Marking Fluid Research Articles

The Effect of a Synthetic Scent on Cheetah Behaviour.

In cheetahs, age at first parturition correlates negatively with reproductive lifespan (asymmetric reproductive aging); therefore, breeding cheetahs at a young age is essential to maximize reproductive performance in this species. However, younger females display a significantly reduced frequency of copulatory behaviour, which negatively affects breeding. Volatile organic compounds (VOCs) are known to regulate appropriate behavioural responses in various species, including reproductive behaviour; moreover, they have proven to play a role in captive breeding methods in cheetahs, as well as mate choice. Therefore, the objective of this study was to evaluate the effect of a synthetic scent (SS) on the frequency of the five oestrous behaviour(s) (sniff, rub, roll, spray, and meow-chirp) known to be indicative of oestrus in female cheetahs. Based on the results of a previous study from our research group, five VOCs, identified in the marking fluid of male cheetahs, and known to be pheromones involved in reproductive behaviour, were used to create the SS. This was accomplished by mixing benzaldehyde, acetophenone, indole, dimethyl disulphide and phenol with (99.9%) ethanol. Seven female cheetahs were then observed for one oestrus cycle without stimulation (control) and then once again while exposed to the SS (treatment), which was sprayed on foil trays placed around the outside of each enclosure. The occurrence of the five oestrous behaviours was recorded and tallied per day of observations. Although the SS did not have a significant effect on the frequency of oestrous behaviours displayed by the females used in this study, five of the seven (71%) did show an increase in their behaviour with the SS when oestrogen concentrations were at their highest (peak oestrus), including three of the four younger females. The SS also significantly increased the sniffing behaviour in general. Although the results of this study do indicate that VOCs influence cheetahs and their behaviour, firm conclusions cannot be drawn due to the low number of animals used, as well as the significant effect the observation methods had on the results. Nonetheless, this study represents the first of this kind in cheetahs, therefore representing an important step in determining the role of VOCs in aiding breeding in captivity.

288 Chemical Characterization of the Marking Fluid of Breeding and non-Breeding Male Cheetahs

Abstract Volatile semiochemicals released, amongst other methods, through marking fluid (MF), and characterized by scent/odor, are involved in communication and identification of an individual. Scent has an important role in reproduction of cheetahs and other felids; in fact, the presence/odor of a male cheetah has been noted to trigger the estrous cycle of females. The objectives of the study were to analyze the MF of male cheetahs from different breeding groups to determine the composition of volatile organic chemicals (VOCs) present. Five males per group (e.g., breeding vs non-breeding) were selected from two facilities. The first facility only had breeding males (B; e.g., animals regularly exposed to females on estrus and actively involved in breeding programs); the second facility, only had non-breeding males (NB). The average age of the B and NB males was of 8.1±2.3 and 6.7±2.8 years respectively. Eighteen MF samples were collected from both groups. VOCs were extracted from the headspace, using solid-phase microextraction techniques and analyzed with gas-chromatography mass-spectrometry. Possible relationships between the frequency of presence/absence of each VOC and the respective groups were evaluated through a contingency test with Fisher’s exact test. Differences in relative concentration (RC) of the identified compounds between groups were analyzed using analysis of variance for repeated measures. Fifty-eight VOCs were identified from the MF, and twelve were commonly identified in the samples collected. The RC of dodecanoic acid and nonanoic acid was significantly greater in the B compared with the NB males; whereas indole tended to be greater in the NB compared with the B group. Although the observed differences could indicate the possible involvement of specific VOCs in reproductive behavior, the different diets and age could have also affected the VOCs RC. Further studies are needed to better understand possible differences in RC of VOCs between breeding groups to confirm their role in reproduction.

Chemical Characterization of the Marking Fluid of Breeding and Non-Breeding Male Cheetahs.

Simple SummaryThe study aimed at chemically characterizing the marking fluid of both breeding and non-breeding male cheetahs (Acinonyx jubatus). Specifically, it focused on identifying potential differences in pheromones related to sexual behavior/attraction in this species. Furthermore, it aimed at providing more information as a basis for future studies, such as the investigation of specific semiochemicals in the reproductive behavior of cheetahs. The results of this study support the hypothesis of differences in the relative concentration of volatile organic chemicals between male cheetahs; however, they highlight the importance of diet and age on the presence of volatile organic chemicals in the marking fluid.Scent is known to play an important role in the reproduction of cheetahs and other felids. In fact, the presence/odor of a male cheetah has been noted to trigger the estrous cycle in females. The objective of this study was to analyze the marking fluid (MF) of male cheetahs from different breeding groups to determine the composition of volatile organic compounds (VOCs) present, with the aim of identifying potential pheromones relating to sexual behavior/attraction in this species. Four breeding (B; age: 8.9 ± 1.3 years old) and four non-breeding (NB; age: 5.5 ± 0.8 years old) males were selected for this study. Samples were collected into a glass beaker, transferred immediately into a 20 mL glass screw-cap vial with a polytetrafluoroethylene (PTFE) coated silicone septum, and stored until analyzed by headspace solid-phase microextraction (HS-SPME) using gas chromatography–mass spectrometry. A contingency test with Fisher’s exact test, using the frequency (FREQ) procedure of SAS 9.4, was conducted to determine the difference between the number of VOCs identified per breeding group; furthermore, differences in relative concentration (RC) of the identified VOCs between breeding groups were analyzed using ANOVA for repeated measures with the GLIMMIX procedure. From the 13 MF samples analyzed, 53 VOCs were identified, and 12 were identified in all the samples. Five of these (dimethyl disulfide, benzaldehyde, acetophenone, phenol, and indole) are known to be involved in attraction/sexual behavior in mammals. Between the two groups, the RC of indole was significantly higher in the NB group, whereas the RC of dodecanoic acid was significantly higher in the B group. Although not significant, the RC of benzaldehyde was higher in the B versus the NB group. The results of this study do support the hypothesis of differences in VOCs’ between B and NB male cheetahs. However, the overlapping of age and breeding status and the diet differences could not be controlled. Still, the evidence of changes in MF composition in male cheetahs necessitates further studies on possible strategies to improve reproduction in captivity.

Characterizing the scent and chemical composition of Panthera leo marking fluid using solid-phase microextraction and multidimensional gas chromatography\u2013mass 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.

Analysis of Odorants in Marking Fluid of Siberian Tiger (Panthera tigris altaica) Using Simultaneous Sensory and Chemical Analysis with Headspace Solid-Phase Microextraction and Multidimensional Gas Chromatography-Mass Spectrometry-Olfactometry.

Scent-marking is the most effective method of communication in the presence or absence of a signaler. These complex mixtures result in a multifaceted interaction triggered by the sense of smell. The objective was to identify volatile organic compound (VOC) composition and odors emitted by total marking fluid (MF) associated with Siberian tigers (Panthera tigris altaica). Siberian tiger, an endangered species, was chosen because its MF had never been analyzed. Solid phase microextraction (SPME) for headspace volatile collection combined with multidimensional gas chromatography-mass spectrometry-olfactometry for simultaneous chemical and sensory analyses were used. Thirty-two VOCs emitted from MF were identified. 2-acetyl-1-pyrroline, the sole previously identified compound responsible for the “characteristic” odor of P. tigris MF, was identified along with two additional compounds confirmed with standards (urea, furfural) and four tentatively identified compounds (3-methylbutanamine, (R)-3-methylcyclopentanone, propanedioic acid, and 3-hydroxybutanal) as being responsible for the characteristic aroma of Siberian tiger MF. Simultaneous chemical and sensory analyses improved characterization of scent-markings and identified compounds not previously reported in MF of other tiger species. This research will assist animal ecologists, behaviorists, and zookeepers in understanding how scents from specific MF compounds impact tiger and wildlife communication and improve management practices related to animal behavior. Simultaneous chemical and sensory analyses is applicable to unlocking scent-marking information for other species.

Chemical Characterization of Territorial Marking Fluid of Male Bengal Tiger, Panthera tigris

The territorial marking fluid of the male Bengal tiger, Panthera tigris, consists of a mixture of urine and a small quantity of lipid material that may act as a controlled-release carrier for the volatile constituents of the fluid. Using gas chromatography and gas chromatography-mass spectrometry, 98 volatile compounds and elemental sulfur were identified in the marking fluid. Another 16 volatiles were tentatively identified. The majority of these compounds were alkanols, alkanals, 2-alkanones, branched and unbranched alkanoic acids, dimethyl esters of dicarboxylic acids, gamma- and delta-lactones, and compounds containing nitrogen or sulfur. Several samples of the marking fluid contained pure (R)-3-methyl-2-octanone, (R)-3-methyl-2-nonanone, and (R)-3-methyl-2-decanone, but these ketones were partly or completely racemized in other samples. The gamma-lactone (S)-(+)-(Z)-6-dodecen-4-olide and the C(8) to C(16) saturated (R)-gamma-lactones and (S)-delta-lactones were present in high enantiomeric purities. The chiral carboxylic acids, 2-methylnonanoic acid, 2-methyldecanoic acid, 2-methylundecanoic acid, and 2-ethylhexanoic acid were racemates. Cadaverine, putrescine, and 2-acetylpyrroline, previously reported as constituents of tiger urine, were not detected. The dominant contribution of some ketones, fatty acids, and lactones to the composition of the headspace of the marking fluid suggests that these compounds may be important constituents of the pheromone. Although it constitutes only a small proportion, the lipid fraction of the fluid contained larger quantities of the volatile organic compounds than the aqueous fraction (urine). The lipid derives its role as controlled-release carrier of the chemical message left by the tiger, from its affinity for the volatiles of the marking fluid. Six proteins with masses ranging from 16 to 69 kDa, inter alia, the carboxylesterase-like urinary protein known as cauxin, previously identified in the urine of the domestic cat and other felid species, were identified in the urine fraction of the marking fluid.

Putative chemical signals of leopard

Abstract A study of marking fluid (MF) of Indian male leopard has been undertaken as a continuation of the earlier work on tiger and cheetah pheromones. The behavioural observations reveal that MF is presumably the primary means of communication in leopard. The chemical analyses reveal the presence of acetic, propionic, isobutyric, butyric, isovarleric, valeric, isohexanoic, hexanoic, isoheptanoic, heptanoic, isooctanoic, octanoic and nonanoic acid in the acidic fraction of steam distillate of MF. Amines like etylenediamine, putrescine, cadaverine and phenylethylamine, dimethylamine and trimethylamine were also detected in the basic fraction of MF. The amount of lipid extracted from MF is 1.15 mg/ml.

The fixative lipid of tiger pheromone

Tigers communicate with one another with the help of Marking Fluid (MF) which is a lipid-rich fluid sprayed upwards and backwards through the urinary channel of both the sexes. The volatile molecules of the MF are made to last longer with the help of lipid ‘fixatives’ the total amount of which is 1–2 mg/ml. This lipid comprises cholesterol ester, wax ester, triglyceride, free fatty acids, diglyceride, monoglyceride, free sterol and phospholipid as revealed by thin layer chromatography. The gas liquid chromatogram of fatty acid methyl esters of the total lipid of the MF, when compared with the fatty acids of groin and body fat of tiger (analysed by other workers), reveals differences in higher proportions of palmitoleic and myristic acids and of highly unsaturated fatty acids. The palmitoleic acid content of total lipid and triglyceride is high in comparison to the wax esters and cholesterol esters of the MF-fat. The unique feature of the alcohol part of the wax esters is a series of saturated straight chain primary alcohols of C 4 to C 20 and these are accompanied by the corresponding monoenoic unsaturates.

Amines in the marking fluid and anal sac secretion of the tiger, Panthera tigris.

Analysis of the marking fluid of two tigers (one Bengal and one Sumatran) by GC using an amine-specific column and a nitrogen-specific detector has shown the presence of the following amines: ammonia, methylamine, dimethylamine, trimethylamine, triethylamine, propylamine, and butane-1,4-diamine (putrescine). In contrast to previously published reports, we were unable to detect 2-phenylethylamine. The anal sac secretion was found to have a similar amine content.

Short Chain Free Fatty Acids as Putative Pheromones in the Marking Fluid of Tiger

Short Chain Free Fatty Acids as Putative Pheromones in the Marking Fluid of Tiger

Fragrant Volatiles in Rice and Marking Fluid of Tiger

Fragrant Volatiles in Rice and Marking Fluid of Tiger

Chin-Ball Application of Permethrin to Cows for Horn Fly (Diptera: Muscidae) Control

Control of horn flies, Haematobia irritans (L.), was obtained with 1.0% permethrin applied to cows in marking fluid by a bull equipped with a chin-ball or bull-point marker. The treatment provided horn fly control 2 days after use of the chin-ball was initiated and remained effective 3 weeks after the bull was removed from the herd.

DHOBIE MARK DERMATITIS

When the personnel of the 20th General Hospital was first exposed to laundered clothes, soon after arrival in the C. B. I. theater, a small epidemic of patchy made its appearance, which in all instances was distressing and in a few was temporarily incapacitating. The exact localization of the circumscribed patches of on that part of the skin in contact with the mark and the course of the lesions made it quite obvious that this represented a contact induced by the marking fluid which the native dhobies or washermen used in making their characteristic laundry marks (fig. 1). Having now completed identification of the causative agent, we make this report with a view of recording dhobie mark dermatitis as an entity which, to the best of our knowledge, has not been reported previously. We believe that our observations suggest a possible derivation of the term