Flow Cytometric Chromosomal Sex Sorting of Stallion Spermatozoa Induces Oxidative Stress on Mitochondria and Genomic DNA

To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville sorting technology using flow cytometry. Improvement of this technology in the equine species requires increasing awareness of the modifications that the sorting procedure induces on sperm intactness. Oxidative stress is regarded as the major damaging phenomenon, and increasing evidence regards handling of spermatozoa – including sex sorting – as basic ground for oxidative damage. The aim of this study was to disclose whether the flow cytometric sorting procedure increases the production of reactive oxygen species (ROS), and to identify if ROS production relates to DNA damage in sorted spermatozoa using specific flow cytometry‐based assays. After sorting, oxidative stress increased from 26% to 33% in pre‐ and post‐incubation controls, to 46% after sex sorting (p < 0.05). Proportions of DNA fragmentation index post‐sorting were approximately 10% higher (31.3%); an effect apparently conduced via oxidative DNA damage as revealed by the oxyDNA assay. The probable origin of this increased oxidative stress owes the removal of enough seminal plasma due to the unphysiological sperm extension, alongside a deleterious effect of high pressure on mitochondria during the sorting procedure.     

The effect of the polymerized bovine haemoglobin solution, Hb-200, on endothelial function in isolated arterial rings from rats

Haemoglobin-based oxygen carriers have been developed as 'blood substitutes'. Early cross-linked haemoglobins caused marked vasoconstriction, however, polymerized haemoglobin solutions, such as the bovine product haemoglobin glutamer-200 (bovine) (Hb-200), are said to have lesser vascular effects. The aim of this study was to quantify the effect of Hb-200 on isolated rat arterial rings. The actions of Hb-200 were investigated using rings of aorta and second-order mesenteric arteries from female Wistar rats. The arterial rings were mounted for isometric tension recording. Addition of Hb-200 (1 microM) to arterial rings, precontracted with phenylephrine (50-70% maximum phenylephrine-induced tone), resulted in vasoconstriction. Addition of Hb-200 to arteries precontracted with phenylephrine and relaxed with acetylcholine (1 microM) also caused contraction. Furthermore preincubation with Hb-200 (1 microM) enhanced the response to phenylephrine (1 nm to 10 microM) and impaired acetylcholine-induced relaxation (10 nm to 10 microM). Responses to Hb-200 were similar to those in response to the nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine methyl ester (L-NAME, 100 microM), alone or in combination with Hb-200. These data demonstrate that Hb-200 has a significant vasoconstrictor effect similar to L-NAME. Thus the vascular actions of Hb-200 are consistent with activity as a NO scavenger. This may have implications for the clinical use of Hb-200.     

Epigallocatechin‐3‐Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm–Zona Pellucida Heterologous Binding

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin‐3‐gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2‐h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm , 500 nm and 5 μm ) and EGCG (10, 20 and 60 μm ), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 μm (but not of 60 μm ) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm , the presence of EGCG at all the concentrations tested (10, 20 and 60 μm ) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone.     

Relationship of Bread Quality to Kernel,Flour, and Dough Properties

This study measured the relationship between bread quality and 49 hard red spring (HRS) or 48 hard red winter (HRW) grain, flour, and dough quality characteristics. The estimated bread quality attributes included loaf volume, bake mix time, bake water absorption, and crumb grain score. The best‐fit models for loaf volume, bake mix time, and water absorption had R² values of 0.78–0.93 with five to eight variables. Crumb grain score was not well estimated, and had R² values ≈0.60. For loaf volume models, grain or flour protein content was the most important parameter included. Bake water absorption was best estimated when using mixograph water absorption, and flour or grain protein content. Bake water absorption models could generally be improved by including farinograph, mixograph, or alveograph measurements. Bake mix time was estimated best when using mixograph mix time, and models could be improved by including glutenin data. When the data set was divided into calibration and prediction sets, the loaf volume and bake mix time models still looked promising for screening samples. When including only variables that could be rapidly measured (protein content, test weight, single kernel moisture content, single kernel diameter, single kernel hardness, bulk moisture content, and dark hard and vitreous kernels), only loaf volume could be predicted with accuracies adequate for screening samples.     

Using Visible and Near‐Infrared Reflectance Spectroscopy and Differential Scanning Calorimetry to Study Starch,Protein, and Temperature Effects on Bread Staling

Starch, protein, and temperature effects on bread staling were investigated using visible and near‐infrared spectroscopy (NIRS) and differential scanning calorimetry (DSC). Bread staling was mainly due to amylopectin retrogradation. NIRS measured amylopectin retrogradation accurately in different batches. Three important wavelengths, 970 nm, 1,155 nm, and 1,395 nm, were associated with amylopectin retrogradation. NIRS followed moisture and starch structure changes when amylopectin retrograded. The amylose‐lipid complex changed little from one day after baking. The capability of NIRS to measure changes in the retrograded amylose‐lipid complex was limited. Two important wavelengths, 550 nm and 1,465 nm, were key for NIRS to successfully classify the starch‐starch (SS) and starch‐protein (SP) bread based on different colors and protein contents in SS and SP. Low temperature dramatically accelerated the amylopectin retrogradation process. Protein retarded bread staling, but not as much as temperature. The starch and protein interaction was less important than the starch retrogradation. Protein hindered the bread staling process mainly by diluting starch and retarding starch retrogradation.     

An Automated Near‐Infrared System for Selecting Individual Kernels Based on Specific Quality Characteristics

An automated sorting system was developed that nondestructively measured quality characteristics of individual kernels using near‐infrared (NIR) spectra. This single‐kernel NIR system was applied to sorting wheat (Triticum aestivum L.) kernels by protein content and hardness, and proso millet (Panicum miliaceum L.) into amylose‐bearing and amylose‐free fractions. Single wheat kernels with high protein content could be sorted from pure lines so that the high‐protein content portion was 3.1 percentage points higher than the portion with the low‐protein kernels. Likewise, single wheat kernels with specific hardness indices could be removed from pure lines such that the hardness index in the sorted samples was 29.4 hardness units higher than the soft kernels. The system was able to increase the waxy, or amylose‐free, millet kernels in segregating samples from 94% in the unsorted samples to 98% in the sorted samples. The portion of waxy millet kernels in segregating samples was increased from 32% in the unsorted samples to 55% after sorting. Thus, this technology can be used to enrich the desirable class within segregating populations in breeding programs, to increase the purity of heterogeneous advanced or released lines, or to measure the distribution of quality within samples during the marketing process.     

Predicting Scab,Vomitoxin, and Ergosterol in Single Wheat Kernels Using Near-Infrared Spectroscopy

Near-infrared spectroscopy (NIRS) was used to detect scab damage and estimate deoxynivalenol (DON) and ergosterol levels in single wheat kernels. Results showed that all scab-damaged kernels identified by official inspectors were correctly identified by NIRS. In addition, this system identified more kernels with DON than did a visual inspection. DON and ergosterol were predicted with standard errors of ≈40 and 100 ppm, respectively. All samples with visible scab had single kernels with DON levels >120 ppm, and some kernels contained >700 ppm of DON. This technology may provide a means of rapidly screening samples for potential food safety and quality problems related to scab damage.     

Automated Color Classification of Single Wheat Kernels Using Visible and Near-Infrared Reflectance

Modification of an existing single kernel wheat characterization system allowed collection of visible and near-infrared (NIR) reflectance spectra (450–1,688 nm) at a rate of 1 kernel/4 sec. The spectral information was used to classify red and white wheats in an attempt to remove subjectivity from class determinations. Calibration, validation, and prediction results showed that calibrations using partial least squares regression and derived from the full wavelength profile correctly classed more kernels than either the visible region (450–700 nm) or the NIR region (700–1,688 nm). Most results showed >99% correct classification for single kernels when using the visible and NIR regions. Averaging of single kernel classifications resulted in 100% correct classification of bulk samples.     

Effect of NaOH on Visible Wavelength Spectra of Single Wheat Kernels and Color Classification Efficiency

A diode-array system, which measures spectral reflectance from 400 to 700 nm, was used to quantify single wheat kernel color before and after soaking in NaOH as a means of determining color class. Wheat color classification is currently a subjective determination and important in determining the end-use of the wheat. Soaking kernels in NaOH and classifying the soaked kernels with the diode-array system resulted in more difficult-to-classify kernels correctly classified (98.1%) than the visual method of classifying kernels (74.8%). Kernel orientation had a slight effect on correct classification, with the side view correctly classifying more kernels than the dorsal or crease view. The diode-array system provided a means of quantifying kernel color and eliminated inspector subjectivity when determining color class.     

High‐Speed NIR Segregation of High‐ and Low‐Protein Single Wheat Seeds

Wheat breeders need a nondestructive method to rapidly sort high‐ or low‐protein single kernels from samples for their breeding programs. For this reason, a commercial color sorter equipped with near‐infrared filters was evaluated for its potential to sort high‐ and low‐protein single wheat kernels. Hard red winter and hard white wheat cultivars with protein content >12.5% (classed as high‐protein, 12% moisture basis) or < 11.5% (classed as low‐protein) were blended in proportions of 50:50 and 95:5 (or 5:95) mass. These wheat blends were sorted using five passes that removed 10% of the mass for each pass. The bulk protein content of accepted kernels (accepts) and rejected kernels (rejects) were measured for each pass. For 50:50 blends, the protein in the first‐pass rejects changed as much as 1%. For the accepts, each pass changed the protein content of accepts by ≈0.1%, depending on wheat blends. At most, two re‐sorts of accepts would be required to move 95:5 blends in the direction of the dominant protein content. The 95:5 and 50:50 blends approximate the low‐ and high‐protein mixture range of early generation wheat populations, and thus the sorter has potential to aid breeders in purifying samples for developing high‐ or low‐protein wheat. Results indicate that sorting was partly driven by color and vitreousness differences between high‐ and low‐protein fractions. Development of a new background specific for high‐ or low‐protein and fabrication of better optical filters for protein might help improve the sorter performance.