Dietary NDF/Starch Ratio Modulates Colonic Microbiota and Metabolites in Lambs before and after Weaning
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn the current manuscript (fermentation-2572619), the authors investigate the effects of different neutral detergent fiber (NDF)/starch ratios on the composition of microbial communities and metabolites in the colon of lambs before and after weaning, as well as changes in colonic environmental homeostasis, using 16S amplicon sequencing and untargeted metabolomics. The results show that feeding a 1.0 NDF/starch ratio starter may be more effective in regulating microbial fermentation, leading to an increase in beneficial microbiota and metabolites and thus improving colonic environmental homeostasis in lambs before and after weaning. The research work has been executed well and the overall premise looks promising. However, there are several concerns need to be addressed as follows:
- It is recommended to avoid using non-standard abbreviations in the manuscript title. It is also important to define any abbreviations used in the manuscript, especially if they are used for the first time.
- In the Abstract, the objective is not clearly stated and the description of the experimental groups is not clear. for example, why is the ratio of NDF/starch 0.5 and the groups A35 and A56?
- Why was the first part of the study (Zhao et al., 2023) not presented in the introduction section, and what is the added value of the current manuscript compared with Zhao et al. (2023)? Clarify in the introduction section
Zhao, H., Lv, F., Liu, G., Pang, X., Han, X., & Wang, X. (2023). Effects of starters with different NDF/starch ratio on rumen fermentation parameters and rumen microorganisms in lambs. Frontiers in Veterinary Science, 10, 1064774..
4. The hypothesis of the study should be clarified at the end of the Introduction section.
5. Lines 87-99: “…….the Regulations of the Administration of Laboratory Animals (Ministry of Science and Technology, revised June 2004).” add to the references list.
6. Line 98:“ ……….Gansu Runmu Biological Engineering Co., Ltd” Provide full details of feed source, ie, company, city, country. A similar issue is found in line 102 . Please elaborate.
7. Table 1: What is the composition of both Mixed energy feed and Mixed protein feed? “Energy and CP ratio” id you intend to express the ratio of Energy to CP? If so, what is the unit of measurement? Also, the analysis methods of the starters should be mentioned in the M&Ms section.
8. Line 115: add to the references list.
9. Line 108: clarify the slaughter method or add a reference.
10. Line 116: the sampling of VFA is missing. Describe also the number of replicates per group (n=?) each time.
11. Line 216: The methods section could be improved by providing more detail on the statistical model used and the post-hoc test used for multiple comparisons.
12. In all Tables and Figs., describe the experimental groups and all abbreviations used in the table's footnotes and figure legends. Describe also the experimental groups and the number of analyzed samples (n=?).
- The discussion section could be improved by providing more context for the results and explaining how they fit into the broader literature. For example, they could discuss how their findings compare to previous studies on the effects of NDF/starch ratios on microbial communities and metabolites in the colon.
- The authors should provide more detail on the implications of their findings for animal nutrition and health.
Minor editing of English language required
Author Response
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions highlighted/in track changes in the re-submitted files.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe study examined the colonic microbiome and metabolome of young goats, but many clear defects in experimental design and data analysis diminish its scientific values.
---Two treatment groups were defined as 0.5 and 1 based on their NDF/Starch ratio. However, the info in Table 1 does not explain how these ratios were calculated. Please provide the composition of mixed energy feed and mixed protein feed as well as the amount of starch from proximate analysis.
---No growth performance data was provided even though animal growth was monitored (such as "each weighing near to the group's average weight" on line 97). Moreover, both Introduction and Discussion mention inflammation, but no data on their inflammation status is presented, which prevent meaningful correlation analysis and discussion.
---The Materials and Methods section is poorly written. For example, "Determination of volatile fatty acids (VFA) used metaphosphorylated rumen fluid" on line 117, while the Results shows the concentrations in colonic digesta (3.1). Also, what is metaphosphorylated rumen fluid?
---Please explain the preparation of the fluid in the sentence of "100 μL of sample was placed in an EP tube and 400 μL of 80% methanol in water" (2.5), which was not mentioned in the other parts of Materials and Methods.
---There are two feeding treatments and two time points (day 35 and day 56). Basically, this is a 2 x 2 factorial design. A correct statistical analysis should be conducted to examine the effects of feeding, age, and feeding-age interaction in Table 2.
---The microbiome model contains 4 groups of samples (Fig. 1e) while the metabolome models are divided by two time points (two Fig. 5) using OPLS-DA, a highly manipulative modeling method. Please construct a model containing all 4 groups of samples together and discuss the effects of time and age accordingly.
---No rigorous effects have been spent to validate the chemical identities of the metabolite markers in the pathway analysis (Figure 6). Many of stated pathways, such as alcohols and derivatives, conjugates, Triterpenoids, Chalcones and dihydrochalcones, Carbonyl compounds, Flavones, Serotonergic synapse, Drug metabolism - cytochrome P450, Taste transduction, are irrelevant to colonic functions and metabolism. Therefore, metabolomic analysis was poorly executed.
Comments on the Quality of English Language---Young goats should be called as kids, not lambs.
---Many grammatical issues.
Author Response
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions highlighted/in track changes in the re-submitted files.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe research titled 'Dietary NDF / Starch Ratio Modulates Colonic Microbiota and Metabolites in lambs before and after weaning' addresses an intriguing topic. However, in my opinion, the present paper falls outside the scope of the journal. Additionally, I believe that it has some shortcomings in its current form:
- I suggest using keywords that are not already included in the title.
- In my opinion, the introduction section could benefit from a more organized structure. I suggest beginning with the significance of proper nutrition during both the pre and post-weaning phases. Clarify the roles of starch and NDF during these periods and their potential impact on the microbiota. What gaps in our knowledge exist concerning these components? Following this, you could elaborate on the importance of the sheep sector and explain why you chose to investigate this specific issue within sheep.- The Materials and Methods section requires rewriting to enhance readability and clarity. I recommend providing a more detailed description of the different diets used. Additionally, including information about milk characteristics and the housing system for the lambs would be beneficial.
- How was the rumen fluid collected?
- I strongly recommend providing a more comprehensive description of the statistical analysis that was conducted.
- If you have any further questions or need clarification, please don't hesitate to ask.
- I recommend expanding the Discussion section to encompass study limitations and practical implications. This will provide a more comprehensive view of the research outcomes and their real-world significance.
Author Response
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions highlighted/in track changes in the re-submitted files.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have revised the manuscript according to my comments, so I suggest it will be accepted.
Author Response
Thank you very much for taking the time to review this manuscript.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have done an excellent job revising this manuscript; however, I still have some specific comments:
In the introduction section, there seem to be some inaccuracies in my opinion. I would suggest improving this paragraph (lines 39-53) by reading and citing paper 10.3390/vetsci10090554, which covers this topics on milk-fed calves.
Line 42: In my view, the term "psychological" is not appropriate. I recommend using the term "social" instead. Nevertheless, if the authors have a better term in mind, they are free to use it.
Line 46: The term "plant-based" is absolutely incorrect (all herbivore diets are plant-based). I suggest using the term "forage" to refer to feeds rich in NDF.
Lines 62-63: The abbreviation "total volatile fatty acids" has already been mentioned in the abstract. Just use "TVFA."
Lines 94 and 108: The term "ad libitum" should be italicized.
Table 1: Remove "2)" in the nutrient levels row.
Statistical Analysis Section: This section needs improvement. If you don't want to include the specific model used, please at least mention which fixed and random effects were considered in the equation.
Author Response
Thank you very much for taking the time to review this manuscript. Below is a detailed response, which we have made in the manuscript in accordance with your revisions and highlighted in red font.
Author Response File: Author Response.pdf
