Buffered Formaldehyde Solution Research Articles

Aspectos reprodutivos das fêmeas de Leporinus lacustris Campos, 1945 (Characiformes, Anostomidae) em uma planície de inundação Neotropical

Abstract: Aim This study evaluates the reproduction and fecundity of Leporinus lacustris in upper Paraná River floodplain environments with the objectives to (i) recognize the germ cells of folliculogenesis and oogenesis; (ii) characterize reproductive phases; (iii) investigate oocyte diameter and evaluate if this species has group synchronous or asynchronous oocyte development and if spawning is total or batch; (iv) estimate batch fecundity; (v) establish the relationship between fecundity/ovary weight, standard length, and total weight; (vi) verify the reproductive areas of this species in the upper Paraná River floodplain. Methods Sampling was carried out using gillnets in rivers, and open and closed lagoons, in March, June, September, and December 2013, 2014, 2015, 2016, 2018, and 2019 and March and September 2017. A total of 442 females were sampled and 111 subsampled, each of which had an ovary removed and fixed in Bouin solution. A subsample of 20 ovaries in the spawning capable phase were fixed in a 4% buffered formaldehyde solution to estimate the diameter of the oocytes and fecundity. The ovaries were dehydrated, embedded in historesin, cut, stained, and evaluated as regards folliculogenesis, oogenesis, development phases, and reproduction areas. Results The germ cells recorded were differentiated oogonia, primary and secondary growth oocytes, fully-grown and mature oocytes, and post-ovulatory follicles that permitted the recognition of the reproductive phases. Oocyte diameter information revealed that the species has group synchronous oocyte development, determinate fecundity and batch spawning, and eliminates an average of 38,490 oocytes. Conclusions The fact that Leporinus lacustris is reproductively successful in the Baía River and in the Fechada, Guaraná, Patos, Garças, Ventura and Ressaco do Pau Véio lagoons strengthens the importance of the areas of environmental protection of the islands and lowlands of the Paraná River, Ilha Grande National Park and Ivinheima River State Park.

Clinical and immunohistochemical evaluation of penile tumors in bulls

Fibropapilloma is the most common neoplasic formation associated with bovine papillomavirus type I. It occurs inside the penis of bulls and causes clinically adverse effects, such as phimosis, paraphimosis, urethral stricture, and urinary retention. This study aimed to conduct a clinical and immunohistochemical evaluation of penile tumors, which are associated with adverse outcomes with regard to breeding value and yield in bulls.A total of 40 bulls of different breeds aged between 10 months to 3 years were included in the study. Tumor tissue samples collected postoperatively were fixed in 10% buffered formaldehyde solution. The avidin–biotin–peroxidase method was used for immunohistochemical staining. Three separate areas were examined under 40X objective lens for each fibropapilloma tissue. The immunoreactivity was classified as none (-), mild (+), moderate (++), and severe (+++). Fibropapillomas ranged from 2 to 10 cm in diameter. Thirty-two papillomas were pedunculated and eight were sessile and attached to the body; 30 were solitary and 10 were multiple. There were 13 relapsed cases and 27 non-relapsed cases. Immunohistochemical examination revealed a statistically significant difference in the binary comparisons of proliferating cell nuclear antigen (PCNA) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) based on positive cell scoring between the relapsed and non-relapsed cases. As a result, it was concluded that the clinical data together with the cancer markers, which were used in the immunohistochemical evaluation and were of great importance in terms of the prognosis of penile tumors that caused significant economic losses, are useful parameters in evaluating the malignancy of penile tumors.

Immunohistochemical Detection of Pro-Inflammatory and Anti-Inflammatory Interleukins in the Lungs of Sheep with Jaagsiekte

Objective: In this study, it was aimed to evaluate the levels of interleukins such as IL-1β, IL-6, IL-10 and IL-12β in Jaagsiekte sheep by immunohistochemical methods. In this way, it will be revealed whether interleukins are effective in the progression of Jaagsiekte and how useful they are in the diagnosis of the disease. 
 Material-Method: The material of current study consisted of lung tissues of 26 sheep (Control, n=6 and Jaagsiekte, n=20) brought to Department of Pathology for routine histopathological diagnosis. Tissue samples taken were fixed in 10% buffered formaldehyde solution. 5 µm-thick sections were taken from the paraffin blocks prepared after routine tissue follow-up procedures. Hematoxylin & Eosin staining was applied to the sections in order to detect histopathological changes. Sections were examined and photographed under a light microscope. The routine streptavidin–biotin peroxidase complex method was used. 
 Results: In sheep with Jaagsiekte, tumoral foci with large and small acinar or papillary growths were observed in the alveolar and bronchiole lumens. The control group was negative for IL-1β, IL-6, IL-10 and IL-12β immunoreactivity. IL 1β-6-10 and 12β levels were dramatically increased in the Jaagsiekte group compared to the control group. 
 Conclusion: It was determined that interleukins were produced from tumoral cells and tumor microenvironment elements, and these interleukins showed pro-inflammatory effects, except for IL-10. Data from the current study show that interleukins are very useful markers for the diagnosis of Jaagsiekte.

Prognostic significance of PCNA, MMP-9 and p53 in bovine ocular squamous cell carcinomas

In this study, we aimed to evaluate PCNA, MMP-9 and p53 expressions according to differentiation degree of BOSCCs by immunohistochemical methods. The material of this study was composed of BOSCC biopsy samples taken from 30 cattle brought to our department. Tissue samples from cattles were fixed in 10% buffered formaldehyde solution, processed routinely, embedded in paraffin and sectioned at 5μm and stained with Hematoxylin & Eosine in order to detect histopathological changes. Sections were examined and photographed under a light microscope. Avidin-Biotin Peroxidase method was used as immunohistochemical method. We observed that the masses were nodular and cauliflower-like appearance. We found that the surfaces of the masses were highly hemorrhagic and ulcerative, sometimes covered with a purulent discharge. We defined cases with excessive and large numbers of keratin pearls, large tumoral islands, and evident squamous differentiation were defined as well-differentiated. In moderately-differentiated cases, we found that the number and size of keratin pearls decreased compared to well-differentiated cases. In addition, we observed that tumoral islets were smaller in these cases, similar to keratin pearls, and the number of poorly differentiated tumor cells increased. In poorly-differentiated cases, we determined that keratinization was either absent or formed in individual cells. As a result of statistical analysis, there was no statistically significant difference between good, moderate and poorly differentiated cases in terms of PCNA and MMP-9 expressions, but we found that the increase in p53 expression correlated with the degree of differentiation of the tumor. In conclusion, we think that p53 is a useful marker in determining the prognosis of BOSCCs.

Immunohistochemical Investigation of Lipid Peroxidation in Renal Coccidiosis of Geese

Objective: In this study, we aimed to evaluate the oxidative damage caused by lipid peroxidation due to renal coccidiosis by histopathological and immunohistochemical methods.Material-Method: The material of this study was made up of tissue samples taken from 139 geese whose average age was 10 weeks, who were brought to our department dead between 2013-2020. Tissue samples taken were fixed in 10% buffered formaldehyde solution. 5 µm-thick sections were taken from the paraffin blocks prepared after routine tissue follow-up procedures. Hematoxylin & Eosin staining was applied to the sections in order to detect histopathological changes. Sections were examined and photographed under a light microscope. Results: Various clinical signs such as fever, respiratory distress, weakness, anorexia, tremors, inability to get up from the ground, balance disorders, rotational movement, diarrhea, wheezing were detected in geese. In systemic necropsies of geese, large and small white nodular structures were detected in the kidney. In histopathological examinations, coccidiosis agents (E. truncata) were found in the tubular epithelium of the kidney. Necrosis and mononuclear cell infiltration were observed in the tubules due to the presence of E. truncata. In addition, edema and hyperemia in the lungs, multifocal necrosis in the liver, cell infiltration in the portal spaces, and enteritis were other important histopathological findings. In one case, aspergillosis was detected together with renal coccidiosis. We observed that MDA expression was more severe in the oocyst stages, which is the mature form of the parasite, compared to other parasitic life stages. Conclusion: Based on the results obtained from this study, it was revealed that renal coccidiosis in geese caused lipid peroxidation / oxidative damage through the increase in MDA expression.

Evaluation of MMP-9 and iNOS expressions in sheep with encephalitic listeriosis

This study aimed to correlate Matrix Metalloproteinase-9 and iNOS expressions with the severity of histopathological findings in tissue samples taken from sheep with encephalitic listeriosis. Thus, the role of these molecules in the pathogenesis of the disease can be elucidated. After systemic necropsy, tissue samples of adult sheeps with meningoencephalitis were investigated by the culture, histopathological and immunohistochemical methods in the presence of Listeria spp. isolation from tissues was performed in accordance with the USDA-FSIS method with some modifications. Tissue samples were fixed in a 10% buffered formaldehyde solution. Following routine procedures, tissue sections at 5 μm were stained with Hematoxylin and Eosin, investigated under light microscope and photographed. Immunohistochemical staining was performed on the tissues using the avidin-biotin immune peroxidase complex method. Listeria spp. were obtained in 20 (83.3%) of 24 tissue samples with the presence of bright grey-black centred smooth colonies on Listeria Selective Agar and identified as Listeria monocytogenes through the phenotypically supportive tests. Liquefaction necrosis, purulent meningoencephalitis, perivascular cuffing, microabscesses and glial nodules were the most important histopathological findings. MMP-9 immunpositive reactions were observed in the cytoplasm of microglial cells and neurons in areas where inflammatory and necrotic areas are concentrated in medulla oblongata and pons. In perivascular cuffing areas, immune reactions in endothelial cells were detected. We detected iNOS positive reactions in the medulla oblangata and pons, especially in inflammatory cells in the microabscesses. Consequently, a positive correlation (p < 0.05) was found between MMP-9 expression and the severity of histopathological findings in sheep with encephalitic listeriosis. expression. In addition, we found that iNOS expression increased in parallel with the increase in MMP-9

Farklı Tipteki Köpek Meme Karsinomlarında PCNA ve MMP-9 Ekspresyonlarının İmmunohistokimyasal Olarak Değerlendirilmesi

The aim of this study was to evaluate reveal whether there is a significant relationship between cell proliferation and invasion-metastasis capacity in various mammary adenocarcinoma subtypes observed in dogs, by means of immunohistochemical markers such as PCNA and MMP-9. The material of this study was composed of tissue samples of mammary carcinomas taken from 6 Kangal and 4 Setter dogs with an average age of 8.4 years brought to our department between 2012-2020 years. Tissue samples from dogs were fixed in 10 % buffered formaldehyde solution. After routine tissue procedures, 5 μm thick sections were taken from paraffin blocks prepared for Hematoxylin and Eosin staining. Sections were examined under light microscope to determine the cancer patterns. Avidin-Biotin Peroxidase method was used as immunohistochemical method. 4 of 10 mammary carcinoma cases were classified as intraductal papillary carcinoma, 4 as carcinoma mixed, 1 as solid carcinoma and 1 as tubular carcinoma. We found the highest mean number of PCNA and MMP-9 positive cells in solid subtype. In conclusion, we thought that a positive relationship between cell proliferation and invasion and metastasis capacity under the solid subtype.

Expression of PCNA, MMP-9, p53, Bax and Bcl-2 in canine transmissible venereal tumors

In this study, we aimed to evaluate the proliferative, metastatic and apoptotic capacities of TVT cases, which are in various phases of development, by using immunohistochemical markers. The material of this study consisted of twelve female and six male dogs diagnosed with TVT brought to our department between 2007 and 2020 years. Diff-quick staining was applied to the smear taken from tumoral masses for cytological examinations. Tumoral tissues from dogs were fixed in a 10% neutral buffered formaldehyde solution. After routine tissue procedures Hematoxylin & Eosin stain was applied to the sections. Tissue sections were investigated under a light microscope and photographed. Immunohistochemical staining was performed on the tissues using the avidin-biotin immune peroxidase complex method. As a result of macroscopic, cytological and histopathological examinations, TVT positive cases were divided into three according to their developmental stages. While the expression of PCNA, MMP-9, mutant p53 and Bcl-2 increased significantly in progressive cases compared to regressive and stable cases, Bax expression increased significantly in regressive cases compared to progressive and stable cases. In conclusion, we thought that the mentioned markers are very useful for understanding the prognosis of TVT, the tumor aggressiveness and the survival of the malignant cells.

Investigation of food products containing garlic or onion for a false positive sulphite response by LC-MS/MS

ABSTRACTIn the US, sulphites must be declared on the label if they are present in concentrations greater than 10 mg/kg (determined as) SO2 because an allergic-like response has been reported in a small subset of the population upon consumption of sulphite-containing products. The most widely used method for sulphite determination, the optimised Monier-Williams (OMW), produces false positive results with vegetables from the Allium (garlic) and Brassica (cabbage) genera due to extraction conditions that are thought to cause endogenous sulphur compounds to release SO2. Recently, an LC-MS/MS method was developed for sulphites but has only been tested with samples that are 100% Allium or Brassica. Since regulatory samples may contain these vegetables as ingredients, additional investigations were necessary to determine the potential extent of false positives. Four blank matrices, chips, phyllo shells, hummus, and quinoa were spiked with various concentrations of onion and garlic powders. The sulphite concentrations were determined using an LC-MS/MS method. The matrix is extracted with a buffered formaldehyde solution, converting free and reversibly bound sulphite to the stable formaldehyde adduct, hydroxymethylsulfonate (HMS). It was determined that even at concentrations up to 8% garlic powder or 2% onion powder, the measured sulphite concentration was below the 10 mg/kg SO2 labelling threshold. Commercial dried garlic powders were evaluated to determine the variation in responses that might be encountered in future regulatory samples. Recovery studies were conducted to determine if these methods would detect added sulphite. The ability to eliminate false positives due to these ingredients will result in a greater reliability in the accurate determination of added sulphite to ensure compliance with labelling requirements.

Accidental Poisoning with Nerium Oleander in Goat dairy in the Santiago Metropolitan Region in Chile

A small dairy goat farm in central Chile stocked their troughs with ornamental plants and grasses, which were obtained after pruning the gardens of neighboring sectors. Twelve hours after the goats ingested this food, the farm registered a death toll of eight goats, all of which were adult females in their productive stage, before entering the milking parlor. The mortality episode lasted 84 hours and produced mortality and morbidity values of 48 and 54 percent, respectively (n = 52). During a visit to the farm, clinic inspections, autopsies, and sample tissues were obtained for microscopic analysis (the tissues specimens were fixed in 10% buffered formaldehyde solution). The clinic inspection and microscopic analysis both confirmed that the animals had cardiovascular disease. The lesions were compatible with those produced by cardiac glycosides. During the site visit, Nerium oleander was found in the troughs, which suggested that the animals were poisoned by eating this plant. There was a high presence of this plant in the poisoned area, and it was not associated with the mortality episode before the on-site visit, which indicates that the authors should conduct training and inform producers about the handling and feeding of their animals

Simultaneous Occurrence of Hemangiosarcoma and Grade II Mast Cell Tumor in a Pit Bull Terrier

Background: The incidence of cutaneous neoplasms in dogs is high and quite variable. Hemangiosarcoma (HSA) and mast cell tumor (MCT) are commonly diagnosed neoplasms in isolation; however, reports of concomitant occurrence in a single patient are rare. HSA is a malignant mesenchymal neoplasm of endothelial origin; the spleen is the most commonly affected organ. MCT is a common neoplasm that may affect any region of the body, without predilection for sex, although some breeds have shown higher incidence. This report describes a case of HSA and MCT in a Pit bull terrier.Case: A 5-year-old white male Pit bull was presented to the Small Animal Clinic of the Federal Rural University of Pernambuco with nodules in preputial region measuring 5,4 x 3,7 cm and an ulcerated nodule in the right lateral thoracic region measuring 23,0 x 19,0 x 5,5 cm. The owner reported surgical excision of two previous nodules one year before the consultation, but neither were submitted for cytopathological or post-surgical histopathological examination. Two months after the procedure, the nodules recurred. Cytopathological examinations of preputial and lateral thoracic nodules were performed, with a suggestive diagnosis for HSA and MCT, respectively. Due to the unfavorable prognosis and the weakness of the animal, euthanasia was elected. Necroscopic examination revealed an ideal body condition score (4/9), hypertrophy of right pre-scapular and axillary lymph nodes, red hepatization in the apical lobe of the right lung, with multiple, soft and pigmented nodules in the spleen, liver, pancreas and testis. Several tissue samples were collected, conditioned in 10% buffered formaldehyde solution, routinely processed for histology, and stained with hematoxylin-eosin and toluidine blue. Microscopically, the lateral thoracic nodule consisted of round cells in cordonal arrangement, with sparse basophilic and discretely granular cytoplasm, and a round, central nucleus with a single evident nucleolus. Some cells were positively marked by toluidine blue, and the neoplasm was classified as grade II MCT. The preputial and digital nodules were confirmed as well-differentiated HSA, with fusiform cells forming vascular structures containing large numbers of erythrocytes and irregular fibrovascular stroma. Metastases of HSA were observed in the lung, liver, spleen, pancreas, and testis. Invasion of mast cells was observed in lymph node.Discussion: Although the etiology of HSA has not yet been clarified, its occurrence in dogs with poorly pigmented skin exposed to ultraviolet light is well established, consistent with observations in this case. MCTs often occur on the pelvic and thoracic limbs, abdomen, and chest. Primary site of HSA may be in any tissue, but it is commonly seen in highly vascular organs such as the spleen. When it occurs on the skin, HSA has more frequently been reported in the preputial and scrotal regions in dogs. For both neoplasms, the locations in this Pit bull corresponded to those commonly reported. MCTs with ulceration, edema, pruritus, and flushing are associated with worse prognosis. Metastasis is a major concern when evaluating oncologic patients. While it is difficult to determine the primary site of HSAs, metastasis often occurs in the lungs and liver. In this case, metastatic lesions were identified in the lungs, liver, spleen, pancreas, and testis by necropsy and histopathology. Fine needle aspiration of neoplasm-suggestive lesions should be performed prior to surgical procedures, especially because of its high sensitivity for MCTs. MCT and HSA are tumors of great significance, due to their high occurrence and variable prognosis. Multidisciplinary diagnostics involving clinical and pathological aspects are essential to guide the prognosis and treatment of these tumors.Mariana Lumack do Monte Barretto, Rômulo Freitas Francelino Dias, Saulo Romero Felix Gonçalves, Órion Pedro da Silva, Roseane Oliveira Feitosa, Stephanie Caroline Gueiros Silva, Valdemiro Amaro da Silva Junior & Andrea Alice da Fonseca Oliveira

Reproductive strategies of deep-sea squid (Mastigoteuthidae, Chiroteuthidae, Batoteuthidae and Cranchiidae)

Reproductive systems of rare adult specimens of the deep-sea squid genera Chiroteuthis, Mastigoteuthis, Liocranchia, and Bathoteuthis were collected in 2006 and 2015 in the different research surveys in the Atlantic Ocean between 46°40′S and 17°11′N. Whole squids were preserved in 4% buffered formaldehyde solution, subsequently transferred into 70% ethanol and studied in laboratory condition providing initial observations on spawning pattern in these animals. The potential fecundity of Ch. cf. joubini was ~ 45,000–50,000, the maximum egg size was 1.6–1.7 mm, while those of M. agassizii were ~ 8000–16,000 and 1.7–1.9 mm respectively. A maturing female of B. skolops had ~ 4800 eggs of which some ~ 1200 were atretic. The ovary of a spent L. reinhardti contained ~ 116,500 post-ovulatory follicles and no residual egg. Mature females of Chiroteuthis and Mastigoteuthis had spematangia implanted externally in the mantle, whereas Liocranchia had a specialised spermatangia receptacle on the inside of the mantle. Reproductive adaptations of these genera are discussed in relation to spawning habits of other deep-sea squids. Synchronous ovulation was found to be a prevailing type of the gonad development with all eggs being spawned as a single batch, with ot without brooding. In some species, this single batch is not released at once but in several consequent portions exhibiting ‘extended synchronous’ spawning.

Comparison of Thermographic Imaging and Other Diagnostic Techniques in Diagnosis of Cattle with Laminitis

Background: Lameness results in major economic losses on dairy farms. The proportion of lameness in cows are between 4-55%, the origin of 90% lameness are caused by foot diseases. Hoof abnormalities such as laminitis are major contributors to lameness. Aim of the study, compared with thermographic imaging (IRT) and radiographic, magnetic resonans (MR), computer tomography (CT), histopathological analysis on the diagnosis of laminitis.Materials, Methods & Results: Preliminary, the animals (20 Holstein-Fresian) were selected by lameness examination for evaluation of the for any obvious signs of disease. The animals were examined when rising and walking of the free stalls. Walking was very painful and clinical symptoms of general distress become present for laminitic group. The cows were scored on a 1 to 5 scale for their locomotion (1 = normal locomotion; 5 = severely lame). Thermographic examinations were performed the all animals after the routine clinical examination methods. The infrared images were taken from the dorsal view of all hoovesto monitor the temperature of coronary band. All images were scanned using a hand-held portable infrared camera (Wahl, Thermal Imager HSI3000 Series), which was calibrated to ambient temperature and absorptive conditions. Incentives radiological examinations, dorso-palmar and latero-medial shooting was done for using of (Regius Model 110 Konica, Minolta) 70 kV and 30 mAs for each claws. MRI images of all isolated digits were performed at the Department of Radiology, by using Siemens Symphony Erlangen, Germany. Continuous series of transversal, sagital and dorsoplantar scan were obtained from all digit. Computer tomography (CT), isolated digits were performed at the Imaging Department of Radiology, by using Siemens Symphony Erlangen, Germany. CT scanner was used to obtain contiguous slices from the region of the proximal sesamoid bones to the toe. Anterior side (laminar and perilaminar tissues) of the claws was uncovered with electric wheel. Tissue samples were taken 10% buffered formaldehyde solution after establishing a routine laboratory procedure following the prepared paraffin blocks 5 micrometers thick sections with hematoxylineosin dye method according to the staining. It was finally examined by light microscopy. The computer soft ware of the infrared thermal camera showed increased local temperature, by 0.5-1.5°C, between the normal and suspected regions for laminitic cases. Clinically, animals suspected of laminitis were showed general stiffness, lameness during walking difficulty. Examination of the claws was demonstrated of rings and plump appearance was evident in paries ungulae. The main damage was seen during trimming of the sole from no hemorrhages or discoloration of control group. The transversal MRI images provided excellent depictions of anatomical structures when compared to their corresponding pictures. Identifiable anatomic structure was labeled on the line drawings of the limb sections and on the corresponding CT images. Twenty out of 30 claws (65%) were shown laminitis statistical with histopathological examination. In histopathological examination; mononuclear cells and neutrophil granulocytes infilrations and edema, haemorrhagia, mild hyperemia in the lamellar region was remarkable in laminitic cases.Discussion: As a results, thermographic examination may have potential as a detection tool for laminitis. MR transversal images provided excellent depiction of anatomical structures and many biometric research in the bovine hoof can be easily investigated. The usefulness of IRT, MRI, CT in evaluating laminitis in the acute patient remains stil open. The use of diagnostic techniques during the initial active phase of laminitis as a means to increase the understanding of the disease and also serves as a justification for the development of an experiment involving live cows induces laminitis.

Influence of Platelet-Poor Plasma on Angiogenesis and Maintenance of Volume in Autogenous Bone Grafts

The aim of this study was to evaluate the effect of different storage media on angiogenesis and maintaining autogenous bone graft volume in rabbits. Two grafts were removed bilaterally from the calvaria of 18 rabbits. One graft was removed and immediately fixed in the right mandibular angle (control group). The other graft was stored for 30minutes in 1 of the following storage media (n=6): saline solution (saline group), air exposure (dry group), or platelet-poor plasma (PPP group) and then retained by a screw in the right mandibular angle in the same animal. Four weeks later the animals were euthanized, and the grafted areas were harvested, fixed in 10% phosphate buffered formaldehyde solution, and embedded in paraffin. The 5-μm semi-serial sections were stained in hematoxylin and eosin and Mallory trichrome. Histologic analysis of all groups showed the bone graft was vascularized and well incorporated into the recipient site. The number of blood vessels decreased in the saline and dry groups compared with the control group (P<.03); in contrast, the number of blood vessels increased in the PPP group (P<.05). There were fewer osteoclasts in the saline group compared with the control group (P<.05). Furthermore, the saline group showed larger numbers of blood vessels than the dry group (P<.01). The PPP group showed larger bone graft volumes compared with the dry and saline groups (P<.01). In addition, the saline group showed larger bone graft volumes than the dry group (P<.01). PPP improved angiogenesis, maintained the volume of the autogenous bone graft, and was a better storage medium during the trans-surgical period than the dry and saline media.

Immunohistochemical assessment of Cox-2, and ki- 67 expression in gastric cancer

Cell proliferation characteristics may reflect the aggressiveness of gastric cancer and their eventual prognosis. The aim of this study was to evaluate whether the proliferative activities determined by Ki-67 labeling index (LI) could be used as a predictor in patients with gastric cancer, and examine the relation between COX-2 and Ki-67 expression and gastric cancer. The expression of COX-2 and Ki-67 were detected in 40 patients with gastric cancer and 30 healthy individuals were assessed by immunohistochemistry. The biopsies were fixed in 10% buffered formaldehyde solution, embedded in paraffin and stained immunohistochemically with anti-human COX-2 and Ki-67 antibodies. Expression of Ki-67 andCOX-2was significantly higher in tumor compared with control samples.A significant correlation was found between COX-2 and Ki-67 protein expression. In conclusion,the expression of COX-2 and Ki-67 proteins was closely connected with gastric cancer.

The first record of the eggs of black wing flyingfish Hirundichthys rondeletii (Valenciennes, 1846) in the Turkish nearshores of the Aegean Sea (Gümüldür)

Hirundichthys rondeletii is among the three flyingfish species showing distribution in the Turkish seas. However, the egg stages of Exocoetidae species found in the Turkish waters have not been documented. Flyingfish eggs were randomly obtained on 16 July 2016 on the coast of Gumuldur, at a depth of approximately 1 m from a sandy bottom. Samples were fixed in a 4% buffered formaldehyde solution. The eggs were described and illustrated. The diameters of spherical fish eggs were between 1.75 mm and 2.05 mm. They have bipolar filaments: a single filament on one pole and 28-33 filaments on the other pole. The maximum length of these filaments was 73 mm. Vitellus in the eggs was homogeneous and did not contain oil globules. The total egg number determined with the gravimetric method was 4319.91 $\pm$ 22. This is the first record of the eggs of black wing flyingfish H. rondeletii found in the Turkish waters.

Structural shift of gut microbiota during chemo-preventive effects of epigallocatechin gallate on colorectal carcinogenesis in mice.

AIMTo investigate the effect of epigallocatechin gallate (EGCG) on structural changes of gut microbiota in colorectal carcinogenesis.METHODSAn azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis mouse model was established. Forty-two female FVB/N mice were randomly divided into the following three groups: group 1 (10 mice, negative control) was treated with vehicle, group 2 (16 mice, positive control) was treated with AOM plus vehicle, and group 3 (16 mice, EG) was treated with AOM plus EGCG. For aberrant crypt foci (ACF) evaluation, the colons were rapidly took out after sacrifice, rinsed with saline, opened longitudinally, laid flat on a polystyrene board, and fixed with 10% buffered formaldehyde solution before being stained with 0.2% methylene blue in saline. For tumor evaluation, the colon was macroscopically inspected and photographed, then the total number of tumors was enumerated and tumor size measured. For histological examination, the fixed tissues were paraffin-embedded and sectioned at 5 mm thickness. Microbial genomic DNA was extracted from fecal and intestinal content samples using a commercial kit. The V4 hypervariable regions of 16S rRNA were PCR-amplified with the barcoded fusion primers. Using the best hit classification option, the sequences from each sample were aligned to the RDP 16S rRNA training set to classify the taxonomic abundance in QIIME. Statistical analyses were then performed.RESULTSTreatment of mice with 1% EGCG caused a significant decrease in the mean number of ACF per mouse, when compared with the model mice treated with AOM/DSS (5.38 ± 4.24 vs 13.13 ± 3.02, P < 0.01). Compared with the positive control group, 1% EGCG treatment dependently decreased tumor load per mouse by 85% (33.96 ± 6.10 vs 2.96 ± 2.86, respectively, P < 0.01). All revealed that EGCG could inhibit colon carcinogenesis by decreasing the number of precancerous lesions as well as solid tumors, with reduced tumor load and delayed histological progression of CRC. During the cancerization, the diversity of gut microbiota increased, potential carcinogenic bacteria such as Bacteroides were enriched, and the abundance of butyrate-producing bacteria (Clostridiaceae, Ruminococcus, etc.) decreased continuously. In contrast, the structure of gut microbiota was relatively stable during the intervention of EGCG on colon carcinogenesis. Enrichment of probiotics (Bifidobacterium, Lactobacillu, etc.) might be a potential mechanism for EGCG’s effects on tumor suppression. Via bioinformatics analysis, principal coordinate analysis and cluster analysis of the tumor formation process, we found that the diversity of gut microbiota increased in the tumor model group while that in the EGCG interfered group (EG) remained relatively stable.CONCLUSIONGut microbiota imbalance might be a potential mechanism for the prevention of malignant transformation by EGCG, which is significant for diagnosis, treatment, prognosis evaluation, and prevention of colorectal cancer.

Effects of staining method and clinician experience on the evaluation of stallion sperm morphology

Evaluation of sperm morphology is part of the assessment of fertility in human and animal reproduction. Analyses can be performed using different techniques, including the use of staining methods In our prospective study, the morphology of equine sperm was evaluated using 3 staining methods Diff-Quik, eosin-nigrosin, and SpermBlue, the latter being a relatively new stain commonly used for human sperm. Our hypotheses were that (1) SpermBlue allows easier assessment of horse sperm morphology and facilitates better identification of sperm abnormalities, and (2) sperm morphology classification differs depending on the evaluator’s experience. Semen was obtained from various horse breeds; 40 samples from stallions between 2 and 15 years of age were collected during the 2016 breeding season and stored in a 2% buffered formaldehyde solution until processing. For each sample, 3 semen smears were made and stained with Diff-Quik, eosin-nigrosin, and SpermBlue. All morphological parameters were then evaluated blindly using a light microscope by a novice evaluator and a more experienced evaluator. For each slide, 200 spermatozoa were examined randomly and classified according to their characteristics. For the identification of morphologically normal spermatozoa, no significant difference between evaluators was found with any of the staining methods used. In contrast, significant differences between evaluators were observed in the classification of some anomalies affecting mainly the midpiece and the tail. Poor dye fixation was observed with SpermBlue.

Chronic anaemia causes degenerative changes in trophoblast cells of the rat placenta

Objectives: Iron deficiency anaemia causes adverse pregnancy outcome. There are few studies on effects of anaemia on the structure of trophoblastic cells which are important in placental function. These data are important for understanding the function and disorders of the placenta. The aim of this study was to investigate the ultrastructural cellular changes associated with iron deficiency anaemia in rat placenta. Methods: Forty-nine female Sprague-Dawley rats were randomly separated into experimental and control groups. The experimental group was rendered anaemic by removing 1.5 ml of blood per bleed on five alternate days, and the placentas were collected on gestational days 17, 19 and 21. For light microscopy, five cubic millimeter segments were fixed in 10% buffered formaldehyde solution; dehydrated in ethanol and embedded in paraffin wax. Five micron thick sections were cut, deparaffinized and stained with Hematoxylin and Eosin. For transmission electron microscopy, 1 mm3 sections were fixed in 2.5% phosphate buffered glutaraldehyde, post fixed in 2% osmium tetroxide, dehydrated in ethanol, cleared in propylene and embedded in epon resin. Ultrathin sections stained with uranyl acetate and lead citrate were examined with JEOL electron microscope. Results: Cytotrophoblast, syncytiotrophoblast and giant trophoblastic cells of placentas of anaemic rats showed cytoplasmic and nuclear vacuolation with loss of cell margins. In addition, there was atrophy of microvilli on the cell surface, as well nuclear chromatolysis, nucleolar degeneration and appearance of dark bodies. Conclusion: Chronic anaemia causes trophoblastic cell degeneration. This may undermine the functional integrity of the cells and constitute part of the mechanism for poor fetal outcome.

PH influences the biocompatibility of methylene blue solutions.

The aim of this study was to investigate the biocompatibility of methylene blue at different pH levels through the method of implantation in subcutaneous tissue. Eighty-four sterilized polyethylene tubes were allocated in the subcutaneous tissue of 28 rats, each one receiving four tubes, set into four groups: group tube (G-T)-empty tube, fibrin group (G-F)-tube filled with fibrin sponge, group methylene blue pH 7 (G-MB/pH 7)-tube filled with fibrin sponge soaked by methylene blue (100μg/ml) at pH 7.0, and group methylene blue pH 1 (G-MB/pH 1)-tube filled with fibrin sponge and soaked by methylene blue (100μg/ml) at pH 1.0. After 7, 15, and 30days, seven animals from each group were euthanized, and the tubes involved by the surrounding tissue were removed and fixed with 4% buffered formaldehyde solution. The collected pieces were processed and histological sections (4μm) were stained with hematoxylin and eosin and analyzed by light microscopy. Scores were assigned to analysis of histopathologic parameters. The results were statistically analyzed by the Kruskal-Wallis test (p≤0.05). At 7 and 30days, the G-MB/pH 1 group showed no significant difference in the G-T control group, while G-MB/pH 7 had a significant increase on tissue reaction, also when compared to G-T. At 15days, there was no statistical difference between the groups. Within the limits of this study, it is concluded that methylene blue at pH 1.0 provides better biocompatibility than at pH 7.0.