Sprayed Urine Emits a Pungent Odor due to its Increased Adhesion to Vertical Objects via Urinary Proteins Rather Than to Changes in its Volatile Chemical Profile in Domestic Cats.

Spraying urine on vertical objects by raising the tail is a commonly observed functional behavior for chemical communication in Felidae species, including domestic cats (Felis silvestris catus). The sprayed urine is recognized as a chemical signal for territorial ownership of their habitats. Previous studies reported that sprayed urine emits a more pungent odor than urine excreted from a squatting position. However, little is known about how sprayed urine acts as a strong scent mark in the environment. Here, we showed that sprayed urine originates only from bladder urine without any secretions, such as anal sac secretions, but it can effectively emit volatile organic compounds (VOCs) when smeared on vertical objects due to its strong adhesion. Chemical profiles of VOCs and odor qualities were similar between fresh sprayed urine and bladder urine sampled immediately after spraying from the same individuals. Meanwhile, feline-specific proteinuria arising from excretion of a carboxylesterase that produces a precursor of cat-specific odorants resulted in reduced surface tension of the urine and increased adhesion to vertical surfaces, which kept sprayed urine on the surfaces and led to the emission of large amounts of VOCs. In conclusion, proteinuria contributes to the emission of a strong odor through its enhanced adhesion to vertical objects without other secretions containing malodorous substances. These findings improve our understanding of the mechanism of scent marking via the spraying of urine for chemical communication in cats.

Serum metabolic profiling in pregnant Holstein cows 3 weeks prior to parturition using two-dimensional gas chromatography time-of-flight mass spectrometry.

This preliminary study explored potential serum biomarkers for predicting the onset of milk fever (MF), a bovine parturient disease with hypocalcemia. We conducted two-dimensional gas chromatography mass spectrometry-based metabolomics in 8 and 17 pregnant Holstein cows that did and did not develop MF 3 weeks later, respectively. In principal component analysis (PCA) applied to a dataset containing 1,498 metabolites, serum metabolites exhibited highly similar chemical profiles between cows with and without MF. PCA with a limited dataset of metabolites containing fatty acids, which had significantly different values between the groups and/or correlation coefficients of >0.5 for the serum calcium concentration, distinguished the two groups. These suggest the possibility of developing serum biomarkers for predicting bovine MF.

Development of the gas chromatography/mass spectrometry-based aroma designer capable of modifying volatile chemical compositions in complex odors.

Many volatile organic compounds (VOCs) are used to produce various commercial products with aromas mimicking natural products. The VOCs responsible for aromas have been identified from many natural products. The current major strategy is to analyze chemical compositions and aroma qualities of individual VOCs using gas chromatography/mass spectrometry (GC/MS) and GC-olfactometry. However, such analyses cannot determine whether candidate VOCs contribute to the characteristic aroma in mixtures of many VOCs. In this study, we developed a GC/MS-based VOC collection/omission system that can modify the VOC compositions of samples easily and rapidly. The system is composed of GC/MS with a switching unit that can change gas flow routes between MS and a VOC collection device. We first applied this system to prepare gas samples for omission tests, and the aroma qualities of VOC mixtures with and without some VOCs were evaluated by panelists. If aroma qualities were different between the 2 samples, the omitted VOCs were likely key odorants. By collecting VOCs in a gas bag attached to the collection device and transferring some VOCs to MS, specific VOCs could be omitted easily from the VOC mixture. The system could prepare omission samples without chemical identification, preparation of each VOC, and laborious techniques for mixing VOCs, thus overcoming the limitations of previous methods of sample preparation. Finally, the system was used to prepare artificial aromas by replacing VOC compositions between different samples for screening of key odorants. In conclusion, the system developed here can improve aroma research by identifying key odorants from natural products.

In situ hybridization analysis of odorant receptor expression in the olfactory organ of the pig-nosed turtle Carettochelys insculpta.

The turtle olfactory organ consists of upper (UCE) and lower (LCE) chamber epithelium, which send axons to the ventral and dorsal portions of the olfactory bulbs, respectively. Generally, the UCE is associated with glands and contains ciliated olfactory receptor neurons (ORNs), while the LCE is devoid of glands and contains microvillous ORNs. However, the olfactory organ of the pig-nosed turtle Carettochelys insculpta appears to be a single olfactory system morphologically: there are no associated glands; ciliated ORNs are distributed throughout the olfactory organ; and the olfactory bulb is not divided into ventral and dorsal portions. In this study, we analyzed the expression of odorant receptors (ORs), the major olfactory receptors in turtles, in the pig-nosed turtle olfactory organ, via in situ hybridization. Of 690 ORs, 375 were classified as class I and 315 as class II. Some class II ORs were expressed predominantly in the posterior dorsomedial walls of the nasal cavity, while other class II ORs and all class I ORs examined were expressed in the remaining region. These results suggest that the pig-nosed turtle olfactory organ can be divided into two regions according to the expression of ORs.

Assessing the safety and suitability of using silver vine as an olfactory enrichment for cats.

Olfactory enrichment is a strategy that can improve welfare among animals managed in captivity, such as household domestic cats. Catnip (Nepeta cataria) and silver vine (Actinidia polygama) that produce iridoids are used as olfactory enrichments for cats, but little is known about the safety or the best plant resources to use that maximize positive cat responses. We report physiological effects and suitable harvest and drying methods for using silver vine as olfactory enrichment. Continuous exposure of cats to silver vine showed no hallmarks of addictive behavior, while blood indicators of stress and hepatic or renal injury showed no increase in cats stimulated with it. Drying the leaves changed the iridoid profile, enhancing the feline response. In conclusion, dried silver vine leaves are the most suitable resource for developing olfactory enrichment that maximizes feline typical response, which would not result in dependence, stress, or toxicity to the liver or kidneys in cats.

Expression of type 1 vomeronasal receptors in the olfactory organ of the African lungfish, Protopterus dolloi.

The vomeronasal organ is an olfactory organ found in amphibians and higher vertebrates. Type 1 vomeronasal receptors, one of the major olfactory receptors in vertebrates, are expressed in the vomeronasal organ in mammals. In amphibians and fish, they are expressed in the olfactory epithelium. The lungfish, which is the species of fish most closely related to amphibians, has a primitive vomeronasal organ: the recess epithelium. Expression of type 1 vomeronasal receptors has been reported in both the olfactory epithelium and the recess epithelium in three species of African lungfish and one species of South American lungfish. However, a previous study suggested that in the African lungfish Protopterus dolloi these receptors are expressed only in the olfactory epithelium. In this study, we identified 21 type 1 vomeronasal receptor genes in P. dolloi and examined the expression sites in the olfactory organ. In P. dolloi, most cells expressing the type 1 vomeronasal receptor were distributed in the olfactory epithelium, but a few were also found in the recess epithelium. This implies that the functions of the olfactory epithelium and the primitive vomeronasal organ are incompletely separated, and that all extant African and South American lungfish share this trait.

In situ hybridization analysis of olfactory receptor expression in the sea turtle olfactory organ.

The olfactory organ of turtles consists of an upper chamber epithelium (UCE) with associated glands, and a lower chamber epithelium (LCE) devoid of glands. The UCE and LCE are referred to as the air-nose and the water-nose, respectively, because the UCE is thought to detect airborne odorants, while the LCE detects waterborne odorants. However, it is not clear how the two are used in the olfactory organ. Odorant receptors (ORs) are the major olfactory receptors in turtles; they are classified as class I and II ORs, distinguished by their primary structure. Class I ORs are suggested to be receptive to water-soluble ligands and class II ORs to volatile ligands. This study analyzed the expression of class I and II ORs in hatchlings of the green sea turtle, Chelonia mydas, through in situ hybridization, to determine the localization of OR-expressing cells in the olfactory organ. Class I OR-expressing cells were distributed mainly in the LCE, implying that the LCE is receptive to waterborne odorants. Class II OR-expressing cells were distributed in both the UCE and LCE, implying that the entire olfactory organ is receptive to airborne odorants. The widespread expression of class II ORs may increase opportunities for sea turtles to sense airborne odorants.

Type 1 vomeronasal receptor expression in juvenile and adult lungfish olfactory organ.

Lungfish are the most closely related fish to tetrapods. The olfactory organ of lungfish contains lamellae and abundant recesses at the base of lamellae. Based on the ultrastructural and histochemical characteristics, the lamellar olfactory epithelium (OE), covering the surface of lamellae, and the recess epithelium, contained in the recesses, are thought to correspond to the OE of teleosts and the vomeronasal organ (VNO) of tetrapods. With increasing body size, the recesses increase in number and distribution range in the olfactory organ. In tetrapods, the expression of olfactory receptors is different between the OE and VNO; for instance, the type 1 vomeronasal receptor (V1R) is expressed only in the OE in amphibians and mainly in the VNO in mammals. We recently reported that V1R-expressing cells are contained mainly in the lamellar OE but also rarely in the recess epithelium in the olfactory organ of lungfish of approximately 30 cm body length. However, it is unclear whether the distribution of V1R-expressing cells in the olfactory organ varies during development. In this study, we compared the expression of V1Rs in the olfactory organs between juveniles and adults of the African lungfish Protopterus aethiopicus and South American lungfish, Lepidosiren paradoxa. The density of V1R-expressing cells was higher in the lamellae than in the recesses in all specimens evaluated, and this pattern was more pronounced in juveniles than adults. In addition, the juveniles showed a higher density of V1R-expressing cells in the lamellae compared with the adults. Our results imply that differences in lifestyle between juveniles and adults are related to differences in the density of V1R-expressing cells in the lamellae of lungfish.

Type 1 vomeronasal receptors expressed in the olfactory organs of two African lungfish, Protopterus annectens and Protopterus amphibius.

Lungfish are the fish related most closely to tetrapods. The olfactory organ of lungfish contains two distinct sensory epithelia: the lamellar olfactory epithelium (OE) and the recess epithelium (RecE). Based on their ultrastructural and histological characteristics, the lamellar OE and the RecE are considered to correspond respectively to the teleost OE and a primitive vomeronasal organ (VNO). In tetrapods, the OE and VNO have been shown to express different families of olfactory receptors; for example, in mammals, the OE expresses odorant receptors and trace amine-associated receptors, while the VNO expresses type 1 (V1Rs) and type 2 (V2Rs) vomeronasal receptors. In the present study, we examined the expression of V1Rs in the olfactory organs of two African lungfish, Protopterus annectens and Protopterus amphibius. RNA sequencing and phylogenetic analyses identified 29 V1R genes in P. annectens and 50 V1R genes in P. amphibius. Most V1Rs identified in these lungfish were classified as the tetrapod-type V1Rs initially found in tetrapods and distinct from fish-type V1Rs. In teleost, which all lack a VNO, all olfactory receptors are expressed in the OE, while in Xenopus V1Rs are expressed exclusively in the OE, and not in the VNO. In situ hybridization analysis indicated that lungfish V1Rs were expressed mainly in the lamellar OE and rarely in the RecE. These results imply that V1R expression in lungfish represents an intermediate step toward the complete segregation of V1R expression between the OE and VNO, reflecting the phylogenetic position of lungfish between teleosts and amphibians.

Cold stress induces malformed tomato fruits by breaking the feedback loops of stem cell regulation in floral meristem.

Fruit malformation is a major constrain in fruit production worldwide resulting in substantial economic losses. The farmers for decades noticed that the chilling temperature before blooming often caused malformed fruits. However, the molecular mechanism underlying this phenomenon is unclear. Here we examined the fruit development in response to cold stress in tomato, and demonstrated that short-term cold stress increased the callose accumulation in both shoot apical and floral meristems, resulting in the symplastic isolation and altered intercellular movement of WUS. In contrast to the rapidly restored SlWUS transcription during the recovery from cold stress, the callose removal was delayed due to obstructed plasmodesmata. The delayed reinstatement of cell-to-cell transport of SlWUS prevented the activation of SlCLV3 and TAG1, causing the interrupted feedback inhibition of SlWUS expression, leading to the expanded stem cell population and malformed fruits. We further showed that the callose dynamics in response to short-term cold stress presumably exploits the mechanism of bud dormancy during the seasonal growth, involving two antagonistic hormones, abscisic acid and gibberellin. Our results provide a novel insight into the cold stress regulated malformation of fruit.

Domestic cat damage to plant leaves containing iridoids enhances chemical repellency to pests.

Catnip (Nepeta cataria) and silver vine (Actinidia polygama) produce iridoids with arthropod-repellent effects. Cats rub and roll against these plants, transferring iridoids to their fur that repels mosquitoes. Cats also lick and chew plant leaves during this response, although the benefit of this additional behavior has remained unknown. Here, we show that feline leaf damage substantially increases iridoid emission from both plants while also diversifying iridoids in silver vine. Cats show an equivalent duration of response to the complex cocktail of iridoids in damaged silver vine and to the much higher level of a single iridoid produced by damaged catnip. The more complex iridoid cocktail produced when silver vine is licked and chewed by cats increases mosquito repellency at low concentration. In conclusion, feline leaf damage contributes by releasing more mosquito-repellent iridoids. Feline olfactory and behavioral sensitivity is fine-tuned to plant-specific iridoid production for maximizing the mosquito repellency gained.

Preliminary study of urinary excretion of liver-type fatty acid-binding protein in a cat model of chronic kidney disease.

Urinary liver-type fatty acid-binding protein (uL-FABP) is a clinically useful biomarker for monitoring chronic kidney disease (CKD) in humans. However, long-term monitoring of uL-FABP in CKD cats has not been reported. The objective of this preliminary study was to investigate whether the urinary excretion of L-FABP could predict the deterioration of renal function in 2 CKD model cats. Urinary liver-type fatty acid-binding protein (uL-FABP) increased before standard renal biomarkers, including serum creatinine, blood urea nitrogen, and symmetric dimethylarginine, in 1 cat with deteriorating renal function, but remained low and relatively stable in another cat with stable renal function. Our results suggest that uL-FABP is a potential clinical biomarker for predicting the progression of CKD in cats, as it is in humans.

La protéine urinaire de liaison aux acides gras de type hépatique (uL-FABP) est un biomarqueur cliniquement utile pour la surveillance de l'insuffisance rénale chronique (MRC) chez l'homme. Cependant, aucune surveillance à long terme de l'uL-FABP chez les chats atteints d'IRC n'a été signalée. L'objectif de cette étude préliminaire était de déterminer si l'excrétion urinaire de L-FABP pouvait prédire la détérioration de la fonction rénale chez deux chats modèles de CKD. La protéine uL-FABP a augmenté avant les biomarqueurs rénaux standards, y compris la créatinine sérique, l'azote uréique sanguin et la diméthylarginine symétrique, chez un chat dont la fonction rénale se détériorait, mais est restée faible et relativement stable chez un autre chat dont la fonction rénale était stable. Nos résultats suggèrent que l'uL-FABP est un biomarqueur clinique potentiel pour prédire la progression de l'IRC chez le chat, comme c'est le cas chez l'homme.(Traduit par Docteur Serge Messier).

The characteristic response of domestic cats to plant iridoids allows them to gain chemical defense against mosquitoes.

Domestic cats and other felids rub their faces and heads against catnip (Nepeta cataria) and silver vine (Actinidia polygama) and roll on the ground as a characteristic response. While this response is well known, its biological function and underlying mechanism remain undetermined. Here, we uncover the neurophysiological mechanism and functional outcome of this feline response. We found that the iridoid nepetalactol is the major component of silver vine that elicits this potent response in cats and other felids. Nepetalactol increased plasma ß-endorphin levels in cats, while pharmacological inhibition of µ-opioid receptors suppressed the classic rubbing response. Rubbing behavior transfers nepetalactol onto the faces and heads of respondents where it repels the mosquito, Aedes albopictus Thus, self-anointing behavior helps to protect cats against mosquito bites. The characteristic response of cats to nepetalactol via the µ-opioid system provides an important example of chemical pest defense using plant metabolites in nonhuman mammals.

Detection of urinary luteinizing hormone in Japanese black cows after administration of gonadotropin-releasing hormone.

The blood luteinizing hormone (LH) surge in cows is well studied. However, little is known about urinary LH in cows. This study examined urinary LH concentrations after administration of gonadotropin-releasing hormone (GnRH) in six Japanese black cows to induce LH secretion from the pituitary gland into the bloodstream. Abrupt rises in plasma and urinary LH were observed after GnRH administration. Plasma and urinary LH peaked at 2 and 5 hr, respectively. A positive correlation was observed between plasma LH concentrations and urinary LH amounts. Ovulation was confirmed in the cows after 48 hr of GnRH administration. These data strongly suggest that urinary LH is derived from plasma LH, which triggers ovulation in cows.

Cobetia sp. Bacteria, Which Are Capable of Utilizing Alginate or Waste Laminaria sp. for Poly(3-Hydroxybutyrate) Synthesis, Isolated From a Marine Environment.

We isolated the Cobetia sp. strains IU 180733JP01 (5-11-6-3) and 190790JP01 (5-25-4-2) from seaweeds and showed that both strains accumulate poly(3-hydroxybutyrate) [P(3HB)] homopolymer in a nitrogen-limiting mineral salt medium containing alginate as a sole carbon source. Genome sequence analysis of the isolated strains showed that they have putative genes which encode enzymes relevant to alginate assimilation and P(3HB) synthesis, and the putative alginate-assimilating genes formed a cluster. Investigation of the optimum culture conditions for high accumulation of P(3HB) showed that when the 5-11-6-3 strain was cultured in a nitrogen-limiting mineral salt medium (pH 5.0) containing 6% NaCl and 3% (w/v) alginate as a sole carbon source for 2 days, the P(3HB) content and P(3HB) production reached 62.1 ± 3.4 wt% and 3.11 ± 0.16 g/L, respectively. When the 5-25-4-2 strain was cultured in a nitrogen-limiting mineral salt medium (pH 4.0) containing 5% NaCl and 3% (w/v) alginate for 2 days, the P(3HB) content and P(3HB) production reached 56.9 ± 2.1 wt% and 2.67 ± 0.11 g/L, respectively. Moreover, the 5-11-6-3 strain also produced P(3HB) in a nitrogen-limiting mineral salt medium (pH 5.0) containing 6% NaCl and freeze-dried and crushed waste Laminaria sp., which is classified into brown algae and contains alginate abundantly. The resulting P(3HB) content and P(3HB) productivity were 13.5 ± 0.13 wt% and 3.99 ± 0.15 mg/L/h, respectively. Thus, we demonstrated the potential application of the isolated strains to a simple P(3HB) production process from seaweeds without chemical hydrolysis and enzymatic saccharification.

Characterization of mouse di-N-acetylchitobiase that can degrade chitin-oligosaccharides.

Di-N-acetylchitobiase (Ctbs) degrades ß-1,4 glycoside bonds of the chitobiose core of free asparagine-linked glycan. This study examined whether Ctbs degrades chitin-oligosaccharides to GlcNAc in mammals. We analyzed Ctbs mRNA and protein expression in mouse tissues and characterized enzymatic activity using recombinant mouse Ctbs expressed in Escherichia coli. Ctbs mRNA and protein were expressed in various tissues of mouse, including the stomach. Optimal conditions for recombinant Ctbs were pH 3.0 and 45°C, and the recombinant enzyme was retained more than 94% activity after incubation at pH 3.0-7.0 and below 37°C. The recombinant Ctbs hydrolyzed (GlcNAc)3 and (GlcNAc)6 at pH 3.0 and produced GlcNAc. The K m of Ctbs was lowest with (GlcNAc)3 as a substrate. k cat/K m was fourfold as high with (GlcNAc)3 and (GlcNAc)4 as substrates than with (GlcNAc)2. These results suggest that Ctbs digests chitin-oligosaccharides or (GlcNAc)2 of reducing-end residues of oligosaccharides and produces GlcNAc in mouse tissues.

Renal expression and urinary excretion of liver-type fatty acid-binding protein in cats with renal disease.

BACKGROUND: Liver-type fatty acid-binding protein (L-FABP) is a biomarker for early detection of renal disease in humans. Liver-type fatty acid-binding protein is cytotoxic oxidation products secreted from proximal tubules under ischemia and oxidative stress. OBJECTIVE: To examine renal expression and quantify urinary excretion of L-FABP in catswith renal disease. ANIMALS: One hundred and thirty-four client-owned cats including 34 cats with serum creatinine (sCre) values >1.6 mg/dL and 10 other cats that died in clinics. METHODS: Tissue expressions of L-FABP were examined by reverse transcription polymerase chain reaction and Western blotting. Urinary L-FABP (uL-FABP) and serum L-FABP (sL-FABP) levels were determined by enzyme-linked immunosorbent assay. Anti-liver-type fatty acid-binding protein antibody immunostained renal sections. RESULTS: Feline kidneys express L-FABP. Strong L-FABP signals were observed in the lumens of proximal tubular cells in 5 cats with high uL-FABP excretion, but not in 5 cats with low uL-FABP excretion. In 9 normal cats, uL-FABP index was <1.2 µg/g urinary creatinine (uCre). High uL-FABP indexes (>10.0 µg/g uCre) were detected in 7 of 100 cats with low sCre (<1.6 mg/dL) and 18 of 44 cats with high sCre (>1.6 mg/dL). There was a weak correlation between L-FABP index and sCre, serum symmetric dimethylarginine (SDMA), or blood urea nitrogen (BUN), and these correlation coefficients were increased by analyzing only data of cats with sCre >1.6 mg/dL. There was a weak correlation between u L-FABP index and sL-FABP in all tested cats, but not in cats with high sCre. CONCLUSIONS AND CLINICAL IMPORTANCE: This study demonstrates correlations between L-FABP and current renal biomarkers for chronic kidney disease in cats, such as sCre and SDMA. Liver-type fatty acid-binding protein may be a potential biomarker to predict early pathophysiological events in feline kidneys.

LC-ESI-MS/MS quantification of carnosine, anserine, and balenine in meat samples.

The histidine-containing imidazole dipeptide carnosine and its methylated analogs anserine and balenine are present at high concentrations in vertebrate tissues. Although the physiological functions of the imidazole dipeptides have not been elucidated yet, it has been suggested that they play significant biological roles in animals. Despite increasing interest, few studies have challenged the quantifications of carnosine, anserine, and balenine by a single HPLC run because they have similar retention times. In this study, we developed a method to quantify these imidazole dipeptides in meat samples using an LC-ESI-MS/MS triple-quadrupole mass spectrometer. We improved the liquid chromatographic separation of the imidazole dipeptides by applying a mix-mode column, which provides both normal phase and ion exchange separations, and developed multiple reaction-monitoring of the transitions for quantification of m/z 227 → 110 for carnosine, m/z 241 → 126 for anserine, m/z 241 → 124 for balenine, and m/z 269 → 110 for L-histidyl-L-leucine (internal standard). The established method met all pre-defined validation criteria. Intra- and inter-day accuracy and precision were ±10.0% and ≤14.8%, respectively. The ranges of quantifications were 14.7 ng/mL to 1.5 mg/mL for carnosine, 15.6 ng/mL to 1.6 mg/mL for anserine, and 15.6 ng/mL to 1.6 mg/mL for balenine. In conclusion, the validated method was successfully applied to the quantification of imidazole dipeptides in biological samples without derivatization.

Temporal changes in urinary excretion of liver-type fatty acid binding protein (L-FABP) in acute kidney injury model of domestic cats: a preliminary study.

Liver-type fatty acid-binding protein (L-FABP) is a biomarker for the early detection of renal diseases in humans. It is secreted along with cytotoxic oxidation products from proximal tubular epithelial cells under conditions of ischemia and/or oxidative stress. This study examined urinary L-FABP excretion under renal ischemia in feline acute kidney injury (AKI) model. L-FABP excretion increased immediately after renal ischemia/reperfusion, despite the absence of obvious structural damage to the kidneys, in the two AKI model cats studied. L-FABP was detected in the renal tubular lumen immediately after renal ischemia/reperfusion in the two cats, but not in a sham surgery cat. These results suggested that high L-FABP excretion is a pathophysiological response associated with antioxidant defense in proximal tubules with renal ischemia and/or oxidative stress in a feline model.

GC × GC-MS-Based Volatile Profiling of Male Domestic Cat Urine and the Olfactory Abilities of Cats to Discriminate Temporal Changes and Individual Differences in Urine.

Domestic cats (Felis silvestris catus) are solitary and territorial, and mark their territories by spraying urine, which emits a strong odor produced by volatile organic compounds (VOCs). Previous studies have focused on identifications of specific VOCs, such as 3-mercpto-3-methyl-1-butanol, a cat-specific VOCs. However, little is known about how whole volatile profiles of their sprayed urine change over time or how the profiles differ among individuals. This study investigated temporal changes and individual differences of volatile profiles produced by whole VOCs in cat urine, and the ability of cats to discriminate between these scent differences. Volatile profiles of fresh and aged cat urine were analyzed by using two-dimensional gas chromatography-mass spectrometry with a VOC preconcentrator comprehensively. Volatile profiles produced by hundreds of VOCs emitted from cat urine were influenced primarily by the age of the urine, and secondarily by individuality. During habituation-dishabituation tests, subjects discriminated between fresh and 24 h-old samples of same individuals, and between odor of different individuals from 0 h-, 3 h-old, and 24 h-old samples. These results strongly suggest that cats can recognize conspecific individuals via olfaction. Since most VOCs varied among individuals but were not stable over time, their urine may contain unknown VOCs that vary among individuals, are stable over time, and act as individual recognition signals.