An improved method for the purification of the trichothecene deoxynivalenol (vomitoxin) from Fusarium graminearum culture

It was hypothesized that a simplified and efficient strategy could be developed for large-scale production and purification of the mycotoxin deoxynivalenol from Fusarium graminearum rice cultures for toxicological studies. F. graminearum R6576 was cultured on rice and extracted with methanol, and the extract was concentrated and subjected to silica gel low-pressure liquid chromatography (LPLC) under a hexane-acetone gradient system. Deoxynivalenol isolation was monitored by thin-layer chromatography, and fractions containing deoxynivalenol were pooled, concentrated, and applied to a second LPLC column under the same conditions. An enriched deoxynivalenol fraction was obtained, which yielded a crystalline material. Repeated crystallization yielded spectroscopically pure deoxynivalenol. The identity of this compound was confirmed by HPLC comparison to an authentic deoxynivalenol standard, FABMS analysis, and comparison of the (1)H and (13)C NMR spectra with published data. This simplified purification scheme eliminated many laborious steps and equipment previously required to obtain gram quantities of crystalline deoxynivalenol for biological testing in animal models.     

Production and specificity of polyclonal antibodies to hexanal-lysine adducts

Hexanal content is a widely used index of lipid oxidation in foods. The objectives of this study were to develop antibodies to hexanal-lysine adducts, devise an ELISA, and characterize antibody specificity. Hexanal was made immunogenic by covalent attachment to lysine side chains of bovine serum albumin via reductive alkylation. Polyclonal antibodies had antiserum titers as high as 6.15 x 10(5). A competitive indirect ELISA was developed with a detection limit of 0.7 ng of hexanal/mL. Antibodies were carrier-independent, reacting with hexanal conjugates of several proteins but not with the corresponding native proteins. Cross-reactivities with chicken serum albumin conjugates of n-heptanal, n-pentanal, and n-octanal were 86. 3, 11.8, and 2.2%, respectively. Antibodies reacted strongly with hexanal-modified lysine and hexanal-modified epsilon-aminocaproic acid but did not recognize free amino acids or free hexanal. It may be feasible to use this ELISA to monitor lipid oxidation in food provided hexanal is alkylated to a carrier protein prior to analysis.     

Studies on the Intracellular Ca2+ Concentration of Frozen–Thawed Dog Spermatozoa: Influence of Equex from Different Sources, Two Thawing Diluents and Post-thaw Incubation in Capacitating Conditions

The addition of 0.5% (v/v) of Equex STM Paste (Nova Chemical Sales, Scituate Inc., MA, USA), whose active ingredient is sodium dodecyl sulphate (SDS), to a Tris–egg yolk extender was demonstrated to improve the longevity of frozen–thawed dog spermatozoa during in vitro incubation at 38°C. The aim of the first experiment was to compare the effects of two SDS‐containing compounds, Equex STM Paste and Equex Pasta (Minitüb, Tiefenbach, Germany), when added to a Tris–egg yolk based extender, on the post‐thaw longevity of dog spermatozoa, as well as on the intracellular Ca²⁺ concentration of spermatozoa, during post‐thaw incubation at 38°C. The post‐thaw sperm survival and longevity, as well as the quality of the sperm movement, were significantly better when using Equex STM Paste. Such prolonged sperm longevity, however, was associated to a higher intracellular Ca²⁺ concentration in a large subpopulation of the live spermatozoa. A second experiment was aimed to evaluate the effects of sperm dilution immediately post‐thaw with a Tris buffer containing glucose or fructose. The two Tris buffers were no different for any of the sperm parameters studied. The aim of a third experiment was to evaluate the sperm longevity, motility patterns and intracellular Ca²⁺ concentration of cryopreserved dog spermatozoa during post‐thaw incubation in capacitating conditions [canine capacitating medium (CCM) with or without 5 μg/ml of heparin]. Heparin had no significant effects on any of the sperm parameters evaluated. During the first 8 h of incubation, the majority of the live spermatozoa had a high intracellular Ca²⁺ content. However, after 8–10 h of incubation, it had significantly declined. The highest proportion of fast motile sperm, and the highest curvilinear velocity, average path velocity and amplitude of lateral head displacement for the total motile sperm were observed during the 2–4‐h incubation period. It was concluded that: (a) the addition of 0.5% (v/v) of Equex STM Paste to a Tris–egg yolk based extender significantly improved the post‐thaw longevity of dog spermatozoa, but the same concentration of Equex Pasta had no significant beneficial effects; (b) sperm dilution after thawing with a Tris buffer containing glucose or fructose made no difference in post‐thaw sperm longevity; (c) the addition of 5 μg/ml of heparin to CCM had no significant capacitating effects on frozen–thawed dog spermatozoa.     

Comparison of radioimmunoassay and enzyme-linked immunosorbent assay for determining aflatoxin M1 in milk

Using a highly specific antibody against aflatoxin M1, a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA) were developed for the quantitation of M1 in milk. RIA was sensitive in the range of 5-50 ng per assay but was subject to interference by whole milk. Extraction and cleanup were therefore necessary for the detection of M1 in milk at 0.5 ng/mL. An ELISA procedure was developed by using an aflatoxin M1-carboxymethyl-horseradish peroxidase conjugate as the ligand. Competitive assays revealed that this system was relatively more sensitive for M1 than for B1, and had a much lower degree of cross-reactivity for aflatoxins B2, G1, G2, B2a, and aflatoxicol. As low as 0.25 ng M1/mL in artificially contaminated milk (raw, whole, skim) could be detected by ELISA in 3 h without extraction or cleanup. Because of its simplicity, sensitivity, and specificity, ELISA is the preferred method for monitoring aflatoxin M1 in milk.     

Analysis of zearalenone in cereal and Swine feed samples using an automated flow-through immunosensor

The development of a sensitive flow-though immunosensor for the analysis of the mycotoxin zearalenone in cereal samples is described. The sensor was completely automated and was based on a direct competitive immunosorbent assay and fluorescence detection. The mycotoxin competes with a horseradish-peroxidase-labeled derivative for the binding sites of a rabbit polyclonal antibody. Control pore glass covalently bound to Prot A was used for the oriented immobilization of the antibody-antigen immunocomplexes. The immunosensor shows an IC(50) value of 0.087 ng mL(-1) (RSD = 2.8%, n = 6) and a dynamic range from 0.019 to 0.422 ng mL(-1). The limit of detection (90% of blank signal) of 0.007 ng mL(-1) (RSD = 3.9%, n = 3) is lower than previously published methods. Corn, wheat, and swine feed samples have been analyzed with the device after extraction of the analyte using accelerated solvent extraction (ASE). The immunosensor has been validated using a corn certificate reference material and HPLC with fluorescence detection.     

Analysis and pharmacokinetics of cyclopiazonic acid in market weight pigs

The pharmacokinetic behavior of cyclopiazonic acid (CPA) was determined in market weight pigs using a competitive indirect ELISA developed for the determination of the mycotoxin in various biological matrices. Sample preparation for corn and skeletal muscle was achieved with a single extraction and recoveries of 53+/-6% over the effective range of the standard curve. The detection limit of CPA was 1 ppb in plasma, which required no extraction, and 20 ppb in corn and skeletal muscle with average intra- and interassay CV of 11 and 23%, respectively. Levels of CPA contamination in corn grown and stored in Michigan were unremarkable compared with published toxicity thresholds; the highest level of CPA found in any sample was 47 ppb. In pigs given a 20-mg i.v. bolus, CPA distributed rapidly among three compartments, with an overall volume of distribution (49 L) nearly equivalent to total body water. Cyclopiazonic acid was eliminated with a half-life of 24 h. Estimates of these pharmacokinetic parameters were supported by the achievement of steady-state plasma CPA levels within 6 d in pigs consuming a diet containing 10 ppm CPA, and by measured concentrations of CPA in plasma (410+/-44 ng/mL) and skeletal muscle (469+/-86 ng/ g). From these and other data, we concluded that the threat of CPA toxicity to livestock from consumption of cereal grains or to humans from consumption of animal products is minimal.     

Bacterial Isolates Associated With Dystocia And Retained Placenta In Iraqi Buffaloes

The present study was conducted on 50 recently calved Iraqi Buffalo cows. Depending on the kind of parturition, buffalo cows were divided into two main groups, the first group had normal unassisted parturition (NP) (26 animals) and the second group with certain periparturent complications (PPC) (24 animals). After 24 h of parturition, these two groups were further subdivided into two groups as cows expel their foetal membranes in <24 h postpartum and referred as non‐retained placenta (NRP) while cows that did not expel their foetal membrane after 24 h referred as retained placenta (RP). Sampling for bacteriology, uterine discharge for polymorphonuclear cells per cent and blood samples for polymorphonuclear neutrophil (PMN) and the enzyme creatine kinase activity were performed at 6, 24 and 48 h postpartum. In PPC group, the most prevalent bacteria after 6 h of calving were Escherichia coli, β‐haemolytic Streptococci and Lactobacillus acidophilus. Total bacterial isolates in the uterus of buffaloes with RP in PPC group after 24 and 48 h were 129 and 183 respectively. Among the isolates, Archanobacterium pyogenes, Fusobacterium necrophorum, Prevotella melaninogenicus and Staphylococcus aureus were the most prevalent isolates after 48 h of RP buffaloes in PPC group. Polymorphonuclear neutrophil were significantly (p < 0.01) increased in the uterine discharge than in blood in buffaloes with RP in both PPC and NP groups. In conclusion, uterine contamination occurs as a result of postpartum ascending contamination by non‐specific environmental organisms. The presence of Lactobacillus sp. in the uterus indicated a healthy uterus. Peripartum complications followed by retention of foetal membranes with the dominance of E. coli in the uterine lumen might favour the colonization of other bacteria including facultative anaerobic and strictly anaerobic in the uterine wall of buffaloes.     

Comparison of the TBARS Assay and BODIPY C11 Probes for Assessing Lipid Peroxidation in Red Deer Spermatozoa

Several methods are used to measure lipid peroxidation (LPO) in spermatozoa. The objective of this study was comparing the thiobarbituric acid reactive species (TBARS) method and the BODIPY 581/591 C₁₁ (B581) and BODIPY 665/676 C₁₁ (B665) fluorescent probes to measure induced peroxidative damage in thawed epididymal spermatozoa from Iberian red deer. Samples from three males were thawed, pooled, diluted in PBS, incubated at room temperature and assessed at 0, 3, 6 and 24 h under different experimental conditions: Control, hydrogen peroxide (H₂O₂) 0.1 mm or 1 mm , or tert‐butyl hydroperoxide (TBH) 0.1 mm or 1 mm . LPO was assessed by the TBARS assay [malondialdehyde (MDA) detection] and by the fluorescence probes B581 and B665 (microplate fluorimeter and flow cytometry). Increasing MDA levels were only detectable at 1 mm of TBH or H₂O₂. Both fluorescence probes, measured with fluorometer, detected significant increases of LPO with time in all treatments, except Control. Flow cytometry allowed for higher sensitivity, with both probes showing a significant linear relationship of increasing LPO with time for all oxidizing treatments (p < 0.001). All methods showed a good agreement, except TBARS, and flow cytometry showed the highest repeatability. Our results show that both B581 and B665 might be used for LPO analysis in Iberian red deer epididymal spermatozoa, together with fluorometry or flow cytometry. Yet, the TBARS method offered comparatively limited sensitivity, and further research must determine the source of that limitation.     

Effects of Cryopreservation on Bull Spermatozoa Distribution in Morphometrically Distinct Subpopulations

Assisted sperm morphometry analysis (ASMA) was used in this study to determine the effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations. Ejaculates were collected from five bulls and were divided. One portion was diluted at 30 degrees C in a skim milk-egg yolk medium, containing glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 200 sperm heads were analysed from each sample by means of the Sperm-Class Analyser (SCA), and the mean measurements recorded. Our results showed that applying the ASMA technology and multivariate cluster analyses, it was possible to determine that three separate subpopulations of spermatozoa with different morphometric characteristics coexist in bull ejaculates (large, average and small spermatozoa). The mean values of each sperm head dimension among the three subpopulations of spermatozoa were significantly different (p < 0.001). Besides, there were significant (p < 0.001) differences in the distribution of these three sperm subpopulations between fresh and thawed samples. Thus, the percentage of representation of the subpopulation that includes those spermatozoa whose dimensions are the biggest, decreased from 52.06% in extended fresh samples to 15.51% in the thawed ones. Contrarily, the percent of representation of the subpopulation containing the smallest spermatozoa, increased from 8.70% in extended fresh samples to 34.04% in the thawed ones. In conclusion, the present study confirms the heterogeneity of sperm head dimensions in bull semen, heterogeneity that vary through the cryopreservation procedure.