Hank's Salt Research Articles

Devitrite (Na2Ca3Si6O16) phase dominated nanostructured 45S5 bioactive glass: exploring its structural and biological properties

This research study is primarily centred around calcination temperature and time influence on phase formation in bioactive glasses (BGs). In the present study, BG with a nominal composition of 45S5 was synthesized through the sol–gel process. The developed BGs then underwent heat treatment for various sintering durations and temperatures. X-ray diffraction (XRD) patterns of the BGs reveals that the sintering process led to the crystallization of both devitrite (Na2Ca3Si6O16) and combeite (Na2Ca2Si3O9) phases. The field emission scanning electron microscopy study divulges morphological alterations, from sheet-like to rod-like structures to eventually transforming into spherical and sheet-like structures. The surface area and Type-IV mesoporous porosity were validated through Brunauer Emmett Teller analysis, highlighting a notable increase in pore volume and mechanical strength at a lower sintering temperature. In vitro apatite formation was carried out in Hank’s balance salt in order to evaluate the bioactivity of the glass. After 7 d of immersion in simulated body fluid (SBF), XRD patterns and scanning electron microscopy micrographs results showed that formation of hydroxyapatite layer on the surface of the BGs. The BG compatibility with erythrocytes (red blood cells) was also studied, and the results revealed that there was only a low 2% lysis, showing good hemocompatibility. The drug loading and release behaviour of the BGs was studied in the in vitro analysis. The findings showed a high drug encapsulation effectiveness of up to 90% and continuous drug release from the BGs for 24 h. The materials biocompatibility was unambiguously confirmed by cytocompatibility and proliferation studies. This study provides compelling evidence for the exceptional efficacy and promise of the distinct 45S5 BGs in advancing the field of regenerative medicine.

Characteristics of Bio-ceramic Coating on Corrosion Rate of Stainless Steel 316L and Titanium (Ti-6Al-4V)

Thermal spray coating improves SS 316L corrosion resistance using biomaterials like hydroxyapatite (HA), zirconium dioxide (ZrO2), and titanium dioxide (TiO2), promoting bone binding and biological fixation. The study examined the impact of bio-ceramic coatings like HA, HA+TiO2, and HA+ZrO2 on the corrosion rate of stainless steel and titanium in Hank's Balance Salt Solution and artificial saliva media. Electrochemical testing with polarization was carried out to determine the performance of biomaterials against corrosion rates. The addition of TiO2 and ZrO2 to HA in both SS316L and Ti6Al4V materials reduced the corrosion rate more than HA. This study revealed that coating with HA, HA + ZrO2, and HA + TiO2 biomaterials results in varying thicknesses and roughness under the same spraying parameters. Bioceramic coatings on implants offer an alternative coating to improve the performance of biomaterials.

Analysis of KATP Channels Opening Probability of Hippocampus Cells Treated with Kainic Acid.

BackgroundKainic acid (KA)-induced seizures may be a valuable tool in the assessment of anti-epileptic drug efficacy in complex partial seizures. This study investigated the effects of KA on ATP-sensitive K+ (KATP) channels opening probability (NPo), which plays a crucial role in neuronal activities.MethodsFor the optimisation and validation protocol, β-cells were plated onto 35 mm plastic petri dishes and maintained in RPMI-1640 media supplemented with 10 mM glucose, 10% FCS and 25 mM of N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES). The treatment effects of 10 mM glucose and 30 μM fluoxetine on KATP channels NPo of β–cells were assessed via cell-attached patch-clamp recordings. For hippocampus cell experiments, hippocampi were harvested from day 17 of maternal Lister-hooded rat foetus, and then transferred to a Ca2+ and Mg2+-free HEPES-buffered Hank’s salt solution (HHSS). The dissociated cells were cultured and plated onto a 25 mm round cover glasses coated with poly-d-lysine (0.1 mg/mL) in a petri dish. The KATP channels NPo of hippocampus cells when perfused with 1 mM and 10 mM of KA were determined.ResultsNPo of β-cells showed significant decreasing patterns (P < 0.001) when treated with 10 mM glucose 0.048 (0.027) as well as 30 μM fluoxetine 0.190 (0.141) as compared to basal counterpart. In hippocampus cell experiment, a significant increase (P < 0.001) in mean NPo 2.148 (0.175) of neurons when applied with 1 mM of KA as compared to basal was observed.ConclusionThe two concentrations of KA used in the study exerted contrasting effects toward the mean of NPo. It is hypothesised that KA at lower concentration (1 mM) opens more KATP channels, leading to hyperpolarisation of the neurons, which may prevent neuronal hyper excitability. No effect was shown in 10 mM KA treatment, suggesting that only lower than 10 mM KA produced significant changes in KATP channels. This implies further validation of KA concentration to be used in the future.

Human Dental Pulp Stem Cells (DPSCs) Therapy in Rescuing Photoreceptors and Establishing a Sodium Iodate-Induced Retinal Degeneration Rat Model.

Different methods have been used to inject stem cells into the eye for research. We previously explored the intravitreal route. Here, we investigate the efficacy of intravenous and subretinal-transplanted human dental pulp stem cells (DPSCs) in rescuing the photoreceptors of a sodium iodate-induced retinal degeneration model. Three groups of Sprague Dawley rats were used: intervention, vehicle group and negative control groups (n = 6 in each). Intravenous injection of 60mg/kg sodium iodate (day 0) induced retinal degeneration. On day 4 post-injection of sodium iodate, the rats in the intervention group received intravenous DPSC and subretinal DPSC in the right eye; rats in the vehicle group received subretinal Hank's balance salt solution and intravenous normal saline; while negative control group received nothing. Electroretinogram (ERG) was performed to assess the retinal function at day 0 (baseline), day 4, day 11, day 18, day 26, and day 32. By the end of the study at day 32, the rats were euthanized, and both their enucleated eyes were sent for histology. No significant differencein maximal ERGa-wave (p = 0.107) and b-wave, (p = 0.153) amplitudewas seen amongst the experimental groups. However, photopic 30 Hz flicker amplitude of the study eye showed significant differences in the 3 groups (p = 0.032). Within the intervention group,there was an improvement in30 Hzflicker ERG responseof all 6 treatedright eyes, which was injected with subretinal DPSC; while the 30Hz flicker ERG of the non-treated left eyes remained flat. Histology showed improved outer nuclear layer thickness in intervention group; however, findings were not significant compared to the negative and vehicle groups. Combination of subretinal and intravenous injection of DPSCs may have potential to rescue cone function from a NaIO3-induced retinal injury model.

Tribocorrosion performance of Nitrogen-doped diamond like carbon coating by high power impulse magnetron sputtering technique

Nitrogen-doped diamond like carbon film is promising in biological applications, studying the synergistic tribocorrosion performance is indispensable. In this paper, Nitrogen-doped diamond like carbon films were deposited on AISI 304L austenitic stainless steels and Si substrate by using the high power impulse magnetron sputtering technique using Ar and N&lt;sub&gt;2&lt;/sub&gt; as precursors at room temperature. The effect of target pulse duration on the structure, mechanical properties, corrosion resistance and tribocorrosion properties in Hank's equilibrium salt solution and the corresponding mechanism were studied. The results of scanning electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy and nano-hardness test showed that the nitrogen-doped diamond like carbon coatings prepared at a target pulse duration of 60 μs showed the sp&lt;sup&gt;3&lt;/sup&gt; bonding content of 33.9% with the hardness of 12.4 GPaand the root mean square roughness of 0.63 nm. With the increase in pulse duration to 90 μs, the sp&lt;sup&gt;2&lt;/sup&gt; bonding increased, meanwhile the surface roughness increased. The results of potentiodynamic polarization indicated that the Nitrogen-doped diamond like carbon coating prepared at 60μs had best corrosion resistance with the corrosion current density of 7.65 × 10&lt;sup&gt;–8&lt;/sup&gt; A·cm&lt;sup&gt;–2&lt;/sup&gt;. The effect of the target pulse duration on tribocorrosionbehaviour of the Nitrogen-doped diamond like carboncoating was investigated in Hank’s solution by a reciprocating tribometer equipped with a three-electrode electrochemical cell.The coatings at 60 μs exhibited excellent tribocorrosion properties with high open circuit potential of 39 mV, low COF of 0.05 without pitting corrosion due to high corrosionresistance, low contact angel and dense microstructure.The results indicated that corrosion can be accelerated by friction, but it also affect the mechanical properties of the Nitrogen-doped diamond like carbon coatings. The increase in pulse duration to 90 μs, leading to the reduction of sp&lt;sup&gt;3&lt;/sup&gt; bonds which can form a cross-linking structure. The degraded cross-linking structure decreased the corrosion resistance of the coating via the increased porosity in the coating, which weakened the interfacial strength of the coating, and ultimately led to failure of the coatingunder the action of wear.

Surface Modifications to the Ti6Al4V Alloy for Improvement as Biomaterial

The present work is a study of the thermally treated [Ti] alloy Ti6Al4V by annealing at two different temperatures [700 and 800°C] and its resistance and corrosion behavior were evaluated using electrochemical techniques such as: polarization curves [CP] and electrochemical impedance spectroscopy [EIS] in a Hank's salt solution. In addition, the samples were characterized by scanning Electron Microscopy [SEM] and microanalysis by X-ray Spectroscopy by energy dispersion [EDS], in their conditions of arrival [untreated] and after thermal treatments [TT] and chemical conversion with cerium and lanthanum salts [CCT]. The results of the electrochemical tests indicate the decrease in the corrosion rate of the samples with CCT and TT with respect to the untreated and the presence of biphasic microstructure constituted by an α - rich [Al - rich] matrix with β [V - rich].

92 DOES BLASTOCENTESIS AFFECT CRYOPRESERVATION SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS?

The cattle industry primarily employs embryo bisection in order to obtain genetic samples for pre-implantation screening and selection of embryos. Although practical and rapid, bisection is invasive and adversely affects embryo viability and cryopreservation. An alternative biopsy approach is to aspirate the blastocoele fluid (referred to as blastocentesis), which not only provides a genetic sample, but also has the potential to improve cryopreservation (Palini et al. 2013 Repro. Biomed. 26, 603–610). This study investigates blastocentesis as a low impact biopsy procedure to rapidly sample bovine blastocysts with limited effect on embryo cryopreservation survival. In vitro-produced embryos were selected at expanded blastocyst stage and placed in a 50-μL drop of holding media on an inverted microscope. The embryo was held using a glass holding pipette attached to a micromanipulator, oriented so that the inner cell mass was toward the bottom of the view. A 7-μm spiked intracytoplasmic sperm injection pipette attached to the other micro-manipulator was used to pierce the blastocoele cavity and aspirate the blastocoele fluid. Once removed, the aspirate was transferred into 4-μL TE buffer for later genetic analysis. Collapsed blastocysts were then vitrified in ~7 μL 16.5% ethylene glycol, 16.5% dimethyl sulfoxide in TCM-199 (Hanks salts) with 20% FCS and 0.5 M sucrose. Embryos were held for a minimum of 1 week and then thawed and assessed for survival. Post-cryopreservation embryo survival was measured as the proportion of embryos that re-expanded after 48 h in culture. One-way ANOVA was used for statistical testing. A total of 181 control (intact) and 182 blastocentesis embryos were vitrified over 6 replicates. In all but one replicate, non-biopsied control embryos had higher re-expansion rates. Overall, the re-expansion rate was significantly (P = 0.05) higher for control embryos (73.5%) than blastocentesis embryos (61.5%) (Table 1). Initial experiments would suggest embryo survival is affected by the biopsy procedure; however, because this was not the case with every replicate, this may be batch or technician/human error dependent. Further study is required to assess full effect of blastocoele fluid aspiration on embryo cryopreservation, particularly investigating effectiveness for in vivo-produced embryos and subsequent effect on pregnancy rates. Likewise, further investigation is required to assess whether the sample collected is sufficient to allow accuracy over a variety of genetic tests. More than 20 embryos can easily be sampled in an hour using this technique, making it a rapid and efficient process. Given the speed and compatibility with cryopreservation, this sampling procedure may offer an alternative to current techniques used for cattle embryo genetic assessment. Table 1. Post-thaw survival rates of in vitro-produced embryos vitrified after blastocentesis1

The effect of freeze-drying media and storage temperature on ultrastructure and DNA of freeze-dried buffalo bull spermatozoa

ObjectiveThe present study intended for displaying the effect of different freeze-drying media and temperature of storage on the ultrastructure and DNA of freeze-dried buffalo bull spermatozoa. The semen samples undergone freeze-drying were raw semen and frozen-thawed semen extended in Tris-Fructose-Egg yolk-Glycerol. MethodsSemen samples were processed in two portions: First portion was cryopreserved with Tris-Fructose-Egg yolk-Glycerol extender to be freeze-dried with the different media used in this study. Second portion was freeze-dried immediately with the different media used in this study. Semen samples were centrifuged in a percoll gradient (45–90%) for 20 min at 700 × g to remove seminal plasma. Subsequently, sperms were washed twice in Tyrode's albumen lactate pyruvate (TALP) to remove percoll remains, and allocated into the four freeze-drying media (media 1, 2, 3 and 4) respectively. The media tested were: medium 1 (EGTA solution), medium 2 (EDTA solution), medium 3 (TCM199 with Hanks salts and 10% FCS) and medium 4 (TCM199 with Hanks salts and 10% FCS and trehalose). For all the media used, samples were diluted, placed in tubes of 1.5 ml and kept at room temperature for 30 min. Then sperm cell suspensions were cooled in liquid nitrogen vapor (approximately −80 °C for 1 h), by keeping the tubes at a distance of 5 cm from liquid nitrogen surface before plunged into it. Frozen samples were immediately inserted into the freeze-drying machine, previously stabilized at (−40 °C) and 350 × 10−3 Mbar pressure. After 12–16 h of freeze-drying, the tubes containing the samples were covered with aluminum foil and stored for 3 months at different temperatures; 4 °C, −20 °C and −80 °C. Freeze-dried sperm samples were re-hydrated by adding 100 μL of milli-Q water at room temperature. To evaluate sperm ultrastructure, transmission electron microscopy was done. For detection of DNA fragmentation, commet assay was performed. ResultsElectron microscopy showed that the sperm cell component most affected by freeze-drying was the plasma membrane, which was destroyed in all media either in raw or frozen thawed sperm. Microtubules organization was also disorganized in the majority of the sperm from freeze-drying medium 2 and 3, diverging from freeze-drying media 1 and 4, in which microtubules were intact. Conversely, the acrosome and mitochondria were well protected in all media. However, the storage temperature has no effect. The freeze-drying medium with EDTA solution exhibited and the lowest percent of DNA damage (9.3) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (13.6) at temperature of storage (4 °C). In contrast, the freeze-drying medium with EGTA solution exhibited the lowest percent of DNA damage (15.3) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (18) at temperature of storage (−20 °C). On the other hand, the freeze-drying medium with EDTA solution exhibited the lowest percent of DNA damage (11.3) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (20) at temperature of storage (−80 °C). Consequently, the freeze-drying medium (TCM without trehalose) exhibited the lowest percent of DNA damage (6.5) while the freeze-drying medium with EDTA solution exhibited the highest percent of DNA damage (13.9) at temperature of storage (4 °C). On the contrary, the freeze-drying medium with EGTA solution exhibited the lowest percent of DNA damage (13.2) while the freeze-drying medium (TCM without trehalose) exhibited the highest percent of DNA damage (20.8) at temperature of storage (−20 °C). Conversely, the freeze-drying medium with EDTA solution exhibited the lowest percent of DNA damage (6) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (12) at temperature of storage (−80 °C). ConclusionFrom the present study, we demonstrated that the freeze-drying medium containing EGTA and EDTA solution were more efficient in avoiding damage to components of buffalo bull sperm, especially the nuclei. Therefore, the medium used for freeze-drying process directly affected sperm nuclear integrity. Also, the storage temperature of freeze-dried sperm affects sperm nuclear integrity.

The Effect of Freeze Drying Using Different Media and Storage Temperatures on Some Parameters of Buffalo Bull Spermatozoa

THE PRESENT study was designed to display the effect of freeze- …..drying (lyophilization) technique using different freeze-drying media and storage temperatures on some parameters of buffalo bull spermatozoa. The semen samples were collected once weekly from five mature buffalo bulls maintained in Animal Reproduction Research Institute, Ministry of Agriculture, Al- Haram, Giza, Egypt. Samples were allocated into two portions; the first one was cryopreserved in Tris-Fructose-Egg yolk-Glycerol, while the second portion was immediately freeze-dried. These two portions were exposed to the same technical procedures. The freeze-dryer set was programmed at -55°C and 0.001 mbar pressure. The media tested were: EGTA solution, EDTA solution, TCM199 with Hanks salts enriched with 10% fetal calf serum (FCS) and TCM199 with Hanks salts enriched with 10% FCS and 0.2 M trehalose. The storage temperatures experienced were (4, - 20, - 80 oC). The efficiency of each medium and storage temperature on some sperm parameters was explored. Our results revealed no motility either in raw or frozen-thawed spermatozoa after freeze-drying. In case of raw and frozen-thawed semen; with no respect to storage temperatures, the best freeze-dried sperm parameters were observed in TCM-Trehalose medium, while the best storage temperature was (-80°C) followed by (-20°C) with no respect to the type of freeze-drying media. From the present study, it was concluded that the freeze-drying medium containing trehalose could preserve efficiently components of buffalo bull spermatozoa, especially the acrosome, while the storage temperature (-80°C) and (- 20°C) were the best for storage of freeze-dried buffalo bull spermatozoa.

Stability of Minimum Essential Medium functionality despite L-glutamine decomposition.

L-Glutamine (L-Gln) instability in liquid media is a well-known fact. Also, negative effect of ammonia, one of the L-Gln degradation products, on viability of many cell cultures and on replication of different viruses has been described. However, negative effects of ammonia have been reported in doses excessively exceeding those that could be generated in regularly used liquid culture media due to spontaneous L-Gln breakdown (below 2mM). Traditional virus vaccine production processes have been established and registered involving L-Gln containing media use. Eventual culture media replacement in the regular production process belongs to the major regulative changes that require substantial financial expenses. The aim of this study was to evaluate the effect of storage of Minimum Essential Media with Hanks salts on their relevant biological functions during virus vaccine production process in relation to L-Gln decrease. Our results show a cell type dependent effect of spontaneous L-Gln degradation during medium storage. They also suggest that for cell cultures used in measles, mumps, and rubella virus production the media retain their functionality in respect to cell viability or virus growth over a certain time window despite L-Gln degradation.

Tumoricidal Effect of Hematite (α-Fe&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;) and SiO&lt;sub&gt;2&lt;/sub&gt; Nanoparticles in Human Rhabdomyosarcoma Cell Line

Nanotechnology provides the opportunity for the development of new materials in the nanometer size range with many potential applications in biological sciences and clinical medicine. RD (muscle cancer cell line) was seeded out in 25 cm2 plastic tissue culture flasks (NuncWiesbaden Germany) individually, in Minimum Essential Medium (MEM) with Hanks salts, containing 10% fetal bovine serum (FBS) and 2 mM L-Glutamine along with some nonessential amino acids and were incubated for 24 h for proper attachment to the substratum and kept at a 96 wells plate, incubated at 37°C and 5% CO2. SEM was employed to the nanoparticles and size of α-Fe2O3 and SiO2 nanoparticles were about 66 nm and 250 nm. Moreover 10-80μg/mL of Hematite (α-Fe2O3) and SiO2 nanoparticles dispersed solution were labeled for each row of 96 wells plate. The present study evaluates the different parameters, e.g. time of incubation, cytotoxicity and cellular viability of the Human Rhabdomyosarcoma cell line (RD) as an experimental model. The viability of cells was determined by means of neutral red assay (NRA) after the cell-exposition to different concentrations of Hematite (α-Fe2O3) and SiO2 nanoparticles into mentioned tumoricidal cells

610 Method of differential breast tumour diagnostics

The present study intended for displaying the effect of different freeze-drying media and temperature of storage on the ultrastructure and DNA of freeze-dried buffalo bull spermatozoa. The semen samples undergone freeze-drying were raw semen and frozen-thawed semen extended in Tris-Fructose-Egg yolk-Glycerol.Semen samples were processed in two portions: First portion was cryopreserved with Tris-Fructose-Egg yolk-Glycerol extender to be freeze-dried with the different media used in this study. Second portion was freeze-dried immediately with the different media used in this study. Semen samples were centrifuged in a percoll gradient (45–90%) for 20 min at 700 × g to remove seminal plasma. Subsequently, sperms were washed twice in Tyrode's albumen lactate pyruvate (TALP) to remove percoll remains, and allocated into the four freeze-drying media (media 1, 2, 3 and 4) respectively. The media tested were: medium 1 (EGTA solution), medium 2 (EDTA solution), medium 3 (TCM199 with Hanks salts and 10% FCS) and medium 4 (TCM199 with Hanks salts and 10% FCS and trehalose). For all the media used, samples were diluted, placed in tubes of 1.5 ml and kept at room temperature for 30 min. Then sperm cell suspensions were cooled in liquid nitrogen vapor (approximately −80 °C for 1 h), by keeping the tubes at a distance of 5 cm from liquid nitrogen surface before plunged into it. Frozen samples were immediately inserted into the freeze-drying machine, previously stabilized at (−40 °C) and 350 × 10−3 Mbar pressure. After 12–16 h of freeze-drying, the tubes containing the samples were covered with aluminum foil and stored for 3 months at different temperatures; 4 °C, −20 °C and −80 °C. Freeze-dried sperm samples were re-hydrated by adding 100 μL of milli-Q water at room temperature. To evaluate sperm ultrastructure, transmission electron microscopy was done. For detection of DNA fragmentation, commet assay was performed.Electron microscopy showed that the sperm cell component most affected by freeze-drying was the plasma membrane, which was destroyed in all media either in raw or frozen thawed sperm. Microtubules organization was also disorganized in the majority of the sperm from freeze-drying medium 2 and 3, diverging from freeze-drying media 1 and 4, in which microtubules were intact. Conversely, the acrosome and mitochondria were well protected in all media. However, the storage temperature has no effect. The freeze-drying medium with EDTA solution exhibited and the lowest percent of DNA damage (9.3) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (13.6) at temperature of storage (4 °C). In contrast, the freeze-drying medium with EGTA solution exhibited the lowest percent of DNA damage (15.3) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (18) at temperature of storage (−20 °C). On the other hand, the freeze-drying medium with EDTA solution exhibited the lowest percent of DNA damage (11.3) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (20) at temperature of storage (−80 °C). Consequently, the freeze-drying medium (TCM without trehalose) exhibited the lowest percent of DNA damage (6.5) while the freeze-drying medium with EDTA solution exhibited the highest percent of DNA damage (13.9) at temperature of storage (4 °C). On the contrary, the freeze-drying medium with EGTA solution exhibited the lowest percent of DNA damage (13.2) while the freeze-drying medium (TCM without trehalose) exhibited the highest percent of DNA damage (20.8) at temperature of storage (−20 °C). Conversely, the freeze-drying medium with EDTA solution exhibited the lowest percent of DNA damage (6) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (12) at temperature of storage (−80 °C).From the present study, we demonstrated that the freeze-drying medium containing EGTA and EDTA solution were more efficient in avoiding damage to components of buffalo bull sperm, especially the nuclei. Therefore, the medium used for freeze-drying process directly affected sperm nuclear integrity. Also, the storage temperature of freeze-dried sperm affects sperm nuclear integrity.

Target of Rapamycin (TOR) Mediates the Transduction of Nutritional Signals into Juvenile Hormone Production

Anautogeny is a reproductive strategy by which females do not reproduce until they feed. Therefore, nutritional signals must inform the reproductive tissues, and cells that the organism has reached a nutritional status suitable for triggering reproductive processes. One of the possible pathways involved in anautogeny is the "target of rapamycin" (TOR) pathway, which has been described as connecting the nutritional status with growth, proliferation, and cancer. The German cockroach, Blattella germanica, is an anautogenous species whose vitellogenesis is governed by juvenile hormone. In the present report, we describe the cloning of TOR cDNA from B. germanica (BgTOR). Expression studies showed that BgTOR is expressed in adult female corpora allata and fat body. BgTOR knockdown using systemic RNAi in vivo produced a severe inhibition of juvenile hormone synthesis in adult female corpora allata, together with a reduction of mRNA levels corresponding to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase-1, HMG-CoA synthase-2, and HMG-CoA reductase. In addition, there was a reduction of vitellogenin mRNA in the fat body, and ovaries did not grow. Analysis of TOR expression in corpora allata of fed and starved females suggested that TOR is not regulated at the transcriptional level. Nevertheless, there was a reduction in HMG-CoA synthases and reductase mRNA in corpora allata (but not in the fat body) of starved females, together with a dramatic reduction of juvenile hormone production and ovary development. Taken together, our results indicate that TOR knockdown mimics starvation in terms of corpora allata activity, and suggest that nutritional signals that activate juvenile hormone biosynthesis and vitellogenin production are mediated by the TOR pathway.

Effect of Nd:Yttrium-aluminum-garnet laser radiation on Ti6Al4V alloy properties for biomedical applications

The effect of Nd:yttrium-aluminum-garnet laser on the microtopography and physicochemical properties of Ti6Al4V alloy are investigated in the view of biomedical applications. The surface roughness and hardness for laser treated samples (LTS) at 140 J cm−2 were measured 7±0.02 and 825 vickers hardness number, respectively. This superior microhardness value is attributed to grain refinement associated with laser melting and rapid solidification. The electrochemical property, mainly pitting corrosion resistance, has been carried out in Hanks salt balanced physiological solution using standard potentiodynamic polarization testing. A higher corrosion potential of −0.21 V was achieved for LTS. At the optimium treating value of laser fluence (140 J cm−2), the energy dispersive x-ray analysis showed about a 30% decrease of vanadium. The contact angle measurements also indicated an improved surface wettability (i.e., hydrophilicity) characteristic at 35°. Finally, the cell culture studies provided a useful tool to investigate the morphology and cell cytotoxicity.

Investigation of glass-forming ability, deformation and corrosion behavior of Ni-free Ti-based BMG alloys designed for application as dental implants

We developed new Ti-based bulk metallic glassy (BMG) alloys in Ti–Zr–Pd–Cu–Sn system without Ni element for application as biomaterials. These BMG alloys have a high potential to be applied as metallic biomaterials in various forms, such as melt-spun ribbons and cylindrical rods with a diameter of 4 mm. We also investigated of new Ti-based BMG alloys with higher glass-forming ability (GFA) for medical market as dental implants. These Ti-based BMG alloys do not contain Ni, Al and Be elements which are well known to be harmful for human body. In particular, a rod sample of the Ti 44.1Zr 9.8Pd 9.8Cu 30.38Sn 3.92Nb 2 BMG alloy with a diameter of 3 mm produced by copper mold casting exhibits a compressive strength of 1990 MPa and a Young's modulus of 99 GPa. In addition, the Ti 44.1Zr 9.8Pd 9.8Cu 30.38Sn 3.92Nb 2 BMG shows a large supercooled liquid region of 62 K and a reduced glass-transition temperature, T rg( = T g / T m) of 0.61. The high thermal stability of the supercooled liquid allowed the fabrication of cylindrical rod specimens up to 5 mm in diameter. Thus the studied alloy exhibits high glass-forming ability (GFA) and a large size enough to be used for dental implants. The Ti 44.1Zr 9.8Pd 9.8Cu 30.38Sn 3.92Nb 2 BMG alloy also has a high corrosion resistance and is passivated at the lower passive current density of approximately 10 − 2 A m − 2 , 10 − 3 A m − 2 and 10 − 2 A m − 2 , in 1 mass% lactic acid, PBS (phosphate-buffered saline without calcium and magnesium salts solution) and HBSS (Hank's balance salt solution without calcium, magnesium and phenol red), respectively, at 310 K, which are lower than those of pure Titanium and Ti–6Al–4V alloy.

Assessment of canine oocyte viability after transportation and storage under different conditions

To improve assisted reproductive technologies in the domestic dog, different transport treatments were evaluated for their ability to maintain viability of canine oocytes, as assessed by esterase activity 8 h after storage or after 48 h of in vitro maturation (IVM) culture. In Experiment 1, ovaries were transported within reproductive tracts or were excised and stored at either 20 or 37 °C in phosphate buffered saline. Oocytes collected from reproductive tracts transported at 37 °C had the greatest viability after storage ( P < 0.05). However, after IVM there were no significant differences among any of the four storage conditions in oocyte viability or meiotic resumption ( P = 0.05). In Experiment 2, isolated oocytes were transported in either TCM-199 with Hank's salts and Hepes buffer or in TL-Hepes at either 20 or 37 °C, or in maturation medium equilibrated with 5% CO 2 at 37 °C. In Experiment 2, oocytes transported in Hepes buffered media at 37 °C had greater viability rates after storage than did those transported in these same media at 20 °C or in sodium bicarbonate buffered medium at 37 °C ( P < 0.001). After IVM, oocytes transported in the 37 °C treatment groups had greater viability rates than did those transported at 20 °C ( P < 0.01). Overall, isolated oocytes transported at 37 °C had greater rates of meiotic resumption than did those transported at 20 °C ( P < 0.05). Taken together, these data indicate that canine oocytes exhibited sensitivity to lesser temperatures and maintained greater rates of viability during transport at 37 °C. Isolated oocytes maintained greater viability than oocytes transported in situ. Hepes buffered media increased viability rates for isolated oocytes transported at 37 °C compared to a similar medium buffered with sodium bicarbonate.

TOTAL ANTI-OXIDANT CAPACITY OF CELL CULTURE MEDIA

1. The composition of synthetic cell culture media is important for the behaviour of cultured cells in vitro and may affect the results of many in vitro experiments. The total anti-oxidant capacity (TAC) of an extracellular medium may be an important factor in cell redox homeostasis. 2. In the present study, the TAC of cell culture media used for the cultivation of mammalian, yeast and bacterial cells (RPMI1640, Iscove's modified Dulbecco's medium, Dulbecco's modified Eagle's medium, minimum essential medium Eagle's 1959 with Earle's salts, Parker medium 199 with Hanks salts, bacterial Luria-Bertani medium, yeast extract-peptone-glucose and yeast nitrogen base media) was estimated using the 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS(.+)) decolourization assay and the ferric ion reducing anti-oxidant power assay. 3. We found that components of the media such as cysteine, tyrosine, tryptophan and Phenol Red are important contributors to the TAC of cell culture media.

Effects of freeze-drying on cytology, ultrastructure, DNA fragmentation, and fertilizing ability of bovine sperm

Freeze-drying sperm is an alternative to cryopreservation. Although sperm from various species has been freeze-dried, there are few reports for bovine sperm. The primary objective of this study was to evaluate the protective effect of various freeze-drying media on the structural and functional components of bovine sperm. The media tested were composed of TCM 199 with Hanks salts supplemented with 10% fetal calf serum (FCS) and TCM 199 with Hanks salts supplemented with 10% FCS and 0.2 M trehalose and EGTA solution. The efficiency of each medium on the preservation of freeze-dried sperm structures was evaluated with conventional and electron microscopy, DNA integrity was analyzed by a TUNEL assay, and fertilizing ability of lyophilized sperm was determined with ICSI. Although the plasma membrane was damaged in all media tested, mitochondria were similarly preserved in all freeze-drying treatments. The acrosome was best preserved in the media that contained trehalose (other treatments also conserved this structure). In contrast, media containing EGTA or trehalose most effectively preserved the nuclei in freeze-dried sperm, with only 2 and 5%, respectively, of cells with fragmented DNA. Furthermore, sperm conserved with these media also had higher ( P < 0.05) rates of sperm head decondensation (32.5 and 27.5%), pronucleus formation (37.5 and 45.0%) and blastocyst formation (19.4 and 18.3%) than medium supplemented with FCS (15.0, 20.0 and 10.2%, respectively). In conclusion, media with EGTA and trehalose adequately protected bovine sperm during freeze-drying by preserving the viability of their nuclei.

145 REESTABLISHMENT OF AN EXTINCT STRAIN OF SHEEP UTILIZING ASSISTED REPRODUCTIVE TECHNOLOGIES

The objective of this study was to reestablish an extinct strain of sheep that exhibits spontaneous X-linked factor VIII deficiency closely mimicking human hemophilia A. Twenty female carriers of the trait, produced in a previous study (Bormann et al. 2006 Reprod. Fertil. Dev. 18, 201–202), were backcrossed using 3 straws of semen from their affected sire using either IVF or multiple ovulation embryo transfer (MOET). Eleven oocyte donors were synchronized with CIDRs (15 days) and superovulated with a declining dose of FSH (204 mg) twice daily for 3.5 days. Nine MOET donors were synchronized using CIDRs (14 days), superovulated with a declining dose of FSH (184 mg) BID for 3 days with pregnant mare serum gonadotropin (PMSG; 200 IU) given with the final dose of FSH, and given 1000 IU of hCG 12 h post-CIDR removal. Recipient ewes were synchronized using sponges (Ovakron, HeriotAgvet, Rowville, Victoria, Australia) containing 30 mg of flugestone acetate (14 days) and given PMSG (400 IU) at sponge removal, followed by 1000 IU of hCG 12 h post-sponge removal. Oocytes were collected via follicular aspiration during midventral laparotomy and matured as previously reported. Semen for IVF was prepared by centrifugation on a Percoll gradient. Oocytes and sperm were incubated in mTALP with 20% estrus sheep serum (modified from Bavister et al. 1977 Bio. Reprod. 16, 228–237) for 20 h, then vortexed to remove cumulus cells, and cultured in G1.3 medium (Vitrolife, Englewood, CO) with BSA until transfer. Embryos were surgically transferred into oviducts of recipients 24 to 48 h following IVF. The 9 MOET donors were surgically inseminated at the uterotubal junction with approximately 1–2.0 � 106 spermatazoa. Oviducts of eight of these ewes were flushed 48 h post-insemination with warm M199 containing Hanks salts, 25 mm HEPES, 10% FBS, and 0.5 µg mL–1 gentamicin. MOET embryos were surgically transferred to synchronized recipients within 5 h. One MOET donor was not flushed due to poor response and did not produce an offspring. Utilizing 140 ova, IVF produced 54 embryos for an embryo/oocyte rate of 38.6%. All IVF embryos were transferred into 15 recipients resulting in 3 lambs for a lamb/embryo rate of 5.5%. The MOET donors produced 38 embryos and 13 apparently unfertilized ova, generating an embryo/oocyte rate of 74.5%. MOET embryos were transferred into 21 synchronized recipients. MOET produced 16 lambs for a lamb/embryo rate of 42.1%. Co-transfer of 1 IVF and 1 MOET embryo into a single recipient produced one offspring. Utilizing multiple reproductive technologies over a two-year period, 8 hemophilic offspring (7 females and 1 male), 6 carrier females, and 6 unaffected males were produced. This strain of sheep will be used to produce affected offspring for stem cell-based therapies.

353 EFFECTS OF CULTURE DURATION AND CUMULUS CELL REMOVAL ON CANINE OOCYTE IN VITRO MATURATION AND FERTILIZATION USING VASAL SPERM

The objective of this study was to test the hypothesis that in vitro maturation (IVM) and fertilization (IVF) rates of canine oocytes could be improved by increasing culture duration or decreasing/increasing cumulus cell contact with the oocytes when using sperm retrieved from the vas deferens. The canine oocyte is ovulated at the germinal vesicle stage, and maturation of the oocyte occurs in the oviduct and requires up to five days. Therefore, an increase in the culture duration may cause an increase in oocyte nuclear maturation. Canine ovaries and testes were collected from a local clinic, placed in warm saline solution, and transported to the laboratory. Two distinct experiments were carried out, one involving IVM (M-II) after cumulus cell removal at 72 h and 96 h for nuclear maturation evaluation, and the second experiment the same but continued up to IVF. The oocytes were recovered from the ovaries by mincing them in warm Medium-199 with Hanks salts, L-glutamine, and HEPES (GIBCO, Grand Island, NY, USA; Invitrogen Co., Carlsbad, CA, USA). Canine oocytes with a dark cytoplasm and at least 2 layers of cumulus cells were cultured in Medium-199 supplemented with Earle&amp;apos;s salts, 2200 mg mL&amp;minus;1 sodium bicarbonate, 25 mM HEPES, 2 mM sodium pyruvate, 5 &amp;micro;g mL&amp;minus;1 progesterone, 100 ng mL&amp;minus;1 epidermal growth factor, 10 IU mL&amp;minus;1 human chorionic gonadotropin (HCG), 5 &amp;micro;g mL&amp;minus;1 insulin, 0.50 mM epinephrine, 10&amp;percnt; estrus bitch serum, 0.01 mM nonessential amino acids, and 20 &amp;micro;g mL&amp;minus;1 gentamicin. The oocytes were cultured for 72, 96, 120, or 144 h at 38.5&amp;deg;C in 5&amp;percnt; CO2 in humidified air. The cumulus cells were removed after either a 72- or 96-h culture period. For the first 48 h, the cumulus&amp;ndash;oocyte complexes were cultured in the modified Medium-199 containing 10 IU mL&amp;minus;1 HCG and then cultured in the same medium free of HCG. The oocytes were denuded by pipetting, stained with Hoechst 33342, and examined for nuclear maturation. ANOVA was used for statistical analysis of the data. The IVM rate (MII) was significantly higher (P &amp;lt; 0.05) at 72 and 96 h compared to 48, 120, and 144 h (15.1&amp;percnt; and 16.9&amp;percnt; vs. 6&amp;percnt;, 12.4&amp;percnt;, and 9.1&amp;percnt;, respectively). The removal of cumulus cells at 72 h and 96 h resulted in 17.9&amp;percnt; (43/240) and 14.8&amp;percnt; (35/236) IVM rate (MII), respectively (P &amp;gt; 0.05). The sperm motility index (SMI &amp;equals; motility percentage &amp;times; sperm activity grade) was significantly higher in sperm retrieved from the vas deferens (vasal sperm) compared to epididymal and testicular sperm (259 vs. 95 and 19.2, respectively, P &amp;lt; 0.05). The mature oocytes were inseminated by vasal sperm following in vitro hyperactivation with HEPES solution supplemented with 3 mg mL&amp;minus;1 bovine serum albumin. The IVF rates of the oocytes following 72 and 96 h of maturation in vitro were 48.2&amp;percnt; and 40&amp;percnt;, respectively (P &amp;gt; 0.05). Sperm penetration was significantly higher at 96 h compared to 72 h, and the number of sperm heads inside the ooplasm was 3.2 for the 72 h group vs. 4.8 for the 96 h group (P &amp;lt; 0.05). In conclusion, increasing the IVM culture period beyond 72 h did not increase the oocyte maturation rates, and increasing the culture time to 96 h without cumulus cells present increased the rate of sperm penetration.