Methodological Challenges Of The Treadmill Maximum Aerobic Capacity Test In Mice

Background: Physical activity is known to induce physiological adaptations that are beneficial to overall health in humans. Studies that aim to unravel the mechanisms by which exercise exerts its effects often use mice submitted to predetermined physical training intensities commonly defined during maximum aerobic exercise testing on rodent-designed treadmills. However, the accurate estimation of mice aerobic capacity must consider the need for familiarization to the treadmill training and to the maximum test itself. Obtaining the most accurate measure within a period of time without promoting training adaptations and, on the other hand, preventing underestimated baseline values might be challenging in rodent models. Objective: We aimed to investigate the variability of the measurements within three consecutive maximum treadmill tests in C57BL/6 mice of both sexes. Methods: We evaluated 10 female (28 weeks old) and 9 male (27 weeks old) mice weighing 23.3±2.0g and 27.6±3.8g, respectively. All animals were subjected to familiarization on a treadmill designed for experimental studies (AVS Projetos®) for five days, at a speed of 5 m/min for 5 minutes, with a constant inclination of 25o, one week before the tests. The maximum effort test consisted of 5 minutes of warming-up at a speed of 6 m/min followed by incremental speed stages of 2 m/min every 2 minutes, keeping the constant inclination of 25o. The test was considered maximum when the mice were unable to, or refused to, run even with electrical or mechanical stimulation. Three tests (T1, T2, T3) were carried out with an interval of 48 hours to verify the differences in speed and distance covered. Comparisons between the tests were performed using two-way ANOVA followed by Tukey post hoc test. Data are presented as the mean ± standard deviation. Results: In the male group, there was no significant distance increment among the tests (p>0.05). However, 67% of the animals showed higher values in the 3rd test compared to the first. In the female group there was a huge percentage increase in the distance covered from T2 to T1 (93.2±40.3%; p <0.05), as well as from T3 to T1 (98.9±62.3%; p <0.01). Conclusion: Our data corroborate the need for rigorous protocols to determine the maximum aerobic capacity prior to interventions. Only females significantly incresead the distance covered among tests. A reliable pre-intervention measure of aerobic capacity warrants a more precise planning of the training load and improves the accuracy and appropriate interpretation of the effects of exercise training in mice.

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