Differential Rumen Microbial Taxa in Charolais Bulls with Divergent Residual Feed Intake
, Peixin Fan 2 and Ibukun M. Ogunade 1,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsMinor revisions:
1. Minor grammatical issue in abstract: “20 operational taxonomic unit (OTU)” should be “20 operational taxonomic units (OTUs).”
2. The last sentence of the abstract could be slightly more specific about the implications for cattle production.
3.Reference [3] is cited multiple times in succession. Consider streamlining or grouping.
4. Clarify the novelty. What gap in the existing microbiome-feed efficiency literature is this filling?
5. From the results, clarify why multiple OTUs within the same genus (e.g., Prevotella) differ in abundance. Do they represent functionally different strains?
6. In the discussion, further elaborate on how butyrate production in PosRFI bulls may be linked to energy partitioning inefficiencies.
7.Ensure consistent formatting of OTU numbers and statistical notation (e.g., "P < 0.05" rather than mixing with p-values like “p-value=0.92”).
8. In line 99, “Washedbetween” should be “washed between”
Author Response
Minor revisions:
1. Minor grammatical issue in abstract: “20 operational taxonomic unit (OTU)” should be “20 operational taxonomic units (OTUs).”
AU: This has been corrected and highlighted in the manuscript on lines 20-21.
The last sentence of the abstract could be slightly more specific about the implications for cattle production.
AU: We have added more material to the end of the abstract from lines 27 to 31.
3.Reference [3] is cited multiple times in succession. Consider streamlining or grouping.
AU: We have corrected this in lines 38-43.
Clarify the novelty. What gap in the existing microbiome-feed efficiency literature is this filling?
AU: We have added clarifying information to address this point. Specifically, the revised manuscript now acknowledges that while previous literature has explored the relationship between the rumen microbiome and feed efficiency, few studies have investigated these associations using genetically informed metrics such as RFI-EPD. This integration could provide seedstock producers with additional tools for selecting cattle based on both microbial composition and genetic potential for feed efficiency. These revisions can be found on lines 63–66 and 70–73.
From the results, clarify why multiple OTUs within the same genus (e.g., Prevotella) differ in abundance. Do they represent functionally different strains?
AU: Yes, they represent different clusters within 16s rRNA Sequences. We have clarified this in both the Results and Discussion sections of the manuscript. Specifically, we now emphasize that while the genus-level classification may be the same, each OTU represents a unique cluster of 16S rRNA sequences with potential taxonomic and functional differences. This is particularly relevant in genera such as Prevotella and Clostridiales, which are highly diverse and known to contain species and strains with distinct metabolic functions.
In the Discussion, we explicitly explain that Prevotella OTUs 109358 and 626329 were differentially abundant in NegRFI and PosRFI bulls, respectively, and may represent functionally distinct populations. Similarly, we address the differential abundance of Clostridiales OTUs 133719 and 207713. However, I have added more information within the discussion to clarify this point regarding OTU differences within 16s rRNA Sequencing and the representation of different OTUs within genera in lines 232-241.
In the discussion, further elaborate on how butyrate production in PosRFI bulls may be linked to energy partitioning inefficiencies.
AU: Thank you for your comment. To further elaborate, we have added more information into the discussion to further elaborate on how the increased abundance of butyrate-producing microbes in PosRFI bulls may be linked to energy partitioning inefficiencies. Specifically, we now discuss that while butyrate is important for maintaining ruminal epithelial integrity, it contributes less directly to systemic energy metabolism compared to propionate, which is the primary gluconeogenic substrate in ruminants. The increased abundance of butyrate-producing microbes, such as Oscillospira and F16, in PosRFI bulls may help explain their positive RFI phenotype, as these bulls consume significantly more feed to achieve the same growth rate as their more efficient (NegRFI) counterparts. In contrast, NegRFI bulls exhibited a greater relative abundance of propionate-associated microbes which may contribute to improved metabolic efficiency and lower feed intake requirements. This distinction in microbial functional potential provides insight into how microbial-driven differences in volatile fatty acid profiles may contribute to variation in feed efficiency. The added content can be found in the revised Discussion on lines 280-295.
7.Ensure consistent formatting of OTU numbers and statistical notation (e.g., "P < 0.05" rather than mixing with p-values like “p-value=0.92”).
AU: This has been corrected this throughout the manuscript, highlighted in lines 20- 22, 196, 198, 200, 203, and 243.
8. In line 99, “Washedbetween” should be “washed between”
AU: This has been corrected in line 107.
Reviewer 2 Report
Comments and Suggestions for AuthorsTitle: Differential rumen microbial taxa in Charolais bulls with divergent residual feed intake
The study focuses to investigate differentially abundant microbial taxa in the rumen of Charolais cattle with divergent RFI values. The study is interesting due to the results obtained. However, it lacks some details and further integration of some sections, such as the Materials and Methods, Results, and Conclusions
Abstract: Add the meaning of the abbreviations RFI and EPD
Add a relevant conclusion from the study in this section
Keywords: No comments
Introduction: No comments
Materials and Methods:
Table 1. Ingredient and chemical composition of the basal diet fed to Charolais bulls
Check if 48.3% DM is correct, as it is a low percentage of dry matter.
Line 124 a 126 “To prevent contamination Kimtech wipes (Fisher scientific; catalog number 06-666, Pittsburg, PA, USA) were used prior to each sample measurement throughout the spectrophotometry quantification process” This section could be removed from the document as a routine process.
Line 155 remove one of the semicolons (;)
Why were more alpha diversity metrics not included?
Provide at least one metric from each of the following complementary alpha diversity categories: Dominance, Information and Phylogenetics.
Why was the analysis of Firmicutes to Bacteroidetes ratio not included?
Results:
Line 166 – 173 Rewrite the results so that the information already shown in Table 2 is not repeated..
Line 173 In the case of p value (P = 0.073 kg/d) it should not have units.
Discussion and Conclusions:
Include a biological interpretation and conclusions for each reported metric, as well as an interpretation of all metrics collectively.
Add to the discussion other studies with similar results to those found.
Add the limitations of the study, as well as mention the need to complement future studies with other omics tools that would provide more information on microbial consortia in the rumen, their mechanisms of action, regulation, and functioning in coordination with the host.
References: Review and standardize the format.
Author Response
Title: Differential rumen microbial taxa in Charolais bulls with divergent residual feed intake
The study focuses to investigate differentially abundant microbial taxa in the rumen of Charolais cattle with divergent RFI values. The study is interesting due to the results obtained. However, it lacks some details and further integration of some sections, such as the Materials and Methods, Results, and Conclusions
- Abstract: Add the meaning of the abbreviations RFI and EPD
AU: This has been added in line 14.
- Add a relevant conclusion from the study in this section
AU: We have added more material to the end of the abstract from lines 27 to 32.
- Table 1. Ingredient and chemical composition of the basal diet fed to Charolais bulls Check if 48.3% DM is correct, as it is a low percentage of dry matter.
AU: Yes, this is correct.
Line 124 a 126 “To prevent contamination Kimtech wipes (Fisher scientific; catalog number 06-666, Pittsburg, PA, USA) were used prior to each sample measurement throughout the spectrophotometry quantification process” This section could be removed from the document as a routine process.
AU: We have removed this section of information.
- Line 155 remove one of the semicolons (;)
AU: We have removed one of the semicolons, found in line 163.
- Why were more alpha diversity metrics not included?
AU: Alpha diversity was found non-significant between cohorts of steers. In our study, we included commonly used indices such as the Shannon and Chao1 (utilized within Microbiomeanalyst.ca) metrics to assess within-sample microbial diversity. However, alpha diversity was not significantly different between the RFI groups, and neither was beta diversity. Given that no global shifts in microbial diversity were observed, we focused our interpretation on the differential relative abundance of specific taxa, which revealed notable differences between the groups. While additional alpha diversity metrics could have been included, we determined that further indices were unlikely to change the outcome or provide additional insight, given the consistent lack of significance across multiple diversity measures. However, we have added more information on why this was done within the manuscript from lines 221-226.
- Provide at least one metric from each of the following complementary alpha diversity categories: Dominance, Information and Phylogenetics.
AU: Thank you for your suggestion. We agree that metrics from the Dominance, Information, and Phylogenetic categories can provide complementary perspectives on microbial community structure. In response to your comment, we conducted additional analyses using representative metrics from each of these categories (Dominance = Berger-Parker index, Information = Shannon and Simpson indices, Phylogenetics = Faith’s Phylogenetic Diversity). However, these metrics did not reveal statistically significant differences between groups and mirrored the patterns already captured by the diversity indices presented in the manuscript. Therefore, to avoid redundancy and preserve clarity, we chose not to include these additional results in the main text. We are happy to provide these results in the supplementary materials upon request.
- Why was the analysis of Firmicutes to Bacteroidetes ratio not included?
AU: The F:B ratio was not included in this study due to no differences in microbial community at the Phylum level. This makes sense, given that the diversity indices were comparable between the bulls divergent in feed efficiency and is not unexpected, as phyla represent broad taxonomic categories encompassing functionally diverse genera. Additionally, genus-level comparisons revealed taxonomic differences between groups that are more likely to reflect biologically relevant shifts related to feed efficiency. Therefore, we focused our interpretation on genus- and OTU-level differences, which provided more insight into the microbial taxa associated with RFI phenotype.
Results:
- Line 166 – 173 Rewrite the results so that the information already shown in Table 2 is not repeated..
AU: I have rewritten this section of the results, so that it is less redundant. This can be found in lines 171-179.
- Line 173 In the case of p value (P = 0.073 kg/d) it should not have units.
AU: This was corrected in the reconstruction of the table 2 results and can be found in lines 174-183.
Discussion and Conclusions:
- Include a biological interpretation and conclusions for each reported metric, as well as an interpretation of all metrics collectively.
AU: we have added a concluding synthesis paragraph to the Discussion section that provides a biological interpretation of the key taxonomic findings and their potential implications for feed efficiency. This addition includes interpretation of individual microbial metrics—such as the differential abundance of genera associated with propionate versus butyrate production—as well as a collective assessment of how these compositional shifts may influence energy metabolism and nutrient partitioning in cattle with divergent RFI phenotypes. While alpha and beta diversity were not significantly different, the genus-level variation and presence of distinct OTUs within shared genera are now discussed in the context of their functional relevance. This can be found in lines 281-301.
- Add to the discussion other studies with similar results to those found.
AU: We have added more information relative to other study results. This can be found in lines 251-254, 281-295.
- Add the limitations of the study, as well as mention the need to complement future studies with other omics tools that would provide more information on microbial consortia in the rumen, their mechanisms of action, regulation, and functioning in coordination with the host.
AU: We have added more information regarding the limitations of this study in lines 325-330.
- References: Review and standardize the format.
AU: I have reviewed, added, and corrected mistakes within the references. They are highlighted in lines 351-352, 355-356, 363-365, 430-438, 447-453, and 4936-503
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsTitle: Differential rumen microbial taxa in Charolais bulls with divergent residual feed intake
The study focuses on investigating differentially abundant microbial taxa in the rumen of Charolais cattle with divergent RFI values. The study is interesting due to the results obtained. However, it lacks some details in Materials and Methods and further integration of Results and Discussion sections.
Abstract: No comments
Keywords: No comments
Introduction: No comments
Materials and Methods:
If the analyses for the variables Dominance = Berger-Parker index, Information = Shannon and Simpson indices, Phylogenetics = Faith’s Phylogenetic Diversity, and Firmicutes to Bacteroidetes ratio were assessed in this study, but they did not reveal statistically significant differences between groups, each metric should be mentioned in the document as “with no differences between groups” and the results should be added to the supplementary documents.
Results:
Lines 177-179: In the following piece of text, “These results indicate that despite reduced intake, NegRFI bulls achieved comparable growth, highlighting their greater metabolic efficiency relative to PosRFI bulls”, the argument can be changed, because the reduced intake is precisely the variable that shows the NegRFI bulls’ greater metabolic efficiency, since they obtained the same weight gain as the PosRFI bulls, who required a greater feed intake.
Were the initial and final weight data included in the analyses as covariables?
Discussion and Conclusions:
Lines 229-239: Which additional methodologies or techniques could be used to reach the species lever without having the limitations mentioned for the 16 rRNA sequencing? In the case other methodologies are considered, these should be mentioned in the document.
Homogenize, throughout the whole document, the abbreviation for volatile fatty acids (VFAs or VFA).
Take into account that the “OTUs commonly associated with fiber degradation” (lines 297-307) produce all the VFAs, mainly acetate, propionate, and butirate, in decreasing proportion, and are generally mentioned as percentages or correlations with a specific VFA during the fermentation process. Therefore, it would probably be better to present in this document fermentation patterns relative to the VFA of interest, instead of “abundance of VFA”. Additionally, it is important to consider that the microbiome produces the VFAs as fermentation byproducts and that the ruminant determines how the available VFAs are going to be used in the energy metabolism, which includes gluconeogenesis. Besides, even when this study used only one breed, there are other studies in which the breed has been reported to define certain associations between the microbiome and the animal and that there can be differences in this associations within the breed, as well as differences in the energy metabolism (partition and use). In conclusion, all the possible variables that could be implied in this study should be considered to have a better understanding of the subject.
References: No comments
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
