Student Conference on Conservation Science-Bengaluru 2016
A basic workshop on:
Use of non-invasive techniques to understand the conservation physiology of free-ranging animals
Sanjeeta Sharma Pokharel1, Sukesh Bhupathi2 1
2
Centre for Ecological Sciences, Department of Molecular Reproduction and Developmental Genetics Indian Institute of Science, Bangalore For further details, contact:
[email protected] [email protected]
Background: Wikelski and Cooke (2006) defined “conservation physiology” as ‘the study of physiological responses of organisms to human alteration of the environment that might cause or contribute to population decline'. Conservation of any concerned taxa requires in-depth knowledge about their ecology, including the key interdependent facets like behavior, habitats and their physiology. Hence, it necessitates understanding and assessing how physiological aspects play a role in providing the holistic frame for the better conservation of a target species. With this rising need of hours- for understanding the behavior of the wild-animals, many conservationists around the world have now started studying the physiological manifestation as well. In general, under the conservation physiology; we study the hormonal profile of the individuals/populations based on the ecological parameters and the anthropogenic factors if any. There are many ways of assessing the hormones and hormone metabolites- either from blood, hair, saliva or urine from the target animal. But, the majority of it comes with this cost of directly handling the individuals, which might alter the interpretation of the study. Hence, we, wildlife endocrinologists, use the non-invasive technique- which is basically collecting fresh feces, urine, saliva, hair samples etc from the target animals which doesn’t involve direct handling of animals and measuring various hormone-metabolites (reproductive as well as stress hormones). The change in hormone metabolite concentration reflects the change in physiological as well as the behavioural state of the individual. In this workshop, we would like to introduce this technique (from sampling to analysis and interpretation) to the young conservationists/researchers who are willing to study physiological aspects of the free-ranging animals. Objectives The main aim of this workshop is to provide the exposure to the young conservationists and researchers about the use of non-invasive tools in understanding the physiological state (both stress and reproductive health) of the free-ranging animals. The main goals of the workshop are: a.
To introduce the researchers about the stress-response and its relation with other
ecological and anthropogenic factors.
b. To introduce the researchers about the reproductive physiology of animals. c.
To provide the technical understanding for the assessment of physiological parameters
using the non-invasive tools.
How-and-where non-invasive techniques can be used: a. Collection and storage of samples: Non-invasive samples are gaining huge acceptance amongst the conservationists who are willing to understand the physiological facets of animals. Non-invasive samples like feces, urine, saliva, hair, etc can be collected from wild animals without even directly handling them. This reduces the chance of sudden physiological changes such as stress-response in animals when we collect the invasive samples.
Fresh fecal sample from wild male elephant stored in zip-lock bag The collected samples should be labeled with the date, time of collection and also the identity and sex of animal if sighted directly. These samples require the proper storage depending upon the type of non-invasive samples. For instances, the fecal samples are collected shortly after defecation and they require the immediate storage in -20-degree temperature to avoid the degradation of fecal metabolites. In some cases, where -20degree refrigeration is not feasible- we can store the samples in ethanol. In such cases, the
concentration of ethanol should be taken care of. The samples are stored until further extraction. b. Processing of samples: The stored samples are then processed to prepare sample extracts. For fecal samples, there are two ways of extracting samples- wet and dry extraction. Wet extraction involves the use of methanol or ethanol for extracting the hormone metabolites. Dry extraction involves drying or freeze-drying the samples to make the powder and then, extracting the metabolites using methanol or ethanol. For hair samples, samples are cut into fine pieces and are powdered using beads. After completion of pulverization, they are mixed up with common alcohols and the hormone metabolites are extracted. The sample extracts are then stored in -20 degree for further analysis.
Pulverizing elephant fecal sample after lyophilization (freeze-drying) c. Analyzing samples: Sample extracts are then analyzed using the most robust techniques like ELISA (Enzymelinked Immunosorbent Assay) or RIA (Radioimmunoassay) or Liquid Chromatography Mass Spectrometry (LCMS) or HPLC (High-Performance Liquid Chromatography) or in some cases, GCMS (Gas Chromatography Mass Spectrometry) as well. Out of all these techniques, ELISA is widely used to detect and quantify the hormones or hormone metabolites. The basic principle behind different types of ELISA or RIA is the interaction between antibody and
antigen of our interests (hormone metabolites). Using these techniques, we will be able to quantify the level of hormones or hormones metabolites present in non-invasive samples. It is essential to analytically and biologically validate the technique that you use for analyzing the hormone or hormone metabolites.
Sample extract from elephant fecal samples after centrifugation
ELISA plate, coated with standards and sample extracts, at the end of color reaction d. Interpretation: The concentration obtained from ELISA techniques are then used for interpreting and monitoring the physiological or reproductive state of a study animal. It is very important to
keep in mind about the gut-passage time. The concentration that you got from the calculation might not be the concentration of hormone metabolites at that particular time of collection of samples but must be depicting the situation or physiological events animal went through 12 hours or 24 hours ago or in some cases, even for the lifetime (for example, hair samples). We can assess the stress-response by measuring glucocorticoids and its metabolites; reproductive states by measuring progesterone, estrogen and their metabolites in the case of females and testosterone and its metabolites in the case of males. If you are following study animals, always keep a track of any physical shreds of evidence for stress or reproductive signals.
A
A.
B
Female fecal hormone (progestogen) metabolites showing the high-rise during
pregnancy in wild white-handed gibbon females (Barelli et al., 2007) B. Elevated glucocorticoid metabolite level after capturing event in wild gray mouse lemurs (Hämäläinen et al., 2014) What we expect from the participants after the completion of the workshop: a. Basic understanding about the stress and reproductive physiology of wild animals. b.
Exposure to the use of non-invasive techniques of measuring hormones and hormones
metabolites.
c.
A broader understanding of how physiological aspects could contribute in conserving
the free-ranging animals. Suggested references: Barelli C, Heistermann M, Boesch C, Reichard UH (2007) Sexual swellings in wild white-handed gibbon females (Hylobates lar) indicate the probability of ovulation. Hormones and Behavior 51: 221–230. Hämäläinen A, Heistermann M, Ella Fenosoa ZS, Kraus C (2014) Evaluating capture stress in wild gray mouse lemurs via repeated fecal sampling: Method validation and the influence of prior experience and handling protocols on stress responses. General and Comparative Endocrinology 195: 68-79. Palme R (2005) Measuring Fecal Steroids: Guidelines for Practical Application. Annals New York Academy of Sciences 1046: 75–80. Palme R, Touma C, Arias N, Dominchin MF, Lepschy M (2013) Steroid extraction: Get the best out of faecal samples. Wiener Tierärztliche Monatsschrift. Veterinary Medicine Austria 100: 238-246. Steven JC, Lawren S, Craig EF, Anthony PF, John B, Martin W, Steven LC (2013) What is conservation physiology? Perspectives on an increasingly integrated and essential science. Conserv Physiol 1 (1): cot001. doi: 10.1093/conphys/cot001
