Review Reports - Metatranscriptome Analysis of Sheep Rumen Reveals Methane Production Changes Induced by <i>Moringa oleifera</i> as a Dietary Supplement

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

please see attached PDF-file

Comments for author File: Comments.pdf

Author Response

Row 74: Please insert the reference here

Reference [10] has been inserted in line 75, as recommended.

Row 96: I recommend to present the reference at the end

The reference has been moved and is now presented at the end of the sentence, in line 98, as suggested.

Row 122-129: Are the nutrients given dry matter based or ref. to 93.87 % DM? Please complete the dimension

The nutritional composition is now explicitly indicated as being on a dry matter (DM) basis in the revised manuscript (line 123)

Fig 3,4: These are – even in a printout - are difficult to read. Please choose another format

All figures have been carefully checked and improved: they were re-exported from R with increased font size within the figures, and high-resolution PNG files were uploaded to the journal platform to ensure clarity.

Row 334: I recommend to give more explana8on on this statement.

The manuscript has been revised to provide a more detailed explanation of the statement on line 334. We added that cells maintain these metabolic pathways active because their expression is conditioned by the environmental context, in this case, the treatment applied. Consequently, transcript abundance varies depending on the treatment, leading to the overexpression of specific genes within these pathways. (Lines 348-353).

Row 369: I do not see the need for Fig.8; the authors may consider to delete this fig.

We thank the reviewer for the suggestion. Figure 8 is provided as an illustrative summary of the differential transcript expression across treatments, specifically highlighting enzymes involved in methane metabolism. While we understand it is not essential, we believe it helps readers visually interpret the treatment effects on methane-related pathways. Nevertheless, we will gladly remove it should the editor consider it unnecessary.

Discussion:

I like to ask for some comments about the basic hypothesis „the dried organic substanve of the tree modifies the poten8al of rumen fermenta8on to leave Methane“. In the current study rumen liquid was harvested from sheep adapted to alfalfa – then this specimen was inserted in the Rusitec. I recommend to give some coments about the limita8ons of the study, e.g. In vivo vs Rusitec; why not to feed the animals with the Moringa product and to take the sample from the rumen of the adapted animal instead from the in vitro system?

We thank the reviewer for this suggestion. In this study, Moringa oleifera was not fed directly to the animals because it is not commonly available in our region. In vitro fermentation allowed evaluation under controlled conditions. This limitation has been clarified in the discussion (lines 585-593) and conclusion (617-624), and future in vivo studies are recommended to confirm its effects on rumen function and methane mitigation

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Global warming has become a significant public health concern, with intensive livestock farming as a major contributor. To mitigate greenhouse gas emissions, strategies such as manipulating the ruminal environment with dietary additives are essential. This study evaluated Moringa oleifera, a globally widespread tree with antioxidant, multivitamin, protein-rich, and anti-inflammatory properties, as a feed additive. Overall, it is an interesting study with some important findings, and data is sufficient. Hence, it can be accepted after a minor revision.

Specific comments

In Abstract, please show the key findings with brief descriptions.

L25 Why set up those ratio treatments? Show the references for the ratios in M&M section.

L26 What is the “Genetic material”?

L213 Show the accession number after uploading the sequencing data.

Check all the figures, because the words in some figures are very unclear, like figure 2, 3, 4, and so on.

Author Response

In Abstract, please show the key findings with brief descriptions.

We thank the reviewer for the suggestion to highlight the key findings with brief descriptions in the abstract. In response, we have added a concise sentence at the end of the abstract that summarizes the main results (Lines 38-39).

L25 Why set up those ratio treatments? Show the references for the ratios in M&M section.

We thank the reviewer for the comment. The ratios of Moringa oleifera used in this study were selected based on the levels of antinutritional compounds reported by Amad, Zentek et al., (2023), which may influence rumen fermentation. This consideration guided the choice of inclusion levels for the in vitro experiment. The rationale is stated in the Materials and Methods section (lines 153–154).

L26 What is the “Genetic material”?

We have clarified that the “genetic material” refers to total RNA extracted for metatranscriptome sequencing (Line 26).

L213 Show the accession number after uploading the sequencing data.

We thank the reviewer for the comment. The sequencing data have now been fully referenced with both the NCBI BioProject and the corresponding SRA accession numbers (lines 213-215).

Check all the figures, because the words in some figures are very unclear, like figure 2, 3, 4, and so on.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The title included the methane, however, I am not find any methane indexes in this manuscript. The author collected the 6,12, 24, and 48 h samples, but only measure for the bacteria and metabolism. How about these data: gas production, methane production, H2, and the IVDMD, IVNDF, and IVADF. These indexes were the basically for in vitro experiment. Especially, the author must provide the methane data before published. In addition, the experimental only 2 biological replicated for each treatment, the sample size is very small and statistically insufficient. The biological replicates need > 6.

Line 56, In vitro

Line 89, the nutrition of the alfalfa feed to the rumen fluid donors.

Line 92, feed ad libitum?

Line 93, How much ruminal fluid collected from each donors. How to collected ? More detail.

Line 97 to 100, the nutrition of the diet.

Line 97, only one Run? Two samples? The samples too small.

Line 101: mL

2.4 Sequencing, data preprocessing, and assembly. Provided the primer. Line 147, the version of the soft.

Provide microbiota diversity richness and evenness

I suggest added family levels for better visual clarity.

I suggest added LEfSe for better clarity.

Figure 2 improved; it is difficult to read at the current quality.

The discussion need improved.

The references need revised according to the author guideline. Very tough! The editor need checked the manuscript before seek the reviewers!!!

Appendix A

5→%

Author Response

Reviewer 1:

Comments and Suggestions for Authors:

The title included the methane, however, I am not find any methane indexes in this manuscript.

We addressed this comment by integrating methane production results from our previously published in vitro fermentation study (Gómez-Chávez et al., 2025) as Appendix E. Methane (% molar) was evaluated at 6, 12, 24, and 48 h, with significantly lower values in the T1 (15% Moringa oleifera) treatment. These results are also discussed in the main text (Section 2.2; lines 146-151 and Discussion; lines 501-504), and supported by metatranscriptomic findings (Figures 6–9), showing differential expression of enzymes involved in methane metabolism. We trust this now sufficiently addresses your concern.

The author collected the 6,12, 24, and 48 h samples, but only measure for the bacteria and metabolism. How about these data: gas production, methane production, H2, and the IVDMD, IVNDF, and IVADF. These indexes were the basically for in vitro experiment. Especially, the author must provide the methane data before published.

We addressed this by clearly specifying in Section 2.2 that samples were collected at 6, 12, 24, and 48 h and that fermentation parameters, including gas production, methane (CH₄), CO₂, digestibility (IVDMD), NDF, and ADF were measured. The complete results from the in vitro fermentation trial are presented in Appendix E and referenced throughout the manuscript from our previously published in vitro fermentation study (Gómez-Chávez et al., 2025). These data support the metatranscriptomic analysis and demonstrate significant differences, particularly in methane production between treatments. Unfortunately, hydrogen gas (H₂) was not measured.

In addition, the experimental only 2 biological replicated for each treatment, the sample size is very small and statistically insufficient. The biological replicates need > 6.

We appreciate your concern regarding the small number of biological replicates per treatment. Indeed, only two biological replicates were included per group due to practical and logistical constraints. We recognize this as a limitation of our study and have stated it clearly in the Conclusions section. To mitigate this limitation, we performed a power analysis using the RNASeqPower package in R, which indicated that the design retained sufficient statistical power (0.95) for detecting differences in gene expression (lines 185-187). Additionally, our interpretation of the results, particularly those regarding microbial composition and methane metabolism, has been made with caution, and we recommend confirmation through studies with greater replication in future research.

Line 56, In vitro

The term in vitro was italicized as appropriate. It was previously on line 56 is now on line 60.

Line 89, the nutrition of the alfalfa feed to the rumen fluid donors

We have now included the nutritional composition of the alfalfa hay used to feed the rumen fluid donor animals during the adaptation period. This information appears in Section 2.2 lines 136-138.

Line 92, feed ad libitum?

We clarified the feeding conditions in Section 2.2. The donor animals were fed alfalfa hay twice daily under a controlled feeding schedule during the adaptation period and had unrestricted access to water. The controlled feeding schedule was chosen to ensure diet uniformity and to standardize rumen conditions prior to fluid collection. (Lines 135-136)

Line 93, How much ruminal fluid collected from each donors. How to collected ? More detail.

We have added more details regarding the volume and method of rumen fluid collection. Specifically, 500 mL of rumen fluid were collected from each fistulated donor animal, filtered through four layers of cheesecloth, pooled, and maintained under anaerobic conditions. This information has been included in Section 2.2, lines 140-141.

Line 97 to 100, the nutrition of the diet

The full nutritional composition of the experimental diets, including dry matter, ash, ether extract, ADF, NDF, crude protein, and minerals (Ca, P, Mg, and K), was previously reported in detail by Gómez-Chávez et al. (2025), cited as reference [14] in our manuscript. In the current version, we have included a summary of the most relevant values for both Moringa oleifera and alfalfa hay in Section 2.2 (lines 114–117 and 136–137) to provide clarity and context. For the complete dataset, including all fermentation parameters, we kindly refer the reader to Gómez-Chávez et al. (2025) [14].

Line 97, only one Run? Two samples? The samples too small.

As previously mentioned, we are aware of the limitations associated with having only two biological replicates per treatment, which were due to practical and logistical constraints. However, we sought to compensate for this by performing a power analysis using the RNASeqPower package in R, which demonstrated that the experimental design retained a sufficient statistical power (0.95) to detect significant differences in gene expression (lines 185-187). We have acknowledged this limitation in the Conclusions section and interpreted our findings with caution. We also emphasize that further studies with a larger number of biological replicates are necessary to confirm and expand upon these results.

Line 101: mL

The correction was made; it can be seen on line 152

2.4 Sequencing, data preprocessing, and assembly. Provided the primer.

Thank you for your suggestion. We confirm that reverse transcription was performed using the random hexamer primers provided in the Illumina TruSeq Stranded Total RNA Library Prep Kit. Unfortunately, the exact sequences of these primers are proprietary to Illumina and not publicly disclosed. We have clarified this point in the revised manuscript accordingly

Line 147, the version of the soft.

R software version added (Lines 206-207)

Provide microbiota diversity richness and evenness

The requested information regarding microbiota diversity, richness, and evenness has been added in lines 265–268 and 413–417, and shown in Appendices D.1 and D.2. These aspects were assessed using the Shannon index, which considers both species richness and their relative abundance (evenness).

I suggest added family levels for better visual clarity.

As suggested, we added Appendix C, which presents the taxonomic classification at the family level by percentage for each treatment to improve visual clarity. Additionally, this information is now discussed in more detail in the Results sections (lines 258–264) and Discussion sections (lines 401-402; 427–433).

I suggest added LEfSe for better clarity.

While LEfSe was not applied, we added complementary information based on the MA plot (Appendix F), which displays differential expression at the phylum level between T0 and T1. This approach revealed significant changes, particularly the increased abundance and activity of Proteobacteria in T1. This method, based on gene expression, provides an alternative way to visualize differential abundance between treatments T1 (Lines 269-271; 446-452).

Figure 2 improved; it is difficult to read at the current quality.

We have improved the quality and resolution of the figure 2 to enhance readability.

The discussion need improved.

We have thoroughly reviewed and revised the Discussion section to minimize speculative statements and ensure that all interpretations are firmly grounded in the experimental data presented. Where relevant, we have clearly distinguished between data-supported conclusions and hypotheses or interpretations derived from the literature, highlighting areas for future research. This comprehensive revision improves the clarity and scientific rigor of the manuscript.

The references need revised according to the author guideline. Very tough! The editor need checked the manuscript before seek the reviewers!!!

We have thoroughly revised the references to ensure full compliance with the journal’s author guidelines. We appreciate the importance of accurate formatting and have carefully checked the entire reference list accordingly.

Appendix A

5→%

The correction was made; it can be seen on line 578 (Appendix A).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

A very interesting subject of the manuscript, dealing with a very important area, and I approached the manuscript itself with great enthusiasm and a desire for new knowledge. However, I must say that the manuscript itself left me somewhat disappointed.

First of all, the summary at the end does not present the real conclusions of the research but is generalized and contains a message that does not relate to the research itself.

The introduction is very short and does not really introduce us to the purpose of the research. In the first part, the plant itself, the subject of the research, is explained, but it is not mentioned as anything more important in the text. Therefore, I think that the introduction itself is too short and insufficient without adequately introducing the reader to the problem.

Material and methods, are a particular problem, and in the first part 2.1. methods are cited (12, 13) that are not actually methods. The authors state that something is described in the literature, but this is not in the references given, and reference is also made to other sources. For example, line 74 states that the proximal analysis contains NDF and ADF, but these units are not part of the proximal analysis of feed. In addition, there is a reference (13) in the manuscript that does not describe it. The materials therefore need a completely new way of presenting and describing everything that was done in the research.

A particular problem with the manuscript is the number of samples, i.e. the number of repetitions. For each treatment we have only two replicates, i.e. n=2. It goes without saying that this number is not sufficient for the study, which is also reflected in the results, where we have a large difference in microbiota within treatment, i.e. between the first and second replicate. We see some regularities in the appearance of the microbiota between the treatments, but there is too much variation within the treatment, which would certainly be avoided with a larger number of replicates.

The conclusion reflects the research. In the sentence (lines 424-426) it says that the addition of Moringa oleifera changes the microbiota, but since we have three treatments in the research, i.e. two levels of supplementation, I think this should also be emphasized somewhere.

In general, a very interesting topic and the research itself, but the execution itself was not done in an appropriate way, and without a significant change in the approach to the problem itself, the interpretation of it in the text, such a manuscript is not acceptable.

Author Response

Reviewer 2:

Comments and Suggestions for Authors:

First of all, the summary at the end does not present the real conclusions of the research but is generalized and contains a message that does not relate to the research itself.

The abstract has been revised to reflect the actual findings of the study, emphasizing the effects of Moringa oleifera on the ruminal microbiota, methane-related pathways, and archaeal activity.

We have substantially expanded this section to better present the significance of rumen fermentation in greenhouse gas emissions and the role of Moringa oleifera as a promising supplement for methane mitigation. The revised introduction now includes detailed information on the plant’s bioactive compounds, mechanisms of action supported by recent studies, and the relevance of metatranscriptomic analysis for this research. The study objective is also clearly stated. We believe these changes address your concerns and improve the clarity and context of the manuscript.

Material and methods, are a particular problem, and in the first part 2.1. methods are cited (12, 13) that are not actually methods.

We clarified that reference 12 (now 14) reports previously published results of the chemical characterization and is not a methods citation. Reference 13 (now 15) was replaced with the correct methodological reference for fiber analysis (Van Soest et al., 1991), adapted for the ANKOM analyzer.

The authors state that something is described in the literature, but this is not in the references given, and reference is also made to other sources. For example, line 74 states that the proximal analysis contains NDF and ADF, but these units are not part of the proximal analysis of feed.

In the revised Methods section, we have clearly distinguished the proximate analysis components (crude protein, dry matter, ash, ether extract) from the fiber fractions (NDF and ADF). The latter were determined following the Van Soest et al. (1991) method [15], which is the standard procedure for fiber analysis and not part of the traditional proximate analysis. Proximate components were analyzed using official AOAC methods [16]. This clarification ensures accurate terminology and appropriate referencing, addressing your concern.

In addition, there is a reference (13) in the manuscript that does not describe it.

Reference 13 (now 15) was replaced with the correct methodological reference for fiber analysis (Van Soest et al., 1991), adapted for the ANKOM analyzer.

The materials therefore need a completely new way of presenting and describing everything that was done in the research.

We have substantially revised sections 2.1 and 2.2 to provide a clearer, more detailed, and logically structured description of the procedures and materials used.

Section 2.1 now includes a comprehensive description of the preparation and chemical characterization of Moringa oleifera, specifying all analytical methods, instrumentation, and references. This clarifies the nutritional composition and the methods used to obtain it.

Section 2.2 has been expanded to detail the ethical approvals, animal management, rumen fluid collection procedures, and the in vitro fermentation experimental design. We also clarified the diet composition and incubation conditions, along with the specific parameters measured and their relevance to the current metatranscriptomic analysis.

The conclusion has been revised to emphasize that two levels of Moringa oleifera supplementation were tested, and that differences were observed not only between the control and supplemented groups, but also between the two dosages. Specifically, the 15% inclusion level (T1) was identified as the most effective.

We appreciate your comment regarding the overall execution and interpretation of the study. In response, we have made substantial revisions throughout the manuscript to clarify and strengthen the rationale, experimental design, and data interpretation. We hope these major changes address your concerns and demonstrate the scientific value of the study. Thank you for your helpful feedback.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

General comments

The manuscript titled "Metatranscriptome analysis of sheep rumen reveal methane production changes induced by Moringa oleifera as a dietary supplement", submitted for publication in Fermentation, investigates the effects of Moringa oleifera as a dietary supplement on ruminal microbial communities and methane metabolism using in vitro fermentation and metatranscriptomic analysis.

The topic is timely and relevant in light of global efforts to mitigate greenhouse gas emissions from livestock production. However, several minor issues must be addressed before the manuscript can be considered for publication. Specific comments are provided below.

- Correct minor grammar issues (e.g., “reveal” → “reveals” in the title).

Introduction

- I suggest adding a dedicated paragraph presenting Moringa oleifera as a dietary supplement. This should include information on its nutritional composition, bioactive compounds, and documented effects on rumen fermentation and microbial communities, particularly in the context of methanogenesis.

- I recommend considering the inclusion of references to other studies that have investigated different dietary supplements used in ruminant nutrition—particularly in sheep—for the mitigation of methane emissions.

Materials & Methods:

- L 74-76: Please include the specific equipment used for the determinations of NDF, ADF, crude protein, dry matter, ash, and fat, as well as the name of the manufacturer (including city and country).

- The manuscript states that chromatography was used for mineral analysis; however, this appears to be unusual, as atomic absorption spectrometry (AAS) or inductively coupled plasma (ICP) techniques are typically the standard methods for mineral determination. Could you please verify this information and correct the description if necessary?

- L 77-78: For the mineral and amino acid analysis, please include a brief description of the analytical methods employed, along with the specific equipment used (including model and manufacturer name, city, and country).

- Although the bromatological profile of Moringa oleifera is referenced from a previous publication [12], I recommend including a concise summary of the key nutritional parameters (e.g., crude protein, NDF, ADF, mineral content) within the current manuscript. This is important in order to better understand its dietary impact.

Results

- I suggest standardize treatment names (T0, T1, T2) throughout to avoid confusion with “Control_1” or “Low_1.”

- Results figures should be accompanied by clearer legends and summary points.

- I suggest addressing the limitations of the study design, particularly the fact that only two biological replicates were used per treatment group. This low level of replication may reduce the statistical power of the analysis and limit the generalizability of the findings.

Discussion

- The Discussion section includes several speculative interpretations (e.g., L 290-291, L302–308) that are not directly supported by the data presented.

I recommend refining the discussion by limiting speculative statements and ensuring that all interpretations are directly supported by the presented data

Author Response

Reviewer 3:

Comments and Suggestions for Authors:

- Correct minor grammar issues (e.g., “reveal” → “reveals” in the title).

The correction was made; it can be seen on line 2

Introduction

Thank you for the suggestion. We have added a dedicated paragraph in the Introduction presenting Moringa oleifera as a dietary supplement, including its nutritional composition, bioactive compounds, and effects on rumen fermentation and methane mitigation, supported by relevant literature and experimental evidence.

To address the suggestion, we incorporated references and discussion of previous studies evaluating Moringa oleifera as a dietary supplement for methane mitigation in ruminants, particularly in sheep. This includes findings on its bioactive compounds, its effects observed in RUSITEC experiments, and its potential inhibitory action on methanogenesis-related enzymes. The information was added in the Introduction (lines 62–81) and further discussed in the Discussion section (lines 527–535).

Materials & Methods:

- L 74-76: Please include the specific equipment used for the determinations of NDF, ADF, crude protein, dry matter, ash, and fat, as well as the name of the manufacturer (including city and country).

We have revised Section 2.1 to include detailed information on the specific equipment and manufacturers (including city and country) used for the determination of NDF, ADF, crude protein, dry matter, ash, and fat. These details now appear in the updated version of the manuscript, in accordance with your request.

We have corrected the description of the mineral analysis method. The revised manuscript now specifies that atomic absorption spectrophotometry (AAS) was used, with the corresponding equipment and manufacturer details included

We have included a brief description of the analytical methods used for both mineral and amino acid analyses. The revised text now specifies the equipment, including model and manufacturer details, as well as the city and country.

In the revised manuscript, we have included a concise summary of the key nutritional parameters of Moringa oleifera, including crude protein, NDF, ADF, ash, ether extract, and mineral content, to better highlight its dietary impact. Additionally, the full results of the in vitro fermentation trial are presented in Appendix E and are referenced throughout the text, based on our previously published study (Gómez-Chávez et al., 2025).

Results

- I suggest standardize treatment names (T0, T1, T2) throughout to avoid confusion with “Control_1” or “Low_1.”

We have standardized the treatment names to T0, T1, and T2 throughout the text, tables, figures, and appendices to ensure consistency and avoid confusion.

- Results figures should be accompanied by clearer legends and summary points.

In the revised manuscript, we have improved the legends of Figures 1, 2, 3, and 6, which were previously less clear. The updated captions now include more detailed descriptions and highlight key summary points to enhance clarity and understanding of the results.

We have addressed this concern in the revised manuscript by acknowledging the limitation of having only two biological replicates per treatment group. This constraint was due to logistical and practical reasons. We have added a clear statement in the Conclusions section, emphasizing that the findings should be interpreted with caution and highlighting the need for further validation with larger sample sizes. Additionally, we applied appropriate statistical analyses to mitigate this limitation and support the observed trends.

Discussion

- The Discussion section includes several speculative interpretations (e.g., L 290-291, L302–308) that are not directly supported by the data presented. I recommend refining the discussion by limiting speculative statements and ensuring that all interpretations are directly supported by the presented data

We thoroughly revised the Discussion section to reduce speculative statements and ensure that all interpretations are clearly supported by our experimental data or, when appropriate, identified as hypotheses based on previous studies. In particular, we revised lines 362–368 and 385–389 to soften speculative language and clarify mechanistic explanations. These changes improve the clarity, scientific rigor, and overall interpretative strength of the manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Line 150, delete 2025.

Line 174 "["

Figure 1, Define the abbreviate in the figure. the same as below.

Again, the references need doubel revised following the author guidline.

Appendix E, add the unit of gas production. How about the DMD?

Author Response

Line 150, delete 2025.

The "2025" has been deleted. The change is now reflected in line 157.

Line 174 "["

The citation format has been corrected to “[20]”. The change now appears in line 181.

Figure 1, Define the abbreviate in the figure. the same as below.

The abbreviations used in Figure 1 have been defined in the text at the beginning of the Results section (lines 224–229) when mentioned for the first time, and they are also included in the figure caption for clarity and ease of interpretation.

Again, the references need doubel revised following the author guidline.

A thorough review of the references was conducted, and all citations have been corrected to follow the journal's author guidelines. Additionally, reference 17, which was previously missing from the main text, has now been properly included (line 159).

Appendix E, add the unit of gas production. How about the DMD?

The unit of gas production has been added in line 636 of Appendix E. Regarding the DMD, the information was already included and is reported in line 634; previously it was referred to using the term “Digestibility”, which has now been clarified as DMD for consistency.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I must admit that the authors have made some improvements according to the commentaries and instructions, but with their additions they have only confirmed my suspicion of errors in the methodology.

All this refers primarily to the in vitro fermentation and the settings of the whole experiment. The methodology mentioned is partially described, but they mainly refer to their previously published work. Some standard methods are mentioned in this paper, but they were not used or explained in detail. The authors feed the rumen fluid donor animals only alfalfa hay. Such a meal contains too much protein (more than 20%) and is probably not energy-rich enough. This probably led to a different number and type of rumen flora in the rumen. In the standard methods, including the ones they refer to, the animals are always fed a balanced meal consisting of both protein and energy feeds. Another thing I don't understand is why rumen fluid is taken after a 24 hour fast. In all standard methods it is recommended to take the fluid 2 hours after the morning feed. It is likely that the authors also came across a small amount of rumen fluid after deprivation. This can be seen from the fact that they only took 500 ml of rumen fluid per sheep. Again, I think this is too little as they used the Daisy incubator for the analyses, and after filtering this is certainly not enough. I say this because they mention 4 incubation times, which can only be done in each individual container, and the rumen fluid content is too low for that. Also, some of the analysis was done in glass tubes. Nowhere is it described whether all analyses were carried out simultaneously or at specific intervals.

When I talk about in vitro analyses, the authors also mention digestibility. The authors have also mentioned digestibility in the introduction, the method is described, but after that we only have a table, no mention of these parameters in the results or in the discussion. Since there is no such data, these analyses and the methodology should not even be mentioned in the manuscript.

And finally, let me come back to my biggest complaint about this manuscript, the number of repetitions. The authors understand this shortcoming and justify it with a logistical problem. Unfortunately, such an explanation cannot be accepted in scientific research. In their constantly referenced paper, they had three replicates, and since this paper is a continuation, one replicate was lost. My concern about the accuracy of the data also stems from the fact that we see that the difference and discrepancy between T1_1 and T1_2 replicates in the table is too large, and other representations of replicates where there are no statistics, and of course with two samples there is no appropriate statistic other than the mean.

Therefore, despite the author's best efforts, I have to conclude that two replicates are not sufficient for a serious scientific paper, and I cannot accept the corrected manuscript as such.

Author Response

Reviewer 2:

Comments and Suggestions for Authors:

The authors have made some improvements according to the commentaries and instructions, but with their additions they have only confirmed my suspicion of errors in the methodology. All this refers primarily to the in vitro fermentation and the settings of the whole experiment. The methodology mentioned is partially described, but they mainly refer to their previously published work. Some standard methods are mentioned in this paper, but they were not used or explained in detail.

We appreciate this observation and have now expanded the description of the in vitro fermentation methodology in the revised manuscript. While we originally referred to our previous study to avoid repetition, we understand the importance of providing a self-contained explanation. We have added more details on the substrate preparation, rumen fluid handling, incubation conditions, and analyses performed to ensure clarity and reproducibility (Section 2.1, lines 145-151).

The authors feed the rumen fluid donor animals only alfalfa hay. Such a meal contains too much protein (more than 20%) and is probably not energy-rich enough. This probably led to a different number and type of rumen flora in the rumen. In the standard methods, including the ones they refer to, the animals are always fed a balanced meal consisting of both protein and energy feeds.

We acknowledge that the diet of rumen fluid donor animals can significantly influence the ruminal microbial composition and fermentation parameters. In our study, donor animals were fed exclusively with alfalfa hay, a choice driven by the need to maintain consistency between the donor diet and the substrates evaluated, as well as reflecting local feeding practices relevant to our production system. This approach allowed us to standardize the rumen environment while providing a microbial inoculum suitable for our exploratory metatranscriptomic analysis.

Another thing I don't understand is why rumen fluid is taken after a 24 hour fast. In all standard methods it is recommended to take the fluid 2 hours after the morning feed. It is likely that the authors also came across a small amount of rumen fluid after deprivation. This can be seen from the fact that they only took 500 ml of rumen fluid per sheep.

The rumen fluid was collected after a 24-hour fasting period to reduce variation associated with postprandial fermentation peaks and to obtain a more stable microbial population. Regarding volume, we pooled fluid from multiple animals to obtain a sufficient total amount for all incubations. We have clarified this in Section 2.1., lines 138-140.

Again, I think this is too little as they used the Daisy incubator for the analyses, and after filtering this is certainly not enough. I say this because they mention 4 incubation times, which can only be done in each individual container, and the rumen fluid content is too low for that. Also, some of the analysis was done in glass tubes. Nowhere is it described whether all analyses were carried out simultaneously or at specific intervals.

The Daisy incubator was specifically used for assessing dry matter disappearance (DM Digestibility) using ANKOM bags, while fermentation parameters, including gas production, were measured in glass tubes. We have clarified the timing of analyses and the specific systems used for each parameter in the revised Methodology section (Section 2.1 lines 145-151).

The in vitro digestibility parameter was included in the current manuscript as part of the experimental design and methodology to provide a complete overview of the study. However, as this variable was already analyzed, discussed, and published in detail in our previous article (Gómez-Chávez et al., 2025), we have limited its mention in this manuscript to avoid redundancy and overlapping publication. In line with another reviewer’s suggestion, we ensured that this information is clearly reported in the table for completeness, but without extensive discussion, as it has already been addressed in the cited work. We believe this approach maintains transparency while respecting the boundaries of previously published results.

We thank the reviewer for highlighting the importance of adequate replication in scientific research. We fully acknowledge that having three or more biological replicates is the standard for robust statistical analysis and increased confidence in the results. Unfortunately, due to logistical and resource constraints specific to this study phase, only two biological replicates were available.

We recognize that this limitation affects the statistical power and interpretation of our data. However, it is important to emphasize that this study was designed as an exploratory metatranscriptomic analysis aimed at identifying potential microbial and transcriptomic changes induced by Moringa oleifera supplementation. The findings serve as a valuable foundation to guide future, more extensive studies with adequate replication.

Furthermore, to maintain transparency and rigor, we have explicitly stated these limitations in the revised manuscript and interpreted the results with appropriate caution, avoiding overgeneralization of conclusions. We believe that despite this constraint, the data provide meaningful insights into the microbial dynamics affected by the treatments.

We appreciate the reviewer’s understanding of the challenges in conducting such advanced analyses and hope that this explanation clarifies the scope and context of the study.