The principles of the 3Rs (Replacement, Reduction, and Refinement) were developed over 50 years ago, providing a framework for performing more humane animal research. Since then, they have been embedded in national and international legislation, regulations on the use of animals in scientific procedures, as well as in the policies of organizations that fund or conduct animal research.
The principle of refinement applies to all aspects of animal use, from how animals are housed and produced to the scientific procedures performed on them1. Examples of refinement in a laboratory setting include providing animals with housing that allows the expression of species-specific behaviors. Evidence suggests that single-housing configurations can alter an animal’s behavior, physiology, and immunology. Such changes can lead to variation in experimental results that impairs both the reliability and repeatability of studies. Another example of refinement is implementing and training staff on procedures to minimize any distress.
Social Housing Configurations, Specifically for Nonhuman Primates
Given that most nonhuman primates (NHPs) are adapted for sociality, social context is important for improving repeatability, reproducibility, and external validity of NHPs in biomedical research. Socialization is widely regarded as the best form of enrichment for NHPs in a laboratory setting, and social group housing is the gold standard because it replicates, as closely as possible, their normal social environment. This results in the healthiest and most normal NHPs, both behaviorally and biologically, and thus optimal welfare and robust study results.
Because regulatory agencies around the world generally require developmental and reproductive toxicity (DART) testing of all new drugs to be used by women of childbearing age or men of reproductive potential, Altasciences examined ways to exceed current housing standards for these complex studies – with the ultimate goal of reducing the number of animals used on studies like these in the future. NHPs are most frequently used for DART testing when rodents and/or rabbits are not a pharmacologically relevant species. For example, NHPs (particularly cynomolgus monkeys) are commonly used for DART studies for large molecule biotherapeutics. Due to a high and variable spontaneous abortion rate, developmental toxicity studies (such as pre- and post-natal development studies (PPND)) require large group sizes to differentiate the inherent variability in pregnancy outcome from test compound effects. In these complex and lengthy studies, incidences of embryo-fetal loss (EFL), still birth (SB), gestation length (GL), and clinical pathology parameters are important endpoints to consider when planning the number of animals needed for enrollment. In the following case study, Altasciences evaluated the potential benefits of group housing female cynomolgus monkeys to determine if this housing configuration would decrease the variability in pregnancy outcome as described above.
- Implementing group housing for socialization of laboratory macaques on reproductive toxicology studies is challenging for several reasons:
- Macaques manage their social systems with more frequent and severe aggression ranging from mild status interactions (including social signaling and minimal contact) to severe bites causing traumas requiring medical treatment, and in extreme cases, death
- Ideal housing configurations may not be feasible given the number or age/sex class of research subjects
- Constraints related to study design, available housing, or clinical condition
- The implementation of social housing requires close coordination between research personnel and individuals who are responsible for behavioral and clinical management of the primates
- The effects of group housing on pregnant females are not well documented and a social housing configuration could potentially impact the inherent variability in pregnancy outcome already seen in this model
- Very little is known about how dams with newborns will behave in social housing configurations and if an increase in aggression will be observed
In a study conducted at Altasciences, 36 pregnant cynomolgus monkeys were socially housed (three per unit) in caging systems that comply with the Animal Welfare Act and recommendations set forth in “The Guide for the Care and Use of Laboratory Animals” (National Research Council). Pregnancy was monitored by ultrasound (embryo-fetal viability check) every 7 to 14 days throughout gestation, and dams were allowed to deliver naturally in their respective social housing units. Study-specific training guides were established for the social housing configuration utilized on this study; behavior and enrichment specialists were continuously involved in the development of procedures and husbandry configurations. Subjects were consistently monitored for aggression, and behavioral assessments were continuously performed throughout the study. Handling of animals was refined to the use of specially designed NHP procedure cages that are used for training and performing procedures, ease of animal handling, and minimizing stress. Historical data from previous PPND studies where females were single-housed was used to compare any changes in aggression, animal behavior, EFL, SB, infant survival ratio, and clinical pathology parameters.
The results of this study were compared to historical data from over 40 developmental and reproductive studies in single-housed cynomolgus monkeys. An increase of aggression amongst group-housed females was not observed throughout the study, nor was there evidence of stereotypical behaviors exhibited by the females before or after birthing in this environment. No changes were observed in clinical pathology parameters collected during the gestation and lactation periods (from maternal and infants) of group-housed females when compared to standard, baseline values for this species. The incidence of EFL decreased by over 50% in group-housed females when compared to historical values of single-housed females (17.1% vs 8.3%) and a similar % decrease in the rate of SB was also observed in group-housed females when compared to single-housed females (14.7% vs 6.1%). Mean gestation length of group-housed females was not affected, whereas infant survival rate at Day 7 significantly improved from 68.6% to 83.3%, when females were group housed.
Studies such as the one described above performed at Altasciences demonstrate that social housing is possible in research settings that were previously believed to require non-social housing. It used to be that NHPs on DART studies were not socially housed because there was a fear that social interaction could lead to injury or even death of newborns, or that stress levels could be elevated given the presence of infants in the same housing with other dams. However, the results of our study strongly suggest that housing female NHPs in a group housing configuration for DART studies provides significant and improved outcomes compared to housing animals individually. The refinement of utilizing low stress training and handling techniques on this study further led to exceptionally low (or in many cases no) levels of aggression and stereotypical behaviors generally seen in NHPs when group housed. By refining our methods, techniques, enrichment, and housing configurations, it is very likely that a reduction of animals on future studies can be achieved given the improved outcome of key parameters, such as EFL, SB, and survival rates (i.e., less animals will need to be enrolled at the start of the study). The evidence presented suggests that changes to social housing matter for the wellbeing of macaques and for their physiology, both of which can impact research outcomes.
At Altasciences, we are committed to the highest standards in animal welfare and the 3Rs. We ensure that studies are performed to the most up-to-date scientific knowledge and incorporate new technologies when relevant, while minimizing the use of animals and/or optimizing the procedures that are required.
1National Centre for the Replacement Refinement & Reduction of Animals in Research. Retrieved from https://www.nc3rs.org.uk/refined-animal-model-multiple-sclerosis.
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