Birds were long assumed to be anosmic or at best microsmatic. Today, it is well known that birds use olfaction in behaviours like foraging, mate choice, and kin recognition. In this context, scientists have started to elucidate the chemical space of avian olfaction to answer the research question which compounds drive olfaction-induced behaviour in birds. Chemical analysis in this field of research so far focused on the most abundant volatile compounds, of which only a few if at all possess an odour to human beings. However, it is not always the substances with high abundances that are most important for olfaction-induced behaviour, but rather trace compounds (see e.g. the mammary pheromone in rabbits 2-methylbut-2-enal). Since odorants are often trace compounds that may be not detected by mass spectrometry, researchers in aroma science apply olfaction-guided approaches, namely gas chromatography – olfactometry (GC-O), a method using the human sense of smell as a detector. In view of this, the overall aim of this thesis was to characterize the chemical composition of zebra finch odour by applying olfaction-guided approaches, which have not been previously used in this field, aiming to identify so far undetected substances that might influence behaviour. Three different sample types, namely preen oil, feathers, and bird’s overall odour (in the following described as whole body odour) were studied to reach this aim and to achieve an understanding of the similarities and differences between the chemical compositions of these sample types. In addition, the aim of this thesis was to summarise the state of knowledge in terms of the volatile composition of preen oil, feathers and whole body odour of different bird species. To elucidate the chemical composition of preen oil and feathers from zebra finches, samples were pooled, a solvent extraction of each sample pool was performed, and afterwards solvent-assisted flavor evaporation (SAFE) was applied for isolation of volatile compounds. The extracts were concentrated via distillation and analysed by gas chromatography – mass spectrometry (GC-MS), GC-O and heart-cut two-dimensional gas chromatography – mass spectrometry coupled with olfactometry (2D- GC-MS/O). The identified substances were divided into the sub-groups odour-active volatile compounds and further volatile compounds not being olfactorily perceived during GC-O. The results showed a substantial overlap in the volatile composition of preen oil and feathers, which is mainly characterized by alkanes, carboxylic acids, aldehydes, ketones, and linear alcohols. In total, 121 compounds were identified in preen oil and feathers out of which 95 respectively 49 have not been identified in zebra finches respectively any bird species before. The application of olfaction-guided approaches and classical GC-MS combined thus made it possible to identify numerous new substances in the preen oil and feather composition. Whole body samples were chosen as the third sample type to evaluate which compounds birds may encounter during real social interactions, where they are exposed not only to preen oil or feathers but to the entire bird, such as when a parent approaches the nest. The samples were collected by sampling the headspace above zebra finches using an open loop system equipped with either activated charcoal or Tenax ® TA as an adsorbent. The samples were eluted with a solvent, pooled, concentrated via distillation and afterwards analysed via GC-MS, GC-O, and 2D-GC- MS/O. Primarily the same substance classes were found as in zebra finch preen oil and feathers. 22 of the identified substances were not identified in zebra finch preen oil and feathers before and 12 of them not in any bird species before. Compounds being exclusively detected in zebra finch whole body samples (n = 22) could either originate from odour sources not sampled with feathers and preen oil, e.g. faeces, or might be detected due to the different work-up methods applied. A comparison of results obtained with the two adsorbents activated charcoal and Tenax ® TA showed overall comparable results. All in all, this study showed that the investigation of feathers and preen oil does not cover all potential candidate substances that could release a behavioural response and that whole body sampling should be used in addition or as an alternative. Additionally, two literature reviews on volatile organic compounds (VOCs) detected in preen oil and feathers and avian chemical signatures both in the volatile and non- volatile fraction of preen oil, feathers and whole body odour were prepared. In total, 39 studies on 41 different species were evaluated regarding VOCs and 43 studies about chemical signatures related to species, genetic distance/compatibility, individual identity, sex, breeding season and age. The substances identified so far include alkanes, alkenes, alcohols, aldehydes, ketones, carboxylic acids, aromatic or cyclic compounds, amines and amides, sulfides, and a few other compounds. Consistencies regarding chemical signatures could be identified in terms of substance classes, but not in terms of specific substances. Additionally, the review showed that chemical signatures are made up of several substances which supports the assumption that not only one substance makes up a chemical signature, but a specific composition of substances. The reviews further show that the composition of preen oil and feathers is basically the same across all bird species in terms of substance classes with differences in individual substances. In sum, the application of olfaction-guided approaches allowed for the identification of several substances, like ethyl octanoate, 1,3-benzothiazole and vanillin, which have not been described in bird odour before, but may play a role in avian behaviour. This work is therefore an important basis for the design of behavioural experiments, in which single substances respectively substance mixtures will be tested regarding their detection by zebra finches and potential behavioural effects. GC-O experiments with birds may complement such experiments, and will help to elucidate, together with other experiments, which substances are odour-active to birds. This thesis further provides a foundation for future studies on chemical signatures linked to individual traits and states in zebra finches, potentially transmitted via airborne communication, and offers reviews that help researchers compare new findings with the state of the art and follow the methodological guidelines provided.
Tatjana Alves Soares (Thu,) studied this question.