Sampling and Analysis of Animal Scent Signals

Summary

We have developed an effective methodology for sampling and analysis of odor signals in order to understand how they may be used in animal communication. Particularly, we use headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry to analyze the volatile components of animal odors and scent-markings.

Abstract

We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry, to understand how they may be used in animal communication. This technique allows the semi-quantitative analysis of the volatile components of odor secretions by enabling the separation and tentative identification of the components in the sample, followed by the analysis of peak area ratios to look for trends that could signify compounds that may be involved in signaling. The key strengths of this current approach are the range of sample types that can be analyzed; the lack of need for any complex sample preparation or extractions; the ability to separate and analyze the components of a mixture; the identification of the components detected; and the capability to provide semi-quantitative and potentially quantitative information on the components detected. The main limitation to the methodology relates to the samples themselves. Since the components of specific interest are volatile, and these could easily be lost, or their concentrations altered, it is important that the samples are stored and transported appropriately after their collection. This also means that sample storage and transport conditions are relatively costly. This method can be applied to a variety of samples (including urine, feces, hair and scent-gland odor secretions). These odors consist of complex mixtures, occurring in a range of matrices, and thus necessitate the use of techniques to separate the individual components and extract the compounds of biological interest.

Introduction

Very little is known about the chemical changes underpinning the olfactory signals in animals¹, also because of methodological challenges in recording and quantifying volatile chemical profiles of odors². There are several potential pitfalls when working with highly complex, chemical matrices; these include when sampling and analyzing the odor samples³.

At the Rosalind Franklin Science Center, University of Wolverhampton, we are undertaking the analysis of odors and scent-marks to understand how they may be used by animals. We combine semiochemistry with behavioral ecol....

Protocol

1. Sample collection

1. Sample odors that are one of the following:
   1. Collect spontaneously released by habituated study subjects (e.g., zoo primates) via scent-marking on sterile filter paper (e.g., scent-gland odor secretions) or directly into vials (e.g., urine).
   2. Collect by rubbing sterile cotton swabs after training study subjects by using positive reinforcement training.
   3. Collect by rubbing sterile cotton swabs after sedation of study subjects.
2. Place samples into sterile 10 mL screw-capped clear glass vials, and seal with screw topped caps incorporating FTFE/silicone septa. Immediately store them at ....

Results

Following this protocol, we tentatively identified a total of 32 volatile chemical compounds from the analysis of 14 ano-genital scent-marks spontaneously released on filter paper by red-ruffed lemurs (Varecia variegata rubra) and compared odor profiles with features of the signaler¹². Naturally occurring volatile compounds, such as hydrocarbons, terpenes, terpene alcohols and ketones, were present within these profiles and included compounds that had previously been found to act as sex p.......

Discussion

The use of control samples, both environmental controls created at the time of sample collection and system blanks, are crucial for the interpretation of the scent-mark samples. Any peaks attributed to the sampling environment or the instrumental system need to be excluded from scent-mark samples so that only the peaks of interest are included in any interpretation. These controls can also play a role in assessing and monitoring the 'health' of the instrumentation.

The protocol includes steps .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 mL autosampler vials	Agilent	5188-5392	10 ml screwtop vials with
18 mm vial caps	Agilent	8010-0139	Magnetic with PTFE/silicone septa
Autosampler	Agilent	GC120 PAL autosampler
Capillary column	Agilent	HP5-MS	30 m x 0.25 mm; 0.25 µm
Data analysis software	Agilent	-	ChemStation
Gas Chromatograph	Agilent	7890B
Inlet septa	Agilent	5182-3442	Merlin microseal
Mass Selective Detector	Agilent	5977A
Reporting software	Microsoft	-	Excel
Spectral library	NIST	-	NIST/EPA/NIH Mass Spectral Library
Spectral library search program	NIST	-	MS Search v.2.2
Splitless Inlet liner	Agilent	5190-4048
SPME fibres	Agilent	SU57345U	65 µm PDMS/DVB fibre