Effect of storage conditions on the LDL effectiveness in ovine sperm cryopreservation.

The present study evaluated the effect of storage conditions on the LDL efficacy for cryopreserving ovine sperm. In this way, we compared egg yolk extender with three different forms for LDL storing, LDL diluted in Tris-glucose extender and stored in frozen (i) and freeze-dried (ii) states and LDL stored pure and added into the extender prior to use (iii). We also tested the effect of two storage temperatures (-20 and -80 °C) and three storage times (30, 60, 120 d). Frozen and freeze-dried extenders containing LDL, as well as LDL stored pure, improved post-thaw sperm quality. Storage temperatures did not influence negatively the cryoprotectiveness of LDL extenders. Furthermore, lyophilised LDL extenders stored at -20 °C were more effective in preserving sperm longevity than the other extenders stored at -20 °C. Finally, LDL extenders stored for 30 and 120 d were more efficient than 60 d in preserving ram sperm freezability.

Effects of packaging, mineral oil coating, and storage time on biogenic amine levels and internal quality of eggs.

This study was carried out with the aim of evaluating the effects of mineral oil application on eggshells and the use of plastic packages with lids on the physical-chemical and microbiological quality and biogenic amine contents of eggs stored under refrigeration for up to 125 d. A total of 1,920 eggs from 46-wk-old Hyline W36 laying hens were randomly distributed into 4 groups soon after classification: (i) 480 eggs were stored in pulp carton tray packages; (ii) 480 eggs were stored in plastic packages with lids; (iii) 480 eggs were stored in carton packages after the application of mineral oil; and (iv) 480 eggs were stored in plastic packages with lids after the application of mineral oil. The internal quality was measured by Haugh units, by the counts of mesophilic and psychrotrophic microorganisms, by the most probable number of total and thermal-tolerant coliforms, by the counts of molds and yeasts, by the analysis of Salmonella spp. and Staphylococcus spp., and by the levels of biogenic amines in the egg yolk and albumen. The application of mineral oil to the eggshell resulted in higher Haugh unit values throughout storage, and the use of plastic packages altered the internal quality. The application of mineral oil and the use of packaging had no effects on the microbiological and biogenic amine results. Microbiological analyses showed the absence of Salmonella spp., Staphylococcus aureus, thermal-tolerant coliforms, and fungi. However, the highest counts of mesophilic (1.1 × 10(7) cfu/g) and psychrotrophic (6.7 × 10(7) cfu/g) microorganisms were recorded. The highest values of biogenic amines detected and quantified were putrescine (2.38 mg/kg) and cadaverine (7.27 mg/kg) in the egg yolk and putrescine (1.95 mg/kg), cadaverine (2.83 mg/kg), and phenylethylamine (2.57 mg/kg) in the albumen. Despite these results, the biogenic amine levels recorded were considered low and would not be harmful to consumer health.

Bioactive amines and internal quality of commercial eggs.

The physicochemical and microbiological qualities of commercial eggs produced by layer hens of different ages (approximately 30 and 60 wk old) were submitted to storage under room temperature or refrigeration for 28 d. A total of 600 eggs were subjected to microbiological analyses of their inner contents and another 600 to a determination of Haugh units (HU) and bioactive amine content. A decrease in the quality of the inner contents of the eggs was observed during the experiment, mainly in the eggs from the 60-wk-old layers, which presented the worst HU values when stored at room temperature (P < 0.05). Microbiological analyses showed an absence of Salmonella spp., Staphylococcus aureus, and coliforms, either total or thermal-tolerant; however, low counts of other Staphylococcus species, Enterobacter spp., Pseudomonas spp., mesophilic aerobic bacteria, and fungi were also recorded. The chromatographic analysis of bioactive amines detected the presence of phenylethylamine in all albumens (38.0 mg/kg) and spermidine in the yolks (1.02 mg/kg). It was concluded that the age of the hens and the time and temperature of storage influenced the quality parameters of the eggs (P < 0.05). Furthermore, despite the low levels of microbial contamination found, phenylethylamine was detected in the albumen. It was not possible to establish index of quality of eggs using bioactive amines present in the yolk and albumen of eggs.

Species-specific detection of venom antigens from snakes of the Bothrops and Lachesis genera.

Venom-insoluble adsorbents were employed to absorb out the cross-reacting antibodies from monovalent polyclonal antivenoms. The absorbed antivenoms were tested by enzyme-linked immunosorbent assay against homologous and heterologous venoms and showed species-specificity throughout a range of venom concentrations. The same absorbed antisera were used in immunoblots under non-reducing conditions as probes to reveal species-specific antigens. In all cases studied this was achieved. The range of mol. wts of specific antigens was between 20,000 and 120,000, approximately. Venoms added to human serum experimentally were specifically detected by their homologous absorbed antivenom antibodies. The work here described could be important in the development of diagnostic assays for envenomings involving snakes from the Bothrops and Lachesis genera.

The effect of stepwise iodination on biological properties of Bothrops jararaca venom.

By titrating 5 mg of native venom with aliquots of a 2 x 10(-2) M iodine monochloride solution, neutralization of lethality by the incorporation of iodine was found with 200 +/- 5 microliters of solution, and above, up to 310 +/- 10 microliters, when saturation with iodine was attained. Doses up to 1500 micrograms (equivalent to 32 LD50 of native venom), where injected i.p. in mice without lethal effects. Proteolytic, phospholipase A2 and esterolytic activities were greatly reduced, but a low activity persisted even in fully iodinated samples. Direct hemolysis was markedly inhibited, and incapacity to coagulate fibrinogen and horse plasma was also observed in the iodinated samples. Hemorrhage and necrosis in rat skin, caused by 20 micrograms of iodinated venom were not elicited by doses up to 120 micrograms of iodinated anavenom. In mice, the myonecrosis that resulted from direct i.m. injection of native venom, and the massive hemorrhage caused by 5 LD50 doses injected i.p. were abolished by venom iodination. Blood congestion in liver, spleen, kidneys, and lungs, almost disappeared with iodination to the level of neutralization, and was barely seen with venom samples iodinated to saturation. The clinical signs of impaired physical activity, appearing in mice injected with 700 to 1500 micrograms of the iodinated anavenom were intensified by captopril and attenuated by epinephrine.

Detoxification of the T2 fraction from a scorpion (Tityus serrulatus, Lutz and Mello) venom by iodination and some immunogenic properties of the derivatives.

The iodination of the T2 fraction abolished its lethal capacity, and doses up to 30 times the LD50 were injected i.p. in mice without noticeable toxic effects. The modified fraction retained its immunological properties. Antibodies generated against the iodinated T2 fraction were also reactive toward the native T2 fraction, T1 fraction and the whole soluble venom.

Development of an ELISA to assess the potency of horse therapeutic polyvalent antibothropic antivenom.

The objective of this study was the search for a suitable venom antigen to be used in an in vitro alternative immunoassay, to the standard antivenom neutralization assay using mice. Bothrops jararaca venom was fractionated in DEAE-Sephacel columns and the fractions were tested for a correlation between antibody capture enzyme linked immunosorbent assay (ELISA) absorbance values and the 'in vivo' antivenom potency. Individual antivenoms from 14 horses and 15 separate FUNED polyspecific Bothrops ampouled antivenoms (final product) were used. Fractions showing the higher correlations were further chromatographed in a Sephadex G-75 column and again tested for the correlation. Two fractions with haemorrhagic activity displayed a correlation of r = 0.77 and r = 0.8 against the individual horse antivenom sera and of r = 0.79 and r = 0.8 for the ampouled antivenom. For all results p < 0.001. Two other fractions with phospholipase A2 activity showed a correlation of r = 0.66 (p < 0.01) and r = 0.56 (p < 0.03) against the individual horse antivenom sera. Electrophoresis results show a similar composition for both antigens with haemorrhagic activity. Results indicate that the fractions purified would be suitable for the desired objective of this study.

Inhibition of mutalysin II, a metalloproteinase from bushmaster snake venom by human alpha2-macroglobulin and rabbit immunoglobulin.

Mutalysin II is a 22.5-kDa zinc endopeptidase isolated from Lachesis muta muta snake venom. In order to determine whether the inhibitors human alpha2-macroglobulin (alpha2-M) and rabbit antibody to mutalysin II share a common mechanism, we have investigated the inhibition of mutalysin II by these two different glycoproteins. The proteolytic activity of mutalysin II with dimethylcasein as substrate was completely inhibited by human alpha2-M and by a purified rabbit antibody to mutalysin II. The protection of fibrin(ogen) digestion by alpha2-M was slightly better than the protection offered by the antibody. In addition, the purified antibody reacted only with the metalloproteinase in bushmaster venom, as demonstrated by immunodiffusion. SDS-PAGE analysis of reduced samples showed that the interaction of mutalysin II with alpha2-M resulted in the formation of high molecular complex ( approximately 180000) and M(r) 90000 fragments generated by the venom enzyme. Also, fragments at 85 and 23 kDa were detected under non-reducing conditions after incubation of rabbit immunoglobulin with enzyme. Proteolysis of dimethylcasein as substrate revealed that the stoichiometry of inhibition was 1.0 mol of human alpha2-M and 1.5 mol of rabbit IgG antimutalysin II per mole of enzyme. Furthermore, dimethylcasein hydrolysis indicated that several viperid snake venoms, including Bothrops atrox, B. alternatus and Trimeresurus flavoviridis cross-reacted with the specific rabbit antibody to varying degrees.

Development of a species-specific ELISA for Brazilian pit-viper venoms.

Antigenic cross-reactivity between venoms of the genus Bothrops has been shown to be an extensive problem. However, some venom components are species-specific. In this study we have produced species-specific antivenoms against some members of the genus Bothrops. Monospecific rabbit antivenoms (IgG) were absorbed on venom affinity adsorbents. The species-specificity was tested by ELISA assays and immunoblots. The results of both assays showed complete species-specificity in some cases and highly increased species-specificity in others. These reagents can be used to determine the envenomating species in snake bite patients as an aid to improved serotherapy.

Generation of protective immune sera by Crotalus durissus terrificus venom detoxified by controlled iodination.

1. Whole soluble venom from the snake Crotalus durissus terrificus was detoxified by controlled iodination. Doses equivalent to 100 LD50 of the native venom were administered to mice, without signs of intoxication. 2. The non-toxic iodinated derivatives were able to stimulate antibodies in rabbits and horses within a short period (6 months) of immunization. Horse antisera attained titers of 0.5 to 0.9 mg/ml for protection against native venom. 3. Horse antisera obtained in horses from native and iodinated venom were run against both native and iodinated venoms, as antigens, in gel immunodiffusion. The precipitation lines showed total identity of the two types of sera.

Determination of the neutralizing potency of horse antivenom against bothropic and crotalic venoms by indirect enzyme immunoassay.

The use of ELISA to determine antisnake venom potency of horse immune sera should provide benefits of costs and reproducibility compared to in vivo assays. In the present investigation we evaluated the correlation between ELISA antibody levels and in vivo neutralization assays. For the indirect ELISA method, 0.016 micrograms/well of Bothrops jararaca or Crotalus durissus terrificus venom were used to coat the plates and 100 microliters/well of each sample of antibothropic or anticrotalic venom sera were used at 1:10,000 dilution. Sheep anti-horse IgG conjugated to peroxidase was added and the substrate H2O2/o-phenylenediamine produced the color that was read at 492 nm. A correlation coefficient of r = 0.97 was found for anticrotalic venom antibodies and no significant correlation was observed for antibothropic venom sera using 16 serum samples from immunized horses. However, when three antibothropic venom sera showing high in vivo neutralization potency and low absorbance in ELISA or high absorbance values and low in vivo protection were not included in the correlation analysis the coefficient value was r = 0.88. The correlation coefficient did not improve for all 16 antibothropic sera when a partially purified Bothrops jararaca venom fraction was used to coat the ELISA plates. The results indicate that ELISA could be used to determine the neutralizing potency of anticrotalic venom sera. For the antibothropic venom sera further studies are needed.

Natural, but not lyophilized, low density lypoproteins were an acceptable alternative to egg yolk for cryopreservation of ram semen.

The objective was to evaluate the suitability of using natural or lyophilized low density lipoproteins (LDL), in lieu of whole egg yolk, in extenders for cryopreserving ram semen. Once extragonadal sperm reserves were depleted in 10 fertile Santa Inês cross rams, two ejaculates per ram were collected for cryopreservation. Nine extenders were used: Tris-16% egg yolk extender with 5% glycerol as a control (T1), and substitution of whole egg yolk with 8, 12, 16 or 20% natural LDL (T2-T5, respectively), or with 8, 12, 16, or 20% lyophilized LDL (T6-T9). Semen was diluted to 100 × 10(6) sperm/mL, packaged into 0.25 mL straws, cooled, held at 5 °C for 3 h, and then frozen in liquid nitrogen vapor. Immediately after thawing (37 °C for 30 s), sperm total and progressive motility, and kinetic parameters were analyzed with computer assisted semen analysis (CASA). Percentage of sperm with plasma membrane functional integrity was assessed by the hypoosmotic swelling test (HOST), sperm membrane physical integrity with propidium iodide (PI), and acrosome integrity with FITC-PSA using an epifluorescent microscope. For all sperm end points, there was no difference between the control and natural LDL treatments (P > 0.05): total motility (T1: 20.9 ± 11.9 and average of T2-T5: 25.9 ± 13.6%; mean ± SD), progressive motility (T1: 6.6 ± 4.2 and average of T2-T5: 11.7 ± 7.5%), HOST(+) (T1: 23.7 ± 6.9 and average of T2-T5: 23.2 ± 8.7 %) and PI(-)/PSA(-) (T1: 13.8 ± 7.8 and average of T2-T5: 18.1 ± 7.8%). However, lyophilization was apparently unable to preserve the protective function of LDL; every sperm end point was significantly worse than in the control and natural LDL groups. We concluded that natural LDL was appropriate for cryopreserving ram semen, as it yielded results similar to those obtained with whole egg yolk.

Avaliação da qualidade interna de ovos de consumo pela pesquisa do teor de aminas bioativas / Evaluation of the internal quality of commercial eggs by searching of the bioactive levels

Com o objetivo de avaliar a qualidade dos ovos de consumo pela pesquisa dos níveis de aminas bioativas, foram coletados, pelos serviços de inspeção oficiais, 224 amostras de ovos provenientes de cinco regiões distintas do estado de Minas Gerais, durante o período de um ano. As aminas biogênicas (putrescina, cadaverina, feniletilamina, histamina e tiramina) e as poliaminas (espermidina e espermina) foram pesquisadas por cromatografia líquida de alta eficiência e detecção ultravioleta (CLAE/UV) após derivação pré-coluna com cloreto de dansila. Os resultados demonstraram que a putrescina estava presente, em baixas concentrações, em todas as amostras de gema e de albúmen. As demais aminas também foram detectadas, porém em menor frequência, e a espermina somente foi encontrada em uma amostra de albúmen. Foi concluído que os ovos de consumo produzidos no estado de Minas Gerais não são uma fonte considerável de poliaminas, importantes para o crescimento e a proliferação celular, e os baixos teores de aminas biogênicas, formadas pela descarboxilação de aminoácidos por enzimas bacterianas, não representam riscos à saúde do consumidor, o que indica que o ovo apresenta boa qualidade, tomando por base o critério de aminas bioativas.

In order to evaluate the quality of commercial eggs by searching the bioactive amine levels, 224 samples of eggs from the five regions of Minas Gerais State were collected during one year by the official inspection service. The biogenic amines (putrescine, cadaverine, phenylethylamine, histamine and tyramine) and the polyamines (spermidine and spermine) were determined by high performance liquid chromatography with ultraviolet detection (HPLC/UV) and pre-column derivatization with dansyl chloride. The results demonstrated the presence of putrescine in all samples of yolk and albumen, but in low concentrations. The other amines were also detected, however, with a lower frequency, and spermine was found only in one sample of albumen. It was concluded that the commercial eggs produced in Minas Gerais State are not a considerable source of polyamines, important for growth and cell proliferation; and low levels of biogenic amine, formed by decarboxylation of amino acids by bacterial enzymes, do not represent risks to consumer health, indicating that it has good quality, based on the bioactive amine criterion.

Validation of an HPLC-UV method for the identification and quantification of bioactive amines in chicken meat / Validação de metodologia por HPLC/UV para identificação e quantificação de aminas bioativas em carne de frango de corte

A high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method was validated for the study of bioactive amines in chicken meat. A gradient elution system with an ultraviolet detector was used after extraction with trichloroacetic acid and pre-column derivatization with dansyl chloride. Putrescine, cadaverine, histamine, tyramine, spermidine, and spermine standards were used for the evaluation of the following performance parameters: selectivity, linearity, precision, recovery, limits of detection, limits of quantification and ruggedness. The results indicated excellent selectivity, separation of all amines, a coefficient of determination greater than 0.99 and recovery from 92.25 to 102.25% at the concentration of 47.2mg.kg-1, with a limit of detection at 0.3mg.kg-1 and a limit of quantification at 0.9mg.kg-1 for all amines, with the exception of histamine, which exhibited the limit of quantification, of 1mg.kg-1. In conclusion, the performance parameters demonstrated adequacy of the method for the detection and quantification of bioactive amines in chicken meat.(AU)

Um método de cromatografia líquida de alta eficiência (CLAE) para pesquisa de aminas bioativas em carne de frango foi validado. Foi utilizado um sistema de gradiente de eluição com detector ultravioleta, após extração com ácido tricloroacético e derivação pré-coluna com cloreto de dansila. Os padrões de putrescina, cadaverina, histamina, tiramina, espermidina e espermina foram utilizados para avaliação dos seguintes parâmetros de desempenho: seletividade, linearidade, precisão, recuperação, limites de detecção, limites de quantificação e robustez. Os resultados mostraram excelente seletividade e separação de todas as aminas, coeficiente de determinação superior a 0,99, recuperação entre 92,25 e 105,25% na concentração 47,2mg.kg-1, limites de detecção de 0,3mg.kg-1 e limite de quantificação de 0,9mg.kg-1 para todas as aminas, com exceção da histamina, que apresentou o limite de quantificação mais alto, de 1mg.kg-1. Foi concluído que os parâmetros de desempenho demonstraram adequação do método para detecção e quantificação de aminas bioativas em carne de frango.(AU)

Diagnóstico do complexo teníase-cisticercose bovina em São João Evangelista, Minas Gerais, Brasil / Diagnosis of Bovine Taeniasis-Cysticercosis Complex in São João Evangelista, Minas Gerais, Brazil

Com o objetivo de diagnosticar a situação do complexo teníase-cisticercose bovina em Minas Gerais, Brasil, foi selecionado o município de São João Evangelista, onde foram coletadas amostras de sangue de 339 bovinos em 15 propriedades rurais, sorteadas aleatoriamente. Em cada propriedade, foi aplicado um questionário socioeconômico para a análise de fatores que favorecem a manutenção do complexo teníase-cisticercose bovina. Foi realizado também o diagnóstico de teníase humana por meio de exame coproparasitológico dos habitantes das propriedades. Encontrou-se a prevalência de 4,1% para cisticercose bovina e a frequência de 2,94% para teníase humana. Entre os fatores de risco para a manutenção do complexo teníase-cisticercose analisados, foi observada uma relação estatisticamente significativa (P=0,042) entre a ocorrência de cisticercose bovina e a ingestão de carne malpassada pelos entrevistados. Concluiu-se que a cisticercose bovina está presente no município de São João Evangelista, MG, em índices considerados endêmicos, sendo o consumo de carne malpassada e não inspecionada o principal fator de risco para a manutenção do complexo teníase-cisticercose, o que reforça a necessidade da adoção de medidas de controle com contínua vigilância epidemiológica e sanitária.

In order to diagnose the situation of bovine taeniasis-cysticercosis complex in the state of Minas Gerais, Brazil, the city of São João Evangelista was selected, and blood samples were collected from 339 cattle in 15 randomly selected farms. A socioeconomic questionnaire was filled in each property for the analysis of the factors that favor the maintenance of the taeniasis-cysticercosis complex. Additionally, there was the diagnosis of human taeniasis verified by stool examinations of the properties' inhabitants. A prevalence of 4.1% for bovine cysticercosis and the frequency of 2.94% for human taeniasis were found. Among the risk factors, a statistically significant relation (p = 0.042) was found between the occurrence of bovine cysticercosis and the ingestion of undercooked meat. It was concluded that bovine cysticercosis is broadly distributed in the city of São João Evangelista, with rates considered endemic, being the consumption of raw and not-inspected meat the main risk factors for the maintenance of complex taeniasis-cysticercosis, reinforcing the need to adopt control measures with continuous epidemiological and health surveillance.

Aminas bioativas e qualidade da carne de frangos de corte / Bioactive amines and meat quality of broiler chickens

Com o objetivo de avaliar a qualidade da carne de frangos de corte mediante pesquisa dos níveis de aminas bioativas, foram coletadas, pelos serviços de inspeção oficiais, 160 amostras de carcaças provenientes de cinco regiões distintas do estado de Minas Gerais, durante o período de um ano. As poliaminas (espermidina e espermina) e as aminas biogênicas (putrescina, cadaverina, histamina, tiramina) foram pesquisadas por cromatografia líquida de alta eficiência e detecção ultravioleta (CLAE/UV). Os resultados encontrados demonstraram a presença das poliaminas, espermidina e espermina, em todas as amostras, em concentrações médias de 3,56mg/100g e 5,72mg/100g, respectivamente. Em todas as amostras foram detectadas, em concentrações muito baixas, as aminas putrescina, cadaverina, histamina e tiramina. Foi concluído que a carne de frangos de corte produzida no estado de Minas Gerais é uma fonte de poliaminas, importantes para o crescimento e a proliferação celular, e que os baixos teores de aminas biogênicas encontrados não representam riscos à saúde do consumidor, indicando que esse tipo de carne apresenta boa qualidade, tomando por base o critério de aminas bioativas.

In order to evaluate the meat quality of broiler chickens by searching the bioactive amines level, 160 samples of carcass from the five regions of the Minas Gerais State, were collected during one year by the official inspection service. The poliamines (spermidine and spermine) and the biogenic amines (putrescine, cadaverine, histamine and tyramine) were determined by high performance liquid chromatography with ultraviolet detection (HPLC/UV). The results demonstrated the presence of polyamines spermidine and spermine in all samples, at mean concentrations of 3.56mg/100g and 5.72mg/100g, respectively. The biogenic amines putrescine, cadaverine, histamine and tyramine were also found, but in low concentrations. It was concluded that the chicken broiler meat produced in Minas Gerais state is a source of polyamines, important for growth and cell proliferation; and that the biogenic amine levels found were low, and do not represent risks to consumer health, indicating that it has good quality, based on the criterion of bioactive amine.

Cryoprotection effectiveness of low concentrations of natural and lyophilized LDL (low density lipoproteins) on canine spermatozoa / Eficiência de baixas concentrações de lipoproteínas de baixa densidade natural e liofilizada na crioproteto de espermatozoides caninos

The aim of this study was to evaluate the use of low concentrations of natural and lyophilized low density lipoprotein (LDL) from hen's egg yolk for cryopreservation of canine semen. Different ammonium sulphate concentrations were tested to extract LDL from egg yolk. The yolk was centrifuged, and LDL was isolated using 10, 20, 40, 45, or 50 percent ammonium sulphate solution (ASS). The LDL-rich floating fraction was collected for chemical characterization. Dry matter content was lowest (P<0.05) in the LDL extracted with the 50 percent ASS. The purification of LDL increased in association with increasing ammonium sulphate concentrations. SDS-PAGE showed that the 50 percent ASS solution yielded a purer fraction of LDL from egg yolk. For semen cryopreservation, TRIS extender was used replacing 20 percent egg yolk (control) by natural or lyophilized LDL using 1, 2, and 3 percent (w/v). Semen was centrifuged (755Xg for 7 min), diluted with one of the extenders, packed into 0.5mL straws (100x106 sperm/mL), and placed in a programmable cryopreservation machine. Thawed semen (37°C/ 30s) was analyzed for sperm motility, morphology, and by the hypoosmotic and epifluorescence tests (CFDA/ PI). Natural LDL extracted with 50 percent ASS was as effective as whole egg yolk to preserve canine frozen sperm when using low concentrations. The lyophilized LDL, mainly in the two higher concentrations tested (2 and 3 percent), was unsuitable to maintain the effectiveness of the LDL cryoprotective effect on dog sperm...

O objetivo deste estudo foi avaliar o uso de baixas concentrações da lipoproteína de baixa densidade (LBD) extraída da gema do ovo de galinha, nas formas natural e liofilizada, na criopreservado do sêmen canino. Diferentes concentrações de sulfato de amônio também foram testadas na extrato da LBD da gema do ovo. A gema foi centrifugada, sendo a LBD isolada usando-se soluto saturada de sulfato de amônio (SSA) nas concentrações de 10, 20, 40, 45 e 50 por cento. A frado rica em LBD foi coletada para caracterizado química. O conteœdo de matéria seca foi menor (P<0,05) na LBD extraída com SSA 50 por cento. A pureza da LBD melhorou medida que se aumentou a concentrado de SSA utilizada. SDS-PAGE mostrou que a SSA 50 por cento produziu uma frado mais pura de LBD oriunda da gema do ovo. Para o congelamento de sêmen, o meio diluidor TRIS teve a gema do ovo a 20 por cento (controle) substituída pela LBD a 1, 2 e 3 por cento (p/v), nas formas natural e liofilizada. O sêmen foi centrifugado (755xg por 7min), diluído em um dos meios diluidores em teste e envasado em palhetas de 0,5mL (100x106 sptz/mL), sendo congelado em máquina de congelamento programável. O sêmen descongelado (37°C/30s) foi analisado quanto motilidade e morfologia espermática e nos testes hiposm-tico e de epifluorescência (DACF/IP). A LBD natural extraída com SSA 50 por cento foi tão eficiente quanto a gema do ovo na preservado do espermatozoide canino congelado nas baixas concentrações testadas. A LBD liofilizada, principalmente as duas maiores concentrações (2 e 3 por cento), não foi adequada para manter o efeito crioprotetor da LBD sobre o espermatozoide canino...

Biocompatibility evaluation of hydroxyapatite/collagen nanocomposites doped with Zn+2.

In this work, novel composites based on calcium phosphates (CaP)/collagen (COL) doped with Zn(+2) have been synthesized. They were characterized by SEM coupled to EDS microprobe in order to evaluate their morphology and chemical composition, respectively. The biocompatibility of these synthetic CaP/COL nanocomposites doped and undoped with Zn(+2) was investigated through osteoblast cell culture assay. Calcium phosphates were produced via aqueous precipitation routes where two different phases were obtained, hydroxyapatite (HAP) and biphasic hydroxyapatite-betatricalcium phosphate (HAPbetaTCP). In the sequence, the type-I collagen (COL) was added to the inorganic phase based on calcium phosphate and the mixture was blended until a homogenous composite was obtained. Zn(+2) aqueous solution (1.0 wt%) was used as the doping reagent. The cell viability and the alkaline phosphatase production of osteoblasts in the presence of the composites were evaluated and compared to control osteoblasts. Also, the biocompatibility of the composite was investigated through cell morphological analysis using optical microscopy of osteoblasts. All experiments were performed in triplicates (n = 3) from three different experiments. They were analyzed by variance test (ANOVA) and Bonferroni's post-test with differences statistically significant at p < 0.05. The results showed that the CaP/COL composites doped and undoped with Zn(+2) did not present alterations in cell morphology in 72 h and had similar cell viability and alkaline phosphatase activity to the control. All the tested CaP/COL composites showed adequate biological properties with the potential to be used in bone tissue replacement applications.

Identification of Staphylococcus spp. isolated during the ripening process of a traditional minas cheese / Identificação de Staphylococcus spp. isolados durante o processo de maturação de um queijo-de-minas tradicional

The population dynamics of Staphylococcus spp. was studied during the ripening of Canastra Minas cheese at three farms located in the State of Minas Gerais, Brazil. The presence of coagulase (coa), thermonuclease (nuc), and enterotoxin (sea, seb, sec, and sed) genes was investigated in Staphylococcus strains isolated during the 60-day cheese-ripening period. The presence of the staphylococcal enterotoxins A, C, and D was also investigated in the cheese samples. Cheese samples that were matured for 0, 7, 15, 30, and 45 days presented staphylococci counts from 10³ to 10(8)cfu/g. All isolates considered coagulase-positive by physiological tests had the coa gene. However, no association was observed between the results obtained with biochemical tests and those obtained by PCR using gene-specific primers for coagulase-negative strains. Coagulase and thermonuclease genes occurred simultaneously in 41.3 percent of Staphylococcus spp. tested. None of the investigated Staphylococcus strains expressed enterotoxins SEA, SEB, SEC, and SED. Enterotoxins A, C, and D were not detected in any of the cheese samples.

Estudou-se a dinâmica das populações de Staphylococcus spp. durante a maturação do queijo Canastra, em três fazendas localizadas no estado de Minas Gerais. A presença dos genes que codificam para a produção das enzimas coagulase (coa), termonuclease (nuc) e produção de enterotoxinas (sea, seb, sec e sed), em linhagens de Staphylococcus isoladas durante os 60 dias de maturação do queijo foi analisada. Também foi investigada a presença de enterotoxina estafilocócica A, C e D nas amostras de queijo. As amostras de queijo com 0, 7, 15, 30 e 45 dias de maturação apresentaram contagens de Staphylococcus spp. entre 10³ e 10(8)ufc / g. Todos os isolados coagulase positivo nos testes fisiológicos apresentaram o gene coa. Não foi observada associação entre os resultados obtidos com os testes bioquímicos e aqueles obtidos com a PCR usando iniciadores gene-específicos para linhagens coagulase negativa. Os genes da coagulase e termonuclease ocorreram simultaneamente em 41,3 por cento dos Staphylococcus spp. testados. Nenhum dos isolados de Staphylococcus apresentou os genes que codificam para a produção das enterotoxinas SEA, SEB, SEC ou SED. As enterotoxinas A, C ou D não foram detectadas em nenhuma das amostras de queijo analisadas.

Stepwise iodination. A general procedure for detoxification of proteins suitable for vaccine development and antiserum production.

By gradual incorporation of stable iodine into toxins and whole venoms it is possible to abolish completely the lesion and lethal properties of the native components. Allergen extracts can be turned anallergic. Physiological proteins with strong biological activity can also be rendered innocuous. Tyrosine and histidine are the reactive groups that incorporate the hapten. Within the same batch of protein, there is a defined ratio hapten/protein to achieve the desired modified properties of the derivative. The iodinating solutions are easily prepared, can be accurately standardized and have unlimited shelf lives. The derivatives are obtained in a short time. The cost of the entire procedure is very low. The method was applied to tityustoxin and whole venom of the scorpion Tityus serrulatus; crotoxin and whole venom of Crotalus durissus terrificus; to five bothropic venoms; to allergenic extracts of Schistosoma mansoni; to cholera and tetanus toxin; and to insulin, kallikrein and tonin. The derivatives obtained were stable, did not show any reversion to toxicity, generated antibodies against the native antigens and gave active protection when injected into animals. No local or systemic side effects were observed, even after prolonged use. The injections were also apparently painless. By extensive haptenization self proteins can be rendered non-self, able to generate antibodies against both the derivative and the native unmodified protein, and iodination was very convenient for this purpose. A new schedule for immunization, only feasible with completely toxoided venoms is presented. It is based on a clonal expansion induced by a small dose, followed by an exponential saturation dose of the same toxoid. The attainment of higher levels of antibodies against the native antigen, in the generated sera is unmatched by other procedures.