Cryopreservation of bovine embryos is used to efficiently implant surrogate mothers. It has been widely accepted that high lipid content in the oocyte interrupts its survival during freeze-thaw cycles. Serum component in the culture medium is thought to increase the embryo`s lipid contents. Conversely, L-carnitine stimulates lipid metabolism by transporting long chain fatty acids into the mitochondria. Objective of this study was to analyze the effect of L-carnitine supplementation in IVM medium and defined IVC medium on the development, lipid contents and the cryosurvival of bovine IVF embryos. 0.0, 1.5, 3.0 and 6.0 mM L-carnitine was supplemented in IVM medium, respectively (IVM-LC 0.0, LC 1.5, LC 3.0 and LC 6.0). Development rate from the 2cell to the morula stages was higher in IVM-LC 3.0 groups than those of IVM-LC 6.0 (p<0.05). But there were no significant differences among the other groups in the blastocyst rates and lipid content results. When 0.0, 1.5, 3.0 and 6.0 mM L-carnitine were supplemented in IVC medium (IVC-LC 0.0, LC 1.5, LC 3.0 and LC 6.0), development competence was not significantly different between those embryos. Lipid contents of embryos treated L-carnitine (IVC-LC 1.5, 3.0 and 6.0) were significantly lower than embryos of non-treated group. L-carnitine was supplemented 0.0, 1.5, 3.0, 6.0 mM during IVM and 3.0 mM during IVC (LC 0.0 - 3.0, LC 1.5 – 3.0, LC 3.0 – 3.0, LC 6.0 – 3.0) and cryosurvival of blastocysts confirmed after freezing-thawing. There were no significant differences on development, but LC 3.0 – 3.0 was significantly lower lipid contents than other groups. And LC 3.0 – 3.0 had better survival rates and hatched rates of blastocysts than LC 0.0 – 0.0. In conclusion, supplementation of L-carnitine in defined IVC medium decreases lipid contents. And L-carnitine supplementation improves cryosurvival and developmental ability of bovine IVF embryos.

The early-onset familial Alzheimer's disease (EOFAD/ FAD), the less common type of Alzheimer's disease (AD) currently affects a vast number of individuals worldwide. This type is being inherited as an autosomal dominant fashion. Missense mutations on Amyloid precursor protein (APP) and Presenilins 1 and 2 (PSEN1 & PSEN2) are known as major genetic factors in FAD. Conversely, missense mutations on microtubule-associated protein tau (MAPT) are also thought to involve. Up to date, several triple-transgenic animal models with muted forms of the human APP, PSENs and MAPT have been reported. Compared to other animals, canines are more emotional and their disease signs can be easily diagnosed. This attempt was to develop a triple transgenic canine model for the AD. We have obtained the coding sequences of APP, PSEN1 and MAPT from Dana-Farber/Harvard Cancer Center DNA resource core at HMS and incorporated several common AD mutations. The transgenic construct is composed of hNSE (ENO2) promoter-driven three AD genes fused together with modified 2A sequences. It was transfected into the canine fetal fibroblasts which were then used to perform somatic cell nuclear transfer (SCNT). The viable transgenic embryos were obtained after in vitro culture and the GFP was detected. In this study, we have successfully produced viable triple transgenic canine cloned embryos using SCNT technique. These transgenic canine embryos will be further developed into canines with FAD. The transgenic canines will be a good candidate in the AD research field.

Various plant extracts were widely used to control plant diseases and insect pests in organic farming system. This study was conducted to evaluate insecticidal activity of plant extracts against three moth in vitro. Insecticidal activity of 21 kinds of farm-made plant extracts including snowbell tree, sweet oleander leaf, sweet oleander and white cedar, collected from farmhouses were tested against diamondback moth (Plutella xypostella) and tobacco cutworm (Spodoptera litura) larvae. In addition, four plant extracts (pyrethrum flower, derris, neem and Sophora flavescens) were tested for insecticidal activity against Potato tuber moth (Phthouimaea operculella) larvae. As a result of insecticidal test on diamondback moth, insecticidal activity of pyrethrum flower extract was found to be 50% or more in diluted 100 times. The insecticidal activities against tobacco cutworm were 92.5% at snowbell tree extract, 77.5% at the sweet oleander leaf and white cedar extracts. Among the plant extracts, insecticidal activity of 300 times-diluted pyrethrum flower and derris extract was 85%, neem and Sophora flavescens extracts were similar to non-treatment. Consequently, pyrethrum flower extract and snowbell tree, sweet oleander leaf, white cedar extracts and pyrethrum flower, derris extracts for controlling diamondback moth and tobacco cutworm and potato tuber moth were selected. We think the selected organic materials can be used to control diamondback moth and tobacco cutworm, potato tuber moth under organic farmhouse condition.

본 연구에서는 국내 유통 중인 농산물 9품목(n = 578)에 대한 납과 카드뮴 함량을 조사하고 이들의 섭취로 인한 위해성을 평가하고자 하였다. 납과 카드뮴의 함량은 마이크로웨이브 분해 후 ICP-MS로 분석하였다. 조사대상 농산물의 납 평균 함량은 각각 보리 0.014 mg/kg, 완두콩 0.010 mg/kg, 강낭콩 0.008 mg/kg, 녹두 0.006 mg/kg, 파인애플 0.008 mg/kg, 살구 0.016 mg/kg, 매실 0.015 mg/kg, 자두 0.021 mg/kg, 대추 0.019 mg/kg이었고, 카드뮴 평균함량은 보리 0.017 mg/kg, 완두콩 0.004 mg/kg, 강낭콩 0.007 mg/kg, 녹두 0.005 mg/kg, 파인애플 0.001 mg/kg, 살구 0.002 mg/kg, 매실 0.002 mg/kg, 자두 0.002 mg/kg, 대추 0.003 mg/kg이었다. 모든 시료의 납, 카드뮴 함량은 EU, CODEX 및 국내 기준보다 낮은 수준이었다. 조사 대상 농산물에 대한 납, 카드뮴의 인체노출량을 산출한 결과, 납은 잠정주간섭취허용량(PTWI, 25 μg/kg b.w./week)의 0.067%, 카드뮴은 월간잠정섭취허용량(PTMI, 25 μg/kg b.w./month)의 0.28%이었다. 이상의 결과는 조사 대상 농산물의 납, 카드뮴 오염도와 이들의 섭취에 의한 위해성이 모두 낮은 수준이라는 것을 보여준다.

Curcumin is an active polyphenolic compound with antioxidant, anti-inflammatory and antitumor properties. Curcumin, however, is highly unstable under physiological conditions due to its low stability in acidic and alkaline conditions. Therefore, the objective of this study was to investigate the effects of enzyme-treated rice starch as a wall material on the stability of curcumin in oil-in-water emulsion under different pH conditions. The rice starch was treated using 4-a-glucanotransferase for different time periods and their molecular weight distribution was measured by HPSEC. Curcumin was encapsulated within lipid droplets of O/W emulsion prepared with Tween 20 and the modified rice starch in the aqueous phase at different concentrations (0, 2.5, 7.5 and 10 wt%). The temperature and pH stability of the system were determined respectively by measuring particle size, zeta potential and retention of the curcumin loaded in the emulsion after one-week storage in the solutions with different pH and temperature conditions. The average molecular weight of the modified starch decreased with treatment time. The 96h treated rice starch had the lowest molecular weight while the 1h treated starch mainly consisted of high molecular weight components. The storage temperature did not significantly influence the stability of curcumin emulsion. However, the particle size of the emulsion with modified starch slightly increased when stored at acidic pH condition, which might be attributed to starch aggregation. The curcumin retention was higher for the samples with the modified starch than the control at all concentrations. The pH stability of the curcumin was also higher than the control at all pH conditions. Specifically, the 1h treated starch showed the best performance regarding curcumin protection in emulsion, which might be attributed to the high viscosity that retarded the curcumin release. Further research needs to be conducted on the mechanism.

Oocyte is the central factor in the bi-directional communication axis in the ovarian follicles. It controls the cumulus or granulosa cells to perform functions which are beneficial for its own development via secreting paracrine growth factors, including GDF9 and BMP15. The aim of this study was to investigate whether the recombinant GDF9 and BMP15 are able to promote meiotic resumption and cumulus expansion of canine COCs during IVM, as well as to demonstrate the actions of GDF9 and BMP15 in regulating the expression of connexin transcripts in the ovarian granulosa cells. As results, GDF9 and BMP15 significantly improved the meiotic resumption rate and cumulus expansion by activating ERK1/2 signaling. Treatments with GDF9 significantly improved the expression of CyclinB1 but inhibited the expression of Cx43 transcripts. In addition, cumulus expansion genes (MAPK1, Ptgs2, Tnfaip6 and Ptx3) were differentially improved by GDF9 and BMP15. In the ovarian granulosa cells, GDF9 suppressed the expression of Cx43 transcripts by binding ALK4/5/7 receptors and activation Smad2/3 signaling, whereas, BMP15 stimulated the expression of Cx43 transcripts by binding ALK2/3/6 receptors and activating Smad1/5/8 signaling. In conclusion, by regulating functions of granulosa/cumulus cells, oocyte has the potential to enhance the growth and maturation of itself.

Oocyte is the central factor in the bi-directional communication axis in the ovarian follicles. It controls the cumulus or granulosa cells to perform functions which are beneficial for its own development via secreting paracrine growth factors, including GDF9 and BMP15. The aim of this study was to investigate whether the recombinant GDF9 and BMP15 are able to promote meiotic resumption and cumulus expansion of canine COCs during IVM, as well as to demonstrate the actions of GDF9 and BMP15 in regulating the expression of connexin transcripts in the ovarian granulosa cells. As results, GDF9 and BMP15 significantly improved the meiotic resumption rate and cumulus expansion by activating ERK1/2 signaling. Treatments with GDF9 significantly improved the expression of CyclinB1 but inhibited the expression of Cx43 transcripts. In addition, cumulus expansion genes (MAPK1, Ptgs2, Tnfaip6 and Ptx3) were differentially improved by GDF9 and BMP15. In the ovarian granulosa cells, GDF9 suppressed the expression of Cx43 transcripts by binding ALK4/5/7 receptors and activation Smad2/3 signaling, whereas, BMP15 stimulated the expression of Cx43 transcripts by binding ALK2/3/6 receptors and activating Smad1/5/8 signaling. In conclusion, by regulating functions of granulosa/cumulus cells, oocyte has the potential to enhance the growth and maturation of itself.

Somatic cell nuclear transfer (SCNT) has been considered for preserving genetically valuable or endangered animals. Sapsaree is a Natianal Monument in Korea to maintian a pure pedigree. The aim of this study was to produce azoospermia Sapsaree using SCNT and identify normal reproductive ability of cloned azoospermia Sapsaree. Ear skin biopsy was performed on a thirteen-year-old azoospermia Sapsaree and ear skin fibroblasts were isolated for SCNT as donor cells. The fibroblasts were injected into enucleated in vivo matured oocytes, the couplets were electrical fused by two pulse of direct current (55 V for 15 μs) using titanium and platinum fusion needle and activated by calcium ionophore. Cloned embryos were surgically transferred into oviducts of natuarally estrus cycle synchronized recipient dogs. The fusion rate of platinum needle was 70%, which was higher than those of titanium needle (64.1%). Developmental rate to the 8 cells and 10 cell stages was higher in platinum needle group (24% and 16%, respectively) than those of platinum needle group (14.8% and 3.1%, respectively). Total 35 SCNT embryos were transferred into oviducts of 3 recipient dogs and one recipient finally delivered a puppy by caesarean section. As results, this study demonstrated that platinum fusion needle could be successfully make the reconstructed embryos and improve the efficiency of canine SCNT. Cloning azoospermia Sapsaree may contribute to conserve genetically valuable and unique pedigree. And further study should be confirm whether cloned live dog is azoospermia.

Prolonged communication between oocytes and the surrounding somatic cells is one of the unique reproductive physiology in canine. Paracrine Kit ligand (KITL) signaling is a well-known communication between granulosa cells and the oocyte. KITL is a cytokine growth factor secreted by granulosa cells that signals via the c-kit receptor expressed by oocytes. Paracrine factors, including growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), exert their effects by binding with the kinase receptors expressed on the granulosa cells. However, the regulations of GDF9 and BMP15 in the canine KITL expression are currently poorly understood. Therefore, we investigated the effects of GDF9 and BMP15 on the expression of KITL in canine ovarian granulosa cells in vitro. In Annexin V assay recombinant GDF9 and BMP15 did not induce apoptosis in the cultured ovarian granulosa cells. When treated, FSH significantly increased KITL expression, and hCG suppressed its expression. When both FSH and hCG were treated, the expression of KITL was affected by GDF9 and BMP15 in dose and time dependent manner in the luteal granulosa cells. GDF9 (10 ng/mL) significantly decreased KITL expression after12 h. BMP15 (10 ng/mL) significantly also decreased KITL expression after 24 h. Western blot and immunochemistry results indicate that GDF9 activated Smad2/3. After blocking ALK 4/5/7 receptors by SB, GDF9 failed to activate Smad2/3, also BMP15 did not activate Smad1/5/8 after blocking ALK 2/3/6 receptors by DM. So GDF9 exerts its effects via using ALK 4/5/7 receptors to activate SMAD2/3 signaling, and BMP15 binds ALK 2/3/6 receptors to activate SMAD1/5/8 signaling. The expression of KITL was not changed by SB or DM treatment. However, the effect of GDF9 and BMP15, which decreased the expression of KITL, was suppressed by SB or DM treatment. In conclusion, this study provides the first evidence that recombinant GDF9 and BMP15 decrease KITL expression in canine ovarian granulosa cells.

The cancer and Parkinson's disease associated protein DJ-1 is multifunctional protein that involves in diverse cellular process. DJ-1 protein has a cellular protective role and promoted cell survival under an oxidative stress. However, the cellular protective mechanism of DJ-1 is not fully understand, and we needs to be further study their functions in novel organisms. In the present study, we investigated the protective role of DJ-1 against induced oxidative stress in canine cell line. On the basis of these experiments, canine DJ-1 overexpressing and null cell lines were established. The stable overexpression and down regulation of DJ-1 efficiency confirmed by the western blot analysis. Subsequently, the DJ-1 gene transfected cell lines and control cells were subjected to induced the oxidative stress, and then cell viability, cell proliferation assay, cellular apoptosis detection analysis (Annexin V and TUNEL assay), intracellular ROS and mitochondrial activity were measured appropriately. The results showed that DJ-1 overexpressed cells were up-regulated cell viability under oxidative stress conditions induced by the rotenone and hydrogen peroxide (H2O2), whereas loss of DJ-1 cells were down-regulated the cell survival activity. Additionally, overexpression of DJ-1 cells increased cell resistance to oxidative stress and inhibited the elevation of cell death and cellular ROS induced apoptosis. Moreover, DJ-1 overexpressed cells was increased mitochondrial functions by using confocal microscopy with MitoTracker staining. On the contrary to this, DJ-1 null cells show defective cellular protection and mitochondria activity against oxidative stress conditions. Our data indicate that canine DJ-1 protein attenuates cellular apoptosis and ROS generation, enhances the cellular survival activity and promote mitochondrial function under the oxidative stress, likewise other mammalian cells. Importantly, DJ-1 overexpression may be an important part of a protective strategy as a sensor for oxidative stress.

RNA interference (RNAi) has been proven as an operative technique for efficient gene silencing in many organisms. In our study, Tetranychus urticae, an extremely polyphagous and rapidly resistance developing mite against acaricides, was screened by double-stranded RNA (dsRNA) delivery method using multi-unit chambers. Among several lethal genes of T.urticae, COPA (the coatomer subunit alpha), a gene involved in membrane transport between the endoplasmic reticulum (ER) and the Golgi complex, showed the highest mortality rate [median lethal time (LT50)=54h]. To investigate the effect of dsCOPA treatment to lysosome formation, we used the Lysotracker green DND26 dye, selective to acidic cellular compartments such as lysosome. The result revealed that the dsEGFP-treated T. urticae has 1.3-fold more of lysosome than that of dsCOPA treated, indicating that downregulation of COPA affected lysosomes function and autophagy, thereby resulting in lethality. To investigate the further detailed toxic mechanism of COPA knockdown, investigation on histological changes in T. urticae fed COPA dsRNA is currently on going.

For more than two decades, the intracytoplasmic sperm injection (ICSI) technique has been used as a valuable tool to provide opportunities for studying fertilization, treating human infertility, and producing transgenic animals. Not only in facilitating fertilization but also in propagating mammalian species, ICSI has enhanced the potential of assisted reproductive technologies in human. Polyspermic fertilization has been one of major problems in pig reproduction, but the ICSI helped to solve the problem, and used widely to generate transgenic piglets. Although the ICSI technique is considered to be a very useful tool in assisted reproductive technologies, including generation of transgenic animals, there are some disadvantages using the technique. In this review, we describe the ICSI technique and its application in animal production and human infertility, and discuss advantage and disadvantage of the technique in mammals.

The purpose of this study was to examine the influences of chronic shoulder pain on the muscle tone in trunk muscles. The study's subjects were 40 men and women in their 30 to 50s, which were divided into two groups. A chronic shoulder pain group consisted of 20 subjects who had been diagnosed with chronic shoulder pain by doctors, and a painless group consisted of 20 subjects who had experienced no such pain. An analysis was performed using electromyography on the muscle tone in the rectus abdominalis, external oblique, internal oblique, and erector spinae muscles under the same conditions between the two groups. The analysis results were as follows. The chronic shoulder pain group exhibited an overall high level of trunk muscle tone than the painless group, along with a statistically significant difference in the rectus abdominalis(p<.05). Moreover, the chronic shoulder pain group showed differences in the trunk muscle tone depending on the affected side. The chronic left shoulder pain group yielded higher levels of muscle tone in the right-side trunk muscles. In particular, the group revealed statistically significant differences in the rectus abdominalis and internal oblique(p<.05). The chronic right shoulder pain group exhibited higher levels of muscle tone in the left-side trunk muscles with a statistically significant difference in the internal oblique(p<.05). The above results suggested that chronic shoulder pain influences increases in the muscle tone in the trunk muscles on the opposite side to the affected shoulder.

The objective of this study was to investigate the effects of NEAA and leptin supplemented to in vitro culture medium on the developmental competence of porcine embryos after intracytoplasmic sperm injection (ICSI), and to modify the culture condition to improve the quality and the development of ICSI-derived porcine embryos in vitro. After ICSI, the putative zygotes were then cultured in PZM-3 medium with/without NEAA or leptin. The proportion of embryos that developed to the blastocyst stage significantly increased when 1% NEAA (24.62%) was added to the medium compared with 2% NEAA and no NEAA group (17.24% and 20.24%, respectively, p<0.05). The effect of different concentration of leptin (0, 10, 100, 500 ng/ml) was evaluated on the development of porcine ICSI embryos cultured in vitro. In case of blastocyst formation, 100 ng/ml group (27.05%) showed significantly higher rate than 10, 500 ng/ml, and control group (23.45%, 17.99%, and 19.68%, respectively, p<0.05). We also evaluated the effects of different NEAA and leptin treatment time on the development of porcine embryos after ICSI. Among groups of embryos cultured in the presence of NEAA or leptin for whole 7 days (D 1-7), first 4 days (D 1-4), the subsequent 3 days (D 5-7), both NEAA (27.13%, 21.17 %, and 17.56%, respectively, p<0.05) and leptin (25.60%, 20.61%, and 16.53%, respectively, p<0.05) showed that supplementation for whole 7 days significantly increased the blastocyst formation rate compared with the other groups of D1-4 and D5-7. We further evaluated the combination effect of 1% NEAA and 100 ng/ml leptin compared with the effect of each supplementation with 1% NEAA or 100 ng/ml leptin or no supplementation on development of embryos. For blastocyst formation, combination group of NEAA and leptin (24.78%) showed significantly higher rate than other three groups (18.37%, 20.44 %, and 13.27%, respectively, p<0.05). We further evaluated the expression of proapoptosis genes such as BAX and BAK and anti-apoptosis genes, BCL-XL and BCL-2 in blastocysts cultured in the presence of 100 ng/ml leptin. RT-PCR analysis revealed that leptin supplementation significantly decreased the expression of pro-apoptosis genes as well as increased the expression of anti-apoptosis genes. These results of present study demonstrate that NEAA and leptin could improve the in vitro development of ICSI- derived porcine embryos with optimal concentration of each reagent. Furthermore, the optimal culture condition could increase the quality of ICSI-derived embryos in vitro.

True hermaphrodites are animals of equivocal sex in which both male and female gonads develop simultaneously. The frequency of true hermaphroditism is higher in pigs than in other domestic animals. Two Korean pigs were diagnosed with true hermaphroditism showing ovotestes, epididymes, penes, and uteri. Histomorphologically, the testicular tissues consisted of Sertoli cells that were devoid of spermatogenic germ cells and showed proliferation of interstitial cells. However, the uteri were of normal architecture and had well-developed uterine endometrial glands. The samples were 38, XX female karyotype without the sex-determining region Y (SRY) gene. The findings of this study could contribute to the understanding of true hermaphroditism in the Korean pig industry. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.