pertusa were used for the extraction of NaCl soluble protein and dried materials of four species, Perphyra suborbiculata, Undaria pinnatifida, Enteromorpha linza and Sargassum fulvellum were used for the extraction of alcohol soluble protein. The frozen and mascerated samples were prepared by the same method described in previous paper (Ryu, 1977). And the dried materials were moistened with alcohol solution before freezing. The effect of solvent concentration on the extractability of NaCl soluble protein differed from species. The extractability of Undaria Pinnatifide, Hizikia fusiforme, Perphyra suborbiculata, Enteromorpha linza, and Ulva pertusa reached maxima at 0.25M NaCl solution while the 1.0M for Sargassum fulvellum, Saygassum kjellmanianum and Codium coarctatum. In case of alcohol soluble proteins, it was shown at $20\%$ ethanol solution for Porphyra suborbiculata, Undaria pinnatifida, Enteromorpha linza, and Sargassum fulvellum. Variation of the ratio of sample vs solvent gave slight effect upon the extractability, but the ratio of 1:30(w/v) seemed most efficient for the extraction of NaCl soluble proteins and 100 ml solvent added to 1 g dried sample was effective in case of alcohol soluble proteins. Extraction time has a minimal effect upon the extraction of alcohol soluble protein, and approximately 21 to $43\%$ of algal protein was extracted within 1 hour. But in case of NaCl soluble protein extraction, the effect of time revealed differently from species to species resulting in that the extraction for 1 hour gave a maximum extractability in Ulva pertusa and Enteromorpha linza, 2 hours in Porphyra suborbiculata, Codium coarctatum and 3 hours in Undaria pinnatifica, Hizikia susiforme, Sargassum fulvellum and Sargassum kjellmanianum. When the NaCl soluble protein of Undaria pinnatifida and Enteromopha linza was extracted at various temperature, the most effective extraction temperature was $40^{\circ}C$ while the temperature was $50^{\circ}C$ for Undaria pinnatifida and $60^{\circ}C$ for Hixikia fusiforme, Sargassum fulvellum, Sargassum kjellmanianum and Codium coarctatum. Bus in case of alcohol soluble extraction, the optimum temperature was $30^{\circ}C$ for Enteromorpha linza and $40^{\circ}C$ for Undaria pinnatifida, Sargassum fulvellum and Porphyra suborbiculata. In the effect of pH on extractability, the maximum extractability of NaCl soluble proteins was obtained at pH 7to 8 and pH 8 to 9 for alcohol soluble protein.

• Applied Biological Chemistry
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• v.13 no.3
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• pp.207-218
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• 1970

Studies were carried out to develope the most economical and practical methods of packaging and preservation of kimchi, so commercialization of kimchi manufacture could proceed rapidly. The results obtained may be summarized as following. (1) It is generally established that the acceptable range of lactic acid content of kimchi is between 0.4% and 0.75%. Based on sensory evaluation, kimchi having lactic acid content below 0.4% and above 0.75% was not edible, and the time of optimum taste corresponded to the vicinity of 0.5% of lactic acid content. For the refrigeration storage with or without preservatives, the packaging kimchi in plastic film must be done at the lactic acid content of 0.45%, for lactic acid fermentation will continue slowly after the packaging. However, for the heat sterilized kimchi the packaging should be done at the 0.5% of lactic acid content for the best because lactic acid fermentation is completely stopped after the packaging. (2) Polyethylene, polypropylene, and polycello were chosen as suitable packaging materials. Polyethylene is cheapest among them but kimchi packaged in this film was damaged frequently in handling process and gave off kimchi flavor. On the other hand polypropylene also gave off kimchi flavor, but its higher mechanical strength gave better protection to kimchi and it had superior display effect due to the transparancy. Therefore polypropylene made much better packaging material. Polycello proved to be the best packaging material from the standpoint of physical characteristics but its price is higher than that of other plastic films. To be effective, the thickness of plastic films for packaging kimchi must exceed 0.08mm. (3) Keeping property of kimchi appeared to be excellent by means of freezing. However, by the time the frozen kimchi was thawed out at room temperature, moisture loss due to drip was extensive, rendering the kimchi too stringy. (4) Preservation of kimchi at refrigerated temperatures proved to be the best method and under the refrigerated condition the kimchi remained fresh as long as 3 months. The best results were obtained when kimchi was held at $0^{\circ}C$. (5) In general, preservatives alone were not too elective in preserving kimchi. Among them potassium sorbate appeared to be most effective with the four fold extension of self-life at $20^{\circ}C$ and two fold extension at $30^{\circ}C$. (6) In heat sterilization the thickness of packaged kimchi product had a geat effect upon the rate of heat penetration. When the thickness ranged from 1.5 to 1.8cm, the kimchi in such package could be sterilized at $65^{\circ}C$ for 20 minutes. Kimchi so heat treated could be kept at room temperature as long as one month without apparent changes in quality. (7) Among combination methods, preservation at refrigerated and heat sterilization could be favorably combined. When kimchi was stored at $4^{\circ}C$ after being sterilized at $65^{\circ}C$ for 20 minutes, it was possible to preserve the kimchi for more than 4 months.

• Korean journal of food and cookery science
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• v.20 no.5
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• pp.436-443
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• 2004

The purpose of this study was to examine the physico-chemical and sensory characteristics of Kakdugi made with mashed red pepper. With regard to the pH of the Kakdugi, those of the juice from Kakdugi with red pepper powder and of the liquid with mashed red pepper were the highest and lowest immediately after preparation, respectively, but thereafter both slightly decreased, but were similar after the fifth week. Generally, the total acidity of Kakdugi liquid was the higher than that of Kakdugi juice. With regard to the L value, that of the Kakdugi juice was higher than that of Kakdugi liquid and that of Kakdugi with mashed red pepper washigher than that of Kakdugi with red pepper powder. From the third week, the 'L' values of all samples generally decreased. The 'a' value of the Kakdugi liquid with mashed red pepper during fermentation was highest During early fermentation, the juice of Kakdugi with red pepper powder showed a higher value than that of Kakdugi with mashed red pepper, but conversely, from the second week that of Kakdugi with mashed red pepper was higher than that of Kakdugi with red pepper powder. The 'b' value of the juice from Kakdugi with red pepper powder was highest until the second week, but from the third week that of Kakdugi with mashed red pepper was highest. With respect to the organic acids contents, those of citric, quinic and malic acids decreased, but those of lactic and acetic acids increased during fermentation progression. In addition, the citric, lacticand malic acids contents of the Kakdugi with mashed red pepper werethe highest, whereas that of quinic acid of the Kakdugi with red pepper powder was the highest. From the forth week, the acetic acid content of the Kakdugi with mashed red pepper was further increased. As a result of the sensory test, Kakdugi with mashed red pepper showed significantly higher values with regard to redness and fresh flavor, but in overall acceptability in the QDA, appearance and taste in the acceptance test. Therefore, our results indicate that mashed red pepper particularly increased the 'a' value and organic acid contents of Kakdugi compared to those of red pepper powder, leading to an increased overall acceptability.

• 한국환경농학회:학술대회논문집
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• 2009.07a
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• pp.273-282
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• 2009

The transmissible spongiform encephalopathies (TSEs) disease group are fatal neurodegenerative disorders affecting a wide range of hosts. The group includes kuru and Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and goats and Bovine spongiform encephalopathy (BSE) in cattle. The exact nature of the infectious agent involved in the transmission of these diseases remains controversial. However, a central event in their pathogenesis is the accumulation in infected tissues of an abnormal form of a host-encoded protein, the prion protein (PrP). Whereas the normal cellular protein is fully sensitive to protease ($PrP^{sen}$), the disease-associated prion protein ($PrP^d$) is only partly degraded ($PrP^{res}$), its amino-terminal end being removed. BSE was first reported in the mid-80s in the UK. Ten years later, a new form of human prion disease, variant CJD (vCJD) developed in the wake of the BSE epidemic, and there is now strong scientific evidence that vCJD was initiated by the exposure of humans to BSE-infected tissues, thus indicating a zoonotic disease. However, the ban on the feeding of animal-derived proteins to ruminants, and the apparent lack of vertical transmission of BSE, have led to a decline in the incidence of the disease within cattle herd and therefore, an assumed decreased risk for human contacting vCJD. The origin of the original case(s) of BSE still remains an enigma even though three hypotheses have been raised. Hypotheses are i) sheep- or goat-derived scrapie-infected tissues included in meat and bone meal fed to cattle, ii) a previously undetected sporadic or genetic bovine TSE contaminating cattle feed or iii) originating from a human TSE through animal feed contaminated with human remains. A host cellular membrane protein ($PrP^C$), which is abundant in central nervous system tissue, appear to be conformationally altered in the diseased host into a prion protein ($PrP^{Sc}$). This $PrP^{Sc}$ is detergent insoluble and partially protease-resistant ($PrP^{res}$). The term $PrP^{res}$ is normally used to describe the protein detected after protease treatment, in techniques such as Western immunoblotting, and enzyme-linked immunosorbant assay using fresh/frozen tissue. Immunohistochemistry may performed with formalin-fixed tissues. Also, clinical signs of the BSE are one of the major diagnostic indicators. Recently, atypical forms (known as H- and L-type) of BSE have appeared in several European countries, Japan, Canada and the United States. An unusual case was also reported in a miniature zebu. The atypical BSE fall into two groups based on the relative molecular mass (Mm) of the unglycosylated $PrP^{res}$ band relative to that of classical BSE, one of the higher Mm (H-type) and the other lower (L-type). Both types have been detected worldwide as rare cases in older animals, at a low prevalence consistent with the possibility of sporadic forms of prion diseases in cattle. This raises the unwelcome possibility that vCJD could increase in the human population. Now, active surveillance program against BSE is going on in Korea. In regional veterinary service lab, ELISA is applied to screen the BSE in slaughter and confirmatory tests by Western immunoblotting and immunohistochemisty are carried out if there are positive or suspect in the screening test. Also, the ruminant feed ban is rigorously enforced. Removal of specified risk materials such as brain and spinal cord from cattle is mandatory process at slaughter to prevent the infected material from entering the human food chain.

• Journal of the Korean Arthroscopy Society
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• v.16 no.2
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• pp.107-113
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• 2012

Purpose: The purpose of this study was to evaluate the posterior cruciate ligament (PCL) reconstruction with single bundle, single-incision technique using Achilles tendon and tibialis anterior allograft with ligament remnant preservation. Materials and Methods: Twenty six patients underwent PCL reconstruction was included. There were 21 males and 5 females. Mean age was 32 years. Used graft was a fresh frozen Achilles tendon allograft (group I, 14 cases) and tibialis anterior allograft (group II, 12 cases). Arthroscopic PCL reconstruction was performed using transtibial, single-incision and single bundle technique with remnant preserving as possible. For clinical evaluation, range of motion, posterior drawer test, Lysholm score, Tegner activity scale, International Knee Documentation Committee (IKDC) grade and posterior stress radiograph were used. The mean follow-up period was 21.6 months (12-40 months). Associated injuries were 5 medial collateral ligament injuries, which were treated by conservative method. Results: Range of motion (ROM) was returned to normal range in 24 cases, but ROM deficit under $10^{\circ}$ flexion was 2 cases at final follow-up period. Preoperative posterior drawer test was 17 cases in grade II and 9 cases in grade III. At final follow-up 13 cases returned within normal grade, 7 cases grade I and 6 cases grade II posterior instability. Lysholm mean score was improved from preoperatively 62 to 90 at final follow-up period. Tegner activity mean scale improved from preoperatively 3.5 to 5.6 at final follow-up period. IDKC grade was grade A was 3 cases, grade B 17 cases, grade C 6 cases. In posterior stress radiograph, posterior displacement was improved from mean 12 mm preoperative to 4.5 mm at final follow-up. There were no statistical differences between two groups in clinical evaluations. There were two cases of re-rupture of graft at the bone-tendon junction in group I. Conclusion: We had successful results of PCL reconstruction with single-incision, single bundle technique using Achilles and tibialis anterior allograft without difference between two groups in patients with PCL injury. There were more re-rupture of graft in Achilles tendon group.

• Journal of Embryo Transfer
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• v.33 no.1
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• pp.17-22
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• 2018

This study was conducted to determine the effect of pentoxifylline levels on sperm motility, survival rate, sperm membrane integrity of frozen semen and fresh-extended equine semen in Jeju cross-bred horses. As a result of sperm characteristic comparison depending on pentoxifylline levles at 30 minutes post-thaw, the progressive motilities were $53.25{\pm}2.87$ (4mM pentoxifylline) and $50.28{\pm}2.14$ (8mM pentoxifylline) and significantly higher compared to the control group($40.09{\pm}5.15$) and other treatment group (16mM pentoxifylline, $41.27{\pm}2.82$). The progressive fast motility were $22.44{\pm}1.62$ (4mM pentoxifylline,) and $22.74{\pm}3.07$ (8mM pentoxifylline) and significantly higher compared to the control group ($13.47{\pm}1.48$) and other treatment group (16mM pentoxifylline, $14.66{\pm}3.68$) (p<0.05). As a result of sperm characteristic comparison depending on pentoxifylline levles at 30 minutes post-thaw were $68.96{\pm}1.64$ (4mM pentoxifylline) and $67.90{\pm}6.72$ (8mM pentoxifylline) and significantly higher compared to the control group ($53.48{\pm}4.84$) and other treatment group (16mM pentoxifylline, $58.14{\pm}2.65$) (p<0.05). In conclusion, these results suggest that treatment groups with 4mM and 8mM pentoxifylline were higher compare to equine seperm mobility and the control group and treatment groups with more than 16mM pentoxifylline has a negative effect on sperm characteristics. After thawing, the total motility in post-thawed equine sperm has increased by 10 percent for 1 hour. these results suggest that pentoxifylline contributes to the improvement of the equine sperm motility and characteristics in post-thawed semen.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.10 no.3
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• pp.151-162
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• 1977

Distribution of marine algae is diverse in Korea and the resource of edible algae is abundant marking 239,037 tons of yearly production in 1976. They have been known as a protein source and used as a supplement in Korean diet. It is necessary to estimate the potentiality and properties of usable algal proteins especially as food resources and studies of extraction and separation of the proteins, therefore, are basically required for this purpose. In this study, the influence of various factors including the sample treatment, extraction time and temperature, sample us extraction solvent ratio and pH upon the extractability of the water soluble protein was determined. And the effect of precipitation treatment for isolation of the algal protein from the extracts was also tested. Nine species of algae, the major ones in consumption as food namely Porphyra suborbiculata, Undaria pinnatifida, Hizikia fusiforme, Sargassum fulvellu, Enteromorpha linza, Codium fragile, Sargassum kjellmanianum and Ulva pertusa were collected as fresh from Kijang, Yangsan Gun, in the vicinity of Busan city. The content of crude protein $(N\times6.25)$ of the algae ranged from $9.46\%\;to\;24.14\% showing the highest value in Porphyra suborbiculata and the minimum in Hizikia fusiforme. In the effort of maceration of blending methods on the extractability, immersion freezing in dry ice-methanol solution appeared most effective yielding 1.5 to 2.5 times extractability than that of the mortar grinding method. The effect of the ratio of sample vs solvent on extractability differed from species. It was enhanced at the ratio of 1:20 (w/v) in Ulva pertusa and Enteromorpha linza while the ratio was 1:30 (w/v) for Cedium fragile, Undaria pinnatifida, Hizikia fusiferme, Sargassum fulvellum and Porphyra suborbiculata and 1:40 for Sargassum kjellmanianum respectively. The effect of extraction time and temperature was revealed differently from species which might be caused by differences in the constitution of algal tissues resulting in that the extraction for 1 hour at $50^{\circ}C$ gave the maximum extractabilily in Ulva pertusa and Enteromorpha linza, 2 hours in Porphyra suborbiculata, Hikikia fusiforme, Undaria pinnatifida, Sargassum kjellmanianum and 3 hours in Codium fragile. And the extractability was higher at $50^{\circ}C$ to $60^{\circ}C$ for the most of the tested samples except Hizikia fusiforme. The optimum pH for the extraction was 9 to 12. The recovery of extractable nitrogen to the total nitrogen was $63\%$ in average with the first extracts and $8.6\%$ with the second extracts respectively. Both extracts were prepared by 2 hour extraction at $50{\pm}1^{\circ}C$ with dry ice-methanol frozen and seasand macerated materials. And these conditions assumed to be an optimum for the extraction of water soluble algal proteins since the nitrogen content after the first extraction covered $90\%$ of the total water extractable nitrogen. In the precipitation of the extracted proteins, Barnstein method and methanol treatment seemed to be more efficient than other precipitation methods.