Linking stress and immunity: Immunoglobulin A as a non-invasive physiological biomarker in animal welfare studies.

As the animal welfare community strives to empirically assess how care and management practices can help maintain or even enhance welfare, the development of tools for non-invasively measuring physiological biomarkers is essential. Of the suite of physiological biomarkers, Immunoglobulin A (IgA), particularly the secretory form (Secretory IgA or SIgA), is at the forefront because of its crucial role in mucosal immunity and links to physical health, stress, and overall psychological well-being. While interpretation of changes in SIgA concentrations on short time scales is complex, long-term SIgA patterns are consistent: conditions that create chronic stress lead to suppression of SIgA. In contrast, when welfare is enhanced, SIgA is predicted to stabilize at higher concentrations. In this review, we examine how SIgA concentrations are reflective of both physiological stress and immune function. We then review the literature associating SIgA concentrations with various metrics of animal welfare and provide detailed methodological considerations for SIgA monitoring. Overall, our aim is to provide an in-depth discussion regarding the value of SIgA as physiological biomarker to studies aiming to understand the links between stress and immunity.

Intermittent pair-housing, pair relationship qualities, and HPA activity in adult female rhesus macaques.

Laboratory rhesus macaques are often housed in pairs and may be temporarily or permanently separated for research, health, or management reasons. While both long-term social separations and introductions can stimulate a stress response that impacts inflammation and immune function, the effects of short-term overnight separations and whether qualities of the pair relationship mediate these effects are unknown. In this study, we investigated the effects of overnight separations on the urinary cortisol concentration of 20 differentially paired adult female rhesus macaques (Macaca mulatta) at the California National Primate Research Center. These females were initially kept in either continuous (no overnight separation) or intermittent (with overnight separation) pair-housing and then switched to the alternate pair-housing condition part way through the study. Each study subject was observed for 5 weeks, during which we collected measures of affiliative, aggressive, anxious, abnormal, and activity-state behaviors in both pair-housing conditions. Additionally, up to three urine samples were collected from each subject per week and assayed for urinary free cortisol and creatinine. Lastly, the behavioral observer scored each pair on four relationship quality attributes ("Anxious," "Tense," "Well-meshed," and "Friendly") using a seven-point scale. Data were analyzed using a generalized linear model with gamma distribution and an information theoretic approach to determine the best model set. An interaction between the intermittent pairing condition and tense pair adjective rating was in the top three models of the best model set. Dominance and rates of affiliation were also important for explaining urinary cortisol variation. Our results suggest that to prevent significant changes in HPA-axis activation in rhesus macaque females, which could have unintended effects on research outcomes, pairs with "Tense" relationships and overnight separations preventing tactile contact should be avoided.

Differential behavior patterns in cynomolgus monkey Macaca fascicularis in home cage in response to human gaze.

BACKGROUND: Non-human primates, when encountering human beings, show wariness and alertness. These behaviors differ when there is direct human gaze vs. when human averts his gaze. METHODS: We observed cynomolgus monkey in their home cage and studied their behaviors in response to human gaze. Four behaviors were analyzed: opening mouth, staring at observer, agitated activity, and approaching observer. RESULTS: Three behaviors appeared to be sensitive to human gaze between when the human observer gazed at the monkey and when the human observer looked away. Individual animals also displayed subpatterns of responses to human gaze. CONCLUSIONS: These results indicate that, even in their home cage, monkeys display a heightened level of awareness when gazed upon by a human observer, suggesting that human gaze may elicit emotional reactions. Further, under the human gaze, distinct behavioral subpatterns were apparent within the monkey cohort in our study, indicative of subgroups within the cohort.

Psychological Distress in Chimpanzees Rescued From Laboratories.

The United States is one of the last countries allowing invasive research on chimpanzees. Biomedical research on chimpanzees commonly involves maternal deprivation, social isolation, intensive confinement, and repetitive invasive procedures. These physically harmful and psychologically traumatic experiences cause many chimpanzees to develop symptoms of psychopathology that persist even after relocation from laboratories to sanctuaries. Through semistructured interviews with chimpanzee caregivers, direct behavioral observations, and consultation of laboratory records, we were interested in qualitatively analyzing symptoms of psychological distress in a sample of 253 chimpanzees rescued from biomedical research now residing at an accredited chimpanzee sanctuary. We present the results of this analysis and include an illustrative case study of one rescued chimpanzee who engages in self-injurious behaviors and meets modified Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, criteria for posttraumatic stress disorder. We discuss our results in light of recent policy changes regarding the use of chimpanzees in biomedical research in the United States and their implications for those involved in the rescue and rehabilitation of chimpanzees from biomedical research.

Comparing options for pair housing rhesus macaques using behavioral welfare measures.

In a biomedical research environment, research or management procedures may render continuous full contact pairing of rhesus macaques (Macaca mulatta) unfeasible. This study aimed to determine whether separation on a frequent basis or housing in adjacent cages with tactile contact interferes with the behavioral benefits of continuous full contact. Behavioral data (1260 hours) were collected from 32 adult females and 16 adult males housed at two National Primate Research Centers. Subjects were studied in four housing conditions: single housing, full contact pair housing, intermittent contact pair housing, and protected contact housing. After introduction, each pair was housed in each of the three social housing conditions in varying order. Among females, but not males, introducing animals into full and intermittent contact reduced levels of abnormal behavior. There was a trend toward this reduction in protected contact. In both females and males, full and intermittent contact was associated with lower levels of anxiety-related behavior, but protected contact was not. Females spent more time inactive in protected contact than either full or intermittent contact, and males showed a trend toward less inactivity following introduction into full contact. Both sexes showed less affiliation in protected contact compared to the other forms of social housing. Agonistic behavior among females was not affected by housing condition; among males, levels were equivalent in full and intermittent contact but were higher in intermittent than protected contact. Frequent separation of pairs does not appear to detract from the behavioral benefits of pair housing. Separation by a barrier permitting tactile contact is inferior to other forms of social housing but showed modest improvements over single housing nonetheless. This study can guide the provision of social contact to rhesus macaques under conditions restricting pairs from continuous full contact.

Gestures and social-emotional communicative development in chimpanzee infants.

Communicative skills of chimpanzees are of significant interest across many domains, such as developmental psychology (how does communication emerge in prelinguistic beings?), evolution (e.g., did human language evolve from primate gestures?), and in comparative psychology (how does the nonverbal communication of chimpanzees and humans compare?). Here we ask about how gestures develop in chimpanzee infants (n = 16) that were raised in an interactive program designed to study skill development. Data on socio-communicative development were collected following 4 hr of daily interaction with each infant, longitudinally from birth through the first year of life. A consistent and significant developmental pattern was found across the contexts of tickle play, grooming, and chase play: Infant chimpanzees first engaged in interactions initiated by others, then they initiated interactions, and finally, they requested others to join them in the interaction. Gestures were documented for initiating and requesting tickle play, for initiating and requesting grooming, and for initiating and requesting chase play. Gestural requests emerged significantly later than gestural initiations, but the age at which gestures emerged was significantly different across contexts. Those gestures related to hierarchical rank relations, that is, gestures used by subordinates in interaction with more dominant individuals, such as wrist presenting and rump presenting, did not emerge in the same manner as the other gestures. This study offers a new view on the development of gestures, specifically that many develop through interaction and communicate socio-emotional desires, but that not all gestures emerge in the same manner.

Periodic stays in a 'playcage' as an environmental enrichment measure for laboratory rats housed in individually ventilated cages: Short report.

Male Zucker Diabetic Sprague Dawley rats were housed in standard individually ventilated cages with floor area of 1500 cm2, and were placed in a 'playcage' (a large open cage) for three visits per week from the age of 16-18 weeks. The playcage was introduced in an attempt to reverse the compulsive behaviour that the rats displayed in the individually ventilated cages, with the purpose to increase their well-being and to provide cognitive as well as physical stimulation. After two weeks of periodic stays in the playcage, the rats' repetitive behaviour in their home cage ceased, and the rats displayed signs of happiness and excitement when they were in the playcage. The observations strongly indicate that periodic stays in a larger playcage can be an alternative environmental enrichment for laboratory rats when housing in a larger home cage is not an option.

Evaluation of reproduction and raising offspring in a nursery-reared SPF baboon (Papio hamadryas anubis) colony.

Baboons (Papio hamadryas anubis) of a conventional breeding colony were nursery-reared to create a specific pathogen-free (SPF) baboon-breeding program. Because the founding generations were nursery-reared until 2 years of age, it was suspected that the SPF baboons would exhibit increased reproductive challenges as adults. Mothering behavior was of interest, because SPF females were not exposed to parental role models during the nursery-rearing process. We compared reproductive data from the SPF baboon breeding program during its first 10 years with data from age-matched baboons during the same period from an established, genetically-similar conventional breeding colony. We also evaluated records documenting mother-infant behaviors within the SPF colony. The average age of menarche in SPF females was 3.3 years. The overall live birth rate of both SPF and conventional females was approximately 90%, with no difference in pregnancy outcome between the two colonies. The average age at first conception for SPF females was earlier (4.2 years) than that of the conventional females (4.7 years). In both colonies, primiparous females were more likely to abort than multiparous females. Similarly, primiparous females were more likely to lose their infants to death or human intervention. A mothering score system was developed in the SPF colony to facilitate intervention of poor mother-infant relationships. Records revealed 70% of SPF mothers were able to raise one or more of their infants successfully to at least 180 days of age, which did not differ from conventional mothers. SPF females returned to post-partum amenorrhea 27 days sooner on average than the conventional females, independent of dam age. The nursery-rearing process used for recruitment into the SPF colony therefore did not have an adverse effect on reproduction or rearing offspring.

Behavioral analysis in laboratory rats: Challenges and usefulness of 50-kHz ultrasonic vocalizations.

Many rodent species emit and detect vocalizations in the ultrasonic range. Rats use three classes of ultrasonic vocalizations depending on developmental stage, experience and the behavioral situation. Calls from one class emitted by juvenile and adult rats, the so-called 50-kHz calls, are typical for appetitive and social situations. This review provides a brief historical account on the introduction of 50-kHz calls in behavioral research followed by a survey of their scientific applications focusing on the last five years, where 50-kHz publications reached a climax. Then, specific methodological challenges will be addressed, like how to measure and report 50-kHz USV, the problem of assignment of acoustic signals to a specific sender in a social situation, and individual variability in call propensity. Finally, the intricacy of interpreting 50-kHz results will be discussed focusing on the most prevalent ones, namely as communicative signals and/or readouts of the sender's emotional status.

Socialization strategies and disease transmission in captive colonies of nonhuman primates.

In captive research environments for nonhuman primates (NHP), social housing strategies are often in conflict with protocols designed to minimize disease transmission. This is particularly true in breeding colonies, and is especially relevant when attempting to eliminate specific pathogens from a population of primates. Numerous strategies have been used to establish such specific pathogen free (SPF) breeding colonies (primarily of macaques), ranging from nursery rearing of neonates to single housing of socially reared yearlings to the rearing of infants in large social groups. All these strategies attempt to balance the effects of the chosen socialization strategy on parameters related to disease transmission, including the ultimate elimination of the target pathogens. Such strategies may affect the overall disease states of NHP breeding colonies through selective breeding processes. This can occur either by creating subpopulations of animals that do not have target diseases (SPF colonies), but may have other issues; or by creating situations in which the "best" animals are sold and the breeding colony is stocked with animals that may be more disease susceptible than those that were sold. The disease states of NHP research colonies also may be affected by selective utilization programs, in which animals removed from the breeding colony for health/behavior reasons, are preferentially chosen for use in scientific investigations. Such utilization criteria raise the question of whether ideal subjects are being chosen for use in research. Finally, captive primate colonies, where both socialization and disease states are intensely managed, may provide opportunities for those testing predictions from models of the interactions of socialization and disease transmission in the evolution of wild populations of NHP. This would be especially true for some extreme conditions of these disease ecology models, given the exceedingly high social densities and levels of pathogen control that exist in many captive nonhuman primate colonies.

Parallels in sources of trauma, pain, distress, and suffering in humans and nonhuman animals.

It is widely accepted that animals often experience pain and distress as a result of their use in scientific experimentation. However, unlike human suffering, the wide range of acute, recurrent, and chronic stressors and trauma on animals is rarely evaluated. In order to better understand the cumulative effects of captivity and laboratory research conditions on animals, we explore parallels between human experiences of pain and psychological distress and those of animals based on shared brain structures and physiological mechanisms. We review anatomical, physiological, and behavioral similarities between humans and other animals regarding the potential for suffering. In addition, we examine associations between research conditions and indicators of pain and distress. We include 4 case studies of common animal research protocols in order to illustrate incidental and experimental factors that can lead to animal suffering. Finally, we identify parallels between established traumatic conditions for humans and existing laboratory conditions for animals.

Caring for nonhuman primates in biomedical research facilities: scientific, moral and emotional considerations.

Animal care for nonhuman primates (NHPs) in biomedical facilities has undergone major changes in the past few decades. Today, most primate facilities have dedicated and highly trained animal care technicians who go to great efforts to ensure the physiological and psychological well being of the primates in their charge. These caretakers work closely with the animals and, as a result, often develop strong relationships with them. Once discouraged and considered a potential threat to scientific objectivity, such positive relationships are now seen as important components to animal care. Positive interactions between caretakers and primates can benefit the primates by reducing their stress and improving their overall well being which can, in turn, help the scientific endeavor. Further, providing the best possible care is our moral responsibility. However, there can also be emotional costs associated with caring for NHPs in research facilities, particularly when animals become ill or have to be euthanized. Facilities can do much to help ease this conflict. High-quality and conscientious animal care is good for the animals, science, and public perception of research facilities.

Perceptions of laboratory animal facility managers regarding institutional transparency.

Institutions that conduct animal research are often obliged to release some information under various legal or regulatory frameworks. However, within an institution, perspectives on sharing information with the broader public are not well documented. Inside animal facilities, managers exist at the interface between the people who conduct animal research and those charged with providing care for those animals. Their perception of transparency may influence their interpretation of the institutional culture of transparency and may also influence others who use these facilities. The objective of our study was to describe perceptions of transparency among animal research facility managers (all working within the same ethical oversight program), and how these perceptions influenced their experiences. Semi-structured, open-ended interviews were used to describe perceptions and experiences of 12 facility managers relating to animal research transparency. Four themes emerged from the participant interviews: 1) communication strategies, 2) impact on participant, 3) expectations of transparency, and 4) institutional policies. Similarities and differences regarding perceptions of transparency existed among participants, with notable differences between participants working at university versus hospital campuses. These results illustrate differences in perceptions of transparency within one institutional animal care and use program. We conclude that institutions, regulators and the public should not assume a uniform interpretation of a culture of transparency among managers, and that sustained communication efforts are required to support managers and to allow them to develop shared perspectives.

The acoustic startle reflex as a tool for assessment of odor environment effects on affective states in laboratory mice.

Apart from self and conspecific odors, odors from other species also influence the affective states in laboratory mice (Mus musculus musculus) in their home cages and during experimental procedures, possibly inducing confusion and inconsistency in experimental data. Thus, it is important to detect the types of animal odors associated with housing, husbandry, and laboratory practice that can arouse different types of affective changes in mice. Here, we aimed to test the effectiveness of the acoustic startle reflex (ASR) in detecting changes in the affective states of laboratory mice due to animal-derived-odor as it has a non-zero baseline, and can be enhanced or attenuated by positive or negative affective shifts, respectively. We used ASR to examine the affective changes in mice that were induced by bedding odors and an alarm pheromone. The odor of bedding obtained from the mice' home cages significantly attenuated the ASR, suggesting positive affective shifts in the test mice, whereas that from bedding obtained from rat cages significantly enhanced the ASR, suggesting negative affective shifts. No significant changes in ASR were observed in mice presented with the odor of bedding obtained from cages of unfamiliar conspecifics. In contrast, there was significant ASR enhancement in mice exposed to volatile components of alarm pheromones trapped in water, suggesting negative affective shifts. Thus, our findings show that ASR may be a valuable tool in assessing the effects of odors on the affective states in laboratory mice.

Laboratory animal welfare: cage enrichment and mouse behaviour.

Mice housed in standard cages show impaired brain development, abnormal repetitive behaviours (stereotypies) and an anxious behavioural profile, all of which can be lessened by making the cage environment more stimulating. But concerns have been raised that enriched housing might disrupt standardization and so affect the precision and reproducibility of behavioural-test results (for example, see ref. 4). Here we show that environmental enrichment increases neither individual variability in behavioural tests nor the risk of obtaining conflicting data in replicate studies. Our findings indicate that the housing conditions of laboratory mice can be markedly improved without affecting the standardization of results.