Estimation of volume densities of hepatocytic peroxisomes in a model fish: Catalase conventional immunofluorescence versus cytochemistry for electron microscopy

Accurately accessing changes in the intracellular volumes (or numbers) of peroxisomes within a cell can be a lengthy task, because unbiased estimations can be made only by studies conducted under transmission electron microscopy. Yet, such information is often required, namely for correlations with functional data. The optimization and applicability of a fast and new technical proceeding based on catalase immunofluorescence was implemented herein by using primary hepatocytes from brown trout (Salmo trutta f. fario), exposed during 96 h to two distinct treatments (0.1% ethanol and 50 µM of 17α‐ethynylestradiol). The time and cost efficiency, together with the results obtained by stereological analyses, specifically directed to the volume densities of peroxisomes, and additionally of the nucleus in relation to the hepatocyte, were compared with the well‐established 3,3'‐diaminobenzidine cytochemistry for electron microscopy. With the immuno technique it was possible to correctly distinguish punctate peroxisomal profiles, allowing the selection of the marked organelles for quantification. By both methodologies, a significant reduction in the volume density of the peroxisome within the hepatocyte was obtained after an estrogenic input. The most interesting point here was that the volume density ratios were quite correlated between both techniques. Overall, the immunofluorescence protocol for catalase was evidently faster, cheaper and provided reliable quantitative data that discriminated in the same way the compared groups. After this validation study, we recommend the use of catalase immunofluorescence as the first option for rapid screening of changes of the amount of hepatocytic peroxisomes, using their volume density as an indicator. Microsc. Res. Tech. 78:134–139, 2015. © 2014 Wiley Periodicals, Inc.     

Timing of artificial insemination using fresh or frozen semen after automated activity monitoring of estrus in lactating dairy cows

The objective of this observational experiment was to determine the association between the time of artificial insemination (AI) and pregnancy per AI (P/AI) in lactating Holstein cows inseminated with either fresh or frozen semen considering different characteristics of an estrus event (i.e., onset, peak, and end) using an automated activity monitoring system. A total of 3,607 AI services based on the alert of an automated activity monitoring system (Heatime; SCR Engineers Ltd., Netanya, Israel) were evaluated from 4 commercial dairy farms in Germany. Pregnancy diagnosis was performed by transrectal palpation 38 ± 3 d after AI or by transrectal ultrasonography 30 ± 3 d after AI. Estrus intensity was categorized based on peak activity of estrus (PAE) into low (35–89 index value) and high (90–100 index value) intensity. The mean (± standard deviation) duration of an estrus event was 14.3 ± 4.6 h. The mean (± standard deviation) interval from onset of estrus (OE; moment where index value was ≥35) to AI was 16.8 ± 8.0 h, from PAE to AI was 11.9 ± 8.1 h, and from end of estrus (EE; moment where index value returned to <35) to AI was 2.5 ± 8.7 h. Primiparous cows had greater P/AI than multiparous cows, whereas first AI postpartum yielded greater P/AI compared with subsequent AI services. Type of semen was not associated with P/AI. Cows with heat stress 1 wk before AI had decreased P/AI. Cows with low estrus intensity (26.0%) were less fertile compared with cows showing high estrus intensity (32.8%). Cows with intermediate 100-d milk yield had decreased P/AI compared with cows with either low or high 100-d milk yield. There was a quadratic effect of the interval from OE to AI on P/AI. At 38 d after AI, P/AI was greatest for cows inseminated from 7 to 24 h after OE, within 18 h after PAE, or from 5 h before EE to 12 h after EE. There was no interaction between the interval from OE to AI and type of semen. There tended to be an interaction between the intervals from PAE to AI and type of semen and from EE to AI and type of semen. Cows inseminated with fresh semen within 5 h before EE had greater P/AI compared with frozen semen, whereas cows inseminated with frozen semen from 13 to 18 h after EE had greater P/AI compared with fresh semen. In conclusion, inseminating cows from 7 to 24 h after OE or 1 to 18 h after PAE yielded greatest P/AI irrespective of type of semen. In addition, high estrus intensity was positively associated with P/AI.     

Toxic Potential of Cerrado Plants on Different Organisms

Cerrado has many compounds that have been used as biopesticides, herbicides, medicines, and others due to their highly toxic potential. Thus, this review aims to present information about the toxicity of Cerrado plants. For this purpose, a review was performed using PubMed, Science Direct, and Web Of Science databases. After applying exclusion criteria, 187 articles published in the last 20 years were selected and analyzed. Detailed information about the extract preparation, part of the plant used, dose/concentration tested, model system, and employed assay was provided for different toxic activities described in the literature, namely cytotoxic, genotoxic, mutagenic, antibacterial, antifungal, antiviral, insecticidal, antiparasitic, and molluscicidal activities. In addition, the steps to execute research on plant toxicity and the more common methods employed were discussed. This review synthesized and organized the available research on the toxic effects of Cerrado plants, which could contribute to the future design of new environmentally safe products.     

Technical note: Validation of an in-house bovine serum enzyme immunoassay for progesterone measurement

Measuring circulating progesterone (P4) of dairy cows is a key component of many research studies dealing with basic and applied reproduction physiology. The gold standard in dairy cows for the measurement of P4 in serum is radioimmunoassay (RIA), but it generates radioactive waste and requires licensed facilities. The purpose of this study was to develop and validate an in-house competitive enzyme immunoassay (EIA) to measure the P4 concentration in serum of dairy cattle. The secondary objective was to validate a commercial EIA. In the present study, a competitive EIA was developed using commercially available antibodies and conjugates. Ninety-six well microtiter plates were coated with the secondary antibody and incubated overnight. Following a washing step, the wells were blocked using the primary antibody. Serum samples were prepared by first extracting P4 using petroleum ether, then diluted in working conjugate solution. Samples were pipetted into the coated and blocked plates, then the matching HRP conjugate label (P4-3-HRP, East Coast Bio, North Berwick, ME) was added. The plates were incubated for 2 h, then washed. The substrate solution was added, and the plate was incubated up to 1 h at room temperature in the dark until a blue color had developed. A stop solution was added, and the optical density measured on a microplate reader was set at 450 nm. The binding proportion was calculated by a visible spectrum absorbance reader, and the amount of P4 was calculated using a log-logit regression line. The commercial EIA was executed as suggested by the manufacturer. The validation of the in-house EIA was done by calculating the inter- and intraassay coefficients of variation (CV) and evaluating the parallelism of diluted samples. The results from the in-house and commercial EIA were also compared with the ones from the RIA graphically (scatterplots and Bland-Altman plots) and statistically, using the Spearman correlation coefficient (r) and the Cohen's kappa statistics using a threshold of 1.0 ng/mL (κ). For the in-house EIA, the intraassay CV were all <10%, but the interassay for samples with small and large P4 concentration had CV of 12.5 and 11.0%, respectively. The correlations between the results from the EIA and the RIA were strong (in-house: r = 0.90; commercial: r = 0.83). At small concentrations (<1.0 ng/mL), however, the correlation with the gold standard was weak (in-house: r = 0.27; commercial: r = 0.14). This was likely due to the lack of accuracy at small concentrations, also shown by the absence of parallelism in samples ≤0.4 ng/mL. In conclusion, results from both the in-house and commercial EIA strongly correlated with the gold standard, but less so at smaller concentrations. The in-house EIA offers good accuracy to measure P4 in samples with a concentration >0.4 ng/mL, and a perfect agreement with RIA using a threshold of 1.0 ng/mL.     

Development of poly(lactic acid) nanostructured membranes for the controlled delivery of progesterone to livestock animals

Solution blow spinning (SBS) is a novel technology feasible to produce nanostructured polymeric membranes loaded with active agents. In the present study, nanofibrous mats of poly(lactic acid) (PLA) loaded with progesterone (P4) were produced by SBS at different P4 concentrations. The spun membranes were characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR). The in vitro releasing of P4 was evaluated using high-performance liquid chromatography (HPLC). Interactions between progesterone and PLA were confirmed by rheological measurements of the PLA/P4 solutions and in the spun mats by microscopy (SEM), thermal (DSC) and spectral (FTIR) analyses. SEM micrographs provided evidences of a smooth and homogeneous structure for nanostructured membranes without progesterone crystals on fiber surface. FTIR spectroscopy indicated miscibility and interaction between the ester of PLA and the ketone groups of the P4 in the nanofibers. X-ray analysis indicated that the size of PLA crystallites increased with progesterone content. Finally, by in vitro release experiments it was possible to observe that the progesterone releasing follows nearly first-order kinetics, probably due to the diffusion of hormone into PLA nanofibers.     

Technological optimization of manufacture of probiotic whey cheese matrices

In attempts to optimize their manufacture, whey cheese matrices obtained via thermal processing of whey (leading to protein precipitation) and inoculated with probiotic cultures were tested. A central composite, face-centered design was followed, so a total of 16 experiments were run using fractional addition of bovine milk to feedstock whey, homogenization time, and storage time of whey cheese as processing parameters. Probiotic whey cheese matrices were inoculated with Lactobacillus casei LAFTIL26 at 10% (v/v), whereas control whey cheese matrices were added with skim milk previously acidified with lactic acid to the same level. All whey cheeses were stored at 7 °C up to 14 d. Chemical and sensory analyses were carried out for all samples, as well as rheological characterization by oscillatory viscometry and textural profiling. As expected, differences were found between control and probiotic matrices: fractional addition of milk and storage time were the factors accounting for the most important effects. Estimation of the best operating parameters was via response surface analysis: milk addition at a rate of 10% to 15% (v/v), and homogenization for 5 min led to the best probiotic whey cheeses in terms of texture and organoleptic properties, whereas the best time for consumption was found to be by 9 d of storage following manufacture.     

Biofuels from waste fish oil pyrolysis: Chemical composition

In a previous study, waste fish oil was converted into bio-oil by a fast pyrolysis process at 525 °C in a continuous pilot plant reactor with 72-73% yield. The bio-oil was distilled to obtain light bio-oil and heavy bio-oil and these biofuels were characterized in terms of their physico-chemical properties. In this study, the chemical composition of light bio-oil and heavy bio-oil was determined using GC-FID, GC-MS, ¹H and ¹³C NMR techniques. The GC-MS analysis of waste fish oil showed the main composition of fatty acids to be the following: C_16:0 (15.87%), C_18:2 (20.96%), C_18:1 (17.29%), C_20:5 (5.11%), C_20:1 (7.59%), C_22:6 (4.53%), C_22:1 (10.42%) and others. The GC-FID analysis of the light bio-oil showed 482 compounds that were PIONA classified as paraffins (4.48%), iso-paraffins (8.31%), olefins (26.56%), naphthenes (6.07%) and aromatics (16.86%). The heavy bio-oil had a similar chromatographic profile as diesel oil, with a high content of carboxylic acids and olefins. These results are in good agreement with those for the gasoline and diesel oil fractions of petroleum.