Proliferation and apoptosis dynamics of the normal canine mammary gland during the oestrous cycle evaluated by Ki-67 and Caspase-3 indexes.

Understanding the normal physiology of the canine mammary gland (CMG) is crucial, as it provides a foundational reference for understanding canine mammary neoplasms. The relation between the Proliferation Index (PI) indicated by Ki-67 expression, along with the Apoptotic Index (AI) determined through Caspase-3 expression during the oestrous cycle, is inadequately documented in existing literature. This study seeks to offer insights into the interplay between PI and AI in the CMG across oestrous cycle phases. An extensive investigation was conducted on a diverse case series of bitches (n = 18). Oestrous cycle stages were determined through vaginal cytology, histological examination of the reproductive tract and serum progesterone and oestradiol concentrations. The entire mammary chain was histologically examined, and proliferation and apoptosis were assessed via double immunohistochemistry employing anti-Ki-67 and Caspase-3 antibodies. PI and AI were evaluated through a systematic random sampling approach, counting a minimum of 200 cells for each cell type. There was a significantly higher PI during early dioestrus in all mammary gland components, with a greater proportion of positive cells observed in epithelial cells compared to stromal cells. The highest PI was detected in epithelial cells within the end buds. Significant differences were found in Ki-67 labelling across the cranial mammary glands. A positive and strong correlation was noted between progesterone concentration and PI in epithelial cells. The AI remained consistently low throughout the oestrous cycle, with few differences observed across histological components. Caspase-3 labelling displayed the highest positivity in caudal mammary pairs. A negative and moderate correlation was identified between progesterone concentration and AI in interlobular mesenchymal cells. This study highlights the influence of endocrine regulation on cell proliferation indices in mammary tissue, emphasizing the need to consider these hormonal variations in toxicopathological studies involving canine mammary gland.

Comparison of the effects of platelet concentrates produced by high and low-speed centrifugation protocols on the healing of critical-size defects in rat calvaria: a microtomographic and histomorphometric study.

The current study evaluated the healing of critical-size defects (CSD) created in rat calvaria treated with platelet concentrates produced by high-speed (Leukocyte- and Platelet-Rich Fibrin - L-PRF) and low-speed (Advanced Platelet-Rich Fibrin - A-PRF) protocols of centrifugation. Twenty-four rats were distributed into three groups: Control, L-PRF, and A-PRF. Five mm diameter CSD were created on the animals' calvaria. The defects of the L-PRF and A-PRF groups were filled with 0.01 ml of L-PRF and A-PRF, respectively. The control group defects were filled with a blood clot only. All animals were euthanized on the 35th postoperative day. Histomorphometric and microtomographic analyses were then performed. The L-PRF and A-PRF groups had significantly higher bone volume and neoformed bone area than those of the control group and lowered bone porosity values (p < .05). No significant differences were observed between A-PRF and L-PRF groups for the analyzed parameters. Therefore, it can be concluded that i) L-PRF and A-PRF potentiated the healing of CSD in rat calvaria; ii) high and low-speed centrifugation protocols did not produce PRF matrices with different biological impacts on the amount of bone neoformation.

Morphological and morphometric characterization of domestic cat epididymal sperm.

Sperm morphometry is the tool that confers objectivity to the morphological evaluation by accurately measuring the dimensions of the gamete and its structures. Thus, the aim of the study was to perform a morphometric characterization of the domestic cat sperm. Therefore, sperm samples were collected from twenty pairs of epididymis in a TRIS extender at 37ºC. An aliquot of the sample was used to make a smear with Rose Bengal solution, and afterwards, the morphology and morphometry were analysed. In the morphology, were quantified the percentage of normal sperm cells, morphological changes of head, midpiece and tail. In morphometry, each normal sperm cell was measured for length, width, area and perimeter of head and midpiece, tail length and total length. The parameters ellipticity, elongation, regularity and rugosity were also determined. The percentage of normal sperm was 67.21%. Of the abnormalities, the curled/folded tail, followed by the curved midpiece, abnormal shaped head and detached head were the most quantified. The sperm head presented 5.56 ± 0.01 µm and 3.10 ± 0.01 µm of length and width, respectively. The head area was 16.94 ± 0.05 µm2 , while the perimeter was 16.16 ± 0.03 µm. In the derived parameters, the values were as follows: ellipticity of 1.81 ± 0.00; elongation of 21.39 ± 0.12; regularity of 0.81 ± 0.00; and rugosity of 0.14 ± 0.00. The midpiece presented length and width of 7.96 ± 0.01 µm and 0.76 ± 0.01 µm, respectively. The mean length of the sperm tail was 45.12 ± 0.06 µm, and the total cell size was 58.67 ± 0.06 µm. Thus, it was concluded that the cat sperm is an elongated cell, with high rugosity and regularity. The spermatic tail represents more than ¾ of the total length of the cell and the midpiece exceeds the length of the head.

Lectin-carbohydrate complex evaluation by chemiluminescence.

In order to characterize the affinity between specific carbohydrate-binding proteins such as lectins, a model is proposed to study these interactions using a polysaccharide membrane to simulate such adsorption. Here, lectin-carbohydrate interactions were chemiluminescently investigated using lectins conjugated to acridinium ester (AE) and polysaccharides composed of their respective specific carbohydrates. The lectin-AE conjugates were incubated with discs (0.0314-0.6358 cm2) of phytagel, chitosan and carrageenan. The complex formation chemiluminescently detected followed the Langmuir isotherm from which constants were estimated. The association constant (Ka) and maximum binding sites on the membranes were 2.4 × 10-7 M-1 ±â€¯0.8 × 10-7 M-1 and 1.3 × 10-3 mol. mg-1 ± 0.3 × 10-3 mol. mg-1 (Con A); 0.9 × 10-6 M-1 ±â€¯0.4 × 10-6 M-1 and 0.021 × 10-3 mol. mg-1 ± 0.003 × 10-3 mol. mg-1 (WGA) and 2.0 × 10-6 M-1 ±â€¯0.9 × 10-6 M-1 and 0.069 × 10-3 mol. mg-1 ± 0.010 × 10-3 mol. mg-1 (PNA). The proposed model might be useful to study binding affinity and estimate the amount of binding not limited by the sugar content in the membrane.

The in vitro antileishmanial activity of essential oil from Aloysia gratissima and guaiol, its major sesquiterpene against Leishmania amazonensis.

Leishmaniases is a tropical disease caused by protozoa of the genus Leishmania for which the current treatment is expensive, besides increasing reports of parasite resistance. This study investigated the anti-Leishmania amazonensis activity of the essential oil from Aloysia gratissima (AgEO) and guaiol, the major sesquiterpene constituent in the oil. Our results showed that AgEO killed promastigotes and intracellular amastigotes at an IC50 of 25 and 0·16 µg mL-1, respectively, while guaiol killed amastigotes at an IC50 of 0·01 µg mL-1. Both AgEO and guaiol were safe for macrophages up to 100 µg mL-1, as evaluated by the dehydrogenase activity, membrane integrity and phagocytic capacity. AgEO and guaiol did not induce nitrite oxide (NO) in resting macrophages and inhibited the production of NO in lipopolysaccharide-stimulated macrophages. The ultrastructural analysis suggested that AgEO and guaiol act directly on parasites, affecting promastigotes kinetoplast, mitochondrial matrix and plasma membrane. Together, these results pointed out that AgEO and guaiol could be promising candidates to develop anti-Leishmania drugs.

Microbial Food-Web Drivers in Tropical Reservoirs.

Element cycling in aquatic systems is driven chiefly by planktonic processes, and the structure of the planktonic food web determines the efficiency of carbon transfer through trophic levels. However, few studies have comprehensively evaluated all planktonic food-web components in tropical regions. The aim of this study was to unravel the top-down controls (metazooplankton community structure), bottom-up controls (resource availability), and hydrologic (water residence time) and physical (temperature) variables that affect different components of the microbial food web (MFW) carbon stock in tropical reservoirs, through structural equation models (SEM). We conducted a field study in four deep Brazilian reservoirs (Balbina, Tucuruí, Três Marias, and Funil) with different trophic states (oligo-, meso-, and eutrophic). We found evidence of a high contribution of the MFW (up to 50% of total planktonic carbon), especially in the less-eutrophic reservoirs (Balbina and Tucuruí). Bottom-up and top-down effects assessed through SEM indicated negative interactions between soluble reactive phosphorus and phototrophic picoplankton (PPP), dissolved inorganic nitrogen, and heterotrophic nanoflagellates (HNF). Copepods positively affected ciliates, and cladocerans positively affected heterotrophic bacteria (HB) and PPP. Higher copepod/cladoceran ratios and an indirect positive effect of copepods on HB might strengthen HB-HNF coupling. We also found low values for the degree of uncoupling (D) and a low HNF/HB ratio compared with literature data (mostly from temperate regions). This study demonstrates the importance of evaluating the whole size spectrum (including microbial compartments) of the different planktonic compartments, in order to capture the complex carbon dynamics of tropical aquatic ecosystems.

Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives.

In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a-h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 10(4) to 1.0 × 10(6) M(-1) and quenching constants from -0.2 × 10(4) to 2.18 × 10(4) M(-1) indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N- (4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties.

Resveratrol is active against Leishmania amazonensis: in vitro effect of its association with Amphotericin B.

Resveratrol is a polyphenol found in black grapes and red wine and has many biological activities. In this study, we evaluated the effect of resveratrol alone and in association with amphotericin B (AMB) against Leishmania amazonensis. Our results demonstrate that resveratrol possesses both antipromastigote and antiamastigote effects, with 50% inhibitory concentrations (IC50s) of 27 and 42 µM, respectively. The association of resveratrol with AMB showed synergy for L. amazonensis amastigotes, as demonstrated by the mean sums of fractional inhibitory index concentration (mean ΣFIC) of 0.483, although for promastigotes, this association was indifferent. Treatment with resveratrol increased the percentage of promastigotes in the sub-G0/G1 phase of the cell cycle, reduced the mitochondrial potential, and showed an elevated choline peak and CH2-to-CH3 ratio in the nuclear magnetic resonance (NMR) spectroscopy analysis; all these features indicate parasite death. Resveratrol also decreased the activity of the enzyme arginase in uninfected and infected macrophages with and without stimulation with interleukin-4 (IL-4), also implicating arginase inhibition in parasite death. The anti-Leishmania effect of resveratrol and its potential synergistic association with AMB indicate that these compounds should be subjected to further studies of drug association therapy in vivo.

Cryopreservation of canine epididymal sperm using ACP-106c and TRIS.

The objective was to cryopreserve sperm recovered from the canine epididymal cauda immediately after an orchiectomy. The sperm was stored for 12h at 4 °C using ACP-106c and TRIS as extenders. Sixty adult male dogs were used. The testis-epididymis complex (TEC) was removed, immersed in 0.9% saline and transported to the laboratory. The 60 TEC were divided into groups according to the 4 °C cooling time (0 h or 12 h) and according to the extender used for sperm recovery (ACP-106c or TRIS), forming 4 experimental groups: G0h-ACP, G12h-ACP, G0h-TRIS and G12h-TRIS. The sperm were recovered from the epididymal cauda using the retrograde flow technique. Next, 1.0 mL of ACP-106c or 1.0 mL of TRIS (preheated to 37 °C for 5 min) was added to the sperm of each epididymis. One week later, the sperm was thawed at 37 °C for 1 min, and its morphology, functionality and total and progressive sperm motilities were analyzed. Other parameters were obtained by Computer Assisted Semen Analysis (CASA). The data were submitted to multivariate analysis of variance (MANOVA) (P<0.05). The total motility values were 52.17 ± 1.78 and 49.8 ± 1.93 for groups G0h-ACP and G12h-ACP and 50.7 ± 2.06 and 43.90 ± 2.51 for groups G0h-TRIS and G12h-TRIS, respectively. A decrease in total sperm motility was observed after 12h of cooling for both extenders (P<0.05). ACP-106c can be used as an extender for freezing canine epididymal sperm, and the freezing procedure must be performed immediately after sperm recovery.

Chick heterophils release DNA extracellular traps (DETs) in vitro and in vivo upon Aspergillus fumigatus conidia exposure.

Aspergillosis is a common fungal disease in avian species, causing high mortality in young chicks in agricultural farms and yards. It is caused by fungi belonging to the genus Aspergillus. Aspergillosis occurs by inhalation of fungal conidia, and in chickens, effective infection control relies on a rapid and large influx of heterophils to the lungs. Heterophils, upon different stimuli, release to the extracellular milieu their chromatin associated with several proteins that ensnare and kill different pathogens similarly to neutrophil extracellular traps. Here, we showed that Aspergillus fumigatus conidia and the peptidogalactomannan (PGM), isolated from the fungus cell wall, induce the release of DNA extracellular traps (DETs) in chicks' blood and lung heterophils. We demonstrated that reactive oxygen species, elastase and peptidyl arginine deiminase (PAD) were involved in DETs extrusion, the occurrence of DETs in the lungs of A. fumigatus-exposed chicks in vivo, and its role in chick survival. These results may contribute to developing more efficient tools for the therapeutic and diagnosis of aspergillosis.

Cyanopeptides occurrence and diversity in a Brazilian tropical reservoir: Exploring relationships with water quality.

Microcystins (MCs) are a class of toxic secondary metabolites produced by some cyanobacteria strains that endanger aquatic and terrestrial organisms in various freshwater systems. Although patterns in MC occurrence are being recognized, divergences in the global data still hamper our ability to predict the toxicity of cyanobacterial blooms. This study aimed (i) to determine the dynamics of MCs and other cyanopeptides in a tropical reservoir, (ii) to investigate the correlation between peptides and potential cyanotoxin producers (iii) identifying the possible abiotic factors that influence the peptides. We analyzed, monthly, eight MC variants (MC-RR, -LA, -LF, -LR, -LW, -YR, [D-Asp3]-RR and [D-Asp3]-LR) and other peptides in 47 water samples collected monthly, all season long, from two sampling sites in a tropical eutrophic freshwater reservoir, in southeastern Brazil. The cyanopeptides were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The biomass of potential cyanobacterial producers and water quality variables were measured. MCs were detected in both sampling sites year-round; the total MC concentration varied from 0.21 to 4.04 µg L-1, and three MC variants were identified and quantified (MC-RR, [D-Asp3]-RR, -LR). Additionally, we identified 28 compounds belonging to three other cyanopeptide classes: aeruginosin, microginin, and cyanopeptolin. As potential MC producers, Microcystis spp. and Dolichospermum circinalis were dominant during the study, representing up to 75% of the total phytoplankton. Correlational and redundancy analysis suggested positive effects of dissolved oxygen, nitrate, and total phosphorus on MC and microginins concentration, while water temperature appeared to favor aeruginosins. A comparison between our results and historical data showed a reduction in total phosphorus and cyanobacteria, suggesting increased water quality in the reservoir. However, the current MC concentrations indicate a rise in cyanobacterial toxicity over the last eight years. Moreover, our study underscores the pressing need to explore cyanopeptides other than MCs in tropical aquatic systems.

Inhibition of Microbicidal Activity of Canine Macrophages DH82 Cell Line by Capsular Polysaccharides from Cryptococcus neoformans.

Cryptococcus neoformans is a lethal fungus that primarily affects the respiratory system and the central nervous system. One of the main virulence factors is the capsule, constituted by the polysaccharides glucuronoxylomannan (GXM) and glucuronoxylomanogalactan (GXMGal). Polysaccharides are immunomodulators. One of the target cell populations for modulation are macrophages, which are part of the first line of defense and important for innate and adaptive immunity. It has been reported that macrophages can be modulated to act as a "Trojan horse," taking phagocytosed yeasts to strategic sites or having their machinery activation compromised. The scarcity of information on canine cryptococcosis led us to assess whether the purified capsular polysaccharides from C. neoformans would be able to modulate the microbicidal action of macrophages. In the present study, we observed that the capsular polysaccharides, GXM, GXMGal, or capsule total did not induce apoptosis in the DH82 macrophage cell line. However, it was possible to demonstrate that the phagocytic activity was decreased after treatment with polysaccharides. In addition, recovered yeasts from macrophages treated with polysaccharides after phagocytosis could be cultured, showing that their viability was not altered. The polysaccharides led to a reduction in ROS production and the mRNA expression of IL-12 and IL-6. We observed that GXMGal inhibits MHC class II expression and GXM reduces ERK phosphorylation. In contrast, GXMGal and GXM were able to increase the PPAR-γ expression. Furthermore, our data suggest that capsular polysaccharides can reduce the microbicidal activity of canine macrophages DH82.

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva.

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

CA 15-3, CRP, and LDH correlates with prognostic parameters in canine mammary neoplasms.

The identification of putative prognostic factors in canine mammary neoplasms (CMNs) has been focused on tissue-specific biomarkers, but the serum biomarkers, including cancer antigen 15-3 (CA 15-3), c-reactive protein (CRP), and lactate dehydrogenase (LDH) have been demonstrated to display clinical application in cases of CMNs. The aim of the study was to evaluate the levels of these serum biomarkers and their association with well-established prognostic factors in CMNs. Samples from 15 female canines with CMNs and 15 clinically healthy ones were collected. The results were evaluated using the Tukey's, Pearson, or Spearman tests. The cut-off point, sensitivity, specificity, and area under curve (AUC) were evaluated using the receiver operating characteristic (ROC) curve analysis in a logistic regression model (P<0.05). The levels of CA 15-3, CRP and LDH were significantly higher in the serum of female dogs with CMNs compared to the healthy ones. Moreover, these factors were positively correlated with ulceration, tumor size, histopathological grade, metastatic lymph node, and clinical staging. Female dogs with CMNs were found to exhibit highest serum levels of CA 15-3, CRP, and LDH. Therefore, they can be applied to improve the efficacy of the diagnosis and prognostic evaluation in casas of CMNs.

Nutrients and not temperature are the key drivers for cyanobacterial biomass in the Americas.

Cyanobacterial blooms imperil the use of freshwater around the globe and present challenges for water management. Studies have suggested that blooms are trigged by high temperatures and nutrient concentrations. While the roles of nitrogen and phosphorus have long been debated, cyanobacterial dominance in phytoplankton has widely been associated with climate warming. However, studies at large geographical scales, covering diverse climate regions and lake depths, are still needed to clarify the drivers of cyanobacterial success. Here, we analyzed data from 464 lakes covering a 14,000 km north-south gradient in the Americas and three lake depth categories. We show that there were no clear trends in cyanobacterial biomass (as biovolume) along latitude or climate gradients, with the exception of lower biomass in polar climates. Phosphorus was the primary resource explaining cyanobacterial biomass in the Americas, while nitrogen was also significant but particularly relevant in very shallow lakes (< 3 m depth). Despite the assessed climatic gradient water temperature was only weakly related to cyanobacterial biomass, suggesting it is overemphasized in current discussions. Depth was critical for predicting cyanobacterial biomass, and shallow lakes proved more vulnerable to eutrophication. Among other variables analyzed, only pH was significantly related to cyanobacteria biomass, likely due to a biologically mediated positive feedback under high nutrient conditions. Solutions toward managing harmful cyanobacteria should thus consider lake morphometric characteristics and emphasize nutrient control, independently of temperature gradients, since local factors are more critical - and more amenable to controls - than global external forces.

[Body and sexuality during pregnancy]. / Corpo e sexualidade na gravidez.

The objective of this ethnographic study was to understand how women experience their body's physiological processes during pregnancy and their effects on sexuality. The study involved seven women living in a poor neighborhood in São Paulo. Data collection was performed through participant observation and interviews using guiding questions. The data were presented in the narrative form and then organized into the following categories: realizing the changes in the body; living with the changes in the body; and feelings and sensations experienced in sexual life during pregnancy and imagining the body after pregnancy. The women referred to the changes in their bodies as discomforts, and expressed their concern that these changes would be permanent. They expressed they hoped that, after childbirth, their body would go back to what it was like before pregnancy and that they would recover their sexual desire. Recognition of these concerns is an essential tool to guarantee appropriate professional practices.

[Nursing care: how nursing assistants perceive it]. / Cuidado de enfermagem: percepção dos enfermeiros assistenciais.

This is a descriptive, exploratory study wih a qualitative approach performed in a University Hospital from the Federal Public Network of the city of Fortaleza, state of Ceará, Brazil. It aimed to understand how nursing assistants perceive nursing care and to know the actions performed by nurses that are recognized as care activities. Eight nurses from the surgical intensive care unit participated in the study. A questionnaire with open questions was used as an instrument of data collection. Analysis was performed through content analysis with the formation of thematic categories and a framework that allowed the best visualization of results. Results show that, although they know and conceptualize nursing care, nurses have difficulties in achieving the larger care, maybe because they are overloaded with technical procedures performed in hospital environments.

Sperm quality and morphometry characterization of cryopreserved canine sperm in ACP-106c or TRIS.

Morphological sperm evaluation supported by the morphometry can be used in the determination of the seminal quality and in the investigation of potential extenders. Although there are studies comparing TRIS and ACP extenders, there are no comparative studies between them for the computerized assisted semen analysis (CASA), sperm viability, membrane functionality and sperm morphometry parameters of cryopreserved canine semen. Hence, we aimed to evaluate the effects of ACP-106c and TRIS on post-freezing canine sperm quality. Five dogs were submitted to semen collection twice with one-week interval. The semen was evaluated within the parameters: total motility, vigor, concentration, viability, plasma membrane functionality, morphology and morphometry. In the morphometric evaluation, the morphologically normal sperm was measured as: length, width, area and perimeter of the head and the midpiece, tail length and total length. The parameters of ellipticity, elongation, regularity and roughness were determined. Then, the semen was divided into two aliquots that were diluted in TRIS or ACP-106c, with the addition of egg yolk and glycerol. The diluted semen was refrigerated and frozen. The thawed samples were evaluated. Total motility, viability, sperm membrane functionality and normal morphology reduced after thawing in both extenders (morphology reduced from 89.60 ± 1.3% to 84.40 ± 1.8 and 84.60 ± 1.1% in TRIS and ACP-106c, respectively). However, it did not differ between TRIS and ACP-106c. In the ACP-106c the sperm head defects in cryopreserved semen were higher compared to fresh semen (P < 0.05). For all the morphometric parameters evaluated, there were no differences between fresh and cryopreserved samples (3.70 ± 0.4% vs. 2.30 ± 0.5%). In kinetics, with an interval of one week statistical differences between the extenders were found only in the parameters ALH and LIN (P < 0.05). Regardless of the extender, there were no changes in the morphometric parameters of sperm after thawing.

Heterologous in vitro fertilization and embryo production for assessment of jaguar (Panthera onca Linnaeus, 1758) frozen-thawed semen in different extenders.

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 106 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.

A Novel Protocol for the Synthesis of 1,2,4-Oxadiazoles Active against Trypanosomatids and Drug-Resistant Leukemia Cell Lines.

Cancer and parasitic diseases, such as leishmaniasis and Chagas disease, share similarities that allow the co-development of new antiproliferative agents as a strategy to quickly track the discovery of new drugs. This strategy is especially interesting regarding tropical neglected diseases, for which chemotherapeutic alternatives are extremely outdated. We designed a series of (E)-3-aryl-5-(2-aryl-vinyl)-1,2,4-oxadiazoles based on the reported antiparasitic and anticancer activities of structurally related compounds. The synthesis of such compounds led to the development of a new, fast, and efficient strategy for the construction of a 1,2,4-oxadiazole ring on a silica-supported system under microwave irradiation. One hit compound (23) was identified during the in vitro evaluation against drug-sensitive and drug-resistant chronic myeloid leukemia cell lines (EC50 values ranging from 5.5 to 13.2 µM), Trypanosoma cruzi amastigotes (EC50 = 2.9 µM) and Leishmania amazonensis promastigotes (EC50 = 12.2 µM) and amastigotes (EC50 = 13.5 µM). In silico studies indicate a correlation between the in vitro activity and the interaction with tubulin at the colchicine binding site. Furthermore, ADMET in silico predictions indicate that the compounds possess a high druggability potential due to their physicochemical, pharmacokinetic, and toxicity profiles, and for hit 23, it was identified by multiple spectroscopic approaches that this compound binds with human serum albumin (HSA) via a spontaneous ground-state association with a moderate affinity driven by entropically and enthalpically energies into subdomain IIA (site I) without significantly perturbing the secondary content of the protein.