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Computer Assisted Sperm Analysis (CASA) in the Critically Endangered Captive Arabian Leopard (Panthera pardus nimr): A Multivariate Clustering Analysis
Abstract
Rationale: Big felids including the Panthera genus are under tremendous stressful conditions that threaten the very existence of wild populations around the world. Survivability is commonly linked to numerous factors such as poaching, habitat fragmentation, inbreeding depression and lack of prey. A crucial element that is used to mitigate endangerment risk is the enhancement of reproductive performance with the use of assisted reproductive technologies. Amongst them is computer assisted sperm analysis (CASA) that digitally evaluates the kinematics of individual spermatozoa. Regrettably, this powerful tool is overlooked in all big felids due to the lack of a universal setting.Objective: To conduct a comparative CASA with several species modules and to deploy it for the first time in the critically endangered Arabian leopard.Results: The progressive motility was variable amongst all settings, whereby the highest in the bovine standard (82.9%), lowest in the stallion setting (12%), subjective (85%) and average at 50.1%. The combination of all motility parametrics, indicate a progressive joining of two minor and two major clusters with a very high distance of 93% and a linkage space of approximately 42%. This in turn demonstrate notable divergence of two important kinematic settings.Conclusion: The current study illustrates the inconsistent and incompatible readings amongst various CASA species modules. This affirms the urgent need to establish CASA exclusively customized for the Panthera genus to maximize the reproductive potential.
... Figure 5 shows representative sperm tracks and kinematic values for rapid progressive sperm of the African lion, Bengal tiger and African leopard. Progress has also been made to adapt different domestic animal settings and establish the correct CASA settings for sperm motility (for example, progressive motility) for the endangered Arabian leopard [41]. The Comparative Spermatology Group at the University of the Western Cape, in conjunction with several other research groups, including the National Zoological Garden in Pretoria as well as Dr. Imke Lueders from Hamburg, studied lion semen and sperm functionality in some 50 male lions in South Africa (some aspects published [40], but mostly unpublished). ...
... Figure 5 shows representative sperm tracks and kinematic values for rapid progressive sperm of the African lion, Bengal tiger and African leopard. Progress has also been made to adapt different domestic animal settings and establish the correct CASA settings for sperm motility (for example, progressive motility) for the endangered Arabian leopard [41]. ...
Simple Summary
In general, wildlife species have been underrepresented, in terms of understanding their reproductive physiology. The artificial propagation of wildlife species, found in aquaculture (e.g., fish) and in protection of endangered species (e.g., black-footed ferret), is pertinent to this discussion. One important approach to addressing this would involve a basic understanding of the structure and function of the gametes of many wildlife species. The focus of this investigation was to provide a better understanding of the physiology of sperm of diverse wildlife species, with special attention given to the assessment of high-quality sperm. Modern approaches using sophisticated microscopy (image analysis) techniques, e.g., computer-aided sperm analysis and sperm flagellar analysis, provided advanced technology to evaluate sperm quality quantitatively. Some of these techniques involved largely automated assessments of many aspects of sperm motility, morphology, vitality, fragmentation and other indirect methods. These modern assessments are fundamental to classify sperm quality. Accordingly, cutting-edge technologies used to define high quality sperm of representative species from most vertebrate animal groups (from fish to primates) were discussed in the present work. These approaches are also important in developing and assessing the best methods to cryopreserve sperm for assisted reproductive technologies in wildlife species.
Abstract
(1) Background: in order to propagate wildlife species (covering the whole spectrum from species suitable for aquaculture to endangered species), it is important to have a good understanding of the quality of their sperm, oocytes and embryos. While sperm quality analyses have mainly used manual assessment in the past, such manual estimations are subjective and largely unreliable. Accordingly, quantitative and cutting-edge approaches are required to assess the various aspects of sperm quality. The purpose of this investigation was to illustrate the latest technology used in quantitative evaluation of sperm quality and the required cut-off points to distinguish the differential grades of fertility potential in a wide range of vertebrate species. (2) Methods: computer-aided sperm analysis (CASA) with an emphasis on sperm motility, 3D tracking and flagellar and sperm tracking analysis (FAST), as well as quantitative assessment of sperm morphology, vitality, acrosome status, fragmentation and many other complimentary technologies. (3) Results: Assessing sperm quality revealed a great deal of species specificity. For example, in freshwater fish like trout, sperm swam in a typical tight helical pattern, but in seawater species sperm motility was more progressive. In amphibian species, sperm velocity was slow, in contrast with some bird species (e.g., ostrich). Meanwhile, in African elephant and some antelope species, fast progressive sperm was evident. In most species, there was a high percentage of morphologically normal sperm, but generally, low percentages were observed for motility, vitality and normal morphology evident in monogamous species. (4) Conclusions: Sperm quality assessment using quantitative methodologies such as CASA motility, FAST analysis, morphology and vitality, as well as more progressive methodologies, assisted in better defining sperm quality—specifically, sperm functionality of high-quality sperm. This approach will assist in the propagation of wildlife species.
... Although the motility values reported here are useful, subjective assessment lacks the precision to consider total motility a relevant indicator of fertility, making the incorporation of computer-assisted sperm analysis (CASA) beneficial for implementing assisted reproduction technologies in lizards. However, access to this tool may be limited in some contexts [45,46]. ...
While most lizard species are polygamous, dominant males typically have greater access to females and copulate more frequently than submissive ones. Several studies suggest that applying assisted reproduction techniques to this taxon could be an effective tool to enhance conservation programs. Therefore, the proper selection of males for assisted breeding programs is crucial for their overall success. In this study, we report for the first time data on semen and sperm quality from dominant males of the Mexican lizard Sceloporus torquatus using techniques commonly employed in assessing mammalian fertility. The semen and sperm characteristics were as follows: volume, 14.0 µL; sperm concentration, 125.7 × 10⁶ cells/mL; wave motion, 2.7; total motility, 87.8%; sperm viability, 89.0%; normal morphology, 88.8%; plasma membrane integrity, 87.7%; low plasma membrane fluidity, 94.9%; capacitation status: uncapacitated (F), 90.5%; capacitated (B), 7.2%; sperm with acrosome reaction (AR), 2.3%; and, acrosome integrity, 88.8%. These results suggest that, according to mammalian fertility parameters, dominant males of S. torquatus are fertile. This series of tests provides a valuable tool for conducting a comprehensive analysis of the functional changes that may occur in sperm handling during assisted processes, and that may contribute—along with the proper selection of individuals—to reproductive success.
... However, to ensure meaningful analysis, specific standardization tailored to the species or group of species under study is essential [27]. Prior studies have shown that sperm motility, concentration, and morphology assessments can vary significantly when different CASA setup used are applied to the same sample, potentially compromising the system's effectiveness [7,28]. Additionally, it is crucial to acknowledge the unique reproductive biology of sharks compared to other fish species, noting that detailed descriptions of key sperm characteristics, such as morphology, are often lacking for the majority of shark species. ...
1. One-third of elasmobranch species are threatened with extinction, and the precise assessment of their sperm parameters and quality is one stage in the development of reproductive technology for animal conservation.
2. Computer-Assisted Sperm Analysis (CASA) is the gold standard for sperm analysis; however, there were not one setup established for elasmobranchs.
3. This study presents a description of a specific setup developed for elasmobranch semen analysis using the CASA system.
4. In addition, it provides a gold-standard technique protocol for elasmobranch sperm quality assessment.
5. The described setup presented an accuracy of more than 90 percent in fourteen distinct species of freshwater and marine sharks, stingrays, and rays, failing only when there were two or more bundled sperm.
... O'Meara et al. [22] found that using different CASA setup variables had an effect on the motility, concentration, and morphology of cattle. Baqir et al. [23] used setups from several species to evaluate Arabian leopard (Panthera pardus nimr) sperm, demonstrating that the divergence in setups between species renders this essential technique ineffective. Therefore, standardizing a CASA setup is a vital step, and addressing it for giant anteaters will enable future research to ensure consistency and repeatability of findings. ...
The giant anteater (Myrmecophaga tridactyla) is a member of the Myrmecophagidae family and is now classified as "Vulnerable" due to the risk of extinction. In spite of this, research on reproductive biotechnologies in the species is rare, and semen collection has only been performed using electroejaculation to date. Pharmaceutical semen collection through urethral catheterization has become a distinct method that is being applied to numerous species of wild animals. In addition to establishing a species-specific setup, we compared semen acquired by urethral catheterization in three wild animals to that obtained in captivity in two animals. The average volume of semen collected was 180,0±24.5 µL, with an average motility of 62±14.7% and a vigor of 3,9 ±0.8. The concentration of sperm was 194.6 ±139.8 x 10 6 per mL. Using pharmaceutical semen collection through urethral catheterization was an effective, practical, and safe way for semen collection and evaluation in captive and free-living giant anteaters, according to the compiled data. We have described a specific setup for analyzing giant anteater semen using the CASA system, eliminating subjectivity from future research.
... It has been proposed that ICSI can serve as a tool to help endangered species , as these populations have low genetic diversity and low sperm quality, especially after cryopreservation procedures (Baqir et al., 2018;Cai et al., 2018;Franklin et al., 2018). It also constitutes a useful technique for the production of genetically modified embryos by coupling with the sperm-mediated gene transfer technique (ICSI-SMGT) (Perry et al., 1999; Pereyra-formation rates have been observed (~50−70%) with the birth of live offspring (Kimura and Yanagimachi, 1995;Hu et al., 2012). ...
Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique mainly used to overcome severe infertility problems associated with the male factor, but in cattle its efficiency is far from optimal. Artificial activation treatments combining ionomycin (Io) with 6-dimethylaminopurine after piezo-ICSI or anisomycin after conventional ICSI have recently increased the blastocyst rate obtained. Compounds to capacitate bovine spermatozoa, such as heparin and methyl-β-cyclodextrin and compounds to destabilize sperm membranes such as NaOH, lysolecithin and Triton X-100, have been assessed, although they have failed to substantially improve post-ICSI embryonic development. Disulfide bond reducing agents, such as dithiothreitol (DTT), dithiobutylamine and reduced glutathione, have been assessed to decondense the hypercondensed head of bovine spermatozoa, the two latter being more efficient than DTT and less harmful. Although piezo-directed ICSI without external activation has generated high fertilization rates and modest rates of early embryo development, other studies have required exogenous activation to improve the results. This manuscript thoroughly reviews the different strategies used in bovine ICSI to improve its efficiency and proposes some alternative approaches, such as the use of extracellular vesicles (EVs) as ‘biological methods of oocyte activation’ or the incorporation of EVs in the in vitro maturation and/or culture medium as antioxidant defence agents to improve the competence of the ooplasm, as well as a preincubation of the spermatozoa in estrous oviductal fluid to induce physiological capacitation and acrosome reaction before ICSI, and the use of hyaluronate in the sperm immobilization medium.
Spermatozoa are highly specialized male cells that are characterized by a unique ability to move, which is a critical factor for successful fertilization. The relative simplicity of motility assessment, especially in livestock, has made it a widely used parameter for evaluating ejaculate quality or cryopreserved semen in the clinical field, and an advanced tool in reproductive physiology and toxicology research. Technological advances in image analysis and computational methods have substantially increased its accuracy through the use of computer-assisted sperm analysis (CASA) to minimize subjective bias in motility assessments. Nevertheless, this more objective method still presents some significant challenges, including variability in the sample preparation, imaging conditions, and analytical parameters. These issues contribute to inconsistency and impair the reproducibility and comparability of data between laboratories. The implementation of standardized protocols, combined with comprehensive training and rigorous evaluation, can serve to mitigate some of the emerging inconsistencies. In addition, the in vitro conditions under which CASA analyses are performed often differ significantly from the natural environment of the female reproductive tract in vivo. This review discusses the methodologies, critical issues, and limitations of sperm motility analyses using CASA, with a particular focus on the boar as an important agricultural and biomedical model species in which this system is widely used.
Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique (ART) mainly used to overcome severe male factor infertility problems in humans and animals. However, in cattle, one of the most demanded species for its meat and milk, the efficiency of this technique is low. The present study compared the effect of the piezoelectric and conventional injection systems on the preimplantational development and quality of bovine embryos generated by ICSI. Evaluations of the conditions for performing the Piezo-ICSI procedure showed that the application of a strong pulse (I4S7) was more effective in damaging the sperm plasma and acrosomal membranes prior to injection, compared to a soft pulse (I2S2, P < 0.05). In addition, Piezo-ICSI embryos without the application of exogenous activators achieved similar levels of development as Piezo-ICSI embryos activated with ionomycin and anisomycin (P > 0.05). When comparing conventional and piezoelectric injection systems, no significant differences in embryo development were observed (P > 0.05). However, embryos generated by Piezo-ICSI showed a higher embryo quality in terms of total cell number (P < 0.05). In addition, Piezo-ICSI embryos showed an expression profile of genes essential for embryonic development similar to IVF embryos (P > 0.05), in contrast to conventional ICSI-derived embryos, which presented overexpression of CASP3 and IFNT2 (P < 0.05). In conclusion, we confirmed that Piezo-ICSI is a more convenient approach than traditional ICSI, since does not require exogenous activation and generate embryos of better quality, regarding the total number of blastomeres and the pattern of gene expression observed.
Elasmobranchs, a group encompassing rays and sharks, display spermatozoa with morphological characteristics markedly distinct from those observed in bony fishes. However, the andrology of these species remains poorly elucidated, with the absence of standardized procedures for semen evaluation representing a significant gap in scientific knowledge. The present study aimed to develop and validate a protocol for the analysis of elasmobranch spermatozoa using the Computer-Assisted Semen Analysis (CASA) system, to establish a chemically defined medium for sperm activation, and to assess the efficacy of diluents for cryopreservation, comparing the cryoprotectants dimethyl sulfoxide (DMSO) and methanol. Semen samples from multiple species of rays and sharks were collected at the São Paulo Aquarium and the Rio de Janeiro Marine Aquarium. The CASA configuration was initially optimized using semen from Potamotrygon rays and subsequently validated in marine rays and sharks using the IVOS II system. Sperm activation was tested in waters with varying salinity levels and later with the commercial activator Actifish. In terms of cryopreservation, the diluents Freezefish and INRA were explored in combination with DMSO and methanol. This work established a protocol for the evaluation of elasmobranch semen, validated across 14 different species, and acknowledged the helical morphology of the sperm head through the use of the CASA system—a significant milestone for the application of reproductive biotechnologies in the conservation of these animals. It was observed that salinity plays a predominant role in sperm activation, with optimal conditions mirroring the natural breeding environments, such as estuaries or marine habitats. Actifish emerges as an effective alternative, surpassing heterogeneous saline solutions. Nonetheless, further research is emphasized for species in which sperm activation has not yet been effectively established. Regarding cryopreservation, future studies are imperative for determining the most suitable diluents and cryoprotectants. Preliminary data suggest that mammalian-based media may be more promising than those based on bony fishes, and DMSO proves to be a more effective intracellular cryoprotectant compared to methanol. These findings establish a foundation for ongoing research in this crucial segment of elasmobranch reproductive biology.
For many people, thoughts of the United Arab Emirates conjure images of ultramodern skyscrapers and rolling sand dunes. However, the Emirates are a rich mosaic of ecosystems and habitats that support surprisingly diverse communities of organisms, and there is growing awareness of the importance of these previously underappreciated natural assets. A Natural History of the Emirates provides a comprehensive overview of the unusual environmental setting of this young nation, and surveys the major ecosystems and the marine and terrestrial organisms occurring across the nation. From freshwater streams in the hyperarid Hajar Mountains to the world’s most temperature-tolerant coral reefs, the UAE is home to an astounding variety of uniquely adapted organisms that are providing insights into climate change and how organisms cope with and respond to extreme environmental conditions. The book closes with a section on human interactions with this unique environment, and proposes initiatives to ensure the protection of these unique natural assets into the future.
This is an open access book.
Establishing and maintaining protected areas (PAs) are key tools for biodiversity conservation. However, this approach is insufficient for many species, particularly those that are wide-ranging and sparse. The cheetah Acinonyx jubatus exemplifies such a species and faces extreme challenges to its survival. Here, we show that the global population is estimated at ∼7,100 individuals and confined to 9% of its historical distributional range. However, the majority of current range (77%) occurs outside of PAs, where the species faces multiple threats. Scenario modeling shows that, where growth rates are suppressed outside PAs, extinction rates increase rapidly as the proportion of population protected declines. Sensitivity analysis shows that growth rates within PAs have to be high if they are to compensate for declines outside. Susceptibility of cheetah to rapid decline is evidenced by recent rapid contraction in range, supporting an uplisting of the International Union for the Conservation of Nature (IUCN) Red List threat assessment to endangered. Our results are applicable to other protection-reliant species, which may be subject to systematic underestimation of threat when there is insufficient information outside PAs. Ultimately, conserving many of these species necessitates a paradigm shift in conservation toward a holistic approach that incentivizes protection and promotes sustainable human–wildlife coexistence across large multiple-use landscapes.
Feline epididymal sperm is commonly used for in vitro fertilization. It also yields the opportunity to conserve genetic material from valuable males that suddenly die. Epididymal sperm quality parameters vary considerably among laboratories, implicating the need for objective evaluation methods. The aim of the present study was to describe reference values of computer-assisted sperm analysis (CASA) parameters of fresh epididymal cat sperm and to assess the effect of prolonged cooled storage (4�C) on various sample characteristics. Epididymides obtained from tomcats after routine orchiectomy (2–4 pairs/replicate) were sliced to release spermatozoa. The sperm suspension was placed on a 2-layer gradient and, after centrifugation, the sperm pellet was recovered. In Experiment 1 (20 replicates), sperm motility parameters were assessed immediately after retrieval (T0) using the Hamilton Thorne analyzer Ceros 12.1 (HTR; Hamilton Thorne Biosciences, Beverly, MA, USA). In Experiment 2, fresh (T0) sperm samples (4 replicates) were evaluated for motility parameters (HTR), acrosomal status (FITC-Pisum sativum agglutinin staining), morphology (eosin/nigrosin (E/N) staining), and membrane integrity (E/N and SYBR�-14-propidium iodide staining; Molecular Probes, Inc., Eugene, OR, USA). After addition (1:2) of a Tris-glucose-citrate diluent containing 20% egg yolk, samples were cooled and reassessed on Days 1 (T1), 3 (T3), 5 (T5), 7 (T7), and 10 (T10). Results were analyzed in a mixed linear model, with replicate as random factor and time as fixed effect (S-PLUS 7.0; Insightful Corp., Seattle, WA, USA). Results of Experiment 1 were as follows (mean � SD): motility (MOT): 80.8% � 23.5; progressive motility (PMOT): 69.9% � 23.2; velocity average pathway (VAP): 98.7 µm s–1 � 24.2; velocity straight line (VSL): 89.3 µm s–1 � 25.4; velocity curved line (VCL): 134.8 µm s–1 � 31.9; amplitude lateral head (ALH): 4.3 µm � 2.0; beat cross frequency (BCF): 34.6 Hz � 7.0; and straightness (STR): 89.6% � 6.6. In Experiment 2, MOT, PMOT, VAP, VSL, VCL, BCF, and the percentage of normal spermatozoa showed a decrease over time (P < 0.05) compared to fresh samples, starting from T1, T3, T5, T7, T5, T3, and T1, respectively. In contrast, STR, ALH, membrane integrity, and the percentage of acrosome-intact spermatozoa were not affected (P > 0.05) by cooled storage. To summarize, we have presented a set of reference values for CASA-parameters of fresh, epididymal cat spermatozoa. Cooled storage impaired most motility parameters and lowered the percentage of normal spermatozoa, but did not influence membrane integrity or acrosomal status. The effect of cooled storage on DNA fragmentation of sperm and its subsequent influence on in vitro embryo development require further investigation.
As a well-studied felid with limited genetic diversity, the cheetah (Acinonyx jubatus) has shaped much of the scientific debate surrounding inbreeding depression. The species survived a population bottleneck ~. 12,000 years ago and was extirpated from >. 75% of its historical range in the last century. Modern cheetahs produce poor-quality semen, a presumed manifestation of inbreeding depression. Within Felidae, a positive association between genetic diversity and semen quality is well supported by pedigree data and inter-species comparisons. However, this relationship has never been examined among individual cheetahs. Furthermore, whether ongoing population declines are exacerbating inbreeding depression in wild or captive cheetah populations is unknown. Using 12 microsatellite markers, we evaluated the relationship between heterozygosity and reproductive traits among wild (n = 54) and captive (n = 43) male cheetahs born from 1976-2007. We tested the hypotheses that genetic diversity has declined over the last ~. 30 years and is positively correlated with semen quality/breeding success in the cheetah. Findings revealed that genetic diversity has decreased in the wild, but not captive, population. Unexpectedly, heterozygosity was lower in proven versus unproven breeders and did not correlate with semen quality. A small proportion of all males (<. 10%) produced relatively high quality ejaculates, with sperm traits similar to those of non-inbred felid species. These data suggest a more complex relationship between inbreeding and male cheetah reproductive traits than previously appreciated. Intensive management of captive cheetahs appears to be minimizing inbreeding, whereas the continued erosion of genetic diversity in wild males is of conservation concern.
The leopard’s (Panthera pardus) broad geographic range, remarkable adaptability, and secretive nature have contributed to a misconception that this species might not be severely threatened across its range. We find that not only are several subspecies and regional populations critically endangered but also the overall range loss is greater than the average for terrestrial large carnivores. To assess the leopard’s status, we compile 6,000 records at 2,500 locations from over 1,300 sources on its historic (post 1750) and current distribution. We map the species across Africa and Asia, delineating areas where the species is confirmed present, is possibly present, is possibly extinct or is almost certainly extinct. The leopard now occupies 25-37% of its historic range, but this obscures important differences between subspecies. Of the nine recognized subspecies, three (P. p. pardus, fusca, and saxicolor) account for 97% of the leopard’s extant range while another three (P. p. orientalis, nimr, and japonensis) have each lost as much as 98% of their historic range. Isolation, small patch sizes, and few remaining patches further threaten the six subspecies that each have less than 100,000 km2 of extant range. Approximately 17% of extant leopard range is protected, although some endangered subspecies have far less. We found that while leopard research was increasing, research effort was primarily on the subspecies with the most remaining range whereas subspecies that are most in need of urgent attention were neglected.
A highly-illustrated feature highlighting the unique features of Khor Kharfot and Wadi Sayq in Dhofar, Oman, calling for protection and greater awareness of their value in preserving a remnant of Arabia's past.
Available in English http://www.wmenews.com/newsletters/1366812925wmenews_V6_I4_eng.pdf
and Arabic
www.wmenews.com/newsletters/1366812925WMENewsV6I4_ar.pdf
This study aimed to evaluate semen from Jungle Cat (Felis chaus) by urethral catheterization (CT) after medetomidine administration that offers feasible and different approaches to obtaining good quality sperm, especially in wild felids. Accordingly, this method was tested in five Jungle Cats. After general anesthesia with the α2-agonist medetomidine (which also stimulates semen release into the urethra) and ketamine, an abdomen ultrasound was performed to locate dilation of the first segment of the urethra (prostatic urethra). A commercial Tom cat urinary catheter 3–5 (depending on the size of the animal) was advanced into the urethra to reach the semen full dilated primary region of the urethra, so as to allow semen collection into the lumen of the catheter by capillary forces. After retraction, sperm volumes between 69 ± 27.92 yielded motility of 77.13 ± 14.15 (mean ± SD) with a mean sperm concentration of 75.13 ± 17.05 million/ml. The results of this study showed that semen collection in jungle cat is feasible, using this method. This study describes a simple, useful in field, inexpensive method which does not require the training of the animal and is better than other methods. Samples have normal pH, suitable color and consolidation, high concentration and lower contamination with excellent motility in Jungle Cat and potentially, other wild felid species, as an alternative to electro-ejaculation.
The Indochinese leopard (Panthera pardus delacouri) is a genetically distinct subspecies that historically occurred throughout mainland Southeast Asia, but might have experienced recent declines in numbers and distribution. This study aimed to determine the current distribution of the Indochinese leopard, and estimate its population size, by reviewing data from camera trap and other wildlife surveys conducted during the past 20 years. Our results showed the Indochinese leopard likely now occurs only in 6.2% of its historical range, with only 2.4% of its distribution in areas of confirmed leopard presence. The leopard is extirpated in Singapore, likely extirpated in Laos and Vietnam, nearly extirpated in Cambodia and China, and has greatly reduced distributions in Malaysia, Myanmar, and Thailand. There are plausibly only two major strongholds remaining, which we consider priority sites: Peninsular Malaysia, and the Northern Tenasserim Forest Complex. We also identified a small isolated population in eastern Cambodia as a third priority site, because of its uniqueness and high conservation value. We estimate a total remaining population of 973–2503 individuals, with only 409–1051 breeding adults. Increased poaching for the illegal wildlife trade likely is the main factor causing the decline of the Indochinese leopard. Other potential contributing factors include prey declines, habitat destruction, and possibly disease. We recommend a separate IUCN assessment for the Indochinese leopard, and that this subspecies be classified as Endangered. Our findings provide important information that can help guide where conservation actions would be most effective in preventing the extinction of this subspecies.