Saturated Humidity Research Articles

Tolerance of codling moth, and apple quality associated with low pressure/low temperature treatments

A combination of low pressure (LP) and low temperature (LT) may serve as a phytosanitary disinfestation treatment for fresh fruit. In this study, different life stages of codling moth (eggs, 2nd to 3rd instar larvae, 5th instar larvae and pupae) were treated in hypobaric chambers maintained at 10°C and 1.33kPa with nearly saturated humidity (>98%). Weight loss, color, firmness, titratable acidity (TA), and soluble solids content (SSC) were selected as quality parameters to evaluate the quality changes of ‘Red Delicious’ apples before and after the LPLT treatment. Results showed that the 5th instar larvae were the most tolerant life stage for codling moth under LPLT treatment conditions. Insect mortality increased with increasing LPLT treatment time to >98% after 12 days of exposure to 10°C temperature and 1.33kPa pressure. Although stored in less than optimum conditions for apples, the measured quality variables of ‘Red Delicious’ were maintained relatively well after 15 days of LPLT treatment. The results suggest that LPLT technology has potential as an alternative, non-chemical disinfestation treatment for apples.

Relationship of moisture content with temperature and relative humidity in concrete

Variations of temperature and moisture content in concrete arise as the effects of climate load in a natural environment; because it can be easily measured, relative humidity is often used as a transition index reflecting moisture content. Determination of the relationship between moisture content and temperature/humidity in concrete is of great significance in accurate prediction of the durability of reinforced concrete structures. Under constant temperature conditions, measurements of both relative humidity and moisture content in concrete are carried out synchronously. Results show that pore water saturation, considered as the characterisation of moisture content, changes non-linearly with relative humidity in concrete; the evolution is significantly influenced by the water–cement ratio and temperature. Finally, based on experimental results and theoretical analysis, a model of the relationship between pore water saturation, temperature and relative humidity in concrete is established, and its feasibility is verified through an independent test.

PM 7/42 (2) Clavibacter michiganensis subsp. michiganensis

Specific scopeThis standard describes a diagnostic protocol for Clavibacter michiganensis subsp. michiganensis.Specific approval and amendmentApproved in 2004‐09.Revision adopted in 2012‐09.

EFFECT AND EFFICACY OF THERMAL ENVIRONMENT PROVIDED BY A NEW BATHING STYLE, “MIST SAUNA BATHING”

Mist sauna is a style of bathing in which hot water vapor is sprayed into a bathroom, establishing an air temperature of 40°C with saturated humidity. Bath heater and dryer equipment with mist sauna function was released onto the Japanese market in 2004. After their introduction, various studies investigated the effects of mist sauna bathing, and it has been demonstrated that mist sauna bathing has various effects and efficacies, not only hyperthermic effects but also other effects including on physical appearance. Mist sauna bathing occurs in a bathroom, usually without a toilet, in which the room temperature is approximately 40°C with 100% relative humidity. It has been shown that the mist sauna causes little hemodynamic change, which ensures its safety during bathing. Therefore, we can enjoy safer physiological bathing in a mist sauna than in traditional hot water immersion bathing. In addition, the mist sauna elicits benefits such as improved skin condition, heat acclimation, and autonomic balance. Since mist sauna bathing does not involve immersion of the body in bathtub water, it is less likely to result in an accident during bathing because of the low impact of hemodynamic changes. Recently, mist sauna bathing has drawn attention in the field of nursing care as a bathing style for the hospitalized elderly that can reduce the burden on care-giving personnel during bathing. It is expected that mist sauna will be adopted by homes and various facilities as a useful approach for various purposes, regardless of the user’s age or gender.

Stabilisation of amorphous drugs under high humidity using pharmaceutical thin films

In this study, the stabilization effects of three polymers on four model drugs (felodipine, fenofibrate, carbamazepine, and celecoxib) under saturated humidity were investigated. Three different types of thin films (solid dispersions, drug films with a polymer film coating and drug films laid on top of polymer coated surfaces) were prepared and compared with films containing the drug alone. ATR-FTIR spectroscopy, polarised light microscopy (PLM), scanning electron microscopy (SEM) and nano-thermal analysis (nano-TA) were performed on the model systems after storage under saturated humidity. The recrystallisation tendency of the drug in the drug containing thin films was found to be strongly related to the intrinsic crystallization tendency of the drug film alone and the strength of drug–polymer interactions. Additionally, under high humidity, the glass transition temperature of the polymer is no longer an indicator of its drug stabilization capability. Instead, it is the hygroscobicity of the polymer that appears to be the most important parameter. Amongst the polymers tested in this study, EUDRAGIT E PO was found to have the greatest inhibitory effect on crystallization, whilst PVP K30 was found to have the least protective effect; presumably because of its hygroscopic nature.

Hydrogen generator system using Ru catalyst for PEMFC (proton exchange membrane fuel cell) applications

High purity hydrogen produced by hydrolysis of sodium borohydride can be used directly in (proton exchange membrane) PEM fuel cells for portable devices and automotive applications. The advantages of high storage capacity, quickly start, controllable reaction and mild condition, hydrogen generation by catalytic hydrolysis of chemical hydride, such as sodium borohydride, has attracted much attention for development recently.Development and optimal design of a hydrogen generator based on (sodium borohydride) NaBH4 using Ru catalyst are reported in the present study. A multi-layer coating process of Ru on Ni foam was employed to enhance its capability for hydrogen generation cycles. The optimal deposition density of Ru particles on Ni foam was confirmed by the SEM (scanning electron microscopy) morphology. The cycling generation for the steady state reaction can be maintained at least 10 times. The activity of Ru catalyst during a hydrogen generation process was examined in a continuous flow reactor. In general, NaBH4 solution cannot be fully utilized through the hydrogen generator, as a result, the effluent from the generator may continue to dissociate, which turns into hydrogen leakage. A secondary reactor was therefore installed after the primary generator to collect the remaining hydrogen. The reactant solution containing 20 wt.% NaBH4 and 3 wt.% NaOH was introduced into the structured catalyst at a constant flow rate using a liquid pump, and H2 rate of 1.72 L min−1 (0.43 L (H2) min−1 g−1 catalyst). The conversion of NaBH4 from flow rates of hydrogen was about 92.2%.The present hydrogen generator was later integrated with a 100 W PEM fuel cell and the performance of the integrated system was investigated. The hydrogen produced from NaBH4 has high purity and saturated humidity; therefore, it can be directly used as the fuel for PEMFCs (proton exchange membrane fuel cells), which in general require humidified hydrogen. It is found that for cell voltage above 0.6 V, the performance of cell using NaBH4 hydrogen is 103.45 W, versus 99.9 W with cylinder hydrogen. The cell performance using the hydrogen from NaBH4 is 3.6% higher than that using cylinder hydrogen.

Enhanced performance of a Nafion membrane through ionomer self-organization in the casting solution

A Nafion membrane with enhanced performance has been prepared by using a novel casting solution containing low-polarity nonsolvent tetrachloroethylene (TCE). TCE induces self-organization of the ionomer and turns the originally transparent Nafion in dimethylformamide (DMF) solution into opaque. The Nafion membrane cast from such opaque solution remains mechanically robust and shows a proton conductivity of 0.07 S cm−1 at room temperature, and 0.21 S cm−1 at 80 °C under saturated humidity, which is an increase of 62% and 103% respectively over the membrane without TCE involvement. Besides, water content and water permeability of the new membrane become higher. The enhanced membrane performance is attributed to larger clusters of sulfonate groups in Nafion, which is revealed by small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM).

254 EFFECTS OF OXYGEN TENSION AND OOCYTE DENSITY DURING IN VITRO MATURATION ON THE LEVELS OF REACTIVE OXYGEN SPECIES IN BOVINE OOCYTES AND MATURATION MEDIUM

The reactive oxygen species (ROS) produced by animal cells and at physiological levels are responsible for several cellular functions. However, when there is an imbalance between ROS production and the antioxidant system in the cell, oxidative stress occurs and causes severe cell damage. In oocytes, ROS can affect the dynamics of maturation and early embryo development processes. Oxygen tension and the density of oocytes by medium volume during in vitro maturation (IVM) can influence ROS production. The aim of this study was to evaluate the influence of the association between oxygen tension (5 or 20%) and different oocyte densities during IVM (1 : 10 or 1 : 20 oocytes µL–1 of medium) on the ROS levels in oocytes and medium. Bovine oocytes (n = 420) were obtained from slaughterhouse ovaries by aspiration of 2- to 8-mm follicles. Quality I and II oocytes (De Loss et al. 1989 Gamete Res. 24, 197–204) were homogeneously distributed into groups of 15 oocytes per treatment: Treatment (T) 1 = 1 : 10 in 5% of O2; T2 = 1 : 10 in 20% of O2; T3 = 1 : 20 in 5% of O2; and T4 = 1 : 20 in 20% of O2. The oocytes were matured in TCM-199 supplemented with 10% oestrous mare serum, 100 µg mL–1 of epidermal growth factor, 50 µg mL–1 of LH, 5 µg mL–1 of FSH, and 22 µg mL–1 of pyruvate for 22 to 24 h at 39°C, in 5% CO2 and saturated humidity. To assay ROS production, denuded oocytes and 60-µL samples of IVM medium were evaluated by the spectrofluorometric method with 2′7′-dichlorofluorescein-diacetate, in which the fluorescence intensity emission was considered an indicator of ROS production and measured by a spectrofluorophotometer. The ROS production in oocytes and in IVM medium was expressed as units of fluorescence (UF); data were analysed by ANOVA and Duncan’s test with a 5% level of significance. Seven replications were performed. In treatment groups T1 and T3, the ROS production in oocytes was higher (P < 0.05) than in oocytes of treatment groups T2 and T4 (13.53 and 18.78 UF v. 7.92 and 6.15 UF, respectively). The ROS production in IVM medium was higher in the T1 (23.86 UF) and T2 (24.12 UF) treatment groups than in the T3 (18.78 UF) and T4 (18.57 UF) treatment groups. These results suggest an increase in ROS production in IVM oocytes under a 5% O2 atmosphere in relation to a 20% O2 atmosphere, irrespective of the oocyte density by volume of IVM medium. On the other hand, the accumulation of ROS in IVM medium seemed higher when the oocyte density was 1 oocyte to 10 µL of IVM medium, independent of the oxygen tension used. A higher level of ROS in 5% O2 tension may be caused by competition for O2 between oocyte and cumulus cells, causing a reduction in O2 levels and changing the availability of O2 to energy generation in oocytes and consequently increasing ROS generation. In this respect, 5% O2 during IVM may contribute to the onset of oxidative stress in oocytes, which may compromise fertilization and early embryo development. Further research is necessary to clarify esterase activity in oocytes and the addition of exogenous peroxidase to validate the assay. Financial support: FAPERGS (1011575) and CNPq (501763/2009).

147 AGGREGATION OF IN VIVO FERTILIZED AND PARTHENOGENETICALLY ACTIVATED MOUSE EMBRYOS

Embryonic chimerism – mixing of cells originated from at least two different fertilizations – has been used as a tool for stem cell pluripotency diagnosis, transgenic rodent production, and organogenesis studies. Additionally, parthenotes are used for studies related to gene imprinting and the ability of their cells to compose the placenta and/or an adult animal. The aim of this work was to validate the production and characterize developmental kinetic of parthenogenetic embryos, obtained from C57BL/6 EGFP mice (EGFP), to determine their potential to produce chimeras, and to localize parthenogenetic cells on the produced blastocyst. Embryos were harvested from superovulated females. For the aggregation, pre-compaction IVF (Swiss Webster/SW strain) or parthenogenetic activated (PGA; SW and EGFP) embryos were used. For PGA, strontium chloride hydrate (5 mM for 6 h) was used. Two experiments were outlined in order to: i) evaluate the development and kinetic from IVF (IVF and in vitro developed; 2-cell stage embryos, n = 53) and from PGA (parthenogenetically activated and in vitro developed; n = 409 oocytes) techniques, both from SW; ii) evaluate the aggregation between pairs of control embryos (C; n = 20, 4-cell in vivo produced embryos from SW) or pairs of 4-cell in vivo produced SW embryos and 4- to 8-cell PGA EGFP embryos (parthenogenetic embryo, PG; n = 40). After manipulation (removal of the zona pellucida and approximation of pairs, for C and PG groups), all the groups were kept in vitro culture (37°C, 5% CO2, and saturated humidity) for 48 to 60 h (C and PG), or up to blastocyst stage (IVF and PGA). Chimerism rate and parthenogenetic cells fluorescence (pre- and post-culture) were evaluated under an inverted microscope with epifluorescence source, and digital images were captured (Eclipse Ti and NIS-Elements, Nikon, Japan, respectively), by merging visible and UV light images. The rate of PGA (EGFP oocytes) was 54.5% (66/121), assessed 6 h post-activation by the presence of at least one pronucleus. The assessment was performed with 20× magnification and Hoffman modulation contrast in an inverted microscope. The rate of blastocyst production between IVF and PGA was significantly different (71.4 and 12.9%, respectively; P < 0.001, Fisher’s exact test). When developmental kinetic was evaluated there a difference in the average time to the majority of embryos reach blastocyst stage for IVF (48 h) and PGA (120 h) was observed. The chimerism rate was assessed by the presence of a single and cohesive cell mass (C) or by the incorporation of EGFP cells in the SW embryo (PG). There was difference (P = 0.006; Fisher’s exact test) between C (55.0%; 11/20) and PG (17.5%; 7/40) chimerism rates, assessed from 48 to 60 h of culture. In PG group, the incorporation of EGFP cells in the obtained chimeras was mainly detected in the trophectoderm although one chimeric blastocyst contained EGFP cells only in the inner cell mass. It was concluded that parthenogenetic embryos have a slower developmental kinetic (until 120 h of culture) than embryos derived from IVF. Acknowledgment to FAPESP for fellowship and funding.

148 MICE BLASTOCYST RECONSTRUCTION BY AGGREGATION OF INNER CELL MASS WITH TROPHECTODERM

In order to reduce fetal losses of bovine clones the replacement of trophectoderm (TE) by microinjection of immunosurgically isolated inner cell mass (ICM) was proposed. It was reported that those techniques could damage both ICM and TE. An alternative to decrease this problem would be the technique of cell aggregation by approximation. Currently it has been used only in pre-compaction embryos. The aims of this study were to evaluate the effectiveness to aggregate post-compaction embryos and to verify the feasibility of reconstructed blastocysts production by ICM and TE approximation. Mice (Swiss Webster, SW; and C57BL/6/EGFP, EGFP) were superovulated to obtain embryos 2.5 to 3.5 days post-coitus. In both control groups (CG; exp. 1 and 2), whole embryos from 8 cells to early morula stages and without zona pellucida were aggregated. In all experiments, bisection was performed with a blade controlled by micromanipulator to produce two fragments according to the requirements of each experiment (i.e. demi embryos in Exp. 1 and Exp. 2 or TE fragment and whole ICM in Exp. 3). The joined fragments were in vitro cultured in microwell (300 µm diameter and based on well of the well system) filled with 400 µL of KSOMaa, covered with mineral oil by 24 h (incubation 37°C, 5% of CO2 and saturated humidity). Embryonic aggregation rate (AR) was evaluated by detection of a single and cohesive cell mass (CG from Exp. 1 and Exp. 2) or by the presence of a typical morphologically blastocyst (all the remaining groups). Only to validate AR evaluation, 25% of the aggregation attempts were performed with distinct phenotype embryos (EGFP and SW) and they could be tracked under epifluorescence. In Exp. 1, the aggregation by approximation was tested to evaluate embryonic stages: 2 demi-blastocysts (2DB; n = 28), demi-morula and demi-blastocyst (DMDB; n = 20) and control group (CG; n = 25). In Exp. 2, an adhesive agent (phytohemagglutinin; PHA) and fragment increasing were used in the groups: 2DB (n = 24), 4 demi-blastocysts (4DB, that is four half-blastocyst) and CG (n = 22). After aggregation validation (Exp. 1 and Exp. 2), blastocyst reconstruction by the approximation of ICM and TE fragments (with PHA presence) was tested on groups: 1 TE fragment and 1 ICM (TI; n = 48) and 2 TE fragments and 1 ICM (2T1I; n = 17). The rates were analyzed by chi-square and Fisher’s exact tests (significance of 5%). In experiments 1 (3.6; 15.0; and 60.0%, respectively to 2DB, DMDB, and CG) and 2 (8.3; 36.4; and 77.3%, respectively to 2DB, 4DB, and CG), the AR differed among groups with exception of 2DB and DMDB (exp. 1; P > 0.05). In Exp. 3, there was no difference on the AR between TI and 2T1I (27.1 and 29.4%, respectively). Despite the low adhesion potential of the trophectoderm cells, is feasible to produce chimeric embryos by aggregation. We infer that the use of an adhesive agent could increase AR maintaining the contact of the embryonic fragments. The proposed technique was practical and effective for blastocyst reconstruction. Additionally, the present model established in mice is about to be tested in cattle for further validation. Financial support: FAPESP.

Assessment of desiccants and their instructions for use in rapid diagnostic tests

BackgroundMalaria rapid diagnostic tests (RDTs) are protected from humidity-caused degradation by a desiccant added to the device packaging. The present study assessed malaria RDT products for the availability, type and design of desiccants and their information supplied in the instructions for use (IFU).MethodsCriteria were based on recommendations of the World Health Organization (WHO), the European Community (CE) and own observations. Silica gel sachets were defined as self-indicating (all beads coated with a humidity indicator that changes colour upon saturation), partial-indicating (part of beads coated) and non-indicating (none of the beads coated). Indicating silica gel sachets were individually assessed for humidity saturation and (in case of partial-indicating silica gels) for the presence of indicating beads.ResultsFifty malaria RDT products from 25 manufacturers were assessed, 14 (28%) products were listed by the “Global Fund Quality Assurance Policy” and 31 (62%) were CE-marked. All but one product contained a desiccant, mostly (47/50, 94%) silica gel. Twenty (40%) RDT products (one with no desiccant and 19 with non-indicating desiccant) did not meet the WHO guidelines recommending indicating desiccant. All RDT products with self- or partial-indicating silica gel (n = 22 and 8 respectively) contained the toxic cobalt dichloride as humidity indicator. Colour change indicating humidity saturation was observed for 8/16 RDT products, at a median incidence of 0.8% (range 0.05%-4.6%) of sachets inspected. In all RDTs with partial-indicating silica gel, sachets with no colour indicating beads were found (median proportion 13.5% (0.6% - 17.8%) per product) and additional light was needed to assess the humidity colour. Less than half (14/30, 47%) IFUs of RDT products with indicating desiccants mentioned to check the humidity saturation before using the test. Information on properties, safety hazards and disposal of the desiccant was not included in any of the IFUs. There were no differences between Global Fund-listed and CE marked RDT products compared to those which were not. Similar findings were noted for a panel of 11 HIV RDTs that was assessed with the same checklist as the malaria RDTs.ConclusionRDTs showed shortcomings in desiccant type and information supplied in the IFU.

Survival of Actinobacillus pleuropneumoniae outside the pig

Transmission of Actinobacillus pleuropneumoniae is primarily thought to be via direct transfer of mucus from pig to pig. For transfer between farms, the organism may need to persist in the wet or dried state to be carried on an inanimate surface. The survival of A. pleuropneumoniae was investigated under controlled laboratory conditions. In aqueous suspension, survival was improved by the presence of NaCl and mucin; it was prolonged at lower temperature. In dry state, it survived best on a hydrophobic surface either under desiccated conditions or saturated humidity. Detectable viability was maintained for 3–4days. When frozen, A. pleuropneumoniae survived for more than 17weeks at −20°C, but the viability declined to 0.01% during that time. Survival at −70°C was effective for long term storage. Results obtained from this investigation would be applicable for sampling method, transport techniques, epidemiological study, and biosecurity implementation.

Numerical simulation of humidification and heating during inspiration within an adult nose

The temperature of inhaled air is highly relevant for the humidification process. Narrow anatomical conditions limit possibilities for in vivo measurements. Numerical simulations offer a great potential to examine the function of the human nose. In the present study, the nasal humidification of inhaled air was simulated simultaneously with temperature distribution during a respiratory cycle. A realistic nose model based on a multislice CT scan was created. The simulation was performed by the Software Fluent(r). Boundary conditions were based on previous in vivo measurements. Inhaled air had a temperature of 20(deg)C and relative humidity of 30%. The wall temperature was assumed to be variable from 34(deg)C to 30(deg)C with constant humidity saturation of 100% during the respiratory cycle. A substantial increase in temperature and humidity can be observed after passing the nasal valve area. Areas with high speed air flow, e.g. the space around the turbinates, show an intensive humidification and heating potential. Inspired air reaches 95% humidity and 28(deg)C within the nasopharynx. The human nose features an enormous humidification and heating capability. Warming and humidification are dependent on each other and show a similar spacial pattern. Concerning the climatisation function, the middle turbinate is of high importance. In contrast to in vivo measurements, numerical simulations can explore the impact of airflow distribution on nasal air conditioning. They are an effective method to investigate nasal pathologies and impacts of surgical procedures.

익산 미륵사지 석탑 사리구의 현황과 매장환경 분석

익산 미륵사지 석탑은 백제 무왕대인 639년에 축조되었고 탑 안에 봉안된 사리유물은 1,370년 동안 석탑 심주석 사리공의 매장환경 속에 존재하였다. 현재까지 우리나라에서 확인된 사리유물은 150여 건에 이른다. 그러나 사리유물의 수습 및 유물에 대한 연구가 진행된 바 있지만 사리유물이 어떠한 환경에 노출되었는지에 대한 데이터 분석은 이루어진 바가 없다. 따라서 본 연구에서는 사리유물의 노출환경에 대한 훼손도를 평가함과 동시에 매장환경에 대한 분석을 진행하고 데이터를 제시하였다. 사리구의 매장환경을 분석한 결과 목탑의 경우는 물의 영향을 가장 많이 받지만 석탑은 포화습도의 영향을 많이 받는다는 것으로 나타났다. 따라서 사리구 내부의 환경은 1~2일 정도 완충된 외부의 온도에 비교적 민감한 영향을 받지만, 습도와는 무관한 반응을 보였다. 그러나 사리구 내부의 포화습도에 도달되는 기간은 사리를 봉안하는 날의 외부습도에 영향을 받는 것으로 나타났다. 본 탑은 30일이 소요되는 시점에 100% 포화습도에 이르렀다. Iksan Mireuksaji Stone pagoda was built A.D. 639 when King Moo was governing Baekje, in which Sarira has been enshrined in a hole dug in the stereobate for 1,370 years. In South Korea, about 150 cases of Sarira have been found so far. In this connection, many studies have been conducted on how to manage it and on its genesis, but there have been no data on environments to which it has been exposed. In this study, accordingly, researches were made into environments to which Sarira has been exposed as well as into how much it has been damaged under the environments. Sarira in wooden pagodas was influenced greatly by water, but that in stone pagodas by saturated relative humidity. Thus, Sarira in the containers was significantly influenced by the outside temperature for 1 to 2 days, but had no any reaction to humidity. The time it took for the humidity in a Sarira container to reach its maximum, varied depending upon the humidity of the day when Sarira was enshrined. In the case of this pagoda, the humidity reached its maximum 30 days later.

Electric-Field-Controlled Flow in Nanoscale-Thin Wetting Films

A novel nanofluidic system based on electroosmotic flow in nanoscale-thin aqueous wetting films is reported. The water films formed spontaneously on mica substrates in a saturation humidity environment. The film thickness was determined to be a few tens of nanometers by optical interference and fluorescence intensity measurements and was consistent with a theoretical evaluation of the thickness of a film based on the competing forces of electrostatic repulsion and capillary pressure. Lateral flow was induced by applying a dc electric field tangential to the film and characterized by tracking the position of a fluorescent probe. The mobilities of the thin fluid layer and the flow marker were lower than the predictions of the electrokinetic theory, which may be a result of adsorption of the fluorescent molecules to the mica. Confinement of the film to two-dimensional "channels" was achieved by microcontact printing of patterned hydrophobic monolayers onto the substrate. This system has the advantage of simple and inexpensive fabrication in comparison to nanofluidic devices made by traditional lithography techniques.

Fabrication of multi-level carbon nanotube arrays with adjustable patterns

Multi-level carbon nanotube (CNT) arrays with adjustable patterns were prepared by a combination of the breath figure (BF) process and chemical vapor deposition. Polystyrene-b-poly(acrylic acid)/ferrocene was dissolved in carbon disulfide and cast onto a Si substrate covered with a transmission electron microscope grid in saturated relative humidity. A two-level microporous hybrid film with a block copolymer skeleton formed on the substrate after evaporation of the organic solvent and water. One level of ordered surface features originates from the contour of the hard templates; while the other level originates from the condensation of water droplets (BF arrays). Ultraviolet irradiation effectively cross-linked the polymer matrix and endowed the hybrid film with improved thermal stability. In the subsequent pyrolysis, the incorporated ferrocene in the hybrid film was oxidized and turned the polymer skeleton into the ferrous inorganic micropatterns. Either the cross-linked hybrid film or the ferrous inorganic micropatterns could act as a template to grow the multi-level CNT patterns, e.g. isolated and honeycomb-structured CNT bundle arrays perpendicular to the substrate.

Effect of Thermal Treatment on Fracture Toughness of Wood Specimen Dried by Modeled High-Temperature Setting Method

The high-temperature setting method may be put to practical use in Japanese sawmills for drying square timbers including pith. In order to prevent splits and cracks, green timber is softened by heating and then rapidly dried at high temperature in low humidity air, so as to generate compression stress on the surface. In this study, we perform wedge splitting tests to obtain the fracture energy, and discuss how thermal treatment affects the timber's fracture toughness.Spruce specimens were curved to perform the wedge splitting test. The curved air-dried specimens impregnated with sufficient distilled water to imitate green wood. Two sets of timber specimens were used : the first set, to test the degradation caused by the initial heating process, and the second set, to examine the further degradation effects attributable to the so-called drying set process. The wet specimens were heated for 12 hours, at one of four temperatures (105°C, 85°C, 75°C and 65°C) in saturated humidity air. For the drying set process, those specimens initially heated at 85°C were dried for 12 hours in four temperature conditions (120°C, 90°C, 75°C and 55°C) in low humidity air (33% of relative humidity).As a result, the fracture toughness of almost all specimens was less than that of the control specimen. In particular, that of heated wet specimen was lower with increasing process temperature. This also seems to be based on the thermal decomposition of wet hemicelluloses, since wet hemicelluloses are thought to decompose at 80-90°C. The specimen dried at 55°C displayed the least toughness of all. As this glass transition temperature of wet lignin is approximately 60°C, this specimen was probably insufficiently softened to prevent splits and cracks from occurring on the surface.

72 USE OF C57BL/6/EGFP MOUSE TESTICULAR CELLS TO TRAIN PERIVITELLINE SPACE MICROINJECTION

The production of embryonic chimeras has been studied as a tool for in vivo pluripotency validation in embryonic stem cells (ESC) as well as to produce transgenic mice. Among the techniques to produce chimeras, one of the most used is microinjection (MI) of ESC into blastocysts or in the perivitelline space (PVS) of the embryos with 4 to 8 cells. A well-established training model for this technique could be very useful when ESC are not available, in which injected cells could be easily identified and their subsequent fate could be tracked. Hence, we aimed to test, in mice, a training model for MI in embryos (Swiss Webster, SW) using a pool of EGFP cells derived from testes of the C57BL/6/EGFP strain. Embryos were recovered from prepubertal female SW (n = 20), superstimulated and mated according to a previously described treatment. The MI was performed in the PVS of 4- to 8-cell embryos (collected at 2.5 dpc). When possible, embryos from the same female were randomly allocated to 3 groups: control (C, n = 17), embryos not subjected to MI; perforated (P, n = 15), embryos submitted to perforation by micropipette, without cell injection; and microinjected (MI, n = 32), embryos perforated and submitted to PVS injection with 6 to 8 cells from EGFP testes. After manipulation, embryos from all groups underwent 24 h of in vitro culture (37°C, 5% CO2 and saturated humidity). The viability and quality of the embryos (according to the IETS Manual 1998) and, in group MI, the fluorescence of testicular cells, were evaluated pre- and post-culture. The results were analysed by chi-square test (total frequency observed) and ANOVA (considering the four replicates) with significance being considered when P < 0.05. There was no difference among mortality rates [i.e. % of viable embryos that died after 24 h of culture, of the groups (5.9, 26.7 and 25.0% for C, P and MI, respectively]. The percentage of embryos that maintained or improved quality after 24 h of culture, in comparison with quality evaluation pre-culture, was different (P < 0.01) among groups C, P and MI (94.1, 73.3 and 43.8%, respectively). One chimeric blastocyst was obtained in the MI group (3.1%, 1/32). Considering the proposed conditions, this model for training of MI of EGFP testicular cells in the PVS was feasible and practical to acquire skills, when ESC are not available. Moreover, the method allows easy identification of injected and, eventually, aggregated cellular components. Financial support was received from FAPESP of Brazil.

71 EFFECT OF CELL QUANTITY IN MICE CHIMERA PRODUCTION BY DEMI-BLASTOCYST AGGREGATION

Eight-cell-stage to pre-compaction morula are the most used embryonic stages to aggregation, because the embryos, in these early stages, synthesise cell adhesion molecules that increase the aggregation chances among them (Vestweber et al. 1987 Develop. Biol. 124, 451–456). Although post-compaction embryos produce reduced aggregation rates, they are not refractory to this process (Nogueira et al. 2010 Transgenic Res. 19, 344–345). Based on the evidence of less permissive aggregation in post-compaction-stage embryos and the need to expose the inner surface of those embryos to improve aggregation rate, the aim of this study was to evaluate, in mice, the influence of cell quantity (i.e. the quantity of half-embryos put together to aggregate themselves) in the chimerism rate of split blastocysts. Embryos, with preferentially different phenotypes, were obtained from C57BL/6/EGFP and Swiss Webster strains. Females ranging from 21 to 45 days old were superstimulated and mated according to Mancini et al. (2008 Transgenic Res. 17, 1015). Eight-cell-stage embryos (8C) and pre-compaction morula (PCM) were recovered (2 to 2.5 days post coitum) and had their zona pellucida removed using pronase treatment (2 mg mL–1 for 15 min), whereas blastocysts (recovered 3.5 dpc) were split with a microblade controlled by micromanipulator in an inverted microscope (NK2; Eppendorf, Hamburg, Germany and Eclipse Ti; Nikon, Tokyo, Japan, respectively). The aggregation groups were a control (C) with 2 pre-compaction whole embryos (8C or PCM, or both) and 2 experimental with post-compaction embryos [i.e. 2 (2DB) or 4 (4DB) demi-blastocysts]. The structures (2 or 4) of the groups were stuck to each other with the use of phytohemagglutinin (1 mg mL–1) and cultured in vitro by 24 h (37°C, 5% CO2 and saturated humidity). After culture, the presence of chimeric embryos was verified by detection of a single, cohesive cell mass or a structure in an 8 shape with more than one-half of its total diameter aggregated. For the 4DB group, a successful aggregation was considered when, at least 2 of 4 DB had aggregated. The results were analysed using chi-square test, Fisher's exact test and Kruskal-Wallis (to compare among groups, between groups and among medians of group replicates, respectively) and significance was considered when P < 0.05. The aggregation rates for the groups C, 2DB and 4DB were, respectively, 77.3a; 8.3b and 36.4%c (P < 0.001). The increasing of the aggregation technique efficacy, in post-compaction stages, would be particularly interesting in farm animals (e.g. bovine species), where it is not feasible to obtain, in vivo, pre-compaction stages embryos (as 8 cells) and when only trophectoderm aggregation is wanted. It was concluded that cell increasing (from 2 to 4 DB) improved the chimerism rate, but not enough to be similar to the control group. Supported by FAPESP of Brazil.

94 USE OF β-MERCAPTOETHANOL AND CYSTEINE FOR IN VITRO MATURATION AND CULTURE OF SHEEP EMBRYOS

During in vitro production (IVP), embryos are sensitive to suffering negative effects from catabolites, such as reactive oxygen species (ROS). Under physiological conditions, the action of the ROS is blocked by antioxidants such as glutathione, but glutathione's concentration is reduced during the main steps of the IVP process. The objective of the present study is to evaluate the effect of the supplementation of the media for in vitro maturation (IVM) and in vitro culture (IVC) with β-mercaptoethanol and cysteine on the rates of embryo development and viability after vitrification in open pulled straws (OPS). Ten IVP routines were conducted for IVP, using ovaries form pubertal sheep collected in a slaughterhouse. The ovaries were kept in a saline/antibiotic solution at 30°C during transport to the laboratory. The cumulus oophurus–oocytes complexes (COC) selected for IVM were allocated to 2 treatments: T1 (control), including no antioxidants in the IVM and IVC media (n = 676); and T2, including 50 μM β-mercaptoethanol and 600 μM cysteine, in the IVM and IVC media (n = 729). The IVM was conducted using the TCM 199 medium including oestradiol, FSH, LH, pyruvate, heat inactivated sheep serum and antibiotics, for 22 to 24 h. Sperm selection was conducted by swim-up in medium with tris-glucose-citric acid with fresh semen. For IVF, conducted for 18 to 22 h, 1 × 106 spermatozoa per mL were used in SOF medium including 2% heat-inactivated sheep serum. Both IVM and IVF were conducted with incubation with 5% CO2 at 39°C with saturated humidity. After IVF, the probable zygotes were denuded and cultured for 8 days in SOF medium with 0.4% BSA, at 39°C, in bags with 3 gases (5% CO2, 90% N2 and 5% O2). The criteria considered for embryo viability were: cleavage rate at Day 2 (cleaved/inseminated), embryo development at Day 7 (blastocysts/cleaved) and the reexpansion rate 24 h post-vitrification. Such frequencies were compared between treatments by the chi-squared test. The cleavage rate did not differ (P > 0.05) for T1 (60.3%) and T2 (64.3%). The rate of embryro development at Day 7 was also similar (P > 0.05) for T1 (33.6%) and T2 (36.6%). The reexpansion rate for T1 (76.9%) and T2 (54.1%) were also similar (P > 0.05). Thus, supplementation of IVM and IVC media with β-mercaptoethanol and cysteine presented no effect in the development and viability of vitrified sheep embryos. CAPES, MARFRIG Group.