Influence of the artificial sodium saccharin sweetener Sucram® on the rumen content and rumen epithelium microbiota in dairy cattle: A pilot study

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1. 

   Version published to 10.1099/acmi.0.000473.v1 on Access Microbiology

   Dec 5, 2023
2. 

   Access Microbiology

   Oct 27, 2022

   Dear Dr Schmitz-Esser, Thank you for your submission, please read and address the reviewers comments. Reviewer one has indicated significant concerns (both methodological and ethically), which while not as strongly worded, are similarly indicated in reviewers two and three. While it is appreciated that this is a pilot study, it is important that, regardless of the scale, the study is carried out to a high standard and that with the use of animals, all appropriate care data is included. Due to this, I have opted for major rather than minor revisions, mostly to allow appropriate time for revisions. Any questions you have, please get in touch. Best wishes, John. This is a study that would be of interest to the field and community. The research you have presented is not scientifically sound. The reviewers have highlighted major …

   Dear Dr Schmitz-Esser, Thank you for your submission, please read and address the reviewers comments. Reviewer one has indicated significant concerns (both methodological and ethically), which while not as strongly worded, are similarly indicated in reviewers two and three. While it is appreciated that this is a pilot study, it is important that, regardless of the scale, the study is carried out to a high standard and that with the use of animals, all appropriate care data is included. Due to this, I have opted for major rather than minor revisions, mostly to allow appropriate time for revisions. Any questions you have, please get in touch. Best wishes, John. This is a study that would be of interest to the field and community. The research you have presented is not scientifically sound. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. The reviewers raise concerns regarding the scientific rigour and experimental design of the work. The reviewers believe the results shown in the manuscript do not support the conclusions presented.

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3. 

   Access Microbiology

   Oct 25, 2022

   Comments to Author

   A novel study with impactful and interesting findings. 1. Methodological rigour, reproducibility and availability of underlying data *Lines 136-140, Perhaps this belongs in results/discussion? It is mentioned in the discussion and seems more appropriate than in methods. *Were any positive or negative controls utilized in library preparation? If so, what were they and how was potential contamination addressed in analysis? *Is there a reason OTU methodology was utilized instead of ASV? Were there any pseudo counts or minimum thresholds added (OTU present in inconsistent in use *Consider adding hypothesis to abstract and subsequently the introduction. 4. Literature analysis or discussion *Perhaps consider adding material to introduction on expected metabolism with saccharin and also a few sentences on …

   Comments to Author

   A novel study with impactful and interesting findings. 1. Methodological rigour, reproducibility and availability of underlying data *Lines 136-140, Perhaps this belongs in results/discussion? It is mentioned in the discussion and seems more appropriate than in methods. *Were any positive or negative controls utilized in library preparation? If so, what were they and how was potential contamination addressed in analysis? *Is there a reason OTU methodology was utilized instead of ASV? Were there any pseudo counts or minimum thresholds added (OTU present in inconsistent in use *Consider adding hypothesis to abstract and subsequently the introduction. 4. Literature analysis or discussion *Perhaps consider adding material to introduction on expected metabolism with saccharin and also a few sentences on "implications" or "reason" for supplementing Sucram. While this becomes evident in the discussion it may help to include 1 line in the abstract and a bit more in the introduction. *Line 385-288: Consider adding in details on differences expected based on digestive tract type and also specific tissue (rumen vs. small intestine vs. large intestine), what might be expected in the small intestine of ruminants? *General comment: Consider offering speculations (or supported) mode of action information when possible. This was excellent when discussion SRB, methanogens, etc.! 5. Any other relevant comments *Consider reminding readers if results being interpreted in the discussion are RCM or REM.

   Please rate the manuscript for methodological rigour

   Good

   Please rate the quality of the presentation and structure of the manuscript

   Good

   To what extent are the conclusions supported by the data?

   Partially support

   Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

   No

   Is there a potential financial or other conflict of interest between yourself and the author(s)?

   No

   If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

   Yes

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4. 

   Access Microbiology

   Sep 28, 2022

   Comments to Author

   This study evaluated the effect of a Saccharin-based sweetner, used to improve intakes and efficiency, on the rumen content and epimural microbiota in canulated dairy cows. The manuscript is well written, the topic is relevant and of interest to the field, and with some revisions and amendments can be accepted for publication. Issues: - I understand this is a pilot study; the fact that the Sucram was added directly through the fistula, does this limit the extent to which these findings can be considered in a "real" scenario, where the animals would consume the additive as part of it's feed? This is discussed briefly but should be expanded on. - There is a repeated theme in the Discussion section of reference to SCFA and SCFA-producing bacteria which were among the differentially abundant OTUs. …

   Comments to Author

   This study evaluated the effect of a Saccharin-based sweetner, used to improve intakes and efficiency, on the rumen content and epimural microbiota in canulated dairy cows. The manuscript is well written, the topic is relevant and of interest to the field, and with some revisions and amendments can be accepted for publication. Issues: - I understand this is a pilot study; the fact that the Sucram was added directly through the fistula, does this limit the extent to which these findings can be considered in a "real" scenario, where the animals would consume the additive as part of it's feed? This is discussed briefly but should be expanded on. - There is a repeated theme in the Discussion section of reference to SCFA and SCFA-producing bacteria which were among the differentially abundant OTUs. However, treatment had no effect on the SCFA, so it's hard to make this jump, based on the data and results at hand. - Some of the data analysis approaches are perhaps not optimal; e.g. using T-tests for non-normal data. ANCOM and ALDEX2 are also far more robust for the analysis of amplicon sequencing data (Nearing et al., 2022; Microbiome differential abundance methods produce different results across 38 datasets. Nature Communications). No P-value multiple testing procedure seems to have been implemented. - More taxonomic detail should be added to the figures; I think re-analysing at a higher taxonomic level might be beneficial here given the relatively modest shifts observed. Minor corrections L53 - "..in abundances of.. L57 - Typo, should be "understudied" L74 - Lactating rather than milking I think L74 - "Several recent..." L81 - You can just say that no research exists, no need for the "little or none" part as you state later in the paper than this is the first study in this area (eg. L109). L86 - the statement about mongastric species should have a reference. L93 - antibiotic not antibiotics L94 - When talking about alternative feed additives you should state the goal of their use; e.g. improving gut health/reducing disease perhaps, and reference appropriately. L102 - I would say "host animal" or simply "host" L104 - What signalling are you talking about here? I think you should make this clearer. L115 - Is it true to say this now? There have been a good number of papers examining the epimural microbes at this point (e.g. https://www.frontiersin.org/articles/10.3389/fmicb.2021.625400/full ; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846140/ ) L118 - I think a stronger statement of rationale is needed here, this one is a bit vague without detail. L142 - "region" not part L188 - Is this an appropriate test? T-tests assume normally distributed data; I don't believe A-div data fits this. Nothing about data transformation here, I would say a non-parametric test like a Kruskal Wallis or similar, depending on the number of groups, would be more appropriate. L194 - Why ANOSIM and not PERMANOVA? The latter performs better. See " PERMANOVA, ANOSIM, and the Mantel test in the face of heterogeneous dispersions: What null hypothesis are you testing?; Walsh et al., 2013" ) L208 - Was any multiple testing procedure (e.g. FDR) applied to this data? L211 - Can just say "Concentrations of acetic, butyric, ....) to be more concise. L235 - Again a T-test; normality? L351 - see above; is this then representative of a "true" effect on the rumen microbes? L375 - highly abundant - from what I can see in Fig. 4, only 2 of the DA OTUs comprised >1% of the total.. can we say they are "highly abundant"? L376-379 - this is a stretch, as you have analysed the data at the OTU level. So you could say that "OTUs belonging to some members..." etc. in that manner. Alternatively (and I suggest this) you could do the analysis at the Genus level rather than OTU. L379-382 - I would include the alpha and beta diversity findings here L399 - degradation of what? L400-405 - this section is a bit vague and rambling and should be presented more clearly. L412 - what pathways? Where are they documented? L422-425 - Ok, discussion about SCFA - but there was no impact on SCFA here? So your statement is not really reflected in your data. L427-8 - I would mention taxonomies when discussing individual OTUs like this. L444 - Ok, so what does this observation suggest in the context of this study? L449-455 - Again we are seeing discussion of SCFA data, but with no changes observed as a result of treatment here, it's hard to make these links. L468 - Prevotella was not the most abundant Genus? You state clearly in the results section that Succinivibrionaceae UCG-001 was the most abundant. In fact the presence of this genus in such high proportions in a couple of your samples is noteworthy, as it is rarely seen in such high abundance in the rumen. You should include discussion of this. Fig. 4 - I think the taxonomy of each OTU needs to be included here;

   Please rate the manuscript for methodological rigour

   Satisfactory

   Please rate the quality of the presentation and structure of the manuscript

   Very good

   To what extent are the conclusions supported by the data?

   Partially support

   Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

   No

   Is there a potential financial or other conflict of interest between yourself and the author(s)?

   No

   If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

   Yes

   Read the original source
5. 

   Access Microbiology

   Sep 6, 2022

   Comments to Author

   1. Methodological rigour, reproducibility and availability of underlying data. No rigor statistically and no full description of methods for reproducibility. Authors have not provided animal care data, since there is a tissue biospy done, this is an ethical issue as well. 2. Presentation of results Poorly organized with significant results in the suppplementary tables 3. How the style and organization of the paper communicates and represents key findings No key findings, no clear information towards a hypothesis provided 4. Literature analysis or discussion Literature is not fully developed and discussion is strictly hypothetically assumptions assembled from other research with no context given to the results presented in this study. 5. Any other relevant comments Please do not start sentences …

   Comments to Author

   1. Methodological rigour, reproducibility and availability of underlying data. No rigor statistically and no full description of methods for reproducibility. Authors have not provided animal care data, since there is a tissue biospy done, this is an ethical issue as well. 2. Presentation of results Poorly organized with significant results in the suppplementary tables 3. How the style and organization of the paper communicates and represents key findings No key findings, no clear information towards a hypothesis provided 4. Literature analysis or discussion Literature is not fully developed and discussion is strictly hypothetically assumptions assembled from other research with no context given to the results presented in this study. 5. Any other relevant comments Please do not start sentences with Because and if you use However it is never after ; but only to start a sentence. Impact: Line 55: what are relevant metabolic processes Line 63: what is the surrounding environment Introduction: Line 76: what are stress conditions Line 91: leaky gut is not for the rumen but for hindgut Line 105: this is from an old reference and has no context in molecular microbiology Line 115: this is not true please check the literature again What is the chemical structure of sucram - how does it breakdown chemically - which metabolic processes could it impact? Why do you think it will impact the microbes only? No hypothesis Methods: Line 122: age, DMI, BW are needed for context Line 122: 5 cows is very low - please perform a power analysis Line 130: what is the sugar content of the diet - this is necessary to understand the current conditions in the rumen Line 134: Why this dosage Line 139: ADG is not for lactating cows - please remove all references Line 142: RCM and SCFA sampling is not described Line 152: bead-beating time and speed Line 172-182: please include year of release, website and date of access for all online platforms and databases Line 181: is not stated above Line 185: If the 2 niches are handled separately why is the subsample depth equal Line 187: Was the data normally distributed? Was it normalized? T-tests require data normality. Line 213: procedures must be described in brief. Results: Line 238-321: Why are microbial populations between baseline and d28 described together but are separated in the Figures. Line 288: This is strange that there are 3.05 million reads on a low diversity population versus 910,228 reads on a high diversity population. Either your DNA extraction was done insufficiently for forage associated bacteria (highly possible and well described in the literature) or there is an error in your samples. Also why are there 30 samples for the REM? 5 cows - 2 time points, 1 niche = 10 samples - similar to what is written in the RCM section. Line 290: 6511 is an excessively high number of OTUs for the REM population - there is an error in your bioinformatics pipeline and an overestimation that started with the 3.05 million sequences. Figure 3: Where is the pvalue of the ANOSIM Figure 5: There are major issues with your mM of acids - please check the literature and you will see that either your method has issues or your have mislabelled your SCFA. Line 347: of Line 350: The authors need a better reason to not take associated production data if they want to compare their results to beef cattle where production changes have taken place. Line 352-369: Please remove. There is no reason to compare small and large intestine in monogastrics to ruminants - this makes no sense and has no context in a paper where fecal samples were not considered. Line 374: Why is this unexpected - the dosage was low and the diet is high starch/sugar content. Line 392: This table needs to be moved to the main manuscript if it will be presented as significant and discussed in the discussion. Line 407-436: The authors have no accompanying data to support the impact of SRB and methanogens in the context of this experiment - the rest is speculation and since no SCFA differences were found, and no Picrust or Cowpie analysis was done - there is no basis for this discussion. Line 458: Again the authors have no digestability data and therefore this is all speculation. Prevotella has a wide range and it seems VERY unlikely that REM is doing hemicellulolytic work if it is firmly adherent to the tissue in a biofilm. The authors do not seem to have a good grasp of the rumen microbiome and how it functions. Conclusion: Not supported by results. Please remove references to animal health as authors have no data to support this.

   Please rate the manuscript for methodological rigour

   Very poor

   Please rate the quality of the presentation and structure of the manuscript

   Very poor

   To what extent are the conclusions supported by the data?

   Not at all

   Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

   No

   Is there a potential financial or other conflict of interest between yourself and the author(s)?

   No

   If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

   No: No animal care approval indicated for major animal work

   Read the original source