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Chasing away accurate results: exhaustive chase protocols underestimate maximum metabolic rate estimates in European perch Perca fluviatilis

Abstract Metabolic rates are one of many measures that are used to explain species' response to environmental change. Static respirometry is used to calculate the standard metabolic rate (SMR) of fish, and when combined with exhaustive chase protocols it can be used to measure maximum metabolic rate (MMR) and aerobic scope (AS) as well. While these methods have been tested in comparison to swim tunnels and chambers with circular currents, they have not been tested in comparison with a no‐chase control. We used a repeated‐measures design to compare estimates of SMR, MMR and AS in European perch Perca fluviatilis following three protocols: (a) a no‐chase control; (b) a 3‐min exhaustive chase; and (c) a 3‐min exhaustive chase followed by 1‐min air exposure. We found that, contrary to expectations, exhaustive chase protocols underestimate MMR and AS at 18°C, compared to the no‐chase control. This suggests that metabolic rates of other species with similar locomotorty modes or lifestyles could be similarly underestimated using chase protocols. These underestimates have implications for studies examining metabolic performance and responses to climate change scenarios. To prevent underestimates, future experiments measuring metabolic rates should include a pilot with a no‐chase control or, when appropriate, an adjusted methodology in which trials end with the exhaustive chase instead of beginning with it.

The oxygen and capacity limited thermal tolerance (OCLTT) hypothesis is the subject of ongoing debate (Jutfelt et al., 2018), but within the theory's framework, aerobic scope (AS) is used to measure the full potential capacity of an individual's oxygen transport system above standard metabolic rate (SMR) and has been linked to individual performance and fitness (Pörtner et al., 2017). Thus, studies examining how aquatic species will physiologically respond to climate change often measure AS. It is typically quantified as the difference between SMR, which is the rate of oxygen consumption in a resting, postabsorptive individual at a given temperature, and the highest rate of oxygen consumption at that same temperature, maximum metabolic rate (MMR). Depending on the research question, however, AS can also be measured in factorial terms (AS factorial = MMR/SMR) (Halsey et al., 2018). In addition to being used to calculate AS, MMR alone has been used to predict future success under climate change scenarios.
For example, a thermally fixed MMR may indicate low adaptive capacity and predict limited success under warmer conditions (Sandblom et al., 2016) while the MMR of an individual compared with others in the same school can help determine spatial positioning within the school and impact food intake (Killen et al., 2012). No matter the framework, for study results to be used to accurately predict the future success of individual species the methods for determining MMR, SMR and subsequently AS must be accurate.
There is a wide range of studies focused on constructing the optimal aquatic respirometer and how to most accurately measure MMR in fishes (Clark et al., 2013;Norin & Clark, 2016;Roche et al., 2013;Rummer et al., 2016;Svendsen et al., 2016). Two well-established respirometer designs are the swim tunnel, in which a fish swims against a current generated within the respirometer, and the static respirometer, in which the fish's movement is largely restricted. Both have advantages and disadvantages depending on the focal species and study design.
The first study to examine the difference between a swim tunnel and a static respirometer with respect to MMR following an exhaustive chase protocol was performed on cod (Reidy et al., 1995). This study showed a higher MMR in cod Gadus morhua L. following a chase protocol but hypothesized that this could be due to excessive stress associated with the protocol and that measurements in the swim tunnel were more comparable to natural conditions (Reidy et al., 1995). Swim tunnel respirometry has since become a popular method for measuring MMR in species with an active lifestyle that involves continuous swimming and is useful in that it allows measurements both during the period of exercise and immediately following it (Clark et al., 2013;Killen et al., 2017;Norin & Clark, 2016). For fish that do not naturally swim for prolonged periods, using an exhaustive chase protocol has become a common alternative (Clark et al., 2013;Killen et al., 2017;Rummer et al., 2016).
Studies have shown these chase protocols produce values within the same range as those produced via burst performance and critical swimming speed protocols in a swim tunnel respirometer (Killen et al., 2007(Killen et al., , 2017Sylvestre et al., 2007, but see Rummer et al., 2016) and that oxygen consumption rate (ṀO 2 ) following exercise is higher than during the exercise itself (Norin & Clark, 2016).
Our focal species European perch Perca fluviatilis L. is routinely exercised using an exhaustive chase protocol to determine MMR since it has been cited as unwilling to swim against the current in a swim tunnel (Brijs et al., 2015;Jensen et al., 2017;Sandblom et al., 2016). In past experiments, P. fluviatilis has been manually chased till exhaustion (qualified as unresponsive to tactile stimuli) over a period of 1-5 min and then placed in a size-matched intermittentflow respirometry chamber where the individual is left to recover to its SMR for between 10 and 48 h (Baktoft et al., 2016;Brijs et al., 2015;Christensen et al., 2017;Jensen et al., 2017;Sandblom et al., 2016). This study aimed to quantify the difference in MMR and AS obtained using exhaustive chase protocols compared to a no-chase control and to establish the most appropriate method to induce maximum oxygen consumption in P. fluviatilis at our experimental temperature of 18 C. We compared two of the most common methods used to elicit MMR outside of a swim tunnel, an exhaustive chase and an exhaustive chase followed by 1-min air exposure (Norin & Clark, 2016), with a no-chase control. We compare the MMR, SMR and AS following the 'exercise' protocols with those from a no-chase protocol in which fish were transferred directly from their home tank to a static respirometry chamber. Although there are studies comparing the efficacy of exhaustive chase protocols with a variety of swim tunnel protocols, to the best of our knowledge no prior study has compared the results of MMR following 'exhaustive chase' methods with MMR achieved following a 'no-chase' control. One week prior to starting metabolic measurements, P. fluviatilis [n = 15, 170 ± 18 mm, 48.9 ± 15 g (means ± S.D.)] were weighed, measured and divided into five groups of three similar-sized individuals which were housed together to simplify size-matching of fish to respirometry chambers and allow easy recapture of individuals tested on the same dates. The water in the tanks in which fish were housed was maintained at 18 ± 0.5 C using thermostat heaters starting 1 week before trials began and for the duration of the experiment.

| MATERIALS AND METHODS
Aside from the stress experienced during chase and chaise + air protocols, stress was minimized for the duration of captivity and over the course of the experiment. Fish were sacrificed at the end of the experiment using an overdose of benzocaine.

| Respirometry setup
Metabolic rates were measured using intermittent flow respirometry based on the protocols described by Clark et al. (2013)  To prevent the build-up of microbes over the course of the experiment, the system was cleaned between each trial using bleach. After removing the oxygen mini sensors, 15 ml of bleach was added to each tank and flushed through the system for 5 min. This was followed by thoroughly rinsing the system with fresh water three times.
After cleaning, the system was refilled with tap water that had been aerated and maintained at 18 ± 0.5 C for a minimum of 24 h.

| Protocol description and experimental schedule
Each individual fish was tested using all three protocols following a blocked Latin square experimental design (Figure 1) to counterbalance any possible effects of treatment order. Fish were fasted for 23-25 h prior to the start of each trial, at which point three fish were removed simultaneously from their home tank using a mesh net and placed in a red opaque 9 l bucket with aerated tap water at 18 ± 0.5 C. One of three protocols, A, B, or C, was then performed on each individual before they were placed in the respirometer at the beginning of the 30 s wait phase. The wait phase is necessary to account for a lag in the system which can result in a nonlinear oxygen curve (Loligo-Systems, 2020) and fish were placed in the chamber during this phase so that the first measurement phase would be linear and thereby included in the analysis. Fish remained in the respirometry chambers in a dark room, overnight for a minimum of 17 h and 32 min. The fourth respirometry chamber remained empty for the duration of the trial in order to measure background respiration.
Protocol A, no chase: The individual was transported from its home tank (submerged in water) and placed directly into the respirometer during the wait phase (Chabot et al., 2016). The individual assigned this protocol was always placed in its respirometry chamber first. Protocol C, chase + air exposure: The individual was chased following the method described in Protocol B. Following the chase, fish were scooped into a mesh net and held out of the water for 1 min (Clark et al., 2012(Clark et al., , 2013Rummer et al., 2016), after which the individual was placed in the respirometry chamber during the wait phase. there is no MMR value and subsequently no AS value due to failure to start the respirometry software before placing the fish in the respirometry chamber (Supporting information Figure S1). R-code for calculations of all metabolic measures is available on the data repository Zenodo, DOI: 10.5281/zenodo.3873396.

| Statistical analyses
We used linear mixed-effects models to analyse the data set using package lme4 (Bates et al., 2015) in R. Factors "treatment" and "treatment order" were set as fixed effects with three levels: treatments A, B and C, treatment orders ABC, BCA and CAB. "Fish identity" was set as a random effect to account for individual variation between fish.
The interaction term Order:Treatment was nonsignificant for all metabolic measures (Supporting Information Table S1) and was therefore not included in the model. Adjusted repeatabilities (R), which control for variance caused by fixed effects Treatment and Order, were calculated for each metabolic measure using rptR (Stoffel et al., 2017) with a bootstrap value of 1000. The normality of residuals was verified with Shapiro-Wilk normality tests in package stats (R Core Team, 2019) and homogeneity of variance was verified using Levene's test in package car (Fox & Weisberg, 2019). A Grubbs test using package outliers (Komsta, 2011) was used to test for outliers. An ANOVA using package car (Fox & Weisberg, 2019) was used to test the significance of "treatment" and "treatment order" using type II Wald F tests with Kenward-Roger df. Finally post hoc pairwise comparisons using Tukey's method within package emmeans (Lenth, 2019) were used to test for differences in metabolic responses between treatments. Raw data was imported using package rMR (Moulton, 2018) and plots were created using ggplot2 (Wickham, 2016). All analyses were performed in R version 3.5.3 (R Core Team, 2019).

| RESULTS
We found a highly significant effect of treatment on MMR and AS (Table 1 and Figure 2b,c). There was a marginally nonsignificant effect of treatment on SMR (Table 1 and Figure 2a). The order in which individual fish received each treatment was not significant for SMR, MMR or AS (Table 1).
Pairwise comparisons using HSD-Tukey post hoc analyses showed that MMR was significantly higher in no-chase compared to both chase and chase + air treatments (Supporting Information - Table S2 and Figure 2b). There was no significant difference in MMR following chase compared to chase + air treatments (Supporting Information Table S2 and Figure 2b). The MMR 3 also show that MMR following no-chase was significantly higher than following chase and chase + air protocols (Supporting Information Table S2), but that there was no significant difference between MMR 3 in chase and chase + air treatments (Supporting Information Table S2).

This significant difference between MMR in different treatments
was reflected by the pairwise comparisons of AS, which also show higher estimates in no-chase treatments compared to chase and chase + air treatments (Supporting Information Table S2 and Figure 2c), but no significant difference in fish oxygen consumption between chase and chase + air treatments (Supporting Information - Table S2 and Figure 2c). Using the chase protocol resulted in a 16% underestimate of MMR and a 21% underestimate of AS compared to the no-chase control (Table 2). Using the chase + air protocol also resulted in underestimates of MMR (16%) and AS (24%) compared to the no-chase control (Table 2). There was no significant difference between the SMR values in no-chase and chase treatments

| DISCUSSION
Measuring oxygen consumption rate (ṀO 2 ) directly following an exhaustive chase protocol is thought to serve as the best measure of MMR using a static respirometry set-up because during this period the fish will be at peak ṀO 2 as it recovers and pays off the oxygen debt created during the chase protocol (Clark et al., 2013). However, we found that, under our study conditions, both chase and chase + air treatments underestimated MMR by an average of 16% and resulted in subsequent underestimates of AS (chase 21% and chase + air 24%) compared to the no-chase control group when calculating MMR based on the global maximum during the trial. The pattern of lower estimates of MMR following chase protocols even occurs when MMR is measured based on the first three measures of ṀO 2 after placing fish in the respirometry chamber (MMR 3 ) (chase 12% and chase + air 8%). Thus, our study serves as a call to thoroughly test that methods intended to elicit maximum oxygen consumption are having their desired effect. Our results suggest that a no-chase protocol should be included in pilot studies for any fish species that is tested using an exhaustive chase protocol, as has been recommended for small or nonathletic species (Clark et al., 2013), benthic species and ambush predators (Norin & Clark, 2016), and any other species unwilling to swim in swim-tunnel style respirometers (Brijs et al., 2015;Killen et al., 2007). A potential limitation in our study design is that our chase protocols using a hand-net could be considered mild compared to studies which chase fish by hand and include tail pinching to provoke a strong swimming response (Mochnacz et al., 2017;Roche et al., 2013;Rosewarne et al., 2016).
Oxygen consumption rates following exhaustive chase protocols were less variable than those following the no-chase protocol.
Supporting Information Figure S1 shows the difference in oxygen T A B L E 1 Output of a linear mixed model with fixed factors Order and Treatment and random factor FishID consumption over time, including the later peaks in ṀO 2 in the nochase treatment (Supporting Information Figure S2), which is assumed to be the result of spontaneous activity. The more limited range of oxygen consumption rates following an exhaustive chase could be the result of stress or a prolonged recovery from anaerobic exercise induced during the chase.
The marginally nonsignificant overestimate of SMR following the chase + air protocol compared to both the no-chase and chase protocols indicates that air exposure may be preventing fish from fully recovering within the timeframe of our experiment. This suggests that chase + air exposure should only be used in cases in which air exposure is an important part of the study question, as opposed to using it to enhance the effect of an exhaustive chase. However, the lack of a significant difference between SMR following the chase compared to no-chase protocols indicates that recovery from an exhaustive chase alone did not prevent fish from reaching their SMR over the duration of the study period, a concern that has been raised previously by Norin and Clark (2016).
We found that when calculating MMR using the global maximum ṀO 2 , the time at which MMR was reached was spread across the 17 h trial (Supporting Information Figure S2). This indicates that future studies should take advantage of the high temporal resolution of modern respirometry set-ups and include measurements from over the course of the entire trial instead of limiting MMR calculations to the measures immediately following the chase. Jensen et al. (2017) found differences in the time MMR was reached between trials at different temperatures. At low water temperatures (5 and 10 C) P. fluviatilis MMR always occurred immediately following the chase protocol, while at higher temperatures (15-27 C) MMR could also occur spontaneously over the course of the trial. To account for these differences, we propose future experiments use a method discussed by Norin and Clark (2016) in which the fish is placed directly in the respirometer for a long series of measures and then removed from the respirometer for a chase protocol and returned for a short measurement period following the chase. By taking the global maximum from both portions of the trial, underestimates from both, an unnecessary chase at high temperatures and a lack of chase at low temperatures could be avoided.
Accurate measurements are needed to make accurate predictions of species success under climate change scenarios, especially when making comparisons between species. Measuring MMR and AS are important aspects of conservation physiology, which seeks to understand and predict the ability of different species to survive under a range of current and future thermal regimes (Cooke et al., 2013).
Many studies have been focused on creating respirometry systems and methods which give accurate estimates of MMR in part so that future predictions within this realm of conservation will be accurate.
Our results suggest that future studies using static respirometry should use a no-chase control during pilot studies to determine whether any exhaustive chase protocol is needed. Additionally, studies with multiple temperatures or multiple species could use a chase protocol at the end of the experiment and use the global maximum ṀO 2 measure to prevent underestimates.

DATA AVAILABILITY STATEMENT
Data including the AutoResp files (cut to 17 h and 32 min) for each fish and R scripts used for analysis are available on the openly accessible repository Zenodo under DOI: 10.5281/zenodo.3873396.