New Sperm Morphology Analysis in Equids: Trumorph® Vs Eosin-Nigrosin Stain

The evaluation of the male fertility potential is based on the analysis of the basic spermatic characteristics of concentration, motility and morphology. Thus, the study of sperm morphology is a fundamental element in the seminal analysis, but its real meaning has been biased by the techniques used for its evaluation. These techniques involve dehydration phases and subsequent staining, which involves the production of artifacts. The aim of the study is to compare two methods for equid semen morphology evaluation, Trumorph® using living sperm vs. eosin-nigrosine stain. A total of 49 ejaculates from stallions and donkeys were used. After semen collection and dilution, an aliquot was placed on the slide and introduced in the Trumorph® device. Then observation was made with a 40x objective and negative phase-contrast microscope. Another aliquot was stained using eosin-nigrosine stain and viewed using 100× magnification. Well-formed sperm were observed, and different abnormalities were identified using Trumorph®. The use of eosin-nigrosin staining method and Trumorph® led to the same results and both techniques can be used for stallion and donkey sperm morphological analysis. However, considering the fact that Trumorph® uses living sperm helps prevent sperm cell alteration during sample preparation. Therefore, Trumorph® can be a good alternative to the conventional staining method, which provides a quick test on live sperm.

An improved clearing and staining protocol for evaluation of arbuscular mycorrhizal colonisation in darkly pigmented woody roots

Arbuscular mycorrhizal fungi (AMF) establish symbiotic interactions with the roots of vascular plants, including grapevines. Verifying AMF colonisation routinely requires establishing the presence of hyphae, arbuscules and vesicles. Clearing roots with potassium hydroxide (KOH) followed by staining with trypan blue has been used previously to visualise fungal structures, however visualisation is difficult with darkly pigmented roots, such as those of grapevines so additional steps are required to ensure clear visualisation. Three fixing and clearing processes were evaluated prior to staining with trypan blue: 1) fixing grapevine roots in 70% v/v ethanol overnight; 2) clearing by heating the roots in either 2% or 10% w/v KOH; and 3) clearing the roots in 3% v/v hydrogen peroxide for 10 min. Roots were examined under a compound light microscope for the presence of AMF. A combination of fixing grapevine roots in 70% ethanol overnight and clearing by autoclaving in 10% KOH produced the greatest enhancement in subsequent staining of grapevine roots with trypan blue overnight. The best method tested enabled the discrimination of arbuscular mycorrhizal structures in fresh roots of grapevines without the use of toxic chemical fixatives.

Experimental Justification of the Use of Colloidal Blood Substitutes for Fat Globulemia

The purpose of the study is to assess in vitro the effect of colloidal blood substitutes on fat globules in the blood of patients with severe polytrauma.Materials and methods. Perftoran, 5% albumin solution, dextran-40, dextran-60, modified gelatin, hydroxyethyl starches 200/0.5 and 130/0.42 were added to the blood of 19 patients with severe polytrauma at ratios 1:20, 1:10, 1:5 and 1:3, respectively, which corresponded to the addition of 0.015 ml, 0.03 ml, 0.06 ml, 0.1 ml of the blood substitute under test to 0.3 ml of blood in a tube. Microscopy of the samples with assessment of the number and area of fat globules was carried out using a transmitted-light medical microvisor mVizo-101 (LOMO, Russia) 30 minutes after the blood and blood substitute exposure and subsequent staining with Sudan IV. The findings were processed using JMicroVision 1.2.7 software.Results. Dextran-60, hydroxyethyl starch 200/0.5 and 130/0.42 in vitro lead to a decrease in fat globules in the blood of patients with severe polytrauma in proportion to the dilution degree. Modified gelatin, dextran-40, 5% albumin solution and Perftoran have a significant additional emulsifying effect on the fat globules. The maximum emulsifying effect was obtained after addition of Perftoran to the blood.Conclusion. The experimental data on the effect of 5% albumin solution, modified gelatin, dextran-40, and Perftoran on fat globules justify the prospect for their further clinical application for prevention and treatment of fat embolism in an extended clinical trials.

Study of the sexual dysfunction secondary to antidepressants in animal models

IntroductionSexual dysfunction is a very important problem in western countries. One of the causes is the treatment with antidepressants; most of the currently available produce sexual dysfunction in men and women (lower libido, anorgasmia, etc.).ObjectiveComparing the nervous system of the animals we expect to find differences to explain the biological substratum of the sexual dysfunction that produce the selective serotonin reuptake inhibitors.MethodTwenty Wistar rats; approximate weight 150 g. It is divided into 4 groups: 2 experimental (paroxetine and agomelatina mouth) and 2 controls. There is a daily conduct. Weighing at the beginning of the study, 14 and 28 days. Is performed sacrifice by decapitation, is extracted from the brain and after fixing paraffin cuts are carried out for their subsequent staining (immunohistochemistry) with their corresponding murine antibody and viewing through optical microscope.ResultsLower immunoreactivity with the antibody anti-TH in the animals treated with paroxetine, at all levels of the dopaminergic activity (tracks mesolimbica, cortical circuit, nigrostriatal pathway and tubero-infundibular). This decrease is reaffirmed after the statistical treatment of data.ConclusionsTreatment with paroxetine in animal models causes a depletion of the dopaminergic system that can be one of the biological bases of sexual dysfunction, altering the reward mechanisms as well as producing hyperprolactinemia.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Study of the skin anatomy with high-frequency (22 MHz) ultrasonography and histological correlation

The present essay is aimed at getting the radiologist familiar with the basic histological skin structure, allowing for a better correlation with sonographic findings. A high-frequency (22 MHz) ultrasonography apparatus was utilized in the present study. The histological analysis was performed after the skin specimens fixation with formalin, inclusion in paraffin blocks and subsequent staining with hematoxylin-eosin. The authors present a literature review showing the relationship between sonographic and histological findings in normal cutaneous tissue, and discuss the technique for a better performance of the sonographic scan. High-frequency ultrasonography is an excellent tool for the diagnosis of different skin conditions. However, as this method is operator-dependent, it is crucial to understand the normal skin structure as well as the correlation between histological and sonographic findings.

Structural Manipulation of Microcone Arrays for Microsurgical Modification of Ophthalmic Tissues

The purpose of this study was to utilize controllable fiber-drawing techniques in order to fabricate glass microcone arrays for use in office-based optical surgery instruments. The cone spacing is controlled via the drawing process while an etching process controls the cone height-to-base ratio. The device viability was tested by imprinting, and subsequent staining, of low-density polyethylene and porcine corneas, resulting in a consistent patterned structure of micron-sized perforations. After imprint, the device was examined and no evidence of microcone fracture or overpenetration was present during the course of these experiments. This research promises to lead to advances in optical surgery for the treatment of recurrent corneal erosions, providing quicker, safer, and more cost-effective procedures with decreased risk of vision loss and scarring associated with current procedures such as anterior stromal puncture. The ease of procedure and micron-sized incisions could potentially replace current techniques and provide a viable treatment alternative for recurrent corneal erosions in the visual axis.

Microscopic Detection of Viable Staphylococcus epidermidis in Peri-Implant Tissue in Experimental Biomaterial-Associated Infection, Identified by Bromodeoxyuridine Incorporation

ABSTRACT Infection of biomedical devices is characterized by biofilm formation and colonization of surrounding tissue by the causative pathogens. To investigate whether bacteria detected microscopically in tissue surrounding infected devices were viable, we used bromodeoxyuridine (BrdU), a nucleotide analogue that is incorporated into bacterial DNA and can be detected with antibodies. Infected human tissue was obtained postmortem from patients with intravascular devices, and mouse biopsy specimens were obtained from mice with experimental biomaterial infection. In vitro experiments showed that Staphylococcus epidermidis incorporated BrdU, as judged from staining of the bacteria with anti-BrdU antibodies. After incubation of bacteria with BrdU and subsequent staining of microscopic sections with anti-BrdU antibodies, bacteria could be clearly visualized in the tissue surrounding intravascular devices of deceased patients. With this staining technique, relapse of infection could be visualized in mice challenged with a low dose of S. epidermidis and treated with dexamethasone between 14 and 21 days after challenge to suppress immunity. This confirms and extends our previous findings that pericatheter tissue is a reservoir for bacteria in biomaterial-associated infection. The pathogenesis of the infection and temporo-spatial distribution of viable, dividing bacteria can now be studied at the microscopic level by immunolabeling with BrdU and BrdU antibodies.

Nuclei of Plants as a Sink for Flavanols

Onion cepa (L.) and Tsuga canadensis (L.) Carr, were investigated histochemically on the association of flavanols to nuclei. The young roots of Onion cepa are totally devoid of flavanol structures. Therefore, the excised roots tips were directly incubated into different solutions of flavanols. After 3 h of incubation a flavanol binding on the nuclei was recognizable, as seen by a yellowishbrown tanning reaction. Still to ensure the presence of flavanols on the nuclei, subsequent staining with the pdimethylaminocinnamaldehyde reagent (DMACA) resulted in an intense blue colouration. Tsuga canadensis has significant amounts of vacuolar flavanol deposits in all parts of the tree as indicated by the DMACA reagent. It is obvious that also the nuclei were associated strongly with flavanols which can be demonstrated particularly elegant in the cells of the seed wings by histochemical methods. However, the mode of flavanol release from the original deposits is not yet clear.

Vacuole Partitioning During Meiotic Division in Yeast

We have examined the partitioning of the yeast vacuole during meiotic division. In pulse-chase experiments, vacuoles labeled with the lumenal ade2 fluorophore or the membrane-specific dye FM 4-64 were not inherited by haploid spores. Instead, these fluorescent markers were excluded from spores and trapped between the spore cell walls and the ascus. Serial optical sections using a confocal microscope confirmed that spores did not inherit detectable amounts of fluorescently labeled vacuoles. Moreover, indirect immunofluorescence studies established that an endogenous vacuolar membrane protein, alkaline phosphatase, and a soluable vacuolar protease, carboxypeptidase Y, were also detected outside spores after meiotic division. Spores that did not inherit ade2- or FM 4-64-labeled vacuoles did generate an organelle that could be visualized by subsequent staining with vacuole-specific fluorophores. These data contrast with genetic evidence that a soluble vacuolar protease is inherited by spores. When the partitioning of both types of markers was examined in sporulating cultures, the vacuolar protease activity was inherited by spores while fluorescently labeled vacuoles were largely excluded from spores. Our results indicate that the majority of the diploid vacuole, both soluble contents and membrane-bound components, are excluded from spores formed during meiotic division.

Biochemical and genetic characterization of esterase-27 (ES-27), the major plasma cholinesterase of the house mouse (Mus musculus)

SummaryEsterase-27A (ES-27A) was characterized in strain A/WySnA by a cascade of seven bands seen after disc electrophoresis of serum and subsequent staining for esterase. ES-27A catalyses the hydrolysis of thiocholine butyrate and is strongly inhibited by 100 μm tetraisopropyl pyrophosphamide (isoOMPA). Hence, the enzyme was concluded to be a cholinesterase EC 3.1.1.8. A heat-labile form termed ES-27B was represented by strain AKR/Han. From a three-point cross (AKR/Han, A/Wy) and a five-point cross (AKR/Han, SEG/1), the gene order on chromosome 3 was concluded to be centromere–Car-2–Es-26–Es-27–Amy-1–Adh-1.