Review Reports

Round 1

Reviewer 1 Report

General Comments

You created several abbreviations that not always you use them throughout the manuscript. If you want to abbreviate some term, please be consistent to use the abbreviation throughout the manuscript according to the journal’s guidelines.

Abstract – Your abstract is somewhat confusing. You need to add how many commercial and self-prepared raw diets you analyzed. Also, you need to be clearer on how you did this trial and what was the intent of using PDSC and the results of it, including the CG analysis. Where there any specific essential fatty acid that was in a higher concentration?

Introduction – your introduction could be shortened and have more details about what you did for your trial. I think you have to focus on 3 key aspects of your work. First would be the commercial vs raw cat foods. Second would be that fat profile of cat foods was not addressed yet. Third the oxidation, there are different methods and you chose 1. Why you chose this one? What are benefits of this one over the others? What are drawbacks of this method? I don’t see the need of talking about how to formulate foods (L72-82) or the fatty acid requirement and how to analyze them (L83-104).

Supplemental file – I would add a table with the guarantee analysis of these commercial foods and the calculated values for the guarantee analysis of the raw diets on a table on a dry matter basis just for a quick comparison. I would also add the brands and manufacturers of the commercial foods you used in this trial. Make sure you have 1 figure or 1 table per page, with the title in the same page as the figure/table. You can use page breaks to help you with this formatting.

Materials and Methods – Why did you decide to make 5 raw foods? What was the criteria you used to create these different foods? This is not clear from what you described in item 2.3. Please provide additional information regarding the authors choice in making 5 diets and their differences. It is not clear when you extracted the fats from the raw foods. Did you prepare the foods and extracted the fats right away? You waited to extract the fats from commercial foods once they expired, you must do the same for the raw foods, otherwise, you CANNOT compare these foods, because it is clear that you will have more oxidation on commercial foods vs raw foods. Since you prepared the raw diets yourself, you need to provide more information about how you prepared them. How were the ingredients mixed? Did you have to grind them or were they provided to you already ground?

Discussion – your discussion is too long and needs to be shortened to a couple pages. You use a lot of other references of things you didn’t test to make the case for the results you found. This is a too far of an extrapolation to be true. It could be the case, but you just cannot make these connections because you didn’t do the necessary work to make these connections. In the specific comments I have highlighted the portions I think you could reduce the text.

Conclusion – most of your conclusions are not supported by your research, rather from other research that you cited in your manuscript. You must conclude about what your research contributed to the scientific knowledge, not what other studies contributed. Please revise them accordingly.

Specific Comments

L26 – you need to describe what the abbreviation GC and PDSC are

L27 – what tool?

L29-31 – this sentence is very similar to the first sentence in the abstract, just written in a different way. Please consider removing it.

L41 – “Raw meat” not “Raw-meat” please revise this throughout your manuscript.

L67 – “for all ingredients used in the…” not “for all using ingredients in the…” please revise

L68-69 – please provide some clarity for this sentence, it is not clear what you what to say here

L72-82 – I think you are contradicting yourself here in this sentence. If “Most of the cat owners feed their pets with commercial cat foods” this mean that most cats are being fed complete and balanced diets. So how now, “a lot of pet owners find useful different kinds of nutrition balancing software and use them to compose home-made cat food”? While the raw food segment is growing rapidly, there is not a lot of people feeding these diets to their cats. You need to make it clear about it. Please revise this sentence. You also mentioned in L55-56 that “specialized knowledge” is required to formulate a diet. While there are some online tools pet owners can use, I think you need to be clear that they can be reliable, if the cat owner has proper knowledge in cat nutrition, which is not the case for the vast majority of pet owners. I don’t think these software are the problem when it comes to the formulation of the diet. I think the problem relies on most pet owners having not the basic understanding of how the tool works and what are the required nutrients their pets require. I think you need to revise this paragraph to address that there are tools to help pet owners to make raw foods for cats; however, how much knowledge (regarding ingredient nutritional composition, formulation, and cat nutrition) the pet owner has to be able to properly formulate a raw cat food?

L95-97 – this last sentence is incomplete, please revise

L103-104 – This sentence is not clear. What do you mean by “structure” and “the species composition”? Please revise this sentence

L123-124 – “Food labels” not “Feeds label” please revise

Table 1 – Please analyze all commercial foods for their dry matter composition. In commercial production there are intervals that are accepted for production, and you need to be more precise in your paper, so please analyze these commercial foods for their moisture content instead of reporting the moisture from the guarantee analysis. In dry extruded commercial pet foods, “crude fat” content needs to be analyzed as acid hydrolyzed fat because of lipid-starch complexes that form during extrusion. How did you analyze the fat content in those diets? If you did the normal fat extraction method, you will need to reanalyze the dry diets again.

L136-142 – please remove these sentences from the manuscript.

L145 – “Food was made” not “Food is made” please revise

L171-172 – what were the “reaction conditions”? You need to describe in a lot more detail how the hydrolysis was done, what were the reagents you used? Is this a standard method? Do you have a reference for this method? Please provide a lot more details about this analysis.

L185-186 – how many times each sample was analyzed? You must analyze all the samples the same number of times to have comparable results.

L209 – why did you have poor fat extraction of CW-2 and CW-3? You could have done multiple extractions to have enough fat for the analysis you did. Please analyze these foods and provide the values for them as well. Also, on L131 you mentioned that the abbreviation for the wet foods would be CW 5-7 and now you have CW 1-3, which one will you use? You must be consistent throughout the manuscript.

Table 2 – Why on Table 1 you use CW-5, CW-6, and CW-7, and on this table you use CW-1? You must be consistent with your treatment abbreviations, please revise this throughout the manuscript. Please add the results from the 2 CW foods. Extract more fat from those foods and analyze them for these fatty acids as well. It is not clear from your Materials and Methods, but if for this table you did an analysis that is different than the GC analysis for table 1, you need to describe this analysis in the Materials and Methods. In the GC analysis you would already have the valued for these fatty acids. Thus, it would be just a matter of computing these numbers from the GC analysis you did for table 1. Please explain better what is the difference between these 2 tables and if you did some special analysis to have the values for table 2.

L231 – I am not sure about “significantly more fat” because there is not statistical analysis performed in this manuscript. The numbers are different. Moreover, I don’t think the fat extraction method you use is the most appropriate for some of the samples you analyzed.

L232-233 – This seems rather unusual for me, since wet pet foods are meat based and the meat used usually have higher fat levels compared to the ingredients used to produce dry extruded foods. I think you have an issue with the extraction method you used to analyze for “crude fat”, you should analyze these samples using the acid hydrolyzed fat method instead.

L234-237 – for me these results are expected. Commercial pet foods have antioxidants added to the product to prevent fat oxidation. Even with the confounding effect you added to the analysis (by waiting for the diets to expire) you still would have the antioxidants effective against the oxidation you are trying to achieve with the PDSC. Since several other methods are used to measure fat oxidation, I would encourage you to analyze your fat samples for oxidation using other methods, like peroxide value, p-anisidine value, and thiobarbituric acid reactive substances. These analyses measure the compounds produced during oxidation rather than force the sample to oxidize, like PDSC.

L239-242 – nutritional profile of a pet food is not largely related to lipids. It is related to all nutrients present in the food, and it is largely related to the essential nutrients, all essential nutrients equally, not just the fatty acids. Please remove this paragraph.

L257-258 – this sentence here makes no sense. Your variation in fat composition of the raw foods is most likely exclusively from differences in composition of the cuts of meats you used vs. the ones used to create the nutrient database in the formulation tool. Differences like latitude, sex, age, season, genetics, diet composition, and activity level would be expected to have a bi impact in diet formulation if you were a big company sourcing ingredients globally throughout the year and trying to produce the same diet in all your facilities globally.

L262-263 -the formulation software you use has an approximation of the values from these meat sources. When you go to a grocery store or a butcher to get meat, you could have got a leaner or fatter cut of the meat and used it to make the diets. I don’t think this is an issue of the software, I think this is an error associated with the production of very small batches of food, you will get a lot of variation in the ingredient’s nutritional composition. Thus, you could discuss as this being a flaw in the self-prepared diets. Moreover, because of their inconsistencies in nutrient composition, it would also be difficult for the pet owner to estimate food intake to the animals and maintain their pets with ideal body weight.

L282-306 – remove these paragraphs. You did not evaluate digestibility of these fat sources. Moreover, it is not important which position the fatty acids are in the triglyceride, because if in positions 1 and 3 they will be digested and absorbed, and if in position 2 it will be absorbed as is. As you mentioned, commercial pet foods have more fat than the requirement, and with a digestibility of 88.8% it would never be a situation of deficiency due to low availability/digestion/absorption.

L3113-314 – I don’t think this is a quality response. The ingredients you used to produce the raw diets have a lot more fat in them compared to the ingredients used to produce the dry foods. Moreover, we know that a lot of omega-6 could create a low chronic inflammatory state in the animal. So, we balance the omega-6/omega-3 in commercial diets to try to limit the low chronic inflammation status that omega-6’s could start.

L314-315 what about food processing? What does it do specifically to AA?

L315-317 – the sole reason this was added to the diet is because of the differences in life stages these diets are intended for. All your commercial diets are formulated for adults at maintenance; therefore, they do not require EPA and DHA. Conversely, your raw foods were formulated for all life stages, which includes growth and reproduction, and these life stages have a requirement for EPA and DHA; thus, the difference in ingredient selection for these diets. You must to address that in your discussion.

L333 – What do you mean by “facilitative”, please revise this sentence.

L344 – “indicated” not “indicate”

L343-355 – I think you need to put this in perspective. EPA and DHA are only required nutrients for growth and reproduction life stages, not for adult cats at maintenance and geriatric cats, which are the vast majority of pet cats. Please revise these sentences to address this properly. EPA and DHA are not required nutrients for the vast majority of the pet population. Yes, they do have benefits even for adults and geriatric cats, but they are not essential for their survival.

L361-362 – please extract more fat from these samples and analyze them again. Likely you didn’t even have a representative sample to analyze for the fatty acid composition in table 1. Moreover, check the abbreviation you are using for these diets. What is it, CW-5 and 6 or CW-2 and 3?

L369-371 – again, I do not think the AA concentration in the diet is a matter of quality of the ingredients used to produce the diets as it is of the composition of these ingredients. Moreover, what does process has to do with the concentration of AA in the diet? This is not clear from your manuscript. Please provide a thorough explanation for the effects of processing on AA.

L371-375 – you need to clearly state that the differences in EPA+DHA of these diets and whole prey is because your raw foods were formulated for all life staged and, thus, had to have fish oil added as a source of these required nutrients for growth, gestation, and lactation. All your commercial foods and prey were evaluated for adult cats, which do not have a requirement for these nutrients. Please revise accordingly.

L381-385 – this sentence is long and somewhat confusing; I would suggest you to split it into more sentences to make the text clearer.

L394-396 – this last sentence needs some clarification as well, please revise it.

L398-400 – this is a proper tool as compared to what? I don’t think this is a conclusion from your work, since this tool was not tested, but it was simply used to formulate the raw foods. Consider removing this sentence.

L401-403 – How do you know that your raw foods “are more beneficial” than commercial diets if you did not feed these diets to cats? You cannot conclude that from the samples you analyzed and the results you have.

L404-408 – like I mentioned before, your raw diets are for a different life stage; therefore, they must have EPA and DHA. The commercial diets are for adult cats a maintenance; thus, there is no requirement for EPA and DHA. As a result, this conclusion you had is a flaw in how you designed the trial.

L409-410 – how is the fatty acid distribution “of particular importance in the digestive process”? You never addressed that in your Materials and Methods, Results, and Discussion. You have not tested anything other than the composition of the fats present in these diets. Not mentioning that most likely you did not have a representative fat extraction of your commercial diets.

L411-412 – The type of meat does impact the fat profile of a given food. The quality of meat not as much. Moreover, you did not test the same meat with different quality to conclude that meat quality has an impact on fat profile.

L416-418 – this is not a conclusion of your study. This is something you can mention in the Discussion section as you did a few times already. This would be an appropriate conclusion if this was a review paper.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Overall opinion: The research presented in the manuscript is actual and interesting. It provides a comparison of lipid fraction characteristics of commercial and self-prepared cat foods. Even the study is very interesting, the methodology appears not relevant or not clearly described. It is not clear how many samples were taken and analyzed from each of the groups. Since there is no statistical method described in the paper, it seems that only one sample was analyzed in each group. If so, there is not enough data in the paper to draw valid and scientifically relevant conclusions. Therefore, it makes no sense to point out other shortcomings that have also been observed.

I hope the authors will not be discouraged by the negative review, given that the idea of ​​the research is really interesting.

Therefore, if they can provide evidence that a statistically relevant number of samples from each group has been analyzed and that the results have been statistically processed, only then the conclusions will be scientifically based. In that case, the paper may be further considered for publication.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Dear Authors,

all comments are given in the article as sticky notes and some parts have strikethrough signs. Generally, the article is good, the methodology is fine as well as the results. My overall comment is that you have compared three different kinds of cat food, according to technological treatment and probably the use of additives (commercial food) that could be a reason for the differences in results. It can be only a review of fatty acid profiles in different cat food and can not be comparable. I recommend revision regarding the discussion of results.

 

Best regards

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

General comments

Abstract – You need to add the commercial diets in the objectives (L9-10). Since in the title of the manuscript you added “commercial cat foods” it is important and must be mentioned in the objectives as well. If you need to decrease your word count, you can remove the sentence “Extracted fat fractions were compares quantitively…” on L13-14 (because in the abstract you don’t mention any data comparing the raw foods with whole prey items) and sentence “Additionally, the FA profile and oxidative stability…” on L18-19 can replace the sentence in L13-14 and include the raw diets. You can just mention that the diets were analyzed for the FA profile and oxidative stability, since this is the data that you reported in your abstract.

Abbreviations – my rules of thumb for abbreviation are (1) the abbreviated term needs to appear in almost all pages of the manuscript and multiple times in some pages (2) no more than 10 abbreviations in a paper. I always try to abbreviate what is used a lot in the manuscript. Abbreviating a term that appears only a hand full of times throughout the manuscript is just an abbreviation that the reader will have to go back and find it to remember what it is. I think you need to review how many you use and what are the terms really important to abbreviate because you use it a lot. For example, 2-MAG you use only twice (L65 and L150)) and the description of this abbreviation comes in the second time it is mentioned in the manuscript (L150).

Introduction – Please remove all portions of what you did in the trial or analyzed in the trial from this session. These are to be added to the Materials and Methods session and must be removed from the Introduction.

Materials and Methods – why did you select these specific commercial cat foods? Was the selection based on the ingredients? Price? You need to have some sentences explaining why you selected these 7 commercial cat foods.

2.8. Statistical analysis – you cannot perform statistical analysis with this data because you do not have replication in this design. In this case, what you called “replications” are subsamples and not samples. They are not independent from each other to be considered replications. They came from the same sample of food. They could be considered samples only if each of the triplicate was from a different lot of cat food. This is a major flaw in your manuscript and this is the cause of the rejection. I think you need to submit this manuscript to a short communication instead, and the sole reason is that you cannot perform statistical analysis other than a mean value and the coefficient of variation of your triplicates.

Results – remove all the “significant differences” you may think you have. You cannot perform statistical analysis with the data you have.

Discussion – it is odd to me that you have a Discussion that does not follow the order that you presented the results. I would encourage you discussing your results in the same order you presented them. You could have made comparisons between dry and wet pet foods in your discussion as well. There are major differences in the ingredient composition of these diets that could explain in part the differences you reported among them.

 

Manuscript specific comments

L19 what a “favorable omega-6 to omega-3 ratio” means?

L34-45 in my view, these sentences could prevent you from publishing your work. You mention in them that there are few studies that evaluated raw cat foods and that more studies are needed. You also mention after the Wilson et al example that they should have fed the diets to cats. Then your study, you didn’t feed the diets to cats. You “just” evaluated the nutrient composition of the diets. Your work is relevant to the field, but I think you need to do some work on these sentences to address the issue without pointing the “flaw” of your study in the introduction. You can mention this limitation of your work in the discussion, saying that more feeding studies are needed to evaluate the diets using cats.

L49-50 This sentence should be removed from the Introduction, because this is Materials and Methods.

L53 EPA and DHA are only essential for some life stages. A common terminology is that EPA and DHA are conditionally essential. Adult and geriatric cats do not have a requirement for these FAs, only gestation and lactation and growing kittens have a requirement for EPA and DHA. Please address this requirement differences here.

L55-56 I don’t think this sentence is needed here. That is one of the ways a nutrient is essential/indispensable or not.

L57-58 Again, this is Materials and Methods and should be removed from the Intro

L58-65 Here you need to explain what the problem when an EFA is in the sn-2 position. If the digestion rate is lower, but 2-MAG is absorbed as is, it means that the cat will have that EFA absorbed and utilized in the body. If the absorption rate is the issue, then this is not clear in any of these sentences. Could you please expand more on the matter?

L65 MAG was not defined. Likely this abbreviation is not needed too.

L66-67 please remove “also” and “which was examined for tested foods” from this sentence

L85 I would change this header to “Commercial cat foods” and move L91-100 and L102 to the header “BARFny kalkulator and diet preparation”

L94 stage of life, not “stage of live”, please revise

L96-97 what were the “two meat ingredients”? Were the diets made out of difference combinations of beef- and poultry-based meats? Please provide some more details about these “meat ingredients”.

L104 I would change the header to “Self-prepared cat foods”

L116 what do you mean by “greater scale”?

Tables 1 and 2 – remove all statistical analysis of these tables. You do not have true replication to be able to perform statistical analysis as you did. Because your work is related to fats, instead of reporting the “Main animal ingredients” you should report the main ingredients in the diets that contribute to the fat of the diet. For example, EPA and DHA contents are not related to the beef, chicken, turkey or duck meats, but because self-prepared diets had salmon oil and cod-liver oil in their formulas.

Figure 2 – Should the added values of these 4 FAs within the same sample add to less than 100%? It is not clear from this figure title is these values reported are the concentration of each reported FA in the position sn-2 or if, for example, 56% of the linoleic acid in TAGs are in the position sn-2 in diet SP-1. Please make the correct revisions to make clear what these values represent.

L225-232 Please revise these sentences, they are not clear. I think part of these sentences not being clear is that figure 2 needs some work as I outlined in my previous comment.

L237-244 I would start your results with this portion here. Also, this should be reported fatty acid composition, not here with the oxidative stability. This is mainly because the data for the crude fata analysis is reported on tables 1 and 2 and it has nothing to do with the oxidative stability data.

Figure 3 – you cannot perform ANOVA analysis with the data you have. You do not have true replication for that.

L260-261 why was only this diet made twice? This may be the only diet that you actually have replication if in your triplicate you have samples from batch 1 and 2.

L276-278 this sentence should be part of your result section and not be here in the Discussion.

L290-297 why there are these differences in EFA content? You need to explain better the differences between prey and diets. For me the key difference is that the diets are formulated according to the known requirements of the cat, while prey is the composition of the whole animal. Another important distinction you need to make between diets and preys is that whole prey has a higher proportion of undigested materials (fur, feathers, etc.) that would dilute the essential nutrients compared to diets.

L299-301 this is not what you mentioned in L62-63 in the Introduction. These sentences contradict your Introduction. You must be consistent. Moreover, you need to add some references here about the formation of insoluble calcium soaps. What is the proportion of FAs that react with calcium? What is the proportion of calcium lost?

L306 EPA and DHA are conditionally essential as I mentioned previously, this should be addressed here too. Please revise this sentence

L321-328 I think you should also mention that self-prepared diets do not have any antioxidant added to them, which would prevent oxidation.

L334-336 you must address how this non-quantitative extraction method used could be representative of the fat profile of the sample. Case in point, how do you know that in the not-extracted fat portion there is a different FA profile?

L337-340 I do not think the differences are due to the “quality” of the ingredients used, but the differences in the ingredients used. You have more fat in raw meats because the rendering process was created specifically to remove moisture and fat of the raw material. Thus, it is obvious that there will be less fat in an animal by-product meal compared to a fresh meat (human grade or not). Please revise this sentence.

347 “high levels” not “high level” please revise

L348 what do you mean by “which rich source are added plants”? please revise

L360 I think “inter alia” should be Italicized.

L370 this is not the case for commercial cat foods. Commercial cat foods have vegetable oils that will impact the FA composition and distribution much more than meats. Moreover, meats are not commonly used in commercial dry pet foods, protein meals are, and the fat content of these protein meals is much smaller on a dry matter basis that the fat content of meats. Please revise this conclusion.

L371-372 you didn’t test PDSC against other methods that evaluate fat oxidation in pet foods; therefore, you cannot conclude that from the data you presented in this manuscript. Please remove this sentence from your conclusion.

Supplemental material comments

What were the commercial diets used? You must provide this information in case someone wants to replicate your study. You can mention in the Materials and Methods that the research and researchers were not sponsored by any of the commercial diet manufacturers and explain how these commercial diets were chosen. Why did you select these ones and not other commercial foods, or why not other different food recipes/flavors from the same (or not) manufacturer?

L28-29 I think “chicken protein hydrolyzed” should be either “hydrolyzed chicken protein” or “chicken protein hydrolysate”, please revise

L44 “yellow millet” instead of “millet yellow”

L54, 61-62 what do you mean by “chicken stomachs”. The “stomach” in the chicken is the proventriculus, which is not something you would find at the grocery stores. What I think you mean here is chicken gizzards, which is a muscular organ responsible for grinding the feed. Gizzards is something that you will find at the stores, just like hearts and livers. Which one did you use proventriculus (the true stomach in a chicken) or gizzard?

L110 please replace “date” by “data”.

L112 “is presented” please revise

Table S1 and S2 are missing the unit. I am assuming these numbers are expressed as percentages, but you need to add the unit on these tables. Why did you select these FAs and not all the EFAs? While if is not clear from your Introduction why the sn-2 position is a problem, I think you should focus on the EFAs in this position, since there seems to be an issue with it. The non-essential FAs will not have an impact in the cat’s health in this position, since they are non-essential and cats can synthesize them when needed and as much as needed. Please revise these tables and explain the issue with an EFA in the sn-2 position in the TAG in the Introduction of your manuscript.

Author Response

Dear Reviewer,

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

I would like to thank the authors for the comments and the effort made to improve the manuscript.

Regarding the new version of the manuscript, it can be seen that more extensive changes have been made, including statistical data processing.

First, I want to emphasize the importance of research methodology, because if research is methodologically flawed, everything else, including results and conclusions, makes no sense. Although the authors stated in their response to the reviewer that two samples were taken from each group, I could not find that information in the text of the manuscript. Namely, in the section 2.8. (Statistical analysis) only stated that each sample was analyzed with three replications. The number of samples per group is not specified, nor is the method of sampling. But this is not a big and irreparable omission (although it is surprising that such important information was not mentioned). The questions are: 1. Why did the authors omit the data on the number of samples per group (such an important data) nad they did not perform statistical data processing in the first version of the manuscript? 2. Are the two samples per group is statistically relevant number? 3. The data in Tables 1 and 2 in the first and second versions of the manuscript are different. Does this mean that the authors subsequently analyzed additional samples (one from each group) and then statistically processed them, or did they take the results of only one sample in the first version of the manuscript, even though they had two?

Furthermore, what seems very important are the results shown in the Table 1 & 2. At the very least, the data in the tables are confusing and unclear. The authors had to know that each table must provide enough data to be able to read independently (without looking at the text of the manuscript), which is not the case with these tables. The meaning of all abbreviations in the table must be clearly explained below the table.

Furthermore, regarding the data in the tables, the meaning of the label ‡ is unclear, as it is placed only in group SP-1 and only for DM and C10:0. Does it means that other data in the tables does not expressed as mean ± SD? Why the SD (if it is) displayed in some cases with one decimal, and in other with two or three decimals? Two decimals are sufficient; Uniformity is needed.

What is the meaning of GDM abbreviation? What are the differences between fat in DM and crude fat in DM? This is not explained in the Material and Methods.

Furthermore, the letters in the table` rows which indicate significant differences are totally confused! If you have five groups in the table (as in Table 1), five different letters (a, b, c, d and e) are enough to indicate differences in case that all groups are statistically different. Eight different letters (from a to h, if I noticed well) were used in Table 1 and the letter designations are confusing and it is impossible to read significant differences between groups from them. Here is an explanation on one of the simpler examples - C12:0 (second FA in the Table 1): From the data and letter designations of this FA it can be concluded that the amounts of C12:0 were similar in groups SP-1, SP-4 and SP-5 (they all marked with letter a), while in groups SP-2 and SP-3 it differed from each other and in relation to all other groups. If so, why are groups SP-2 and SP-3 marked by the letters de and bc, respectively? Why not with the letters b and c? In case that the letter designations refer to both tables, ie if they indicate differences between all 12 groups (tables 1 & 2), then it is not surprising that the results are displayed very confused and inconspicuous. Differences between groups arranged in two tables cannot be shown in such a way. A better way to mark statistical differences needs to be found.

Figure 3: why the letters to indicate differences between groups do not start with a? You've sorted them by size, but they're less clear.

Table 3: what is the purpose of Table 3? Since the composition of whole pray items was not the subject of the study, why do you compare that data (also in the text too)?

General suggestion: Too much emphasis is placed on the method of composing (balancing) meals through the text, ie the use of BARFny kalkulator. The study is about the differences in the chemical (lipid fraction) characteristics of commercial and homemade cat food, not about the different method of cat meal composing (balancing). Therefore, the method of composition of meals can be mentioned in the methods chapter, but further it is important to clearly present the obtained results and to logically explain the identified differences, and to support everything with appropriate references. This is not done in this manuscript.

Author Response

Dear Reviewer,

Please see the attachment.

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

I was the reviewer that requested you to perform statistics. However, the way you performed your statistics is wrong. Doing the extraction 3 times does not mean having 3 independent observations; therefore, you do not have replication. For you to have 3 independent samples, the foods should have been prepared 3 times, in the case of self-prepared diets, or you should have purchased 3 different lots of the same commercial foods. In your study, the food is the experimental unit, not the fat extracted from that food. Therefore, it is essential that you capture the variability in the food and not the variability form the person that analyzed the samples. For the commercial foods, you need to have at least 2 different lots of the same food for you to have at least 2 experimental units of that food. For your home prepared diets, ideally you would need to purchase the ingredients that contain fat again and make the diets again with these new ingredients. This is the correct way for you to analyze the differences of these diets, because then you are measuring the variability of the diets themselves.

I completely understand that the pandemic makes research a lot more complicated and have caused a lot of problems along the way for all researchers. However, that does not excuse something being done the wrong way. My suggestion to you is to evaluate the differences in fat composition of the different types of diets (dry vs. wet vs. self-prepared diets). Since these diets are from different manufacturers/recipes (in the case of commercial foods) or were prepared several times (in the case of self-prepared diets), then you have independent observations. This way you can perform statistical analysis. Even though you don’t have a balanced design, I see this is the best option you have to perform statistical analysis properly. Otherwise, if you continue analyzing your data the way it is now, I have no choice than recommend to the editor to reject your manuscript.

 

I don’t think it is debatable if EPA and DHA are essential nutrients for adult cats. As outlined by the NRC (2006), on chapter 15, they outlined 4 different nutrient categories: minimum requirements, adequate intake, recommended allowance, and safer upper limit. The minimum requirement “is defined as the minimal concentration that will support a defined physiological state”. And there is not a requirement for EPA+DHA on table 15-12. The adequate intake “is defined as the concentration in the diet of amount required by the animal of a nutrient that is presumed to sustain a given life stage when no minimum requirement has been demonstrated. The adequate intake is based on published data demonstrating the adequacy of a concentration or amount of nutrient for a give life stage in the target species, which is supported, in some cases, by comparative data from studies in other species.” Therefore, because there are no studies determining the requirement of EPA+DHA for adult cats, they used values from other species. Now, the recommended allowance “is defined as the concentration of amount of a nutrient in a diet formulated to support a given physiological state. The recommended allowance is mased on the minimum requirements and, where applicable, includes a safety factor for nutrients with uncertain bioavailability. If no minimum requirement is available, the recommended allowance is based on the adequate intake.” In addition, as you mentioned, commercial diets are based on AAFCO and FEDIAF for US and Canada and Europe. Because AAFCO and FEDIAF do not have a requirement for EPA+DHA and the majority of cat foods intended for adult cats at maintenance are not supplemented with these fatty acids, this is sufficient empirical evidence that these 2 fatty acids are not required nutrient by adult cats at maintenance. So please, revise your manuscript accordingly, EPA+DHA are conditionally essential, but they are not required by adult cats at maintenance.

 

L127 because you added this sentence here, I would not see any issues in reporting the manufacturers of the commercial foods you selected for this study. Again, this information is essential if someone would like to repeat your work or even use those foods as reference points.

Author Response

Dear Reviewer,

please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear authors,

since you have answered the doubts and clarified the ambiguites, I will recomended to editor tha publishing of your manuscript with a smal remark: the explanation of FA abreviations below the Table 1 is not necesser, since they are generally known.

Kind regards

Author Response

Dear reviewer,

Thank you very much for the time you spent reviewing our manuscript. We also thank you for all valuable comments. Following your suggestion, the explanation of FA abbreviations below the Table 1 have been removed.

Best regards