Exercise evaluation with metabolic and ventilatory responses and blood lactate concentration in mice

This study aimed to clarify the differential exercise capacity between 2-month-old and 10-month-old mice using an incremental running test. Metabolic and ventilatory responses and blood lactate concentration were measured to evaluate exercise capacity. We examined whether incremental running test results reflected metabolic and ventilatory responses and blood lactate concentration observed during the steady-state running test. Metabolic response significantly declined with age, whereas ventilatory response was similar between the groups. A low-intensity/moderate exercise load of 10/min in an incremental running test was performed on both mice for 30minutes. They showed a characteristic pattern in ventilatory response in 10-month mice. The results of incremental running tests didn't necessarily reflect the steady-state metabolic and ventilatory responses because some parameters showed an approximation and others did not in incremental and steady-state tests, which changed with age. Our study suggests metabolic and ventilatory responses depending on age and provides basic knowledge regarding the objective and quantitative assessment of treadmill running in an animal model.


Introduction
Exercise is known to promote the health and physical performance of individuals and prevent the development of disease (Warburton et al., 2006;Wen et al., 2011;Gremeaux et al., 2012).Exercise can also help reduce morbidity risk and repair the physiological damage caused by cerebrovascular diseases (Saunders et al., 2014), cardiovascular diseases (Fiuza-Luces et al., 2018), respiratory diseases (Gloeckl et al., 2013), and dementia (Livingston et al., 2017).Several animal model studies have been conducted to elucidate the effects of exercise on these diseases.
When exercise prescription is considered in animal model studies, selecting measuring parameters that are used in human studies, such as heart rate, blood pressure, arterial oxygen saturation, oxygen consumption, or ventilation, would be preferable to compare (Voss et al., 2013).To perform a quantitative assessment of the effects of exercise, the incremental running test has been widely used not only in humans but also in rodents (Schefer and Talan, 1996;Kemi et al., 2002;Ferreira et al., 2007;Hoydal et al., 2007;Balady et al., 2010;Ayachi et al., 2016;Picoli et al., 2018).Although aging is considered to affect physical performance and proper exercise prescription, comparisons between the above seven studies are not possible since the incremental running test was not the same.Kemi et al. (2002) used 2-month-old male and female C57BL/6 J mice and a regular 20-minute warm-up at 40-50% of V˙O2 max before the V˙O2 max protocol, which was conducted by increasing speed by 1.8 m/min every 2 min (Kemi et al., 2002).Ferreira et al. (2007) used 5-month-old male C57BL/6 J mice, and V˙O2 max was obtained by increasing speed by 3 m/min every 3 min without warm-up (Ferreira et al., 2007).Schefer and Talan (1996) used 12-and 24-month-old male C57BL/6 J mice but did not apply the incremental running test (Schefer and Talan, 1996).Instead, each mouse was tested with only one of the following treadmill speeds: 3, 4, 8, 12, 15, 17, or 20 m/min.Based on several indirect evidences, they suggested that the oxygen consumption (V˙O 2 ) values obtained during running at the speed of 25 m/min for 12-month-old mice and at the speed of 20 m/min for 24-month-old mice can be considered maximal V˙O 2 .In summary, the effects of aging on parameters obtained with the incremental running test remain unknown.Furthermore, all these studies did not measure respiratory parameters, such as respiratory frequency (f R ) and tidal volume (V T ), which could be affected by aging.
Two-month-old rodents are frequently used in science research, which has been reported in studies focusing on the age of model animals (Jackson et al., 2017).Several animal models of diseases, such as stroke, cardiovascular disease, chronic obstructive pulmonary disease, and dementia, have often used young adult animals (Hasenfuss, 1998;Durukan and Tatlisumak, 2007;Gotz and Ittner, 2008;Jones et al., 2017).The life history of mice is considered as follows: 3-6 months of age is considered mature, 10-14 months as middle-aged, and 18-24 months as old (Flurkey et al., 2007).Focusing on the motor system, the motor function of mice is better in 2-3-month-old mice, and eventually, when they are past six months, the motor performance levels begin to decline.Some specific examples include the following reports from previous studies: Spontaneous physical activity (SPA), distance traveled, and speed of locomotion started to decrease in 6-month-old C57BL/6 mice.The reduction in SPA became more evident, and energy expenditure decreased from 8 months of age (Benfato et al., 2017).In open-field studies, significant reductions in voluntary movement, reduced approaches to novel objects, and reduced spatial cognitive performance have been observed in mice at around 12 months of age (Yanai and Endo, 2021).In the rotor lot test, exercise capacity decreased from around six months, and a significant decrease in exercise capacity was observed in 10-month mice compared to 2-month mice (Shoji et al., 2016;Yanai and Endo, 2021).Based on these previous studies, the occurrence of physiological change between mature-aged and middle-aged mice may be inferred.Therefore, it is necessary to consider the age of the animal models, physical function, and exercise capacity when examining the effects of exercise on animal models.
Thus, this study had two aims.First, this study aimed to evaluate the differences in exercise capacity of different aged mice using the incremental running test.To achieve this goal, pulmonary gas exchange, ventilatory responses, and blood lactate concentrations during the incremental running test on the treadmill were measured in 2-month-old (2 mo) and 10-month-old (10 mo) C57BL/6 J mice.Second, this study aimed to evaluate whether the metabolic and ventilatory responses observed at the treadmill speed of 10 m/min during the incremental running test results can serve as reference values for a steady-state running speed test at a speed of 10 m/min for both 2 mo and 10 mo mice.In our previous study, 2 mo mice ran on the treadmill at the speed of 10 m/min for 30 min, and the metabolic index during this running was not significantly different from the 10 m/min value in the incremental running test (Yoshikawa et al., 2022).However, the ventilatory parameter was not evaluated in our previous study.Therefore, we evaluated whether the metabolic and ventilatory responses observed during the incremental running test could be used as an indicator of steady-state running by performing the same 10 m/min exercise intensity not only on 2 mo mice but also on 10 mo mice in this study.

Ethical approval
All experimental protocols were approved by the Showa University Animal Experiment Committee and were based on the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (ethical number: 09013, 02106, 03051).The investigators understand the ethical principles under which Respiratory Physiology & Neurobiology operates, and the work complies with the animal ethics checklist used by the journal.

Animals
Male C57BL/6 J mice were purchased from Sankyo Labo Service Corporation (Tokyo, Japan).After purchase, the middle-aged mice were bred in the Showa University Laboratory of Animal Experiments until the experiment.They were subsequently bred in our animal care facility under conventional holding conditions, housed in cages with 12-hour light/dark cycles, and received water and food (LabDiet 5058) ad libitum.

Overview of the experimental protocol
This study comprised three experiments (Table 1).The purpose of experiment 1 was to investigate exercise capacity in both 2 mo and mo mice using an incremental running test.In this experiment, the pulmonary gas exchange and ventilation patterns were measured during the running test.The purpose of experiment 2 was to investigate exercise capacity in both animals and the same test in a similar way, but blood lactate measurement was used to assess this test.Blood lactate concentration was used as a marker of exercise intensity and training status, and we used this method as an evaluation of exercise intensity (Brooks, 1986;Billat, 1996;Morishita et al., 2020).The purpose of experiment The study consisted of three experiments.Exercise capacity was compared between 2 mo and 10 mo mice by performing the incremental treadmill running test in experiment 1. V˙O2 (mL/min/kg), V˙CO2 (mL/min/kg), RER (V˙CO2/V˙O2), fR (breaths/min), VT (mL/10 g), and V˙I (mL/min/10 g) were measured and analyzed during this test.Exercise capacity was also compared between 2 mo and 10 mo mice focusing on the blood lactate concentration (mmol/L) during the same test in experiment 2. Finally, experiment 3 was conducted to determine whether the values of the incremental treadmill running test are reflected in a steady-state running exercise.2 mo, 2-month-old mice; 10 mo, 10-month-old mice; V˙O 2 , oxygen consumption; V˙CO 2 , carbon dioxide output; RER, respiratory exchange ratio; f R , respiration frequency; V T , tidal volume; V˙I, minute ventilation.
A. Yoshikawa et al. was to investigate whether the incremental running test results could serve as reference values for a steady-state running speed test in both 2 mo and 10 mo mice.Pulmonary gas exchange, ventilation patterns, and blood lactate concentrations were measured.The mice used in these three experiments were different.

Treadmill chamber equipment: the measurement of pulmonary gas exchange and ventilatory response during running
The mice were tested using an airtight modular treadmill (width × depth × height of 70 × 440 × 75 mm; 5 • upslope: MK-680AT/02 M, Muromachi Kikai Co., Ltd., Tokyo, Japan).The chamber was subjected to continuous airflow (1 L/min) via positive-pressure ventilation to obtain oxygen/carbon dioxide (O 2 /CO 2 ) pulmonary gas metabolism measurements (Oxymax ver 4.7x, Columbus Instruments, Columbus, OH, USA), f R (breaths/min), V T (mL), and minute ventilation (V˙I: mL/ min/10 g) as ventilatory parameters (Biosystem XA for Windows; Buxco Electronics, Sharon, CT, USA) (Fig. 1A).In this whole body plethysmography measurement of ventilation volume, V T is generally calculated from the inspiratory volume since expiration often is of longer duration, and the expiratory volume could be affected by baseline shift (Hernandez et al., 2012).Our previous study reported that the ventilation of mice while running on the treadmill was measured with this device, and V T and V˙I were calculated from inspiratory volume (Izumizaki et al., 2013).Because this device calculates ventilation through inspiration, this study also calculated V T and V˙I based on inspiration.Experiments 1, 2, and 3 were performed on this set treadmill device.Therefore, all treadmill angles were set at 5 • and airflow at 1 L/min.

Incremental running test on a treadmill
This test was adopted to evaluate the exercise capacity of animals using a scientific evaluation method that has also been used in human experiments.Exercise capacity was determined by O 2 consumption (V˙O 2 ; mL/min/kg), CO 2 production (V˙CO 2 ; mL/min/kg), f R (breaths/ min), V T (mL/10 g), and V˙I (mL/min/10 g).The respiratory exchange ratio (RER) was also a parameter of exercise capacity, calculated by dividing V˙CO 2 by V˙O 2 .In this study, exercise capacity was further assessed by oxygen consumption efficiency estimation using the ventilatory equivalent (V˙I/V˙O 2 or V˙I/V˙CO 2 ) from the pulmonary gas exchange and ventilatory response (Redkva et al., 2018;Phillips et al., 2020).
By the day before the incremental running test, mice were acclimatized to treadmill running for 30 min at a speed of 10 m/min per day for a few days as a pre-training and then subjected to the incremental running test for the estimation of the exercise capacity and physiological responses in each mouse (2 mo or 10 mo) using step-up treadmill speeds.Before beginning running, the mice were kept in a treadmill chamber for approximately 30 min to acclimatize to their surroundings, and a period of 5 min before running was defined as rest.
The protocol for this test was based on our previous study (Yoshikawa et al., 2022) (Fig. 1B).Briefly, the mice ran at 1, 3, 5, 10, 15, and 20 m/min speed for 1 min each, and the speed was then increased by 2 m/min every 1 min.We aimed to avoid spending more than 20 min to reach the maximum running ability because young mice can often run at a maximum speed of 40-50 m/min (Kregel et al., 2006;Hoydal et al., 2007).In addition, the sudden movement of the stationary treadmill belt considered the stress on the mice and the fear of contact with electrical stimulation with the treadmill apparatus.Previous studies have devised a way to eliminate this stress response by starting with a few minutes of starting running and then increasing the speed (Billat et al., 2005;Carvalho et al., 2005).Although not the same as these previous studies, we used the first 3 min at low speed as a familiarization period (thus 1, 3, and 5 m/s for 1 min), after which the treadmill speed was gradually increased to 20 m/min by 5 m/min.Then, the treadmill speed was increased by 2 m/min to evaluate the metabolic and ventilatory responses in more detail.
The treadmill chamber contained stimulus grids on its rear side.The time immediately before the running speed at which the mice could no longer run and stayed on the stimulus grids was defined as the maximum running ability, and V˙O 2 peak , V˙O 2 , V˙CO 2 , f R , V T , and V˙I were expressed as the average for every 1 min.

Blood lactate concentrations
Blood lactate levels were measured to assess exercise intensity in both 2 mo and 10 mo mice during running exercise.The higher the exercise intensity, the higher the blood lactate concentration level (Ferreira et al., 2007;Cunha et al., 2009).A blood drop was collected from the tail vein (using the tail snap method), placed on a test strip, and inserted into a lactate pro-LT device (Lactate Pro, Arkray, Inc., Kyoto, Japan) (Izumizaki et al., 2013;Ayachi et al., 2016;Morishita et al., 2020).In experiment 2, the blood lactate concentration was measured when the mice began running and every 4 and 2 min in 2 mo and 10 mo mice, respectively.In experiment 3, blood lactate concentrations before and after 30 min of running exercise were evaluated.A. Yoshikawa et al.

Definition of ventilatory and lactate thresholds
The ventilatory threshold (VT) and lactate threshold (LT), as markers of anaerobic threshold, were examined based on pulmonary gas exchange, ventilatory response, and blood lactate levels obtained in experiments 1 and 2. By examining this, treadmill speed can be used to determine whether the exercise is high or low in intensity for the animal.The VT and LT were examined by detecting inflection points.The inflection point is that at which the rate of increase in V˙CO 2 exceeds the rate of increase in V˙O 2 in VT (Beaver et al., 1986), and the blood lactate concentration levels increase rapidly in LT (Cunha et al., 2009;Abreu et al., 2016;Cerezuela-Espejo et al., 2018).The inflection points of both VT and LT were analyzed using piecewise regression and two-segmented linear models in the regression library of SigmaPlot (SigmaPlot 13; Systat Software Inc., San Jose, CA, USA) used in our previous studies (Izumizaki et al., 2011;Tsukada et al., 2017;Yoshikawa et al., 2022).

Evaluation of pulmonary gas exchange and ventilatory response at a steady treadmill speed
We decided to confirm whether the incremental running test results could serve as reference values for a steady-state running speed test in both 2 mo and 10 mo mice in experiment 3 (Table 1).By the day before a steady-state running test, mice were acclimatized to treadmill running using the same acclimatization protocol as that in the incremental running test.After this acclimatization, mice ran at a treadmill speed of 10 m/min for 30 min in a steady-state running test in accordance with our previous study.Pulmonary gas exchange and ventilatory response during running were monitored.

Statistical analysis
In this study, we statistically compared (1) 2 mo and 10 mo animals in experiments 1 and 2 and (2) the results of the incremental running test and steady-state running test within each group.The results are expressed as mean ± standard error.Age, weight, and maximum treadmill speed were compared using Student's t-test (SPSS ver.26, IBM Corp., Armonk, NY, USA).V˙O 2 , V˙CO 2 , RER, f R , V T , V˙I, and the ventilatory equivalent were compared for each treadmill speed and interaction (age × metabolic and respiratory variables) using two-way repeated measures ANOVA and post hoc Tukey's honestly significant difference (HSD) (JMP® Pro ver.15. 2.0, SAS Institute Inc., Cary, NC, USA).The blood lactate concentrations at the same treadmill speed were also compared using Student's t-test with SPSS software.Finally, the average values of the pulmonary gas exchange (V˙O 2 , V˙CO 2 , and RER), ventilatory parameters (f R , V T , and V˙I), ventilatory equivalent, and blood lactate concentration after performing the incremental and steady-state running tests were statistically analyzed and compared using Student's t-test with SPSS software.For statistical analysis, we calculated the average values of the 30-minute steady-state running test.Only the blood lactate concentration values obtained after performing the incremental and steady-state running tests were used and compared.The statistical significance level was set at p < 0.05.
V˙O 2 and V˙CO 2 gradually increased in both groups and achieved exercise capacity in 10 mo mice, while in 2 mo mice, they were once in a steady state from approximately 20 m/min to 30 m/min and then increased further (Fig. 2B, C).The values of both V˙O 2 and V˙CO decreased in 10 mo mice compared to those in 2 mo mice, and significant difference from 3 m/min to maximal exercise capacity in V˙O 2 and from resting time to maximal exercise capacity in V˙CO 2 respectively (Fig. 2B, C).V˙O2 in the maximal exercise capacity were 138.15 ± 5.32 mL/min/kg and 87.84 ± 7.96 mL/min/kg in 2 mo and 10 mo mice, respectively.In addition, there could be a significant interaction between age and V˙O 2 and V˙CO 2 (Fig. 2B, C).The RER was not significantly different both 2 mo and 10 mo mice in ANOVA and post hoc tests; however, a significant interaction was shown between age and RER (Fig. 2D).The RER (V˙CO 2 /V˙O 2 ) in 2 mo mice showed a transient increase from rest to 3 m/min, gradually decreasing until the treadmill speed reached 15 m/min and then gradually increasing further.The mo mice showed a transient increase from rest to 5 m/min, then plateaued until 15 m/min, and then gradually increased further.This means that Δ V˙CO2 was higher than Δ V˙O2 in the 10 mo mice compared to 2 mo mice at the same treadmill exercise speed.As a result, an interaction was found between the two age groups.These metabolic results show that 10 mo mice had significantly lower V˙O 2 and V˙CO capacities per kilogram than 2 mo mice.
Regarding the ventilatory parameters in f R , and V T , the response pattern to incremental running was interestingly shown to be similar and was not significantly interact between 10 mo and 2 mo mice; in addition, there was no significant difference in each treadmill speed with two groups of mice (Fig. 2E, F).This pattern in f R and V T was divided into four phases.The first phase was observed at steps between rest and 5 m/min.During this phase, the increase in f R exceeded the decrease in V T .The second phase was observed at steps between 5 and 20 m/min.During this phase, f R did not change consistently in either group, but V T appeared to decrease slightly in 2 mo mice not 10 mo mice.The third phase was observed at steps between 20 and 30 m/min.During this phase, the VT increase exceeded the fR decrease in both groups.All 10 mo mice reached exhaustion in the third phase.From 30 m/min onwards, the fourth phase was observed only in 2 mo mice.During this phase, f R increases again while V T remains almost constant.When the changing pattern in V˙I obtained by multiplying this fR and V T was evaluated, there was a significant interaction in both groups (Fig. 2G).In the first phase, V˙I of the 2 mo mice increased rapidly, whereas that of the 10 mo mice increased less.In the second phase, V˙I of the 2 mo mice showed a decreasing trend, while that of the 10 mo mice plateaued.In the third phase, both groups showed an increase in V˙I.The results showed that 2 mo mice had significantly higher V˙I from the first phase to the beginning of the second stage than 10 mo mice.

VT and LT in 2 mo and 10 mo mice during incremental treadmill running
To reveal the significance of aerobic/anaerobic metabolism during exercise, we examined VT and LT from V˙O 2 and V˙CO 2 and blood lactate concentration levels, respectively.VT and LT were indicated as inflection points and inflection points were calculated using a segmented linear regression model consisting of two segments (Izumizaki et al., 2011;Tsukada et al., 2017;Yoshikawa et al., 2022).VT was calculated from V˙O 2 and V˙CO 2 and was 113.77 ± 2.66 in V˙O 2 and 94.28 ± 2.59 in V˙CO 2 , while 75.25 ± 6.47 in V˙O 2 and 65.70 ± 7.53 in V˙CO 2 in 2 mo and 10 mo animals, respectively (Fig. 3A).One animal in the 2 mo groups was used as missing data because this data did not match a segmented linear regression model consisting of two segments, but V˙O 2 and V˙CO were significantly lower in 10 mo mice than those in 2 mo mice (V˙O 2 : t [15] = − 6.10, p < 0.0001 and V˙CO 2 : t [1] = − 6.51, p < 0.0001) (Fig. 3A).By applying these VT values to treadmill speeds, it was found

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A. Yoshikawa et al. that the speeds were approximately 15 m/min in 10 mo and 20 m/min in 2 mo mice (Fig. 2B, C).
LT was evaluated as another method of examining whether the aerobic or anaerobic metabolic system was significant (Table 1: experiment 2).Table 2 summarizes the data obtained from both groups of mice.The data on blood lactate concentration was collected every 4 and 2 min in 2 mo and 10 mo mice, respectively, and the timings of the treadmill speeds were at rest and at 10, 24, 32, and 40 m/min and at rest and at 3, 10, 20, 24, and 28 m/min, respectively.Because the 10 mo groups reached the limit of exercise capacity at approximately half the time of the 2 mo group from experiment 1 (Fig. 2), the timing of blood collection was set at half the time (every 2 min) instead of every 4 min.The blood lactate concentration was significantly higher in 10 mo mice than that in 2 mo mice at 10 and 24 m/min, although there was no significant difference between these values at rest (Table 2).LT was defined by using a segmented linear regression model.The inflection points of treadmill speed and blood lactate concentration were 23.95 ± 2.34 m/min and 3.36 ± 0.48 mmol/L, respectively, in 2 mo mice, while they were 20.00 ± 1.56 m/min and 5.85 ± 0.82 mmol/L, respectively, in 10 mo mice (Fig. 3B).

Ventilatory equivalents in 2 mo and 10 mo mice during incremental treadmill running
To clarify the ventilatory efficiency in 2 mo and 10 mo mice, the ventilatory equivalents for oxygen (V˙I/V˙O 2 ) (Fig. 4A) and carbon dioxide (V˙I/V˙CO 2 ) (Fig. 4B) were calculated.V˙I/V˙O 2 and V˙I/V˙CO 2 did not significantly differ between the two groups, indicating no difference in ventilatory efficiency.The response patterns of V˙I/V˙O 2 and V˙I/V˙CO 2 to the incremental running test were similar between the two groups.Thus, V˙I/V˙O 2 and V˙I/V˙CO 2 decreased from rest to 20-24 m/min, after which they increased.However, since the difference in the amount of change was greater with 2 mo mice than with 10 mo mice, there was an interaction between the two groups.

Pulmonary gas exchange and ventilatory response in 2 mo and 10 mo mice during running on a treadmill with a steady-state running speed test
In our previous study, we indicated that the representation of quantitative exercise intensity was important for animal model studies (Yoshikawa et al., 2022).Therefore, experiment 3 was used to verify whether the results of the incremental treadmill running test were applied as a reference value for steady-state running exercise in 10 mo mice as well as in 2 mo mice (Table 1: experiment 3).Fig. 5 shows the temporal changes in the parameters of pulmonary gas exchange (V˙O 2 , V˙CO 2 , and RER) and ventilatory parameters (f R , V T , and V˙I) during treadmill running for 30 min at 10 m/min in 2 mo and 10 mo mice.
The response patterns of V˙O 2 and V˙CO 2 in this steady-state running Fig. 2. Comparison of running capacity during maximal treadmill running speed, pulmonary gas exchange, and ventilatory pattern between the 2 mo and 10 mo mice during the incremental treadmill running. 2 mo (blue) and 10 mo (red) mice entered the airtight treadmill chamber and stayed there for approximately 30 min to acclimatize to their surroundings.The airtight treadmill chamber was inclined at 5 • and received a continuous airflow (1 L/min).The rest corresponds to the average value for five minutes immediately before running.The mice ran at 1, 3, 5, 10, 15, and 20 m/min for 1 min each, and the speed was increased by 2 m/min for every 1 min.The treadmill chamber contained stimulus grids on its rear side.The time immediately before the running speed, at which the mice could no longer run and stayed on the stimulus grids, was defined as the maximum running capacity.(A) Relationship between running ability and the number of mice.(B) Results of V˙O 2 (mL/min/kg), (C) V˙CO 2 (mL/min/kg), (D) RER (V˙CO 2 /V˙O 2 ), (E) f R (breaths/min), (F) V T (mL/10 g), and (G) V˙I (mL/min/10 g).The horizontal axes indicate treadmill speeds.All values are presented as mean ± standard error.Variables were compared between 2 mo and 10 mo mice at each treadmill speed using two-way repeated measures ANOVA post hoc Tukey's HSD.p value indicated the results of the ANOVA test.The p value of the interactions was indicated as Int.Asterisk was indicated the significantly different between 2mo and 10 mo mice for each treadmill speed.* p < 0.05; * * p < 0.01.V˙O 2 , oxygen consumption; V˙CO 2 , carbon dioxide output; RER, respiratory exchange ratio; f R , respiration frequency; V T , tidal volume; V˙I, ventilation.speed test were similar between the two groups.In both groups, V˙O 2 and V˙CO 2 gradually increased for the first several minutes, peaked at 4-6 min, and then gradually decreased (Fig. 5A, B).The resultant RER (V˙CO 2 /V˙O 2 ) increased for the initial several minutes and then decreased to steady values (Fig. 5C).The response patterns for f R and V T differed between the two age groups.Although the f R values did not show marked changes during steady running in 2 mo mice after the initial increase, they gradually decreased in 10 mo mice (Fig. 5D).In 2 mo mice, V T decreased instantly, after which it did not show marked changes during the steady-state running speed test.In contrast, in 10 mo mice, V T decreased and reached the lowest value 7 min after the starting time; thereafter, it gradually increased (Fig. 5E).V˙I, the multiplication result of f R and V T , decreased slightly during the steady-state running speed test compared to that at rest and did not show marked changes in 2 mo mice.Similarly, 10 mo mice presented decreased V˙I values during the steady-state running speed test (Fig. 5F).The ventilatory equivalents of oxygen (V˙I/V˙O 2 ) (Fig. 5G) and carbon dioxide (V˙I/V˙CO 2 ) (Fig. 5H) were similar between the two groups.Both parameters decreased until approximately 5 min after the start of running and then remained at an almost steady state.
From these results, we compared the incremental running test results with the average values of pulmonary gas exchange, ventilatory parameters, and blood lactate concentration during 30-minute steady-state running at 10 m/min (Table 3).In 10 mo mice, there was no significant difference between the incremental running test and steady speed running in all parameters.In 2 mo mice, V˙O 2 showed no significant difference between the incremental running test and steady speed running.V˙CO 2 and RER were significantly lower, while V T , V˙I, V˙I/V˙O 2 , and V˙I/V˙CO 2 were significantly higher in the steady-state than those in the incremental running test in 2 mo mice.The results showed that V˙O 2 at 10 m/min during the incremental running test could be used as a reference for the intensity of exercise at 10 m/min of steady-state running speed in both 2 mo and 10 mo mice.

Discussion
To examine the different exercise capacities in 2 mo and 10 mo mice, pulmonary gas exchange, ventilatory responses, and blood lactate concentrations were assessed during the incremental running test.The incremental running test consists of a graded exercise test in which the treadmill speed and angle are gradually increased (Petrosino et al., 2016), or the treadmill speed is gradually increased while the treadmill angle remains constant.The incremental running test was adopted in this study in which the treadmill angle was always set at 5 • .
As the results of the incremental running test, V˙O 2 in the maximal exercise capacity were 138.15 ± 5.32 mL/min/kg in 2 mo and 87.84 ± 7.96 mL/min/kg in 10 mo mice, and these results were similar to those of previous young (Kemi et al., 2002;Petrosino et al., 2016) and aged (Ayachi et al., 2016) mice's studies.The previous study evaluated metabolism during exercise in 24-month-old mice compared to 12-month-old mice and showed age-related metabolic decline (Schefer and Talan, 1996).Our results and those of several previous studies reveal that aged mice have significantly reduced oxygen consumption compared to young mice.
Regarding the relationship between the incremental running test and steady-state running, the exercise load below LT/VT is often used for steady-state running exercise.These opinions have been introduced in human participants, especially in patients with heart disease (Garber et al., 2011;Group, 2014).This study examined LT and VT with the incremental running test and confirmed that the anaerobic threshold (AT) is important for determining the intensity of steady-state running exercise based on the incremental running test.It has been reported that exercise intensity before AT can stabilize exercises, while exercise intensity after AT can increase the intensity of exercise (Faude et al., 2009;Abreu et al., 2016).As the results of both VT and LT, 2 mo mice showed the treadmill speed at approximately 20 m/min in VT and 24 m/min in LT.On the other hand, 10 mo mice showed the treadmill speed at approximately 15 m/min in VT and 20 m/min in LT.Previous studies reported that the LT value was approximately 20 m/min (Okamoto et al., 2015) and 18 m/min (Billat et al., 2005) in young-aged mice, indicating some variation.On the other hand, there are no reports on VT values in young-aged mice because there have been few reports of incremental running tests focusing on ventilatory response.Few studies of LT and VT in older animals have been reported.In human studies, VT and LT have been reported to be consistent (Pallares et al., 2016;Cerezuela-Espejo et al., 2018).Studies using rodents do not address these aspects, but previous studies have questioned the reliability of the measurement of blood lactate levels in mice (Lonbro et al., 2019).The mechanism of the difference in these values could not be elucidated in this study and requires further validation.
From the value of both VT and LT, 10 m/min was set as the exercise intensity in this study.V˙O 2 of 10 m/min were 101 ± 3.25 mL/min/kg and 62 ± 4.29 mL/min/kg for 2 mo and 10 mo mice, respectively, which were 73% and 71% of % V˙O 2 in the maximal exercise capacity, respectively.Fernando et al. developed a prediction equation to predict submaximal oxygen consumption (Fernando et al., 1993).From this prediction equation, 10 m/min of treadmill speed was indicated as approximately 74% V˙O 2 max, and our values were found to be similar to this prediction equation.This was interestingly similar to the values in mice aged 10 months as well as those aged two months.Our and Fernando's study indicated that the treadmill speed at 10 m/min was before LT and VT points in both 2 mo and 10 mo mice, which was low/moderate exercise intensity for both aged mice.The exercise intensity at 10 m/min on treadmill speed for 30 min benefitted the disease model mice (Ma et al., 2018;Wang et al., 2020;Yoshikawa et al., 2022); hence, we consider that the results of this study can provide useful information for studies to verify the effects of exercise on these model animals.
We considered the exercise intensity at 10 m/min to be low/moderate at approximately 70% of V˙O 2 in the maximal exercise capacity for mice at 2 and 10 mo.
In this study, there was no significant difference in V˙O 2 values between the incremental running test and steady-state running exercise in both 2 mo and 10 mo mice; however, in 2 mo mice, significant differences were found in V˙CO 2 and RER.This result indicated that the value was significantly lower in steady-state running than in the incremental running test.Alternatively, blood lactate concentration levels did not differ between the two running situations.Blood lactate concentration levels have been shown to remain unchanged when an exercise load is set below LT/VT (Faude et al., 2009;Abreu et al., 2016).The level of non-significant blood lactate concentration suggests that the 10-m/min exercise load during steady-state running may be similar to or milder than that of the incremental running test and suggests that V˙CO 2 and RER may therefore be significantly lower in steady-state running than those in the incremental running test.
The respiratory parameters, such as f R and V T, during the incremental running test have not been reported in previous studies.In other words, this study can be considered the first to assess ventilatory response during an incremental running test.In a typical ventilation pattern for an incremental running test in humans, V T increases at lower exercise workloads, then plateaus, whereas f R increases linearly with increasing exercise workloads during the incremental running test (Sheel and Romer, 2012).On the other hand, the ventilation pattern in mice was the opposite of that in humans, with an increase in f R at lower exercise workloads and a decrease in breathing rate at higher exercise workloads.In addition, V T tended to decrease at lower exercise workloads and to increase as the exercise load increased.This ventilation pattern in mice was similar in all the participating mice, suggesting that the result of our study might be a typical ventilation pattern in the incremental running test in mice.Focusing on comparing the results of the incremental running test and the steady-state running test for ventilation patterns, the ventilatory pattern of 2 mo mice showed a significant increase in V T and V˙I during the 30-minute steady-state running compared with that of the incremental running test.Ten mo mice showed no significant differences in these values; however, the f R pattern gradually decreased while V T increased, and V˙I remained constant.This ventilatory response during steady movement was clearly different from that in 2 mo mice, and it is a very interesting phenomenon as a pattern of ventilatory response during exercise performed by aging mice.Hyperpnea during running is principally supported by an upregulation of the respiratory rhythm generator in the brainstem (Del Negro et al., 2018).In 2 mo mice, this function may have worked and promoted respiration regarding the specific ventilation pattern.In 10 mo mice, complex respiratory control mechanisms may be involved.For example, organic changes in the lungs and airways showed decreased lung volume and airway resistance changes with age (Huang et al., 2007;Elliott et al., 2016).It is possible that these lung parenchymal changes occur peripherally with simultaneous changes in the respiratory center.Because central nerve cells undergo neuronal cell death and network changes as they age (Castelli et al., 2019), the age-related respiratory center function begins to decline in older animals compared to that in younger animals.However, age-related changes in the neurons in the respiratory center have not been reported, and the functions of these neurons remain unclear.On the other hand, there is an interesting previous study on limb movement and respiratory control during running.This study has reported that respiratory control and limb movement operate without phasic constraints (Herent et al., 2020).This suggests that there is a more complex control system for the relationship between running and breathing, and this study suggests that this may vary with age.
The ventilatory response has been reported to be affected by body temperature and room temperature (Mortola and Maskrey, 2011).
However, this study failed to control temperature changes in mice during exercise or at room temperature in an airtight modular treadmill chamber.Future studies will examine respiratory responses in animals during exercise while monitoring body and room temperature.
Exercise elicits a variety of responses in our organs.For example, the role of exercise in our body is diverse, including improving physical fitness and health maintenance, mental stability, prevention of disease, and promotion of recovery from disease (Silverman and Deuster, 2014).The wide variety of effects of exercise and the mechanisms by which exercise exerts its effects remain unclear.Animal studies are essential to clarify the mechanism of exercise in the body.Therefore, rodents are the animal species used especially in scientific research.Most of the rodents used are relatively young animals, as young as approximately ten weeks (2 months) old (Jackson et al., 2017).The reason for using these young-aged animals was that (i) younger animals are often used in previous studies, and it is easier to compare the results with those of previous studies; (ii) older animals show greater variability in biological responses; and (iii) the cost of purchasing and raising animals is higher (Jackson et al., 2017).While these perspectives are important, age is an important part of disease research as age must be considered to transfer model animals closer to human physical conditions to elucidate diseases and treatments.The differences in metabolic and respiratory responses shown in our results, as well as the differences in exercise performance reported in previous studies (Graber et al., 2015;Shoji et al., 2016;Yanai and Endo, 2021;Reiber et al., 2022) may be important influencing factors in disease model animal studies.From these findings, our study's measurement methods of pulmonary gas exchange, ventilatory responses, and blood lactate concentrations, which have similarities in animals and humans (Voss et al., 2013), were the strength of our study.This study was only a quantitative method of exercise intensity assessment when prescribing exercises.This study suggests that quantifying exercise provides useful data to objectively verify the effects of exercise prescription on biological functions in animal models.

Conclusions
In this study, 2 mo and ten mo mice were evaluated for exercise capacity using the incremental running test and determine whether these test results can serve as reference values for a steady-state running speed test.Pulmonary gas exchange, ventilatory responses, and blood lactate concentrations were monitored during these tests in both animal groups, and different responses were indicated.The results of incremental running tests do not necessarily reflect the steady-state metabolic and ventilatory responses because some parameters show an approximation, and others do not in incremental and steady-state tests.Our study suggests metabolic and ventilatory responses depending on age and provides basic knowledge regarding the objective and quantitative assessment of treadmill running in an animal model.

Funding
This work was supported by Grants-in-Aid for Scientific Research (grant numbers 17K13069 and 20K11292, AY, and 16H05443, HO) from the Japan Society for the Promotion of Science. A. Yoshikawa et al.

Fig. 1 .
Fig. 1.Schematic overview of the experimental apparatus and protocol of the incremental running test.(A) Illustration of the treadmill and measurement equipment used in this study to measure pulmonary gas exchange and ventilatory responses during running on treadmill.(B) Illustration showing graded treadmill speed in the incremental treadmill running test.

Fig. 3 .
Fig.3.Ventilatory and lactate thresholds in 2 mo and 10 mo mice during incremental treadmill running.VT from the relationship between V˙O 2 and V˙CO 2 and LT were analyzed using a piecewise regression model and two-segmented linear model in the SigmaPlot regression library.The results of 2 mo and 10 mo (red) are presented in VT (A).The results of 2 mo and 10 mo (red) are presented in LT (B).All these values are presented as mean ± standard error.Arrowheads, blue and red indicate the inflection point as VT and LT in 2 mo and 10 mo mice, respectively.In VT, the values of V˙O 2 (x-axis) and V˙CO 2 (y-axis) are shown in parentheses, and treadmill speed (x-axis) and blood lactate concentration (y-axis) are shown in parentheses in the LT.VT, ventilatory threshold; LT, lactate threshold; V˙O 2 , oxygen consumption; V˙CO 2 , carbon dioxide output.

Fig. 4 .Fig. 5 .
Fig. 4. Evaluation of the ventilatory equivalents and the relationship between oxygen consumption and carbon dioxide output in mice with different ages when performing the incremental running test on a treadmill.To clarify the ventilatory efficiency in 2 mo (blue) and 10 mo (red) mice, the ventilatory equivalents for oxygen (V˙I/V˙O 2 : mL/min/kg) and carbon dioxide (V˙I/V˙CO 2 : mL/min/kg) were evaluated.(A) Results for V˙I/V˙O 2 and (B) V˙I/V˙CO 2 are presented.Statistical analysis was used to compare between the 2 mo and 10 mo mice at each treadmill speed using two-way repeated measures ANOVA post hoc Tukey's HSD.p value indicated the results of the ANOVA test.The p value of the interactions was indicated as Int.Asterisk was indicated the significantly different between 2mo and 10 mo mice for each treadmill speed.* p < 0.05; * * p < 0.01.

Table 1
Schematic overview of this experiment.

Table 2
Animal information and blood lactate concentration in 2 mo and 10 mo mice after performing the incremental running test.
The blood lactate concentration was measured before running and every 4 and 2 min thereafter in 2 mo and 10 mo mice, respectively.All values are presented as mean ± standard error.Statistical analyses were performed to compare variables between the 2 mo and 10 mo groups at rest, 10 m min-1, and 24 m min-1 using Student's t-test.*p< 0.05; * * p < 0.01.A.Yoshikawa et al.