Impact of Heat Stress on Rumen Fermentation Patterns and Microbiota Diversity and Its Association with Thermotolerance in Indigenous Goats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript explores the ruminal fermentation and microbiota response to heat stress in two indigenous goat breeds (Nandidurga and Bidri), using a climate chamber model and 16S rRNA sequencing. While the topic is relevant and timely in the context of climate change and livestock resilience, the study suffers from critical limitations in experimental design, statistical rigor, microbial data interpretation, and figure quality. Substantial revision is required before the manuscript can be considered for publication.
1. The sample size per group (n = 6; microbiota n = 4) is insufficient to robustly support biological conclusions, especially for microbiome comparisons. Power analysis should be performed to validate the sample size.
2. Functional prediction using PICRUSt is not explored in depth, and no validation is offered for the inferred pathways, especially those relevant to heat stress adaptation (e.g., SCFA biosynthesis, hydrogenotrophic metabolism).
3. Figures 2–5 and Figures 9–11 are low resolution and fragmented. They should be merged into composite multi-panel figures for clarity and space efficiency.
4. The contrasting responses between the two breeds deserve deeper discussion, possibly involving host genetics, baseline rumen microbial differences, or epigenetic factors.
5. The manuscript inconsistently uses “P,” “p,” and italic/regular formatting for p-values. All statistical significance should follow journal style, preferably italicized P.
6. Table 2 (correlation matrix) can be placed in supplementary material, with the main findings described in the results.

Author Response

response to heat stress in two indigenous goat breeds (Nandidurga and Bidri), using a climate chamber model and 16S rRNA sequencing. While the topic is relevant and timely in the context of climate change and livestock resilience, the study suffers from critical limitations in experimental design, statistical rigor, microbial data interpretation, and figure quality. Substantial revision is required before the manuscript can be considered for publication.

Response: We appreciate the efforts from the reviewer to review this manuscript.

Comment 1: The sample size per group (n = 6; microbiota n = 4) is insufficient to robustly support biological conclusions, especially for microbiome comparisons. Power analysis should be performed to validate the sample size.

Response: We understand the reviewer’s concern. The study being experimentally set up (not a field study) and controlled well aided in maintaining the segregation between the groups. Additionally it becomes difficult to increase the sample size for the climate chamber induced study as our facilities can host a maximum of 24 goats (therefore sample size per group =6).

Comment 2: Functional prediction using PICRUSt is not explored in depth, and no validation is offered for the inferred pathways, especially those relevant to heat stress adaptation (e.g., SCFA biosynthesis, hydrogenotrophic metabolism).

Response: We understand your concern on this point. This is not to provide an excuse, however as researchers we all understand that it is difficult to analysis all possible variables in a proposed research work. One of the major reasons being the financial constraint and difficult in managing the required laboratory activities. We firmly believe that the present study in itself in voluminous, 15 day interval collection of rumen liquor to assess an elaborate list of rumen variables, day 45 rumen metagenomics. All of these being just a small fraction of the total variables recorded in this project (intended to be published in different manuscript).

We agree performing a rumen metabolomics especially to validate the metagenomics finding would increase the quality of our research. However we regret for not being performing this because of the constraint associated with research support (both in terms of technical support and funding). We will try to overcome this challenge in our future work.

Comment 3: Figures 2–5 and Figures 9–11 are low resolution and fragmented. They should be merged into composite multi-panel figures for clarity and space efficiency.

Response: The initial figures that were inserted were of high quality, as they occupied larger space in the manuscript we had to reduce its size. We have re-inserted the figures.

Comment 4: The contrasting responses between the two breeds deserve deeper discussion, possibly involving host genetics, baseline rumen microbial differences, or epigenetic factors.

Response: Thank you for your suggestion we have worked on discussing the findings from the rumen metagenomics data with rumen variables between the two breeds which can be viewed in certain parts of the discussion. The lack of documented literature on genomic characterization between the two breeds limits are access to discuss further between the two breeds’ genetics.

Comment 5: The manuscript inconsistently uses “P,” “p,” and italic/regular formatting for p-values. All statistical significance should follow journal style, preferably italicized P.

Response: Thank you for pointing this inconsistency. We have addressed the concern which is reflected in the revised manuscript

Comment 6: Table 2 (correlation matrix) can be placed in supplementary material, with the main findings described in the results.

Response: Thank you for your suggestion, we have placed the table 2 as a supplementary table 2 and revised all table numbers accordingly

Reviewer 2 Report

Comments and Suggestions for Authors

This research paper titled "Unravelling the Heat Stress Response in Goats Based on Changes in the Rumen Fermentation Pattern and Microbiota Diversity (fermentation-3713715)" appears more like a review or meta-analysis based on its title. I have the following comments and suggestions:

The 2-4 Line title seems ambitious and significant, but it is apparently unreasonable to draw conclusions or describe objectives based solely on a single experimental trial with only six replicates per group. Therefore, it is suggested that the authors modify the title to reflect the impact/effect of heat stress on...of ... style.

On Line 13, given that goats have such strong adaptability to the environment, is it appropriate to use goats to study the correlation between heat stress and rumen fermentation characteristics and microbiota?

On Line 25, the V3-V4 region in 16S rRNA sequencing is too outdated. Given current research, at least the 16S rRNA third-generation full-length sequencing depth should be used.

On Line 34, it is recommended that the keywords here be arranged in alphabetical order. Is "Adaptation" appropriate here? I think the focus of this study should be the impact of heat stress on rumen fermentation and microbiota. Is discussing adaptability deviating from the original research focus of this paper?

On Lines 87-88, mentioning that "The animals were stall-fed with a standard maintenance diet," is it representative to study heat stress using a diet based on maintenance requirements? This has a significant impact on the interpretation of the subsequent results, and the authors need to provide a reasonable explanation here.

On Line 90, is there any basis for simulating heat stress only from 10 a.m. to 4 p.m. for six hours? More surprisingly, on Line 92, it is mentioned that the meteorological parameters in the artificial climate chamber are recorded only twice a day. This is unreasonable. A simple temperature and humidity recorder can record data continuously and in real-time. How can recording only twice a day accurately reflect the actual temperature and humidity? Since the focus of this paper is heat stress, the temperature and humidity data must be complete and accurate.

What is the correlation between the recording time points of 8 a.m. and 2 p.m. mentioned on Lines 96-97 and the previous 10 a.m. and 4 p.m.?

On Lines 109-110, check whether there is any problem with the description of the treatments for the NC and BC groups.

On Lines 116-117, if animals under grazing conditions will preferentially choose areas under heat stress, the significance of this paper seems diminished.

On Line 178, OTU clustering has many disadvantages. The current trend is ASV.

On Line 179, is it version 1 or 2 of the PICRUSt program? This is crucial for result prediction. Additionally, the description in this paragraph is too brief. The authors are requested to supplement the analysis methods for the raw data and the instruments and version numbers used.

On Line 198, the THI is 95, which seems unlikely. Generally, a THI above 82 is already considered severe heat stress. What degree of heat stress does a THI above 95 represent? The authors are requested to verify the data.

On Line 205, in the description of the results of 3.2. Rumen Liquor Variables, the authors list data for 0, 15, 30, and 45 days for each indicator. What is the intention of listing so much data?

On Line 206, it is ammoniacal nitrogen, not NH₃.

The results described on Lines 206-307 are suggested to be integrated into one paragraph. The current description is too redundant, making it difficult to understand the intended meaning of the results.

The data in Figure 8 on Lines 544-547 is unnecessary in the main text. I would like to know what meaning corresponding to the research objective can be expressed with this figure?

On Line 686, it is stated that "As a first-time study." In fact, there have been many studies on heat stress in goats. It is unreasonable for the authors to focus on a specific local breed of goat here. There are many goat breeds worldwide. Does this mean that using each breed is innovative? If so, the current research results will not be very representative. I think the descriptions on Lines 686-691 are not suitable for the conclusion section. The authors should concisely highlight the innovative findings and significance of this study here.

On Line 718, regarding the references, there are many studies on heat stress in meat sheep worldwide. The authors only mention a small part in the introduction. It is suggested that the authors re-search and enrich the research progress, and add relevant literature in the discussion section to enhance the interpretability of the paper's results.

Author Response

General comment: This research paper titled "Unravelling the Heat Stress Response in Goats Based on Changes in the Rumen Fermentation Pattern and Microbiota Diversity (fermentation-3713715)" appears more like a review or meta-analysis based on its title. I have the following comments and suggestions

Response: Thank you for your suggestion, we have made an attempt to modify the title, depicting the major finding from the study (Line 4).

Comment 1: The 2-4 Line title seems ambitious and significant, but it is apparently unreasonable to draw conclusions or describe objectives based solely on a single experimental trial with only six replicates per group. Therefore, it is suggested that the authors modify the title to reflect the impact/effect of heat stress on...of ... style.

Response: Thank you for your suggestion, as mentioned in the response to the previous comment, we have made an attempt to modify the title, depicting the major finding from the study (Line 4).

Comment 2: On Line 13, given that goats have such strong adaptability to the environment, is it appropriate to use goats to study the correlation between heat stress and rumen fermentation characteristics and microbiota?

Response: We appreciate the reviewer for expression his/her concern. We agree that when compared to other ruminants, several literatures highlight the superior adaptability of goats. However, there are sparse reports assessing the molecular adaptation in goats. Furthermore, past studies from our laboratory have also revealed differences among the indigenous breeds. Hence this was one of the few objectives of our work group based on this a project. As it would confuse the readers to elaborate all these details, we focused on highlighting our primary objective which was to assess the heat stress responses exhibited by the concerned two breeds of goat using a less explored approach of rumen metagenomics. We mentioned a few statements to explain this concept in the introduction which is between lines 52-58.

Comment 3: On Line 25, the V3-V4 region in 16S rRNA sequencing is too outdated. Given current research, at least the 16S rRNA third-generation full-length sequencing depth should be used.

Response: We authors thank the reviewer for providing this suggestion. We understand that the field of biotechnology is advancing and we will make a note of this for our future projects. Unfortunately, this project is completed and at the time of the study we had provisions to opt for 16s V3-V4 rRNA sequencing and shotgun metagenomics only. The financial requirement to perform a shotgun metagenomics would out pass our budget and hence we had to chose this option which technically is well accept still in our field.

We hope the reviewer understands the practical and financial constraints associated with such research.

Comment 4: On Line 34, it is recommended that the keywords here be arranged in alphabetical order. Is "Adaptation" appropriate here? I think the focus of this study should be the impact of heat stress on rumen fermentation and microbiota. Is discussing adaptability deviating from the original research focus of this paper?

Response: Thank you for placing your suggestion. The word ‘Adaptation’ has been deleted and the words ‘Cellulose’ has been added in the keywords (Line 36)

Comment 5: On Lines 87-88, mentioning that "The animals were stall-fed with a standard maintenance diet," is it representative to study heat stress using a diet based on maintenance requirements? This has a significant impact on the interpretation of the subsequent results, and the authors need to provide a reasonable explanation here.

Response: It is a standard practice to follow a maintenance diet for goats under the present age group. Furthermore, the dietary requirement for each goat was addressed based on its body weight.

Comment 6: On Line 90, is there any basis for simulating heat stress only from 10 a.m. to 4 p.m. for six hours? More surprisingly, on Line 92, it is mentioned that the meteorological parameters in the artificial climate chamber are recorded only twice a day. This is unreasonable. A simple temperature and humidity recorder can record data continuously and in real-time. How can recording only twice a day accurately reflect the actual temperature and humidity? Since the focus of this paper is heat stress, the temperature and humidity data must be complete and accurate.

Response: Thank you for expressing your concern. Our intention of simulation the heat stress from 10 am to 4 pm was to mimic a natural scenario wherein the livestock are sent out for grazing. Livestock farming in India is predominately led by small scale farmers who take their animals for grazing on open field/public spaces/pasturelands during this period.

The climate chamber has its data recorder which records the ambient temperature and weather variables. However apart from this we used a dry bulb and wet bulb thermometer and thermo hygrometer to record the weather variables for the calculation of the THI. As this formula needed the dry bulb and wet bulb measurements which the automated data recorder installed in the climate chamber could not provide.

Comment 7: What is the correlation between the recording time points of 8 a.m. and 2 p.m. mentioned on Lines 96-97 and the previous 10 a.m. and 4 p.m.?

Response: The purpose of setting the chambers to the simulated temperature and humidity from 10 am to 4 pm is to simulate heat stress in the animals. Here the duration of heat stress is the main focus. The two point weather recording and THI computation, at 8 am and 2pm, is to consider a cumulative impact of heat stress.

Comment 8: On Lines 109-110, check whether there is any problem with the description of the treatments for the NC and BC groups.

Response: Thank for pointing the typing error, we deleted the word ‘two’ thereby making the sentence clear (lines 112-114).

Comment 9: On Lines 116-117, if animals under grazing conditions will preferentially choose areas under heat stress, the significance of this paper seems diminished.

Response: We apologize for the confusion. We did not intend to state that the animals would preferably choose areas under heat stress. Rather we wanted to relate to the field situation wherein livestock are taken for grazing during the day. Livestock are not housed indoors during the day in majority of the farms (small scale) in India. When taken out for grazing, often they face the harsh climate due to lack of shelter at the grazing land. Therefore we felt instead of subjecting the animals to a constant temperature, a simulated model will bridge the differences of an experimental and field study.

Comment 10: On Line 178, OTU clustering has many disadvantages. The current trend is ASV.

Response: Thanking you so much for sharing your concern. As stated earlier, the sequencing and analysis for this project was already completed making it difficult to incorporate such suggestions, however, as mentioned we will make a note of this in our upcoming projects. I hope the reviewer understands the present situation.

Comment 11: On Line 179, is it version 1 or 2 of the PICRUSt program? This is crucial for result prediction. Additionally, the description in this paragraph is too brief. The authors are requested to supplement the analysis methods for the raw data and the instruments and version numbers used.

Response: For the present analysis the PICRUSt version 1 was used (line 184).

Comment 12: On Line 198, the THI is 95, which seems unlikely. Generally, a THI above 82 is already considered severe heat stress. What degree of heat stress does a THI above 95 represent? The authors are requested to verify the data.

Response: We understand that the THI values are high and hence we verified the data before the analysis. As the afternoon temperatures in the heating chamber was set to 40 °C, the THI calculation using McDowell (1972) equation resulted in the average afternoon THI of 95.

Comment 13: On Line 205, in the description of the results of 3.2. Rumen Liquor Variables, the authors list data for 0, 15, 30, and 45 days for each indicator. What is the intention of listing so much data?

Response: One of the purpose of this detailed description is to provide the readers an overview of the variations (if any) in the rumen variables over the period of time in the four groups.

Comment 14: On Line 206, it is ammoniacal nitrogen, not NH₃.

Response: We appreciate the suggestion. The term NH₃ was replaced with ammoniacal nitrogen throughout the manuscript.

Comment 15: The results described on Lines 206-307 are suggested to be integrated into one paragraph. The current description is too redundant, making it difficult to understand the intended meaning of the results.

Response: We have reduced the above stated contents and the edits made can be seen between lines 219-228 and 235-260 of the revised manuscript

Comment 16: The data in Figure 8 on Lines 544-547 is unnecessary in the main text. I would like to know what meaning corresponding to the research objective can be expressed with this figure?

Response: For studies on rumen metagenomics, it is a usual practice to assess the Venn diagram so as to get an idea of the number of common and shared microorganism across the different taxonomical classes in the groups. The intention of the data on figure 8 was for the same reason.

Comment 17: On Line 686, it is stated that "As a first-time study." In fact, there have been many studies on heat stress in goats. It is unreasonable for the authors to focus on a specific local breed of goat here. There are many goat breeds worldwide. Does this mean that using each breed is innovative? If so, the current research results will not be very representative. I think the descriptions on Lines 686-691 are not suitable for the conclusion section. The authors should concisely highlight the innovative findings and significance of this study here.

Response: We understand that there is relatively good quantum of literature on heat stress in goats, however there are very scanty reports assessing the impact of heat stress on rumen microbial diversity using metagenomics in goats. To our knowledge there is only one documented report on dairy goats (Li et al 2024). Thus we believe our study is novel, not only is this study a first time attempt to assess rumen metagenome diversity in heat stress goats, its also unique for comparing the differences among goat breeds.

Comment 18: On Line 718, regarding the references, there are many studies on heat stress in meat sheep worldwide. The authors only mention a small part in the introduction. It is suggested that the authors re-search and enrich the research progress, and add relevant literature in the discussion section to enhance the interpretability of the paper's results.

Response: We authors wanted prioritized citing articles on goats first and only on sections wherein no such information were there we opted for other species.

Reviewer 3 Report

Comments and Suggestions for Authors

 Review, paper no. fermentation-3713715 entitle „Unravelling the Heat Stress Response in Goats Based on Changes in the Rumen Fermentation Pattern and Microbiota Diversity”. The authors have used the standard journal format in manuscript writing.

This study represents the first investigation into the effects of extreme heat stress on rumen fermentation and microbiota in indigenous goat breeds (Nandidurga and Bidri). This aspect constitutes the value of the manuscript. However, the practical application should be specified precisely.

Specific comments:

Abstract: Is sufficiently presented (methods, results, general conclusions).

Strengthen the introduction by pointing out a specific research gap and potential applications of the results.

Simplify and improve the syntax, e.g.: "Furthermore, these alterations were predicted to impair functional pathways..."

Add a strong final sentence that emphasizes the significance of the results and possible practical applications (clearly point to a goat breed better suited for HS).

Introduction: The introduction section is sufficient and analytically and adequately covers the need for the study.

Methods: The methodology is sufficiently presented. However, it has a few inaccuracies.

Randomization was performed, but no details were provided — such as how the randomization was conducted, or whether body weight, body condition, or physiological status were taken into account.

The animals were exposed to heat stress for 6 hours per day, but the conditions during the remaining hours of the day were not described.

The feed ratio (60%/40%) was provided, but no information was given regarding the chemical composition, protein content, fiber, or energy value. The composition and nutritional value of the diet should be described in detail.

Line 110: “Sheep” ??? — This requires clarification.

Was there an adaptation period for the animals to the climatic chamber conditions?

Were clinical or physiological examinations performed on the animals?

Section 2.4: Rumen enzymes assay — It is not stated whether the measurements were performed in replicates.

Section 2.5: Volatile fatty acids — The procedure for extracting VFAs from rumen fluid was not described.

Statistics — There is no mention of testing for data normality. A better statistical approach would be to use a Mixed Model. The Tukey HSD test provides better control for multiple comparisons and false-positive errors than Duncan’s test.

Supplementary

Figure S2 – S4. Provide the units of relative abundance of each type of rumen microorganisms.

Result and discussion

The results of the study are analytically presented. Figures are adequate explain the findings of the study. The results of study are sufficiently discussed.

The influence of heat stress on the rumen microbiome in both goat breeds studied has been demonstrated. Although no significant decrease in rumen pH was observed, it remains unclear whether the organism’s acid–base balance was disrupted, for instance, due to the potential production of substantial amounts of lactic acid by certain bacterial species. A broader discussion is needed regarding the observed decrease in ammonia concentration and its possible link to disturbances in acid–base homeostasis under heat stress conditions.

Could authors define possible limitations of the study?

Conclusion: The conclusion is well structured, clearly presenting the main conclusions and their significance.

Author Response

General comment 1: Review, paper no. fermentation-3713715 entitle „Unravelling the Heat Stress Response in Goats Based on Changes in the Rumen Fermentation Pattern and Microbiota Diversity”. The authors have used the standard journal format in manuscript writing.

Response: we authors sincerely thank the reviewer for the efforts taken to revise the manuscript.

General comment 2: This study represents the first investigation into the effects of extreme heat stress on rumen fermentation and microbiota in indigenous goat breeds (Nandidurga and Bidri). This aspect constitutes the value of the manuscript. However, the practical application should be specified precisely.

Response: We have modified our results and discussion to highlight the breed differences and thereby provide a statement about the better breed. An additional sentence in the introduction was also added to address the reviewer’s comment (Line 66-67)

Comment 1: Abstract: Is sufficiently presented (methods, results, general conclusions).

Response: Thank you for your efforts taken to review the manuscript.

Comment 2: Strengthen the introduction by pointing out a specific research gap and potential applications of the results.

Response: Thank you for your suggestion, due to the word limit constrain in the abstract, we could not include an elaborate justification, however we have improvised a statement to address this comment which can be viewed between lines 14-15 of the revised manuscript.

Comment 3: Simplify and improve the syntax, e.g.: "Furthermore, these alterations were predicted to impair functional pathways..."

Response: Thank you for pointing out the typing error we have rectified this sentence (Lines 31-32).

Comment 4: Add a strong final sentence that emphasizes the significance of the results and possible practical applications (clearly point to a goat breed better suited for HS).

Response: Thank you for your suggestion. We have incorporated your suggestion which can be viewed between lines 34-35 of the revised manuscript.

Comment 5: Introduction: The introduction section is sufficient and analytically and adequately covers the need for the study.

Response: We authors extend our gratitude for your feedback

Comment 6: Methods: The methodology is sufficiently presented. However, it has a few inaccuracies.

Randomization was performed, but no details were provided — such as how the randomization was conducted, or whether body weight, body condition, or physiological status were taken into account.

Response: As our goats were of the same age group and of the nearly similar body weight, we did not have to consider these criteria stringently. However, upon randomly allotting the animals in each group, we did check if the average body weight for each group (especially for each breed) before the start of the experiment were similar.

Comment 7: The animals were exposed to heat stress for 6 hours per day, but the conditions during the remaining hours of the day were not described.

Response: The climate chambers were turned off everyday after 4pm and turned on the next day before 10 am. We missed mentioning this point and have now indicated it in the revised manuscript. (lines 96-97)

Comment 8: The feed ratio (60%/40%) was provided, but no information was given regarding the chemical composition, protein content, fiber, or energy value. The composition and nutritional value of the diet should be described in detail.

Response: Thank you for your suggestion. We have added this information in the supplementary data (supplementary table 2)

Comment 9: Line 110: “Sheep” ??? — This requires clarification.

Response: We apologize for the typing error. We have rectified the mistake which can be viewed on line 114

Comment 10: Was there an adaptation period for the animals to the climatic chamber conditions?

Response: Yes the animals were acclimatized for a period of 14 days in the climate chamber before starting the experiment

Comment 11: Were clinical or physiological examinations performed on the animals?

Response: Yes, physiological variables were recorded for this data, however that part of the data is aligned as another chapter intended to be used as a separate manuscript hence we could not include the data in the present manuscript

Comment 12: Section 2.4: Rumen enzymes assay — It is not stated whether the measurements were performed in replicates.

Response: We would like to clarify that the this particular analysis for conducted in three replicates. This was mentioned in section 2.4.

Comment 13: Section 2.5: Volatile fatty acids — The procedure for extracting VFAs from rumen fluid was not described.

Response: Thank you for pointing this, we have cited a reference study from our laboratory to explain the detail about the methodology associated with VFA extraction. This edit can be viewed between lines 140-141.

Comment 14: Statistics — There is no mention of testing for data normality. A better statistical approach would be to use a Mixed Model. The Tukey HSD test provides better control for multiple comparisons and false-positive errors than Duncan’s test.

Response: We appreciate your suggestion. The rumen variables (pH, ammoniacal nitrogen, enzymes and fermentation variables) were re-analysis using mixed model in SAS. The Tukey test was adopted for post hoc analysis. The revision in the materials and methods and results are indicated in the revised manuscript under the respective sub-section.

Comment 15: Supplementary-Figure S2 – S4. Provide the units of relative abundance of each type of rumen microorganisms.

Response: The heat maps were generated based on the same data as the relative abundance. Hence the unit of relative abundance is as percentage. The legend on the right hand side provides a ideal of the values as inserting the values for each component may impair the quality of the figure. We have indicated the unit in the legend for these figures which can be observed in the revised manuscript.

Comment 16: Result and discussion- The results of the study are analytically presented. Figures are adequate explain the findings of the study. The results of study are sufficiently discussed.

Response: Thank you for your comment.

Comment 17: The influence of heat stress on the rumen microbiome in both goat breeds studied has been demonstrated. Although no significant decrease in rumen pH was observed, it remains unclear whether the organism’s acid–base balance was disrupted, for instance, due to the potential production of substantial amounts of lactic acid by certain bacterial species. A broader discussion is needed regarding the observed decrease in ammonia concentration and its possible link to disturbances in acid–base homeostasis under heat stress conditions.

Response: We agree with the reviewer’s comment. The modified statistical analysis (which was suggested by Reviewer 3) slightly changed some of the significance values for the rumen variables estimated. The discussion was therefore modified based on the revised finding. Attempts were also made to address the link between the estimated rumen variables and metagenomics profile.

Comment 18: Could authors define possible limitations of the study?

Response: We authors are of the opinion that no study can be perfect. We were of the opinion that assessing a time-lag study on the rumen microbial diversity could provide even deeper insight into the rumen dynamics right from the start of the study until its end. This however would involve sequencing higher number of samples, being a major financial constraint. We have also mentioned a statement for proposed future study to strengthen the study considering other aids (Line).

Comment 19: Conclusion: The conclusion is well structured, clearly presenting the main conclusions and their significance.

Response: Thank you, we appreciate your efforts in reviewing the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed most of my previous concerns appropriately, and the manuscript has improved. However, the figures still require further improvement. Currently, they are too fragmented and scattered. I recommend merging related panels where appropriate and improving image resolution and formatting to enhance clarity and readability.

Author Response

General comment: The authors have addressed most of my previous concerns appropriately, and the manuscript has improved. However, the figures still require further improvement. Currently, they are too fragmented and scattered. I recommend merging related panels where appropriate and improving image resolution and formatting to enhance clarity and readability.

Response: We thank the reviewer for acknowledging our revision as appropriate. We have tried our level best to improve the quality of the figures. We feel since the figures are in word looking very small they appear not clear. We have merged related panels and tried improving the resolution to enhance the clarity and readability. The qualities of the figures are as per the requirement and we feel in online published version it will come properly.

Reviewer 2 Report

Comments and Suggestions for Authors

I have reviewed the revised version and response letter provided by the authors. I found that the authors have ignored many of the concerns I raised previously and have not made any modifications or improvements in areas where data could be re-analyzed. Below are my specific comments:

The title in lines 2-4 is overly assertive regarding the significance of the study. With only six replicates per group, such a conclusion cannot be drawn. The revised title merely adds “Indication Towards Altered Cellulose Degradation,” which is self-evident, as stress (including heat stress) is known to alter fiber degradation in ruminants. This limits the significance of the study. In the previous Comment 5, the authors need to provide a reasonable explanation for the design to explore heat stress using a diet based on maintenance requirements.

In the previous Comment 6, the authors could use the Temperature Humidity Index (THI), which can be calculated based on temperature and relative humidity alone, to provide more accurate data. I am still puzzled by the reason mentioned in the previous Comment 7. Regarding Comment 10, the authors simply need to conduct an ASV analysis on the raw sequencing data, which is not difficult. Additionally, the authors should upload the raw data to a public database, such as NCBI, and at least list the accession number in the Data Availability Statement section.

For Comment 11, the PICRUSt version 1 used by the authors is outdated, and many metabolic pathways have been overlooked. The authors should re-analyze the data using PICRUSt version 2 for functional prediction analysis.

Figure 6 and Figure 7 can be combined for comparison of the four groups together using LEfSe.

Regarding Comment 16, instead of using a Venn diagram to show shared microbes between groups, it would be better to directly list the differentially abundant microbes. If the authors insist on including the Venn diagram, it could be placed in the supplementary materials.

The first sentence of the revised conclusion in Comment 17 is inappropriate. There are numerous reports on the use of metagenomics and metabolomics to study heat stress in goats. The authors only found one, which is not objective. Since the journal does not allow citing specific references, I would suggest that the authors use comprehensive databases for their literature search. They should then improve the introduction section as mentioned in Comment 18.

Author Response

General comment: I have reviewed the revised version and response letter provided by the authors. I found that the authors have ignored many of the concerns I raised previously and have not made any modifications or improvements in areas where data could be re-analyzed. Below are my specific comments:

Response: There were four reviewers comments provided to us and we substantially revised the manuscript based on all four reviewers comments. We tried our best to address the comments wherever possible. May be we have not clarified few of this reviewer comments satisfactorily. We tried our best to address those comments in depth in this version of the manuscript.

Comment 1: The title in lines 2-4 is overly assertive regarding the significance of the study. With only six replicates per group, such a conclusion cannot be drawn. The revised title merely adds “Indication Towards Altered Cellulose Degradation,” which is self-evident, as stress (including heat stress) is known to alter fiber degradation in ruminants. This limits the significance of the study. In the previous Comment 5, the authors need to provide a reasonable explanation for the design to explore heat stress using a diet based on maintenance requirements.

Response: The title was now changed appropriately reflecting the contents of the manuscript. I hope the reviewer will agree to this title now. As far as the diet is concerned, we would like to clarify that the animals were fed on the basis of 3% body weight on dry matter basis and our institute follow this standard. This information was modified in the revised manuscript. Further, we would like to clarify that the feeding pattern was similar across all four groups so we authors believe the differences were nullified and the effect obtained could be solely due to heat stress.

Comment 2: In the previous Comment 6, the authors could use the Temperature Humidity Index (THI), which can be calculated based on temperature and relative humidity alone, to provide more accurate data. I am still puzzled by the reason mentioned in the previous Comment 7. Regarding Comment 10, the authors simply need to conduct an ASV analysis on the raw sequencing data, which is not difficult. Additionally, the authors should upload the raw data to a public database, such as NCBI, and at least list the accession number in the Data Availability Statement section.

Response: The THI indicated in the manuscript at 8:00 h and 14:00 h are to show the diurnal variation. Since it is confusing we have now provided the temperature and relative humidity data for each hour starting from 10:00 h to 16:00 h. We believe this avoid the confusion and provide more clarity about the heat stress the animals were exposed at each hour between 10.00 h to 16:00 h. inside the climate chamber. We like to clarify that we outsourced for bioinformatics analysis and now the firm denied reanalysis as the project has already been completed therefore we are not in a position to do the ASV analysis. Although ASV analysis is updated methodology, even now researchers use OTU cluster based analysis. We would like to clarify that the raw data from this experiment was already included in public database and the accession number is provided in the data availability statement.

Comment 3: For Comment 11, the PICRUSt version 1 used by the authors is outdated, and many metabolic pathways have been overlooked. The authors should re-analyze the data using PICRUSt version 2 for functional prediction analysis.

Response: We like to clarify that we outsourced for bioinformatics analysis and now the firm denied reanalysis as the project has already been completed. Therefore, it is not possible to change the PICRUSt version 1 analysis to PICRUSt version 2. However, we authors still believe PICRUSt version 1 analysis gives a fair bit of analysis providing useful information. Moreover other three reviewers did not question this analysis. Therefore we authors request this reviewer to allow us to go with PICRUSt version 1 analysis itself.

Comment 4: Figure 6 and Figure 7 can be combined for comparison of the four groups together using LEfSe.

Response: Thank you for placing your suggestion. We have combined figure 6 and 7 for comparing the four groups and altered the figure numbers accordingly both in the headings and in textr of he revised manuscript.  

Comment 5: Regarding Comment 16, instead of using a Venn diagram to show shared microbes between groups, it would be better to directly list the differentially abundant microbes. If the authors insist on including the Venn diagram, it could be placed in the supplementary materials.

Response: As per the suggestion of the reviewer the Venn diagrams are shifted to supplementary figures and the changes were made in the text.

Comment 6: The first sentence of the revised conclusion in Comment 17 is inappropriate. There are numerous reports on the use of metagenomics and metabolomics to study heat stress in goats. The authors only found one, which is not objective. Since the journal does not allow citing specific references, I would suggest that the authors use comprehensive databases for their literature search. They should then improve the introduction section as mentioned in Comment 18.

Response: Thank you for expressing your concern. We have now modified the first sentence in the conclusion as suggested by the reviewer. Further few more sentences were included by thoroughly reviewing the literature to improve the introduction section. Three new references were included in reference section.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

I can understand that it is challenging for the author to re-optimize the analysis of sequencing data. Nevertheless, the author has made a great effort. Here are some suggestions:

The term “Adaptation” in lines 2 - 4 is ambiguous and can lead to misunderstandings. It is suggested that the author further optimize it.

The newly added research progress described in lines 62 - 73 is rather awkward. These studies are indeed relevant to the content of this research and should be expressed in a logical language. In addition, the current three references are only a part of the studies on heat stress and goats. More relevant ones should also be mentioned. The current total number of references is 28, which is lower than the required 30 for research papers by the journal. It is suggested to increase it.

The red font in lines 103 - 104 does not need to be italicized.

Line 177 should be 16S.

For lines 504 - 505 regarding Figure 6, I mean to put the four groups in one figure so that the size of each box is uniform. The current two figures have different scales on the x - axis. The interval on the left figure is 1, while that on the right figure is 2. This needs to be unified.

In lines 701 - 704, it is mentioned that there should theoretically be 24 rumen fluid samples for sequencing. However, I found through the accession number PRJNA787712 that there are only 16 Biosamples. What is the reason here?

In terms of presenting the results, at least two of PCA/PCoA/NMDS/ANOSIM in beta - diversity should be displayed. Please supplement them.

As long as there is the raw sequencing data, these analyses are easy to perform. The author is expected to further improve and present a more comprehensive research outcome to the readers.

Author Response

General comment: I can understand that it is challenging for the author to re-optimize the analysis of sequencing data. Nevertheless, the author has made a great effort. Here are some suggestions:

Response: We thank the reviewer for his appreciation for our efforts in revising the manuscript.

Comment 1: The term “Adaptation” in lines 2 - 4 is ambiguous and can lead to misunderstandings. It is suggested that the author further optimize it.

Response: We addressed this particular comment of the reviewer and changed the word “adaptation” ’to “thermotolerance” in the title of the revised manuscript.

Comment 2: The newly added research progress described in lines 62 - 73 is rather awkward. These studies are indeed relevant to the content of this research and should be expressed in a logical language. In addition, the current three references are only a part of the studies on heat stress and goats. More relevant ones should also be mentioned. The current total number of references is 28, which is lower than the required 30 for research papers by the journal. It is suggested to increase it.

Response: This suggestion of the reviewer was well taken and accordingly these sentences were modified and additional three more references were included to a total 31 references in the paper. The numbering of the references were adjusted accordingly.  

Comment 3: The red font in lines 103 - 104 does not need to be italicized.

Response: This mistake was rectified in the revised manuscript.

Comment 4: Line 177 should be 16S.

Response: This mistake was rectified in the revised manuscript

Comment 5: For lines 504 - 505 regarding Figure 6, I mean to put the four groups in one figure so that the size of each box is uniform. The current two figures have different scales on the x - axis. The interval on the left figure is 1, while that on the right figure is 2. This needs to be unified.

Response: This suggestion of the reviewer was considered and the figure was altered again using the same scale to compare the results across the breeds.  

Comment 6: In lines 701 - 704, it is mentioned that there should theoretically be 24 rumen fluid samples for sequencing. However, I found through the accession number PRJNA787712 that there are only 16 Biosamples. What is the reason here?

Response: We like to clarify that 24 samples were used only for rumen fermentation variables estimation. But for 16S V3-V4 rRNA sequencing only 16 samples were used. This was clearly mentioned in the materials and methods section in sub heading 2.6. Rumen Metagenomics: 16S V3-V4 rRNA sequencing. To avoid confusion in data availability statement also 16 number of samples was indicated.

Comment 7: In terms of presenting the results, at least two of PCA/PCoA/NMDS/ANOSIM in beta - diversity should be displayed. Please supplement them.

Response: We only have PCoA analysis result which was included as Supplementary figure S2 in the revised manuscript. We will take care of other analysis in future experiments.  

Comment 8: As long as there is the raw sequencing data, these analyses are easy to perform. The author is expected to further improve and present a more comprehensive research outcome to the readers.

Response: We thank the reviewer for this suggestion and we assure that in our future experiments we will incorporate all his suggestions in the work pipeline for bioinformatics analysis.  We felt the comments by this reviewer was very useful for our ongoing research and we understand all the comments is to fine tune our analysis to present a comprehensive research outcome. These suggestions will be followed in our future experiments.