Somatic cell nuclear transfer (SCNT) is the technique which generates embryos by transferring diploid nucleus into an enucleated oocyte, it has produced specific animals successfully in a variety of species. However, the developmental capacity of SCNT embryos is still relatively lower than that of embryos produced in vivo. Oocyte is a kind of lipid rich cells, its quality limits the efficiency of embryo production. L-carnitine is a co-enzyme facilitating the transportation of long chain fatty acids across the inner mitochondria membrane where fatty acids are used for generating adenosine triphosphate (ATP) via beta-oxidation. It also has antioxidant actions which may protect mitochondrial membranes and DNA against damage induced by reactive oxygen species (ROS). Whether L-carnitine is functional in bovine SCNT embryos are unknown. Therefore, the objective of this study was to examine the effects of L-carnitine on oocyte maturation and developmental competence of subsequent SCNT embryos. L-carnitine was supplemented during IVM, then intracellular ROS and GSH levels, mitochondrial activity, gene expression of COCs were analyzed at the end of IVM. SCNT embryos were produced subsequently, apoptosis detection and gene expression evaluation were performed in blastocysts. In the results, treatments with 1.5 mM and 3 mM L-carnitine significantly improved maturation rates (P<0.05). Treatments with 3 mM L-carnitine effectively induced improvement in nuclear maturation, intracellular GSH levels and mitochondrial activity, as well as a reduction in intracellular ROS levels (P<0.05). mRNA levels of CPT1A, ACAA1, ACAA2, AREG, EREG, SOD1, GPX4, GLUT1 and CDC2 transcripts were effectively up-regulated by 3 mM L-carnitine treatments (P < 0.05). Similarly, 3mM L-carnitine induced an increase in blastocyst developmental rates and an improvement in blastocyst quality (P<0.05). Our study indicates that L-carnitine treatment during IVM improves oocyte nuclear maturation and subsequent SCNT embryo development.

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.

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 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.

The aim of this study was to examine the effect of acteoside (the cyclin-dependent kinase inhibitor) on the SCNT efficiency with adult fibroblasts in dog. Canine adult fibroblasts were obtained from muscle and cell cycle of fibroblasts was synchronized by culturing to confluency, serum starvation and treating with 30 μM acteoside for 48 h. Cell cycle stages, cell cytotoxicity (apoptosis) and, prduction of reactive oxygen species (ROS) were analyzed using flow cytometry. The canine cells, prepared by confluent-cell culture or treating with 30 μM acteoside for 48 h, were injected into enucleated in vivo matured oocytes, the couplets were electrical fused and activated by calcium ionomycin. SCNT embryos using acteoside-treated fibroblasts were surgically transferred into oviducts of estrus cycle synchronized recipient dogs. In cell cycle synchronization (G0/G1), there was no significant difference between serum starvations (83.9%) and acteoside treated groups (81.3%) that were higher than confluent group (78.5%). In production of apoptosis, confluent and acteoside treated groups (4.3 and 4.5%, respectively) were generated less than serum starvation group (21.8%). In case of ROS, serum starvation group was induced a significantly higher than other groups. After synchronization of the donor cell cycle, either confluent or acteoside treated, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into naturally estrus-synchronized surrogates. Fusion and cleavage rate of acteoside treated group were 64.1 and 41.5%, which were higher than those of confluent group (53.9 and 20.6%, respectively). The reconstructed embryo development rates to 4-cell and 8-cell in acteoside treated group were 29.5 and 14.8%, respectively, while confluent group showed 11.1 and 3.2%, respectively. Total 54 SCNT embryos using acteoside-treated fibroblasts were transferred into oviducts of 2 recipient dogs and one recipient finally delivered one puppy, whereas din`t detected pregnancy on transfer of cloned embryos reconstructed with confluent cells in 6 surrogate dogs. In conclusion, the results of the current study demonstrated that canine fibroblasts could be successfully arrested at the G0/G1 stage with reduced the formation of ROS and apoptosis after acteoside treatment. This results may contribute to improve the effi-ciency of canine SCNT. * This research was supported by iPET (Grants 110056-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Acteoside acts as an anti-oxidative activity and anti-apoptosis in the cells. But, it has been not studied on maturation and development of porcine oocytes. The aims of the present study were to examine the effects of acteoside on the morphological progress of meiosis, developmental competence, and ROS in porcine oocytes. Oocytes were matured in tissue culture medium-199, supplemented with acteoside at various concentrations: 0 (control), 10, 30 and 50 μM. The oocytes maturation rates of groups supplemented with acteoside were no significantly different (81.13, 85.96, 82.95 and 83.68%, respectively). Level of ROS was significantly decreased in acteoside treated group. Furthermore, the parthenogenetic blastocyst rate was significantly improved in 10 μM acteoside treated group compared with control group (44.83 vs. 27.75%). And we investigated effect of acteoside on the oocytes condition represented by cytoplasmic maturation by homogeneous distribution and formation of cytoplasmic organelles and regulation of apoptosis-related genes. In the results. during IVM, 10 μM acteoside treated oocytes showed that the mitochondria and lipid droplet were smaller and homogeneous distribution in cytoplasm compare with control oocytes. And reverse transcription polymerase chain reaction (RTPCR) of parthenogenetic blstocysts revealed that acteoside increased the anti-apoptotic genes (Mcl-1, Bcl-2 and Bcl-xL), whereas reduced the expression of pro-apoptotic genes (Bax and Bak). In conclusion, based on the results, the effect of acteoside on IVM was not attractive. However, in acteoside treated group, cytoplasmic maturation seemed to be improved with morphologically uniform distribution of cytoplasmic organelles. Furthermore, embryonic development in acteoside treated group was significantly highly increased than that of non-treated group. Our results represents that addition of acteoside to the IVM medium has a beneficial effect in physiology of porcine oocytes, providing a improved method for porcine oocytes in vitro. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.

The objective of the current study was to describe in vitro embryo production in Hanwoo, analyzing oocytes yield and embryo production. The effects of oocytes production and the number of OPU procedures per animal on embryo production were also evaluated. OPU was done every 3～4 days during experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, we compared the recovery rate of oocytes based on OPU session (Experiment 1). The average of collected oocytes was calculated from every 10 session. The average number of total oocytes recovered per animalonsessionwas 5.16 (mean). Second, we compared the recovery rate base on collection period of OPU (Experiment 2). The following results show the difference of the number of recovered oocytes in every month during the procedure between the months of session. Every animal shows the constant number of recovered oocytes for the first 5 months. However, the recovery rate of oocytes was decreased from month 6 to 8. Third, we compared the developmental rate to blastocyst in two groups (Experiment 3). Oocytes by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24～48 h after in vitro fertilization (IVF) was 75.8% and blastocyst development rate was 18.8% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (61.1%), blastocyst development rate was higher compared with needle puncture group (28.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio- technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being.

The objective of this study was to evaluate in vitro production of bovine embryos in Hanwoo. Oocytes were collected by ovum pick up (OPU) from ovaries of genetically high-value Hanwoo or by needle puncture from ovaries of slaughtered cattle. OPU was done every 3 4 days duing experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, We compared the in vitro maturation rate in two groups (Experiment 1). 545 oocytes were recoverd from 4 females by 32 trials of OPU and then 433 oocytes were shown MⅡ stage after in vitro maturation (79.4%). In case of needle puncture group, 1905 oocytes were collected and then 1420 oocytes were matured to MⅡ stage during in vitro culture(74.5%). Second, we compared the developmental rate to blastocyst in two groups (Experiment 2). 1420 oocyte by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24 48 h after in vitro fertilization (IVF) was 88.6% and blastocyst development rate was 20.5% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (84.8%), blastocyst development rate was higher compared with needle puncture group (26.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio-technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being. This research was suppoted by Imsil-gun agricultural technology service center.