Researchers should always follow institutional guidelines for example, the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition) or ARRIVE [2] or European Communities Council Directive (86/609/EEC) [3], in conducting experiments with animals, and seek approval from local Institutional Animal Care and Use Committee, for example [4], especially in cases where care and handling of animals deviate from standard practices. Mice in general are maintained on 12 h light:dark cycles [5, 6], for example, lights on from 6 am to 6pm [7] or 7 am to 7 pm [8] or occasionally 14 light–10 dark cycle [9].

The laboratory mouse has unique thermoregulatory characteristics, and it is marked by differences in metabolic rate, thermal conductance, variable core temperature, and preference for warm temperatures. These factors set mice apart from humans in the extrapolation of biomedical data. Thus, the thermal physiology of mice presents unique challenges to the biomedical researcher because changes in temperature can affect the interpretation and translation of data to humans.

Temperature regulation specifically affects autonomic and behavioral effectors, including pharmacokinetics and metabolism of drugs and other chemicals. When considering the temperature-dependence of all biochemical reactions, any significant difference in body temperature is anticipated to impact drug and chemical efficacy and metabolism directly.

In general, the housing temperature for laboratory mice in research facilities is between 20–26 °C [10, 11] or 18–23 °C [12], or more narrowly, 20–22 °C (70 ± 2 °F) [13] or 22.8°C [14] ; while the thermoneutral ambient temperature for mice is 30–31°C. Significant differences between mice housing under 20–26 °C vs. 30–31°C have been observed, in terms of metabolism level [15], thermoregulatory responses to lipopolysaccharide [16], obesity phenotype in uncoupling protein 1 (UCP1)-deficient mice [17], and immunity and tumor growth [18, 19]. For instance, mice at 30°C have mean diurnal energy expenditure rates 1.8 times higher than basal metabolism, similar to the human situation; while mice under 20°C display energy expenditure 3.1 times basal metabolism [15], therefore, mice studied at thermoneutrality (~ 30 °C) are thought to be a better model for human energy homeostasis [20].

In addition to housing temperature, Makowska IJ et al investigated the segregation of nesting and elimination areas of housing cages and proposed to use a system with multiple cages to house mice [21].

The following discusses specific considerations about handling experimental animals. Researchers have also tried to control the stress variation through other means. A Muller et al listed several practices such as conducting no experiments on the day of cage change, injection and measurement habituation [22].

In a 2012 article titled "Playful handling by caretakers reduces fear of humans in the laboratory rat" [23], the authors tested four scenarios regarding rat handling, including (1) minimal handling; (2) daily exposure to a passive hand for 2 min; (3) daily tickling for 2 min; and (4) daily hand restraint for 2 min (similar to pinning by a dominant rat). The authors found that rats responded well to both restraint and passive exposure treatments, and responded best to daily tickling. A standardized tickling protocol has been published [24].

Another study indicates that gently handling of mice reduced their innate defensive responses to visual threats [25] and reduced swimming immobility in the forced swim test - see more details below [1]. Cloutier S et al. evaluated the effect of tickling on responses to repeated intraperitoneal injections in laboratory rats and found tickling could mitigate the aversiveness of the procedure [26].

There are different methods for restraining mice, depending on the purpose of the experiment. The standard protocol to handle laboratory mice is to pick them up from the tail in the middle part of the tail and not from the tip. Through this simple handling specificity, the mice could be transferred to another cage or a balance, and be examined for instance for sex determination, or lifted directly onto the cage wire lid for close examination with less anxiety. Thus far, animal handlers were unaware that the standard procedure of handling mice could affect the results of their experiments, and that picking up the mice from the tail-tip, for example, could generate anxiety and stress in the mice.

In a study by Jane Hurst and Rebecca West from the Institute of Integrative Biology (University of Liverpool, UK), the authors identified two alternative methods to tail handling; the use of tunnels, and open-hand handling. More gentle handlings reduced aversion and high anxiety and led to a voluntary approach, low anxiety, and acceptance of physical restraints. The first method the authors described involves a cage tunnel that is normally present in all cages. This method works well when the handler brings the tunnel towards the animal without direct contact. The second is to "cup" the mouse in hand and allow it to walk over the handler's hand without any restraint. The "cupping" technique may be successfully utilized without fear that the mouse may immediately jump away by first holding the mouse in a closed hand for up to 30 seconds to familiarize the mouse with the handler [27].

Picking up the mice by the tail is routinely used in the laboratory. For this reason, animal handlers oversee the anxious response in mice that could affect the result of the experiments because these responses are often evaluated as normal. However, one study showed that the way of handling mice has profound effects on animal behavior, and it could induce anxiety and stress responses that influence the results of animal experiments considerably. The researchers also concluded that when a strong anxiety response is required for specific experiments, the cage tunnel and "cupped" on open methods are not recommended. Thus, carrying mice in tunnels or "cupping" in hand are important alternative handling methods that may reduce the intra-experimental variability which is of critical importance since it will help reduce experimental variability and reproducibility [28].

The way of handling laboratory mice is of critical importance since it can have negative effects on the mice behavior, and induce fear, anxiety responses [27], and anhedonia - a core symptom of clinical depression which can be measured in rodents by assessing how they consume a sucrose solution [29]. These studies indicate that animal experiments could be altered and that it is important to refine laboratory procedures and practices in order to ensure high standards of animal welfare and scientific data quality.

In a study where the responses of the three handling methods in male and female mice in different strains (BALB/c, C57BL/6 and ICR (CD-1)) were assessed, different anxiety and stress responses were observed. Normally urination and defecation are used as a parameter to measure anxiety or stress in mice. The researchers assessed the responses of the three handling methods using both male and female mice since they can have different responses in anxiety and stress. They evaluated anxiety-related behavior immediately before and after daily handling, and they also assessed the anxiety after the mice become familiar with one of the handling methods. The researchers showed that mice of all strains and sex developed a consistent voluntary interaction in the ninth daily handling session among all the three handling methods – the tail, home cage tunnel, or "cupped" on open hand [28]. Results showed that the tail approach yielded the lowest voluntary interaction with the handler and induced more urination and defecation during the handling process. The responses were comparable in the light and dark phases of the diurnal cycle and were not dependent on the experience of the handler. The researchers showed different advantages in using these new methods. If the handler first picked up the mice and placed them on the hand by the tunnel or cupping method, the anxiety and stress behavior was reduced during tail handling to examine for instance ventral surface and anogenital area of mice (abdominal inspection). They also noticed the same response in mice after being restrained by the scruff of the neck.

[enlarge]

Figure 1. Differences in immobility during FST test in mice handled gently, aggressively, or in the control group. Reproduced from Figure 1 of [1].

In studies that examined the effects of different handling techniques on depressive-like behavior, the benefits of gentle handling were established. In one study that utilized the Porsolt forced swim test (FST) in adult C57BL/6J male mice, the authors established the importance of consistent gentle handling in order to achieve less challenging behavioral testing, better data collection, and overall improved animal welfare [1]. The FST is used to measure depressive behaviors in rodents [30]. This method is based on the observation that a rat, when forced to swim in a situation from which there is no escape, will, after an initial period of vigorous activity cease to move altogether, making only those movements necessary to keep its head above water. This immobile behavior is thought to indicates a state of despair in which the rat has learned that escape is impossible and resigns itself to the experimental conditions. The experiment described in the study involved researchers that handled the mice in a gentle, aggressive, or minimal (control) fashion over approximately two weeks prior to testing. These were defined in the following manner: 1) The gently handled mice were individually removed from their home cages, placed in the palm of the experimenter's hand, and stroked on the left and right flanks and head for 90 seconds. Researchers also permitted head movement between their hands. 2) The aggressively handled mice were individually removed from their home cages, grasped at the proximal end of their tails, and suspended in the air approximately 15 cm from the surface of a biosafety cabinet for 90 seconds. These mice were not allowed to latch onto any surrounding equipment or attempt to grasp the experimenter's fingers. 3) The control mice were handled only by husbandry staff during routine cage changes [1]. When these mice were tested for depressive-like behavior by the FST, the results demonstrated a significant beneficial effect on gentle handling (Figure 1). In Another study, gentle handling reduced the swimming immobility in the FST compared to mice that were aggressively or minimally handled, and suggest that gentle handling can reduce the stress and despair in the animals [27]. In conclusion, studies demonstrate the benefits of gentle handling of mice that undergo depression-based testing and indicate that a gentle handling technique is effective when interacting with mice prior to and during behavioral testing. These observations also point to the need to standardize the handling procedures in order to reduce the depressive symptoms in mice.

Human-based conditioning that was first described by Pavlov and by Gantt, Newton, Royer, and Stephens (1966), may also have important implications for animal research in a variety of settings. In a study that evaluated this factor in laboratory mice, the authors reported that the laboratory rats consistently chose a familiar human over an unfamiliar human following fourteen and five 10-min exposures and even following a single 10-min exposure. Furthermore, this preference was retained in the absence of additional contact for at least five months [31]. These results confirm that laboratory rats can tell individual humans apart, a prerequisite for associating them with hedonic events. Furthermore, there is evidence that familiarity with the animal handlers increases reproducibility in animal tests. In a study that investigated the effect of identity of the experimenter and familiarity to their test animals on the results obtained from a standard test of anxiety, the authors found that having different experimenters perform the same test (i.e. elevated plus maze) using the same equipment and rats from the same breeding colony within the same room of the same laboratory - significantly affects the results if the experimenters were unfamiliar with the animals, but not if they were familiar with the animals [32]. Thus, the familiarity of the test animals with their experimenters is anticipated to increase the reproducibility in the results with animal tests and should be taken into consideration when planning behavioral studies in animals. Such trend is already apparent in behavioral studies where familiarity with the animals is described in the material and methods section: "mice were allowed to habituate to different holding rooms for behavioral experiments for two weeks before testing, and to experimenters for at least three days before experiments [33].

There is evidence supporting the influence of experimenter gender on a variety of psychological and physiological variables [34]. In a policy paper that investigated how the gender of the experimenter affects experimental findings, the authors emphasized the importance of reporting and controlling for experimenter gender in future research since this factor appears to affect the physiological data. For example, in one study, exposure of mice and rats to male but not female experimenters produces pain inhibition [35]. Interestingly, male handlers' stimuli induced a robust physiological stress response that resulted in stress-induced analgesia. Analgesia is the phenomenon of pain suppression upon exposure to unconditioned or conditioned stressful stimuli, also known as stress-induced analgesia [36]. This analgesic effect could be replicated with T-shirts worn by men, bedding material from gonadally intact, and unfamiliar male mammals, and presentation of compounds secreted from the human axilla. Experimenter sex can, therefore, affect apparent baseline responses in behavioral testing.

These studies and others have helped increase the awareness for experimenter gender considerations, and in behavioral studies it is becoming more common to encounter a specific mention about the experimenter gender in the Materials and Methods section as in the following study under the Behavioral Experiment section: "Behavioral experiments were performed on 3- to 9-month-old male mice by a male experimenter, except for the Barnes maze, where a male experimenter performed intraperitoneal injections, and a female experimenter performed the experiments" [33] or others [37].

In conclusion, the importance of handling the mice can be summarized by the words of Professor Hurst: "The routine handling of laboratory animals is essential, so it is important that we do all we can to reduce any stress and anxiety [27] ". Using methods that minimize anxiety also reduces confounding factors, improves the responses during experiments, and lead to more robust scientific outcomes.

The article was extensively re-written and updated by Dr. Goldi Kozloski in July 2019.