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.

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.

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.

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.

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.

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 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/ ).

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.

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.

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.

Flow cytometric detection of oxidative DNA damage in fish spermatozoa exposed to cadmium - Short communication.

The aim of the present pilot study was to apply a flow cytometric assay, the so-called OxyDNA test, to determine the level of oxidative DNA damage in fish spermatozoa exposed to different concentrations (0.01-10,000 mg/L) of cadmium. Milt was collected from three randomly selected Prussian carp (Carassius auratus gibelio) males. Oxidative DNA damage was assessed with the OxyDNA kit and using flow cytometry. The ratio of OxyDNA-positive events increased significantly at higher cadmium concentrations. The results indicate that direct contact of fish spermatozoa with cadmium-polluted water initiates genotoxic damage.

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.

Effects of high ambient temperature on fish sperm plasma membrane integrity and mitochondrial activity - A flow cytometric study.

Local extreme climatic conditions occurring as a result of global climate change may interfere with the reproduction of animals. In the present study fish spermatozoa were incubated at different temperatures (20, 25, 30 and 40 °C) for 10 and 30 minutes, respectively and plasma membrane integrity and mitochondrial membrane potential changes were evaluated with flow cytometry using SYBR-14/PI and Mitotracker Deep Red FM fluorescent dyes. No significant differences were found in plasma membrane integrity at either incubation temperatures or time points. Mitotracker Deep Red FM histogram profiles indicating mitochondrial activity showed significant (p < 0.001) alterations in all cases of higher (25, 30 and 40 °C) temperature treatments as compared to the samples incubated at 20 °C. Our results indicate that fish spermatozoa exposed to high temperatures suffer sublethal damage that cannot be detected with conventional, vital staining techniques.

A comparison of alternative assays to measure DNA damage in stallion spermatozoa: TUNEL test versus 'Nicoletti assay'.

The aberrations of sperm DNA may cause various problems and have negative consequences on fertility. These influence embryonic development or might lead to early embryo loss. Sperm Chromatin Structure Assay (SCSA) is the flow cytometric method most often used for the detection of DNA lesions; however, some studies using that method reached confusing conclusions. The aim of this pilot study was to adjust and compare two alternative tests, namely the TUNEL test and the Nicoletti assay. The above-mentioned two flow cytometric methods capable of detecting the fragmented DNA of sperm were tested on 12 frozen-thawed stallion semen samples. The TUNEL test demonstrated much higher DNA fragmentation ratio than the Nicoletti assay (mean ± SD: 30.77 ± 13.03% vs. 1.93 ± 0.89%, respectively). A fluorescent microscopic check of the samples showed that TUNEL labelled the plasma membrane and the mitochondria in a nonspecific way, rather than detecting only the fragmented DNA, thus eventually resulting in a false positive sign. The Nicoletti assay is simpler, quicker and does not detect nonspecific binding; however, further analyses are required to determine its diagnostic value.

Effect of feeding different oils on plasma corticosterone in broiler chickens.

A study was conducted to examine the effects of different oils on the plasma corticosterone concentrations of broiler chickens fed ad libitum or deprived of feed for 24 hours. A total of 36 Ross broilers were randomly assigned to one of three dietary treatments at 10 days of age and fed a grower diet supplemented with 60 g/kg soybean oil (rich in linoleic acid, C18:2n-6), linseed oil (rich in a-linolenic acid, C18:3n-3) or fish oil (rich in C14:0, C16:0, C16:1n-7, C20:1n-9; eicosapentaenoic acid and docosahexaenoic acid, EPA, C20:5n-3 and DHA, C22:6n-3), respectively, for 18 days. Dietary supplementation of fish oil resulted in lower (P < 0.05) baseline plasma corticosterone levels of chickens fed ad libitum for 18 days compared to soybean and linseed oil supplementations. Feed deprivation for 24 h induced a significant (P < 0.05) increase in corticosterone concentration in every treatment group compared to the ad libitum-fed birds. The hormone levels of feed-deprived birds did not differ significantly among groups fed diets supplemented with different oils.

In Vitro Gene Conservation Status and the Quality of the Genetic Resources of Native Hungarian Sheep Breeds.

Studies revealed a global loss of genetic resources for local sheep breeds. Therefore, the current study aimed to introduce and highlight the progress made on Hungary's existing gene conservation program (small Gene Bank). Furthermore, we evaluated breed (Tsigai, Cikta, and Racka), season, and individual variabilities (n = 24) of the pre-freeze and post-thaw semen stored in the Gene Bank to enhance the gene conservation of the breeds. The samples were cryopreserved manually, and post-thaw spermatozoa were analyzed for motility (CASA), viability, chromatin structure, and morphometry of the sperm nuclei. Ejaculate volume, spermatozoa concentration, subjective motility and standard motility, kinematic parameters, and spermatozoa's head area standard deviation of the post-thaw samples differed significantly among breeds (p < 0.05). Season affected ejaculate volume, total spermatozoa number/ejaculate, STR, BCF, and ALH. We observed a significant (p < 0.001; 0.05) breed and season interaction on concentration, total spermatozoa number/ejaculate, VCL, LIN, WOB, spermatozoa's head average perimeter and nucleus length (Tsigai and Cikta differed but were statistically the same as Racka). Similarly, season significantly (p < 0.05) affected the proportion of ejaculate suitable for freezing. There was a significant (p < 0.05) difference in kinematic parameters and viability among the rams across the breeds. The spermatozoa's head morphometry of the Tsigai and Cikta breeds differed significantly (p < 0.05) among the rams. There were individual and breed differences in many spermatozoa quality parameters. The stored samples are of good quality, with more than 40% having intact membranes and low abnormal chromatin condensation.

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.

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.

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.

The red mud accident in Ajka (Hungary): characterization and potential health effects of fugitive dust.

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.