2004 TECHNICAL MEETING

"Analysis of hormones in droppings and egg-yolk in birds"

The organizers were Dr. Susi Jenni-Eiermann and Dr. Wolfgang Goymann, and the meeting was held at the Max Planck Institute of Ornithology in Seewiesen (Germany) from 21-22 October 2004.

The proceedings are published by the New York Academy of Sciences:

Bird Hormones and Bird Migrations: Analyzing Hormones in Droppings and Egg-Yolks and Assessing Adaptations in Long-Distance Migration. 2005. Wolfgang Goymann, Susanne Jenni-Eiermann & Ulf Bauchinger (Eds.), Annals of the New York Academy of Sciences 1046.

The articles are available online under http://www.nyas.org (if subscription to the Annals).

Alternatively, individual articles may be requested from the authors or the editors of the volume (see the item "Participants" to get their addresses).

Contents: 1. Bauchinger, U. & Goymann, W. 2005. Two workshops in Ornithology - A general introduction and dedication to Ebo Gwinner. 2. Goymann, W. & S. Jenni-Eiermann. 2005. Introduction to the ESF Technical Meeting: Analysis of hormones in droppings and egg-yolk of birds. 3. Klasing, K. C. 2005. Potential impact of nutritional strategy on non-invasive measurements of hormones in birds. 4. Möstl, E., S. Rettenbacher & R. Palme. 2005. Measurement of corticosterone metabolites in birds' droppings: An analytical approach. 5. Goymann, W. 2005. Non-invasive monitoring of hormones in bird droppings: physiological validation, sampling, extraction, sex differences, and the influence of diet on hormone metabolite levels. 6. Touma, C. & R. Palme. 2005. Measuring fecal glucocorticoid metabolites in mammals and birds: the importance of a physiological validation. 7. Palme, R. 2005. Measuring fecal steroids: guidelines for a practical application. 8. Baltic, M., Jenni-Eiermann, S., Arlettaz, R. & Palme, R. 2005. A non-invasive technique to evaluate human-generated stress in the Black Grouse. 9. Thiel, D., S. Jenni-Eiermann & R. Palme. 2005. Measuring corticosterone metabolites in droppings of Capercaillies (Tetrao urogallus). 10. Wasser, S. K. & K. Hunt. 2005. Noninvasive measures of reproductive function and disturbance in the barred owl, great horned owl, and northern spotted owl. 11. Hirschenhauser, K., K. Kotrschal & E. Möstl. 2005. A synthesis of measuring steroid metabolites in goose feces. 12. Scheiber, I., S. Kralj & K. Kotrschal. 2005. Sampling effort/frequency necessary to infer individual acute stress responses from fecal analysis in Greylag Geese (Anser anser). 13. Groothuis, T.G.G. & N. von Engelhardt. 2005. Investigating maternal hormones in avian eggs: Measurement, manipulation and interpretation. 14. von Engelhardt, N. & T.G.G. Groothuis. 2005. Measuring steroid hormones in avian eggs. 15. Rettenbacher, S., Möstl, E., Hackl, R. & Palme, R. 2005. Corticosterone in chicken eggs. 16. Benowitz-Fredericks, Z.M., Kitaysky, A. S. & Wingfield, J. C. 2005. Steroids in allantoic waste – an integrated measure of steroid exposure in ovo.

           

Preamble

The analysis of hormones in faeces (mammals) or excrements (birds) is a new non-invasive method to get insight into an animal‘s physiology. Endocrinological cycles of free-living animals can be studied without the difficulty of capture and the corresponding stress and disturbance. Hence, the technique may be helpful in a wide range of research fields relating to physiology, behavioral ecology, ethology and wildlife conservation. However, there are inconsistencies in the methods published. One main problem is the fact, that hormones are extensively metabolised before excretion. Steroids, for instance, are not only metabolized in the liver, but also by intestinal bacteria which can affect the structure of the metabolites. The correct metabolite and antibody have first to be determined. Other problems are connected with the sampling of the excrements, especially in free-living animals. A quite substantial amount of studies gained experience with domestic and zoo animals and with free-living mammals. However in birds, new problems might occur, because birds excrete a mixture of urine and faeces and – compared to mammals – mostly in small amounts only. These practical problems have raised the idea to discuss the method and its application in a group of experts and to write up a report of the meeting.

Summary 

Twelve presenting scientists of 5 European countries (Austria 6, Germany 2, Great Britain 1, Switzerland 1, The Netherlands 2), 3 presenting scientists from the United States and 5 non-presenting scientists (from the MPIO) have met at the Max Planck Institute for Ornithology in Seewiesen, Germany to participate in the ESF technical meeting: “Analysis of hormones in droppings and egg-yolk in birds” (20 - 23 October 2004). The analysis of hormones in excrements of birds is a relatively new, non-invasive method to obtain insights into an animal‘s physiology without causing stress due to handling. However, there are inconsistencies in the methods published, both in the biochemical procedures and in the sampling protocols. Therefore, the aim of the workshop was to discuss the method and its application within a group of experts and to evaluate the best procedures.

The diverse research and educational background of the participants enabled to cover the entire range of topics and aspects that are all relevant for these methods. Evolutionary and behavioural ecologists, conservation biologists, ethologists, biochemists as well as a specialist in immunology and avian nutrition presented their results under a methodological aspect, each during a 30-45 min talk. Three main topics were covered: (1) biochemical and organismal aspects of the method, (2) sampling methodology and assay techniques and (3) excrements, egg yolk, steroids and what lies beyond...

The small size of the group as well as the friendly atmosphere allowed an open, effective and frank discussion. Moreover, as a side-effect, co-operations between laboratories for new projects were agreed upon during the many informal discussions. In a final discussion it was agreed to publish proceedings of this meeting with an introductory chapter, the results presented in the talks and a reference list of the most relevant publications. Last but not least, the participants agreed to publish guidelines on how to determine hormones in droppings and egg-yolk of birds. The journal  “Annals of the New York Academy of Sciences” has agreed to publish the proceedings of this technical meeting (deadline 6 January 2005).

Scientific content of and discussion at the event

Recently the use of non-invasive methods to measure hormones in excrements of birds has gained substantial importance. The quantification of hormones provides insights into the reproductive stage, behaviour and responses to disturbances in the life cycle of an animal. Hence, the method is of interest for a wide range of research disciplines, both basic and applied, such as physiology, ethology, behavioural ecology, stress research, veterinary research, reproductive and zoo biology, and conservation biology.

These non-invasive methods have the major advantage that samples can be collected repeatedly without any handling stress. Traditionally, hormones are analysed from blood samples of animals, a procedure which usually implies the capture of the animal. Thus, blood sampling is usually restricted to one or few samples per individual, as repeated catching and sampling may represent a considerable stressor causing disturbance and change in behaviour. Another advantage of the non-invasive methods is that hormones can be analysed retrospectively over the last hours. 

However, there are inconsistencies in the methods and sampling procedures published. One main problem is the fact, that hormones are extensively metabolised before excretion. Another difficulty is the sampling of the excrements, especially in free-ranging animals. Many studies gained experience with domestic and zoo animals and with free-ranging mammals. In birds, however, additional problems might occur, because birds excrete a mixture of urine and faeces and – compared to mammals – mostly in small amounts. Also, researchers should be aware of endogenuous rhythms, both daily and annual, that may differ between species, sexes and individuals. 

          The aim of the technical meeting was to gather and discuss these inconsistencies and problems. Procedures and sampling protocols providing the best and most consistent measurements were evaluated.  

The first part of the meeting covered the subject: Biochemical and organismal aspects of the method. The effect of the diet and the digestive strategy on the decomposition of the steroid hormones was discussed. Nutrition and digestion have an important impact on the catabolism of the hormones and their detectability in droppings, especially in birds, because birds excrete urine and faeces together into the cloaca. Moreover urine is flowing back into the rectum up to the caeca for reabsorption of water. Hence, urine and faeces are mixed and the excretion of the hormones via urine and faeces cannot be distinguished. Other factors influenced by diet and digestion are the frequency of voiding and the amount of the droppings which might change the biochemical structure of the metabolised hormone. The pulsative secretion of the bile may result in qualitative and quantitative differences of hormone metabolites from dropping to dropping. Besides these chemical aspects of hormone metabolism, individual and sex differences, behaviour or social status need to be considered. These factors potentially influence the concentration of the hormone metabolites. It became clear, that - before applying these methods – the biology of the species studied has to be considered carefully. 

The second part of the first day was devoted to the subject: Sampling methodology and assay techniques. In several talks data were presented demonstrating the influence of a variety of environmental factors on the catabolism of hormones. Ambient temperature, air pressure or precipitation might change the concentration of hormone metabolites. It was shown that these difficulties can be handled with a strict sampling protocol, the collection of a sufficient amount of droppings and a thorough homogenisation of the droppings. The fact that hormone concentrations fluctuate in a species specific diurnal and annual rhythm also has to be taken into consideration when collecting and assaying droppings.

The question whether assays could be standardized (same antibodies, same assay protocol) to allow a comparison of the concentrations between different laboratories was discussed. This, however, was considered to be too complex. It became clear that a standardisation of the assays is not the first priority, because most research groups work with different species which might show species-specific concentrations. For most studies relative concentrations reflecting the effect to an experimental protocol are sufficient. It was emphasized, however, that these limitations may make it difficult to do absolute and relative comparisons across species and populations when they were measured with different antibodies.

 Another issue was the influence of diet and metabolic rates on the excretion of hormones. There are only few studies regarding these aspects and as far as the participants knew, not a single published study on birds. Related to this topic it was discussed whether there are alternatives to the commonly used wet or dry weight as a reference standard. For laboratory studies the excreted amount of steroid metabolites per time period sometimes may represent an alternative, for studies of free-ranging animals, this reference standard may be difficult to apply.

The second day of the meeting was devoted to the subject: Excrements, egg yolk, steroids and what lies beyond... Several studies were presented about the effects of hormones in the egg on growth and behaviour of the chick. Naturally occurring hormone concentrations were measured and related to patterns of growth and behaviour. It became evident, that many fundamental questions are not resolved yet, especially whether the secretion of the hormones represents an active or passive transfer from the mother to the egg. Another problem is raised by the fact, that the hormones are not equally distributed in the egg, but are found in layers. Hence it is not clear, whether the embryo is exposed to different hormone concentrations at different developmental stages or whether the yolk is mixed when the capillary growth of the embryo starts and the yolk is infiltrated. Moreover, one study showed that hormones infuse the albumen when dipped into a hormone solution, hence the albumen also might play a role. Measuring the whole egg content might resolve this problem. A promising new approach was presented by a study about hormone analyses in the allantoic waste, thus measuring the metabolites of maternal and embryonic hormones retrospectively after hatching.

Studies injecting hormones into the egg also face the problems mentioned above. It is unknown in which layer of the egg-yolk the hormone is injected and whether this might have an impact on the development of the embryo. It was also pointed out, that only physiological concentrations should be injected, hence, that the naturally occurring concentration should be known before the experiment. Otherwise measured effects might reflect nothing but pharmacological effects.

So far, mainly steroids such as corticosterone and sex hormones were measured in bird excrements or egg-yolk. One future aspect was whether it would soon be possible to measure catecholamines in droppings, hormones that respond so quickly to stress that it is impossible to measure basal concentration in the blood by conventional blood sampling. One study showed that catecholamines are excreted in chickens. However, the concentrations were so low that an assay for these metabolites cannot be expected in the near future.

Another question for future developments was whether other tissues than droppings, egg yolk or allantoic waste are suited to measure hormones. In birds feathers might offer new possibilities, however, no validation studies were done so far.

         In a final discussion it became clear that the determination of hormones in droppings and egg-yolk is not as simple and straight-forward to apply as it might appear to some investigators. However, given a careful validation, the non-invasive methods offer the potential to study a variety of questions of high interest which could otherwise not be studied. A consensus among the participants was, that it is absolutely necessary to make this point clear to all who intend to apply these methods. Otherwise potentially erroneous results will end in a discredit of the whole method. Therefore all participants agreed to publish not only the results presented in the talks, but also guidelines of the critical validation steps and a reference list of the published literature in the “Annals of the New York Academy of Sciences”.  

Assessment of the results and impact of the event on the future direction of the field

For the first time a workshop about the non-invasive methods of measuring hormone metabolites in excrements and egg-yolk was carried out. Reports of completely different research directions using the same method were given, all under the aspect of the methodology. The different experimental designs, the variety of bird species used and the differing working conditions (field and lab experiments) provided a complete overview of the current knowledge about the non-invasive methods. Non-invasive sampling is often very easy, as the collection of droppings does not require special permits or capture of the animals, and thus might tempt some researchers to underestimate the tricks and pitfalls of the technique. Biological validations are essential and many technical factors related to sampling and the biology of the species studied need to be taken into account before the method can be used. The strong request of the participants of this workshop was that all scientists using this method in the future should be aware of and follow the necessary validation instructions. If properly validated, the non-invasive technique to determine hormones is a promising technique with great potential. To encourage other scientists and pass on the knowledge that was gathered at this workshop, it was decided to write guidelines about how to evaluate the method and to list the most relevant publications. The proceedings including these guidelines and most relevant publications will be published in the “Annals of the New York Academy of Sciences.

Wednesday 20 October 2004       Arrival in Seewiesen 

Due to construction works in Seewiesen, our guest room facilities in Seewiesen are temporarily closed. Thus, housing will be outside of Seewiesen, in the guest house of the Beamtenfachschule in Herrsching, about 10km from Seewiesen. Busses will be available for your transportation.

 After the get-together the busses will bring you to the guest house and they will pick you up from there the following morning at 8:10h in the morning. After the afternoon session the busses will bring you back to the guest house so that you have a bit of time to rest before dinner.

 Breakfast will be served at the guest house and lunch will be provided at the Mensa of the Institute. For dinner, we will go to a local Gasthof, so that you get the opportunity to taste true Bavarian food and (maybe even more important) beer. The costs for accommodation, breakfast and lunch are covered by the meeting grant, the dinners are not included. 

Thursday 21 October 2004

Biochemical and organismal aspects of the method

Sampling methodology and Assay techniques 

Summary of first day and general discussion

Friday 22 October 2004

Excrements, egg-yolk, steroids and what lies beyond....

Saturday 23 October 2004