Effects of Adding Sodium Diacetate and Aspergillus oryzae to TMR Diets on Lactating Camel Production Performance, Milk Quality, and Fecal Microbiota

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors This paper presents a thorough and highly interesting study on a topic of importance in
animal production in regions of the world where camels are a significant source of milk.
The authors provide a detailed introduction outlining the benefits of using camel milk and
describing some metabolic aspects of this species. The materials and methods are precisely
described, and the results are presented in great detail using appropriate figures and
graphs. This presentation of results allows for an understanding of the benefits of the
additive used and the characteristics of the microbiota of the treated animals.
The discussion is well-structured and based on recent literature published in
peer-reviewed journals. One aspect that should be considered in this discussion
(and also in the introduction) is that camelids are not true ruminants, and if a
comparison is made with these animals due to a lack of information on camelids, this
must be taken into account. Similarly, the text mentions the rumen, and camelids
do not have a rumen homologous to that of ruminants, even though they have a
compartmentalized stomach.
Minor aspects

Line 219 change microbial flora by microbiota

Line 27 change physiological structure by physiological characteristics

Author Response

Q1:Line 219 change microbial flora by microbiotaLine 27 change physiological structure by physiological characteristics.

A1：We have made significant revisions to the entire text; all changes are highlighted in red. Thank you for your approval.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The topic is relevant and potentially valuable for advancing knowledge on nutritional strategies for lactating Bactrian camels. However, several aspects of the work need substantial clarification and revision before the manuscript can be considered for publication. Below, I provide detailed comments and suggestions organized by section.

1. Abstract

The conclusions in the abstract are too general. Please clarify that only specific bioactive components (e.g., vitamin E, vitamin C, certain fatty acids) were affected, while milk yield and most conventional milk composition parameters were unchanged.

Include key experimental details such as number of animals, trial duration, and treatments.

Avoid overinterpretation of microbiota results, given the small sample size (n = 3 per group).

Suggestion: state that microbiota findings are exploratory.

2. Introduction

There is substantial inconsistency in the terminology used for the Aspergillus oryzae additive (e.g., “kojimaase,” “kojimab,” “omethoate enzyme”).

Recommendation: standardize terminology throughout the manuscript as Aspergillus oryzae (AO).

The rationale for the study requires strengthening. Include more background literature on camel nutrition and previous use of sodium diacetate (SDA) and A. oryzae in ruminants.

Explicitly identify the knowledge gap and explain why evaluating these additives in camels is necessary.

The final paragraph should clearly state the specific objective(s) of the study.

3. Materials and Methods

There is a major discrepancy in SDA dosage (150 g/day in text vs. 1000 mg/kg in Table).

Action required: correct and standardize the dose across all sections.

The description of fecal sampling and handling is incomplete.

Suggestion: specify how samples were collected, pooled, stored, and processed for DNA extraction.

The statistical analysis is not appropriate for repeated measures.

Recommendation: use or justify a repeated-measures mixed model and clearly specify fixed and random effects.

It is unclear whether feed intake was monitored or measured. This is highly relevant for nutrition studies.

Provide more details on housing conditions, pen size, and management practices.

4. Results

Several parts of the Results section include interpretation, which should be moved to the Discussion.

Table formatting is inconsistent; the use of superscript letters indicating statistical differences is unclear.

Standardize the notation (e.g., use lowercase letters consistently) and explain in each caption.

Several values (e.g., unusually high SEM for certain fatty acids) should be rechecked for accuracy.

Figures lack essential information such as sample size (n), statistical tests used, and variance explained (for PCA plots).

Revise all figure captions accordingly.

5. Discussion

Some interpretations appear overstated relative to the data, particularly regarding microbiota effects.

Suggestion: moderate claims and emphasize the limitations imposed by the small sample size.

Statements about “improved milk quality” are not fully supported, as only specific components changed.

Recommendation: revise conclusions to reflect the limited scope of observed effects.

Strengthen the link between your results and previous literature in camels and other ruminants.

6. Conclusions

Conclusions are too broad.

Specify that SDA increased vitamin E and C, while most milk traits were unaffected, and microbiota responses were limited.

Include study limitations such as sample size for microbiota and lack of intake measurements.

7. Figures and Tables

Improve resolution of figures and correct typographical errors in tables (e.g., “p-vaule”).

Ensure that all legends include: sample size (n), statistical test used, meaning of superscripts or letters.

Standardize treatment names (TMR, SDA, AO) across all tables and figures.

Comments on the Quality of English Language

The English language throughout the manuscript requires improvement to ensure clarity and professional scientific presentation. Several sentences are grammatically incorrect, overly vague, or difficult to follow, and terminology is used inconsistently (e.g., multiple names for Aspergillus oryzae). I recommend a thorough revision by a fluent English speaker or a professional editing service. Consistent terminology, clearer sentence structure, and correction of grammatical errors will greatly improve readability and comprehension.

Author Response

Q1: The conclusions in the abstract are too general. Please clarify that only specific bioactive components (e.g., vitamin E, vitamin C, certain fatty acids) were affected, while milk yield and most conventional milk composition parameters were unchanged.Include key experimental details such as number of animals, trial duration, and treatments.

A1:We have made significant changes to the abstract and added further information as you suggested.The specific changes are as follows: Camel milk is highly valued for its nutritional and therapeutic properties. However, extensive management systems often lead to inconsistent milk quality. This study aimed to evaluate the effects of sodium diacetate (SDA) and Aspergillus oryzae (AO) as dietary additives on the milk composition and fecal microbiota of lactating Bactrian camels. Thirty camels of similar parity were randomly assigned to three groups: a control group (TMR), an SDA group (1000 mg/kg DM), and an AO group (40 g/d). The trial lasted 45 days, including a 15-day acclimation period. Routine milk components were analyzed every 10 days, while fatty acids and fecal microbiota were assessed on day 30. Results showed that SDA supplementation significantly increased the concentrations of Vitamin E, Vitamin C, and unsaturated fatty acids (UFA) in milk without affecting milk yield or routine components. Microbiota analysis indicated that SDA treatment significantly increased the abundance of the fungal genus Melanocarpus, although no broad shifts in microbial community structure were observed across groups. In conclusion, dietary SDA (1000 mg/kg) effectively enhances bioactive substances in camel milk while maintaining stable milk quality. These findings suggest that SDA is a viable "green" additive for improving the functional value of camel milk in intensive production systems.

Q2:There is substantial inconsistency in the terminology used for the Aspergillus oryzae additive (e.g., “kojimaase,” “kojimab,” “omethoate enzyme”).Recommendation: standardize terminology throughout the manuscript as Aspergillus oryzae (AO).The rationale for the study requires strengthening. Include more background literature on camel nutrition and previous use of sodium diacetate (SDA) and A. oryzae in ruminants.Explicitly identify the knowledge gap and explain why evaluating these additives in camels is necessary.The final paragraph should clearly state the specific objective(s) of the study.

A2:We have made significant revisions to the introduction, specifically lines 47-52, adding information about the benefits of camel milk bioactive substances for the human body, and lines 73-85, adding information about the application effects of sodium diacetate and amylase in ruminants, and summarizing the objectives of this experiment. 

Q3: There is a major discrepancy in SDA dosage (150 g/day in text vs. 1000 mg/kg in Table).Action required: correct and standardize the dose across all sections.The description of fecal sampling and handling is incomplete.Suggestion: specify how samples were collected, pooled, stored, and processed for DNA extraction.The statistical analysis is not appropriate for repeated measures.Recommendation: use or justify a repeated-measures mixed model and clearly specify fixed and random effects.It is unclear whether feed intake was monitored or measured. This is highly relevant for nutrition studies.Provide more details on housing conditions, pen size, and management practices.

A3:In lines 97-101, we modified the dosage used; lines 134-151 provide supplementary information on fecal sample analysis; Table 3 includes additional data on daily feed intake; lines 92-95 include supplementary data on the housing conditions; and lines 124-127 describe the experimental procedure.

Q4: Several parts of the Results section include interpretation, which should be moved to the Discussion.Table formatting is inconsistent; the use of superscript letters indicating statistical differences is unclear.Standardize the notation (e.g., use lowercase letters consistently) and explain in each caption.Several values (e.g., unusually high SEM for certain fatty acids) should be rechecked for accuracy.Figures lack essential information such as sample size (n), statistical tests used, and variance explained (for PCA plots).Revise all figure captions accordingly.

A4: We have revised and highlighted in red the captions for all figures and tables in the full text.

Q5: Some interpretations appear overstated relative to the data, particularly regarding microbiota effects.Suggestion: moderate claims and emphasize the limitations imposed by the small sample size.Statements about “improved milk quality” are not fully supported, as only specific components changed.Recommendation: revise conclusions to reflect the limited scope of observed effects.Strengthen the link between your results and previous literature in camels and other ruminants.

A5:We have made significant revisions to the discussion section, including lines 271-275, 281-288, 289-312, and 326-332.  These revisions include a more thorough discussion of the data, comparisons with previous studies, appropriate scientific hypotheses, and suggestions for future research directions.

Q6:Conclusions are too broad.Specify that SDA increased vitamin E and C, while most milk traits were unaffected, and microbiota responses were limited.Include study limitations such as sample size for microbiota and lack of intake measurements.

A6:We have rewritten the conclusions to make them concise and clear.Line334-337.

Q7:Improve resolution of figures and correct typographical errors in tables (e.g., “p-vaule”).Ensure that all legends include: sample size (n), statistical test used, meaning of superscripts or letters.

A7：We have corrected the errors throughout the text, and all changes are highlighted in red.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

1. The abstract requires major changes. No objective described, results described in very general terms, no substantive summary.
2. The introduction requires significant changes, as it is written in very general terms. Especially in the first paragraph, several important bioactive substances that are important for human health should be mentioned (the description is very general), and the aim or research hypothesis should be described. A substantive description should be provided of which ingredients may be important for increasing milk yield. The article does not contain specific information (much of which can be found in the literature), but only describes the problem in very general terms. The authors should familiarise themselves better with the available scientific literature.
3. Materials and methods. It is necessary to add the age of the camels, which calf it was, and whether milk samples were taken before the start of the observation (baseline level). Whether the cows were milked or fed their offspring. A detailed description of animal husbandry.
4. Results described correctly, well documented with tables, graphs and figures
5. The discussion requires a change, referring the obtained results to relevant literature. What is the significance of the research for practice? Is it rather cognitive research

Comments for author File: Comments.pdf

Author Response

Q1:The abstract requires major changes. No objective described, results described in very general terms, no substantive summary

A1:We have made significant changes to the abstract and added further information as you suggested.The specific changes are as follows: Camel milk is highly valued for its nutritional and therapeutic properties. However, extensive management systems often lead to inconsistent milk quality. This study aimed to evaluate the effects of sodium diacetate (SDA) and Aspergillus oryzae (AO) as dietary additives on the milk composition and fecal microbiota of lactating Bactrian camels. Thirty camels of similar parity were randomly assigned to three groups: a control group (TMR), an SDA group (1000 mg/kg DM), and an AO group (40 g/d). The trial lasted 45 days, including a 15-day acclimation period. Routine milk components were analyzed every 10 days, while fatty acids and fecal microbiota were assessed on day 30. Results showed that SDA supplementation significantly increased the concentrations of Vitamin E, Vitamin C, and unsaturated fatty acids (UFA) in milk without affecting milk yield or routine components. Microbiota analysis indicated that SDA treatment significantly increased the abundance of the fungal genus Melanocarpus, although no broad shifts in microbial community structure were observed across groups. In conclusion, dietary SDA (1000 mg/kg) effectively enhances bioactive substances in camel milk while maintaining stable milk quality. These findings suggest that SDA is a viable "green" additive for improving the functional value of camel milk in intensive production systems.

Q2:The introduction requires significant changes, as it is written in very general terms. Especially in the first paragraph, several important bioactive substances that are important for human health should be mentioned (the description is very general), and the aim or research hypothesis should be described. A substantive description should be provided of which ingredients may be important for increasing milk yield. The article does not contain specific information (much of which can be found in the literature), but only describes the problem in very general terms. The authors should familiarise themselves better with the available scientific literature.

A2:We have made significant revisions to the introduction, specifically lines 47-52, adding information about the benefits of camel milk bioactive substances for the human body, and lines 73-85, adding information about the application effects of sodium diacetate and amylase in ruminants, and summarizing the objectives of this experiment.

Q3:Materials and methods. It is necessary to add the age of the camels, which calf it was, and whether milk samples were taken before the start of the observation (baseline level). Whether the cows were milked or fed their offspring. A detailed description of animal husbandry.

A3:In lines 97-101, we modified the dosage used; lines 134-151 provide supplementary information on fecal sample analysis; Table 3 includes additional data on daily feed intake; lines 92-95 include supplementary data on the housing conditions; and lines 124-127 describe the experimental procedure.

Q4:Results described correctly, well documented with tables, graphs and figures

A4:We have verified and revised the tables and images.

Q5:The discussion requires a change, referring the obtained results to relevant literature.

What is the significance of the research for practice? Is it rather cognitive research.

A5:We have made significant revisions to the discussion section, including lines 271-275, 281-288, 289-312, and 326-332.  These revisions include a more thorough discussion of the data, comparisons with previous studies, appropriate scientific hypotheses, and suggestions for future research directions.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The mansucript can be accepted

Author Response

Thank you for your recognition; it is the greatest encouragement for my work.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been substantially improved and now presents a clear, well-structured, and scientifically sound study on the effects of sodium diacetate and Aspergillus oryzae supplementation in lactating camels. The research question is relevant, particularly given the limited number of nutrition and microbiota studies in camels, and the experimental design is appropriate for the stated objectives.

The methodology is adequately described, and the results are clearly presented and logically discussed. The authors appropriately avoid overinterpretation of non-significant effects on milk yield and routine milk components, while clearly highlighting the positive effects of sodium diacetate on bioactive milk components (vitamin E, vitamin C, and fatty acid profile). The discussion is balanced and places the findings in the context of existing literature on other ruminant species, while acknowledging the unique physiological characteristics of camels.

The authors also appropriately acknowledge the limitation related to the relatively small sample size used for microbiota analysis, which strengthens the overall credibility of the manuscript.

Only minor revisions are recommended. These mainly concern editorial and clarity issues, including correction of typographical errors (e.g., “p-vaule” to “p-value”), consistency in section numbering, and minor adjustments to wording in the microbiota PCA interpretation to avoid overly strong statements given the sample size. Additionally, brief clarification regarding KEGG pathway annotations (e.g., “Systemic lupus erythematosus”) would improve clarity for readers.

Comments on the Quality of English Language

The English language is clear and generally well written. Only minor grammatical and stylistic revisions are needed to improve fluency and readability. No major language editing is required.

Author Response

Q1: Modify the p-value format throughout the article.

A1:I apologize for the misspelling of the English word. I have corrected the entire text and changed "vauel" to "value".

Q2:Consistency in section numbering

A2:The chapter numbers have been corrected. Apologies for the incorrect chapter labeling in the Materials and Methods section.

Q3:Minor adjustments to wording in the microbiota PCA interpretation to avoid overly strong statements given the sample size.

A3:Line269-273，We have revised the statements, focusing on predictive evaluation.

Q4:Brief clarification regarding KEGG pathway annotations (e.g., “Systemic lupus erythematosus”) would improve clarity for readers.

A4:Line364-387，We provided a brief overview of the pathway and noted that it involves microbial samples.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Accept

Author Response

Thank you for your recognition; it is the greatest encouragement for my work.