To improve the developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos, this study compared the developmental rates to blastocyst stage in the SCNT embryos using donor fibroblasts treated with 5-azacytidine (5AC) and S-adenosylhomocysteine (SAH) at different concentrations. Their reprogramming efficiency level was investigated with level of telomerase activity. Donor fibroblasts isolated from adult ear skin of a cow were exposed to 5AC and SAH at different concentrations during 2 passages. After nuclear transfer into enucleated recipient oocytes, the cleavage and developmental rates were significantly (p<0.05) decreased in the SCNT embryos using 5AC-treated fibroblasts (5AC-SCNT embryos), compared with those of non-treated control (control-SCNT embryos) and SAH-treated fibroblasts (SAH-SCNT embryos). The developmental rates to blastocyst stage tended to be slightly increased in the SAH-SCNT embryos at each of the concentrations, and especially, the developmental rates in the SCNT embryos using 1.0 mM SAH-treated fibroblasts were significantly (p<0.05) higher than that of control SCNT embryos. The mean numbers of total and ICM cell in blastocysts were also significantly (p<0.05) decreased in the 5AC-SCNT embryos, compared with those of other SCNT blastocysts. Further, the level of telomerase activity was also significantly (p< 0.05) decreased in the 5AC-SCNT embryos than those of control and SAH-SCNT embryos. Whereas, a significantly (p<0.05) up-regulated telomerase activity was observed in SAH-SCNT embryos, compare with that of control-SCNT embryos. In conclusion, SCNT embryos using hypomethylated donor cells with SAH, not 5AC, may improve the developmental potential and reprogramming efficiency.

Bovine papillomaviruses (BPVs) are DNA oncogenic viruses that belong to the Papillomavirus genus. The purposes of the present study were to histopathologically diagnose skin warts on Korean native cattle from Jeju Island and to detect the presence of BPVs from tissue samples of the skin warts, using the polymerase chain reaction (PCR) assay. The sites of 10 skin wart involvement were the head (40%: 10% were in both the head and neck), shoulder (30%), neck (20%), and tail (20%), including the perineum. All skin warts were confirmed as fibropapillomas, as characterized by the abundant proliferating fibrous tissue in the dermis and increased epithelial covering of variable thickness. According to the PCR analysis, five out of 10 (50%) cattle were positive for BPV-1, and six (60%) cattle were positive for BPV-2. Two cattle were positive for both BPV-1 and BPV-2. To the best of our knowledge, this is the first study of BPV-1 and BPV-2 infections of skin warts from Korean native cattle in Korea.

본 연구에서는 다양한 처리 온도와 압력 조건에 따라 초임계 이산화탄소(SC-CO2) 처리한 동결 건조 소간의 이화학적 특성을 비교분석하였다. 무기성분은 SC-CO2 처리에 의해 다소 감소한 Cu를 제외하고는 대체적으로 증가하는 경향을 보였다. Vit A는 SC-CO2처리에 의해 다소 감소하였지만, vit B3는 처리에 의해 모든 조건에서 증가하였으며, 특히 45℃, 450bar에서는 13.20mg/100g으로 가장 높게 나타났으며, 처리 전 7.16mg/100g에 비해 크게 증가하였다. 소간 내 지방의 추출 수율은 SC-CO2 처리 시 온도와 압력이 증가함에 따라 높아지는 경향을 보였다. SC-CO2 처리에 의해 취득한 소간유의 지방산 조성을 분석한 결과, 주요 지방산은 Palmitic acid(43.20~55.82%), Linoleic acid(9.25~16.80%) 그리고 Myristic acid(5.16~10.54%)로 나타났다. 또한, 17종의 아미노산을 분석한 결과, 45℃에서 SC-CO2를 처리하였을 때 아미노산의 손실이 적게 일어났었다. 이상의 결과를 종합하여 보면 SC-CO2는 낮은 수분함량에서도 이화학적 특성의 변화를 최소화 하였다. 이에 따라 SC-CO2 처리는 식품산업에서 부산물인 소간을 이용하여 미생물의 오염으로부터 안전한 고부가가치 식품으로써 다양한 가능성을 열 수 있을 것으로 판단된다.

To date, most serodiagnostic methods for brucellosis screening are based on antibodies against lipopolysaccharides of Brucella spp. However, this approach has the drawback of yielding false-positive results due to cross-reactivity with lipopolysaccharides of other related pathogens, especially Yersinia enterocolitica O:9. In this study, Brucella abortus AspC was cloned and expressed by PCR amplification into a pCold TF expression system to obtain recombinant AspC (rAspC). The immunogenicity of rAspC was confirmed by western blotting of Brucella-positive bovine serum. rAspC-based ELISA was performed to determine whether rAspC could be used in the serodiagnosis of bovine brucellosis. rAspC reacted strongly with anti-Brucella antibodies in positive sera in the tube agglutination test (TAT), but did not show strong reaction with most negative samples. In particular, the average OD492 value at the highest TAT titer showed a 1.4-fold increase with respect to the cutoff value. The accuracy, specificity, and sensitivity of rAspC were 71.88%, 78.33%, and 68%, respectively. These findings suggest that rAspC might be valuable for the serological diagnosis of bovine brucellosis.

Luteolysis is a cyclical regression of the corpus luteum in many non-primate mammalian species. Prostaglandin F2α (PGF2α) from the uterus and ovary induces functional and structural luteolysis in bovine. The action of PGF2α is mediated by PGF2α receptor located on the luteal steroidogenic and endothelial cell membranes. PGF2α plays an important role in regulating nitric oxide production in endothelial cells of the bovine corpus luteum. Nitric oxide production and nitric oxide synthase activity are stimulated and induced by PGF2α in luteal endothelial cells. Moreover, the reactive oxygen species inhibits progesterone secretion in bovine luteal cells and induces apoptosis. Thus, the interaction between PGF2α and reactive oxygen species provides important aspects in physiology of the corpus luteum forfunctional and structural luteolysis.

Transfection is a gene delivery tool that is a popular means of manipulating cellular properties, such as induced pluripotent stem cell (iPSC) generation by reprogramming factors (Yamanaka factors). However, the efficiency of transfection needs to be improved. In the present study, three transfection protocols - non-liposomal transfection (NLT), magnetofection and electroporation - were compared by analysis of their transfection efficiencies and cell viabilities using human dental pulp cells (hDPC) and bovine fetal fibroblasts (bFF) as cell sources. Enhanced green fluorescent protein gene was used as the delivery indicator. For magnetofection, Polymag reagent was administrated. NLT, FuGENE-HD and X-treme GENE 9 DNA transfection reagents were used for NLT. For electroporation, the NeonTM and NEPA21TM electroporators were tested. NeonTM electroporation showed highest transfection efficiency when compared with NLT, magnetofection, and NEPA21TM electroporation, with transfection efficiency of about 33% in hDPC and 50% in bFF, based on viable cell population in each cell type. These results suggest that transfection by NeonTM electroporation can be used to deliver foreign genes efficiently in human and bovine somatic cells.

This study was designed to evaluate the effect of bovine serum albumin (BSA) in a maturation medium on oocyte maturation and embryonic development in pigs. Immature pig oocytes were matured for 44 h in a medium supplemented with 0.4% (w/v) BSA, 0.1% (w/v) polyvinyl alcohol (PVA), or 10% (v/v) pig follicular fluid (PFF). After IVM, oocytes reached metaphase II stage were activated for parthenogenesis (PA) or used as cytoplasts for somatic cell nuclear transfer (SCNT). Nuclear maturation (89.5%, 90.7% and 91.3% for BSA, PVA and PFF, respectively) and intraoocyte glutathione contents (1.20, 1.16 and 1.00 pixels/oocyte for BSA, PVA and PFF, respectively) were not altered by the macromolecules added to maturation medium. IVM of oocytes in a medium containing BSA (21.4%) and PVA (20.7%) showed significantly lower blastocyst formation after PA than culture in medium with PFF (39.2%). After SCNT, oocytes matured in medium with BSA showed decreased embryonic development to the blastocyst stage (9.2%) compared to those matured in medium with PFF (28.9%), while 23.6% of SCNT oocytes matured in medium with PVA developed to the blastocyst stage. When the effect of BSA in a maturation medium during the first 22 h and the second 22 h of IVM in combination with PFF or PVA was examined, PVA-BSA showed a higher nuclear maturation (94.1%) than BSA-PFF (84.5%). However, there was no significant difference in the blastocyst formation among tested combinations (47.3, 52.2, 50.0, 44.4 and 49.0% for PFF-PFF, PFF-BSA, PVA-BSA, BSA-PVA and BSA-PFF, respectively). Our results demonstrate that BSA and PVA added to maturation medium can support oocyte maturation comparable to PFF-supplemented medium. However, maturation of oocytes in a BSA-containing medium decreases embryonic development after PA and SCNT when compared with the medium supplemented with PFF.

We investigate the effect of L-glutathione (GSH), an antioxidant, treatment during the somatic cell nuclear transfer (SCNT) procedures on the in vitro development and DNA methylation status of bovine SCNT embryos. Bovine in vitro matured (IVM) oocytes were enucleated and electrofused with a donor cell, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine. The recipient oocytes or reconstituted oocytes were treated with 50 μM GSH during these SCNT procedures from enucleation to activation treatment. The SCNT embryos were cultured for 7 days to evaluate the in vitro development, apoptosis and DNA methylation in blastocysts. The apoptosis was measured by TUNEL assay and caspase-3 activity assay. Methylated DNA of SCNT embryos at the blastocyst stages was detected using a 5-methylcytidine (5-MeC) antibody. The developmental rate to the blastocyst stage was significantly higher (P<0.05) in GSH treatment group (32.5±1.2%, 78/235) than that of non-treated control SCNT embryos (22.3±1.8%, 50/224). TUNEL assay revealed that the numbers of apoptotic cells in GSH treatment group (2.3±0.4%) were significantly lower (P<0.05) than that of control (3.8±0.6%). Relative caspase-3 activity of GSH treated group was 0.8±0.06 fold compared to that of control. DNA methylation status of blastocysts in GSH treatment group (13.1±0.5, pixels/ embryo) was significantly lower (P<0.05) than that of control (17.4±0.9, pixels/embryo). These results suggest that antioxidant GSH treatment during SCNT procedures can improve the embryonic development and reduce the apoptosis and DNA methylation level of bovine SCNT embryos, which may enhance the nuclear reprogramming of bovine SCNT embryos.

Antioxidants, as reactive oxygen species scavengers, are one of the beneficial additives in serum-free defined culture medium. In this study, three separate experiments were performed to determine the effects of 3-hyroxyflavone added to the culture medium on the developmental competence of follicular bovine oocytes during in vitro maturation (IVM) and/or in vitro culture (IVC). The rate of blastocyst developed from oocytes cultured in IVM medium with 3 hyroxyflavone was significantly higher than that from control oocytes (39.0% vs. 26.3%, p<0.001), respectively. However, oocytes cultured in the medium with addition of 3-hyroxyflavone only at IVC period did not show significance in the blastocyst development when compared with control. When 3-hyroxyflavone was added to both IVM and IVC media, the rate of blastocyst formation was even significantly lower (21.1%) than control (26.5%; p<0.05). The present findings suggested that antioxidative activity of 3-hydroxyflavone added to only IVM medium beneficially affected the developmental competence of follicular bovine

The aim of the present study was to compare two different serum-free media, modified synthetic oviduct fluid (mSOF) and modified potassium simplex optimization medium (mKSOM) containing 20% RD (RPMI1640 + DMEM, 1:1 v/v) (RD-mKSOM), for in vitro culture (IVC) of bovine embryos. After in vitro maturation and fertilization, the presumptive zygotes were cultured in two different serum-free conditions for 7 days and 9 days to evaluate blastocyst formation and hatching, respectively. Serum supplemented conventional CR2 medium was used as control. After 7 day of culture, there was no significant difference in cleavage and blastocyst formation rates among three groups (mSOF, 59.3 and 30.1%; RD-mKSOM, 65.0 and 41.5%; control, 51.6 and 38.0%, respectively). Hatching rate was significantly higher in control (69.0%) than other experimental groups (mSOF, 22.0%; RD-mKSOM, 39.5%) (P＜0.0001 and P＜0.001, respectively). Although both serum-free conditions showed lower hatching rates than serum-added control, in serum-free groups, RD-mKSOM showed significantly higher hatching rate than mSOF (P＜0.001). In addition, one-step using RD-mKSOM may facilitate IVC procedure than two-step culture system. In conclusion, the results indicate that one-step RD-mKSOM is more suitable defined culture system for IVC of bovine embryos than two-step mSOF.

Programmed cell death or apoptosis is associated with changes in K+ concentration in many cell types. Recent studies have demonstrated that two-pore domain K+ (K2P) channels are involved in mouse embryonic development and apoptotic volume decrease of mammalian cells. In cerebellar granule neurons that normally undergo apoptosis during the early developmental stage, TASK-1 and TASK-3, members of K2P channels, were found to be critical for cell death. This study was performed to identify the role of K+ channels in the H2O2-induced or cryo-induced cell death of mouse and bovine embryos. Mouse and bovine two-cell stage embryos (2-cells) exposed to H2O2 for 4 h suffered from apoptosis. The 2-cells showed positive TUNEL staining. Treatment with high concentration of KCl (25mM) inhibited H2O2-induced apoptosis of 2-cells by 19%. Cryo-induced death in bovine blastocysts showed positive TUNEL staining only in the cells near the plasma membrane. Cryoprotectant supplemented with 25 mM KCl reduced apoptosis slightly compared to cryoprotectant supplemented with 5 mM KCl. However, the combination of antioxidants (β-mercaptoethanol) with 25 mM KCl significantly decreased the rate of H2O2-induced and cryo-induced apoptosis compared to treatments with only antioxidants or 25 mM KCl. These results show that blockage of K+ channel efflux for a short-time reduces H2O2- and cryo-induced apoptosis in mouse and bovine embryos. Our findings suggest that apoptosis in mouse and bovine embryos might be controlled by modulation of K+ channels which are highly expressed in a given cell type.

K+ channels are involved in the regulation of a variety of physiological functions, including proliferation, apoptosis and differentiation, in mammalian cells. Our previous study demonstrated that the blockage of K+ channels inhibits mouse early embryonic development. This study was designed to identify the effect of K+ channels during bovine embryonic development. K+ channel blockers (tetraethylammonium (TEA), BaCl2, quinine, ruthenium red and fluoxetine) were added to the culture medium during in vitro fertilization (IVF) for 6 h to first identify the short-term effect of these chemicals. Among K+ channel blockers, fluoxetine, which is used as a selective serotonin reuptake inhibitor, significantly increased the blastocyst formation rate by approximately 6% when compared to control. During the in vitro maturation (IVM) of immature oocytes and the in vitro culture (IVC) of embryos, the oocytes and embryos were exposed to fluoxetine for either a short-term (6 h) or a long-term (24 h) to compare the embryonic development in response to exposure time. The 6 h exposure to fluoxetine during IVM did not affect the blastocyst formation rate, but the rate of blastocyst formation was reduced after the 24 h exposure. On the other hand, embryonic development increased approximately 10% in both groups of embryos exposed to fluoxetine for 6 and 24 h during IVC. Taken together, fluoxetine treatment during IVF and IVC, but not IVM, enhances bovine embryonic development. These results suggest that fluoxetine-modulated signals in oocytes and embryos could be an important factor towards enhancing bovine embryonic development.

This study was carried out to investigate the effects of tissue inhibitor of matalloproteinase-1 (TIMP-1), Activin A and Heparin binding epidermal growth factor (HB-EGF) on in vitro production of bovine embryos. In experiment 1, presumptive zygotes were cultured in the medium supplemented with TIMP-1 (0.5 μg/ml), Activin A (100 ng/ml), or HB-EGF (100 ng/ml) at 39 ℃ in a humidified atmosphere of 5% (v/v) CO2, 5% (v/v) O2 and 90% (v/v) N2. In experiment 2, TIMP-1 + HB-EGF or Activin A + HB-EGF combinations were supplemented in the culture medium. The developmental rate to blastocysts, hatching rate and total cell numbers of the blastocysts were evaluated in both experiments. The embryos cultured in medium without growth factor supplementation was used as control group. In experiment 1, the embryos cultured in medium supplemented with TIMP-1 and Activin A showed significantly higher developmental rate to blastocysts than those cultured with HB-EGF and control (36.9%, 34.1%, 21.2% and 23.1%, respectively) (P<0.0001). However, the hatching rate of blastocyst was significantly higher in embryos with HB-EGF than those with TIMP-1, Actvin A and Control groups (84.4%, 58.8%, 51.4% and 49.3%, respectively) (P<0.001). Total cell number per blastocyst was also significantly higher in embryos with HB-EGF group (174.3±2.5) than those with TIMP-1, Activin A (149.7 and 150.0, respectively) (P<0.05) and Control (119.0) (P<0.001). In experiment 2, embryos cultured with combined treatment of Activin A and HB-EGF resulted in significantly higher rates of blastocysts formation (48.0%), hatching rate (89.7%) and total cell number in blastocyst (182.3±2.1) than those with TIMP-1 and HB-EGF combination group (32.0%, P<0.001; 76.6%, P<0.05; 165.7±4.2, P<0.001, respectively). Our data demonstrate that in vitro production of bovine embryos could be improved by combined supplementation of Activin A and HB-EGF in culture medium.

Matrix Metalloproteinases (both MMP2 and -9) play a pivotal role of the embryos hatching and implantation. Therefore, the objective of this study was carried out to investigate the influence of MMP2 and MMP9 on embryo development potential and subsequent effect at molecular level. There was no significant difference of cleavage rate among the groups. The development competence of blastocyst was significantly higher (P<0.05) in MMP9 treatment (39.81±16.61) than that to the combined treatment of MMP2 and –9 (23.68±0.27), but there was no significant difference among the control vs. MMP2 vs. MMP9 (35.05±2.74 vs. 32.71±6.18 vs. 39.81±16.61, respectively). On the other hand, the hatching rate of blastocysts was significantly lower (P<0.05) in combined group of MMP2 and –9 (12.55±0.09) (Table1). The expression level of MMP2 and MMP9 was significantly lower (P<0.05) in the entire treatment groups than that in the control group. But the expression of MMP9 was significantly higher (P<0.05) when compared in the entire treatment groups. The relative expression embryonic developmental gene, IFNt expression level significantly lower (P < 0.05) in the MMP9 embryos. The placenta establishment genes, PLAC8 and SSLP1, expression were significantly higher (P < 0.05) in the MMP2 embryos compared to other groups. Transcription regulation gene, HNRNPA2B1, was higher (P < 0.05) in the combined group of MMP2+MMP9 than that in the other groups. In conclusion, our results suggest that MMPs to culture medium improves the blastocyst development rate and further impact on target gene expression analysis.

Matrix Metalloproteinases (MMP)-2 and -9 are participated in embryo development, implantation, remodeling of epithelial cell and ovulation. The objective of this study is to evaluate an impact of MMP2 and MMP9 on embryonic developmental competence as well as gene expression profiles of in vitro-produced bovine embryos. After in vitro fertilization, embryos of all groups were transferred into IVC-2 medium treated with MMP2 and MMP9 to check the optimum concentration on the basis of embryo development competence and cell numbers. The optimum concentrations for MMP2 and 9 were 1,200 ng/ml and 300 ng/ml. The blastocyst development competence was not different among 1,200 ng/ml of MMP2 vs. 300 ng/ml of MMP9 vs. combined MMP2 + 9 vs. control groups (41.46 ± 10.66 vs. 37.73 ± 8.92 vs. 45.11 ± 11.41% vs. 41.59 ± 11.88, respectively). Furthermore, the developmental competences to hatching and hatched blastocysts were not also different among the same groups (79.84 ± 12.63 vs. 83.3 ± 17.46 vs. 78.55 ± 14.48% vs. 72.02 ± 14.09). In addition, total cell number was significantly (p<0.05) greater in blastocyst treated with MMP9 300 ng/ml among all treatment groups. On the other hand, there was no significant difference of ICM vs. TE ratio in all groups. The expression of five out of six genes (i.e., MMP2, MMP9, IFNt, SSLP1 and HNRNPA2B1) was different among the groups. The expression of IFNt and HNRNPA2B1 genes was significantly greater in MMP9 (p<0.05), but there was no difference of MMP9 expression between MMP2 and MMP9 group (p>0.05). The normalized expression of MMP2 and SSLP1 was greater in MMP2 than other groups (p<0.05). In conclusion, MMPs treatment during IVC-2 medium was remarkably effected on blastocyst developmental competence and gene expression profiles that are related to embryo quality and implantation.

Lacticin NK34 is a small nisin-like bacteriocin present in the supernatants of an isolate of Lactococcus lactis from jeotgal, a salted and fermented Korean food made with seafood such as shrimp, oysters, and fish. Recently, we demonstrated that a partially purified NK34 is highly effective against various Staphylococcus species in a murine infection model. In this study, the two major bacterial pathogens associated with bovine mastitis, Streptococcus aureus and S. agalactiae, were evaluated for their susceptibility to NK34 in vitro using a standard teat-dip assay as well as in vivo using mastitic cows infected with one of these pathogenic strains. The experimental analyses showed a significant decrease (up to 98 times) in the bacterial numbers between the NK34-treated and -untreated teats. Moreover, a dramatic reduction in somatic cell counts was observed at 3 days post-treatment with 10 ml of NK34 administered directly into the mastitic cows. Neither S. aureus nor S. agalactiae were recovered. Taken together, these results imply that lacticin NK34 is an alternative antimicrobial substitute for the treatment of bovine mastitis, caused especially by either S. aureus or S. agalactiae.

A cell line of bovine origin was immortalized to isolate foot-and-mouth disease virus (FMDV). The immortalization was performed by infection of bovine primary epithelial cells with a recombinant retrovirus that overexpressed the human telomerase (hTERT), after primary culture of fetal bovine kidney tissue and removal of fibroblasts. After cloning the immor- talized cell line into single cells, the cloned cell lines were named JNUBK-1, JNUBK-2, JNUBK-3 and JNUBK-4, according to their characteristics. To confirm the epithelial phenotype of the cell lines JNUBK-3 and JNUBK-4, which showed stable proliferation capability over 35 generations after immortalization, the expression of cytokeratin and fibronectin was measured. Finally, the FMDV titer in the JNUBK-3 and JNUBK-4 cell lines was measured and was 800∼2,000 times higher than that of the currently used cell line IRBS-2. In conclusion, more sensitive isolation and production of FMDV became possible through the use of the immortalized JNUBK-3 and JNUBK-4 cell lines.

Oxygen consumption is a useful parameter for evaluating mammalian embryo quality, since individual bovine embryos was noninvasively quantified by scanning electrochemical microscopy (SECM). Recently, several approaches have been used to measure the oxygen consumption rates of individual embryos, but relationship between oxygen consumption and pregnancy rates of Hanwoo following embryo transfer has not yet been reported. In this study, we measured to investigate the correlation between oxygen consumption rate and pregnancy rates of Hanwoo embryo using a SECM. In addition to, the expression of pluripotent gene and anti-oxidant enzyme was determined using real-time PCR by extracting RNA according to the oxygen consumption of in vivo embryo. First, we found that the oxygen consumption significantly increased in blastocyst-stage embryos (blastocyst) compared to early blastocyst stage embryos, indicating that oxygen consumption reflects the embryo quality (Grade I). Oxygen consumption of blastocyst was measured using a SECM and total cell number of in vitro blastocyst was enumerated by counting cells stained by propidium iodide. The oxygen consumption or GI blastocysts were significantly higher than those of GII blastocysts (10.2 × 1015/mols—1 versus 6.4 × 1015/mols—1, p<0.05). Total cell numbers of in vitro blastocysts were 74.8, 90.7 and 110.2 in the oxygen consumption of below 10.0, 10.0∼12.0 and over 12.0∼1015/mols—1, respectively. Pregnant rate in recipient cow was 0, 60 and 80% in the transplantation of embryo with the oxygen consumption of below 10.0, 10.0∼12.0 and over 12.0 × 1015/mols—1, respectively. GPX1 and SOD1 were significantly increased in over —10.0 group than below 10.0 groups but in catalase gene, there was no significant difference. On the other hand, In OCT-4 and Sox2, pluripotent gene, there was a significant difference (p<0.05) between the below-10.0 (0.98 ± 0.1) and over 10.0 (1.79 ± 0.2). In conclusion, these results suggest that measurement of oxygen consumption maybe help increase the pregnant rate of Hanwoo embryos.

In mammal, unfertilized oocytes remain in the oviduct or under in vitro culture, which is called "oocyte aging". This asynchrony negatively affects fertilization in pre- and post-implantation embryo development. Caffeine a phos-phodiesterase inhibitor is known to rescue oocyte aging in several species. The objective of this study is to determine the cytoskeleton distribution in aged oocytes and the embryo developmental ability of aged oocytes in the present or absence of caffeine during maturation. Caffeine treatment increased the incidence of normal spindle assembly of aged oocytes (treatment, 67.57±4.11% aging, 44.61±6.4%) and no significant differences compared to control group. Fluorescence values were compared using ROS (Reactive oxidation species) stain. Fluorescence values appear of con-trol group intensity rate (51.53.±3.80), aging group (68.10±5.54) and treatment of caffeine (45.04±2.98). Aged oocytes that were derived from addition of caffeine to the IVM (in vitro maturation) medium had significantly increased 2-cell that developed to the blastocyst stage compared to the aging group. Blastocysts, derived from caffeine treatment group, significantly increased the total cell number compare aging (90.44±10.18 VS 67.88±7.72). Apoptotic fragments of genomic DNA were measured in individual embryo using TUNEL assay. Blastocyst derived from caffeine treatment group decreased significantly the apoptotic index compared to blastocyst derived from aging group. In conclusion, we inferred that the caffeine treatment during oocyte aging can improve the developmental rate and quality in bovine embryos developing in vitro

Many countries have implemented genetic evaluation for fertility traits in recent years. In particular, reproductive trait is a complex trait and need to require a system-level approach for identifying candidate genes related to the trait. To find the candidate gene associated with reproductive trait, we applied a weighted gene co-expression network ana-lysis from expression value of bovine genes. We identified three co-expressed modules associated with reproductive trait from bovine microarray data. Hub genes (ZP4, FHL2 and EGR4) were determined in each module; they were topologically centered with statistically significant value in the gene co-expression network. We were able to find the highly co-expressed gene pairs with a correlation coefficient. Finally, the crucial functions of co-expressed modules were reported from functional enrichment analysis. We suggest that the network-based approach in livestock may an important method for analyzing the complex effects of candidate genes associated with economic traits like repro-duction.