In vivo acridine orange human spermatozoa staining—A new perspective for RNA detection and spermatozoa morphology evaluation

Significant increases in male infertility and the still unresolved questions on the compatibility and interpretation of current methods in infertility diagnostics call for new protocols. Morphology, genome damage, RNA content and quantity are currently in practice as the major parameters in evaluation of sperm quality. However, results of various methods are not always in mutual concordance. In this study, in vivo acridine orange (AO) staining, which is presently in application in the estimation of genome damage in reticulocytes, was adjusted for spermatozoa staining. Ten men suffering from oligoasthenoteratozoospermia (OAT) and 10 healthy fertile men were analysed using in vivo AO staining. Microscopic analysis was performed by fluorescent and confocal fluorescent microscopy. Our results show that this method preserves spermatozoa membranes, which enables new insight into spermatozoa genome damage, RNA content in residual cytoplasm, damage of neck area with mitochondrion and tail pathology. The introduced method explains the difference between results of sperm DNA fragmentation assay and the globally used AO staining and opens new options for the development of automated systems. In conclusion, the results of our study offer (a) an innovative approach to the analysis of spermatozoa pathology, (b) enable localization and quantification of RNA in residual cytoplasm, (c) a significant contribution to research of aetiology of infertility in men, (d) open new perspectives for the automatization of sperm quality estimation and (e) improve the personalized approach in the selection of in vitro fertilization protocols.     

Kinetics of abamectin disposition in blood plasma and milk of lactating dairy sheep and suckling lambs

Abamectin (ABM) has been used worldwide as an anthelmintic drug in veterinary medicine and as an agricultural pesticide. Its pharmacokinetics and permeation into milk was evaluated in dairy sheep after subcutaneous administration. ABM elimination half-lives and mean residence times were 1.7 and 3.7 days for blood plasma and 1.9 and 3.8 days for milk, respectively. The ABM milk to plasma concentration ratio (0.89) primarily depends on milk fat content. Transfer of ABM residues to suckling lambs was evaluated by determination of ABM concentration time courses in lambs' plasma. Mean maximal concentration in lambs was 1.6 microg L(-1) at 3.3 days, and elimination half-life was 2.7 days. In ewes' plasma and milk, ABM was detected up to 23 days. Because of different pharmacokinetics, ABM exposure in lambs was almost 10% of the exposure in ewes, although the amount excreted in milk was only 1.0% of the dose.     

Particle and structure characterization of fulvic acids from agricultural soils

Purpose

Structural studies on fulvic acids (FAs) are of significant importance since FAs are involved in many environmentally important processes, such as adsorption and transportation of nutrients, trace elements, and organic pollutants. Interactions between suspended and dissolved soil components are controlled by a variety of attractive and repulsive interparticle forces influenced partially by FA properties. The aim of this paper is a detailed characterization of FAs derived from agricultural soils varied with physicochemical properties.

Materials and methods

Forty topsoils (pH_KCL?=?3.8–7.8, clay content?=?0–6%, and TOC?=?7.0–187.2 g kg^?1) were collected from rural area. Fulvic acids (FAs) were isolated according to the IHSS method. The overall FA solutions were purified by nonionic macroporous acrylic ester resin (DAX-8), and the organic carbon content in FA fraction (FA-OC) was determined by a liquid C–N analyzer. The particle size diameter (PSD) and polydispersity (PDI) were analyzed by a dynamic light scattering technique, while the zeta potential (ZP) was measured using an electrophoretic light scattering method. Spectroscopic properties of FAs, including occurrence and distribution of functional groups, were investigated by near-IR spectroscopy.

Results and discussion

Agricultural soils differed substantially, with FA-OC content ranging from 0.6 to 8.8 g kg^?1 that accounted for 0.5 to 22.6% of TOC. The PSD exhibited wide range of particle size (0.2 to 69.6 nm) and was characterized by different polydispersity (14–183.1%). The ZP described the behavior, and surface charge of FA particles varied from ??1.7 to +?3.3 mV. Low ZP characterized 77% of FAs and indicated the ease of aggregate formation and intermolecular connections. The measured ZP also showed that suspended organic particles of FAs had both positive and negative charges, which was confirmed by the spectroscopic analysis. The presence of negative charges on FA particle surfaces was connected with the occurrence of phenol and carboxyl groups while positive charge with amine.

Conclusions

Detailed characterization of FAs from agricultural soils confirms their heterogeneous and complex nature. The results indicate that FAs mainly exist as small molecules that form molecular aggregates or associations in solutions. FA in a solution of a similar ionic strength may be positively or negatively charged due to its chemical structure and aggregate behavior which affects their properties in the soil.

     

Assessment of the potential for dredged material dispersal from dumping sites in the Gulf of Gdańsk

Purpose

Environment-friendly management of sites used for disposal of locally generated sedimentary material involves designation of an optimal dumping site location which will render the dredged material re-usable for beneficial purposes. The objective of this research was to determine whether wind, waves, and currents can induce transport of sediment from offshore dumping sites located at intermediate depths in the southern Baltic.

Materials and methods

The problem was addressed by exploring potential sediment transport from two sites located in the Gulf of Gdańsk at depths of about 20 m. A total of 29 combinations of hydrodynamic variables, representing the most extreme possible situations in the area, including eight theoretical uniform wind fields over the entire Baltic Sea from the W, NW, N, NE, E, SE, S, and SW sectors, the wind speed of 30 m s^?1, as well as 21 historical extreme storms, retrieved from the HIPOCAS project database, were used.

Results and discussion

The bottom velocities resulting from waves and currents at the dumping sites considered were computed using wave models (WAM, SWAN) and the M3D hydrodynamic model (based on the POM model). To estimate the velocities critical for bedload transport, formulae developed by Soulsby (1997) and Sawamoto and Yamashita (Proc Coastal Sediments 87:415–423, 1987) were used. The volumetric bedload transport was computed based on Meyer-Peter and Müller (1948). The model simulations demonstrated that, for the storm conditions analyzed, the current velocity in the area of the two dumping sites would be so low that it would practically not affect the magnitude of the bottom sediment transport. Thus, the resultant volume of bedload transported would be equal to that generated by the wave action. For the heaviest historical storm, the maximum transport is about 3?×?10^?5 m² s^?1.

Conclusions

Under conditions of theoretical storms, the bottom orbital velocities would be higher and the resultant sediment transport would reach almost 7?×?10^?5 m² s^?1 for northerly winds. However, this value is still very low compared with the volume of sediment being dumped. The findings of this study may prove useful in designation of future dumping sites.

     

A blind climber: The first evidence of ultrasonic echolocation in arboreal mammals

The means of orientation is studied in the Vietnamese pygmy dormouse Typhlomys chapensis, a poorly known enigmatic semi‐fossorial semi‐arboreal rodent. Data on eye structure are presented, which prove that Typhlomys (translated as “the blind mouse”) is incapable of object vision: the retina is folded and retains no more than 2500 ganglion cells in the focal plane, and the optic nerve is subject to gliosis. Hence, Typhlomys has no other means for rapid long‐range orientation among tree branches other than echolocation. Ultrasonic vocalization recordings at the frequency range of 50–100 kHz support this hypothesis. The vocalizations are represented by bouts of up to 7 more or less evenly‐spaced and uniform frequency‐modulated sweep‐like pulses in rapid succession. Structurally, these sweeps are similar to frequency‐modulated ultrasonic echolocation calls of some bat species, but they are too faint to be revealed with a common bat detector. When recording video simultaneously with the ultrasonic audio, a significantly greater pulse rate during locomotion compared to that of resting animals has been demonstrated. Our findings of locomotion‐associated ultrasonic vocalization in a fast‐climbing but weakly‐sighted small mammal ecotype add support to the “echolocation‐first theory” of pre‐flight origin of echolocation in bats.     

Impact of individual training parameters and manner of taking breath odor samples on the reliability of canines as cancer screeners

The aim of this study was to assess the progress in operant conditioning of dogs during 3 training phases up to a level where the dogs performed a minimum of 40 correct indications without false alerts and hesitations in at least 100 trials in each phase, and to compare the training results with the working phase. Breath samples from 57, 45, and 118 patients with breast cancer, melanoma, and lung cancer, respectively, were used as patterns, with samples from 305 healthy volunteers as controls. Six dogs in 2 age-groups (20 months vs. 6 months old) were used for the training. In phase I, the dogs were trained to indicate by sitting or lying down before the breath samples taken from cancer patients (pattern odor) coupled with food odor and placed among 4 blank samples (a lineup of 5 samples). In phase II, a pattern sample without food odor was placed among blank samples. In phase III as well as in the working phase, a pattern sample was placed among control samples. Significant individual differences were found in dogs’ performance during the training. The percentage of trials with prompts to achieve self-dependent work decreased in older dogs in consecutive training phases, but increased in phase II in the younger dogs. The increasing level of challenge in consecutive training phases resulted in a higher percentage of false alerts and misses, and in younger dogs, decreased motivation for sniffing the odor samples was observed in training phase III. Nonsignificant and negative correlations between the training phases and working phase in the percentage of correct indications and false alerts show that it is difficult to predict the performance of fully trained dogs on the basis of the results achieved during the training. Fully trained dogs discriminated pattern breath samples (cancer) from healthy controls with 79% sensitivity and 78% specificity, or with 50% sensitivity at the probability of correct response by chance of 50% and 20%, respectively. The sensitivity decreased to 68% and 37%, respectively, when each sample was tested only once, although being better than by chance (P < 0.05). The percentage of false alerts and misses is difficult to reduce during the training; thus, the percentage of correct indications tended to decrease in the course of training phases II and III. Odor samples taken in hospitals may contain components of “hospital odors,” which may be a confounding factor for dogs’ indications. Using trained dogs for cancer screening on the basis of detection of odor markers still requires further study.     

Quantifying the spatial relationship between bird species’ distributions and landscape feature boundaries in southern Ontario, Canada

Understanding what features of the landscape affect species distribution is critical to effectively implement conservation strategies. This study investigates how a boundary analysis framework can be used to characterize the spatial association between boundaries (i.e., spatial locations of high rates of change) in bird species?? distributions and landscape features at the regional scale. The study area covers 92,000?km² in southern Ontario (Canada) and extends from the Great Lakes-St. Lawrence biome to the southern Canadian Shield biome. Landcover composition was derived from Ontario Land Cover data (1991?C1998; 7 types) and elevation data were derived from the Canada3D digital elevation model. Bird distributions were estimated using indicator kriging based on point counts obtained from the Ontario Breeding Bird Atlas data (2001?C2005; 60 species). Boundaries were delineated for both data types using a 10?×?10?km cell resolution. Spatial boundary overlap statistics were used to quantify the spatial relationship between landscape features and bird boundaries and tested using a randomization procedure. There was significant positive association and spatial overlap between delineated landscape feature boundaries and bird boundaries. The number of spatially overlapping cells between the two boundary types was 67 out of 164 (41?%) and 76?% of cells were within 11.42?km of each other. These results were statistically significant (P?<?0.001) and suggest a strong spatial relationship between high rates of change in landscape features and bird species?? distributions at the regional scale. A boundary analysis framework could be used to identify boundary shifts in response to climate change and anticipate changes in species distributions.     

Protein kinase A activity and NO are involved in the regulation of crucian carp (Carassius carassius) red blood cell osmotic fragility

Fish Physiology and Biochemistry - Activation of the cAMP pathway by β-adrenergic stimulation and cGMP pathway by activation of guanylate cyclase substantially affects red blood cell (RBC)...