RESEARCH ARTICLE
A three-criteria performance score for rats
exercising on a running treadmill
Juan Gabriel Rı´os-Kristja´nsson1☯, David Rizo-Roca1☯, Karen Mist Kristja´nsdo´ttir1,2,
Cristian Andre´s Nu´ñez-Espinosa1,3, Joan Ramon Torrella1, Teresa Pagès1,
Gine´s ViscorID1*
1 Department of Cell Biology, Physiology & Immunology, Faculty of Biology, University of Barcelona,
Barcelona, Spain, 2 Department of Biotechnology and Chemical Engineering, Aarhus University School of
Engineering, Aarhus N, Denmark, 3 School of Medicine, University of Magallanes, Casilla, Punta Arenas,
Chile
☯ These authors contributed equally to this work.
* gviscor@ub.edu
Abstract
In this study, we propose a novel three-criteria performance score to semiquantitatively clas-
sify the running style, the degree of involvement and compliance and the validity of electric
shock count for rats exercising on a treadmill. Each score criterion has several style-marks
that are based on the observational registry of male Sprague-Dawley rats running for 4–7
weeks. Each mark was given a score value that was averaged throughout a session-registry
and resulting in a session score for each criterion, ranging from “0” score for a hypothetical
“worst runner”, to score “1” for a hypothetical “perfect runner” rat. We found significant differ-
ences throughout a training program, thus providing evidence of sufficient sensitivity of this
score to reflect the individual evolution of performance improvement in exercise capacity
due to training. We hypothesize that this score could be correlated with other physiological
or metabolic parameters, thus refining research results and further helping researchers to
reduce the number of experimental subjects.
Introduction
A significant amount of scientific literature on exercise sciences is based on experimental
research involving laboratory rats, being motor-driven treadmill running, voluntary wheel
running and swimming the most common types of exercise [1–6]. Although a marked differ-
ence has been reported on the exercise capacity of rats in relation to different strains [7–9], in
most published work using treadmill training the mention of problems with the rats whilst car-
rying out the training sessions is absent [10–13]. This could lead to the assumption that all or
much of the rats have been trained in the same extent by the end of a training session within
each study. However, the natural proneness of rat for short voluntary running bursts [14] sug-
gests that, if given the choice, it would run with recurrent breaks; unless, perhaps, if running
away from a perceived danger. Thus, the expectancy of good habituation to continuous run-
ning on a treadmill, throughout a relatively long exercise session, might not be realistic from a
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OPEN ACCESS
Citation: Rı´os-Kristja´nsson JG, Rizo-Roca D,
Kristja´nsdo´ttir KM, Nu´ñez-Espinosa CA, Torrella
JR, Pagès T, et al. (2019) A three-criteria
performance score for rats exercising on a running
treadmill. PLoS ONE 14(7): e0219167. https://doi.
org/10.1371/journal.pone.0219167
Editor: Clemens Fu¨rnsinn, Medical University of
Vienna, AUSTRIA
Received: February 19, 2019
Accepted: June 18, 2019
Published: July 9, 2019
Copyright: © 2019 Rı´os-Kristja´nsson et al. This is
an open access article distributed under the terms
of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files.
Funding: This study was supported by DEP2010-
22205-C02-01 (GV) and DEP2013-48334-C2-1-P
(GV & JRT) grants from the Plan Nacional I+D+i
(The Spanish Ministry of Economy and
Competitiveness). JGRK was supported by a
predoctoral grant (BES-2011-044293) from The
Spanish Ministry of Economy and
Competitiveness. The funders had no role in study
biological point of view, especially at the beginning of a training period. Obviously, the rats
can still be gradually trained to run, but the fact the rats finished a training session does not
guarantee that all of them did the same training volume or equivalent intensity. Few published
experiments reported on the different performance between individual rats when running. For
instance, Arnold et al. (2014) described a protocol to select aged rats for an exercise protocol,
arguing that not all animals are equally prone to run in a treadmill [15], while in a paper from
Ferraresso et al. (2012) the authors specifically stated that they differentiated animals that ran
voluntarily and animals that refused to ran in order to distribute the rodents among the differ-
ent experimental groups [16]. However, this kind of information is usually omitted in experi-
mental papers.
Many treadmills for rats promote running by means of a light electric shock stimulus via
the touch of a metal grid. A monitoring apparatus processes the input of the electric shock
count with the related accumulating duration; useful to estimate how successfully a rat has
been running. However, this setup has two potential problems: 1) the equipment cannot differ-
entiate between a rat that is continuously touching the grid and e.g. a piece of faeces stuck on
it; 2) when rats run on the treadmill, a marked majority tend to avoid running in many differ-
ent ways. Fig 1 (marks B-N) describes evasive behaviours, which compromise compliance, to
avoid running or electrical shock punishment [17]. Thus, the experimenter needs to be aware
of these possible artefacts when using a treadmill.
Accepting the likelihood of all rats not running equally, or being unequally prone to run on
a treadmill, leads to the question of how different qualitative running traits can be represented
and how to quantify that. Assumption of equal performance and compliance for the same
workload of all the animals in a training protocol is unrealistic. Indeed, if the training itself is
important, also the training type is important; e.g. continuous vs. interval training or forced vs.
spontaneous exercise. Subsequently, the fundamental questions should address the manner
how the rats diverge in training and how this is reflected in the data, and address a systematic
approach to determine those deviations.
Here we propose a system based on marks aimed to semiquantitatively assess the rat run-
ning style on a treadmill, and quantify this assessment with a score value, eventually leading to
an average score. Thus, the main goal of this work is to develop and apply a semiquantitative
scoring system to assess rat running compliance to an exercise protocol in a motorized tread-
mill. This score allows to “classify” the rats, which can serve as a critical factor for further phys-
iological data interpretation. The score could also be used to compare the treadmill running
performance of rats between different experimental conditions. Moreover, from an ethical
point of view, and according to principles of the Three Rs (reduction, refinement and replace-
ment), it is necessary to apply data tools that allow researchers to obtain the best from the
experimental animals with which they are working. A vast majority of researchers simply dis-
card animals that do not show ability to run on the treadmill, but the selected group might not
be representative of the “normal” animal population. On the other hand, it certainly becomes
an ethical issue if the selection of the experimental animals relies on discarding them, rather
than refining the number of rats in the experiment from the onset.
Material and methods
Animals
All procedures were performed in accordance with the internal protocols of our laboratory,
authorized by the University of Barcelona’s Ethical Committee for Animal Experimentation
and ratified, in accordance with current Spanish legislation, by the Departament de d’Agricul-
tura, Ramaderia, Pesca i Alimentacio´ of Generalitat de Catalunya (file #8784). The score
Performance score for running rats
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design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
developed and presented in this article derived as a secondary observational registry from 130
male Sprague Dawley rats (strain: RjHan:SD, Janvier Labs, France). The animals were used in
a primary experiment on the study of the recovery of induced skeletal muscle damage in the
hind limbs of trained rats [18–21] in the framework of a research project approved by the
Fig 1. Schematic setup of the AEY performance score. The top section explains the setup of a treadmill diagram,
followed by three sections for each score criteria: A, E and Y. The score criteria-sections have subsections (from left to
right): (1) Mark, a registry note for the described style; (2) Diagram, a schematic drawing (if applicable); (3)
Description, the characteristics are described; and (4) Score, the determined score value for further calculations.
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Institutional Experimental Animal Ethics Committee. The experimental protocol required sev-
eral weeks of previous conditional exercise training in a running treadmill and 1 day of
exhaustive downhill exercise followed by different recovery interventions. All the rats used
were males and started the training program at 6 weeks of age (body weight: 154 ± 8 g) in
order to fulfil the requirements of the primary study, were housed at maximum 3 animals per
cage (215×465×145 mm) and were fed standard diet (15-mm diameter granulates) and water
ad libitum. The facility’s room temperature and relative humidity ranged between 20–25˚C
and 45–55% respectively. All the rats were regularly checked for stress signs judging from their
physical appearance and body weight and received treatment fulfilling the National and Euro-
pean directives for the care of animal uses for scientific purposes [22].
Instrumental
An encased five lane treadmill and its accompanying treadmill controller (LE 8710, Harvard
Apparatus, United States) with an adjustable plane (5˚/ramp-setting) from −15˚ to 25˚ was
used to carry out the exercise training. An adjustable electric shock stimulator, ranging from
0.2–2.0 mA, discharged when animal contacted the metal grids behind the back end of the
treadmill belt. The treadmill encasing, separating the lanes, with front and back wall air holes
for each lane, enabled uncontrolled airflow inside it. For each lane, the monitor on the tread-
mill controller displayed the number of electric shocks generated, the accumulated time of
electric shocks and the calculated distance considering the set velocity and the time lapsed,
deducting the time spent receiving a shock. Speed adjustments affected collectively all the five
lanes of the treadmill belt. Furthermore, the experimenters encouraged the rats to run with a
light push or a sound to minimise the experience of excessive electric shocks, especially during
the first days of habituation to exercise on the running treadmill.
The three-criteria performance score
The score’s style code is based in three domains, each giving their own score value. These 3 cri-
teria/scores are: 1) the running “attitude” (A), 2) a clue of the “endurance” (E), and 3) the
gross “yield” (Y) provided by the electric shock count. Henceforward, the three-criteria perfor-
mance score can be summarized as the AEY-score. More specifically, the criteria for the A-
score concerns the physical positioning and actions of the rat whilst running (or trying to
avoid running). The E-score concerns the position of the rat’s tail as an indicator of tiredness
based on the effort to hold it up to avoid receiving an electric shock from the grid. The Y-score
addresses the potential artefacts and problems considering the digital representation of the
electric shock count (the sole insight usually considered of the rat performance in the running
treadmill).
The development of the score was carried out in 2 stages: (a) the initial stage (55 rats) where
different situations and characteristics were documented and the criteria for the scores were
defined; and (b) the quantification stage (75 rats) where the defined criteria were systematically
registered for each rat throughout every training session.
Fig 1 presents illustrative style diagrams and text explanations relative to the three domains
of the score [A;E;Y] along with its corresponding marks and score values used for registering
and quantifying during a training session. The experimental objective was to train the rats
towards their best potential, recording the individual trends along the way. If it was needed
and physically possible, the experimenters interfered with a rat’s unwanted styles by outlasting
its determination. Relatively low treadmill speed (in the first days of habituation as part of the
protocol) partially led to unfavourable running styles in some rats; hindering the development
a continuous “proper” running (style A). Rats that demonstrated style B and E had no major
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issues with exercising and needed to be lightly pushed or pulled back onto four legs. Styles C,
H and I represented rats that normally had high shock counts and needed an extra physical
push to achieve a favourable positioning on the belt. Style D came across as a display of tired-
ness (or a sense of outwitting) by periodically stopping until almost touching (or fully touch-
ing) the shock grid and then quickly moving to the front of the treadmill. In styles F and G, the
rat cleverly discovered that, by lightly touching its lane-sidewalls, enough resistance was pro-
vided to slide on the treadmill belt without moving towards the grid. Among styles E, F and G,
the rat was equally inactive regarding the exercise; hence, all styles were later assigned the same
quantification score, leaving the AEY-score open to take on board similar circumstances with-
out major changes. Style J represented distracted or stressed rats (not necessarily “bad run-
ners”) seeming susceptible to disrupting stimuli outside the treadmill. Styles K, M and N,
normally occurred when rats were exercising to exhaustion; with style M prevailed on a down-
ward slope. Style L is specific to treadmills where the belt does not cover the edge (e.g. within
the two peripheral lanes of a multi-lane treadmill; a design flaw to be solved). In our case, a
style L-rat supported the fore and hind limbs of the same body side onto the non-moving part,
whilst the other limbs moved. The occurrence of style L was further minimised by rotating the
lane positions between sessions during the first days of the training protocol.
Quantifying the assessment
Each domain of the score had a maximal value of “1” that was considered for the “ideal run-
ner” rat. The running styles or situations that were considered unfavourable would reduce that
value towards the score floor of “0” for each part of the score. Thus, the more a trait compro-
mised the animal’s compliance to the exercise protocol (to run continuously at 0.45 m/s for 30
min, see Exercise Protocols below), the more negative score was assigned to that trait.
For the A-score, the rat was given the roof value of “1” by default. Only one style was con-
sidered to represent the “ideal runner” (style A) and reduced this “1” by the value of “0” (i.e.
no effect). Meanwhile, the other styles, having unfavourable traits of various degree, were
given negative values (Fig 1, score column). Many of the unfavourable traits were visually dif-
ferent, although a similar negative value was assigned.
For the E-score (Fig 1), the value was applied directly with only four possible style assigna-
tions: between the most favourable, with the value of “1”, and the most unfavourable, with the
value of “0.25”. The value of “0” was only applied if no registry was obtained during the registry
period.
The Y-score was assigned the default value of “1” if no observation was registered. The 4
major types of observations (Fig 1), generally followed by a detailed description in the registry
notes, were considered all equally influential and assigned the same value of “–0.15”. Each type
was only counted once per registry period giving the lowest possible score of “0.4”. The Y-
score therefore, served as an indicator for the underlying incidences. However, during registra-
tion, if some incidence was considered having a major effect on the monitor-readings, or con-
tinuously reoccurred, the score could be assigned with a “0” with the representative mark (Fig
1), to differentiate numerically from the lowest possible calculated score of “0.4”.
Averaging out the scores
The running styles of each rat were registered with the corresponding marks for each of the
particular score values (Fig 1) during all registry periods of a training session. As each registry
period (5 min) could contain various styles registered, the notion of a dominant style (the
underlined mark in the registry) was considered as having occurred at least twice (in A and E-
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domains). A representative registry form used in the semiquantitative assessment, with three
different 5-min registry periods, is displayed in Fig 2 (Step 1).
For each domain of the score criteria (A, E and Y), within each individual registry period,
the score values were calculated (Fig 2, Step 2) as:
Aresulting in a training-session score for each rat (Fig 2, Step 3). The average training-session
scores were further calculated to represent periods of several sessions and the standard devia-
tions of these training-session scores were used to evaluate the variability between different
rats of the same experimental group.
Representation of the AEY score
The score should be represented as [A;E;Y], where a theoretical best rat would have [1;1:1] and
the theoretical worst rat [0;0;0]. The score should be accompanied by detailed information
about the training setup, including: (i) defined experimental period or duration; (ii) training
day-frequency per week; (iii) training sessions per day, type of session and resting period
between sessions (if applicable); and (iv) session time length (t), set speed (v), treadmill slope
angle (θ). The average score can be calculated to represent different temporary stages, i.e.: a
training day, a week of training, a general training program score, or a more extended-overall
score such as a rat life-span score.
Exercise protocols
All exercise sessions started with a 5-min warmup (not included in the session time length), to
gradually reach a target speed. The rats trained for 29 days over the period of 7 weeks, training
5 days/week (except when noted below), never training on the weekends. They trained either 1
session/day (always around 09:00 h) or 2 session/day (latter session around 17:00 h), as contin-
uous-training sessions.
Week 1 consisted of three consecutive training days before the weekend, 1 session/day
(except 2 session/day the last day), with the gradual changes of: v = 0.30–0.34 m/s, t = 10–25
min/session (θ = 0˚). Week 2 consisted of 2 session/day with the gradual changes of: v = 0.35–
0.45 m/s, t = 30–32 min/session (θ = 0˚).
Week 3 and 4 consisted of 2 session/day with v = 0.45 m/s, t = 30 min/session (θ = 0˚). Day
D (downhill protocol) consisted of 2 sessions with v = 0.55 m/s, t = until exhaustion (90
min) and 45 min/session (former and latter, respectively) and θ = −15˚. This day was the 2nd
day of week 5, were the 1st day was a resting day. Weeks 5, 6 and 7 consisted of 1 session/day
(around 13:00 h) at v = 0.30 m/s, t = 15 min and θ = +5˚, but only for one of the experimental
groups during the muscle injury recovery period. Week 7 consisted of only two consecutive
training days straight after the weekend.
Statistical analysis
Data in Figs 3 and 4 are represented as box plots. The box represents the interquartile range and
shows the first and the third quartiles, which are separated by the median. Black triangles repre-
sent the mean. Whisker end points represent the standard deviation, and the black circles repre-
sent outlier values. Scores between Week 1 –Week 4 and between Week 5 –Week 7 were
statistically compared using a Kruskal-Wallis test followed by Dunn’s multiple comparison post
hoc test. The Mann–Whitney U test was used to compare morning vs. afternoon scores within
each week and to compare Week 4 vs. D. Statistical significance was considered when P<0.05.
Results
Fig 3 presents the average score and standard deviation obtained throughout the entire train-
ing period, where each week, albeit with different count of training sessions, is separated with
at least a weekend of rest. The A-score significantly increases after the first week showing a
progressive lower dispersion trend. From day D until week 7, a similar pattern is observed,
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although starting from lower score values. Throughout weeks 1 to 4, the E-score follows an
opposite trend to the A-score, reflected in significant decreasing mean values. Day D shows a
marked and significant drop in mean values and the greatest deviation. Thereafter, the average
level increases with a simultaneous deviation decrease. The Y-score appears as quite stable
throughout the entire experimental period with a significant drop in the average at day D
accompanied by increased deviation and, furthermore, a slight non-significant decrease on
week 7 along with a greater deviation.
Fig 4 focuses on the first four weeks (training period), each separated by a weekend-rest,
where the two daily training sessions (at morning and afternoon) are displayed separately.
Whilst the overall weekly tendencies are similar in all three parts of the score, when comparing
Fig 3. AEY Score along an exercise program. A box plot representation of the weekly average values for the 3 parts of the rat AEY performance score
throughout a 7-week treadmill-training programme. On week 1 and 2 the rats (N = 75) carried out a gradual preconditioning training in preparation for a steady
training on week 3 and 4. On week 5, 6 and 7 the rats (N = 27, 12 and 6, respectively) carried out light rehabilitation exercise after one day of downhill exhaustion
exercise (N = 65) marked specifically as day D when commencing week 5. Statistically significant differences are indicated as follows:  vs. Week 1; # vs. Week 2;
† vs. Week 4; ‡ vs. Week 5. One, two, and three repeated symbols correspond to P<0.05, P<0.01, and P< 0.001, respectively.
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morning and afternoon mean values, the magnitude of the tendencies varies in A- and E-
scores. The A-score of morning session is significantly lower than the score of afternoon ses-
sion in the first two weeks. The opposite occurs with E-score, where the afternoon session-val-
ues are significantly lower over the 4 weeks. The Y-score shows a marked difference between
the morning and the afternoon sessions.
Discussion
General considerations
The main goal of this paper was to develop a semiquantitative tool for an extensive and sensi-
tive assessment of the exercise performance on a running treadmill- of laboratory rats. It is
easy to design a scoring system only based on “good runners” rats, but running ability is highly
Fig 4. AEY Score comparison at two different daytimes. A box plot representation of the morning and afternoon session-average values for the 3 parts of the rat
AEY performance score for 75 rats throughout the first four weeks of a 7-week treadmill-exercise programme. On week 1 and 2 the rats carried out a gradual
preconditioning training in preparation for a steady training on week 3 and 4. Statistically significant differences are indicated as follows:  vs. Morning within the
same week. One, two, and three repeated symbols correspond to P<0.05, P<0.01, and P< 0.001, respectively.
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variable. Surprisingly, in most published work, problems during habituation to run and per-
formance heterogeneity among trained rats are not mentioned, maybe because most of the
animals finally run relatively well or because researchers discard “bad runners”, which finally
are used for sedentary or control groups. In any case, it is a fact that the contingency of being a
“good runner rat” can be categorised. From our experience, some rats like running from the
very beginning of the experiment and they cope well with the run as the speed is increased.
Other rats appear putting much more attention on avoiding the shock grid than on forward
running, and others continuously touch the grid and need a long time to become continuous
runners. The question posed here is whether the score itself can semiquantitatively differenti-
ate between these types of running rats and if AEY score correlates to the qualitative differ-
ences observed when they are monitored during the training sessions.
Whilst the criteria within the A-score have been quantified in accordance with an impact
on the theoretical best style of running (mark A), the simple underlying mathematical design
does not allow an average score to be directly back-traced to the dominant style/mark regis-
tered. That sort of evaluation requires looking at the actual registry and using different sta-
tistical approach. All the accumulative average-calculations render the back tracing-style
estimation improbable. On the other hand, as the E-score focuses exclusively on the tail’s posi-
tioning as a sign of tiredness, its marks are more descriptive and correlate better with the actual
score value they give. The assessment for the Y-score is different since it serves rather as a qual-
ity control index for the data output from the treadmill monitoring apparatus.
Application of the AEY-score in different exercise protocols
We repeatedly noted that the rats caught up differently with the exercise on the running tread-
mill during the first training sessions. In Fig 3, this progression is reflected in the greatest devi-
ation in the A-score on week 1, which progressively decreases until week 4. Simultaneously,
the E-score demonstrates an increasing variation, suggesting a dissociation between the “skill”
to achieve a continuous running style and the endurance of the animal. Even more, it can be
hypothesized that as rats learn to run more continuously, they are more prone to fatigue prob-
ably because 1) the real ran distance increases; and 2) rat metabolism and anatomy is prepared
for short voluntary running bursts instead of continuous speed [14]. The exercise sessions at
day D are completely different; being a downhill running-to-exhaustion protocol, designed to
induce muscle damage instead of a continuous running at constant slope and speed. During
that protocol, a more A-score variety took place, which was reflected in a lower average A-
score with greater underlying variation; and the E-score dropped significantly, as expected in a
protocol designed to be extenuating. These results indicate that the AEY score is indeed able to
discriminate between good and bad performance: during regular, moderate training (weeks 3
and 4) most animals perform well, obtaining scores close to 1. On the other hand, at day D the
performance significantly decreased as a consequence of the exhaustive downhill running pro-
tocol. Indeed, a significant increase in the plasmatic concentration of muscle damage biomark-
ers creatine kinase-MM and myoglobin was found in a subgroup of animals sacrificed 24 h
after day D [23], suggesting that the alterations in the AEY score had a physiological basis.
Conversely, during the subsequent weeks (active recovery period from week 5 to week 7),
the exercise protocol was very light. However, despite the reduced speed and duration of these
sessions, rats exhibited significantly lower A-score the first week after the downhill running-
to-exhaustion protocol. This A-score decrease could be associated to muscle damage [23],
which could hinder rat’s ability to exercise continuously due to compromised muscle function,
soreness or pain. Thus, these results suggest that this tool is sensitive enough to discern
between healthy and animals with eccentric exercise-induced muscle damage.
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In general, it is likely that motor control memory plays a role in the success of a good A-
score; frequent similar sessions might improve it whilst less frequent or different sessions do
not. Perhaps the training time in relation to circadian and internal biorhythms is also a factor
of considerable importance. Comparing the morning and afternoon sessions (Fig 4), there is a
significant difference in the A-score in the first two weeks. Indeed, since rats are nocturnal ani-
mals, the lower A-score obtained in morning sessions it is not surprising. Nocturnal animals
are more prone to perform physical activity (search of food, mating, exploration) during cre-
puscular and night hours, which could explain their better performance during the afternoon
sessions. It could be hypothesized that the presence of more artefacts, faeces and urine drops
contacting the metal grid (reflected by the lower Y-score) during the morning sessions could
be due to a physiological stress response [24] as a consequence of the intrinsic circadian bio-
rhythm disruption in these animals. Furthermore, some learning effect and habituation to the
motorized treadmill could contribute to the better afternoon scores. Conversely, due to a rela-
tively short resting period before the afternoon session, the E-score (representing tiredness)
decreased.
Finally, as can be observed in Fig 3, the Y-score remained constant regardless of the degree
of habituation to the treadmill (week 1 vs. week 4) and the intensity and duration of the exer-
cise, which ranged from exhaustive (D) to light (weeks 5–7). Thus, parameters directly related
only to electric shocks count should not be used in rat performance and compliance evalua-
tion, but could serve as a good quality control indicator of training session.
Limitations and advantages
Probably the most adverse aspect of the proposed score is the non-automated assessment,
although it gets easier with practice. There are many calculations involved, albeit simple, and
setting them up in a software programme such as Microsoft Excel is one way to work automat-
ically through them. The main benefits, besides the pinpointing of potential outliers, are that
the method provides a systematic and sensitive way to compare rats in different treatments or
experimental conditions and even among different studies. Still, the assessment leading up to
an individual AEY-score will always be subjected to some bias. In terms of future develop-
ments, the next step for the AEY score would be to correlate it with physiological representa-
tive parameters of exercise performance, such as VO2 measurements, in a similar way to the
widely used Borg’s scale for rating perceived exertion in humans [25,26]. This could either
reinforce our style-score correlation or shed a new light on it. Perhaps it is difficult and unreal-
istic to get a %VO2max correlation with specific running styles, but it could correlate with our
0–1 scale in either or both the A- and E-score. Moreover, this and other physiological measure-
ments would be needed to further assess the validity of the proposed tool. Additionally, this
score system is open enough to be applied to different strains and age. However, because we
do not have tested this tool in these cases, one should empirically check if the AEY-score needs
any adjustments, especially regarding the running style. For instance, it is plausible that healthy
but aged rats would be unable to keep running with an A-style for a given speed and duration,
or that an specific running behaviour would be more prevalent in certain conditions. In these
cases, experimenters could decide to modify the scores and classifications to better fit their
experimental conditions.
Conclusion
As an inexpensive tool relying on the experimenter’s observational capacity, we have suggested
the three-part AEY score is a good method to assess and describe the laboratory rat perfor-
mance capacity during exercise on a running treadmill in a semiquantifiable manner. This
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would be useful for considering and correlating the sample dispersion with other physiological
or anatomical parameters and, moreover, to obtain the best from the animals in question, thus
contributing to promoting the principles of the 3Rs (Replacement, Reduction and Refinement)
in the use of experimental animals.
Supporting information
S1 File. RawDataAEYScore. This database contains the registered data and calculations for
the exercise style of each rat on a running treadmill according to the descriptions in Fig 1 and
as reflected in the sheet 1 labelled as “Styles”. The procedure was applied in four distinct phases
of a higher scale project designed for the study the effects of different interventions for skeletal
muscle recovery after injury induced by forced eccentric exercise in trained rats. This phases
were: a two weeks habituation to treadmill exercise period (sheet 2 “Habit”), a four weeks of
exercise training period (sheet 3 “Training”), a day of two sessions (4 hours of break) of down-
hill exercise until exhaustion (sheet 4 “DH Day”), and a rehabilitation period of 21 days (sheet
5”Rehab”). Pooled total data are available in a whole dataset (sheet 6 “Pool”).
(XLSX)
Acknowledgments
This study was supported by DEP2010-22205-C02-01 and DEP2013-48334-C2-1-P grants
from the Plan Nacional I+D+i (The Spanish Ministry of Economy and Competitiveness). The
authors are grateful to Elizabeth Radley, Inês Ferreira, Sergi Montero, Daniel Ramos, Maria
Tsimpidi, Sara Martı´nez, Marien Espino, Carla Lo´pez-Grado, Alexandra Giraldo and Fer-
nando Garzo´n for their technical cooperation. The authors declare no conflict of interest.
Author Contributions
Conceptualization: Juan Gabriel Rı´os-Kristja´nsson, Karen Mist Kristja´nsdo´ttir.
Data curation: Juan Gabriel Rı´os-Kristja´nsson, David Rizo-Roca, Cristian Andre´s Nu´ñez-
Espinosa.
Formal analysis: Juan Gabriel Rı´os-Kristja´nsson, Karen Mist Kristja´nsdo´ttir.
Funding acquisition: Gine´s Viscor.
Investigation: David Rizo-Roca, Karen Mist Kristja´nsdo´ttir, Cristian Andre´s Nu´ñez-Espinosa,
Joan Ramon Torrella, Teresa Pagès, Gine´s Viscor.
Methodology: Juan Gabriel Rı´os-Kristja´nsson, David Rizo-Roca, Karen Mist Kristja´nsdo´ttir,
Cristian Andre´s Nu´ñez-Espinosa.
Project administration: Gine´s Viscor.
Resources: Teresa Pagès, Gine´s Viscor.
Software: Juan Gabriel Rı´os-Kristja´nsson, Karen Mist Kristja´nsdo´ttir.
Supervision: Juan Gabriel Rı´os-Kristja´nsson, Joan Ramon Torrella, Teresa Pagès, Gine´s
Viscor.
Validation: Cristian Andre´s Nu´ñez-Espinosa, Joan Ramon Torrella.
Visualization: David Rizo-Roca, Gine´s Viscor.
Writing – original draft: Juan Gabriel Rı´os-Kristja´nsson, Karen Mist Kristja´nsdo´ttir.
Performance score for running rats
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Writing – review & editing: David Rizo-Roca, Cristian Andre´s Nu´ñez-Espinosa, Joan Ramon
Torrella, Teresa Pagès, Gine´s Viscor.
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Performance score for running rats
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A three-criteria performance score for rats exercising on a running treadmill.
Ríos-Kristjánsson, Juan Gabriel,Rizo-Roca, David,Kristjánsdóttir, Karen Mist,Núñez-Espinosa, Cristian Andrés,Torrella, Joan Ramon,Pagès, Teresa,Viscor, Ginés