Dolphin-WET-Development of a Welfare Evaluation Tool for Bottlenose Dolphins (Tursiops truncatus) under Human Care.

Ensuring high standards of animal welfare is not only an ethical duty for zoos and aquariums, but it is also essential to achieve their conservation, education, and research goals. While for some species, animal welfare assessment frameworks are already in place, little has been done for marine animals under human care. Responding to this demand, the welfare committee of the European Association for Aquatic Mammals (EAAM) set up a group of experts on welfare science, cetacean biology, and zoo animal medicine across Europe. Their objective was to develop a comprehensive tool to evaluate the welfare of bottlenose dolphins (Tursiops truncatus), named Dolphin-WET. The tool encompasses 49 indicators that were either validated through peer review or management-based expertise. The first of its kind, the Dolphin-WET is a species-specific welfare assessment tool that provides a holistic approach to evaluating dolphin welfare. Inspired by Mellor's Five Domains Model and the Welfare Quality®, its hierarchical structure allows for detailed assessments from overall welfare down to specific indicators. Through combining 37 animal-based and 12 resource-based indicators that are evaluated based on a two- or three-level scoring, the protocol offers a detailed evaluation of individual dolphins. This approach allows for regular internal monitoring and targeted welfare management, enabling caretakers to address specific welfare concerns effectively.

Effects of Storage Time and Thawing Method on Selected Nutrients in Whole Fish for Zoo Animal Nutrition.

Piscivores in human care receive whole fish that were frozen, stored and thawed before feeding. Nutrient losses have been documented, but exact changes during storage and with different thawing methods are unknown. Primarily, it was hypothesized that frozen fish lose different vitamins and trace minerals during a storage period of six months. Secondly, that different thawing methods have a significant influence on the degree of vitamin loss. Three fish species, herring (Clupeus harengus), mackerel (Scomber scombrus) and capelin (Mallotus villosus) were analyzed at four time points within a storage period of 6 months at -20 °C. At each time point, three thawing methods were applied: thawing in a refrigerator (R), thawing at room temperature (RT), and thawing under running water (RW). The following nutrients were analyzed: vitamin A, B1, D3 and E, iron (Fe), copper (Cu), zinc (Zn) and selenium (Se). The statistical method used was a linear mixed effect model. Cu was below detection limits in all analyzed samples, vitamin B1 in most analyzed herring (44/48 samples) and capelin (in 25/36 samples), respectively. In addition, the vitamin D3 concentration was also below detection limits in half of the capelin samples (18/36). No concentration changes of Fe (p = 0.616), Zn (p = 0.686) or Se (p = 0.148) were observed during a storage period of six months, in contrast to a significant decrease in vitamin A (p = 0.019), D3 (p = 0.034) and E (p = 0.003) concentrations. Thawing fish with different thawing methods did not result in concentration changes of Fe (p = 0.821), Zn (p = 0.549) or Se (p = 0.633), but in a significant concentration change of vitamin A (p = 0.002). It is essential to supplement vitamins B1 and E in diets containing whole fish to avoid deficiencies in piscivorous species, and care should be taken not to store fish longer than six months, due to the depletion of vitamins A, D3 and E.

A pilot study about assisted reproduction in harpy eagles (Harpia harpyja) in the course of species conservation including collection, storage, and analysis of semen.

Currently, 52% of all raptor species demonstrate a decreasing population tendency, and the American harpy eagle (Harpia harpyja) has been categorized as "near threatened" by the IUCN. Habitat loss, persecution, and subsequent reduction of genetic diversity are regarded as major threats to the world's strongest eagle. Captive breeding and reintroduction into protected habitats are approaches of species conservation projects, but captive propagation is difficult due to low ex-situ numbers and scarce successful breeding pairs. The aim of the present study was to collect, analyze, and store semen from harpy eagles and to use aliquots for artificial insemination to increase the number of offspring and to include more individuals into the ex-situ gene pool. First, semen collection and semen availability were assessed in four males during the course of 1 year in European zoos. Second, these experiences were transferred to ex-situ breeding programs in Brazilian zoos to attempt semen collection in 13 male eagles. Semen collection was successful in 51.7% of the attempts and in 8/13 males (individual success rates 20-100%) using electro-stimulation. Most commonly, whey-like to milky, whitish semen samples were collected, regularly containing urate impurities (67.7%). The median semen volume was 106 µl and the median sperm concentration 5,000 sperm/µl (750-22,500 sperm/µl). Mean values for pH were 6.7, for sperm motility 27.7 ± 22.6%, for progressive motility 2.9 ± 5.6%, and for sperm viability 46.6 ± 16.3%. Using semen extenders, a sperm motility of 8% was maintained for 27 h in the refrigerator. Artificial insemination was performed in one female, but the success of fertilization could not be assessed due to egg destruction. In this study, methods for assisted reproduction were refined for use in harpy eagles, and the first semen samples were evaluated as a start to establish species-specific orientation values.

Comparison of Two Different Feather Sampling Methods to Measure Corticosterone in Wild Greater Flamingos (Phoenicopterus roseus) and Wild Mallards (Anas platyrhynchos).

This research project had the aim to validate the possible alternative and less-painful sampling method of cutting feathers close to the skin instead of plucking them for subsequent feather corticosterone analysis, confirming recently-published results for other species in captivity. Analyzing CORTf is often used in animal welfare studies in combination with behavioral monitoring. The background of this idea was to act in the sense of animal welfare and reduce the burden of animal studies according to the 3-R-Principle (Replacement, Reduction, and Refinement) by refining procedures. To confirm the hypothesis that the sampling method itself has no influence on CORTf levels measured, plucked and cut samples of the respective bird were collected. Birds of two wild species were used: the Mallard (Anas platyrhynchos) and the Greater Flamingo (Phoenicopterus roseus). The CORTf was measured by using an enzyme-linked immunosorbent assay (ELISA). The determined values were inspected for their mean values, standard deviation (SD), and average differences. Afterwards, the CORTf levels of both species were compared, according to the sampling method, with the concordance correlation coefficient (CCC). In the Bland-Altman (BA) plot the differences of the methods were displayed against the mean values. Additionally, sex, as a possible factor influencing CORTf, was analyzed using the Mann-Whitney U test. The values of CCC showed poor agreement in the comparability of the two methods, whereas the concordance of the BA plot was decent. The average differences between the methods were marginal for both species (Mallards: -0.16 pg/mm, Flamingos -0.13 pg/mm). In summary, all anomalies or differences between the methods were negligible. Therefore, the alternative sampling method seems to be as suitable as the common standard method. No significant difference was found between females and males. Nevertheless, our results suggest that CORTf should not be interpreted in just considering the values themselves, but the results they should be analyzed in the context of a wider set of parameters. Hence, further studies are encouraged to create a larger data pool.

Feather Corticosterone Measurements and Behavioral Observations in the Great White Pelican (Pelecanus onocrotalus) Living under Different Flight Restraint Conditions in German Zoos.

The pinioning of birds was previously one of the most-accepted forms of mutilation in zoos. Despite a lack of knowledge on the effects of deflighting procedures with regard to the well-being of deflighted birds, pelicans are often reversibly deflighted by feather-clipping to keep them in open enclosures, including those with ponds without netting. In the present study, we focused on the welfare implications of flight restraint on one of the most commonly kept types of birds in German zoos, the great white pelican. A combination of behavioral observations and feather corticosterone concentrations (CORTf) of pelicans with different deflighting statuses (i.e., irreversibly deflighted, reversibly deflighted, and airworthy) was used to evaluate the effects of deflighting status on pelican welfare. We observed 215 individuals in 21 different German zoos. The pelicans lived in differently designed exhibits. An ethogram for these species was developed and their behavior was evaluated by scan sampling. Feather samples from 182 individuals were collected to determine if different deflighting conditions influenced the CORTf and therefore stress levels. The hypothesis was that the CORTf values of airworthy pelicans differ from those of deflighted pelicans. Tendencies with regard to the flight status groups were found. Conversely, reversibly deflighted pelicans had higher CORTf levels than irreversible deflighted and airworthy pelicans. Tendencies with regard to CORTf values and the group size of the kept pelicans were observed. The CORTf values were lower in groups consisting of more than 10 animals. In addition, the frequency of fluttering behavior was positively associated with CORTf values. Pelicans that frequently showed fluttering had higher CORTf values. Therefore, fluttering behavior might be considered a sign of stress levels in pelicans. This study is one of the first important steps in assessing the impact of deflighting procedures on the welfare of great white pelicans kept in zoos.

Activity Budget Comparisons Using Long-Term Observations of a Group of Bottlenose Dolphins (Tursiops truncatus) under Human Care: Implications for Animal Welfare.

Zoos and aquaria must provide optimal husbandry conditions and the highest welfare standards for their animals. How the welfare state of an animal or a group of animals can be precisely assessed is currently under debate, and new approaches are necessary to reliably evaluate changes in welfare. One particular measure that is easily applicable is behavioral observations. However, for dolphins and other cetaceans under human care, reliable behavior-based measures are rare. Using long-term observations of a group of bottlenose dolphins, we investigate how their activity budgets and different behaviors changed over time and are impacted by construction noise. Additionally, we investigate whether behavioral differences are also reflected in changes in the dolphins' performance during daily training sessions. Our results show that construction noise significantly alters the dolphins' behavior. Play behavior decreases during construction periods; most importantly, the frequency of fast swimming activities significantly increased, and at the same time, a decrease in training performance is found. Additionally, inter- and intraindividual behavioral differences are attributed to factors, such as age or weaning. Significant changes in a dolphin's activity budget can also pose potential welfare concerns. Thus, this study highlights the importance of regularly assessing and analyzing the behavior of dolphins under human care. Behavioral observations are essential welfare indicators and can-when complemented with other measures, such as assessment of training performance-provide zoo staff with important information about each individual's state of welfare.

Behavioural Diversity Study in Bottlenose Dolphin (Tursiops truncatus) Groups and Its Implications for Welfare Assessments.

In the recent past, animal welfare studies have tried to determine the best animal welfare measures and indicators. Expression of behavioural diversity is considered a potential positive welfare indicator, and to the authors' knowledge, it has not been validated nor studied in cetaceans. For the first time, a behavioural diversity study on bottlenose dolphins (Tursiops truncatus) groups was conducted at six European facilities. The study was carried out by the animal care staff, biologists and veterinarians and included 54 dolphins housed in several group compositions at the different participating facilities. The goal of our study was to analyse behavioural diversity in bottlenose dolphins at the group level to investigate how particular factors might impact the diversity of behaviours within the group and to discuss its implications for dolphin welfare assessments. Eight factors (i.e., "observer location", "number of individuals", "age class", "sex", "social grouping", "presence/absence of leading male", "presence/absence of visitors" and "enrichment provision") impacted the behavioural diversity of the observed groups, while no significant impact of the factors "time of day" and "activity before/after observation" could be found. Our study showed the feasibility of this kind of approach for cetaceans under professional care and the relevance to considering this parameter in dolphin welfare studies, despite certain limitations that warrant further research.

Analysis of hair steroid hormones in polar bears (Ursus maritimus) via liquid chromatography-tandem mass spectrometry: comparison with two immunoassays and application for longitudinal monitoring in zoos.

Analysis of hair cortisol concentrations (HCCs) is a promising method for monitoring long-term stress in mammals. However, previous measurements of HCCs in polar bears (Ursus maritimus) have yielded highly variable results, which are likely due to different methodological approaches. In this study, hair samples of zoo-housed polar bears were analyzed for cortisol with two independent immunoassays [an enzyme-linked immunoassay (EIA) and a chemiluminescence assay (CLIA)] and liquid chromatography-tandem mass spectrometry (LC-MS/MS). HCC measurements depended significantly on assay type applied, sample processing (cutting vs. powdering hair) and their interaction. Best agreement was observed between LC-MS/MS and CLIA (R2 = 0.81 for powdered hair) and sample processing had a minor, albeit significant, effect on obtained HCC measurements in these assays (R2 > 0.9). EIA measurements were consistently higher than with the other assays. HCC measurement was validated biologically for CLIA and LC-MS/MS in one male polar bear that experienced considerable stress for a prolonged period of time (> 18 weeks). Subsequently, by using the validated LC-MS/MS the measurement of cortisol could be complemented by the analysis of other steroids including cortisone, testosterone and progesterone levels from hair samples collected over a 9-month period (5-13 months) from six zoo-housed polar bears (five males, one female). No seasonal steroid variation was observed except in male progesterone levels. For all steroids except cortisone, a strong body region effect (neck or paw) was observed. Cortisol and cortisone, as well as progesterone and testosterone, concentrations were positively correlated. We show that hair steroid concentrations can be used to longitudinally measure stress and reproductive hormone axes in polar bears. The data established herein provide important basic information regarding methodology and study design for assessing hair steroid hormones.

Saliva and Blood Cortisol Measurement in Bottlenose Dolphins (Tursiops truncatus): Methodology, Application, and Limitations.

A central task of zoos and aquaria is the frequent and accurate assessment of their animals' welfare. Recently, important steps have been made, such as the introduction of animal welfare evaluation tools and welfare decision trees. To determine animal welfare, it is not only important to collect life history data, such as longevity and reproductive success, but also for experienced observers or caretakers to conduct behavioral observations on a regular basis to assess animals' emotional state. To physiologically validate welfare observations, glucocorticoid levels are usually assessed, as they are a common indicator of stress. While, for many animals, these levels can be easily determined via fecal or hair samples, for cetaceans, the levels are usually determined via blood samples. As blood samples cannot be taken very frequently and the process may cause stress to the animals (if the samples are not taken following medical training), other techniques, such as the measurement of health biomarkers (especially cortisol, which can be measured in saliva), have become the focus of cetacean stress research. However, there are two problems associated with saliva measurements in cetaceans: saliva might either be diluted with pool water or be contaminated by fodder fish, as frozen fish usually contains high levels of cortisol. In our study, we investigated how saliva cortisol levels are connected to blood cortisol levels and how saliva cortisol can be influenced by fodder fish. We examined saliva and blood samples in eleven bottlenose dolphins (Tursiops truncatus) kept in an outdoor and indoor facility in Germany. Furthermore, we assessed the cortisol levels of different kinds of fodder fish. Our data show that, although saliva cortisol values are elevated under stress and arousal, they seem not to be correlated with blood cortisol values. We also show that, after feeding, saliva cortisol values are increased up to 100-fold. Our results suggest that saliva cortisol measurements in dolphins have to be conducted and considered with care, as they can easily be contaminated. Moreover, it is important to use the right laboratory method in order to specifically detect cortisol; in our study, we conducted reliable tests, using LC-MS/MS.

Validation of an Alternative Feather Sampling Method to Measure Corticosterone.

The most common feather sampling method for feather corticosterone measurement is by plucking the feathers from the bird's skin. This procedure performed on living, restrained birds is qualified as an animal experiment according to German/European legislation, which has to be applied for from the competent authorities. The Directive 2010/63/EU requires the full implementation of the 3-R Principle of Russel and Burch in animal experiments, which means not only to replace the use of animals, but also to reduce the number of animals used and to refine procedures whenever possible. In response to this issue, the aim of this study was to validate an alternative, less invasive sampling method by cutting feathers close to the skin in comparison to the gold standard of plucking them. For this proof-of-principle study, a conventional poultry husbandry with trial groups of geese (Anser anser domesticus) and ducks (Anas sterilis) was selected. All birds were kept under the same living conditions to standardize the influencing factors regarding husbandry, and thus, their stress levels. Feather samples were collected between the shoulders from 46 geese and 51 ducks, both by cutting as well as by plucking, directly after slaughter for meat production. Feather corticosterone levels were measured with Enzyme-Linked Immunosorbent Assay (ELISA). Results were compared using Bland-Altman plots and concordance correlation coefficients (CCC). It could be seen that concordance between corticosterone levels in cut and plucked feathers was rather poor: 0.38 for Anser, and 0.57 for Anas. However, comparing the mean corticosterone values in pg/mm of each species with their respective standard deviations, the differences between the methods were negligible. As the results showed that the differences between the individuals were markedly greater than the differences between the methods, the determination of corticosterone levels in cut feathers is valid compared to using plucked feathers. The validation tests of ELISA showed only acceptable repeatability and reliability. Hence, the results should be verified in further studies. In conclusion, it is recommended for future research to use cut instead of plucked feathers for corticosterone measurement.

Faecal glucocorticoid metabolites as a measure of adrenocortical activity in polar bears (Ursus maritimus).

Analysis of faecal glucocorticoid metabolites (FGMs) is frequently applied to assess adrenocortical activity in animal conservation and welfare studies. Faecal sample collection is non-invasive and feasible under field conditions. FGM levels are also less prone to circadian rhythms, episodic fluctuations and short acute stressors than glucocorticoid (GC) levels obtained from other matrices, for example blood or saliva. To investigate the suitability of FGM measurement in polar bears (Ursus maritimus), a species listed as Vulnerable by the IUCN (International Union for Conservation of Nature), a cortisol enzyme immunoassay (EIA) was biologically validated by demonstrating a significant increase in FGMs after five zoo-to-zoo transports. In addition to validating the method, the study also documented an average delay of 7 h until the first occurrence of food colorants in the monitored polar bears, which provides essential information for future studies. After validation, the assay was applied to measure FGM concentrations of five polar bears over a 1-year period. Several pre-defined potentially stressful events were recorded in an event log to measure their effect on FGM concentrations. A mixed model analysis revealed significant increases in FGM concentrations after social tension and environmental changes, whereas season and sex had no significant effect. The study demonstrates that the applied cortisol EIA is suitable for measuring FGM levels in polar bears and that using a carefully validated assay for FGM analysis in combination with a detailed sampling protocol can serve as a valuable tool for evaluating mid- to long-term stress in polar bears. FGM levels can be used to monitor stress in captive polar bears in order to optimize housing conditions but also to elucidate stress responses in wild populations for targeted conservation measures.

Feather Corticosterone Measurements of Greater Flamingos Living under Different Forms of Flight Restraint.

Deflighting zoo birds is a practice that receives increasing criticism due to its presumed incompatibility with animal welfare. To our knowledge, this is the first approach to address this problem in a scientific way. To do this, we compared feather corticosterone (CORTf) from Greater Flamingos (Phoenicopterus roseus, n = 151) of different flight status (i.e., pinioned, feather clipped, airworthy) from twelve different zoological institutions. To complement the hormone measurements, behavioral observations (scan samplings) were conducted prior to feather sampling within the presumed time frame of feather growth. We hypothesized that CORTf of the deflighted flamingos would differ from CORTf of their airworthy conspecifics. No significant difference in CORTf was found between the three groups, and our hypothesis was rejected. However, the impact of the institution itself (i.e., the housing conditions) proved to be the most dominant variable (variance between the institutions = 53.82%). Due to high variability, the behavioral observations were evaluated descriptively but did not give rise to doubt the findings in CORTf. Therefore, we assume that the method of flight restraint of Greater Flamingos does not have a measurable effect on CORTf. We consider this model for evaluating animal welfare of zoo birds a useful tool and provide ideas for further adjustments for consecutive studies.

Vitamin blood concentration and vitamin supplementation in bottlenose dolphins (Tursiops truncatus) in European facilities.

BACKGROUND: As fish eaters bottlenose dolphins (Tursiops truncatus) in human care need to receive daily vitamin supplementation, because whole thawed fish lacks certain vitamins. However, the exact concentration of supplementation has not been established and is a matter of discussion. To ensure adequate vitamin supplementation in pets, vitamin blood concentrations are measured. This is not a common practice in dolphins. The objective of the present study was to collect information about vitamin supplementation in bottlenose dolphins and on vitamin blood concentrations of healthy animals in European facilities. In addition, these results were compared with blood levels of wild animals. Conclusions on how to provide bottlenose dolphins in human care with an effective vitamin supplementation will then be drawn. Initially, fish-handling techniques and vitamin supplementation were evaluated by questionnaire, which was sent to 25 European facilities that house bottlenose dolphins. Secondly, blood samples from 57 dolphins living in 10 facilities were taken and sent by mail to a reference laboratory. They were analysed for retinol, thiamine pyrophosphate, cobalamin, calcidiol and tocopherol. The blood concentrations were then correlated with vitamin supplementation, fish handling techniques and pre-existing blood concentrations of free-ranging dolphins. Finally, the data was subjected to a standard analysis of variance techniques (ANOVA) and a linear model analysis. RESULTS: Fish was mainly thawed in a refrigerator. Further, the 95 % confidence interval for retinol blood concentrations was 0.048 to 0.059 mg/l and for tocopherol 17.95 to 20.76 mg/l. These concentrations were 27 and 53 %, respectively, higher than those found in free-ranging animals. In contrast, calcidiol concentrations (143.9-174.7 ng/ml) of the dolphins in human care were lower than in blood found for free-ranging animals. Regarding thiamine pyrophosphate and cobalamin, concentrations ranged between 0.42 and 0.55 mg/l and 175.55 and 275.22 pg/ml respectively. No reference concentrations for free-ranging Tursiops truncatus were found. CONCLUSIONS: These findings suggest an over-supplementation of retinol (vitamin A) and tocopherol (vitamin E) in bottlenose dolphins (Tursiops truncatus) housed in human care. Therefore, vitamin A supplementation should not exceed 50,000 IU per animal per day and vitamin E supplementation should be around 100 IU per kg fed fish per day.

Zebra-borne equine herpesvirus type 1 (EHV-1) infection in non-African captive mammals.

Equine herpesvirus type 1 (EHV-1) was detected in an Indian rhinoceros (Rhinoceros unicornis), which was euthanized because of severe neurological disease. Encephalitis was suspected and EHV-1 DNA was detected in brain, lung, and spleen tissues. The viral IR6 protein was detected in lung tissues by Western blot analysis. Phylogenetic analyses of EHV-1 sequences amplified from various tissues was nearly identical to one recently described that resulted in both non-fatal and fatal encephalitis in polar bears. This represents transmission of EHV-1 to a species that is not naturally sympatric with the natural host of the virus and broadens the host range to Asian non-equid perissodactyls.

Biochemical and immunohistochemical characterization of feline spongiform encephalopathy in a German captive cheetah.

Feline spongiform encephalopathy (FSE) is a transmissible spongiform encephalopathy that affects domestic cats (Felis catus) and captive wild members of the family Felidae. In this report we describe a case of FSE in a captive cheetah from the zoological garden of Nuremberg. The biochemical examination revealed a BSE-like pattern. Disease-associated scrapie prion protein (PrP(Sc)) was widely distributed in the central and peripheral nervous system, as well as in the lymphoreticular system and in other tissues of the affected animal, as demonstrated by immunohistochemistry and/or immunoblotting. Moreover, we report for the first time the use of the protein misfolding cyclic amplification technique for highly sensitive detection of PrP(Sc) in the family Felidae. The widespread PrP(Sc) deposition suggests a simultaneous lymphatic and neural spread of the FSE agent. The detection of PrP(Sc) in the spleen indicates a potential for prion infectivity of cheetah blood.