# Chemical Characterization of the Marking Fluid of Breeding and Non-Breeding Male Cheetahs
###### Abstract  
Keywords:The study aimed at chemically characterizing the marking fluid of both breeding and non-breeding male cheetahs (_Acinnay 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. Footnote †: journal: _Animals_**2022**, 12, 2284. [[https://doi.org/10.3390/anil2172284](https://doi.org/10.3390/anil2172284)]([https://doi.org/10.3390/anil2172284](https://doi.org/10.3390/anil2172284))  
and tail, as well as urine. Regarding scent deposition from their bladder, all felines exhibit the same two behaviors [1; 2; 3]. Normal urination is one method that involves squatting above the ground before the urine is released [4]. Spraying of urine, termed spray marking behavior, develops around puberty [3; 5] and involves the release of one or more streams of urine onto an object [3]. There has been no evidence to prove that other chemical compounds are released into the urine before it is sprayed as a marking fluid [3]. Therefore, it can be assumed that the chemical composition of the urine is the same as that of the marking fluid. A scent mark forms from the mixture of semiochemicals in the marking fluid, which is left behind on the object to transmit certain messages or signals that are detectable to other animals at a later stage [6; 7]. Lipids are fixed to the semiochemicals to increase the persistence time of the scent mark [8]. The definition of semiochemicals is \"chemical signal vehicles that carry information between organisms\", and pheromones are a class of semiochemicals that act as chemical signals between individuals of the same species (intraspecific, [9]). The purpose of scent marks is to mark territory, identify neighbors, detect bigger predators and nearby food/prey, signal alarms and attract members of the opposite sex [6; 10; 11; 12; 13]. Furthermore, information regarding the age, sex, and reproductive status of an individual can also be obtained from their scent mark [6]. Although several studies have been conducted on the role of semiochemicals in wild and domestic fields [3; 5; 6; 7; 10; 14; 15; 16], little is known about their function in cheetahs. In fact, it has been speculated that the lack of interest in cheetah urine among scientists is most likely due to the very little odor it emits [17], which leads to further speculation that the concentration of VOCs potentially related to breeding behavior could be low [17]. A relatively recent study used the voided urine of various male cheetahs to prove the olfactory role of semiochemicals in mate choice by female cheetahs [18], thus proving the involvement of semiochemicals in the urine of male cheetahs in attracting the opposite sex. Furthermore, two studies [11; 17] analyzed the volatile organic chemicals (VOCs) in cheetahs' urine, collected from both male and female cheetahs, with gas chromatography-mass spectrometry (GC-MS) to identify the VOC composition. [PERSON] (2002) analyzed the urine of four wild male cheetahs located in Namibia, along with three captive males and one captive female located in South Africa. Similarly, [PERSON] et al. (2006) analyzed the urine of two wild males, four captive males, and one captive female located in different facilities in South Africa. Combined, these studies were successful in identifying 53 VOCs, of which 35 were identified in both studies. Despite their success, these studies only compared sexes rather than individuals or specific groups.