Evolution of the ancestral mammalian karyotype and syntenic regions.

Decrypting the rearrangements that drive mammalian chromosome evolution is critical to understanding the molecular bases of speciation, adaptation, and disease susceptibility. Using 8 scaffolded and 26 chromosome-scale genome assemblies representing 23/26 mammal orders, we computationally reconstructed ancestral karyotypes and syntenic relationships at 16 nodes along the mammalian phylogeny. Three different reference genomes (human, sloth, and cattle) representing phylogenetically distinct mammalian superorders were used to assess reference bias in the reconstructed ancestral karyotypes and to expand the number of clades with reconstructed genomes. The mammalian ancestor likely had 19 pairs of autosomes, with nine of the smallest chromosomes shared with the common ancestor of all amniotes (three still conserved in extant mammals), demonstrating a striking conservation of synteny for ∼320 My of vertebrate evolution. The numbers and types of chromosome rearrangements were classified for transitions between the ancestral mammalian karyotype, descendent ancestors, and extant species. For example, 94 inversions, 16 fissions, and 14 fusions that occurred over 53 My differentiated the therian from the descendent eutherian ancestor. The highest breakpoint rate was observed between the mammalian and therian ancestors (3.9 breakpoints/My). Reconstructed mammalian ancestor chromosomes were found to have distinct evolutionary histories reflected in their rates and types of rearrangements. The distributions of genes, repetitive elements, topologically associating domains, and actively transcribed regions in multispecies homologous synteny blocks and evolutionary breakpoint regions indicate that purifying selection acted over millions of years of vertebrate evolution to maintain syntenic relationships of developmentally important genes and regulatory landscapes of gene-dense chromosomes.

Taphonomy of harpy eagle predation on primates and other mammals.

The goal of this study is to provide a taphonomic analysis of bone fragments found in harpy eagle nests in the Brazilian Amazonia, utilizing the largest sample of prey remains collected to date. Harpy eagle kill samples were collected from nine nests, between June 2016 and December 2020 in Mato Grosso, Brazil. We identified the specimens, calculated the number of identified specimens (NISP) and minimum number of individuals (MNI). These metrics were used to estimate bone survivability and fragmentation. A total of 1661 specimens (NISP) were collected, representing a minimum number of 234 individuals (MNI). We identified at least nine species of primates, which represent 63.8% of the individuals in the kill sample. Harpy eagles preyed mostly on the medium-sized capuchin and bearded saki monkeys (28.2% of the MNI), and two-toed sloths (17.7% of the MNI). The large woolly monkeys also represented a significant portion of the sample (11.5% of the MNI). Three distinct patterns of bone survivability were found, one characterizing two-toed sloths, another characterizing medium-sized monkeys, and a third typical of woolly monkeys. We conclude that harpy eagle predation leaves an identifiable signature on the prey with a bone survivability pattern specific to each taxon. The intertaxon variations observed in the taphonomic signatures of harpy eagle kills should be taken into account when evaluating the potential influence of these raptors as accumulators of bone material in both paleontological and neontological assemblages.

REPRODUCTIVE CYCLE STAGE ASSESSMENT IN THE TWO-TOED SLOTH (CHOLOEPUS HOFFMANNI), PRELIMINARY RESULTS.

The number of two-toed sloths (Choloepus hoffmanni) has significantly decreased in the last years. Deepening the knowledge of this tropical mammal's reproductive physiology is essential to improve captive breeding within conservation programs for this species. However, several aspects of its reproductive biology remain unexplored and have not been described sufficiently. The aim of this work was to describe the estrous cycle and reproductive physiology of two adult female C. hoffmanni by vaginal cytology, appearance of the external genitalia, and behavior. Vaginal cytology assay showed that the average duration of the estrous cycle was 15.1 ± 4.53 d. Positive correlations (P < 0.05) were found between the peak presence of superficial cells (estrous phase) and four parameters: aggressive behavior, pursuing behavior, vulvar swelling, and vaginal discharge. This pilot study, conducted on just two animals, forms a basis for a study design that may be employed for a more comprehensive assessment of the two-toed sloth reproductive physiology and behavior.

FIRST REPORT OF CHEMICAL IMMOBILIZATION AND PHYSIOLOGICAL PARAMETERS FOR FREE-RANGING MANED SLOTH (BRADYPUS TORQUATUS), USING A COMBINATION OF KETAMINE AND MEDETOMIDINE.

The maned sloth (Bradypus torquatus) is an endemic and endangered species of two Brazilian states, with much unknown biological information needed to direct conservation actions. Other sloth species have been studied regarding anesthesia; however, there is a lack of anesthesia research for the maned sloth. Anesthetic data were collected from 12 free-range maned sloths that were immobilized for a field examination. Individuals were anesthetized using a combination of ketamine (4.0 mg/kg) and medetomidine (0.03 mg/kg), and antagonized with atipamezole (0.1 mg/kg). Time to induction and recovery were recorded and compared with sex and age classes. After the induction and until antagonist administration, physiological parameters (rectal temperature, heart rate, respiratory rate, and oxygen saturation) were recorded every 10 min during anesthesia and were statistically evaluated over time. Induction was fast (3.21 ± 0.76), but recovery was longer (113.3 ± 18) when compared to other studies. Induction and recovery times were not different across sex or age classes. Rectal temperature, heart rate, and oxygen saturation remained stable throughout the procedure. Respiratory rate significantly decreased over time, from 18.25 ± 7.03 to 13.17 ± 3.66 movements per minute. Our results indicate that the described combination of ketamine and medetomidine is a safe and effective choice for anesthesia of maned sloths.

Differences in fungal communities in the fur of two- and three-toed sloths revealed by ITS metabarcoding.

Sloths have dense fur on which insects, algae, bacteria and fungi coexist. Previous studies using cultivation-dependent methods and 18S rRNA sequencing revealed that the fungal communities in their furs comprise members of the phyla Ascomycota and Basidiomycota. In this note, we increase the resolution and knowledge of the mycobiome inhabiting the fur of the two- (Choloepus hoffmanni) and three-toed (Bradypus variegatus) sloths. Targeted amplicon metagenomic analysis of ITS2 nrDNA sequences obtained from 10 individuals of each species inhabiting the same site revealed significant differences in the structure of their fungal communities and also in the alpha-diversity estimators. The results suggest a specialization by host species and that the host effect is stronger than that of sex, age and animal weight. Capnodiales were the dominant order in sloths' fur and Cladosporium and Neodevriesia were the most abundant genera in Bradypus and Choloepus, respectively. The fungal communities suggest that the green algae that inhabit the fur of sloths possibly live lichenized with Ascomycota fungal species. The data shown in this note offer a more detailed view of the fungal content in the fur of these extraordinary animals and could help explain other mutualistic relationships in this complex ecosystem.

Evidence of artefacts made of giant sloth bones in central Brazil around the last glacial maximum.

The peopling of the Americas and human interaction with the Pleistocene megafauna in South America remain hotly debated. The Santa Elina rock shelter in Central Brazil shows evidence of successive human settlements from around the last glacial maximum (LGM) to the Early Holocene. Two Pleistocene archaeological layers include rich lithic industry associated with remains of the extinct giant ground sloth Glossotherium phoenesis. The remains include thousands of osteoderms (i.e. dermal bones), three of which were human-modified. In this study, we perform a traceological analysis of these artefacts by optical microscopy, non-destructive scanning electron microscopy, UV/visible photoluminescence and synchrotron-based microtomography. We also describe the spatial association between the giant sloth bone remains and stone tools and provide a Bayesian age model that confirms the timing of this association in two time horizons of the Pleistocene in Santa Elina. The conclusion from our traceological study is that the three giant sloth osteoderms were intentionally modified into artefacts before fossilization of the bones. This provides additional evidence for the contemporaneity of humans and megafauna, and for the human manufacturing of personal artefacts on bone remains of ground sloths, around the LGM in Central Brazil.

The functional significance of aberrant cervical counts in sloths: insights from automated exhaustive analysis of cervical range of motion.

Besides manatees, the suspensory extant 'tree sloths' are the only mammals that deviate from a cervical count (CC) of seven vertebrae. They do so in opposite directions in the two living genera (increased versus decreased CC). Aberrant CCs seemingly reflect neck mobility in both genera, suggesting adaptive significance for their head position during suspensory locomotion and especially increased ability for neck torsion in three-toed sloths. We test two hypotheses in a comparative evolutionary framework by assessing three-dimensional intervertebral range of motion (ROM) based on exhaustive automated detection of bone collisions and joint disarticulation while accounting for interacting rotations of roll, yaw and pitch. First, we hypothesize that the increase of CC also increases overall neck mobility compared with mammals with a regular CC, and vice versa. Second, we hypothesize that the anatomy of the intervertebral articulations determines mobility of the neck. The assessment revealed that CC plays only a secondary role in defining ROM since summed torsion (roll) capacity was primarily determined by vertebral anatomy. Our results thus suggest limited neck rotational adaptive significance of the CC aberration in sloths. Further, the study demonstrates the suitability of our automated approach for the comparative assessment of osteological ROM in vertebral series.

Muscle architectural properties indicate a primary role in support for the pelvic limb of three-toed sloths (Bradypus variegatus).

Tree sloths evolved below-branch locomotion making them one of few mammalian taxa beyond primates for which suspension is nearly obligatory. Suspension requires strong limb flexor muscles that provide both propulsion and braking/support, and available locomotor kinetics data indicate that these roles differ between fore- and hindlimb pairs. Muscle structure in the pelvic limb is hypothesized to be a key anatomical correlate of function in braking/support during suspensory walking and propulsion and/or support during vertical climbing. This expectation was tested by quantifying architecture properties in the hindlimb limb musculature of brown-throated three-toed sloths (Bradypus variegatus: N = 7) to distinguish the roles of the flexor/extensor functional muscle groups at each joint. Measurements of muscle moment arm (rm ), mass, belly length, fascicle length, pennation angle, and physiological cross-sectional area (PCSA) were taken from n = 45 muscles. Overall, most muscles studied show properties for contractile excursion and fast joint rotational velocity. However, the flexor musculature is more massive (p = 0.048) and has larger PCSA (p = 0.003) than the extensors, especially at the knee joint and digits where well-developed and strong flexors are capable of applying large joint torque. Moreover, selected hip flexors/extensors and knee flexors have modified long rm that can amplify applied joint torque in muscles with otherwise long, parallel fascicles, and one muscle (m. iliopsoas) was capable of moderately high power in B. variegatus. The architectural properties observed in the hip flexors and extensors match well with roles in suspensory braking and vertical propulsion, respectively, whereas strong knee flexors and digital flexors appear to be the main muscles providing suspensory support in the pelvic limb. With aid in support by the forelimbs and the use of adaptive slow locomotion and slow muscle fiber recruitment patterns, structure-function in the tensile limb systems of sloths appears to collectively represent an additional mechanism for energy conservation.

Pump the brakes! The hindlimbs of three-toed sloths decelerate and support suspensory locomotion.

Modern tree sloths are one of few mammalian taxa for which quadrupedal suspension is obligatory. Sloth limb musculature is specialized for slow velocity, large force contractions that stabilize their body below branches and conserve energy during locomotion. However, it is unknown whether two- and three-toed sloths converge in their use of limb kinetics and if these patterns are comparable to how primates perform arboreal suspensory locomotion. This study addressed this need by collecting limb loading data in three-toed sloths (Bradypus variegatus; N=5) during suspensory walking. Sloths performed locomotor trials at their preferred speed on an instrumented beam apparatus with a force platform as the central supporting segment. Peak forces and impulses of the forelimb and hindlimb were recorded and analyzed in three dimensions. The hindlimbs of B. variegatus apply large braking forces greater in magnitude than peak forces generated by the forelimbs in propulsion, a pattern consistent with that observed in two-toed sloths. However, B. variegatus exhibits hindlimb-biased body weight support in vertical peak forces and impulse, with appreciable laterally directed forces in each limb pair, both of which vary from limb loading distributions in two-toed sloths. Moreover, body weight distribution between limb pairs is opposite to that employed by primates during quadrupedal suspension. Thus, there appear to be multiple strategies for achieving suspensory locomotion in arboreal mammals. These differences may be attributable to anatomical variation or phylogenetic position, but as of yet an explanation remains unknown. Future EMG analyses are expected to provide insight into how specific hindlimb muscle groups contribute to braking forces and stabilizing the center of mass of sloths during suspension.

Zoonotic Giardia duodenalis assemblage A in northern sloth from Brazilian Amazon.

BACKGROUND: The parasite Giardia duodenalis infects a wide range of vertebrate hosts, including domestic and wild animals as well as humans. Giardia is genotyped into eight assemblages (A-H). Zoonotic assemblages A and B have already been identified in humans and wild and domestic animals (non-human primates and cats) from Brazilian Amazon and in the world. Due to its zoonotic/zooanthroponotic nature, surveillance initiatives and the definition of Giardia assemblages are important in order to characterise the epidemiological scenario and to implement further control measures. OBJECTIVES: Determine assemblages of G. duodenalis in sloths from the Brazilian Amazon Region. METHODS: Faecal parasitological examination of sloths from Amazonas State. Polymerase chain reaction (PCR) targeting the beta giardin (BG), and genes from multilocus sequence typing (MLST) scheme, amplicon sequencing and phylogenetic analysis. FINDINGS: Here, we identified, by microscopy, Giardia in two northern sloths (Bradypus tridactylus). These two samples were submitted to molecular assays and it was revealed that both were infected by G. duodenalis assemblage A. Phylogenetic analysis showed that they belong to assemblage A within sequences from humans and wild and domestic animals. CONCLUSION: Therefore, besides showing, by the first time, the current presence of this parasite in sloths, our findings reveals that this wild animal species would be part of the zoonotic/zooanthroponotic scenario of this parasite in the Brazilian Amazon.

The Yale Geochronometric Laboratory and the Rewriting of Global Environmental History.

Beginning in the nineteenth century, scientists speculated that the Pleistocene megafauna-species such as the giant ground sloth, wooly mammoth, and saber-tooth cat-perished because of rapid climate change accompanying the end of the most recent Ice Age. In the 1950s, a small network of ecologists challenged this view in collaboration with archeologists who used the new tool of radiocarbon dating. The Pleistocene overkill hypothesis imagined human hunting, not climate change, to be the primary cause of megafaunal extinction. This article situates the Pleistocene overkill hypothesis in a broader history of the emergence of historical ecology as a distinct sub-discipline of paleoecology. Tracing the work of the Yale Geochronometric Laboratory and an interdisciplinary research network that included Paul Sears, Richard Foster Flint, Edward Deevey, Kathryn Clisby, and Paul S. Martin, it reveals how both the methods and the meaning of studying fossil pollen shifted between the 1910s and 1960s. First used as a tool for fossil fuel extraction, fossil pollen became a means of envisioning climatic history, and ultimately, a means of reimagining global ecological history. First through pollen stratigraphy and then through radiocarbon dating, ecologists reconstructed past biotic communities and rethought the role of humans in these communities. By the 1980s, the discipline of historical ecology would reshape physical environments through the practice of ecological restoration.

Fecal microbiota transplants modulate the gut microbiome of a two-toed sloth (Choloepus didactylus).

The microbes inhabiting an animal's gastrointestinal tracts, collectively known as the gut microbiome, are vital to animal health and wellbeing. For animals experiencing gut distress or infection, modulation of the gut microbiome, for example, via fecal microbiota transplant (FMT), provides a possible disease prevention and treatment method. The beneficial microbes present in the donor's transplanted feces can help combat pathogens, assist in digestion, and rebalance the recipient's microbiota. Investigating the efficacy of FMTs in animal health is a crucial step toward improving management strategies for species under human care. We present a case study of the use of FMTs in a two-toed sloth experiencing abnormally large, clumped, and frequent stools. We used 16 S rRNA amplicon sequencing of fecal samples to (a) compare the microbiomes of the FMT donor, a healthy, cohoused conspecific, and the FMT recipient and (b) assess the influence of multiple rounds of FMTs on the recipient's microbiome and stool consistency and frequency over time. In response to the FMTs, we found that the recipient's microbiome showed trends toward increased diversity, shifted community composition, and altered membership that more resembled the community of the donor. FMT treatment was also associated with marked, yet temporary, alleviation of the recipient's abnormal bowel movements, suggesting a broader impact on gut health. Our results provide valuable preliminary evidence that FMT treatments can augment the recipient's gut microbiome, with potential implications for animal health and management.

FATAL GASTRIC DILATATION AND VOLVULUS IN THREE CAPTIVE JUVENILE LINNAEUS'S TWO-TOED SLOTHS (CHOLOEPUS DIDACTYLUS).

Linnaeus's two-toed sloth (Choloepus didactylus) is one of two extant neotropical species of the family Megalonychidae. Despite their being commonly kept under managed care, the digestive physiology of sloths remains poorly understood. Gastrointestinal disease has been reported as a primary or contributing cause of morbidity and mortality in captive two-toed and three-toed (Bradypus spp.) sloths. Gastric dilatation due to gas accumulation ("bloat") has been reported in sloths; however, a literature search failed to yield any published mention of gastric volvulus in any sloth species. Following an inquiry sent to the electronic mailing lists of the American Association of Zoo Veterinarians, the European Association of Zoo and Wildlife Veterinarians, and the LatinVets community, three cases of fatal gastric dilatation and volvulus (GDV) were identified in one male and two female Linnaeus's two-toed sloths from institutions in the United States, Canada, and Germany. All cases occurred in juvenile sloths <1 yr of age. Two animals were primarily hand reared, whereas one was reared primarily by its dam. Two animals were found dead with no overt premonitory signs, whereas one animal died following a 3-wk course of waxing and waning clinical signs consistent with gastric gas accumulation. In all cases, GDV was diagnosed on postmortem examination. Similar to other species, the condition is likely subsequent to a combination of host- and husbandry-related contributing factors. Further research into sloth husbandry is required in order to take an evidence-based approach to their management.

Antibiotic-producing Micrococcales govern the microbiome that inhabits the fur of two- and three-toed sloths.

Sloths have a dense coat on which insects, algae and fungi coexist in a symbiotic relationship. This complex ecosystem requires different levels of controls; however, most of these mechanisms remain unknown. We investigated the bacterial communities inhabiting the hair of two- (Choloepus Hoffmanni) and three-toed (Bradypus variegatus) sloths and evaluated their potential for producing antibiotic molecules capable of exerting control over the hair microbiota. The analysis of 16S rRNA amplicon sequence variants revealed that the communities in both host species are dominated by Actinobacteriota and Firmicutes. The most abundant genera were Brevibacterium, Kocuria/Rothia, Staphylococcus, Rubrobacter, Nesterenkonia and Janibacter. Furthermore, we isolated nine strains of Brevibacterium and Rothia capable of producing substances that inhibited the growth of common mammalian pathogens. The analysis of the biosynthetic gene clusters of these nine isolates suggests that the pathogen-inhibitory activity could be mediated by the presence of siderophores, terpenes, beta-lactones, Type III polyketide synthases, ribosomally synthesized and post-translationally modified peptides, non-alpha poly-amino acids like e-Polylysine, ectoine or non-ribosomal peptides. Our data suggest that Micrococcales that inhabit sloth hair could have a role in controlling microbial populations in that habitat, improving our understanding of this highly complex ecosystem.

Myology of the pelvic limb of the brown-throated three-toed sloth (Bradypus variegatus).

Tree sloths rely on their limb flexors for bodyweight support and joint stability during suspensory locomotion and posture. This study aims to describe the myology of three-toed sloths and identify limb muscle traits that indicate modification for suspensorial habit. The pelvic limbs of the brown-throated three-toed sloth (Bradypus variegatus) were dissected, muscle belly mass was recorded, and the structural arrangements of the muscles were documented and compared with the available myological accounts for sloths. Overall, the limb musculature is simplified by containing muscles with generally long and parallel fascicles. A number of specific and informative muscle traits are additionally observed in the pelvic limb of B. variegatus: well-developed hip flexors and hip extensors each displaying several fused bellies; massive knee flexors; two heads of the m. adductor longus and m. gracilis; robust digital flexors and flexor tendons; m. tibialis cranialis muscle complex originating from the tibia and fibula and containing a modified m. extensor digitorum I longus; appreciable muscle mass devoted to ankle flexion and hindfoot supination; only m. extensor digitorum brevis acts to extend the digits. Collectively, the findings for tree sloths emphasize muscle mass and organization for suspensory support namely by the hip flexors, knee flexors, and limb adductors, for which the latter two groups may stabilize suspensory postures by exerting appreciable medially-directed force on the substrate. Specializations in the distal limb are also apparent for sustained purchase of the substrate by forceful digital flexion coupled with strong ankle flexion and supination of the hind feet, which is permitted by the reorganization of several digital extensors. Moreover, the reduction or loss of other digital flexor and ab-adductor muscles marks a dramatic simplification of the intrinsic foot musculature in B. variegatus, the extent to which varies across extant species of two- and three-toed tree sloths and likely is related to substrate preference/use.

Does a suspensory lifestyle result in increased tensile strength? Organ-level material properties of sloth limb bones.

The material composition of vertebrate connective tissue is highly conserved across taxa. Existing data suggest that the compressive and tensile strength of limb bones are very similar despite marked variation in limb posture and locomotor patterns. However, the material properties of limb bone tissue from suspensory taxa have not been formally evaluated. Sloths are nearly obligatory in their use of below-branch suspensory locomotion and posture, thus placing their limb bones and associated soft tissue structures under routine tensile loading. It is possible that sloth limb bones are modified for enhanced tensile strength, perhaps at the expense of compressive strength. Forelimb and hindlimb bones of two-toed (Choloepus hoffmanni) and three-toed (Bradypus variegatus) sloths were tested in compression and bending to evaluate this hypothesis. Strength and elastic (Young's) modulus were similarly lower in sloth limb bones during both compression and bending, as compared with pronograde taxa. Ratios of peak bending strength to compressive strength additionally were elevated (sloths: 1.4-1.7; upright taxa: 0.6-1.2) for sloth limb bones. Overall, the material properties measured from the limb bones of tree sloths support our hypothesis of predicted function in a tensile limb system. Future studies should aim to directly test bones in tension to confirm indications of elevated axial tensile strength. Nevertheless, the results herein expand understanding of functional adaptation in mammalian tissue for a range of locomotor/postural behaviors that were previously unexplored.

Low-cost thermoregulation of wild sloths revealed by heart rate and temperature loggers.

Arboreal herbivores require large digestive tracts for leaf fermentation and detoxification; however, they must also have a low body mass that allows them to reach the foliage. The three-toed sloth, Bradypus tridactylus, experiences this trade-off, as leaves comprise 97.2% of its diet. Their calorie intake is extremely low owing to the low available caloric density of leaves and slow digestive processes related to leaf fibre fermentation and secondary compound detoxification. Sloths may require a high body temperature to assist fermentation; however, thermogenesis is energy-consuming. To investigate how sloths accomplish thermoregulation using marginal energy, we attached heart rate (HR) and temperature loggers to wild B. tridactylus individuals inhabiting the Amazon rainforest and recorded their HR and body surface temperature (Tskin). Tskin changed with ambient temperature (Ta) but was higher than Ta in 99.2% of cases. Increases in Tskin and HR did not coincide, suggesting that the increases were not caused by thermogenesis. Instead, they may passively increase Tskin by selecting warmer microhabitats and sunbathing. Consequently, 90.5% of Tskin were within 27.6-36.0 °C while the Ta fluctuated between 21.5 and 42.9 °C. This low-cost thermoregulation results in a low HR. In this study, the mean HR during observation was approximately 38.4% of the expected value based on the mammalian allometric relationship between body mass and HR. Thus, these properties may contribute to the low metabolic rates of sloths, alleviating their restricted energy intake.

Sri Lankan Sloth Bear (Melursus ursinus inornatus) Attacks on Humans.

The sloth bear (Melursus ursinus) is an omnivore that has been reported around most parts of South Asia. Although rare, sloth bear attacks can inflict potentially life-threatening injuries. This cross-sectional study analyzed 10 patients who had been mauled by sloth bears and who presented to rural hospitals in the Anuradhapura district of Sri Lanka between 2015 and 2019. All of the patients were male farmers. The human-bear encounters occurred in the jungle during the daytime. Ten victims sustained multiple soft tissue and bone injuries, mainly to the face and arms. The injuries ranged from minor abrasions to extensive deep lacerations and bone fractures. All of the patients were managed in the tertiary care hospital by multidisciplinary surgical teams. Sloth bear-inflicted wounds must be treated as major trauma. There is a need to formulate guidelines and train medical officers in managing animal-inflicted injuries in Sri Lanka.

Dietary neutral detergent fibre and lignin contents affect intake, digestibility and digesta retention in captive sloths (Bradypus variegatus).

We evaluated food intake, digestibility, digesta retention and digestible energy (DE) intake in four three-toed sloths (Bradypus variegatus, body mass 2.86 ± 0.10 kg) fed two leaf-based diets containing different neutral detergent fibre (NDF) and lignin contents. Total dry matter intake (DMI), and intake in relation to body mass and metabolic body weight were higher for the mixed diet with lower NDF content based on Pterondon sp. + Inga sp. (47 g day-1 , 16 g kg-1 day-1 and 21 g kg-0.75 day-1 ) compared to the treatment with higher content of NDF from only Cecropia pachystachya leaves (37 g day-1 , 14 g kg-1 day-1 and 18 g kg-0.75 day-1 ). The digestibility of dry matter (dDM) and neutral detergent fibre (dNDF) were higher in the mixed diet (60% and 61% respectively). There was a higher supply of DE and metabolisable energy (ME) on the mixed diet, at 221 and 199 kJ kg-0.75 day-1 , meeting the average energy requirement of 185 kJ ME kg-0.75 day-1 ME estimated for sloths in this study. In contrast, the diet with C. pachystachya generated a deficit of 31 kJ ME kg-0.75 day-1 . There was a correlation between DMI and dNDF (r2 = 0.89), and between dNDF and dDM (r2 = 0.98) across treatments. The mean retention times for a liquid and a particle marker were lower on the mixed diet with the higher intake at 133 h (passage rate = 0.75% h-1 ) and 181 h (0.55% h-1 ), and longer on the single-species diet with lower intake at 204 h (0.49% h-1 ) and 261 h (0.38% h-1 ). The results suggest that it may be beneficial for sloths to be offered a variety of browse from which they can choose low-NDF components. Further, we suggest that these sloths perform 'digesta washing' to increase the microbial yield in the stomach to maximise the digestion of NDF and dry matter.