Maria Madalena Pessoa Guerra

Cytotoxic effect and apoptosis induction by Bothrops leucurus venom lectin on tumor cell lines

Neoplastic transformation is the abnormal proliferation of cells. These transformations are often related to changes in cell surface glycoconjugates which can be detected by lectins. We evaluated the anti-tumor potential of BlL, a galactoside-binding lectin isolated from Bothrops leucurus venom as well as its cytotoxicity and hemolysis activity. The phosphatidylserine externalization and mitochondrial membrane potential were also determined. BlL exhibited cytotoxic activity against all tumor cell lines tested by induced phosphatidylserine externalization and mitochondrial depolarization, indicating cell death by apoptosis.

Vitamin E (Trolox) addition to Tris-egg yolk extender preserves ram spermatozoon structure and kinematics after cryopreservation

Several studies reveal that vitamin E acts as a cellular stabilizer of unsaturated lipids against oxidative deterioration, thus maintaining structural and functional integrity at the subcellular level. The objective of this study was to evaluate Vitamin E (Trolox) addition to freezing extender for ram spermatozoa. Semen samples were diluted in Tris-yolk egg medium without antioxidant (control group) and with Trolox in different concentrations (30, 60 and 120μM). After thawing (37°C/30s), samples were subjected to analysis for plasma membrane integrity (PMi), acrosome integrity (Aci), mitochondrial membrane potential (MMP), sperm kinematics, and ultrastructural integrity. The Trolox 60 and 120μM groups showed higher percentages of iPMs (P<0.05) when compared to the control group. Differences were observed among groups in sperm kinematic indicators (progressive motility, linearity, straightness, oscillation index, straight-line velocity and average path velocity), with higher values (P<0.05) for the Trolox 60 and 120μM groups. On ultrastructural assessment, Trolox addition at the three concentrations preserved spermatozoon head plasma membranes, while for the spermatozoon tail, plasma membrane preservation at 60μM was higher (P<0.05) than the other groups. The Trolox 60 and 120μM groups presented more mitochondrial ultrastructural preservation than the other groups (P<0.05). These results indicate that Trolox addition to Tris-egg yolk at 60 and 120μM provides greater structural integrity (plasma membrane and mitochondria) and kinematics for ram spermatozoa after cryopreservation.

The expression and localization of leptin and its receptor in goat ovarian follicles.

Leptin, a hormone that was originally identified in adipocytes, has been implicated in the regulation of ovarian folliculogenesis through endocrine, autocrine and/or paracrine mechanisms. The aim of this study was to investigate the expression patterns of leptin (LEP) and its receptor (LEPRb) in different types of ovarian follicular cells from goats. In small follicles, the expression levels of LEP were higher (P<0.001) in granulosa cells than in theca cells, cumulus cells and oocytes. The expression of LEP in granulosa cells was higher (P<0.001) in small follicles than in large follicles. In large follicles, the expression of LEPRb was higher (P<0.05) in granulosa cells than in theca cells, cumulus cells and oocytes. Higher expression (P<0.05) of LEPRb was detected in granulosa cells isolated from large follicles than in granulosa cells isolated from small follicles. Immunohistochemical analyses revealed the presence of the LEP and LEPR proteins in follicles at all stages of development. The most intense staining for LEP and LEPR was observed in the cytoplasm of oocytes and the surrounding granulosa cells. In conclusion, it was demonstrated that leptin and its receptor are expressed at both the mRNA and protein levels in goat ovarian follicles. Furthermore, the presence of a leptin signaling system in the caprine ovary suggests a potential regulatory role for leptin in follicular development and the maturation of goat oocytes.

Efeito da adição de trolox e pentoxifilina na motilidade, integridade do acrossoma e do DNA de espermatozoides equinos após descongelação

Three stallions were used to study the effect of trolox and pentoxifylline addition on the motility and integrity of acrossome and DNA equine spermatozoa after thawing. Tris-egg-yolg diluent with glycerol (5%) were used to freeze the semen samples in a freezing machine. The samples were thawed at 37oC during 30 seconds and treated with: T1=150µL of semen + 150µL of Tris; T2= 150µL of semen + 150µL of Tris +150mM/mL of trolox; T3= 150µL of semen + 150µL Tris +3.5mM of pentoxifylline; and T4= 150µL of semen + 150µL of Tris + 3.5mM of pentoxifylline + 150mM of trolox. After 0, 60, and 120 minutes of incubation (37oC), the samples were analyzed to motility, vigor, and integrity of acrossome and DNA. There was no difference (P>0.05) among treatments considering 0 and 60 minutes of incubation in all studied parameters. After 120 minutes of incubation, it was observed higher percentage (P<0.05) of cells with total and progressive motility in the samples of T2. It can be concluded that the trolox addition after thawing of equine semen preserved total and progressive motility of the sperm incubated at 37oC during 120 minutes.

Avaliação histomorfométrica do parênquima testicular de ratos adultos tratados com diferentes doses de ivermectina

The aim of this work was to evaluate the ivermectin effect on the testicular parenchyma through the daily spermatic production and the efficiency of the spermatogenesis in adult Wistar rats treated with different dosages (200, 400 and 600µg/kg) of ivermectin. Based on the histomorfometric evaluation, ivermectin had no deleterious effect on the testicular parenchyma and spermatogesis, which one was confirmed through the maintenance of the daily spermatic input and intrinsic income of spermatogenesis (PED/g/t), as well as by the maintenance of the testicular parenchyma structure. Based on the quantitative and qualitative results of spermatogenesis, it is possible to conclude that ivermectin does not have toxic-degenerative effect on the testicular parenchyma of adult Wistar rats.

Antimicrobial, Antiproliferative and Proapoptotic Activities of Extract, Fractions and Isolated Compounds from the Stem of Erythroxylum caatingae Plowman

In the study, we have examined the antitumor and antimicrobial activities of the methanol extract, the fractions, a fraction of total alkaloids and two alkaloids isolated from the stem of Erythroxylum caatingae Plowman. All test fractions, except the hexane fractions, showed antimicrobial activity on gram-positive bacteria and fungi. The acetate: methanol (95:5), acetate, chloroform and hexane fractions show the highest cytotoxicity activity against the NCI-H292, HEp-2 and K562 cell lines using MTT. The absence of hemolysis in the erythrocytes of mice was observed in these fractions and 6β-Benzoyloxy-3α-(3,4,5- trimethoxybenzoyloxy) tropane (catuabine B). Staining with Annexin V-FITC and JC-1 was used to verify the mechanism of action of the compounds of E. caatingae that showed cytotoxicity less than 30 μg/mL in leukemic cells. After 48 h of incubation, we observed that the acetate: methanol (95:5), acetate, and chloroform fractions, as well as the catuabine B, increased in the number of cells in early apoptosis, from 53.0 to 74.8%. An analysis of the potential of the mitochondrial membrane by incorporation of JC-1 showed that most cells during incubation of the acetate: methanol (95:5) and acetate fractions (63.85 and 59.2%) were stained, suggesting the involvement of an intrinsic pathway of apoptosis.

Prevalence and risk factors associated with Leptospira spp. infection in technified swine farms in the state of Alagoas, Brazil: risk factors associated with Leptospira spp. in swine farms.

The aim of this study was to determine the prevalence and to identify the risk factors associated with Leptospira spp. infection in technified pig farms in the state of Alagoas, Brazil. To compose sample for the prevalence study, 342 pigs were used (312 sows and 30 boars) proceeding from seven swine farms distributed in five districts of the state of Alagoas, Brazil. The infections serological diagnosis was performed by microscopic agglutination test. The risk factors analysis was performed using research questionnaires consisting of objective questions related to the breeder, the general characteristics of the property, and the productive, reproductive and sanitary management. Prevalence of 16.1% (55/342) of pigs seropositive was obtained. The associated risk factors were not performing quarantine (P = 0.003, OR = 5.43, CI = 1.79-16.41) and the use of artificial insemination (P = 0.023, OR = 3.38, CI = 1.18-9.66). A significant association of sow infection with the increased number of stillborn and mummified foetuses was found, as well as with the increased frequency of oestrus recurrence and the increased weaning-to-oestrus interval of seropositive sows. One might state that Leptospira spp. infection is disseminated in technified pig farms in the State of Alagoas, favouring reproductive failures and the impairment of zootechnical performance in these properties. The risk factors identified in this study are facilitators in the infecting agent dissemination and should be adjusted to control the disease in the herds studied.

Prevalência e fatores de risco associados à infecção por Toxoplasma gondii em granjas suinícolas tecnificadas no Estado de Alagoas

This study was aimed to determine the prevalence and to identify risk factors associated with infection by Toxoplasma gondii in pigs from commercial swine farms in the state of Alagoas, Brazil. To compose the sample size of prevalence 342 pigs were used, with 312 sows and 30 boars, from of seven swine farms and distributed in five districts of the state of Alagoas. The serological examination for the detection of anti-Toxoplasma gondii antibodies was achieved by Indirect Immunofluorescence (IFAT), using anti-pig IgG-conjugated to fluorescein isothiocyanate. The analysis of risk factors were performed by the application of questionnaires consisting of objective questions relating to the designer, the general characteristics of the property, the production, reproductive and health management. The prevalence was of 26,9% (92/342) of seropositive pigs. The associated factor was the boars introduction on farms in the last five years (p=0,014; OR=1,83; CI=1,13-2,96). It is concluded that infection with Toxoplasma gondii is widespread in commercial pig farms in the state of Alagoas, Brazil. The results suggest the control of cat population, the performance of cleaning and sanitation practices and laboratory testing to detect of infection by T. gondii in animals to be placed on the farms as measures to reduce the infection rates on region studied.

Oral administration of an anti-inflammatory does not compromise the efficacy of intra-testicular injection of zinc gluconate as a contraceptive for dogs

The objective was to determine whether the efficacy of zinc gluconate (Testoblock(®)) as a chemical contraceptive in male dogs was compromised in the presence of metamizole sodium (a nonsteroidal antiinflammatory/analgesic agent). Ten sexually mature mongrel dogs were assigned to two groups, a control group (n = 5) and a treated group (n = 5). Testoblock(®), a proprietary zinc-gluconate (13.1 mg zinc/ml) solution in a physiological vehicle, was injected into each testis (0.2-1.0 ml/testis, based on testis width). Half of the dogs (treated group) were also given metamizole sodium (also known as sodium dipyrone) orally (25mg/kg three times a day for 2 days), starting 2-3 h after testis injection. A physical examination and assessment of testis width, hematology, clinical chemistry (hepatic and renal function) and semen characteristics, were done immediately after treatment and then every 2 months for 180 days. There was no post-treatment scrotal biting or licking, although there was transient testicular swelling in both control and treated dogs during the first 3 days after injection. At 60 days after injection, all dogs were azoospermic. At 120 and 180 days, seven dogs had azoospermia and the remaining three (two control and one treated) had apparent aspermia (no ejaculate could be collected). There were no significant differences between groups for clinical findings or any aspect of hematology, renal, or hepatic function. In conclusion, giving metamizole sodium concurrent with an intra-testicular injection of a zinc-based solution did not interfere with chemical sterilization and it improved animal welfare.

Use of Fluorescent Microscopy for Sperm Quality of Penaeids

The present study is the first attempt to evaluate the use of fluorescent microscopy as a tool for determining the sperm qualitity in penaeid shrimp species. The probes propidium iodide and 6-carboxyfluorescein diacetate (CFDA) were used in combination to assess sperm quality of Litopenaeus vannamei captive broodstock and wild stocks of Farfantepenaeus subtilis and Litopenaeus schmitti. L. vannamei showed a significant higher amount of live cells (80.87%) after 93 days in captivity, when compared to animals entering (day 0) the maturation system (61.03%). The percentages of live sperm cells for wild-caught F. subtilis and L. schmitti were above 50% over the 12-month sample period in northeastern Brazil. These results demonstrate that the fluorescent microscopy can be used as a tool to determine the sperm quality in penaeid, allowing the evaluation of male performance in aquaculture systems, as well as to determine their reproductive cycle in fisheries research.