The Impact of Food Enrichment on the Behavior of Cownose Ray (Rhinoptera bonasus) Kept under Human Care

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall remarks:

The need of enrichment towards fish in public aquaria is becoming more and more important to increase natural behaviour and overall welfare. This study is a nice example of different techniques of enrichment. 

What I missed in this study is a controle. A comparance with normal feeding routine and their affiliated behaviour. This makes it possible to say something about the use of enrichment opposed to normal feeding. In Materials and Methods this was mentioned pre-research was done. It would strengthen the study if this controle can be included within the results and analyses and thus strengthen the conclusions.

What I understand from the paper is that the four first quadrants are the same size, but the 5th is half if the display. This makes comparances between the 5 areas not possible in the way it's done in this study.

It's not clear how the enrichment items were managed. How long were they in the tank, were the eaten (partly)? How is the learning curve of the animals within the study

Small remarks and discussion points per sentence number:

74: size and age of the animals?

78: Itajara = itajar

82: which international animal welfare standards?

111: why suddenly a written reference in stead of a number used in other refs

116: It's not clear what quadrant 5 is. Is this the whole deeper area? Why is choosen for a larger quadrant 5 compared to the other 4? This makes it more difficult to compair the 5 quadrants. It's better to have 8 quadrants of the same size. And/or compaire the shallow and deep area.

135: describe depth in figure 2. and make clear quadrant 5

145: what was the diet defined by the aquarium team and what food items are used within the 4 enrichment items?

150: where were the enrichment items placed within the tank and where was the normal feeding station?

179: please define "enrichment type*Individual" in the table heading

181: is studyperiod the same as pre-feeding, during feeding and after feeding? In the notes after the table its called pre-, enrichment and postenrichment. Use same descriptions all over paper

233: how much of the food is eaten by the rays during the observations? Might this effect the post enichment activity?

250: it's not clear where the food/enrichment items are offered to the rays. Since this may also affect the swimming behaviour. this is first time you descibe this and still it's not clear. Why on different places, why creating an extra unknown factor within your research?

256: Is increase of fouraging in the post-enrichment phase an affect of the foodenrichment or lack of food eaten and food smell they encoutered? Would this be the same for normal food?

288: 'Captivity' should not be used throughtout the paper. Please change to ex situ, in aquaria, or under human care.

 

Author Response

Reviewer 1

 

Overall remarks:

The need of enrichment towards fish in public aquaria is becoming more and more important to increase natural behaviour and overall welfare. This study is a nice example of different techniques of enrichment. 

What I missed in this study is a controle. A comparance with normal feeding routine and their affiliated behaviour. This makes it possible to say something about the use of enrichment opposed to normal feeding. In Materials and Methods this was mentioned pre-research was done. It would strengthen the study if this controle can be included within the results and analyses and thus strengthen the conclusions.

Response: thank you for your suggestion. In fact, the control group is the animals during the baseline (pre-feeding phase in our paper; we changed pre-feeding for baseline, feeding for enrichment and pos-feeding for post-enrichment to maintain the same nomenclature throughout the text). They function as their own controls in environmental enrichment studies, since we follow the ABA protocol. The pre-research you mentioned was the preliminary observation period, where we constructed our ethogram e made the adjustments needed for the official data collection. Thus, our experimental delineation is correct for the study.

What I understand from the paper is that the four first quadrants are the same size, but the 5th is half if the display. This makes comparances between the 5 areas not possible in the way it's done in this study.

Response: thank you for your comment. The tank was divided in this way because of its configuration. The quadrants 1-4 were the shallowest while the quadrant 5 was the deepest. Rays normally did not use quadrant 5, but the records in there were due to one individual being expelled from quadrants 1-4 by another individual. This was inserted in the discussion section (lines 368-372). However, we agree with you and reanalyzed data dividing the tank only in 2 quadrants of the same size. We made all adjustments accordingly (in red on the paper).

It's not clear how the enrichment items were managed. How long were they in the tank, were the eaten (partly)? How is the learning curve of the animals within the study

Response: more information about how enrichment were managed were inserted in the text (all marked in red). Enrichment were eaten entirely most of the times by the rays, but sometimes parts of the enrichment were left by the rays and were retrieved from the tank. We did not registered a learning curve of the animals since this is not common for this type of study. We can say that the rays were habituated with the enrichment items since the beginning, exploring it immediately after their availability inside the tank.

Small remarks and discussion points per sentence number:

74: size and age of the animals?

Response: this information was inserted in the Methods section. See lines 100-103, in red.

78: Itajara = itajar

Response: thank you for that. The name was corrected (Epinephelus itajara, Atlantic goliath grouper). The specific epithet had the first letter capitalized, and this has been corrected.

 

.82: which international animal welfare standards?

Response: the citation was inserted. World Organisation for Animal Health’s “Aquatic Animal Code, Section 7 Animal Welfare”,  2023).

111: why suddenly a written reference in stead of a number used in other refs

Response: Sorry for this mistake. Reference adjusted to a number.

116: It's not clear what quadrant 5 is. Is this the whole deeper area? Why is choosen for a larger quadrant 5 compared to the other 4? This makes it more difficult to compair the 5 quadrants. It's better to have 8 quadrants of the same size. And/or compaire the shallow and deep area.

Response: Thank you for your comment. Yes, quadrant 5 was the whole deeper area. We changed this analysis following your previous suggestions. It makes much more sense this way. Now, we only have 2 quadrants of the same size: the shallow and the deep quadrants. The text was adjusted accordingly (in red).

135: describe depth in figure 2. and make clear quadrant 5

Response: changed accordingly.

145: what was the diet defined by the aquarium team and what food items are used within the 4 enrichment items?

Response: this information was added to the text (in red). The food inside the enrichment items were those designated for the ray’s diet.

150: where were the enrichment items placed within the tank and where was the normal feeding station?

Response: this information was added to the text In red).

179: please define "enrichment type*Individual" in the table heading

Response: done

181: is studyperiod the same as pre-feeding, during feeding and after feeding? In the notes after the table its called pre-, enrichment and postenrichment. Use same descriptions all over paper

Response: Yes, you are right. As said before, we have standardized the terms to avoid confusion and clarify the text (pre-feeding = baseline; feeding = enrichment; after feeding = post-enrichment).

233: how much of the food is eaten by the rays during the observations? Might this effect the post enichment activity?

Response: normally, rays ate all the food contained in the enrichment items. Many studies report an increase in activity and foraging after the use of environmental enrichment. This is a good long-term effect of the enrichment items on the behavior of animals. We believe that this was the case in our study. We inserted in the text the information about the consumption of the food present in the enrichment items to clarify. Also, we inserted a sentence about it in the Discussion section (in red).

250: it's not clear where the food/enrichment items are offered to the rays. Since this may also affect the swimming behaviour. this is first time you descibe this and still it's not clear. Why on different places, why creating an extra unknown factor within your research?

Response: we have included this information in the methodology (lines 168-194) and improved this description in the discussion. In fact, the items were always offered in the same places in quadrant 1. The items were always offered in pairs to reduce competition for the lanes. This is commonly done in enrichment studies. We have included some videos as supplementary material to make it clearer how and where the items were offered to the rays.

256: Is increase of fouraging in the post-enrichment phase an affect of the foodenrichment or lack of food eaten and food smell they encoutered? Would this be the same for normal food?

Response: It was probably an effect of the environmental enrichment, since the food was offered daily to the rays. It was the same for normal food since the food items were the same, only offered in different ways when inside the enrichment items.

288: 'Captivity' should not be used throughtout the paper. Please change to ex situ, in aquaria, or under human care.

Response: ok, thank you. Changed throughout the paper.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

 

Thank you for submitting this manuscript that explores the behaviour of cownose rays when provided with feeding enrichment. It is great to see some research on this fascinating species, and the work could aid in garnering further attention to elasmobranch research in zoos and aquaria.

At current however, there seem to be some large revisions required in the manuscript to ensure the work is scientifically robust. I have attached the PDF version of the manuscript with specific comments. Additionally, please consider the following points: 

1. Literature. Currently there are gaps in the explanation of the literature: the number of sources being cited is relatively few, and this has resulted in a limited overview of the natural feeding behaviour of the cownose ray. This means that the depth of knowledge as to what natural behaviour looks like is similarly limited. There is also some deficit in the explanation of behavioural research methods, food presentation in zoos and aquaria (and its effects on behaviour) and space use in aquariums. These gaps need to be substantially filled if the results are to be well interpreted.

2. Methods repeatability. Currently there are many gaps in the methods including the following: It is unclear when observations took place, how vegetables and enrichment was prepared, where enrichment was distributed and how fast behaviours were observed using an instantaneous focal sampling method. It is not clear what happened if the focal animal disappeared from viewing. Please provide all this information clearly in the methods as all of the above points affect repeatability.

3. Space use. Currently space use is poorly considered, and there is no graphical demonstration as to how rays use the space differently in different conditions. The space use information provides limited useful information in its current state: I recommend looking at Electivity Index or SPI or similar, as this would allow meaningful comparison of space use between conditions or between individuals whilst also accounting for the unequal space use point that has not yet been addressed.

4. Behaviour. Please provide clear graphical demonstrations of behaviour change in the conditions (e.g. activity budget or similar). Please explain how fast behaviours such as biting were observed (surely they were unlikely to occur exactly at the observation point in instantaneous sampling).

These are considerable changes, but ultimately the underlying project is useful. I hope these points are useful in substantisally redeveloping this manuscript for use by others.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

 

Dear Authors,

 

Thank you for submitting this manuscript that explores the behaviour of cownose rays when provided with feeding enrichment. It is great to see some research on this fascinating species, and the work could aid in garnering further attention to elasmobranch research in zoos and aquaria.

At current however, there seem to be some large revisions required in the manuscript to ensure the work is scientifically robust. I have attached the PDF version of the manuscript with specific comments.

Response: thank you for this positive feedback. We agreed with your suggestions and all of them were applied to the text, both the ones listed above and those on the pdf file. These changes improved the quality of the paper. Thank you very much.

Additionally, please consider the following points: 

1. Currently there are gaps in the explanation of the literature: the number of sources being cited is relatively few, and this has resulted in a limited overview of the natural feeding behaviour of the cownose ray. This means that the depth of knowledge as to what natural behaviour looks like is similarly limited. There is also some deficit in the explanation of behavioural research methods, food presentation in zoos and aquaria (and its effects on behaviour) and space use in aquariums. These gaps need to be substantially filled if the results are to be well interpreted.

Response: we inserted paragraphs about the food presentation in zoos and aquaria and its effect on behavior and space use in aquaria (in red in the introduction section). Also, we inserted more references about the cownose behavior, as suggested.

2. Methods repeatability. Currently there are many gaps in the methods including the following: It is unclear when observations took place, how vegetables and enrichment was prepared, where enrichment was distributed and how fast behaviours were observed using an instantaneous focal sampling method. It is not clear what happened if the focal animal disappeared from viewing. Please provide all this information clearly in the methods as all of the above points affect repeatability.

Response: thank you for your suggestions. Many of them was also suggested by reviewer 1. We inserted all these information in the text. Please, see all insertions in red, in the Methods section.

3. Space use. Currently space use is poorly considered, and there is no graphical demonstration as to how rays use the space differently in different conditions. The space use information provides limited useful information in its current state: I recommend looking at Electivity Index or SPI or similar, as this would allow meaningful comparison of space use between conditions or between individuals whilst also accounting for the unequal space use point that has not yet been addressed.

Response: Thank you very much for this suggestion. We changed the analysis evaluating space use by treatment and individuals, as well as calculating the SPI index.

4. Please provide clear graphical demonstrations of behaviour change in the conditions (e.g. activity budget or similar). Please explain how fast behaviours such as biting were observed (surely they were unlikely to occur exactly at the observation point in instantaneous sampling).

Response: thank you. We inserted on the text an explanation about the fast behaviors (please, see lines 147-151). We inserted a figure showing the behavioral changes during the phases. See figure 3. Some behaviors were so infrequently recorded that the graphs did not provide meaningful insights. Therefore, for these behaviors, the statistical results were presented alongside the text.

These are considerable changes, but ultimately the underlying project is useful. I hope these points are useful in substantisally redeveloping this manuscript for use by others.

Response: thank you for your invaluable suggestions. All of them were applied (in red on the text).

 

About the suggestions made directly in the pdf. The English was converted for the American style. Almost all suggestions were accepted. The only suggestion not accepted is explained above:

1 - Regarding the use of bibliographic references on rays, we agree with you on the preference for studies focusing on rays. However, your mentioned studies primarily examine animals in their natural habitat, specifically focusing on foraging behavior in natural environments. No studies on rays have reported findings similar to ours, nor have any studies been conducted on sharks. We did find one study involving bony fish, which we have now cited. Studies employing environmental enrichment for cartilaginous fish are exceedingly rare and do not provide information on post-item removal effects on fish behavior. Therefore, in addition to incorporating this reference on bony fish, we have included a statement highlighting this knowledge gap and suggesting the need for further research."

2 - About he colors of the figures: they have no signification other than differentiate the treatments showed on the figure

3 - Regarding the capitalization of scientific names in the references, we attempted to modify this, but it was an automatic change made by the reference management software (Zotero). Before submission, we manually corrected all the references, but we are unsure if upon opening the file, they will revert to the incorrect spelling. We apologize for any inconvenience this may cause.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the extensive changes made by the authors.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for submitting a revised copy of your manuscript. It is clear that all concerns have been addressed in full; this work is now scientifically more robust. Your revisions are clear throughout the manuscript, and I look forward to seeing this work in print!