Rats are typically used to simulate human physical conditions associated with exercise. However, the direct application of animal study results to humans requires an exact methodology. Exercise in rats is generally “forced,” wherein several means are employed to keep the animals moving. Translational applications from rat studies necessitate adequate control over the type, intensity, and duration of exercise.

Evidences suggest intrinsic differences between the forced and voluntary exercise models¹. For example, forced exercise exacerbates a stress response as compared to voluntary exercise². Thus, it may be difficult to establish sufficient parallel findings in humans using these models and presents a need for a more precise model that can translate and extend findings in animal models to humans.

Aerobic fitness is typically determined by maximal oxygen uptake (VO_2max). As the equipment used for the estimation of VO_2max in rats is expensive, alternative methods are followed for determination of the effort intensity. Anaerobic threshold, or the aerobic-anaerobic transition phase, is a widely applied method for the determination of the extent of an animal’s effort during physical exercise^3-6. The investigation of changes in the blood lactate concentration during incremental power test (INCP) and the use of gold standard protocol of maximal lactate steady state (MLSS) are precise methods for the identification of anaerobic threshold⁷ and, as a result, the intensity of the animal’s effort during physical exercise^{4, 5}.

Changes in parameters other than blood lactate concentration are observed as a response to the animal’s effort to maintain homeostasis during physical exercise. Depending upon the exercise type and intensity, the concentration of stress hormones such as corticosterone— a measure of the hypothalamic-pituitary-adrenal (HPA) axis activity— increases following physical exercise^{8 10}. The increase in corticosterone level in response to exercise induced stress is attributed to its capacity to supply energy. Further, the analysis of blood glucose, serum free fatty acid, blood lactate, and serum corticosterone concentrations immediately after physical exercise seems to be highly useful in the assessment of exercise-induced stress¹¹.

Forced running wheel (FRW) enables researchers to determine the intensity and duration of exercise without using a stimulator. Given its complex movement patterns, the physical exercise in this ergometer offers many benefits for studying the memory of healthy and unhealthy rat models^{1, 12, 13}. However, FRW exhibits limitations in terms of the application of the desired intensity^{1, 14, 15}. The primary aim of this study was to examine blood lactate levels and biochemical markers of stress following INCP and MLSS protocols. We hypothesize that changes in stress biomarkers at intensities above lactate threshold (LT) are higher than those at intensities below and around LT.

Our study showed a correlation between exercise intensity and MLSS in Wistar rats running on FRW. These findings are important for examining exercise intensity and performance in murine studies because it allows researchers to identify the intensity of physical activities associated with MLSS²¹, which is regarded as a standard method for the analysis of aerobic capacity as well as for the detection of highest aerobic power⁷. In this study, MLS intensity was determined to be 16 m/min on FRW (Figure 2), according to a protocol similar to that established by Contarteze et al⁸.

The MLSS intensity determined in our study corresponded to the blood lactate concentration of 4.1 ± 0.4 mM. Manchado et al. reported similar values (3.9 ± 0.3 mM at the speed of 20 m/min) in rats running on a treadmill¹⁶. Given the sensitivity of lactate production in the muscles used during exercise and the movement pattern, the observed difference in the intensity of MLSS can be related to the ergometer used in studies²². On treadmill, the movement is directed forward on a flat and linear path, whereas running on FRW necessitates the animal to grab the bars. Thus, it can be argued that the complexity of the wheel impels the animal to put more efforts for maintaining its speed of running. Furthermore, the INCP test results indicated a sudden rise (> 1 mM) in blood lactate concentration at 15 m/min. Hence, the concentration equivalent to LT was visually determined to be 3.6 ± 0.45 mM at this speed.

Pilis et al.⁵ reported an LT value of 4 mM at 25 m/min in rats running at increasing speed on the treadmill. Thus, the type of the exercise (treadmill versus FRW) as well as the protocol may influence LT. In Wistar rat, LT identified by an INCP test is usually similar to its MLSS²³. This is contradictory to results observed in our study. The absence of any previous knowledge of FRW parameters such as the speed at, and duration of, each stage in the INCP test may have affected the LT value.

Ferreira et al. found a correlation between exercise intensity and MLSS and showed that MLSS occurred at 60% of maximal speed in the INCP test²⁴. Our result showed that the intensity of MLSS on FRW correlated to 65% of the peak speed obtained from the INCP test. Given the dependence of MLSS intensity on aerobic capacity and the sensitivity of aerobic exercises⁴, this value can be used to design training programmes of aerobic exercises and analyze endurance performance of healthy rats running on forced wheel.

The elevated secretion of adrenocorticotropic hormone in response to stress (especially when the need for vital substrates increases) results in the rise in the activity of HPA axis, thereby increasing the concentration of glucocorticoids such as corticosterone²⁵. Glucocorticoids break down amino acids and fats from storage sites to release glucose as an energy source for other tissues¹¹. Thus, the increase in blood glucose, NEFA, and lactate levels during exercise can be attributed to the rise in corticosterone level. We observed a significant increase in the concentration of blood glucose in all exercise groups as compared with that in the control (resting) group. The increase in glucocorticoids was associated with 6-10 times increase in liver glycogenolysis and reduction in the rate of cellular glucose consumption, thereby increasing the blood glucose levels^{26, 27}. Therefore, the increased rate of gluconeogenesis and reduction in cellular glucose consumption at exercise intensities higher than MLSS may increase the blood glucose concentration. Although no significant difference was observed between the corticosterone concentrations in 10 m/min and control groups, it was surprising that the blood glucose concentration reported for the 17.5 m/min group (intensity above MLSS) was significantly higher than that in the 10 m/min group. Glucocorticoids also stimulate the release of free fatty acids either directly or indirectly via the parasympathetic system²⁸. The NEFA concentration reported in our study was significantly higher in all groups as compared to 10 m/min and control groups.

Although we have studied only the stress-related biomarkers in response to acute exercise in rats, it should be noted that stress induction with high-intensity exercise may elicit muscle damage. The increase in the CK activity in healthy subjects following high-intensity or prolonged exercise is an indicator of exercise-induced muscle damage²⁹. We observed an increase in the CK activity in 17.5 m/min group as compared to that in other groups (Figure 4D). The CK activity fails to reflect the amount of muscle mass involved in exercise. However, alterations in the CK activity was observed with the accumulation of blood lactate, glucose, NEFA, and corticosterone, indicative of the increased performance pressure on muscles during high-intensity exercise. It therefore seems that a little change in the exercise intensity around MLSS (16-17.5 m/min) may significantly affect the physiological response to exercise on FRW. Further studies for the determination of precise intensity should be carried out at 16 m/min or below at the start of the aerobic training program or above 16 m/min for interval exercise in healthy untrained rats using FRW.

A well-designed exercise protocol is of paramount importance in exercise studies. We performed a direct measurement of the exertion in animals during exercise. Researchers have traditionally applied low-to-moderate or high-intensity interval protocols, owing to the uncertainty regarding the exertion of rats using FRW^{1, 14, 15}. While studies have shown that rats are reluctant to run at speed ≥ 20 m/min on FRW, literature suggests anaerobic threshold in rats running on treadmill at around 20 m/min^{5, 16}. Researchers have incorporated changes in the FRW platform to encourage animals to run for 1 h at ≥ 20 m/min^{30, 31}. However, any change in the movement pattern alters the sport-specific mass of working muscle²¹ and may affect both the perceived exertion and exercise intensity. Given some of animals in our study were unable to complete the CP test at 17.5 m/min, indicating that untrained rats require extra effort at speed above LT. Therefore, it could be argued that the animal’s reluctance to run at such high velocities for a long time is related to the exercise-induced fatigue, not the ergometer deficiency.

Our study provides a measure of intensity of exercise at a given running speed on FRW. The exercise at different intensities based on aerobic-anaerobic transition phase induced endocrine metabolic stress responses (measured as an increase in corticosterone concentration) in animals. The speed above of 16 m/min on FRW resulted in increased physiological demands and muscle damage in untrained healthy Wistar rats. We suggest that researchers use an initial speed of ≤16 m/min for aerobic training or >16 m/min during interval training of untrained rat, using FRW, to achieve expected adaptations to exercise.