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Article
Peer-Review Record

Posture as a Non-Invasive Indicator of Arousal in American Toads (Anaxyrus americanus)

J. Zool. Bot. Gard. 2021, 2(1), 1-9; https://doi.org/10.3390/jzbg2010001
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Reviewer 3: Anonymous
J. Zool. Bot. Gard. 2021, 2(1), 1-9; https://doi.org/10.3390/jzbg2010001
Received: 28 October 2020 / Revised: 21 November 2020 / Accepted: 4 December 2020 / Published: 7 January 2021
(This article belongs to the Special Issue Recent Advances in the Science of Zoo and Aquarium Animal Welfare)

Round 1

Reviewer 1 Report

This is a well written paper which links body posture with rates of throat expansion in American toads, which the authors posit can be a welfare indicator of arousal. The paper is easy to read and organized well.

However, I don't feel the authors have made a convincing argument linking body posture to arousal. For instance, they state that throat expansion is related to oxygen intake, which they even caution later on it does not always relate since frogs can use many types of breathing. However, even if it is related, the authors haven't made it very clear how this is related to arousal and welfare.

Moreover, even if it is related, the biggest question I have is why use posture? I applaud the authors for coming up with a novel method development and a well thought out Table 1. However, it seems as if posture is just a proxy for throat expansion because a frog needs to adjust its posture to make room and expand their throat. So if this is the case, why look at posture at all? Isn't it much quicker and less confusing to just looking at the throat expansion? I think the authors need to make it clearer why posture should be used instead of checking their throats.

I also think everything else about arousal that was studied is actually more interesting, such as the presence/absence of conspecifics, enriched vs basic housing, and time of day. I believe this should actually be the focus of the paper and then follow and go into how its related to posture. But that's just a suggestion. If the authors make the previous issues much clearer, it would drastically improve the manuscript.

Smaller issues are line 136 needs to be connected with 137. Also in Table 1, it is not easy to see a difference between 3 and 5. Perhaps a better picture or description?

Author Response

Reviewer 1:

This is a well written paper which links body posture with rates of throat expansion in American toads, which the authors posit can be a welfare indicator of arousal. The paper is easy to read and organized well.

However, I don't feel the authors have made a convincing argument linking body posture to arousal. For instance, they state that throat expansion is related to oxygen intake, which they even caution later on it does not always relate since frogs can use many types of breathing. However, even if it is related, the authors haven't made it very clear how this is related to arousal and welfare.

Our response: Thank you for pointing out that the argument wasn’t fully clear. We expressed caution in our interpretation of how throat expansion relates to arousal because of the complicated nature of anuran breathing patterns. However, there is little doubt that more throat expansions do indicate higher arousal since more throat expansions signifies that there is a greater likelihood that oxygen being delivered to the body. We changed the wording in the introduction (lines 58-59) and discussion (lines 356-358 and 363-365) to reflect the high degree of certainty that there is a positive relationship between throat expansion rates and arousal while still encouraging readers to consider the nuances of anuran breathing. We make the link between arousal and welfare clearer by reinforcing what was stated in the introduction (lines 29-30), that arousal is one of two components of welfare in addition to valence, by adding statements in the introduction (line 77) and in the discussion (line 300).

Moreover, even if it is related, the biggest question I have is why use posture? I applaud the authors for coming up with a novel method development and a well thought out Table 1. However, it seems as if posture is just a proxy for throat expansion because a frog needs to adjust its posture to make room and expand their throat. So if this is the case, why look at posture at all? Isn't it much quicker and less confusing to just looking at the throat expansion? I think the authors need to make it clearer why posture should be used instead of checking their throats.

Our response: Thank you for raising this question, we have added a sentence in lines 74-77 to address this point: “Although throat expansion rates may be informative on their own, posture is potentially a more practical measure of welfare in the field because it can be assessed instantaneously and when a toads’ throat may be out of sight of the observer.” Furthermore, we changed the wording in lines 58-59 that suggested that the focus of our paper may be developing breathing rates on their own as an indicator of welfare. From our experience, toads’ throat expansions were visible even from the lowest posture, so we don’t have reason to believe that toads raise themselves off the substrate in order to make more room for their throat.

I also think everything else about arousal that was studied is actually more interesting, such as the presence/absence of conspecifics, enriched vs basic housing, and time of day. I believe this should actually be the focus of the paper and then follow and go into how its related to posture. But that's just a suggestion. If the authors make the previous issues much clearer, it would drastically improve the manuscript.

Our response: Thank you for this suggestion, we agree that those are exciting findings as well. As the reviewer has indicated this is only a suggestion, we have opted to make the previous issues clearer as requested and plan to focus a second manuscript on the influences mentioned. We are confident that the present work introduces an important, novel tool that can aid researchers studying amphibian welfare.

Smaller issues are line 136 needs to be connected with 137.

Our response: Corrected.

Also in Table 1, it is not easy to see a difference between 3 and 5. Perhaps a better picture or description?

Our response: We added a descriptive clause describing posture 5 in line 160 beyond what is listed in Table 1 to further clarify what this posture looks like: “Posture 5, characterized by forelimbs and hindlimbs extended away from the body wall, was excluded because it did not occur frequently enough to generate a set of photos for inter-reliability testing. ” We agree that better photos of posture 5 would be ideal. Unfortunately, because this posture was so rare (occurring only twice in the entire study as mentioned in lines 210-211), the photos in Table 1 are the only photos we have of toads displaying this posture. Despite not having another option we still feel that the photos show an accurate representation of the posture (angle of >95 degrees between upper and lower limb, fully visible throat and chest, most of the underbelly lifted from substrate).The photos for posture 3 are the most representative that we have as it demonstrates an angle between 55 and 95 degrees between the upper and lower arm, a fully visible chest, and body weight distribution mostly on the lower ¾ of the toad’s body.

Reviewer 2 Report

This is a very interesting study investigating how to use posture in captive toads as a tool for animal welfare evaluation. The manuscript is very well written and the experiments are well planned, analysed and discussed. Besides a few small suggestions (see “detailed comments”), I only have some remarks regarding the figures and tables: Figure 2 and Table S1 are missing! (this probably just happened by accident and the authors have them already prepared). I further find Table 2 rather unnecessary in the main text, I think it should be shifted to the supplementary materials. If all figures and tables are added and placed right, this is all in all a very nice manuscript that is well worth publishing.

  • Line 36: Apoda is more commonly called Gymnophiona in scientific literature
  • Line 78: Toads are common all over the world. Please name the species you are working with here
  • Line 81, 88, 92 & 93: Please use the metric system in a scientific journal. Most readers are not familiar with feet, gallons and “
  • Line 121: Table S1 is not mentioned under supplementary materials (and I had no access to it, see above)
  • Line 146: better: “collected by a single observer starting at 11AM and 14PM”
  • Line 288-291: sentence seems to be missing a word

Author Response

Reviewer 2:

This is a very interesting study investigating how to use posture in captive toads as a tool for animal welfare evaluation. The manuscript is very well written and the experiments are well planned, analysed and discussed. Besides a few small suggestions (see “detailed comments”), I only have some remarks regarding the figures and tables:

Figure 2 and Table S1 are missing! (this probably just happened by accident and the authors have them already prepared).

Our response: Thank you, we have addressed this. We originally included a Figure 2 to visualize the Tukey test results, but much of it was redundant with Figure 1 and it did not add any clarity to the written results. We unintentionally left in a reference to the omitted Figure 2 which has now been removed from line 226. Table S1 was a printable version of Table 1 which we have now decided is unnecessary. We removed the reference to it from lines 128-129.

I further find Table 2 rather unnecessary in the main text, I think it should be shifted to the supplementary materials. If all figures and tables are added and placed right, this is all in all a very nice manuscript that is well worth publishing.

Our response: We agree, and in response to this comment and the similar comment of Reviewer 3, we have now removed Table 2 and included the only unique piece of information that was in Table 2 (the AICc value) has been added directly to the results text in section 3.2 Model Significance on line 236.

Line 36: Apoda is more commonly called Gymnophiona in scientific literature

Our response: Line 36 changed from Apoda to Gymnophiona.

Line 78: Toads are common all over the world. Please name the species you are working with here

Our response: Changed from “toads” to “American toads” in line 85 to reflect that the following information was all from sources specifically about American toads and not toads in general.

Line 81, 88, 92 & 93: Please use the metric system in a scientific journal. Most readers are not familiar with feet, gallons and “

Our responses: Completed. In lines 88-89 (what was referred to as line 81 )we changed several hundred square feet to “over 30 square meters”. In line 115 (what was referred to as line 88) we changed 55-gallon tank to “208.2-liter tank”. In line 118 (what was referred to as line 92) we changed 5.5’’ x 5’’ x 4’’ to 14 cm. x 13 cm. x 10 cm. In line 120 (what was referred to as line 93) we changed 3’’ to 8 cm.

Line 121: Table S1 is not mentioned under supplementary materials (and I had no access to it, see above)

Our response: Table S1 was a printable version of Table 1 which we have now decided is unnecessary following reviewer comments. We removed the reference to it from lines 128-129.

Line 146: better: “collected by a single observer starting at 11AM and 14PM”

Our response: Line 187 (what was referred to as line 146) 2:30PM changed to 14:30PM

Line 288-291: sentence seems to be missing a word

Our response: In lines 387-388 (what was referred to as lines 288-291) we added the following words in brackets for clarity: Another method [for determining emotional valence] is comparing animals’ postures in environments that are known to be favorable and unfavorable.

Reviewer 3 Report

The reviewer thanks the authors for an interesting study investigating a poorly researched topic: amphibian welfare. The article is well-written, although some additional information is requested. Specific line comments and suggestions are listed below:

“Throat”: suggest replacing with “gular region” throughout manuscript (see: Hui J, Sharma S, Rajani S, Singh A. The Specific Molecular Composition and Structural Arrangement of Eleutherodactylus Coqui Gular Skin Tissue Provide Its High Mechanical Compliance. International journal of molecular sciences. 2020 Jan;21(16):5593.)

Materials & Methods

Suggest briefly mentioning that toads are common captive animals in zoological institutions in the Study Species section--this would underscore the importance of your study from a welfare perspective

Lines 116-118: “The scores from the real-time and slow-motion videos were strongly correlated, indicating high accuracy for real-time coding of throat expansion rates (Pearson correlation, r = 0.97, p < 0.001).“ This should be placed in the Results section.

Lines 137-139: “The scores from the original and naive observer based on still images demonstrated good agreement (Cohen’s kappa, k=0.63).” This should be placed in the Results section.

It is currently unclear how many throat excursion measurements were performed (in total) for each toad over the 5 weeks of the study. Please define.

Lines 208-209: Recommend reporting the mean difference (along with the 95% CI of the difference and p values ) between the postures where there was statistically significant difference as this provides more practical information than P values alone. Example: “The difference in throat expansion rates for postures 4 and 2 was XX min (95%CI : XX to XX; P = XX)."

Figures and Tables

Line 210: could not locate Figure 2

Table 2: this stable is bit confusing to understand and may be better suited as supplementary data.

Author Response

Reviewer 3:

The reviewer thanks the authors for an interesting study investigating a poorly researched topic: amphibian welfare. The article is well-written, although some additional information is requested. Specific line comments and suggestions are listed below:

“Throat”: suggest replacing with “gular region” throughout manuscript (see: Hui J, Sharma S, Rajani S, Singh A. The Specific Molecular Composition and Structural Arrangement of Eleutherodactylus Coqui Gular Skin Tissue Provide Its High Mechanical Compliance. International journal of molecular sciences. 2020 Jan;21(16):5593.)

Our response: After thorough discussion among authors, we chose to keep the terminology “throat” instead of “gular region”. Of the anuran breathing papers we cited that mention a name for the region in question, one primarily uses “buccal floor” (Jones 1982), one uses throat (Jongh & Gans 1969), and one uses “floor of the buccal cavity” (Gargalioni & Milsom 2007). While there doesn’t seem to be unified terminology for this part of a toad’s anatomy, in all cases it appears to be defined the same way that we define it on line 149: “the area that expands during ventilation”. We feel that the paper mentioned by Hui et al. (2020) was justified in using the more technical term “gular region” as the paper was about the physiological mechanics of anuran breathing. However, we prefer the more accessible term “throat” since the focus of our paper is developing an accessible welfare tool.

Materials & Methods

Suggest briefly mentioning that toads are common captive animals in zoological institutions in the Study Species section--this would underscore the importance of your study from a welfare perspective

Our response: Done. We added the bolded phrase to lines 86-87: American toads are common throughout their native range in the Northeastern and Midwestern United States and parts of Canada [17] and are commonly housed in zoological institutions.

Lines 116-118: “The scores from the real-time and slow-motion videos were strongly correlated, indicating high accuracy for real-time coding of throat expansion rates (Pearson correlation, r = 0.97, p < 0.001).“ This should be placed in the Results section.

Our response: Done. We added a new section called “3.1. Validation of Methods” on line 229 and moved the aforementioned statement to this section.

Lines 137-139: “The scores from the original and naive observer based on still images demonstrated good agreement (Cohen’s kappa, k=0.63).” This should be placed in the Results section.

Our response: Done. We added a new section called “3.1. Validation of Methods” on line 229 and moved the aforementioned statement to this section.

It is currently unclear how many throat excursion measurements were performed (in total) for each toad over the 5 weeks of the study. Please define.

Our response: We have now clarified. The following sentence was added to lines 197-199: Because some toads were visible more often than others, the total throat expansion rates analyzed per toad ranged from 4 measurements to 43 measurements with an average of 23 measurements analyzed per toad.

Lines 208-209: Recommend reporting the mean difference (along with the 95% CI of the difference and p values ) between the postures where there was statistically significant difference as this provides more practical information than P values alone. Example: “The difference in throat expansion rates for postures 4 and 2 was XX min (95%CI : XX to XX; P = XX)."

Our response: Thank you, we have now added the mean difference and 95% CIs as recommended in lines 275-278.

Figures and Tables

Line 210: could not locate Figure 2

Our response: Corrected. As mentioned above, we unintentionally left in a reference to the omitted Figure 2 which has now been removed from line 226 (what was referred to as line 210).

Table 2: this stable is bit confusing to understand and may be better suited as supplementary data.

Our response: We agree, and in response to this comment and the similar comment of Reviewer 2, we have now removed Table 2 and included the only unique piece of information that was in Table 2 (the AICc value) has been added directly to the results text in section 3.2 Model Significance on line 234.

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