Effects of ghrelin on in vitro nuclear maturation and subsequent embryo development of immature bovine oocytes

Development of efficient culture system to support embryonic development would be valuable when percentage of produced embryos reaching to the blastocyst stage is important. However, the rate of bovine embryo production in vitro is still lower than expected. Present study was performed to investigate the effect of ghrelin on nuclear maturation and subsequent bovine embryo development in vitro. Cumulus-oocyte-complexes were collected from slaughterhouse ovaries and randomly allocated in each treatment groups. Five different concentrations of ghrelin (0, 5, 50, 500 and 1000 ng mL-1) were added to the in vitro maturation medium (Hepes-buffered medium 199+fetal calf serum+gonadotrophins+insulin+antibiotics). The proportion of oocytes developed to metaphase II stage was significantly increased at 5 and 50 ng mL-1 ghrelin (86.32±3.38 and 89.77±2.92%, respectively). The result also indicated that adding high concentration of ghrelin adversely affect (p<0.05) the nuclear maturation rates of bovine oocytes. However, the subsequent embryo development was not significantly affected by addition of ghrelin to the IVM medium. This study showed that inclusion of 5-50 ng mL-1 ghrelin in maturation medium may have beneficial effects on nuclear maturation of bovine oocytes in vitro

A comparative study of MTA solubility in various media

INTRODUCTION: Solubility of root filling materials is heavily influenced by the environment they are in contact with. This study compared the solubility of ProRoot MTA in deionized water and synthetic tissue fluid. MATERIALS &amp; METHODS: Forty specimens of prepared MTA were immersed in deionized water and synthetic tissue fluid (20 samples each). The solubility was assessed after 7 and 28 days. Scanning electron microscope observation was also performed. The mean weight loss was evaluated using a digital scale. Data were analyzed using one-way ANOVA. Tukey test was performed for multiple comparisons. RESULTS: MTA solubility in synthetic tissue fluid was significantly lower than deionized water after 7 and 28 days (P&lt;0.05). Secondary electron detectors revealed the presence of lumps and platelets on the surfaces of both specimens. Also, more voids were observed in specimen stored in deionized water. CONCLUSION: MTA dissolved faster in deionized water than synthetic tissue fluid. Despite this, the solubility of this material in both media was acceptable.

The role of metalloproteinase and hypoxia conditions in endometrial cells and embryo implantation

In the process of implantation, metalloproteinase enzymes play a key role in basement membrane degradation and endometrial extracellular matrix. The activity of these enzymes is impeded by binding Tissue Inhibitors of Metalloproteinase (TIMP). The oxygen concentration in the mammalian uterus at the time of implantation is about 2-5%. It is seen that the imposition of hypoxia on cancer cells increases the expression of metalloproteinase enzymes and reduces the expression of metalloproteinase inhibitors, resulting in increased cell invasion. To know the effect of Hypoxia-Inducible Factor (HIF) and other related factors, we decided to evaluate hypoxic conditions on endometrial epithelial cells of the uterus and roll of matrix metalloproteinases (MMPs) on angiogenesis and invasion of the embryo during implantation. In this study, human and mouse endometrial epithelial cells were incubated for 24-48 hours in hypoxic conditions. Subsequently, the expression level of TIMP-1 was measured in mouse and human epithelial cells by Real-Time PCR technique. The cell viability in hypoxic conditions was evaluated by MTT assay. Our results demonstrated that hypoxia reduced the quantitative gene expression of TIMP-1 in the human and mouse endometrial epithelial cells compared to the control group. It can be concluded that applying hypoxic conditions by reducing the TIMP-1 expression and consequently increasing MMP expression, may improve the embryo implantation rate

Selection of immature bovine oocytes using Brilliant Cresyl Blue enhances nuclear maturity after vitrification

Beside cooling/warming rates and composition of vitrification solution, developmental stage of immature oocytes may also affect their vitrification outcome. The aim of the present study was to evaluate the selection effect of developmentally competent immature bovine oocytes by Brilliant Cresyl Blue (BCB) on maturity of oocytes after vitrification. Oocytes were obtained from slaughterhouse ovaries. Only oocytes with 4-5 layers of cumulus cells and homogenous cytoplasm were used. After exposure to BCB stain, immature oocytes were divided into colored (BCB+) and colorless (BCB-) cytoplasm groups. Immature oocytes were equilibrated in VS1 (7.5 Ethylene Glycol (EG)+7.5% DMSO) for 10-12 min and then exposed to VS2 (15% EG+ 15% DMSO+0.5M sucrose) for 1 min. Thereafter, oocytes were loaded on Cryotop and directly plunged into liquid nitrogen. After warming, oocytes were examined for presence of polar body and nuclear maturity. Higher number of oocytes in BCB+group extruded first polar body in comparison with other vitrified groups but not significantly (p>0.05). Compared to the BCB- oocytes, there was significantly lower percentage of degeneration for BCB+oocytes (p<0.05). Within vitrified groups, reaching to the MII stage was significantly higher in BCB+group (51.5%) compared with BCB and vitrified-control groups (27.9 and 40.3%, respectively). These results indicated that selection of potent immature bovine oocytes using brilliant cresyl blue improved the nuclear maturity of immature oocytes after vitrification. In addition, this selection can be a valuable tool to improve the vitrification outcome

Cryotop device enhances vitrification outcome of immature Bovine Oocytes.

The aim of this study was to evaluate the effectiveness of different cryodevices (Open Pulled Straw (OPS), Electron Microscopy Grid (EMG) and cryotop for vitrification of immature bovine oocytes. Polar body, MII stage, survivability and subsequent developmental rates were compared. Only oocytes with 4-5 layers of cumulus cells were used. Oocytes were equilibrated in the first vitrification solution (VS1; HS+10% DMSO+10% Ethylene Glycol (EG)) for 30-45 sec and then in the second vitrification solution (VS2; 20% DMSO+20% EG+0.5 M Sucrose) for 25 sec. Within 30 sec they were mounted on one of the cryodevices and directly plunged into Liquid Nitrogen (LN2) for 10 days. Immature oocytes vitrified using cryotop represented higher rate of polar body extrusion and nuclear maturity (p<0.05). The highest survivability resulted from cryotop and EMG groups and no significant difference found between them. Vitrified oocytes in cryotop group had highest cleavage and blastocyst rates. All of the mean measured rates for vitrified/warmed immature oocytes were significantly lower than that of control group (p<0.05). In conclusion, results of this study showed the superiority of cryotop device for vitrification of immature bovine oocytes which resulted in higher viability and subsequent embryo development

Sequence and phylogenetic analysis of the non-structural 3A and 3B protein-coding regions of foot-and-mouth disease virus subtype A Iran 05

The A Iran 05 foot-and-mouth disease virus (FMDV) subtype was detected in Iran during 2005 and has proven to be highly virulent. This study was undertaken to focus on molecular and phylogenetic analysis of 3A and 3B coding-regions in the A Iran 05 field isolate. To assess the genetic relatedness of A Iran 05 isolate the nucleotide and predicted amino acid sequences of the 3AB region of type A FMDV isolates were compared with twenty previously described type A FMDV isolates. The phylogenetic tree based on the 672 bp 3AB gene sequences of type A FMDV from thirteen different locations clustered them into five distinct lineages. The A Iran 05 isolate clustered in lineage A along with four type A variants and was closely matched with viruses isolated in Turkey and Pakistan during 2005~2006. The number of protein sequence differences exhibited by each of the isolates revealed that A Iran 05 isolate contains three amino acid substitutions at positions 47 and 119 of 3A and 27 of the 3B coding region. The nucleotide identity between A Iran 05 and the other four isolates of lineage A was estimated to be 98%

Types of neural guides and using nanotechnology for peripheral nerve reconstruction

Peripheral nerve injuries can lead to lifetime loss of function and permanent disfigurement. Different methods, such as conventional allograft procedures and use of biologic tubes present problems when used for damaged peripheral nerve reconstruction. Designed scaffolds comprised of natural and synthetic materials are now widely used in the reconstruction of damaged tissues. Utilization of absorbable and nonabsorbable synthetic and natural polymers with unique characteristics can be an appropriate solution to repair damaged nerve tissues. Polymeric nanofibrous scaffolds with properties similar to neural structures can be more effective in the reconstruction process. Better cell adhesion and migration, more guiding of axons, and structural features, such as porosity, provide a clearer role for nanofibers in the restoration of neural tissues. In this paper, basic concepts of peripheral nerve injury, types of artificial and natural guides, and methods to improve the performance of tubes, such as orientation, nanotechnology applications for nerve reconstruction, fibers and nanofibers, electrospinning methods, and their application in peripheral nerve reconstruction are reviewed

Biological activity (antibacterial, antifungal, antiviral and cytotoxic) of extract from Dysidea spp.

Sponges are the most primitive of the multicellular, These organisms don’t have any mechanical defense system, so their early appearance in evolution has given them alot of time for the development of advanced secondary metabolites as chemical defense system. Sponges have the potential to provide drugs from chemical components against diseases. In this investigation the sponge samples, which it is Dysidea spp. , were collected at depth of 15- 20 meter, from locations on the coastline of Island Hengam in Persian Gulf of Iran. For identifying natural components, methanolic and diethyletter were used as extraction solvents, after removal of the solvents, the GC/MS spectra of the fraction were obtained. Then in vitro cytotoxic, antimicrobial, antifungal and antiviral activities were identified. In vitro cytotoxity screening, by XTT assay, against KB/ C152 and HUT-78/ C185 cell line, was conducted in this study in 1 - 500 µg/ml . IC50 for diethyletter and methanolic extract was 200 µg/ml in HUT-78 , IC50 for diethyletter extract was 325µg/ml and methanolic extract 325µg/ml in KB. In vitro antimicrobial activity by Broth Dilution Methods against clinical gram-positives and gram negatives (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus و subtilis Bacillus). The results conducted that the MIC values of methanol and diethyletter extract for Escherichia coli 20mg/ml, Bacillus subtilis 10mg/ml and 2mg/ml for Staphylococcus aureus. The MBC values of the diethyletter extracts for Bacillus subtilis 30 mg/ml) and S. aureus aureus 10mg/ml. In vitro antifungal activity by Broth Dilution Methods against clinical pathogens; Candida albicans and Aspergillus fumigatus. The results conducted that the aqueous extracts didn’t have any antifungal activities on pathogens, minimum inhibitor concentrations (MIC) of the diethyletter extract on C. albicans 0/75mg/ml, MFC 5 mg/ml and methanolic extract 0.5mg/ml and MFC 5 mg/ml on A. fumigatus In vitro antiviral activities by XTT assay against MT-2 cell line. The results conducted that IC50 for diethyletter extract 500µg/ml and methanolic extract 475 µg/ml

Effect of equilibration temperature on in vitro viability and subsequent embryo development of vitrified-warmed immature bovine oocytes

Problem statement: Vitrification is replacing conventional slow freezing to cryopreserve gametes and embryos especially for in vitro production of embryo in domestic animal species. However, the results are still not satisfactory. The aim of this experiment was to study the effect of different equilibration temperatures on in vitro viability of immature bovine oocytes after vitrification. Approach: Oocytes were obtained from slaughterhouse ovaries. Only grade one oocytes were used. Oocytes were equilibrated in three different temperatures: 32, 37, or 41°C. Immature oocytes were equilibrated in VS1 (7.5 Ethylene Glycol (EG) + 7.5% DMSO) for 10-12 min and then exposed to VS2 (15% EG + 15%DMSO + 0.5M sucrose) for one min. Thereafter oocytes were loaded on hand-made Cryotop and directly plunged into liquid nitrogen. After warming, oocytes were examined for viability, maturation, cleavage and blastocyst production. Results: Oocytes that were equilibrated at 37°C had significantly higher (p<0.05) viability than 41°C, but there were no significant difference between 37 and 41 with 32°C. Maturation rate in 37°C group was significantly higher compared with other groups. The highest percentage of degenerated and germinal vesicle stage oocytes were obtained from 41°C than 32 and 37°C. Cleavage rate of 37°C group (38.77%) was greater than other groups (30.84 and 28.95% for 32 and 41°C, respectively). The highest blastocyst rate was also produced when oocytes equilibrated at 37°C (6.45%). Conclusion: In conclusion, these results indicated that immature bovine oocytes can be equilibrated successfully at 37°C while higher or lower temperature can significantly decrease their subsequent viability and development