본 연구는 광질이 장미의 꽃잎 착색에 미치는 영향에 대한 기초자료를 얻고자 수행했다. 꽃눈 분화가 시작된 미니 장미 5품종 ‘Berigamo’(yellowish white), ‘Elpaso Yellow’(yellow), ‘Elpaso’ (bicolor, yellow+orange), ‘Meggiore’(pink), ‘Maasland’(red)를 대상으로 3주간 광질처리를 실시하였다. 광질처리는 300μmol·m-2·s-1 백 색광(W) 대조구와 210μmol·m-2·s-1 백색광에 적색(R), 녹색(G), 청색(B), 원적외선(FR) 및 자외선 B(UVB)을 각 90μmol·m-2·s-1 보광하여 대조구와 동일한 PPFD로 설정하였다. 광질의 꽃잎착색 효과는 품종 특이적으로 R, FR, UVB 보광은 특히 분홍색 품종에서 각각 19.0%, 28.8%, 6.4% 착색을 증진시킨 반면, G와 B 보광은 안토시아닌 함량을 각각 17.8%, 19.0% 감소시켰 다. 황색 품종에서는 UVB보광으로 카로티노이드 함량이 19.6% 증가하였다.

Cryopreservation is mainly used for preservation of boar sperm. However, this method stresses the sperm by reactive oxygen species (ROS), and the conception rate and the litter size are not more efficient than the liquid preservation of spermatozoa. Therefore, we use chitosan which is a natural product derived antioxidant compound. We used GnHA (chitosan+hyaluronic acid) and GnHG (chitosan hydrogel) as chitosan complexes to cryopreserve boar sperm for improve sperm metabolism and function. Sperm parameter (sperm motility, progressive motility, path velocity, straight-line velocity, curvilinear velocity) is measured by computer-assisted sperm analysis (CASA) using frozen sperm with GnHA or GnHG (0, 0.25, 0.5, 1 mg/mL), respectively. Also, lipid peroxidation analysis using malondialdehyde (MDA) is performed to confirm the antioxidative effect of chitosan in frozen spermatozoa. CASA analysis showed GnHA and GnHG are effective against cryopreserved boar sperm. And antioxidant effect is measured by lipid peroxidation analysis. GnHA and GnHG, which is chitosan complex are effective for boar sperm cryopreservation by antioxidant effect.

In the present study of this experiment was to understand the expression of apoptotic gene expression in the ovary of miniature pigs and pigs on the 15th day of estrus. Also the compare and analyze of programmed cell death type(Apoptosis and autophagy) expression pattern during mature oocyte on the miniature and normal pig cells. Analysis of mRNA gene expression of ovary in miniature and normal pigs on the 15th day of estrus showed that the expression of genes related to Autophagy (ATG13, MAP1LC3, Beclin1) was high in normal pigs but the expression of ATG1 and ATG5 genes was low. In addition, the expression of genes related to apoptosis (Casp-3, BAX) was high in the mini pigs, and the gene related to the LH hormone was high in the miniature pigs, whereas the expression of the gene related to the FSH hormone was high in the normal pigs. On the other hand, the result of muture oocyte on the miniature and normal pig cells is the expression of Casp-3 protein was moust high from treatment of FL+rapa (FSH+LH and Rapamycin) of the oocyte on the miniature pig cell. However, MAP1LC3 expression was higher in the oocytes of treatment of rapanycine treatment on the nomal pig cells. There was no gene expression in cumulus cells of matured oocytes in mini pig cells, whereas MAP1LC3 expression was higher in oocyte cumulus cells matured in normal pig cells. It was confirmed that the miniature and normal pigs showed different programmed cell death patterns, In the case of oocytes matured in miniature pig cells, MAP1LC3 gene expression was found to be low in spite of treatment with Autophagy regulator.

Cryopreservation is mainly used for preservation of boar sperm. However, this method stresses the sperm by reactive oxygen species (ROS), and the conception rate and the litter size are not more efficient than the liquid preservation of spermatozoa. Therefore, we use chitosan which is a natural product derived antioxidant compound. We used GnHA and GnHG as chitosan complexes to cryopreserve boar sperm for improve sperm metabolism and function. Sperm parameter (sperm motility, progressive motility, path velocity, straight-line velocity, curvilinear velocity) is measured by computer-assisted sperm analysis (CASA) using frozen sperm with GnHA or GnHG (0, 0.25, 0.5, 1 mg/mL). Also, lipid peroxidation analysis using malondialdehyde (MDA) is performed to confirm the antioxidative effect of chitosan in frozen spermatozoa. Sperm motility was higher in GnHA 0.25 mg/mL and GnHG 0.5 mg/mL compared to control. In addition, GnHG 0.5 mg/mL was significantly decreased in lipid peroxidation analysis. The results suggest that GnHA and GnHG are effective for boar sperm cryopreservation by antioxidant effect.

Phalaenopsis ‘Blanc Rouse’ 품종은 국립원예특작과학원에서 2013년도에 육성한 신품종이다. 이 품종은 2007년에 백색 바탕에 분홍색의 소형 P. ‘KV 600’ 품종을 모본으로 하고 진한 핑크색의 P. ‘Kang 1’ 품종을 부본으로 교배시킨 후대 계통 중에서 선발하였다. 2010년에 화색, 초장, 화경 및 식물체의 생육상태 등을 고려하여 1차 선발 후 2013년에 2차 특성검정을 통하여 품종의 안정성과 균일성을 확인하고 ‘Blanc Rouse’로 명명하였다. 이 품종은 흰색바탕에 자주색 순판을 가졌으며 꽃 가운데 분홍색의 줄무늬가 크게 형성되어 있는 품종이다. 화형은 꽃잎과 꽃받침이 평편한 모양이고 꽃의 길이와 폭은 4.3cm, 4.8cm로 소형이다. 꽃대가 평균 2개 발생하며 복총상 화서로 화서당 꽃 수가 39.4개로 볼륨이 있어 소형분화에 적합하다. 자연 개화시기는 11월 하순으로 개화가 빠른 조생종 이다. 잎의 길이와 폭은 각각 17.3, 6.2cm이며 엽형은 수평이다. 기내 증식력이 높고 변이가 거의 없으며 내병성이 강하여 재배관리가 용이하다.

본 연구에서는 6-benzylaminopurin(BA)처리가 미니 P. ‘Jiaho’s Pink Girl’와 D. ‘Coffe’의 개화에 미치는 영향을 살펴 보고, 개화 품질 향상을 위한 BA적정농도와 처리시기를 구명 하고자 하였다. 1차 실험은 BA적정농도를 구명하고자 농도 100, 200, 400mg·L-1로 50mL씩 엽면 살포하여 처리하였다. 두 품종 모두 농도가 증가함에 따라 출현화경수가 증가하였 고, 특히 400mg·L-1에서 무처리보다 2배 증가하였다. 그러나 BA처리구 중 P. ‘Jiaho’s Pink Girl’ 품종은 농도 400mg·L-1, D. ‘Coffee’는 농도 200과 400mg·L-1에서 출현화경 중 개화하 지 못하는 화경이 증가하였다. D. ‘Coffee’의 경우 BA농도가 증가함에 따라 화경길이, 화수, 꽃크기가 감소하였으나, P. ‘Jiaho’s Pink Girl’은 BA처리시 화수가 유의적으로 증가하였 다. 2차 실험은 적정 BA처리시기를 구명하고자 무처리(BA 0mg·L-1)를 제외하고 농도 100mg·L-1 를 50mL씩 저온처리 일을 기준으로 4시점에 처리(-7, 0, +7, +14일)하였다. P. ‘Jiaho’s Pink Girl’은 저온 7일 후 BA처리시 출현화경수와 화 수가 유의적으로 증가하였고 화경출현소요일수는 8일 촉진되 었다. 반면에 D. ‘Coffee’는 출현화경수와 화수에서 처리시기 에 따른 유의적인 차이는 없었고, 저온 14일 후 처리를 제외 하고 BA처리 실험구에서 개화화경수가 감소하였다. 따라서, P. ‘Jiaho’s Pink Girl’ 품종의 경우 저온 7일 후 BA농도 100mg·L-1 처리가 품질향상에 효과적인 반면, D. ‘Coffee’ 품 종은 더 많은 화경출현이 오히려 상품성을 떨어뜨리는 결과를 가져왔다. 즉 BA처리가 미니 팔레놉시스와 도리테놉시스의 화경출현을 촉진시켜 화경수를 증가시키는데 기여하지만 품 종과 환경에 따라 반응이 상이하여 BA사용시 부작용 여부 등 을 고려할 필요가 있다.

Pigs have been extensively used as mediators of xenotransplantation research. Specifically, the Massachusetts General Hospital (MGH) miniature pig was developed to fix major histocompatibility antigens for use in xenotransplantation studies. We generated transgenic pigs for xenotransplantation using MGH pigs. However, it has not been studied yet whether these pigs show similarity of reproductive physiological characteristics to wild types of MGH miniature pig. In this study we analyzed the estrous cycles and pregnancy characteristics of wild type (WT) and transgenic MGH miniature pigs, which were α1,3-galactosyltransferase (GalT) heterozygous and homozygous knock-out, and membrane cofactor protein (MCP) inserted in its locus, GalT-MCP/+ and GalT-MCP/-MCP pigs. Estrous cycles of WT, GalT-MCP/+ and GalT-MCP/-MCP pigs were 20.9±0.74, 20.1±1.26, and 17.3±0.87 days, respectively, and periods of estrous were 3.2±0.10, 3.1±0.12, and 3.1±0.11 days. The periods of gestation of WT, GalT-MCP/+ and GalT-MCP/-MCP pigs were 114.2±0.37, 113.3±0.67, and 115.4±0.51 days, respectively. Litter sizes of WT, GalT-MCP/+ and GalT-MCP/-MCP pigs were 4.8±0.35, 4.8±1.11 and 3.0±0.32 respectively. There were no significant differences on estrous cycle, periods of estrous and gestation, and litter size among WT, GalT-MCP/+ and GalT-MCP/-MCP pigs, meaning that GalT knock-out and additional expression MCP of the MGH miniature pig did not effect on reproduction traits. These results provide relevant information to establish breeding system for MGH transgenic pig, and for propagation of GalT-MCP/-MCP pig to supply for xenotransplantation research.

The cryopreservation of sperm has become the subject of research for successful artificial insemination technologies. Antifreeze proteins (AFPs), one of the factors necessary for effective cryopreservation, are derived from certain Antarctic organisms. These proteins decrease the freezing point of water within these organisms to below the temperature of the surrounding seawater to protect the organism from cold shock. Accordingly, a recent study found that AFPs can increase the motility and viability of spermatozoa during cryopreservation.To evaluate this relationship, we performed cryopreservation of boar sperm with AFPs produced in the Arctic yeast Leucosporidium sp. AFP expression system at four concentrations (0, 0.01, 0.1, and 1 μg/ml) and evaluated motility using computer assisted sperm analysis. DNA damage to boar spermatozoa was measured by the comet assay, and sperm membrane integrity and acrosome integrity were evaluated by flow cytometry. The results showed that motility was positively affected by the addition of AFP at each concentration except 1 μg/ml (p<0.001).Although cryopreservation with AFP decreased the viability of the boar sperm using, the tail DNA analyses showed that there was no significant difference between the control and the addition of 0.1 or 0.01 μg/ml AFP. In addition, the percentage of live sperm with intact acrosomes showed the least significant difference between the control and 0.1 μg/ml AFP (p<0.05), but increased with 1 μg/ml AFP (p<0.001). Our results indicate that the addition of AFP during boar sperm cryopreservation can improve viability and acrosome integrity after thawing.

Our objective was to evaluate the function of treahlose and erythritol in reducing ROS concentrations, which is associated with a general improvement in the quality of frozen-thawing miniature pig sperm. Semen was mixed in modified Modena B extender, added to cooling media and freezing media, followed by the supplement of 100 mM trehalose and/or 100 mM erythritol with spermatozoa (1000x 109cells/straw). The trehalose plus erythritol (TE) added group had less intracellular H2O2 than did control and trehalose (36.6±1.6 vs. 49.0±5.8 and 48.8±7.9; P<0.05). The percentage of viable acrosome-intact sperm (FITC-PNA-/PI-) was higher in erythritol and TE than controls (57.0±5.5% and 62.5±4.3% vs. 45.4±5.4%; P<0.05 and P<0.001). The percentage of sperm with high fragmented DNA was observed in control group when compared with erythritol and TE also trehalose (65.5±1.3% vs 59.3±0.7% and 59.0±0.3% vs 62.2± 0.8%; P<0.001). The percentage of sperm LPO was higher in control and trehalose than erythritol (4.4±0.5% and 5.0±0.5% vs. 3.5±0.2; P<0.01 and P<0.001), and was lowest in the TE (control and trehalose vs. TE: P<0.001, erythritol vs. TE: P<0.05). Also, we performed that surgical insemination based on above data to evaluate the function of new cryoprotectant such as trehalose plus erythritol in vivo. Finally, 1 pregnant gilt showed natural estrus was allowed to go to term and 8 live piglets were born. In conclusion, miniature pig sperm was successfully cryopreserved with trehalose plus erythritol provided the increasing the sperm quality and reducing the ROS.

Cryopreservation of miniature pig sperm is essential because of high demand of organ transplant in mass production. However, miniature pig sperm are vulnerable to oxidative stress more than other mammals. Erythritol is a naturally occurring sugar alcohol with powerful antioxidant property. Thus, the aim of our study is to verify if erythritol could reduce lipid peroxide and enhance viability of frozen thawed miniature pig sperm. Ejaculated semen samples were frozen with cryoprotectant subjected to erythritol treatment (0, 10, 100, 500 mM). After frozen thawed, spematozoa viability were examined using the computer assisted sperm analysis (CASA) system. The product of lipid peroxidation, malondialdehyde (MDA) were quantified using spectrophotometer with DPPH and ABTS assays as ROS scavenger markers. Our result showed that erythritol enhanced sperm viability (p<0.05), reduced lipid peroxides significantly (p<0.05), proving the concentration of 100 mM erythritol to be an effective for lowing oxidative damage. Data from our study suggest that erythritol exhibits significant lipid peroxidation scavenging characteristics which may prevent oxidative damage, enhance viability of frozen thawed sperm and thus could be a effective additive as cryoprotectant.

This study investigated the effects of L-carnitine (LC) and nicotinic acid (NA) on sperm viability during liquid storage at 18℃ in miniature pigs. 10 μM LC and 30 mM NA, combined LC and NA (LN) were treated in fresh semen for 3, 7, and 10 days. In results, sperm survival increased in NA- and LN-treated semen on 7 and 10 days (p<0.05), mitochondrial integrity of live sperm increased in LN-treated semen on 7 days (p<0.05), but not NA-treated semen. In addition, we examined the acrosome reaction of sperm in miniature pigs. LC and NA did not influence on acrosome reaction of boar sperm. In conclusion, LC and NA effectively maintained the viability and quality of sperm during long-term storage in miniature pigs, suggesting that the combined LN may be useful for improving the semen extender for long-term liquid storage in pigs.

Xenotransplantation involves multiple steps of immune rejection. The present study was designed to produce nuclear transfer embryos, prior to the production of transgenic pigs, using fibroblasts carrying transgenes human complement regulatory protein hCD59 and interleukin-18 binding protein (hIL-18BP) to reduce hyperacute rejection (HAR) and cellular rejection in pig-to-human xenotransplantation. In addition to the hCD59-mediated reduction of HAR, hIL-18BP may prevent cellular rejection by inhibiting the activation of natural killer cells, activated T-cell proliferation, and induction of IFN-γ. Transgene construct including hCD59 and ILI-18BP was introduced into miniature pig fetal fibroblasts. After antibiotic selection of double transgenic fibroblasts, integration of the transgene was screened by PCR, and the transgene expression was confirmed by RT-PCR. Treatment of human serum did not affect the survival of double-transgenic fibroblasts, whereas the treatment significantly reduced the survival of non-transgenic fibroblasts (p<0.01), suggesting alleviation of HAR. Among 337 reconstituted oocytes produced by nuclear transfer using the double transgenic fibroblasts, 28 (15.3%) developed to the blastocyst stage. Analysis of individual embryos indicated that 53.6% (15/28) of embryos contained the transgene. The result of the present study demonstrates the resistance of hCD59 and IL-18BP double-transgenic fibroblasts against HAR, and the usefulness of the transgenic approach may be predicted by RT-PCR and cytolytic assessment prior to actual production of transgenic pigs. Further study on the transfer of these embryos to surrogates may produce transgenic clone miniature pigs expressing hCD59 and hIL-18BP for xenotransplantation.

동물의 장기를 인간에게 이식하게 되면 초급성거부반응(Hyperacute rejection, HAR)이 일어난다. 초급성거부반응은 면역계의 구성요소 중 보체(complement)에 의해 일어나는 거부반응으로 돼지의 혈관세포 표면에 있는 Galα(1,3)Gal 당분자에 인간의 항체가 즉각 반응하기 때문에 일어나며, α1,3-galactosyltransferase(α1,3-GT) 유전자는 돼지 혈관세포 표면의 Galα(1,3)Gal 당분자 생성에 관여한다. 따라서 인간에게 돼지의 장기를 이식하기 위해서는 α1,3-galactosyltransferase 유전자를 제거하는 것이 필요한 것으로 알려져 있다. 본 연구실의 이전 연구에서, 시카고 미니돼지 귀체세포에서 상동 재조합(Homologous recombination)을 통해 α1,3-galactosyltransferase 유전자가 제거된 체세포를 개발한 바 있으며, 이 체세포를 통하여 α1,3-GT 유전자가 제거된 돼지도 생산된 바 있다. 본 연구에서는, human serum 처리 시 돼지 세포를 보호해 준다고 보고되고 있는 human complement regulator인 human Decay-accelerating factor(hDAF)와 human α1,2-fucosyltransferase(hHT)유전자를 α1,3-GT 유전자 위치에 gene targeting하여 동시에 hDAF와 hHT가 발현하는 체세포를 개발하였다. Knock-in vector는 hDAF와 hHT 두 유전자가 발현할 수 있도록 IRES로 연결하였으며, α1,3-GT 유전자의 start codon을 이용하여 발현할 수 있도록 구축하였다. 구축한 vector는 electroporation을 통해 미니돼지 체세포에 도입하였으며, PCR 결과, α1,3-GT 유전자 위치에서 상동 재조합이 일어났음을 확인하였다. Positivenegative 선별 방법을 통해 얻은 gene targeting 된 체세포는 RT-PCR에 의해 hDAF와 hHT 유전자의 발현이 확인되었으며, 대조군(NIH minipig)에 비해 α1,3-GT 유전자의 발현이 감소하였다. 또한 이들 세포에 100% human complement serum을 처리하였을 때 knock-in 세포가 대조군에 비해 30% 정도 더 높은 생존율을 보였다. 따라서 개발된 체세포는 이종간 장기이식을 위한 돼지 생산과 함께 이를 이용한 이종간의 장기 이식 시 초급성 거부반응을 억제하는 데 사용될 수 있을 것으로 생각된다.

Objective of this study was to investigate the effect of nicotinic acid (NA) on the characteristics in fresh semen of miniature pig. We evaluated viability, acrosome reaction and mitochondrial integrity of sperm on 0, 3, 7 and 10 days during storage period with nicotinic acid. As results, the survival rate of sperm in 15 mM NA (day 3, 87.8 ± 1.2%; day 5, 84.0 ± 2.7%; day 7, 82.2 ± 0.9%) and 30 mM NA (day 3, 87.7 ± 0.3%; day 5, 84.4 ± 2.5%; day 7, 82.3 ± 0.7%) groups were higher than control and 5 mM NA groups in 3, 7 and 10 days of semen storage. The NA-treated sperm on 10 day was used day for observing acrosome integrity. The survival sperm with acrosome reaction was higher in 30 mM NA group (day 3, 2.7 ± 0.2%; day 5, 3.3 ± 0.6%; day 7, 11.4 ± 0.3%) than in the control, significantly (P<0.05). Moreover, the live sperm with mitochondrial integrity was higher in whole treatment groups of NA than control group, significantly (P<0.05). Specially, most mitochondrial integrity on 10 day of semen storage was significantly higher in 30 mM NA group (90.2 ± 1.6%) than other treatment groups (control, 81.8 ± 3.1%; 5 mM NA, 83.4 ± 3.0%; 15 mM NA, 89.1 ± 0.7%, P<0.05). In conclusion, supplement of NA in liquid semen of miniature pig can improve and maintain semen quality, such as viability, acrosome reaction, and mitochondria integrity.

L-Carnitine is an antioxidant for the transport of fatty acids in mitochondria and breakdown of lipids for metabolic energy. Some studies have suggested that carnitine improves sperm motility in mammals. The objective of this study was to investigate the effect of L-carnitine on the characteristics in fresh semen of miniature pigs. The collected fresh semen was stored in modena B medium with L-carnitine (0, 1.0, 2.0, and 4.0 mg/ml) for 10 days at 18℃. The semen quality of viability, acrosome reaction and mitochondria integrity was analyzed on 0, 3, 7, and 10 day of semen storage. The percentages of live and dying sperm were not different among treatment groups with different concentrations of L-carnitine during the storage period. In acrosome reaction analysis, when the sperm stored for 7 day, the percentages of live sperm with acrosome reaction were significantly (p<0.05) lower in 1 (9.0±0.9%), 2 (7.6±0.2%) or 4mg/ml (7.9±0.8%) L-carnitine-treated groups than the control group (0 mg/ml L-carnitine) (11.12±0.2%). However, there were no difference in percentages of live sperm with acrosome reaction for 3 and 10 days of storage with each concentrations of L-carnitine. When sperm was stored for 3 and 10 days, the percentages of live sperm with mitochondria integrity were significantly higher in 2 mg/ml of L-carnitine-treated group than control group (p<0.05). In conclusion, the L-carnitine has a positive effect on acrosome reaction and mitochondria integrity in liquid state of fresh semen in miniature pigs.

Although the majority of surviving pigs cloned by somatic cell nuclear transfer (SCNT) appear to be physiologically normal, there is a general lack of detailed hemato-physiologic studies for the period of early adulthood to substantiate this claim. In the present study, we investigated variation in blood chemistry and endocrinological parameters between mesenchymal stem cells (MSCs) derived from cloned and normal age-matched female and male miniature pigs. Cloned females and males showed normal ranges for complete blood count assessments. Biochemical assessments showed that γ-GGT, ALT and cholesterol levels of male and female clones were significantly (P<0.05 or P<0.01, respectively) higher than that of age-matched control miniature pigs. Variations in insulin and IGF-1 were higher in female clones than in male clones and controls. Thus, although female and male cloned miniature pigs may be physiologically similar to normal animals, or at least within normal ranges, a greater degree of physiological and endocrinological variation was found in cloned pigs. The above variation must be taken into account before considering cloned female or male miniature pigs for various biomedical applications.

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.