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Similar reliability and equivalent performance of female and male mice in the open field and water‐maze place navigation task

Although most nervous system diseases affect women and men differentially, most behavioral studies using mouse models do not include subjects of both sexes. Many researchers worry that data of female mice may be unreliable due to the estrous cycle. Here, we retrospectively evaluated sex effects on coefficient of variation (CV) in 5,311 mice which had performed the same place navigation protocol in the water‐maze and in 4,554 mice tested in the same open field arena. Confidence intervals for Cohen's d as measure of effect size were computed and tested for equivalence with 0.2 as equivalence margin. Despite the large sample size, only few behavioral parameters showed a significant sex effect on CV. Confidence intervals of effect size indicated that CV was either equivalent or showed a small sex difference at most, accounting for less than 2% of total group to group variation of CV. While female mice were potentially slightly more variable in water‐maze acquisition and in the open field, males tended to perform less reliably in the water‐maze probe trial. In addition to evaluating variability, we also directly compared mean performance of female and male mice and found them to be equivalent in both water‐maze place navigation and open field exploration. Our data confirm and extend other large scale studies in demonstrating that including female mice in experiments does not cause a relevant increase of data variability. Our results make a strong case for including mice of both sexes whenever open field or water‐maze are used in preclinical research.

not common practice (Kokras & Dalla, 2017;Leger & Neill, 2016;Zucker & Beery, 2010). Even in 2015, of the 71 research articles that used rodents published in the journal Pain, only 3 affirmed the use of both sexes (Mogil, 2016). Our own informal PubMed survey of 100 studies published in 2016 that used the water-maze place navigation task to study spatial learning and memory of mice revealed a similar result. Only 12 studies had included subjects of both sexes with 6 stating that they had added sex as a factor to the statistical model. A total of 8 studies had used only female subjects and 69 only males.
There are several reasons why researchers are so reluctant to include female mice in their studies (Mogil, 2016;Prendergast, Onishi, & Zucker, 2014), the most important being the worry that due to their estrous cycle, inclusion of female mice in a study will increase variability of results and necessitate the testing of more subjects. It is often believed that females must be tested at each stage of the estrous cycle to generate reliable data. For tests of pain sensitivity, this has been disproved by a retrospective analysis in 7,875 mice (Mogil & Chanda, 2005), clearly demonstrating that data are equally reliable in female and male mice-also if females are tested at random stages of the estrous cycle. In addition, the study showed that, reproducing human sex differences in pain sensitivity, female mice overall had significantly shorter tail-withdrawal latencies. A recent meta-analysis extracted variability measures from 293 published articles, in which female mice tested at random stages of the estrous cycle were compared with males with regard to behavioral, physiological, morphological, and molecular traits (Prendergast et al., 2014). The study concluded that variability was not significantly greater in females than males for any endpoint and was substantially greater in males for several traits.
Even in 2015, of the 71 research articles that used rodents published in the journal Pain, only 3 affirmed the use of both sexes. Our own informal PubMed survey of 100 studies published in 2016 that used the water-maze place navigation task to study spatial learning and memory of mice revealed a similar result.
To our knowledge, retrospective behavioral studies as rigorous as Mogil and Chanda (2005) have not been conducted in other domains of behavior. In particular, comparable analyses are lacking for the important domains of learning and memory as well as for exploration and anxiety. The water-maze place navigation task (Morris, 1981;Morris, Garrud, Rawlins, & O'Keefe, 1982) is one of the most common behavioral tests for assessing spatial learning and memory in mice (D'Hooge & De Deyn, 2001;Schoenfeld, Schiffelholz, Beyer, Leplow, & Foreman, 2017;Wolfer, Colacicco, & Welzl, 2013). The open field test is very often used to measure activity, exploration, and anxiety-related responses in a novel environment. Both tests have been used during several decades in our laboratory with standardized protocols. This has accumulated a large body of data which we use in this report for a systematic retrospective analysis.
The large number of subjects available for the present study provides not only sufficient power to detect small differences but also permits to test for equivalence. Therefore, in our retrospective analysis we addressed two questions: (i) is the behavior of female mice in the water-maze and open field equally reliable or more variable than that of males? (ii) Do male mice perform better in the place navigation task Reflecting the scientific focus of the lab, most animals included in this retrospective analysis were from experiments using genetically modified mouse models to study normal function and diseases of the brain. A number of strain comparison experiments were included as well. Table 1 gives an overview of the study population. About 70% of the animals had a heterogeneous genetic background, mostly F2-3 or partial backcross generations of crosses between substrains of C57BL/6 and 129. Thirty percent were either inbred (mostly C57BL/6) or F1 hybrids. Median age of the animals was 3.4 months with an interquartile range of 2.5-4.7 months. Six percent of the mice had an age of 12 months or older.

| Water-maze place navigation
The round white poly-propylene pool had a diameter of 150 cm with 68 cm high walls. It was filled with water (24-26°C, depth 15 cm) which was rendered opaque by addition of 1 L of milk (UHT whole milk 3.5% fat, Coop., Switzerland). The white quadratic goal platform (14 × 14 cm) was made of metallic wire mesh and painted white. It was hidden 0.5 cm below the water surface in the center of one of the four quadrants, approximately 30 cm from the side wall. Salient extra-maze cues were placed on the walls of the testing room. Animals performed 30 training trials (max. duration 120 s), 6 per day with intertrial intervals of 30-60 min and varying starting positions. During the first 18 trials the hidden platform was held in the same position (acquisition phase) and then moved to the opposite quadrant for the remaining 12 trials (reversal phase). The first 60 s of the first reversal trial served as probe trial to test for spatial retention.

| Open field
The round arena had a diameter of 150 and 35 cm high sidewalls made of white polypropylene. Each subject was released near the wall and observed for 10 min on 2 subsequent days.

| Video-tracking
During all experiments, the path of moving mice was tracked using a video-   (Wolfer, Madani, Valenti, & Lipp, 2001) and converted to a consistent format.

| Water-maze place navigation, training
Escape performance during training was assessed by computing the standard measures escape latency, swim path length, and swim speed (excluding floating episodes). Spatial orientation was further evaluated using cumulative search error (sum of distances to target measured at 1 s intervals minus value that would be obtained for an ideal direct swim) (Gallagher, Burwell, & Burchinal, 1993), Whishaw's error adapted to mice and pool size (% path outside a 0.1856 m wide corridor connecting release point and goal) (Whishaw, 1985), path efficiency (% path during which speed vector component toward goal is 75% or more) (Poirier et al., 2007), and average distance from target (proximity). Further, we evaluated time spent floating (episodes of immobility or decelerations with speed minimum <0.06 m/s), the number of times mice jumped off the goal platform instead of climbing and remaining on it, the time they spent within a 7 cm zone from the wall as a measure of thigmotaxis, and the number of times they approached and contacted the wall. Body weight was recorded before and after training in a subset of animals. Body weight is known to be strongly and reliably sex-dependent in mice. Therefore, we included this measure as a reference for comparison with other sex effects.
To facilitate analysis of the large study sample, all measures were averaged across training trials to obtain a single value per subject.

| Water-maze, probe trial
Spatial retention was assessed using % time spent swimming in the former goal quadrant and in a circular target zone comprising 12.5% of the pool surface, annulus crossing index (crossings over target minus average of crossings over annuli in adjacent quadrants divided by distance swum), proximity (average distance to trained target) (Gallagher et al., 1993), and polar error (average angle between two lines connecting the pool center to the subject and trained target, respectively).

| Open field
Locomotor activity was evaluated using the standard measure of distance moved. To further characterize locomotion, we segmented the recorded path into bouts of walking (>8.5 cm/s) and episodes of lingering or resting (Madani et al., 2003). This permitted to determine % time walking and walking speed. Anxiety related parameters were % time spent in the center field (50% of area) and average distance to center. To assess the efficiency of exploration, we divided the arena into quadratic tiles of 7 × 7 cm and determined the % of tiles in which the mice had shown lingering activity. When moving toward the center mice typically walk slowly and accelerate considerably when returning toward the wall. Therefore we also calculated the difference between out and inbound speed. 2), that is differences between males and females amounted to less than one fifth of the standard deviation. According to common practice, effects with |d| > 0.2 were classified as small, medium, and large as shown in Figure 1. Effects with 0.20 < |d| < 0.25 were considered marginal.
To compare the variability of measurements between sexes, we calculated the coefficient of variation (CV, standard deviation divided by mean) separately for females and males in each group. complemented with the packages effsize and ggplot2 (Wickham, 2009).

| Open field
The open field test is primarily used to assess locomotor activity in a novel environment. In the present study, we assessed three measures of activity: distance moved, time spent walking, and walking speed. In all three, females has significantly higher scores than males (Figure 1a    Even though slightly larger CV in female mice was found or could not be ruled out for part of the measures, the results of this study clearly demonstrate that inclusion of female Our study makes a strong case for the inclusion-together with male mice-of females without testing for estrous cycle in studies of spatial memory, anxiety-related behavior, and locomotor activity with mouse models of nervous system disease. This will render studies more representative (Clayton, 2016;Miller et al., 2017;Shansky & Woolley, 2016) and provide opportunities to study sexspecific aspects of disease, provided that they are biologically determined (Eliot & Richardson, 2016). Using only male mice will not make results more reliable. Rather, efforts should be maximized to control more relevant sources of variability, such as genetic background (Crusio, Goldowitz, Holmes, & Wolfer, 2009;Lipp & Wolfer, 2003;Magara et al., 1999;Mohajeri et al., 2004;Wolfer, Muller, Stagliar-Bozizevic, & Lipp, 1997) and laboratory environment (Crabbe, Wahlsten, & Dudek, 1999).

| Equivalent learning performance and exploration in female and male mice
In addition to evaluating CV as a measure of data variability, we also directly compared mean scores of female and male mice. The only measurements to show sex differences that were both statistically significant and of relevant size were body weight and the number of fecal boli deposited in the open field, both higher in male mice.
While the observation of a significant advantage of male mice in the place-navigation task confirmed our expectations, the small, in fact negligible, size of this effect may appear surprising. We speculate that the observed sex effects are the vestiges of a former biologically relevant sexual dimorphism in spatial navigation during dispersal. In wild mice, dispersal is mainly driven by aggressive behavior, which is more intense among males than females.
However, whether or not males have to venture new territories depends on complex interactions of factors such as age and hierarchical status of the individual, population density, food availability and abiotic environmental factors (Latham & Mason, 2004). Under laboratory conditions, these factors are largely nonexistent and sex specific variations in spatial navigation are present but do not reach significant effect size. The lack of sex differences in anxiety-and exploration related open field measures found here is in line with a recent study in wild Mus musculus performing the same task (Bimova, Mikula, Macholan, Janotova, & Hiadlovska, 2016), which found neither sex differences nor an effect of estrus phase on female exploratory behavior. Alternatively, the negligible effect size in sex-specific spatial navigation in laboratory mice could be the consequence of generally reduced aggressive behavior in males by the lack of selective pressure on this trait over many generations of breeding in a laboratory environment.
The observation of a significant advantage of male mice in the water-maze place navigation task is of interest also in view of the ample evidence in human psychology documenting superior performance of men in spatial tasks, while women perform better in other cognitive domains. This effect is particularly well known and reliable in tasks involving mental rotation of objects (Voyer, 2011;Voyer, Voyer, & Bryden, 1995), but a male advantage has also been observed repeatedly in studies involving navigation of a virtual water-maze (Astur et al.,1998;Astur, Purton, Zaniewski, Cimadevilla, & Markus, 2016;Astur, Tropp, Sava, Constable, & Markus, 2004;Korthauer, Nowak, Frahmand, & Driscoll, 2017;Newhouse, Newhouse, & Astur, 2007). Performance differences between women and men in spatial tasks may be strongly confounded with socialization and gender-biased observation of congruent sex differences in water-maze navigation between mice and human subjects is nevertheless remarkable.

| CONCLUSION
Our retrospective analysis of sex differences in a large number of mice tested in the water-maze place navigation task and in the open field confirms and extends other large scale studies in demonstrating that including female mice in experiments does not cause a relevant increase of data variability. Further, performance of female and male mice in these tests is equivalent. Our results make a strong case for including mice of both sexes whenever these tests are used in preclinical research. This will be necessary for adequate modeling of the human population and to capture sex-dependent aspects of disease mechanisms.

ACKNOWLEDGMENTS
We would like to express our gratitude-without naming them individuallyto the many staff members and students who over the years have contributed to collecting behavioral data. We thank Gene Fisch for helpful advice with statistical analysis and Lutz Slomianka for critical reading of the manuscript. The studies have been supported by numerous grants of the Swiss National Science Foundation as well as by the 6th and 7th Framework Programmes of the European Union. IA and DPW are members of the Neuroscience Center Zurich (ZNZ). DPW is also a member of the Zurich Center for Integrative Human Physiology (ZIHP).