Review Reports - Dietary Guanidinoacetic Acid Improves Meat Tenderness and Antioxidant Capacity in Rabbits via Modulating Muscle Fiber Characteristics and Fat Metabolism

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors

After careful evaluation of the manuscript, I would like to offer several comments. While the study has both notable strengths and identifiable limitations, it is not suitable for publication in its current form. Several issues require clarification and correction; therefore, I recommend a major revision.

The Materials and Methods (M&M) section lacks critical methodological detail necessary for reproducibility and proper evaluation of the study (see detailed comments). The authors state that preliminary trials evaluated guanidinoacetic acid (GAA) supplementation at 0, 50, 100, and 150 mg/kg and concluded that 100 mg/kg was optimal. However, no data are provided to substantiate this claim. These results should be presented, at a minimum, as supplementary material to support the selection of the experimental treatment level.

Furthermore, based on Figure 1 and the tabulated data, the study follows a 2 × 2 factorial design with two factors: diet (CON vs. GAA) and age (40 d vs. 60 d). Under this design, using one-way ANOVA is statistically inappropriate. A two-way ANOVA, including the interaction term between diet and age, is required to correctly assess both main effects and their interaction. Consequently, the statistical analyses should be recalculated using an appropriate model. Given this fundamental issue, interpretation of the current results is unreliable, and a reassessment of the Results section is necessary following reanalysis.

The tables and figures are generally well presented and clear. The Discussion section is well written and demonstrates good scientific reasoning.

Specific comments:

LL83–89: The novelty of the study should be more explicitly articulated and emphasized.

L95: Please specify the genotype or breed of the rabbits. It appears on the figures, but should be presented in M&M main text and in the Abstract.

Additionally, provide justification for selecting 40 and 60 days of age, as commercial slaughter age in most production systems typically ranges from 70 to 80 days.

Table 1: Units are missing for several dietary components (e.g., alfalfa hay, corn) and should be added for clarity.

L145: Provide detailed information on the slaughter procedure. Were all animals slaughtered on the same day? – if not, instead of a two-way ANOVA you need a mixed model, and a correction for the day of slaughter.

L155: The anatomical term should be corrected to longissimus thoracis et lumborum and formatted in italics throughout the manuscript.

LL155–161: The methodological descriptions are insufficient and must be expanded to ensure reproducibility:

pH measurement: Specify calibration procedures (buffers used, calibration frequency), whether temperature compensation was applied, and the number of measurements per muscle.

Drip loss: Provide a step-by-step protocol and include an appropriate reference. Number of measurements per muscle??

Texture analysis: Specify the equipment used, sample preparation and dimensions, methodological approach (e.g., Warner–Bratzler or alternative), and instrumental parameters (pre-test and post-test speed, load cell capacity, trigger force, etc.). Once again, number of repetitions per muscle should be added.

Similar detail is required for all other analytical methods to allow replication by other researchers.

Table 3: Include a clear description in the M&M section of how the dressing percentage was calculated.

Figure 2: The methods corresponding to these measurements are not described in the M&M section and should be included.

Author Response

Response to Reviewer

We are very grateful for the important suggestions and critical comments you have put forward once again. Based on the concerns of the reviewers, we have revised the manuscript. The following is our point-by-point response to your comments. Our responses are directly shown in a different color (blue). The changes made during the revision of the manuscript are highlighted in a different color (red).

1) The Materials and Methods (M&M) section lacks critical methodological detail necessary for reproducibility and proper evaluation of the study (see detailed comments). The authors state that preliminary trials evaluated guanidinoacetic acid (GAA) supplementation at 0, 50, 100, and 150 mg/kg and concluded that 100 mg/kg was optimal. However, no data are provided to substantiate this claim. These results should be presented, at a minimum, as supplementary material to support the selection of the experimental treatment level.

Response: Thank you for this rigorous and constructive comment, which is of great value to improve the methodological rigor, reproducibility and scientific integrity of our manuscript. We fully acknowledge the omission of the preliminary trial data and methodological details in the original manuscript, and have made comprehensive supplements and revisions as requested. We have collated all raw data from the preliminary experiments in this study and compiled them into Supplementary Table S1, which will be submitted as supplementary material alongside the revised manuscript. In parallel, a corresponding mention of this supplementary dataset has been added in Section 2.4 (Experimental Design) of the revised manuscript. (line 140-142)

2) Furthermore, based on Figure 1 and the tabulated data, the study follows a 2 × 2 factorial design with two factors: diet (CON vs. GAA) and age (40 d vs. 60 d). Under this design, using one-way ANOVA is statistically inappropriate. A two-way ANOVA, including the interaction term between diet and age, is required to correctly assess both main effects and their interaction. Consequently, the statistical analyses should be recalculated using an appropriate model. Given this fundamental issue, interpretation of the current results is unreliable, and a reassessment of the Results section is necessary following reanalysis.

Response: Thank you for this critical and professional comment on our statistical analysis. We fully acknowledge the inappropriate use of one-way ANOVA for our 2×2 factorial design, and we have strictly followed your suggestion to complete the reanalysis of all data with the correct statistical model, with corresponding revisions to the manuscript. The details are as follows:

We have reanalyzed all datasets using two-way analysis of variance via the general linear model (GLM) procedure in SPSS 27.0, with diet (CON vs. GAA) and initial age (40 d vs. 60 d) as fixed factors, and the diet × age interaction term included in the model. Tukey’s multiple range test was used for post hoc comparisons.

We have updated all tables and figure notes in the Results section, supplementing the P-values for the main effects of diet, main effects of age, and diet × age interaction for all measured indicators.

We have revised the statistical method description in Section 2.9, updated all statistical results in the Results section, and optimized the relevant discussion in Section 4 based on the reanalysis results, to ensure all statements are fully supported by the correct statistical analysis.

3) LL83–89: The novelty of the study should be more explicitly articulated and emphasized.

Response: Thank you for this valuable suggestion to highlight the novelty of our work. We have comprehensively revised the manuscript to explicitly articulate and emphasize the core innovations of this study. We have rewritten this paragraph to clearly define the critical research gap in existing literature, and explicitly state the innovative objectives of this study. We have strengthened the elaboration of the study's innovation and application value in the Abstract and the Conclusion section, to make the novelty of the work more prominent and clear throughout the manuscript. (line 83-94)

4) L95: Please specify the genotype or breed of the rabbits. It appears on the figures, but should be presented in M&M main text and in the Abstract.

Response: Thank you for this careful and constructive comment. We sincerely apologize for the omission of the rabbit breed information in the main text of the manuscript, and we have supplemented and standardized this information in all relevant sections as requested. We have supplemented the breed information at the first mention of experimental animals in the Abstract. We also have rewritten M&M main text to explicitly specify the breed. In addition, we have rechecked the full manuscript, to ensure the breed name "New Zealand White rabbits" is consistently and accurately presented in all relevant sections, fully consistent with the information shown in the figures. (line15 line27-28 line89 line100 line478)

5) Additionally, provide justification for selecting 40 and 60 days of age, as commercial slaughter age in most production systems typically ranges from 70 to 80 days.

Response: Thank you for your question regarding the choice of initial ages in the experiment. Rabbits starting at 40 days of age reached the standard slaughter age and body weight after 45 days of feeding, which aligns with the mainstream commercial slaughter cycle. Given that consumers in some areas of Fujian Province, where this study was conducted, prefer larger rabbits, we additionally included a 60-day-old group to complement the study. Rabbits in this group met the preferences of those consumers at 105 days of age. Moreover, the two initial ages represent different physiological stages of meat rabbits, allowing the age‑dependent effects of GAA to be validated. The rationale for this selection has been further clarified and supplemented in the Introduction and Discussion sections of the revised manuscript. (line 89-92 line 353-358)

6) Table 1: Units are missing for several dietary components (e.g., alfalfa hay, corn) and should be added for clarity.

Response: Thank you for your careful check and constructive comment on the clarity of our table. We sincerely apologize for the missing units of dietary components in Table 1, which may have caused ambiguity in reading and reproducibility. We have revised the table thoroughly as requested. We have revised the column header of Table 1 from the original "Contents" to "Contents, %", and simultaneously optimized the layout of the table in the revised manuscript. (line 121)

7) L145: Provide detailed information on the slaughter procedure. Were all animals slaughtered on the same day? – if not, instead of a two-way ANOVA you need a mixed model, and a correction for the day of slaughter.

Response: Thank you for this rigorous and professional comment, which is critical to ensure the standardization of our experimental procedures and the reliability of our statistical analysis. We sincerely apologize for the insufficient description of the slaughter procedure in the original manuscript, and have supplemented the detailed information. We have added the full standardized slaughter process in the revised manuscript: All blood collection and slaughter were performed at 9 a.m. on the same day. Rabbits were stunned by electrical stunning (120 V, 50 Hz, 3 s) to minimize pain, followed by rapid and complete exsanguination via carotid artery and jugular vein incision for 5 min. (line 159 line162-164)

8) L155: The anatomical term should be corrected to longissimus thoracis et lumborum and formatted in italics throughout the manuscript.

Response: Thank you for this professional and rigorous correction of the anatomical terminology. We sincerely apologize for the non-standard use of the muscle anatomical term in the original manuscript, and have completed a full correction and standardization throughout the manuscript as requested. Meanwhile, we have double-checked the full text to eliminate any residual non-standard expressions. (line 168 line 175-198 line 479)

9) LL155-161: The methodological descriptions are insufficient and must be expanded to ensure reproducibility

pH measurement: Specify calibration procedures (buffers used, calibration frequency), whether temperature compensation was applied, and the number of measurements per muscle.

Drip loss: Provide a step-by-step protocol and include an appropriate reference. Number of measurements per muscle??

Texture analysis: Specify the equipment used, sample preparation and dimensions, methodological approach (e.g., Warner-Bratzler or alternative), and instrumental parameters (pre-test and post-test speed, load cell capacity, trigger force, etc.). Once again, number of repetitions per muscle should be added.

Similar detail is required for all other analytical methods to allow replication by other researchers.

10) Table 3: Include a clear description in the M&M section of how the dressing percentage was calculated.

Response: Thank you for this careful and constructive comment to improve the clarity and reproducibility of our study. We sincerely apologize for the omission of the detailed calculation method of dressing percentage in the original Materials and Methods (M&M) section, and we have supplemented the complete, standardized description as requested. We have added the detailed definition of carcass weight and the standardized calculation formula of dressing percentage in Section 2.5 Sample collection of the revised manuscript. (line162-166 line172)

11) Figure 2: The methods corresponding to these measurements are not described in the M&M section and should be included.

Response: Thank you for this careful and constructive comment. We sincerely apologize for the omission of the methodological description for organ index measurement in the original Materials and Methods section, and we have supplemented the complete, standardized protocol in the revised manuscript as requested. (line 166-168 line 173)

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General comments:

This manuscript by Yanhui Liang et al. entitled “Dietary guanidinoacetic acid improves meat tenderness and antioxidant capacity in rabbits via modulating muscle fiber characteristics and fat metabolism”, concluded that dietary guanidinoacetic acid supplementation enhances meat tenderness and antioxidant capacity without compromising growth performance, highlighting guanidinoacetic acid as a promising feed additive for improving the quality of rabbit meat, in line with the growing consumer demand for healthier and more sustainably produced animal products. While some of the findings in this manuscript are interesting, there are many major issues that must be addressed by the authors. Specific comments are provided below.

-Grammatical Error in Simple Summary Line 13: "this study was conducted to investigated" should be corrected to "this study was conducted to investigate."

-The study only detected serum antioxidant indexes but did not measure antioxidant parameters in longissimus dorsi muscle and liver as mentioned in section 2.7. Please supplement the missing tissue antioxidant data or revise the relevant description in Materials and Methods to maintain consistency.

-Section 2.9 is titled "Meat quality and histological analysis," which is identical to the title of Section 2.6 (line 154). Based on the content (lines 194–197), Section 2.9 should be titled "Statistical Analysis." This appears to be a copy-paste error.

-Organ indices, serum biochemical parameters, antioxidant capacity, and gene expression were all measured with only n = 3 biological replicates. This is insufficient for reliable statistical inference given the variability visible in the figures. The authors should provide a power analysis justifying this sample size, or acknowledge it as a significant limitation. The discrepancy between the total animal number (960 rabbits, line 27) and the analytical sample size (n = 3) must be explicitly explained.

-The significant reduction in lung index in 40-day-old GAA-supplemented rabbits (Figure 2D) is noted in the Results but receives only a brief, speculative mention in the Discussion (lines 293–299). A reduced lung-to-bodyweight ratio could reflect either reduced inflammation or allometric growth differences. This finding warrants a more substantive mechanistic discussion.

Author Response

Response to Reviewer

1) -Grammatical Error in Simple Summary Line 13: "this study was conducted to investigated" should be corrected to "this study was conducted to investigate."

Response: Thank you for your careful check and correction of this grammatical error. We sincerely apologize for this inadvertent writing mistake in the original manuscript, and we have completed the strict correction and full-text grammar check as requested. (line 13)

2) -The study only detected serum antioxidant indexes but did not measure antioxidant parameters in longissimus dorsi muscle and liver as mentioned in section 2.7. Please supplement the missing tissue antioxidant data or revise the relevant description in Materials and Methods to maintain consistency.

Response: Thank you for your careful and rigorous check of our manuscript, and we sincerely apologize for the inconsistency between the methodological description and the actual experimental content in the original manuscript. We have strictly revised the relevant content to ensure full consistency between the Materials and Methods section and the actual experimental data. We have revised the description in Section 2.7 Serum Biochemical Indexes and Antioxidant Capacity, and clearly specified that all antioxidant parameters (SOD, CAT, MDA, T-AOC) in this study were detected only in serum samples, which is fully consistent with the data presented in the Results section. (line 217-221)

3) -Section 2.9 is titled "Meat quality and histological analysis," which is identical to the title of Section 2.6 (line 154). Based on the content (lines 194–197), Section 2.9 should be titled "Statistical Analysis." This appears to be a copy-paste error.

Response: Thank you for your careful check and correction of this structural error in our manuscript. We sincerely apologize for this copy-paste error that led to duplicate and mismatched section titles, which may have caused confusion in reading. We have corrected the title of Section 2.9 from the duplicate "Meat quality and histological analysis" to the accurate and content-matching title "Statistical Analysis". We have simultaneously conducted a comprehensive check of all section titles, numbering, and corresponding content throughout the full manuscript, to eliminate any other potential structural errors, ensuring the entire manuscript has a standardized, logical, and clear structure. (line 244)

4) -Organ indices, serum biochemical parameters, antioxidant capacity, and gene expression were all measured with only n = 3 biological replicates. This is insufficient for reliable statistical inference given the variability visible in the figures. The authors should provide a power analysis justifying this sample size, or acknowledge it as a significant limitation. The discrepancy between the total animal number (960 rabbits, line 27) and the analytical sample size (n = 3) must be explicitly explained.

Response: Thank you for this rigorous, responsible and highly valuable comment on the statistical reliability of our study. We fully acknowledge the critical issue of insufficient biological replicates for the relevant detection indicators, and sincerely apologize for the lack of explicit explanation for the discrepancy between the total animal number and the analytical sample size in the original manuscript. We have addressed this issue comprehensively in the revised manuscript. We fully agree that n=3 biological replicates are insufficient for reliable statistical inference for indicators with high individual variability, which is a significant limitation of this study. Furthermore, this limitation has been acknowledged in the Discussion, and Conclusion sections of the manuscript. (line 465-468 line 483-484)

5) -The significant reduction in lung index in 40-day-old GAA-supplemented rabbits (Figure 2D) is noted in the Results but receives only a brief, speculative mention in the Discussion (lines 293–299). A reduced lung-to-bodyweight ratio could reflect either reduced inflammation or allometric growth differences. This finding warrants a more substantive mechanistic discussion.

Response: We sincerely appreciate your rigorous and thorough comments. However, in accordance with another reviewer’s suggestion on statistical methodology, we reanalyzed all data using a 2 × 2 factorial design. The reanalysis revealed that dietary supplementation with guanidinoacetic acid (GAA) had no significant main effect on either lung index or kidney index (P > 0.05), and the diet × age interaction was also not significant (P > 0.05). The significant differences originally detected by one-way ANOVA were false positives resulting from an inappropriate statistical model, rather than a genuine biological effect of GAA. Based on these findings, the manuscript has been comprehensively and accordingly revised (line 267-269 line 371-377).

Reviewer 3 Report

Comments and Suggestions for Authors

The authors evaluated the effects of dietary guanidinoacetic acid addition on rabbits, the manuscript is well structured and the findings are constructive for rabbits production, however, the following concerns are suggested to be addressed.

1.Why the studies were separated into two different age treatment groups instead of one study with a duration across from age of 40 days to age of 60 days?
2.The breed of the rabbits should be stated, and the 45d duration is less or longer than the finishing period? If less than the finishing period, will the meat tenderness reduce when the rabbits achieve their market weight?
3.“60-day-old rabbits” in line 32 and “40-day-old rabbits ” is not accurately expressed, did authors measure the meat shear force of rabbits with an age of 60 days and the muscle fibre area of rabbits with an age of 40 days?, check through the text please.
4.Information of GAA application in rabbit feeding should be included in “introduction section” and then clarify what drive the authors to perform this study.
5.Economic profits is the important index for farmers considering whether use GAA or not, so the economic profit analysis would be suggested to include.
6.Check the format of references and then standardize according to the requirements of the Journal.

Author Response

Response to Reviewer

1.Why the studies were separated into two different age treatment groups instead of one study with a duration across from age of 40 days to age of 60 days?

Response: Thank you for this professional and in-depth question about our experimental design. The core rationale for setting two independent age treatment groups (rather than a single continuous trial spanning 40 to 60 days of age) is based on our scientific objectives, statistical design requirements. The primary scientific goal of this design is to systematically verify whether the biological effects of GAA are dependent on the growth stage of rabbits, and to quantify the main effects of diet, main effects of age, and the critical diet×age interaction effect. By setting two independent age groups with an identical 45-day feeding cycle, we completely eliminated the confounding factor of feeding duration, and can accurately attribute the differences in GAA effects to the initial growth stage of rabbits. In contrast, a single continuous trial spanning 40 to 60 days of age cannot separate the effects of growth stage and feeding duration, nor can it support the factorial analysis to verify the interaction effect, which would make it impossible to clarify the optimal supplementation window of GAA. In addition, Rabbits at 40 days and 60 days of age have fundamentally different physiological and metabolic states: 40-day-old rabbits have immature organ function, active muscle fiber hyperplasia, and weak antioxidant system; 60-day-old rabbits have mature metabolic function, stable muscle fiber development, and active fat deposition. The identical 45-day feeding cycle for both groups ensures that we can directly compare the differential response to GAA in these two distinct physiological stages. Another reason is that, given the varying consumer preferences for rabbit size, we designed the experiment with two groups of different ages. Additionally, we have also explained the rationale for our grouping in the Introduction and Discussion sections of the revised manuscript. (line 89-92 line 353-358)

2.The breed of the rabbits should be stated, and the 45d duration is less or longer than the finishing period? If less than the finishing period, will the meat tenderness reduce when the rabbits achieve their market weight?

Response: Thank you for your careful and constructive comments. First, we have supplemented and standardized the rabbit breed information throughout the revised manuscript as required: we clearly specified that the experimental animals are New Zealand White rabbits at the first mention of experimental animals in the Abstract, and rewrote the relevant content in the main text of the Materials and Methods section to explicitly confirm the breed information, ensuring full consistency across the full manuscript. Second, the 45-day feeding duration in this study is fully matched with, or even slightly longer than, the conventional finishing period in mainstream commercial meat rabbit production systems. All rabbits in both age groups reached the standard market slaughter weight and age at the end of the 45-day feeding trial, with no situation where the feeding duration was shorter than the finishing period. All meat tenderness measurements were completed when the rabbits fully achieved their market weight, so the data directly reflect the real meat quality state of commercial rabbits at the time of marketing, and there is no risk of meat tenderness reduction when the rabbits reach the market weight. (line15 line 27-28 line 89 line100 line 478 line 353-358)

3.“60-day-old rabbits” in line 32 and “40-day-old rabbits ” is not accurately expressed, did authors measure the meat shear force of rabbits with an age of 60 days and the muscle fibre area of rabbits with an age of 40 days?, check through the text please.

Response: Thank you for your careful and rigorous check of our manuscript, and we sincerely apologize for the inaccurate and ambiguous age-related expressions in the original manuscript, which caused misunderstandings to your review. We have clarified the core definition and completed a full-text standardization revision of all relevant expressions. The "40-day-old rabbits" and "60-day-old rabbits" in the original manuscript refer to the initial age of rabbits when they were enrolled in the trial, not the age when the indicators were measured. All meat quality, muscle fiber characteristics, antioxidant capacity and other related indicators were measured at the end of the 45-day feeding trial, when the rabbits were slaughtered. In the revised manuscript, we have provided a clearer definition by replacing “40-day-old rabbits” with “40-day-old group” and “60-day-old rabbits” with “60-day-old group” to eliminate ambiguity. (line 134)

4.Information of GAA application in rabbit feeding should be included in “introduction section” and then clarify what drive the authors to perform this study.

Response: Thank you for your valuable suggestions. We have revised and refined the Introduction section. After reviewing the research progress on guanidinoacetic acid (GAA) in other livestock and poultry species, we highlighted the notable scarcity of studies on GAA application in rabbit production. Based on this, we have clearly articulated the primary objective of the present study. (line 72-94)

5.Economic profits is the important index for farmers considering whether use GAA or not, so the economic profit analysis would be suggested to include.

Response: We fully agree that economic profit is a key practical indicator for farmers to evaluate the application value of GAA. However, the GAA used in this study was provided free of charge by the manufacturer for scientific research purposes, and there is currently no formal market pricing for this product. Therefore, it is impossible to conduct an accurate and reliable cost-benefit analysis at this stage. We have clearly explained this limitation in the Discussion section, and noted that a comprehensive economic profit evaluation can be performed in future studies once the official market price of GAA is confirmed under commercial production conditions. (line 468-473)

6.Check the format of references and then standardize according to the requirements of the Journal.

Response: Thank you for your careful check and constructive comment on the standardization of our references. We sincerely apologize for the non-standard reference format in the original manuscript, which may have caused inconvenience to your review and the editorial work.

We have strictly followed the latest author guidelines and reference format requirements of the Journal, and completed a comprehensive full-text check and standardized revision of all references. (line 515-634)

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript addresses the use of guanidinoacetic acid as a nutritional strategy to improve meat quality in rabbits. The experimental question is appropriate and the authors evaluated several important outcomes. The graphical summary and general organization are positive aspects of the document.

However, in my opinion, the manuscript should be rejected primarily because the effective sample size for the main biological endpoints is insufficient to support the conclusions. Although the study initially included 960 rabbits, the key measurements appear to be based on only 5 animals per group for carcass/meat quality traits and 3 per group for organ indices, serum biochemical parameters, antioxidant markers, and gene-expression analyses. The small n severely limits reliability, and is especially problematic because the authors make broad mechanistic claims regarding antioxidant capacity, muscle fiber remodeling, and lipid metabolism from very limited subsamples.

Additional major problems further support rejection. The statistical analysis is inadequate for the experimental design, as the study involves two ages and two dietary treatments but does not properly test age, treatment, or age × treatment interaction. Several methodological details are insufficient for reproducibility, including shear force procedures, histological quantification, qPCR validation, antioxidant assays, and slaughter conditions. The conclusions are also overinterpreted. Finally, the manuscript contains inconsistently reported outcomes, and several language and formatting issues that indicate the paper requires substantial restructuring before it could be reconsidered.

Author Response

Dear Reviewer,

We sincerely thank you for taking the time to review our manuscript. We appreciate your recognition of our research topic, the important outcomes, the graphical abstract, and the overall organization of this work. Your highly constructive and professional comments are of great value and have guided us to substantially improve the quality of this paper. We fully understand all the core concerns you have raised, and have carried out comprehensive, rigorous and targeted revisions and improvements to the manuscript in response to every deficiency you pointed out. We sincerely hope that you can reconsider our manuscript and give us an opportunity for re-evaluation after revision. The following is our point-by-point response to your comments. Our responses are directly shown in a different color (blue). The changes made during the revision of the manuscript are highlighted in a different color (red).

(1) However, in my opinion, the manuscript should be rejected primarily because the effective sample size for the main biological endpoints is insufficient to support the conclusions. Although the study initially included 960 rabbits, the key measurements appear to be based on only 5 animals per group for carcass/meat quality traits and 3 per group for organ indices, serum biochemical parameters, antioxidant markers, and gene-expression analyses. The small n severely limits reliability, and is especially problematic because the authors make broad mechanistic claims regarding antioxidant capacity, muscle fiber remodeling, and lipid metabolism from very limited subsamples.

Response: Thank you for this rigorous and responsible comment on the statistical reliability of our study, which is critical for ensuring the academic rigor of the manuscript. We fully acknowledge the issue of insufficient biological replicates for relevant indicators and sincerely apologize for the overinterpretation of mechanistic claims based on limited subsamples in the original manuscript. We have systematically revised the full manuscript to address this issue comprehensively. We have clearly clarified the distinction between the total number of experimental animals and the actual sample size used for analysis in the revised manuscript. In this study, a total of 960 New Zealand rabbits were used, with four treatment groups and 240 rabbits per group. Each treatment had 10 replicates, and each replicate consisted of 24 rabbits. For growth performance parameters, the mean value of each replicate was used as one experimental unit for statistical analysis (Line 153-154). For slaughter sampling, we adopted a random sampling strategy by randomly selecting 5 rabbits per group from all replicates to analyze slaughter performance, meat quality, and organ indices (Line 156). For serum biochemical parameters, antioxidant indices, and gene expression, 3 rabbits were further randomly selected from the 5 slaughtered rabbits in each group for measurement (Line 210 Line 223). This study was carried out based on a commercial breeding trial, and limited by the test cost and the field conditions of large-scale commercial breeding, we had to control the sample size for subsequent laboratory testing, for which we deeply apologize. We have made a clear and rigorous statement of this limitation in the discussion section in the revised manuscript (Line 465-467). Meanwhile, we have clearly stated in the revised manuscript that in follow-up studies, we will further expand the sample size for core indicator detection, optimize the experimental design, and conduct systematic and rigorous verification and improvement of the preliminary results obtained in this study (Line 250-253, Line 467-468 and Line 483-484). Additionally, we have thoroughly revised the Discussion and Conclusion sections to avoid overinterpretation of the results (Line 371-378, Line 391-393, Line 403-419, Line 423-425 and Line 439-445).

(2) Additional major problems further support rejection. The statistical analysis is inadequate for the experimental design, as the study involves two ages and two dietary treatments but does not properly test age, treatment, or age × treatment interaction.

Response: Thank you for this rigorous and professional comment on our statistical analysis. We sincerely apologize for the inappropriate statistical method in the original manuscript. We have fully addressed this issue with a comprehensive revision of the entire statistical analysis workflow in the revised manuscript. We have re-analyzed all the experimental data using the 2×2 two-way ANOVA method, systematically evaluated the independent effects of age, dietary treatment, and the age × treatment interaction effect on each detected indicator. We have revised the description of the new statistical methods in the statistical analysis section (Line 245-249). At the same time, based on the re-analyzed statistical results, we have comprehensively checked, revised and updated the relevant contents in the results and discussion sections of the manuscript, to ensure that the statistical method is completely matched with the experimental design, and the interpretation of the statistical results is rigorous and accurate (line 255-475).

(3) Several methodological details are insufficient for reproducibility, including shear force procedures, histological quantification, qPCR validation, antioxidant assays, and slaughter conditions. The conclusions are also overinterpreted.

Response: Thank you for this rigorous and constructive comment, which is critical to ensure the reproducibility and academic standardization of our study. We sincerely apologize for the insufficient methodological details in the original manuscript, and have comprehensively supplemented and standardized the complete, replicable protocols for all the experimental procedures you mentioned in the Materials and Methods section of the revised manuscript. Regarding the shear force measurement procedure, we have added detailed descriptions in the revised manuscript, including the equipment used, sample preparation method, sample dimensions, and the detection method (Line 186-192). In addition, we have also supplemented the number of replicate measurements for each muscle type (Line 193). For the histological quantification, we have supplemented the detection method, which involves imaging the stained tissue sections with an optical microscope, followed by quantitative measurement of muscle fiber diameter, number, total area, and fiber density using image analysis software (Line 201-207). For the qPCR validation, we have supplemented the complete reaction system composition and amplification program settings in the revised manuscript (Line 229-237). For the antioxidant analysis, we have specified the exact samples used and the sources of the assay kits in the revised manuscript (Line 217-221). Regarding the slaughter conditions, we have clarified in the revised manuscript the slaughter time, the electrical stunning procedure before slaughter, and the bleeding procedure (Line 157-158 and Line 160-162). In addition, we have also provided detailed information for other analytical methods, including the calibration procedure and temperature compensation during pH measurement, the number of measurements per muscle (Line 177-183), and the step-by-step experimental procedure for drip loss determination as well as the number of replicates (Line 183-186), etc.

(4) Finally, the manuscript contains inconsistently reported outcomes, and several language and formatting issues that indicate the paper requires substantial restructuring before it could be reconsidered.

Response: Thanks for your comments. We have systematically checked the entire manuscript. The one‑way ANOVA results originally present in the Results section have been removed, and all statistical analyses have been uniformly replaced with the main effects and interaction effects derived from two‑way ANOVA (Line 256-352). The Discussion section has also been thoroughly revised accordingly (Line 354-476). All sample sizes and units have been standardized in the corresponding tables and figures. In the revised manuscript, all data and conclusions are consistent throughout.

Once again, we sincerely thank you for your professional and detailed comments, which have greatly improved the academic rigor and quality of our manuscript. We have comprehensively revised the manuscript in response to all the questions and suggestions you raised, and earnestly request you to re-review the revised manuscript and give us an opportunity for resubmission and re-evaluation. We cherish your valuable comments very much, and are fully willing to cooperate with you to complete any further revisions and improvements to the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has greatly improved. I have no other suggestions; therefore, in my opinion, it can be accepted in the present form.

Author Response

We sincerely thank you for your positive evaluation and for recommending acceptance of our manuscript in its current form. We greatly appreciate your time and expertise in reviewing our work.

In addition, following further suggestions from the Academic Editor, we have added more detailed information regarding the number of replicates and the total number of animals slaughtered in the Materials and Methods section (Lines 113–115, 143–145, 162–165, 220–232). The changes made during the revision of the manuscript are highlighted in a different color (red). These minor clarifications do not affect the scientific content or conclusions of the manuscript.

Thank you again for your support and constructive comments.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript can be accepted in the current form.

Author Response

Thank you very much for your positive evaluation and for recommending acceptance. In response to additional requests from the Academic Editor, we have now clearly specified the number of replicates and the total number of animals slaughtered in the Materials and Methods section (Lines 113–115, 143–145, 162–165, 220–232). The changes made during the revision of the manuscript are highlighted in a different color (red).

Reviewer 3 Report

Comments and Suggestions for Authors

No additional comments

Author Response

Thank you for your assessment. Following the Academic Editor’s requests, we have now specified replicate and animal numbers in the Materials and Methods (Lines 113–115, 143–145, 162–165, 220–232). The changes made during the revision of the manuscript are highlighted in a different color (red). Thank you again.

Reviewer 4 Report

Comments and Suggestions for Authors

Although the authors have improved the statistical description and provided additional methodological details, the central concern remains unresolved. The main biological endpoints supporting the mechanistic conclusions are still based on only three animals per group. This sample size is not sufficient to provide reliable estimates of biological variability or to support robust inference regarding antioxidant capacity, serum biochemical responses, and gene-expression regulation. This is not merely a reporting limitation that can be addressed by more cautious wording; it is a technical limitation of the experimental design. Therefore, the revised manuscript still does not provide sufficient evidential support for the proposed conclusions.

Author Response

We fully acknowledge this limitation. As clarified in the revised manuscript, the sample size was not identical for all indicators: most measurements were based on five animals per group, whereas several other indicators (serum parameters and gene expression analyses) were based on three animals per group. We agree that the relatively limited sample size reduces the precision of the estimates and limits the generalizability of the findings. Accordingly, we have explicitly acknowledged this limitation in the manuscript and moderated the wording of the mechanistic conclusions.

At the same time, we respectfully note that the observed differences for the relevant indicators were sufficiently pronounced to be detected under the statistical analyses (Two-way ANOVA) applied in this study. Despite the relatively small sample size for certain indicators, the observed differences were statistically significant (e.g., shear force: p_(diet) < 0.01; SOD: p_(diet) < 0.01; T‑AOC: p_(diet) < 0.05), and the results were consistent across multiple independent measurements (e.g., tenderness, antioxidant capacity, and gene expression). This consistency supports the biological validity of the findings. We therefore believe that the revised manuscript still provides scientifically meaningful evidence, while recognizing that further studies with a larger sample size would be valuable for confirmation (Line 476-477).

In addition, our trial was approved by the Fujian Agriculture and Forestry University Animal Care and Use Committee (Approval ID: PZCASFAFU24122). And we clearly specified the number of replicates and the total number of animals slaughtered in this revised manuscript (Line 113-115, 143-145, 162-165, 220-232). We have also explicitly acknowledged this limitation in the revised manuscript (Discussion section, lines 473–479). The changes made during the revision of the manuscript are highlighted in a different color (red).

Once again, we sincerely thank you for your critical comments and remain open to further revisions.