Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “Evaluation of nutritional value and rumen degradation rate of six unconventional feeds using in vitro and in situ methods” is interesting and falls within the scope of Fermentation Journal. However, several issues must be addressed before further consideration, as follows:

 

Abstract:

L20–21: Should amino acids and PUFA also be mentioned as nutrients?

L22: Clarify “batch culture.”

L23: MCP is not a conventional abbreviation; please define.

L24: Rephrase “distinct differences” for clarity.

L26: What do LS and LBBL refer to? All abbreviations must be explained at first use, even in the abstract.

Some P-values should be included in the abstract.

L37–40: Avoid repeating results in the conclusion statement.

 

Introduction:

Ensure that species names such as Lycium barbarum and Cyperus esculentus are italicized throughout.

L89: Clarify the hypothesis; replace “have certain effects…” with a more precise statement.

L92: One important objective seems to be full characterization of the products; the authors should explicitly address this.

 

Materials and Methods:

L101: Use “protocol” instead of numbers, since an alphanumeric description is provided.

L106: Remove “such as.”

L113: Indicate size in mm (probably around 0.5 mm).

L116: Only one decimal is sufficient.

L118: Include pen measure and other specifications.

L121: Remove extra space.

Table 1: Ensure units are included; review CaHPO3. Which salt? NaCl?.

L134: Review use of “I.”

L135: Verify reference.

L139: Check “cm².”

L147: Clarify “measurement.”

L150: Rephrase for clarity.

L152: Add model, make, city, and country per journal guidelines.

L156: This section is confusing; add details about vials, syringes, tubes, caps, gassing, and gas release during the 72 h incubation to make the method reproducible.

L164: Review text for clarity.

Table 2: Must be self-explanatory; add reference for buffer.

L171: Verify (method 934.01) throughout.

L172: Review procedures, especially crude ash (942.05).

L178: Provide HPLC conditions and equipment specifications throughout. All equipment should include model, make, city, and country.

L195: Review reference style and formulas.

L206: This information can be removed.

Justify the use of Duncan’s test over other post-hoc tests.

L209–210: Clearly define significance thresholds (e.g., P ≤ 0.05 or 0.05 < P < 0.10); include 0.05 as effect or trend.

 

Results:

Tables must be self-explanatory; indicate number of samples (n) used for SD calculations.

Table 3: Clarify whether DM refers to original material or air-dried samples; confirm units for all variables (e.g., %DM, g/DM). Remove “DM basis” from the title if not expressed on a DM basis.

L242–249: Define “low” and “high” amounts.

L250: Review all tables for clarity.

Figure 1: Add space between variable and “(%)”; ensure italics are correct throughout.

Table 7: Confusing; remove SDs and use a single column for SEM.

Figure 3: Include error bars.

Figures must be self-explanatory, with treatment descriptions and statistical statements needed.

Table 8: Same issues as Table 7.

 

Discussion:

Avoid repeating results. Statements such as “high,” “low,” “great,” or “less” must be supported by references.

L322–332: This section is purely results; move or rewrite.

L334: Example cited is not relevant; find a study more closely related.

L336, L345, L357, L379–382: Add references.

L338–339: Discuss based on your own results.

L394–415: Paragraph too long; break into two.

L426: Clarify the evidence for “a more porous wall architecture.”

Discussion should include practical implications of increasing product levels—how much should producers use?

 

Conclusion:

Translate results from % inclusion to practical units.

The supplementary tables were provided as images/figures, which is not appropriate.

Please add SEM to Tables 1–4.

There is no footnote explaining the meaning of the superscript letters.

Table S5: indicate whether values are % DM, and use a space between the variable and unit.

Tables 4–10: review the use of blue font.

All tables must be self-explanatory so that readers can understand them independently if extracted from the manuscript.

Use italic for species.

 

Author Response

Comments 1: L20–21: Should amino acids and PUFA also be mentioned as nutrients?

Response 1: Thank you for this valuable suggestion. We agree that amino acids and polyunsaturated fatty acids (PUFA) are crucial nutrients. We have revised the abstract to include them in the list of analyzed components for better comprehensiveness. Located at line 23.

Comments 2: L22: Clarify “batch culture.”

Response 2: We thank the reviewer for this suggestion to improve clarity. The term has been revised to "in vitro batch fermentation culture" to more accurately describe the methodological approach. Located at line 25.

Comments 3: L23: MCP is not a conventional abbreviation; please define.

Response 3: We appreciate this comment. As suggested, we have provided the full name of MCP as "microbial crude protein" upon its first occurrence in the abstract. Located at line 27.

Comments 4: L24: Rephrase “distinct differences” for clarity.

Response 4: Thank you for pointing this out. We have replaced the subjective term "distinct differences" with the more standard statistical term "significant differences" to enhance precision. Located at line 28.

Comments 5: L26: What do LS and LBBL refer to? All abbreviations must be explained at first use, even in the abstract.

Response 5: We sincerely thank the reviewer for this careful observation. Although the full names are provided in the methods section, we have now supplemented the full terms for LS (Licorice Straw) and LBBL (Lycium Barbarum Branches and Leaves) upon their first mention in the abstract to ensure it is self-contained. Located at line 17.

Comments 6: Some P-values should be included in the abstract.

Response 6: We appreciate this suggestion to strengthen the abstract. We have added key P-value information (e.g., P < 0.001) for the most significant findings related to degradation rates and fermentation parameters to provide a clearer indication of statistical robustness. Located at line 33,37,39.

Comments 7: L37–40: Avoid repeating results in the conclusion statement.

Response 7: We sincerely thank the reviewer for this valuable suggestion. We fully understand the concern about avoiding redundancy between the Results and the Conclusion sections. However, our study involved the evaluation of six unconventional feed resources, and we employed both in vitro gas production and in situ rumen degradability methods to comprehensively assess their nutritional value. One of the main objectives of our work was to identify the optimal substitution levels of each feed that could be practically recommended in animal production. For this reason, we summarized these optimal substitution ratios in the abstract conclusion as the key outcome of our research. Nevertheless, we carefully revised the text to make the conclusion more concise and avoid unnecessary repetition, while still retaining the practical implications that are essential for producers and readers.

Comments 8: Ensure that species names such as Lycium barbarum and Cyperus esculentus are italicized throughout.

Response 8: Thank you for this important reminder. We have rechecked the entire manuscript and ensured that all scientific species names are consistently presented in italics.

Comments 9: L89: Clarify the hypothesis; replace “have certain effects…” with a more precise statement.

Response 9: We appreciate this feedback. We have rephrased the hypothesis to be more precise and objective. It now reads: "Different types of feed and their usage ratios can affect the rumen degradation and in vitro gas production in sheep." Located at line 95-96.

Comments 10: L92: One important objective seems to be full characterization of the products; the authors should explicitly address this.

Response 10: We sincerely thank the reviewer for this helpful suggestion. We agree that one of the key objectives of our study is to fully characterize the unconventional feed resources. In the revised manuscript, we have made this objective explicit by stating that our study not only evaluated nutrient composition, amino acids, PUFA, and bioactive compounds, but also assessed their rumen degradability and fermentation characteristics at different substitution levels. This comprehensive approach allows us to determine their appropriate substitution levels in sheep diets.

Revised Text:
This study evaluated the feed characteristics of six unconventional feed resources using in vitro fermentation and in situ rumen degradability methods, and determined their appropriate substitution levels in sheep diets. Located at line 101-104.

Comments 11: L101: Use “protocol” instead of numbers, since an alphanumeric description is provided.

Response 11: Thank you for this correction. We have replaced "numbers" with "protocol" as suggested. Located at line 111.

Comments 12: L106: Remove “such as.”

Response 12: We have removed "such as" as per the your 's suggestion. Located at line 119.

Comments 13: L113: Indicate size in mm (probably around 0.5 mm).

Response 13: Thank you for the suggestion. We have specified the sieve size in millimeters (0.425 mm, which corresponds to a 40-mesh sieve) for clarity. Located at line 126.

Comments 14: L116: Only one decimal is sufficient.

Response 14: We have adjusted the number of decimal places for the animal weight to one, as recommended. Located at line 129.

Comments 15: L118: Include pen measure and other specifications.

Response 15: We thank the reviewer for this suggestion to improve methodological detail. We have added the specific pen dimensions and features (1.5 meters × 1.2 meters, with slatted flooring and a separate feeder). Located at line 131-132.

Comments 16: L121: Remove extra space.

Response 16: The extra space has been removed. Thank you.

Comments 17: Table 1: Ensure units are included; review CaHPO3. Which salt? NaCl?.

Response 17: We appreciate the reviewer's careful review of the table. We have ensured all units are included. The compound CaHPO3 has been corrected to CaHPO₄·2H₂O, and the salt is specified as NaCl. Located at Table 1.

Comments 18: L134: Review use of “I.”

Response 18: The unnecessary "I" at the beginning of the sentence has been removed. Thank you for catching this error. Located at line 149.

Comments 19: L135: Verify reference.

Response 19: We sincerely thank the reviewer for pointing this out. We have re-checked the reference and confirmed its accuracy. The missing details have now been supplemented in the revised manuscript to ensure the citation is complete. Located at line 150-152.

Comments 20: L139: Check “cm².”

Response 20: We have verified and confirmed that "cm²" is the correct unit for the surface area of the nylon bags. Located at line 156

Comments 21: L147: Clarify “measurement.”

Response 21: We have changed "measurement" to the more precise term "nutrient analysis". Located at line 165.

Comments 22: L150: Rephrase for clarity.

Response 22: We have rephrased the sentence for better clarity: "...replace the corn stover in the basal diet with the aforementioned six materials at substitution levels of 0%, 25%, 50%, 75%, and 100%.". Located at line 169-171.

Comments 23: L152: Add model, make, city, and country per journal guidelines.

Response 23: We have added the required details for the grinder (KX-500A, Beijing Zhongxing Ltd., Beijing, China) and the water bath constant temperature oscillator device (DSHZ-300A, Taicang Experimental Equipment Co., Jiangsu, China) as per the journal guidelines. Located at line 172.

Comments 24: L156: This section is confusing; add details about vials, syringes, tubes, caps, gassing, and gas release during the 72 h incubation to make the method reproducible.

Response 24: We greatly appreciate this detailed feedback to improve reproducibility. We have completely rewritten this section to provide a more precise and sequential description of the in vitro gas production method, including details on syringes, vials, incubation conditions, gas measurement procedures, and steps taken to maintain anaerobiosis, based on the method of Menke et al. Located at line 178-189.

Comments 25: L164: Review text for clarity.

Response 25: We have re-examined the indicated text and made adjustments to improve its clarity and flow. Located at line 178-189.

Comments 26: Table 2: Must be self-explanatory; add reference for buffer.

Response 26: Thank you for this suggestion. We have made Table 2 self-explanatory by adding a clear title and a reference to the original source of the buffer composition (Menke et al.). Located at line 198.

Comments 27: L171: Verify (method 934.01) throughout.

Response 27: Thank you for your reminder. We have consulted additional references to verify the methods for DM and Ash determination in AOAC, confirming that the title of 930.15: Loss on Drying (Moisture) for Feeds pertains to the determination of dry matter in feeds and raw materials (135 ℃, 2 h). It is located under the reference number 4.1.06 in the 22nd Edition. We have made corrections based on this information. Located at line 203-204.

Comments 28: L172: Review procedures, especially crude ash (942.05).

Response 28: We have re-examined the section on the determination of crude ash in the AOAC 22nd Edition, Method Number: 942.05, Title: Ash of Animal Feed, Locator: 4.1.10 (corresponding to "942.05 → 4.1.10" in the Index of Method Numbers), Section ID: C4-3, applicable for the determination of crude ash in animal feed (including roughage, concentrates, mixed rations, etc.).  The measured crude ash represents the total amount of inorganic matter (minerals) in the sample, commonly used for evaluating the nutritional value of feed and standard analysis.  The less rigorous parts of the measurement method have been modified, incinerated to constant weight. Located at line 204-205.

Comments 29: L178: Provide HPLC conditions and equipment specifications throughout. All equipment should include model, make, city, and country.

Response 29: We have supplemented the manuscript with the HPLC conditions and the full equipment specifications (model: Agilent 1260 Infinity II, make: Agilent Technologies, city: Waldbronn, country: Germany) as requested. Located at line 210-212.

Comments 30: L195: Review reference style and formulas.

Response 30: We have standardized the presentation of formulas and symbols throughout the manuscript to ensure consistency and correctness. Located at line 229.

Comments 31: L206: This information can be removed.

Response 31: The indicated redundant information has been removed from the manuscript. Located at line 237-238.

Comments 32: Justify the use of Duncan’s test over other post-hoc tests.

Response 32: We have supplemented the justification for using Duncan's test by stating that it is widely applied in animal nutrition studies and provides greater statistical power for detecting differences with relatively small sample sizes compared to more conservative tests like Tukey's. Located at line 245-247.

Comments 33: L209–210: Clearly define significance thresholds (e.g., P ≤ 0.05 or 0.05 < P < 0.10); include 0.05 as effect or trend.

Response 33: We have clearly defined the significance thresholds in the statistical analysis section as follows: P ≤ 0.05 indicates statistical significance, and 0.05 < P ≤ 0.10 is considered a trend. Located at line 248-249.

Comments 34: Tables must be self-explanatory; indicate number of samples (n) used for SD calculations.

Response 34: We have supplemented the information indicating the sample size (n) used for calculating the average standard error of the mean (SEM) in the table. Located at line 264.

Comments 35: Table 3: Clarify whether DM refers to original material or air-dried samples; confirm units for all variables (e.g., %DM, g/DM). Remove “DM basis” from the title if not expressed on a DM basis.

Response 35: Thank you for this clarification. We confirm that all nutrient values in Table 3 are presented on a dry matter (DM) basis. The table heading has been revised to clearly state "[%, on dry matter basis, mean ± SEM, n=3]". Located at line Table 3.

Comments 36: L242–249: Define “low” and “high” amounts.

Response 36: We have replaced the subjective terms "low" and "high" with specific numerical descriptions or ranges to provide objective information. Located at line 290-291.

Comments 37: L250: Review all tables for clarity.

Response 37: We have thoroughly reviewed all tables again to ensure that all abbreviations are explained in the footnotes, units are correct, and the overall presentation is clear and consistent.

Comments 38: Figure 1: Add space between variable and “(%)”; ensure italics are correct throughout.

Response 38: We have redrawn Figure 1, adding a space between the variables and "(%)" and ensuring that species names are italicized correctly. Located at Figure 1.

Comments 39: Table 7: Confusing; remove SDs and use a single column for SEM.

Response 39: Thank you for your suggestion. We have changed the format of Table 7 to use a single-column SEM. Located at line Table 7.

Comments 40: Figure 3: Include error bars.

Response 40: We have supplemented explanations and descriptions for the error bar section in Figure 3. Located at line 355-356.

Comments 41: Figures must be self-explanatory, with treatment descriptions and statistical statements needed.

Response 41: We have supplemented the article's table with processing descriptions and statistical statements. Located at line 322-327.

Comments 42: Table 8: Same issues as Table 7.

Response 42: Thank you very much for your feedback. We apologize if our table was not clear enough and led to any misunderstanding. As our objective is to determine the optimal proportions of six different materials in sheep diets to facilitate the selection of these six feeds in production, we do not compare the six materials against each other. We have revised Table 8 to present the information in a clearer format and, following your suggestion, have adopted the use of single-column SEM. Located at Table 8-(1-6).

Comments 43: Avoid repeating results. Statements such as “high,” “low,” “great,” or “less” must be supported by references.

Response 43: Thank you very much for your suggestion. We have revised and edited the repetitive parts in the discussion that overlap with the results. Located at line 390-396.

Comments 44: L322–332: This section is purely results; move or rewrite.

Response 44: Thank you for your suggestion. We have reorganized and rewritten this section of the content. Located at line 389-409.

Comments 45: L334: Example cited is not relevant; find a study more closely related.

Response 45: Thank you for your suggestion. However, we regret to inform you that we did not find any information regarding the references in L334 and its adjacent lines.  It might be due to the garbled text caused by the file viewing software we used, leading to a misunderstanding. If you are referring to the original references 21 and 22, we have already removed that sentence, as we determined, based on the opinions of various experts, that its presence could easily cause misunderstanding among readers. Located at line 401-404.

Comments 46: L336, L345, L357, L379–382: Add references.

Response 46: We have added appropriate references to support the statements at the indicated lines. The results mentioned at L336 are from our present study, while relevant literature citations have been provided for L345, L357, and L379-382. Located at line 411,423,450.

Comments 47: L338–339: Discuss based on your own results.

Response 47: We greatly appreciate your feedback. We have recognized that L338–339 indeed lacks strong relevance to the findings of this study and could potentially lead to misunderstandings. Therefore, we have removed the description in this section. Located at line 401-404.

Comments 48: L394–415: Paragraph too long; break into two.

Response 48: We have split the long paragraph into two shorter paragraphs to improve readability and logical flow, as suggested. Located at line 452-469.

Comments 49: L426: Clarify the evidence for “a more porous wall architecture.”

Response 49: We agree that this claim required support. We have added a relevant reference (Zhong et al., 2021) that discusses how lignin composition and cell wall structure (including porosity) affect degradability, thereby providing evidence for the statement. Located at line 509.

Comments 50: Discussion should include practical implications of increasing product levels—how much should producers use?

Response 50: We greatly appreciate your valuable feedback. Your Comments regarding the discussion section should include practical application recommendations for the product (such as specific usage amounts for producers) is indeed crucial, as it can significantly enhance the applied value of the research. In this paper, our research objective primarily focuses on systematically evaluating the nutritional value, bioactive components, and fermentation characteristics in the rumen of these six unconventional feeds. Based on the results of in vitro fermentation and rumen degradation rate measurements, we have preliminarily proposed an appropriate inclusion ratio range for each feed in sheep diets, providing producers with an initial, evidence-based reference for usage amounts. At the same time, we fully agree with your viewpoint that the conclusions from in vitro studies ultimately need to be validated through animal experiments. We would like to inform you that the animal feeding experiment aimed at verifying the effectiveness of these recommended addition ratios has been successfully completed This study conducted a detailed evaluation of the effects of these feeds on sheep's growth performance, blood indices, immune-antioxidant properties, and rumen microorganisms, with the results strongly supporting the preliminary conclusions of this paper. We are currently preparing a more in-depth follow-up research paper and plan to submit it for publication as soon as possible.

Comments 51: Translate results from % inclusion to practical units.

Response 51: Thank you for your feedback. We have converted this content unit to the actual unit of g/Kg. Located at line 556-560.

Comments 52: The supplementary tables were provided as images/figures, which is not appropriate.

Response 52: We have reformatted all supplementary tables as proper editable tables (e.g., in Word) rather than images, ensuring they meet the journal's requirements.

Comments 53: Please add SEM to Tables 1–4.

Response 53: We thank the reviewer for this suggestion. Table 1 presents the daily feed formulation without SEM; Table 2 is derived from the preparation of solutions in the reference; Tables 3 and 4 display the data as mean ± SEM, n=3, with the tables already described. Located at Tables 3 and 4.

Comments 54: There is no footnote explaining the meaning of the superscript letters.

Response 54: We have added a standard footnote to all relevant tables explaining that "Different superscript letters (a, b, c, ...) within a row indicate significant differences among treatments (P ≤ 0.05)."

Comments 55: Table S5: indicate whether values are % DM, and use a space between the variable and unit.

Response 55: We have corrected Table S5 (and all other supplementary tables) to clearly indicate the basis of expression (dry matter basis where applicable) and to include a space between the variable and its unit. Located at Table S5.

Comments 56: Tables 4–10: review the use of blue font.

Response 56: We have removed the use of blue font throughout all tables (4-10 and supplementary tables) and ensured consistent, black text formatting. Located at tables 4–10

Comments 57: All tables must be self-explanatory so that readers can understand them independently if extracted from the manuscript.

Response 57: We have thoroughly reviewed all tables to ensure they are self-explanatory. This includes clear titles, fully defined abbreviations in footnotes, explicit units, and necessary statistical annotations.

Comments 58: Use italic for species.

Response 58: We have ensured that all scientific species names throughout the manuscript, including in tables and figures, are italicized consistently.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript brings up an interesting subject in animal nutrition. However, some explanations must be addressed before further considerations.

Abstract:

Does not split the text into sections.

It is hard to understand which kind of feedstuff the authors are working on. Please, revise it. There is no information on the type of ingredients.

Line 25: Does not start a sentence by using an acronym.

Line 23: What does MCP mean? Please, give the entire word before acronyms.

 

Material and Methods

 

Line 117-118: How was the individual feeding management since the animals were in the same pen?

 

Table 1: Were the nutrients presented in percentage? Please, be clear.

Line 134: The letter “I” at the beginning of the sentence?

Line 134-135: Doe Van Soest reported CP analysis?

Lines 158-160: Fix the verbal time. “The artificial saliva “is” prepared by” the right is ”was”.

How many batches did the authors run? Which were the experimental units?

Please, revise the superscript letters throughout the entire manuscript.

Table 3: Please, give the footnote below the table. Format it. Same for all tables. They are a really mess.

The tables should be self-explanatory. The reader needs to be familiar with the meanings of the acronyms.

Figure 1: What about the time zero? It is the extent of the disappearance, not the rate, right? Rate would be %/h…

Figure 2: Where is the statistical approach for this figure? Same for Figure 3.

Line 315: Please, fix the entire manuscript. It is not correct to use the word “significantly”; if the P-value was below 5% (as stated) it was significant. Improve it.

 Table 8: I am unsure of the meanings of the acronyms in Table 8. Please, add a footnote. A regression analysis must be considered.

Discussion

The data analysis does not support the discussion section. Some statistical analysis needs to be run before it.

Conclusion

How did the authors find that these percentages were better than any statistical approach that was performed?

Comments on the Quality of English Language

The English needs to be improved to achieve a better flow.

Author Response

Comments 1: Does not split the text into sections.

Response 1: Thank you for this suggestion. We have revised the Abstract by dividing it into four clear sections (Objective, Methods, Results, Conclusion) to improve clarity and readability. Located at line16,22,28,42.

Comments 2: It is hard to understand which kind of feedstuff the authors are working on. Please, revise it. There is no information on the type of ingredients.

Response 2: We appreciate this valuable comment. We have revised the Abstract to clearly specify the six unconventional feed resources investigated, including , pepper residue, grape marc, pepper straw, Lycium barbarum branches and leaves, licorice straw, and Cyperus esculentus leaves. Located at line 17-19.

Comments 3: Line 25: Does not start a sentence by using an acronym.

Response 3: Thank you for pointing this out. We have revised the sentence by replacing the abbreviation CP with its full name, ensuring it starts with the full name rather than the abbreviation. Located at line 30.

Comments 4: Line 23: What does MCP mean? Please, give the entire word before acronyms.

Response 4: Thank you for your valuable comments. We have clarified the meaning of MCP upon its first occurrence, and supplemented its full name as microbial crude protein at line 27. 

Comments 5: Line 117-118: How was the individual feeding management since the animals were in the same pen?

Response 5: Thank you for your careful observation. We have clarified this point in the "Materials and Methods" section, stating that the sheep were housed individually, managed separately, and supplemented with information on the dimensions of the pen based on expert opinions. Located at line 130-132.

Comments 6: Table 1: Were the nutrients presented in percentage? Please, be clear.

Response 6: We appreciate the reviewer’s suggestion. We have clarified in the table note that all nutrient values are expressed on a dry matter basis and presented as percentages. Located at line 145-146.

Comments 7: Line 134: The letter “I” at the beginning of the sentence?

Response 7: Thank you for Commentsing out this typographical error. We have corrected it by removing the unnecessary “I.” Located at line149.

Comments 8: Line 134-135: Doe Van Soest reported CP analysis?

Response 8: Thank you for your feedback. We have corrected this point, clarifying that the NDF and ADF analyses were conducted using the Van Soest method, while DM and CP were determined according to the AOAC methods 942.05 and 984.13. Located at line 150-152.

Comments 9: Lines 158-160: Fix the verbal time. “The artificial saliva “is” prepared by” the right is ”was”.

Response 9: Thank you for your correction. To maintain consistency, we have changed the present tense to the past tense and have rephrased this paragraph based on expert opinions. Located at line 178-190.

Comments 10: How many batches did the authors run? Which were the experimental units?

Response 10: Thank you for this important question. We have clarified the design for both the in situ and in vitro experiments: In the in situ nutrient digestibility trial, each rumen-cannulated sheep was considered an experimental unit; In the in vitro fermentation trial, all incubations were performed in three independent batches, with each incubation bottle serving as an experimental unit. Located at line 183-184.

Comments 11: Please, revise the superscript letters throughout the entire manuscript.

Response 11: Thank you for this careful suggestion. We have rechecked all tables and figures, and corrected the superscript letters to ensure consistency with statistical significance.

Comments 12: Table 3: Please, give the footnote below the table. Format it. Same for all tables. They are a really mess.

Response 12: We appreciate this important comment. We have reformatted all tables to include standardized footnotes, ensuring they are self-explanatory and clearly indicate statistical comparisons.

Comments 13: The tables should be self-explanatory. The reader needs to be familiar with the meanings of the acronyms.

Response 13: Thank you for pointing this out. We have added full explanations of all acronyms in the footnotes of each table.

Comments 14: Figure 1: What about the time zero? It is the extent of the disappearance, not the rate, right? Rate would be %/h…

Response 14: Thank you for your valuable comments. In Figure 1, the 0 time point on the X-axis refers to the moment when the nylon bag was placed into the rumen fistula of the sheep (the official start time of the experiment). Regarding the Y-axis, it represents the percentage of DM, CP, ADF, and NDF degraded in the sheep's rumen, where 0 indicates no degradation, referring to the extent of disappearance rather than the rate. Located at Figure 1.

Comments 15: Figure 2: Where is the statistical approach for this figure? Same for Figure 3.

Response 15: Thank you very much for your feedback.  We have supplemented this section in the data analysis part of the materials and methods. Located at line 241-242.

Comments 16: Line 315: Please, fix the entire manuscript. It is not correct to use the word “significantly”; if the P-value was below 5% (as stated) it was significant. Improve it.

Response 16: Thank you for raising this important suggestion. We have carefully revised the article by supplementing the P-values for the "significant" related content in the results section derived from this experiment, or removing unnecessary wording.

Comments 17: Table 8: I am unsure of the meanings of the acronyms in Table 8. Please, add a footnote. A regression analysis must be considered.

Response 17: Thank you for pointing this out. We have added footnotes to Table 8 to explain all acronyms. Additionally, regarding Table 8, we apologize if the way our table was presented caused any misunderstanding. Since our objective is to determine the optimal proportions of six different materials in sheep diets to facilitate the selection of these six feeds in production, we do not compare the six materials against each other. We have revised Table 8 to present the information in a clearer format. Located at line382-385.

Comments 18: The data analysis does not support the discussion section. Some statistical analysis needs to be run before it.

Response 18: Thank you for this comment. We have thoroughly re-examined the Discussion section to ensure that all interpretations are directly supported by the statistical results (ANOVA and Duncan's test, P < 0.05) presented in the Results section. Our discussion of differences between feeds and their optimal substitution levels is strictly based on these significant findings. We have revised the text to strengthen this link and believe the discussion is now fully grounded in our data analysis.

Comments 19: How did the authors find that these percentages were better than any statistical approach that was performed?

Response 19: Thank you for pointing out this issue. We have revised the content of this section, converting the percentage units into actual units of g/Kg. Located at line556-560.

Comments 20: Comments on the Quality of English Language

The English needs to be improved to achieve a better flow.

Response 20: We greatly appreciate your suggestions, and we have invited a professional English teacher from our school to review and improve this article.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Line  Comment

16   composition

23 is MCP a standard abbreviation, or does it need to be defined?

25 Need to know what LS, LBBL, GM etc. are common and scientific name and plant part

26  use gm/kg, no more than 3 significant digits, because no lab measurement is more accurate.

29, 30   in situ degradation

32 3 significant digits

57 scientific name, also need plant part being used and abbreviation

59 due to being rich in

71  use common name such as wolf berry or are you referring to the fruit gogi berry or to plant foliage?

83 common name tiger nut and plant part, nut or foliage?

100 Need to mention cannulation was done at veterinary hospital?  Animal welfare committee approval for cannulation and how cannula was maintained such as weekly cleaning of area.  Also how big was the diameter of the cannula and how many bags were inserted at a time.

106  Would it be appropriate to call these byproducts or alternative feedstuffs?

110 air dried

Table 1 assume % need to so designate 3 digits is adequate.  Corn stover is referenced later is MS where is it in the table?

130 reference for ME calculation

 134 disappearance

157 why 72 hour? Menke used 24 hours.

164 delete measured for

162 72 hour incubation

204 .08% is very slow.  Did you mean 8%?

217 What is microbial C?

Table 4 table heading is g/100 g.  is this g/100g protein or g/100g DM? former would be better in my opinion.

Table 3 3 significant digits is adequate.

Table 7 three significant digits only and lines will fit better

Table 8  why not include pH, ammonia and MCP ?

331 indicating a wide range in protein level among alternative feedstuffs

337 although lignin is a component of ADF, lower ADF does not necessarily mean lower lignin.

348 why not compare levels of vitamins to animal requirements? Also realize thag B vitamins may be degraded in the rumen.

356 if amino acids are reported based on g/100 g protein, this is a better way of evaluating adequacy and ability to provide supplementary amino acids.

379  It must be realized that the rumen will hydrogenate most of these unsaturated fatty acids, especially at the level that they will be consumed.

417 degradability has been observed to…

426 can’t say anything about lignin since it was not measured unless someone else has lignin analysis for PR

431 cannot allude to high lignin feedstuffs since you did not measure lignin

436 high proportion of slowly degradable

439 reference this statement, please

There were very extensive evaluation of feedstuffs by laboratory analysis, but no actual feeding trial done of any sort.  Laboratory analysis do not tell the whole story about the suitability of these materials for use in rations. The lack of some  kind of feeding or investigation of in vivo digestibility is a very serious flaw in the study.

Line  Comment

16   composition

23 is MCP a standard abbreviation, or does it need to be defined?

25 Need to know what LS, LBBL, GM etc. are common and scientific name and plant part

26  use gm/kg, no more than 3 significant digits, because no lab measurement is more accurate.

29, 30   in situ degradation

32 3 significant digits

57 scientific name, also need plant part being used and abbreviation

59 due to being rich in

71  use common name such as wolf berry or are you referring to the fruit gogi berry or to plant foliage?

83 common name tiger nut and plant part, nut or foliage?

100 Need to mention cannulation was done at veterinary hospital?  Animal welfare committee approval for cannulation and how cannula was maintained such as weekly cleaning of area.  Also how big was the diameter of the cannula and how many bags were inserted at a time.

106  Would it be appropriate to call these byproducts or alternative feedstuffs?

110 air dried

Table 1 assume % need to so designate 3 digits is adequate.  Corn stover is referenced later is MS where is it in the table?

130 reference for ME calculation

 134 disappearance

157 why 72 hour? Menke used 24 hours.

164 delete measured for

162 72 hour incubation

204 .08% is very slow.  Did you mean 8%?

217 What is microbial C?

Table 4 table heading is g/100 g.  is this g/100g protein or g/100g DM? former would be better in my opinion.

Table 3 3 significant digits is adequate.

Table 7 three significant digits only and lines will fit better

Table 8  why not include pH, ammonia and MCP ?

331 indicating a wide range in protein level among alternative feedstuffs

337 although lignin is a component of ADF, lower ADF does not necessarily mean lower lignin.

348 why not compare levels of vitamins to animal requirements? Also realize thag B vitamins may be degraded in the rumen.

356 if amino acids are reported based on g/100 g protein, this is a better way of evaluating adequacy and ability to provide supplementary amino acids.

379  It must be realized that the rumen will hydrogenate most of these unsaturated fatty acids, especially at the level that they will be consumed.

417 degradability has been observed to…

426 can’t say anything about lignin since it was not measured unless someone else has lignin analysis for PR

431 cannot allude to high lignin feedstuffs since you did not measure lignin

436 high proportion of slowly degradable

439 reference this statement, please

There were very extensive evaluation of feedstuffs by laboratory analysis, but no actual feeding trial done of any sort.  Laboratory analysis do not tell the whole story about the suitability of these materials for use in rations. The lack of some  kind of feeding or investigation of in vivo digestibility is a very serious flaw in the study.

Line  Comment

16   composition

23 is MCP a standard abbreviation, or does it need to be defined?

25 Need to know what LS, LBBL, GM etc. are common and scientific name and plant part

26  use gm/kg, no more than 3 significant digits, because no lab measurement is more accurate.

29, 30   in situ degradation

32 3 significant digits

57 scientific name, also need plant part being used and abbreviation

59 due to being rich in

71  use common name such as wolf berry or are you referring to the fruit gogi berry or to plant foliage?

83 common name tiger nut and plant part, nut or foliage?

100 Need to mention cannulation was done at veterinary hospital?  Animal welfare committee approval for cannulation and how cannula was maintained such as weekly cleaning of area.  Also how big was the diameter of the cannula and how many bags were inserted at a time.

106  Would it be appropriate to call these byproducts or alternative feedstuffs?

110 air dried

Table 1 assume % need to so designate 3 digits is adequate.  Corn stover is referenced later is MS where is it in the table?

130 reference for ME calculation

 134 disappearance

157 why 72 hour? Menke used 24 hours.

164 delete measured for

162 72 hour incubation

204 .08% is very slow.  Did you mean 8%?

217 What is microbial C?

Table 4 table heading is g/100 g.  is this g/100g protein or g/100g DM? former would be better in my opinion.

Table 3 3 significant digits is adequate.

Table 7 three significant digits only and lines will fit better

Table 8  why not include pH, ammonia and MCP ?

331 indicating a wide range in protein level among alternative feedstuffs

337 although lignin is a component of ADF, lower ADF does not necessarily mean lower lignin.

348 why not compare levels of vitamins to animal requirements? Also realize thag B vitamins may be degraded in the rumen.

356 if amino acids are reported based on g/100 g protein, this is a better way of evaluating adequacy and ability to provide supplementary amino acids.

379  It must be realized that the rumen will hydrogenate most of these unsaturated fatty acids, especially at the level that they will be consumed.

417 degradability has been observed to…

426 can’t say anything about lignin since it was not measured unless someone else has lignin analysis for PR

431 cannot allude to high lignin feedstuffs since you did not measure lignin

436 high proportion of slowly degradable

439 reference this statement, please

There were very extensive evaluation of feedstuffs by laboratory analysis, but no actual feeding trial done of any sort.  Laboratory analysis do not tell the whole story about the suitability of these materials for use in rations. The lack of some  kind of feeding or investigation of in vivo digestibility is a very serious flaw in the study.

Line  Comment

16   composition

23 is MCP a standard abbreviation, or does it need to be defined?

25 Need to know what LS, LBBL, GM etc. are common and scientific name and plant part

26  use gm/kg, no more than 3 significant digits, because no lab measurement is more accurate.

29, 30   in situ degradation

32 3 significant digits

57 scientific name, also need plant part being used and abbreviation

59 due to being rich in

71  use common name such as wolf berry or are you referring to the fruit gogi berry or to plant foliage?

83 common name tiger nut and plant part, nut or foliage?

100 Need to mention cannulation was done at veterinary hospital?  Animal welfare committee approval for cannulation and how cannula was maintained such as weekly cleaning of area.  Also how big was the diameter of the cannula and how many bags were inserted at a time.

106  Would it be appropriate to call these byproducts or alternative feedstuffs?

110 air dried

Table 1 assume % need to so designate 3 digits is adequate.  Corn stover is referenced later is MS where is it in the table?

130 reference for ME calculation

 134 disappearance

157 why 72 hour? Menke used 24 hours.

164 delete measured for

162 72 hour incubation

204 .08% is very slow.  Did you mean 8%?

217 What is microbial C?

Table 4 table heading is g/100 g.  is this g/100g protein or g/100g DM? former would be better in my opinion.

Table 3 3 significant digits is adequate.

Table 7 three significant digits only and lines will fit better

Table 8  why not include pH, ammonia and MCP ?

331 indicating a wide range in protein level among alternative feedstuffs

337 although lignin is a component of ADF, lower ADF does not necessarily mean lower lignin.

348 why not compare levels of vitamins to animal requirements? Also realize thag B vitamins may be degraded in the rumen.

356 if amino acids are reported based on g/100 g protein, this is a better way of evaluating adequacy and ability to provide supplementary amino acids.

379  It must be realized that the rumen will hydrogenate most of these unsaturated fatty acids, especially at the level that they will be consumed.

417 degradability has been observed to…

426 can’t say anything about lignin since it was not measured unless someone else has lignin analysis for PR

431 cannot allude to high lignin feedstuffs since you did not measure lignin

436 high proportion of slowly degradable

439 reference this statement, please

There were very extensive evaluation of feedstuffs by laboratory analysis, but no actual feeding trial done of any sort.  Laboratory analysis do not tell the whole story about the suitability of these materials for use in rations. The lack of some  kind of feeding or investigation of in vivo digestibility is a very serious flaw in the study.

Author Response

Comments 1: 16 composition

Response 1: Thank you for the suggestion. We have revised the word “composition” to “chemical composition” to improve clarity. Located at line 23.

Comments 2: 23 is MCP a standard abbreviation, or does it need to be defined?

Response 2: Thank you for pointing this out. We have added the full term at its first mention: microbial crude protein (MCP). Located at line 27.

Comments 3: 25 Need to know what LS, LBBL, GM etc. are common and scientific name and plant part

Response 3: We are grateful for this valuable feedback. Incorporating the suggestions from you and another expert, we have supplemented the abstract with the full names of these materials (Pepper residue (PR), Grape marc (MC), Pepper straw (PS), Lycium barbarum branches and leaves (LBBL), Licorice straw (LS), and Cyperus esculentus leaves (CES)), to include the common names, scientific names, and plant parts of each feed. Located at line 17-19.

Comments 4: 26 use gm/kg, no more than 3 significant digits, because no lab measurement is more accurate.

Response 4: Thank you for your correction. We have standardized all nutritional data to grams per kilogram (on a DM basis), and we have retained only two significant decimal places. Perhaps the file viewing software we used is different, which may have caused the garbled text. Located at line 31.

Comments 5: 29, 30 in situ degradation

Response 5: Thank you for the suggestion. We have revised the wording to consistently use “in situ ruminal degradation.” Located at line 25.

Comments 6: 32 3 significant digits

Response 6: Thank you for your suggestion.  We noticed that we did not retain the decimal places for "42,400 mg/kg," and we have now revised it to "4.24 gm/kg."

Comments 7: 57 scientific name, also need plant part being used and abbreviation

Response 7: Thank you for your suggestions. Based on your and another expert's feedback, we have supplemented the full names of the materials used in the abstract section, including the plant parts utilized, and annotated the abbreviations in parentheses upon their first mention. Additionally, we have reiterated this information in the materials and methods section. Located at line 17-19.

Comments 8: 59 due to being rich in

Response 8: Thank you for the comment. We have revised "due to their rich" to "due to being rich in". Located at line 63.

Comments 9: 71 use common name such as wolf berry or are you referring to the fruit gogi berry or to plant foliage?

Response 9: Thank you for your clarification request. We have stated that "Lycium barbarum branches and leaves"(LBBL)" was used in this study. We have found two expressions: "wolfberry branches and leaves" and "Lycium barbarum branches and leaves". We have chosen to use one of them. Located at line 61-62.

Comments 10: 83 common name tiger nut and plant part, nut or foliage?

Response 10: Thank you for pointing this out. We have clarified that only the leaves of Cyperus esculentus (commonly called tiger nut) were used. Located at line 62.

Comments 11: 100 Need to mention cannulation was done at veterinary hospital?  Animal welfare committee approval for cannulation and how cannula was maintained such as weekly cleaning of area. Also how big was the diameter of the cannula and how many bags were inserted at a time.

Response 11: Thank you for this important suggestion. We have revised the Materials and Methods to include details of the surgical procedure, approval, and post-operative care. Located at line 111-114.

Comments 12: 106 Would it be appropriate to call these byproducts or alternative feedstuffs?

Response 12: Thank you for identifying this issue. Compared to conventional forage such as corn stalks, alfalfa, and wheat straw, the six materials we use are not widely utilized or are minimally used in production. We hope these materials can serve as alternatives to conventional forage. Additionally, all the materials we use are by-products derived from food processing, brewing, and other industries.

Comments 13: 110 air dried

Response 13: Thank you for pointing this out. We have standardized the description by changing "naturally dried" to "air dried". Located at line 122.

Comments 14: Table 1 assume % need to so designate 3 digits is adequate.  Corn stover is referenced later is MS where is it in the table?

Response 14: Thank you for your suggestion. "Corn stover" in Table 1 is the "Corn stover" we have, and we have unified it throughout the text. We have only retained two significant decimal places. Perhaps the file viewing software we use is different, which may have caused the garbled characters. Located at line 169，520，Table 1.

Comments 15: 130 reference for ME calculation

Response 15: Thank you for your feedback. Our calculation of ME is based on the "Nutritional Requirements of Meat Sheep in China," as referenced in the 13th entry of the full-text references. We apologize for the confusion caused by the placement of the reference and have revised the location where the reference is inserted. Located at line 144.

Comments 16: 134 disappearance

Response 16: Thank you for pointing this out. We have corrected the word to “disappearance.” Located at line 149.

Comments 17: 157 why 72 hour? Menke used 24 hours.

Response 17: Thank you for your insightful comments. We have clarified that the 72-hour time point was selected to better capture the asymptotic phase of fiber degradation, aiming to capture the comprehensive and sustained effects of the treatment. Our pilot studies indicate that the phenotypic differences observed within 24 hours are preliminary, whereas more pronounced and stable responses can be formed within 72 hours. A longer duration is crucial for evaluating the long-term efficacy of the model. Therefore, we extended the cultivation time to 72 hours. Located at line 194-196.

Comments 18: 164 delete measured for

Response 18: Thank you for your reminder. We have revised the description of this text based on your input and that of another expert. Located at line 194-196.

Comments 19: 162 72 hour incubation

Response 19: Thank you for your clarification.  We have confirmed that the cultivation period is 72 hours, with gas production recorded at regular intervals. Due to slight discrepancies in the number of rows and columns, we have also revised "72 h of cultivation" to "72 hour incubation.". Located at line 194.

Comments 20: 204 .08% is very slow. Did you mean 8%?

Response 20: Thank you for your explanation. We apologize for the extra "%" in "k = 0.08%/h"; it should actually be "k = 0.08/h". In consideration of another expert's opinion, we have removed this section of the text. Located at line 236-237.

Comments 21: 217 What is microbial C?

Response 21: Thank you for your reminder. We apologize for mistakenly writing "Vitamin C" as "Microbial C," and we have corrected it. Located at line 255.

Comments 22: Table 4 table heading is g/100 g.  is this g/100g protein or g/100g DM? former would be better in my opinion.

Response 22: Thank you for the comment. We have revised the heading to clarify that amino acids are expressed as g/100 g protein, which is more appropriate for nutritional evaluation. Located at table 4.

Comments 23: Table 3 3 significant digits is adequate.

Response 23: Thank you for your suggestion. We have only saved two decimal places, and perhaps the file viewing software we use is different, which may have caused the garbled text.

Comments 24: Table 7 three significant digits only and lines will fit better

Response 24: Thank you for your suggestion. We have only saved two decimal places, and perhaps the file viewing software we use is different, which may have caused the garbled text.

Comments 25: Table 8 why not include pH, ammonia and MCP ?

Response 25: Thank you for your valuable suggestions. Since the data for pH, ammonia, and MCP have already been plotted in Figure 3, to avoid redundancy, we have not included pH, ammonia, and MCP in Table 8 again.

Comments 26: 331 indicating a wide range in protein level among alternative feedstuffs

Response 26: Thank you for your suggestions. We will revise this sentence by integrating the descriptions of CP and ADF, NDF to enhance conciseness and clarity. Located at line 396-397.

Comments 27: 337 although lignin is a component of ADF, lower ADF does not necessarily mean lower lignin.

Response 27: Thank you for your reminder. We have rephrased this sentence to enhance its rationality. Located at line 406-409.

Comments 28: 348 why not compare levels of vitamins to animal requirements? Also realize thag B vitamins may be degraded in the rumen.

Response 28: We appreciate the reviewer's insightful comment. We agree that comparing vitamin levels with animal requirements is an important consideration. In this study, our primary objective was to conduct a comparative characterization of the six unconventional feedstuffs themselves, rather than assessing the adequacy of complete diets. More importantly, as the reviewer correctly pointed out, most B vitamins are either degraded or synthesized by rumen microbes, which may complicate and potentially mislead direct comparisons between dietary content and host animal requirements. Additionally, we have revised the discussion on vitamins (Section 4.1.1) to clarify this critical point. We have added a sentence stating, "It should be noted that B vitamins undergo significant degradation and microbial synthesis in the rumen, which complicates the direct equivalence of their dietary content to post-ruminal availability.". Located at line 422-424.

Comments 29: 356 if amino acids are reported based on g/100 g protein, this is a better way of evaluating adequacy and ability to provide supplementary amino acids.

Response 29: Thank you for the suggestion. We have reformatted amino acid results as g/100 g protein and revised the discussion accordingly.

Comments 30: 379  It must be realized that the rumen will hydrogenate most of these unsaturated fatty acids, especially at the level that they will be consumed.

Response 30: We appreciate the reviewer for raising this critical point.  We fully agree and have revised the discussion in Section 4.1.3 to incorporate this important information. Located at line 465-468.

Comments 31: 417 degradability has been observed to…

Response 31: Thank you for your feedback. We have made modifications to address this issue. Located at line 493.

Comments 32: 426 can’t say anything about lignin since it was not measured unless someone else has lignin analysis for PR

Response 32: We sincerely appreciate the issue raised by the reviewer. You are correct that we did not directly measure the lignin content, and therefore cannot make definitive assertions about it. We have strictly followed your advice and revised this section accordingly. Located at line 505-508.

Comments 33: 431 cannot allude to high lignin feedstuffs since you did not measure lignin

Response 33: We appreciate the reviewer for raising this coherent and effective point. In accordance with your suggestion and following the same principle as revision comment 32, we have removed the implication regarding the CEL lignin content and revised this section of the text. Located at line 512.

Comments 34: 436 high proportion of slowly degradable

Response 34: Thank you very much for your feedback. We have revised this section of the description based on your comments. Located at line 518.

Comments 35: 439 reference this statement, please

Response 35: Thank you for the suggestion. We have added an appropriate reference to support the statement. Located at line 512.

Comments 36: There were very extensive evaluation of feedstuffs by laboratory analysis, but no actual feeding trial done of any sort.  Laboratory analysis do not tell the whole story about the suitability of these materials for use in rations. The lack of some  kind of feeding or investigation of in vivo digestibility is a very serious flaw in the study.

Response 36: We greatly appreciate the valuable insights you have provided. Your observation regarding the fact that laboratory analyses cannot fully indicate the suitability of these materials for dietary use is highly significant, and we fully concur with this point. In this paper, our research objective primarily focuses on systematically evaluating the nutritional value, bioactive components, and rumen fermentation characteristics of these six unconventional feedstuffs themselves. Based on the results of in vitro fermentation and rumen degradation rate determinations, we have preliminarily proposed an appropriate inclusion range for each feedstuff in sheep diets, providing producers with an initial, in vitro evidence-based reference for dosage.  Simultaneously, we completely agree with your perspective that the conclusions from in vitro studies ultimately need to be validated through animal experiments. We would like to inform you that the animal feeding experiment aimed at verifying the effectiveness of these recommended inclusion ratios has been successfully completed.  This study thoroughly evaluated the effects of these feeds on the growth performance, blood parameters, immune-antioxidant capacity, and rumen microbiota of sheep, with the results strongly supporting the preliminary conclusions of this paper. We are currently preparing a more in-depth follow-up research paper and plan to submit it for publication as soon as possible.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors did an excellent job revising the manuscript, addressing most of the suggestions made during the first review round. However, several points still require attention before the paper is ready for publication. Also, the use of the track changes tool made it somewhat difficult to read the revised manuscript at times. I recommend only highlighting the modifications in a different color instead of using full track changes to improve readability. The following suggestions are provided:

 

L155: cm.

L149-150: This method must be reviewed. For instance, in my AOAC (2016) book, the method 942.05 is for determining ash in animal feed.

L164: Still mesh.

L171: Still mesh.

L181: Vaseline

Write numbers up to 9 throughout.

Were the animals adapted before sampling? Were the rumen content samples collected from 12 animals individually or as a pooled (mixed) sample?

This is Menke et al (1979): “One part of liquor was mixed with two parts of a medium consisting of (added in order) 400 ml H2O, 0-1 ml solution A (13-2 g CaCl2.2H2O, 10-0 g MnClg. 4H2O, 1 -0 g CoCl2. 6H2O, 8-0 g FeCl3. 6H2O and made up to 100 ml with H2O), 200 ml solution B (39 g NaHC3/l H2O), 200 ml solution C (5-7 g Na2HPO4, 6-2 g KH2PO4, 0-6 g MgSO4.7H2O and made up to 1000 ml with H2O), 1 ml resazurine (0-1 %, w/v) and 40 ml reduction solution (95 ml H2O, 4 ml lN-NaOH and 625 mg Na2S.9H2O).” Thus, something is off.

L236-237: Review the formula ED=a+b×c/（k+c）and its description.

L244-245: you do not need to justify in the manuscript.

In the statistical analysis section, please describe the model used in detail, specifying which factors were considered fixed and which were random. Additionally, indicate whether data normality was tested and describe any procedures used for identifying or handling outliers and influential observations.

L263: SEM or SD?

L273: The absence of any variation in three test samples is quite unlikely to occur. Please review these data carefully.

The tables must be cited in the text. Review all of them.

Table 5: Is there a test comparing these values or this is descriptive?

L289: is it “g/kg”? If yes, review wherever is used.

L354: Are you sure those are SEM error bars?

The discussion still bringing many results statements (i.e. Substantial variations in chemical composition and bioactive ingredients were observed among the six unconventional by-products). Please review.

Author Response

We sincerely appreciate the reviewers' recognition and suggestions. We have removed the "Track Changes" marks and highlighted all modifications in red font. Additionally, we have marked the changes made in this revision with yellow highlights to facilitate your reading of the manuscript.

Comments 1: L155: cm.

Response 1: Thank you for noticing this detail. We have revised the text to use the correct unit abbreviation “cm” consistently throughout the manuscript. Located at line 155.

Comments 2: L149-150: This method must be reviewed. For instance, in my AOAC (2016) book, the method 942.05 is for determining ash in animal feed.

Response 2: Thank you for your feedback. We have revisited the method for determining Ash and located the relevant section on Ash determination in the AOAC 22th edition at the following URL: https://academic.oup.com/aoac-publications/book/45491/chapter-abstract/445504661? redirectedFrom=fulltext. Below is a screenshot of the relevant page from this URL.

Comments 3: L164: Still mesh.

Response 3: We appreciate the expert's meticulous review. We have verified the sieve specifications and uniformly revised it to "0.425 mm sieve" throughout the text to maintain consistency. Located at line 164.

Comments 4: L171: Still mesh.

Response 4: Thank you for the expert's correction. We have once again reviewed the entire text and uniformly revised it to "0.425 mm sieve" to maintain consistency. Located at line 172.

Comments 5: L181: Vaseline

Response 5: We appreciate the expert's advice. The Vaseline used here is petroleum jelly, and we have revised "Vaseline" to "petroleum jelly" in the text to enhance clarity. Located at line 182.

Comments 6: Write numbers up to 9 throughout.

Response 6: Thank you very much for your input, but I truly apologize that I didn't fully grasp the meaning. Could you please clarify if it is in section 2.3.1, for example, 1.DM, 2.Ash?

Comments 7: Were the animals adapted before sampling? Were the rumen content samples collected from 12 animals individually or as a pooled (mixed) sample?

Response 7: We appreciate the experts' attention to the experimental details. All sheep underwent a 15-day acclimatization period before sampling, and the rumen content samples were collected as mixed samples from 12 sheep. We have added this detail in the manuscript for clarification. Located at line 173-174.

Comments 8: This is Menke et al (1979): “One part of liquor was mixed with two parts of a medium consisting of (added in order) 400 ml H2O, 0-1 ml solution A (13-2 g CaCl2.2H2O, 10-0 g MnClg. 4H2O, 1 -0 g CoCl2. 6H2O, 8-0 g FeCl3. 6H2O and made up to 100 ml with H2O), 200 ml solution B (39 g NaHC3/l H2O), 200 ml solution C (5-7 g Na2HPO4, 6-2 g KH2PO4, 0-6 g MgSO4.7H2O and made up to 1000 ml with H2O), 1 ml resazurine (0-1 %, w/v) and 40 ml reduction solution (95 ml H2O, 4 ml lN-NaOH and 625 mg Na2S.9H2O).” Thus, something is off.

Response 8: We appreciate the experts for pointing out the inconsistencies. We have corrected the formulation of the Menke et al. (1979) buffer in Table 2, accurately cited the literature, and supplemented the specific method for solution preparation in lines 185-189 of the text, ensuring consistency with the original method and making it easier to understand.

Comments 9: L236-237: Review the formula ED=a+b×c/（k+c）and its description.

Response 9: We appreciate this correction. The formula has been verified and revised to the standard format “ED=a+(b×c)/(c+k)” with an explanatory definition added for all parameters.

Comments 10: L244-245: you do not need to justify in the manuscript.

Response 10: Thank you for the suggestion. The justification text for using Duncan’s test has been removed to streamline the section and maintain focus on analytical methods. Located at line 258-260.

Comments 11: In the statistical analysis section, please describe the model used in detail, specifying which factors were considered fixed and which were random. Additionally, indicate whether data normality was tested and describe any procedures used for identifying or handling outliers and influential observations.

Response 11: We appreciate the expert's valuable feedback. We have supplemented the model description in the "Statistical Analysis" section, clarified the fixed and random effects, and explained the normality test and the approach to handling outliers. Located at line 250-255.

Comments 12: L263: SEM or SD?

Response 12: Thank you for the expert's reminder. We have already stated in line 257 of the text that all tabular data are expressed as mean ± SEM, and the SEM you pointed out in line 263, Table 3, is also SEM.

Comments 13: L273: The absence of any variation in three test samples is quite unlikely to occur. Please review these data carefully.

Response 13: We appreciate the experts for pointing this out. Upon re-examining the raw data, we identified a rounding issue during the export process. However, since the content in Table 4 pertains to the amino acid content of six raw materials, the detected values themselves are very small. Consequently, when calculating the standard error of the mean (SEM), the SEM values were found to be extremely small, less than 0.01. Therefore, the rounded SEM values are displayed as 0.00 in this context.

Comments 14: The tables must be cited in the text. Review all of them.

Response 14: Thank you for the expert's reminder. We have re-examined the entire text and found that Table 5 and Tables 8-(1-6) were not cited in the article. We have supplemented the relevant citations in lines 292 and 371-380 of the article.

Comments 15: Table 5: Is there a test comparing these values or this is descriptive?

Response 15: We appreciate the reviewer’s observation. Table 5 presents the fatty acid composition of the six unconventional by-products as descriptive data only, with no statistical comparison among treatments. This table is intended to provide an overview of fatty acid profiles rather than to assess significant differences. In this article, Table 4 is in the same situation. I would like to inquire if it can be processed in the same manner as Table 5, by only displaying the average values to simplify the table?

Comments 16: L289: is it “g/kg”? If yes, review wherever is used.

Response 16: Thank you for the expert's reminder. We initially used the unit mg/kg in the text, and then revised it to gm/kg in the first modification based on another expert's suggestion. Currently, we have unified all units in the text to g/kg.

Comments 17: L354: Are you sure those are SEM error bars?

Response 17: Thank you for the observation. We have confirmed that the error bars represent SEM and clarified this in the figure caption.

Comments 18: The discussion still bringing many results statements (i.e. Substantial variations in chemical composition and bioactive ingredients were observed among the six unconventional by-products). Please review.

Response 18: Thank you very much for your suggestions. We have deleted the sentences that repeated the results in lines 401-402 as you exemplified. Additionally, we have re-examined the entire discussion section and found that there was also a repetition of the results in line 410, which we have rewritten in line 411 of the article.