Open access peer-reviewed chapter
Abstract
The search for the quality of life of animals in captivity has been carried out in laboratories and animal facility in Brazil and around the world. Most of them today use different practices daily, highlighting the environmental enrichment that promotes animal welfare. This practice is of great importance and can happen in a physical, sensory, cognitive, social, and food way, adapted for mice for more reliable results in research, since the animals start to behave in a more similar way to the natural way. The use of animal models such as mice is of great value to scientific knowledge that is continually expanding, proving the importance of using environmental enrichment that tends to promote a more pleasant environment for laboratory animals in various institutions across the country.
Keywords
- mouse
- environmental enrichment
- animal welfare
- animal facility
- refinement
1. Introduction
The relationship between humans and animals and the maintenance of animals in captivity have existed for a long time, but it was only in the last three decades that the concern for animal welfare and the possibility for animals to express their natural behaviors were recognized as a science [1].
Among the animals used in animal facility, mice are the most common in scientific experiments and research, as approximately 99% of human genes have been mapped in them, creating a relationship between humans and animals for laboratory experiments. Associated with this, they have several characteristics that make them an important ally in health sciences, including the fact that they are small, docile, and have many physiological similarities when compared to other animal models, such as amphibians, insects, birds, and other mammals [2].
Currently, the most used animal models in scientific research are mice. Mice are induced to show expected and desired behavioral patterns during an experiment, but the literature sometimes describes several undesirable behavioral patterns, such as anxiety, stress, depression, aggression, eating disorders, and compromised immunity [3, 4].
Since mice raised in captivity spend most of their lives in cages, it is extremely important that environmental enrichment is implemented to meet the physiological and ethological needs of these animals. This allows them to be subjected to potentially stressful procedures without any alteration in the experimental results.
The legislation regarding animal welfare in Brazil requires that, when used in experiments, a minimum of discomfort is preserved, thus generating positive benefits for both the animal’s welfare and the research results. According to the European legislation, the environmental enrichment of animal housing compartments must be adapted to the needs of the individual and the species to which it belongs. The enrichment strategies of the facilities must be reviewed and updated regularly [5].
This work aims to present the possibilities for improvement in the pursuit of the welfare of mice that are outside their natural habitat and housed in friendly experimental facilities. This is achieved through the implementation of environmental enrichment, aiming for the quality of life and the maintenance of the natural behavior of these animals in captivity.
2. Theoretical framework
2.1 Environmental enrichment
The maintenance of animals in captivity is an activity that has existed for thousands of years, accompanying the history of the relationship between humans and animals. According to Andrade et al. [6], before Christ, philosophers like Aristotle and Hippocrates, at the beginning of animal experimentation, observed the functioning of animal and human organs and noted similarities and differences, contributing to the start of the science of animal experimentation. Since scientists were prohibited from performing autopsies on human cadavers, studies were conducted on animals that provided a closer approximation of human physiological functions [6].
In 1925, Yerkes recognized the importance of animal welfare through environmental enrichment, along with Hedge, in the years 1950–1969, by describing the importance of the animal having the opportunity to express its natural behavior with the implementation of objects added to its environment [7]. It was only from 1970 that the implementation of environmental enrichment began in zoos and animal facilities [8]. And, concomitantly, some ethical debates were about the conduct of experiments on animals. However, this topic had already been questioned between the years 1710 and 1776, when the English physiologist Ferguson warned about the cruel treatment to which animals were subjected in experiments without the major ethical implications. For him, all torture and suffering should be avoided as much as possible [9].
According to Saad et al. [8], science has recognized and mentioned animal welfare and the importance of environmental enrichment because animals are beings capable of having feelings such as joy, pain, and suffering [10]. The perception that experimental animals are sentient beings directly influenced the development of environmental enrichment strategies, aiming not only to minimize pain and suffering but also to promote the physical and emotional well-being of animals used for scientific purposes [2, 11].
Henriques and Sampaio mention the publication of “The Principle of Humane Experimental Technique” by two English researchers, Russell and Burch [12], where the concept of the 3Rs was introduced in 1959, with the goal of changing the view of the time and leading to the reflection that the use of animals in experiments could be minimized. Thus, the 3Rs term referred to considerations about:
Replacement: The substitution of one species for another lower on the zoological scale or for microorganisms, or, if possible, for non-biological material. This should be understood as a substitute for procedures involving the use of in vitro methods, physicochemical techniques, computational or mathematical modeling, or the use of organisms that are known to have limited consciousness. Human studies include epidemiology, post-marketing surveillance, and the ethically approved use of human volunteers, as well as any other method developed based on or using molecular or cellular mechanisms of the phenomenon to be studied.
Reduction: The reduction in the number of animals used. Studies involving animals must ensure that the results are statistically valid and obtained with the smallest possible number of individuals. Researchers should, whenever possible, seek guidance from the biostatistician at their institution in project preparation, so they know in advance how the data will be analyzed. Another point that should be considered is the sample size. A sample that is too small will not allow the studied effect to be detected with any degree of reliability. Meanwhile, exposure that is too large leads to the unnecessary use of animals. The sample size should be determined using a formal statistical method.
Refinement: The refinement of techniques to minimize the level of stress and pain caused to the animal during experimentation. The use of analgesia and anesthesia for pain relief and the defined criteria for the application of a humane endpoint should be carefully carried out, evaluated, and described during the investigation. All scientific teaching and research activities must include the environmental enrichment program to be implemented [2, 12, 13, 14].
According to Martins et al. [15], in ethical terms, animal welfare and environmental enrichment have become fundamental for laboratory animals, especially related to the issue of refinement.
For Broom and Molento [10], welfare is defined by a set of responses and concepts that encompass the psychological and behavioral state expressed by animals, such as fear, stress, and health, among other reactions. Thus, well-being is related to the animal’s various attempts to adapt to the environment it is subjected to.
According to Refs. [6, 16], the evaluation of animal welfare is carried out through two parameters: behavioral and physical, with the behavioral one being the most important in captive animals. Thus, it is observed that animal welfare is closely linked to a correct response to the experimental results.
Environmental enrichment helps with the physical and psychological issues of the animals, allowing them to express their natural behavior. It also contributes to motor stimulation and social interactions, always aiming to improve the biological functions of animals living in captivity [17]. Enrichment can be considered not only as an external means that promotes stimuli but also as a source capable of generating patterns of brain function, since each animal will have different experiences in its enriched environments. There are various techniques used in environmental enrichment to achieve greater stimulation of these animals, such as the insertion of stimuli like those that exist in nature to avoid possible stereotyped and stress behaviors [18].
Elidio et al.’s [19] research demonstrated that with environmental enrichment, mice showed less stress during handling, reduced aggression, better health conditions, less loss of offspring, a higher mating rate, and lower cortisol circulation. According to Lambert et al. [20], with the implementation of environmental enrichment, brain changes correlated with memory and learning were observed, thus showing greater cognitive potential [6]. In older mice, an increase in presynaptic protein levels in the hippocampus was found [20], since brain plasticity is responsive at any age, thus reducing the risks of neurodegenerative changes [18].
Environmental enrichment can enhance certain actions in the immune system, such as increasing the activity of macrophages and the number of innate cells, as described by Souza [21].
However, the authors highlight the importance of considering that biological systems will not always act in the same way. Modifications both in the individual’s own organism and in the environment where they live corroborate that certain results are passive to oscillations. Another point raised is that the absence of structures that stimulate the animals’ natural behavior decreases well-being and causes stress, reducing the credibility of the study, since stressed animals do not produce reliable results and contradict the principle of the 3Rs [12, 22].
Mieske et al. [11] demonstrated that a stimulating environment can be considered essential for the development of natural behavior and animal welfare of research rodents. Although boredom has never been studied directly and is rarely mentioned, it clearly reflects many studies on the effects of improving living conditions. Chronic boredom, a consequence of living in a barren and confined environment, can pose a health risk to laboratory animals, limiting their validity as model organisms for biomedical research. A stimulating living environment promotes the well-being of laboratory workers, regardless of age and sex.
Among all the advantages generated for studies, some authors question the implementation of environmental enrichment due to its influence on the standardization of research. According to Newberry [17], if there is no correct understanding of species behavior, an inconsistent and inadequate interpretation of the experiment’s results will occur. Likewise, the implementation of environmental enrichment will not be applicable when the animal shows behavioral changes due to stress and especially aggression in males, to avoid possible interferences in the result of the experiment.
2.2 Environmental enrichment items
Acrylic igloos (Figure 1) are also considered a form of environmental enrichment. In experiments, some of their advantages have been reported, including increased locomotion in non-acrylic-housed animals, a reduction in feeding behavior, and an increase in natural self-grooming behavior. However, aggressive behavior with territorial marking was observed in males [22]. The use of a transparent acrylic material with multiple, red-colored entries facilitates the visualization of animal corpses, as the animals themselves are enclosed in black [23]. It is important to highlight that mice perceive red as a shade of black, which can contribute to a greater sense of security, reducing stress and improving the comfort and well-being of the animals inside the red igloo [24].
Figure 1.
Acrylic igloo environmental enrichment item for mice. Animal Facility of the Carlos Chagas Pavilion at the Oswaldo Cruz Institute’s Center for Animal Experimentation - Fiocruz.
The running wheel was implemented to stimulate physical activity in mice. However, its use is controversial because it can trigger obsessive behavior, leading animals to exercise more than they would in their natural habitat [2]. According to Souza [21], 4 weeks of exercise with a running wheel improved neuroplasticity, cognitive functions, and neurotrophin expression in the organs studied.
Dallagno [25] reported that adult mussels increased their intake of pelleted food without gaining body mass, whereas older mussels showed no such increase or body mass gain. According to National Council for the Control of Animal Experimentation (CONCEA) [2], while a running wheel stimulates physical activity, it can also lead to undesirable obsessive behaviors, causing mice to exercise beyond what is natural for them.
Studies have shown that the sensory, motor, and cognitive stimulation from exercise produces positive results by inducing neuroplasticity mechanisms, as evidenced by increased expression of neurotrophic factors and neurotransmitter receptors in various organs. The neurochemical and neuroanatomical results observed in animals with similar exercise-like enrichments have been associated with changes in anxiety-like and activity-related social behaviors. Recent work demonstrated that physical exercise provided cognitive and emotional regulation benefits and improved contextual fear conditioning in Gfap-hsv-tk transgenic mice [26].
The chew ball is a common item for domestic pets (dogs and cats). The interaction it provides is expected to be stimulated by its shape and the sound it makes when moved. It has shown satisfactory interaction results with younger animal groups. This structure is highly resistant and durable, but it has the disadvantage of a high cost and difficulty of sterilization [15]. Adult murine mice showed less interest in using the chew ball when first introduced to it and after a five-day interaction period [27].
Polyvinyl chloride (PVC) tubes are durable and help protect insects during the day [2]. According to Santos and Fontes [28], the introduction of PVC tubes in some experiments led to an increase in the number of litters in cages, indicating the improved aspects of animal reproduction. Oliveira [3] reported that PVC tubes were used to reduce episodes of aggression and fighting among groups of animals in a cage, serving a protective role (Figure 2).
Figure 2.
Enrichment item for Mice: PVC Tube. From the Animal Facility, Carlos Chagas Pavilion, Center for Animal Experimentation, Oswaldo Cruz Institute – Fiocruz.
Absorbent paper (Figure 3) immediately piques the interest of mice, stimulating nesting behaviors as well as individual and social interactions. Although it is inexpensive and easy to introduce, its use in cages must be time-limited because it quickly loses its structural integrity as an environmental enrichment item [15].
Figure 3.
Environmental enrichment item: absorbent paper for mice. From the Animal Facility, Carlos Chagas Pavilion, Center for Animal Experimentation, Oswaldo Cruz Institute – Fiocruz.
According to National Council for the Control of Animal Experimentation (CONCEA) [2], adding nesting boxes made from absorbent paper is highly valuable for stimulating the typical nesting behaviors of birds. To maintain its effectiveness, this item should be replaced as soon as it becomes damp with urine or drinking water. However, if the paper towels or lids remain dry and their structure is intact, they can stay in the cages for up to 15 days [27].
Cotton flakes (Figure 4) are associated with reproduction, specifically with nest building. They represent a low-cost physical enrichment material, easy to handle, and can be replaced during cage cleaning or, alternatively, sterilized by autoclaving. Furthermore, they do not cause harm to newborns, have been shown to reduce pre-weaning mortality, and contribute to increased pup body weight [28]. In addition to their benefits for offspring, cotton flakes may also be used as nesting material. According to Rosa et al. [29], cotton was the preferred nesting material among LG/J females, possibly due to its superior nest-building capacity. However, as a shelter/protective object, it was not the preferred option when compared with paper rolls, across females at different life stages (Pubertal, Virgin Adult, and Adult).
Figure 4.
Environmental enrichment item: cotton flakes. Animal Facility, Carlos Chagas Pavilion, Center for Animal Experimentation, Oswaldo Cruz Institute – Fiocruz.
The sounds of the tropical forest are characterized as auditory enrichment and may promote increased physical activity, enhanced brain functions, and lifespan extension. These effects were observed both with lower- and higher-range sound exposure, in comparison with the control group. Studies in rodents have indicated that music can increase physiological stress [30, 31]. However, other studies have demonstrated that music induces positive behavior [32] and physiological benefits in rodents [32, 33, 34]. It has been suggested that constant background noise throughout the day may provide certain benefits in the housing of these animals, by decreasing excitability and reducing startle responses to sudden noises [35].
To assess the effects of classical music on the general motor activity of unrestrained mice, Cruz et al. [36] employed the open field test. In this assay, no statistical differences were observed in the rhythm of spontaneous movements; however, a reduction in inactivity time was recorded in groups exposed to classical music, indicating an increase in motor activity. Moreover, mice exposed to Mozart’s music displayed enhanced exploratory activity, suggesting its potential role in environmental enrichment for captive rodents.
Additional enrichment artifacts, beyond those already mentioned, include paper tubes, shredded paper, wire-free disposable masks, wooden balls, golf balls, bats, and blocks of softwood [2]. Any item used for environmental enrichment should be time-limited to preserve novelty and stimulation, while also preventing the development of stress signals [1].
The type of bedding/flooring used constitutes a fundamental component of the rodent environment, with significant implications for animal health, and should therefore be considered when selecting environmental enrichment strategies. The quality of bedding/flooring material is closely related to its moisture retention properties, as well as to microbial, traumatic, and aeration effects. Wood shavings have recently been preferred as laboratory animal bedding due to their low cost. However, alternative bedding materials that are dust-free, reduce allergen spread, and minimize ammonia production within cages are available on the market. Despite these advantages, such materials are not always financially viable and/or considered comfortable for mice, and they generally do not impact ammonia generation, differing mainly in urine absorption capacity. In addition to the material itself, the volume of bedding provided is an important factor, since rodents use it in nest building alongside other enrichment items [37, 38].
2.2.1 Sensory indicator
Sensory enrichment encompasses the stimulation of all five senses in animals. These stimuli include visual, auditory, olfactory, tactile, and gustatory inputs (Figure 5A and B) [28]. In some experiments, music was introduced as an enrichment item aimed at reducing group stress [32, 33, 34]. Naturally, animals emit sounds related to courtship, aggression, defense, and maternal care, which can be influenced by ambient noises [28]. Furthermore, environmental sounds may alter physiological and behavioral parameters as well as circadian rhythms in animals [39].
Figure 5.
Sensory environmental enrichment item for mice: hay. Animal Facility, Carlos Chagas Pavilion, Center for Animal Experimentation, Oswaldo Cruz Institute – Fiocruz.
2.2.2 Nutritional/feeding indicator
Food enrichment (Figure 6) represents the most immediate way of promoting animal welfare. Providing mice with a varied diet stimulates their natural foraging behavior [17]. However, the standardization of feeding schedules through environmental enrichment may trigger a physiological stress response in animals when alterations occur at any given time [40]. CONCEA [2] emphasized that the diversity of food offerings may interfere with experimental outcomes due to the lack of standardization and the impracticality of feeding management throughout the study. A seed mixture can be scattered on the cage floor; however, mice tend to consume mainly sunflower seeds while disregarding others. Quality control of these seeds is essential, as there is a potential risk of chemical or biological contamination. This practice may be contraindicated for animals used in nutritional or toxicological studies [2].
Figure 6.
Environmental enrichment item: seed mixture for feeding. Center for Animal Experimentation, Oswaldo Cruz Institute – Fiocruz.
2.2.3 Social indicator
Mice are highly social species and exhibit improved welfare when housed in pairs or groups. They display more natural behaviors when paired or grouped from the time of weaning, with groups preferably established prior to puberty to prevent stereotypic behaviors and to allow the development of their own social and territorial organization. Positive social stimulation functions as environmental enrichment, enabling mice to express natural behaviors such as grooming and jumping with specifics, thereby expressing emotions and rapidly reducing potential signs of stress [40]. Particular attention must be given to groups composed of male mice. The older the animals, the greater the likelihood of aggressive behaviors. As a rule, same-sex groups should be established before puberty. Territorial and social organization also vary among different mouse strains. Adult Swiss males exhibit greater intolerance in territory establishment and display higher levels of aggression compared to other strains. The addition or removal of an individual may compromise the welfare of the entire group [2].
2.2.4 Human-Animal relationship
According to National Council for the Control of Animal Experimentation (CONCEA) [2], animal handling can be characterized as a form of environmental enrichment (Figure 7). This requires clearly described procedures, accessible to all staff involved in animal care, to ensure the positive interactions with animals during cage changes and experimental manipulations, thereby habituating them to routine procedures and consequently maintaining high levels of animal welfare. Refined handling practices that prioritize habituation and socialization of laboratory animals with animal care personnel have a positive impact on welfare and, consequently, on scientific quality [19]. Vilela et al. [41] also reported that animals accustomed to contact with caretakers during physical restraint in the cage and intraperitoneal inoculation exhibited significantly reduced intraspecific aggression (confrontations, attacks, and bites) following these procedures, thereby ensuring animal welfare and ultimately improving the quality of research outcomes.
Figure 7.
Environmental enrichment item: human–animal interaction with mice during cage changes. Animal Facility, Carlos Chagas Pavilion, Center for Animal Experimentation, Oswaldo Cruz Institute – Fiocruz.
3. Conclusions
The present study demonstrated that the outcomes of implementing environmental enrichment and promoting animal welfare should be analyzed in a comprehensive and continuous manner, considering both the short- and long-term benefits for animals maintained outside their natural habitat.
The introduction of environmental enrichment should be maintained consistently to provide the ongoing improvements in mouse behavior, thereby reducing negative interventions in response to experimental procedures.
The use of mice according to the 3Rs directly facilitated responses to surveys and indirectly contributed to the welfare of these animals, allowing them to express their natural behaviors without stress and free from abnormal or stereotypic behaviors in cages, enabling interaction with the environmental enrichment items.
Implementing animal welfare through environmental enrichment makes the environment more pleasant and closer to the animals’ natural habitat, allowing mice to express their innate behaviors. This, in turn, improves their mental and immune states and consequently enhances research outcomes, thereby preventing the unnecessary use and disposal of mice during research projects.
Acknowledgments
We would like to dedicate this work to the memory of Roseli Lopes Simões, a dear colleague and friend, whose dedication, enthusiasm, and companionship left a profound mark on everyone who had the privilege of working alongside her. Her passion for work, generosity, and contagious joy remain an inspiration to us all. She will be deeply missed, but her memory and legacy will continue to live on in every step of our professional and personal journey.
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