Development of collagenous scaffolds for wound healing: characterization and in vivo analysis.

The development of wound dressings from biomaterials has been the subject of research due to their unique structural and functional characteristics. Proteins from animal origin, such as collagen and chitosan, act as promising materials for applications in injuries and chronic wounds, functioning as a repairing agent. This study aims to evaluate in vitro effects of scaffolds with different formulations containing bioactive compounds such as collagen, chitosan, N-acetylcysteine (NAC) and ε-poly-lysine (ε-PL). We manufactured a scaffold made of a collagen hydrogel bioconjugated with chitosan by crosslinking and addition of NAC and ε-PL. Cell viability was verified by resazurin and live/dead assays and the ultrastructure of biomaterials was evaluated by SEM. Antimicrobial sensitivity was assessed by antibiogram. The healing potential of the biomaterial was evaluated in vivo, in a model of healing of excisional wounds in mice. On the 7th day after the injury, the wounds and surrounding skin were processed for evaluation of biochemical and histological parameters associated with the inflammatory process. The results showed great cell viability and increase in porosity after crosslinking while antimicrobial action was observed in scaffolds containing NAC and ε-PL. Chitosan scaffolds bioconjugated with NAC/ε-PL showed improvement in tissue healing, with reduced lesion size and reduced inflammation. It is concluded that scaffolds crosslinked with chitosan-NAC-ε-PL have the desirable characteristics for tissue repair at low cost and could be considered promising biomaterials in the practice of regenerative medicine.

Mutagenicity, hepatotoxicity, and neurotoxicity of glyphosate and fipronil commercial formulations in Amazon turtles neonates (Podocnemis expansa).

Pesticides are considered one of the main causes of the population decline of reptiles worldwide, with freshwater turtles being particularly susceptible to aquatic contamination. In this context, we investigated the potential mutagenic, hepatotoxic, and neurotoxic effects in neonates of Podocnemis expansa exposed to substrate contaminated with different concentrations of glyphosate and/or fipronil during embryonic development. Eggs collected from the natural environment were artificially incubated in sand moistened with pure water, water added with glyphosate Atar 48® at concentrations of 65 and 6500 µg/L (groups G1 and G2, respectively), water added with fipronil Regent® 800WG at 4 and 400 µg/L (groups F1 and F2, respectively) and, water added with the combination of 65 µg/L glyphosate and 4 µg/L fipronil or with 6500 µg/L glyphosate and 400 µg/L fipronil (groups GF1 and GF2, respectively). For mutagenicity analysis, we evaluated the frequency of micronuclei (MN) and other erythrocyte nuclear abnormalities (ENAs), while for evaluation of hepatotoxicity and neurotoxicity, livers and encephalon were analyzed for histopathological alterations. Exposure to pesticides, alone or in combination, increased the frequency of erythrocyte nuclear abnormalities, particularly blebbed nuclei, moved nuclei, and notched nuclei. Individuals exposed to fipronil exhibited congestion and inflammatory infiltrate in their liver tissue, while, in the encephalon, congestion, and necrosis were present. Our study confirms that the incubation of eggs in substrate polluted with glyphosate and fipronil causes histopathological damage and mutagenic alteration in P. expansa, highlighting the importance of using different biomarkers to evaluate the ecotoxicological effects of these pesticides, especially in oviparous animals.

Zinc oxide nanoparticles functionalized with curcumin supplementation during in vitro embryo culture impaired in a concentration-dependent-manner blastocyst production in cattle.

This work investigated the effect of zinc oxide nanoparticles functionalized with curcumin (ZnO(np) + CUR) supplementation during the in vitro embryo culture (IVC) on the bovine in vitro embryo production, and the cellular antioxidant response. The cumulus-oocyte complexes (COCs) were matured, fertilized and then the presumptive zygotes were cultured in the medium in the absence (0 µM-control) or presence of different concentrations of ZnO(np) + CUR (3, 6 or 12 µM). After IVC, the embryos were destined either to assay intracellular ROS levels and mitochondrial membrane potential. The results demonstrated that only the addition of 12 µM ZnO(np) + CUR during IVC decreased intracellular ROS production and the rate of blastocyst production when compared to the control (p < .05). In conclusion, ZnO(np) + CUR addition during the IVC impaired in concentration-dependent-manner bovine in vitro embryo production.

Sperm head morphometry and chromatin alterations in bulls with different conception rates.

The objective of this investigation was to analyze timed-AI conception rates (CRs) of different sires in light of their conventional semen quality parameters, sperm head morphometry, and chromatin alterations. Semen was collected in the field from six Angus bulls and used for the timed-AI of 890 suckled multiparous Nellore cows at a single farm. Semen batches were evaluated on the following in vitro parameters: sperm motility, concentration, and morphology, sperm head morphometry, and chromatin alteration types. The overall CR was 49% and Bulls 1 (43%) and 2 (40%) presented reduced (P < 0.05) pregnancies per AI compared to Bull 6 (61%), even though no differences were observed between their conventional semen quality parameters. Bull 1, however, presented higher (P = 0.0001) shape factor, smaller (P = 0.0025) antero-posterior symmetry, and elevated (P = 0.0141) Fourier 1 parameter, whereas Bull 2 exhibited a higher (P = 0.0023) percentage of chromatin alteration along the central axis of the sperm head. In conclusion, bulls with varying CRs may present sperm head morphometric differences and/or chromatin alterations while not presenting differences in conventional in vitro semen quality parameters. Although further studies are needed to elucidate the concrete implications of chromatin alterations on field fertility, sperm morphometric differences and chromatin alterations may be at least partially causative of the lower pregnancies per timed-AI of certain sires.

L-arginine supplementation minimizes aging-induced changes in the sperm chromatin of roosters.

Arginine is the main amino acid that constitutes the sperm protamine of roosters, named galline, which complexes with sperm DNA, allowing high compaction of its chromatin. Arginine supplementation has positive effects on semen quality in aged roosters, but this supplementation is not known to limit the progressive worsening of sperm chromatin compaction. This work aimed to verify whether L-arginine supplementation in the feed improve or maintain sperm chromatin quality since aging in roosters is usually accompanied by worsening chromatin quality. Four groups of 52-wk-old Ross AP95 lineage roosters were used, of which 6 semen samples per group were evaluated, totaling 24 samples. Another 24 samples, 6 per group, were evaluated after 6 wk of supplementation when one group was not supplemented (control) and the other 3 were supplemented with 1.15 kg (treatment 1), 2.17 kg (treatment 2), and 3.18 kg (treatment 3) of L-arginine/ton of feed. Computer image analysis of semen smears stained with toluidine blue pH 4.0 was used for sperm chromatin evaluation. Sperm chromatin was evaluated for compaction heterogeneity and compaction intensity by percentage decompaction relative to standard heads and by integrated optical density (IOD), which was used for the first time to identify sperm chromatin changes. Sperm head morphology was also evaluated by means of area and length. The IOD proved to be more efficient in identifying changes in rooster sperm chromatin compaction than the percentual decompaction. In general, chromatin compaction was positively influenced by the supplementation with L-arginine, being better in the supplementation with the highest levels tested. This was corroborated by the smaller average of the variables referring to the size of the spermatozoa heads of the animals that received feed with a higher content of L-arginine, since better compacted heads naturally tend to be smaller. Finally, arginine supplementation was able to limit or even improve sperm chromatin decompaction during the experimental period.

Roles of viable but non-culturable state in the survival of Campylobacter jejuni.

Despite being considered fragile and fastidious, Campylobacter jejuni is the most prevalent cause of foodborne bacterial gastroenteritis, and chicken meat is considered the main vehicle of transmission to humans. This agent can survive adverse conditions in the form of biofilms, but extreme stress (nutritional, oxidative and thermal) promotes the acquisition of a state called viable but not culturable (VBNC). The emergence of this pathogen worldwide and the recent international requirements in its control instigated us to qualitatively and quantitatively estimate the time required for the acquisition of the VBNC form in 27 strains of C. jejuni, characterize morphological aspects, determine its adaptive and invasive potential and perform comparative metabolomic evaluation. Extreme stress promoted the complete acquisition of the VBNC form in a mean time of 26 days. Starting from an average initial count of 7.8 log CFU/mL, the first four days determined the greatest average reduction of the culturable form of 3.2 log CFU/mL. The scanning and transmission image analyses showed a transition from the typical viable form (VT) to the VBNC form, with initial acquisition of the straight rod shape, followed by loss of the flagella and subdivision into two to 11 imperfect cocci arranged in a chain and rich in cellular content, until their individual release. RT-PCR identified the presence of ciaB and p19 transcripts in the 27 cultivable C. jejuni strains, a character maintained in the VBNC form only for p19 and in 59.3% (16/27) of the VBNC strains for the ciaB gene. The average inoculation of 1.8 log CFU/mL of C. jejuni VBNC into primary chicken embryo hepatocyte cells promoted the occurrence of apoptosis processes significantly after 24 hours of contact by one of the strains tested. In C. jejuni VBNC, we detected higher expression of metabolites linked to protective and adaptation mechanisms and of volatile organic precursor compounds indicative of metabolism interruption. The oscillations in the time of acquisition of the VBNC form together with the presence of transcripts for ciaB and p19, the identification of cell lysis and metabolites that ensure the maintenance of the pathogen alert to the fact that C. jejuni VBNC remains virulent and adapted to stress, which makes evident the potential danger of this latent form, which is not detectable by official methodologies.

Physical Training Improves Cardiac Structure and Function of Rats After Doxorubicin-Induced Cardiomyopathy

Abstract Background: The use of doxorubicin in chemotherapy has been associated with cardiotoxicity and heart failure. Physical exercise produces favorable morphofunctional adaptations in the cardiovascular system and may reverse cardiac dysfunction in patients undergoing chemotherapy. Objective: To assess the effects of physical training on myocardial structure, cardiac function, and exercise tolerance in Wistar rats initiated after the onset of cardiotoxicity-induced cardiotoxicity. Methods: This study investigated 30 adult male Wistar rats randomly divided into four groups: control (C), exercise (EX), doxorubicin (DX), and doxorubicin and exercise (DXEX). The DX and DXEX groups received six doses of doxorubincin from 1.25 mg/kg body weight up to a cumulative dose of 7.5 mg/kg. Injections were administered intraperitoneally three times a week for two weeks; after this stage, the EX and DXEX groups started physical training (swimming) sessions three times a week with a load of 5% of their body weight. Echocardiography and exercise tolerance tests were performed. Generalized linear models were used in statistical analysis, and a p<0.05 was set as statistically significant. Results: Left ventricular shortening fraction and ejection fraction were reduced in the DX group compared to C, EX, and DXEX. The DXEX group showed greater tolerance to effort when compared to the DX and C groups. Conclusion: Physical training, initiated after the onset of doxorubicin-induced cardiotoxicity, improved cardiac function and exercise tolerance in rats.

Physiological Changes in Chicken Embryos Inoculated with Drugs and Viruses Highlight the Need for More Standardization of this Animal Model.

Several studies have been developed using the Gallus gallus embryo as an experimental model to study the toxicity of drugs and infections. Studies that seek to standardize the evaluated parameters are needed to better understand and identify the viability of CEs as an experimental model. Therefore, we sought to verify whether macroscopic, histopathological, blood count, metabolites and/or enzymes changes and oxidative stress in CE of different ages are specific to the model. To achieve this goal, in ovo assays were performed by injecting a virus (Gammacoronavirus) and two drugs (filgrastim and dexamethasone) that cause known changes in adult animals. Although congestion and inflammatory infiltrate were visible in the case of viral infections, the white blood cell count and inflammation biomarkers did not change. Filgrastim (FG) testing did not increase granulocytes as we expected. On the other hand, CE weight and red blood cell count were lower with dexamethasone (DX), whereas white blood cell count and biomarkers varied depended on the stage of CE development. Our work reinforces the importance of standardization and correct use of the model so that the results of infection, toxicity and pharmacokinetics are reproducible.

First Report of Genetic Variability of Erysipelothrix sp. Strain 2 in Turkeys Associated to Vero Cells Morphometric Alteration.

Erysipelas is a disease caused by the Erysipelothrix genus, whose main species is the E. rhusiopathiae, the causative agent of animal erysipelas and human erysipeloid. We isolated Erysipelothrix sp. strain 2 (ES2) from turkey's organs during an outbreak in Brazilian commercial and breeder flocks with sepsis and high mortality levels. We studied 18 flocks, accounting for 182 samples, being eight flocks (84 samples) as ES2 positive with individuals demonstrating clinical symptoms and high mortality. We obtained the genetic variability of 19 samples with PFGE and found two clones, both from the same flock but different samples, and two clusters. Interestingly, we found 15 strains with high genetic variability among and within flocks. We have found a positive association between the proximity of ES2 positive turkey flocks and commercial swine sites through epidemiological analysis. We infected Vero cells with two different isolates and three distinct concentrations of ES2. After performing the morphometry, we recorded enlargement of the nucleus and nucleolus. Moreover, we performed fluorescence assays that resulted in apoptotic and necrotic cells. We demonstrated that ES2 could multiply in the extracellular medium and invade and survive inside Vero cells. For the first time, our finds show that ES2 may have similar behavior as E. rhusiopathiae as a facultative intracellular microorganism, which may represent a hazard for humans.

Classification of the sperm chromatin compaction alterations in bulls (Bos taurus) and its correlation with the efficiency of the in vitro production of embryos

This paper proposes to classify the sperm chromatin compaction alterations in bulls, according to the affected area location and its objective is evaluating the correlation of the intensity, the heterogeneity and these kinds of chromatin decompaction with the rates of cleavage and the formation of blastocysts of in vitro production of embryos (IVPE). It was used several subfertile animals sperm samples, which were evaluated using the toluidine blue staining and computer image analysis, making possible the categorization of the chromatin decompaction according to their location. The percentages of chromatin decompaction and heterogeneity were also evaluated. IVPEs were done and the rates of cleavage and of blastocysts were correlated with the chromatin characteristics. It made possible the classification of the chromatin decompaction according to the head affected part in at least four types: base decompaction, basal half decompaction, central axis decompaction, total decompaction. Based on the correlation, it can be implicated that each type of classification has different influences on the bull fertility. It made possible understanding that sperms amount with 5% or more of chromatin decompaction intensity interferes in the bull fertility and this condition can be featured as an uncompensable defect, while the heterogeneity of chromatin is not an important factor in the IVPE results.

Sperm head morphometry and chromatin condensation are in constant change at seminiferous tubules, epididymis, and ductus deferens in bulls.

The aim of this study was to evaluate the sperm head morphometry and chromatin condensation at different regions of the reproductive tract in bulls. Sperm smears of seminiferous tubules (ST), epididymis head (EH), body (EB), and tail (ET), and ductus deferens (DD) were stained with toluidine blue. Afterwards, the sperm head morphometry and chromatin alteration types were evaluated by a computational image analysis. Overall, spermatozoa of ST had lower (P < 0.05) area (A), perimeter (P), width (W), length (L), ellipticity (E), and Fourier harmonics (F0, F1, and F2). The chromatin decondensation (CD) and heterogeneity (CH) were higher (P < 0.05) in the ST region and decreased (P < 0.0001) during the migration along the reproductive tract (ST - DD direction). Considering the factors extracted (Factors 1 and 2) by the principal component analysis, the parameters A, P, W, L, and F0 were responsible for ∼36% of the Factor 1, while the E, F0, F1, and anterior-posterior symmetry (APS) contributed ∼27% to Factor 2. Both, CD and CH were associated with Factor 1 in the EH and ET regions and Factor 2 in the ST. Also, a well-defined difference between sperm heads collected from the ST and DD regions was observed by canonical analysis. The distribution of each chromatin alteration type was recorded. The proportion of normal sperm was lower (P < 0.05) in ST compared to other regions. Moreover, the chromatin influenced the morphometry and sperm heads with whole chromatin alteration type showed a smaller (P < 0.05) A, P, W, L, and E. In summary, the epididymal maturation is important for chromatin compaction and final morphometry of the sperm head. Also, the identification and quantification of the sperm chromatin condensation in different regions of reproductive tract can be used as potential biomarkers to predict the fertility in bulls.

Gene expression of FOXP3, indoleamine 2,3-dioxygenase (IDO), IL10 and CSF1 in the uterus of cows that received an intrauterine infusion of maternal and paternal antigens / Expressão gênica de FOXP3, indoleamina 2,3-dioxigenase (IDO), IL10 e CSF1 em útero de vacas que receberam infusão intrauterina de antígenos paternos e maternos

Sensitization with conceptus antigens has been shown to be useful for improving reproductive performance facilitating maternal acceptance of an allogeneic embryo through the induction of cytokines and immunoregulatory cells in the uterine microenvironment. As FOXP3, IDO, IL10 and CSF1 in the uterus are important on the recognition and development of embryos during early pregnancy, this study aimed to determine whether simultaneous or isolated administration of paternal (semen) and maternal (PBMCs) antigens in the uterus of cow, on the day of estrus, influence the gene expression of these cytokines. Forty crossbred cows were divided into four treatments: T0: Control; T1: Semen; T2: PBMCs (peripheral blood mononuclear cells) from another cow and T3: PBMCs+Semen. Antigens were administered into the uterine body on the estrus day (D0). Uterine biopsies designed for molecular analysis of gene expression were collected in vivo seven (D7) and fourteen (D14) days after immunostimulation. Transcripts from FOXP3, IDO, IL-10 and CSF-1 were detected in all RNA samples extracted from uterine biopsies. The semiquantitative analysis showed that none of the treatments caused significant increase in the expression of these genes. Furthermore, on D14 all treatments led to a decline in the number of CSF-1 transcripts; moreover, treatment with PBMCs+Semen also led to a drop in the abundance of IL-10 transcripts. Such results suggest that isolated or simultaneous administration of both antigens would not increase maternal tolerance to embryo alloantigens, nor would it create favorable conditions to its growth and pre-implantation development, at least regarding the effects mediated by these genes on D7 and D14 of the estrous cycle.

A sensibilização intrauterina com antígenos do concepto tem-se mostrado útil para melhorar o desempenho reprodutivo, facilitando a aceitação materna de um embrião alogênico por meio da indução de citocinas e células imunorreguladoras no microambiente uterino. Como FOXP3, IDO, IL10 e CSF1 no útero são importantes no reconhecimento e desenvolvimento de embriões durante a gestação inicial, este estudo teve como objetivo determinar se a administração simultânea ou isolada de antígenos paterno (sêmen) e materno (PBMCs) no útero de vacas, no dia do estro, influenciam a expressão gênica dessas citocinas. Quarenta vacas mestiças foram divididas em quatro tratamentos: T0: Controle; T1: Sêmen; T2: PBMCs (células mononucleares do sangue periférico) e T3: PBMCs + Sêmen. Os antígenos foram administrados no corpo do útero no dia do estro (D0). Biópsias uterinas projetadas para análise molecular da expressão gênica foram coletadas in vivo sete (D7) e catorze (D14) dias após imunoestimulação. Transcritos de FOXP3, IDO, IL-10 e CSF-1 foram detetados em todas as amostras de RNA extraídas de biópsias uterinas. A análise semiquantitativa mostrou que nenhum dos tratamentos causou um aumento significativo na expressão desses genes. Além disso, no D14, todos os tratamentos levaram a um declínio na quantidade de transcritos do CSF-1; Além disso, o tratamento com PBMCs + sêmen também levou a uma queda na abundância de transcritos de IL-10. Estes resultados sugerem que a administração isolada ou simultânea de ambos os antígenos não deve aumentar a tolerância materna aos aloantígenos do embrião, nem deve criar condições favoráveis ao seu crescimento e desenvolvimento pré-implantação, pelo menos em relação aos efeitos mediados por esses genes nos dia sete e catorze do ciclo estral.

Can Bos indicus cattle breeds be discriminated by differences in the changes of their sweat gland traits across summer and winter seasons?

This study addresses the hypothesis that Bos indicus cattle breeds can be discriminated by the changes that occur in their sweat gland traits between summer and winter seasons in tropical conditions. Samples of the skin were taken from six Bos indicus cattle breeds (eight subjects per breed), including Nellore, Cangaian, Gyr, Guzerat, Punganur, and Sindhi in winter and summer. The sweat gland epithelium (µm), glandular portion length (µm), sweat gland duct length (µm), gland depth (µm), and sweat gland density (cm2) were determined. Principal component analyses were performed to address the overall structure of breed's group, together with confirmatory analyses by the least squares procedures. Exploratory analysis showed that cattle breeds presented patterns of dissimilarity in the changes in their skin and sweat glands traits between winter and summer seasons. Breeds were separated into three groups under the two principal components, which represented 77.26% of the total variance. The first group was composed of Sindh and Guzerat cattle, which did not present modifications in the parameters assessed between seasons. The most visible alterations were observed in Gyr cattle (third group). In fact, confirmatory analyses showed that glandular portion length, sweat gland duct length, gland depth, and sweat gland density of the Gyr cattle increased (P < 0.05) during the summer season. In conclusion, the results of this investigation demonstrated that morphological traits of the skin and sweat glands associated with seasonal changes in tropical conditions were able to discriminate among Bos indicus cattle breeds.

Nanocomposite of Ag-Doped ZnO and AgO Nanocrystals as a Preventive Measure to Control Biofilm Formation in Eggshell and Salmonella spp. Entry Into Eggs.

Salmonella spp. is an important foodborne agent of salmonellosis, whose sources in humans often include products of avian origin. The control of this bacterium is difficult especially when Salmonella spp. is organized into biofilms. We hypothesized that the novel nanocomposites of ZnO nanocrystals doped with silver (Ag) and silver oxide (AgO) nanocrystals (ZnO:Ag-AgO) synthesized by the coprecipitation method could control or prevent the formation of Salmonella Enteritidis (SE) and Salmonella Heidelberg (SH) biofilm and its entry into turkey eggs. The diffraction characteristics of ZnO and AgO showed sizes of 28 and 30 nm, respectively. The Zn to Ag substitution into the ZnO crystalline structure was evidenced by the ionic radius of Ag+2 (1.26 Å), which is greater than Zn+2 (0.74 Å). For the SE analyses post-biofilm formation, the ZnO:Ag-AgO was not able to eliminate the biofilm, but the bacterial load was lower than that of the control group. Additionally, SE was able to infiltrate into the eggs and was found in both albumen and yolk. For the SH analyses applied onto the eggshells before biofilm formation, the ZnO:Ag-AgO treatment prevented biofilm formation, and although the bacterium infiltration into the eggs was observed in all treated groups, it was significantly smaller in ZnO:Ag-AgO pre-treated eggs, and SH could not reach the yolk. There was no difference in pore size between groups; therefore, the inhibition of biofilm formation and the prevention of bacterium entry into the egg were attributable to the use of ZnO:Ag-AgO, which was not influenced by the egg structure. Although the amount of Ag and Zn in the shell of the ZnO:Ag-AgO group was greater in relation to the control, this difference was not detected in the other egg components. In the search for new measures that are effective, safe and viable for controlling microorganisms in poultry farming, the application of a nanocomposite of Ag-doped ZnO and AgO nanocrystals appears as an alternative of great potential to prevent Salmonella sp biofilms in eggshells and other surfaces.

Three-dimensional levitation culture improves in-vitro growth of secondary follicles in bovine model.

RESEARCH QUESTION: Does a three-dimensional culture system based on magnetic levitation with nanoparticles assembly maintain the follicular structure and viability with adequate growth rates leading to oocyte maturation after long-term culture? DESIGN: Randomized-controlled trial of treatments in a bovine model. Secondary follicles (n = 213) isolated from bovine ovaries were cultured in a two-dimensional system (two-dimensional control) or three-dimensional levitation system with different concentrations (three-dimensional 50 µl/ml, 100 µl/ml and 200 µl/ml) of magnetic nanoparticles. Follicular growth (diameter, daily growth and growth patterns), morphology (normal, degenerated and extruded follicles), antrum formation, oocyte viability and chromatin configuration were assessed. RESULTS: Secondary follicles of three-dimensional 200-µl/ml treatment showed higher viability, antrum formation and lower degeneration rates than two-dimensional control. Also, follicles cultured in the three-dimensional 200-µl/ml treatment presented a most homogenous daily growth rate as shown by the lowest variance and standard deviation. Compared with the two-dimensional control, the proportion of non-growing and slow-growing follicles were 3.8-fold lower and 1.6-fold higher, respectively, in the three-dimensional 200-µl/ml treatment. After in-vitro maturation, the three-dimensional 200-µl/ml had a greater proportion of viable oocytes (1.7-fold) and meiotic resumption rates (2.4-fold) than the two-dimensional control treatment. CONCLUSION: The three-dimensional levitation culture system improves the viability of in-vitro development of bovine secondary follicles, antrum formation and lower extrusion and degeneration rates and adequate growth rate leading to relevant oocyte viability and meiotic resumption after in-vitro maturation. This approach does not require a specific medium, and has the potential as an alternative method to in-vitro follicle culture in several species, including humans.

Micro-RNAs differentially expressed in comparative analysis of sperm samples with high and low efficiency in the in vitro production of bovine (Bos Taurus) embryos / Micro-rnas diferencialmente expressos em análise comparativa de amostras de espermatozoides com alta e baixa efi ciência na produção in vitro de embriões bovinos (bos taurus )

Infertility or subfertility in bovine males may be related to spermatic microRNAs (miRNAs), whose function seems to be associated with the regulation of gene expression, degradation orstorage of messenger RNAs (mRNAs) for later translation into early embryonic development. Thus, the purpose of this study was to identify differentially expressed miRNAs in semen samples from bulls (Bos taurus) with low and high efficiency in the in vitro embryo production (IVEP) and to evaluate if they can be used as markers of semen efficiency for IVEPs. In order to identify miRNA markers of semen efficiency in thein vitro embryo production, eight semen samples from each animal, one bull with high and two bulls with low efficiency in IVEPs were used to perform the RNAseq technique for miRNAs. Initially the samples were washed with PBS to remove the extender semen and subsequently were submitted to RNA extraction protocols performed according to procedures described by mirVana™ miRNA Isolation Kit. Then, the amplification of the miRNAs was carried out, not to mention the preparation of the library (Ion Total RNA-Seq Kit v2), the PCR emulsion reaction, enrichment, as well as the injection of the sample on the chip by the Ion Chef equipment. The sequencing was done on Ion Proton equipment. The comparison between the samples was established using two methodologies for searching for targets to increase the robustness of the analytical procedure: the miRanda program using as cutoff minimum free energy of the hybridization -20 kcal/Mol, 100% of identity between nucleotides 2 and 8 of the miRNA, and the RNAhybrid program, using as cutoff minimum free energy of hybridization -20 kcal/mol. In sum, 1306 miRNAs were identified in the samples. The bta-miR-380-5p, bta-miR-155, bta-miR-30c and bta-miR-34a genes were identified by the Bioinformatics as being strongly differentially expressed between the groups, indicating that these genes may present themselves as possible efficiency markers. However, it has become clear that there is no single miRNA that marks different types and causes of fertility problems.

A infertilidade ou subfertilidade em machos bovinos pode estar relacionada a microRNAs espermáticos (miRNAs), cuja função parece estar associada à regulação da expressão gênica, degradação ou armazenamento de RNAs mensageiros (mRNAs), para posterior tradução no desenvolvimento embrionário inicial. Assim, o objetivo deste estudo foi identificar miRNAs diferencialmente expressos em amostras de sêmen de touros (Bos taurus) com baixa e alta eficiência na produção in vitro de embriões (PIVE) e avaliar se eles podem ser utilizados como marcadores de eficiência do sêmen em PIVEs. Para identificar miRNA marcadores da eficiência de sêmen em PIVE, oito amostras de sêmen de cada animal, sendo um touro com alto e dois touros com baixa eficiência, foram utilizados para realizar a técnica de RNAseq para miRNAs. Inicialmente as amostras foram lavadas com PBS para remover o diluente do sêmen e, posteriormente, foram submetidas a protocolos de extração de RNA realizados de acordo com os procedimentos descritos pelo Kit de isolamento de miRNA mirVana ™. Em seguida, foi realizada a amplificação dos miRNAs, a preparação da biblioteca (Ion RNA-Seq Kit v2), a reação de emulsão de PCR, enriquecimento e a injeção das amostras no chip apropriado utilizando o equipamento Ion. Chef. O sequenciamento foi realizado no equipamento Ion Proton. A comparação entre as amostras foi estabelecida utilizando duas metodologias de busca de alvos para aumentar a robustez do procedimento analítico: o programa miRanda utilizando como valor de corte a energia mínima livre de hibridização -20 kcal / Mol e 100% de identidade entre os nucleotídeos 2 e 8 do miRNA, e o programa RNAhybrid, utilizando como valor de corte a energia mínima livre de hibridização -20 kcal / mol. Em suma, 1306 miRNAs foram identificados nas amostras. Os genes bta-miR-380-5p, bta-miR-155, bta-miR-30c e bta-miR-34a foram identificados pela bioinformática como sendo fortemente diferencialmente expressos entre os grupos, indicando que esses genes podem se apresentar como possíveis marcadores de eficiência. No entanto, ficou claro que não existe um único miRNA que marque diferentes tipos e causas de problemas de fertilidade.

Ecotoxicological effects of the insecticide fipronil in Brazilian native stingless bees Melipona scutellaris (Apidae: Meliponini).

Melipona scutellaris Latreille, 1811 (Hymenoptera, Apidae) is a pollinator of various native and cultivated plants. Because of the expansion of agriculture and the need to ensure pest control, the use of insecticides such as fipronil (FP) has increased. This study aimed to evaluate the effects of sublethal doses of FP insecticide on M. scutellaris at different time intervals (6, 12, and 24 h) after exposure, via individually analyzed behavioral biomarkers (locomotor activity, behavioral change) as well as the effect of FP on different brain structures of bees (mushroom bodies, antennal cells, and optic cells), using sub-individual cell biomarkers (heterochromatin dispersion, total nuclear and heterochromatic volume). Forager bees were collected when they were returning to the nest and were exposed to three different concentrations of FP (0.40, 0.040, and 0.0040 ng a.i/bee) by topical application. The results revealed a reduction in the mean velocity, lethargy, motor difficulty, paralysis, and hyperexcitation in all groups of bees treated with FP. A modification of the heterochromatic dispersion pattern and changes in the total volume of the nucleus and heterochromatin were also observed in the mushroom bodies (6, 12, and 24 h of exposure) and antennal lobes (6 and 12 h) of bees exposed to 0.0040 ng a.i/bee (LD50/100). FP is toxic to M. scutellaris and impairs the essential functions required for the foraging activity.

Positive effect of resveratrol against preantral follicles degeneration after ovarian tissue vitrification.

This study aimed to evaluate whether the addition of resveratrol to vitrification/thawing medium improves the cryotolerance of preantral follicles enclosed in bovine ovarian fragments. Ovarian fragments were obtained from bovine fetuses and distributed to the following groups: fresh ovarian fragments (control), vitrified (VIT), and vitrified with resveratrol (VIT + RESV). Overall, the mean percentage of normal follicles was greater (P < 0.05) in the VIT + RESV compared to the VIT group. Moreover, the probability of finding normal follicles was 2.5 greater (P < 0.05) in the VIT + RESV group. In class comparison, the primordial and transitional follicles have ∼3.0 times (P < 0.05) greater odds of being normal after vitrification compared to the secondary follicles. Additionally, a negative association (P < 0.05) was observed between the proportion of viable follicles and the stage of follicular development. ROS levels were similar (P > 0.05) between the VIT and VIT + RESV groups, and both were lower (P < 0.05) than the control group. The tissue viability in the VIT + RESV group was similar (P > 0.05) to the control group. In summary, the resveratrol provided greater ovarian tissue viability and has a positive effect against degeneration of preantral follicles enclosed in ovarian fragments.

Sperm chromatin alterations in fertile and subfertile bulls.

Alterations in sperm chromatin have been related with subfertility in several mammals. In this study, chromatin alteration types (Base, Basal half, Central axis, Dispersed, and Whole) were assessed by toluidine blue (TB) staining, 6-diamidino-2-fenilindole (DAPI) and anti-protamine 1 antibody (anti-PR1) labeling in sperm samples of fertile and subfertile bulls. Semen samples were obtained from bulls kept in Artificial Insemination Center (fertile bulls) or from bulls subjected to scrotal insulation (subfertile bulls). The percentage of chromatin alterations identified by TB was similar (P > 0.05) in semen samples of fertile and subfertile bulls. In contrast, a greater (P < 0.01) chromatin decondensation and heterogeneity were recorded in semen samples of subfertile bulls. In DAPI and anti-PR1 methods, the subfertile bulls samples had a higher (P < 0.05) percentage of alteration in the base as well as overall chromatin alterations (P < 0.05). Moreover, the chromatin alterations recorded with TB, DAPI, and anti-PR1 were compared in semen samples of fertile and subfertile bulls. In fertile bulls, the overall chromatin alterations were similar (P > 0.05) among the methods In contrast, semen samples of subfertile bulls had a higher (P < 0.05) percentage of overall chromatin alterations when labeled with DAPI. In conclusion, our findings shown that all dye tested had specific sperm stainability and can be feasible to monitor subfertility condition in bulls. Also, different chromatin alteration types in sperm samples of fertile and suberftile bulls were recorded.

Ambiente web para citomorfometria usando imagens 3D / Web environment for histomorphometry using 3D images

Softwares de bioimage são utilizados para a análise de imagens microscópicas e auxiliam os usuários em suas tomadas de decisões, a usabilidade pode limitar o seu uso. OBJETIVOS: Desenvolver um software de bioimage que pode ser acessado em um navegador web. Auxiliar patologistas e outros usuários na tomada de decisão, minimizando a subjetividade de suas avaliações. MÉTODOS: Foram realizados estudos nos softwares de bioimage open sources, o qual foram identificadas as características positivas e negativas, permitindo a escolha das tecnologias apropriadas para desenvolver o ambiente. RESULTADOS: O trabalho proveu um software de bioimage para análise citomorfométrica usando imagens tridimensionais que pode ser acessado por meio de um navegador web. CONCLUSÃO: O ambiente proposto é capaz de subsidiar os usuários com informações sobre as estruturas das células para tomada de decisão, fornecendo dados quantitativos e permitindo a exploração por meio de uma cena tridimensional.

Bioimage softwares are used for the analysis of microscopic images and assist users in their decision making,usability can limit its use. PURPOSES: Develop a bioimage software that can be accessed through a web browser. Assist pathologists and other users in decision making, minimizing the subjectivity of its evaluations. METHODS: Studies were carried out in the bioimage open sources softwares, which have been identified the positive and negative characteristics, allowing the choice of the appropriate technologies to developing the environment. RESULTS: The work provided abioimage software for histomorphometric analysis using three-dimensional images that can be accessed through a webbrowser. CONCLUSION: The proposed environment can subsidize the users with information on the structures of cells to decision making, providing quantitative data and allowing the exploration of a three-dimensional scene.