Cryopreservation of canine semen using a coconut water extender with egg yolk and three different glycerol concentrations

Semen was collected by digital manipulation from six adult dogs. The second fraction of the ejaculate was used in this study. The semen was assessed by macroscopic and microscopic criteria 1 min after collection, first dilution, cooling, glycerol addition and thawing. Experiments were conducted to compare the effect of three different concentrations of glycerol in coconut water extender. The freezing method employed was that one described for caprine semen with slight changes. Semen was thawed at 37 degrees C for 1 min. Spermatozoal motility after thawing was 49.2+/-26, 44.2+/-18.3 and 35.8+/-26.8% for groups with 4, 6 and 8% glycerol, respectively. The vigor after thawing was 2.6+/-1.1, 2.7+/-1.0 and 2.1+/-1.2 for these groups, respectively. There was no difference among groups in motility and vigor. However, a smaller percentage of total and secondary abnormalities was observed using 6% glycerol in coconut water extender. In conclusion, the three glycerol concentrations (4, 6 or 8%) can be used successfully in cryopreservation of canine semen using a coconut water extender.     

Organic and inorganic compound variations in haemolymph, epidermal tissue and cuticle over the molt cycle in Scylla serrata (Decapoda)

Analysis of organic and inorganic compounds in haemolymph, epidermal tissue and cuticle, allowed the correlation of their content variation during the molt cycle in Scylla serrata (Decapoda). The extrusion of carbohydrate, chitin, H(2)O- and NaOH-soluble proteins of the epidermal tissue from D2 stage on suggested an early formation of the organic fraction in the new cuticle. The additional secretion of HCl-soluble protein, glycosaminoglycans, calcium, phosphorus and sulfur in D2-D3 stages suggests mineral nucleation shortly before or at ecdysis. This is consistent with the dominant content of proteins and chitin associated with peaks of carbohydrates and GAG-sulfur groups detected in the cuticle immediately after ecdysis. Furthermore, the maximal phosphorus content at this moment suggests calcium, magnesium and manganese phosphate deposits. After an intense accumulation of calcium, chitin and sharp decrease of protein from A to B stages, a constant rate of their deposition in intermolt and dissolution in premolt occurred. The variations concerning proteins, carbohydrates, glycosaminoglycans, calcium, magnesium and sulfur in the haemolymph, before and after ecdysis, suggest a transitory phenomenon for calcium binding and releasing. Other major elements such as, sodium, potassium and chloride may function as osmotic regulators in the haemolymph and in epidermal compartments. The copper profile presented an indicator role for variations of osmolality over the molt cycle.     

The role of eicosanoids on Rhodnius heme-binding protein (RHBP) endocytosis by Rhodnius prolixus ovaries

The participation of eicosanoids and second messengers on the regulation of RHBP endocytosis by the ovaries was investigated, using [(125)I]RHBP in experiments in vivo and in vitro. Addition of PGE(2) (one of the products of the cyclooxygenase pathway) decreased in vitro the uptake of RHBP by 35%. The rate of RHBP endocytosis increased in the presence of indomethacin, a potent cyclooxigenase inhibitor, up to 50% in vitro and up to 55% in vivo, thus giving support to the role of cyclooxygenase derivatives on endocytosis regulation. The amount of PGE(2) secreted to the culture medium by the cells of Rhodnius prolixus ovaries was 1.1 ng/ovary following RHBP uptake assay. The amount of PGE(2) decreases approximately 25% in the presence of 5 microM indomethacin. Using a scanning electron microscope we have observed that neither the surface area nor the patencies of follicle cells were affected by treatment with indomethacin, thus suggesting that, its effect is elicited in the oocyte. Finally, we have identified two ovarian peptides that were dephosphorylated after the indomethacin treatment (18 and 25 kDa). Taken together these data show that local mediators such as eicosanoids act upon the oocytes controlling RHBP endocytosis, perhaps using the protein phosphorylation signal transduction pathway.     

Pseudomonas aeruginosa toxin ExoU induces a PAF-dependent impairment of alveolar fibrin turnover secondary to enhanced activation of coagulation and increased expression of plasminogen activator inhibitor-1 in the course of mice pneumosepsis

Background

ExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A₂ activity, was shown to induce vascular hyperpermeability and thrombus formation in a murine model of pneumosepsis. In this study, we investigated the toxin ability to induce alterations in pulmonary fibrinolysis and the contribution of the platelet activating factor (PAF) in the ExoU-induced overexpression of plasminogen activator inhibitor-1 (PAI-1).

Methods

Mice were intratracheally instilled with the ExoU producing PA103 P. aeruginosa or its mutant with deletion of the exoU gene. After 24 h, animal bronchoalveolar lavage fluids (BALF) were analyzed and lung sections were submitted to fibrin and PAI-1 immunohistochemical localization. Supernatants from A549 airway epithelial cells and THP-1 macrophage cultures infected with both bacterial strains were also analyzed at 24 h post-infection.

Results

In PA103-infected mice, but not in control animals or in mice infected with the bacterial mutant, extensive fibrin deposition was detected in lung parenchyma and microvasculature whereas mice BALF exhibited elevated tissue factor-dependent procoagulant activity and PAI-1 concentration. ExoU-triggered PAI-1 overexpression was confirmed by immunohistochemistry. In in vitro assays, PA103-infected A549 cells exhibited overexpression of PAI-1 mRNA. Increased concentration of PAI-1 protein was detected in both A549 and THP-1 culture supernatants. Mice treatment with a PAF antagonist prior to PA103 infection reduced significantly PAI-1 concentrations in mice BALF. Similarly, A549 cell treatment with an antibody against PAF receptor significantly reduced PAI-1 mRNA expression and PAI-1 concentrations in cell supernatants, respectively.

Conclusion

ExoU was shown to induce disturbed fibrin turnover, secondary to enhanced procoagulant and antifibrinolytic activity during P. aeruginosa pneumosepsis, by a PAF-dependent mechanism. Besides its possible pathophysiological relevance, in vitro detection of exoU gene in bacterial clinical isolates warrants investigation as a predictor of outcome of patients with P. aeruginosa pneumonia/sepsis and as a marker to guide treatment strategies.     

Quantitative trait loci affecting lactose and total solids on chromosome 6 in Brazilian Gir dairy cattle

Fourteen Brazilian Gir sire families with 657 daughters were analyzed for quantitative trait loci (QTL) on chromosome 6 affecting lactose and total solids. Cows and sires were genotyped with 27 microsatellites with a mean spacing between markers of 4.9 cM. We used a 1% chromosome-wide threshold for QTL qualification. A QTL for lactose yield was found close to marker MNB66 in three families. A QTL for total solid yield was identified close to marker BMS2508 in three families. A QTL for lactose percentage, close to marker DIK1182, was identified in two families. A QTL for total solid percentage, close to marker MNB208, was identified in four families. These QTLs could be used for selection of animals in dairy production systems.     

Esterase polymorphisms for analysis of genetic diversity and structure of soybean (Glycine max) cultivars

We used native polyacrylamide gel electrophoresis to identify polymorphism levels in α- and β-esterase loci from leaf tissues of Brazilian soybean cultivars for the analysis of population genetic diversity and structure, and to investigate relationships between conventional and genetically modified cultivars. The cultivars included lines developed by a soybean-grower cooperative (CD), by EMBRAPA (BR), and "Roundup Ready" (RR) cultivars. Esterase isozymes recorded with α-naphthyl acetate and β-naphthyl acetate were produced from 14 loci. Two to three allelic variants were detected in leaves from 420 plants of 21 CD, BR, and RR cultivars at Est-1, Est-2, Est-3, Est-5, and Est-14 loci. The estimated proportion of polymorphic loci in CD cultivars was 21.4%, and in BR and RR cultivars it was 28.6%. High and low H(O) and H(E) values were observed within CD and BR cultivars and a very high cultivar differentiation level was evident in the plants of the 21 CD, BR, and RR cultivars (F(ST) = 0.3865). A low level of differentiation (F(ST) = 0.0289) was detected between conventional and RR cultivars. Plants from cultivar BR37 had the highest level of genetic differentiation compared to the other cultivars. The genetic basis of BR cultivars (0.5538-0.9748) was found to be broader than the genetic basis of CD cultivars (0.7058 for CD205 and CD209 and 0.9995 for CD205 and CD208). Higher genetic identity was detected between plants of CD and CDRR cultivars (I = 0.9816). Understanding the genetic structure of these populations can help provide specific culture strategies for each cultivar, depending on its level of heterozygosity.     

Acidocalcisomes as calcium- and polyphosphate-storage compartments during embryogenesis of the insect Rhodnius prolixus Stahl

Background

The yolk of insect eggs is a cellular domain specialized in the storage of reserve components for embryo development. The reserve macromolecules are stored in different organelles and their interactions with the embryo cells are mostly unknown. Acidocalcisomes are lysosome-related organelles characterized by their acidic nature, high electron density and large content of polyphosphate bound to several cations. In this work, we report the presence of acidocalcisome-like organelles in eggs of the insect vector Rhodnius prolixus.

Methodology/Principal findings

Characterization of the elemental composition of electron-dense vesicles by electron probe X-ray microanalysis revealed a composition similar to that previously described for acidocalcisomes. Following subcellular fractionation experiments, fractions enriched in acidocalcisomes were obtained and characterized. Immunofluorescence showed that polyphosphate polymers and the vacuolar proton translocating pyrophosphatase (V-H⁺-PPase, considered as a marker for acidocalcisomes) are found in the same vesicles and that these organelles are mainly localized in the egg cortex. Polyphosphate quantification showed that acidocalcisomes contain a significant amount of polyphosphate detected at day-0 eggs. Elemental analyses of the egg fractions showed that 24.5±0.65% of the egg calcium are also stored in such organelles. During embryogenesis, incubation of acidocalcisomes with acridine orange showed that these organelles are acidified at day-3 (coinciding with the period of yolk mobilization) and polyphosphate quantification showed that the levels of polyphosphate tend to decrease during early embryogenesis, being approximately 30% lower at day-3 compared to day-0 eggs.

Conclusions

We found that acidocalcisomes are present in the eggs and are the main storage compartments of polyphosphate and calcium in the egg yolk. As such components have been shown to be involved in a series of dynamic events that may control embryo growth, results reveal the potential involvement of a novel organelle in the storage and mobilization of inorganic elements to the embryo cells.