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.

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.

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.

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.