Deep Learning for Face Detection and Pain Assessment in Japanese macaques (Macaca fuscata).

Facial expressions have increasingly been used to assess emotional states in mammals. The recognition of pain in research animals is essential for their well-being and leads to more reliable research outcomes. Automating this process could contribute to early pain diagnosis and treatment. Artificial neural networks have become a popular option for image classification tasks in recent years due to the development of deep learning. In this study, we investigated the ability of a deep learning model to detect pain in Japanese macaques based on their facial expression. Thirty to 60 min of video footage from Japanese macaques undergoing laparotomy was used in the study. Macaques were recorded undisturbed in their cages before surgery (No Pain) and one day after the surgery before scheduled analgesia (Pain). Videos were processed for facial detection and image extraction with the algorithms RetinaFace (adding a bounding box around the face for image extraction) or Mask R-CNN (contouring the face for extraction). ResNet50 used 75% of the images to train systems; the other 25% were used for testing. Test accuracy varied from 48 to 54% after box extraction. The low accuracy of classification after box extraction was likely due to the incorporation of features that were not relevant for pain (for example, background, illumination, skin color, or objects in the enclosure). However, using contour extraction, preprocessing the images, and fine-tuning, the network resulted in 64% appropriate generalization. These results suggest that Mask R-CNN can be used for facial feature extractions and that the performance of the classifying model is relatively accurate for nonannotated single-frame images.

Endodontic Treatment of a Maxillary Incisor Tooth in a Chimpanzee (Pan troglodytes).

Chimpanzees (Pan troglodytes) with teeth severely damaged by dental caries and/or periodontal disease are often managed with medication and/or tooth extraction. A common endodontic treatment for severely decayed teeth in a 26-year-old female chimpanzee is reported. The left maxillary central incisor tooth had lost its crown, probably due to trauma that was not recent, and it had a fistula most likely due to chronic apical periodontitis. The diagnosis was confirmed radiographically before treatment. To treat the infected root canal, endodontic treatment used in humans was adapted for a chimpanzee. After the treatment, the tooth was sealed using an adhesive resin composite. At 11-years post-treatment, there were no signs of recurrence of the lesion or of failure of the tooth seal. The results of this case report suggest that common endodontic treatments used in humans are also effective in chimpanzees.

Housing relocation does not have to induce a significant stress response in captive Japanese macaques (Macaca fuscata).

BACKGROUND: Previous studies suggest that housing relocation may be stressful for captive non-human primates. Our study investigated the stress levels of Japanese macaques during a housing relocation by measuring their daily fecal cortisol metabolites, which are often used as an indicator of stress. METHODS: Ten adult Japanese macaques, single-housed for research purposes, were relocated to a new facility. Fecal samples were collected daily for 7 days. Cortisol metabolite concentrations were determined via enzyme immunoassay. RESULTS: No significant differences in cortisol metabolite levels were found in 7 days, but concentration levels showed that the highest median was associated to the relocation day. CONCLUSIONS: The minimal cortisol metabolite increase suggests that there was a slight activity increase in the hypothalamic-pituitary-adrenal axis. Techniques encouraging cooperation of the monkeys, the short time duration of the relocation, and consistency in the environment may have contributed to the minimal stress levels observed.

Annual two-dose tetanus toxoid vaccination induces protective humoral immunity to all age groups of rhesus macaques.

A tetanus outbreak occurred during 2014-2015 in the rhesus macaques reared in an open enclosure in our facility. As the soil of the facility was suspected to be contaminated with Clostridium tetani spores, there was a risk of further tetanus occurring among the macaques. To protect them from tetanus, a tetanus toxoid vaccination was recommended; however, the vaccinated elderly animals might not be effectively protected due to insufficient humoral immune responses. Hence, we evaluated the dynamics of antibody responses among rhesus macaques of all age groups vaccinated with two-dose tetanus toxoid at a 1-year interval during a 3-year follow-up study. The vaccination developed anti-tetanus toxin-specific antibodies in animals of all age groups, the antibody levels peaked 1 year after the second vaccination, and the peak levels decreased with age. However, the levels among elderly individuals (aged ≥13 years) were still higher than the threshold level, which was supposed to protect them from tetanus development. Although the rhesus macaques in our facility had a risk of occasional exposure to the spores due to the outbreak, no incidence of tetanus has ever occurred to date. These results indicate that the vaccination protocol is effective in protecting not only younger but also older animals from tetanus.

The Japan Monkey Centre Primates Brain Imaging Repository of high-resolution postmortem magnetic resonance imaging: The second phase of the archive of digital records.

A comparison of neuroanatomical features of the brain between humans and our evolutionary relatives, nonhuman primates, is key to understanding the human brain system and the neural basis of mental and neurological disorders. Although most comparative MRI studies of human and nonhuman primate brains have been based on brains of primates that had been used as subjects in experiments, it is essential to investigate various species of nonhuman primates in order to elucidate and interpret the diversity of neuroanatomy features among humans and nonhuman primates. To develop a research platform for this purpose, it is necessary to harmonize the scientific contributions of studies with the standards of animal ethics, animal welfare, and the conservation of brain information for long-term continuation of the field. In previous research, we first developed a gated data-repository of anatomical images obtained using 9.4-T ex vivo MRI of postmortem brain samples from 12 nonhuman primate species, and which are stored at the Japan Monkey Centre. In the present study, as a second phase, we released a collection of T2-weighted images and diffusion tensor images obtained in nine species: white-throated capuchin, Bolivian squirrel monkey, stump-tailed macaque, Tibet monkey, Sykes' monkey, Assamese macaque, pig-tailed macaque, crested macaque, and chimpanzee. Our image repository should facilitate scientific discoveries in the field of comparative neuroscience. This repository can also promote animal ethics and animal welfare in experiments with nonhuman primate models by optimizing methods for in vivo and ex vivo MRI scanning of brains and supporting veterinary neuroradiological education. In addition, the repository is expected to contribute to conservation, preserving information about the brains of various primates, including endangered species, in a permanent digital form.

Emergence of an erythroid cell-specific regulatory region in ABO intron 1 attributable to A- or B-antigen expression on erythrocytes in Hominoidea.

A- and B-antigens are present on red blood cells (RBCs) as well as other cells and secretions in Hominoidea including humans and apes such as chimpanzees and gibbons, whereas expression of these antigens on RBCs is subtle in monkeys such as Japanese macaques. Previous studies have indicated that H-antigen expression has not completely developed on RBCs in monkeys. Such antigen expression requires the presence of H-antigen and A- or B-transferase expression in cells of erythroid lineage, although whether or not ABO gene regulation is associated with the difference of A- or B-antigen expression between Hominoidea and monkeys has not been examined. Since it has been suggested that ABO expression on human erythrocytes is dependent upon an erythroid cell-specific regulatory region or the + 5.8-kb site in intron 1, we compared the sequences of ABO intron 1 among non-human primates, and demonstrated the presence of sites orthologous to the + 5.8-kb site in chimpanzees and gibbons, and their absence in Japanese macaques. In addition, luciferase assays revealed that the former orthologues enhanced promoter activity, whereas the corresponding site in the latter did not. These results suggested that the A- or B-antigens on RBCs might be ascribed to emergence of the + 5.8-kb site or the corresponding regions in ABO through genetic evolution.

Effect of sedatives or anesthetics on the measurement of resting brain function in common marmosets.

Common marmosets are promising laboratory animals for the study of higher brain functions. Although there are many opportunities to use sedatives and anesthetics in resting brain function measurements in marmosets, their effects on the resting-state network remain unclear. In this study, the effects of sedatives or anesthetics such as midazolam, dexmedetomidine, co-administration of isoflurane and dexmedetomidine, propofol, alfaxalone, isoflurane, and sevoflurane on the resting brain function in common marmosets were evaluated using independent component analysis, dual regression analysis, and graph-theoretic analysis; and the sedatives or anesthetics suitable for the evaluation of resting brain function were investigated. The results show that network preservation tendency under light sedative with midazolam and dexmedetomidine is similar regardless of the type of target receptor. Moreover, alfaxalone, isoflurane, and sevoflurane have similar effects on resting state brain function, but only propofol exhibits different tendencies, as resting brain function is more preserved than it is following the administration of the other anesthetics. Co-administration of isoflurane and dexmedetomidine shows middle effect between sedatives and anesthetics.

Investigating subtle changes in facial expression to assess acute pain in Japanese macaques.

Changes in facial expression provide cues for assessing emotional states in mammals and may provide non-verbal signals of pain. This study uses geometric morphometrics (GMM) to explore the facial shape variation in female Japanese macaques who underwent experimental laparotomy. Face image samples were collected from video footage of fourteen macaques before surgery and 1, 3, and 7 days after the procedure. Image samples in the pre-surgical condition were considered pain-free, and facial expressions emerging after surgery were investigated as potential indicators of pain. Landmarks for shape analysis were selected based on the underlying facial musculature and their corresponding facial action units and then annotated in 324 pre-surgical and 750 post-surgical images. The expression of pain is likely to vary between individuals. Tightly closed eyelids or squeezed eyes and lip tension were the most commonly observed facial changes on day 1 after surgery (p < 0.01974). A good overall inter-rater reliability [ICC = 0.99 (95% CI 0.75-1.0)] was observed with the method. The study emphasizes the importance of individualized assessment and provides a better understanding of facial cues to pain for captive macaque care.

The effect of different body positions on the cardiothoracic ratios obtained by chest radiography in Japanese macaques (Macaca fuscata) and rhesus macaques (Macaca mulatta).

BACKGROUND: Although some studies have reported cardiac diseases in macaques, an adequate screening method for cardiac enlargement has not yet been established. This study aimed to evaluate the positioning of macaques for radiographs and establish reference intervals for the cardiothoracic ratio (CTR). MATERIALS AND METHODS: We developed a device for chest radiography in the sitting position and performed chest radiography in 50 Japanese and 48 rhesus macaques to evaluate the CTR and chest cavity size. RESULTS: In Japanese and rhesus macaques, the thorax height was significantly larger, the heart width was significantly smaller, and the mean CTR was significantly smaller in the sitting position than in the prone position. The reference intervals for CTR in the sitting position were 51.6 ± 4.6% and 52.2 ± 5.1% in Japanese and rhesus macaques, respectively. CONCLUSION: Thoracic radiographic images obtained in a sitting position resulted in a smaller CTR and a larger thorax height, which could be useful for detecting pulmonary and cardiac abnormalities.

CalliFACS: The common marmoset Facial Action Coding System.

Facial expressions are subtle cues, central for communication and conveying emotions in mammals. Traditionally, facial expressions have been classified as a whole (e.g. happy, angry, bared-teeth), due to automatic face processing in the human brain, i.e., humans categorise emotions globally, but are not aware of subtle or isolated cues such as an eyebrow raise. Moreover, the same facial configuration (e.g. lip corners pulled backwards exposing teeth) can convey widely different information depending on the species (e.g. humans: happiness; chimpanzees: fear). The Facial Action Coding System (FACS) is considered the gold standard for investigating human facial behaviour and avoids subjective interpretations of meaning by objectively measuring independent movements linked to facial muscles, called Action Units (AUs). Following a similar methodology, we developed the CalliFACS for the common marmoset. First, we determined the facial muscular plan of the common marmoset by examining dissections from the literature. Second, we recorded common marmosets in a variety of contexts (e.g. grooming, feeding, play, human interaction, veterinary procedures), and selected clips from online databases (e.g. YouTube) to identify their facial movements. Individual facial movements were classified according to appearance changes produced by the corresponding underlying musculature. A diverse repertoire of 33 facial movements was identified in the common marmoset (15 Action Units, 15 Action Descriptors and 3 Ear Action Descriptors). Although we observed a reduced range of facial movement when compared to the HumanFACS, the common marmoset's range of facial movements was larger than predicted according to their socio-ecology and facial morphology, which indicates their importance for social interactions. CalliFACS is a scientific tool to measure facial movements, and thus, allows us to better understand the common marmoset's expressions and communication. As common marmosets have become increasingly popular laboratory animal models, from neuroscience to cognition, CalliFACS can be used as an important tool to evaluate their welfare, particularly in captivity.

Primate veterinarians' knowledge and attitudes regarding pain in macaques.

BACKGROUND: Assessment of pain in macaques is challenging. The aims of this study were (1) to investigate current knowledge and attitudes of primate veterinarians concerning acute pain in macaques; (2) to synthesise current knowledge and opinion to facilitate pain assessment. A primary question of interest was whether more confident individuals differ in their knowledge and attitudes from less-confident individuals. METHODS: An online survey was conducted amongst primate veterinarians serving both laboratories and zoos/sanctuaries. The questionnaire consisted of demographic information, attitudes towards pain, pain rating and analgesics, pain recognition and confidence in recognising pain and sources of information used. RESULTS AND CONCLUSIONS: There was generally good use of analgesia by respondents. More confident individuals reported that they recognise pain both behaviourally and in facial expressions, rated all pain signs more highly and used more analgesics. Specialist support networks aimed at increasing veterinarian confidence in macaque pain assessment could be beneficial.

Prevalence of antibodies against human respiratory viruses potentially involving anthropozoonoses in wild bonobos.

One of the current threats to the bonobo (Pan paniscus), a highly endangered ape species only found in the Democratic Republic of the Congo, are anthropozoonoses caused by human respiratory viruses. To date, epidemiological information regarding respiratory viral infections in bonobos is limited. In this study, we examined fecal immunoglobulin A antibodies against human respiratory viruses in bonobos, which may help estimating the viral prevalence. A substantial proportion of bonobos were positive for the antiviral antibodies, including those against parainfluenza virus, respiratory syncytial virus, influenza virus, rhinovirus, and mumps virus. The prevalence of the antibodies was found to depend on the viral species and bonobo populations, suggesting that the bonobos had been exposed to these respiratory viruses. These results may indicate the need for an epidemiological evidence-based action plan for the protection of bonobos from anthropozoonoses.

Physiological variation in Japanese macaques (Macaca fuscata) housed in different outdoor cages evaluated using the metabolic profile test.

Captive primates require environmental enrichment to minimize physical and mental stress. However, only a few objective evaluations have been performed to assess environment-induced physiological variations in these animals. In this study, we evaluated the usage of the metabolic profile test (MPT) to assess the influences of the housing environment on the physiology of Japanese macaques. Five male macaques were housed in an old type of cage (old cage group), in which the macaques were exposed to wind (except for a shelter box), and four males were in a new -type of cage (new cage group), which had a sub-room with a waterer that was surrounded by insulating panels. Blood samples were collected bimonthly for a year from the two groups to determine the complete blood count and blood biochemistry. The increase in the body weight of the macaques in both groups was suppressed during the cold season. Furthermore, this suppression was more pronounced in the old cage group, suggesting that the energy expenditure was higher in the old cage group than in the new cage group. Moreover, the red blood cell count and hematocrit values were higher during the cold season in both cages than during the warm season, suggesting that macaques were dehydrated during the cold season. Dehydration tendency was more pronounced in the macaques from the old cage group than in those from the new cage group, suggesting that their water intake decreased during the cold season. Our results suggest that the MPT can be used to evaluate environment-induced physiological variations in Japanese macaques.

Faecal proteomics as a novel method to study mammalian behaviour and physiology.

Mammalian faeces can be collected noninvasively during field research and provide valuable information on the ecology and evolution of the source individuals. Undigested food remains, genome/metagenome, steroid hormones, and stable isotopes obtained from faecal samples provide evidence on diet, host/symbiont genetics, and physiological status of the individuals. However, proteins in mammalian faeces have hardly been studied, which hinders the molecular investigations into the behaviour and physiology of the source individuals. Here, we apply mass spectrometry-based proteomics to faecal samples (n = 10), collected from infant, juvenile, and adult captive Japanese macaques (Macaca fuscata), to describe the proteomes of the source individual, of the food it consumed, and its intestinal microbes. The results show that faecal proteomics is a useful method to: (i) investigate dietary changes along with breastfeeding and weaning, (ii) reveal the taxonomic and histological origin of the food items consumed, and (iii) estimate physiological status inside intestinal tracts. These types of insights are difficult or impossible to obtain through other molecular approaches. Most mammalian species are facing extinction risk and there is an urgent need to obtain knowledge on their ecology and evolution for better conservation strategy. The faecal proteomics framework we present here is easily applicable to wild settings and other mammalian species, and provides direct evidence of their behaviour and physiology.

Extending the MaqFACS to measure facial movement in Japanese macaques (Macaca fuscata) reveals a wide repertoire potential.

Facial expressions are complex and subtle signals, central for communication and emotion in social mammals. Traditionally, facial expressions have been classified as a whole, disregarding small but relevant differences in displays. Even with the same morphological configuration different information can be conveyed depending on the species. Due to a hardwired processing of faces in the human brain, humans are quick to attribute emotion, but have difficulty in registering facial movement units. The well-known human FACS (Facial Action Coding System) is the gold standard for objectively measuring facial expressions, and can be adapted through anatomical investigation and functional homologies for cross-species systematic comparisons. Here we aimed at developing a FACS for Japanese macaques, following established FACS methodology: first, we considered the species' muscular facial plan; second, we ascertained functional homologies with other primate species; and finally, we categorised each independent facial movement into Action Units (AUs). Due to similarities in the rhesus and Japanese macaques' facial musculature, the MaqFACS (previously developed for rhesus macaques) was used as a basis to extend the FACS tool to Japanese macaques, while highlighting the morphological and appearance changes differences between the two species. We documented 19 AUs, 15 Action Descriptors (ADs) and 3 Ear Action Units (EAUs) in Japanese macaques, with all movements of MaqFACS found in Japanese macaques. New movements were also observed, indicating a slightly larger repertoire than in rhesus or Barbary macaques. Our work reported here of the MaqFACS extension for Japanese macaques, when used together with the MaqFACS, comprises a valuable objective tool for the systematic and standardised analysis of facial expressions in Japanese macaques. The MaqFACS extension for Japanese macaques will now allow the investigation of the evolution of communication and emotion in primates, as well as contribute to improving the welfare of individuals, particularly in captivity and laboratory settings.

Pharmacokinetics and effects on clinical and physiological parameters following a single bolus dose of propofol in common marmosets (Callithrix jacchus).

The objectives of this study were (a) to establish a population pharmacokinetic model and (b) to investigate the clinical and physiological effects of a single bolus dose of propofol in common marmosets. In Study 1, pharmacokinetic analysis was performed in six marmosets under sevoflurane anaesthesia. 8 mg/kg of propofol was administrated at a rate of 4 mg kg-1  min-1 . Blood samples were collected 2, 5, 15, 30, 60, 90, 120 or 180 min after starting propofol administration. Plasma concentration was measured, and population pharmacokinetic modelling was performed. A two-compartment model was selected as the final model. The population pharmacokinetic parameters were as follows: V1  = 1.14 L, V2  = 77.6 L, CL1  = 0.00182 L/min, CL2  = 0.0461 L/min. In Study 2, clinical and physiological parameters were assessed and recorded every 2 min after 12 mg/kg of propofol was administrated at a rate of 4 mg kg-1  min-1 . Immobilization was sustained for 5 min following propofol administration without apparent bradycardia. While combination of propofol and sevoflurane caused apnoea in Study 1, apnoea was not observed following single administration of propofol in Study 2. These data provide bases for further investigation on intravenous anaesthesia using propofol in common marmosets.

Evaluation of anaesthetic and cardiorespiratory effects after intramuscular administration of alfaxalone alone, alfaxalone-ketamine and alfaxalone-butorphanol-medetomidine in common marmosets (Callithrix jacchus).

BACKGROUND: Anaesthesia is often required in common marmosets undergoing various procedures. The aim of this study was to evaluate anaesthetic and cardiopulmonary effects of alfaxalone, alfaxalone-ketamine and alfaxalone-butorphanol-medetomidine in common marmosets. METHODS: The following treatments were repeatedly administered to seven female common marmosets: Treatment A, alfaxalone (12 mg kg-1 ) alone; treatment AK, alfaxalone (1 mg animal-1 ) plus ketamine (2.5 mg animal-1 ); treatment AMB, alfaxalone (4 mg kg-1 ), medetomidine (50 µg kg-1 ) plus butorphanol (0.3 mg kg-1 ); and treatment AMB-Ati, AMB with atipamezole at 45 minutes. RESULTS AND CONCLUSIONS: Marmosets became laterally recumbent and unresponsive for approximately 30 minutes in A and AK and for approximately 60 minutes in AMB. The animals showed rapid recovery following atipamezole injection in AMB-Ati. The decrease in heart rate and SpO2 was significantly greater in AMB compared to A and AK. Oxygen supplementation, anaesthetic monitors and atipamezole should be available especially when AMB is administered.

The profile of lipid metabolites in urine of marmoset wasting syndrome.

Marmoset wasting syndrome (MWS) is clinically characterized by progressive weight loss. Although morbidity and mortality of MWS are relatively high in captive marmosets, its causes remain unknown. Lipid mediators are bioactive metabolites which are produced from polyunsaturated fatty acids, such as arachidonic acid (AA) and eicosapentaenoic acid. These lipid metabolites regulate a wide range of inflammatory responses and they are excreted into the urine. As urinary lipid profiles reflect systemic inflammatory conditions, we comprehensively measured the levels of 141 types of lipid metabolites in the urines obtained from healthy common marmoset (Callithrix jacchus) (N = 7) or marmosets with MWS (N = 7). We found that 41 types of metabolites were detected in all urine samples of both groups. Among them, AA-derived metabolites accounted for 63% (26/41 types) of all detected metabolites. Notably, the levels of AA-derived prostaglandin (PG) E2, PGF2α, thromboxane (TX) B2 and F2-isoprostanes significantly increased in the urine samples of marmosets with MWS. In this study, we found some urinary lipid metabolites which may be involved in the development of MWS. Although the cause of MWS remains unclear, our findings may provide some insight into understanding the mechanisms of development of MWS.