Gastrointestinal Parasites Affecting Non-Human Primates That Are Kept Ex Situ and Their Handlers in Different Brazilian Institutions: Diagnosis and Analysis of Risk Factors.

AIM: Determining gastrointestinal parasites' frequency in non-human primates (NHPs) and handlers in different Brazilian institutions, and associate it with management information. METHODS: Different institutions in São Paulo (A), Brasília (B), Rio de Janeiro (C), Pará (D) and Santa Catarina (E) were included in the study. Fecal samples were processed by using coproparasitological techniques; information about NHP handling and professionals' routine were collected through the use of questionnaires. RESULTS: In total, 33.1% of 511 NHP samples were positive for parasites; the Entamoebidae Group and Strongyloides sp.-compatible eggs were the most detected protozoa and helminths, respectively. The Entamoebidae Group was mainly diagnosed in Alouatta from Institutions E and D, and was also the only parasite detected in handlers. Strongyloides-compatible eggs were mostly evident in fecal samples collected from Cebidae from Institutions B and D. Some risk factors associated with parasite infection were a high number of animals in the same enclosure and their use for research protocol purposes, whereas quarantine, a post-infection vacant period in enclosures and antiparasitic supply were categorized as protective factors against these agents' infection. CONCLUSIONS: Parasites showing different transmission routes but concomitantly circulating in NHP institutions located in different Brazilian regions were identified in the current study, with an emphasis on those presenting zoonotic potential.

Lessons from a Multilaboratorial Task Force for Diagnosis of a Fatal Toxoplasmosis Outbreak in Captive Primates in Brazil.

Toxoplasmosis is an important zoonotic disease caused by the parasite Toxoplasma gondii and is especially fatal for neotropical primates. In Brazil, the Ministry of Health is responsible for national epizootic surveillance, but some diseases are still neglected. Here, we present an integrated investigation of an outbreak that occurred during the first year of the COVID-19 pandemic among eleven neotropical primates housed at a primatology center in Brazil. After presenting non-specific clinical signs, all animals died within four days. A wide range of pathogens were evaluated, and we successfully identified T. gondii as the causative agent within four days after necropsies. The liver was the most affected organ, presenting hemorrhage and hepatocellular necrosis. Tachyzoites and bradyzoite cysts were observed in histological examinations and immunohistochemistry in different organs; in addition, parasitic DNA was detected through PCR in blood samples from all specimens evaluated. A high prevalence of Escherichia coli was also observed, indicating sepsis. This case highlights some of the obstacles faced by the current Brazilian surveillance system. A diagnosis was obtained through the integrated action of researchers since investigation for toxoplasmosis is currently absent in national guidelines. An interdisciplinary investigation could be a possible model for future epizootic investigations in animals.

Insights into the evolutionary history of the most skilled tool-handling platyrrhini monkey: Sapajus libidinosus from the Serra da Capivara National Park.

Sapajus libidinosus members of the Pedra Furada group, living in the Serra da Capivara National Park, use stone tools in a wider variety of behaviors than any other living animal, except humans. To rescue the evolutionary history of the Caatinga S. libidinosus and identify factors that may have contributed to the emergence and maintenance of their tool-use culture, we conducted fieldwork seasons to obtain biological samples of these capuchin monkeys. UsingCYTBsequences, we show a discrete but constant population growth from the beginning of the Holocene to the present, overlapping the emergence of the Caatinga biome. Our habitat suitability reconstruction reports the presence of plants whose hard fruits, seeds, or roots are processed by capuchins using tools. TheS. libidinosusindividuals in the Caatinga were capable of dynamically developing and maintaining their autochthonous culture thanks to: a) cognitive capacity to generate and execute innovation under selective pressure; b) tolerance favoring learning and cultural inheritance; c) an unknown genetic repertoire that underpins the adaptive traits; d) a high degree of terrestriality; e) presence and abundance of natural resources, which makes some places "hot spots" for innovation, and cultural diversification within a relatively short time.

Exploring the diversity of AVPR2 in Primates and its evolutionary implications.

The current study focuses on the investigation of AVPR2 (VTR2C) protein-coupled receptor variants specific to different primate taxa. AVPR2 is activated by the neurohormone AVP, which modulates physiological processes, including water homeostasis. Our findings reveal positive selection at three AVPR2 sites at positions 190, 250, and 346. Variation at position 250 is associated with human Congenital Nephrogenic Diabetes Insipidus (cNDI), a condition characterized by excessive water loss. Other 13 functional sites with potential adaptive relevance include positions 185, 202, 204, and 252 associated with cNDI. We identified SH3-binding motifs in AVPR2's ICL3 and N-terminus domains, with some losses observed in clades of Cercopithecidae, Callitrichinae, and Atelidae. SH3-binding motifs are crucial in regulating cellular physiology, indicating that the differences may be adaptive. Co-evolution was found between AVPR2 residues and those in the AVP signal peptide/Neurophysin-2 and AQP2, other molecules in the same signaling cascade. No significant correlation was found between these Primates' taxon-specific variants and the bioclimatic variables of the areas where they live. Distinct co-evolving amino acid sequences in functional sites were found in Platyrrhini and Catarrhini, which may have adaptive implications involving glucocorticoid hormones, suggesting varied selective pressures. Further studies are required to confirm these results.

IgM antibody responses against Plasmodium antigens in neotropical primates in the Brazilian Atlantic Forest.

Introduction: Zoonotic transmission is a challenge for the control and elimination of malaria. It has been recorded in the Atlantic Forest, outside the Amazon which is the endemic region in Brazil. However, only very few studies have assessed the antibody response, especially of IgM antibodies, in Neotropical primates (NP). Therefore, in order to contribute to a better understanding of the immune response in different hosts and facilitate the identification of potential reservoirs, in this study, naturally acquired IgM antibody responses against Plasmodium antigens were evaluated, for the first time, in NP from the Atlantic Forest. Methods: The study was carried out using 154 NP samples from three different areas of the Atlantic Forest. IgM antibodies against peptides of the circumsporozoite protein (CSP) from different Plasmodium species and different erythrocytic stage antigens were detected by ELISA. Results: Fifty-nine percent of NP had IgM antibodies against at least one CSP peptide and 87% against at least one Plasmodium vivax erythrocytic stage antigen. Levels of antibodies against PvAMA-1 were the highest compared to the other antigens. All families of NP showed IgM antibodies against CSP peptides, and, most strikingly, against erythrocytic stage antigens. Generalized linear models demonstrated that IgM positivity against PvCSP and PvAMA-1 was associated with PCR-detectable blood-stage malaria infection and the host being free-living. Interestingly, animals with IgM against both PvCSP and PvAMA-1 were 4.7 times more likely to be PCR positive than animals that did not have IgM for these two antigens simultaneously. Discussion: IgM antibodies against different Plasmodium spp. antigens are present in NP from the Atlantic Forest. High seroprevalence and antibody levels against blood-stage antigens were observed, which had a significant association with molecular evidence of infection. IgM antibodies against CSP and AMA-1 may be used as a potential marker for the identification of NP infected with Plasmodium, which are reservoirs of malaria in the Brazilian Atlantic Forest.

Bifidobacteria define gut microbiome profiles of golden lion tamarin (Leontopithecus rosalia) and marmoset (Callithrix sp.) metagenomic shotgun pools.

Gut microbiome disruptions may lead to adverse effects on wildlife fitness and viability, thus maintaining host microbiota biodiversity needs to become an integral part of wildlife conservation. The highly-endangered callitrichid golden lion tamarin (GLT-Leontopithecus rosalia) is a rare conservation success, but allochthonous callitrichid marmosets (Callithrix) serve as principle ecological GLT threats. However, incorporation of microbiome approaches to GLT conservation is impeded by limited gut microbiome studies of Brazilian primates. Here, we carried out analysis of gut metagenomic pools from 114 individuals of wild and captive GLTs and marmosets. More specifically, we analyzed the bacterial component of ultra filtered samples originally collected as part of a virome profiling study. The major findings of this study are consistent with previous studies in showing that Bifidobacterium, a bacterial species important for the metabolism of tree gums consumed by callitrichids, is an important component of the callitrichid gut microbiome - although GTLs and marmosets were enriched for different species of Bifidobacterium. Additionally, the composition of GLT and marmoset gut microbiota is sensitive to host environmental factors. Overall, our data expand baseline gut microbiome data for callitrichids to allow for the development of new tools to improve their management and conservation.

Genetic diversity in ex situ populations of the endangered Leontopithecus chrysomelas and implications for its conservation.

Leontopithecus chrysomelas, the Golden-headed Lion Tamarin (GHLT), is an endangered and endemic Neotropical primate from the Atlantic Forest of Brazil that has suffered a reduction of its habitat and population size in the wild. Ex situ populations have been established as a relevant alternative to safeguard the species and retain its genetic diversity and evolutionary potential. This study evaluated the genetic diversity and structure of the two main Brazilian captive populations of GHLT, which have been under human care at the Primatology Center of Rio de Janeiro (CPRJ) and the Zoological Park Foundation of São Paulo (FPZSP). Our results revealed levels of genetic diversity overall comparable to those observed for other Leontopithecus species and for ex situ and in situ populations of GHLT previously studied. Bayesian and principal coordinate analyses showed a moderate differentiation between CPRJ and FPZSP populations. Both populations presented observed heterozygosity values higher than expected heterozygosity values for most of the microsatellites used in this study, suggesting that the management has been efficient in avoiding an increase in homozygosity. However, simulations point to a significant loss of genetic diversity in the next 100 years, mainly in the FPZSP population. Such data are relevant for further decision-making on the metapopulation management of L. chrysomelas in captive conditions and for integrating in situ and ex situ conservation plans.

Detection of Plasmodium simium gametocytes in non-human primates from the Brazilian Atlantic Forest.

BACKGROUND: Plasmodium species of non-human primates (NHP) are of great interest because they can naturally infect humans. Plasmodium simium, a parasite restricted to the Brazilian Atlantic Forest, was recently shown to cause a zoonotic outbreak in the state of Rio de Janeiro. The potential of NHP to act as reservoirs of Plasmodium infection presents a challenge for malaria elimination, as NHP will contribute to the persistence of the parasite. The aim of the current study was to identify and quantify gametocytes in NHP naturally-infected by P. simium. METHODS: Whole blood samples from 35 NHP were used in quantitative reverse transcription PCR (RT-qPCR) assays targeting 18S rRNA, Pss25 and Pss48/45 malaria parasite transcripts. Absolute quantification was performed in positive samples for 18S rRNA and Pss25 targets. Linear regression was used to compare the quantification cycle (Cq) and the Spearman's rank correlation coefficient was used to assess the correlation between the copy numbers of 18S rRNA and Pss25 transcripts. The number of gametocytes/µL was calculated by applying a conversion factor of 4.17 Pss25 transcript copies per gametocyte. RESULTS: Overall, 87.5% of the 26 samples, previously diagnosed as P. simium, were positive for 18S rRNA transcript amplification, of which 13 samples (62%) were positive for Pss25 transcript amplification and 7 samples (54%) were also positive for Pss48/45 transcript. A strong positive correlation was identified between the Cq of the 18S rRNA and Pss25 and between the Pss25 and Pss48/45 transcripts. The 18S rRNA and Pss25 transcripts had an average of 1665.88 and 3.07 copies/µL, respectively. A positive correlation was observed between the copy number of Pss25 and 18S rRNA transcripts. Almost all gametocyte carriers exhibited low numbers of gametocytes (< 1/µL), with only one howler monkey having 5.8 gametocytes/µL. CONCLUSIONS: For the first time, a molecular detection of P. simium gametocytes in the blood of naturally-infected brown howler monkeys (Alouatta guariba clamitans) was reported here, providing evidence that they are likely to be infectious and transmit P. simium infection, and, therefore, may act as a reservoir of malaria infection for humans in the Brazilian Atlantic Forest.

Isolation and genetic characterization of Toxoplasma gondii from a captive black-and-gold howler monkey (Alouatta caraya Humboldt, 1812) in Brazil.

Toxoplasma gondii was isolated in mice from different tissues of a captive black-and-gold howler monkey (Alouatta caraya) kept in a colony at the Primatology Center of Rio de Janeiro State, Brazil, and it was genotypically characterized based on using PCR-RFLP and Microsatellite Analysis (MS), later on. T. gondii was successfully isolated from inocula deriving from heart, liver and tissue pool (heart, liver, lungs, axillary lymph nodes and cerebellum) samples. The isolate was named TgBgHmBrRJ1. The high virulence of the aforementioned strain was observed in infected mice. Non-archetypal genotype (ToxoDB PCR-RFLP #206) was obtained through PCR-RFLP. This genotype had been previously described in 12 isolates from different hosts, also in Southeastern Brazil, a fact that indicates likely high circulation of this genotype in this region. The isolate was also classified as non-archetypal, based on MS genotyping, as well as presented genotypic identity close to that of strains isolated from free-range non-symptomatic chickens (TgCkBr244,245,278,279) in Espírito Santo State. It is worth emphasizing that despite the large number of reports about clinical toxoplasmosis in neotropical primates in Brazil, this is just the second isolate of this parasite ever reported in this group of animals.

Pulmonary Granuloma Is Not Always the Tuberculosis Hallmark: Pathology of Tuberculosis Stages in New World and Old World Monkeys Naturally Infected with the Mycobacterium tuberculosis Complex.

We present the pathology of monkeys naturally infected with Mycobacterium tuberculosis complex from five different colonies in Rio de Janeiro, Brazil. On the basis of gross and histopathological findings, the lesions were classified into chronic-active, extrapulmonary, early-activation or latent-reactivation stages. Typical granulomatous pneumonia was seen in 46.6% of cases (six rhesus monkeys [Macaca mulatta] and one Uta Hick's bearded saki [Chiropotes utahickae]). The absence of pulmonary granulomas did not preclude a diagnosis of tuberculosis (TB): classical granulomatous pneumonia was observed in the chronic-active and latent-reactivation stages but not in the extrapulmonary and early-activation stages. The early-activation stage was characterized by interstitial pneumonia with a predominance of foamy macrophages and molecular and immunohistochemical evidence of M. tuberculosis complex infection. TB should be considered as a cause of interstitial pneumonia in New World Monkeys. We recommend the use of immunohistochemistry and molecular analysis for diagnosis of TB, even when typical macroscopic or histological changes are not observed.

Neotropical Sylvatic Mosquitoes and Aedes aegypti Are Not Competent to Transmit 17DD Attenuated Yellow Fever Virus from Vaccinated Viremic New World Non-Human Primates.

Beside humans, thousands of non-human primates (NHPs) died during the recent outbreak caused by the yellow fever virus (YFV) in Brazil. Vaccination of NHPs against YFV with the YF 17DD attenuated virus has emerged as a public health strategy, as it would reduce sylvatic transmission while also preserving endangered susceptible species. The hypothesis of establishing an uncontrolled transmission of this attenuated virus in nature was raised. We assessed vector competence of four sylvatic mosquito species, Haemagogus leucocelaenus, Haemagogus janthinomys/capricornii, Sabethes albiprivus, and Sabethes identicus, as well as the urban vector Aedes aegypti for YF 17DD attenuated vaccine virus when fed directly on eleven viremic lion tamarins or artificially challenged with the same virus. No infection was detected in 689 mosquitoes engorged on viremic lion tamarins whose viremia ranged from 1.05 × 103 to 6.61 × 103 FFU/mL, nor in those artificially taking ≤ 1 × 103 PFU/mL. Low viremia presented by YF 17DD-vaccinated New World NHPs combined with the low capacity and null dissemination ability in sylvatic and domestic mosquitoes of this attenuated virus suggest no risk of its transmission in nature. Thus, vaccination of captive and free-living NHPs against YFV is a safe public health strategy.

Subacute multisystemic toxoplasmosis in a captive black-and-gold howler monkey (Alouatta caraya) indicates therapy challenging.

A 10-year-old black howler monkey presented with a 36-day subacute clinicopathological picture of fever, prostration, inappetence, intestinal hypomotility, and emaciation. Therapy was trimethoprim-sulfamethoxazole with streptomycin. The liver, lungs, lymph nodes, and spleen presented lesions. Toxoplasma gondii isolation and PCR determined the diagnosis, and indirect fluorescent antibody tests confirmed an increase in antibody titers.

Metastatic endometrioid carcinoma in a hybrid Leontopithecus sp.

Primary female reproductive neoplasms in Platyrrhines species are few reported. We present the gross, histological, and immunohistochemical findings of metastatic endometrioid carcinoma in the uterus, urinary bladder, jejunum, and rectum of a Leontopithecus sp. The neoplastic endometrial cells expressed strong cytoplasmic immunolabeling of cytokeratin 7.

The gut microbiome of exudivorous marmosets in the wild and captivity.

Mammalian captive dietary specialists like folivores are prone to gastrointestinal distress and primate dietary specialists suffer the greatest gut microbiome diversity losses in captivity compared to the wild. Marmosets represent another group of dietary specialists, exudivores that eat plant exudates, but whose microbiome remains relatively less studied. The common occurrence of gastrointestinal distress in captive marmosets prompted us to study the Callithrix gut microbiome composition and predictive function through bacterial 16S ribosomal RNA V4 region sequencing. We sampled 59 wild and captive Callithrix across four species and their hybrids. Host environment had a stronger effect on the gut microbiome than host taxon. Wild Callithrix gut microbiomes were enriched for Bifidobacterium, which process host-indigestible carbohydrates. Captive marmoset guts were enriched for Enterobacteriaceae, a family containing pathogenic bacteria. While gut microbiome function was similar across marmosets, Enterobacteriaceae seem to carry out most functional activities in captive host guts. More diverse bacterial taxa seem to perform gut functions in wild marmosets, with Bifidobacterium being important for carbohydrate metabolism. Captive marmosets showed gut microbiome composition aspects seen in human gastrointestinal diseases. Thus, captivity may perturb the exudivore gut microbiome, which raises implications for captive exudivore welfare and calls for husbandry modifications.

Infection by Prosthernorchis elegans (Diesing, 1851) in captive Callithrix aurita (É. Geoffroy, 1812) and Leontopithecus rosalia (Linnaeus, 1766) from Rio de Janeiro, Brazil.

This study reports on infection by Prosthernorchis elegans of Callithrix aurita and Leonthopithecus rosalia through biometry on adults and by molecular biology. Seventy-eight helminths were recovered from the animals' intestine. This is a detailed morphological description and the first molecular characterization of P. elegans in animals from Brazil.

Uniparental genetic markers to investigate hybridization in wild-born marmosets with a mixed phenotype among Callithrix aurita and invasive species.

The native marmoset of the Southeastern Atlantic Forest in Brazil is among the 25 most endangered primates of the world. Hybridization with alien species is one of its main threats registered since the early 2000s based on phenotype, so far, without genetic confirmation. Using uniparental molecular markers, we analyzed 18 putative hybrids, captured from 2004 to 2013 in different localities of the Atlantic Forest. A nine base pair deletion in the SRY gene of C. aurita was used to investigate paternal ancestry. Maternal ancestry was assessed by DNA sequencing of ca. 455 bp from the COX2 gene. Hybridization was confirmed for 16 out of the 18 marmosets since they inherited COX2 haplotypes of the alien C. penicillata or C. jacchus and the SRY deletion specific to C. aurita. Two individuals inherited both parental lineages of C. aurita, which is probably related to backcrossing or hybrid interbreeding. The direction of hybridization of females with the matrilineal lineage of invasive species with males descending from the native lineage was predominant in our sampling. This is the first time that hybridization between C. aurita and invasive species has been confirmed through genetic analysis.

Spontaneous disseminated T-cell lymphoma in a buff-headed capuchin (Sapajus xanthosternos).

Reports of spontaneous hematopoietic neoplasms in Platyrrhines species are scarce. We present the gross, histological, and immunohistochemical findings of disseminated T-cell lymphoma in a male 25-year-old Sapajus xanthosternos kept in a Brazilian conservation center. No molecular evidence of betaherpesvirus or gammaherpesvirus was associated with the occurrence of this neoplasm.

The genome of the zoonotic malaria parasite Plasmodium simium reveals adaptations to host switching.

BACKGROUND: Plasmodium simium, a malaria parasite of non-human primates (NHP), was recently shown to cause zoonotic infections in humans in Brazil. We sequenced the P. simium genome to investigate its evolutionary history and to identify any genetic adaptions that may underlie the ability of this parasite to switch between host species. RESULTS: Phylogenetic analyses based on whole genome sequences of P. simium from humans and NHPs reveals that P. simium is monophyletic within the broader diversity of South American Plasmodium vivax, suggesting P. simium first infected NHPs as a result of a host switch of P. vivax from humans. The P. simium isolates show the closest relationship to Mexican P. vivax isolates. Analysis of erythrocyte invasion genes reveals differences between P. vivax and P. simium, including large deletions in the Duffy-binding protein 1 (DBP1) and reticulocyte-binding protein 2a genes of P. simium. Analysis of P. simium isolated from NHPs and humans revealed a deletion of 38 amino acids in DBP1 present in all human-derived isolates, whereas NHP isolates were multi-allelic. CONCLUSIONS: Analysis of the P. simium genome confirmed a close phylogenetic relationship between P. simium and P. vivax, and suggests a very recent American origin for P. simium. The presence of the DBP1 deletion in all human-derived isolates tested suggests that this deletion, in combination with other genetic changes in P. simium, may facilitate the invasion of human red blood cells and may explain, at least in part, the basis of the recent zoonotic infections.

Evolutionary analysis of the anti-viral STAT2 gene of primates and rodents: Signature of different stages of an arms race.

STAT2 plays a strategic role in defending viral infection through the signaling cascade involving the immune system initiated after type I interferon release. Many flaviviruses target the inactivation or degradation of STAT2 as a strategy to impair this host's line of defense. Primates are natural reservoirs for a range of disease-causing flaviviruses (e.g., Zika, Dengue, and Yellow Fever virus), while rodents appear less susceptible. We analyzed the STAT2 coding sequence of 28 Rodentia species and 49 Primates species. Original data from 19 Platyrrhini species were sequenced for the SH2 domain of STAT2 and included in the analysis. STAT2 has many sites whose variation can be explained by positive selection, measurement by two methods (PALM indicated 12, MEME 61). Both evolutionary tests significantly marked sites 127, 731, 739, 766, and 780. SH2 is under evolutionary constraint but presents episodic positive selection events within Rodentia: in one of them, a moderately radical change (serine > arginine) at position 638 is found in Peromyscus species, and can be implicated in the difference in susceptibility to flaviviruses within Rodentia. Some other positively selected sites are functional such as 5, 95, 203, 251, 782, and 829. Sites 251 and 287 regulate the signaling mediated by the JAK-STAT2 pathway, while 782 and 829 create a stable tertiary structure of STAT2, facilitating its connection with transcriptional co-activators. Only three positively selected sites, 5, 95, and 203, are recognized members who act on the interface between STAT2 and flaviviruses NS5 protein. We suggested that due to the higher evolutionary rate, rodents are, at this moment, taking some advantage in the battle against infections for some well-known Flaviviridae, in particular when compared to primates. Our results point to dynamics that fit with a molecular evolutionary scenario shaped by a thought-provoking virus-host arms race.

Comparison between three dosages of intramuscular alfaxalone and a ketamine-dexmedetomidine-midazolam-tramadol combination in golden-headed lion tamarins (Leontopithecus chrysomelas).

OBJECTIVES: To characterize the cardiopulmonary and anesthetic effects of alfaxalone at three dose rates in comparison with a ketamine-dexmedetomidine-midazolam-tramadol combination (KDMT) for immobilization of golden-headed lion tamarins (GHLTs) (Leontopithecus chrysomelas) undergoing vasectomy. STUDY DESIGN: Prospective clinical trial. ANIMALS: A total of 19 healthy, male, wild-caught GHLTs. METHODS: Tamarins were administered alfaxalone intramuscularly (IM) at 6, 12 or 15 mg kg-1, or KDMT, ketamine (15 mg kg-1), dexmedetomidine (0.015 mg kg-1), midazolam (0.5 mg kg-1) and tramadol (4 mg kg-1) IM. Immediately after immobilization, lidocaine (8 mg kg-1) was infiltrated subcutaneously (SC) at the incision site in all animals. Physiologic variables, anesthetic depth and quality of immobilization were assessed. At the end of the procedure, atipamezole (0.15 mg kg-1) was administered IM to group KDMT and tramadol (4 mg kg-1) SC to the other groups; all animals were injected with ketoprofen (2 mg kg-1) SC. RESULTS: A dose-dependent increase in sedation, muscle relaxation and immobilization time was noted in the alfaxalone groups. Despite the administration of atipamezole, the recovery time was longer for KDMT than all other groups. Muscle tremors were noted in some animals during induction and recovery with alfaxalone. No significant differences were observed for cardiovascular variables among the alfaxalone groups, whereas an initial decrease in heart rate and systolic arterial blood pressure was recorded in KDMT, which increased after atipamezole administration. CONCLUSIONS AND CLINICAL RELEVANCE: Alfaxalone dose rates of 12 or 15 mg kg-1 IM with local anesthesia provided good sedation and subjectively adequate pain control for vasectomies in GHLTs. KDMT induced a deeper plane of anesthesia and should be considered for more invasive or painful procedures. All study groups experienced mild to moderate hypothermia and hypoxemia; therefore, the use of more efficient heating devices and oxygen supplementation is strongly recommended when using these protocols.