Ethical statement

All experimental procedures were performed in accordance with the Manual for Care and Use of Laboratory Animals, and were approved by the Ethics Committee of the Macedonian Center for Bioethics at the University of St. Kiril and Metodij, in Skopje, North Macedonia (protocol number 2401-592, 2017). The biomedical research recommendations for animal use was issued by the Council of International Organizations for Medical Sciences. The anesthetics were applied following the EU Directive's standards, Directive 86/609/ EEC.

Experimental animals

This experimental study was conducted at the Faculty of Natural Sciences and Mathematics of the Institute of Biology in Skopje, North Macedonia. In this study, experimental animals included female white laboratory rats from the Wistar strain (n = 64) at the age of 4 - 5 months with a body mass of 220 ± 20 g. During the study, the animals were exposed to a standard food and water diet regimen (ad libitum) and were kept in a room under a constant light regime of 12 h (06:00 to 18:00) of light and 12 h of the dark at a thermoneutral temperature of 26°С. The total number of animals used in this study was 20, and they were divided into two groups of 10 animals randomly. The first group was the control group of rats (n = 10), which were not subjected to the forced swim tests, and the second group of rats (n = 10) were subjected to forced swim tests. The gender was specific with only female rats. Exclusion criteria were rats older than 5 months and rats with physical impairments.

Experimental procedure

The experimental group of animals (n = 10) was exposed to forced swim tests for 21 days. For the remaining 7 days up to the end of the experiment, 5 rats from the experimental group continued to swim until day 21 while 5 rats were allowed to rest. The water temperature was measured before and after swimming. To determine the level of cortisol, the blood was collected from the tail of the rats on the 1, 7, and 14 days of the experiment. On day 21, the blood from the abdominal aorta was collected. The serum analyses were made using a Mouse/Rat Cortisol ELISA protocol. The duration of the entire experiment was 22 days.

Forced swimming test

Rats were exposed to a forced swim test, with the swimming program consisting of two phases: adaptation and training. In the first week (adaptation), the rats swam 60 - ­­90 min per day for 6 days (1 week). The training period commenced at the beginning of the second week of swimming, and its duration was progressively increased from 50 min to approximately 90 min per day. Rats were exposed to the swimming test between 08:30 and 12:00 AM. This intensity was maintained until the end of the training program, which lasted 6 days per week for a total of three weeks (21 days) from Monday-Saturday (on Sunday, there were no activities for the experimental rats).

Forced swimming stress was induced in the rats by forcing them to swim in a cylindrical swim tank which was filled with tepid water (± 34°С). All swimming protocols were recorded via a video camera and a photo camera (Figure 1). The length of the cylindrical swim tank was approximately 80 – 90 cm. The tank was filled with water to a depth of 50 – 60 cm, a depth which exceeded the length of the rats, including their tail (the length of the rats were approximately 40 cm). The width of the tank was 60 cm.

Body temperature was measured rectally using a digital thermometer before and after the forced swim test each day on days 5 – 10, 13 – 18, 20, and 21. Additionally, a description of statistics for temperature was made for each day. During the procedure for measuring body temperature before the swim test, the rats were immobilized with one hand, with their tails displaced, and the thermometer was penetrated into the rectum for approximately 10 s. The same procedure was applied after the swim test to measure their body temperature (Figure 2).

Collection and preparation of blood serum and plasma

The procedure for collecting blood was always performed in the morning and before the forced swim tests. Blood was taken from the rats' tail vein on days 1, 7 and 14, and then the rats were awakened; there was no need for anesthesia. In brief, a small nick over the lateral tail vein using a sterile scalpel blade was made approximately 2 - 3 mm from the tip of the tail. When the nick was deep enough, blood started welling up from the nick immediately. The blood was collected by allowing it to drop into a collection tube. The tail was gently stroked from the tail base toward the tail end to encourage blood flow. The amount of blood collected was 1.5 mL, and it was allowed to clot for 30 min at room temperature. The serum was separated by centrifugation at 3000 rpm for 15 min. After centrifugation, the amount of serum collected was 0.75 mL and it was subsequently stored at -20°С. To measure the concentration of cortisol, we used a Mouse/Rat Cortisol kit (Sigma-Aldrich, St. Louis, MO, USA), according to its manufacturer’s protocol (ELISA SIG-ALD SE120082). On day 21, all experimental rats were sacrificed, and blood was collected from the abdominal artery. Whole blood was collected into centrifuge tubes containing potassium-EDTA (as an anticoagulant) and centrifuged immediately for 3000 rpm for 15 min. After centrifugation, the amount of plasma collected was 5-6 mL, and the sample was subsequently stored at -20°С. The plasma samples were then subjected to the cortisol estimation similarly using the Mouse/Rat Cortisol ELISA.

Statistical analyses

Data were analyzed using the SPSS 22.0 software package for Windows (SPSS Inc., Chicago, IL, USA). Basic descriptive statistics were computed. The normality distribution of the data was determined using the skewness, kurtosis, and Kolmogorov-Smirnov method. One-Way Repeated-Measures ANOVA was used to analyze differences in variables, and the least significant difference (LSD) test was performed.

Temperature

Based on the temperature measurements obtained before and after the completion of the forced swim tests, the following results were obtained, which were statistically processed. As shown in Figure 3, the rectal temperature of the rats before the onset of physical stress was in the interval of normal values, but there was a noticeable decrease in the temperature after exposure to physical stress. According to the figure, there was a difference between the rectal temperature measured on the 21 day for the same rats. Moreover, as shown in Table 1, there were statistically significant differences in all comparisons.

Cortisol

According to Table 2, the values ​​of skewness of the cortisol variable in the control group (Sk = −0.04) of rats that swam for 21 days (Sk = −0.55) and in rats that rested from the 14 to 21 day (Sk = −0.02) were within the limits of the recommended values (​from −1 to + 1), indicating that the distribution of the results was approximately symmetric. Mild negative asymmetry was observed for rats that swam until day 7 (Sk = −1.37).

Based on the calculated coefficients of variability, the homogeneity at all collected timepoints was satisfactory. The lowest level of homogeneity was observed on the 7 day of swimming (CV = 5.56), whereas the highest level of dispersion of results was observed on the 21 day after swimming (CV = 43.82).

Based on the standard deviations (SD) at all timepoints, there was no statistically significant deviation of the results from the arithmetic mean. The standard error values indicated a minimum dispersion because there was no significance in relation, proportionally, to the corresponding standard deviation.

According to the results from the Kolmogorov−Smirnov method, as shown in Table 3, in all groups the results were normally distributed (p > 0.20). Although this was a small selective sample of subjects, the results of the analysis indicated that there was a normal distribution at all timepoints. Thus, we concluded that the degree of normality of the distributions of the applied variables satisfied the necessary methodological and statistical criteria for the application of correct and justified parametric statistical procedures for further processing of the data. Indeed, this created conditions for sufficient precise scientific determination, analysis, and comparison of data.

As shown by the values ​​in Table 4, there was no statistically significant influence of time since Wilks' Lambda = 0.348 and the degrees of freedom was F(3.6) = 63.221, which provided statistical significance at the level of Q = 0.079. The size of the partial effect of the determinants was at a high value of 0.652.

To determine whether there were statistically significant differences among the investigated groups at the univariate level for the cortisol variable, post-hock tests (LSD test) were applied. Our analyses of the tests are shown in Table 5 and Figure 4. According to the arithmetic means and statistical significance level, any statistically significant differences were observed only between the control group and the group of rats that swam for 21 days (p < 0.05).

The arithmetic means indicated that rats who swam for 21 days had lower serum cortisol values (0.34) ​​than the control group of rats (0.43). There were no statistically significant differences between the other groups.

According to the arithmetic means, the rats that swam for 7 days (0.39) and the rats that rested from day 14 until day 21 showed lower serum cortisol values (0.36) ​​relative to the control group.