Endometrial claudin-4 and leukemia inhibitory factor are associated with assisted reproduction outcome

Background: Claudin-4 (CLDN4) is one of several proteins that act as molecular mediators of embryo implantation. Recently, we examined immunolabeling of leukemia inhibitory factor (LIF) in the endometrial tissue of 52 IVF patients, and found that LIF staining intensity was strongly correlated with successful pregnancy initiation. In the same set of patients, we have now examined endometrial CLDN4 expression, to see how expression intensity may vary with LIF. We examined CLDN4 in the luteal phase of the menstrual cycle, immediately preceding IVF treatment. Our aim was to compare expression of LIF and CLDN4 in the luteal phase, and document these patterns as putative biomarkers for pregnancy. Methods: Endometrial tissue was collected from women undergoing IVF. Endometrial biopsies were obtained during the luteal phase preceding IVF, and were then used for tissue microarray (TMA) immunolabeling of CLDN4. Previously published LIF expression data were then combined with CLDN4 expression data, to determine CLDN4/LIF expression patterns. Associations between successful pregnancy after IVF and combined CLDN4/LIF expression patterns were evaluated. Results: Four patterns of immunolabeling were observed in the endometrial samples: 16% showed weak CLDN4 and strong LIF (CLDN4(-)/LIF(+)); 20% showed strong CLDN4 and strong LIF (LIF(+)/CLDN4(+)); 28% showed strong CLDN4 and weak LIF (CLDN4(+)/LIF(-)); and 36% showed weak CLDN4 and weak LIF (CLDN4(-)/LIF(-)). Successful implantation after IVF was associated with CLDN4(-)/LIF(+)(p = 0.003). Patients showing this endometrial CLDN4(-)/LIF(+) immunolabeling were also 6 times more likely to achieve pregnancy than patients with endometrial CLDN4(+)/LIF(-) immunolabeling (p = 0.007). Conclusion: The combined immunolabeling expression of CLDN4(-)/LIF(+) in endometrial tissue is a potential biomarker for predicting successful pregnancy in IVF candidates.

Influence of Caffeine and Chondroitin Sulfate on Swine Sperm Capacitation and In Vitro Embryo Production

Background: The establishment of an in vitro production (IVP) of embryo in swine allows the generation of embryos with the same quality as in vivo produced embryos with less costs and time. In order to achieve successful fertilization under normal circumstances in vivo, mammalian spermatozoa must first undergo capacitation and then acrosome reaction. The purpose of this study was compared the efficacious of IP/CFDA fluorescence and Coomassie Blue G (CB) staining to detect capacitated sperm cells in refrigerated and fresh semen. Morever, it was investigated the efficacious of caffeine and chondroitin sulphate to promote in vitro sperm capacitation and in vitro embryo produced (IVP) of swine embryos. Materials, Methods & Results: A sperm-rich fraction from ejaculate was obtained using the gloved-hand method and the gel-free fraction was separated using sterile gauze. The semen was diluted in BTS at a final concentration of 1.5 x 10(8) cells/mL. The sperm suspension was incubated for 2 h at 25 degrees C, refrigerated and maintained for 1 h at 15-18 degrees C (refrigerated group) or used immediately (fresh group). Sperm capacitation was assessed by IP/CFDA fluorescence and CB staining for both fresh and refrigerated semen. For PI/CFDA evaluation, a final solution containing 1.7 mM formaldehyde, 7.3 mM PI and 20 mM CFDA in 950 mu L saline was prepared. In the dark, 40 mu L PI/CFDA final solution was added to 10 mu L semen and after 8 min, slides were analyzed on epifluorescence microscopy. For CB evaluation, sperm cells were fixed in 4% paraformaldehyde for 10 min and centrifuged twice at 320 x g in ammonium acetate pH 9 for 8 min. A smear was made and stained with 2.75 mg/mL CB in solution containing 12.5% methanol, 25% glacial acetic acid and 62.5% water, for 2 min. The smear was washed in running water, air dried and sealed with Permount (R), diluted 2:1 in xilol to avoid staining oxidation. Our results showed that refrigeration did not affect sperm capacitation and comparing staining methods, the PI/CFDA combination was more efficient to detect capacitated sperm, when compared to CB staining. In experiment 2, we evaluated the effect of different incubation time (1 - 5 h) with chondroitin sulfate and caffeine on sperm capacitation. For in vitro fertilization, oocytes were obtained from slaughterhouse ovaries. Oocytes with a thick and intact cumulus oophurus layer and cytoplasm with homogenous granules were selected for in vitro maturation for 44 h. According to the results of experiment 2, it was used for in vitro fertilization refrigerated semen was capacitated with 50 mu g/mL chondroitin sulfate for 2 h or capacitated with 5 mu g/mL caffeine for 3 h. Six hours after insemination, cumulus oophorus cells were mechanically removed and oocytes were washed and incubated in microdrops of culture medium. Embryo development after fertilization with sperm capacitated with caffeine or chondroitin sulfate was evaluated on days 3, 5 and 7 of culture. No differences were observed in days 3 or 5 of in vitro culture. However, it was observed an increase on blastocyst rate on Day 7 of culture when caffeine was used as the capacitor agent. Discussion: Molecular basis of sperm capacitation is still poor understood. Sperm capacitation can occur in vitro spontaneously in defined media without addition of biological fluids. We observed that sperm capacitation increased as incubation period enlarged and it was observed using Coomassie blue G and PI/CFDA for fresh semen and for refrigerated semen. It can be concluded that the cooling of semen did not change their pattern of sperm capacitation and this is best assessed by IP/CFDA than by CB. In addition to the use of caffeine in sperm capacitation produces more blastocysts than the chondroitin sulfate after in vitro fertilization.

Effect of Hormonal Supplementation Periods and In Vitro Maturation Media on Developmental Competence of Pig Oocytes

Background: The oocyte ability to undergo successful fertilization, cleavage and embryonic development depends on meiotic maturation and developmental competence acquisition. In vitro maturation (IVM) protocols currently use eCG, hCG or a combination of both, the effect of these gonadotrophins during IVM and subsequent embryonic development is still controversial. Several media have been used for IVM of porcine oocytes: TCM199, Whitten's and NCSU23 have also been shown to support pig oocyte IVM. This study was designed to determine the effect of hormonal supplementation period and maturation media during in vitro maturation of pig oocytes (1) and subsequent embryonic development (2). Materials, Methods & Results: Oocytes with intact cumulus oophurus layers and homogeneous cytoplasm were collected from prebubertal gilts. IVM was subjected in NCSU23, TCM199 or Whitten's media supplemented with 10 IU/mL eCG and 10 IU/mL hCG for the first 24 or 48 h of IVM. In each replicate the oocytes were fixed every 4 h from 32 to 48 h IVM or the past 48 h after IVM, oocytes were fertilized in vitro in mTBM medium for six hours and cultured in NCSU23 medium for nine days. Cleavage, blastocyst and hatching rates were evaluated at 48 h (day 2), 168 h (day 7) and 216 h (day 9), respectively. The addition of eCG and hCG during the first 24 h IVM increased the proportion of oocytes that reached MII stage at 44 h of maturation in NCSU23 medium. This effect was also observed in Whitten medium at 44 and 48 h (P < 0.05). However, it was not observed in the TCM199 medium. No effect of maturation medium on oocyte nuclear maturation (P > 0.05) was observed in oocytes matured in the presence of eCG and hCG during the first 24 h IVM or during 48 h IVM. A progressive increase of maturation indexes was observed on oocytes matured with hormonal supplementation in Whitten media for 24 h. Higher indexes were obtained at 44 and 48 h. When NCSU23 media was used, no difference after 36 h of maturation was observed. The same result was observed in TCM199. A progressive increase of maturation indexes was observed on oocytes matured with hormonal supplementation for 48 h in Whitten media. Higher indexes were obtained in 36 and 40 h. When NCSU23 or TCM199 were used, no difference was observed. No effect of IVM media on the percentage of fertilized oocytes and polyspermic oocytes or number of spermatozoa per fertilized oocytes was observed. Also, no effect of IVM media on cleavage and blastocyst rates was seen. However, the proportion of hatched blastocysts was lower in NCSU23 compared to Whitten or TCM199. Discussion: Similar results were reported by Marques et al. [13], that it no differences between hormonal supplementation for 22 or 44 h were observed. Therefore, more studies are needed to elucidate the role of these hormones in nuclear in vitro maturation in pig oocytes. In conclusion, no effect of maturation media on meiotic progression was observed. However, the proportion of oocytes that reached metaphase II (MII) stage was higher when eCG + hCG were added for 24 h than 48 h mainly at the 44 h of maturation. In addition, no differences were observed in cleavage and blastocyst rates of the cultured embryos. However, embryos cultured in NCSU23 showed lower rates of hatching compared to other media. These results indicated no effect of maturation media on the fertilization and embryonic development even in the presence of cysteine, PFF and EGF, except for hatched embryos that these rates were lower in NCSU23.

Consequences for the Bovine Embryo of Being Derived from a Spermatozoon Subjected to Post-Ejaculatory Aging and Heat Shock: Development to the Blastocyst Stage and Sex Ratio

The objective was to determine whether aging of sperm caused by incubation at normothermic (38.5 C) or heat shock (40 C) temperatures for 4 h prior to oocyte insemination affects sperm motility, fertilizing ability, competence of the resultant embryo to develop to the blastocyst stage and blastocyst sex ratio. In the first experiment, the percent of sperm that were motile was reduced by aging (P<0.001) and the reduction in motility was greater for sperm at 40 C compared to sperm at 38.5 C (P<0.01). In the second experiment, oocytes were inseminated with aged sperm. A smaller percent of oocytes fertilized with sperm aged at either temperature cleaved by Day 3 after insemination than oocytes fertilized with fresh sperm (P<0.05). There was no effect of sperm aging on the percent of oocytes or cleaved embryos that developed to the blastocyst stage. Aging of sperm before fertilization at 38.5 C reduced the percent of blastocysts that were male (P=0.08). In the third experiment, incubation of sperm at 38.5 C or 40 C for 4 h did not reduce fertilizing ability of sperm as determined by pronuclear formation at 18 h post insemination. In conclusion, aging of sperm reduced cleavage rate and the percent of blastocysts that were males but had no effect on the developmental capacity of the. embryo. The effect of aging on cleavage rate may represent reduced motility and errors occurring after fertilization and pronuclear formation. Aging at a temperature characteristic of maternal hyperthermia had little additional effect except that polyspermy was reduced. Results indicate that embryo competence for development to the blastocyst stage is independent of sperm damage as a result of aging for 4 h at normothermic or hyperthermic temperatures.

Imprinted gene expression in in vivo- and in vitro-produced bovine embryos and chorio-allantoic membranes

Cloning by nuclear transfer is often associated with poor results due to abnormal nuclear reprogramming of somatic donor cells and altered gene expression patterns. We investigated the expression patterns of imprinted genes IGF2 and IGF2R in 33- to 36-day bovine embryos and chorio-allantoic membranes derived from in vivo- and in vitro-produced embryos by somatic cell nuclear transfer (SCNT), parthenogenetic activation, and in vitro fertilization (IVF). There was a lower IGF2 expression rate in the SCNT (0.19) and parthenogenetic (0.02) groups when compared to in vivo and IVF embryos (2.01; P < 0.05). In the chorio-allantoic membranes, IGF2 showed a baseline expression pattern (P < 0.05) in parthenotes (0.001) when compared to in vivo, IVF (3.13), and SCNT (0.98) groups. IGF2R was less expressed (P < 0.05) in SCNT chorio-allantoic membranes (0.25) when compared to the in vivo group. The low expression of IGF2 in parthenogenetic embryos and chorio-allantoic membranes confirms its imprinted status in cattle. Alterations in the relative frequency of IGF2 and IGF2R transcripts were observed in SCNT-derived bovine embryos and chorioallantoic membranes, respectively, supporting the hypothesis that abnormalities in the expression of imprinted genes are causes of the low efficiency of SCNT procedures in this species.

Historical carbon emissions and uptake from the agricultural frontier of the Brazilian Amazon

Tropical ecosystems play a large and complex role in the global carbon cycle. Clearing of natural ecosystems for agriculture leads to large pulses of CO(2) to the atmosphere from terrestrial biomass. Concurrently, the remaining intact ecosystems, especially tropical forests, may be sequestering a large amount of carbon from the atmosphere in response to global environmental changes including climate changes and an increase in atmospheric CO(2). Here we use an approach that integrates census-based historical land use reconstructions, remote-sensing-based contemporary land use change analyses, and simulation modeling of terrestrial biogeochemistry to estimate the net carbon balance over the period 1901-2006 for the state of Mato Grosso, Brazil, which is one of the most rapidly changing agricultural frontiers in the world. By the end of this period, we estimate that of the state`s 925 225 km(2), 221 092 km(2) have been converted to pastures and 89 533 km(2) have been converted to croplands, with forest-to-pasture conversions being the dominant land use trajectory but with recent transitions to croplands increasing rapidly in the last decade. These conversions have led to a cumulative release of 4.8 Pg C to the atmosphere, with similar to 80% from forest clearing and 20% from the clearing of cerrado. Over the same period, we estimate that the residual undisturbed ecosystems accumulated 0.3 Pg C in response to CO2 fertilization. Therefore, the net emissions of carbon from Mato Grosso over this period were 4.5 Pg C. Net carbon emissions from Mato Grosso since 2000 averaged 146 Tg C/yr, on the order of Brazil`s fossil fuel emissions during this period. These emissions were associated with the expansion of croplands to grow soybeans. While alternative management regimes in croplands, including tillage, fertilization, and cropping patterns promote carbon storage in ecosystems, they remain a small portion of the net carbon balance for the region. This detailed accounting of a region`s carbon balance is the type of foundation analysis needed by the new United Nations Collaborative Programmme for Reducing Emissions from Deforestation and Forest Degradation (REDD).

The Amazon Frontier of Land-Use Change: Croplands and Consequences for Greenhouse Gas Emissions

The Brazilian Amazon is one of the most rapidly developing agricultural frontiers in the world. The authors assess changes in cropland area and the intensification of cropping in the Brazilian agricultural frontier state of Mato Grosso using remote sensing and develop a greenhouse gas emissions budget. The most common type of intensification in this region is a shift from single-to double-cropping patterns and associated changes in management, including increased fertilization. Using the enhanced vegetation index (EVI) from the Moderate Resolution Imaging Spectroradiometer (MODIS) sensor, the authors created a green-leaf phenology for 2001-06 that was temporally smoothed with a wavelet filter. The wavelet-smoothed green-leaf phenology was analyzed to detect cropland areas and their cropping patterns. The authors document cropland extensification and double-cropping intensification validated with field data with 85% accuracy for detecting croplands and 64% and 89% accuracy for detecting single-and double-cropping patterns, respectively. The results show that croplands more than doubled from 2001 to 2006 to cover about 100 000 km(2) and that new double-cropping intensification occurred on over 20% of croplands. Variations are seen in the annual rates of extensification and double-cropping intensification. Greenhouse gas emissions are estimated for the period 2001-06 due to conversion of natural vegetation and pastures to row-crop agriculture in Mato Grosso averaged 179 Tg CO(2)-e yr(-1),over half the typical fossil fuel emissions for the country in recent years.

Total soil carbon and chemical attributes under different land uses in the Brazilian savanna

Total soil carbon and chemical attributes under different land uses in the Brazilian savanna. The Brazilian savanna region (Cerrado) is one of the largest cultivated areas of the world. The different land uses in the region can effectively change the quantities of soil organic matter and the cycling of nutrients. I-lie objective of this study was to evaluate the effect of different land use management systems on the relationship between soil organic carbon and the soil chemical attributes of a Red Latosol (Oxisol) under Cerrado in Rio Verde (Goias state). The treatments studied were native vegetation (cerrado), low-productivity pasture, conventional tillage with soybean, and no-tillage with soybean and maize. The smallest values for pH, available P, K, Ca and Mg were observed for the Cerradao treatment, even if the relatively high C levels increased the potential soil cation exchange capacity. The pasture, conventional tillage and no-tillage treatments showed higher K, Ca, Mg, available 13, and S concentrations in the soil. In the areas where soil tillage did not take place and lime and fertilizers were applied superficially, the stratification of the soil organic carbon provides the retention of the elements near to the surface, with significance correlations with the soil chemicals attributes.

BORON UPTAKE AND DISTRIBUTION IN FIELD GROWN CITRUS TREES

In low fertility tropical soils, boron (B) deficiency impairs fruit production. However, little information is available on the efficiency of nutrient application and use by trees. Therefore, this work verified the effects of soil and foliar applications of boron in a commercial citrus orchard. An experiment was conducted with fertigated 4-year-old `Valencia` sweet orange trees on `Swingle` citrumelo rootstock. Boron (isotopically-enriched 10B) was supplied to trees once or twice in the growing season, either dripped in the soil or sprayed on the leaves. Trees were sampled at different periods and separated into different parts for total B contents and 10B/11B isotope ratios analyses. Soil B applied via fertigation was more efficient than foliar application for the organs grown after the B fertilization. Recovery of labeled B by fruits was 21% for fertigation and 7% for foliar application. Residual effects of nutrient application in the grove were observed in the year after labeled fertilizer application, which greater proportions derived from the soil supply.

Comparison of univariate and multivariate calibration for the determination of micronutrients in pellets of plant materials by laser induced breakdown spectrometry

The application of laser induced breakdown spectrometry (LIBS) aiming the direct analysis of plant materials is a great challenge that still needs efforts for its development and validation. In this way, a series of experimental approaches has been carried out in order to show that LIBS can be used as an alternative method to wet acid digestions based methods for analysis of agricultural and environmental samples. The large amount of information provided by LIBS spectra for these complex samples increases the difficulties for selecting the most appropriated wavelengths for each analyte. Some applications have suggested that improvements in both accuracy and precision can be achieved by the application of multivariate calibration in LIBS data when compared to the univariate regression developed with line emission intensities. In the present work, the performance of univariate and multivariate calibration, based on partial least squares regression (PLSR), was compared for analysis of pellets of plant materials made from an appropriate mixture of cryogenically ground samples with cellulose as the binding agent. The development of a specific PLSR model for each analyte and the selection of spectral regions containing only lines of the analyte of interest were the best conditions for the analysis. In this particular application, these models showed a similar performance. but PLSR seemed to be more robust due to a lower occurrence of outliers in comparison to the univariate method. Data suggests that efforts dealing with sample presentation and fitness of standards for LIBS analysis must be done in order to fulfill the boundary conditions for matrix independent development and validation. (C) 2009 Elsevier B.V. All rights reserved.

Phytomass production and nitrogen accumulation in maize cultivated with different doses of (15)N-urea

This work aimed to study the possible alterations in production, accumulation of the vegetative phytomass and nitrogen efficiency use of the maize crop, in different doses of N applied in the fertilization, by using the technique of isotopic dilution of (15)N. The completely randomized block experimental design was adopted, with 5 treatments and 4 replicates. The following treatments were constituted in the doses in covering: 0, 50, 100, 150 and 200 kg ha(-1) of N, with fertilization of N-urea, respectively. Comparisons among the treatments had been run for crop productivity; nitrogen accumulation for the plant, and use of the nitrogen of the urea-(15)N for the crop. The increase of the dose of N-fertilizer resulted in increase of the dry matter mass, of the dry matter yield crop tax, of the productivity and accumulation of N in the maize plants.

Contribuition of nitrogen from biological nitrogen fixation, nitrogen fertilizer and soil nitrogen on the growth of the common bean and cowpea

Biological nitrogen fixation (BNF) constitutes a valuable source of this nutrient for the common bean Phaseolus vulgaris L and cowpea Vigna unguiculata (L.) Walp., being its avaibility affected by mineral N in the soil solution. The objectives of this work were to evaluate the effects of nitrogen rate, as urea, on symbiotic fixation of N(2) in common bean and cowpea plants, using the isotopic technique, and quantifying the relative contributions of N sources symbiotic N(2) fixation, soil native nitrogen and urea N on the growth of the common bean and cowpea. Non nodulating soybean plants were used as standard. The research was carried out in greenhouse, using pots with 5 kg of soil from a Typic Haplustox (Dystrophic Red Yellow Latosol). The experimental design was completely randomized blocks, with 30 treatments and three replications, arranged in 5x3x2 factorial outline. The treatments consisted of five N rates: 2, 15, 30, 45 and 60 mg N kg(-1) soil; three sampling times: 23, 40 and 76 days after sowing (DAS) and two crops: common bean and cowpea. The BNF decreased with increase N rates, varying from 81.5% to 55.6% for cowpea, and from 71.9% to 55.1% for common bean. The symbiotic N(2) fixation in cowpea can substitute totally the nitrogen fertilization. The nitrogen absorption from soil is not affected by nitrogen fertilizer rate. The N recovery from fertilizer at 76 DAS was of 60.7% by common bean, and 57.1% by cowpea. The symbiotic association in common bean needs the application of a starting dose (40 kg N ha(-1)) for economically acceptable yields.

Seed phytate and protein content in beans depending on the application of basalt powder

Seed phytate and protein content in beans depending on the application of basalt powder. The content of phytate in the grains is correlated with the supply of phosphorus to the plant, but there is a lack of knowledge as to possible effect of slower availability of nutrients in the soil. The objectives of this study were to assess the effect of rock powder, alone or combined with cattle manure, on the productivity, levels of phosphorus, protein and phytate content in beans. The experiment was carried out in a randomized blocks design, with four replications. The treatments were control (limestone, granite and natural phosphate); conventional fertilization; powder basalt (2.5, 5.0, 10.0 and 20.0 ton. ha(-1)); cattle manure, and doses of powder basalt with cattle manure. In the treatment with conventional fertilizer, the total phosphorus content in grain was higher than the control, but the application of powder of basalt did not show a difference significant. Increase in the doses of basalt powder increased the phosphorus content, but phytate content remained constant. Basalt powder proved to be an alternative to maintain low levels of phosphorus in the form of phytate in the grains.

Fractionation of Zn, Cd and Pb in a Tropical Soil After Nine-Year Sewage Sludge Applications

A long-term field experiment was carried out in the experiment farm of the Sao Paulo State University, Brazil, to evaluate the phytoavailability of Zn, Cd and Pb in a Typic Eutrorthox soil treated with sewage sludge for nine consecutive years, using the sequential extraction and organic matter fractionation methods. During 2005-2006, maize (Zea mays L.) was used as test plants and the experimental design was in randomized complete blocks with four treatments and five replicates. The treatments consisted of four sewage sludge rates (in a dry basis): 0.0 (control, with mineral fertilization), 45.0, 90.0 and 127.5 t ha(-1), annually for nine years. Before maize sowing, the sewage sludge was manually applied to the soil and incorporated at 10 cm depth. Soil samples (0-20 cm layer) for Zn, Cd and Pb analysis were collected 60 days after sowing. The successive applications of sewage sludge to the soil did not affect heavy metal (Cd and Pb) fractions in the soil, with exception of Zn fractions. The Zn, Cd and Pb distributions in the soil were strongly associated with humin and residual fractions, which are characterized by stable chemical bonds. Zinc, Cd and Pb in the soil showed low phytoavailability after nine-year successive applications of sewage sludge to the soil.

Carbon and nitrogen cycling in a tropical Brazilian soil cropped with sugarcane and irrigated with wastewater

Carbon (C) and nitrogen (N) dynamics in agro-systems can be altered as a consequence of treated sewage effluent (TSE) irrigation. The present study evaluated the effects of TSE irrigation over 16 months on N concentrations in sugarcane (leaves, stalks and juice), total soil carbon (TC), total soil nitrogen (TN), NO(3)(-)-N in soil and nitrate (NO(3)(-)) and dissolved organic carbon (DOC) in soil solution. The soil was classified as an Oxisol and samplings were carried out during the first productive crop cycle, from February 2005 (before planting) to September 2006 (after sugarcane harvest and 16 months of TSE irrigation). The experiment was arranged in a complete block design with five treatments and four replicates. Irrigated plots received 50% of the recommended mineral N fertilization and 100% (T100), 125% (T125), 150% (T150) and 200% (T200) of crop water demand. No mineral N and irrigation were applied to the control plots. TSE irrigation enhanced sugarcane yield but resulted in total-N inputs(804-1622 kg N ha(-1)) greater than exported N (463-597 kg N ha(-1)). Hence, throughout the irrigation period, high NO(3)(-) concentrations (up to 388 mg L(-1) at T200) and DOC (up to 142 mg L(-1) at T100) were measured in soil solution below the root zone, indicating the potential of groundwater contamination. TSE irrigation did not change soil TC and TN. (C) 2009 Elsevier B.V. All rights reserved.