The effects of neem oil (Azadirachta indica A. JUSS) enriched with different concentrations of azadirachtin on the integument of semi‐engorged Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) females

Several studies searching for methods to control Rhipicephalus sanguineus s.l., (dog tick) infestations have been developed aiming to minimize the damages caused by these ectoparasites to the hosts and the environment, which is harmed by the indiscriminate use of toxic acaricide products. In this scenario, neem oil has been used as a natural alternative against ticks, once this chemical has repellent properties and interferes in the growth regulation of these ectoparasites, inhibiting ecdysis. The present study evaluated the effects of azadirachtin‐enriched neem oil on the integument of semi‐engorged R.sanguineus s.l., females through morphohistological techniques. The results showed the occurrence of significant morphological and histochemical alterations, mainly in the females exposed to higher concentrations, which demonstrates the dose‐dependent action of the chemical. A decrease in the cuticle thickness was observed, as well as a modification in the distribution of the epithelial cells, which displayed pyknotic and fragmented nuclei, and intensely vacuolated cytoplasm, indicating that these cells would be undergoing death processes. These morphological alterations observed in the integument of the females exposed to the azadirachtin‐enriched neem oil encourage the use of this chemical as a strategy to control these ectoparasites.     

Morphological alterations in the synganglion and integument of Rhipicephalus sanguineus ticks exposed to aqueous extracts of neem leaves (Azadirachta indica A. JUSS)

Currently, the necessity of controlling infestation by ticks, especially by Rhipicephalus sanguineus, has led researchers and public health managers around the world to search for new and more efficient control methods. This way, we can highlight neem (Azadirachta indica A. Juss) leaf, bark, and seed extracts, which have been very effective on tick control, and moreover causing less damage to the environment and to the host. This study showed the potential of neem as a control method for R. sanguineus through morphological and morphometric evaluation of the integument and synganglion of females, in semiengorged stage. To attain this, routine techniques of optical microscopy, scanning electron microscopy and morphometry of the cuticle and subcuticle of the integument were applied. Expressive morphological alterations were observed in both organs, presenting a dose‐dependent effect. Integument epithelial cells and nerve cells of the synganglion showed signs of cell vacuolation, dilated intercellular boundaries, and cellular disorganization, alterations not previously reported in studies with neem. In addition, variations in subcuticle thickness were also observed. In general, the effects of neem are multiple, and affect the morphology and physiology of target animals in various ways. The results presented in this work are the first evidence of its effects in the coating and nervous system of ticks, thus allowing an indication of neem aqueous extracts as a potential control method of the brown dog tick and opening new perspectives on acaricide use. Microsc. Res. Tech. 77:989–998, 2014. © 2014 Wiley Periodicals, Inc.     

Cytotoxic effects of extract of Acmella oleraceae (Jambú) in Rhipicephalus microplus females ticks

The present study analyzed the effects of different concentrations of the hexane extract of A. oleraceae (HEAO) (Jambú) on the germ cells of semi‐engorged Rhipicephalus microplus female ticks, through a morpho‐histological study, evaluating the effectiveness of the extract in the genesis of the individuals. To perform this analysis, 100 semi‐engorged females were divided into five groups with 20 individuals each: groups I and II, respectively constituted by distilled water control and 50% ethanol + 1% DMSO, and groups III, IV, and V constituted by treatment with HEAO in the concentrations of 12.5, 25.0 and 50.0 mg/mL, respectively. All the ticks were immersed in the different concentrations of the extract or in distilled water for 5 minutes, dried and conditioned in BOD incubator for 7 days. The individuals of the treatment groups revealed the action of this extract showing alterations in the germ cells of the females from the different groups when compared with those from the groups I and II (control groups). These alterations were mainly related to the size and shape of the oocytes; number of yolk granules; presence, number, size and location of vacuoles in the cytoplasm of all the germ cells; and the presence of nuclear alterations in these cells as well. Thus, it was demonstrated that the concentrations of HEAO affected the germ cells of R. microplus ticks. The effects of the extract are similar to those caused by renowned and efficient chemical products used to control these ticks. Microsc. Res. Tech. 79:744–753, 2016. © 2016 Wiley Periodicals, Inc.     

Action of andiroba oil (Carapa guianensis) on Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi‐engorged females: Morphophysiological evaluation of reproductive system

Because of the increasing medical‐veterinary importance of ticks, the development of alternative control methods, less aggressive to the host and the environment has become the target of several researches. In this sense, the present study analyzed the action of different concentrations (5, 10, and 20%) of andiroba seed oil (Carapa guianensis) on the reproductive system of Rhipicephalus sanguineus females, through histochemical techniques and the quantification of the reproductive efficiency index. The results showed that andiroba oil is a potent natural agent, able to cause several changes in the oocytes of this species, impairing the reproductive success, once this natural product induces great physiological changes in the oocytes in all development stages, such as drastic reduction in proteins, polysaccharides, and lipids in these cells, and these components are essential for the viability of the embryo. In addition, it was observed that this product stimulate the oviposition, mainly at the concentration of 20%. This higher production of eggs represents a defense mechanism developed by the organism in order to ensure the reproductive success of the species, even in the presence of the toxic agent. However, the results obtained suggested that the laid eggs would not be viable, due to the great changes undergone by the oocytes. Thus, the present study showed that the use of this vegetal product would be an alternative way to control the ticks, bringing benefits similar to the ones obtained through the use of synthetic acaricides; however, with less damage to nontarget organisms and the environment as well. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

Ultrastructure of differentiating oocytes and vitellogenesis in the giant freshwater prawn, Macrobrachium rosenbergii (de man)

The ultrastructure of oogenesis in Macrobrachium rosenbergii, with reference to vitellogenesis, has not been reported. We used light and electron microscopy, as well as vitellin (Vn) purification and antibody production, to study the temporal and spatial production of Vn in the ovary by immunofluorescence. Histologically, the ovary is subdivided into cone‐shaped ovarian pouches with a central core containing layers of oogonia. These divide to produce oocytes that migrate outwardly and differentiate into mature oocytes. During the course of differentiation, oocytes undergo modifications, including the rearrangement of nuclear chromatin, the accumulation of ribosomes, rough endoplasmic reticulum (RER), and lipid, and the formation of secretory and yolk granules, resulting in four stages. Ultrastructurally, early previtellogenic oocytes (Oc₁) are characterized by the accumulation of new ribosomal aggregates, translocated from the nucleus. Late previtellogenic oocytes (Oc₂) show nuclear heterochromatin with a “clock face” pattern, the presence of RER, and three types of secretory granules. Follicular cells occupy the intercellular spaces and surround the Oc₂. Early vitellogenic oocytes (Oc₃) are larger, with nuclei containing predominantly decondensed euchromatin, and cytoplasm with yolk and secretory granules, and few lipid droplets. Late vitellogenic oocytes (Oc₄) are characterized by completely euchromatic nuclei, an indistinct plasma membrane, yolk platelets and secretory granules, and abundant lipid. Vitellogenin (Vg) in ovaries of M. rosenbergii consist of two main bands at MW 90 and 102 kDa. Our data indicates that Vn is present, and probably synthesized in Oc₃ and Oc₄, but there may be some undetected exogenous Vg production. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Ultrastructure of oocytes of the Urostreptus atrobrunneus (Diplopoda,Spirostreptida, Spirostreptidae): A potential urban centers plague

The knowledge of the process of egg formation is indispensable for understanding the mechanisms involved in the reproduction of different species. In this context, the objective of this work was to describe the ultrastructure of the oocytes of Urostreptus atrobrunneus (Spirostreptida), a potential plague of urban centers in different locations of São Paulo State. The lack of knowledge about the morphology, physiology, and the reproductive behavior of the species have hindered an effective control of it. The oocytes of U. atrobrunneus presented three development stages: young oocyte or type I; intermediary oocyte or type II; and mature oocyte or type III. During the oocyte development, the cytoplasm become filled with several globules of protein, drops of lipids, and sphaerocrystals, and it was not observed in many organelles in the oocytes with exception of mitochondria, abundant, principally in young oocytes. The vitelline membrane is also deposited in a discontinuous form and the chorion does not present differentiation of layers. The follicular epithelium alters its shape according to the development phase of the oocyte. Part of the vitellus is from exogenous origin and part is endogenous. Before this, only two studies about the ultrastructural analysis of the female germ cells of diplopods were published. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.     

Antiproliferative effect of extracts of <i>Sida rhombifolia</i> L. (Malvaceae) on the <i>Allium cepa</i> cell cycle

Field collected roots of four populations of Sida rhombifolia were used for preparing aqueous decoctions at two concentrations: 4g/L; and 16g/L. Afterwards, we used three groups of six onion (Allium cepa) bulbs for testing each population. Slides were made with all bulbs through the smashing technique. Cells in all phases of the cell cycle of A. cepa were analyzed. The mitotic index (% of cells in mitosis) was calculated, and the statistical analysis through the χ2 test was carried out at 5% probability. The results showed that the aqueous extracts of S. rhombifolia have antiproliferative activity at high concentrations. Practically no chromosomal aberrations were induced by treatments.     

Genotoxic effect of Physalis angulata L. (Solanaceae) extract on human lymphocytes treated in vitro

Physalis angulata L (Solanaceae) is a medicinal plant from North of Brazil, whose different extracts and infusions are commonly used in the popular medicine for the treatment of malaria, asthma, hepatitis, dermatitis and rheumatism. However, the genotoxic effects of P. angulata on human cells is not well known. The main purpose of the present study was to evaluate the in vitro genotoxic effects of aqueous extract of P. angulata using the comet assay and the micronucleus assay in human lymphocytes provided from 6 healthy donors. Treatments with P. angulata extracts were performed in vitro in order to access the extent of DNA damage. The comet assay has shown that treatments with P. angulata at 0.5, 1.0, 2.0, 3.0 and 6.0 microg/mL in culture medium were genotoxic. Lymphocytes treated with P. angulata at the concentrations of 3.0 and 6.0 microg/mL in culture medium showed a statistically significant increase in the frequency of micronucleus (p<0.05), however, the cytokinesis blocked proliferation index (CBPI) was not decreased after P. angulata treatment. In conclusion, the present work demonstrated the genotoxic effects of P. angulata extract on human lymphocytes in vitro.     

A method for abolishing the nonspecific binding artifacts produced by the peroxidase-antiperoxidase complex (PAP) in immunostained mucous epithelia

The presence of nonspecific staining artifacts is a potential problem in ultrastructural immunocytochemistry. In the course of staining for lysozyme in the human and rabbit endocervix, the peroxidase-antiperoxidase complex (PAP) was found to bind to mucous granules in a selective and nonspecific manner. Sections etched in 10% aqueous H₂O₂, incubated for 5 minutes in 1:50 PAP in Tris-buffered saline (TBS) and subsequently treated with 3, 3′-diaminobenzidine-H₂O₂, revealed a staining precipitate within the matrix of mucous granules. The same selective and nonspecific staining could also be visualized when horseradish peroxidase (1 mg/ml) was substituted for PAP in the immunocytochemical sequence. Thus, this staining method made the reliable demonstration of the desired antigen difficult. The affinity of PAP for mucous granules can be completely eliminated by subjecting sections, previously etched in H₂O₂ to expose the antigenic sites, to a 3-minute incubation in immunoglobulin (1:10 human IgA, 1:5 human IgG, or 1:5 antihuman IgG) immediately prior to using PAP in the standard immunocytochemical staining sequence. The use of this modification to Sternberger's unlabeled antibody-enzyme method is recommended because it allows for the elimination of all nonspecific staining artifacts without interfering with specific localization by the primary antibody. The mechanism that causes nonspecific binding of peroxidase to mucous granule constituents is unclear, although carbohydrate binding may play a role in the interaction between mucous granules and peroxidase.     

Ultrastructural and developmental toxicity of potato and tomato leaf extracts to beet armyworm,Spodoptera exigua (lepidoptera: noctuidae)

Beet Armyworm, Spodoptera exigua is a herbivorous moth and a serious pest of many economically important plants, which are used as food sources. Because of rigorous standards of food quality, usage of synthetic insecticides in crop protection, against pests, is limited. Solanaceae plant extracts may be a relatively cheap source of efficient natural insecticides that can limit usage of synthetic substances. Their biological activity is not fully known. In particular, ultrastructural studies, using transmission electron microscopy, are not usual. In the present article we describe the effects of sublethal concentrations of tomato and potato leaf extracts against S. exigua. Acute lethal effects were not observed. Both extracts exerted similar effects within midgut and fat body cells. Midgut cells were not significantly altered while fat body cells showed prominent swelling of nuclear envelope and endoplasmic reticulum, vacuolization of mitochondria and fusion of fat droplets. These changes were much more intensive within groups exposed to potato than tomato extracts at highest concentration at least. Light microscopy was used to observe and document developmental alterations of S. exigua exposed to potato and tomato leaf extracts. Potato leaf extracts significantly decreased hatching success and caused morphological malformations of imagoes. Among them, malformations of wings were the most prominent. Interestingly, these effects were not observed within populations exposed to tomato extracts at highest concentration at least.     

Antiproliferative and genotoxic effects of <i>Mikania glomerata</i> (Asteraceae)

Mikania glomerata is a plant used in Brazilian traditional medicine, known as ‘guaco’. It possesses anti-inflammatory properties and the aqueous extracts of its leaves are indicated for the treatment of diseases of the respiratory tract. This study aimed at evaluating the antiproliferative and genotoxic effect of Mikania glomerata leaf infusions on the cell cycle of onion. The material used was collected in the native environment from Rio Grande do Sul State, Brazil. Aqueous extracts through infusions were prepared in two concentrations: 4g/L (usual concentration) and 16g/L (4x more concentrated) of each of the populations. Two groups of four onion bulbs for each plant population were used plus a control group. The rootlets were fixed in ethanol-acetic acid (3:1), conserved in ethanol 70% and slides were prepared using the squashing technique colored with orcein 2%. The cells were observed and analyzed during cell cycle. Per group of bulbs, 2000 cells were analyzed, and the mean values of the cell number of each of the phases of the cell cycle were calculated, determining the mitotic index (MI). Statistic analyses of the data were carried out by the x2 (p= 0.05) test. We conclude that M. glomerata presents both antiproliferative and genotoxic activity.     

Mouse oocyte differentiation during antral follicle development

During antral follicle development mouse oocytes undergo rearrangement of granulosa cell interactions and the oocytes released from follicles at the beginning or at the end of antral development are either devoid of denuded oocytes (DO) or strictly associated with cumulus-intact (CI) cumulus cells. In this study, these two oocyte classes were analyzed before germinal vesicle (GV) and after in vitro maturation (IVM) to evaluate (a) the ultrastructural aspect of oolemma microvilli by scanning electron microscopy analysis and (b) specific morphological markers of differentiation (chromatin organization, mitochondria, cortical granules, microfilaments, and spindle of metaphase II- MII-). At GV-stage, CI oocytes exhibited remarkable differences (a) in the oolemma microvillar ultrastructure and distribution with respect to DO and (b) in the chromatin organization that was typical of meiotically competent germ cells. By contrast, homogeneous patterns of distribution of mitochondria, cortical granules, and microfilaments characterized both the oocyte classes. At the end of culture, CI oocytes, even when matured without cumulus cells, reached more efficiently the MII stage and acquired an ultrastructural microvillous configuration different from DO. In addition, MII-arrested DO had a higher percentage of meiotic spindles with abnormal morphology in comparison with preovulatory oocytes, while cortical granule and microfilament patterns revealed no appreciable differences between the groups. With regard to mitochondria, a polarized distribution of these organelles was found in 82% of DO and in 97% of CI oocytes. These observations suggested that the achievement of the full antral follicle development is a condition for the acquisition of specific qualitative properties that are essential for the production of fertilizable oocytes, both in in vivo and in vitro models as well.     

The bioactive compound carvacrol as a potential acaricide: An assessment of its effects on the integument of female Rhipicephalus sanguineus sensu lato ticks

Studies seeking control methods for infestation of Riphicephalus sanguineus sensu lato (s.l.) ticks (dog ticks) have been carried out in order to minimize damage to both the tick's hosts and the environment, the latter due to the misuse of acaricide products. In this regard, carvacrol has been used as a natural alternative against ticks as it displays several properties including acaricidal. In this context, the present study aimed to evaluate the ultramorphology and morphohistochemistry of the integument of semi-engorged R. sanguineus s.l. females exposed to different carvacrol concentrations. The findings indicate that the integument surface of females exposed to the highest carvacrol concentration (25 μl/ml) became wrinkled, suggesting dehydration or the result of integument cuticular and epithelial layer disorganization in response to the toxic product. Morphohistochemical integument layer alterations were more significant and intense in females exposed to the highest carvacrol concentration (25 μl/ml), confirming dose-dependent carvacrol action. Among other cell and tissue alterations, changes in epithelial cell shape, size, and arrangement (epidermal layer) were noted, alongside altered and pyknotic-shaped nuclei, suggesting a death process for these cells. This epithelium changed from simple cubic to stratified, also in response to the presence of the evaluated bioactive compound. Thus, the findings reported herein demonstrate that carvacrol may be an alternative for an efficient and more sustainable tick control in the near future.     

Detection of sulfur in fixed and embedded rat peritoneal mast cell granules by X-ray energy dispersive spectroscopy

Purified rat peritoneal mast cells contained 3.3 × 10^?5 gm SO₄ and 2.2 × 10^?8 gm Ca/10⁶ cells. The molar ratio of S/Ca in the whole cell was 600:1. Frozen thin sections of unfixed mast cells contained only sulfur (S) in the granules when examined by X-ray energy dispersive spectroscopy (EDS). Mast cells fixed in 3% glutaraldehyde and 1.5% formaldehyde in 75% ethanol (Et/Ald) or in mixed buffered aldehydes and embedded in Epon 812 or the low viscosity resin diepoxyoctane (DEO) contained S in all granules and Ca in some of the granules measured. Neither element was found in the nucleus, cytoplasm, or resin. Isolated, Et/Ald fixed and embedded granules also contained S. The presence of Ca in the granules was artifactual in that the Ca was absorbed from water in the trough of the diamond knife and/or from the filter paper used to blot the sections dry. This phenomenon was investigated further. Sections of Et/Ald fixed and embedded mast cells were incubated with 5 × 10^?6 to 10^?2 M CaCl₂. Ca was detectable in 100% of the granules incubated at concentrations ≥ 10^?4 M and reached a constant S/Ca ratio of 2.0 at concentrations ≥ 10^?3 M. Ca was not detectable in the nucleus, cytoplasm, or resin at 10^?2 M. A plot of S versus Ca counts from the granules of cells incubated with 10^?2 M CaCl₂ was linear with a slope of 2.0 and a correlation coefficient of 0.88. Et/Ald fixed cells incubated with distilled H₂O had fewer granules containing Ca, 10%, than unincubated cells, 77%. Further, H₂O removed all Ca from Et/Ald fixed cells embedded in DEO. These studies show that S, which is present as SO₄ on the proteoglycan heparin, is readily detectable by X-ray EDS in fixed and embedded cells. An artifact of the technique is that weak anionic sites, which are most probably carboxyl groups on the proteoglycan, can bind the divalent cation Ca and cause spurious localization.     

Role of LIBS in Elemental Analysis of Psidium guajava Responsible for Glycemic Potential

Abstract

The present study deals with the detection of elements responsible for glycemic potential of ripe and unripe fruit peel aqueous extracts of Psidium guajava (P. guajava). Treatment with the aqueous extract of unripe fruit peel showed a significant fall of 17.5% (p<0.001) in blood glucose levels (BGLs) of normal rats during fasting blood glucose (FBG) test with a dose of 400 mg/kg bw. In sub‐diabetic rats, a fall of 19.8% (p<0.001) was observed with the same dose during a glucose tolerance test (GTT). The significant fall observed in FBG, post prandial glucose (PPG) and urine sugar levels of severely diabetic rats was 20.7%, 17.5% (p<0.05), and 66.6% (p<0.01), respectively. On the contrary, the effect of ripe fruit peel aqueous extract showed a regular rise of 24.4% (p<0.01) in BGL of normal rats and of 90.0% (p<0.001) in sub‐diabetic rats. Laser Induced Breakdown Spectroscopy (LIBS) has been used for the identification of elements responsible for the glycemic potential of fruit peel aqueous extracts of P. guajava. Concentration of Mg was found higher in unripe fruit peel aqueous extract than in the ripe fruit peel aqueous extract whereas the concentration of K was found lower in the extract of unripe fruit peel than in the extract of ripe fruit peel. Thus, the LIBS results help in defining the role of Mg and K in diabetes management. However, the concentrations of other minerals, like Na, N, O, and C, are nearly the same in both of the extracts.     

Physiological and histopathological responses of Porcellio laevis (Isopoda,Crustacea) as indicators of metal trace element contamination

This study was designed to assess the impact of the mixture of cadmium (Cd) and zinc (Zn) on the bioaccumulation and the ultrastructural changes in the hepatopancreas of Porcellio laevis (Latreille, 1804) after 4 weeks of exposure to contaminated Quercus leaves under laboratory conditions. For each metal, four concentrations were used with four replicates for each concentration. Metal concentrations in the hepatopancreas and the rest of the body were determined using atomic absorption spectrometry. From the first week until the end of the experiment, a weight gain in P. laevis was observed particularly between the first and the end of exposure from 93.3 ± 18.22 mg fw to 105.22 ± 16.16 mg fw and from 106.4 ± 22.67 mg fw to 125.9 ± 23.9 mg fw for Mix1 and Mix4, respectively. Additionally, the determined metal trace elements (MTE) concentrations in the hepatopancreas were considerably higher compared to those in the rest of the body and seem to be dose‐dependent. Using transmission electron microscopy (TEM), some alterations were highlighted in the hepatopancreas. The main observed alterations were (a) the destruction of the microvilli border in a considerable portion of cells, (b) the increase of the lipid droplets with different shapes and sizes, (c) the increase in the number of the mitochondria, and (d) the appearance of TE in the form of B‐type granules. The obtained results confirmed the ability of P. laevis to deal with high amounts of MTE, suggesting its possible use in future soil's biomonitoring programs.     

Cytochemistry of fat body trophocytes and ovaries of workers and queens of Melipona quadrifasciata anthidioides (Hymenoptera: Apidae: Meliponini) during vitellogenesis

ABSTRACT The fat body (FB) of insects is where yolk proteins are synthesized. Therefore, relationships between the FB and oogenesis were studied in nurse workers, virgins, and physogastric queens of Melipona quadrifasciata anthidioides, a stingless bee in which the workers produce and lay eggs while provisioning brood cells. The relationships between FB and oogenesis, as well as the routes of materials from hemolymph to the oocytes, were studied through the cytochemical detection of lipids by osmium imidazole (OI), carbohydrates by ruthenium red (RR) and basic proteins by ammoniacal silver (AS). The results show differences in the presence of the studied materials in FB trophocytes and ovary of the classes of females studied and oogenesis phases. Material that tested positive for the treatments was detected among the classes of individuals studied in both, trophocytes and oocytes, and in the route of those materials from hemolymph to the oocytes. The differences found among the individual classes indicate relationships with the nutrition and adaptation to the parsimonious use of nutrients in the metabolism of reproduction. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

Perivitelline space: does it play a role in blocking polyspermy in mammals?

The perivitelline space of mammalian oocytes changes in size and composition during preimplantation development. Often overlooked in the past, this space contains a hyaluronan-rich extracellular matrix prior to fertilization and a cortical granule envelope following release of the cortical granules at fertilization. The hyaluronan-containing matrix of unfertilized oocytes is well developed in some species such as opossums and humans but is scant in rodents including the hamster and mouse. The significance of the hyaluronan-rich matrix, which attaches to the plasma membrane of the oocytes, is not fully understood. However, hyaluronan, which can inhibit membrane fusion, is present in the perivitelline space (PVS) of unfertilized oocytes and must be negotiated by the fertilizing sperm. Following fertilization, the cortical granule envelope forms as the cortical granules disperse, thereby causing the PVS to increase significantly in size. Calcium is important in the dispersion of the cortical granules following exocytosis. Once formed, the cortical granule envelope in some species is about the same thickness as the zona pellucida, but it is not readily visualized unless it is stained with fluorescent probes or examined ultrastructurally after following stabilization with ruthenium red. The envelope contains proteins that remain in the PVS until the time of blastocyst hatching. Although little work has been done on the functions of the cortical granule envelope, several studies are consistent with the idea that it plays a role in blocking polyspermy. While nicotine increases polyspermy in sea urchins, its effects on polyspermy in human smokers have not been characterized, but could be addressed in human in vitro fertilization labs.     

The effect of water on the acid-base properties of new and used IC engine lubricating oils

The absolute values of total base number (TBN) were determined for several sets of both fresh and used engine oil samples. Basic and acidic compounds from these oils have been extracted into water, 7% sea water and ethanol-water (1:1, v/v) mixtures. The extracts have been determined by potentiometric titration as pH extracts, TBN (water extract) and alkalinity. The oil formulations are not resistant to the presence of water but basic additives in fresh oil are more resistant than those for used oils. The percentage of TBN extractable into water significantly increases with service life. The presence of significant amounts of water in lubricating oil is serious and should form part of any lubricant condition monitoring system. The case is argued for both the use of dehydration canisters in oil systems and for the development of a new method of water content determination for used formulated lubricating oils which is quick, accurate and suitable for automated condition monitoring systems. The pH of aqueous extracts for condition monitoring of lubricating oils is shown to be only meaningful for TBN values of 2 or less.     

Ultrastructure of the bullfrog (Lithobates catesbeianus‐Shaw, 1802) oviduct in different seasons

The bullfrog (Lithobates catesbeianus) is an exotic animal that adapted perfectly to the climatic conditions of Brazil after introduction in the country. These climatic conditions favor the reproduction and fattening of bullfrogs. However, the development of the bullfrog is significantly reduced in colder regions because low temperatures affect the secretion of gonadotropins and change the sensitivity of the germinal epithelium to gonadotropic hormones. Thus, the temperature and photoperiod are abiotic factors that influence the life cycle of this species, especially with regard to reproduction. Samples from different regions of the oviducts (the infundibulum, magnum, and isthmus) of 9 bullfrogs were collected in the different seasons of the year. The samples were fixed and processed for analysis with scanning and transmission electron microscopy. A comparison of the morphology of the oviducts collected during different seasonal periods suggests that the frogs have greater reproductive potential in the spring due to the development of the structures related to the oviduct, presence of developed cilia, and electron‐dense granules. However, seasonal changes were noted in the animal throughout the year in preparation for reproduction during the spring and summer. Microsc. Res. Tech. 76:523–532, 2013. © 2013 Wiley Periodicals, Inc.