Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa     

Letícia Z. Oliveira  Vera F. M. Hossepian de Lima  Marcelo A. Levenhagen  Ricarda M. dos Santos  Terezinha I. Assump??o  José O. Jacomini  André F. C. de Andrade  Rubens P. de Arruda  Marcelo E. Beletti 《Journal of veterinary science (Suw?n-si, Korea)》2011,12(3):267-272

The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.  相似文献   

Molecular and serological detection of Babesia spp. in neotropical and exotic carnivores in Brazilian zoos     

André MR  Adania CH  Teixeira RH  Allegretti SM  Machado RZ 《Journal of zoo and wildlife medicine》2011,42(1):139-143

Large and small piroplasms have been observed in the blood smears of various wild carnivores, but few studies utilizing molecular characterization have been done. The goal of this present study was to investigate the presence of Babesia sp. by molecular and serologic techniques in exotic and neotropical carnivores maintained in captivity at Brazilian zoos. Blood and sera samples were collected from 146 Brazilian wild felids, 21 exotic felids, 1 genet (Genetta tigrina), 3 European wolves (Canis lupus), and 94 Brazilian wild canids in Brazilian zoos in the S?o Paulo and Mato Grosso states and in the Federal District. A total of 53 wild felids (31.74%) and 10 wild canids (10.31%) were seropositive for Babesia canis by Indirect Fluorescent Antibody Test (IFAT). Antibodies were detected in ocelots, little-spotted cats, margays, pampas cats, jaguars, pumas, jaguarundis, crab-eating foxes, and bush dogs. Babesia sp. DNA, with high similarity to B. leo, was detected in one pampas cat and one genet.  相似文献   

A simple and efficient method for isolating small RNAs from different plant species     

FlordeFátima Rosas-Cárdenas  Noé Durán-Figueroa  Jean-Philippe Vielle-Calzada  Andrés Cruz-Hernández  Nayelli Marsch-Martínez  Stefan de Folter 《Plant methods》2011,7(1):4

Background  

Small RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species.  相似文献   

Determination of trinexapac in wheat by liquid chromatography-electrospray ionization tandem mass spectrometry     

Hiemstra M  de Kok A 《Journal of agricultural and food chemistry》2003,51(20):5855-5860

A quantitative and confirmatory method for the analysis of trinexapac (free acid metabolite of trinexapac-ethyl) in wheat is described. Residues were extracted from wheat with acetonitrile in aqueous phosphate buffer (pH 7) overnight. The extract was directly injected into the HPLC system. Chromatographic separation was achieved on an octadecylsilica column, and detection was performed by negative ion electrospray ionization tandem mass spectrometry. The precursor ion of trinexapac [M - H](-) at m/z 223 was subjected to collisional fragmentation with argon to yield two intense diagnostic product ions at m/z 135 and 179, respectively. Accuracy and specificity for routine analysis of trinexapac were demonstrated. The validated concentration range was 10-200 microg/kg based on a 0.10 g/mL wheat sample extract. Recoveries were within the range of 71-94%, with associated relative standard deviations better than 10%. The limit of detection for trinexapac in wheat was estimated at 5 microg/kg. The method has been applied to a survey of 100 samples of wheat. In 46% of the samples analyzed, a quantifiable amount of trinexapac was detected, ranging from 10 to 110 microg/kg. It has been demonstrated that analyses of trinexapac accurately reflect the total amount of residues of the plant growth regulator, trinexapac-ethyl, in the wheat samples following field application. No residues of the parent compound, trinexapac-ethyl, in wheat were detected.  相似文献   

Rapid liquid chromatography tandem mass spectrometry assay to quantify plasma (-)-epicatechin metabolites after ingestion of a standard portion of cocoa beverage in humans     

Roura E  Andrés-Lacueva C  Jáuregui O  Badia E  Estruch R  Izquierdo-Pulido M  Lamuela-Raventós RM 《Journal of agricultural and food chemistry》2005,53(16):6190-6194

A rapid liquid chromatography electrospray ionization tandem mass spectrometry with negative ion detection method was developed and validated to determine cocoa flavonoid metabolites in human plasma and urine after the intake of a standard portion of a cocoa beverage. A chromatographic run time of only 9 min provided clear separation of all metabolites and internal standards. Samples were analyzed in a product-ion scan of m/z 289, 369, and 465 to identify the metabolites and in multiple reaction monitoring acquisition mode to quantify (-)-epicatechin ((-)-Ec) (289/ 245), (-)-epicatechin-glucuronide ((-)-EcG) (465/289), and (-)-epicatechin-sulfate ((-)-EcS) (369/289). One (-)-Ec-G and three (-)-Ec-S were identified and confirmed in urine as the major metabolites, and one (-)-Ec-G was the only metabolite present in plasma volunteers (n = 5) at a mean concentration of 625.7 +/- 198.3 nmol/L at 2 h after consumption of a cocoa beverage containing 54.4 mg of (-)-Ec.  相似文献   

Extracellular prolyl endoprotease from Aspergillus niger and its use in the debittering of protein hydrolysates     

Edens L  Dekker P  van der Hoeven R  Deen F  de Roos A  Floris R 《Journal of agricultural and food chemistry》2005,53(20):7950-7957

The observation that the bitterest peptides from casein hydrolysates contain several proline residues led us to hypothesize that a proline-specific protease would be instrumental in debittering such peptides. To identify the desired proline-specific activity, a microbiological screening was carried out in which the chromogenic peptide benzyloxycarbonyl-glycine-proline-p-nitroanilide (Z-Gly-Pro-pNA) was used as the substrate. An Aspergillus niger (A. niger) strain was identified that produces an extracellular proline-specific protease with an acidic pH optimum. On the basis of sequence similarities, we conclude that the A. niger-derived enzyme probably belongs to the S28 family of clan SC of serine proteases rather than the S9 family to which prolyl oligopeptidases belong. Incubating the overexpressed and purified enzyme with bitter casein hydrolysates showed a major debittering effect. Reversed phase HPLC analysis revealed that this debittering effect is accompanied by a significant reduction of the number of hydrophobic peptides present.  相似文献   

Discrimination of recombinant and pituitary-derived bovine and porcine growth hormones by peptide mass mapping     

Pinel G  André F  Le Bizec B 《Journal of agricultural and food chemistry》2004,52(3):407-414

Somatotropins, which are used in cattle for growth and lactating performances, are difficult to reliably detect because no direct method exists. Reversed-phase high-performance liquid chromatography (RP-HLC) coupled to electrospray ionization quadrupole mass spectrometry (ESI/MS) has been developed to separate and characterize the N-terminal peptides resulting from tryptic cleavage of natural and recombinant growth hormones from different species (bovine, porcine, and human) and suppliers. Conditions for tryptic digestion were optimized. This technique was found to be optimal to cleave efficiently the N-terminal peptide of the proteins without releasing too much noise from the matrix. Characterization of the peptides through ESI(+)-MS allowed natural and recombinant growth hormones from bovine and porcine species with N-terminal amino acid sequences differing from one amino acid residue to be discriminated. However, the studied human growth hormones had similar primary sequences that did not permit any discrimination between recombinant and natural forms, thus confirming the known identity of these hormones. Protein digestions with pepsin and chymotrypsin were also compared but were not conclusive due to the too small N-terminal peptides released after proteolysis.  相似文献   

Identification on commercialized products of AFLP markers able to discriminate slow- from fast-growing chicken strains   总被引：2，自引：0，他引：2  

Fumière O  Dubois M  Grégoire D  Théwis A  Berben G 《Journal of agricultural and food chemistry》2003,51(5):1115-1119

The European chicken meat market is characterized by numerous quality marks: "Label de Qualité Wallon" in Belgium, "Label Rouge" in France, denominations of geographical origin, organic agriculture, etc. Most of those certified productions have specifications requiring the use of slow-growing chicken strains. The amplified fragment length polymorphism (AFLP) technique has been used to search molecular markers able to discriminate slow-growing chicken strains from fast-growing ones and to authenticate certified products. Two pairs of restriction enzymes (EcoRI/MseI and EcoRI/TaqI) and 121 selective primer combinations were tested on individual DNA samples from chicken products essentially in carcass form that were ascribed as belonging to either slow- or fast-growing strains. Within the resulting fingerprints, two fragments were identified as type-strains specific markers. One primer combination gives a band (333 bp) that is specific for slow-growing chickens, and another primer pair generates a band (372 bp) that was found to be characteristic of fast-growing chickens. The two markers were isolated, cloned, and sequenced. The effectiveness and the specificity of the two interesting determinants were assessed on individuals of two well-known strains (ISA 657 and Cobb 500) and on commercialized products coming from various origins.  相似文献   

Contamination environnementale par les métaux et maladie de Parkinson     

J. Zayed  P. André  J. C. Panisset  S. Ducic  G. Campanella  M. Roy  G. Kennedy  C. Delisle 《Water, air, and soil pollution》1990,49(1-2):197-203

Parkinson's disease (PD) has been proposed to result from the interaction of aging and environment in susceptible individuals. The prevalence of PD in Quebec was 84 per 100 000 in 1985. The regional variation observed has been linked with several environmental factors. We intended to verify the existence of a relation between the development of PD with exposure to 3 metal pollutants: Fe, Mn, and Al. The choice of these metals rests on their potential presence in the environment caused by the industrial activity in the region studied, and on the fact that it theoretically favors the prevalence of the illness. Environmental exposure to these metals and the resulting risks were evaluated using red spruce as a bio-indicator. The metal concentrations in tree rings were determined by neutron activation analysis. These concentrations permitted a temporal estimation of contamination in each of the territories studied, from 1938 to 1987. The risk of environmental exposure was assumed to be proportional to the concentration of the metal. The calculated risks combined with the history of residence allowed a comparison between parkinsonians and controls. It appeared that environmental exposure to the metals was not significantly different between those two groups.  相似文献   

Comparative study of methods for DNA preparation from olive oil samples to identify cultivar SSR alleles in commercial oil samples: possible forensic applications     

Breton C  Claux D  Metton I  Skorski G  Bervillé A 《Journal of agricultural and food chemistry》2004,52(3):531-537

Virgin olive oil is made from diverse cultivars either mixed or single. Those ensure different tastes and typicity, and these may be also enhanced by the region of production of cultivars. The different olive oil labels correspond to their chemical composition and acidity. Labels also may correspond to a protected origin indication, and thus, such oils contain a given composition in cultivars. To verify the main cultivars used at the source of an olive oil sample, our method is based on DNA technology. DNA is present in all olive oil samples and even in refined oil, but the quantity may depend on the oil processing technology and oil conservation conditions. Thus, several supports were used to retain DNA checking different techniques (silica extraction, hydroxyapatite, magnetic beads, and spun column) to prepare DNA from variable amounts of oil. At this stage, it was usable for amplification through PCR technology and especially with the magnetic beads, and further purification processes were checked. Finally, the final method used magnetic beads. DNA is released from beads in a buffer. Once purified, we showed that it did not contain compounds inhibiting PCR amplification using SSR primers. Aliquot dilution fractions of this solution were successfully routinely used through PCR with different SSR primer sets. This enables confident detection of eventual alien alleles in oil samples. First applied to virgin oil samples of known composition, either single cultivars or mixtures of them, the method was verified working on commercial virgin oil samples using bottles bought in supermarkets. Last, we defined a protocol starting from 2 x 40 mL virgin olive oil, and DNA was prepared routinely in about 5 h. It was convenient to genotype together several loci per sample to check whether alleles were in accordance with those of expected cultivars. Thus, forensic applications of our method are expected. However, the method needs further improvement to work on all oil samples.  相似文献