Journal of Applied Physiology -- eLetters for Copp et al., 106 (4) 1072-1078

Reply to Ole J. Kemi and Ulrik Wisloff (manuscript number JAPPL-00240-2009)

Reproducibility of endurance capacity and O₂_peak in male Sprague-Dawley rats.

Steven W. Copp, Robert T. Davis, David C. Poole, and Timothy I. Musch

Departments of Kinesiology, Anatomy & Physiology, Kansas State University, Manhattan, Kansas,

TO THE EDITOR: We wish to thank Ole Kemi and Ulrik Wisloff for their rapid, almost hasty, response to our paper. Their highly laudatory comments regarding previous publications from our group are appreciated. Whilst agreeing that the literature is “sparse” regarding establishing and standardizing reproducible exercise protocols in the rat – the stated purpose of Copp et al’s paper (1) - Kemi and Wisloff proceed to make several critical statements including the suggestion that the authors were not aware of their publications. As evidenced below these suggestions are without substance and, where such critical statements are based in fact, rather than opinion, we are happy to rebut them, as follows:

Criticism: The statement that “no rigorous analysis of exercise performance reproducibility has been performed in the rat” does “injustice to the rigorous analysis of exercise performance, including measures of O₂peak that has been undertaken by our group for more than a decade.” “It should be easy to find what has been previously published in the field.”

Rebuttal: We are well aware of the work of Kemi, Wisloff and colleagues and have taken care to cite such when appropriate (e.g., Musch, 2006, ref. 5). Moreover their practice of rewarding rats after running with chocolate is now standard in our laboratory. Furthermore, it should be acknowledged that Wisloff and colleagues have demonstrated excellent reproducibility between submaximal O₂ measurements (see figure 1; Wisloff et al., 2001, ref. 7). However, neither previously nor now could justification be found for citing them regarding the specific issue of within-animal reproducibility of either endurance capacity or O₂peak i.e., the stated purpose of the Copp et al. (1) paper (please note the specific title of our manuscript). Specifically, the two papers at issue that concern rats rather than mice (i.e., Wisloff et al., 2001, ref. 7 and Hoydal et al., 2007, ref. 2: Kemi et al., 2002, ref. 4, is wholly mice and, as such, irrelevant to the present intercourse) present only group mean data, rather than coefficients of variation (CV), for O₂ max in control animals. This type of data does not assess the variability within individual animals – a sentinel parameter in exercise studies particularly where the strongest study design is repeated measures using each rat as its own control. We argue that our demonstration of very low within-animal CV’s for endurance capacity and O₂peak at multiple points across ~5 weeks is original and was not assessed by Kemi et al. (4) nor Wisloff et al. (7). Indeed, it was not presented in the results or conclusions in their above-cited papers. Rather, almost all the data and text in those papers relates to protocol issues and the training of rats. Data similar to that of Copp et al. (1) do not appear anywhere in the manuscripts and the description of what Kemi and Wisloff may be referring to is cursory, “The reproducibility of O₂max …was examined in a third set of rats (6 female). A 1-day rest period separated tests.” The Results section is bereft of key data from these (group III) rats though in the legend to Figure 4 (ref. 7) it is stated that “sedentary animals only performed pre- and posttests.” However, mean data from 3 O₂max tests is presented which, again, lends no insight into within-rat reproducibility.

Criticism: Issue of ‘proper’ allometric scaling to ‘prevent’ growth-related decreases in O₂ peak.

Rebuttal: We are well aware of the issue and controversy surrounding allometric scaling within and between species (e.g., Weibel and Hoppeler, 2005, ref. 6). Accordingly, we elected to present the unadulterated data for the readers’ inspection and interpretation rather than manipulate it in this fashion.

Criticism: “…the presented evidence hardly implies any anaerobic component of the suggested endurance test, and that the ease with which rats may be instrumented does not make it the ideal experimental model.”

Rebuttal: Might we respectfully suggest that Kemi and Wisloff read the extensive critical power literature which, as addressed in Copp et al. (1), underlies the compartmentalization of the exercise energetics. Whilst, as explicitly stated by Copp et al. (1), critical velocity has not been directly demonstrated in the rat (and needs to be!), it has in every species and modality investigated from man to the horse, mouse, and salamander. This suggests that the hyperbolic character of the time-to-fatigue vs. speed (or power) achieved at such workloads is comprised of aerobic and so-called anaerobic energetic components and constitutes a fundamental property of muscular systems (e.g., rev. Jones et al. 2008, Ref. 3). Additionally, our statement, “… the rat is better-situated for invasive… catheters, and this fact should be considered when selecting the most appropriate and feasible experimental model” in no way implies that the ease of instrumentation makes the rat the ideal experimental model in all investigations. Kemi and Wisloff’s contention that it does is indeed perplexing.

Finally, the humane and scientific issue of reducing animal groups and numbers was specifically addressed by Copp et al. (1) in regards to measuring O₂peak and endurance capacity in rats. It is our sincere hope that other groups will continue to address this concern in future studies. In closing, we are happy to recognize the eminent position of the Kemi and Wisloff group in advancing the science and practice of running and training healthy and compromised rats on the motor driven treadmill. Any review of this area (e.g., Musch et al., 2006, ref. 5) would certainly be incomplete without reference to their work. However, as ratified by the reviewers the stated hypotheses of Copp et al. (1) could not be addressed by the data from Kemi et al. (4) or Wisloff et al. (7) or the extant literature.

Copp SW, Davis RT, Poole DC, Musch TI. Reproducibility of endurance capacity and O₂_peak in male Sprague-Dawley rats. J Appl Physiol , 2009, in press.
Hoydal MA, Wisloff U, Kemi OJ, Ellingsen O. Running speed and maximal oxygen uptake in rats and mice: practical implications for exercise training. Eur J Cardiovasc Prev Rehabil 14: 753-760, 2007.
Jones AM, Wilkerson DP, DiMenna F, Fulford J, Poole DC. Validation of the ‘critical power’ concept for human exercise using 31P magnetic resonance spectroscopy. Am J Physiol Regul Integr Comp Physiol 294: R585-R593, 2008.
Kemi OJ, Loennechen JP, Wisloff U, Ellingsen O. Intensity-controlled treadmill running in mice: cardiac and skeletal muscle hypertrophy. J Appl Physiol 93: 1301-1309, 2002.
Musch TI. Exercise protocols using rats and mice. In: Resource Book for the Design of Animal Exercise Protocols. Kregel KC, Allen DL, Booth FW, Fleshner MR, Henriksen EJ, Musch TI, O’Leary DS, Parks CM, Poole DC, Ra’anan, Sheriff DD, Sturek MS, Toth LA. American Physiological Society, Bethesda, MD, 2006, pgs 1-137.

6. Weibel ER, Hoppeler H. Exercise-induced maximal metabolic rate scales with muscle aerobic capacity. J Exp Biol 208:1635-1644, 2005.

7. Wisloff U, Helgerud J, Kemi OJ, Ellingsen O. Intensity-controlled treadmill running in rats: O₂max and cardiac hypertrophy. Am J Physiol Heart Circ Physiol 280: H1301-H1310, 2001.