This study examine the costs of increased brood size in terms of oxidative balance and telomere length in captive passerines breeding at low temperatures. It shows no effect of the brood size manipulation, although sample size might be limited to conclude on the small effects expected (esp. on TL). However, interesting changes were found between incubation and nestlings feeding. This study is timely and provides new evidence in the hot debate on the oxidative costs of reproduction, by trying to add environmental constraints in a captive setting. I however have to main concerns. First, there was no control for the temperature manipulation and the authors do not sufficiently discuss why they were confident that such manipulation would increase the effect of brood size manipulation and not mask it. Second, the authors do not impartially present both sides of the methodological debate around the measure of reproductive costs, that is whether reproductive costs should be studied between non-breeding vs. breeding individuals or by modulating reproductive effort in breeding individuals. I think that their study does provide interesting information in this debate and should thus be discussed in that light.
Some sections (esp. the end of the first paragraph of introduction and the statistics section in M&M) could probably be made clearer with some re-writing, and with proof-reading by a native English speaker (as I am not one myself, I am not sure whether some sentences are correct or not).
l. 63-66 These sentences are poorly referenced and lack precision: which costs of reproduction are detected (in terms of body condition? survival?), in which experimental settings? Please explain in more details.
l. 70 “both processes may independently mediate” instead of “both processes independently may mediate”
l. 72 “the inevitable”
l. 80 Are you sure this reference is appropriate? The main point in Speakman and Garratt 2014 on the links between oxidative stress and aging is that, citing them: “Evidence is also accumulating to suggest that oxidative stress is not the predominant cause of ageing”
l. 81 In which direction and on which component of senescence (reproductive, actuarial)? Some cited references do not support this particular statement (Wiersma et al. 2004 does not measure senescence).
l. 81-84 I do not follow the logic between the two parts of the sentence, especially since some of the references study reproductive senescence and not actuarial (survival) senescence. Please rephrase to make that clearer.
l. 84-89 Please provide references for these statements.
l. 100-103 Here you should probably discuss the “oxidative shielding” hypothesis and how it might or not apply to your protocol (Blount, J. D. et al. 2015 Oxidative shielding and the cost of reproduction. Biol. Rev. doi:10.1111/brv.12179).
l. 109 As Descamps et al. 2009 manipulated breeding conditions by submitting birds to an immune challenge, could you better explain the rationale behind your manipulation of temperature? Indeed, Beamonte-Barrientos & Verhulst 2013 showed that increased metabolic rate through decreased temperature was not associated with higher oxidative damages (Beamonte-Barrientos, R. & Verhulst, S. 2013 Plasma reactive oxygen metabolites and non-enzymatic antioxidant capacity are not affected by an acute increase of metabolic rate in zebra finches. J. Comp. Physiol. doi:10.1007/s00360-013-0745-4). Moreover, in the context of your brood size manipulation, as individuals with increased reproductive effort might also produce more thermal energy, thermoregulation costs of low temperature could be partly compensated in the enlarged-brood group.
l. 137-142 It should be clearly stated that a donor nest gave nestlings to more than one recipient nest. Was there any effect of the nest of origin on nestling condition and survival?
l. 144-146 Could a nestling from a donor nest then be swapped and end up in a control nest, or were only original nestlings from enlarged nests swapped with the control nest?
l. 170-171 Could you provide the range and distribution of DNA concentration, 260/230 and 260/280 ratios?
l. 176 Why did you choose GADPH as the control gene?
l. 192-195 As requested by the MIQE guidelines, “repeatability” should not be expressed as the CV of Ct values (due to the distribution of Ct and the inherent inter-plate variation), but as the CV of the eventual quantification measure (here TL ratio). See Bustin, S. A. et al. 2009 The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem for details. Could you also provide proper repeatability estimates of TL ratios to evaluate the range of technical variation compared to between-samples variation?
l. 224 “mM” instead of “mmol”. Please use consistent notations between text and figures (either mmol/L or mM).
l. 224-226 Here again, as the scale is very different for the two measures, could you provide a repeatability estimate?
l. 239-260 It is not clear to me whether the models for adult body mass on one side and TL, dROMs and OXY on the other differed, as the effects on the latter variables are not explicitely mentionned. Also, why did you not check body mass before treatment as you did for the other variables ? Overall, rewriting this section to better highlight the fixed and random effects in each model would improve readability.
l. 309 Although they “sacrificed” individual offspring condition, the total reproductive output of enlarged broods (total body mass) was still higher, so parents seem to gain from brood enlargement without suffering more damages. Do you have data on nestlings survival after independence and future reproductive success (or from the literature) to assess whether parents of enlarged brood have any interest in increasing their effort?
l. 327-342 You should discuss here the actual meaning of the dROM and OXY measures. Indeed, dROM cannot be simply interpreted as a measure of ROS, as several antioxidants act early in the oxidative cascade, before ROMs formation, and OXY does not directly measure enzymatic antioxidant activities, but rather non-enzymatic activities.
Have you tested that the dynamics of TL and oxidative parameters with time did not differ between males and females? If so, please rephrase the statistics and results sections to make that clearer.
l.338 Do you have evidence that metabolic rate is higher during nestling feeding than during incubation in similar laboratory settings?
l. 343-351 Please cite some references in this paragraph.
l. 353-355 and 381 Could telomere loss also be interpreted as a cost of reproduction? How does it compare to telomere loss in non-breeding individuals over the same length of time?
l. 358-361 Do you have enough power to detect the small effect found in other studies?
l. 376-379 You might rather discuss that effect of age in the second paragraph of the introduction, when you discuss opportunistic vs. seasonal breeders.