Simultaneously evaluating postthaw viability and acrosome integrity of spermatozoa by flow cytometry would provide a valuable testing tool in both research and routine work. In the present study, a new triple-stain combination was developed for the simultaneous evaluation of viability and acrosome integrity of bovine sperm processed in egg yolk-based extender by flow cytometer. SYBR-14 and propidium iodide (PI) enabled the discrimination of sperm cells from egg yolk and debris particles, which was instrumental for the flow cytometric analyses of frozen-thawed bovine sperm, because it implied that washing steps to remove egg yolk were no longer required. In addition, phycoerythrin-conjugated peanut agglutinin (PE-PNA) was used to discriminate acrosome-damaged/reacted sperm cells from acrosome-intact cells. Repeatability was calculated using two processed ejaculates of 10 bulls. Three straws per batch were analyzed in duplicate measurements. Method-agreement analysis between the SYBR-14/PE-PNA/PI and fluorescein isothiocyanate (FITC)-conjugated PNA was performed, with FITC-PNA/PI staining being carried out on 14 frozen-thawed semen samples immediately after thawing and after a 3-h incubation at 37 degrees C. The British Standards Institution repeatability index of the SYBR-14/PE-PNA/PI combination was 2.6%. On average, the FITC-PNA/PI method showed a 6.3% overestimation of the live and acrosome-intact sperm cell subpopulation. In conclusion, the new triple-stain combination is highly repeatable and easy to use in routine application, and it provides a more precise estimate for the rate of sperm cells with intact head membrane and acrosome compared to the generally used and validated FITC-PNA/PI staining.

As a result of a tragic industrial accident, a highly alkaline red mud sludge inundated settlements and agricultural areas near Ajka, Hungary on October 4, 2010. One of the major concerns about the aftermaths of the accident is the potential health effects of vast amounts of fugitive dust from red mud sediment. Thus, we studied the chemical and physical properties of particles of red mud and its respirable fugitive dust, and performed toxicity measurements. Under unfavorable meteorological conditions dry red mud sediment could emit very high amounts of respirable alkaline particles into the air. The number size distribution of fugitive dust peaks above 1 μm aerodynamic diameter; therefore, its inhalation is unlikely to affect the deep regions of the lungs. No significant mineralogical or elemental fractionation was observed between the sediment and dust, with the major minerals being hematite, cancrinite, calcite, and hydrogarnet. Although the high resuspension potential and alkalinity might pose some problems such as the irritation of the upper respiratory tract and eyes, based on its size distribution and composition red mud dust appears to be less hazardous to human health than urban particulate matter.

Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of 'bench-top' flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa.

The aim of this study was to investigate the effect of the swim up and Percoll methods to select frozen-thawed bull spermatozoa with high quality membrane and acrosomal integrity and final concentration. Semen samples from six Holstein-Friesian bulls were examined. The whole experiment was repeated three times. Before and after both treatments, spermatozoa were subjected to a double-staining method and evaluated by brightfield light microscope using 40x dry, or 100x oil immersion objectives. The concentration of spermatozoa evaluated by haemocytometer was 8.8 x 10(7)/ml after thawing, and the percentage of live cells with intact acrosome was 45.8%. Both treatments significantly increased the proportion of live spermatozoa compared with no treatment, and the use of Percoll gradient resulted in a significantly higher percentage of living cells with an intact acrosome (88.2%) than the swim up method (69.4%). The concentration of spermatozoa after Percoll separation (9.3 x 10(6)/ml) was higher than that after the swim up method (5.8 x 10(6)/ml). These results indicate that spermatozoa with a higher viability and acrosome integrity can be obtained by Percoll separation than by the swim up method. Therefore the use of Percoll-treated spermatozoa in IVF systems can be more expedient.

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