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Thank you for making those adjustments in your revision.
I believe that you have successfully addressed the reviewer's concerns regarding the lack of comparison to freezing alone. It is now clear that your study does not comment on the efficacy of freezing alone and therefore cannot and should not be used as evidence against freezing alone as a method of euthanasia.
While this study does not address the entire question of best practice for cockroach euthanasia, it does represent a worthwhile study and deserves to be published. Therefore the caveats as stated above and within the body of your manuscript are necessary for publication.
Because you have taken the reviewer's concerns into account and clearly outlined the limitations of the study I now believe that this paper is ready for publication. Congratulations!
[# PeerJ Staff Note - this decision was reviewed and approved by Nigel Andrew, a PeerJ Section Editor covering this Section #]
Thank you for your resubmission to PeerJ.
This revision was reviewed by one reviewer who has highlighted again that there is a flaw in the experimental design or introduction of this paper. Please address the points raised about freezing as a method of humane killing.
It would be best if you can experimentally show the effects of freezing alone. If you cannot do that, then you need to make it abundantly clear in the intro and the discussion that you did not test freezing alone and therefore there is no evidence to suggest that freezing alone is inhumane.
The implication that freezing alone is inhumane will, as the reviewer stated, have an incorrect influence on institutions like animal ethics. We need to be really careful of the downstream impacts of animal welfare experiments and how that can affect animal ethics procedures and potentially harm practitioners is critical and needs to be carefully and thoughtfully addressed in this manuscript before we can approve it for publication here at PeerJ.
clearly written. relevant references.
Research question flawed given no evidence that freezing as a primary method of euathansia is distressful.
Line 61-64 implying that exposure to freezing temperatures could impart prolonged aversive behavior and potentailly distress and suffering is misleading and not supported by the references listed. Freezing as a primary method of euthanasia should be evaluated before suggesting use of isoflurane risking personnel exposure to waste anesthetic gas. Publishing this manuscript implying that freezing is inhumane without ruling out freezing as an acceptable primary method of euthanasia will incorrectly influence institutions to unecessarily use isoflurane.
Thank you for submitting this manuscript to PeerJ. Your manuscript has been reviewed by two experts in the field, and overall they found your manuscript to be well-written, convincing and important.
I generally agree with Reviewer 2, in that you really should be evaluating freezing alone as a method of humane killing for cockroaches, because that is standard protocol for many invertebrates. If you cannot test freezing alone, you need to provide a strong rationale for why not, with a statement saying that it is still an acceptable mode of humanely killing these animals.
Please see and respond to the reviewer's comments in your next submission and provide a detailed rebuttal letter indicating where you have made the corresponding changes in the manuscript.
Thank you and I look forward to reading the next submission.
Introduction and background clearly define the context the authors are working in and provide an appropriate presentation of the study. References are complete and relevant.
Structure conforms to PeerJ standards.
The Research Proposal Form for North Carolina Zoo has been duly filled in and has been approved by the NCZ IACUC (Institutional Animal Care and Use Committee). All relevant information are included.
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This is an original primary research that falls within Scope of the journal.
The goal of the research is well defined, relevant and justified; it is clearly stated that this research will fill some knowledge gaps about euthanasia in four cockroaches species.
The experiment protocol is rigorously described with all needed details.
Ethical standards are carefully considered in all steps of the protocol.
The novelty of the study is stated and the practical impact of its results are properly supported.
Statistical analysis supports the structure of the study and the validity of the results obtained.
Tables clearly summarizes the results obtained in the study.
The article is well written, clear, easily readable and understandable.
Line 339: "and" should be probably deleted
Good except for the recommendation not to use freezing given limitations of exposure and study design (See comments below).
Since freezing adult cockroaches in a commissary-kitchen freezer at 0 degrees F (-18C) is the most common euthanasia method used in zoos and museums, the authors should evaluate this method as a one-step method until 100% of all species are dead. Ideally, the cockroaches should be observed during the freezing process for aversive behaviors. Recognizing real time observations may not be feasible, the authors should at least check for unconsciousness/ death/ no movement at one-hour intervals as done in Table 1 as well as lack of recovery at room temp . Freezing should be also similarly be evaluated for all species as a secondary method at 30 minute to 1-hour intervals before “not recommending freezing” as an acceptable primary or secondary method of euthanasia.
LOTS of excellent tarcking of microenvironmental parameters and time to detah.
The authors should have evaluated efficacy of freezing of all 4 species over 30 minute to hourly intervals before not recommending freezing as an acceptable secondary method. The authors should also evaluate freezing as a primary method of euthanasia.
In the attached PDF, I've made some suggestions in blue font and deletions in red font especially when implying invertebrates experience pain or distress. Best to use "aversive behaviors". For the abstract, I also deleted reference to cockroaches in houses as readers may think the authors are implying pest cockroaches should experience a good death "euthanasia" as described in the publication.
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