Evaluation of domestic animal sperm head morphology via flow cytometric DNA labelling and pulse shape analysis using bull and stallion spermatozoa as model species.

The aim of the present study was to test a rapid, robust flow cytometric technique for the detection of sperm head abnormalities of domestic bulls and stallions. The so-called PulSA approach detects the pulse profiles of propidium-iodide labelled spermatozoa. In the first experiment, species-specific threshold values were established on sperm samples that were tested for sperm head abnormalities with a classic visual morphology analysis. In the second experiment, serial mixtures of bull and stallion spermatozoa mimicking different percentages of sperm head abnormalities were analysed. Non-metric multidimensional scaling showed a clear separation between the normal and mixed samples. The PulSA approach may be a useful tool in identifying sub- or infertile breeding males as well as in studying the evolutionary aspects of sperm morphology and morphometry.

Glycerol-Free Equilibration with the Addition of Glycerol Shortly before the Freezing Procedure: A Perspective Strategy for Cryopreservation of Wallachian Ram Sperm.

This study investigated the effect of glycerol added in different phases of sperm equilibration on CASA and flow cytometry parameters of thawed ram spermatozoa. Sperm was collected from adult Wallachian rams. The freezing extender was glycerol-free ANDROMED® (Minitub GmbH, Tiefenbach, Germany) supplied by 6% exogenous glycerol at different stages of the cryopreservation process. The purpose of this study was to compare two strategies of glycerol addition for sperm cryopreservation. The first strategy included the use of a glycerol-free extender for the procedure of glycerol-free equilibration and chilling, with the glycerolation of the extender by 6% glycerol shortly before sperm slow freezing (GFA). The second strategy included the use of a freezing extender already glycerolated by 6% glycerol before the equilibration and chilling of sperm and following slow freezing (GA). Sperm samples were analyzed after equilibration (but before freezing) and after thawing (at T0, T1 h, and T2 h time points). iSperm® mCASA (Aidmics Biotechnology Co., LTD., Taipei, Taiwan) was used for the evaluation of sperm kinematics. Flow cytometry was used to measure sperm viability (plasma membrane/acrosome intactness) and mitochondrial membrane potential. The obtained results significantly demonstrated that the glycerol-free equilibration with the addition of glycerol shortly before freezing is a perspective strategy for cryopreservation of Wallachian ram sperm.

A Pilot Study on the Uptake of Propidium Iodide and YO-PRO-1 Iodide through the Pannexin Channels in Wallachian Frozen-Thawed Ram Spermatozoa.

Propidium iodide (PI) and YO-PRO-1 (YPI) dyes are routinely used to determine sperm viability in many livestock species. It is commonly accepted that these dyes penetrate only sperm cells with damaged plasma membranes. Recently, however, the mechanism of dye uptake unrelated to damaged plasma membranes, but instead related to pannexin channels in dog and stallion sperm cells was demonstrated. This pilot study aimed to evaluate the role of pannexins in the uptake of PI and YPI dyes on Wallachian frozen-thawed ram spermatozoa by flow cytometry using probenecid, a specific inhibitor of pannexin channels. Additionally, the expression of pannexins in Wallachian sperm was evaluated directly (by qRT-PCR). The results demonstrate the active role of pannexin channels in the uptake of PI and YPI dyes on frozen-thawed Wallachian ram sperm. In conclusion, when using the PI or YPI exclusion assay to determine Wallachian frozen-thawed ram sperm viability, the danger of overestimating the number of spermatozoa with the damaged plasma membrane must be considered. The observed breed-specific, and more importantly, individual differences in gene expression as well as in dye uptake indicate the need for further studies.

Protected areas have a mixed impact on waterbirds, but management helps.

International policy is focused on increasing the proportion of the Earth's surface that is protected for nature1,2. Although studies show that protected areas prevent habitat loss3-6, there is a lack of evidence for their effect on species' populations: existing studies are at local scale or use simple designs that lack appropriate controls7-13. Here we explore how 1,506 protected areas have affected the trajectories of 27,055 waterbird populations across the globe using a robust before-after control-intervention study design, which compares protected and unprotected populations in the years before and after protection. We show that the simpler study designs typically used to assess protected area effectiveness (before-after or control-intervention) incorrectly estimate effects for 37-50% of populations-for instance misclassifying positively impacted populations as negatively impacted, and vice versa. Using our robust study design, we find that protected areas have a mixed impact on waterbirds, with a strong signal that areas managed for waterbirds or their habitat are more likely to benefit populations, and a weak signal that larger areas are more beneficial than smaller ones. Calls to conserve 30% of the Earth's surface by 2030 are gathering pace14, but we show that protection alone does not guarantee good biodiversity outcomes. As countries gather to agree the new Global Biodiversity Framework, targets must focus on creating and supporting well-managed protected and conserved areas that measurably benefit populations.

Setting priorities for climate change adaptation of Critical Sites in the Africa-Eurasian waterbird flyways.

Despite their importance for biodiversity and ecosystem services, wetlands are among the most threatened ecosystems globally. The conservation of many migratory waterbirds depends on the conservation of a network of key sites along their flyways. However, the suitability of these sites is changing under climate change, and it is important that management of individual sites in the network adapts to these changes. Using bioclimatic models that also account for changes in inundation, we found that projected climate change will reduce habitat suitability for waterbirds at 57.5% of existing Critical Sites within Africa-Eurasia, varying from 20.1% in Eastern Europe to 87.0% in Africa. African and Middle East sites are particularly threatened, comprising 71 of the 100 most vulnerable sites. By highlighting priority sites for conservation and classifying Critical Sites into Climate Change Adaptation Strategy (CCAS) classes, our results can be used to support the climate change adaptation of both individual sites and the entire site network.

Cycle Length Evaluation in Persistent Atrial Fibrillation Using Kernel Density Estimation to Identify Transient and Stable Rapid Atrial Activity.

PURPOSE: Left atrial (LA) rapid AF activity has been shown to co-localise with areas of successful atrial fibrillation termination by catheter ablation. We describe a technique that identifies rapid and regular activity. METHODS: Eight-second AF electrograms were recorded from LA regions during ablation for psAF. Local activation was annotated manually on bipolar signals and where these were of poor quality, we inspected unipolar signals. Dominant cycle length (DCL) was calculated from annotation pairs representing a single activation interval, using a probability density function (PDF) with kernel density estimation. Cumulative annotation duration compared to total segment length defined electrogram quality. DCL results were compared to dominant frequency (DF) and averaging. RESULTS: In total 507 8 s AF segments were analysed from 7 patients. Spearman's correlation coefficient was 0.758 between independent annotators (P < 0.001), 0.837-0.94 between 8 s and ≥ 4 s segments (P < 0.001), 0.541 between DCL and DF (P < 0.001), and 0.79 between DCL and averaging (P < 0.001). Poorer segment organization gave greater errors between DCL and DF. CONCLUSION: DCL identifies rapid atrial activity that may represent psAF drivers. This study uses DCL as a tool to evaluate the dynamic, patient specific properties of psAF by identifying rapid and regular activity. If automated, this technique could rapidly identify areas for ablation in psAF.

Setting population-size targets for geese causing socio-economic conflicts.

Most European goose populations have increased exponentially, and this has increasingly brought them into conflict with human activities. To manage this conflict, we used multi-criteria decision analysis to help set population targets for a super-abundant population of greylag geese (Anser anser). We relied on expert elicitation to assess the consequences of varying goose abundance on nine ecological, economic, and societal objectives. Representatives from national governments and from non-governmental organizations then weighted the objectives based on their perceived relative importance, and we used a consensus-convergence model to reach stakeholder agreement on the tradeoffs among objectives. The preferred population targets for two management units represent about a 20% reduction from current abundances, which from a management perspective would require considerable effort above and beyond current population-control measures. We believe that multi-criteria decision analysis can provide a systematic and transparent framework for building consensus among diverse stakeholders in a wide array of human-wildlife conflicts.

Andrological and cytogenetic investigations of an infertile Przewalski's stallion.

The case of an 8-year-old, sexually active but infertile Przewalski's stallion (Equus ferus przewalskii) was studied. Besides the infertility, the stallion also showed permanent problems with its body condition, being obviously weaker than all the other group members. The horse was kept in a separate place for two years with 12 mares in its harem group (six mares had foals earlier); however, none of the mares covered got pregnant. Andrological and cytogenetic investigations revealed underdeveloped testes, arrested spermatogenesis, azoospermia, and XY/XXY/X0 mosaicism. We classify the case as a mosaic Klinefelter syndrome, the first reported case in Przewalski's horse.

A 65-Day Fumonisin B Exposure at High Dietary Levels Has Negligible Effects on the Testicular and Spermatological Parameters of Adult Rabbit Bucks.

A 65-day study was undertaken to test the effects of two doses (10 and 20 mg/kg) of dietary fumonisin Bs (FB) on the rabbit male reproduction system. Body and testicular weight was not affected by the intoxication, neither the fatty acid composition of the testicular total phospholipids; the testis histological analysis failed to reveal any toxic effect. The FBs increased the testicular concentration and activity of reduced glutathione and glutathione peroxidase and decreased initial phase lipid peroxidation (conjugated dienes and trienes) in a dose dependent manner. Sperm morphology and chromatin condensation were monitored on Feulgen-stained smears. No significant differences were observed between the treatment groups and between sampling time points. The live cell ratio in the sperm (as assessed with flow cytometry) was not different among groups at any of the five sampling timepoints and was also identical within groups. Similarly, the spermatozoa membrane lipid profile was also identical in all three groups after the total intoxication period. In summary, it was demonstrated that FBs in an unrealistic and unjustified high dose still do not exert any drastic harmful effect on the leporine, male reproduction system, meanwhile slightly augmenting testicular antioxidant response.

Effects of Acute Heat Stress on a Newly Established Chicken Hepatocyte-Nonparenchymal Cell Co-Culture Model.

Heat stress is one of the most important issues in broiler flocks impairing animal health and productivity. On a cellular level, excess heat exposure can trigger heat shock response acting for the restoration of cell homeostasis by several mechanisms, such as affecting heat shock protein synthesis, redox homeostasis and pro-inflammatory cytokine production. The major aim of this study was to establish a novel avian hepatocyte-nonparenchymal cell co-culture as a model for investigating the cellular effects of heat stress and its interaction with inflammation in chicken liver. Cell fractions were isolated by differential centrifugation from a freshly perfused chicken liver, and hepatocyte mono-cultures as well as hepatocyte-nonparenchymal cell co-cultures (with cell ratio 6:1, hepatocytes to nonparenchymal cells, mimicking a milder hepatic inflammation) were prepared. Isolated and cultured cells were characterized by flow cytometry and immunocytochemistry applying hepatocyte- and macrophage-specific antibodies. Confluent cell cultures were exposed to 43 °C temperature for 1 or 2 h, while controls were cultured at 38.5 °C. The metabolic activity, LDH enzyme activity, reactive oxygen species (H2O2) production, extracellular concentration of heat shock protein 70 (HSP70), and that of the pro-inflammatory cytokines interleukin (IL-)6 and IL-8 were assessed. Shorter heat stress applied for 1 h could strongly influence liver cell function by significantly increasing catabolic metabolism and extracellular H2O2 release, and by significantly decreasing HSP70, IL-6, and IL-8 production on both cell culture models. However, all these alterations were restored after 2 h heat exposure, indicating a fast recovery of liver cells. Hepatocyte mono-cultures and hepatocyte-nonparenchymal cell co-cultures responded to heat stress in a similar manner, but the higher metabolic rate of co-cultured cells may have contributed to a better capability of inflamed liver cells for accommodation to stress conditions. In conclusion, the established new primary cell culture models provide suitable tools for studying the hepatic inflammatory and stress response. The results of this study highlight the impact of short-term heat stress on the liver in chickens, underline the mediatory role of oxidative stress in acute stress response, and suggest a fast cellular adaptation potential in liver cells.

An atypical Bacillus anthracis infection in a bull-A potential occupational health hazard.

Bacillus anthracis infecting cattle is usually identified based on the typical symptom: sudden death. Bacillus anthracis causing atypical symptoms may remain undiagnosed and represent a potential occupational health hazard for, that is veterinarians and producers, butchers and tanners. In the year 2004, one case of sudden death in a dairy farm in southern Finland was diagnosed as bovine anthrax. Four years later 2008, an atypical case of anthrax was diagnosed in the same holding. The bull was taken to the Production Animal Hospital of the Faculty of Veterinary Medicine, University of Helsinki because of fever, loss of appetite and a symmetrically swollen scrotal sac. Penicillin treatment cured the fever but not the swollen scrotum. Before the intended therapeutic castration, a punctuate consisting of 10 ml fluid collected into a syringe from the scrotal sac was cultivated on blood agar at 37°C. After 24 hr, an almost pure culture of a completely non-hemolytic Bacillus cereus-like bacteria was obtained. The strain was identified as B. anthracis using Ba-specific primers by the Finnish Food Safety Authority (RUOKAVIRASTO). After the diagnosis, the bull was euthanized and destroyed, the personnel were treated with prophylactic antibiotics and the clinic was disinfected. In this particular case, treatment with water, Virkon S and lime seemed to be effective to eliminate endospores and vegetative cells since no relapses of anthrax have occurred in 10 years. This case is the last reported anthrax case in Finland.

Evaluation of a new algorithm for tracking activation during atrial fibrillation using multipolar catheters in humans.

BACKGROUND: Conventional mapping techniques during atrial fibrillation (AF) are difficult to apply because of cycle length irregularity. Mapping studies are usually restricted to short durations of AF in limited regions because of the laborious manual annotation of local activation time (LAT). The purpose of this study was to test an automated algorithm to map activation during AF, with comparable accuracy to manual annotation. METHODS: Left atrial (LA) mapping was performed using a 20-pole double loop catheter (AFocusII) in 30-second data segments from 16 patients. The new algorithm (RETRO-Mapping) was designed to detect wavefront propagation between electrodes, and display activating wavefronts on a two-dimensional representation of the catheter. Activation patterns were validated against their bipolar electrograms and with isochronal maps. The mapping protocol was approved by the research ethics committee (13/LO1169 and 14/LO1367). RESULTS: During AF, uniform wavefront activation direction (mean ± SD, degrees) from manually constructed isochronal maps was comparable to RETRO-Propagation Map (RETRO-PM) and RETRO-Automated Direction (RETRO-AD): 1 ± 6.9 for RETRO-PM; and 2 ± 6.6 for RETRO-AD. There was no significant difference in activation direction assigned to 1373 uniform wavefronts during AF when comparing RETRO-PM with RETRO-AD (Bland-Altman mean difference: -0.1 degrees; limits of agreement: -8.0 to 8.3; 95% CI -0.4 to 0.2; (r = 0.01) R2 = < 0.005; P = .77). CONCLUSION: We have developed and validated a new technique to map activation during AF. This technique shows comparable accuracy to that of conventional isochronal mapping with careful manual adjustment of LAT.

A splice donor variant in CCDC189 is associated with asthenospermia in Nordic Red dairy cattle.

BACKGROUND: Cattle populations are highly amenable to the genetic mapping of male reproductive traits because longitudinal data on ejaculate quality and dense microarray-derived genotypes are available for thousands of artificial insemination bulls. Two young Nordic Red bulls delivered sperm with low progressive motility (i.e., asthenospermia) during a semen collection period of more than four months. The bulls were related through a common ancestor on both their paternal and maternal ancestry. Thus, a recessive mode of inheritance of asthenospermia was suspected. RESULTS: Both bulls were genotyped at 54,001 SNPs using the Illumina BovineSNP50 Bead chip. A scan for autozygosity revealed that they were identical by descent for a 2.98 Mb segment located on bovine chromosome 25. This haplotype was not found in the homozygous state in 8557 fertile bulls although five homozygous haplotype carriers were expected (P = 0.018). Whole genome-sequencing uncovered that both asthenospermic bulls were homozygous for a mutation that disrupts a canonical 5' splice donor site of CCDC189 encoding the coiled-coil domain containing protein 189. Transcription analysis showed that the derived allele activates a cryptic splice site resulting in a frameshift and premature termination of translation. The mutated CCDC189 protein is truncated by more than 40%, thus lacking the flagellar C1a complex subunit C1a-32 that is supposed to modulate the physiological movement of the sperm flagella. The mutant allele occurs at a frequency of 2.5% in Nordic Red cattle. CONCLUSIONS: Our study in cattle uncovered that CCDC189 is required for physiological movement of sperm flagella thus enabling active progression of spermatozoa and fertilization. A direct gene test may be implemented to monitor the asthenospermia-associated allele and prevent the birth of homozygous bulls that are infertile. Our results have been integrated in the Online Mendelian Inheritance in Animals (OMIA) database ( https://omia.org/OMIA002167/9913/ ).

An Evaluation of Boar Spermatozoa as a Biosensor for the Detection of Sublethal and Lethal Toxicity.

A novel, objective, and rapid computed motility inhibition (CMI) assay was developed to identify and assess sublethal injury in toxin-exposed boar spermatozoa and compared with a subjective visual motility inhibition (VMI) assay. The CMI values were calculated from digital micrographic videos using a custom MATLAB® script by contrasting the motility index values of each experiment with those of the background and control experiments. Following a comparison of the CMI and VMI assays results, it was determined that their agreement depended on the shape of the dose-response curve. Toxins that exhibited a steep slope were indicative of good agreement between the assays. Those depicted by a gentle decline in the slope of the dose-response curve, the CMI assay were shown to be two times more sensitive than the VMI assay. The CMI assay was highly sensitive to the inhibition of mitochondrial function and glucose transport activity by sublethal doses of toxins and to disruption of cellular cation homeostasis by carrier ionophoric toxins, when compared to the cytotoxicity and lethal toxicity assays (i.e., that evaluated the inhibition of cell proliferation in somatic cell lines (FL, PK-15, and MNA cells)) and disruption to spermatozoa membrane integrity. The CMI assay recognized subtle sublethal toxicity changes in metabolism, manifested as a decrease in boar spermatozoa motility. Thus, it was feasible to effectively compare the objectively-measured numerical values for motility inhibition using the CMI assay against those reflecting lethal damage in the spermatozoa cells and somatic cell lines using a cytotoxicity assay.

Ejaculated boar spermatozoa displaying a rare multivesicular defect.

Two cases of a previously unreported sperm defect appearing in boar studs in Finland are presented. Spermatozoa showed small particles scattered on their surface with a prevalence decreasing with boar age. Semen samples, either stained with eosin-nigrosin or examined with phase contrast optics on formaldehyde-fixed spermatozoa, revealed the presence of multiple particles attached to the surface of spermatozoa counted as dead cells at fixation. Transmission electron microscopy revealed these were multivesicular and multilamellar vesicles, built up by phospholipid membranes. The case is classified as a post-epididymal multivesicular sperm defect with a favorable prognosis.

Eco- and genotoxicity profiling of a rapeseed biodiesel using a battery of bioassays.

Biodiesel is considered an important renewable energy source but still there is some controversy about its environmental toxicity, especially to aquatic life. In our study, the toxicity of water soluble fraction of biodiesel was evaluated in relatively low concentrations using a battery of bioassays: Vibrio fischeri bioluminescence inhibition, Sinapis alba root growth inhibition, Daphnia magna immobilization, boar semen live/dead ratio and DNA fragmentation and Unio pictorum micronucleus test. While the S. alba test indicated nutritive (stimulating) effect of the sample, the biodiesel exerted toxic effect in the aquatic tests. D. magna was the most sensitive with EC50 value of 0.0226%. For genotoxicity assessment, the mussel micronucleus test (MNT) was applied, detecting considerable genotoxic potential of the biodiesel sample: it elucidated micronuclei formation already at low concentration of 3.3%. Although this test has never been employed in biodiesel eco/genotoxicity assessments, it seems a promising tool, based on its appropriate sensitivity, and representativity.

Successful conservation of global waterbird populations depends on effective governance.

Understanding global patterns of biodiversity change is crucial for conservation research, policies and practices. However, for most ecosystems, the lack of systematically collected data at a global level limits our understanding of biodiversity changes and their local-scale drivers. Here we address this challenge by focusing on wetlands, which are among the most biodiverse and productive of any environments and which provide essential ecosystem services, but are also amongst the most seriously threatened ecosystems. Using birds as an indicator taxon of wetland biodiversity, we model time-series abundance data for 461 waterbird species at 25,769 survey sites across the globe. We show that the strongest predictor of changes in waterbird abundance, and of conservation efforts having beneficial effects, is the effective governance of a country. In areas in which governance is on average less effective, such as western and central Asia, sub-Saharan Africa and South America, waterbird declines are particularly pronounced; a higher protected area coverage of wetland environments facilitates waterbird increases, but only in countries with more effective governance. Our findings highlight that sociopolitical instability can lead to biodiversity loss and undermine the benefit of existing conservation efforts, such as the expansion of protected area coverage. Furthermore, data deficiencies in areas with less effective governance could lead to underestimations of the extent of the current biodiversity crisis.

Quick cytogenetic screening of breeding bulls using flow cytometric sperm DNA histogram analysis.

The aim of the present study was to test the FXCycle PI/RNase kit for routine DNA analyses in order to detect breeding bulls and/or insemination doses carrying cytogenetic aberrations. In a series of experiments we first established basic DNA histogram parameters of cytogenetically healthy breeding bulls by measuring the intraspecific genome size variation of three animals, then we compared the histogram profiles of bulls carrying cytogenetic defects to the baseline values. With the exception of one case the test was able to identify bulls with cytogenetic defects. Therefore, we conclude that the assay could be incorporated into the laboratory routine where flow cytometry is applied for semen quality control.