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.     

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.     

Skin biopsies predict acute graft-versus-host disease after small bowel transplantation in pigs

Intestinal transplantation is being increasingly performed to treat patients with irreversible intestinal failure. The major cause of intestinal graft failure is graft-versus-host disease (GVHD) that represents a life-threatening complication after small bowel transplantation (Itx). The purpose of this study was to assess the diagnostic and prognostic value of skin biopsy histological changes for acute GVHD after Itx in pigs. Thirty-four Large White pigs were divided into three groups: Group 1 with Itx only, Group 2 with Itx and donor bone marrow infusion (Itx BM) and Group 3 (control group - before the operation). Animals received tacrolimus-based immunosuppression from day 0 to day 30 postoperatively. Skin and small bowel biopsies were histologically assessed, analysed and classified from grade 1 to 4 on postoperative days 15, 30, 45 and 60. There was a strong correlation between the histological grading values of skin biopsy changes and the histological grading values of small bowel biopsy changes (Kendall's tau_b is 0.855 for the Itx group and 0.730 for the Itx BM group). The significant correlation found between skin and small bowel histological changes suggests the prognostic value of skin biopsies after Itx. In conclusion, our findings emphasise the diagnostic and prognostic value of skin biopsy analysis for acute GVHD after Itx.     

Immune defense of wild‐caught Norway rats is characterized by increased levels of basal activity but reduced capability to respond to further immune stimulation

Studies of wild animals' immunity often use comparison with laboratory‐raised individuals. Using such an approach, various data were obtained concerning wild Norway rat's immunity. Lower or higher potential of immune system cells to respond to activation stimuli were shown, because of analysis of disparate parameters and/ or small number of analyzed individuals. Inconsistent differences between laboratory and wild rats were shown too, owing to great response variability in wild rats. We hypothesized that wild rats will express more intense immune activity compared to their laboratory counterparts which live in a less demanding environment. To test this, we analyzed the circulating levels of inflammatory cytokine interleukin‐6 (IL‐6), a mediator which has a central role in host immune defense. In addition, we examined the activity of the central immune organ, the spleen, including cell proliferation and production of pro‐inflammatory cytokines interferon‐γ (IFN‐γ) and interleukin‐17 (IL‐17), which are major effectors of cellular adaptive immune response. In order to obtain reasonable insight into the immunity of wild Norway rats, analysis was conducted on a much larger number of individuals compared to other studies. Higher levels of plasma IL‐6, higher spleen mass, cellularity and basal IFN‐γ production concomitantly with lower basal production of anti‐inflammatory cytokine interleukin‐10 (IL‐10) revealed more intense immune activity in the wild compared to laboratory rats. However, lower responsiveness of their spleen cells' proinflammatory cytokine production to concanavalin A (ConA) stimulation, along with preserved capacity of IL‐10 response, might be perceived as an indication of wild rats' reduced capability to cope with incoming environmental stimuli, but also as a means to limit tissue damage.     

Reducing sampling intensity in order to investigate spatial variability of soil pH,organic matter and available phosphorus using co-kriging techniques. A case study of acid soils in Eastern Croatia

Soil pH, hydrolytic acidity (HA), organic matter (OM) and plant available phosphorus (AP) are factors controlling the environmental-friendly soil management in agroecosystems. These parameters are highly variable in space. The objective of this work is to study spatial variability of pH, HA, OM and AP using several interpolation methods in Eastern Croatia. A total of 1004 (0–30 cm) soil samples were collected, and several univariate and multivariate interpolation performances were tested. The results showed that soils of the study area had high HA and AP, while pH and OM were low. The variogram analysis revealed different spatial structures among studied soil properties and demonstrate a need for variable-rate management. Soil pH and OM had lower spatial variability compared to AP and HA. Ordinary kriging was the most accurate method to estimate the studied variables. The incorporation of auxiliary variables increased the precision of the estimations for HA. Soil AP and OM showed different results for spatial prediction obtained by co-kriging. Overall, the incorporation of pH as auxiliary variable increased the prediction of the models. However, more co-variates should be incorporated in further models, in order to identify with more precision areas that need to be restored.     

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.