Early life experience and sex influence acoustic repertoire use in wild-born, but hand-reared, captive cheetahs (Acinonyx jubatus).

Early deprivation of adult influence is known to have long-lasting effects on social abilities, notably communication skills, as adults play a key role in guiding and regulating the behavior of youngsters, including acoustic repertoire use in species in which vocal production is not learned. Cheetahs grow up alongside their mother for 18 months, thus maternal influences on the development of social skills are likely to be crucial. Here, we investigated the impact of early maternal deprivation on vocal production and use in 12 wild-born cheetahs, rescued and subsequently hand-reared either at an early (less than 2 months) or a later stage of development. We could distinguish 16 sound types, produced mostly singly but sometimes in repeated or multitype sound sequences. The repertoire of these cheetahs did not differ fundamentally from that described in other studies on adult cheetahs, but statistical analyses revealed a concurrent effect of both early experience and sex on repertoire use. More specifically, early-reared males were characterized by a high proportion of Purr, Meow, and Stutter; early-reared females Mew, Growl, Hoot, Sneeze, and Hiss; late-reared males Meow, Mew, Growl, and Howl; and late-reared females mostly Meow. Our study demonstrates therefore the long-term effects of maternal deprivation on communication skills in a limited-vocal learner and its differential effect according to sex, in line with known social differences and potential differential maternal investment. More generally, it emphasizes the critical importance to consider the past history of the subjects (e.g., captive/wild-born, mother/hand-reared, early/late-mother-deprived, etc.) when studying social behavior, notably acoustic communication.

Captive rearing of orphaned African wild dogs (Lycaon pictus) in Namibia: A case study.

African wild dogs (AWDs; Lycaon pictus) are an endangered canid species facing drastic decline throughout their range due to habitat fragmentation and persecution by humans over livestock depredation, resulting in dens destroyed and adult members of packs and pups often being killed. Breeding of captive AWDs is challenging due to high juvenile mortality, only marginally improved from wild conditions, thus both in situ and ex situ conservation remains critical. As a result of human-wildlife conflict, between 2017 and 2018, the Namibian Ministry of Environment, Forestry and Tourism confiscated three litters of orphaned AWD pups from rural farmers who had destroyed the dens in Eastern Namibia and placed the pups with the Cheetah Conservation Fund. Seventeen of the 18 pups were successfully reared to yearlings with 15 individuals translocated for eventual soft release into a private game reserve. This case study provides information on the successful rearing of three litters of orphaned wild dog pups on behavior, housing, husbandry, diet, growth and medical issues as limited information is available for rearing orphaned pups from the age of 2.5 weeks old.

The global decline of cheetah Acinonyx jubatus and what it means for conservation.

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.

Regional variation in the cheetah (Acinonyx jubatus) revisited: Morphology of wild and captive populations.

The cheetah (Acinonyx jubatus) is listed as a vulnerable species by the International union for the conservation of nature (IUCN), including two critically endangered subspecies, the Saharan cheetah, and the Iranian cheetah, so it is imperative that we understand variation in cheetah morphology to make good decisions regarding the conservation of this species. Here, we aim to determine whether northeastern African cheetahs have smaller body sizes than southern African cheetahs. This study also adds to our knowledge of cheetah morphology from two cheetah populations that do not yet have comprehensive published data: Kenya, and northeastern Africa, including captive individuals. We calculated means and standard deviations on cranial and body measurements of live or in few cases, freshly dead, cheetahs from the aforementioned populations, plus previously published data on Namibian and Botswanan cheetahs and compared them to one another using multivariate analysis of variance. Results show that northeastern African cheetahs have smaller body sizes than southern and eastern African populations. We also found that captive cheetahs retain the morphological characteristics of their ancestral population- captive cheetahs from southern Africa have similar body sizes to wild southern African cheetahs and larger body sizes than captives from northeastern Africa. Other analyses regarding cheetah growth agree with previous studies on Namibian and Botswanan cheetah populations rates. As such, this study can serve as a baseline for the care of captive cheetah populations to maintain healthy weights and body proportions.

Conservation Genetics of the Cheetah: Lessons Learned and New Opportunities.

The dwindling wildlife species of our planet have become a cause célèbre for conservation groups, governments, and concerned citizens throughout the world. The application of powerful new genetic technologies to surviving populations of threatened mammals has revolutionized our ability to recognize hidden perils that afflict them. We have learned new lessons of survival, adaptation, and evolution from viewing the natural history of genomes in hundreds of detailed studies. A single case history of one species, the African cheetah, Acinonyx jubatus, is here reviewed to reveal a long-term story of conservation challenges and action informed by genetic discoveries and insights. A synthesis of 3 decades of data, interpretation, and controversy, capped by whole genome sequence analysis of cheetahs, provides a compelling tale of conservation relevance and action to protect this species and other threatened wildlife.

Age-associated and deslorelin-induced declines in serum anti-Müllerian hormone concentrations in female cheetahs, Acinonyx jubatus.

Anti-Müllerian hormone (AMH) is widely used in human medicine to non-invasively estimate the size of the ovarian follicle reserve and to predict the ovarian response to gonadotropin stimulation in the context of assisted reproductive technologies (e.g., IVF). These applications of AMH testing have recently expanded to non-human mammals, with production animals, such as cows, goats and sheep being the primary focus of AMH research. However, few investigations have involved exotic species, and in particular carnivores. In this study, we measured AMH concentrations (0.078-3.078ng/mL) in archived serum samples that had been collected from 36 adult female cheetahs across their reproductive lifespan (2-15years of age). Similar to other mammals, AMH concentration in cheetahs declined with age, and its variability among females of the same age was considerable. The rates at which AMH declined over time in individual cheetahs were also highly variable. Five cheetahs had been contracepted with the long-acting GnRH agonist deslorelin for 6-18months prior to sample collection, and their AMH concentrations were relatively low compared to untreated females. In this first study of AMH in an exotic carnivore, the findings demonstrate that the age-associated decline in AMH is highly variable and that deslorelin appears to suppress AMH concentration in serum. Owing to the increased use of assisted reproductive technologies in ex situ populations of threatened and endangered species, such as cheetahs, the present study's findings will need to be taken into consideration if AMH is to be used successfully to optimize breeding management decisions in exotic species.

CARDIORESPIRATORY EFFECTS OF DEXMEDETOMIDINE-BUTORPHANOL-MIDAZOLAM (DBM): A FULLY REVERSIBLE ANESTHETIC PROTOCOL IN CAPTIVE AND SEMI-FREE-RANGING CHEETAHS (ACINONYX JUBATUS).

Multiple anesthesia protocols have been used in the cheetah ( Acinonyx jubatus ). Twenty healthy, captive cheetahs were immobilized with dexmedetomidine (15.8 ± 1.9 µg/kg), butorphanol (0.22 ± 0.03 mg/kg), and midazolam (0.18 ± 0.03 mg/kg) by intramuscular injection. Induction, recumbency, and recovery times were recorded, and physiologic parameters were monitored. Anesthesia was antagonized with atipamezole (0.125 ± 0.02 mg/kg) and naltrexone (0.1 ± 0.014 mg/kg) intramuscularly. All cheetahs were safely anesthetized with this protocol. Cheetahs were laterally recumbent by 8 ± 3.5 min. Cardiorespiratory values were stable throughout the length of anesthesia. Moderate hypertension, with systolic blood pressure ranging from 178 ± 19.8 mm Hg, was initially observed but decreased over time. There was a statistical decreasing trend in temperature; SpO2; and systolic, mean, and diastolic blood pressure, but not in heart rate and end-tidal CO2. Recoveries were rapid, with cheetahs standing by 11.3 ± 5.7 min postreversal administration. This is the first report of a dexmedetomidine-butorphanol-midazolam anesthetic combination in cheetahs. Overall, this anesthetic protocol proved to be safe and effective.

Serum Amyloid A Protein Concentration in Blood is Influenced by Genetic Differences in the Cheetah (Acinonyx jubatus).

Systemic amyloid A (AA) amyloidosis is a major cause of morbidity and mortality among captive cheetahs. The self-aggregating AA protein responsible for this disease is a byproduct of serum amyloid A (SAA) protein degradation. Transcriptional induction of the SAA1 gene is dependent on both C/EBPß and NF-κB cis-acting elements within the promoter region. In cheetahs, 2 alleles exist for a single guanine nucleotide deletion in the putative NF-κB binding site. In this study, a novel genotyping assay was developed to screen for the alleles. The results show that the SAA1A (-97delG) allele is associated with decreased SAA protein concentrations in the serum of captive cheetahs (n = 58), suggesting genetic differences at this locus may be affecting AA amyloidosis prevalence. However, there was no significant difference in the frequency of the SAA1A (-97delG) allele between individuals confirmed AA amyloidosis positive versus AA amyloidosis negative at the time of necropsy (n = 48). Thus, even though there is evidence that having more copies of the SAA1A (-97delG) allele results in a potentially protective decrease in serum concentrations of SAA protein in captive cheetahs, genotype is not associated with this disease within the North American population. These results suggest that other factors are playing a more significant role in the pathogenesis of AA amyloidosis among captive cheetahs.

TREATMENT OF CHRONIC HERPESVIRAL DERMATITIS IN A CAPTIVE CHEETAH (ACINONYX JUBATUS) IN NAMIBIA.

A 9-yr-old male cheetah (Acinonyx jubatus) housed at the Cheetah Conservation Fund in Namibia developed cutaneous lesions consisting of alopecia, erythema, ulceration, and crusting on the left fore and hind limbs. Histopathology of skin biopsies in conjunction with indirect fluorescent antibody and polymerase chain reaction testing confirmed a diagnosis of feline herpesvirus-1 dermatitis; microbial culture indicated secondary bacterial infection. Therapy included targeted systemic antimicrobial and antiviral treatment, topical medications, and repeated cryotherapy. Lesions exhibited varying degrees of clinical improvement but, overall, progressed in extent, size, and severity during the subsequent 2.5 yr of intense treatment. The cheetah was ultimately euthanized due to a guarded prognosis and concerns about poor quality of life. Potential factors initiating or contributing (or both) to the severity and nonhealing nature of the cutaneous lesions include chronic unidentified stress, altered immune system function, and other environmental influences.

COMPARISON OF TWO α2-ADRENERGIC AGONISTS ON URINE CONTAMINATION OF SEMEN COLLECTED BY ELECTROEJACULATION IN CAPTIVE AND SEMI-FREE-RANGING CHEETAH (ACINONYX JUBATUS).

Alpha2-adrenergic agonists are used to immobilize many veterinary species, but use has been infrequently linked to urine contamination of semen collected via electroejaculation. The objective of the study was to compare the α2-agonists medetomidine and dexmedetomidine on urine contamination of semen in anesthetized cheetahs (Acinonyx jubatus) during electroejaculation procedures. From 2009-2012, a retrospective medical record review revealed 21 anesthesia events in 12 adult male cheetahs. Animals were immobilized with combinations of Telazol® (2.33±0.43 mg/kg) and ketamine (2.38±1 mg/kg); Telazol (1.17±0.14 mg/kg), ketamine (1.17±0.14 mg/kg), and medetomidine (0.012±0.0017 mg/kg); or Telazol (1.59±0.1 mg/kg), ketamine (1.59±0.1 mg/kg) and dexmedetomidine (0.01±0.001 mg/kg). Semen was successfully collected in all animals; four animals anesthetized with medetomidine had urine contamination (P=0.037). Medetomidine may contribute to urine contamination; however, further investigation is needed to determine significance in cheetahs.

Response of woody vegetation to bush thinning on freehold farmlands in north-central Namibia.

Bush encroachment affects much of the Namibian woodland landscape, causing significant loss of open savannah habitat and farm profits. Thinning of the trees/shrubs is recommended; however, research is required to identify the overall efficacy and effects of this method on the woodland habitat. We aimed to examine the effect of the thinning strategy applied on the vegetation structure of encroaching tree/shrub species, as well as the sighting lines of the habitat. Vegetation surveys were done on three freehold farms in north-central Namibia. The study utilised a combination of a blocked and split-plot study design: each block consisted of a pair of thinned and non-thinned plots with multiple subplots. Thinned plots had been manually thinned, with a post-thinning age of three years or more. Results revealed that tree/shrub abundance differed between species; thinned areas had the least abundance and overall species-treatment interactions were significant. Thinning caused a significant reduction in overall tree/shrub densities, settling within the recommended range for the area. Thinning also significantly reduced the average tree/shrub height, canopy area, medium-sized trees/shrubs, and increased sighting lines. This confirms a bush encroachment mitigation strategy that favours grass cover, and wildlife that rely on longer sighting lines for safety or when hunting.

Different patterns of metabolic cryo-damage in domestic cat (Felis catus) and cheetah (Acinonyx jubatus) spermatozoa.

Felid spermatozoa are sensitive to cryopreservation-induced damage, but functional losses can be mitigated by post-thaw swim-up or density gradient processing methods that selectively recover motile or structurally-normal spermatozoa, respectively. Despite the importance of sperm energy production to achieving fertilization, there is little knowledge about the influence of cryopreservation or post-thaw processing on felid sperm metabolism. We conducted a comparative study of domestic cat and cheetah sperm metabolism after cryopreservation and post-thaw processing. We hypothesized that freezing/thawing impairs sperm metabolism and that swim-up, but not density gradient centrifugation, recovers metabolically-normal spermatozoa. Ejaculates were cryopreserved, thawed, and processed by swim-up, Accudenz gradient centrifugation, or conventional washing (representing the 'control'). Sperm glucose and pyruvate uptake, lactate production, motility, and acrosomal integrity were assessed. Mitochondrial membrane potential (MMP) was measured in cat spermatozoa. In both species, lactate production, motility, and acrosomal integrity were reduced in post-thaw, washed samples compared to freshly-collected ejaculates. Glucose uptake was minimal pre- and post-cryopreservation, whereas pyruvate uptake was similar between treatments due to high coefficients of variation. In the cat, swim-up, but not Accudenz processing, recovered spermatozoa with increased lactate production, pyruvate uptake, and motility compared to controls. Although confounded by differences in non-specific fluorescence among processing methods, MMP values within treatments were positively correlated to sperm motility and acrosomal integrity. Cheetah spermatozoa isolated by either selection method exhibited improved motility and/or acrosomal integrity, but remained metabolically compromised. Collectively, findings revealed a metabolically-robust subpopulation of cryopreserved cat, but not cheetah, spermatozoa, recovered by selecting for motility rather than morphology.

Diagnosis-based treatment of helminths in captive and wild cheetahs (Acinonyx jubatus).

This study was designed to identify endoparasites in captive cheetahs (Acinonyx jubatus) living in a seminatural captive environment in north-central Namibia. Results were used to assess the need for anthelmintic treatment and for the selection of an appropriate drug. The study assessed fecal parasite excretion qualitatively and quantitatively using a fecal flotation method during the winter of 2009. Four different species of parasites (two nematodes and two coccidias) were identified. Parasite excretion rates were found to be significantly lower than that of wild cheetahs living in the same area. Samples of the wild cheetahs were obtained at the time of anesthesia or were attributed to the wild individuals using genetic profiling. Captive cheetahs were dewormed with fenbendazole, whereas wild cheetahs were treated using ivermectin. Efficacy of these treatments was demonstrated at the end of the study.

Rewilding Apex Predators Has Effects on Lower Trophic Levels: Cheetahs and Ungulates in a Woodland Savanna.

The restoration of ecosystems through trophic rewilding has become increasingly common worldwide, but the effects on predator-prey and ecosystem dynamics remain poorly understood. For example, predation pressure may impose spatiotemporal behavioural adjustments in prey individuals, affecting herbivory and predation success, and therefore potentially impinging on the long-term success of trophic rewilding through apex predator reintroduction. Predation risk might have detrimental effects on prey through displacement from water or other vital resources. We investigated how five species of African ungulates responded behaviourally to changes in predation risk, following cheetah releases in the system. We grouped ungulates by body size to represent preferred prey weight ranges of the cheetah and examined changes in visitation rates, duration of stay, and activity patterns at waterholes with and without cheetah presence. During cheetah presence, visitation rates of ungulates were low for medium-sized species but high for large-sized species, suggesting that the species within the cheetah's preferred prey weight range adjusted behaviourally to minimize waterhole visits. Visits to waterholes were longer for small- and large-sized ungulates with cheetah presence, possibly indicating increased vigilance, or a strategy to maximize water intake per visit while minimizing visits. We did not detect significant differences in circadian or seasonal activity in waterhole visits, which may be attributable to the need of ungulates to access water year-round in our semi-arid study system and where migration was impeded due to physical barriers (fencing). We recommend further research into the long-term behavioural consequences of trophic rewilding on prey populations and trophic cascades to assist the success of recovery programs and to minimize potential detrimental effects at target sites.

Chronic Stress-Related Gastroenteric Pathology in Cheetah: Relation between Intrinsic and Extrinsic Factors.

The rapid decline of cheetah (Acinonyx jubatus) throughout their range and long-term studies of captive breeding has increased conservation action for this species including the study of chronic diseases. Gastritis is one of the captive diseases that leads to high mortality presented with symptoms including vomiting, diarrhea, anorexia, and weight loss. The disease presents different histological lesions in the gastrointestinal tract that are characterized by inconstant and different clinical appearance in captive and free-range cheetahs. The aim of this review is to summarize the causes of chronic gastritis in the cheetah. Factors including diet, living conditions, infections with gastric Helicobacter-like organisms (GHLOs), the lack of genetic polymorphism and the cheetah's specific-immunocompetence are analyzed. All studies on gastroenteric cheetah pathologies, conducted between 1991 (to the best of our knowledge, the first report on online databases) and 2021, are included in this review, highlighting the possible correlation between stress-related captive conditions and chronic gastric pathology.

Categorising cheetah behaviour using tri-axial accelerometer data loggers: a comparison of model resolution and data logger performance.

BACKGROUND: Extinction is one of the greatest threats to the living world, endangering organisms globally, advancing conservation to the forefront of species research. To maximise the efficacy of conservation efforts, understanding the ecological, physiological, and behavioural requirements of vulnerable species is vital. Technological advances, particularly in remote sensing, enable researchers to continuously monitor movement and behaviours of multiple individuals simultaneously with minimal human intervention. Cheetahs, Acinonyx jubatus, constitute a "vulnerable" species for which only coarse behaviours have been elucidated. The aims of this study were to use animal-attached accelerometers to (1) determine fine-scale behaviours in cheetahs, (2) compare the performances of different devices in behaviour categorisation, and (3) provide a behavioural categorisation framework. METHODS: Two different accelerometer devices (CEFAS, frequency: 30 Hz, maximum capacity: ~ 2 g; GCDC, frequency: 50 Hz, maximum capacity: ~ 8 g) were mounted onto collars, fitted to five individual captive cheetahs. The cheetahs chased a lure around a track, during which time their behaviours were videoed. Accelerometer data were temporally aligned with corresponding video footage and labelled with one of 17 behaviours. Six separate random forest models were run (three per device type) to determine the categorisation accuracy for behaviours at a fine, medium, and coarse resolution. RESULTS: Fine- and medium-scale models had an overall categorisation accuracy of 83-86% and 84-88% respectively. Non-locomotory behaviours were best categorised on both loggers with GCDC outperforming CEFAS devices overall. On a coarse scale, both devices performed well when categorising activity (86.9% (CEFAS) vs. 89.3% (GCDC) accuracy) and inactivity (95.5% (CEFAS) vs. 95.0% (GCDC) accuracy). This study defined cheetah behaviour beyond three categories and accurately determined stalking behaviours by remote sensing. We also show that device specification and configuration may affect categorisation accuracy, so we recommend deploying several different loggers simultaneously on the same individual. CONCLUSION: The results of this study will be useful in determining wild cheetah behaviour. The methods used here allowed broad-scale (active/inactive) as well as fine-scale (e.g. stalking) behaviours to be categorised remotely. These findings and methodological approaches will be useful in monitoring the behaviour of wild cheetahs and other species of conservation interest.

Effect of a Probiotic Mixture in Captive Cheetahs (Acinonyx Jubatus) with Gastrointestinal Symptoms-A Pilot Study.

Cheetahs (Acinonyx jubatus) are classified as "vulnerable" species due to the low numbers persisting in the wild. Gastrointestinal diseases are very common in this species when they are kept in captivity, in particular gastritis. Clinical signs are predominantly characterized by vomiting, diarrhea, weight loss and anorexia. In this study, we evaluated the efficacy of a multi-strain probiotic in two groups of cheetahs: Group A (n = 4)-rescued cheetahs housed at the Cheetah Conservation Fund (Otjiwarongo, Namibia); Group B (n = 9)-captive cheetahs housed in Italian zoos. Animals showed gastrointestinal signs of different severity, and were positive for Helicobacter spp., detected by PCR in stool samples. Two sachets of probiotic formulation were administered to all cheetahs once a day for 21 consecutive days. Clinical conditions (appetite loss, vomiting, stool consistency and Body Condition Score) before (T0) and after 21 days of probiotic administration (T1) were then compared using a simplified Feline Chronic Enteropathy Activity Index (FCEAI) score. A slight but not significant improvement in the scores was observed in Group A, which had mild intestinal symptoms, while a significant decrease in vomiting and stool consistency (**p < 0.01) scores was observed in Group B, which had more pronounced symptoms. Results suggest that high concentrations of live probiotics can be of help in managing gastrointestinal signs in cheetahs.

Increasing age influences uterine integrity, but not ovarian function or oocyte quality, in the cheetah (Acinonyx jubatus).

Although the cheetah (Acinonyx jubatus) routinely lives for more than 12 yr in ex situ collections, females older than 8 yr reproduce infrequently. We tested the hypothesis that reproduction is compromised in older female cheetahs due to a combination of disrupted gonadal, oocyte, and uterine function/integrity. Specifically, we assessed 1) ovarian response to gonadotropins; 2) oocyte meiotic, fertilization, and developmental competence; and 3) uterine morphology in three age classes of cheetahs (young, 2-5 yr, n = 17; prime, 6-8 yr, n = 8; older, 9-15 yr, n = 9). Ovarian activity was stimulated with a combination of equine chorionic gonadotropin and human chorionic gonadotropin (hCG), and fecal samples were collected for 45 days before gonadotropin treatment and for 30 days after oocyte recovery by laparoscopy. Twenty-six to thirty hours post-hCG, uterine morphology was examined by ultrasound, ovarian follicular size determined by laparoscopy, and aspirated oocytes assessed for nuclear status or inseminated in vitro. Although no influence of age on fecal hormone concentrations or gross uterine morphology was found (P > 0.05), older females produced fewer (P < 0.05) total antral follicles and oocytes compared to younger counterparts. Regardless of donor age, oocytes had equivalent (P > 0.05) nuclear status and ability to reach metaphase II and fertilize in vitro. A histological assessment of voucher specimens revealed an age-related influence on uterine tissue integrity, with more than 87% and more than 56% of older females experiencing endometrial hyperplasia and severe pathologies, respectively. Our collective findings reveal that lower reproductive success in older cheetahs appears to be minimally influenced by ovarian and gamete aging and subsequent dysfunction. Rather, ovaries from older females are responsive to gonadotropins, produce normative estradiol/progestogen concentrations, and develop follicles containing oocytes with the capacity to mature and be fertilized. A more likely cause of reduced fertility may be the high prevalence of uterine endometrial hyperplasia and related pathologies. The discovery that a significant proportion of oocytes from older females have developmental capacity in vitro suggests that in vitro fertilization and embryo transfer may be useful for "rescuing" the genome of older, nonreproductive cheetahs.

Oxidative phosphorylation is essential for felid sperm function, but is substantially lower in cheetah (Acinonyx jubatus) compared to domestic cat (Felis catus) ejaculate.

Compared with the normospermic domestic cat, sperm metabolic function is compromised in the teratospermic cat and cheetah, but the pathway(s) involved in this deficiency are unknown. Glycolysis is essential for sperm motility, yet it appears to function normally in spermatozoa of either species regardless of structural morphology. We conducted a comparative study to further understand the mechanisms of energy production in felid spermatozoa, with the hypothesis that oxidative phosphorylation is required for normal sperm function and is impaired in teratospermic ejaculates. Electroejaculates from both species were stained with MitoTracker to quantify mitochondrial membrane potential (MMP) or were incubated to assess changes in sperm function (motility, acrosomal integrity, and lactate production) after mitochondrial inhibition with myxothiazol. Sperm midpiece dimensions also were quantified. Sperm mitochondrial fluorescence (directly proportional to MMP) was ~95% lower in the cheetah compared with the normospermic and teratospermic cat, despite the cheetah having a 10% longer midpiece. In both species, MMP was increased 5-fold in spermatozoa with retained cytoplasm compared with structurally normal cells. Inhibition of oxidative phosphorylation impaired sperm function in both species, but a 100-fold higher inhibitor concentration was required in the cat compared with the cheetah. Collectively, findings revealed that oxidative phosphorylation was required for sperm function in the domestic cat and cheetah. This pathway of energy production appeared markedly less active in the cheetah, indicating a species-specific vulnerability to mitochondrial dysfunction. The unexpected, cross-species linkage between retained cytoplasmic droplets and elevated MMP may reflect increased concentrations of metabolic enzymes or substrates in these structures.

Glycolytic enzyme activity is essential for domestic cat (Felis catus) and cheetah (Acinonyx jubatus) sperm motility and viability in a sugar-free medium.

We have previously reported a lack of glucose uptake in domestic cat and cheetah spermatozoa, despite observing that these cells produce lactate at rates that correlate positively with sperm function. To elucidate the role of glycolysis in felid sperm energy production, we conducted a comparative study in the domestic cat and cheetah, with the hypothesis that sperm motility and viability are maintained in both species in the absence of glycolytic metabolism and are fueled by endogenous substrates. Washed ejaculates were incubated in chemically defined medium in the presence/absence of glucose and pyruvate. A second set of ejaculates was exposed to a chemical inhibitor of either lactate dehydrogenase (sodium oxamate) or glyceraldehyde-3-phosphate dehydrogenase (alpha-chlorohydrin). Sperm function (motility and acrosomal integrity) and lactate production were assessed, and a subset of spermatozoa was assayed for intracellular glycogen. In both the cat and cheetah, sperm function was maintained without exogenous substrates and following lactate dehydrogenase inhibition. Lactate production occurred in the absence of exogenous hexoses, but only if pyruvate was present. Intracellular glycogen was not detected in spermatozoa from either species. Unexpectedly, glycolytic inhibition by alpha-chlorohydrin resulted in an immediate decline in sperm motility, particularly in the domestic cat. Collectively, our findings reveal an essential role of the glycolytic pathway in felid spermatozoa that is unrelated to hexose metabolism or lactate formation. Instead, glycolytic enzyme activity could be required for the metabolism of endogenous lipid-derived glycerol, with fatty acid oxidation providing the primary energy source in felid spermatozoa.