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.     

Effects of antioxidants on rapeseed oil oxidation in an artificial digestion model analyzed by UHPLC-ESI-MS

A normal diet contains large quantities of oxidized fatty acids, glycerolipids, cholesterol, and their cytotoxic degradation products because many foods in the diet are fried, heated, or otherwise processed and consumed often after long periods of storage. There is also evidence that the acid medium of the stomach promotes lipid peroxidation and that the gastrointestinal tract is a major site of antioxidant action, as demonstrated by various colorimetric methods. The identity and yields of specific products of lipid transformation have seldom been determined. The present study describes the molecular species profiles of all major gastrointestinal lipids formed during digestion of autoxidized rapeseed oil in an artificial digestion model in the presence of L-ascorbic acid, 6-palmitoyl-O-L-ascorbic acid, 3,5-di-tert-butyl-4-hydroxytoluene (BHT), DL-α-tocopherol, and DL-α-tocopheryl acetate. Differences in oxidized lipid profiles were detected in the samples digested in the presence of different antioxidants, but none of them could prevent the formation of oxidized lipids or promote their degradation in a gastric digestion model. The lack of effect is attributed to the inappropriate nature of the gastrointestinal medium for the antioxidant activity of these vitamins and BHT. A fast ultrahigh performance liquid chromatographic-electrospray ionization-mass spectrometric method was developed for the analysis of lipolysis products, including epoxy, hydroperoxy, and hydroxy fatty acids, and acylglycerols, utilizing lithium as ionization enhancer.     

To jump or not to jump? Strategies employed by leisure and sport horses

Show jumping is one of the most popular equestrian disciplines although strategic jumping per se is seldom used by free-living horses when negotiating obstacles that they might otherwise avoid. To establish if horses are naturally motivated to jump, we examined horses in a free-choice situation when negotiating various obstacles under test conditions. Eighteen leisure horses (LHs) and 16 sport horses (SHs) participated in a number of 2-choice tests. First, horses were presented with 2 optional routes during a “free-choice test” to reach a food stimulus. The choices were a shorter route (over an obstacle) or a longer route (around the obstacle). During 8 consecutive trials, the horses encountered an increase in obstacle height on every second trial as follows: 0, 20, 35, and 50 cm. In the “ridden test” after an initial conditioning period, the horses were tested with a Y-maze formation during 3 consecutive trials, where one element of the maze contained an obstacle arm (OA). The horses walked or trotted over the obstacle most frequently (59.9%) and jumped the obstacle in only 10.7% of the cases. For both the LH and SH groups, the horses' motivation to traverse the obstacle decreased as the obstacle height increased and only 44.1% of horses actually negotiated the 50-cm obstacle as compared with the 20-cm obstacle (85.3%). The LH group preferred to go around the obstacle significantly more often than the SH group (24 trials, 16.7% vs. 9 trials, 7.0%, for SH and LH, respectively; χ² = 6.81, N = 33, P = 0.0090). The LH group used a jumping strategy to clear the obstacle far less frequently (6 trials, 4.2%) than the SH group (23 trials, 18.0%; χ² = 9.96, N = 29, P = 0.0023). In the Y-maze trials, the LH group exhibited preference (40 per 54 trials, 74.1%; χ² = 12.5, P < 0.0001), and the SH group failed to exhibit any preference (20 per 48 trials; χ² = 1.33, P = 0.3123). There was no evidence of any correlation between the motivation to clear the obstacle and total number of OA choices in either the LH group (r_s = 0.13, P = 0.9594) or the SH group (r_s = ?0.25, P = 0.3492). The findings from the present study indicate that SHs are motivated and willing to jump obstacles more often than are LHs under similar conditions. However, the apparent reluctance of the horses overall to continue jumping as the obstacle height increases suggests that, in general, many horses could easily encounter excessive demands (overfacing) in sport. This issue should be carefully monitored in terms of equine training, competition, and welfare.     

Assessment of antioxidant activity and rheological properties of wheat and buckwheat milling fractions

This study examined the antioxidant properties of the ethanolic extracts of wheat milling fractions (wheat flour type 500 and type 850, and bran) and their polyphenol and tocopherol content, and rheological characteristics of wheat dough supplemented with buckwheat flours (light and wholegrain). The results obtained in this study were correlated with our previously published data on wheat flour type 400, wholegrain wheat flour and buckwheat flours.Buckwheat flours exhibited significantly higher (P < 0.05) antiradical activity on hydroxyl (OH), superoxide anion (O₂⁻) and (1,1-diphenyl-2-picrylhydrazyl) DPPH radicals, antioxidant activity and reducing power than all investigated wheat milling fractions when their corresponding IC₅₀ values were compared.The rheological parameters of wheat dough supplemented with light and wholegrain buckwheat flour (0-50%) were obtained by using Mixolab. Results indicated changes in protein and starch properties of dough.The obtained results indicate the benefit of using buckwheat flours in wheat-based food products, i.e. their contribution in functional and tailor-made-food production.     

Effect of Climate Change on Wheat Quality and HMW Glutenin Subunit Composition in the Pannonian Plain

The primary goal of this study is to improve our understanding of the extent of influence of climatic factors in Serbia and high‐molecular‐weight glutenin subunit (HMW‐GS) composition upon wheat end‐use quality. In‐depth analyses were performed on four bread wheat cultivars that are the most common in agricultural practice in Serbia. Total glutenin content showed significant difference between the production years, in opposition to gliadins. Cluster analysis of different percentages of glutenin and gliadin subunit molecular weight ranges (<40,000, 40,000–80,000, 81,000–120,000, and >120,000) indicated that the year of production and the cultivar did not have a significant effect on the percentage ranges for glutenins. However, they had a considerable impact on the percentage ranges for gliadins. Production year and the interaction of year and cultivar had the strongest influences on the percentage of SDS‐unextractable polymeric proteins. A synergistic effect of the HMW‐GS composition and climatic conditions revealed that all eight samples with HMW‐GS composition 2*, 5 + 10, 7 + 9 along with the highest Glu 1 score of 9 (out of a maximum of 10) produced in the year 2011 belonged to two clusters with the best wheat end‐use quality. Furthermore, the climate conditions in 2011 made it possible for the wheat cultivars with HMW‐GS composition –, 2 + 12, 7 + 9 to possess similar qualities as cultivars with HMW‐GS composition 2*, 5 + 10, 7 + 9 produced in 2012.     

Prevalence of antibodies against Leptospira sp. in snakes,lizards and turtles in Slovenia

Background

Leptospiral infections in poikilothermic (cold blooded) animals have received very little attention and the literature concerning natural infections of these animals is limited. The aim of this study was to determine the prevalence of leptospiral antibodies in reptiles, imported into Slovenia and intended to be pets in close contact with humans. A total of 297 reptiles (22 snakes, 210 lizards and 65 turtles) were tested for specific antibodies against serovars of Leptospira interrogans sensu stricto using the microscopic agglutination test (MAT). Live cultures of different serovars were used as antigens. MAT was performed according to standard procedures and the degree of reaction was interpreted by estimating the percentage of agglutinated leptospires. Samples showing titres of ≥ 50 against one or more serovars were considered as positive.

Results

Antibodies against seven pathogenic serovars of L. interrogans sensu stricto were detected in 46 of 297 reptiles. Among 22 snakes, specific antibodies against pathogenic serovars of three Leptospira species (L. interrogans, L. kirschneri and L. borgpetersenii) at titre levels from 1:50 to 1:400 were detected in 6 snakes. In 31 of 210 lizards, specific antibodies were found in titres from 1:50 to 1:1000 and, finally, among 65 turtles (terrapins and tortoises), 9 had specific antibodies at titre levels between 1:50 and 1:1600. Animals imported from non-EU countries showed significantly higher prevalence (25.0%; 95 confidence interval: 16.7–33.3%) than animals from EU member states (10.4%; confidence interval: 6.1–14.7%).

Conclusions

Reptiles may be considered as potential reservoirs of L. interrogans sensu stricto. Origin of the animals is a risk factor for presence of leptospiral antibodies, especially in lizards. Special attention should be focused on animals from non-EU member states.     

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.