A multidisciplinary approach to analyze the antimicrobial resistance in natural ecosystems.

Antimicrobial Resistance (AMR) poses a global threat to both human health and environmental well-being. Our study delved into Costa Rican wildlife reserves, uncovering a substantial human impact on these ecosystems and underscoring the imperative to pinpoint AMR hotspots. Embracing a One Health perspective, we advocated for a comprehensive landscape analysis that intricately intertwined geographic, climatic, forest, and human factors. This study illuminated the link between laboratory results and observed patterns of antimicrobial use, thereby paving the way for sustainable solutions. Our innovative methodology involved deploying open-ended questions to explore antimicrobial usage across livestock activities, contributing to establishing a comprehensive methodology. Non-invasive sampling in wildlife emerged as a critical aspect, shedding light on areas contaminated by AMR. Feline species, positioned at the apex of the food chain, acted as sentinels for environmental health due to heightened exposure to improperly disposed waste. Regarding laboratory findings, each sample revealed the presence of at least one antimicrobial resistance gene (ARG). Notably, genes encoding resistance to tetracyclines dominated (94.9%), followed by beta-lactams (75.6%), sulfonamides (53.8%), aminoglycosides (51.3%), quinolones (44.9%), phenicols (25.6%), and macrolides (20.5%). Genes encoding polymyxins were not detected. Moreover, 66% of samples carried a multi-resistant microbiome, with 15% exhibiting resistance to three antimicrobial families and 51% to four. The absence of a correlation between forest coverage and ARG presence underscored the profound human impact on wildlife reserves, surpassing previous estimations. This environmental pressure could potentially modify microbiomes and resistomes in unknown ways. As not all antimicrobial families encoding ARGs were utilized by farmers, our next step involved evaluating other human activities to identify the primary sources of contamination. This comprehensive study contributed crucial insights into the intricate dynamics of AMR in natural ecosystems, paving the way for targeted interventions and sustainable coexistence.

Spheroid formation and luteinization of granulosa cells of felids in a long-term 3D culture.

In the present study, granulosa cells (GCs) from domestic cats and Persian leopard were cultured and characterized from selected days. The culture period was divided into two phases: maintenance, which lasted for 7 days, and luteinization, which followed for up to 11 days. Luteinization was performed on ultra-low attachment plates, supporting the formation of spheroids in a medium supplemented with insulin, forskolin, and luteinizing hormone (LH). GCs of domestic cat produced estradiol (E2) and progesterone (P4) during the maintenance phase. The gene expressions of some proteins involved in steroidogenesis were stable (STAR, HSD3B1) or decreased over time (CYP11A1, HSD17B1, CYP17A1, and CYP19A1), which was similar to the expressions of gonatropin receptors (LHCGR and FSHR). During the luteinization phase, P4 concentration significantly increased (P < 0.05), and E2, in contrast to the proliferation phase, was below detection range. The expression of genes of proteins involved in steroidogenesis (STAR, CYP11A1, HSD3B1, HSD17B1, CYP17A1, and CYP19A1) and of gonadotropin receptors (LHCGR and FSHR) significantly increased during the luteinization period, but some expressions exhibited a decrease at the end of the phase (LHCGR, FSHR, HSD17B1, CYP19A1). The morphology of the luteinized GCs of domestic cat resembled large luteal cells and had numerous vacuole-like structures. Also, the GCs of Persian leopard underwent luteinization, shown by increasing P4 production and HSD3B1 expression. This study confirms that GCs from felids can be luteinized in a 3D spheroid system which can be a basis for further studies on luteal cell function of felids. Additionally, we could show that the domestic cat can serve as a model species for establishing cell culture methods which can be transferred to other felids.

Detection of Trypanosoma evansi in jaguars (Panthera onca): insights from the Brazilian Pantanal wetland.

Trypanosoma evansi is a widespread and neglected zoonotic parasite that affects domestic and wild animals, causing a disease commonly known as "surra." The Brazilian Pantanal wetland is recognized as an enzootic area for this protozoan, yet recognizing the importance of reservoir hosts also in order to prevent zoonotic outbreaks. This study aimed to assess the occurrence of T. evansi in jaguars (Panthera onca) from the Brazilian Pantanal wetland and explore associated clinical and hematological manifestations. A total of 42 animals were screened by PCR and sequenced for species identification when positive. Trypanosoma evansi was detected in six free-ranging jaguars (six positive animals of 42 captures and 16 recaptures), representing the first molecular evidence of such infection in this animal species. Our findings suggest that jaguars may act as reservoir hosts of T. evansi in the Brazilian Pantanal wetland. The better understanding of the role of wildlife in the epidemiology of T. evansi is also of importance to future reintroduction and translocation programs toward wildlife conservation efforts.

Do not disturb: Visitors' effect on the behavior and welfare of female and male big cats in zoos.

The effect of visitor presence on animal behavior needs to be evaluated to ensure the welfare and improve the husbandry of the individuals in zoos. This study aims to assess the effect of visitor presence on the behavior and welfare of pairs of Amur tiger, snow leopard, and Eurasian lynx at Parco Natura Viva, Italy. The study was made of two periods: the baseline, in which the zoo was closed, and the visitor presence period, in which the zoo was opened. Per period and per subject 12 30-min observations were done. Continuous focal animal sampling method was used to collect behavior duration of the big cats. The main results of the study highlighted that, when visitors were present, all felids except for the female lynx were significantly more inactive than in the baseline. Moreover, despite interindividual and inter-species variability in the significance level of findings, natural behaviors such as attentive behavior, exploration/marking, locomotion, and positive social interactions were performed more in the baseline than in the visitor presence period. Finally, when visitors were present, as the studied subjects experienced daily longer exposure to visitors, inactivity increased whereas individual species-typical behaviors (e.g., locomotion) and positive social interactions decreased. Thus, visitors seem to partially alter the behavioral time-budgets of the study big cats, increasing inactivity and decreasing the performance of species-specific behavior, at least in some individuals.

The perception of felid welfare by zookeepers in North America and the implications for zoo managers.

Zookeepers working with felids were asked to complete an online survey to examine their perceptions of animal welfare. A total of 121 zookeepers in an AZA-accredited zoo completed the survey. Results from this study suggest that institutional offerings of professional development programming in animal welfare have the greatest potential to influence zookeeper perceptions of animal welfare. Participants also identified four areas of improvement for felid welfare, including 1) larger and more dynamic spaces, 2) increased attention to behavioral husbandry, 3) more unique diet presentations, and 4) the ability for the animal to remain out of the view of the public. In addition, there appear to be differences in perception between traditionally described "small cat" and "large cat" keepers in terms of the ability to deliver the Five Freedoms.

Genetic and population diversity of Toxocara cati (Schrank, 1788) Brumpt, 1927, on the basis of the internal transcribed spacer (ITS) region.

The present investigation was aimed to study the sequence, phylogenetic and haplotype analyses of Toxocara cati based on the ITS region, along with the genetic diversity, demographic history and population-genetic structure. The maximum likelihood tree based on Kimura 2-parameter model was constructed using the complete ITS region of all the nucleotide sequences (n = 57) of Toxocara spp. and other related ascarid worms available in the GenBank™. It placed all the sequences of T. cati into four major clades designated as T. cati genotypes 1-4 (TcG1-G4). A total of 66 signature nucleotides were identified in the ITS region between genotypes. The median-joining haplotype network displayed a total of 24 haplotypes, with China exhibiting the highest number of haplotypes (h = 20) followed by India (h = 4), and Japan and Russia (h = 1). It indicated a clear distinction between all the four genotypes. The pairwise FST values between all the genotypes indicated huge genetic differentiation (> 0.25) between different T. cati genotypes. Moreover, the gene flow (Nm) between T. cati genotypes was very low. Results of AMOVA revealed higher genetic variation between genotypes (92.82%) as compared to the variation within genotypes (7.18%). The neutrality indices and mismatch distributions for the G1-G4 genotypes, Indian isolates and the overall dataset of T. cati indicated either a constant population size or a slight population increase. The geographical distribution of all the genotypes of T. cati is also reported. This is the first report of genotyping of T. cati on the basis of the ITS region.

The influence of welfare and bonds with animals on the job satisfaction of felid keepers in North America.

The current study set out to examine the perceptions of felid welfare in zoos and elucidate the factors that influence an individual animal care professional's acceptance and recognition of welfare. A total of 121 felid keepers employed in an Association of Zoos & Aquariums facility were included in analysis. Results demonstrate job satisfaction is directly linked to access to training on welfare topics. The relationship between job satisfaction and the fulfillment of the Five Freedoms is positively correlated. Additionally, overall job satisfaction does not appear to influence the bond with felids, but it does with other zoo animals. Those with lower reported job satisfaction demonstrate an overall lower bond with nonfelids, where this not observed in felids. Participants in this study demonstrate the direct connection between job satisfaction and perceived fulfillment of the Five Freedoms. Participants who were provided access to training and information on animal welfare often demonstrated a higher job satisfaction, more positive perception of their employer, and overall view of zoos meeting the Five Freedoms with captive felids.

Changes in Serum Amyloid A Level in Domestic Cats during Pregnancy.

Reproduction of endangered species in captivity is an urgent problem for the conservation and restoration of biodiversity. For mammals, including felids, assessing and monitoring of pregnancy progression is fundamental for successful breeding. For the first time, changes in the concentrations of serum amyloid A (SAA), the main protein of the acute phase of inflammation in felines, were assessed during pregnancy in a domestic cat. It was found that changes in SAA concentrations in pregnant females are consistent: an increase to the middle of pregnancy (day 30) and a decrease to day 60. After parturition, the SAA concentrations in the blood of domestic cats increase. The litter size significantly affected the dynamic of SAA concentrations during the experiment, in particular, after parturition, the increase in its level was significantly higher in the females that gave birth to larger litters (from four to seven kittens).

Exploiting genomic synteny in Felidae: cross-species genome alignments and SNV discovery can aid conservation management.

BACKGROUND: While recent advances in genomics has enabled vast improvements in the quantification of genome-wide diversity and the identification of adaptive and deleterious alleles in model species, wildlife and non-model species have largely not reaped the same benefits. This has been attributed to the resources and infrastructure required to develop essential genomic datasets such as reference genomes. In the absence of a high-quality reference genome, cross-species alignments can provide reliable, cost-effective methods for single nucleotide variant (SNV) discovery. Here, we demonstrated the utility of cross-species genome alignment methods in gaining insights into population structure and functional genomic features in cheetah (Acinonyx jubatas), snow leopard (Panthera uncia) and Sumatran tiger (Panthera tigris sumatrae), relative to the domestic cat (Felis catus). RESULTS: Alignment of big cats to the domestic cat reference assembly yielded nearly complete sequence coverage of the reference genome. From this, 38,839,061 variants in cheetah, 15,504,143 in snow leopard and 13,414,953 in Sumatran tiger were discovered and annotated. This method was able to delineate population structure but limited in its ability to adequately detect rare variants. Enrichment analysis of fixed and species-specific SNVs revealed insights into adaptive traits, evolutionary history and the pathogenesis of heritable diseases. CONCLUSIONS: The high degree of synteny among felid genomes enabled the successful application of the domestic cat reference in high-quality SNV detection. The datasets presented here provide a useful resource for future studies into population dynamics, evolutionary history and genetic and disease management of big cats. This cross-species method of variant discovery provides genomic context for identifying annotated gene regions essential to understanding adaptive and deleterious variants that can improve conservation outcomes.

Cloprostenol, a synthetic analog of prostaglandin F2α induces functional regression in cultured luteal cells of felids†.

In the present study, we investigated the effect of the synthetic analog of prostaglandin F2α (PGF2α)-cloprostenol-on cultured steroidogenic luteal cells of selected felid species over a 2-day culture period. The changes induced by cloprostenol were measured based on progesterone concentration and mRNA expression analysis of selected genes. Cloprostenol significantly reduced concentration of progesterone in cell culture medium of small luteal cells isolated from domestic cat corpora lutea (CL) at the development/maintenance stage (P < 0.05), but did not influence progesterone production in cultured cells from the regression stage. A decrease or complete silencing of progesterone production was also measured in cultured luteal cells of African lion (formation stage) and Javan leopard (development/maintenance stage). Gene-expression analysis by real-time PCR revealed that treatment with cloprostenol did not have an influence on expression of selected genes coding for enzymes of steroidogenesis (StAR, HSD3B, CYP11A1) or prostaglandin synthesis (PTGS2, PGES), nor did it effect hormone receptors (AR, ESR1, PGR, PTGER2), an anti-oxidative enzyme (SOD1) or factors of cell apoptosis (FAS, CASP3, TNFRSF1B, BCL2) over the studied period. Significant changes were measured only for expressions of luteinizing hormone (P < 0.05), prolactin (P < 0.05) and PGF2α receptors (P < 0.005) (LHCGR, PRLR, and PTGFR). The obtained results confirm that PGF2α/cloprostenol is a luteolytic agent in CL of felids and its impact on progesterone production depends on the developmental stage of the CL. Cloprostenol short-term treatment on luteal cells was associated only with functional but not structural changes related to luteal regression.

Molecular and Serological Survey of the Cat-Scratch Disease Agent (Bartonella henselae) in Free-Ranging Leopardus geoffroyi and Leopardus wiedii (Carnivora: Felidae) From Pampa Biome, Brazil.

The genus Bartonella comprises emerging bacteria that affect humans and other mammals worldwide. Felids represent an important reservoir for several Bartonella species. Domestic cats are the main reservoir of Bartonella henselae, the agent of cat scratch disease (CSD). It can be transmitted directly by scratches and bites from infected cats and via cat fleas. This study aims to investigate the circulation of Bartonella spp. in free-ranging Neotropical wild felids from Southern Brazil using serological and molecular methods. In this study, 53 live-trapped free-ranging wild felids were sampled, 39 Leopardus geoffroyi and 14 Leopardus wiedii, from five municipalities in the Rio Grande, do Sul state, southern Brazil. All captured animals were clinically healthy. Two blood samples of L. geoffroyi were positive, by PCR, for the presence of B. henselae DNA. Conversely, none of L. wiedii blood samples were positive when tested using PCR. Indirect immunofluorescence assay (IFA) showed that 28% of serum samples of wild felids were reactive (seropositive) for B. henselae by immunofluorescence, with titers ranging from 64 to 256. The results presented here provide the first evidence of a Bartonella-enzootic cycle involving L. geoffroyi and L. wiedii, which may account for the spillover of the emerging zoonotic pathogen B. henselae for the indigenous fauna in Southern Brazil.

Effects of somatic tissue cryopreservation on puma (Puma concolor L, 1771) tissue integrity and cell preservation after in vitro culture.

Somatic resource banks play a crucial role in the conservation of genetic diversity, allowing for the preservation of biological samples from different populations. Puma somatic cells can be recovered from these banks and used in assisted techniques toward enhancing their multiplication and conservation. In response to the population reduction of this ecologically importance species, we aimed to evaluate the capacity of cryopreservation of somatic tissues on the maintenance of the integrity and quality of the cells recovered after culture, with the aim of establishing a somatic tissue bank that will allow for the safeguarding of a wide genetic sampling of pumas. Cryopreservation increased the thickness of the corneum layer in the tissues, and the number of perinuclear halos and empty gaps. Nevertheless, cryopreservation was able to maintain normal fibroblast patterns, even showing an increase in the percentage of collagen fibers. Cryopreservation maintained the proliferative potential of the tissues and the parameters evaluated during in vitro culture, mainly regarding the viability, proliferative activity, and apoptosis levels. Nevertheless, cells from cryopreserved tissues showed decreased metabolism and mitochondrial membrane potential when compared to cells from non-cryopreserved tissues. In summary, we demonstrated for the first time that puma somatic tissues subjected to cryopreservation are viable and maintain tissue integrity, featuring minimal changes after warming. Although viable somatic cells are obtained from these tissues, they undergo alterations in their metabolism and mitochondrial membrane potential. Improvements in the conservation conditions of somatic samples are needed to increase the quality of somatic tissue banks in this species.

Cell survival after cryopreservation of dissociated testicular cells from feline species.

Testicular cell suspension (TCS) can be cryopreserved for male germ-line preservation and fertility restoration. We aimed to validate a cryopreservation protocol for TCS of domestic cat to be applied in endangered felids species. Testis tissue from adult domestic cats was enzymatically dissociated and spermatogenic cells were enriched. The resulting TCS was diluted in 7.5% or 15% Me2SO based medium. Slow and fast freezing methods were tested. We examined the effects of freezing approaches using two combinations of fluorescent dyes: Calcein-AM with Propidium iodide (C/PI) and SYBR14 with Propidium iodide (S/PI). Ploidy analysis of domestic cat fresh TCS revealed that the majority of testicular cells were haploid cells. Based on microscopic observation, two size populations (12.3 ± 2.3 µm and 20.5 ± 4 µm in diameter) were identified and presumed to be mainly spermatids and spermatocytes, respectively. Both evaluation methods proved higher viability of aggregated cells before and after cryopreservation compared with single cells, and superiority of low concentration of Me2SO (7.5%) in association with slow freezing to preserve viability of testicular cells. However, S/PI resulted in a more precise evaluation compared with the C/PI method. The combination of 7.5% Me2SO-based medium with slow freezing yielded post thaw viability of S/PI labeled aggregated (49.8 ± 20%) and single cells (31.5 ± 8.1%). Comparable results were achieved using testes of a Cheetah and an Asiatic golden cat. In conclusion, TCS from domestic cat can be successfully cryopreserved and has the potential to support fertility restoration of endangered felids species.

Influence of different extenders on morphological and functional parameters of frozen-thawed spermatozoa of jaguar (Panthera onca).

Due to the global decrease in jaguar population, conservation strategies are essential and the development of effective semen cryopreservation protocols would contribute to the formation of germplasm banks. Therefore, the objectives were to (1) evaluate the use of TRIS and ACP-117c extenders for jaguar semen freezing, (2) describe the ultrastructural changes in sperm after cryopreservation, and (3) evaluate the binding capacity of the thawed sperm. Eight ejaculates from five mature individuals were collected by electroejaculation, extended in TRIS or a coconut based-extender (ACP-117c), and frozen in liquid nitrogen. Samples were evaluated for sperm motility, vigor, membrane functionality, mitochondrial activity, morphology (using light microscopy, scanning electron microscopy - SEM and transmission electron microscopy - TEM), sperm kinetic parameters (by computerized analysis - CASA), and sperm binding capability using an egg yolk perivitelline membrane assay. Samples preserved in TRIS presented better post-thaw motility (46.0 ±â€¯7.7%) and membrane functionality (60.5 ±â€¯4.2%) and higher mitochondrial activity (21.5 ±â€¯3.7%) than those preserved in ACP-117c (20.9 ±â€¯5.4% motile sperm; 47.1 ±â€¯2.5% functional membrane; 11.8 ±â€¯1.7% mitochondrial activity). Regarding ultrastructural evaluations, SEM showed that both extenders were able to preserve the superficial membrane of the sperm, but TEM revealed the occurrence of nuclear electron lucent points, especially in samples extended in ACP-117c. Additionally, TRIS also provided a higher number of sperm bound to the perivitelline membrane (29.5 ±â€¯3.3%) in comparison to samples diluted in ACP-117c (18.6 ±â€¯1.5%). Overall, we suggest the use of a TRIS-based extender for cryopreservation of jaguar semen.

Oral Lesions in Captive Nondomestic Felids With a Focus on Odontogenic Lesions.

Odontogenic lesions are well described in domestic cats, but published literature describing these lesions in nondomestic felids is limited. This study reports oral lesions in 109 captive, non-domestic felids. Ten cases of odontogenic lesions were diagnosed, including 9 with fibromatous epulis of periodontal ligament origin (FEPLO) and one odontogenic cyst in a cougar. FEPLO was common in lions. FEPLO did not recur after surgical removal in any of the 3 cases for which follow-up information was available. Increased occurrences of oral papillomas in snow leopards and eosinophilic granulomas in tigers were identified, which is consistent with the reported literature. With the exception of oral papillomas in snow leopards and FEPLO in lions, the spectrum of oral lesions in nondomestic felids was similar to what is reported in domestic cats, with squamous cell carcinoma being the most common oral malignancy, and stomatitis/gingivitis/glossitis accounting for approximately one third of all cases. Rare diagnoses with one case each included hemangioma, fibrosarcoma, melanoma, cleft palate, and glossal amyloidosis.

First confirmed case of Alaria alata mesocercaria in Eurasian lynx (Lynx lynx) hunted in Latvia.

Trematode Alaria spp. have a complex life cycle that includes canids, mustelids, and felids as definitive hosts. However, several mammal species, besides acting as definitive hosts, may also act as paratenic hosts. Alaria spp. life cycles are well studied. Still, there is a lack of knowledge on one host significance as both-the paratenic and definitive host, especially in the case of Eurasian lynx (Lynx lynx). Previous studies have described Eurasian lynx as a definitive host for A. alata although the prevalence has always been low. The aim of the present study was to analyze the occurrence of A. alata in Eurasian lynx in Latvia. The viscera (n = 231) and muscle samples (n = 234) from Eurasian lynx were collected during the hunting seasons from 2004/2005 to 2017/2018. The prevalence of the adult A. alata in Eurasian lynx was 1.7%. However, mesocercariae infection in Eurasian lynx was detected for the first time. These findings suggest that the Eurasian lynx may serve both as paratenic and definitive host for A. alata.

Assisted reproduction for felid species conservation-Sperm competences at risk.

Cryobanking of gametes in combination with artificial insemination is an essential option to support conservation programmes for endangered and threatened species. About two-thirds of the felid species are classified as 'near threatened', 'vulnerable' or 'endangered' (www.cites.org), and mostly, epididymal sperm are collected from euthanized or castrated male felids and cryopreserved. However, epididymal compared with ejaculated and cryopreserved compared with fresh sperm have a limited potential to fertilize if vaginal non-surgical insemination is applied in feline species. Missing or highly diluted seminal fluid in epididymal and cryopreserved sperm, as well as a potential interference of extender ingredients with the natural interactive properties of sperm in the female genital tract is discussed as potential drawback which hampers a proper sperm transit and fertilization besides the limited longevity of cryopreserved feline sperm. Individual components in seminal fluid as well as cryoextenders may adversely alter sperm properties and have a different impact on fertility and preservation success. The identification and investigation of beneficial as well as detrimental components is a precondition to deduce options for improving the process of cryopreservation in felids, particularly, if only epididymal sperm are available.

Distribution of uterine histological changes in aged captive cheetahs (Acinonyx jubatus).

The histological effect on the felid uterus of sterilization, via ovariectomy or salpingectomy, is currently unknown. To investigate the association of ovariectomy or salpingectomy with uterine health, it is first necessary to establish if changes are distributed evenly throughout the uterus. Both laparoscopic ovariectomy and salpingectomy with concurrent sampling of the tip of the uterine horn are possible in the cheetah. Currently accepted practice for histopathological screening of the uterus utilizes four biopsy samples. It is not known whether this method accurately reflects the status of the entire uterus. In this study we histologically examined the uteri of six older cheetahs (one 7-year-old and five 10-10.5-year-old animals) via 21 tissue samples (three samples from seven different anatomical regions) per cheetah to determine overall uterine health. Although no defined lesions were detected, mild endometrial gland dilation, assumed to be of no functional consequence, was observed in multiple samples. The odds of observing this dilation was lowest in the uterine body and progressively increased in a cranial direction, being significantly higher at the tip of the uterine horns (OR = 11.5; 95% CI, 2.0-65.1; p = 0.006). This supported the reliability of sampling the tip of the uterine horn to screen for endometrial gland dilation.

Type X strains of Toxoplasma gondii are virulent for southern sea otters (Enhydra lutris nereis) and present in felids from nearby watersheds.

Why some Toxoplasma gondii-infected southern sea otters (Enhydra lutris nereis) develop fatal toxoplasmosis while others have incidental or mild chronic infections has long puzzled the scientific community. We assessed robust datasets on T. gondii molecular characterization in relation to detailed necropsy and histopathology results to evaluate whether parasite genotype influences pathological outcomes in sea otters that stranded along the central California coast. Genotypes isolated from sea otters were also compared with T. gondii strains circulating in felids from nearby coastal regions to assess land-to-sea parasite transmission. The predominant T. gondii genotypes isolated from 135 necropsied sea otters were atypical Type X and Type X variants (79%), with the remainder (21%) belonging to Type II or Type II/X recombinants. All sea otters that died due to T. gondii as a primary cause of death were infected with Type X or X-variant T. gondii strains. The same atypical T. gondii strains were detected in sea otters with fatal toxoplasmosis and terrestrial felids from watersheds bordering the sea otter range. Our results confirm a land-sea connection for virulent T. gondii genotypes and highlight how faecal contamination can deliver lethal pathogens to coastal waters, leading to detrimental impacts on marine wildlife.

Analysis of metabolic flux in felid spermatozoa using metabolomics and 13C-based fluxomics†.

Spermatozoa from three feline species-the domestic cat (Felis catus), the cheetah (Acinonyx jubatus), and the clouded leopard (Neofelis nebulosa)-were analyzed using metabolomic profiling and 13C-based fluxomics to address questions raised regarding their energy metabolism. Metabolic profiles and utilization of 13C-labeled energy substrates were detected and quantified using gas chromatography-mass spectrometry (GC-MS). Spermatozoa were collected by electroejaculation and incubated in media supplemented with 1.0 mM [U13C]-glucose, [U13C]-fructose, or [U13C]-pyruvate. Evaluation of intracellular metabolites following GC-MS analysis revealed the uptake and utilization of labeled glucose and fructose in sperm, as indicated by the presence of heavy ions in glycolytic products lactate and pyruvate. Despite evidence of substrate utilization, neither glucose nor fructose had an effect on the sperm motility index of ejaculated spermatozoa from any of the three felid species, and limited entry of pyruvate derived from these hexose substrates into mitochondria and the tricarboxylic acid cycle was detected. However, pathway utilization was species-specific for the limited number of individuals (four to seven males per species) assessed in these studies. An inhibitor of fatty acid beta-oxidation (FAO), etomoxir, altered metabolic profiles of all three felid species but decreased motility only in the cheetah. While fluxomic analysis provided direct evidence that glucose and fructose undergo catabolic metabolism, other endogenous substrates such as endogenous lipids may provide energy to fuel motility.