Assessing the exploratory and anxiety-related behaviors of mice. Do different caging systems affect the outcome of behavioral tests?
Introduction
The caging environment of laboratory rodents is important not only for their health and welfare, but also for the reliability and reproducibility of experimental results. Housing systems have technologically evolved over the last decade, and questions have arisen concerning their effect(s) on laboratory animals' welfare. Various housing systems have been developed to standardize the factors that may influence the health, welfare, and behavior of laboratory animals [1], [2], [3], [4], [5], [6], [7], [8]. The standard housing systems that are widely used for laboratory mice include Open Top Cages (OTC), Individually Ventilated Cages (IVC), and Motor Free Ventilated Cages (MFVC).
The sides and bottoms of OTC are solid and are usually manufactured from transparent plastic material. The cage top (cover) is constructed of thin metal bars, which allows the free exchange of air. For this reason, the intracage or microenvironment of these cages, namely the temperature, the relative humidity, the concentrations of carbon dioxide and ammonia, is more directly influenced by the animal room's environment (macroenvironment), the frequency of bedding changes, and the housing density. OTC housing also allows cage-to-cage and room-to-cage transmission of airborne pathogens and does not protect staff from exposure to animal-related aeroallergens [9], [10], [11], [12]. The food hopper and water bottle are located in the front upper part of these cages.
When used for housing laboratory mice, IVCs have been shown to provide a significant barrier to the spread of infectious agents between cages [13], [14]. IVCs protect staff from animal-related aeroallergens and zoonotic agents and animals from pathogens, can be used to quarantine animals of unknown health status, such as genetically modified rodents which are exchanged between laboratories [15], [16], improve the air quality in the cage due to high-efficiency particulate filtration of the incoming air (HEPA), decrease the accumulation of microbiological contaminants and chemical pollutants [14], and reduce the frequency of cage and bedding changes [17]. Air is introduced through an inlet located either on the lid or at the rear wall of the body of the cage and exits through an outlet located on top of the cage. Flexible silicone hoses connect the cage rack to the air handling unit, and air is circulated by supply and exhaust fans. Negative or positive intracage pressure can be defined and ventilation rates may vary from 25 to 120 air changes per hour (ACH) [5], [15], [16], [18]. The food hopper and water bottle are located in the front upper part of these cages. Notwithstanding these advantages, IVCs do have some disadvantages including their high purchase cost, technical maintenance, and complexity [19]. High intra-cage ventilation rate also produce noise and may induce stress and heat loss to the cage residents [5].
In MFVC housing units, air enters the cage through an inlet in the cage's front wall and exits through an outlet in the cage's rear wall. The air outlet of the cage rack is connected directly to the exhaust of the room's heating, ventilation, and air conditioning (HVAC) unit. The advantage of MFVC is protection of the staff against zoonotic agents and animal-related aeroallergens as well as animals against pathogens. Similar to IVCs, MFVCs decrease the accumulation of microbiological contaminants and chemical pollutants [14], with the added bonus of a lower purchase cost. On the other hand, MFVC operate only under negative pressure and their ventilation rates, which are approximately 20–25 ACH, can't be modified [20]. The food hopper and water bottle are located along the right side inside the cage.
Behavioral scientists frequently use laboratory mice to investigate the neural basis of behavior. Although behavioral tests are often conducted outside the home cage and in macroenvironments other than the areas where the animals were reared, housing conditions could play an important role on the behavioral testing outcomes [21], [22], [23]. Since each of the abovementioned caging system creates a different microenvironment, we undertook a study to investigate whether the different housing conditions of OTCs, IVCs and MFVCs influence commonly assessed behaviors of caged mice. For this purpose, we employed the open field and the elevated plus maze tests. The open field is widely used to assess novel environment exploration, general locomotor activity (distance travelled over a defined period of time) and anxiety-related behavior (time spent in the center vs. close to the arena walls (thigmotaxis) is inversely proportional to the anxiety/stress of being in a novel, brightly lit, unprotected environment). The elevated plus maze is the gold standard for measuring anxiety-related behaviors in mice where increased time spent/number of entries in open arms is indicative of reduced anxiety-like behavior. Mice from the three different caging systems were individually examined and groups were compared for possible differences in behavioral performance in the open field and elevated plus maze [24], [25]
Section snippets
Material and methods
The study was performed in the animal facility and behavioral phenotyping laboratory of the Biomedical Research Foundation of the Academy of Athens. The facility is registered as a “breeding” and “user” establishment according to the Greek Presidential Decree 56/2013, which harmonizes Greek national legislation on animal experimentation with the European Community Directive 2010/63 on the Protection of Animals Used for Experimental and Other Scientific Purposes [26].
Open field test
The caging system had no effect on the locomotor activity of mice in the three housing systems, as measured by the total distance travelled in 30 min [F(2, 27) = 0.228, p = 0.798]. The locomotor activity of all mice progressively decreased over time [F(5135) = 31.204, p < 0.001] with no differences observed between the three groups (Fig. 1). Although, IVC-housed (211.61 ± 77.31) and MFVC-housed mice (218.66 ± 58.73) tended to spend more time in the central area of the arena than OTC-housed mice (167.62 ±
Discussion
Nowadays, a repertoire of different caging systems for mice is available. The most common standard housing systems used in the majority of animal facilities are OTC, IVC, and MFVC systems. The main differences between these caging systems are the ventilation rate, the method and rate of air exchange, the shape of the individual cages, and the cage location of the food and water hoppers.
Although, most behavioral studies focus on the influence different conditions may have on the physiology and
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