Population Of Rhesus Macaques Research Articles

Genetic diversity of 1,845 rhesus macaques improves genetic variation interpretation and identifies disease models

Understanding and treating human diseases require valid animal models. Leveraging the genetic diversity in rhesus macaque populations across eight primate centers in the United States, we conduct targeted-sequencing on 1845 individuals for 374 genes linked to inherited human retinal and neurodevelopmental diseases. We identify over 47,000 single nucleotide variants, a substantial proportion of which are shared with human populations. By combining rhesus and human allele frequencies with established variant prediction methods, we develop a machine learning-based score that outperforms established methods in predicting missense variant pathogenicity. Remarkably, we find a marked number of loss-of-function variants and putative deleterious variants, which may lead to the development of rhesus disease models. Through phenotyping of macaques carrying a pathogenic OPA1:p.A8S variant, we identify a genetic model of autosomal dominant optic atrophy. Finally, we present a public website housing variant and genotype data from over two thousand rhesus macaques.

Rhesus Macaques: VNTR Polymorphism of the FCGRT Gene.

Variable number tandem repeat (VNTR) polymorphisms of the human neonatal IgG Fc receptor α-chain gene (FCGRT) are known to influence the expression levels of FCGRT and IgG in serum. Monkeys are considered to be a relevant biological model for studying the effects of immunobiological drugs. The study determined the functional VNTR polymorphisms of the FCGRT gene in 109 male rhesus macaques from the nursery of the Kurchatov Complex of Medical Primatology. PCR amplification of samples was carried out followed by electrophoretic separation of DNA fragments in a 2% agarose gel. Individual DNA amplification products were sequenced (according to Sanger system) in forward and reverse directions to confirm the specificity. The genotyping showed that the VNTR polymorphism of the FCGRT gene in the studied population of rhesus macaques is presented by 9 variants. The frequency of the VNTR5 allele associated with lower IgG levels was 14.2%, and the most common one was the VNTR7 allele (25.2%). We also identified alleles that have not been previously reported: VNTR3, VNTR4, VNTR6, VNTR8, and VNTR9. The study allows to consider rhesus macaques as a potential model for studying the immunological response depending on the genetic VNTR variant of FCGRT.

Skeletal age during hurricane impacts fluctuating asymmetry in Cayo Santiago rhesus macaques.

As natural disasters become more frequent due to climate change, understanding the biological impact of these ecological catastrophes on wild populations becomes increasingly pertinent. Fluctuating asymmetry (FA), or random deviations from bilateral symmetry, is reflective of developmental instability and has long been positively associated with increases in environmental stress. This study investigates craniofacial FA in a population of free-ranging rhesus macaques (Macaca mulatta) that has experienced multiple Category 3 hurricanes since the colony's inception on Cayo Santiago, including 275 individuals from ages 9 months to 31 years (F = 154; M = 121). Using geometric morphometrics to quantify FA and a linear mixed-effect model for analysis, we found that sex, age, and decade of birth did not influence the amount of FA in the individuals included in the study, but the developmental stage at which individuals experienced these catastrophic events greatly impacted the amount of FA exhibited (p = .001). Individuals that experienced these hurricanes during fetal life exhibited greater FA than any other post-natal developmental period. These results indicate that natural disasters can be associated with developmental disruption that results in long-term effects if occurring during the prenatal period, possibly due to increases in maternal stress-related hormones.

Landscape genomics analysis provides insights into future climate change-driven risk in rhesus macaque

Climate change significantly affects the suitability of wildlife habitats. Thus, understanding how animals adapt ecologically and genetically to climate change is important for targeted species protection. Rhesus macaques (Macaca mulatta) are widely distributed and multi-climatically adapted primates. This study explored how rhesus macaques adapt to climate change by integrating ecological and genetic methods and applying species distribution models (SDMs) and a gradient forest (GF) model. The findings suggested that temperature seasonality primarily affects habitat suitability and indicated that climate change will have a dramatic impact on macaque populations in the future. We also applied genotype-environment association (GEA) analyses and selection signature analyses to identify genes associated with climate change and provide possible explanations for the adaptation of rhesus macaques to climatic environments. The population genomics analyses suggested that the Taihang population has the highest genomic vulnerability with inbreeding and low heterozygosity. Combined with the higher ecological vulnerability, additional conservation strategies are required for this population under higher risk of climate change. Our work measured the impact of climate change and enabled the identification of populations that exhibit high vulnerability to severe climate change. Such information is useful for selecting populations of rhesus macaques as subject of long-term monitoring or evolutionary rescue under future climate change.

Age-Related Differences in Ocular Features of a Naturalistic Free-Ranging Population of Rhesus Macaques.

Rhesus macaques (Macaca mulatta) are the premier nonhuman primate model for studying human health and disease. We investigated if age was associated with clinically relevant ocular features in a large cohort of free-ranging rhesus macaques from Cayo Santiago, Puerto Rico. We evaluated 120 rhesus macaques (73 males, 47 females) from 0 to 29 years old (mean ± SD: 12.6 ± 6.4) from September to December 2021. The ophthalmic evaluation included intraocular pressure (IOP) assessment, corneal pachymetry, biomicroscopy, A-scan biometry, automated refraction, and fundus photography after pupil dilation. The associations of age with the outcomes were investigated through multilevel mixed-effects models adjusted for sex and weight. On average, IOP, pachymetry, axial length, and automated refraction spherical equivalent were 18.37 ± 4.68 mmHg, 474.43 ± 32.21 µm, 19.49 ± 1.24 mm, and 0.30 ± 1.70 diopters (D), respectively. Age was significantly associated with pachymetry (β coefficient = -1.20; 95% confidence interval [CI], -2.27 to -0.14; P = 0.026), axial length (β coefficient = 0.03; 95% CI, 0.01 to 0.05; P = 0.002), and spherical equivalent (β coefficient = -0.12; 95% CI, -0.22 to -0.02; P = 0.015). No association was detected between age and IOP. The prevalence of cataracts in either eye was 10.83% (95% CI, 6.34-17.89) and was significantly associated with age (odds ratio [OR] = 1.20; 95% CI, 1.06-1.36; P = 0.004). Retinal drusen in either eye was observed in 15.00% (95% CI, 9.60-22.68) of animals, which was also significantly associated with age (OR = 1.14; 95% CI, 1.02-1.27; P = 0.020). Rhesus macaques exhibit age-related ocular associations similar to those observed in human aging, including decreased corneal thickness, increased axial length, myopic shift, and higher prevalence of cataract and retinal drusen.

Coming to the Caribbean-acclimation of Rhesus macaques (Macaca mulatta) at Cayo Santiago.

To investigate whether the Cayo Santiago, Puerto Rico (Latitude: 18.1564°N; temperature range 19°C to 32°C) rhesus macaque population has acclimated to their tropical island conditions since arriving from Lucknow, India (Latitude: 26.8470°N; temperature range 8°C to 41°C) in 1938. Using the derived skeletal collection, measurements were taken of long bone lengths, diaphyseal circumference, and body weight using 635 (237 males and 398 females) skeletally mature individuals. Measurements sampled colony members born over a 51-year time span at Cayo Santiago, from 1951 to 2002. Results demonstrated that body weights and diaphyseal circumferences significantly declined in both males and females. Long bone lengths relative to body weight and diaphyseal circumference also increased in females. Whereas body weight, long bone length and diaphyseal circumference declined at near parallel rates in males. The population has acclimated to homogenous, tropical, conditions of the Caribbean island since their arrival over 80 years ago. Trends in both sexes aligned with Bergmann's rule, though females displayed a greater decline in body weight, as well as greater affinity with Allen's rule, than did males. Buffering effects related to male competition may be responsible for this discrepancy. Overall, the Cayo Santiago populations, as shown over a significant period (1951-2002) of their history, have acclimated to their island conditions by decreasing in size and altering body proportions.

Extensive prevalence and significant genetic differentiation of Blastocystis in high- and low-altitude populations of wild rhesus macaques in China

BackgroundBlastocystis is a common intestinal protist with a wide range of hosts. Thus far, 38 subtypes have been identified. In recent years, wild animals have been confronted with habitat fragmentation as well as an increasing risk of zoonotic disease transmission due to human disturbance. Only limited data are available on Blastocystis infection and subtype distribution in wild rhesus macaques in China. The aim of the present study was to investigate the prevalence and genetic diversity of Blastocystis in nine wild rhesus macaque populations in China.MethodsA total of 276 faecal samples were collected from five high-altitude populations (high-altitude group [HAG]; 2800–4100 m a.s.l.) and four low-altitude populations (low-altitude group [LAG]; 5–1000 m a.s.l) of rhesus macaques. PCR-based analysis, using a new primer pair for the amplification of a 1690-bp sequence of the small subunit ribosomal RNA (SSU rRNA) gene, was used for prevalence and genetic diversity analysis.ResultAnalysis of faecal samples revealed that Blastocystis infection was common in rhesus macaques, with an infection positivity rate of 80.1% (n = 221/276 samples). There was no significant difference (P = 0.121) in positivity rate between the LAG (84.3%) and HAG (76.8%). Overall, 33 haplotypes were obtained and classified into four subtypes (STs), of which three were potentially zoonotic subtypes (ST1, 29.7%; ST2, 16.7%; ST3, 31.9%) and one that was first identified in this study and named ST39 (12.0%). The STs were distributed differently among the rhesus macaque populations, except for ST3, which was found in all populations. Phylogenetic analyses revealed two major divergent clades of ST3 for the HAG and LAG. Genetic diversity analysis showed a high genetic diversity of ST3 (haplotype diversity: 0.846; nucleotide diversity: 0.014) in the rhesus macaques, but a high genetic differentiation (FST > 0.25) and a low gene flow (Nm = 0.09) between the HAG and LAG.ConclusionOur study, which is the first investigation on Blastocystis infection in multiple wild rhesus macaque populations in China, indicates a potential risk of zoonotic transmission of Blastocystis in the study areas. Blastocystis ST3 showed high genetic diversity in wild rhesus macaques and significant genetic differentiation between the HAG and LAG. Our results provide fundamental information on the genetic diversity and prevalence of Blastocystis in wild rhesus macaque populations.Graphical

Within-individual changes reveal increasing social selectivity with age in rhesus macaques

Accumulating evidence in humans and other mammals suggests older individuals tend to have smaller social networks. Uncovering the cause of these declines can inform how changes in social relationships with age affect health and fitness in later life. While age-based declines in social networks have been thought to be detrimental, physical and physiological limitations associated with age may lead older individuals to adjust their social behavior and be more selective in partner choice. Greater selectivity with age has been shown in humans, but the extent to which this phenomenon occurs across the animal kingdom remains an open question. Using longitudinal data from a population of rhesus macaques on Cayo Santiago, we provide compelling evidence in a nonhuman animal for within-individual increases in social selectivity with age. Our analyses revealed that adult female macaques actively reduced the size of their networks as they aged and focused on partners previously linked to fitness benefits, including kin and partners to whom they were strongly and consistently connected earlier in life. Females spent similar amounts of time socializing as they aged, suggesting that network shrinkage does not result from lack of motivation or ability to engage, nor was this narrowing driven by the deaths of social partners. Furthermore, females remained attractive companions and were not isolated by withdrawal of social partners. Taken together, our results provide rare empirical evidence for social selectivity in nonhumans, suggesting that patterns of increasing selectivity with age may be deeply rooted in primate evolution.

Sociability in a non-captive macaque population is associated with beneficial gut bacteria.

The relationship between social behaviour and the microbiome is known to be reciprocal. Research in wild animal populations, particularly in primate social groups, has revealed the role that social interactions play in microbial transmission, whilst studies in laboratory animals have demonstrated that the gut microbiome can affect multiple aspects of behaviour, including social behaviour. Here we explore behavioural variation in a non-captive animal population with respect to the abundance of specific bacterial genera. Social behaviour based on grooming interactions is assessed in a population of rhesus macaques (Macaca mulatta), and combined with gut microbiome data. We focus our analyses on microbiome genera previously linked to sociability and autistic behaviours in rodents and humans. We show in this macaque population that some of these genera are also related to an individual's propensity to engage in social interactions. Interestingly, we find that several of the genera positively related to sociability, such as Faecalibacterium, are well known for their beneficial effects on health and their anti-inflammatory properties. In contrast, the genus Streptococcus, which includes pathogenic species, is more abundant in less sociable macaques. Our results indicate that microorganisms whose abundance varies with individual social behaviour also have functional links to host immune status. Overall, these findings highlight the connections between social behaviour, microbiome composition, and health in an animal population.

Reduced injury risk links sociality to survival in a group-living primate

Sociality has been linked to a longer lifespan in many mammals, including humans. Yet, how sociality results in survival benefits remains unclear. Using 10 years of data and over 1,000 recorded injuries in rhesus macaques (Macaca mulatta), we tested two injury-related mechanisms by which social status and affiliative partners might influence survival. Injuries increased individual risk of death by 3-fold in this dataset. We found that sociality can affect individuals' survival by reducing their risk of injury but had no effect on the probability of injured individuals dying. Both males and females of high social status (measured as female matrilineal rank and male group tenure) and females with more affiliative partners (estimated using the number of female relatives) experienced fewer injuries and thus were less likely to die. Collectively, our results offer rare insights into one mechanism that can mediate the well-known benefits of sociality on an individual's fitness.

Concentrations of urinary neopterin, but not suPAR, positively correlate with age in rhesus macaques

Identifying biomarkers of age-related changes in immune system functioning that can be measured non-invasively is a significant step in progressing research on immunosenescence and inflammaging in free-ranging and wild animal populations. In the present study, we aimed to investigate the suitability of two urinary compounds, neopterin and suPAR, as biomarkers of age-related changes in immune activation and inflammation in a free-ranging rhesus macaque (Macaca mulatta) population. We also investigated age-associated variation in gene transcription from blood samples to understand the underlying proximate mechanisms that drive age-related changes in urinary neopterin or suPAR. Neopterin was significantly positively correlated with age, and had a moderate within-individual repeatability, indicating it is applicable as a biomarker of age-related changes. The age-related changes in urinary neopterin are not apparently driven by an age-related increase in the primary signaler of neopterin, IFN-y, but may be driven instead by an age-related increase in both CD14+ and CD14− monocytes. suPAR was not correlated with age, and had low repeatability within-individuals, indicating that it is likely better suited to measure acute inflammation rather than chronic age-related increases in inflammation (i.e., “inflammaging”). Neopterin and suPAR had a correlation of 25%, indicating that they likely often signal different processes, which if disentangled could provide a nuanced picture of immune-system function and inflammation when measured in tandem.

Variation and heritability of retinal cone ratios in a free-ranging population of rhesus macaques.

A defining feature of catarrhine primates is uniform trichromacy—the ability to distinguish red (long; L), green (medium; M), and blue (short; S) wavelengths of light. Although the tuning of photoreceptors is conserved, the ratio of L:M cones in the retina is variable within and between species, with human cone ratios differing from other catarrhines. Yet, the sources and structure of variation in cone ratios are poorly understood, precluding a broader understanding of color vision variability. Here, we report a large‐scale study of a pedigreed population of rhesus macaques (Macaca mulatta). We collected foveal RNA and analyzed opsin gene expression using cDNA and estimated additive genetic variance of cone ratios. The average L:M ratio and standard error was 1.03:1 ± 0.02. There was no age effect, and genetic contribution to variation was negligible. We found marginal sex effects with females having larger ratios than males. S cone ratios (0.143:1 ± 0.002) had significant genetic variance with a heritability estimate of 43% but did not differ between sexes or age groups. Our results contextualize the derived human condition of L‐cone dominance and provide new information about the heritability of cone ratios and variation in primate color vision.

Identifying the environmental and anthropogenic causes, distribution, and intensity of human rhesus macaque conflict in Nepal

Reducing conflict between humans and wildlife is considered a top conservation priority. However, increasingly human-induced disturbances across natural landscapes have escalated encounters between humans and wildlife. In Nepal, forests have been destroyed, fragmented, and developed for human settlements, agricultural production, and urban centers for decades. As a result, human-wildlife conflict, in the form of crop-raiding, livestock predation, and injuries to humans and wildlife, is common throughout the country. In particular, crop-raiding by macaques is an increasingly common form of human-wildlife conflict. Rhesus macaques (Macaca mulatta) have been identified as a top ten crop-raiding wildlife species in Nepal. In order to better understand the nationwide distribution and intensity of human-rhesus macaque conflict (HRMC), we conducted an extensive literature review of reported incidences of HRMC during the period 2000 to 2021 in Nepal. We also created an online survey to obtain nationwide data on the location and severity of HRMC, and modeled the set of ecological factors that affect habitat suitability for rhesus macaques. An ensemble of three different species distribution model (SDM) algorithms were used to analyze these data. We found that almost 44% of Nepal's land area contains suitable habitat for rhesus macaques, with less than 8% of all suitable habitat located in protected national parks. As humans continue to alter and fragment natural landscapes, HRMC in Nepal has intensified. At present, nearly 15% of the country's land area in which human settlements are permitted, is characterized by moderate or high rates of HRMC. We argue that prioritizing programs of forest restoration, strategic management plans designed to connect isolated forest fragments with high rhesus macaque population densities, creating government programs that compensate farmers for income lost due to crop-raiding, and educational outreach that informs local villagers of the importance of conservation and protecting biodiversity, offer the most effective solutions to reduce HRMC in Nepal.

Prevalence and new genotypes of Enterocytozoon bieneusi in wild rhesus macaque (Macaca mulatta) in China: A zoonotic concern

Enterocytozoon bieneusi is a zoonotic pathogen with a wide range of animal host. There are only few reports of E. bieneusi infection in wild Chinese rhesus macaques. Here, we determined the prevalence of E. bieneusi in nine wild rhesus macaque populations and assessed their zoonotic potential by performed genotype of ITS gene. A total of 324 fecal samples of rhesus macaque were collected in nine geographical populations from five Chinese provinces (Sichuan, Chongqing, Qinghai, Tibet and Hainan). 38 samples (11.7%) were found to be infected with E. bieneusi, and 11 genotypes were identified including three known genotypes (D, EbpC and SCC-2) and eight novel genotypes named Mul6∼13. Genotype D (63.2%) was the most prevalent, being observed in seven populations except XZ-2 and QH, and other genotypes were identified only in a single area. According to the phylogenetic analysis, Mul6∼9, Mul11∼13 and zoonotic genotype D were clustered into Group 1, indicating that these genotypes may be potentially zoonotic. Among nine populations, population SC-3 had the highest infection rate (26.3%), and the lowest was the wild QH population without infection, but the difference of infection rate among the nine populations is not significant. It is concluded that, rhesus macaque populations are generally infected E. bieneusi in many areas of China, and there may be a risk of cross infection with E. bieneusi in some areas found having zoonotic genotypes, and these areas should be paid more attention to prevent.

Genetic Homogeneity of the Population of Male Rhesus Macaques by the Polymorphisms of Genes oprm1, npy, maoa, crh, 5-htt as Determined by Cluster Analysis of Blood Count Data

Genetic Homogeneity of the Population of Male Rhesus Macaques by the Polymorphisms of Genes oprm1, npy, maoa, crh, 5-htt as Determined by Cluster Analysis of Blood Count Data

Introduced Monkey Populations of Florida - History, Status, and Potential Impacts

At least nine populations of non-human primates have established in Florida since the 1930s. These include three species: rhesus macaques (Macaca mulatta), vervet monkeys (Chlorocebus sabaeus), and squirrel monkeys (Saimiri sp.). Invasion success has varied among the three species. As of 2021, a single, growing population of rhesus macaques was present in central Florida; a single, stable population of vervet monkeys was present in south Florida; and only two squirrel monkeys remained of a previous population on a private property in south Florida. Demonstrated and potential impacts of the three species also varied. Continued monitoring and research of introduced primate populations in Florida is critical to better understand impacts and management needs.

Experiencing a Natural Disaster Accelerates Aging of the Immune System

Extreme adverse events such as natural disasters can accelerate disease progression and promote chronic inflammation. These phenotypes also increase in prevalence with age, suggesting that experiencing adversity might accelerate aging of the immune system. Adversity can induce persistent gene regulatory changes which may mechanistically explain the immune similarities between aging and adversity. To test how immune system aging is accelerated following a natural disaster, we measured the impact of Hurricane Maria on peripheral blood immune cell gene expression in a population of free-ranging rhesus macaques (Macaca mulatta) from before (n=435) versus after (n=108) Hurricane Maria. Experiencing Hurricane Maria altered the expression of 260 genes (FDR<10%), which were primarily involved in the inflammatory response. There was significant overlap in these hurricane-affected and age-associated genes with 40% (n=104) being associated with both the hurricane and aging, more than double the expected amount (Fisher’s Exact Test OR=3.7, p=4.06 x 10–21). The effects of the hurricane and aging on gene expression were also significantly correlated (rho=0.23, p=1.33 x 10-84), suggesting that they alter similar molecular pathways in the immune system. Further, we found that animals that experienced the hurricane had a gene expression profile that was, on average, 1.6 years older than animals that did not experience the hurricane (the equivalent of 6–7 years in a human lifespan, p=0.003). Together, our results provide some of the first evidence that extreme natural disasters mechanistically accelerates aging in the immune system.

Effects of Age and Social Adversity on Immune Cell Populations in a Non-Human Primate Model of Human Aging

Significant hallmarks of aging are immune function decline and rising cumulative inflammation. These immunosenescent signatures are also found in individuals who experience chronic social adversity, independently of age. However, no studies to date have examined how social adversity alters immune function across the lifespan –data that are essential to identify the molecular routes through which social adversity might lead to increased aging-related disease. Over a two-year period, we investigated how age and social adversity (quantified by low social status) affected immunity. We measured immune cell proportions at baseline and their gene regulation after in vitro stimulation with pathogen molecules that stimulated both Th1 and Th2 immune responses in a population of free-ranging rhesus macaques. We first performed flow cytometry on peripheral whole blood to quantify changes on immune cell proportions across the lifespan (n=235) and in animals of different social statuses (n=141). We found significant decreases in CD20+ B cells and CD3+/CD4+ T cell proportions with age, suggesting diminished antibody production and adaptive immune responses in older individuals. Age-associated increases in CD3+/CD8+, CD3+/CD4+/CD25+ T regulatory cells and CD14-/CD16+/HLA-DR+ non-classical monocytes indicated heightened baseline inflammation in older animals. Social adversity recapitulated the effects of aging in CD14+/CD16-/HLA-DR+ classical monocytes, indicating immune deficits in phagocytosis and pathogen clearance in older and lower status individuals. Using RNA-seq, our stimulations (n=1,320) will allow us to identify molecular immune pathways that are disrupted by age and social adversity, similarities in response between age and adversity, and how the effect of adversity varies across the lifespan.

Pre and Post Sterilization Behaviour Studies of Rhesus macaque (Macaca mulatta) in Himachal Pradesh, India

Abstract: Rhesus Macaque (Macaca mulatta) population in India has been on a rise due to their high breeding rate and ability to acclimatize to various environments (Imam 2013). They are often seen in Human habitations especially in agricultural fields, raiding crops and becoming a nuisance to the farmers. According to agricultural department, the marauding macaques have raided agricultural crops worth Rs 185 crore and horticulture crops worth Rs 38 crore in various districts of HP in 2014. To address this menace, sterilization programme is carried out at respective Monkey sterilization centre (MSC’s) established at 8 monkey affected districts across Himachal Pradesh. This study aims to investigate and understand the various behavioural changes that may occur (intra specific, social , feeding, stress/aggression ) in the period of post sterilization of Rhesus macaque. The proposed study site for this project is a Village called Gopalpur located in Kangra District of Himachal Pradesh. For pre-sterilization monitoring, a virgin Troupe was identified and selected for capture based on the MSC’s record on capture locations and sterilized monkeys. The virgin troupe was monitored for their behaviour which included changes and their usual aggression, Feeding, grooming, sleeping, and mating and home range, for a period of 3 weeks. 100 % capture of this troupe was aimed for sterilization at a time. But failure of the same, the remaining population was to be monitored for their behavioural changes due to separation from their parent troupe. Capturing Rhesus of Chamunda Area near the addressed site was challenging due to many reasons which are discussed in the report. Three monkeys were caught successfully out of which two escaped and one turned out to be pregnant. The identified troupe couldn’t be captured and sterilized to study their post behavioural changes. The pregnant monkey was released back at the point of capture as it can’t be sterilized. This study on presterilization behaviour will form a baseline data for future studies on their post-sterilization behavioural changes. The study will also help in streamlining the monkey capturing process. Keywords: Rhesus macaque, Behaviour, Monkey sterilisation centre, MSC, Sterilisation program.

Factors influencing the success of male introductions into groups of female rhesus macaques: Introduction technique, male characteristics and female behavior.

In captive populations of rhesus macaques, novel adult males are commonly introduced to female groups every few years to prevent inbreeding, which mimics male dispersal in wild macaque populations. However, introducing adult males is challenging because macaques are aggressive to newcomers, which can result in serious injuries. Efforts to reduce trauma risk during the introduction process and increase the probability of success are needed. Here we investigate the impact of multiple factors, including male attributes (e.g., age, weight, rank, and experience), introduction method (punctuated vs. continual exposure to females), and female behavior, on males' trauma risk and integration success. We studied eight introductions of multimale cohorts (3-7 males each; N = 36 total) into existing female groups of rhesus macaques at the Yerkes National Primate Research Center. Four cohorts were introduced using the punctuated exposure method where adult males were moved each morning from run housing to the females' indoor enclosure and returned to run housing in the afternoon, and four cohorts were introduced using the continual exposure method where adult males were moved to an introduction enclosure attached to the females' outdoor compound, allowing males to live in protected contact next to the female group continuously. Generalized linear mixed models fitted to trauma risk (e.g., latency to first trauma; total trauma count) and success or failure to integrate (i.e., continual residence within the female group for greater than 53% of days within a 28-day window after first overnight stay) showed that continual exposure to females in the introduction enclosure reduced male trauma risk and increased the likelihood of successful integration compared to punctuated exposure. Males received less trauma when they received a higher rate of grooming from females. Male attributes had no effect. These findings highlight the importance of introduction technique and female behavior in the process of males' social integration into female groups.