The open-field test: What does it really tell us about animal temperament? Thank you all very much for coming today. In my presentation I’m going to talk about mesuring animal temperament. In my opinion, some bases on which temperament is relied on are sometimes unclear so I will suggest that to improve the results of these tests we need to investigate the biological meanings of the metrics derived from the tests by means of independent behavioural essays. Dani Perals Bertomeu Tutor: Dr. Dani Sol

Interest Denis Réale UQAM, Canada Andrew Sih UC Davis, USA In recent years there has been an explosion of interest in the study of animal temperament.

Open-field test Open-field cage Start cage This increase of the interest is mostly due to the easiness of measuring animal behaviours by means of the OFT. This test involves measuring the behaviour of an animal after it enters on its own accord on an open field cage, often filled with novel objects.

Shortcomings Open-field cage Start cage EXPLORATION Exploration NEOPHOBIA Neophobia Despite the progress, less attention has been paid to assess if the experiments really(rielly) measure the behavioural traits they are intended to measure. - Besides measuring exploration, the test could also be interpreted as measuring neophobia, shyness and so on. These behaviours could also be correlated forming behavioural syndromes which should be taken into account. SHYNESS

Objective Assess the validity and reliability of the open-field test by comparing the results of an open-field test with those of independent tests.

Materials 58 common mynas (Acridotheres tristis) 2 populations: Canberra and Newcastle (Sol et al., 2011; Sol, Bartomeus, & Griffin, 2012). Our subjects were 58 common mynas(mainas) (Acridotheres tristis) from two populations introduced to Australia, Camberra and Newcastle.

Methods Start cage Open-field cage VARIABLES OF THE OPEN-FIELD TEST BEHAVIOURAL TRAITS OF INDEPENDENT TESTS EXPLORATION TIME TO ENTER AT THE OPEN FIELD CAGE TIME TO EXAMINE THE OPEND FIELD CAGE NUMBER OF BEAK PECKS TO NOVEL OBJECTS NEOPHOBIA In our test, the bird was released in the start cage and after 5-min of habituation it was allowed to enter the novel space. We measured a variety of metrics including the time(taime) to enter the open field cage, the time to examine the new space and its novel objects. All of them are used to estimate exploration The results of the open field test were then compared with independent measurements of exploration, shyness, motivation, activity and neophobia. These independent measurements were taken from three experiments carried out with the same individuals the days before the OFT. The independent measurements of the behaviour traits are relevant due to the validity of the myna behaviours across contexts and to avoid any test dependent correlation. SHYNESS MOTIVATION ACTIVITY

Repeatability Name Coefficient Confidence interval 95% Neophobia axis 0.407 (0.603-0.368) Activity axis 0.443 (0.579-0.368) Shyness axis 0.312 (0.465-0.231) Motivation axis 0.532 (0.674-0.438) Exploration axis 0.441 (0.592-0.311) Our measurements of exploration, shyness, activity, neophobia and motivation were all repeatable across contexts, a prerequisite to be considered personality traits.

Results 1. Are different behaviors expressed independently in the different tasks? Regarding the results, firstly, we wondered if the different behaviors were expressed independently(independéntly) in the different tasks

General Linear Mixed Model 1. Are different behaviors expressed independently in the different tasks? -0.01 -0.24 General Linear Mixed Model -0.51 -0.03 -0.01 0.24 EXPLOR SHY MOTIV ACTIV NEOPH Due to the fact that the possible existence of correlations between behavioural traïts may alter the interpretation of the results, we checked if behaviours in common mynas were organized in syndormes with a multi-response General Linear Mixed Model with each of the five behavioural axes (activity, shyness, motivation, neophobia and exploration). -0.33 -0.82 -0.33 p < 0.05 p > 0.05 Perals et al. (in press) 0.64

General Linear Mixed Model 1. Are different behaviors expressed independently in the different tasks? General Linear Mixed Model SHY ACTIV The intra-class coefficient for individuals suggested that activity and shyness were correlated with shier individuals being less active and bolder ones more active. -0.82 p < 0.05 p > 0.05 Perals et al. (in press)

Often behaviours are organized in syndromes 1. Are different behaviors expressed independently in the different tasks? Often behaviours are organized in syndromes According to the results of the General linear Mixed Model we must take into account the possible existence of syndormes when measuring behavior in the Open field Test.

Results 2. Do all the behaviours performed in the open-field test only reflect the exploration trait of the individuals? Secondly, we wondered if all the behaviours performed in these tests only reflect exploration of the individuals

Latency to enter at the open-field cage 2. Do all the behaviours performed in these tests only reflect the exploration trait of the individuals? Survival analysis EXPLOR SHY MOTIV ACTIV NEOPH -0.07 0.23 -0.10 0.12 0.11 To this porpouse, one behavioural variable used to asses the exploration of the individuals is the time needed by the individual to enter in the open field cage. In our Open field test, to assess which behaviours distinguish the individuals that cross earlier we carried(carrid) out a survival analysis (análisis) with this variable as a function(fancshian) of their behavioural axes which showed that the time to enter at the open field was only consistently affected by their shyness axis. p < 0.05 p > 0.05 Latency to enter at the open-field cage Perals et al. (in press)

Latency to enter at the open-field cage 2. Do all the behaviours performed in these tests only reflect the exploration trait of the individuals? Survival analysis SHY 0.23 Resulting that shier individuals took longer to cross than bolder individuals. p < 0.05 p > 0.05 Latency to enter at the open-field cage Perals et al. (in press)

Number of Pecks (Open-field test) 2. Do all the behaviours performed in these tests only reflect the exploration trait of the individuals? General Linear Model EXPLOR SHY MOTIV ACTIV NEOPH - 0.22 0.26 0.16 0.14 -0.39 Another used variable to asses the exploration of the individuals is the number of pecks they perform at the novel objects. In our test, to determine (detérmin) which behavioural axes distinguish the few individuals that pecked, we modelled a General Linear Model with this variable as a function(fancshian) of their behavioural axes. Which showed that the only behavioural axis that consistently affected the number of pecks variable was their exploratory behaviour. p < 0.05 p > 0.05 Number of Pecks (Open-field test) Perals et al. (in press)

2. Do all the behaviours performed in these tests only reflect the exploration trait of the individuals? General Linear Model EXPLOR The number of beak pecks delivered to the novel object in the independent test. About the exploration axis, it was formed by the number of beak pecks delivered to the novel object in the independent test, so higher values of this axis mean more beak pecks delivered No or less beak pecks delivered More beak pecks delivered

Number of Pecks (Open-field test) 2. Do all the behaviours performed in these tests only reflect the exploration trait of the individuals? General Linear Model EXPLOR Num. Pecks independent test. 0.22 Thus, individuals that pecked the novel object more frequently during the independent test also pecked more the novel objects during the OFT. p < 0.05 p > 0.05 Number of Pecks (Open-field test) Perals et al. (in press)

Time to examine the open-field cage 2. Do all the behaviours performed in these tests only reflect the exploration trait of the individuals? Survival analysis EXPLOR SHY MOTIV ACTIV NEOPH - - 0.12 0.01 0.25 0.06 0.03 Another variable used to assess exploration is the time to examine the four corners and the perch of the open field cage. In our test, to determine which behavioural trait distinguishes the individuals that finished sooner from the ones that didn’t finish we carried(carrid) out a survival analysis with the time mynas spend to examine (examin) the open field cage as a function of their behavioural axis which showed that the time to examine the open field was only consitently affected by their exploratory axis. . p < 0.05 p > 0.05 Time to examine the open-field cage Perals et al. (in press)

Time to examine the open-field cage 2. Do all the behaviours performed in these tests only reflect the exploration trait of the individuals? Survival analysis EXPLOR Num. Pecks independent test. - 0.12 Being the individuals that peck more the novel objects in the independent test the faster ones to examine the open field cage p < 0.05 p > 0.05 Time to examine the open-field cage Perals et al. (in press)

2. Do all the behaviours performed in these tests only reflect the exploration trait of the individuals? A. Not all behaviours performed only reflect exploration SHY 0.23 Our study confirms that the latency to enter the open field was unrelated to exploration propensity measured in an independent test, it rather reflected variation in shyness-boldness Latency to enter at the open-field cage

2. Do all the behaviours performed in these tests only reflect the exploration trait of the individuals? B. Two behavioural variables predict more reliably the exploration of the individuals. Num. Pecks independent test. EXPLOR - 0.12 0.22 Moreover, our cross-contextual analyses identified two behaviour variables that reliably predicted exploration in the OFT. According to our experiments, individuals that examined faster the open field cage, pecked more the novel objects both in the independent test and the open field test. This conclusion was not affected by the existence of behavioural syndromes Time to examine the open-field cage Number of Pecks (Open-field test)

Conclusion Our analyses highlight that the open-field test is a valid approach if one measures the appropriate behaviours and checks (rather than assume) their validity and reliability for the study aim with multiple independent tests. In conclusion, our analyses highlight that the OFT is a valid approach if one measures the appropriate behaviours and test (rather than assume) their validity and reliability for the study organism with multiple independent tests.

Implications Standardize the open field test Avoid discrepances and contradictions Make results more interpretable and easier to compare Improve the estimation of temperament Improve risk analysis for biological invasions Facilitate re-introduction of endangered species Predict species response to climate change

Thanks for your attention If you have any questions about the speech, I’ll be happy to answer them.