Study of the Relationship Between Natural Mating Expression and Intestinal Resistance Genes in Captive Adult Giant Pandas

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The submitted manuscript titled “Study of the relationship between natural mating expression and intestinal resistance genes in captive adult giant pandas” investigates the potential relationship between mating behavior and intestinal antibiotic resistance genes in giant pandas. The authors have applied extensive genetic and metagenomic analyses to link gut microbiota composition and genetic factors with the expression of natural mating behavior.

Despite the comprehensive statistical analyses and models used, the conclusions remain largely hypothetical, as there is no direct connection between the observed microbiological data and physiological parameters of the animals. I recommend:

1. Linking the findings with blood or hormonal data – if not measured by the authors, these could at least be discussed in the Discussion section with reference to previous studies. This would strengthen the biological relevance of the observed associations.

2. Expanding the discussion on the role of sex hormones, which are a key determinant of mating behavior in mammals, and integrating this with microbiome data to provide a more robust interpretation.

3. Discussing potential mechanisms by which gut microbiota could influence the synthesis or modulation of sex hormones and pheromones, and their impact on mating behavior in giant pandas.

Overall, the manuscript presents an interesting and innovative study, but it would benefit significantly from linking microbiome and genetic data with physiological and hormonal indicators to support the proposed conclusions more convincingly.

Author Response

We thank you very much for giving us an opportunity to revise our manuscript, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Study of the relationship between natural mating expression and intestinal resistance genes in captive adult giant pandas”. (ID: microbiolres-4115760).

- General comments:

Comment 1: The submitted manuscript titled “Study of the relationship between natural mating expression and intestinal resistance genes in captive adult giant pandas” investigates the potential relationship between mating behavior and intestinal antibiotic resistance genes in giant pandas. The authors have applied extensive genetic and metagenomic analyses to link gut microbiota composition and genetic factors with the expression of natural mating behavior.

Response to comment 1: Thank you very much for your recognition of our study. Thank you for your review of our paper and your valuable suggestions. The following are our responses to all your comments.

Comment 2: Overall, the manuscript presents an interesting and innovative study, but it would benefit significantly from linking microbiome and genetic data with physiological and hormonal indicators to support the proposed conclusions more convincingly.

Response to comment 2: Thank you for the reviewers' suggestions. We will incorporate the combination of physiological indicators and microbiome data in our subsequent experiments to further support our research conclusions.

- Specific questions:

Question 1: Despite the comprehensive statistical analyses and models used, the conclusions remain largely hypothetical, as there is no direct connection between the observed microbiological data and physiological parameters of the animals. I recommend:

Linking the findings with blood or hormonal data – if not measured by the authors, these could at least be discussed in the Discussion section with reference to previous studies. This would strengthen the biological relevance of the observed associations.

Response to Question 1: In accordance with the reviewers' suggestions, we have reorganized the discussion section of the article and added relevant content. “Research into the intestinal microbiota had revealed that severe psychological stress can disrupt the equilibrium of the human gut flora. Such dysbiosis, driven by shifts in the taxonomic composition of the gut microbiota-particularly the Firmicutes to Bacteroidetes ratio-results in a reduction of certain microbial metabolites, such as short-chain fatty acids. This reduction subsequently impairs the brain’s neurotransmitter system and exacerbates psychological stress [37]. Previous work had identified significant correlations in giant pandas between fecal levels of Clostridium tetani and s_Clostridium_sp_MSJ_8 and the synthesis of the short-chain fatty acid propionate and its metabolite methylmalonate [38]. Importantly, psychological stress induced by suboptimal captive environments has been established as a critical factor disrupting the natural mating behavior of giant pandas [16,39]”.

Question 2: Expanding the discussion on the role of sex hormones, which are a key determinant of mating behavior in mammals, and integrating this with microbiome data to provide a more robust interpretation.

Response to Question 2: In accordance with the reviewers' suggestions, we have reorganized the discussion section of the article and added relevant content. “Research into the role of sex hormones in mammalian mating behavior had continued to advance. Yi and Cha (2022) revealed that gut microbiota modulate sexual attractiveness and mating preferences in mammals by influencing individual-specific odors mediated by sex pheromones. Their work suggests that gut microbiota alter the abundance of these odorant pheromones, which in turn shapes the mating decisions of animals [4]”.

Question 3: Discussing potential mechanisms by which gut microbiota could influence the synthesis or modulation of sex hormones and pheromones, and their impact on mating behavior in giant pandas.

Response to Question 3: In accordance with the reviewers' suggestions, we have reorganized the discussion section of the article and added relevant content. “We therefore hypothesized that alterations in the gut microbiota-driven by environmental factors such as antibiotic administration, which might promote the enrichment of ARGs-could facilitate the production of key chemical signaling molecules in the perianal glands via microbial metabolites. Alternatively, microbial-derived short-chain fatty acids and other metabolites might influence giant panda neurodevelopment and psychological states through the gut-brain axis, ultimately disrupting natural communication and mating behavior [44]”.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The article was written clearly and concisely, within the scope of the journal. Generally, the study is an impressive study that makes a strong contribution to the field of microbiota. It suggests that changes in the abundance of gut microbiota and ARGs are correlated with the manifestation of normal mating behavior in captive adult male giant pandas. But the manuscript needs critical improvements especially for materials and methods and results sections.

Comments for author File: Comments.pdf

Author Response

We thank you very much for giving us an opportunity to revise our manuscript, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Study of the relationship between natural mating expression and intestinal resistance genes in captive adult giant pandas”. (ID: microbiolres-4115760).

- General comments:

Comment 1: The article was written clearly and concisely, within the scope of the journal. Generally, the study is an impressive study that makes a strong contribution to the field of microbiota. It suggests that changes in the abundance of gut microbiota and ARGs are correlated with the manifestation of normal mating behavior in captive adult male giant pandas. But the manuscript needs critical improvements especially for materials and methods and results sections.

Response to Comment 1: Thank you very much for your recognition of our study. Thank you for your review of our paper and your valuable suggestions. The following are our responses to all your comments.

-Special Questions：

Abstract:

Question 1: The introduction sets up a link between gut microbiota and mating, but your results focus heavily on antibiotic resistance genes (ARGs). Please clarify the core hypothesis and the rationale for ARGs.

Response to Question 1: As with the reviewers' suggestions, we have added the relevant contents in Abstract “We hypothesize that antibiotic treatment-induced residual antibiotic resistance genes in captive giant pandas may disrupt intestinal microbiota homeostasis, diminish the abundance of beneficial microorganisms involved in short-chain fatty acid synthesis, and consequently impair nervous system function via the gut-brain axis. The ensuing physiological stress is likely to suppress innate mating behavior and compromise pheromone synthesis, thereby reducing an individual's attractiveness to potential mates”.

Question 2: Change "Normal" vs. "Abnormal" Mating Behavior to natural and non-natural.

Response to Question 2: As the reviewers' suggestions, we have corrected "normal" and "abnormal" to “natural” and “non-natural”.

Question 3: Terms like "substantial reduction" and "remarkable increase" in lines 24-26 are qualitative. Please, add quantitative descriptors or at least consistent statistical terminology.

Response to Question 3: As the reviewers' suggestions, we have revised the wording, uniformly changing it to "significantly" and added the statistical p-values.

Introduction:

Question 4: You state that in line 55-56, “Recently, it has recently been reported that gut microbiota dysbiosis can 55 reduce the sexual attraction of female mice to male mice (Mus)”, please delete recently.

Response to Question 4: As the reviewers' suggestions, we have deleted this word.

Question 5: More information about giant pandas can improve your introduction.

Response to Question 5: As the reviewers' suggestions, we have added the relevant contents in Introduction “Giant pandas are highly selective, solitary foragers [18]. In the brief spring estrus period, wild individuals primarily utilize chemical cues from perianal gland secretions and urine to disseminate reproductive signals and facilitate mate choice [19,20]. Consequently, mate selection represents a critical stage in the giant panda mating process, directly influencing the likelihood of successful natural copulation between males and females. This process holds significant implications for the management of captive populations and the recovery of small, isolated wild populations [21,22]”.

Question 6: The main hypothesis in your introduction is that “gut microbiota can influence the synthesis of sex pheromones.” Why do you not test the relation between gut microbiota and sex pheromones?

Response to Question 6: We fully agree with the reviewer's suggestion. Our experiment indeed lacks the detection of metabolomics and the related research. This is a regret of this experiment. We hope that in the subsequent research, we can consider correlating the data of metabolites and physiological immune indicators with the data of microorganisms to better prove our conclusion.

Question 7: The sentence in line 100, “These pandas exhibit both normal and abnormal mating behaviors and are housed separately.” Is repeated, please delete it.

Response to Question 7: As with the reviewers' suggestions, we have deleted this sentence.

Question 8: State why profiling the resistome (the collection of ARGs) is a critical and logical metric for assessing the microbial dysbiosis, you hypothesize is linked to mating behavior in a captive.

Response to Question 8: As the reviewers' suggestions, we have added the relevant contents in Introduction “The administration of antibiotics can perturb the composition of the gut microbiota, thereby disrupting its homeostatic equilibrium and fostering the expansion of drug-resistant bacterial populations. Such perturbations not only compromise the protective role of the microbiota but also heighten host susceptibility to drug-resistant pathogens, diminish microbial diversity, and alter the abundance and metabolic activity of bacterial communities [10]. Furthermore, antibiotic usage promotes the dissemination of antimicrobial resistance genes and adversely affects the structural and functional integrity of the gut microbiota. For example, β-lactam antibiotic therapy is frequently associated with a marked reduction in microbial diversity and metabolic functionality [11]. Antibiotic-induced dysbiosis can also severely impair the metabolic and immunomodulatory functions of the gut microbiota. Clinical observations indicate that such dysbiosis leads to a decline in beneficial bacteria alongside an expansion of pathogenic species [12]. This imbalance correlates with decreased concentrations of short-chain fatty acids and tryptophan, as well as elevated levels of purines.”.

Question 9: Define your goals more specifically.

Response to Question 9: As the reviewers' suggestions, we have rewrite this sentence in Introduction “We therefore hypothesize that the intensive administration of antibiotics in captive environments may induce gut microbiota dysbiosis in giant pandas, fostering the expansion of pathogenic bacteria while diminishing the abundance of microbial taxa involved in the synthesis of intestinal metabolites, such as short-chain fatty acids. These alterations are posited to impact nervous system function via the gut-brain axis, elicit psychological stress, and consequently suppress natural mating behavior and disrupt the synthesis of chemical pheromones, ultimately reducing an individual’s sexual attractiveness to potential mates. To test this hypothesis, we selected captive adult male giant pandas exhibiting either normal or abnormal mating behavior from the Chengdu Research Base of Giant Panda Breeding as our study cohort. Metagenomic sequencing of fecal samples was performed to elucidate the relationship between gut microbial composition, antibiotic resistance gene abundance, and mating behavior from an intestinal microbiological perspective. This approach offers a novel avenue for uncovering the physiological mechanisms through which captive conditions influence mate choice and natural mating behavior in giant pandas”.

Materials and Methods:

Question 10: You have 11 pandas but 31 fecal samples. This indicates multiple samples per panda, turning the study into a repeated-measures design. This creates a severe statistical problem: the samples from the same individual are not independent. Failing to account for this in the analysis invalidates most statistical comparisons, unless a repeated-measures or mixedmodel approach is used.

Response to Question 10: We fully agree with the reviewer's suggestions. It is precisely because the number of giant panda samples is limited that we adopted the method of repeated sampling from individual animals to meet the requirements of microbiome detection. However, this approach often leads to non-independent data when analyzing the differences between sample groups. Additionally, the small sample size results in non-normally distributed data with unequal variances. To address this issue, we employed the Wilcoxon rank test, a non-parametric test, to compare the differences between groups.

Question 11: The chemical analysis and the composition ratios of the bamboo diet are needed.

Response to Question 11: It is very regrettable that we were unable to conduct a component analysis of the bamboo that the giant pandas were feeding on during the experiment at that time.

Question 12: The sentence “the animals were categorized into two groups: Natural mating group (NM group), five had successful natural mating experience; Artificial insemination group (AI group), and another six male giant pandas did not have successful natural mating experience.” Is not clear and needs to be enhanced.

Response to Question 12: As the reviewers' suggestions, we have rewrite this sentence in Materials and Methods “the pandas were divided into two groups: the NM group, consisting of five male adult giant pandas with successful natural mating experience (able to produce offspring through natural mating after reaching adulthood) The artificial insemination (AI) group comprised six male adult giant pandas without successful natural mating experience (unable to produce offspring through natural mating after reaching adulthood)”.

Question 13: Why were the natural mating group 5 animals vs 6 animals for the artificial insemination group?.

Response to Question 13: Due to the limited number of captive pandas, there are no more than 20 adult male pandas available for natural mating in global captive institutions. As the second largest captive panda population in the world, Chengdu Panda Base has only five adult male pandas capable of natural mating. The number of experimental animals we selected for the experiment is already the maximum possible.

Question 14: Why were fecal samples from pandas collected for three consecutive days?

Response to Question 14: To ensure that the samples collected from each experimental giant panda are all gathered in the same seasonal environment as much as possible, because the endocrine hormones and intestinal flora of giant pandas vary greatly in different seasons.

Question 15: You state that “All fecal samples of giant pandas were gathered during the estrus period in spring 2023 (February).” February is Winter season not Spring season. Winter in China begins from December 21 to March 20.

Response to Question 15: Due to the overall global climate change, the estrus period of the giant pandas in the panda base is generally concentrated from January to March. Therefore, the fecal samples of the giant pandas we collected in February are exactly from the estrus period. In addition, we also need to consider the behavioral manifestations. During the sampling process, the adult male giant pandas frequently displayed estrus behaviors such as marking and rubbing their private parts, which can also prove that they were in the estrus period.

Question 16: You stated, “Specifically, fecal samples from four pandas were collected for three consecutive days, whereas samples from Xiang Ge was collected for one day.” Explain why "Xiang Ge" had only one sample.

Response to Question 16: According to the breeding plan of that year, after Xiang Ge's sample was collected once, it was sent back to the Wolong Panda Center. Therefore, no samples of Xiang Ge were collected later.

Question 17: Explain if males that successfully achieved and not achieved natural copulation during the 2023 breeding season or all breeding life. Re-structure for narrative flow. Group steps by purpose.

Response to Question 17: We have made improvements to the Materials and Methods section.

Question 18: There is no section dedicated to the statistical design and tests used for group comparisons.

In general, the materials and methods section contains some inaccurate and missing information, and the details should be included in this section. Please review it carefully.

Response to Question 18: We have made improvements to the Materials and Methods section “Anosim analysis was a non-parametric test used to determine whether the differences between groups were significantly greater than those within groups, thereby assessing the significance of the grouping. Metastats and Linear Discriminant Analysis (LDA) effect size (LEfSe) analysis were used to search for species differences between groups. Metastats analysis was carried out to perform a permutation test between groups at each taxonomic level and obtain a p-value. Subsequently, the White and Nagarajani method was used to correct the p-value, yielding a q-value [32]. To screen for species biomarkers with significant differences between groups, first, the rank sum test method was used to detect differentially expressed species among different groups, and then LDA was employed to reduce dimensions and evaluate the influence size of the differentially expressed species, thereby obtaining the LDA score. To ascertain the impact of the gut microbiota on the natural mating behavior of adult male giant pandas in captivity, we employed the LEfSe algorithm. This was based on the relative abundances of gut microbiota in the NM and AI groups of giant pandas. The algorithm was utilized to identify significant difference groups with LDA scores exceeding 2 [33]. It was precisely because the number of giant panda samples is limited that we adopted the method of repeated sampling from individual animals to meet the requirements of microbiome detection. However, this approach often lead to non-independent data when analyzing the differences between sample groups. Additionally, the small sample size results in non-normally distributed data with unequal variances. To address this issue, we employed the Wilcoxon rank test, a non-parametric test, to compare the differences between groups”.

Results:

Question 19: The presentation is extremely confusing and contradictory, primarily due to a severe misinterpretation of statistical findings and a lack of a clear, logical narrative. This section requires a major rewrite for clarity and accuracy.

Response to Question 19: We have made improvements to the Results section.

Question 20: In line 271-272: you report NO significant difference in beta-diversity of gut microbiota (Adonis: R=0.082, p=0.073). You then dedicate a full section (3.3) to describing significant differences in the abundance of 315 genera and 2234 species in lines 283-291 and figures. Later, you again claim significant differences in ARG composition (Fig 6E).

Response to Question 20: We have made the modifications.

Question 21: Reporting the upregulation of "olfactory transduction" and "taste transduction" pathways in the gut microbiome is biologically confounding. These are host neural pathways.

Response to Question 21: Beta diversity measures the overall compositional differences between different sample communities, taking into account the changes in both the types and abundances of all species (including genera). A significant difference in genus abundance usually refers to the fact that through differential analysis (such as DESeq2, edgeR, etc.), the relative abundance of certain genera shows statistically significant differences between different groups at the genus level. These two results may seem contradictory, but they actually reflect the scale differences in the changes of microbial communities: Non-significant beta diversity: This means that from the perspective of the overall community structure, the differences in microbial composition among the treatment groups have not reached a statistically significant level. Possible reasons include: Although the abundance of a few genera changes significantly, they account for a small proportion in the community and have a limited impact on the overall structure. There are a large number of low-abundance "rare species" in the community, and their fluctuations may be "diluted" by the high-abundance dominant genera. There are strong individual differences or environmental noise among the samples, which mask the differences in the overall structure. Significant differences in genus abundance: This indicates that at a specific functional or taxonomic unit, the experimental treatment has indeed produced detectable biological effects. For example, the abundance of a certain probiotic or pathogen significantly increases or decreases in the treatment group, which may be a direct result of the experimental intervention.

Question 22: Different tests are used without justification (Adonis, Metastats, Wilcoxon, LEfSe)..

Response to Question 22: We have made improvements to the Materials and Methods section “Anosim analysis was a non-parametric test used to determine whether the differences between groups were significantly greater than those within groups, thereby assessing the significance of the grouping. Metastats and Linear Discriminant Analysis (LDA) effect size (LEfSe) analysis were used to search for species differences between groups. Metastats analysis was carried out to perform a permutation test between groups at each taxonomic level and obtain a p-value. Subsequently, the White and Nagarajani method was used to correct the p-value, yielding a q-value [32]. To screen for species biomarkers with significant differences between groups, first, the rank sum test method was used to detect differentially expressed species among different groups, and then LDA was employed to reduce dimensions and evaluate the influence size of the differentially expressed species, thereby obtaining the LDA score. To ascertain the impact of the gut microbiota on the natural mating behavior of adult male giant pandas in captivity, we employed the LEfSe algorithm. This was based on the relative abundances of gut microbiota in the NM and AI groups of giant pandas. The algorithm was utilized to identify significant difference groups with LDA scores exceeding 2 [33]. It was precisely because the number of giant panda samples is limited that we adopted the method of repeated sampling from individual animals to meet the requirements of microbiome detection. However, this approach often lead to non-independent data when analyzing the differences between sample groups. Additionally, the small sample size results in non-normally distributed data with unequal variances. To address this issue, we employed the Wilcoxon rank test, a non-parametric test, to compare the differences between groups”.

Question 23: P-values are sometimes reported, sometimes not.

Response to Question 23: We have added the p-values to the contents involved in the results.

Question 24: In line 348: Reporting a PCA/Adonis result with p=0.146 and calling it a "significant difference" is statistically incorrect. A p-value > 0.05 is not significant.

Response to Question 24: We have corrected this sentence.

Question 25: Ensure every figure reference points to the correct panel and that the text description matches the figure caption exactly.

Response to Question 25: As the reviewer’s suggestion, we have checked all the figures.

Question 26: Figures need improvement.

Response to Question 26: As the reviewer’s suggestion, We have already replaced all the figures with clear ones.

Discussions:

Question 27: The discussion reads more like a descriptive summary than a persuasive argument.

Response to Question 27: We have reorganized the Discussion section and added some necessary content.

Question 28: Failure to directly address and explain core contradictions, for example, “in lines 387-401: you spend considerable effort explaining why there is no difference in beta-diversity (stable diet, environment). However, the central finding of your paper is that there are differences in specific taxa and functions linked to mating.

Response to Question 28: We have reorganized the Discussion section and added some necessary content “Research into the intestinal microbiota had revealed that severe psychological stress can disrupt the equilibrium of the human gut flora. Such dysbiosis, driven by shifts in the taxonomic composition of the gut microbiota-particularly the Firmicutes to Bacteroidetes ratio-results in a reduction of certain microbial metabolites, such as short-chain fatty acids. This reduction subsequently impairs the brain’s neurotransmitter system and exacerbates psychological stress [37]. Previous work had identified significant correlations in giant pandas between fecal levels of Clostridium tetani and s_Clostridium_sp_MSJ_8 and the synthesis of the short-chain fatty acid propionate and its metabolite methylmalonate [38]. Importantly, psychological stress induced by suboptimal captive environments has been established as a critical factor disrupting the natural mating behavior of giant pandas [16,39].”.

Question 29: The annotation of microbial genes and ARGs to host neural sensory pathways requires cautious interpretation.

Response to Question 29: In the KEGG analysis of the functional metagenome of the intestinal microbiota, if the "olfactory transduction" and "taste transduction" pathways are found to be upregulated, it may suggest that the genes or metabolites related to these pathways have undergone significant changes in the intestinal environment. It is worth noting that although olfactory receptors are not only present in the olfactory epithelium but also expressed in various non-olfactory tissues and participate in inflammatory responses and metabolic processes, most studies on this topic have focused on the structural and functional mechanisms of olfactory receptors rather than their roles in the intestinal microbiota. Therefore, if the "olfactory transduction" and "taste transduction" pathways are upregulated in the KEGG analysis, it may be necessary to further combine metabolomics data or other multi-omics analyses to explore their potential biological significance. The upregulation of these pathways may indicate that the intestinal microbiota has an underappreciated potential in regulating host perception or metabolism.”.

Question 30: Several sentences restate well-established knowledge without clearly linking it to what is new in your study.

Many sentences are long and complex, which can obscure the main point.

Response to Question 30: We have reorganized the Discussion section and added some necessary content.

Conclusion: Should be improved, including limitations and future perspectives. Focus on the main conclusion.

Response: We have reorganized the Conclusion section and added some necessary content “Nevertheless, the present findings, which relied exclusively on fecal microbiome data, remained speculative in the absence of validation via targeted metabolomic profiling or neurophysiological evaluations. Furthermore, constrainted imposed by the endangered status of giant pandas and ethical considerations resulted in a limited sample size. Future studies employing multi-omics methodologies and larger cohorts would be necessary to corroborate these initial observations.”.

Question 31: It makes an overly strong and dangerously specific recommendation that is not supported by your data

The antibiotic recommendation is unjustified and potentially harmful. Remove the specific antibiotic class suggestions entirely.

Response to Question 31: We have reorganized the Conclusion section and added some necessary content “This study presented the initial evidence suggesting that the changes in the abundance of gut microbiota and resistance genes might be associated with the manifestation of normal mating behavior in captive adult male giant pandas. The findings of this study offered novel perspectives on how the captive environment influenced the decline in natural mating behavior among adult male giant pandas.”.

 

Reviewer 3 Report

Comments and Suggestions for Authors This study links the gut microbiota of male giant pandas to their mating behavior.
The topic is very interesting, especially considering the importance of captive breeding
for this species.
The methodology is very well described and is suitable for achieving the proposed
objectives. The results are presented clearly using good graphs and tables.
However, the introduction and discussion need to be rethought. In particular,
I believe the introduction should delve deeper into the background information
found in mammals and minimize that derived from studies in Drosophila, given the
enormous differences that exist in sexuality between insects and mammals.
Regarding the discussion, I also believe that the comparative analysis with results from
insects should be minimized. Furthermore, I think the discussion should be expanded upon
in reference 38, as it is a study similar to the present one and is important clarify the diffeerences
to clearly demonstrate the differences between the two papers. On another note, I believe it would be interesting to explore the possibility of a
method to modify the microbiota and consequently improve mating ability, as an
alternative to the use of antibiotics.    

 

Author Response

We thank you very much for giving us an opportunity to revise our manuscript, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Study of the relationship between natural mating expression and intestinal resistance genes in captive adult giant pandas”. (ID: microbiolres-4115760).

- General comments:

Comment 1: This study links the gut microbiota of male giant pandas to their mating behavior. The topic is very interesting, especially considering the importance of captive breeding for this species. The methodology is very well described and is suitable for achieving the proposed objectives. The results are presented clearly using good graphs and tables.

Response to Comment 1: Thank you very much for your recognition of our study. Thank you for your review of our paper and your valuable suggestions. The following are our responses to all your comments.

Comment 2: However, the introduction and discussion need to be rethought. In particular, I believe the introduction should delve deeper into the background information found in mammals and minimize that derived from studies in Drosophila, given the enormous differences that exist in sexuality between insects and mammals. Regarding the discussion, I also believe that the comparative analysis with results from insects should be minimized. Furthermore, I think the discussion should be expanded upon:

Response to Comment 2: As with the reviewers' suggestions, we have removed all the information related to the pheromones of the fruit fly Drosophila from the "Introduction" and "Discussion" sections, and revised and added relevant content.

Comment 3: in reference 38, as it is a study similar to the present one and is important clarify the differences to clearly demonstrate the differences between the two papers. On another note, I believe it would be interesting to explore the possibility of a method to modify the microbiota and consequently improve mating ability, as an alternative to the use of antibiotics.

Response to Comment 3: Thank you for the reviewers' suggestions. A key distinction between the present study and the work of Yan et al. (2024) is that this research not only examines the influence of gut microbiota on the natural mating behavior of captive giant pandas but also seeks to elucidate the relationship between gut microbiota, associated resistance genes, and behavior. This investigation provides a novel perspective on the connection between gut microbiota and natural mating behavior.

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript still has several points to improve.

Comments for author File: Comments.pdf

Author Response

We thank you very much for giving us an opportunity to revise our manuscript, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Study of the relationship between natural mating expression and intestinal resistance genes in captive adult giant pandas”. (ID: microbiolres-4115760).

- General comments:

Comment: The article was written clearly and concisely, within the scope of the journal. Generally, the study is an impressive study that makes a strong contribution to the field of microbiota. It suggests that changes in the abundance of gut microbiota and ARGs are correlated with the manifestation of normal mating behavior in captive adult male giant pandas. However, the manuscript needs critical improvements, especially for the materials and methods and results sections.

Response to Comment: Thank you very much for your recognition of our study. Thank you for your review of our paper and your valuable suggestions. The following are our responses to all your comments.

-Special Questions：

Abstract:

Question 1: You mentioned in lines 31 and 35 “(q < 0.05)”, it is P, not q.

Response to Question 1: Modified.

Question 2: Change "Normal" vs. "Abnormal" Mating Behavior to natural and non-natural in all manuscript not the abstract.

Response to Question 2: As the reviewers' suggestions, we have corrected "normal" and "abnormal" to “natural” and “non-natural”.

Materials and Methods:

Question 3: You have 11 pandas but 31 fecal samples. This indicates multiple samples per panda, turning the study into a repeated-measures design. This creates a severe statistical problem: the samples from the same individual are not-independent. Failing to account for this in the analysis invalidates most statistical comparisons, unless a repeated-measures or mixedmodel approach is used.

I do not agree with your response. You can use the repeated measurements test, mixedeffects models, or the Friedman test (non-parametric test). The Wilcoxon rank test is not suitable for more than two samples from the same animal.

Response to Question 3: The main comparison method we used was to compare the differences between groups (NM group and AI group). In fact, it mainly involved the comparison between two groups, so we used the Wilcoxon rank test. According to the reviewers' suggestions, we also analyzed the data using the method provided by the reviewers. The results were the same as those obtained in this experiment and did not affect the conclusion.

Question 4: The chemical analysis and the composition ratios of the bamboo diet are needed.

Response to Question 4: This information cannot be provided. The unavailability of this information does not compromise the experimental outcomes, as all bamboo supplied to the pandas was standardized in composition and nutrient content throughout the study.

Question 5: You state that “All fecal samples of giant pandas were gathered during the estrus period in spring 2023 (February).” February is the winter season, not the spring season. Winter in China begins from December 21 to March 20. Just modified the spring season to the winter season.

Response to Question 5: Modified.

Question 6: Explain if males that successfully achieved and not achieved natural copulation during the 2023 breeding season or throughout their breeding life.

Response to Question 6: The original statement was revised to enhance clarity, specifying that in 2023, one to two giant pandas within the NM group successfully completed natural mating and gave birth for the first time, whereas the remaining individuals were reproductively experienced. This adjustment was made to eliminate potential ambiguity. The central point remains that all giant pandas categorized under the NM group represent adult males that have demonstrated the capability to achieve natural mating and successful reproduction at least once.

Question 7: Re-structure for narrative flow. Group steps by purpose.

There is no section dedicated to the statistical design and tests used for group comparisons.

In general, the materials and methods section contains some inaccurate and missing information, and the details should be included in this section. Please review it carefully.

Response to Question 7: We have made every effort to sort out some of the content.

Results:

Question 8: The presentation is extremely confusing and contradictory, primarily due to a severe misinterpretation of statistical findings and a lack of a clear, logical narrative. This section requires a major rewrite for clarity and accuracy.

Response to Question 8: We have made improvements to the Results section. The main comparison method we used was to compare the differences between groups (NM group and AI group). In fact, it mainly involved the comparison between two groups, so we used the Wilcoxon rank test. According to the reviewers' suggestions, we also analyzed the data using the method provided by the reviewers. The results were the same as those obtained in this experiment and did not affect the conclusion.

Question 9: In line 271-272: you report NO significant difference in beta-diversity of gut microbiota (Adonis: R=0.082, p=0.073). You then dedicate a full section (3.3) to describing significant differences in the abundance of 315 genera and 2234 species in lines 283-291 and figures. Later, you again claim significant differences in ARG composition (Fig 6E).

Response to Question 9: Beta diversity measures the overall compositional differences between different sample communities, taking into account the changes in both the types and abundances of all species (including genera). A significant difference in genus abundance usually refers to the fact that through differential analysis (such as DESeq2, edgeR, etc.), the relative abundance of certain genera shows statistically significant differences between different groups at the genus level. These two results may seem contradictory, but they actually reflect the scale differences in the changes of microbial communities: Non-significant beta diversity: This means that from the perspective of the overall community structure, the differences in microbial composition among the treatment groups have not reached a statistically significant level. Possible reasons include: Although the abundance of a few genera changes significantly, they account for a small proportion in the community and have a limited impact on the overall structure. There are a large number of low-abundance "rare species" in the community, and their fluctuations may be "diluted" by the high-abundance dominant genera. There are strong individual differences or environmental noise among the samples, which mask the differences in the overall structure. Significant differences in genus abundance: This indicates that at a specific functional or taxonomic unit, the experimental treatment has indeed produced detectable biological effects. For example, the abundance of a certain probiotic or pathogen significantly increases or decreases in the treatment group, which may be a direct result of the experimental intervention.

Question 10: Reporting the upregulation of "olfactory transduction" and "taste transduction" pathways in the gut microbiome is biologically confounding. These are host neural pathways.

Response to Question 10: This is indeed the result presentation of KEGG functional analysis. In the KEGG analysis of the functional metagenome of the intestinal microbiota, if the "olfactory transduction" and "taste transduction" pathways are found to be upregulated, it may suggest that the genes or metabolites related to these pathways have undergone significant changes in the intestinal environment. It is worth noting that although olfactory receptors are not only present in the olfactory epithelium but also expressed in various non-olfactory tissues and participate in inflammatory responses and metabolic processes, most studies on this topic have focused on the structural and functional mechanisms of olfactory receptors rather than their roles in the intestinal microbiota. Therefore, if the "olfactory transduction" and "taste transduction" pathways are upregulated in the KEGG analysis, it may be necessary to further combine metabolomics data or other multi-omics analyses to explore their potential biological significance. The upregulation of these pathways may indicate that the intestinal microbiota has an underappreciated potential in regulating host perception or metabolism.”. 

Question 11: Again you mentioned “(q < 0.05)”, it is P, not q.

Response to Question 11: Modified.

Question 12: Ensure every figure reference points to the correct panel and that the text description matches the figure caption exactly.

➢ Figures need improvement.

Response to Question 12: We have made every effort to modify and improve the relevant content.

Discussions:

Question 13: The discussion reads more like a descriptive summary than a persuasive argument.

➢ Several sentences restate well-established knowledge without clearly linking it to what is new in your study.

➢ Many sentences are long and complex, which can obscure the main point.

Response to Question 13: We have made every effort to modify and improve the relevant content. We would be most grateful if the reviewers could offer specific suggestions for improvement and paragraph revisions. We will humbly accept them and make the necessary adjustments.