Sampling and Analysis of Animal Scent Signals

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.

The role played by odor in animal communication is currently underestimated and so understudied. In particular very little is known about the chemical changes underpinning the olfactory signals in animals including humans. And these is due also to methodological challenges, recording and quantifying chemical compounds of odors used by animals to communicate.

There are several potential challenges when working with highly complex chemical mixtures and these include also when sampling and analyzing the odor samples. We are conducting the analysis of odors used by animals in order to understand how they may be used by them when they communicate between each other. We combined Cytochemistry with Bioecology endocrinology and cytology to improve our understanding of the role played by odor in animal communication.

For our current methodology in Wolverhampton, we have adopted the technique of Headspace solid phase microstructure coupled with gas chromatography mass spectrometry, built upon and an unseen Of the methodology are used in the past. Sample odors are collected in one of the following ways. One, spontaneously released sample odors from habituated study subjects.

For example, zoo primates collect scent gland odor secretions by scent marking on sterile filter paper or urine samples directly into vials. Two, after training study subjects by using positive reinforcement collect odor secretions by rubbing scent glance with sterile cotton swabs. Three, after sedation of study subjects collect odor secretions by rubbing scent glands with sterile cotton swabs, by samples into sterile 10 milliliter screw capped, clear glass vials and seal with screw topped caps incorporating P T F E silicone sector.

Immediately store the vials at minus 20 degrees Celsius. Note, it is vital that clean personal protective equipment such as nitrile gloves is used during sampling. Change gloves frequently avoid direct skin contact with samples and vials.

It is preferred to use brand new vials. However, in case of used vials, it is vital to pre-clean the vials, and then use the same protocol. Take environmental blanks every time scent marks are collected.

Do this by exposing an unused filter paper or cotton swab and vials to the environment while sampling is undertaken. Samples are prepared in the field. For a scent marked filter paper cut an approximate 10 millimeter square from the paper and place into a 10 milliliter screw topped head space vial.

For a swab, cut off the swab head at the very end of the swab shaft and place into a head space vial. After each sample has been prepared dispose of or clean the blade use to cut the sampling media, with an appropriate antibacterial wipe and or alcohol and dry thoroughly. Store all samples at minus 20 degrees celsius.

Note, samples should be stored at minus 20 degrees celsius however, in the field store at the lowest is temperature possible and transfer to minus 20 degrees Celsius at the earliest opportunity. Prior to analysis remove samples from the freezer and allowed to walk naturally to room temperature for at least one hour. Set up the analytical methods on the GCMS as follows for SPMS analysis conditions follow the manufacturer's directions to condition SBME fibers before first use.

It is vital that the SBME assembly is correctly installed in the auto sampler and that it is aligned to the auto sampler trays fiber conditioning unit and GC inlet port. Incorrect alignment could result in damage or destruction of the Sbme fiber. Ensure that the perch gas supply to the fiber conditioning unit is turned on.

To improve between sample retention time consistency, the analytical method is retention time locked add the sample vials to the auto sampler tray place an empty head space vial to act as a system blank in the first position of the auto sampler tray. Place the environmental blank in the second position and then place all the remaining samples for analysis in the subsequent positions of the auto sampler tray. Create an analytical sequence to analyze each sample within the sample tray.

In the mass hunter home screen, select sequence for the menu bar and then load sequence from the dropdown menu. A blank sequence table will be displayed complete the sequence table for all blanks and samples by inserting the appropriate information save as completed sequence table. Note, the exact information for the sequence table will be dependent on the laboratories formatting of the table.

Minimum information would normally include sample type sample name, file location, and number analytical method and data file location and name allocation of a data file name that matches the sample name aged future data processing. Additional samples can be added to the sequence during the analysis. Run the sequence by selecting sequence from the menu bar and then run sequence.

After analysis returned samples to the freezer as soon as possible. Note, it may be possible to reanalyze samples. But it should be noted that some volatile components may have been totally extracted during the initial analysis and some compound may have undergone thermal and bacterial decomposition at 40 degrees Celsius.

Thus, the result in chromatogram may not be thoroughly representative of the original scent marking. Initial data analysis includes the integration of chromatograms to obtain retention time and peak area data together with tentative identification of peaks using cam station software and nest mass spectral databases. Data analysis can be carried out either manually or through a semi-automated method.

If the semi-automated method is used then it is sometimes beneficial to undertake a degree of manual data analysis to verify tentative identifications. To start data analysis open the data file by clicking on the appropriate file in the left-hand navigation bar. The total ion chromatogram TIC will be displayed in the top window of the data analysis screen.

To integrate the TIC using the RTE integrator, firstly select chromatogram from the menu bar and then select integrator from the dropdown menu a pop up box opens and choose the RTE integrator. Go back to the chromatogram menu and now select integrate. To adjust integration parameters so that peaks or to greater than three times baseline noise are integrated, again select chromatogram from the menu bar and then MS Signal integration parameters from the dropdown menu.

In the pop up box, adjust the minimum peak area as appropriate 1.0 produces acceptable results in our examples. To identify peaks and generate a summary report, select export reports from the menu bar and then library search results report to XLS. Note, the spectral libraries to be searched together with the number of library matches to be displayed need to be preset within the software before a library search can be undertaken.

The resulting spreadsheet report contains integration data for each peak and a tentative spectral library match to assign identity. Typically the library quality or library match should be greater than 80 to accept the tentative identification. Save the spreadsheet.

To identify a peak directly from the TIC, choose the peak of interest. If the peak is small zoom in by drawing a box around the peak, by holding the left-hand mouse button down stretch the box over the peak and release the mouse button. Place the cursor line so it is at the highest point of the peak or just after.

Double click, the right hand mouse button and the mass spectrum for the peak will appear in the low window of the data analysis screen. To search a spectral library, move the cursor anywhere in the spectral window and double click the right hand mouse button the library search results will appear in a new window. To remove the background noise from a spectrum of interest first, double click the right hand mouse button on the peak in question then click the right hand mouse button in an area with no peaks immediately in front of the peak of interest click on the subtract button in the menu ribbon or select chromatogram for the menu bar and then subtract spectra from the dropdown menu.

Subtracted spectrum will display in the lower window of the data analysis screen and will display a dash next to the scan data in the window header. Following this protocol, we tentatively identified a total of 32 volatile chemical compounds from the analysis of 14 a genital scent marks spontaneously released on filter paper by red ruffed lemurs and compared odor profiles with features of the signaler naturally occurring volatile compounds such as hydrocarbons, terpenes, Turpin alcohols and ketones were present within these profiles and include the compounds that had previously been found to act as sex hormones and cues to fitness in other animal species. The compounds that have been tentatively identified are listed in table one.

Representative chromatograms one from a control and one from a Lima scent mark are shown in figure one. The number and relative abundance of the components varied from sample to sample across different study subjects. However, six compounds labeled A to F on the chromatogram were present in all samples.

These compounds were respectively bends out to height to E's isle one hexanal paracrystal six paramentha two eight dye one two pinene four and pentodecane. The results from this study suggested that red ruffed lemurs use sent marking to convey information about sex and female age with a no genital marking playing a role in socio sexual communication. Another representative outcome following the use of this protocol was our study of fertility advertisement by female olive baboons.

We identified a total of 74 volatile compounds from the analysis of 395 female baboon vaginal odor samples. These included a range of naturally occurring odorous volatile compounds such as ketones, alcohols, aldehydes, terpenes, volatile fatty acids and hydrocarbons. Typical chromatograms use to compare blank troll and female baboon vaginal odor samples from fertile and non fertile periods are shown in figure two.

We examine the relationships between vaginal odor profiles and sexual receptivity of female baboons. Our results showed that the total amount of vaginal odor differs with fertility suggesting that odor might play a role in signaling female baboon fertility. We also found differences in vaginal odor between group types but we could not distinguish the effects of group composition, female age imparity.

It is the combined advantages of the sampling and use of the GCMS. So using head space solid phase micro extraction enables a wide range of different samples to be analyzed using GCMS. We're able to separate out the components of these complex markings and then identify each of those individual components using the mass spectrometer.

And then, so the whole combined technique is very powerful and giving us a lot of information about these scent markings, which in the past would there would have been a lot of sample preparation extraction required to be able to obtain the results.