An improved method for specific-target preamplification PCR analysis of single blastocysts useful for embryo sexing and high-throughput gene expression analysis

Gene expression analysis in preimplantation embryos has been used for answering fundamental questions related to development, prediction of pregnancy outcome, and other topics. Limited amounts of mRNA in preimplantation embryos hinders progress in studying the preimplantation embryo. Here, a method was developed involving direct synthesis and specific-target preamplification (STA) of cDNA for gene expression analysis in single blastocysts. Effective cell lysis and genomic DNA removal steps were incorporated into the method. In addition, conditions for real-time PCR of cDNA generated from these processes were improved. By using this system, reliable embryo sexing results based on expression of sex-chromosome linked genes was demonstrated. Calibration curve analysis of PCR results using the Fluidigm Biomark microfluidic platform (Fluidigm, South San Francisco, CA) was performed to evaluate 96 STA cDNA from single blastocysts. In total, 93.75% of the genes were validated. Robust amplification was detected even when STA cDNA from a single blastocyst was diluted 1,024-fold. Further analysis showed that within-assay variation increased when cycle threshold values exceeded 18. Overall, STA quantitative real-time PCR analysis was shown to be useful for analysis of gene expression of multiple specific targets in single blastocysts.     

Mercury content of some marine fish from the southern Caribbean Sea

The mercury concentration in muscle and liver of 63 specimens, comprising 17 species of edible fish from coastal waters of the Caribbean Sea near Cumaná, was determined by cold vapour atomic absorption spectrophotometry. The Hg content of edible muscle tissue, expressed on a fresh weight basis, varied from 0.048 microgram g-1 in lisa (Mugil curema) to 0.190 microgram g-1 in tajali (Trichiurus lepturus), with an average value of 0.096 +/- 0.042 microgram g-1. The corresponding values for liver varied from 0.135 microgram g-1 in lebranche (Mugil brasiliensis) to 0.361 micrograms g-1 in tajali (Trichiurus lepturus), with an average value of 0.269 +/- 0.059 microgram g-1. Of the 17 species analysed, seven had a mercury concentration greater than 0.10 microgram g-1 and the remainder contained less than 0.08 microgram g-1 in edible muscle tissue.     

Interactions of human chorionic gonadotropin with genotype and parity on fertility responses of lactating dairy cows

Fertility-promoting effects of treatment of lactating dairy cattle with human chorionic gonadotropin (hCG) after artificial insemination (AI) have been variable. Here, we tested whether fertility response to hCG in lactating Holstein cows interacts with genotype and parity. Primiparous (n = 538) and multiparous (n = 613) cows were treated with hCG (3,300 IU) or vehicle 5 d after AI. Pregnancy was diagnosed on d 32 and 60 after AI. A subset of cows (n = 593–701) was genotyped for 4 single nucleotide polymorphisms (SNP) previously associated with fertility. Treatment with hCG increased progesterone concentration on d 12 after AI regardless of genotype or parity. Pregnancy per AI was improved by hCG in primiparous cows but not in multiparous cows. Moreover, hCG treatment interacted with a SNP in coenzyme Q9 (COQ9) to affect fertility. Fertility of cows treated with vehicle was greatest for the AA allele, whereas fertility was lowest for the same genotype among cows treated with hCG. Pregnancy per AI was also affected by genotype for heat shock protein A1-like (HSPA1L) and progesterone receptor (PGR), but no interactions were observed with treatment. Genotype for a SNP in prostate androgen-regulated mucin-like protein 1 (PARM1) was not associated with fertility. Overall, results show that variation in response to hCG treatment on fertility depends on parity and interacts with a SNP in COQ9.     

Embedding strength in joints of fast-growing Argentinean Eucalyptus grandis with dowel-type fasteners. Analysis according to the criterion adopted by European standards

The present paper reports on the results of an investigation regarding the determination of the embedding strength of sawn timber of Argentinean Eucalyptus grandis with dowel-type fasteners. The importance of accurately knowing this property has increased with the increasing demand for this material for structural purposes. An empirical research project with 294 specimens prepared for testing under the embedding action of rigid fasteners (nail, bolt and dowel) was carried out according to the procedures prescribed in EN 383 (2007). The results showed no significant influence of pre-drilling on the embedding strength for nailed joints, which is not in line with the European experience and the corresponding criterion adopted by the Eurocode?5 (2005). The characteristic values of the embedding strength estimated based on the timber density and by applying the corresponding equation from the Eurocode?5 (2005) for bolted joints were 45% and 176% greater than those obtained in this project for the specimens loaded parallel and perpendicular to the grain, respectively, and similar percentages were found for dowelled joints (45% and 126%). These results showed that the reliability of structural design may be strongly affected if the embedding strength of this timber species is estimated by following the procedures adopted by the Eurocode?5 (2005) for dowel-type connections.     

Involvement of the oestrogenic receptors in superior mesenteric ganglion on the ovarian steroidogenesis in rat

Oestradiol (E(2)) is a key hormone in the regulation of reproductive processes. The aims of this work were a) to examine the distributions of oestrogen receptor α (ERα) and ERβ in the neurons of the superior mesenteric ganglion (SMG) in the oestrus stage by immunohistochemistry, b) to demonstrate whether E(2) in the SMG modifies progesterone (P(4)), androstenedione (A(2)) and nitrite release in the ovarian compartment on oestrus day and c) to demonstrate whether E(2) in the ganglion modifies the activity and gene expression in the ovary of the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD). The ex vivo SMG-ovarian nervous plexus-ovary system was used. E(2), tamoxifen (Txf) and E(2) plus Txf were added in the ganglion to measure ovarian P(4) release, while E(2) alone was added to measure ovarian A(2) and nitrites release. Immunohistochemistry revealed cytoplasmic ERα immunoreactivity only in the neural somas in the SMG. E(2) increased ovarian P(4) and A(2) release at 15, 30 and 60?min but decreased nitrites. The activity and gene expression of 3β-HSD increased, while the activity and gene expression of 20α-HSD did not show changes with respect to the control. Txf in the ganglion diminished P(4) release only at 60?min. E(2) plus Txf in the ganglion reverted the effect of E(2) alone and the inhibitory effect of Txf. The results of this study demonstrate that ERα activation in the SMG has an impact on ovarian steroidogenesis in rats, thus providing evidence for the critical role of peripheral system neurons in the control of ovarian functions under normal and pathological conditions.     

Turbidimetric Behavior of Colloidal Particles in Beer Before Filtration Process

The effect of colloidal particles and yeast on turbidity of Pilsen beer before the filtration process was studied in this work. The colloidal particles are mainly composed of polysaccharides, representing 96.89 %, in second place proteins with a concentration close to 2 %, and polyphenols less than 0.3 %. There is also a very low concentration of yeast (<0.25 %). The presence of different types of particles in the sample caused multimodal histogram in the particle size distribution and four distinct zones were identified: (1) very small individual particles ( $ \overline D = 0.0{\text{6}}\mu {\text{m}} $ ), (2) yeast ( $ \overline D = {\text{3}}\mu {\text{m}} $ ), (3) colloidal aggregates ( $ \overline D = {\text{17}}\mu {\text{m}} $ ), and (4) a zone with a high dispersion of size, with two $ \overline D $ values (101 and 200 μm). Particles size counts well correlate with both the scanning electron microscopy (SEM) digital image analysis, and the turbidity determination. The fractal dimension (D _f) of the aggregates was determined by analyzing the SEM images with the Variogram method, obtaining D _f?>?2.4 values. Those values are typical of aggregates formed by rapid flocculation or diffusion limited aggregation. Results of this study support the formulation of a model valid for the prediction of colloidal particles concentration in beer.     

Perspectives on allelopathy in mexican traditional agroecosystems: A case study in tlaxcala

Agroecosystems in Tlaxcala, Mexico, are surrounded by trees and water channels and have a great variety of cultivated and noncultivated plants. The main results of a study carried out on a traditional agroecosystem in Santa Inés, Tlaxcala are presented. Some ecological aspects of polycultures, plant covers (dry leaves ofAlnus firmifolia, Berula erecta, andJuncus sp.), and the allelopathic potential of crops and noncultivated plants (fresh and dry material) were analyzed. The main plants (trees, shrubs, and herbs) present in the agroecosystem were identified. The total number of weeds in plots where plant covers were added was reduced. The number of nodules ofRhizobium phaseoli and the production of bean and squash increased with plant covers. Corn, beans, and squash showed a clear allelopathic effect, as well asChenopodium murale, Tradescantia crassifolia, Melilotus indicus, andAmaranthus hybridus, among other weeds. The contribution of allelopathy in studies of traditional agroecosystems is of great importance for the management of species in space and time. Allelopathy can be the basis of biological control of pests and weeds and of the discovery of new useful substances.     

Quality assessment of dehydrated red bell pepper using tempering drying cycles

Quality assessment of red bell pepper dices preserved through batch dehydration introducing tempering cycles were performed in this work. The parameters considered were: the percentages of ascorbic acid and final antioxidant activity retention, as well as the influence of calcium chloride on diffusivity and colour values. Although, a similar final moisture content was reached through different red bell pepper dehydration processes, but the one with tempering cycles had better qualities than those without tempering. Ascorbic acid and final antioxidant activity retention showed higher values and so were colour intensities compared to control samples in all cases. In respect to the effective diffusivity coefficients, there was an increment as the calcium chloride concentration increased. As of the treatments with tempering cycles, the best scheme was 15 min drying time and 30 min tempering time at 60 °C.     

Effect of wood species on corrosion behavior of carbon steel and stainless steels in black liquors

Black liquors produced by pulping different wood species can have different corrosivity towards carbon steel equipment in pulp mills. Inorganic constituents of black liquor alone do not correlate well with the corrosivity of different black liquors. Organic constituents of the black liquor have significant effect on determining its corrosivity. However, understanding of the role of individual organic constituents or group of constituents of black liquors in overall black liquor corrosivity is generally lacking. The objective of this study was to determine the effect selected wood species on the corrosivity of their black liquors which contained similar inorganic constituents. The role of black liquor constituent, pyrocatechols, whose presence in the black liquor has been associated with an increase in corrosion of carbon steel, was also studied. Results from electrochemical tests point to the mechanisms by which the catechols may participate in the accelerated corrosion of carbon steels. This study demonstrates the role of organic constituents of the black liquor in determining its corrosivity.