To enhance the legibility of the results, the habituation phase was marked as the H mean score from habituation trials 5 to 7, which served as a reference value for further analyses, while the test trials were marked as T1, T2, and T3, respectively. Novelty, i.e. addition or change of objects in zone C, was introduced in the first test trial T1.
The initial four habituation trials have not been presented here, as they served only as a habituation phase and not as an element of the comparative analysis of the animals’ response to novelty.
The data was analysed using a General Linear Model procedure GLM, with repeated measurements H, T1, T2, T3 as within-subject factors, followed by an LSD PostHoc test which involved a comparison of the habituation phase H with the three test trials T1, T2 and T3. Bonferroni correction for multiple comparisons was employed. Differences were considered significant for p ≤ 0.05. Data analysis was carried out using JASP v. 0.14.1 software, an open-source project supported by the University of Amsterdam.
Time spent in the transporter
The amount of time spent in the transporter, excluding the latency to leave the transporter (that is, the amount of time from the moment the transporter was opened until the rat first entered the experimental apparatus), was measured for each group.
In the ADD group, the analysis showed a significant main effect of trial: F(3, 39) = 5.033, p = 0.005, Eta2 = 0.279 (Wilks’ Lambda). A post-hoc analysis showed a significant decrease in the time spent in the transporter in the first and third test trials compared to the habituation phase (T1: p = 0.008, d = 1.090; T3: p = 0.017, d = 0.982).
In the CMPLX group, the analysis showed a significant main effect of trial: F(3, 36) = 8.695, p < 0.001, Eta2 = 0.420 (Wilks’ Lambda). A post-hoc analysis showed a significant decrease in the time spent in the transporter in all test trials compared to the habituation phase (T1: p < 0.001, d = 1.564; T2: p = 0.03, d = 1.327; T3: p = 0.027, d = 0.965).
In the SIZE group, the analysis showed a significant main effect of trial: F(3, 36) = 11.934, p < 0.001, Eta2 = 0.499 (Wilks’ Lambda). A post-hoc analysis showed a significant decrease in the time spent in the transporter in all test trials compared to the habituation phase (T1: p < 0.001, d = 1.726; T3: p = 0.024, d = 0.986).
Time spent in the unchanged zone of the chamber
In the ADD group, the analysis showed a significant main effect of trial: F(3, 39) = 15.421, p < 0.001, Eta2 = 0.543 (Wilks’ Lambda). A post-hoc analysis showed a significant decrease in the time spent in the unchanged zone of the chamber in all test trials compared to the habituation phase (T1: p < 0.001, d = 3.775; T2: p = 0.043, d = 0.850; T3: p < 0.001, d = 1.441).
In the CMPLX group, the analysis showed a significant main effect of trial: F(3, 36) = 14.825, p < 0.001, Eta2 = 0.553 (Wilks’ Lambda). A post-hoc analysis showed a significant decrease in the time spent in the unchanged zone of the chamber in the first test trial compared to the habituation phase (T1: p < 0.001, d = 2.345). Then, a significant increase was observed in the second and third trials compared to the first test trial (T2: p = 0.003, d = 1.291; T3: p = 0.003, d = 1.305).
In the SIZE group, the analysis showed a significant main effect of trial: F(3, 36) = 9.605, p < 0.001, Eta2 = 0.445 (Wilks’ Lambda). A post-hoc analysis showed no significant changes in the time spent in the unchanged zone of the chamber between the habituation phase and the first test trial. However, an increase was observed in the second test trial compared to the habituation phase (T2: p = 0.036, d = 0.924). There were also differences between the first test trial and subsequent test trials (T2: p = 0.007, d = 1.176; T3: p = 0.020, d = 1.010).
Time spent in the changed zone of the chamber
In the ADD group, the analysis showed a significant main effect of trial: F(3, 39) = 21.277, p < 0.001, Eta2 = 0.621 (Wilks’ Lambda). A post-hoc analysis showed a significant increase in the time spent in the changed zone of the chamber in all test trials compared to the habituation phase (T1: p < 0.001, d = 2.341; T2: p < 0.001, d = 1.409; T3: p < 0.001, d = 2.092).
In the CMPLX group, the analysis showed a significant main effect of trial: F(3, 36) = 46.825, p < 0.001, Eta2 = 0.796 (Wilks’ Lambda). A post-hoc analysis showed a significant increase in the time spent in the changed zone of the chamber in all test trials compared to the habituation phase (T1: p < 0.001, d = 3.634; T2: p < 0.001, d = 2.180; T3: p < 0.001, d = 1.851). Then, a significant decrease was observed in the second and third trials compared to the first test trial (T2: p = 0.020, d = 1.013; T3: p = 0.012, d = 1.088).
In the SIZE group, the analysis showed a significant main effect of trial: F(3, 36) = 32.268, p < 0.001, Eta2 = 0.729 (Wilks’ Lambda). A post-hoc analysis showed a significant increase in the time spent in the changed zone of the chamber only in the first test trial compared to the habituation phase (T1: p < 0.001, d = 3.203). Then, a significant decrease was observed in the second and third trials compared to the first test trial (T2: p < 0.001, d = 1.737; T3: p < 0.001, d = 2.294).
Frequency of moving between the chamber zones (left/right/transporter)
In the ADD group, the analysis showed a significant main effect of trial: F(3, 39) = 5.336, p = 0.004, Eta2 = 0.291 (Wilks’ Lambda). A post-hoc analysis showed a significant decrease in the frequency of moving between the zones of the chamber only in the second test trial compared to the habituation phase (T2: p = 0.004, d = 1.201).
In the CMPLX group, the analysis showed a significant main effect of trial: F(3, 36) = 11.567, p < 0.001, Eta2 = 0.491 (Wilks’ Lambda). A post-hoc analysis showed a significant decrease in the frequency of moving between the zones of the chamber in the first and second test trials compared to the habituation phase (T1: p = 0.001, d = 1.461; T2: p = 0.003, d = 1.319).
In the SIZE group, the analysis showed a significant main effect of trial: F(3, 36) = 12.171, p < 0.001, Eta2 = 0.504. A post-hoc analysis showed a significant increase in the frequency of moving between the zones of the chamber only in the third test trial compared to the habituation phase (T3: p = 0.005, d = 1.214). A significant increase was also observed between the first and third test trials (p < 0.001, d = 1.575) and between the second and third test trials (p = 0.005, d = 1.228).
Time spent on contact with the tunnels in the unchanged zone of the chamber
In the ADD group, the analysis showed a significant main effect of trial: F(3, 39) = 9.252, p < 0.001, Eta2 = 0.416 (Wilks’ Lambda). A post-hoc analysis showed a significant decrease in the time spent on contact with the tunnels in the unchanged zone of the chamber in all test trials compared to the habituation phase (T1: p < 0.001, d = 1.400; T2: p < 0.001, d = 1.413; T3: p = 0.002, d = 1.304).
In the CMPLX group, the analysis showed a significant main effect of trial: F(3, 36) = 11.032, p < 0.001, Eta2 = 0.479 (Wilks’ Lambda). A post-hoc analysis showed a significant decrease in the time spent on contact with the tunnels in the unchanged zone of the chamber in the first test trial compared to the habituation phase (T1: p < 0.001, d = 1.836). Then, a significant increase was observed in the second and third trials compared to the first test trial (T2: p = 0.008, d = 1.153; T3: p = 0.007, d = 1.182).
In the SIZE group, Mauchly’s test indicated that the assumption of sphericity had been violated (χ2(5) = 11.860, p = 0.038), so the degrees of freedom were corrected using Greenhouse–Geisser estimates of sphericity (ε = 0.65). The analysis showed a significant main effect of trial: F(1.949, 23.392) = 19.403, p < 0.001, Eta2 = 0.618. A post-hoc analysis showed a significant increase in the time spent on contact with the tunnel in the unchanged zone of the chamber in the second and third test trials compared to the habituation phase (T2: p < 0.001, d = 1.674; T3: p = 0.002, d = 1.369). Then, a significant increase was observed in the second and third trials compared to the first test trial (T2: p = 0.002, d = 1.42; T3: p = 0.006, d = 1.185).
Frequency of contact with the tunnels in the unchanged zone of the chamber
In the ADD group, the analysis showed a significant main effect of trial: F(3, 39) = 5.592, p = 0.003, Eta2 = 0.301 (Wilks’ Lambda). A post-hoc analysis showed a significant decrease in the frequency of contact with the tunnels in the unchanged zone of the chamber in the first and third test trials compared to the habituation phase (T1: p = 0.014, d = 1.003; T3: p = 0.005, d = 1.155).
In the CMPLX group, the analysis showed a significant main effect of trial: F(3, 36) = 7.953, p < 0.001, Eta2 = 0.399 (Wilks’ Lambda). A post-hoc analysis showed a significant decrease in the frequency of contact with the tunnels in the unchanged zone of the chamber in the first and second test trials compared to the habituation phase (T1: p < 0.001, d = 1.547; T2: p = 0.004, d = 1.249).
In the SIZE group, the analysis showed a significant main effect of trial: F(3, 36) = 3.960, p = 0.015, Eta2 = 0.248. A post-hoc analysis showed a significant increase in the frequency of contact with the tunnel in the unchanged zone of the chamber only between the first and third test trials (p = 0.042, d = 0.901).
Time spent on contact with the tunnels in the changed zone of the chamber
In the ADD group, the analysis showed a significant main effect of trial: F(3, 39) = 25.771, p < 0.001, Eta2 = 0.665 (Wilks’ Lambda). A post-hoc analysis showed a significant increase in the time spent on contact with the tunnels in the changed zone of the chamber in all test trials compared to the habituation phase (T1: p < 0.001, d = 3.107; T2: p < 0.001, d = 1.627; and T3: p < 0.001, d = 2.458).
In the CMPLX group, the analysis showed a significant main effect of trial: F(3, 36) = 58.170, p < 0.001, Eta2 = 0.829 (Wilks’ Lambda). A post-hoc analysis showed a significant increase in the time spent on contact with the tunnels in the changed zone of the chamber in all test trials compared to the habituation phase (T1: p < 0.001, d = 4.372; T2: p < 0.001, d = 2.101; T3: p < 0.001, d = 2.308). Then, a significant decrease was observed in the second and third trials compared to the first test trial (T2: p = 0.011, d = 1.104; T3: p = 0.008, d = 1.160).
In the SIZE group, the analysis showed a significant main effect of trial: F(3, 36) = 51.999, p < 0.001, Eta2 = 0.812 (Wilks’ Lambda). A post-hoc analysis showed a significant increase in the time spent on contact with the tunnel in the changed zone of the chamber in all test trials compared to the habituation phase (T1: p < 0.001, d = 4.757; T2: p = 0.005, d = 1.220; T3: p = 0.012, d = 1.096). Then, a significant decrease was observed in the second and third trials compared to the first test trial (T2: p < 0.001, d = 1.614; T3: p < 0.001, d = 3.428).
Frequency of contact with the tunnels in the changed zone of the chamber
In the ADD group, the analysis showed no significant main effect of trial: F(3, 39) = 2.169, p = 0.107 (Wilks’ Lambda). In the CMPLX group, the analysis showed no significant main effect of trial: F(3, 36) = 2.098, p = 0.118 (Wilks’ Lambda). In the SIZE group, the analysis showed no significant main effect of trial: F(3, 36) = 2.249, p = 0.099 (Wilks’ Lambda). Table 1 shows the descriptive statistics of all behavioural measures.
Effect size analysis
In order to achieve a result analysis design compatible with our previous study (2019), we conducted a similar analysis with regard to the effect size. Table 2 shows all the dependent variables collected in this study across the three experimental groups together with Eta2 values. Kruskal–Wallis ANOVA (Eta2 value by group) showed no differences between the groups (H = 0.962, df = 2, p = 0.618).
Summary of the results
ADD group
The rats from this group responded to the addition of new tunnels with a significant behavioural shift involving increased exploration of the newly installed tunnels. This effect was manifested by a major increase in the time spent in the changed zone of the chamber (in all test trials compared to the habituation phase), as well as the duration of contact with the tunnels in the changed zone of the chamber. What is more, a decrease was observed in the amount of time spent in the unchanged zone, in the duration of contact with the tunnels in this zone and in the length of stay in the transporter (in the first and third test trials; it could be observed in each trial in our previous study). Furthermore, there was a significant decrease in the frequency of moving between the zones of the chamber in the second test trial and in the frequency of contact with the tunnels in the unchanged zone of the chamber. It should be noted that the effects described above were stable across all three test trials. This characteristic is important in the context of comparisons with the other two experimental manipulations.
SIZE group
The rats from this group initially reacted with a behavioural shift towards the enlarged tunnel (trial T1) and then towards the unchanged one (trial T2, T3), simultaneously decreasing the amount of time spent in the transporter. This effect was manifested by a significant increase in the time spent in the changed zone of the chamber in the first test trial and in the amount of time spent on contact with the tunnel in the changed zone of the chamber in all test trials (a significant decrease, however, was observed in both cases in the second and third trials). Consequently, the rats from this group exhibited an increase in the amount of time spent in the unchanged zone of the chamber in the second test trial. In addition, there was a significant increase in the time spent on contact with the tunnel in the unchanged zone of the chamber in the second and third test trials. A significant increase was also observed in the frequency of contact with the tunnel in the unchanged zone of the chamber in the third test trial. Moreover, there was a significant increase in the frequency of moving between the zones of the chamber in the third test trial.
CMPLX group
The rats from this group exhibited a strong response to the increased complexity of the objects with a profound behavioural shift towards the complex tunnel and then, to a limited extent, towards the unchanged one, with a significant decrease in the amount of time spent in the transporter. This effect was also manifested by a significant increase in the time spent in the changed zone of the chamber, as well as in the time spent on contact with the tunnels in the changed zone of the chamber in all test trials. However, there was a significant (albeit not very steep) decline in the second and third test trials compared to the first test trial (both in the time spent in the zone and on contact with the tunnels). Consequently, a significant decrease was observed in the frequency of contact with the tunnels in the unchanged zone of the chamber in the first and second test trials, in the time spent in the unchanged zone of the chamber in the first test trial (in the case of the latter, an increase was then observed in the second and third trials compared to the first one). Moreover, the rats showed a decrease in the time spent in the unchanged zone of the chamber in the first test trial, and an increase in the second and third trials compared to the first test trial. Furthermore, a decrease was observed in the frequency of moving between the zones of the chamber in the first and second test trials. There was also a significant decrease in the time spent in the transporter in all test trials.
As regards the effect size, no differences were found in any of the three experimental groups, which suggests that all manipulations had similar impact.
Source: Ecology - nature.com
