Breaking the spell: Changes in the behavior of two zoo-kept tigers (Panthera tigris) after exposure to a distinct feast-and-fast feeding regime.

The behavior of zoo carnivores has received intense attention due to their propensity for locomotor stereotypies. We observed two adult male tiger (Panthera tigris) siblings kept together for the duration of 104 days by round-the-clock video observation. The period consisted of three baseline periods with the zoo's regular feeding regime of five feeding days per week interrupted by two individual fasting days, with feeding occurring in the evening (B1-B3 of 14 days each). These periods were interrupted by two intervention periods (I1: randomized feeding times, 28 days; I2: gorge-feeding with three 10-day fasting periods, 34 days). As expected, day and night-time behavior was different, with the majority of sleep occurring at night. Pacing, which was mainly considered anticipatory, significantly decreased from 88 ± 132 min/day during B1 to 20 ± 33 min/day during B3. Pacing did not increase during the fasting days of I2. Over the course of whole study, lying time decreased and nonpacing locomotion increased. A major difference was observed between gorge-feeding and the subsequent first fasting days: during gorge-feeding, tigers spent a large part of the day feeding and locomoting (and less sleeping); on the subsequent day, they locomoted about 4.5 h less and slept about 4.3 h more. We suggest that interrupting routines by fasting periods of several days may be effective for reducing regular anticipatory behavior and creates an across-day structure that may correspond to the evolved psychological disposition of large carnivores.

Time series cluster analysis reveals individual assignment of microbiota in captive tiger (Panthera tigris) and wildebeest (Connochaetes taurinus).

Fecal microbiota variability and individuality are well studied in humans and also in farm animals (related to diet- or disease-specific influences), but very little is known for exotic zoo-housed animals. This includes a wide range of species that differ greatly in microbiota composition and variation. For example, herbivorous species show a very similar and constant fecal microbiota over time, whereas carnivorous species appear to be highly variable in fecal microbial diversity and composition. Our objective was to determine whether species-specific and individual-specific clustering patterns were observed in the fecal microbiota of wildebeest (Connochaetes taurinus) and tigers (Panthera tigris). We collected 95 fecal samples of 11 animal individuals that were each sampled over eight consecutive days and analyzed those with Illumina MiSeq sequencing of the V3-V4 region of the 16SrRNA gene. In order to identify species or individual clusters, we applied two different agglomerative hierarchical clustering algorithms - a community detection algorithm and Ward's linkage. Our results showed that both, species-specific and individual-specific clustering is possible, but more reliable results were achieved when applying dynamic time warping which finds the optimal alignment between different time series. Furthermore, the bacterial families that distinguish individuals from each other in both species included daily occurring core bacteria (e.g., Acidaminococcaceae in wildebeests or Clostridiaceae in tigers) as well as individual dependent and more fluctuating bacterial families. Our results suggest that while it is necessary to consider multiple consecutive samples per individual, it is then possible to characterize individual abundance patterns in fecal microbiota in both herbivorous and carnivorous species. This would allow establishing individual microbiota profiles of animals housed in zoos, which is a basic prerequisite to quickly detect deviations and use microbiome analysis as a non-invasive and cost-effective tool in animal welfare.

Development of A Tool for Assessing the Reputation of Zoos: The Zoo Ethical Reputation Survey (ZERS).

Nowadays, most zoos have taken prominent and active positions in endangered species conservation and educating visitors about the value of biodiversity. However, to be effective and trusted in their mission, they must act ethically and have a good reputation. Yet, the drivers that can influence their reputation are still little investigated, and there are still few studies focused on assessing the reputation of these institutions. In the present work, we report the development of a tool, the Zoo Ethical Reputation Survey (ZERS), and its pilot application to assess the opinions of the visitors of two zoos, one in Italy and one in Germany, on drivers that may influence the ethical reputation of zoos. Preliminary results based on the answers of 274 respondents show that visitors' opinions on zoos acting with ethical responsibility are correlated with emotional appeal and familiarity with these institutions. The application of ZERS can help zoos identify weaknesses in their reputation and develop new strategies to improve people's attitudes towards them, bringing many benefits to the individual zoo and zoological institutions in general.

Diurnal and Nocturnal Behaviour of Cheetahs (Acinonyx jubatus) and Lions (Panthera leo) in Zoos.

Mammals are constantly exposed to exogenous and endogenous influences that affect their behaviour and daily activity. Light and temperature, as well as anthropogenic factors such as husbandry routines, visitors, and feeding schedules are potential influences on animals in zoological gardens. In order to investigate the effects of some of these factors on animal behaviour, observational studies based on the analyses of activity budgets can be used. In this study, the daily and nightly activity budgets of six lions (Panthera leo) and five cheetahs (Acinonyx jubatus) from four EAZA institutions were investigated. Focused on the influencing factor light and feeding, we analysed these activity budgets descriptively. Behaviour was recorded and analysed during the winter months over an observation period of 14 days and 14 nights using infrared-sensitive cameras. Our results show that lions and cheetahs exhibit activity peaks at crepuscular and feeding times, regardless of husbandry. Thus, lions in captivity shift nocturnal behaviour familiar from the wild to crepuscular and diurnal times. In cheetahs, in contrast, captive and wild individuals show similar 24 h behavioural rhythms. The resting behaviour of both species is more pronounced at night, with cheetahs having a shorter overall sleep duration than lions. This study describes the results of the examined animals and is not predictive. Nevertheless, the results of this study make an important contribution to gaining knowledge about possible factors influencing the behaviour of lions and cheetahs in zoos and offer implications that could be useful for improving husbandry and management.

Introducing the Software CASE (Cluster and Analyze Sound Events) by Comparing Different Clustering Methods and Audio Transformation Techniques Using Animal Vocalizations.

Unsupervised clustering algorithms are widely used in ecology and conservation to classify animal sounds, but also offer several advantages in basic bioacoustics research. Consequently, it is important to overcome the existing challenges. A common practice is extracting the acoustic features of vocalizations one-dimensionally, only extracting an average value for a given feature for the entire vocalization. With frequency-modulated vocalizations, whose acoustic features can change over time, this can lead to insufficient characterization. Whether the necessary parameters have been set correctly and the obtained clustering result reliably classifies the vocalizations subsequently often remains unclear. The presented software, CASE, is intended to overcome these challenges. Established and new unsupervised clustering methods (community detection, affinity propagation, HDBSCAN, and fuzzy clustering) are tested in combination with various classifiers (k-nearest neighbor, dynamic time-warping, and cross-correlation) using differently transformed animal vocalizations. These methods are compared with predefined clusters to determine their strengths and weaknesses. In addition, a multidimensional data transformation procedure is presented that better represents the course of multiple acoustic features. The results suggest that, especially with frequency-modulated vocalizations, clustering is more applicable with multidimensional feature extraction compared with one-dimensional feature extraction. The characterization and clustering of vocalizations in multidimensional space offer great potential for future bioacoustic studies. The software CASE includes the developed method of multidimensional feature extraction, as well as all used clustering methods. It allows quickly applying several clustering algorithms to one data set to compare their results and to verify their reliability based on their consistency. Moreover, the software CASE determines the optimal values of most of the necessary parameters automatically. To take advantage of these benefits, the software CASE is provided for free download.

Nature Interest Scale - Development and Evaluation of a Measurement Instrument for Individual Interest in Nature.

Interest is an important factor for successful learning that has been the subject of intensive research for decades. Although interest in nature is of great importance for environmental education, to date there is no valid and reliable measurement tool. Therefore, the purpose of this study was to develop and test a scale for interest in nature, the Nature Interest Scale (NIS). In study 1, nine items were selected based on the three dimensions of the psychological interest construct to represent interest in nature. The factor structure of this new measurement instrument, was tested using confirmatory factor analyses. The results show that the instrument represents the three dimensions of the interest construct well. In study 2 the validity (discriminant and convergent validity) as well as the reliability (internal consistency, composite reliability, test-retest reliability) of the NIS were demonstrated. In study 3, the applicability of the NIS was tested with a different target group, students with learning disabilities. The results of this factor analysis also confirm the factor structure of the scale. Thus, this study provides a valid and reliable measurement tool for individual interest in nature that can be used for future research.

Discrimination of Acoustic Stimuli and Maintenance of Graded Alarm Call Structure in Captive Meerkats.

Animals living in human care for several generations face the risk of losing natural behaviors, which can lead to reduced animal welfare. The goal of this study is to demonstrate that meerkats (Suricata suricatta) living in zoos can assess potential danger and respond naturally based on acoustic signals only. This includes that the graded information of urgency in alarm calls as well as a response to those alarm calls is retained in captivity. To test the response to acoustic signals with different threat potential, meerkats were played calls of various animals differing in size and threat (e.g., robin, raven, buzzard, jackal) while their behavior was observed. The emitted alarm calls were recorded and examined for their graded structure on the one hand and played back to them on the other hand by means of a playback experiment to see whether the animals react to their own alarm calls even in the absence of danger. A fuzzy clustering algorithm was used to analyze and classify the alarm calls. Subsequently, the features that best described the graded structure were isolated using the LASSO algorithm and compared to features already known from wild meerkats. The results show that the graded structure is maintained in captivity and can be described by features such as noise and duration. The animals respond to new threats and can distinguish animal calls that are dangerous to them from those that are not, indicating the preservation of natural cooperative behavior. In addition, the playback experiments show that the meerkats respond to their own alarm calls with vigilance and escape behavior. The findings can be used to draw conclusions about the intensity of alertness in captive meerkats and to adapt husbandry conditions to appropriate welfare.

Unraveling differences in fecal microbiota stability in mammals: from high variable carnivores and consistently stable herbivores.

BACKGROUND: Through the rapid development in DNA sequencing methods and tools, microbiome studies on a various number of species were performed during the last decade. This advance makes it possible to analyze hundreds of samples from different species at the same time in order to obtain a general overview of the microbiota. However, there is still uncertainty on the variability of the microbiota of different animal orders and on whether certain bacteria within a species are subject to greater fluctuations than others. This is largely due to the fact that the analysis in most extensive comparative studies is based on only a few samples per species or per study site. In our study, we aim to close this knowledge gap by analyzing multiple individual samples per species including two carnivore suborders Canoidea and Feloidea as well as the orders of herbivore Perissodactyla and Artiodactyla held in different zoos. To assess microbial diversity, 621 fecal samples from 31 species were characterized by sequencing the V3-V4 region of the 16S rRNA gene using Illumina MiSeq. RESULTS: We found significant differences in the consistency of microbiota composition and in fecal microbial diversity between carnivore and herbivore species. Whereas the microbiota of Carnivora is highly variable and inconsistent within and between species, Perissodactyla and Ruminantia show fewer differences across species boundaries. Furthermore, low-abundance bacterial families show higher fluctuations in the fecal microbiota than high-abundance ones. CONCLUSIONS: Our data suggest that microbial diversity is significantly higher in herbivores than in carnivores, whereas the microbiota in carnivores, unlike in herbivores, varies widely even within species. This high variability has methodological implications and underlines the need to analyze a minimum amount of about 10 samples per species. In our study, we found considerable differences in the occurrence of different bacterial families when looking at just three and six samples. However, from a sample number of 10 onwards, these within-species fluctuations balanced out in most cases and led to constant and more reliable results.

Deep learning-based pose estimation for African ungulates in zoos.

The description and analysis of animal behavior over long periods of time is one of the most important challenges in ecology. However, most of these studies are limited due to the time and cost required by human observers. The collection of data via video recordings allows observation periods to be extended. However, their evaluation by human observers is very time-consuming. Progress in automated evaluation, using suitable deep learning methods, seems to be a forward-looking approach to analyze even large amounts of video data in an adequate time frame.In this study, we present a multistep convolutional neural network system for detecting three typical stances of African ungulates in zoo enclosures which works with high accuracy. An important aspect of our approach is the introduction of model averaging and postprocessing rules to make the system robust to outliers.Our trained system achieves an in-domain classification accuracy of >0.92, which is improved to >0.96 by a postprocessing step. In addition, the whole system performs even well in an out-of-domain classification task with two unknown types, achieving an average accuracy of 0.93. We provide our system at https://github.com/Klimroth/Video-Action-Classifier-for-African-Ungulates-in-Zoos/tree/main/mrcnn_based so that interested users can train their own models to classify images and conduct behavioral studies of wildlife.The use of a multistep convolutional neural network for fast and accurate classification of wildlife behavior facilitates the evaluation of large amounts of image data in ecological studies and reduces the effort of manual analysis of images to a high degree. Our system also shows that postprocessing rules are a suitable way to make species-specific adjustments and substantially increase the accuracy of the description of single behavioral phases (number, duration). The results in the out-of-domain classification strongly suggest that our system is robust and achieves a high degree of accuracy even for new species, so that other settings (e.g., field studies) can be considered.

Impact of biological education and gender on students' connection to nature and relational values.

The new concept of relational values (RVs) is gaining more and more attention in environmental research, but empirical analyses are still rare. However, this type of research is necessary because the RVs have an influence on environmental behavior. To evaluate the impact of biological education on attributing higher importance to RVs and connectedness to nature, we compared the connection to nature scores (using the inclusion of nature scale (INS) and connectedness to nature scale (CNS)) and RV scores of biologically interested high school students (n = 417) with first year (n = 593) and advanced biology (n = 223) students. While high school students showed significant lower connection to nature scores than university students, there was no significant difference in RVs between the test groups. These results suggest that there is a lack of factors in the university study of biology that can change RVs. The gender comparison of RVs and connection to nature showed a significant higher RV score for females while INS and CNS did not show a gender difference. Thus, the study makes an important contribution to the research, as it was able to prove that gender has an influence on a person's RVs but not on their connection to nature.

Connecting High School Students With Nature - How Different Guided Tours in the Zoo Influence the Success of Extracurricular Educational Programs.

Zoos attract millions of visitors every year, many of whom are schoolchildren. For this reason, zoos are important institutions for the environmental education of future generations. Empirical studies on the educational impact of environmental education programs in zoos are still rare. To address this issue, we conducted two studies: In study 1, we investigated students' interests in different biological topics, including zoos (n = 1,587). Data analysis of individual topics revealed large differences of interest, with advanced students showing less interest in zoos. In study 2, we invited school classes of this age group to visit different guided tours at the zoo and tested connection to nature before and after each educational intervention (n = 608). The results showed that the guided tours are an effective tool to raise students' connection to nature. Add-on components have the potential to further promote connection to nature. The education programs are most effective with students with a low initial nature connection.

Exploring the Construct of Relational Values: An Empirical Approach.

In recent environmental research, relational values (RVs) have emerged as a new group of values to explain environmental behavior. Although this new concept is attracting attention, empirical studies on the subject are still rare. On this basis, we have conducted three studies to analyze an existing measurement tool for RVs and compared the construct with the concept of connection to nature. In study 1, we confirmed convergent and discriminant validity of the RV scale by comparing it with the Two Factor Model of Environmental Values (2-MEV) model using a sample of n = 350 university students. Additionally, study 1 verified reliability using test-retest reliability on three different groups of students (n 1 = 53; n 2 = 37; n 3 = 48). In study 2, principal component analyses were performed to examine the structure of RVs and to compare it to the concept of connection to nature by reusing the sample 350 university students from study 1. The results show that RVs and connection to nature are not fundamentally distinct constructs, but overlap. However, if the structure of the RV measurement is forced to a single factor, no perfect fit is found, making a multidimensional solution more likely. A third study was conducted to review the results from study 2 using confirmatory factor analysis on a new sample of 878 university and high school students. Study 3 confirmed RVs as a multidimensional construct with three factors: care, community, and connection. It also proved the overlap of the connection to nature and RV concepts to some extent.

Diurnal variation of salivary cortisol in captive African elephants (Loxodonta africana) under routine management conditions and in relation to a translocation event.

The present study assessed the diurnal variation in salivary cortisol in captive African elephants during routine management (baseline) and in relation to a potential stressor (translocation) to evaluate to what extent acute stress may affect diurnal cortisol patterns. Under baseline conditions, we collected morning and afternoon saliva samples of 10 animals (three zoos) on different days in two study periods (n = 3-10 per animal, daytime and period). Under stress conditions, we sampled the transported cow (newcomer) and the two cows of the destination zoo before and after the transport in the morning and afternoon (n = 3-9 per animal, daytime and transport phase), as well as after the first introduction of the newcomer to the bull (n = 1 per animal). Cortisol was measured in unextracted samples by enzyme immunoassay. Under baseline conditions, we observed the expected diurnal variation with higher cortisol levels in the morning than in the afternoon. Under stress conditions, neither a significant difference between pre- and posttransport, nor between morning and afternoon levels was found. The percentage difference between morning and afternoon cortisol after the transport, however, was remarkably lower than before the transport in the newcomer potentially indicating a stress response to familiarization. Saliva samples taken immediately after the introduction of the newcomer to the bull revealed a marked cortisol increase. Our findings indicate that stressors may disturb the diurnal cortisol rhythm. Furthermore, provided that samples can be collected promptly, salivary cortisol is a useful minimally invasive measure of physiological stress in the African elephant.

Functional properties and cell type specific distribution of I(h) channels in leech neurons.

The hyperpolarisation-activated cation current (I(h)) has been described in many vertebrate and invertebrate species and cell types. In neurons, I(h) is involved in rhythmogenesis, membrane potential stabilisation and many other functions. In this work, we investigate the distribution and functional properties of I(h) in identified leech neurons of intact segmental ganglia. We found I(h) in the mechanosensory touch (T), pressure (P) and noxious (N) neurons, as well as in Retzius neurons. The current displayed its largest amplitude in P neurons and we investigated its biophysical and pharmacological properties in these cells. I(h) was half-maximally activated at -65 mV and fully activated at -100 mV. The current mutually depended on both Na(+) and K(+) with a permeability ratio p(Na)/p(K) of ∼0.21. The reversal potential was approximately -35 mV. The time course of activation could be approximated by a single time constant of ∼370 ms at -60 mV, but required two time constants at -80 mV of ∼80 and ∼560 ms. The current was half-maximally blocked by 0.3 mmol l(-1) Cs(+) but was insensitive to the bradycardic agent ZD7288. The physiological function of this channel could be a subtle alteration of the firing behaviour of mechanosensory neurons as well as a stabilisation of the resting membrane potential.

Swelling-activated chloride channels in leech Retzius neurons.

During periods of high activity neurons are expected to swell due to the uptake of Cl(-). To find out whether leech Retzius neurons possess swelling-activated Cl(-) channels that facilitate Cl(-) efflux and, hence, volume recovery, we exposed the cells to hypotonic solutions. In hypotonic solutions, the cells slowly swelled but did not undergo a regulatory volume decrease. However, the cell volume increased less than predicted for an ideal osmometer, suggesting the action of a compensatory mechanism. The cell swelling was paralleled by a marked decrease in the input resistance as well as by the activation of a membrane current with a reversal potential close to the Cl(-) equilibrium potential. This current was substantially diminished by removing bath Cl(-), by applying the Cl(-) channel blocker DIDS, or by treating the cells with the tubulin polymerization inhibitor colchicine. Furthermore, in the presence of colchicine or vinblastine, the cell swelling was substantially increased. It is concluded that leech Retzius neurons possess swelling-activated Cl(-) channels that require an intact microtubule system for activation. The channels may help to restore cell volume after periods of high neuronal activity.

Ionic mechanism of ouabain-induced swelling of leech Retzius neurons.

By using electrophysiological and microfluorimetric methods, we found that leech Retzius neurons swell after inhibition of the Na(+)-K(+) pump by the cardiac glycoside ouabain. To explore the mechanism of this swelling, we measured the effect of ouabain on [Na(+)](i), [K(+)](i), and [Cl(-)](i), as well as on the membrane potential, by applying triple-barrelled ion-sensitive microelectrodes. As shown previously, ouabain induced a marked [Na(+)](i) increase, a [K(+)](i) decrease, and a membrane depolarization, and it also evoked an increase in [Cl(-)](i). The analysis of the data revealed a net uptake of NaCl, which quantitatively explained the ouabain-induced cell swelling. In the absence of extracellular Na(+) or Cl(-), NaCl uptake was excluded, and the cell volume remained unaffected. Likewise, NaCl uptake and, hence, cell swelling did not occur when the Na(+)-K(+) pump was inhibited by omitting bath K(+). Also, in K(+)-free solution, [Na(+)](i) increased and [K(+)](i) dropped, but [Cl(-)](i) slightly decreased, and after an initial, small membrane depolarization, the cells hyperpolarized for a prolonged period. It is concluded that the ouabain-induced NaCl uptake is caused by the depolarization of the plasma membrane, which augments the inwardly directed electrochemical Cl(-) gradient.

Ca2+ influx into identified leech neurons induced by 5-hydroxytryptamine.

The role of 5-hydroxytryptamine (5-HT, serotonin) in the control of leech behavior is well established and has been analyzed extensively on the cellular level; however, hitherto little is known about the effect of 5-HT on the cytosolic free calcium concentration ([Ca(2+)](i)) in leech neurons. As [Ca(2+)](i) plays a pivotal role in numerous cellular processes, we investigated the effect of 5-HT on [Ca(2+)](i) (measured by Fura-2) in identified leech neurons under different experimental conditions, such as changed extracellular ion composition and blockade of excitatory synaptic transmission. In pressure (P), lateral nociceptive (N1), and Leydig neurons, 5-HT induced a [Ca(2+)](i) increase which was predominantly due to Ca(2+) influx since it was abolished in Ca(2+)-free solution. The 5-HT-induced Ca(2+) influx occurred only if the cells depolarized sufficiently, indicating that it was mediated by voltage-dependent Ca(2+) channels. In P and N1 neurons, the membrane depolarization was due to Na(+) influx through cation channels coupled to 5-HT receptors, whereby the dose-dependency suggests an involvement in excitatory synaptic transmission. In Leydig neurons, 5-HT receptor-coupled cation channels seem to be absent. In these cells, the membrane depolarization activating the voltage-dependent Ca(2+) channels was evoked by 5-HT-triggered excitatory glutamatergic input. In Retzius, anterior pagoda (AP), annulus erector (AE), and median nociceptive (N2) neurons, 5-HT had no effect on [Ca(2+)](i).

L-type Ca2+ channel antagonists block voltage-dependent Ca2+ channels in identified leech neurons.

We investigated the effect of L-type Ca2+ channel antagonists on the Ca2+ influx through voltage-gated Ca2+ channels in leech Retzius, Leydig, AP, AE, P, and N neurons. The efficacy of the antagonists was quantified by monitoring their effect on the increase in the intracellular free Ca2+ concentration ([Ca2+]i; measured by Fura-2) that was induced by depolarizing the cell membrane by raising the extracellular K+ concentration. This K+-induced [Ca2+]i increase was blocked by the phenylalkylamines verapamil, gallopamil, and devapamil, the benzothiazepine diltiazem, as well as by the 1,4-dihydropyridine nifedipine. The blocking effect of the three phenylalkylamines was similar, being most pronounced in P and N neurons and smaller in Leydig, Retzius, AP, and AE neurons. Contrastingly, diltiazem and nifedipine were similarly effective in the neurons investigated, whereby their efficacy was like that of the phenylalkylamines in Retzius, Leydig, AP, and AE neurons. Depending on cell type and blocking agent, the concentrations necessary to suppress the K+-induced [Ca2+]i increase by 50% were estimated to vary between 5 and 190 microM. At high concentrations, the phenylalkylamines and diltiazem by themselves caused a marked [Ca2+]i increase in Leydig, P, and N neurons, which is probably due to activation of the caffeine-sensitive ion channels present in the plasma membrane of these cells. Together with previous observations, the results indicate a distant relationship of the voltage-gated Ca2+ channels present in many if not all leech neurons to vertebrate L-type Ca2+ channels.

Potentiometric measurement of cell volume changes and intracellular ion concentrations under voltage-clamp conditions in invertebrate nerve cells.

Because changes in cell volume might disturb the normal function of animal cells, most cells are endowed with volume-regulating mechanisms. Experimentally induced changes in cell volume are often paralleled by changes in the membrane potential, which might affect a variety of transport processes across the cell membrane and, in turn, volume regulation [1, 2]. We have shown previously that multi-barrelled ion-selective microelectrodes are useful tools for measurement of cell volume and the intracellular concentrations of ions that might be relevant to volume regulation [1]. To investigate whether voltage-dependent transport processes are involved in cell-volume regulation we combined the potentiometric technique of ion-selective microelectrodes with the voltage-clamp technique. This combination enables simultaneous recording of cell volume, relevant intracellular ion concentrations, and ion currents across the cell membrane at a fixed membrane potential.