Characterizing the scent and chemical composition of Panthera leo marking fluid using solid-phase microextraction and multidimensional gas chromatography–mass spectrometry-olfactometry

Lions (Panthera leo) use chemical signaling to indicate health, reproductive status, and territorial ownership. To date, no study has reported on both scent and composition of marking fluid (MF) from P. leo. The objectives of this study were to: 1) develop a novel method for simultaneous chemical and scent identification of lion MF in its totality (urine + MF), 2) identify characteristic odorants responsible for the overall scent of MF as perceived by human panelists, and 3) compare the existing library of known odorous compounds characterized as eliciting behaviors in animals in order to understand potential functionality in lion behavior. Solid-phase microextraction and simultaneous chemical-sensory analyses with multidimensional gas-chromatography-mass spectrometry-olfactometry improved separating, isolating, and identifying mixed (MF, urine) compounds versus solvent-based extraction and chemical analyses. 2,5-Dimethylpyrazine, 4-methylphenol, and 3-methylcyclopentanone were isolated and identified as the compounds responsible for the characteristic odor of lion MF. Twenty-eight volatile organic compounds (VOCs) emitted from MF were identified, adding a new list of compounds previously unidentified in lion urine. New chemicals were identified in nine compound groups: ketones, aldehydes, amines, alcohols, aromatics, sulfur-containing compounds, phenyls, phenols, and volatile fatty acids. Twenty-three VOCs are known semiochemicals that are implicated in attraction, reproduction, and alarm-signaling behaviors in other species.

Interfacing solid phase-microextraction (SPME) and multidimensional chromatography with olfactometry provides a unique opportunity to address the knowledge gaps in chemical sensory analysis development for lion MF and to identify the chemicals responsible for the characteristic odor of lion MF. The emissions of volatiles from MF (defined here as a simultaneous and mixed excretion of MF and urine) was analyzed in totality, MF was not separated from urine, in order to improve understanding of the perceived odor of gases emitted from lion MF. We did not analyze fecal excretions, a common form of scent-marking, because in lions defecation can be done at random 1 . This is indicating its potentially lower order in the hierarchy of scentmarkings. Although the scope of this study was limited to lion's MF, the same approach could be used for other species, excretions, environments, and with behavioral studies. Once odor and odor-causing compounds in territorial markings are known, this knowledge can be exploited to determine the effects of specific compounds on animal behavioral. Future studies can develop behavioral assays and compare changes in chemical composition of scent-markings to the behavioral responses during the introduction of the odorous compounds identified in this study to lions. This unique and novel methodology combining SPME and MDCG-MS-O could be used to further understand the way animals perceive scent-markings, and potentially prevent the eradication of many endangered species.
Chemosensory cues play a large role in the reproductive behavior and proliferation of many species. Understanding the role of odors in scent-markings has proven to be integral in the conservation research of a plethora of endangered species. The focus of this work has been to increase reproduction in and out of captivity. Odors within scent-markings have been proven to influence male ejaculation in various animals including giant pandas (Ailuropoda melanoleuca) 2 , Drosophila melanogaster and Pieris rapae [3][4][5] , Zosterisessor ophiocephalus and Gobius niger 6 , Gallus gallus 7 and Microtus pennsylvanicus 8 . Males tend to ejaculate in the presence of competitive males in an effort to preserve their genetic influence and survival within their species. In the case of giant pandas it is hypothesized that chemosensory cues from potential rivals "increase male pandas' sexual motivation towards females, and enhance their territorial behavior" 2 . The lack of competition in captive environments can potentially be inhibiting reproduction of endangered species unless knowledge of chemosensory cues is expanded 2 .
Often, studies are able to equate behaviors with scent-markings, but do not identify specifically the roles of individual compounds in animal behavior nor attempt to understand how the animals are perceiving these scents. The ability of elephants to detect cyclohexanone in musth has led scientists to suspect that some musth signal messages in elephants may be single compounds 9 . More research on the roles of individual scents and chemical compounds within markings is needed in order to gain an understanding of the influence each has on eliciting behaviors.
Solid phase-microextraction is a solvent-free, one step sampling/sample preparation technique that has been limitedly used in the sample preparation of large mammalian scentmarkings 10,11 . Since its conception in the late 1980s, it has proven to be one of the superior sample preparation techniques available for analytical work in the area of fundamental analytical chemistry, environmental analysis, pharmaceutical, food and forensic analyses 10,[12][13][14][15][16] . SPME is a reusable technique that combines sampling and sample preparation and is suitable for laboratory and field environmental work 17 . The SPME process is facilitated on a polymeric coating that has a high affinity for organic compounds. SPME has been used for sampling of volatile compounds in air 18 , livestock odor, breath of animals 19,20 , volatiles inside rumen 21 , volatiles emitted by decaying animal mortalities 22 , and insect-induced plant volatiles among other applications 23 . Enrichment associated with SPME often leads to significantly improved method detection limits and elimination of artifacts from solvents compared with other sampling and preparation methods 24 .
Multidimensional-GC-MS-O is one of the most advanced methods for simultaneous chemical and sensory analysis, enabling volatile organic compound separation and isolation of odor-active compounds. Precise and advanced capabilities to detect trace levels of components is due to its multi-column system which allows for a better separation and identification of volatiles 25 many of which are odorous [26][27][28] . The olfactometry is enabled by a sniff port which gives odor panelists an opportunity to characterize each separated compound as it is being eluted through one of the selected GC columns. This feature allows for the determination and verification of compounds through chemical (GC column retention times, MS spectral matches) and, simultaneous odor matching confirmation using trained odor panelists and published scentto-compounds link libraries 29 . There is limited working knowledge of how mammals process odor signals 30,31 . Therefore, the human nose is considered ideal in understanding odor perception in animals because the human sense of smell is capable of distinguishing and recognizing a diverse range of characteristics of volatile compounds 32 . Headspace-SPME and MDGC-MS-O was used in the identification of VOCs from Panthera tigris altaica MF 33 . This use of SPME in conjunction with MD-GC-MS-O allowed for aroma recognition and chemical confirmation of 2-AP, which was previously considered one of the characteristic odor compounds of P. tigris tigris MF, but could not be identified previously using solely chemical analysis with GC-FID and GC-MS 33 . The objectives of this study were to: 1) develop a novel method for the simultaneous chemical and scent identification of lion MF in its totality, 2) identify the characteristic odorants responsible for the overall scent of lion MF as perceived by human panelists, and 3) compare the existing library of known odorous compounds characterized as eliciting behaviors in animals in order to understand their functionality in lion behavior.